PMID- 10725950 TI - Outcomes of primary and secondary treatment of pericardial effusion in patients with malignancy. AB - OBJECTIVE: To evaluate the treatment strategies for primary and secondary management of malignancy-related pericardial effusions. PATIENTS AND METHODS: Retrospective review of Mayo Clinic Rochester charts and external records of patients with pericardial effusion associated with malignant disease who required treatment between February 1979 and June 1998 was performed. Telephone interviews with patients, their families, or their physicians were conducted to determine the outcomes of treatment. Recurrence of pericardial effusion and survival were the main outcome measures. RESULTS: Of 1002 consecutive pericardiocenteses performed during the period under study, 341 were performed in 275 patients with confirmed malignant disease. Patients were followed up for a minimum of 190 days, unless death occurred first. Of 275 patients, recurrence of pericardial effusion or persistent drainage necessitated secondary management in 59 (43 of 118 simple pericardiocenteses, 16 of 139 pericardiocenteses with extended catheter drainage, and 0 of 18 pericardial surgery following temporizing pericardiocentesis). Recurrence was strongly and independently predicted by absence of pericardial catheter for extended drainage, large effusion size, and emergency procedures. Recurrence after secondary management occurred in 12 patients: 11 underwent successful pericardiocentesis with extended catheter drainage, and 1 had pericardial surgery. Median survival of the cohort was 135 days, and 26% survived the first year after diagnosis of pericardial effusion. Male sex, positive fluid cytology for malignant cells, lung cancer, and clinical presentation of tamponade or hemodynamic collapse were independently associated with poor survival. CONCLUSION: Echocardiographically guided pericardiocentesis with extended catheter drainage appears to be safe and effective for both primary and secondary management of pericardial effusion in patients with malignancy. PMID- 10725951 TI - John Northrop--definitive study of enzymes. PMID- 10725952 TI - Clinical preventive medicine in primary care: background and practice: 2. Delivering primary preventive services. AB - Strategies to incorporate preventive services into primary care settings have been underutilized. The first component of delivering preventive services in the primary care setting is the health risk assessment followed by establishment of practice guidelines and protocols for preventive services--who is eligible for what service (based on age, sex, and other clinical characteristics) and when. A computerized reminder system can be useful to track past and currently due preventive services for each patient and can also serve as a follow-up system for test results. Well-trained paramedical personnel can perform appropriate patient counseling and education. The goal of counseling and education is to change patient behavior. The first step in this difficult process is once again to ascertain health risks and then to determine the patient's stage of readiness- defined as precontemplation, contemplation, preparation, action, and maintenance. The counselor assists in identification of target behavior, advocates and commends behavior change, reinforces health benefits of behavior change, offers resources, strategies, and support, and creates a plan of action and monitoring mechanisms. Improved implementation of preventive services in primary care could have a major impact on the health of the population. PMID- 10725953 TI - von Hippel-Lindau disease. AB - An autosomal dominant tumor predisposition syndrome, von Hippel-Lindau disease (VHL) is characterized by the presence of benign and malignant tumors. Hallmark lesions include retinal angiomas, hemangioblastomas of the cerebellum and spinal cord, and renal cell carcinomas. Affected persons may also have angiomatous or cystic lesions of the kidneys, pancreas, and epididymis, as well as adrenal pheochromocytomas. In this article, we discuss the clinical features and diagnostic criteria for this clinically underdiagnosed condition. An update on recent findings regarding the molecular genetics of VHL is provided, including a discussion of the evolving understanding of genotype-phenotype correlations. Understanding the molecular and functional aspects of this condition will lead to the development of strategies for the management and treatment of inherited and sporadic VHL-associated tumors. PMID- 10725954 TI - Occupational exposure to trace concentrations of waste anesthetic gases. AB - The relationship between exposure to trace concentrations of waste anesthetic gases in the operating room and the possible development of adverse health effects has concerned health care professionals for numerous years. Results of studies have been conflicting. In the late 1960s and early 1970s, some US and European epidemiological studies of operating room personnel showed an increase in the incidence of adverse health effects, including spontaneous abortion and development of congenital abnormalities in offspring. However, subsequent analysis of these studies by 2 independent groups showed that the apparent increase in adverse health effects was most likely due to flaws in these studies' methods and data collection. A later prospective study showed no causal relationship between exposure to trace concentrations of waste anesthetic gases and adverse health effects. Each institution should have a waste anesthetic gas management program that includes scavenging of waste anesthetic gases, work practices to reduce contamination, documented maintenance and regular checking of all equipment, and education of all personnel on this subject. A mechanism for reporting work-related health problems should be in place in each institution. PMID- 10725955 TI - Management of difficult-to-control hypertension. AB - Hypertension is a primary risk factor for heart disease and stroke, the first and third most common causes of death in the United States. The National Health and Nutrition Examination Survey (NHANES) revealed an increase in awareness of hypertension from 51% to 73%, and, among persons with hypertension, the treatment rate has increased from 31% to 55% (from 1976-1980 vs 1988-1991). Of importance, the rate of those achieving goal blood pressure (< 140/90 mm Hg) has only improved from 10% in NHANES-II (1976-1980) to 29% in NHANES-III (1988-1991). Thus, more than 70% of persons with hypertension in whom good blood pressure control has not been achieved are termed "difficult hypertensives." Failure to achieve treatment blood pressure goals of less than 140/90 mm Hg is usually attributed to the presence of resistant hypertension, a resistant physician, secondary causes of hypertension such as renovascular disease, medication adverse effects, or a nonadherent patient. A practical understanding of the pathophysiology of resistant hypertension, appropriate screening techniques for secondary forms of hypertension, and alternative management strategies for a chronic disease such as hypertension can result in treatment goals being achieved in most difficult hypertensives. PMID- 10725956 TI - Laryngospasm and paradoxical bronchoconstriction after repeated doses of beta 2 agonists containing edetate disodium. AB - A 22-year-old woman with mild intermittent asthma, who had no previous history of an adverse reaction to an albuterol metered-dose inhaler, developed paradoxical bronchoconstriction after inhalation of the fourth dose of an albuterol nebulizer solution. She experienced the same symptoms and laryngospasm with repeated inhalations of albuterol and metaproterenol nebulizer solutions that contained edetate disodium. Each episode responded to racemic and subcutaneously administered epinephrine. To our knowledge, this is the first report of paradoxical bronchoconstriction and laryngospasm due to repeated doses of beta 2 agonist solutions with edetate disodium. PMID- 10725957 TI - Diffuse stromal Leydig cell hyperplasia: a unique cause of postmenopausal hyperandrogenism and virilization. AB - A 60-year-old woman presented with diffuse scalp alopecia, hirsutism, and clitorimegaly, and the mean serum testosterone levels were greater than 200 ng/dL. Findings on computed tomography of both adrenal glands were normal. After bilateral oophorectomy, a unique histological picture consisting of diffuse stromal Leydig cell hyperplasia was found. Reinke crystals were present, but neither hilus cell hyperplasia nor stromal hyperthecosis was noted. Sequencing of the 11 exons of the gene for the luteinizing hormone receptor revealed no abnormality. Relevant data suggest that treatment of the postmenopausal woman with hyperandrogenism and virilization is bilateral laparoscopic oophorectomy if she has no pronounced ovarian enlargement or adrenal tumor on imaging. In this setting, an intensive endocrine evaluation or a search for metastatic disease seems to be unnecessary. PMID- 10725958 TI - Sarcoidosis presenting as spiculated breast masses. AB - A 67-year-old woman sought medical treatment of cardiomyopathy, which had been diagnosed 2 years earlier; the causative factor was sarcoidosis. A screening mammogram revealed multiple spiculated masses in both breasts. A review of previous films obtained elsewhere showed that these masses had been increasing in prominence during the past 3 years. The patient had no visible axillary nodal abnormalities. Sarcoidosis was considered a diagnostic possibility, and a large core needle biopsy was done with stereotactic guidance. The histological diagnosis was non-necrotizing granulomatous inflammation, consistent with sarcoidosis. PMID- 10725959 TI - Traumatic carotid artery dissection causing blindness. AB - A case of delayed postoperative visual loss due to bilateral traumatic carotid artery dissection is presented. In patients with a major craniofacial injury due to a high-speed motor vehicle accident, we suggest that carotid artery duplex ultrasonography be used in the initial evaluation for possible carotid artery dissection. Magnetic resonance imaging of the head and neck with magnetic resonance angiography should be performed subsequently if indicated. Early diagnosis and initiation of therapy can minimize complications. PMID- 10725960 TI - 76-year-old man with retroperitoneal bleeding after prostatectomy. PMID- 10725961 TI - History of the development of antiemetic guidelines at Mayo Clinic Rochester. AB - This article describes the historic experience of the development of antiemetic guidelines for patients taking chemotherapy drugs at Mayo Clinic Rochester. The initial guidelines for the use of serotonin (5-hydroxytryptamine3) receptor antagonists for the prevention of chemotherapy-induced nausea and vomiting were developed in early 1995 and implemented in September 1995. In February 1997, the guidelines were reviewed and modified. In the spring of 1998, major changes were made based on new data from the literature and discussions with antiemetic authorities in the United States. These guidelines were implemented in July 1998. The guidelines were again reviewed and modified in December 1998. In addition, we compared costs associated with the 1997 guidelines and the December 1998 guidelines. The developed guidelines, utilizing clinically available agents, seem to provide high-quality patient care at a reasonable cost. PMID- 10725962 TI - Short-term international medical service. AB - There are many opportunities for short-term medical service internationally. Prerequisite preparation must include consideration of motivation, flexibility, culturally sensitive health care, and problems in communication and patient approach. Practical considerations require recognizing the importance of choice of locale, lack of materials and equipment, health concerns, travel, and expenses. A reference for opportunities available and a list of articles suggested as supplementary reading are provided. PMID- 10725963 TI - Lyme disease and pseudotumor. PMID- 10725964 TI - Hostility and cardiac rehabilitation. PMID- 10725965 TI - Fibromyalgia and pain management. PMID- 10725966 TI - Exacerbation of multiple sclerosis following wasp stings. PMID- 10725967 TI - Aggrenox: a combination of antiplatelet drugs for stroke prevention. PMID- 10725968 TI - Epirubicin for adjuvant therapy in node-positive breast cancer. PMID- 10725969 TI - Sirolimus (Rapamune) for transplant rejection. PMID- 10725970 TI - Tobacco use among middle and high school students--United States, 1999. PMID- 10725971 TI - Update: influenza activity--United States, 1999-2000 season. PMID- 10725972 TI - Progress toward poliomyelitis eradication--Chad, 1996-1999. PMID- 10725973 TI - Outbreak of Shigella sonnei infections associated with eating a nationally distributed dip--California, Oregon, and Washington, January 2000. PMID- 10725974 TI - Update: penicillin G availability. PMID- 10725975 TI - Therapy for venous thrombosis: guidelines for a competent and cost-effective approach. PMID- 10725976 TI - Aberrations of the tissue factor pathway in patients positive for lupus anticoagulant. AB - To evaluate the relationship between the tissue factor (TF) pathway and lupus anticoagulant (LA), in the present study, we measured the plasma levels of TF antigen and TF pathway inhibitor (TFPI) antigen in patients positive for LA. Plasma TF and TFPI levels in LA-positive patients were significantly higher than levels in healthy volunteers (p < 0.01). In LA-positive patients, there were no significant differences in plasma TF and TFPI levels between patients with and without thrombosis. In patients with thrombosis, there was no significant difference in the plasma TF level between LA-positive and LA-negative patients; however, the plasma TFPI level in LA-positive patients was significantly lower than that in LA-negative patients (p < 0.01). We also examined the TF pathway in human umbilical venous endothelial cells (HUVEC) incubated with plasma of LA positive patients, LA-negative patients, and healthy volunteers. TF activity was significantly higher (p < .05) in HUVECs incubated with the plasma of LA-positive patients than in cells incubated with the plasma of the other two groups (p < .01). However, there was no significant difference in TFPI antigen levels among the media of HUVECs incubated with the plasma of all groups. The viability of HUVEC incubated with the plasma of LA-positive patients with thromboses, LA positive patients without thromboses, and LA-negative patients with thromboses were significantly lower than that of HUVECs incubated with the plasma of healthy volunteers (p < .01). These findings suggest that abnormalities of the TF pathway plays an important role in the mechanism of hypercoagulability in LA-positive patients. LA may affect vascular endothelial cells causing thrombogenesis. PMID- 10725977 TI - Cytoskeletal organization and incorporation of beta 3 integrin in thrombin stimulated platelets: effect of acetylsalicylic acid. AB - Platelet stimulation by agonists is followed by changes in cytoskeletal organization that includes actin polymerization and association of the membrane skeleton (which is connected with the integrin alpha IIb beta 3) with the underlying cytoplasmic actin filaments. The effect of orally administered acetylsalicylic acid to healthy volunteers on incorporation of contractile protein and beta 3 integrin into the cytoskeletal core of thrombin-stimulated platelets was studied. Stimulation was followed by increased contractile protein and beta 3 incorporation into the cytoskeleton. Acetylsalicylic acid intake resulted in decreased incorporation of myosin and actin (32% and 20%, respectively), and a decrease (36%) in the association of beta 3 integrin with the cytoskeletal elements was evident. In conclusion, we have shown that acetylsalicylic acid, besides the known inhibitory effect on thromboxane synthesis, promotes changes in the cytoskeletal organization of thrombin stimulated platelets that could limit thrombus formation. PMID- 10725978 TI - Elevated plasma vascular endothelial cell growth factor and thrombomodulin in juvenile diabetic patients. AB - The major cause of morbidity and mortality in patients with type 1 diabetes mellitus is vascular disease and the death rate in this group of patients can be up to six times that of the general population. Elevated levels of blood glucose can cause endothelial cell damage, and markers of endothelial damage such as von Willebrand factor (vWF) and thrombomodulin (TM) have been reported to increase in adult diabetic patients. Growth factors are strongly linked to smooth muscle cell proliferation that contributes significantly to the vascular occlusive process and it has been shown that vascular endothelial cell growth factor (VEGF) stimulates release of vWF from endothelial cells. Vascular endothelial cell growth factor levels have been shown to be increased in vitreous fluid from the eyes of diabetic patients with proliferative retinopathy compared to those without. In this study we have shown that plasma levels of both TM and VEGF were significantly increased in juvenile diabetic patients with no clinical evidence of vascular disease compared to normal age and sex-matched control subjects. Median TM levels were 45.5 ng/mL (I.Q.R. 34 to 56 ng/mL) and 61 ng/mL (I.Q.R. 41 to 72 ng/mL) in the control group and in the diabetic patients respectively (p = .0005) and median levels of VEGF were 19.6 pg/mL (I.Q.R. 15.9 to 28.1 pg/mL) in the control group and 37.1 pg/mL (I.Q.R. 22.1 to 50.3 pg/mL) in the diabetic patients (p = .027 Mann-Whitney U test). This suggests that microvascular disease begins in childhood and can be detected using laboratory tests before any clinical changes are apparent. PMID- 10725979 TI - Regional thrombolysis with urokinase for central venous catheter-related thrombosis in patients undergoing high-dose chemotherapy with autologous blood stem cell rescue. AB - Fifty-one of 300 patients undergoing high-dose chemotherapy with (n = 245) or without (n = 55) autologous stem cell rescue developed central venous catheter related thrombosis diagnosed by Doppler sonography or contrast venography. Eighteen of these individuals underwent regional thrombolysis defined as the infusion of urokinase into a superficial vein of the ipsilateral upper extremity in a dose not sufficient to produce systemic fibrinolysis by laboratory criteria. Urokinase was administered at a dose of 75,000-150,000 U/hour for 24 to 96 hours and contrast venography was performed to assess response. All individuals had a partial or complete resolution of clinical signs and symptoms. Fifty percent of patients also achieved a partial radiographic response defined as clot lysis with irregular canalization of the vein. Therapeutic doses of heparin for 5 to 7 days and warfarin for at least 3 months were commenced at the conclusion of urokinase therapy. Twelve catheters were salvaged and utilized subsequently until no longer required. Six catheters were removed because of poor catheter function or rethrombosis. The median interval from diagnosis of the thrombus until extraction of the 12 salvaged catheters was 3 months (range 1-8 months). Only a single patient who developed gastrointestinal bleeding required discontinuation of urokinase. Regional thrombolysis is safe, easy to administer, effective in many instances, less costly than the doses of antifibrinolytic agents required to induce systemic fibrinolysis, and should be considered in patients receiving high dose chemotherapy with autologous stem cell rescue who develop central venous catheter-related thrombosis. PMID- 10725980 TI - Normal life expectancy and thrombosis-free survival in aspirin treated essential thrombocythemia. AB - Focusing on platelet-mediated erythromelalgia as a specific, presenting, and single sign of essential thrombocythemia (ET) we were able to define the characteristic peripheral blood and bone marrow findings in ET patients. From 1974 to 1986 we treated 20 symptomatic ET patients with microvascular circulation disturbances including erythromelalgia (N = 18), atypical or typical transient ischemic attacks (N = 6), or acute coronary ischemic syndrome (N = 3) with aspirin and one course of busulfan. The mean platelet counts before and after busulfan treatment were 1,009 (range 545-1,525) and 241 (range 159-315) x 10(9)/L, respectively. After induction of a complete remission, treatment with busulfan and aspirin was discontinued until symptoms returned. All 20 patients remained asymptomatic as long as ET was in maintained remission (platelet < 350 x 10(9)/L) for 4 to 61 (mean 36) months. Eight patients became symptomatic and 4 patients remained asymptomatic at relapse of ET after a follow-up period of 19 to 61 and 31 to 46 months, respectively. Platelet counts at time of symptomatic relapse in the 8 patients were 410, 450, 490, 500, 515, 545, 548, and 635 x 10(9)/L and at time of asymptomatic relapse in 4 ET patients 577, 600, 648, and 725 x 10(9)/L. Based on these observations, since 1986 we followed the strategy to treat ET patients with aspirin as long as the platelet count was between 400 and 1,000 up to 1,250 x 10(9)/L. Clear indications to reduce the platelet count were bleeding, aspirin side effects, and platelets counts above 1500 x 10(9)/L. This nonleukemogenic or least toxic approach in ET is the rationale behind the normal life expectancy and subsequent thrombosis-free survival in 68 ET patients after a mean follow-up period of 6.2 years. PMID- 10725981 TI - Circadian interrelationships among levels of plasma fibrinogen, blood platelets, and serum interleukin-6. AB - Circadian (24 h) rhythms of fibrinogen, interleukin-6 (IL-6), and platelet levels were studied in 11 males ages 46 to 72 years. Since there is a known circadian rhythm for fibrinogen and IL-6, we postulated that the peak level (acrophase) of fibrinogen would follow the acrophase of IL-6, based on the fact that IL-6 is the stimulus for fibrinogen production in the liver. Platelet levels were measured to show whether there was any correlation with the IL-6 acrophase because it has been reported that IL-6 affects megakaryocytes and platelets in dogs. We found that the acrophase for IL-6 occurred at 02:03 h and the acrophase for fibrinogen occurred at 09:16 h. Platelet counts peaked at 16:56 h. Thus, there was a positive correlation between IL-6 and fibrinogen acrophases and a negative correlation of each with the acrophase for platelets. The positive linkage of IL 6 with fibrinogen in this study suggests that suppression of IL-6 production would lower those peak fibrinogen levels that occur in the morning in association with arterial ischemic events. This could result in fewer arterial ischemic events, especially in the morning. PMID- 10725982 TI - Soluble plasma thrombomodulin levels in patients with chronic myeloproliferative disorder. AB - The plasma levels of soluble thrombomodulin (TM) were measured in 44 patients with chronic myeloproliferative disorder, 15 with polycythemia vera (PV), 29 with essential thrombocythemia (ET), and a group of 62 matched healthy controls. The younger patients had significantly lower TM levels (mean: 15.6 +/- 4.8 ng/mL) than the older patients (mean: 28.6 +/- 8.2 ng/mL, p < .001). Moreover, a significant negative correlation between platelet counts and plasma TM levels in healthy persons was noted (r = 0.317, p < .05). The only significant difference we found in plasma TM levels between patients and controls or among patients was between the young patients with ET (mean: 29.0 +/- 19.2 ng/mL) and young healthy controls (mean: 15.6 +/- 4.8 ng/mL). It is possible that younger ET patients with more active platelets are more susceptible to earlier vascular damage. The lack of any significant difference compared with the older patient population supports this hypothesis. PMID- 10725983 TI - Asymptomatic homozygous nt 20210 G to A prothrombin polymorphism in two blood donors belonging to two different kindreds. AB - The nucleotide (nt) 20210 G to A prothrombin polymorphism has been associated with an increased incidence of thrombosis, particularly venous thrombosis. The increased incidence of thrombosis resulted from an increase in prothrombin levels. Two homozygous patients with this abnormality were found to be completely asymptomatic. In one of the two cases there was no thrombosis even after the additional risk factor of oral contraceptive therapy for several months. Prothrombin activity and antigen as assayed by several methods were at the upper limits of normalcy in one case and slightly elevated in the other. The two cases described in this article do not rule out the possibility that this prothrombin abnormality be associated, given special acquired circumstances, with thrombosis. However, they indicate that extreme caution is needed to avoid an inaccurate conclusion. The fact that homozygous patients may remain asymptomatic indicates that the prothrombotic defect is very mild, if any. PMID- 10725984 TI - Heparin-induced thrombocytopenia: clinical considerations of alternative anticoagulation with various glycosaminoglycans and thrombin inhibitors. AB - Heparin-induced thrombocytopenia (HIT), the most common complication of heparin therapy, is also the most common form of the drug-induced thrombocytopenias. HIT is classified as type I and type II, the first being benign and the latter severe. HIT type II is attributed to an immune response characterized by complexes of heparin and platelet factor (PF) 4. Enzyme-linked immunosorbent assays allow easy and simple determination of these antibody titers; however, because specificity and sensitivity is not optimal, there is concern that the clinical relevance may be low. In clinical trials many patients were shown to form HIT-IgG in response to heparin without developing manifestations of HIT type II. Therefore, routine screening of clinically asymptomatic patients for antiheparin/PF 4 antibodies is not recommended. HIT type II is a clinico pathologic syndrome that ideally should be confirmed by laboratory testing. If any clinical suspicion arises, however, heparin and low molecular weight heparin therapy should be discontinued and an alternative anticoagulant therapy started. Alternative drugs have been evaluated in significant numbers of patients including danaparoid and thrombin inhibitors. In the case of danaparoid, it is highly recommended that an in vitro test for cross-reactivity be performed before the onset of therapy. If testing cannot be performed, immediate administration of a thrombin inhibitor is preferred. PMID- 10725985 TI - Effects on hemostasis after two-year use of low dose combined oral contraceptives with gestodene or levonorgestrel. AB - We studied 67 healthy women who were randomly allocated to receive third generation gestodene (Gynera) or second generation levonorgestrel (Microgynon 30) combination of low-dose estrogen oral contraceptives (OCs) for their hemostatic effects over 2 years. Hemostatic changes were apparent within 3 months of OC use. Hematocrit (Hct) was not affected, but hemoglobin (Hb) concentration decreased by 18 months. Shortened prothrombin time (PT) and activated plasma thromboplastin time (APTT) were associated with elevated fibrinogen within the 12-month use of both OCs. Factor VII was reduced only in Micro 30 during the 18 months of use. Enhanced thrombin-antithrombin (TAT)-complex level was seen at 18 months of Gynera use. Prothrombin fragment1+2 (F1+2) rise was seen at 3 months with Micro 30. Reduced antithrombin III (ATIII) activity was seen at 18 months with Gynera and at 24 months with Micro 30. Increased protein C activity was seen at 3 months and reduced protein S occurred at 18 months of Gynera use. Tissue plasminogen activator (t-PA) activity was enhanced for 6 months in both OCs with raised D dimer levels for 12 months with Gynera and 6 months with Micro 30. Decreased t-PA antigen was seen at 18 months and decreased urokinaselike plasminogen activator (u-PA) antigen occurred throughout the 24 months of both OCs use. Enhanced u-PA activity was only seen in Gynera users. Elevated plasminogen levels were apparent throughout both OCs use. PAI-1 levels were significantly decreased with Micro 30. With Gynera, the decreased PAI-1 activity was seen only at 18 months and PAI-1 antigen at 12 months. No change in platelets and von Willebrand factor (vWF) were seen in long-term OC use except that beta-thromboglobulin (beta-TG) showed decreased trends reaching statistical significance by 18 and 24 months of Micro 30 use and by 24 months of Gynera use. A further significant decrease in beta-TG, u-PA antigen, ATIII, and protein S levels were seen 3 months after pill stoppage compared with pretreatment levels. Activated protein C resistance (APCR) was negative in all subjects before and during OC use. The study indicated dynamic balance between coagulation and fibrinolysis with no endothelial activation. However, because some hemostatic markers showed wide fluctuations during OC use, a longer term study is warranted to investigate any adverse hemostatic changes that might enhance the risks of venous thromboembolism in Asian subjects known to be less prone to thrombosis. PMID- 10725986 TI - Anticoagulant therapy of pregnant patients with prosthetic heart valves: rationale for a clinical trial of low molecular weight heparin. AB - The management of anticoagulant therapy for the prevention of thromboembolism from prosthetic heart valves in the pregnant patient is far from ideal and deserves more clinical research. Warfarin therapy given alone throughout pregnancy poses high risks to the fetus because it crosses the placental barrier. Heparin alone throughout pregnancy poses high risks to the mother, which might be lessened somewhat by more intensive therapeutic regimens and careful monitoring of the heparin level or anticoagulated state of the blood. Because of the major risk of embryopathy in the fetus during the first trimester and latter half of the third trimester, heparin therapy during those times has been recommended. In the opinion of some experts, high-risk cases may benefit from low-dose aspirin in addition to anticoagulant therapy. The fact that warfarin is contraindicated during pregnancy (according to the pharmaceutical company that markets it) poses some problems for the physician who prescribes it. For the above reason, alternative drugs are worthy of consideration and should be given clinical trials. Low molecular weight heparin has the potential for greatly reducing drug related risk for the fetus while providing greater safety for the mother. PMID- 10725987 TI - Thrombogenesis in myocardial infarction and related syndromes: the role of molecular markers in diagnosis and management. AB - Anticoagulant therapy has undergone some major developments in recent years. Conventional drugs that produce anticoagulant effects such as heparin and oral anticoagulants are no longer considered the only candidates for the anticoagulant/antithrombotic management of patients. Recombinant hirudin, glycoproteins IIb/IIIa targeting antibodies, synthetic peptides such as Hirulog and efegatran are being tested for their efficacy. These drugs produce their effects at different sites. To monitor their overall effects on the hemostatic system, molecular markers offer a practical and reliable approach. Markers of thrombin generation are useful for the monitoring of antithrombin drugs whereas, the efficacy of antiplatelet drugs can be assessed by monitoring the platelet release products. Furthermore, polytherapy using several anticoagulant and antithrombotic drugs in combination has been considered. In these situations, the use of molecular markers may also prove to be invaluable. The introduction of simple technology such as the test strip or particle agglutination methods may be available for the measurement of many of these markers in the near future. This will be useful for ready availability and reduced cost for individual marker testing. Furthermore, this type of technology can be used at bedside, off-site, and in doctor's offices. It is clear that the molecular marker profiling provides useful information on the nature of pathophysiology of a given thrombotic disorder. However, for practical use, a cost-effective and simpler assay-based approach will enhance their use, and these tests will be readily accepted at the laboratory and clinical levels. PMID- 10725988 TI - Chemotherapy and thrombosis in gynecologic malignancy. AB - Thromboembolism is a severe and frequent problem in gynecologic malignancy. The average DVT incidence during chemotherapy of 5% might represent the lower range of incidence because < 55% of thrombotic complication manifest clinical signs. However, it seems likely that in addition to chemotherapy other risk factors such as menopausal status, BMI of patients, or type of preceding surgery must coincide before thrombosis manifests. While monitoring of patients using sophisticated coagulation tests did not identify patients' risk for DVT during chemotherapy, an evaluation of the coagulation status before initiating chemotherapy is recommended. Patients with a venous access device (e.g., indwelling central venous catheter or with port cart) are at a particularly high risk for DVT. This has to be considered when cytoreductive therapy is given. Thrombosis prophylaxis, orally or subcutaneously, should only be considered in a subpopulation of patients who offer a combination of the aforementioned risk factors. Thrombosis prevention trials during chemotherapy found a significant reduction of DVT in patients treated with anticoagulants. PMID- 10725989 TI - Acquired APC resistance in neurosurgical patients may not be a risk factor for postoperative deep vein thrombosis. AB - Acquired resistance to activated protein C has been reported during oral contraception and pregnancy. Its thrombogenic potential was studied in 41 neurosurgical patients who were enrolled in the placebo group of a thromboprophylaxis trial. Normalized activated protein C sensitivity ratio (nAPC SR), clotting activity of factors V and VIII, and levels of protein C antigen were measured prior to and at days 3 and 7 after surgery. Bilateral venography was done in all patients at days 8-10 to demonstrate deep vein thrombosis. A lowered nAPC-SR was found in 76% (baseline), 80% (day 3), and 88% (day 7) of patients. It was inversely related to factor VIII clotting activity (p = .0003) and protein C antigen, (p = .02). Deep vein thrombosis was demonstrated in 30% of patients with a normal nAPC-SR and in 23% of patients with a lowered nAPC-SR. Pulmonary embolism was not observed. Multivariate analysis did not identify a lowered nAPC-SR as a thrombotic risk factor, in contrast with gender (women, p = .02) and Quetelet index (> or = 25 kg/m2, p = .006). Our data provide no evidence that acquired activated protein C resistance, frequently found in neurosurgical patients, contributes to their high risk of postoperative deep vein thrombosis. PMID- 10725990 TI - Outcome of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome in Japan. AB - We examined 159 patients with thrombotic thrombocytopenic purpura and hemolytic uremic syndrome in Japan. The subjects were divided in three groups; 90 patients with thrombotic thrombocytopenic purpura, 51 patients with verotoxin-induced hemolytic uremic syndrome, and 18 patients with drug-induced hemolytic uremic syndrome. Eighty-two percent of the patients with thrombotic thrombocytopenic purpura had associated neurological disorders and 78% of drug-induced hemolytic uremic syndrome associated with pulmonary edema. Renal insufficiency was noted in the 69% cases with both hemolytic uremic syndrome groups. Seventeen patients with thrombotic thrombocytopenic purpura had systemic lupus erythematosus and 6 were pregnant. Autoantibody were positive in 53% of thrombotic thrombocytopenic purpura. Seventy-seven percent of patients with thrombotic thrombocytopenic purpura received plasma exchange at 4,000 mL/day three times a week, 71% antithrombotic agents, and 78% steroid administration, respectively. However, 27% of the patients with hemolytic uremic syndrome were treated by hemodialysis in addition to antithrombotic agents. When drug-induced hemolytic uremic syndrome was diagnosed, the drug was immediately discontinued and the patients were treated with antiplatelet agents. Seventy-four percent of the patients with thrombotic thrombocytopenic purpura were alive at 26 weeks compared with 95% of those with hemolytic uremic syndrome. As thrombotic thrombocytopenic purpura had a high mortality rate in Japan, we should carry out early diagnosis and early treatment. PMID- 10725991 TI - Increased activated protein C: protein C inhibitor complex and decreased protein C inhibitor levels in patients with chronic renal failure on maintenance hemodialysis. AB - Hemostatic abnormalities were examined in 55 patients during maintenance hemodialysis (HD). Before HD, plasma protein C and protein S antigens were almost within the normal range, while plasma thrombin-antithrombin III complex (TAT III) and plasmin-plasmin inhibitor complex (PPIC) levels in HD patients were increased slightly, and plasminogen activator inhibitor 1 level was significantly increased, compared to that in normal volunteers. Plasma activated protein C (APC) and protein C inhibitor (PCI) complex and APC alpha 1 antitrypsin (alpha 1AT) complex were not detected in normal volunteers; however, plasma APC-PCI complex was increased in 36 of the patients and plasma APC-alpha 1AT complex was increased in 25 patients. Plasma PCI levels in these patients before HD were significantly decreased. Plasma TAT, PPIC, and tissue type plasminogen activator levels were significantly higher before HD than after 1 hour HD and at the end of HD, while the changes in plasma protein C antigen, protein S antigen, PCI antigen, APC-PCI complex, and APC-alpha 1AT complex were not significant after 1 hour of HD or at the end of HD compared to levels before HD. Plasma PCI levels were correlated with APC-PCI complex, suggesting that decreased PCI levels might be caused by the activation of protein C. PMID- 10725992 TI - Evaluation of the Clot Signature Analyzer as a hemostasis test in healthy volunteers exposed to low doses of aspirin. AB - Several variables affect bleeding time that make it difficult to obtain consistent measurements. The Clot Signature Analyzer (CSA) has been developed to assess in vitro hemostasis using well-controlled flow chambers. In this study, the equivalencies in the CSA parameters with the conventional bleeding time or platelet aggregation methods were evaluated in subjects exposed to aspirin. The CSA parameters, platelet hemostasis time (PHT) and collagen-induced thrombus formation (CITF), were compared to bleeding time (Surgicutt2) and collagen induced platelet aggregation, respectively. Fifty-three healthy volunteers were given two doses of aspirin (81 and 243 mg) in one day. Following the baseline period, the volunteers took 81 mg of aspirin and then took 243 mg 2 hours later. The changes in each value from the baseline to that at either aspirin dose (2 hours after dosing) were evaluated. Platelet hemostasis time and CITF correlated well with bleeding time and aggregation, respectively, but PHT was not significantly increased after 81 mg of aspirin, whereas bleeding time was significantly increased. The variation in PHT was slightly higher than that of bleeding time. At 81 mg, CITF was significantly increased but aggregation was not, even though the variation was comparable. This suggests that PHT and CITF can simulate the changes in bleeding time and aggregation, respectively, but the sensitivity of PHT for detecting the changes in bleeding time was no better than the conventional method. Also, CITF was more sensitive than aggregation in detecting platelet response to collagen. In conclusion, the proposed CSA is not always suitable for detecting hemostatic abnormalities. PMID- 10725993 TI - The effect of antiplatelet drugs, heparin, and preanalytical variables on platelet function detected by the platelet function analyzer (PFA-100). AB - The platelet function analyzer (PFA)-100 is a newly developed instrument that provides a rapid, in vitro, quantitative measurement of platelet adhesion and aggregation in whole blood flowing through a small aperture under high shear conditions. Thirty patients undergoing percutaneous transluminal coronary angioplasty (PTCA) and ten normal individuals were included in this study. In vitro and in vivo studies were conducted to discern the effect of combinations of antiplatelet drugs (aspirin, ticlopidine, abciximab) and heparin on the performance of the device as well as the effects of preanalytical variables, such as method of sample collection and ex vivo anticoagulants. Studies were also conducted examining the effect of aperture size (standard 150 microns vs. smaller 120 microns) on the ability of the device to detect the effect of antiplatelet drugs. There was no difference in mean PFA-100 closure time with citrate versus PPACK anticoagulants or with venipuncture vs. sheath sampling. Closure times did not vary with heparin administration. Closure times were slightly longer for patients taking aspirin plus ticlopidine compared to aspirin alone (p = NS). In contrast adenosine disphosphate (ADP) induced platelet aggregation was significantly less in patients that took aspirin plus ticlopidine vs. aspirin alone (p = .0005). In vitro, there was a dose-dependent increase in closure time for both aperture sizes with increasing abciximab concentration. Although both cartridges showed infinite closure times at an abciximab concentration of 2.25 micrograms/mL, there was a slight benefit to using the 120 microns aperture cartridges at abciximab concentrations of 1.75 to 2.0 micrograms/mL. In ten patients who were followed during abciximab therapy to assess the effect of aperture size, the PFA-100 was able to detect in vivo platelet inhibition by abciximab, but detection of recovery from abciximab-induced platelet dysfunction was slightly better for the PFA-100 with the 120 microns aperture compared to the standard 150 microns aperture collagen/ADP cartridge. PMID- 10725994 TI - Aspirin seems as effective as myelosuppressive agents in the prevention of rethrombosis in essential thrombocythemia. AB - The course of essential thrombocythemia (ET) is complicated by bleeding, major thrombosis, and microvascular complications. Because about one-half of ET patients remain asymptomatic long term, the decision to use aspirin acetylsalicylic acid, (ASA) or myelotoxic drugs has not yet been clearly established. While vasomotor symptoms are improved by small doses of ASA, higher doses (900 mg/day) induce an unacceptable rate of serious hemorrhagic complications in patients with polycythemia vera. This retrospective study evaluates the utility of therapy in preventing thrombosis in ET and the efficacy and a safety of 100 mg/day of ASA in these patients. One hundred ninety-five consecutive patients with ET diagnosed in agreement with the Polycythemia Vera Study Group (PVSG) criteria are evaluated. All vascular complication before, at, or after diagnosis were recorded and related to the treatment used: no therapy, ASA alone, myelosuppressive agents or both. All treated patients had a significant reduction of thrombotic complications without increased hemorrhagic complications, in spite of therapy adopted. In addition, a significant reduction of rethrombosis was obtained in 60 patients with a previous thrombosis. A low rate of thrombosis (5.1%) was observed during the follow-up of the 135 patients previously asymptomatic for major complications. No difference appears to exist between the use of ASA and cytotoxic drugs in preventing thrombosis and rethrombosis in ET patients. However, the possible increase of cancer and leukemia with myelosuppressive drugs is minimized in patients treated with ASA. A low dose of ASA would seem to be a safe and effective agent in ET. PMID- 10725995 TI - Combined factor V and factor VII deficiency due to an independent segregation of the two defects. AB - A patient with combined factor V and factor VII deficiency is described together with a family study. The propositus appeared to be double heterozygous for factor V and factor VII deficiency. Since the patient showed a parallel decrease of activity and antigen, he appeared to be double heterozygous for a true deficiency. The patient had inherited the factor V defect from the mother and the factor VII defect from the father. The parents of the propositus were not consanguineous. Other family members were found to have isolated factor V or factor VII deficiency. This is the third family so far described with this peculiar combined defect but the first to be investigated by clotting and immunologic assays. PMID- 10725996 TI - Functional diagnosis of activated protein C resistance in patients with antiphospholipid antibodies. PMID- 10725997 TI - Plasma levels of TPA, PAI-1 and thrombospondin in patients with systemic vasculitis. PMID- 10725998 TI - Pseudothrombocytopenia and platelet antigens. PMID- 10725999 TI - Acquired von Willebrand disease due to increasing platelet count can readily explain the paradox of thrombosis and bleeding in thrombocythemia. AB - The arterial microvascular thrombotic events in thrombocythemia including erythromelalgia, atypical and typical cerebral and ocular transient ischemic attacks, and acute coronary syndromes already occur at platelet counts > 400 x 10(9)/L and are sensitive to low-dose aspirin, which does not elicit bleedings at platelet counts < 1,000 x 10(9)/L. An increasing platelet count in thrombocythemia to above 1,000 x 10(9)/L is accompanied by the acquisition of a von Willebrand factor deficiency due to the loss of intermediate and large von Willebrand factor multimers. The arterial thrombotic condition in thrombocythemia changes into an overt spontaneous bleeding tendency at mean platelet counts of about 2,000 +/- 1,000 x 10(9)/L due to an acquired von Willebrand disease type II with normal values for von Willebrand factor antigen concentration but absence of intermediate and large von Willebrand factor multimers in plasma. At platelet counts between 1,000 and 2,000 x 10(9)/L, thrombosis and bleeding frequently occur in sequence or paradoxically, and low-dose aspirin does prevent thrombotic complications but aggravates or may elicit bleeding symptoms. Reduction of the platelet count < 1,000 x 10(9)/L significantly restores the von Willebrand factor deficiency with the reappearance of the intermediate and some of the large von Willebrand factor multimers and the disappearance of the bleeding tendency, but the thrombotic tendency persists as long as platelet counts are above the upper limit of normal. The acquisition of von Willebrand factor deficiency at increasing platelet counts can readily explain the paradox of thrombosis and bleeding in thrombocythemia and has important clinical implications. PMID- 10726000 TI - Acquired von Willebrand disease in monoclonal gammapathies: effectiveness of high dose intravenous gamma globulin. AB - The reported underlying lymphoproliferative disorders associated with acquired von Willebrand disease (AvWD) include benign monoclonal gammapathy, multiple myeloma, Waldenstrom disease, chronic lymphocytic leukemia, non-Hodgkin lymphoma, and hairy cell leukemia. The AvWD in patients with a monoclonal gammapathy and/or a lymphoproliferative disorder is featured by a prolonged bleeding time, normal platelet count, and a decreased or absent ristocetine-induced platelet aggregation in combination with a prolonged aPTT and normal PT due to low levels of factor VIII/von Willebrand factor (vWF) parameters in the absence of a factor VIII inhibitor in the Bethesda assay. In vitro and vivo experiments consistently showed that the anti-vWF autoantibodies in monoclonal gammapathies cause a rapid clearance of the factor VIII/vWF complex from the circulation after DDAVP and factor VIII/vWF concentrate infusion. Multimeric analysis of the vWF usually show a type II-like AvWD due to the absence of large vWF multimers as the consequence of the rapid clearance of the anti-vWF-factor VIII/vWF complex from the circulation. There is a poor response to intravenous DDAVP and factor VIII/vWF concentrate infusion, but high dose intravenous gamma globulin (1 g/kg for 2 days) usually induces a transient correction of the factor VIII/vWF parameters for 1 to a few weeks. PMID- 10726001 TI - The G20210A polymorphism in the 3'-untranslated region of the prothrombin gene in Mexican mestizo patients with primary antiphospholipid syndrome. AB - In an effort to identify alleles associated with an increased risk of venous thrombosis in patients with primary antiphospholipid syndrome, we studied the G20210A polymorphism (the G-->A mutation at nucleotide position 20210) in the 3' untranslated region of the prothrombin gene in a group of 14 patients with primary antiphospholipid syndrome. We did not find any patient with the mutated gene. Since the prothrombin mutation is more prevalent in white populations, this finding may be related with the genetic composition of the Mexican mestizos, in whom the white component is low. The polymorphism of the prothrombin gene in Mexican mestizo patients with antiphospholipid syndrome does not seem to be related to the thrombophilia observed in these patients. PMID- 10726002 TI - Diminished platelet aggregation in patients with iron deficiency anemia. AB - To evaluate platelet function in iron deficiency anemia, using impedance (in whole blood) and optic (in platelet-rich plasma methods, platelet aggregation analyses were performed in 42 children with iron deficiency anemia at the time of diagnosis and after iron therapy. Collagen-induced platelet aggregation was decreased in patients before therapy compared to after therapy and control levels as indicated by the two methods while adenosine diphosphate (ADP)-induced platelet aggregation was decreased only by the whole blood method. Platelet aggregation with epinephrine, performed only by the optic method, was not altered in patients with iron deficiency anemia. Platelet counts were found to be increased in patients with iron deficiency anemia, and decreased with therapy. These results indicate that some platelet functions are decreased in patients with iron deficiency anemia. These decreased functions may be compensated by increased platelet numbers. PMID- 10726003 TI - R-hirudin as anticoagulant in regular hemodialysis therapy: finding of therapeutic R-hirudin blood/plasma concentrations and respective dosages. AB - Recently heparin-induced thrombocytopenia type II has been diagnosed more frequently and does not exclude hemodialysis patients. Up to now, recombinant hirudin is the only available anticoagulant showing no immunologic cross reactions with heparin. However, the use of r-hirudin in hemodialysis patients with different degrees of residual renal functions is impossible using standard dosages because elimination of r-hirudin varies depending on the degree of residual renal function. Therefore the first study was carried out using consecutive r-hirudin anticoagulated hemodialyses to determine the appropriate dose of r-hirudin. Ten hemodialysis patients with creatinine clearance values ranging between 0 and 13 mL/min/1.73m2 were anticoagulated with r-hirudin. An initial bolus of 0.1 mg/kg bwt before the first hemodialysis, resulted in an average r-hirudin blood concentration of 305 ng/mL at the end of treatment. The dose for each of the following four hemodialyses was adjusted individually to reach the minimum therapeutic r-hirudin blood concentration. At the end of these treatments the mean blood r-hirudin concentration was 422 ng/mL. The necessary mean doses ranged between 0.008 and 0.125 mg/kg bwt correlating to the creatinine clearance values of the patients. All hemodialyses of the study were effective and safe. Bleeding times determined during r-hirudin anticoagulation were significantly lower than control values measured 2 days after a heparin administration. The study proved that r-hirudin may be an efficient and safe heparin alternative as a hemodialysis anticoagulant when the individual's residual renal function is noted for dosage and dose adjustment and is controlled by drug monitoring using the ecarin clotting time. PMID- 10726004 TI - Exclusion and diagnosis of deep vein thrombosis by a rapid ELISA D-dimer test, compression ultrasonography, and a simple clinical model. AB - The classical clinical signs of deep vein thrombosis (DVT) are unspecific and may be found in several other conditions besides DVT. Therefore, patients suspicious of DVT are subjected to elaborate invasive or noninvasive evidence-based procedures that actually confirm DVT in only 20% to 30% of patients in this setting. However, simple laboratory tests and noninvasive strategies to exclude and diagnose DVT are becoming available in the clinical emergency setting of outpatients. In the presented literature, a sound basis is provided for quantifying clinical judgment for the diagnosis of acute proximal DVT. The number of positive clinical findings at time of first suspicion of DVT appears to correlate directly with the probability of acute proximal DVT. The modified clinical model of Landefeld and Wells for DVT allows reasonable accurate classification of patients into low, moderate, and high probability for suffering DVT. The rapid automated enzyme-linked immunoabsorbant assay (ELISA) VIDAS D dimer presently available can be rapidly performed in daily practice and emergency situations and is accurate to a high degree, especially in ruling out ongoing venous thromboembolic processes. The sequential use of the rapid ELISA VIDAS D-dimer test and compression ultrasonography in a well-designed clinical setting using a simple clinical model predicts a significant improvement due to a high sensitivity near 100% for the exclusion and diagnosis of DVT in the majority of outpatients with suspect DVT. A prospective decision analysis management study is proposed to exclude and diagnose DVT based on the rapid ELISA VIDAS D-dimer test and compression ultrasonography within the context of a ready-to-use simple clinical model. The proposed simple model of a rational diagnosis of deep vein thrombosis (RADIA DVT) has to be tested in a large multicenter study of more than 1,000 outpatients with suspected DVT. This model would be less expensive, easy to perform, and likely yield a significant simplification and improvement of highly accurate evidence-based exclusion or diagnosis of DVT on the basis of which clear cut indications of anticoagulation could be appropriately initiated or safely withheld. PMID- 10726005 TI - Serum cardiolipin antibodies in cancer patients with thromboembolic events. AB - This study was undertaken to investigate a possible association of anticardiolipin antibodies (ACLAs) in cancer patients with thromboembolic events. Twenty-five patients with solid tumors complicated with acute thrombosis, 36 cancer patients without any thrombotic events, and a group of 20 healthy volunteers without thrombosis or malignancy were included. The mean age of the cancer patients with and without thrombosis and healthy subjects were 50 years (range 20-75), 45 years (range 23-66), and 40 years (range 20-68), respectively. Deep venous thrombosis (n = 16) and thrombosis of the central venous port catheter systems (n = 9) were confirmed by Doppler sonography in all patients. IgG and IgM isotypes of ACLAs were quantitated by enzyme-linked immunosorbent assay with normal levels of < 23 GPL and < 11 MPL, respectively. Mean values of IgG ACLAs were found similar in cancer patients with acute thrombosis (13.8 +/- 4.9 GPL), without thrombosis (12.8 +/- 5.4 GPL) or in healthy subjects (14.8 +/- 5.5 GPL). Although the mean values of IgM ACLAs were within normal limits in all groups, cancer patients with thrombotic events had higher levels of IgM ACLAs (mean = 10.5 +/- 2.2 MPL) than cancer patients without thrombosis (mean = 4.6 +/- 2.4 MPL) (p = .01). Healthy subjects also had lower levels of IgM ACLAs (mean = 7.1 +/- 3.2 MPL) than cancer patients with thrombosis (p = .16). In addition, a higher percentage of cancer patients with or without thrombosis had IgM and IgG ACLA levels above normal limits compared with healthy controls. In conclusion, our study suggests an association between ACLAs or IgG and particularly IgM isotypes and venous thrombosis in malignancy. Identification of cancer patients who are at higher risk for developing thromboembolic events might lead to a better selection of patients for prophylactic anticoagulant therapy. PMID- 10726006 TI - Thrombotic risk of muscular dystrophy: protein C deficiency, factor V Leiden, and myotonic dystrophy. AB - While patients with hereditary muscular disorder are more sedentary and believed to be at risk of thrombosis, there is little literature on thrombosis associated with hereditary muscular disorder. We report a rare case of double heterozygous type I protein C deficiency and factor V Leiden in a patient with classic myotonic dystrophy. PMID- 10726007 TI - Thrombosis involving the major veins with heterozygote factor V Leiden mutation as the only risk factor. AB - A 27-year-old man was admitted to our hospital with the complaints of swelling of his face and lower limbs. Echocardiography showed minimal pericardial effusion accompanied by disordered diastolic function. Cardiac catheterization was performed to rule out constrictive pericarditis. Normal pressure tracings of the right heart rule out constrictive pericarditis, however, a narrowing of the inferior vena cava was observed. Venographies of the inferior and superior vena cavae showed extensive thrombotic involvement of these great veins. Protein C, protein S, anticardiolipin antibodies, fibrinogen, antithrombin-III, activated protein C resistance, and factor V levels were in normal limits. Heterozygosity for factor V Leiden mutation was detected. We conclude that factor V Leiden mutation can cause extensive thrombotic involvement of major veins and should be considered in idiopathic thrombosis of them. PMID- 10726008 TI - Central retinal vein thrombosis associated with prothrombin 20210G/A gene variant. AB - We describe a patient with central retinal vein thrombosis associated with the prothrombin gene 20210G/A. The associated risk factors and the patient's personal and family history of thrombosis are dealt with. In addition, the literature on the cases reported up to now is reviewed as is the prevalence of this new mutation in patients with central retinal vein thrombosis. PMID- 10726009 TI - Effects of aprosulate, a novel synthetic glycosaminoglycan, on coagulation and platelet function parameters: a prospective, randomized phase I study. AB - In a phase I clinical trial the effect of the highly sulfated polyanion "Aprosulate" was studied in healthy volunteers using different coagulation and platelet function parameters. Eighteen healthy volunteers aged 21 to 30 years received two single subcutaneous doses of aprosulate (0.5 mg/kg body weight; 1.0 mg/kg body weight), or unfractionated heparin (Calciparin 7,500 IU). The washout period between the different drugs/doses was at least 7 days. Coagulation and platelet function parameters (activated partial thromboplastin time, Heptest, fibrinogen, von Willebrand factor, ristocetin cofactor, platelet adhesion to siliconized glass, and platelet-induced thrombin generation time [a new method for measuring thrombin generation in platelet-rich plasma in the presence of platelets]) were assessed during 24 hours after each injection. Aprosulate led to a significant and dose-dependent prolongation of activated partial thromboplastin time and Heptest. This effect lasted for 4 hours (activated partial thromboplastin time) to 8 hours (Heptest). Activated partial thromboplastin time was not prolonged after the injection of unfractionated heparin (7,500 IU). Fibrinogen, von Willebrand factor, and ristocetin cofactor remained unchanged with both drugs. Platelet induced thrombin generation time was slightly prolonged and platelet adhesion was slightly diminished up to 2 hours using 0.5 mg/kg aprosulate, and up to 4 hours using 1.0 mg/kg aprosulate while the platelet induced thrombin generation time system was not influenced by the subcutaneous injection (7,500 IU) of unfractionated heparin. Both drugs and doses were well tolerated. Plasma transaminase concentrations alanin aminotransferase and aspartate aminotransferase serum values were slightly increased in some volunteers but returned to normal during or after the study (< 4 weeks). Further clinical trials will have to establish whether aprosulate is an effective drug for the prophylaxis of deep venous thrombosis. PMID- 10726010 TI - Low molecular weight heparin as thromboprophylaxis throughout pregnancy in heritable thrombophilic women. PMID- 10726011 TI - Low incidence of venous thrombosis in homozygous patients with NT 20210 G to a prothrombin polymorphism. PMID- 10726012 TI - Warfarin therapy: a review of the literature since the Fifth American College of Chest Physicians' Consensus Conference on Antithrombotic Therapy. AB - Evidence-based medicine is currently a fashionable term. The evidence that warfarin is safe, effective, and cost beneficial in preventing stroke in AF, DVT treatment, and DVT prophylaxis is mounting. However, the evidence that warfarin remains underutilized in these conditions is also mounting. There is new evidence that supports conservative management of overanticoagulation without bleeding. The amount of time, if any, that patients are off warfarin for various procedures is being reduced. Warfarin interactions with other agents continue to be reported so that practitioners can avoid or treat them. Even the contraindication of warfarin in pregnancy is being reexamined. Those with expertise in anticoagulation therapy have an imperative to inform colleagues about the evidence in favor of warfarin. PMID- 10726013 TI - Exclusion of deep vein thrombosis with rapid ELISA D-dimer testing: from theory to daily practice. PMID- 10726014 TI - Diagnosis of deep vein thrombosis by the use of a rapid ELISA D-dimer test, CUS, and a clinical model: a cost-effectiveness analysis. PMID- 10726015 TI - Diagnosis of venous thromboembolism by a rapid ELISA D-dimer test, clinical model and noninvasive imaging techniques. PMID- 10726016 TI - A prospective clinical study on the feasibility and safety of a combined noninvasive/pulmonary angiography strategy in patients with suspected pulmonary embolism: an interim analysis. PMID- 10726017 TI - The role of spiral computed tomography in the assessment of patients with suspected pulmonary embolism. PMID- 10726018 TI - Value of thrombin-antithrombin III complexes in major orthopedic surgery: relation to the onset of venous thromboembolism. AB - This study evaluated (a) the possible changes of plasma levels of thrombin antithrombin III complexes during hospitalization to predict venous thromboembolism in patients undergoing elective total hip replacement and (b) the sensitivity and specificity of thrombin-antithrombin III complexes in the late incidence of deep vein thrombosis when these patients are discharged from the hospital. In 50 consecutive patients (18 men, mean age = 63 +/- 8 years) a venous blood sample was obtained from each patient before surgery and postsurgery on days 5 +/- 2, 9 +/- 2, and 45 to evaluate the thrombin-antithrombin III complexes by the enzyme-linked immunosorbent assay as a part of a larger surveillance program. Six of 50 patients developed deep vein thrombosis, diagnosed by phlebography on the 45th day postsurgery. From the day before until the ninth day after surgery, mean values of the thrombin-antithrombin III complexes increased to a greater extent in patients with deep vein thrombosis than in those without, although the differences were not significant (from 14.8 +/- 11.2 ng/mL to 36.2 +/- 19.1 ng/mL in the former group and from 13.6 +/- 3.3 ng/mL to 22.4 +/- 5.1 ng/mL in the latter, p = NS). On the 45th day after surgery the mean value of the thrombin-antithrombin III complexes reduced less in patients with deep vein thrombosis (up to 9.9 +/- 1.9 ng/mL and to 25.2 +/- 17.2 ng/mL, respectively, p = NS). In addition, thrombin-antithrombin III complexes remained over the level reached on the fifth day only in the patients who developed deep vein thrombosis. On the 45th day after surgery, thrombin-antithrombin III complexes exhibited a sensitivity of 17%, a specificity of 86%, and an accuracy of 78% in differentiating the presence and absence of deep vein thrombosis as compared with phlebography. We conclude that after total hip replacement (a) serial measurement of the thrombin-antithrombin III complexes does not appear helpful in predicting venous thromboembolism during hospitalization, and (b) measurement of thrombin antithrombin III complexes has a low diagnostic accuracy in diagnosing delayed deep vein thrombosis. However, the greater and persistent increase of thrombin antithrombin III complexes level in patients who developed deep vein thrombosis may deserve further investigations. PMID- 10726019 TI - Do hemophilia A and von Willebrand disease protect against carotid atherosclerosis? A comparative study between coagulopathics and normal subjects by means of carotid echo-color Doppler scan. AB - Atherosclerosis is a multifactorial disease caused by genetic and environmental factors with important clinical sequelae. The aim of this study was to evaluate the degree of carotid atherosclerosis by echo-color Doppler scan in a group of patients affected by hemophilia A and von Willebrand disease versus a group of normal subjects apparently free of atherosclerotic risk factors. All coagulopathics and normal patients who came to our Internal Medicine Department (Padua Hospital) underwent physical exam, blood analysis, standard electrocardiogram, chest x-ray, echo-color Doppler scan, and a thorough history. We examined 156 subjects, 76 coagulopathics (46 men, 30 women) and 77 normals (37 men, 40 women). Coagulopathics were affected by hypertension in 28.9% of cases, diabetes mellitus in 6.5%, dislipidemia in 17.1%, smoke in 39.4%, and obesity in 36.8% (p < .05). Echo-color Doppler scan revealed carotid plaques in 27.2% of control patients versus 13.1% of coagulopathics (p < .05). Hemophilics and subjects with von Willebrand disease with a more serious illness had fewer plaques than those with lighter defects. Coagulopathics showed 23.6% of the plaques we revealed on the whole, versus 76.3% of control subjects (p < .01), with a lighter degree of stenosis (p < .01). Our data demonstrate that patients with hemophilia A and von Willebrand disease have fewer carotid plaques and a smaller degree of carotid stenosis than normal subjects of the same sex and age. These data seem to strengthen the hypothesis that blood coagulation defects may allow protection against carotid atherosclerosis and its sequelae. PMID- 10726020 TI - Mild myocardial stunning affects platelet aggregation and certain hemostatic factors in swine. AB - Myocardial stunning is characterized by transient contractile dysfunction occurring subsequent to an episode of ischemia followed by reperfusion. Platelet activation and hemostatic abnormalities have been described in patients with unstable angina and acute myocardial infarction, however, their role in the pathogenesis of myocardial stunning is unknown. The purpose of this study was to determine if platelet aggregation and certain hemostatic factors change during myocardial stunning following brief coronary arterial occlusion. Nine Yorkshire swine underwent left anterior descending coronary artery occlusion for 8 minutes followed by 90 minutes of reperfusion. Blood samples were obtained at baseline, at 4 and 8 minutes of occlusion, and at 60 and 90 minutes of reperfusion. Platelet aggregability and concentrations of antithrombin III, protein C, protein S, fibronectin, endothelin 1, and the stable metabolites of thromboxane (TxB2) and prostacyclin (6-keto-PGF1a) were measured in systemic circulation. The occlusion phase was associated with a decline of endothelin 1 (-13.6%), and TxB2 (-19.6%), and elevation of antithrombin III (+40.2%) and protein C (+22.9%). Mild myocardial stunning was associated with a significant increase in platelet aggregation (+33.7%), endothelin-1 (+24.7%), 6-keto-PGF1a (+41.5%), TxB2 (+11.9%), and protein C (+42.3%) during the reperfusion phase. There were no changes in plasma fibronectin and total protein S. Thus, mild myocardial stunning following brief coronary artery occlusion is associated with substantial dynamic changes in platelet aggregability and certain hemostatic factors. These results may be relevant to understanding the mechanisms determining myocardial stunning and coronary arterial patency following reperfusion. PMID- 10726021 TI - Effects of ticlopidine or ticlopidine plus aspirin on platelet aggregation and ATP release in normal volunteers: why aspirin improves ticlopidine antiplatelet activity. AB - Aspirin and ticlopidine are used to prevent arterial thrombosis. In some clinical settings ticlopidine is administered with aspirin for improving antithrombotic effect. We administered aspirin (100 mg/day), ticlopidine (500 mg/day), or ticlopidine and aspirin for 7 days to healthy volunteers. Platelet aggregation and ATP release induced by sodium arachidonate, ADP, or a combination of both were measured. Sodium arachidonate (0.25 mmol/L), which produces no platelet aggregation, combined with adenosine diphosphate (1 mumol/L), which produced a reversible platelet aggregation of 20% after ticlopidine, resulted in a synergistic platelet aggregation response in normal (74.6 +/- 9.2%) and in ticlopidine platelet-rich plasma (59.1% +/- 14.9%, p < 0.0001). Synergism after sodium arachidonate (0.75 mmol/L) plus adenosine diphosphate (4 mumol/L) fell from 75.8% +/- 11.0% and 59.1% +/- 15.6% after ticlopidine or aspirin, respectively, to 14.8% +/- 18.0% (p < 0.0001) after ticlopidine plus aspirin. Aspirin and ticlopidine alone did not inhibit adenosine triphosphate release as thoroughly as did aspirin plus ticlopidine. Aspirin or ticlopidine does not adequately prevent platelet activity as ticlopidine plus aspirin do. Addition of aspirin to treatment with ticlopidine improves their antiplatelet activity and better results could be obtained in arterial thrombotic prevention strategies. PMID- 10726022 TI - Aspirin and platelet-lowering agents for the prevention of vascular complications in essential thrombocythemia. AB - Low-risk essential thrombocythemia patients include patients aged 18 to < 80 years with no vascular risk factor or previous thrombosis, no associated disease, a normal life expectancy, and a platelet count between 400 and 1,000 x 10(9)/L up to 1,500 x 10(9)/L. Asymptomatic essential thrombocythemia patients may be at risk for microvascular circulation disturbances. The indication for low-dose aspirin in asymptomatic essential thrombocythemia patients is uncertain, therefore randomization for aspirin 50 mg versus placebo is recommended. Symptomatic essential thrombocythemia patients with erythromelalgia and its ischemic complications, atypical transient ischemic attacks, minor stroke, visual disturbances and "superficial thrombophlebitis" in the absence of bleeding, vascular risk factors, or vascular disease have a clear indication for aspirin in a regular dose. To determine whether 50 mg/day is as effective as 100 mg/day for the prophylaxis of microvascular circulation disturbances in essential thrombocythemia, a randomized trial comparing low-dose aspirin 50 mg versus 100 mg at platelet counts between 400 and 1,000 up to 1,500 x 10(9)/L is recommended. To address the question whether reduction of the platelet count to normal (< 350 x 10(9)/L) is as effective as low-dose aspirin for the long-term relief of microvascular circulation disturbances, a randomized study comparing low-dose aspirin with the correction of platelet count to normal by anagrelide is recommended. High-risk essential thrombocythemia patients have a clear indication for platelet reductive therapy, including: (a) platelets > 1,500 x 10(9)/L, history of major thrombosis (myocardial infarction, stroke, peripheral occlusive vascular disease), or presence of vascular disease (e.g., arteriosclerosis); (b) history or presence of spontaneous or major bleedings, bleedings elicited by low dose aspirin for the secondary prevention of vascular complications in essential thrombocythemia at platelet counts < 1500 x 10(9)/L, and side effects of long term aspirin treatment such as gastritis; and progression from low- to high-risk essential thrombocythemia patients during follow-up or progressive myeloproliferative disease such as significant splenomegaly, myelofibrosis, leukocytosis, etc. To address the question of optimal treatment of high-risk essential thrombocythemia patients, randomization for anagrelide versus interferon at < 65 years of age and anagrelide versus hydroxyurea at an age > 65 years is recommended. PMID- 10726023 TI - Simultaneous monitoring of argatroban and its major metabolite using an HPLC method: potential clinical applications. AB - Argatroban is a peptidomimetic inhibitor of thrombin that is currently undergoing extensive clinical trials as a heparin substitute for thrombotic complications. Argatroban is readily metabolized into a major derivative, M1, that has pharmacological characteristics distinct from its parent compound. The currently available clot-based assays measure the cumulative anticoagulant effect of argatroban and its metabolite(s). Available HPLC methods do not differentiate between argatroban and M1-metabolite. A modified method was developed to simultaneouly quantitate M1-metabolite and argatroban in biological fluids. Initial validation studies for the method included clinical trials of argatroban in patients with heparin-induced thrombocytopenia, (ARG 911 Study) and coronary interventional procedures (ARG 310 Study). Plasma samples were extracted with acetonitrile and reconstituted in a mobile phase. Calibration curves were prepared by running known standards of argatroban and M1-metabolite in normal human plasma. Ultraviolet detection was made at 320 nm. The retention times for argatroban and M1-metabolite peaks were found to be 10.5 +/- 0.3 minutes and 3.9 +/- 0.1 minutes, respectively. The extraction efficiency was > 95% (r2 = 0.99). In heparin-induced thrombocytopenia patients with major bleeding complications (n = 30), the relative increase in M1-metabolite compared to argatroban varied widely (two- to eight-fold). The mean concentration of argatroban during the steady infusion period was found to be 0.7 +/- 0.35 microgram/mL, and for M1 metabolite, it was 5.5 +/- 2.8 micrograms/mL. Proportionate results were not seen when higher dosages of argatroban were administered (coronary angioplasty studies). Argatroban and M1-metabolite levels also compared well with the results in global clotting assays. Owing to the simultaneous quantitation of argatroban and M1-metabolite, this method provides a rapid assessment of the pharmacokinetics and pharmacodynamics of argatroban. The differential quantitation may be useful in the assessment of relative metabolic turnover of argatroban that can be related to the hepatic and renal functions in a given patient. PMID- 10726024 TI - Synthetic pentasaccharides do not cause platelet activation by antiheparin platelet factor 4 antibodies. AB - A synthetic selective inhibitor of factor Xa, the pentasaccharide SR90107A/Org31540 is in clinical development for the prophylaxis of postsurgical deep vein thrombosis. Another synthetic pentasaccharide with even more sustained inhibition of factor Xa, SanOrg34006, has also been developed. Both of these agents were tested in comparison to unfractionated heparin and a low molecular weight heparin (enoxaparin) for their relative platelet activation potential in heparin-induced thrombocytopenia assays. Sera from patients (n = 30) with heparin induced thrombocytopenia were pooled and validated for heparin-dependent aggregation responses. Using heparin-platelet factor 4 Sepharose columns, antibodies to heparin-platelet factor 4 were purified from the same pool. The effects of heparin, enoxaparin, SR90107A/Org31540, and San-Org34006 were evaluated in a platelet aggregation assay using platelet donors (n = 10). At comparable concentrations, heparin and enoxaparin consistently produced platelet activation, whereas both pentasaccharides failed to produce a response at a concentration up to 100 micrograms/mL (approximately 50 microM). Similarly, in the 14C-serotonin release and flow cytometric assays, heparin and enoxaparin produced positive responses (n = 30), whereas the two pentasaccharides consistently failed to produce any effect. These observations suggest that the two pentasaccharides with highly selective anti-Xa activity are devoid of generating antiheparin-platelet factor 4 antibody, do not produce heparin-induced thrombocytopenic responses and may inhibit active heparin-induced thrombocytopenia antibody platelet activation. PMID- 10726025 TI - Direct measurement of unfractionated heparin using a biochemical assay. AB - A number of investigations have noted that functional biological assays for heparin are not always reliable and may not reflect the actual biochemical level of heparin in patients receiving anticoagulant therapy. This creates the possibility that patients receiving anticoagulant treatment may have an excess or deficiency of circulating levels of heparin. To address this problem, we have developed a direct biochemical measurement of heparin. The heparin assay uses fluorophore-assisted carbohydrate electrophoresis (FACE) to directly measure the predominate disaccharide of unfractionated heparin. In this study, unfractionated heparin was measured in vitro throughout a wide range of heparin concentrations in plasma. Seven in vivo pharmacokinetic studies in five normal subjects given 3,000 USP units of unfractionated heparin intravenously showed a three-phase elimination process with higher peak plasma levels and shorter elimination times than predicted from previous studies. At these doses, heparin is largely eliminated intact through urinary excretion. Body weight has a significant effect on heparin kinetics. When we compared the direct biochemical assay with two biological clotting assays, we found the latter can overestimate biochemical heparin concentrations. The FACE assay, due to its sensitivity, is also able to measure circulating levels of endogenous heparin in plasma and urine. Direct heparin measurement using the FACE technique is practical and useful for studies of the correlation of biochemical and biological activities. PMID- 10726026 TI - Familial association of hypoplasminogenemia and heterozygous factor V deficiency. AB - The coinheritance of hypoplasminogenemia and heterozygous factor V deficiency in a relative with thrombotic disease and no hemorrhagic tendency is described. The proposita, a 28-year-old woman, suffered from neurologic disturbances due to two ischemic cerebral lesions confirmed by nuclear magnetic resonance scan. She was found to be affected with heterozygous plasminogen deficiency in a coagulation study for inherited thrombophilia. Moreover, she disclosed a prolongation of prothrombin time and activated partial thromboplastin time, which was compatible with heterozygous factor V deficiency. Her father, with a history of deep vein thrombosis, was also affected with plasminogen deficiency, as well as three brothers and one sister who were asymptomatic. The mother of the proposita showed borderline or slightly decreased factor V levels and normal plasminogen levels; she was therefore considered to be heterozygous for factor V deficiency. Heterozygous factor V deficiency was also found in one brother and one sister of the proposita, and they were both asymptomatic. Among the other available family members, one brother and one sister of the proposita, all asymptomatic for either thrombotic or bleeding events, showed a normal clotting and fibrinolytic profile. To our knowledge, this is the first case of combined heterozygous plasminogen and factor V deficiency in the same family. Two of six patients with hypoplasminogenemia showed thrombotic events, and in one of these symptomatic cases the coexistence of factor V deficiency did not prevent the occurrence of thrombosis. As expected, no hemorrhagic tendency was observed in patients with heterozygous factor V deficiency, who may be mildly symptomatic only in 10% of cases. PMID- 10726027 TI - Thrombosis and hemorrhage in chronic myeloproliferative disorders. PMID- 10726028 TI - A further case of homozygous G20210A prothrombin gene mutation without thromboembolic events. PMID- 10726029 TI - Heparin-induced thrombocytopenia: historical review. AB - Heparin-induced thrombocytopenia (HIT) and heparin-induced thrombosis (HITT), recognized as clinical entities only in the past 30 years, have become a major concern for all who work in the field of thromboembolism, its prevention, and treatment. Studies of the 1930 to 1970 era were primarily devoted to the interaction of heparin with platelets in vivo with no knowledge that such interaction could lead to serious morbidity and mortality in as many as 3-5% of patients receiving the drug. When this was recognized in the 1970s, concerns arose regarding recognition and diagnoses. Clinicians, laboratory investigators, and drug companies entered into many research collaborations. The advent of low molecular weight heparin (LMWH) and heparinoids led to studies of their interaction with the IgG antibody that was identified as the mechanism for heparin-induced thrombocytopenia of the immune type. Newer, more sensitive test systems were developed and applied to studies comparing the new low LMWHs and heparinoids. These studies showed a lower incidence of immune-mediated thrombocytopenia with LMWH. The specificity of the antibody for platelet factor 4 heparin complexes led to the development of new tests in the early 1990s. This was soon followed by experiments with antithrombin drugs such as hirudin and argatroban as replacements for heparin in the HIT-HITT syndrome. Shorter courses of heparin therapy and the use of LMWH in place of heparin had become more standard practice in the 1990s. PMID- 10726030 TI - Clinical aspects of heparin-induced thrombocytopenia and thrombosis and other side effects of heparin therapy. AB - Heparin, first used to prevent the clotting of blood in vitro, has been clinically used to treat thrombosis for more than 50 years. Although several new anticoagulant drugs are in development, heparin remains the anticoagulant of choice to treat acute thrombotic episodes. The clinical effects of heparin are meritorious, but side effects do exist. Bleeding is the primary untoward effect of heparin. Major bleeding is of primary concern in patients receiving heparin therapy. However, additional important untoward effects of heparin therapy include heparin-induced thrombocytopenia, heparin-associated osteoporosis, eosinophilia, skin reactions, allergic reactions other than thrombocytopenia, alopecia, transaminasemia, hyperkalemia, hypoaldosteronism, and priapism. These side effects are relatively rare in a given individual, but given the extremely widespread use of heparin, some are quite common, particularly HITT and osteoporosis. Although reasonable incidences of many of these side effects can be "softly" deduced from current reports dealing with unfractionated heparin, at present the incidences of these side effects with newer low molecular weight heparins appear to be much less common. However, only longer experience will more clearly define the incidence of each side effect with low molecular weight preparations. PMID- 10726031 TI - Inadequacy of current prevention strategies for heparin-induced thrombocytopenia. AB - Heparin-induced thrombocytopenia is one of the most difficult problems facing clinicians today. Despite recent understanding of the pathophysiology of this disorder, there are many unresolved issues about diagnosis, prevention, and treatment. In this article, difficulties physicians encounter when faced with a suspected heparin-induced thrombocytopenia patient will be reviewed as well as our experience in 113 patients with heparin-induced thrombocytopenia which highlights the failure of current preventive strategies for heparin-induced thrombocytopenia. The experience of using warfarin in 51 patients with heparin induced thrombocytopenia will also be reviewed. PMID- 10726032 TI - Laboratory diagnosis of heparin-induced thrombocytopenia. AB - The characteristics of the currently available platelet function assays (platelet aggregation, serotonin release, and flow cytometry) and enzyme-linked immunosorbent assays that quantitate antiheparin-platelet factor 4 antibody titers were studied using sera collected from clinically diagnosed heparin induced thrombocytopenia patients, patients without heparin-induced thrombocytopenia, patients with platelet immune disorders other than heparin induced thrombocytopenia, and normal individuals. The platelet aggregation assay was less sensitive than the serotonin release assay, which was less sensitive than the enzyme-linked immunosorbent assay (p < 0.001). Yet heparin-induced thrombocytopenia was identified by platelet aggregation assay in cases where the serotonin release assay and/or the enzyme-linked immunosorbent assay were negative. Patients with heparin-induced thrombocytopenia and thrombosis were more often positive than heparin-induced thrombocytopenia patients without thrombosis (p < 0.05). Positive platelet aggregation assay and serotonin release assay results were generally associated with a higher antibody titer; however, a minimum critical titer could not be identified. Over a 30-day period the percentage of positive responses did not change significantly even though clinical symptoms corrected in most heparin-induced thrombocytopenia patients. Multiple testing over several days enhanced the chance of detecting a positive, and combined results of the three assays further enhanced the positive response (p < 0.005). In patients without heparin-induced thrombocytopenia, false-positive results were obtained with the enzyme-linked immunosorbent assay. These data demonstrate that there is no direct correlation between the positive responses of these assays, that clinically positive patients can be missed by all assays, and the presence of antibody alone does not determine clinical heparin-induced thrombocytopenia. With these limitations, the combination of aggregation, serotonin release, and enzyme-linked immunosorbent assay testing with multiple samples offers the best chance of identifying a positive heparin-induced thrombocytopenia patient. Caution is advised for all assays as none is optimal. PMID- 10726033 TI - Generation and pathogenicity of anti-platelet factor 4 antibodies: diagnostic implications. AB - Recent studies have elucidated the target antigens for heparin-dependent antibodies. The major one is generated in the presence of platelet factor 4 (PF4) and heparin at a well-defined concentration that allows formation of heparin-PF4 complexes and induces an alteration of the PF4 molecule. It then exposes neoepitopes, which bind the heparin-dependent antibodies. These antibodies are generated only in some heparin-treated patients, and only a subgroup of them develops thrombocytopenia (HIT) sometimes associated with thrombosis (HITT). The major challenge is to understand how antibodies can be generated and why only some of them are pathogenic. The presence of the IgG isotype at a high concentration is an important factor for developing HIT/HITT. The clinical context, which induces accumulation and activation of platelets at pathological sites, is also important for disease occurrence. The presence of antibodies (mainly the IgG isotype) enhances cell-cell interactions and platelet activation followed by platelet accumulation at these sites. Generation and expression of the autoantigen, HPF4 complexes, is a key feature for triggering antibody binding and pathogenicity. Factors contributing to antibody generation and its persistence, and factors contributing to the expression of the autoantigen triggering the disease development are discussed. PMID- 10726034 TI - Functional heterogeneity of antiheparin-platelet factor 4 antibodies: implications in the pathogenesis of the HIT syndrome. AB - Heparin-induced thrombocytopenia represents one of the most severe drug-induced disorders of platelets. This syndrome is believed to be mediated through antibodies generated against a heparin-platelet factor 4 complex. Complexation of a sulfated mucopolysaccharide chain of heparin with a platelet granular protein (platelet factor 4) produces an allosteric modification of platelet factor 4 resulting in neoepitope formation and the generation of antiheparin-platelet factor 4 antibodies. These antibodies are capable of activating platelets by binding to heparin, platelet factor 4 and the Fc receptor on platelets, resulting in a complex pathophysiology involving ischemic, thrombotic, and inflammatory processes. To characterize this antibody, IgG fractions were obtained from the serum of patients with heparin-induced thrombocytopenia using ammonium sulphate precipitation and heparin-platelet factor 4-sepharose 4B affinity chromatography methods. With the affinity purification, two major components, peaks I and II, with high antiheparin-platelet factor 4 antibody titers were eluted. The purity of all the fractionated immunoglobulins was established by sodium dodecylsulphate polyacrylamide gel electrophoretic analyses. While peak I did not induce 14C serotonin release from platelets in the heparin-dependent assay for heparin induced thrombocytopenia antibodies (14C-serotonin release assay), peak II and the IgGs obtained with the ammonium sulphate precipitation method exhibited a strong and concentration-dependent activation in the presence and absence of heparin and low molecular weight heparin. These immunoglobulins were treated with heparinase, a cationic ion-exchange resin (Heparsorb), or dialyzed to remove traces of heparin, and when tested in the 14C-serotonin release assay, showed the same high degree of activity. These data are suggestive of the generation of heparin-induced thrombocytopenia antibodies capable of activating platelets directly in a nonheparin-dependent manner. These observations underscore the complex pathophysiology of heparin-induced thrombocytopenia syndrome and suggest that the severity of this syndrome in some patients may be due to the generation of "super-active" heparin-induced thrombocytopenia antibodies capable of activating platelets without the requirement of heparin. This could explain why the cessation of heparin in patients does not necessarily correct the symptoms of heparin-induced thrombocytopenia or associated thrombosis. PMID- 10726035 TI - Soluble adhesion molecules in the HIT syndrome: pathophysiologic role and therapeutic modulation. AB - Heparin-induced thrombocytopenia pathophysiology is now known to be a complex process that involves platelets, vascular endothelium, and leukocytes/lymphocytes. The activation products from these sites also contribute to the activation of coagulation and fibrinolytic deficit. While many of the markers of hemostatic activation processes have been found to be increased during the acute phase of heparin-induced thrombocytopenia syndromes, the circulating levels of soluble adhesion molecules such as the P, E, and L selectins, and intracellular and vascular cell adhesion molecules have not been reported. Since the pathophysiology of heparin-induced thrombocytopenia involves the activation of platelets, endothelium, and leukocytes, it is expected that the activation products related to these hemostatic systems including soluble selectins and cellular adhesion molecules will also be increased in circulating blood. These alterations may also provide an index of the pathophysiologic process. With the availability of highly sensitive enzyme-linked immunosorbent assays for soluble P, E, and L selectins, intracellular and vascular cell adhesion molecules, it is now possible to measure these adhesion molecules in biological fluids. This study reports on the circulating levels of various adhesion molecules in patients with heparin-induced thrombocytopenia and their modulation after therapeutic interventions by the use of direct thrombin inhibitors. With the availability of recombinant hirudin, it is now possible to treat these patients with alternate antithrombin agents. However, the immunoactivation of platelets and other cells as shown here indicates the possible need for additional adjunct therapeutic approaches to suppress their participation in the thrombotic process. The reported increase in the circulating levels of the soluble adhesion molecules during the heparin-induced thrombocytopenia and heparin-induced thrombocytopenia with thrombosis syndrome suggests that the antiheparin platelet factor 4 antibody is capable of modulating their regulation. The prognostic role of these mediators in the management of heparin-induced thrombocytopenia syndrome warrants further investigation. PMID- 10726036 TI - Antithrombin agents: the new class of anticoagulant and antithrombotic drugs. AB - Antithrombin drugs represent a wide group of natural agents, recombinant agents equivalent to some of the naturally occurring proteins, and synthetic agents. This group of drugs is characterized by marked structural and functional heterogeneity. Several of these drugs are currently in various phases of development. Argatroban represents the first clinically approved antithrombin agent, which was made available in Japan several years ago. Two recombinant hirudin preparations, Revasc (Novartis) and Refludan (Aventis), are available for postsurgical DVT prophylaxis and alternate anticoagulant use in patients with heparin-induced thrombocytopenia. A synthetic antithrombin agent based on the combined structures of hirudin and antithrombin peptides, hirulog (Bivalirudin), is undergoing clinical trials in cardiovascular indications. Additional studies on the hirudins are being carried out to test their efficacy as surgical and interventional anticoagulants as replacements for heparin. However, the need for a proper antagonist is one of the limiting factors for the optimal development of hirudin in this indication. Several of the synthetic thrombin inhibitors are also being developed for oral use for the prophylaxis of DVT in surgical patients. Since the therapeutic index of thrombin inhibitors is narrower than that of heparin, this route may not be an optimal approach for the development of these agents. Despite several unresolved developmental issues, the thrombin inhibitors provide a useful alternative to heparin anticoagulation and may prove to be useful in validated clinical use. PMID- 10726037 TI - Heparin-induced thrombocytopenic potential of GAG and non-GAG-based antithrombotic agents. AB - We have undertaken these studies of the heparin-like, or glycosaminoglycan, and nonglycosaminoglycan-based antithrombotics in an effort to add to the understanding of the pathophysiologic mechanism of heparin-induced thrombocytopenia by investigations of how glycosaminoglycan-related agents interact with the heparin-induced thrombocytopenia antibodies. The low molecular weight heparins, originally thought to be useful alternatives to heparin because of their smaller size, show platelet activation and aggregation responses in platelet heparin-induced thrombocytopenia serum systems (P-selectin expression, microparticle formation, serotonin release, platelet aggregation). Although the molecular mass and sulfation of the heparinoid Lomoparan is similar to that of heparin and low molecular weight heparins, its chemical structure is different and probably is not recognized by the heparin-induced thrombocytopenia antibodies. The heparin-related pentasaccharide did not show a positive reaction in any system of platelet activation/aggregation. These studies have shown that the antibodies produced in patients with heparin-induced thrombocytopenia are reactive to highly sulfated glycosaminoglycans and nonglycosaminoglycan agents and less dependent on the molecular mass of these agents; whether the agent is a heparin or nonheparin compound was not critical. A combination of a moderate sulfation but low molecular mass in a heparin-like molecule was sufficient to prevent interaction with the heparin-induced thrombocytopenia antibodies. However, a chemical structure that is different from heparin (e.g., a heparinoid or a thrombin inhibitor) will also be nonreactive to platelet activation by heparin-induced thrombocytopenia antibodies. PMID- 10726038 TI - Low molecular weight heparins: differences and similarities in approved preparations in the United States. AB - There is adequate preclinical data to support the differential biochemical and pharmacological behavior of the currently approved low molecular weight heparins (LMWHs) in the United States. Initial studies on the anti-Xa, anti-IIa, and U.S. Pharmacopoeial (USP) potencies have clearly demonstrated differences among these products. Furthermore, the ratios between the anti-X and anti-IIa activities vary from one product to another. This is primarily due to the composition of each product manufactured by using different patented methods. Studies in pharmacologic animal models, using gravimetric dosages or adjusted anti-Xa dosages of the LMWHs, produce product-specific results. The pharmacokinetics and pharmacodynamics of each product also vary markedly and are not predictable on the basis of any pharmacopoeial potency designation. These agents are capable of releasing tissue factor pathway inhibitor (TFPI), an inhibitor of the coagulation process. Its release is also dependent on the type of LMWH. In the United States enoxaprin, dalteparin, and ardeparin have been approved for DVT prophylaxis. Only enoxaparin and dalteparin have been approved for the acute coronary syndrome. Recently the clinical differentiation among these LMWHs has been demonstrated in the treatment of acute coronary syndrome. Similarly, when these drugs are used at high dosages, they are expected to produce product-specific pharmacodynamic effects. It must be noted that while these drugs may be interchangeable at clinically optimized/approved dosages, these drugs are not interchangeable at equivalent anti-Xa dosages. Even at optimized dosages, the clinical provile of each drug may be different. Thus, each of the LMWHs should be considered a distinct entity and their use in a given clinical situation should be validated in proper clinical trials. PMID- 10726039 TI - Long-term outpatient dalteparin (fragmin) therapy for arterial and venous thrombosis: efficacy and safety--a preliminary report. AB - The availability of low molecular weight heparin (LMWH), with associated ease of home delivery and enhanced efficacy and safety, has rendered the long-term outpatient use of LMWH feasible. Although warfarin is usually used for long-term secondary prevention, it is not the drug of choice for a variety of conditions such as antiphospholipid thrombosis syndrome, sticky platelet syndrome, and patients with malignancy and other medical conditions who have failed adequate warfarin doses. We assessed the long-term efficacy and safety of outpatient dalteparin in a series of patients with conditions associated with prior warfarin failure or potential to warfarin therapy (antiphospholipid syndrome). The results of this study, detailed herein, demonstrate that long-term dalteparin is highly effective and safe when used as long-term therapy for secondary prevention in selected prothrombotic disorders. PMID- 10726040 TI - Low molecular weight heparins and heparin-induced thrombocytopenia. AB - Heparin-induced thrombocytopenia (HIT) and HIT thrombosis syndrome (HITTS) are immune-mediated complications of clinical use of unfractionated heparin (UFH). The antibody/antigen complex is composed of heparin and platelet factor 4. This complex not only activates platelets but also the clotting system leading to thrombin generation. This explains the thrombosing tendency of these patients, and venous and arterial thromboembolisms are encountered with a morbidity and mortality of about 25-37%. The incidence of HIT is about 3% when UFH is administered therapeutically. The diagnosis is at this time based on clinical observations, especially a sudden, unexplained drop in platelet counts without other reasons. Laboratory tests can be used to confirm the clinical diagnosis, but none of the available tests is 100% reliable. There is no test that will predict HIT and no test that will signal the development of HITTS. Treatment consists of discontinuation of UFH in any form and anticoagulation with danaparoid or r-hirudin, if needed. The use of low molecular weight heparins instead of UFH could largely (not totally) alleviate the problem. PMID- 10726041 TI - Vascular damage correlates between heparin-induced thrombocytopenia and the antiphospholipid syndrome. AB - Antibody-mediated disorders of heparin-induced thrombocytopenia and antiphospholipid antibody syndrome have remarkably similar clinical presentations, both of which can progressively result in severe vascular and thrombotic disorders. We hypothesized that the mechanism of platelet activation as occurs in heparin-induced thrombocytopenia may also occur in antiphospholipid antibody syndrome particularly at the vascular wall, that endothelial injury may be similar in heparin-induced thrombocytopenia and antiphospholipid antibody syndrome, and that these alterations may be caused by related antibodies. Antibody titers and vascular endothelial damage in patients with heparin-induced thrombocytopenia and antiphospholipid antibody syndrome were studied in plasma samples collected from normals (n = 17), heparin-induced thrombocytopenia patients (n = 15), antiphospholipid antibody syndrome patients (n = 30), and patients clinically diagnosed with antiphospholipid antibody syndrome and heparin induced thrombocytopenia (n = 8). Diagnosis of heparin-induced thrombocytopenia was confirmed by 14C-serotonin release assay or positive antiheparin-platelet factor 4 antibody titer, and antiphospholipid antibody syndrome was confirmed by positive anti-beta 2-glycoprotein (GP) 1/cardiolipin (IgG or IgM) antibody titer. The antiheparin-platelet factor 4 antibody was not detected in any patient with antiphospholipid antibody syndrome. Patients with heparin-induced thrombocytopenia did not have elevated IgG anti-beta 2-GP1 titers, but three (20%) patients had low-positive IgM anti-beta 2-GP1 titers. The endothelial damage markers of soluble thrombomodulin, soluble P-selectin (p < 0.05 vs. normal), plasminogen activator inhibitor-1 and tissue factor were elevated in heparin-induced thrombocytopenia and antiphospholipid antibody syndrome patients. The soluble E-selectin was elevated only in the patients with both heparin induced thrombocytopenia and antiphospholipid antibody syndrome (p < 0.05 vs. normal). Levels of soluble L-selectin and von Willebrand factor were not different from normals. The pathogenesis of heparin-induced thrombocytopenia and antiphospholipid antibody syndrome appears to be due to two distinct antibodies but associated with similar damage to the vascular endothelium in both diseases. PMID- 10726042 TI - Thrombolytic drugs in acute myocardial infarction. PMID- 10726043 TI - Elevated tissue factor levels in leukemic cell homogenate. AB - Tissue factor (TF) antigen and activity were measured in leukemic cell homogenates. In leukemic cell homogenate, especially that of acute promyelocytic leukemia (APL), both TF antigen and activity were significantly higher than these levels in the mononuclear cells obtained from healthy volunteers. Both TF antigen and activity were significantly higher in myelocytic leukemia than in lymphocytic leukemia cells. In leukemic cell homogenates, there was a close correlation between TF antigen and TF activity. The TF activity/TF antigen ratio was significantly higher in myelocytic leukemia than in lymphocytic leukemia cells. As the TF activity was not increased in lymphocytic leukemia cell homogenates to which were added phospholipids, the decrease in TF activity in lymphocytic leukemia might not be due to phospholipid in the leukemic cell membrane. Values for TF activity, TF antigen, and the TF activity/TF antigen ratio in leukemic cell homogenate from patients with disseminated intravascular coagulation (DIC) were significantly higher than those in patients without DIC. Therefore, the measurement of TF antigen and activity in leukemic cells could be useful for the prediction of DIC. PMID- 10726044 TI - Statins induce hypothrombotic states? PMID- 10726045 TI - The antithrombotic factor singlet oxygen/light (1O2/h nu). AB - Activated phagocytes (especially polymorphonuclear granulocytes (PMNs)) by respiratory oxidative/photonic burst (activation of NADPH-oxidase and myeloper oxidase) generate large amounts of oxidants of the hypochlorite-/chloramine-type, which are physiologic sources for singlet oxygen (1O2), a nonradical-excited (photon (h nu) emitting) oxygen species [Weiss SJ, NEJM 1989;320:365-376]. In vitro experiments show that 1O2 (1) inhibits coagulation by inactivation of thrombocytes, fibrinogen, factor V, factor VIII, and factor X and (2) activates fibrinolysis by inactivation of the main fibrinolysis inhibitors plasminogen activator inhibitor (PAI)-1 and alpha-2-antiplasmin, and by activation of single chain urokinase by plasmin and oxidized fibrin. Additionally, this work suggests that 1O2/h nu acts antithrombotically, inducing selective thrombolysis in vivo (i.e., thrombolysis induced by 0.1 to 0.5 mmol/l chloramine within 30 to 60 minutes without changes of the plasmatic hemostasis system). 1O2 might activate flowing to (on the endothelium) rolling PMN, increasing their chance to get in contact with fibrin/platelet aggregates deposited on the endothelial layer. Via 1O2 generation, the thrombus-activated phagocytes might call for (acute, physiologic) inflammation/fibrinolysis amplification, resulting in the "moving front" of PMN, which infiltrates and destroys the thrombus. 1O2 seems to (partially) participate in the reactivity of nitric oxide, another prooxidative agent. The inhibition of physiologic amounts of 1O2 by blood cholesterol might be involved in the pathogenesis of atherothrombosis. Consequently, it is suggested that activated PMNs modulate hemostasis, shifting it into an antithrombotic state; this cellular part of fibrinolysis seems to be of greater physiologic importance than the plasmatic one. Impaired PMN function (e.g., as occurring in patients with antineutrophil cytoplasmic antibodies or under cytostatic treatments) often results in serious thrombotic complications. Light is the only signal whose origin can be immediately recognized by a fast moving cell in the (dark) blood stream. The cell signal action of 1O2/h nu (e.g., released by chloramines such as taurine-chloramine or vancomycin, by fiberoptic, by photodynamic therapy, or by so-called redox-cycling drugs such as quinones or tetracyclines) might be a new and physiologic principle for pharmacologic intervention in atherothrombosis. PMID- 10726046 TI - Essential thrombocythemia in young adults: major thrombotic complications and complications during pregnancy--a follow-up study in 68 patients. AB - OBJECTIVES: Although essential thrombocythemia (ET) is usually primarily considered a disorder of middle age, it has been observed in children and young adults. However, the real risk for thrombosis in these patients has not been clearly established. DESIGN: Prospective analysis of consecutive patients younger than 40 at the time of the diagnosis of ET and followed in our department between 1980 and 1998. SUBJECTS: Sixty-eight patients (28 males and 40 females, median follow-up 99.14 months) affected by ET diagnosed in agreement with the Polycythemia Vera Study Group criteria. INTERVENTIONS: Asymptomatic ET patients were not treated. In contrast, patients with associated atherosclerotic risk factors, microvascular disturbances, or a previous major arterial thrombosis were given acetyl salicylic acid (ASA 100 mg/day). Only patients with major thrombotic complications and a platelet count > 1,000 x 10(9)/L received cytoreductive therapy. OUTCOME MEASURES: (1) to evaluate thrombotic complications in young patients with ET, (2) to relate thrombotic risk to the presence of general atherosclerotic risk factors, and (3) to adopt treatment, and (4) to report the outcome of the pregnancies monitored in our population. RESULTS: Fifteen patients had major thrombosis, 11 of which were the presenting features of ET. No rethrombosis has been observed. Only one patient with thrombotic complications was under efficient treatment. Atherosclerotic risk factors are more common in patients with major arterial thrombosis than in asymptomatic subjects. Thirteen normal babies were delivered out of 16 pregnancies, 6 of the pregnant women were on ASA therapy. CONCLUSIONS: Most thrombosis in young ET patients occurred at the time of the diagnosis, and venous thrombotic events represent one-third of total thrombosis. Cardiovascular risk factors seem to be concurrent stimuli for arterial thrombosis in ET. The thrombotic complication rate was 2.6/100 patients/year ASA reduces microvascular disturbances, thrombosis, and rethrombosis and possibly reduces obstetric complications in women with ET. PMID- 10726047 TI - Combined heterozygous plasminogen deficiency and factor V Leiden defect in the same kindred. AB - Combined plasminogen deficiency and resistance to activated protein C defect (factor V Leiden) have been described in a few families and associated with a variable occurrence of thrombotic events. Here we describe a new family with thrombophilia and the presence of hypoplasminogenemia and factor V Leiden mutation. In addition, a brief review of the literature is presented. Nine patients belonging to this kindred underwent coagulation study for hereditary thrombophilia, which included plasminogen antigen and activity assays, an activated protein C resistance test, and genetic analysis for factor V Leiden mutation and for prothrombin variant 20210A. The proposita, a 40-year-old asymptomatic female with a family history of thrombotic diathesis, was affected by heterozygous plasminogen deficiency. Hypoplasminogenemia was found also in her two sisters, in one instance associated with factor V Leiden mutation. The mother was the putative carrier of hypoplasminogenemia, but she refused to be studied. The symptomatic father was heterozygous for factor V Leiden mutation, but presented with normal plasminogen levels. Among the available siblings investigated from the paternal side, resistance to activated protein C due to factor V Leiden mutation was found in three patients, one of whom experienced venous thromboembolism. Another uncle with a history of thrombotic disease showed no coagulation abnormalities. These findings together with the data from literature confirm the role of factor V Leiden as an independent risk factor for venous thromboembolism, whereas isolated hypoplasminogenemia does not seem to increase the risk for thrombosis. There is no clear evidence that the coinheritance of these two defects may be associated with an additional risk for thrombosis compared with the presence of factor V Leiden mutation alone. PMID- 10726048 TI - The Leiden mutation of coagulation factor V in Hungarian SLE patients. AB - We studied the prevalence and the effect of coagulation factor V Leiden mutation on the occurrence of thrombotic episodes in 120 Hungarian patients having systemic lupus erythematosus (SLE) with or without antiphospholipid antibody. The frequency of the factor V Leiden mutation in Hungarian SLE patients was 13%, which is comparable with those found previously in a healthy Caucasian population. The incidence of venous thrombosis among factor V Leiden carriers has been found to be higher (odds ratio [OR] 1.7) than it is in patients without Leiden mutation (38% vs 29%). In addition, the frequency of venous thrombosis in the heterozygous SLE patients (OR 8.4 [confidence interval (CI) 0.8-83.9] P = 0.06) is dependent on the coexistence of other risk factors, such as antiphospholipid antibody. Moreover, among heterozygous factor V SLE patients, the Leiden mutation could explain the tendency to have significantly higher prevalence of fetal losses (OR 3.9 [CI 1.2-12.0] P = 0.02) and higher prevalence of cerebrovascular lesions, cardiac valvular abnormalities, and Raynaud's syndrome than that found in individuals without factor V Leiden mutation of those having antiphospholipid antibody. Systemic lupus erythematosus patients with combined defects suffer more severely from thrombosis than those with a single risk factor do, suggesting that thrombophilia is a multifactorial disorder in SLE, also. Although, the factor V Leiden mutation does not seem to be a significant risk factor for venous thrombosis in SLE, these data demonstrate that Leiden mutation can be regarded as an additive thrombogenic factor providing higher predisposition to several vasoocclusive disorders in SLE. PMID- 10726049 TI - Exclusion and diagnosis of pulmonary embolism by a rapid ELISA D-dimer test and noninvasive imaging techniques within the context of a clinical model. AB - A negative rapid ELISA D-dimer test alone in out-patients with a low to moderate clinical probability (CP) on pulmonary embolism (PE) is predicted to safely exclude pulmonary embolism. The combination of a negative rapid ELISA D-dimer test and a low to moderate CP on PE followed by compression ultrasonography (CUS) for the detection of deep vein thrombosis (DVT) is safe and cost-effective as it reduces the need for noninvasive imaging techniques to about 50% to 60% of outpatients with suspected PE. A high probability ventilation-perfusion (VP) scan or a positive spiral CT consistent with PE and the detection of DVT by CUS are currently considered to be clear indications for anticoagulant treatment. Subsequent pulmonary angiography (PA) is the gold standard diagnostic strategy to exclude or diagnose PE in suspected outpatients with a negative CUS, a positive rapid ELISA D-dimer test, and a nondiagnostic VP scan or negative spiral CT to prevent overtreatment with anticoagulants. However, the willingness of clinicians and the availability of resources to perform PA is restricted, a fact that has provided an impetus for clinical investigators to search for alternative noninvasive strategies to exclude or detect venous thromboembolism (VTE). Serial CUS testing for the detection of DVT in patients with a low to moderate CP on PE and a nondiagnostic VP scan or negative spiral CT is predicted to be safe and will reduce the need for PA to less than 10% or even less than 5%. This noninvasive serial CUS strategy restricts the need for invasive PA to a minor group of patients (< 5%) with the combination of a low CP on PE and high probability VP scan or the combination of a nondiagnostic VP scan or negative spiral CT and a high CP on PE. Prospective evaluations are warranted to implement and to validate the advantages and the disadvantages of the various combinations of noninvasive strategies and to compare serial CUS testing versus PA in randomized clinical management studies of outpatients with suspected pulmonary embolism. PMID- 10726050 TI - Dose relation in the prevention of proximal vein thrombosis with a low molecular weight heparin (tinzaparin) in elective hip arthroplasty. AB - This is a review of a double-blind, prospective study comparing the thromboprophylactic efficacy and safety of two different prophylactic regimens of a low molecular weight heparin (tinzaparin) in 250 consecutive patients (aged < or 18) undergoing primary elective hip arthroplasty. Regimen 1: 75 U anti-Xa/kg BW (actual range 63 to 91) once daily started 12 hours before operation; and regimen 2: 50 U anti-Xa/kg BW (actual range 41 to 71) once daily started 2 hours before operation. Both regimens were administered in a weight-adjusted fashion and were continued for 7 days after operation or until full mobilization. Efficacy was evaluated by occurrence of postoperative deep vein thrombosis (DVT) diagnosed by bilateral ascending phlebography on day 7 +/- 2 after operation, and the venograms were evaluated in an assessor blind fashion by a panel of three expert radiologists. Safety was evaluated by the amount of blood lost and transfusion requirements during and after the operation; all bleeding complications, reoperations, adverse events and deaths were observed during the study. A 3-month follow-up on survival and occurrence of thromboembolism was performed on all randomized patients. The result was a significantly better protective effect against proximal DVT by regimen 1 compared with regimen 2. This was achieved with improved safety in terms of a significantly decreased need for blood transfusions during operation and fewer wound complications in the postoperative period in favor of regimen 1. Therefore, tinzaparin administered in a dosage of 75 U anti-Xa/kg BW 12 hours before surgery is significantly more protective against proximal DVT and safer than the standard regimen of 50 U anti Xa/kg BW started 2 hours before surgery in patients undergoing primary elective hip arthroplasty. PMID- 10726051 TI - Prevalence of hepatitis B and C in Egypt and Africa. AB - Hepatitis B and C are, and will remain for some time, major health problems in Egypt and the entire continent of Africa. Both infections can lead to an acute or silent course of liver disease, progressing from liver impairment to cirrhosis and decompensated liver failure or hepatocellular carcinoma (HCC) in a 20-30 year period. In addition, hepatitis B and C infection rates differ in different settings, and prognosis may be worse in conjunction with schistosomiasis in Egypt, malaria in Sudan and human immunodeficiency virus (HIV) in other African populations. Unlike hepatitis B virus (HBV), for which the prospects for controlling the spread of infection by vaccination are promising, prospects for development of an effective vaccine against hepatitis C virus (HCV) are limited. As well as screening of blood for transfusion and using sterile needles for injection, preventive measures should be undertaken to reduce the risk of contact (often described as community-acquired infection). Until more is known about the unidentified routes of transmission in tropical and subtropical settings it will be difficult to be specific about the kind of measures which may be effective. Success may largely depend on changing habits within the population. Prevention should be the main goal of current efforts until low-cost, effective therapies become available. PMID- 10726052 TI - The immunopathogenesis of HBV infection. AB - Clinical manifestations of hepatitis B virus (HBV) infection are a balance between viral and host factors. The immune response against any virus consists of a coordinated defence of innate immunity and acquired, virus-specific immunity. In acute HBV, immune responses associated with recovery include vigorous, polyclonal CD4 T cells directed against multiple epitopes within HBV; antibodies directed against surface envelope proteins (anti-HBs), the development of which requires the presence of a CD4 response; and HBV-specific cytotoxic T lymphocytes (CTLs). HBV-specific CTLs can induce death of infected hepatocytes as well as produce cytokines. Most individuals with acute HBV recover without evidence of massive liver destruction; this, plus evidence from transgenic animal models, suggests that these cytokines produced by T cells play an important role in controlling HBV replication. Individuals who fail to mount a vigorous response in acute HBV develop chronic infection. In these cases, the persisting ineffective immune response appears to be responsible for liver damage and is likely to initiate the process of hepatic fibrosis. Based on our current understanding of the immune response in acute and chronic HBV, several groups are investigating the prospect of manipulating the immune response in chronic HBV. PMID- 10726053 TI - Pathogenesis of chronic hepatitis C virus infection. AB - HCV-infected patients with chronic liver disease have evidence of circulating HCV specific antibodies and a polyclonal, multi-specific T cell response. CD4+ proliferative responses and HCV-specific CTLs directed against one or more viral antigens are readily detected in those individuals who develop chronic HCV infection and appear to compartmentalize within the liver. Cytokines, which are produced locally within the liver and systemically, may play an important role in controlling viral replication and contributing to hepatocellular damage. However, neither the humoral nor cellular immune response, nor the cytokine response appears sufficient to eradicate infection in most patients. In its attempt to clear the virus from the liver, the immune system contributes to the hepatocellular injury seen in the majority of chronically infected patients. A better understanding of the host's immune response may provide further insight on the pathogenetic mechanisms involved in development of chronic hepatitis and aid the development of better therapeutic strategies. PMID- 10726054 TI - Hepatitis delta virus: molecular biology, pathogenesis and immunology. AB - Hepatitis delta virus (HDV) is a unique infectious agent which causes severe liver disease in those infected with its helper virus, hepatitis B virus. No effective antiviral therapy for HDV exists. This review covers recent advances in the molecular biology, pathogenesis and immunology of HDV, with an emphasis on potential targets for the development of successful antiviral agents. PMID- 10726055 TI - Molecular tools for the treatment of hepatitis C. AB - The treatment of chronic hepatitis C is aimed at eliminating viral replication in order to prevent further evolution towards cirrhosis and hepatocellular carcinoma. Virological parameters include hepatitis C virus (HCV) genotype, qualitative viraemia, quantitative viral load and the characteristics of HCV quasi-species heterogeneity. These parameters can be used to predict and monitor the response to therapy, in order to help clinicians to tailor treatment of chronic hepatitis C and to better understand the molecular mechanisms underlying HCV resistance to antiviral drugs. Current knowledge on these various issues is reviewed in the present article. PMID- 10726056 TI - Transgenic mice as a chemotherapeutic model for hepatitis B virus infection. AB - Many transgenic mice carrying either portions of the hepatitis B virus (HBV) genome, or the complete genome, have been developed as models because HBV does not infect any other organisms besides humans and chimpanzees to cause a productive infection and disease. Some of these models have been useful in evaluating chemotherapeutic agents such as interferon-alpha, interleukins-2 and 12, other cytokines and nucleoside analogues for efficacy against HBV. A recently developed transgenic mouse (Guidotti et al., Journal of Virology 69:6158-6169.) which supports the replication of high levels of infectious HBV, provides the opportunity to evaluate the effect of antiviral drugs on various portions of the HBV life cycle in the whole animal. Evaluation of lamivudine, zidovudine and interferon-alpha B/D (IFN-alpha) in this HBV transgenic mouse model are described. Lamivudine and IFN-alpha were highly efficacious in reducing serum HBV DNA. As might be predicted, zidovudine was not efficacious. IFN-alpha was more effective in reducing virus titres in male mice as compared to female mice. This gender difference was not due to lower ability of female mice to express the virus. One anticipates that as this high level HBV transgenic-expressing mouse becomes more fully developed as a chemotherapeutic model, questions about the efficacy of different agents, routes of administration, synergy of antiviral combinations and novel drug therapies will be answered. PMID- 10726057 TI - Molecular virology of hepatitis C virus: an update with respect to potential antiviral targets. AB - Hepatitis C virus (HCV), a positive-strand enveloped RNA virus, is a major cause of chronic liver disease worldwide. Cis-acting RNA elements and virus-encoded polypeptides required for HCV replication represent attractive targets for the development of antiviral therapies. Internal ribosome entry site-directed translation of HCV genome RNA produces a long polyprotein which is co- and post translationally processed to yield at least 10 viral proteins. A host signal peptidase is responsible for maturation of the structural proteins located in the N-terminal one-third of the polyprotein. Thus far, four enzymatic activities encoded by the non-structural (NS) proteins have been reported. The NS2-3 region encodes an autoproteinase responsible for cleavage at the 2/3 site. The N terminal one-third of NS3 functions as the catalytic subunit of a serine proteinase which cleaves at the 3/4A, 4A/4B, 4B/5A and 5A/5B sites, and NS4A is an essential cofactor for some of these cleavages. NS3 also encodes an RNA stimulated NTPase/RNA helicase at its C terminus, and NS5B has been shown to possess an RNA-dependent RNA polymerase activity. To date, no functions have been reported for NS4B or NS5A in RNA replication, however, NS5A has been implicated in modulating the sensitivity of HCV to interferon. Sequence and structural conservation within the 3' terminal 98 bases of genomic RNA suggest a functional importance in the virus life-cycle and hence another target for antiviral intervention. Recently, HCV infection was shown to be initiated in chimpanzees following intrahepatic inoculation of RNA transcribed from cloned HCV cDNA. The ability to generate large quantities of infectious HCV RNA may facilitate the development of reliable cell culture replication systems useful for the evaluation of antiviral drugs. PMID- 10726058 TI - Targets for inhibition of hepatitis C virus replication. AB - Considerable progress has been made in characterizing the proteins involved in hepatitis C virus (HCV) replication, despite the lack of a cell culture system. A number of systems have been developed to examine the processes involved in viral replication, including the initiation and processing of the viral proteins required for RNA replication, the unwinding activities of the RNA helicase and the synthesis of RNA by the viral polymerase. These processes have been examined using individually cloned proteins expressed in various in vitro systems, which may be suitable targets for antiviral agents. The viral helicase and protease domains have now been crystallized, which may enable the rational design of specific inhibitors. The recent developments in HCV research in understanding the function of the viral non-structural proteins and the establishment of in vitro screening assays may aid in the development of new antiviral agents. PMID- 10726059 TI - Helicase, a target for novel inhibitors of hepatitis C virus. AB - Virus-encoded enzymes of hepatitis C virus (HCV) were identified from its genome sequence. This allows application of drug discovery strategies which rely on inhibition of enzymes unique to HCV. Discovery of high-affinity inhibitors is facilitated by knowledge of the target enzyme's three-dimensional structure. For development of inhibitors of the HCV helicase, which belongs to a class of enzymes for which little structural information is available, the impact of structural information on the drug discovery process is greater than for targets belonging to well-characterized classes of enzyme. Here the structure of the HCV helicase is described. Regions required for enzymatic activity, which are also the preferred sites of drug interaction, are highlighted. PMID- 10726060 TI - The hepatitis C virus NS3 proteinase: structure and function of a zinc-containing serine proteinase. AB - The hepatitis C virus (HCV) NS3 protein contains a serine proteinase domain implicated in the maturation of the viral polyprotein. NS3 forms a stable heterodimer with NS4A, a viral membrane protein that acts as an activator of the NS3 proteinase. The three-dimensional structure of the NS3 proteinase complexed with an NS4A-derived peptide has been determined. The NS3 proteinase adopts a chymotrypsin-like fold. A beta-strand contributed by NS4A is clamped between two beta-strands within the N terminus of NS3. Consistent with the requirement for extraordinarily long peptide substrates (P6-P4'), the structure of the NS3 proteinase reveals a very long, solvent-exposed substrate-binding site. The primary specificity pocket of the enzyme is shallow and closed at its bottom by Phe-154, explaining the preference of the NS3 proteinase for cysteine residues in the substrate P1 position. Another important feature of the NS3 proteinase is the presence of a tetrahedral zinc-binding site formed by residues Cys-97, Cys-99, Cys-145 and His-149. The zinc-binding site has a role in maintaining the structural stability and guiding the folding of the NS3 serine proteinase domain. Inhibition of the NS3 proteinase activity is regarded as a promising strategy to control the disease caused by HCV. Remarkably, the NS3 proteinase is susceptible to inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A/NS4B, NS4B/NS5A and NS5A/NS5B cleavage sites. The Ki values of the inhibitory products are lower than the K(m) values of the respective substrates and follow the order NS4A < NS5A < NS4B. Starting from the observation that the NS3 proteinase undergoes product inhibition, very potent, active site-directed inhibitors have been generated using a combinatorial peptide chemistry approach. PMID- 10726061 TI - Preclinical investigation of L-FMAU as an anti-hepatitis B virus agent. AB - Preclinical aspects of a potent anti-hepatitis B virus (HBV) L-nucleoside, 1-(2 fluoro-5-methyl-beta-L-arabino-furanosyl)uracil (L-FMAU) are described. L-FMAU was prepared from L-ribose derivatives via either L-xylose or L-arabinose. L-FMAU shows potent antiviral activity against hepatitis B virus (EC50 5.0 microM in H1 cells) with high selectivity in vitro. L-FMAU is not incorporated into mitochondrial DNA and no significant lactic acid production was observed in vitro. L-FMAU is phosphorylated by thymidine kinase as well as deoxycytidine kinase, ultimately to the triphosphate, which inhibits HBV DNA polymerase as the mechanism of antiviral action. Preliminary in vivo toxological studies suggest no apparent toxicity for 30 days at 50 mg/kg/day in mice and for 3 months in woodchucks (10 mg/kg/day). L-FMAU also has respectable bioavailability in rats. L FMAU shows potent anti-HBV activity in vivo against woodchuck hepatitis virus in chronically infected woodchucks and there is no significant virus rebound after cessation of the drug treatment. PMID- 10726062 TI - Hepatitis C virus in the transplant setting. AB - Hepatitis C virus (HCV)-associated end-stage liver disease is a leading diagnosis in patients undergoing liver transplantation, accounting for approximately 20-25% of transplantations in many centres. In spite of universal viral recurrence, early post-transplantation infection generally results in indolent disease with good graft and patient survival, at least for the first 5-7 years, comparable to those observed in other patients undergoing transplantation for non-viral end stage liver disease. The full consequences of HCV recurrence are however beginning to be delineated with development of progressive liver failure observed with longer follow-up in a still unknown proportion of patients. Factors which may influence the outcome include viral load at transplantation and the type/amount of immunosuppression. Currently, the only available drugs are interferon and ribavirin, alone and in combination, used either therapeutically when the disease has fully developed, or prophylactically early after transplantation. Unfortunately, interferon has been used with limited success and with concerns about toxicity. Ribavirin monotherapy has been ineffective in producing meaningful results. Preliminary results on combination therapy are promising, both when administered before hepatitis develops or when histologic disease is present. Current antiviral therapy is however unable to eliminate HCV in the liver transplant setting, suggesting that indefinite treatment designed to suppress the effects of virus may be necessary. Major therapeutic advances for HCV infection are awaited. PMID- 10726063 TI - Coinfection by hepatitis B virus and hepatitis C virus. AB - Coinfection by hepatotropic viruses can occur due to the fact that hepatitis B virus (HBV) and hepatitis C virus (HCV) share similar routes of transmission. Different clinical features of liver disease can be observed in infected patients, ranging from fulminant, acute and chronic hepatitis to hepatocellular carcinoma (HCC). The relative role of the infecting viruses in determining the final clinical picture is not yet well defined. Several reports indicate that clinical and pathological severity of liver disease among coinfected patients is increased and in patients with HCC, co-occurrence of both viruses is a common event. The potential mechanism of tumour development still remains speculative, although direct and indirect roles for both HBV and HCV have been proposed. At the molecular level, reciprocal interference of virus replication has been repeatedly described and the extent of interference is influenced by the infecting HCV genotype, genotype 1 of HCV having more efficient inhibitory activity on HBV than genotype 2. Sequence similarities between an arginine-rich nucleocapsid motif of both viruses could support these clinical observations. Concerning response rates to interferon therapy, no satisfactory results have been achieved to date, although identification of effective therapeutic schemes, based on virological status of both viruses are warranted. PMID- 10726064 TI - Relationship between infection with hepatitis C virus and hepatocellular carcinoma in Japan. AB - The number of deaths due to HCC is increasing in Japan. Males in particular have seen a marked increase in the past 20 years, and HCV infection accounts for this increase. In patients treated with IFN and followed up for more than 12 months, HCV clearance was achieved in 29.8% and sustained normalization of ALT levels was achieved in 41.4% following IFN therapy. The incidence of HCC following IFN treatment is dependent on the effect of IFN on ALT levels. The incidence of HCC was low in patients with sustained normalization of ALT levels following IFN therapy, regardless of whether or not HCV had been eradicated. In the future, in addition to the development of therapies to eradicate HCV, treatments to lower ALT levels will also be important. PMID- 10726065 TI - Use of symptom questionnaires in the assessment and follow-up of men with benign prostatic disease. AB - The assessment of lower urinary tract symptoms is an essential component of the evaluation of men with benign prostatic disease in clinical practice and research. Symptom evaluation is necessary for the formal evaluation of the effectiveness of treatments in randomized controlled trials and in the assessment of outcome in individuals, and can also be useful in determining the numbers of individuals in the community who might potentially require or benefit most from treatment. Increasingly, symptom assessments are being undertaken using a small number of questionnaires that are self-completed by patients and have been tested for validity and reliability. PMID- 10726066 TI - Sexual dysfunction in patients with benign prostatic hyperplasia. AB - Two of the most common problems presenting to urologists are benign prostatic hyperplasia and sexual dysfunction, with an increasing number of patients presenting for treatment as a result of the proliferation of less invasive therapies. How such therapies for lower urinary tract symptoms affect sexual function in men is important to both urologists and their patients, and is the focus of this review. PMID- 10726067 TI - The use of voiding studies (flowmetry and urodynamics) in the assessment and follow-up of patients. AB - The role of urinary flow measurement and pressure-flow studies in the diagnostic work-up of men presenting with lower urinary tract symptoms suggestive of benign prostatic obstruction is still an unresolved issue. In this paper, evidence from the peer-reviewed literature of the last 12 months is critically reviewed in the light of the recent recommendations of the 4th International Consultation on benign prostatic hyperplasia. PMID- 10726068 TI - Aspects of imaging in the assessment and follow up of benign prostatic hyperplasia. AB - For assessing patients suffering from benign prostatic hyperplasia and monitoring subsequent treatment, symptom questionnaires, uroflowmetry, prostate volume measurements, postvoiding residual urine volume measurements and pressure-flow studies may be used. This review highlights aspects of imaging in the assessment of benign prostatic hyperplasia, including volume determinations of prostate and postvoiding residual urine, texture imaging and biopsy guidance. Future developments are also briefly discussed. PMID- 10726069 TI - Diagnostic research in benign prostatic hyperplasia--from sensitivity to neural networks. AB - Establishing the diagnosis in patients with lower urinary tract symptoms is complicated. Several tests have been developed, but the importance of each of these tests in the diagnostic process is not well understood. This paper describes the methods used to evaluate diagnostic tests, ranging from traditional sensitivity and specificity to more sophisticated techniques such as logistic regression and neural networks. PMID- 10726070 TI - Alternative methods for management of prostatic outflow obstruction. AB - During the past year no new therapies for prostatic outflow obstruction were introduced. Instead, research focused on refinement of treatments previously available. Further reports on the efficacy and safety of alpha 1-adrenergic receptor subtype selective agents have appeared. The long-term effects of finasteride on the natural history bladder outflow obstruction have become clearer. The clinical efficacy of device therapy has evolved, and information regarding intermediate-term outcomes is now available. PMID- 10726071 TI - Bacterial and fungal infections after kidney transplantation. AB - Infectious diseases in renal transplant recipients are a major issue both from the medical and from the economical point of view. The major risk factor is the immunosuppressive therapy. Infections observed during the first weeks after transplantation are not different from those observed in nonimmunosuppressed patients after surgery. Infections with opportunistic pathogens, however, emerge at the beginning of the second month and are mainly determined by the allograft function. PMID- 10726072 TI - Human immunodeficiency virus: post-exposure prophylaxis. AB - Post-exposure prevention is a combined modality approach to reducing HIV transmission. The recommendations are based on the risk of local prevalence and on the likelihood of transmission. In the light of the severe consequences of HIV infection, post-exposure prophylaxis after occupational exposure should be recommended. The medication, consisting of a potent antiretroviral combination (two nucleoside analogues and one reverse transcriptase inhibitor), should be available within two hours. PMID- 10726073 TI - Short-term therapy of acute uncomplicated cystitis. AB - Short-term therapy, including single-dose treatment and up to 3-day courses, can be considered the treatment of choice in premenopausal women with acute uncomplicated cystitis, because of similar effectiveness, better tolerance and compliance, and lower cost as compared with conventional therapy. Several studies with trimethoprim alone or in combination with a sulfonamide, usually sulphamethoxazole, and with fluoroquinolones with moderately long half-lives, such as ciprofloxacin, enoxacin, lomefloxacin and ofloxacin, suggested that results obtained with a single dose may be inferior to those with a 3-day course, and therefore that the latter may be better. Longer therapy was not considered to be necessary. On the other hand, fluoroquinolones with longer half-lives, such as fleroxacin, pefloxacin and rufloxacin, as single-dose therapy may be as effective as other standard regimens. Fosfomycin trometamol may also be suitable for single dose therapy. Agents that have not been shown effective in any of these short term regimens should no longer be used for the treatment of acute cystitis. PMID- 10726074 TI - Antimicrobial resistance in Neisseria gonorrhoeae. AB - Despite a declining incidence of gonorrhea in much of the industrialized world, antimicrobial resistance continues to present a challenge to the treatment of gonococcal infections. Although no clinically significant resistance to the broad spectrum cephalosporins has been identified, fluoroquinolone-resistant gonococcal strains are now prevalent in Australia and much of the Far East. Ongoing surveillance for antimicrobial resistance is crucial to obtain the information needed to choose effective empiric therapies for gonorrhea. PMID- 10726075 TI - Bibliography. Current world literature. Benign prostatic hyperplasia. PMID- 10726076 TI - Bibliography. Current world literature. Urinary tract infections and sexually transmitted diseases. PMID- 10726077 TI - Renal medicine and renal transplantation. PMID- 10726078 TI - Renal calculi. AB - This past year produced some remarkable reports on renal (and other urinary) calculi. Randall's plaques have returned, phosphate relates to insulin and lipid metabolism, and sialic acid is out. Risk factors for astronauts, cystinuric individuals, older and indinavir patients achieve importance. Discovery by spiral computed tomography advances, teleconsultation emerges and shot-gun therapy with potassium-magnesium citrate succeeds. Endoscopic or shock wave lithotripsy vie for which is best, and both attempt to eliminate open surgery. Yet open surgery still has its place. PMID- 10726079 TI - Obstructive uropathy. AB - There have been a significant number of advances in the understanding of the pathophysiology of obstructive uropathy. Of particular interest are the molecular biologic advances with respect to apoptosis, TGF-beta and the definition of the expression of certain genes. Research in the developing kidney in the fetus as it is affected by obstructive uropathy has had no significant advances in the past year. PMID- 10726080 TI - Renovascular hypertension. AB - Approximately 5% of all hypertensive patients have renovascular hypertension, although its true incidence is unknown. The pathophysiology of renovascular hypertension has been linked to other intrarenal systems, the lipoxygenase pathway, and renin angiotensin. Many advances have been made in this field, but emphasis is now being placed on using less invasive or non-invasive tests to identify functionally significant lesions with a high degree of accuracy. The treatment modalities have shifted from aggressive surgical revascularization to less invasive management. The use of arterial stents has simplified the management of patients with renovascular hypertension, but long-term results are not yet available. PMID- 10726081 TI - Living donor nephrectomy. AB - The need for more organs for kidney transplantation is increasing. Cadaver sources for these organs are stable, therefore living donation must increase if the need is to be met. Less perfect kidneys are now being transplanted. The pool of potential donors is being expanded. The process of kidney donation is being made easier in an effort to increase the number of donors. The donor work-up is being streamlined. Laparoscopic donor nephrectomy has been introduced, and appears to be promising as a technique of lessening donor pain and suffering, while maintaining excellent graft results. PMID- 10726082 TI - Renal transplantation. AB - There has been an increase in the transplantation of kidneys from living, genetically unrelated donors and from extended criteria cadaver donors. The past policies about paid renal donors are being reconsidered. Techniques have been developed to reduce morbidity for the living renal donor. The variety of immunosuppressants allows individuation of therapy. Guidelines for conception and pregnancy have been established. PMID- 10726083 TI - Radiological approach to renal cysts and the Bosniak classification system. PMID- 10726084 TI - Helical computed tomography of renal lesions. AB - Helical computed tomography is a new technique that acquires a volumetric data set of both kidneys within a single breath-hold. It has proved to be superior to conventional computed tomography. Multiphasic helical computed tomography after the administration of contrast material shows advantages concerning the detection and characterization of renal lesions and the preoperative staging of renal cell carcinomas. The proper scan protocol for a sufficient examination of the kidneys is described as well as its diagnostic value. The characteristic computed tomography morphology of benign and malignant renal lesions is reviewed and the advantages of helical computed tomography are illustrated. PMID- 10726085 TI - Computed tomography evaluation of adrenal masses. AB - Computed tomography is the first line modality in the evaluation of patients with adrenal gland masses, and has the potential to be very accurate in the localization of adrenal gland masses in patients with diseases associated with hyperfunctioning conditions of the adrenal gland. Computed tomography allows a specific diagnosis of acute adrenal haemorrhage, adrenal myelolipoma, and adrenal cysts. It is also helpful in the assessment of patients with Addison's disease, particularly the subacute form secondary to granulomatous diseases. Quantitative evaluation of adrenal masses on unenhanced or delayed-enhanced computed tomography has been shown to be highly accurate in distinguishing adrenal adenomas from non-adenomas. Attenuation of 18 HU or less on unenhanced computed tomography scans indicates adenoma with a high specificity and acceptable sensitivity. On delayed-enhanced computed tomography scans, adrenal adenomas exhibit a greater washout of contrast material than do adrenal non-adenomas. Therefore, adrenal non-adenomas have significantly higher attenuation than adenomas on delayed-enhanced computed tomography scans obtained at arbitrarily chosen times (3-60 min) after the initiation of contrast material administration. PMID- 10726086 TI - Magnetic resonance: evaluation of adrenal lesions. AB - Magnetic resonance imaging provides a detailed display of adrenal anatomy. The development of new imaging sequences, especially chemical shift imaging, has vastly enhanced the specificity of magnetic resonance imaging. This review outlines current magnetic resonance imaging strategies for assessing disease affecting the adrenal glands. Particular emphasis is placed on the characterization of adrenal masses. PMID- 10726088 TI - Bibliography. Current world literature. Renal medicine and renal transplantation. PMID- 10726087 TI - Computed tomographic angiography in the preoperative evaluation of potential renal transplant donors. AB - The preoperative screening of potential renal transplant donors has undergone a major evolution with the introduction of computed tomographic angiography. With computed tomographic angiography, the radiologist is able to provide the surgeon with precise and valuable preoperative details of renal arterial and venous anatomy. In addition, computed tomographic angiography provides valuable information regarding the renal parenchyma and adjacent organs. This helps reduce the risks and complications associated with transplant surgery and improves the chances of a successful outcome. Helical computed tomographic angiography is rapidly replacing intravenous urography and conventional angiography as the imaging modality of choice for evaluating renal donors. PMID- 10726089 TI - Bibliography. Current world literature. Imaging, interventional techniques. PMID- 10726090 TI - Interstitial brachytherapy for prostate cancer--just an expensive variant of 'watchful waiting'? AB - As a result of the recent resurgence of interest in interstitial brachytherapy, extended follow-up studies of early experience in this field are now being reported. However, such outcomes must be evaluated within the context of recent comprehensive assessments of the age-specific natural history of conservatively managed prostate cancer ('watchful waiting', i.e. no curative intervention). This raises issue with the contention that treating patients with low-grade cancer using brachytherapy alone results in a treatment-derived, extended, clinical progression-free survival. The necessity for proposed randomized trials of brachytherapy (compared with conservative management in low-grade disease, and brachytherapy plus external beam radiotherapy) is discussed in this context. There is no consensus regarding the best modality for the treatment of localized prostate cancer, since no well-accepted, adequately powered randomized trial of competing modalities for this disease has ever been completed. Consequently, it has become common while counseling patients with the disease to place substantial emphasis on 'quality of life' considerations and the 'outcomes uncertainty management' issues associated with each option of therapy. There is even a question of whether every patient who is diagnosed with prostate cancer should be treated. However, while the importance of the variable biologic risk is generally widely accepted by experienced physicians caring for such patients, it is extremely difficult for the individual patient to weigh the evidence of the relative risks of progression dispassionately at the time of diagnosis. In this review, I wish to highlight several papers that have been published within the past year that are important in addressing this ongoing clinical problem. Any physician counseling patients about localized prostate cancer treatment should read and become familiar with the data herein: papers addressing the issues of interstitial brachytherapy outcomes, and the natural history of prostate cancer itself. PMID- 10726091 TI - Role for retroperitoneal lymphadenectomy for testis cancer. AB - There continue to be several controversies surrounding the role for retroperitoneal lymphadenectomy (RPL) in the management of patients with germ cell cancer of the testis. The initial treatment options for those with clinical stage I disease are surveillance (orchiectomy only), RPL or chemotherapy. Survival rates are similar with RPL and surveillance. Surgical morbidity has been reduced as techniques for RPL continue to improve. The likelihood of early or late (> 2 years) recurrence in the retroperitoneum is almost eliminated by RPL. Fewer follow-up computerized tomography scans of the abdomen are required and there are opportunities to reduce the duration and methods of follow-up, compared with surveillance. For patients with stage II disease, chemotherapy and RPL are equally effective initial treatment options but many patients require a combined approach. Initial RPL should be reserved for patients with smaller volume disease and possibly with lower preoperative marker levels. With RPL, patients are accurately staged and cured most of the time without double treatment. Approximately 30% of those with larger masses will have residual disease after initial chemotherapy and will require RPL as a second treatment. The third indication for RPL is to excise residual retroperitoneal masses following primary chemotherapy. Models to predict the presence of residual viable tumor, rather than necrosis only, at the time of surgery have been developed. If the orchiectomy specimen contained no teratoma, the tumor markers normalize after three or four courses of chemotherapy, and if the residual mass on computerized tomography scan is less than 2 cm in diameter, the rate of viable tumor may be low enough to omit RPL. In this way, the greater morbidity often associated with post-chemotherapy RPL may be avoided. PMID- 10726092 TI - Periurethral collagen injections for incontinence following radical prostatectomy: does the patient benefit? AB - Promising early results have been reported with periurethral collagen injections in the treatment of incontinence after radical prostatectomy. However, a significant proportion of patients does not benefit from this minimally invasive option. Recently, the results of longer follow-up studies have become available in some series. Investigators have also attempted to identify the prognostic factors of success. This review reports the latest advances on periurethral collagen injections in the treatment of incontinence after radical prostatectomy. PMID- 10726093 TI - Surgical options in adrenalectomy: laparoscopic versus open surgery. AB - Small hormone-active benign tumors are considered as clear indication for laparoscopic adrenalectomy. Laparoscopy resection of pheochromocytomas is still a controversial issue, but recent data have shown that the specific risks of pheochromocytoma surgery are not increased by the laparascopic approach. The majority of endoscopic adrenalectomies are performed via the transperitoneal route, but there is growing interest in the retroperitoneoscopic approach. The advantages and disadvantages of each endoscopic approach have to be weighed carefully, but the final decision will also depend on the experience of the surgeon. Several retrospective studies have compared laparoscopy with open surgery. There is general agreement that laparoscopy is superior to open surgery since it is associated with less pain, a shorter hospital stay, and more rapid return to normal activities, and also yields the best cosmetic and long-term results. Partial adrenalectomy may be indicated for bilateral pheochromocytoma, and also has advantages for patients with aldosterone-producing adenomas. The feasibility of laparoscopic partial adrenalectomy has been demonstrated. Laparoscopic intraoperative ultrasonography is valuable in selected cases. The already low morbidity of laparoscopic adrenalectomy can be reduced further by using needlescopic techniques. PMID- 10726094 TI - Laparoscopic donor nephrectomy: standard of care or unnecessary risk of organ loss? AB - Laparoscopic donor nephrectomy was developed to remove disincentives to live donation. It has been demonstrated to decrease the length of hospitalization, postoperative pain, time to convalescence and activity, while providing an optimal cosmetic result. Initial reports suggest that laparoscopic donor nephrectomy is feasible and equivalent to open donor nephrectomy. PMID- 10726095 TI - What's topical in functional reconstruction and trauma--with particular reference to urethroplasty. PMID- 10726096 TI - Diagnosis and management of trauma to the kidney. AB - Because trauma is an unplanned event, guidelines for managing patients sustaining upper urinary tract injury must already exist in the surgeon's mind before laying eyes on the injured patient. In order to simplify and standardize the evaluation and treatment of such patients, urologists have applied a system of staging paradigms and treatment algorithms, analogous to systems successfully used in the less chaotic arena of, for example, urologic oncology. Advancements in imaging modalities, improvements and renovations of surgical technique, and critical review of outcomes data have impacted on the management of genitourinary trauma and will be likely to influence the way we treat trauma patients in the future. PMID- 10726097 TI - Reconstructive surgery using urological prostheses. AB - Urological prostheses have been available and widely used for the past two decades. The use of penile prostheses, artificial urinary sphincters, and testicular implants for genitourinary reconstruction for erectile dysfunction, incontinence, and orchiectomy have been successful, widely used and of low morbidity. More recently designed devices, innovations in implantation techniques, and improved intraoperative and postoperative care have resulted in the successful worldwide use of these prosthetic devices. PMID- 10726098 TI - Bladder substitution. AB - Preliminary data have shown that composite reservoirs consisting of gastric segments in combination with ileum or colon may reduce metabolic problems. Orthotopic neobladders are the most natural way to reconstruct the urinary bladder and are a safe option in selected male and female patients. Experimental and preliminary clinical data about new methods using autologous muscle transfer and tissue engineering are promising. PMID- 10726099 TI - What is new in urinary diversion. AB - There have been some suggestions for changes in technique and investigations of the quality of life. As experience has grown, there have been increasing numbers of reports of complications. Careful attention to technique, especially in nerve sparing cystectomy and orthotopic cystoplasty may reduce the rate of incontinence. Increasing awareness of quality of life issues should improve preoperative counselling of patients, especially those whose underlying condition is not life-threatening. PMID- 10726101 TI - Bibliography. Current world literature. Urological surgery, surgical techniques, technology. PMID- 10726100 TI - Contemporary management of urethral trauma and the post-traumatic stricture. AB - This review discusses the contemporary management of urethral trauma and post traumatic urethral strictures occurring in both the anterior and posterior urethra. Experience in the full spectrum of urethral reconstructive procedures is important. The initial management of patients relies upon adequate drainage of the bladder and management of any other associated acute life-threatening injuries. The principles of urethral reconstruction in both the anterior and posterior urethra are discussed, with reference to the recent literature on the subject. PMID- 10726102 TI - Bibliography. Current world literature. Functional reconstruction and trauma. PMID- 10726103 TI - Nutritional determinants of serum triglycerides. PMID- 10726104 TI - Markers of intestinally-derived lipoproteins: application to studies of altered diet and meal fatty acid compositions. AB - BACKGROUND AND AIM: The atherogenic potential of dietary derived lipids, chylomicrons (CM) and their remnants (CMr) is now becoming more widely recognised. To investigate factors effecting levels of CM and CMr and their importance in coronary heart disease risk it is essential to use a specific method of quantification. Two studies were carried out to investigate: (i) effects of increased daily intake of long chain n-3 polyunsaturated fatty acid (LC n-3 PUFA), and (ii) effects of increasing meal monounsaturated fatty acid (MUFA) content on the postprandial response of intestinally-derived lipoproteins. The contribution of the intestinally-derived lipoproteins to total lipaemia was assessed by triacylglycerol-rich lipoprotein (TRL) apolipoprotein B-48 (apo B-48) and retinyl ester (RE) concentrations. METHODS AND RESULTS: In a randomised controlled crossover trial (placebo vs LC n-3 PUFA) a mean daily intake of 1.4 g/day of LC n-3 PUFA failed to reduce fasting and postprandial triacylglycerol (TAG) response in 9 healthy male volunteers. Although the pattern and nature of the apo B-48 response was consistent with the TAG response following the two diets, the postprandial RE response differed on the LC n-3 PUFA diet with a lower early RE response and a delayed and more marked increase in RE in the late postprandial period compared with the control diet, but the differences did not reach levels of statistical significance. In the meal study there was no effect of MUFA/SFA content on the total lipaemic response to the meals nor on the contribution of intestinally derived lipoproteins evaluated as TAG, apo B-48 and RE responses in the TRL fraction. In both studies, the RE and apo B-48 measurements provided broadly similar information with respect to lack of effects of dietary or meal fatty acid composition and the presence of single or multiple peak responses. However the apo B-48 and RE measurements differed with respect to the timing of their peak response times, with a delayed RE peak, relalive to apo B-48, of approximately 2-3 hours for the LC n-3 PUFA diet (p = 0.002) study and 1 1.5 hours for the meal MUFA/SFA study. CONCLUSIONS: It was concluded that there are limitations of using RE as a specific CM marker, apo B-48 quantitation was found to be a more appropriate method for CM and CMr quantitation. However it was still considered of value to measure RE as it provided additional information regarding the incorporation of other constituents into the CM particle. PMID- 10726105 TI - Effects of insulin therapy and glycemic control on distribution of HDL alpha and pre-beta subfractions in non insulin-dependent diabetic subjects. AB - BACKGROUND AND AIM: The heterogeneous population of HDL particles can be separated into six classes which may provide a more sensitive index of cardiovascular risk than HDL cholesterol. The aim of this study was therefore to evaluate HDL distribution in NIDDM. METHODS AND RESULTS: Twenty-five individuals were treated with diet and sulfonylurea and 14 with insulin; both groups had similar plasma lipid profiles and BMI, but the insulin-treated subjects were older and their diabetes was of longer duration. The major findings were a greater proportion of apoA1 in alpha 1-HDL in the insulin-treated group (24.3 +/- 5.2% vs 20.8 +/- 4.0%), which is a desirable profile, and a greater proportion of pre-beta 1-HDL, which are the initial acceptors of cellular cholesterol. CONCLUSIONS: Our results suggest a beneficial influence of insulin treatment on reverse cholesterol transport. Moreover, glycemic control (HbA1c) also related to alpha 3-HDL significantly (r = 0.51; p < 0.01), indicating an association between poorer glycemic control and an adverse HDL profile. PMID- 10726106 TI - Modifications of alpha-tocopherol and fatty acid concentrations in blood and adipose tissue of obese patients during a weight loss programme. AB - BACKGROUND AND AIM: The aim of the study was to describe qualitative and/or quantitative modifications of lipoproteins, including their fatty acid composition, in obese patients during a hypocaloric diet, and determine whether the variations observed paralleled modifications of alpha-tocopherol concentration in adipose tissue and blood. METHODS AND RESULTS: 15 healthy, obese volunteers (5 males, 10 females; age: 32-69 yr; BMI: 28.4-60.5 kg/m2) were given a 3-week hypocaloric diet (3.9 MJ (941 kcal)). Adipose tissue and blood samples were taken at the beginning and at the end of this period. At baseline and after 3 weeks measurements were made for alpha-tocopherol and fatty acids in total serum, lipoproteins and adipose tissue; thiobarbituric acid-reactive substances (TBARS) in serum. A significant drop in cholesterol-rich particles (LDL and HDL) was observed, in parallel to a significant enrichment of n-6 polyunsaturated fatty acids (PUFA) at the expense of both saturated and monounsaturated fatty acids in serum. A drop in alpha-tocopherol concentration (expressed as mumol alpha-tocopherol/g lipid) in serum and lipoprotein fractions paralleled the decrease in cholesterol-rich lipoproteins. CONCLUSIONS: Our results suggest that a hypocaloric diet not only decreases cholesterol-rich particle levels in serum, but also leads to a significant modification of fatty acid composition which may reflect improvement of insulin sensitivity. We did not observe any modification in adipose tissue after diet with regard to both alpha-tocopherol and fatty acid concentrations. Despite a drop in alpha-tocopherol concentration and an increase in n-6 PUFA content in serum, we did not find any enhancement of serum lipid peroxidation level evaluated by the thiobarbituric acid-reactive substance (TBARS) assay. If we assume that dietary intakes of alpha-tocopherol were not modified during this diet, it can be supposed that adipose tissue released alpha tocopherol without any specific regulation, in parallel to the release of fatty acids. PMID- 10726107 TI - Increased levels of factor VII, fibrinogen and activity of plasminogen activator inhibitor during postprandial triglyceridemia in patients with ischemic heart disease confirmed by angiography. AB - BACKGROUND AND AIM: Impaired elimination of triglycerides (TG) after postprandial lipemia is often observed in patients with ischemic heart disease (IHD). Longer retention of TG-rich lipoproteins in the circulation may in turn be the cause of hemostatic disturbances in the form of hypercoagulation. The aim of this study was to evaluate the influence of postprandial lipemia on the hemostatic system in normolipemic patients with angiographically documented atherosclerotic changes of coronary arteries. METHODS AND RESULTS: 20 males under 60 years of age with IHD and 10 healthy males matched for age were studied. Hemostatic and lipid parameters were determined in blood collected before (fasting) and after (2, 4, 6 and 8 hours) an oral fat load (200 ml of 30% cream). A statistically significant increase in factor VII (FVIIc-%) was found during postprandial lipemia in comparison with fasting. Although in patients with IHD the area under the curve for FVIIc was not significantly different than in the control group, it was observed that FVIIc in the IHD group exceeded the fasting level earlier (4, 6 and 8 h) than in controls (8 h). Levels of FVIIc reached for the IHD group: fasting value = 96.3 +/- 16.3; postprandial 4 h = 103.2 +/- 21.3, 6 h = 105.2 +/- 21.8, 8 h = 106.0 +/- 21.4; for the controls: fasting value = 96.8 +/- 17.2; postprandial 8 h = 107.7 +/- 22.0. In the IHD group, unlike in controls, postprandial lipemia was accompanied by a statistically significant rise in fibrinogen levels (Fb-g/l) as compared with fasting (fasting value = 3.69 +/- 0.59; postprandial 2 h = 3.89 +/- 0.65, 4 h = 3.93 +/- 0.63). Mean values for PAI-1 were lower in the IHD than in the control group, but PAI-1 activity during postprandial lipemia rose earlier in the IHD group (4 and 6 h) than in the controls (6 h). PAI-1 levels were in the IHD: fasting values = 1.4 +/- 1.2; postprandial 4 h = 2.3 +/- 1.6, 6 h = 2.5 +/- 1.7; in the control: fasting value 2.9 +/- 1.7; postprandial 6 h = 3.9 +/- 2.0. The level of fragment 1 + 2 (F1 + 2) during postprandial lipemia decreased in both groups. CONCLUSIONS: The lipemic stimulus in fasting normolipemics induces a hypercoagulant effect with greater changes in patients with ischemic heart disease than in healthy subjects. Longer periods of postprandial lipemia might constitute an additional factor promoting thrombus formation in patients with dysfunction of the vascular endothelium. PMID- 10726108 TI - Plasma Lp(a) values in familial hypercholesterolemia and its relation to coronary heart disease. AB - BACKGROUND AND AIM: To analyze plasma Lp(a) levels and examine different risk factors and coronary heart disease (CHD) in a sample of genetically diagnosed familial hypercholesterolemia (FH) patients. METHODS AND RESULTS: Ninety heterozygous FH patients and 41 non-FH relatives were enrolled in a study to evaluate their plasma and lipoprotein cholesterol, as well as their triglyceride and Lp(a) levels. We found no differences in plasma Lp(a) levels and log transformed values between 90 FH subjects and their 41 unaffected relatives (22.3 mg/dl +/- 19.4 vs 17.7 mg/dl +/- 21.3 and 1.12 +/- 0.5 vs 0.96 +/- 0.54) nor between null allele and defective allele FH subjects (log Lp (a) levels 2.013 +/- 0.282 vs 1.959 +/- 0.151). FH CHD+ were significantly older, and had higher mean systolic and diastolic blood pressure and higher mean plasma triglyceride levels than FH CHD-. No differences in mean and log transformed Lp(a) plasma concentrations were found. CONCLUSIONS: Plasma Lp(a) levels are not related to LDL receptor status and class mutations, nor to the presence of CHD in FH patients. PMID- 10726109 TI - Prevention of obesity: the role of the food industry. AB - The food industry is a critical factor in any potentially successful long-term strategy to prevent obesity. By producing new products low in energy density and improving the nutritional quality (and reducing the energy content) of existing products, as well as through advances in responsible marketing and labeling, the food industry can provide foods that enable consumers to achieve lower energy intakes without going short of essential nutrients. The food industry is not the sole factor and government policies regarding agriculture, prices and subsidies are equally essential. Caterers and retailers are yet another important cog in the wheel for progress in obesity prevention. Intensive collaboration between all these players will only be attained if obesity prevention is given the priority it deserves in future public health planning. PMID- 10726110 TI - Dietary vitamin B6 supplementation prevents ethanol-induced hypertension in rats. AB - BACKGROUND AND AIMS: All known pathways of ethanol metabolism result in the production of acetaldehyde, a highly reactive compound. Acetaldehyde has been shown to deplete vitamin B6 in chronic alcoholics. It also binds with sulfhydryl groups of membrane proteins, altering membrane Ca2+ channels and increasing vascular cytosolic free calcium, peripheral vascular resistance and blood pressure. The aldehyde-binding thiol compound, N-acetyl cysteine, attenuates elevated blood pressure and associated adverse changes in ethanol-induced hypertensive rats. Vitamin B6 supplementation increases the level of endogenous cysteine. Aim of this work was thus to investigate whether a dietary supplementation of vitamin B6 can prevent ethanol-induced hypertension and associated changes in Wistar-Kyoto (WKY) rats. METHODS AND RESULTS: Starting at 7 weeks of age, WKY rats were divided into three groups of six animals each. The control group received a normal vitamin B6 diet (regular chow) and normal drinking water, the ethanol group, the same diet plus 1% ethanol in the drinking water, and the ethanol + vitamin B6 group a high vitamin B6 diet (20 times normal diet) and 1% ethanol in the drinking water. After 14 weeks, systolic blood pressure, platelet [Ca2+]i and kidney and aortic aldehyde conjugate levels were significantly higher in the ethanol group. These rats also showed smooth muscle cell hyperplasia in the small arteries and arterioles of the kidneys. Dietary vitamin B6 supplementation prevented these changes. CONCLUSIONS: Dietary vitamin B6 supplementation prevented ethanol-induced hypertension and associated changes in WKY rats by normalizing tissue aldehyde conjugate levels. PMID- 10726111 TI - Similar effects of diets high in oleic or linoleic acids on coagulation and fibrinolytic factors in healthy humans. AB - BACKGROUND AND AIM: Monounsaturated fatty acids (MUFA) have been shown to be beneficial. Their haemostatic effects, however, are poorly known. We compared the effects of oleic acid (OA) and linoleic acid (LA) on variables related with coagulation and fibrinolysis in healthy subjects. METHODS AND RESULTS: Thirty eight healthy volunteers (20 women, 18 men; mean age 27) consumed a saturated fat baseline diet for four weeks and were then switched to either a high LA diet (11.5 en%) or a high OA diet (18.0 en%) for another four weeks when nearly all food was provided for the whole day. A control group of 13 subjects consumed their habitual diet throughout the study. No differences between the OA and LA diets were found in the plasma levels of fibrinogen, plasminogen activator inhibitor, antithrombin III, von Willebrand factor antigen or D-dimers. Factor FVII coagulant activity was significantly lower after the OA diet. CONCLUSIONS: The results indicate largely similar effects for OA and LA on factors related with coagulation and fibrinolysis in humans. The effects of dietary fatty acid composition on FVII coagulant activity should be further studied. PMID- 10726112 TI - Effects of a 3-month treatment with terazosin on fasting and postprandial glucose and lipid metabolism in type 2 diabetic patients with hypertension. AB - BACKGROUND AND AIM: The effects of the alpha-1-adrenergic blocker terazosin on blood pressure and fasting and postprandial glucose and lipid metabolism were assessed in type 2 diabetic patients with hypertension. METHODS AND RESULTS: In this single-blind, randomized, crossover, placebo-controlled pilot study, thirteen patients were given terazosin for three months. Blood pressure and metabolic parameters were measured after a 14-hr overnight fast. In addition, a 800-calorie test meal was administered after placebo and terazosin to evaluate blood glucose and lipid changes following a standardized physiological stimulus. Blood pressure was significantly reduced and HDL-cholesterol significantly increased after terazosin. A significant decrease at fasting with a smaller reduction after the meal test was observed for free fatty acids when terazosin was given in comparison to placebo, suggesting an improvement in insulin resistance. A slight decrease in fasting and postprandial triglycerides was also observed. Cardiovascular risk, calculated according to the Framingham formula, was significantly reduced at the end of the terazosin treatment. CONCLUSIONS: Antihypertensive treatment with terazosin is effective, has no adverse effects on fasting and postprandial glucose and lipid metabolisms, and appears to improve the cardiovascular risk profile of hypertensive patients with associated metabolic diseases. PMID- 10726113 TI - Early markers of cardiovascular damage in obese subjects. AB - Cardiovascular disease remains a frequent cause of morbidity and mortality in industrialized countries, particularly in subjects with hypertension, diabetes mellitus, and dyslipidemia, conditions frequently associated with central obesity. Identification of early morphological and/or functional alterations of the cardiovascular system may help target individuals most likely to benefit from preventive measures. The literature data and our own experience suggest that parameters that are direct expressions of cardiovascular damage, can be identified at an early stage. For example, diastolic dysfunction may precede the clinical expression of several cardiac diseases, left ventricular hypertrophy is one of the first manifestations of cardiac involvement in hypertension, central obesity and diabetes mellitus, and a carotid plaque may point to concomitant coronary artery disease. Other early manifestations of cardiovascular involvement are microalbuminuria and endothelial dysfunction. Insulin resistance and alterations of the renin-angiotensin-aldosterone system play an important physiopathogenic role in the development of cardiovascular damage in obese subjects, and their association with risk and cardiovascular disease has been confirmed in numerous studies. Since all these changes generally precede overt clinical manifestations and are closely related to cardiovascular morbidity, they may help identify individuals at the highest risk of cardiovascular events. PMID- 10726114 TI - Dietary fat and the etiology of type 2 diabetes: an epidemiological perspective. PMID- 10726115 TI - Postprandial lipaemia and monounsaturated fat. PMID- 10726116 TI - [Atrial fibrillation as risk for thrombosis]. PMID- 10726117 TI - [Cellulite--how does one deal with it?. Interview by Dr. Beate Schumacher]. PMID- 10726118 TI - [ABC of osteoporosis therapy. Which drugs for prevention and therapy?]. AB - Medical Prevention and Therapy Fractures in the elderly, in particular of the proximal femur, may give rise to functional impairment with loss of independence and mobility. Apart from falls, the most common cause of fractures is osteoporosis or other impairments of mechanical stability of bone. In addition to the avoidance of falls and their sequelae, and the minimization of risk factors where possible, optimization of the mechanical stability of the bone is an important point of attack for interventional strategies. Current data on medical possibilities of osteoporosis prevention and treatment are presented and evidence for their effectiveness in reducing fractures discussed. Recommendations based on confirmed data are made, and possible future therapeutic approaches discussed. PMID- 10726119 TI - [Therapy of osteoporosis: native vitamin D or as hormone? Advantages of activated vitamin D in secondary osteoporosis]. AB - Vitamin D and its active metabolite (D-Hormone) are major weapons in the therapeutic arsenal available for the treatment of osteoporosis. With regard to native vitamin D, controlled studies have confirmed the prophylactic effect of treatment with vitamin D (+ calcium) in the area of nonvertebral fractures, in particular in elderly women with vitamin D deficiency. The widespread prophylactic use of this form of treatment, which is both inexpensive and largely free of side effects, would presumably save costs by greatly reducing the incidence of fractures of the femur. Treatment with D-hormone (calcitriol) or the pro-hormone 1 alpha-hydroxy-vitamin D (alfacalcidol) is a specific form of treatment of osteoporosis that has been shown to prevent fractures, in particular of the vertebrae, in a number of controlled prospective studies. The D-hormone is of particular value in the treatment of secondary forms of osteoporosis (induced by glucocorticoids or chronic inflammatory disease). Although the incidence of severe side effects is low, monitoring of serum calcium ist nevertheless recommended. PMID- 10726120 TI - [Poisoning by Citrullus colocynthis. Unknown to us, a frequent poisonous plant in foreign travel]. PMID- 10726121 TI - [Hypericum special extract. Effectiveness in the elderly and in chronic disease]. PMID- 10726122 TI - [Hyperhidrosis. 1: Clinical manifestations]. PMID- 10726123 TI - [Eruptions "overnight". Eruptive xanthomas in secondary type IV hyperlipoproteinemia caused by diabetes mellitus]. PMID- 10726124 TI - [Dyspepsia caused by food allergy]. PMID- 10726126 TI - [Confusion syndrome in alcoholism. Faulty assessment can produce deleterious course]. AB - Confusion is a frequent syndrome in alcoholism caused by a large number of neuropsychiatric and somatic disorders with severe alcohol intoxication, pathological intoxication, alcohol dementia and hepatic encephalopathy among them. Possible pathophysiologic mechanisms involved and therapeutic strategies are discussed. PMID- 10726125 TI - [Alcohol withdrawal syndrome and delirium tremens. Diagnosis and therapy]. AB - The alcohol withdrawal syndrome can be classified into three degrees of severity on the basis of the symptomatology, autonomic withdrawal, predelirium and delirium tremens. In American literature the severity of withdrawal is recorded using the CIWA-A scale (Clinical Institute Withdrawal Assessment--Alcohol). The pathophysiological causes lie in an imbalance between the inhibitory and excitatory neurotransmitters after giving up alcohol. This results in predomination by the excitatory system. Therapeutic intervention is possible here. Clomethiazole has effective sedative actions, stabilises the autonomic nervous system, and is an anticonvulsant. It is the drug of choice for autonomic withdrawal and predelirium. The benzodlazepines have a similar effect, but cannot be controlled so accurately. Carbamazepine can prevent withdrawal convulsions and progression of delirium. Clonidine acts on autonomic withdrawal and, together with neuroleptics and benzodiazepines, is easy to use parenterally for delirium tremens, while parenteral clomethiazole harbours dangers. PMID- 10726127 TI - [Therapy of alcoholism--a crux also for the physician]. PMID- 10726128 TI - [The goal of antihypertensive therapy: preventing left heart dysfunction]. PMID- 10726129 TI - [Hyperhidrosis. 2: Idiopathic hyperhidrosis, therapy]. PMID- 10726130 TI - [Young woman with fluctuating hypo- and hyperthyroidism. Autoimmune thyroiditis]. PMID- 10726131 TI - [Asthma therapy by the general practitioner--new approaches and progress. Why inhaled steroids and delayed action beta-2 adrenergic drugs should be combined early on]. AB - Early treatment with high-dose inhaled steroids can significantly improve the prognosis of asthma. Inhaled steroids used to treat inflammation of the mucosa, and hyperreactivity may be reduced only under careful surveillance. Hig-dose initial treatment must be followed by low-dose maintenance therapy. The best therapeutic results are obtained with a combination of inhaled steroids and long acting beta-2-adrenergic agents. Careful titration of the dose and therapeutic effects is a major task for the family doctor. Bronchial inflammation and reactivity are dependent on external factors, and are rarely stable. The most important therapeutic basis is, therefore, continuous management involving both the patient and the family doctor. The patient should be provided with relevant information on his/her disease and its treatment. A prerequisite for the effective management of asthma is the provision of individual peak-flow-adjusted and emergency plans. PMID- 10726132 TI - [Educating the asthma patient for routine illness management. Assuring long-term therapeutic success]. AB - Although education programs for adult asthmatics promotes improved knowledge of the disease, it has been found that this alone does not result in any change in patient behavior. Against this background, the National Asthma Education Program Expert Panel (NAEP) recommends adapting the contents of the education programs more closely to the needs of the individual patient. This is to be achieved through self-monitoring, more influence on environmental factors, improved knowledge of the disease process and self-medication skills, and greater self sufficiency on the part of the patient. Other studies have discussed various possible ways of implementing these recommendations, including the training of physical self-awareness, consideration and correction of subjective notions of the disease, dealing with disease-related emotions, and providing concrete advice on coping with the situations met with in daily life. A new patient education program developed by the Alpenklinik in Oberjoch serves to illustrate how the NAEP recommendations can be implemented. PMID- 10726133 TI - [Erythema and scaly skin--what is the origin? Morphologically and descriptively oriented dermatologic diagnosis (5)]. PMID- 10726134 TI - [Skin manifestations after sun tanning. Determining the etiology]. PMID- 10726135 TI - [Vaccinations for physicians and medical personnel]. PMID- 10726136 TI - [Arthralgia with erythema nodosum. Lofgren syndrome in acute pulmonary sarcoidosis with erythema nodosum]. PMID- 10726137 TI - [Encouraging study results. HIV from birth on--virostatic drugs can decrease the risk]. PMID- 10726139 TI - [Fatigue syndrome--a kind of grief?]. PMID- 10726138 TI - [Influenza becomes treatable. Breakthrough against the last great epidemic?]. PMID- 10726140 TI - [Helicobacter test: not from the pharmacy]. PMID- 10726141 TI - [Fibrillating hearts. What is the value of reestablishing sinus rhythm in atrial fibrillation]. PMID- 10726142 TI - [HIV: interleukin 2 supports the immune system]. PMID- 10726143 TI - [Obesity is not a character weakness. Leptin and twin research show: fat control is genetically regulated]. AB - The results of recent research have confirmed the leptin control system. Leptin is produced in fatty tissue, crosses the blood-brain barrier, and signals repletion of fat stores. This in turn triggers a reduction in food intake and an increase in energy expenditure. While neuropeptide Y is known to play a central nervous mediator role, it is not certain whether leptin plays a marginal or a main role in the energy balance. Of at least equal, and possibly even greater practical/clinical importance are the results of research on the family and twins. For these reveal a tendency for weight increase and abdominal storage of fat and increased intake of calories to have a strong genetic link. The conclusions are a lowering of the expectations placed in reduction dieting, and an increase in efforts aimed at prevention. PMID- 10726144 TI - [How safe are the new obesity drugs? Indications and contraindications of orlistat and sibutramine]. AB - Sibutramine and Orlistat are suitable "supporting drugs" for use in patients trying to lose weight. Orlistat reduces the absorption of fat from the intestine by about one-third. Over the long term too, the weight loss achieved under Orlistat (9%) has been greater than that seen under placebo (6.5%). Increased fat losses via the stools are associated with side effects and abandonment of treatment. Sibutramine inhibits the uptake of serotonin and noradrenaline in the synaptic gap, thus enhancing the CNS effects of these two transmitters, and prolonging the sensation of satiety. The most common side effects of sibutramine are dry mouth, headache and fatigue. The effects of sibutramine on weight reduction are similar to those of orlistat. For both drugs, the indications have been defined, and in the case of sibutramine, interactions with other medications have to be taken into account. PMID- 10726145 TI - [Obesity stresses blood pressure and energy metabolism. What weight reduction really accomplishes]. AB - The increase in morbidity and mortality associated with distinct obesity is based on the one hand on the direct effects of overweight on circulatory and pulmonary function, and on the other on a higher prevalence of subsequent sequelae. The latter are, in particular, arterial hypertension and metabolic disturbances, which either arise due to overweight, or which are fostered by it. This means that weight reduction is of particular importance for the treatment of the sequelae of metabolic disorders. PMID- 10726146 TI - [Antibiotics in therapy of bacterially infected diabetic foot. Results in 191 patients]. PMID- 10726147 TI - [Treatment of ascites. Goal is symptom remission and not cure of ascites]. PMID- 10726148 TI - [Anticoagulation for diabetic patients. Thrombosis ABC, 8: diabetes mellitus]. PMID- 10726149 TI - [Antibiotic therapy of bronchial infections. 2: Community acquired and nosocomial pneumonias]. PMID- 10726150 TI - [Vertigo and syncope in a 61-year-old patient. Villous adenoma with severe electrolyte and fluid loss]. PMID- 10726151 TI - [Taking the peak out of blood pressure. Diabetics can also live spontaneously]. PMID- 10726152 TI - [Blind spots of the diagnostic hemostasis screen]. AB - The most powerful instrument to establish the presence or absence of a coagulation disorder is the history of the patient. In addition, screening laboratory tests (consisting of the platelet count, bleeding time and global clotting assays, such as the prothrombin time and the activated partial thromboplastin time) may be helpful to support the diagnosis. In two patients, a 21-year-old man and a 10-year-old girl, with a marked history of enhanced bleeding normal screening laboratory tests were found. The male patient had a congenital alpha 2-antiplasmin deficiency and the girl had a homozygous deficiency of factor XIII. Some defects in the coagulation system (such as defects in fibrin network formation and fibrinolysis, but also mild Von Willebrand disease) are indeed not detected by screening laboratory tests. In patients with a strong suspicion of a coagulation disorder such defects should be specifically tested for. PMID- 10726153 TI - [Postoperative wound infections: a useful indicator of quality of care?]. AB - An indicator can be defined as a measurable element of care that gives an impression of the quality of care. It can be used for screening on potential quality problems, for monitoring of well-defined processes and for a check after the introduction of quality improvement activities. Although the notion of an indicator appears to be an attractive concept in quality management, some questions have to be answered before a specific indicator can be used. The first question regards the validity of the indicator: to what extent does the indicator reflect the quality of the care? Next is the question of registration: can the indicator be measured in a valid and reliable way? The third question is whether appropriate activities will be initiated after the indicator has given a signal. Postoperative wound infections appears to be a valid outcome indicator because of the relationship between process of care (infection prevention policy) and outcome of care (the number of infections). The weak point lies in the reliability of the registration of wound infections. PMID- 10726154 TI - [Disorders of bone metabolism in gastrointestinal and hepatic diseases]. AB - Gastrointestinal and hepatic diseases are frequently associated with metabolic bone disorders, especially osteoporosis and osteomalacia. Malabsorption of vitamin D and calcium are important aetiological factors, although other factors may contribute, like the use of glucocorticosteroids in the treatment of inflammatory bowel disease. Treatment is first of all focused on correction of calcium and vitamin D deficiency. In severe osteoporosis additional treatment with bisfosfonates needs serious consideration. PMID- 10726155 TI - [Tanning beds: effect on skin cancer risk unclear]. AB - Recent estimations show that over 25% of the Dutch population make regular use of tanning equipment. This use is still increasing, in spite of improving knowledge on the potential hazards of ultraviolet radiation. There are different motivations to use the tanning equipment. Younger women are largely represented in the group of sunbed users. Recent studies have brought the testimony that intermittent sun exposure (e.g. during holidays) is an important risk factor for skin cancer (notably basal cell carcinoma and melanoma). The investigations have not provided convincing evidence on the relation between the use of artificial devices and the development of skin cancer. This is partly caused by the fact that sunbed users are generally very motivated to get a tan. It is therefore difficult to distinguish between the effect of natural sun and of artificial UV radiation. In the Netherlands, the analyses of scientific data provide the basis for recommendations concerning sun exposure and use of sunbeds. There is an effort to provide the general public with qualified, professional information on the responsible way of tanning. PMID- 10726156 TI - [Disseminated intravascular coagulation]. AB - Disseminated intravascular coagulation is characterised by systemic activation of blood coagulation, resulting in formation of intravascular thrombi and impaired organ perfusion. Simultaneously, the ongoing consumption of platelets and coagulation factors may lead to bleeding. Disseminated intravascular coagulation is seen in septicaemic infections, trauma, malignancies, obstetrical complications, vascular diseases, toxic and immunological reactions. In summary, the systemic deposition of fibrin is caused by enhanced thrombin generation, simultaneous depression of physiological anticoagulant mechanisms and diminished fibrin degradation due to inhibition of fibrinolysis. The increased insight into the pathogenesis of disseminated intravascular coagulation provides a solid basis for development of improved management strategies for patients with this complication. Therapy may include anticoagulants, platelet and plasma transfusion, concentrates of coagulation inhibitors and antifibrinolytic agents. PMID- 10726157 TI - [Frequency of postoperative wound infections: an unsuitable parameter for comparison of hospitals]. AB - OBJECTIVE: To evaluate the reliability of registration of wound infections and their contribution to total postoperative morbidity. DESIGN: Prospective. METHOD: During the period from 1 July 1997 through 30 June 1998 of all patients treated in the department of Surgery of the Diaconessen Hospital, Utrecht, the treatment related complications were recorded during the hospital stay as well as at the first outpatient follow-up. The clinical registration forms were checked and supplemented after discharge, during the transfer and by the hospital hygienist. The precision of registration, the implications of the registered wound infections, and the share of this complication in overall postoperative morbidity were studied. RESULTS: There were 2172 admissions (2004 patients): 996 males and 1176 females (mean age at admission 55 years). The response of registration was 100% during hospital stay and 59.5% in the outpatient department. Postoperative course was complicated in 506 patients (23% of the admissions). Of the 735 registered complications 583 (79%) were recorded during clinical stay. Airway and wound infections were the most frequently registered complications (in 4.4% and 4.3% of the admissions respectively). Of the wound infections 45% were recorded after discharge and 88% fully recovered by conservative means. Both the frequency of wound infections in relation to other postoperative complications and the severity of the registered wound infections varied with the reason of admission. CONCLUSION: The incidence of postoperative wound infections was not reliably measured as a result of low response of registration after hospital discharge. The contribution of wound infections to overall postoperative morbidity and the severity of these infections varied with the reason of admission. PMID- 10726158 TI - [Management of treatment refusal by a psychiatric patient with a life-threatening infectious form of pulmonary tuberculosis]. AB - A Somalian man aged 26 was admitted to a general hospital because of haemophthisis. A severe infectious and life threatening pulmonary tuberculosis was diagnosed. Thereafter, the patient denied having any disease, refused any therapy or diagnostic procedure and wanted to leave the hospital. He was isolated by virtue of the Wet Bestrijding Infectieziekten ('WBI': Bill on Management of Infectious Diseases). Permission for treatment without consent was arranged by virtue of the Wet op de Geneeskundige Behandelingsovereenkomst ('WGBO': Decree on the Medical Contract). The use of measures like fixation and involuntary administration of medication could also be arranged by virtue of the WGBO. The Wet Bijzondere Opnemingen Psychiatrische Ziekenhuizen ('Wet BOPZ': Bill on Compulsory Admission to Psychiatric Hospitals), which exclusively concerns involuntary treatment exclusively of psychiatrics patients, was of no value in this case. Analysis of the different aspects of these three laws led to the conclusion that in case of a somatic disease, whether or not complicated by a psychiatric diagnosis, a treatment without consent must be arranged by virtue of the WGBO. PMID- 10726159 TI - [Does medical education still train doctors?]. PMID- 10726160 TI - The impact of histochemistry--a historical perspective. AB - Histochemistry has a remarkable long-term impact on cell and tissue biology, embryology, and pathology and remains at the forefront of research in these disciplines. Histochemical techniques, and particularly immunohistochemical and in situ hybridization techniques, are now more widely used than ever and, in conjunction with new developments in microscopical imaging and analysis, will continue to have an important position in the life sciences and medicine. However, histochemistry is often mistakenly perceived as an archaic discipline, and its contributions to cell and molecular biology are not always given the credit it deserves. PMID- 10726161 TI - In situ fluorescence visualization of bromouridine incorporated into newly transcribed nucleolar RNA. AB - Bromouridine-triphosphate is commonly used for in situ immunocytochemical labeling of newly synthesized RNA in living cells. While extranucleolar transcripts do not require special conditions for visualization, special treatment prior to fixation (e.g. incubation with alpha-amanitine) is necessary for immunofluorescence detection of bromouridine-labeled nucleolar RNA in previous studies. We show in the present investigation that bromouridine triphosphate is efficiently used by both extranucleolar and nucleolar RNA polymerases in living cultured cells. The failure to detect incorporated bromouridine within nucleoli is entirely due to improper treatment of cells after bromouridine incorporation. When methanol/acetone fixation is used, fluorescence signals within nucleoli can be routinely found. PMID- 10726162 TI - Hemolysis of human erythrocytes with saponin affects the membrane structure. AB - Incubation of cells and tissues with saponin makes the lipid bilayer permeable to macromolecules. Ghosts (membrane preparations) of saponin-lysed erythrocytes do not reseal, thus indicating an irreversible damage of the lipid bilayer. We investigated the influence of disturbance of the lipid bilayer on membrane proteins by comparing ghosts of saponin-lysed erythrocytes with ghosts of cells lysed in hypotonic buffer. Transmission electron microscopy revealed destruction of the lipid bilayer and emergence of multilamellar buds in saponin-lysed ghosts. Freeze-fracture electron microscopy showed regions with crystalline lipids and an increase in particle-free areas on fracture faces. The number of protein sulfhydryl groups and the binding of hemoglobin were diminished in saponin-lysed ghosts. A Scatchard plot of hemoglobin binding revealed the decrease of high affinity binding sites. All these results indicate an aggregation of band 3 protein also demonstrated by laser scanning microscopy after incubation of cells labelled with eosin-5-maleimide with sublytic concentration of saponin. Hemolysis with saponin also affected the interaction between transmembrane proteins and the cytoskeleton. Dissociation of peripheral membrane proteins by incubation of ghosts in low salt buffer or by blocking sulfhydryl groups was increased and the association of spectrin with spectrin-depleted vesicles was decreased. The increased incorporation of the fluorescent probe Merocyanine 540 into saponin lysed ghosts and the increased relative fluorescence quantum yield confirmed the perturbation of the lipid bilayer and the changed interaction between membrane lipids and intrinsic membrane proteins. Our results suggest that permeabilization of the lipid bilayer with saponin to admit the access of antibodies to the cytoplasmic surface of cells can aggregate transmembrane proteins and affect the immunocytochemical localization of associated proteins of the cytoskeleton. PMID- 10726163 TI - Lobular distribution pattern of lactate dehydrogenase and 6-phosphogluconate dehydrogenase activity in rat liver. AB - Lactate dehydrogenase (LDH) and 6-phosphogluconate dehydrogenase (6-PGDH) activities were measured in lobular areas expanding between 3 portal tracts and an efferent central vein in the livers of male Wistar rats, using a Lowry technique. The maximum of LDH activity was found in a nearly uniform broad area in the lobular periphery. From that area values decreased along periportal/septal ->perivenous gradients, but only slightly within that area along the periportal- >septal axis of the vascular septum. Maximum values of 6-PGDH activity were present in an intermediate area close to the central vein demonstrating a rather inhomogeneous distribution pattern without a clear definition of zonal limits. Our data on the distribution pattern of LDH are in agreement with the concept of the metabolic lobulus and are supported by a recent evaluation of the vascular architecture in rat liver. The lobular distribution pattern of 6-PGDH cannot be interpreted without doubt in accordance with that concept. PMID- 10726164 TI - Immunocytochemical visualization of transfected DNA in cultured cells. AB - Nonviral transfection is one of the modern methods for the incorporation of foreign genes into cells. This process involves uptake of foreign genetic material by the cell and further trafficking through the cytoplasm to the nucleus. Elucidation of cytoplasmic pathways of transfection complexes can be useful to improve already existing gene delivery systems or to establish new systems. To monitor transfection complexes in the cell during transfection, we elaborated a method for the visualization of transfection complexes by introducing digoxigenin-labelled nucleotides into foreign DNA followed by detection of digoxigenin label with the use of antibodies directed against digoxigenin. This procedure allowed the visualization of DNA in transfection complexes and to monitor these complexes in cells during transfection. PMID- 10726165 TI - Cyclosporine A affects the organization of cytoskeletal fibrillar proteins in rat thymus. AB - We have evaluated whether cyclosporine A affects cell structure and cytoskeletal proteins of the thymus of Wistar rats. Immunohistochemical analysis showed that expression of the cytoskeletal proteins vimentin and desmin was much higher in epithelial cells, dendritic cells and lymphocytes in the thymus of treated rats than in untreated controls. Protein expression was observed as a positive condensation in a distinct area near the nucleus with a capping-like configuration. An ultrastructural study showed that the amount of cytoskeletal fibrillar structures was increased in the treated rats. The structures were assembled in a limited area of the cell with a nuclear capping-like configuration which was in agreement with the light microscopical observations. Immunoblotting analysis demonstrated that vimentin and desmin had a lower molecular weight in treated rats than in controls (57 and 53 kDa versus 55 and 51 kDa, respectively). The results clearly indicate that cyclosporine A affects the structure of the cytoskeleton suggesting that this could be the first step in its immunosuppressive effects by altering nucleus/cytoplasm signaling. PMID- 10726166 TI - Expression of CD146 adhesion molecules (MUC18 or MCAM) in the thymic microenvironment. AB - The thymic microenvironment is thought to play a critical role in T-lymphocyte development, providing signals both via cell surface molecules such as adhesion molecules and soluble molecules. The present investigation is focused on immunoelectron microscopical analysis of distribution patterns of CD146 adhesion molecules (MUC18 or MCAM) in the microenvironment of normal human thymus, using the pre-embedding indirect immunoperoxidase technique. The anti-CD146 monoclonal antibody (mAb) revealed strong membraneous labelling of immature thymocytes at both the light and electron microscopical level. Proliferating thymocytes, most of the epithelial cells, macrophages, endothelial cells and smooth musle cells of small vessels and capillaries showed both membraneous and cytoplasmic labelling with anti-CD146 mAb as was demonstrated by electron microscopy. In contrast, these cells displayed a strong cytoplasmic immunoreactivity at the light microscopical level. The extracellular matrix was also stained with the anti CD146 mAb. No labelling was observed in interdigitating cells. Interestingly, the CD146 molecule was strongly expressed on apical and lateral membranes of endothelial cells as was demonstrated electron microscopically. This selective CD146 labelling of capillary endothelium mainly localized at the cortico medullary junction may be manifestations of lymphocyte transmembrane migration and lymphocyte homing. In conclusion, the present study suggests that CD146 is expressed by most elements of the microenvironment of normal human thymus. Therefore, it may be a pan-antigen which is essential for the maintenance of thymic architecture and function. PMID- 10726167 TI - DNA in situ hybridization (interphase cytogenetics) versus comparative genomic hybridization (CGH) in human cancer: detection of numerical and structural chromosome aberrations. AB - DNA in situ hybridization techniques for cytogenetic analyses of human solid cancers are nowadays widely used for diagnostic and research purposes. The advantage of this methodology is that it can be applied to cells in the interphase state, thereby circumventing the need for high-quality metaphase preparations for karyotypic evaluation. In situ hybridization (ISH) with chromosome specific (peri)centromeric DNA probes, also termed "interphase cytogenetics", can be used to detect numerical changes, whereas comparative genomic hybridization (CGH) discloses chromosomal gains and losses, i.e. amplifications and deletions. We wanted to compare both methods in human solid tumors, and for this goal we evaluated ISH and CGH within a set of 20 selected prostatic adenocarcinomas. Chromosomes 7 and 8 were chosen for this analysis, since these chromosomes are frequently altered in prostate cancer. ISH with chromosome 7 and 8 specific centromeric DNA probes was applied to standard, formalin-fixed and paraffin-embedded, histological sections for numerical chromosome analysis. CGH with DNA's, extracted from the same histologic area of the archival specimens, was used for screening of gains and losses of 7 and 8. ISH with centromeric probes distinguished a total of 26 numerical aberrations of chromosome 7 and/or 8 in the set of 20 neoplasms. In the same set CGH revealed a total of 35 losses and gains. CGH alterations of 7 and 8 were seen in twenty-two of the 26 chromosomes (85%) that showed aberrations in the ISH analysis. Concordance between ISH and CGH was seen in 11 (of 26; 42%) chromosomes. Eight chromosomes were involved in gains (5 x #7, 3 x #8), three in losses (3 x #8). This included both complete (3/11) and partial (8/11) CGH confirmation of the numerical alteration. Partial CGH confirmation was defined as loss or gain of a chromosome arm with involvement of the centromeric region. In the majority of these cases it concerned a whole chromosome arm, mostly the long arm. We conclude that generally a fair correlation was found between ISH and CGH in interphase preparations of a series of prostate cancers. However, when specified in detail, most of the numerical ISH aberrations were only partly represented in the CGH analysis. On the one hand, it suggests that CGH does not adequately discriminate numerical abnormalities. On the other hand, it likely implies that not all numerical changes, as detected by interphase cytogenetics, are truly involving the whole chromosome. A part of these discrepancies might be caused by structural mechanisms, most notably isochromosome formation. PMID- 10726168 TI - Nucleolar coefficient of granulocyte precursors and granulocytes after visualization of nucleoli by two different methods. AB - To clarify differences in the incidence and number of nucleoli in the granulopoietic lineage, these nuclear components were studied in human and rabbit granulocyte precursors and granulocytes after visualization by 2 widely employed cytochemical procedures, i.e. a procedure for the demonstration of RNA and the silver reaction for the demonstration of nucleolar silver stainable proteins (SSPs). In early stages of the granulocyte proliferating compartment, substantial differences were not found between specimens in which nucleoli were visualized by both procedures. However, in contrast to specimens stained with the silver reaction, the number of cells without nucleoli was substantially larger in advanced stages of granulocyte development in specimens stained for RNA. The number of nucleoli per cell as expressed by the nucleolar coefficient was generally larger in specimens stained with the silver reaction for nucleolar SSPs. These differences were significant starting with the stage of myelocytes. Moreover, in specimens stained with the silver reaction, most of human mature granulocytes did not contain nucleoli but nucleoli were present in all mature granulocytes of rabbits. Such differences were not observed in specimens stained for RNA in which most granulocytes were without RNA-containing nucleoli. Thus, the evaluation of the presence or absence of nucleoli in specimens depended on the visualization procedure. It is likely that in micronucleoli which are characteristic for terminal differentiation of the granulocytic lineage, RNA containing structures may be lost or are below the detection limit of the light microscope. In addition, differences in the presence of nucleoli exist apparently between human and rabbit granulocytes in specimens stained for SSPs but not in those stained for RNA. PMID- 10726169 TI - Chromophobe cell carcinoma and renal cell neoplasms with mucin-like changes. AB - Hale's colloidal iron staining of 8 chromophobe cell carcinomas (CCC) was compared with that of non-chromophobe renal cell carcinomas (RCC), renal oncocytomas, and renal adenomas. Six non-chromophobe RCC showing diffuse and moderate cytoplasmic staining contained extensive areas with translucent cytoplasm as observed in CCC. Seventeen of 25 conventional RCC of the clear cell variant (randomly chosen from 130 cases), 21 of 26 RCC with areas of chromophilic cytoplasm, and 16 of 20 papillary RCC, 7 of 14 adenomas and 14 of 16 oncocytomas displayed focal areas with mild to moderate staining of the cytoplasm. Hale's colloidal iron staining was partially reduced by digestion with neuramidase but not with hyaluronidase. This positive staining demonstrated glycoproteins containing sialylated glycoconjugates, probably a type of acid epithelial mucin. We suggest that there is a spectrum of mucin-like changes in typical CCC representing RCC with extensive and marked "mucin-like changes". The eosinophilic variant of CCC and some RCC with extensive chromophobe cell features represent renal neoplasms with moderate changes. The other RCC, oncocytomas and papillary renal neoplasms with mild to moderate staining with Hale's colloidal iron represent renal neoplasms with focal mucin-like changes. RCC with extensive chromophobe cell features may pose a differential diagnostic problem with CCC. PMID- 10726171 TI - [Morphology and phenomenology of death by trampeling]. PMID- 10726170 TI - [Occupation-related aspects of homicidal acts]. AB - Several homicides characterized by peculiarities related to the occupation of the perpetrator are reported and compared with observations from literature. Common characteristics of the cases are determined by the following factors: availability of the equipment (i.e. drugs, poisons, tools) and application of special knowledge, skills or techniques provided by professional education (i.e. pharmacology, injection and dissection techniques in physicians and related professions, use of extraordinary, self-constructed weapons by technicians). Remarkable features of some cases are the long period of meticulous preparation and professional attempts to abolish or disguise suspicious findings. Thus, these homicides often present with uncommon modes of commitment and unusual forensic findings which are difficult to recognize and assess. Supplementing own experience by the cases reported in literature is an indispensable prerequisite to examine these cases properly and to provide professional and competent expert opinion for the police authorities. PMID- 10726172 TI - [Suicide by a locomotive with unusual transport of suicide victims]. AB - Two suicides are described in which the victims were transported for 400 m and 16 km (!) hanging from the locomotive's hook coupling. According to the Deutsche Bahn AG, it would be possible for a body caught on a coupling to be dragged for almost 600 km without being discovered. Upon discovery of such a body, the police must search long stretches of the railroad tracks. PMID- 10726173 TI - [Microscopic study of powders of hallucinogenic mushrooms--Psilocybe sp]. AB - The paper presents simple methods for microscopic examination and basic microchemical testing for the identification of suspect mushroom powders. The microscopic features of the most commonly cultivated and trafficked hallucinogenic genus Psilocybin are described and may serve for the decision whether any suspect material consists of such mushroom powder (and is therefore to be subjected to further analysis) or not. PMID- 10726174 TI - [An unusual case of self-harm with petroleum distillate]. AB - After surgical treatment of an inguinal hernia, a 26-year-old male was discharged from hospital with a well healing surgical wound. Four weeks later he was admitted again with a major localized painful swelling of the soft tissues, whereas the clinical examination revealed no further local or systemic signs of inflammation. The immediate operation disclosed no abscess formation, but a large colliquation necrosis of the subcutaneous tissue with an outstanding petroleum like odour. On toxicological examination of the necrotic tissue, a respective petroleum distillate was found. The patient was confronted with the suspicion of having injected the petroleum preparation himself to prolong the wound healing and finally admitted the self-mutilating behaviour. The pathophysiology, clinical history and morphological findings are discussed in comparison with case reports of chemically induced self-mutilation. PMID- 10726175 TI - [Fatal child neglect in East Germany 1 January 1985 to 2 October 1990. Results of a multicenter study]. AB - No reliable data are available on cases of lethal child neglect in the area of the former German Democratic Republic. In a multicenter study we therefore examined the police and court records for such cases occurring in the period 1 January 1985 to 2 October 1990 in the entire area of the former German Democratic Republic. RESULTS: A total of 9 cases were reported to the study center. This does not include undetected cases, whose number, though indeterminate, is probably small due to the general obligation to perform autopsies on all children dying before their 16 birthday. Just over half of all victims were less than one year old, the oldest was 3 1/2 years old. Starvation and severe dehydration were by far the most common causes of death; in half of cases these occurred in combination with hypothermia. Most often mother killed their children by neglect, either alone or together with the victim's father. In the majority of cases no close bond existed between the parents and the child. Seventy percent of the perpetrators were chronic alcoholics. All 10 of the perpetrators were sentenced to imprisonment for periods ranging from one year to life long. Mitigating circumstances were presented at the sentencing phase of the trial on behalf of 20% of those convicted. The experience of legal medical and pathological institutes in the former German Democratic Republic underscores the need to perform an autopsy on all deceased infants and young children. Only this can ensure that no cases of lethal child neglect are overlooked. PMID- 10726176 TI - [Cervical findings and petechial hemorrhages in falls from high positions]. AB - Cases of combined suicide are infrequent findings, especially cases in which each method can be lethal. In interpretation of cases with a multitude of serious injuries it might be impossible to discover minor injuries which had occurred in the course of a preceding confrontation, because they may be masked by the general traumatization. In two cases of suicidal fall from a height neck injuries and petechial bleedings were found. The first case (woman 53 years) was a combination suicide (strangulation by ligature, deep incisions into her wrists, jump out of the window). The intensive petechial bleedings in the face could be explained as a result of ligature strangulation and the fracture of a lower thyroid horn as an indirect fracture resulting of the head traumatization. In the second case (girl, 14 years) petechial bleedings in the face were found. Furthermore the victim had marks on her neck-skin, consisting of well-lined bleedings and scratches, presenting a clear pattern, which could be related to a necklace. An explanation of these injuries as a result of a direct impact could not be given. Something must have happened prior the fall. Informations concerning the last hours before her suicide could not be obtained but a strangulation-attempt (by herself? by others?) must be ascertained. The histological investigation of the skin of this region arised a negative vital reaction, therefore a very short interval between that event and the death was assumed. PMID- 10726177 TI - [The diagnosis of cystic fibrosis in the adult]. PMID- 10726178 TI - [The validation of a portable 3-channel recording system (Oxyflow, Edentec) for the diagnosis of the sleep apnea syndrome]. AB - OBJECTIVE: To evaluate the usefulness of a portable recording device (Oxyflow, EdenTec) to measure oronasal airflow, oxygen saturation and arterial pulse for diagnosing sleep apnea syndrome (SAS) using conventional polysomnography as the gold standard. METHODS: Sixty-two subjects suspected of having SAS were studied prospectively by simultaneously recording conventional polysomnography and Oxyflow data in the sleep laboratory. Two different investigators, blinded to each other's findings, interpreted the data from each method. The apnea-hypopnea index (AHI) cut-off points used were?? 10, 15 and 30. The sensitivity, specificity, positive predictive value and negative predictive value of the Oxyflow indices for each AHI cut-off point were calculated. Both computer generated and manually collected data from the Oxyflow device were analyzed. Manual readings were recorded by two independent investigators and interobserver agreement was calculated. The usefulness of both automatic and manual analyses for SAS diagnosis was assessed using receiver operating characteristic curves (ROC). RESULTS: Fifty-eight (93.5%) men and 4 (6.5%) women with a mean age (+/- SD) of 53 +/- 11 years (29-73) were enrolled. An AHI > or = 10 was observed in 58% of the patients and mean AHI was 25 +/- 28 (0-125). The index of respiratory disturbance per hour of analysis with desaturation events > or = 4% (RDI4%) was the parameter with the largest area under the ROC curve (0.90 for AHI > or = 10; 0.94 for AHI > or = 15 and 0.96 for AHI > or 30). Manual reading was practical and reproducible (agreement 0.93, kappa coefficient 0.82) but its efficiency was no greater than that of automatic analysis. CONCLUSIONS: The Oxyflow device may be a useful diagnostic tool for SAS. Its portability and simplicity makes it potentially useful for in-home studies. PMID- 10726179 TI - [The characteristics of the mechanical activity of the respiratory muscles during the diaphragmatic respiration technic]. AB - Noteworthy among breathing training techniques is so-called diaphragmatic breathing. In spite of the technique's name, however, little is known of the functional characteristics of this ventilatory method. OBJECTIVE: To asses the mechanics of respiratory muscles, particularly diaphragm muscles, during diaphragmatic breathing in patients with severe chronic obstructive pulmonary disease (COPD). METHODS: Ventilatory pattern and respiratory pressures (abdominal [Pga], intrathoracic [Pes] and transdiaphragmatic [Pdi]) were studied in 10 patients with severe COPD in stable phase (age 69 +/- 6 years, FEV1 33 +/- 12% ref) at baseline and during deep breathing with spontaneous muscle recruitment (SMR) and during breathing training. Measurements were taken with the patient seated and in supine decubitus position. RESULTS: In seated position ventilatory pattern was similar with SMR and during breathing training. Mean Pdi during airflow, however, was greater during breathing training than with SMR (34.8 +/- 8.0 and 29.3 +/- 9.3 cmH2O, respectively, p < 0.05) for similar levels of Pes. Mechanical effectiveness of the diaphragm expressed as Vt/Pdi) was less during breathing training, however (36.1 +/- 10.4 and 49.5 +/- 15.8 cc/cmH2O, p < 0.05), with no changes in overall efficacy of respiratory muscles (Vt/Pes). In supine decubitus position, ventilatory patterns of SMR and breathing training were similar, although Vt and T1 were slightly higher in the latter (1,065 +/- 305 vs. 1,211 +/- 314 cc, p < 0.01; and 2.76 +/- 1.32 vs. 3.07 +/- 1.23 sec, p < 0.05). Pdi was also higher during breathing training (29.7 +/- 10.2 and 38.0 +/- 10.5 cmH2O, p < 0.05), although accompanied in this case by a higher Pes (21.2 +/- 7.5 to 26.4 +/- 8.4 cmH2O, p < 0.005). In supine decubitus position, the effectiveness of both diaphragm muscles and respiratory muscles overall was similar for both ventilatory modes. CONCLUSIONS: Breathing training truly involves greater use of the diaphragm, both in seated and supine decubitus positions. Breathing training does not provide greater ventilatory efficacy than SMR, however, in COPD patients. PMID- 10726180 TI - [Obtaining samples of the human diaphragm during upper laparotomy. A structural analysis]. AB - The diaphragm seems to undergo adaptive structural change in chronic obstructive pulmonary disease. The possibility of obtaining muscle specimens is limited, however, particularly when respiratory function is severely affected. OBJECTIVE: To assess the viability of a new technique for obtaining diaphragm muscle samples appropriate for structural assessment even from patients with severe functional change, and to study the size of fibers in relation to severity of disease. METHODS: Fifteen muscle specimens were obtained from patients (aged 57 +/- 15 years) by abdominal laparotomy. All had undergone full lung function testing. Muscle samples were taken during surgery using a new technique involving formation of a tobacco pouch with dome biopsy. The method had been previously validated in animal models. Later, the biopsies were processed to evaluate fiber proportions and sizes (ATPase dyes at different levels of pH). RESULTS: The 15 patients had a wide range of lung function results (FEV1 22-120% ref); 4 were severely affected (FEV1 < or = 50% ref). Nutritional status was normal in all cases; FEV1/FVC was 67 +/- 13%, RV was 134 +/- 55% ref, maximal mouth pressure (PImmax) was -75 +/- 27 cmH2O, transdiaphragmatic pressure (PIdimax) was 96 +/- 26 cmH2O, DLCO was 87 +/- 26% ref and PaO2 was 89 +/- 14 mmHg. We were able to obtain specimens valid for structural analysis from all patients with no complications. Light type I fibers predominated (54 +/- 9%) and size was normal overall (57 +/- 9 microns minimum diameter [Dm] atrophy index 195 +/- 243, and hypertrophy index 66 +/- 78), with no differences between the two fiber subtypes (Dm 58 +/- 8 microns for type I and 61 +/- 8 microns for type II). Overall size correlated inversely with static volumes (e.g. Dm with RV, r = -0.729, p < 0.01). CONCLUSIONS: The laparoscopic technique described is simple and safe for use in humans to obtain diaphragm muscle specimens that are valid for morphometric analysis, allowing us to enlarge the range of subjects that can be enrolled for this type of study. The fiber muscles studied are smaller when functional involvement is greater in chronic obstructive pulmonary disease. PMID- 10726181 TI - [A comparison of several measurement scales for assessing dyspnea in their daily activities in patients with chronic obstructive lung disease]. AB - The objective of this study was to compare two instruments for measuring dyspnea to functional capacity, defined as maximal oxygen uptake in a stress test limited by symptoms. We carried out a descriptive study to determine correlation between the methods. Subjects with severe chronic obstructive pulmonary disease were enrolled and dyspnea during daily activities was evaluated using the Medical Research Council (MRC) scale and the chronic respiratory disease health perception questionnaire (CRQ). Dyspnea was also assessed on a visual analog scale (VAS) and the Borg scale. Patients performed a progressive treadmill test limited by symptoms to measure peak VO2 and VE. Correlation analysis of the two measures of dyspnea and the objective measures of functional capacity (peak VO2 and VE) showed that the clinical dyspnea measures (CRQ and MRC) correlated well with peak aerobic capacity and with maximal ventilation, whereas the VAS and Borg scale were not significantly related to the aforementioned variables. We also observed that the clinical scales for dyspnea correlated with each other, as did the VAS and Borg scale, but that there was no correlation between the two types of measurement. We conclude that clinical dyspnea indexes (CRQ and MRC) correlate well with maximal aerobic capacity and with each other. However, the VAS and Borg scale do not correlate with functional capacity measured by peak VO2 and VE. PMID- 10726183 TI - [Guideline for the use of inhaled drugs. The Working Group of SEPAR: the Nursing Area of the Sociedad Espanola de Neumologia y Cirugia Toracica]. PMID- 10726182 TI - [The prognostic factors of invasive pulmonary aspergillosis in patients with chronic pneumopathy]. AB - OBJECTIVES: a) To determine in patients with chronic respiratory disease the risk factors for death due to semi-invasive and invasive pulmonary aspergillosis (SIPA), and b) to describe the clinical features of SIPA in such patients. METHOD: Twenty-one patients with chronic respiratory disease were enrolled (9 with chronic obstructive pulmonary disease, 2 asthmatics and 3 with bronchiectasis, 5 with post-tubercular sequelae and 2 mixed cases). A diagnosis of SIPA was established in our hospital when, in a patient with a clinical picture consistent with such a diagnosis, the fungus was isolated in bronchial secretions or parenchymal pulmonary specimens were obtained during autopsy. RESULTS: The most common symptoms were dyspnea (81%), cough (67%) and expectoration (62%) increasing over the levels usual for patients with chronic respiratory disease. Hemoptysis was present in only 14%. Eight patients (38%) died as a result of SIPA. A comparison of those surviving and non-surviving patients revealed that the latter had significantly higher LDH levels and white cell counts, and significantly lower total plasma protein and platelet counts. CONCLUSIONS: a) Low protein levels and high LDH levels and white cell counts with thrombopenia are indicators of poor prognosis in chronic respiratory disease patients with SIPA, and b) such patients do not usually present signs or symptoms that lead to a suspicion of SIPA given that such signs are typical of failing compensatory mechanisms in the disease itself. PMID- 10726184 TI - [The applicability of stress tests in pneumology]. PMID- 10726185 TI - [Hyoid fracture and traumatic subcutaneous cervical emphysema from an attempted hanging. Apropos a case]. AB - We describe a patient with a history of psychiatric disorder who was brought to our hospital after attempted suicide by hanging. Severe subcutaneous facial, palpebral and cervical emphysema was present, with dysphonia, dysphagia and slight respiratory difficulty. Fiberoptic bronchoscopy revealed upper airway obstruction due to edema in an intact airway. Successive CAT scans gave evidence of hyoid fracture and laryngocele, in addition to the corresponding emphysema of the subcutaneous area and pneumomediastinum. Given the persistence of dysphagia, we ordered esophageal tests, which showed functional alteration of the upper esophageal sphincter. Suprasternal cervicotomy to drain the pneumomediastinum and laryngeal microsurgery to treat the laryngocele resolved the problem. PMID- 10726186 TI - [Pneumothorax as the clinical presentation of bronchogenic carcinoma. A report of 3 cases]. AB - The association of pneumothorax and lung cancer is rare and diagnosis is complex in such cases. Clinical suspicion of cancer must be based on radiological findings and the existence of risk factors. We discuss the mechanisms involved in the development of pneumothorax in patients with lung cancer, the clinical significance of the association, and the recommended diagnostic approach and therapeutic guidelines. PMID- 10726187 TI - [A paratracheal pericardial diverticulum]. PMID- 10726188 TI - [The concept of down-staging in bronchopulmonary carcinoma and its translation to Spanish]. PMID- 10726189 TI - [Chronic necrotizing aspergillosis and cystic fibrosis]. PMID- 10726190 TI - [Idiopathic spontaneous hemothorax]. PMID- 10726191 TI - [The monitoring of antitubercular drugs. Is it clinically useful?]. PMID- 10726192 TI - [Severe emphysema with incapacitating dyspnea: volume-reduction surgery or lung transplant?]. PMID- 10726193 TI - [Small-cell nonanaplastic bronchogenic carcinoma. The new stage I]. AB - OBJECTIVE: To validate updated guidelines for stage I classification of patients with differentiated small-cell bronchogenic carcinoma. METHODS: Seven hundred seventeen tumors of differentiated small-cell bronchogenic carcinoma were resected in our hospital and given a TNM classification of stage I based on guidelines recently issued by the Spanish Society of Pneumology and Chest Surgery (SEPAR). Survival was calculated using the Kaplan-Meier method and curves were compared with a log-rank test. The Cox proportional hazards model was used to analyze multiple variables. RESULTS: One hundred forty-two cases were classified as stage IA and 575 as stage IB. Survival was significantly longer for stage IA than for stage IB (p = 0.0021). The prognosis was significantly better for stage IA patients who were asymptomatic (p = 0.0380) or who had tumors < or = 2 cm in diameter (p = 0.0431). In stage IB, histologic grade (p = 0.0104) and tumor diameter (p = 0.0002) significantly affected survival. A noteworthy finding was the 82% survival at five years in a group of 66 patients with a maximum tumor diameter of 3 cm classified as T2N0M0 due to invasion of the visceral pleura or to proximal involvement of a lobar bronchus at a site > 2 cm from the carina; that survival rate was not significantly different from survival for stage IA (p = 0.1573). Multivariate analysis showed that tumor diameter (p = 0.0272) was of prognostic importance in stage IA, while tumor diameter (p = 0.0005) and histologic grade (p = 0.0092) were relevant in stage IB. CONCLUSION: The new staging guidelines for differentiated small-cell bronchogenic carcinoma are nearer to prognostic reality given that survival for stage IA patients is significantly longer than for stage IB patients. However, the method continues to have shortcomings in that it fails to achieve one of its main objectives, namely prognostic homogeneity for each subgroup, as indicated by problems related to variables of tumor extension such as diameter, involvement of the visceral pleura or bronchial location, apart from other factors that affect survival. PMID- 10726194 TI - [Maximum inspiratory flows in healthy children and asthmatics 4 to 8 years old. The implications for the inhalation of drugs in powder form]. AB - Twenty-two asymptomatic asthmatic children and twenty-one healthy controls aged between 4 and 8 years old were studied in order to determine peak inspiratory flow (PIF) in baseline conditions through a Turbuhaler powder aerosol device. PIF was measured randomly on one of three days using computerized spirometry. In healthy children, baseline PIF ranged from 129 to 244 L/min; PIF through the Turbuhaler ranged from 72 to 99 l/min. In asthmatics, the ranges were 122 to 236 l/min and 79 to 102 l/min for baseline and Turbuhaler PIF, respectively. PIF was significantly related to age (p < 0.05). The difference between PIF through the Turbuhaler for asthmatic and healthy children was not significant. PIF of healthy and asthmatic Chilean children between 4 and 8 years of age exceeded the level required to obtain an optimum dose from this type of device. This study suggests that the aerosol powder doses produced by devices such as that used in this study can be used by children from age 4 and older. PMID- 10726195 TI - [The evaluation of the efficacy of a multidisciplinary treatment in a group of obese patients with a BMI > or = 35 and a change in lung function]. AB - OBJECTIVE: To assess the efficacy of an interdisciplinary protocol for treating obesity in a group of patients with BMI > or = 35 and with altered respiratory function that was not necessarily related to obesity or not. PATIENTS AND METHOD: Forty obese individuals between 18 and 60 years of age with altered respiratory function were enrolled. Spirometric values, plethysmograph volumes, arterial blood gases, and nighttime respiratory polygraphs were recorded. Following psychological and nutritional evaluation, the patients commenced year-long treatment for obesity involving a personalized diet and psychological counseling. Follow-up was weekly and individualized at first; in later sessions, patients were grouped. Lung function tests were repeated after loss of 5 kg. Sleep polygraphy was repeated after loss of 10 kg. RESULTS: Weight loss over 15 kg was achieved by 48.6% of the patients. Respiratory function variables: FVC, FEV1, RV, ERV, PaO2 and SatO2 after treatment changed significantly from initial levels. Significant differences were also seen in the severity of sleep apnea and pressures needed for continuous positive airway pressure. Uric acid, glucose and triglyceride blood levels became normal in 89%, 61% and 50% of the patients, respectively, after weight loss. No characteristic psychological profile was identified for severe obesity, although levels of anxiety, eating behavior, marital adjustment and perception of body image were aspects that were fundamentally altered. CONCLUSIONS: In the difficult group of obese patients with BMI > or = 35, interdisciplinary treatment has proven effective for achieving substantial weight loss, while improving respiratory function and severity of sleep disorder. This therapy, which is at present viable for few centers, deserves consideration in the interest of benefiting the increasing number of obese patients. PMID- 10726196 TI - [Chronic obstructive pulmonary disease and the seasons of the year]. AB - OBJECTIVE: To study the existence of differences among patients with chronic obstructive pulmonary disease (COPD) hospitalized for exacerbation during different seasons of the year. PATIENTS AND METHOD: DESIGN: transversal study. SUBJECTS: all patients admitted to the short-stay medical ward for exacerbation of COPD over a period of one year. MEASUREMENTS: demographic variables, disease history, signs and symptoms, baseline walking test, baseline function tests, baseline blood gases, length of stay and death while hospitalized. INTERVENTIONS: patient variables were described and the characteristics of autumn/winter admissions were compared to those of spring/summer admissions. RESULTS: Two hundred thirty-two patients with a mean age of 70.9 +/- 9.4 years (87.9% male). Eighty-four (36.2%) were admitted in winter, 65 (28%) in spring, 30 (12.9%) in summer and 53 (22.8%) in autumn. Twenty-one percent were being treated with corticoids, 27% were using domiciliary oxygen therapy and 36% had cor pulmonale. Baseline lung function test results were as follows: FEV1 44%, FEV1/FVC 48%, pO2 65.9 mmHg and pCO2 43.5 mmHg. Mean length of stay was 4.9 +/- 6 days; 9 patients (3.9%) died. Only PCO2 upon admission showed seasonal autumn-winter versus spring summer differences (44.3 versus 49.5, p = 0.0008). Statistically significant differences were not observed for other variables. CONCLUSIONS: Clear seasonal variation can be observed in the number of patients requiring admission due to COPD exacerbation, but we found no relevant seasonally-related differences in disease characteristics. PMID- 10726197 TI - [The internal consistency and content validity of the Spanish version of the Asthma Autonomy Questionnaire]. AB - The Asthma Autonomy Questionnaire (AAQ) was designed to evaluate asthmatics' desire to learn about their disease and to make decisions. The AAQ consists of 26 items distributed in two scales: Preferences in the Search for Information (PSI, 8 items) and Preferences in Decision Making (PDM, 6 general items and 12 related to 3 scenarios depicting asthma in stable phase, during mild exacerbation and during severe exacerbation). The aim of this study was to analyze the internal consistency (Cronbach's-coefficient) and content validity (factorial analysis of principal components) of the AAQ. After translation and back translation, the Spanish version of the AAQ was administered to 115 adult asthmatics of both sexes and differing levels of severity. The alpha coefficients for the two scales and 3 scenarios ranged from 0.42 (PSI) to 0.73 (stable phase scenario); only for the stable-phase scenario were values high or statistically acceptable. Factorial analysis reproduced the content of the scales only approximately, with some items proving to relate to factors that were different from the scale they originally belonged to. These results indicate that, in its current formulation, the AAQ presents important measurement problems and revision is advisable. PMID- 10726198 TI - [Muscle changes in chronic obstructive pulmonary disease: the theory of compartments]. PMID- 10726199 TI - [Tracheobronchomegaly: an exceptional predisposing factor for pulmonary aspergillomas and massive hemoptysis]. AB - Mounier-Kuhn syndrome--or tracheobronchomegaly--is a rare congenital disorder characterized by significant dilation of the trachea and main bronchi. It is accompanied by ineffective cough and is often complicated by recurrent lung infections and bronchiectasis. Clinical presentation varies widely, ranging from forms with scarce involvement of functional capacity to others that progress to respiratory failure that can prove fatal. We report an exceptional case of massive hemoptysis secondary to the presence of pulmonary aspergillomas and bilateral bronchiectasis in which bronchial arteriography with embolization and surgical resection were insufficient for resolving the complications. PMID- 10726200 TI - [Sequential double lung transplant in Kartagener's syndrome]. AB - Very few cases of lung transplantation have been described for patients with Kartagener's syndrome. We report the first case to be published in Spain. A 15 year-old girl with complete Kartagener's syndrome underwent sequential transplantation of both lungs. Due to the unusual distribution of the organs in this syndrome, the bronchial stumps of donor and recipient had to be distributed differently. With the initial technical difficulties overcome, the patient now leads a normal life two years after transplantation. PMID- 10726201 TI - [Pediatricians and childhood tuberculosis]. PMID- 10726202 TI - [The present decisive influence of environmental mycobacteria on the prevalence of tuberculosis infection in Spain]. PMID- 10726203 TI - [The epidemiology of childhood tuberculosis]. PMID- 10726204 TI - [Massive pleural effusion as the first manifestation of chronic pancreatitis]. PMID- 10726205 TI - [Isoniazid-induced fever]. PMID- 10726206 TI - An estimate of rapid cytoplasmic calcium buffering in a single smooth muscle cell. AB - Cytoplasmic calcium increments in the absence of sarco (endo) plasmic reticulum function were measured with a low-affinity fluorophore Indo-1FF in single isolated smooth muscle cells from guinea-pig urinary bladder. To evaluate the Ca(2+)-buffering properties of the myoplasm, Ca2+ influx, measured as time integral of the Ica (integral of Ica), was compared with corresponding free Ca2+ increments (delta [Ca2+]i) in the cytoplasm. The ratio between integral of ICa and delta [Ca2+]i (integral Ica/delta [Ca2+]i), reflecting the Ca2+ buffering properties of the cytosol, was in the range of 4.9-9.3 pC/microM (mean 6.2 +/- 1.2, n = 12). It remained approximately constant (6.4 +/- 1.4 pC/microM, n = 8) during recordings lasting up to 25 min, suggesting that cytoplasmic Ca2+ binding does not change markedly during cell dialysis and that the endogenous Ca2+ buffer is not significantly washed out of the cell through the patch pipette. Wash-in or wash-out of BAPTA, a mobile high-affinity Ca2+ buffer, into or from the cell markedly changed the relationship between Ca2+ influx through Ca2+ channels and delta [Ca2+]i within minutes. Changes in integral of ICa/delta [Ca2+]i during the sequence of depolarizing steps, which increased free [Ca2+]i up to 5 microM, suggested lower limits for the apparent affinity of a rapid Ca2+ buffer (16 microM) and for the total buffer concentration (530 microM). Introduction of 4 mM DPTA (Kd for Ca2+ = 81 microM) into the cell more than doubled the total cytoplasmic Ca2+ buffer capacity. These results suggest that cytoplasmic Ca2+ buffer in smooth muscle cells has a low affinity for free Ca2+. The Ca(2+) binding ratio of the cytoplasm in most cells was estimated to be between 30 and 40. The Ca(2+)-binding ratio did not differ markedly between cells isolated from neonatal (< or = 5 days) and adult animals. PMID- 10726207 TI - Expression level of inositol trisphosphate and inositol tetrakisphosphate receptors and their influence on Ca2+ release in permeabilized HL-60 and T15 cells. AB - To try to further define the mechanism of action of the putative second messenger inositol 1,3,4,5-tetrakisphosphate (InsP4), we have studied its effects in permeabilized cells expressing different levels of inositol trisphosphate receptor (InsP3R) types I and III and of the GTPase-activating protein GAP1IP4BP. During the growth curve of human HL-60 cells and mouse T15 cells there was an increase in these proteins, which was further increased by differentiation (HL 60) and, marginally, by transformation (T15). T15 cells entering the stationary phase showed much lower concentrations of these proteins and expression was below detection in apoptotic HL-60 cells. Rasp21 showed a different pattern of expression. The ratios of InsP3R subtypes seem to affect the dose-response curve for inositol 2,4,5-trisphosphate Ins(2,4,5)P3. In permeabilized T15 cells the curve was approximately 5-fold to the right of that obtained using HL-60 cells. However, permeabilized untreated and differentiated HL-60 cells and T15 cells all showed a comparable synergistic effect of InsP4 on Ca2+ release stimulated by a concentration of Ins(2,4,5)P3, releasing approximately 20% of the Ins(1,4,5)P3 sensitive Ca2+ pool. The data indicate that under these conditions InsP4 is acting independently of cell type, of the ratio of inositol trisphosphate receptor subtypes, and of the concentration of GAP1IP4BP. PMID- 10726208 TI - GABA- and glutamate-mediated network activity in the hippocampus of neonatal and juvenile rats revealed by fast calcium imaging. AB - In the rat hippocampus, during the first postnatal week, network activity is characterized by GABA-driven giant depolarizing potentials (GDPs) associated with calcium signals that are readily blocked when the GABAA antagonist bicuculline is applied to the bath. Towards the end of the first postnatal week, in concomitance with the shift of GABA responses from the depolarizing to the hyperpolarizing direction, functional glutamatergic connections start appearing. At this developmental stage, application of bicuculline blocks GABAA-mediated inhibition and induces the appearance of interictal epileptiform discharges. In the present experiments, we have used a high spatio-temporal resolution imaging system to compare, on a time scale of tens of ms, the onset and propagation of fast calcium transients generated within a GABAergic or glutamatergic network. We found that, during the first postnatal week, calcium signals associated to evoked GDPs arise from the activation of a local circuitry of neurons spanning the stratum radiatum and the pyramidal layer. Similar activation patterns were elicited by focal application of GABA in the presence of kynurenic acid, a broad spectrum ionotropic glutamatergic antagonist, and were blocked by bicuculline. During the second postnatal week, in the presence of bicuculline, calcium signals associated with interictal discharges evoked by stimulation of glutamatergic fibres propagated along the well-defined three-synaptic pathway from the dentate gyrus to the CA1 hippocampal area. PMID- 10726209 TI - Calcium signals in prostate cancer cells: specific activation by bone-matrix proteins. AB - Cancer of the prostate commonly metastasizes to bony sites where cells acquire an aggressive, rapidly proliferating, androgen-independent phenotype. The interaction between bone and prostate, thus, becomes a key factor in disease progression. Fluctuations in intracellular ionized Ca2+ [Ca2+]i are rapid, regulated signal transduction events often associated with cell proliferation. Hence, Ca2+ signals provide a convenient measure of early events in cancer cell growth. This study developed single cell fluorescent imaging techniques to visualize Ca2+ signals in Fura-2 loaded prostatic cancer cell lines of various metastatic phenotypes. Solubilized bone fractions containing extracellular matrix and associated proteins were tested for the ability to trigger Ca2+ signals in prostate cancer cell lines. Fractions representing the complete repertoire of non collagenous proteins present in mineralized bone were tested. Results demonstrated that two bone fractions termed D3b- and D4a-triggered Ca2+ signals in prostate cancer cells derived from bone (PC-3), but not brain (DU-145) metastases of prostate cancer. Lymph-node derived LNCaP cells also did not produce a Ca2+ signal in response to addition of soluble bone matrix. No other bone fractions produced a Ca2+ signal in PC-3 cells. It is of interest that bone fractions D3b and D4a contain a number of non-collagenous matrix proteins including osteonectin (SPARC) and osteopontin (OPN), as well as prothrombin. Moreover, antibody LM609 that recognizes the alpha v beta 3 integrin, blocks the ability of OPN to trigger a Ca2+ transient in PC-3 cells. These studies support a conclusion that bone-matrix proteins play a role in the growth and progression of metastatic prostate cancer, and that prior growth in bone may be associated with development of a bone-matrix-responsive phenotype. PMID- 10726210 TI - Mobilization of Ca2+ stores in individual pancreatic beta-cells permeabilized or not with digitonin or alpha-toxin. AB - The concentration of free Ca2+ in the cytoplasm and organelles of individual mouse pancreatic beta-cells was estimated with dual wavelength microfluorometry and the indicators Fura-2 and furaptra. Measuring the increase of cytoplasmic Ca2+ resulting from intracellular mobilization of the ion in ob/ob mouse beta cells, most organelle calcium (92%) was found in acidic compartments released when combining the Ca2+ ionophore Br-A23187 with a protonophore. Only 3-4% of organelle calcium was recovered from a pool sensitive to the Ca(2+)-ATPase inhibitor thapsigargin. Organelle Ca2+ was also measured directly in furaptra loaded beta-cells after controlled plasma membrane permeabilization. The permeabilizing agent alpha-toxin was superior to digitonin in preserving the integrity of intracellular membranes, but digitonin provided more reproducible access to intracellular sites. After permeabilization, the thapsigargin-sensitive fraction of Ca2+ detected by furaptra was as high as 90%, suggesting that the indicator essentially measures Ca2+ in endoplasmic reticulum (ER). Both alpha toxin- and digitonin-permeabilized cells exhibited ATP-dependent uptake of Ca2+ into thapsigargin-sensitive stores with half-maximal and maximal filling at 6-11 microM and 1 mM ATP respectively. Most of the thapsigargin-sensitive Ca2+ was mobilized by inositol 1,4,5-trisphosphate (IP3), whereas caffeine, ryanodine, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate lacked effects both in beta-cells from ob/ob mice and normal NMRI mice. Mobilization of organelle Ca2+ by 4-chloro-3-methylphenol was attributed to interference with the integrity of the ER rather than to activation of ryanodine receptors. The observations emphasize the importance of IP3 for Ca2+ mobilization in pancreatic beta-cells, but question a role for ryanodine receptor agonists. PMID- 10726212 TI - Safety should be priority during clerkships. PMID- 10726211 TI - Characteristics of collagen-induced Ca2+ mobilization in bovine platelets. AB - We characterized the collagen-induced increase in cytosolic Ca2+ ([Ca2+]i) of bovine platelets loaded with the Ca2+ indicator Fura-PE3/AM. Collagen (10 micrograms/ml)-induced increase in [Ca2+]i was only partially inhibited by aspirin, a cyclooxygenase inhibitor, or adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS, a P2Y1 receptor antagonist), while in human platelets it was almost completely suppressed by aspirin. Collagen-induced increase in [Ca2+]i of bovine platelets was inhibited by U73122 (0.3-5 microM), a phospholipase C inhibitor. Collagen (10 micrograms/ml) increased production of inositol 1,4,5-trisphosphate, which was prevented by pretreatment with U73122 (5 microM). Collagen (10 micrograms/ml) accelerated Mn2+ entry, since the rate of Fura-PE3 quenching by Mn2+ was enhanced by 13-fold following stimulation with collagen. U73122 inhibited the acceleration of Mn2+ entry induced by collagen. PGE1 (2.5 microM) partially inhibited the collagen (50 micrograms/ml)-induced increase in [Ca2+]i in bovine platelets but not in human platelets. The data suggest that collagen induced Ca2+ mobilization in bovine platelets is mediated by phospholipase C. The Ca2+ mobilization in bovine platelets is different from that in human ones as to the dependency on arachidonic acid metabolites and sensitivity to PGE1. PMID- 10726213 TI - Faculty workload assessment proves to be beneficial. PMID- 10726214 TI - Community practice experience developed for third-year residents. PMID- 10726215 TI - Survey garners data on numbers of faculty members. PMID- 10726216 TI - Multicultural curricula in family practice residencies. AB - BACKGROUND AND OBJECTIVES: In 1985, results from a national survey indicated that 25% of family practice residencies taught about multicultural issues in their programs. Our current study identified the current status and content of the curricula and determined facilitating and impeding factors to multicultural curricula. METHODS: In 1998, the Society of Teachers of Family Medicine's Group on Multicultural Health Care and Education conducted a cross-sectional mail survey of all 476 family practice residency programs. RESULTS: With a 59% response rate, 58% of responding programs have an informal curriculum on multicultural issues, 28% have a formal curriculum, and 14% have no curriculum. Programs with a formal curriculum teach more content, employ more educational methods, use more evaluation techniques, and feel more successful than programs with an informal curriculum. Important factors that facilitate curricula include cultural diversity of communities and residents, multicultural interests of faculty and residents, and faculty's multicultural expertise. Factors that impede curricula include lack of time, money, resources, faculty expertise, and cultural diversity in the community. Programs with formal, informal, and no curriculum identify different facilitators and impediments. CONCLUSION: There was a marked increase in the prevalence of multicultural curricula in family practice residencies from 1985 to 1998. PMID- 10726217 TI - Rural residency tracks in family practice: graduate outcomes. AB - BACKGROUND: Because the distribution of physician services in the rural United States continues to be a problem, rural residency tracks in family practice have been developed as a strategy to acculturate residents into a rural practice model. Residents complete the first year of training in an urban-based program and the last 2 years in a rural community. METHODS: We surveyed all 77 graduates of 13 nationally distributed rural training tracks that had graduates between 1988 and 1997. RESULTS: The response rate was 83% (n = 64). Seventy-six percent of respondents practice in a rural community, and 61% practice in federally designated health professional shortage areas. A total of 69% of respondents admitted patients to rural hospitals, 67% provided labor and delivery services, and 48% performed Cesarean sections. Existing physician groups were major influences on practice location. Thirty-nine percent were near their hometown, and 45% were near the community in which they completed residency training. Only 14% had a financial obligation to the community, and 94% reported that their rural training was adequate or better. CONCLUSIONS: Most graduates of rural training tracks have located their practice sites in rural communities, and most graduates provide labor and delivery services. Location decisions were associated with existing physician groups, hospitals, hometowns, and proximity to training sites. PMID- 10726218 TI - Complementary/alternative medicine: comparing the view of medical students with students in other health care professions. AB - OBJECTIVE: We compared the opinions, knowledge, and attitudes of final-year medical, physiotherapy, occupational therapy, nursing, and pharmacy students about complementary/alternative medicine (CAM). METHODS: A cross-sectional study questionnaire (n = 442) was administered on site at the University of Western Ontario and the University of Toronto to fourth-year health professions students. Outcome measures were self-reported knowledge, attitude, and perceived usefulness of CAM therapies, the perceived importance of scientific inquiry for the acceptance of CAM, and educational exposure to the topic. RESULTS: Educational exposure to CAM was correlated with the perceived usefulness of CAM. Medical students reported the least amount of education about CAM and viewed CAM therapies as less useful than did their health professions student peers. Medical students and pharmacy students were more likely than the other health professions students to view traditional scientific forms of evidence as necessary before accepting CAM therapies. CONCLUSIONS: Perceptions differed among the different health professions student groups about the usefulness of CAM therapies and the kind of evidence needed before they should be incorporated into standard care. This may have important implications for multidisciplinary care. PMID- 10726219 TI - Evaluating faculty development outcomes by using curriculum vitae analysis. AB - BACKGROUND AND OBJECTIVES: Many faculty development (FD) programs depend on external private and public sponsors that routinely require systematic studies on FD outcomes. The results of evaluation studies can influence whether or not programs continue to be funded. To better evaluate program outcomes on academic productivity, this paper presents and illustrates an evaluation method that uses the curricula vitae (CVs) of FD program graduates. METHODS: The evaluation method is implemented by first preparing a record-review template of coding categories that is applied to FD graduates' updated CVs. Next, semi-structured interviews are held with subjects to resolve uncertain CV codes. Finally, coded data are entered into standardized forms and analyzed to yield descriptive findings. The method was piloted with two groups of FD graduates (n = 17) to determine its utility and limitations. RESULTS: Results show excellent inter-rater reliability (Cohen's Kappa = .79). There was an overall increase in productivity, measured by the CV, during and after the FD program. CONCLUSIONS: CV analysis can be a useful method for assessing FD program outcomes. Several limitations of the method, such as incomplete CVs and self-report bias, must be considered. PMID- 10726220 TI - How to write a case report. AB - BACKGROUND: Since before Hippocrates, case reports have provided a rich resource for teaching and research in medicine. Case reports are published by many prominent journals--more than 140,000 case reports are indexed in MEDLINE from 1996 to present--and a number of narrative guidelines for the preparation of case reports have appeared in the medical literature. To facilitate the preparation of case reports, we reviewed the existing guidelines and a random sampling of published case reports and created a fill-in-the-blanks worksheet for physicians to use to capture unique scientific observations. Although originally developed to assist family practice residents to write case reports, the case report worksheet can be used by physicians in any practice setting and any discipline to collect and report interesting, unusual, or newsworthy cases. PMID- 10726222 TI - Understanding patients from the former Soviet Union. AB - BACKGROUND: Increasing numbers of immigrants from the former Soviet Union (FSU) are coming to the United States. Educated and resilient, this population has many ethnic origins, but all have shared a common experience under the Soviet system and the deterioration of health care since the collapse of the Soviet Union. An ethnomedical approach was used to review published work and integrate material obtained in interviews with physicians in the FSU. Information is organized into concepts of causality, therapists, and forms of therapy. FSU medicine incorporates many Western treatments but also uses natural and spa remedies. Mainstream FSU physicians make diagnoses and use therapies that are unknown in the West. In addition, the active traditions of folk medicine and magical curing persist. US health care practitioners need to go to extra lengths to understand the perspectives and experiences of these patients and must explain basic concepts of health care in this country that will be new. An open approach to the patients' non-Western beliefs and remedies will support a successful patient practitioner interaction. PMID- 10726221 TI - Changing practices in the use of pneumococcal vaccine. AB - BACKGROUND AND OBJECTIVES: An important issue facing primary care practices is how to best improve preventive services to patients. We sought to determine if an intervention designed by a continuous quality improvement (CQI) process (reminder sticker and patient education sign in each examining room) or a patient education intervention (sign only) could increase the rate of pneumococcal vaccination. METHODS: These two interventions were administered over a 6-month period in a controlled, prospective study design in a family practice residency program clinic. The study targeted patients ages 65 and older and patients ages 2-64 with diabetes mellitus who had never received the pneumococcal vaccine. The main outcome measure was the vaccination rate in the targeted population. RESULTS: A total of 1,647 patient encounters involving 778 patients were documented during the study period. Overall, the reminder and sign module had higher percentages of pneumococcal vaccination in this target population (20% versus 11% for sign only, versus 7% control). Chi-square analysis revealed a statistically significant difference for this group, compared with placebo, but not for the sign-only group. CONCLUSIONS: An intervention designed from a CQI process to impact the office patterns of primary care physicians can produce measurable changes in pneumococcal vaccination rates. PMID- 10726223 TI - Proceedings of an international workshop on polyethylene glycol (PEG) solutions in the treatment of chronic constipation. Rome, Italy. PMID- 10726224 TI - Need of the ideal drug for the treatment of chronic constipation. AB - Bulking agents are not always effective and are not well tolerated by many patients. Non-bulking laxatives are not devoid of side-effects. A satisfactory treatment for functional constipation is not yet available and an ideal drug to efficiently treat these patients is needed. Unlike commonly used laxatives, low doses of polyethylene glycol solutions have few side-effects and appear to be beneficial to the patients. PMID- 10726225 TI - Classification, pharmacology, and side-effects of common laxatives. AB - No definition of the term laxative is satisfactory since their mode of actions affects multiple mechanisms, absorption/secretion and motor activity. Laxatives are very heterogeneous chemically. They act by either holding water inside the bowel lumen (dietary fibre, osmotic laxatives), by inhibition of water absorption or stimulation of secretion (stimulant laxatives), or by stimulation of colonic motility (stimulant laxatives, 5HT4 agonists). Most laxatives have side-effects but these are usually mild. PMID- 10726226 TI - Polyethylene glycol as a non-absorbable prokinetic agent in the lower gastrointestinal tract. AB - In the rabbit isolated colon, intraluminal infusion of polyethylene glycol facilitates peristaltic activity. The primary target of polyethylene glycol appears to be the intrinsic sensory neurons, and not the excitatory motor neurons. It would appear that polyethylene glycol activates the excitatory pathways of the peristaltic reflex releasing tachykinins and acetylcholine at the level of the intrinsic sensory neurons. PMID- 10726227 TI - Mechanisms of action of low doses of polyethylene glycol in the treatment of functional constipation. AB - Different doses of polyethylene glycol have different effects on intestinal function. In normal volunteers, low doses increase stool weight without modifying oro-anal transit time. In constipated patients, low doses decrease stool consistency, increase stool frequency, and facilitate stool evacuation without modifying stool weight and colonic transit time. Low doses of polyethylene glycol may be efficient in the treatment of functional constipation by effecting dilution water in the faeces and, thus, reducing stool consistency. PMID- 10726228 TI - Use of low dose polyethylene glycol solutions in the treatment of functional constipation. AB - A brief review is made of trials which used low doses of polyethylene glycol (13 30 g/day) solutions (125-500 ml/day) in the treatment of chronic functional constipation. Most of these were short-term studies, and confirmed that polyethylene glycol solution increased bowel frequency, improved defaecation and decreased stool consistency. Three studies reported that polyethylene glycol electrolyte solution accelerated transit through the large bowel. One long-term study observed remission of constipation-related symptoms in more than 70% of the polyethylene glycol electrolyte solution treated patients, and the efficacy of the treatment was maintained over a 6-month period, despite progressive reduction of daily dosage. PMID- 10726229 TI - New polyethylene glycol electrolyte solution for the treatment of constipation and faecal impaction. AB - Results from a controlled clinical trial indicate that low doses of polyethylene glycol solutions are more effective and have fewer side-effects than lactulose. Large doses of polyethylene glycol are effective in the treatment of faecal impaction. Resolution of faecal impaction was obtained in all patients, and in 25/30 patients with three administrations in a three-day period. PMID- 10726230 TI - Clinical subgroups of chronic constipation: exploring the potential of polyethylene glycol. AB - This brief review has explored the rationale for therapy of polyethylene glycol electrolyte solutions in subgroups of patients with chronic constipation. There appears to be a good rationale for their use in patients with colonic inertia or those with extrinsic denervation. However, first-line therapies such as dietary fibre and bulking agents should be tried initially prior to resorting to osmotic agents such as polyethylene glycol electrolyte solutions. PMID- 10726231 TI - Use of polyethylene glycol solution in slow transit constipation. AB - Patients with long-standing functional slow-transit constipation were treated with low daily doses of polyethylene glycol solutions. Bowel frequency, stool consistency and colonic transit time improved markedly during the treatment. No relevant side-effects were reported during the study period. PMID- 10726232 TI - Polyethylene glycol solution in subgroups of chronic constipation patients: experience in obstructed defaecation. AB - Low doses of polyethylene glycol solutions were administered in patients with long-standing constipation and in a subgroup with severe symptoms of obstructed defaecation and in whom anorectal surgery was considered. During treatment all patients with constipation improved and nineteen of the twenty-one patients with dyschezia became asymptomatic. Only two patients were submitted to surgery. PMID- 10726233 TI - Use of polyethylene glycol solution in functional and organic constipation in children. AB - A brief review is made of the use of high dose polyethylene glycol solution in the treatment of functional constipation and encopresis in children. Experience with low dose polyethylene glycol for the treatment of neurogenic constipation in children with severe brain damage is also reported. Treatment with polyethylene glycol caused a significant increase in bowel frequency and a decrease in gastrointestinal transit time. Side-effects, consisting of nausea, vomiting and irritability, have limited the use of this treatment in a few children. PMID- 10726234 TI - Zolpidem, a valuable alternative to benzodiazepine hypnotics for chronic insomnia? AB - Sleep quality and anxiety levels were examined using questionnaires and polysomnographic recordings in 22 chronic insomnia patients who regularly used benzodiazepines to treat their sleeping problems. After abruptly discontinuing their benzodiazepine medication, patients were randomly allocated to receive either a placebo or zolpidem 10 mg for 1 week, after which they entered an open extension phase, receiving zolpidem 10 mg for 3 weeks. Subjectively, sleep quality was considered mediocre during the use of a benzodiazepine hypnotic. One week after the discontinuation, an increase in sleep latency was observed in the placebo group, whereas zolpidem induced a significant decrease in sleep latency. Deterioration of other sleep variables (probably rebound) was not suppressed by zolpidem. An explanation for this could be the selective pharmacological profile of zolpidem. Polysomnographic differences between placebo and benzodiazepine and between placebo and zolpidem were not reflected by the subjective data on sleep and anxiety. Changes of sleep structure caused by hypnotics seem not always to be felt as such by patients. After 3-4 weeks of zolpidem treatment, the percentage of non-rapid eye movement-4 sleep increased significantly, corresponding with a significant subjective improvement of sleep quality. This indicates that zolpidem may restore physiological sleep. PMID- 10726235 TI - Comparative study of the efficacy and safety of trazodone versus clorazepate in the treatment of adjustment disorders in cancer patients: a pilot study. AB - The efficacy of trazodone (mean once-daily dose 111.5 +/- 36.3 mg) versus clorazepate (mean once-daily dose 17.5 +/- 7.5 mg) to relieve anxious and depressive symptoms in 18 patients undergoing treatment for breast cancer was investigated in a 28-day randomized, double-blind study. Efficacy was evaluated using the Hospital Anxiety and Depression Scale, the Revised Symptom Checklist and the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire. A successful response to treatment was achieved in 91% (10/11) of patients who received trazodone and 57% (four of seven) of patients who were administered clorazepate (P = 0.1373). Bayesian analysis revealed that the prior probability of making a wrong decision in prescribing trazodone rather than clorazepate reduced from 26% to 8%. Assessment of the clinical scales suggested a benefit of trazodone compared with clorazepate, although the differences were not significant. Safety of both treatments was similar. Trazodone is devoid of an abuse risk and dependence and, therefore, could be a valuable alternative to clorazepate in the treatment of adjustment disorders in cancer patients. PMID- 10726236 TI - Mizolastine is effective and well tolerated in long-term treatment of perennial allergic rhinoconjunctivitis. Riperex Study Group. AB - The aim of this study was to determine the long-term efficacy and safety of mizolastine, a new second-generation antihistamine with European approval, in the treatment of perennial allergic rhinoconjunctivitis. In this study, 141 patients were treated with once-daily mizolastine 10 mg or 15 mg in a 5-month open-label extension of a 1-month double-blind, placebo-controlled trial, which assessed once-daily mizolastine 10 mg. Mizolastine significantly reduced the nasal subscore (sneezing, rhinorrhoea, itch; end-baseline +/- SD, -2.5 +/- 6.3), nasal obstruction (-1.2 +/- 2.6) and rhinoscopy scores (-1.3 +/- 2.6), and improved ocular and total nasal scores after 6 months' treatment. Improvement was maintained for the duration of the study with no loss of drug efficacy. Adverse effects were mild with no specific effects associated with prolonged use. These results clearly demonstrate that mizolastine is effective and well tolerated in the long-term treatment of perennial allergic rhinoconjunctivitis. The significant clinical improvement in nasal blockade may reflect mizolastine's histamine/5-lipoxygenase dual inhibition. PMID- 10726237 TI - Evaluation of growth in children using quantitative bone scintigraphy. AB - Our aim was to assess the value of quantitative bone scintigraphy for evaluating long-bone growth and to establish the normal uptake patterns of the growth plate in children. Subjects (180 girls, 154 boys) of normal weight and height, aged 2 20 years, were investigated using skeletal scintigraphy. Regions of interest were outlined over posterior images of the distal femoral growth plate and femoral diaphysis. The average number of counts per fixel in each region of interest was determined. The ratio of uptake in the distal femoral growth plate to that in the femoral diaphysis (GP:D ratio) was calculated for all subjects, and its relationship with age was determined. Peak GP:D ratios in girls and boys were reached at ages 11 and 13 years, respectively (8.26 in girls and 8.18 in boys), corresponding to the periods of most rapid growth. Our findings suggest that determining the scintigraphic GP:D ratio may be useful for evaluating growth and development in children if normal standard GP:D ratios are established. PMID- 10726238 TI - The effect of hyaluronic acid-carboxymethylcellulose in reducing adhesion reformation in rabbits. AB - The effect of hyaluronic acid-carboxymethylcellulose film (Seprafilm) in reducing postoperative adhesion formation was examined in a rabbit induced-adhesion model. During laparotomy, the ileocaecal region was mechanically and chemically abraded to induce lesions. After a 28-day recovery period, adhesions were lysed by microsurgery and Seprafilm was applied to the lysed lesion in 10 rabbits and six rabbits received physiological saline. A third laparotomy was performed 10-14 days later and the area of adhesion reformation was compared to that found prior to application of test materials. The area of adhesion reformation with Seprafilm decreased to (mean +/- standard deviation) 11.71 +/- 10.97% of the originally lysed lesion and, the area was significantly reduced compared with controls treated with physiological saline. These results suggest that the use of Seprafilm may be a valuable new anti-adhesion material for abdominal or pelvic surgery and may be superior to existing anti-adhesion materials and techniques. PMID- 10726239 TI - The efficacy, safety and pharmacokinetics of intravenous ciprofloxacin in patients with lower respiratory tract infections. AB - The efficacy and safety of intravenous ciprofloxacin 200 mg every 8 or 12 h and 300 mg every 12 h in treatment lasting 3-14 days were investigated in patients with lower respiratory tract bacterial infections. Patients presented with pneumonia, bronchiectasis with infection, previous pulmonary tuberculosis with infection and diffuse panbronchiolitis. Clinical efficacy was seen in six of eight patients, with apparent recovery in terms of chest radiographs, fever reduction and laboratory findings. Pharmacokinetic analysis in one patient treated with intravenous ciprofloxacin 300 mg showed that at 0.5 h after the first dose, ciprofloxacin serum and sputum concentrations were equivalent (2.45 micrograms/ml and 2.25 micrograms/ml, respectively). Adverse events were recorded in only two patients and involved a slight elevation in liver function tests and eosinophilia. This study indicates that intravenous ciprofloxacin is useful in the treatment of lower respiratory tract infections. PMID- 10726240 TI - Catamenial epilepsy: in search of a clinical entity and its prevalence. AB - The prevalence of catamenial epilepsy has been difficult to determine for several reasons, including menstrual cycle variability and randomness of seizure occurrence, a high prevalence of seizure clustering in males and nonmenstruating females with epilepsy, and the lack of a definition of catamenial epilepsy in previous studies. Recently, a definition describing three distinct patterns that may provide a sensible nomenclature for future research on catamenial epilepsy has been proposed, and it may provide standardized criteria for inclusion in catamenial epilepsy studies. Such a definition should allow estimates of true prevalence of this disorder to be more precise and prospective treatment trials to be designed. PMID- 10726241 TI - Multiple sclerosis pathways: an innovative nursing role in disease management. AB - Multiple sclerosis (MS), a chronic disease of the central nervous system, is characterized by a variable and unpredictable course. The most common pattern of the disease is the relapsing-remitting form in which clearly defined relapses (also called exacerbations) are followed by complete or incomplete recovery. Interferon beta-1b (Betaseron), a drug that affects the natural course of the disease, was developed for the treatment of relapsing-remitting MS. Multiple Sclerosis Pathways (MSP), a disease management program, was developed to provide comprehensive and personal support to MS patients taking interferon beta-1b and to serve as an information resource for all people with MS, their families, and healthcare professionals. The MSP program includes personal patient assistance, reimbursement services, a 24-hour nurse hotline, training program, educational resources, and injection supplies. The nurse hotline counselor (NHC) utilizes the nursing process in a unique telephone nursing practice in this program. The positive impact of education and support on adherence to therapy has been validated by training and nurse hotline data. PMID- 10726242 TI - A qualitative study of life after stroke. AB - The purpose of this qualitative, descriptive exploratory study was to enhance understanding about quality of life after a stroke from the patient's own perspective. The guiding theoretical perspective was Parse's human becoming theory. Loosely structured interviews aimed at eliciting descriptions of quality of life were scheduled during the acute care stay and at one and three months after stroke onset. A total of 32 interviews were conducted with 13 participants, including 9 men and 4 women, aged 40-91 years. Through a process of analysis synthesis, four themes representing participants' descriptions were created: (1) suffering emerges amid unaccustomed restrictions and losses, (2) hopes for endurance mingle with dreams of new possibilities, (3) appreciation of the ordinary shifts perspectives, and (4) consoling relationships uplift the self. When interpreted in light of the human becoming theory, the themes expand understanding of what it is like to live with a stroke and provide insights that may enhance the quality of care. PMID- 10726243 TI - The needs of the family of critically ill neurosurgical patients: a comparison of nurses' and family members' perceptions. AB - In this study, researchers identified the important needs of family members of critically ill neurosurgical patients and explored the relationship between needs and unmet needs as perceived by nurses and family members. A total of 52 family members and 36 nurses in three neurosurgical special care units in Hong Kong were asked to complete the Chinese version of the 45-item Critical Care Family Needs Inventory. The rank order of most important needs reported by family members indicates that the majority of needs are related to assurance; needs for support and comfort were much less important. When rating needs, nurses underrated most of the needs considered important by family members. Needs for proximity were also underrated in importance by nurses when compared to family ratings, and needs for support were heavily overrated by nurses. The needs for proximity were least met. An inverse relationship between nurses' ratings of importance and the frequency of unmet needs was demonstrated. The most important need that was also largely unmet was having a specific person call when unable to visit. The findings of this study indicate areas of unmet need that require additional nursing interventions. PMID- 10726244 TI - Recruiting and retaining research participants for a clinical intervention study. AB - This article describes strategies investigators can use to recruit and retain research participants in a clinical intervention study. Although specific issues and examples focus on family caregivers of stroke survivors, many strategies discussed here are applicable to other populations. PMID- 10726245 TI - Building a mission for quality care. AB - Clearly, there is a benefit to the group process in helping to establish teamwork. Teamwork and cooperation can assist with promoting effective communication, improving work quality, and building a sense of well-being within the group. With this cooperation, setting goals and looking toward the future can become a reality. Once goals are set, then developing a professional image can begin. Developing a mission statement can be an effective means to help create that professional image. Having the opportunity to develop a mission for a patient care area and articulating it through a mission statement coalesces the values, beliefs, and philosophy of a group of neuroscience staff. The following is the mission statement developed by the neurosciences unit at the University of Wisconsin Hospital and Clinics: We, the staff of UWHC Neuroscience unit embrace a vision of excellence in health care for all. Our mission is to deliver consistent quality patient care, while fostering our own professional growth. As caring healers, teachers and patient advocates in an ever-changing health care environment, we are empowered by the code for nurses. Within our scope of practice, we strive to maintain a balance of basic human respect and dignity for patients and their families in their quest for wellness, adaptation, rehabilitation or comfort care. It is our hope that patients and families will work with the health care team to construct a plan of care that best meets the patient's needs and goals. We are committed to accommodate special communication, religious or cultural needs of patients and their families. Our final acknowledgment is to ourselves, as members of the health care team. We celebrate the dignity of the staff by recognizing each individual as a special person capable of making unique and significant contributions to the unit. PMID- 10726246 TI - Health-related quality of life in clinical practice. AB - This month's question addressed something that many of us perhaps still have not formally incorporated into clinical practice, although we all are interested in our patients' health-related quality of life and want our inventions to result in improvements within this area. This view is exemplified by the response from Australia, which is one of several similar, unpublished, replies from the International Panel on this month's question (others came from Germany and Sweden). As mentioned in the introduction, health-related quality of life is becoming increasingly important as an outcome measure in clinical trials of new therapeutic interventions and several new measures have been and are developed. It is interesting and encouraging to hear about the new developments within this area that are being made by neuroscience nurses in different countries and within different subspecialties around the globe. As reported from the International Panel in here, new HRQL measures are currently developed in Canada and the UK focusing on patients with brains injuries and Huntington's disease (HD), respectively. In contrast to most established measures, the Canadian study has primarily been aimed at the positive aspects of life and not merely absence of the negative ones. Steve Smith in the UK has recently started developing a scale for use in clinical management of patients with HD. As far as I have been able to determine, this is the first measure of this kind to be developed for HD. Anyone who would like to know more about Steve's work or take part thereof is encouraged to contact him at this address above. Despite the fact that there already are several HRQL measures available, there is still a need for new instruments reflecting new aspects of health and disease. In addition, tools need to be designed for challenging conditions not readily addressed by existing measures. The replies from Canada and the UK represent these needs. It will be very interesting to eventually take part of the results from these colleagues' work. Finally, the terms HRQL and quality of life are often used interchangeable, indicating that focus also is needed on definition and conceptual construct within this field. There is still a great deal of controversy about what quality of life is and, as a result, how it is best measured and by whom. PMID- 10726247 TI - Duplex scan aids in determining need for carotid endarterectomy. PMID- 10726248 TI - Acute pain management protocol. PMID- 10726249 TI - Mild hypothermia for severely head-injured patients with low intracranial pressure (ICP). PMID- 10726250 TI - [Combination systems for SPECT, coincidence, PET and CT. Technical spectrum, operating assumptions and possible areas of application]. PMID- 10726251 TI - [Is the post-therapeutic dosimetry of patients with short-term hospitalization after 131I therapy sufficiently reliable?]. AB - AIM: According to the new recommendations of the Federal German Radiation Protection Committee (SSK) for patient discharge, that were published in April 1997, patients can be discharged after radioiodine therapy with a radiation exposure of less than 1 mSv per year in 2 m distance. The aim of this study was to evaluate whether the measurement of the achieved dose was different 48 hours after application of I-131 and after an interval of one week. The study was planned in order to ensure quality management in the follow-up of the patients. METHOD: In a prospective study 115 patients were followed, that were treated for a benign thyroid disease. The patients were discharged 48 hours after the intake of I-131 after falling short of the exposure limit. All patients were measured at discharge and about 8 days later with the same uptake facility. RESULTS: Because of similar results focal and disseminated forms of benign thyroid diseases could be analysed together. The calculated doses 8 days after the discharge were higher than the values at the time of discharge. The doses were underestimated about 4% with an standard error of 15%. CONCLUSION: The error in early dosimetric measurements is small in comparison to the overall uncertainty in therapy and uptake dosimetry. A valid dosimetry and sufficient quality management can be guaranteed even for an early discharge. PMID- 10726252 TI - [Clinical implications of a new TSH receptor antibody assay (DYNOtest TRAKhuman) in autoimmune thyroid diseases]. AB - AIM: Conventional radioreceptor-antibody-assays (RAAs) fail in the detection of TSH-receptor antibodies (TRAKs) in 10-30% of patients with Graves' disease (GD). The aim of this study was the evaluation of the diagnostic and clinical impact of a new RRA (DYNOtest TRAKhuman) which uses the human recombinant TSH-Receptor in the diagnosis of autoimmune thyroid disease. METHODS: Sera from 142 consecutive patients (GD: n = 50, autoimmune thyroiditis/AIT: n = 92) and from 55 controls (31 patients without any thyroid disease and 14 with euthyroid goiter) were evaluated both with the DYNOtest TRAKhuman-assay and a conventional RRA (TRAK Assay). Thyroid in vitro parameters and thyroid sonography were performed in all patients. RESULTS: The DYNOtest TRAK-assay was significantly superior to the conventional RRA in the diagnosis of GD (p < 0.00012), especially in those who were treated by thionamides (p < 0.003) and in the diagnosis of TRAK-positive patients with AIT (p < 0.003). The majority of TRAK-positive AIT-patients suffered from hypothyroidism. One false positive result in patients with euthyroid goiter was found in the TRAK-Assay as well as in the DYNOtest TRAKhuman Assay. Therefore the specificity of the DYNOtest TRAKhuman was not inferior compared with the conventional assay. CONCLUSION: The DYNOtest TRAK-assay is superior in the diagnostic work up of Graves' disease compared with a conventional TRAK-assay and offers an equal specificity. PMID- 10726253 TI - [Relationship between thyroglobulin and reliability of thallium 201 scintigraphy in differentiated thyroid cancer]. AB - AIM: Tumor scintigraphy with 201-TlCl is an established diagnostic method in the follow-up of differentiated thyroid cancer. We investigated the relationship between thyroglobulin (Tg) level and tumor detectability. SUBJECT AND METHODS: We analyzed the scans of 122 patients (66 patients with proven tumor). The patient population was divided into groups with Tg above (N = 33) and below (N = 33) 5 ng/ml under TSH suppression or above (N = 33) and below (N = 33) 50 ng/ml under TSH stimulation. Tumor detectability was compared by ROC-analysis (True-Positive Fraction test, specificity 90%). RESULTS: There was no significant difference (sensitivity 75% versus 64%; p = 0.55) for patients above and below 5 ng/ml under TSH suppression and a just significant difference (sensitivity 80% versus 58%; p = 0.04) for patients above and below 50 ng/ml under TSH stimulation. In 18 patients from our sample with tumor, Tg under TSH suppression was negative, but 201-TlCl-scan was able to detect tumor in 12 patients. CONCLUSION: Our results demonstrate only a moderate dependence of tumor detectability on Tg level, probably without significant clinical relevance. Even in patients with slight Tg elevation 201-TlCl scintigraphy is justified. PMID- 10726254 TI - [Incidence of familial non-medullary thyroid carcinoma in the patient register of the Clinic and Polyclinic of Nuclear Medicine, University of Wurzburg]. AB - AIM: In this study the incidence rate of familial non-medullary thyroid carcinoma was investigated in the first and second grade relatives of patients registered at the Clinic and Polyclinic for Nuclear Medicine, University of Wurzburg. PATIENTS AND METHODS: In this study 596 patients with differentiated thyroid carcinoma were enclosed, who were treated between 01.01.81 and 31.12.95. The data concerning a familial occurrence were studied by a retrospective survey-based analysis. These data were compared to a literature analysis for familial non medullary thyroid carcinoma. RESULTS: 14 patients of the 596 patients treated showed a familial occurrence (2.3%). All these patients suffered from papillary thyroid carcinoma. According to the prognostic factors (tumor state, lymph node involvement, metastatic disease) no differences could be evaluated in the different groups (sporadic versus familial non-medullary thyroid disease). CONCLUSION: A familial occurrence of differentiated thyroid carcinomas is not frequently observed, but should be considered due to further genetic diseases. PMID- 10726255 TI - Comparison of HIG scintigraphy and bloodpool scintigraphy using HDP in arthritic joint disease. AB - AIM: Because of a similar tracer accumulation, we assumed to get the same information about synovitis in arthritic joint disease with HIG scintigraphy and bloodpool scintigraphy using HDP. Therefore, we compared retrospectively 23 patients. METHODS: In HIG scintigraphy, synovitis was diagnosed according to increasing activity from early to late image. In bloodpool scintigraphy according to an increased activity in comparison to the surrounding tissues. RESULTS: In 694 joints comparison of both scintigraphic modalities was possible, resulting in a 2 x 2 kappa coefficient of 0.93 or 0.97 by using late-phase bone scintigraphy as an anatomical marker. For intra- and interobserver agreement, 2 x 2 kappa coefficients of 0.93 and 0.88 in HIG scintigraphy, respectively 0.96 and 0.90 in blood-pool scintigraphy were calculated. CONCLUSION: This study shows an excellent agreement in the visualization of synovitis by HIG and bloodpool scintigraphy. Because of its higher objectivity and lower cost, investigation of synovitis should be performed by bloodpool scintigraphy. PMID- 10726256 TI - [Metastasizing follicular thyroid carcinoma with intracranial iodine 131 uptake in brain edema due to a frontal meningioma]. PMID- 10726257 TI - I-131 whole body scintigraphy in a patient with micronodular pulmonary metastases from papillary thyroid carcinoma. PMID- 10726258 TI - [Diagnostic imaging of a TSH-producing pituitary adenoma associated with the "empty sella" by somatostatin and dopamine D2 receptor scintigraphy]. PMID- 10726259 TI - Bilateral renal metastasis in follicular thyroid carcinoma. PMID- 10726260 TI - Apparent tolerance of Plasmodium falciparum in infants in a highly endemic area. AB - The incidence of fever among infants in the village of Idete in the Morogoro region of Tanzania was analyzed in relation to densities of Plasmodium falciparum parasites in the peripheral blood. Parasite densities in both fever cases and in asymptomatic infants, were compared and a Bayesian non-parametric mixture decomposition algorithm was used to estimate the proportion of fevers attributable to malaria and hence the incidence of clinical malaria. Age group specific densities of peripheral parasitaemia showed little seasonality, but the clinical malaria incidence showed a clear peak in the wet season in children aged less than 9 months. Estimates of the parasitaemia-specific incidence of clinical malaria were used to quantify apparent tolerance of parasites, and indicated that clinical episodes occurred on average at lower parasite densities during the wet season than in the dry season. These patterns could reflect differences in levels of anti-toxic immunity, but the nature of the seasonal differences supports the alternative explanation that the variation in apparent tolerance may be an effect of changes in the ratio of peripheral parasite densities to the sequestered mass. PMID- 10726261 TI - Monkeys of the rainforest in French Guiana are natural reservoirs for P. brasilianum/P. malariae malaria. AB - Monkey blood samples were collected from 214 monkeys relocated as part of the wildlife rescue organized in French Guiana during the filling of the Petit Saut Dam on the Sinnamary River. These samples were tested for malaria parasites by microscopy of thick blood filsm and by nested PCR for small subunit rRNA genes (SSUrRNA). Parasitic blood forms similar to Plasmodium brasilianum were detected in 4 monkey species: Alouatta seniculus macconnelli, Saguinus midas midas, Pithecia pithecia and Ateles paniscus paniscus, with the highest prevalence in Alouatta monkeys. PCR was more sensitive than the conventional method for detecting low-grade parasitaemia in positive monkeys. The examination of blood films indicated that 5.6% of the animals carried parasites whereas the nested PCR for ribosomal DNA indicated a prevalence of 11.3%. The P. brasilianum SSUrRNA gene sequence was analysed and aligned with those from P. malariae, P. falciparum and P. vivax. This suggested that P. brasilianum and P. malariae are very closely related. Similar results were obtained from analysis of the sequences in P. malariae and P. brasilianum isolates of a polymorphic gene fragment analogous to the merozoite surface protein-1 (MSP-1) gene of P. falciparum. The P. brasilianum/P. malariae sequences were more similar to those of P. vivax than to those of P. falciparum, at least in the gene region examined. The high degree of DNA homology in the sequences of the SSUrRNA and msp1-like genes is consistent with other characterizations demonstrating a taxonomic relationship between P. brasilianum and P. malariae species. Our results provide further evidence that P. brasilianum and P. malariae are virtually identical and should probably be considered to be a single malaria species. This raises the question as to whether monkeys living in the rainforest are natural reservoirs for both simian and human malaria. These results have implications for the interpretation of the current epidemiological situation in French Guiana. PMID- 10726262 TI - Clustering of Brugia malayi infection in a community in South-Sulawesi, Indonesia. AB - The present study was undertaken in village Karondang in South-Sulawesi, Indonesia, to investigate the influences of genetic, household and environmental factors on Brugia malayi infection. Infection status was determined by measuring both microfilariae in night blood and anti-filarial IgG4, as a marker for detection of active filarial infection. A total of 171 residents participated in the study; familial relationships between subjects were registered to construct pedigrees and distances between households were measured. The data were analysed using a test statistic for familial aggregation. For distribution of microfilariae over the study population a genetic influence on infection susceptibility was favoured over the household and environmental effects. For anti-filarial IgG4, all 3 clustering models gave significant results, suggesting that genetic, household and/or environmental factors influence specific IgG4 antibodies. PMID- 10726263 TI - Absence of lipophosphoglycan-like glycoconjugates in Entamoeba dispar. AB - Invasive amoebiasis is the result of infection of Entamoeba histolytica. The closely related Entamoeba dispar can colonize the human gut but does not cause invasive disease. In this study, E. dispar was analysed for the presence of the lipophosphoglycan-like (LPG) glycoconjugate known to be present on the cell surface of E. histolytica. E. dispar cells were radio-isotope labelled with [3H]galactose or [3H]inositol. The acidic glycoconjugates were extracted and analysed by hydrophobic chromatography over phenyl-Sepharose and by sodium dodecyl sulphate polyacrylamide gel electrophoresis. No LPG-like molecules could be identified in E. dispar in contrast to E. histolytica, suggesting that these molecules may be absent in the non-pathogenic species. PMID- 10726264 TI - Exposure, infection and immune responses to Schistosoma haematobium in young children. AB - Behavioural, parasitological and immunological data were obtained from 48 children up to 6 years old, resident in a Schistosoma haematobium endemic area in Zimbabwe. The children averaged more than 1 contact with infective water bodies every 3 days and all showed immunological evidence of exposure (an anti-cercarial and/or anti-egg antibody response). IgM was the dominant isotype and appeared in the youngest children, followed by IgA, IgE and IgG3. However, only 38 children showed evidence of infection (an anti-egg response or eggs in urine) and only 14 were excreting eggs. The best estimates from these data are that less than 1 in 100 contacts results in infection and less than 1 in 1000 result in egg output. This suggests that there may be substantial attrition of invading cercaria even in naive individuals. PMID- 10726265 TI - Muscles or testes? Comparative evidence for sexual competition among dioecious blood parasites (Schistosomatidae) of vertebrates. AB - Schistosomes, which live as parasitic adults in the blood vessels of mammals, birds and crocodiles, are unique among trematodes because they have separate sexes. Several studies have shown that the sex ratio is often biased towards males. Sexual dimorphism can be very important in several genera (Schistosoma, Heterobilharzia, Schistosomatium), as larger males grip smaller females in a gynecophoric canal. But not all schistomatid species have developed a gynecophoric canal. It is also striking that the number of testes varies widely, from a small number of testes reported (2-7) to a very high number (> 500). We tested the hypothesis of trade-off investment by males: muscle (as estimated by the length of the gynecophoric canal) in order to sequester one or a few females, or testes in order to inseminate numerous females. We used recent comparative methods, which need phylogenetic information of the analysed species. We employed a morphological cladistic analysis of the Schistosomatidae and a construction of a phylogenetic supertree of Schistosoma based on available molecular information. Our phylogenetic analysis supports previous hypotheses on the origin and diversification of the schistosomes. The family seems to have originated as parasites of crocodiles (or possibly other related poikilotherms) and to have secondarily evolved within the Aves and then mammals. Our phylogenetic analyses show that (1) the possession of a gynecophoric canal is a plesiomorphic character, and several reductions of this character have occurred during the diversification of the Schistosomatidae; (2) a small number of testes is a plesiomorphic stage, and an increase of testes number has occurred several times. Finally, our comparative analysis suggests that there is a trade-off between investment in muscle or in number of testes (other variables being controlled for). Male competition for access to females seems to follow two ways: investment in testes in order to fertilize a high number of females or investment in muscle in order to hold and sequester one (Schistosoma) or a few females (Heterobilharzia). We show the existence of the trade-off between muscles and testes in connection with a male biased sex ratio. The development of dioecy cannot explain the trade-off between muscles and testes, which instead appears to be a result of sexual selection possibly driven by the male biased sex ratio. PMID- 10726266 TI - Increased susceptibility of salmonids to the monogenean Gyrodactylus salaris following administration of hydrocortisone acetate. AB - Gyrodactylus salaris infects numerous salmonid species, ranging from the fully susceptible (Norwegian strains of Salmo salar), through species which, though initially susceptible, eventually eliminate their infections (Salvelinus alpinus and S. fontinalis) to entirely resistant (Salmo trutta) species. Here we describe experiments in which Salvelinus alpinus, S. fontinalis and Salmo trutta, implanted with hydrocortisone acetate to simulate stress-induced immunosuppression, were challenged with G. salaris. With previously uninfected Salvelinus fontinalis, G. salaris infections on fish treated with hydrocortisone acetate grew larger, and for longer, than on sham-treated controls. A similar result was obtained with S. trutta. Patterns of infection on Arctic charr, Salvelinus alpinus, were more complex, because individual fish varied from susceptible to highly resistant. Fish were therefore initially infected with G. salaris, and the most highly resistant group of individuals identified and disinfected. After 6 months recovery from this primary infection, hydrocortisone acetate was administered to half the fish, and all were challenged with G. salaris. Parasite populations on the hydrocortisone-treated individuals were consistently larger than those on the sham-treated controls, exceeding 30 parasites per fish after 5 weeks, in comparison with less than 10 parasites per fish on controls. These results indicate that hydrocortisone administration can lead to enhanced gyrodactylid populations on a range of salmonids. This suggests that the response to G. salaris is mediated by the immune system, and that the spectrum of responses observed in different species are, at least in part, due to the same mechanism. At a practical level, stress-induced immunosuppression during handling and transport of cultured salmonids may prove an important factor in the dissemination of G. salaris between watersheds. PMID- 10726267 TI - Sexual competition in an acanthocephalan parasite of fish. AB - Acanthocephalans are polygamous parasites of vertebrates and some species are known to aggregate in sexual congress to mate. Such a reproductive behaviour could lead to male-male competition for access to females and could have consequences for sexual selection. We dissected 87 gobiid fish, Gobius bucchichii, harbouring 891 acanthocephalans, Acanthocephaloides propinquus. The parasites were sexed and their body sizes were measured. Testicular volume was also evaluated in 82 males in order to establish their phenotypic sexual investment in relation to the estimated sex ratio. We found that parasite intensity (i.e. the number of individuals/fish) was not correlated with fish size, but that parasite size was significantly related to host size. Our results showed that there was a significant relationship between the mean female body size and their number within one host. We found that when the percentage of male parasites in a host increased, presumably increasing male-male competition for access to females, males had a larger testicular volume. We discuss these results in terms of energy allocation, sexual and sperm competition. We conclude that competition for space should be less important for males than competition for access to females. Moreover, increasing testis size should confer advantages to males especially for their reproductive success when sperm competition occurs. PMID- 10726268 TI - The neuropeptide F (NPF) encoding gene from the cestode, Moniezia expansa. AB - Neuropeptide F (NPF) is an abundantly expressed neuropeptide in platyhelminth nervous systems, and exhibits a moderate, myogenic effect on muscle preparations of parasitic flatworms. NPF displays structural similarities to peptides from molluscs and vertebrate members of the neuropeptide Y (NPY)-superfamily of peptides. NPY is one of the most abundant and highly conserved neuropeptides within vertebrates and similarities between the gene organization of NPY, pancreatic polypeptide (PP) and peptide tyrosine tyrosine (PYY), suggest a common evolutionary origin of this peptide family. Dual localization analyses coupled with confocal scanning laser microscopy revealed a close spatial relationship between NPF-containing nerves and muscle fibres in M. expansa. Molecular cloning techniques identified the M. expansa NPF (mxNPF) precursor and characterized the isolated transcript which encodes an open reading frame of 57 amino acids. The transcript possesses a 17 amino acid signal peptide and the mature NPF sequence (39 amino acids) followed by a carboxyterminal glycyl extension. Sequence analysis of genomic DNA identified a product which possessed a 54 base pair intron with consensus sequences for 5' and 3' splice sites. The M. expansa npf gene possesses a phase 2 intron within the penultimate arginyl residue, a characteristic feature of NPY superfamily peptide-genes. The intron-exon organization of the npf gene, coupled with the abundant expression of NPF within the nervous systems of flatworms, suggests an early evolutionary origin for this peptide transmitter family within the nervous systems of basal bilaterian metazoans. PMID- 10726269 TI - Nematode neuropeptide modulation of the vagina vera of Ascaris suum: in vitro effects of PF1, PF2, PF4, AF3 and AF4. AB - Ascaris suum possesses a large number of FMRFamide-related peptides (FaRPs) of which KNEFIRFamide (AF1), KHEYLRFamide (AF2) and KSAYMRFamide (AF8/PF3) have been shown to modulate the intrinsic, rhythmic activity of the vagina vera of A. suum in vitro. In the present study, the effects of the nematode FaRPs, SDPNFLRFamide (PF1), SADPNFLREamide (PF2) and KPNFIRFamide (PF4) (from Panagrellus redivivus) and AVPGVLRFamide (AF3) and GDVPGVLRFamide (AF4) (from A. suum) on the in vitro activity of the vagina vera were examined. The effects of each of the peptides were qualitatively and quantitatively distinct. All 3 FaRPs from P. redivivus were inhibitory, causing a cessation of contractions. PF2 was 3 times more potent than PF1, with a threshold of 1 nM. Although PF4 was the least potent (threshold, 10 nM), its effects at > or = 10 nM were quantitatively the greatest. Both AF3 and AF4 (1 microM) induced complex, multiphasic responses consisting of an initial contraction and spastic paralysis followed by a return of contractile activity of increased amplitude. AF3 was 3 times more potent than AF4. The effects of these peptides had some similarities to those observed on A. suum somatic body wall muscle in vitro, with PF1, PF2 and PF4 being inhibitory and AF3 and AF4 being excitatory. PMID- 10726270 TI - A radiometric analysis of nitric oxide synthase activity in Hymenolepis diminuta. AB - The free radical nitric oxide (NO) is a neuronal messenger which is synthesized from L-arginine and O2 by nitric oxide synthase (NOS). In the synthesis NO and L citrulline are produced in a stoichiometric 1:1 relation. The activity of NOS was analysed in homogenates of the rat tapeworm Hymenolepis diminuta by measuring the formation of L-[3H]citrulline after incubation with L-[3H]arginine. The nature of NOS in H. diminuta was determined by studying the effect of 3 types of NOS inhibitors: (1) L-NAME, (2) EGTA, (3) 7-nitro-indazole. All inhibitors caused a significant but not complete reduction in the formation of L-[3H]citrulline. The results are discussed against the background of nerve cells and fibres positive for NADPH-diaphorase staining in H. diminuta. PMID- 10726271 TI - The role of lambs in louping-ill virus amplification. AB - In some areas of Scotland, the prevalence of louping-ill virus has not decreased despite the vaccination of replacement ewes for over 30 years. The role of unvaccinated lambs in viral persistence was examined through a combination of an empirical study of infection rates of lambs and mathematical modelling. Serological sampling revealed that most lambs were protected by colostral immunity at turnout in May/June but were fully susceptible by the end of September. Between 8 and 83% of lambs were infected over the first season, with seroconversion rates greater in late rather than early summer. The proportion of lambs that could have amplified the louping-ill virus was low, however, because high initial titres of colostral antibody on farms with a high force of infection gave protection for several months. A simple mathematical model suggested that the relationship between the force of infection and the percentage of lambs that became viraemic was not linear and that the maximum percentage of viraemic lambs occurred at moderately high infection rates. Examination of the conditions required for louping-ill persistence suggested that the virus could theoretically persist in a sheep flock with over 370 lambs, if the grazing season was longer than 130 days. In practice, however, lamb viraemia is not a general explanation for louping-ill virus persistence as these conditions are not met in most management systems and because the widespread use of acaracides in most tick affected hill farming systems reduces the number of ticks feeding successfully. PMID- 10726272 TI - Population dynamics of Plasmodium falciparum in an unstable malaria area of eastern Sudan. AB - The Plasmodium falciparum population in Asar village, eastern Sudan, where malaria transmission is markedly seasonal, was monitored monthly over a period of 15 months. A cohort of infected patients was treated and then followed monthly throughout the dry season until the next transmission season. Parasitaemia detected by microscopy among the cohort reduced dramatically following treatment, but remained sporadic during the dry season, and reappeared following the onset of the next wet season. However between 40 and 50% of the cohort retained a persisting parasitaemia detectable by PCR throughout the dry season. These parasites were genetically complex, consisting of multiple clones with a large repertoire of alleles of the studied genes. While the number of clones per host dropped significantly following treatment of acute cases during the transmission season, drug treated people nevertheless maintained an average of one clone throughout the dry season. Allele frequencies of MSP-1, MSP-2 and GLURP showed slight, statistically insignificant, fluctuations between the dry and wet seasons. A higher frequency of inbreeding was estimated among the parasites that survived the dry season compared to the wet season. PMID- 10726273 TI - RFLP analysis of PCR-amplified small subunit ribosomal DNA of three fish microsporidian species. AB - The phylogenetic relationships of the microsporidian species Microgemma caulleryi, Pleistophora finisterrensis and Tetramicra brevifilum were investigated on the basis of restriction fragment length polymorphism (RFLP) analysis of PCR-amplified small-subunit rDNA (SSUrDNA). Using PCR primers specific for microsporidian SSUrDNA, a single product was obtained from each species, and heteroduplex analysis indicated a high degree of sequence homology among the 3 products. In RFLP analysis of the PCR-amplified SSUrDNA, the enzymes AluI and DdeI gave restriction patterns that differed among all 3 species. Phylogenetic analysis using restriction patterns as differential characters indicated that Microgemma caulleryi and Tetramicra brevifilum are more closely related to each other than to Pleistophora finisterrensis. PMID- 10726274 TI - The spatial pattern of trypanosomosis prevalence predicted with the aid of satellite imagery. AB - Information on the spatial pattern of African animal trypanosomosis forms a prerequisite for rational disease management, but few data exist for any country in the continent. The present study describes a raster or grid-based Geographic Information System for Togo, a country representative of subhumid West Africa, with data layers on tsetse, trypanosomosis, animal production, agriculture and land use. The paper shows how trypanosomosis prevalence and packed cell volume (PCV) map displays may be predicted from correlations between representative field data and environmental and satellite data acquired from the National Oceanographic and Atmospheric Administration (NOAA) and Meteosat platforms. Discriminant analytical methods were used to assess the relationship between the amount of field data used and the accuracy of the predictions obtained. The accuracy of satellite derived predictions decreases from tsetse abundance to trypanosomosis prevalence to PCV value. The predictions improve when eco-climatic and epidemiological predictors are combined. In Togo, and probably elsewhere, the patterns of trypanosomosis prevalence and PCV are much influenced by animal husbandry and other anthropogenic factors. Additional predictor variables, incorporating these influences might therefore further improve the models. PMID- 10726275 TI - High prevalence of Echinococcus multilocularis in urban red foxes (Vulpes vulpes) and voles (Arvicola terrestris) in the city of Zurich, Switzerland. AB - Over a period of 26 months from January 1996 to February 1998, 388 foxes from the city of Zurich, Switzerland, were examined for intestinal infections with Echinococcus multilocularis and other helminths. The prevalence of E. multilocularis in foxes sampled during winter increased significantly from 47% in the urban to 67% in the adjacent recreational area, whereas prevalence rates of other helminths were similar in both areas. Seasonal differences in the prevalence of E. multilocularis were only found in urban subadult male foxes which were significantly less frequently infected in summer than in winter. The distribution of the Echinococcus biomass, as expressed by worm numbers per fox was overdispersed in 133 infected foxes randomly sampled in winter. Ten of these foxes (8%) were infected with more than 10,000 specimens and carried 72% of the total biomass of E. multilocularis (398,653 worms). Prevalences did not differ significantly in these foxes in regard to age and sex but worm burdens were significantly higher in subadult foxes as compared with adult foxes. In voles (Arvicola terrestris) trapped in a city park of Zurich, E. multilocularis metacestodes were identified by morphological examination and by PCR. The prevalence was 20% among 60 rodents in 1997 and 9% among 75 rodents in 1998. Protoscoleces occurred in 2 of the cases from 1997. The possible risk for human infection is discussed with respect to the established urban E. multilocularis cycle. PMID- 10726276 TI - Onchocerca-Simulium complexes in Venezuela: can human onchocerciasis spread outside its present endemic areas? AB - The compatibility between sympatric and allopatric combinations of Onchocerca volvulus-anthropophilic species of Simulium was studied in the north-eastern focus of human onchocerciasis as well as in a densely populated locality of the Amazonas State in Venezuela. The objectives were to test the conjecture that local adaptation exists between the parasite and its vectors (the Onchocerca Simulium complex hypothesis), and assess the possibility of the infection spreading from its present distributional range. For the homologous combination, O. volvulus-S. metallicum cytospecies E in Anzoategui State (north-eastern focus), parasite yield was 45% in contrast to 1% for the heterologous, southern parasite-S. metallicum infection. This was significantly lower than the parasite yield (4-10%) expected after allowing for the effect of density-dependent limitation of infective larval output described in this paper for S. metallicum. The population of S. exiguum s.l. from southern Venezuela allowed no larval development beyond the L1 stage of either northern or southern parasites. Mechanisms for such refractoriness probably operate at the level of the thoracic muscles, not affecting microfilarial uptake or migration out of the bloodmeal. The parasite yield of southern O. volvulus in S. oyapockense s.l. flies biting man at Puerto Ayacucho (Amazonas) was about 1%, in agreement with the figures recorded for highly compatible sympatric combinations such as O. volvulus-S. ochraceum s.l. in Guatemala. No infective larval development of the northern parasite was observed in southern S. oyapockense. These results, together with considerations of typical worm burdens in the human host, presence/absence of armed cibaria in the simuliids, parasite-induced vector mortality, and fly biting rates, suggest a lower potential for onchocerciasis to spread between the northern and southern endemic areas of Venezuela than that between Amazonian hyperendemic locations and settlements outside this focus with high densities of S. oyapockense s.l. PMID- 10726277 TI - Stochastic simulation and the detection of immunity to schistosome infections. AB - In this paper we address the question of detecting immunity to helminth infections from patterns of infection in endemic communities. We use stochastic simulations to investigate whether it would be possible to detect patterns predicted by theoretical models, using typical field data. Thus, our technique is to simulate a theoretical model, to generate the data that would be obtained in field surveys and then to analyse these data using methods usually employed for field data. The general behaviour of the model, and in particular the levels of variability of egg counts predicted, show that the model is capturing most of the variability present in field data. However, analysis of the data in detail suggests that detection of immunity patterns in real data may be very difficult even if the underlying patterns are present. Analysis of a real data set does show patterns consistent with acquired immunity and the implications of this are discussed. PMID- 10726278 TI - A survey of genes expressed in adults of the human hookworm, Necator americanus. AB - Hookworms are gut-dwelling, blood-feeding nematodes that infect hundreds of millions of people, particularly in the tropics. As part of a program aiming to define novel drug targets and vaccine candidates for human parasitic nematodes, genes expressed in adults of the human hookworm Necator americanus were surveyed by the expressed sequence tag approach. In total 161 new hookworm genes were identified. For the majority of these, a function could be assigned by homology. The dataset includes proteases, protease inhibitors, a lipid binding protein, C type lectins, an anti-bacterial factor, globins and other genes of interest from a drug or vaccine development viewpoint. Three different classes of small, secreted proteins were identified that may be involved in the host-parasite interaction, including potential potassium channel blocking peptides. One third of the genes were novel. These included highly expressed, secreted (glyco)proteins which may be part of the excretory-secretory products of these important pathogens. Of particular interest are a family of 9 genes with similarity to the immunomodulatory protein, neutrophil inhibitory factor, that may play a role in establishing an immunocompromised niche for this successful parasite. PMID- 10726279 TI - Inferring infection processes of a parasitic nematode using population genetics. AB - The distribution of genetic differentiation in a population of the parasitic nematode Strongyloides ratti divided between rat hosts was determined. We applied population genetic theory to these data to determine the source of new infections. We estimate the rate at which a rat acquires a new infection from (a) the existing subpopulation of parasites within that rat ('self-reinfection') versus (b) the wider environment ('immigration'). We find that the observed levels of genetic diversity and differentiation in the study population are consistent with low to moderate rates of self-reinfection and inconsistent with high rates of self-reinfection. PMID- 10726280 TI - Physiological status of wild European eels (Anguilla anguilla) infected with the parasitic nematode, Anguillicola crassus. AB - The effect of the parasitic swimbladder nematode, Anguillicola crassus, on the physiological status of wild European eels (Anguilla anguilla) was investigated during an 18 month survey (February 1995 until September 1996), and compared with that of wild uninfected eels collected over the same time-period. Despite the occurrence of up to 15 blood-feeding adults in the swimbladder lumen of the infected eels and as many as 25 additional larvae in the swimbladder wall, there were no major differences in hormonal, metabolic or osmoregulatory status of the 2 groups of eels. Wild European eels appear to adapt to chronic parasitism with Anguillicola crassus. The possible adverse effects of additional simultaneous stressors are discussed. PMID- 10726281 TI - Changes in the lipophilicity of the surfaces of Meloidogyne incognita and Haemonchus contortus during exposure to host signals. AB - The surfaces of plant and animal parasitic nematodes share certain lipids, which seem to be important in the infection process. The surfaces of 2 parasitic nematodes, Meloidogyne incognita and Haemonchus contortus, were activated by different pH buffers to allow the insertion of different fluorescent probes. The lipid analogue PKH26 and the surface charge indicator, cationized ferritin, were used as probes with these nematodes but labelled only the retaining 2nd-stage moulted cuticle of H. contortus 3rd-stage larvae (L3). Shedding of the second moult of H. contortus L3 was also visualized with PKH26 and cationized ferritin. The fluorescent anionic lipid probe 5-N-(octadecanoyl)-aminofluorescein (AF18) was inserted into the epicuticle layer of M. incognita 2nd-stage juveniles (J2) and H. contortus L3, and also of the second moult of H. contortus L3. Incubation with tomato root diffusate caused modifications of the M. incognita surface allowing the insertion of AF18. Fluorescence with AF18 was significantly decreased after treating M. incognita J2 with amiloride, a potent blocker of hydrogen and sodium (H+/Na+) antiporter. No surface fluidity was observed in M. incognita J2 and H. contortus L3 pre-treated with alkaline buffer when the lipid analogue AF18 was used in fluorescence recovery after photobleaching experiments. The significance of these findings to host infection processes is discussed. PMID- 10726283 TI - Panting desert ants--and how they navigate. PMID- 10726282 TI - The ABA-1 allergen of Ascaris lumbricoides: sequence polymorphism, stage and tissue-specific expression, lipid binding function, and protein biophysical properties. AB - The ABA-1 protein of Ascaris lumbricoides (of humans) and Ascaris suum (of pigs) is abundant in the pseudocoelomic fluid of the parasites and also appears to be released by the tissue-parasitic larvae and the adult stages. The genes encoding the polyprotein precursor of ABA-1 (aba-1) were found to be arranged similarly in the two taxa, comprising tandemly repeating units encoding a large polyprotein which is cleaved to yield polypeptides of approximately 15 kDa which fall into 2 distinct classes, types A and B. The polyprotein possibly comprises only 10 units. The aba-1 gene of A. lumbricoides is polymorphic, and the majority of substitutions observed occur in or near predicted loop regions in the encoded proteins. mRNA for ABA-1 is present in infective larvae within the egg, and in all parasitic stages, but was not detectable in unembryonated eggs. ABA-1 mRNA was confined to the gut of adult parasites, and not in body wall or reproductive tissues. Recombinant protein representing a single A-type unit for the A. lumbricoides aba-1 gene was produced and found to bind retinol (Vitamin A) and a range of fatty acids, including the pharmacologically active lipids lysophosphatidic acid, lysoplatelet activating factor, and there was also evidence of binding to leukotrienes. It failed to bind to any of the anthelmintics screened. Differential Scanning Calorimetry showed that the recombinant protein was highly stable, and unfolded in a single transition at 90.4 degrees C. Analysis of the transition indicated that the protein occurs as a dimer and that the dimer dissociates simultaneously with the unfolding of the monomer units. PMID- 10726284 TI - Diagnosis of acute pulmonary embolism: an update. AB - Considerable progress has been made in pulmonary embolism diagnosis during the last ten years. New diagnostic instruments such as plasma D-dimer measurement and lower venous compression ultrasonography have been developed. Clinical evaluation of the likelihood of pulmonary embolism has been rehabilitated and proven accurate and valid. The interpretation of lung scan results has become more simple and clear to clinicians. Finally, two diagnostic strategies have been validated in large scale outcome studies. Both rely on a sequential combination of the above mentioned instruments and have been able to safely manage more than 90% of patients without a pulmonary angiogram. The 3-month venous thromboembolic risk in patients without pulmonary embolism and, hence, not anticoagulated was less than 1% in both studies. In the absence of a formal comparison of their respective cost-effectiveness, choosing between these strategies may rest on local preferences or logistics. Finally, spiral CT scan is highly promising and may considerably modify the diagnostic workup of pulmonary embolism in the near future. However, it is insufficiently validated and its position in a rational algorithm for diagnosing pulmonary embolism is not yet defined. PMID- 10726285 TI - Sleep and breathing. AB - This paper reviews some of the recent advances in the sleep apnoea/hypopnoea syndrome. Sleep apnoea has recently been shown to be familial and in some of these patients this is due to retroposition of the maxilla and mandible. There is now good evidence that individuals with sleep apnoea have an increased risk of road accidents and increasing, although not yet 100% robust, evidence that they have an increased frequency of systemic hypertension. The last few years have seen a large number of randomised controlled trials of CPAP therapy which have shown clear evidence that CPAP improves symptoms, subjective sleepiness, objective sleepiness, cognitive function, IQ, mood, quality of life and driving ability. Evidence that CPAP influences cardiovascular and cerebrovascular outcomes is awaited. PMID- 10726286 TI - [The future of asthma therapy]. AB - Today bronchial asthma is regarded as a chronic inflammatory disease of the airways, which is modulated and controlled by immunological mechanisms. This new understanding of its pathogenesis is reflected in novel asthma treatment strategies. Newer treatments no longer aim at blocking effects of single mediators or at bronchodilation, but try to intervene in specific immunological mechanisms. An overview of future developments in asthma therapy is presented, with special emphasis on the anti-IgE concept of E-25. PMID- 10726287 TI - [Tuberculosis in Switzerland--a millenium problem?]. AB - In Switzerland a family physician sees a case of tuberculosis only every 4-6 years. While tuberculosis does not seem to be a major medical challenge in western countries, it is a real problem in the developing world. Nevertheless, special situations do arise in the western hemisphere where the standard therapy needs to be changed. HIV-positive patients are at greater risk of contracting tuberculosis after exposure, and therapy must be adjusted to the patient's immunological status. Multidrug-resistant tuberculosis is a major challenge to the attending physician, and takes us back to the time before antitubercular drugs existed. Standard medication is ineffective and we have to fall back on alternative drugs. Compliance of patient and physician are crucial for effective treatment, as in the case of all long-term therapy. Medical staff, and especially hospital personnel in emergency and tuberculosis departments, are at high risk of infection. Younger staff are no longer immunised by vaccination or earlier infection as they were a few decades ago. Multidrug-resistant bacteria pose a real threat for these groups and effective protection against transmission is important. Discovering a new vaccine which provides adequate immunisation is the only way to tackle the problem of tuberculosis worldwide. New drugs must also be developed to treat those already suffering from the disease. PMID- 10726289 TI - [37-year-old man with cough, thoracic pain and hemoptysis]. PMID- 10726288 TI - [SAPALDIA: past, present and future]. AB - The cross-sectional part of SAPALDIA studied the associations between respiratory symptoms or functional abnormalities and chronic exposure to a polluted environment. The results showed an elevated risk of developing respiratory symptoms in regions with higher levels of particulate matters (PM10). The same was observed with passive smoking exposure. Decreased spirometric parameters (especially FVC) were measured in areas with the highest levels of PM10. While the functional alteration is clinically minimal in the individual, the effect is significant when one considers the prevalence of an abnormal functional parameter in a general population. Exposure to pollutants at the workplace increases bronchial reactivity to non-specific stimuli, with atopy acting as amplifier of this effect. The longitudinal part of SAPALDIA examined the associations between the dynamics of respiratory symptoms or functional abnormalities and average levels, as well as short-term fluctuations, of atmospheric pollutants. It was observed that symptomatic episodes tend to last longer in more polluted regions, whichever marker of pollution is used (PM10 or NO2). Passive smoking tends to prolong respiratory symptoms and shorten asymptomatic intervals. Short-term increases in outdoor pollutants are associated with an increase in incidence of peak flows below individual baseline values. Numerous questions remain regarding the relationship between respiratory health and environment. A SAPALDIA II study is under preparation which will follow the evolution of SAPALDIA I participants with regard to respiratory symptoms, pulmonary function, bronchial reactivity and markers of atopy. PMID- 10726290 TI - [Perspectives in geriatrics: a good time for reorientation?]. PMID- 10726291 TI - [Pursuit of the older job seeker: the advice from SER]. PMID- 10726292 TI - [Is 'attention' important for the results of dementia screening? Relation among Digit Span Test, CST amd ADS in elderly patients]. AB - This study focused on the relationship between attention and dementia screening test performance, using the adapted Wechsler's Digit Span test for elderly patients, the Cognitive Screening Test (CST) and the Amsterdam Dementia Screening Test (ADS6). Participants were dementia patients and psychiatric patients (n = 147). In both groups no floor-effect was found on the Digit Span test. Principal components analysis showed that CST and ADS6-scores had relatively high loadings on one factor, in contrast to digit span scores that loaded on a second factor. On average, psychiatric patients did hardly worse than normal controls. Attention deficits were more apparent in dementia patients. Considering a maximum of r = .41, these more or less subtle deficits were only moderately related to dementia screening test performance. It is concluded that the adapted Digit Span test is suitable for measuring attention deficits in elderly patients. However, Digit Span predicts performance on dementia screening test only to a modest degree. PMID- 10726293 TI - [Suboptimal health and memory performance of elderly]. AB - The influence of health-related incidents on memory test performance and subjective memory ratings was assessed in a) a group of healthy older adults recruited via the register of population (group 1: N = 117, mean age 62 years, range 46-89) and b) a group of healthy older subjects having memory complaints and applying for memory training (group 2: N = 111, mean age 63 years, range 45 85). The study tries to cross-validate a previous study, where the presence and frequency of health-related incidents were related to cognitive performance. The presence of 9 categories of health-related incidents (consultation of a neurologist, systemic diseases, repeated mild concussions, repeated anaesthesia, use of psychotropic medication, alcohol use, other neurotoxic factors, such as exposure to organic solvents, psychiatric disorders, birth complications or developmental problems) was assessed in a semi-structured interview. Memory performance was assessed with a battery covering different aspects of memory. Memory self-ratings were assessed with questionnaires asking for frequencies of memory failures and a general judgement of memory capacity. Health-related incidents occurred in about half of both subject groups and were not related to age or other demographic characteristics. In both subject groups, the presence, nor the number of health-related incidents was related to memory performance or memory complaints and there was no interaction with age. The results are not in agreement with the notion that health-related indices explain age differences in cognitive performance. PMID- 10726294 TI - [Measurement of quality of life in patients with dementia of Alzheimer type and their caregivers: Schedule for the Evaluation of Individual Quality of Life (SEIQoL)]. AB - Twelve patients with mild to moderate Alzheimer's Disease (AD) and their caregivers were interviewed with the SEIQoL. The SEIQoL measures quality of life by taking into account the relevant determinants for a particular individual. The subject rates 5 areas in life most important to the quality of life. The relative contribution of each area to the overall quality of life is then calculated with a multiple regression analysis programme developed for the purpose. Next the SEIQoL Index score, validity and reliability are computed. One patient was unable to complete the interview. The remaining (8 women, 3 men, mean age 71.3 years) had a mean SEIQoL Index score of 79.9 (median: 85.4), which is comparable to healthy Dutch elderly. The caregivers (10 spouses, 2 daughters; mean age 67.4 years), on the other hand, had a lower SEIQoL Index score: 62.2 (median: 63.8). Validity and reliability were good for both groups. Thus, caregivers in this pilot study experienced a lower quality of life than AD patients and healthy Dutch elderly. The SEIQoL allows quantitative measurement of completely individualised quality of life for AD patients and their caregivers. PMID- 10726295 TI - [Fluid intake by healthy old people? A literature survey]. AB - This literature search investigates the amount of fluid that a healthy elderly person drinks if he is able to drink according to his needs. The answer to this physiological question may be used by clinicians if they make decisions about how much fluid they need to administer to their patients. The literature that offered an answer to this question was limited. The total number of patients that were examined, however, was high (2866). It appeared that healthy elderly people drink approximately 2100 ml. of fluid per day. This is (probably) no less than the average fluid intake of young adults. Clinicians often think that healthy elderly persons drink less than healthy young adults. However, there is no evidence to be found in the literature to support this opinion. The tendency to underestimate the fluid intake level of elderly patients should be taken into account when determining the quantity of fluid that should be administered to an individual patient. PMID- 10726296 TI - Detection of an Ehrlichia bovis-like organism in cultured buffalo monocytes. AB - Monocytes from a buffalo were cultured in RPMI 1640 medium following separation of plasma by the erythrocyte sedimentation technique and subsequent separation of mononuclear cells by density gradient centrifugation. Growth of an organism considered to be Ehrlichia bovis was noticed in the cultured monocytes after 10 days. The inclusions were considered to be those of E. bovis from their morphology, staining characteristics and growth characteristics in culture, and by indirect immunofluorescence examination with an anti-E. canis serum. The utility of peripheral blood monocyte cultures opens the possibility of diagnosing the carrier status of ehrlichiosis in animals. PMID- 10726297 TI - The effect of oral administration of polyethylene glycol on faecal helminth egg counts in pregnant goats grazed on browse containing condensed tannins. AB - Thirty yearling F1 Anglo-Nubian x Mubende goats, averaging 21 +/- 0.45 kg, kept on free-range feeding in the Ankole range land, Uganda, were screened for health and nutritional status, effectively treated against helminth parasites, mated, and randomly divided into two equal groups during a 3-month preparatory phase. During the 6 months that followed, the goats in one group received a daily oral dose (50 g/goat) of poly(ethylene glycol) (PEG), while the other group acted as the control (no PEG). The goats were monitored for faecal nematode egg counts and body weight gains, along with the quality of their diet, nematode contamination of the pasture, and the prevailing climatic factors in the area. Goats treated with PEG had significantly (p < 0.05) higher faecal helminth egg loads. The mean nematode eggs per gram of faeces (epg) of the PEG group (290 epg) was more than double that of the control group (129 epg). All the PEG-treated goats exhibited moderate to severe infections at the end of the experiment. The gain in body weight during gestation was lower (p < 0.05) in the PEG group (70.4 g per goat per day) than in the control group (91.8 g per goat per day). The PEG group lost 2.3 g per goat per day in the fifth month. PEG deactivates condensed tannins, and it was concluded that condensed tannins play a significant role in reducing the negative effects of gastrointestinal helminth burdens in the natural free-range feeding system of the Ankole range land in Uganda. Selective feeding on such range lands might expose goats to optimal concentrations of dietary condensed tannins with resultant beneficial effects. PMID- 10726298 TI - Impact of tsetse control on the age-specific prevalence of trypanosomosis in village cattle in southeast Uganda. AB - The prevalence of trypanosomosis, mean packed cell volume and anti-trypanosomal antibody levels in village cattle of different age groups (< 0.5 year, 0.5-2 years, > 2-5 years and > 5 years) in the areas with tsetse control were compared with those of corresponding age groups in areas without tsetse control in Tororo, southeast Uganda. The prevalence of trypanosomosis in cattle in the age groups of 0.5-2 years, > 2-5 years and > 5 years in the areas with tsetse control was significantly lower than in cattle in similar age groups in the areas without tsetse control (p < 0.5). Trypanosoma vivax was the most predominant Trypanosoma species in the areas with tsetse control, while T. congolense was the most predominant species in the areas without tsetse control. The mean Trypanosoma antibody levels in cattle in the age groups < 0.5 year, 0.5-2 years and > 2-5 years in the areas with tsetse control were significantly lower than those of the similar age groups in the areas without tsetse control (p < 0.5). The mean PCV values for cattle in the age groups 0.5-2 years, > 2-5 years and > 5 years from the areas with tsetse control were significantly higher than those of the similar age groups in the areas without tsetse control. Tsetse control appeared to have a considerable impact on the prevalence of trypanosomosis, distribution of Trypanosoma species, specific antibody levels and the packed cell volume of cattle in the different age groups. PMID- 10726299 TI - The hydropericardium syndrome and inclusion body hepatitis in domestic fowl. AB - Hydropericardium syndrome, an emerging disease of poultry, has recently been detected in some countries of Asia and America, particularly in broiler birds aged 3-6 weeks. The disease is characterized by its sudden occurrence with high mortality of up to 80% in broilers and low mortality of under 10% in layers, associated with hydropericardium. Its course is of 7-15 days under natural conditions. The causative agent is probably fowl adenovirus serotype 4, belonging to group I aviadenovirus genus of the family adenoviridae, which can be cultivated in primary cell cultures of chicken kidney and embryo liver cells. The transmission of disease occurs laterally by the oral-faecal route. The livers of affected birds show necrotic foci, and basophilic intranuclear inclusion bodies fill the entire enlarged nucleus of some of the hepatocytes. The disease can be diagnosed from its gross lesions, histopathological changes in the liver and by serological tests, such as agar gel diffusion, counter immunoelectrophoresis, indirect haemagglutination and ELISA. It has been brought under control by inactivated liver organ vaccines (0.25 ml/bird) or inactivated cell culture vaccines (10(3.5) LD50/bird) given by the subcutaneous route at 10-15 days of age. The vaccine is effective in the face of an outbreak and significantly reduces the mortality. PMID- 10726300 TI - Costs and returns of camels, cattle and small ruminants in pastoral herds in eastern Ethiopia. AB - Two questionnaire surveys (2 x 44) were conducted among pastoral households, using three grazing management systems. The average number of Tropical Livestock Units (250 kg) was 4.0 per member of the household. Milk production was the most important source of revenue (66% of the total) followed by sale of livestock (17%) and transport (16%). High mortality rates were recorded for all livestock. About 27% of the milk was sold fresh or as butter. Sedentary and transhumant grazing management systems showed similar levels of income, but nomads had a 2.6 fold higher overall net income. The average total gross income from the entire herd amounted to US$ 6382 per household per year. The calculated costs were 29% of the gross returns. The contribution to the total gross revenues of camels, cattle and small ruminants was 58%, 25% and 17%, respectively. PMID- 10726301 TI - A comparative study on the oestrous response to PGF2 alpha analogue treatment, and conception rates according to time of artificial insemination, in Zebu (Bos indicus) and Baoule (Bos taurus) cattle. AB - The oestrous response, interval and conception rates were studied after synchronization with a prostaglandin analogue (cloprostenol) and artificial insemination (AI) performed at different times in 50 Zebu (Bos indicus) and 83 Baoule (Bos taurus) cattle indigenous to Burkina Faso. The overall proportion of cows responding to synchronization was 70% (93/133). Although the response was higher for the Baoule cattle, at 73.5% (61/83), than for the Zebu, at 64% (32/50), the difference was not statistically significant (p > 0.05). The mean oestrous interval from treatment to the onset of oestrus (TOI) was shorter in the Zebu (54.1 h, SD 6.7) than in Baoule (65.2 h, SD 12.9) cattle (p < 0.001). Of the Zebu (n = 32) that responded, 65.7% presented oestrus over a period of 12 h ranging from 48 h to 60 h after treatment. For the Baoule cows, the highest proportion of animals in oestrus over a period of 12 h was 41% between 60 h and 72 h after treatment. The frequency distribution of onset of the oestrus indicated that up to 64.5% of the Zebu and 79.5% of the Baoule cattle showed onset of oestrus during the daytime. For Zebu and Baoule cows inseminated 13 h or 18 h after the onset of oestrus, conception rates were 56% and 57% (p > 0.05) and 33% and 64% (p < 0.05), respectively. Based on these findings, it appears that the oestrous response to synchronization was adequate for both Zebu and Baoule cattle and that the time to onset of oestrus varied according to genotype. It was also concluded that conception rates were satisfactory for both genotypes but that, for Baoule cattle, AI performed 18 h after oestrus significantly increased conception rates compared to AI at 13 h after oestrus. PMID- 10726302 TI - The effect of house-roof type on ambient temperature, feed intake, egg production and egg size in commercial layers during the summer months in south-eastern Botswana. PMID- 10726303 TI - [Lectins and recognition of protein ligands]. AB - The general principles of protein-carbohydrate lectin interactions were discussed. Experimental evidences of the molecular mimicry between carbohydrate and protein lectin ligands were analyzed. The interdomain interactions in lectins as well as the interactions between ligands and lectin domains other than carbohydrate-binding domains were considered. PMID- 10726304 TI - [Membrane mechanisms of regulating Ca ion concentration in smooth muscle cells]. AB - The aim of this review is to summarize some current concepts on the membrane mechanisms of energy-dependent Ca2+ transport in the smooth muscles. The emphasis is placed on the properties and mechanisms of regulation of plasma membrane and endoplasmic reticulum calcium pumps, sarcolemmal sodium/calcium exchanger and mitochondrial Ca2+ transport. PMID- 10726305 TI - [Effect of low temperature on electron transport in plant mitochondria respiratory chain]. AB - The etiolated 2.5-day winter wheat sprouts were chilled at 3 degrees C during 24 to 144 hours. After 24 h cooling, shoot intact mitochondria showed a high degree of activation of the alternative oxidase, which was measured as sodium azide and benzohydroxamate sensitivity of the organelles respiration with succinate as a substrate. The role of the alternative oxidase in limiting the level of reactive oxygen species produced in the stressed plant tissues is discussed. PMID- 10726306 TI - [Variability of RNA in morphogenesis of sugar beet and sunflower leaves]. AB - We investigated the developmental gene expression patterns of Beta vulgaris L. using DNA: RNA hybridization reactions. We found that 2,100 and 900 diversive poly(A)+mRNA were synthesized while 430 and 4,000 diversive poly(A)+mRNA were disappeared during greening in cells of cotyledons and during senescence in cells of yellowing leaves respectively. In the other periods of sugar beet vegetation differential poly(A)+mRNA transcription failed to be revealed in the cells of green leaves within the limits of sensitivity of this method. On the average in the sugar beet green leaves 34,600 poly(A)+mRNA are synthesized, whereas in the sunflower cells cytoplasm (Helianthus annuus L.) about 94,000 diverse mRNA are synthesized. The comparison with some literary data testifies about the species specificity of gene expression pattern during the morphogenesis of the cultural plants. PMID- 10726307 TI - [Effect of hydrocortisone on composition of polyphosphoinositides in liver cell nuclear membrane and rat brain]. AB - The action of hydrocortisone in vivo was shown to cause changes both in the total amount of phospholipids and polyphosphoinositides of rat liver and brain nuclear membranes. The hormone increased the content of five from seven phospholipid fractions including the fraction of monophosphoinositide when acting in concentration and exposition leading to activation of biosynthetic processes. The increasing of monophosphoinositides amount was accompanied by the decreasing of triphosphoinositides content which indicated the redistribution among the phosphoinositides fractions under the steroid hormone action in both cell types. PMID- 10726308 TI - [Minimal amino acid sequence, recognized by anibodies to the peptide GPQPPQPPQP from the proline-rich region of pertactin]. AB - Three antiserum samples obtained from rabbits immunized by the conjugate KLH-10P (keyhole limpet hemocyanine-decapeptide GPQPPQPPQP) were used to study antigenic structure of 10P. Antigenic properties of conjugates 6P (PGPQPP) and 4P (PQPP) with ovalbumine were studied by an indirect immunoassay (ELISA). Also 4P, 6P, PQP and QPP peptides were used for a competition assay. It was found that antibodies to 10P have demonstrated different specificity to short sites. Antibodies recognized such shot peptides as PQP and QPP in the competition assay. The efficiency of serum antibodies reaction with those peptides increased from QPP and PQP to PGPQPP. Only one serum sample had no antibodies to glutaraldehyde. Gly glutaraldehyde-Gly hapten-like ligand was used to inhibit activity of antibodies to cross-linking agent into two samples. It is allowed to improve analysis of antibodies, which recognize shot sequences PQP and QPP. PMID- 10726309 TI - [Features of plasminogen activation in a complex with antiplasminogen monoclonal antibody IV-1C]. AB - Antiplasminogen monoclonal antibody IV-1c (IV-1c) with antigenic determinant in V709-G718 site of plasminogen (Pg) protease domain (Druzhina N.N. et al. 1996.) can induce catalytic activity in Pg moiety of the complex. Catalytic activity appeared in Pg-IV-1c complex after approximately 2 h lag-period. Rate of Lys-Pg activation was higher then that of Glu-Pg. Amidolytic activity of Pg-SK equimolar complex was completely inhibited by IV-1c at 2:1 = Pg:IV-1c molar ratio. At constant Glu-Pg concentration increasing of the IV-1c concentration to equimolar of Pg accelerated Pg activation. Subsequent increase of IV-1c concentration inhibited the Pg activation sharply. Increasing of Glu-Pg concentration at constant IV-1c one did not inhibit Glu-Pg activation in Pg-IV-1c complex. The rate dependence of Pg activation from Glu-Pg-IV-1c complex concentration curve had bell-shaped form with maximum at 500 nM. Electrophoretic analysis of components of Glu-Pg-IV-1c complex showed that Lys-Pg and Lys-Pm were not observed at 100 nM complex concentration for 6 h period of reaction. At 680 nM concentration Glu-Pg-IV-1c complex these forms appeared in initial moments of reaction activation after lag-period. Kinetic scheme and peculiarities of Pg activation reaction in Pg-IV-1c complex are discussed. PMID- 10726310 TI - [Plasminogen binding with decapeptide and polypeptide fragments of streptokinase]. AB - The plasminogen binding with streptokinase decapeptides, modeling the primary structure of molecule, and chymotryptic fragments of streptokinase have been investigated. The immunoenzymatic assay has shown that plasminogen binds to all streptokinase fragments with the decreasing affinity in the set of fragments: 36 > 30 > 17 > 7 > 11 kDa. Location of the binding sites in streptokinase primary structure was performed using the immobilized decapeptides on plastic pins adopted to IEA. In the presence of 10 mM 6-aminohexanoic acid 11 sites for human Glu- and mini-plasminogens, pig and bovine plasminogens binding have been found. They were of the same location for human, bovine and pig plasminogens. 3 sites were located in plasminogen alpha-domain--T43-A72, N113-T126, Q133-V158, 5 sites in beta-domain--T163-L188, A203-S222, Q239-I264, Y275-L294, T315-L340, and 3 sites in gamma-domain--T361-R362, N377-E392, T397-N410. Participation of linear part of streptokinase polypeptide chain in plasminogen--streptokinase complex formation is suggested. PMID- 10726311 TI - Obtaining immunologically active bovine viral diarrhea virus (BVDV) proteins for enzyme linked immunosorbent assay (ELISA). AB - We report here the preparation of BVDV antigen (Ag) to obtain adequate quantities of pure active viral proteins from Madin Darby Bovine kidney (MDBK) cell culture infected with BVDV cytophatic Oregon C24V strain. SDS-PAGE, Immunodot, ECL Western blot and ELISA showed the best specificity and activity of BVDV Ag obtained from infected cells only by mild experimental conditions. BVDV Ag preparations showed the peculiar BVDV sensitivity to proteases, nonionic surfactants and tend for protein degradation and irreversible loss of conformational antigenic determinants with subsequent inability to detect antibodies against viral Ag in hyperimmune sera. The widest panel of immunologically active and specific polypeptides, that elicit Ab production in hyperimmune sera, was obtained by ultrasonication and subsequent purification on 20%-50% sucrose cushion. We observed that BVDV tend to remain in the infected cells, to associate with components of serum and cellular origin--this is of crucial importance towards the specificity of ELISA. BVDV antigenic properties are determined by the labile conformational antigenic epitopes. PMID- 10726312 TI - [Mitogenic effects of staphylococcal antigenic substances on irradiated lymphocytes from Wistar rats]. AB - Proliferation activity of lymphocytes from rats was studied using a micromethod of blasttransformation reaction after X-ray irradiation (absorbed doses for animals were 0.5 and 1 Gy). Staphylococcus protein A and peptidoglycan stimulated the proliferation of irradiated lymphocytes. It was assumed that staphylococcus antigen substances held radiation protective effects. Staphylococcus protein A may be used for stabilization of lymphopoesis and functional activity of humoral and cellular immunity in irradiated animals due to T- and B-mitogenic properties. PMID- 10726313 TI - [Hemoglobin from newborn calves during artificial changes in the acid-base parameters of blood]. AB - The modelling of metabolic acidosis and alcalosis states proves that AAB is capable to influence on the haemoglobin parameters and its oxiform levels in the blood of newborn animals. The quantitative redistribution of the indicated haemoglobin forms in blood of the experimental animal is estimated as compensator process and is explained by their buffer properties. The investigated regularities revealed some aspects of adaptive mechanisms manifested during the abnormal exit of the newborn organism from the respiratory-metabolic acidosis state. PMID- 10726314 TI - [Interrelation between the cation-anion correlation of cow's blood and colostrum and acid-base state in newborn calves]. AB - The neonatal period of the development is characterized by metabolic and functional reorganization in the organism including the acid-alkaline state, that is the important maintain for the metabolic homeostasis. The disturbances in the cation-anion correlation, the decrease of the base reserve in the cows blood and collostrum influence to the cation-anion balance and to the normalization of the acid-alkaline state in the newborn calves that is the reason of decreasing their adaptive abilities. PMID- 10726315 TI - [Effect of vitamin D3 on free-radical oxidation of lipids in low density lipoproteins in vitamin D deficiency]. AB - The lipid free radical oxidation in low density lipoproteins was investigated on D-deficiency model in vivo. The processes of lipid free radical oxidative activation in low density lipoproteins at D-deficiency occurred. The chemiluminescence kinetic parameters: the maximum intensity of the first flash and inclination angle tangent of an ascending branch of the second flash grew at D-deficiency in comparison with control group (p < 0.02 and p < 0.05, respectively). At the same time, the vitamin D3 introduction to the experimental animals diet failed result statistically reliable inclination angle tangent of an ascending branch of the second flash was reduced (p < 0.02). Increase of the products reacting with thiobarbituric acid content in low density lipoproteins in D-deficiency conditions (p < 0.001) was found. Vitamin D3 introduction to the diet reduced quantity of products reacting with thiobarbituric acid in low density lipoproteins (p < 0.01). However, their level remained higher than for the control animals (p < 0.01) as established. PMID- 10726316 TI - [Antiradical and antioxidant properties of nonsteroidal anti-inflammatory agents- derivatives of pyridinecarboxylic acid]. AB - A number of pyridincarboxylic acid derivatives PV-1-4, 7 and emoxypine preparation antioxidative activity in yolk lipoprotein suspension was studied by a method of Fe(2+)-initiated biochemiluminescence. Lipid peroxidation in suspension was effectively inhibited by the studied compounds in various concentration ranges. PV 1, 3, 4, 7 inhibited lipid peroxidation at the concentrations 100-fold, then those of PV 2 and emoxypine. Antiradical activity of the studied compounds was demonstrated by their forming a complex with the stable diphenylpickrylhydrazyl radical. The effect of these compounds as antioxidants is discussed. PMID- 10726317 TI - [Effect of biogenic stimulators--aloe extract and biotrite--on lipid peroxidation processes in saliva in inflammatory periodontal disease]. AB - The influence of biostimulators of vegetative origin, such as aloes and biotritis, on the process of lipid peroxidation in parodontium tissues, was studied. Biotritis has a more considerable parodontoprotective effect than aloes. PMID- 10726318 TI - [Metabolic changes in blood in pulmonary tuberculosis patients from various blood groups]. AB - 285 Patients suffering from the respiratory tract organs primary tuberculosis were subjected to the tests on definition of blood groups according to AB0 system, as well as its total proteolytic activity, alpha 1-antitrypsin, ceruloplasmin, general haptoglobin, lysozyme, malonic dialdehyde and diene conjugates levels were estimated. The sick persons as compared with the healthy ones were defined to reveal a tendency to increase of the persons with 0(I)- and B(III)-blood groups and decrease of those ones with A(II)-groups. Independently on the pulmonary tuberculosis patients phenotypic index their tested blood biochemical indices levels increase. As an exception is the proteolytic activity of the persons with B(III)- blood group, and also alpha 1-antitrypsin and lysozyme--with 0(I) and AB(IV) groups, in this case their rates failed to exceed the norm confidential interval. The blood metabolic parameters were defined as independent on its group. PMID- 10726319 TI - [Effect of chronic emotional stress and low dose ethanol on the level of neuronal cell adhesion molecule and glial fibrillary acidic protein in rat brain]. AB - The effect of chronic emotional stress and ethanol on NCAM and GFAP levels in cerebral cortex, hippocampus, striatum, cerebellum and medulla-ponts was investigated. We report about increase of NCAM and GFAP concentrations in the cerebral cortex and decline of the total protein contents in the investigated brain areas of middle-sleep rats under the stress conditions. Ethanol in the dose of 0.5 g/kg during 7 days evoked opposite changes of NCAM and GFAP concentration and elevation of the total protein level in medulla-pons. In the other brain areas level changes of only one (any) of the two investigated neurospecific proteins were observed. Ethanol injections to the stressed rats normalized the relative weights of adrenals and the level of total protein in the brain areas but didn't normalize the behavioral activity in an "open field" test. Besides, we observed a dramatic increase of GFAP level (over 10 times) in the medulla-pons which may be connected with glioses. These results suggest the specific changes of NCAM and GFAP contents under the chronic emotional stress which don't correlate with changes in the hypophysis-adrenals system. PMID- 10726320 TI - [Characteristics of biologically active mannan substances, isolated from fodder yeast]. AB - The component composition and antiviral properties of the mannan-containing preparations were studied. These preparations were extracted from some laboratory and commercial specimens of the fodder yeast cultivated on different substrates. It was shown, that the main component of pure preparation was the mannan which had molecular mass near 13 kDa. The monosaccaride composition of mannan component was varied depending on the source, degree of purification, methods of purification of the preparations. However, the crude and pure mannan preparations activity relatively to VTV was approximately equal. PMID- 10726321 TI - [New systems of coordinates in the study of ligand-receptor interaction]. AB - Two new coordinate systems that allow to determine the parameters of ligand receptor interaction are suggested. These coordinate systems principally differ from the well-known coordinates of Klotz and Scatchard. It was shown that suggested coordinates were simpler and more convenient then coordinates of Klotz and Scatchard and in some cases was more informative. The case when a ligand interacts with two classes of non-identical independent receptors was also considered. PMID- 10726322 TI - [Mechanism of eosin Y action of activity of solubilized Ca2+, Mg2+- ATPase from smooth muscle sarcolemma]. AB - The eosin Y inhibitory effect on the activity of smooth muscle plasma membrane Ca(2+)-transporting ATPase was studied: effect of this inhibitor on the maximal initial rate of ATP-hydrolase reaction, catalyzed by Ca2+, Mg(2+)-ATPase, on the affinity of enzyme for the reaction reagents (Ca2+, Mg2+, ATP). Dependence of eosin Y inhibitory effect on some physicochemical factors of incubation medium was studied too. It was determined that eosin Y inhibited reversibly and with high specificity purified Ca2+, Mg(2+)-ATPase solubilized from myometrial cell plasma membrane (Ki--0.8 microM), decreased the turnover rate of this enzyme determined both by Mg2+, ATP and Ca2+. This inhibitor had no effect on the enzyme affinity for Ca2+, increased affinity for Mg2+ and decreased affinity for ATP. It was determined that inhibition of Ca2+, Mg(2+)-ATPase by eosin Y depended on pH and dielectric permeability of the incubation medium: increasing of pH from 6.5 to 8.0 reduced the apparent Ki, decreasing of dielectric permeability from 74.07 to 71.19 increased the apparent Ki. PMID- 10726323 TI - [Effect of alpha-tocopherol and phospholipids with omega-3 fatty acids on membrane properties]. AB - As a result of the investigations conducted it was displayed, that alpha tocopherol and phospholipids including into their composition omega-3-acids, differed in their influencing the composition of heart microsomes membranes lipids. The insufficient quantity of vitamin E in the animals ration was defined as leading to the cardiac microsomes lisophospholipids (lisophosphatidylcholin, lisophospatidylethanolamin), diphosphatidylglycerol increase as well as to the tendency to sphingomyeline and phosphatidylethanolamin decrease. While administrating both alpha-tocopherol and the complex of phospholipids with omega 3-fatty acids, the correction of the phospholipids composition microsomes membranes is observed as tending towards their stabilization, however the marine phospholipids complex is more active than alpha-tocopherol. Administration of phospholipids with omega-3-fatty acids during the period of 30 days provided for the increase of relationship: polyunsaturated fatty acids to saturated fatty acids in the cardiac microsomal membranes, evidencing about increasing the unsaturated cellular membranes. While administrating the phospholipids, into the cardiac microsomes the eicozepentaenic acid was identified, failing to be in the norm, docozahexaenic acid content increased. The results obtained testify, that at the pathology there are changes in the quantitative relationship of membrane phospholipids and fatty acids, being a result of changing the biomembranes permeability as well as their functions disturbances. The adverse effect of E deficiency to the membrane structure was revealed as capable to be regulated by the marine phospholipid complex, including omega-3-fatty acids. PMID- 10726325 TI - [Prions and serpins: structural analogs and their consequences. I. Protease transport hypothesis]. AB - The structural analogy between prions pathogenic form and serine proteinase inhibitors (serpins) was laid into the basis of explaining the prion diseases main peculiarities--nucleic-acid-free transfer of infection, neurodegenerative processes, existence of the minimal infective dose, long-term latent period and some others. The assumption about pathogenic prions as the proteins supplying the extracellular proteinases transport into the intracellular space permits to bring the pathogenesis of prion diseases to the known and partially proved processes order regarding the case of prion diseases. PMID- 10726324 TI - [Modification of phospholipid composition of rat isolated heart tissue using N stearoylethaonolamine (NSE) in a model of acute ischemia-reperfusion]. AB - The phospholipid composition of the myocardial tissue of the isolated rat heart under acute ischemia and effect of NSE on the level of phospholipid were studied. It was shown that the level of phospholipids and PI under ischemia increased by 15% and 25% respectively, but the level of DPG decreased by 14%. The addition of NSE into the perfusion medium prevents the alterations of phospholipids and DPG levels. This suggests that NSE has some cardioprotective properties. PMID- 10726326 TI - Acute renal failure--more than kidney ischemia? PMID- 10726327 TI - [Anaphylactic shock]. AB - IgE-dependent and IgE-independent hypersensitivity reactions, the latter due to physical, chemical or hyperosmolar stimuli, may evolve as anaphylaxis or an anaphylactoid reaction, by an escalating release of mediators from mast cells and basophils. Without immediate treatment, anaphylaxis goes along with substantial morbidity (shock, multiple organ failure) and mortality; within minutes this explosive clinical response can be fatal. The severity of anaphylactic/anaphylactoid reactions is graded from stages 0 to IV in order to guide the management of this disease, stage III corresponding to anaphylactic shock. Severe anaphylactic reactions may take a progressive course despite adequate therapy; even in the case of an initial favourable response to treatment measures life-threatening symptoms may recur; there may be late-phase reactions 6 to 12 hours after the initial reaction. For the initial emergency management a differentiation between IgE-mediated and IgE-independent anaphylactoid reactions is not required. These are the pertinent principles of therapy in hypotensive and hypoxic patients: removal of the likely noxious agent at the site of introduction, provision of a patent airway, 100% oxygen supplementation, intravenous fluid therapy and pharmacological support with catecholamines. After primary care the monitoring and therapy of the patient with anaphylactic shock has to be continued on the intensive care unit. Guidelines for management of acute anaphylaxis referring to both the stage of disease including shock and the main clinical manifestation (cutaneous, pulmonary, cardiovascular) have been established by a German interdisciplinary consensus conference and were published in 1994; consensus guidelines for emergency medical treatment have been communicated by the ILCOR (1997) and the Project Team of the Resuscitation Council (UK) (1999). PMID- 10726328 TI - Impairment of renal function in patients resuscitated from cardiac arrest: frequency, determinants and impact on outcome. AB - OBJECTIVE: To assess frequency, determinants, and impact on outcome of impaired renal function in cardiac arrest survivors. PATIENTS AND METHODS: In a retrospective analysis 87 patients admitted after cardiac arrest to an emergency department between 1 March 1994 and 31 October 1995 were evaluated; patients were followed until 31 December 1998 or death. Serum creatinine was measured on arrival, 24 hours, 3 and 7 days after cardiac arrest. Impaired renal function was subclassified according to severity differentiating in between cases with an elevation of serum creatinine level > 1.4 mg/dl to 2 mg/dl and > 2 mg/dl. We examined the association between prearrest history and CPR data, collected according to the "Utstein Style", and renal function. RESULTS: Patients were followed for a median of 1199 days (IQR 16 to 1427). Impaired renal function (serum creatinine level > 1.4 mg/dl) was found on admission in 36 patients (41%), at 24 hours in 24 (31%), at 3 days in 13 (19%) and on day seven in 9 patients (16%) respectively. History of congestive heart failure and duration of low flow state (from the beginning of basic and/or advanced life support until restoration of spontaneous circulation) were significantly associated with elevated serum creatinine (> 1.4 mg/dl) at 24 hours after the event. The occurrence of impaired renal function was also more frequent in patients with diabetes mellitus and hypertension, but this did not reach statistical significance. The relative risk for death was 2.8 (95% confidence interval 1.3-5.8) for a serum creatinine level of > 1.4 mg/dl to 2.0 mg/dl and 5.4 (95% confidence interval 2.4-12.1) for values > 2 mg/dl. CONCLUSION: Transient impaired renal function is common in patients surviving cardiac arrest. Congestive heart failure and low flow time are independent predictors for the development of impaired renal function. There is a positive association in between increased serum creatinine levels and risk of death. PMID- 10726329 TI - Effect of verapamil on baroreflex sensitivity and on cardiovascular variability. AB - OBJECTIVE: Epidemiological evidence indicates that depressed baroreflex sensitivity and heart rate variability are associated with reduced survival secondary to coronary heart disease as well as with an increased risk of developing coronary heart disease. In view of the conflicting data in the literature concerning the effect of calcium channel antagonists on autonomic balance, we evaluated the effect of verapamil on heart rate and blood pressure variability, and on baroreflex sensitivity. METHODS: Baroreflex sensitivity was studied in 11 rabbits (27 series) under slight sedation induced by pentobarbital infusion (5 mg/kg/hour), both with a steady-state method using phenylephrine induced blood pressure ramps, and by spectral analysis estimating the transfer function from mean arterial blood pressure to heart rate. Mean arterial blood pressure in the femoral artery, heart rate, and a microphotoelectric plethysmogram of the capillary network of rabbit's ears were simultaneously recorded during the entire experiment. Baroreflex sensitivity was measured before and after 30 min of verapamil infusion (20 micrograms/kg/min). RESULTS: Verapamil reduced baroreflex sensitivity measured by steady-state (2.6 +/- 0.2-1.7 +/- 0.2 beats/min/mmHg, mean +/- SEM) and transfer function methods (19.0 +/- 3.1-5.3 +/- 0.9; control vs. verapamil infusion, p < 0.001), and increased cardiovascular variability as estimated both by standard deviation in mean arterial blood pressure (2.0 +/- 0.1-4.0 +/- 0.4 mm Hg) and standard deviation in heart rate (6.5 +/- 1.0-9.8 +/- 1.1 bpm; p < 0.05). Verapamil increased heart rate (+3%; p < 0.05), reduced systemic mean arterial blood pressure (-12%; p < 0.05), and mean arterial blood pressure swings induced by increasing doses of phenylephrine bolus injections (-6% to -15%; p < 0.05). The reduction was larger for larger blood pressure ramps and exceeded the systemic arterial pressure reduction induced by verapamil infusion. A nonsignificant trend towards an increase in microcirculation was observed. CONCLUSIONS: Besides the direct cardiodepressant and vasodilatatory action of verapamil, its suppressive effect on baroreflex sensitivity should be taken into account, since this sensitivity might contribute to an increased risk of cardiac morbidity and mortality. PMID- 10726330 TI - Outcome in patients with intracerebral hemorrhage: predictors of survival. AB - BACKGROUND AND PURPOSE: The aim of this retrospective study was to determine in hospital mortality and morbidity secondary to intracerebral hemorrhage and to analyse variables considered to be significantly associated with survival in these patients. PATIENTS AND METHODS: The study cohort consisted of 135 consecutive patients with intracerebral hemorrhage, admitted to a large community hospital in the urban area of Vienna. The diagnosis of intracerebral hemorrhage was established in all cases with axial computed tomography. The following variables were analysed: age, sex, Glasgow coma score on admission, location of hematomas, intraventricular hemorrhage, neurosurgical interventions and medical complications. RESULTS: Sixty-seven (49.6%) of the 135 patients died, 50 (37%) of them during the first 4 days after the acute event, 13 within the 1st week and 4 within one month. In a multivariate analysis the risk of death was significantly increased by the presence of intraventricular hemorrhage (p < 0.01), a Glasgow coma score of 6 or less (p < 0.0001) and age greater than 60 years (p < 0.001). Gender, medical complications and surgical removal of hemorrhage with or without additional ventriculostomy did not correlate significantly with outcome while an infratentorial location of hematoma showed a trend (p < 0.15) towards a higher mortality. CONCLUSION: A Glasgow coma score of 6 or less on admission, age greater than 60 years and the presence of intraventricular hemorrhage appear to be predictors of mortality in patients with intracerebral hemorrhage. PMID- 10726331 TI - Acute renal failure and rhabdomyolysis after inadvertent intra-arterial infusion of excessive doses of epinephrine during cardiopulmonary resuscitation. AB - Severe renal dysfunction or even acute renal failure necessitating renal replacement therapy are rather infrequent observations in patients following cardiopulmonary resuscitation. A low flow situation alone does not seem to be sufficient for renal breakdown and in addition other factors, such as preexisting renal disease, severe infections or congestive heart failure must be present. We report a patient, in whom during cardiopulmonary resuscitation a central venous catheter was placed which inadvertently was located in the aortic arch. Through this malpositioned line increasing and finally excessive amounts of epinephrine (in total 150 mg) were injected because of inadequate therapeutic response. After finally successful resuscitation the patient developed rhabdomyolysis and acute renal failure, which required hemodialyis therapy. Intraarterial infusion of the vasoconstrictor catecholamine obviously caused a critical reduction in renal and skeletal muscle perfusion. Nevertheless, the patient was discharged from hospital in good neurologic condition and with normal renal function. PMID- 10726332 TI - [Nonlinear dynamics, chaos theory and wavelet analysis of the heart]. AB - Over the past years, techniques of nonlinear dynamics have gained increasing attention in cardiology. This kind of partly interdisciplinary research is supposed to provide a better understanding of cardiac disease, especially in the field of tachyarrhythmia. This article intends to introduce the principles of diagnostic procedures in terms of nonlinear dynamics, chaos theory and stochastic processes related to cardiology. An introduction to a new technique of wavelet analysis is given, and possible applications in the quantitative classification of high-risk patients for sudden cardiac death are discussed. PMID- 10726333 TI - [Primary stability of 2 PLIF (posterior lumbar interbody fusion) techniques--a biomechanical and finite element analysis]. AB - The purpose of this study was to compare the initial stiffness of two techniques for posterior interbody lumbar fusion by biomechanical and finite element analysis. Initial stiffness was tested under compression, torsion and shearing forces. The effect of an increasing initial stability by additional posterior instrumentation is proven by the biomechanical analysis and the finite element method. PMID- 10726334 TI - [Thoracic intervertebral disk displacement: a rare differential diagnosis of segmental irritation--guidelines for use of chiropractic therapy]. AB - Thoracic disc herniations rarely occur; they vary with respect to the clinical symptomatic and do not always lead to neurological deficit. A patient is being reported on with first symptoms of unspecific back pain. The patient has been frequently treated within 12 months (at regular intervals) exclusively by chiropractic manipulation without a considerable success. It is the objective of this case report that in persistent and therapy resistant back pain without obvious neurological deficite beside native radiographs further diagnostic investigation (MRI) is indicated. Concerning chiropractic manipulations (mobilisation techniques with impulse) this paper recommends testing maneuvers, the consideration of contraindications (structural lesions, neurological deficite) and emphasizes the use of atraumatic manipulation techniques. PMID- 10726335 TI - [Radiometric study of postoperative development of skull shape and cranial volume in children with craniosynostoses]. AB - Preoperative CT diagnostics seem indispensible for evaluation of hard and soft tissue situation in children with craniosynostoses. Regular postoperative control is limited because of general anaesthesia and radiation exposure. Therefore, cranial growth of these children is hardly metrically examined. A method is looked for to replace CT for this indication and to allow differentiated analysis of skull shape and intracranial volume. 15 children (7 trigonocephalies, 4 plagiocephalies, 4 syndromal craniosynostoses) were operated on using a standardized bilateral frontoorbital advancement. Using conventional pre- and postoperative skull roentgenograms, a craniometric analysis of skull length, height and broadth according to the modified method of Ebel [9] and Schmid et al. [22] was carried out for assessment of the skull shape and the intracranial volume. Skull shape and volume showed good development (depending on the synostosis form). The bilateral frontoorbital advancement proved to be an effective operation method. Validation of results was given by comparison with normal and patient populations known from the published literature. The presented easy, safe and exposure-reduced method of analysis should be used regularly in preoperative diagnostics and routine postoperative control of craniosynostoses. PMID- 10726336 TI - Factors influencing shunt dependency after aneurysmal subarachnoid haemorrhage. AB - In this retrospective analysis of 138 patients treated for ruptured aneurysms the development of shunt dependent hydrocephalus was evaluated regarding possible predictive factors. In 15 patients (11%) ventriculo-atrial shunt was implanted due to hydrocephalus. One predictive factor was the localisation of aneurysms as patients with hydrocephalus had PcoA aneurysms in 40% compared to 20% in the group of patients without hydrocephalus and only 7% compared to 28% MCA aneurysms. An other predictive factor was the severity of the subarachnoid haemorrhage (SAH) as 7 patients out of the 15 were graded Fisher IV on admission. Furthermore, an important predictive factor was the presence of acute hydrocephalus as 13 out of the 15 patients (87%) with shunt dependent hydrocephalus had acute hydrocephalus requiring external ventricular drainage. An other possible factor was the intraoperative opening of the lamina terminalis as in 73% of the patients with shunt dependent hydrocephalus compared to 82% in the group of patients without hydrocephalus this procedure was performed during surgery. The results suggest that shunt dependency is more likely after severe SAH especially in the presence of an acute hydrocephalus and in patients with aneurysms located in the basal cisterns. Therefore treatment of the acute hydrocephalus and possible the opening of the lamina terminalis could have a positive effect on the development of shunt dependent hydrocephalus after SAH. PMID- 10726337 TI - The role of MRI in the surgical selection of cerebral metastases. AB - BACKGROUND: About one third of patients operated on for a "single" cerebral metastasis diagnosed by CT scan have probably more than one lesion. In fact, Gd DTPA enhanced MRI has proved to be more sensitive than CT in detecting the number of cerebral metastases, reducing the number of patients candidate to surgery. METHODS: Thirty-five patients with a CT scan picture of a single cerebral neoplasm of presumable metastatic nature performed a Gd-DTPA enhanced cerebral MRI. In 25 cases MR images confirmed a single lesion, while in other 10 (28.6%) showed a number of metastases ranging from 2 to 6. In order to assess the utility of MRI in surgical selection of patients and then to reconsider the concept of operability limited to single brain metastases only, we compared the survival of the 25 cases who preoperatively performed MRI (MRI group) with that of other 25 consecutive patients operated on in pre-MR era (during a 24-month period), with a CT picture of single cerebral metastasis (non-MRI group). RESULTS: The median survival was 36 +/- 5 weeks for MRI group and 40 +/- 15 for non-MRI group. The Kaplan-Meier product-limit survival analysis and the log-rank test do not reveal any statistically significative difference of survival between the two groups. CONCLUSIONS: Even if the number of patients is limited for definitive considerations, our preliminary results seem to suggest that the further selection of surgical cases obtained with MRI could not impact significatively the survival. Therefore, in selected patients with multiple cerebral metastases a surgical treatment could be reasonable. PMID- 10726338 TI - Primary intradural pontocerebellar chordoma metastasizing in the subarachnoid spinal canal. AB - Chordomas that are entirely extraosseous and intradural are rare. Additionally subarachnoid spinal implantation from such a cranial, intradural chordoma has never been reported before. The authors present a case of a widespread primary intradural chordoma in the basal cisterns of a 48-year-old woman which shows seeding of neoplastic cells to the spinal leptomeninges. It is concluded that also in cases of intradural and intracranial chordomas a tumor staging should include the search for spinal subarachnoid metastases. PMID- 10726339 TI - [The laparoscopic treatment of ovarian formations]. PMID- 10726340 TI - [The corticosteroid therapy of premature rupture of the fetal membranes]. AB - Antenatal corticosteroids are now administered for the purpose of hastening maturation of the preterm infant's organs and tissues, thus reducing neonatal mortality and the incidence of respiratory distress syndrome (RDS). Our purpose was to determine the efficacy of maternal corticosteroid therapy on fetal maturation in cases with preterm premature rupture of the membranes. The rates of RDS and survival in low birth weight infants treated with corticosteroids is influenced by the duration of the premature rupture of the membranes and the duration of the corticosteroid therapy, as well as by the gestational age. PMID- 10726341 TI - [The diagnosis and management in suspected retained placenta in the late puerperal period]. AB - The aim of this retrospective study is to find out the reasons for RICU in late postpartum period and the reliability of methods for diagnosis of retained placental pieces. We investigated the medical records of all cases with RICU from 24th ours postpartum up to 2 weeks after it. The rate is 0.9% of all births. Hystological confirmation of retained placental tissue there are in 25% of cases. Our data shows that the anamnesis does not help in the diagnosis of retained placental tissue in late puerperium. Most often the curettage is undertaken because of hemorrhage and ultrasound data of retained placental tissue. At the same time the transabdominal ultrasound is of low diagnostic value for residua post partum. The febrility, the subinvolution of the uterus and the number of symptoms in given case are of no importance in the diagnosis of retained placental tissue. The suspicion in the integrity of the placenta after its birth is a reason for RICU in spite of well contracted uterus and lack of hemorrhage. As the diagnosis of retained placental tissue in late puerperium is uncertain the RICU should be undertaken after failed conservative management. PMID- 10726342 TI - [Single-layer or double-layer suturing of the uterine incision in cesarean section? The late prognosis]. PMID- 10726343 TI - [Human recombinant erythropoietin in the treatment of the severe anemia following labor (a preliminary report)]. AB - BUT: The present clinical study was undertaken to evaluate the efficacy, safety and tolerability of r-HuEPO in the treatment of severe anaemia post partum. MATERIALS AND METHODS: Five women received Eprex (Cilag), for five days, and iron and folic acid twice daily. Haematological indices were investigated on the 2d, 5th, 14th and 30th day. RESULTS: On the 30th day the mean haemoglobin concentration was 110 g/l. A rapid haematopoietic effect was shown by an increase in the number of reticulocytes. The peak reticulocytes counts was achieved on the fifth day. The blood pressure at the course of the puerperium was normal. There was no changes in serum electrolytes during treatment with Eprex. The women noted no side effect from rHuEPO. CONCLUSIONS: The use of rHuEPO may decrease the need for red cell transfusions in severe anaemia postpartum. PMID- 10726344 TI - [The active management of the placental period with Remestyp]. AB - The aim of this prospective study is to asses, the possibility to use Remestyp after the delivery of the head as a prophylaxis of blood lost during the third stage of labor. The material includes 209 cases: 82 with bolus dose of 0.2 mg Methergin, 54 cases with 10 ME Oxytocin, 32 parturition with 200 mg Remestyp, and 41 controls without any uterostonics during the placental period. The blood lost is assessed by gravimetric method. The results show that the total blood lost from the delivery of the neonate to two hours after it is significantly lower in active management of labor than in expectant one. The type of uterostonic is not essential in regard of total blood lost. The prophylactics of blood lost with Remestyp has best results in cases of stimulated with Oxytocin infusion labor. The active management of third stage of labor with Methergin or Remestyp has less complication than are used oxytocin or without uterostonics. The mean time for delivery of placenta is shortest in the group with Remestyp. The three uterostonics in mansion doses do not influenced significantly the artery pressure. Our experience demonstrates that Remestyp can be used for active management of third stage of labor and the results are as those with Methergin. Remestyp is preferable when are expected significant lacerations of the birth canal. PMID- 10726345 TI - [The use of hysterosonography and transvaginal sonography in combination with the progesterone test in asymptomatic women in risk groups in the postmenopause]. AB - The hysterosonography allows evaluation of the endometrial cavity by endouterine administration of sterile physiologic solution. We wanted to assess the role of the hysterosonography and the transvaginal ultrasonography in asymptomatic women in post-menopause in combination with progesterone test. The progesterone test was performed by border of the endometrial depth of 4 mm, we used progesterone tablets Primolut Nor for 10 days., by depth 5 mm and over. Under the border of 4 mm we assessed the endometrium as atrophic. When bleeding occurred we used transvaginal sonography in combination with hysterosonography and in this way were better visualised the sub-mucous uterine fibroids nodes and also very small lesions., and endometrial cancers. The transvaginal sonography allowed to measure better the endometrial depth and in combination with the hysterosonography provided very good information. One hundred postmenopausal women from the risk groups with diabetes, blood hypertension, obesity and treated with Tamoxiphen were subjected to transvaginal sonography by endometrial depth of 4 mm we used progesterone test. -so it was used by 5 mm and over When bleeding occurred we performed abrasio probatoria separata The hysterosonography was used as additional method for clearing and determining of some polyps and very small lesions. The sensitivity of the transvaginal sonography was compared with the hysterosonography and was nearly the same--91% resp. 94%. The specificity was 31% for the transvaginal sonography. The combining of the transvaginal sonography and hysterosonography increased the sensitivity up to 96% and the specificity up to 98%. The hysterosonography can be additional method for diagnose of focal lesions, because of the good shape and forms of the lesions, especially in some risk groups receiving Tamoxiphen, with diabetes mellitus, blood hypertension and obesity patients. PMID- 10726346 TI - [Gynecological interventions and cesarean section without suturing of the peritoneum (visceral and parietal)]. AB - The authors reveal advantages of sewing off technics of parietal and visceral peritoneum in gynaecologic operations and Caesarean sections compared with common routine technics of sewing up. This study is prospective, controlled and includes two years period. 170 cases of gynaecologic operations and 45 Caesarean sections are investigated. It is confirmed the role of preoperative microbiologic investigations of cervical secret and vaginal secret. On the base of results similar to world experience in this field authors recommend described technics to gynaecologists and obstetricians. PMID- 10726347 TI - [The most suitable tests for studying chlamydial infection in women with sterility]. AB - The study was carried out on 280 infertile women (185 with primary and 95 with secondary infertility), patients of the University Clinic of Gynecologic Endocrinology and Infertility, Sofia. The present study was designed to estimate which is the most suitable test for detection of chlamydial infection in women with infertility. Evidence of past chlamydial infection was determined by the presence of antichlamydial IgG antibodies by commercial ELISA test. From 196 of the patients examined, 101 were positive for antichlamydial IgG antibodies (62.7%). 84 women were examined by means of direct antigen test for Chlamydia trachomatis from the endocervix (DIF. ELISA). Tubal patient was examined by means of hysterosalpingography and/or laparoscopy. In 34 women with unilateral or bilateral tubal occlusion the endocervical direct antigen test was positive in 4 (11.8%) and in 30 women with patient tubes 2 were positive by this test (6.7) In 38 women with tubal occlusion 29 of the women with unilateral or bilateral tubal occlusion had a presence of IgG antichlamydial antibodies in their sera (76.3%) compared to 17 of 38 women with patient tubes (44.7%). The results of the present study indicate that the examination antichlamydial IgG antibodies in the sera of women with infertility has a better predictive value with regard to tubal pathology compared to the direct antigen test for detection Chlamydia trachomatis in the endocervix. Examination for latent infection or reinfection has to be carried out before the administration of more invasive methods for the management of infertility. PMID- 10726348 TI - [Sex behavior and contraception among the population of Romany origin]. AB - The modern and reliable methods for contraception are not sufficiently used in our country, especially among the Gypsy population, where the abortions and the births are relatively higher. Through a standardised interview information about the fertility and the familial status of the Gypsy population of Sofia, Pleven [correction of Eleven] and Sliven regions was gathered. The aim of the study is analysis of the sexual behaviour of the population in the given regions and the need of adequate contraception avoidance of unwanted gravidity. The results show that only 12% of the population know how to protect themselves of sexually transmitted diseases, 55% of the women have not knowledge about their own gravidity, 57.3% of them give birth of their first child at the age of 15-17, 78.1% do not use contraceptive means and 3.6% use harmful "protective" methods. PMID- 10726349 TI - [The treatment of hirsutism in adolescent girls with spironolactone]. AB - OBJECTIVE: The purpose of this study was to determine the antiandrogenic properties of spironolactone on hirsute adolescents. MATERIAL AND METHODS: Thirty four adolescents between 15-18 years were studied. The hirsute adolescents were treated with spironolactone from 4th to the 22d day of the menstrual cycle for 8 months. RESULTS: Clinical improvement became evident usually 3 months after initiation of treatment. After 8 months, hirsutism has improved in 24 (70.6%) of 34 adolescents. A decrease in testosterone levels occurred in all patients, even those with initially normal values. CONCLUSIONS: The encouraging results obtained with spironolactone suggest that it may proved to be the appropriate drug for treatment of hirsute adolescents. PMID- 10726350 TI - [The postcoital test: clinico-laboratory observations]. AB - The choice of optimal time, the technique and limitations of the post-coital test (PCT) have been described. They are of important significance for getting reliable information both concerning the cervical mucus, quality and migration of spermatozoa in female genital tract. It has been stressed that a negative PCT does not necessary exclude pregnancy. There are shown data for prognostic value of the PCT. The observation of more than 20 motility spermatozoa per HPF and higher frequency of pregnancy is common finding in comparison to low motility and number of spermatozoa in cervical mucus. The data support the hypothesis of "filtering" mechanism, excluding entrance of abnormal spermatozoa. Observing "shaking" phenomenon directs to searching immunological factor (local or systemic) against male gametes. PMID- 10726351 TI - [Inflammatory gynecological diseases as a social problem in women of reproductive age]. AB - Gynecological infections in women of reproductive age are connected with considerable economic losses both for diagnosis and treatment and for temporary disability. There are annually 12,000-15,000 pelvic infections in women of reproductive age in Bulgaria. Late sequel include infertility, ectopic pregnancy, chronic disability. The management of all these complications exceed considerably the expenses if proper diagnosis and treatment of the pelvic infections has been administrated. About 25% of the women who have had salpingitis are expected to be affected by infertility. The age group of women 20-25 years are the most affected. Most of these women have not accomplished their reproductive potential. The most frequent causes for pelvic infections are sexually transmitted pathogens and intrauterine manipulations--dilatation and curettage, IUDs. The necessity for control programs for prevention of sexually transmitted infections as well as education for diminishing unwanted pregnancy and abortions is pointed out. PMID- 10726352 TI - [Extra-neoplastic epithelial changes in cancer of the vulva]. AB - A retrospective morphological research is made on the material of 78 cases from vulva for a three years period of time. They are identified 35 cases with invasive carcinoma, 2 c. with noninvasive carcinoma, 19 c. with vulvar dystrophia and squamous intraepithelial neoplasia (VIN). The invasive carcinomas are divided: keratinizing squamous cell carcinoma--31 c.; nonkeratinizing squamous cell carcinoma--3 c; adenoid squamous cell carcinoma--1. In all operated patients are examined the changes in the tissues surrounding the tumor and in the underlying tissue. They are divided in: lichen sclerosus, squamous cell hyperplasia, mixed dystrophia and squamous intraepithelial neoplasia. The morphological variants of squamous carcinoma, the epithelial changes and the patient's age are paralleled. PMID- 10726353 TI - [A comparative efficacy study of the preoperative use of GnRH agonists in women with uterine fibromyomas]. AB - The aim of the study is to establish the efficacy of preoperative use of GnRH agonists in women with uterine fibromyomas. The study is a randomized prospective one and includes 34 patients, divided in two groups: group I--with preoperative application of GnRH agonists--Zoladex and group II--without medication. In Zoladex group amenorrhea was achieved in 76% of patients after 7-8 weeks of treatment. After a 3-months treatment with Zoladex Hb levels increased from 8.9 +/- 0.9 gl/l to 11.7 +/- 1.2 g/l; levels of serum Fe--from 7.3 +/- 4 mumol/l to 18.5 +/- 5 mumol/l. Total uterine volume decreased by 30% before surgery (from 328 +/- 85 ml to 233 +/- 61 ml), while myoma volume decreased by 39% (from 178 +/ 62 ml to 109 +/- 44 ml). Mean blood loss during surgery (hysterectomy) is definitely less in patients, treated with Zoladex--194 +/- 75 ml., compared to 287 +/- 102 ml in control group. The significant reduction in myoma volume in 6 patients due to presurgical treatment with Zoladex made smaller operation- myomectomy, possible. Side effects, related to GnRH agonist Zoladex, are well tolerated and transitory and did not lead to retreatment from the trial. PMID- 10726354 TI - [The correlation between the presence of antisperm antibodies and autoimmune processes in sterility]. PMID- 10726355 TI - [Myasthenia and pregnancy]. AB - The myasthenia is a neurological autoimmune disease affecting women in reproductive age. The combination of myasthenia and gravidity has two important aspects; reflection of gravidity on the neurological illness and the influence of the myasthenia on the course of the gravidity and the delivery. The authors present one case of myasthenia and gravidity and offer some practical advises: 1. narrow collaboration between the patient and the different specialists. 2. good information about the myasthenia evolution during the gravidity. 3. adequate therapeutic approach to the given case. PMID- 10726356 TI - [Diagnostic ultrasound and the intrauterine pessary]. AB - The aim of the authors is an analysis of the results in application of ultrasound technique in patients with intrauterine device (IUD). Using the ultrasound criteria for a correct and incorrect position of IUD, the criteria for an elevated risk for expulsion and contraindications for IUD application, the authors have used ultrasound technique for diagnosis in 65 patients with IUD. The causes for the "wrong" diagnosis were discussed: imperfection of the apparatuses, non-observance of the conditions for ultrasound examinations and lack of experience. Practical conclusions and recommendations for an increase of the effectively of this seemingly easy method for diagnosis and follow-up of the IUD were made. PMID- 10726357 TI - [The treatment of acute mycotic colpitis with ciclopiroxolamine (Dafnegin-CSC) in pregnant patients in the 10th lunar month]. AB - Thirty-four patients at the end of pregnancy were treated with ciclopiroxolamine (Dafnegin) vaginally for the period from January to October 1998. The effect of therapy was evaluated. One hundred women were examined and 34 (34%) of them had vaginal candidiasis. All patients were treated with ciclopiroxolamine (Dafnegin) vaginal creme once time daily for 14 days. Thirty-two patients (94.11%) were healthy, in two patients (5.88) candidiasis was reduced to minimal colonies. PMID- 10726358 TI - [The use of bromocryptin--Richter for the suppression of the onset of lactation after the interruption of pregnancy between the 14th and 28th gestational weeks- a preliminary report]. AB - The effect and side reactions of bromocriptin of the firm "Richter" were evaluated in this study. Fifteen women with interrupted pregnancy between 14 and 28 gestational week were explored. To prevent the onset of lactation treatment was started within a few hours after abortion in dosage 2.5 mg (one tablet) twice daily, for 14 days. The prolactin-lowering action began to take effect 2 hours after ingestion of the first tablet, peak after 5 hours, and maintained for 8-12 hours. In course of treatment 14 days in dosage two times daily, the lactation was inhibited in 100% of the cases, in 65% of them she was stopped at the first three days of therapy, in 15% of the cases she was stopped between 3rd and 7th day, and in 20%--after the 7th day of the treatment. The adverse reactions nausea, dizziness, vomiting, diarrhoea, constipation, headache and orthostatic hypotension were transitory. They were observed rarely, and during the first 3-5 days of treatment disappeared. PMID- 10726359 TI - [Laparoscopy-assisted vaginal hysterectomy--the surgical technic]. PMID- 10726360 TI - A gelatin sponge implantation model in swine for the determination of local immune reactions. AB - For in vivo determination of innate and memory immune reactions we have implanted sterile gelatin sponges subcutaneously in swine for the monitoring of the following immunological parameters: 1. Analysis of local cell population phenotypes after vascularization of the gelatin sponges using flow cytometry. 2. Comparative analysis of sponge-infiltrating cells after loading with viral antigen in primed as well as naive animals. 3. Performance of reverse transcription quantitative competitive PCR (RT-qcPCR) for the detection of porcine cytokine mRNA indicative for IFN-gamma, IL-2, IL-4, IL-8 and IL-10. The in vitro analysis, e.g. by re-exposure to viral antigens, allows the determination of cytokine reaction patterns of sponge derived cells, draining lymph node cells as well as PBMC of the same individual. Studies of innate reactions and modulation of cellular infiltration in transplanted gelatin sponges are possible. Functional assays, e.g. cell-mediated cytotoxicity, antigen specific cell proliferation, using sponge-derived cells will provide additional information about the suitability of the model for example in vaccine potency tests. PMID- 10726361 TI - [Food as a potential vector for antibiotic resistances. 2: Relevance of lactic acid bacteria]. AB - Transfer of antibiotic resistances via the food chain is possible through the ingestion of resistant parts of the original food microflora. Lactic acid bacteria (especially glycopeptide resistant enterococci) are considered as important vectors because of their ability to transfer resistances by genetic mechanisms. Therefore a literature review and own investigations concerning the incidence and the resistance profile of enterococci from fresh meat were performed. The isolates harboured in part resistances relevant for human medicine. However, they could be isolated only sporadically and could not be demonstrated quantitatively in most cases. The resistance profile differed from those of human clinical origin. These results were confirmed by other authors. Additionally other investigators could prove molecular differences compared to clinical strains. Therefore food can only be considered as a vector if resistance transfer from food isolates to pathogenic microorganisms is possible. Such a transfer could be shown only in very low frequencies. In conclusion so far lactic acid bacteria cannot be considered as the main source for the incidence of antibiotic resistances in man. PMID- 10726362 TI - [Relevance of infection with equine herpesvirus 1 (EHV-1) in a German thoroughbred stud: vaccination, abortion and diagnosis]. AB - The aim of the present study was to clarify whether an EHV-1 induced abortion can be prognosticated by an increase of antibody titres, virus shedding and/or viraemia and whether the current abortion diagnostic is suitable. In this context the immune response post immunization and a possible reactivation were of great interest. For this purpose blood samples of 32 mares between the ages of 5-21 years were regularly investigated during a period of two years before and after vaccination and pregnancy. Neutralization tests, indirect immunofluorescence tests as well as PCR and virus isolation were used for EHV-1 diagnostics. It could be shown that the horses reacted individually to vaccination. In 14 cases a EHV-1-reactivation was suggested. An abortion prognosis was not possible even using serological, virological and molecular biological parameters. In addition, virus shedding and antibody titres were individual. An acute infection was detectable by a significant rise of antibodies and viraemia as well as virus shedding in the secretions. For the abortion diagnostics the antigen detection in combination with virus isolation and PCR from fetal lungs gave reliable results. In addition, the virological and serological investigation of the mare is recommendable. For prophylaxis we would advise a regular vaccination and strict hygiene. PMID- 10726363 TI - [Effect of a high single subcutaneous dose of unfractionated heparin on platelet function in dogs]. AB - The influence of heparin on different tests of platelet function was investigated in 5 healthy dogs receiving subcutaneously 1000 I.E./kg BW of a commercial unfractionated Sodium heparin preparation. Blood samples were collected before and 4 hours after heparin injection. Besides plasma activity of heparin platelet count, capillary bleeding time with two different methods, platelet aggregation according to Breddin and to Born with the inductors ADP, collagen, and thrombin as well as in vitro bleeding time were measured. The activity of heparin four hours after the subcutaneous heparin application was 1.20 +/- 0.11 I.E./ml. Compared to the starting values no significant influence of this heparin level could be demonstrated on platelet count, platelet aggregation according to Born with the inductors ADP and collagen, platelet aggregation according to the Breddin method as well as in vitro bleeding time (p > 0.05). Only one of the two methods used for measuring the capillary bleeding time showed a slight prolongation compared to the starting value (mean = 77 sec) to 88 sec (p < 0.05). The most significant influence was seen on the platelet aggregation induced by 1 I.U./ml thrombin, whereby the aggregation maximum decreased from 92.0% (mean) to 12.2% (p < 0.001). Whereas the latter result has to be interpreted primarily as a consequence of the anti-thrombin effect of heparin, the results of the other tests in summary illustrate the clinical unimportant influence of heparin on platelet function in dogs. PMID- 10726364 TI - Exclusion of strain 670/89 as type strain for serovar 20 of Riemerella anatipestifer. AB - Classical phenotypic characterisation and numeric analysis of whole-cell fatty acid patterns of twenty-one type strains of hitherto reported Riemerella anatipestifer serovars revealed that the type strain of serovar 20 does not belong to the species Riemerella anatipestifer sensu stricto and, therefore, it has to be excluded as a representative Riemerella anatipestifer serotype strain. PMID- 10726365 TI - [Action of the vaccination of sows against E. coli infections with a subsequent postpartum booster]. AB - In an eastern european pig productions unit with high prevalence of suckling piglets diarrhoea during late lactation the following trial was conducted: The sows were assigned to an experimental and to a control group and were treated as follows: Group one (15 sows) were vaccinated with a single 2 ml dose of Porcovac Plus (Hochst Roussel Vet.) during their late pregnancy (gilts were vaccinated twice). Booster vaccination was performed between day 2-7 p.p. Group two (15 sows) were vaccinated during their late pregnancy the same was as the sows in the group one, but received no p.p. Booster. The following parameter were evaluated. A: Preweaning diarrhoea B: Preweaning mortality C: Three weeks weaning litter weights D: Postweaning mortality E: Average weight gain during the first 3 weeks postweaning The results revealed a marked difference between the groups (group 1: 16.1% vs. group 2: 23.3%) regarding preweaning diarrhoea (parameter A). A similar difference was to be seen regarding parameter B (preweaning mortality) between the groups (group 1: 7.5% vs. group 2: 10.7%). In spite of this there was a non significant difference as regards weaning litter weights (parameter C) between group 1: 59.2 +/- 2.4 kg and group 2: 57 +/- 2.2 kg. Postweaning parameters showed better results in the booster vaccinated group regarding both evaluated parameter as well. Regarding piglet mortality (parameter D) there was a marked difference between the group 1 (0.67%) and group 2 (2.1%) to be seen. Regarding average daily gain (parameter E) between group 1 (470 +/- 11 gr) and group 2 (380 +/- 9 gr) there was a significant difference (p < 0.05) to be discovered. It is the opinion of the authors that p.p. Booster vaccination of the sows is an economically important tool in large and small pig production units. PMID- 10726366 TI - Caprine-associated Q fever in Newfoundland. PMID- 10726367 TI - Reptile-associated salmonellosis--selected states, 1996-1998. PMID- 10726368 TI - Restaurant-associated outbreak of Salmonella typhimurium phage type 1 gastroenteritis--Edmonton, 1999. PMID- 10726369 TI - Update: respiratory syncytial virus activity--United States, 1998-1999 season. PMID- 10726370 TI - Outbreak of Yersinia pseudotuberculosis in British Columbia--November 1998. PMID- 10726371 TI - Direct costs attributed to chickenpox and herpes zoster in British Columbia--1992 to 1996. PMID- 10726373 TI - Progress towards global poliomyelitis eradication, as of May 1999. PMID- 10726372 TI - First isolation of Lyme disease spirochete, Borrelia burgdorferi, from blacklegged tick, Ixodes scapularis, removed from a bird in nova Scotia, Canada. PMID- 10726374 TI - [The progression of hepatic fibrosis in chronic hepatitis C]. AB - The natural history of liver fibrosis in 47 untreated patients with chronic hepatitis C and no cirrhosis, as well as the factors associated with its progression, were evaluated by examining two consecutive liver biopsies, separated by an average interval of 64.8 +/- 62.9 months (12-244). In all the biopsies, the degree of necroinflammation and the stage of fibrosis were determined on a scale from 0+ to 4+. In 53% of the patients, liver fibrosis did not progress. The interval between biopsies was significantly higher in those who progressed from one stage to another (85 +/- 77 months) than in those who did not (41 +/- 27 months), p = 0.014. Two factors were independently associated with a greater risk of fibrosis progression: a history of daily alcohol intake > or = 80 g (p = 0.02) and having acquired an infection through a known parenteral mechanism such as blood transfusion, major surgery or hemodialysis (p = 0.018). Necroinflammation was significantly diminished in the second biopsy due to a lesser necrosis and inflammation of the lobules. IN CONCLUSION: a) liver fibrosis progression is independent of necroinflammation; b) progression is related to the duration of the disease and with the mechanism of transmission, and c) it is aggravated by excessive alcohol consumption. PMID- 10726375 TI - [Fatal infectious mononucleosis during azathioprine treatment in Crohn's disease]. AB - The benefits of immunosuppressive agents in inflammatory bowel disease have been the subject of controversy. However, extensive clinical data have conclusively proved their efficacy with respect to potential toxicity. Azathioprine is one of the most widely used immunosuppressive agents in the treatment of Crohn's disease and its effectiveness has been proved in corticosteroid dependent, corticosteroid refractory and fistulizing Crohn's disease. We present a 24-year-old male, treated with azathioprine for corticosteroid dependent Crohn's disease who was admitted to hospital with constant fever, lymphadenopathy and liver function abnormalities secondary to infectious mononucleosis after a primary infection with Epstein-Barr virus. The patient developed constitutional symptoms and progressive jaundice and 14 days after hospital admission died because of massive upper gastrointestinal bleeding due to hemorrhagic erosive gastropathy. PMID- 10726376 TI - [Multiple hepatic focal nodular hyperplasia: its presentation in childhood and atypical evolution]. AB - Multiple focal nodular hyperplasia is an uncommon benign liver tumor although its incidence has been increasing in the last few years. A case of focal nodular hyperplasia in a young woman is described, which was discovered in infancy and which presented two nodules in each lobe. The diagnosis was subsequently confirmed by large surgical biopsy. Conservative therapy was given for 16 years during which time there was progressive tumor growth, increase of pain and cholestatic enzymes. The unusual presentation this benign lesion may have, a strategy for its diagnosis and the generally conservative management that is currently favored are discussed. PMID- 10726377 TI - [The prolonged administration of intravenous immunoglobulins as a treatment for refractory fistulous Crohn's diseases]. AB - Fistulating Crohn's disease is present in 17-35% of non-surgically treated patients and in up to 45% of surgically treated ones. Among the several therapeutic alternatives for this disease is intravenous immunoglobulin administration. We present a 28-year-old woman with refractory fistulating Crohn's disease who improved after prolonged immunoglobulin administration (32 months). PMID- 10726378 TI - [Pseudoachalasia: a diagnosis to consider in the assessment of dysphagia]. AB - The diagnosis of achalasia is based on the patient's symptoms and on manometric studies although other diseases present similar symptomatology. To present an elderly woman clinically and manometrically diagnosed with achalasia of the cardia who was treated by dilatation. When the patient relapsed and was surgically treated she was found to have and esophageal adenocarcinoma. When considering a diagnosis of achalasia, alternative diagnoses (especially neoplasias) should be borne in mind, especially in unusual situations, such as rapid onset or in the elderly. PMID- 10726379 TI - [Antineutrophil cytoplasmic antibodies (ANCA) in chronic inflammatory intestinal disease]. PMID- 10726380 TI - [Prolonged survival following limited surgery in cholangiocarcinoma]. PMID- 10726381 TI - [The remission of autoimmune hepatitis during pregnancy]. PMID- 10726382 TI - [Barium-induced peritonitis. An infrequent but persistent picture]. PMID- 10726383 TI - [Steatosis and nonalcoholic steatohepatitis. A comparative analysis]. AB - Liver biopsies with a main histological diagnosis of steatosis were selected from 3,422 liver biopsies carried out in our department between January 1995 and December 1998. Patients with known risk factors for steatosis, such as excessive alcohol consumption, hepatitis C infection, treatment with amiodarone, perhexiline maleate, tamoxifen, antiviral drugs (didanosine, zidovudine) methotrexate, sodium valproate or total parenteral nutrition, Wilson's disease and organ transplant were subsequently excluded. Of the 43 liver biopsies finally included in the study, 23 showed simple steatosis and 20 steatohepatitis. Eighty one per cent of the patients were male (mean age of 44 years) and the majority were asymptomatic. The most frequent indication for liver biopsy was hypertransaminasemia. No differences were observed between the two groups in terms of frequency and severity of classical risk factors for steatosis (diabetes mellitus, dyslipemia and obesity). Thirty-five percent of patients with steatohepatitis and 26% of those with simple steatosis had none of these risk factors. Patients with steatohepatitis were older than those with simple steatosis. They presented more severe symptomatology, the degree of steatosis was more intense and laboratory investigations showed greater alterations. These results suggest that simple steatosis and nonalcoholic steatohepatitis are two different phases of the same disease. The difficulty in clinical differentiation justifies carrying out liver biopsy, especially in patients with more severe symptomatology whose laboratory results show greater alterations, since these patients present more marked histological lesions, are at risk of developing liver cirrhosis and require therapy. PMID- 10726384 TI - [Does eliminating Helicobacter pylori mean the healing of the duodenal ulcer? The results of a prospective study in Spain]. AB - AIM: To evaluate the predictive value of Helicobacter pylori eradication in the healing of duodenal ulcer. METHODS: A prospective study of 92 duodenal ulcer patients (diagnosed by endoscopy) with H. pylori infection demonstrated by rapid urease test, positive histology and culture. They were treated with a 7-day regimen of lansoprazole, clarithromycin and amoxicillin. Healing was evaluated in a follow-up gastroscopy performed one month after treatment had finished. Eradication was defined as negative urease test, histology and culture at 30 days and negative urea breath test at 60 days. RESULTS: Duodenal ulcer healing was observed in 85 patients (92.4%, CI 85-96.9). Eradication of H. pylori infection was the only variable independently associated with ulcer healing. Healing was observed in 97.2% of patients with H. pylori eradication versus 75% of those with persistent infection (p < 0.01; OR = 11.6; CI 95% = 2.06-65.9). CONCLUSION: Eliminating H. pylori infection favors duodenal ulcer healing and, from a clinical point of view, confirmation of H. pylori eradication almost always means healing of duodenal ulcer. PMID- 10726385 TI - [The prediction of the endoscopic diagnosis in the dyspepsia patient: the value of the predominating presenting symptom and the initial clinical presumption]. AB - AIM: To assess, in our patient population, whether upper gastrointestinal symptoms can be used to predict the presence of relevant pathology on endoscopy. PATIENTS AND METHODS: In the first retrospective phase, 311 gastroscopies, indicated for dyspeptic symptoms were selected. The gastroscopies were performed by the same endoscopist from April 1998 to March 1999. Patients were divided into four groups according to the main presenting symptom (epigastralgia, epigastralgia with pyrosis, pyrosis with nonspecific symptoms). The positive and negative predictive value, as well as the overall sensitivity and specificity of each group of symptoms for the presentation of a relevant pathology were calculated. In the second, prospective, phase an attempt was made, based on the clinical history taken in the initial consultation, to predict the results of the gastroscopies of 77 dyspeptic patients. There were four possibilities: normal or not relevant, esophagitis, ulcer and neoplasia. The positive predictive value, negative predictive value, sensitivity and specificity of the diagnostic hypothesis were calculated. RESULTS: a) Retrospective phase: the overall positive predictive value of dyspeptic symptoms to predict relevant pathology was 0.38. The highest positive predictive value corresponded to symptoms of pyrosis (0.48). Dividing the patients into groups according to age (older or younger than 45 years) did not improve the predictive value of symptoms. b) Prospective phase: the highest positive predictive value corresponded to "pathology without relevance" (0.7). The prediction of ulcer only gave a positive predictive value of 0.55. CONCLUSION: The main presenting symptom is not a good predictor of whether relevant pathology will be found on gastroscopy. The initial diagnostic hypothesis is not a sufficiently effective tool to accurately detect which patients will most benefit from gastroscopy. PMID- 10726386 TI - [The clinicopathological characteristics and prognosis of tumors of the gastrointestinal stroma]. AB - Gastrointestinal stromal tumors form a small percentage of digestive tract tumors. They have recently been the subject of a new histopathologic classification made possible by immunohistochemical techniques. A series of criteria has been proposed which enables the identification of the most malignant and clinically aggressive tumors. We present five patients who were diagnosed with gastrointestinal stromal tumor, each of which had distinct characteristics. The different diagnostic tests performed, the difficulties of reaching a diagnosis as well as the treatment and distinct behavior of these tumors are discussed. PMID- 10726387 TI - [Lymphocytic colitis: hypopotassemia as a complication and an association with toxic multinodular goiter]. AB - Lymphocytic colitis is a rare clinicopathologic syndrome, characterized by chronic watery diarrhea, diffuse inflammatory changes in the colonic mucous in spite of normal findings on colonoscopy and marked intraepithelial lymphocytic infiltration on biopsy. Although the physiological mechanism of diarrhea is not clear, patients do not usually present hydroelectrolytic alterations and the results of routine laboratory investigations are usually normal. The association between lymphocytic colitis and thyroid disease, possibly autoimmune, in the form of hypo- or hyperthyroidism is relatively common. We report a 61-year-old woman with a history of multinodular toxic goiter, whose previously uninvestigated chronic diarrhea became more acute and led to the diagnosis of lymphocytic colitis. Results of laboratory investigations revealed only a significant hypokalemia with an associated nonfunctioning bilateral adrenal incidentaloma. The patient evolved well when treated with sulfasalazine. Hypokalemia as a complication of lymphocytic colitis and an association between lymphocytic colitis and toxic multinodular goiter does not seem to have been previously described. PMID- 10726388 TI - [Acute necrotizing esophagitis]. AB - Acute necrotizing esophagitis is a rare disease. Its pathogenesis is influenced by situations of low systemic perfusion, such as hypertension, heart failure or sepsis, in which other factors, such as the application of a nasal tube, infections or drugs also play a role. We present a case of acute necrotizing esophagitis in a patient with copious vomiting, renal failure, gastric hemorrhage due to Mallory-Weiss syndrome and esophageal infection due to Actinomyces. The patient was undergoing coadjuvant chemotherapy for a surgically-treated colonic neoplasia. Maintenance therapy produced favorable evolution with restoration of esophageal epithelium and no stenotic complications. PMID- 10726389 TI - [Esophageal perforation and postoperative fistulae of the upper digestive tract treated endoscopically with the application of Tissucol]. AB - We present three patients with serious gastro-esophageal complications which were treated with Tissucol. The first patient developed a rare postoperative oesophago pleural fistula. The second suffered a traumatic esophageal perforation (possibly iatrogenic) that was discovered at the end of the removal procedure of an alimentary bolus impacted in the distal esophagus. Attempts to close the high output oesophago-pleural fistula with standard treatment were unsuccessful. It was closed with Tissucol at the third attempt, in conjunction with oesophago jejunal stenosis by means of endoscopic dilatation. In the second patient, early Tissucol application after detection of pneumomediastinum was an effective complementary treatment to the conservative approach and rapidly closed the perforation. The third patient developed a low debit postoperative gastro cutaneous fistula that did not resolve with conservative treatment. It was closed with only one session of Tissucol sealing. We consider that the endoscopic application of fibrin glue should become the first step in the conservative treatment of small esophageal perforations or postoperative esophageal pleural fistulae, especially in cases of high output fistulae. The success of this technique depends on the localization and selective catheterization of the fistula and on brushing the fistular opening. Total resolution of any distal stenosis is necessary to prevent reopening of the fistula. PMID- 10726390 TI - [Finally anti-inflammatory agents less damaging to the stomach?]. PMID- 10726391 TI - [The cell line associated with ulcer and trefoil peptides: a system of gastrointestinal mucosal repair]. PMID- 10726392 TI - [Helicobacter pylori and ischemic cardiopathy]. PMID- 10726393 TI - [The sequelae following thrombolysis in mesenteric embolism]. PMID- 10726394 TI - [Duodenal diverticulitis]. PMID- 10726395 TI - [Dysfunction and manometry of Oddi's sphincter]. PMID- 10726396 TI - [The 1998 Federal Health Survey. Experiences, results, perspectives]. PMID- 10726397 TI - [The Federal Health Survey: response, composition of participants and non responder analysis]. AB - The first German Health Survey was carried out from October 1997 to March 1999. In the survey, 7,124 subjects of a representative sample of the residential population aged between 18 and 79 years were interviewed and medically examined. The response rate was 61.4%; 0.4% of the sample participated only partly in the study. Moreover, 16.0% of the non-respondents filled in a short questionnaire. This results in (although partly limited) information from 77.8% of the sample. The sample also comprises aliens living in Germany. More than 15% of the non respondents could not be addressed to participate in the study because they were never reached personally. According to the statements in the short questionnaire, non-respondents and respondents differ in particular items such as e.g. education. The differences between the individual answers concerning the important question of the general health status can be classified as negligible. Weighting factors were calculated to arrive at representative information on the 18 to 79 year-old population. PMID- 10726398 TI - [Process and results of field work concerning the Federal Health Survey]. AB - "I + G Gesundheitsforschung" was responsible for data acquisition and data processing of the survey, which included the production of the survey documents and the conceptualization and production of the operation manual. Furthermore, the institute's task was sample design and sample drawing. This article reports on the continuity and the results of the field work and includes experiences and realizations resulting from the field work. These might be helpful for similar surveys in the future and contribute to the improvement of the procedures. PMID- 10726399 TI - [External quality control in the Federal Health Survey: concept and site visits]. AB - The results of the German National Health Interview and Examination Survey will be of great importance for health policy and research. Therefore, internal quality management was supplemented by an external quality control which was carried out by a private company (Bernhard Schwertner Feld organisation, Augsburg). The interviewer training, sampling and response, field work and data management were included into the external quality control. For each of these four areas, measures of control and detailed check-lists were provided by the external quality control. The aim was to find sources of potential and real errors and to arrive at recommendations for internal quality management. The general concept of external quality control is briefly presented and experiences and results from the quality control of the field work are described in the present paper. PMID- 10726400 TI - [Incidence of myocardial infarct in Germany: prevalence, incidence trends, East West comparison]. AB - The German National Health Interview and Examination Survey, as a descriptive cross-sectional study, allows the recording of post-myocardial infarct cases, e.g. the number (prevalence) of survivors after an infarct has occurred (non lethal myocardial infarcts). The 18 to 79 year old residential population in Germany had a lifetime prevalence of 2.45% for conditions after a heart attack. The age-specific lifetime prevalence values increase with increasing age for both men and women. Between the ages of 30 and 59 there are more than 4 male heart attack victims for every woman; between the ages of 60 and 79 this relation is only 1 to 1.5. The 30-< 80 year old population in Germany has around 1,450,000 post-myocardial infarct cases (heart attack victims). There are about 100,000 male post-myocardial infarct cases among relatively young adults between 30 and 49 years of age. There were hardly any cases in the female population in this age group. In regard to post-myocardial infarcts there were no significant morbidity differences between the eastern and western German states in 1997/98. A comparison of the lifetime prevalence rates between 1997/98 and 1990/92 shows the following trend: the number of post-myocardial infarcts in the German population 25-< 70 years old decreased; in Western Germany the lifetime prevalence rates also declined, in Eastern Germany the prevalence rates among both men and women increased in this period of time. In the period 1997/98 there were around 190,000 non-lethal myocardial infarcts in the 18 to 79 year old German population in a full 12 month period (incidence cases of survivors after the occurrence of an acute first and/or acute reinfarcts). PMID- 10726401 TI - [Stroke: prevalence, incidence, trends, East-West comparison. Initial results of the 1998 Federal Health Survey]. AB - The prevalence of survival after a stroke is dependent on the incidence and the fatality rate, whereby the incidence and the fatality rate are influenced by different factors. A survey can only include the minor cases of post-stroke conditions. The prevalence figures thus only reflect the less serious or rehabilitated strokes. Despite this underrepresentation of strokes in the survey we can observe an underestimation of the stroke problem in Germany. Whereas we previously assumed 440,000 to 500,000 strokes in Germany, the projection from the data of the German National Health Interview and Examination Survey amounted to around 945,000 cases (only strokes with "minor" motor, sensory and cognitive losses and restrictions which allow a participation in the survey). The lifetime prevalence rate of the 18-< 80 year old female population is somewhat higher than the male population of the same age (n.s.). The 50 to < 60 year old men have a relatively high prevalence rate. The corresponding age specific prevalence rates increase with increasing age. There are no significant differences in morbidity between the former East and West German states, the prevalence rates of men are somewhat higher in the East and those of women in the West. We can see the following trends in a comparison of the lifetime prevalence rates between 1997/98 and 1990/92: the number of post-stroke conditions among German men 25-< 70 years old declined significantly, among women they increased slightly (n.s.); the prevalence as a whole also declined significantly among men in western Germany, among women they increased slightly (n.s.) in contrast to former West Germany the prevalence rates among men in eastern Germany increased slightly, among women they were almost cut in half. 32.8% of the population with "minor" post-stroke conditions is characterized by sensory disruptions, 32.1% by impairments when walking, 31.3% by paralyses, 20.5% by speech impairments, 17.1% by cognitive disorders and 3.1% by disturbances of consciousness. PMID- 10726402 TI - [Prevalence of diabetes mellitus in the adult German population]. AB - In the German National Health Interview and Examination Survey 7,124 subjects of a representative sample of the 18 to 79 year old population having their residence in Germany were interviewed and medically examined. Using a self administered questionnaire as well as a subsequent personal interview by a physician, the participants were questioned regarding past and present diseases. Based on the data of the physicians' interviews, the prevalence rate for diabetes was 4.7% for men and 5.6% for women in the examined age-group. There is a strong increase of the prevalence with age. According to the present data nearly every fifth women in the age range from 70 to 79 years is suffering from diabetes mellitus. The disease is much more frequent in the new federal states (former GDR) than in the old ones (former West Germany). About one fourth of the diabetics uses insulin, far more than 40% are on oral antidiabetics. According to the interrogation of the physicians, about half of the diabetics not using any drugs are not even on a diet (ca. 15%). The portion of undetected diabetics in the examined population is estimated to be about 1% considering the values of blood and urine parameters (glucose in serum and urine, fructosamine, HbA1c). PMID- 10726403 TI - [Blood pressure in Germany--current status and trends]. AB - The blood pressure measurements performed in the course of the German National Health Interview and Examination Survey 1998 showed higher levels in men compared to women and higher levels in the Eastern part of Germany as compared to the West. Generally, blood pressure increased with age. The prevalence of hypertension was higher in men, reaching almost 30%, as compared to women (26.9%), and higher in the East compared to the West. A comparison of data of 1998 and data of the Health Examination Survey East-West 1991 reveals an increase in the incidence of hypertension in the West and a decrease in the East. This seems to imply a convergence of the two regions on a high level. PMID- 10726404 TI - [Prevalence of hay fever in Germany--East-West comparison and temporal trends]. AB - In recent years several studies in children and adults have shown an increase in prevalence of atopic diseases in East and West Germany. The observed frequency of allergic diseases, however, was significantly lower in the East compared to the West. Using data of the German National Health Interview and Examination Survey 1998 and of National Surveys from 1990/92 it was examined, whether the reported increase in prevalence could be confirmed for the total population and whether the differences between East and West are still present. In a self-administered questionnaire, study participants were asked whether they have ever had hay fever. Additionally, in a physician's interview, subjects were asked whether a physician had ever diagnosed hay fever. The questionnaire data were used in comparison with the previous national surveys for the calculation of time trends. A total of 6974 persons filled in the questionnaire and 7099 persons took part in the interview. Physician-diagnosed hay fever was reported by 15% of the total study population. Clear differences in the prevalence rates between East and West Germany are still existing; 11% in the East and 17% in the West suffer from hay fever. In both parts of the country prevalence decreases with increasing age. The highest rates were found among those aged 20-29 and 30-39 years. Based on the questionnaire data the morbidity rose from about 10% in 1990/92 to 17% in 1998. The overall relative increase is quite comparable in East and West Germany. Stratification by age and gender shows considerable differences. In young women from East Germany the increase in prevalence is substantially higher and in women aged 40 years or older much lower than in West German women. In men this pattern has not been observed. PMID- 10726405 TI - [Incidence of allergic diseases in East and West Germany]. AB - Using data of the German National Health Interview and Examination Survey 1998, a remarkable difference in the prevalence of hay fever between East and West Germany has been observed. Several studies in children and adults have also shown a considerable East-West divergence in other allergic diseases and in sensitisation rates. The aim of this investigation was to examine whether in a representative sample of the adult German population East-West differences in the frequency of asthma, atopic dermatitis, food allergy, urticaria, contact dermatitis and "other allergies" can be found. The calculations base on data of a physician's interview in which study participants were asked whether a physician had ever diagnosed one of the above mentioned diseases. A higher prevalence of all allergic diseases has been observed in West compared to East Germany and women from both parts of the country have higher morbidity rates than men. At least one physician-diagnosed allergy was reported by 40% of the study participants, whereas in East Germany about 30% and in the West 43% suffer from an allergic disease. The prevalence in women is 47% and in men 33%. Extremely high allergy rates were found among West German women at the age of 30-39 years (62%). Although the frequency of allergies decreases with increasing age, considerably high morbidity rates were ascertained even in the oldest age groups. The prevalence in participants aged 70-79 years amounts to 25%. Clear differences between East and West could be demonstrated in this age group, too (West 27% and East 14%). PMID- 10726406 TI - [Vaccination status and illnesses in foreign travelers]. AB - Millions of German citizens travel outside Europe every year. How they prepare themselves for these travels from a medical viewpoint, or whether they fall ill during these travels is inadequately researched. The German National Health Interview and Examination Survey showed that 11% of the people questioned travelled to Africa, Asia, South or Central America within the last three years. The highest proportion of these travellers was in the 20-59 year age group. Men travelled more frequently than women. The travellers also had better vaccination coverage against tetanus and polio compared to those who had not travelled. 30% of the travellers to endemic areas for malaria had received malaria prophylaxis and 30% who travelled to endemic areas for yellow fever were protected by vaccination. 28% of those questioned said they experienced general health problems related to their travel, mostly due to mild diarrhea. PMID- 10726407 TI - [Prevalence of antibodies to hepatitis A, hepatitis B and hepatitis C viruses in the German population]. AB - Representative random samples were tested for hepatitis A, hepatitis B and hepatitis C infections within the framework of the German National Health Interview and Examination Survey. The laboratory parameters included determination of anti-HAV, anti-HBc, anti-HBs, HbsAg, anti-HCV and hepatitis C virus RNA. The prevalence rate for anti-HAV was 46.5% with a definite age dependence. The infection rates for hepatitis B of 7.7% in former West Germany and of 4.3% in former East Germany were obtained. This is equivalent to a total of 7% prevalence rate. The HbsAg carrier rate was 0.6%. Hepatitis C virus antibodies showed a prevalence rate of 0.4%. PMID- 10726408 TI - [Anthropometric data and obesity]. AB - In the German National Health Interview and Examination Survey 1998 several anthropometric data were obtained from 7124 men and women, aged 18-79 years. These data were analysed and compared with 1990/92 survey data. On average, people form the Western part of Germany are somewhat taller than those from the Eastern part, the differences being smallest in the youngest age group. With the use of the Body Mass Index (BMI) as the criterion, the prevalence of overweight (BMI > or = 25 kg/m2) ranges from 52% for West German women to 67% for West German men, and of obesity (BMI < or = 30 kg/m2) from 18% for West German men to 24.5% for East German women. Generally, overweight is more prevalent in the East than in the West. In the male population, aged 25-69 years, the prevalence of obesity increased by 5.9% in the East and by 11.5% in the West during the last decade. Among females the prevalence of obesity increased by 6.4% in the West, but decreased by 6.3% in the East. Still obesity is more prevalent among East German females. Since obesity is a key health risk, these recent German prevalence figures are alarming. PMID- 10726409 TI - [Smoking behavior in Germany]. AB - In a representative sample, 7,124 men and women in the age of 18 to 79 years were interviewed regarding their smoking habits. In 1998, one third of the participants, 37% of the men and 28% of the women, were smokers. The highest proportion of smokers was found in the youngest age group of 18 to 24 years--49% males and 44% females. The mean cigarette consumption of male smokers was 20 cigarettes per day, that for female smokers was 16. About one third of the smokers have tried to stop smoking at least once during the past year. 86% of the men and 80% of the women started their habit under the age of 20. Compared to a previous survey in 1990/92, the proportion of male smokers dropped by 3% in West Germany, whereas in East Germany the figure remained the same. The proportion of female smokers rose by 1% in West Germany and by 8% in East Germany (from 21% to 29%). The latter change means an increase of the smoking prevalence by 42% in East German women during the short period of only 7 years. PMID- 10726410 TI - [Physical activity]. AB - The questionnaire data of the German National Health Interview and Examination Survey 1998 were used to determine the actual physical activity level in Germany. With the use of similar data from the National Health Surveys 1990/92 changes in activity level over time were estimated. At present, a large part of the population is sedentary during leisure time. Among men this proportion is larger in the eastern part and among older women in the western part of Germany. The proportion of young sedentary women is, however, smaller in the eastern part of Germany. The proportion of men engaged in sport for two hours per week or more, is larger in the western part of Germany. Among West and East German women this proportion is almost equal, although among younger women more are active in the eastern part. During the last seven years, the proportion of sedentary men younger than 50 years of age has grown, whereas the same proportion has declined among those over 50 years of age. Among women a similar decline is apparent above 30 years of age and more pronounced as it is for men. In general, the proportion of active men and women engaged in sport during leisure time for more than 2 hours per week has increased. PMID- 10726411 TI - [Intake of dietary supplements and nutritional behavior]. AB - In recent years there has been a significant increase in the use of dietary supplements in Germany. Using data of the German National Health Interview and Examination Survey and the Nutrition Survey 1998, the differences in usual dietary intake between regular supplement users and others were examined. Regular supplement users have, on average, a more nutrient rich dietary pattern with a higher vitamin and mineral content. This was observed even after adjustment for age and total energy intake. Additional differences in other health relevant characteristics like body mass index, smoking habits, level of physical activity and social economic status between regular supplement users and others, indicate that the regular users show in general a more health conscious behaviour. PMID- 10726412 TI - [Utilization of medical services]. AB - About 90% of all Germans are seeing their doctor at least once a year. Half of the population has consulted a doctor during the past four weeks. More than 95% of the patients have been satisfied with these consultations. On average, a medical practitioner was consulted 11 times a year. Half of the consultations were caused by 20% of population. In most cases, dentists and general practitioners are contacted. More than 90% of the members of the german official health insurance system (Gesetzliche Krankenversicherung) have a family doctor already today. Annually, 12 working days are lost for illness. The average duration of a stay in hospital is rising with age, from 6 days for the youngest to 28 days for the oldest. For 2/3 of cures, the duration was 4 weeks. PMID- 10726413 TI - [Satisfaction with living conditions and health]. AB - In the present publication, contentment with different life areas like work, housing, financial situation, health, familial situation and with life in general is described. The analysis is based on data of the German National Health Interview and Examination Survey 1998, in which a representative sample of the German residential population between 18 and 79 years of age participated in a standardized interview with regard to health relevant topics as well as in a medical examination. In general, women are more content with their life than men. In all age classes, contentment with life is higher in the West than in the East. Highest contentment is observed with family (over 70%) and housing, and lowest with financial situation. The East German residents show the highest degree of dissatisfaction in the age-group of 20 to 29 years regarding their financial situation (20%). In comparison to 1991, contentment with life is reduced negligibly in the East, and in the West it approximately remained the same. Men and women in both parts of Germany are more content with their health today than ten years ago. In contrast, contentment with the financial situation as well as the work situation is lower than ten years ago. PMID- 10726414 TI - [Subjective statements regarding use of selected drug groups--initial results of the 1998 Federal Health Survey]. AB - This publication presents data concerning the daily frequency of usage for 34 selected drug-groups. The results are based on standardized medical interviews done with a representative sample of the German resident population aged 18 to 79 years during the German National Health Interview and Examination Survey 1998. Nearly 52% of the interviewed men and women report the daily use of drugs at least from one of the above mentioned 34 groups during the last year before the interview. In both sexes the prevalence of drug-usage rises steadily with age. Women are more often drug-users than men with approximately doubled daily drug usage in some medication groups. With worsening of the health status the sex specific differences of drug-usage diminish. For females aged 18 to 45 years the rank-order of drug-usage is as follows: oral contraceptives (33.0%), thyroid drugs (11.5%), vitamins (7.6%). For males of the same age group the rank order in the 'new Bundeslander' is as follows: antihypertensives (4.7%), drugs against common cold and grippe (3.8%) and vitamins (3.8%). In the 'old Bundeslander' the rank-order for males is the following: vitamins (5.0%), antihypertensives (2.1%) and thyroid drugs (1.9%). For those study participants older than 45 years, drug groups for therapy of cardiovascular diseases become prominent and are the most often used medications with daily use. This could be observed for males and females. The results give a first impression concerning the drug-usage pattern in an adult resident population in Germany. The discussion of the results has to consider, that the answers given in the interviews strongly depend on the memory of the study participants and their lay-understanding of the different groups of the questionnaire. Further it has to be considered that the 34 drug-groups did not cover the whole medication pattern. PMID- 10726415 TI - [Environmental noise and satisfaction with area of residence]. AB - In the German National Health Interview and Examination Survey, more than one third, i.e. 36.2% of persons interviewed on the matter of noise nuisance in their homes attributed to outdoor noise confirmed the occurrence of noise. Regarding this result only small and statistically insignificant differences have been assessed in East and West Germany. The frequency of affirmative answers given by German women, ages 20-39, was significantly higher as compared to men in the same ages bracket. Regarding the dominant noise effects in their home, 87% of persons affected mentioned traffic noise as a source. In the second place neighbour noise has been reported by 32%. Aircraft and railway noises percepted by 20%, are in the third and fourth place. Particularly severe indoor noise effects, i.e. persisting noise during the night, are accompanied with clearly increased discontent regarding the home and living area. PMID- 10726416 TI - [Utilization of health screening studies and measures for health promotion]. AB - Health-conscious behaviour of a population may be measured by the utilization rate in screening programs and health promotion measures. In the German National Health Interview and Examination Survey 1998, 7124 respondents were asked for their individual participation. The utilization in free health check-ups (1997) was 26.7% for men and 24.5% for women. Health related medical advice was given to 70.9% of men and 67.8% of women in the wake of the check-up. Annual early cancer screening test were taken by 22.6% of men and 36.5% of women. Cancer-related medical advice was reported by 42.4% of men and 43% of women. 10.5% of all respondents participate in health promotion measures, women two times more often than men (13.8% vs. 7%). The ranking according to the type of measures is: 44% for back (muscle) training, followed by nutrition consultation (13%), weight reduction (10%) and anti-smoking, -drinking and -substance use measures (4%). The results show differences in utilization rates for early cancer diagnosis, health checkups and health promotion programmes according to age, region, social status and health insurance type. PMID- 10726417 TI - [Social class and health]. AB - This publication deals with the German social class and their interactions with health or illness. The analysis uses the data of the German National Health Interview and Examination Survey 1998. The results are as follows: During the last decade the proportion of inhabitants of lower social class has decreased in favour of the proportions of middle and upper social classes. The formerly observed differences between the eastern part (former GDR) and the western part of Germany have diminished or have even levelled to zero. Even today men in Germany belong more often to the upper social class than women. For the risk factors smoking, massive obesity and inactivity in sports a distinct gradient concerning the social class can be observed. Those belonging to lower social class are more often smokers, have significantly more often massive obesity and show more seldom activities in sports. Hypertension and hypercholesterolemia are more often observed in men of the upper social class than in those belonging to lower class while for women both mentioned risk factors are more often seen in the lower social class. With respect to morbidity different patterns can be observed. NIDDM, chronic bronchitis and gastric and duodenal ulcer are examples for higher prevalence data in the lower social class while allergic rhinitis can be observed more often in the higher class. The level of complaints is higher in the lower class than in the upper class. By differentiating according to the eastern or western part clear differences emerge concerning social class especially in the 'old Bundeslander' (western part). Members of the upper class estimates their health status clearly to be better than those study participants belonging to the lower class. This perhaps can be explained by their lower level of complaints. The contentedness concerning live and health-status is higher in the upper than in the lower class. Respecting the highest level of education, class-specific differences concerning drug utilization are observed doubly frequent in the 'old Bundeslander' compared to the 'new Bundeslander'. According to the level of complaints and the prevalence of diseases most drug groups are used more often in the lower than in the upper class whereas drugs with presumed preventive potential are clearly more often consumed in the upper class. PMID- 10726418 TI - [Social class index in the Federal Health Survey]. AB - Since the first scaling of social status for the German National Health Interview and Examination Survey, relevant socio-economical circumstances and constraints have changed. This prompted an examination of relevant developments such as changes in income distribution and increasing income in the West and since 1990 in the East of Germany, changes in educational levels, and changes in social prestige of professional groups. In spite of these changes we must assume that the relations between social strata remained constant over time. The task is to assess how and to what extend the developments mentioned above have been reflected and to examine the necessity of bringing social status scaling in line with these developments, as well as to adjust social status scaling. PMID- 10726419 TI - [The SF-36 in the Federal Health Survey--description of a current normal sample]. AB - The German National Health Interview and Examination Survey 1998 included the Short Form 36-Questionnaire as an instrument for measuring health-related quality of life. As a result of the subjective assessment by 6964 survey participants aged between 18 and 80 years a description of a new German normative population sample is given. Using the same statistical parameters as in the description of the normative German sample from 1994 a time comparison can be made. PMID- 10726420 TI - [The SF-36 in the Federal Health Survey--possibilities and requirements for application at the population level]. AB - The SF-36 Questionnaire is an internationally accepted instrument for measuring therapeutic success by subjective assessment of health-related quality of life, done by patient groups. It remains to be seen to what extent this instrument is suitable for measuring the subjective health status of population groups or changes thereof. The answers to the SF-36 Questionnaire in the German National Health Examination and Interview Survey together with information about other health parameters allow deeper evaluation of the features of this instrument. A new approach to establishing a model revesling the association between age, social status, morbidity and the SF-36 Scales is discussed. PMID- 10726421 TI - [Nutrition in Germany 1998]. AB - In addition to the German National Health Interview and Examination Survey, the German Nutrition Survey 1998 (GeNuS) was conducted in a subsample of 4030 participants. Among these persons, a comprehensive dietary interview was performed with use of the software DISHES 98. The proportion of 33-34% energy from fat is considerably less than the 40% energy from fat which was estimated about ten years ago. In general, the supply of most vitamins, minerals and trace elements is sufficient. For a part of the population, the intake of dietary fibre, vitamins D and E, folate, zinc, iodine and, among women, also vitamins B1, B2, B6, iron and phosphorus is on a suboptimal level. Observed differences in dietary habits in the eastern and western part of Germany did not have an obviously more favourable dietary pattern in any part of Germany. PMID- 10726422 TI - [Folic acid intake of women in childbearing age]. AB - An optimal maternal folate status reduces the risk of neural tube defects in the offspring. In the German National Health Interview and Examination Survey, red cell folate and serum folate levels have been measured in 1,266 women aged between 18 and 40 years. Applying the dietary assessment software DISHES 98 made it possible to estimate the individual folate intake. A high proportion of the participants showed sub-optimal red cell folate levels as well as folate intakes below the recommendations. PMID- 10726423 TI - [1998 environment survey--initial results]. AB - In close connection with the German National Health Interview and Examination Survey 1998, a third round of the German Environmental Survey (GerES III) was carried out using a random subsample of about 4,500 subjects, which are representative for the German population aged 18 to 69 years. The GerES was carried out by the Federal Environmental Agency on behalf of the Federal Ministry for Environment, Nature Conservation and Reactor Safety. The participants had to undergo an examination in human-biomonitoring. An environmental questionnaire was used to get exposure-related information. Preliminary results indicate a reduction of the body burden with metals in 1998 compared with 1990/92. However, some individuals showed elevated values. For those people health effects are possible or cannot be excluded with sufficient certainty. Using the results of GerES III it will be possible for the first time to establish reference values for organochlorine compounds on a representative data basis. The examination of the tap water used in the subjects households shows that the limit and guideline values of the German Drinking Water Ordinance have not always been met in 1998. This holds especially for lead, copper, and zinc which are being used as pipe material for domestic plumbing. PMID- 10726424 TI - [Affective, somatoform and anxiety disorders in Germany--initial results of an additional federal survey of "psychiatric disorders"]. AB - BACKGROUND AND AIMS: The paper informs about methods and field survey procedures used in the German National Health Interview and Examination Survey--Mental Health Supplement (GHS-MHS)--and provides 12-month prevalence estimates of affective, anxiety and somatoform disorders in the general population. Such data have previously not been available for Germany on a nationwide level. METHODS: Findings are based on a two-stage design: In the first stage all the 7124 participants of the German National Health Interview and Examination Survey (core survey) completed the Composite International Diagnostic-Screener (CIDS) for mental disorders. In the second stage 4181 probands (all screen-positive and a random sample of 50% of the screen-negative subjects) were interviewed with the full Composite International Diagnostic Interview (DIA-X-M-CIDI) by clinical interviewers. The overall response rate was 87.6%. RESULTS: Affective (12-months prevalence estimate: 6.3%), anxiety (9%) and somatoform disorders (7.5%) are widespread in all age groups (18 to 65 years) of the German population; women are significantly more often affected than men. The prevalence rates of somatoform and affective disorders were found to be significantly lower in the former East German Lander as compared to the former West, whereas no such difference was found with regard to anxiety disorders. All disorders resulted in considerable reduction of work productivity during the past month; affective disorders reported on average 1.3 days/month total impairment and 7.2 days/month partial impairment, indicated significantly higher reduction in work productivity than anxiety and somatoform disorders. CONCLUSIONS: Affective, somatoform and anxiety disorders are highly prevalent mental disorders (total: 17.3%) in the German population. The result of a lower morbidity of affective and somatoform disorders in former East Germany was unexpected and requires further clarification. The findings on impairment of work productivity emphasize the economic impact of psychiatric disorders on society. PMID- 10726425 TI - In vitro apoptosis of lymphocytes after exposure to levels of corticosterone observed following submaximal exercise. AB - BACKGROUND: To evaluate the in vitro effects of corticosterone, equivalent to blood levels measured after a single submaximal treadmill exercise, on apoptosis and necrosis of mouse thymic and peripheral lymphocytes. METHODS: EXPERIMENTAL DESIGN: analysis of variance with independent factors of time (0, 90, 210 minutes of incubation) and concentration of corticosterone (0, 150, 250, 450, 850 ng/ml). MEASURES: percentage of apoptotic, necrotic, and viable thymocytes and splenocytes determined by flow cytometry using Annexin V-FITC antibody and propidium iodide. RESULTS: There was a significantly higher % of apoptotic thymocytes at 210 min at the higher corticosterone concentrations but not in % of apoptotic splenocytes at the same time point. For both lymphoid populations, corticosterone incubation was associated with a higher % of necrotic cells at 210 minutes but not at 0 or 90 minutes. For thymocytes, the interaction (time x concentration) was significant, with greater % necrosis observed at the lower (150 and 250 ng/ml) concentrations of corticosterone. CONCLUSIONS: The data suggest that in vitro exposure to corticosterone, at physiological concentrations (< or = 450 ng/ml) observed after a moderate exercise stress, induces apoptosis in thymocytes and necrosis in both thymocytes and splenocytes. The implications for exercise-mediated glucocorticoid regulation of immune function remain to be investigated. PMID- 10726426 TI - Relationship between moderate intensity endurance training volume and natural killer cell cytolytic activity. AB - BACKGROUND: This study was designed to examine the relationship between endurance training volume and natural killer cell (NK) cytolytic activity. We hypothesized that a dose dependent relationship exists between forced treadmill training volume and training induced increases in NK cell cytolytic activity. METHODS: EXPERIMENTAL DESIGN: female, Swiss Webster mice were assigned to treadmill control (TC) or treadmill trained groups (n = 10 per group). Trained mice ran at 12 m per min. (8 degrees grade) for: 15 (EX15), 30 (EX30), or 60 minutes (EX60) per day, five days per week for 11 weeks. Splenic NK cell activity was expressed as median lytic unit (LU), median LU per asialo GM1 (AsGM1+) cell, and median LU per spleen. RESULTS: Median NK activity was not significantly increased by training volume. A trend toward greater median LU per AsGM1+ cell was observed in EX30 group versus TC (p = 0.1). Training volumes less than or greater than this level produced smaller increases in NK cytolytic activity. CONCLUSIONS: These data provide preliminary evidence indicating that training induced increases in splenic NK cell cytolytic activity do not exhibit a dose dependent relationship with treadmill training volume. PMID- 10726427 TI - Resting serum antioxidant status is positively correlated with peak oxygen uptake in endurance trained runners. AB - BACKGROUND: This study tested the hypothesis that the ability to scavenge free radicals in serum was compromised in trained runners. METHODS: EXPERIMENTAL DESIGN: peak VO2, the ability to scavenge free radicals in serum and the plasma concentration of malondialdehyde (MDA) were assessed in 18 male runners. PARTICIPANTS: subject characteristics (mean +/- SEM) were height 1.77 +/- 0.01 m, mass 71.4 +/- 1.2 kg, age 31 +/- 1 years and weekly training distance 45 +/- 5 km.week-1. MEASURES: venous blood samples were collected at rest. Serum total antioxidant capacity (TAC) was determined using a chemiluminescent technique. This involved the oxidation of luminol, in a reaction catalysed by horseradish peroxidase. Serum antioxidant protection was quantified relative to a soluble vitamin E analogue (Trolox) and expressed as Trolox equivalents (Trolox Eq.). MDA was determined using a highly specific assay, using HPLC with fluorimetric detection. Peak VO2 was determined from expired gas measurements collected during an incremental running test on a motorised treadmill. Data were analysed using Pearson correlations. RESULTS: Serum TAC was 500 +/- 26 mumol Trolox Eq.l-1, with a plasma MDA concentration of 1.5 +/- 0.1 mmol.l-1 and serum urate concentration of 274 +/- 12 mmol.l-1. Peak VO2 was 63 +/- 1 ml.kg-1.min-1. Significant correlations were observed between peak VO2 and serum TAC (r = 0.365, p < 0.05); peak VO2 and serum urate (r = 0.463, p < 0.05) and serum urate and serum TAC (r = 0.807, p < 0.001). Plasma MDA and serum TAC were not significantly correlated (r = 0.026, p > 0.05). CONCLUSIONS: These data demonstrate that the ability to quench free radicals in serum in increased in relation to the maximum ability to consume oxygen, however this response does not appear to provide any additional protection against peroxidative damage at rest. PMID- 10726428 TI - Relationship between strength qualities and performance in standing and run-up vertical jumps. AB - BACKGROUND: The purpose of this investigation was to determine the relationships between the strength qualities of the leg extensor musculature and performance in vertical jumps (VJ) performed from a standing position and a run-up. METHODS: Twenty-nine males with experience in jumping activities were tested for vertical jumping capacities with a standing VJ (double leg takeoff) and run-up jumps from a 1, 3, 5 and 7 stride approach (single leg takeoff). The speed-strength and maximum strength qualities of the leg extensors were assessed by tests involving concentric, stretch-shortening cycle (SSC) and isometric muscular actions. Pearson's correlations and stepwise multiple regression was performed to describe the relationships with jumping performance. RESULTS: The speed-strength tests correlated significantly with both jump types (r = 0.55-0.82), but maximum strength did not. A drop jump test considered to measure reactive strength correlated more strongly with the run-up jump than the standing VJ. The standing VJ was best predicted by a low stretch load SSC test, whereas the run-up jump was best predicted by a model that also including the test of reactive strength. CONCLUSION: The role of maximum strength in jumping performance was not clear but speed-strength qualities were considered important. It was concluded that reactive strength is relatively more important for jumping from a run-up than for the standing VJ, and this should be reflected by appropriate training methods and test protocols for the assessment of athletes who jump. PMID- 10726429 TI - The effects of rest interval on quadriceps torque and perceived exertion in healthy males. AB - BACKGROUND: The ability to generate maximal muscular force is dependent upon time mediated mechanisms of fatigue. Interset rest interval length may not only affect force production but may also manifest changes in ratings of perceived exertion (RPE). The purpose of this study was to examine the effects of rest interval on quadriceps torque and RPE during multiple sets of resisted knee extensions. METHODS: 14 healthy male volunteers were assessed for concentric isokinetic quadriceps peak torque, total work, and average power on the Biodex System II Isokinetic Dynamometer at a preset angular velocity of 180 deg.sec-1. Perceived exertion was measured with a modified category-ratio scale (CR-10). Under isometric conditions the perceptual range was anchored with one high and two low anchors. Subjects were randomly assigned to one of two groups: short rest interval (Group 1, 40 sec) and long rest interval (Group 2, 160 sec). All subjects performed 4 sets of 20 maximal isokinetic contractions with an inter-set rest interval that corresponded to their group assignment. Following the completion of each set subjects were asked to assign a rating out of 10 to the feelings in their quadriceps by visually observing a chart of the CR-10 scale. RESULTS: The results demonstrated a significantly greater decline in quadriceps peak torque (p < 0.05), total work (p < 0.05), and average power (p < 0.05) by Group 1 as compared to Group 2. Changes in RPE across the 4 sets of exercise did not show a significant difference between the 2 groups. CONCLUSIONS: The generation of quadriceps torque is a function of rest interval length, whereas perceived exertion appears to be unaffected. PMID- 10726430 TI - Evaluating a test protocol for predicting maximum lactate steady state. AB - BACKGROUND: Maximum lactate steady state (MLSS) is defined as the highest steady state exercise level one can maintain while also maintaining an equilibrium between the elimination of blood lactate and the diffusion of lactate into the blood. MLSS is an excellent tool for assessing fitness level, predicting endurance performance, and designing training programs. METHODS: This investigation assesses the validity of the Lactate Minimum Test (LMT), which consists of inducing lactic acidosis through a VO2peak test, followed by an eight minute walking recovery and an incremental exercise test, to determine if the running velocity associated with the minimum lactate value predicts the MLSS velocity. Following this LMT, two constant velocity 28-minute runs were performed, one at the predicted MLSS velocity (trial 1) and the other 0.13 m sec 1 (4-8%) above the predicted MLSS velocity (trial 2). Ten active female subjects participated (32 +/- 7 yrs (mean +/- SD); 65.7 +/- 16.4 kg; VO2peak 40.0 +/- 7.5 ml.kg-1.min-1). RESULTS: During trial 1, there was a -0.6 +/- 0.3 mmol l-1 (mean +/- SE) change in lactate. Based on a definition of lactate steady state (LSS) as less than a 0.5 mmol.l-1 increase, this value signified LSS. A similar comparison during trial 2 revealed a 1.8 +/- 0.3 mmol.l-1 increase in lactate, signifying a workload above LSS and therefore confirming trial 1 as the maximum LSS (MLSS). CONCLUSIONS: These results suggest that the test protocol accurately predicted the MLSS velocity. PMID- 10726431 TI - The cardiovascular work of competitive dinghy sailing. AB - BACKGROUND: Hiking is the special manoeuvre, which the dinghy sailor uses to counterbalance the capsizing effect of the wind on the boat. In the present research the work required of the heart by this exercise was studied in the laboratory using a boat simulator. METHODS: Seven Laser male sailors selected from those in the first places in the junior national rank participated in this study. Their endurance, at different levels of isometric hiking efforts, was measured. Energy expenditure due to hiking was estimated from measurements of oxygen consumption, carbon dioxide elimination and blood lactate concentration. The cardiac load was evaluated by measuring blood pressure using the conventional method and heart rate measured by electrocardiography. Cardiac output was measured using the CO2 re-breathing method. Left ventricular work was then calculated as cardiac output multiplied by mean arterial pressure. RESULTS: The most relevant result was that, while whole body cost of hiking was relatively low (about 1 IO2 min-1), the power of the heart was very significant: cardiac output almost doubled with respect to that at rest and arterial mean pressure rose from 12.5 kPa (rest) to 18.5 kPa (hiking). Thus, left ventricular power rose from 1.2 Watt to 3.2 Watt, which is a typical cardiovascular response to muscular isometric contraction. CONCLUSIONS: These results assume relevance when a person's eligibility for sailing sports is evaluated. PMID- 10726432 TI - Temporal patterns of physical activity in Olympic dinghy racing. AB - BACKGROUND: The objective of the present study was to determine the temporal patterns of physical activity in four classes of Olympic racing dinghy. METHODS: EXPERIMENTAL DESIGN: Descriptive. SETTING: A field (on-water) study. PARTICIPANTS: Nineteen elite New Zealand sailors (fifteen male and four female). INTERVENTION: Not applicable. MEASURES: The temporal pattern (duration and frequency) and nature of the physical activities of each sailor during each leg of simulated races were recorded on video tape and subsequently systematically quantified and categorised using notational analysis. The accumulated percentage of total leg time spent sitting (upright or leaning backwards), hiking (upright or fully extended) whilst trimming and whilst pumping the mainsheet and for the time spent on rig adjustments, tacking and gybing were calculated for both up wind and off-wind sailing. RESULTS: When sailing up-wind, the most time was spent hiking upright (average 29-66% of total leg time) while trimming the mainsheet. During off-wind sailing, sailors spent the most time sitting upright while trimming the mainsheet (average 29-55% total leg time). Hiking upright while trimming the mainsheet was executed the greatest number of times (average 15.8 23.9) when sailing up-wind and sitting upright while trimming was executed the most times (average 3.5-7.4) when sailing off-wind. The most lengthy continuous activity was hiking upright while trimming the mainsheet when sailing up-wind (9 18 seconds) and sitting upright while trimming the mainsheet when sailing off wind (17-34 seconds). CONCLUSIONS: The most physically demanding aspect of Olympic yacht racing is hiking. It occurs for the majority of up-wind legs when the wind starts to exceed approximately 8 knots. The only respite that the sailor gets from hiking is during tacking, rig adjustments or sitting in-board for brief periods when the wind is low. Sustained hiking tends to last for no more than approximately 20 seconds before the sailor changes to either a more extended or more upright hiking posture. The physical demands during off-wind sailing are generally less, except for a greater requirement for power in the arms and shoulders to pump the mainsheet in order to assist the dinghy in accelerating down waves. The findings of the present study are directly applicable to the design of sailing specific physical conditioning programmes for Olympic class sailors. PMID- 10726433 TI - Physical activity level during a round of golf on a hilly course. AB - BACKGROUND: Regular physical activity plays a role in preventive medicine. Our study aimed at establishing the duration of different levels of exercise intensity during a round of golf. METHODS: PARTICIPANTS: we studied 21 male and 9 female golfers (mean age 53 +/- 11 and 54 +/- 13 years respectively) volunteering for a round of golf on a hilly course. MEASURES: we recorded mean heart rate (HR) of every 15 seconds. Blood pressure was taken on each tee. Maximum HR (HRmax) reserve of each subject was calculated from the difference between pre-exercise and maximum HR attained during a test to volitional exhaustion on a cycle ergometer. A percentage of this value was added to the resting HR and was expressed as a percentage of HRmax reserve. RESULTS: Before start mean HR (+/- SD) was 86 +/- 11 beats per minute (BPM), during play 113 +/- 18, and during rest after play 100 +/- 24 BPM. Mean maximal HR of holes were 135 +/- 21 BPM. Mean systolic blood pressure was 145 +/- 30 before play, 137 +/- 31 on tees during play and 119 +/- 15 mmHg after play. A mean of 82 +/- 51 minutes was spent at 50-74% of HRmax reserve. 21 +/- 27 and 23 +/- 38 minutes were spent in the two higher intensity classes. An average of 106 +/- 77 minutes were spent at or above the individual heart rate equivalent of 100 W, the mean heart rate for this time was 128 +/- 17 BPM. Creatine kinase (+47%; p < 0.001), uric acid (+9%; p < 0.001) and HDL-cholesterol (+6%; p < 0.05) increased, triglycerides decreased by 18% (p < 0.01). CONCLUSIONS: The HR level during the golfround not using an electric cart relative to the maximum attained on the ergometer reaches the exercise intensity of 50 to > 85% HRmax reserve for a mean of over 2 hours, much longer than the 20-60 min recommended for endurance training. PMID- 10726434 TI - Physiologic study of pressure point techniques used in the martial arts. AB - BACKGROUND: Study physiologic changes occurring during "knockouts" produced by application of pressure point techniques during martial arts demonstrations. METHODS: EXPERIMENTAL DESIGN: prospective analysis of physiologic variables during and immediately following an acute event. SETTING: martial arts demonstration carried out at a medical center hospital. SUBJECTS: 12 normal volunteers participating in a martial arts demonstration. INTERVENTIONS: application of various pressure point techniques that have been observed to produce states of unresponsiveness in volunteers. MEASURES: continuous ECG and video/EEG monitoring with measurements of blood pressure and oxygen saturation. Qualitative analysis of EEG and ECG recordings and quantitative comparison of heart rate, blood pressure, and oxygen saturation measurements before during and after the period of induced unconsciousness. RESULTS: No significant changes in blood pressure, oxygen saturation, cardiac rate or rhythm, or electroencephalogram are noted during the knockouts produced by application of pressure point techniques. There was only variable inability for subjects to remember words spoken to them during the episode of apparent unresponsiveness. CONCLUSIONS: The mechanism for the state of unresponsiveness produced by application of pressure point techniques is not related to a significant cardiac or pulmonary process. There is no evidence of reduced cerebral blood flow during this time or of other dangerous physiologic changes. The exact mechanism for this phenomenon remains uncertain. PMID- 10726435 TI - Physical Activity Scale for the Elderly (PASE): the relationship with activity measured by a portable accelerometer. AB - BACKGROUND: The purpose of this study was to examine the validity of the Physical Activity Scale for the Elderly (PASE) by comparing PASE scores with physical activity assessed by a portable accelerometer. METHODS: EXPERIMENTAL DESIGN: Correlational-mean activity level over a 3-day period was correlated with PASE scores. SETTING: Physical activity of participants was monitored over 3 days during normal daily activity. PARTICIPANTS: Twenty healthy adult volunteers, 67 80 years of age, participated in this study. MEASURES: Physical activity from 09:00 to 21:00 hours was assessed over 3 consecutive weekdays (Wednesday-Friday) using a Computer Science and Applications, Inc. (CSA) portable accelerometer. Following completion of the 3-day monitoring period physical activity was assessed with the PASE. RESULTS: PASE scores were significantly correlated with average 3-day CSA readings (r = 0.49, p < 0.05) in the total sample and in those over age 70 years (r = 0.64, p < 0.5). CONCLUSIONS: These results add to the literature supporting the validity of the PASE as a measure of physical activity in older individuals. PMID- 10726436 TI - The effects of various intensities and durations of exercise with and without glucose in milk ingestion on postexercise oxygen consumption. AB - BACKGROUND: The purpose of this study was to compare the 2-hr excess postexercise oxygen consumption (EPOC), respiratory exchange ratio (RER), and rectal temperature (RT) after four-exercise bouts of varying intensity and duration, with and without glucose in milk (GM) ingestion. METHODS: EXPERIMENTAL DESIGN: subjects completed the exercise tests in random order 4 times on the same weekday. The four experimental exercise conditions were low intensity, long duration with GM (LL & GM), low intensity, long duration without GM (LL & NGM), high intensity, short duration with GM (HS & GM), and high intensity, short duration without GM (HS & NGM). PARTICIPANTS: ten male college students (20.8 +/- 0.6) yrs participated voluntarily. Measures. Two-way repeated measures ANOVA (exercise condition x time) was used to compare the variables. RESULTS: Mean EPOC for the 2-hr postexercise period for HS & GM (211.5 ml O2/kg) was significantly greater than EPOC for HS & NGM (154.8 ml O2/kg), LL & GM (140.4 ml O2/kg) and LL & NGM (125.2 ml O2/kg). Mean recovery oxygen uptakes were 6.4, 5.7, 4.5, and 4.2 ml/min/kg with HS & GM, HS & NGM, LL & GM and LL & NGM, respectively. Mean RERs for high intensity exercises were significantly lower than RERs for low intensity exercises during the recovery period. Mean RT for HS & GM (37.60 degrees C) and HS & NGM (37.43 degrees C) were significantly higher than RT for LL & GM (37.19 degrees C) and LL & NGM (37.15 degrees C) during the recovery period. CONCLUSION: These results suggest that preexercise intake of GM increases EPOC above that observed in the fasting condition, and high intensity short duration exercise increases fat oxidation during recovery period more than low intensity long duration exercise. PMID- 10726437 TI - The effects of swimming and running on energy intake during 2 hours of recovery. AB - OBJECTIVE: To determine energy intake in the 2 hrs after swimming (S) and running (R) at the same relative exercise intensity and duration (71.8 +/- 2.5% VO2max; 45 min) to evaluate whether a difference in recovery energy intake could explain the greater body fat observed in swimmers relative to runners. METHODS: EXPERIMENTAL DESIGN: this was a randomized crossover design. SETTING: running exercise was conducted on a motorized treadmill (Quinton) while swimming was conducted in a 45.7 m pool. PARTICIPANTS: eight well-trained competitive male triathletes participated in this investigation. INTERVENTIONS: subjects were blinded to the purpose of the study and swam and ran on separate occasions for 45 min at 71.8 +/- 2.5% of VO2max. Subjects were then placed in a room with a variety of foods and beverages for 2 hrs after R and S. MEASURES: energy intake (kJ/2 hrs and kcal/2 hrs) was determined by weighing and measuring the food remaining in the room after 2 hrs of postexercise recovery. Expired gases, heart rates, and Ratings of Perceived Exertion were obtained at 15 min intervals throughout exercise. Blood samples for serum glucose and lactate were obtained preexercise and immediately, 15 min, and 135 min postexercise. Perceived hunger and thirst ratings were obtained after the subjects were seated in the room containing the food. RESULTS: Serum glucose was significantly (p < or = 0.05) higher after R compared to S immediately after exercise (5.4 +/- 0.3 mmol/L for R and 4.2 +/- 0.1 mmol/L for S) but no significant differences were observed for hunger using a five point Likert scale (3.3 +/- 0.3 for R and 3.4 +/- 0.3 for S), energy intake (4584 +/- 611 kJ/2 hrs; 1095 +/- 146 kcal/2 hrs for R and 4383 +/- 484 kJ/2 hrs; 1047 +/- 116 kcal for S) or blood lactate. CONCLUSION: The type of exercise, swimming or running, did not significantly influence energy intake during 2 hours of postexercise recovery. PMID- 10726438 TI - The contribution of anthropometric characteristics to performance scores in elite female gymnasts. AB - BACKGROUND: Aims of this study were: a) to identify anthropometric variables correlated with gymnastic performance, and b) to predict performance scores from a combination of anthropometric dimensions. METHODS: EXPERIMENTAL DESIGN: correlational analysis and a stepwise multiple regression were used. SETTING: Subjects were participants at the 24th World Championships Artistic Gymnastics, Rotterdam, The Netherlands, in 1987. PARTICIPANTS: A total of 168 female gymnasts (mean age: 16.5 +/- 1.8 years) were investigated. Each gymnast participated in all events. MEASURES: An extensive battery of anthropometric dimensions was taken on each athlete. The somatotype was estimated. Skeletal maturation of the hand wrist was assessed. Competition scores for the four individual gymnastic events (balance beam, floor exercise, vault, uneven bars) and a composite score for each gymnast were the dependent variables. RESULTS: Moderately high, significant correlations (p < 0.01) were observed between skinfolds and endomorphy, and gymnastics performance scores, r varying from -0.38 to -0.60, for biceps skinfold and the score on balance beam, and for endomorphy and the total score, respectively. The correlations suggest that gymnasts with more subcutaneous fat and higher endomorphy have lower performance scores. About 32% to 45% of the variance in gymnastic performance scores could be explained by anthropometric dimensions and/or derived variables, but endomorphy and chronological age are the most important predictors. CONCLUSIONS: There is a relatively strong relationship between several anthropometric variables and gymnastic performance in a sample of elite female gymnasts, but the associations are not sufficiently high to predict performance scores on an individual basis. PMID- 10726439 TI - Exercise-induced bronchospasm in nonasthmatic obese and nonobese boys. AB - BACKGROUND: In this study it was aimed to compare the degree and frequency of exercise-induced bronchospasm (EIB) in nonasthmatic obese boys with those in nonasthmatic nonobese boys. METHODS: PARTICIPANTS: Fifty boys (24 obese and 26 control), aged 11 to 15 years, with no history of asthma or other atopic diseases, took part in the study. MEASURES: Anthropometric measurements and spirometry were performed at rest and spirometry was repeated at 5 and 15 minutes after an eight-minute submaximal exercise on cycle ergometer. RESULTS: The falls in ratio of forced expiratory volume in one second (FEV1) to forced vital capacity (FVC) and expiratory rates at 5 and 15 minutes after the exercise were significant in obese boys. Only significant change was the fall in FEV1/FVC at 5th minutes in controls. There was no significant difference between obese group and controls in the number of EIB-positive cases. Pulmonary function changes after the exercise had significant negative correlation with body mass index, subscapular and biceps skinfolds. CONCLUSIONS: Since the fall in pulmonary function was severe in obese boys and these falls were correlated with body mass index and skinfolds, it is concluded that diagnosis and management of EIB may improve aerobic exercise performance and participation in obese children. PMID- 10726440 TI - [The correlation between idiopathic leukocytospermia, embryo quality and outcome in the FIVET and ICSI procedures]. AB - BACKGROUND: An adverse effect of leukocytospermia in seminal fluid on motility and fertilizing power of spermatozoa has been described. This detrimental effect could be mediated by radical oxygen species (ROS). Recently, a direct effect on nuclear DNA of sperm induced by ROS has been described, although the chance of fertilization did not seem altered during ICSI procedure. Aim of this prospective case-control study was to compare the outcome of results in term of fertilization rate and embryo quality in patients with and without idiopathic leukocytospermia during IVF and ICSI cycles. METHODS: Seventy-two patients selected for a program of IVF and ICSI were admitted in the study. Fourty-two patients underwent IVF procedure, 14 with idiopathic leukocytospermia and 28 without, and thirty underwent ICSI procedure, 16 with leukocytospermia and 14 without leukocytospermia. RESULTS: Statistical significant differences on cleavage rate of embryos between leukocytospermia and control group in IVF cycles were observed. In ICSI procedure a low fertilization, cleavage rate and percentage of good embyros in the presence of leukocytospermia were evidenced. CONCLUSIONS: The presence of a significant number of leukocytes in semen, even in idiopathic condition, could affect the results of IVF and ICSI procedure. An adverse effect of lipoperoxidation process of plasma membrane and damage of nuclear chromatin of sperm, as result of leukocyte contamination could be hypothesized and future studies needed in order to verify the role of ROS on sperm functions. PMID- 10726441 TI - [The effects of menopausal replacement therapy in women with uterine myomas]. AB - BACKGROUND: The aim of this study was to compare the effects of two different therapies in menopausal women with uterine fibroids. METHODS: Thirty menopausal women with uterine fibroids were evaluated. They were randomized into two groups: the patients included in Group A were treated with transdermal hormonal therapy with 50 micrograms of ethynilestradiol and 5 mg of MAP; the patients in Group B with 2.5 mg of tibolone. The patients of both the groups have been treated for one year. Plasmatic levels of E2, FSH and LH were evaluated before and after treatment and the volume of fibroids was compared by transvaginal ultrasonography at the beginning of the study, after 6 months and after 1 year. RESULTS AND CONCLUSIONS: The results suggest that transdermal hormonal therapy increases significantly the volume of the fibroids while tibolone does not. PMID- 10726442 TI - [The rationalization of the follow-up of hormonal replacement treatment in menopause]. AB - BACKGROUND: Decision making about the opportunity of starting or continuing hormone replacement therapy (HRT) in menopause should rely on an overall evaluation of its risks and benefits for the women's health; the evaluation of HRT cost-benefit ratio, however, should include its possible outcomes from an economical point of view. In this view, and with the certainty that menopausal patients should be protected by proper treatments, our case series has been evaluated in order to improve the quality of our clinical schedules for both their access to HRT and the treatment follow-up. METHODS: Two groups of patients have been considered the first one consisted of 560 women observed during '97 for climacteric symptoms and candicated to begin HRT. The second one consisted of 100 women on HRT for 1 to 6 years. In the first group we considered which test and with which frequency were responsible for stopping or delay the beginning of therapy; while in the second group we evaluated the reasons for stopping treatment. RESULTS AND CONCLUSIONS: On the basis of our experience, the exams required before starting HRT seem to be the following: patient history, mammography, densitometry and endometrial sample as well as the parameters of glucose lipidic, coagulative and hepatic metabolism. Densitometry is useful in the annual follow-up only in patients with bone alterations from the beginning. The same exams seem required for the follow-up, with the exception of bone densitometry which should be performed yearly only in patients with bone demineralization. PMID- 10726443 TI - [Fertilization in vitro and microinsemination. 6 years of experience in the Ospedale Microcitemico of Cagliari]. AB - BACKGROUND: In this study the results of six years experience (1993-1998) in IVF, ICSI and assisted hatching on 442 sterile couples for a total of 868 cycles are reported. Since 1997 ICSI has also been carried out in cases of azoospermia extracting mobile spermatozoa from the epididymal (MESA) or from the testicle (TESE). METHODS: Ages ranged from 20 to 48 and mean years sterility was 5.868 cycles were carried out of which 153 (17.62%) were for IVF, 705 (81.22%) for ICSI and 10 (1.15%) for MESA and TESE. Assisted hatching was performed through "partial zona dissection" in 329 cases on a total of 987 embryos. RESULTS: The pregnancy rates per embryo transfer in IVF and ICSI cycles were 22.4 and 19.96% respectively. Results considering patients age were: 33.87% below 35, 29.55% between 35 and 38 and 6.60% above 38. In the group of 189 assisted hatching patients the evolutive pregnancy rate rose from 15.78 to 26.40% and multiple pregnancies from 28.63 to 42%. CONCLUSIONS: ICSI has offered high rates of fertilization and pregnancy even in extreme cases of oligoasthenospermia or cases of azoospermia adopting MESA or TESE techniques. A decisive factor on pregnancy rates is age, very low over 38 years. Assisted Hatching further increased the pregnancy rates. PMID- 10726444 TI - [Chemotherapy in the treatment of ovarian carcinosarcoma]. AB - Carcinosarcoma of the ovary is a rare neoplasm representing 1% of this organ malignancies. The disease appears almost exclusively in advanced stage having an unfavourable prognosis. Three patients affected by carcinosarcoma (MMMT) of the ovary admitted to our Operative Unit have been treated. All patients underwent surgery and subsequently chemotherapy. Two patients were affected by heterologous MMMT and were stage IV and IIIc respectively, the other one, affected by homologous MMMT, was stage IIIc. Stage IV patient was submitted to 6 cycles of CARBO + IFX + CDDP, second look and further 6 cycles of TAX. After 23 months she was submitted to colostomy for intestine occlusion. At the 35 months she died for cachexia and intestine occlusion. Stage IIIc heterologous patient was submitted to 6 cycles of CDDP + EPI + IFX + MESNA for 3 days; at 6 months from diagnosis she did not present any sign of disease. Stage IIIc homologous patient, affected by chronic renal insufficiency and submitted to dialysis, underwent 5 cycles of TAX and at 11 months from diagnosis presented partial response. Carcinosarcoma of the ovary, because of its rarity, and of the poor record of cases in the literature, is a much debated topic in particularitis complementary therapy. Opinions of the several authors are in contrast regarding the use of CHT + RT at the same time. Only RT after surgery does not seem to improve the survival of these patients. Personal experience, with the reported outlines, compared with survival, seems to confirm the use of CDDP and of IFX and to give new horizons to TAX, waiting for further findings. PMID- 10726445 TI - [Peritoneal cyst. A case report]. AB - Cystic mesothelioma is a rare benign tumor of the abdominal and pelvic peritoneum, consisting of solitary or multiple cysts. No more than 130 cases are reported. Several risk factors such as chronic peritoneal irritation, caused by foreign bodies, infection or endometriosis, were hypothesized but the pathogenesis is still unknown. A 51-year menopausal woman was submitted to ultrasonography because of abnormal uterine bleeding. The scan revealed a right ovarian cyst (size 81 x 64 mm) with the feature of serous cyst. In the anamnesis a cystectomy of the right ovary and appendectomy were reported. At laparoscopy, then converted in laparotomy, a cyst arising from peritoneum of the posterior surface of the uterus was found. The right ovary was normal. The histopathological finding was: serous simple cyst of peritoneum. Ultrasonographic diagnosis was not confirmed by surgery; in fact, sometimes, it may be difficult to establish the origin of pelvic cystic mass, from ovary or peritoneum, by ultrasonography. It is mandatory to suggest a laparoscopy and/or laparotomy in case of pelvic cystic mass that does not regress in the time even after administration of oral contraceptives. PMID- 10726446 TI - [The role of the gynecologist in first aid in cases of sexual violence. A protocol proposal]. AB - The aim of this report is to provide a medical form to use in case of sexual assault, prepared taking into consideration the information lacking at present in most medical reports on the subject. This call for the necessity to conform the medical approach to the same protocol which may also be used from the forensic medicine point of view. More precise information should be obtained about the assault and the injuries inflicted, and also an immediate psychological support should be given to the victim. This information will then serve as a guide to the medical staff. PMID- 10726447 TI - Cleft lip: a histochemical and ultrastructural analysis of lip muscles. AB - AIMS: In order to evaluate the pathogenesis of cleft-lip in relation to both the anatomical and structural anomalies of the mesenchymal tissues, the authors concluded that the presence of structural anomalies in the examined tissues could not explain the malformation, but might be a consequence of it. Delayed muscular development, asymmetrical distribution of the muscular fibres and their anomalous insertion suggest that the anatomical/functional loss clinically detectable in the orbicular muscle could be the result of a perinatal dysmorphological process rather than of a simple mesenchymal hypoplasia. METHODS: Schendel et al. suggested that a metabolic defect in the mitochondrial function could cause a deficiency in cell migration and proliferation responsible for the malformation in question. To establish whether the pathogenesis of the cleft-lip is associated with an alteration in mitochondrial functionality, eight patients affected by unilateral cleft-lip were subjected to a biopsy of the orbicular muscle during the course of reparative surgery. RESULTS: The results obtained showed: 1) a great variation in the size of muscle fibres; 2) the absence of ragged red fibres; 3) a normal oxidative function in the muscle fibres examined; 4) the absence of typologically significant groupings positive for myofibral ATPases. Furthermore, the morphology of the mitochondria was preserved in all cases and neither inclusions nor morphological or volumetric changes were detected. CONCLUSIONS: This preliminary data did not confirm the constant presence of mitochondrial pathology responsible for the malformation in question. In our opinion, the growth deficiency of the maxillary segment could be ascribed to the cicatrization of the surgical repair of the cleft-lip. PMID- 10726448 TI - [A comparative evaluation of the efficacy of the excisional new attachment procedure (ENAP) relative to root planing in the etiological phase of periodontal therapy]. AB - BACKGROUND: In the present study, the effectiveness of root planing has been compared to the excisional new attachment procedure (ENAP) during the etiological phase of periodontal therapy, after the supragingival scaling, in order to establish if a technique offering an easy access to the subgingival areas could reduce the need for a surgical phase in the periodontal treatment. METHODS: Twenty-seven patients, affected by moderate periodontitis, participated in this study; in each of them root planing was performed in a half of the oral cavity (control site) and the ENAP in the other half (test site). The main clinical parameters of periodontal health (probing depth--PD-, attachment loss--AL-, plaque index--PlI- and gingival index--GI-) were evaluated before and 1, 2 and 6 months after the periodontal treatment. RESULTS: The results of the study showed that the parameters related with the amount of plaque and with the conditions of the marginal gingival tissue were not influenced by the different treatments used. Better improvements were found in PD and AL values in teeth treated by ENAP compared to those treated by root planing; this result is explained by a better access to the roots offered by the ENAP. CONCLUSIONS: We can conclude that, within the limits of the present study, the ENAP can reduce the need for a further surgical treatment of the periodontal patient. PMID- 10726449 TI - [Morphological and positional changes in the disk after nonsurgical therapy. The MRI documentation and clinical correlations]. AB - AIMS: To evaluate the position and shape of the disc using MNR before and after non-surgical therapy and to correlate MNR findings with other signs and symptoms. METHODS: MNR imaging of 26 joints belonging to a selected sample of 13 patients (7 F, 6 M, mean age 27 years) with signs and symptoms were compared with MNR performed after a period of non-surgical treatment that included a combination of splint therapy, psychoeducational advice, drugs and physiotherapeutic aids, including manipulation. The criteria for selection were monolateral symptoms and bilateral nature at the initial NMR showing reducible and non-reducible dislocation not associated with degenerative changes and/or effusion. The interval between diagnostic NMR and control imaging was approximately 12 months. The interval between splint application and control NMR was approximately 8 months. NMR included a sagittal study with the mouth open and shut and a coronal study only with the mouth shut using 3 mm-wide cuts. Post-therapy RMN was performed without the splint in position. RESULTS: The comparison between NMR images showed that 17 of the 26 joints studied had a stationary pathological situation and 9 were in evolution. In spite of these pathological NMR images, the signs and symptoms were found to have regressed or improved. CONCLUSIONS: These findings can be interpreted in two ways: the disc dislocation and deformation are compatible with the lack of signs and symptoms, or many dislocations should be regarded as an anatomical variation rather than an abnormality. PMID- 10726450 TI - Calcium phosphates produced by physical methods in the treatment of dentin hypersensitivity. AB - BACKGROUND: The aim of this paper is to study the properties of innovative materials based on defective calcium phosphates produced by physical methods in the therapy of dentin hypersensitivity. METHODS: In particular, the effects of gels, aqueous solutions and toothpastes containing the above mentioned materials on dentinal permeability measured as dentin hydraulic conductance have been studied. The calcium phosphates have been characterized by X-ray powder diffraction (Rietveld analysis) and Fourier transform infra-red analysis. In addition, scanning electron microscopy has been performed to study the surface of dentin and enamel after treatment with the phosphates. In particular sound occlusal dentin, sound cervical dentin, carious occlusal dentin, sound buccal enamel and carious buccal enamel have been observed. RESULTS: The results have shown that these biocompatible materials can be produced with chemical and physical characteristics very similar to dentin and/or enamel. By forming a protective layer inside and outside the dentin tubuli, the calcium phosphates significantly reduce the dentinal hypersensitivity. CONCLUSIONS: These phosphates seem to be a promising material for clinical application. PMID- 10726451 TI - [An analysis of the psychophysiological components in subjects with temporomandibular disorders. Myographic tension and the management of aggressiveness]. AB - BACKGROUND: The stomatognathic literature doesn't sufficiently stress the role of aggressiveness as a modulator of muscular tensions. Our study focused on the hypothesis that 1) original psychological problems related to the modulation of aggressivity could produce 2) chronic muscular tension of the oral district 3) they can become the basis of the malocclusive phenomenon. The investigation was conducted in the laboratory of Clinical Psychophysiology of the University of Rome "La Sapienza". METHODS: We have examined the myographic activity of the temporal and masseter muscles of both sides in 20 women with temporomandibular dysfunction and in the control group comparable by age. The myographic tension was examined in three "stimulus situations": a) closed lips without any teeth contact; b) with light teeth contact; c) with compression of the dental arcade. Moreover, we have examined the style of aggressiveness modulation through Rosenzweig's PFS test. Features of the experimental group were. 1) age between 20 and 30 yrs; 2) presence of condylomeniscal uncoordination; 3) deviation of the median line of incisors; 4) lateral deviation in the opening and/or closing mandible. RESULTS: The experimental group showed a higher score of myographic amplitude for the above-mentioned muscles of both sides in three "stimulus situations". Moreover, it presented at the Rosenzweig's test more elevated scores for the Dominance of Obstacle (O-D); b) less elevated scores for Ego Defence (E D). CONCLUSIONS: Results are commented on the basis of an integrated psychophysiological model. PMID- 10726452 TI - [The effects of drugs on the oral cavity]. AB - As shown by the growing numbers of users attending the public drug addiction services, drug abuse is a phenomenon that is constantly spreading. It is important that dentists are aware of the oral problems linked to drug abuse. This study examines the general effects and oral implications of the illegal substances used by the majority of drug addicts. The main dental complications of cannabinoids are the increased incidence of squamous cell carcinomas of the oral cavity, the presence of xerostomia and severe gingivitis. Depending on how it is taken, cocaine may cause ischemic necrosis of the palate, inflammation, ulceration and gingival retraction, as well as an increased incidence of bruxism. Hallucinogens have few direct oral effects, but among these it is worth recalling xerostomia, increased bruxism and oral problems linked to malnutrition caused by ecstasy. Turning to the opioids, heroin is the drug primarily used by the majority of drug addicts. Its oral effects mainly take the form of dental decay, showing a particular form and extent linked either directly or indirectly to heroin use. This results in "typical" or "atypical" caries pathologies directly linked to the effects of heroin. Given the extent of this phenomenon, it is important that dentists are aware of the problems linked to drug abuse that they may have to treat. PMID- 10726453 TI - [New therapies in rheumatoid arthritis. Stopping joint deterioration]. PMID- 10726454 TI - [Specific COX-2 inhibitors. Also effective in arthritis?]. PMID- 10726455 TI - ["Wonder cures" for tinnitus. Much noise about nothing?. Interview by Dr. med. Cornelia Herberhold]. PMID- 10726456 TI - [Detecting phobic vertigo!]. AB - The syndrome of phobic postural vertigo is characterized by a combination of non rotational dizziness, subjective disturbance in the upright stance and gait, despite normal clinical balance tests. Phobic postural vertigo most frequently occurs in form of attacks. It may be accompanied by anxiety and panic symptoms. It is almost invariably associated with particular constellations of perceptual stimuli or social situations. There is a tendency for rapid conditioning, generalization, and avoidance behavior to develop. Frequently, onset of condition follows periods of particular psychosocial stress. Despite a considerable rate of improvement in vertigo complaints, a psychiatric comorbidity in a subgroup of patients must be respected during the course of illness. Educative information on the mechanism and possible releasing factors and behavioural therapeutic techniques with self-controlled exposure behaviour are the main therapeutic approaches. PMID- 10726457 TI - [Psychogenic vertigo incapacitates patients longer. What psychiatric illnesses might manifest as vertigo]. AB - Dizziness is a common symptom that, despite extensive medical evaluation, often remains unexplained. Psychiatric or psychosomatic disorders underlie the condition in 30-55% of the cases. The most common disturbances are phobic and anxiety disorders, followed by dissociative, depressive and somatoform disorders. The assessment of psychiatric and psychosomatic symptoms should always be included in the neuro-otological examination of dizziness. Early interdisciplinary treatment should be initiated with the aim of preventing chronification of psycho-genetic vertigo. PMID- 10726458 TI - [Regulating blood pressure and energy metabolism... vital for prognosis of diabetic patients]. PMID- 10726459 TI - [Chemical peeling. Impressive results in acne scars and aging skin]. AB - The dividing line between dermatology and cosmetology is sometimes fluid. At dermatological congresses, cosmetological questions often take up a considerable amount of time. A connection between dermatology and cosmetology are the therapeutic procedures, now known as chemical peeling, aimed in particular at treating photodamaged skin, and hyperpigmentation. PMID- 10726460 TI - [Counseling before and in pregnancy. 7: Drug dependence, alcohol and nicotine use]. PMID- 10726461 TI - [Acute dyspnea in diabetes mellitus. Anterior wall infarct]. PMID- 10726462 TI - [Obtaining legal rights for the severely handicapped patient. What the social security office really wants to know from you]. PMID- 10726463 TI - [With the internet doctor through the jungle of medicine. Diagnosis determined by patients now independently]. PMID- 10726464 TI - [Antidepressive therapy. Hypericum extract Ll 160 highly effective]. PMID- 10726465 TI - [Organ protection in hypertensive type 2 diabetic patients. Double chance with AT1 blockers]. PMID- 10726466 TI - [V alpha 14 NKT cells as a novel immune system]. PMID- 10726467 TI - [Beta 1 integrin molecule and its immunoregulatory function of T cells]. PMID- 10726468 TI - [B cell signaling]. PMID- 10726469 TI - [Pathogenesis and treatment of atopic dermatitis]. PMID- 10726470 TI - [Recent advances in immunodeficiencies]. PMID- 10726471 TI - [Current status of gene therapy and prospect of its clinical application]. PMID- 10726472 TI - [Treatment of arthritis with cyclin-dependent kinase inhibitor p 16INK4a gene]. PMID- 10726473 TI - [Novel gene therapy for rheumatoid arthritis]. PMID- 10726474 TI - [Study on abnormal expression of T cell receptor-CD3 complex in the circulating blood T cells of patients with systemic lupus erythematodes]. PMID- 10726475 TI - [Cyclosporin A reverses steroid-resistance induced by P-glycoprotein in patients with SLE]. PMID- 10726476 TI - [Treatment of interstitial pneumonia associated with polymyositis/dermatomyositis with cyclosporin A]. PMID- 10726477 TI - [Gene therapy for attenuating chronic rejection of cardiac allografts]. PMID- 10726478 TI - [Immunomodulation after organ grafting by novel immunosuppressive drug and gene transfection]. PMID- 10726479 TI - [Bone marrow chimerism and tolerance in organ transplantation]. PMID- 10726480 TI - [Weaning of immunosuppression in living-related liver transplantation]. AB - In our experience of living donor liver transplantation (LDLT), some recipients could be successfully weaned from any immunosuppressant in case of various situations like severe infectious complications, posttransplant lymphoproliferative disorder, and noncompliance. Based on such unexpected experiences, we started the intentional weaning of immunosupressants after LDLT. The patients were selected among the recipients who had been followed up longer than two years with stable normal graft function, and without any rejection episodes in the latest one year. So far, 26 cases were enrolled into this program, and 6 cases could be intentionally weaned from any immunosuppressants. Four recipients unfortunately had a rejection episode during the weaning. Immunosuppressants were again returned to the pre-weaning level in these recipients, and the rejection could be treated without any persistent dysfunction of the graft. Clear discrimination of the recipients who will be or will not be able to be weaned from immunosuppression is still impossible. Mixed lymphocyte reaction showed tendency of low response to the donor, but the result is not stationary in all recipients. Identification of suitable parameters to promise the possibility of weaning is a big problem to be solved in the future. PMID- 10726481 TI - [Control of rheumatoid arthritis (RA) by induction of apoptosis]. PMID- 10726482 TI - [Development of immunotherapy against HER2/neu: hydrophobized polysaccharides/oncoprotein complexes and human dendritic cells]. PMID- 10726483 TI - [Gene-immunotherapy using dendritic cells]. PMID- 10726484 TI - [Vaccination of chronic myelogenous leukemia patients with peptide-pulsed dendritic cells]. PMID- 10726485 TI - [Interaction of H. pylori infection with the progression of autoimmune gastritis in the neonatally thymectomized BALB/c mice]. PMID- 10726486 TI - [Oral tolerance and its clinical application]. PMID- 10726487 TI - [Mechanism of leukocytapheresis effect in the treatment of ulcerative colitis]. AB - To solve adverse effects of high dose steroid administration for patients with moderately severe and severe ulcerative colitis (UC), additional use of leukocytapheresis (LCAP) was tried to settle colonic inflammation. We evaluated immunological changes in the treatment of LCAP using leukocyte removal filter for UC patients. We then assessed the clinical effectiveness of LCAP compared with that of high dose of steroid therapy. LCAP removed monocytes, granulocytes, and lymphocytes presenting CD 11 b+, CD 11 c+, and HLADR+, selectively from the patients. Proinflammatory cytokine productions measured such as TNF alpha, IL-1 beta, and IL 8 reduced and IL 10 increased immediately after LCAP compared with before perfusion. Improved rate was about 70% for LCAP group and about 40% for high dose steroid group (Refer J Gastroenterol). Selective removal of granulocyte, monocytes, and activated lymphocytes inhibits proinflammatory cytokine production and increases immune modulating cytokine productions (Refer Therapeutic Apheresis). Then quick inhibition of several inflammatory deteriorated factors simultaneously controls the activity and clinical symptoms of UC with less severe adverse effects. It can be considered one option for treatment of UC. PMID- 10726488 TI - [Intestinal epithelial cell-derived IL-7 mediated immune responses in mucosal immunity and intestinal inflammation]. PMID- 10726489 TI - [Implication of M cells in the occurrence of early lesions of inflammatory bowel disease]. PMID- 10726490 TI - [Susceptibility loci to autoimmune arthritis in MRL/lpr mice]. PMID- 10726491 TI - [SLE susceptibility genes]. PMID- 10726492 TI - Journal 2000, honoring our past, shaping our future. PMID- 10726494 TI - The role of vertical ramus osteotomy in the management of TMJ internal derangements. PMID- 10726493 TI - Interdisciplinary team approach to the dental management of craniofacial fibrous dysplasia. PMID- 10726495 TI - Case of the month. Pyogenic granuloma. PMID- 10726496 TI - Neuralagia-inducing cavitational osteonecrosis. A case presentation. PMID- 10726497 TI - Unilateral coronal synostosis treated by internal forehead distraction. AB - A 1-year-old infant with left hemicoronal synostosis was treated by distraction osteogenesis of the craniofacial skeleton using an internal distraction device. Surgery was performed through a coronal incision. The frontal bone and upper half of both orbits were first osteotomized en bloc after minimal epidural dissection of the supraorbital area and no epidural dissection around the coronal osteotomy site. The lateral one fourth of the frontal bone, including the right lateral half of the orbit, was left intact. The internal distraction device was fixed in the left temporal area. A 0.5-mm per day rate of distraction was performed up to an elongation of 17 mm after a 5-day latency period. The distraction device was removed after a consolidation period of 2 months. The results obtained were satisfactory, with symmetry of the forehead, orbit, and nose achieved without complications. The merits of this procedure are no extradural dead space after the operation (which prevents infection), shortened operative time, reduced blood loss, filling in the bone gap created by advancement with new bone, acceptable cosmesis by the parents during distraction, and no fixation device left after the second operation. PMID- 10726498 TI - Coincident metopic synostosis and deformational plagiocephaly. AB - A male infant is described in whom coincident pathologies of metopic synostosis and deformational plagiocephaly were observed. The role in causation of localized pressure (in particular, extreme constraint) is addressed. PMID- 10726499 TI - The mandibular foramen: an anatomic study and its relevance to the sagittal ramus osteotomy. AB - The sagittal split ramus osteotomy is the most commonly used procedure to reposition the mandible surgically. Because it is more technically difficult and associated with a higher incidence of complications compared with other mandibular osteotomies, thorough knowledge of the anatomy of the mandibular ramus is a prerequisite. Anatomic measurements related to the mandibular foramen were obtained from 57 formalin-preserved non-Asian hemimandibles. As shown in previous reports, great variability was noted in the position of the mandibular foramen. However, these studies utilized Asian mandibles with a clear discrepancy in key anatomic measurements in comparison with the authors' data. This brings into question the validity of these earlier studies when applying their data to non Asian groups. The "fade-out" point of the internal oblique ridge was found not to be a reliable anatomic reference for placement of the horizontal osteotomy along the medial ramus. Thus, familiarity with the described relationships of the mandibular foramen will assist in performing properly a sagittal split of the ramus and will reduce the chance for an unfavorable split. PMID- 10726500 TI - A longitudinal three-dimensional evaluation of the growth pattern in hemifacial microsomia treated by mandibular distraction osteogenesis: a preliminary report. AB - This study analyzed in three dimensions the longitudinal growth pattern of young patients with hemifacial microsomia (HFM) before and after mandibular distraction osteogenesis (DO). Six individuals with HFM (five boys and one girl; age at distraction, 12.5 +/- 2.4 years) were treated with similar procedures (surgery, type and direction of distraction, no functional orthodontic treatment before and after DO). Two individuals who did not undergo DO until late in their growth were used to compare growth patterns. In addition, one individual besides the six previously chosen sample in whom no DO was performed was also used to compare longitudinal growth patterns. Lateral and posteroanterior (PA) cephalograms were utilized preoperatively, spanning a period of 9 years. Computerized three dimensional models were constructed from the lateral and PA cephalograms using a vector intercept algorithm. In the comparison group, for a period of 8 years on the affected side, the ramus height, body length, and total mandibular length increased at an average rate of 1.3, 1.9, and 3.0 mm per year respectively. On the unaffected side, the ramus height increased by 2.1 mm per year, 1.9 mm in the body, and 2.9 mm per year in total mandibular length. On average, the gonial angle on the affected side was increased by 1 deg per year, yet decreased by 1 deg per year on the unaffected side. The proportions between the affected to the unaffected side were maintained. In the six individuals 18 months after DO, it was found that the ramus height was reduced by 1.0 mm, whereas the body was found to resume its growth with a faster rate on the distracted side, maintaining its proportion. Angular changes demonstrated closing of the gonial angle on both the unaffected (0.5 deg) and distracted (3.5 deg) sides. Observed in three dimensions were the following: (1) On average, unoperated patients with isolated HFM tend to maintain their asymmetrical facial proportions and do not worsen substantially with time. (2) Different treatment effects were seen on the ramus, body, and total length of the mandible: changes in body length > ramus height > total length. (3) Eighteen months after DO, the correction was stable but with some degree of settling back from the initial overcorrection (< 5%). (4) Eighteen months after DO the mandibular body was found to have greater growth than the ramus. (5) Evaluating changes in three dimensions provides an improved understanding of the growth pattern and distraction effects on the mandible and its structural components. (6) Additional studies on the effect of mandibular distraction on other conditions involving mandibular deformities are required. In addition, the effect of various distractor devices should also be evaluated. Three-dimensional evaluation is recommended for improved accuracy. PMID- 10726501 TI - Pericranial flap correction of superior sulcus depression in the anophthalmic orbit. AB - Distortion of the supratarsal sulcus of the upper eyelid after orbital trauma is a well-recognized and troublesome problem. This is particularly true of the anophthalmic orbit. The authors present two patients in whom this deformity has been addressed using a pedicled pericranial flap. They found this technique provides abundant, well-vascularized tissue that is manipulated easily to conform to the demands of the defect. In addition, the vascularity of the tissue provides predictability of the result when compared with other described techniques such as fat and dermis-fat grafts. PMID- 10726502 TI - Key points in the fixation of the craniofacial skeleton with absorbable biomaterial. PMID- 10726503 TI - The use of ultrasound-assisted liposuction in the treatment of an involuted hemangioma. AB - The authors report a case of a large, residual involuted hemangioma of the facial region in a 10-year-old girl. Ultrasound-assisted liposuction was used to debulk the tumor mass and to avoid the possibility of inadvertent iatrogenic damage to the facial nerve during surgical excision. PMID- 10726504 TI - Sequential cleft management with the sliding sulcus technique and alveolar extension palatoplasty. AB - Conventional methods of cleft lip repair deprive the anterior (buccolingual) alveolar mucoperiosteum of blood supply from the facial-internal maxillary arcade. Six months later, at palatoplasty, lingual incisions permanently isolate the lingual mucoperiosteum from its blood supply--the greater palatine artery. The osteogenic alveolar mucoperiosteum is thus converted from a richly supplied boundary zone between the two angiosomes into an isolated tissue dependent on osseus backflow. Cleft-sided growth disturbance is considered from this perspective. Subperiosteal techniques that preserve the blood supply to this tissue are considered in a sequential plan of cleft management. PMID- 10726505 TI - LactoSorb resorbable osteosynthesis after sagittal split osteotomy of the mandible: a 2-year follow-up. AB - Twenty patients had sagittal split osteosynthesis performed with three or four 2.5-mm resorbable screws on each side without additional fixation. The screws consisted of a poly-L-latic/polyglycolic copolymer (LactoSorb). Of the 20 patients, 17 had mandibular advancement, two had mandibular setback, and one underwent unilateral mandibular advancement after previous condylectomy. Six patients underwent simultaneous Le Fort I osteotomy and four patients had simultaneous genioplasty. Postoperative training elastics were maintained for an average of 2.5 weeks. Six of the patients have been followed for 2 years, and 14 have been followed between 1 and 2 years without observable relapse and without clinical, radiological, or histological signs of healing complications. The resorbable osteosynthesis utilized in this series of sagittal split osteotomy seems to offer reliable stability during the period of bony healing without any adverse tissue reactions. PMID- 10726506 TI - Bilateral Tessier No. 9 cleft. AB - The authors describe a patient with bilateral Tessier no. 9 cleft. This is the rarest of the rare craniofacial clefts. It is an upper lateral orbital cleft with a soft-tissue deformity that extends from the lateral third of the upper eyelid, through the lateral end of the eyebrow to the temporal scalp. The bony disruption extends from the superolateral orbital rim across the greater wing of the sphenoid to the upper squamous temporal area. The clinical and radiological examinations are reported. No surgical correction was necessary. PMID- 10726507 TI - A destructive calcium pyrophosphate dihydrate deposition disease of the temporomandibular joint. AB - A case of calcium pyrophosphate dihydrate (CPPD) crystal deposition arthropathy of the temporomandibular joint is reported. The patient presented a 10-year history of swelling and pain of the left preauricular region. Magnetic resonance imaging showed a calcified mass filling the joint space and destroying the roof of the joint. Radiographs showed chondrocalcinosis of other joints. The authors discuss the diagnosis of this arthropathy and the reason why the temporomandibular joint is more affected than the other joints in the patient reported. PMID- 10726508 TI - Extracranial cephalic schwannomas: a series of 15 patients. AB - The aim of this study was to report a series of extracranial cephalic schwannomas. Fifteen patients with extracranial schwannomas treated between 1981 and 1999 are presented, and their clinical course during a median follow-up of 4.1 years is discussed. There is a female predominance. No specific factors have been identified. Their diagnosis is often delayed (median, 2.6 years). There is no predominant side. The orbit represents the most frequent location of schwannomas (26%). The trigeminal nerve is the most often affected (53%). Computed tomography and magnetic resonance imaging contribute to the diagnosis. Macroscopically, the schwannoma is a well-defined tumor of ovoid form and brownish color. It is formed of soft tissues and is fragmented easily. Diagnosis is often evident on microscopic examination. The only treatment is surgery. It consists of enucleation after opening the epineurium using an operating microscope, without interruption of the continuity of the nerve. The authors have observed only two relapses (the first two patients operated without a microscope). Total excision allows recovery. Nerve injuries have variable prognosis. It is necessary in juvenile populations to search for neurofibromatosis. All schwannomas required surgical treatment using an operating microscope to obtain total recovery. PMID- 10726509 TI - The thickness of the skull in Korean adults. AB - New instrumentation and techniques for skeletal fixation have been developed in response to concerns regarding variability of the skull base and the calvaria. The fear of intracranial penetration has limited the use of internal or external fixation to the skull base and the calvaria. Despite potential clinical usefulness, limited anthropometric data have been reported on the skull base and the calvaria. The purpose of this study was to measure the average thickness of various points of the skull base and the calvaria of Korean adults. Fifty-one Korean adult skull bases and calvaria, which were cut 2 cm above the supraorbital margin anteriorly and 2 cm above the inion posteriorly, were measured in this study. Thickness of 13 clinically important points were measured with a Teclock GM-21 Calipergauge (Teclock Co., Tokyo, Japan). The mean thickness of the two opposite sides were compared using paired t-tests. They did not differ significantly from the others except for three points. Frontal bone A (located 1 cm above the supraorbital margin and 1.5 cm lateral to the midline) was 6.35 +/- 2.88 mm, frontal bone B (located at the midpoint of the frontal midpoint and the frontoparietal suture on the cut edge) was 5.24 +/- 1.50 mm, frontal bone C (located at the midpoint of the vertex and frontal bone B) was 6.63 +/- 1.77 mm, the pterion was 3.19 +/- 0.85 mm, the midpoint of the squamous temporal was 1.96 +/- 0.65 mm, parietal bone A (located 1.5 cm below the middle meningeal groove) was 3.99 +/- 1.11 mm; parietal bones B and C (divided at the distances between the frontoparietal suture and the occipital midpoint with a ratio of 1:3 and 2:3 respectively on the cut edge) were 4.71 +/- 1.06 mm and 5.35 +/- 0.91 mm respectively, occipital bone A (located 1.5 cm posterolateral from the foramen magnum) was 2.31 +/- 1.00 mm, occipital bone B (located 1.5 cm lateral to the midline and 0.5 cm above the inion) was 5.41 +/- 1.50 mm, occipital bone C (located occipital to the midpoint and 2 cm above the inion) was 8.2 +/- 1.67 mm, the orbital roof was 1.37 +/- 1.08 mm, and the orbital lateral wall was 2.25 +/- 1.06 mm. The data give practical information for determining the position and depth for safe and effective internal or external fixation during craniofacial procedures. PMID- 10726510 TI - A biomechanical analysis of the orbital zygomatic complex in human cadavers: examination of load sharing and failure patterns after fixation with titanium and bioresorbable systems. PMID- 10726511 TI - Biomaterials in the face: benefits and risks. AB - An extensive review of biomaterials in the face was conducted in an American Society of Maxillo-facial Surgeons-sponsored biomaterials symposium. The symposium was held in Boston, MA, immediately preceding the 1998 annual meeting of the ASPRS/PSEF. The scope of the symposium extended from current reconstructive techniques for the facial skeleton, including autogenous bone and biomaterials, to potential application of new techniques in molecular biology that may enable the body's own tissues to be engineered to provide bone and cartilage to reconstruct the facial skeleton. The authors review the presentations and relevant literature on biomaterials in the face. The following topics are reviewed: current reconstructive techniques using autogenous bone grafts, methyl methacrylate cranioplasty, demineralized bone, and hydroxyapatite; biomaterials used for rigid fixation, including metallic and bioabsorbable implants; biomaterials used for facial augmentation, including porous polyethylene, hard-tissue replacement, and ceramic biomaterials; biofilm, or a layered polysaccharide matrix secreted by bacteria on the surface of implants; and potential means of inducing bone formation by directing the body's own tissues through cytokine interaction, gene transfer, and tissue engineering. PMID- 10726512 TI - The sliding sulcus procedure: simultaneous repair of unilateral clefts of the lip and primary palate--a new technique. AB - During embryogenesis the clefting event unleashes a series of processes that act in concert to deform the soft tissue envelope into a "dysfunctional matrix" within which bone and cartilage subsequently form. Much of secondary cleft deformity results from failure to solve this pathologic soft tissue-bone equation at the primary repair. The sliding sulcus procedure uses the subperiosteal plane to create a centralization of the soft tissue matrix, alleviate tension, and redistribute the forces of growth in a more normal pattern. By incising at the level of the alveolus, this approach preserves the blood supply to the osteogenic cells of the buccolabial periosteum. This approach has interesting theoretical implications for the preservation of alveolar growth potential. Arch stabilization and elimination of fistula may be of long-term benefit to the orthodontist. PMID- 10726513 TI - Obstructive sleep apnea in a growing patient. AB - Sleep apnea syndrome (SAS) is a complex respiratory disorder that is very difficult to diagnose and to treat surgically as well as medically. SAS can affect growing patients as well as adults. SAS shows a central, an obstructive, and a mixed form. Diagnosis is based on clinical examination of the patient and instrumental examinations such as teleradiography, nuclear magnetic resonance imaging (NMR), three-dimensional computed tomography, polysomnography, rhinomanometry, and spirometry. The patient presented has an obstructive form of SAS in addition to Crouzon's disease. He underwent a Le Fort III osteotomy to obtain an advancement of the orbitomaxillary complex, allowing an increase in volume of posterior airway space at the level of the hypopharynx. PMID- 10726514 TI - Craniofacial reconstruction of a giant cystlike temporal encephalocele. AB - A 4-year-old girl underwent craniofacial reconstruction for giant cystlike encephalocele deriving from the temporo-maxillary region and giving impression of the duplicated head. The case of temporal encephalocele in this report is especially unusual in the extent of encephalocele, the degree to which it had expanded the zygomatic arch, mandible, cranial vault, and the radiologically undetectable bony defect. The use of craniofacial principles in the resection and reconstruction of the temporal encephalocecle are described. PMID- 10726515 TI - Reduction of zygomatic arch fracture using a towel clip. AB - The authors describe a simple technique for reducing zygomatic arch fractures with a towel clip. The orbital rim and zygomatic arch are outlined with a marking pen. The exact fracture site is then marked by palpation. The depressed fracture site is held with a towel clip and pulled outward gently. A clicking noise may be heard. The clip is then released. The contour of the zygomatic arch is compared with the other side. Using this technique the authors reduced 11 fractured zygomatic arches (arch only, displaced medially) in 11 patients and the results were satisfactory in all patients. The towel clip method is one procedure to reduce zygomatic arch fracture. PMID- 10726516 TI - Juvenile ossifying fibroma of the midface. AB - Juvenile ossifying fibroma is an unusual maxillofacial fibro-osseous lesion characterized by cell-rich osteoid strands. A 7-year-old girl presenting with a massive juvenile ossifying fibroma of the maxilla resulting in facial deformity, orbital displacement, and extension into the anterior skull base is discussed. The importance of combining clinical, radiographic, and histopathological findings for the diagnosis and management of the tumor is discussed. PMID- 10726517 TI - Combining single- and double-tooth osteotomies with traditional orthognathic surgery. AB - Combining large-segment orthognathic surgery and unitooth or small-segment surgery is an effective approach to deal with a wide range of dentofacial deformities that have minor to severe occlusal problems. The indications for combining single- or double-tooth osteotomies with traditional orthognathic surgery were patients with dentofacial deformities and malocclusion requiring stable correction within a short overall treatment period. From 1991 to 1998, a total of 36 patients underwent combination single- or double-tooth-segment osteotomy with traditional orthognathic procedures performed at Chang Gung Memorial Hospital. The indications for surgery were maxillary protrusion (N = 5), bimaxillary protrusion (N = 19), mandibular prognathism with maxillary protrusion (N = 11), and noncleft maxillary retrusion (N = 1). The types of osteotomies performed were the Le Fort I, the anterior segmental osteotomies of the maxilla or the mandible, the palatal split, and the posterior segment in combination with single-tooth or double-tooth segments. Follow-up ranged from 12 months to 6 years and showed stability in the movements with no complications. There was no loss of any "osteotomized" segment. The average overall treatment time was approximately 18 months--5 months preoperative and 13 months postoperative orthodontic treatment. This was at least 6 months shorter in duration compared with traditional orthognathic surgery. Combining traditional orthognathic surgery with single- or double-tooth segments allows us to treat complex dentofacial deformities in the vertical, transverse, and sagittal dimensions with differential repositioning of all segments, either major or minor, simultaneously. The authors' experience with 36 consecutive patients evidence good results and demonstrate the procedure to be safe with minimal complications. PMID- 10726518 TI - Extracranial optic nerve decompression: a 10-year review of 92 patients. AB - Over a 10-year period the authors have performed 92 transethmoidal optic nerve decompressions for the treatment of visual loss due to various pathological processes, including 45 cases of trauma, 32 cases of neoplasm, 2 cases of bacterial pansinusitis, 5 cases of sphenoethmoidal mucocele, 4 cases of aspergillosis (all were immunocompetent patients), 2 cases of Wegener's granulomatosis, and 2 cases of sarcoidosis. Forty-eight patients (52%) had preoperative visual acuity of light perception or better, and in 44 patients (48%) the preoperative vision was no light perception. Sixty-five patients (71%) achieved improvement of vision postoperatively. Twenty-four patients (26%) had no change in vision and 3 patients (3%) had deterioration of vision after surgery. The mean percentage of improvement was 40.7% +/- 6.9% in the trauma group, 61.6% +/- 23.2% in the neoplasm group, 66.4% +/- 25.2% in the infectious/mucocele group, and only one patient in the inflammatory group had slight visual improvement from no light perception to counting fingers. Extracranial optic nerve decompression can result in the improvement of visual function in some patients with optic nerve injury from various causes. PMID- 10726519 TI - Effects of camphorated parachlorophenol on human periodontal ligament cells in vitro. AB - To date, there has been very little research into the possible effects of endodontic therapy on regeneration of a lost periodontal attachment. The objective of this study was to examine the effects of the endodontic medication, camphorated parachlorophenol (CMCP), on human periodontal ligament cells in vitro. The cytotoxic effects of CMCP were evaluated by 3-(4,5-dimethylthiazol-2 yl)-2,5-diphenyl tetrazolium bromide colorimetric assay and cell proliferation using a [3H]thymidine incorporation assay. CMCP inhibited the human periodontal ligament cells viability and proliferation in a dose-dependent manner (p < 0.05). These data indicate that the use of CMCP in a root canal could cause periodontium damage. Although this study was conducted in vitro, the findings suggest that it may not be advisable to use CMCP as an interim medication when a periodontal surgical procedure, especially an attempt at regeneration or a new attachment procedure, is being considered in tissues adjacent to the endodontically involved tooth. PMID- 10726520 TI - In vitro antimicrobial effect of RC-Prep within dentinal tubules. AB - The antimicrobial effect of RC-Prep and its components--10% urea peroxide, 15% EDTA, and glycol--were evaluated individually, in vitro, within dentinal tubules. Standardized bovine incisor root specimens that had been infected with either Staphylococcus aureus or Streptococcus peltzer were incubated with each agent for 10 and 45 min. After exposure to the agent, dentin samples were collected from within the lumina using ISO 23 to 31 burs. Dentin powder samples were incubated with bacterial broth, and the amount of bacteria present was assessed by spectrophotometry. All agents tested were more effective in killing bacteria after incubation for 45 min than after 10 min. PMID- 10726521 TI - In vitro disinfection of dentinal tubules by various endodontics irrigants. AB - Effectiveness of endodontic irrigants within dentinal tubules of human teeth was evaluated. Mid-sections of single-rooted teeth were prepared into dentin wedges. The pulpal sides of the sections were exposed to Micrococcus luteus or Bacillus megaterium that grew into the tubules. Irrigants used in the study included: 0.525% NaOCl, 0.12% chlorhexidine, RC Prep, 0.5% betadine iodine, and sterile H2O (as a control). Pulpal surfaces were exposed to an irrigant and then rinsed in sterile water. The samples were then cracked, exposing a fresh surface. Culture of the exposed dentin surfaces showed that selected irrigants reached to the far ends of the dentinal tubules in a concentration sufficient to kill 100% of the M. luteus. However B. megaterium was neither killed nor apparently inhibited by any irrigant. We conclude that endodontic irrigants permeate throughout dentinal tubules, but their effectiveness is dependent on the type of bacteria found within the tubules. PMID- 10726522 TI - Confocal and scanning electron microscopic study of teeth restored with fiber posts, metal posts, and composite resins. AB - Forty-two single-rooted lower premolars, extracted for periodontal reasons, were endodontically treated and divided into 7 groups of 6 teeth each. In five of the groups, three different types of carbon fiber posts, quartz fiber posts, and titanium posts were used in combination with All Bond 2 dental adhesive. In two groups, two types of carbon fiber posts were also cemented with Panavia 21 dental adhesive. After 3 wk storage in saline, the teeth were longitudinally sectioned; one half was observed using confocal microscopy and the other by scanning electron microscopy. The specimens were evaluated for the presence of a resin dentin interdiffusion zone for the presence of voids at post-resin-dentin interfaces and for the determination of the fiber posts' structure. Upon examination with the confocal microscope, the interfaces of teeth restored with All Bond 2 showed a higher percentage (p < 0.05) of resin dentin interdiffusion zone than those treated with panavia. The fiber size and the post structure were similar in all the fiber posts observed. Some voids were present inside the fiber post structure. PMID- 10726524 TI - Shaping and cleaning the root canal system: a scanning electron microscopic evaluation of a new instrumentation and irrigation technique. AB - The purpose of the present scanning electron microscopic study was to investigate the efficacy of a combination of EDTA, NaOCl, and surface-active irrigating solutions during and after root canal preparation with ProFile nickel-titanium rotary instruments. Thirty canals were divided into three groups, instrumented and irrigated as follows: 5% NaOCl and a final flush with 17% EDTA were used for group A; group B specimens were irrigated using 17% EDTA, followed 15 s later by 1% TRITON X-100 (tensioactive agent) and then by 5% NaOCl; and group C specimens were irrigated with the same combination, but once shaping procedures were completed the irrigating sequence was repeated three times. After scanning electron microscopic evaluation, group C specimens exhibited the most effective debridement of the root canals. Results showed that tensioactive agent contributed to enhanced debridement. Cleaning was significantly improved once shaping procedures were completed. PMID- 10726523 TI - Inflammatory cytokine production and specific antibody responses to lipopolysaccharide from endodontopathic black-pigmented bacteria in patients with multilesional periapical periodontitis. AB - We examined the induction of the cytokines interleukin (IL)-1 beta, IL-6, and IL 8 by lipopolysaccharides (LPSs) from several species of possible endodontopathic black-pigmented bacteria. Studies were conducted in human whole blood cultures from six patients (two from each group) with differing numbers of periapical periodontitis lesions (i.e. patients with radiographically clear periapical lesions in 10 or more teeth (high-lesion group, n = 4), in one or two teeth (low lesion group, n = 6), and six healthy volunteers with no periapical lesions (no lesion group)). LPS from Prevotella intermedia ATCC 25611, Porphyromonas gingivalis 381, and Prophyromonas endodontalis ATCC 27067 induced a higher IL-8 response in the subjects of the high-lesion group, compared with the subjects of the other two groups. To ascertain the degree of sensitization by test bacteria, we examined the reactivities of antibodies in serum and saliva from the subjects to different bacterial species. LPS from P. gingivalis reacted strongly with sera from the high-lesion group. Thus, LPS from black-pigmented bacteria may be involved in multilesional periapical periodontitis by inducing particular cytokines and/or humoral immune responses. PMID- 10726526 TI - A comparison of molar root canal preparation using stainless-steel and nickel titanium instruments. AB - Fifty-four extracted human mandibular molars were embedded and sectioned at two levels. The reassembled mesial root canals were prepared with stainless-steel hand K-files (Flexofiles) and either Nitiflex or Mity nickel-titanium hand K files using a push-pull anticurvature filing technique. Each of the three experimental groups contained 36 mesial canals randomly distributed. Superimposed pre- and post-instrumentation cross-sectional root images were magnified using a stereomicroscope and transferred to a computer for measurement and statistical analysis. The direction and extent of canal center movement were evaluated. At the apical level, the groups produced no significant difference of direction of canal center movement. In cervical sections, all groups tended to move in a distolingual direction. The three groups, however, produced no significant difference in the cervical sections in the extent of canal center movement. In apical sections, Nitiflex produced the least canal center movement. PMID- 10726525 TI - Comparison of acetaminophen, ibuprofen, and nabumetone therapy in rats with pulpal pathosis. AB - The purpose of this study was to determine if daily treatment with acetaminophen, ibuprofen, or nabumetone would alter prostaglandin E2 (PGE2)/leukotriene B4 (LTB4) synthesis in exposed, infected dental pulp and periradicular tissue in rats. Dental pulp of the bilateral first and second mandibular molars were exposed for 15 or 24 days. Rats were divided into four groups. A daily pharmacological oral dose of one of the medications or the suspending solution alone was administered to the designated group. Eicosanoids were extracted from pulpal and periradicular tissues and assayed. PGE2 was significantly elevated in pulp-exposed, nontreated rats and was significantly reduced in the ibuprofen- and nabumetone-treated groups. LTB4 was significantly increased in all pulp-exposed groups at 15 days when compared with control nonexposed groups. Results showed that only ibuprofen reduced LTB4 in the exposed dental pulp at 24 days, although it did not do so at 15 days. Repetitive treatment with acetaminophen did not suppress PGE2/LTB4 in pulp-exposed molars. PMID- 10726527 TI - Evaluation of the cytotoxicity of calcium phosphate root canal sealers by MTT assay. AB - The purpose of this study was to evaluate the cytotoxicity of some calcium phosphate-based sealers (Sankin apatite root canal sealers (SARCS) types 1 to 3) in comparison with currently used sealers (CRCS, Ketac Endo, AH26, and Endomethasone) by using MTT assay on L929 cells. Monolayer cell cultures were prepared on 96-well plates. After incubation at 37 degrees C in a humidified 5% CO2-containing air atmosphere for 24 h in the presence of each sealer extracts, 25 microliters of 5 mg/ml of MTT in saline were added into each well and incubated a further 3 h at 37 degrees C. A solubilization buffer consisting of 23% sodium dodecyl sulfate in 50% N,N-dimethylformamide (pH 4.7) was used to dissolve formazan precipitate. The optical densities of the plates were then read by a microplate spectrophotometer at 570 nm. Greater magnitude of optical density due to intense blue coloring is regarded as showing a higher percentage of cell viability. Among the different types of sealers, SARCS types 1 to 3 and CRCS did not exert any cytotoxic effects, whereas AH26, Ketac Endo, and Endomethasone produced some cytotoxicity. PMID- 10726528 TI - Results of endodontic retreatment: a randomized clinical study comparing surgical and nonsurgical procedures. AB - Information of "success" rates after surgical or nonsurgical endodontic retreatment is abundant but inconclusive. Reported healing frequencies vary between 45% and 90%. The present study was designed to find any systematic difference between the methods. Nonsurgical and surgical retreatment was randomly assigned to 95 endodontically "failed" cases. The outcome of the procedures was clinically and radiographically recorded, and followed for 4 years. At the 12 month recall, a statistically significant (p < 0.05) higher healing rate was observed for cases surgically retreated. At the final 48-month examination, no such difference was found. These findings may be explained by (a) slower healing dynamics in the nonsurgical group and (b) the event of late "failures" in the surgical group. Within the latter category, four cases classified as healed after 1 yr failed at the final follow-up. Conclusively, this study failed to show any systematic difference in the outcome of surgical and nonsurgical endodontic retreatment. Surgical retreatment seems to result in more rapid periapical bone fill, but also may imply a higher risk of "late failures." From a scientific point of view, the length of the follow-up period is very important and may strongly influence the conclusions made. PMID- 10726529 TI - Long-term radiographic and histological observations of endodontically treated teeth. AB - Endodontic therapy was performed on 14 teeth in juveniles with inflamed or necrotic pulps. The patients were recalled at 6-month intervals for radiological and clinical examinations. After 1 yr, periapical surgery was performed on six teeth. After 18 months, 5 more patients were subjected to periapical surgery and, after 2 1/2 years, 3 more patients had periapical surgery. A small block section containing the root tip and surrounding tissues was removed from all of the patients. Radiographs showed reductions in size, but not elimination, of periapical lesions. Histological examinations revealed that most root canals were overfilled. Inflammation persisted around zinc oxide-eugenol particles beyond the tooth apexes. In addition, gutta-percha overfilling enhanced the proliferation of cell rests of Malassez. In some cases, dentin filings were found at and beyond the tooth apexes. The formation of new hard tissue was stimulated by the presence of dentin chips. PMID- 10726530 TI - A survey of filling methods, intracanal medications, and instrument breakage. AB - The present survey was conducted to obtain answers to some basic questions regarding the use of intracanal medications, root canal filling methods, and the intracanal breakage of instruments. A letter was sent to 300 endodontists listed in the 1995-1996 membership roster of the American Association of Endodontists. Eighty-five replies were received. Calcium hydroxide as an intracanal medication was used by 91.7% of the responding endodontists. With regard to the root canal filling technique, 52.9% used the lateral condensation method. When intracanal breakage occurred, 95.3% of the respondents informed the patient. The results of this survey provide useful information for the education of undergraduate dental students. PMID- 10726531 TI - Management of the latex hypersensitive patient in the endodontic office. AB - This case report documents the treatment of an endodontic patient who experienced a type 1 hypersensitivity reaction to latex. The dental, medical, and environmental aspects of treating latex allergic patients are reviewed. Because gutta-percha and latex rubber are similar compounds, the possible cross reactivity of these materials is discussed. PMID- 10726532 TI - Periradicular scars: a sometime diagnostic conundrum. AB - This paper presented illustrations of two different clinical cases that developed residual bone scars. The first case dealt with the evolution of a bone scar after surgical removal of a large cyst with a "thru-and-thru" defect, not an uncommon event. The second case showed a typical periradicular lucency associated with a nonvital pulp, which evolved into a bone scar after nonsurgical root canal therapy. PMID- 10726533 TI - Adherence of enamel matrix derivatives on root-end filling materials. AB - It was recently shown that application of enamel matrix derivatives (EMDs) on denuded root dentin promotes periodontal regeneration. EMD is shown to adhere to the etched dentin, but its adherence to root-end filling materials is not known. The purpose of this study was to evaluate the adherence of a commercially available EMD product to root-end filling materials. Dentin sections were embedded in blocks made of acrylic resin. Cavities were prepared in similar acrylic resin blocks and were filled with amalgam, IRM, or composite resin. EMD was labeled with radioactive iodine and applied to the surfaces of the dentin sections, freshly made fillings, or acrylic resin controls. The specimens were rinsed, and the amount of radioactive iodine was determined in a gamma counter. Substantial amounts of EMD adhered to dentin sections. EMD adherence to amalgam and IRM was significantly less than to dentin or composite resin. PMID- 10726534 TI - Concentration of eugenol apically released from zinc oxide-eugenol-based sealers. AB - This study investigated the amount of eugenol released from a zinc oxide-eugenol based sealer at the apex of teeth filled according to two techniques: the single cone and the Thermafil. The crown of 10 maxillary central incisors was removed, and the canal was prepared with ProFile to a size 30 under NaOCl irrigation. The patency of the apex was checked with a #8 K-file between each ProFile. Five roots were filled with a Thermafil #30 and 0.03 g of Sealite; five roots were filled using a Lentulo with 0.07 g of sealer and a gutta-percha cone #30. The powder/liquid ratio of the sealer was of 5/1. The concentration of eugenol released in phosphate-buffered saline was spectrofluorimetrically determined immediately after sealing, after 1 day, and after 1 month of storage. The roots filled with the single-cone technique released significantly more eugenol than these filled with Thermafil immediately after sealing (p = 0.002); but, after 1 day or 1-month storage, there was no difference. For both techniques, eugenol concentration decreased over time (p = 0.01): the immediate concentration was higher than the 1-day concentration (p = 0.04). Eugenol concentration after 1 month storage was undetectable. The results of this work show that the level of eugenol released from a zinc oxide-eugenol-based sealer beyond the apex is very low and decreases over time. PMID- 10726535 TI - A comparison of the effects of three chelating agents on the root canals of extracted human teeth. AB - The effects of EDTA were compared in vitro with the effects of two other chelating agents: succimer and trientine HCl. Thirty extracted human teeth with single canals were used. Groups of 10 were treated by the three agents and then examined under the scanning electron microscope. The results showed that EDTA widened the dentinal tubules. Trientine HCl also produced widening of the dentinal tubules very similar to EDTA, whereas succimer produced more extensive widening. PMID- 10726536 TI - Pulpal exposure alters neuropeptide levels in inflamed dental pulp and trigeminal ganglia: evaluation of axonal transport. AB - Dental pulp is richly innervated with neurons containing calcitonin gene-related peptide (CGRP) or substance P (SP). Prior studies have demonstrated that inflammation alters these pulpal neuropeptides. In this study, we used a radioimmunoassay to evaluate the specificity of this response and the contribution of axonal transport. Rat mandibular molars were exposed and immunoreactive CGRP (iCGRP) and immunoreactive SP were measured. At 7 to 14 days after exposure, both pulpal iCGRP (73%) and immunoreactive SP (135%) displayed peak increases above control levels. This response was somatotopically restricted, with no changes observed in contralateral (intact) molars, or in ipsilateral mandibular molars after exposure of maxillary molars. Transection of the inferior alveolar nerve on day 13 significantly reduced pulpal levels of iCGRP on day 14. Collectively, these studies indicate that pulpal inflammation evokes a selective alteration in neuropeptide levels, due at least in part to alterations in transport or synthesis of neuropeptides in the trigeminal ganglion. PMID- 10726537 TI - Development of a clindamycin-impregnated fiber as an intracanal medication in endodontic therapy. AB - The effectiveness of traditional endodontic intracanal medications in reducing bacterial numbers and preventing acute flare-ups and pain continues to be questioned. In the present study, a new local delivery device was developed that releases a substantive dose of clindamycin into root canals. Clindamycin impregnated ethylene vinyl acetate (EVA) fibers were produced, and the sensitivity of common endodontic microbes to the fibers were established. An in vitro model was developed to persistently infect 32 extracted human teeth with endodontic pathogens to test the efficacy of the clindamycin/EVA fibers in reducing the number of colony-forming units. The clindamycin/EVA fibers were shown to be effective in reducing growth of common endodontic microbes on blood agar plates, and in significantly reducing growth of Prevotella intermedia, Fusobacterium nucleatum, and Streptococcus intermedius in extracted human teeth, thus indicating merit in further exploring the potential of these fibers as intracanal medications. PMID- 10726538 TI - Reaction of dogs' teeth to root canal filling with mineral trioxide aggregate or a glass ionomer sealer. AB - This study was conducted to observe the reaction of apical tissues of dogs' teeth after root canal filling with gutta-percha and mineral trioxide aggregate (MTA) or a glass ionomer (Ketac-Endo) as a sealer. The root canals were instrumented and filled by the lateral condensation technique with the sealers studied. Animals were killed 6 months later, and the specimens were removed and prepared for histological analysis. Results showed no inflammatory reaction of apical tissue and total closure of the apical foramen of all the teeth sealed with MTA. The teeth sealed with Ketac-Endo showed two cases of partial closure and different degrees of chronic inflammatory reaction. In conclusion, MTA exhibited better biological properties than Ketac-Endo. PMID- 10726539 TI - Leakage in roots coronally sealed with different temporary fillings. AB - This study was aimed at determining the ability of different coronal temporary fillings to prevent corono-apical penetration of bacteria. A total of 103 human teeth, including three controls, were instrumented, obturated with gutta-percha, coronally sealed with either Cavit, Intermediate Restorative Material (IRM), glass-ionomer cement, Cavit/glass-ionomer cement, or IRM/glass-ionomer cement, respectively. Each root was fixed with wax between two chambers: the coronal chamber harboring soy broth with 10(8) colony-forming units of Streptococcus mutans/ml, the apical chamber containing sterile soy broth. The latter was checked daily for turbidity, indicating corono-apical penetration of bacteria. The Cavit group, the IRM group, and the Cavit/glass-ionomer cement group showed significantly more leakage than the glass-ionomer cement group of the IRM/glass ionomer cement group. If a sample leaked, all except one (IRM/glass-ionomer cement) leaked before day 12. This in vitro study seems to indicate that only glass-ionomer cement and IRM combined with glass-ionomer cement may prevent bacterial penetration to the periapex of root-filled teeth over a 1-month period. PMID- 10726540 TI - Comparison of the cleaning efficacy of passive sonic activation and passive ultrasonic activation after hand instrumentation in molar root canals. AB - The purpose of this study was to compare the cleaning efficacy of passive ultrasonic activation with that of passive sonic activation after hand instrumentation. Sixty curved molar canals were hand-instrumented to size 35 and divided into three groups. Group 1 received no further treatment. Group 2 received 3 min of passive sonic activation. Group 3 received 3 min of passive ultrasonic activation. The roots were split and photomicrographs (x20) were made of the apical 6 mm of canal. A transparent grid was placed over projected images, and the total number of squares covering the apical 6 mm of canal space and the number of squares containing debris were counted. A debris score was calculated for each specimen by dividing the number of squares with debris by the total number of squares. The mean debris scores were 31.6% for hand instrumentation only, 15.1% for the sonic group, and 16.7% for the ultrasonic group. The debris scores for the sonic and ultrasonic activation groups were significantly lower than that for the hand instrumentation only group (p < 0.01); however, there was no significant difference between the sonic and ultrasonic activation groups. Passive sonics after hand instrumentation produces a cleaner canal than hand instrumentation alone and is comparable with that of passive ultrasonics. PMID- 10726542 TI - Scanning electron microscopic study of the interface of glass ionomer cement sealers and conditioned bovine dentin. AB - This in vitro study characterized the interface of glass ionomer cement root canal sealers and dentin conditioned by common endodontic irrigants. The interface of Ketac-Endo and two experimental glass ionomer cement sealers (KT-308 and ZUT) with bovine dentin--conditioned with either distilled water, 2.6% NaOCl, or 17% EDTA, followed by 2.6% NaOCl--was investigated. Cylinders of each of the sealers were formed on the conditioned dentin surface and allowed to set for 90 min. They were first shear tested to failure to determine their adhesive bond strength. Then three dentin specimens from each group were processed for scanning electron microscopic study. The test-failed surface of the strongest and weakest bonded specimens was examined. A third specimen, of intermediate bond strength, was cross-sectioned and the interface was also examined. Failures in all of the specimens were found to be mainly cohesive in nature. Specimens conditioned with 2.6% NaOCl only revealed the presence of a hybrid layer at the interface composed of dentin and whichever of the sealers were tested. PMID- 10726541 TI - Resistance of experimental glass ionomer cement sealers to bacterial penetration in vitro. AB - This study evaluated penetration of Enterococcus faecalis in canals filled with glass ionomer cement sealers. Canals of 40 extracted teeth in four equal experimental groups were prepared and filled with KT-308 (experimental sealer) and a single gutta-percha cone (SC), ZUT (KT-308 with an antibacterial agent) and SC, Kerr sealer and vertically compacted gutta-percha (VC), or Kerr sealer with the antibacterial agent and VC. Eight additional teeth served as positive controls and four as negative controls. Pulp chambers were inoculated with E. faecalis, and bacterial penetration through the filled canals was assessed over a period of 90 days. Incidence of bacterial penetration was significantly higher for ZUT/SC than for Kerr sealer/VC (p < 0.05). KT-308/SC did not differ significantly from any other material. The positive controls showed significantly faster penetration than all the other groups (p < 0.05). It was concluded that KT 308 effectively prevented penetration of E. faecalis, whereas ZUT required further development. PMID- 10726543 TI - Modulation of chronic enteric infection by distant oral infection. AB - This investigation determined the effects of a distant oral infection (Porphyromonas gingivalis) on a concurrent local enteric infection (Echerichia coli). A modified murine subcutaneous tissue chamber model was used. Subcutaneously implanted chambers with different microbial makeups were monitored for sloughing and their contents assayed for prostaglandin E2. Bilaterally implanted chamber experiments revealed that a distant "chronic" (immunization with heat-killed organism, followed by live challenge) P. gingivalis infection offered protection against a local chronic E. coli infection, as evidenced by delaying the time for 50% of the chambers to reject from day 19 to day 25 and a statistically significant prostaglandin E2 decrease from 529.4 +/- 176.6 ng/ml to 191.5 +/- 100.9 ng/ml (p < 0.01) (Mann-Whitney test). An acute (live challenge only) distant P. gingivalis infection or immunization with the heat-killed organism alone had no effect on "chronic" E. coli infection in this model. These data suggest that the presence of low-grade chronic oral infection may modify the responses to other infectious challenges. PMID- 10726544 TI - Effect of rotational speed on nickel-titanium file distortion. AB - Separation of instruments while performing root canals is something that has plagued all practitioners. The purpose of this study was to evaluate the effect of rotational speed on nickel-titanium file distortion and separation. Forty extracted human molars were instrumented with Profile (Dentsply Tulsa Dental Products, Tulsa, OK) ISO .04 rotary nickel-titanium instruments at 166.67 or 333.33 rpm. The instruments were evaluated after instrumentation for separation and/or distortion. The results of this study were statistically significant and indicate that file distortion and/or separation is four times as likely to occur at 333.33 rpm vs. 166.67 rpm. PMID- 10726545 TI - Use of the microscope in endodontics: a report based on a questionnaire. AB - A survey regarding operating microscope (OM) usage and training was mailed to 3356 active members of the American Association of Endodontists (AAE) in the United States. Data from 2061 questionnaires indicate that 52% of the endodontists surveyed have access to and use the OM in their practice. The frequency of use as a function of years since completing endodontic training was as follows: < 5 yr, 71%; 6 to 10 yr, 51%; and > 10 yr, 44%. The OM was used most often for retrieval of separated instruments, root-end preparations, and root-end fillings. Of the respondents who use the OM, 36% report they do not use it as often as anticipated. The most common reasons reported were positional difficulties, inconvenience, and increased treatment time. The information collected from the survey reveals that the more training the operator has on the microscope, the more likely he or she is to use it for all procedures. PMID- 10726546 TI - Effects of simulated clinical conditions on nickel-titanium rotary files. AB - When rotary nickel-titanium files are used in highly curved canals, there is a significant risk of separation. This study exposed files to saline or 5.25% NaOCl, while freely rotating in axial motion with a range of approximately equal to 6 mm at 150 rpm for 30 s in a 1.5 mm inner diameter, thin-walled metal tube of 15 mm length with a 90 degrees curve and a 5 mm radius of curvature. Files of 0.04 taper; 25 mm length; and in ISO sizes 25, 30, and 35 were each used three times. Groups were sterilized in a Chemiclave at 132 degrees C for 30 min after each use or tested without sterilization. Controls were not subjected to simulated use. Torsional moment (N-cm) and angular deflection (degrees) at failure was determined on a Torsiometer/Memocouple. Data (n = 5) were analyzed by analysis of variance. Tukey-Kramer intervals at the 0.05 significance level were determined. Exposure to cyclic fatigue, irrigants, and sterilization did not consistently affect torsional moment or angular deflection, compared with controls. Torsional moment significantly increased with an increase in size, regardless of condition (unsterilized vs. sterilized) or solution (saline vs. NaOCl). PMID- 10726547 TI - Thermafil retreatment using a new "System B" technique or a solvent. AB - The solid plastic carrier in the Thermafil obturation system must be removed to facilitate retreatment. The purpose of this study was to compare the efficacy and time required to retreat canals obturated with Thermafil with plastic carriers using a new technique based on the System B HeatSource or a solvent. Fifty-two extracted human mandibular premolars with single canals were instrumented and then obturated with Thermafil with plastic carriers. After 2 wk storage at 22 degrees C and 100% humidity, they were randomly divided into 2 groups of 26 teeth each. Group 1 teeth were retreated using chloroform and hand files, whereas teeth in group 2 were retreated with a new technique using the System B HeatSource. The end point of retreatment was defined as complete removal of the plastic carrier. The time required for retreatment was recorded. Then, the apical 5 mm segment of each root was sectioned horizontally at 1 mm intervals and each section digitally imaged. The total area of the canal and the area of the canal occupied by gutta percha and sealer were measured using NIH image software. Data were analyzed using an unpaired t test. The mean time for retrieval of the plastic carrier was significantly less for the System B technique (1.8 min) than for the solvent technique (3.6 min) (p < 0.001). The difference between the two groups in the amount of filling material (carrier, gutta-percha, and sealer) removed was not significant (p > 0.05). PMID- 10726548 TI - An in vitro comparison of two modern apex locators. AB - Two apex locators were compared regarding their ability to accurately locate the apical constriction in the presence of various canal fluids at different meter readings. Forty-one root canals were filled with 1% NaOCl, 3% H2O2, and 0.9% NaCl, respectively. Electronic working length (EWL) measurements were recorded with the apex locators Root ZX (meter readings: "Apex", "0.5", and "1") and Apit (meter readings: "Apex" and "3"). The deviation of the EWL from the apical constriction was determined. The proportion of measurements within +/- 0.5 mm of the apical constriction ranged between 0.76 and 0.85 for Root ZX at the meter readings "Apex" and "0.5," regardless of the canal contents. Apit consistently displayed shorter measurement values than Root ZX and reached the highest proportions at the meter reading "Apex": 0.59 (1% NaOCl), 0.61 (3% H2O2), and 0.68 (0.9% NaCl). In the presence of NaOCl, Root ZX provides the more accurate EWL measurements at the meter reading "0.5" and "Apex." PMID- 10726549 TI - A conservative biopsy technique for periapical lesions. AB - This paper presented a conservative technique for biopsy of periapical lesions. The case report demonstrated the insertion of a flexible microsurgical biopsy forceps through a labial sinus tract into a lesion for removal of specimens for histopathological evaluation. This limited tissue removal seemed to induce a change from a cyst to a granuloma, as well as stimulate a degree of repair. PMID- 10726550 TI - Hepatitis C virus: emergence of a silent pathogen. AB - Hepatitis C is a relatively common blood-borne disease with risk considerations for health professionals, parenteral drug users, and other at-risk people. Medical and dental professionals have had to familiarize themselves with the rapidly emerging information about this form of hepatitis in order to treat infected patients and protect themselves, their coworkers, and others from exposure. This article summarizes the current status of scientific and clinical evidence with regard to hepatitis C. PMID- 10726551 TI - Oral medicine: advances in diagnostic procedures. AB - In the latter part of the 20th century, the computer and molecular biology have facilitated great scientific progress in medicine and dentistry. In dentistry, emerging clinical methods based in molecular biology and digital technology have the potential to improve the early diagnosis of dental caries, periodontal disease, and oral cancer. In addition, saliva shows potential as a convenient substitute for blood in diagnostic testing for systemic and oral diseases. DNA chip technology, a new system that combines these two technologies, has potential diagnostic value in dentistry as well as medicine. For each of the three common oral disease processes, emerging diagnostic procedures are discussed, with an emphasis on their potential utility for the practicing dentist of the 21st century. PMID- 10726552 TI - Intraosseous anesthesia: a review. AB - The recent introduction of intraosseous injection devices has renewed interest in the modality of local anesthesia. Three devices currently available are the Stabident System, the Hypo Brand Intraosseous Needle, and the Cyberjet System. The Stabident System is the most popular and the only one for which published research is available. Primary intraosseous anesthesia is 45 percent to 93 percent effective but of short duration. Supplemental intraosseous anesthesia is 80 percent to 90 percent effective and provides profound anesthesia of long duration (60 minutes or longer). It is used when a prior conventional infiltration or nerve block is inadequate. During use of an anesthetic solution with a vasoconstrictor for intraosseous anesthesia, 46 percent to 100 percent of patients reported an increase in heart rate. There was a 2 percent to 27 percent incidence of moderate and sometimes severe pain during the intraosseous procedure. Postoperative complications occurred in 2 percent to 15 percent of patients and lasted one to 14 days. PMID- 10726554 TI - Workforce issues in pediatric dentistry: recommendations of a recent conference. AB - A recent conference of the California Society of Pediatric Dentists discussed issues surrounding the shortage of pediatric dentists in the state. Several contributing factors were identified and recommendations made regarding ways of working with the dental schools to help increase the number of practitioners. This article frames the problem regarding servicing the state's children and discusses some recommended actions. PMID- 10726553 TI - Newer approaches to preventing dental caries in children. AB - Although the incidence of dental caries has shown a general decline during the past few decades, it still remains a significant health problem in children. The role of mutans streptococci in the caries process is discussed, including its transmission from mother to child during a discrete "window of infectivity." Anticipatory guidance--an approach used to better intercept the caries process to prevent it from progressing--is discussed. This program is introduced during infancy and is adapted to the child's particular needs as he or she matures. Anticipatory guidance allows for the implementation of some newer preventive strategies. Following the determination of mutans streptococci levels in at-risk infants and their mothers, a prevention program can be provided to both. Through proper education, various forms of topical fluoride supplementation, and antimicrobial therapy, it is hoped that newer preventive strategies can more effectively reduce the threat of caries at a much younger age than previously possible. PMID- 10726555 TI - Pediatric oral conscious sedation: changes to come. AB - Recent media attention has focused the public's attention on issues surrounding pediatric oral conscious sedation. Under a law passed in 1998 and taking affect on Jan. 1, 2000, California dentists will be subject to certification and procedural provisions designed to ensure the educational qualification of the provider and the standards under which the procedure is performed. This article discusses the history of concern and regulation regarding sedation of children in the dental office. PMID- 10726556 TI - Effects of materials used in pediatric dentistry on the pulp: a review of the literature. AB - This paper reviews a selection of materials used in treating children's teeth. Often, the success of the material is determined by its effect on the underlying pulp tissue, either by virtue of its direct effect or in its ability to prevent ingress of contaminants. The materials reviewed will include some of those used for cavity liners, bases, restorations, pulp capping, and pulpotomies. PMID- 10726557 TI - Reporting child abuse and neglect: responding to a cry for help. AB - Dentists, registered dental hygienists, and registered dental assistants are designated by law as mandated reporters who are required to report suspected cases of abuse and neglect while in their professional capacities. By taking a proactive role in the detection and reporting of child abuse and neglect, dental mandated reporters may save the lives of young victims and assist agencies in helping families in the community. It is vitally important that mandated reporters become aware of their legal obligations regarding the reporting of abuse and neglect. This article is designed to serve as a dental team in-service training program for the detection and reporting of child abuse and neglect. Many of the concepts discussed can be applied to the other forms of family violence. PMID- 10726558 TI - Digital X-rays are here; why aren't you using them? AB - Digital imaging is here. It is cost-effective and helps dentists glean more useful information to make treatment decisions. Many more choices of digital systems are available for dentists to adopt than when the technology was introduced. The hardware is less costly than it was even one year ago, and image storage is now very inexpensive. Technical time is reduced, and no special training is required if the dentist or auxiliary has used a paralleling system. Insurance companies are gearing up to accept image files attached to claims. Why, then, are dentists not buying these systems as fast as manufacturer's can build them? This article explores that question and discusses the false assumptions behind perceived obstacles. PMID- 10726559 TI - Digital radiography--not if, but when. AB - Digital radiography can enhance the dental practice by facilitating diagnosis, enabling orderly filing and archiving, and allowing better communication with patients. Although the initial investment in equipment is substantial, it is quickly repaid and provides both a substantive and fiscal benefit. There are challenges involved in implementation, but they are quickly being overcome. It is only logical for dentistry to move along with the rest of society into the digital age and take advantage of its benefits. PMID- 10726560 TI - Digital radiography in dentistry: what it should do for you. AB - Digital radiology will become an important part of dental practice. Manufacturers should develop more sophisticated tools, including software for digital subtraction; image processing routines for the diagnosis of caries, periodontitis and periapical disease; tools for three-dimensional viewing of the teeth and supporting structures; and analysis of bone trabecular pattern for early detection of systemic disease. Hardware improvements should include increased dynamic range and sensitivity to radiation, and improved resolution. Sensors should be made the size of film, and components should be interchangeable across manufacturers. The true opportunity offered by digital imaging, computer-aided diagnosis, should continue to develop with particular attention to development of tools that add value for solving diagnostic problems and ease of use for the dentist and patient. PMID- 10726561 TI - Dental imaging centers. AB - Dedicated dental imaging centers have been providing valuable imaging services to the dental community for many years. The centers feature specialized and sophisticated imaging equipment and highly trained personnel and provide photographic and radiographic services for the community. This article discusses selected sophisticated imaging equipment found in dental imaging centers and discusses current and anticipated future services provided by the centers. These future services include the construction of patient-specific interactive three dimensional models to be used for diagnosis, treatment planning, treatment simulations, communication, and evaluation of treatment outcomes. PMID- 10726562 TI - Commercial applications of Tuned Aperture Computed Tomography. AB - Tuned Aperture Computed Tomography will allow the creation of three-dimensional images of dental structures from layers of digital information that can be gathered in the dental office. These three-dimensional images will give a fuller view of the structures, thereby providing more information from which to make a better diagnosis. Unlike similar medical tomosynthetic imaging techniques, TACT should be easily accommodated into dental practice needs. PMID- 10726563 TI - Digital radiography and California third parties. AB - Acceptance of digital radiography is increasing but has not yet progressed to the mainstream. A key factor in widespread use of this advancement is acceptance- both theoretical and technological--by dental health plans for claim submissions. This article details a survey of California third parties to examine their practices, concerns, and plans regarding digital dental radiography. PMID- 10726564 TI - Martin Van Butchell (1735-1814): the eccentric, "kook" dentist of old London. AB - This article is a thumbnail sketch of the life and times of Martin Van Butchell (1735-1814), an eccentric, "kook" advertising dentist of Old London. Van Butchell earned these descriptive labels by displaying an unorthodox lifestyle, an outrageous personal appearance and outlandish, extreme and socially unacceptable personal and professional behaviors. While the general populace seemed to be fascinated by his strange ways, dentists and physicians were generally alienated by them. Nevertheless, he was considered a good dentist for his time, and he was extremely popular with his patients. Martin practiced dentistry for 23 years, and he practiced medicine as well, specializing in the treatment of ruptures and anal fistulas. Van Butchell interacted greatly with both John and William Hunter, who became two of the most famous and talented physicians, surgeons, anatomists and biologists of all time. When his first wife, Mary died, Martin arranged for her body to be embalmed and publicly displayed in his dental office for advertising purposes. Her preserved body was shown at the Museum of the Royal College of Surgeons (London), until it was destroyed by a German fire bomb in May, 1941. Mary's remains were on public display for a total of 166 years. PMID- 10726565 TI - The life and work of Dr. George Byron Hayes, pioneer maxillofacial surgeon. AB - When the First World War broke out in April of 1914, an American dentist practicing in Paris offered his services to the French government. He was Dr. George Byron Hayes, a native of a small upstate New York village and a graduate of the dental school of the University of Michigan. The new type of trench warfare led to facial wounds on a scale never before experienced in any conflict. But the military medical services had no experience or skill in dealing with these new and devastating wounds. Dr. Hayes stepped into the breach and performed such exceptional and often miraculous surgery that he was hailed by the entire Western world. The French government made him a Chevalier of the Legion of Honor and the U.S. government awarded him the Distinguished Service Medal, seldom awarded to a civilian. The most prominent surgeon of the day, Dr. Harvey Cushing, brought a team of physicians from Harvard to observe the treatment rendered wounded soldiers in the American Hospital in Neuilly, France, and, in lauding Dr. Hayes' work, stated that his contributions were the most remarkable of all, and that dental surgery should now be recognized as a specialty, the equal of any in medicine. PMID- 10726566 TI - Gleanings about dentistry from the world of literature (nineteenth in a series). PMID- 10726567 TI - Dr. Chamberlen's anodyne teething necklaces. PMID- 10726568 TI - How it evolved--the lancet. PMID- 10726569 TI - The mystery of John Wilkes Booth's dentist. AB - For many years there has been much speculation over the identity of the dentist of President Abraham Lincoln's assassin, John Wilkes Booth. Some have considered Dr. William Merrill (1833-1918), a rather prominent Washington, D.C. dentist, as the man who restored two of Booth's teeth with gold a few days before the assassination. Who was the mysterious Dr. Merrill and what evidence do we have that he ever treated Booth? PMID- 10726570 TI - Nicolas Dubois de Chemant and the use of porcelain in dental art. PMID- 10726571 TI - Edward Hartley Angle--in pursuit of perfection. PMID- 10726572 TI - Perspectives on root-end resection. PMID- 10726573 TI - Cosmetic oral and maxillofacial surgery. AB - The dental specialty of oral and maxillofacial surgery over the last fifteen years has been intimately involved in not only altering the bony facial skeleton, but also the esthetic defects of the facial soft tissue drape. These procedures are currently being taught, in many of our residency programs, along side the art and science of dentoalveolar surgery. When one combines the modification of both the skeletal and soft tissues, the resultant effect can be greater than the sum of its parts. Should one take this a step further and add to the list, proper arch form as prepared by the orthodontist along with cosmetic shape and appearance of the dentition by the restorative dentist using the latest in cosmetic dental techniques, what transforms is a result that has never been seen heretofore in the annals of dentofacial care. To follow are a number of case reports illustrating some salient points. PMID- 10726574 TI - Your fee schedule. AB - For doctors to be able to establish and protect their fees, it is imperative that they understand exactly what a fee is, as well as what it is not. This article will define the myriad of terms associated with fees and will illustrate the methods used by third-party payers to determine benefit amounts payable. This should help doctors better understand and protect the inherent value of annually updated fees. PMID- 10726575 TI - A comparison of occlusal amalgam margin angle and clinical appearance of amalgam restorations. PMID- 10726576 TI - Oral health status of children in Tennessee: a survey of caries prevalence and oral health needs. PMID- 10726578 TI - A clinical approach to dental nutrition among the elderly: a description and discussion of geriatric dental nutrition. AB - This paper describes the role of nutrition in dentistry, especially as it relates to elderly patients. The nutritional aspects are well understood, but seldom considered in treatment plan formulation and execution, even in an academic health setting. When dental school programs are shown to have clinical relevance to a target population and are faculty supported, they are most successful. This is the case at the University of Tennessee College of Dentistry in Memphis. The role of the Nutritionist focuses on the periodontal and dental aspects of geriatric patient exposure to disease causing agents in the diet. Nutritionist, dentist, student and patient all participate in a team effort to lower patient exposures and the development of a healthful diet. In this clinical approach to geriatric patient dental nutrition, all parties contribute to the success of the whole. PMID- 10726577 TI - The effect of finishing method on the microleakage of Class V microfilled composite resin restorations. PMID- 10726579 TI - Resin composites for foundation restorations. AB - This study focused on the compressive properties of two core buildup materials, Ti-Core, (EDS, Inc.) and FluoroCore, (L.D. Caulk). Specimens were 0.6 x 1.2 cm cylinders, Prior to testing, the specimens were aged in a moist environment for one, seven, or 28 day(s). The properties of interest were compressive strength, modulus of elasticity and toughness. The data set for each of the three compressive properties was subjected to a separate two-way analysis of variance. Scheffe F-tests were used to compare means. Whereas the effect of material on each of the three properties was significant (p < 0.002), the effects of specimen age (p > 0.400) and material x specimen age (p > 0.350) were not significant. FluoroCore was stronger and tougher than Ti-Core. On the other hand, Ti-Core displayed the highest stiffness, but ruptured brittley. The moderately low strength of Ti-Core, and the insufficient elastic modulus of FluoroCore, avert favorable recommendations pertaining to the construction of foundation restorations. PMID- 10726580 TI - A review of the current status of vital bleaching. PMID- 10726581 TI - Trigeminal neuralgia--a dental diagnosis challenge. PMID- 10726582 TI - Goals and strategies for improving dental care in the nursing home: Part Two. PMID- 10726583 TI - Troubleshooting vinyl polysiloxane impressions. AB - For consistent results in impressions and restorations, the following four rules make a handy checklist: 1. Exclude as many variables as possible from your technique. 2. Review materials and techniques on a routine basis. 3. Discuss with laboratory technicians what they observe as they work with your impressions. 4. Pursue the satisfaction of keeping your skills at their highest level. PMID- 10726585 TI - A survey of luting cements preferred by dental practitioners in the Commonwealth of Virginia. PMID- 10726584 TI - Dental erosion: a review and new concerns for dental practitioners in Virginia. PMID- 10726586 TI - The use of automatic defibrillators in the dental office. PMID- 10726587 TI - Replacement of missing single molar with single wide-diameter implant or double standard endosseous implants. PMID- 10726588 TI - Childhood chronic inflammatory demyelinating polyneuropathy. AB - Chronic inflammatory demyelinating polyneuropathy (CIDP) is a chronic disorder of the peripheral nervous system with sensory and motor involvement, and insidious onset over a period of months. In children and adults, both proximal and distal muscles are affected. Muscle stretch reflexes are absent or depressed. Laboratory findings include elevated cerebrospinal fluid protein with no increase of mononuclear cells. Electrophysiological and pathological studies show evidence of demyelination. No control studies of the efficacy of immunomodulating therapy in childhood CIDP are available. However, several studies have indicated clinical improvement after treatment with prednisolone, plasmapheresis and intravenous immunoglobulin, but disappointing results with other immunosuppressive agents. While some children have a monophasic course, with complete recovery, others have a protracted course, with either a slowly progressive or a relapsing-remitting course, resulting in prolonged morbidity and disability. PMID- 10726589 TI - Closed spinal dysraphism: a review on diagnosis and treatment in infancy. AB - This article reviews the clinical presentation, pathophysiology, diagnostic strategies, and therapeutic management of closed spinal dysraphism in infancy. Four groups of symptoms are distinguished: (1) cutaneous abnormalities, (2) lower motor neuron dysfunction due to congenital spinal and nerve root abnormalities, (3) upper motor neuron dysfunction due to tethering of the spinal cord, and (4) symptoms caused by associated malformations. Guidelines are proposed concerning timing and type of diagnostic investigations in infancy. This essentially encompasses a neurological assessment, including spinal ultrasonography and nuclear magnetic resonance imaging of the spine and the brain, and a urological assessment, including ultrasonography of kidneys and bladder, cystourethrography and a urodynamic study. As to the tethered cord syndrome it is concluded that first, already in infancy, a progressive neurological dysfunction can be detected; second, surgical untethering should be performed only upon appearance of upper motor neuron signs or upon progression of lower motor neuron signs. Despite its frequently asymptomatic course, the diagnosis of a congenital lumbosacral lipoma, and in the more general sense, of a closed spinal dysraphism, implies a periodic, multidisciplinary follow-up for life. PMID- 10726590 TI - Apnoeic attacks as an isolated manifestation of epileptic seizures in infants. AB - Apnoea as an isolated manifestation of seizures is well described in neonates but is only occasionally observed in infants. We present data from four infants, with apnoea as the sole manifestation of seizures, documented by polygraphic ictal electroencephalogram (EEGC) and video recording. The four infants, after normal pregnancy and delivery at term, showed the first apnoea at the age of 2-11 months. The interictal EEG was normal. The ictal EEG and video recording showed in all infants a focal rhythmic alpha or theta activity with or without generalization, which lasted 40-120 seconds. The apnoea appeared a few seconds after the beginning of rhythmic activity and the heart rate remained unchanged during the apnoea. At 2 years' follow-up, three children are seizure-free under anti-epileptic therapy with normal psychomotor development in two, and a slight delay in the third infant. The fourth child has partial seizures and is severely retarded. PMID- 10726591 TI - Serum neuron specific enolase: a marker for neuronal dysfunction in children with continuous EEG epileptiform activity. AB - Non-convulsive status epilepticus (NCSE) is a common complication of the childhood epileptic encephalopathies. An essential feature for the diagnosis of non-convulsive status epilepticus is a continuous epileptiform activity on the electroencephalogram (EEG). Dementia is thought to be a possible long-term sequel of non-convulsive status epilepticus, the mechanism of which has remained elusive. Neuron specific enolase is a marker of neuronal damage. The serum concentration of neuron specific enolase (sNSE) has been measured in 17 children with continuous epileptiform activity on the EEG and in 16 children with epilepsy but without a continuous dysrhythmia. There was a significant difference in the concentration of sNSE between the two groups. PMID- 10726592 TI - Vitamin K deficiency--late onset intracranial haemorrhage. AB - A retrospective study is presented of the clinical features and outcome of late onset haemorrhagic disease due to vitamin K deficiency in 11 babies who were admitted to the emergency or child neurology unit during a 4-year period (January 1994-December 1997). The disease occurred in infants between 30 and 119 days of age (mean: 56+/-24 days). None of them received vitamin K after birth and all were breastfed. The presenting complaints were seizures (91%), drowsiness (82%), poor sucking (64%), vomiting (46%), fever (46%), pallor (46%), acute diarrhoea (27%), irritability and high-pitched cry (18%). On examination, tense or bulging fontanelle (73%), anisocoria (36%), weak neonatal reflexes (18%), cyanoses (18%) were the most frequent findings. The localizations of the intracranial haemorrhage were as follows: intracerebral (91%), subarachnoid (46%), subdural (27%), and intraventricular (27%). No fatality was observed. However, after a follow-up period ranging from 6 to 48 months (mean: 21+/-13 months), only three (27%) infants remained neurologically normal. Seizure disorders (73%), severe psychomotor retardation (46%), cerebral palsy (46%) and microcephaly (46%) were observed in the remainder. Hydrocephalus developed in three (27%) babies but none of them required shunt replacement. The value is emphasized of vitamin K prophylaxis in the newborn to reduce the incidence of late onset intracranial haemorrhage and handicap in children. PMID- 10726593 TI - Childhood dermatomyositis: clinical course of 36 patients treated with low doses of corticosteroids. AB - Thirty-six patients with juvenile dermatomyositis, seen consecutively between 1983 and 1996 and treated initially with low doses of corticosteroids (prednisolone 1 mg/kg/day), were studied retrospectively to evaluate their long term evolution and to identify factors predictive of the functional outcome. After a mean follow-up of 4.9 years, 28 (78%) of the patients were well without functional impairment; five patients had inactive disease but with persisting disabilities; and three patients had active disease despite several years of treatment. Fifteen children (42%) developed dystrophic calcifications which, in five of these patients, interfered with functions. These patients treated for juvenile dermatomyositis with a low dose corticosteroid regimen had an evolution identical to that of the published series of patients treated with higher doses and probably had a better quality of life. The best predictors of good functional recovery and minimal calcinosis were early treatment after the onset of symptoms and low creatine kinase serum level at the time of diagnosis. PMID- 10726594 TI - Reversible cortical atrophy and cognitive decline induced by valproic acid. AB - An 18-year-old male suffered from familial progressive myoclonic epilepsy from the age of 7 years. In addition to seizures, there was a marked decline in school performance. At the age of 14 years, sodium valproate was started as add-on therapy; 2 weeks later he was hospitalized in a stuporous state. The serum level of valproate was within the therapeutic range. Cognitive evaluation disclosed moderate mental retardation. No metabolic abnormalities were detected. Valproate was discontinued and during the 4 following months, a slow but significant improvement was documented in cognitive functions. Repeated assessment was within the range of mild mental retardation. Initially, magnetic resonance imaging (MRI) showed mild cortical atrophy. A subsequent MRI study performed 2 years later was normal. PMID- 10726595 TI - Differential diagnosis of disorders of biogenic amines metabolism. PMID- 10726596 TI - Neuropsychological functioning in depressed versus nondepressed participants with Alzheimer's disease. AB - Differences in cognitive functioning between participants with Alzheimer's Disease (AD) reporting depressive symptomatology (AD-Dep; n = 37) and a control group of nondepressed participants with AD (AD-Con; n = 98) were investigated based on hypothesized impairments of attention/concentration, psychomotor speed, and simple motor speed. Groups did not differ in age, education, overall severity of dementia, auditory comprehension, or use of psychotropic medications. AD-Dep participants performed significantly more poorly than AD-Con participants on 3 of the 13 measures on which they were hypothesized to exhibit greater impairment (WAIS-R Block Design, WAIS-R Digit Symbol, and speeded motor programming); and there were trends toward poorer performance on four additional measures (WAIS-R Object Assembly, WAIS-R Picture Arrangement, WAIS-R Digit Span-backward, and letter fluency). There was only one significant effect for the 13 measures on which no group differences were hypothesized; the AD-Dep participants unexpectedly obtained better WMS-R Logical Memory delayed recall scores than the AD-Con participants. Finally, AD-Dep participants exhibited an unexpected pattern of greater right hand advantage on the Finger Tapping Test. PMID- 10726597 TI - The role of cognition, depression, and awareness of deficit in predicting geriatric rehabilitation patients' IADL performance. AB - Fifty consecutive patients aged 60 years or older admitted to an inpatient rehabilitation program were studied to determine whether demographic, cognitive, affective, and awareness of deficit data had predictive value in determining performance-based Instrumental Activity of Daily Living (IADL) capacity. Correlational and hierarchical regression analysis found that cognition, awareness of deficit, and years of education accounted for 70% of IADL variance. Of particular note, cognition contributed unique variance above and beyond that accounted for by demographic variables. Additionally, the Awareness Interview (AI) contributed unique variance above and beyond that accounted for by demographic and cognitive variables. This study provides support for the assessment of cognitive functioning and awareness of deficit in rehabilitation settings to assist in outcome prediction. PMID- 10726598 TI - Normative data on computerized paced auditory serial addition task performance. AB - Computerized testing has increased in popularity over the past two decades. The present study examined properties of the computerized version of the Paced Auditory Serial Addition Task (PASAT). The PASAT was administered to 168 college students ages 17 to 48 years with no history of neurological or emotional problems. Analysis of variance showed no significant effects of gender or age. With increasing rate of presentation, participants gave fewer correct responses. The number of errors decreased rather than increased across PASAT trials, reflecting participants' tendency to "skip" items. Additional normative data are presented. Performance on the computerized PASAT appears similar to published norms on the PASAT version presented via audiocassette. It is suggested that the computerized and audiocassette versions of the PASAT may be used as alternate forms in well-educated adults. PMID- 10726599 TI - Normative data for 4-year-old children on the California Verbal Learning Test Children's Version. AB - This study presents normative data for 4-year-old children on the California Verbal Learning Test-Children's Version (CVLT-C), a measure of verbal learning and memory. Norms are currently available for children 5 years and older; however, normative data are unavailable for this younger population. Forty males and 40 females comprise this normative sample of 4-year-old children. The mean number of words recalled increased from the first to the fifth learning trial, and a consistent level of recall was maintained across delay recall trials. Extra list intrusion responses were common and these responses were more frequent than correct responses on cued but not free recall conditions. Finally, yes/no recognition testing resulted in the greatest mean number of words remembered compared to the other trials. Overall, the pattern of performance across the learning and memory variables in this younger population was similar to that of older children, but at a lower level. These data suggest that 4-year-old children are able to perform this task, making possible the use of the CVLT-C in normal and clinical populations in this age group. PMID- 10726600 TI - Practice effects on commonly used measures of executive function across twelve months. AB - Fifty men (age M = 32.50; education M = 14.98 years) were administered the Wisconsin Card Sorting Test (WCST), Ruff Figural Fluency Test (FFT), Verbal Concept Attainment Test (VCAT) Trail Making Test, Parts A and B (TMT), and F-A-S Verbal Fluency at baseline and 12 months later. WCST, FFT, and VCAT scores improved significantly over a 12-month interval. In contrast, TMT and F-A-S scores did not change. Level of intellectual ability failed to moderate the effect of previous testing upon performance. Suggestions concerning the use of these measures in longitudinal research designs and clinical follow-up examinations are offered, and reliable change indices concerning these measures are included. PMID- 10726602 TI - A comparison of computerized and standard versions of the Wisconsin Card Sorting Test. AB - The manual version of the Wisconsin Card Sorting Test (WCST) is the most frequently used neuropsychological instrument for differentiating normal from populations with frontal-lobe impairments. A number of computerized versions have become available, and their ability to accurately detect frontal-lobe dysfunction is based on the assumption of equivalence with the standard manual version. Accordingly, comparisons of the distribution properties of central tendency, variability, and shape between the manual version with four computerized versions of the WCST were performed (n = 22 per condition). None of the computerized versions were found to be equivalent to the manual version on all assessment measures. Given the discrepancies between the manual and computerized versions, it is concluded that the norms provided for the standard manual presentation method should not be used for the computerized versions, and for continued use of the computerized versions, new norms for computer versions need to be established. Thus, clinicians and experimenters must use caution when basing conclusions on scores from computer versions of the WCST. PMID- 10726601 TI - Effects of education in dementia assessment: evidence from standardizing the Korean-Dementia Rating Scale. AB - This study established norms and examined validity and reliability of the recently developed Korean version of the Dementia Rating Scale (K-DRS; Chey, 1998) for the elderly Korean population. The K-DRS was administered to 148 normal elderly Korean people and 20 patients with dementia of the Alzheimer's type (DAT). Norms were developed a priori for two age groups (55-64 and 65-84 years). Education (p < .0001) and age (p < .05) proved significant factors in the performance of the K-DRS, whereas gender did not. Accordingly, norms were further specified into two educational levels (6 years or fewer, and more than 6 years). Education was recognized as an important factor in evaluating dementia in a population with limited education. These implications are significant for dementia studies done in developing countries where educational opportunities have been limited for elderly people. Evidence supporting the validity and reliability of the scale was also found. K-DRS total scores correlated well with diagnostic status (r(pb) = .63) and mental status exam scores (r = .80). Test retest reliability was .96, and interrater reliability was .99. PMID- 10726603 TI - Trail making test in chronic toxic encephalopathy: performance and discriminative potential. AB - The objective of this study was to examine the influence of chronic toxic encephalopathy (CTE) on Trail Making Test (TMT) performance, with special focus on the discriminative potential of this test. We assessed TMT performance in patients diagnosed with CTE, patients with similar symptoms but no diagnosis, and healthy participants. Inferior performance was seen in CTE, and increasing age had a negative effect on TMT performance only for the CTE group. This effect was most pronounced in TMT-B. However, the ability of the TMT to identify CTE was low, whereas all healthy participants were identified as healthy. Thus, the sensitivity of TMT alone was low, but it succeeded in correctly classifying normal subjects. The pattern of results indicates that normal TMT performance may be seen in individuals with mild to moderate brain syndromes, such as CTE, whereas poor performance should not be expected in healthy individuals. PMID- 10726604 TI - A normative study of Nelson's (1976) modified version of the Wisconsin Card Sorting Test in healthy older adults. AB - A modified version of the Wisconsin Card Sorting Test (mWCST) proposed by Nelson (1976) was administered to 229 healthy community-dwelling older adults, composed of 97 men and 132 women, ages 45 to 91. Investigating the impact of demographic factors such as age, education, and gender on mWCST performance, results indicated that mWCST performance was significantly affected by both age and education. Unlike the standard WCST, however, gender did not significantly influence mWCST performance. Because demographic factors appear to exert a significant influence on mWCST performance of normal older adults, demographically-corrected norms were calculated according to the procedure described by Heaton, Grant, and Matthews (1991). In addition, longitudinal analysis of mWCST performance revealed that significantly fewer nonperseverative errors were committed at retest approximately one year later. Number of categories completed and perseverative errors did not appear to demonstrate significant practice effects in this sample. Accounting for demographic influences and the inspection of practice effects on serial administration of the mWCST may improve upon its sensitivity and specificity for use in the clinical assessment of executive function in older adults. PMID- 10726605 TI - Construct and concurrent validity of the Hopkins Verbal Learning Test-revised. AB - The present investigation examined the validity of the revised Hopkins Verbal Learning Test (HVLT-R). In a principal components analysis with varimax rotation, measures of new learning and delayed recall loaded on a single factor distinguishable from measures related to general cognitive function and visual memory. The HVLT-R also correlated most strongly with other tests of verbal memory and relatively weakly with a test of general intelligence. Group comparisons showed that normal controls performed better than age- and education matched patients with probable Alzheimer's disease (AD) or vascular dementia (VaD). Discriminant function analyses and Bayesian statistics revealed high classification accuracies for dementia patients versus controls. When scores on the HVLT-R and other neuropsychological tests were subjected to discriminant function analyses, performance on the HVLT-R delayed recognition task was found to be the most useful in discriminating patients with AD from those with VaD. We conclude that the HVLT-R is a valid test of verbal learning and memory that is best suited for use with elderly patients suspected of dementia. PMID- 10726606 TI - Odd/Even short forms of the Boston Naming Test: preliminary geriatric norms. AB - It has been suggested that the 30-item version of the Boston Naming Test (BNT), in which either the odd or even items from the standard 60-item test are given, is the most psychometrically sound short form. However, no normative data are available for this version. We administered the Odd/Even BNT to 30 community dwelling elderly individuals (age M = 72.93, range 61-84; education M = 13.73) in order to collect normative data. Odd and even forms were equivalent. The combined mean total correct score was 27.13 (SD = 2.06), a score consistent with that derived by retrospective extraction in the original odd/even test construction study. Each form discriminated normals from age- and education-matched patients with probable Alzheimer's disease, suggesting criterion-related validity. PMID- 10726608 TI - The behavioural assessment of the dysexecutive syndrome as a tool to assess executive functions in schizophrenia. AB - Recent research into the cognitive dysfunctions in schizophrenia has focused on executive deficits. This study investigates performance of patients with schizophrenia on the recently developed Behavourial Assessment of the Dysexecutive Syndrome (BADS). Matched groups of 24 patients with schizophrenia and 17 healthy volunteers were administered the BADS, the Modified Card Sorting Test (MCST), the Tower of London (TOL), a test of general intelligence, and measures of daily functioning. Performance of the schizophrenic group was significantly below that of the control group on the BADS and the MCST, but not on the TOL. The BADS correlated weakly with the MCST. Both tests showed a modest correlation with daily functioning. The BADS appears to offer a useful contribution to the assessment of executive deficits in schizophrenia. PMID- 10726607 TI - Normative study of the Korean-California Verbal Learning Test (K-CVLT). AB - As a normative study of the Korean version of the California Verbal Learning Test (K-CVLT), the present study examined the K-CVLT performances of 357 neurologically intact Koreans (181 males and 176 females) who were selected by stratified sampling reflecting the recent Korean census data. The factor analysis of the K-CVLT showed that the K-CVLT was a valid and useful tool for qualitatively assessing a number of theoretically meaningful processes and strategies underlying verbal memory. Norms were developed on the 22 memory indices separately for gender and age groups. Implications for the K-CVLT's factor analytic results are discussed, and the K-CVLT's normative data is compared with that of the CVLT. PMID- 10726609 TI - Variables moderating cultural and ethnic differences in neuropsychological assessment: the case of Hispanic Americans. AB - Problems associated with the use of culture and ethnicity as independent variables in neuropsychological assessment research are reviewed. Culture and ethnicity are complex multidimensional constructs that have defied operational definition. There are no clear criteria for separating cultural and ethnic groups. Cognitive differences between culture and ethnic groups encourage speculations of cultural or ethnic superiority and race-norming. An alternative approach is to focus upon measurable psychological variables that differ between cultural and ethnic groups and potentially impact neuropsychological test scores. To illustrate, research with Hispanic Americans is reviewed to show that English language fluency, length of residence within the United States, years of education, and persistence of poverty all impact test performance. PMID- 10726610 TI - Radiation related prognostic factors in radiation oncology. AB - 1. The outcome of a course of radiotherapy is very dependent on the dose per fraction. The smaller the dose per fraction, as a general rule, the better the sparing of the late reacting normal tissues. 2. Overall treatment time is important, especially for tumours with a rapid doubling time. In such a case, the ideal of small doses per fraction (to save late reacting tissues) as well as a short overall treatment time (to offset the effect of repopulating) can be achieved by small doses per fraction applied two to three times per day, including Saturdays or weekends. 3. The BED (biologically effective dose) is a simple to use formula indicating the effects of fractionation. The most important term in the formula is the alpha/beta ratio which is available from experimental work for many tumours and tissues and can be looked up. As a guide, an alpha/beta ratio of 10 for early (acute) reaction and for tumour effects, and an alpha/beta ratio of 2 for late effects plus normal tissue complications can be used. 4. The application of the BED demonstrates that for HDR intracavitary therapy for cervical carcinoma, the biologically relevant dose lateral to point M(A) falls very much more rapidly than the nominal dose. Line sources are shown by comparison with other published reports, not to be intrinsically inferior to tandem ring/tandem ovoid systems and may have advantages the more cumbersome systems do not have, and may have the large advantage of allowing multiple small fractions without anaesthesia. For the particular line source system under discussion, water in a 40 cm3 Foleys bulb is used as the protecting medium for the posterior bladder wall and the anterior rectum. This particular system allows fraction sizes far smaller than 9.1 Gy at point (M)A, e.g. 3 Gy, which bestows an even greater benefit in terms of the therapeutic ratio according to BED10 and BED2 calculations. PMID- 10726611 TI - Saving the lymph nodes. AB - We need improved imaging and staging techniques to identify metastatic disease in the lymph nodes. Once we are able to do this accurately, then we can save the lymph nodes. Until that time, however, gynecologic oncologists should give careful thought as to which nodes they choose to remove and how lymph node removal may affect short-term and long-term morbidity. PMID- 10726612 TI - Development, characterization and distribution of adoptively transferred peripheral blood lymphocytes primed by human papillomavirus 18 E7--pulsed autologous dendritic cells in a patient with metastatic adenocarcinoma of the uterine cervix. AB - We describe a 27-year-old woman with systemic chemoresistant and radioresistant metastatic disease secondary to a recurrence of human papillomavirus (HPV) 18 infected cervical adenocarcinoma of the uterine cervix who received adoptive transfer of peripheral blood T cells stimulated with HPV 18 E7-pulsed autologous dendritic cells (DC). Extensive in vitro characterization of the DC-activated T cells derived from peripheral blood mononuclear cells (PBMC) included phenotypic analysis, cytotoxicity and intracellular cytokine production. High cytotoxicity activity was observed by CD8+T cells against autologous tumor cells, but not against NK-sensitive K562 cells, autologous Con-A lymphoblasts, or autologous Epstein-Barr virus-transformed lymphoblastoid cells. Blocking studies demonstrated that lytic activity was significantly inhibited by pretreatment of tumor targets with MAb specific for HLA class I as well as that of effector cells with anti-CD8, anti-LFA-1, but not anti CD3 MAb. Two-color flow cytometric analysis of the cytotoxic T cells revealed that a significant proportion of CD8+ cells was also CD56+. These double positive CTLs were thymically derived, as shown by expression of heterodimeric CD8 molecules (alpha/beta CD8) and were endowed with high cytotoxic activity against tumor cells. Analysis of intracellular cytokine expression showed that the striking majority of E7-pulsed DC activated CD8+ T cells strongly expressed IFN-gamma, TNF-alpha and IL-2 but not IL-4. The patient received two infusions of cytotoxic tumor-specific T cells at 2 week intervals, and in vivo distribution of the T cells was followed by 111 oxine labeling and serial gamma camera imaging. Persistent accumulation of radioactivity in the lungs, which harbored extensive metastatic disease, was detected up to 120 hrs after the infusion. Taken together, these results illustrate the potential of E7-specific and tumor-specific CTL-based immunotherapy for the treatment of patients with invasive cervical cancer. PMID- 10726614 TI - Mitotically active haemorrhagic cellular (apoplectic) leiomyoma. AB - Apoplectic leiomyoma is a distinctive smooth muscle tumour usually occurring in women either taking oral contraceptives or who are pregnant or recently postpartum. Most of these tumours show 0-2 mitoses per 10 high power fields, but a mitotic index of up to 8 per 10 high power fields is allowed in such tumours. We describe an apoplectic leiomyoma with a number of atypical features including a high mitotic index (up to 20 per 10 high power fields) in a 47-year-old woman. Follow-up clinically and by computerised tomography (CT) for 3 years demonstrates no recurrence. PMID- 10726613 TI - Use of the ultrasound surgical aspirator in ovarian cancer: a case-control study. AB - BACKGROUND: This study was undertaken to evaluate the use of the ultrasound surgical aspirator in ovarian cancer and to determine if cytoreduction was improved with its use. METHODS: The study is a retrospective case control trial. Twenty-six consecutive ovarian cancer patients who had the ultrasound surgical aspirator used during their surgery were retrospectively compared to 25 consecutive ovarian cancer patients who did not have the ultrasound surgical aspirator used during their surgeries. The latter group had their surgeries immediately before the ultrasound surgical aspirator was introduced into the hospital. Both groups were similar in age, stage, histology type, grade, and median number of chemotherapy cycles. RESULTS: Patients that had the ultrasound surgical aspirator used had a 69% optimal cytoreduction rate compared to 16% in the control group (p = .001). This was statistically significant (p = 0.001). Survival time was equal in both groups. CONCLUSIONS: Results of the study showed that use of the ultrasound surgical aspirator may permit more patients to be optimally cytoreduced. PMID- 10726616 TI - Papnet-assisted, primary screening of cervico-vaginal smears. AB - PURPOSE: The Papnet system was initially designed for rescreening negative Pap tests but may also be an effective primary screener. METHODS: A set of 2,200 archival slides diagnosed by conventional, manual screening as 2,000 (90%) WNL, 47 (2.1%) carcinomas, 50 (2.3%), HSIL, 50 (2.3%) LSIL, and 53 (2.4%) ASCUS/AGUS were compared to the results of Papnet-assisted, primary screening. Following Papnet scanning, the digitized images were triaged and classified as abnormal or negative. All abnormals had a full manual screening, whereas negatives had a limited screening. Results by each screening method were compared and discordant cases were peer reviewed for a consensus result. Screening efficacy by each method was measured against a standard result composed of the concordant and consensus results. RESULTS: There were 101 concordant and 181 discordant abnormal results. The standard result for the slide set was 1,953 (88.9%) WNL, 87 (3.9%) ASCUS/AGUS, 52 (2.4%) LSIL, 62 (2.8%) HSIL, 39 (1.8%) carcinomas, and 5 (0.2%) unsatisfactory. Papnet versus manual sensitivity rates were 87.6% vs 72.3% at the ASCUS/AGUS threshold, 85.6% vs 82.4% at the LSIL threshold, and 89.1% vs 90.1% at the HSIL threshold. CONCLUSIONS: Papnet-assisted, primary screening equals conventional, manual screening in the detection of a wide range of cell abnormalities and is more effective in the detection of abnormalities at the lower end of the abnormal spectrum. PMID- 10726617 TI - Benign glandular and squamous metaplastic-like cells seen in vaginal Pap smears of post hysterectomy patients: incidence and patient profile. AB - We have observed benign glandular cells and squamous metaplastic-like cells in vaginal Pap smears of post hysterectomy patients (PHP). Vaginal Pap smears from 1,547 PHP were retrieved. In 2% of these smears (Group A) glandular cells were observed, with the majority of the smears revealing squamous metaplastic-like cells (47%). Mucinous endocervical columnar-like cells were seen in 9% of the cases, glandular cells not resembling endocervical cells in 13%, and a combination of the former two categories in 31%. Group A patients were compared with other PHP without these cells in their vaginal smears (Group B). Several clinical and surgical parameters were evaluated. A distinctive clinical profile was not identified for either group of patients (A or B). Of patients in group A 49.8% had a history of a previous gynecologic malignancy (Group B: 19%). Based on our study, we postulate that in the absence of a clinically identifiable source of these cells, the most likely source of origin is probably vaginal adenosis not associated with DES exposure in utero or a metaplastic phenomenon perhaps related to therapy. These cells do not seem to be related to imminent neoplasia or dysplasia. PMID- 10726615 TI - The presence of HPV 16, 18 and p53 immunohistochemical staining in tumor tissue of Israeli Jewish women with cervical and vulvar neoplasia. AB - The incidence of cervical neoplasia in Israeli Jewish women is persistently lower, while that of vulvar carcinoma is comparable to that in other populations. The aim of the present investigation was to assess the prevalence of HPV and of immunohistochemically detected mutant p53 in Israeli Jewish women with cervical and vulvar neoplasia compared with other populations. Tissue sections from formalin-fixed paraffin-embedded blocks of ten patients with CIN III, 29 with invasive squamous cell carcinoma, three with adenocarcinoma and 14 with invasive vulvar carcinoma, were examined for the presence of HPV 16 and HPV 18 DNA by PCR amplification, and for mutant p53 protein by immunohistochemical staining. HPV negative cases were re-examined with a sensitive primer. HPV DNA was detected in eight patients with CIN III and in 23 patients with invasive squamous carcinoma. In the remaining cervical squamous neoplasia tissue analysis with the sensitive primer could not be done. HPV DNA was also detected in two patients with adenocarcinoma and in nine (64.2%) patients with vulvar carcinoma. Positive p53 immunohistochemical staining was found only in one CIN III patient, in six (20.7%) squamous carcinoma and in 11 (78.6%) vulvar carcinoma patients. Of the p53 immunohistochemical staining positive tissues, two with cervical carcinoma and six with vulvar carcinoma were also HPV-positive. The prevalence of HPV and of positive p53 immunohistochemical staining in our series of Israeli Jewish women with cervical and vulvar neoplasia is similar to that in other populations, suggesting that the etiological factors are probably also alike. PMID- 10726618 TI - Glandular abnormalities on cervical smear: a study to compare the accuracy of cytological diagnosis with underlying pathology. AB - The interpretation of glandular abnormalities detected by cervical smear provides a diagnostic dilemma. This study aims to compare the accuracy of cytological diagnosis with underlying pathology so that guidelines for the investigation and management of abnormal glandular smears may be formulated. A retrospective review of 150 women with glandular abnormalities reported on cervical smear collected over 12 months from 1996 in a University hospital was performed. Smears were graded by the initial report into 3 groups, dependent on the severity of abnormality. Investigation, treatment and subsequent 3-year follow-up were recorded. The accuracy of prediction for a significant neoplastic or preneoplastic glandular pathology only was 0% with mild, 9% (3/35) with moderate, and 24% (9/38) with severe abnormalities. When squamous lesions were included, the chance of finding any dysplastic squamous or glandular abnormality was 16% (12/77), 51% (18/35) and 82% (31/38), respectively, following a smear showing a suspected glandular abnormality only. Our results highlight the poor specificity of predicting glandular neoplasia or preneoplasia from cervical smears, with a final diagnosis of high grade CIN in 35% (17/49) of patients with dyskaryotic glandular cytological changes only and 83% (20/24) where concomitant squamous dyskaryosis was reported. The reporting of reactive or minor changes in endocervical cells was of no diagnostic value. Management protocols for moderate and severe glandular abnormalities should include visualisation and biopsy of the uterine cavity to exclude endometrial neoplasia. PMID- 10726620 TI - Bilateral Krukenberg tumor of the ovary during pregnancy. AB - This case report concerns a 35-year-old woman suffering from gravidic cholestasis, thrombocytosis and iterative vomiting episodes who underwent an elective cesarean section at week 35 because of recent herpetic vulvitis. Large bilateral ovarian tumors were observed which were interpreted as pregnancy luteomas. Nevertheless a biopsy of the right ovary was performed. Histologic examination revealed massive luteinization of the ovarian stroma. In addition, large tumor cells were found dispersed throughout the ovary as well as in vascular spaces as either isolated or clustered signet-ring cells. In search of the primary tumor, gastroscopy revealed a gastric ulcer in the antrum. The biopsies of the ulcer margins as well as those taken at distance demonstrated signet-ring cells in the lamina propria. Bilateral salpingo-oophorectomy and total gastrectomy were performed. In spite of postoperative chemotherapy, the patient died of disease 5 months after diagnosis. PMID- 10726619 TI - Apoptosis and apoptosis-related proteins (Fas, Fas ligand, Blc-2, p53) in lymphoid elements of human ovarian tumors. AB - Different types of lymphocytes have different roles in tumor suppression. Thus, their expression of apoptosis-related proteins (ARP - Fas and Fas ligand, bcl-2, p53) in lymphocytes and their apoptosis were analyzed immunohistochemically in ovarian tumors of different grades. Ovaries without oncologic disorders had few lymphocytes, mainly T cells, and no ARP. Benign cysts presented features of weak immune reaction: small lymphoid infiltration and few lymphocytes. The ARP were present in 13.7% to 23.5% of the lymphocytes, and apoptosis was rare. In borderline tumors, expansion of lymphoid infiltrates and increased density of lymphocytes resulted in a tenfold rise in total lymphocytes, reflecting intensification of the immune response. Most lymphocytes were T cells (92%) predominated by CD8+ cells that were in direct contact with tumor epithelial cells. ARP species were found in 47% to 65% of the lymphocytes, and apoptosis in 2.2%. In carcinomas with ligh lymphoid infiltration, lymphocytes were 2.5 times more abundant, and the apoptotic index as well as the number of CD20+ and CD25+ lymphocytes rose sharply, whereas bcl-2 positive lymphocytes decreased to 8% of their number in borderline tumors. In carcinomas with low lymphoid infiltration, the total lymphocyte count decreased eightfold compared to carcinomas with high lymphoid infiltration, reflecting the deep subcompensation of the lymphoid system. Few p53-positive lymphocytes were found in the carcinomas. In conclusion, we found a positive correlation between apoptosis and the numbers of CD4+ or CD8+ lymphocytes in epithelial ovarian tumors. This correlation could reflect the antitumor activity of T cells. However, the high expression of ARP studied by immune cells at the vicinity of the tumor ARP reveals the lymphoid vulnerability to apoptosis, resulting in devastation of the lymphoid tissue, and consequently in tumor progression. PMID- 10726621 TI - Leiomyosarcoma of the vulva: a brief communication. PMID- 10726622 TI - Recurrence of Sertoli-Leydig cell tumour in contralateral ovary. Case report and review of literature. PMID- 10726623 TI - Serum markers as prognostic factors in epithelial ovarian cancer: an overview. AB - A comprehensive review of the literature and the authors' personal experience on serum determination of tumour markers in epithelial ovarian cancer can be summarised as follows: CA 125 is the most reliable marker for monitoring the course of epithelial ovarian cancer; CA 125 assay is not an adequate screening test for this malignancy but it can represent an useful adjunct to clinical examination and ultrasound in the differential diagnosis of ovarian masses in postmenopausal women; Serial measurements of CA 125 are useful in monitoring the response to chemotherapy and follow-up. In patients with preoperative positive CA 125 assay, the concomitant determination of other tumour markers does not add further information when compared to CA 125 alone. Conversely in patients with preoperative negative assay the measurement of one or more of other antigens could be of clinical relevance. PMID- 10726624 TI - Comparison of CA 125 after three courses of chemotherapy and results of second look surgery. AB - OBJECTIVE: To compare CA 125 levels after three courses of cisplatin-based chemotherapy and the results of second-look surgery. METHODS AND MATERIALS: From January 1990 to December 1996, the medical records of 72 patients diagnosed with epithelial ovarian cancer were reviewed. After initial staging surgery, all patients received cisplatin-based chemotherapy. Prior to each course of chemotherapy, patients underwent physical exams and serum CA 125 was obtained. After 6 courses of chemotherapy, if CA 125 levels were normal (< or = 35 IU/ml) and there was no clinical evidence of disease, the patient was offered second look surgery. The sensitivity, specificity, and negative predicative value of CA 125 levels after 3 courses of chemotherapy and results of second-look surgery were calculated. Survival curves were constructed using Kaplan-Meier actuarial methods. RESULTS: Seventy-two patients were enrolled in the study. After completing 3 courses of chemotherapy, 43 out of 72 patients were reported to have normal CA 125 levels and were offered second-look surgery. Forty-six out of 72 patients underwent second-look surgery, 28 patients (60%) were reported to have positive second-look surgery. Of the patients with normal CA 125 levels after 3 courses of chemotherapy, 23 patients (57.5%) had a positive second-look surgery. The sensitivity and specificity of CA 125 values after 3 courses of chemotherapy were 17.9% and 94.7%, respectively and the negative predicative value was 43.9%. Patients with normal CA 125 values after 3 courses of chemotherapy had a significantly improved survival compared to those who failed to normalized their CA 125 levels after three courses of chemotherapy. CONCLUSION: Normalization of CA 125 after 3 courses of chemotherapy is a poor predicator of findings at second look surgery. PMID- 10726625 TI - Use of the Cornier pipelle as the only means of presurgical histologic diagnosis in endometrial carcinoma: agreement between initial and final histology. AB - OBJECTIVE: To test the reliability of the Cornier pipelle as a diagnostic tool for submitting endometrial carcinoma patients directly to surgery, without the additional performance of dilatation and curettage. METHODS: In this prospective study, 56 consecutive patients with the presumptive diagnosis of endometrial carcinoma, based on the analysis of material sampled by means of microcurettage using the Cornier pipelle, were submitted to hysterectomy. The findings of the final pathological report were compared with those of the previous microcurettage. Both were analysed at the same Pathology laboratory throughout the study. The presence of endometrial carcinoma was confirmed intraoperatively in all cases, and, as a consequence, a staging laparotomy was completed in all of them. RESULTS: Although the initial diagnosis of carcinoma was confirmed in every case (100% sensitivity), the final pathological report revealed discordant histological subtypes in 6 out of the 56 cases (10.7%). Only two, however, represented higher histological risk than initially stated. Discrepant grading values were also found in 3 out of 41 (7.3%) cases of the endometrioid subtype for which tumor grade been established in the microcurettage specimen. Not performing dilatation and curettage under general anesthesia prior to surgery resulted in a significant saving, both in hospitalization costs and bed occupation. CONCLUSION: When endometrial sampling by means of the Cornier pipelle yields the diagnosis of carcinoma, it can be confidently relied upon. However, some high-risk cases can be missed due to discordance between initial and final histology, and this could eventually lead to the choice of an inadequate surgical strategy. PMID- 10726626 TI - Trends in breast cancer incidence in Thrace, Greece: an epidemiological assessment. AB - PURPOSE: In the present study we describe the epidemiologic characteristics of breast cancer in relation to certain risk factors affecting the two major ethnic groups (Christian Orthodox and Muslims) in the area of Thrace, Greece. METHOD: We performed a cross-sectional study of 196 consecutive patients, aged 28-85 years, with breast cancer, who were referred to our clinic for treatment from January 1986 to June 1998. All patients were submitted to clinical, laboratory and mammographic control. Ultrasound examination and aspiration cytology were performed on cystic-like lesions. Epidemiologic characteristics of the patients were abstracted from medical charts. To evaluate the results, we used the direct standardization method (1995 Eur. Population) and chi2 test. RESULTS: Breast cancer incidence for the two study populations (Christian Orthodox and Muslims) was 20.9 and 2.3/100,000, respectively. We did not notice statistically significant differences in most epidemiologic characteristics between the two subject groups. CONCLUSIONS: Although the cumulative breast cancer incidence in Thrace, Greece is comparatively low, the discrepancy observed in the incidence of Christian Orthodox and Muslims suggests that differences in lifestyle between these two ethnic groups might be determinant factors influencing the prevalence of the disease. The appearance of the disease in a considerable number of young and elderly women, make the extension of modern screening methods in the above mentioned age groups necessary for the improvement of breast cancer prevention and incidence rates. PMID- 10726627 TI - ASCUS: comparative follow-up results related to previous SIL diagnosis. AB - The clinical significance of ASCUS (atypical squamous cells of undetermined significance) remains undetermined. In a variety of cases, it is possible to identify an underlying neoplastic squamous lesion. With the aim of establishing some rationale basis for management, we have evaluated the history and the follow up of 137 woman diagnosed with ASCUS. These woman were distributed into two groups, with or without history of SIL (30 and 107 woman, respectively); 38 woman did not come to the control. In general, the rate was 30.3% for low grade SIL (squamous intraepithelial lesions) and 6.1% for high grade SIL. In both groups the rate of low and high grade SIL was similar. In our opinion, women that are diagnosed with ASCUS must be submitted to colposcopic exams independently of their history. PMID- 10726628 TI - The addition of topotecan to carboplatin and paclitaxel as first-line therapy for advanced ovarian cancer; is it possible only with peripheral blood stem cell support? AB - A phase I study was performed in order to evaluate the tolerability of the combination of fixed doses of carboplatin and paclitaxel and escalated doses of topotecan as first line chemotherapy for advanced epithelial ovarian cancer. Three stage III and one stage IV patients entered the study. The dose limiting toxicity (neutropenia and thrombocytopenia) was reached at the first dose level: paclitaxel 175 mg/m2 on day 1, carboplatin AUC 5 on day I and topotecan 0.5 mg/m2 daily from day 1 to day 3. We conclude that it is not possible to add topotecan to standard regimens of carboplatin and paclitaxel without bone marrow support. PMID- 10726629 TI - The effects of tamoxifen therapy on the endometrium. AB - From January 1992 to December 1998, 219 women (aged between 30 and 81 yrs; average 55 years) affected by breast cancer were treated. These women in addition to the usual adjuvant therapy, were treated with TAM 20 mg/day, for a period between 24 and 72 months (average 40). In this group there were 84 postmenopausal and 31 premenopausal women. In 8 fertile patients ovarian activity was suppressed with GnRh analogue therapy and one patient underwent attinic castration. Before performing TAM therapy, a hysteroscopic exam was done and patients were followed up with an annual check-up. None had any endometrial side-effects after the first check-up. After two years, 31 women (26.9%) complained of endometrial alterations (hyperplasia, polyps and endometrial cancer). One women only after 6 years of follow-up, had metrorrhagia; an endometrial adenocarcinoma was found. We would like to point out the necessity of monitoring these patients with an annual check up (transvaginal sonography and/or hysteroscopy). PMID- 10726630 TI - Use of hydroxyurea and alpha-interferon in chronic myeloid leukemia during pregnancy: a case report. PMID- 10726631 TI - Different patterns of postoperative bleeding following cytoreductive surgery for gynecological cancer. AB - PURPOSE OF INVESTIGATION: To study the possible causes of postoperative bleeding following maximal cytoreductive surgery for gynecological cancers. METHOD: We have retrospectively reviewed all our cases of postoperative bleeding following major abdominal and pelvic cytoreductive surgery within a 48-hour period. In the postoperative period, replacement therapy was ineffective in achieving hemodynamic stability. During re-operation, the entire abdominal cavity was evaluated for bleeding sites that were adequately ligated or electrocoagulated. RESULTS: Of 942 women undergoing major cytoreductive surgery 22 women (2.3%) were re-operated for postoperative bleeding after a mean of 14.2 hours. Bleeding was either localized from a vessel in 9 women (40.9%) or diffuse (capillary oozing) in 13 women (59.1). Operative deaths have been as high as 36.8%. CONCLUSION: Postoperative bleeding following cytoreductive surgery can be from a single group of vessels or a capillary oozing from the edges or denuded areas of excised peritoneum. PMID- 10726632 TI - Endometrial cancer: preoperative evaluation of myometrial infiltration magnetic resonance imaging versus transvaginal ultrasonography. AB - From January 1996 to December 1998, 33 patients with endometrial carcinoma were preoperatively examined in our department; 30 women underwent transvaginal ultrasonography (TVUS) and magnetic resonance imaging (MRI), and 3 only TVUS. Diagnosis was obtained by histopathological examination of the tissue removed by hysteroscopically controlled biopsy or by curettage of the uterine cavity. TVUS and MRI were performed a few days before surgery. After surgery the uterus was histopathologically examined by a pathologist in order to evaluate the depth of myometrial invasion. The results were compared with TVUS and MRI data to determine sensibility and specificity, positive predictive value (PPV) and negative predictive value (NPV) of the two methods. According to the results of the present study we conclude that: TVUS is a low cost, easily performed and reliable method in a high percentage of cases if carried out by a skilled ecographist. MRI, is more expensive and has a lower specificity and sensibility index; it is a valid method if the cervical canal is involved and/or myometrial invasion is > 50% (M2) and if lymphatic invasion has to be investigated. PMID- 10726633 TI - Pure dysgerminoma of the ovary: a review of 45 well staged cases. AB - PURPOSE: To evaluate the significance of meticulous surgical staging, and whether type of initial surgery or adjuvant therapy impacted on survival in cases of pure ovarian dysgerminoma. METHODS: Retrospective chart review of 45 patients treated for pure ovarian dysgerminoma at a single institute. Survival analysis with the Kaplan Meier and log rank test and the chi-square test for the comparison of categorical variables were used. RESULTS: Of the 45 patients subjected to a surgical staging procedure 30 (67%) had stage I disease, 2 (4%) had stage II, 9 (20%) had stage III, and 4 (9%) had stage IV pure ovarian dysgerminoma. Thirteen of these 45 patients were referred to have seemingly stage I disease. Restaging within 2 months proved stage IIIC disease in 3 (23%) of them. With a median follow-up of 61 months, the overall survival rate for ovarian dysgerminoma in this series was 84%. Significantly lower survival rates were found in patients with advanced stage (stage III-IV) ovarian dysgerminoma (53.9%), when compared with earlier stages (96.9%). Twenty-one patients with unilateral disease and fertility desire were treated with conservative surgery, 19 patients with nonconservative surgery, and in 5 suboptimal debulking could be carried out. As regards recurrence or survival rate, no significant difference was found between patients who were treated conservatively or nonconservatively, whether or not adjuvant chemotherapy or radiotherapy was given. Of the 21 patients treated with conservative surgery, 11 (52%) achieved one or more pregnancies. CONCLUSION: After careful surgical staging and confirming unilateral disease, conservative surgery, followed if necessary by adjuvant chemotherapy, seems to be the ideal treatment in cases of pure ovarian dysgerminoma. PMID- 10726634 TI - An advanced case of double primary malignancy involving the breasts and uterine cervix: satisfactory response to non-surgical management. AB - Double primary cancers are fairly rare. We report here a case of metachronous lesions; advanced cancer of the breasts and squamous cell carcinoma of the cervix. What is peculiar in this case is the rather unusual positive response to conservative treatment. Despite widespread metastases even to the liver, the patient is well and active more than six years after breast cancer was first diagnosed. Treating such advanced cases may be rewarding at times. PMID- 10726635 TI - PRRSV, the virus. AB - Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-strand RNA virus that belongs to the Arteriviridae family. PRRSV grows in primary alveolar macrophages and in monkey kidney cell lines. The genomic RNA is approximately 15 kb. The genome encodes the RNA replicase (ORF1a and ORF1b), the glycoproteins GP2 to GP5, the integral membrane protein M, and the nucleocapsid protein N (ORFs 2 to 7). A comparison of nucleotide sequences of different strains indicates that European and North American strains represent two distinct antigenic types. Various PRRSV-specific monoclonal antibodies and recombinant structural proteins have been produced. Well-defined PRRSV mutants can be generated with the recently developed infectious cDNA clone of PRRSV. PMID- 10726636 TI - A review of evidence for immunosuppression due to porcine reproductive and respiratory syndrome virus. AB - Accounts of field disease and experimental studies involving porcine reproductive and respiratory syndrome (PRRS) are reviewed for evidence of immunomodulation or immunosuppression by the causative virus. The conclusion is that immunomodulation through infection of alveolar macrophages is likely to occur, but that it is transient and at a local level, in the lung. There is some evidence for more subtle effects via more disseminated replication or induction of apoptosis with some isolates, but more definitive studies are needed. There is some emerging evidence of interaction between PRRSV and different cells of the immune system, but its significance for the course of disease or pig health are unclear. Likewise, the current experimental evidence for any interaction of PRRSV with other pathogens is ambiguous and therefore no firm conclusions can yet be drawn. Strains of PRRSV do vary in pathogenicity, which may be related to their degree of ability to cause overt respiratory disease in the absence of other agents. Experimentally, varying degrees of interstitial pneumonia are a common histological finding. There is, as yet, no firm evidence of general immunosuppression--in fact, some contrary evidence exists in the form of observations of a transient enhancement of humoral response, possibly through polyclonal B cell activation. The basis of pathogenicity of PRRSV and of any interaction with other agents is still unknown and is likely to remain unclear. Virus interaction with the pig's immune system must be addressed before any assessment of virulence of any known or emergent strains of PRRSV can be made. PMID- 10726637 TI - Ultrastructural pathogenesis of the PRRS virus. AB - Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) was first isolated in swine alveolar macrophages (SAMs) and has subsequently been reported to replicate in other cell lines. Entry of the virus inside the cell takes place by receptor mediated endocytosis. Following the entry of the virus into the cell, several not completely understood changes take place. PRRSV has been reported to be an apoptotic-inductor virus both in vivo and in vitro. Interestingly, it has been suggested that PRRSV-induced apoptosis occurs in cells other than those in which PRRSV replicates by a bystander mechanism. In this paper the ultrastructural pathogenesis of PRRSV will be reviewed. PMID- 10726638 TI - A brief review of procedures and potential problems associated with the diagnosis of porcine reproductive and respiratory syndrome. AB - Experience has shown that, for a number of reasons, a diagnosis of porcine reproductive and respiratory syndrome (PRRS) is sometimes difficult. In this review we discuss: (1) field observations and laboratory tests that are useful in arriving at a definitive diagnosis; (2) the impact of so-called atypical PRRS on diagnostic procedures in North America; (3) the means by which diagnostic problems can often be circumvented by appropriate sample selection; and (4) methods used for presumptive identification of PRRS virus strains. PMID- 10726639 TI - The "colorful" epidemiology of PRRS. AB - The paper describes the specifics of the epidemiology of the Porcine Reproductive and Respiratory Syndrome (PRRS), that is its "behavior" as a communicable disease in porcine populations, and compares them to the general epidemiological characteristics of communicable diseases. This analysis shows that infection with the PRRS virus "behaves" epidemiologically both as an epidemic and as an endemic disease: on the one hand it can spread like an epidemic in naive populations, and on the other it seems to linger on infinitely in an affected population with its clinical expression varying from farm to farm like an endemic disease. The paper tries to draw "epidemiological" conclusions on the general methods for controlling and/or eradicating the disease, and to identify areas of further research. PMID- 10726640 TI - Aujeszky's disease (pseudorabies) virus: the virus and molecular pathogenesis- state of the art, June 1999. AB - Considerable progress has been made during the last years in understanding the molecular basis of protein function in pseudorabies virus (PrV), the causative agent of Aujeszky's disease (AD). Major topics have been the identification and functional characterisation of viral envelope glycoproteins and cellular virus receptors, elucidation of viral proteins involved in neurovirulence and neuropathogenesis, detection and characterisation of attenuating mutations present in and leading to successful attenuated live vaccines, and the near completion of the genomic sequence of PrV DNA. This review, which follows an article prepared for the 1993 AD symposium in Budapest, Hungary, will briefly summarise those recent developments and update the reader on the current state of the art in PrV research. PMID- 10726641 TI - Aujeszky's disease virus: opportunities and challenges. AB - Since its description in 1902, Aujeszky's disease (AD) has become one of the most thoroughly examined viral diseases of swine. The causative agent, Aujeszky's disease virus (ADV), is a neurotropic alphaherpesvirus that produces fatal encephalitis in newborn pigs and a milder syndrome in older animals. In several instances this virus has been used as a test case to examine novel vaccine concepts in swine, including the honor of being the first genetically modified vaccine used in the field. Furthermore, the examination of the immune response to infection or vaccination with this virus has revealed important information about the function of the porcine immune system, including evidence on the existence of a dichotomy between the humoral and cellular immune response in swine. This review presents a summary of research where ADV has been a valuable tool for the development of novel vaccines and has provided information to better understand the immune response of swine to infectious agents. PMID- 10726642 TI - Challenges of the final stages of the ADV eradication program. AB - The authors were chairpersons in the session on epidemiology and control of Aujeszky's disease (AD). In this document, they focus on several issues, such as vaccination, compliance and surveillance, which influence the eradication programs. Also, some research topics which may need attention in the future are indicated. The main conclusion is that eradication programs for AD virus have made good progress in different parts of the world and that we have the knowledge and tools to do the job. It must be realized, however, that setbacks can occur. As prevalence declines, susceptibility increases and producers may let their guard down so that the virus may spread again in susceptible areas. PMID- 10726643 TI - NIMH/APPC workgroup on behavioral and biological outcomes in HIV/STD prevention studies: a position statement. PMID- 10726644 TI - Evaluation of patient-administered tampon specimens for Chlamydia trachomatis and Neisseria gonorrhoeae. AB - BACKGROUND: The patient-administered tampon specimen has proven to be an easy and sensitive method for the diagnosis of genital Chlamydia trachomatis and Neisseria gonorrhoeae infections in women by polymerase chain reaction (PCR). This method avoids the need for endocervical sampling and stringent criteria for transport. GOAL: To evaluate two commercial amplification systems for the detection of C trachomatis and N gonorrhoeae from tampon specimens. STUDY DESIGN: A group of 400 positive and negative tampon specimens tested by an in-house PCR method were selected from a pool of more than 2,000 previously collected tampons. Overall, 93 C trachomatis-positive and 77 N gonorrhoeae-positive specimens were evaluated. Each specimen was tested by Roche Cobas Amplicor and Abbott LCx (LCR), and results were compared to the in-house PCR method. RESULTS: Detection of C trachomatis by both assays was not significantly different from the in-house PCR assay. Fewer tampons were positive for N gonorrhoeae by LCR than either the in house assay (P = 0.0001) or by Roche Amplicor (P = 0.01). However, tampon specimens tested by Roche Amplicor required DNA extraction to achieve comparative sensitivity. CONCLUSION: Both commercial assays can be applied to tampon collected specimens for automated detection of sexually transmitted diseases. The detection of C trachomatis was similar to the in-house PCR test for both assays (P = 0.73, 0.68). Detection of N gonorrhoeae resulted in fewer positive tampon specimens when tested by ligase chain reaction than both Roche Amplicor and in house PCR. PMID- 10726645 TI - Epidemiologic trends of sexually transmitted diseases in China. AB - OBJECTIVE: To describe the current epidemiology of sexually transmitted diseases (STDs), we analyzed the time trends in distribution of reported cases of STDs in China from 1989 to 1998. STUDY DESIGN: Based on the computerized database of the National System of STD Surveillance, a retrospective study of STD cases reported from 1989 to 1998 was carried out. RESULTS: From 1989 to 1998, STD incidence increased significantly in men and women and demonstrated nonlinear growth trends, with the exception of gonorrhea incidence. Between 1990 and 1998, the incidence increased more in females (4.20 times) than that in males (3.79 times). Syphilis incidence increased approximately 20 times during this period at an average annual rate of 52.7%. Gonorrhea incidence increased 2.6 times during this period at an average annual rate of 11.4%, which reached a plateau from 1994 to 1997. Extramarital transmission as source of STD infection has significantly increased from 1995 to 1998. CONCLUSION: The incidence of STDs has continued to increase in China from 1989 to 1998. The epidemic patterns are different from rates in developed counties in which behavior interventions have been successfully implemented, suggesting the need for active behavioral-intervention programs in China. PMID- 10726646 TI - Sexually transmitted diseases in the People's Republic of China in Y2K: back to the future. PMID- 10726647 TI - Urine screening for gonococcal and chlamydial infections at community-based organizations in a high-morbidity area. AB - BACKGROUND: Increasing availability of urine testing for Neisseria gonorrhoeae (GC) and Chlamydia trachomatis (CT) offers expanded opportunities to collaborate with community-based organizations (CBOs) to screen high-risk populations for sexually transmitted diseases (STDs). GOAL: To determine the prevalence and correlates of genital tract gonococcal and chlamydial infection among CBO clients, and to assess the feasibility of implementing widespread community-based STD screening programs. DESIGN: Free, voluntary, confidential first-catch urine screening was conducted at 20 CBOs serving disadvantaged populations in St. Louis, MO. Brief demographic, behavioral, and sexual contact data were obtained from all participants. Urine samples were tested by ligase chain reaction (LCR). Persons testing positive were promptly notified and directed to seek treatment. RESULTS: A GC and/or CT infection was identified in 24 of 277 persons (8.7%) screened; 2 persons were infected with GC only, 17 with CT only, and 5 with GC and CT. Treatment was documented for 22 persons (91.7%) testing positive. The highest rates of infection were found at shelters (12.3%) and residence facilities (11.1%). Costs of screening were $38 per sample collected and $453 per case identified. CONCLUSION: Community-based urine testing successfully identified GC and CT infections, and was well accepted by community members and CBOs. Community-based screening can significantly impact STD epidemiology by facilitating early detection, treatment, and interruption of transmission. PMID- 10726648 TI - Artificial nodules of the penis: case report of an Indonesian man. PMID- 10726649 TI - Ambulatory STD management in an inner-city emergency department: descriptive epidemiology, care utilization patterns, and patient perceptions of local public STD clinics. AB - BACKGROUND AND OBJECTIVES: In conjunction with a lack of primary and preventive care, many patients are treated in emergency departments (EDs) for sexually transmitted diseases (STDs). GOAL: To epidemiologically characterize patients accessing an inner-city ED for treatment of STDs. DESIGN: One hundred patients 17 years or older who were treated for an STD or who had conditions that were given syndromic or presumptive diagnoses of an STD participated in this prospective case series. Cases were identified on the basis of history and physical examination. Patients were interviewed to collect information on demographics, drug and alcohol use, current symptoms, self-medication, and health care access. RESULTS: A total of 98% of patients who were approached participated. Two thirds of the participants were female. Women were more likely than men to have health insurance, Medical Assistance, and a regular source of health care. Injecting drug use was associated with a 5.3 increase in the odds ratio of delayed treatment seeking (P = 0.038). Effort to self-treat was associated with a 3.2 increase in the odds of delayed treatment seeking (P = 0.015). Being female was associated with a 4.1 increase in the odds of self-treatment (P = 0.009). CONCLUSION: This study identifies several potential barriers to appropriate health care access and use. The study also identifies the ED as a potential source of intervention for more comprehensive health care and an entry into the health care system for a difficult-to-reach patient population. PMID- 10726651 TI - Evaluation of chlamydia and gonorrhea screening criteria: San Francisco sexually transmitted disease clinic: 1997 to 1998. AB - BACKGROUND AND OBJECTIVES: The advent of more sensitive diagnostic testing technologies and competition in public healthcare spending have resulted in a reevaluation of sexually transmitted disease (STD) screening practices in an attempt to target populations at greatest risk. Screening among populations with a < 2% prevalence of chlamydia and a < 1% prevalence of gonorrhea may not be cost effective. GOAL: To identify subpopulations with a low prevalence of chlamydia or gonorrhea. STUDY DESIGN: The prevalence of genital chlamydia and gonorrhea among asymptomatic STD patients screened from 1997 to 1998 at San Francisco City Clinic was stratified by demographic and behavioral risk factors. RESULTS: The prevalence of chlamydia and gonorrhea was 3.4% and 1.1% among asymptomatic women and 4.0% and 1.0% among asymptomatic men, respectively. Two low-prevalence subpopulations identified among asymptomatic patients were women older than 29 years (chlamydia, 1.2%) and men who have sex with women (gonorrhea, 0.8%). CONCLUSIONS: These data identified low-prevalence subpopulations among asymptomatic STD patients. As a result, the STD screening criteria at San Francisco City Clinic were changed accordingly. PMID- 10726650 TI - Prevalence and risk factors for human herpesvirus 8 infection in northern Cameroon. AB - BACKGROUND: The modes of transmission of HHV-8 are still unclear. GOAL: To evaluate the distribution and transmission of HHV-8 infection. DESIGN: Serosurvey conducted in a Cameroon hospital among 292 persons, including children (5-10 years), adolescents (15-20 years), and adults (30-40 years). Antibodies against lytic and latent antigens to HHV-8 were detected by immunofluorescence assay; antibodies against Epstein-Barr virus viral antigens were detected by enzyme linked immunoabsorbent assay. RESULTS: The prevalence of HHV-8 antilytic antibodies remained stable and was 39.8% among children, 51.5% among adolescents, and 61.8% among adults. Epstein-Barr virus seroprevalence was high among children, and remained stable among adolescents and adults. A history of sexually transmitted diseases was an independent determinant of HHV-8 infection (adjusted odds ratio 2.47; 95% CI 1.09-4.91). CONCLUSION: The high prevalence of HHV-8 infection among children indicates nonsexual modes of transmission in Cameroon, with sexual transmission occurring among adolescents and adults. PMID- 10726652 TI - Primary and secondary syphilis in the metropolitan area of Nashville and Davidson County, Tennessee: 1996 to 1998 epidemic described. AB - BACKGROUND: Metropolitan Nashville, Tennessee experienced a syphilis epidemic from 1996 to 1998. GOAL: This study describes the syphilis epidemic during this period. DESIGN: Descriptive analyses of syphilis surveillance data (1988-1998) were performed, with emphasis on the current epidemic (1996-1998), and were stratified by age, gender, race, and census tracts. RESULTS: Five features were observed regarding the 1996 to 1998 syphilis epidemic: (1) males and females were almost equally affected, with a delay in diagnosis in female patients; (2) the 30 39-year age group was most affected; (3) the 15-19-year age group had the highest percentage increase in incidence; (4) African Americans were the most affected population subgroup; and (5) downtown Nashville and the surrounding areas were most affected. CONCLUSION: Although talk of eradicating syphilis has surfaced nationally, Nashville has continually faced syphilis as a public health problem. During each of the past 3 years (1996-1998), the incidence of syphilis in this community has reached epidemic proportions. PMID- 10726654 TI - Prevalence of hepatitis G virus in patients with hemophilia and their steady female sexual partners. AB - BACKGROUND: Hepatitis G virus (HGV), also known as GB virus C, is a newly discovered Flavivirus that is transmissible by blood transfusion and other possible routes. OBJECTIVE: To study the risk of sexual transmission of HGV in female sexual partners of men with hemophilia (n = 161 couples). METHODS: Blood samples obtained from 11 medical centers were analyzed for (1) HGV RNA by polymerase chain reaction; (2) antibodies to HGV by enzyme immunoassay; and (3) other viruses and T-cell counts by routine laboratory tests. Subjects completed a questionnaire that assessed sexual intercourse frequency, number of sexual partners, condom usage, sexually transmitted diseases, illicit drug usage, and needlestick or broken-glass injuries. RESULTS: The HGV infection (RNA +/- antibody positive) prevalence was 48% among men and 21% among women. Prevalence of hepatitis C virus, hepatitis B virus, and HIV among men was 99%, 94%, and 86%, compared with 3%, 11%, and 12% among women, respectively. The odds ratio for HGV infection for women with an HGV-positive male sexual partner was 2.14 (P = 0.06) without adjustment, and 2.77 (P = 0.03) with adjustment for other variables, none of which were independently significant. CONCLUSION: These results suggest a low level of HGV sexual transmission. PMID- 10726653 TI - High prevalence of asymptomatic STDs in incarcerated minority male youth: a case for screening. AB - BACKGROUND AND OBJECTIVES: To assess STD prevalence among a sample of incarcerated minority male youth in a southern US city. METHODS: A consecutive entrant, cross-sectional study of 284 minority males 14 to 18 years was performed. All adolescents were screened for gonorrhea and chlamydia using ligase chain reaction tests. RESULTS: Eighteen percent of youth were identified as having either gonorrhea, chlamydia, or both. Approximately 84% of those with sexually transmitted diseases (STDs) self-reported having no symptoms. Failure to use condoms in the past month was significantly associated with a positive test result for STDs (odds ratio = 1.9, 95% CI = 1.1-3.3). CONCLUSIONS: The findings indicate an urgent need for routine STD screening and STD-prevention programs for adolescent males in detention facilities. A study of 284 detained black male adolescents revealed 18% prevalence of gonorrhea, chlamydia, or both. Approximately 84% of those with sexually transmitted diseases self-reported not having symptoms. PMID- 10726655 TI - Infection of the macrophage cell line NR8383 with Mycobacterium tuberculosis (H37Ra) leads to an increase in oligodeoxynucleotide accumulation. AB - Mycobacterium tuberculosis infection continues to be a daunting clinical challenge. Although it may well be one of the most studied bacteria in history, several aspects of its pathology remain a mystery. The resurgence of drug resistant M. tuberculosis strains and with its unusual pathology have promoted a renewed basic and clinical research interest in developing new therapies to combat this pathogen. The primary localization site for M. tuberculosis is within alveolar macrophages. Drug delivery strategies and novel therapeutic agents designed to target alveolar macrophages may lead to efficient destruction of M. tuberculosis. Oligodeoxynucleotides (ODN) are short segments of nucleic acids that can interfere with transcription and translation processes. In this report, a monocyte-macrophage cell line was characterized in regard to ODN transport in the presence or absence of M. tuberculosis infection. The cells accumulated ODN in a time-dependent and concentration-dependent manner, regardless of the presence of serum. After 4 hours of incubation with M. tuberculosis (multiplicity of infection [MOI] 10:1), infected NR8383 cells demonstrated 1.5-7-fold increase in fluorescein isothiocyanate (FITC)-labeled phosphorothioate ODN accumulation as measured by flow cytometry. The increase in uptake was associated only with fluorescent-labeled ODN and not labeled markers of fluid phase endocytosis (e.g., tetramethylrhodamine isothiocyanate [TRITC], FITC-labeled dextran). NR8383 cells activated by phytohemagglutinin (PHA) did not demonstrate a significant increase in the uptake of either FITC-labeled dextran or FITC-labeled ODN. These studies demonstrate that NR8383 cells that have been infected with M. tuberculosis can specifically accumulate ODN, and this route of accumulation may lead to a means of drug targeting to mycobacteria-containing cells. PMID- 10726656 TI - In vivo evaluation of a morpholino antisense oligomer directed against tumor necrosis factor-alpha. AB - Morpholino antisense oligomers directed against the cytokine tumor necrosis factor (TNF-alpha) can specifically inhibit production of TNF-alpha by macrophages in vitro. To evaluate the efficacy of morpholino antisense in vivo, we characterized a mouse model of increased pulmonary TNF-alpha production and inflammation in response to aerosolized endotoxin. Pretreatment of mice by intranasal (i.n.) insufflation of oligomers (30 microl of 100 microM/ml) 12 hours prior to lipopolysaccharide (LPS) exposure resulted in specific and consistent inhibition of TNF-alpha production by the oligomer MAS-2, whereas no effect was observed with a sequence-scrambled control (% inhibition 31.5 +/- 3.5 vs. 1.3 +/- 8.0, respectively, p < 0.005). Dose-response analysis showed similar efficacy for MAS-2 at 25-100 microM/ml and diminished effects with lower concentrations. Inhibition of TNF-alpha did not alter the increase in neutrophils seen in bronchoalveolar lavage (BAL) fluid, a result consistent with observations using i.n. administration of neutralizing anti-TNF-alpha antibody or TNF receptor knockout mice. The results establish that morpholino oligomers directed against cytokine targets can function in vivo. Additional studies of other targets and administration protocols to improve efficacy are warranted. PMID- 10726657 TI - The roles of E6-AP and MDM2 in p53 regulation in human papillomavirus-positive cervical cancer cells. AB - The p53 tumor suppressor is regulated by the MDM2 oncoprotein through a negative feedback mechanism. MDM2 promotes the ubiquitination and proteasome-dependent degradation of p53, possibly by acting as a ubiquitin ligase. In cervical cancer cells containing high-risk human papillomaviruses (HPV), p53 is also targeted for degradation by the HPV E6 oncoprotein in combination with the cellular E6-AP ubiquitin ligase. In this report, we describe the identification of efficient antisense oligonucleotides against human E6-AP. The roles of MDM2 and E6-AP in p53 regulation were investigated using a novel E6-AP antisense oligonucleotide and a previously characterized MDM2 antisense oligonucleotide. In HPV16-positive and HPV-18 positive cervical cancer cells, inhibition of E6-AP, but not MDM2, expression results in significant induction of p53. In HPV-negative tumor cells, p53 is activated by inhibition of MDM2 but not E6-AP. Furthermore, treatment with both E6-AP and MDM2 antisense oligonucleotides in HPV-positive cells does not lead to further induction of p53 over inhibition of E6-AP alone. Therefore, E6-AP mediated degradation is dominant over MDM2 in cervical cancer cells but does not have a significant role in HPV-negative cells. PMID- 10726658 TI - The influence of antisense gene location on target gene suppression in the fission yeast Schizosaccharomyces pombe. AB - A fission yeast model was employed to investigate the influence of antisense gene location on the efficacy of antisense RNA-mediated target gene suppression. Fission yeast transformants were generated that contained the target lacZ gene at a fixed position and a single copy antisense lacZ gene integrated into various genomic locations, including the same locus as the target gene. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus compared with other genomic locations, indicating that target and antisense gene colocalization is not a critical factor for efficient antisense RNA-mediated gene expression in vivo. Instead, increased lacZ downregulation correlated with an increase in antisense dose, with the steady-state levels of antisense RNA being dependent on genomic position effects and transgene copy number. PMID- 10726659 TI - Absorption of antisense oligonucleotides in rat intestine: effect of chemistry and length. AB - An in situ single-pass perfusion model was used to assess the effect of chemical modification and length on permeability and absorption of various oligonucleotides in rat intestine. Phosphorothioate oligodeoxynucleotides (PS ODN) were compared with oligoribonucleotides with 2'-methoxyethyl (MOE) or 2'-O methyl (OMe) modifications. A 25-mer PS-OMe-modified oligonucleotide showed relatively poor permeability in this model, as did unmodified 20-mer PS-ODN (permeability coefficient [P(eff)] = 2-8 X 10(-6)cm/sec). Modifying some or all of the oligonucleotides with 2'-MOE groups on deoxyribose and 5'-methylation of the cytosines substantially increased intestinal permeability of oligonucleotides. Both partially and fully modified PS-MOE oligonucleotides showed a (2-4)-fold increase in permeability as compared with unmodified PS-ODN. The presence of a phosphodiester backbone in MOE-modified compounds led to further increases in intestinal permeability. PS-MOE composed of 6, 8, 10, 12, 14, 16, 18, 20, and 22 nucleotides were also examined. It was found that the permeability of these oligonucleotides increased linearly with decreasing length. PMID- 10726660 TI - Fluorescence resonance energy transfer analysis of RNase L-catalyzed oligonucleotide cleavage. AB - A method is described for monitoring the cleavage of an oligoribonucleotide substrate by the 2-5A-dependent RNase L based on fluorescence resonance energy transfer (FRET). The oligoribonucleotide, rC11U2C7, was labeled covalently at its 5'-terminus with fluorescein and at its 3'-terminus with rhodamine to provide a substrate for RNase L. On cleavage, the fluorescence at 538 nm (with 485 nm excitation) increased by a factor of 2.8, allowing real-time quantitation of the reaction progress. The method was performed easily in a 96-well plate format and allowed quantitative high throughput analyses of RNase L activity with different activators. PMID- 10726661 TI - Nucleotides -1 to -4 of hepatitis delta ribozyme substrate increase the specificity of ribozyme cleavage. AB - In the past, the use of delta ribozyme as a therapeutic tool was limited because substrate specificity was thought to be determined by only 8 nucleotides. Recently, we have accumulated evidence suggesting that the substrate sequence upstream of the cleavage site, which is not involved in the binding with the delta ribozyme, appears to be essential in the selection of an appropriate cleavage site. To understand the role of this region in efficient cleavage, we synthesized a collection of small substrates that possessed single and multiple mutations in positions -1 to -4 and determined the kinetic parameters of their cleavage using a model antigenomic delta ribozyme. Some substrates were found to be uncleavage, whereas others showed >60-fold difference in relative specificity between the least and most efficiently cleaved substrates. The base at each position from -1 to -4 contributes differently to the ability of a substrate to be cleaved. An optimal sequence for positions -1 to -4 was determined to be 1HRHY(-4) (H = U, C, or A). These results shed light on new features that contribute to the substrate requirement of delta ribozyme cleavage and should increase interest in the use of this unique ribozyme. PMID- 10726662 TI - The field of clinical psychology: past, present, and future. 1945. PMID- 10726663 TI - The field of clinical psychology: a response to Thorne. AB - This article begins with a retrospective review of the editorial written in 1945 by Frederick C. Thorne at the inauguration of his new publication, the Journal of Clinical Psychology. It first considers the predictions made by Thorne about the future of clinical psychology and which of these turned out to be accurate, partially accurate, or inaccurate. At the time, the editorial caused its author to be accused of anti-Semitism and thus got the journal off to a rather bad start, from which it may have recovered only recently. The article then moves on to consider the past, present, and future of the field of clinical psychology from a contemporary vantage-point. PMID- 10726664 TI - Medical responsibility for training in clinical psychology. 1949. PMID- 10726665 TI - The Kubie "solution": an idea before its time? AB - Interpreted in historical context, Kubie's 1948 proposal to move the professional aspects of clinical psychology training and the awarding of the doctorate into the medical-school environment was an attempt to remedy a critical shortage of psychotherapists while maintaining medical control over the professional practice of psychology. The proposal failed, in part, because medicine had no legitimate claim to either training or regulating clinical psychologists. A parallel was drawn between that post-war situation and the current one in which many psychological practitioners are pressing for prescribing privileges in regard to psychoactive drugs, and similarities and important differences are noted between the two conditions. The requirement that medicine be involved importantly in the psychopharmacologic training of psychologists poses an acute problem concerning the maintenance of professional independence. PMID- 10726666 TI - On training clinical psychologists in psychotherapy. 1949. PMID- 10726667 TI - The evolution of psychotheroapy training: reflections on manual-based learning and future alternatives. AB - Although psychotherapists-in-training may rely significantly on their clinical intuitions when first beginning to practice therapy, they quickly discover that much more is required to conduct effective treatment. Increasingly over the past two decades, training manuals have been used to impart explicit guidelines to beginning psychotherapists. In addition, manuals offer a means for helping more experienced therapists to learn new approaches, while also allowing careful research on the relative efficacy of different psychotherapies. In some instances, manuals have been designed specifically to address troublesome transference and countertransference issues (e.g., Time-Limited Dynamic Psychotherapy; Strupp & Binder, 1984). But even manualized treatments that target hostile interactions between therapist and patient are sometimes of limited use in teaching therapists to work with difficult patients, as illustrated by the Vanderbilt II Psychotherapy Research Project. Experimental research on the distinctions between implicit learning and explicit learning (e.g., Lee & Vakoch, 1996) helps explain the constraints of manualized treatments. By relying on explicit, clearly articulated guidelines, treatments conducted in accordance with manuals may interfere with tacit reasoning processes. As a consequence of the strong emphasis that manuals place on explicit rules for treatment, this type of training can hinder the development of complex clinical judgments. Future generations of novice psychotherapists may benefit from training experiences that are designed to promote an integration of implicit and explicit learning. PMID- 10726668 TI - Report of roundtable on internship and training of clinical psychologists. 1947. PMID- 10726669 TI - Training the scientist-practitioner for the 21st century: putting the bloom back on the rose. AB - The field of clinical psychology has been characterized over the past five decades as shaped by social, economic, and political forces outside of it. Even before the Boulder Conference, meetings and writings evidenced the underpinnings of the scientist-practitioner model that was to be developed in a welcoming climate (Dosier, 1947; Kubie, 1949; Luchins, 1949). Difficulties in implementation, however, produced a schism between the scientist and practitioner aspects. Our thesis is that the current environment dictated by managed care necessitates a reaffirmation of the scientist-practitioner model. We believe that clinical psychologists are in an unparalleled position to intercede among forces in the current climate because of their sensitivity to clinical and research issues, and we offer suggestions for how clinicians, researchers, and training programs can close the gap between research and practice. PMID- 10726670 TI - The future of diagnostic testing in clinical psychology. 1946. PMID- 10726671 TI - Visions of clinical assessment: then, now, and a brief history of the future. AB - Perspectives on the discipline of psychological assessment are provided through reflections on a classic article by Hunt (1946), review of current issues, and a projection of changes likely to occur over the next fifty years. Topics covered include the place of idiosyncratic qualitative responses, test design, clinical judgment, managed care, financial efficacy, over emphasis on pathology, treatment planning, status of projectives, ecological validity, relationship between theory and tests, innovations in scale construction, and computer-assisted assessment. Future predictions relate to innovations in computer-assisted assessment, practitioner roles in an increasingly automated environment, the future of traditional tests, and human-rights issues. PMID- 10726672 TI - The dynamics of "structured" personality tests. 1945. PMID- 10726673 TI - Dynamics of personality test responses: the empiricist's manifesto revisited. AB - This article revisits the classic empirical manifesto written by Meehl in 1945, and examines subsequent developments in structured personality assessment. The current status of personality assessment from an empirical-scale-development perspective is presented, with examples drawn from recent work on the MMPI-2. Meehl's heuristic defense of empirically based personality-scale construction was reexamined and the lasting influences of these views were highlighted. Meehl's early conceptualization of the relative unimportance of item content in personality-test construction and several alternative views were summarized, and Meehl's modified position was described. The role that test-taking attitudes can play in personality assessment was discussed in the 1945 article, and Meehl's views on the need for appraisal of invalidating conditions have been reaffirmed in contemporary test development. Finally, the so-called "dynamics" of a structured personality item response were discussed from a contemporary perspective, and some recent research was included to illustrate the continued importance of anchoring test interpretation in empirical correlates. PMID- 10726674 TI - The Rorschach test in clinical diagnosis. 1947. PMID- 10726675 TI - The Rorschach test in clinical diagnosis: a critical review, with a backward look at Garfield (1947). AB - The present article comments on a classic study by Garfield (1947) then reviews research on the Rorschach and psychiatric diagnoses. Despite a few positive findings, the Rorschach has demonstrated little validity as a diagnostic tool. Deviant verbalizations and bad form on the Rorschach, and indices based on these variables, are related to Schizophrenia and perhaps to Bipolar Disorder and Schizotypal Personality Disorder. Patients with Borderline Personality Disorder also seem to give an above-average number of deviant verbalizations. Otherwise the Rorschach has not shown a well-demonstrated relationship to these disorders or to Major Depressive Disorder, Posttraumatic Stress Disorder (PTSD), anxiety disorders other than PTSD, Dissociative Identity Disorder, Dependent, Narcissistic, or Antisocial Personality Disorders, Conduct Disorder, or psychopathy. PMID- 10726676 TI - Using the Rorschach properly in practice and research. AB - Proper use of the Rorschach Inkblot Method (RIM) in practice and research requires (a) well-founded expectations concerning what the RIM should be expected to do, and (b) appropriate methods for examining its validity in achieving the purposes for which it is intended. The RIM is a personality-assessment instrument, and its validity should be judged from its substantial correlations with observed behaviors that are conceptually linked to personality processes. Knowledge about personality functioning gleaned from Rorschach data may contribute to diagnostic formulations, but associations between Rorschach indices and psychometrically shaky DSM diagnostic categories have little bearing on the utility of the instrument for achieving its intended purposes. Adequate conceptual formulation of this kind is as necessary as solid empirical verification in the development and use of psychological assessment instruments. PMID- 10726678 TI - Prophylactic sclerotherapy in children with esophageal varices: long-term results of a controlled prospective randomized trial. AB - BACKGROUND/PURPOSE: Experience using endoscopic prophylactic sclerotherapy (PS) is restricted to adult patients and has led to conflicting results. There has not been a randomized, controlled study on the use of PS in children. The purpose of this study is to evaluate prospectively the value of PS to prevent the first hemorrhage from esophageal varices in children with portal hypertension and to assess the effect of PS on survival rate. METHODS: In a controlled, prospective, computer-based randomized trial, the effectiveness of PS was analyzed in 100 consecutive children allocated to a group receiving sclerotherapy (n = 50) or to a control group (n = 50) subjected only to regular clinical and endoscopic examinations. Clinical characteristics in both groups were similar. The minimum follow-up period was at least 18 months after the cessation of the sessions of sclerotherapy. RESULTS: After a median follow-up of 4.5 years, PS eliminated the esophageal varices in 47 of 50 (94%) patients but only 38 (76%) of them do not present upper digestive hemorrhage. Before complete obliteration of the varices, upper gastrointestinal bleeding occurred in 12 patients (24%). Six children (12%) had gastric varices, 3 of 6 of whom (50%) bled. Congestive hypertensive gastropathy was observed to occur in 8 (16%) patients, 4 of 8 of which (50%) had hemorrhagic episodes. Two patients bled from undetermined cause. In the control group, only 29 (58%) children remained free from esophageal variceal bleeding and 26 (52%) from any upper gastrointestinal bleeding (P<.05). During the follow-up period, the development of gastric varices was observed in 5 (10%) patients (P>.05) and of congestive hypertensive gastropathy in only 3 (6%) patients (P<.05), but none of them bled. PS does not improve survival rate. CONCLUSIONS: In children with cirrhotic and noncirrhotic portal hypertension, PS reduces the overall incidence of bleeding from esophageal varices that were eradicated in 94% of cases. The source of bleeding has been different in each group, being predominantly from esophageal varices in the control group and from the stomach in the prophylaxis group. When applied with appropriate technique, PS is a safe procedure with a low incidence of minor complications. PS does not change the incidence of gastric varices but increases the development of congestive hypertensive gastropathy. PS increases the risk of bleeding from the naturally formed gastric varices and from congestive hypertensive gastropathy. PS does not affect survival rate. PMID- 10726677 TI - A nonreviewer's comment: On the Rorschach and baseball. PMID- 10726679 TI - RNA differential display of scarless wound healing in fetal rabbit indicates downregulation of a CCT chaperonin subunit and upregulation of a glycophorin-like gene transcript. AB - BACKGROUND/PURPOSE: Scars form as wounds heal in adult organisms. In addition to disrupting cosmetic appearance, scar tissue can cause significant morbidity, and even death if it blocks vital organ function. Previous work has established that fetal wounds, especially in early to midgestation, can heal without scarring. Because such inherent physiological mechanisms ultimately are under genetic control, a study was initiated to elucidate the differences in gene expression that produce scarless wound healing in the mammalian fetus but scarring in postnatal wounds. Reverse transcription polymerase chain reaction (RT-PCR) differential display (DD) was used to detect differentially expressed mRNA transcripts in a rabbit model of wound healing. METHODS: Adult and 21-day fetal full-thickness rabbit skin specimens from wounded and unwounded sites were harvested 12 hours postwounding. RNA extracted from the tissue was used as a template in DD reactions using anchoring and random primers to generate tissue specific gene expression fingerprints. The over 2,000 resulting amplimers (gene transcripts) were screened for differential expression among the 4 types of specimens: fetal control (unwounded), fetal wound, adult control, and adult wound. Selected bands distinctly upregulated or downregulated in fetal wound lanes on the DD gels were excised, and the cDNA was extracted, reamplified, cloned into vectors, and sequenced. DD results were confirmed by limiting dilution RT-PCR using sequence-specific primers. RESULTS: Differential display (DD) showed 22 amplimers that were significantly upregulated in all fetal wound samples as compared with little or no expression in fetal control, adult control, or adult wound tissues. Conversely, 5 transcripts were downregulated in the fetal wound specimens but highly expressed in the 3 comparison tissues. Reamplification of selected transcripts by PCR, followed by cloning and DNA sequencing, yielded 7 distinct sequences, each representing a gene expressed differently in fetal wound than in the other 3 tissues. A transcript that was downregulated in fetal wound showed very high sequence homology to part of the human gene for the eta subunit of the hetero-oligomeric particle CCT (the chaperonin containing T-complex polypeptide 1 or TCP-1). An upregulated amplimer showed significant DNA sequence homology to glycophorins A and B. One sequence was identified as 28S rRNA. The remaining 4 candidate sequences showed no significant homology to known genes, but 1 had high homology to expressed sequence tags of unknown function. CONCLUSIONS: With careful experimental design and proper controls and verifications, differential display of RNA expression is a potentially powerful method of finding genes that specifically regulate a particular physiological process such as fetal wound healing. No a priori knowledge of what genes might be involved, or why, is necessary. This study indicates that downregulation of a gene that codes for a chaperonin subunit and upregulation of several other genes may be involved in the striking scarless character of wound healing in the mammalian fetus. Results suggest the hypothesis that downregulation of the CCT chaperonin in fetal wound may inhibit the formation of myofibroblasts, a cell type that correlates highly with scarring in postnatal wound healing, by preventing the folding of sufficient alpha-smooth muscle actin to form the stress fibers characteristic of these cells. PMID- 10726680 TI - Intralesional corticosteroid therapy in proliferating head and neck hemangiomas: a review of 155 cases. AB - PURPOSE: The purpose of this paper is to review the effect of intralesional corticosteroid therapy in the treatment of 155 head and neck hemangiomas. METHODS: In the past 10 years, we have treated 155 proliferating head and neck hemangiomas with intralesional corticosteroid injections. Three to 6 injections of triamcinolone acetonide (10 mg/mL) in monthly intervals were given. Using slides and chart review, the results were assessed 1 month after completion of the treatment. RESULTS: Eighty-five percent of the lesions showed greater than 50% reduction in volume. Varied treatment response was noted in different classes of hemangioma. Eighty percent of the superficial, 75% of the deep, and 60% of the combined hemangiomas show more than 50% reduction in volume. Further growth was not found after treatment. The postinjection complication rate was 6.4% in this series. There were 2 patients with cushingoid appearance, 5 with cutaneous atrophy, and 3 suffered from anaphylactic shock. We found that lesions showing less than 50% reduction in volume were located mostly in the perioral area. CONCLUSIONS: Intralesional corticosteroid injections are safe and effective in arresting hemangioma proliferation. Superficial hemangiomas yield the best results. PMID- 10726681 TI - Surgical and pathological basis for interval appendicectomy after resolution of appendicular mass in children. AB - BACKGROUND/PURPOSE: The role of appendicectomy after the resolution of appendicular mass is debatable. A study was conducted to evaluate whether surgical and pathological features of the excised appendices favor the operation in the quiescent period. METHODS: During a 60-month period, 59 patients were admitted in our unit with a diagnosis of appendicular mass and were treated initially with conservative management. Five patients failed to respond to this management and they were operated on immediately. RESULTS: Fifty-four patients recovered fully, and relatives were advised to bring them back to the hospital for appendicectomy on a scheduled date after 6 weeks. Fifteen of 54 (27.7%) patients did not return, but the other 7 came back because they had symptoms of recurrent appendicitis. The remaining 32 patients underwent appendicectomy as scheduled. The surgical findings and pathological features of excised appendices showed various abnormalities. CONCLUSIONS: Considering these features it could be concluded that delayed appendicectomy is unjustified in patients with absent appendix or with its lumen obliterated, whereas the other remaining patients who harbored normal, thickened, fibrotic, transected, stump, and appendix with chronic inflammation or containing fecal casts would benefit from operation. Because we have no method to date to distinguish between these variants "in situ" delayed appendicectomy seems beneficial for all the patients who respond well to the initial management of appendicular mass. PMID- 10726682 TI - A comparison of neonatal and adult multiorgan failure in a rat model. AB - BACKGROUND/PURPOSE: To establish a neonatal animal model of multiorgan failure (MOF) for the histological study of the sequence and severity of neonatal MOF in comparison to a model of adult MOF. METHODS: Neonatal and adult Sprague-Dawley rats received a single intraperitoneal injection of the inflammatory agent Zymosan. Rats were weighed; randomly killed on days 1 through 6; and heart, lung, liver, kidney, spleen, and ileocecum harvested for histological examination. RESULTS: Neonatal animals receiving Zymosan showed a significant increase in total body weight not seen in adults. The sequence and severity of MOF-induced organ damage were strikingly different in adult and neonatal animals. Mild lung damage was seen as early as day 1 in adult rats receiving Zymosan. This progressed to moderate damage by day 2 and severe damage by day 6. Lungs of neonatal rats receiving Zymosan showed only mild damage by day 4, which had progressed no further by day 6. Mortality rate was not significantly different between adult and neonatal animals receiving Zymosan. CONCLUSIONS: Zymosan can be used in a neonatal animal model to incite MOF. In the neonatal animal model of MOF there is (1) substantial early capillary leak as shown by increased body weight; (2) a unique progression of organ involvement-liver, kidney, lung compared with adult animals with MOF-lung, liver, kidney; and (3) relative sparing of the lung from injury. These findings are consistent with previous clinical observations of a difference in neonatal and adult MOF. PMID- 10726683 TI - Determination of imbalance between MMP-2 and TIMP-2 in human neuroblastoma by reverse-transcription polymerase chain reaction and its correlation with tumor progression. AB - BACKGROUND/PURPOSE: The matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs) have been implicated in tumor invasion and metastasis. Net matrix degradation and proteolysis depend on the critical local balance between MMPs/TIMP-2. We attempted to determine their expression balance and to evaluate its importance with tumor progression. METHODS: Expressions of MMP-2, MMP-9, and TIM P-2 mRNAs was quantified by reverse-transcription polymerase chain reaction (RT-PCR) in tumor tissues from 25 neuroblastoma patients. RESULTS: MMP 2, MMP-9, and TIMP-2 expression was observed in all the samples but with different trends. Increased expressions of MMP-2 mRNA was evident in advanced stages (Evans' stage III and IV; P = .02; unpaired ttest), and in patients who died of progressive disease (P = .0001). Whereas, the expressions of MMP-9 and TIMP-2 had no such significant association with clinical stages and prognosis. The ratio of MMP-2/TIMP-2 mRNAs was significantly higher in the advanced stages versus early stages (mean +/- SD = 1.66+/-0.65 and 1.11+/-0.34, respectively; P = .02) and in patients who died of progressive disease versus alive patients (mean = 2.13+/-0.78 and 1.21+/-0.36, respectively; P = .0006). CONCLUSIONS: Coexpression of MMPs and TIMP-2 in neuroblastoma indicates the need to evaluate their expression balance. Significantly higher expression of MMP-2 mRNA and increased ratio of MMP-2 and TIMP-2 mRNAs in advanced stages or patients who have died of progressive disease suggests an association between elevated MMP-2 expression and poor prognosis. To establish the role of enzyme to inhibitor mRNA ratio as a reliable predictor, cohort studies with significant number of cases may be carried out. PMID- 10726684 TI - A histopathological study of esophageal atresia and tracheoesophageal fistula. AB - BACKGROUND/PURPOSE: Histopathologic examination of the esophagus has not been reported often in esophageal atresia (EA) and tracheoesophageal fistula (TEF). Abnormal esophageal motility has been reported after successful repair of EA and TEF, although the exact etiology of this motility disorder remains unclear. Histological evaluation of the fistulous portion of the esophagus has been performed to document any abnormality that may be responsible for the abnormal motility. METHODS: Histopathological study of fistulous segment of esophageal atresia and distal TEF was conducted on 65 patients, which included autopsy material from 3 patients in whom the entire esophagus was examined by serial section. Special stains were administered to each specimen to look for tracheobronchial remnants and the arrangement of muscle fibers. Clinical evaluation was performed in 27 patients who were available for review 6 months to 9 years after successful surgical treatment. These patients were categorized into 3 groups according to their clinical status. RESULTS: These findings showed that the majority of these patients have tracheobronchial remnants in the esophagus in the form of abnormal mucous glands and ducts, abnormal mucin secretion, presence of cartilage, and a disorganized muscle coat. CONCLUSIONS: The presence of abnormal mucous glands and abnormal mucin secretion has not been highlighted before. Although tracheobronchial remnants and a disorganized muscle coat are common findings in the fistulous portion of the esophagus, these may not reflect the structure of the remaining esophagus. PMID- 10726685 TI - Renal enlargement in the fetus and newborn with congenital diaphragmatic hernia: a refuted hypothesis. AB - BACKGROUND/PURPOSE: Congenital diaphragmatic hernia (CDH) is one of the most frequent causes of neonatal death because of lung hypoplasia (LH). The literature mentions a relationship between renal and pulmonary development, with higher kidney weight in presence of LH. The aims of this study were to evaluate the relationship between lung and kidney weight and to test the hypothesis of renal enlargement in fetuses and newborns with CDH. METHODS: Body weight (BW), combined kidney weight (KW), and lung weight (LW) of 52 CDH cases were established; 52 morphologically normal fetuses or newborns, matched by BW, served as a control population. Comparisons were done by covariance analysis, and a P value of less than .05 was considered as significant. RESULTS: Excluding renal abnormalities, adjusted mean kidney weights were 22.0 g (+/-1.8 SE) in CDH cases and 20.5 g (+/ 1.5 SE) in controls (F = 1.05; P = .308). KW to BW ratio was lower in CDH cases than in controls (P = .023). LW was significantly lower in CDH cases than in controls. CONCLUSIONS: No significant difference between KW of CDH cases and controls could be observed. The current study provides enough evidence to reject the hypothesis of renal enlargement in cases of LH and CDH. PMID- 10726686 TI - Elevation of serum interleukin-18 levels and activation of Kupffer cells in biliary atresia. AB - BACKGROUND/PURPOSE: Interleukin-18 (IL-18)/interferon-gamma-inducing factor (IGIF) is a novel proinflammatory cytokine that can induce interferon gamma (IFN gamma). In addition, IL-18 enhances intracellular adhesion molecule-1 (ICAM-1) expression as well as Fas ligand (FasL) expression, and induces apoptosis in hepatic injury. The aim of this study was to clarify the potential role of IL-18 in the pathogenesis of the progressive inflammation and fibrosis in biliary atresia (BA). METHODS: Six children with BA before hepatic portoenterostomy (HPE), 13 with BA including 7 without jaundice and 6 with persistent jaundice after HPE, and 16 healthy controls were examined. Blood samples were obtained preoperatively from 6 patients, after HPE from 13, and after liver transplantation from 4. The IL-18 level was determined by an enzyme-linked immunosorbent assay (ELISA). Immunohistochemically, liver specimens from BA patients were studied using a monoclonal antibody to macrophage-associated antigen (CD68). RESULTS: IL-18 levels were elevated in the patients before HPE compared with those of the controls (349+/-54 pg/mL v. 138+/-13 pg/mL, P<.0001). After HPE, extremely high concentrations of IL-18 were observed in patients with persistent jaundice (532+/-95 pg/mL, P<.0001), and the IL-18 levels were significantly high even in the patients without jaundice (249+/-29 pg/mL, P<0.005). The high IL-18 level lasted for a long time even in the patients without jaundice after HPE. In contrast, the IL-18 levels immediately decreased after liver transplantation. Immunohistochemically, the number of CD68-positive Kupffer cells was significantly higher, and the size was larger in the livers of the patients than in the controls. The proliferation of CD68-positive cells was much more conspicuous in the liver specimens obtained during liver transplantation than in those at the time of HPE. CONCLUSIONS: Our findings showed elevation of serum IL-18 levels and activation of Kupffer cells in BA. IL 18 released from activated Kupffer cells might play an important role in the pathophysiology of the progressive inflammation and fibrosis in BA. Furthermore, IL-18 level may be related to the prognosis in patients with BA. PMID- 10726687 TI - Manganese deposits in patients with biliary atresia after hepatic porto enterostomy. AB - PURPOSE: The aim of this study was to determine if there is latent manganese toxicity in patients with biliary atresia. METHODS: Fifteen children with biliary atresia were examined postoperatively with regard to whole-blood manganese levels using brain magnetic resonance imaging (MRI) and I-123 iodoamphetamine (IMP) per rectal portal scintigraphy. RESULTS: Nine (60%) of the 15 had high whole-blood manganese levels (mean, 4.1 microg/dL; range, 1.2 to 9.6; normal, 0.5 to 2.5), and these 9 had hyperintense globus pallidus on T1-weighted images, with no corresponding signal change in T2 sequences. I-123 IMP per rectal portal scintigraphy was done for 13 patients to evaluate portosystemic shunt flow. 12 (92%) of these patients had an increased flow. Mean shunt ratio was estimated to be 41% (range, 0.6 to 98; normal, <5%). Encephalopathy was evident in only 1 patient. CONCLUSIONS: Some patients with biliary atresia in the postoperative period have manganese deposits in globus pallidus on T1-weighted images and high whole-blood manganese levels, possibly caused by increased portsystemic shunt, and a latent or subclinical encephalopathy is also present. PMID- 10726688 TI - Pexis of the great vessels for patients with tracheobronchomalacia in infancy. AB - BACKGROUND/PURPOSE: Aortopexy has been established as a surgical treatment for tracheobronchomalacia in infancy. However, the effects in patients with bronchial involvement remain controversial. The authors performed aortopexy and pexis of the pulmonary artery trunk (PApexy) for patients with tracheobronchomalacia. METHODS: Between 1992 and 1997, 14 infants with tracheobronchomalacia were treated. Patients ranged in age from 4 months to 7 years (median, 3.5 years). Using patients' records, intraoperative bronchofiberscopic results and clinical outcomes were analyzed retrospectively. RESULTS: Concerning tracheomalacia, aortopexy eliminated airway collapse as shown by intraoperative fiberscopic results and clinical outcome in 4 patients. Regarding tracheobronchomalacia, aortopexy eliminated collapse of the trachea and the right main bronchus in 3 patients but did not improve collapse of the lower half of the left main bronchus in 3 patients, which was eliminated by PApexy. Aortopexy eliminated airway collapse of the left main bronchus in 1 of 3 patients with bronchomalacia. PApexy eliminated collapse of the lower half of the left main bronchus in 1 patient. CONCLUSIONS: These results indicate that intraoperative bronchofiberscopic results were closely correlated to the clinical outcome, and suggest that aortopexy is helpful for eliminating collapse of the right main bronchus and the proximal half of the left main bronchus and that PApexy eliminates collapse of the distal half of the left main bronchus. PMID- 10726689 TI - The effects of intraamniotic human neonatal urine and meconium on the intestines of the chick embryo with gastroschisis. AB - BACKGROUND/PURPOSE: Urinary waste products in the amniotic fluid has been implicated as a cause of intestinal damage (ID) in gastroschisis based on the fact that fetus urinates physiologically into the amniotic cavity. However, experimental and clinical data suggest that intrauterine defecation is a physiological event, thus gastrointestinal waste products also may be responsible for ID in gastroschisis. An experimental study was performed to investigate the effects of intraamniotic human neonatal urine and diluted meconium on the intestines of chick embryo with gastroschisis. METHODS: Five-day-old fertilized chick eggs (Gallus domesticus) were used. Gastroschisis was created through amniotic cavity without opening the allantoic cavity. Sterile urine and meconium were obtained from newborn humans, and 1% meconium suspension was prepared. The eggs were divided in to 3 groups. In the first group, gastroschisis was created, and amniotic fluid was reinstilled without changing its composition (control group). Equal amounts of amniotic fluid and urine mixture was instilled into the amniotic cavity in second group (urine group) and 1% meconium suspension was instilled in similar fashion in the third group after creation of gastroschisis (meconium group). RESULTS: Histopathologic features of the intestines of the urine group did not differ from the intestines of the control group. The meconium group's bowel showed serosal thickening, inflammation, focal fibrin, and collagen deposits. Histopathologic changes of intestines induced by intraamniotic diluted meconium are consistent with the ones described for human gastroschisis specimens. CONCLUSION: Gastrointestinal waste products seem responsible for the ID in gastroschisis rather than urinary waste products. PMID- 10726690 TI - Quality of life for children with fecal incontinence after surgically corrected anorectal malformation. AB - PURPOSE: The aim of this study was to investigate influences of fecal incontinence on children's quality of life after surgically corrected anorectal malformation. METHODS: Seventy-one children with anorectal malformation underwent follow-up for 8 to 16 years postoperatively. They were divided into good, fair, and poor groups by assessment of anal function. The quality of life was investigated by self-structured disease impact questionnaires and Achenbach's Child Behavior Check List (CBCL). A quality-of-life scoring criteria was established to assess quality of life of the 71 children with operated anorectal malformation and controls. RESULTS: Owing to fecal incontinence, 12 (16.9%) children reported that there were problems in peer relationships; school absences happened in 13 (18.3%) children; 17 (23.9%) children had to restrict themselves to certain food. Based on CBCL, 13 (18.3%) children had behavior problems. The behavior problems in children with poor fecal continence (66.7%) were significantly higher than those of children with good fecal continence (8.6%). According to the quality-of-life scoring criteria, the scores of children with operated anorectal malformation (9.4+/-3.7) were significantly lower than those of the controls (11.2+/-0.9). The scores of children with poor fecal continence (4.8+/-1.4) were significantly lower than those of children with good fecal continence (10.9+/-1.2). CONCLUSIONS: The children with poor fecal continence have poor quality of life caused by fecal dysfunction. Attention should be paid to the rehabilitation of fecal continence after surgery, such as bowel training and biofeedback therapy. The somatic and psychological care and long-term follow up are necessary to improve the quality of life. PMID- 10726691 TI - The effects of serum depletion and dexamethasone on growth and differentiation of human neuroblastoma cell lines. AB - BACKGROUND/PURPOSE: Neuroblastoma is the most solid common extracranial malignancy in childhood. Despite multimodality treatment, high-risk disease continues to carry a poor prognosis. Glucocorticoids have been shown previously to induce differentiation in murine neuroblastoma cell lines, but no such effect has been documented in human neuroblastoma cells. Glucocorticoids are known to be active in the differentiation process of the neural crest. These studies describe the effects of dexamethasone on 6 human neuroblastoma cell lines. METHODS: Dexamethasone was added to cultured neuroblastoma cell lines (LA1-5S, LA1-15N, BE[2]S, BE[2]N, SH-EP-1, SH-SY5Y) maintained in media supplemented with either normal serum or charcoal-depleted serum. Proliferation assays were performed, and flow cytometry was used to assess alterations in cell cycle. Cells were closely monitored for morphological changes with serial phase-contrast microscopy. Immunohistochemistry (3F8, NF-1, TRK-A) of cultured cells was used to evaluate differentiation. Glucocorticoid receptor levels was assessed using immunoblotting. RESULTS: Dexamethasone decreased the rate of cellular proliferation in both standard and charcoal-depleted conditions. Flow cytometry showed a G1 accumulation. Increased expression of the differentiation-associated antigens was found in cells cultured in charcoal-depleted media, and a further augmentation was seen with the addition of dexamethasone. In standard media, dexamethasone had no detectable effect on the expression of these antigens. Glucocorticoid receptor expression was found to be comparable in all cell lines. CONCLUSIONS: Human neuroblastoma cells are sensitive to the differentiating effects of dexamethasone in an environment of charcoal-depleted serum. This phenomenon may be caused by the existence of growth and mitogenic factors in serum that are inhibiting differentiation. PMID- 10726692 TI - Ceramide induces apoptosis in neuroblastoma cell cultures resistant to CD95 (Fas/APO-1)-mediated apoptosis. AB - BACKGROUND/PURPOSE: Spontaneous tumor regression is a well-known characteristic in neuroblastomas. Because preliminary reports have shown that regression may be caused by apoptosis (a lethal cascade mediated by the CD95 (APO-1/Fas)-receptor), we analyzed the expression of CD95-receptors in 5 human neuroblastoma cell lines. Ceramides (known stimuli of apoptosis downstream from the CD95-receptor complex) also were used to test whether apoptosis would be induced in neuroblastoma cell cultures resistant to CD95-mediated programmed cell death. METHODS: The expression of the CD95-receptor was assessed by flow cytometry after incubation with either fluorisothiocyanate-conjugated (FITC) anti-CD95-antibody (UB2) or CD95-ligand for 16 hours. Apoptotic cell death was detected via microscopy, cell viability testing (MTT, 3-[4,5 dimehylthiazole-2-yl]-2,5 diphenyltetrazoliumbromide), and flow cytometric analysis after propidium iodide staining of the DNA. RESULTS: CD95-receptor expression was found on all neuroblastoma cell lines. Stimulation of the CD95-receptor of the malignant glioblastoma cell line LN229 (positive control) with either anti-CD95-antibody or CD95-ligand induced apoptosis. Apoptosis was not seen, however, in any of the neuroblastoma cell lines when the CD95-receptor was stimulated with anti-CD95 antibody or the CD95-ligand. Significant apoptosis was detected in all neuroblastoma cell lines after the addition of 25 micromol/L C2- and C6-ceramide. CONCLUSIONS: CD95-receptors are present on neuroblastoma cell lines, and these cells are resistant to apoptosis stimulated by anti-CD95-antibody or CD95-ligand. Apoptosis is induced, however, when these cells are treated with ceramide. A signal blockage downstream from the CD95-receptor complex and upstream of ceramide may account for this finding, and the "cellular FLICE inhibitory protein" (cFLIP) may be primarily responsible. PMID- 10726693 TI - Phenotype, intestinal morphology, and survival of homozygous and heterozygous endothelin B receptor--deficient (spotting lethal) rats. AB - BACKGROUND/PURPOSE: Spotting lethal (sl) rats, a model for Hirschsprung's disease, recently have been found to carry a deletion in the endothelin B (ET(B)) gene, causing functional lack of ET(B) receptors. The ET(B) receptor mediates, together with and in counterbalance to the ET(A) receptor, endothelin actions on vessels, cell proliferation, and migration. The authors investigated the effect of homozygosity (sI/sI) or heterozygosity (+/sl) on phenotype, intestinal morphology, and survival. METHODS: Weight, circumference, and serum albumin were measured. Histological tests of major organs and immunoperoxidase reaction for Peripherin, glial fibrillary acid protein (GFAP), and S-100 in small and large intestine were performed. Peripherin-immunostained sections of colon and jejunum were analyzed morphometrically. Screening for sepsis included search for enterocolitis, bacterial infection, endotoxin, and iNOS mRNA. RESULTS: Sl/sl rats died within 4 weeks of life, showing an early and a later death group. Serum albumin levels were decreased in sl/sl rats, whereas signs of sepsis were rare. Immunostaining uncovered alterations in nerve and glial cells in the whole gut of sl/sl rats, and to a subtle degree also in +/sl rats, which appear clinically normal. Morphometric quantification yielded statistically significant alterations in sl/sl rats only. No obvious abnormalities were found in other organs. CONCLUSIONS: Sl/sl rats die from malnutrition rather than sepsis, too early for ischemic complications to occur. Rats of the later death group are a suitable model for studying the ET8 receptor in vivo. Subtle abnormalities in the enteric nervous system of heterozygous rats underline the critical role of the "gene dose" for functional compensation. PMID- 10726694 TI - Prediction of outcome by computer-assisted analysis of lung area on the chest radiograph of infants with congenital diaphragmatic hernia. AB - BACKGROUND/PURPOSE: Pulmonary hypoplasia is a major cause of mortality and morbidity in infants with congenital diaphragmatic hernia (CDH). Pulmonary hypoplasia is characterized by low volume lungs, and affected infants are likely to have a low lung area on their chest radiograph. The authors assessed whether, in CDH infants, computer-assisted analysis of the chest radiograph lung area gave an accurate indication of lung volume, and if a low lung area was a better predictor of poor outcome (death or oxygen dependency at 28 days) than other test results. METHODS: Comparisons were made of the radiographic lung area derived by computer-assisted analysis and lung volume, assessed by measurement of functional residual capacity (FRC) on day 1 before surgical intervention and on the first postoperative day. Compliance was measured, and the maximum and modified ventilation indices and maximum Paco2 also was noted. Twenty-five CDH infants with a median gestational age of 38 weeks were studied; 18 had FRC measurements preoperatively. RESULTS: Both preoperatively and postoperatively, the lung areas and FRCs correlated significantly (r = 0.51, P<.05; r = 0.76, P<.02, respectively). Eleven infants had a poor outcome (5 infants died without an operation); that group preoperatively differed significantly from those with a good outcome with respect to having a lower compliance (P<.02) and higher maximum ventilation index (P<.01) and maximum modified ventilation index (P<.05). Only postoperatively did infants with a poor outcome versus good outcome have a significantly lower lung area (P<.05); they also had a lower increase in lung area preoperatively to postoperatively (P<.01). Receiver operator characteristic curves were constructed; comparison of the areas under the curves showed that preoperatively, a low compliance and high ventilation index were the best predictors of poor outcome. Postoperatively, a low lung area performed as well as the ventilation indices. CONCLUSION: Computer-assisted analysis of the lung area on the chest radiograph is useful in predicting outcome in CDH infants postoperatively but not preoperatively. PMID- 10726695 TI - Mathieu and 'V' incision sutured (MAVIS) results in a natural glanular meatus. AB - BACKGROUND: The Mathieu hypospadias repair for distal hypospadias produces a horizontal bucket handle meatus, which is cosmetically unacceptable. A new surgical technique, the Mathieu and "V" Incision Sutured (MAVIS), is described. METHODS: In 52 children, with a mean age of 16.5 months, distal hypospadias was repaired using the vascularized parameatal-based flip flap repair. A "V" incision at the apex of the flap, and suturing of each side of the V to the glans flaps followed by glanuloplasty achieved a natural vertical slit meatus. RESULTS: The mean period of follow-up has been 19.7 months. Fistula occurred in 2 patients (3.9%) and was repaired successfully. CONCLUSIONS: This technique of MAVIS provides a cosmetically acceptable natural vertical slit glanular meatus. A generous vascular pedicle on the flap covers the lateral suture lines to prevent fistulas. PMID- 10726696 TI - Gliomatosis peritonei as a complication of a ventriculoperitoneal shunt: case report and review of the literature. AB - Gliomatosis peritonei, the implantation of neuroglial tissue upon the peritoneal surfaces, is a rare event most often associated with solid or immature teratomas of the ovary in young girls. The authors report a case of a 10-month-old girl with a ventriculoperitoneal shunt (VPS) who presented with bilateral inguinal hernias. Herniorrhaphy was uneventful. Microscopic examination of the hernia sacs showed exuberant mesothelial hyperplasia containing multiple nests of differentiated glial tissue. Subsequent computed tomography and laparoscopy disclosed normal ovaries with no evidence of intraabdominal or pelvic abnormalities. Gliomatosis peritonei in this case was attributed to transport of glial tissue from the cerebrospinal fluid into the peritoneal cavity via the shunt. With the exclusion of an ovarian germ cell neoplasm and in the presence of a VPS, the clinical course with regard to the glial implants in these children is uneventful. If it is appreciated that gliomatosis peritonei may be a complication of a VPS, an extensive clinical evaluation generally is unnecessary. PMID- 10726697 TI - Nonoperative management of newborn splenic injury: a case report. AB - Traumatic injury of the spleen is rare in newborns. Nonoperative management of pediatric splenic injuries is now recognized as the treatment of choice, but there is scant experience with the problem in neonates. The authors report their experience with a neonatal splenic rupture, managed nonoperatively. PMID- 10726698 TI - Infarction of the choledochus, liver, gallbladder, and pancreas: a unique complication of the hemolytic uremic syndrome. AB - Hemolytic uremic syndrome (HUS) is associated with multiple nonrenal manifestations. A unique case is described of a 3-year-old boy who presented with a classic diarrheal prodrome followed by massive necrosis of the biliary tree and common bile duct, pancreas, and the left lobe of his liver. This complication of HUS has not been reported in the English-language literature. PMID- 10726699 TI - Acquired ileal atresia and spontaneous reconstitution of intestinal continuity in a premature infant with necrotizing enterocolitis. AB - An 849-g (26-week gestation) premature infant had pneumoperitoneum on the 20th day of life after having normal stools and accepting partial enteric alimentation. Percutaneous penrose drainage had to be performed on 2 consecutive days at 2 different sites (right lower quadrant, left lower quadrant), at which time she stabilized. Eleven days later, she started to pass stool, and oral feeding was begun (1 to 2 mL every 4 hours). Enteral intake could not be advanced because of repeated bouts of abdominal distension, despite having regular bowel motions. Gastrointestinal contrast radiographic investigation suggested a stricture of the ileum. At laparotomy (at age 2 months) ileal atresia with a "V" shaped defect in the mesentery was found. Surprisingly, intestinal continuity was established via an ileoileal fistula. After resection and anastomosis, she recovered fully. Mesenteric and enteric vascular ischemia (necrotizing enterocolitis) produced acquired ileal atresia-a rare occurrence. More rare is the reestablishment of intestinal continuity by fistulization. PMID- 10726700 TI - Splenic littoral cell angioma in an infant. AB - A 1-year-old girl presented with fever, asthenia, and splenomegaly with hypersplenism. Abdominal ultrasound scan and magnetic resonance imaging showed multiple nodular cystic masses in an enlarged spleen. The histological examination of the resected spleen showed a novel type of vascular tumor called littoral cell angioma. The histopathologic and immunohistochemical features of this rare lesion are described. Distinction from other splenic vascular tumors is stressed because the clinical behavior of this new entity seems to be benign. PMID- 10726701 TI - Partial nephrectomy in cystic partially differentiated nephroblastoma. AB - Cystic partially differentiated nephroblastoma (CPDN) is an uncommon renal neoplasm in children. It is now recognized as a tumor with low but definite malignant potential. The authors report a patient that was treated with partial nephrectomy and chemotherapy with successful outcome. The literature on CPDN is briefly discussed. PMID- 10726702 TI - Early diagnosis of fetal bladder outlet obstruction. AB - Prenatal ultrasonography has facilitated early diagnosis of in utero bladder outlet obstruction. This represents one of the earliest diagnoses of prune-belly syndrome and sheds light on the natural history of the bladder outlet obstruction in the fetus. PMID- 10726703 TI - Sacrococcygeal extraspinal ependymomas: the role of coccygectomy. AB - BACKGROUND: Ependymomas, the common glial tumors of the spinal cord, occur occasionally outside the central nervous system and are called exstraspinal ependymomas (EEP). EEPs are found primarily in sacrococcygeal region during childhood. The pathogenesis and the treatment of the sacrococcygeal (SC) ependymomas are still controversial. Therefore, we present our case with metaanalysis of other case reports to determine the optimal treatment modality for SC EEPs. METHODS: A metaanalysis of case reports of SC EEPs, including the current case, was conducted. Also all available case reports of EEPs, without age limit, were analyzed to determine the distribution of EEPs localization. RESULTS: EEPs usually are found in teratoma localizations such as the SC area, ovary, paraovarian structures, and medastinum. The distribution of EEPs localization differs with age. Local recurrence rate of EEPs after coccyx excision is zero, however, it increases to 71% when the coccyx was left behind. CONCLUSION: The identical clinical characteristics of the SC teratomas and EEPs imply that the SC EEPs may be monophasic teratomas as their ovarian counterparts are named. Coccyx excision is an important part of the surgical treatment of these tumors, with an apparent decrease in the recurrence rate. PMID- 10726704 TI - Diagnostic difficulties in neuronal intestinal dysplasia and segmental colitis. AB - An 11-month-old girl with a prolonged history of bloody, mucoid diarrhea is presented. Although the initial diagnosis given by the rectosigmoid biopsy obtained during laparotomy was neuronal intestinal dysplasia, accompanying findings including mixed inflammatory cell infiltration of the mucosa and submucosa with mucosal ulcerations suggested nonspecific colitis. The subsequent biopsy specimen that was obtained after performing colostomy and treating with broad-spectrum antibiotics and rectal irrigations showed improvement in the structure of ganglion cells and submucous and myenteric plexuses. Although the mucosal ulcerations and inflammatory reaction improved, the colonic stricture persisted, so the Duhamel procedure was performed, and the patient had an uneventful outcome. It is claimed that inflammatory disease of the rectosigmoid colon of unknown etiology and neuronal intestinal dysplasia have occurred together in the current case or that one disease might cause the other in time. PMID- 10726705 TI - Recurrent posttraumatic urethrovaginal fistula: a new application for ASTRA. AB - The authors describe the results of an application of the surgical technique called ASTRA (anterior sagittal transrectal approach) in the case of a 16-year old girl with a posttraumatic urethrovaginal fistula. The young girl with a posttraumatic urethrovaginal fistula had recurrence after 4 surgical attempts at closure performed by the vaginal approach before it was definitively corrected with the ASTRA. Two years after the ASTRA operation the patient is well with a complete restitution of function confirmed by a voiding cystourethrogram and urodynamic and rectal manometric tests. The successful recovery confirmed that ASTRA may be an excellent method of treating the developmental or acquired anomalies of the perineal region. PMID- 10726706 TI - Adenocarcinoma of the rectum with associated colorectal adenomatous polyps in tuberous sclerosis: a case report. AB - The authors present the case of a 17-year-old girl with tuberous sclerosis (TS) who presented with symptoms of intussusception. Although endoscopically diagnosed with multiple colonic polyps, presumed to be hamartomas, and an invasive rectal adenocarcinoma, postoperative pathology findings confirmed the rectal cancer and showed multiple colonic adenomas. Multiple colonic adenomatous polyps in a young girl with tuberous sclerosis is extremely rare. Furthermore, we believe that this is the first report of an invasive adenocarcinoma of the large intestine occurring in a patient with TS. PMID- 10726707 TI - Congenital adrenal hyperplasia associated with maternal pregnancy luteoma and the Antley-Bixler syndrome. AB - The authors report on a child with indifferent external genitalia consisting of severe micropenis with penile urethra leading to the tip of the glans and bilateral cryptorchidism. Diagnostic workup findings showed a female karyotype, homozygous 21-hydroxylase deficiency, and excessive testosterone exposure prenatally as a consequence of maternal pregnancy luteoma, altogether causing this unusual phenotype. In addition, the girl suffered from skeletal anomalies consistent with the diagnosis of Antley-Bixler syndrome. Our case shows that, although the association of congenital adrenal hyperplasia with other syndromes is rare, and even if other possible reasons for in utero virilization are present, complete diagnostic workup including karyotyping and hormonal status should be done in all patients with ambiguous genitalia, especially in cases of an unusual phenotype. The authors report on the diagnostic procedures and discuss the surgical approach in this particular case, never described before in the literature. PMID- 10726708 TI - The role of the 11beta-hydroxysteroid dehydrogenase type 2 in human hypertension. AB - The 11 beta-hydroxysteroid dehydrogenase type 2 (11 PHSD2) enzyme inactivates 11 betahydroxy steroids in sodium-transporting epithelia such as the kidney, thus protecting the non-selective mineralocorticoid receptor (MR) from occupation by cortisol in humans. Inhibition by xenobiotics such as liquorice or mutations in the HSD11 B2 gene, as occur in the rare monogenic hypertensive syndrome of apparent mineralocorticoid excess (AME), result in a compromised 11 betaHSD2 enzyme activity, which in turn leads to overstimulation of the MR by cortisol, sodium retention, hypokalaemia, low plasma renin and aldosterone concentrations, and hypertension. Whereas the first patients described with AME had a severe form of hypertension and metabolic derangements, with an increased urinary ratio of cortisol (THF+5alphaTHF) to cortisone (THE) metabolites, more subtle effects of mild 11 beta HSD2 deficiency on blood pressure have recently been observed. Hypertension with no other characteristic signs of AME was found in the heterozygous father of a child with AME, and we described a girl with a homozygous gene mutation resulting in only a slightly reduced 11 beta HSD2 activity causing 'essential' hypertension. Thus, depending on the degree of loss of enzyme activity, 11 beta HSD2 mutations can cause a spectrum of phenotypes ranging from severe, life-threatening hypertension in infancy to a milder form of the disease in adults. Patients with essential hypertension usually do not have overt signs of mineralocorticoid excess, but nevertheless show a positive correlation between blood pressure and serum sodium levels, or a negative correlation with potassium concentrations, suggesting a mineralocorticoid influence. Recent studies revealed a prolonged half-life of cortisol and an increased ratio of urinary cortisol to cortisone metabolites in some patients with essential hypertension. These abnormalities may be genetically determined. A genetic association of a HSD11 B2 flanking microsatellite and hypertension in black patients with end-stage renal disease has been reported. A recent analysis of a CA-repeat allele polymorphism in unselected patients with essential hypertension did not find a correlation between this marker and blood pressure. Since steroid hormones with mineralocorticoid action modulate renal sodium retention, one might hypothesize that genetic impairment of 11 beta HSD2 activity would be more prevalent in salt-sensitive as compared with salt-resistant subjects. Accordingly, we found a significant association between the polymorphic CA-microsatellite marker and salt-sensitivity. Moreover, the mean ratio of urinary cortisol to cortisone metabolites, as a measure for 11betaHSD2 activity, was markedly elevated in salt-sensitive subjects. These findings suggest that variants of the HSD11 B2 gene may contribute to the enhanced blood pressure response to salt in some humans. PMID- 10726709 TI - Hypertension and breast cancer risk in a 19-year follow-up study (the DOM cohort). Diagnostic investigation into mammarian cancer. AB - BACKGROUND: To investigate whether hypertension and the use of anti-hypertensive drugs are associated with breast cancer risk. METHODS: This was a prospective study of 11, 011 women living in Utrecht, the Netherlands, aged 50-65 years at enrolment in a breast cancer screening project (DOM cohort). Women attended screening rounds between 1974 and 1985 at which blood pressure was measured and information on drug use and breast cancer risk factors was ascertained. Since 1974 (median follow-up time 19 years), information on breast cancer occurrence and death has been registered. Hypertension was defined as a systolic blood pressure > 160 mmHg or a diastolic blood pressure > 95 mmHg or current use of drugs for the indication hypertension. Cox's regression analysis was used to investigate the association between hypertension (treated or untreated) and subsequent breast cancer risk. Analyses were adjusted for age, body mass index, height, parity, familial breast cancer, smoking and oral contraceptive use. RESULTS: A total of 523 women were diagnosed with breast cancer. Hypertensive women experienced a statistically significant increased breast cancer risk of 23% (age-adjusted hazard ratio (HRa) = 1.23; 95% confidence interval (CI) 1.01 1.49). After adjustment for all confounders, the increase was 14% (HR = 1.14; 95% CI 0.93-1.40). The decline in risk was mainly attributable to the effect of BMI. The risk was similar in treated (HR = 1.22; 95% CI 0.91-1.63) and untreated hypertensive women (HR = 1.13; 95% CI 0.91-1.40). CONCLUSION: These results do not support an association between hypertension and breast cancer, and if there is a link, it is likely to be positive and relatively small in size (+14%). This relation, if present, is not attributable to anti-hypertensive drugs, since the relation is also present in non-drug users. PMID- 10726710 TI - Blood pressure levels and obesity trends in hypertensive and normotensive Finnish population from 1982 to 1997. AB - OBJECTIVE: To assess the trends in blood pressure (BP) and in body mass index (BMI) in the hypertensive and normotensive population in Finland during 1982 1997. DESIGN: Four independent cross-sectional standardized population surveys were conducted in 1982, 1987, 1992 and 1997. SETTING: The provinces of North Karelia and Kuopio in eastern Finland and the region of Turku-Loimaa in southwestern Finland. PARTICIPANTS: Men and women aged 25-64 years were selected randomly from the national population register. The participants were classified into four groups according to their BP level and treatment status: normotensive, unaware hypertensive, aware but untreated hypertensive and treated hypertensive. The total number of participants was 24,083. MAIN OUTCOME MEASURES: The means of systolic BP (SBP), diastolic BP (DBP) and BMI, as well as the distribution of BMI among the four study groups were measured. RESULTS: Mean SBP decreased significantly in all groups. The fall in DBP was significant only in drug-treated hypertensive men and women (P< 0.001). Mean BMI increased significantly in all groups except in aware hypertensive women receiving no antihypertensive drug treatment The proportion of obese subjects (BMI > 30 kg/ m2) increased most in aware hypertensive men and in drug-treated hypertensive women. CONCLUSIONS: The prevalence of obesity has increased significantly in normotensive and particularly in hypertensive Finns during the past 15 years. There is an urgent need for more effective measures for weight reduction in obese hypertensive patients in primary healthcare, and for the prevention and control of obesity in the whole population. PMID- 10726711 TI - Evaluation of heterodimeric guanylyl cyclase genes as candidates for human hypertension. AB - OBJECTIVE: Both physiologic and pharmacological data have implicated the nitric oxide (NO) signaling cascade in the regulation of blood pressure in humans and its impairment in the pathogenesis of hypertension. In biological systems, the principal receptor for NO is NO-stimulated guanylyl cyclase. NO-stimulated guanylyl cyclases are obligate heterodimers (alpha/beta). The genes for guanylyl cyclase subunits alpha1, beta, and beta2 are likely candidates for causing hypertension in the Dahl rat as their expression is altered and their gene loci are closely linked to known quantitative trait loci for blood pressure in Dahl rat crosses. The objective of the current study was to test whether markers near guanylyl cyclase subunit genes were linked to hypertension in Caucasians. DESIGN: To test for linkage of genetic markers in or near the guanylyl cyclase genes to hypertension in Caucasians, a sample of 124 Utah hypertensive sib pairs was genotyped. RESULTS: Four highly polymorphic markers in or near the human guanylyl cyclase subunits homologous to the rat alpha1 (human chromosome 8), rat beta1 (human chromosome 4), and rat beta2 (human chromosome 13) genes showed no evidence of excess allele sharing in the set of hypertensive sibships. CONCLUSION: We conclude that the heterodimeric guanylyl cyclase subunit loci do not appear to be linked to hypertension in Caucasians. PMID- 10726712 TI - Evaluation of three polymorphisms in the promoter region of the angiotensin II type I receptor gene. AB - BACKGROUND: Angiotensin II induces vasoconstriction and growth via stimulation of the AT1 receptor. A genetic variant (+1166A/C) in the 3' untranslated region of this gene had been found to be associated with arterial hypertension, aortic stiffness and coronary artery disease. OBJECTIVE: In order to evaluate further the potential implications of the genetic variability of the AT1 gene we explored three newly characterized single nucleotide polymorphisms (SNPs) in its promoter in a Caucasian population-based sample (n = 623). One of these (-2228G/A) is in complete linkage disequilibrium with six additional SNPs in the region such that, indirectly, potential functional implications of these sites were assessed as well. For comparison, we genotyped the previously described +1166A/C variant RESULTS: The allele frequencies of the -2228G/A, -1424C/ G and -521 C/T SNPs were 0.82/0.18, 0.963/0.037 and 0.64/0.36, respectively. Statistical analysis by one factor ANOVA revealed no significant relationship of any allele, genotype or haplotype with age, sex, body mass index, heart rate, systolic or diastolic blood pressure, hypertension, the intake of antihypertensive medication or left ventricular mass. Likewise, renin, angiotensinogen, angiotensin-converting enzyme, aldosterone or atrial natriuretic peptide levels were not found to be associated with any of these SNPs. Surprisingly, the -2228 A allele was found to be overrepresented in subjects with diabetes mellitus (n = 25, P = 0.006). However, this result could not be confirmed when additional individuals with diabetes mellitus (n = 45) were analysed. A weak linkage disequilibrium was observed between the -2228 A allele and the +1166 C allele (chi2 1 3.1; P = 0.010). CONCLUSION: From the present data it is unlikely that any one of the nine newly characterized SNPs in the promoter region of AT1 gene is associated with arterial hypertension. PMID- 10726714 TI - Assessment of arterial and cardiopulmonary baroreflex gains from simultaneous recordings of spontaneous cardiovascular and respiratory variability. AB - OBJECTIVES: In usual models of cardiovascular regulation, arterial pressure drives RR interval through a simple baroreflex, and the influence of respiration is dismissed. We examined the applicability of a trivariate autoregressive model to obtain separate values of the gain of the arterial and non-arterial, i.e. cardiopulmonary, components of the lumped baroreflex, employing spontaneous RR interval, systolic arterial pressure and respiration variability. DESIGN: We studied 30 normal subjects (age 37 +/- 1 years), both at rest and during standing, a condition known to enhance sympathetic activity while reducing venous return. Electrocardiogram was obtained by telemetry, arterial pressure by Finapres and respiration with a piezoelectric respiratory belt Data were acquired with a PC and processed with an ad hoc Windows program. METHODS: We employed an additive and a linear multivariate approach to approximate overall gain of the arterial pressure-heart beat period baroreflex (alphalumped) and of its arterial (alphaart) and non-arterial, i.e. cardiopulmonary (alphacp), components, from continuous beat-by-beat series of RR interval, systolic arterial pressure variability and respiration, without using any non-physiological intervention. RESULTS: The overall baroreflex gain at rest (alphalumped = 23.7 +/- 3.4 ms/mmHg) was subdivided into arterial (alphaart = 5.2 +/- 1.0 ms/mmHg) and cardiopulmonary (alphacp = 18.5 +/- 3.2ms/mmHg) components. During active orthostatism, alphaluumped was diminished to 10.0 +/- 2.2 ms/ mmHg. In addition, standing selectively reduced alphacp to 4.8 +/- 1.3 ms/mmHg, while alphaart was not significantly changed. CONCLUSIONS: A trivariate autoregressive model, that considers explicitly the influence of respiration, can subdivide overall, lumped, arterial pressure-heart period baroreflex gain, into two separate components, alphaart and alphacp. Only the latter is reduced by active orthostatism. PMID- 10726713 TI - Receptor- and non-receptor-mediated clearance of big-endothelin and endothelin-1: differential effects of acute and chronic ETA receptor blockade. AB - AIMS: The aims of this study were to define and characterize the different mechanisms and sites of clearance of plasma endothelin-1 (ET-1) and big endothelin-1 (BigET-1) and evaluate possible effects of ETA versus combined ETA and ETB receptor blockade or endothelin converting enzyme (ECE) inhibition. METHODS: Time courses and sites of clearance were evaluated in Wistar-Kyoto rats after bolus injection of radiolabelled peptides into the carotid artery before or after treatment with LU1 35252 (ETA) and bosentan (ETA and ETB) as receptor antagonists or the ECE inhibitor phosphoramidon. RESULTS: The study shows that differential clearance of 125I-ET-1 and 125I-BigET-1 is mediated by distinct tissue-specific, receptor- and non-receptor-mediated mechanisms. Low levels of plasma ET-1 are rapidly cleared, mainly in the pulmonary circulation, through a low-capacity saturable ETB receptor-linked mechanism. In contrast, BigET-1 clearance is markedly slower, confined largely to liver and kidneys, is essentially non-receptor-mediated and is independent of converting enzyme activity. Acute inhibition of both ETA and ETB receptors with bosentan dramatically prolonged 125I-ET-1 plasma half-life and shifted tissue uptake from lung to liver and kidneys. Pulmonary clearance of 125I-ET-1 was decreased by chronic but not acute treatment with the specific ETA receptor antagonist LU135252. In contrast, 125I-Big-ET-1 clearance and tissue uptake were essentially unchanged by all treatments. CONCLUSIONS: Plasma levels and clearance studies on ET-1 and BigET-1 may provide differential information regarding pathological changes in their separate uptake mechanisms. Such data could have diagnostic or prognostic value in pulmonary, hepatic and renal pathophysiology or future therapeutic monitoring of treatment efficacy following administration of selective receptor antagonists. PMID- 10726715 TI - Inhibitory effect of insulin on bradykinin-induced venodilation. AB - BACKGROUND: Excessive insulin is one of the risk factors of hypertension and arteriosclerosis despite its vasodilative properties shown in recent studies. Although many vasoactive substances contribute and interact with each other in the development of hypertension, the interactions between insulin and other vasoactive substances have yet to be elucidated. OBJECTIVE: To assess the effect of insulin on the action of bradykinin. METHODS: The vasodilating effect of bradykinin was evaluated, with or without coadministration of insulin, in human dorsal hand veins of healthy volunteers. In cultured porcine aortic endothelial cells, the bradykinin-induced increase of intracellular calcium was also investigated before and after insulin administration. RESULTS: Insulin significantly attenuated bradykinin-induced increase in intracellular calcium and venodilation in cultured endothelial cells and human dorsal hand veins, respectively. CONCLUSIONS: These findings suggest that insulin attenuates the bradykinin-induced calcium elevation in endothelial cells and may decrease the production of vasodilative substances from endothelial cells, resulting in the reduction of vasodilation. This effect may contribute to the development of hypertension in patients with hyperinsulinaemia. PMID- 10726716 TI - Change in common carotid artery diameter, distensibility and compliance in subjects with a recent history of impaired glucose tolerance: a 3-year follow-up study. AB - OBJECTIVE: To assess the development of common carotid artery properties (diameter, distensibility and compliance) in a cohort of 140 subjects (mean age 65.8 years, SD 7.5 years) originally diagnosed as impaired glucose tolerant in a population-based study, and to explore determinants of changes observed. DESIGN: An observational, longitudinal study over a 3-year-period. METHODS: Vessel wall movement detector system based on ultrasonography, linear generalized estimating equations. RESULTS: Carotid artery diameter rose from 6.87-7.02 mm (+ 2.2%, P<0.001). Distensibility decreased from 11.8 to 10.9 x 10-3 kPa-1 (-8.3%, P=0.009). Compliance decreased from 0.44-043 mm2 kPa-1 (P=0.17). Changes in blood pressure level were negatively associated with changes in distensibility and compliance. Baseline fasting glucose levels were positively associated with changes in diameter, while fasting insulin levels were negatively associated with changes in distensibility and compliance in men, but not in women. CONCLUSIONS: In subjects with a recent history of impaired glucose tolerance, we observed an increase in carotid artery diameter and a decrease in distensibility. Change in blood pressure level and baseline fasting glucose and HbA1c levels were positively related to the increase in diameter. In men, but not in women, baseline fasting insulin levels were associated with an acceleration of these changes. PMID- 10726717 TI - Ambulatory blood pressure and neuroendocrine control after diet-assisted gastric restrictive surgery. AB - BACKGROUND: Long-term weight control after conventional diet is disappointing but may be improved when diet is assisted by gastric restrictive surgery (GRS). OBJECTIVE: To determine the effects of GRS on ambulatory blood pressure (ABP) and neuroendocrine BP control in 28 morbidly obese subjects. METHODS: A BP and heart rate were recorded every 10 min for 25 h before and 4 months after GRS. Effects of marked reductions in body weight on the renin-angiotensinaldosterone system, on plasma insulin and on sympathetic activity were also determined. RESULTS: Body mass index decreased from 43 +/- 1 to 34 +/- 1 kg/m2 and systolic (S) BP decreased by 7 +/- 2 mmHg during daytime (P=0.01) and by 8 +/- 3 mmHg during the night (P=0.02). Pulse pressure, a marker of reduced arterial compliance, decreased by 5 +/- 1 mmHg throughout the 24 h period (P < 0.001). Diastolic BP remained unchanged. Heart rate decreased more during the night (-13 +/- 2 bpm, P<0.0001) than during daytime (-5 +/- 2 bpm, P=0.03). Reductions in SBP were largest in subjects with highest initial BP values (r = -0.63, P<0.001) but were unrelated to weight loss. GRS decreased fasting glycaemia, plasma insulin, plasma C peptide and 24 h urine sodium (n=20) and noradrenaline (n=19) excretion (P<0.01). CONCLUSIONS: Diet-assisted GRS favourably affects neuroendocrine BP control in obese patients. Reductions in sodium intake, insulin levels and sympathetic tone combined with possible improvements in arterial compliance induce persistent 24 h reductions in SBP and pulse BP. Reductions in BP are largest in subjects with highest initial BP values and are unrelated to the amount of weight loss, thereby emphasizing the importance of even moderate reductions in weight on BP control. PMID- 10726718 TI - Doxazosin modifies Bcl-2 and Bax protein expression in the left ventricle of spontaneously hypertensive rats. AB - BACKGROUND: Increased apoptosis has recently been reported in the heart of spontaneously hypertensive rats (SHRs). OBJECTIVE: To investigate the molecular basis of apoptosis in the left ventricle of SHRs in terms of the expression of Bcl-2 protein (which protects from apoptosis) and Bax protein (which acts as an apoptotic promoter). In addition, we analysed the involvement of alpha adrenergic receptors in the left ventricular apoptosis of SHRs. METHODS: The study was performed in untreated SHRs (n=16) and SHRs that were orally treated with doxazosin (10 mg/kg body weight per day, for 15 days), a selective alpha1 receptor blocker (n=16). A group of Wistar-Kyoto (WKY) rats (n=16) was used as the control. RESULTS: The left ventricles of untreated SHRs showed a significant increase in Bcl-2 protein expression and a reduced presence of Bax protein. The ratio of Bcl-2:Bax in SHRs was higher than in WKY rats, suggesting an anti apoptotic state. Paradoxically, both the number of apoptotic cardiac cells and the cleavage of an 85-kDa fragment of the poly (ADP-ribose) polymerase (PARP), a marker of caspase-3 activity, were higher in the left ventricle of SHRs than in WKY rats, suggesting an apoptotic situation. Bax promotes cell apoptosis when it is bound to Bcl-2. We then determined the abundance of Bax-Bcl-2 complexes in the left ventricle of the two groups of animals. Bax-Bcl-2 complexes were more abundant in SHRs than WKY rats. In a second set of experiments, we analysed the role of alpha1-adrenergic blockade by doxazosin in the above-described mechanisms. Doxazosin treatment reduced the formation of Bax-Bcl-2 complexes in the left ventricle of SHRs, and this was accompanied by a decrease in the levels of 85kDa PARP and a reduction in apoptotic left ventricular cells. CONCLUSIONS: The present work suggests that the presence of Bax-Bcl-2 complexes in the left ventricle could be a more reliable marker of the apoptotic state than the determination of the absolute expression of Bcl-2 and Bax proteins. Moreover, the inhibition of alpha1 -adrenergic receptors by doxazosin decreased the abundance of BaxBcl-2 complexes and promoted a reduction of apoptosis in the left ventricle of SHRs. PMID- 10726719 TI - Fixed low-dose combination therapy in hypertension--a dose response study of perindopril and indapamide. AB - OBJECTIVE: To establish the optimal dose of the perindopril/indapamide combination (Per/Ind) in the treatment of mild or moderate hypertension. DESIGN: This was a randomized, double-blind, placebo-controlled, seven-way parallel group, dose-ranging study, set in multicenter, outpatient offices/clinics in Europe and Canada. PATIENTS: A total of 438 patients aged between 18 and 75 years whose supine diastolic blood pressure was between 95 and 114 mmHg were randomly assigned to an 8-week double-blind treatment with either placebo, Per 2/Ind 0.625, Per 4/Ind 1.25, Per 8/Ind 2.5, Per 0/Ind 1.25, Per 2/Ind 1.25 or Per 8/Ind 1.25 mg. MAIN OUTCOME MEASURES: Systolic and diastolic blood pressure measured in the clinic approximately 24 h after dosing. RESULTS: There was a linear dose response relationship (P<0.001) for doubling the dose of Per 2/Ind 0.625 mg up to Per 8/Ind 2.5 mg with a progressive fall in supine diastolic blood pressure (-9.3 to -15.0 mmHg). Combining 1.25 mg Ind with increasing doses of Per (0, 2, 4 and 8 mg) also showed a linear dose-response relationship (P<0.001), with supine diastolic blood pressure falling by -8.0 to -12.0 mmHg compared with a fall of 5.2 mmHg for the placebo group. Similar findings were noted for supine systolic blood pressure, standing blood pressure and ambulatory blood pressure. Hypokalemia was more common (9.7%) in the Per 8/Ind 2.5 mg group than in the groups receiving other doses (0-4.6%). CONCLUSION: The combinations of Per 2/Ind 0.625 mg and Per 4/Ind 1.25 mg were effective in reducing blood pressure without producing clinically important side effects. PMID- 10726720 TI - Long-term efficacy of a new, fixed, very-low-dose angiotensin-converting enzyme inhibitor/diuretic combination as first-line therapy in elderly hypertensive patients. AB - OBJECTIVE: To determine the long-term efficacy and safety of a fixed, very-low dose tablet combining one-half the standard dose of perindopril with one-quarter the standard dose of indapamide as first-line treatment in elderly patients. DESIGN: Double-blind, randomized, placebo-controlled study in an outpatient setting. PATIENTS AND INTERVENTIONS: Following a single-blind, placebo run-in period of 4 weeks, patients [65-85 years, with mild-to-moderate essential hypertension or isolated systolic hypertension (ISH)] were randomized to receive one tablet of perindopril 2 mg/indapamide 0.625 mg (Per/ Ind) (n=193) or placebo (n=190), daily for 12 weeks. After this first 12-week period, all patients on Per/Ind (n=138) and patients responding to placebo (n=61) were maintained on their previous regimen for a further 48 weeks. Patients in the placebo group whose blood pressure was not normalized, were switched to Per/Ind (n=60). MAIN OUTCOME MEASURE: The primary endpoint was the proportion of patients with blood pressure that normalized between weeks 0 and 60. RESULTS: After 1 year of treatment (intention-to-treat) supine systolic and diastolic blood pressure decreased by 23.0 +/- 15.3 mmHg and 13.3 +/- 94 mmHg with Per/Ind (n=253: 193 from randomized Per/Ind group and 60 from the placebo group switched at week 12). The mean decreases in systolic blood pressure were similar in essential hypertension and ISH (systolic blood pressure 23.2 versus 22.7 mmHg, respectively). Per/Ind treatment (n=253) achieved an initial normalization of blood pressure in 96.2% [95% confidence interval (CI) 93.6-98.9%; Kaplan-Meier estimate] of Per/Ind-treated patients; 79.8% (95% CI 74.1-85.5%) of these maintained a normalized blood pressure throughout the 1 -year follow-up. The incidence of adverse events was similarly low in the placebo and active therapy groups. Efficacy and safety results for the over 75 years subgroup were similar to those for the younger elderly subjects CONCLUSIONS: The fixed, very low-dose combination of perindopril 2 mg/indapamide 0.625 mg results in sustained blood pressure control when used as first line treatment of elderly hypertensive patients over 1-year, and is well-tolerated. PMID- 10726721 TI - Comparison of antihypertensive and metabolic effects of losartan and losartan in combination with hydrochlorothiazide--a randomized controlled trial. AB - INTRODUCTION: Losartan is an angiotensin II receptor blocker indicated for treatment of hypertension. It also inhibits platelet agreggation through blockade of thromboxane A2/ prostaglandin H2 receptors, and has a uricosuric effect We determined the effect on ambulatory blood pressure (ABP) of 100 mg losartan monotherapy (L100) versus 50 mg losartan/12.5 mg hydrochlorothiazide (HCTZ) combination therapy (L50H12.5C), in patients uncontrolled on 50 mg losartan. We also assessed the effects of losartan on platelet aggregation and serum urate at these clinically relevant doses. METHODS: This was a randomized, double-blind trial of L100 versus L50H12.5C, in moderate hypertensives (sitting diastolic blood pressure (DBP) >or = 95 mmHg and < 120 mmHg). After 4 weeks of placebo run in, patients received 50 mg losartan for 6 weeks; patients uncontrolled (sitting DBP > or = 95 mmHg) were randomized to L100 or L50H12.5C for a further 6 weeks. Platelet function was assessed by measuring percentage inhibition of platelet aggregation, and serum uric acid was also measured. RESULTS: Monotherapy with 50 mg losartan reduced ABP by 16.0/9.9 mmHg during the day and 9.8/5.5 mmHg at night However, 16 out of 24 (66%) patients had uncontrolled blood pressure on this treatment L50H12.5C further reduced daytime ABP by 10.7(10.7)/8.4(6.5) mmHg mean (SEM) compared with L100 (-5.3(9.7)/-2.3(4.8), P = 0.013). 50 mg losartan and L100 did not affect platelet function or uric acid levels beyond placebo values; treatment with L50H12.5C was associated with a significant rise in serum urate above levels obtained on 50 mg losartan (366.9(67.6) versus 331.6(65.0), P=0.006), to levels similar to placebo (358.8(80.9)). CONCLUSION: L50H12.5C is an effective antihypertensive regimen in patients with moderate hypertension that is uncontrolled on 50 mg losartan monotherapy, and is the preferred treatment option in these patients compared with increasing the dose of losartan. The additional benefit of losartan on platelet inhibition was not evident in our population at these doses; however, there was evidence to suggest that the uricosuric effects of losartan might ameliorate the uric acid retention effects of therapy with hydrochlorothiazide. PMID- 10726722 TI - Regarding the accuracy of generalized transfer functions for estimating central aortic blood pressure when calibrated non-invasively. PMID- 10726723 TI - Physiological relevance of nitric oxide-angiotensin II interplay in the cardiovascular system. PMID- 10726724 TI - The skin as a target for allergic diseases. PMID- 10726725 TI - Immunologic mechanisms in hypersensitivity reactions to metal ions: an overview. AB - Metal ions such as Ni2+, Co2+, Cu2+, or Cr3+ are haptens with a high immunogenic potential, as contact dermatitis caused by ionic metals occurs in about 10-15% of the human population. Since alloys containing Ni2+, Co2+, and Cr3+ are components of implants in replacement surgery, dentures, orthodontic wires, and various other devices, adverse reactions to metal ions create serious problems in practical medicine as incompatibility reactions to metal-containing biomaterials. On the other hand, contact dermatitis to metal ions such as Ni2+ is a well established model for studying the molecular mechanisms involved in the recognition of haptens by the immune system. Although many investigations have been performed to elucidate the molecular interactions causing contact hypersensitivity in man, many aspects remain to be clarified. This review will focus on the experimental data accumulated so far on the immunologic mechanisms responsible for the recognition of metal ions by T cells and eliciting adverse immune reactions causing contact dermatitis. PMID- 10726726 TI - Consensus statement on the treatment of allergic rhinitis. European Academy of Allergology and Clinical Immunology. PMID- 10726727 TI - Survey of recognition and utilization of guidelines for the diagnosis and management of bronchial asthma in Japan. AB - BACKGROUND: In Japan in 1993, the Japanese Society of Allergology (JSA) developed guidelines for diagnosis and management of asthma (JGL), which were based on the concept that asthma is a chronic inflammatory disorder of the airway. METHODS: This survey study was intended to investigate the recognition and utilization of JGL among physicians who had treated asthma. The survey comprised two methods: a quantitative mail survey and a qualitative door-to-door survey conducted by trained interviewers. RESULTS: In the mail survey, a total of 1028 physicians responded; 552 members of the JSA and 476 nonmembers. Ninety-four percent of JSA members were aware of adult asthma management guidelines, while 53% of nonmembers were aware of them. Although approximately half of the physicians, both members and nonmembers, found differences between the asthma management policies in JGL and their previous policies, most of them utilized JGL once they read it. In the qualitative door-to-door survey, 80-90% of physicians evaluated JGL as good after they read it. CONCLUSIONS: JGL was recognized and utilized by most JSA members, but only half of nonmember physicians were aware of JGL, although they utilized JGL after they read it. Further action to implement JGL among nonspecialist physicians is needed to improve management of asthma. PMID- 10726728 TI - Protein sequence analysis and mapping of IgE and IgG epitopes of an allergenic 98 kDa Dermatophagoides farinae paramyosin, Der f 11. AB - BACKGROUND: A 98-kDa mite paramyosin (Der f 11) from Dermatophagoides farinae (Df) is highly allergenic, and its cDNA (Df642) has been cloned. This paper describes the sequence characteristics and the mapping of the immunodominant human IgE and IgG epitopes of Der f 11. METHODS: The protein sequence analysis was performed with a combination of FASTA, GCG, and CLUSTAL W computing packages. The whole cDNA insert and its PCR-derived DNA fragments were generated and expressed in E. coli. These overlapping recombinant peptides (F1 to F5) were used for B-cell epitope mapping with 18 mite-allergic sera by dot immunoassays. RESULTS: Df642 cDNA encodes a partial sequence that contains the 2nd to 26th 28 residue repeats and lacks the N-terminus and the C-terminus. The sequence identity of Der f 11 with other known paramyosins is 34-60%. The dominant IgE epitopes are located in peptides F1 and F4, whereas the dominant IgG epitopes are located in peptides F1 and F2. These peptides are more reactive than whole rDf642. CONCLUSIONS: Mite paramyosin is very similar to other known paramyosins. The human IgE and IgG epitopes are scattered throughout the entire molecule. Data also indicate the presence of unique IgE and IgG epitopes in Der f 11. PMID- 10726729 TI - Quantitative, rather than qualitative, differences in CD69 upregulation in human blood eosinophils upon activation with selected stimuli. AB - BACKGROUND: The study aimed to investigate whether CD69 expression on granulocytes is subject to specific regulation by inflammatory mediators, and, if so, to identify these factors in relation to eosinophil activity markers such as the EG2 epitope and ECP release. METHODS: Peripheral blood leukocytes from healthy donors were used. The surface and intracellular distribution of CD69 was investigated with a whole-blood cell-membrane permeabilization technique, the FOG method, and flow cytometry. In vitro stimulation was performed with GM-CSF, IL-5, IL-5 plus eotaxin, LPS, and fMLP. RESULTS: A preformed intracellular pool of CD69 was demonstrated in both eosinophils and neutrophils, but not in monocytes. Almost no resting eosinophils, neutrophils, or monocytes expressed CD69 on the cell surface. However, in vitro stimulation with selected stimuli increased the proportion of CD69-positive eosinophils to various extents, with GM-CSF being the most and fMLP the least efficient stimulus. The neutrophils did not respond under these conditions. Increased expression of the EG2 epitope and initiation of degranulation preceded CD69 upregulation. CONCLUSIONS: Eosinophils and neutrophils from healthy donors have a preformed intracellular pool of CD69, which is mobilized on the cell surface on eosinophils, but not on neutrophils, to various extents by selected stimuli. Monocytes, however, do not have a preformed intracellular pool of CD69. Our data indicate that a kinetic order exists among the EG2 expression, the degranulation process, and CD69 upregulation. Due to a quantitative, rather then a qualitative, upregulation of CD69 by stimuli associated with both allergic and bacterial inflammation, CD69 may be a potential activity marker of clinical value. PMID- 10726730 TI - Asthma in the desert: spectrum of the sensitizing aeroallergens. AB - BACKGROUND: Bronchial asthma is common in Kuwait, a desert country, but the sensitizing allergens are uncertain. This study investigated the sensitizing allergens in Kuwaiti patients with extrinsic asthma. METHODS: A total of 553 asthmatics (male:female ratio: 1.4; mean age: 31.7 years [range 3-76 years]) and 112 matched controls were studied. Sera from all patients/subjects were tested by the CAP-RAST method for specific IgE to 14 locally relevant inhalant allergens. RESULTS: Specific IgE to at least one allergen was detected in 87.2% of the patients compared with 24.1% of controls. Among the confirmed extrinsic asthmatics, the sensitization rates for the allergen groups were as follows: pollens (87.1%), house dust (76.1%), and molds (30.3%). The three most prevalent sensitizing pollens were from Chenopodium (70.7%), Bermuda grass (62.9%), and Prosopis (62.7%), all of which are horticultural plants imported for the purpose of "greening" the desert. For all allergens, except the molds, the prevalence rate was higher in males than females, but age had only a weak effect. Severe asthma occurred significantly more frequently among mold-sensitized patients. CONCLUSIONS: These results show that even in a desert environment, pollens and house-dust allergens may be important sensitizing allergens. They also illustrate how practices that "green" the desert can affect public health. PMID- 10726731 TI - Indoor air pollutants in schools: nasal patency and biomarkers in nasal lavage. AB - BACKGROUND: There is growing concern about the respiratory health aspects of the indoor air quality in schools. METHODS: A standardized investigation, including nasal lavage (NAL), measurement of the nasal cavity by acoustic rhinometry, and hygienic measurements of airborne pollutants, was performed in classrooms, outside the pollen season. All 279 school personnel working in the main buildings of 12 randomly selected primary schools in an urban community in central Sweden (Uppsala) were invited to enroll in the study; 234 (84%) participated. Eosinophil cationic protein (ECP), myeloperoxidase (MPO), lysozyme, and albumin were analyzed in NAL fluid. Crude statistical analysis, as well as multiple regression analysis, was performed, controlling for room temperature, age, sex, current smoking, and a history of atopy. RESULTS: Most classrooms (83%) did not meet the Swedish ventilation standards. A lower degree of nasal patency was found at higher concentrations of respirable dust, nitrogen dioxide (NO2), formaldehyde, and total molds, and in the presence of Aspergillus spp. in the classroom air. The most consistent findings were observed for formaldehyde, NO2, and Aspergillus spp., related to both decreased nasal patency and increase of ECP and lysozyme in NAL. The presence of yeast was associated with an increase of ECP and lysozyme in NAL, but was not related to nasal patency. CONCLUSIONS: Ventilation flow was below current hygienic standards in the classrooms. Air pollutants in the classroom air may influence nasal patency and inflammatory response in the nasal mucosa. PMID- 10726732 TI - Study of the Th1/Th2 balance, including IL-10 production, in cultures of peripheral blood mononuclear cells from birch-pollen-allergic patients. AB - BACKGROUND: Excessive production of interleukin (IL)-4, IL-5, IL-10, and IL-13 is thought to be important in the development of allergy and asthma. The objective of this investigation was to study Th1/Th2-like cytokine profiles in vitro in seven patients allergic to birch pollen and six nonallergic controls during the birch-pollen season. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated and cultured with birch-pollen extract (BPE) or tetanus toxoid (TT) for 7 days, harvested, and restimulated with the mitogens phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA) for 24 h. Cytokine production was determined by ELISA, and logarithmic cytokine ratios were compared between the two groups and between the antigens. RESULTS: In the allergic group, the cultures prestimulated with BPE had a more Th2-like cytokine response than the TT prestimulated cultures; i.e., lower IFN-gamma and higher IL-10 production (P<0.05), as well as higher IL-5/IFN-gamma and IL-13/ IFN-gamma ratios (P<0.05). There were also significantly higher IL-4/IFN-gamma (P<0.005) and IL-5/IFN-gamma (P<0.05) ratios in BPE-stimulated cultures in the allergic group than in the control group. The IL-4 and IL-13 production in vitro correlated with the specific serum IgE levels. CONCLUSIONS: BPE stimulation induces a Th2-like cytokine response by PBMC isolated during the pollen season from birch-pollen allergic patients, indicating a Th2-type immune response to birch pollen in vivo. PMID- 10726733 TI - Indoor viable mold spores--a comparison between two cities, Erfurt (eastern Germany) and Hamburg (western Germany). AB - BACKGROUND: In eastern Germany, the prevalence of allergies is lower than in western Germany for both children and adults. Several reasons for this fact have been discussed, although it is still not completely understood. One purpose of the epidemiologic study "Indoor and genetic factors in asthma and allergy" (INGA) is to compare exposure to mold spores in two German cities. Therefore, 405 homes in Erfurt (east) and Hamburg (west) were visited twice by trained investigators between June 1995 and May 1997. METHODS: Samples of settled dust were taken by vacuuming from the carpet in the living room. Sieved house dust was diluted and plated on DG18 agar. The analyses were carried out in duplicate in the same laboratory. RESULTS: No significant difference could be shown for the total and for single genera (Alternaria, Aspergillus, Cladosporium, and Penicillium) in concentration of spores of viable fungi in settled house dust between Erfurt and Hamburg. Seasonal variation of the mold picture, with highest values in August, could be identified both indoors and outdoors. CONCLUSIONS: Because outdoor concentration is the main influence on indoor concentration of mold spores from June to October, we recommend sampling from November to May to evaluate exposure to indoor mold spores. PMID- 10726734 TI - Evaluation of IgE antibodies to recombinant pollen allergens (Phl p 1, Phl p 2, and Phl p 5) in a random sample of patients with specific IgE to Phleum pratense. AB - BACKGROUND: We measured specific IgE levels against the recombinant allergens (RAs) rPhl p 1, rPhl p 2, and rPhl p 5 in patients allergic to grass pollen, and examined the existence of different patterns of IgE production to RAs. The seasonal variations of IgE levels to rPhl p 1, rPhl p 2, and rPhl p 5 were considered, too. METHODS: Blood was taken from 276 consecutive patients with allergy to grass pollen diagnosed by patient history and skin prick testing. Total and specific serum IgE was measured by the immunoenzymatic CAP FEIA System. Eosinophil cationic protein (ECP) and myeloperoxidase (MPO) were assessed by radioimmunoassay according to the instructions of the manufacturers. RESULTS: We observed eight different patterns of IgE production to rPhl p 1, rPhl p 2, and rPhl p 5 in patients with specific IgE to timothy grass. A significant difference between the values of IgE levels to timothy and the sum of each level of specific IgE to individual RAs was found (P = 0.039, Wilcoxon matched pairs test) in the whole population (n = 276 subjects). In four subgroups of patients, the sum of each level of specific IgE to individual RAs was equal to the levels of specific IgE to timothy grass extract. In one subgroup, the sum of IgE to RAs was lower than the levels of IgE to the natural counterpart (P = 0.013). A lack of subjects in two subgroups did not permit comparison at all. Finally, three subjects with specific IgE to timothy did not show specific IgE to RAs. Out of 276 patients, blood was taken from two different groups of subjects at different time points: November-January and May-July, respectively. The median values were as follows: total IgE = 139 kU/l, IgE to timothy = 10.2 kUA/l; IgE to rPhl p 1 = 3.6 kUA/l, to rPhl p 2 = <0.35 kUA/l, and to rPhl p 5 = 1.1 kUA/l; ECP = 8.25 microg/l; MPO = 303.08 microg/l (before exposure to grass pollen); total IgE = 159 kU/l, IgE to timothy = 57.2 kUA/l; IgE to rPhl p 1 = 22.1 kUA/l, to rPhl p 2 = 5.9 kUA/l, and to rPhl p 5 = 3.9 kUA/l; ECP = 16.21 microg/l; MPO = 413.09 microg/l (during the pollen season). There were significant variations of specific IgE levels between the patients exposed to pollen and the unexposed patients. Moreover, there were statistical differences in the IgE, ECP, and MPO levels in sera before and during the pollen season P<0.035, P<0.017, and P<0.0062, respectively. CONCLUSIONS: The results suggest that RAs allow establishment of the patient's IgE-reactivity profile, encourage future research, and encourage manufacturers to produce further RAs for precise diagnosis and substantially improved immunotherapy injection of only those allergens against which significant amounts of specific IgE are produced. PMID- 10726735 TI - Effect of current exposure to Der p 1 on asthma symptoms, airway inflammation, and bronchial hyperresponsiveness in mite-allergic asthmatics. AB - The existence of a dose-response relationship between indoor allergen exposure and sensitization has been widely described, but the effect of allergen exposure on asthma activity (symptoms, bronchial hyperresponsiveness [BHR], and inflammation) is not clear. Our aim was to determine the existence of an association among current exposure to mite allergens and symptoms, BHR, and airway inflammation assessed in blood and sputum from asthmatic patients sensitized to Dermatophagoides pteronyssinus. We selected 31 mild and recently diagnosed (12-24 months) asthma patients sensitized to D. pteronyssinus. Allergenic exposure (Der p 1, Der 2) was assessed by a commercial assay based on monoclonal antibodies (mAb), carried out on the dust samples collected from patients' beds in a standardized way. Patients completed an asthma symptom questionnaire and underwent skin tests, methacholine bronchial challenge, and sputum induction. Sputum cell profile was analyzed and eosinophil cationic protein (ECP), tryptase, albumin, and interleukin(IL)-5 levels were quantified in sputum supernatant. Total eosinophil numbers and ECP levels were measured in blood samples. Most patients were exposed to Der p 1 levels under 2 microg/g of dust. Der p 1 exposure was higher among the subjects with positive sputum tryptase detection (P = 0.020). Der p 1 levels showed a trend toward correlation with asthma symptoms (P = 0.066, r = 0.36) and correlated with sputum tryptase levels (P = 0.032, r = 0.42). No relationship between BHR, eosinophilic inflammation, and allergenic exposure was found. Our results suggest that asthma symptoms and lung mast-cell activation are at least partially dependent on current allergen exposure. The lack of correlation between mite exposure, eosinophilic inflammation, and BHR supports the role of other factors that enhance the immunologic response initiated by allergen, increasing the activity of asthma. PMID- 10726736 TI - Severe hypersensitivity syndrome to lamotrigine confirmed by lymphocyte stimulation in vitro. AB - BACKGROUND: The anticonvulsant lamotrigine has been associated with severe adverse events such as the hypersensitivity syndrome and severe bullous reactions. So far, specific immunologic tests have rarely been performed to demonstrate specific sensitization. METHODS: A 36-year-old man suffering from epilepsy was concomitantly treated with high doses of sodium valproate and lamotrigine. About 1 month later, a severe hypersensitivity syndrome occurred affecting skin, lymph nodes, and liver. Three months later, skin tests and lymphocyte stimulation tests with anticonvulsants were performed. RESULTS: Skin tests were negative with all drugs; lymphocyte stimulation tests were twice positive with lamotrigine. Later re-exposure to sodium valproate was tolerated. CONCLUSIONS: Lamotrigine may elicit a severe hypersensitivity syndrome. Particularly high initial doses and concomitant treatment with sodium valproate increase the risk of cutaneous reactions. The lymphocyte stimulation test was used to identify the culprit drug. Lymphocyte sensitization to the drug or a metabolite may be involved in the pathogenesis. PMID- 10726737 TI - Very late reactions to allergen-specific immunotherapy caused by physical exercise. PMID- 10726738 TI - Allergy to anis. PMID- 10726739 TI - Latex allergy in a glove plant. PMID- 10726740 TI - Anaphylaxis to paramethasone with tolerance to other corticosteroids. PMID- 10726741 TI - Contact urticaria due to cyclopentolate hydrochloride. PMID- 10726742 TI - Anaphylaxis to sesame. PMID- 10726743 TI - Lettuce allergy. PMID- 10726744 TI - Hazards in the use of diphencyprone. PMID- 10726745 TI - Successful treatment of delayed pressure urticaria with montelukast. PMID- 10726746 TI - Electrophoretic mobility of concentrated spheres with a charge-regulated surface. AB - The electrophoretic behavior of a concentrated spherical colloidal particle is modeled theoretically under the Debye-Huckel condition. The surface of a particle contains dissociable functional groups, the dissociation of which yields negative fixed charges. The model derived is applicable to an arbitrarily thick double layer. We show that the absolute surface potential decreases with the increase in kappa(a); kappa and a are the reciprocal Debye length and the radius of a particle, respectively. Moreover, the variation of the absolute electrophoretic mobility as a function of kappa(a) has a maximum. PMID- 10726747 TI - Electrophoretic properties of dodecyltrimethylammonium bromide micelles in KBr solution. AB - Charge in ionic micelles determines the trends of their stability and their practical applications. Charge can be calculated from zeta potential (zeta) measurements, which, in turn, can be obtained by Doppler microelectrophoresis. In this study, the electrophoretic properties of dodecyltrimethylammonium bromide (DTAB) in KBr aqueous solution (0-6 mM) were determined by Doppler microelectrophoresis. At very low surfactant concentrations (up to 6 mM), zeta potential was quite constant and due to the ionized monomers (DTA+). Above 6 mM, zeta potential increased to a maximum at surfactant concentrations still below the critical micellar concentration (CMC). This increase could be explained by a formation of nonmicellar aggregates of DTAB. Then, above the CMC, zeta potential underwent an abrupt reduction, which was dependent qualitatively and quantitatively on KBr concentration, and which could be due to an increase of the number of counterions adsorbed on the micelle surface. Calculation of effective micellar charge from zeta potential gave the surface charge density. Comparing this value with the theoretical, obtained from geometrical considerations, a fraction of 0.29 of charged micellar headgroups was obtained when DTAB was in aqueous solution, which is consistent with the value obtained by conductivity measurements. PMID- 10726748 TI - Ultrasensitive fluorescence protein detection in isoelectric focusing gels using a ruthenium metal chelate stain. AB - SYPRO Ruby IEF Protein Gel Stain is an ultrasensitive, luminescent stain optimized for the analysis of protein in isoelectric focusing gels. Proteins are stained in a ruthenium-containing metal complex overnight and then rinsed in distilled water for 2 h. Stained proteins can be excited by ultraviolet light of about 302 nm (UV-B transilluminator) or with visible light of about 470 nm. Fluorescence emission of the dye is maximal at approximately 610 nm. The sensitivity of the SYPRO Ruby IEF protein gel stain is superior to colloidal Coomassie blue stain and the highest sensitivity silver staining procedures available. The SYPRO Ruby IEF protein gel stain is suitable for staining proteins in nondenaturing or denaturing carrier ampholyte isoelectric focusing and immobilized pH gradient gel electrophoresis. The stain is compatible with N,N' methylenebisacrylamide or piperazine diacylamide cross-linked polyacrylamide gels as well as with agarose gels and high tensile strength Duracryl gels. The stain does not contain extraneous chemicals (formaldehyde, glutaraldehyde, Tween-20) that frequently interfere with peptide identification in mass spectrometry. Successful identification of stained proteins by peptide mass profiling is demonstrated. PMID- 10726749 TI - Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution. AB - SYPRO Tangerine stain is an environmentally benign alternative to conventional protein stains that does not require solvents such as methanol or acetic acid for effective protein visualization. Instead, proteins can be stained in a wide range of buffers, including phosphate-buffered saline or simply 150 mM NaCl using an easy, one-step procedure that does not require destaining. Stained proteins can be excited by ultraviolet light of about 300 nm or with visible light of about 490 nm. The fluorescence emission maximum of the dye is approximately 640 nm. Noncovalent binding of SYPRO Tangerine dye is mediated by sodium dodecyl sulfate (SDS) and to a lesser extent by hydrophobic amino acid residues in proteins. This is in stark contrast to acidic silver nitrate staining, which interacts predominantly with lysine residues or Coomassie Blue R, which in turn interacts primarily with arginine and lysine residues. The sensitivity of SYPRO Tangerine stain is similar to that of the SYPRO Red and SYPRO Orange stains - about 4-10 ng per protein band. This detection sensitivity is comparable to colloidal Coomassie blue staining and rapid silver staining procedures. Since proteins stained with SYPRO Tangerine dye are not fixed, they can easily be eluted from gels or utilized in zymographic assays, provided that SDS does not inactivate the protein of interest. This is demonstrated with in-gel detection of rabbit liver esterase activity using alpha-naphthyl acetate and Fast Blue BB dye as well as Escherichia coli beta-glucuronidase activity using ELF-97 beta-D-glucuronide. The dye is also suitable for staining proteins in gels prior to their transfer to membranes by electroblotting. Gentle staining conditions are expected to improve protein recovery after electroelution and to reduce the potential for artifactual protein modifications such as the alkylation of lysine and esterification of glutamate residues, which complicate interpretation of peptide fragment profiles generated by mass spectrometry. PMID- 10726750 TI - Identification of phage antibodies toward the Werner protein by selection on Western blots. AB - A procedure was established for selecting phage antibodies (phage-abs) from phage displayed antibody repertoires by panning against proteins, separated by sodium dodecyl phosphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes (Western blots). This immobilization strategy is applicable for secondary rounds of panning in selections against semipurified proteins, and directs the selection toward antibodies suitable as immunochemical reagents in Western blots. In model experiments, enrichment factors as high as 1.9x10(5) were obtained in a single round of panning. Furthermore, we demonstrate the application of this approach by selection of phage-abs recognizing the human Werner protein, which is defective in a premature aging syndrome. PMID- 10726752 TI - A method to produce Ponceau replicas from blots: application for Western analysis. AB - We describe a fast and simple method to produce print-quality like Ponceau replicas from blots. These have many applications for Western analysis, primarily to alleviate localization of antigens in complex protein patterns generated by two-dimensional electrophoresis. PMID- 10726751 TI - Quantitative analysis of relative protein contents by Western blotting: comparison of three members of the dystrophin-glycoprotein complex in slow and fast rat skeletal muscle. AB - We have developed a method for accurate quantitative analysis and statistical comparison of the relative contents of the dystrophin-glycoprotein complex (DGC) in skeletal muscle. This method was applied to compare DGC contents in slow (soleus) and in fast (extensor digitorum longus, EDL) rat skeletal muscles. The quantitative analysis combines a modified bicinchoninic acid (BCA) assay with Western blotting and enhanced chemiluminescence (ECL). This combination allows the use of high levels of detergents and reducing reagents essential for extracting DGC. In addition, the evaluation of the total amount of proteins in each sample makes it possible to have a reference and to accurately compare relative protein levels without using a specific standard. With a large gradient gel, we could concomitantly compare two groups (n = 9) and quantify all protein contents differing highly in their molecular masses (from 35 kDa to 427 kDa). Each experiment was triplicated and normalized; the two muscles were compared using the Mann-Whitney test (P<0.001) to establish their protein content. The DGC relative levels for the slow muscle soleus and the fast muscle EDL differed significantly: dystrophin, beta-dystroglycan, and gamma-sarcoglycan levels were 130%, 110% and 120% higher in the soleus, respectively. The differences observed in the expression level of cytoskeletal associated protein (dystrophin) and transmembranous anchorage components may correspond to a physiological response of the muscle fibers to duration, magnitude, and frequency of the imposed mechanical loading. PMID- 10726753 TI - High-efficiency passive elution of bacterial lipopolysaccharides from polyacrylamide gels. AB - We recently described a method for recovering polyacrylamide-gel-separated bacterial lipopolysaccharides (LPS) based on the sensitive on-gel LPS detection (1-10 ng/band) with zinc-imidazole followed by passive elution from 32 microm average size gel microparticles into water. With this procedure, the recovery of rough- or semismooth-type LPS after 3 h elution is about 70-80%, while that of smooth LPS is only about 10%. Here we evaluated whether a simple replacement of water with other eluents would enhance the passive diffusion of LPS. We found that solutions of the detergents sodium dodecyl sulfate (SDS), sodium deoxycholate (DOC) and Triton X-100, or mixtures of the organic solvents acetonitrile and triethylamine and water, increased the recovery of a smooth LPS band from Vibrio cholerae O1 in a concentration-dependent manner. Furthermore, a quantitative recovery of rough or smooth LPS from V. cholerae O1, Escherichia coli O111:B4, E. coli K-235, or Serratia marcescens was feasible in 1% SDS or DOC after 3 h or in 5% triethylamine after only 2 min. A simple dilution of SDS or DOC or evaporation of triethylamine rendered the eluted LPS preparations compatible with biochemical activity determination, as tested by Limulus amebocyte lysate assay. Thus, this improved micropurification method may be a suitable interface between analytical gel electrophoresis and further characterization or use of LPS. PMID- 10726754 TI - Nuclear integrations of mitochondrial DNA in primates: inference of associated mutational events. AB - To infer the possible mutational events taking place along the interorganellar transfer of genetic material from mitochondria to the nucleus, four integrations of mitochondrial DNA (mtDNA) in the human genome were characterized together with their flanking nuclear sequences. By determining their presence/absence status in different primate species, these integrations were inferred to have occurred on the lineages leading to catarrhines (Old World monkeys and hominoids), to hominoids and to humans, respectively. In case of a polymorphic state, with respect to its presence in a certain species, each preintegration sequence was either cloned in the same species or in a primate taxon that branched off before the transfer of the mtDNA to the nucleus took place. For the four mtDNA integrations presented here, random mobilization of the mtDNA and differing mechanisms for generating free ends in the nuclear target sequences can be inferred. Additionally, no common sequence features at the preintegration sites could be observed for these integrations. Moreover, the comparisons of the sites before and after integration suggest different ways of integration. Thus, mtDNA integrations represent unique molecular recombinations in the evolutionary history and can, according to their presence/absence status in different species, help to determine the branching order in phylogenetic trees. PMID- 10726755 TI - Rapid identification of the ABO genotypes by their single-stand conformation polymorphism. AB - The ABO locus on chromosome 9 contains many more alleles than are currently used routinely in forensic science. The use of single-strand conformation polymorphism (SSCP) can separate sequence polymorphisms that differ by only one base. The SSCP process used allows for both single- and double-stranded polymerase chain reaction (PCR) products to be visualized. The six ABO genotypes can be differentiated by the allele-specific B and O SSCP patterns. The double-stranded DNA produced 'hybrid' bands due to heterozygous samples and allowed sequence diversity to be detected between alleles of heterozygotes. These 'hybrid' bands are valid markers to confirm genotypes of specimens. PMID- 10726756 TI - Analysis of the NF1 gene by temperature gradient gel electrophoresis reveals a high incidence of mutations in exon 4b. AB - A total of 196 unrelated patients with neurofibromatosis type 1 (NF1) was screened for mutations in exons 4a-c of the NF1 gene by temperature gradient gel electrophoresis (TGGE) of polymerase chain reaction (PCR)-amplified genomic DNA fragments using intron-based primers. DNA samples with abnormal TGGE band patterns were subjected to sequence analysis. Sequence alterations were identified in ten patients (5.1%): 496delGT (1), 499delTGTT (4), T528A = D176E (2), T539A = L180X (1), 540insA (1), C574T = R192X (1). Thus, a total of six different mutations was identified in exon 4b but none in exons 4a and 4c. Only the missense mutation D176E, which we assume to be a nonpathogenic polymorphism, and the 4-base pair (bp) deletion 499delTGTT have been described before. The reason for the high incidence of mutations in exon 4b is obviously a tetranucleotide tandem repeat comprising nucleotides 495-502 (TGTTTGTT) that may give rise to slipped mispairing and subsequent deletion of one repeat unit during replication. Additionally, the recurrent 4 bp deletion was found as a second hit in a malignant schwannoma of a further NF1 patient, suggesting that microlesions may be as frequent among somatic as among germline mutations. This is the first report of a systematic study of NF1 exons 4a-c in a large group of NF1 patients. PMID- 10726757 TI - Estimation of total ribonucleic acid quantity from dilute samples by nondenaturing electrophoresis and silver staining. AB - Often, the amount of RNA that can be isolated from a defined tissue is very small. A method consisting of nondenaturing polyacrylamide gel electrophoresis and silver staining is described that can be used to evaluate the concentration of very dilute RNA samples. The method is a good starting point for assays dealing with small amounts of RNA, such as semiquantitative reverse transcription polymerase chain reaction (RT-PCR), making it possible to perform parallel assays from similar amounts of total RNA when quantitation by other methods is too insensitive. The method has been used successfully to monitor the amount of total RNA isolated from rat sciatic nerve and the rat C6 glioma cell line. PMID- 10726758 TI - Extremely high levels of human mitochondrial DNA heteroplasmy in single hair roots. AB - For many years it has been assumed that the vast majority of mitochondrial genomes of a single individual are identical, both in the same tissue and within different tissues. Incidences of heteroplasmy (i.e., the occurrence of two or more codominating types of molecules within the mitochondrial DNA population of the same individual) were thought to be extremely rare. This study strongly supports the thesis that heteroplasmy is a principle, rather than an exception, in mitochondrial DNA genetics. During direct sequencing of the first hypervariable segment of the human mitochondrial control region (HV1) in 100 single hair roots obtained from 35 individuals, 24 different heteroplasmic positions were identified. Unusually high levels of heteroplasmy (up to six positions in the HV1 region) were encountered in two individuals. Two individuals related in maternal lineage shared the same heteroplasmic positions. Moreover, highly variable levels of heteroplasmy were observed even among roots from the same individual. The most probable mechanisms involved in generating so many mismatches are mutations occurring presumably in the female germline, followed by differential segregation of mitotypes during the development of individual hairs. Generally, heteroplasmy complicates sequence comparisons in mitochondrial DNA testing performed for forensic purposes, but in some cases it can substantially increase the discriminating power of the analysis. PMID- 10726759 TI - Low probe concentration can cause problems in multilocus DNA fingerprinting (cautionary notes III). AB - The power of multilocus DNA fingerprinting depends on the reliability with which the uniqueness of an individual's profile can be demonstrated. This cautionary note stresses the importance of the probe concentration in this procedure. In case of a probe shortage, DNA fragments rich in tandem repeats have the potential to impede hybridization in other parts of the gel, and thus interfere with bands that are part of a DNA fingerprinting profile. PMID- 10726760 TI - Automatic calibration in capillary electrophoresis. AB - A direct, completely automated calibration procedure in capillary electrophoresis (CE) is presented. The manual calibration operations involved in analytical methods, such as external calibration, standard addition, and internal standard, were accomplished with a continuous flow system (CFS) coupled to commercial capillary electrophoresis equipment via a programmable arm. The system was managed by the proper CE microprocessor and allowed automatic calibration and direct quantification of the analytes in the sample without any manual pretreatment, therefore avoiding or minimizing manually associated errors. The whole system including peristaltic pumps and the programmable arm was connected via an electronic interface and completely controlled by a computer using a program written in GW-BASIC. The potential of this new CFS-CE arrangement was assessed by applying it to samples containing selected compounds. PMID- 10726761 TI - Resolution of overlapped peaks of amino acid derivatives in capillary electrophoresis using multivariate curve resolution based on alternating least squares. AB - The application of chemometric techniques to the resolution of overlapped peaks in capillary electrophoresis (CE) is described. When a physical separation can not be completely accomplished, chemometrics might still resolve the determination of the analytes mathematically. CE with diode array detection can provide a large amount of data consisting of spectra registered over time. In this study, the capillary electrophoretic separation of 1,2-naphthoquinone-4 sulfonate derivatives of amino acids is studied. Most of the common amino acid derivatives can be separated at 30 kV in a fused-silica capillary by using a 40 mM sodium tetraborate + isopropanol (3:1 v/v) solution as background electrolyte. However, peaks of certain derivatives (Phe, His, Leu and Ile) still overlap. A multivariate curve resolution method based on an alternating least squares optimization procedure is used for the resolution of the overlapped electrophoretic peaks. The method takes advantage of spectral and electrophoretic differences of analytes to recover their pure electrophoretic and spectral profiles. In addition, each analyte in the mixture can be quantified using the corresponding standards. PMID- 10726762 TI - Coupling of solvent semimicroextraction with capillary electrophoresis using ethyl acetate as sample matrix. AB - This paper reports a strategy to couple liquid-liquid semimicroextraction (LLsME) with capillary electrophoresis (CE) based on a newly introduced on-column decomposable sample matrix, ethyl acetate (EA). LLsME was performed in volumetric flasks of 100 mL. Samples containing an aqueous phase were first saturated with EA. Then, an extra 500 microL of EA was added to extract the samples. Sample injection in CE could be made in hydrodynamic mode by dipping the injection end of the capillary into the organic (EA) phase in the volumetric flasks. As a demonstration, alkylphenones in water samples were extracted by LLsME into EA and subjected to separation by micellar electrokinetic chromatography (MEKC). Alkylphenones of C8-C12 with concentrations of about 10 ppb can be concentrated and detected after extraction; extraction efficiencies range from 72- to 334 fold. Linearity of extraction was determined and the effect on reproducibility by spiking an internal standard was studied. The method developed is time-saving and requires no further special experimental device other than a basic CE setup. Therefore, it would be readily acceptable for routine analysis, especially in analytical laboratories dealing with environmental samples. PMID- 10726763 TI - Liquid-phase microextraction and capillary electrophoresis of acidic drugs. AB - Vial liquid-phase microextraction (LPME) combined with capillary electrophoresis (CE) was evaluated for the determination of the acidic drugs ibuprofen, naproxen, and ketoprofen present in water samples and in human urine. The 2.5 mL samples containing the drugs were filled into conventional vials and subsequently acidified by 250 microL of 1-10 M HCl. Porous hollow fibers of polypropylene containing 25 microL of an aqueous solution of 0.01-0.1 M NaOH (acceptor solution) and with dihexyl ether immobilized in the pores of the wall were placed into each of the samples. The acidic drugs were extracted from the acidified sample solutions into the dihexyl ether phase, in the pores of the hollow fiber, and further into the alkaline acceptor solution forced by high partition coefficients. The drugs were extracted almost quantitatively (75-100% extraction efficiency) from the 2.5 mL samples and into the 25 microL acceptor solutions, providing 75-100 times preconcentration. The acceptor solutions were collected for automated CE analysis, which enabled the drugs to be detected down to the 1 ng/mL level. PMID- 10726764 TI - Compensation of the siphoning effect in nonaqueous capillary electrophoresis by vial lifting. AB - Increasing the sample load in nonaqueous capillary electrophoresis through the use of wide-bore capillaries is a good way to scale up analytical separations to semipreparative level. However, obtaining high efficiency requires the use of special instrumentation to eliminate siphoning. When wide-bore capillaries are employed, relatively large solvent volumes are transported from inlet to outlet vial, and due to the difference in liquid levels a siphoning flow from outlet to inlet is established. Siphoning induces a deviation from the plug-like flow profile and adversely affects the separation efficiency. In this study the use of wide-bore capillaries in nonaqueous capillary electrophoresis was examined with compensation for siphoning by lifting of the inlet vial. The inlet vial is raised at a speed appropriate for maintaining equal levels of liquid in the inlet and outlet vials. The optimal lift rate was determined empirically from a series of runs in which the lift rate was varied. As well, a simple theoretical model was devised for the calculation of lift rates. The model was successfully applied for the 200 microm and 320 microm ID capillaries but for the 530 microm ID capillary the predicted optimal lift rate was too low. Evidently this was because the theory was unable to account for the effect of siphoning on the migration times. Three model compounds, bumetanide, furosemide and ethacrynic acid, were separated using an acetonitrile-ethanol mixture (50:50, v/v) with potassium acetate (1 mM) or ammonium acetate (5 mM) as electrolyte. Good separation of bumetadine and ethacrynic acid was obtained even with a 530 microm ID capillary when the lift rate was carefully optimized. Without elimination of siphoning the peaks would not have been detectable. The viscosities and electrical conductivities of the electrolyte solution measured at different temperatures showed that viscosity as well as conductivity decreased with increasing temperature. The temperature dependence of the conductivity was used to estimate the temperature inside the CE capillary. PMID- 10726765 TI - Influence of electric field strength and capillary dimensions on the separation of DNA. AB - This work is a continuation of earlier studies on the influence of polymer concentration and polymer composition on the capillary electrophoretic separation of DNA. The focus is on the capillary dimensions and the electric field strength as factors influencing the resolution. The aim was to establish optimum conditions for the separation of single-stranded DNA in capillaries and derive strategies for the construction of micromachined separation devices. PMID- 10726766 TI - Synthetic oligopeptides as isoelectric point markers for capillary isoelectric focusing with ultraviolet absorption detection. AB - Sixteen peptides (trimers to hexamers) were designed for use as a set of pI markers for capillary isoelectric focusing (CIEF). Each peptide contains one tryptophan residue for detection by UV absorption and other amino acid residues having ionic side chains, which are responsible for focusing to its pI. The pIs of these peptides were determined by slab-gel IEF using commercial carrier ampholytes. The focused peptides in the gel were detected by absorption measurement at 280 nm using a scanning densitometer and the pH gradient was determined by measuring the pH of the gel using an oxidized metal membrane electrode. The pI values of the peptides ranged from 3.38 to 10.17. The obtained values agreed well with the predicted ones, which were calculated based on amino acid compositions, with root mean square differences of 0.15 pH unit. The peptides were detected at 280 nm as very sharp peaks when separated by CIEF. The pI values of some standard proteins were redetermined by CIEF by using this set of peptide pI markers and the values agreed closely with those reported previously. The sharp focusing, stability, high purity and high solubility of these synthetic pI markers should facilitate the profiling of a pH gradient in a capillary and the determination of the pI values of proteins. PMID- 10726767 TI - New carrier electrolytes for the separation of chlorophenols by capillary electrophoresis. AB - Optimum conditions for the separation of positional isomers of chlorophenols by capillary zone electrophoresis (CZE) were established. The behavior of five volatile electrolytes (L-cysteic acid, 3-amino-1-propanesulfonic acid, aminomethanesulfonic acid, diethylmalonic acid, and ammonium acetate) was compared. The best performance based on low electrophoretic current and high separation efficiency was obtained for diethylmalonic acid as working electrolyte. The influence of pH on the separation, using both uncoated fused silica capillaries and modified capillaries (NaAMPS from EKT) with anionic coating, was discussed. Moreover, the effect of electrolyte concentration and applied voltage using fused-silica capillaries was studied. The optimum CZE conditions that allowed the separation of 16 chlorophenols were 20 kV, 30 mM diethylmaIonic acid, pH 7.25, and uncoated fused-silica capillary. Figures of merit such as run-to-run and day-to-day precision, linearity, and limits of detection were calculated. PMID- 10726768 TI - Capillary electrophoresis behavior of water-soluble anionic porphyrins in the presence of beta-cyclodextrin and its O-methylated derivatives. AB - The electrophoretic behavior of water-soluble anionic porphyrins, such as meso tetrakis(4-carboxyphenyl) porphyrin (TCPP), meso-tetrakis(4-sulfonatophenyl) porphyrin (TSPP) and its zinc(II) and copper(II) complexes (ZnTSPP and CuTSPP, respectively) has been studied by capillary zone electrophoresis using fused silica capillaries. The selectivity of the separation is strongly dependent on the type and concentration of betacyclodextrin (betaCD) or the O-methylated derivatives added to the background electrolyte. CuTSPP and TSPP can be separated using a pH 2.5 aqueous sodium phosphate buffer in the presence of 1 mM betaCD. Resolution is poorer or absent employing alkylated betaCDs, such as the heptakis (2,6-di-O-methyl)-beta-cyclodextrin or the heptakis(2,3,6-triO-methyl)-beta cyclodextrin, as additives. On the other hand, separation of TSPP from its copper and zinc complexes has been achieved using a pH 7.0 aqueous sodium phosphate buffer, in the presence of 0.75 mM betaCD and 20% dimethyl sulfoxide (DMSO) as organic modifier. Under such conditions, the calibration curve for quantitative analysis of copper(II) was obtained. A rationale for the observed behavior will be presented and discussed on the basis of binding and protonation equilibria and a simple mathematical model. PMID- 10726769 TI - Capillary zone electrophoresis and micellar electrokinetic chromatography of solution of polyaniline particles. AB - The behavior of two different fractions of the electrically conducting polymer polyaniline (PANI) was studied in order to characterize them using fast, simple, and cheap methods of capillary zone electrophoresis. In contradistinction to the expectations that the particles will bear a positive charge on their surface, in the milieu of electrolytes used for electrophoresis (0.01-0.05 M buffers of pH 4 8.5), the surface was demonstrated to be slightly negatively charged, which led to a slow migration to the anode. An evident charge was given to the particles after their interaction with micelles formed from surface active components, cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS). An active electrophoretic migration and full separation of the PANI fractions was achieved by micellar electrokinetic chromatography (MEKC) with negatively charged SDS micelles. With this method, a reliable qualitative as well as quantitative analysis was performed in the concentration range of 0.1-1.6 mg/mL with a reproducibility of migration times better than 1.5%. PMID- 10726770 TI - Determination of cow's milk and ripening time in nonbovine cheese by capillary electrophoresis of the ethanol-water protein fraction. AB - A novel method is reported for analyzing adulteration of goat and ewe cheeses with cow's milk: capillary zone electrophoresis (CZE) in isoelectric, acidic buffers (50 mM imino diacetic acid, IDA, pH = pI 2.3). The cheese samples were extracted with a 20:80 v/v ethanol-water mixture in presence of 3 M urea and 1% beta-mercaptoethanol for 1 h. After centrifugation and lipid extraction, the samples were dissolved in 50 mM IDA, 6 M urea and 0.5% hydroxyethyl cellulose and analyzed by CZE at 700 V/cm. A total of 18 characteristic peaks were resolved among the three types of cheeses and 18 variables were defined as their respective areas. There was excellent similarity among the electrophoretic patterns obtained with cheeses of a given type of milk, while cheeses made with different types of milk were easily distinguishable. Most peaks were common to all cheeses, but the profile differed depending on the type of milk used. Principal component analysis, linear discriminant analysis, and partial least squares regression (PLS) were used for statistical analysis of the data obtained by CZE. In particular, by using PLS multivariate regression, the contents of cow's milk in presumably pure goat and ewe cheeses, as well as in binary and ternary mixtures, could be predicted with relative standard deviations of ca. 6 7%. In addition, the ripening time in goat and ewe cheeses could also be predicted. PMID- 10726771 TI - Solubilization of delipidated macrophage membrane proteins for analysis by two dimensional electrophoresis. AB - Two-dimensional electrophoresis of proteins is often precluded due to the lack of solubilization of cell membrane extracts in an aqueous medium. Various additives and detergents have been used to circumvent the problem, but their efficacy may not be satisfactory. In this study, the removal of lipidic components of the cell membrane extract with chloroform-methanol was used to achieve solubilization. Optimal delipidation was obtained with acetone washings. This procedure increased solubilization of membrane proteins from a murine macrophage cell line, thus showing a substantial improvement in gel resolution. The two-dimensional gels loaded with delipidated extract proved to be free of smearing and horizontal streaking. In addition, other protein spots were revealed that were not detected in the gels loaded with undelipidated cell membrane extract. PMID- 10726772 TI - Protein patterns of gel-entrapped Escherichia coli cells differ from those of free-floating organisms. AB - The two-dimensional electrophoretic patterns of cellular proteins from gel entrapped Escherichia coli cells were compared to those of exponential- and stationary-phase free-floating organisms. The amounts of several proteins in immobilized cells were significantly different from those in free bacteria. Immobilized organisms rapidly produced a high level of dipeptide permease and a single-strand binding protein, and progressively accumulated an aldehyde dehydrogenase. Immobilization also induced a decrease in the levels of two proteins, i.e., the YFID protein and a DNA-binding, stationary-phase protein. The possible role of these proteins in the high resistance of immobilized bacteria to stresses is discussed. PMID- 10726773 TI - Two-dimensional electrophoretic analysis of Corynebacterium glutamicum membrane fraction and surface proteins. AB - An improved protocol for the two-dimensional analysis of proteins of the Corynebacterium glutamicum cytoplasmic membrane fraction is described. By use of increased 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) concentrations (2-4%) and an optimized electrophoresis protocol, horizontal streaking of proteins of the cytoplasmic membrane fraction was almost completely avoided. More important, in contrast to a previously published method, both a sample tray and IPG-phor isoelectric focusing unit can be used for the in-gel application of proteins. The described protocol was also found to be suitable for hydrophilic cytoplasmic proteins. Additionally, the preparation and analysis of C. glutamicum cell surface proteins is described. Proteins were extracted with lauroyl sarcosinate and 100-120 spots were separated on two-dimensional (2-D) gels in comparison to 18-20 spots observed previously by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). C. glutamicum proteins can now be separated into three distinct fractions resembling different functional units of the bacterial cell. PMID- 10726774 TI - Differential expression of phosphatidylethanol-amine-binding protein in rat hepatoma cell lines: analyses of tumor necrosis factor-alpha-resistant cKDH-8/11 and -sensitive KDH-8/YK cells by two-dimensional gel electrophoresis. AB - To determine intracellular factors influencing the sensitivity of cancer cells to tumor necrosis factor-alpha (TNF-alpha), we studied the expression of intracellular proteins in TNF-alpha-resistant cKDH-8/11 and -sensitive KDH-8/YK rat hepatoma cell lines using the technique of two-dimensional gel electrophoresis (2-DE). From the 2-DE patterns, it was demonstrated that TNF alpha-resistant cKDH-8/11 cells had increased levels of protein of molecular weight (Mr) 22 500 and isoelectric point (pI) 5.2, compared with TNF-alpha sensitive KDH-8/YK cells. Therefore, we excised cyanogen bromide (CNBr) fragments of proteins in the spot for N-terminal sequencing. Microsequencing for the CNBr fragments identified the protein as rat phosphatidylethanolamine-binding protein. These findings suggest that the intracellular phosphatidylethanolamine-binding protein could be one of the factors responsible for the resistance of cKDH-8/11 cells to TNF-alpha-induced cell death. PMID- 10726775 TI - Calcium-dependent secretion in human neutrophils: a proteomic approach. AB - Bactericidal, proteolytic and signal proteins released by activated neutrophils play a major role in infection fighting and inflammatory processes. These proteins are mainly stored in organelles called granules until induction of their controlled exocytosis. The present work deals with the characterization of the proteins which are secreted upon activation of human neutrophils by ionomycin and calcium. Proteins were separated by two-dimensional gel electrophoresis and identified by peptide mass fingerprinting. Almost all the previously described soluble components of neutrophil granules could be identified. Moreover, several additional proteins were shown to be secreted by activated neutrophils, namely calgranulins, human cartilage glycoprotein of 39 kDa (HC gp-39), chitotriosidase, and annexin XI. Their subcellular localization and possible functions are discussed. PMID- 10726776 TI - Differences in protein level between neonatal and adult brain. AB - Age is a physiological condition that affects protein expression. We investigated differences in protein level between newborn and adult brains. Brain tissue extracts from male and female adult and neonatal rats were analyzed by two dimensional gel electrophoresis. Gel comparison revealed the presence of many differences, of qualitative and quantitative nature, between a neonatal and adult brain. The most significant age-related difference concerned alpha-fetoprotein, which was detected in the brain of neonatal rats only. The levels of 22 proteins, including dihydropyrimidinase-related proteins 1,3, and 4 and 14-3-3 proteins, were higher in the neonatal brain, whereas the levels of 28 proteins, including dihydropyrimidinase-related protein 2, dynamin-1 and many enzymes were higher in the adult brain. We did not detect a consistent sex-related difference in the brain proteins. An inconsistency was observed in the location of the spot representing glial fibrillary acidic protein in the male brain. PMID- 10726777 TI - Identification of gel-separated tumor marker proteins by mass spectrometry. AB - Two-dimensional gel electrophoresis with subsequent analysis by mass spectrometry was applied to study differences in protein expression between benign and malignant solid tumors from human beast, lung and ovary cells. Cells from freshly resected clinical material were lysed and the extracts were subjected to isoelectric focusing with immobilized pH gradients followed by second-dimensional separation on 10-13% sodium dodecyl sulfate (SDS)/polyacrylamide gels. Polypeptides were identified using matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry after in-gel protein digestion. Some of the upregulated polypeptides in malignant cells are of potential importance as markers of tumor proliferation. Twenty such proteins were identified, ten constituting novel identifications and ten sequence verifications of previously gel-matched proteins. The proteins identified span a wide range of functions, but several cases of protein truncation were found. Truncated forms of cytokeratins 6D and 8, and of cathepsin D were identified. Truncated froms of these over-expressed proteins support the presence of proteolytic processing steps in tumor material. The protein processing and the difference between protein and mRNA abundancies in tumors of different malignancy and origin suggest that studies at the protein level are important for an understanding of tumor phenotypes. PMID- 10726778 TI - Ocular injuries caused by fireworks. 25 years of experience with preventive campaigns in Denmark. PMID- 10726779 TI - Dissection and cotransplantation of large pieces of RPE and neural retina; effect of protease K on the development. AB - PURPOSE: This study attempts to cotransplant large pieces of the RPE and neural retina in the subretinal space of rabbits by using protease K for dissection of the donor tissue, and to investigate the effect of dissection technique on the development of the grafts. METHODS: Eyes from 15-day-old pigmented rabbit embryos were partly digested by protease K to assist dissection of sclera and the choroid from RPE and neural retina. Large pieces of RPE and the neural retina thus obtained were cotransplanted into the eyes of adult albino rabbits who were allowed to survive for up to 63 days. The transplants were examined under light microscope. RESULTS: It was possible to transplant large sheets of RPE and neural retina together. Both the RPE and the neural retina survived after cotransplantation. Retinal pigment epithelium survived in layers, but at places formed clusters. In cotransplants neural retina formed rosettes, developed gliosis, and photoreceptors failed to develop outer segments, possibly due to the action of protease K. CONCLUSION: Proteases seem to be injurious for the development of the neural retina. PMID- 10726780 TI - Comparability of three-dimensional optic disc imaging with different techniques. A study with confocal scanning laser tomography and raster tomography. AB - PURPOSE: We wanted to compare optic nerve head topography measurements and discrimination between normal and glaucomatous eyes with two entirely different three-dimensional optic disc imaging techniques, confocal scanning laser tomography (Heidelberg Retina Tomograph, Heidelberg Engineering) and raster tomography (Glaucoma-Scope, Ophthalmic Imaging Systems). METHODS: Both eyes of 225 normal subjects and 229 eyes of 166 patients with glaucoma at different stages were imaged with the Heidelberg Retina Tomograph and the Glaucoma-Scope. Optics discs were analysed in 15 degrees sectors around the circumference. Depth measurements were calibrated on objects with known dimensions. RESULTS: We observed no significant differences in absolute measurements of maximum cup depth and cup area between the two instruments. We observed small differences in absolute measurements of disc anti rim area between the two instruments. Discrimination between normal and glaucomatous eyes was close to identical for the two instruments. Both instruments had the same ability to distinguish glaucomatous regional alterations of optic nerve head topography from normal disc configuration. CONCLUSION: Both instruments while using entirely different principles for three-dimensional optic disc imaging gave very similar results. This correspondence of results implies that the same optic disc parameters can be analysed, and the results interpreted similarly for both methods, and probably with other three-dimensional imaging instruments. It may also indicate that results of studies with one of the instruments have general validity. PMID- 10726781 TI - A comparison between one- and two-field 60 degree fundus photography when screening for diabetic retinopathy. AB - PURPOSE: To compare the severity level of diabetic retinopathy obtained when assessed from two versus only one 60 degrees photographic field using colour transparencies and red-free, black-and-white photographs. To compare the areal coverage of these two photographic strategies to that of seven-field 30 degrees photography. METHODS: Two ophthalmologists graded photographs of 74 eyes of 74 type I and II diabetes patients. Inter-method agreement was expressed in percentages and using kappa statistics and scatter-diagrams. The comparison of the approximate photographic areal coverage was done from diagrams using planimetry. RESULTS: The severity level of retinopathy when judged from two photographic fields was more severe in 13.5% (Grader 1) and in 16.2% (Grader 2) from colour transparencies and in 13.5% (Grader 1) and in 14.9% (Grader 2) from red-free black-and-white prints, as compared to assessments from only one field. Kappa values (0.84-0.86) for inter-method agreement for five pooled retinopathy levels revealed good agreement. Neither grader missed retinopathy requiring clinical assessment or treatment (levels > or = 47) when minimal retinopathy (levels 14-20) was detected using only one 60 degrees colour slide or red-free photograph. A second optic disc-centred field provided valuable additional information when more severe retinopathy lesions (levels > or = 30) were detected in the macula-centred field. One macula-centred 60 degrees photograph covered 60% and two 60 degrees photographs 80% of the area covered by seven-field 30 degrees photography. Two-field 60 degrees photography covers areas left outside seven field 30 degrees photography. CONCLUSION: We propose the use of one macula centred 60 degrees photograph when screening for the first lesions of diabetic retinopathy. After they have been found two-field 60 degrees photography is recommended. PMID- 10726782 TI - Spotting of disc haemorrhages on fundus pictures; on Kodachrome slides and on digitally enhanced images. AB - PURPOSE: To find out whether digital processing can facilitate interpretation of fundus photographs. METHODS: Digitised fundus pictures were manipulated and enhanced by a process called histogram equalisation. Essentially, the gamma values for red (R), green (G), and blue (B) were separately modified. The principle is explained and exemplified. MATERIAL AND RESULTS: Small disc haemorrhages, which may normally be hard to detect, became more visible after histogram equalisation, which is demonstrated in test series. CONCLUSION: Digital histogram equalisation offers a new way of looking at fundus pictures, which, alone or combined with other capabilities of digital processing, has great potential for diagnosis, as well as for follow up studies. The possibility to decide the true nature of suspect spots after applying the enhancement procedure is likewise of great value. PMID- 10726783 TI - The influence of near-work on development of myopia among university students. A three-year longitudinal study among engineering students in Norway. AB - PURPOSE: The aim of this study was to investigate the effect of near-work on development and progression of myopia among adults exposed to high educational demands. METHODS: A three-year longitudinal refraction study was performed among 224 Norwegian engineering students (mean age 20.6 years, 117 females and 107 males) measuring their refraction at the beginning and the end of the period. The examinations included automated and clinical refraction in cycloplegia and a questionnaire regarding time spent on different kinds of near-work was filled in by the participants. A total of 192 students (100 females and 92 males) completed the study. RESULTS: The mean refractive change of -0.51+/-0.49 D (n=192) during the three-year period was statistically significant (p=0.0001). A significant relationship between refractive change towards myopia and time spent on reading scientific literature (p< or =0.001) and on practical near-work (p< or =0.05), respectively, was found. Also, a significant relationship between refractive change towards myopia and time spent at lectures was revealed (p< or =0.001). No relationship was found between refractive change and time spent at working with video display terminals (VDT) or watching television, respectively. CONCLUSIONS: The results indicate that intensive near-work could initiate myopia or lead to its progression in young adults. The time spent on near-work seems to play a significant role in that process. PMID- 10726784 TI - Color Doppler ultrasonography in ocular Behcet's disease. AB - PURPOSE: The aim of this prospective study was to assess the ocular hemodynamic changes in patients with Behcet's disease who had involvement of the posterior segment by color Doppler ultrasonography. METHODS: The present study was carried out in 32 eyes of 24 patients with ocular Behcet's disease and 42 eyes of 21 persons as a healthy age-matched control group. In order to detect the hemodynamic changes in ocular Behcet's disease, peak systolic and end-diastolic velocities (PSV, EDV; cm/sec), resistive and pulsatile indices (RI, PI), and PSV/EDV ratio of central retinal artery (CRA), short posterior ciliary artery (PCA) and ophthalmic artery (OA) were determined. RESULTS: The mean PSV and EDV (8.1+/-2.6 and 2.7+/-0.1 cm/sec, respectively) in the CRA were significantly lower in patients with Behcet's disease than in healthy controls (11.5+/-2.2 and 3.5+/-1.0 cm/sec, respectively, p<0.001). For the PCA, these values (12.7+/-4.2 and 4.3+/-1.1 cm/sec, respectively) were significantly lower in the patient group than in the control the group (18.2+/-4.2 and 6.1+/-2.5 cm/sec, respectively, p<0.001). In the patient group, a statistically significant decrease compared to the control group was detected in the mean PSV of the OA (31.8+/-8.2 and 35.8+/ 0.6 cm/sec, respectively, p<0.02). However, no significant difference in the other parameters of these vessels was observed between groups. CONCLUSIONS: These results demonstrate the presence of some circulatory changes in the flow velocities of the CRA, PCA and OA in patients with Behcet's disease when compared with control subjects. PMID- 10726785 TI - Prolonged persistence on the ocular surface of fortified gentamicin ointment as compared to fortified gentamicin eye drops. AB - PURPOSE: A comparative study on the elimination of gentamicin from the ocular surface and the concentration of gentamicin in the anterior chamber following application of either an ointment or eye drops containing equal concentrations (1.5%) of gentamicin. METHODS: A disc-diffusion test was used to determine the concentration of gentamicin in fornix inferior of 10 persons. The anterior chamber concentration of gentamicin was determined in 5 cataract patients by the TDX analyzer, Abbot Laboratories, II., USA. RESULTS: Ten minutes following application, the concentration of gentamicin was significantly higher in the eyes receiving ointment (310.6 mg/L) compared to drops (45 mg/L) (p<0.01). Furthermore, gentamicin could be detected 40 minutes after application in the eyes receiving ointment compared to 10 minutes in the eyes receiving drops. The anterior chamber concentration of gentamicin after application of either drops or ointment was lower than 0.6 mg/L and thus below detection limit. CONCLUSIONS: The persistence of gentamicin ointment was significantly longer on the ocular surface as compared to gentamicin eye drops. Gentamycin ointment may thus provide a means to reduce the high application frequency presently in use with eye drops to treat bacterial keratitis and thereby reduce patient inconvenience, especially during nighttime. PMID- 10726786 TI - Short-term effect of latanoprost and timolol eye drops on tear fluid and the ocular surface in patients with primary open-angle glaucoma and ocular hypertension. AB - PURPOSE: To investigate and compare the short term effects of topical latanoprost and timolol on the tear fluid and ocular surface condition in patients with bilateral primary open angle glaucoma or ocular hypertension. METHODS: Thirty seven patients were included in this randomized, double-masked, parallel group study. Patients received either latanoprost 0.005% (n= 18) or timolol 0.5% (n= 19) instilled once daily in the morning for a treatment period of 27 days. Routine ophthalmic examinations, including intraocular pressure measurement, as well as tests to evaluate tear fluid and the ocular surface were performed. RESULTS: After one drop of medication, tear secretion was significantly reduced by timolol, but not by latanoprost. At the end of the study the break-up time (BUT) was significantly decreased in the timolol group but not in the latanoprost group. The BUT still remained in the normal range in both groups, although it is important to note that timolol was administered at half the clinical dose. Both latanoprost and timolol tended to increase Rose-Bengal staining of the cornea and conjunctiva after one month of treatment but no statistically significant difference was found between the groups. Corneal sensitivity was within the normal range for all patients during the study. CONCLUSION: Regarding ocular surface effects, no clinically important differences between latanoprost and timolol were observed as all the effects remained in the normal range. PMID- 10726787 TI - Comparison of endothelial cell density estimated by contact and non-contact specular microscopy. AB - PURPOSE: To compare a contact and a non-contact specular microscope in the determination of endothelial cell density. SUBJECTS AND METHODS: One hundred and twenty-one eyes from 70 patients who had undergone various degrees of photorefractive keratectomy for myopia were included. The endothelium was imaged by contact (Konan Clinical Specular Microscope) and non-contact (Topcon SP-1000) specular microscopy and the endothelial cell density estimated. RESULTS: The average endothelial cell density achieved by the contact specular microscope was 3011+/-298 cells/mm2 (mean+/-SD, n=121) and by the non-contact specular microscope 3015+/-265 cells/mm2 (n= 121). The difference in endothelial cell density between the contact and the non-contact specular microscope (contact minus non-contact) was -4+/-175 cells/mm2 (t=0.26, 2p>0.05 in a paired t-test). The sampling error on the estimated endothelial cell density was 76 cells/mm2 for the contact specular microscope and 74 cells/mm2 for the non-contact specular microscope. CONCLUSION: The average endothelial cell density and the precision of the measuring technique were similar for the contact and the non-contact specular microscope. Furthermore, the endothelial cell densities estimated by the two instruments at various values of anterior central corneal refractive power and central corneal thickness were similar. The two instruments can be used interchangeably. PMID- 10726788 TI - Corneal epithelial changes due to adenovirus type 8 infection. A non-contact photomicrographic in vivo study in the human cornea. AB - PURPOSE: To investigate corneal epithelial changes in human adenovirus type 8 (Ad8) infection, and compare them with herpes simplex virus type 1 (HSV1) changes. MATERIAL AND METHOD: 12 patients with Ad8 infection: in 10 verified by tissue culture, in 2 clinically. The corneae were photographed by non-contact photomicrography. RESULTS: Within the first days the epithelium showed multiple elevations measuring about 20-30 microm in diameter, and from about day 5 large numbers of scattered subsurficial abnormal cells. At the end of the first week larger (about 150-200 microm in diameter) epithelial foci started to appear. No focal ulcerative changes were observed. CONCLUSIONS: The subsequent stages of Ad8 cytopathic effect are difficult to discern. The primary virus target seems to be the deeper epithelial layers. The damaged cells are probably successively transported toward the surface and desquamated. The etiology of larger focal changes is unclear. The presence (HSV1) or absence (Ad8) of ulcerative changes differentiates these two infections. PMID- 10726789 TI - Optical coherence tomography and localized defects of the retinal nerve fiber layer. AB - PURPOSE: To test the capability of the optical coherence tomography (OCT) to demonstrate and quantitate retinal nerve fiber layer (RNFL) defects. METHODS: The authors examined 6 eyes of 6 chronic open angle glaucoma patients with the OCT. The patients had abnormal Humphrey 30-2 visual fields which corresponded to RNFL defects visible in monochromatic fundus images taken with a digital imaging system. The RNFL images were used for directing the OCT scans to areas where most information was believed to be obtainable. Several linear scans of different lengths across healthy and abnormal RNFL regions were made. RESULTS: When the OCT images were compared to RNFL photographs, the defective areas showed reduced backscattering with the OCT, being distinctly different from the adjacent normal RNFL. Except for one case the RNFL thickness values were smaller in the areas of abnormal appearance compared to areas of normal appearance. CONCLUSION: This preliminary study suggests that the OCT examination results of the RNFL are in good agreement with the RNFL appearance in monochromatic fundus images. PMID- 10726790 TI - Impaired dark adaptation in polycythemia. Improvement after treatment. AB - PURPOSE: To determine if dark adaptation is reduced in individuals with polycythemia and if so whether there is any improvement in dark adaptation after treatment. METHODS: Dark adaptation was recorded monocularly by automatic dark adaptometry in ten consecutive patients with polycythemia before and after treatment. Analogue investigations were performed in 31 healthy control subjects. RESULTS: Dark adaptation was markedly impaired in the patients as compared with the control subjects. After reduction of the red cell count and normalization of the hematocrit and hemoglobin the dark adaptation was markedly improved. There was no significant change in dark vision in the control subjects negating a confounding learning effect. CONCLUSION: The findings indicate a sustained but reversible neuronal hypofunction secondary to polycythemia. As the rheological abnormality was normalized, dark adaptation was improved, probably secondary to normalized microcirculation within the retina or the brain, or both, possibly with reactivation of formerly inactive neuronal cells. PMID- 10726791 TI - Image analysis to improve diagnosis of exfoliation. AB - PURPOSE: To improve visualization of exfoliation material in standard colour photographs by image analysis. METHODS: Standard anterior segment colour photographs taken from 17 patients with known exfoliation were subjected to film scanning with a maximum accuracy of 3175 dpi. RESULTS: Before image scanning, visualization of exfoliation was classified as barely visible in 10 (59%) patients and clearly visible in 7 (41%) patients. After the image scanning, exfoliation material became clearly visible in 13 (76%) patients and could easily be demonstrated. CONCLUSION: Diagnosis of exfoliation material can be improved by standard film scanning. PMID- 10726792 TI - Eye injuries from fireworks in Western Sweden. AB - PURPOSE: To investigate the extent and type of as well as the circumstances connected with, ocular injuries caused by pyrotechnical products in the Western region of Sweden (1 750 000 inhabitants). METHODS: A three-year prospective observational study was carried out to register, interview and follow-up all patients with ocular injuries caused by fireworks. The patients were reported by all ophthalmologists in the area. RESULTS: 52 patients (72 injured eyes) were reported, 49 males and 3 females. 39 of 52 (75%) were younger than 18 years. 15 patients (including the three females) were bystanders. The injuries ranged from conjunctivitis to complete loss of the eye. The pyrotechnical device had been manipulated or misused in 19 (37%) of the 52 accidents. Seriousness of injury could not be correlated to type of device or to whether the injured person was active in launching the firework or not. 20 (38%) of the 52 patients had permanent damage to the eye. CONCLUSION: Ocular injuries from fireworks remain a problem. Preventive measures such as protective glasses and legislation are being discussed. PMID- 10726793 TI - Glaucoma following congenital cataract surgery: an 18-year longitudinal follow up. AB - PURPOSE: To report the occurrence of postoperative glaucoma and to evaluate risk factors. METHODS: Children born in four of the western counties of Sweden who were diagnosed with congenital cataracts formed a cohort (n=137). The following parameters were evaluated: age at cataract surgery; type of surgery; visual outcome; postoperative IOP; optic disc abnormalities; date of onset of the complication; number of reoperations, including treatment for secondary cataract; presence of systemic anomalies; microphthalmus; and eye-related anomalies. RESULTS: A diagnosis of glaucoma was recorded for 12% of the eyes. The mean follow-up time was 9.6 years. There is a relationship between surgery before the age of 10 days and development of glaucoma. Microphthalmus is an important risk factor as well. CONCLUSIONS: Despite modern surgical techniques the incidence of aphakic glaucoma is 10% or higher. No time-dependent increase in the incidence of late-onset glaucoma between the 1980s and the 1990s could be proven. PMID- 10726794 TI - Latanoprost: experience of 2-year treatment in Scandinavia. AB - PURPOSE: The aim of the study was to assess efficacy and side effects of latanoprost during two years of treatment. METHODS: The study was a randomized, parallel group, double-masked, multicenter comparison between latanoprost and timolol in patients with open angle glaucoma or ocular hypertension, followed by an open-label 18-month extension during which all patients were treated with latanoprost. RESULTS: Latanoprost caused a marked and sustained reduction of the intraocular pressure (IOP). IOP was reduced from baseline levels 25.1+/-3.5 mm Hg (mean+/-SD) in 183 patients initially randomized to treatment with latanoprost to 17.4+/-2.9 mm Hg (n=66) after 24 months of treatment. For patients initially randomized to treatment with timolol the corresponding figures were 24.3+/-2.3 mm Hg (n=72) and 17.4+/-2.6 (n=41) mm Hg after 18 months of treatment with latanoprost. Two patients were withdrawn because of uncontrolled IOP and 11 patients required additional timolol treatment to maintain an adequate IOP control. Patients initially treated with timolol and switched to latanoprost had a further reduction of the IOP of 1.0 mm Hg after 6 months of treatment with latanoprost (p<0.005). 46 patients were withdrawn from the study, mostly due to increased iris pigmentation or an iris color with known high risk of developing increased pigmentation. 22 patients developed increased pigmentation of the iris. The follow-up revealed no previously unknown ocular or systemic side effects. CONCLUSION: Once daily applications of latanoprost cause a marked and sustained reduction of the IOP. The only clinically significant side effect noted was the increased pigmentation of the iris, most frequently seen in irides with a mixture of brown and blue/gray or green colors. No systemic side effect was observed. PMID- 10726795 TI - Comparative evaluation of early vs. deferred vitrectomy in Eales' disease. AB - METHODS: 40 cases of vitreous haemorrhage secondary to Eales' disease were taken up for vitrectomy. Depending upon duration of vitreous haemorrhage patients were divided into two groups: Group I (20 eyes) - early vitrectomy group with duration between 3-6 months; Group II (20 eyes) - deferred vitrectomy group with duration of more than 6 months. All patients were followed up for a minimum period of 3 months following vitrectomy. RESULTS: Eyes in Group I showing a preoperative ultrasonic picture of complete posterior vitreous detachment, less mobility of organised vitreous haemorrhage and mid vitreous organisation on kinetic echography achieved a final visual acuity of 6/9 or better in 13 (65%) eyes as compared to 4 (20%) eyes in Group II (p<0.01). Poor visual outcome in the deferred group was secondary to cystoid macular oedema, macular scar, macular pucker formation and macular degeneration. CONCLUSION: Improved visual outcome in the early vitrectomy group was probably because the haemorrhagic blood and its toxic products had less time to damage the macula, a lesser incidence of macular traction and cystoid macular oedema. PMID- 10726796 TI - Helical and conventional CT in the imaging of metallic foreign bodies in the orbit. AB - PURPOSE: To compare helical CT to conventional CT imaging in the assessment of orbital metallic foreign bodies with regard to image quality, scanning time, and radiation exposure. METHODS: Twenty-four standardized metallic foreign bodies, placed into the orbit (anterior, epibulbar, posterior) of cadaver heads were studied. Helical CT scanning in the axial plane with multiplanar reconstruction of coronal and sagittal images was performed as well as conventional CT imaging with direct scanning in the axial and the coronal planes (Tomoscan SR 7000, Philips, The Netherlands). Two masked observers consensually graded the studies using 5 predetermined criteria. Radiation dose delivered to the lens and scanning time were measured for the helical and the conventional CT imaging workup. RESULTS: Helical CT imaging scored statistically significantly better with regard to overall accuracy of foreign body localization and presence of beam-hardening streak artifacts from dental fillings. Conventional CT scored significantly better with regard to stair-step artifacts. The radiation dose delivered to the lens was 35.4 mGy for helical CT imaging and 73.9 mGy for conventional CT workup (axial and coronal scanning). Total scanning time was 18 seconds for helical CT axial scanning and 104 seconds for conventional CT axial and coronal scanning. CONCLUSION: Helical CT is superior to conventional CT imaging, because it can provide adequate information about orbital metallic foreign bodies with a single acquisition, thus reducing both the number of examinations and the radiation exposure for the patient. PMID- 10726797 TI - Uveitis in children: population-based study in Finland. AB - PURPOSE: To investigate the incidence and prevalence rates of different types of uveitis in children, and to compare them with the rates in adults. SUBJECTS AND METHODS: For this population-based retrospective study the medical records of all residents of the district of Turku University Hospital with a diagnosis of uveitis seen at the Eye Clinic of Turku University Hospital during the years 1980 1982 and 1988 were reviewed. RESULTS: A total of 1122 uveitis cases were identified, and 55 (4.9%) of them were children under 16 years. The incidence per 100 000 population per year for all uveitis cases in children was 4.3 (95% confidence interval [CI], 2.2 to 6.4), and the prevalence was 27.9 (95% CI, 17.1 to 38.6), which was significantly lower compared with the rates in adults (p=0.001 for incidence and prevalence). The vast majority of children, 50 (90.9%), had anterior uveitis (AU), and the incidence and prevalence rates of AU were significantly lower than in adults (p=0.001 for incidence and prevalence). Three (5.5%) children had posterior uveitis (PU), but there was no significant difference in the incidence and only marginally significant difference in the prevalence rate of PU in children compared with the rates in adults (p=0.33 for incidence, and p=0.07 for prevalence). Only one case (1.8%) was found with intermediate and one with panuveitis, but no new cases. The commonest diagnostic groups in children were AU associated with juvenile rheumatoid arthritis, idiopathic acute anterior uveitis, and idiopathic chronic anterior uveitis. Toxoplasmic retinochoroiditis was found in all of the PU cases with the incidence 0.3, and the prevalence 1.1, which did not differ significantly from the rates in adults (p=1.0 for incidence, and p= 0.48 for prevalence). CONCLUSION: Uveitis is rarer in children than in adults. However, in contrast to studies from tertiary referral centers, the distribution of different forms of uveitis in children in this population-based study seems to resemble the distribution in adults. PMID- 10726798 TI - The acoustic lighthouse effect. An ultrasonic response met in eyes after vitreoretinal surgery with silicone oil. AB - PURPOSE: To discuss and possibly explain the ultrasonic lighthouse effect, a metaphor suggested for an artifact-like phenomenon sometimes found by B-scan in eyes having had intra-ocular perfluorocarbon and/or silicone oil injected as part of vitreoretinal surgery. METHOD: Analysis of the ultrasonic findings with a view to the specific gravities of the above injected substances. Compared to water, perfluorocarbon has a higher and silicone oil a lower gravity; they are heavy and light, respectively. RESULTS: The elicited lighthouse effect may appear localized or 'mobile'. When evoked only from specific positions on the globe, most likely it is explained by a lens-like accumulation of material trapped at the vitreous base just under the transducer. When mobile it depends on head position; there is a gravitational shift in position of the material, as evident in particular when becoming visible in the anterior chamber. CONCLUSIONS: In case reports in literature slit-lamp verified anterior segment perfluorocarbon remnants have caused acoustic disturbances of a similar nature. In this study we found evidence that also silicone oil can underlie the acoustic lighthouse effect. PMID- 10726799 TI - Vogt-Koyanagi-Harada syndrome in a pregnant patient treated with high-dose systemic corticosteroids. AB - PURPOSE: Vogt-Koyanagi-Harada (VKH) syndrome is usually treated with high-dose systemic corticosteroids. However, in a pregnant patient with VKH syndrome, the effects of high-dose corticosteroids on the fetus are controversial. We discuss treating VKH syndrome during pregnancy, systemic corticosteroids, and abnormalities in the delivered infant. METHODS: Case report and literature review. RESULTS: A 26-year-old Japanese woman in the 16th week of gestation was diagnosed with VKH syndrome and treated with high-dose systemic prednisolone after the 18th week of gestation. Although the patient's ocular findings gradually improved, a low-birth-weight infant was delivered with epibulbar dermoid, lipodermoids, and preauricular appendages. Although low birth weight might result from systemic prednisolone administration, the anomalies of the infant may have been inherited rather than caused by VKH syndrome and high-dose systemic corticosteroid administration. CONCLUSION: In this case and previously reported cases, VKH syndrome and systemic corticosteroids administered during pregnancy may not precipitate abortion and congenital malformation. PMID- 10726800 TI - Choroidal Langerhans' cell histiocytosis. AB - PURPOSE: To report a patient with choroidal Langerhans' cell histiocytosis. METHODS: A solitary tumor was found in the left eye of a 49-year-old male who had no definite history of systemic disorders, but had observed visual disturbances for a period of 1 month. Ultrasonography, fluorescein angiography, and indocyanine green angiography were performed and the eyeball was enucleated. We prepared the specimen for microscopic examinations. RESULTS: Fluorescein angiographic findings of the lesion were mottled hyperfluorescence in the arteriovenous phase and strong hyperfluorescence in the late phase. Hypofluorescence in both early and late phases showed on indocyanine green angiogram. The lesion of choroid was widely infiltrated by histiocytes, though no extraocular invasion was found. Immunohistochemical studies including S-100 and CD 68 staining revealed characteristic features of Langerhans' cell histiocytosis. Electron microscopic examination of the histiocytes showed histiocytosis X body (Birbeck granule) in the cytoplasm and indented nucleus. CONCLUSION: We consider that this is a case of choroidal Langerhans' cell histiocytosis with no evidence of systemic lesions. PMID- 10726801 TI - Atrial septal defect with interatrial aneurysm and Axenfeld-Rieger syndrome. AB - PURPOSE: The aim of this presentation is to report a rare association between interatrial aneurysm and Axenfeld-Rieger syndrome. METHODS: We present a 20-year old girl with Axenfeld-Rieger syndrome, in whom the diagnosis was confirmed by the facial dysmorphic appearance, slit-lamp examination, applanation tonometer, gonioscopy, and cardiovascular examination. RESULTS: The patient had prominent Schwalbe's line in all quadrants, iris atrophy, and secondary glaucoma in both eyes. The echocardiogram revealed an atrial septal defect with interatrial aneurysm. CONCLUSION: This is the first case report of coexisting Axenfeld-Rieger syndrome and interatrial aneurysm. We would like to stress that aneurysm can be a potential occult source of cerebral embolism. PMID- 10726802 TI - Pigmented spindle cell naevus of reed presenting in the conjunctiva. AB - PURPOSE: This study aimed to present the clinical and histopathological features of a pigmented spindle cell naevus (PSCN) appearing in the conjunctiva. METHODS: Histopathological examination of the lesion following excision and review of the pertinent literature. RESULTS: The features were consistent with those previously recognized in the skin as the PSCN of Reed. CONCLUSION: The PSCN can appear in the conjunctiva and should not be confused with conjunctival melanoma. PMID- 10726803 TI - Acremonium keratitis in a patient with herpetic neurotrophic corneal disease. AB - Fungi belonging to the genus Acremonium Link ex Fries 1821 are ubiquitous environmental contaminants and soil saprophytes, but are infrequent pathogens in humans. These filamentous fungi (previously known as Cephalosporium) are an uncommon cause of mycotic keratitis. As in the case of other filamentous fungi, corneal trauma with contaminated matter is the most frequent risk factor for the infection. We report in this paper a case of keratomycosis caused by Acremoniumpotronii, in a patient with a history of herpetic keratitis. Medical treatment with amphotericin B was unsuccessful and the infection eventually resolved with penetrating keratoplasty. PMID- 10726804 TI - Lacrimal canalicular stenosis associated with systemic 5-fluorouacil therapy. AB - PURPOSE: Ocular side effects of systemic 5-fluorouracil therapy include excessive lacrimation which often resolves on cessation of therapy. Permanent stenosis of lacrimal puncta and canaliculi is rare. This report highlights this uncommon complication of 5-fluorouracil therapy. METHODS: Report of two cases and review of literature. RESULTS: Severe stenosis of puncta and canaliculi may be associated with prolonged systemic 5-fluorouracil therapy. CONCLUSION: It is important for both the oncologist and ophthalmologist to be aware of the potential ocular toxicity of 5-fluorouracil. Surgical management to relieve the epiphora is often challenging, and early intervention may be beneficial. PMID- 10726805 TI - Spontaneous involution of a large retinal arterial macroaneurysm. AB - BACKGROUND: Acquired retinal macroaneurysms are round dilatations of retinal arterioles that occur in the posterior fundus. They tend to rupture before they grow large and cause a visual decrease. The average diameter of macroaneurysms in the previous reports is 281 micrometer. METHODS: A 63-year-old man complained of a sudden decrease of visual acuity in the left eye. Fundus examination of his left eye revealed a large protruded mass, 2 disc diameters in size, at the first bifurcation of the inferotemporal retinal artery. Preretinal vitreous bleeding and serous retinal detachment were observed around the lesion. Indocyanine green (ICG) angiography showed this mass to be continuous with the inferotemporal retinal artery and ICG dye leaked from the retinal artery into the lumen of the mass, with pulsation. RESULTS: Six months later, the large macroaneurysm underwent spontaneous involution. CONCLUSION: As shown in this patient, retinal macroaneurysms may grow extremely large. ICG angiography was useful to diagnose this large retinal arterial macroaneurysm. PMID- 10726806 TI - Uncommon ophthalmologic findings associated with Wolfram syndrome. PMID- 10726807 TI - Strategies for cardiac marker measurement. PMID- 10726808 TI - Statistical techniques for evaluating the diagnostic utility of laboratory tests. AB - Clinical laboratory data is used to help classify patients into diagnostic disease categories so that appropriate therapy may be implemented and prognosis estimated. Unfortunately, the process of correctly classifying patients with respect to disease status is often difficult. Patients may have several concurrent disease processes and the clinical signs and symptoms of many diseases lack specificity. In addition, results of laboratory tests and other diagnostic procedures from healthy and diseased individuals often overlap. Finally, advances in computer technology and laboratory automation have resulted in an extraordinary increase in the amount of information produced by the clinical laboratory; information which must be correctly evaluated and acted upon so that appropriate treatment and additional testing, if necessary, can be implemented. Clinical informatics refers to a broad array of statistical methods used for the evaluation and management of diagnostic information necessary for appropriate patient care. Within the realm of clinical chemistry, clinical informatics may be used to indicate the acquisition, evaluation, representation and interpretation of clinical chemistry data. This review discusses some of the techniques that should be used for the evaluation of the diagnostic utility of clinical laboratory data. The major topics to be covered include probabilistic approaches to data evaluation, and information theory. The latter topic will be discussed in some detail because it introduces important concepts useful in providing for cost effective, quality patient care. In addition, an example illustrating how the informational value of diagnostic tests can be determined is shown. PMID- 10726810 TI - Methods for improving clinical trials. AB - Over the past decades, the randomized controlled trial has entered an era of continuous improvement and has gradually become accepted as the most effective way of determining the relative efficacy and toxicity of new therapies because it controls for placebo and time effects. However, even sensitive and properly designed and executed trials do not always confirm hypotheses to be tested, and conclusions are not always confirmed by subsequent trials. Although the former may be due to wrong hypotheses, the latter is likely to be due to the presence of certain imperfections within the design and execution of the trial itself. In this opinion paper, while focusing on such imperfections, the author searched for methods for further improvement of controlled trials, particularly clinical trials. The examples used in this paper are obtained from literature search as well as recent studies performed by the Netherlands Working Group on Cardiovascular Research (WCN). Methods for improvement could include: 1. making every effort to avoid asymmetries in the treatment groups; 2. emphasis on statistical power rather than just null-hypothesis testing; 3. adjusting for asymmetries not only of patient characteristics but also of outcome variables; 4. accounting routinely for type III errors; 5. routinely weighing benefits of a new drug against risks. PMID- 10726809 TI - Magnesium in disease: a review with special emphasis on the serum ionized magnesium. AB - This review deals with the six main clinical situations related to magnesium or one of its fractions, including ionized magnesium: renal disease, hypertension, pre-eclampsia, diabetes mellitus, cardiac disease, and the administration of therapeutic drugs. Issues addressed are the physiological role of magnesium, eventual changes in its levels, and how these best can be monitored. In renal disease mostly moderate hypermagnesemia is seen; measuring ionized magnesium offers minimal advantage. In hypertension magnesium might be lowered but its measurement does not seem relevant. In the prediction of severe pre-eclampsia, elevated ionized magnesium concentration may play a role, but no unequivocal picture emerges. Low magnesium in blood may be cause for, or consequence of, diabetes mellitus. No special fraction clearly indicates magnesium deficiency leading to insulin resistance. Cardiac diseases are related to diminished magnesium levels. During myocardial infarction, serum magnesium drops. Total magnesium concentration in cardiac cells can be predicted from levels in sublingual or skeletal muscle cells. Most therapeutic drugs (diuretics, chemotherapeutics, immunosuppressive agents, antibiotics) cause hypomagnesemia due to increased urinary loss. It is concluded that most of the clinical situations studied show hypomagnesemia due to renal loss, with exception of renal disease. Keeping in mind that only 1% of the total body magnesium pool is extracellular, no simple measurement of the real intracellular situation has emerged; measuring ionized magnesium in serum has little added value at present. PMID- 10726811 TI - Infrared analysis of urinary stones: a trial of automated identification. AB - A Search algorithm included in the Opus software of Bruker (Germany) was evaluated for analysis of urinary stones. Three reference libraries containing respectively 85 (single components), 1,059 (binary mixtures) and 4,565 (ternary mixture) digitized spectra were created and used to identify unknown spectra (n=320), applying the automatic procedure. Identification of the major component was correct in 83% of cases but the percentage of identification significantly decreased for the second and the third components. In cases of identification of the two first components, quantitative assessment was correct within tolerance limits +/- 15%. The computer results are judged unsatisfactory with regard to pathology because computer-aided identification is not sufficiently sensitive and specific to differentiate species with similar spectral pattern, even for the identification of main component, and also to detect minor components. It can be of assistance to guide spectral analysis, but it cannot replace human identification. PMID- 10726812 TI - Evaluation of a direct alpha-amylase assay using 2-chloro-4-nitrophenyl-alpha-D maltotrioside. AB - We present the adaptation of an IFCC method for alpha-amylase using 2-chloro-4 nitro-phenyl-alpha-D-maltotrio-side as substrate (1) suited for routine work at 37 degrees C. In the assay, a constant proportion of substrate, i. e. 92%, is directly converted to 2-chloro-4-nitrophenol and maltotriose. The method is based on multi- and univariate optimization leading to following measurement conditions: substrate, 2.25 mmol/l; chloride, 310 mmol/l; calcium 5.0 mmol/l; 4 morpholinoethanesulphonic acid, 50 mmol/l; pH 6.28. The assay may be carried out manually or by mechanized procedures, with substrate or sample start, and it shows these analytical properties in measuring amylase activity of sera: no lag phase, detection limit 2.9 U/l, linear range < or = 820 U/l (for 300 s) or < or = 1450 U/l (for 120 s of measurement), and total manual imprecision 3.2% (CV) at 46 U/l. Bilirubin < or = 630 micromol/l, haemoglobin < or =6 g/l, triacylglycerols < or =30 mmol/l, heparin < or =100 kU/l, and glucose < or =120 mmol/l do not interfere. For adults, we established a preliminary 0.95-reference interval of 30 90 U/l not dependent on sex or age. A close association with the IFCC method demonstrates the reliable transfer of its measurement conditions to a robust routine method with minimal changes. PMID- 10726813 TI - Accurate platelet counting in an insidious case of pseudothrombocytopenia. AB - Anticoagulant-induced aggregation of platelets leads to pseudothrombocytopenia. Blood cell counters generally trigger alarms to alert the user. We describe an insidious case of pseudothrombocytopenia, where the complete absence of Coulter counter alarms both in ethylenediaminetetraacetic acid blood and in citrate or acid citrate dextrose blood samples was compounded by the fact that the massive aggregates were exclusively found at the edges of the blood smear. Non recognition of pseudothrombocytopenia can have serious diagnostic and therapeutic consequences. While the anti-aggregant mixture citrate-theophylline-adenosine dipyridamole completely failed in preventing pseudothrombocytopenia, addition of iloprost to anticoagulants only partially prevented the aggregation. Only the prior addition of gentamicin to any anticoagulant used resulted in a complete prevention of pseudothrombocytopenia and enabled to count accurately the platelets. PMID- 10726814 TI - A new liquid homogeneous assay for HDL cholesterol determination evaluated in seven laboratories in Europe and the United States. AB - We evaluated a new liquid homogeneous assay for the direct measurement of high density lipoprotein cholesterol (HDL-C Plus) in seven laboratories. The assay includes two reagents which can be readily used in most available clinical chemistry analyzers. The total CVs of the new method were below 4.6% and the bias in relation to the designated comparison method was below 3.9%. The total error ranged between 4 to 7%. HDL-C values determined by this method were in good agreement with those obtained by the old homogeneous assay using lyophilized reagents, and other homogeneous and precipitation assays (0.944 < r < 0.996). The assay was linear up to at least 3.89 mmol/l HDL-C. Hemoglobin did not interfere, whereas in icteric samples slight deviations were observed. Lipemia up to 11.3 to 22.6 mmol/l triglycerides did not interfere with this homogeneous HDL-C assay. In samples of patients with paraproteinemia, discrepant results were seen. This liquid homogeneous HDL-C assay was easy to handle and produced similar results in all laboratories participating in this study. This method will enable clinical laboratories to reliably measure HDL-C for risk assessment of coronary heart disease. PMID- 10726815 TI - Tissue release of cardiac markers: from physiology to clinical applications. AB - The early release of cardiac markers is influenced by a variety of factors, the most important influence being their intracellular compartmentation. In contrast to the release of cytosolic proteins, the release of structurally bound proteins requires both a leaky plasma membrane and a dissociation or degradation of the subcellular structure, which is a slower process. Another major impact is the susceptibility to the degradation by cytosolic proteases, such as the calpains. The lysosomes are stable within the first 3-4 hours after onset of ischemia, and, therefore, their enzymes are not involved in the early degradation of structurally bound proteins. Troponin I and troponin T are substrates of micro calpain. Current experimental as well as clinical results suggest that the molecular mass seems to be of minor importance for the pattern of appearance of myocardial proteins in blood after myocardial infarction. However, within the family of molecules with a certain intracellular compartmentation, the molecular mass is an influence on the appearance in blood, because heavier molecules diffuse at a slower rate, and particularly smaller molecules, such as myoglobin, may enter the vascular system to an even larger extent directly via the microvascular endothelium. The higher the concentration gradient of a marker between the cardiomyocytes and the interstitial space, the faster a parameter will translocate from sarcoplasma to the interstitial space as soon as the plasma membrane permeability is increased. Another influence is local blood and lymphatic flow. Recent experimental studies showed that reperfusion causes a true acceleration of cellular protein leakage by an acute manifestation of plasmalemmal disruptions and not just an enhanced wash out. Marker protein time courses after myocardial damage are also markedly influenced by their disappearance rate from blood. Most proteins appear to be catabolized in organs with a high metabolic rate, such as liver, pancreas, kidneys, and the reticuloendothelial system. Smaller molecules, such as myoglobin, also pass the glomerular membranes of the kidneys and are reabsorbed and subsequently metabolized in tubular epithelial cells. PMID- 10726816 TI - The specificity of biochemical markers of cardiac damage: a problem solved. AB - This paper reviews the tissue specificity of cardiac troponin I (cTnl), cardiac troponin T (cTnT) and creatine kinase (CK) MB in human and animal heart and skeletal muscles. Studies reveal that CK-MB can be expressed up to 20% of total CK activity in human skeletal muscle; and therefore is not 100% specific for the heart. One cTnl isoform has been described and shown to be 100% specific for the heart. While one to four cTnT isoforms are expressed in diseased and regenerating human skeletal muscle, these isoforms are not the same as the cTnT isoforms expressed in the human heart and are not detected by the cTnT diagnostic assays used in clinical practice. Representative cases are described demonstrating the role of monitoring cardiac troponins in blood for differentiating false positive CK-MB increases due to skeletal muscle injury. Further, sufficient reactivity and tissue specificity of cTnl and cTnT assays are demonstrated for use as markers of myocardial injury in laboratory animals. Monitoring cTnl and cTnT concentrations in the circulation appears poised as the new standards for detection of myocardial injury. PMID- 10726817 TI - Biochemical factors influencing measurement of cardiac troponin I in serum. AB - Troponin I (cTnI), a sensitive and reliable marker of damaged cardiac tissue, is now widely used in clinics. But the existence of different cTnI assays with a wide variety of cut-off values and discrepancies between the results of measurements of one and the same sample by different assays is puzzling for clinicians. The most urgent issue at the moment is the development of the international standard, which can be used for the calibration of different assays, thus decreasing between assay biases. But another important item, which should be considered by manufacturers, is the standardisation of the epitopes of the antibodies used for the assay development. The importance of such standardisation originates from the complicated biochemical nature of cTnI. Here we briefly try to analyse the main factors that can influence antigen recognition by different antibodies and formulate principles of antibody selection for assay development. PMID- 10726818 TI - The sensitivity of cardiac markers: an evidence-based approach. AB - The aim of this study was to determine whether, using an evidence-based approach, the results of the papers found in the literature are valid and sufficiently scientifically rigorous to be used to definitely address the problem of cardiac marker sensitivity in detection of acute myocardial infarction. In particular, the diagnostic sensitivities of myoglobin, creatine kinase (CK)-MB isoenzyme, determined as mass concentration, CK-MB isoforms, and of the two cardiac troponins, troponin I and troponin T, were reviewed using a priori formulated inclusion/exclusion criteria for judging the eligibility of studies to be included in the analysis. A clear final message derived from this systematic analysis is the unacceptably poor diagnostic sensitivity of all evaluated markers at patient admission, with substantial failure rate to rule out myocardial infarction at this time. Myoglobin is at present the most sensitive of the markers studied for excluding early AMI with an optimum timing of sampling at patient presentation and approximately 4 h later. However, this marker cannot be used by itself as a proportion of patients admitted to the hospital with a late infarction could be missed. The early rate of rise of CK-MB mass and troponin T is similar. Maximum sensitivity of these two parameters is achieved by the analysis of a second sample 6 to 12 h after admission. Additional larger studies are needed to address the question which troponin shows earlier release after myocardial damage, and to clarify the role of CK-MB isoforms as a possible early marker of myocardial infarction. PMID- 10726819 TI - Risk stratification and therapeutic decision making in patients with acute coronary syndrome--the role of cardiac troponin T. AB - Patients with chest pain represent an inhomogeneous group with greatly varying severity of coronary artery disease and cardiac risk. The proper selection of different treatment strategies in these patients requires reliable risk assessment. Patients with definitive myocardial infarction: in patients with ST segment elevation on ECG, a positive troponin T (cTnT) on admission identifies a group of patients having a threefold higher mortality rate than patients with a negative cTnT test. The differences in risk based on cTnT are found for patients treated with thrombolytic as well as mechanical recanalization therapy. These differences in mortality based on admission cTnT may be explained by more severe coronary artery disease, worse left ventricular function, and less efficient microvascular reperfusion in the cTnT-positive patients. Patients with rest angina: in patients with angina at rest, a positive cTnT value on admission identifies a subgroup having a threefold higher cardiac event rate than cTnT negative patients. The cTnT-positive patients seem to benefit from treatment with low molecular weight heparin and fibrinogen receptor antagonists, while cTnT negative patients do not. The differences in risk and response to therapy may be due to more severe coronary artery disease, more critical coronary artery stenoses, and a higher rate of intracoronary thrombus formation in the cTnT positive versus negative patients. Low risk chest pain patients: in low risk chest pain patients, (i.e. no rest angina, no ECG-changes) cTnT-positive patients on admission have a twofold higher cardiac event rate than cTnT-negative patients. The proper treatment strategy for the low risk cTnT-positive patients remains to be determined. Troponin T versus troponin 1: many of the findings on cTnT also relate to troponin I. However, there is a high interassay variability of troponin I assays, which has to be taken into consideration. PMID- 10726821 TI - EC4 European syllabus for post-graduate training in clinical chemistry. Version 2 -1999. European Communities Confederation of Clinical Chemistry, EC4 Register Commission. PMID- 10726820 TI - Cardiac markers: centralized or decentralized testing? AB - Testing for the diagnosis of acute myocardial infarction and other diseases included in the spectrum of the "acute coronary syndrome" is rapidly changing from the traditional enzymatic assays to mass measurement of more specific and sensitive markers (cardiac troponins, CK-MB and myoglobin). Several questions have arisen since the introduction of these new markers into the clinical setting: the choice of strategies for optimizing the utilization of biochemical assays combining different (early and specific) markers, a rationale for sampling specimens and the identification of clinically useful turnaround times. In particular, for achieving the last goal, attention has been directed toward near patient testing for cardiac markers in addition to, or as a replacement for, traditional diagnostic methodologies. While qualitative methods for measuring cardiac markers at the bedside have some limitations which compromise their clinical usefulness, new quantitative devices offer a real alternative to decentralized testing. Regulatory and quality management issues related to near patient testing, as well as the performance of recently introduced devices for a decentralized measurement of cardiac markers are reviewed. PMID- 10726822 TI - Plasma malondialdehyde and obesity: is there a relationship? PMID- 10726823 TI - Reference intervals: are interlaboratory differences appropriate? PMID- 10726824 TI - Clinical aspects of neurofibromatosis 1. PMID- 10726825 TI - Acute encephalitis in Swiss children: aetiology and outcome. AB - Since published data on the course and prognosis of encephalitis in Central Europe is limited, we retrospectively evaluated 104 children with either acute strict sense encephalitis (n = 80) or acute cerebellar ataxia (n = 24) treated at the Department of Pediatrics, University of Bern, Switzerland, between 1980 and 1991. Of the 80 patients with strict sense encephalitis, four (5%) died acutely and 28 (36%) of 78 followed up had sequelae - eight patients with severe, six with moderate and 14 with mild sequelae. Young age and seizures were shown to correlate with poor outcome. Among the 24 patients with acute cerebellar ataxia, there was no fatal outcome and none developed severe residua, but six had mild and one had moderate sequelae. Initial cerebrospinal fluid white cell count was significantly higher in these children with sequelae compared with those without any sequelae after acute cerebellar ataxia. PMID- 10726826 TI - Neurophysiological findings in a case of carbohydrate-deficient glycoprotein (CDG) syndrome type I with phosphomannomutase deficiency. AB - The carbohydrate-deficient glycoprotein (CDG) syndromes are multisystemic disorders involving the glycosylation pathway. The most common subtype is CDG syndrome type I (CDG I). In most CDG I patients a phosphomannomutase (PMM) deficiency has been recognized as the basic defect. We made a neurophysiological evaluation in an 8-year-old boy affected by CDG I with PMM deficiency. The evaluation included central and peripheral nervous system assessment [electroencephalogram (EEG), multimodal evoked potentials (MEP), somatosensory evoked potentials (SEP), visual evoked potentials (VEP), auditory brainstem response (ABR), electroretinogram (ERG) and motor and sensory nervous conduction velocity (NCV)]. We found a peculiar electrophysiological pattern characterized by slowly and mildly progressive motor NCV reduction; progressive impairment of ERG and VEP; slowing of background activity and sharp waves at the EEGs; late sensorineural abnormality of ABR; decreased amplitude and increased latency of SEP. To our knowledge this is the first report involving the neurophysiological aspects both at onset and during follow-up of a case of CDG I with proven PMM deficiency. PMID- 10726827 TI - Familial spastic paraplegia, axonal sensory-motor polyneuropathy and bulbar amyotrophy with facial dysmorphia: new cases of Troyer-like syndrome. AB - We studied two Libyan siblings, born to healthy consanguineous parents, who had suffered from a progressive neurological disorder, characterized by facial dysmorphia, ataxia, spastic paraplegia and an axonal sensory-motor polyneuropathy, since the age of 3 years. The clinical picture progressed slowly over a 6-year period to involve also bulbar and distal limb muscles. Interestingly, we found unusual tubulofilamentous inclusions in peripheral nerves and presynaptic buttons at the neuromuscular junctions. Describing the clinical picture of this presumably new disorder, we comment on the difference from similar conditions. PMID- 10726828 TI - Elevation of serum creatine kinase as the only manifestation of an intragenic deletion of the dystrophin gene in three unrelated families. AB - This study reports three children from three unrelated families, aged from 9 to 12 years, who were investigated because of the incidental finding of elevated serum creatine kinase (CK) levels and were found to have a dystrophinopathy. The molecular defect consisted of a deletion of variable extent within the central rod domain of the dystrophin gene, involving either exons 32-44 or 48-51 or 48 53. In each family we found the same deletion in at least one adult male relative aged from 40 to 77 years, who was either completely asymptomatic or had very mild muscle involvement (thin muscles and/or mild scoliosis), with normal or borderline CK levels. This study suggests once again that deletions of the central rod domain of dystrophin may be associated with elevation of serum CK as the only manifestation and that prediction of the clinical severity based solely on the molecular findings should be interpreted with caution. PMID- 10726829 TI - Chemotherapy with Adriamycin (doxorubicin) and CCNU (lomustine) in four children with recurrent craniopharyngioma. AB - BACKGROUND: The prognosis of craniopharyngioma in children after subtotal surgical removal, followed by irradiation of remaining tumour with 50 Gy, is better than usually reported. In our subjects we found a relapse rate of 5% in the last 20 years. The treatment of recurrences forms a special problem because the possibilities of adjuvant radiotherapy are restricted. We report on a chemotherapeutic treatment after multiple or very rapid recurrences of craniopharyngioma in four children. METHODS: Four children experienced their first tumour recurrence at respectively 3, 8, 50 and 59 months after the initial treatment. New neurosurgical attempts to remove the recurring tumour, and in one patient a second course of radiotherapy, were performed, but there were two or more recurrences in these children, resulting in further restriction of surgical or radiotherapeutical possibilities. Chemotherapy was given, consisting of five intravenous ambulatory courses of Adriamycin (doxorubicin) (33 mg/m2/day, continuously over 3 days) together with oral CCNU (lomustine) (80 mg/m2 at day 1) at 6-weeks intervals. RESULTS: After the chemotherapy there was no further tumour recurrence after 12, 10, 3 and 3 years respectively. In the third patient a cystic relapse occurred after 3 years' remission. In the fourth patient a complete regression was observed of the cystic part of the tumour. The side effects of the chemotherapy consisted of alopecia and bone marrow depression. No signs of cardiomyopathy have been found. CONCLUSION: Treatment of recurrent craniopharyngioma in children by chemotherapy with anthracyclines and nitrourea derivates may be effective. PMID- 10726830 TI - McArdle's disease in childhood: report of a new case. AB - McArdle's disease (glycogenosis type V) is an inherited glycogen storage disease characterized clinically by myalgia, cramps and sometimes myoglobinuria, triggered by exercise. The onset of exercise intolerance is usually in late childhood or adolescence and diagnosis is exceptionally established during infancy. We report the case of a 6-year-old girl who had been complaining of aching muscles for a long time, and who presented after a near-drowning incident, with extensive muscle necrosis, probably secondary to myophosphorylase deficiency induced cramps. These unusual manifestations led to the diagnosis of this rare disorder. We compare the clinical findings of this case to nine previous reports. This highlights the heterogeneous spectrum of this disease in childhood and supports the distinction of three clinical pictures in childhood: a neonatal form rapidly fatal, a milder form with congenital myopathic symptoms and a benign classical form with myalgia, cramps and pigmenturia. PMID- 10726831 TI - Neuroborreliosis and isolated trochlear palsy. AB - We report here for the first time a child with isolated trochlear palsy and neuroborreliosis. IgG and IgM antibodies against Borrelia burgdorferi were highly positive in serum and cerebrospinal fluid respectively. The symptoms resolved completely after initiation of antibiotic treatment with ceftriaxone. PMID- 10726832 TI - Post-traumatic mutism in children: clinical characteristics, pattern of recovery and clinicopathological correlations. AB - Among the numerous clinical syndromes observed after severe traumatic head injury, post-traumatic mutism is a disorder rarely reported in adults and not studied in any detail in children. We report seven children between the ages of 3 1/2 and 14 years who sustained severe head injury and developed post-traumatic mutism. We aim to give a precise clinical characterization of this disorder, discuss differential diagnosis and correlations with brain imaging and suggest its probable neurological substrate. After a coma lasting from 5 to 25 days, the seven patients who suffered from post-traumatic mutism went through a period of total absence of verbal production lasting from 5 to 94 days, associated with the recovery of non-verbal communication skills and emotional vocalization. During the first days after the recovery of speech, all patients were able to produce correct small sentences with a hypophonic and monotonous voice, moderate dysarthria, word finding difficulties but no signs of aphasia, and preserved oral comprehension. The neurological signs in the acute phase (III nerve paresis in three of seven patients, signs of autonomic dysfunctions in five of seven patients), the results of the brain imaging and the experimental animal data all suggest the involvement of mesencephalic structures as playing a key role in the aetiology of post-traumatic mutism. PMID- 10726833 TI - Electroclinical correlation in neonatal seizures. AB - A dissociation between clinical and electroencephalographic phenomena is often observable in neonatal seizures. This finding raises important questions, including those related to the management of these patients. Another characteristic of neonatal convulsions is represented by their increased tendency towards status epilepticus. In order to examine the electroclinical correlation and its possible relationship to the occurrence of status epilepticus, recorded video-electroencephalograms of 17 newborns were submitted to detailed analysis. Time of onset, duration and other characteristics of all clinical and electrical events were noted. Five degrees of correlation were observed, from constant concurrence to complete dissociation. Examining the role of different parameters, it was observed that the incidence of electric discharges related significantly to the occurrence of electroclinical dissociation. PMID- 10726834 TI - Rett syndrome: critical examination of clinical features, serial EEG and video monitoring in understanding and management. AB - We studied data on seizures, vacant spells and breathing dysrhythmia from the British Rett Survey and 150 electroencephalographic records from 78 classic cases, including 23 with prolonged synchronous recordings of EEG, respiration and movement. The proportion of abnormal records increased from 6 of 18 (33%) during the first 6 months of the regression period to 44 of 59 (75%) in the later period to 6 years, the increase in abnormality following rather than preceding the onset of regression. In young girls the EEG abnormality increased in sleep but decreased during episodic hyperventilation and breath-holding. Epileptogenic activity was commonly present without clinical seizures. Eleven vacant spells were monitored and were not epileptic but related to the breathing abnormality. Full monitoring is essential when supposed seizures are intractable. The intermittent EEG abnormality and behavioural changes indicate abnormal fluctuating arousal possibly of midbrain or brainstem origin. PMID- 10726835 TI - Subcortical somatosensory evoked potentials after median nerve stimulation in children. AB - We report our normative data of subcortical somatosensory evoked potentials (SEPs) after median nerve stimulation from a group of 55 children 4-15 years of age and 18 young adults 18-29 years of age. We recorded near-field potentials from the brachial plexus, the cervical cord and the somatosensory cortex. The far field potentials P13, P14 and N18 from the brainstem were recorded from the scalp electrodes, when a non-cephalic reference at the contralateral Erb's point or an ear reference was used. The N9 (brachial plexus), N13a (dorsal horn), P13 (caudal medulla oblongata), N18 (medulla oblongata) and N20 (somatosensory cortex) were present in all subjects. The N13b (dorsal column near the foramen magnum or cuneate nucleus) was observed in all children and in 16 adults, P14 (medial lemniscus) in 52 children and 17 adults. The median nerve SEPs provide reliable information about the function of the somatosensory pathway from the upper limb. The subcortical median nerve SEPs should be particularly useful to detect lesions of the upper cervical cord and the cervicomedullary junction. The subcortical SEPs remain unchanged during sleep and facilitate reliable SEP recordings, when sedation is necessary in infants and children. PMID- 10726836 TI - Somatosensory evoked potentials after posterior tibial nerve stimulation- normative data in children. AB - We report normative data of somatosensory evoked potentials to posterior tibial nerve stimulation from 47 children 4-15 years of age. We recorded near-field potentials from the peripheral nerve, the cauda equina, the lumbar spinal cord and the somatosensory cortex. Far-field potentials were recorded from the scalp electrodes with a reference at Erb's point and on the earlobe. The near-field potentials N8 (peripheral nerve) and P40 (cortex) were present in all children. N20 (near-field from the cauda equina) was recorded in 38 subjects. N22 (near field from the lumbar spinal cord), P30 and N37 ( both far-field waveforms probably generated in the brainstem) were recorded in 46 subjects each. The latencies and the peripheral conduction time (N8-N22) increased with age, while the central conduction time (N22-P40) and the intracranial conduction time (P30 P40) both decreased with age (up to about 10 years of age). The spinal conduction time (N22-P30) was relatively independent of age. The interpeak latencies allow the assessment of specific portions of this pathway. The subcortical posterior tibial nerve-somatosensory evoked potentials are of particular interest in children when the cortical peaks are influenced by sedation and sleep, or by anaesthesia. PMID- 10726837 TI - Magnetic resonance imaging findings in mild mucopolysaccharidosis II (Hunter's syndrome). PMID- 10726838 TI - Acute cerebellitis with near-fatal cerebellar swelling and benign outcome under conservative treatment with high dose steroids. AB - Acute cerebellar swelling is an emergency because of brainstem compression as well as upward or downward cerebellar herniation. Few childhood cases are on record, with fatal outcome in three out of six. We report a girl with probable Epstein-Barr virus-associated cerebellar swelling who recovered completely with steroid treatment after a stormy course. Review of the literature showed that all three patients, including our own, who recovered fully, received high-dose steroids in contrast to none of the four patients who died or survived with sequelae. Neuroimaging and evoked potential studies are useful for early diagnosis and disease monitoring. We conclude that for the time being high-dose steroid treatment is advocated in patients with acute infectious or parainfectious cerebellar swelling. PMID- 10726839 TI - Epilepsy (update). PMID- 10726840 TI - Some influences on cognition in early life: a short review of recent opinions. AB - Many factors can affect a child's ability to learn, and these may operate before, during, and after birth. Some of these are considered, and are hopefully important, but not necessarily the most obvious. The intrauterine environment may not be so influential as the infant's genetic endowment, but nevertheless is of considerable importance. For example, malnutrition resulting from placental insufficiency leading to small-for-date babies can impair brain development. Also a relationship between birth weight and cognitive function in early adult life has been demonstrated; and the babies' condition at birth can be a risk factor for various disabilities. Lack of stimulation in infancy, for example if postnatal depression interferes with the mother's interaction with her baby, can significantly affect the infant's learning capacity. A good paradigm is the development of language, which starts with the way mothers 'talk' to their babies; and this continues into childhood. The importance of nutrition also continues, and is one of the factors which favours breast feeding against formula foods. The whole subject has to be viewed against the background of normal development, and the great loss of neurons and synapses that occur in early life. If neural circuits are formed at this time, and these neurons and synapses are not lost, the easier it will be to exploit them. This emphasizes the importance of early education, which is not always sufficiently acknowledged. PMID- 10726841 TI - Neurodevelopmental profile in low-risk preterm infants at 5 years of age. AB - The goal of this study is to determine the neurodevelopmental profile of a group of low-risk preterm infants and to determine whether the potentially unfavourable outcome is due to a few infants with moderate to severe impairments or to a majority of infants with only slight impairments. In a prospective study 44 low risk preterm infants, i.e. infants with a neonatal risk score indicating a favourable outcome, born between 25-34 weeks gestational age, and 18 healthy term infants were examined neurologically and tested neuropsychologically at 5 years of age. The more unfavourable outcome in the group of low-risk preterm infants compared with the term infants was largely attributable to a poorer outcome in 12 of the 44 low-risk preterm infants. The remaining low-risk preterm infants showed similar test scores compared with the term infants. From these results we conclude that the unfavourable neurodevelopmental outcome of low-risk preterm infants is due to moderate to severe impairment in a few low-risk preterm infants, rather than slight impairment in the majority. The low-risk preterm infants with an unfavourable outcome showed particular impairment on measures of visual-motor integration, concentration and auditory memory in combination with integrative functions. PMID- 10726842 TI - An early onset muscular dystrophy with diaphragmatic involvement, early respiratory failure and secondary alpha2 laminin deficiency unlinked to the LAMA2 locus on 6q22. AB - We present four subjects from one family and one subject (with an affected sibling who had died) from a second, unrelated family, with early onset, Duchenne like, muscular dystrophy who presented with proximal girdle weakness, calf and generalized muscle hypertrophy, selective wasting of the sternomastoid muscles, rigidity of the spine and contractures of the tendo Achilles. Intellect was normal. Serum creatine kinase was grossly elevated and the muscle biopsies showed a dystrophic picture. All five subjects have developed early respiratory failure due to severe diaphragmatic involvement; two have already died aged 4 and 7 years of age and the remaining three are dependent on night time ventilation. There has been very little deterioration over time in the skeletal muscle function, and the survivors remain ambulant, the oldest being 11 years. Immunocytochemical studies of the muscle biopsy showed a normal pattern for dystrophin and the dystrophin associated glycoproteins, but a reduction of the laminin alpha2 chain of merosin. Magnetic resonance imaging of the brain was normal. The disease did not link to the LAMA2 locus for laminin alpha2 on chromosome 6q, so that these families seem to represent a new form of autosomal recessive muscular dystrophy with a secondary merosin deficiency. The primary protein deficiency has not yet been identified. PMID- 10726843 TI - Excessive production of tumour necrosis factor alpha by peripheral blood mononuclear cells in X-linked adrenoleukodystrophy. AB - Previous studies have suggested that cytokine production may play a role in the cerebral demyelination and phenotypical variations of X-linked adrenoleukodystrophy (ALD). At initial evaluation, the serum titre of tumour necrosis factor alpha (TNFalpha) but not interleukin IL-1alpha, IL-1beta, IL-6, IL-4 and interferon INF-gamma of 12 ALD patients with cerebral demyelination, was higher than that of controls and of six ALD patients without cerebral demyelination. However, TNFalpha was not detected in the cerebrospinal fluid of the same patients with cerebral demyelination. In a serial study of 15 patients over 2-5 years, the level of serum TNFalpha paralleled the progression of demyelination. Peripheral blood mononuclear cells from 15 symptomatic ALD patients, irrespective of their clinical phenotype, produced higher levels of TNFalpha than controls after in vitro stimulation by lipopolysaccharide. The production of other cytokines was normal. Abnormal production of TNFalpha was observed in two of six asymptomatic ALD patients but was absent in both of two bone marrow transplanted ALD patients. These data suggest that monocytes of ALD patients have an intrinsic alteration in the regulatory pathway of TNFalpha production. PMID- 10726844 TI - Cerebrospinal fluid free choline in movement disorders of paediatric onset. AB - We measured free choline in cerebrospinal fluid (CSF) of 78 patients with movement disorders of paediatric onset and various controls as a putative index of central phospholipid metabolism. Most of the disorders studied were myoclonic disorders, such as progressive myoclonus epilepsy, the opsoclonus-myoclonus syndrome, and essential myoclonus, but other movement disorders, interictal seizure disorders, and different neurological and nonneurological disorders were also included. There were no significant differences in CSF choline concentrations in myoclonic disorders or other movement disorders compared with controls. The CSF choline levels were lowest in children with seizure disorders including progressive myoclonus epilepsy. In progressive myoclonus epilepsy, the CSF choline values resembled other epileptic disorders rather than other myoclonic disorders. When all the data were analysed collectively, no significant relation of CSF choline was found to patient age, gender, aliquot of CSF measured, or the length of time the sample was stored at -70 degrees C. Separate analyses of data from children and adults showed a trend toward a biphasic relation between patient age and CSF choline which could be pursued in developmental studies of normal subjects. Reduced CSF choline may indicate increased choline incorporation into brain phospholipids, disturbances of choline metabolism, decreased choline release, or non-neural factors. PMID- 10726845 TI - Muscle-eye-brain disease: clinical features, visual evoked potentials and brain imaging in 20 patients. AB - Clinical features, magnetic resonance imaging and visual evoked potentials were analysed and correlated in 20 Finnish patients with muscle-eye-brain disease. Significantly enhanced visual evoked potentials were found in 15 patients (giant in 14 of them). Magnetic resonance images were available in 17 cases. The images of 12 patients with giant visual evoked potentials showed typical brain malformation pachygyria with a nodular cortical surface i.e. cobblestone cortex, midline defect and hypoplastic pons but no significant abnormalities in the grey white matter. One male had typical structural changes but flat visual evoked potentials. His extreme hydrocephalus with optic nerve compression may explain the findings. No structural changes on magnetic resonance images were found in the remaining four patients; however, in two of them marked alterations in the white matter were found. Three of these patients showed normal and one flat visual evoked potentials. Only one patient with giant visual evoked potentials and typical structural findings on magnetic resonance imaging had changes in a large area in the white matter (several attacks of status epilepticus might have caused the alterations in the white matter). Thus, the combination of giant visual evoked potentials and typical structural changes on magnetic resonance imaging with normal intensities of white matter and deep grey matter seems to be a good marker for patients with muscle-eye-brain disease. PMID- 10726846 TI - X-linked myotubular myopathy--a long-term follow-up study. AB - X-Linked myotubular myopathy is a well delineated congenital myopathy, with a high neonatal and early childhood mortality. Only a single gene, mapping to Xq28 has been implicated and has recently been characterized. Phenotypic variability, both inter- and intrafamilial, has been recorded. Its severest expression is a uniform disease with polyhydramnios due to prenatal (neuromuscular) swallowing disorder, and partial inability to expand the lungs postnatally leading to early postnatal death in all. The mildest expression appears to be represented by the first family reported in the literature in which intrafamilial phenotypic variability was marked. There was neonatal asphyxia, but recovery took place in most affected patients and very mild expression permitting normal life into adulthood has been found in two patients. A long-term follow-up is given on both these families. Results emphasize the importance of the family history when trying to prognosticate in an individual case. PMID- 10726847 TI - Intravenous immunoglobulin treatment in children with Guillain-Barre syndrome. PMID- 10726848 TI - The Second Congress of the European Paediatric Neurology Society: Maastricht 8-11 October 1997. PMID- 10726849 TI - Chiral monophosphines as ligands for asymmetric organometallic catalysis. AB - Chelating chiral diphosphines are often used as ligands of organometallic complexes. However monophosphines, or more generally ligands with one phosphorus linked to one or several heteroatom, may also be useful. This review gives the main results obtained in that area, by considering the classes of monodentate chiral ligands bearing one P(III) atom and involved in asymmetric catalysis with organometallic complexes. PMID- 10726850 TI - High entrapment of insulin and bovine serum albumin into neutral and positively charged liposomes by the remote loading method. AB - Remote loading of insulin and bovine serum albumin (BSA) into neutral or positively-charged liposomes by incubation under a transmembrane pH gradient or non-pH gradient was investigated. Trapping efficiencies in several incubation conditions were compared with those of the conventional reverse-phase evaporation vesicle method (c-REV method). For neutral liposomes, insulin could not be effectively loaded into the liposomes by incubation, regardless of the incubation conditions. The trapping efficiency of insulin into positively-charged liposomes was higher than that of neutral liposomes, especially by the pH-gradient method. Insulin could be loaded into positively-charged liposomes about twofold more efficiently than by the pH-gradient method, compared with the c-REV method. Insulin distributed more on the surface of liposomes by the pH-gradient method than by the c-REV method. BSA showed significantly higher affinity to positively charged liposomes than to neutral ones by various methods. However, the transmembrane pH-gradient method did not increase BSA loading into liposomes, compared with the c-REV and the non-pH-gradient methods. Our results suggest that the pH-gradient method, combining electrostatic interactions, may be useful for preparation of liposomal insulin and that the high hydrophobicity of BSA may not increase the remote loading of BSA into liposomes by the pH-gradient method. PMID- 10726851 TI - Characterization of the activity of L-ascorbic acid 2-[3,4-dihydro-2,5,7,8 tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-be nzopyran-6-yl-hydrogen phosphate] potassium salt in hydroxyl radical elimination. AB - The effect of L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12 trimethyltridecyl)-2H -1-benzopyran-6-yl-hydrogen phosphate] potassium salt (EPC K1) on hydroxyl radical (*OH) elimination was studied using electron spin resonance (ESR) and spectrophotometric experiments. The addition of EPC-K, and *OH scavengers eliminated the *OH generated from Cu2+/H2O2, Fe2+/H2O2 and H2O2/UV irradiation reaction systems. However, in competitive reactions using different concentrations of a spin-trap agent, the addition of the *OH scavenger altered the IC50 values, whereas the addition of EPC-K1 and a metal chelater did not change the value in the Cu2+/H2O2 and Fe2+/H2O2 reaction systems. The addition of EPC-K1 and metal chelater changed the ESR signal for free Cu2+. The spectrophotometric experiments confirmed that the addition of EPC-K1 and metal chelater altered the absorption spectra due to CuCl2 and FeSO4, whereas the *OH scavenger did not alter the spectra. Therefore, it was demonstrated that EPC-K, has the ability both to scavenge *OH directly and to inhibit the generation of *OH by the chelation of Cu2+ and Fe2+. PMID- 10726852 TI - Chemical constituents of Micromelum minutum. Isolation and structural elucidation of new coumarins. AB - The chemical constituents of an acetone extract of the stems of Micromelum minutum Wight et Arn (Rutaceae), collected at Nakorn-Rachasima province in Thailand, were studied. Six new coumarins, named micromarin-A (1), -B (2), -C (3), -F (4), -G (5), and -H (6), were isolated along with six known coumarins, and their structures were elucidated by chemical and spectroscopic methods. PMID- 10726854 TI - Mechanism of C-2 hydroxylation during the biosynthesis of 20-hydroxyecdysone in Ajuga hairy roots. AB - Feeding synthetic [2beta-2H]- and [2alpha-2H]-cholesterols to the hairy roots of Ajuga reptans var. atropurpurea and 2H-NMR analysis of the biosynthesized 20 hydroxyecdysone revealed that hydroxylation at C-2 proceeds with retention of configuration. Feeding [2alpha,3alpha-2H2]cholesterol followed by 2H-NMR analysis of the 2,3,22-triacetate of the resulting 20-hydroxyecdysone ruled out a mechanism which involves a partial loss of the 2alpha-hydrogen. The steric course of C-2 hydroxylation in Ajuga hairy roots is identical with that reported in the insect, Schistocerca gregaria. PMID- 10726853 TI - Chemical constituents of Avicennia alba. Isolation and structural elucidation of new naphthoquinones and their analogues. AB - Three new naphthoquinones and their analogues, named avicequinone-A (1), -B (2), C (3), and avicenol-A (4), -B (5), -C (6), respectively, were isolated from the stem bark of Avicennia alba (Avicenniaceae) collected in Singapore, and their structures were elucidated by means of spectral methods. Gillan and co-workers have proposed that the structures of the new phytoalexins isolated from Avicennia marina are 1,2-naphthoquinones 8 and 9. Our synthetic and spectrometric studies showed that these structures should be revised respectively to 1,4 naphthoquinones 2 and 3, named avicequinone-B and -C by us. PMID- 10726855 TI - Structure-specificity relationship of cardiac glycosides as a substrate for glucohydrolase II. AB - Cardenolide glucohydrolase II (CGH II) is a cardenolide-specific glucohydrolase obtained from Digitalis lanata leaves. We investigated the structure-specificity relationship of several cardenolide disaccharides as a substrate for CGH II. Conformation analysis of the substrates was performed using molecular mechanics calculations. The sugar chain conformation of two inert glycosides was significantly different from that of the other glycosides. The other two glycosides, which were weak substrates of CGH II, were suggested to have an intramolecular hydrogen bond between the sugar groups. It was deduced that this hydrogen bond restricts the conformational change of the sugar chain and prevents the glycosides from enzymatic recognition. PMID- 10726856 TI - Sesquiterpenoid derivatives from Ferula ferulaeoides [correction of ferulioides]. IV. AB - Four novel prenyl-furocoumarin type sesquiterpenoid derivatives, 2,3-dihydro-7 hydroxy-2S*,3R*-dimethyl-3-[4,8-dimethyl-3(E),7-nonadie nyl]-furo[3,2-c]coumarin, 2,3-dihydro-7-hydroxy-2R*,3R*-dimethyl-3-[4,8-dimethyl-3(E),7-nonadie nyl] furo[3,2-c]coumarin, 2,3-dihydro-7-hydroxy-2S*,3R*-dimethyl-3-[4-methyl-5-(4 methyl-2-furyl)- 3(E)-pentenyl]-furo[3,2-c]coumarin, and 2,3-dihydro-7-methoxy 2S*,3R*-dimethyl-3-[4,8-dimethyl-3(E),7-nonadie nyl]-furo]3,2-c]coumarin were isolated from the roots of Ferula ferulaeoides [corrected]. Their structures were established by detailed spectral analysis and the biosynthetic pathway leading to these prenyl-furocoumarin type sesquiterpenoids is proposed based on these structures. PMID- 10726857 TI - The constituents of the root and stem of Aristolochia heterophylla Hemsl. AB - Seven new compounds, sodium aristolochate-VII (1), aristolactam-CIV (2), madolin I (3), -J (4), -K (5), -L (6) and -M (7) together with 71 known compounds were isolated and characterized from the fresh root and stem of Aristolochia heterophylla Hemsl. Their structures were determined by spectral methods. Compound 8 was revised as aromadendrane-4beta, 10beta-diol by spectral data and single-crystal X-ray analysis. PMID- 10726858 TI - Steroidal oligoglycosides from the seeds of Allium tuberosum. AB - Three new spirostanol steroidal oligoglycosides, together with a known oligoglycoside, were obtained from the seeds of Allium tuberosum after enzymatic hydrolysis of furostanol saponin fraction by beta-glucosidase. On the basis of spectroscopic analysis, the structure of new spirostanol oligoglycosides were elucidated as (25S)-spirost-5-ene-2alpha,3beta-diol 3-O-alpha-L-rhamnopyranosyl (1-->4)-[alpha-L-rhamnopyranosyl-(1-->2)]-be ta-D-glucopyranoside, (25S) spirostane-3beta,5beta,6alpha-triol 3-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D glucopyranoside, and (25S)-5beta-spirostane-3beta,6alpha-diol 3-O-alpha-L rhamnopyranosyl-(1-->4)-beta-D-glucopyranoside. PMID- 10726859 TI - Preparation and characterization of two crystalline forms of 4-amino-5-chloro-2 methoxy-N-[(2S,4S)-1-ethyl-2-hydroxymethyl-4-pyrroli dinyl]benzamide (TKS159). AB - For 4-amino-5-chloro-2-methoxy-N-[(2S,4S)-1-ethyl-2-hydroxymethyl-4-pyrrolid inyl]benzamide (TKS159), two polymorphs, forms alpha and beta, were prepared and characterized by means of X-ray powder diffractometry, thermal analysis, infrared spectroscopy and 13C-NMR spectroscopy, both in the solution and solid phases. The X-ray powder diffraction analysis gave different patterns for forms alpha and beta. In the thermogravimetry and differential thermal analysis profiles, form beta exhibited characteristic endo- and exothermic peaks at 112.7 degrees C and 116.2 degrees C, respectively, due to the partial melting-induced phase transition to form alpha without accompanying weight loss, and these were followed by an additional endothermic peak at 138.2 degrees C due to fusion. For form alpha, only an endothermic peak at 137.8 degrees C due to fusion was observed. The IR spectroscopic analyses of forms alpha and beta gave different absorption bands assigned to N-H and O-H stretching, N-H bending, and C=O stretching vibrations. From the data obtained by thermal analysis, form alpha was shown to be thermodynamically more stable than form beta. PMID- 10726860 TI - Quaternary isoquinoline alkaloids from Stephania cepharantha. AB - From the quaternary alkaloidal fraction of the dried tubers of Stephania cepharantha, two new isoquinoline alkaloids, stecepharine and tetradehydroreticuline have been isolated along with the known compounds, magnoflorine, menisperine, steponine, cyclanoline, oblongine, cis-N methylcapaurine and 2'-N-methylisotetrandrine. cis-N-Methylcapaurine (=9-O methylstecepharine) was isolated as a natural product for the first time. Their structures were determined on the basis of spectroscopic evidence. PMID- 10726861 TI - Structural studies of C-amidated amino acids and peptides: crystal structures of Z-Gly-Phe-NH2, Tyr-Lys-NH2, and Asp-Phe-NH2. AB - As part of the series investigating the structural features of C-terminal amidated amino acids and peptides, three crystal structures of Z-Gly-Phe-NH2, Tyr Lys-NH2, and Asp-Phe-NH2 were analyzed by the X-ray diffraction method, and their molecular conformations and intermolecular interactions were investigated. Although the respective dipeptides exhibited an energetically allowable torsion angle concerning each backbone or side chain, the observed extended (Z-Gly-Phe NH2, Asp-Phe-NH2) and folded (Tyr-Lys-NH2) conformations were considerably different from those of the corresponding unamidated peptides, due to the conformational flexibility of the respective dipeptides. The comparison between the crystal packings of the amidated and unamidated dipeptides indicated that the C-terminal amides tend to associate with the same neighboring group through hydrogen bonds, in which both the amide NH and O=C groups participate, while the unamidated peptides prefer a linear molecular connection, where both or either of the two carboxyl oxygens participate in the hydrogen bond formation. The difference in hydrogen bonding ability between the C-terminal amide and carboxyl groups has been considered to be based on the structural data of the related peptides analyzed so far. PMID- 10726862 TI - A novel class of inhibitors for human steroid 5alpha-reductase: synthesis and biological evaluation of indole derivatives. II. AB - In a search for novel nonsteroidal inhibitors of human prostatic 5alpha reductase, we found a new series of indole derivatives that showed potent inhibitory activities for the human enzyme. Among them, 4-[(1-benzyl-1H-indol-5 yl)oxyl-3-chlorobenzoic acid (2d, YM-32906) showed more potent inhibitory activity than finasteride with an IC50 value of 0.44 nM. 3-Chloro-4-[[1-(4 phenoxybenzyl)-1H-indol-5-yl]oxy]benzoic acid (2m) showed inhibitory activities for both human and rat prostatic 5alpha-reductase with IC50 values of 2.1 and 73 nM, respectively. The synthesis and structure-activity relationships of these indole derivatives are presented. PMID- 10726863 TI - Stereochemistry of cis- and trans-hinokiresinol and their estrogen-like activity. AB - Naturally occurring phenylpropanoids, hinokiresinol (trans-hinokiresinol) and nyasol (cis-hinokiresinol) were found to possess appreciable estrogen receptor binding activity. Strong differences in activity were observed between the geometrical isomers and enantiomers. Among these, (3S)-cis-hinokiresinol displayed the highest activity, one order of magnitude greater than the activity of genistein. Furthermore, cis- and trans-hinokiresinol stimulated the proliferation of estrogen-dependent T47D breast cancer cells, and their stimulatory effects were blocked by an estrogen antagonist, indicating that the compounds are estrogen agonists. In addition, the absolute configuration of C-3 in (+)-cis-hinokiresinol has been assigned as S by comparison with the circular dichroism spectra of the hydrogenated products prepared from cis and trans ((3S) trans-hinokiresinol: previously assigned) isomers. These results incidentally provide us with an unambiguous answer to contradictory reports regarding the assignment of the full stereochemisry of cis- and trans-hinokiresinol that have existed in the literature for more than two decades. PMID- 10726864 TI - Quantification of the surface morphologies of lactose carriers and their effect on the in vitro deposition of salbutamol sulphate. AB - Application of the scanning probe microscopy technique for quantitative measurement of the surface roughness of lactose carriers was evaluated. The roughness values of four different lactose carriers were related to the in vitro deposition results of the drug, salbutamol sulphate. The rugosity values of the lactose carriers were represented by Ra values which were in the order of DCL 40>DCL-11>lactose 325M>lactose 200 M. In vitro deposition results using a twin impinger showed that rougher carrier surfaces generally allowed more drug particles to be emitted from the capsules and inhaler but the availability of the drug to stage 2 was reduced, as detachment of drug particles from the carrier surfaces was more hindered. There was an optimum Ra value for greater delivery of the drug particles to stage 2 of the twin impinger. A balance between adherence and detachment of the drug from the carrier surface was needed in order to optimize the delivery of a drug to the desired target sites using a dry powder inhaler. PMID- 10726865 TI - Convenient synthesis of 2,3,9,10-tetraoxygenated protoberberine alkaloids and their 13-methyl alkaloids. AB - New and convenient synthesis of 2,3,9,10-tetraoxygenated protoberberine alkaloids and their 13-methyl alkaloids through the same intermediates was developed. Acylation of the brominated benzylphenethylamine (13) with alpha-chloro-alpha (methylthio)acetyl chloride, followed by cyclization with stannic chloride, furnished the key intermediates 4-methylthio-3-phenethylisoquinolin-3-ones (14), which were methylated to provide their methyl derivatives (17). Both isoquinolin 3-ones (14, 17) were easily transformed into protoberberine alkaloids (16) and their 13-methyl alkaloids (21) in good yield. PMID- 10726866 TI - Studies on Lewis acid-mediated intramolecular cyclization reactions of allene-ene systems. AB - The Lewis acid-mediated reactions of allene-ene compounds, derived from 3 methylcitronellal or dimethyl malonate, were carried out using various Lewis acids such as ethylaluminum dichloride, diethylaluminum chloride, titanium chloride, zinc chloride etherate, or boron trifluoride etherate, affording unexpectedly intramolecular [2+2]cycloaddition products under some particular reaction conditions without any formation of intramolecular ene reaction products. PMID- 10726867 TI - Convenient synthesis of 5-trifluoroacetylated imidazoles by ring transformation of mesoionic 1,3-oxazolium-5-olates. AB - Mesoionic 4-trifluoroacetyl-1,3-oxazolium-5-olates (1), obtained from the reaction of N-acyl-N-alkylglycines (2) with trifluoroacetic anhydride, react with amidines to give 5-trifluoroacetylimidazoles (3) in moderate yield. The novel ring transformations of 1 into 3 occur via an initial attack of amidines on the C 2 position of the ring. PMID- 10726868 TI - A tetrahydroisoquinoline-monoterpene glucoside and an iridoid glucoside from Alangium kurzii. AB - From the leaves of Alangium kurzii, a new tetrahydroisoquinoline-monoterpene glucoside, 6-O-methyl-N-deacetylipecosidic acid and a new iridoid glucoside, 10-O benzoyladoxosidic acid, were isolated along with alangiside, demethylalangiside, 6''-O-beta-D-glucosylhenryoside, uridine and four known flavonoid glycosides. The structures of new glucosides were determined on the basis of spectroscopic and chemical methods. PMID- 10726869 TI - Influence of production variables on the sphericity of melt pellets. AB - This paper reports the influence of post-melt impeller speed, post-melt processing time, binder concentration and particle size of bulk material on the sphericity of pellets produced by melt pelletization in a high shear mixer. Lactose was used as the bulk material with polyethylene glycol 3000 as a meltable binder. The sphericity of pellets was found to be affected by post-melt impeller speed and post-melt processing time. Binder concentration and particle size of bulk material had a lesser effect on pellet sphericity. The melt pelletization process can be divided into two spheronization phases (a fast initial rate, followed by a slower rate). The change in the spheronization rate was associated with the pellet size, porosity and flow pattern of the processing material. The present study established a bi-exponential mathematical model to relate the pellet sphericity with post-melt specific energy consumption. The relationship of pellet sphericity with post-melt specific energy consumption was independent of the effects of the production variables. PMID- 10726870 TI - Cimiracemoside a: A new cyclolanostanol xyloside from the rhizome of Cimicifuga racemosa. AB - A new 9,19-cyclolanostane-type triterpene xyloside (1), from the rhizomes of Cimicifuga racemosa, has been isolated together with four known saponins; cimiaceroside A, 25-O-methylcimigenol-3-O-beta-D-xylopyranoside, 27-deoxyactein and 23-O-acetylshengmanol-3-O-beta-D-xylopyranoside. The structure of the new compound was established as 16beta,23:22beta,25-diepoxy-12-acetoxy-3beta,23,24b eta-trihydroxy-9,19-cyclolanost-7-ene-3-O-beta-D-xylopyranoside . For the structure elucidation, 1D- and 2D-NMR experiments and high resolution electrospray ionization Fourier transformation mass spectrometry (HRESIFTMS) were used. PMID- 10726871 TI - Novel potassium channel activators. III. Synthesis and pharmacological evaluation of 3,4-dihydro-2H-1,4-benzoxazine derivatives: modification at the 2 position. AB - A new series of 3,4-dihydro-2H-1,4-benzoxazine derivatives, where various substituents were introduced into one of the geminal dimethyl groups at the 2 position, were synthesized and their potassium channel-activating activity was evaluated. Introduction of a hydroxyl group, as in compound 5, resulted in good solubility in water and a long duration of action compared with the parent compound 1. Introduction of a nitrato group, as in compound 8, produced typical nitrate activity such as exhibited by nitroglycerine in addition to potassium channel-activating activity. X-ray structural analysis of compound 5 showed that the sum of the bond angles around the N atom at the 4 position was 357.8 degrees, suggesting that the N atom had an approximately sp2-like planar bond configuration. PMID- 10726872 TI - Indonesian medicinal plants. XXIII. Chemical structures of two new migrated pimarane-type diterpenes, neoorthosiphols A and B, and suppressive effects on rat thoracic aorta of chemical constituents isolated from the leaves of Orthosiphon aristatus (Lamiaceae). AB - Two novel migrated pimarane-type diterpenes named neoorthosiphols A (1) and B (2) were isolated from the water decoction of the leaves of Orthosiphon aristatus (Lamiaceae), which has been prescribed in Javanese traditional medicine (jamu) for the treatment of hypertension, etc. The absolute chemical structures have been elucidated on the basis of physicochemical properties. It has been found that two migrated pimarane-type diterpenes (1, 2), four isopimarane-type diterpenes (3, 4, 5, 6), three benzochromenes (7, 8, 9) and two flavones (12, 13) exhibit a suppressive effect on contractile responses in rat thoracic aorta, among thirteen chemical constituents (1-13) isolated from the leaves. PMID- 10726873 TI - Synthesis of 2-substituted 3-nitro-1,2-dihydropyridines by heterocyclic annulation reactions of a sec-nitrodienamine with aldehyde compounds. AB - The reaction of a sec-nitrodienamine 3 with aldehyde compounds afforded 2 substituted 3-nitro-1,2-dihydropyridines 5, providing a heterocyclic annulation reaction. PMID- 10726874 TI - A novel cytotoxic C-methylated biflavone from the stem of Cephalotaxus wilsoniana. AB - Bioassay-directed fractionation of an ethanolic extract of Cephalotaxus wilsoniana has resulted in the isolation of a novel C-methylated biflavone, taiwanhomoflavone-A (1). Its structure was elucidated on the basis of spectroscopic analysis. Taiwanhomoflavone-A is cytotoxic with ED50 values of 3.4, 1.0, 2.0 and 2.5 microg/ml, respectively, against KB epidermoid carcinoma of nasopharynx, COLO-205 colon carcinoma, Hepa-3B hepatoma, and Hela cervix tumor cells. PMID- 10726875 TI - O-(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate-N hydroxysuccinimide-CuCl2: a facile and reliable system for racemization-free coupling of peptides having a carboxy-terminal N-methylamino acid. AB - Simultaneous use of N-hydroxysuccinimide (HOSu) and CuCl2 with a HOSu-based uronium coupling reagent, O-(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate, has been found to eliminate the racemization of the carboxy terminal N-methylamino acid residue during the segment condensation. PMID- 10726876 TI - Novel catalytic asymmetric sulfoxidation in water using the hypervalent iodine reagent iodoxybenzene. AB - A new catalytic asymmetric oxidation of sulfides to sulfoxides in water using the hypervalent iodine(V) reagent iodoxybenzene (PhIO2) has been developed. This methodology is distinctly different from the previously reported use of cyclodextrins or biological catalysts and provides a new route to asymmetric sulfoxidation in water. PMID- 10726877 TI - Labeling conditions using a 2-aminobenzamide reagent for quantitative analysis of sialo-oligosaccharides. AB - During 2-aminobenzamide (2-AB)-labeling of sialo-oligosaccharides the authors encountered a significant loss of sialic acids under the reported conditions, at 65 degrees C for 2 h. By examination of the relationship between labeling temperature and recovery of tetra-sialylated oligosaccharides, 2-AB-labeling at 37 degrees C for 16 h resulted in a maximum yield with practically no loss of sialic acid. Under the newly optimized conditions, the reproducibility of 2-AB labeling was examined in triplicate with three lots of recombinant human erythropoietin (r-HuEPO). The present conditions gave analytical data comparable to those obtained by the established NaB[3H]4 labeling procedure. The modified method has been applied to oligosaccharide structural analysis of sialoglycoprotein therapeutics. PMID- 10726878 TI - Selection of mobile phase in high-performance liquid chromatographic determination for medicines. AB - A convenient method was developed to select mobile phase to separate drugs commonly used in clinical therapy, using high-performance liquid chromatography (HPLC). The separation conditions determined by this method were similar for each compound. Two kinds of mobile phase, a mixture of acetonitrile-phosphate buffers, pH 4.2 and 2.5, was used and the composition of mobile phase used for a drug in HPLC analysis was systematically determined as follows: (1) the retention time of a drug was measured under the gradient condition, (2) this retention time value was applied to the regression equation which was determined from the retention time values of p-hydroxybenzoate derivatives under the gradient condition and the concentration of acetonitrile in mobile phase, giving about 5 min of retention time, (3) percentage of acetonitrile in the isocratic condition was calculated from this regression equation and a target drug was eluted with the mobile phase consisting of the calculated concentration of acetonitrile. According to the proposed method, the composition of mobile phase, with which retention time value was between 4 to 8 min in the isocratic condition, was examined for 75 drugs clinically used. Seventy-two of the 75 drugs were analyzed well in the mobile phase, their composition was calculated by the regression equation, and the peak shape of each compound was observed to be sharp. Using this method, the time required for not only the setting of HPLC conditions but also the analysis will be shortened. PMID- 10726879 TI - Role of MerT and MerP from Pseudomonas K-62 plasmid pMR26 in the transport of phenylmercury. AB - To investigate the individual role of MerT and MerP encoded by Pseudomonas K-62 pMR26 in the transport of phenylmercury, a series of mutants with a specific point mutation in merT and/or genetic deletion in merP were constructed and transformed into Escherichia coli XL1-Blue. Transport of phenylmercury across the cytoplasmic membrane of E. coli mediated by MerT and MerP was inhibited by NaCN and by cold temperatures. Deletion of merP reduced, but did not completely abolish the C6H5Hg+-hyperuptake and -hypersensitive phenotypes suggesting that transport of phenylmercury into the cytoplasm of E. coli is still occurring. Mutations of the vicinal cysteine residues (Cys24 and Cys25) in the first transmembrane region of MerT to serine caused complete loss of Hg2+-hyperuptake and -hypersensitivity, whereas the mutations did not affect the C6H5Hg+ hyperuptake and -hypersensitive phenotypes. In addition, no additive effect on the C6H5Hg+-hyperuptake and -hypersensitive phenotypes was found, when mutations of the two cysteines in MerT to serine were further introduced in the merP deleted mutants. These results clearly demonstrated that the vicinal cysteine residues of MerT are not involved in the transport of C6H5Hg+, but indeed are involved in the transport of Hg2+ as previously reported. PMID- 10726880 TI - Crystallin proteins in lenses of hereditary cataractous rat, ICR/f. AB - ICR/f mutation in rat, an inherited disorder, is characterized by the development of cataracts. In this study, we analyzed and compared the crystallins in normal and cataractous rat lenses using gel filtration and two-dimensional gel electrophoresis, and determined the transglutaminase activities and Ca2+ content in the mutant and normal lenses. The Ca2+ content about 10-fold and the activity of transglutaminase was about 1.8-fold higher in the cataractous lenses than in the normal lenses. Analysis of the cataractous lens proteins showed a remarkable decrease in gamma-, betaB1-, betaA3-, and betaA4-crystallin content, accompanied with some increase in alpha-crystallin (or its aggregate). Higher molecular weight proteins were also observed in the cataractous lenses, with molecular masses which correspond to those of cross-linked dimers (43 to 55 kDa) of beta crystallins. We consider that the mutation accelerates the aggregation of the crystallins, which is associated with their cross-linking by transglutaminase. PMID- 10726881 TI - Effects of natural product extracts on contraction and mechanical properties of fibroblast populated collagen gel. AB - Fibroblast-populated collagen gel cultures have been used as a dermal model of wound contraction and granulation in the wound healing process and as an in vitro model of dermal tissue. We evaluated the effects of various natural product extracts on collagen gel contraction-promoting activity and mechanical properties (relaxation time) using this model, and observed that some natural product extracts, such as Hibamata extract (from Fucus vesiculosus) promoted gel contraction and increased the relaxation time of the gels. In addition, we investigated the mechanism of the promotion of the gel contraction, noting increased expression of integrin alpha2 and beta1 subunit molecules on the surface of the fibroblasts, suggesting that some extracts, such as Hibamata extract, promote gel contraction by increasing the expression of integrin molecules on the fibroblasts surface. For other types of natural product extracts, other mechanisms of the gel contraction-promoting activity, which were independent of an increase of integrins, were suggested. Although the mechanisms of promotion of gel contraction by these extracts are still unclear, it is at least clear that, more than one mechanism appears to be present. Therefore, more effective drug regimens for improving dermal tissues and wound healing may be achieved by combining drugs which increase integrin molecules and drugs with other mechanisms of action. PMID- 10726882 TI - Pharmacokinetics of ginsenoside deglycosylated by intestinal bacteria and its transformation to biologically active fatty acid esters. AB - Ginsenosides are deglycosylated by intestinal bacteria to active forms after oral administration. The present study demonstrated the pharmacodynamics of 20-O-beta D-glucopyranosyl-20(S)-protopanaxadiol (M1), an intestinal bacterial metabolite of ginsenosides, and the in vitro and in vivo antitumor activities of M1 metabolites in comparison with M1 using C57BL/6 mice and Wistar rats. M1 was selectively accumulated into the liver soon after its intravenous administration to mice, and mostly excreted as bile; however, some M1 was transformed to fatty acid ester (EM1) in the liver. EM1 was isolated from rats in a recovery dose of approximately 24 mol%. Structural analysis indicated that EM1 comprised a family of fatty acid mono-esters of M1. Because EM1 was not excreted as bile as M1 was, it was accumulated in the liver longer than M1. Although the cytotoxicity of M1 against B16-F10 melanoma cells was attenuated by fatty acid esterification, EM1 inhibited tumor growth more than M1 in vivo. These results suggest that the fatty acid M1 esters may be the real active principles of ginsenosides in the body. PMID- 10726883 TI - Zinc protection of mercury-induced hepatic toxicity in mice. AB - The actions and interactions of heavy metals on certain organ functions have been of concern, since occupational exposure to certain metals results in impairment of functions. Studies were carried out to determine the effects of zinc (Zn) and mercury (Hg) on murine liver. CD-1 male mice were administered 4 ppm HgCl2, 800 ppm ZnCl2, 4 ppm HgCl2+800 ppm ZnCl2 or deionized water in their drinking water for 12 weeks. Histological evaluation of the liver confirmed the toxic effects of Hg, as well as the normal morphology of the Zn-exposed animals. A combined treatment of both metals resulted in protection of the Hg-induced liver damage by Zn. The results of this experiment indicate that Hg has a toxic effect on liver, while Zn has a protective action against such toxic effects. PMID- 10726884 TI - Antioxidant and hepatoprotective actions of the medicinal herb Artemisia campestris from the Okinawa Islands. AB - The antioxidant action of Artemisia campestris was examined in vitro and in vivo. A water extract of A. campestris showed a strong scavenging action of 1,1 diphenyl-2-picrylhydrazyl (DPPH), hydroxyl and superoxide anion radicals. When the extract was given intraperitoneally to mice prior to carbon tetrachloride (CCl4) treatment, CCl4-induced liver toxicity, as seen by an elevation of serum aspartate aminotransferase and alanine aminotransferase activities, was significantly reduced. Depression of the elevation of serum enzyme levels after CCl4-treatment was also observed by oral administration of the extract. In that case, CCl4-derived lipid peroxidation in the liver was decreased by the extract treatment. These results suggest that the extract of A. campestris scavenges radicals formed by CCl4 treatment resulting in protection against CCl4-induced liver toxicity. PMID- 10726885 TI - Adhesive strength of autologous fibrin glue. AB - To establish an easy and rapid method for measuring the adhesive strength of fibrin glue and to clarify the factor(s) most affecting the strength, a study was made on the effect of the concentration of plasma components on the strength of cryoprecipitate (Cryo) prepared from a subject's own autologous plasma to be used as fibrin glue. The adhesive strength of the Cryo was measured with various supporting materials instead of animal skin using a tester of tension and compression. The results were as follows: (1) the strength of Cryo applied to ground flat glass (4 cm2) was significantly greater than that applied to clear glass, clear plastic, or smooth and flat wood chips; (2) the adhesive strength of Cryo depended on the concentration of thrombin with the optimal concentration being 50 units/ml; (3) the concentration of CaCl2 did not affect the adhesive strength of Cryo; (4) the adhesive reaction was dependent on the temperature and the adhesive strength more quickly reached a steady state at 37 degrees C than at lower temperature; (5) the adhesive strength was correlated well with the total concentration of fibrinogen and fibronectin. These results indicate that the adhesive strength of Cryo can be easily and quickly evaluated using a tester and ground glass with thrombin at 50 units/ml, and that the adhesive strength of Cryo can be predicted from the total concentration of fibrinogen and fibronectin. PMID- 10726886 TI - Positively charged liposomes containing tumor necrosis factor in solid tumors. AB - The antitumor effects against solid tumors, such as Meth A sarcoma, MH-134 hepatoma and colon 26 adenocarcinoma, were examined after intratumoral administration of liposomes and Tumor Necrosis Factor (TNF) solution. The antitumor effects of liposomes against solid tumors were superior to those of TNF solution. In particular, the antitumor effect of positively charged (decyl amine) liposomes was superior to that of negatively charged liposomes and TNF solution. Further, positively charged liposomes containing a higher dose of TNF than the solution could be administered without killing the mice, because of reduced side effects. After intratumoral (Meth A sarcoma) administration, the TNF plasma concentration was determined in order to estimate the systemic side-effects of TNF. The area under curve (AUC) after administration of positively charged liposomes containing 6 times dose of TNF was about 1/30 the AUC after the administration of TNF solution. After administration of positively charged liposomes, TNF was mainly retained locally. Positively charged liposomes exhibited a stronger antitumor effect than the solution and had a lower AUC (about 1/180) than the solution. Consequently, some solid tumors could be completely cured by positively charged liposomes, because of their increased antitumor effect and reduced toxicity. PMID- 10726887 TI - Usefulness of liposomes as an intranasal dosage formulation for topical drug application. AB - The potential of liposomes as an intranasal dosage formulation for topical application was investigated in rats. When 5(6)-carboxyfluorescein (CF), a model absorbable drug, dissolved in phosphate-buffered saline (PBS) was administered intranasally, CF was rapidly absorbed into the systemic circulation and no adhesion of CF to the nasal mucosa was observed. The fraction of CF absorbed from the nasal mucosa reached about 48% 1 h after administration. On the other hand, only 3% of the dose was absorbed when CF was encapsulated in liposomes consisting of dipalmitoylphosphatidylcholine and cholesterol (DPPC-liposomes). In addition, the amount of CF adhering to the nasal mucosa after administration as DPPC liposomes was 20- to 28-fold greater than that in PBS solution. In particular, positively charged liposomes markedly enhanced the adhesion of CF to the nasal mucosa. Differences in the lipid composition of liposomes did not affect the absorption of CF. However, the ability of liposomes to adhere to the nasal mucosa was consistent with the fluidity of the liposomal membrane. Furthermore, the action of liposomes on the anti-histaminic effect of diphenhydramine hydrochloride (DH) was studied in rats by measuring the amount of protein leaking into the nasal cavity under quasi-allergic conditions. The anti-histaminic effect of DH was strong but of short-duration when DH was administered as a PBS solution. However, liposomes prolonged the anti-histaminic effect of DH, suggesting that liposomes may adhere to the nasal mucosa and release DH slowly. In conclusion, liposomes suppress drug absorption into the systemic circulation and concurrently increase drug retention in the nasal cavity. PMID- 10726889 TI - Species differences in oral bioavailability of methotrexate between rats and monkeys. AB - The contributions of incomplete absorption and a first-pass effect to the low bioavailability (BA) of methotrexate (MTX) were evaluated pharmacokinetically in rats and monkeys which respectively have a lower and higher aldehyde oxidase (AO) activity than humans. Plasma concentration profiles of MTX in rats showed linear and nonlinear pharmacokinetics respectively after intravenous (i.v.) and oral dosing of 0.1, 0.5 or 2.5 mg/kg MTX. In rats, most of the dose was excreted as the parent compound into bile and urine after i.v. dosing of 0.5 mg/kg MTX, while the radioactivity was largely eliminated in expired air after oral dosing of 0.5 mg/kg 14C-MTX. Elimination in expired air fell markedly following antibiotics treatment. 7-Hydroxymethotrexate (7-OH-MTX), formed from MTX by AO, was detected in monkey plasma after i.v. and oral dosing of 0.5 mg/kg MTX, but not in rat plasma. The ratio of the cumulative urinary excretion of 7-OH-MTX to MTX in monkeys was higher after oral dosing than after i.v. dosing. The low BA in rats (10% at 0.5 mg/kg) was shown to be mainly due to incomplete absorption, including limited absorption and degradation to 2,4-diamino-N10-methylpteroic acid (DAMPA) and glutamic acid (Glu) by the carboxypeptidase of intestinal bacteria. The low BA in monkeys (5% at 0.5 mg/kg) was shown to be mainly due to the extensive first pass effect, including metabolism to 7-OH-MTX. PMID- 10726888 TI - Effects of lipid composition on the transcorneal penetration of liposomes containing disulfiram, a potential anti-cataract agent, in the rabbit. AB - We previously demonstrated that topical treatment with disulfiram (DSF) prevented the development of cataracts in sodium selenite-injected rat pups. In biological systems, DSF is rapidly reduced to diethyldithiocarbamate (DDC), a potent antioxidant. In this study, we investigated the effect of altering the lipid composition of liposomes containing DSF on the transcorneal transit of DDC. Liposomes containing DSF were prepared with various molar ratios of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) and cetylpyridinum chloride (CPC) by reverse-phase evaporation. Liposomes with a DMPC to DPPC molar ratio of 5:5, examined by differential scanning calorimetry, had the highest enthalpy of transition and the presence of one molar ratio of CPC further enhanced the enthalpy value. The addition of bovine serum albumin or a homogenate of rabbit cornea to the incubation buffer resulted in the release of DDC, but not DSF from the liposomes. The amount of DDC present in the aqueous humor of rabbit eyes following topical administration increased with increase in DMPC to DPPC ratios and was also enhanced by the addition of CPC to the liposomes. The results of this study suggest that liposome formulations are effective for transcorneal drug delivery of anticataract agents such as DSF. DSF in liposomes consisting of DMPC, DPPC, and CPC with a molar ratio of 8:2:1 may be a potential drug formulation for the prevention and/or treatment of cataracts. PMID- 10726890 TI - Effects of caffeine and theophylline on the development of dental caries in rats. AB - The purpose of this study was to evaluate the influence of caffeine and theophylline on the development of dental caries in rats. Six Wistar dams (spf), mutans streptococci free, were obtained, each with six male pups. The dams were infected by Streptococcus sobrinus 6715 and divided into three groups which received during the lactating period: (1) diet 2000; (2) diet 2000 plus caffeine (2 mg/100 g) and (3) diet 2000 plus theophylline (0.57 mg/100 g). After weaning, the pups were infected by S. sobrinus, placed in a Konig-Hofer programmed feeder machine, and received 17 meals daily at hourly intervals, for five weeks. During this time the pups were fed with the same diet that their dams were. The percentage of S. sobrinus relative to total flora was significantly higher in the theophylline group. The results for slight (Ds) and moderate (Dm) dentine lesions, for smooth-surface and sulcal scores were statistically higher for the theophylline group than the other groups. Salivary assays did not demonstrate significant inorganic alterations in salivary composition. Caffeine and theophylline groups showed the highest ulcer score. It is concluded that caffeine does not affect the cariogenic potential of the diet, however theophylline can increase the development of dental caries, and this effect may be related to organic alterations of salivary composition. PMID- 10726891 TI - Antitumor effects of a novel lipophilic platinum complex (SM-11355) against a slowly-growing rat hepatic tumor after intra-hepatic arterial administration. AB - The antitumor effects of cis[((1R,2R)-1,2-cyclohexanediamine-N,N')bis(myristato)] platinum(II) (SM-11355) were evaluated in a rat hepatic tumor model, and were compared with those of cisplatin (CDDP). A novel slowly-growing rat hepatic tumor model was established by the successive transplantation of rat AH109A tumor into the liver. The drugs, which were suspended in Lipiodol, were administered into the proper hepatic artery of tumor-bearing rats. Tumor growth was suppressed in the group that received SM-11355 suspended in Lipiodol (SM-11355/Lipiodol). Mean tumor growth rates in the groups administered 20 microl of Lipiodol containing 0, 0.02, 0.04, 0.1, 0.2, or 0.4 mg of SM-11355 were 244, 86, 110, 81, 51, and 40%, respectively, 1 week after treatment. Those in the groups administered 20 microl of Lipiodol containing 0.1, 0.2, or 0.4 mg of CDDP were 240, 110, and 45%, respectively. In the groups administered 0.2 and 0.4 mg of SM-11355 or 0.4 mg of CDDP, massive necrosis was observed in the tumor tissue 1 week after drug administration, and the tumors disappeared 4 weeks after drug administration. Serum glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) levels were measured as markers of liver damage one day after the drug was administered into the hepatic artery of rats. The minimum toxic dose, which raised serum GOT and GPT levels significantly compared with Lipiodol alone, was 0.2 mg for SM-11355/Lipiodol and 0.1 mg for CDDP/Lipiodol, respectively. The results demonstrated that SM-11355/Lipiodol exerted antitumor activity at a dose that showed no hepatic toxicity in the rat model, but CDDP/Lipiodol did not. PMID- 10726892 TI - Identification of basic fibroblast growth factor-responsive genes by mRNA differential display in an immortalized neural stem cell line. AB - Basic fibroblast growth factor (bFGF) has been shown to stimulate proliferation and differentiation of neural stem cells through regulation of gene expressions. To clarify the roles of bFGF during early neurogenesis, we performed a series of differential display with mRNAs from an immortalized neural stem cell line treated with bFGF for different periods. We isolated ten independent cDNAs whose mRNA levels were regulated by bFGF. Some of these cDNA were identical to known genes, including calmodulin and thrombospondin 1, while others were unknown genes. One of these unknown genes up-regulated by bFGF (clone 2C) was specifically expressed in the brain among various rat tissues. It is expected that further analysis of clone 2C will reveal important roles of bFGF in the regulation of brain development. PMID- 10726893 TI - Effects of Dai-kenchu-to on levels of 5-hydroxytryptamine (serotonin) and vasoactive intestinal peptides in human plasma. AB - We examined the effects of Dai-kenchu-to (DKCT) on the levels of vasoactive intestinal peptide (VIP) and 5-hydroxytryptamine (serotonin; 5-HT) in plasma taken from 6 healthy subjects. A single oral administration of 7.5 g DKCT caused significant increases in plasma VIP at 30, 60 to 90 and 120 min (3.5-5.5 pg/ml), compared with the response in a placebo group (about 1.0 pg/ml). DKCT also caused significant increases in plasma 5-HT at 30 (121.8+/-7.3 ng/ml) and 60 (156.5+/ 8.0 ng/ml) min, compared with the response in the placebo group (about 101 ng/ml). These results indicate that the stimulatory effect of DKCT on VIP immunoreactive substance (VIP-IS) secretion is due, at least in part, to increased 5-HT levels in the abdomen. As a consequence, increased VIP-IS may improve feelings of coldness in the abdomen. PMID- 10726894 TI - Cytotoxic effects of pyridino[2,3-f]indole-4,9-diones on human tumor cell lines. AB - The cytotoxicities of pyridino[2,3-f]indole-4,9-dione derivatives were examined against human lung tumor cell lines (A 549), human ovarian tumor cell lines (SK OV-3), human melanoma tumor cell lines (SK-MEL-2), human CNS tumor cell lines (XF 498) and human colon tumor cell lines (HCT 15) in vitro using a Sulforhodamine B assay. 3-Ethoxycarbonyl-1-(2-methoxyethyl)-2-methyl-1H-pyridino[2,3-f]ind ole-4,9 dione (5) showed excellent cytotoxicity against XF 498 and HCT 15. The ED50 values of 5 were 0.006 microg/ml against XF 498 and 0.073 microg/ml against HCT 15, while those of doxorubicin were 0.012 and 0.264 microg/ml, respectively. 1 Benzyl-3-ethoxycarbonyl-2-methyl-1H-pyridino[2,3-f]indole-4,9-di one (7) (ED50 value 0.065 microg/ml) was also significantly more cytotoxic against HCT 15 compared with doxorubicin. PMID- 10726895 TI - Inhibitory effect of herbal medicines on rotavirus infectivity. AB - The inhibitory effect of some traditional herbal medicines on the infectivity of rotavirus, which predominantly occurs in sporadic diarrhea in infants and young children, was investigated. Among the 34 kinds of herbal medicines tested, the fruit of Citrus aurantium had the most potent inhibitory activity on rotavirus infection. The active components of the fruit of Citrus aurantium were neohesperidin and hesperidin. Their 50% inhibitory concentrations were 25 and 10 microM, respectively. PMID- 10726896 TI - Perilla citriodora from Taiwan and its phytochemical characteristics. AB - Perilla citriodora Nakai, the wild species of Perilla, was collected from Taiwan and its essential oil analyzed. GC-MS analysis of its oil showed that it has a novel composition of limonene (23.5%) and elemicin (17.8%). In Japanese Perilla, a monoterpene (limonene) and a phenylpropanoid (elemicin) have not been detected in the same plant. To compare the sequence similarity of a secondary metabolic enzyme between P. frutescens and P. citriodora, the nucleic acid sequence of the limonene synthase in this P. citriodora was analyzed using the reverse transcript polymerase chain reaction (RT-PCR) method. Primers for PCR were designed by employing the known sequence of the limonene synthase cloned from P. frutescens. It was found that the limonene synthase in P. citriodora and that in P. frutescens share a high sequence identity, probably indicating that both enzymes evolved from a common ancestor. PMID- 10726897 TI - Anti-herpes virus activity of Solanum steroidal glycosides. AB - Since some Solanum-genus plants have traditionally been used for anti-cancer and anti-herpes agents from olden times, we examined anti-herpes simplex virus type 1 (HSV-1) activity of typical steroidal glycosides with the frameworks of spirostane (including nuatigenin glycoside), furostane, solasodane, tomatidane and ergostane (including dimer) obtained from Solanum plants. Among these steroidal glycosides, the spirostanol glycosides were most effective. An inclination was observed for the potency of activity to decrease in the order of spirostane, tomatidane, ergostane, solasodane, nuatigenin type, dimer of ergostane and furostane. It was also suggested that the activity depends on the kind of oligosacchride moiety. PMID- 10726898 TI - Antiinflammatory effect of Forsythia suspensa V(AHL) and its active principle. AB - This study was carried out to elucidate the antiinflammatory active principles obtained from 70% methanol extract of the dried fruit of Forsythia suspensa V(AHL) (F. suspensa). The methanol extract was then partitioned between n-hexane and water, and then the n-hexane fraction was evaporated to dryness under vacuum. The n-hexane fraction was chromatographed (Frs. I--V), Fr. IV was rechromatographed (Frs. VI--VIII), and then Fr. VII was rechromatographed (Frs. IX--XI) by silica gel column chromatography. The antiinflammatory activity of these fractions was investigated on acetic acid-induced vascular permeability in rats. The n-hexane fraction showed an antiinflammatory effect and these activities shifted successively to Fr. IV, Fr. VII and Fr. X. The chemical structure of the active principle obtained from Fr. X was identified as 3beta acetoxy-20,25-epoxydammarane-24-ol. These results suggest that the antiinflammatory and an analgesic effect of 70% methanol extract of F. suspensa may be the result of the compound that it contains. PMID- 10726899 TI - Quantification of a very low level of serum human immunodeficiency virus type 1 (HIV-1) RNA using competitive PCR at intervals and the clinical course. AB - Quantification of human immunodeficiency virus type 1 (HIV-1) genomic RNA levels below the detection limit (400 copies/ml) of commercially available quantification kits is possible by increasing the amount of serum samples using competitive PCR conditions. We evaluated disease prognosis in patients without symptoms for a long period after infection (long-term non-progressors) and patients in whom the virus was controlled to a low level by administration of anti-HIV drugs. For 102 of the 414 serum samples stored at -80 degrees C which showed HIV-1 RNA below 400 copies/ml by competitive PCR using 100 to 200 microl sera, an increase in the sample volume to 500-2000 microl, extraction of HIV-1 RNA, and quantitative detection by competitive PCR was performed. Follow-up quantification of serum HIV-1 RNA from 4 patients indicated that this method is useful in assessing prognosis in the early stage of the disease in patients without symptoms for a long period after infection (long-term non-progressors) and patients in whom the virus in the serum was controlled by administration of anti-HIV drugs to below the detection limit of commercially available quantification kits. Quantification of low level serum HIV-1 RNA was also considered useful as a parameter of treatment. PMID- 10726900 TI - Novel drug delivery system using autologous fibrin glue--release properties of anti-cancer drugs. AB - To clarify the release properties of anti-cancer drugs from fibrin glue, a study was performed using several anti-cancer drugs with remarkably different physical properties. Concentrated fibrinogen, fibronectin, and coagulation factor XIII were prepared from healthy human plasma according to the cryoprecipitate method. Fibrin glue containing anti-cancer drugs was prepared as follows; the cryoprecipitate was mixed with each anti-cancer drug and aprotinin, then thrombin was added. These glues were incubated in PBS containing plasminogen and urokinase at 37 degrees C for seven days, and the medium was then sampled several times after centrifugation. The drug concentration in each sample was measured using HPLC. Fibrin glue without aprotinin was quickly hemolyzed and disappeared after 2 -4 h. That with aprotinin was only slightly hemolyzed and more than half remained after 7 days. Mitomycin C and fluorouracil were quickly released from the glue regardless of the presence or absence of aprotinin. However, enocitabine was gradually released from glue with aprotinin although quickly released from that without. The rate of release of each drug from the glue with aprotinin correlated well with its hydrophobicity. Thus, to establish a sustained release system using fibrin glue, one should use the more lipophilic anti-cancer drugs and a fibrinolytic enzyme inhibitor. PMID- 10726901 TI - Inhibitory effects of tetrahydroisoquinoline derivatives on Ca2+ and Na+ channels in crude nerve endings. AB - Semi-synthetic tetrahydroisoquinoline derivatives prepared from natural alkaloids, possess Ca2+ antagonistic properties. These derivatives significantly blocked KCl-stimulated Ca2+ uptake (In chick and rat crude nerve endings) which can be partially inhibited by the selective N-type Ca2+ channel blocker omega conotoxin GVIA or the selective P-type Ca2+ channel blocker omega-agatoxin IVA. Moreover, PX42 (10 microM; for the tetrahydroisoquinoline compounds in this study) could inhibit the activity of calmodulin-dependent phosphodiesterase and block veratridine-induced (or tetrodotoxin-sensitive) Na+ uptake. The possible mechanism(s) of non-selective inhibition of ion channels of PX42 is discussed. PMID- 10726902 TI - Impact of insulin resistance on lipoprotein subpopulation distribution in lean and morbidly obese nondiabetic women. AB - The purpose of this study was to examine the effects of insulin resistance on the lipoprotein subpopulation distribution of very-low-density, low-density, and high density lipoproteins (VLDL, LDL, and HDL) in lean and morbidly obese nondiabetic women. Lean women (body mass index [BMI], 20 to 27 kg/m2) stratified by BMI were divided into insulin-sensitive (SL, n = 12) and insulin-resistant (RL, n = 8) groups according to Bergman's minimal model, SI. A group of obese women (BMI, 30 to 53 kg/m2), also stratified by BMI, were divided into insulin-sensitive (SO, n = 10) and insulin-resistant (RO, n = 11) groups in a similar fashion. Resistant groups were similar to sensitive groups (SL v RL and SO vRO) in age, weight, percent body fat, and waist circumference, ie, total and regional adiposity. VLDL, LDL, and HDL subpopulation distributions were determined in fasting plasma samples by nuclear magnetic resonance (NMR) spectroscopy. The average particle sizes of all 3 classes of lipoproteins were similar for the SL and RL groups. In contrast, RO subjects had larger VLDL, smaller LDL, and smaller HDL, than SO subjects (P < .05). Lower concentrations of large LDL and large HDL were found in RO compared with SO subjects (P < .05). In obese women, but not in lean women, VLDL size was associated with plasma insulin (r = .60, P < .005), while LDL size and HDL size were negatively correlated with plasma insulin (r = -.39, P < .05 and r = -.38, P < .05) and positively correlated with SI (r = .54, P < .01 and r = .42, P < .05). These results suggest that in obese women, insulin resistance may be involved in the formation of lipoprotein subpopulation distributions that are associated with vascular disease. PMID- 10726903 TI - No evidence of linkage between the very-low-density lipoprotein receptor gene and fasting serum insulin or homeostasis model assessment insulin resistance index: the National Heart, Lung, and Blood Institute Family Heart Study. AB - A major gene effect on the fasting insulin level and insulin resistance has been suggested in previous studies. Several candidate genes for insulin resistance in rare syndromes have been proposed. However, there has been limited success in finding genes for common forms of insulin resistance. There is accumulating evidence of a relationship between insulin resistance and a disturbance of free fatty acid (FFA) metabolism. The very-low-density lipoprotein (VLDL) receptor, which is associated with FFA metabolism, could serve as a possible candidate gene for insulin resistance. We performed linkage analyses between the VLDL receptor gene and fasting insulin and the homeostasis model assessment (HOMA) insulin resistance index (fasting insulin x fasting glucose/22.5) in 1,050 sibpairs participating in the phase II physical examination of the National Heart, Lung, and Blood Institute Family Heart Study (FHS). Data analyses were completed using the SIBPAL component of the SAGE software package (SAGE, Statistical Analysis for Genetic Epidemiology, Version 3.1; Computer program package available from the Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, OH, 1997). We did not find evidence for linkage of the fasting insulin or the HOMA insulin resistance index with a polymorphic marker at the VLDL locus (P = .316 and .402, respectively). Adjustment of fasting insulin and the HOMA insulin resistance index for the body mass index (BMI) did not change the results (P = .319 and .472, respectively). In conclusion, no evidence was found for a linkage between a locus controlling the fasting insulin level or HOMA insulin resistance index and a VLDL polymorphism in the present study. Additional adjustment of fasting insulin or the HOMA insulin resistance index for the BMI did not change the linkage results significantly. PMID- 10726904 TI - A genetic study of dehydroepiandrosterone sulfate measured before and after a 20 week endurance exercise training program: the HERITAGE Family Study. AB - Familial aggregation and possible major gene effects were evaluated for the baseline serum dehydroepiandrosterone sulfate (DHEAS) level and the change in DHEAS in response to a 20-week exercise training program in a sample of 481 individuals from 99 Caucasian families who were sedentary at baseline and who participated in the HERITAGE Family Study. Baseline DHEAS levels were not normally distributed, and were therefore logarithmically transformed and adjusted for the effects of age and sex prior to genetic analysis. The DHEAS response to training was computed as the simple difference, post-training minus baseline, and was adjusted for the baseline DHEAS level, age, and sex. Maximal (genetic and familial environmental) heritabilities (using a familial correlation model) reached 58% and 30% for the baseline and the response to training, respectively. Our estimate for the baseline is generally in agreement with previous reports, suggesting that the magnitude of the familial effect underlying this phenotype in these sedentary families is similar to that in the general population. However, segregation analysis showed no evidence for a multifactorial familial component in data for either the baseline or the response to training. Rather, a major additive gene controlling the baseline was found. For the response to training in the complete sample, transmission of the major effect from parents to offspring was ambiguous, but in a subset of 56 "responsive" families (with at least 1 family member whose response to training was greater than 1 standard deviation) this major effect was Mendelian in nature. The putative major genes accounted for 50% and 33% of the variance for the baseline and the response to training, respectively. The novel finding in this study is that the baseline DHEAS level and the change in DHEAS in response to training may be influenced by major gene effects. PMID- 10726905 TI - Morning cortisol levels and glucose effectiveness. AB - Anabolic corticosteroids have been reported to enhance glucose effectiveness (SG). In experimental models of long-term cortisol infusion in diabetic dogs, the maintenance of normal SG during chronic hypercortisolemia prevented a significant deterioration of glucose tolerance. We hypothesized that in analogy with exogenous corticosteroids, endogenous cortisol might influence SG. We aimed to study the influence of serum cortisol on SG prior to a frequently sampled intravenous glucose tolerance test (FSIVGTT) in 18 otherwise healthy men. The serum cortisol level or free cortisol index (ratio of cortisol to cortisol binding globulin [CBG]) were not associated with the body mass index (BMI), waist to hip ratio (WHR), fasting insulin, or insulin sensitivity ([SI] all r < .20, P = NS). Conversely, SG correlated with serum cortisol levels measured prior to the FSIVGTT (r = .60, P = .008) and with the free cortisol index (r = .48, P = .03). The association was stronger in lean subjects (BMI < 25 kg/m2, r = .90, P = .002, n = 8). Men with a pre-FSIVGTT serum cortisol level above the median (431 nmol/L) were similar by age, BMI, WHR, and SI to the subjects with cortisol levels below the median, but the latter presented a significantly decreased SG (0.0014 +/- 0.006 v 0.022 +/- 0.007 min(-1), P = .03). In multiple linear regression analysis, fasting glucose (P = .02) and serum cortisol (P = .027) independently predicted SG, contributing to 26% of its variance. In summary, our findings suggest that the prevailing cortisol level appears to be associated with SG. The lower cortisol levels usually found in abdominally obese men could contribute to their altered glucose tolerance, perhaps via decreased SG. PMID- 10726906 TI - Troglitazone enhances glycolysis and improves intracellular glucose metabolism in rat mesangial cells. AB - To clarify the action of the new antidiabetic agent, troglitazone, on rat mesangial cells, we assessed its effect on the uptake and intracellular metabolism of glucose. Troglitazone increased the uptake of 2-deoxyglucose (2DOG) in a dose-dependent manner with an upregulation of glucose transporter 1 (GLUT1) mRNA, whereas it had no effect on the uptake of alpha-methyl glucoside (AMG). This troglitazone-induced glucose uptake was not suppressed by phlorizin. In 5 mmol/L glucose, 2 microg/mL (4.5 micromol/L) troglitazone increased glucose consumption 2.9-fold, similar to that in 20 mmol/L glucose. Troglitazone increased the production of pyruvate and lactate as a consequence of the increase in glycolysis, but did not increase the cellular ATP content. Troglitazone improved the high-glucose-induced accumulation of intracellular sorbitol and fructose and elevation of the cellular redox potential. These data suggest that troglitazone enhances glucose uptake through GLUT1 with an acceleration of glycolysis, and improves the abnormal intracellular glucose metabolism under high glucose conditions in rat mesangial cells. PMID- 10726907 TI - Synergistic effects of insulin-like growth factor-I and human chorionic gonadotropin in the rat ovary. AB - Insulin and low doses of lutenizing hormone (LH) activity (human chorionic gonadotropin [hCG]) act synergistically in the rat to produce anovulation, large ovarian cysts, and elevated plasma androstenedione levels. Further, both insulin and insulin-like growth factor-I (IGF-I) affect the ability of gonadotropins to enhance both ovarian theca and granulosa cell function in vitro. The present series of experiments were performed to determine if recombinant human IGF-I (rhIGF-I) can act in a manner similar to insulin when combined with subovulatory doses of hCG in adult normally cycling rats. Fifty-four female Sprague-Dawley rats were randomly assigned to the following treatment groups at the age of 64 days: (A) vehicle alone (controls, phosphate-buffered saline containing 0.09% pig gelatin), (B) twice-daily subcutaneous injections of 0.5 to 3.0 U insulin, (C) twice-daily subcutaneous injections of 1.5 U hCG, (D) both insulin and hCG, (E) twice-daily subcutaneous injections of rhIGF-I (2.5 mg/kg/d), and (F) both hCG and rhIGF-I. After 22 days of treatment, the animals were killed on day 23, trunk blood was collected, and the ovaries were excised for histological study. Eight of 9 control rats and 5 or 6 of 9 rats treated with insulin, hCG, or rhIGF-I alone displayed normal estrus cycles throughout the in vivo treatment period as assessed by daily vaginal smears. In marked contrast, only 1 animal treated with hCG + insulin and 2 animals treated with hCG + rhIGF-I continued to display vaginal smears indicative of normal cycling. Multiple large ovarian follicular cysts were found only in these latter 2 groups (3 of 9 animals in each group). Mean serum testosterone levels were significantly elevated in animals receiving insulin + hCG (0.72 +/- 0.28 v 0.17 +/- 0.03 ng/mL in controls, P = .05). Mean serum androstenedione levels were significantly elevated in animals receiving hCG and animals receiving rhIGF-I + hCG (5.57 +/- 0.99 and 2.39 +/- 0.68 ng/mL, respectively, v0.14 +/- 0.14 ng/mL in controls, P< .01 and P< .05, respectively). We conclude that rhIGF-I and insulin act synergistically with subovulatory doses of hCG to disrupt normal reproductive cycling, elevate serum androgen concentrations, and induce large ovarian cysts in intact adult rats. PMID- 10726908 TI - Gluconeogenesis in non-obese diabetic (NOD) mice: in vivo effects of vandadate treatment on hepatic glucose-6-phoshatase and phosphoenolpyruvate carboxykinase. AB - The contribution of gluconeogenesis to hyperglycemia in non-obese diabetic (NOD) mice has been investigated using oral vanadate administration. Vanadate compounds have been shown to mimic many actions of insulin; however, the exact mechanism is poorly understood. The aims of the present study were (1) to elucidate vanadate's action in vivo, and to assess the possibility that its glucose-reducing effect is dependent on the presence of a minimal concentration of insulin; and (2) to evaluate the effects of vanadate administration on the key hepatic gluconeogenesis enzymes, glucose-6-phosphatase (G-6-Pase) and phosphoenolpyruvate carboxykinase (PEPCK), as well as glucose-6-phosphate dehydrogenase (G-6-PDH). Vanadate caused a significant reduction in blood glucose but failed to normalize it, despite effective serum vanadate concentrations (26.2 +/- 1.6 micromol/L). Two weeks after initiation of treatment, blood glucose levels were 26.0 +/- 1.8, 21.7 +/- 3.0, 16.0 +/- 1.6, and 14.3 +/- 2.3 mmol/L in the control (C), insulin (I), vanadate (V), and combined vanadate and insulin (V + I) groups, respectively (P < .001). G-6-Pase activity was significantly reduced by vanadate (622 +/- 134 v365 +/- 83 nmol/min/mg protein in C vV, P < .05). PEPCK activity was also significantly reduced (844 +/- 370, 623 +/- 36, 337 +/- 43, and 317 +/- 75 nmol/min/mg in the C, I, V, and V + I groups, respectively, P < .001). No significant differences in the hepatic glycogen stores and G-6-PDH activity were noted between treatment groups. Our study suggests that the inhibition of hepatic G-6-Pase and PEPCK activity by vanadate plays an important role in reducing blood glucose levels in NOD mice. PMID- 10726909 TI - Downregulation of leptin by free fatty acids in rat adipocytes: effects of triacsin C, palmitate, and 2-bromopalmitate. AB - Free fatty acid (FFA) has been reported to decrease leptin mRNA levels in 3T3-L1 adipocytes. When using this cell line, it is difficult to determine the protein levels because a very small amount of leptin is secreted into the medium. The effect of FFA on leptin secretion from adipocytes has not yet been determined. In addition, in vivo studies have failed to demonstrate a FFA-induced decrease in plasma leptin levels. To clarify the effect of FFA on leptin production, we investigated the leptin protein level in the medium and the mRNA level in primary cultured rat adipocytes treated with triacsin C, which is a potent inhibitor of acyl-coenzyme A (CoA) synthetase, palmitate, and 2-bromopalmitate. Triacsin C (0 to 5 x 10(-5) mol/L) decreased leptin concentrations in the culture medium in a dose-dependent manner. Leptin mRNA levels were decreased to 10% of the control in the presence of triacsin C. The concentration of triacsin C needed to suppress leptin production was similar to the Ki value (approximately 10(-5) mol/L) for inhibition of acyl-CoA synthetase. Both palmitate and 2-bromopalmitate decreased leptin concentra-tions but did not affect the triacsin C-induced decrease in leptin additively. In conclusion, both protein and mRNA levels of leptin were decreased by triacsin C and FFA in primary cultured rat adipocytes. Our findings suggest that FFA is involved in the regulation of leptin production in adipocytes. PMID- 10726910 TI - Bezafibrate reduces blood glucose in type 2 diabetes mellitus. AB - The clinical efficacy of bezafibrate was examined with special reference to glucose metabolism in patients with type 2 diabetes mellitus (DM2). In protocol 1, 342 patients with DM2 and hyperlipidemias were randomly divided into 2 groups, 16-week bezafibrate treatment (n = 174) and no bezafibrate treatment (n = 168). In protocol 2, 20 DM2 patients were randomly divided into 2 groups, 8-week bezafibrate treatment (n = 10) and no bezafibrate treatment (n = 10), and a meal tolerance test (MTT) was performed. In protocol 1, bezafibrate treatment significantly reduced the fasting levels of triglyceride (TG) by 50% +/- 1.6%, total cholesterol (TC) by 12% +/- 1.1%, plasma glucose (PG) from 151.3 +/- 3.5 to 128.6 +/- 3.4 mg/dL, and hemoglobin A1c (HbA1c) from 7.2% +/- 0.1% to 6.9% +/- 0.1%, and significantly increased high-density lipoprotein cholesterol (HDL-C) by 20% +/- 0.8%. In protocol 2, fasting TG, PG, and insulin levels were significantly reduced by bezafibrate treatment. Moreover, in the MTT, postprandial increments of TG were significantly blunted after bezafibrate treatment, whereas postprandial PG and insulin levels were not significantly changed. Leptin levels were significantly decreased, while tumor necrosis factor alpha (TNF-alpha) levels were not changed. In conclusion, both hyperglycemia and hyperlipidemia can be improved by bezafibrate treatment in DM2. PMID- 10726911 TI - Leptin levels in humans are acutely suppressed by isoproterenol despite acipimox induced inhibition of lipolysis, but not by free fatty acids. AB - Leptin secretion is complexly regulated in humans. Insulin has been shown to stimulate leptin secretion, whereas in vitro data suggest that catecholamines and free fatty acids (FFAs) inhibit leptin secretion. To dissect differential effects on leptin secretion, we performed two experimental protocols in 11 lean healthy subjects in addition to a saline infusion plus oral acipimox to suppress lipolysis (SAL + ACX) as a control experiment: (1) isoproterenol (approximately 30 ng/kg x min, to increase the heart rate by approximately 50 bpm) plus oral acipimox (ISO + ACX, 240 minutes) and (2) Intralipid (Pharmacia & Upjohn, Erlangen, Germany) plus heparin (LIP, 420 minutes). During SAL + ACX, FFAs decreased from 0.44 +/- 0.04 to 0.06 +/- 0.02 mmol/L (P = .001), while serum insulin and leptin remained unchanged. During ISO + ACX, FFAs decreased similarly from 0.41 +/- 0.13 to 0.09 +/- 0.02 mmol/L (P= .001), while insulin increased from 47 +/- 8 to a maximum of 116 +/- 15 pmol/L (P= .001) and serum leptin decreased acutely from 6.4 +/- 2.1 to a minimum of 5.4 +/- 1.8 ng/mL after 90 minutes (P = .003 vSAL + ACX). After 150 minutes, leptin returned to control levels. During LIP, the elevation of FFAs from 0.34 +/- 0.04 to 1.71 +/- 0.19 mmol/L (P = .001) had no effect on serum insulin or leptin concentrations (both P = nonsignificant). In conclusion, our results show that in humans, isoproterenol acutely suppresses leptin levels independently of increased FFAs, and elevated FFAs have no acute effect on leptin levels. The fact that an inhibition of leptin secretion occurred despite conditions that are known to suppress intracellular cyclic adenosine monophosphate (cAMP) levels, as demonstrated by suppressed lipolysis, suggests that signaling mechanisms other than those mediated by cAMP must be involved in modulating leptin secretion. PMID- 10726912 TI - Amelioration of insulin resistance but not hyperinsulinemia in obese mice overexpressing GLUT4 selectively in skeletal muscle. AB - The effects of gold-thioglucose (GTG) treatment were examined in mice overexpressing GLUT4 selectively in skeletal muscle (MLC-GLUT4 mice) and in age matched controls. Groups of MLC-GLUT4 and control mice were injected with GTG or saline at 5 weeks of age. At 12 weeks following the injections, GTG-treated control mice exhibited a 35% increase in body weight versus saline-treated controls. Similarly, a 30% increase in body weight was observed in GTG-treated MLC-GLUT4 mice compared with saline-treated MLC-GLUT4 mice 12 weeks after the injections. In saline-treated lean MLC-GLUT4 and control mice, intraperitoneal injection of insulin decreased blood glucose in 1 hour by 63% and 38%, respectively. Insulin also decreased blood glucose by 40% in GTG-treated obese MLC-GLUT4 mice after 1 hour. However, insulin did not reduce blood glucose levels in GTG-treated obese control mice. The ability of insulin to clear blood glucose in GTG-treated obese MLC-GLUT4 mice is associated with increased skeletal muscle GLUT4 content and white adipose tissue (WAT) GLUT4 content as compared with GTG treated obese controls. However, fasting blood glucose levels in GTG-treated obese MLC-GLUT4 and control mice were elevated by approximately 30% compared with saline-treated groups. Lastly, although GTG-treated obese MLC-GLUT4 mice exhibited improved glucose clearance in response to insulin, they nevertheless remained as hyperinsulinemic as GTG-treated obese control mice. These results suggest that genetic overexpression of GLUT4 in skeletal muscle may ameliorate the development of insulin resistance associated with obesity but cannot restore normal glucose and insulin levels. PMID- 10726913 TI - Inhibition of progressive reduction of islet beta-cell mass in spontaneously diabetic Goto-Kakizaki rats by alpha-glucosidase inhibitor. AB - The Goto-Kakizaki (GK) rat, an animal model of type 2 diabetes, exhibits mild hyperglycemia with a reduction of beta-cell mass. The mechanism for islet structural changes in this model and whether the changes are affected by metabolic control are not known. In the present study, we examined the process of islet changes in male GK rats aged 6, 8, 12, 24, and 36 weeks. Treatment effects with an alpha-glucosidase inhibitor (Voglibose; Takeda, Osaka, Japan) for 24 weeks (12 to 36 weeks of age) were also evaluated. The beta-cell mass increased until 8 weeks of age in both GK and control rats, but the increase was significantly (P < .01) smaller in GK rats versus at 8 weeks of age. Thereafter, the beta-cell mass decreased in GK rats, whereas it remained constant in controls. Voglibose treatment significantly (P < .01) inhibited the reduction of beta-cell mass in GK rats. Proliferative activity of beta cells as measured by bromodeoxyuridine (BrdU) uptake was significantly (P < .05) lower in GK rats versus control rats at 6 and 8 weeks, but the difference disappeared after 12 weeks of age, regardless of Voglibose treatment. The present study thus demonstrates a progressive loss of beta cells in GK rats that was mitigated by Voglibose treatment. We consider that the beta-cell loss in GK rats was due to an early impairment in proliferative activity and reduced survival. Voglibose did not appear to stimulate beta-cell proliferation, but exerted its effect via a reduction of hyperglycemia. PMID- 10726914 TI - Activation of transforming growth factor-beta1 in diabetic kidney disease. AB - Recent data have suggested that certain growth factors and cytokines are involved in the development of diabetic nephropathy. The aim of this study was to investigate whether circulating transforming growth factor beta 1 (TGF-beta1) and tumor necrosis factor alpha (TNF-alpha) are associated with diabetic kidney disease. Serum levels of active and total TGF-beta1 and TNF-alpha were measured in type 2 diabetic patients with nephropathy (n = 23) or without (n = 35) and normoglycemic controls (n = 12). Serum levels of circulating active TGF-beta1 were significantly higher in patients with diabetic nephropathy (0.43 +/- 0.06 ng x mL(-1)) compared with diabetic patients without renal involvement (0.23 +/- 0.03 ng x mL(-1), P = .002) and healthy controls (0.24 +/- 0.03 ng x mL(-1), P= .001), whereas the levels of total (active + latent) TGF-beta1 were not different between the subgroups. Active TGF-beta1 concentrations were correlated with urinary albumin excretion (r = .49, P < .003) and serum creatinine (r= .55, P < .01). Sera from patients with type 2 diabetes contained significantly more TNF alpha than sera from normoglycemic controls (3.07 +/- 0.24 v 1.65 +/- 0.20 pg x mL(-1), P = .001). However, the comparison of serum TNF-alpha concentrations between microalbuminuric and normoalbuminuric diabetic patients showed no significant difference (3.21 +/- 0.28 v 2.97 +/- 0.34 pg x mL(-1), P = .12). In conclusion, type 2 diabetic patients with diabetic nephropathy exhibit increased activation of TGF-beta1, in serum, suggesting an association between circulating TGF-beta1 activity and the development of renal disease. PMID- 10726915 TI - Comparison of dual-energy X-ray absorptiometry to four other methods to determine body composition in underweight patients with chronic gastrointestinal disease. AB - Assessment of body composition may provide important information about the nutritional status. The applicability of two safe and convenient methods for body composition analysis, bioelectrical impedance analysis (BIA) and dual-energy x ray absorptiometry (DXA), in underweight patients with chronic gastrointestinal disease has been sparsely elucidated. Our objective was to compare measurements by DXA with four other methods. Furthermore, we compared total body water (TBW) by BIA using three different BIA equations with measurement of TBW by tritium dilution (TBW-3H2O). Nineteen clinically stable underweight patients with chronic gastrointestinal disease were included in the study (body mass index [BMI], 19.3 +/- 1.2 kg/m2). Body composition was assessed using total body potassium (TBK), isotope dilution of tritium (3H2O), anthropometry (skinfold thickness [SF]), BIA, and DXA. Fat-free mass (FFM) by DXA was in reasonable agreement with body composition measurements by TBK (mean difference(TBK-DXA) = -1.61 kg, r = .88, standard error of the estimate [SEE] = 4.66 kg) and 3H2O (mean difference(3H2O DXA) = 0.98 kg, r = .93, SEE = 3.34 kg). Although mean values for FFM by DXA differed significantly versus BIA and SF, we found highly significant correlations between the measurements (r = .97 and r = .97, respectively). The mean TBW by BIA was overestimated by 1.9 and 3.1 L compared with TBW-3H2O when prediction equations for normal-weight subjects were used. We conclude that the DXA method is a valuable addition to the list of methods available for body composition studies in clinically stable underweight patients. Our data show that BIA equations for normal-weight subjects overestimated TBW in the patients studied. PMID- 10726916 TI - Evidence for impaired glucose effectiveness in cirrhotic patients after liver transplantation. AB - To evaluate the impact of acute and chronic liver disease and single immunosuppression (cyclosporine A [CSA] or FK506) on insulin sensitivity and glucose effectiveness in liver-grafted patients, we performed a frequently sampled intravenous glucose tolerance test (FSIGTT) in nondiabetic patients after orthotopic liver transplantation (OLT) with acute liver failure ([ALF] group, n = 9, with CSA therapy), in patients after OLT with chronic liver disease (CSA group, n = 8; FK506 group, n = 8), and in 9 healthy control subjects. Insulin sensitivity and glucose effectiveness were determined by analyzing glucose and insulin data from the FSIGTT with Bergman's minimal model technique for glucose. The intravenous glucose tolerance index ([KG] ie, the slope of the regression of the logarithm of blood glucose concentration) was not different between the ALF group (2.17 +/- 0.16 min(-1)) and controls (2.29 +/- 0.13 min(-1)), but was lower (P < .05) in both groups with chronic liver disease (CSA group, 1.46 +/- 0.1; FK506 group, 1.61 +/- 0.11 min(-1)) compared with the ALF group (P < .05). A positive relation for the KG and glucose effectiveness was found in all liver grafted patients and controls. Insulin sensitivity was not different between all liver-grafted patients and controls. The body mass index (BMI) was the overall determinant of insulin sensitivity in all groups. Single immunosuppressive therapy does not impair insulin sensitivity in liver-grafted patients. The lower glucose effectiveness in liver-grafted patients with chronic liver disease but not in patients after ALF points to a defect in the regulation of glucose mediated glucose uptake in peripheral tissue. PMID- 10726917 TI - Lack of effect of sodium nitroprusside on insulin-mediated blood flow and glucose disposal in the elderly. AB - Insulin increases skeletal muscle blood flow in healthy young subjects by a nitric oxide (NO)-dependent mechanism. Impairment of this mechanism may contribute to the insulin resistance of normal aging, a state characterized by reduced endothelial production of NO, an attenuated effect of insulin on skeletal muscle blood flow, and resistance to insulin-mediated glucose uptake (IMGU). We tested the hypothesis that the NO donor sodium nitroprusside (SNP) would augment insulin-mediated vasodilation and thus increase IMGU in healthy elderly subjects. Experiments were performed with young (n = 9; age, 25 +/- 1 years; body mass index [BMI], 24 +/- 1 kg/m2) and old (n = 10; age, 78 +/- 2 years; BMI, 25 +/- 1 kg/m2) healthy subjects. Each group underwent two studies in random order. In one study (control), insulin was infused using the euglycemic clamp protocol for 240 minutes at a rate of 40 mU/m2/min (young) and 34 mU/m2/min (old). In the other study (SNP), SNP was coinfused with insulin from 120 to 240 minutes. At regular intervals in each study, blood samples were obtained and calf blood flow was measured using venous occlusion plethysmography. Glucose and insulin values were similar in control and SNP studies in both age groups. In the young, SNP had no effect on blood flow to the calf, but its action in calf resistance vessels augmented insulin-mediated vasodilation, since incremental calf vascular conductance was greater during SNP infusion (control v SNP, 0.027 +/- 0.002 v 0.040 +/- 0.008 mL/100 mL/min/mm Hg, P< .0001). However, SNP had no effect on insulin-mediated glucose disposal. In the elderly, SNP reduced the blood flow to the calf, but this was countered by its effect on calf resistance vessels such that vascular conductance was unaffected (control v SNP, 0.012 +/- 0.003 v 0.011 +/- 0.003 mL/100 mL/min/mm Hg, P = nonsignificant [NS]). Steady-state (180 to 240 minutes) glucose disposal (control v SNP, 7.47 +/- 0.47 v 6.54 +/- 0.56 mg/kg/min, P < .01) rates were significantly lower during SNP infusion. In summary, systemic infusion of SNP did not increase insulin-mediated glucose disposal in either young or old subjects. Thus, the present findings do not support the concept that increasing NO availability will enhance glucose disposal in either age group. However, because the incremental increases in IMGU during SNP infusion paralleled the changes in blood supply to the calf rather than calf vascular conductance, any potential benefits on NO delivery in elderly subjects may have been offset by the direct or reflex effects of systemic hypotension. Other stimuli to NO production that do not cause hypotension must be tested before this therapeutic strategy can be considered as a potential means for enhancing the metabolic actions of insulin in the elderly. PMID- 10726918 TI - Disposition of dietary ethanol carbons in rats: effects of gender and nutritional status. AB - Dietary ethanol is an important contributor to total caloric intake and has been associated with gender-specific alterations in body weight and the risk for coronary heart disease. To understand the metabolic basis of these effects, it is important to first clarify the effects of gender and nutritional state on the metabolic fate of dietary ethanol. Tracer studies were therefore performed using 14C-labeled ethanol in fasted or fed male and female Sprague-Dawley rats (N = 64) previously unexposed to ethanol. 1-(14C)-ethanol (4.5 microCi) was mixed with unlabeled ethanol (for a total ethanol dose equal to 10% of total daily caloric intake) and a 3-kcal liquid meal and administered through gastric feeding tubes. 14CO2 production was measured over the subsequent 8 hours. The 14C content of skeletal muscle, liver, adipose tissue, gastrointestinal (GI) tract, brain, heart, kidney, and serum was determined at 4 time points following tracer administration (20 minutes and 3, 8, and 24 hours; n = 4 at each time point). Tracer content on a whole-body level was significantly greater in skeletal muscle compared with liver in all groups (1.32 +/- 0.02 x 10(6) v 0.27 +/- 0.02 x 10(6) dpm, P < .001). Skeletal muscle tracer content decreased rapidly after 3 hours, whereas liver tracer content remained fairly constant throughout the study period. Fed female rats were the exception, with a significant increase in the tracer content of total liver and liver lipid at 8 hours. The tracer content was higher in the lipid extracts in liver from fed rats compared with fasted rats (1.08 +/- 0.19 x 10(5) v 0.48 +/- 0.08 x 10(5) dpm, P = .002). While male rats exhibited a fairly constant tracer content in adipose tissue throughout the 24 hour period, female rats showed an increase in adipose tissue tracer content at 8 and 24 hours, with levels 3 to 4 times those of the male animals (5.91 +/- 1.42 x 10(4) v 1.55 +/- 0.42 x 10(4) dpm, P = .02). These results demonstrate that (1) skeletal muscle plays an important role in the metabolism of dietary ethanol, (2) the fed state appears to favor the conversion of ethanol-derived carbons to lipid, and (3) female rats have a greater propensity to convert ethanol-derived carbons to lipid and to store these carbons in adipose tissue. PMID- 10726919 TI - Glucoregulation during and after intense exercise: effects of alpha-adrenergic blockade. AB - In intense exercise (>80% maximal oxygen consumption [VO2 max]), the 7- to 8-fold increase in glucose production (Ra) is tightly correlated with the greater than 14-fold increase in plasma norepinephrine (NE) and epinephrine (EPI). To distinguish the relative roles of alpha- and beta-adrenergic receptors, the responses of 12 control (C) lean, healthy, fit young male subjects to 87% VO2 max cycle ergometer exercise were compared with those of 7 subjects (at 83% VO2max) receiving intravenous phentolamine (Ph). The Ph group received a 70-microg/kg bolus and then 7 microg/kg/min from -30 minutes, during exercise and for 60 minutes of recovery. The data were analyzed by comparing exercise responses to exhaustion in Ph subjects (11.4 +/- 0.6 min) with those at both 12 minutes and at exhaustion in C subjects (14.6 +/- 0.3 min) and during recovery. There were no significant differences between groups in the plasma glucose response during exercise, but values were higher in C versus Ph subjects during the first 40 minutes of postexercise "recovery." The Ra response during the first 12 minutes of exercise was not different by repeated-measures ANOVA, reaching 10.6 +/- 1.3 mg/kg/min in C and 9.6 +/- 1.5 in Ph subjects at 12 minutes. However, in C subjects, Ra increased significantly to 14.1 +/- 1.2 mg/kg/min by exhaustion, and remained higher versus Ph subjects until 15 minutes of recovery. The Rd during recovery was not different between groups; thus, the higher Ra in C subjects in early recovery was responsible for the greater hyperglycemia observed in C subjects. Ph subjects showed a more rapid, marked increment (P = .002) in both plasma NE (to 64 v38 nmol/L) and EPI at exhaustion, and catecholamine concentrations remained higher in Ph versus C subjects during recovery. Whereas plasma insulin (IRI) declined in the C group, it increased 3-fold (P = .001) in the Ph group during exercise and until 15 minutes of recovery. Ph had no effect on glucagon (IRG). Thus, the glucagon to insulin ratio decreased in Ph subjects from baseline levels during exercise and early recovery, but increased in C subjects. The increase in Ra among Ph subjects despite the decrease in the glucagon to insulin ratio supports our earlier evidence that these hormones are not principal regulators of the Ra in intense exercise. The shorter time to exhaustion and markedly higher catecholamine levels in Ph subjects limited our ability to isolate the effects of alpha-adrenergic receptors on the Ra.alpha Adrenergic receptors appear to have little influence on the Rd. PMID- 10726920 TI - Delayed effects of exercise on the plasma leptin concentration. AB - Recent studies have concluded that a single exercise session has no immediate effect on the plasma concentration of leptin, a putative satiety factor. We tested the hypothesis that an increase in energy expenditure would decrease the leptin concentration but the effects would be manifest in a 48-hour period following exercise. Eleven active males completed two treadmill exercise sessions with different energy expenditure (800 or 1,500 kcal) at 70% maximal O2 consumption (Vo2max). Subjects maintained constant energy intake on the day before, the day of, and 2 days after exercise, as verified by dietary recall. Compared with preexercise in either exercise session, there were no differences in plasma leptin concentrations following exercise (0 and 24 hours postexercise) except at 48 hours postexercise, where an approximately 30% decrease (P < .05) was observed. With either duration of exercise, plasma glucose increased about 10% (P < .05), insulin decreased 35% to 46% (P < .05), and cortisol increased 41% to 50% (P < .05, 1,500 kcal only) immediately following exercise, but returned to preexercise values at 24 and 48 hours postexercise. A statistically significant correlation was observed between the changes in leptin and insulin (r = .49, P < .0001). Single exercise sessions of varying energy expenditure decreased the plasma leptin concentration after 48 hours in association with a preceding decrease in insulin. PMID- 10726921 TI - Metabolic effects of vanadyl sulfate in humans with non-insulin-dependent diabetes mellitus: in vivo and in vitro studies. AB - To investigate the efficacy and mechanism of action of vanadium salts as oral hypoglycemic agents, 16 type 2 diabetic patients were studied before and after 6 weeks of vanadyl sulfate (VOSO4) treatment at three doses. Glucose metabolism during a euglycemic insulin clamp did not increase at 75 mg/d, but improved in 3 of 5 subjects receiving 150 mg VOSO4 and 4 of 8 subjects receiving 300 mg VOSO4. Basal hepatic glucose production (HGP) and suppression of HGP by insulin were unchanged at all doses. Fasting glucose and hemoglobin A1c (HbA1c) decreased significantly in the 150- and 300-mg VOSO4 groups. At the highest dose, total cholesterol decreased, associated with a decrease in high-density lipoprotein (HDL). There was no change in systolic, diastolic, or mean arterial blood pressure on 24-hour ambulatory monitors at any dose. There was no apparent correlation between the clinical response and peak serum level of vanadium. The 150- and 300-mg vanadyl doses caused some gastrointestinal intolerance but did not increase tissue oxidative stress as assessed by thiobarbituric acid-reactive substances (TBARS). In muscle obtained during clamp studies prior to vanadium therapy, insulin stimulated the tyrosine phosphorylation of the insulin receptor, insulin receptor substrate-1 (IRS-1), and Shc proteins by 2- to 3-fold, while phosphatidylinositol 3-kinase (PI 3-kinase) activity associated with IRS-1 increased 4.7-fold during insulin stimulation (P = .02). Following vanadium, there was a consistent trend for increased basal levels of insulin receptor, Shc, and IRS-1 protein tyrosine phosphorylation and IRS-1-associated PI 3-kinase, but no further increase with insulin. There was no discernible correlation between tyrosine phosphorylation patterns and glucose disposal responses to vanadyl. While glycogen synthase fractional activity increased 1.5-fold following insulin infusion, there was no change in basal or insulin-stimulated activity after vanadyl. There was no increase in the protein phosphatase activity of muscle homogenates to exogenous substrate after vanadyl. Vanadyl sulfate appears safe at these doses for 6 weeks, but at the tolerated doses, it does not dramatically improve insulin sensitivity or glycemic control. Vanadyl modifies proteins in human skeletal muscle involved in early insulin signaling, including basal insulin receptor and substrate tyrosine phosphorylation and activation of PI 3 kinase, and is not additive or synergistic with insulin at these steps. Vanadyl sulfate does not modify the action of insulin to stimulate glycogen synthesis. Since glucose utilization is improved in some patients, vanadyl must also act at other steps of insulin action. PMID- 10726922 TI - The novel hypoglycemic agent YM440 normalizes hyperglycemia without changing body fat weight in diabetic db/db mice. AB - To determine the relationship between hypoglycemic activity and body weight gain induced by insulin sensitizers, we compared the effects of thiazolidinedione analogs (troglitazone and pioglitazone) and the oxadiazolidinedione analog (Z) 1,4-bis4[(3,5-dioxo-1,2,4-oxadiazolidin-2-yl)methyl]phen oxy?but-2-ene (YM440) in diabetic db/db mice. Oral treatment with YM440(100 mg/kg) for 28 days decreased the blood glucose concentration (control v YM440, 418 +/- 12 v243 +/- 44 mg/dL). The hypoglycemic activity of this agent was comparable to that of troglitazone (300 mg/kg) and pioglitazone (100 mg/kg). There were no changes in food intake among the groups. Troglitazone and pioglitazone, but not YM440, significantly increased body weight gain during treatment (control, 7.2 +/- 0.5 g; YM440, 7.5 +/- 0.8 g; troglitazone, 10.9 +/- 0.8 g; and pioglitazone, 14.5 +/- 1.1 g). To further assess whether the increase in body weight by troglitazone or pioglitazone was due to adipogenesis, the weight of intraabdominal fat tissue (epididymal, retroperitoneal, and perirenal) was determined. There were no differences in the total weight of visceral fat between the control and YM440 treatment (3.53 +/- 0.23 and 3.60 +/- 0.16 g). In contrast, troglitazone and pioglitazone significantly increased the fat weight (4.31 +/- 0.13 and 4.66 +/- 0.19 g). Thiazolidinediones are known as ligands for peroxisome proliferator activated receptor gamma (PPARgamma), a nuclear receptor responsible for adipogenesis. Troglitazone and pioglitazone activated PPARgamma and increased triglyceride accumulation and mRNA expression of fatty acid-binding protein (FABP) in 3T3-L1 cells. However, YM440 had no effect on these indices for adipocyte differentiation. These results suggest that the mechanism is different for the hypoglycemic action of YM440 versus the thiazolidinediones. YM440 ameliorates hyperglycemia without changing PPARgamma activity, adipocyte differentiation, or fat weight. Thus, YM440 could be a useful hypoglycemic agent for the treatment of non-insulin-dependent diabetes mellitus (NIDDM) without affecting body weight. PMID- 10726923 TI - Insulin-mediated glucose disposal is decreased in normal subjects with relatively low plasma magnesium concentrations. AB - The relationship between the plasma magnesium (Mg) concentration and steady-state plasma insulin (SSPI) and glucose (SSPG) concentrations at the end of a 180 minute infusion of octreotide, insulin, and glucose was determined in 98 healthy nondiabetic subjects. For the purposes of data analysis, the population was divided into tertiles on the basis of the plasma Mg concentration: I, plasma Mg 0.83 mmol/L; II, plasma Mg 0.84 to 0.91 mmol/L; and III, plasma Mg 0.92 mmol/L. The three groups were identical in terms of age, gender distribution, and degree of obesity. However, both fasting plasma insulin (P < .05) and SSPG (P < .05) concentrations were significantly higher in the tertile (I) with the lowest plasma Mg concentration. Furthermore, there was a significant inverse correlation between plasma Mg and SSPG concentrations (r = -.27, P < .01) in the entire population. These results indicate that variations in the plasma Mg concentration have a relatively modest but significant effect on insulin-mediated glucose disposal in healthy subjects, with lower plasma Mg concentrations associated with increased insulin resistance. PMID- 10726924 TI - IC202B and C, new siderophores with immunosuppressive activity produced by Streptoalloteichus sp. 1454-19. AB - IC202B (1) and C (2) were isolated from the culture filtrate of Streptoalloteichus sp. 1454-19. The structures were elucidated by various NMR spectral analyses including 1H-15N HMBC and FAB-MS experiments. IC202B and C showed immunosuppressive activity on a mixed lymphocyte culture reaction at the same IC50 value of 1.6 microg/ml. PMID- 10726925 TI - Decatromicins A and B, new antibiotics produced by Actinomadura sp. MK73-NF4. I. Taxonomy, isolation, physico-chemical properties and biological activities. AB - New antibiotics designated decatromicins A and B were isolated from the culture broth of Actinomadura sp. MK73-NF4. They were purified by butyl acetate extraction, silica gel column chromatography, silica gel TLC and Sephadex LH-20 column chromatography. Decatromicins A and B inhibited growth of Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). PMID- 10726926 TI - Decatromicins A and B, new antibiotics produced by Actinomadura sp. MK73-NF4. II. Structure determination. AB - The structures of decatromicins A and B that strongly inhibit the growth of MRSA were elucidated by the analysis of various NMR experiments. The planar structure was determined by 1H, 13C, COSY, HMQC and HMBC NMR spectra. The relative configuration of aglycone was elucidated by NOESY experiments and the absolute structure was determined by application of the modified Mosher's method. The absolute structure of glycosyl moiety was determined by X-ray analysis of the O (p-bromobenzoyl) derivative. PMID- 10726927 TI - Inhibition of plasminogen activator inhibitor-1 by 11-keto-9(E),12(E) octadecadienoic acid, a novel fatty acid produced by Trichoderma sp. AB - We have recently found a novel fatty acid, 11-keto-9(E),12(E)-octadecadienoic acid (KOD), that enhances fibrinolytic activity of endothelial cells. The mechanism of action of KOD has been investigated. KOD increased 2-fold the plasmin activity of bovine aortic endothelial cells at 250 microM. The stimulation was dependent on plasminogen and was inhibited by anti-urokinase, whereas KOD did not enhance the urokinase-catalyzed plasminogen activation and the resulting plasmin activity in a cell-free system. Neither the production of urokinase nor the conversion of pro-urokinase to the active, two-chain form was elevated by KOD, but it decreased plasminogen activator inhibitor-1 (PAI-1) activity of cells and inactivated PAI-1 irreversibly in a purified system. These results demonstrated that the KOD enhancement of endothelial fibrinolytic activity was mediated, at least in part, by inactivation of PAI-1. PMID- 10726928 TI - The decrease of cytochrome c oxidase activity by 15-deoxyspergualin results in enhancement of XTT reduction in cultured cells. AB - Exposure to immunosuppressant, 15-deoxyspergualin (DSG) induced enhanced formazan producing activity from XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H tetrazolium-5-carb oxanilide, sodium salt) in cultured cells, but not from MTT (3 (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Formazan generation from XTT is known to be stimulated by cytochrome c oxidase (COX) inhibitors such as KCN and NaN3, whereas MTT reduction is not affected by them. So, the effect of DSG on COX was examined. DSG did not directly inhibit the enzyme, but the cellular enzyme activity was decreased by exposure to DSG. It was thought that stimulation of XTT reduction by DSG resulted from the decrease of cellular cytochrome c oxidase activity. PMID- 10726929 TI - Enzymatic 1-N-acetylation of paromomycin by an actinomycete strain #8 with multiple aminoglycoside resistance and paromomycin sensitivity. AB - An actinomycete strain #8 with multiple aminoglycoside (AG) resistance and paromomycin (PRM) sensitivity was examined for its capability of enzymatic modification of AGs. Cell free extracts from the strain converted all of the examined AGs including PRM in the presence of acetyl CoA. PRM was completely modified to at least two products (major and minor spots upon TLC) without significant reduction of the antibiotic activity of the reaction mixture. The structure determination and antibiotic assay of the purified major product revealed l-N-acetylPRM and its antibiotic activity (12% activity of PRM), indicating the existence of AAC(1). It was thus obvious that the 1-N-acetylation of PRM did not cause PRM resistance. Apramycin, the substrate of the known AAC(1), was not readily acetylated, suggesting that the AAC(1) of strain #8 is a new type. Two diacetylated products (1,2'-di-N-acetylPRM and 1,6"'-di-N acetylPRM) were found in the minor spot, suggesting the existence of additional AACs. PMID- 10726931 TI - F-11334s, new inhibitors of membrane-bound neutral sphingomyelinase. PMID- 10726930 TI - Structure elucidation of roselipins, inhibitors of diacylglycerol acyltransferase produced by Gliocladium roseum KF-1040. AB - The structures of roselipins 1A, 1B, 2A and 2B were elucidated by spectroscopic studies including 1H-1H COSY, 13C-1H COSY, 13C-1H HMQC and 13C-1H HMBC NMR experiments, and degradation experiments. They have the common skeleton of 2,4,6,8,10,12,14,16,18-nonamethyl-5,9,13-trihydroxy-2E,6E, 10E-icosenoic acid modified with a D-mannose and a D-arabinitol. Roselipin A and B groups were stereoisomers at the arabinitol moiety, which esterified the fatty acid from the different terminal hydroxy residue. Roselipin 2 group was 6"-O-acetyl roselipin 1 group. PMID- 10726932 TI - Preparation and in vitro antibacterial activity of 6-O-methylerythromycin D. PMID- 10726933 TI - Solid-phase synthesis of cyclooctadepsipeptide N-4909 using a cyclization cleavage method with oxime resin. PMID- 10726934 TI - Circumcision in men and the prevention of HIV infection: a 'meta-analysis' revisited. AB - There is debate on the role of male circumcision in HIV transmission. Most case control and cohort studies from Africa have shown an association between a lack of circumcision and an increased risk of HIV infection in men. The evidence is conflicting, however, with cross-sectional surveys from Tanzania and Rwanda either showing no relationship or an association in the opposite direction. A recent review and meta-analysis of the literature concluded that the risk of HIV infection was lower in uncircumcised men (combined odds ratio 0.94, 95% confidence interval 0.89 to 0.99). However, the analysis was performed by simply pooling the data from 33 diverse studies, which is an inappropriate method for combining studies. We re-analysed the data, stratifying by study, and found that an intact foreskin was associated with an increased risk of HIV infection: combined odds ratio 1.43 (1.32 to 1.54) with a fixed effect model and 1.67 (1.25 to 2.24) with a random effect model. There was significant between-study heterogeneity (P<0.0001) which was partly explained by stronger associations in studies in high-risk groups. The results from this re-analysis thus support the contention that male circumcision may offer protection against HIV infection, particularly in high-risk groups where genital ulcers and other STDs 'drive' the HIV epidemic. A systematic review is required to clarify this issue. Such a review should be based on an extensive search for relevant studies, published and unpublished, and should include a careful assessment of the design and methodological quality of studies. Much emphasis should be given to the exploration of possible sources of heterogeneity. In view of the continued high prevalence and incidence of HIV in many countries in sub-Saharan Africa, the question of whether circumcision could contribute to prevent infections is of great importance, and a sound systematic review of the available evidence should be performed without delay. PMID- 10726935 TI - Vaccine characteristics and acceptability of HIV immunization among adolescents. AB - HIV immunization programmes will only be effective if sufficient numbers of persons accept the vaccine. Our aims were to evaluate HIV vaccine acceptability among adolescents and to examine how vaccine characteristics influence acceptability. We recruited 661 adolescents from community health clinics in Indianapolis, Indiana, USA to complete either written or computerized questionnaires, both of which assessed HIV vaccine acceptability as a function of efficacy, cost, type of vaccine, mode of delivery, and parental permission for immunization (required or not required). For both the written and computer methods, efficacy had the strongest effect on acceptability, followed by type of vaccine and cost. Low efficacy, high cost, and live-attenuated vaccines were associated with lower acceptability. These findings suggest that as efforts to develop HIV vaccines continue, it will be important, in parallel, to anticipate potential obstacles to vaccine acceptance, including the belief that a less efficacious HIV vaccine is unacceptable. PMID- 10726936 TI - Diagnosis of bacterial vaginosis by wet mount identification of bacterial morphotypes in vaginal fluid. AB - In order to develop a more practical way of diagnosing bacterial vaginosis (BV), we evaluated a scoring system, weighting small bacterial morphotypes versus lactobacillary morphotypes in wet mounts, assessed criteria for BV and normalcy from this scoring, and then evaluated their reproducibility and accuracy. We examined 754 women for pH, homogeneous vaginal discharge, amine odour, clue cells and the composite clinical diagnosis. We also examined wet mounts for small bacterial morphotypes and lactobacillary morphotypes, and weighted their quantitative presence as a bacterial morphotype score. The term 'small bacterial morphotypes' denotes a group of small bacillary forms comprising coccobacilli, tiny rods, and mobile curved rods. The different characteristics of BV were all gradually associated with increased bacterial morphotype scoring. We deemed a score of 0-1 as normal, 2-4 as intermediate phase, grade I, 5-6 as intermediate phase, grade II, and 7-8 indicative of BV. Reproducibility of the interpretation was high, both for the new grading system (weighted Kappa 0.90 in women perceiving and 0.81 in women not perceiving abnormal vaginal discharge) and for the new criterion for BV (non-weighted Kappa 0.91 and 0.84 in the 2 groups of women). The new criterion also proved highly concurrent with the composite clinical diagnosis (Kappa 0.91 and 0.81 in the 2 groups). In conclusion, the wet mount bacterial morphotype scoring is valid for grading of the disorder of the vaginal microbial ecosystem, and the new criterion for BV a more practical option than existing diagnostic methods. PMID- 10726937 TI - Psychosocial determinants for sexual partner referral in Uganda: qualitative results. AB - In order to understand and elicit the psychosocial factors influencing compliance with sexual partner referral, 10 focus group discussions (FGDs) and 40 individual interviews based on the attitude-social influence-self efficacy (ASE) model were held in Mbarara District of Uganda. The focus groups were derived from both rural and urban areas. Informal interviews were held with 20 men and 20 women presenting with symptoms of a sexually transmitted disease (STD) at the outpatient department of Mbarara Hospital. The emerging outcome of attitudinal beliefs regarding health consequences of partner referral were mainly positive. However, outcome beliefs relating to the relationship with the partners were mainly negative. Social influence for sexual partner referral was from health care providers, partner(s), friends and relatives. The self-efficacy beliefs were mainly negative especially among women. Barriers hampering sexual partner referral were related to poor quality of health care, gender relations and type of sexual partners. There is a need to target these beliefs and self-efficacy expectations in health education and STD counselling and for more research to evaluate the psychosocial determinants of sexual partner referral quantitatively. PMID- 10726938 TI - An acceptability study of female-controlled methods of protection against HIV and STDs in south-western Uganda. AB - We aimed to assess the acceptability of a variety of formulations of female controlled methods of protection against HIV and STDs among men and women in south-western Uganda. Pilot interviews were carried out with 50 men and 55 women and 25 focus group discussions (FGDs) were held with 138 women and 42 men. The female condom, foaming tablets, sponge, foam, gel and film were demonstrated to 146 women and 35 of their male partners, who then tried out 2 of the products. They were interviewed 7 times during the course of 5 months. At the end experiences were evaluated during a second series of FGDs. Sixty-five (45%) women completed the trial. The main reasons for non-completion were related to geographical mobility. Product preference after the initial demonstration was similar to that at the end of the trial. The most popular formulations were the sponge (25% of the women), foaming tablets (23%), and the female condom (19%). The foam was of medium popularity (16%). The gel (9%) and film (7%) were least popular. Ten per cent of the women and 14% of the men reported products interfering with sexual enjoyment; 24% of the women and 67% of the men said products increased enjoyment. 'Dry sex' is not popular in this area and increased lubrication was an important determinant of acceptability. Age, level of education and location did have some effect on preference. Although secrecy was a dominant theme in the FGDs, 87% of the women had informed their partners by the end of the trial. The products were generally well received. Female control was an important issue for both sexes. Male attitudes were ambivalent because female ownership of products increased women's control. Although they have clear preferences, women appear to accept the products generally and might use a single available product just as readily if choice was limited, as long as it conforms to general cultural preferences, such as those relating to wet/dry sex. PMID- 10726940 TI - Attitudes regarding vaccinations of STDs and other diseases. AB - The purpose of this study was to examine, among 2 groups of individuals with different risk profiles, the similarity of their attitudes towards vaccines for sexually transmitted diseases (STDs) and non-STDs. Subjects were recruited from an undergraduate psychology class at an urban university (n=518) or were participants in genital herpes vaccine trials (n=87). The participants were asked to complete a questionnaire regarding their attitudes about vaccines for selected diseases. The results of this study revealed that, in general, both groups supported vaccination for most diseases. There were differences, however, between groups regarding 3 diseases: measles, genital warts, and chlamydia. The vaccine trial participants were more likely to accept vaccines for measles and the college students were more likely to accept vaccines for chlamydia and genital warts. The results of this study suggest that negative attitudes regarding vaccination to control STDs may not be a significant barrier to use. PMID- 10726939 TI - Is it evidence-based practice? Prophylactic antibiotics for termination of pregnancy to minimize post-abortion pelvic infection? AB - Sexually transmitted infections (STIs) causing upper genital tract problems after termination of pregnancy (TOP) is well recognized. We undertook this study to assess the local prevalence of Chlamydia trachomatis infection and to estimate the potential benefits of introducing screening. The prevalence rate of C. trachomatis was 6%. Nine sexual contacts of the index cases were identified. They were symptom free, but all had non-specific urethritis (NSU). Four of them were positive for C. trachomatis. We conclude that screening for chlamydial infection is essential and routine prophylactic antibiotic cover may not be beneficial. PMID- 10726942 TI - Ureaplasma urealyticum colonization in the vaginal introitus and cervix of human immunodeficiency virus-infected women. AB - Ureaplasma urealyticum colonization was examined in paired cervical and introital specimens from 56 untreated HIV-seropositive women. Specimens were tested for U. urealyticum by polymerase chain reaction (PCR). Peripheral blood was examined for CD4 lymphocyte counts and HIV RNA concentration. U. urealyticum was detected in the cervix of 38 (69.1%) women. Introital U. urealyticum was present in 16 (28.6%) women, all of whom were cervical-positive. Cervical motion pain was present in 40.0% of women with U. urealyticum in the introitus and cervix, 23.8% of those with only cervical U. urealyticum and 5.9% of women negative for this organism (P=0.03). There was no relation between U. urealyticum colonization and CD4 lymphocyte count. However, 64.3% of women with introital U. urealyticum had circulating HIV RNA levels > 10,000 copies per ml as compared with 28.6% of women with only cervical U. urealyticum and 7.1% of women negative for this organism (P < 0.01). PMID- 10726941 TI - HIV testing in gay sex clubs. AB - The purpose of this study was to evaluate a programme of human immunodeficiency virus (HIV) antibody testing at gay sex clubs. Conducting secondary analyses with 2 datasets, we evaluated HIV-testing preferences of patrons at 2 sex clubs and compared their risks to testers at a standard testing clinic. Sex club testers had significantly more partners and were significantly older than their clinic peers. Sixteen per cent of sex club testers reported that they would not test if testing were not available at the sex club. Gay sex clubs offer an opportunity to reach men at high risk for HIV, some who otherwise may not test. PMID- 10726943 TI - Detecting cervical infection among family planning clients: difficulties at the primary health-care level in Indonesia. AB - In developing and testing an operational model for the integration of reproductive tract infection/sexually transmitted disease (RTI/STD) management into existing family planning (FP) services in Indonesia, this study allowed for assessment of disease prevalence and evaluation of diagnostic methods for detection of endocervicitis caused by chlamydial infection and/or gonorrhoea. Data were collected over 28 weeks in 1997 at 2 FP clinics in the low-income harbour neighbourhood of North Jakarta. Among 486 consenting female FP clients, prevalence of chlamydial infection was 9.3%, gonorrhoea 1.2%, trichomoniasis 4.5% and syphilis 0.8%. Clinically observed abnormal vaginal discharge, cervical inflammation and vaginal lesions/ulcers were all associated with cervical infection (P<0.05), but insufficiently sensitive (<60%). Clinical diagnosis for cervical infection had 48.8% sensitivity, 75.4% specificity, but only 18.3% positive predictive value (PPV). On-site Gram stains for gonorrhoea were 83.3% sensitive and 94.5% specific, but had only 16.1% PPV. Presence of mucopurulent cervicitis was only 39.6% sensitive for cervical STD, with PPV of only 16.3%. Development of an affordable and accurate detection tool for chlamydial infection remains the main obstacle to effective RTI/STD management in this population. PMID- 10726944 TI - Symptomatic and asymptomatic chlamydial non-gonococcal urethritis in Jamaica: the potential for HIV transmission. AB - To determine the contribution of Chlamydia trachomatis to non-gonococcal urethritis (NGU) in men attending sexually transmitted disease (STD) clinics in Jamaica we studied men with NGU (n=339), and control groups including asymptomatic men who were STD contacts (n=61), asymptomatic men who were not STD contacts (n=32) and men with gonococcal urethritis (GU) (n=61). Urethral specimens were examined for C. trachomatis and Neisseria gonorrhoeae. Serological tests for syphilis (STS) and HIV-1 infection were also performed. C. trachomatis accounted for 63% of cases of NGU but high prevalences were also found in asymptomatic STD contacts (59%), asymptomatic STD non-contacts (78%) and men with GU (48%). The prevalence of C. trachomatis in men with GU differed significantly from that in men with NGU and asymptomatic STD non-contacts (P<0.05). C. trachomatis infection in men with NGU was associated with multiple sex partners (71% vs 58%; chi2=4.78; odds ratio (OR)=1.76; P<0.05) and previous history of gonococcal infection (83% vs 42%; chi2=59.8; OR=6.8; P<0.0001). Concomitant infection with HIV-1 occurred in 5.2% of cases of NGU and 50% and 90%, respectively, of the HIV-positive men had chlamydia or reactive STS. As a cost effective strategy in the control of STD and HIV we recommend presumptive treatment for C. trachomatis in men seeking STD treatment in Jamaica. PMID- 10726945 TI - Sexual dysfunction in genitourinary medicine clinics. PMID- 10726946 TI - An audit of antiretroviral medication offered to patients with HIV attending a district general hospital for outpatient care. AB - In order to determine if antiretroviral prescribing for patients with HIV infection attending the Central Middlesex Hospital is according to current UK guidelines and effective at reducing the serum HIV viral load, 71 case notes were reviewed. All patients eligible for treatment according to the British HIV Association (BHIVA) guidelines were currently being offered triple therapy. The most recent serum HIV viral loads of patients taking at least 3 antiretrovirals were undetectable in 75% of the 20 patients on their first established regimen and 36% of 14 patients who had failed at least one drug according to previous surrogate marker results. Such work allows an individual clinic to monitor its antiretroviral prescribing practices in the face of constantly updated information and guidelines. PMID- 10726947 TI - Opportunistic testing for chlamydial infection in people attending a sexual medicine clinic for HIV tests. AB - Patients presenting for an HIV test alone represent an opportunity for screening for Chlamydia trachomatis. This audit was designed to assess the uptake of chlamydial screening by urinary ligase chain reaction (LCR) in adults attending a genitourinary clinic. All patients requesting an HIV test were offered full genital screening in the first audit period, if they declined they were offered chlamydial LCR. During the second period patients who refused full screening were asked to provide a urine sample for LCR testing unless they declined. Nine hundred and forty-two patients presented for HIV tests alone during the first audit period. Two hundred (22%) agreed to provide a urine sample for LCR testing, 7 (3.5%) were positive for chlamydia. During the second audit period 794 patients presented for HIV tests alone, 426 (55.1%) provided urine for LCR testing, 15 (3.4%) were positive. The uptake of urinary LCR, a non-invasive test for a potentially serious infection remained disappointingly low despite changes to the audit protocol. Reasons for this are discussed. PMID- 10726948 TI - Not every penile lump is a wart! Schwannoma of the penis. PMID- 10726949 TI - Pseudobulbar palsy recovery following HSV encephalitis in AIDS. PMID- 10726950 TI - Distant healing. PMID- 10726951 TI - Termination of pregnancy. PMID- 10726952 TI - Sensitivity of chlamydial diagnostic tests. PMID- 10726953 TI - HIV and HTLV-1 co-infection. PMID- 10726954 TI - Non-heart-beating liver donors: towards increasing the liver supply. PMID- 10726955 TI - Morphological mechanisms for regulating blood flow through hepatic sinusoids. AB - This review summarizes what is known about the various morphological sites that regulate the distribution of blood flow to and from the sinusoids in the hepatic microvascular system. These sites potentially include the various segments of the afferent portal venules and hepatic arterioles, the sinusoids themselves, and central and hepatic venules. Given the paucity of smooth muscle in the walls of these vessels, various sinusoidal lining cells have been suggested to play a role in regulating the diameters of sinusoids and influencing the distribution and velocity of blood flow in these vessels. While sinusoidal endothelial cells have been demonstrated to be contractile and to exhibit sphincter function, attention has recently focused on the perisinusoidal stellate cell as the cell responsible for controlling the sinusoidal diameter. A very recent study, however, suggested that the principal site of vasoconstriction elicited by ET-1 was the pre-terminal portal venule. This raised the question of whether or not the diameters of sinusoids might decrease due to passive recoil when inflow is reduced or eliminated and intra-sinusoidal pressure falls. In more recent in vivo microscopic studies, clamping of the portal vein dramatically reduced sinusoidal blood flow as well as the diameters of sinusoids. The sinusoidal lumens rapidly returned to their initial diameters upon restoration of portal blood flow suggesting that sinusoidal blood pressure normally distends the sinusoidal wall which can recoil when the pressure drops. Stellate cells may be responsible for this reaction given the nature of their attachment to parenchymal cells by obliquely oriented microprojections from the lateral edges of their subendothelial processes. This suggests that care must be exercised when interpreting the mechanism for the reduction of sinusoidal diameters following drug administration without knowledge of changes occurring to the portal venous and hepatic inflow. PMID- 10726956 TI - Ecstasy-induced toxicity in rat liver. AB - BACKGROUND/AIMS: The aim of the present study was to examine the effects of single and repeated administration of 3,4-methylenedioxyme-thamphetamine (MDMA, "ecstasy") on rat liver. METHODS: Animals were given an acute (20 mg/kg) and repeated (20 mg/kg, b.i.d., for 4 consecutive days) intraperitoneal dose of MDMA, and at various times after administration the hepatic and serum determinations were made. RESULTS: The effect of acute MDMA administration included increased triglyceride and cholesterol levels and an increase in all enzyme activities 6 h post administration. The toxic effect of MDMA was also observed in other hepatic processes. Glycogen content showed a marked decrease, which was accompanied by a decrease in serum glucose levels. No significant changes in lipid peroxidation and hepatic GSH content were observed. In contrast, multiple MDMA administration produced some evidence of oxidative stress, namely, increased MDA content and decreased GSH content, a small decrease in liver glycogen at 3 h recovering 6 h post dose, no effect on blood glucose and increased AST and ALP activities but no effects on ALT activity. Seven days after the last MDMA injection a tendency towards recovery was shown. CONCLUSION: Our results show that the liver toxicity caused by MDMA administration involves several mechanisms. PMID- 10726957 TI - A study of graft versus host disease using bile duct implants under the kidney capsule. AB - BACKGROUND/AIMS: Graft versus host disease (GVHD) following histoincompatible bone marrow transplantation may be modelled experimentally using irradiated metallothionein promoter-H-2Kb transgenic mice (MET-Kb mice), reconstituted with syngeneic non-transgenic spleen and lymph node (LN) cells. In this model, inflammation peaks at 3 weeks post-reconstitution, but resolves by 3 months, and is focussed on portal tracts and bile ducts (BD). The aim of this study was to determine if transgene-expressing hepatocytes play a role in the immune response, why portal tracts are selectively targeted, and which cell types are involved. METHODS: Intrahepatic BD (IHBD) with attached hepatocytes, or extrahepatic BD (EHBD) devoid of hepatocytes, were isolated from MET-Kb mice and implanted under the kidney capsule of transgenic (syngeneic) and congenic (allogeneic) mice. Three weeks post-implantation, BD were scored histologically for rejection or survival, and stained for various cell-surface molecules. RESULTS: Generally, IHBD survived better than EHBD, and T cells were the predominant infiltrating cell type in both implants. Both types of implants undergoing rejection expressed intercellular adhesion molecule-1 (ICAM-1) and leukocyte function antigen-1 (LFA 1) at high density; BD and the underlying kidney parenchyma also expressed class I and II major histocompatibility complex (MHC). CONCLUSIONS: The rejection of both groups of implants by congenic recipients suggests that BD from MET-Kb mice express the transgene, but the reason for the selective targeting of portal tracts rather than transgene-expressing hepatocytes remains unclear. One possible explanation is that dendritic cells/antigen-presenting cells (DC/APC) in portal tracts, which express high levels of MHC and co-stimulatory molecules, are the primary targets, and that BD are infiltrated and destroyed as 'bystanders'. PMID- 10726958 TI - Bile salt independent flow during bile salt-induced choleresis and cholestasis in the rat: role of biliary thiol secretion. AB - AIMS/BACKGROUND: Previous studies have shown that the generation of the so-called "bile salt-independent flow" (BSIF) may be partly dependent on the hepatic availability and rate of canalicular secretion of osmotically active substances such as glutathione (GSH) and derived thiols. This study examined the role of common bile salts (BS) on the BSIF formation under both choleretic and cholestatic conditions, and on the relationship of the BSIF to the biliary thiol secretion. METHODS: Experiments were carried out in adult male Sprague-Dawley rats both in situ in the isolated perfused rat liver and in vivo. The effect of choleretic and cholestatic doses of BS on the biliary BS secretion rate (BSSR), BS-dependent flow (BSDF), and BSIF was evaluated. RESULTS: In the perfused rat liver, the infusion of low and physiological doses of taurocholic acid stimulated the biliary BSSR, BSDF, and BSIF. This was associated with increased biliary thiol secretion and thiol-dependent bile flow. In vivo administration of taurocholic acid, taurochenodeoxycholic acid or taurolithocholic acid in step wise increasing doses leading to cholestasis showed that the onset of cholestasis was not accompanied by a significant decline in the BSSR or BSDF but rather by a marked inhibition of the apparent BSIE During cholestasis, the three BS produced a significant reduction of biliary thiol secretion, with a marked decrease in thiol-dependent bile flow sufficient to account for a major proportion of BSIF inhibition. This decline in thiol secretion occurred before the drop in biliary BS secretion and was more pronounced than the reduction in BS output. No change in hepatic thiol content was observed. Administration of free or glyco-conjugated BS also resulted in a significant decrease of BSIF during the cholestatic period, suggesting a common role for BSIF inhibition in BS-induced cholestasis. CONCLUSION: The changes in bile flow during BS-induced choleresis and cholestasis are mediated by changes in the portion of the BSIF regulated by the thiol secretion. PMID- 10726959 TI - Biliary excretion of bile acids and organic anions in zone 1- and zone 3-injured rats. AB - AIMS: Hepatocytes in zone 1 of the hepatic lobule play a role in the uptake and biliary excretion of bile acids and organic anions under physiological conditions, and those in zone 3 may play a role only with a high-dose load. To further elucidate the role of hepatic zonation on the handling of bile acids and organic anions, biliary excretion of these compounds was studied in zone 1- and zone 3-injured rats. METHODS: Biliary excretion of bile acids and organic anions was studied in zone 1- and zone 3-injured rats, caused by allyl alcohol and bromobenzene, respectively. RESULTS: Biliary excretion of a tracer dose of taurocholate was unchanged in the injury in both zones, but that of leukotriene C4 was decreased in zone 1 injury. The excretory maximum of taurocholate was decreased with zone 1 and the zone 3 injuries. Biliary excretion of deoxycholate metabolites was decreased in zone 3 injury, although the profile of metabolites in the bile was unchanged. Sulfobromophthalein excretion was decreased in zone 1 injury, but unchanged in zone 3 injury. CONCLUSIONS: These findings indicate that zone 1 is very important for biliary excretion of both organic anions and bile acids. In contrast, zone 3 is considered not to have a role in biliary excretion of organic anions, but to play a role in the excretion of bile acids. PMID- 10726960 TI - Reduced hepatic content of dehydroepiandrosterone sulphotransferase in chronic liver diseases. AB - AIMS/BACKGROUND: Dehydroepiandrosterone sulphotransferase (DHEA ST) is the enzyme responsible for sulphation of lithocholic acid and other potentially hepatotoxic steroids. We have previously shown that DHEA ST activity is reduced in cytosol of liver from miscellaneous patients with chronic liver disease. The aim of this study was to investigate the cause of diminished sulphotransferase activity in order to further our understanding of whether a reduction in the ability to sulphate potentially hepatotoxic bile acids might play a role in the aetiology of primary cholestatic liver disease. METHODS: We quantified DHEA ST in human liver cytosol from groups of patients with chronic liver diseases and normal subjects using a semiquantitative sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE)/ immunoblotting method, and an enzyme-linked immunosorbent assay (ELISA). We determined DHEA ST enzyme activity and correlated it with its immunoreactive concentration in 87 samples of human liver tissue. RESULTS: DHEA ST activity and concentration were significantly reduced in primary biliary cirrhosis, primary sclerosing cholangitis, chronic active hepatitis and alcoholic cirrhosis but not in cryptogenic cirrhosis when compared to normal liver. There were no significant differences among disease groups. In all groups enzyme activity and cellular concentration correlated, suggesting that no aberrant non-functional enzyme was produced. CONCLUSION: These results confirm that DHEA ST activity is diminished in liver disease and that the reduction is due to diminished enzyme presence. Further studies are required to show whether the reduction has any pathogenetic significance or is merely a consequence of disease. PMID- 10726961 TI - Central and systemic haemodynamic effects of terlipressin in portal hypertensive patients. AB - AIMS/BACKGROUND: Cirrhotic patients exhibit a hyperdynamic and hyporeactive circulation with central hypovolaemia which may influence the course of the disease. As terlipressin, a vasopressin analogue, may modify systemic haemodynamics in these patients, the aim of the present study was to assess the acute effects of terlipressin on central and systemic haemodynamics. METHODS: Sixteen patients with alcoholic cirrhosis and portal hypertension had their systemic, central, and splanchnic haemodynamics determined at baseline and after a blind randomised bolus infusion (2 mg) of terlipressin/placebo. RESULTS: After terlipressin, the arterial blood pressure and the systemic vascular resistance increased by 26% and 61%, respectively (both p<0.001), and the cardiac output, heart rate, and arterial compliance decreased by 18%, 11%, and 32%, respectively (all p<0.001). The central circulation time increased by 36% (p<0.001), whereas the central and arterial blood volume only increased by 4% (p= 0.07). As expected, both portal pressure and hepatic blood flow decreased (17% and 29%, both p<0.001). The decrease in portal pressure after terlipressin was significantly related to the increase in systemic vascular resistance (r=-0.52, p<0.05) and the central circulation time (r=-0.80, p<0.0001). CONCLUSIONS: Terlipressin significantly attenuates the hyperdynamic circulation in portal hypertensive patients without a further contraction of the central and arterial blood volume. The systemic haemodynamic response to terlipressin is moreover associated with the decrease in portal pressure. Terlipressin may therefore have potentially beneficial effects on the hyperdynamic circulation in cirrhosis in addition to its effects on portal pressure. PMID- 10726962 TI - Systemic and splanchnic endothelin-1 plasma levels in liver cirrhosis before and after transjugular intrahepatic portosystemic shunt (TIPS). AB - AIMS/BACKGROUND: Endothelin-1 (ET-1) may be a mediator for portal hypertension in liver cirrhosis. The aim of the present study was to determine the concentrations of ET-1 in the systemic and splanchnic circulation before and after reduction of portal hypertension by transjugular intrahepatic portosystemic shunt implantation (TIPS). METHODS: Plasma concentrations of immunoreactive ET-1 were measured in peripheral venous blood samples from 25 patients with liver cirrhosis before and at 1, 3, 9 and 15 months after TIPS. Furthermore, acute effects of TIPS on ET-1 were studied in plasma samples from the hepatic vein, the portal vein 30 minutes before and after TIPS and in the femoral artery (only after TIPS) in a subgroup of 15 patients. In addition, the portocaval pressure gradient was determined before and after TIPS. RESULTS: Before TIPS peripheral venous plasma ET-1 concentrations (n=25; median 4.2 pg/ml; range 1.9-14.7) were significantly increased in patients with refractory ascites (n=7; median 7.8, range 3.5 14.7) compared to patients with repetitive bleeding (n=18; median 3.4; range 1.9-7.1) (p=0.003). Furthermore, peripheral ET-1 concentrations correlated with the degree of liver dysfunction according to the Child-Pugh classification (Spearman's r=0.46; p=0.02). Following TIPS, peripheral ET-1 concentrations remained unchanged during a follow-up of 15 months. Before TIPS, a positive gradient of ET 1 concentrations from portalvenous to hepatovenous and peripheral venous levels was found (p<0.03). Immediately after TIPS, arterial ET-1 concentrations reached markedly increased levels in individual patients (88, 92 and 103 pg/ml). Severe systemic reactions to these high levels were not observed. Peripheral venous, hepatovenous and portalvenous ET-1 concentrations did not correlate with portocaval pressure gradients. CONCLUSION: Cirrhotic patients demonstrated unchanged peripheral venous ET-1 concentrations up to 15 months after TIPS. Portal congestion was associated with increased ET-1 levels in the prehepatic splanchnic area. The effect of portal decompression on splanchnic and systemic ET 1 levels deserves further investigation. PMID- 10726963 TI - Kupffer cell depletion by CI2MDP-liposomes alters hepatic cytokine expression and delays liver regeneration after partial hepatectomy. AB - BACKGROUND: Although Kupffer cells (KCs) are capable of producing important growth-stimulating cytokines, their role in liver regeneration following partial hepatectomy (PH) remains poorly understood. METHODS: In the present study liver regeneration was studied after KC-depletion by intravenous administration of liposome-encapsulated dichloromethylene-diphosphonate (C12MDP), a method known to physically eliminate KCs. Furthermore, splenectomy was performed one week prior to PH to exclude the effect of C12MDP-liposomes on macrophage populations in the spleen. RESULTS: KC-depletion was confirmed in cryostat liver sections stained with the monoclonal antibody ED2, a marker for resident tissue macrophages. Forty eight hours after PH, the cumulative hepatocyte DNA synthesis, as determined in liver sections by the hepatocyte bromodeoxyuridine labeling index, was significantly decreased in KC-depleted rats when compared to control-rats. The weight of the remnant liver, expressed as a percentage of the initial liver weight, was significantly less at 96 h after PH in KC-depleted rats. KC-depletion abolished the hepatic interleukin-6 (IL-6) and interleukin-10 (IL-10) mRNA synthesis and decreased hepatic expression of tumor necrosis factor-alpha (TNF alpha), hepatocyte growth factor (HGF) and transforming growth factor-beta1(TGF beta1) mRNA after PH, as was assessed by reverse-transcriptase polymerase chain reaction (RT-PCR). Moreover, at 4 h after PH the systemic release of IL-6 was significantly decreased in KC-depleted rats. CONCLUSION: We conclude that KCs are important for hepatocyte regeneration after PH. Delayed liver regeneration in KC depleted rats can be explained, at least in part, by an imbalanced hepatic cytokine expression, thereby suppressing important growth-stimulating cytokines. PMID- 10726964 TI - Ursodeoxycholic acid reduces expression of heat shock proteins in primary biliary cirrhosis. AB - BACKGROUND: To identify injured cells in the liver of patients with primary biliary cirrhosis (PBC) and to determine the effects of ursodeoxycholic acid (UDCA) on these cells, we examined the cellular expression of heat shock proteins (HSPs) in PBC both before and after treatment with UDCA. METHODS: Expression of HSP70 and ubiquitin in PBC livers (n=34) was evaluated immunohistochemically as well as by immunoblot analysis, and compared with chronic viral hepatitis type C (n= 9), primary sclerosing cholangitis (n=8), and controls (n=7). RESULTS: Immunoblot analysis demonstrated a marked expression of HSP70 and ubiquitin in PBC. Immunohistochemical staining for both HSP70 and ubiquitin was observed to be strong in biliary epithelial cells (BECs) and moderate in both hepatocytes and arteries in PBC. Cellular labelling rates for HSP70 and ubiquitin of bile ducts in PBC were significantly higher (p<0.01) than those in chronic viral hepatitis type C, primary sclerosing cholangitis, or controls. The labelling rates for HSP70 and ubiquitin in bile ducts and in hepatocytes were significantly decreased (p<0.01) after treatment with UDCA in PBC. CONCLUSIONS: The present data suggest that BECs and hepatocytes significantly express HSPs even in the early stages of PBC, and that UDCA treatment significantly improves their condition. The immunohistochemical evaluation of HSPs is a valid and sensitive means to identify injured cells in PBC. PMID- 10726965 TI - Haemodynamic responses to a ring-deleted analogue of atrial natriuretic peptide in rats with cirrhosis. AB - AIMS: In cirrhosis, the effects of selective activation of atrial natriuretic peptide (ANP) R2-receptors on haemodynamics, endogenous ANP (ANP1-28) and sodium excretion are unknown. The aim of the present study was to examine the effects of selective activation of ANP-R2 receptors by ANP4-23 (a ring-deleted analogue of endogenous ANP) on haemodynamics, plasma ANP1-28 concentrations and urinary sodium excretion in conscious cirrhotic and normal rats. METHODS: Haemodynamics and sodium excretion were measured prior to and following administration of ANP4 23. Plasma ANP1-28 concentrations were also measured. RESULTS: In cirrhotic rats, ANP4-23 significantly decreased portal pressure and tributary blood flow by 15% and 25% respectively but significantly increased portal territory vascular resistance by 30%. Systemic and renal haemodynamics were not altered by ANP4-23. In normal rats, ANP4-23 did not significantly change splanchnic, renal and systemic haemodynamics. In both groups of rats, ANP4-23 increased plasma ANP1-28 concentrations but did not change sodium excretion. CONCLUSIONS: ANP4-23 administration induced splanchnic vasoconstriction in cirrhotic but not in normal rats. ANP4-23-elicited vasoconstriction caused a portal hypotensive effect in cirrhotic rats. Finally, in both groups, ANP4-23 caused an increase in plasma ANP1-28 concentrations but did not increase sodium excretion. PMID- 10726966 TI - Subfulminant hepatitis requiring liver transplantation following ibuprofen overdose. PMID- 10726967 TI - The role of cleavage of cell structures during apoptosis. AB - In this work we have studied the behavior of some cell structures, such as actin, tubulin and chromatin during apoptosis induced in F9 cells after retinoic acid treatment. In this experimental model, all defined steps of morphological changes described for apoptosis are observed. The correlation between a partial maintenance of F-actin and microtubular structures and the spatial distribution of F-actin suggests a possible relationship between this molecule and the characteristic shape changes observed in apoptosis. Additionally, the disposition of monomeric G-actin suggests a possible relationship between the fragmentation of this molecule and the cleavage of DNA. The analysis of the U2af1-rs1 specific sequence shows that the internucleosomal fragmentation observed in this gene is randomly produced during apoptosis and is not dependent of demethylation status. The results obtained confirm that specific cleavage of these cell structures is inherent to the development of the apoptotic process and do not exclude the possibility that proteolysis of key actin and/or tubulin molecules or the cleavage of specific chromatin sequences other than the ones analyzed here, could control the different phases of the apoptotic process. PMID- 10726968 TI - Apoptotic behaviour of hepatic and extra-hepatic tumor cell lines differs after Fas stimulation. AB - Fas-induced apoptosis is one form of programmed cell death responsible for hepatocyte demise. However, the role of this cell surface receptor in the death of tumoral hepatic cells is still being debated. It has been shown that some hepatoma cell lines may escape apoptosis because of abnormal Fas localization correlated with non-functionality of the Fas protein or dysfunctionality in the Fas pathway cascade. The aim of this study was to investigate the behaviour of four hepatoma cell lines, HepG2, Hep3B, SKHep1 and Chang-Liver and two extrahepatic cell lines, MCF7, a mammary tumoral cell line and OVCAR-3, an ovarian tumoral cell line, when they were treated with an agonistic anti-Fas antibody alone, with interferon gamma (IFNgamma), an up-regulator of Fas protein expression, alone or with a combination of both agents. We first performed immunofluorescence and flow cytometry to confirm that Fas was present on the cell surface of each cell line in the normal state. Apoptosis was then investigated after induction with the various treatments, by DAPI staining, agarose gel DNA electrophoresis and PARP cleavage. Caspase 8 and 3 expression, as well as two anti-apoptotic proteins Bcl-2 and HSP70, and one proapoptotic protein Bax were also investigated by immunoblot allowing identification of several apoptotic pathways based on the behaviour of the different studied proteins. HepG2 and OVCAR-3 cells were sensitive to the anti-Fas antibody alone. Hep3B was resistant to Fas-induced apoptosis but sensitive to IFNgamma-induced apoptosis. MCF7 was resistant to anti-Fas antibody and IFNgamma Chang-Liver and SKHep1 were sensitive to IFNgamma and anti-Fas antibody but at different degrees. Chang-Liver used the Fas and IFNgamma pathways, while SKHep1 involved mostly the Fas pathway. These results show that each tumor cell line is characterized by different apoptotic behaviour in relation to Fas and/or IFNgamma-induced apoptosis. In addition, despite the high level of Bcl-2 and HSP70 proteins in the tumoral cells investigated here, they were not fully protected against apoptosis, except for MCF7. This emphasizes the necessity to analyse the different proteins responsible for apoptosis to adapt anti-tumoral therapeutics. PMID- 10726969 TI - Tumour necrosis factor alpha (TNFalpha): role in suppression of apoptosis by the peroxisome proliferator nafenopin. AB - The peroxisome proliferator (PPs) class of non-genotoxic rodent hepatocarcinogens induce mouse hepatocyte DNA synthesis and suppress apoptosis. This phenotype can be reproduced in vitro using exogenous tumour necrosis factor alpha (TNFalpha), suggesting a role for TNFalpha in mediating the liver growth response to PPs. In hepatocytes isolated from the peroxisome proliferator activated receptor alpha (PPARalpha) null mouse, PPs are unable to stimulate DNA synthesis or to suppress either spontaneous or TGFbeta1-induced apoptosis. However, the ability of TNFalpha to modulate hepatocyte survival and growth is unaltered, suggesting that TNFalpha acts independently or downstream of PPARalpha to mediate the growth changes associated with PPARalpha activation. Since PPARalpha is a ligand activated transcription factor, we determined if TNFalpha gene expression was altered by PP treatment during an early time window preceding PP-induced growth changes. However there was no induction of TNFalpha expression by nafenopin over the constitutive levels noted in control cultured cells. In summary, TNFalpha acts downstream or independently of PPARalpha to mediate the suppression of apoptosis and induction of DNA synthesis by PPs. In this in vitro model, the PP nafenopin do not appear to mediate de novo TNFalpha gene expression suggesting that the response to nafenopin may be mediated by bioactivation or release of pre existing TNFalpha protein from Kupffer cells. PMID- 10726970 TI - Developmental changes in antioxidant enzymes and oxidative damage in kidneys, liver and brain of bcl-2 knockout mice. AB - While programmed cell death is induced by a variety of internal and external stimuli, including reactive oxygen species, the anti-apoptotic protein Bcl-2 is involved in opposing cell death and affects the antioxidant status of cells. Since the exact mechanism of its action is uncertain, in this study we examined the role of Bcl-2 using a loss of function model, Bcl-2 knockout mice. The consequence of Bcl-2 knockout was assessed in kidneys, liver and brain, using protein carbonyls and cellular levels of antioxidant enzymes as markers of oxidative stress. Kidney extracts from 8 days-old Bcl-2-knockout mice had 59% higher content of protein carbonyls relative to the wild type, but similar levels of oxidized proteins at the age of 30 days. By marked contrast, in liver and brain, levels of protein carbonyls were similar at 8 days but by 30 days the liver of knockout animals (and brains, as we have shown previously) show 36% higher protein carbonyls. Measures of glutathione reductase (GRX), glutathione transferase (GST) and catalase revealed significantly higher levels in kidneys of 8 days old Bcl-2-knockout mice compared to wild type. By 30 days activities of glutathione-related enzymes and catalase increased and abolished the differences between the knockout and wild type. At 8 days, in liver there were no significant differences in activities of all enzymes between the mice, however by 30 days, the specific activity of GRX was significantly higher in Bcl-2-knockout mice, relative to controls. From day 8 to day 30 there was an increase in liver catalase activity that resulted in significantly higher levels in Bcl-2-knockout animals. Catalase activity in brains of Bcl-2-knockout, 8 days old mice was significantly higher compared to the wild type, and significantly lowers at 30 days. Taken together our findings indicate that Bcl-2 knockout results in significant perturbations of oxidative metabolism and antioxidant status of in kidney, liver and brain. Such changes are tissue specific with respect to age, magnitude and type of enzyme affected. PMID- 10726971 TI - Protective effect of a caspase inhibitor in models for cerebral ischemia in vitro and in vivo. AB - In primary neuronal-astrocyte cultures from mouse brain, ischemic conditions were simulated by combined oxygen-glucose deprival (OGD) for 2 hrs. This treatment resulted in near complete neuronal damage 24 hrs. later and was accompanied by DNA degradation and apoptotic nuclear morphology. Since caspases are key enzymes in the propagation and execution of programmed cell death, we evaluated the effect of the caspase inhibitor z-VAD-fmk. Damage following 2 hrs. OGD could be reduced by up to 56% with z-VAD-fmk (p<0.05). DNA-fragmentation and caspase activation has been also reported in an in vivo model of cerebral ischemia imitating human stroke. In this model the middle cerebral artery (MCA) is permanently occluded resulting in focal cerebral ischemia and subsequent infarction. Since z-VAD.fmk does not penetrate the blood-brain barrier it was applied intraventricularly as a bolus injection given 30 min. before MCA occlusion which was followed by 24 hrs. of infusion. Infarct volume was determined 48 hrs. after MCA occlusion by means of in vivo magnetic resonance imaging. Z-VAD.fmk dose dependently reduced infarct volume reaching a significant decrease of the cortical infarct by 45% when given as a 120 ng bolus followed by 40 ng/hr. infusion (p<0.05). In summary, our study supports the concept that caspase inhibitors are beneficial in brain ischemia. PMID- 10726972 TI - DNA laddering and caspase 3-like activity in the spinal cord of a mouse model of familial amyotrophic lateral sclerosis. AB - Transgenic mice with several copies of a mutated human superoxide dismutase 1 (Gly93-Ala substitution) gene, i.e. a mutation responsible for the development of familial amyotrophic lateral sclerosis (ALS), integrated into the mouse genome, develop a slowly progressive paralysis of the hind-limbs accompanied by a corresponding degeneration of spinal cord neuronal tissue. We have used two different lines of these transgenic mice [a low (approximately 12 copies) or a high (approximately 24) copy number of the mutated human superoxide dismutase 1 gene] to find evidence of programmed cell death in affected spinal cord tissue at distinct age groups. Hallmarks of programmed cell death, i.e. DNA laddering and an increase in caspase 3-like activity, were found in the spinal cord of both lines of mice. Behavioural evaluation of the mice indicated that the hallmarks of programmed cell death were mainly, but not exclusively found in symptomatic animals just before or at end-stage. These data suggest that programmed cell death may play a role in the disease process of familial ALS particularly in its terminal phase. PMID- 10726973 TI - Insulin-like growth factor-I inhibits the progression of human U-2 OS osteosarcoma cells towards programmed cell death through interaction with the IGF I receptor. AB - Insulin-like growth factor-I exerts potent mitogenic effects through the type I IGF receptor, a member of the insulin receptor family, and exhibits at the same time some insulin-like metabolic activities. We have questioned whether IGF-I presents moreover a modulatory effect upon programmed cell death (PCD)(apoptosis) in serum-deprived human osteosarcoma U-2 OS cells, a cell line synthesizing IGF II and exhibiting an increased DNA synthesis following treatment with IGF-I. U-2 OS cells were cultured in a medium containing 0.8% FCS and growth arrest was induced by transfer to serum-free growth conditions. PCD was measured using a commercially available DNA degradation ELISA while viable cell numbers were counted microscopically after trypan exclusion to estimate net proliferative activity. Following serum withdrawal for 24 hrs., the level of PCD in U-2 OS cells was increased six-fold while cell number was reduced by approximately 35% compared to cells grown in the presence of 15% serum. Incubation with recombinant human IGF-I for 24 hrs. caused a dose-dependent inhibition of the level of programmed cell death. Co-incubation with an IGF-I receptor monoclonal antibody (alphaIR3) dose-dependently blocked the effects of 10 ng/ml IGF-I on PCD, with an ED50 of 1-10 ng/ml of alphaIR3 immunoglobulin. Conversely IGF-1 provoked a significant cell number increase, an effect blocked by addition of alphaIR3. The addition of an inhibitor of caspase 1 (ICE) had little effect on PCD but resulted in a net increase in the number of viable cells. In summary, IGF-I treatment of U 2 OS cells at the same time inhibits the induced programmed cell death and increases the cell number, effects which are blocked by addition of IGF-I receptor antibodies. These data support the hypothesis that IGF-I affects cells in a dual way, both by enhancing proliferative responses and by suppressing programmed cell death. The differential response between PCD and cell number to ICE inhibitors suggests the existence of independent control systems for these processes although the role of IGF-I in this study has yet to be determined. PMID- 10726974 TI - Expression of apoptosis and cell cycle related genes in proliferating and colcemid arrested cells of divergent lineage. AB - Progression through the cell cycle and redirection of cells towards programmed cell death (apoptosis) are tightly inter-related processes. However the requirement for tissue and cell type specificity suggests that a wide variety of mechanisms are used to achieve the same purpose. To examine this issue, we investigated cell cycle (c-myc, p53, p21/WAF) and apoptosis related (bcl-2, bcl X(L), bax-alpha) gene expression in two cell lines of very different origin under proliferating and apoptosis-inducing conditions. Transformed human osteosarcoma cells (MG63) and non-transformed human kidney embryonal fibroblasts (293-0) were kept in culture in medium containing 10% FCS and growth arrest was induced by the addition of 50 ng/ml colcemid. Colcemid treatment caused growth arrest and elevated expression of cyclin B1 protein in both cell lines. Apoptosis was significantly elevated in both cell lines after colcemid exposure for at least one cell cycle. However the pattern of expression of cell cycle and apoptosis related genes, determined by RT-PCR, was quite different between the two cell lines during exponential growth and cell cycle arrest. Colcemid treatment did not markedly influence c-myc, p53 and p21/WAF expression in MG63 cells but did suppress c-myc and increase p21/WAF in 293-0 cells. Furthermore colcemid treated MG63 cells exhibited elevated bcl-2 and bax-alpha expression while similar treatment of 293-0 cells resulted in decreased bcl-X(L) and slightly increased bax-alpha expression. While growth arrest and apoptosis were induced in both MG63 and 293 cells following colcemid treatment, the differences in gene expression suggest that the mechanism by which these cells determine cell fate is quite different and may determine the sensitivity of different cell populations to anti neoplastic drug therapy. The distinct patterns of gene expression should be carefully defined before mechanisms of apoptotic cell death are studied. PMID- 10726975 TI - Programmed cell death in post-traumatic bone callus. AB - Some osteoblasts in the expanded population of periosteal cells that occurs following bone injury are removed from the callus by apoptosis. Our objective was to study whether the consequences of activation of the death program could include feedback control of the healing response. Transforming growth factor beta and interleukin-1beta were delivered together continuously to a standardized tibial defect in rats for 3 days using implanted micro-osmotic pumps. The bones were recovered at 1, 2, 3, 5, 7, 10 and 14 days after injury (n = 6 in each treated and control group) and concentrations of proliferating cells, osteoblasts and apoptotic bodies were determined. The injury-induced apoptotic component of the healing response was shifted in time due to the combined cytokines, compared with vehicle only, with the result that the peak in the concentration of apoptotic bodies occurred 2-3 days earlier in the treated animals. Neither osteoprogenitor proliferation nor osteoblast concentration was affected by addition of the cytokines. The results suggested that activation of apoptosis during injury repair was not necessarily a passive consequence of the cellular response to injury. Programmed cell death could therefore have an active role in modulating bone repair. PMID- 10726976 TI - The significance of programmed cell death or apoptosis and matrix vesicles in atherogenesis. AB - Programmed cell death or its current synonym, apoptosis, is considered a genetically controlled biological process of cell deletion complementary to cell replication. Apoptosis is most likely a self-regulatory mechanism whereby a genetically determined biochemical pathway to death is initiated in cells sustaining irreparable damage particularly of DNA. Initiating factors in each instance need to be established. Identified in arteries, apoptosis correlates with the localization and severity of atherosclerosis. Granulovesicular disintegration of vascular smooth muscle cells leads to abundant vesicular debris mostly in the intima and increasing with age and hypertension and such matrix vesicle production is a major pathogenetic feature of atherosclerosis. The abundant debris produced accumulates lipid and minerals as usually occurs in non phagocytosed cell debris. Similar vesiculation in erythrocytes under haemodynamic stress supports the contention that vascular cells under haemodynamic biomechanical stresses in spontaneous and experimental atherosclerosis degenerate due to depletion of cytoplasm, DNA fragmentation and oxidative damage with some cells inevitably undergoing terminal apoptosis. Evaluation of apoptosis must take into account the concomitant changes in the whole vessel wall and its matrix. Currently generalizations about therapeutic or pathogenetic roles for apoptosis in any disease are speculative and unwarranted. PMID- 10726977 TI - Different vitamin D analogues induce sphingomyelin hydrolysis and apoptosis in the human keratinocyte cell line HaCaT. AB - Sphingomyelin hydrolysis seems to be a ubiquitous pathway generating ceramide, an important cell response modifier. Upon agonist-stimulation this pathway is linked to biological responses as inhibition of proliferation, promotion of differentiation and induction of apoptosis. One of the agonists described is 1alpha,25-dihydroxyvitamin D3. Recently, we could demonstrate the existence of sphingomyelin hydrolysis in human primary keratinocytes as well as in the human keratinocyte cell line HaCaT after treatment with 1alpha,25-dihydroxyvitamin D3. In the present study we tested four vitamin D analogues on HaCaT keratinocytes for their ability to inhibit cell proliferation and to induce sphingomyelin hydrolysis. These analogues, calcipotriol, EB 1213, GS 1500 and tacalcitol inhibit cell growth after 48 hrs. of incubation and trigger the hydrolysis of sphingomyelin. Moreover, all analogues tested induce apoptotic cell death in HaCaT keratinocytes after 24 hrs. of incubation. This study indicates that sphingomyelin hydrolysis, subsequently leading to the elevation of cellular ceramide levels, may represent an important signal transduction pathway for 1alpha,25-dihydroxyvitamin D3 and its analogues in human keratinocytes. Possible differences of the mechanism underlying vitamin D-induced sphingomyelin hydrolysis has to be studied in more detail and may contribute to the antipsoriatic action of these analogues. PMID- 10726978 TI - Resistance of senescent keratinocytes to UV-induced apoptosis. AB - Irradiation with ultraviolet (UV) triggers programmed cell death (apoptosis) in keratinocytes. This process is believed to protect against skin carcinogenesis since the cells with damaged DNA are selectively removed, limiting the likelihood of the development of a malignant keratinocyte clone. The p53 protein is able to detect mutation-bearing DNA fragments and is thus indispensable for the UV induced apoptosis in the epidermis. Since age is a risk factor for the development of skin tumors we investigated whether ultraviolet induces apoptosis and p53 activation in senescent keratinocytes. Cultured senescent keratinocytes were irradiated with broad-band ultraviolet, apoptosis was assessed using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) technique and the p53 activation pattern was determined with Western blotting and immunofluorescent staining with a panel of anti-p53 antibodies recognising different conformational forms of the protein (PAb 122, PAb 240, DO-7). In senescent keratinocytes arrested in the G1 phase of cell cycle, ultraviolet irradiation (100-2000 J/m2) caused accumulation and nuclear translocation of p53. However, in contrast to young cells where UV induces apoptotic cell death in G1, apoptosis was not detected in senescent cells. There were subtle differences in the p53 activation pattern between senescent keratinocytes and known patterns in young keratinocytes and other cell types. In senescent keratinocytes a constitutional nuclear expression of p53 (conformational form recognized by PAb 240) was present and the p53 induction in response to ultraviolet radiation was rapid. Suppression of apoptosis in senescent keratinocytes may be an important mechanism responsible for enhanced skin carcinogenesis in old age. PMID- 10726979 TI - Apoptosis-inducing activity of vitamin C and vitamin K. AB - Apoptosis-inducing activity of vitamins C and K and of their analogs are reviewed. Vitamin C shows both reducing and oxidizing activities, depending on the environment in which this vitamin is present. Higher concentrations of vitamin C induce apoptotic cell death in various tumor cell lines including oral squamous cell carcinoma and salivary gland tumor cell lines, possibly via its prooxidant action. The apoptosis-inducing activity of ascorbate is stimulated by Cu2+, lignin and ion chelator, and inhibited by catalase, Fe3+, Co2+ and saliva. On the other hand, at lower concentrations, ascorbic acid displays an antioxidant property, preventing the spontaneous and stress or antitumor agent-induced apoptosis. Sodium 5,6-benzylidene-L-ascorbate, intravenous administration of which induces degeneration of human inoperable tumors and rat hepatocellular carcinoma in vivo, induces apoptotic or non-apoptotic cell death, depending on the types of target cells. On the other hand, elevation of intracellular concentration of ascorbic acid by treatment with ascorbate 2-phosphate or dehydroascorbic acid makes the cells resistant to the oxidative stress-induced apoptosis. Vitamin K2, which has a geranylgeranyl group as a side chain,and vitamin K3 induces apoptosis of various cultured cells including osteoclasts and osteoblasts, by elevating peroxide and superoxide radicals. Synergistic apoptosis inducing actions have been found between vitamins C and K, and between these vitamins and antiproliferative agents. The possible therapeutic application of these vitamins is discussed. PMID- 10726980 TI - The fibronectin-derived anti-adhesive peptide III14-2 suppresses adhesion and apoptosis of leukemic cell lines through down-regulation of protein-tyrosine phosphorylation. AB - We previously found that fibronectin (FN) has a cryptic functional site (YTIYVIAL, #1848-1855) opposing to cell adhesion to extracellular matrix (ECM). The present study demonstrates that the FN peptide containing this anti-adhesive site, termed peptide III14-2, affects programmed cell death (PCD) (apoptosis) as well as cell adhesion by down-regulating protein-tyrosine phosphorylation. Peptide III14-2 suppressed the integrin alpha5beta1-mediated adhesion of leukemic cell lines (K562 and HL60), and protein tyrosine phosphatase inhibitor, 1 microM phenylarsine oxide (PAO) blocked the anti-adhesive effect of peptide III14-2. These leukemic cells underwent PCD when exposed to PAO at the higher concentration (5 microM), as judged by nuclear and DNA fragmentations, and which was reversed by tyrosine kinase inhibitor, genistein. Peptide III14-2 suppressed the PAO-induced PCD, whereas a control peptide in which the anti-adhesive sequence YTIYVIAL is scrambled, was inactive. Western blotting showed that PAO stimulated the tyrosine phosphorylation of cellular proteins including focal adhesion kinase and that peptide III14-2 inhibited them, suggesting that protein tyrosine phosphorylation represents a common early signal for the adhesion and PCD. The anti-adhesive site of FN molecule may play a crucial role also in a variety of cellular processes other than adhesion and PCD by down-regulating protein-tyrosine phosphorylation. PMID- 10726981 TI - Morphological features of apoptosis in hematopoietic cells belonging to the T lymphoid and myeloid lineages. AB - Taking into account that apoptosis plays a pivotal role in shaping normal hematopoiesis, morphological features of apoptosis were investigated in both primary cells and continuous cell lines committed towards the T-lymphoid and the myeloid lineages. Apoptosis was induced using: dexamethasone (10(-7) M) for primary rat thymocytes; infection with the T-lymphotropic human herpesvirus 7 (HHV-7) for peripheral blood CD4+ T-cells; staurosporine (1 microM) for MOLT4 CD4+ lymphoblastoid T-cells, HL60 human promyelocytic and U937 human monoblastoid cells; and using senescence of the culture for primary human megakaryocytes. Cell morphology was examined by both transmission electron microscopy and in situ nick translation (NT) revealed by laser scanning confocal microscopy. In spite of the use of different apoptotic agonists, the morphological aspects of apoptosis were similar within the T-lymphoid and the myeloid lineage. While chromatin condensation characterized the early apoptotic events in both lineages, late apoptoses were mainly characterized by further nuclear condensation in lymphoid cells and by production of micronuclei in myeloid cells. Moreover, NT analysis clearly showed that the micronuclei derived from HL60 undergoing apoptosis were composed of both degraded and intact DNA. Thus, T-lymphocytes and myeloid cells showed a lineage-related behavior characterizing the late morphological aspects of apoptosis. PMID- 10726982 TI - High cell density provides potent survival signals for resting T-cells. AB - Activated T-cells are susceptible to apoptosis through two particularly important pathways: ligation of CD95 (Fas/Apo-1) or cytokine deprivation. Resting T-cells have until recently been considered to be relatively resistant to apoptosis. In this report we show that resting T-cells die rapidly by apoptosis when deprived of serum or cell contact. Primed CD45RO+ cells were more susceptible than naive CD45RA+ cells, consistent with their relative expression of Bcl-2. CD4+, CD8+ and gammadelta T-cells were equally prone to apoptosis under all studied conditions. A linear relationship between cell survival and serum concentration was observed for cells cultured between 0.5-2x10(6)/ml. T-cells cultured at low density died even in high concentrations of serum. However, resting T-cells cultured at high cell density (4x10(6)/ml) survived for extended periods in the absence of serum or other survival factors. This effect was mediated by the production of soluble factors and independent of integrin mediated signals. These results suggest that T-cells at sites of high density such as the lymph node paracortex are independent of external survival factors, while those trafficking through the peripheral circulation are highly dependent on serum derived factors for survival. PMID- 10726983 TI - Expression and subcellular redistribution of Bax during TGF-beta1-induced programmed cell death of HC11 mouse mammary epithelial cells. AB - The effect of TGF-beta1, an auto/paracrine antiproliferative and apoptogenic factor on Bax transcript level (RT-PCR), subcellular distribution of Bax protein (immunoelectron microscopy), Bcl-2 protein level and apoptotic cell number (flow cytometry with FITC-conjugated monoclonal anti-Bcl-2 antibody and DNA stained with DAPI) in HC11 mouse mammary epithelial cells was examined. TGF-beta1 increased Bax transcript level (evaluated by Bax mRNA/GAPDH mRNA ratio) and stimulated Bax protein movement from cytosol to organellar membranes, mainly mitochondrial, during 60 min. The new observation is the presence of Bax on channel membranes of Golgi apparatus and translocation of Bax from cytosol to the fibrous nucleoplasm via nuclear envelope pores (especially after 120 min. of cell exposure to TGF-beta1). Prolactin protected HC11 cells against TGF-beta1-induced PCD, which could occur at two levels: 1) TGF-beta1 expression, through the decrease of TGF-beta1 transcript content, and 2) Bax/Bcl-2 checkpoint, through down-regulation of Bax and up-regulation of Bcl-2. In conclusion, Bax and Bcl-2 proteins are implicated in the mechanism of TGF-beta1-induced PCD and antiapoptotic action of prolactin in HC11 mouse mammary epithelial cells. The activation of transcription and redistribution of Bax from cytosol to organellar membranes and nucleus constitute the early events in the cellular response to TGF beta1. PMID- 10726984 TI - Morphological and biochemical analysis of cell death in human ejaculated spermatozoa. AB - We have analyzed the type of cell death occurring in human normal ejaculated spermatozoa. Sperm cells were prepared either by centrifugation alone (group 1) or by density gradient centrifugation (group 2) and were cultured for 24 hrs. Cells were examined after 4 and 24 hrs. By comparison unprepared spermatozoa were used as a control group. Necrosis was investigated by intra-cellular vital stain penetration and electron microscopy. Apoptosis was researched by DAPI staining, annexin V-binding, electron microscopy, DNA fragmentation and PARP cleavage. In group 1, after 4 hrs., there was a mixture of spermatozoa dead either by necrosis or apoptosis while after 24 hrs., necrosis was prominent. Similar findings were observed in the control group. In contrast, in group 2 apoptosis was the major form of cell death of spermatozoa after 24 hrs. of culture. These findings suggest that apoptosis can be an important factor when spermatozoa are used for assisted reproductive technologies. PMID- 10726985 TI - Modulation of programmed cell death by medicinal plants. AB - Programmed cell death (apoptosis), a form of cell death, described by Kerr and Wyllie some 20 years ago, has generated considerable interest in recent years. The mechanisms by which this mode of cell death (seen both in animal and plant cells), takes place have been examined in detail. Extracellular signals and intracellular events have been elaborated. Of interest to the clinician, is the concentrated effort to study pharmacological modulation of programmed cell death. The attempt to influence the natural phenomenon of programmed cell death stems from the fact that it is reduced (like in cancer) or increased (like in neurodegenerative diseases) in several clinical situations. Thus, chemicals that can modify programmed cell death are likely to be potentially useful drugs. From foxglove, which gave digitalis to the Pacific Yew from which came taxol, plants have been a source of research material for useful drugs. Recently, a variety of plant extracts have been investigated for their ability to influence the apoptotic process. This article discusses some of the interesting data. The ability of plants to influence programmed cell death in cancerous cells in an attempt to arrest their proliferation has been the topic of much research. Various cell-lines like HL60, human hepatocellular carcinoma cell line (KIM-1), a cholangiocarcinoma cell-line (KMC-1), B-cell hybridomas, U937 a monocytic cell line, HeLa cells, human lymphoid leukemia (MOLT-4B) cells and K562 cells have been studied. The agents found to induce programmed cell death (measured either morphologically or flow cytometrically) included extracts of plants like mistletoe and Semicarpus anacardium. Isolated compounds like bryonolic acid (from Trichosanthes kirilowii var. Japonica, crocin (from saffron) and allicin (from Allium sativum) have also been found to induce programmed cell death and therefore arrest proliferation. Even Chinese herbal medicine "Sho-saiko-to" induces programmed cell death in selected cancerous cell lines. Of considerable interest is the finding that Panax ginseng prevents irradiation-induced programmed cell death in hair follicles, suggesting important therapeutic implications. Nutraceuticals (dietary plants) like soya bean, garlic, ginger, green tea, etc. which have been suggested, in epidemiological studies, to reduce the incidence of cancer may do so by inducing programmed cell death. Soy bean extracts have been shown to prevent development of diseases like polycystic kidneys, while Artemisia asiatica attenuates cerulein-induced pancreatitis in rats. Interestingly enough, a number of food items as well as herbal medicines have been reported to produce toxic effects by inducing programmed cell death. For example, programmed cell death in isolated rat hepatocytes has been implicated in the hepatitis induced by a herbal medicine containing diterpinoids from germander. Other studies suggest that rapid progression of the betel- and tobacco-related oral squamous cell carcinomas may be associated with a simultaneous involvement of p53 and c-myc leading to inhibition of programmed cell death. Several mechanisms have been identified to underlie the modulation of programmed cell death by plants including endonuclease activation, induction of p53, activation of caspase 3 protease via a Bcl-2-insensitive pathway, potentiate free-radical formation and accumulation of sphinganine. Programmed cell death is a highly conserved mechanism of self-defense, also found to occur in plants. Hence, it is natural to assume that chemicals must exist in them to regulate programmed cell death in them. Thus, plants are likely to prove to be important sources of agents that will modulate programmed cell death. PMID- 10726986 TI - About true science and false words: "oxidase", "cryostat" and "apoptosis". AB - The author examines the concept's deviations due to some erroneous constructions of words, by considering the words oxidase, cryostat and apoptosis. But he insists much more on the bandy-legged structures linked to these words which deviate totally from any significance attached "officially" to them and leading to false deductions. The author conveys all scientists to use: oxydase, cryotome and Programmed Cell Death, or PCD, forgetting all other queer non-sense words. He suggests to use the word "cytoptosis". PMID- 10726987 TI - Rapid detection and quantification of Cryptosporidium baileyi oocysts in feces and organs of chickens using a microscopic slide flotation method. AB - A simple semiquantitative microscopic slide flotation (MSF) method using modified Sheather's sugar solution (MSSS) is presented for the rapid detection and quantification of Cryptosporidium baileyi oocysts in the feces and mucosal and/or organ scrapings of chickens. Oocyst shedding was evaluated by examination of the surface of coverslips, and the average quantitative score (0-5) recorded for 10 microscopic fields (magnification x250) is reported. The equivalence between these scores and the actual number of oocysts counted per gram of feces was assessed (rs = 0.89; P < 0.001). The applicability of this method was tested by comparison of the kinetics of oocyst shedding in feces of inoculated chickens with those reported by other authors working under similar conditions. In organs the MSF method was compared to histology. Fewer false-negative results were obtained using MSF versus the histology method. The MSF method was particularly more efficient in tracheae with low levels of infection and in the lungs, regardless of the level of infection. The MSF method was also very efficient in detecting oocysts in air sacs from chickens with aerosacculitis. It provides a specific and sufficiently sensitive, simple, rapid, reliable, and low-cost means of diagnosing C. baileyi in the feces and organs of chickens. This method can be used in the routine diagnosis of cryptosporidia in chickens, and it could be extended to other avian species and used in epidemiology studies to evaluate the prevalence of cryptosporidiosis in fowl. PMID- 10726988 TI - The crucial role of granulocytes in the early host defense against Strongyloides ratti infection in mice. AB - The involvement of granulocytes in the host early defense against the nematode, Strongyloides ratti, was studied. It was confirmed that granulocytes were effectively depleted for 4 days by anti-granulocyte monoclonal antibody (anti-Gr 1). To examine the involvement of granulocytes in the host defense against migrating larvae, 2,000 S. ratti infective larvae (L3) were inoculated subcutaneously 1 day after antibody treatment. The number of S. ratti eggs secreted in feces (EPG) was higher in the granulocyte-depleted group than in the control group. The number of migrating larvae also increased in the granulocyte depleted group in accordance with the increase in EPG. Therefore granulocytes are crucial for the host early defense against migrating larvae of S. ratti. Next, the involvement of granulocytes in the intestinal early defense was examined. Mice were treated with the antibody on day 3 post-infection. On that day, almost all inoculated larvae reached the intestine and molted to become adults. EPG on day 5 post-infection was increased by the antibody treatment, but no effect was observed on intestinal worm numbers. The fecundity (EPG/worm number) of S. ratti adult worms in the granulocyte-depleted group was higher than that in the control group. Thus granulocytes are also involved in the intestinal early defense through suppressing fecundity of the adult worms. On the other hand, the depletion of granulocytes had no effect on the late adaptive response against S. ratti adult worms (e.g. number of intestinal mucosal mast cells, time of worm expulsion). These results suggest that granulocytes are mainly involved in the host early defense against parasites. PMID- 10726989 TI - In vivo activities of the new anthelmintic depsipeptide PF 1022A. AB - PF 1022A is a member of a new class of cyclic depsipeptides with antiparasitic activity. Following in vitro and laboratory animal studies it was tested for its anthelmintic efficacy in companion and livestock animals against a wide spectrum of intestinal nematodes and lungworms. Studies were carried out in rats, dogs, horses, sheep, and cattle. Animals were either naturally or experimentally infested. The efficacy of PF 1022A was investigated against the following parasite species: Strongyloides ratti and Nippostrongylus brasiliensis in rats, Ancylostoma caninum in dogs, small strongyles (cyathostomes) in horses, Trichostrongylus colubriformis and Haemonchus contortus in sheep, and Dictyocaulus viviparus in cattle. Doses varied from 1 to 10 mg/kg body weight for oral, subcutaneous or intravenous application in companion and livestock animals. High degrees of efficacy were found in all the above-cited examinations, and no clinical signs of intolerability were observed. PMID- 10726991 TI - Pathological effects and reduced survival in Rhipicephalus appendiculatus ticks infected with Theileria parva protozoa. AB - Pathological effects have been demonstrated in a number of arthropod species as a consequence of parasitic infection. This is usually manifest as reduced arthropod survival and/or fecundity. This paper describes the detrimental effects the protozoan parasite, Theileria parva has on Rhipicephalus appendiculatus ticks. R. appendiculatus ticks were dissected and sectioned at regular intervals during their nymph to adult moult after detaching from a T. parva infected calf, and assessed by light and electron microscopy. The reproductive capacity of the T. parva infected ticks was also compared with that of control, uninfected ticks. The number of T. parva forms seen during the ticks' moult were recorded and showed a substantial reduction as the moult progressed. A non-linear relationship between piroplasm ingestion by the engorged nymph and eventual adult salivary gland infection levels was shown. Tick gut and salivary gland pathology was noted at various stages throughout the moult and correlated with the parasite burdens in the affected organs at those timepoints. The reproductive performance of infected ticks was greatly impaired compared to controls. Infected female ticks had longer bloodmeal engorgement times, reduced bloodmeal volumes, smaller egg batch weights and greatly decreased egg hatching success. The pathological effects are discussed in relation to parasite population dynamics within the ticks and compared with similar examples of pathology evidenced with other parasite infected arthropod species. PMID- 10726990 TI - Removal of hydrogen peroxide by a 1-cysteine peroxiredoxin enzyme of the filarial parasite Dirofilaria immitis. AB - Prior studies have shown that filarial nematodes can effectively metabolize hydrogen peroxide in excess of that generated by activated host cells. However, the mechanisms of H2O2 removal by the filarial parasites are unclear. Herein we report the results of studies carried out on the biochemical activity and on immunolocalization of a recombinant peroxiredoxin (Prx) enzyme from the dog filarial parasite Dirofilaria immitis. A full-length cDNA encoding a 1-Cys Prx enzyme from the dog heartworm D. immitis was expressed in Escherichia coli as a recombinant polyhistidine fusion protein (rDiPrx-1). rDiPrx-1 was capable of reducing H2O2 in the presence of dithiothreitol. The apparent kinetic constants determined for DiPrx-1 using H2O2 as a substrate were a Michaelis constant (Km) of 16.28 mM and a maximal velocity (Vmax) of 16 micromol/min(-1). Consistent with the enzyme activity, D. immitis adult worms could detoxify exogenously added H2O2 in vitro. Antibodies to rDiPrx-1 identified a 27-kDa native antigen in parasite extracts and larval and adult excretory-secretory products. The antibodies were used to localize the native antigen to the lateral hypodermal chords of both male and female worms by immunohistochemistry. In addition, labeling was seen in the afibrillar muscle cells in male worms and in some areas of the uterine wall in female worms. Thus, DiPrx-1 is the first parasite Prx to be shown to detoxify exogenously added H2O2 in an in vitro system. PMID- 10726992 TI - Redistribution of plasma-membrane surface molecules during formation of the Leishmania amazonensis-containing parasitophorous vacuole. AB - Leishmania amazonensis presents two developmental stages that gain access to the host macrophage through phagocytosis. The protozoan resides in a membrane-bound compartment, the parasitophorous vacuole (PV), which can fuse with the endocytic system. For evaluation of the parasite/host-cell interaction process and of PV biogenesis, the two parasite forms or host-cell membrane whose surface had previously been labeled with specific probes for lipids, proteins, and sialoglycoconjugates were allowed to interact for periods varying from 5 to 15 min for adhesion and from 30 to 60 min for PV formation. The fate of fluorescent probes was followed by confocal laser scanning microscopy. In host cells previously labeled with PKH26, DTAF and FITC-thiosemicarbazide, which label membrane lipids, proteins, and sialoglycoconjugates, respectively, interaction with both protozoan forms revealed that adhesion to the macrophage was sufficient for labeling of the parasite surface. In addition, recently formed PVs displayed strongly labeled intravacuolar parasites, except for amastigote-macrophage interaction in a DTAF-labeled macrophage that displayed slight labeling of intravacuolar parasites, with the membrane lining the PV evidently being stained. Therefore, the vacuole modulation presents some particularities such that different host-cell membrane components may be selected, depending on the protozoan form involved. Thereafter, amastigotes labeled with the probes mentioned above displayed a diffuse labeling pattern after interaction with unlabeled macrophages, suggesting the spreading of Leishmania surface molecules during the initial parasite-invasion stages. In particular, intravacuolar DTAF labeled amastigotes showed a delineating halo around the PV, with the intravacuolar parasite being partially labeled. Promastigotes could not be labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) or with fluorescein 5-thiosemicarbazide, but promastigotes labeled with PKH26 lost the fluorescent probe during the invasion process such that slightly labeled promastigotes were seen inside the PV. These observations indicate the existence of a dynamic process of exchange of membrane-associated glycoproteins and lipids between the parasite and the host cell. PMID- 10726993 TI - Galactose-specific adhesin and cytotoxicity of Entamoeba histolytica. AB - We examined the molecular mechanisms of the cytotoxicity of Entamoeba histolytica, using the loss of transepithelial electrical resistance (TER) of monolayers of Madin-Darby canine-kidney (MDCK) cells on their incubation with axenic trophozoites of the HM1-IMSS strain. Such loss of TER occurs very early (in 2-5 min) and is caused by the opening of tight junctions and the detachment of cells. We used specific inhibitors for three of the four molecules currently accepted as being responsible for cytotoxicity: galactose-specific adhesin(s), phospholipase A, and cysteine proteinases. We also used inhibitors of calcium channels. Axenic trophozoites of E. histolytica strain HM1-IMSS were preincubated with the different inhibitors for 1 h prior to their coincubation with MDCK-cell monolayers. The only inhibitor that effectively blocked the loss of TER caused by the parasite was galactose. We suggest that in this experimental model, galactose specific adhesin(s) are essential for amebic cytotoxicity. PMID- 10726994 TI - Immune response to Trichinella spiralis larvae after treatment with the anti allergic compound ketotifen. AB - Ketotifen was used as an anti-allergic agent to study the relationship between eosinophil-related responses and IgG1 and IgG2a antibody responses in BALB/c mice infected with Trichinella spiralis. The results showed that leukocyte and eosinophil numbers and interleukin-5 (IL-5) concentrations in the peritoneal fluid increased after exposure to nematodes and the increases were slightly greater in animals treated with ketotifen. A decreased concentration of eosinophil peroxidase and an elevation in IgG1 accompanied the muscle phase of infection. In mice treated with ketotifen, antibody-mediated recognition of muscle larvae was delayed. The retardation of IgG1 and IgG2a responses may have been responsible for the ineffective immune response against larvae migrating into the muscle. The activation of eosinophils was accompanied by changes in IL-5 concentration, but these changes were not associated with differences in protection against infection. PMID- 10726995 TI - Nitric oxide is not involved in the killing of Trypanosoma cruzi by chicken macrophages. AB - It is known that chicken macrophages derived in vitro from blood monocytes have the capacity to destroy Trypanosoma cruzi, but Toxoplasma gondii can survive within these cells. This study was performed to determine the involvement of nitric oxide (NO) in the killing of T. cruzi by chicken macrophages. Activated (by interferon-gamma and lipopolysaccharide) mouse peritoneal macrophages were used as controls. Macrophages were infected with T. cruzi and T. gondii; after 2, 24, and 48 h, NO was assayed using the Griess reagent. Respiratory-burst involvement, revealed by the reduction of nitroblue tetrazolium (NBT), was determined in chicken macrophages. Chicken macrophages did not produce NO; mouse macrophages were capable of producing NO with no multiplication of parasites. Reduction of NBT could be detected in chicken macrophages that interacted with T. cruzi but was absent in those that interacted with T. gondii. These results demonstrate that chicken macrophages do not use NO as a microbicidal agent when infected with T. cruzi or T. gondii. PMID- 10726996 TI - Creatine kinase and lactate dehydrogenase levels as potential indicators of Trypanosoma cruzi infectivity and histotropism in experimental Chagas' disease. AB - The results of the present study reveal an early increase in activity levels of creatine kinase and lactate dehydrogenase in the plasma of mice infected with Trypanosoma cruzi strains K-1, X-1, and Tulahuen as compared with uninfected control mice. An increase in creatine kinase activity was detected earlier in K-1 and X-1-infected mice than in Tulahuen-infected mice. Moreover, an increase in lactate dehydrogenase activity occurred at 1.5 days after infection with the X-1 and Tulahuen strains and at 3.5 days after infection with the K-1 strain. Generally, the highest activity levels were found in the plasma of mice infected with the most virulent and lethal Tulahuen strain as compared with the less virulent and nonlethal K-1 and X-1 strains. A significant decrease in creatine kinase levels occurred later in the tissues than in the plasma of K-1- and X-1 infected mice but did not vary significantly in any of the tissues from Tulahuen infected mice. Similarly, the specific activity of lactate dehydrogenase in tissues from K-1- and X-1-infected mice dropped at a later stage than did the activity in plasma, but infection with the Tulahuen strain caused an earlier reduction in the activity of lactate dehydrogenase in the heart and skeletal muscle. The activity levels of both enzymes in plasma and tissues showed a linearly negative and statistically significant correlation. The present study reveals that levels of creatine kinase and lactate dehydrogenase activity in plasma could be early indicators of and suitable tools for monitoring of the infectivity of these strains of T. cruzi and might reflect their inherent histotropism during experimentally acute Chagas' disease. PMID- 10726997 TI - Cloning of the chaperonin t-complex polypeptide 1 gene from Schistosoma mansoni and studies of its expression levels under heat shock and oxidative stress. AB - The protein TCP-1 (t-complex polypeptide 1) is a subunit of the hetero-oligomeric complex CCT (chaperonin containing TCP- 1) present in the eukaryotic cytosol. Chaperone function may be critical for the development and survival of the different life stages of Schistosoma mansoni, a parasite that is exposed to drastic environmental changes during its development. We isolated a full-length S. mansoni TCP-1 cDNA (SmTCP-1A) encoding a protein highly homologous with TCP-1. The deduced SmTCP-1A amino-acid sequence shows up to 65% identity with other eukaryotic CCT family members. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the mRNA expression levels of SmTCP-1A in adult S. mansoni were down-regulated in worms subjected to heat shock and oxidative stress conditions. This down-regulation of SmTCP-1A mRNA may reflect a switch in CCT subunits as an adaptive response to heat shock and oxidative stress conditions. PMID- 10726998 TI - Unexpectedly high variability of the histone H4 gene in Leishmania. AB - The sequence of the cDNA of the histone H4 gene of Leishmania tarentolae is reported herein. The predicted 100-amino-acid-long protein has the highest degree of identity with the histone H4 gene of L. infantum and shares with it a 5' region that shows a very low degree of identity with the corresponding region of histone H4 genes from other organisms. However, between these two genes is a 7.7% nucleotide difference that results in seven different amino acids, located in the 5', central, and 3' regions of the coding sequence. Such a divergence in the H4 gene, which is considered to be one of the most highly conserved genes, between closely related members of the genus Leishmania is unexpected and may reflect some unusual features of these important proteins in kinetoplastid flagellates. PMID- 10726999 TI - Diagnostic follow-up of well-differentiated thyroid carcinoma: historical perspective and current status. AB - Well-differentiated (follicular and papillary) thyroid carcinoma accounts for 80% to 90% of the approximately 28,000 new cases each year and the estimated 376,000 existing cases of primary thyroid cancer in Europe and the United States. It is among the most curable neoplasms, but 5% to 20% of survivors develop local or regional recurrences, and <5% to 10% distant metastases, generally in the first years of follow-up, but sometimes after many years. Outcome of patients with recurrent or metastatic disease is highly dependent on the size and extent of neoplastic foci when detected. Because of the long-term risk of recurrence and the importance of timely detection, diagnostic follow-up of well-differentiated thyroid carcinoma is life-long and must be very sensitive. The past three decades have seen great progress in improving the safety, efficacy and convenience of the diagnostic follow-up of well-differentiated thyroid cancer. Three major innovations account for this progress: 1) increased understanding of prognostic factors for disease recurrence and individualization of follow-up according to these factors; 2) the emergence of serum thyroglobulin (Tg) measurement as the principal modality in diagnostic follow-up; 3) and most recently, the introduction of recombinant human thyroid-stimulating hormone (rhTSH) to provide TSH stimulation during thyroid hormone suppression therapy (THST) and to avoid THST withdrawal for Tg testing or iodine-131 (I-131) whole-body scanning. Continued work in these three areas and in new areas will allow the thyroidology community to build on, and patients to benefit from recent progress. PMID- 10727000 TI - Recombinant human thyroid-stimulating hormone (rhTSH): clinical development. AB - Recombinant human thyroid-stimulating hormone (rhTSH) was developed to safely provide exogenous TSH stimulation in patients on thyroid hormone suppression therapy (THST), which is integral to long-term management of well-differentiated thyroid cancer. Such stimulation allows detection of thyroid remnant and neoplastic tissue by serum thyroglobulin (Tg) testing and/or diagnostic iodine 131 (I-131) imaging, sparing patients THST withdrawal and resultant metabolic impairment, discomfort and morbidity needed to obtain endogenous TSH stimulation. An extensive clinical development process including nearly a decade of multinational, multicentre study or other follow-up of over 500 patients has demonstrated that: 1) rhTSH is safe and well-tolerated, with the main side effects transient, mild to moderate nausea in approximately 11% or headache in approximately 7% of patients. Of note, no antibodies to TSH were detected in any patient, even in 27 patients who have received multiple treatments; 2) in patients on THST, rhTSH effectively provides TSH stimulation that allows I-131 diagnostic imaging to detect persistent or recurrent disease with a generally equivalent sensitivity and image quality to those observed after THST withdrawal; 3) rhTSH increases the sensitivity of Tg testing in patients on THST; 4) rhTSH administration allows patients to remain euthyroid and obviates THST withdrawal; therefore, rhTSH administration avoids the significantly lower quality of life and greater discomfort and morbidity due to hypothyroidism during withdrawal, according to patients' and caregivers' ratings on validated instruments. These safety and efficacy findings have led to regulatory approval of rhTSH for diagnostic use in the United States in December 1998; regulatory approval is pending in the European Union. PMID- 10727001 TI - Clinical experience with recombinant human thyroid-stimulating hormone (rhTSH): whole-body scanning with iodine-131. AB - Whole-body scanning (WBS) with iodine-131 (I-131) is currently used together with serum thyroglobulin (Tg) measurement in the diagnostic follow-up of well differentiated thyroid carcinoma. One of the main disadvantages of I-131 WBS is its requirement of repeated weeks-long withdrawal of thyroid hormone suppression therapy (THST) to raise endogenous thyroid-stimulating hormone (TSH) production. This results in hypothyroidism and associated abnormalities, discomfort and morbidity. Recently, however, a series of multicentre clinical studies established the efficacy, safety, non-antigenicity, and quality of life benefits of recombinant human TSH (rhTSH, Thyrogen, thyrotropin alfa, Genzyme Corporation, Cambridge, MA, USA) in promoting radioiodine uptake and permitting sensitive I 131 WBS in patients on THST after initial therapy of well-differentiated thyroid cancer. Thus in everyday practice, rhTSH administration may in many cases supersede THST withdrawal as a preparative method for I-131 imaging. With the use of rhTSH, as whenever I-131 WBS is performed, useful and accurate imaging requires meticulous attention to good scanning practices. These include use of appropriate equipment, proper timing, sufficient scanning time, vigilance against artifacts and iodine contamination, and consideration of additional imaging in the case of ambiguous 48-hour scans. Whole-body retention of I-131 is approximately 50% greater during hypothyroidism after THST withdrawal than during euthyroidism on THST and rhTSH. Therefore, it is important to use an adequate diagnostic activity of > or =4 mCi (148 MBq) to compensate for the faster radioiodine clearance in the euthyroid state permitted by rhTSH administration. Ongoing dosimetric research eventually may provide more specific guidance regarding radioiodine activities for diagnostic, and, particularly, therapeutic purposes, with the use of rhTSH. PMID- 10727002 TI - Clinical experience with recombinant human thyroid-stimulating hormone (rhTSH): serum thyroglobulin measurement. AB - Only normal or neoplastic thyroid follicular cells produce and secrete the prohormone, thyroglobulin (Tg). For some 25 years, elevated serum concentrations of Tg therefore have been employed as a post-operative marker for well differentiated thyroid carcinoma. The aim of serum Tg measurement is to identify patients requiring: A) further testing to confirm the presence and to determine the stage, site and functionality of tumour; and/or B) further treatment. Serum Tg testing has the advantages of superior sensitivity to radioiodine whole-body scanning (WBS), lack of false positive readings, simplicity, speed, low cost, precision and wide availability. However, major limitations of serum Tg testing are interference by serum anti-Tg antibodies and decreased sensitivity during THST. To obviate this last drawback, the use of recombinant human thyroid stimulating hormone (rhTSH) has been clinically investigated as a preparative adjunct to serum Tg testing in the diagnostic follow-up of well-differentiated thyroid cancer. A large, multicentre international Phase III study now has confirmed evidence from earlier Phase I/II and Phase III trials and established the safety and efficacy of rhTSH in stimulating Tg release by residual and neoplastic thyroid tissue. This confirmatory study has clearly shown that 1) rhTSH administration significantly increases sensitivity of serum Tg measurement in patients on THST; and 2) by permitting sensitive diagnostic follow-up with serum Tg measurement and/or radioiodine WBS during THST, rhTSH administration improves patient comfort and quality of life compared to THST withdrawal. Thus use of the drug in diagnostic follow-up recently has received regulatory approval in the United States, and such approval is pending in the European Union. With regulatory approval, rhTSH is likely to gain an important role as a preparative adjunct to serum Tg testing in everyday practice. PMID- 10727003 TI - Recombinant human thyroid-stimulating hormone (rhTSH) in the radioablation of well-differentiated thyroid cancer: preliminary therapeutic experience. AB - Iodine-131 (I-131) ablation of thyroid remnant and/or persistent, recurrent or metastatic tumour is part of the initial and subsequent management of well differentiated thyroid carcinoma. Key to optimizing the safety and efficacy of radioablation is maximizing the selective uptake of radioiodine by normal or neoplastic thyroid tissue. This is achieved by ensuring adequate serum concentrations of thyroid-stimulating hormone (TSH). Exogenous TSH administration obviates the thyroid hormone suppression therapy withdrawal that is necessary for endogenous TSH elevation. It also avoids the marked morbidity, discomfort, and impairment in professional and educational pursuits and quality of life that often result from such withdrawal. Multicentre clinical studies have documented the safety and efficacy of recombinant human TSH (rhTSH) in promoting radioiodine uptake in the diagnostic scanning of well-differentiated thyroid cancer. Study of the use of rhTSH to facilitate radioablation of remnant and malignant thyroid tissue is at an earlier stage, with formal clinical investigation underway. Since April 1995, however, rhTSH has been employed as a radioablative adjunct in over 100 patients in the manufacturer's Compassionate Use Program. Twelve of these cases, reported or reviewed in the present paper, provide preliminary evidence that rhTSH is safe and effective in the radioablation setting. More data are needed to confirm these observations and to provide guidelines for optimal radioiodine dosing, and should be furnished by ongoing clinical investigation. rhTSH is the only acceptable treatment option in a subgroup of patients with well differentiated thyroid cancer, including those with hypopituitarism, ischaemic heart disease, a history of "myxoedema madness," debilitation due to very advanced disease or inability to produce TSH due to continued production of thyroxine by thyroid remnant or metastatic tumour. Therapeutic use of rhTSH may be considered in an increasing number of other cases. PMID- 10727004 TI - The role of recombinant thyroid-stimulating hormone (rhTSH) in the detection and management of well-differentiated thyroid carcinoma: a roundtable discussion. AB - A Roundtable meeting of European endocrinologists and nuclear medicine physicians recently examined the role of recombinant human thyroid-stimulating hormone (rhTSH, Thyrogen, thyrotropin alfa, Genzyme Corporation, Cambridge, MA, USA) in the detection and management of well-differentiated thyroid carcinoma. The meeting sought to review clinical work to date and to provide guidance and perspective on the agent's application in everyday practice. The Roundtable included a panel discussion centering on three major topics: 1) rationale for use of exogenous versus endogenous TSH stimulation of radioiodine uptake and thyroglobulin (Tg) production; 2) impact and use of rhTSH in diagnostic follow up; and 3) role of rhTSH in radioablation. The panel concluded that obtaining TSH stimulation of radioiodine uptake and Tg production exogenously with rhTSH obviates the weeks-long withdrawal from thyroid hormone suppression therapy (THST) needed to obtain such stimulation endogenously. rhTSH administration thereby eliminates morbidity, discomfort and uncertainties of compliance and timing associated with THST withdrawal. Thus compared to withdrawal, rhTSH administration offers important advantages in safety, quality of life, reliability, predictability and convenience to patients and care providers. rhTSH therefore represents a major advance in the diagnostic follow-up of well differentiated thyroid cancer. By increasing the sensitivity of serum Tg testing during THST, rhTSH administration also improves the sensitivity of diagnostic follow-up. In addition, heightening the sensitivity of Tg testing may permit more selective application of iodine-131 whole body scanning, decreasing the invasiveness of diagnostic follow-up. Clinical studies are underway to confirm the preliminary promise of rhTSH as an adjunct to radioablation of normal or neoplastic thyroid tissue, with emphasis on the therapeutic setting. PMID- 10727005 TI - Cloning and characterization of repressory and stimulatory DNA sequences upstream the Na/I-symporter gene promoter. AB - To investigate the existence of potential enhancer or silencer elements in the 5' flanking region of the human Na+/I-symporter (NIS) gene, we cloned 2,512 bp of genomic DNA further upstream of the previously defined proximal promoter. When tested in reporter gene assays, this sequence had no transcriptional activity per se, but was able to repress the activity of the heterologous SV40 promoter. Conversely, when fused to the homologous NIS gene promoter and thus comprising 3,800 bp 5'-flanking region, the transcription of the proximal NIS promoter was stimulated in the human cell lines FTC-133 (from thyroid) and HeLa, but inhibited in the rat thyroid cell line FRTL-5. This might be due to differences between the upstream regions of the rat and human NIS gene. Comparative analysis with standard promoters (SV40) led to the conclusion that the 5'-flanking region of the human NIS gene also exhibited transcriptional activity in non-thyroid cells. Thyroid-stimulating hormone (TSH) had a moderately stimulating effect on the full length NIS reporter gene construct in FRTL-5 cells. This stimulation is presumably mediated by a putative cAMP responsive element found in the first half of the cloned sequence. PMID- 10727007 TI - Decrease of TSH levels and epithelium/colloid ratio in rat thyroid glands following administration of proadrenomedullin N-terminal peptide (12-20). AB - Adrenomedullin (AM) exerts a potent and long-lasting hypotensive effect and is considered to be an important hormone in blood pressure control. AM is a 52-amino acid peptide synthesized as part of a 185-amino-acid preprohormone that also contains 20-amino-acid residues in the N-terminus, which has similar biological activity. This sequence is named a proadrenomedullin N-terminal 20 peptide (PAMP). Also, proadrenomedullin N-terminal peptide (PAMP)(12-20) exerts vasodepressor response, however this response is 3-fold less potent than the effect evoked by full-sequence peptide. Both AM and PAMP controls secretory activity of the pituitary gland and adrenal cortex, however, their action on the other endocrine glands is not recognized. Therefore, the aim of the study was to examine whether PAMP(12-20) is able to affect the structure and function of the rat thyroid gland. In adult female rats, subcutaneous PAMP(12-20) administration (1 or 4 nmol/rat/day for 6 days, autopsy 60 min after the last injection) had no effect on the weight of the thyroid gland. Peptide administration however, resulted in a dose-dependent increase in the volume of thyroid colloid, and lowered epithelium/colloid ratio in the gland (3.76 +/- 0.49, 2.66 +/- 0.27, 2.38 +/- 0.26, means +/- SE, n = 6, control, 1 and 4nmol PAMP/rat, respectively). PAMP administration changed neither the length of thyroid capillaries per unit area of surface nor their diameter. Lower dose of PAMP(12-20) significantly lowered blood TSH concentration (p < 0.01) while total and free T3 and T4 concentrations remained unchanged. Collectively, these findings suggest that PAMP(12-20) exerts a mild inhibitory effect on secretory activity of the rat thyroid gland. PMID- 10727006 TI - Topical glucose and accumulation of excitotoxic and other amino acids in ischemic cerebral cortex. AB - Pre-ischemic hyperglycemia aggravates brain damage due to transient global ischemia as demonstrated by exacerbation of brain lesions. Lactacidosis and elevated glutamate levels have been implicated as mechanisms of the increased damage. Our objective was to determine the effects of different levels of glucose (0, 66.5, 450 mg/dL) in cortical superfusates on the ischemia/reperfusion-evoked release of amino acids from the rat cerebral cortex. Physiologic levels of glucose significantly reduced the amount of aspartate, glutamate and gamma aminobutyric acid and the supra-physiologic levels of glucose reduced the amount of aspartate and phosphoethanolamine released from the cortex during ischemia/reperfusion in comparison with no glucose. The decrease in glutamate release may be due to increased availability of glucose for glycolysis with the subsequent formation of ATP and lactate, which has been shown to act as an energy source for neurons. The decreased levels may also reflect the continued energy dependent uptake of glutamate by glial cells. PMID- 10727008 TI - Multiple prolactin-releasing activity in the bovine hypothalamic extract. AB - The prolactin (PRL)-releasing activity (PRA) in the bovine hypothalamic extract (BHE) was compared to that of known substances with PRA and further characterized by gel filtration and reversed-phase high performance liquid chromatography (RP HPLC). Crude BHE produced marked dose-dependent stimulation of PRL secretion from the cultured rat adenohypophysial cells. Among the synthetic substances examined, vasoactive intestinal peptide (VIP), thyrotropin-releasing hormone (TRH) and beta endorphin (END) showed significant PRA. However, the flatter dose-response slope for TRH compared with BHE or the small amounts of VIP and END in BHE suggested that these peptides could not account for the major active elements of BHE. Oxytocin and interleukin-1beta were also tested, but they exhibited no PRA in our assay system. Gel filtration of BHE on the Sephadex G-100 column yielded two peaks of PRA distinct from TRH, VIP and END. One eluted in the void and the other in more retarded fractions. The latter fractions were pooled and subjected to the two-step RP-HPLC. The PRA was separated into three peaks designated peaks I, II and III in the first RP-HPLC experiment. Furthermore, the second RP-HPLCs with finer resolution revealed that peak II as well as peak III consisted of three peaks, while peak I eluted as a single peak. Most of these seven PRA peaks exhibited different RP-HPLC profiles from those of the newly characterized PRL releasing peptides. These findings again provide confirmatory evidence that BHE contained unique factors different from the above known substances. PMID- 10727009 TI - Kinetic studies on rabbit liver glucocorticoid 5alpha-reductase. AB - The serum concentration of active glucocorticosteroids depends not only on adrenal synthesis but also on enzymatic activation of 11-dehydro-glucocorticoids in the liver by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). In order to define the respective involvement of other regulative enzymes in the metabolism of 11-dehydro-glucocorticoids in the liver, the objective of this study was to evaluate the kinetic behavior of NADPH:delta 4-3-ketosteroid-5alpha reductase (5alpha-reductase, EC 1.3.99.5). The interrelations to liver 11beta HSD1 will be discussed. The kinetic properties of 5alpha-reductase of the rabbit liver were measured by a radioenzymatic assay and characterized with respect to protein-, substrate-, cosubstrate-, and pH-dependence. Michaelis-Menten enzyme kinetic parameters (Km and Vmax) were obtained for the formation of 5alpha reduced 11-dehydrocorticosterone and corticosterone metabolites. We found that both 11-dehydrocorticosterone (Km 4.2 x 10(-6) mol/l, Vmax 2,600 pmol x min(-1) x mg(-1)) and corticosterone (Km 0.5 x 10(-6) mol/l, Vmax 38 pmol x min(-1) x mg( 1)) exhibit a high affinity to 5alpha-reductase. With respect to cosubstrate-, pH dependence and finasteride inhibition, it is likely that 11-dehydrocorticosterone metabolism is primarily controlled by isoenzyme 5alpha-reductase type 1. This study shows that the deactivation of GCS especially of 11-dehydro-glucocorticoids via 5alpha-reductase is an important metabolic pathway in the liver. The metabolic activation of GCS by 11beta-HSD could possibly lead to an excess of GCS in the hepatocytes. Due to 5alpha-reductase activity this excess can be limited - on the level of CORT as well as of 11-DHC. PMID- 10727010 TI - Desaturation function does not decline after menopause in human females. AB - Aging appears to decrease delta6-desaturase activity in males, but in females it is uncertain. delta6- and delta5-desaturase functions were investigated in pre- and post-menopausal women who were normoglycemic or had type 2 diabetes (2 x 2 factorial, n = 37). Subjects were compared for indicators of diabetic control, estrogen levels, fatty acid profiles and indices of delta6- and delta5-desaturase activity. Diet intakes that were compared to determine whether results were a function of dietary factors known to influence desaturase activity revealed no differences (P>0.05). Post-menopausal women with type 2 diabetes had more 18:2 n6 in serum phospholipids (P<0.05) than did the pre- and post-menopausal control subjects. Fatty acid ratios of 18:3 n6/18:2 n6 indicated greater delta6 desaturase activity for women with type 2 diabetes, but differences were not found between pre- and post-menopausal groups. Significant correlation (P < 0.05) indicates an association between diabetic status and desaturase function, but function did not appear to be affected by menopausal status. In contrast to reports using male subjects, we found no evidence that desaturase function decreased in aging females, as reported for males, or increased as hypothesized in this study. PMID- 10727011 TI - Mutation in bombesin receptor subtype-3 gene is not a major cause of obesity in the Japanese. AB - Bombesin receptor subtype-3 (BRS-3) is one of the candidate genes of obesity. The mice lacking BRS-3 have been shown to develop mild obesity. These mice also showed hypertension and impaired glucose metabolism, supporting these mice as a good model for human obesity. We screened 104 Japanese obese men (BMI > 26.4, 26.5-44.1) to investigate whether there is any genetic defect in BRS-3 gene. The DNA fragments containing each exon of BRS-3 gene were amplified by polymerase chain reaction (PCR) and were directly sequenced. No mutation, nor polymorphism was found in the coding region of BRS-3, suggesting that mutation of this gene is not a major cause of obesity in humans. PMID- 10727012 TI - Peripheral glucose metabolism in patients with essential hypertension. AB - The present study was designed to determine the effect of essential hypertension on peripheral glucose metabolism during the postabsorptive state and after an oral glucose challenge. Ten normal subjects and nine patients with essential hypertension were studied after an overnight fast (12-14 h) and for 3 h after the ingestion of 75 g of glucose. Peripheral glucose metabolism was analyzed by the forearm technique to estimate muscle exchange of substrate combined with indirect calorimetry. Decreased forearm glucose uptake was observed in hypertensive patients compared to normal subjects (4.9+/-0.6 vs. 8.6+/-0.5 mmol x 100 ml forearm(-1) x 3 h(-1)) with diminished nonoxidative glucose metabolism (2.7+/-0.5 vs. 6.9+/-0.6 mmol x 100 ml forearm(-1) x 3 h(-1)). Muscle glucose oxidation did not differ significantly between groups. Both serum free fatty acid levels and lipid oxidation rates were similar in the normal subjects and the hypertensive patients, and declined in a similar fashion after glucose ingestion. Basal serum insulin levels did not differ significantly between normal and hypertensive patients, whereas the insulinemic response to glucose load was greater among the patients with essential hypertension. These data show that insulin resistance occurring in patients with essential hypertension is accompanied by impaired muscle glucose uptake and nonoxidative metabolism. PMID- 10727013 TI - Oxidative protein damage in plasma of type 2 diabetic patients. AB - In this study, we evaluated protein oxidation in 84 patients with Type 2 diabetes with no complications and in 61 healthy volunteers who formed the control group, whose ages matched those of the patients. We determined plasma carbonyl and plasma thiol levels as markers of oxidative protein damage and erythrocyte glutathione, plasma ceruloplasmin and transferrin as markers of free radical scavengers. The concentrations (mean +/- SD) of both of plasma carbonyl (1.24 +/- 0.46 vs. 0.72 +/- 0.17 nmole/mg protein; p < 0.0001) and lipid hydroperoxides (1.8 +/- 0.63 vs. 1.3 +/- 0.21 micromole/l; p < 0.0001) were increased, and the concentration of plasma transferrin (3.85 +/- 0.65 vs. 4.59 +/- 0.79 g/l; p < 0.05) was decreased, respectively, in Type 2 diabetic patients compared with those of the controls. There were no significant differences in the concentrations of plasma thiol (0.0064 +/- 0.001 vs. 0.0068 +/- 0.001 micromole/mg protein), erythrocyte glutathione (2.54 +/- 0.57 vs. 2.65 +/- 0.56 mg/g Hb), plasma ceruloplasmin (548 +/- 107.30 vs. 609 +/- 93.34 mg/l) between the patients and the controls. These changes observed in diabetic patients contribute to the imbalance in the redox status of the plasma. We attribute this imbalance to oxidative protein damage in Type 2 diabetic patients clinically free of complications. PMID- 10727015 TI - Identification and characterization of five new subunits of TRAPP. AB - TRAPP (transport protein particle), a multiprotein complex containing ten subunits, plays a key role in the late stages of endoplasmic reticulum to Golgi traffic in the yeast Saccharomyces cerevisiae. We previously described the identification of five TRAPP subunits (Bet5p, Trs20p, Bet3p, Trs23p and Trs33p). Now we report the identification of the remaining five subunits (Trs31p, Trs65p, Trs85p, Trs120p and Trs130p) as well as an initial characterization of the yeast complex and its human homologue. We find that three of the subunits are dispensable for growth and a novel sequence motif is found in Bet3p, Trs31p and Trs33p. Furthermore, biochemical characterization of both yeast and human TRAPP suggests that this complex is anchored to a Triton X-100 resistant fraction of the Golgi. Differences between yeast and human TRAPP as well as the relationship of TRAPP subunits to other docking/tethering factors are discussed. PMID- 10727014 TI - Plasma leptin in prepubertal patients with glycogen storage diseases. PMID- 10727016 TI - The GTP-binding protein RhoA localizes to the cortical granules of Strongylocentrotus purpuratas sea urchin egg and is secreted during fertilization. AB - The sea urchin egg has thousands of secretory vesicles known as cortical granules. Upon fertilization, these vesicles undergo a Ca2+-dependent exocytosis. G-protein-linked mechanisms may take place during the egg activation. In somatic cells from mammals, GTP-binding proteins of the Rho family regulate a number of cellular processes, including organization of the actin cytoskeleton. We report here that a crude membrane fraction from homogenates of Strongylocentrotus purpuratus sea urchin eggs, incubated with C3 (which ADP-ribosylates specifically Rho proteins) and [32P]NAD, displayed an [32P]ADP-ribosylated protein of 25 kDa that had the following characteristics: i) identical electrophoretic mobility in SDS-PAGE gels as the [32P]ADP-ribosylated Rho from sea urchin sperm; ii) identical mobility in isoelectro focusing gels as human RhoA; iii) positive cross reactivity by immunoblotting with an antibody against mammalian RhoA. Thus, unfertilized S. purpuratus eggs contain a mammalian RhoA-like protein. Immunocytochemical analyses indicated that RhoA was localized preferentially to the cortical granules; this was confirmed by experiments of [32P]ADP-ribosylation with C3 in isolated cortical granules. Rho was secreted and retained in the fertilization membrane after insemination or activation with A23187. It was observed that the Rho protein present in the sea urchin sperm acrosome was also secreted during the exocytotic acrosome reaction. Thus, Rho could participate in those processes related to the cortical granules, i.e., in the Ca2+-regulated exocytosis or actin reorganization that accompany the egg activation. PMID- 10727017 TI - Dissection of functional domains by expression of point-mutated profilins in Dictyostelium mutants. AB - Profilin is a ubiquitous cytoskeletal protein whose function is fundamental to the maintenance of normal cell physiology. By site-directed mutagenesis of profilin II from Dictyostelium discoideum the point mutations K114E and W3N were generated by PCR thus changing actin and poly-(L)-proline-binding activity respectively. W3N profilin is no longer able to bind to poly-(L)-proline concomitant with a slight reduction in actin binding. The K114E profilin exhibited a profound decrease in its ability to interact with actin, whereas binding to poly-(L)-proline was essentially unchanged. Binding to phospholipids was indistinguishable from the wild-type profilin. The in vivo properties of the point-mutated profilins were studied by expressing either W3N or K114E in profilin-minus D. discoideum mutants which have defects in the F-actin content, cytokinesis and development (Haugwitz et al., Cell 79, 303-314, 1994). Expression of K114E or W3N displayed a reduction in the F-actin content, normal cell morphology, and the transformants were capable of undergoing complete development. Interestingly, only cells that drastically overexpressed W3N could restore the aberrant phenotype, whereas the mutant protein K114E with its fully functional poly-(L)-proline binding and its strongly reduced actin-binding activities rescued the phenotype at low concentrations. Wild-type and both mutated profilins are enriched in phagocytic cups during uptake of yeast particles. These data suggest a) that a functional poly-(L)-proline-binding activity is more important for suppression of the mutant phenotype than the G actin binding activity of profilin, and b) that the enrichment of profilin in highly active phagocytic cups might be independent of either poly-(L)-proline or actin-binding activities. PMID- 10727018 TI - The glycolytic enzyme enolase is present in sperm tail and displays nucleotide dependent association with microtubules. AB - We examined the expression and localisation of enolase (2-phospho-D-glycerate hydrolase) in differentiating rat spermatogenic cells. We found that enolase is most abundant in mature spermatozoa and in residual cytoplasmic bodies detached from elongating spermatids with little to no enolase detected in meiotic primary spermatocytes and round spermatids. We localised enolase mostly to the tail of mature spermatozoa by immunoblotting and by immunofluorescence. RT-PCR analysis of differentiating spermatogenic cells detected only the alpha isoform of enolase. As several glycolytic enzymes are known to associate with microtubules prepared from brain, we investigated the association of enolase with brain and testis microtubules. We found that only a small fraction of testis and brain derived cytosolic enolase (4.9% and 11.2%, respectively) co-sediments with microtubules stabilised in the presence of taxol. In the presence of certain nucleotides in excess (3 mM ATP, CTP, GTP and ITP) the association of enolase with microtubules was disrupted, however, this was not the case for UTP. This observation is consistent with the finding that in the presence of 0.5 mM AMP PNP, a nonhydrolysable analogue of ATP, there is an increased association of enolase with microtubules. We propose that the nucleotide-dependent association of enolase with microtubules regulates enzyme activity by linking energy production to utilisation. PMID- 10727019 TI - Mesenchymal entactin-1 (nidogen-1) is required for adhesion of peritubular cells of the rat testis in vitro. AB - Epithelial-like Sertoli cells isolated from immature rat testis aggregate to form tubule-like structures when cultured on a monolayer of mesenchyme-derived peritubular cells. At the end of this morphogenetic process both cell types are separated by a basement membrane. In this study the gene expression of monocultures and direct cocultures of peritubular cells and Sertoli cells was examined using DD-RT-PCR. One of the isolated cDNA clones showed high homology to the cDNA encoding the basement membrane component entactin-1 (nidogen-1). Even though the entactin-1 (nidogen-1) gene is transcribed in peritubular cells, Sertoli cells, and in direct cocultures, the mRNA is translated only by the peritubular cells. No entactin-1 (nidogen-1) was detected in the Sertoli cells by Western blotting. Moreover, peritubular cell monocultures and cocultures showed the presence of one single band at 152 kDa in the supernatant, whereas in cell lysates two bands were detectable at 152 kDa and 150 kDa. Perturbation experiments using monoclonal antibodies directed against entactin-1 (nidogen-1) were performed with peritubular cells and Sertoli cells, respectively, and demonstrated loss of cell adhesion of the peritubular cells, while the Sertoli cells remained adherent. From these data we conclude that entactin-1 is exclusively produced and secreted by mesenchymal peritubular cells, and affects adhesion of peritubular cells in an autocrine manner. PMID- 10727020 TI - Suppression of transcription factor NF-kappaB activity by Bcl-2 protein in NIH3T3 cells: implication of a novel NF-kappaB p50-Bcl-2 complex for the anti-apoptotic function of Bcl-2. AB - Bcl-2 can suppress apoptosis by controlling genes that encode proteins required for programmed cell death and by interference with peroxidative damage. Overexpression of Bcl-2 in NIH3T3 cells can prevent GSNO-induced (S nitrosoglutathione-induced) apoptosis. The experimental results indicated that activation of NF-kappaB by GSNO is involved in inducing apoptosis. Surprisingly, we found that Bcl-2 delayed the release of IkB by formation of a Bcl-2-NF-kappaB complex (p50-p65-IkappaB) in the cytoplasm during cell apoptosis. Furthermore, a novel Bcl-2-p50 complex was found in the nucleus. These features were only observed in Bcl-2-transfected cells but not in the parental NIH3T3 cells. Overexpression of Bcl-2 suppressed the levels of c-myc, a target gene of NF kappaB, and influenced the DNA-binding activity of NF-kappaB during GSNOinduced apoptosis. We suggest that the Bcl-2-p50 complex inhibits NF-kappaB DNA-binding activity by competing with the p65-p50 heterodimer for the DNA-binding site in the nucleus. Finally, it has been demonstrated that the anti-apoptotic potential of Bcl-2 may be attributed to its complexing with p50 in the nucleus that leads to blockage of nuclear gene expression. PMID- 10727021 TI - An oncogenic form of the Flt-1 kinase has a tubulogenic potential in a sinusoidal endothelial cell line. AB - We have previously reported a constitutively activated form of the Flt-1 kinase (BCR-FLTm) molecularly engineered based on the structural backbone of the activated tyrosine kinase BCR-ABL. Here we show that it can induce not only growth stimulation but also tubulogenic differentiation of non-tubulogenic NP31 (non parenchymal) sinusoidal endothelial cells of rat liver in basement membrane matrix. Tubules formed in vitro were accompanied by fenestration structures and allowed circulation when transplanted into syngeneic animals. This biological response was not observed in other activated forms of kinases constructed in a similar fashion, which include Trk (BCR-TRK), KDR (BCR-KDR), and the parental BCR ABL. Interestingly, formation of fine tubules was accomplished with lower but not higher expression levels of BCR-FLTm. Compared to NP cells in primary culture NP31 is deficient in expression of alpha1 integrin subunit, which was restored by expression of BCR-FLTm that had tubulogenic ability. Matrix-induced tyrosine phosphorylation of an adaptor protein Shc with recruitment of Grb-2 was observed even when tubulogenesis was nearly completed at G1 stage of the cell cycle in 2-3 weeks. Activation of matrix metalloproteinase 2 (MMP-2) and expression of urokinase type plasminogen activator (uPA) was observed with cellular invasion into matrix at the depth of 200-300 microm. Inhibitors for MAP kinase activator MEK1 and for serine proteases showed deleterious effects on the tubulogenesis. We suppose that matrix ligand-induced integrin signals cooperate with a low level of Flt-1 kinase activity to promote tubulogenic behaviors of endothelial cells in this system. PMID- 10727022 TI - Expression of the green fluorescent protein in Paramecium tetraurelia. AB - In this paper we describe the expression of green fluorescent protein (GFP) as a reporter in vivo to monitor transformation in Paramecium cells. This is not trivial because of the limited number of strong promoters available for heterologous expression and the very high AT content of the genomic DNA, the consequence of which is a very aberrant codon usage. Taking into account differences in codon usage we selected and modified the original GFP open reading frame (ORF) from Aequorea victoria and placed the altered ORF into the Paramecium expression vector pPXV. Injection of the linearized plasmid into the macronucleus resulted in a cytoplasmic fluorescence signal in the clonal descendants, which was proportional to the number of copies injected. Southern hybridization indicated the establishment and replication of the plasmid during vegetative growth. Expression was also monitored by Northern and Western analysis. The results indicate that the modified GFP can be used in Paramecium as a reporter for transformation as an alternative to selection with antibiotics and that it may also be used to construct and localize fusion proteins. PMID- 10727023 TI - Correct evaluation of reporter assays in different cell lines by direct determination of the introduced plasmid amount. AB - Transfection efficiency in reporter gene assays is usually determined by cotransfection of a reference reporter gene under the control of a constitutively active strong promoter and determination of the reference enzyme activity. The SV40 promoter-driven beta-galactosidase reporter plasmid is frequently used as the reference reporter plasmid. Here we show that the beta-galactosidase expression in different cell lines does not correctly reflect the amount of plasmid taken up by cells and thus is not an accurate measure of transfection efficiency. The direct determination of introduced plasmid concentration in lysates of transfected cells is suitable for monitoring the transfection efficiency in reporter gene assays even if different cell lines are compared. PMID- 10727024 TI - Subclinical hypothyroidism and hyperthyroidism in elderly subjects: should they be treated? PMID- 10727025 TI - Should we treat the somatopause? PMID- 10727026 TI - Effects of steroid hormones on myelin proteins of the peripheral nervous system. PMID- 10727027 TI - Estrogen receptor-beta: a novel mediator of estrogen action in brain and reproductive tissues. Morphological considerations. PMID- 10727028 TI - The adrenal cortex in physiological and pathological aging: issues of clinical relevance. PMID- 10727029 TI - Dehydroepiandrosterone as neurosteroid: neuroendocrine effects in post-menopausal women. PMID- 10727030 TI - Acute oral administration of dehydroepiandrosterone in male subjects: effect of age on bioavailability, sulfoconjugation and bioconversion in other steroids. PMID- 10727031 TI - Dehydroepiandrosterone substitution in women. PMID- 10727032 TI - Dehydroepiandrosterone-sulfate (DHEA-S) restores the release of IGF-I from natural killer (NK) immune in old patients with dementia of Alzheimer's type (DAT). PMID- 10727033 TI - DHEA-based preparations in Italy, Europe and USA: the regulations, the active ingredients, the formulations. PMID- 10727034 TI - Altered laboratory thyroid parameters in elderly people. PMID- 10727035 TI - Age-related changes of thyroid function in both sexes. PMID- 10727036 TI - The "low-T3 syndrome" in unselected elderly home-dwellers: an epidemiological study in Dicomano, Italy. PMID- 10727037 TI - Screening for hypothyroidism in institutionalized elderly people with cognitive and functional impairment. PMID- 10727038 TI - Atrial fibrillation and thyroid function in elderly hospitalized subjects. PMID- 10727039 TI - Hyperthyroidism in the elderly: differences in the clinical findings compared with younger patients. PMID- 10727040 TI - Differentiated thyroid carcinoma (DTC) in the elderly: disease outcome, therapeutic approach, and long-term results in a group of 314 patients. PMID- 10727041 TI - Blood selenium levels and thyroid function in subjects aged 80 years and over. PMID- 10727042 TI - IGF-I levels in obesity: their relationship to blood pressure levels. PMID- 10727043 TI - Two-year treatment with rhGH of one patient with chronic congestive heart failure and growth hormone deficiency. PMID- 10727044 TI - The predictive role of insulin-like growth factor-I on insulin-mediated glucose uptake in the elderly. PMID- 10727045 TI - Growth hormone, Alzheimer's disease and normal aging in the oldest old subjects. PMID- 10727046 TI - Young adults with Down's syndrome show increased corticotrope responsiveness to hCRH or hexarelin, a peptidyl GH secretagogue. PMID- 10727047 TI - The IGF-I response to very low rhGH doses is preserved in human aging. PMID- 10727048 TI - The GH response to GHRH in normal adults is not affected by enalapril or furosemide while is blunted by digoxin. PMID- 10727049 TI - Activity of GH/IGF-I axis in patients with dilated cardiomyopathy. PMID- 10727050 TI - Testosterone, gonadotropins, prolactin and sex hormone-binding globulin in healthy centenarians. PMID- 10727051 TI - Leptin changes in normal weight and obese women in pre- and post-menopausal conditions. PMID- 10727052 TI - The contribution of androgen decline to the aging-related changes of body composition and lipoprotein in the healthy man. PMID- 10727054 TI - Post-menopausal estrogen supplementation only partially blunts the sympathoadrenal response to mental stress. PMID- 10727053 TI - Effects of 5 alpha-reductase inhibition by finasteride on lipoproteins and body composition in males affected by benign prostatic hyperplasia (BPH). PMID- 10727055 TI - Evaluation of adrenal function in aging. PMID- 10727056 TI - Serum dehydroepiandrosterone sulfate (DHEA-S) and total antioxidant capacity (TAC) in healthy centenarian subjects. PMID- 10727057 TI - Serum profile of dehydroepiandrosterone (DHEA) concentrations in DHEA supplemented elderly subjects. PMID- 10727058 TI - Sensitivity of bone remodeling markers in elderly male patients chronically treated with glucocorticoids. PMID- 10727059 TI - Dehydroepiandrosterone sulfate serum levels: no significance in diagnosing Alzheimer's disease. PMID- 10727060 TI - Corticotroph and adrenal responsiveness to hCRH, hexarelin and ACTH in young and elderly subjects. PMID- 10727062 TI - Adult-onset type 1 diabetes mellitus with Alzheimer's disease: case report. PMID- 10727061 TI - Cushing's syndrome: psychiatric involvement as important aspect of the clinical picture in old age. PMID- 10727063 TI - Beta-cell behavior, evaluated as basal and mixed meal-stimulated insulin, and proinsulin secretion, in elderly diabetic patients with "secondary failure". PMID- 10727064 TI - Hepatic osteodystrophy in elderly: role of insulin-like growth factor-I and osteocalcin. PMID- 10727065 TI - Melatonin and aging: relevance for clinical approach? PMID- 10727066 TI - Nitric oxide biomarkers increase during exercise-induced vasodilation in the forearm. AB - The purpose of the study was to determine if exercise-induced vasodilation was associated with an increase in forearm plasma levels of nitric oxide (NO) biomarkers (NO2- + NO3- and L-citrulline). Twelve healthy subjects (27+/-6 yrs) performed incremental rhythmic forearm exercise with the nondominant hand for 6 min each at 15, 30 and 45% of maximal voluntary contraction (MVC). Forearm blood flow (FBF) was determined in the exercise arm using venous occlusion plethysmography. Blood samples were obtained from the antecubital vein of the exercise and nonexercise arms for the measurement of NO biomarkers. In the exercise arm, FBF increased by a mean of 150%, 335% and 585% above baseline at 15, 30 and 45% of MVC, respectively. (ANOVA, P= 0.0001). Venous plasma NO2- + NO3 levels increased from 24+/-4 micromol/L at baseline, to 29+/-5, 32+/-4 and 3+/-4 micromol/L (ANOVA, P = 0.0001). Venous plasma L-citrulline levels increased from 31+/-5 micromol/L at baseline to 58+/-10, 87+/-7 and 141+/-15 micromol/L (ANOVA, P = 0.0001). There was a linear relationship between FBF and venous plasma NO2- + NO3- (slope= 0.38+/-0.10, P=0.0007) and between L-citrulline, (slope= 5.1 +/-1.3, P = 0.0004). Venous plasma levels of NO2- + NO3- and L-citrulline in the nonexercise arm were unchanged. These results demonstrate that exercise-induced vasodilation in the forearm is associated with forearm plasma levels of NO2- + NO3- and L-citrulline, in vivo markers of NO production. PMID- 10727067 TI - A comparison of the indirect estimate of mean arterial pressure calculated by the conventional equation and calculated to compensate for a change in heart rate. AB - The standard equation used to calculate mean arterial pressure (MAP) assumes that diastole persists for 2/3 and systole for 1/3 of each cardiac cycle. This ratio is altered when heart rate increases, and therefore we investigated the efficacy of predicting MAP during exercise using non-invasive indirect methods. Eight subjects exercised on a cycle ergometer for 3 minute intervals to elicit heart rates between 100-110, 120-130, 140-150, 160-170, and 180-190 beats/min. In the last minute of each 3 min interval an ECG recording was taken and systolic (SP) and diastolic (DP) blood pressure was measured by manual auscultation. MAP was calculated for each heart rate interval by: MAP=DP+1/3(SP-DP) (method A), and MAP= DP + Fs(SP- DP) (method B), where Fs is the fraction of the cardiac cycle comprising systole, measured from the ECG. Fs increased from 0.35+/-0.049 at rest to 0.47+/-0.039 at a heart rate of 180-190 beats/min. MAP measured by method B was consistently greater than MAP calculated by method A at all heart rates greater than resting heart rate (p<0.01). The error incurred when using the standard MAP equation (method A) to derive MAP during exercise (measured as the percentage difference between method A and B) increased linearly with heart rate (r=0.98). The standard MAP equation should not be applied during exercise, as it does not account for the change in the systolic: diastolic period ratio as heart rate increases. PMID- 10727068 TI - Comparison of physiological strain and muscular performance of athletes during two intermittent running exercises at the velocity associated with VO2max. AB - The purpose of this study was to examine physiological strain and muscular performance responses of well trained athletes during two intermittent running exercise protocols at the velocity associated with VO2max. Ten national level middle-distance runners (VO2max 69.4+/-5.1; mean+/-SD) performed in random order two 28 min treadmill running exercises: 14 bouts of 60 s runs with 60 s rest (IR60) and 7 bouts of 120 s runs with 120 s rest between each run (IR120). During IR120 peak oxygen uptake (12%), peak heart rate (3%) and peak blood lactate (79%) were significantly higher than during IR60 (P< 0.001) and almost the same as in the VO2max test. In IR120 the relative aerobic energy release calculated on the basis of the accumulated oxygen deficit during the running bouts was significantly higher than in IR60 (81.5+/-2.7 vs. 70.2+/-2.6%, P<0.001) likewise the sum oxygen consumption during the 14 min running (P< 0.001), while during the 14 min recovery it was as much lower (P < 0.001). There were no changes either during or between the IR60 and IR120 protocols with regard to the muscular performance parameters, stride length or height of maximal vertical jumps. In conclusion, during intermittent running at the velocity associated with VO2max doubling the duration of work and rest bouts from 60 s to 120s increased the physiological strain of well trained athletes to the same level as at exhaustion in the VO2max test but the muscular performance variables were not influenced. PMID- 10727069 TI - Reproducible voluntary muscle performance during constant work rate dynamic leg exercise. AB - During constant intensity treadmill or cycle exercise, progressive muscle fatigue is not readily quantified and endurance time is poorly reproducible. However, integration of dynamic knee extension (DKE) exercise with serial measurement of maximal voluntary contraction (MVC) force of knee extensor muscles permits close tracking of leg fatigue. We studied reproducibility of four performance indices: MVC force of rested muscle (MVC(rest)) rate of MVC force fall, time to exhaustion, and percentage of MVC(rest) (%MVC(rest)) at exhaustion in 11 healthy women (22+/-1 yrs) during identical constant work rate 1-leg DKE (1 Hz) on 2 separate days at sea level (30 m). Means+/-SD for the two test days, and the correlations (r), standard estimate errors and coefficients of variation (CV%) between days were, respectively: a) MVC(rest)(N), 524+/-99 vs 517+/-111, 0.91, 43.0, 4.9%; b) MVC force fall (N x min(-1)), -10.77+/-9.3 vs -11.79+/-12.1, 0.94, 3.6, 26.5 %; c) Time to exhaustion (min), 22.6+/-12 vs 23.9+/-14, 0.98, 2.7, 7.5 %; and d) %MVC(rest) at exhaustion, 65+/-13 vs 62+/-14, 0.85, 7.8, 5.6%. There were no statistically significant mean differences between the two test days for any of the performance measures. To demonstrate the potential benefits of evaluating multiple effects of an experimental intervention, nine of the women were again tested within 24hr of arriving at 4,300 m altitude using the identical force, velocity, power output, and energy requirement during constant work rate dynamic leg exercise. Low variability of each performance index enhanced the ability to describe the effects of acute altitude exposure on voluntary muscle function. PMID- 10727070 TI - Controlled lengthening or shortening contraction-induced damage is followed by fiber hypertrophy in rat skeletal muscle. AB - To study the hypothesis that more severe damage, caused by controlled lengthening (L) contractions, results in greater myofiber hypertrophy compared to increase in fiber size followed shortening (S) contractions, tibialis anterior muscles of anesthesized male Wistar rats were subjected to 240 either L or S contractions. The highest increase in muscle beta-glucuronidase activity, an indicator of muscle damage, was observed in L (7.1-fold) 4 days and in S (2.6-fold) 8 days postexercise. Dystrophin- and desmin-negative as well as fibronectin-positive fibers (signs of the early phase of damage) were observed immediately after exercise in the L group. At 4 days, massive myofiber injury was visible, and internally localized nuclei were present at 15-80 days after exercise in the L group. The shift towards more glycolytic fiber types (p<0.05 in L and S) and an increased mean cross-sectional area of type IIX/B fibers (p < 0.001 in L and S) at 80 days were observed in both groups. The observed minor damage with unchanged myofiber structures following S induced, however, an increase in myofiber cross sectional area of nearly the same magnitude as that following L, which was more damaging. The results do not support the hypothesis that fiber hypertrophy depends on the extent of the myofiber damage upon the exercised muscles. PMID- 10727071 TI - Prediction of change in maximal aerobic power by the 2-km walk test after walking training in middle-aged adults. AB - The aim was to study the 2-km walk test's accuracy in predicting maximal aerobic power (VO2max) and its changes during 15-week walking training in 108 healthy middle-aged adults. Training prescription was 65-75% of VO2max 50 min/session, four times weekly. VO2max was measured walking on a treadmill and predicted from the 2-km walk test using gender-specific equations including age, body mass index, performance time and heart rate. The difference in VO2max between the walk test and the maximal exercise test before training was -0.9 ml x min(-1) x kg(-1) (SD 4.4) in men and -2.2 (3.5) in women. The total error was from 4.1 to 4.5 ml x min(-1) x kg(-1). After training the increase in measured VO2max was 2.9 ml x min(-1) x kg(-1) more in the walkers compared to the controls. The difference between the predicted and measured changes in VO2max during training ranged from 1.8 to -1.0 ml x min(-1) x kg(-1) between the study groups except in walking men, for whom the walk test overestimated the difference. The total error was from 3.1 to 4.9 ml x min(-1) x kg(-1). The 2-km walk test can be used as a reasonably accurate field test to predict changes in VO2max due to aerobic training in healthy nonathletic adults. PMID- 10727072 TI - Cardiovascular fitness in premenarcheal girls and young women. AB - Maximal oxygen uptake (VO2max) in females, expressed as ml x kg(-1) x min(-1), declines steadily during the first three decades of life. The contribution of diminished cardiovascular function to this apparent fall in aerobic fitness is unknown. Cardiac responses to maximal cycle exercise were compared in 24 premenarcheal females (mean age 11.7 years) and 17 young adult women (mean age 27.4 years) using Doppler echocardiography. Mean VO2max was 40 ml x kg(-1) x min( 1) and 34.7 ml x kg(-1) x min(-1) in the girls and women, respectively (p < 0.05). When VO2max was expressed relative to allometrically-derived mass(0.52), however, no significant difference was observed in aerobic fitness between the two groups. Similar allometric analyses revealed no significant differences in average maximal cardiac output (10.50 vs 10.07 L x min(-1) BSA(-1.11) for girls and women, respectively) nor maximal stroke volume (53 vs 56 ml BSA(-1.13) respectively). These findings suggest that 1) allometric scaling is important in eliminating the effects of body size on VO2max, 2) body dimension differences can account for variations in VO2 in young females, and 3) cardiac functional reserve is similar in premenarcheal girls and young adult women. PMID- 10727073 TI - In fencing, does intensive practice equally improve the speed performance of the touche when it is performed alone and in combination with the lunge? AB - Global movements are generally composed of several simple movements. In this study we tested the hypothesis that the effects of practice on the performance of a simple movement cannot be highlighted when it is performed alone but only when it is part of a global movement. For this purpose we examined the performance of the touche movement in fencing, in terms of maximal speed of the foil, when it is performed alone and in combination with the lunge. The touche can be assimilated to a pointing task towards a target while the lunge can be assimilated to a forward stepping movement. Four international class fencers and five novices were tested. The results show that 1) the performance in the isolated touche condition was comparable between the two test populations, 2) the performance was higher in the expert population compared to the novice population in the sequential touche + lunge condition, and 3) the velocity of the centre of mass at the time of the peak velocity of the touche was higher in the expert population than in the novice population in the sequential touche + lunge condition. In line with Bernstein's concepts on the effects of practice on motor control it is suggested that the experts exploit to advantage the forces developed during the lunge whereas the novices do not. PMID- 10727074 TI - Alterations in running economy and mechanics after maximal cycling in triathletes: influence of performance level. AB - The effects of the triathlon performance level on the metabolic and mechanical alterations in running after an exhaustive cycling exercise were studied. Eight elite and 18 middle-level triathletes completed two 7 min runs on a treadmill at a velocity corresponding to that sustained during a triathlon before and after maximal cycling exercise. Energy cost of running was quantified during the last minute of each run from the net oxygen uptake. External mechanical cost was quantified during the last minute of each run from displacements of the centre of mass using a kinematic arm. The effect of cycling on the running energy cost differed when comparing the elite (from 4.01+/-0.46 to 3.86+/-0.34J x kg(-1) x m( 1)) and the middle-level triathletes (from 3.67+/-0.37 to 3.76+/-0.39 x kg(-1) x m(-1) (P<0.01). The effect of cycling on the respiratory muscle O2 was more important (P<0.05) for the middle-level (from 120.1+/-27.2 to 166.4+/-47.8 ml x min(-1)) than for elite triathletes (from 124.5 +/- 24.5 to 143.7 +/- 28.9 ml x min(-1)). A tendency to a decrease of the mechanical cost and of the vertical displacement of the centre of mass during the braking phase was observed for the elite triathletes, suggesting a better leg stiffness regulation than for their less successful counterparts. PMID- 10727075 TI - How valid is the determination of hematocrit values to detect blood manipulations? AB - The aim of this paper is a critical reflection of the practice in competitive cycling to use the hematocrit value (Hct) as an indirect control measure for doping with erythropoietin. To demonstrate the individual physiological variation of Hct values, five different studies were performed: 1) Eight subjects were observed (i) during 23 h after a 1 h lasting bout of cycle exercise at 60% of maximum performance and (ii) during 24h under control conditions. 2) Seven subjects were exposed to a 20 min period of -7 head down tilt (HDT), which was followed by 15 min in sitting position. 3) From four subjects blood samples were taken in a sitting position up to 60 min after they had ingested 1 liter isotonic saline solution. 4) Ten subjects performed a vita maxima test on a cycle ergometer, starting at 100W and increasing the workload by 17W every minute. 5) Four elite cyclists participated in a 10 days competition (1,700 km). RESULTS: 1) During the 24h observation period Hct decreased during the night from 45.3+/-3.1 % to 42.9+/-1.5% and returned to the initial values in the morning. This diurnal variation was even more pronounced after submaximal exercise (-4.1 %). 2) Due to fluid shifts from the interstitial into the intravasal compartment, HDT was accompanied by a 3.1+/-0.5% lower Hct. 3) Drinking of the isotonic saline solution also reduced the hematocrit by 3.3+/-0.5% after one hour. 4) Maximum cycle exercise increased the Hct from 46.8+/-2.4 % to 51.3+/-1.9% which was due to a 15 % decrease in plasma volume. 5) Repeated bouts of cycle-exercise reduced the Hct from 46.4+/-1.5% to 41.3+/-1.6%. CONCLUSIONS: All experiments demonstrate that the Hct is not a constant value but can be considerably changed by physiological measures. Clinical studies show that brain oxygen supply decreases with increasing Hct-values, which are also associated with a higher risk of stroke accidents. We therefore recommend to use a Hct-limit solely under strongly controlled standardized conditions to protect professional cyclists from hazardous manoeuvre until more appropriate methods to detect EPO-doping are developed. PMID- 10727076 TI - Effect of exogenous creatine supplementation on muscle PCr metabolism. AB - 31P NMR was used to assess the influence of two weeks creatine supplementation (21g x d(-1)) on resting muscle PCr concentration, on the rate of PCr repletion (R(depl)), and on the half-time of PCr repletion (t 1/2). Body mass (BM) and volume of body water compartments were also estimated by impedance spectroscopy. Fourteen healthy male subjects (20.8+/-1.9 y) participated in this double-blind study. PCr was measured using a surface coil placed under the calf muscle, at rest and during two exercise bout the duration of which was 1 min. They were interspaced by a recovery of 10 min. The exercises comprised of 50 plantar flexions-extensions against weights corresponding to 40% and 70% of maximal voluntary contraction (MVC), respectively. Creatine supplementation increased resting muscle PCr content by approximately 20% (P= 0.002). R(depl) was also increased by approximately 15% (P< 0.001) and approximately 10% (P = 0.026) during 40% and 70% MVC exercises, respectively. No change was observed in R(repl) and t1/2. BM and body water compartments were not influenced. These results indicate that during a standardized exercise more ATP is synthesized by the CK reaction when the pre-exercise level in PCr is higher, giving some support to the positive effects recorded on muscle performance. PMID- 10727077 TI - Nutrition antioxidant status and oxidative stress in professional basketball players: effects of a three compound antioxidative supplement. AB - Professional basketball players of the First Spanish League (ACB) are highly trained athletes performing high training loads and competition sessions. As intense physical activity has been suggested to increase free radical production, we examined the effects of a vitamin antioxidant mixture on the degree of oxidative stress in these athletes. Subjects received either 600 mg alpha tocopherol, 1,000 mg vitamin C and 32 mg beta-carotene, or a placebo over 32 days during a regular competition season. Plasma concentrations of alpha-tocopherol, vitamin C, beta-carotene, retinol, lipoperoxides (LPO), and the total antioxidant status (TAS) were analysed before and after treatment. Lipoperoxide plasma levels decreased significantly (p < 0.05) in the vitamin antioxidant supplemented group. The LPO/TAS ratio decreased about 15.3% in this group indicating a reduction in oxidative stress. Vitamin C dropped dramatically (p < 0.03, mean 15.4 micromol/L) in the placebo group leading to a marginal plasma vitamin C concentration (from 11 to less than 28 micromol/L). The results of this study suggest that the administered vitamin antioxidant mixture decreases oxidative stress and avoids the development of a marginal vitamin C status in professional basketball player during habitual training. PMID- 10727078 TI - Identification of epi-1 locus as a laminin alpha chain gene in the nematode Caenorhabditis elegans and characterization of epi-1 mutant alleles. AB - A new genetic locus, epi-1, has been identified and mapped, which affects epithelialization of various tissues in the nematode, Caenorhabditis elegans. Seven independent epi-1 mutant alleles have been obtained. These mutants have a wide spectrum of abnormalities, all seem to be caused by a primary defect of basement membrane. We have identified the epi-1 gene as a structural gene of laminin alpha chain. The sequence analyses of the gene and cDNAs revealed that the gene consists of 15 exons and encodes a protein of 3704 amino acids in an open reading frame of 11115 base pairs. The nematode alpha chain is similar to its vertebrate and fly orthologs in the domain structure. The mRNA is trans spliced to SL1 leader RNA as many of the nematode mRNAs. Mutation sites have been identified in four alleles. Two alleles have nonsense mutations and produce truncated proteins lacking the domain necessary for the formation of a heterotrimeric laminin molecule. The other two alleles have missense mutations in domains VI and IIIb, respectively. PMID- 10727079 TI - Analysis of mouse intron 7 DNA sequence of the APP gene: comparison with the human homologue. AB - Mutations in the beta-amyloid precursor protein gene (APP) cause Alzheimer disease (AD) in certain families. The mature protein (APP) exists in several different isoforms resulting from alternative splicing of the primary transcript. Several lines of evidence indicate that particular isoform(s) of APP may contribute to the etiology of AD. One of the isoforms, APP695, lacks the Kunitz protease inhibitor (KPI) domain encoded by exon 7. APP695 is expressed predominantly in neurons, whereas the KPI domain containing isoforms, APP751 and APP770, are expressed ubiquitously. The ratio of APP751/APP695 mRNA tends to increase in the brain of AD patients. Furthermore, this ratio in mouse brain is much lower than that in human brain, and mice are resistant to the spontaneous development of beta-amyloidosis. In addition, transgenic mice that develop pathological changes similar to those of AD expressed more KPI-domain containing APP mRNA than transgenic mice without the changes. Previous studies imply that the controlling elements exist in the flanking sequences of the alternatively spliced exons. Therefore, we have determined the DNA sequences of intron 7 and made a comparison between mouse and human DNA sequences of intron 7. Mouse intron 7 shares about 50% sequence identity with the human homologue, with higher sequence identity (approximately 85%) mainly in the 5' end (approximately 250 bp) of the intron. A palindromic sequence was found in both human and mouse intron 7 and showed subtle differences in their structure between the two species. Whether this sequence plays any roles in regulating alternative splicing of exon 7 remains to be determined. Human intron 7 contains a Alu element, which possesses potential retinoic acid and thyroid hormone responsive elements that might be involved in the regulation of alternative splicing. Mouse intron 7 sequence also contains a few repeat sequences which are specific to the genome of mice and rats. Homologies shared between human and mouse intron 7 sequences may contribute to the common characteristics of neuron-specific splicing of APP in both species. The unique features of the intron may account for differences between human and mouse brain in fine tuning of alternative splicing of the APP transcript, which may lead to their different susceptibilities to beta-amyloidosis. PMID- 10727080 TI - Structure and polymorphism of two stress-activated protein kinase genes centromeric of the MHC: SAPK2a and SAPK4. AB - As MHC genes are potent determinants of susceptibility to immunopathological diseases, the mapping of SAPK2a (CSBP) and SAPK4 to chromosome 6p 21.2-21.3 suggested that these genes may mediate the effects of the MHC on disease. Here we describe the genomic structure and localisation of both genes approximately 2.3Mb centromeric of HLA-DP. Examination of the complete coding region and selected intronic regions of SAPK2a and SAPK4 from 22 human EBV-transformed B-cell lines of different MHC haplotypes and racial background revealed complete sequence conservation. There were no notable differences in levels of expression of SAPK2a and SAPK4 mRNA in cell lines of different MHC haplotypes or racial origin. Examination of the SAPK2a and SAPK4 sequences from two chimpanzees revealed 3 nucleotide differences between human and chimpanzee in each gene resulting in only one amino acid change in SAPK4, and 6 nucleotide substitutions plus 2 deletions in 600bp of intronic sequence from SAPK4. This highlights the selective pressure placed on these genes to maintain their protein sequence, but does not favour a role in genetic regulation of disease or provide evidence of linkage disequilibrium with the MHC. PMID- 10727081 TI - DENS (differential extension with nucleotide subsets): application to the sequencing of human genomic DNA and cDNA. AB - Here we describe further development of our method of DNA sequencing by Differential Extension with Nucleotide Subsets (DENS) and its application to the sequencing of human genomic DNA and full-insert cDNA. Essentially, DENS is primer walking without custom primer synthesis; instead, DENS uses a presynthesized library of octamer primers degenerate in two positions (4,096 tubes/sequences for a complete library). DENS converts an octamer selected from this library into a long primer on the template, at the intended site only. This is done using a two step procedure which starts with a limited extension of the octamer (at 20 degrees C) in the presence of only two of the four possible dNTPs. The primer is extended by five bases or more at the intended priming site, which is deliberately selected to maximize the extension length (as are the two-dNTP set and the primer itself). The subsequent termination reaction at 60 degrees C then accepts the primer extended at the intended site, but not at alternative sites, where the initial extension (if any) is generally much shorter. This paper presents a set of rules for selection of DENS priming sites. We also compare different ways of template preparation for DENS sequencing. The data were obtained from primer walking on three human genomic DNA subclones of 3 to 4 kbp and four cDNA clones containing inserts of 1.9, 2.3, 3.8, and 4.9 kbp. Full length sequences were obtained from both strands of each subclone by automated dye-terminator fluorescent DNA sequencing using DENS with degenerate octamer primers. We compared the following types of DNA templates: single-stranded and double-stranded phagemid DNA, double-stranded PCR products, asymmetric PCR products, and single-stranded DNA produced by digestion with Lambda Exonuclease of double-stranded PCR product phosphorylated at one end (Exo-PCR). While all of the preps were found to work, the best results were obtained with Exo-PCR and phagemid single-stranded DNA. Exo-PCR directly from overnight bacterial culture with no plasmid prep of any kind yielded templates for DENS as good as Exo-PCR from purified DNA. We found that the Tm of the differentially extended octamers is an important factor in the success of DENS. Clustering of successful reactions was clearly distinguished in the Tm range of 50-66 degrees C, with success rates of 70% for Exo-PCR and 65% for ss phagemid templates. PMID- 10727082 TI - Identification of a novel mRNA species of the LKB1/STK11 Peutz-Jeghers serine/threonine kinase. AB - Germline mutations in the LKB1/STK11 serine/threonine kinase cause Peutz-Jeghers syndrome and this gene is also mutated at a moderate frequency in a wide variety of sporadic tumours. The translated region of LKB1/STK11 (1302bp) codes for a serine/threonine kinase of otherwise unknown function. We report a novel LKB1/STK11 mRNA species which is found at variable levels in all tissues examined. The novel mRNA, which we believe may be an unusual splice variant, consists of a 444bp in-frame deletion of exons 5-7 and part of exon 8. This deletion removes a large part of the kinase domain and comparison with other LKB1/STK11 mutations shows that kinase function is undoubtedly abolished. The role of the novel mRNA species remains unclear, but it retains a putative cAMP dependent kinase phosphorylation site and may play some regulatory role. PMID- 10727083 TI - A 356-Kb sequence of the subtelomeric part of the MHC Class I region. AB - The subtelomeric part of the MHC Class I region contains 11 of the 21 genes described on chromosome 6 at position 6p21.3. The general organization of those and other genes resident in the region was revealed by determining a 356,376 bp sequence. Potential exons for new genes were identified by computer analysis and a large number of ESTs were selected by testing the sequence by the BLAST algorithm against the GenBank nonredundant and EST databases. Most of the ESTs are clustered in two regions. In contrast, the whole HLA-gene region is crammed with LINE and SINE repeats, fragments of genes and microsatellites, which tends to hinder the identification of new genes. PMID- 10727084 TI - Isolation and characterisation of wheat cDNA clones encoding PR4 proteins. AB - Two cDNA clones encoding the previously characterised PR4 proteins wheatwin 1 and wheatwin2 from wheat (Triticum aestivum cv. S. Pastore) have been identified and named wPR4a and wPR4b, respectively. The clones have been isolated by screening a cDNA library with a specific cDNA probe obtained by RT-PCR. The wPR4a and wPR4b cDNAs contain open reading frames of 441 and 447 bp that encode for wheatwin1 and wheatwin2, respectively. PMID- 10727085 TI - Cloning of an ORF with homology to Mycobacterium echA1, encoding the enoyl-CoA hydratase, in Rhodococcus fascians. AB - An open reading frame encoding a polypeptide of significant homology (55.7% identity) with the enoyl-CoA hydratase encoded by the gene echA1 from Mycobacterium tuberculosis has been found in the genome of the plant-pathogen bacteria Rhodococcus fascians strain NRRL-B-15096. Sequence alignments showed that it possesses several conserved blocks common to E. coli, M. tuberculosis and human mitochondria. One of such blocks includes a glutamate residue located at position 149, corresponding to the glutamate 139 of Escherichia coli. This glutamate was previously shown to be the catalytic residue of enoyl-CoA hydratase in the multienzyme complex of fatty acid oxidation from E. coli. Our results provide additional information on the conserved domains of this enzyme. Significant homologies in other genome regions between R. fascians and M. tuberculosis confirm their phylogenetic relationship. PMID- 10727086 TI - Sequence analysis of the transcription control region upstream of the human FGF-3 gene. AB - With the purpose of studying the transcriptional regulation of the human FGF-3 gene, we have cloned and determined the nucleotide sequence of the 11-kbp region flanking its 5' end. Analysis of the sequence disclosed the presence of multiple repetitive elements. Remarkably, all of them were found to have inserted in the same orientation as the FGF-3 gene, suggesting that the whole upstream region could play a role in the control of its transcription. Unique regions within the sequence were scanned for the presence of transcriptional regulatory elements. A potential "Initiator" sequence preceded by several motifs homologous to binding sites for transcription factors pinpointed a putative promoter, 6 kbp upstream of the ATG codon for the FGF-3 protein. A 250-nt sequence stretch surrounding the "Initiator" was found to display punctate homology with the first (P1) of the three promoters (P1, P2 and P3) of the mouse Fgf-3/int-2 gene, specifically in the region of the transcriptional start sites. These data should be useful in studying the mechanisms of regulation of the FGF-3 transcription unit. PMID- 10727087 TI - cDNA sequence of bovine thioredoxin. AB - In this paper, we report the cDNA sequence of bovine thioredoxin. We determined the full-length cDNA sequence of bovine thioredoxin by RT-PCR, 5'-RACE and 3' RACE methods. Currently, the thioredoxin cDNA sequences of only five mammalian species (human, macaca, mouse, ovine and rat) are registered in the GenBank database. We performed sequence comparisons on the total cDNA sequence and the coding region, and produced a multialignment between the amino acid sequences of bovine and other mammalian thioredoxins. The amino acid sequences of thioredoxins are highly conserved among mammalian species, for example, only one difference exists between the amino acid sequences of bovine and ovine thioredoxin. PMID- 10727088 TI - Nucleotide sequence of the feline Bmi-1 coding region. AB - The bmi-1 gene was discovered as a clonal integration site of the Moloney murine leukaemia virus in B-cell lymphomas. The Bmi-1 protein contains the RING finger motif and is homologous to two Drosophila proteins known to be part of a multimeric protein complex involved in repressing gene transcription. A similar role for the highly conserved Bmi-1 protein in mammalian cells has been suggested. The coding regions for the mouse and human genes are known and are 92% homologous. This study involved PCR amplification, cloning, and sequencing of the 980bp feline bmi-1 coding region which was shown to be 92% and 97% homologous to the mouse and human genes respectively. From the open reading frame the feline protein is 326 amino acids in length and is 99% homologous to the human protein and 97% homologous to the mouse protein. This data is consistent with the closer relationship between the feline and human genomes and provides another experimental system in which to analyse Bmi-1 function. PMID- 10727089 TI - Sequence of the gene encoding hsp90e from Cryptosporidium parvum. AB - A composite 2364 nt DNA sequence with an open reading frame (ORF) encoding an endoplasmic reticulum-associated heat shock protein 90 (CpHsp90e) was determined from clones isolated from genomic libraries constructed from the KSU-1 isolate of Cryptosporidium parvum. Transcription was verified by isolation of a clone from a cDNA library with a similar restriction map to that observed with genomic DNA. The predicted protein consists of 787 amino acids, has a predicted molecular size of 89.2 kDa, and was found to share strong homology with other endoplasmic reticulum-associated hsp90 proteins. PMID- 10727090 TI - Comparison of gene arrangements of chloroplasts between two centric diatoms, Skeletonema costatum and Odontella sinensis. AB - We have cloned and sequenced 3.4 kbp, 2.5 kbp, 1.9 kbp, 1.6 kbp and 0.5 kbp segments of a marine centric diatom, Skeletonema costatum, chloroplast DNA. These segments contain 28 genes. The genes which are not encoded on chloroplast genomes of chlorophyll a+b plants are found such as the psaD, ycf33, ycf35 and ycf47 genes. The gene sequences were compared with that of Odontella sinensis. At nucleic acid level, the ycf genes have lower homologies (69-87%) with O. sinensis than the other genes (78-100%), and some differences in the gene arrangement are found between two centric diatoms, O. sinensis and S. costatum. PMID- 10727091 TI - Molecular analysis of a tryptophan-2-monooxygenase gene (IaaM) of Agrobacterium vitis. AB - Tryptophan-2-monooxygenase genes occur in a number of bacteria and encode the conversion of tryptophan to the plant hormone precursor indole-3-acetamide. The role of these genes in the plant-bacteria interaction is often unclear. However, their function as a virulence determinant is established for Pseudomonas savastanoi and Agrobacterium tumefaciens. Some members of the Agrobacteria, such as Agrobacterium vitis have a limited host range. We have characterized the tryptophan-2-monooxygenase (iaaM) gene of A. vitis strain AG162 and show it is different from other A. vitis strains and related to iaaM of A. rhizogenes. The sequence of AG162 iaaM was deposited in the Genbank database under the accession number AF142716. PMID- 10727092 TI - On the information highway, or sitting on the curb? PMID- 10727093 TI - Reconfiguring a curriculum for the new millennium: the process of change. AB - Over the past decade, changing demographics, new technology, an increased focus on health promotion, and radical shifts in health care reimbursement have significantly impacted the nursing role. The purpose of this article is to review the process of change at one university as its curriculum evolved from an integrated acute-care focus to a community-based, health promotion framework. A review of the literature and survey data from students, alumnae, faculty, other universities, and curriculum consultants laid the groundwork for this process. Curriculum generation followed guided by data analysis, template design and revision, values clarification, sacrificing "sacred cows," and consensus building. Implications for nursing and nursing education are explored. PMID- 10727094 TI - Toward a narrative-centered curriculum for nurse practitioners. AB - This paper discusses various alternative and nontraditional teaching strategies currently used in nurse practitioner curricula. These instructional strategies include case-study analysis (Ryan-Wenger & Lee, 1997) and problem-based learning/practice-based learning (Barrows, 1994). We suggest a further evolution, using principles and practices of a narrative pedagogy (Diekelmann, 1995) to allow convergence of these several narratively-focused inductive and interpretive approaches. This combination of ways of learning has led us toward a narrative centered curriculum for family nurse practitioners (FNPs). Specific ways to use narrative in the FNP curriculum are presented to demonstrate how to take the curriculum beyond traditional ways of teaching and learning. PMID- 10727095 TI - An integrative review illuminates curricular applications of primary health care. AB - The International Council of Nurses supported primary health care and advocated its acceptance by other intersectorial stakeholders. Nurse educators involved in curriculum development have respected this direction. To pursue the primary health care vision and influence the adoption of primary health care delivery systems, all principles of primary health care must be implemented simultaneously. An integrative review was conducted to ascertain the interpretation of primary health care held by nurse authors. A content analysis of the nursing literature revealed that 184 of the 254 articles retrieved (72%), applied primary health care in a manner that is consistent with the World Health Organization (WHO) definition. Through curricular applications of different interpretations of the primary health care principles, differences in the created learning environment are discussed. The Declaration of Alma Ata, evolving from beliefs and values shared by 134 member nations present at the Assembly, delineated that primary health care was the accepted infrastructure to guide policies that direct health care delivery systems regardless of the context in the country (WHO, 1978). Even with this overwhelming endorsement, very few countries have made changes in policies and infrastructure for implementing the underlying values and beliefs ascribed in primary health care. Changes that have occurred have not been organized around the vision or the principles of primary health care. This delay has had, and will continue to have, a huge impact on nursing education, practice, and research. Nurses are interested in helping people attain, maintain, or regain health; primary health care has the potential to achieve health for all citizens of the world. PMID- 10727096 TI - Student voice in curriculum making. AB - Undergraduate student nurses (n = 117) were asked to reflect critically on their psychiatric clinical learning experience and identify strengths and weaknesses not only in the actions and behaviors of others, but also in their own. A questionnaire was specifically constructed to encourage the voicing of issues, concerns, actions, and behaviors that centered around the concept of quality in relation to four predetermined categories: clinical practice, clinical nursing staff, clinical facilitators, and students. Themes, inductively derived from the collected information within each category, were organized into clusters and then into frequency distributions to facilitate interpretation. The study generated information that should be useful in planning and supervising effective and mutually satisfying clinical practicums in any psychiatric context. Moreover, the responses gave voice to matters that otherwise may have gone unrecognized in the curriculum. The study confirms that student voice must become an integral part of the alternatives from which curriculum-making choice is made. PMID- 10727097 TI - Alternative and complementary therapies in nursing curricula. AB - The growing interest in and use of alternative and complementary therapies by health care professionals and laypersons, the incorporation of these therapies in medical curricula and practice, and the greater acceptance of the legitimacy of such treatment methods require that nurse educators consider how this content may be incorporated into curricula. The authors propose that prelicensure students learn the premises that support such healing practices and develop skill in eliciting and evaluating patients' use of alternative therapies. In-depth study of one or more complementary therapies may be the focus of continuing education or elective courses. Students in graduate programs may investigate the safety and efficacy of nonorthodox therapies. PMID- 10727098 TI - Community-based curricula: new issues to address. AB - Although the issues that may arise when developing and implementing a community based curriculum may seem overwhelming, such issues should not deter programs from continuing their efforts in that direction. Rarely is a new curriculum perfect. Most come with their inherent problems and issues, and community-based curricula are no exception. Nursing education is being presented with a challenge to design curricula that allows nursing to make a difference in patient outcomes in every setting. As nursing educators we need to welcome that challenge. PMID- 10727099 TI - Depicting death: lessons on writing and professional development in nursing. PMID- 10727100 TI - Design and evaluation of benzophenone-containing conformationally constrained ligands as tools for photoaffinity scanning of the integrin alphaVbeta3-ligand bimolecular interaction. AB - Integrins are cell-surface adhesion molecules involved in mediating cell extracellular matrix interactions. High-resolution structural data are not available for these heterodimeric receptors. In order to generate tools for photoaffinity scanning of the RGD-binding site of human integrin alphaVbeta3. new conformationally constrained ligands were designed. The ligands were based on five different cyclic peptidic or peptidomimetic scaffolds with high affinity for alphaVbeta3. A single photoreactive group (a benzophenone moiety) was introduced at different positions relative to the RGD triad. In addition, an 125I or a biotin group was introduced as a reporting tag. Twenty-four cyclic ligands were prepared and their binding affinity for alphaVbeta3 was determined. In most cases, the modifications resulted in a 5- to 500-fold decrease in affinity relative to the unmodified scaffold. Analogs representing three of the five families were screened for their cross-linking efficiency. Ligands with submicromolar affinities cross-linked efficiently and specifically to the integrin receptor, whereas ligands with weaker affinities gave specific cross linking, but with lower efficiency. Almost all of the screened ligands cross linked predominantly to the beta3 subunit. PMID- 10727101 TI - Highly fluorescent protein labeling using dendritic peptide derivatives. AB - Non-specific fluorescent dyes and photosensitizers are routinely used in clinical practice for the photodetection and photoablation of superficial lesions. Future applications in photomedicine are likely to rely on the selective delivery of photoactive compounds to diseased areas, using specific targeting agents such as antibodies. This fact underlines the need for methods that allow the chemically defined conjugation of several photoactive molecules to a single protein 'vehicle', with full retention of binding affinity. Here, we present methods for the site-specific fluorescent labeling of proteins using dendritic peptides, which had been chemically modified with multiple molecules of fluorescein. Branched peptide derivatives can be stably conjugated to proteins either by reaction with suitable free reactive groups or by using the high-affinity non covalent interaction between calmodulin and a specific binding peptide. Chemical modification of proteins with one, two or four molecules of fluorescein resulted in a proportional increase in protein fluorescence. PMID- 10727102 TI - Peptide analogs from E-cadherin with different calcium-binding affinities. AB - Cadherins are a family of calcium-dependent cell-surface proteins that are fundamental in controlling the development and maintenance of tissues. Motif B of E-cadherin seems to be a crucial calcium-binding site as single point mutations (D134A and D134K) completely inactivate its adhesion activity. We analyzed peptide models corresponding to motif B (amino acids 128-144) as well as selected mutations of this motif. Our NMR studies showed that this motif B sequence is actually an active calcium-binding region, even in the absence of the rest of the cadherin molecule. We found that the binding affinity of this motif is very sensitive to mutations. For example, our peptide P128-144 with the native calcium binding sequence has an affinity of Kd 0.4 mM, whereas the mutants P128-144/ D134A and P128-144/D134K containing the replacement of Asp134 by Ala and Lys, have Kd values of only 1.5 and 11 mM, respectively. Removing Asp at position 134, which correlates with the loss of adhesion activity, decreases calcium-binding affinity 20-fold. Ala132, along with residues Asp134, Asp136 and Asn143, is involved in calcium binding in solution. We also demonstrated that the calcium binding affinity can be increased 3-fold when an additional Asp is introduced at position 132. In 50% organic solvent, this binding affinity of peptide P128 144/A132D (17-mer) from E-cadherin is similar to that of peptide P72-100/C73-77 91A (29-mer) from alpha-lactalbumin. PMID- 10727104 TI - Estimation of the deamidation rate of asparagine side chains. AB - Statistical analysis of data from the literature concerning the deamidation reaction of asparagine side-chains in short peptides reveals that the logarithm of rate constants can be solved into a constant plus contributions from the residues closest to asparagine. A table of amino acid contributions has been derived, from which deamidation rate constants can be estimated with good approximation. Assuming the contribution of glycine to be zero, the mean of the absolute values of the contributions for the residues following asparagine is approximately seven times that for the preceding residues. In both positions residues with no bulk side chains or with functional side groups contribute markedly to the increase in the rate constant. PMID- 10727103 TI - Two MSA 2 peptides that bind to human red blood cells are relevant to Plasmodium falciparum merozoite invasion. AB - Plasmodium falciparum merozoite membrane surface antigen 2 (MSA2) has been associated with the development of protective immunity against malaria. MSA2 antibodies were able to inhibit in vitro merozoite invasion. In our search for experimental evidence concerning the participation of MSA2 in merozoite invasion, 40 peptides were synthesized according to sequences reported for the CAMP and FC27 prototype Plasmodium strains. These peptides were purified, 125I radiolabeled and tested for their ability to bind to erythrocytes. Two MSA2 synthetic peptides with high specific binding to human erythrocytes were found. The peptide coded 4044 (KNESKYSNTFINNAYNMSIR), located in the MSA2 N-terminal conserved region, has an affinity coefficient of 72 nM and showed a positive cooperativity for the receptor-ligand interaction. The other peptide, coded 4053 (NPNHKNAETNPKGKGEVQKP) and located in the central variable region of MSA2, has an affinity coefficient of 49nM and also showed a positive cooperativity for the receptor-ligand interaction. The binding capacity of these peptides is affected by erythrocytes treated with neuraminidase and trypsin, but it is not affected by chymotrypsin. Both of these sequences inhibit in vitro erythrocyte parasite invasion by up to 95% suggesting that they have an important role in the parasite's invasion process. Furthermore, as published previously [A. Saul et al. (1992) J. Immunol., 148, 208-211], a protective B epitope is included in the 4044 peptide sequence. PMID- 10727105 TI - 3-(3'-fluorenyl-9'-oxo)-L-alanine: a novel photoreactive conformationally constrained amino acid. AB - Photoaffinity scanning of the ligand-G-protein-coupled receptor bimolecular interface is a direct approach to mapping the interactions of ligands and receptors. Such studies are an important first step toward generating an experimentally based model of the ligand-receptor complex. The synthesis and spectroscopic characterization of Boc-3-(3'-fluorenyl-9'-oxo)-L-alanine and 9 fluorenone-3-carboxylic acid are described. Incorporation of these two photophores into the parathyroid hormone (PTH) molecule yields potent agonists. These photoreactive analogs cross-link specifically with the recombinant human PTH1 receptor stably expressed in human embryonic kidney cells. The availability of the 9-fluorenone (a conformationally constrained derivative of benzophenone, the abundantly used photophore) for photoaffinity scanning provides an important tool to probe the effect of conformational flexibility of the photophore on the selection of the cross-linking site in the macromolecular acceptor. PMID- 10727106 TI - Differential contribution of the conserved tyrosine residues to activity and structural stability of Ophiophagus hannah alpha-neurotoxins. AB - Two alpha-neurotoxins, Oh-4 and Oh-7, from king cobra (Ophiophagus hannah) venom were subjected to Tyr modification with tetranitromethane. Selective nitration of Tyr4 in Oh-4 caused a slight decrease in lethal toxicity of 11% and a decrease in nicotinic acetylcholine receptor (nAchR)-binding activity of 28%, whereas nitration of Tyr4 in Oh-7 resulted in an approximately equal 60% decrease in lethality and nAchR-binding activity. When the Tyr23 in Oh-4 or Tyr22 in Oh-7 appears to be 'buried' in the toxin following further modification, the toxins lost their biological activity and conformational change concurrently. Nevertheless, the dinitrated Oh-4 retained a beta-sheet structure as revealed by CD spectra and exhibited a precipitin reaction with anti-Oh-4 sera. These results indicate that both Tyr4 and Tyr22 play a crucial role in the neurotoxicity of Oh 7, whereas intact Tyr23 is involved in the manifestation of the toxicity of Oh-4 to a greater extent. In contrast to Oh-4, the conformational stability of Oh-7 depends heavily upon the integrity of Tyr22. PMID- 10727107 TI - Maurotoxin and the Kv1.1 channel: voltage-dependent binding upon enantiomerization of the scorpion toxin disulfide bridge Cys31-Cys34. AB - Maurotoxin (MTX) is a 34-amino acid polypeptide cross-linked by four disulfide bridges that has been isolated from the venom of the scorpion Scorpio maurus palmatus and characterized. Maurotoxin competed with radiolabeled apamin and kaliotoxin for binding to rat brain synaptosomes and blocked K+ currents from Kv1 channel subtypes expressed in Xenopus oocytes. Structural characterization of the synthetic toxin identified half-cystine pairings at Cys3-Cys24, Cys9-Cys29, Cys13 Cys19 and Cys31-Cys34 This disulfide bridge pattern is unique among known scorpion toxins, particularly the existence of a C-terminal '14-membered disulfide ring' (i.e. cyclic domain 31-34), We therefore studied structure activity relationships by investigating the structure and pharmacological properties of synthetic MTX peptides either modified at the C-terminus ?i.e. MTX(1-29), [Abu31,34]-MTX and [Cys31,34, Tyr32]D-MTX) or mimicking the cyclic C terminal domain [i.e. MTX(31-34)]. Unexpectedly, the absence of a disulfide bridge Cys31-Cys34 in [Abu 31,34]-MTX and MTX(1-29) resulted in MTX-unrelated half-cystine pairings of the three remaining disulfide bridges for the two analogs, which is likely to be responsible for their inactivity against Kv1 channel subtypes. Cyclic MTX(31-34) was also biologically inactive. [Cys31,34, Tyr32]D-MTX, which had a 'native', MTX-related, disulfide bridge organization, but a D-residue-induced reorientation of the C-terminal disulfide bridge, was potent at blocking the Kv1.1 channel. This peptide-induced Kv1.1 blockage was voltage-dependent (a property not observed for MTX), maximal in the low depolarization range and associated with on-rate changes in ligand binding. Thus, the cyclic C-terminal domain of MTX seems to be crucial for recognition of Kv1.3, and to a lesser extent, Kv1.2 channels and it may contribute to the stabilization and strength of the interaction between the toxin and the Kv1.1 channel. PMID- 10727108 TI - Modifications of the cyclic mu receptor selective tetrapeptide Tyr-c[D-Cys-Phe-D Pen]NH2 (Et): effects on opioid receptor binding and activation. AB - The previously described cyclic mu opioid receptor-selective tetrapeptide Tyr-c[D Cys-Phe-D-Pen]NH2 (Et) (JOM-6) was modified at residues 1 and 3 by substitution with various natural and synthetic amino acids, and/or by alteration of the cyclic system. Effects on mu and delta opioid receptor binding affinities, and on potencies and efficacies as measured by the [35S]-GTPgammaS assay, were evaluated. Affinities at mu and delta receptors were not influenced dramatically by substitution of Tyr1 with conformationally restricted phenolic amino acids. In the [35S]-GTPgammaS assay, all of the peptides tested exhibited a maximal response comparable with that of fentanyl at the mu opioid receptor, and all showed high potency, in the range 0.4-9nM. However, potency changes did not always correlate with affinity, suggesting that the conformation required for binding and the conformation required for activation of the opioid receptors are different. At the delta opioid receptor, none of the peptides were able to produce a response equivalent to that of the full delta agonist BW 373,U86 and only one had an EC50 value of less than 100nM. Lastly, we have identified a peptide, D-Hat-c[D-Cys-Phe-D-Pen]NH2 (Et), with high potency and > 1,000-fold functional selectivity for the mu over delta opioid receptor as measured by the [35S]-GTPgammaS assay. PMID- 10727109 TI - (alphaMe)Aun: a highly lipophilic, chiral, Calpha-tetrasubstituted alpha-amino acid. Incorporation into model peptides and preferred conformation. AB - Using a chemo-enzymatic approach we prepared the highly lipophilic, chiral, Calpha-methylated alpha-amino acid (alphaMe)Aun. Two series of terminally protected model peptides containing either D-(alphaMe)Aun in combination with Aib or L-(alphaMe)Aun in combination with Gly were synthesized using solution methods and fully characterized. A detailed solution conformational analysis, based on FT IR absorption, 1H NMR and CD techniques, allowed us to determine the preferred conformation of this amino acid and the relationship between chirality at its alpha-carbon atom and screw sense of the helix that is formed. The results obtained strongly support the view that D-(alphaMe)Aun favors the formation of the left-handed 3(10)-helical conformation. PMID- 10727110 TI - Equations of state for POPX lipids at the air/water interface. A comprehensive study. AB - We have investigated monomolecular fluid-like films of palmitoyl oleoylphosphatidyl lipids with choline, glycerol and serine head groups, respectively. Conventional Langmuir trough experiments have been evaluated towards a thermodynamic analysis applying a novel approach that was recently developed in this laboratory. Our work involves elaborate efforts to exclude possible error sources of the basic measuring parameters. By means of pertinent mass conservation plots it could then be shown that the present lipids form a practically insoluble monolayer. Relative deviations of the lateral pressure from its ideal (gaseous) value are seen to be a very pronounced linear function of the surface concentration (between 1 and about 35 mN/m). They reveal a clearly manifested Boyle point around 4 mN/m, indicating formation of aggregates in the very low pressure range. The results are discussed in terms of a rather simple quantitative formulation of the underlying equation of state including fit curves of the related partial molecular area and Gibbs free energy. PMID- 10727111 TI - A potential role for sterol carrier protein-2 in cholesterol transfer to mitochondria. AB - Mitochondrial cholesterol oxidation rapidly depletes cholesterol from the relatively cholesterol-poor mitochondrial membranes. However, almost nothing is known regarding potential mechanism(s) whereby the mitochondrial cholesterol pool is restored. Since most exogenous cholesterol enters the cell via the lysosomal pathway, this could be a source of mitochondrial cholesterol. In the present study, an in vitro fluorescent sterol transfer assay was used to examine whether the lysosomal membrane could be a putative cholesterol donor to mitochondria. First, it was shown that spontaneous sterol transfer from lysosomal to mitochondrial membranes was very slow (initial rate, 0.316 +/- 0.032 pmol/min). This was due, in part, to the fact that 90% of the lysosomal membrane sterol was not exchangeable, while the remaining 10% also had a relatively long half-time of exchange t(1/2) = 202 +/- 19 min. Second, the intracellular sterol carrier protein-2 (SCP-2) and its precursor (pro-SCP-2) increased the initial rate of sterol transfer from the lysosomal to mitochondrial membrane by 5.2- and 2.0 fold, respectively, but not in the reverse direction. The enhanced sterol transfer was due to a 3.5-fold increase in exchangeable sterol pool size and to induction of a very rapidly (t(1/2) = 4.1 +/- 0.6 min) exchangeable sterol pool. Confocal fluorescence imaging and indirect immunocytochemistry colocalized significant amounts of SCP-2 with the mitochondrial marker enzyme cytochrome oxidase in transfected L-cells overexpressing SCP-2. In summary, SCP-2 and pro SCP-2 both stimulated molecular sterol transfer from lysosomal to mitochondrial membranes, suggesting a potential mechanism for replenishing mitochondrial cholesterol pools depleted by cholesterol oxidation. PMID- 10727112 TI - Monomolecular organization of the main tetraether lipid from Thermoplasma acidophilum at the water-air interface. AB - The monomolecular organization of the main tetraether phospholipid from the archaeon Thermoplasma acidophilum was studied by means of a Langmuir film balance integrated into a fluorescence microscope. After transfer to solid surfaces at different pressures the films were further investigated by ellipsometry, small angle X-ray scattering and atomic force microscopy. In order to complete former results about the main tetraether phospholipid of T. acidophilum [Strobl, C., Six, L., Heckmann, K., Henkel, B., Ring, K., 1985. Z. Naturforsch. 40c, 219-222], the thickness and the two-dimensional organization of the monomolecular films were investigated. Two mean heights values were determined, one of 1.5-1.8 nm and another one of 4-5 nm, indicative for two different molecular arrangements. The former one is interpreted as a 'horseshoe' organization with two polar endings in the aqueous subphase, whereas the latter appears to represent the upright population of molecules with one polar end in the subphase and the other one in the air. In freshly spread and compressed films small domains of the upright lipid population are initially observed, which enlarge with increasing pressure. These domains are no longer existent after 12 h of spreading without compression. PMID- 10727113 TI - Derivatised lipids in membranes. Physico-chemical aspects of N-biotinyl phosphatidylethanolamines, N-acyl phosphatidylethanolamines and N-acyl ethanolamines. AB - The physical properties of N-biotinyl phosphatidylethanolamines, N-acyl phosphatidylethanolamines and of N-acyl ethanolamines, in aqueous dispersions, are reviewed. Emphasis is placed on the calorimetric (i.e. chain melting) properties, the thermotropic phase behaviour, certain aspects of the structure and dynamics, and the miscibility with other membrane lipids. In the case of N biotinyl phosphatidylethanolamines, the specific binding of avidin, and in the case of N-acyl ethanolamines, the function of the third chain, is also considered. All of these properties are relevant to the role of these rather unusual lipids in membranes. PMID- 10727114 TI - Formation and properties of Langmuir and Gibbs monolayers: a comparative study using hydrogenated and partially fluorinated amphiphilic derivatives of mannitol. AB - The synthesis and surface behavior of a series of nine new hydrogenated nonionic surfactants and their fluorinated analogs, derived from D-mannitol are described. Adsorption monolayers (Gibbs monolayers) were studied by surface pressure (H) measurements as a function of time. For the spread monolayers (Langmuir monolayers), the measurements of surface pressure versus molecular area (A) were performed. For the most hydrophobic amphiphiles at low concentrations, the adsorption at the air/water interface from the bulk solution required extremely long times to attain equilibrium. The A values for two compounds which could be studied in both adsorbed and spread monolayers provided data allowing a direct comparison of the properties of the two types of films formed at the air/water interface. In spite of different mechanisms of formation of Langmuir and Gibbs monolayers, their characteristic parameters were identical, proving the equivalence of these two types of structures. PMID- 10727115 TI - Mechanisms for ion formation during the electron impact-mass spectrometry of picolinyl ester and 4,4-dimethyloxazoline derivatives of fatty acids. AB - Mass spectral studies have been conducted with isotopically stable labelled and fluorinated picolinyl esters and 4,4-dimethyloxazoline (DMOX) derivatives of fatty acids in order to establish mechanisms of ion formation. Reciprocal hydrogen transfer is shown to be involved in the formation of the ion at m/z 126 with dimethyloxazoline derivatives and for the ion at m/z 164 with picolinyl esters. Inclusion of a fluorine atom alpha to the carboxyl of a fatty acid has been demonstrated to enhance rearrangements for expulsion of internal chain fragments with both methyl ester and dimethyloxazoline derivatives. When two fluorine atoms are inserted into the alpha position a similar rearrangement has been shown to occur with picolinyl esters, although not nearly to the same extent as that observed with either of the other derivatives. Mechanisms for such rearrangements are proposed and discussed. With fatty acid dimethyloxazoline derivatives the M-15 ion arises solely from the loss of a methyl radical from the ring and the M-43 ion has at least three different mechanisms of formation. Such rearrangements make it difficult to establish the identity of the terminal moiety of the alkyl chain. In mass spectrometry terms the picolinyl ester would seem to be the superior derivative for structural characterisation of fatty acids. PMID- 10727116 TI - Synthesis of structured lipids and etherlipids with antioxidants: combination of a selena fatty acid and a selena fatty alcohol with a carotenoic acid in glyceride molecules. AB - Selenium and carotenoids show similar and complementary properties and protect against a variety of pathological processes. Mixtures of both compounds are found in nutritional supplements and are used to prevent several diseases. The synthetic connection of carotenoids with selenium in glycerols may increase the chemopreventive activity of the individual compounds. Beta-apo-8'-carotenoic acid and 7-selenacapryloic acid were esterified with glycerol to highly unsaturated stable di- and triglycerides. Intramolecular selenium:carotenoid ratios of 1:1, 2:1 and 1:2 were obtained for 1-(7-selenaoctanoyl)-3-(3beta-apo-8 -carotenoyl) glycerol, 1,3-di-(beta-apo-8'-carotenoyl)-2-(7-selenaoctanoyl)-glycero l and 1,2 di-(7-selenaoctanoyl)-3-(beta-apo-8'-carotenoyl)-glycero l, respectively. The carotenoic acid was likewise connected to the pharmacologically interesting 11 selenalaurylglycerolether forming an alkyl-acylglyceride: 1-(11-selenadodecyl)-3 (beta-apo-8'-carotenoyl)-glycerol. PMID- 10727117 TI - Statistical optimization of a reversed-phase liquid chromatographic method for the analysis of amiloride and hydrochlorothiazide in tablets. AB - A method has been developed for the separation of hydrochlorothiazide and amiloride by high-performance liquid chromatographic (HPLC) method on a C18 column with detection at 280 nm. The optimal conditions of separation were determined with the aid of 'window diagram' technique of Laub and Purnell. The effect of simultaneously varying the pH, proportion aqueous acetic acid and methanol in the mobile phase were studied to optimize the separation. A response surface diagram was used to optimize the experimental conditions for the separation. The mobile phase composition that provides an acceptable resolution hydrochlorothiazide and amiloride in a short elution time is water:methanol (60:40) and pH 3.2 (pH adjusted to 3.2 with CH3COOH). A method is applied for the quantitative analysis of Moduretic tablets (Merck Sharp & Dokme International). The powdered tablets are extracted with methanol, containing caffeine as the internal standard, and assayed by comparison of peak areas after liquid chromatography. PMID- 10727118 TI - Indirect atomic absorption spectrometric determination of pindolol, propranolol and levamisole hydrochlorides based on formation of ion associates with manganese thiocyanate and potassium ferricyanide. AB - A new simple, accurate, precise and sensitive indirect method for the determination of pindolol HCl (1), propranolol HCl (2) and levamisole HCl (3) using atomic absorption spectrometry has been developed. The method is based on precipitation of the ion associates formed from the reaction of (1), (2) or (3) with manganese thiocyanate and/or potassium ferricyanide. The solubility of the solid complexes at the optimum conditions of pH and ionic strength values have been studied. Saturated solutions of each ion-associate were prepared under the optimum conditions and the metal ion content in the supernatant was determined. The method has been used for the determination of 1.14-17.07, 1.18-17.75 and 1.08 16.24 microg/ml of (1), (2) and (3), respectively using manganese thiocyanate and 1.71-25.60, 1.77-26.62 and 1.62-24.36 microg/ml of (1), (2) and (3), respectively using potassium ferricyanide. The method developed applied for analysis of bulk drugs and some of their pharmaceutical preparations. PMID- 10727119 TI - Spectrophotometric determination of loperamide hydrochloride by acid-dye and charge-transfer complexation methods in the presence of its degradation products. AB - Two simple, sensitive and accurate spectrophotometric methods for the determination of loperamide hydrochloride (lop. HCl) are described. The first method is based on the formation of ion-pair association complex (1:1) with bromothymol blue (BTB), bromophenol blue (BPB) and naphthol blue black B (NBB). The coloured products are extracted into chloroform, and measured spectrophotometrically at 414 (BTB), 415 (BPB) and 627 nm (NBB). Beer's law was obeyed in the ranges of 5-35, 5-30 and 0.8-11.2 microg ml(-1) for BTB, BPB and NBB methods, respectively. The method was found to be specific for the analysis of the drug in presence of its degradation products which can be detected by HPLC procedure. The second method is based on the reaction of the basic loperamide with iodine in chloroform to give molecular charge-transfer complex with intense bands at 295 and 363 nm. Beer's law was obeyed in the ranges 2.5-17.5 and 2.5 22.5 microg ml(-1) for the method at 295 and 363 nm, respectively. Optimization of the different experimental conditions are described for both methods. The proposed methods have been applied successfully for the analysis of the drug in pure form and in its dosage forms. The methods have the advantage of being highly sensitive and simple for the determination of a small dose drug, which is also a weak UV-absorbing compound. PMID- 10727120 TI - Simultaneous interaction of steroidal drugs with gamma- and hydroxypropyl-beta cyclodextrin studied by charge-transfer chromatography. AB - The simultaneous interaction of 15 steroidal drugs with tau-cyclodextrin (tauCD) and hydroxypropyl-beta-CD (HPbetaCD) was determined by charge transfer chromatography and the relative strength of interaction was calculated for each drug-tauCD-HPbetaCD ternary complex. The mixture of CDs interacted with each steroidal drugs decreasing the lipophilicity of the guest molecules. The chemical structure of steroidal drugs markedly influenced their capacity to interact with the mixture of CDs, the more lipophilic compounds formed stronger complexes with CDs. In the overwhelming majority of cases the stability of drug-tauCD-HPbetaCD system was higher than those of binary (drug-tauCD and drug-HPbetaCD) system indicating the probability of ternary complex formation. The data indicated that the ternary complex formation has to be taken into consideration in pharmaceutical formulations containing more than one type of CD or CD derivatives. PMID- 10727122 TI - Spectrophotometric methods for the determination of lansoprazole and pantoprazole sodium sesquihydrate. AB - Spectrophotometric procedures for determination of two irreversible proton pump inhibitors, lansoprazole (I) and pantoprazole sodium sesquihydrate (II) are presented. Two methods were based on charge transfer complexation reaction of these drugs, where they act as n-donors, with either pi acceptor 2,3-dichloro-5,6 dicyano-1,4-benzoquinone (DDQ) and with sigma acceptor as iodine. A third method was also investigated depending on ternary complex formation with eosin and copper (II). The colored products were quantified spectrophotometrically using absorption bands at 457 nm for DDQ (method A) at 293 and 359 nm for iodine (method B) and at 549 nm using ternary complex formation (method C), for both drugs. The molar combining ratio and the optimum assay conditions were studied. These methods determined the lansoprazole in concentration ranges from 10 to 90, 1.48 to 6.65 and 3.69 to 16.61 microg ml(-1) with mean percentage recovery 99.63% for DDQ, 99.71%, 99.18% for iodine and 99.76% for ternary complex and with relative standard deviation 0.11, 0.24, 0.13 and 0.36%, respectively. For pantoprazole, the concentration ranges were 10-60, 17.7-141.6 and 4.3-25.9 microg ml(-1) with mean percentage recovery 99.51, 98.97, 99.84 and 99.46% and relative standard deviation 0.53, 1.21, 0.65, 0.81% for the three mentioned methods, respectively. Investigation of the formed complexes was made with respect to its composition, molar ratio of the reaction, association constant K(C)AD, molar absorptivity epsilon(lambda)AD and free energy change deltaG for methods (A) and (B). The proposed methods have been applied successfully to the analysis of the cited drugs either in pure form or in pharmaceutical formulations, with good accuracy and precision, compared statistically with those given by the reported methods. They are recommended for quality control and routine analysis. PMID- 10727121 TI - A subnanogram API LC/MS/MS quantitation method for depsipeptide FR901228 and its preclinical pharmacokinetics. AB - A highly sensitive and specific atmospheric pressure ionization (API) liquid chromatographic-tandem mass spectrometric (LC/MS/MS) method for the quantitation of depsipeptide FR901228 (NSC-630176, FR), a naturally occurring antitumor agent, was developed and validated. FR was extracted from human or rat plasma along with the internal standard, t-Boc-Met-Leu-Phe (BMLP) with ethyl acetate. Components in the extract were separated on a 5-microm C8 Spherisorb 50 x 4.6 mm i.d. column by isocratic elution with methanol/acetonitrile/12 mM ammonium acetate (60:10:30, v/v/v). The liquid flow was passed through a presource splitter and 5% of the eluate was introduced into the API source. The components were analyzed in the multiple-reaction monitoring (MRM) mode to enhance specificity. Linear calibration curves were obtained in the range of 0.1-100.0 ng/ml with 0.5 ml human plasma and 0.5-100.0 ng/ml with 0.1 ml rat plasma. The limit of quantitation (LOQ) was 0.1 ng/ml using 0.5 ml human plasma and 0.5 ng/ml using 0.1 ml rat plasma. The overall within-day precision was below 12% in human plasma and below 7% in rat plasma; and the between-day precision was below 10.2% in human plasma and 7.2% in rat plasma. The accuracy at low, medium and high levels ranged from 99.3 to 111.7% in human plasma and 96.2-107.3% in rat plasma. The high sensitivity permitted pharmacokinetic study of FR in the rat at a single i.v. dose as low as 1 mg/kg. At this dose, plasma FR levels declined biexponentially with a mean terminal t(1/2) of 187.7 min (n = 6) and were detectable up to 24 h. After an oral dose at 5 mg/kg, plasma FR levels were highly erratic and yielded a mean bioavailability of 1.6% (n = 6). At a higher oral dose of 50 mg/kg, a mean bioavailability of 10.6% was obtained, both being estimated by a non-crossover method. PMID- 10727123 TI - Fast determination of paracetamol by using a very simple photometric flow-through sensing device. AB - A simple flow-through UV optosensing device was developed for the determination of paracetamol based on its transient retention and concentration on a suitable active solid support (Sephadex QAE A-25 anion-exchange resin) packed in the flow cell and the continuous monitoring of its native absorbance on the solid phase at 264 nm. The sample was injected into a 0.08-M NaCl carrier stream at pH 11.0 by using a simple monochannel FIA manifold. After developing the analytical signal, paracetamol was desorbed from the solid support by the carrier solution itself. A very good linear response was found in the concentration range 0.5-8.0 microg ml( 1) with a RSD (%) of 1.24, a detection limit of 0.022 microg ml(-1) and a sampling rate of 40 h(-1). A strong increase in sensitivity as well as a very much higher selectivity were achieved as compared with the conventional flow injection method as a consequence of the separation of the analyte from the sample plug and its retention on the active solid support placed in the detection area. Applicability of the proposed sensor to direct determination of paracetamol in pharmaceuticals (to solve the sample being the only treatment) was successfully demonstrated. PMID- 10727124 TI - Electrochemical studies of the interaction of adriamycin to DNA. AB - An electrochemical investigation of the interaction of adriamycin (DXH) with DNA on a Hg electrode is reported. In weakly acidic media of pH 4.0-7.0, the addition of DNA to DXH solution results in the decrease of redox peak currents. In the presence of DNA, no new peak appears and the standard rate constant k(s) is not significantly changed. The binding of DXH to DNA by electrostatic attraction and intercalation forms a kind of supramolecular complex DXH-DNA, which is electrochemically non-active. The equilibrium constant for the complex is calculated. The decrease in peak current is proportional to DNA concentration and can be used to determine DNA concentration. PMID- 10727125 TI - Comparison of the composition of some petroleum samples which may be applied for skin and mucous membrane treatment. AB - A particular Croatian petroleum (sample P1) and its three derivatives (samples P2, P3 and P4), potentially applied as healing preparations for skin and mucous membrane treatment, were studied in order to learn their composition and to discriminate them according to two criteria: composition of natural petroleum compounds, and lacking aromatics. Elemental (C, H, N and S) and group composition (by LC, UV/VIS, IR and 1H NMR) were determined and the single component distributions were analyzed (by GC) and identified (by GC-MS). Focussed saturated compounds (n-alkanes, pristane and phytane, drimanes/eudesmanes, steranes and hopanes) were studied in order to emphasize the preservation or destruction of genuine petroleum structures in derivatives. Samples P2 (petroleum-brownish color, petroleum like smell) and P3 (colorless, transparent, slight pine-like odor), were found, now constituting petroleum, to still be composed of the components of their native structure. Compared to sample P1, they were missing light and heavy compounds. While sample P2 contained different compound classes, sample P3 comprised exclusively saturated hydrocarbons, satisfying pharmacopoeia's requirement regarding the low aromatics content. Almost a half of sample P3 was composed of cyclic moieties, including terpenoids, possibly responsible for the odor. Samples P1, P2 and P3 were found rather rich in steranes. Sample P4 (colorless, transparent, no smell) was found denaturalized. In spite of high similarity in bulk properties to sample P3, it comprised no detectable amount of n-alkanes, pristane and phytane, or drimanes/eudesmanes, steranes and hopanes (although found rich in oligocycles). PMID- 10727126 TI - Chemiluminescence determination of thioridazine hydrochloride by flow-injection analysis. AB - Hydrogen peroxide, potassium permanganate, potassium dichromate, potassium hexacyanoferrate(III), cerium(IV) sulphate and sodium peroxidisulphate have been tested as potential reagents for chemiluminescence generation from the oxidation of phenothiazine derivatives. Only with potassium permanganate in acidic medium were satisfying results achieved. A total of 13 different phenothiazine derivatives produce luminescence of different intensities on oxidation. Thioridazine hydrochloride was chosen to develop a rapid and simple method for its determination in pharmaceutical formulations. The limit of detection is 1.2 x 10(-6) mol l(-1), and 110 samples per hour can be determined. PMID- 10727127 TI - LC procedure with SPE for quantification of indobufen enantiomers: pharmacokinetic studies. AB - A rapid and selective liquid chromatography (LC) with solid phase extraction (SPE) to quantify indobufen (INDB) enantiomers is described. The INDB enantiomers and internal standard (racemic flurbiprofen) are extracted from a small volume of acidified serum (0.2 ml) by means of SPE using cartridges with octadecyl chemically bound phase and analysed on reversed phase (RP), C18 column with ultraviolet detection at 275 nm. Recovery of both INDB enantiomers was in the range 92.1-94.3%. The mobile phase is composed of acetonitrile-potassium dihydrogen phosphate (pH 4.75; 10 mM) (38:62, v/v). The linear range of the standards curves was from 0.25 to 25.00 microg ml(-1) in serum for both enantiomers. The limit of quantification and detection for both INDB enantiomers in serum were 0.25 microg ml(-1) (CV < or = 10%), and 0.1 microg ml(-1), respectively. Both intra- and inter-day accuracy and precision of the calibration curves were determined and their CV values did not exceed 10%. The validated method has been successfully applied for chiral pharmacokinetics studies of INDB from tablets and intramuscular injections in man. PMID- 10727128 TI - Simultaneous determination of tiopronin and its metabolites in rat blood by LC ESI-MS-MS using methyl acrylate for stabilization of thiol group. AB - A sensitive and reproducible HPLC-electrospray tandem mass spectrometric method has been developed for the analysis of tiopronin (TP) and its metabolites, 2 mercaptopropionic acid (2-mpa) and S-methylated TP (SA13), in rat blood using methyl acrylate (MA) for the stabilization of a thiol group. The thiol groups of TP and 2-mpa in rat blood were immediately derivatized by the addition of MA acetonitrile solution in 0.1 M Tris HCl (pH 9.1). The purification of the derivatives was accomplished by a simple liquid-liquid extraction procedure involving protein precipitation step. The analysis was performed on a Zorbax SB C18 analytical column by a gradient elution with methanol-0.05 M acetic acid (15:85 and 7:3, v/v). Negative ion electrospray ionization with selected reaction monitoring was employed for the detection of analytes. Linearity of calibration was observed over the range of 0.5-1000 ng/ml for TP and 2-mpa, and 2-1000 ng/ml for SA13. The intra- and inter-assay variability for all analytes at the limit of quantitation (LOQ) level ranged from 5.47 to 16.75% and 4.95 to 7.23%, respectively. The LOQs estimated for TP, 2-mpa and SA13 were 0.5, 0.5 and 2 ng/ml, respectively. This assay method was successively applied to a pharmacokinetics study after an oral administration of TP (10 mg/kg) to rats. PMID- 10727129 TI - LC assay method for oxfendazole and oxyclozanide in pharmaceutical preparation. AB - A method has been developed for the simultaneous determination of oxfendazole and oxyclozanide in a pharmaceutical preparation. The method involves reversed phase chromatography with isocratic elution of the mobile phase and detection at 300 nm. The range of quantification for oxfendazole and oxyclozanide was found to be 2-7 microg ml(-1) and 3-10 microg ml(-1), respectively. The validity of the method was evaluated in terms of linear regression analysis, precision, specificity and accuracy. PMID- 10727130 TI - Analysis of potassium counter ion and inorganic cation impurities in pharmaceutical drug substance by capillary electrophoresis with conductivity detection. AB - Capillary electrophoresis with conductivity detection is a selective and quantitative method for the analysis of counter cations, such as potassium, in pharmaceutical drug substances. It is also a sensitive and specific technique for screening and measuring inorganic impurities in drug substance samples. Both counter ion and inorganic impurities can be measured in the same test. PMID- 10727132 TI - Semi-automated 96-well liquid-liquid extraction for quantitation of drugs in biological fluids. AB - A semi-automated liquid-liquid extraction (LLE) technique for biological fluid sample preparation was introduced for the quantitation of four drugs in rat plasma. All liquid transferring during the sample preparation was automated using a Tomtec Quadra 96 Model 320 liquid handling robot, which processed up to 96 samples in parallel. The samples were either in 96-deep-well plate or tube-rack format. One plate of samples can be prepared in approximately 1.5 h, and the 96 well plate is directly compatible with the autosampler of an LC/MS system. Selection of organic solvents and recoveries are discussed. Also, precision, relative error, linearity and quantitation of the semi automated LLE method are estimated for four example drugs using LC/MS/MS with a multiple reaction monitoring (MRM) approach. The applicability of this method and future directions are evaluated. PMID- 10727131 TI - A sensitive LC-MS/MS method for the quantification of fluticasone propionate in human plasma. AB - A sensitive and selective LC-(APCI) MS/MS method capable of quantifying fluticasone propionate (FP) at levels down to 10 pg ml(-1) in human plasma is reported. The method was validated over a linear range from 10 to 1000 pg ml(-1) using a previously published solid-phase extraction procedure with a 13C3-labeled internal standard. The inter and intra batch precision (coefficient of variation) and accuracy (% bias) of the quality controls samples (20, 25, 50, 100, 200, 500 and 1000 pg ml(-1)) were less than 15 and 11%, respectively. The method is robust, rapid (analysis time of 2 min), selective and hence is ideally suited for pharmacokinetic investigations involving inhalation of therapeutic doses of FP. PMID- 10727133 TI - Identification and quantitation assays for intact tablets of two related pharmaceutical preparations by reflectance near-infrared spectroscopy: validation of the procedure. AB - Quantitative analysis based on near-infrared (NIR) spectroscopy uses individual calibration equations for each sample studied because of the need to model all possible chemical and physical variability sources. A NIR method, using a fibre optical probe, for the analysis of two different and related pharmaceuticals from two different production steps (cores and tablets) is studied. Both pharmaceuticals have the same active principle and similar excipients, but with different nominal concentration values. The pharmaceuticals are identified by comparison using a second-derivative spectral library; an identification procedure consisting of two-steps (cascade) library: correlation coefficient followed by maximum distance in wavelength space is proposed. Once a sample has been positively identified, the active principle is quantified with partial least squares regression (PLSR) using a sole and global calibration. The proposed method was validated for use as a control method, and for this purpose selectivity of the identification process, and the repeatability, intermediate precision, accuracy, linearity, and robustness of the active principle quantitation, were assessed. PMID- 10727134 TI - Re-evaluation of anion-exchange HPLC for the analysis of acidic compounds. AB - A number of weak and strong anion-exchange HPLC phases have been evaluated for the analysis of acidic drugs and related compounds. Using eluents with high organic content, reasonable chromatography could be obtained and widely differing compounds easily separated in isocratic mode. Retention was related to solute pKa and concentration of ammonium acetate in the eluent. All the phases showed an unexplained loss in retention with repeated use, although with one material; nucleosil SAX, this was low (15%) and considered acceptable. This material was also shown to be relatively stable under typical bioanalytical conditions. PMID- 10727135 TI - A RIA combined with SPE for the determination of a dual D2-receptor and beta2 adrenoceptor agonist, AR-C68397XX, in human plasma. AB - A radioimmunoassay has been developed for the determination of AR-C68397XX, a dual D2-receptor and beta2-adrenoceptor agonist, in human plasma. The method incorporates solid phase sample extraction and is suitable for the determination of the analyte at pg ml(-1) concentrations. The antiserum was raised in Suffolk cross sheep following primary and booster immunisations with an immunogen prepared by conjugating a carboxyphenylmethyl derivative of AR-C68397XX, to bovine serum albumin. The radioligand was prepared by the 125I-labelled iodination of a derivative of AR-C68397XX. The solid phase extraction procedure, using octadecyl sorbent, was introduced to remove matrix interferences in the plasma and to enhance method sensitivity. The calibration range is 20-500 pg ml( 1), using 0.5 ml of undiluted human plasma sample. PMID- 10727136 TI - Structure elucidation of methylidene malonate 2.1.2 cyclic trimers with mass spectrometry, liquid chromatography and nuclear magnetic resonance investigations. AB - The anionic polymerization of methylidene malonate 2.1.2. is able to yield four different molecule types. The major product obtained at pH 5.5 corresponds to a trisubstituted cyclohexanone formed by a back-biting reaction. A mixture of four diastereoisomer couples due to the presence of three asymmetric carbon atoms was produced in the same proportion. These results were deduced from mass spectrometry (MS), liquid chromatography MS (LC-MS) and 1H nuclear magnetic resonance (NMR) investigations. PMID- 10727137 TI - Determination of nimesulide in pharmaceutical dosage forms by second order derivative UV spectrophotometry. AB - In this study, nimesulide which has been used as an analgesic, antipyretic and anti-inflammatory agent, was analyzed by using second order derivative UV spectrophotometry. The solvent, the degree of derivation, ranges of wavelength and n-value were chosen in order to optimize the conditions. The concentration of nimesulide in its solutions in ethanol and chloroform were determined between the wavelength ranges of 200 and 500 nm (n = 6, delta lambda = 21) and in the linearity ranges of 2.0-90.0 microg ml(-1) in ethanol and 2.0-50.0 microg ml(-1) in chloroform by using the values obtained from the second derivative UV spectrum of the substance. The developed second derivative UV spectrophotometric method was applied to the pharmaceutical preparations such as tablet, sachet (granule) and suspension. Tablet and sachet were analysed in ethanol while the suspension was analysed in chloroform. The results obtained from derivative UV spectrophotometry were compared with those obtained by using HPLC. It was found that the difference was not statistically important between these methods. It was concluded that developed derivative UV spectrophotometric method was accurate, sensitive, precise, reproducible and could be applied directly and easily to the pharmaceutical preparations. PMID- 10727138 TI - The potential replacement of HPLC by 125I-RIA for the characterization of cyclosporin A: a bioavailability study after oral administration in healthy human subjects. AB - A significant overestimation of cyclosporin A (CsA) by a radioimmunoassay using 125I-labeled monoclonal antibody (125I-RIA), compared to the reference HPLC method, has been reported for a limited number of samples from transplant patients. However, the extent of the discrepancy, with respect to bioavailability parameters, has not been examined for the case of the oral administration of a single dose CsA to healthy subjects where a number of factors which might be involved in this overestimation (e.g. under steady state condition and a significant accumulation of CsA metabolites) would be absent. Therefore, the objective of this study was to assess the effect of potential difference manifested by the two analytical procedures, 125I-RIA and HPLC, on the bioavailability analysis of CsA. An oral CsA formulation was administered to 22 healthy male subjects and the blood samples were analysed by both 125I-RIA and HPLC. Significant discrepancies in the estimated CsA concentrations by the two methods (paired t-test, P < 0.001) were found. The difference (bias) increased with increasing concentrations of blood CsA (P < 0.001). However, despite the bias in CsA estimations, the AUC and Cmax, obtained by 125I-RIA and HPLC methods showed only small differences (i.e. 2% for AUC and 7% for Cmax). Thus, our results suggest that the bias of the 125I-RIA vis-a-vis the HPLC method in the estimation of CsA blood levels may not, in practice, affect the bioavailability analysis (e.g. bioequivalence study) of CsA in a situation where a single dose CsA is orally administered to healthy subjects. PMID- 10727139 TI - Spectrophotometry assay of imipramine and desipramine using ammonium metavanadate and its application to pharmaceutical preparations. AB - Simple and sensitive method for determination of imipramine and desipramine is reported. The procedure is based on the oxidation of the drugs by ammonium metavanadate. Linear calibration graphs were obtained in the concentration range 0.6-40 microg ml(-1) of imipramine and 0.7-35 microg ml(-1) of desipramine with a relative standard deviation (RSD) less than 0.5%. The method was applied to the determination of the drugs in pharmaceutical preparations and compared favourably with independent official methods. PMID- 10727140 TI - Method development for the determination of the immunosuppressive drug mycophenolic acid in serum with MECC. PMID- 10727141 TI - Libration motion of guest spin probe molecules in organic glasses: CW EPR and electron spin echo study. AB - Echo-detected (ED) EPR spectra of nitroxide spin probes dissolved in glassy materials provide evidence that guest molecules in these media undergo fast librational motion. Theory of spin relaxation of a librating molecule is presented. The mean squared amplitude, , of this motion which can be derived from continuous wave (CW) EPR spectral splitting is found to depend linearly on temperature in the low temperature region. This may be ascribed to thermal harmonic vibrations. The slope of the linear dependence varies from glass to glass and seems to correlate with the strength of the intermolecular bonds and with a degree of the fragility of the glass. Above the glass transition temperature increases sharply. Different applications are discussed: study of molecular properties of glass, intracellular glass formation in plant tissues, structural investigations. PMID- 10727142 TI - Utilization of characteristics of fast linear prediction based on the lattice algorithm in an analysis of time domain magnetic resonance. AB - We examined details of the fast linear prediction (FLP) analysis of time domain data. The FLP method is introduced by Gesmar and Hansen [J. Magn. Reson., Ser. A 106 (1994) 236] to improve computational efficiency of the LP analysis. We focused on two characteristic features of FLP inherited from the lattice algorithm. The first is bi-directional prediction. One can obtain both forward and backward prediction models by single execution of FLP. It is found that distances between the forward and backward prediction roots in a complex plane can be used to determine a number of resonance lines. We showed that the method utilizing the distance is as effective as that using the singular value of the singular value decomposition (SVD) analysis. Secondly, the FLP method gives prediction models for all of smaller prediction orders than some given value. This character enables one to examine a prediction order dependence of spectral parameters estimated by the analysis. We found that there were significant differences in the order dependence of the estimated frequencies between true and false resonance signals. PMID- 10727143 TI - Time resolved electron spin resonance of spin correlated micelle confined radical pairs: shape of the anti-phase structure. AB - A new approach to the theory of the EPR of spin correlated radical pairs (SCRP) is formulated. It is based on spectral exchange methods as applied to the two site model and takes into account explicitly both the motion of one quantum (transverse magnetization) and two quantum coherences (polarizations) of SCRPs. This innovation allows for the interpretation of anti phase structure (APS) spectral shape asymmetry and for the transformation of the initially created ST0RPM polarization into the APS spectral pattern. The results of this method as applied to micellized spin correlated radical pairs have been compared with the exact numerical solution of the corresponding stochastic Liouville equation and is commented on. PMID- 10727144 TI - Simulation of EPR and time resolved EPR lineshapes in partially ordered glasses. AB - Simulation of magnetic resonance spectra of probes in partially ordered glasses requires in principle a numerical integration on the full set of three Euler angles omega=(alpha beta gamma) from a laboratory fixed to a molecule fixed reference frame. It is shown that it is possible to manage efficiently this problem by using the algebraic properties of the Wigner matrix elements. This analysis is applied to time resolved EPR (TREPR) spectra of a series of bis adducts of C60 in the ordered glass of a nematic liquid crystal solvent. A paramagnetic triplet state is created by light excitation and TREPR spectra are obtained with the external magnetic field set parallel or perpendicular to the director n of the mesophase. The preferred orientation in the mesophase of the triplet state zero field tensor is determined. PMID- 10727145 TI - Frequency dependent study of the correlation functions in EPR spectroscopy--the Cole-Davidson approach 1. Perdeuterated 2,2,6,6-tetramethyl-4-piperidone N-oxide in toluene. AB - EPR linewidth measurements of PD-Tempone in toluene at 1 (L-Band), 4 (S-Band), 9 (X-Band) and 34 GHz (Q-Band) microwave frequencies indicate the presence of a distribution of relaxation times. The empirical response parameter introduced by Cole-Davidson for the analysis of dielectric relaxation in liquids has been used for the analysis of EPR relaxation data in the low frequency region. The Cole Davidson parameter can assume values in the range 0 < beta < or = 1. When beta = 1, one obtains the Debye-type spectral density. The calculated linewidth data at 1 GHz agrees with a Cole-Davidson distribution function with a width parameter 0.83 +/- 0.04 for a spherical solute. Beta < 1 at L-band suggests the presence of an asymmetrical distribution of relaxation times associated with different modes of relaxation mechanisms or internal molecular motions. This study shows EPR experiments at low microwave frequencies are more sensitive to the shape of the correlation function. PMID- 10727146 TI - The role of the Mg2+ cation in ATPsynthase studied by electron paramagnetic resonance using VO2+ and Mn2+ paramagnetic probes. AB - The electron paramagnetic resonance (EPR), electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) spectra of Mg2+-depleted chloroplast F1-ATPase substituted with stoichiometric VO2+ are reported. The ESEEM and HYSCORE spectra of the complex are dominated by the hyperfine and quadrupole interactions between the VO2+ paramagnet and two different nitrogen ligands with isotropic hyperfine couplings /A1/ = 4.11 MHz and /A2/ = 6.46 MHz and nuclear quadrupole couplings e2qQ1 approximately 3.89-4.49 MHz and e2qQ2 approximately 1.91-2.20 MHz, respectively. Aminoacid functional groups compatible with these magnetic couplings include a histidine imidazole, the epsilon-NH2 of a lysine residue, and the guanidinium group of an arginine. Consistent with this interpretation, very characteristic correlations are detected in the HYSCORE spectra between the 14N deltaM1 = 2 transitions in the negative quadrant, and also between some of the deltaM1 = 1 transitions in the positive quadrant. The interaction of the substrate and product ADP and ATP nucleotides with the enzyme has been studied in protein complexes where Mg2+ is substituted for Mn2+. Stoichiometric complexes of Mn x ADP and Mn x ATP with the whole enzyme show distinct and specific hyperfine couplings with the 31P atoms of the bonding phosphates in the HYSCORE (ADP, A(31Pbeta) = 5.20 MHz: ATP, A(31Pbeta) = 4.60 MHz and A(31Pgamma) = 5.90 MHz) demonstrating the role of the enzyme active site in positioning the di- or triphosphate chain of the nucleotide for efficient catalysis. When the complexes are formed with the isolated alpha or beta subunits of the enzyme, the HYSCORE spectra are substantially modified, suggesting that in these cases the nucleotide binding site is only partially structured. PMID- 10727147 TI - Spin glass formation in La0.9Sr0.1CoO3 catalyst for flameless combustion of methane. AB - Two samples of composition La0.9M0.1CoO3 (M = Sr, Ce) have been compared as catalysts for the flameless combustion of methane. The former showed a lower activity than the latter and this difference was enhanced at lower temperature. Aiming at understanding the origin of this behaviour, EPR analysis was carried out at temperatures down to 100 K. At T < 245 K a zero-field intense feature appeared with the M = Sr sample only, characterized by opposite phase with respect to the g approximately 2 line. This zero-field line was attributed to microwave absorption by spin glass formed by cobalt- and oxygen-based paramagnetic ions. The tendency to strong interaction among these species could also be a reason of the low oxygen availability for the catalytic methane oxidation at higher temperature. PMID- 10727148 TI - Electron paramagnetic resonance study of V2O5 deactivation in the course of catalytic oxidation of SO2 to SO3. AB - The influence of reagents interactions with V2O5 catalyst on its activity in the reaction of SO2 oxidation has been investigated by electron paramagnetic resonance (EPR) method. It was found that the deactivation of V2O5 is caused by the diminishing of the number of adsorption centers able to interact with reagent molecules and with the creation of elements of the VOSO4 phase. PMID- 10727150 TI - Electron paramagnetic resonance characteristics of some non-heme low-spin iron(III) complexes. AB - We have recorded the powder EPR-spectra of some near octahedral iron(III) complexes with tridentate ligands donors and analysed their spectra with simple ligand field analysis and for some cases with the angular overlap model (AOM). We have determined the electron praramagnetic resonance (EPR) characteristic of bis 1,4,7-triazacyclonane iron(III)chloride at 4 K and found that it was similar to the characteristics of the so-called 'highly anisotropic low spin' complexes. We have recorded the powder spectra of bis (2,6-bis(benzimidazoly-2-yl)pyridine) iron(III) perchlorate and made an AOM-analyses of the structural similar complex bis-(2,6 (N-carbamoyl)-pyridine) iron(III). With a combination of ligand field analyses and AOM, we could determine the pi-donor properties of these ligands. The same approach have been used to determine the pi-donor properties of the hydroperoxo ligand. Finally we have recorded the powder EPR-spectrum of [Fe(CN)6]3- doped in K3[Co(CN)6] and [Co(NH3)6][Co(CN)6] at 4 and 100 K and in water at 4 K. The spectra are interpreted as the effect of a dynamic Jahn-Teller distortion. PMID- 10727149 TI - Magnetic resonance study of Rh complexes in AgCl microcrystals. AB - X-ray or UV irradiation at room temperature of Rh3+ doped AgCl emulsion powders leads to the production of three paramagnetic Rh2+ related centres, labeled R4, R5 and R6. A combined X and Q band electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) study allowed the determination of a nearly complete structural model for these centres. In the X band ENDOR spectra of R4 and R5 interactions of the unpaired electron with nearby protons have been identified, indicating that for these centres Cl- ligands have been exchanged by H2O or OH-. The R6 centre, identified as a (RhCl6)4- complex, has been found to be fundamentally different from the dominant centre in large Rh2+ doped AgCl single crystals grown from the melt. The results are compared with recent work by other researchers in the same field. PMID- 10727151 TI - Cu(II) complexes in bacterial growth medium: electron spin resonance study. AB - In this study we report a spectroscopic investigation on the structure and stability of Cu(II)-complexes that are formed in a minimum growth medium (MM), normally used for Bacillus subtilis cultures. As other transition metals, Cu(II) compounds are toxic to this bacterium and the toxicity depends on the Cu(II) concentration. MM contained NH4+ ions and asparagine (asn) as the source of inorganic and organic nitrogen. Both ESR and electronic spectra demonstrated the very important role played by the amino acid asparagine in the coordinative behaviour of Cu(II). In particular, three different complexes were evidenced: Cu(H2O)6(2+); Cu(asn)+ and Cu(asn)2. The relative amount of these three species strongly depended on pH, on Cu:asn ratio and on the presence of the phosphate ions. They were identified and evaluated quantitatively by extensive simulation of the electron spin resonance (ESR) spectra recorded in different experimental conditions. The bis-complex was found to be more stable in MM than in an asparagine-containing water solution with the same Cu:asn ratio. A comparison of the spectroscopic results with microbiological investigations is also made. PMID- 10727152 TI - Charge-transfer photochemistry of bis(diethyl-diselenocarbamato)copper(II). AB - The photochemical reactions of bis(diethyl-diselenocarbamato)copper(II), Cu(Et2dsc)2, complex have been studied in toluene, CH2Cl2, CHCl3 and chloroalkane/EtOH mixed solvents. Charge-transfer irradiation induces intramolecular oxidation of the ligand and reduction of copper(II) to copper(I) as evidenced by EPR and UV-Vis spectra of the complex as well as quantum yield results. When photolysis is carried out in CHCl3 or CH2Cl2 or in the solvent mixture CHCl3/EtOH resp. CH2Cl2/EtOH of lower than 1:1 EtOH content, the primary photoproduct CuI(Et2dsc) is further oxidised in a dark reaction with the chloroalkane producing the corresponding paramagnetic mixed-ligand CuII(Et2dsc)Cl complex in equilibrium with its chloride-bridged and EPR silent, dimeric form Cu2(Et2dsc)2Cl2. At low concentration of EtOH the equilibrium is shifted to the dimeric form whereas at higher than 1:1 EtOH content in the mixed solvent CHCl3/EtOH it is shifted to CuII(Et2dsc)Cl. A reaction mechanism is proposed and the role of ethanol is discussed. PMID- 10727153 TI - In situ EPR and UV-vis spectroelectrochemistry of hole-transporting organic substrates. AB - A newly developed in situ electron paramagnetic resonance (EPR)/ultraviolet visible (UV-vis) spectroelectrochemical cell equipped with a laminated indium-tin oxide (ITO) working electrode was used in the investigation of various organic substrates which are potential hole-transporting materials. The experiment demonstrated the possibility of using such a technique for examining redox behavior of conducting polymers (polypyrrole, PPy), oligomers (thiophene dimmer and quarterthiophene) and bis-anilines (N,N,N',N'-tetraphenylbenzidine, TPB). All investigated structures formed stable paramagnetic intermediates in the first oxidation step characterised with UV-vis spectra in the region 400-600 nm. In the second oxidation step EPR-silent di-cationic structures are formed with broad vis bands in the region 600-1000 nm. The measurement of the reference UV-vis spectra direct in the EPR cavity was possible using a specially-constructed non-contacted ITO plate in the spectroelectrochemical cell in the case of polypyrrole. PMID- 10727154 TI - Filling factor of a paramagnetic sample in a rectangular cavity: theory and application. AB - A computational method is presented for calculating the filling factor of an electron paramagnetic resonance (EPR) tube in a rectangular TE102 cavity. The algorithm employs the conventional finite element method. In addition to the filling factor, the algorithm allows to calculate the quality factor and the reflection coefficient of the loaded cavity. This method allows calculating very accurately the EPR signal intensities from which the spin concentration of paramagnetic samples can be determined. A comparison between the predicted EPR signal intensities to several experimental results was found to be satisfactory. The method also allows optimizing the EPR tube dimensions and its glass quality to improve measurement sensitivity. PMID- 10727155 TI - Quantitative electron paramagnetic resonance and spectrophotometric determination of the free radical 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy. AB - The first quantitative estimation of the stable free radical 4-hydroxy-2,2,6,6 tetramethylpiperidinyloxy (TEMPOL) simultaneously performed by electron paramagnetic resonance (EPR) and visible spectrophotometric titration is reported. The obtained molar absorptivity of TEMPOL 13.2 +/- 0.1 l(-1) m(-1) cm( 1) at 22,900 cm(-1) may be used as the future criterium about the purity of the EPR active substance. The studied compound may be considered as the second (after 1,1-diphenyl-2-pikrilhydrazil) material calibrated as the primary standard for quantitative EPR spectrometry. PMID- 10727156 TI - Electron paramagnetic resonance (EPR) studies on stable and transient radicals in humic acids from compost, soil, peat and brown coal. AB - Quantitative EPR method was applied to characterise four types of humic acids (HA) derived from composts, soil, peat and soft brown coal. For each sample of HA the level of native (indigenous) radicals was estimated. Interactions of the HA with various gaseous agents and organic solvents were investigated. Strong effects of gaseous ammonia and aliphatic amines on spin concentration enhancement were observed; additionally, higher values of g-value were found to be associated with the formed 'transient' radicals. Correlation of copper(II) ions uptake by different HA with effect diminishing primary spin concentration was established. It was recognised that the radical centres, which are enhanced by 'ammonia effect' are quenched in the formed HA-Cu(II) complexes. The both opposite effects are competitive from each other, where 'copper(II) quenching effect' prevails. Reaction of nitrogen dioxide with the humic acids was also examined. The presence of diketones and/or other compounds with active methylene group results in formation of the iminoxy radicals; these radicals are immobilized in the solid (macromolecular) matrix of the humic acids. PMID- 10727157 TI - Decomposition study of the electron paramagnetic resonance spectrum of irradiated alanine. AB - Recent Electron Paramagnetic Resonance (EPR) studies on alanine powders as a function of irradiation dose and temperature on the one hand and single crystal Electron Nuclear DOuble Resonance (ENDOR) studies on the other hand, showed the presence of at least three radicals contributing to the total alanine EPR spectrum. The latter spectrum obtained after irradiation at room temperature (RT), is dominated by the well-known stable-alanine-radical (SAR) CH3C*HCOO-, also denoted R1. Appropriate heating of irradiated alanine causes the relative contribution of R1 to decrease, resulting in a spectrum mainly caused by the H abstraction radical CH3C*(NH3)COO-, denoted R2. Although the EPR spectrum of these two radicals could be satisfactorily simulated, their influence on dose reconstruction has not been reported yet. Therefore, a detailed Maximum Likelihood Common Factor Analysis (MLCFA) study has been performed on EPR spectra from polycrystalline alanine samples, after irradiation and heat treatments. Conclusions concerning the number of contributing radicals and their influence on the RT irradiated alanine EPR spectrum will be made. PMID- 10727158 TI - Study of relaxation rates of stable paramagnetic centers in gamma-irradiated alanine. AB - The stable L-alanine radical induced by gamma-irradiation was examined by electron paramagnetic resonance (EPR), transfer saturation EPR and electron nuclear double resonance (ENDOR) in the temperature region of fast motion of the methyl group (180-320 K). From the obtained spectral line broadening and spectral intensity the correlation time for the methyl rotation was estimated. The complex processes determining the relaxation rate were examined in the same temperature interval. It was shown that important contributions to the relaxation rate arise from non-secular and pseudo-secular types of contributions. The non-secular contribution involves intramolecular dynamics while the pseudo-secular contribution originates from intermolecular motions. The obtained values for the dynamical parameters have been compared with those obtained by pulse EPR methods and by proton nuclear magnetic resonance (NMR) on undamaged crystals. PMID- 10727159 TI - Identification of irradiation treatment of aromatic herbs, spices and fruits by electron paramagnetic resonance and thermoluminescence. AB - Electron paramagnetic resonance and thermoluminescence signals induced by gamma irradiation in some herbs, spices and fruits were systematically studied in order to detect the treatment. Using European protocols the validity and effectiveness of these two techniques are compared in regard to time of storage after irradiation. PMID- 10727160 TI - Electron paramagnetic resonance for everybody--MICROspec-X--a new class of electron paramagnetic resonance spectrometer. AB - The electron paramagnetic resonance spectroscopy is the only method for detecting free radicals. Free radicals have an increased importance in our daily life. A small transportable EPR spectrometer is presented for the popularisation of the EPR method. The technical construction and some applications are illustrated which show the usability of the spectrometer. PMID- 10727161 TI - Contact allergy, irritancy and 'danger'. AB - Conventional models of the immune response are based on distinguishing self and non-self. However, we consider that the more recently proposed 'danger' model may be an illuminating alternative for studying allergic contact dermatitis. In this model, an antigenic signal on its own would tend to produce tolerance. In contrast, in the presence of a 'danger' signal, which, in the case of allergic contact dermatitis, we suggest is usually cutaneous irritancy, the immune system would become activated, leading first to the induction of sensitization and then subsequently to the elicitation of a contact hypersensitivity response. In most cases, both the antigenic signal and irritant signal will come from the hapten, although, e.g., in an occupational setting, traumiterative dermatitis would be the source of the 'danger' signal. Typically, the irritant signal tends to be more concentration-dependent and thus is the overriding factor in the determination of the effective sensitizing and eliciting concentrations of the hapten. A further prediction of this hypothesis is that successful experiments demonstrating low-dose tolerance with contact allergens may be explained by the loss of the irritant effect at lower dilutions, whilst an antigenic stimulus remains present. PMID- 10727162 TI - Dental gold alloys and contact hypersensitivity. AB - A total of 172 patients referred to the Norwegian National Adverse Reaction Group were patch-tested with a dental series. Of these, 25% showed a positive reaction to gold sodium thiosulfate or potassium dicyanoaurate. There was a statistically significant correlation (p=0.0019) between the presence of dental gold and a positive patch test to gold. There was a statistically significant correlation between ear piercing and a positive patch test to gold (p=0.04). In most cases, we did not find clinical correlates to positive patch tests to gold. 2 patients with objective and subjective oral/perioral and general symptoms are described as case reports. Their symptoms disappeared when gold restorations were removed. We conclude that there is an overrepresentation of gold allergies among those with dental restorations containing gold. PMID- 10727163 TI - Racial differences in acute and cumulative skin irritation responses between Caucasian and Asian populations. AB - Japanese, and Chinese subjects. In this 2nd study, no differences were seen in the acute or cumulative irritation responses between the Japanese and Caucasian subjects. The Chinese subjects showed a heightened response to 10% acetic acid (after 4 h of exposure), but otherwise displayed a similar response profile in the acute irritation test. They showed a slower and less severe response in the cumulative irritation test compared to the Caucasian or Japanese subjects. These divergent results underscore the difficulty in ascribing true population-based differences in skin reactivity based upon studies in limited subject populations. It may be possible to detect differences in individual studies, but repeat testing may fail to confirm a consistent trend. These findings may be indicative of the wide variation in skin responsiveness across human subjects in general. PMID- 10727164 TI - Stability of corticosteroid patch test preparations. AB - This study investigated the stability of tixocortol pivalate, budesonide, and hydrocortisone-17-butyrate (Hc-17-B) when present in a mix with petrolatum and when the corticosteroids were kept separately in petrolatum. The concentrations chosen for the corticosteroids were the same as those used in a study within the European Environmental Contact Dermatitis Research Group (EECDRG), in which 2 corticosteroid mixes (1 with a high concentration and 1 with a low concentration) and the 3 individual constituents, each at 2 concentrations, were patch tested. Ethanolic solutions of each corticosteroid, as well as 2 mixtures of these 3 corticosteroids, were also made up at corresponding concentrations. The preparations were kept at room temperature, refrigerated, and deep frozen, and repeatedly for 1 year, investigations to check stability by high performance liquid chromatography were carried out. A decrease of < or =20% of the initial value at time 0 was used as the threshold for stability. The petrolatum preparations and the ethanolic solutions of budesonide and tixocortol pivalate were stable for at least the whole investigative period, irrespective of storage conditions, while Hc-17-B 1.0% in ethanol kept deep frozen was stable at least during the same period. The latter corticosteroid when kept at room temperature was stable for 3 months only. PMID- 10727165 TI - Direct emission of the allergen primin from intact Primula obconica plants. AB - The allergen primin and its precursor miconidin were collected by dynamic headspace technique from Primula obconica Hance and quantified and identified by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The primin concentrations in leaves/stems and flowers (including pedicel and calyx) of P. obconica were 262 microg and 531 microg (g fresh plant material)(-1), respectively, whereas miconidin concentrations were 92 microg (g fresh plant material)(-1) in leaves/stems and 194 microg (g fresh plant material)(-1) in flowers. The amounts of primin released from unchopped leaves/stems and flowers were 65.3 ng and 18.8 ng (g fresh plant material)(-1) h(-1), respectively. Dynamic headspace analysis of intact P. obconica plants showed that primin is also directly emitted from undamaged plants, although in smaller concentrations (6.2 ng (g fresh plant material)(-1) h(-1)), whereas miconidin was only emitted in minute amounts (less than 0.4 ng (g fresh plant material)(-1) h(-1)) both from intact plants and from unchopped plant parts. The possibility that primin from P. obconica could be a potential airborne allergen and therefore a source of airborne contact dermatitis is discussed. PMID- 10727166 TI - Quantitative structure-activity relationships: sulfonate esters in the local lymph node assay. AB - The biological activity of skin-sensitizing chemicals is related to their ability to react, either directly or after metabolic activation, with appropriate skin proteins. For direct acting electrophilic compounds, this ability can be modelled, using the RAI (relative alkylation index) approach, by a combination of electrophilicity and hydrophobicity parameters. The development of predictive quantitative structure-activity relationships (QSAR) models of skin sensitization, using mechanism-based physicochemical parameters, has been greatly facilitated by the introduction of the murine local lymph node assay (LLNA), which is able to describe the extent of the biological response in objective and quantitative terms. In the present work, sensitization response data in the LLNA is generated for a series of 6 sulfonate esters. An RAI-based hybrid QSAR/dose response relationship is derived using a negative hydrophobicity coefficient in the RAI expression, to model the effect of retention of the hydrophobic test chemicals in the stratum corneum. Dose-response analyses are used to estimate EC3 and EC20 values as quantitative indices of skin sensitization potential for each compound, and regression analysis is applied to develop QSARs correlating these EC3 and EC20 values with an RAI-based parameter. The high statistical quality of these QSARs demonstrates both the consistency of the LLNA method for generating high quality skin sensitization data, and the value of the RAI approach in development of mathematical models for skin sensitization. PMID- 10727167 TI - Foot dermatitis due to non-disperse azo dyes. PMID- 10727168 TI - Occupational contact dermatitis from headphones containing diethylhexyl phthalate. PMID- 10727169 TI - Failure of vehicle to influence local lymph node assay response to benzocaine. PMID- 10727170 TI - Flare-up of skin tests to amoxycillin and ampicillin. PMID- 10727171 TI - Contact dermatitis due to flurbiprofen. PMID- 10727172 TI - Axillary dermatitis from farnesol in a deodorant. PMID- 10727173 TI - Sensitive skin as subclinical expression of contact allergy to nickel sulfate. PMID- 10727174 TI - Occupational allergic contact urticaria from maleic anhydride. PMID- 10727175 TI - Contact leukoderma following nickel dermatitis elicited by TENS electrode plates. PMID- 10727176 TI - Reproducibility of the atopy patch test with whole house dust mite bodies in atopic dermatitis. PMID- 10727177 TI - Statistics on occupational contact dermatitis from (meth)acrylates in dental personnel. PMID- 10727178 TI - Allergic contact dermatitis from sodium metabisulfite in an antihemorrhoidal cream. PMID- 10727179 TI - Coexistence of immediate and delayed-type allergy to natural rubber latex. PMID- 10727180 TI - Concurrent Type I and Type IV natural rubber latex hypersensitivity? PMID- 10727181 TI - Patch test reactions to natural rubber latex: irritant or allergic? PMID- 10727182 TI - Allergic contact dermatitis from acrylamide in a chemical mixer. PMID- 10727183 TI - Concordance and discordance between TRUE Test and Finn Chamber. PMID- 10727184 TI - Routine patch testing with fragrance chemicals in The Netherlands. PMID- 10727186 TI - Motor and perceptual-motor competence in children with Down syndrome: variation in performance with age. AB - The aim of this study was to determine whether perceptual-motor competence in school-age children with Down syndrome was generally delayed or varied as a function of type of action. Twenty-two children with Down syndrome (13 males, 9 females), aged between 4.5 and 14 years were assessed on two standardized tests, the Movement Assessment Battery for Children (Movement ABC), a test of motor competence assessing gross and fine motor coordination, and on the Developmental Test of Visual-Motor Integration, a test focusing on shape copying. In order to obtain a profile of each child's performance on the different items contained in the Movement ABC, the test was used in its extended form. This involves testing any child who failed an item appropriate for his/her level, at progressively lower levels until a base-line measure was obtained. All the children obtained scores below the 5th percentile for their age on both tests. However, superimposed on this delay, we found distinct variation as a function of task. Whereas some aspects of gross motor function showed age development with delayed but regular acquisitions, all the aspects of fine motor skills assessed were more severely impaired and showed little development with age. Accuracy and timing of tasks requiring bimanual coordination were most impaired in our sample while balance and ball skills showed more variability. These results suggest that intervention in the motor domain should be varied according to each child's particular profile of performance. PMID- 10727185 TI - Adenylosuccinase deficiency: possibly underdiagnosed encephalopathy with variable clinical features. AB - Adenylosuccinase deficiency is an autosomal recessive inherited defect of purine synthesis. In enzyme deficient patients, two normally undetectable compounds, succinylaminoimidazole carboxamide riboside and succinyladenosine, accumulate in urine, cerebrospinal fluid and, to a minor extent, in plasma. Analysing 150 highly selected urine specimens from patients with unidentified neurogenerative disorders we discovered the first two German cases of adenylosuccinase deficiency. The deficiency causes moderate to severe mental retardation, often accompanied by epileptic seizures and/or autistic features, and is occasionally associated with growth retardation and muscular hypotonia. Of the two German cases we present here, one patient fits into the clinical picture outlined by previous reports. The other patient, however, shows a pattern of symptoms so far undescribed: severe early infantile epileptic encephalopathy with reduced myelination. On mutation analysis this patient is the first to reveal a 39 base pair deletion in the adenylosuccinase gene in contrast to the point mutations detected in previous cases. Adenylosuccinase deficiency may be an underdiagnosed metabolic disorder with variable expression. This should be taken into consideration in patients with unclassified neurological conditions. PMID- 10727187 TI - Magnetic resonance imaging cranial and cerebral dimensions: is there a relationship with Chiari I malformation? A preliminary report in children. AB - The pathogenesis of idiopathic Chiari I malformation (CM I) remains poorly understood, although it is thought to be due to disproportion between hindbrain structures and the posterior fossa (PF) dimensions. This study was undertaken to determine whether CM I and its possible complications are associated with disproportion between cranial and cerebral dimensions. Surface of the vermis, PF and supratentorial (ST) area were measured on magnetic resonance imaging midsagittal sections and were correlated with age in 19 children with CM I and 43 controls. The ratio of the PF/ST area and of the vermis/PF area were also plotted against age in the two groups and against length of the tonsils herniation in the CM I malformation group. Only the vermis midsagittal area and the vermis/PF ratio were found to be significantly higher in CM I malformation subjects. There was no correlation between the length of tonsils herniation and the vermis/PF area or the PF/ST area ratio in this group. The most severe complications (large syrinx and hydrocephalus) were not associated with particular measurements or calculated ratios in the children with CM I malformation except for the relatively small PF area in a neonate. We conclude that factors responsible for the appearance of clinical signs remain largely unknown. In particular, cranial and brain measurements alone do not seem to predict complications or unfavourable clinical evolution. PMID- 10727188 TI - Periventricular haemorrhagic infarct in a preterm neonate. AB - Magnetic resonance imaging (MRI) was performed on an infant born, at 28 weeks gestational age who suffered a sudden episode of bradycardia and desaturation on the 3rd day of life. Imaging demonstrated bilateral germinal layer haemorrhage and intraventricular haemorrhage, with parenchymal involvement in a fan-shaped pattern in the periventricular white matter on the left. These appearances are consistent with a combination of intravascular thrombi and perivascular haemorrhage along the course of the medullary veins. We believe that this is the first report of the MRI appearance of an acute periventricular haemorrhagic infarct associated with a germinal layer haemorrhage/intraventricular haemorrhage in a preterm neonate. PMID- 10727189 TI - A case of paroxysmal non-kinesigenic dyskinesia associated with lesions of the globus pallidus and substantia nigra. PMID- 10727190 TI - Familial pontocerebellar hypoplasia type I with anterior horn cell disease. AB - We report the association of pontocerebellar hypoplasia and anterior horn cell disease in three female siblings. One child presented with the classical clinical and neuropathological features of pontocerebellar hypoplasia with associated anterior horn cell disease, described by Barth as pontocerebellar hypoplasia type I. This patient showed polyhydramnios, congenital contractures, respiratory insufficiency, hypotonia, areflexia, listlessness and myoclonic seizures. Postmortem examination revealed a loss of neurons and reactive gliosis in the pontocerebellum and in addition anterior horn cell atrophy resembling Werdnig Hoffmann disease. Another sibling demonstrated the same clinical symptoms. However neuropathological findings showed evidence for pontocerebellar hypoplasia only. The third sibling was examined after induced fetal abortion because of prenatally diagnosed arthrogryposis. Anterior horn cell disease was obvious histologically whereas pontocerebellar hypoplasia could not be demonstrated due to cerebral autolysis. The similar clinical and neuropathological findings in the three reported siblings suggest a common genetic defect with different patterns of pontocerebellar hypoplasia and associated anterior horn cell disease. The gene defect of this rare disorder is still unknown. The 'survival motor neuron' gene of spinal muscular atrophy was not found in these three siblings. PMID- 10727191 TI - Adult and paediatric movement disorders. PMID- 10727192 TI - Neuronal ceroid lipofuscinoses. PMID- 10727193 TI - What's in a name? Muscular dystrophy revisited. PMID- 10727194 TI - 'Anonymous'--an organization for families of children with undiagnosed brain diseases. PMID- 10727195 TI - Depressive disorder in children and adolescents. AB - Depression is a relatively common health issue in children and adolescents. Different pathogenetic factors are implied: genetic, biological, psychological and environmental. The core symptoms of depression are the same for children, adolescents and adults but the prominence of characteristic symptoms changes with age. The clinical picture of depression according to age level is described in different types of mood disorders (major depressive disorder, dysthymia, bipolar disorder) and in mental retardation. Over half of the youths with depression have comorbid conditions: anxiety disorders, other mood disorders, attention deficit/hyperactivity disorder, and conduct disorders. Different factors affect the natural course and risk of suicide. Assessment procedures of depression and comorbid conditions include a psychiatric evaluation of the depressed subject and his family, structured interviews and specific rating scales. A comprehensive treatment strategy, psychoeducational, psychotherapeutic and psychopharmacological, is proposed. PMID- 10727196 TI - Nitric oxide in developing brain. AB - The role of NO in the neonatal brain, particularly during hypoxia and ischaemia has been studied extensively in animal models of focal and global ischaemia. The n-NOS and i-NOS activation have been found to be harmful whereas e-NOS activation has a neuroprotective effect in focal ischaemia (Fig. 1). The findings following global ischaemia are somewhat more controversial. Although all these studies clearly demonstrate that NO has an important role in the neonatal brain, it may be difficult to apply the results to humans for it is not clear when and which isoform of NOS gets activated following ischaemia in newborn infants. Also it is hard to determine the timing of intervention to inhibit or stimulate the production of NO in humans since we still do not know all the details about protective mechanisms of the human body. Some interventions may have a deleterious effect on some of those mechanisms. In addition, the relatively selective NOS inhibitors which are now used in animal experiments are not appropriate for human studies. New studies regarding the production of NO following ischaemia will be needed in newborns, together with the development of selective NOS inhibitors which can be used in humans. If the NO production follows the same pattern in humans as in animals at least the effects of i-NOS may be prevented either by selective inhibitors or by neuroprotective agents. PMID- 10727197 TI - Childhood epilepsy with neuropsychological regression and continuous spike waves during sleep: epilepsy surgery in a young adult. AB - We describe the case of a man with a history of complex partial seizures and severe language, cognitive and behavioural regression during early childhood (3.5 years), who underwent epilepsy surgery at the age of 25 years. His early epilepsy had clinical and electroencephalogram features of the syndromes of epilepsy with continuous spike waves during sleep and acquired epileptic aphasia (Landau Kleffner syndrome), which we considered initially to be of idiopathic origin. Seizures recurred at 19 years and presurgical investigations at 25 years showed a lateral frontal epileptic focus with spread to Broca's area and the frontal orbital regions. Histopathology revealed a focal cortical dysplasia, not visible on magnetic resonance imaging. The prolonged but reversible early regression and the residual neuropsychological disorders during adulthood were probably the result of an active left frontal epilepsy, which interfered with language and behaviour during development. Our findings raise the question of the role of focal cortical dysplasia as an aetiology in the syndromes of epilepsy with continuous spike waves during sleep and acquired epileptic aphasia. PMID- 10727198 TI - Iron supplementation aggravates periventricular cystic white matter lesions in newborn mice. AB - Periventricular leukomalacia (PVL) is the main cause of neurological impairment in premature newborns. The pathogenesis of PVL remains unclear but may involve glutamate excitotoxicity and free radical production. Oxygen and iron, which are widely used in premature newborns, are oxidizing agents with a potential for promoting free radical production. We previously described a mouse model of excitotoxic neonatal white matter lesions mimicking several aspects of human PVL. In the present study, we used this mouse model to investigate whether iron pretreatment or 100% oxygen exposure worsened excitotoxic lesions. We found that iron pretreatment but not hyperoxia significantly increased white matter lesions, suggesting that high doses of iron may aggravate PVL in premature newborns. PMID- 10727199 TI - A paradoxical rise of neonatal seizures after treatment with vitamin B6. AB - We report the case of a newborn with intractable epileptic seizures developing a paradoxical rise of seizure frequency and electroencephalogram alterations after administration of vitamin B6. We have been unable to determine the aetiology of this disorder. In a newborn presenting with drug-resistant epileptic seizures, the first therapeutic option remains the application of intravenous pyridoxine, but the physician should be aware of the risk of an increase in seizure frequency. PMID- 10727200 TI - Can mebendazole cause lateralized occipital seizures? PMID- 10727201 TI - Closed spinal dysraphism. PMID- 10727202 TI - Doped rare gas solids as model systems for chromophore-matrix interactions. AB - Molecular electronic states are extremely sensitive to changes in their environment. Therefore, by inserting probe molecules into a host material and monitoring the ensuing changes in the widths and spectral positions of the electronic absorption bands, interactions of probe and host molecules can be studied. A statistical mechanical model used to analyze these data can also be employed to extract from pressure-induced changes of the band shape information about the local environment of the probe molecule. Dye-doped solid rare gases are ideal model systems to test the basic assumptions of the statistical model, revealing its potential and limitations. PMID- 10727203 TI - Two-state reactivity as a new concept in organometallic chemistry. AB - It is proposed that spin-crossing effects can dramatically affect reaction mechanisms, rate constants, branching ratios, and temperature behaviors of organometallic transformations. This phenomenon is termed two-state reactivity (TSR) and involves participation of spin inversion in the rate-determining step. While the present analysis is based on studies of transition metals under idealized conditions, several recent reports imply that TSR is by no means confined to the gas phase. In fact, participation of more than a single spin surface in the reaction pathways is proposed as a key feature in organometallic chemistry. PMID- 10727204 TI - Oligothienylenevinylenes as a new class of multinanometer linear pi-conjugated systems for micro- and nanoelectronics. AB - This Account deals with the synthesis and characterization of monodisperse soluble oligothienylenevinylenes with chain lengths up to 100 A. The chain length dependence of the electronic and electrochemical properties both in solution and in the solid state are analyzed and discussed in the context of the potential use of oligothienylenevinylenes as molecular wires. Problems related to interchain interactions are illustrated by the analysis of the effects of structure on the reversible dimerization of cation radicals and by the synthesis of new series of end-substituted oligomers. PMID- 10727205 TI - Recovery of carbonyl compounds from N,N-dialkylhydrazones. AB - Deprotonation of enantiomerically pure hydrazones and subsequent trapping with suitable electrophiles generates new stereogenic centers with excellent stereoselectivity. To liberate the original carbonyl functionality in the final products, it is necessary to cleave the hydrazone moiety. In recent years, many reagents have been developed to regenerate carbonyl compounds from the corresponding dialkylhydrazones which are compatible with a wide range of functionalities. This has allowed the use of hydrazones in the total synthesis of complex natural products. This Account is meant to be an overview of methods which are classified as oxidative, hydrolytic, and reductive cleavage procedures. PMID- 10727206 TI - Catalytic macrocyclizations of thietanes and thiiranes by metal carbonyl complexes. AB - This Account summarizes our recent studies of the catalytic transformations of thietanes and thiiranes by metal carbonyl complexes of the third row of the transition elements. The goal of the work has been to prepare polythioether macrocycles (thiacrowns) catalytically. This has been achieved with high success for certain thietane reactions. Thiiranes, on the other hand, tend to yield macrocyclic polydisulfides, but it has recently been found that these reactions can be redirected toward the thiacrown products by using certain alkyne carboxylates as cocatalysts. Vinylthiiranes are transformed into 3,6-dihydro-1,2 dithiins catalytically in excellent yield via W(CO)(5)(vinylthiirane) complexes. PMID- 10727207 TI - Mass spectrometry in viral proteomics. AB - Mass spectrometry is a valuable tool in structural and functional viral proteomics, where it has been used to identify viral capsid proteins, viral mutants, and posttranslational modifications. Further, mass-based approaches combined with time-resolved proteolysis (mass mapping) have revealed the dynamic nature of viral particles in solution; this method is contributing to an understanding of the dynamic domains of the viral capsid which may have significant value in developing new approaches for viral inactivation. As a result of these experiments, and by comparison with complementary data from X-ray crystallography, a new dimension to viral protein structure and function is emerging. PMID- 10727208 TI - Crystal and molecular simulation of high-performance polymers. AB - Single-crystal X-ray analyses of oligomeric models for high-performance aromatic polymers, interfaced to computer-based molecular modeling and diffraction simulation, have enabled the determination of a range of previously unknown polymer crystal structures from X-ray powder data. Materials which have been successfully analyzed using this approach include aromatic polyesters, polyetherketones, polythioetherketones, polyphenylenes, and polycarboranes. Pure macrocyclic homologues of noncrystalline polyethersulfones afford high-quality single crystals-even at very large ring sizes-and have provided the first examples of a "protein crystallographic" approach to the structures of conventionally amorphous synthetic polymers. PMID- 10727209 TI - Identification of 12-lipoxygenase interaction with cellular proteins by yeast two hybrid screening. AB - The platelet isoform of 12-lipoxygenase (12-LOX) is expressed in a variety of human tumors. 12-LOX metabolizes arachidonic acid to 12(S)-hydroxyeicosateraenoic acid (12(S)-HETE), which induces a number of cellular responses associated with tumor progression and metastasis. Little is known about 12-LOX regulation and no direct regulators of 12-LOX activity have been identified. To identify potential regulators of 12-LOX, we isolated cDNAs encoding 12-LOX interacting proteins using the yeast two-hybrid system. We screened a yeast two-hybrid interaction library from human epidermoid carcinoma A431 cells and identified four cellular proteins that interact specifically with 12-LOX. We identified type II keratin 5, lamin A, the cytoplasmic domain of integrin beta4 subunit and a phosphoprotein C8FW as 12-LOX interacting proteins. Here, we demonstrated that keratin 5, a 58 kD protein required for formation of 8 nm intermediate filaments, binds to 12-LOX in human tumor cells and may contribute to the regulated trafficking of 12-LOX. We also showed that lamin A binds 12-LOX in human tumor cells. These proteins provide the first candidate regulators of 12-LOX. PMID- 10727210 TI - Imaging and mapping heparin-binding sites on single fibronectin molecules with atomic force microscopy. AB - Fibronectin is composed of multiple homologous repeats and contains many functional domains. Two major heparin-binding domains have previously been identified: the Hep I site near the amino terminus and the Hep II site near the carboxyl terminus. The Hep II site has been considered the high-affinity heparin binding site based on studies of fibronectin fragments. However, few studies have been carried out on heparin binding by intact fibronectin. We imaged single fibronectin molecules as well as heparin-coated gold particles bound to whole dimeric plasma fibronectin molecules with tapping mode atomic force microscopy. We observed heparin-gold particles preferentially bound at two locations that correspond to the Hep I and Hep II sites. Quantitative analysis of images of individual fibronectin-heparin-gold complexes showed that almost twice as many heparin-gold particles bound to the N-terminal Hep I site compared to the Hep II site. In contrast to previous findings with fibronectin fragments, these results suggest that the Hep I site has a binding affinity higher than or comparable to the Hep II site in the intact fibronectin molecule. PMID- 10727211 TI - Molecular cloning and functional analysis of apoxin I, a snake venom-derived apoptosis-inducing factor with L-amino acid oxidase activity. AB - We previously purified apoxin I, an apoptosis-inducing factor with L-amino acid oxidase (LAO) activity, from Western diamondback rattlesnake venom. To determine the primary structure of apoxin I, we cloned its cDNA. The amino acid sequence showed that apoxin I has an FAD binding domain and shares homology with L-amino acid oxidase (LAO) from Neurospora crassa, human monoamine oxidase B, and mouse interleukin 4-induced F1G1 protein. The full-length apoxin I has an N-terminal signal sequence that is processed in mature apoxin I in venom. When the apoxin I gene was transfected into human 293T cells, the recombinant protein was expressed in the cells, and a significant amount of apoxin I was secreted into the medium. The secreted recombinant apoxin I protein showed LAO and apoptosis-inducing activity, but the recombinant protein in the cells did not, suggesting that maturation and secretion of the apoxin I protein is needed for its activity. Treating the transfected cells with tunicamycin inhibited the secretion and LAO activity of the recombinant apoxin I. In addition, deleting the amino-terminal region flanking the signal sequence, the FAD-binding domain and the carboxy terminal region abolished the secretion and LAO activity of the recombinant proteins. These results indicate that in order for apoxin I to become active, these regions and posttranslational modification, such as N-glycosylation, are required. PMID- 10727212 TI - Fetal Alz-50 clone 1 (FAC1) protein interacts with the Myc-associated zinc finger protein (ZF87/MAZ) and alters its transcriptional activity. AB - Transcription factors mediate their regulatory effects through interaction with DNA and numerous nuclear proteins. The fetal Alz-50 clone 1 (FAC1) protein, a novel DNA-binding protein with the capacity to repress transcription, is likely to function through a similar mechanism (1). Using the two-hybrid yeast screen, we have shown that FAC1 interacts with the myc-associated zinc finger protein (ZF87/MAZ). This association was confirmed in vitro with recombinant protein. The ZF87/MAZ interaction domain was mapped to the region containing a putative nuclear localization signal (NLS) and nuclear export sequence (NES) of FAC1, using deletion mutants of the FAC1 protein. FAC1, on the other hand, recognizes a conformational interface that includes the proline/alanine-rich domain of ZF87/MAZ and the first zinc finger. Cotransfection of NIH3T3 cells with ZF87/MAZ and a luciferase reporter containing the SV40 promoter and enhancer results in an increase in transcriptional activation, suggesting ZF87/MAZ is able to recognize its consensus binding site present in the SV40 promoter. Cotransfection with FAC1 reduces the level of ZF87/MAZ-induced activation of the SV40 promoter in a dose dependent manner. A mutant FAC1, lacking the ZF87/MAZ interaction domain, does not alter ZF87/MAZ activation of the SV40 promoter. These data demonstrate that interaction between FAC1 and ZF87/MAZ alters the transactivation capacity of ZF87/MAZ. By immunoblot analysis, FAC1 and ZF87/MAZ exhibit similar tissue distribution and co-localize to pathologic structures in Alzheimer's disease brain. Coexpression of FAC1 and ZF87/MAZ suggest that interaction of these two proteins will have biological implications for gene regulation in neurodegeneration. PMID- 10727213 TI - Thermus thermophilus contains an eubacterial and an archaebacterial aspartyl-tRNA synthetase. AB - Thermus thermophilus possesses two aspartyl-tRNA synthetases (AspRSs), AspRS1 and AspRS2, encoded by distinct genes. Alignment of the protein sequences with AspRSs of other origins reveals that AspRS1 possesses the structural features of eubacterial AspRSs, whereas AspRS2 is structurally related to the archaebacterial AspRSs. The structural dissimilarity between the two thermophilic AspRSs is correlated with functional divergences. AspRS1 aspartylates tRNA(Asp) whereas AspRS2 aspartylates tRNA(Asp), and tRNA(Asn) with similar efficiencies. Since Asp bound on tRNA(Asn) is converted into Asn by a tRNA-dependent aspartate amidotransferase, AspRS2 is involved in Asn-tRNA(Asn) formation. These properties relate functionally AspRS2 to archaebacterial AspRSs. The structural basis of the dual specificity of T. thermophilus tRNA(Asn) was investigated by comparing its sequence with those of tRNA(Asp) and tRNA(Asn) of strict specificity. It is shown that the thermophilic tRNA(Asn) contains the elements defining asparagine identity in Escherichia coli, part of which being also the major elements of aspartate identity, whereas minor elements of this identity are missing. The structural context that permits expression of aspartate and asparagine identities by tRNA(Asn) and how AspRS2 accommodates tRNA(Asp) and tRNA(Asn) will be discussed. This work establishes a distinct structure-function relationship of eubacterial and archaebacterial AspRSs. The structural and functional properties of the two thermophilic AspRSs will be discussed in the context of the modern and primitive pathways of tRNA aspartylation and asparaginylation and related to the phylogenetic connexion of T. thermophilus to eubacteria and archaebacteria. PMID- 10727214 TI - Structure and function of procollagen C-proteinase (mTolloid) domains determined by protease digestion, circular dichroism, binding to procollagen type I, and computer modeling. AB - Procollagen C-proteinase-2 (pCP-2, mTld) is derived from the longest splicing variant of the gene encoding bone morphogenetic protein 1 (BMP-1). The variants have identical amino terminal signal peptides, prodomains and astacin-like protease domains. However, they differ in the length of their carboxy terminal part, which in pCP-2 has the composition CUB1, CUB2, EGF-like1, CUB3, EGF-like2, CUB4, CUB5, and C-tail. In the shorter form, pCP-1 (i.e., BMP-1), the sequence ends after the CUB3-domain. Using a combination of mutagenesis and structural approaches, we have investigated the structure and function of subfragments of pCP-2. The full-length latent recombinant enzyme and its N-terminally truncated form lacking the prodomain were tested for their enzymic activity. The intact protein showed only partial processing of procollagen type I, whereas the truncated form expressed enzymic activity indistinguishable from its native counterpart purified from chick embryo tendons. These results clearly demonstrated that the prodomain is required for the latency of the enzyme but not for its correct folding. Limited proteolysis of the recombinant protein with alpha-chymotrypsin produced four discrete fragments revealing the location of cleavage sites between the repetitive CUB/EGF domains. The results provide evidence that the CUB sequences form independently folded modules that are stabilized by two pairs of internal disulfide bridges. The modules are linked to each other by more flexible, hinge-like peptides. Solid-phase binding assays with isolated CUB domains and immobilized procollagen type I demonstrated that the first three but not the last two CUB domains specifically bound to the substrate. To define putative sites for CUB-CUB or CUB-substrate interactions, we generated molecular models for pCP-2 CUB domains. The models were obtained using as a template the structure of CUB domain in zona pellucida adhesion protein PSP-I/PSP II from porcine sperm. The predicted conformations for homology models were, subsequently, confirmed by circular dichroism spectroscopy of polypeptide domains isolated following limited proteolysis with alpha-chymotrypsin. PMID- 10727215 TI - An engineered blockage within the ammonia tunnel of carbamoyl phosphate synthetase prevents the use of glutamine as a substrate but not ammonia. AB - The heterodimeric carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP. The enzyme catalyzes the hydrolysis of glutamine within the small amidotransferase subunit and then transfers ammonia to the two active sites within the large subunit. These three active sites are connected via an intermolecular tunnel, which has been located within the X-ray crystal structure of CPS from E. coli. It has been proposed that the ammonia intermediate diffuses through this molecular tunnel from the binding site for glutamine within the small subunit to the phosphorylation site for bicarbonate within the large subunit. To provide experimental support for the functional significance of this molecular tunnel, residues that define the interior walls of the "ammonia tunnel" within the small subunit were targeted for site-directed mutagenesis. These structural modifications were intended to either block or impede the passage of ammonia toward the large subunit. Two mutant proteins (G359Y and G359F) display kinetic properties consistent with a constriction or blockage of the ammonia tunnel. With both mutants, the glutaminase and bicarbonate-dependent ATPase reactions have become uncoupled from one another. However, these mutant enzymes are fully functional when external ammonia is utilized as the nitrogen source but are unable to use glutamine for the synthesis of carbamoyl-P. These results suggest the existence of an alternate route to the bicarbonate phosphorylation site when ammonia is provided as an external nitrogen source. PMID- 10727216 TI - Structure and thermodynamics of the extraordinarily stable molten globule state of canine milk lysozyme. AB - Here, we show that an unfolded intermediate of canine milk lysozyme is extraordinarily stable compared with that of the other members of the lysozyme alpha-lactalbumin superfamily, which has been studied previously. The stability of the intermediate of this protein was investigated using calorimetry, CD spectroscopy, and NMR spectroscopy, and the results were interpreted in terms of the structure revealed by X-ray crystallography at a resolution of 1.85 A to an R factor of 17.8%. On the basis of the results of the thermal unfolding, this protein unfolds in two clear cooperative stages, and the melting temperature from the intermediate to the unfolded states is about 20 degrees C higher than that of equine milk lysozyme. Furthermore, the (1)H NMR spectra of canine milk lysozyme at 60 degrees C, essentially 100% of which exists in the intermediate, showed that small resonance peaks that arise from ring-current shifts of aliphatic protons are still present in the upfield region from 0 to -1 ppm. The protein at this temperature (60 degrees C) and pH 4.5 has been found to bind 1-anilino naphthalene-8-sulfonate (ANS) with enhancement of the fluorescence intensity compared with that of native and thermally unfolded states. We interpret that the extraordinarily stable intermediate is a molten globule state, and the extraordinary stabilization of the molten globule state comes from stronger protection around the C- and D-helix of the aromatic cluster region due to the His-21 residue. The conclusion helps to explain how the molten globule state acquires its structure and stability. PMID- 10727217 TI - Crystal structure at 1.9 A resolution of the apoovotransferrin N-lobe bound by sulfate anions: implications for the domain opening and iron release mechanism. AB - Several lines of functional evidence have shown that anion binding to a nonsynergistic site is a prerequisite for the anion-mediated iron release mechanism of transferrins. We report here structural evidence of the location of sulfate anion binding sites of the ovotransferrin N-lobe via the 1.90 A resolution apo crystal structure. The crystals were grown in an ammonium sulfate solution and belonged to space group P6(3)22 with the following unit cell dimensions: a = b = 125.17 A and c = 87.26 A. The structural determination was performed by isomorphous replacement, using Pt and Au derivatives. The structure refinement gave an R-factor of 0.187 in the resolution range of 7.0-1.90 A for the final model. From the electron density map, the existence of four bound SO(4)(2)(-) anions was detected. Three of them that exhibited reasonably low B factors were all located in the opened interdomain cleft (sites 1-3). In site 1, the bound anion directly interacts with an Fe(3+)-coordinating ligand; SO(4) O1 and SO(4) O3 form hydrogen bonds with His250 NE2. Oxygen atom O3 of the same sulfate anion makes a hydrogen bond with Ser91 OG in a hinge strand. The sulfate anion in site 2 partially occupies the synergistic anion binding sites; SO(4) O2 and SO(4) O3 are hydrogen bonded to Arg121 NE and NH2, respectively, both of which are consensus anchor groups for CO(3)(2)(-) anion in holotransferrins. The former oxygen atom of SO(4)(2)(-) is also hydrogen bonded to Ser122 N, which forms a hydrogen bond with Fe(3+)-coordinating ligand Asp60 OD2 in holotransferrins. Some of the SO(4)(2)(-) oxygen atoms in sites 1 and 2 interact indirectly through H(2)O molecules with functionally important protein groups, such as the other Fe(3+)-coordinating ligands, Tyr92 OH and Tyr191 OH, and a dilysine trigger group, Lys209 NZ. In site 3, SO(4) O1 and SO(4) O4 form hydrogen bonds with Ser192 OG and Tyr191 N, respectively, and SO(4) O2 forms hydrogen bonds with Ser192 N and Ser192 OG. These structural data are consistent with the view that the anion bindings to the interdomain cleft, especially to sites 1 and 2, play crucial roles in the domain opening and synergistic carbonate anion release in the iron release mechanism of the ovotransferrin N-lobe. PMID- 10727218 TI - Kinetic and crystallographic studies on the active site Arg289Lys mutant of flavocytochrome b2 (yeast L-lactate dehydrogenase). AB - Flavocytochrome b(2) from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction. The crystal structure of the native yeast enzyme has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 837-863] as well as that of the sulfite adduct of the recombinant enzyme produced in Escherichia coli [Tegoni, M., and Cambillau, C. (1994) Protein Sci. 3, 303-313]; several key active site residues were identified. In the sulfite adduct crystal structure, Arg289 adopts two alternative conformations. In one of them, its side chain is stacked against that of Arg376, which interacts with the substrate; in the second orientation, the R289 side chain points toward the active site. This residue has now been mutated to lysine and the mutant enzyme, R289K-b(2), characterized kinetically. Under steady-state conditions, kinetic parameters (including the deuterium kinetic isotope effect) indicate the mutation affects k(cat) by a factor of about 10 and k(cat)/K(M) by up to nearly 10(2). Pre-steady-state kinetic analysis of flavin and heme reduction by lactate demonstrates that the latter is entirely limited by flavin reduction. Inhibition studies on R289K-b(2) with a range of compounds show a general rise in K(i) values relative to that of wild-type enzyme, in line with the elevation of the K(M) for L-lactate in R289K-b(2); they also show a change in the pattern of inhibition by pyruvate and oxalate, as well as a loss of the inhibition by excess substrate. Altogether, the kinetic studies indicate that the mutation has altered the first step of the catalytic cycle, namely, flavin reduction; they suggest that R289 plays a role both in Michaelis complex and transition-state stabilization, as well as in ligand binding to the active site when the flavin is in the semiquinone state. In addition, it appears that the mutation has not affected electron transfer from fully reduced flavin to heme, but may have slowed the second intramolecular ET step, namely, transfer from flavin semiquinone to heme b(2). Finally, the X-ray crystal structure of R289K-b(2), with sulfite bound at the active site, has been determined to 2.75 A resolution. The lysine side chain at position 289 is well-defined and in an orientation that corresponds approximately to one of the alternative conformations observed in the structure of the recombinant enzyme-sulfite complex [Tegoni, M., and Cambillau, C. (1994) Protein Sci. 3, 303-313]. Comparisons between the R289K-b(2) and wild-type structures allow the kinetic results to be interpreted in a structural context. PMID- 10727219 TI - Diethyl pyrocarbonate modification abolishes fast electron accepting ability of cytochrome b561 from ascorbate but does not influence electron donation to monodehydroascorbate radical: identification of the modification sites by mass spectrometric analysis. AB - Cytochrome b(561) from bovine adrenal chromaffin vesicles contains two heme B prosthetic groups and transports electron equivalents across the vesicle membranes to convert intravesicular monodehydroascorbate radical to ascorbate. To elucidate the mechanism of the transmembrane electron transfer, effects of the treatment of purified cytochrome b(561) with diethyl pyrocarbonate, a reagent specific for histidyl residues, were examined. We found that when ascorbate was added to the oxidized form of diethyl pyrocarbonate-treated cytochrome b(561), less than half of the heme iron was reduced but with a very slow rate. In contrast, radiolytically generated monodehydroascorbate radical was oxidized rapidly by the reduced form of diethyl pyrocarbonate-modified cytochrome b(561), as observed for untreated cytochrome b(561). These results indicate that the heme center specific for the electron acceptance from ascorbate was perturbed by the modification of amino acid residues nearby. We identified the major modification sites by mass spectrometry as Lys85, His88, and His161, all of which are fully conserved and located on the extravesicular side of cytochrome b(561) in the membranes. We suggest that specific N-carbethoxylation of the histidyl ligands of the heme b at extravesicular side abolishes the electron-accepting ability from ascorbate. PMID- 10727220 TI - Inhibitory copper binding site on the spinach cytochrome b6f complex: implications for Qo site catalysis. AB - The isolated cytochrome (cyt) b(6)f complex from spinach is inhibited by Cu(2+) with a K(D) of about 1 microM at pH 7.6 in the presence of 1.6 microM decyl plastoquinol (C(10)-PQH(2)) as a substrate. Inhibition was competitive with respect to C(10)-PQH(2) but noncompetitive with respect to horse heart cyt c or plastocyanin (PC). Inhibition was also pH-sensitive, with an apparent pK at about 7, above which inhibition was stronger, suggesting that binding occurred at or near a protonatable amino acid residue. Equilibrium binding titrations revealed ca. 1.4 tight Cu(2+) binding sites with a K(D) of about 0.5 microM and multiple (>8) weak (K(D) > 50 microM) binding sites per complex. Pulsed electron paramagnetic resonance (EPR) techniques were used to identify probable binding sites for inhibitory Cu(2+). A distinct enhancement of the relaxation time constant for the EPR signal from bound Cu(2+) was observed when the cyt f was paramagnetic. The magnitude and temperature-dependence of this relaxation enhancement were consistent with a dipole interaction between Cu(2+) and the cyt f (Fe(3+)) heme at a distance of between 30 and 54 A, depending upon the relative orientations of Cu(2+) and cyt f heme g-tensors. Two-pulse electron spin-echo envelope modulation (ESEEM) and 4-pulse 2-dimensional hyperfine sublevel correlation (2D HYSCORE) measurements of Cu(2+) bound to isolated cyt b(6)f complex indicated the presence of a weakly coupled nitrogen nucleus. The nuclear quadrupole interaction (NQI) and the hyperfine interaction (HFI) parameters identified one Cu(2+) ligand as an imidazole nitrogen of a His residue, and electron-nuclear double resonance (ENDOR) confirmed the presence of a directly coordinated nitrogen. A model of the 3-dimensional structure of the cytochrome b(6)f complex was constructed on the basis of sequences and structural similarities with the mitochondrial cyt bc(1) complex, for which X-ray structures have been solved. This model indicated three possible His residues as ligands to inhibitory Cu(2+). Two of these are located on the "Rieske" iron-sulfur protein protein (ISP) while the third is found on the cyt f protein. None of these potential ligands appear to interact directly with the quinol oxidase (Q(o)) binding pocket. A model is thus proposed wherein Cu(2+) interferes with the interaction of the ISP protein with the Q(o) site, preventing the binding and subsequent oxidation of plastoquinonol. Implications for the involvement of ISP "domain movement" in Q(o) site catalysis are discussed. PMID- 10727221 TI - Pathways of energy transformation in antenna reaction center complexes of Heliobacillus mobilis. AB - The conversion of excitation energy in the antenna reaction center complex of Heliobacillus mobilis was investigated at 10 K as well as at 275 K by means of time-resolved absorbance difference spectroscopy of isolated membranes in the (sub)picosecond time range. Selective excitation of the primary electron acceptor, chlorophyll (Chl) a 670, and of the different spectral pools of bacteriochlorophyll (BChl) g (BChl g 778, BChl g 793, and BChl g 808) was applied. At 10 K, excitation at 770 or 793 nm resulted on the one hand in rapid energy transfer to BChl g 808 and on the other hand in fast charge separation from excited BChl g 793 ( approximately 1 ps). Once the excitations were on BChl g 808, the bleaching band shifted gradually to the red, from 806 to 813 nm, and charge separation from excited BChl g 808 occurred by a very slow process ( approximately 500 ps). The main purpose of our experiments was to answer the question whether an "alternative" pathway for charge separation exists upon excitation of Chl a 670. Our measurements showed that the amount of oxidized primary donor (P798(+)) relative to that of excited BChl g produced by excitation of Chl a 670 was considerably larger than upon direct excitation of BChl g. This indicates the existence of an alternative pathway for charge separation that does not involve excited antenna BChl g. This effect occurred at 10 K as well as at 275 K. The mechanism for this process is discussed in relation to different trapping models; it is concluded that charge separation occurs directly from excited Chl a 670. PMID- 10727222 TI - New insights on the proton pump associated with cytochrome b6f turnovers from the study of H/D substitution effects on the electrogenicity and electron transfer reactions. AB - We have studied the effect of protium/deuterium substitution on different kinetics associated with the turnovers of cytochrome b(6)f complex in whole cells of Chlamydomonas reinhardtii. Both the oxidation of cytochrome f and the reduction of hemes b were only little affected by the isotopic substitution. Contrasting with this, the initial slope of the electrogenic phase associated with cytochrome b(6)f turnover was slowed by a factor of 4 by H(2)O/D(2)O substitution. Whereas in the presence of H(2)O the electrogenic phase developed concomitantly with cytochrome b reduction, it lagged for a few hundreds of microseconds after cytochrome b reduction in the presence of D(2)O. We propose that a proton pump is triggered by the oxidation of plastoquinol at the Q(o) site. The proton transfer is specifically delayed upon isotopic substitution, accounting for the lack of significant effect on the electron-transfer reaction as well as for the strong decrease of the initial rate of the electrogenic phase. PMID- 10727223 TI - Site-directed mutagenesis identifies residues in uncoupling protein (UCP1) involved in three different functions. AB - Using site-specific mutagenesis, we have constructed several mutants of uncoupling protein (UCP1) from brown adipose tissue to investigate the function of acidic side chains at positions 27, 167, 209, and 210 in H(+) and Cl(-) transport as well as in nucleotide binding. The H(+) transport activity was measured with mitochondria and with reconstituted vesicles. These mutant UCPs (D27N, D27E, E167Q, D209N, D210N, and D209N + D210N) are expressed at near wt levels in yeast. Their H(+) transport activity in mitochondria correlates well with the reconstituted protein except for D27N (intrahelical), which shows strong inhibition of H(+) transport in the reconstituted system and only 50% decrease of uncoupled respiration in mitochondria. In the double adjacent acidic residues (between helix 4 and helix 5), mutation of D210 and of D209 decreases H(+) transport 80% and only 20%, respectively. These mutants retain full Cl(-) transport activity. The results indicate that D210 participates in H(+) uptake at the cytosolic side and D27 in H(+) translocation through the membrane. Differently, E167Q has lost Cl(-) transport activity but retains the ability to transport H(+). The separate inactivation of H(+) and Cl(-) transport argues against the fatty acid anion transport mechanism of H(+) transport by UCP. The mutation of the double adjacent acidic residues (D209, D210) decreases pH dependency for only nucleoside triphosphate (NTP) but not diphosphate (NDP) binding. The results identify D209 and D210 in accordance with the previous model as those residues which control the location of H214 in the binding pocket, and thus contribute to the pH control of NTP but not of NDP binding. PMID- 10727224 TI - A novel glycolipid and phospholipid in the purple membrane. AB - A novel glycolipid of mass 1935 and a phospholipid of mass 1522 are the main residual lipids (along with traces of PGP-Me, S-TGD-1, and PG) specifically associated with "delipidated" bacteriorhodopsin fractions BR I and BR II, prepared by Triton X-100 treatment of purple membrane (PM), from a genetically engineered strain (L33) of Halobacterium salinarum, and chromatography on phenyl Sepharose CL-4B. The novel glycolipid and phospholipid are components of the PM matrix not previously described. The TLC isolated and purified novel glycolipid and phospholipid were shown, by chemical degradation, mass spectrometry, and NMR analyses, to have the structure, respectively, of a phosphosulfoglycolipid, 3 HSO(3)-Galp-beta1,6Manp-alpha1,2Glcp-alpha1,1-[sn-2, 3-di-O-phytanylglycerol]-6 [phospho-sn-2,3-di-O-phytanylglycero l], and of a glycerol diether analogue of bisphosphatidylglycerol (cardiolipin), sn-2,3-di-O-phytanyl-1-phosphoglycerol-3 phospho-sn-2, 3-di-O-phytanylglycerol. PMID- 10727225 TI - Role of glutamate-268 in the catalytic mechanism of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans. AB - Nonphosphorylating nicotinamide adenine dinucleotide (phosphate)- [NAD(P)-] dependent aldehyde dehydrogenases share a number of conserved amino acid residues, several of which are directly implicated in catalysis. In the present study, the role of Glu-268 from nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans was investigated. Its substitution by Ala resulted in a k(cat) decrease by 3 orders of magnitude. Pre-steady-state analysis showed that, for both the wild-type and E268A GAPNs, the rate-limiting step of the reaction is associated with deacylation. The pH dependence of the rate of acylation of wild-type GAPN is characterized by the contributions of distinct enzyme protonic species with two pK(a)s of 6.2 and 7.5. Substitution of Glu-268 by Ala resulted in a monosigmoidal pH dependence of the rate constant of acylation with a pK(a) of 6.2, which suggested the assignment of pK(a) 7.5 to Glu 268. Moreover, the E268A substitution did not significantly affect the efficiency of acylation of GAPN, showing that Glu-268 is not critically involved in the acylation, which includes Cys-302 nucleophilic activation and hydride transfer. On the contrary, the drastic decrease of the steady-state rate constant for the E268A GAPN demonstrated the essential role of Glu-268 in the deacylation. At basic pH, the solvent isotope effect of 2.3, characterized by a unique pK(a) of 7.7, and the linearity of the proton inventory showed that the rate-limiting process for deacylation is associated with the hydrolysis step and suggested that the glutamate form of Glu-268 acts as a base catalyst in this process. Surprisingly, the double-sigmoidal form of the pH-steady-state rate constant profile, characterized by pK(a) values of 6.1 and 7.4, revealed the high efficiency of the deacylation even at pH lower than 7.4. Therefore, we propose that the major role of Glu-268 is to promote deacylation through activation and orientation of the attacking water molecule, and in addition to act as a base catalyst at basic pH. From these results in relation to those recently described [Marchal, S., and Branlant, G. (1999) Biochemistry 38, 12950-12958], a scenario for the chemical catalysis of GAPN is proposed. PMID- 10727226 TI - Single-turnover kinetic analysis of Trypanosoma cruzi S-adenosylmethionine decarboxylase. AB - Trypanosoma cruzi S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the pyruvoyl-dependent decarboxylation of S-adenosylmethionine (AdoMet), which is an important step in the biosynthesis of polyamines. The time course of the AdoMetDC reaction under single-turnover conditions was measured to determine the rate of the slowest catalytic step up to and including decarboxylation. Analysis of this single-turnover data yields an apparent second-order rate constant for this reaction of 3300 M(-1) s(-1) in the presence of putrescine, which corresponds to a catalytic rate of >6 s(-1). This rate is minimally 100-fold faster than the steady-state rate suggesting that product release, which includes Schiff base hydrolysis, limits the overall reaction. AdoMetDC exhibits an inverse solvent isotope effect on the single-turnover kinetics, and the pH profile predicts a pK(a) of 8.9 for the basic limb. These results are consistent with a Cys residue functioning as a general acid in the rate-determining step of the single-turnover reaction. Mutation of Cys-82 to Ala reduces the rate of the single turnover reaction to 11 M(-1) s(-1) in the presence of putrescine. Further, a solvent isotope effect is not observed for the mutant enzyme. Reduction of the wild-type enzyme with cyanoborohydride traps the Schiff base between the enzyme and decarboxylated substrate, while little Schiff base species of either substrate or product was trapped with the C82A mutant. These data suggest that Cys-82 functions as a general acid/base to catalyze Schiff base formation and hydrolysis. The solvent isotope and pH effects are mirrored in single-turnover analysis of reactions without the putrescine activator, yielding an apparent second-order rate constant of 150 M(-1) s(-1). The presence of putrescine increases the single-turnover rate by 20-fold, while it has relatively little effect on the affinity of the enzyme for product. Therefore, putrescine likely activates the T. cruzi AdoMetDC enzyme by accelerating the rate of Schiff base exchange. PMID- 10727227 TI - Oxidation-reduction properties of the regulatory disulfides of sorghum chloroplast nicotinamide adenine dinucleotide phosphate-malate dehydrogenase. AB - Oxidation-reduction midpoint potentials (E(m)) have been measured for the thioredoxin-dependent, reductive activation of sorghum nicotinamide adenine dinucleotide phosphate- (NADP-) dependent malate dehydrogenase (MDH) in the wild type enzyme and in a number of site-specific mutants. The E(m) value associated with activation of the wild-type enzyme, -330 mV at pH 7.0, can be attributed to the E(m) of the C365/C377 disulfide present in the C-terminal region of the enzyme. The C24/C29 disulfide, located in the N-terminal region of the enzyme and the only other disulfide present in oxidized, wild-type MDH, has a E(m) value of 280 mV at pH 7.0. A third regulatory disulfide, C24/C207, that is absent in the oxidized enzyme but is thought to be formed during the activation process, has an E(m) value at pH 7.0 of -310 mV. E(m) vs pH profiles suggest pK(a) values for the more acidic cysteine involved in the formation of each of these disulfides of 8.5 for C24/C29; 8.1 for C24/C207; and 8.7 for C365/C377. The results of this study show that the N-terminal disulfide formed between C24 and C29 has a more positive E(m) value than the two other disulfides and is thus is likely to be the "preregulatory disulfide" postulated to function in activating the enzyme. PMID- 10727228 TI - Catalytic activity of the D38A mutant of 3-oxo-Delta 5-steroid isomerase: recruitment of aspartate-99 as the base. AB - 3-oxo-Delta(5)-steroid isomerase (KSI) from Comamonas (Pseudomonas) testosteroni catalyzes the isomerization of beta,gamma-unsaturated 3-oxosteroids to their conjugated isomers through an intermediate dienolate. Residue Asp-38 (pK(a) 4.57) acts as a base to abstract a proton from C-4 of the substrate to form an intermediate dienolate, which is then reprotonated on C-6. Both Tyr-14 (pK(a) 11.6) and Asp-99 (pK(a) >/= 9.5) function as hydrogen-bond donors to O-3 of the steroid, helping to stabilize the transition states. Mutation of the active-site base Asp-38 to the weakly basic Asn (D38N) has previously been shown to result in a >10(8)-fold decrease of catalytic activity. In this work, we describe the preparation and kinetic analysis of the Ala-38 (D38A) mutant. Unexpectedly, D38A has a catalytic turnover number (k(cat)) that is ca. 10(6)-fold greater than the value for D38N and only about 140-fold less than that for wild type. Kinetic studies as a function of pH show that D38A-catalyzed isomerization involves two groups, with pK(a) values of 4.2 and 10.4, respectively, in the free enzyme, which are assigned to Asp-99 and either Tyr-14 or Tyr-55. A mechanism for D38A is proposed in which Asp-99 is recruited as the catalytic base, with stabilization of the intermediate dienolate ion and the flanking transition states provided by hydrogen bonding from both Tyr-14 and Tyr-55. This mechanism is supported by the lack of detectable activity of the D38A/D99N, D38A/Y14F, and D38A/Y55F double mutants. PMID- 10727229 TI - 3-hydroxy-3-methylglutaryl-coenzyme A synthase reaction intermediates: detection of a covalent tetrahedral adduct by differential isotope shift 13C nuclear magnetic resonance spectroscopy. AB - Binding of [1,2-(13)C]acetyl-CoA to wild-type 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase is characterized by large upfield shifts for C1 (184 ppm, Deltadelta = 20 ppm) and C2 (26 ppm, Deltadelta = 7 ppm) resonances that are attributable to formation of the covalent [1,2 -(13)C]acetyl-S-enzyme reaction intermediate. NMR spectra of [1, 2-(13)C]acetyl-S-enzyme prepared in H(2)(16)O versus H(2)(18)O indicate a 0.055 ppm upfield shift of the C1 resonance in the presence of the heavier isotope. The magnitude of this (18)O-induced (13)C shift suggests that the 184 ppm resonance is attributable to a reaction intermediate in which C1 exhibits substantial carbonyl character. No significant shift of the C2 resonance occurs. These observations suggest that, in the absence of second substrate (acetoacetyl-CoA), enzymatic addition of H(2)(18)O to the C1 carbonyl of acetyl-S-enzyme occurs to transiently produce a tetrahedral species. This tetrahedral adduct exchanges oxygen upon backward collapse to re-form the sp(2) hybridized thioester carbonyl. In contrast with HMG-CoA synthase, C378G Zoogloea ramigera beta-ketothiolase, which also forms a (13)C NMR-observable covalent acetyl-enzyme species, exhibits no (18)O-induced shift. Formation of the [(13)C]acetyl-S-enzyme reaction intermediate of HMG-CoA synthase in D(2)O versus H(2)O is characterized by a time-dependent isotope-induced upfield shift of the C1 resonance (maximal shift = 0. 185 ppm) in the presence of the heavier isotope. A more modest upfield shift (0.080 ppm) is observed for C378G Z. ramigera beta ketothiolase in similar experiments. The slow kinetics for the development of the deuterium-induced (13)C shift in the HMG-CoA synthase experiments suggest a specific interaction (hydrogen bond) with a slowly exchangeable proton (deuteron) of a side chain/backbone of an amino acid residue at the active site. PMID- 10727230 TI - Heterogeneous glycosylation of immunoglobulin E constructs characterized by top down high-resolution 2-D mass spectrometry. AB - Posttranslational glycosylation is critical for biological function of many proteins, but its structural characterization is complicated by natural heterogeneity, multiple glycosylation sites, and different forms. Here, a top down mass spectrometry (MS) characterization is applied to three constructs of the Fc segment of IgE: Fcepsilon(3-4) (52 kDa) and Fcepsilon(2-3-4)(2) (76 kDa) disulfide-bonded homodimers. Fourier transform MS of a reduced sample of Fcepsilon(2-3-4) gave molecular masses of 37 527, 37 689, 37 851, and 38 014 Da, directly characterizing multiple glycoforms (hexose = 162 Da) without chromatographic separation. Limited proteolysis of the nonreduced Fcepsilon(2-3 4)(2) protein yielded a peptide mixture with molecular weight values that agreed with those expected from the DNA sequence. The single glycosylation site in these constructs was identified, and quantities were determined of five glycoforms that agreed within +/-2% of the molecular ion values. The 2-D mass spectrum of two glycosylated peptides showed these to have high-mannose structures, -GlcNAc (hex)(n)(), demonstrating that Fcepsilon(2-3-4) has a single such structure of n = 5-9. For a mutated sample of Fcepsilon(3-4), in addition to five glycoforms, MS showed a molecular discrepancy that could be assigned with proteolysis and 2-D mass spectra to the oxidation of two methionines and an additional residue difference. PMID- 10727231 TI - DNA sequence dependent and independent conformational changes in multipartite operator recognition by lambda-repressor. AB - Binding of regulatory proteins to multipartite DNA binding sites often occurs with protein-protein interaction, resulting in cooperative binding. The operators of bacteriophage lambda have several pairs of repressor binding sites (O(R)1 O(R)2, O(R)2-O(R)3, O(L)1-O(L)2, and O(L)2-O(L)3) separated by a variable number of base pairs, and thus, bacteriophage lambda is a model system for studying multipartite operator recognition by DNA-binding proteins. Near-UV circular dichroism spectra show that the DNA is distorted in O(R)1-O(R)2 and O(L)2-O(L)3 but much less so in O(R)2-O(R)3. Upon titration of lambda-repressor with single operator sites O(R)1, O(R)2, and O(R)3, it was observed that the tryptophan fluorescence quenches to different degrees, suggesting different conformations of the protein in the three DNA-protein complexes. Acrylamide quenching of tryptophan fluorescence of lambda-repressor bound to these single operators also shows different Stern-Volmer constants, supporting the above conclusions. Titration of lambda-repressor with oligonucleotides containing pairs of operator sites also causes different degrees of fluorescence quenching. In particular, fluorescence quenching induced by O(R)1-O(R)2 binding is less than the quenching induced by either of the single operators alone, suggesting additional conformational changes upon establishment of protein-protein contact. Stern Volmer constants obtained from acrylamide quenching of tryptophan fluorescence of lambda-repressor bound cooperatively to pairs of operator sites are different from those of the single-operator-site-bound repressors. For example, O(R)2-O(R)3 bound repressor has significantly higher acrylamide quenchable components than either of the O(R)2- or O(R)3-bound proteins, again suggesting additional conformational changes upon establishment of protein-protein contact. We conclude that the strategy of recognition of multipartite operator by lambda-repressor is complex and varied, involving conformational changes in both DNA and protein that are determined by the separation of the binding sites as well as the nucleic acid sequence. PMID- 10727232 TI - Necessity of conserved asparagine residues in the leucine-rich repeats of platelet glycoprotein Ib alpha for the proper conformation and function of the ligand-binding region. AB - The polypeptides of the platelet von Willebrand factor (vWf) receptor, the GP Ib IX-V complex, each contain tandem repeats of a sequence that assigns them to the leucine-rich repeat protein family. Here, we studied the role of conserved Asn residues in the leucine-rich repeats of GP Ib alpha, the ligand-binding subunit of the complex. We replaced the Asn residue in the sixth position of the first or sixth leucine-rich repeat (of seven) either with a bulky, charged Lys residue or with a Ser residue (sometimes found in the same position of other leucine-rich repeats) and studied the effect of the mutations on complex expression, modulator dependent vWf binding, and interactions with immobilized vWf under fluid shear stress. As predicted, the Lys substitutions yielded more severe phenotypes, producing proteins that either were rapidly degraded within the cell (mutant N158K) or failed to bind vWf in the presence of ristocetin or roll on immobilized vWf under fluid shear stress (mutant N41K). The binding of function-blocking GP Ib alpha antibodies to the N41K mutant was either significantly reduced (AK2 and SZ2) or abolished (AN51 and CLB-MB45). Ser mutations were tolerated much better, although both mutants demonstrated subtle defects in vWf binding. These results suggest a vital role for the conserved asparagine residues in the leucine-rich repeats of GP Ib alpha for the structure and functions of this polypeptide. The finding that mutations in the first leucine-rich repeat had a much more profound effect on vWf binding indicates that the more N-terminal repeats may be directly involved in this interaction. PMID- 10727233 TI - Interaction between 6-hydroxydopamine and transferrin: "Let my iron go". AB - The dopamine analogue 6-hydroxydopamine (6-OHDA) is selectively toxic to catecholaminergic neurons. Because of its selectivity for neuroblastic cells in the sympathetic nervous system lineage, 6-OHDA has been suggested as a chemotherapeutic agent for targeted treatment of patients with neuroblastoma. We tested the hypothesis that the toxicity of 6-OHDA is caused by its interaction with serum ferric transferrin (Fe-TF) resulting in release of iron. We further hypothesized that this iron, through its redox-cycling by 6-OHDA, triggers generation of reactive oxygen species. 6-OHDA-induced release of iron from Fe-TF was demonstrated by: (1) low-temperature EPR spectroscopic evidence for decay of the characteristic Fe-TF signal (g = 4.3) and appearance of the high-spin signal from iron chelated by 6-OHDA oxidation products; (2) spectrophotometric detection of complexing of iron with the Fe(2+) chelator ferrozine; (3) redox-cycling of ascorbate yielding EPR-detectable ascorbate radicals; and (4) generation of hydroxyl radicals as evidenced by EPR spectroscopy of their adduct with a spin trap, 5, 5'-dimethylpyrroline oxide (DMPO) (DMPO-OH). Our low-temperature EPR studies showed that in human plasma, 6-OHDA caused iron release only under nitrogen gas but not under air or oxygen. The absence of a 6-OHDA effect in plasma under aerobic conditions was most likely due to its ferroxidase activity [with consequent reuptake of Fe(III) by apoTF] and catalytic oxidation of 6-OHDA by ceruloplasmin. Modeling of these plasma activities by a stable nitroxide radical, 2,2,6, 6-tetramethyl-1-piperidinyloxy (TEMPOL), resulted in protection of plasma Fe-TF against iron release under nitrogen. Parenteral administration of 6-OHDA to mice resulted in iron release from Fe-TF as evidenced by transformation of the Fe-TF low-temperature EPR signal that was indistinguishable from that seen in in vitro models. In addition, administration of the iron chelator deferoxamine (DFO) to mice prior to administration of toxic doses of 6-OHDA resulted in a decrease in activity impairment of mice as compared to that seen with 6-OHDA alone. These findings underscore the physiological and pharmacological relevance of 6-OHDA-mediated iron release from Fe-TF and suggest that iron chelators (DFO) may be used for prevention of 6-OHDA toxicity. PMID- 10727234 TI - CC chemokine MIP-1 beta can function as a monomer and depends on Phe13 for receptor binding. AB - The reported structures of many CC chemokines show a conserved dimer interface along their N-terminal region, raising the possibility that the quaternary arrangement of these small immune proteins might influence their function. We have produced and analyzed several mutants of MIP-1 beta having a range of dimer K(d) values in order to determine the significance of dimerization in receptor binding and cellular activation. NMR and analytical ultracentrifugation were used to analyze the oligomeric state of the mutants. Functional relevance was determined by receptor binding affinity and the ability to invoke intracellular calcium release from CHO cells transfected with the MIP-1 beta receptor CCR5. The monomeric N-terminally truncated mutant MIP(9) was able to bind the CCR5 receptor with a K(i) of 600 pM but displayed weak agonistic properties, while the monomeric mutant P8A still retained the ability to tightly bind (K(i) = 480 pM) and to activate (EC(50) = 12 nM) the receptor. These data suggest that the MIP-1 beta dimer is not required for CCR5 binding or activation. In addition, we identified Phe13, the residue immediately following the conserved CC motif in MIP 1 beta, as a key determinant for binding to CCR5. Replacement of Phe13 by Tyr, Leu, Lys, and Ala showed the aromatic side chain to be important for both binding to CCR5 and chemokine dimerization. PMID- 10727235 TI - The choline binding site of phospholipase C (Bacillus cereus): insights into substrate specificity. AB - The phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLC(Bc)) is a 28.5 kDa enzyme with three zinc ions in its active site. The roles that a number of amino acid residues play as zinc ligands and in binding and catalysis have been elucidated. Recent mechanistic studies indicate that the rate of the reaction is limited by a proton-transfer step during chemical hydrolysis and not substrate binding or product release. An X-ray structure of PLC(Bc) complexed with a phosphonate inhibitor related to phosphatidylcholine revealed that the three amino acid residues Glu4, Tyr56, and Phe66 comprise the choline binding pocket. However, because the contributions that these three residues make to substrate recognition and specificity were unknown, a series of site-specific mutants for Glu4, Tyr56, and Phe66 were constructed by PCR mutagenesis. On the basis of a comparison of their respective CD spectra and melting temperatures, it appears that the mutants adopt folded structures in solution that are virtually identical to that of wild-type PLC(Bc). The kinetic parameters k(cat) and K(m) for the hydrolysis of the three soluble substrates 1, 2-dihexanoyl-sn-glycero-3 phosphocholine (C6PC), 1, 2-dihexanoyl-sn-glycero-3-phosphoethanolamine (C6PE), and 1, 2-dihexanoyl-sn-glycero-3-phospho-L-serine (C6PS) at concentrations below their corresponding critical micelle concentration (cmc) values were determined for each mutant. Replacement of Phe66 with a nonaromatic residue dramatically decreased k(cat) (approximately 200-fold) and reduced PLC(Bc) activity toward C6PC, C6PE, and C6PS, whereas changes to Glu4 and Tyr56 typically led to much more modest losses in catalytic efficiencies. Mutations of Glu4 had relatively little effect upon k(cat) and K(m) for C6PS, but they significantly influenced K(m) for C6PC and C6PE. Replacing Tyr56 with nonaromatic residues also affects catalytic efficiency, albeit to a much lesser degree than the corresponding changes at position 66. However, the presence of an aromatic residue at position 56 seems to confer some substrate selectivity for C6PC and C6PE, which bear a positive charge on the headgroup, relative to C6PS, which has no net charge on the headgroup; this increase in specificity arises largely from a reduced k(cat) for C6PS. PMID- 10727236 TI - Role of a NifS-like protein from the cyanobacterium Synechocystis PCC 6803 in the maturation of FeS proteins. AB - In Azotobacter vinelandii and Escherichia coli NifS or NifS-like proteins are involved in FeS protein assembly by mobilizing sulfur from free cysteine. This sulfur together with Fe(2+) is then incorporated into apo-FeS proteins to form an FeS center. A different activity termed C-DES [for cyst(e)ine desulfurylase] was recently isolated from the cyanobacterium Synechocystis PCC 6714 which also mobilized sulfur and which was able to incorporate the FeS center into apoferredoxin. In the genome of the cyanobacterium Synechocystis PCC 6803, there are three open reading frames (orfs) that are similar to NifS and one that is similar to C-DES, indicating that this bacterium might contain both activities, NifS and C-DES. One orf from Synechocystis PCC 6803 encoding a NifS-like protein, slr0387, was overexpressed in E. coli and purified. The molecular mass of the recombinant protein was determined to be about 82 kDa, indicating that it is a homodimer. The absorption spectrum was typical for PLP-containing proteins with an absorption maximum at 390 nm at pH 9.0 and at 425 nm at pH 6.5. The pH dependence of the absorption spectrum correlated with enzyme activity. Maximal activity measured as sulfide production was observed between pH 8.5 and 10. The activity decreased at lower pH values and was undetectable at pH 5.5. pH dependent changes in the absorption spectrum and activity were attributed to protonation of the Schiff base formed by a lysine side chain and the PLP cofactor. Studies on substrate specificity demonstrated that cysteine derivatives other than cysteine methyl ester and cysteine-sulfinic acid could not serve as substrates for this enzyme. In particular, cystine was not a substrate for the Synechocystis NifS-like protein, whereas it is the best substrate for C-DES. In the presence of Fe(2+), cysteine, and a reductant, the NifS-like protein was able to produce holoferredoxin from apoferredoxin. The implications of two different activities for FeS center biosynthesis in Synechocystis are discussed. PMID- 10727237 TI - Activation of the human P-glycoprotein ATPase by trypsin. AB - The human MDR1 gene product, P-glycoprotein (Pgp), a tandemly duplicated molecule containing two putative ATP- and perhaps two drug-binding sites, is responsible for multidrug resistance in tumors. In this report, we characterized the effects of trypsinization of Pgp on its ATPase function. Incubation of Pgp-containing membranes with trypsin at a ratio of 1000:1 (w/w) resulted in a gradual increase in the basal- and the drug-stimulated ATPase activities of Pgp in a time dependent manner. The maximal basal-, verapamil-, and vinblastine-stimulated ATPase activities of the trypsinized Pgp were approximately 1.8-, 1.5-, and 1.75 fold higher than the activities of the native Pgp, respectively. Increased basal- and drug-stimulated ATPase activities of the Pgp were also observed when the ratio of membrane protein to trypsin in the incubation mixtures was raised to 10:1 (w/w). Immunoblotting analysis of Pgp tryptic digests using Pgp-specific NH(2)11, C219, and C494 antibodies together revealed the degradation of full length Pgp and formation of at least eight peptides migrating in the 36-60 kDa range. Immunoprecipitation reactions using NH(2)11 and C494 antibodies have suggested that the peptides originating from the NH(2) half of Pgp are in strong association with the COOH half of the peptide. These findings suggest that while Pgp fragments together exhibit the ATPase functional characteristics, Pgp possesses a cleavage activation site or region, and its cleavage leads to the activation of basal ATPase function of Pgp. PMID- 10727238 TI - Apolipoprotein C-II39-62 activates lipoprotein lipase by direct lipid-independent binding. AB - Apolipoprotein C-II (apoC-II) is an exchangeable plasma apolipoprotein and an endogenous activator of lipoprotein lipase (LpL). Genetic deficiencies of apoC-II and overexpression of apoC-II in transgenic mice are both associated with severe hyperlipidemia, indicating a complex role for apoC-II in the regulation of blood lipid levels. ApoC-II exerts no effect on the activity of LpL for soluble substrates, suggesting that activation occurs via the formation of a lipid-bound complex. We have synthesized a peptide corresponding to amino acid residues 39-62 of mature human apoC-II. This peptide does not bind to model lipid surfaces but retains the ability to activate LpL. Conjugation of the fluorophore 7-nitrobenz-2 oxa-1,3-diazole (NBD) to the N-terminal alpha-amino group of apoC-II39-62 facilitated determination of the affinity of the peptide for LpL using fluorescence anisotropy measurements. The dissociation constant describing this interaction was 0.23 microM, and was unchanged when LpL was lipid-bound. Competitive binding studies showed that apoC-II39-62 and full-length apoC-II exhibited the same affinity for LpL in aqueous solution, whereas the affinity for full-length apoC-II was increased at least 1 order of magnitude in the presence of lipid. We suggest that while the binding of apoC-II to the lipid surface promotes the formation of a high-affinity complex of apoC-II and LpL, activation occurs via direct helix-helix interactions between apoC-II39-62 and the loop covering the active site of LpL. PMID- 10727239 TI - Measurements of interbilayer forces and protein adsorption on uncharged lipid bilayers displaying poly(ethylene glycol) chains. AB - Poly(ethylene glycol) (PEG)-stabilized liposomes were recently shown to exhibit differences in cell uptake that were linked to the liposome charge. To determine the differences and similarities between charged and uncharged PEG-decorated liposomes, we directly measured the forces between two supported, neutral bilayers with terminally grafted PEG chains. The measurements were performed with the surface force apparatus. The force profiles were similar to those measured with negatively charged PEG conjugates of 1, 2-distearoyl-sn-glycero-3 phosphatidyl ethanolamine (DSPE), except that they lacked the longer ranged electrostatic repulsion observed with the charged compound. Theories for simple polymers describe the forces between end-grafted polymer chains on neutral bilayers. The force measurements were complemented by surface plasmon resonance studies of protein adsorption onto these layers. The lack of electrostatic forces reduced the adsorption of positively charged proteins and enhanced the adsorption of negatively charged ones. The absence of charge also allowed us to determine how membrane charge and the polymer grafting density independently affect protein adsorption on the coated membranes. Such studies suggest the physical basis of the different interactions of charged and uncharged liposomes with proteins and cells. PMID- 10727240 TI - An endogenous sulfated inhibitor of neuronal inositol trisphosphate receptors. AB - In cerebellum, inositol trisphosphate- (InsP(3)-) gated Ca channels play a key role in learning, though they exhibit a low sensitivity to InsP(3) compared to peripheral tissues. In the present study, the cerebellar InsP(3) receptor is shown to be associated with a novel inhibitor of InsP(3) binding. (3)H-InsP(3) binding studies indicated that this inositol trisphosphate receptor inhibitor (IRI) could completely inhibit InsP(3) binding to the purified cerebellar InsP(3) receptor and acted as a competitive inhibitor. Gel filtration of IRI showed a predominant peak at 6500 Da, though this peak appeared to be an aggregate (with a monomeric molecular mass of approximately 1500 Da). Mass spectrometry of IRI showed a predominant peak at 1635 m/z, consistent with this low molecular mass estimate. The inhibitory activity of IRI was prevented by pretreatment with aryl sulfatase, suggesting the presence of a critical sulfo ester in IRI. IRI was insensitive to proteases and organic extraction but bound to concanavalin A, suggesting that IRI is a sulfated glycan. IRI was present in cerebellum but below the level of detection in aorta. IRI was also present in the neuronal cell line N1E115 (which exhibits a low sensitivity to InsP(3)). We conclude that IRI is a novel endogenous sulfated inhibitor of the InsP(3) receptor that modulates the sensitivity of the InsP(3) receptor and thus may explain the low InsP(3) sensitivity of neurons. PMID- 10727241 TI - Active site determination of Gpi8p, a caspase-related enzyme required for glycosylphosphatidylinositol anchor addition to proteins. AB - Glycosylphosphatidylinositol (GPI) anchors are attached to newly synthesized proteins in the ER by a transamidation reaction during which a C-terminal GPI attachment signal is replaced by a preformed GPI precursor lipid. This reaction depends on GAA1 and GPI8, the latter belonging to a novel cysteine protease family. Homologies between this family and other Cys proteinases, such as caspases, pointed to Cys199 and His157 as potential active site residues. Indeed, gpi8 alleles mutated at Cys199 or His157 are nonfunctional, i.e., they are unable to suppress the lethality of Deltagpi8 mutants. The overexpression of these nonfunctional alleles in wild-type cells leads to the accumulation of the free GPI precursor lipid CP2, delays the maturation of the GPI protein Gas1p, and arrests cell growth. The dominant negative effect of the Cys199 mutant cannot be overcome by the simultaneous overexpression of Gaa1p. Most GPI8 alleles mutated in other conserved regions of the protein can complement the growth defect of Deltagpi8, but nevertheless accumulate CP2. CP2 accumulation, a delay in Gas1p maturation and a slowing of cell growth can also be observed when Gpi8p is depleted to 50% of its normal level in wild-type cells. The dominant negative effect of nonfunctional and partially functional mutant alleles can best be explained by assuming that Gpi8p works as part of a homo- or heteropolymeric complex. PMID- 10727242 TI - Effect of an alternative disulfide bond on the structure, stability, and folding of human lysozyme. AB - Human lysozyme has four disulfide bonds, one of which, Cys65-Cys81, is included in a long loop of the beta-domain. A cysteine-scanning mutagenesis in which the position of Cys65 was shifted within a continuous segment from positions 61 to 67, with fixed Cys81, has previously shown that only the mutant W64CC65A, which has a nonnative Cys64-Cys81 disulfide, can be correctly folded and secreted by yeast. Here, using the W64CC65A mutant, we investigated the effects of an alternative disulfide bond on the structure, stability, and folding of human lysozyme using circular dichroism (CD) and fluorescence spectroscopy combined with a stopped-flow technique. Although the mutant is expected to have a different main-chain structure from that of the wild-type protein around the loop region, far- and near-UV CD spectra show that the native state of the mutant has tightly packed side chains and secondary structure similar to that of the wild type. Guanidine hydrochloride-induced equilibrium unfolding transition of the mutant is reversible, showing high stability and cooperativity of folding. In the kinetic folding reaction, both proteins accumulate a similar burst-phase intermediate having pronounced secondary structure within the dead time of the measurement and fold into the native structure by means of a similar folding mechanism. Both the kinetic refolding and unfolding reactions of the mutant protein are faster than those of the wild-type, but the increase in the unfolding rate is larger than that of the refolding rate. The Gibbs' free-energy diagrams obtained from the kinetic analysis suggest that the structure around the loop region in the beta-domain of human lysozyme is formed after the transition state of folding, and thus, the effect of the alternative disulfide bond on the structure, stability, and folding of human lysozyme appears mainly in the native state. PMID- 10727243 TI - Effects of mutations on the thermodynamics of a protein folding reaction: implications for the mechanism of formation of the intermediate and transition states. AB - We have measured changes in heat capacity, entropy, and enthalpy for each step in the folding reaction of CD2.d1 and evaluated the effects of core mutations on these properties. All wild-type and mutant forms fold through a rapidly formed intermediate state that precedes the rate-limiting transition state. Mutations have a pronounced effect on the enthalpy of both the intermediate and folded states, but in all cases a compensatory change in entropy results in a small net free-energy change. While the enthalpy change in the folded state can be attributed to a loss of van der Waals interactions, it has already been shown that changes in the stability of the intermediate are dominated by changes in secondary structure propensity [Lorch et al. (1999) Biochemistry 38, 1377-1385]. It follows that the thermodynamic basis of beta-propensity is enthalpic in origin. The effects of mutations on the enthalpy and entropy of the transition state are smaller than on the ground states. This relative insensitivity to mutation is discussed in the light of theories concerning the nature of the rate limiting barrier in folding reactions. PMID- 10727244 TI - Disulfide linkage of the b and delta subunits does not affect the function of the Escherichia coli ATP synthase. AB - The ATP synthase of Escherichia coli is believed to act through a rotational mechanism in which the b(2)delta subcomplex holds the alphabeta hexamer stationary relative to the rotating gamma and epsilon subunits. We have engineered a disulfide bond between cysteines introduced at position 158 of the delta subunit and at a position just beyond the normal C-terminus of the b subunit. The formation of this disulfide bond verifies that the C-terminal region of b is proximal to residue 158 of delta. The disulfide bond does not affect the ability of the F(1)F(0) complex to hydrolyze ATP, couple ATP hydrolysis to the establishment of a proton gradient, or maintain a proton gradient generated by the electron transport chain. These results are consistent with a permanent association of b(2) with delta as suggested by the rotational model of enzyme function. PMID- 10727245 TI - Two-dimensional structure of beta-amyloid(10-35) fibrils. AB - Beta-amyloid (Abeta) peptides are the main protein component of the pathognomonic plaques found in the brains of patients with Alzheimer's disease. These heterogeneous peptides adopt a highly organized fibril structure both in vivo and in vitro. Here we use solid-state NMR on stable, homogeneous fibrils of Abeta(10 35). Specific interpeptide distance constraints are determined with dipolar recoupling NMR on fibrils prepared from a series of singly labeled peptides containing (13)C-carbonyl-enriched amino acids, and skipping no more that three residues in the sequence. From these studies, we demonstrate that the peptide adopts the structure of an extended parallel beta-sheet in-register at pH 7.4. Analysis of DRAWS data indicates interstrand distances of 5.3 +/- 0.3 A (mean +/- standard deviation) throughout the entire length of the peptide, which is compatible only with a parallel beta-strand in-register. Intrastrand NMR constraints, obtained from peptides containing labels at two adjacent amino acids, confirm the secondary structural findings obtained using DRAWS. Using peptides with (13)C incorporated at the carbonyl position of adjacent amino acids, structural transitions from alpha-helix to beta-sheet were observed at residues 19 and 20, but using similar techniques, no evidence for a turn could be found in the putative turn region comprising residues 25-29. Implications of this extended parallel organization for Abeta(10-35) for overall fibril formation, stability, and morphology based upon specific amino acid contacts are discussed. PMID- 10727246 TI - Proximal and distal effects on the coordination chemistry of ferric Scapharca homodimeric hemoglobin as revealed by heme pocket mutants. AB - The ferric form of the homodimeric hemoglobin from Scapharca inaequivalvis (HbI) displays a unique pH-dependent behavior involving the interconversion among a monomeric low-spin hemichrome, a dimeric high-spin aquomet six-coordinate derivative, and a dimeric high-spin five-coordinate species that prevail at acidic, neutral, and alkaline pH values, respectively. In the five-coordinate derivative, the iron atom is bound to a hydroxyl group on the distal side since the proximal Fe-histidine bond is broken, possibly due to the packing strain exerted by the Phe97 residue on the imidazole ring [Das, T. K., Boffi, A., Chiancone, E. and Rousseau, D. L. (1999) J. Biol. Chem. 274, 2916-2919]. To determine the proximal and distal effects on the coordination and spin state of the iron atom and on the association state, two heme pocket mutants have been investigated by means of optical absorption, resonance Raman spectroscopy, and analytical ultracentrifugation. Mutation of the distal histidine to an apolar valine causes dramatic changes in the coordination and spin state of the iron atom that lead to the formation of a five-coordinate derivative, in which the proximal Fe-histidine bond is retained, at acidic pH values and a high-spin, hydroxyl-bound six-coordinate derivative at neutral and alkaline pH values. At variance with native HbI, the His69 --> Val mutant is always high-spin and does not undergo dissociation into monomers at acidic pH values. The Phe97 --> Leu mutant, like the native protein, forms a monomeric hemichrome species at acidic pH values. However, at alkaline pH, it does not give rise to the unusual hydroxyl bound five-coordinate derivative but forms a six-coordinate derivative with the proximal His and distal hydroxyl as iron ligands. PMID- 10727247 TI - Novel human TEF-1 isoforms exhibit altered DNA binding and functional properties. AB - The transcriptional enhancer factor-1 (TEF-1) is a member of the TEA/ATTS domain family. TEF-1 binds to GT-IIC (GGAATG), SphI (AGTATG), SphII (AGCATG), and M-CAT (GGTATG) response elements and is involved in the transactivation of a variety of genes, including the SV40 large T antigen, mammalian muscle-specific genes, and human chorionic somatomammotropin genes. Also, TEF-1 acts as a transcriptional repressor in placental cells, possibly through interaction with the TATA binding protein (TBP), preventing TBP binding to the TATA box. Here we describe the cloning, tissue-specific expression pattern, and functional characterization of two novel TEF-1 isoforms, TEF-1beta and TEF-1gamma. These isoforms most likely arise from alternative splicing of mRNA transcribed from a single gene and involve substitutions and/or insertions in a region immediately following the DNA binding domain. TEF-1beta appears to be widely distributed like the prototypic TEF-1, designated TEF-1alpha, whereas TEF-1gamma exhibits a narrower tissue specific expression pattern that includes pancreas, kidney, and skeletal and heart muscle. The relatively limited sequence alterations among these isoforms cause significant changes in their DNA binding and transcriptional activities. TEF-1beta and TEF-1gamma bind to GT-IIC sequences with higher affinity and repress hCS promoter more efficiently than TEF-1alpha. These results suggest that each TEF-1 isoform may play unique regulatory roles in various tissues. PMID- 10727248 TI - Expression and stereochemical and isotope effect studies of active 4 oxalocrotonate decarboxylase PMID- 10727249 TI - Paracrine regulation of epithelial progesterone receptor by estradiol in the mouse female reproductive tract. AB - Regulation of progesterone receptor (PR) by estradiol-17beta (E(2)) in mouse uterine and vaginal epithelia was studied. In ovariectomized mice, PR expression was low in both vaginal stroma and epithelium, but high in uterine epithelium. E(2) induced PR in vaginal epithelium and stroma, but down-regulated PR in uterine epithelium. Analysis of estrogen receptor alpha (ERalpha) knockout (ERKO) mice showed that ERalpha is essential for E(2)-induced PR expression in both vaginal epithelium and stroma, and for E(2)-induced down-regulation, but not constitutive expression of PR in uterine epithelium. Regulation of PR by E(2) was studied in vaginal and uterine tissue recombinants made with epithelium and stroma from wild-type and ERKO mice. In the vaginal tissue recombinants, PR was induced by E(2) only in wild-type epithelium and/or stroma. Hence, in vagina, E(2) induces PR directly via ERalpha within the tissue. Conversely, E(2) down regulated epithelial PR only in uterine tissue recombinants constructed with wild type stroma. Therefore, down-regulation of uterine epithelial PR by E(2) requires stromal, but not epithelial, ERalpha. In vitro, isolated uterine epithelial cells retained a high PR level with or without E(2), which is consistent with an indirect regulation of uterine epithelial PR in vivo. Thus, E(2) down-regulates PR in uterine epithelium through paracrine mechanisms mediated by stromal ERalpha. PMID- 10727250 TI - Paracrine regulation of epithelial progesterone receptor and lactoferrin by progesterone in the mouse uterus. AB - The objective of this study was to determine whether uterine stromal and/or epithelial progesterone receptor (PR) is required for the antagonism by progesterone (P(4)) of estradiol-17beta (E(2)) action on expression of PR and lactoferrin in uterine epithelium. Uterine tissue recombinants were prepared with epithelium (E) and stroma (S) from wild-type (wt) and PR knockout (PRKO) mice: wt S+wt-E and PRKO-S+wt-E. P(4) action on epithelial PR expression was studied in wt S+wt-E and PRKO-S+wt-E tissue recombinants. E(2) down-regulated epithelial PR in both types of tissue recombinants, but P(4) blocked E(2)-induced down-regulation of epithelial PR only in wt-S+wt-E tissue recombinants. Thus, P(4) requires stromal PR to inhibit E(2)-induced down-regulation of epithelial PR. Epithelial PR is not sufficient in itself. The inhibitory effect of P(4) on lactoferrin expression was studied in 4 types of tissue recombinants (wt-S+wt-E, PRKO-S+wt-E, wt-S+PRKO-E, and PRKO-S+PRKO-E). E(2) induced lactoferrin in all 4 types of tissue recombinants. P(4) blocked E(2)-induced lactoferrin expression only in wt S+wt-E tissue recombinants. In wt-S+PRKO-E tissue recombinants, P(4) inhibited lactoferrin expression only partially. P(4) failed to block E(2)-induced lactoferrin expression in PRKO-S+wt-E and PRKO-S+PRKO-E tissue recombinants. Thus, both epithelial and stromal PR are essential for full P(4) inhibition of E(2)-induced lactoferrin expression. PMID- 10727251 TI - Ruminant relaxin in the pregnant one-humped camel (Camelus dromedarius). AB - We have determined the cDNA sequence of preprorelaxin in the pregnant one-humped camel by employing reverse transcription- and rapid amplification of cDNA ends polymerase chain reaction. Camel preprorelaxin consisted of 600 base pairs (bp) encoding a protein of 199 amino acids (aa) with a signal peptide of 25 aa (75 bp), a B domain of 28 aa (84 bp), a C domain of 121 aa (366 bp), and an A domain of 24 aa (72 bp). The N terminus of the C domain of camel prorelaxin contained the unique proline-rich repetitive sequence (-RPAP)(3)-(-K/RPAL-)(2), and within the B domain the classical -GRELVR- receptor binding motif was found. Camel preprorelaxin showed highest homology with porcine (74.6%) and equine (65.4%) relaxin. The ovary and the uteroplacental unit were a dual source of relaxin in the pregnant dromedary. Within the ovary, weak expression of relaxin was detected in large luteal cells of the mature corpus luteum. In the ovarian follicles, immunoreactive relaxin, but not relaxin mRNA, was detected in the granulosa and theca interna cell layer. Beginning at around Day 93 of gestation and coinciding with increasing interdigitation of the fetal villus with the underlying maternal endometrium, uterine luminal epithelial cells in the uteroplacental tissue expressed relaxin. Weak expression of immunoreactive relaxin, but not relaxin mRNA, was observed in villous trophoblast cells. Pseudostratified trophoblast cells at the base of the placental villi and multinucleate giant cells did not express relaxin. PMID- 10727252 TI - Energy metabolism in preimplantation bovine embryos derived in vitro or in vivo. AB - This study was an investigation of metabolism during bovine preimplantation development from the oocyte up to the hatched blastocyst derived in vitro or in vivo. Metabolism was determined by estimating the consumption of radiolabeled glucose, pyruvate, or lactate during a 4-h incubation period in a closed noninvasive system with NaOH as trap for the continuous collection of CO(2). The postincubation medium was analyzed for the presence of lactate. Embryonic metabolism from the matured oocyte to the 12-cell stage was more or less constant, with pyruvate being the preferred substrate. The first marked increase in oxidation of glucose occurred between the 12- and 16-cell stage. Compaction of morula and blastocyst expansion was accompanied by significant increases in oxidation of all three energy substrates. The incorporation of glucose increased steadily 15-fold from the 1-cell to the blastocyst stage. In general, the pattern of metabolism was similar between the embryos derived in vitro and in vivo but with some distinct differences. The most apparent feature of glucose metabolism by in vitro-produced embryos was a 2-fold higher rate of aerobic glycolysis as compared to that in their in vivo counterparts. In vitro-matured oocytes produced measurable amounts of lactate, whereas in vivo-matured oocytes exhibited a significantly lower metabolic activity and did not produce any lactate. When in vivo-collected embryos were preexposed to culture conditions, lactate production increased significantly and at the hatched blastocyst stage matched that of their in vitro counterparts. In vitro-produced embryos up to the 8-cell stage oxidized significantly higher amounts of lactate and had a lower ratio of pyruvate-to lactate oxidation than the in vivo-obtained embryos. The results of this study show that under our culture conditions, important differences exist at the biochemical level between bovine embryos produced in vitro and those generated in vivo that may well affect the developmental capacity. PMID- 10727253 TI - Modification of survival rate of mouse embryos developing in heterozygous females for ovum mutant gene. AB - The DDK syndrome (polar infertility) is caused by an incompatibility system due to the ovum mutant (Om) locus. For brevity, the following gene symbols are used in the present report: DDK allele, Om; C57BL/6Cr allele, +. In this investigation, we first attempted to introduce the Om allele of DDK strain into the genetic background of C57BL/6Cr strain. The attempt resulted in the production of no young at the third generation of successive backcrosses. Secondly, mating experiments were performed with heterozygous (Om/+) females having background genes of C57BL/6Cr and DDK strains in the ratios 1:1(B1D), 3:1(B3D), 7:1(B7D), and 15:1(B15D). The survival rate of the embryos as judged by the percentage number of live fetuses/number of corpora lutea at Day 12 of pregnancy was 41.3 +/- 3.2%, 27.3 +/- 3. 2%, 16.4 +/- 3.3%, and 11.3 +/- 3.2% (mean +/- SEM) in the B1D, B3D, B7D, and B15D females, respectively, when they were mated with C57BL/6Cr males. Furthermore, the increased embryonic mortality in the heterozygous (Om/+) females with more background genes of C57BL/6Cr strain was found to be due to a failure in blastocyst formation, as in the DDK syndrome. The parallelism between the proportion of C57BL/6Cr background genes and embryonic mortality has led to a hypothesis proposing the participation of a modifier gene, namely that a mechanism similar to allelic exclusion may be working in the synthesis of cytoplasmic factor of eggs and that only the Om allele is activated during oogenesis to produce DDK-type cytoplasmic factor in heterozygous (Om/+) females having a modifier gene in the homozygous state. PMID- 10727254 TI - Regulation of steady-state luteinizing hormone messenger ribonucleic acid levels, de novo synthesis, and release by sex steroids in primary pituitary cell cultures of male African catfish, Clarias gariepinus. AB - Primary pituitary cell cultures from sexually mature adult male African catfish, Clarias gariepinus, were used to study the regulation of LH biosynthesis by sex steroids. The cell cultures were exposed to testosterone (T), estradiol (E(2)), or 5alpha-dihydrotestosterone (DHT), a nonaromatizable analogue of T, and to the likewise nonaromatizable 11-ketotestosterone (KT) and 11beta hydroxyandrostenedione (OHA), physiologically relevant androgens in fish. Both T and E(2) elevated glycoprotein alpha (GPalpha) and LHbeta steady-state mRNA levels (quantified by RNase protection assay), de novo synthesis (metabolic incorporation of radioactive amino acids and subsequent immune precipitation of LH), and release of preferentially newly synthesized LH, while DHT had no effect. Inhibiting the aromatase activity abolished the stimulatory effects of T. The effects of E(2) on LH mRNA levels and de novo synthesis were dose dependent. Incubation with 10 ng/ml KT elevated GPalpha and LHbeta mRNA levels, while other concentrations of KT or all concentrations of OHA tested had no effect. The amount of newly synthesized LH, on the other hand, was decreased dose-dependently by OHA but not by KT. Since this OHA-induced decrease did not change the specific activity (dpm immune precipitable [(3)H]-LH/ng immune-reactive LH) of LH, we hypothesize that OHA exerted its effect by activating a crinophagic breakdown of secretory granules in catfish gonadotrophs. Electron microscopic examination of gonadotrophs after in vitro exposure to 50 ng OHA/ml revealed that breakdown organelles had increased in size significantly. We conclude that the balanced production of aromatizable (mainly stimulatory) and 11-oxygenated androgens (mainly inhibitory) may be an important factor in regulating the amounts of LH available for secretion in male African catfish. PMID- 10727255 TI - A specific inhibitor of p34(cdc2)/cyclin B suppresses fertilization-induced calcium oscillations in mouse eggs. AB - Fertilization-induced Ca(2+) oscillations in mouse eggs cease at the time of pronuclear formation when maturation-promoting factor (MPF) is inactivated, but the Ca(2+) oscillations are ceaseless if eggs are arrested at metaphase by colcemid, which maintains the activity of MPF. To determine the possible role of MPF in regulation of cytoplasmic Ca(2+) excitability, roscovitine, a specific inhibitor of p34(cdc2)/cyclin B kinase, was used to inactivate MPF, and its effect on fertilization-induced Ca(2+) oscillations was investigated. Our results showed that roscovitine at >/= 50 microM suppressed fertilization-induced Ca(2+) oscillations in normal and colcemid-treated metaphase II (MII) eggs after the first 1-2 Ca(2+) spikes. Roscovitine inhibition of fertilization-induced Ca(2+) oscillations could be reversed by extensive washing of the eggs. Histone H1 kinase activity in colcemid-treated MII eggs was similarly inhibited by roscovitine, which suggested that the cessation of fertilization-induced Ca(2+) oscillations is due to the inactivation of MPF. Thimerosal-induced Ca(2+) oscillations in Ca(2+)-, Mg(2+)-free medium was also suppressed by roscovitine, suggesting a general inhibitory effect of roscovitine on Ca(2+) oscillations. The inhibition may be achieved by disruption of Ca(2+) release and refilling of the calcium store. Thapsigargin, an inhibitor of the endoplasmic reticulum Ca-ATPase, induced significantly less Ca(2+) release in roscovitine-treated eggs than in the non-drug-treated eggs. Taken together, our results suggest that MPF plays an important role in regulation of the cytoplasmic Ca(2+) excitability in mouse eggs. PMID- 10727256 TI - Macrophage migration inhibitory factor in the bovine corpus luteum: characterization of steady-state messenger ribonucleic acid and immunohistochemical localization. AB - Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine produced by T cells and macrophages. A number of tissues also produce MIF during states of active differentiation and/or proliferation. The purpose of this study was to determine whether MIF is present in the corpus luteum (CL). The steady state mRNA for MIF was examined in CL by Northern analysis on Day 5, Days 9-12, and Day 18 of the estrous cycle and at 0.5, 1, 4, 12, 24, and 36 h after a luteolytic injection of prostaglandin F(2alpha) (PGF(2alpha)) (n = 4 CL per time point). The greatest amount of MIF mRNA was observed in Day 5 CL compared with midcycle and Day 18 CL. Messenger RNA for MIF in CL collected 0.5 h post PGF(2alpha) was greater than in midcycle and all other regressing CL. Immunohistochemical analysis (n = 4) revealed that MIF was present in the bovine CL throughout the estrous cycle and appeared to be localized to large luteal cells. It was concluded that MIF is produced within the bovine CL, mRNA expression is maximal in the early CL, and the protein is primarily localized to large luteal cells. The functional significance of MIF remains to be determined. PMID- 10727257 TI - Development of time-resolved immunofluorometric assays for rat follicle stimulating hormone and luteinizing hormone and application on sera of cycling rats. AB - The aim of this study was to develop time-resolved immunofluorometric assays (TR IFMA) for measuring rat (r)FSH and rLH. The advantages of these IFMAs are higher sensitivity due to lower background values, higher specificity as only intact molecules of FSH and LH can be measured, and a very long shelf life of the nonradioactive biotin antigens compared with radiolabeled iodine antigens. For rFSH, IFMAs are lacking, while for rLH, if present, the resources for antibodies are scarce or the mouse monoclonal antibodies (mMAbs) against LHalpha are inactive with FSH. Thus specific antibodies need to be obtained. With the final TR-IFMAs, rFSH and rLH levels were assessed during the estrous cycle and compared with those obtained with the more classical RIAs and fluoroimmunoassays (FIAs). Two IFMAs for rFSH were developed with mMAbs against the recombinant human (rec h)FSHbeta subunit (FSH56A) attached to the wall and two different rabbit polyclonal antibodies (PAbs) against the alpha subunit of rec hFSH (R93-2705) or recombinant rat (rec r)LH (R95-2715) conjugated with biotin as signal antibody. With both IFMAs, rFSH holo-molecules can be measured. Rat FSH standards could be assessed between 0.02 and 10 ng/ml with a detection limit of 0.05 and 0.24 ng/ml in buffer and serum, respectively. These detection limits in four IFMAs were 8- to 16-fold lower than those in RIAs and FIAs. This detection level allowed the measurement of FSH levels in serum of hypophysectomized (HYPEX) rats at 0.18 ng/ml. In serum of cycling rats, the FSH levels of the IFMA were 2-fold lower than those of the FIA, while in ovariectomized (OVX) rats the IFMA levels were comparable. A peak level of FSH was found during proestrus of Day 2 and gestation with both RIA and FIA, but with IFMAs at gestation only. An IFMA for rLH was set up with mMAb (hCG77A) reacting with rLHbeta as capture and rabbit PAb to rec rLHalpha (R95-2712) as signal antibody. Rat LH standard could be assessed between 0.001 and 10 ng/ml with a detection limit of 0.012 and 0.1 ng/ml in buffer and serum, respectively, which was 8-fold lower than that in RIA/FIA. In serum of HYPEX rats, LH was undetectable (< 0.04 ng/ml), whereas a high background level of 2.5 ng/ml was measured in the FIA. In serum of cycling rats, only a very low LH level of 0.14 ng/ml was measured, which strongly deviated from the level of 3.46 ng/ml with an FIA. The load of LH in serum of OVX rats was 2.91 ng/ml, which was 12-fold lower than that for the FIA. The peak level of LH was detected on proestrus Day 2 with RIA, FIA, and IFMA. In conclusion, two IFMAs for rFSH and one for rLH have been developed with high sensitivity and specificity for intact gonadotropins. The LH pattern during the estrous cycle was comparable between IFMA, RIA, and FIA, although the overall level in the IFMA was much lower, as were HYPEX levels. The FSH pattern differed only on proestrus Day 2 in the IFMA from that of RIA/FIA, showing a peak level with RIA/FIA and a basal level with the IFMA. This implies that in RIA/FIA measurements, proteins other than intact FSH and LH interfere with the analysis at proestrus Day 2 for FSH and in HYPEX, cycling, and OVX rats for LH. PMID- 10727258 TI - Urokinase redistribution from the secreted to the cell-bound fraction in granulosa cells of rat preovulatory follicles. AB - Plasminogen activators (PAs) have been shown to be synthesized in ovarian follicles of several mammalian species, where they contribute to the ovulation process. The type of PA secreted by granulosa cells is species-specific. In fact, whereas in the rat, gonadotropins stimulate tissue-type PA (tPA) production, the same hormonal stimulation induces urokinase PA (uPA) secretion in mouse cells. To investigate in more detail the hormonal regulation of this system, we used the rat ovary as a model in which we analyzed the production of PAs by theca interstitial (TI) and granulosa cells obtained from preovulatory follicles after gonadotropin stimulation. In untreated rats, uPA was the predominant enzyme in both TI and granulosa cells. After hormonal stimulation, an increase in uPA and tPA activity was observed in both cell types. Surprisingly, only tPA mRNA increased in a time-dependent manner in both cell types, while uPA mRNA increased only in TI cells and actually decreased in granulosa cells. These divergent results between uPA enzyme activity and mRNA levels in granulosa cells were explained by studying the localization of the enzyme. Analysis of granulosa cell lysates showed that after hormonal stimulation, 60-70% of the uPA behaved as a cell-associated protein, suggesting that uPA, already present in the follicle, accumulates on the granulosa cell surface through binding to specific uPA receptors. The redistribution of uPA in granulosa cells and the differing regulation of the two PAs by gonadotropins in the rat ovary suggest that the two enzymes might have different functions during the ovulation process. Moreover, the ability of antibodies anti-tPA and anti-uPA to significantly inhibit ovulation only when coinjected with hCG confirmed that the PA contribution to ovulation occurs at the initial steps. PMID- 10727259 TI - A role for hepatocyte growth factor during early postimplantation growth of the placental lineage in mice. AB - Hepatocyte growth factor (HGF) is implicated in placental development; hgfr and hgf null mutant embryos develop placental insufficiency and lethality at 11.5 days (E11.5) after fertilization. The function of HGF in placentation at implantation (E4.5) has not been studied. Using reverse transcription-polymerase chain reaction, we detected HGF receptor (HGFR) mRNA in preimplantation embryos and in cultured blastocyst outgrowths. HGFR protein was detected in trophoblast cells in blastocyst outgrowths. HGF mRNA was not detected at these stages but was detected in the uterus at E5.5. Using in situ hybridization, we detected HGF mRNA in the mesometrial uterus, near the embryo, from E6.5 through E8.5. At E8.5, HGFR mRNA was detected in the chorionic placenta, and HGF mRNA was detected in the allantois. The expression for HGF and HGFR suggested a maternal-to-embryonic communication before the development of the allantois. To test this, blastocyst outgrowths were cultured with HGF. HGF stimulated the outgrowth of trophoblasts in a time-dependent manner and stimulated the expression of proliferating cell nuclear antigen, but it did not scatter trophoblasts. HGF stimulated an increase in the trophoblast cell number, but caused a decrease in the total number of terminally differentiated trophoblasts expressing placental lactogen-1 protein. These data suggest that HGF stimulates the cell division, but not the differentiation, of trophoblast cells during implantation. PMID- 10727260 TI - Roles of gap junctional communication of cumulus cells in cytoplasmic maturation of porcine oocytes cultured in vitro. AB - Cumulus cells of the oocyte play important roles in in vitro maturation and subsequent development. One of the routes by which the factors are transmitted from cumulus cells to the oocyte is gap junctional communication (GJC). The function of cumulus cells in in vitro maturation of porcine oocytes was investigated by using a gap junction inhibitor, heptanol. Cumulus-oocyte complexes (COCs) were collected from the ovaries of slaughtered gilts by aspiration. After selection of COCs with intact cumulus cell layers and uniform cytoplasm, they were cultured in a medium with 0, 1, 5, or 10 mM of heptanol for 48 h. After culture in vitro, one group of oocytes was assessed for nuclear maturation and glutathione (GSH) content, and another group was assigned to in vitro fertilization and assessed for the penetrability of oocytes and the degree of progression to male pronuclei (MPN) of penetrated spermatozoa. At the end of in vitro maturation, the oocytes reached metaphase II at a high rate (about 80%) regardless of the presence of heptanol at various concentrations. Cumulus cell expansion and the morphology of oocytes cultured in the medium with heptanol were similar to those of control COCs matured without heptanol. The amount of GSH in cultured oocytes tended to decrease as the concentration of heptanol in the medium was increased. Although there was no difference in the rates of penetrated oocytes cultured in media with different concentrations of heptanol, the proportion of oocytes forming MPN after insemination decreased significantly (P < 0.01) at all concentrations tested. A higher rate of sperm (P < 0.01) failed to degrade their nuclear envelopes after penetration into the oocytes that were treated with heptanol. GJC between the oocyte and cumulus cells might play an important role in regulating the cytoplasmic factor(s) responsible for the removal of sperm nuclear envelopes as well as GSH inflow from cumulus cells. PMID- 10727261 TI - Selection of the dominant follicle in cattle: role of two-way functional coupling between follicle-stimulating hormone and the follicles. AB - The functional coupling between the declining portion of the FSH surge and the growing follicles of a wave was studied by treating heifers with a minimal dose of estradiol to decrease FSH concentrations without an associated change in LH concentrations. Estradiol treatment when the largest follicle reached >/= 6.0 mm (Hour 0) resulted in depression of both FSH concentrations and diameter of the largest follicle by Hour 8. The smaller follicles were also inhibited. These results supported the hypothesis that FSH continues to be needed by the growing follicles even when the FSH concentrations are decreasing during the declining portion of the FSH surge. Estradiol treatment when the largest follicle was >/= 8.5 mm (expected time of follicular deviation) also resulted in a transient decrease in both FSH concentrations and diameter of the largest follicle, but the diameters of the smaller follicles were not affected. These results supported the hypothesis that the low concentrations of FSH at the expected time of deviation, although inadequate for the smaller follicles, were required for continued growth of the largest follicle. In another study, ablation (Hour 0) of the largest follicle was done at >/= 7.5 mm vs. >/= 8.5 mm. The mean FSH concentrations for the 8.5-mm groups were greater for the ablation group than for the control group at Hours 8 and 12, but there was no difference between the 7.5-mm groups at any hour. These results supported the hypothesis that by the time the largest follicle reaches the expected beginning of deviation it has developed a greater capacity for suppressing FSH. It is postulated that the essence of the selection of a dominant follicle is a close two-way functional coupling between changing FSH concentrations and follicular growth. PMID- 10727262 TI - Trout ovulatory proteins: site of synthesis, regulation, and possible biological function. AB - The mRNA transcripts for trout ovulatory proteins (TOPs) are dramatically up regulated at the time of ovulation. Previous studies indicated that TOPs were produced by the ovaries and were also present in the coelomic fluid that bathes ovulated eggs. In the present study, Western analysis indicated that TOPs were not present in the coelomic fluid prior to ovulation and therefore must be secreted into the coelomic fluid in large quantities during and after ovulation. Using in situ hybridization and immunocytochemistry, TOP mRNA and proteins were localized to the granulosa cell layer of the postovulatory follicle. A whole follicle in vitro incubation system was used to look at the effects of various mediators on TOP mRNA and protein levels. Results of several different secondary messenger agonists suggest that TOPs are regulated through a G protein-mediated pathway that does not involve cAMP but may involve the activation of protein kinase C. Other agonists that had significant effects on TOP RNA and/or protein included transforming growth factor alpha (TGF-alpha), serine proteases, corticosteroids, bacterial lipopolysaccharide, and the nitric oxide generator SNAP ([+/-]-S-nitroso-N-acetylpenicillamine). Overall, while several compounds caused significant effects, none were able to reproduce the increase in TOP RNA and protein that occurs in vivo, suggesting that the natural mediator of TOPs may still be untested, or that a combination of mediators may be involved. Finally, coelomic fluid inhibited the growth of the Gram negative bacterium, P. aeruginosa, and this inhibition was lost following immunoprecipitation of TOPs. This suggests that one function of TOPs may be to protect ovulated eggs from bacterial infection. PMID- 10727263 TI - Studies in humans on the mechanism of potent spermicidal and apoptosis-inducing activities of vanadocene complexes. AB - We previously demonstrated that bis-cyclopentadienyl (Cp) complexes of vanadium(IV) (vanadocenes) are potent spermicidal and apoptosis-inducing agents. To gain further insight into the structure-function relationships controlling these two properties of vanadocenes, we have synthesized analogues in which the bis-Cp rings were substituted with one or five electron-donating methyl groups. The three complexes included vanadocene dichloride (VDC), bis(methylcyclopentadienyl) vanadium dichloride (VMDC), and bis(pentamethylcyclopentadienyl) vanadium dichloride (VPMDC). The concentration dependent effect of these vanadocenes on sperm-immobilizing activity (SIA), mitochondrial membrane potential (DeltaPsim), axonemal dynein ATPase activity, and tyrosine phosphorylation of global and axoneme-specific sperm proteins was assessed by computer-assisted sperm analysis, flow cytometry, colorimetry, and immunoblotting, respectively. Apoptosis-inducing ability was quantitated by the two-color flow cytometric terminal dideoxynucleotidyl transferase-based assay that labels 3'-hydroxyl ends of fragmented DNA. All three vanadocenes induced rapid sperm immobilization (T(1/2) < 15 sec). Substitution of the bis-Cp rings by five methyl groups augmented the SIA of VDC by 10-fold. The EC(50) values (50% inhibitory concentration) for VDC, VMDC, and VPMDC were 7.5 microM, 4.3 microM, and 0.7 microM, respectively. Whereas SIA of vanadocenes was apparent at low micromolar concentrations, the apoptosis-inducing property was evident only at higher micromolar concentrations. The concentrations of VDC, VMDC, and VPMDC required for 50% apoptosis were 49 microM, 67 microM, and 153 microM, and for 50% reduction in sperm DeltaPsim were 435 microM, 173 microM, and 124 microM, respectively. Spermicidal activity of vanadocenes was not dependent on the inhibition of ATPase or tyrosine phosphorylation of global and sperm axonemal proteins. Due to the ability of these vanadocene complexes to rapidly generate hydroxyl radicals in the presence of oxidant, our findings provide unprecedented evidence for a novel mechanism of action for spermicidal vanadocenes. The differential concentration-dependent spermicidal and apoptosis-inducing properties of vanadocenes gives them particular utility as a new class of vaginal contraceptives. PMID- 10727264 TI - Biochemical characterization of two ram cauda epididymal maturation-dependent sperm glycoproteins. AB - Rabbit polyclonal antibodies were raised against ram cauda epididymal sperm proteins solubilized by N-octyl-beta-D-glucopy-ranoside (anti-CESP) and against proteins of the fluid obtained from the cauda epididymidis (anti-CEF). The anti CESP polyclonal antibody reacted with several bands from 17 to 111 kDa with different regionalization throughout the epididymis. The strongest epitopes at 17 kDa and 23 kDa were restricted to the cauda epididymidis. The anti-CEF polyclonal antibody reacted mainly with a 17-kDa and a 23-kDa compound in the cauda sperm extract. These cauda epididymal 17- and 23-kDa proteins disappeared after orchidectomy, but they reappeared in the same regions after testosterone supplementation, indicating that they were secreted by the epithelium. The fluid and membrane 17- and 23-kDa antigens had a low isoelectric point and were glycosylated. The fluid 17- and 23-kDa proteins had hydrophobic properties: they were highly enriched in the Triton X-114 detergent phase and could be extracted from the cauda epididymal fluid by a chloroform-methanol mixture. These proteins were further purified, and their N-terminal sequences did not match any protein in current databases. A polyclonal antibody against the fluid 17-kDa protein recognized the protein in the cauda epididymal sperm extract and immunolocalized it on the sperm flagellum membrane and at the luminal border of all cells in the cauda epididymal epithelium. These results indicated that secreted glycoproteins with hydrophobic properties could be directly integrated in a specific domain of the sperm plasma membrane. PMID- 10727265 TI - Human decidual natural killer cells express the receptor for and respond to the cytokine interleukin 15. AB - The natural killer (NK) cells that are present in the uterine mucosa (decidua) during early pregnancy have a distinctive phenotype, CD56(bright) CD16(-). These cells have previously been shown to proliferate and be activated by interleukin (IL)-2. However, IL-2 is absent from the decidua and placenta, and we have therefore investigated whether IL-15 is present in the uterus and can act on decidual NK cells. Both IL-15 mRNA and protein were found in a variety of cells but particularly in decidual macrophages. IL-15 induced a proliferative response in decidual NK cells that was blocked by anti-IL-15 and was augmented by stem cell factor. The cytolytic activity of decidual NK cells against K562 was augmented. Interestingly, in contrast to IL-2, although activation with IL-15 resulted in some killing of JEG-3 choriocarcinoma cells, normal trophoblast cells remained resistant to lysis. These findings suggest that IL-15 is a candidate cytokine responsible for NK cell proliferation in vivo in the progesterone dominated secretory endometrium and early decidua. PMID- 10727266 TI - Translation of maternal messenger ribonucleic acids encoding transcription factors during genome activation in early mouse embryos. AB - Embryonic genome activation (EGA) in mice is sensitive to treatment with cycloheximide, indicating that protein synthesis plays an important role in mediating EGA. We hypothesized that regulated maternal mRNA recruitment may control the time of EGA by controlling the time of appearance of certain transcription factors (TFs). We also hypothesized that synthesis of other TFs may contribute to EGA independently of controlling the timing of EGA. To test these hypotheses, we used sucrose density gradient fractionation coupled to a quantitative reverse transcription-polymerase chain reaction method to compare polysomal mRNA abundances of specific TF mRNAs between metaphase II oocytes, 1 cell-stage embryos, and 2-cell-stage embryos. We observed a 2-cell-stage-specific increase in polysomal abundance of mouse TEA DNA binding domain 2 (mTEAD-2) mRNA, coincident with the first appearance of mTEAD activity in the early embryo. The mRNAs encoding Sp1, TATA binding protein, and cyclic AMP response element binding protein did not undergo translational recruitment, but exhibited differences in polysomal abundance. We also observed a continuous, high proportion in the polysomal fraction for the mRNA encoding ribosomal protein L23 mRNA, which contrasted with the patterns observed for other maternal transcripts. These observations are consistent with the hypothesis that regulated recruitment of maternal TF mRNAs may control the time of activation of some genes during EGA, and that continuous synthesis of other TFs, like Sp1, may facilitate EGA. PMID- 10727267 TI - Relationship between p62 and p56, two proteins of the mammalian cortical granule envelope, and hyalin, the major component of the echinoderm hyaline layer, in hamsters. AB - Mammalian cortical granules contain two polypeptides (p62 and p56) that are incorporated into the cortical granule envelope after fertilization and function in cleavage of the zygote and the preimplantation blastomeres. Since the echinoderm hyaline layer and mammalian cortical granule envelope are analogous, and since the hyaline layer protein, hyalin, functions in early echinoderm embryogenesis, this study was done to determine whether p62 and p56 and/or other components of the mammalian cortical granule envelope are related to hyalin. A polyclonal antibody (IL2) against purified S. purpuratus hyalin was shown by confocal scanning laser microscopy to bind to hamster cortical granules and to the cortical granule envelope of fertilized hamster oocytes and preimplantation embryos up to the blastocyst stage. In immunoblots, IL2 bound only to 62- and 56 kDa cortical granule proteins that were incorporated into the cortical granule envelope after fertilization. IL2 binding antigens appeared to be resynthesized by preimplantation embryos starting at the 2-cell stage of development. In vivo treatment of 2-cell-stage hamster embryos with IL2 inhibited blastomere cleavage, but treatment of morulae did not inhibit blastocyst implantation. These results support the idea that the mammalian cortical granule envelope proteins, p62/p56, share a common antigenic epitope(s) with echinoderm hyalin, and that p62/p56, like hyalin, play a role in early embryogenesis. PMID- 10727268 TI - Expression of matrix metalloproteinase-2, -9, and -14, tissue inhibitors of metalloproteinase-1, and matrix proteins in human placenta during the first trimester. AB - Matrix metalloproteinases (MMPs) are implicated in the degradation of extracellular matrix; they play important roles in the invasion of the trophoblast cell into the maternal endometrium during placentation. Previous studies have concentrated on comparison of MMP expression in trophoblast cells between the first and third trimester. But the dynamic expression of MMPs during the first trimester has not been reported. In the present study, the expression of MMP-2, -9, and -14 (membrane-type MMP-1) and the production of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) by cultured human cytotrophoblast cells from 6 to 11 wk of gestation were investigated. The cells were cultured under serum-free conditions. There was no MMP-9 secretion by the cells at Week 6, but from Week 7 to 11 the MMP-9 secretion increased gradually. Week 11 cells secreted more than 10-fold as much MMP-9 (167.7 +/- 18.8 ng/ml) as Week 7 (14.7 +/- 3.9 ng/ml) cultures. However, MMP-2 production declined from Week 6 to Week 11, and the production at Week 11 (32.3 +/- 8.1 ng/ml) was about one sixth that at Week 6 (205.7 +/- 27.2 ng/ml). The expression of mRNA transcripts for MMP-2 and MMP-9 correlated with enzyme secretion; we did not detect any MMP-9 mRNA signal in 20 microg total RNA extracted from cultured cells at Weeks 6, 7, and 8 of pregnancy, but a signal was apparent in Weeks 9 and 11. MMP-2 mRNA was expressed throughout the 6- to 11-wk period and exhibited a remarkable decline during this period. MMP-14 mRNA transcripts remained relatively stable from 6 to 11 wk. Significantly more TIMP-1 (P < 0.01) was detected in Week 9 (87.5 +/- 15.0 ng/ml) and Week 11 (169.1 +/- 30.2 ng/ml) media compared to Week 6 media (23.5 +/- 4.8 ng/ml), but we did not detect any TIMP-2 in the media of the tested cells. This study demonstrated that first-trimester human cytotrophoblast cells were able to produce abundant laminin, fibronectin, and vitronectin. However, we did not observe detectable secretion of collagen I and collagen IV. These data indicated that human trophoblast-derived MMPs and their inhibitors are intrinsically and developmentally regulated. The same cytotrophoblast cells that produced MMPs could also secrete various substrates for these enzymes. PMID- 10727269 TI - Effects of estrogens and xenoestrogens on androgen production by Atlantic croaker testes in vitro: evidence for a nongenomic action mediated by an estrogen membrane receptor. AB - The short-term effects of estrogens and xenoestrogens on testicular androgen production were investigated in an in vitro incubation bioassay system using testicular tissue from the Atlantic croaker (Micropogonias undulatus). Incubation of testicular tissue fragments with estradiol over the concentration range of 37 nM to 37 microM caused concentration-dependent decreases in gonadotropin stimulated 11-ketotestosterone (11-KT) production. The effect was specific for estrogens; progesterone, cortisol, and the synthetic androgen mibolerone did not significantly alter 11-KT production at similar concentrations. Diethylstilbestrol, the antiestrogen ICI 182,780, and several xenoestrogens including Kepone (chlordecone), 4-nonylphenol, and a hydroxylated polychlorinated biphenyl metabolite also significantly decreased gonadotropin-stimulated 11-KT production. The action of estradiol was rapid (<5 min) and was not blocked by actinomycin D and cycloheximide, inhibitors of transcription and translation, respectively. Moreover, estradiol conjugated to BSA, which cannot pass through the cell membrane, also caused a decrease in 11-KT production. In addition, an estrogen-binding moiety was identified in testicular membrane preparations that had a single class of high-affinity (K(d) 1.6 nM), saturable (1.2 nM), displaceable, finite (B(max) 0.03 nM, 26 fmol/g testis) binding sites specific for estrogens and exhibited rapid association (t(1/2) = 5 min), characteristics typical of steroid membrane receptors. Overall the relative binding affinities of estrogens, other steroids, antiestrogens, and xenoestrogens for the membrane preparation correlated with their activities in the androgen production bioassay, thereby satisfying the final criteria for the designation of this estrogen binding moiety as a steroid membrane receptor. The results demonstrate that estrogens and also probably xenoestrogens can act on the cell surface via a nongenomic mechanism to alter testicular androgen production in this vertebrate species. PMID- 10727270 TI - Full induction of rat myometrial 11beta-hydroxysteroid dehydrogenase type 1 in late pregnancy is dependent on intrauterine occupancy. AB - The 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD-1) enzyme catalyses the conversion of the biologically inert glucocorticoid 11-dehydrocorticosterone to active corticosterone (11-oxoreductase activity) in vivo, and it is dramatically up-regulated in uterine myometrium in the days leading up to parturition. 11beta-HSD-1 is likely to enhance local concentrations of glucocorticoid within the myometrium and thus facilitate uterine contractility, but the stimulus for the increase in myometrial 11beta-HSD-1 is unknown. The objective of the present study was to test whether the induction of myometrial 11beta-HSD-1 is dependent on uterine occupancy or systemic hormonal signals of late pregnancy. This involved use of a unilateral pregnancy (ULP) model in which the gravid and nongravid uterine horns are both exposed to the normal systemic hormonal milieu of pregnancy. Western blot analysis showed that the 11beta-HSD-1 signal was only partially induced in the nongravid horn of ULP rats on Day 22 of pregnancy (term: Day 23). Moreover, artificial distension of this nongravid horn had no effect on myometrial 11beta-HSD-1 immunoreactivity or bioactivity at either Day 16 or Day 22 of pregnancy. Removal of fetuses and placentas on Day 18 reduced myometrial 11beta-HSD-1 bioactivity 4 days later, and this effect was not overcome by artificial maintenance of uterine distension. In contrast, after fetectomy at Day 18 (i.e., removal of the fetus but not placenta), myometrial 11beta-HSD-1 bioactivity was largely maintained on Day 22, indicative of placental support for myometrial 11beta-HSD-1 over this period. In conclusion, our data show that full induction of myometrial 11beta-HSD-1 expression and associated 11-oxoreductase bioactivity late in rat pregnancy is dependent upon intrauterine occupancy. Although the hormonal milieu of late pregnancy appears to stimulate myometrial 11beta-HSD-1 marginally, full induction clearly requires an additional stimulus. Manipulations involving fetectomy and artificial uterine distension indicate that the placenta provides at least part of this stimulus, but uterine stretch does not appear to play a role. PMID- 10727271 TI - Physiological state of bull sperm affects fucose- and mannose-binding properties. AB - In cattle, sperm are stored in a reservoir in the caudal isthmus of the oviduct until the time of ovulation approaches. Bull sperm are trapped in the reservoir by binding to fucosylated molecules on the oviductal epithelium. Capacitated sperm lose binding affinity for the epithelium; therefore this study was undertaken to determine whether this occurs because capacitated bull sperm lose binding affinity for fucose. BSA conjugated to alpha-L-fucopyranosylphenyl isothiocyanate and fluorescein isothiocyanate (fuc-BSA-FITC) was used in conjunction with flow cytometry to monitor the capacity of bull sperm to bind fucose. Dead sperm were identified using ethidium homodimer and were excluded from analysis. BSA-FITC conjugated with mannose (man-BSA-FITC) and BSA-FITC were used as controls. When examined by epifluorescence microscopy, motile bull sperm that exhibited labeling by any of the probes were fluorescent over the acrosomal region of the plasma membrane. By flow cytometry, labeling of live sperm was greatest for sperm that had been washed in TALP medium and probed with fuc-BSA FITC (mean +/- SD:167 +/- 6.0 relative fluorescence units, collected in logarithmic mode). Labeling by fuc-BSA-FITC was lower in unwashed sperm (60 +/- 2.7) and in washed sperm with seminal plasma added back (56 +/- 8.0). Labeling was also reduced by centrifuging washed sperm through a Percoll step gradient (103 +/- 6.3) and by capacitating washed sperm in medium containing 10 microg/ml heparin (50 +/- 4.4). BSA-FITC labeling was barely detectable in all treatments. Man-BSA-FITC produced little labeling of washed sperm (22 +/- 0.6), as expected; however, intense labeling appeared over the acrosomal region of sperm incubated under capacitating conditions (128 +/- 21.6). It was concluded that removal of seminal plasma exposes fucose-binding sites, which are then lost or modified during capacitation, thereby allowing the release of sperm from the reservoir. At that time, mannose-binding sites are revealed or activated, which might serve to bind sperm to the zona pellucida. PMID- 10727272 TI - Cloning of a glucose phosphate isomerase/neuroleukin-like sperm antigen involved in sperm agglutination. AB - The mouse monoclonal antibody (mAb) A36 produced by us and shown to induce extensive, "tangled" sperm agglutination was used to isolate cDNAs encoding its cognate antigen. Three overlapping cDNA clones specifically recognized by the mAb were isolated from a human testis cDNA expression library in lambdagt11. Sequencing of these cDNAs yielded the complete nucleotide sequence of a 3 kilobase cDNA that encodes the mAb-related polypeptide, designated sperm antigen 36 (SA-36), composed of 558 deduced amino acids. SA-36 cDNA contained a 5' untranslated region of 234 nucleotides (nt), an open reading frame of 1674 nt, and a 3' untranslated region of 1138 nt. SA-36 cDNA displayed > 99% homology to glucose phosphate isomerase (GPI)/neuroleukin (NLK) mRNA. This surprising homology was confirmed in Western blots demonstrating that mAb A36 reacted specifically with GPI obtained from rabbit muscle and from baker's yeast. Moreover, polyclonal, monospecific antibodies produced against beta galactosidase/SA-36-3 fusion protein stained human spermatozoa and caused intensive agglutination of these cells in a manner similar to that with the mAb. Taken together, the data presented here demonstrated that mAb A36 cognate sperm surface antigen, encoded by SA-36 cDNA, is a GPI/NLK-like protein involved in sperm agglutination. PMID- 10727273 TI - Nucleolar proteins and nuclear ultrastructure in preimplantation bovine embryos produced in vitro. AB - The aim of the present investigation was to describe the basic cell biology of the postfertilization activation of rRNA genes using in vitro-produced bovine embryos as a model. We used immunofluorescence confocal laser scanning microscopy and transmission electron microscopy to study nucleolar development in the nuclei of embryos up to the fifth postfertilization cell cycle. During the first cell cycle (1-cell stage), fibrillarin, upstream binding factor (UBF), nucleolin (C23), and RNA polymerase I were localized to distinct foci in the pronuclei, and, ultrastructurally, compact spherical fibrillar masses were the most prominent pronuclear finding. During the second cell cycle (2-cell stage), the findings were similar except for a lack of nucleolin and RNA polymerase I labeling. During the third cell cycle (4-cell stage), fibrillarin, UBF, nucleophosmin, and nucleolin were localized to distinct foci. Ultrastructurally, spherical fibrillar masses that developed a central vacuole over the course of the cell cycle were observed. Early in the fourth cell cycle (8-cell stage), fibrillarin, nucleophosmin, and nucleolin were localized to small bodies that with time developed a central vacuole. UBF and topoisomerase I were localized to clusters of small foci. Ultrastructurally, spherical fibrillar masses with a large eccentric vacuole and later small peripheral vacuoles were seen. Late in the fourth cell cycle, nucleophosmin and nucleolin were localized to large shell like bodies; and fibrillarin, UBF, topoisomerase I, and RNA polymerase I were localized to clusters of small foci. Ultrastructurally, a presumptive dense fibrillar component (DFC) and fibrillar centers (FCs) were observed peripherally in the vacuolated spherical fibrillar masses. Subsequently, the presumptive granular component (GC) gradually became embedded in the substance of this entity, resulting in the formation of a fibrillo-granular nucleolus. During the fifth cell cycle (16-cell stage), a spherical fibrillo-granular nucleolus developed from the start of the cell cycle. In conclusion, the nucleolar protein compartment in in vitro-produced preimplantation bovine embryos is assembled over several cell cycles. In particular, RNA polymerase I and topoisomerase I are detected for the first time late during the fourth embryonic cell cycle, which coincides with the first recognition of the DFC, FCs, and GC at the ultrastructural level. PMID- 10727274 TI - Regulation of calcitonin gene-related peptide expression in dorsal root ganglia of rats by female sex steroid hormones. AB - Calcitonin gene-related peptide (CGRP), a potent vasodilator primarily synthesized in dorsal root ganglia (DRG) neurons, has been shown to decrease vascular resistance and thus regulate blood flow to a variety of organs in rats. Serum CGRP levels in the human have been reported to increase with pregnancy and decrease postpartum. It has been suggested that female sex steroid hormones play a role in cardiovascular function, but the mechanisms are unknown. In this study, we examined the effects of estradiol-17beta (E(2)) and progesterone (P(4)) on the expression of CGRP in DRG in adult rats both in vivo and in vitro. Ovariectomized (ovx) animals were injected s.c. with 5 microg E(2), 4 mg P(4), or 5.0 microg E(2) + 4 mg P(4) in 0.5 ml sesame oil or with oil only, and groups of 4 rats were killed at 0, 24, or 48 h. DRGs were then removed and analyzed for CGRP mRNA and immunoreactive (i-)CGRP content by Northern blotting and RIA, respectively. Primary cultures of DRG neurons from adult female rats were used to assess the effects of varying doses of E(2) (1, 10, 100 nM), P(4) (10, 100, 1000 nM), or E(2) (10 nM) + P(4) (100 nM) in the absence or presence of nerve growth factor (NGF; 20 ng/ml); and CGRP mRNA content in the cells and i-CGRP in the medium were quantitated at 24 or 48 h after incubation. Results of in vivo studies showed that E(2) caused a significant increase in CGRP mRNA at 24 h (1.8-fold) and in i CGRP levels both at 24 h (2. 8-fold) and at 48 h (3.4-fold) in DRG of ovx rats. P(4) also stimulated expression of both CGRP mRNA and i-CGRP. In the in vitro studies, either E(2) or P(4) alone or the two in combination were without effect on CGRP expression in cultured DRG neurons at all the doses tested. However, in the presence of NGF, both CGRP mRNA and peptide levels were significantly enhanced by E(2), P(4), and E(2)+P(4) in a time-dependent (2.0- to 2.8-fold at 24 h, 3.0- to 5. 0-fold at 48 h) and dose-dependent manner, with maximal effects achieved at 1.0 nM (E(2)) and 100 nM (P(4)) at 24 h of incubation. In summary, both E(2) and P(4), either alone or in combination, stimulate CGRP peptide synthesis in DRG neurons through increasing CGRP mRNA. The effects of these steroid hormones are mediated through amplifying the NGF-induced synthesis of CGRP in these neurons. Thus, we propose that the cardiovascular functions of female sex steroid hormones may be mediated, at least in part, by the up regulation of neuronal CGRP synthesis, via NGF-mediated mechanisms. PMID- 10727275 TI - Regulation of tissue inhibitor of metalloproteinases-1 in rat Sertoli cells: induction by germ cell residual bodies, interleukin-1alpha, and second messengers. AB - In the testis, FSH has been shown to induce the expression and secretion of tissue inhibitor of metalloproteinases-1 (TIMP-1) from Sertoli cells in vitro. This study was performed to elucidate further the cellular origin of testicular TIMP-1 and its expression by hormonal and paracrine factors. This is the first report on the expression of testicular TIMP-1 in vivo. TIMP-1 mRNA in whole testis was decreased after hypophysectomy and strongly increased by the injection of FSH-S17 to hypophysectomized rats. Primary cultures of both peritubular and Sertoli cells showed basal expression of TIMP-1 mRNA. In contrast, we were unable to detect TIMP-1 mRNA in Leydig cells, freshly isolated immature germ cells (primary spermatocytes and spermatids), or residual bodies. We further show that treatment of Sertoli cells with 8-(4-chlorophenyl)thio-cAMP (8-CPTcAMP) in combination with 12-O-tetradecanoylphorbol 13-acetate (TPA) or Ca(2+) inducers (calcium ionophore A23187 or thapsigargin) had additive (TPA) and synergistic effects (Ca(2+)) on the level of TIMP-1 mRNA and secreted protein. We also show that both the level of TIMP-1 mRNA and secreted protein from Sertoli cells were strongly increased by residual bodies, as well as by the cytokine interleukin 1alpha. TIMP-1 was not up-regulated by either 8-CPTcAMP or interleukin-1alpha in peritubular cells. In contrast to the regulated secretory fraction of TIMP-1, we also detected constitutively expressed immunoreactive TIMP-1 in the nucleus of Sertoli cells, suggesting a role of nuclear TIMP-1 in these cells. In conclusion, our data show that secretion of TIMP-1 from Sertoli cells is highly regulated by hormonal and local processes in the testis, indicating that TIMP-1 is of physiological importance during both testicular development and spermatogenesis. PMID- 10727276 TI - Effect of dose of prostaglandin F(2alpha) on steroidogenic components and oligonucleosomes in ovine luteal tissue. AB - To determine whether prostaglandin (PG) F(2alpha) had a dose-dependent effect upon secretion of progesterone, oligonucleosome formation, or loss of luteal weight, ewes on Day 9 or 10 of the estrous cycle were administered 0, 3, 10, or 30 mg PGF(2alpha) per 60 kg BW (i.v.), and luteal tissue was collected 9 and 24 h after injection. All doses of PGF(2alpha) decreased (P < 0. 05) concentrations of progesterone in sera by 9 h; however, in ewes treated with 3 mg PGF(2alpha), concentrations of progesterone were similar to control values at 24 h and higher (P < 0.05) than those in the 10- or 30-mg groups. Concentrations of progesterone in sera over all dose levels were highly correlated to luteal concentrations of mRNA encoding steroidogenic acute regulatory protein (P < 0.001), cytochrome P450 side-chain cleavage (P < 0.02), and 3beta-hydroxysteroid dehydrogenase (P < 0.01). Corpora lutea collected at 24 h from ewes treated with the 10- and 30-mg doses of PGF(2alpha) weighed less (P < 0.05) than those from controls. Oligonucleosomes were not present in luteal tissues from control ewes. Surprisingly, all doses of PGF(2alpha)-induced oligonucleosomes in a majority of animals at 9 h and in a majority of ewes treated with 10 and 30 mg of PGF(2alpha) at 24 h. In conclusion, 3 mg of PGF(2alpha) per 60 kg BW transiently decreased serum concentrations of progesterone and induced oligonucleosome formation, but did not result in reduced luteal weight. The 10- and 30-mg doses of PGF(2alpha) decreased secretion of progesterone and induced oligonucleosome formation and luteolysis. PMID- 10727277 TI - Olfactory bulbectomy blocks mating-induced ovulation in musk shrews (Suncus murinus). AB - In many species, reproductive function can be modified by olfactory inputs. We employed bilateral olfactory bulbectomy (BULBX) to examine the effects of disruption of olfactory inputs on mating behavior and ovulation in female musk shrews. On several measures, sexual behavior was delayed in BULBX females compared to controls. When females were mated on five consecutive days, the majority of unoperated and sham-operated (SHAM) shrews ovulated; only one female subjected to BULBX ovulated. Administration of GnRH induced ovulation in the majority of females. We performed immunocytochemistry to assess the effects of bulbectomy on mating-induced responses of the neural GnRH system. In BULBX and SHAM females, the numbers of cells containing proGnRH immunoreactivity in the medial septum (MS)/diagonal band (DB) were significantly elevated 1 h after mating. Bulbectomy increased the numbers of GnRH-immunoreactive peptide containing cells in the preoptic area, but it reduced neuron numbers in the MS/DB, as compared with those in SHAM controls. In addition, the GnRH immunoreactive fiber area in the median eminence was greater in BULBX than in SHAM females. In sum, female musk shrews can display receptivity and engage in copulation without olfactory inputs. However, the olfactory system is essential for mating-induced ovulation. PMID- 10727278 TI - Intrabursal injection of clodronate liposomes causes macrophage depletion and inhibits ovulation in the mouse ovary. AB - To investigate the role of the ovarian macrophage population in ovulation, we examined the effect of depleting this population using liposome-encapsulated clodronate. Clodronate liposomes, saline liposomes, or saline alone was injected under the ovarian bursa in gonadotropin-primed adult mice, either 84 h (Day -3) or 36 h (Day -1) before ovulation. Ovulation rates were determined by counting the number of oocytes released. The numbers of graafian follicles and corpora lutea were also counted immediately before and after ovulation. Macrophage distribution within the theca and stroma of preovulatory ovaries was examined by immunohistochemistry with specific monoclonal antibodies to the macrophage antigens macrosialin, major histocompatability complex class II (Ia), and F4/80. Injection of clodronate liposomes on Day -1 did not affect ovulation rates, whereas administration on Day -3 caused a significant reduction in ovulation rate (mean oocytes ovulated = 5. 25 +/- 0.6 from clodronate liposome-treated ovaries and 9.13 +/- 0.9 from saline-treated ovaries, respectively, P < 0.05). The numbers of macrosialin-positive macrophages present in the theca at ovulation were reduced by treatment with clodronate liposomes on Day -1, and treatment on Day -3 reduced the numbers of Ia-positive and macrosialin-positive macrophages present in the theca. When the subsequent ovarian cycles were examined by vaginal smearing, the metestrous-2/diestrous stage was found to be extended in clodronate liposome-treated animals (7.5 +/- 1.3 days vs. 3.4 +/- 0.4 days for saline liposome-treated animals, P < 0.05). These results suggest that thecal macrophages may be involved in the regulation of follicular growth and rupture, as well as being important for the normal progression of the estrous cycle. PMID- 10727279 TI - Follicle-stimulating hormone mediated calcium signaling by the alternatively spliced growth factor type I receptor. AB - Ovarian granulosa cell and testicular Sertoli cell functions are regulated by the tropic action of the pituitary follicle-stimulating hormone (FSH), which may exert pleiotropic effects using a variety of signaling pathways. The effects of FSH on the mobilization of Ca(2+) into granulosa and Sertoli cells have been widely studied, but whether all the effects of the hormone are mediated by the single G-protein-coupled (G(s)) receptor with the seven-transmembrane structure (R1) has remained an enigma. With the object of resolving this mystery, we have compared the hormonal responses of HEK 293 cells transfected with three different cloned FSH receptor cDNAs of testis/ovary, designated R1 (G(s)), R2 (similar to R1 but having a shorter carboxyl terminus), and R3, a novel FSH receptor exhibiting a growth factor type I receptor motif. The latter two that use the same DNA segment for alternative splicing of the single large 80- to 100-kilobase gene create different structural motifs and carboxyl termini. Of the three receptors, only the FSH-R3 type induced a significant rise in intracellular free calcium concentration ([Ca(2+)](i)), as measured by single cell fluorescence digital imaging with the Ca(2+) sensitive dye fura-2AM. FSH induced a rapid [Ca(2+)](i) response that was concentration dependent. The response was hormone specific, as neither its individual alpha/beta subunits nor the related glycoprotein hormone LH were effective. To determine whether the [Ca(2+)](i) response was due to Ca(2+) influx or to intracellular Ca(2+) mobilization, cells were exposed to Ca(2+)-free buffer and to the Ca(2+)-channel blocker diltiazem (10(-5) M). FSH-Induced [Ca(2+)](i) responses were inhibited in Ca(2+)-free buffer and abrogated in the presence of diltiazem. These novel data demonstrate that FSH can increase [Ca(2+)](i) through L-type voltage-dependent Ca(2+) channels via the growth factor type 1 receptor. Our findings support the concept that different receptor motifs act to integrate intracellular signaling events. PMID- 10727280 TI - Human villous macrophage-conditioned media enhance human trophoblast growth and differentiation in vitro. AB - In human chorionic villi, numerous macrophages, so-called Hofbauer cells, are located adjacent to trophoblasts. To determine the role of the macrophages in the proliferation and differentiation of trophoblasts, cytotrophoblast cells were cultured in serum-free culture-conditioned media of villous macrophages (VMCM), peritoneal macrophages (PMCM), and villous fibroblasts (VFCM). In VMCM, proliferation of cytotrophoblast cells was detected at 24 h by immunocytochemistry with Ki-67-antibody. A large number (P < 0.001) of multinucleated syncytia was formed in VMCM. In VMCM, cytotrophoblast cell fusion was completed by 96 h, which coincided with the peak of hCG secretion and initiation of human placental lactogen (hPL) release. Levels of hCG (P < 0.001) and hPL (P < 0. 001) secretion from syncytial cells were significantly higher in VMCM than in PMCM or in VFCM. Concentrations of macrophage colony-stimulating factor (M-CSF) and vascular endothelial growth factor (VEGF) analyzed by ELISA were greater in VMCM than in PMCM or in VFCM, whereas monocyte chemoattractant protein-1 (MCP-1) concentration was high in PMCM. The expression patterns of M CSF, VEGF, and MCP-1 in villous macrophages and peritoneal macrophages by reverse transcriptase-polymerase chain reaction were similar to their secretion patterns. Thus, villous macrophages have a greater ability to stimulate hCG and hPL secretion than do peritoneal macrophages. This study suggests that macrophages within the villous stroma may stimulate the growth and differentiation of trophoblasts through their secreted substances. PMID- 10727281 TI - Rab3A triggers the acrosome reaction in permeabilized human spermatozoa. AB - The acrosome reaction is a regulated exocytotic process leading to a massive fusion between the outer acrosomal membrane and the cell membrane. In spite of the great amount of information available related to the acrosome reaction in several species, there is a remarkable paucity about the role of monomeric guanosine triphosphatases (GTPases) of the Rab family-well-established participants in exocytosis in other cell types-in the acrosome reaction. Western blot and immunofluorescence analysis indicate that Rab3A is present in human spermatozoa and localizes to the acrosomal region in the sperm head. One difficulty in studying the role of proteins in intact cells is the fact that they are unable to cross the cell membrane. Therefore, we established a working model of streptolysin O-permeabilized human spermatozoa. Permeabilized spermatozoa were able to respond in a regulated way to different stimuli, such as G protein activators and calcium. An acrosomal reaction was also triggered by a Rab3A peptide corresponding to the effector region. More important, recombinant Rab3A protein in the GTP-bound form caused acrosome exocytosis. The same protein loaded with GDP or Rab11 in the GTP-bound form was inactive. Also, recombinant GDI (GDP dissociation inhibitor)-a protein that releases Rab proteins from membrane inhibited a GTPgammaS-stimulated acrosome reaction. Our results indicate that 1) permeabilized spermatozoa can be used to study the role of macromolecules in the acrosome reaction, 2) Rab3A is present in human spermatozoa, and 3) Rab3A or another Rab3 isoform is involved in the exocytosis of the acrosomal granule in human spermatozoa. PMID- 10727282 TI - Ovarian expression of a disintegrin and metalloproteinase with thrombospondin motifs during ovulation in the gonadotropin-primed immature rat. AB - Mammalian ovulation is a dynamic process that requires degradation of the collagenous connective tissue in the thecal layers of a mature follicle. In this reverse transcription-polymerase chain reaction differential display study, gonadotropin-primed immature rats were used to detect ovarian expression of a relatively new type of disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-1) that is known to cleave extracellular matrix in acutely inflamed tissues. Immature Wistar rats were primed with 10 IU eCG s. c., and the temporal pattern of expression of the ADAMTS-1 gene was delineated by extracting ovarian RNA at 0, 2, 4, 8, 12, and 24 h after induction of ovulation by injecting the primed animals with 10 IU hCG s.c. The differential display data, Northern analyses, and in situ hybridization micrographs all showed significant up regulation of ADAMTS-1 gene expression by 8 h after hCG administration. The in situ data indicated that the ADAMTS-1 mRNA was in the granulosa layer of mature follicles. Expression reached a peak at 12 h and remained elevated at 24 h after hCG. ADAMTS-1 gene expression was impaired by the antiprogesterone agent epostane, but this inhibition could be overcome by exogenous progesterone. ADAMTS 1 expression was not affected when ovulation was blocked by treatment of the animals with the anti-eicosanoid agent indomethacin. In conclusion, the temporal pattern of expression of this gene, and its apparent regulation by progesterone, suggests that ADAMTS-1 has a significant role in the inflammatory events of the ovulatory process. PMID- 10727283 TI - Association between expression of reproductive seasonality and alleles of the gene for Mel(1a) receptor in the ewe. AB - To determine whether a link exists between reproductive seasonality and the structure of the gene for melatonin receptor Mel(1a), the latter was studied in two groups of Merinos d'Arles (MA) ewes previously chosen for their genetic value, which took into account their own out-of-season ovulatory activity adjusted by environmental parameters and that of their relatives. The genomic DNA of 36 ewes found regularly cycling in spring (group H) and that of 35 ewes never cycling in spring (group L) during the 2-3 yr before the present study was prepared, and the cDNA corresponding to almost all exon II was amplified and checked for the presence of MnlI restriction sites. The presence (+) or absence ( ) of an MnlI site at position 605 led to genotypes "++", "+-", and "--", whose frequencies differed significantly (P < 0.001) between the H and L groups: 52.8%, 47.2%, and 0% vs. 28.5%, 42.9%, and 28.5%, respectively. Sequencing of exon II cDNA in group L ewes with genotype -- showed the presence of only one allele - with 4 mutations, while that in ewes with genotype ++ showed different types of alleles unrelated to the H or L groups. These + alleles exhibited a combination of 1 to 7 of the 8 mutations recorded in the part of exon II studied. The genotyping of 29 ewes from the more seasonal Ile-de-France breed indicated that 38% of animals had a -- genotype and exhibited the same mutations as in the MA ewes. Finally, a comparison of (125)I-melatonin binding to membrane preparations of pars tuberalis showed a lower number of binding sites (P < 0. 0005) in MA ewes with genotype ++ than in those with genotype -- (43. 2 +/- 4.4 vs. 75.4 +/- 8.4 fmol/mg protein in genotype ++ and genotype --, respectively). In conclusion, the data show an association between genotype -- for site MnlI at position 605 and seasonal anovulatory activity in MA ewes. PMID- 10727284 TI - 2,3,7,8-tetrachlorodibenzo-p-dioxin decreases estradiol production without altering the enzyme activity of cytochrome P450 aromatase of human luteinized granulosa cells in vitro. AB - This study was designed to examine the in vitro effects of 2,3,7, 8 tetrachlorodibenzo-p-dioxin (TCDD) on steroid production in human luteinizing granulosa cells (hLGC). TCDD (10 nM) or its solvent was added at the time of changing medium, directly to the cells, every 48 h for 8 days. To test the hypothesis that TCDD reduces estradiol (E(2)) synthesis by an effect on cytochrome P450 aromatase, aromatase protein and aromatase activity were evaluated. E(2) decreased without changing either aromatase protein or its enzyme activity, suggesting that the target of toxicity of TCDD is upstream of aromatase in the steroidogenic pathway. When hLGC were incubated in the presence of labeled E(2), no changes in the metabolism of E(2) by treatment were observed. Since TCDD did not change progesterone or 17alpha-hydroxyprogesterone, the inhibition of E(2) synthesis by TCDD would seem not to involve steps such as cholesterol transport. Furthermore, the TCDD effect on E(2) concentration in these cells disappeared in the presence of excess androgens. We conclude that the inhibition of E(2) secretion by TCDD involves intermediate steps, specifically, the provision of androgens for aromatization. PMID- 10727285 TI - Contributions of immunohistochemistry and in situ hybridization to the functional analysis of pituitary adenomas. AB - Immunohistochemistry (IHC) and recently in situ hybridization (ISH) have elucidated various aspects of human pituitary adenomas, i.e., functional differentiation and classification, transcription factors and mechanism of hormone production, regulation of hormone secretion, and processing of prohormones. Recently, the use of tyramide (catalyzed signal amplification; TSA or CSA) and RT-PCR has been effective for detection of trivial amount of proteins (peptides) and mRNA, respectively. Immunomolecular histochemistry is expected to further clarify the function and biology of human pituitary adenomas. PMID- 10727286 TI - New frontiers in gold labeling: symposium overview. AB - The Symposium New Frontiers in Gold Labeling was held at the Fifth Joint Meeting of the Japan Society of Histochemistry and Cytochemistry and the United States Histochemical Society. Speakers described technological developments in this area that improved localization of cellular components. Each presentation is summarized in this overview, and complete articles follow that describe these results in more detail. PMID- 10727287 TI - Introduction of a novel HRP substrate-Nanogold probe for signal amplification in immunocytochemistry. AB - Amplification of immunological signals with catalyzed reporter deposition (CARD) allows improved detection of scarce tissue antigens in light and electron microscopy. The technique takes advantage of the oxidation ability of horseradish peroxidase (HRP), in the presence of hydrogen peroxide, to yield the accumulation of one of its specific reporter-tagged substrates. This immunocytochemical approach continues to be improved by the introduction of new reporter molecules tagged to tyramine or to other HRP substrates. In this study we introduced a novel HRP substrate tagged to Nanogold particles. The amplification protocol is based on the application of a specific primary antibody, a biotinylated secondary antibody, streptavidin-HRP, and an HRP substrate coupled to Nanogold, followed by silver intensification. In addition to amplification of immunological signals of high resolution, direct accumulation of Nanogold particles at target sites by enzymatic activity of HRP improves the efficiency of the technique compared to other amplification protocols. Moreover, this approach combines the CARD amplification potentials with the ultrasmall gold probe and the silver intensification method. Immunolabeling obtained by light and electron microscopy, as well as immunodot assay using this new amplification strategy, appear to be highly sensitive, specific, and of enhanced intensity. PMID- 10727288 TI - New frontiers in gold labeling. AB - Recent advances in gold technology have led to probes with improved properties and performance for cell biologists: higher labeling density, better sensitivity, and greater penetration into tissues. Gold clusters, such as the 1.4-nm Nanogold, are gold compounds that can be covalently linked to Fab' antibody fragments, making small and stable probes. Silver enhancement then makes these small gold particles easily visible by EM, LM, and directly by eye. Another advance is the combination of fluorescent and gold probes for correlative microscopy. Chemical crosslinking of gold particles to many biologically active molecules has made possible many novel probes, such as gold-lipids, gold-Ni-NTA, and gold-ATP. PMID- 10727289 TI - FluoroNanogold is a bifunctional immunoprobe for correlative fluorescence and electron microscopy. AB - We applied a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to immunocytochemistry on ultrathin cryosections. FNG has the properties of both a fluorescent dye-conjugated antibody for fluorescence microscopy and a gold particle-conjugated antibody for electron microscopy. Therefore, this bifunctional immunoprobe permits correlative microscopic observation of the same cell profiles labeled in a single labeling procedure by these two imaging methods. We demonstrate the utility of FNG as a secondary antibody for immunocytochemical labeling of myeloperoxidase (a marker protein for azurophilic granules) in ultrathin cryosectioned human neutrophils. Its detection requires high spatial resolution because neutrophils contain many cytoplasmic granules. There was a one-to-one relationship between fluorescent structures labeled with FNG and organelle profiles labeled with the same silver-enhanced FNG in ultrathin cryosections. Use of FNG immunocytochemistry on ultrathin cryosections is an ideal methodology for high-resolution correlative fluorescence and electron microscopy and can provide unique information that may be difficult to obtain with a single imaging regimen. PMID- 10727290 TI - Enhanced labeling efficiency using ultrasmall immunogold probes: immunocytochemistry. AB - Detection of antigen-antibody interactions in immunocytochemistry relies on a reporter system. The most commonly employed reporter systems used are fluorochromes, enzymes, and particulate probes. This article considers the advantages and disadvantages associated with ultrasmall immunogold particles as the reporter system in immunocytochemical applications. PMID- 10727291 TI - A practical technique to postfix nanogold-immunolabeled specimens with osmium and to embed them in Epon for electron microscopy. AB - Nanogold is a tiny gold probe, freely diffusible in cells and tissues, and is suitable for pre-embedding immunohistochemistry. However, it is necessary to develop Nanogold to a larger size so that it can be observed by conventional transmission electron microscopy. Silver enhancement is usually used for visualizing Nanogold, but the silver shell produced is unstable in OsO(4) and often becomes invisible after OsO(4) postfixation, which is necessary for good visualization of ultrastructure. We used silver enhancement with silver acetate, followed by gold toning with chloroauric acid, to replace the silver shell with a more stable gold in order to observe Nanogold after osmium fixation and Epon embedding. This technique is applicable to various intra- and extracellular antigens. For correlative observation of immunolabled specimens by light and electron microscopy, specimens adhered to slideglasses were embedded in Epon under non-adhesive plastic film. By heating the Epon sheets after polymerization, these supports were removed without difficulty and provided easy correlative observation. PMID- 10727292 TI - Freeze-drying allows double nonradioactive ISH and antigenic labeling. AB - Because tissue freeze-drying is an excellent way to preserve antigenic conformation, we have tested the feasibility of this technique to reveal nonradioactive in situ hybridization (ISH) of tissue mRNA. We have compared mRNA detection after different methods of tissue preservation, freeze-drying, cryosectioning, and formaldehyde or methanol fixation. Our results show that nonradioactive ISH is more sensitive for tissues preserved by freeze-drying than for other tissue preparations. We have demonstrated that freeze-drying allows combination of ISH and immunohistochemistry for simultaneous detection of mRNA and antigen because with this technique of tissue preservation ISH does not affect the sensitivity or the amount of the detected antigens. This work underscores the fact that tissue freeze-drying is an easy, convenient, and reliable technique for both ISH and immunohistochemistry and achieves excellent structural conditions for nonradioactive detection. PMID- 10727293 TI - Metabolic imaging in multicellular spheroids of oncogene-transfected fibroblasts. AB - Four rat embryo fibroblast (REF) cell lines with defined oncogenic transformation were used to study the relationship between tumorigenic conversion, metabolism, and development of cell death in a 3D spheroid system. Rat1 (spontaneously immortalized) and M1 (myc-transfected) fibroblasts represent early nontumorigenic transformation stages, whereas Rat1-T1 (T24Ha-ras-transfected Rat1) and MR1 (myc/T24Ha-ras-co-transfected REF) cells express a highly tumorigenic phenotype. Localized ATP, glucose, and lactate concentrations in spheroid median sections were determined by imaging bioluminescence. ATP concentrations were low in the nonproliferating Rat1 aggregates despite sufficient oxygen and glucose availability and lack of lactate accumulation. In MR1 spheroids, a 50% decrease in central ATP preceded the development of central necrosis at a spheroid diameter of around 800 micrometer. In contrast, the histomorphological emergence of cell death at a diameter of around 500 micrometer in Rat1-T1 spheroids coincided with an initial steep drop in ATP. Concomitantly, reduction in central glucose and increase in lactate before cell death were recorded in MR1 but not in Rat1-T1 spheroids. As shown earlier, myc transfection confers a considerable resistance to hypoxia of MR1 cells in the center of spheroids, which is reflected by their capability to maintain cell integrity and ATP content in a hypoxic environment. The data obtained suggest that small alterations in the genotype of tumor cell lines, such as differences in the immortalization process, lead to substantial differences in morphological structure, metabolism, occurrence of cell death, and tolerance to hypoxia in spheroid culture. PMID- 10727294 TI - Cell wall-associated protein A as a tool for immunolocalization of Staphylococcus aureus in infected human airway epithelium. AB - Staphylococcus aureus is a common human pathogen involved in non-bronchial diseases and in genetic and acquired bronchial diseases. In this study, we applied an immunolabeling approach for in vivo and in vitro detection of S. aureus, based on the affinity of staphylococcal protein A (SpA) for the Fc region of immunoglobulins, especially IgG. Most strains of S. aureus, including clinical strains, can be detected with this labeling technique. The approach can be used for detection and localization with transmission electron microscopy or light fluorescence microscopy of S. aureus in infected tissues such as human bronchial tissue from cystic fibrosis (CF) patients. The methodology can also be applied to cell culture models with the aim of characterizing bacterial adherence to epithelial cells in backscattered electron imaging with scanning electron microscopy. Application to the study of S. aureus adherence to airway epithelium showed that the bacteria did not adhere in vivo to intact airway epithelium. In contrast, bacteria adhered to the basolateral plasma membrane of columnar cells, to basal cells, to the basement membrane and were identified beneath the lamina propria when the epithelium was injured and remodeled, or in vitro when the epithelial cells were dedifferentiated. PMID- 10727295 TI - Human bronchial epithelial cells secrete laminin 5, express hemidesmosomal proteins, and assemble hemidesmosomes. AB - Epithelial cells attach to the basement membrane through adhesive contacts between the basal cells of the epithelium and the proteins of the extracellular matrix (ECM). The hemidesmosome (HD) is a specialized cell-ECM contact, that mediates the attachment of the epithelial cell basal surface to the ECM. In bronchial epithelial cells, the protein components that constitute the HD have not been demonstrated. Using immunohistochemical techniques, we determined that normal human bronchial epithelial (NHBE) cells express the HD cell surface integrin alpha6beta4 and produce laminin 5, the ECM protein associated with HDs. Furthermore, expression of the HD-associated structural proteins, bullous pemphigoid antigens 1 (BPAG 1) and 2 (BPAG 2), was demonstrated in NHBE cells by immunofluorescence microscopy and immunoblot analyses. In addition, we confirmed the presence of laminin 5 in the basement membrane (BM) of bronchial epithelial biopsy specimens and of BP230, BP180, and the alpha6beta4 integrin heterodimer at the site of bronchial epithelial cell-ECM interaction in vivo. Finally, using electron microscopy, we were able to demonstrate intact HDs in a glutaraldehyde fixed NHBE cell monolayer. These findings suggest that bronchial epithelium forms HDs and that the laminin 5-alpha6beta4 integrin interaction may be important in stabilizing epithelial cell adhesion to the BM in the lung. PMID- 10727296 TI - Ultrastructural localization of the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) receptor-2 (FLK-1, KDR) in normal mouse kidney and in the hyperpermeable vessels induced by VPF/VEGF-expressing tumors and adenoviral vectors. AB - Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) interacts with two high-affinity tyrosine kinase receptors, VEGFR-1 and VEGFR-2, to increase microvascular permeability and induce angiogenesis. Both receptors are selectively expressed by vascular endothelial cells and are strikingly increased in tumor vessels. We used a specific antibody to localize VEGFR-2 (FLK 1, KDR) in microvascular endothelium of normal mouse kidneys and in the microvessels induced by the TA3/St mammary tumor or by infection with an adenoviral vector engineered to express VPF/VEGF. A pre-embedding method was employed at the light and electron microscopic levels using either nanogold or peroxidase as reporters. Equivalent staining was observed on both the luminal and abluminal surfaces of tumor- and adenovirus-induced vascular endothelium, but plasma membranes at interendothelial junctions were spared except at sites connected to vesiculovacuolar organelles (VVOs). VEGFR-2 was also localized to the membranes and stomatal diaphragms of some VVOs. This staining distribution is consistent with a model in which VPF/VEGF increases microvascular permeability by opening VVOs to allow the transendothelial cell passage of plasma and plasma proteins. PMID- 10727297 TI - Intercellular adhesion molecule-1 and hair follicle regression. AB - Although the intercellular adhesion molecule-1 (ICAM-1) is recognized for its pivotal role in inflammation and immune responses, its role in developmental systems, such as the cyclic growth (anagen) and regression (catagen) of the hair follicle, remains to be explored. Here we demonstrate that ICAM-1 expression in murine skin is even more widespread and more developmentally regulated than was previously believed. In addition to endothelial cells, selected epidermal and follicular keratinocyte subpopulations, as well as interfollicular fibroblasts, express ICAM-1. Murine hair follicles express ICAM-1 only late during morphogenesis. Thereafter, morphologically identical follicles markedly differ in their ICAM-1 expression patterns, which become strikingly hair cycle-dependent in both intra- and extrafollicular skin compartments. Minimal ICAM-1 and leukocyte function-associated (LFA-1) protein and mRNA expression is observed during early anagen and maximal expression during late anagen and catagen. Keratinocytes of the distal outer root sheath, fibroblasts of the perifollicular connective tissue sheath, and perifollicular blood vessels exhibit maximal ICAM-1 immunoreactivity during catagen, which corresponds to changes of LFA-1 expression on perifollicular macrophages. Finally, ICAM-1-deficient mice display significant catagen acceleration compared to wild-type controls. Therefore, ICAM-1 upregulation is not limited to pathological situations but is also important for skin and hair follicle remodeling. Collectively, this suggests a new and apparently nonimmunological function for ICAM-1-related signaling in cutaneous biology. PMID- 10727299 TI - Alopecia areata update. AB - Alopecia areata (AA) is a nonscarring hair loss condition. Among the many factors under investigation in the pathogenesis of AA, the main areas of concentration have been genetic constitution as well as nonspecific immune and organ-specific autoimmune reactions. Treatment with intralesional corticosteroid injections for localized patchy AA and topical immunotherapy for extensive AA have proven successful in the majority of patients, although all treatments are palliative and do not change the prognosis of the disease. PMID- 10727298 TI - A new approach to three-dimensional reconstructed imaging of hormone-secreting cells and their microvessel environments in rat pituitary glands by confocal laser scanning microscopy. AB - There has been considerable interest in the relationship between hormone- secreting endocrine cells and their microvessels in human pituitary gland. However, microcirculatory networks have rarely been studied in three dimensions (3D). This study was designed to visualize and to reveal the relationship between hormone-secreting endocrine cells and their microvessel environment in 3D, using rat pituitary glands under various (hyper/hypo) experimental conditions by confocal laser scanning microscopy (CLSM). Female adult Wistar rats were used after bilateral adrenalectomy or ACTH administration for 2 weeks. Clear 3D reconstructed images of ACTH cells, the microvessel network and counterstained nuclei were obtained at a maximal focus depth of 1 mm by CLSM without any background noise. In the hyperfunctional state, slender cytoplasmic processes of hypertrophic stellate ACTH cells frequently extended to the microvessels. In the hypofunctional state, ACTH cells appeared atrophic and round with scanty cytoplasm, and cytoplasmic adhesions to microvessel network patterns were inconspicuous. Therefore, 3D reconstructed imaging by CLSM is a useful technique with which to investigate the microvessel environment of hormone-secreting cells and has the potential to reveal dynamic hormone-secreting pathways. PMID- 10727300 TI - UVB-induced acantholysis in endemic Pemphigus foliaceus (Fogo selvagem) and Pemphigus vulgaris. AB - BACKGROUND: The autoantibody-mediated disease in fogo selvagem (FS) is aggravated by sunlight exposure. OBJECTIVE: The aim of this study was to test whether UVB exposure of uninvolved skin of patients with FS (n = 21) enhances epidermal acantholysis and in vivo binding of FS autoantibodies as compared with a control group (n = 4). Another control group included 8 patients with pemphigus vulgaris (PV). RESULTS: Exposure of uninvolved skin of FS to UVB induces acantholysis (15/21 patients) with in vivo binding of IgG and C3 to the epidermal intercellular spaces (17/21 patients). Similar results were found in PV (6/8 patients). The test was negative in the control group. CONCLUSION: Sunlight exposure is harmful in the clinical course of patients with FS and those with PV. Protection from sunlight should be part of the treatment of these patients. PMID- 10727302 TI - Depth of cutaneous analgesia after application of a eutectic mixture of the local anesthetics lidocaine and prilocaine (EMLA cream). AB - BACKGROUND: EMLA cream, a eutectic mixture of lidocaine and prilocaine, is a topical anesthetic, frequently used to avoid pain during venipuncture and superficial surgery. However, the depth of analgesia needs further exploration. OBJECTIVE: Our purpose was to investigate the depth of cutaneous analgesia after application of EMLA cream. METHODS: In a single-blind crossover study, EMLA cream was applied under occlusion to the left thighs of 16 subjects. Eleven had application times of 60 and 120 minutes. Five subjects had application times of 3 to 4 hours. Approximately 10 minutes after removal of the EMLA cream, a skin biopsy punch (diameter 4 mm) was inserted to a depth of 1 mm into the skin and the subject rated the pain intensity on a visual analogue scale. If there was no pain or if pain was assessed as acceptable, a new biopsy punch was inserted at a new site to the depth of 2 mm. In this manner, the insertion depth was gradually increased in steps of 1 mm down to a maximum of 6 mm. No skin specimens were removed. RESULTS: After 60 and 120 minutes of EMLA application the mean insertion depths with acceptable pain were 2.9 and 4.5 mm (P <.01), respectively. After 3 to 4 hours of application, 6-mm deep insertions were made with acceptable pain in all 5 subjects. CONCLUSION: Skin biopsy punch insertions in steps of 1 mm appear adequate for assessing the depth of cutaneous analgesia. Biopsy punch insertions with acceptable pain can be made to depths of 1 to 2 mm after 60 minutes, to 2 to 3 mm after 120 minutes, and to 6 mm after 3 to 4 hours of EMLA cream application. PMID- 10727301 TI - IgG4 and IgE are the major immunoglobulins targeting the NC16A domain of BP180 in Bullous pemphigoid: serum levels of these immunoglobulins reflect disease activity. AB - BACKGROUND: Bullous pemphigoid (BP) is an autoimmune blistering disease associated with autoantibodies against the hemidesmosomal glycoprotein BP180. The noncollagenous (NC)16A domain of BP180 has recently been shown to harbor major antigenic sites recognized by BP sera. OBJECTIVE: The purpose of this study was to characterize the subclass distribution and fine specificities of autoantibodies to BP180 NC16A present in the circulation of patients with BP before, and during the course of, therapy for this disease. METHODS: Eighteen BP sera were analyzed by immunoblotting and enzyme-linked immunosorbent assay for the presence of IgG1, IgG2, IgG3, IgG4, and IgE reactive with various sites on the BP180 NC16A domain. The sera were collected before treatment was started and at 4- and 8-week time points after initiation of treatment. RESULTS: We identified IgG4 and IgE as the major immunoglobulins that preferentially react with two distinct epitopes (MCW-1 and MCW-2) within BP180 NC16A. Levels of these autoantibodies correlated with disease activity in BP. During the course of disease, no change was observed with regard to the immunoglobulin subclass predominantly reacting with BP180 NC16A or the specific epitopes within this domain. CONCLUSION: Our data demonstrate that remission of BP is paralleled by a decrease of serum levels of IgE and the different IgG subclasses reactive with BP180 NC16A. PMID- 10727303 TI - Clinical and histopathologic characteristics of trichrome vitiligo. AB - BACKGROUND: The term trichrome vitiligo describes lesions that have a tan zone of varying width between normal and totally depigmented skin, which exhibits an intermediate hue. However, the pathogenesis and the histopathologic characteristics of trichrome vitiligo are unknown. OBJECTIVE: Our purpose was to investigate the clinical and histopathologic characteristics and the pathogenesis of trichrome vitiligo. METHODS: Four punch biopsy specimens were taken from 21 patients with trichrome vitiligo; they were from vitiliginous skin, light brown skin, perilesional normal skin, and normal skin as far as 5 cm from the nearest vitiligo spot. The sections were stained with hematoxylin-eosin; in selected cases, we performed immunohistochemical staining with S-100 protein and CD1a. RESULTS: Trichrome vitiligo occurred most frequently on the trunk in active vitiligo vulgaris. Focal vacuolar degeneration of the basal cell layer and mild inflammatory cell infiltration of the epidermis and dermis were more prominent in light brown skin and perilesional normal skin than in vitiliginous skin and normal skin. The number of melanocytes was decreased in light brown skin compared with perilesional normal skin (P <.05) and in vitiliginous skin compared with light brown skin (P <.05); a few melanocytes were observed even in skin affected by trichrome vitiligo. The number of Langerhans cells was increased in the epidermis of light brown skin and perilesional normal skin compared with vitiliginous and normal skin (P <.05). PUVA therapy yielded excellent repigmentation. CONCLUSION: Trichrome vitiligo is a variant of active vitiligo. The changes of melanocytes, keratinocytes, and Langerhans cells may be involved in the pathogenesis of depigmentation in trichrome vitiligo. PMID- 10727304 TI - Blue in the skin. AB - BACKGROUND: The most prevalent theory explaining blue colors in skin is that of bluish scatter. If this accounted for blue perception, it would be measurable since a predominance of scattered blue light should emerge as a measurable reflectance. When objects are viewed with chromatic surround, there may be an increased perception of the hue complementary to their background. This effect, known as color contrast, is a well-established phenomenon and depends on the size, lightness, and intensity of the surrounding color. OBJECTIVE: The purpose of this article is to determine the effect of background on the perception of blue color from a superficial vein. METHODS: Fifteen subjects were asked to observe the color of skin overlying a vein on the hand. This was performed with a variety of achromatic backgrounds and with variable amounts of surrounding normal skin. Color was also measured by direct matching with standardized Munsell chips and then estimated with normal skin surround. RESULTS: Color matching revealed the hue of the vein to be yellowish-gray, and not blue or green. Although few subjects observed green or blue with neutral backgrounds, as the vein was viewed with progressively larger areas of normal surrounding skin, there was an increasing perception of these colors. CONCLUSION: The bluish color of superficial veins is more clearly explained by color contrast than by scattering. Contrast may also account for the color of certain blue nevi and may help explain some purplish colors in the skin. PMID- 10727305 TI - The value of positron emission tomography scanning in the detection of subclinical metastatic melanoma. AB - We have undertaken a retrospective analysis of all positron emission tomography (PET) scans carried out at St Thomas' Hospital, London, since 1994 to establish the sensitivity and specificity of this radiologic technique in cutaneous malignant melanoma. In particular, we have identified those patients with primary cutaneous malignant melanoma in whom PET scanning revealed in-transit or regional spread to nodes and those patients with known regional spread in whom PET scanning revealed distant metastases. We defined our false-negative results as a negative scan result with positive histology or subsequent clinical progression of disease. False-positive results were defined as a suspect scan with negative histology or no subsequent progression of disease. PET scanning had an overall sensitivity of 78% and specificity of 87%; however, subset analysis (M. D. Anderson staging system) showed a sensitivity of 50% for stage I disease (34 patients and 35 scans) and 33% for stage II disease (9 patients and 9 scans) with specificities of 87% and 100%, respectively. For stage III disease (16 patients and 17 scans), PET showed a sensitivity of 93% and specificity of 50%. Overall, 35% of patients with true-positive scans had their disease restaged. We can conclude therefore that PET is valuable as a staging procedure in patients with known regional spread but is suboptimal in the prediction of outcome in stage I or stage II disease. PMID- 10727306 TI - Delayed-type hypersensitivity skin reactions caused by subcutaneous unfractionated and low-molecular-weight heparins: tolerance of a new recombinant hirudin. AB - BACKGROUND: Eczema-like infiltrated plaques at subcutaneous heparin injection sites are well-documented side effects of these anticoagulants. However, surgical interventions may be problematic if heparin is urgently needed in these patients. OBJECTIVE: The aims of this study were to perform extensive allergy skin testing in 24 patients, including a pregnant woman in whom subcutaneous infiltrated plaques developed after subcutaneous heparin injections, and to find safe therapeutic alternatives for this group of patients. METHODS: Patch, intradermal, and subcutaneous tests were performed with a panel of unfractionated heparins (UFHs), low-molecular-weight heparins (LMWHs), and heparinoids. Since 1997, we have also been performing allergy studies in 8 patients with lepirudin, a new recombinant heparinoid; tolerance of lepirudin was investigated by means of subcutaneous and intravenous injections. The allergy investigations in the pregnant woman were limited to patch tests with heparins and intradermal and subcutaneous tests with pentosanpolysulfate, which are not contraindicated during pregnancy. RESULTS: In our study population 19 of 23 patients were sensitized to all the UFHs and LMWHs tested when intracutaneous and subcutaneous test results were read at up to 96 hours. LMWH was found to be a possible substitute in 4 patients. Five patients were also sensitized to the heparinoid pentosanpolysulfate. Sensitization to the heparinoid danaparoid was observed in 12 of the 13 patients who were tested with this substance. The administration of an intravenous bolus containing a therapeutic dose of lepirudin after negative subcutaneous provocation was tolerated without any side effects in all 8 patients. The pregnant woman was sensitized to LMWH but tolerated subcutaneous pentosanpolysulfate without any side effects. CONCLUSION: Extensive allergy skin testing should be performed to find safe alternatives. With few exceptions, all patients react to both UFHs and LMWHs, as well as to danaparoid. The subcutaneous provocation test is the most reliable diagnostic measure. Intravenous lepirudin, and in some cases subcutaneous pentosanpolysulfate, appears to be a safe alternative in patients with eczema-like infiltrated plaques at subcutaneous heparin-injection sites. PMID- 10727307 TI - Sex-specific differences in the treatment of severe psoriasis. AB - BACKGROUND: Psoriasis treatment requires consideration of patient-specific concerns in addition to the severity of skin involvement. There may be sex specific differences in the treatment of severe psoriasis. OBJECTIVE: The purpose of this study was to determine whether there are sex-specific differences in the treatment of severe psoriasis. METHODS: We analyzed the medications prescribed to patients with a primary and only diagnosis of psoriasis recorded in the 1990-1994 National Ambulatory Medical Care Survey. RESULTS: There were 8.5 million visits to physicians for the treatment of psoriasis in the years 1990-1994. These visits were made by approximately 4.3 million women and 4.1 million men. Only 39% of patients receiving systemic treatments were women. Women received less methotrexate (23% women) and etretinate (35% women) than men, but more psoralen photochemotherapy (PUVA) (63% women) and isotretinoin (100% women) than men. In contrast, there was no notable difference by sex in the potency of topical corticosteroid agents prescribed. CONCLUSION: For mild disease treated with topical agents alone, there is no notable difference in the treatment of men and women. Men are more likely than women to receive intensive treatments for severe psoriasis, at least in part because of the teratogenic potential of these treatments. There is a need for development of new treatments for severe psoriasis that are safe for women of childbearing potential. PMID- 10727308 TI - Periorbital comedones and their relationship to pitch tar: a cross-sectional analysis and a review of the literature. AB - BACKGROUND: Comedones, often in a periorbital distribution, have been claimed to be associated with pitch tar. OBJECTIVE: This cross-sectional study compares the incidence of periorbital comedones between a group of individuals exposed to pitch tar and a control group. METHODS: The incidence of periorbital comedones was determined in workers (n = 55) at a tar distillation plant. A comparison group (n = 55) not exposed to pitch tar was also examined for the presence of facial comedones. The respective incidence rates were compared by using the Fisher exact test. RESULTS: Eighteen percent (10/55) of the study group and 4% (2/55) of the control group had periorbital comedones. The study group had a significantly greater incidence of periorbital comedones (P =.01). CONCLUSION: The incidence of periorbital comedones is significantly greater in individuals exposed to pitch tar. Further studies are needed to elucidate the potential relationship between pitch tar and periorbital comedones. PMID- 10727309 TI - Screening for azathioprine toxicity: a pharmacoeconomic analysis based on a target case. AB - The risk of azathioprine-induced myelosuppression can be predicted by detecting patients with intermediate or low thiopurine methyltransferase (TPMT) activity. Population studies have shown that 89% of whites have high TPMT activity, 11% have intermediate TPMT activity, and 0.3% have low TPMT activity. Three specific mutations in the TPMT gene that cause decreased TPMT activity have recently been identified. Patients homozygous for the TPMT mutations have low TPMT activity, and patients heterozygous for TPMT mutations have intermediate TPMT activity. This has led to the development of a technique for TPMT genotype analysis that will identify patients at risk of azathioprine-induced myelosuppression. We report a case of a patient with bullous pemphigoid who experienced azathioprine induced myelosuppression and who was later found to be homozygous for TPMT mutant alleles. Using the cost of treatment required for this patient and the estimated population prevalence of TPMT mutations, we examined the cost impact of screening for TPMT mutations in all patients being considered for azathioprine therapy. We calculated that screening is cost-neutral assuming patients homozygous for TPMT mutations experience myelosuppression, and that it is cost-beneficial assuming patients heterozygous for TPMT mutations also experience myelosuppression while receiving azathioprine. Screening patients for TPMT mutations will reduce the risk of azathioprine-induced myelosuppression, and our study suggests that it may also be a cost-attractive strategy. PMID- 10727310 TI - Multiple familial cutaneous glomangioma: a pedigree of 4 generations and critical analysis of histologic and genetic differences of glomus tumors. AB - BACKGROUND: Glomangiomas are benign tumors arising from neuromyoarterial cells surrounding cutaneous arteriovenous anastomoses that serve as temperature regulators. They exist as solitary or multiple types, occurring sporadically or in a familial pattern, the latter of which is rare. OBJECTIVE: We describe a 4 generation pedigree of familial cutaneous glomangioma, in addition to the 3 other well-documented pedigrees reported in the literature to date, and we clarify ways in which to distinguish the different types of glomus tumors. METHODS: Nodular skin lesions of 4 affected family members were analyzed by histologic, immunohistologic, and electron microscopic methods. To elucidate the gene defect in this family, we searched for a linkage to a candidate locus on chromosome 11q23 previously identified in paragangliomas, one form of glomus tumor, in 16 family members of 4 generations by using polymorphic markers. RESULTS: The diagnosis of disseminated cutaneous glomangiomas was confirmed histologically in 4 family members of 3 different generations. Glomangiomas were transmitted in an autosomal dominant pattern via the paternal line. Genetic linkage analysis of the affected family members excluded linkage to chromosome 11q23. CONCLUSION: An autosomal dominant pattern of inheritance has been described for glomus tumors of the paraganglioma type originating from the APUD cell system, the underlying genetic defect of which has been mapped to chromosome 11q23. In contrast, we show that the genetic defect in disseminated cutaneous glomus tumors of the glomangioma type deriving from smooth muscle cells or pericytes is not linked to chromosome 11. Thus we suggest that the common term glomus tumor, used for both paragangliomas and glomangiomas in the current literature, is misleading and should be avoided because these tumors have different histologic derivation and genetic origin. PMID- 10727311 TI - Genital melanotic macules: clinical, histologic, immunohistochemical, and ultrastructural features. AB - BACKGROUND: Genital melanotic macules are poorly recognized lesions, which appear as isolated discrete macules. Their occurrence, usually as new pigmented lesions in adult life, can cause concern because they can mimic early melanoma. OBJECTIVE: Our purpose was to define the clinical, histologic, immunohistochemical, and electronmicroscopic features of genital melanotic macules. METHODS: History and clinical features of 10 patients (5 female, 5 male) were assessed in detail. Histologic findings were reviewed in 5 cases, and immunohistochemistry, with the use of the HMB-45 antibody, in 4 cases and electron microscopy in 3 cases. RESULTS: Clinically the lesions varied in color, tan to dark brown/black, and size (0.5-2 cm). Histologic findings showed increased basal pigmentation without atypical features. HMB45 antibody staining was negative. Electron microscopy showed normal morphology and number of melanocytes but increased melanosomes and dermal melanophages. CONCLUSION: Genital melanotic macules are benign, asymptomatic, discrete areas of hyperpigmentation that occur equally in men and women. Histologic, immunohistochemical, and electronmicroscopic study confirms their benign nature. PMID- 10727312 TI - Management of porphyria cutanea tarda in the setting of chronic renal failure: a case report and review. AB - The treatment of porphyria cutanea tarda (PCT) in patients with chronic renal failure poses a therapeutic challenge. In the absence of renal failure, phlebotomy and oral antimalarials have been the standard of care for PCT. However, in the presence of renal failure, associated chronic anemia often precludes the use of phlebotomy, and oral antimalarials are usually ineffective. We describe a patient with severe symptomatic PCT and chronic renal failure whose disease was successfully managed with a combination of high-dose erythropoietin and small volume phlebotomy. We also review several previously reported approaches to management of PCT in the setting of renal failure, which include small repeated phlebotomy, erythropoietin, deferoxamine, chloroquine, plasma exchange, high-efficiency/high-flux hemodialysis, cholestyramine, charcoal hemoperfusion, and kidney transplantation. An algorithm for the management of these patients is proposed. PMID- 10727313 TI - A body-weight-independent dosing regimen of cyclosporine microemulsion is effective in severe atopic dermatitis and improves the quality of life. AB - BACKGROUND: Cyclosporine has shown to be effective in severe atopic dermatitis. Little has been reported on the new microemulsion form (Sandimmune, Neoral) in the treatment of this disease. Also, it has not been investigated whether a body weight-independent dosing regimen of cyclosporine is appropriate for the treatment of atopic dermatitis. OBJECTIVE: The goal of this study was to investigate a body-weight-independent dosing regimen of cyclosporine microemulsion in severe atopic dermatitis by comparing high and low starting doses of treatment. METHODS: A total of 106 adults with severe atopic dermatitis were enrolled in this double-blind study and randomized to receive a starting dose of either 150 mg (low) or 300 mg (high) of cyclosporine microemulsion daily. After 2 weeks the dose could be reduced by 50% if the clinical symptom score was reduced by 50% or more. After 8 weeks the responders entered a 4-week follow-up phase and were randomized to either stop treatment or to continue on their last effective dose every second day. RESULTS: After 2 weeks of treatment the total symptom score decreased from 59.0 to 39.3 with 150 mg and from 60.7 to 33.2 with 300 mg cyclosporine (P <.05). Until week 8 there was a further decrease in the clinical symptom score to 30.8 with low-dose therapy and 25.5 with high-dose therapy. Similar positive effects could be observed in assessments of affected body surface area, itching, sleep loss, and quality of life. At week 2, there was an increase of 0.6% in serum creatinine in patients receiving 150 mg, and 5.8% in the 300 mg group (P <.01). At week 8, the effect on serum creatinine was similar, with a 1.1% rise in the low dose group and a 6.0% increase in the high dose group. Body weight had no influence on efficacy or tolerability in this study. CONCLUSION: Body-weight-independent dosing with cyclosporine seems to be feasible in the short-term treatment of severe atopic dermatitis. Although the starting dose of 300 mg/day is more effective than 150 mg/day, the 150 mg dose would be preferable for the initiation of therapy because of its excellent renal tolerability. PMID- 10727314 TI - Pendulaser carbon dioxide resurfacing laser versus electrodesiccation with curettage in the treatment of isolated, recalcitrant psoriatic plaques. AB - BACKGROUND: Treatment of recalcitrant psoriatic plaques located on certain areas of the body remains problematic despite the many therapeutic options available. OBJECTIVE: This study was conducted to determine the efficacy of Pendulaser carbon dioxide (CO(2)) resurfacing laser to that of electrodesiccation with curettage in the treatment of recalcitrant psoriatic plaques. METHODS: A single psoriatic plaque was divided into thirds, one part treated with CO(2) resurfacing laser, another with electrodesiccation and curettage, and the third left untreated. The psoriatic epidermis and papillary dermis were removed by the two treatment modalities. RESULTS: CO(2) resurfacing laser and electrodesiccation with curettage produced similar therapeutic effects in terms of improvement of psoriasis and were significantly better than the control 4 months later, but not at 6 months. CONCLUSION: For limited recalcitrant psoriatic plaques, CO(2) resurfacing laser and electrodesiccation with curettage may provide an alternative short-term treatment; however, caution must be exercised and the moderately high risk of scarring carefully weighed against the potential benefits. PMID- 10727315 TI - Pulsed dye laser treatment of port-wine stains in pigmented skin. AB - BACKGROUND: Little information is available on pulsed dye laser treatment of darker Fitzpatrick skin types. METHODS: We retrospectively reviewed our clinical experience treating mainly facial (12/13) port-wine stains (PWSs) in 13 patients of skin type V, whose ages ranged from 3 to 65 years. RESULTS: The response was excellent in 2 patients, good or moderate in 4 each, slight in 1, and poor in 2. Hyperpigmentation occurred in 6 patients and hypopigmentation in 1, which improved despite continuing treatment. Limited atrophic scarring developed in two patients. Although these results were less good and side effects more frequent than described in skin types I to IV, 6 patients (46%) achieved a good or excellent response and 85 (87.6%) of the total 97 laser treatments were not associated with significant problems. CONCLUSION: Patients of skin type V with a PWS should not be excluded from pulsed dye laser therapy, provided that treatment expectations and risks are fully discussed. PMID- 10727316 TI - Is the incidence of malignant melanoma decreasing in young Australians? AB - Recent reports have detected a decrease in the incidence of malignant melanoma (MM) in young Australian women, but an increase in older Australians. The incidence rates were calculated for the entire population and no adjustments were made to take into account the change in the racial composition of the Australian population. In the past 25 years there has been an influx of immigrants from Asia, the Pacific Islands, and the Middle East who are at low risk for MM. The migrants are relatively young and there are more Asian women than men. The large increase in the percentage of the population that has a low risk for MM may be an important reason why the incidence has fallen in some population groups. PMID- 10727317 TI - Surgical pearl: use of the cutaneous punch for the removal of lipomas. PMID- 10727318 TI - From the literature: intralesional 5-FU in the treatment of hypertrophic scars and keloids: clinical experience. PMID- 10727319 TI - Cutaneous schistosomiasis: report of a case and review of the literature. AB - Cutaneous disease is a previously reported but unusual presentation for schistosomiasis. We report a case of Schistosoma haematobium infection that appeared 3 years after exposure, with skin lesions as the sole manifestation. The diagnosis was made on the basis of a routine skin biopsy and the patient did well after therapy with praziquantel. Dermatologists should be aware of this presentation of schistosomiasis when evaluating patients with unusual skin lesions who have traveled in areas where schistosomiasis is endemic. PMID- 10727320 TI - Acquired brachial cutaneous dyschromatosis: a common pigmentary disorder of the arm in middle-aged women. AB - We studied 20 Caucasian middle-aged patients, mostly women, who had asymptomatic, gray-brown patches with geographic borders, occasionally interspersed with hypopigmented macules, on the dorsum of the forearms, mostly bilaterally. The distal aspect of the arms was also involved in 2 patients; the face was always spared. Civatte's poikiloderma was associated with this hyperpigmentation in 9 patients. Epidermal atrophy, basal layer hyperpigmentation, elastosis and angiectases were histopathologic features. Pregnancy, menopause, local inflammation, and cosmetics were not predisposing factors. Undue sun exposure, oral contraceptives or estrogens were found only occasionally. Sixty-five percent of patients had hypertension and had been taking antihypertensive drugs, especially angiotensin-converting enzyme inhibitors, for years before pigmentation began. Acquired brachial cutaneous dyschromatosis (ABCD) is the suggested title of this disorder, which is not rare. Its prevalence in postmenopausal women, the hypopigmented macules, and the absence of a relation with estrogens, pregnancy, or hormone replacement therapy, all help to distinguish ABCD from melasma. An association with Civatte's poikiloderma as well as hypertension and/or antihypertensive drugs is suggested. PMID- 10727321 TI - Hyperreactivity of pseudoxanthoma elasticum-affected dermis to vitamin D3. AB - A 68-year-old woman with pseudoxanthoma elasticum (PXE) was treated with oral vitamin D(3). After 2 weeks, new papules developed on the pre-existing plaques. Biopsy specimens of a fresh papule showed more abundant calcium deposition than before therapy. Electron microscopy revealed electron-lucent areas, which suggested unusual mineralization. From these findings, we propose that in patients with PXE, certain cutaneous tissues are highly sensitive to vitamin D(3), resulting in microdeposition of calcium salts. PMID- 10727322 TI - Treatment of severe atopic dermatitis by topical immune modulation using dinitrochlorobenzene. AB - We present the results of a small uncontrolled pilot trial which suggest that contact sensitization to dinitrochlorobenzene and repeated weekly applications significantly improve the clinical status of severe atopic dermatitis in adults. Although no changes were noted in circulating levels of either lymphocyte subset populations or the serum cytokines assayed in this trial, our observations may be due to topical immune modulation by dinitrochlorobenzene. Larger controlled studies of dinitrochlorobenzene treatment in atopic dermatitis are warranted. PMID- 10727323 TI - Sarcoidosis of the scalp: a case series and review of the literature. AB - Sarcoidosis of the scalp is a rare manifestation of cutaneous sarcoidosis. We report 4 cases, all in African-American women with systemic sarcoidosis. A review of the English-language literature reveals only 24 reported cases, most in African-American women with systemic involvement and other cutaneous lesions. Scalp sarcoidosis may exhibit variable morphologies and therefore must be kept in the differential diagnosis of plaques or nodules of the scalp as well as both cicatricial and noncicatricial alopecia. PMID- 10727324 TI - Clear cell hidradenoma in a young girl. AB - Clear cell hidradenoma was diagnosed in a girl at 3(1/2) years of age. Only one case of hidradenoma has been documented in the first decade of life, although it remains unclear whether it was clear cell hidradenoma. This case demonstrates that clear cell hidradenoma is a rare differential diagnosis of cutaneous tumors even in young children, which is of special interest, because malignant transformations can occur. PMID- 10727325 TI - The case for micrographically controlled skin surgery. PMID- 10727326 TI - Phototoxicity profile of PUVA-oil bath therapy. PMID- 10727327 TI - Prurits. PMID- 10727329 TI - Mucocutaneous reactions to chemotherapy. PMID- 10727330 TI - Dermatomyositis and cancer: East and West. PMID- 10727331 TI - Surface Complexation of Calcium Minerals in Aqueous Solution. AB - The complexation of Alizarin Red S (ARS) at the surface of hydrous fluorite particles has been investigated by means of potentiometric titrations, adsorption experiments, and zeta-potential measurements in 0.1 mol dm(-3) KCl ionic medium at 25.0 degrees C, as well as by UV/visible specular reflectance, FT-IR, and FT Raman spectroscopy. Chemical reaction models describing the equilibria of ARS (HA(2-)) at the aqueous fluorite surfaces ( identical withX) have been established as follows: Experimental data were evaluated using the computer program FITEQL on the basis of a constant capacitance model for the electric double layer. Surface complexation mechanisms involving the R-SO(3)(-), R-beta OH, and R-alpha-OH active groups of the ARS molecule are proposed to describe coordination to the fluorite surface. Copyright 2000 Academic Press. PMID- 10727332 TI - An Evaluation of Cellular Analyte-Receptor Binding Kinetics Utilizing Biosensors: A Fractal Analysis. AB - A fractal analysis is presented for cellular analyte-receptor binding kinetics utilizing biosensors. Data taken from the literature can be modeled by using (a) a single-fractal analysis and (b) a single- and a dual-fractal analysis. Case (b) represents a change in the binding mechanism as the reaction progresses on the biosensor surface. Relationships are presented for the binding rate coefficient(s) as a function of the fractal dimension for the single-fractal analysis examples. In general, the binding rate coefficient is rather sensitive to the degree of heterogeneity that exists on the biosensor surface. For example, for the binding of mutagenized and back-mutagenized forms of peptide E1037 in solution to salivary agglutinin immobilized on a sensor chip, the order of dependence of the binding rate coefficient, k, on the fractal dimension, D(f), is 13.2. It is of interest to note that examples are presented where the binding coefficient (k) exhibits an increase as the fractal dimension (D(f)) or the degree of heterogeneity increases on the surface. The predictive relationships presented provide further physical insights into the binding reactions occurring on the surface. These should assist us in understanding the cellular binding reaction occurring on surfaces, even though the analysis presented is for the cases where the cellular "receptor" is actually immobilized on a biosensor or other surface. The analysis suggests possible modulations of cell surfaces in desired directions to help manipulate the binding rate coefficients (or affinities). In general, the technique presented is applicable for the most part to other reactions occurring on different types of biosensors or other surfaces. Copyright 2000 Academic Press. PMID- 10727333 TI - Low Molecular Weight Gelators for Organic Fluids: Gelation Using a Family of Cyclo(dipeptide)s. AB - Simple cyclo(dipeptide)s consisting of diverse amino acids are able to cause physical gelation in a wide variety of organic fluids, including edible oils, glyceryl esters, alcohols, and aromatic molecules. Minimum gel concentrations, FTIR spectroscopy, NMR spectroscopy, and electron micrograph are used to characterize gel phenomenon. The intermolecular hydrogen bonding between N-H and C=O in cyclo(dipeptide)s plays an important role in gelation. FTIR and X-ray diffraction data suggest that the aggregate responsible for gel is an assembly of hydrogen-bonded molecular ladders, which are initially formed from numerous molecules through intermolecular hydrogen bonding. The ladder-like aggregates are intertwined and interlocked, and finally immobilize organic fluids. The gelation ability is discussed in connection with the three-component solubility parameters of solvents. Copyright 2000 Academic Press. PMID- 10727334 TI - Bubble Oscillations in a Closed Cell. AB - A theoretical analysis is given to describe the behavior of an oscillating bubble in a closed measuring cell taking into account the finite liquid compressibility and the cell deformation. The results show that the behavior of a closed liquid cell can differ significantly from that of open cells. For example, under the same conditions two stable meniscus positions can be obtained in a closed cell. In a closed cell a meniscus larger than a hemisphere can be stable even when the gas compartment is open, while in an open cell such a meniscus is always unstable. For closed cells the meniscus can jump between the two equilibrium positions either randomly or under the influence of regular factors, such as external pressure, temperature, and surface tension changes. Relationships are obtained for the description of the pressure response on external harmonic perturbations which make it possible to determine the complex dilatational elasticity as a function of frequency. It is shown that the measured signal can depend on the cell properties, which should be taken into account in the interpretation of experimental data. Copyright 2000 Academic Press. PMID- 10727335 TI - Self-Assembly and Surface Structure of an Amphiphilic Graft Copolymer, Polystyrene-graft-omega-Stearyl-Poly(ethylene oxide). AB - Surface properties of the polystyrene-graft-omega-stearyl-poly(ethylene oxide) (PS-g-SPEO) have been characterized by X-ray photoelectron spectroscopy (XPS), differential scanning calorimetry (DSC), contact angle, and spin probe techniques. The XPS results indicate that the surface and bulk composition of the PS-g-SPEO copolymers differ remarkably from each other. The stearyl and EO components enrich at the copolymer/air interfaces due to the self-assembly of the stearyl groups. At the PS-g-SPEO-72.6 surface (the x in PS-g-SPEO-x indicates the bulk density of the SPEO in wt%), the self-assembly of the hydrophobic stearyl groups is strong enough to form a stable liquid crystalline phase as indicated by DSC. At polymer/water interfaces, PS-g-SPEO-72.6 presents a hydrophilic surface with low PEO mobility, whereas PS-g-SPEO-50.6 and PS-g-SPEO-31.0 present the hydrophobic surface with high PEO mobility. The two different types of the surfaces, with different characters in surface energy, surface mobility of PEO, and surface architecture of SPEO, will be quite valuable as models for detecting the synergistic action of the PEO chains and the stearyl groups (specific ligand for albumin binding) in protein solutions. Copyright 2000 Academic Press. PMID- 10727336 TI - IR Spectra of CH(4) Physisorbed on an Active Carbon at Low Temperature. AB - The IR absorption band of methane adsorbed on an active carbon was obtained at 153 K. The IR nu(1) band of methane (inherently IR inactive in the gaseous phase) adsorbed on the active carbon was induced by the electric field of surface groups, even though the adsorbent was known to be macroscopically hydrophobic. The source of the electric field was suggested to be a hydrogen-bonding surface carboxyl or phenolic OH group. The strength of the electric field was estimated to be 1.13 x 10(5) esu, although the number of sites bearing an electric field was very small. A strong electric field (1.75 x 10(5) esu) was created on the carbon surface by oxidation, and its origin was suggested to be carbonyl groups. Copyright 2000 Academic Press. PMID- 10727337 TI - Adsorption of Gemini and Conventional Cationic Surfactants onto Montmorillonite and the Removal of Some Pollutants by the Clay. AB - The adsorption of a series of gemini surfactants, [C(n)H(2n+1)N(+) (CH(3))(2) CH(2)CH(2)](2).2Br(-), where n = 10, 12, 14, and 16, on clay (Na-montmorillonite) from their aqueous solution in 0.01 M KBr and the effect of this adsorption on the removal of 2-naphthol and 4-chlorophenol have been studied. Compared to those of conventional cationic surfactants with similar single hydrophilic and hydrophobic groups (C(n)H(2n+1)N(+)(CH(3))(3).Br(-), where n = 10, 12, 14, and 16), the molar adsorptions of the gemini and conventional surfactants are almost identical. This indicates that only one of the hydrophilic groups in the gemini molecule is adsorbed onto the clay and that the second hydrophilic is presumably oriented toward the aqueous phase, in contrast to the adsorption of the conventional surfactants, where the hydrophobic group is oriented toward the aqueous phase. Stability studies on dispersions of clay treated with the two types of surfactants confirm this. The slight increase in the moles of surfactant to values above the CEC of the clay with an increase in the carbon number of the hydrophobic chain indicates that adsorption through hydrophobic group interaction occurs in addition to the major ion exchange. Adsorption studies of the pollutants onto the clay treated by either the gemini or the conventional surfactants show that the former are both more efficient and more effective at removing the pollutants from the aqueous phase. Copyright 2000 Academic Press. PMID- 10727338 TI - The Structure of Adsorbed CO(2) in Slitlike Micropores at Low and High Temperature and the Resulting Micropore Size Distribution Based on GCMC Simulations. AB - The Monte Carlo method is used in its grand ensemble variant in combination with CO(2) experimental isotherm data at low (195.5 K) and high temperatures (at 298 and 308 K, i.e., slightly below and above the CO(2) critical temperature, respectively) to characterize microporous carbons and obtain the corresponding pore size distribution (PSD). Specifically, the CO(2) density inside a single, slit-shaped, graphitic pore of given width is found on the basis of grand canonical Monte Carlo (GCMC) simulations for a predefined temperature and different relative pressures. The simulation results provide useful insights concerning the densification process in the micropores and the structure of the CO(2) molecules packing in the individual pores as the temperature or pressure changes from 195.5 K to ambient or from very low to 70 bar, respectively. Effects of temperature, pore size, quadrupole interactions, and molecule elongation on the local density profile within the pore are examined and discussed. In an additional step, we determine the optimal PSD for which the best match is obtained between computed and measured CO(2) isotherms. Comparisons are made between the PSDs found for the same carbon sample at low and high temperatures and conclusions are drawn concerning the applicability of the method and the reliability of the resulting micropore size distributions. Copyright 2000 Academic Press. PMID- 10727339 TI - Competitive Adsorption of Ferricyanide and Ferrocyanide on gamma-Al(2)O(3) Surface. AB - In the past 3 decades, research has proven the significance of competitive adsorption in the equilibrium of pollutants between solid and liquid phases. However, studies on the competitive adsorption of complex ions are very limited in spite of its important role in transporting pollutants in the natural environment. The objective of this study is to derive the thermodynamic parameters of the competitive adsorption between ferricyanide and ferrocyanide from the modified Langmuir isotherm and the triple-layer model (TLM) to determine the location of adsorption. The effects of pH, temperature, and ion concentration on competitive adsorption onto gamma-Al(2)O(3) were investigated. The results demonstrate that ferrocyanide is more competitive than ferricyanide. By comparing the derived K(app) with K(int), we inferred that the adsorption of ferricyanide and ferrocyanide onto gamma-Al(2)O(3) was achieved through outer-sphere complexation. The negative DeltaH degrees indicated that the adsorption was exothermic. The positive entropy (Delta S degrees ) was caused by the replacement and release of a greater number of smaller surface ions by adsorbed ferricyanide and ferrocyanide ions of larger size. Copyright 2000 Academic Press. PMID- 10727340 TI - Bubble Motion in Aqueous Surfactant Solutions. AB - This paper presents the results of numerical simulations of the unsteady motion of a single bubble that is released or injected into water. The governing equations are solved with a finite difference method using an adaptive boundary fitted coordinate system. Results are shown for bubbles in the size range 0.72 to 1.5 mm. The effects of surfactants on the motion and shape of the bubble are investigated. When the surfactant concentration is sufficiently small, the bubble attains a maximum velocity before slowing down to its steady-state velocity. Although the steady-state velocity is nearly independent of surfactant concentration, the maximum velocity can be comparable to steady-state velocity in pure water. This behavior is observed even when the bubble is allowed to equilibrate prior to releasing it. The formation of an immobilized surfactant cap is observed soon after the bubble is released. The effect of the injection velocity on the bubble velocity profile is investigated. The effects of the sorption rate constants and the bulk surfactant concentration on the behavior of the bubble are investigated. The feasibility of using experimental measurements and simulations of unsteady bubble velocities to estimate sorption rate constants is discussed. Copyright 2000 Academic Press. PMID- 10727341 TI - In Situ Observation of Growth Process of alpha-L-Glutamic Acid with Atomic Force Microscopy. AB - The growth kinetics of the (111) and (001) faces of an alpha-L-glutamic acid crystal was investigated from the measurements of the growth rates, coupling with in situ surface observation using atomic force microscopy (AFM). The data of the growth rates were examined by applying the theoretical equations of the BCF and NaN models. The results indicate that the growth mechanism is not due to the screw dislocation but to the two-dimensional nucleation, i.e., the NaN model. It was confirmed by AFM observation that both the (111) and (001) faces grew with the "nucleus above nucleus" (NaN) mechanism. However, the difference of the two dimensional nucleation behavior was observed between the faces. The growing surface of the (111) face was also observed in the presence of L-phenylaline (L Phe) and the pinning effect by L-Phe on the growing step on the (111) face was confirmed. This result supports the mechanism of the additive effect, which was proposed previously. Copyright 2000 Academic Press. PMID- 10727342 TI - The Influence of Alkali Metal Ions on Homogeneous Nucleation of Al(OH)(3) Crystals from Supersaturated Caustic Aluminate Solutions. AB - Homogeneous nucleation of Al(OH)(3) crystals from synthetic, optically clear, caustic aluminate solutions and the influence of alkali metal ion (Na(+) versus K(+)) have been investigated under isothermal, batch crystallization conditions. The nucleation kinetics showed a seventh-order dependence upon Al(III) relative supersaturation and a strong temperature effect. Activation energy of 160 kJ mol( 1) and interfacial energy of 33 mJ m(-2) were estimated and found to be independent of alkali ion, as was the Al(OH)(3) equilibrium solubility. The nucleation rate, however, was faster in aging sodium than in potassium aluminate solutions. It appears that Na(+) ions provide greater stability for the formation and densification of Al(III)-containing, supramolecular clusters which grow more rapidly into Al(OH)(3) crystallites than do K(+) ions. The development of the Al OH octahedral structure of Al(OH)(3) nuclei is an alkali metal ion-mediated, chemical reaction-controlled condensation process, displaying specific gibbsite (gamma-Al(OH)(3))-bayerite (alpha-Al(OH)(3)) dimorphism. Furthermore, significant differences in the level of alkali ion incorporation, reflecting in the purity and morphology of the crystalline product, were observed. Copyright 2000 Academic Press. PMID- 10727343 TI - Electrocatalytic Oxidation of Coenzyme NADH at Carbon Paste Electrodes, Modified with Zirconium Phosphate and Some Redox Mediators. AB - Carbon paste electrodes (CPE), modified with zirconium phosphate (ZrP) along with some redox mediators, were prepared and shown to be active in electrocatalytic oxidation of coenzyme NADH. Nile blue (NB), methylviologen (MV), and benzylviologen (BV) were added to ZrP-containing CPE, showing a shift of their redox potentials toward the positive direction, ranging from ca. 0.20 to 0.35 V. This shift was interpreted in terms of different complexation abilities of oxidized and reduced forms of mediators with the host matrix. The modified electrodes prepared showed electrocatalytic activity for electrooxidation of NADH. The i,c-dependencies for the modified electrodes were shown to follow Michaelis-Menten kinetics. For C:ZrP:NB and C:ZrP:MV electrodes, Michaelis constants of 0.870 +/- 0.012 and 0.153 +/- 0.015 mM, respectively, were obtained. The sensitivity of C:ZrP:NB and C:ZrP:MV electrodes to NADH varied from 19.2 to 60.8 and from 18.4 to 50.9 uA/mM c cm(2), respectively, by changing the working potential from -0.2 to 0.2 V vs. SCE. Copyright 2000 Academic Press. PMID- 10727344 TI - Metallomicellar Catalysis Hydrolysis of p-Nitrophenyl Picolinate Catalyzed by Copper(II), Nickel(II), and Zinc(II) Complexes of Long Alkyl Pyridine Ligands in Micellar Solution. AB - The ternary complex kinetic model for metallomicellar catalysis has been proposed in this paper. The catalytic effects of bivalent metal ion (Cu(2+), Zn(2+), and Ni(2+)) complexes of long alkyl pyridine ligands upon the hydrolysis of p nitrophenyl picolinate (PNPP) have been studied kinetically in aqueous buffer of pH ranging from 5.0 to 8.5 at 30 degrees C. The effect of pH on the reactivity is discussed. The results indicate that the metallomicelles formed by pyridine ligands promote the hydrolysis of PNPP, and the order is Cu(II) system>Ni(II) system>Zn(II) system. A stereochemical modification of the complex in the CTAB micelle is suggested as a likely explanation for the observed phenomena. Copyright 2000 Academic Press. PMID- 10727345 TI - Ellipsometry Studies of Lipoprotein Adsorption. AB - The adsorption of a number of lipoproteins, i.e., low-density lipoprotein (LDL), oxidized LDL (oxLDL), high-density lipoprotein (HDL), and lipoprotein (a), at silica and methylated silica as well as at the latter surface modified through adsorption of proteoheparan sulfate, was investigated with in situ ellipsometry at close to physiological conditions. It was found that LDL, oxLDL, HDL, and lipoprotein (a) all adsorbed more extensively at silica than at methylated silica. Upon exposure of the methylated silica surface to proteoheparan sulfate, this proteoglycan adsorbs through its hydrophobic moiety, thereby forming a layer similar to that in the biological system, with the polysaccharide chains forming brushes oriented toward the aqueous solution. Analogous to the biological system, both lipoprotein (a) and LDL were found to deposit at such surfaces, the latter particularly in the simultaneous presence of Ca(2+). After HDL pre-exposure, however, no LDL deposition was observed, even at high LDL and Ca(2+) concentrations. These findings correlate well with those obtained from clinical investigations on risk factors for atherosclerosis. Copyright 2000 Academic Press. PMID- 10727346 TI - Reduction of Iron(III) Ion by Activated Carbon Fiber. AB - The mechanisms of adsorption of iron(II) ion, iron(III) ion, and reduced iron(III) ion onto an activated carbon fiber and the ability of carbon fibers to reduce iron(III) ion were investigated on the basis of the amounts of iron ion adsorbed. The amount of iron(II) ion adsorbed onto the activated carbon fiber increased with increasing adsorption temperature. Iron(II) ion was more easily removed by the activated carbon fiber than iron(III) ion. Iron(III) ion was adsorbed onto the activated carbon fiber after being reduced to iron(II) ion. The reduction ability of A-20 was stronger than that of A-10 because the hydrophilic groups of A-20 were larger than those of A-10. It is concluded that the activated carbon fiber has a reduction effect on iron(III) ion and that the reduction effect of the activated carbon fiber depended on the number of hydrophilic groups on the activated carbon fiber. Copyright 2000 Academic Press. PMID- 10727347 TI - Partial Wetting in Capillary Liquid Absorption by Limestones. AB - We report measurements of the capillary absorption (imbibition) of water, n heptane, n-decane, n-dodecane, methanol, ethanol, propan-2-ol, and ethanol-water mixtures into a number of dry calcitic limestones. The data are analyzed on the basis of unsaturated flow theory to give values of sorptivity S for water and for each organic liquid at different temperatures. The results show that for the organic liquids S varies as (surface tension/viscosity)(1/2) but that for the water S is anomalously low, indicating partial wetting. The wettability of these limestones is discussed in relation to natural contamination of the pore surface. Copyright 2000 Academic Press. PMID- 10727348 TI - Formation Process of MCM-41 Precursor and Porous Texture of MCM-41. AB - The formation process of the MCM-41 precursors (silicate/surfactant complex) was investigated on the basis of the pH titration curves of Na(4)SiO(4) in the presence of [C(16)H(33)N(CH(3))(3)]Cl. Measurements of the pH titration curves were carried out using the computer-controlled gravimetric titrator constructed in our laboratory. The white precipitate (MCM-41 precursor) was abruptly formed at pH 11.1 (298 K) and at pH 9.9 (343 K). Formation of the MCM-41 precursor can be explained by coagulation of the rod-like micelle colloids whose surface is covered by the condensed silicate anions of (HSiO(3))(n)(n-). The porous texture of the MCM-41 samples whose precursors were synthesized under different conditions was analyzed on the basis of the adsorption isotherms of nitrogen at 77 K. It has been shown that the MCM-41 sample whose precursor was prepared at pH 9.9 and 343 K shows one sharp peak (r(p) = 1.65 nm) in the pore size distribution curve, but the MCM-41 samples whose precursors were prepared at pH 6.5-5.0 and 343 K give two peaks (r(p) = 1.66 nm and r(p) = 2.12-2.36 nm). The appearance of the second peak (r(p) = 2.12-2.36 nm) has been considered to be in connection with the destruction of the MCM-41 precursor into small fragments in acidic medium. Copyright 2000 Academic Press. PMID- 10727349 TI - Spectroscopic Identification of Adsorbed Intermediates Derived from the CO+H(2)O Reaction on Zeolite-Encapsulated Gold Catalysts. AB - Identification of reaction intermediates in the water-gas shift reaction (WGSR: H(2)O+CO-->H(2)+CO(2)) on Au(n+) (110 um) and the mesoscopic (<10 um) length scale properties of the material. Complementary information from nitrogen adsorption, on the mesoscopic scale, and NMR imaging, on the macroscopic scale, is used in conjunction with the mercury porosimetry data to provide a more accurate structural representation of a porous medium. The model is therefore able to probe spatial geometric changes in pellet structural architecture over many length scales during processes such as catalyst manufacture and the deactivation of catalysts by coke deposition. Copyright 2000 Academic Press. PMID- 10727352 TI - A Model for Overlapped EDL Fields. AB - A model to determine the electrical potential and ionic concentration distributions in the overlapped EDL fields between two infinitely large flat plates was developed in this paper. It was found that the treatment in the classical theory may lead to an inaccurate description due to the misuse of the Boltzmann equation. New governing equations for such an overlapped EDL field were derived. The potential distribution and ionic concentration distributions for inorganic oxide-aqueous solution systems were calculated. The results were compared with the predictions of the classical theory. Differences between our model and the classical treatment were found, especially at small separation distance. Copyright 2000 Academic Press. PMID- 10727353 TI - Local Structure Analysis of Strontium Sorption to Hydrous Manganese Oxide. AB - To develop mechanistic models of contaminant distribution processes, we conducted an X-ray absorption fine structure analysis of strontium sorption to hydrous manganese oxide (HMO). Sr K-edge measurements were performed at 298, 220, and 77 K, and at sample loadings from 10(-4) to 10(-2) mol Sr/g HMO. Results from fitting the first shell in the sorbed samples indicate that strontium is surrounded by 10-12 oxygen atoms at an average distance of 2.58 A. This coordination environment is consistent with the strontium atom remaining hydrated upon sorption to the oxide, where in water hydrated strontium has approximately 9 atoms of oxygen at 2.62 A. Furthermore, the temperature dependence of the strontium-oxygen bond also suggests physical adsorption due to the large contribution of the dynamic component of the Debye Waller factor. Although second shell data are consistent with either 3 manganese atoms at 4.12 A or 6 strontium atoms at 3.88 A, both the near-edge and fine structure data for the manganese K edge indicate that the local coordination environment of the manganese ion remains intact as a function of time or strontium sorption. Furthermore, the local structure of amorphous manganese oxide is highly ordered. Copyright 2000 Academic Press. PMID- 10727354 TI - A Low-Frequency Infrared Study of the Reaction of Methoxymethylsilanes with Silica. AB - A thin film infrared technique is used to observe bands due to hydrogen-bonded and chemisorbed methoxymethylsilanes on fumed silica in the low-frequency region below 1300 cm(-1). The low-frequency region contains the characteristic bands due to Si-O-Si, Si-O, Si-C, Si-CH(3), and SiO-C modes. Band assignments are aided by ab initio calculations and comparison to thin film experiments of adsorbed chloromethylsilanes. The spectral interpretation was expected to be more complicated than that of the corresponding chlorosilanes because the strong SiO-C alkoxy bands lie in the same region as the Si-O-Si bands. However, the SiO-C bands are weak in intensity when participating in hydrogen-bonding interactions enabling easy detection of the Si-O-Si bands due to chemisorbed species. By combining the low-frequency data with the spectral information for the hydroxyl region, a clearer picture of the nature of the bonding to the surface is obtained. When adsorbed at room temperature, all methoxy groups participate in hydrogen bonding with the surface hydroxyl groups. When the reaction is performed at 150 degrees C, the silanes are chemisorbed via a Si-O-Si bond and the remaining methoxy groups of the chemisorbed species are hydrogen bonded to the surface hydroxyl groups. At reaction temperature of 400 degrees C there is no evidence of hydrogen bonding but the spectra are complicated by the reaction of methanol with the surface. Copyright 2000 Academic Press. PMID- 10727355 TI - Self-Assembly of Hexagonal Rod Arrays Based on Capillary Forces. AB - A series of well-ordered, extended mesostructures has been generated from hexagonal polyurethane rods (15x3.2 mm) by self-assembly using capillary forces. The surface of one or more sides of the rods was rendered hydrophilic by exposure to an oxygen plasma. This modification determined the pattern of hydrophobic and hydrophilic faces; the hydrophobic sides were coated with a thin film of a hydrophobic lubricant. Agitation of the rods in an approximately isodense aqueous environment resulted in their self-assembly, in a process reflecting the action of capillary forces, into an array whose structure depends on the pattern of hydrophobic sides; capillarity also aligned the ends of the rods. We also carried out experiments in reaction chambers that restricted the motion of the rods; this restriction served to increase the size and regularity of the assemblies. Copyright 2000 Academic Press. PMID- 10727356 TI - Charge Measurement in the Modified Maxwell Displacement Current Method. AB - A modified Maxwell displacement current (MDC) measurement is described. The distance between water surface and a suspended electrode is changed sinusoidally at an angular frequency omega=omega(t), hence the MDC and the induced charge on the suspended electrode are modulated. The dc MDC current and the induced charge could be detected simultaneously and independently. This simplifies considerably charge measurements and will be helpful in the study of monolayers, e.g., the determination of the dipole moment. Copyright 2000 Academic Press. PMID- 10727357 TI - Patient-assessed health outcomes in peripheral arterial disease. PMID- 10727358 TI - Internal carotid artery stenosis: rate of progression. AB - OBJECTIVES: to assess the incidence and the rate of progression of internal carotid artery (ICA) stenosis and to determine the related risk factors. DESIGN: open prospective study. MATERIALS AND METHODS: between 1988-1997, 442 carotid arteries with various degrees of stenosis were followed using colour duplex ultrasonography every 6 months. Of these arteries, 290 (66%) were asymptomatic, 62 (14%) had caused transient ischaemic attack and 90 (20%) a stroke. In 145 cases (33%), there was concomitant coronary artery disease (CAD), in 134 (30%) diabetes mellitus, in 248 (56%) hypertension, in 139 (31%) hypercholesterolaemia and in 370 (84%) history of smoking. Of the plaques, 44 (10%) were uniformly echolucent, 19 (4%) haemorrhagic, 136 (31%) predominantly echolucent, 146 (33%) predominantly echogenic and 97 (22%) uniformly echogenic. RESULTS: significant progression of stenosis occurred in 82 cases (19%). The mean progression rate in these cases was 15% annually (range: 5-50%). There was no statistically significant correlation between the progression of the ICA stenosis and initial neurological status, age, gender, diabetes mellitus, hypertension, hypercholesterolaemia and smoking habit. Stenosis progression was correlated only with CAD and the ultrasonographic characteristics of the plaques. Patients with CAD as well as those with uniformly echolucent plaques presented a higher incidence and rate of stenosis progression (p<0.05). CONCLUSIONS: progression of internal carotid artery stenosis occurred in 19% of cases. The mean progression rate in these patients was 15% annually and was correlated with CAD and the ultrasonographic characteristics of the plaque. PMID- 10727359 TI - Computer-assisted carotid plaque analysis: characteristics of plaques associated with cerebrovascular symptoms and cerebral infarction. AB - OBJECTIVE: to correlate the echostructural characteristics of carotid plaques with neurological symptoms and cerebral infarcts. MATERIALS: one hundred and five plaques were studied in 74 patients by colour-flow duplex ultrasound: 39 were symptomatic and 37 were associated with brain infarction on CT scanning. METHODS: the images were digitalised for computer-assisted image standardisation and divided in homogenous (n=67) and heterogenous (n=38) groups. Parameters of the plaque image were analysed. RESULTS: global echogenicity was significantly lower in symptomatic and in CT(+)plaques (p<0.05). For homogenous plaques, an echogenic cap (EC) was visualised in 8.3% of symptomatic vs. 33.9% of asymptomatic (p<0. 05) and in 7.7% for plaques that were CT(+)vs. 37.7% for CT(-)(p<0. 05). Surface disruption was visualised in 50% of symptomatic vs. 8. 5% of asymptomatic (p=0.002) and in 46% of CT(+)vs. 9.4% of CT(-)plaques (p=0.002). For heterogenous plaques, the echolucent region was juxtaluminal in 67% of symptomatic and CT(+)plaques vs. 33% in asymptomatic and CT(-)(p<0.01). CONCLUSION: echolucent plaques are associated with a higher neurological risk. In homogenous plaques the absence of an echogenic cap and disruption of the plaque surface also correlates with symptoms. In heterogenous plaques, juxtaluminal location of the echolucent region is an additional marker of increased risk. PMID- 10727360 TI - Maintaining carotid flow by shunting during carotid endarterectomy diminishes the inflammatory response mediating ischaemic brain injury. AB - OBJECTIVES: to assess whether shunting during carotid reconstruction affects the release of inflammatory mediators from the ipsilateral hemisphere. MATERIALS AND METHODS: a catheter was placed in the ipsilateral jugular bulb during carotid endarterectomy (CEA) in 20 patients. Eight patients with ICBP (internal carotid backpressure) <40 mmHg received a shunt during CEA and 12 patients with ICBP >40 mmHg were operated upon without a shunt. Four patients with a carotid body tumour were used as controls. Blood was taken from the catheter as well as from the radial artery; before clamping, 5, 15, 30 min after clamping and 5 min after declamping. The oxygen extraction (AVO(2)) was calculated. Plasma concentrations of interleukin-1beta (IL-1beta), phospholipase A(2)(PLA(2)), thromboxane B(2)(TXB(2)), 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha) and prostaglandin E(2)(PGE(2)) were measured by enzyme-linked immunosorbent assay (ELISA) technique. RESULTS: all patients had a normal postoperative course except for one patient in the no-shunt group, who suffered a stroke 1 h later due to occlusion of the endarterectomy site. The AVO(2)extraction increased during clamping in patients operated upon without a shunt (p <0.05). This increase was partly recovered to pre-clamp levels 5 min after reperfusion. The extraction remained stable in the non-shunted patients and the control group. The increased extraction in the non-shunted group correlated with increased levels of IL-1beta during clamping ( p <0.05) and reperfusion ( p <0.01). PLA(2)also increased during reperfusion in the non-shunted group ( p <0.05). An increased ratio between TXB(2)and 6-keto-PGF1alpha was noted during clamping ( p <0.05) and further increased during reperfusion. The levels of PGE(2)remained stable in both CEA groups. The PLA(2)levels, as well as TXB(2), 6-keto-PGF1alpha and PGE(2)levels, remained unchanged during the procedure in the control group. CONCLUSIONS: there is a metabolic response to carotid cross-clamping when no shunt is used. However, the clinical significance of this is unclear, since there were no intraoperative strokes. PMID- 10727361 TI - Optimisation of the non-invasive assessment of critical limb ischaemia requiring invasive treatment. AB - OBJECTIVE: to assess the optimal cut-off values of toe blood pressure (TBP) and transcutaneous oxygen pressure (TcpO(2)) in the supine and sitting positions, in order to accurately detect the presence of severe leg ischaemia requiring invasive treatment. METHODS: in 49 consecutive patients (65 legs) with severe ischaemia according to clinical symptoms of Fontaine III or IV and a lowered ankle blood pressure, TBP and TcpO(2)were measured in the supine and sitting positions. Treatment within 6 weeks after the diagnosis was classified as either conservative or invasive (revascularisation or amputation). RESULTS: of the 65 legs, 38 (58%) required invasive treatment. The mean ankle pressure for this group was 70 mmHg. The optimal cut-off value for TBP was 38 mmHg and for TcpO(2)35 mmHg. A TBP of 0.1) decline in the donor ABPI postoperatively, but no symptoms (subclinical steal). The clinical stage of the donor limb did not deteriorate further during the follow-up period (median 8 months) in these seven patients. The decline in donor ABPI correlated with the FABP and the IVUS measurements, but not to duplex scanning or arteriography. FABP after papaverine injection and IVUS examination showed equal sensitivities and and specificities. CONCLUSION: a clinical steal phenomenon following femoro-femoral bypass surgery seems relatively uncommon, although a subclinical steal is more frequent. Both can be predicted by FABP or IVUS. Further follow-up is required to evaluate whether subclinical steal has any consequences in the long term. PMID- 10727369 TI - Ruptured abdominal aortic aneurysm: a novel method of outcome prediction using neural network technology. AB - BACKGROUND: reported survival following emergency surgery for ruptured abdominal aortic aneurysm (RAAA) varies widely between institutions. This is largely attributable to differences in case mix. The aim of this study was to identify and evaluate a set of prognostic variables that would accurately predict outcome for individual patients from perioperative indices. METHODS: perioperative factors associated with subsequent mortality at our institution were identified by retrospective review of 102 consecutive operations for RAAA over a 7-year period (January 1990 to January 1997). Logistic regression analysis was used to select the most significant variables associated with subsequent mortality. These were used to construct, train, and validate a neural network designed to predict survival from surgery in individual cases on a prospective basis. RESULTS: the 30 day mortality rate was 53%. Multivariate analysis identified four highly significant independent predictors of mortality; preoperative hypotension, intraperitoneal rupture, preoperative coagulopathy, and preoperative cardiac arrest. Using these inputs, the neural network correctly predicted outcome in 82.5% of individual cases. CONCLUSION: a neural network based on just four perioperative variables can accurately predict outcome of RAAA. Prognostic variables should be reported in studies as a measure of the effect of case mix on survival data. Neural networks have potential to aid decision-making relating to outcome for individual cases. PMID- 10727370 TI - Treatment of ruptured abdominal aortic aneurysm, a permanent challenge or a waste of resources? Prediction of outcome using a multi-organ-dysfunction score. AB - OBJECTIVES: in a retrospective study, attempts have been made to identify individual organ-dysfunction risk profiles influencing the outcome after surgery for ruptured abdominal aortic aneurysms. METHODS: out of 235 patients undergoing graft replacement for abdominal aortic aneurysms, 57 (53 men, four women, mean age 72 years [s.d. 8.8]) were treated for ruptured aneurysms in a 3-year period. Forty-eight preoperative, 13 intraoperative and 34 postoperative variables were evaluated statistically. A simple multi-organ dysfunction (MOD) score was adopted. RESULTS: the perioperative mortality was 32%. Three patients died intraoperatively, four within 48 h and 11 died later. A significant influence for pre-existing risk factors was identified only for cardiovascular diseases. Multiple linear-regression analysis indicated that a haemoglobin <90 g/l, systolic blood pressure <80 mmHg and ECG signs of ischaemia at admission were highly significant risk factors. The cause of death for patients, who died more than 48 h postoperatively, was mainly MOD. All patients with a MOD score >/=4 died (n=7). These patients required 27% of the intensive-care unit (ICU) days of all patients and 72% of the ICU days of the non-survivors. CONCLUSION: patients with ruptured aortic aneurysms from treatment should not be excluded. However, a physiological scoring system after 48 h appears justifiable in order to decide on the appropriateness of continual ICU support. PMID- 10727371 TI - Dilatation of the proximal neck of infrarenal aortic aneurysms after endovascular AAA repair. AB - OBJECTIVES: to assess size changes of the proximal aortic neck after endograft placement. METHODS: since 1994, 54 consecutive patients have undergone abdominal aortic aneurysm (AAA) repair with the Endovascular Technologies (EVT) endograft. The study group comprised the 33 patients who had completed at least six months of the prospective follow-up protocol. The pre-, postoperative and follow-up helical computed tomography (CT) angiograms (CTAs) were processed on a workstation. The proximal neck dimensions were measured perpendicular to the central lumen line of the aortic neck. The cross-sectional area was measured at the proximal attachment system and at 1 cm proximal to the renal arteries. RESULTS: while the dimensions of suprarenal aorta did not change, a significant dilatation of the proximal neck was found. The median increase was 10.3% at 6 months and 15.5% at 12 months. No correlation could be found between the amount of dilatation and pre- or postoperative neck-size, graft diameter and amount of graft-oversizing. CONCLUSION: the infrarenal aortic neck demonstrates continued dilatation during follow-up after endograft placement. PMID- 10727372 TI - Contained rupture of an infrarenal abdominal aortic aneurysm treated by endoluminal repair. PMID- 10727373 TI - Endovascular treatment of vascular emergencies and complications following previous vascular surgery. PMID- 10727374 TI - Preoperative B mode ultrasound mapping of upper limb veins prior to vascular access surgery. PMID- 10727375 TI - Ureteric injury as a complication of chemical sympathectomy. PMID- 10727376 TI - "Iodide mumps" after angioplasty. PMID- 10727377 TI - Evolution of Lyman Break Galaxies beyond z = 4. AB - The formation rate of luminous galaxies seems to be roughly constant from z approximately 2 to approximately 4 from the recent observations of Lyman break galaxies (LBGs). The abundance of luminous quasars, on the other hand, appears to drop off by a factor of more than 20 from z approximately 2 to z approximately 5. The difference in evolution between the two classes of objects in the overlapping, observed redshift range (z=2-4) can be explained naturally if we assume that quasar activity is triggered by mergers of luminous LBGs and one quasar lifetime is approximately 107-108 yr. If this merger scenario holds at higher redshift, for the evolutions of these two classes of objects to be consistent at z>4, the formation rate of luminous LBGs is expected to drop off at least as rapidly as exp-&parl0;z-4&parr0;6&solm0;5 at z>4. PMID- 10727378 TI - Metallicity Evolution in the Early Universe. AB - Observations of the damped Lyalpha systems provide direct measurements on the chemical enrichment history of neutral gas in the early universe. In this Letter, we present new measurements for four damped Lyalpha systems at high redshift. Combining these data with [Fe/H] values culled from the literature, we investigate the metallicity evolution of the universe from z approximately 1.5 to 4.5. Contrary to our expectations and the predictions of essentially every chemical evolution model, the N(H i)-weighted mean [Fe/H] metallicity exhibits minimal evolution over this epoch. For the individual systems, we report tentative evidence for an evolution in the unweighted [Fe/H] mean and the scatter in [Fe/H], with the higher redshift systems showing lower scatter and lower typical [Fe/H] values. We also note that no damped Lyalpha system has &sqbl0;Fe&solm0;H&sqbr0;<-2.7 dex. Finally, we discuss the potential impact of small number statistics and dust on our conclusions and consider the implications of these results on chemical evolution in the early universe. PMID- 10727379 TI - Detection and Mapping of Decoupled Stellar and Ionized Gas Structures in the Ultraluminous Infrared Galaxy IRAS 12112+0305. AB - Integral field optical spectroscopy with the INTEGRAL fiber-fed system and Hubble Space Telescope optical imaging are used to map the complex stellar and warm ionized gas structure in the ultraluminous infrared galaxy IRAS 12112+0305. Images reconstructed from wavelength-delimited extractions of the integral field spectra reveal that the observed ionized gas distribution is decoupled from the stellar main body of the galaxy, with the dominant continuum and emission-line regions separated by projected distances of up to 7.5 kpc. The two optical nuclei are detected as apparently faint emission-line regions, and their optical properties are consistent with being dust-enshrouded weak [O i] LINERs. The brightest emission-line region is associated with a faint (mI=20.4), giant H ii region of 600 pc diameter, in which a young ( approximately 5 Myr) massive cluster of about 2x107 M middle dot in circle dominates the ionization. Internal reddening toward the line-emitting regions and the optical nuclei ranges from 1 to 8 mag in the visual. Taking the reddening into account, the overall star formation in IRAS 12112+0305 is dominated by starbursts associated with the two nuclei and corresponds to a star formation rate of 80 M middle dot in circle yr 1. PMID- 10727381 TI - The Discovery of an Embedded Cluster of High-Mass Stars near SGR 1900+14. AB - Deep I-band imaging to I approximately 26.5 of the soft gamma-ray repeater SGR 1900+14 region has revealed a compact cluster of massive stars located only a few arcseconds from the fading radio source thought to be the location of the soft gamma-ray repeater (SGR). This cluster was previously hidden in the glare of the pair of M5 supergiant stars (whose light was removed by point-spread function subtraction) proposed by Vrba et al. as likely associated with SGR 1900+14. The cluster has at least 13 members within a cluster radius of approximately 0.6 pc based on an estimated distance of 12-15 kpc. It is remarkably similar to a cluster found associated with SGR 1806-20. That similar clusters have now been found at or near the positions of the two best studied SGRs suggests that young neutron stars, which are thought to be responsible for the SGR phenomenon, have their origins in proximate compact clusters of massive stars. PMID- 10727380 TI - Dust Emission from High-Redshift QSOs. AB - We present detections of emission at 250 GHz (1.2 mm) from two high-redshift QSOs from the Sloan Digital Sky Survey sample using the bolometer array at the IRAM 30 m telescope. The sources are SDSSp 015048.83+004126.2 at z=3.7 and SDSSp J033829.31+002156.3 at z=5.0; the latter is the third highest redshift QSO known and the highest redshift millimeter-emitting source yet identified. We also present deep radio continuum imaging of these two sources at 1.4 GHz using the Very Large Array. The combination of centimeter and millimeter observations indicate that the 250 GHz emission is most likely thermal dust emission, with implied dust masses approximately 108 M middle dot in circle. We consider possible dust heating mechanisms, including UV emission from the active galactic nucleus (AGN) and a massive starburst concurrent with the AGN, with implied star formation rates greater than 103 M middle dot in circle yr-1. PMID- 10727382 TI - Gamma-Ray Burst Host Galaxies Have "Normal" Luminosities. AB - The galactic environment of gamma-ray bursts can provide good evidence about the nature of the progenitor system, with two old arguments implying that the burst host galaxies are significantly subluminous. New data and new analysis have now reversed this picture: (1) Even though the first two known host galaxies are indeed greatly subluminous, the next eight hosts have absolute magnitudes typical for a population of field galaxies. A detailed analysis of the 16 known hosts (10 with redshifts) shows them to be consistent with a Schechter luminosity function with R*=-21.8+/-1.0, as expected for normal galaxies. (2) Bright bursts from the Interplanetary Network are typically 18 times brighter than the faint bursts with redshifts; however, the bright bursts do not have galaxies inside their error boxes to limits deeper than expected based on the luminosities for the two samples being identical. A new solution to this dilemma is that a broad burst luminosity function along with a burst number density varying as the star formation rate will require the average luminosity of the bright sample (>6x1058 photons s-1 or>1.7x1052 ergs s-1) to be much greater than the average luminosity of the faint sample ( approximately 1058 photons s-1 or approximately 3x1051 ergs s-1). This places the bright bursts at distances for which host galaxies with a normal luminosity will not violate the observed limits. In conclusion, all current evidence points to gamma-ray burst host galaxies being normal in luminosity. PMID- 10727384 TI - Chandra Observations of the Crab-like Supernova Remnant G21.5-0.9. AB - Chandra observations of the Crab-like supernova remnant G21.5-0.9 reveal a compact central core and spectral variations indicative of synchrotron burn-off of higher energy electrons in the inner nebula. The central core is slightly extended, perhaps indicating the presence of an inner wind-shock nebula surrounding the pulsar. No pulsations are observed from the central region, yielding an upper limit of approximately 40% for the pulsed fraction. A faint outer shell may be the first evidence of the expanding ejecta and blast wave formed in the initial explosion, indicating a composite nature for G21.5-0.9. PMID- 10727383 TI - The Galactic Worm GW 123.4-1.5: A Mushroom-shaped H i Cloud. AB - The Dominion Radio Astrophysical Observatory's Synthesis Telescope provides the highest resolution data (1&arcmin; and 0.82 km s-1) to date of an H i worm candidate. Observed as part of the Canadian Galactic Plane Survey, mushroom shaped GW 123.4-1.5 extends only a few hundred parsecs, contains approximately 105 M middle dot in circle of neutral hydrogen, and appears unrelated to a conventional shell or chimney structure. Our preliminary Zeus two-dimensional models use a single off-plane explosion with a modest ( approximately 1051 ergs) energy input. These generic simulations generate, interior to an expanding outer blast wave, a buoyant cloud whose structure resembles the morphology of the observed feature. Unlike typical model superbubbles, the stem can be narrow because its width is not governed by the pressure behind the blast wave or the disk scale height. Using this type of approach, it should be possible to more accurately model the thin stem and other details of GW 123.4-1.5 in the future. PMID- 10727385 TI - A Broad 22 Micron Emission Feature in the Carina Nebula H ii Region. AB - We report the detection of a broad 22 um emission feature in the Carina Nebula H ii region by the Infrared Space Observatory (ISO) short-wavelength spectrometer. The feature shape is similar to that of the 22 um emission feature of newly synthesized dust observed in the Cassiopeia A supernova remnant. This finding suggests that both of the features are arising from the same carrier and that supernovae are probably the dominant production sources of this new interstellar grain. A similar broad emission dust feature is also found in the spectra of two starburst galaxies from the ISO archival data. This new dust grain could be an abundant component of interstellar grains and can be used to trace the supernova rate or star formation rate in external galaxies. The existence of the broad 22 um emission feature complicates the dust model for starburst galaxies and must be taken into account correctly in the derivation of dust color temperature. Mg protosilicate has been suggested as the carrier of the 22 um emission dust feature observed in Cassiopeia A. The present results provide useful information in studies on the chemical composition and emission mechanism of the carrier. PMID- 10727386 TI - A Single Circumbinary Disk in the HD 98800 Quadruple System. AB - We present subarcsecond thermal infrared imaging of HD 98800, a young quadruple system composed of a pair of low-mass spectroscopic binaries separated by 0&farcs;8 (38 AU), each with a K-dwarf primary. Images at wavelengths ranging from 5 to 24.5 um show unequivocally that the optically fainter binary, HD 98800B, is the sole source of a comparatively large infrared excess on which a silicate emission feature is superposed. The excess is detected only at wavelengths of 7.9 um and longer, peaks at 25 um, and has a best-fit blackbody temperature of 150 K, indicating that most of the dust lies at distances greater than the orbital separation of the spectroscopic binary. We estimate the radial extent of the dust with a disk model that approximates radiation from the spectroscopic binary as a single source of equivalent luminosity. Given the data, the most likely values of disk properties in the ranges considered are Rin=5.0+/ 2.5 AU, DeltaR=13+/-8 AU, lambda0=2+4-1.5 um, gamma=0+/-2.5, and sigmatotal=16+/ 3 AU2, where Rin is the inner radius, DeltaR is the radial extent of the disk, lambda0 is the effective grain size, gamma is the radial power-law exponent of the optical depth tau, and sigmatotal is the total cross section of the grains. The range of implied disk masses is 0.001-0.1 times that of the Moon. These results show that, for a wide range of possible disk properties, a circumbinary disk is far more likely than a narrow ring. PMID- 10727387 TI - Ages of Late Spectral Type Vega-like Stars. AB - We have estimated the ages of eight late-type Vega-like stars by using standard age-dating methods for single late-type stars, e.g., location on the color magnitude diagram, Li lambda6708 absorption, Ca ii H and K emission, X-ray luminosity, and stellar kinematic population. With the exception of the very unusual pre-main-sequence star system HD 98800, all the late-type Vega-like stars are the same age as the Hyades cluster (600-800 Myr) or older. PMID- 10727388 TI - J-Band Infrared Spectroscopy of a Sample of Brown Dwarfs Using NIRSPEC on Keck II. AB - Near-infrared spectroscopic observations of a sample of very cool, low-mass objects are presented with higher spectral resolution than in any previous studies. Six of the objects are L dwarfs, ranging in spectral class from L2 to L8/9, and the seventh is a methane or T dwarf. These new observations were obtained during commissioning of the near-infrared spectrometer (NIRSPEC), the first high-resolution near-infrared cryogenic spectrograph for the Keck II 10 m telescope on Mauna Kea, Hawaii. Spectra with a resolving power of R approximately 2500 from 1.135 to 1.360 um (approximately J band) are presented for each source. At this resolution, a rich spectral structure is revealed, much of which is due to blending of unresolved molecular transitions. Strong lines due to neutral potassium (K i) and bands due to iron hydride (FeH) and steam (H2O) change significantly throughout the L sequence. Iron hydride disappears between L5 and L8, the steam bands deepen, and the K i lines gradually become weaker but wider because of pressure broadening. An unidentified feature occurs at 1.22 um that has a temperature dependence like FeH but has no counterpart in the available FeH opacity data. Because these objects are 3-6 mag brighter in the near-infrared compared with the I band, spectral classification is efficient. One of the objects studied (2MASSW J1523+3014) is the coolest L dwarf discovered so far by the 2 Micron All-Sky Survey (2MASS), but its spectrum is still significantly different from the methane-dominated objects such as Gl 229B or SDSS 1624+0029. PMID- 10727389 TI - 2 Micron Spectroscopy within 0&farcs;3 of Sagittarius A* AB - We present moderate- (R approximately 2700) and high-resolution (R approximately 22,400) 2.0-2.4 um spectroscopy of the central 0.1 arcsec2 of the Galaxy obtained with the facility near-infrared spectrometer (NIRSPEC) for the Keck II telescope. The composite spectra do not have any features attributable to the brightest stars in the central cluster; i.e., after background subtraction, W12CO&parl0;2 0&parr0;<2 A. This stringent limit leads us to conclude that the majority, if not all, of the stars are hotter than typical red giants. Coupled with previously reported photometry, we conclude that the sources are likely OB main-sequence stars. In addition, the continuum slope in the composite spectrum is bluer than that of a red giant and is similar to that of the nearby hot star IRS 16NW. It is unlikely that they are late-type giants stripped of their outer envelopes because such sources would be much fainter than those observed. Given their inferred youth (tauage<20 Myr), we suggest the possibility that the stars have formed within 0.1 pc of the supermassive black hole. We find a newly identified broad line component (VFWHM approximately 1000 km s-1) toward the 2.2178 um [Fe iii] line located within a few arcseconds of Sagittarius A*. A similar component is not seen in the Brgamma emission. PMID- 10727390 TI - Hot Stars and Cool Clouds: The Photodissociation Region M16. AB - We present high-resolution spectroscopy and images of a photodissociation region (PDR) in M16 obtained during commissioning of the near-infrared spectrometer (NIRSPEC) on the Keck II telescope. PDRs play a significant role in regulating star formation, and M16 offers the opportunity to examine the physical processes of a PDR in detail. We simultaneously observe both the molecular and ionized phases of the PDR and resolve the spatial and kinematic differences between them. The most prominent regions of the PDR are viewed edge-on. Fluorescent emission from nearby stars is the primary excitation source, although collisions also preferentially populate the lowest vibrational levels of H2. Variations in density-sensitive emission-line ratios demonstrate that the molecular cloud is clumpy, with an average density n=3x105 cm-3. We measure the kinetic temperature of the molecular region directly and find that TH2=930 K. The observed density, temperature, and UV flux imply a photoelectric heating efficiency of 4%. In the ionized region, ni=5x103 cm-3 and THii=9500 K. In the brightest regions of the PDR, the recombination line widths include a nonthermal component, which we attribute to viewing geometry. PMID- 10727391 TI - Infrared Spectroscopy of a Massive Obscured Star Cluster in the Antennae Galaxies (NGC 4038/9) with NIRSPEC. AB - We present infrared spectroscopy of the Antennae galaxies (NGC 4038/9) with the near-infrared spectrometer (NIRSPEC) at the W. M. Keck Observatory. We imaged the star clusters in the vicinity of the southern nucleus (NGC 4039) with 0&farcs;39 seeing in the K band using NIRSPEC's slit-viewing camera. The brightest star cluster revealed in the near-IR [MK&parl0;0&parr0; approximately -17.9] is insignificant optically but is coincident with the highest surface brightness peak in the mid-IR (12-18 um) Infrared Space Observatory image presented by Mirabel et al. We obtained high signal-to-noise ratio 2.03-2.45 um spectra of the nucleus and the obscured star cluster at R approximately 1900. The cluster is very young ( approximately 4 Myr), massive (M approximately 16x106 M middle dot in circle), and compact (with a density of approximately 115 M middle dot in circle pc-3 within a 32 pc half-light radius), assuming a Salpeter initial mass function (0.1-100 M middle dot in circle). Its hot stars have a radiation field characterized by Teff approximately 39,000 K, and they ionize a compact H ii region with ne approximately 104 cm-3. The stars are deeply embedded in gas and dust (AV approximately 9-10 mag), and their strong far-ultraviolet field powers a clumpy photodissociation region with densities nH greater, similar105 cm-3 on scales of approximately 200 pc, radiating LH21-0S&parl0;1&parr0;=9600 L middle dot in circle. PMID- 10727392 TI - Discovery of an Obscured Broad-Line Region in the High-Redshift Radio Galaxy MRC 2025-218. AB - This Letter presents infrared spectra taken with the newly commissioned near infrared spectrometer (NIRSPEC) on the Keck II telescope of the high-redshift radio galaxy MRC 2025-218 (z=2.63). These observations represent the deepest infrared spectra of a radio galaxy to date and have allowed for the detection of Hbeta, [O iii] lambdalambda4959, 5007, [O i] lambda6300, Halpha, [N ii] lambdalambda6548, 6583, and [S ii] lambdalambda6716, 6713. The Halpha emission is very broad (FWHM=9300 km s-1) and luminous (2.6x1044 ergs s-1), and it is very comparable to the line widths and strengths of radio-loud quasars at the same redshift. This strongly supports active galactic nucleus unification models linking radio galaxies and quasars, although we discuss some of the outstanding differences. The line [O iii] lambda5007 is extremely strong and has extended emission with large relative velocities toward the nucleus. We also derive that if the extended emission is due to star formation, each knot has a star formation rate comparable to a Lyman-break galaxy at the same redshift. PMID- 10727394 TI - Erratum. PMID- 10727393 TI - The Rest-Frame Optical Spectrum of MS 1512-cB58. AB - Moderate-resolution, near-IR spectroscopy of MS 1512-cB58 is presented, obtained during commissioning of the near-infrared spectrometer (NIRSPEC) on the Keck II telescope. The strong lensing of this z=2.72 galaxy by the foreground cluster MS 1512+36 makes it the best candidate for detailed study of the rest-frame optical properties of Lyman-break galaxies. In 80 minutes of on-source integration, we have detected Halpha, [N ii] lambdalambda6583, 6548, [O i] lambda6300, He i lambda5876, [O iii] lambdalambda5007, 4959, Hbeta, Hgamma, [O ii] lambda3727, and a strong continuum signal in the range of 1.29-2.46 um. A redshift of z=2.7290+/ 0.0007 is inferred from the emission lines, in contrast to the z=2.7233 calculated from UV observations of interstellar absorption lines. Using the Balmer line ratios, we find an extinction of E(B-V) = 0.27. Using the line strengths, we infer a star formation rate (SFR) of 620+/-18 M middle dot in circle yr-1 (H0=75, q0=0.1, and Lambda=0), which is a factor of 2 higher than that measured from narrowband imaging observations of the galaxy but is a factor of almost 4 lower than the SFR inferred from the UV continuum luminosity. The width of the Balmer lines yields a mass of Mvir=1.2x1010 M middle dot in circle. We find that the oxygen abundance is 13 solar, in good agreement with other estimates of the metallicity. However, we infer a high nitrogen abundance, which may argue for the presence of an older stellar population. PMID- 10727396 TI - Cloning, characterization and mapping of the human ATP5E gene, identification of pseudogene ATP5EP1, and definition of the ATP5E motif. AB - A cDNA encoding the epsilon subunit of human ATP synthase, ATP5E, was isolated from heart, skeletal muscle and spleen cDNA libraries respectively. Its genome structure was characterized as comprising three exons and two introns within a stretch of 5 kb, according to the genomic sequence AL109840. The gene was mapped to human chromosome 20q13.3 between marker D20S173 and 20qter using the radiation hybrid GB4 panel. Northern blot analysis showed that the ATP5E gene was expressed as a single 0.6 kb transcript in all 16 human tissues tested, with a high level present in heart and skeletal muscle. A new conserved motif composed of 24 residues, termed the ATP5E motif [W(R/K)X(5)YX(2)(Y/F)X(3)(C/A)X(4)RX(3)K], was defined on the basis of sequences of ATP synthase epsilon subunits from ten different organisms. In addition, a pseudogene ATP5EP1 was also identified on the basis of genomic sequence AC004066, localized on human chromosome 4q25. By analysing these results combined with the Southern blot patterns of human DNA hybridized with bovine ATP5E cDNA reported previously [Vinas, Powell, Runswick, Iacobazzi and Walker (1990) Biochem. J. 265, 321-326], we provide evidence of yet further homologous sequences (either gene or pseudogene) of ATP5E, in addition to ATP5E and ATP5EP1 in the human genome. PMID- 10727395 TI - Tetrahydrobiopterin biosynthesis, regeneration and functions. AB - Tetrahydrobiopterin (BH(4)) cofactor is essential for various processes, and is present in probably every cell or tissue of higher organisms. BH(4) is required for various enzyme activities, and for less defined functions at the cellular level. The pathway for the de novo biosynthesis of BH(4) from GTP involves GTP cyclohydrolase I, 6-pyruvoyl-tetrahydropterin synthase and sepiapterin reductase. Cofactor regeneration requires pterin-4a-carbinolamine dehydratase and dihydropteridine reductase. Based on gene cloning, recombinant expression, mutagenesis studies, structural analysis of crystals and NMR studies, reaction mechanisms for the biosynthetic and recycling enzymes were proposed. With regard to the regulation of cofactor biosynthesis, the major controlling point is GTP cyclohydrolase I, the expression of which may be under the control of cytokine induction. In the liver at least, activity is inhibited by BH(4), but stimulated by phenylalanine through the GTP cyclohydrolase I feedback regulatory protein. The enzymes that depend on BH(4) are the phenylalanine, tyrosine and tryptophan hydroxylases, the latter two being the rate-limiting enzymes for catecholamine and 5-hydroxytryptamine (serotonin) biosynthesis, all NO synthase isoforms and the glyceryl-ether mono-oxygenase. On a cellular level, BH(4) has been found to be a growth or proliferation factor for Crithidia fasciculata, haemopoietic cells and various mammalian cell lines. In the nervous system, BH(4) is a self protecting factor for NO, or a general neuroprotecting factor via the NO synthase pathway, and has neurotransmitter-releasing function. With regard to human disease, BH(4) deficiency due to autosomal recessive mutations in all enzymes (except sepiapterin reductase) have been described as a cause of hyperphenylalaninaemia. Furthermore, several neurological diseases, including Dopa-responsive dystonia, but also Alzheimer's disease, Parkinson's disease, autism and depression, have been suggested to be a consequence of restricted cofactor availability. PMID- 10727397 TI - Identification of N(omega)-carboxymethylarginine as a novel acid-labileadvanced glycation end product in collagen. AB - Collagen undergoes continuous non-enzymatic glycation during its long life period. The products resulting from the glycation reaction, so-called advanced glycation end products (AGEs), were regarded as potential pathogens of various diseases such as diabetic complications. Although several AGEs were identified from acid hydrolysates of glycated collagen, the major AGE(s) responsible for the diseases have not yet been fully characterized. Moreover, acid-labile constituents were decomposed during acid hydrolysis. To investigate these AGEs, we used the enzymatic hydrolysis method [Bensusan, Dixit and McKnight (1971) Biochim. Biophys. Acta 251, 100-108]. As a result, an acid-labile unknown compound was discovered from the digested glycated collagen. We identified this compound as N(omega)-carboxymethylarginine (CMA) by matrix-assisted laster desorption ionization-MS and NMR. CMA gradually increased in collagen during incubation with glucose and the yield reached about 8 mol/mol of collagen, which is 100 times higher than that of pentosidine. This result suggests that CMA is a major AGE in collagen. PMID- 10727398 TI - Phosphorylation of Oct-2 at sites located in the POU domain induces differential down-regulation of Oct-2 DNA-binding ability. AB - We compared the effects of phosphorylation of Oct-2 protein on its binding to the consensus octamer sequence (ATGCAAAT) and two non-canonical sequences present in human (AAGCAAAT) and murine (AAACAAAT) promoters of the BLR1 (Burkitts' lymphoma receptor 1) gene encoding chemokine receptor CXCR5 (CXC-chemokine receptor 5). The latter cis-acting elements represent low-affinity recognition sequences for the octamer transcription factors. Okadaic acid was found to induce hyperphosphorylation of Oct-2 specifically in cells of lymphoid lineage. Potentially phosphorylated amino acid residues localized to the POU-specific domain of Oct-2. Whereas binding of Oct-2 to the octamer site from the human BLR1 promoter or to the consensus octamer sequence was unaffected by phosphorylation of this factor, a strong reduction of Oct-2 binding to the octamer site from the murine BLR1 promoter was observed. This finding correlates well with the down regulation of expression of the BLR1 gene in murine splenic cells but not in lymphoid cells of human origin treated with okadaic acid. These data support the hypothesis that phosphorylation of Oct-2 may be a mechanism by which activities of the promoters containing non-canonical octamer sequences are differentially regulated in response to extracellular stimuli. PMID- 10727399 TI - Physical characterization of the MUC5AC mucin: a highly oligomeric glycoprotein whether isolated from cell culture or in vivo from respiratory mucous secretions. AB - We have isolated the high-M(r) mucins from growth medium of the early stage of an HT-29 cell culture by gel chromatography and isopycnic density gradient centrifugation. The mucins (buoyant density 1.34-1.44 g/ml) were reactive with an anti-peptide antiserum (MAN-5ACI) raised against a sequence from within the MUC5AC mucin. Similar antisera raised against the MUC2 and MUC5B mucins were not reactive. The MUC5AC reduced-mucin subunits exhibited a homogeneous charge distribution on anion-exchange chromatography, but appeared as two bands, one major and one more minor, after agarose gel electrophoresis. The unreduced mucins had an average M(r) in excess of 40 MDa and were visualized in the electron microscope as large, fine filamentous threads (many microns in length) that after reduction were greatly reduced in size (number average length 570 nm). Agarose gel electrophoresis of unreduced MUC5AC mucins identified a major band just entering the gel with evidence of a 'ladder' of faster-migrating minor bands. Partial reduction of the mucins increased the proportion of the faster bands and at least 16 could be discriminated. M(r) measurements showed that these bands differed by single monomer units. The mucins behaved as very stiff extended structures in solution and this characteristic might explain the poor separation of different-sized oligomers in sedimentation-rate experiments. The cell-culture mucin preparation had similar characteristics of charge and buoyant density to MUC5AC mucins from respiratory secretions in vivo. In addition the MUC5AC mucin from respiratory tract secretions exhibited similar behaviour, reduced and unreduced on agarose gel electrophoresis, indicating that the mucin has a similar molecular phenotype in vivo and in vitro. PMID- 10727400 TI - Tissue variation in the control of oxidative phosphorylation: implication for mitochondrial diseases. AB - Metabolic control analysis has often been used for quantitative studies of the regulation of mitochondrial oxidative phosphorylations (OXPHOS). The main contribution of this work has been to show that the control of mitochondrial metabolic fluxes can be shared among several steps of the oxidative phosphorylation process, and that this distribution can vary according to the steady state and the tissue. However, these studies do not show whether this observed variation in the OXPHOS control is due to the experimental conditions or to the nature of the mitochondria. To find out if there actually exists a tissue variation in the distribution of OXPHOS control coefficients, we determined the control coefficients of seven OXPHOS complexes on the oxygen-consumption flux in rat mitochondria isolated from five different tissues under identical experimental conditions. Thus in this work, only the nature of the mitochondria can be responsible for any variation detected in the control coefficient values between different tissues. The analysis of control coefficient distribution shows two tissue groups: (i) the muscle and the heart, controlled essentially at the level of the respiratory chain; and (ii) the liver, the kidney and the brain, controlled mainly at the phosphorylation level by ATP synthase and the phosphate carrier. We propose that this variation in control coefficient according to the tissue origin of the mitochondria can explain part of the tissue specificity observed in mitochondrial cytopathies. PMID- 10727401 TI - PTRF (polymerase I and transcript-release factor) is tissue-specific and interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc finger transcription factor which binds to the two mouse type-I collagen gene promoters. AB - We have used the yeast two-hybrid system to clone the protein that interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc-finger transcription factor that was cloned previously as the factor that binds to the two mouse proximal promoters of the type-I collagen genes. We utilized as bait the N-terminal domain of BFCOL1 that includes the zinc-finger DNA-binding domain. One cDNA contained a potential open reading frame for a polypeptide of 392 amino acids and was identical to PTRF (polymerase I and transcript-release factor), which is involved in transcription termination of the RNA polymerase I reaction. Northern-blot analysis revealed that the pattern of mRNA expression was similar to that of the type-I collagen gene. In addition, we detected the mRNA expression only in a fibroblast cell line and two bone cell lines, but not in other blood and neuronal cell lines. Recombinant protein was shown to enhance the binding of BFCOL1 to its binding site in the mouse proalpha2(I) collagen proximal promoter in vitro. The transient-transfection experiment showed that PTRF had a suppressive effect on the mouse proalpha2(I) collagen proximal promoter activity. We speculate that PTRF might play a role in the RNA polymerase II reaction as well as that of RNA polymerase I. PMID- 10727403 TI - Biosynthesis of heparin/heparan sulphate: mechanism of epimerization of glucuronyl C-5. AB - In the biosynthesis of heparin and heparan sulphate, D-glucuronic acid residues are converted into L-iduronic acid (IdoA) units by C-5 epimerization, at the polymer level. The reaction catalysed by the epimerase occurs by reversible abstraction and readdition of a proton at C-5 of target hexuronic acid residues, through a carbanion intermediate, with or without an inversion of configuration at C-5 [Prihar, Campbell, Feingold, Jacobsson, Jensen, Lindahl and Roden (1980) Biochemistry 19, 495-500]. Incubation of chemically N-sulphated capsular polysaccharide from Escherichia coli K5 ([4GlcAbeta1-4GlcNSO(3)alpha1-](n)), or of O-desulphated heparin (predominantly [4IdoAalpha1-4GlcNSO(3)alpha1-](n)) with purified C-5 epimerase from bovine liver, resulted in the interconversion of glucuronic acid and IdoA residues, which reached equilibrium (30-40% IdoA/total hexuronic acid) after approx. 1 h of incubation. Similar incubations performed in the presence of (3)H(2)O resulted in progressive labelling at C-5 of the target hexuronic acid units of either substrate polysaccharide. Contrary to chemical D gluco/L-ido equilibrium, established within 1 h of incubation, the accumulation of (3)H label continued for at least 6 h. This isotope effect suggests that the second stage of the reaction, i.e. the re-addition of a proton to the carbanion intermediate, is the rate-limiting step of the overall process. Analysis of the 5 (3)H-labelled polysaccharide products showed that the (3)H was approximately equally distributed between glucuronic acid and IdoA units, irrespective of incubation time (from 15 min to 72 h) and of the relative proportions of the two epimers in the substrate. This finding points to a catalytic mechanism in which the abstraction and re-addition of C-5 protons are effected by two polyprotic bases, presumably lysine residues. Previous experiments relating to the biosynthesis of dermatan sulphate were similarly interpreted in terms of a two base epimerization mechanism but differed from the present findings by implicating one monoprotic and one polyprotic base function [Hannesson, Hagner McWhirter, Tiedemann, Lindahl and Malmstrom (1996) Biochem. J. 313, 589-596]. PMID- 10727402 TI - 5-Hydroxytryptamine1A receptor/Gibetagamma stimulates mitogen-activated protein kinase via NAD(P)H oxidase and reactive oxygen species upstream of src in chinese hamster ovary fibroblasts. AB - The hypothesis of this work is that the 'serotonin' or 5-hydroxytryptamine (5 HT)(1A) receptor, which activates the extracellular signal-regulated kinase (ERK) through a G(i)betagamma-mediated pathway, does so through the intermediate actions of reactive oxygen species (ROS). Five criteria were shown to support a key role for ROS in the activation of ERK by the 5-HT(1A) receptor. (1) Antioxidants inhibit activation of ERK by 5-HT. (2) Application of cysteine reactive oxidant molecules activates ERK. (3) The 5-HT(1A) receptor alters cellular redox properties, and generates both superoxide and hydrogen peroxide. (4) A specific ROS-producing enzyme [NAD(P)H oxidase] is involved in the activation of ERK. (5) There is specificity both in the effects of various chemical oxidizers, and in the putative location of the ROS in the ERK activation pathway. We propose that NAD(P)H oxidase is located in the ERK activation pathway stimulated by the transfected 5-HT(1A) receptor in Chinese hamster ovary (CHO) cells downstream of G(i)betagamma subunits and upstream of or at the level of the non-receptor tyrosine kinase, Src. Moreover, these experiments provide confirmation that the transfected human 5-HT(1A) receptor induces the production of ROS (superoxide and hydrogen peroxide) in CHO cells, and support the possibility that an NAD(P)H oxidase-like enzyme might be involved in the 5-HT mediated generation of both superoxide and hydrogen peroxide. PMID- 10727404 TI - The effect of inositol 1,3,4,5-tetrakisphosphate on inositol trisphosphate induced Ca2+ mobilization in freshly isolated and cultured mouse lacrimal acinar cells. AB - Earlier reports have shown a remarkable synergism between InsP(4) and InsP(3) [either Ins(1,4,5)P(3) or Ins(2,4,5)P(3)] in activating Ca(2+)-dependent K(+) and Cl(-) currents in mouse lacrimal cells [Changya, Gallacher, Irvine, Potter and Petersen (1989) J. Membr. Biol. 109, 85-93; Smith (1992) Biochem. J. 283, 27-30]. However, Bird, Rossier, Hughes, Shears, Armstrong and Putney [(1991) Nature (London) 352, 162-165] reported that they could see no such synergism in the same cell type. A major experimental difference between the two laboratories lies in whether or not the cells were maintained in primary culture before use. Here we have compared directly the responses to inositol polyphosphates in freshly isolated cells versus cells cultured for 6-72 h. In the cultured cells, Ins(2,4,5)P(3) at 100 microM produced a robust stimulation of K(+) and Cl(-) currents, as much as an order of magnitude greater than that observed in the freshly isolated cells. However, the freshly isolated cells could be restored to a sensitivity similar to cultured cells by the addition of InsP(4) at a concentration two orders of magnitude lower than that of Ins(2,4,5)P(3). We discuss the implications of this with respect to the actions of InsP(4), including the possibility that disruption of the cellular structure during the isolation of the cells exposes an extreme manifestation of a possible physiological role for InsP(4) in controlling calcium-store integrity. PMID- 10727405 TI - The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain. AB - Tetranectin is a homotrimeric plasma and extracellular-matrix protein that binds plasminogen and complex sulphated polysaccharides including heparin. In terms of primary and tertiary structure, tetranectin is related to the collectin family of Ca(2+)-binding C-type lectins. Tetranectin is encoded in three exons. Exon 3 encodes the carbohydrate recognition domain, which binds to kringle 4 in plasminogen at low levels of Ca(2+). Exon 2 encodes an alpha-helix, which is necessary and sufficient to govern the trimerization of tetranectin by assembling into a triple-helical coiled-coil structural element. Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6-15) are shown to be of primary importance in heparin binding. PMID- 10727406 TI - Interleukin-6-induced STAT3 transactivation and Ser727 phosphorylation involves Vav, Rac-1 and the kinase SEK-1/MKK-4 as signal transduction components. AB - In the present study, signal transducer and activator of transcription 3 (STAT3) Ser(727) phosphorylation and transactivation was investigated in relation to activation of mitogen-activated protein (MAP) kinase family members including extracellular-signal-regulated protein kinase (ERK)-1, c-Jun N-terminal kinase (JNK)-1 and p38 ('reactivating kinase') in response to interleukin (IL)-6 stimulation. Although IL-6 can activate ERK-1 in HepG2 cells, STAT3 transactivation and Ser(727) phosphorylation were not reduced by using the MAP kinase/ERK kinase (MEK) inhibitor PD98059 or by overexpression of dominant negative Raf. IL-6 did not activate JNK-1 in HepG2 cells and STAT3 was a poor substrate for JNK-1 activated by anisomycin, excluding a role for JNK1 in IL-6 induced STAT3 activation. However, SEK-1/MKK-4 [where SEK-1 stands for stress activated protein kinase (SAPK)/ERK kinase 1, and MKK-4 stands for MAP kinase kinase 4] was activated in response to IL-6 and overexpression of dominant negative SEK-1/MKK-4(A-L) reduced both IL-6-induced STAT3 Ser(727) phosphorylation as well as STAT3 transactivation. Subsequently, the SEK-1/MKK-4 upstream components Vav, Rac-1 and MEKK were identified as components of a signal transduction cascade that leads to STAT3 transactivation in response to IL-6 stimulation. Furthermore, inhibition of p38 kinase activity with the inhibitor SB203580 did not block STAT3 Ser(727) phosphorylation but rather increased both basal as well as IL-6-induced STAT3 transactivation, indicating that p38 may act as a negative regulator of IL-6-induced STAT3 transactivation through a presently unknown mechanism. In conclusion, these data indicate that IL-6-induced STAT3 transactivation and Ser(727) phosphorylation is independent of ERK-1 or JNK-1 activity, but involves a gp130 receptor-signalling cascade that includes Vav, Rac 1, MEKK and SEK-1/MKK-4 as signal transduction components. PMID- 10727407 TI - Characterization of heterosubunit complexes formed by the R1 and R2 subunits of herpes simplex virus 1 and equine herpes virus 4 ribonucleotide reductase. AB - We report on the separate PCR cloning and subsequent expression and purification of the large (R1) and small (R2) subunits from equine herpes virus type 4 (EHV-4) ribonucleotide reductase. The EHV-4 R1 and R2 subunits reconstituted an active enzyme and their abilities to complement the R1 and R2 subunits from the closely related herpes simplex virus 1 (HSV-1) ribonucleotide reductase, with the use of subunit interaction and enzyme activity assays, were analysed. Both EHV-4 R1/HSV 1 R2 and HSV-1 R1/EHV-4 R2 were able to assemble heterosubunit complexes but, surprisingly, neither of these complexes was fully active in enzyme activity assays; the EHV-4 R1/HSV-1 R2 and HSV-1 R1/EHV-4 R2 enzymes had 50% and 5% of their respective wild-type activities. Site-directed mutagenesis was used to alter two non-conserved residues located within the highly conserved and functionally important C-termini of the EHV-4 and HSV-1 R1 proteins. Mutation of Pro-737 to Lys and Lys-1084 to Pro in EHV-4 and HSV-1 R1 respectively had no effects on subunit assembly. Mutation of Pro-737 to Lys in EHV-4 R1 decreased enzyme activity by 50%; replacement of Lys-1084 by Pro in HSV-1 R1 had no effect on enzyme activity. Both alterations failed to restore full enzyme activities to the heterosubunit enzymes. Therefore probably neither of these amino acids has a direct role in catalysis. However, mutation of the highly conserved Tyr-1111 to Phe in HSV-1 R1 inactivated enzyme activity without affecting subunit interaction. PMID- 10727408 TI - Human amiloride-sensitive epithelial Na+ channel gamma subunit promoter: functional analysis and identification of a polypurine-polypyrimidine tract with the potential for triplex DNA formation. AB - The mRNA for the epithelial Na(+) channel gamma subunit (gammaENaC) is regulated developmentally in the lung, colon and distal nephron and in response to Na(+) deprivation and systemic corticosteroids in the distal colon. Because such regulation is likely to be at the level of gene transcription, we examined the function of the promoter and other 5' flanking elements of the human gammaENaC gene. The proximal 5' flanking region contains two GC boxes but does not contain a TATA box. A 450 bp human gammaENaC fragment (-459 to +40) directed the expression of luciferase in H441 cells and primer extension analysis in transfected cells confirmed the correct initiation of human gammaENaC-luciferase chimaeric transcripts. By deletional analysis, GC boxes at -21 and -52 were found to be critical for this promoter activity. To begin to identify transcription factors that bind to the core promoter, a double-stranded oligonucleotide that corresponded to this region was synthesized and tested in a gel mobility-shift assay. Incubation of this radiolabelled oligonucleotide with nuclear extracts from H441 and FRTL5 cells resulted in the formation of four specific and distinct DNA-protein complexes. On the basis of antibody 'supershift' assays, one of these factors corresponds to Sp1, whereas the other three correspond to Sp3. Further upstream, an approx. 300 nt (-1143 to -839) polypurine-polypyrimidine tract (PPy tract) containing internal mirror repeats was identified. When contained in a supercoiled plasmid, the approx. 1200 nt 5' flanking region was sensitive to S1 endonuclease, which was consistent with the formation of an intramolecular triplex DNA ('H-DNA') structure with an unpaired single strand. High-resolution mapping with S1 endonuclease and sequencing of S1-generated clones confirmed that all S1-sensitive sites were within the PPy tract. Finally, a negative regulatory element was identified between -1525 and -1296 that functioned in lung, colon and collecting duct cell lines. PMID- 10727409 TI - Nucleotides of the tRNA D-stem that play an important role in nuclear-tRNA export in Saccharomyces cerevisiae. AB - Nuclear export of tRNA in Saccharomyces cerevisiae involves Los1p and Arc1p. Los1p facilitates tRNA translocation across the nuclear pore complex whereas Arc1p plays a role in delivering some species of tRNA exiting the nucleus to their cognate aminoacyl-tRNA synthetases. Here, we show that mutations of C11 and G24 of the D-stem of the yeast tyrosine amber-suppressor tRNA have different effects on nuclear export of the tRNA. Changing G24 had no effect on export of the tRNA to the cytoplasm. In contrast, mutating C11 resulted in nuclear retention of the tRNA. Nuclear retention of the tRNA mutants was not due to lack of processing, since only the mature forms of the tRNA mutants were found. The fact that mutations of G24 did not affect export of the tRNA also indicates that the effect of mutating C11 is not due to gross alteration of the tertiary structure resulting from disruption of the C11/G24 base pair. Expression of Los1p and the mammalian tRNA export receptor exportin-t rescued nuclear export of the tRNA with changes at position 11. The export-defective mutations of the tRNA mutants were suppressed by introducing the complementary nucleotides at position 24. Taken together, these findings suggest that C11 is important for binding of the tRNA to the export receptor, and that this binding is influenced by the conformation of the base. Finally, the export-defective tRNA mutants described can be used as reporters to identify eukaryotic proteins involved in the nuclear tRNA export process, and characterize the molecular interactions between known receptors and the tRNA substrate. PMID- 10727410 TI - Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P4, P3, P2' and P3' variations in extended substrates. AB - We have determined the kinetic parameters for the hydrolysis by papain, cathepsin B and cathepsin L of internally quenched fluorescent peptides derived from the lead peptides Abz-AAFRSAQ-EDDnp [in which Abz and EDDnp stand for o-aminobenzoic acid and N-(2,4-dinitrophenyl)ethylenediamine respectively], to map the specificity of S(4) and S(3) subsites, and Abz-AFRSAAQ-EDDnp, to identify the specificity of S(2)' and S(3)'. Abz and EDDnp were the fluorescent quencher pair. These two series of peptides were cleaved at the Arg-Ser bond and systematic modifications at P(4), P(3), P(2)' and P(3)' were made. The S(4) to S(2)' subsites had a significant influence on the hydrolytic efficiencies of the three enzymes. Only papain activity was observed to be dependent on S(3)', indicating that its binding site is larger than those of cathepsins B and L. Hydrophobic amino acids were accepted at S(4), S(3), S(2)' and S(3)' of the three enzymes. The best substrates for cathepsins L and B had Trp and Asn at P(2)' respectively; variations at this position were less accepted by these enzymes. The best substrates for papain were peptides containing Trp, Tyr or Asn at P(3)'. Basic residues at P(3) and P(4) were well accepted by cathepsin L and papain. We also explored the susceptibility of substrates Abz-AFRSXAQ-EDDnp, modified at P(2)' (X), to human cathepsin B mutants from which one or two occluding loop contacts had been removed. The modifications at His(111) (H111A) and His(110) (H110A) of cathepsin B led to an increase in k(cat) values of one or two orders of magnitude. The hydrolytic efficiencies of these cathepsin B mutants became closer to those of papain or cathepsin L. PMID- 10727411 TI - Intracellular maturation and localization of the tumour necrosis factor alpha convertase (TACE). AB - Tumour necrosis factor alpha convertase (TACE) is a metalloprotease/disintegrin involved in the ectodomain shedding of several proteins, a process thought to be important in inflammation, rheumatoid arthritis and murine development. The characterization of the intracellular maturation and subcellular localization of endogenous TACE is decribed in the present study. Similarly to other proteolytically active metalloprotease/disintegrins, two forms of TACE are found in cells; a full-length precursor and a mature form lacking the prodomain. Prodomain removal occurs in a late Golgi compartment, consistent with the proposed role of a furin type proprotein convertase in this process. An additional form of TACE, lacking the pro and cytoplasmic domains, is detected when cell lysates are prepared in the presence of EDTA instead of a hydroxamate based metalloprotease inhibitor or 1,10-phenanthroline. This form appears to be generated by mature TACE cleaving its own cytoplasmic tail and may explain why little mature TACE has been detected in previous studies. In cell-surface labelling experiments, mature TACE was detected on the cell surface but immunofluorescence data indicate that TACE is predominantly localized to a perinuclear compartment similar to that described for tumour necrosis factor (TNF)alpha. This raises the possibility that TACE-mediated ectodomain shedding may occur in an intracellular compartment in addition to the cell surface. PMID- 10727412 TI - Involvement of the theta-type protein kinase C in translocation of myristoylated alanine-rich C kinase substrate (MARCKS) during myogenesis of chick embryonic myoblasts. AB - The phosphorylation pattern of numerous proteins in the soluble extracts of chick embryonic muscle cells changes dramatically during myogenesis. One of these proteins, the 63 kDa protein, whose phosphorylation state declines during the differentiation process, was identified as the myristoylated alanine-rich C kinase substrate (MARCKS), a major, specific substrate of protein kinase C (PKC). This decrease in the phosphorylation state of MARCKS was due to a decrease in the level of protein in the cytosol with a simultaneous increase in its level in the membrane fraction. Immunostaining of the cultured myoblasts also revealed that MARCKS translocated from the cytosol to the plasma membrane and to the peripheral region of nuclei as the mononucleated myoblasts fused to form multinucleated myotubes. Immunoprecipitation with an anti-PKC-theta antibody, but not with the antibodies against the other PKC isoforms, such as conventional PKC-alpha, novel PKC-delta, and novel PKC-epsilon, inhibited phosphorylation of MARCKS. Moreover, expression of PKC-theta was found to be down-regulated during the course of myogenic differentiation. In addition, treatment of the cells with PMA, which activates PKC-theta and hence increases the phosphorylation state of MARCKS, reversibly inhibited both MARCKS translocation and myoblast fusion. These results suggest that MARCKS is preferentially phosphorylated by PKC-theta in cultured myoblasts and that the down-regulation of PKC-theta; is partly responsible for MARCKS translocation during myogenesis. These results also suggest that PKC-theta controlled MARCKS translocation is associated with, or a requisite event for, myoblast fusion. PMID- 10727413 TI - Identification and characterization of cis-acting elements conferring insulin responsiveness on hamster cholesterol 7alpha-hydroxylase gene promoter. AB - Bile acid biosynthesis occurs primarily through a pathway initiated by the 7alpha hydroxylation of cholesterol, catalysed by cholesterol 7alpha-hydroxylase (encoded by CYP7A1). Insulin down-regulates CYP7A1 transcription. The aim of our study was to characterize the sequences of hamster CYP7A1 promoter, mediating the response to insulin. We therefore performed transient transfection assays with CYP7A1 promoter/luciferase chimaeras mutated at putative response elements and studied protein-DNA interactions by means of gel electrophoresis mobility-shift assay. Here we show that two sequences confer insulin responsiveness on hamster CYP7A1 promoter: a canonical insulin response sequence TGTTTTG overlapping a binding site for hepatocyte nuclear factor 3 (HNF-3) (at nt -235 to -224) and a binding site for HNF-4 at nt -203 to -191. In particular we show that the hamster CYP7A1 insulin response sequence is part of a complex unit involved in specific interactions with multiple transcription factors such as members of the HNF-3 family; this region does not bind very strongly to HNF-3 and as a consequence partly contributes to the transactivation of the gene. Another sequence located at nt -138 to -128 binds to HNF-3 and is involved in the tissue-specific regulation of hamster CYP7A1. The sequence at nt -203 to -191 is not only essential for insulin effect but also has a major role in the liver-specific expression of CYP7A1; it is the target of HNF-4. Therefore the binding sites for liver-enriched factors, present in the hamster CYP7A1 proximal promoter in close vicinity and conserved between species, constitute a regulatory unit important for basal hepatic expression and tissue restriction of the action of hormones such as insulin. PMID- 10727414 TI - Regulation of the gene promoter for extracellular signal-regulated protein kinase 2 by transcription factors NF-Y and Sp3. AB - We have previously shown that the maximal promoter activity of the gene for extracellular signal-regulated protein kinase 2 (ERK2; also known as p42 mitogen activated protein kinase) resides in the 371 bp 5'-flanking sequence. In the present study we defined roles for a CCAAT box and two adjacent GC boxes in the activity of this promoter. Deletion analysis and DNase I footprinting of this 371 bp region indicated that the CCAAT box at -64 and GC boxes at -86 and -39 are crucial for promoter activity. Electrophoretic mobility-shift assays showed that transcription factor NF-Y/CBF binds to the CCAAT box. Sp1 and Sp3, members of the Sp family of transcription factors, bind to the GC boxes of the ERK2 promoter. The binding of Sp3 was predominant over that of Sp1. Disruption by mutation of any of the CCAAT box and GC boxes similarly decreased promoter activity. These three cis elements exhibited a moderate synergy in promoter function. The transactivating role of NF-Y was corroborated by the finding that a dominant negative form of NF-YA diminished the promoter activity. These results provide clues for refining our understanding of not only the regulation of expression of the gene for ERK2 but also mechanisms by which NF-Y and Sp1/Sp3 regulate transcription. PMID- 10727415 TI - Function of the farnesyl moiety in visual signalling. AB - The aim of this work was to search for the biological function of protein isoprenylation. For this purpose, peptides were synthesized and, by using a convenient protocol, were farnesylated or geranylated at the thiol group of the C terminal cysteine. The interaction of these peptides with photoactivated rhodopsin (Rho*, which is functionally equivalent to metarhodopsin II) was studied with the use of sheep rod outer segments. The sheep rod outer segments, although chosen because of the unavailability of bovine material in the U.K., had favourable optical properties for the direct determination of spectral changes in membrane suspensions. At 20 degrees C and pH 8.0, the t((1/2)) of the conversion of metarhodopsin II (Meta II) (lambda(max) 389 nm) into Meta III (lambda(max) 463 nm) was 3.2 min (less than 1.5 min at 37 degrees C). The t((1/2)) was unaltered in the presence of non-farnesyl peptides but increased by approx. 20% with farnesyl-N-acetylcysteine, by approx. 60% with farnesyl peptide containing residues 544-558 of rhodopsin kinase and by approx. 140% with farnesyl peptide corresponding to residues 60-71 of the gamma-subunit of visual transducin. The effect of various peptides on the activities of bovine and sheep rhodopsin kinase was also studied. In this assay the non-farnesyl peptides and common detergents were found to be inactive; however, all the farnesyl peptides inhibited the activity to various extents. Cumulatively, the results show that, whereas the farnesyl peptides as well as a number of membrane-disrupting detergents affected the conversion from Meta II into Meta III, the inhibition of the activity of rhodopsin kinase was achieved only by the farnesyl peptides. The results are interpreted as showing that Meta II possesses a binding site for the recognition of the farnesyl group that can be used either by the farnesyl moiety of rhodopsin kinase or transducin to make the initial encounter, which can then develop into multivalent interactions characterized by the structure, and the desired function, of each protein. PMID- 10727416 TI - Endogenous oxygen radicals modulate protein tyrosine phosphorylation and JNK-1 activation in lectin-stimulated thymocytes. AB - Molecular events mediating the T-lymphocyte response to lectins are still incompletely understood, although much evidence suggests that both the mitogenic and the death-promoting effects of these agents involve the biochemical cascade initiated by the CD3/T-cell antigen receptor (TCR) complex. Reactive oxygen species (ROS) and in particular H(2)O(2) have been shown to have a role in cell response to cytokines and growth factors. Here we report that the proliferation of mouse thymocytes in response to the mitogenic lectin concanavalin A (ConA) is strongly and selectively inhibited by the intracellular ROS scavenger N acetylcysteine (NAC) and by diphenyleneiodonium (DPI), a potent inhibitor of NADPH-dependent membrane oxidases activated by surface receptors. A rapid 'burst' of intracellular oxygen radicals was observed in mouse thymocytes stimulated by ConA, with kinetics that paralleled the appearance of tyrosine-phosphorylated proteins. This burst was abrogated by the pretreatment of cells with NAC or DPI. Only a modest increase in intracellular oxygen species was found in thymocytes stimulated by strong cross-linking of TCR together with CD4 or CD28. Pharmacological interference with ROS production in ConA-stimulated thymocytes resulted in a decreased tyrosine phosphorylation of multiple protein species, including a 38 kDa band able to recruit the adapter protein Grb2 and corresponding to the recently identified transducer LAT (linker for activation of T-cells), a molecule involved in linking activated TCR to the production of interleukin 2 and the proliferation of T-cells. Furthermore, ROS inhibition markedly attenuated the activation of stress-activated protein kinase/JNK-1 (c Jun N-terminal kinase 1) in response to lectins. Taken together, these results identify ROS as important modulators of the signalling cascade initiated by mitogenic lectins in thymocytes and, by extension, as a novel class of mediators downstream of antigen receptors. PMID- 10727417 TI - Farnesylcysteine analogues inhibit store-regulated Ca2+ entry in human platelets: evidence for involvement of small GTP-binding proteins and actin cytoskeleton. AB - We have investigated the mechanism of Ca(2+) entry into fura-2-loaded human platelets by preventing the prenylation of proteins such as small GTP-binding proteins. The farnesylcysteine analogues farnesylthioacetic acid (FTA) and N acetyl-S-geranylgeranyl-L-cysteine (AGGC), which are inhibitors of the methylation of prenylated and geranylgeranylated proteins respectively, significantly decreased thrombin-evoked increases in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in the presence, but not in the absence, of external Ca(2+), suggesting a relatively selective inhibition of Ca(2+) entry over internal release. Both these compounds and N-acetyl-S-farnesyl-L-cysteine, which had similar effects to those of FTA, also decreased Ca(2+) entry evoked by the depletion of intracellular Ca(2+) stores with thapsigargin. The inactive control N-acetyl-S-geranyl-L-cysteine was without effect. Patulin, an inhibitor of prenylation that is inert with respect to methyltransferases, also decreased store-regulated Ca(2+) entry. Cytochalasin D, an inhibitor of actin polymerization, significantly decreased store-regulated Ca(2+) entry in a time dependent manner. Both cytochalasin D and the farnesylcysteine analogues FTA and AGGC inhibited actin polymerization; however, when evoking the same extent of decrease in actin filament formation, FTA and AGGC showed greater inhibitory effects on Ca(2+) entry, indicating a cytoskeleton-independent component in the regulation of Ca(2+) entry by small GTP-binding-protein. These findings suggest that prenylated proteins such as small GTP-binding proteins are involved in store regulated Ca(2+) entry through actin cytoskeleton-dependent and cytoskeleton independent mechanisms in human platelets. PMID- 10727418 TI - Electrostatic interactions affecting the active site of class sigma glutathione S transferase. AB - We have shown previously that the solvent-induced equilibrium unfolding mechanism of class Sigma glutathione S-transferase (GST) is strongly affected by ionic strength [Stevens, Hornby, Armstrong and Dirr (1998) Biochemistry 37, 15534 15541]. The protein is dimeric and has a hydrophilic subunit interface. Here we show that ionic strength alone has significant effects on the conformation of the protein, in particular at the active site. With the use of NaCl at up to 2 M under equilibrium conditions, the protein lost 60% of its catalytic activity and the single tryptophan residue per subunit became partly exposed. The effect was independent of protein concentration, eliminating the dissociation of the dimer as a possibility for the conformational changes. This was confirmed by size exclusion HPLC. There was no significant change in the secondary structure of the protein according to far-UV CD data. Manual-mixing and stopped-flow kinetics experiments showed a slow single-exponential salt-induced change in protein fluorescence. For equilibrium and kinetics experiments, the addition of an active site ligand (S-hexylglutathione) completely protected the protein from the ionic strength-induced conformational changes. This suggests that the change occurs at or near the active site. Possible structural reasons for these novel effects are proposed, such as the flexibility of the alpha-helix 2 region as well as the hydrophilic subunit interface, highlighting the importance of electrostatic interactions in maintaining the structure of the active site of this GST. PMID- 10727419 TI - C-terminal half of tetanus toxin fragment C is sufficient for neuronal binding and interaction with a putative protein receptor. AB - Tetanus neurotoxin (TeNT) is a powerful bacterial protein toxin that cleaves VAMP/synaptobrevin, an essential protein of the synaptic vesicle fusion machinery, and consequently blocks neurotransmission. The extreme neurospecificity of TeNT is determined by the binding of its C-terminal domain (fragment C or H(C)) to neuronal receptors. Whereas polysialogangliosides are known acceptors for the toxin, the existence of additional protein receptors has also been suggested. We have reported previously on a 15 kDa cell-surface glycoprotein that interacts with TeNT in neuronal cell lines and motoneurons [Herreros, Lalli, Montecucco and Schiavo (2000) J. Neurochem., in the press]. Here, on the basis of the structural information provided by the crystallization of fragment C of TeNT, we have expressed its C-and N-terminal halves as recombinant proteins and analysed their binding abilities to rat phaeochromocytoma (PC12) cells differentiated with nerve growth factor. We found that the C-terminal subdomain of the fragment C of TeNT is necessary and sufficient for cell binding and for the interaction with the 15 kDa putative receptor. In contrast, the N-terminal half showed a very poor interaction with the cell surface. These results restrict the binding domain of TeNT to the C terminal half of the fragment C and highlight the importance of this domain for the neurospecific interaction of the toxin with the synapse. Furthermore, these findings support the use of this portion of TeNT as a neurospecific targeting device, pointing to an involvement of the N-terminal subdomain in later steps of the intoxication pathway. PMID- 10727420 TI - Novel inhibitors of the condensing enzymes of the type II fatty acid synthase of pea (Pisum sativum). AB - The type II fatty acid synthases (FASs) of higher plants (and Escherichia coli) contain three condensing enzymes called beta-ketoacyl-ACP synthases (KAS), where ACP is acyl-carrier-protein. We have used novel derivatives of the antibiotic thiolactomycin to inhibit these enzymes. Overall de novo fatty acid biosynthesis was measured using [1-(14)C]acetate substrate and chloroplast preparations from pea leaves, and [1-(14)C]laurate was used to distinguish between the effects of the inhibitors on KAS I from those on KAS II. In addition, the activities of these enzymes, together with the short-chain condensing enzyme, KAS III, were measured directly. Six analogues were tested and two, both with extended hydrocarbon side chains, were found to be more effective inhibitors than thiolactomycin. Incubations with chloroplasts and direct assay of the individual condensing enzymes showed that all three compounds inhibited the pea FAS condensing enzymes in the order KAS II > KAS I > KAS III. These results demonstrate the general activity of thiolactomycin and its derivatives against these FAS condensation reactions, and suggest that such compounds will be useful for further detailed studies of inhibition and for use as pharmaceuticals against Type II FASs of pathogens. PMID- 10727421 TI - Ca2+-bound calmodulin forms a compact globular structure on binding four trifluoperazine molecules in solution. AB - Small-angle X-ray scattering (SAXS), which determines the radius of gyration, R(g), and the pair distance distribution function, was used to investigate the conformational changes of calmodulin (CaM) on binding to an antagonist, trifluoperazine (TFP), with or without Ca(2+) in solution. We previously applied this SAXS method to CaM complexed with N-(6-aminohexyl)-5-chloro-1 naphthalenesulphonamide (W-7) [Osawa, Kuwamoto, Izumi, Yap, Ikura, Shibanuma, Yokokura, Hidaka and Matsushima (1999) FEBS Lett. 442, 173-177] and found that the binding of two W-7 TFP molecules to one Ca(2+)-saturated CaM molecule induces structural changes from a 'dumb-bell' shape to a compact globular shape. We report here that the most compact globular shape whose size is consistent with that of the 1:2 Ca(2+)-saturated CaM-W-7 complex is formed by the binding of four TFP molecules to one Ca(2+)-saturated CaM molecule. Even in the absence of Ca(2+), the conformational changes of CaM occur on TFP binding, giving a slightly smaller R(g) than Ca(2+)-free CaM alone. PMID- 10727422 TI - Ras effector pathway activation by epidermal growth factor is inhibited in vivo by exoenzyme S ADP-ribosylation of Ras. AB - We have examined the functional consequences of ADP-ribosyltransferase modification of Ras by the exoenzyme S (ExoS) protein of Pseudomonas aeruginosa. ExoS has been shown previously to ADP-ribosylate a number of proteins, including members of the Ras superfamily, which play an essential role in the processes of cell proliferation, differentiation, motility and cell division. HeLa and NIH3T3 cells were infected with ExoS protein, which was delivered via the type III secretion system of the heterologous host Yersinia pseudotuberculosis. Infection of mammalian cells with ExoS results in a change in the ratio of GTP/GDP bound directly to Ras in vivo. This ADP-ribosylation of Ras in vivo is mediated by the C-terminal domain of ExoS. Further, ExoS ADP-ribosylation of Ras in vivo inhibits activation of Ras and the ability to interact with the Ras binding domain of Raf upon stimulation with epidermal growth factor (EGF). In the present study, we show that ExoS activity does not interfere with EGF receptor phosphorylation itself, nor with the formation of a Grb2-activated Shc complex upon EGF stimulation, consistent with ExoS blockage of this mitogenic signalling pathway at the level of Ras. This is further supported by our observation of a substantial inhibition of extracellular signal-regulated kinase and protein kinase B/Akt kinase activation in response to EGF upon ExoS infection. In conclusion, in the present study, the consequences of ExoS infection on Ras effector pathway in vivo have been defined. PMID- 10727423 TI - Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras-related GTPases. AB - Here we report the molecular cloning and biochemical characterization of Rem2 (for Rem, Rad and Gem-related 2), a novel GTP-binding protein identified on the basis of its homology with the Rem, Rad, Gem and Kir (RGK) family of Ras-related small GTP-binding proteins. Rem2 mRNA was detected in rat brain and kidney, making it the first member of the RGK family to be expressed at relatively high levels in neuronal tissues. Recombinant Rem2 binds GTP saturably and exhibits a low intrinsic rate of GTP hydrolysis. Surprisingly, the guanine nucleotide dissociation constants for both Rem2 and Rem are significantly different than the majority of the Ras-related GTPases, displaying higher dissociation rates for GTP than GDP. Localization studies with green fluorescent protein (GFP)-tagged recombinant protein fusions indicate that Rem2 has a punctate, plasma membrane localization. Deletion of the C-terminal seven amino acid residues that are conserved in all RGK family members did not affect the cellular distribution of the GFP fusion protein, whereas a larger deletion, including much of the polybasic region of the Rem2 C-terminus, resulted in its redistribution to the cytosol. Thus Rem2 is a GTPase of the RGK family with distinctive biochemical properties and possessing a novel cellular localization signal, consistent with its having a unique role in cell physiology. PMID- 10727424 TI - Cytokine-inducible enhancer with promoter activity in both the rat and human manganese-superoxide dismutase genes. AB - Diverse pro-inflammatory mediators regulate transcription of the gene (MnSOD) encoding the mitochondrial anti-oxidant protein manganese-superoxide dismutase. Understanding the regulation of this gene is crucial to comprehending its role in cytoprotection. In transfected lung epithelial cells, a human-growth-hormone reporter gene system was utilized to identify a potential enhancer in the MnSOD genomic fragment previously shown to contain multiple DNase-I-hypersensitive sites. Northern analysis demonstrated a 10-20-fold increase in response to pro inflammatory mediators. Inclusion of the MnSOD genomic fragment in reporter constructs was necessary to mimic these stimulus-dependent endogenous levels. The inducible enhancer element was localized to a 260 bp fragment in intron 2, coinciding with a previously defined DNase-I-hypersensitive site. This element functions in an orientation- and position-independent manner as well as with the heterologous thymidine kinase promoter. In addition, we have demonstrated that a homologous sequence within the human MnSOD gene exhibits identical enhancer activity. A novel characteristic of the rat and human enhancer elements involves the ability to promote cytokine-inducible transcription in the absence of a classical promoter. PMID- 10727425 TI - Acidocalcisomes and a vacuolar H+-pyrophosphatase in malaria parasites. AB - Plasmodium berghei trophozoites were loaded with the fluorescent calcium indicator, fura-2 acetoxymethyl ester, to measure their intracellular Ca(2+) concentration ([Ca(2+)](i)). [Ca(2+)](i) was increased in the presence of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase inhibitor, thapsigargin. Trophozoites also possess a significant amount of Ca(2+) stored in an acidic compartment. This was indicated by: (1) the increase in [Ca(2+)](i) induced by bafilomycin A(1), nigericin, monensin, or the weak base, NH(4)Cl, in the nominal absence of extracellular Ca(2+), and (2) the effect of ionomycin, which cannot take Ca(2+) out of acidic organelles and was more effective after alkalinization of this compartment by addition of bafilomycin A(1), nigericin, monensin, or NH(4)Cl. Inorganic PP(i) promoted the acidification of a subcellular compartment in cell homogenates of trophozoites. The proton gradient driven by PP(i) collapsed by addition of the K(+)/H(+) exchanger, nigericin, and eliminated by the PP(i) analogue, aminomethylenediphosphonate (AMDP). Both PP(i) hydrolysis and proton transport were dependent upon K(+), and Na(+) caused partial inhibition of these activities. PP(i) hydrolysis was sensitive in a dose-dependent manner to AMDP, imidodiphosphate, sodium fluoride, dicyclohexylcarbodi-imide and to the thiol reagent, N-ethylmaleimide. Immunofluorescence microscopy using antibodies raised against conserved peptide sequences of a plant vacuolar pyrophosphatase (V H(+)-PPase) suggested that the proton pyrophosphatase is located in intracellular vacuoles and the plasma membrane of trophozoites. AMDP caused an increase in [Ca(2+)](i) in the nominal absence of extracellular Ca(2+). Ionomycin was more effective in releasing Ca(2+) from this acidic intracellular compartment after treatment of the cells with AMDP. Taken together, these results suggest the presence in malaria parasites of acidocalcisomes with similar characteristics to those described in trypanosomatids and Toxoplasma gondii, and the colocalization of the V-H(+)-PPase and V-H(+)-ATPase in these organelles. PMID- 10727426 TI - PfPK6, a novel cyclin-dependent kinase/mitogen-activated protein kinase-related protein kinase from Plasmodium falciparum. AB - We have isolated a novel protein kinase cDNA, PfPK6, by differential display RT PCR (DDRT-PCR) of mRNA obtained from different asexual erythrocytic stages of Plasmodium falciparum, which shows sequence similarity to both cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK) family members. The 915 bp open reading frame (ORF) is interrupted by seven introns and encodes a 305 residue polypeptide with a predicted molecular mass of 35848 Da. Several cDNA clones with some of the intron sequences were isolated, indicating alternate or defective splicing of PfPK6 transcripts because the gene seems to be a single copy located on chromosome 13. The similarity of the catalytic domain of PfPK6 to those of CDK2 and MAPK is 57.3% and 49.6%, respectively. The signature PSTAIRE (single-letter amino acid codes) CDK motif is changed to SKCILRE in PfPK6. The TXY residues that are phosphorylated in MAPKs for their activation are T(173)PT in PfPK6. Three size classes of PfPK6 transcripts of 6.5, 2.0 and 1.1 kb are up regulated during the transition of P. falciparum from ring to trophozoite. Western blot analysis suggested the expression of a 35 kDa polypeptide in trophozoites and schizonts. Immunofluorescence studies indicated both nuclear and cytoplasmic localization of PfPK6 in trophozoite, schizont and segmenter stages. In vitro, recombinant PfPK6 phosphorylated itself and also exogenous substrates, histone and the small subunit of the malarial ribonucleotide reductase (R2). The kinase activity of PfPK6 is sensitive to CDK inhibitors such as olomoucine and roscovitine. PfPK6 showed a preference for Mn(2+) over Mg(2+) ions as a cofactor. The Lys(38)-->Arg mutant is severely defective in its interaction with ATP and bivalent cations and somewhat defective in catalytic rate for R2 phosphorylation. PMID- 10727427 TI - Large induction of the chemotactic cytokine RANTES during cutaneous wound repair: a regulatory role for nitric oxide in keratinocyte-derived RANTES expression. AB - We investigated the role of NO on expressional regulation of the chemotactic cytokine RANTES (regulated upon activation, normal T-cell expressed and secreted) during tissue regeneration using an excisional wound-healing model in mice. Wound repair was characterized by a large and sustained induction of RANTES expression, and inhibition of inducible nitric oxide synthase (iNOS) during repair only slightly decreased RANTES expression levels. Immunohistochemical analysis revealed keratinocytes of the wound margins and the hyperproliferative epithelium to be the main RANTES-expressing cell type within the wound. Therefore we analysed the regulation of RANTES expression in vitro in cultured human keratinocytes of the cell line HaCaT. Here we demonstrate that NO very efficiently suppressed interleukin-1beta- and tumour-necrosis-factor-alpha induced RANTES expression in keratinocytes. Furthermore, down-regulation of cytokine-induced RANTES mRNA in keratinocytes was dependent on endogenously produced NO, as inhibition of the co-induced iNOS by L-N(G)-monomethyl-L-arginine increased cytokine-triggered RANTES expression in the cells. Moreover, we observed strongest RANTES-immunopositive labelling in epithelial areas which were characterized by a NO-mediated low cellularity. Thus our data implicate NO as a negative regulator of RANTES expression during wound repair in vivo, as decreased numbers of keratinocytes observed in the absence of wound-derived NO might compensate for the high levels of RANTES expression which are associated with normal repair. PMID- 10727428 TI - Specific role of the extracellular signal-regulated kinase pathway in angiotensin II-induced cardiac hypertrophy in vitro. AB - Although MAP (mitogen-activated protein) kinases are implicated in cell proliferation and differentiation in many cell types, the role of MAP kinases in cardiac hypertrophy remains unclear. We examined the role of extracellular signal regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAP kinase in angiotensin II (Ang II)-induced hypertrophy compared with phenylephrine induced hypertrophy in neonatal rat cardiac myocytes. Both Ang II and phenylephrine activated ERKs to a similar extent, whereas phenylephrine caused stronger and more sustained activation of JNK and p38 than Ang II. PD98059, a specific inhibitor of MAPK/ERK kinase (MEK),inhibited Ang II-induced, but not phenylephrine-induced, expression of atrial natriuretic factor (ANF) at both the mRNA and polypeptide levels. SB203580, a specific inhibitor of p38 and some JNK isoforms, did not show significant effects on ANF expression induced by Ang II or phenylephrine. Although PD98059 and dominant-negative MEK1 blocked Ang II-induced activation of the ANF promoter, SB203580 or dominant-negative MEK kinase 1 (MEKK1) showed no effect. Phenylephrine-induced ANF promoter activation was significantly inhibited by SB203580 and dominant-negative MEKK1, but not by PD98059 or dominant-negative MEK1. Dominant-negative Ras inhibited both ERK activation and ANF up-regulation by Ang II, whereas constitutively active forms of Ras and MEK were sufficient to activate the ANF promoter. Dominant-negative Ras also partly inhibited the phenylephrine-induced activation of ANF promoter. PD98059 did not affect other markers of Ang II-induced hypertrophy, such as skeletal alpha-actin and c-fos expression, increases in the rate of protein synthesis or rapid sarcomeric actin organization. These results suggest that Ang II uses ERK for ANF expression, whereas phenylephrine uses other pathways. The Ras/ERK pathway selectively mediates ANF expression in various phenotypes observed in Ang II-induced hypertrophy. The ERK pathway mediates an agonist specific and phenotype-specific response in cardiac hypertrophy. PMID- 10727429 TI - Protein kinase C-beta contributes to NADPH oxidase activation in neutrophils. AB - We have analysed the involvement of the beta isotype of the protein kinase C (PKC) family in the activation of NADPH oxidase in primary neutrophils. Using immunofluorescence and cell fractionation, PKC-beta is shown to be recruited to the plasma membrane upon stimulation with phorbol ester and to the phagosomal membrane upon phagocytosis of IgG-coated particles (Fcgamma-receptor stimulus). The time course of recruitment is similar to that of NADPH oxidase activation by these stimuli. The PKC-beta specific inhibitor 379196 inhibits the response to PMA as well as to IgG-coated bacteria. Partial inhibition occurs between 10 and 100 nM of inhibitor, the concentration at which PKC-beta, but not other PKC isotypes, is targeted. Neutrophils isolated from a mouse that lacks PKC-beta also showed an inhibition of NADPH oxidase activation by PMA and IgG-coated particles. The level of inhibition is comparable to that achieved with 379196 in human neutrophils. Thus the PKC-beta isotype mediates activation of NADPH oxidase by PMA and by stimulation of Fcgamma receptors in neutrophils. PMID- 10727430 TI - Human cystathionine gamma-lyase: developmental and in vitro expression of two isoforms. AB - Cystathionine gamma-lyase (CGL) is the last enzyme of the trans-sulphuration pathway, which converts methionine into cysteine. To study the possible differences in enzymic activity of the two human cystathionine gamma-lyase isoforms characterized earlier, these were separately expressed in human kidney embryonic 293T cells. Furthermore, developmental changes in the expression of the two mRNA forms as well as the enzymic activity in human liver were studied, as it has been postulated that a change in the relative expression of CGL isoforms causes the postnatal increase in CGL activity. Transfection with the longer isoform increased the CGL activity 1.5-fold, while the activity of the cells transfected with the shorter form did not differ from the basal activity. In human liver samples, CGL activity was only detected in adult tissue (68+/-9 nmol of cysteine/h per mg of protein), whereas activity in fetal, premature and full term neonatal liver tissue was undetectable. In contrast, strong mRNA expression of both mRNA isoforms was detected from the 19th gestational week onwards and the longer form of CGL appeared to be predominant. The expression of the two mRNA forms varied in parallel. In conclusion, we have shown that only cells overexpressing the longer form of CGL have increased activity, and CGL appears to be regulated at the post-transcriptional level during development. PMID- 10727431 TI - Properties of a polyamine transporter regulated by antizyme. AB - The regulation of polyamine transport by antizyme, a protein that is involved in the rapid degradation of ornithine decarboxylase (ODC), was studied in FM3A mouse cells overproducing ODC. Both artificial (Z1) and natural antizymes not only inhibited polyamine uptake but also stimulated polyamine excretion. The properties of the polyamine transporter regulated by antizyme were characterized. The uptake of radiolabelled polyamines was inhibited by excess acetylpolyamines and a protonophore, CCCP (carbonyl cyanide m-chlorophenylhydrazone), whereas the excretion of radiolabelled polyamines was stimulated by unlabelled polyamines, acetylpolyamines and CCCP in the medium. Furthermore, it is shown that polyamines and acetylpolyamines are excreted from cells. On the basis of the results, it is discussed how antizyme regulates polyamine transport negatively. PMID- 10727432 TI - Partial purification and characterization of a wortmannin-sensitive and insulin stimulated protein kinase that activates heart 6-phosphofructo-2-kinase. AB - A wortmannin-sensitive and insulin-stimulated protein kinase (WISK), which phosphorylates and activates cardiac 6-phosphofructo-2-kinase (PFK-2), was partially purified from perfused rat hearts. Immunoblotting showed that WISK was devoid of protein kinase B (PKB), serum- and glucocorticoid-regulated protein kinase and protein kinase Czeta (PKCzeta). Comparison of the inhibition of WISK, PKCalpha and PKCzeta by different protein kinase inhibitors suggested that WISK was not a member of the PKC family. In addition, WISK contained no detectable phosphoinositide-dependent protein kinase-1 (PDK1) activity. WISK phosphorylated recombinant heart PFK-2 in a time-dependent manner to the extent of 0.4 mol of phosphate incorporated/mol of enzyme subunit, and increased the V(max) of PFK-2 twofold, without affecting the K(m) for fructose 6-phosphate. WISK phosphorylated Ser-466 to a greater extent than Ser-483 in recombinant heart PFK-2, and both sites were demonstrated to be phosphorylated to the same extent by PKB. Gel filtration and in-gel kinase analysis indicated that WISK was a monomer with a M(r) of 56500. Treatment of WISK with protein phosphatase 2A (PP2A) catalytic subunits reversed the effect of insulin, suggesting the involvement of an upstream activating kinase. Indeed, PDK1 was able to partially reactivate the PP2A-treated WISK and this reactivation was not enhanced by PtdIns(3,4,5)P(3) containing vesicles. Moreover, a single 57000-M(r) band was labelled on incubation of the dephosphorylated WISK preparation with PDK1 and [gamma (32)P]ATP. These findings provide evidence for the existence of a new protein kinase in the insulin signalling pathway, probably downstream of PDK1. PMID- 10727433 TI - Regulation of urokinase plasminogen activator gene transcription in the RAW264 murine macrophage cell line by macrophage colony-stimulating factor (CSF-1) is dependent upon the level of cell-surface receptor. AB - Macrophage colony-stimulating factor (CSF-1) binds to a receptor (CSF-1R) encoded by the c-fms proto-oncogene and activates transcription of the urokinase plasminogen activator (uPA) gene in murine bone-marrow-derived macrophages. This article demonstrates that the murine macrophage cell line RAW264 responds to CSF 1 with inducible phosphorylation of cytoplasmic proteins on tyrosine residues but fails to induce transcription of uPA. The defect was correlated with a selective failure to maintain CSF-1Rs on the cell surface, whereas all RAW264 cells contained abundant CSF-1Rs within the presumptive Golgi/endoplasmic reticulum compartment. Transfection with a CSF-1R expression plasmid permitted CSF-1 dependent activation of the signalling pathway targeting an Ets/AP1 (activator protein 1) element in the uPA promoter that has been shown previously to be a target of oncogenic ras and protein kinase C pathways. Mutation of the expressed CSF-1R at either Y807 or Y559, sites of receptor tyrosine phosphorylation implicated in signal transduction, reduced but did not abolish uPA promoter activation by CSF-1. Activation by mutant CSF-1R plasmids was additive; there was no evidence of mutual complementation. The results indicate that maintenance of elevated uPA transcription by CSF-1 requires new receptors emerging continuously on the cell surface. Parallel, partly redundant, signalling pathways arising from phosphorylated tyrosines on the CSF-1R activate multiple cis-acting elements on the complex uPA promoter. PMID- 10727434 TI - The Pl(A2) allele and cardiovascular disease: the pro(33) and con. PMID- 10727435 TI - Old concepts and new developments in the study of platelet aggregation. PMID- 10727436 TI - The lipid-laden foam cell: an elusive target for therapeutic intervention. PMID- 10727437 TI - Dendritic cells: at the clinical crossroads. PMID- 10727438 TI - Benchmarks for antiretroviral therapy. PMID- 10727439 TI - Massive xanthomatosis and altered composition of atherosclerotic lesions in hyperlipidemic mice lacking acyl CoA:cholesterol acyltransferase 1. AB - Inhibitors of acyl CoA:cholesterol acyltransferase (ACAT) have attracted considerable interest as a potential treatment for atherosclerosis. Currently available inhibitors probably act nonselectively against the two known ACATs. One of these enzymes, ACAT1, is highly expressed in macrophages in atherosclerotic lesions, where it contributes to foam-cell formation. In this study, we examined the effects of selective ACAT1 deficiency in two mouse models of atherosclerosis. In the setting of severe hypercholesterolemia caused by deficiency in apoE or the LDL receptor (LDLR), total ACAT1 deficiency led to marked alterations in cholesterol homeostasis and extensive deposition of unesterified cholesterol in the skin and brain. Bone marrow transplantation experiments demonstrated that ACAT1 deficiency in macrophages was sufficient to cause dermal xanthomas in hyperlipidemic LDLR-deficient mice. ACAT1 deficiency did not prevent the development of atherosclerotic lesions in either apoE-deficient or LDLR-deficient mice, despite causing relatively lower serum cholesterol levels. However, the lesions in ACAT1-deficient mice were atypical in composition, with reduced amounts of neutral lipids and a paucity of macrophages in advanced lesions. Although the latter findings may be associated with increased lesion stability, the marked alterations in cholesterol homeostasis indicate that selectively inhibiting ACAT1 in the setting of severe hyperlipidemia may have detrimental consequences. PMID- 10727440 TI - Regulation of inflammation by collagen-binding integrins alpha1beta1 and alpha2beta1 in models of hypersensitivity and arthritis. AB - Adhesive interactions play an important role in inflammation by promoting leukocyte attachment and extravasation from the vasculature into the peripheral tissues. However, the importance of adhesion molecules within the extracellular matrix-rich environment of peripheral tissues, in which cells must migrate and be activated, has not been well explored. We investigated the role of the major collagen-binding integrins, alpha1beta1 and alpha2beta1, in several in vivo models of inflammation. mAb's against murine alpha1 and alpha2 were found to significantly inhibit effector phase inflammatory responses in animal models of delayed-type hypersensitivity (DTH), contact hypersensitivity (CHS), and arthritis. Mice that were alpha1-deficient also showed decreased inflammatory responses in the CHS and arthritis models when compared with wild-type mice. Decreased leukocyte infiltration and edema formation accompanied inhibition of antigen-specific models of inflammation, as nonspecific inflammation induced by croton oil was not inhibited. This study demonstrates the importance in vivo of alpha1beta1 and alpha2beta1, the collagen-binding integrins, in inflammatory diseases. The study also extends the role of integrins in inflammation beyond leukocyte attachment and extravasation at the vascular endothelial interface, revealing the extracellular matrix environment of peripheral tissues as a new point of intervention for adhesion-based therapies. PMID- 10727441 TI - Transgenic mice overexpressing insulin-like growth factor-II in beta cells develop type 2 diabetes. AB - During embryonic development, insulin-like growth factor-II (IGF-II) participates in the regulation of islet growth and differentiation. We generated transgenic mice (C57BL6/SJL) expressing IGF-II in beta cells under control of the rat Insulin I promoter in order to study the role of islet hyperplasia and hyperinsulinemia in the development of type 2 diabetes. In contrast to islets from control mice, islets from transgenic mice displayed high levels of IGF-II mRNA and protein. Pancreases from transgenic mice showed an increase in beta-cell mass (about 3-fold) and in insulin mRNA levels. However, the organization of cells within transgenic islets was disrupted, with glucagon-producing cells randomly distributed throughout the core. We also observed enhanced glucose stimulated insulin secretion and glucose utilization in islets from transgenic mice. These mice displayed hyperinsulinemia, mild hyperglycemia, and altered glucose and insulin tolerance tests, and about 30% of these animals developed overt diabetes when fed a high-fat diet. Furthermore, transgenic mice obtained from the N1 backcross to C57KsJ mice showed high islet hyperplasia and insulin resistance, but they also developed fatty liver and obesity. These results indicate that local overexpression of IGF-II in islets might lead to type 2 diabetes and that islet hyperplasia and hypersecretion of insulin might occur early in the pathogenesis of this disease. PMID- 10727442 TI - Gene transfer of the neuronal NO synthase isoform to cirrhotic rat liver ameliorates portal hypertension. AB - Reduced production of nitric oxide (NO) in the cirrhotic liver results from a defect in hepatic endothelial cell nitric oxide synthase (ecNOS) and appears to contribute to the high intrahepatic resistance and portal hypertension typical of cirrhosis. Therefore, we postulated that targeting a heterologous NOS isoform to sinusoidal endothelial cells or other perisinusoidal cells, such as hepatic stellate cells, would counter the defect in NO production and reduce resistance to blood flow. Recombinant adenovirus (Ad) carrying the neuronal NOS gene (nNOS) targeted liver sinusoidal endothelial cells, stellate cells, and hepatocytes more efficiently than the corresponding cells in cirrhotic livers, but transduction rates were substantial even in cirrhotic animals. Expression of nNOS in each liver cell type, whether from normal or injured liver, caused increased NO production and inhibited endothelin-1-induced contractility of perisinusoidal stellate cells. Finally, in 2 different in vivo models of cirrhosis and portal hypertension, transduction of livers with recombinant Ad.nNOS significantly reduced intrahepatic resistance and portal pressure. The data highlight the feasibility of gene transfer to diseased liver and hepatic cells and demonstrate the potential of a novel therapy for portal hypertension caused by cirrhosis. PMID- 10727443 TI - Accelerated puberty and late-onset hypothalamic hypogonadism in female transgenic skinny mice overexpressing leptin. AB - Excess or loss of body fat can be associated with infertility, suggesting that adequate fat mass is essential for proper reproductive function. Leptin is an adipocyte-derived hormone that is involved in the regulation of food intake and energy expenditure, and its synthesis and secretion are markedly increased in obesity. Short-term administration of leptin accelerates the onset of puberty in normal mice and corrects the sterility of leptin-deficient ob/ob mice. These findings suggest a role for leptin as an endocrine signal between fat depots and the reproductive axis, but the effect of hyperleptinemia on the initiation and maintenance of reproductive function has not been elucidated. To address this issue, we examined the reproductive phenotypes of female transgenic skinny mice with elevated plasma leptin concentrations comparable to those in obese subjects. With no apparent adipose tissue, female transgenic skinny mice exhibit accelerated puberty and intact fertility at younger ages followed by successful delivery of healthy pups. However, at older ages, they develop hypothalamic hypogonadism characterized by prolonged menstrual cycles, atrophic ovary, reduced hypothalamic gonadotropin releasing hormone contents, and poor pituitary luteinizing hormone secretion. This study has demonstrated for the first time to our knowledge that accelerated puberty and late-onset hypothalamic hypogonadism are associated with chronic hyperleptinemia, thereby leading to a better understanding of the pathophysiological and therapeutic implication of leptin. PMID- 10727444 TI - Interaction between succinyl CoA synthetase and the heme-biosynthetic enzyme ALAS E is disrupted in sideroblastic anemia. AB - The first and the rate-limiting enzyme of heme biosynthesis is delta aminolevulinate synthase (ALAS), which is localized in mitochondria. There are 2 tissue-specific isoforms of ALAS, erythroid-specific (ALAS-E) and nonspecific ALAS (ALAS-N). To identify possible mitochondrial factors that modulate ALAS-E function, we screened a human bone marrow cDNA library, using the mitochondrial form of human ALAS-E as a bait protein in the yeast 2-hybrid system. Our screening led to the isolation of the beta subunit of human ATP-specific succinyl CoA synthetase (SCS-betaA). Using transient expression and coimmunoprecipitation, we verified that mitochodrially expressed SCS-betaA associates specifically with ALAS-E and not with ALAS-N. Furthermore, the ALAS-E mutants R411C and M426V associated with SCS-betaA, but the D190V mutant did not. Because the D190V mutant was identified in a patient with pyridoxine-refractory X-linked sideroblastic anemia, our findings suggest that appropriate association of SCS-betaA and ALAS-E promotes efficient use of succinyl CoA by ALAS-E or helps translocate ALAS-E into mitochondria. PMID- 10727445 TI - Th2 responses induced by epicutaneous or inhalational protein exposure are differentially dependent on IL-4. AB - Atopic individuals are predisposed to mounting vigorous Th2-type immune responses to environmental allergens. To determine the factors responsible, animal models that closely mimic natural modes of allergen exposure should prove most informative. Therefore, we investigated the role of IL-4, a known Th2-promoting cytokine, in generation of Th2 responses after exposure of either the skin or airway to soluble protein. Compared with wild-type (WT) mice, IL-4-deficient (IL 4(-/-)) mice showed markedly impaired Th2 activation after primary exposure to inhaled ovalbumin (OVA), with decreased OVA-specific IgG1 and IgE, and significantly fewer eosinophils in bronchoalveolar lavage (BAL) fluid after airway challenge. In contrast, IL-4(-/-) mice initially exposed to epicutaneous (e.c.) OVA mounted Th2 responses equivalent to responses in WT mice, with high numbers of eosinophils in BAL fluid. Because Th2 responses were not induced by e.c. OVA exposure in Stat6(-/-) mice (mice lacking signal transducer and activator of transcription 6), the role of IL-13 was tested. In vivo depletion of IL-13 prevented Th2 responses induced by e.c. OVA exposure in IL-4(-/-) mice. These data demonstrate a marked difference in the IL-4 dependence of Th2 responses generated at two anatomic sites of natural allergen encounter and identify the skin as a particularly potent site for Th2 sensitization. PMID- 10727446 TI - Predicting the duration of antiviral treatment needed to suppress plasma HIV-1 RNA. AB - Effective therapeutic interventions and clinical care of adults infected with HIV 1 require an understanding of factors that influence time of response to antiretroviral therapy. We have studied a cohort of 118 HIV-1-infected subjects naive to antiretroviral therapy and have correlated the time of response to treatment with a series of virological and immunological measures, including levels of viral load in blood and lymph node, percent of CD4 T cells in lymph nodes, and CD4 T-cell count in blood at study entry. Suppression of viremia below the limit of detection, 50 HIV-1 RNA copies/mL of plasma, served as a benchmark for a successful virological response. We employed these correlations to predict the length of treatment required to attain a virological response in each patient. Baseline plasma viremia emerged as the factor most tightly correlated with the duration of treatment required, allowing us to estimate the required time as a function of this one measure. PMID- 10727447 TI - A revised model of platelet aggregation. AB - In this study we have examined the mechanism of platelet aggregation under physiological flow conditions using an in vitro flow-based platelet aggregation assay and an in vivo rat thrombosis model. Our studies demonstrate an unexpected complexity to the platelet aggregation process in which platelets in flowing blood continuously tether, translocate, and/or detach from the luminal surface of a growing platelet thrombus at both arterial and venous shear rates. Studies of platelets congenitally deficient in von Willebrand factor (vWf) or integrin alpha(IIb)beta(3) demonstrated a key role for platelet vWf in mediating platelet tethering and translocation, whereas integrin alpha(IIb)beta(3) mediated cell arrest. Platelet aggregation under flow appears to be a multistep process involving: (a) exposure of vWf on the surface of immobilized platelets; (b) a reversible phase of platelet aggregation mediated by the binding of GPIbalpha on the surface of free-flowing platelets to vWf on the surface of immobilized platelets; and (c) an irreversible phase of aggregation dependent on integrin alpha(IIb)beta(3). Studies of platelet thrombus formation in vivo demonstrate that this multistep adhesion mechanism is indispensable for platelet aggregation in arterioles and also appears to promote platelet aggregate formation in venules. Together, our studies demonstrate an important role for platelet vWf in initiating the platelet aggregation process under flow and challenge the currently accepted view that the vWf-GPIbalpha interaction is exclusively involved in initiating platelet aggregation at elevated shear rates. PMID- 10727448 TI - The Pl(A2) polymorphism of integrin beta(3) enhances outside-in signaling and adhesive functions. AB - Genetic factors are believed to influence the development of arterial thromboses. Because integrin alpha(IIb)beta(3) plays a crucial role in thrombus formation, we analyzed receptor adhesive properties using Chinese hamster ovary and human kidney embryonal 293 cells overexpressing the Pl(A1) or Pl(A2) polymorphic forms of alpha(IIb)beta(3). Soluble fibrinogen binding was no different between Pl(A1) and Pl(A2) cells, either in a resting state or when alpha(IIb)beta(3) was activated with anti-LIBS6. Pl(A1) and Pl(A2) cells bound equivalently to immobilized fibronectin. In contrast, significantly more Pl(A2) cells bound to immobilized fibrinogen in an alpha(IIb)beta(3)-dependent manner than did Pl(A1) cells. Disruption of the actin cytoskeleton by cytochalasin D abolished the increased binding of Pl(A2) cells. Compared with Pl(A1) cells, Pl(A2) cells exhibited a greater extent of polymerized actin and cell spreading, enhanced tyrosine phosphorylation of pp125(FAK), and greater fibrin clot retraction. These adhesion differences appear to depend on a signaling mechanism sensitive to receptor occupancy. Thus, the Pl(A2) polymorphism altered integrin-mediated functions of adhesion, spreading, actin cytoskeleton rearrangement, and clot retraction. PMID- 10727449 TI - Genetic immunization of outbred mice with thyrotropin receptor cDNA provides a model of Graves' disease. AB - We performed genetic immunization of outbred NMRI mice, using a cDNA encoding the human thyrotropin receptor (TSHr). All mice produced antibodies capable of recognizing the recombinant receptor expressed at the surface of stably transfected Chinese hamster ovary (CHO) cells, and sera from most of the immunized mice blocked TSH-dependent stimulation of cAMP accumulation in cells expressing the TSHr. Five out of 29 female mice showed sign of hyperthyroidism including elevated total T4 and suppressed TSH levels. The serum of these mice contained thyroid-stimulating activity, as measured in a classic assay using CHO cells expressing recombinant TSHr. In contrast, only 1 male out of 30 had moderately elevated serum total T4 with undetectable TSH values. The hyperthyroid animals had goiters with extensive lymphocytic infiltration, characteristic of a Th2 immune response. In addition, these animals displayed ocular signs reminiscent of Graves' ophthalmopathy, including edema, deposit of amorphous material, and cellular infiltration of their extraocular muscles. Our results demonstrate that genetic immunization of outbred NMRI mice with the human TSHr provides the most convincing murine model of Graves' disease available to date. PMID- 10727450 TI - Antigen presenting cells expressing Fas ligand down-modulate chronic inflammatory disease in Fas ligand-deficient mice. AB - We assessed the effect of modified antigen presenting cells (APCs) expressing high levels of Fas ligand (APC-FasL) on post-viral chronic inflammatory disease. FasL-deficient B6-gld/gld mice infected with murine cytomegalovirus (MCMV) cleared the virus from their lungs, kidneys, and livers within 2 weeks of infection. However, inflammation persisted in these organs for more than 8 weeks, with a chronically increased T-cell response to MCMV-infected APCs and production of autoantibodies. Administration of APC-AdFasL at 4 weeks suppressed this inflammation and diminished the T-cell response and autoantibody production. APC AdFasL that had been transfected with ultraviolet-irradiated MCMV were more effective than uninfected APC-AdFasL in ameliorating the chronic inflammation. APC-AdFasL migrated preferentially to the spleen, where they triggered apoptosis of lymphocytes in the marginal zone of the spleen. These results confirm that Fas mediated apoptosis is not required for clearance of virus, but is required for down-modulation of the virally induced chronic inflammatory response. This organwide effect of APC-AdFasL appears to be mediated by elimination of activated T lymphocytes in the spleen before their emigration to the target organs. PMID- 10727451 TI - Prostaglandin G/H synthase-2 is required for maximal formation of osteoclast-like cells in culture. AB - We examined the effect on osteoclast formation of disrupting the prostaglandin G/H synthase genes PGHS-1 and-2. Prostaglandin E(2) (PGE(2)) production was significantly reduced in marrow cultures from mice lacking PGHS-2 (PGHS-2(-/-)) compared with wild-type (PGHS-2(+/+)) cultures. Osteoclast formation, whether stimulated by 1,25-dihydroxyvitamin D(3) (1,25-D) or by parathyroid hormone (PTH), was reduced by 60-70% in PGHS-2(-/-) cultures relative to wild-type cultures, an effect that could be reversed by providing exogenous PGE(2). Cultures from heterozygous mice showed an intermediate response. PGHS inhibitors caused a similar drop in osteoclast formation in wild-type cultures. Co-culture experiments showed that supporting osteoblasts, rather than osteoclast precursors, accounted for the blunted response to 1,25-D and PTH. This lack of response appeared to result from reduced expression of RANK ligand (RANKL) in osteoblasts. We cultured spleen cells with exogenous RANKL and found that osteoclast formation was 50% lower in PGHS-2(-/-) than in wild-type cultures, apparently because the former cells expressed high levels of GM-CSF. Injection of PTH above the calvaria caused hypercalcemia in wild-type but not PGHS-2(-/-) mice. Histological examination of bone from 5-week-old PGHS-2(-/-) mice revealed no abnormalities. Mice lacking PGHS-1 were similar to wild-type mice in all of these parameters. These data suggest that PGHS-2 is not necessary for wild-type bone development but plays a critical role in bone resorption stimulated by 1,25 D and PTH. PMID- 10727452 TI - Mature dendritic cells boost functionally superior CD8(+) T-cell in humans without foreign helper epitopes. AB - We have recently shown that a single injection of mature, antigen-pulsed, human dendritic cells (DCs) rapidly elicits CD4(+) and CD8(+) T-cell immunity in vivo. The DCs were pulsed with 2 foreign proteins, keyhole limpet hemocyanin (KLH) and tetanus toxoid (TT), as well as an HLA A2.1-restricted influenza matrix peptide (MP). Responses to all 3 antigens peaked at 30-90 days after immunization and declined thereafter. To determine if the foreign helper proteins (TT and KLH) were essential for CD8(+) T-cell responses to the viral peptide, we reinjected 3 of the HLA-2.1 subjects with mature DCs pulsed with MP alone. All 3 volunteers showed a rapid boost in MP-specific immunity, and freshly sampled blood from 1 contained cytolytic T cells. In all 3 subjects, CD8(+) T-cell responses to booster DCs were faster and of greater magnitude than the responses to the first DC injection. Importantly, the T cells that proliferated after booster DC treatment secreted interferon-gamma upon challenge with much lower doses of viral peptide than those elicited after the first injection, indicating a higher functional avidity for the ligand. These data begin to outline the kinetics of T cell immunity in response to DCs and demonstrate that booster injections of mature DCs enhance both qualitative and quantitative aspects of CD8(+) T-cell function in humans. PMID- 10727453 TI - CD1, tuberculosis, and the evolution of major histocompatibility complex molecules. PMID- 10727455 TI - The formation of immunogenic major histocompatibility complex class II-peptide ligands in lysosomal compartments of dendritic cells is regulated by inflammatory stimuli. AB - During their final differentiation or maturation, dendritic cells (DCs) redistribute their major histocompatibility complex (MHC) class II products from intracellular compartments to the plasma membrane. Using cells arrested in the immature state, we now find that DCs also regulate the initial intracellular formation of immunogenic MHC class II-peptide complexes. Immature DCs internalize the protein antigen, hen egg lysozyme (HEL), into late endosomes and lysosomes rich in MHC class II molecules. There, despite extensive colocalization of HEL protein and MHC class II products, MHC class II-peptide complexes do not form unless the DCs are exposed to inflammatory mediators such as tumor necrosis factor alpha, CD40 ligand, or lipoplolysaccharide. The control of T cell receptor (TCR) ligand formation was observed using the C4H3 monoclonal antibody to detect MHC class II-HEL peptide complexes by flow cytometry and confocal microscopy, and with HEL-specific 3A9 transgenic T cells to detect downregulation of the TCR upon MHC-peptide encounter. Even the binding of preprocessed HEL peptide to MHC class II is blocked in immature DCs, including the formation of C4H3 epitope in MHC class II compartments, suggesting an arrest to antigen presentation at the peptide-loading step, rather than an enhanced degradation of MHC class II-peptide complexes at the cell surface, as described in previous work. Therefore, the capacity of late endosomes and lysosomes to produce MHC class II-peptide complexes can be strictly controlled during DC differentiation, helping to coordinate antigen acquisition and inflammatory stimuli with formation of TCR ligands. The increased ability of maturing DCs to load MHC class II molecules with antigenic cargo contributes to the >100-fold enhancement of the subsequent primary immune response observed when immature and mature DCs are compared as immune adjuvants in culture and in mice. PMID- 10727454 TI - Activation-induced inhibition of interleukin 6-mediated T cell survival and signal transducer and activator of transcription 1 signaling. AB - The cytokines interleukin (IL)-2, IL-4, IL-6, IL-7, and IL-15 have all previously been shown to inhibit resting T cell death in vitro. We have found a difference in the response of T cells to IL-6, depending on the activation status of the cells. IL-6 inhibited the death of naive T cells, but had no effect on the death of either superantigen-activated T cells, or T cells bearing memory markers. This was true even when the resting and activated T cells were isolated from the same animal; thus, the determining factor for IL-6 insensitivity was the activation status or activation history of the cell, and not the milieu in the animal from which the cells were isolated. Activated T cells expressed lower levels of IL-6 receptors on their surfaces, yet there were sufficient levels of receptors for signaling, as we observed similar levels of signal transducer and activator of transcription (Stat)3 phosphorylation in resting and activated T cells treated with IL-6. However, there was profound inhibition of IL-6-induced Stat1 phosphorylation in activated T cells compared with resting T cells. These data suggest that there is activation-induced inhibition of IL-6 receptor signaling in T cells. This inhibition appears to be specific for some but not all of the IL-6 mediated signaling cascades in these cells. PMID- 10727456 TI - Self-recognition of CD1 by gamma/delta T cells: implications for innate immunity. AB - The specificity of immunoglobulins and alpha/beta T cell receptors (TCRs) provides a framework for the molecular basis of antigen recognition. Yet, evolution has preserved a separate lineage of gamma/delta antigen receptors that share characteristics of both immunoglobulins and alpha/beta TCRs but whose antigens remain poorly understood. We now show that T cells of the major tissue gamma/delta T cell subset recognize nonpolymorphic CD1c molecules. These T cells proliferated in response to CD1+ presenter cells, lysed CD1c+ targets, and released T helper type 1 (Th1) cytokines. The CD1c-reactive gamma/delta T cells were cytotoxic and used both perforin- and Fas-mediated cytotoxicity. Moreover, they produced granulysin, an important antimicrobial protein. Recognition of CD1c was TCR mediated, as recognition was transferred by transfection of the gamma/delta TCR. Importantly, all CD1c-reactive gamma/delta T cells express V delta 1 TCRs, the TCR expressed by most tissue gamma/delta T cells. Recognition by this tissue pool of gamma/delta T cells provides the human immune system with the capacity to respond rapidly to nonpolymorphic molecules on professional antigen presenting cells (APCs) in the absence of foreign antigens that may activate or eliminate the APCs. The presence of bactericidal granulysin suggests these cells may directly mediate host defense even before foreign antigen specific T cells have differentiated. PMID- 10727457 TI - Role of lipooligosaccharide in Opa-independent invasion of Neisseria gonorrhoeae into human epithelial cells. AB - Lipooligosaccharide (LOS) has been implicated in the adhesion and invasion of host epithelial cells. We examined the adhesive and invasive abilities of isogenic gonococcal opacity-associated outer membrane protein-negative, pilus positive (Opa-Pil+) Neisseria gonorrhoeae strains expressing genetically defined LOS. Strain F62 (Opa-Pil+), expressing the lacto-N-neotetraose and the galNac lacto-N-neotetraose LOS, and its isogenic derivative that expressed only the lacto-N-neotetraose LOS (F62 Delta lgtD), adhered to, and invaded, to the same extent the human cervical epidermoid carcinoma cell line, ME180. While the adhesive abilities of Opa-Pil+ isogenic strains that express LOS molecules lacking the lacto-N-neotetraose structure were similar to that seen for F62, their invasive abilities were much lower than the strains expressing lacto-N neotetraose. Fluorescence microscopy studies showed that the adherence of F62, but not the strains lacking lacto-N-neotetraose, induced the rearrangement of actin filaments under the adherent sites. Electron microscopy studies demonstrated that F62, but not the strains lacking lacto-N-neotetraose, formed extensive and intimate associations with epithelial cell membranes. Thus, in the absence of detectable Opa protein, the lacto-N-neotetraose LOS promotes gonococcal invasion into ME180 cells. The data also suggest that LOS is involved in the mobilization of actin filaments in host cells, and in the formation of a direct interaction between the bacterial outer membrane and the plasma membrane of ME180 cells. PMID- 10727458 TI - Multiple genetic alterations cause frequent and heterogeneous human histocompatibility leukocyte antigen class I loss in cervical cancer. AB - The nature and frequency of human histocompatibility leukocyte antigen (HLA) class I loss mechanisms in primary cancers are largely unknown. We used flow cytometry and molecular analyses to concurrently assess allele-specific HLA phenotypes and genotypes in subpopulations from 30 freshly isolated cervical tumor cell suspensions.Tumor-associated HLA class I alterations were present in 90% of the lesions tested, comprising four altered pheno/genotype categories: (a) HLA-A or -B allelic loss (17%), mostly associated with gene mutations; (b) HLA haplotype loss, associated with loss of heterozygosity at 6p (50%). This category included cases with additional loss of a (third) HLA-A or -B allele due to mutation, as well as one case with an HLA class I-negative tumor cell subpopulation, caused by a beta2-microglobulin gene mutation; (c) Total HLA class I antigen loss and retention of heterozygosity (ROH) at 6p (10%); and (d) B locus or HLA-A/B downregulation associated with ROH and/or allelic imbalance at 6p (10%). Normal HLA phenotypes and ROH at 6p were observed in 10% of the cases. One case could not be classified (3%). Altered HLA class I antigen expression occurs in most cervical cancers, is diverse, and is mainly caused by genetic changes. Combined with widespread tumor heterogeneity, these changes have profound implications for natural immunity and T cell-based immunotherapy in cervical cancer. PMID- 10727459 TI - Blockade of the Bcr-Abl kinase activity induces apoptosis of chronic myelogenous leukemia cells by suppressing signal transducer and activator of transcription 5 dependent expression of Bcl-xL. AB - Bcr-Abl-expressing leukemic cells are highly resistant to apoptosis induced by chemotherapeutic drugs. Although a number of signaling molecules have been shown to be activated by the Bcr-Abl kinase, the antiapoptotic pathway triggered by this oncogene has not been elucidated. Here, we show that the interleukin 3 independent expression of the antiapoptotic protein, Bcl-xL, is induced by Bcr Abl through activation of signal transducer and activator of transcription (Stat)5. Inhibition of the Bcr-Abl kinase activity in Bcr-Abl-expressing cell lines and CD34(+) cells from chronic myelogenous leukemia (CML) patients induces apoptosis by suppressing the capacity of Stat5 to interact with the bcl-x promoter. Interestingly, after inhibition of the Bcr-Abl kinase, the expression of Bcl-xL is downregulated more rapidly in chronic phase than in blast crisis CML cells, suggesting an involvement of this protein in disease progression. Overall, we describe a novel antiapoptotic pathway triggered by Bcr-Abl that may contribute to the resistance of CML cells to undergo apoptosis. PMID- 10727460 TI - Cytokine-induced Src homology 2 protein (CIS) promotes T cell receptor-mediated proliferation and prolongs survival of activated T cells. AB - Members of the suppressor of cytokine signaling (SOCS) family were discovered as negative regulators of cytokine signaling by inhibition of the Janus kinase signal transducer and activator of transcription (Jak-STAT) pathway. Among them, cytokine-induced Src homology 2 (SH2) protein (CIS) was found to inhibit the interleukin 3- and erythropietin-mediated STAT5 signaling pathway. However, involvement of SOCS proteins in other signaling pathways is still unknown. This study shows that the expression of CIS is selectively induced in T cells after T cell receptor (TCR) stimulation. In transgenic mice, with selective expression of CIS in CD4 T cells, elevated CIS strongly promotes TCR-mediated proliferation and cytokine production in vitro, and superantigen-induced T cell activation in vivo. Forced expression of CIS also prolongs survival of CD4 T cells after TCR activation. Molecular events immediately downstream from the TCR are not changed in CIS-expressing CD4 T cells, but activation of mitogen-activated protein (MAP) kinase pathways by TCR stimulation is significantly enhanced. Together with the increased MAP kinase activation, a direct interaction of CIS and protein kinase Ctheta was also demonstrated. These results suggest that CIS is one of the important regulators of TCR-mediated T cell activation. The functions of CIS, enhancing TCR signaling and inhibiting cytokine signaling, may be important in the regulation of immune response and homeostasis. PMID- 10727461 TI - Signal transducer and activator of transcription 6 is essential in the induction of contact hypersensitivity. AB - Contact hypersensitivity (CHS) is thought to be mainly associated with the activation of T helper type 1 (Th1) cells. However, there is also evidence that Th2 cells or Th2 cytokines play a role in the development of CHS. To analyze the functional contribution of Th2 cytokines interleukin (IL)-4 and IL-13, signal transducer and activator of transcription 6 (STAT6)-deficient (STAT6(-/)-) and wild-type (wt) control C57BL/6 mice were contact sensitized with 5% 2,4,6 trinitrochlorobenzene (TNCB), 0.5% 2,4-dinitrofluorobenzene, or 5% 4-ethoxyl methylene-2-phenyl-2-oxazolin-5-one, and any skin reactions were examined. Ear swelling was significantly reduced with a delayed peak response in STAT6(-/)- mice compared with wt mice.A histological analysis revealed that the infiltration of both eosinophils and neutrophils in the skin challenged after 24 h in STAT6( /)- mice decreased substantially compared with that in wt mice. The expression of Th2 cytokines (IL-4, IL-5) in TNCB-challenged skin tissues and the supernatants from T cells stimulated by 2,4,6-trinitrobenzene sulfonate-modified spleen cells, as well as the immunoglobulin (Ig)E and IgG1 response after challenge, were also profoundly reduced in STAT6(-/)- mice, whereas the expression of interferon gamma was the same in STAT6(-/)- and wt mice after challenge. Furthermore, adoptive transfer experiments revealed that STAT6(-/)- mice induced CHS after injection of lymph node cells obtained from sensitized wt mice. Our data suggest that the STAT6 signal plays a critical role in the induction phase of CHS. PMID- 10727462 TI - Deregulated E2F transcriptional activity in autonomously growing melanoma cells. AB - Inactivation of the retinoblastoma tumor suppressor protein (pRb) has been implicated in melanoma cells, but the molecular basis for this phenotype has not yet been elucidated, and the status of additional family members (p107 and p130, together termed pocket proteins) or the consequences on downstream targets such as E2F transcription factors are not known. Because cell cycle progression is dependent on the transcriptional activity of E2F family members (E2F1-E2F6), most of them regulated by suppressive association with pocket proteins, we characterized E2F-pocket protein DNA binding activity in normal versus malignant human melanocytes. By gel shift analysis, we show that in mitogen-dependent normal melanocytes, external growth factors tightly controlled the levels of growth-promoting free E2F DNA binding activity, composed largely of E2F2 and E2F4, and the growth-suppressive E2F4-p130 complexes. In contrast, in melanoma cells, free E2F DNA binding activity (E2F2 and E2F4, to a lesser extent E2F1, E2F3, and occasionally E2F5), was constitutively maintained at high levels independently of external melanocyte mitogens. E2F1 was the only family member more abundant in the melanoma cells compared with normal melanocytes, and the approximately fivefold increase in DNA binding activity could be accounted for mostly by a similar increase in the levels of the dimerization partner DP1. The continuous high expression of cyclin D1, A2, and E, the persistent cyclin dependent kinase 4 (CDK4) and CDK2 activities, and the presence of hyperphosphorylated forms of pRb, p107, and p130, suggest that melanoma cells acquired the capacity for autonomous growth through inactivation of all three pocket proteins and release of E2F activity, otherwise tightly regulated in normal melanocytes by external growth factors. PMID- 10727463 TI - Regulation of fas ligand expression during activation-induced cell death in T cells by p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase. AB - Activation-induced cell death (AICD) is a mechanism of peripheral T cell tolerance that depends upon an interaction between Fas and Fas ligand (FasL). Although c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) may be involved in apoptosis in various cell types, the mode of regulation of FasL expression during AICD in T cells by these two MAPKs is incompletely understood. To investigate the regulatory roles of these two MAPKs, we analyzed the kinetics of TCR-induced p38 MAPK and JNK activity and their regulation of FasL expression and AICD. We report that both JNK and p38 MAPK regulate AICD in T cells. Our data suggest a novel model of T cell AICD in which p38 MAPK acts early to initiate FasL expression and the Fas-mediated activation of caspases. Subsequently, caspases stimulate JNK to further upregulate FasL expression. Thus, p38 MAPK and downstream JNK converge to regulate FasL expression at different times after T cell receptor stimulation to elicit maximum AICD. PMID- 10727464 TI - Gene dose-dependent maturation and receptor editing of B cells expressing immunoglobulin (Ig)G1 or IgM/IgG1 tail antigen receptors. AB - Conserved differences between the transmembrane and cytoplasmic domains of membrane immunoglobulin (Ig)M and IgG may alter the function of antigen receptors on naive versus memory B cells. Here, we compare the ability of these domains to signal B cell allelic exclusion and maturation in transgenic mice. A lysozyme binding antibody was expressed in parallel sets of mice as IgM, IgG1, or a chimeric receptor with IgM extracellular domains and transmembrane/cytoplasmic domains of IgG1. Like IgM, the IgG1 or chimeric IgM/G receptors triggered heavy chain allelic exclusion and supported development of mature CD21(+) B cells. Many of the IgG or IgM/G B cells became CD21(high) and downregulated their IgG and IgM/G receptors spontaneously, resembling memory B cells and B cells with mutations that exaggerate B cell antigen receptor signaling. Unlike IgM transgenic mice, "edited" B cells that carry non-hen egg lysozyme binding receptors preferentially accumulated in IgG and IgM/G mice. This was most extreme in lines with the highest transgene copy number and diminished in variant offspring with fewer copies. The sensitivity of B cell maturation to transgene copy number conferred by the IgG transmembrane and cytoplasmic domains may explain the diverse phenotypes found in other IgG-transgenic mouse strains and may reflect exaggerated signaling. PMID- 10727466 TI - An essential role for thymic mesenchyme in early T cell development. AB - We show that the mesenchymal cells that surround the 12-d mouse embryo thymus are necessary for T cell differentiation. Thus, epithelial lobes with attached mesenchyme generate all T cell populations in vitro, whereas lobes from which mesenchyme has been removed show poor lymphopoiesis with few cells progressing beyond the CD4(-)CD8(-) stage of development. Interestingly, thymic mesenchyme is derived from neural crest cells, and extirpation of the region of the neural crest involved results in impaired thymic development and craniofacial abnormalities similar to the group of clinical defects found in the DiGeorge syndrome. Previous studies have suggested an inductive effect of mesenchyme on thymic epithelial morphogenesis. However, we have found that mesenchyme-derived fibroblasts are still required for early T cell development in the presence of mature epithelial cells, and hence mesenchyme might have a direct role in lymphopoiesis. We provide an anatomical basis for the role of mesenchyme by showing that mesenchymal cells migrate into the epithelial thymus to establish a network of fibroblasts and associated extracellular matrix. We propose that the latter might be important for T cell development through integrin and/or cytokine interactions with immature thymocytes. PMID- 10727465 TI - The interleukin 7 receptor is required for T cell receptor gamma locus accessibility to the V(D)J recombinase. AB - Defects in the interleukin (IL)-7 signal transduction pathway lead to severe immunodeficiency in humans and in mice. In IL-7 receptor-deficient (IL-7R-/-) mice, lymphoid precursors show a reduced survival rate and variable/diversity/joining region V(D)J recombination is variously affected in different loci, being arrested in the T cell receptor (TCR)-gamma locus, aberrant in the immunoglobulin heavy chain (IgH) locus, and delayed in the TCR-beta locus. Here, we analyze the recombination defect of the TCR-gamma locus. Using ligation mediated polymerase chain reaction, we sought intermediates of the recombination process. In the absence of the IL-7 signal, no initiation of recombination of the TCR-gamma locus was observed, whereas recombination intermediates at the TCR-beta locus could be detected. Thus, the failure to rearrange the TCR-gamma locus is due to a failure to initiate cleavage rather than a failure to religate broken DNA ends. V(D)J recombination was previously thought to begin at the pro-T2 stage of T cell development after the arrest of IL-7R-/- thymocytes at the pro-T1 stage. However, here we show that both TCR-gamma and -beta recombination intermediates are readily detectable in normal T1 cells, but only TCR-beta intermediates were detected in IL-7R-/- T1 cells, supporting a mechanistic role for IL-7 in TCR-gamma locus rearrangement. Since reduced recombination activating gene (rag) expression has been reported in the absence of the IL-7 signal, we directly tested whether the TCR-gamma locus is accessible to cleavage by recombinant Rag proteins in vitro. We found a reduction in chromatin accessibility for Rag-mediated cleavage in IL-7R-/- thymocytes compared with wild type. Thus, IL-7 controls recombination at the TCR-gamma locus by regulating locus accessibility. PMID- 10727467 TI - Invariant chain controls H2-M proteolysis in mouse splenocytes and dendritic cells. AB - The association of invariant (Ii) chain with major histocompatibility complex (MHC) class II dimers is required for proper antigen presentation to T cells by antigen-presenting cells. Mice lacking Ii chain have severe abnormalities in class II transport, T cell selection, and B cell maturation. We demonstrate here that H2-M, which is required for efficient class II antigenic peptide loading, is unexpectedly downregulated in splenocytes and mature dendritic cells (DCs) from Ii(-/-) mice. Downregulation reflects an increased rate of degradation in Ii(-/-) cells. Degradation apparently occurs within lysosomes, as it is prevented by cysteine protease inhibitors such as E64, but not by the proteasome inhibitor lactacystin. Thus, Ii chain may act as a lysosomal protease inhibitor in B cells and DCs, with its deletion contributing indirectly to the loss of H2-M. PMID- 10727468 TI - Internalization of Leishmania mexicana complex amastigotes via the Fc receptor is required to sustain infection in murine cutaneous leishmaniasis. AB - We show here that maintenance of Leishmania infections with Leishmania mexicana complex parasites (Leishmania amazonensis and Leishmania pifanoi) is impaired in the absence of circulating antibody. In these studies, we used mice genetically altered to contain no circulating antibody, with and without functional B cells. This experimental design allowed us to rule out a critical role for B cell antigen presentation in Leishmania pathogenesis. In addition, we show that mice lacking the common gamma chain of Fc receptors (FcgammaRI, FcepsilonRI, and FcgammaRIII) are similarly refractory to infection with these parasites. These observations establish a critical role for antibody in the pathogenesis associated with infection by members of the L. mexicana complex. PMID- 10727470 TI - Identification of a human follicular dendritic cell molecule that stimulates germinal center B cell growth. AB - The initial interaction between B cells and follicular dendritic cells (FDCs) appears to be essential for germinal center (GC) formation. To identify molecules regulating this interaction, we generated FDC-staining monoclonal antibodies (mAbs) and screened them for their ability to block FDC-mediated costimulation of growth and differentiation of CD40-stimulated B cells. Using one of the inhibitory mAbs, 8D6, we expression cloned the cDNA encoding the 8D6 antigen (Ag) from a human FDC line, HK. The 8D6 Ag is a novel protein of 282 amino acids that is expressed abundantly on FDCs. Monolayers of COS cells transiently transfected with the 8D6 Ag cDNA stimulate B cell growth. The mAb 8D6 blocks the costimulatory function completely. The inhibitory activity of the mAb 8D6 was demonstrated to be due to an inhibition of cell cycle progression of CD40 ligand stimulated GC B cells. In addition, the mAb 8D6 inhibits the growth of a lymphoma of GC origin, L3055, which depends on FDCs or HK cells for its growth. These findings suggest that the primary function of FDCs in the GC is to stimulate B cell growth. An FDC signal molecule, 8D6 Ag, may be an important molecule to mediate this function. PMID- 10727471 TI - Pharmacoeconomics and motor neuron disease. PMID- 10727469 TI - T1/ST2-deficient mice demonstrate the importance of T1/ST2 in developing primary T helper cell type 2 responses. AB - We have generated mice with a deficiency in T1/ST2 expression to clarify the roles of T1/ST2 in T helper cell type 2 (Th2) responses. Using immunological challenges normally characterized by a Th2-like response, we have compared the responses of T1/ST2-deficient mice with those generated by wild-type mice. Using a primary pulmonary granuloma model, induced with Schistosoma mansoni eggs, we demonstrate that granuloma formation, characterized by eosinophil infiltration, is abrogated in T1/ST2-deficient mice. Furthermore, we clearly demonstrate that in the absence of T1/ST2 expression, the levels of Th2 cytokine production are severely impaired after immunization. Thus, in a secondary pulmonary granuloma model, draining lymph node cells from the T1/ST2-deficient animals produced significantly reduced levels of IL-4 and IL-5, despite developing granulomas of a magnitude similar to those of wild-type mice and comparable antigen-specific immunoglobulin isotype production. These data clearly demonstrate that T1/ST2 expression plays a role in the development of Th2-like cytokine responses and indicate that effector functions are inhibited in its absence. PMID- 10727472 TI - Skull fractures and mild head injury. PMID- 10727473 TI - A window on the role of dopamine in addiction disorders. PMID- 10727475 TI - Value of radiological diagnosis of skull fracture in the management of mild head injury: meta-analysis. AB - OBJECTIVES: Head injury is a common event. Most patients sustain a mild head injury (MHI), and management depends on the risk of an intracranial haemorrhage (ICH). The value of a plain skull radiograph as a screening tool for ICH is controversial. The aim of this meta-analysis was to estimate and explain differences in reported sensitivity and specificity of the finding of a skull fracture for the diagnosis of ICH, in order to assess the value of the plain skull radiograph in the investigation of patients with MHI, and to estimate the prevalence of ICH in these patients. METHOD: After a systematic literature search 20 studies were selected that reported data on the prevalence of ICH after MHI and/or data on the diagnostic value of skull fracture for the diagnosis of ICH. The mean prevalence of ICH weighted for the sample size was determined. The sensitivity and specificity of different studies were combined using a summary receiver operator characteristic curve. Correlation analysis was used to determine factors that could explain the reported differences between studies. RESULTS: The weighted mean prevalence of ICH after MHI is 0.083. The potential for verification bias and the percentage of patients who had suffered loss of consciousness or post-traumatic amnesia were the most significant factors explaining interstudy differences in sensitivity and specificity. Based on studies wherein at least 50% of patients had a CT study of the brain, the estimated sensitivity of a radiographic finding of skull fracture for the diagnosis of ICH is 0.38 with a corresponding specificity of 0.95. CONCLUSION: The plain skull radiograph is of little value in the initial assessment of MHI patients. PMID- 10727474 TI - Japanese encephalitis. PMID- 10727476 TI - Hedonistic homeostatic dysregulation in patients with Parkinson's disease on dopamine replacement therapies. AB - Hedonistic homeostatic dysregulation is a neuropsychological behavioural disorder associated with substance misuse and addiction. The disorder has been recognised as a consequence of dopamine replacement therapy (DRT) in 15 patients with Parkinson's disease. The syndrome typically develops in male patients with early onset Parkinson's disease, and can occur with orally and subcutaneously administered DRT. These patients take increasing quantities of their DRT, despite increasingly severe drug induced dyskinesias, and may develop a cyclical mood disorder with hypomania or manic psychosis. There is impairment of social and occupational functioning. Tolerance develops to mood elevating effects of DRT and a negative affective withdrawal state occurs if the drugs are withdrawn or doses decreased. The clinical features and guidelines for managing this syndrome are discussed. A set of diagnostic criteria for further investigating this condition is proposed. PMID- 10727477 TI - Assessment of voiding dysfunction in Parkinson's disease by the international prostate symptom score. AB - OBJECTIVES: To find the incidence of voiding dysfunction in Parkinson's disease and to examine the relation between the voiding dysfunction and various indices of the disease (disease severity, disease duration, age, sex, and treatment with antiparkisonian drugs), the presence of voiding dysfunction was quantitatively estimated in patients sampled on the unselected (consecutive) basis. METHODS: Using the international prostate symptom score, lower urinary tract symptoms were quantitatively evaluated in all patients with Parkinson's disease visiting this neurological clinic during 1 month. RESULTS: Of the 203 patients who had completed the questionnaire, 55 (27%) were considered to have symptomatic voiding dysfunction. The degree of lower urinary tract symptoms in these patients was well correlated with the severity of the disease rather than with the disease duration or the age. Thirty three (16%) patients had irritative symptoms alone, whereas three (1.5%) patients had obstructive symptoms alone. The irritative and obstructive symptoms were concomitant in 13 (6%) patients. Quality of life was disturbed by lower urinary tract symptoms, and this disturbance paralleled the severity of the disease. The influence of antiparkisonian drugs on the lower urinary tract symptoms was uncertain. The incidence of lower urinary tract symptoms seemed to be independent of sex, but obstructive symptoms were prevalent in male patients. CONCLUSIONS: This study suggests that voiding dysfunction in patients with Parkinson's disease progressively develops at advanced stages (> or =Hoehn and Yahr stage 3 of the disability). The International prostate symptom score is useful in evaluating the voiding dysfunction of neurodegenerative disease in both men and women, not only reflecting prostatic symptoms. PMID- 10727478 TI - What clinical features are most useful to distinguish definite multiple system atrophy from Parkinson's disease? AB - OBJECTIVES: Few studies have attempted to identify what premortem features best differentiate multiple system atrophy (MSA) from Parkinson's disease (PD). These studies are limited by small sample size, clinical heterogeneity, or lack of postmortem validation. We evaluated the sensitivity and specificity of different clinical features in distinguishing pathologically established MSA from PD. METHODS: One hundred consecutive cases of pathologically confirmed PD and 38 cases of pathologically confirmed MSA in one Parkinson's disease brain bank were included. All cases had their clinical notes reviewed by one observer (AH). Clinical features were divided into two groups: those occurring up to 5 years after onset of disease and those occurring up to death. Statistical analysis comprised multivariate logistic regression analysis to choose and weight key variables for the optimum predictive model. RESULTS: The selected early features and their weightings were: autonomic features (2), poor initial levodopa response (2), early motor fluctuations (2), and initial rigidity (2). A cut off of 4 or more on the ROC curve resulted in a sensitivity of 87.1% and specificity of 70.5%. A better predictive model occurred if the following features up to death were included: poor response to levodopa (2), autonomic features (2), speech or bulbar dysfunction (3), absence of dementia (2), absence of levodopa induced confusion (4), and falls (4). The resulting ROC curve based on individual scores showed a best cut off score of at least 11 of 17 (sensitivity 90.3%, specificity 92.6%). CONCLUSIONS: Predictive models may help differentiate MSA and PD premortem. Hitherto poorly recognised features, suggestive of MSA, included preserved cognitive function and absence of psychiatric effects from antiparkinsonian medication. Diagnostic accuracy was higher in those models taking into account all clinical features occurring up to death. Further studies need to be based on new incident cohorts of parkinsonian patients with subsequent neuropathological evaluation. PMID- 10727479 TI - Recovery from optic neuritis is associated with a change in the distribution of cerebral response to visual stimulation: a functional magnetic resonance imaging study. AB - OBJECTIVES: Recovery to normal or near normal visual acuity is usual after acute demyelinating optic neuritis, despite the frequent persistence of conduction abnormalities as evidenced by the visual evoked potential (VEP). This raises the possibility that cortical adaptation to a persistently abnormal input contributes to the recovery process. The objective of this study was to investigate the pattern of cerebral response to a simple visual stimulus in recovered patients in comparison to normal subjects. METHODS: Functional magnetic resonance imaging (fMRI) was used to study the brain activation pattern induced by a periodic monocular 8Hz photic stimulus in seven patients who had recovered from a single episode of acute unilateral optic neuritis, and in seven normal controls. VEPs and structural optic nerve MRI were performed on patients. RESULTS: Stimulation of either eye in controls activated only the occipital visual cortex. However, in patients, stimulation of the recovered eye also induced extensive activation in other areas including the insula-claustrum, lateral temporal and posterior parietal cortices, and thalamus; stimulation of the clinically unaffected eye activated visual cortex and right insula-claustrum only. The volume of extraoccipital activation in patients was strongly correlated with VEP latency (r = 0.71, p = 0.005). CONCLUSIONS: The extraoccipital areas that were activated in patients all have extensive visual connections, and some have been proposed as sites of multimodal sensory integration. The results indicate a functional reorganisation of the cerebral response to simple visual stimuli after optic neuritis that may represent an adaptive response to a persistently abnormal input. Whether this is a necessary part of the recovery process remains to be determined. PMID- 10727480 TI - Disability outcome measures in therapeutic trials of relapsing-remitting multiple sclerosis: effects of heterogeneity of disease course in placebo cohorts. AB - OBJECTIVES: Recent phase III clinical trials of immunomodulatory therapies in relapsing-remitting multiple sclerosis have shown significant benefits of active treatment on relapse related end points, but effects on disability outcomes have been inconsistent. These apparent discrepancies could be due to differences in the clinical end points employed, the behaviour of placebo cohorts, or both. METHODS: Disability data from the placebo cohorts of two large phase III studies, the United States glatiramer acetate trial (Copolymer 1 Multiple Sclerosis Study Group) and the multinational interferon beta-1a trial (PRISMS Study Group) were combined and masked (n = 313). Two groups of disability outcome measures were assessed. Firstly, measures of disability change (2 year EDSS difference and area under the EDSS/time curve, AUC) were calculated. Secondly, conventional disease progression end points ("confirmed progression" and "worsening to EDSS 6.0") were evaluated by using Kaplan-Meier analysis and compared with a categorical classification based on EDSS trends. RESULTS: The average increase in disability for the entire cohort as assessed by mean 2 year EDSS change (<0.5 EDSS point) or mean AUC (+0.57 EDSS-years) was small. For the "confirmed progression" end points, increasing the stringency of the definition lowered their incidence (from 32% with 1.0 point at 3 months, to 9% with 2.0 points at 6 months), but did not improve the positive predictive accuracy for "sustained progression" maintained to the end of the study. The error rate for this outcome was about 50%. Worsening to EDSS 6.0 was a more reliable end point, but had even lower sensitivity (incidence <10%). EDSS trend analysis showed markedly heterogeneous disease courses, which were then categorised into "stable" (26%), "relapsing-remitting" (59%), and "progressive" (15%) courses. Patients with the last course had deteriorated considerably by the end of 2 years (mean worsening of 2.0 EDSS points). CONCLUSION: In relapsing-remitting multiple sclerosis treatment trials, the conventional measure of mean EDSS change has low sensitivity, whereas the widely applied confirmed progression end points have high error rates regardless of their definition stringency. Alternative methods with better data utilisation include AUC summary measures and categorical disease trend analysis. The heterogeneity of disability outcomes in short trials, combined with unreliable clinical end points, diminishes the credibility of therapeutic claims aimed at reducing irreversible neurological deficits. The behaviour of patients treated with placebo should be carefully analysed before conclusions are drawn on the efficacy of putative treatments. PMID- 10727481 TI - Stepping before standing: hip muscle function in stepping and standing balance after stroke. AB - OBJECTIVE: To compare the pattern of pelvic girdle muscle activation in normal subjects and hemiparetic patients while stepping and maintaining standing balance. DESIGN: Group comparison. METHOD: Seventeen patients who had regained the ability to walk after a single hemiparetic stroke were studied together with 16 normal controls. Median interval between stroke and testing was 17 months. Amplitude and onset latency of surface EMG activity in hip abductors and adductors were recorded in response to sideways pushes in either direction while standing. Similar recordings were made in the same subjects during gait initiation and a single stride. RESULTS: In the standing balance task, normal subjects resisted a sideways push to the left with the left gluteus medius (74 ms) and with the right adductor (111 ms), and vice versa. In hemiparetic patients, the amplitude of activity was reduced in the hemiparetic muscles, the onset latencies of which were delayed (gluteus medius 96 ms, adductor 144 ms). Contralateral, non-paretic, adductor activity was increased after a push towards the hemiparetic side of patients with stroke and the latency was normal (110 ms). During self initiated sideways weight shifts at gait initiation, hemiplegic muscle activation was impaired. By contrast, the pattern and peak amplitude of hip muscle activation in stepping was normal in both hemiparetic and non hemiparetic muscles of the subjects with stroke. CONCLUSIONS: In ambulant patients with stroke, a normal pattern of activation of hemiparetic muscles is seen in stepping whereas the response of these muscles to a perturbation while standing remains grossly impaired and is compensated by increased activity of the contralateral muscles. This suggests that hemiparetic patients should be able to step before regaining standing balance. PMID- 10727482 TI - Pattern of premature degenerative changes of the cervical spine in patients with spasmodic torticollis and the impact on the outcome of selective peripheral denervation. AB - OBJECTIVES: To characterise the pattern of and risk factors for degenerative changes of the cervical spine in patients with spasmodic torticollis and to assess whether these changes affect outcome after selective peripheral denervation. METHODS: Preoperative CT of the upper cervical spine of 34 patients with spasmodic torticollis referred for surgery were reviewed by two radiologists blinded to the clinical findings. Degenerative changes were assessed for each joint separately and rated as absent, minimal, moderate, or severe. Patients were clinically assessed before surgery and 3 months postoperatively by an independent examiner using standardised clinical rating scales. For comparison of means a t test was carried out. To determine whether an association exists between the side of degenerative changes and type of spasmodic torticollis a chi(2) test was used. Changes in severity, disability, and pain before and after surgery were calculated using a Wilcoxon matched pairs signed ranks test. RESULTS: Fourteen out of 34 patients had moderate or severe degenerative changes. They were predominantly found at the C2/C3 and C3/C4 level and were significantly more likely to occur on the side of the main direction of the spasmodic torticollis (p = 0.015). There was no significant difference in age, sex, duration of torticollis, overall severity, degree of disability, or pain between the group with either no or minimal changes and the group with moderate or severe changes. However, in the second group the duration of inadequate treatment was longer (10.1 v 4.8 years; p=0.009), head mobility was more restricted (p = 0.015), and head tremor was more severe (p = 0.01). At 3 months postoperatively, patients with n or minimal degenerative changes showed a significant improvement in pain and severity whereas no difference was found in those with moderate or severe changes. CONCLUSIONS: Patients with spasmodic torticollis have an increased risk of developing premature degenerative changes of the upper cervical spine that tend to be on the side towards which the head is turned or tilted and compromise outcome after surgery. Effective early treatment of spasmodic torticollis with botulinum toxin seems to have a protective effect. Patients with spasmodic torticollis and restricted head mobility who do not adequately respond to treatment should undergo imaging of the upper cervical spine. Patients with imaging evidence of moderate or severe degenerative changes seem to respond poorly to selective peripheral denervation. PMID- 10727483 TI - Myopathy with anti-Jo-1 antibodies: pathology in perimysium and neighbouring muscle fibres. AB - OBJECTIVE: To evaluate muscle pathology and clinical characteristics in patients with a myopathy and serum antibodies to the Jo-1 antigen (histidyl t-RNA synthetase). BACKGROUND: Anti-Jo-1 antibodies occur in syndromes that may include muscle weakness and pain, Raynaud's phenomenon, interstitial lung disease, arthritis, and a skin rash different from that seen in dermatomyositis. The muscle pathology is not well defined. METHODS: Case series. Review of charts, muscle biopsies, and laboratory records. Features of myopathology in 11 patients with anti-Jo-1 antibody associated myopathies were compared with other types of inflammatory myopathies. RESULTS: Myopathology in patients with anti-Jo-1 antibodies consistently included fragmentation of, and macrophage predominant inflammation in, perimysial connective tissue. Perifascicular myopathic changes, including atrophy, regenerating muscle fibres, and some muscle fibre necrosis, were most common in regions near the connective tissue pathology and were most prominent in patients with more severe weakness. Unlike many other inflammatory myopathies, inflammation in endomysial and perivascular regions was uncommon. By contrast with dermatomyositis, capillary density was normal. CONCLUSIONS: Myopathological changes in the anti-Jo-1 antibody syndrome include perimysial connective tissue fragmentation and inflammation, with muscle fibre pathology in neighbouring perifascicular regions. Myositis with anti-Jo-1 antibodies may result from an immune mediated disorder of connective tissue. PMID- 10727484 TI - Treatment of paraneoplastic neurological syndromes with antineuronal antibodies (Anti-Hu, anti-Yo) with a combination of immunoglobulins, cyclophosphamide, and methylprednisolone. AB - OBJECTIVES: To evaluate the effect of a combination of immunoglobulins (IVIg), cyclophosphamide (CTX), and methylprednisolone (MP) on the clinical course of patients with paraneoplastic neurological syndrome (PNS) and antineuronal antibodies (Abs). METHODS: Seventeen patients with paraneoplastic encephalomyelitis/sensory neuropathy (PEM/SN) with anti-Hu Abs (n = 10) or cerebellar degeneration (PCD) with anti-Yo Abs (n = 7) received one to nine cycles (mean 3.5) of a combination of IVIg (0.5 g/kg/day from days 1 to 5), CTX (600 mg/m2 at day 1) and MP (1g/day from day 1 to 3). The Rankin scale (RS) was used to evaluate the response. A positive response was considered as either improvement or stabilisation in patients who were still ambulatory (RS< or =3) at the onset of treatment, whereas only improvement, and not stabilisation, was considered a therapeutic benefit in bedridden patients (RS> or =4). RESULTS: Tolerance was good and no patient experienced grade 3/4 toxicity (World Health Organisation). Sixteen patients were evaluable for response. Of the seven patients with RS> or =4, none improved. Of the nine patients with RS< or =3, none improved but three (two SN and one PCD) stabilised for 4, 35, and 16 months. CONCLUSIONS: This study suggests that vigorous immunosuppressive treatment is not useful in severely disabled PNS patients with antineuronal Abs. In a minority of patients (mainly with SN) who are not severely disabled at the onset of treatment, a transient stabilisation is possible and deserves further evaluation. PMID- 10727485 TI - Clinical syndromes associated with tomacula or myelin swellings in sural nerve biopsies. AB - OBJECTIVES: To describe the neuropathological features of clinical syndromes associated with tomacula or focal myelin swellings in sural nerve biospies and to discuss possible common aetiopathological pathways leading to their formation in this group of neuropathies. METHODS: Fifty two patients with sural nerve biopsies reported to show tomacula or focal myelin swellings were reviewed, light and electron microscopy were performed, and tomacula were analysed on teased fibre studies. Molecular genetic studies were performed on those patients who were available for genetic testing. RESULTS: Thirty seven patients were diagnosed with hereditary neuropathy with liability to pressure palsies (HNPP), four with hereditary motor and sensory neuropathy type I (HMSN I) or Charcot-Marie-Tooth disease type 1 (CMT1), four with HMSN with myelin outfolding (CMT4B), three with IgM paraproteinemic neuropathy, three with chronic inflammatory demyelinating polyneuropathy (CIDP), and one with HMSN III (CMT3). CONCLUSIONS: Most of these syndromes were shown to be related to genetic or immunological defects of myelin components such as peripheral myelin protein 22 (PMP22), myelin protein zero (P0), or myelin associated glycoprotein (MAG). These proteins share the HNK-1 epitope which has been implicated in cell adhesion processes. Impaired myelin maintenance may therefore contribute to the formation of tomacula and subsequent demyelination. PMID- 10727486 TI - Detection of intracranial aneurysms with unenhanced and echo contrast enhanced transcranial power Doppler. AB - OBJECTIVE: To assess transcranial power Doppler and the effect of an intravenous ultrasonic contrast agent for detecting intracranial aneurysms in patients admitted with a subarachnoid haemorrhage METHODS: 203 patients with subarachnoid haemorrhage were examined with the operator blind to the results of the admission CT and subsequent digital subtraction angiogram (DSA). In 98 cases patients were imaged using unenhanced transcranial power Doppler, and in 105 cases, patients were imaged both using unenhanced and echo contrast enhanced (Levovist, Schering AG) transcranial power Doppler. RESULTS: DSA detected 168 intracranial aneurysms in 128 patients (24 patients had multiple lesions) and 75 patients had an aneurysm negative DSA. There was agreement between DSA and transcranial power Doppler in 87 intracranial aneurysms of which 20 were only detected after infusion of contrast agent. Without contrast infusion, transcranial power Doppler showed a sensitivity of 40% and specificity of 91%. A significant increase in detection was noted when using a contrast agent (chi(2) = 9.49, p<0.001). With a contrast study the sensitivity increase to 55% with a specificity of 83%. Intracranial aneurysm detection by transcranial power Doppler was significantly dependent on position using a contrast infusion (chi(2) = 15.87, p<0.05). A positive correlation was also found between the size of intracranial aneurysms measured by transcranial power Doppler and DSA (r = 0.55, p<0.05). The transcranial power Doppler sensitivity and specificity increased with larger aneurysms and reached 100% for intracranial aneurysms greater than 12 mm diameter. Detection by transcranial power Doppler was also dependent on morphology (chi(2) = 14.46, p<0.001). CONCLUSIONS: The detection of intracranial aneurysm by transcranial power Doppler is dependent on aneurysm location, size, and morphology and is enhanced with the use of an intravenous contrast agent. PMID- 10727487 TI - Vasculitic neuropathy in a patient with inactive treated lepromatous leprosy. AB - A 46 year old Asian male with previously treated lepromatous leprosy developed a stepwise multifocal sensory disturbance 25 years later. Neurophysiology demonstrated marked deterioration from previous studies. Sural nerve biopsy disclosed a vasculitic process superimposed on inactive lepromatous leprosy. Immunocytochemical stains for mycobacterial antigen showed deposits within nerve and vessel walls. A delayed vasculitic neuropathy precipitated by persisting mycobacterial antigen is proposed. PMID- 10727488 TI - Diffusion tensor imaging demonstrates deviation of fibres in normal appearing white matter adjacent to a brain tumour. AB - The objective was to study fibre orientation in the cerebral white matter of a patient with a brain tumour using diffusion tensor imaging (DTI). A patient with a mild left hemiparesis and a tumour in the right frontal lobe and 20 healthy volunteers were scanned with a DTI sequence. The scans were spatially normalised and the fibre orientation in the patient compared with the fibre orientation in normal controls. DTI disclosed a change of the orientation of fibres in the patient compared with normal controls. In the normal appearing white matter adjacent to the tumour fibres deviated from the normal superior inferior orientation in the corona radiata by about 30 degrees. This finding was consistent with a displacement by distant mass effect rather than a destruction of fibres, in agreement with the neurological examination. In conclusion, DTI demonstrated a deviation of fibres in normal appearing white matter adjacent to a tumour. The technique will improve understanding of the effects of structural abnormalities on fibres. This will assist the interpretation of clinical findings and functional imaging studies and guide neurosurgical interventions. PMID- 10727489 TI - Clinical, electrophysiological, and molecular genetic studies in a new family with paramyotonia congenita. AB - OBJECTIVES: To characterise the clinical and electrophysiological features and to determine the molecular genetic basis of pure paramyotonia congenita in a previously unreported large Irish kindred. METHODS: Clinical and neurophysiological examination was performed on three of the five affected family members. Five unaffected and three affected members of the family were available for genetic testing. Direct sequence analysis of the SCN4A gene on chromosome 17q, was performed on the proband's DNA. Restriction fragment length polymorphism (RFLP) analysis was used to screen other family members and control chromosomes for the SCN4A mutation identified. RESULTS: Each affected member had clinical and examination features consistent with pure paramyotonia congenita. Electrophysiological studies disclosed a 78% drop in compound muscle action potential (CMAP) amplitude on cooling to 20 degrees C. DNA sequence analysis identified a heterozygous point mutation G4367A in exon 24 of the SCN4A gene which segregated with paramyotonia and was absent in 200 control chromosomes. The mutation is predicted to result in a radical amino acid substitution at a highly conserved position within the voltage sensing fourth transmembrane segment of the fourth repeated domain of the sodium channel. CONCLUSIONS: The G4367A mutation is likely to be pathogenic and it associates with a pure paramyotonia phenotype. In keeping with other paramyotonia mutations in this region of the skeletal muscle sodium channel, it is predicted that this mutation will impair voltage sensing or sodium channel fast inactivation in a temperature dependent fashion. This study provides further evidence that exon 24 in SCN4A is a hot spot for paramyotonia mutations and this has implications for a DNA based diagnostic service. PMID- 10727490 TI - An arachnoid cyst presenting as an intramedullary tumour. AB - A case of thoracic intradural extramedullary arachnoid cyst is presented in which an intramedullary low grade glioma was suspected preoperatively. The cyst was widely fenestrated and postoperatively, the patient experienced considerable improvement in her symptoms. As postoperative MRI studies also showed resolution of the intramedullary changes we regard the intramedullary changes as a result of the cyst, without the existence of primary medullary pathology. To our knowledge an arachnoid cyst, to date, has not been described as the cause of syringomyelia. As radiological findings can be misleading, extramedullary pathology, located more cranially, should be ruled out when treating cystic medullary changes. PMID- 10727491 TI - Illusory limb movements in anosognosia for hemiplegia. AB - To clarify the relation between anosognosia for hemiplegia and confabulation, 11 patients with acute right cerebral infarctions and left upper limb hemiparesis were assessed for anosognosia for hemiplegia, illusory limb movements (ILMs), hemispatial neglect, asomatognosia, and cognitive impairment. Five of 11 patients had unequivocal confabulation as evidenced by ILMs. The presence of ILMs was associated with the degree of anosognosia (p = 0.002), with hemispatial neglect (p<0.05), and with asomatognosia (p<0.01). The results confirm that a strong relation exists between anosognosia for hemiplegia and confabulations concerning the movement of the plegic limb. There is also a strong relation between ILMs and asomatognosia. PMID- 10727492 TI - Epidural compression of the cauda equina caused by vertebral osteoblastic metastasis of prostatic carcinoma: resolution by hormonal therapy. AB - A 59 year old man with prostatic carcinoma developed epidural compression of the cauda equina caused by bony expansion from a vertebral osteoblastic metastasis. For medical reasons he could not undergo radiation or surgery. Hormonal therapy alone relieved his low back pain and restored ambulation and urinary function. Postmyelography CT showed that the bony expansion from the vertebra had completely disappeared after treatment. This is the first report of remarkable improvement due to hormonal therapy alone. PMID- 10727493 TI - Modulation of single motor unit discharges using magnetic stimulation of the motor cortex in incomplete spinal cord injury. AB - OBJECTIVES: Motor evoked potentials (MEPs) and inhibition of voluntary contraction to transcranial magnetic stimulation (TMS) of the motor cortex have longer latencies than normal in patients with incomplete spinal cord injury (iSCI) when assessed using surface EMG. This study now examines the modulation of single motor unit discharges to TMS with the aim of improving resolution of the excitatory and inhibitory responses seen previously in surface EMG recordings. METHODS: A group of five patients with iSCI (motor level C4-C7) was compared with a group of five healthy control subjects. Single motor unit discharges were recorded with concentric needle electrodes from the first dorsal interosseus muscle during weak voluntary contraction (2%-5% maximum). TMS was applied with a 9 cm circular stimulating coil centred over the vertex. Modulation of single motor unit discharges was assessed using peristimulus time histograms (PSTHs). RESULTS: Mean (SEM) threshold (expressed as percentage of maximum stimulator output (%MSO)) for the excitatory peak (excitation) or inhibitory trough (inhibition) in the PSTHs was higher (p<0.05) in the patients (excitation = 47.1 (5.9) %MSO; inhibition = 44.3 (3.2) %MSO) than in controls (excitation=31.6 (1.2) %MSO; inhibition = 27.4 (1.0) %MSO). Mean latencies of excitation and inhibition were longer (p<0.05) in the patients (excitation=35 (1.8) ms; inhibition = 47.1 (1.8) ms) than in the controls (excitation = 21.1 (1.6) ms; inhibition = 27 (0.4) ms). Furthermore, the latency difference (inhibition-excitation) was longer (p<0.05) in the patients (10.4 (2.1) ms) than in the controls (6.2 (0.6) ms). CONCLUSION: Increased thresholds and latencies of excitation and inhibition may reflect degraded corticospinal transmission in the spinal cord. However, the relatively greater increase in the latency of inhibition compared with excitation in the patients with iSCI may reflect a weak or absent early component of cortical inhibition. Such a change in cortical inhibition may relate to the restoration of useful motor function after iSCI. PMID- 10727494 TI - Postmortem study of ataxia with retinitis pigmentosa by mutation of the alpha tocopherol transfer protein gene. AB - A new syndrome of ataxia and retinitis pigmentosa with vitamin E deficiency caused by the missense mutation of alpha-tocopherol transfer protein (alpha-TTP) gene was recently proposed. After studying the first postmortem case with this mutation pathologically and biochemically, whether the symptoms can be treated by supplementation of vitamin E or not is discussed. The major pathological findings were retinal atrophy; severe dying back-type degeneration of the posterior column; and massive accumulation of lipofuscin in neurons including dorsal root ganglion (DRG) cells, which were almost identical to those in vitamin E deficient animals and patients with fat malabsorption. Also, mild loss of Purkinje cells was noted. Because robust expression of alpha-TTP was detected in the cerebellum as well as in the liver and the tissue concentration of vitamin E in the cerebellum was still low even after oral supplementation, the mild Purkinje cell loss might be related to the mutant alpha-TTP in the cerebellum. By contrast, in the DRG, thought to be mainly responsible for ataxia, no expression of alpha-TTP was detected, and the tissue concentration of vitamin E increased to normal after supplementation. It is therefore considered that oral supplementation of vitamin E should effectively counteract the progression of ataxia. PMID- 10727495 TI - Reversal sign after cardiopulmonary arrest. PMID- 10727496 TI - Progressive dementia and gait disorder in a 78 year old woman. PMID- 10727498 TI - Neurosurgical treatment of movement disorders PMID- 10727497 TI - MRI of the brain. Normal anatomy and normal variants PMID- 10727500 TI - Russell and Rubinstein's pathology of tumors of the nervous system. Sixth edition PMID- 10727499 TI - Transcranial doppler ultrasonography PMID- 10727501 TI - Neurosurgical management of aneurysmal subarachnoid haemorrhage PMID- 10727502 TI - Headache PMID- 10727503 TI - Lecture notes on neurology. Seventh edition PMID- 10727505 TI - Fifty neurological cases from the national hospital PMID- 10727504 TI - Parkinson's disease: the treatment options PMID- 10727507 TI - An atlas of orbitocranial surgery PMID- 10727506 TI - Peripheral neuropathy in childhood. Second edition PMID- 10727508 TI - Editorial PMID- 10727509 TI - Protein kinases as mediators of phosphoinositide 3-kinase signaling. PMID- 10727510 TI - Regulation of the cellular localization and signaling properties of the alpha(1B) and alpha(1D)-adrenoceptors by agonists and inverse agonists. AB - The regulation of the cellular distribution and intracellular signaling properties of the alpha(1B)- and alpha(1D)- adrenoceptor (alpha(1)-AR) subtypes was examined in stably transfected Rat 1 fibroblasts. In unstimulated cells, alpha(1B)-AR expression was noted primarily on the cell surface. Treatment with phenylephrine induced internalization of the alpha(1B)-AR and promoted association with arrestin 2. The internalized alpha(1B)-AR colocalized with the transferrin receptor, an endosomal marker. In unstimulated fibroblasts, the alpha(1D)-AR was detected in a perinuclear orientation and was colocalized with arrestin 2 in a compartment also containing the transferrin receptor. After treatment with prazosin, which exhibits inverse agonist properties, the alpha(1D) AR was redistributed from intracellular sites to the cellular periphery and was no longer associated with the transferrin receptor or arrestin 2. alpha(1D)-AR expressing cells exhibited a high degree of basal activity for both inositol phosphate formation and extracellular signal regulated kinase (ERK), which was reduced by treatment with prazosin. In these cells, phenylephrine induced a dose dependent increase in inositol phosphate formation but had no effect on ERK activity. In alpha(1B) -AR-expressing cells, phenylephrine stimulated both inositol phosphate formation and ERK activity. These data show that: 1) there are differences in the cellular localization of the alpha(1)-AR subtypes; 2) the alpha(1B)-AR exhibits expected G protein-coupled receptor activity regarding cellular localization, agonist-mediated internalization, and coupling to second messengers; and 3) the alpha(1D)-AR is constitutively active and, as a result, is localized to intracellular compartments involved in receptor recycling. PMID- 10727511 TI - Epidermal growth factor receptor agonists increase expression of glutamate transporter GLT-1 in astrocytes through pathways dependent on phosphatidylinositol 3-kinase and transcription factor NF-kappaB. AB - The glial glutamate transporter GLT-1 may be the predominant Na(+)-dependent glutamate transporter in forebrain. Expression of GLT-1 correlates with astrocyte maturation in vivo and increases during synaptogenesis. In astrocyte cultures, GLT-1 expression parallels differentiation induced by cAMP analogs or by coculturing with neurons. Molecule(s) secreted by neuronal cultures contribute to this induction of GLT-1, but little is known about the signaling pathways mediating this regulation. In the present study, we determined whether growth factors previously implicated in astrocyte differentiation regulate GLT-1 expression. Of the six growth factors tested, two [epidermal growth factor (EGF) and transforming growth factor-alpha] induced expression of GLT-1 protein in cultured astrocytes. Induction of GLT-1 protein was accompanied by an increase in mRNA and in the V(max) for Na(+)-dependent glutamate transport activity. The effects of dibutyryl-cAMP and EGF were additive but were independently blocked by inhibitors of protein kinase A or protein tyrosine kinases, respectively. The induction of GLT-1 in both EGF- and dibutyryl-cAMP-treated astrocytes was blocked by inhibitors targeting phosphatidylinositol 3-kinase (PI3K) or the nuclear transcription factor-kappaB. Furthermore, transient transfection of astrocyte cultures with a constitutively active PI3K construct was sufficient to induce expression of GLT-1. These data suggest that independent but converging pathways mediate expression of GLT-1. Although an EGF receptor-specific antagonist did not block the effects of neuron-conditioned medium, the induction of GLT-1 by neuron conditioned medium was completely abolished by inhibition of PI3K or nuclear factor-kappaB. EGF also increased expression of GLT-1 in spinal cord organotypic cultures. Together, these data suggest that activation of specific signaling pathways with EGF-like molecules may provide a novel approach for limiting excitotoxic brain injury. PMID- 10727512 TI - New advances in the transport of doxorubicin through the blood-brain barrier by a peptide vector-mediated strategy. AB - Many therapeutic drugs are excluded from entering the brain, due to their lack of transport through the blood-brain barrier (BBB). To overcome this problem, we have developed a novel method in which short, naturally derived peptides (16-18 amino acids) cross the cellular membranes of the BBB with high efficiency and without compromising its integrity. The antineoplastic agent doxorubicin (dox) was coupled covalently to two peptides, D-penetratin and SynB1. The ability of dox to cross the BBB was studied using an in situ rat brain perfusion technique and also by i.v. injection in mice. In the brain perfusion studies, we first confirmed the very low brain uptake of free radiolabeled dox because of the efflux activity of P-glycoprotein at the BBB. By contrast, we have demonstrated that when dox is coupled to either the D-penetratin or SynB1 vectors, its uptake was increased by a factor of 6, suggesting that the vectorized dox bypasses P glycoprotein. Moreover, using a capillary depletion method, we have shown that vectorization of dox led to a 20-fold increase in the amount of dox transported into brain parenchyma. Intravenous administration of vectorized dox at a dose of 2.5 mg/kg in mice led to a significant increase in brain dox concentrations during the first 30 min of postadministration, compared with free dox. Additionally, vectorization led to a significant decrease of dox concentrations in the heart. In summary, our results establish that the two peptide vectors used in this study enhance the delivery of dox across the BBB. PMID- 10727513 TI - Identification of distinct carboxyl-terminal domains mediating internalization and down-regulation of the hamster alpha(1B)- adrenergic receptor. AB - The roles of the carboxyl-terminal tail of the alpha(1B)-adrenergic receptor in its expression, function, and regulation were investigated by site-directed mutagenesis. The receptor construct truncated after residue 363 seemed not to be properly expressed. In contrast, the receptor truncated after residue 366 and all of the longer receptor constructs were properly expressed and exhibited agonist and antagonist binding and activation of phosphoinositide hydrolysis similar to the wild-type receptor. Agonist-induced sequestration of receptors within the plasma membrane, endocytosis into intracellular vesicles, and eventual down regulation were all absent in the receptor truncated after residue 366. A series of sequential truncations and a deletion mutation identified a critical role for residues 403 to 425, which include the previously identified sites for G protein coupled receptor kinase phosphorylation, in agonist-induced internalization of the receptor. Similar studies identified a critical role for residues 367 to 380 in agonist-induced down-regulation. Individual point mutations converting either cysteine 367 or serine 369 to alanine selectively eliminated down-regulation, thus identifying two specific amino acid residues required for down-regulation. Importantly, several of the mutated receptors that failed to show rapid agonist induced internalization nonetheless exhibited normal agonist-induced down regulation. In addition to identifying specific regions and individual residues of the alpha(1B)-adrenergic receptor involved in internalization and down regulation, these studies provide mutated receptors that internalize but do not down-regulate, that down-regulate without internalization, and that are defective in both internalization and down-regulation, all of which should be useful tools for further studies of the specific cellular compartments and molecular mechanisms involved in receptor internalization and down-regulation. PMID- 10727514 TI - Interaction of deoxyguanosine nucleotide analogs with human telomerase. AB - To maintain the telomeres at the ends of the chromosomes, telomerase in human cells adds a repeating sequence of nucleotides (TTAGGG) to the 3'-end of each chromosome using an RNA component of the enzyme as the template for DNA synthesis. Because of the selective expression of this enzyme in cancer cells, we have evaluated the interaction of human telomerase with several deoxyguanosine nucleotides of clinical importance. 2',3'-dideoxyguanosine 5'-triphosphate, 6 thio-2'-deoxyguanosine 5'-triphosphate (T-dGTP), carbovir 5'-triphosphate, and D carbocyclic-2'-deoxyguanosine 5'-triphosphate (D-CdG-TP) inhibited telomerase activity by 50% when these analogs were present at only 2 to 9 times the dGTP concentration. The L-enantiomer of CdG-TP was far less inhibitory, thereby demonstrating the stereoselectivity of telomerase for nucleotide substrates. T dGTP was incorporated into the DNA by telomerase in the absence of dGTP, but unlike dGTP there was little extension of the DNA chain after its incorporation. These results indicate that the metabolites of three clinically useful agents (6 mercaptopurine, 6-thioguanine, and Abacavir) can inhibit human telomerase activity, and it is possible that the effect of these nucleotides on telomerase activity or telomere function could contribute to the mechanism of action of these agents. PMID- 10727515 TI - Preactivation permits subsequent stimulation of phospholipase C by G(i)-coupled receptors. AB - In the complex signal transduction networks involving G protein-coupled receptors there are numerous examples where G(i)-linked receptors augment G(q)-dependent signals. The mechanistic basis of such occurrences is thought to entail signal convergence at phospholipase Cbeta (PLCbeta) via the G protein betagamma-dimers. Herein, we explored the possibility that augmentation by betagamma-dimers requires preactivation of PLCbeta. COS-7 cells were transiently cotransfected with cDNAs encoding various combinations of receptors and G protein subunits. The G(i)-coupled delta- and kappa-opioid receptors could not stimulate PLCbeta unless they were coexpressed with Galpha(16). The opioid-induced response was dose dependent and partially inhibited by pertussis toxin or coexpression with transducin, indicating the involvement of betagamma-subunits released from the G(i) proteins. When PLCbeta was preactivated by constitutively active mutants of Galpha(16), Galpha(q), or Galpha(14), opioids enhanced the activity by 80 to 300% and such responses were mostly pertussis toxin-sensitive. The opioid-induced enhancement was dose-dependent and could not be blocked by staurosporin, a protein kinase C inhibitor. Other G(i)-coupled receptors that were ineffective on their own also acquired the ability to stimulate PLCbeta in the presence of a constitutively active mutant of Galpha(q). Coactivation of endogenous or exogenous G(q)-coupled receptors with the delta-opioid receptor produced strong stimulations of PLCbeta and such responses could be partially blocked by pertussis toxin. These results show that enhancement of G(q)-dependent signals by G(i)-coupled receptors requires activated PLCbeta and is mediated via the betagamma-dimer. PMID- 10727516 TI - Nitric oxide up-regulates aldose reductase expression in rat vascular smooth muscle cells: a potential role for aldose reductase in vascular remodeling. AB - Acceleration of the polyol pathway under hyperglycemia is among the mechanisms implicated in the pathogenesis of diabetic complications. Although aldose reductase (AR), the rate-limiting enzyme in this pathway, is a target for pharmacological intervention of diabetic complications, the clinical efficacy of AR inhibitors has not been consistently proved. Because nitric oxide (NO) plays important roles in vascular hemodynamics and inflammatory responses that are affected under diabetic conditions, the interaction of NO with AR was investigated with rat aortic smooth muscle cells. Spontaneous NO donors, S nitroso-N-acetylpenicillamine (SNAP) and 3-(2-hydroxy-1-methyl-2 nitrosohydrazino)-N-methyl-1-propanamin e, elicited a dose-dependent increase in AR mRNA to a maximum of 7-fold in 12 h. The activity of AR was elevated after 10 h of SNAP treatment. These effects of NO donors were suppressed by the addition of 2-(trimethylammoniophenyl)-4,4,5, 5-tetramethylimidazoline-1-oxy 3-oxide, a scavenger of NO. Induction of AR mRNA by SNAP was completely abolished by actinomycin D or cycloheximide, but unaffected by guanylate cyclase inhibitors or genistein, a tyrosine kinase inhibitor. Pretreatment of the cells with N-acetyl-L cysteine significantly suppressed the SNAP-induced up-regulation of AR mRNA. Under normal glucose conditions, inclusion of the AR inhibitor ponalrestat augmented the cytotoxic effect of SNAP on the cells. The level of AR mRNA also was elevated in a murine macrophage cell line RAW 264.7 stimulated with lipopolysaccharide and interferon-gamma. Inhibition of NO synthesis completely abolished the increase in AR mRNA in the stimulated cells. The up-regulation of AR by NO in the vascular lesions may modulate NO-induced cell death and the ensuing vascular remodeling during inflammatory responses. PMID- 10727517 TI - Hexapeptide and cyclic pentapeptide endothelin antagonists directly activate pituitary gonadotropin-releasing hormone receptors. AB - In the course of our studies toward the development of novel analogs of the decapeptide gonadotropin releasing hormone (GnRH), we have examined a hexapeptide that is an antagonist of endothelin (ET). It was found that this peptide, Ac-D Trp-Leu-Asp-Ile-Ile-Trp (peptide 1), binds specifically to the pituitary GnRH receptor. Moreover, peptide 1 exhibits a GnRH agonistic activity (i.e., it induces luteinizing hormone release from rat pituitary). This activity is mediated directly by the GnRH receptor and is suppressed by a GnRH antagonist. Removal of the acetyl group of peptide 1 results in a hexapeptide (peptide 2) with binding properties similar to those of GnRH but with a diminished affinity toward the ET receptor. Several other ET antagonists were screened for a potential interaction with the GnRH receptor. Two of these, the hexapeptide PD145065 and the cyclic pentapeptide BQ-123, expressed GnRH agonistic activity at micromolar concentrations in vitro. BQ-123, previously approved for trials on humans as an ET antagonist, is demonstrated to act in vivo as a GnRH agonist, in a dose that was demonstrated previously as the minimal required dose for significant ET antagonism. The GnRH agonistic activity of ET antagonists may therefore result in interference with the physiological control of the reproductive system. Such effects may be most severe when ET antagonists are used chronically. Thus, the major practical message of this study is the need to circumvent the potential side effects of ET antagonist-based drugs. PMID- 10727518 TI - Antisense inhibition of delta-opioid receptor gene function in vivo by peptide nucleic acids. AB - Peptide nucleic acids (PNA) are synthetic analogs of DNA that hybridize to complementary oligonucleotide sequences with exceptional affinity and target specificity. The stability of PNA in biological fluids together with the unique hybridization characteristics of these structures suggests that PNA may have considerable potential as antisense agents for experimental use in vivo. To test this hypothesis, we attempted to modulate supraspinal delta-opioid receptor function in rats using PNA sequences designed to be complementary to a region of the rat delta-opioid receptor. Repeated i.c.v. administration of PNA over a period of 5 days significantly inhibited the antinociceptive response and locomotor response to selective delta-opioid receptor agonists. PNA attenuated delta-opioid receptor function in a sequence-specific, target-specific, and reversible manner characteristic of the functional inhibition caused by an antisense mechanism. There were no apparent toxicities arising from the PNA treatment based on the behavior of the animals and inspection of the treated tissues. Saturation binding studies on brain homogenates did not reveal any significant difference in receptor B(max) between treatment groups. However, [(35)S]guanosine-5'-O-(3-thio)triphosphate binding assays demonstrated a significant decrease in agonist efficacy in homogenates prepared from antisense treated rats. Taken together, these results demonstrate that peptide nucleic acids are effective antisense agents in vivo and suggest that PNA may be a useful alternative to phosphodiester or phosphorothioate oligonucleotides, or variants thereof, for determination of gene function in vivo. PMID- 10727519 TI - Rifampicin is not an activator of glucocorticoid receptor. AB - Rifampicin, an antibiotic widely used in tuberculosis therapy, is known to exert psychotropic side effects in some patients. Recently, rifampicin has been reported to activate the glucocorticoid receptor (GR) in human hepatocytes. Because there is evidence that increased levels of glucocorticoids may induce cognitive impairment, sometimes culminating in depression, the side effects of rifampicin may result from GR activation in central nerve cells. Therefore, we used reporter gene assays to determine whether rifampicin displays glucocorticoid like effects in human neuroblastoma SK-N-MC cells or mouse hippocampal HT22 cells. Rifampicin was unable to elicit any detectable transactivation of GR in both cell types, whereas cortisol or dexamethasone led to a potent transcriptional response. Rifampicin was also inactive in the same HepG2 cell line that was originally used to demonstrate the effect of rifampicin on GR. Moreover, rifampicin was unable to compete with dexamethasone for binding to GR. Finally, by blocking the multidrug resistance P-glycoprotein transporter (a xenobiotic extrusion pump) with verapamil or cyclosporin A, we excluded the possibility that the lack of effect by rifampicin was due to its export from the cell. Our results establish that rifampicin does not activate GR, and rule out the hypothesis that the psychotropic side effects of rifampicin treatment are a consequence of GR activation. PMID- 10727520 TI - Differential inhibition of multiple cAMP phosphodiesterase isozymes by isoflavones and tyrphostins. AB - A series of isoflavone and tyrphostin compounds were found to inhibit the degradation of cAMP by several cyclic nucleotide phosphodiesterase (PDE) isozymes. Specific hydroxyl groups on the isoflavone structure were critical for PDE isozyme-selective inhibition. Replacement of the C-7 hydroxyl group of the isoflavone with a methoxy group raised the IC(50) for PDE1, PDE3, and PDE4. The absence of the C-5 hydroxyl group raised the IC(50) from 5 to >100 microM for PDE4, but actually lowered the IC(50) for PDE3 and PDE1. Replacement of the C-4' hydroxyl group with a methoxy group raised the IC(50) for PDE3 and PDE1, yet only slightly changed the IC(50) for PDE4. Various tyrphostins were also potent inhibitors of PDE1, PDE3, and PDE4. The four-carbon side chained tyrphostins were much less potent; however, a very interesting pattern was observed in which removal of phenolic hydroxyls on the tyrphostin structure increased the potency for PDE1 and PDE3, but not PDE4. These results may help to explain some of the therapeutic and intracellular signaling effects of isoflavones and tyrphostins. Moreover, the isozyme selectivity demonstrated by the isoflavones and tyrphostins can serve as a pharmacophore for the design of specific PDE inhibitors. PMID- 10727521 TI - Differential superactivation of adenylyl cyclase isozymes after chronic activation of the CB(1) cannabinoid receptor. AB - Many types of cells exhibit increased adenylyl cyclase (AC) activity after chronic agonist treatment of G(i/o)-coupled receptors. This phenomenon, defined as AC superactivation or sensitization, has mostly been studied for the opioid receptors and is implicated in opiate addiction. Here we show that this phenomenon is also observed on chronic activation of the CB(1) cannabinoid receptor. Moreover, using COS-7 cells cotransfected with CB(1) receptor and individual AC isozymes, we could show selective superactivation of AC types I, III, V, VI, and VIII. The level of superactivation was dependent on the concentration of agonist and time of agonist exposure and was not dependent on the AC stimulator used. No superactivation of AC types II, IV, or VII was observed in COS-7 cells cotransfected with CB(1). The superactivation of AC type V was abolished by pretreatment with pertussis toxin and by cotransfection with the carboxy terminus of beta-adrenergic receptor kinase, which serves as a scavenger of G(betagamma) dimers, implying a role for the G(i/o) proteins and especially G(betagamma) dimers in the cannabinoid-induced superactivation of AC. PMID- 10727522 TI - Molecular cloning and characterization of a lysophosphatidic acid receptor, Edg 7, expressed in prostate. AB - Two G protein-coupled receptors (Edg-2) and (Edg-4) for the lysolipid phosphoric acid mediator lysophosphatidic acid have been described by molecular cloning. However, the calcium-mobilizing receptor Edg-4 is not expressed in some cell lines that exhibit robust calcium responses to this ligand, thus predicting the existence of additional receptor subtypes. We report here on the characterization of a third human lysophosphatidic acid receptor subtype, Edg-7, which mediates lysophosphatidic acid-evoked calcium mobilization. In a rat hepatoma Rh7777 cell line that lacks endogenous responses to lysophosphatidic acid, this lipid mediator, but not others, evokes calcium transients when the cells have been transfected with Edg-7 or Edg-4 DNAs. Furthermore, frog oocytes exhibit a calcium mediated chloride conductance in response to mammalian-selective lysophosphatidic acid mimetics after injection of Edg-7 mRNA. Edg-7-expressing Rh7777 cells do not show inhibition of forskolin-driven rises in cAMP in response to lysophosphatidic acid. However, membranes from HEK293T cells cotransfected with Edg-7 and G(i2)alpha protein DNAs show lysophosphatidic acid dose-dependent increases in [gamma-(35)S]GTP binding with an EC(50) value of 195 nM. When we used this assay to compare various synthetic LPA analogs at Edg-2, Edg-4, and Edg-7 receptors, we found that ethanolamine-based compounds, which are full LPA mimetics at Edg-2 and Edg-4, exhibit little activity at the Edg-7 receptor. Edg-7 RNA was detected in extracts of several rat and human tissues including prostate. Together, our data indicate that Edg-7 is a third lysophosphatidic acid receptor that couples predominantly to G(q/11)alpha proteins. PMID- 10727523 TI - Interactions of the human multidrug resistance proteins MRP1 and MRP2 with organic anions. AB - The human multidrug resistance protein MRP1 and its homolog, MRP2, are both suggested as being involved in cancer drug resistance and the transport of organic anions. We expressed MRP1 and MRP2 in Spodoptera frugiperda ovarian cells and compared their ATP-dependent transport properties and vanadate-sensitive ATPase activities in isolated membrane vesicles. Both MRP1 and MRP2 actively transported leukotriene C(4) and N-ethylmaleimide glutathione (NEM-GS), although the relative affinity of MRP2 for these substrates was found to be significantly lower than that of MRP1. Methotrexate was actively transported by both proteins, although more efficiently by MRP2. ATP-dependent NEM-GS transport by MRP1 and MRP2 was variably modulated by organic anions. Probenecid and furosemide inhibited, whereas under certain conditions sulfinpyrazone, penicillin G, and indomethacin greatly stimulated, MRP2-mediated NEM-GS uptake. Vanadate-sensitive ATPase activity in isolated membranes containing MRP1 or MRP2 was significantly stimulated by NEM-GS and reduced GS, although these compounds acted only at higher concentrations in MRP2. ATP hydrolysis by MRP2 was also effectively stimulated by methotrexate. Probenecid, sulfinpyrazone, indomethacin, furosemide, and penicillin G all significantly increased MRP2-ATPase activity, whereas these compounds acted more as ATPase inhibitors on MRP1. These results indicate that MRP1 is a more efficient transporter of glutathione conjugates and free glutathione than MRP2, whereas several anions are preferred substrates for MRP2. Our data suggest that MRP2 may be responsible for the active secretion of pharmacologically relevant organic anions, such as diuretics and antibiotics, and indicate different modulation possibilities for MRP1 or MRP2 in drug-resistant tumor cells. PMID- 10727524 TI - Loss of cyclosporin and azidopine binding are associated with altered ATPase activity by a mutant P-glycoprotein with deleted phe(335). AB - In this study, we further characterize a mutant P-glycoprotein (P-gp) that has a deletion of Phe(335) and is resistant to inhibition by cyclosporins. Photoaffinity labeling with [(3)H]cyclosporine and [(3)H]azidopine revealed markedly decreased binding to the mutant P-gp compared with wild-type P-gp. Expression of the mutant P-gp in multidrug-resistant variant cell line MES-SA/DxP (DxP) cells was associated with a 2-fold higher basal ATPase activity relative to multidrug-resistant cell line MES-SA/Dx5 (Dx5) cells with wild-type P-gp. Cyclosporine inhibited ATPase activity in both cell types, whereas the cyclosporin D analog valspodar (PSC 833), vinblastine, and dactinomycin stimulated ATPase activity in Dx5 but not in mutant DxP cells. Moreover, the cell lines differed in their responses to verapamil, which produced greater stimulation of ATPase in Dx5 than DxP cells. Verapamil significantly reversed the [(3)H]daunorubicin accumulation defect in wild-type Dx5 cells, but it had no significant effect on [(3)H]daunorubicin accumulation in the mutant DxP cells. Verapamil was not transported by cells expressing either mutant or wild-type P gp. Vanadate trapping of azido-ATP was markedly impaired in mutant P-gp. In conclusion, our data demonstrate that Phe(335) of transmembrane 6 is an important amino acid residue for the formation of cyclosporine and azidopine drug-binding site(s). Phe(335) also plays a role in the coupling of verapamil binding and modulation of daunorubicin intracellular accumulation in wild-type P-gp. In addition, Phe(335) in transmembrane 6 may play a role in coupling drug binding to ATPase activity. The deletion of Phe(335) results in a significant increase in the basal ATPase activity with a concomitant decrease in its ability to trap ATP and transport some P-gp substrates. PMID- 10727525 TI - Agonist-dependent modulation of G protein-coupled receptor kinase 2 by mitogen activated protein kinases. AB - A variety of G protein-coupled receptors (GPCRs) are phosphorylated by G protein coupled receptor kinase 2 (GRK2). This event promotes the binding of regulatory proteins termed beta-arrestins to GPCRs, leading to uncoupling from G proteins and receptor internalization. Recent data indicate that GRK2 and beta-arrestins also play an important role in the stimulation of the extracellular signal regulated kinases (ERK)/mitogen-activated protein kinase (MAPK) cascade by GPCRs. In this report, we have investigated the existence of functional interactions between GRK2 and MAPK. We show that activation of beta(2)-adrenergic receptors (beta(2)-AR) promotes the rapid association of GRK2 and MAPK in living cells, as assessed by coimmunoprecipitation experiments in COS-7 cells transfected with beta(2)-AR, GRK2, and an epitope-tagged MAPK. Coimmunoprecipitation of MAPK and GRK2 is blocked by inhibition of the MAPK cascade and is not observed upon activation of MAPK in the absence of beta(2)-AR stimulation, thus indicating that both an active MAPK and agonist occupancy of GPCR are required for the association to occur. Interestingly, we have found that purified ERK1/MAPK can directly phosphorylate the C-terminal domain of GRK2, and that the phosphorylation process is favored by the presence of Gbetagamma-subunits or an activated receptor. Furthermore, GRK2 phosphorylation by MAPK leads to a decreased activity of GRK2 toward GPCR. Taken together, our results suggest that stimulation of GPCRs promotes the rapid association of GRK2 and MAPK leading to modulation of GRK2 functionality, thus putting forward a new feedback mechanism for the regulation of GPCR signaling. PMID- 10727526 TI - Mutations at arg486 and glu571 in human topoisomerase IIalpha confer resistance to amsacrine: relevance for antitumor drug resistance in human cells. AB - Human topoisomerase II, a nuclear protein involved in chromosome segregation, is the target of amsacrine and other clinically important anticancer drugs. The enzyme is expressed as alpha and beta isoforms whose mutation/down-regulation has been implicated in drug resistance. To understand the role of target mutations in cellular drug resistance, we have used yeast to select and characterize plasmid borne human topoisomerase IIalpha mutants resistant to amsacrine. Single point changes of Glu571 to Lys (E571K) or Arg486 to Lys (R486K) in the conserved PLRGK motif, both of which reside in the GyrB homology domain of human topoisomerase IIalpha, were frequently selected and could be shown in vivo to confer >25-fold and >100-fold resistance, respectively, to amsacrine and approximately 3-fold cross-resistance to etoposide. Highly purified E571K and R486K human topoisomerase IIalpha proteins required 100-fold higher levels of amsacrine to induce DNA cleavage similar to that of wild-type protein, consistent with a resistance mechanism involving reduced cleavable complex formation. Our functional studies of the R486K mutation, previously identified in two amsacrine resistant human cell lines and in human biopsy material, establish unequivocally that it confers resistance, and suggest mechanisms for its phenotypic expression in vivo. These results differ significantly from previous work using yeast topoisomerase II as a model system: introduction of the equivalent mutation to R486K (R476K) into the yeast enzyme did not give amsacrine resistance. We conclude that species-specific differences in topoisomerase II enzymes can affect the drug resistance phenotype of particular mutations and highlight the need to study the relevant human homolog. PMID- 10727527 TI - Down-regulation of ceramide production abrogates ionizing radiation-induced cytochrome c release and apoptosis. AB - Previous work has demonstrated that down-regulation of ceramide production after selection of cells with N-oleoylethanolamine (OE), an inhibitor of ceramidase, results in resistance to DNA damage-induced apoptosis. We report here that acute exposure of WEHI-231 cells (murine B-cell lymphoma) to OE activates neutral sphingomyelinase, induces ceramide production and increases intracellular reactive oxygen species. OE exposure also induces mitochondrial permeability, cytochrome c release, and apoptosis. Cells selected for resistance to OE exhibit little if any change in reactive oxygen species and cytochrome c release when exposed either to OE or to toxic doses of ceramide. Importantly, the OE resistant cells are also resistant to ionizing radiation-induced cytochrome c release and apoptosis. These findings demonstrate that down-regulation of neutral sphingomyelinase activity is associated with decreased DNA-damage-induced apoptosis. In addition, the data suggests that agents that modify extranuclear targets responsible for ceramide production select for cells resistant to ionizing radiation-induced apoptosis through alterations in mitochondrial function. PMID- 10727528 TI - Aspirin and sodium salicylate inhibit endothelin ETA receptors by an allosteric type of mechanism. AB - Aspirin is a commonly used drug with a wide pharmacological spectrum including antiplatelet, anti-inflammatory, and neuroprotective actions. This study shows that aspirin and sodium salicylate, its major blood metabolite, reverse contractile actions of endothelin-1 (ET-1) in isolated rat aorta and human mammary arteries. They also prevent the intracellular Ca(2+) mobilizing action of ET-1 in cultured endothelial cells but not those of neuromedin B or UTP. Inhibition of the actions of ET-1 by salicylates is apparently competitive. Salicylates inhibit (125)I-ET-1 binding to recombinant rat ETA receptors. Salicylic acid promotes dissociation of (125)I-ET-1 ETA receptor complexes both in the absence and the presence of unlabeled ET-1. It has no influence on the rate of association of (125)I-ET-1 to ETA receptors. Salicylates do not promote dissociation of (125)I-ET-1 ETB receptor complexes. Salicylates potentiate relaxing actions of receptor antagonists such as bosentan. It is concluded that salicylates are allosteric inhibitors of ETA receptors. The results also suggest that: 1) irreversible ET-1 binding probably limits actions of receptor antagonists in vivo, and 2) an association of salicylates and ETA receptor antagonists should be used to evaluate the physiopathological role of ET-1 and may be of therapeutic interest in the treatment of ischemic heart disease. PMID- 10727529 TI - A molecularly identified P2Y receptor simultaneously activates phospholipase C and inhibits adenylyl cyclase and is nonselectively activated by all nucleoside triphosphates. AB - We recently cloned and expressed a novel P2Y receptor (tp2y receptor) from a turkey cDNA library. Expression of this receptor in 1321N1 human astrocytoma cells confers nucleotide-dependent stimulation of phospholipase C activity; however, as we demonstrate here, it also confers nucleotide-dependent inhibition of adenylyl cyclase. Both the phospholipase C and adenylyl cyclase responses were promoted by receptor agonists over a similar range of concentrations. Moreover, not only did UTP and ATP activate the avian receptor but ITP, GTP, xanthosine 5' triphosphate, and CTP were also agonists, with EC(50) values ranging between 0.1 and 1 microM. Similar potencies, rank-order, and selectivity of nucleotide agonists were also demonstrated for intracellular Ca(2+) mobilization measured during a 30-s stimulation under constant superfusion conditions. This observation indicates that receptor activation by nucleoside 5'-triphosphates is not produced by interconversion of these nucleotides into ATP or UTP. Pretreatment of cells with pertussis toxin completely abolished the inhibitory effect of nucleotide agonists on adenylyl cyclase, whereas the activation of phospholipase C was only partially inhibited. These results demonstrate that the avian P2Y receptor is a nucleoside triphosphate receptor of broad agonist selectivity that interacts with both pertussis toxin-insensitive and -sensitive G proteins to activate phospholipase C and to inhibit adenylyl cyclase. This is the first cloned P2Y receptor that is clearly Gi/adenylyl cyclase-linked. PMID- 10727530 TI - The multifunctional deoxynucleoside kinase of insect cells is a target for the development of new insecticides. AB - The antiherpetic agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was found to be an efficient substrate for recombinant Drosophila melanogaster deoxyribonucleoside kinase with a K(m) of 4.5 microM and a V(max) of 400 nmol/microg protein/h compared with 1.3 microM and 62.5 nmol/microg protein/h, respectively, for the natural substrate thymidine. Mammalian cytosolic thymidine kinase-1 does not recognize BVDU as a substrate. In sharp contrast to mammalian cells, the insect D. melanogaster and Spodoptera frugiperda (Sf) embryonic cells proved highly sensitive to the cytostatic action of BVDU. BVDU was efficiently metabolized to its 5'-mono-, 5'-di- and 5'-triphosphate derivatives in the insect cell cultures and abundantly incorporated into the insect cell DNA. BVDU prevented the D. melanogaster cells to initiate the S phase of their cell cycle, and exposure of S. frugiperda cells to BVDU led to a dose-dependent retardation of the insect cells in the S phase of their cell cycle. Both inhibition of nucleic acid synthesis (through the 5'-triphosphate of BVDU) and inhibition of thymidylate synthase (through the 5'-monophosphate of BVDU) would account for the cytostatic activity of BVDU against the insect cells. Because of the virtual lack of cytotoxicity of BVDU against mammalian cells, the drug should be considered highly selective in its cytostatic action against the insect cells. When added to the food of S. frugiperda larvae, BVDU caused a remarkable decrease in the weight gain of the larvae and heavily compromised the transformation of the larvae to the pupae and their subsequent adult (moth) phase. Our data indicate that insect multifunctional deoxyribonucleoside kinase should be considered an entirely novel and attractive target in the development of new nucleoside types of highly selective insecticidal drugs. PMID- 10727532 TI - Selective regulation of Gq signaling by G protein-coupled receptor kinase 2: direct interaction of kinase N terminus with activated galphaq. AB - In this study, we investigated the regulation of different G protein-coupled receptor (GPCR)-stimulated signaling pathways by GPCR kinase 2 (GRK2). We used thyrotropin receptor, which is coupled to different G proteins, to investigate the regulation of Galphas- and Galphaq-mediated signaling (assessed by cAMP and inositol phosphate production, respectively). In transfected cells, both pathways were desensitized by GRK2. However a kinase-dead GRK2 mutant (GRK2-K220R) only decreased inositol phosphate production, indicating that GRK2 could regulate Galphaq signaling through a phosphorylation-independent mechanism. Similar results were obtained with serotonin receptor 5-hydroxytryptamine(2C), which is coupled to Galphaq. This effect was mimicked by the N-terminal domain of GRK2 (GRK2-Nter), but not by the C-terminal domain. In cells transfected with Galphaq, direct activation of Galphaq signaling (by AlF(4)(-)) was desensitized by GRK2 Nter, indicating an effect at the Galpha-level. For comparison, in parallel samples we studied a protein regulator of G protein signaling RGS4 and we found a similar regulatory profile. We therefore hypothesized that the GRK2-Nter could directly interact with the Galphaq subunit to regulate its signaling, as demonstrated for several RGS proteins. This hypothesis is further supported by the presence, within the GRK2-Nter, of an RGS homology domain. In direct binding experiments, we found that GRK2-Nter interacts with Galphaq (only when activated) but not with Galphas and Galphao. We conclude that GRK2, besides desensitizing the GPCR by phosphorylation, is able to selectively bind to Galphaq and to regulate its signaling. PMID- 10727533 TI - NDT online PMID- 10727531 TI - Alterations in detergent solubility of heterotrimeric G proteins after chronic activation of G(i/o)-coupled receptors: changes in detergent solubility are in correlation with onset of adenylyl cyclase superactivation. AB - Prolonged G(i/o) protein-coupled receptor activation has been shown to lead to receptor internalization and receptor desensitization. In addition, it is well established that although acute activation of these receptors leads to inhibition of adenylyl cyclase (AC), long-term activation results in increased AC activity (especially evident on removal of the inhibitory agonist), a phenomenon defined as AC superactivation or sensitization. Herein, we show that chronic exposure to agonists of G(i)-coupled receptors also leads to a decrease in cholate detergent solubility of G protein subunits, and that antagonist treatment after such chronic agonist exposure leads to a time-dependent reversal of the cholate insolubility. With Chinese hamster ovary and COS cells transfected with several G(i/o)-coupled receptors (i.e., mu- and kappa-opioid, and m(4)-muscarinic), we observed that although no overall change occurred in total content of G(alphai)- and beta(1)-subunits, chronic agonist treatment led to a marked reduction in the ability of 1% cholate to solubilize G(betagamma) as well as G(alphai). This solubility shift is exclusively observed with G(alphai), and was not seen with G(alphas). The disappearance and reappearance of G(alphai) and G(betagamma) subunits from and to the detergent-soluble fractions occur with similar time courses as observed for the onset and disappearance of AC superactivation. Lastly, pertussis toxin, which blocks acute and chronic agonist-induced AC inhibition and superactivation, also blocks the shift in detergent solubility. These results suggest a correlation between the solubility shift of the heterotrimeric G(i) protein and the generation of AC superactivation. PMID- 10727534 TI - Angiotensin II type 2 receptors in the kidney: evidence for endothelial-cell mediated renal vasodilatation. PMID- 10727535 TI - STOPPING at the CAPPP of good HOPE. PMID- 10727536 TI - Should the results of the HOPE study affect nephrological practice? For the HOPE investigators. PMID- 10727537 TI - Cardiovascular risk in uraemic patients-is it fully explained by classical risk factors? PMID- 10727538 TI - The patient's appraisal of side-effects: the blind spot in quality-of-life assessments in transplant recipients. PMID- 10727539 TI - What's new in vitamin D for the nephrologist? PMID- 10727540 TI - Oxidative stress occurs in absence of hyperglycaemia and inflammation in the onset of kidney lesions in normotensive obese rats. AB - BACKGROUND: Several factors favour the development of kidney lesions. We examined the role of oxidative stress in the onset of renal alterations that occur in Zucker obese (ZO) fa/fa rats. METHODS: Kidney structure, biological data, glycation parameters, advanced glycation end products (AGE), thiobarbituric acid reactive substances (TBARS), circulating antibodies anti-malondialdehyde (MDA) modified low-density lipoprotein (LDL), antioxidant defenses (Cu/Zn and Mn superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) activities, glutathione level), were determined in plasma and/or kidney of young and old ZO rats and lean (ZL) Fa/fa littermates. RESULTS: Renal lesions and functional decline appeared at 3 months in hyperlipidaemic, hyperinsulinaemic, normotensive ZO rats, independently of any macrophage-ED(1)(+)-cell infiltration. At 6 months and thereafter, kidney lesions and functional impairment worsened while numerous ED(1)(+)-cells invaded the interstitium. At 3 and 9 months, TBARS level in the LDL/very low-density lipoprotein fraction and in the kidney was higher in ZO than in ZL rats. Anti-MDA-LDL antibodies were increased in ZO rats. At 3 months, renal activity of Cu/Zn SOD was higher, and activities of catalase and GPx lower in ZO than in ZL rats, leading to an accumulation of hydrogen peroxide (H(2)O(2)). At 9 months, a decrease in Cu/Zn SOD activity and an increase in glutathione level were observed. Blood glucose and glycated proteins, as well as AGE in kidney, remained similar in both ZL and ZO rats, whatever their age. CONCLUSION: These data suggest that oxidative stress triggers, at an early age, the onset of kidney lesions and functional impairment in ZO rats, in absence of hyperglycaemia, hypertension and inflammation. PMID- 10727541 TI - A novel frameshift mutation (2436insT) produces an immediate stop codon in the autosomal dominant polycystic kidney disease 2 (PKD2) gene. AB - BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder that can be caused by mutations in at least three different genes. Several mutations have been identified in PKD1 and PKD2 genes. Most of the mutations found in PKD2 gene are predicted to cause premature termination of the protein. METHODS: We analysed an Argentinian family characterized previously as PKD2. The PKD2 gene was amplified from genomic DNA using 17 primer pairs and the products were analysed by heteroduplex analysis. PCR products that showed a variation by heteroduplex analysis were sequenced directly. The mutation was confirmed by sequencing relatives. The segregation of the mutation in this family was verified by restriction endonuclease digestion of PCR products obtained from genomic DNA of all family members. Results and conclusions. Here, we report a novel mutation present in an Argentinian family characterized as PKD2 by linkage analysis. The mutation, shared by all affected members of the family, is a thymidine insertion at position 2436 of the gene, which results in a translation frameshift and creates an immediate stop codon. This mutation is expected to lead to a truncated protein that lacks the interacting domain with the PKD1 gene product. The thymidine insertion abolished a Ddel restriction site, allowing a rapid test for detection of PKD2 carriers in the family. PMID- 10727542 TI - Angiotensin I-converting enzyme gene polymorphism in non-diabetic renal disease. AB - BACKGROUND: The insertion/deletion (I/D) polymorphism of the angiotensin converting enzyme (ACE) gene determines the concentration of ACE in serum and local tissues. The role of this polymorphism in progressive chronic renal disease is still not fully clear. METHODS: We analysed the impact of the D/D polymorphism on the rate of decline in renal function in patients with non-diabetic, chronic progressive renal insufficiency. Seventy non-diabetic patients, aged 21-69 years at baseline, with moderately advanced renal insufficiency due to primary chronic renal disease were followed for an average of 3 years with repeated measurements of their glomerular filtration rate (GFR). Their mean GFR at baseline was 41 ml/min/1.73 m(2) body surface area (BSA). The polymerase chain reaction (PCR) amplification method was used to detect the I/D polymorphism of the ACE gene. GFR was measured as the clearance of (51)Cr-EDTA and the individual rate of progression was calculated using linear regression. RESULTS: The distributions of the genotypes were: D/D 30%, I/D 49%, and I/I 21%. The rates of progression in the three ACE genotype groups were an annual decline in renal function of -4.2 (SD 4.6) ml/minx1.73 m(2) BSA in the D/D group, -2.7 (SD 3. 4) in the I/D group and -1.7 (SD 3.4) in the I/I group (ANOVA P=0. 12). In patients with proteinuria below 3.5 g/24 h, the D/D group had a significantly higher rate of progression than patients with the I allele. The same was found in a separate analysis when only patients with normal apoliprotein B (below 155 mg/dl) levels were analysed. Furthermore, the D/D genotype was a significant predictor of a more rapid decline in renal function in male, but not female, patients. CONCLUSION: The results in this study in non-diabetic patients with chronic renal disease indicate that the presence of the D allele in the ACE genotype may be of particular importance as a predictor of a high rate of progression in male patients who otherwise do not have a major burden of documented and important prognostic factors for progressive renal insufficiency. PMID- 10727543 TI - The Irbesartan type II diabetic nephropathy trial: study design and baseline patient characteristics. For the Collaborative Study Group. AB - BACKGROUND: Diabetic nephropathy is the most common cause of end-stage renal disease in the developed world. Angiotensin-converting enzyme inhibitors have been demonstrated to be renoprotective in type I diabetes and are now the standard of care for both hypertensive and non-hypertensive type I diabetic patients with any level of proteinuria. The role of blockade of the renin angiotensin system in type II diabetic patients is not defined. The Collaborative Study Group has initiated the Irbesartan Type II Diabetic Nephropathy Trial (IDNT), studying the effect of the angiotensin II receptor antagonist irbesartan on progression of renal disease and mortality in type II diabetic patients with overt nephropathy and hypertension. Here we report the study design and baseline patient characteristics. METHODS: To qualify, hypertensive type II patients, age 30-70 years, must have a 24 h urinary protein excretion of >900 mg and a serum creatinine 90-265 micromol/l (1.0-3. 0 mg/dl) in women and 110-265 micromol/l (1.2-3.0 mg/dl) in men. Three treatment arms include irbesartan, placebo and amlodipine, with every attempt made to achieve similar blood pressure levels in all treatment arms. A total of 1650 patients will be enrolled utilizing approximately 225 clinics worldwide. The primary outcome measure is time to event to the composite end-point of doubling of serum creatinine, end-stage renal disease or death. The secondary outcome measure is time to composite end-point of fatal or non-fatal cardiovascular events. The average length of patient follow-up is expected to be approximately 36 months. RESULTS: The baseline characteristics of the study subjects are: age 59+/-8 years, duration of diabetes 15+/-9 years, height 168+/-11 cm (5 ft 6 in), weight 87+/-19 kg (192 lb), body mass index 31+/ 7 kg/m(2), blood pressure 156+/-18 mmHg/85+/-11 mmHg, serum creatinine 150+/-53 micromol/l (1.7+/-0.6 mg/dl), creatinine clearance 66+/-34 ml/min and 24 h urine protein 4.0+/-3.5 g/day. PMID- 10727544 TI - Altered IgG(4) renal clearance in patients with inflammatory bowel diseases. Evidence for a subclinical impairment of protein charge renal selectivity. AB - BACKGROUND: A loss of intestinal glycosaminoglycans (GAGs) has been shown in inflammatory bowel diseases (IBD). Since GAGs are involved in the regulation of renal protein filtration and GAGs disruption is associated with anionic proteinuria, we examined whether changes in the selectivity of renal protein filtration occur in IBD. METHODS: From 46 patients with IBD (17 with Crohn's disease (CD), and 29 with ulcerative colitis (UC)) and 21 healthy subjects, urine and serum samples were obtained. Albumin, total IgG and IgG(4) clearances were measured using sensitive methods. Serum p-ANCA and TNF-alpha were tested. RESULTS: Median IgG(4) clearance was 0.041 ml/ min/10(-3) in patients with UC and 0.10 ml/ min/10(-3) in CD patients, both significantly higher than in controls (0.03 ml/min/10(-3)) (P<0.03). IgG(4) clearance was above the upper normal limit in 9/17 CD (53%) and in 10/29 UC (34.5%). Eighteen of 19 patients showing abnormal IgG(4) clearance were taking mesalazine. In patients on maintenance oral mesalazine, IgG(4) clearance was higher than that in patients off treatment (0.12 vs 0.03 ml/min/10(-3), P=0.04). No clinical/laboratory sign of renal dysfunction was documented in patients with altered IgG(4) clearance and maintained on mesalazine treatment. CONCLUSION: Renal protein charge permselectivity is impaired in 40% of patients with IBD with no overt proteinuria. Our data suggest that altered IgG(4) clearance may represent a subclinical marker of renal involvement in IBD. PMID- 10727545 TI - B lymphopenia in uremia is related to an accelerated in vitro apoptosis and dysregulation of Bcl-2. AB - BACKGROUND: Lymphopenia has been described in patients with chronic renal failure (CRF). It is postulated that the decline in lymphocytes is due to accelerated apoptosis. We investigated whether dysregulation of programmed cell death plays a role in the immunodeficiency described in CRF. METHODS: Peripheral blood lymphocytes (PBL) from pre-dialysis uraemic patients (nHD) and haemodialysed patients (HD) were cultured with no stimulus for 96 h. Apoptosis of lymphocytes was measured by propidium iodide staining and flow cytometry. Expression of Fas and Bcl-2 was also analysed by flow cytometry. RESULTS: Peripheral blood B cells were significantly lower in pre-dialysis and haemodialysis uraemic patients compared to control. Lymphocytes from both groups of patients had a higher rate of apoptosis in vitro than those from healthy controls. This effect was more pronounced in B lymphocytes and a significant correlation between the B lymphopenia and the percentage of apoptotic B cells after 48 h of culture without stimulus was observed. The increased lymphocyte apoptosis in CRF was accompanied by a significantly lower in vitro Bcl-2 expression. However, Fas did not seem to play a role in spontaneous lymphocyte apoptosis in end-stage renal disease. CONCLUSIONS: Our data indicate that B lymphopenia in CRF may be partially attributed to an increased susceptibility to cell death by apoptosis that is associated with a decreased expression of Bcl-2. PMID- 10727546 TI - Intradialytic glucose infusion increases polysulphone membrane permeability and post-dilutional haemodiafiltration performances. AB - INTRODUCTION: During real-time monitoring of the ultrafiltration coefficient (Kuf) in haemodiafiltration (HDF), it was noticed that the ultrafiltration performance of polysulphone membrane dialysers increased when hypertonic glucose (D50%) was administered through the venous blood return. METHODS: This observation was explored in six non-diabetic chronic dialysis patients during 48 HDF sessions using 1.8 m(2) polysulphone membrane dialysers. In all six patients, 24 sessions were performed with glucose supplementation (as a continuous D50% (500 g/l) infusion at 40 ml/h) and 24 sessions without supplementation. RESULTS: Glucose supplementation led to a marked increase in Kuf from 22.8+/-2.2 (without D50%, n=24) to 32. 1+/-3.9 ml/h/mmHg (with D50%, n=24) (P<0.0001). An increase in percentage reduction ratios for urea and creatinine were also consistently observed during the sessions with glucose administration (from respective mean values of 75+/-5 and 68+/-4% to 79+/-4 and 74+/-10%). Mean double-pool Kt/V, calculated from serum urea concentrations, rose from 1.65+/-0.24 (n=24) to 1.86+/ 0.24 (n=24) (P<0.005). Similar results were observed in a subgroup of 18 HDF sessions (nine with glucose and nine without) monitored with an on-line urea sensor of spent dialysate. No detrimental effects were induced at any time. CONCLUSIONS: We conclude that intravenous glucose administration during high-flux HDF using polysulphone membranes increases significantly both ultrafiltration capacity and dialysis dose delivery. PMID- 10727547 TI - A new method of post-dialysis blood urea sampling: the 'stop dialysate flow' method. AB - BACKGROUND: A standardized practical method of post-dialysis blood sampling is required to improve the precision of using urea kinetics in the evaluation of haemodialysis dose and to permit comparative audit. The methods recommended in the Renal Association and Dialysis Outcomes Quality Initiative (DOQI) guidelines reduce the blood pump speed to a low rate at the end of haemodialysis before blood sampling after 10 and 15 s respectively. However, these 'low flow' methods compensate only partially for cardiopulmonary recirculation and may be impractical in routine practice because they involve sequential steps and require accurate timing of sampling. Therefore we have evaluated an alternative method of stopping only the dialysate flow at the end of the haemodialysis session before performing post-dialysis blood sampling. METHODS: The study was performed in three phases. Serial measurements of blood urea were obtained from arterial and venous samples taken at times 0, 30, 60, 120, 180, 240, 300 and 360 s after stopping dialysate flow and leaving the extracorporeal blood flow rate unchanged at the end of the haemodialysis session in 10 patients. A peripheral venous sample was also taken from the contralateral arm at 0 s to reflect body water urea concentration at the end of dialysis without the effect of access recirculation and with a minimal effect of cardiopulmonary recirculation. The same haemodialysis prescription was repeated in the same 10 patients using the Renal Association method to permit comparison between the two methods. The practical use of the 'stop dialysate flow' method was then evaluated in 117 regular haemodialysis patients undergoing routine monthly assessment of dialysis adequacy and compared with sampling immediately post-dialysis. RESULTS: Within 4 min of stopping the dialysate flow there was no difference between the blood urea concentrations of arterial and venous samples, indicating cessation of diffusion across the dialysis membrane. Also the blood urea concentrations in all of the arterial and venous samples between 4 and 6 min were constant and were equivalent to the blood urea concentration of the peripheral venous sample taken at 0 s. These data suggest that post-dialysis blood sampling may be performed 5 min after stopping dialysate flow at the end of the haemodialysis session. In contrast, the blood urea concentration in the post-dialysis samples obtained using the Renal Association method were lower than the contralateral arm blood urea concentration taken at 0 s (0.31+/-0.42; P<0.05) and consequently the percentage URR was higher (1.35+/-1.84%). In 117 patients the post-dialysis blood urea sample 5 min after stopping dialysate flow averaged 5.49+/-2.11 mmol/1 compared with 5.07+/-2.05 mmol/l immediately after the end of the haemodialysis session (P<0. 0001). This was equivalent to a reduction in URR from 71.7+/-8.3% with sampling immediately post-dialysis to 69.1+/-9.3% with the 'stop dialysate flow' method. CONCLUSIONS: This study shows that there is a window period between 4 and 6 min after stopping dialysate flow at the end of the haemodialysis session when the blood urea concentration in a sample taken from any part of the extracorporeal circuit remains constantly within the co-efficient of variation of laboratory measurement, and is equivalent to a peripheral venous sample taken immediately at the end of the dialysis session. A 'stop dialysate flow' method with blood sampling after 5 min offers several advantages over 'slow flow' methods, since it allows for cardiopulmonary as well as access recirculation, does not require precise timing of blood sampling, and is simple to perform in a busy renal unit. For these reasons the 'stop dialysate flow' method may be used for routine monitoring of the adequacy of delivered haemodialysis and for comparative audit among haemodialysis centres. PMID- 10727548 TI - Treatment with different doses of folic acid in haemodialysis patients: effects on folate distribution and aminothiol concentrations. AB - BACKGROUND: Hyperhomocysteinaemia is highly prevalent among haemodialysis patients and may contribute to their increased cardiovascular risk. Treatment with pharmacological doses of folic acid lowers the plasma homocysteine concentration in these patients. The purpose of the present study was to expand the knowledge about such treatment by testing the effects of stepwise increases in the dose of folic acid on the concentrations of plasma and red blood cell folate as well as the total plasma concentrations of homocysteine (tHcy), cysteine (tCys), and glutathione (tGSH) in patients on chronic hemodialysis. METHODS: Fourteen stable haemodialysis patients completed the study which consisted of four consecutive periods, each of 6 weeks duration: (i) no treatment with folic acid (control period); (ii) 5 mg of folic acid three times per week (15 mg/week); (iii) 5 mg of folic acid daily (35 mg/week); (iv) 10 mg of folic acid daily (70 mg/week). RESULTS: Neither plasma or red cell folate nor plasma aminothiol concentrations changed significantly during the control period. The mean red cell folate concentration doubled during the administration of folic acid at the dose of 15 mg/week but at higher doses the further rise was only marginal. The mean folate concentration in plasma increased steeply especially at the higher doses of folic acid. During treatment with 15 mg/week of folic acid, tHcy fell by a mean of 36%, tGSH increased by a mean of 34%, but tCys was unaffected. Increases in the dose of folic acid did not augment these responses. CONCLUSIONS: The maximal effect on tHcy seemed to be obtained already at the lowest given dose of folic acid (15 mg/week). At that dose, the red blood cells approached folate saturation, which may reflect the situation in other cells that participate in homocysteine metabolism and explain why further increases in the dose of folic acid are not effective from a tHcy-lowering point of view. PMID- 10727550 TI - Stent treatment for pseudocoarctation and refractory hypertension in an elderly patient with Takayasu's arteritis. PMID- 10727549 TI - Interstitial expression of heat shock protein 47 and alpha-smooth muscle actin in renal allograft failure. AB - BACKGROUND: Tubulointerstitial inflammation and fibrosis are the main pathological features of chronic renal allograft rejection, which is characterized by accumulation of extracellular matrix protein. Heat shock protein 47 (HSP47), known as a collagen-specific stress protein, is thought to be a molecular chaperone during the processing and/or secretion of procollagen. HSP47 is thought to be involved in the progression of fibrosis, but its expression in chronic renal allograft rejection is still unknown. METHODS: We examined the expression of HSP47 together with that of alpha-smooth muscle actin (alpha-SMA), a marker of myofibroblasts, and CD68, a marker of macrophages, by immunohistochemistry in allograft kidney tissues. Uninvolved portions of surgically removed kidneys with tumours served as control tissue. RESULTS: Expression of HSP47 was detected in the interstitium of fibrotic regions of allograft kidneys. Cells positive for HSP47 were also stained for alpha-SMA and type I collagen, and the expression of HSP47 correlated with the degree of interstitial fibrosis. Furthermore, the expression of HSP47 correlated with the number of infiltrating macrophages. In contrast, HSP47 and alpha-SMA were not expressed in the control tissues, sections of 1 h post-transplantation biopsy specimens and acute allograft rejection without fibrosis. CONCLUSION: Our results suggest that HSP47 may contribute to the progression of interstitial fibrosis in allograft renal tissues. PMID- 10727551 TI - Parvus tardus waveform suggesting renal artery stenosis-remember the more proximal stenosis. PMID- 10727552 TI - The patient with low back pain and acute oliguric renal failure. PMID- 10727553 TI - Diagnosis of tuberous sclerosis in a patient who presented with polycystic kidney disease. PMID- 10727554 TI - Errors in ultrasonographic measurements of the kidney. PMID- 10727555 TI - A stony history. PMID- 10727556 TI - Consistent timing of the post-dialysis blood sample is necessary to prevent undertreatment in single needle dialysis. PMID- 10727557 TI - Dialysate sodium concentration and blood pressure control in haemodialysis patients. PMID- 10727558 TI - Long-term effects of sevelamer hydrochloride on the calcium x phosphate product and lipid profile of haemodialysis patients PMID- 10727559 TI - Iron deficiency anaemia-a clinical challenge. PMID- 10727560 TI - Does this elderly patient have iron deficiency anaemia, and what is the underlying cause? AB - Important implications for the recognition of iron deficiency anaemia include diagnosis and correction of underlying causes, most of which are identifiable, in the older patient, by means of conventional upper gastrointestinal endoscopy, and by colonoscopy. The aetiological search may, however, have to be widened to include enteroscopic examination of the jejunum and ileum and, in some instances, investigation of potential non-gastrointestinal foci of chronic blood loss. A substantial minority may defy even the most thorough search for the underlying cause. PMID- 10727563 TI - Doctor or nurse? The patients' choice. AB - This project's aim was to assess patients perceived need for a specialist nurse in inflammatory bowel disease. A letter was sent to 64 patients with a summary of the potential role of a specialist nurse. Patients were asked to complete a questionnaire of 10 scenarios on who would be their preferred provider on the range of issues. There were 35 (55%) replies. When comparing the results between the consultant and the specialist nurse patients preferred to see a specialist nurse significantly in four scenarios, and patients preferred to see the consultant significantly in two scenarios. The results indicate that patients feel specialist nurses would have more time to discuss issues and they do not want to bother the busy doctors with them. But they do want doctors to carry out the medical aspects of their care. PMID- 10727561 TI - Primary biliary cirrhosis: new perspectives in diagnosis and treatment. AB - Primary biliary cirrhosis (PBC) is a chronic autoimmune disease characterised by cholestatic liver function tests, antimitochondrial antibodies, and abnormal liver histology. Early descriptions of a rare rapidly progressive disease no longer reflect the more indolent progress often seen today. Many patients have significant long term morbidity through symptoms such as fatigue and itch with a minority progressing to liver failure and need for transplantation. The current data on the diagnosis, clinical progression, and treatment of PBC are reviewed. PMID- 10727562 TI - Management and outcome of brain abscess in renal transplant recipients. AB - Although infection is the commonest central nervous system complication following renal transplantation, brain abscess is uncommon. Over the last 11 years, five renal transplant recipients who had brain abscesses were treated by computed tomography (CT)-guided stereotactic aspiration. Three patients had a fungal abscess, one a tuberculous abscess and the other had a methicillin-resistant Staphylococcus aureus abscess. One patient required a craniotomy for the excision of a fungal abscess which was persistent after two CT-guided stereotactic aspirations. The survivors in this group are the patient with a tuberculous abscess who is alive and well 5 years after diagnosis, and another with a dematiaceous fungal abscess (phaeohyphomycosis). CT-guided stereotactic surgery is minimally invasive, and can safely be performed in these patients. It often leads to an aetiological diagnosis in renal transplant recipients with brain abscesses. Specific antibiotic management directed towards the causative organism rather than empirical treatment can be instituted following the procedure. Although the ultimate prognosis in these patients is bleak even with specific antibiotic therapy, an occasional patient might have a good outcome with prompt and appropriate therapy. PMID- 10727564 TI - Influenza and pneumococcal vaccination: patient perceptions. AB - The efficacy of the influenza vaccine in reducing mortality and hospital admissions is established, particularly in the elderly. However, up to 50% of those at risk do not receive the vaccine. These patients are also at risk from pneumococcal infection and there is considerable overlap between the target group for each vaccine. This study sought to identify at risk individuals from consecutive admissions to an acute geriatric unit and to gain an insight into their perceptions with regard to vaccination. The awareness of each vaccine was recorded, together with the vaccination history. Seventy four per cent of the final cohort had heard of the influenza vaccine, while only 13% had heard of the pneumococcal vaccine. Fifty per cent perceived themselves to be at risk from influenza and its complications and 87% of the cohort believed it to be a serious infection. Influenza vaccine was judged to confer good protection by 72% of the sample and yet up to 50% believed that the vaccine can make the recipient ill. Influenza is perceived as a serious infection by patients and yet many do not believe themselves to be at particular risk. Although influenza vaccination is believed to confer protection, the decision whether, or not, to accept the vaccine is coloured by many factors, including popular myths and anecdotal information from friends and relatives. The uptake of influenza vaccine is suboptimal and the awareness of the pneumococcal vaccine certainly in the elderly is poor. The need for a comprehensive nationwide education campaign promoting both influenza and pneumococcal vaccine is highlighted. PMID- 10727565 TI - Audit of the investigation of iron deficiency anaemia in a district general hospital, with sample guidelines for future practice. AB - Iron deficiency anaemia commonly presents in patients who are asymptomatic. In the absence of published guidelines the search for a cause in such cases is frequently uncoordinated, and risks delay in the diagnosis of pathologies requiring urgent attention. This audit was undertaken to determine how thoroughly patients referred to the gastrointestinal unit in a district general hospital between 1990 and 1995 had been investigated, and to draw up guidelines for future practice on the basis of its results. From the case notes of 334 patients examined endoscopically for anaemia 126 were identified as having both proved iron deficiency and a lack of clinical pointers to its cause. The percentage and details of diagnoses made during initial study and a median follow up period of 28 months were ascertained, together with the certified diagnoses of patients who had died. A cause of iron deficiency was identified in 48 (38%) of patients, 22 with cancer. Ten others received a diagnosis during follow up, of whom three died from the condition to which their anaemia had been attributed. Death certificates supplied diagnoses of potential relevance in three further cases. The main gaps in endoscopic coverage consisted of omitting duodenal biopsy or colonoscopy after negative upper gastrointestinal endoscopy. Moreover, diagnosis of certain extraintestinal pathologies, including cancers, was sometimes delayed for lack of liaison between gastroenterologists and other specialists. These and other points have been addressed in the guidelines now proposed. PMID- 10727566 TI - Delivery of surgical care in a district general hospital without high dependency unit facilities. AB - BACKGROUND: Many hospitals lack the facilities for high dependency care, and patients requiring this level of care are nursed on the surgical ward. The aim of this study was to assess the extent of this problem in a district general hospital, looking at the impact of providing high dependency unit (HDU) care at ward level. METHODS: A 28 bed surgical ward was studied for 39 consecutive days. Patients were assessed as being either appropriately placed (routine) or inappropriately placed (HDU). Nursing interventions and observations over each 24 hour period were recorded for the most dependent patient in each group. RESULTS: Data were collected for a total of 1092 bed days. Median bed occupancy was 22 patients/day (78%). Inappropriately placed HDU patients accounted for 55 bed days (5%, mean 1.4 patients/day). These patients required more nursing intervention than routine patients. HDU patients received more observations during a 24 hour period than routine patients (mean 11.3 and 4.2 respectively, p<0.005). The number of observations recorded for a routine patient in a 24 hour period fell when a HDU patient was nursed concurrently on the ward (mean 5.1/24 hours, falling to 3.8 /24 hours in the presence of an HDU patient, p<0.02). CONCLUSIONS: HDU patients require more intensive nursing care than routine surgical patients, and the nursing of HDU patients on the ward adversely affects the quantity of care available for less dependent patients. High dependency care should therefore be provided in dedicated units. HDU is an essential facility for all surgical patients, including those who require intensive nursing, and the routine surgical patient whose nursing is compromised by the failure to provide comprehensive postoperative care. PMID- 10727567 TI - A new cause of 'non-responsiveness' in coeliac disease? AB - A 42 year old man presented with gluten-responsive coeliac disease and secondary pancreatic insufficiency. Subsequently his symptoms relapsed and repeat small intestinal biopsy showed villous atrophy and infiltration by leukaemic cells, despite continuation of a gluten-free diet. Serious causes of relapse and non responsiveness in coeliac disease include enteropathy-associated T-cell lymphoma, ulcerative jejunitis and an end-stage hypoplastic mucosa. This is the first report of non-responsiveness due to infiltration by leukaemia. PMID- 10727568 TI - Sweet's syndrome and subacute thyroiditis. AB - A 63 year old woman developed biopsy documented lesions of acute febrile neutrophilic dermatosis (Sweet's syndrome) one week after the onset of subacute thyroiditis. This is only the second reported case of such an association. The role of cytokines in the development of both subacute thyroiditis and Sweet's syndrome may be the link between these two conditions. PMID- 10727569 TI - Addison's disease in type 1 diabetes presenting with recurrent hypoglycaemia. AB - Primary adrenal insufficiency (Addison's disease) often develops insidiously. Although a rare disorder, it is more common in type 1 diabetes mellitus. A 19 year old male with type 1 diabetes and autoimmune hypothyroidism experienced recurrent severe hypoglycaemia over several months, despite a reduction in insulin dose, culminating in an adrenal crisis. Recurrent severe hypoglycaemia resolved after identification and treatment of the adrenocortical insufficiency. In type 1 diabetes, undiagnosed Addison's disease can influence glycaemic control and induce severe hypoglycaemia. PMID- 10727570 TI - Galactorrhoea and pituitary mass: a typical prolactinoma? AB - A 21 year old woman presenting with galactorrhoea, hyperprolactinaemia, and a pituitary mass on magnetic resonance imaging (MRI) is described who was referred to us before planned pituitary surgery. Although a thorough history did not suggest hypothyroidism, laboratory studies revealed profound primary hypothyroidism. At that time, pituitary MRI showed homogeneous enlargement of the pituitary gland consistent with pituitary hyperplasia due to primary hypothyroidism. With thyroid hormone replacement therapy the galactorrhoea resolved, concentrations of prolactin and thyroid hormones returned to normal, and the pituitary shrunk to normal size within two months. This case illustrates that primary hypothyroidism can present only with galactorrhoea and pituitary mass, and should therefore be considered in the differential diagnosis of hyperprolactinaemia and pituitary enlargement. PMID- 10727571 TI - Advanced testicular cancer presenting with phlegmasia cerulea dolens. AB - A case of fulminating deep venous thrombosis secondary to invasion of the inferior vena cava is described in a 45 year old man presenting with a germ cell tumour. Despite aggressive supportive care and emergency chemotherapy his late presentation caused his death. The case highlights the necessity for increased public education of the attendant risks in delayed presentation with a testicular lump. PMID- 10727572 TI - Fever and a rash in a 61 year old man. PMID- 10727573 TI - Painful knee: a dilemma. PMID- 10727574 TI - Back pain and dyspnoea in a middle aged diabetic male. PMID- 10727575 TI - A man with abdominal pain. PMID- 10727576 TI - A shot in the dark. PMID- 10727577 TI - Pyrexia and pancytopenia with unusual host immune response. PMID- 10727578 TI - Abdominal colic after vigorous exercise in a middle aged man. PMID- 10727580 TI - Painful knee: a dilemma PMID- 10727581 TI - Back pain and dyspnoea in a middle aged diabetic male PMID- 10727579 TI - Fever and rash in a 61 year old man PMID- 10727582 TI - A man with abdominal pain PMID- 10727583 TI - A shot in the dark PMID- 10727584 TI - Pyrexia and pancytopenia with unusual host immune response PMID- 10727585 TI - Abdominal colic after vigorous exercise in a middle aged man PMID- 10727586 TI - Venlafaxine-induced serotonin syndrome with relapse following amitriptyline. AB - A case of venlafaxine-induced serotonin syndrome is described with relapse following the introduction of amitriptyline, despite a 2-week period between the discontinuation of one drug and the commencement of the other. Electroencephalography may play an important part in diagnosis. With the increasing use of selective serotonin re-uptake inhibitors, greater awareness of the serotonin syndrome is necessary. Furthermore, the potential for drug interactions which may lead to the syndrome needs to be recognised. PMID- 10727587 TI - Abnormal myocardial phosphorus-31 nuclear magnetic resonance spectroscopy in women with chest pain but normal coronary angiograms. AB - BACKGROUND: After hospitalization for chest pain, women are more likely than men to have normal coronary-artery angiograms. In such women, myocardial ischemia in the absence of clinically significant coronary-artery obstruction has long been suspected. Most methods for the detection of the metabolic effects of myocardial ischemia are highly invasive. Phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy is a noninvasive technique that can directly measure high-energy phosphates in the myocardium and identify metabolic evidence of ischemia. METHODS: We enrolled 35 women who were hospitalized for chest pain but who had no angiographically significant coronary-artery obstructions and 12 age- and weight matched control women with no evidence of heart disease. Myocardial high-energy phosphates were measured with 31P-NMR spectroscopy at 1.5 tesla before, during, and after isometric handgrip exercise at a level that was 30 percent of the maximal voluntary grip strength. We measured the change in the ratio of phosphocreatine to ATP during exercise. RESULTS: Seven (20 percent) of the 35 women with chest pain and no angiographically significant stenosis had decreases in the phosphocreatine:ATP ratio during exercise that were more than 2 SD below the mean value in the control subjects without chest pain. There were no significant differences between the two groups with respect to hemodynamic variables at rest and during exercise, risk factors for ischemic heart disease, findings on magnetic resonance imaging and radionuclide perfusion studies of the heart, or changes in brachial flow during the infusion of acetylcholine. CONCLUSIONS: Our results provide direct evidence of an abnormal metabolic response to handgrip exercise in at least some women with chest pain consistent with the occurrence of myocardial ischemia but no angiographically significant coronary stenoses. The most likely cause is microvascular coronary artery disease. PMID- 10727588 TI - Reactivation of genital herpes simplex virus type 2 infection in asymptomatic seropositive persons. AB - BACKGROUND: Most persons who have serologic evidence of infection with herpes simplex virus (HSV) type 2 (HSV-2) are asymptomatic. Historically, it has been assumed that these persons have less frequent viral reactivation than those with symptomatic infection. METHODS: We conducted a prospective study to investigate genital shedding of HSV among 53 subjects who had antibodies to HSV-2 but who reported having no history of genital herpes, and we compared their patterns of viral shedding with those in a similar cohort of 90 subjects with symptomatic HSV 2 infection. Genital secretions of the subjects in both groups were sampled daily and cultured for HSV for a median of 94 days. RESULTS: HSV was isolated from the genital mucosa in 38 of the 53 HSV-2-seropositive subjects (72 percent) who reported no history of genital herpes, and HSV DNA was detected by the polymerase chain-reaction assay in cultures prepared from genital mucosal swabs in 6 additional subjects. The rate of subclinical shedding of HSV in the subjects with no reported history of genital herpes was similar to that in the subjects with such a history (3.0 percent vs. 2.7 percent). Of the 53 subjects who had no reported history of genital herpes, 33 (62 percent) subsequently reported having typical herpetic lesions; the duration of their recurrences in these subjects was shorter (median, three days vs. five days; P<0.001) and the frequency lower (median, 3.0 per year vs. 8.2 per year; P<0.001) than in the 90 subjects with previously diagnosed symptomatic infection. Only 1 of these 53 subjects had no clinical or virologic evidence of HSV infection. CONCLUSIONS: Seropositivity for HSV-2 is associated with viral shedding in the genital tract, even in subjects with no reported history of genital herpes. PMID- 10727589 TI - Risk of persistent growth impairment after alternate-day prednisone treatment in children with cystic fibrosis. AB - BACKGROUND: It is uncertain whether the growth impairment that occurs in children during long-term treatment with glucocorticoids persists after the medication is discontinued and ultimately affects adult height. METHODS: We evaluated growth six to seven years after alternate-day treatment with prednisone had been discontinued in 224 children 6 to 14 years of age with cystic fibrosis who had participated in a multicenter trial of this therapy from 1986 through 1991. Of the children, 151 had been randomly assigned to receive prednisone (either 1 or 2 mg per kilogram of body weight) and 73 to receive placebo. We obtained data on growth up to 1997 from the Cystic Fibrosis Foundation Patient Registry and standardized the data to sex- and age-specific norms from the National Center for Health Statistics. We used z scores to compare growth patterns among treatment groups. RESULTS: In 1997, 68 percent of the patients were 18 years of age or older. The z scores for height declined during prednisone therapy; catch-up growth began two years after treatment with prednisone was discontinued. Among the boys, the z scores for height in those treated with prednisone remained lower than the scores for those who received placebo (P=0.02). The mean heights for boys 18 years of age or older were 4 cm less in the prednisone groups than in the placebo group, an equivalent of 13 percentile points (P=0.03). Among the girls, differences in height between those who were treated with prednisone and those who received placebo were no longer present two to three years after prednisone therapy was discontinued. CONCLUSIONS: Among children with cystic fibrosis who have received alternate-day treatment with prednisone, boys, but not girls, have persistent growth impairment after treatment is discontinued. PMID- 10727590 TI - Images in clinical medicine. Ischemic retinopathy caused by severe megaloblastic anemia. PMID- 10727592 TI - Spontaneous pneumothorax. PMID- 10727591 TI - A prospective study of holiday weight gain. AB - BACKGROUND: It is commonly asserted that the average American gains 5 lb (2.3 kg) or more over the holiday period between Thanksgiving and New Year's Day, yet few data support this statement. METHODS: To estimate actual holiday-related weight variation, we measured body weight in a convenience sample of 195 adults. The subjects were weighed four times at intervals of six to eight weeks, so that weight change was determined for three periods: preholiday (from late September or early October to mid-November), holiday (from mid-November to early or mid January), and postholiday (from early or mid-January to late February or early March). A final measurement of body weight was obtained in 165 subjects the following September or October. Data on other vital signs and self-reported health measures were obtained from the patients in order to mask the main outcome of interest. RESULTS: The mean (+/-SD) weight increased significantly during the holiday period (gain, 0.37+/-1.52 kg; P<0.001), but not during the preholiday period (gain, 0.18+/-1.49 kg; P=0.09) or the postholiday period (loss, 0.07+/ 1.14 kg; P=0.36). As compared with their weight in late September or early October, the study subjects had an average net weight gain of 0.48+/-2.22 kg in late February or March (P=0.003). Between February or March and the next September or early October, there was no significant additional change in weight (gain, 0.21 kg+/-2.3 kg; P=0.13) for the 165 participants who returned for follow up. CONCLUSIONS: The average holiday weight gain is less than commonly asserted. Since this gain is not reversed during the spring or summer months, the net 0.48 kg weight gain in the fall and winter probably contributes to the increase in body weight that frequently occurs during adulthood. PMID- 10727593 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 9-2000. A 41-year-old man with multiple bony lesions and adjacent soft-tissue masses. PMID- 10727594 TI - Chest pain in women with normal coronary angiograms. PMID- 10727596 TI - Introduction PMID- 10727597 TI - Clinical radioimmunotherapy. AB - Radioimmunotherapy (RIT) is a promising new therapy for the treatment of a variety of malignancies. General principles of RIT are discussed, including important considerations in the selection of monoclonal antibodies (MAb) and radionuclides for RIT. Results of clinical trials using RIT for the treatment of lymphoma, leukemia, and solid tumors are summarized. The results from many of these trials are promising, especially for the treatment of lymphohematopoietic malignancies, in which a variety of MAb, radionuclides, and study designs have resulted in high response rates with a number of durable responses. Encouraging results have also been obtained using RIT to treat some solid tumors, primarily in patients with relatively low tumor burdens. RIT is generally well tolerated, with the primary toxicity being transient reversible myelosuppression in most nonmyeloablative studies. Nonhematologic toxicity, especially at nonmyeloablative doses, has been minimal in most studies. Approaches for increasing the therapeutic index of RIT are reviewed, which may further potentiate the efficacy and decrease the toxicity of RIT. PMID- 10727595 TI - Growth in children with chronic lung disease. PMID- 10727599 TI - Use of radionuclides for the palliation of bone metastases. AB - Pain palliation with bone-seeking radiopharmaceuticals is an effective and cost effective management tool in patients with advanced cancer metastatic to bone. Strontium-89 ((89)Sr) (Metastron) and samarium-153 ((153)Sm) EDTMP (Lexidronam) are licensed for use in patients in the United States. Patients with a positive bone scan using technetium 99m methylene diphosphonate ((99m)Tc MDP) are eligible for treatment, and indications and contraindications for use are now well defined. The evidence in the literature now suggests that the radiopharmaceuticals can significantly reduce pain and analgesic requirements, can improve quality of life, can reduce lifetime radiotherapy requirements and management costs, and may slow the progression of painful metastatic lesions. Retreatment is safe and effective. Rhenium-186 ((186)Re) HEDP and Tin-117m diethylenetriaminepenta-acetic acid (DTPA) are in phase II/III trials to evaluate efficacy and compare efficacy with the licensed agents. Phosphorus-32 ((32)P) has been reassessed in two trials evaluating efficacy in comparison with (89)Sr and safety. Toxicity is reversible myelosuppression, which may be significant, and the treatments should not be given to patients with suspected disseminated intravascular coagulation. PMID- 10727598 TI - Systemic radiation therapy with unsealed radionuclides. AB - Systemic unsealed radiation therapy is achieved when a radioactive substance is administered orally or parenterally and that material is concentrated in an organ or site for sufficient time to deliver a therapeutic dose of radiation. The radioactive material usually emits beta particles. In general, there is intense local radiation of the abnormal tissues, and normal organs, which do not trap the radioactive material, are exposed to a small radiation dose. The most frequent treatments involve radioiodine (131)I for hyperthyroidism and differentiated thyroid cancer. Other applications include treatment of painful skeletal metastases, polycythemia vera, malignant cysts, and neuroendocrine tumors. The treatments are usually well tolerated and not associated with long-term effects, such as cancer or infertility. PMID- 10727600 TI - Physical and chemical properties of radionuclide therapy. AB - As more radionuclide therapies move from laboratory feasibility studies into clinical reality, it becomes increasingly important for the labeling chemistry to produce consistently a stable radiopharmaceutical that remains intact under the challenge of human catabolism. Similarly, once proof of principle is established to bring a radionuclide conjugate into clinical therapy trials, dosimetric estimates should be made to select the appropriate radionuclide properties, which are based on animal-specific or patient-specific pharmacokinetics and match a set of specific clinical endpoints. These properties may include the radionuclide physical half-life, radiolabeled conjugate biological uptake and clearance, product-specific activity, range and type of emissions, and resultant effects on tumor and normal tissue cellular survival. The immunologist and labeling chemist have now produced a variety of strategies that have potential to increase the therapeutic ratio (tumor-to-normal tissue dose ratio). The advent of normal tissue clearing agents, fragmented or chimerized carriers to improve targeting, and the method of bispecific or two-step and three-step targeting agents has increased the need for realistic modeling of the carrier in vivo to guide prospectively the competitive development of these radiopharmaceuticals. In this article, examples have been taken from the literature to elucidate the benchmark of success that careful experimental design has fostered to bring these agents into clinical practice by creative and logical methodologies. PMID- 10727602 TI - Review of low-dose-rate radiobiology for clinicians. AB - The use of therapeutic modalities that employ low-dose-rate (LDR) radiation is becoming increasingly prevalent in the clinic (eg, systemic targeted radiation therapy and brachytherapy). A natural tendency for radiation oncologists as they become familiar with these new therapies is to make predictions regarding efficacy and toxicity based on extrapolations from high-dose-rate radiobiology. If unfounded, these extrapolations could be misleading. This article discusses general principles of LDR radiobiology applicable to radioimmunotherapy. PMID- 10727601 TI - Internal dosimetry for systemic radiation therapy. AB - The key to effective use of the medical internal radiation dose (MIRD) schema in radioimmunotherapy (RIT) is to understand how it works and what the essential data input requirements are. The fundamental data are acquired from medical imaging. Image interpretation involves (1) collecting data to determine the source-organ activities, (2) plotting the source-organ time-activity curves, (3) integrating the time-activity curves for an estimate of the residence time, and (4) applying the residence time values (for each important source organ) within the MIRD schema to calculate the tissue absorbed dose to target organs and tumors of interest. This article reviews methods for calculating internal dose. It also describes methods for selecting sampling times, integrating the area under the data curves, and customizing a dose assessment for a patient who does not resemble the MIRD phantom. A sample dose assessment is given, together with common mistakes to avoid. Three approaches to red marrow dosimetry are described. With the increased use of RIT agents for cancer treatment, a solid understanding of internal dose methods is essential for treatment planning and follow-up evaluations. PMID- 10727603 TI - Radioimmunotherapy: designer molecules to potentiate effective therapy. AB - The evolution of monoclonal antibody forms for radioimmunotherapy and other antibody-based applications has been driven by a series of problems that each new form has introduced. Ehrlich was the first to present the concept that antibodies could be exploited in such a manner. Four decades were required before technological advances allowed the exploration of the potential of antibodies for radioimaging and radioimmunotherapeutic applications. Advances in DNA technology have led to the ability to tailor and manipulate the immunoglobulin molecule for specific functions and in vivo properties. This article discusses the use of monoclonal antibodies for radiotherapy with an emphasis on the problems that have been encountered and the subsequent solutions. PMID- 10727604 TI - Experimental radioimmunotherapy. AB - Experimental radioimmunotherapy (RIT) studies in animal models have contributed significantly to the design of clinical RIT protocols, although the results have not always been directly translated. Reviewed in this article are current areas of active research in experimental RIT to increase the therapeutic ratio that are likely to have a significant impact on the design of future clinical studies. Approaches for increasing the therapeutic efficacy of RIT include the development of new targeting molecules (genetically engineered monoclonal antibodies, antibody fragments, single-chain antibodies, diabodies and minibodies, fusion toxins, or peptides); improved labeling chemistry; novel radionuclide use and fractionation; locoregional administration; pretargeting; use of biological response modifiers or gene transfer techniques to increase target receptor expression; bone marrow transplantation; and combined modality therapy with external-beam radiation therapy, chemotherapy, or gene therapy. Further research with these new experimental approaches in preclinical animal models is necessary to contribute to advances in the treatment of cancer patients using radiolabeled antibodies and peptides. PMID- 10727605 TI - Functional role of cysteine residues in the (Na,K)-ATPase alpha subunit. AB - The structural-functional roles of 23 cysteines present in the sheep (Na,K) ATPase alpha1 subunit were studied using site directed mutagenesis, expression, and kinetics analysis. Twenty of these cysteines were individually substituted by alanine or serine. Cys452, Cys455 and Cys456 were simultaneously replaced by serine. These substitutions were introduced into an ouabain resistant alpha1 sheep isoform and expressed in HeLa cells under ouabain selective pressure. HeLa cells transfected with a cDNA encoding for replacements of Cys242 did not survive ouabain selective pressure. Single substitutions of the remaining cysteines yielded functional enzymes, although some had reduced turnover rates. Only minor variations were observed in the enzyme Na(+) and K(+) dependence as a result of these replacements. Some substitutions apparently affect the E1<-->E2 equilibrium as suggested by changes in the K(m) of ATP acting at its low affinity binding site. These results indicate that individual cysteines, with the exception of Cys242, are not essential for enzyme function. Furthermore, this suggests that the presence of putative disulfide bridges is not required for alpha1 subunit folding and subsequent activity. A (Na,K)-ATPase lacking cysteine residues in the transmembrane region was constructed (Cys104, 138, 336, 802, 911, 930, 964, 983Xxx). No alteration in the K(1/2) of Na(+) or K(+) for (Na,K)-ATPase activation was observed in the resulting enzyme, although it showed a 50% reduction in turnover rate. ATP binding at the high affinity site was not affected. However, a displacement in the E1<-->E2 equilibrium toward the E1 form was indicated by a small decrease in the K(m) of ATP at the low affinity site accompanied by an increase in IC(50) for vanadate inhibition. Thus, the transmembrane cysteine-deficient (Na,K)-ATPase appears functional with no critical alteration in its interactions with physiological ligands. PMID- 10727606 TI - Glucose induces a Na(+),K(+)-ATPase-dependent transient hyperpolarization in human sperm. I. Induction of changes in plasma membrane potential by the proton ionophore CCCP. AB - When human sperm was incubated in medium deprived of glucose, glucose restoration caused a transient hyperpolarization of the plasma membrane. This hyperpolarization was also induced by fructose but not by 2-deoxyglucose, a substrate that cannot be metabolized. The hyperpolarization was inhibited by NaF, a glycolysis inhibitor, but not by mitochondrial inhibitors (cyanide, rotenone and antimycin), suggesting that it depended on glycolysis. Furthermore, the hyperpolarization was still induced in medium containing a high concentration of KCl and was insensitive to the K(+) channel blocker TEA and the Cl(-) channel blocker niflumic acid, but it was blocked by ouabain. This suggested that upon glucose addition, there was an increase in the concentration of ATP, that in turns increased the Na(+),K(+)-ATPase activity. Since this pump is electrogenic (2K(+)/3Na(+)) the plasma membrane hyperpolarized. On the other hand, CCCP, a proton ionophore, inhibited the hyperpolarization induced by glucose. When CCCP was added to glucose-treated hyperpolarized sperm, it caused a depolarization that triggered a Ca(2+) influx sensitive to nickel, an inhibitor of voltage dependent calcium channels. Moreover, CCCP caused hyperpolarization in sperm incubated in medium without calcium, a known condition that depolarizes sperm. This indicated that CCCP induced proton permeability in the plasma membrane that was able to change the membrane potential to a value corresponding to the E(H) and that was also able to clamp it, so that it prevented the hyperpolarization induced by glucose. PMID- 10727607 TI - Molecular weight determination of membrane proteins by sedimentation equilibrium at the sucrose or nycodenz-adjusted density of the hydrated detergent micelle. AB - The determination of the molecular weight of a membrane protein by sedimentation equilibrium is complicated by the fact that these proteins interact with detergents and form complexes of unknown density. These effects become marginal when running sedimentation equilibrium at gravitational transparency, i.e., at the density corresponding to that of the hydrated detergent micelles. Dodecyl maltoside and octyl-glucoside are commonly used for dissolving membrane proteins. The density of micelles thereof was measured in sucrose or Nycodenz. Both proved to be about 50% lower than those of the corresponding non-hydrated micelles. Several membrane proteins were centrifuged at sedimentation equilibrium in sucrose- and in Nycodenz-enriched solutions of various densities. Their molecular weights were then calculated by using the resulting slope value at the density of the hydrated detergent micelles, i.e. at gravitational transparency, and the partial specific volume corrected for a 50% hydration of the membrane protein. The molecular weights of all measured membrane proteins, i.e. of photosystem II complex, reaction center of Rhodobacter sphaeroides R26, spinach photosystem II reaction center (core complex), bacteriorhodopsin, OmpF-porin and rhodopsin from Bovine retina corresponded within +/-15% to those reported previously, indicating a general applicability of this approach. PMID- 10727608 TI - Radiation inactivation studies of hepatic sinusoidal reduced glutathione transport system. AB - Sinusoidal transport of reduced glutathione (GSH) is a carrier-mediated process. Perfused liver and isolated hepatocyte models revealed a low-affinity transporter with sigmoidal kinetics (K(m) approximately 3.2-12 mM), while studies with sinusoidal membrane vesicles (SMV) revealed a high-affinity unit (K(m) approximately 0.34 mM) besides a low-affinity one (K(m) approximately 3.5-7 mM). However, in SMV, both the high- and low-affinity units manifested Michaelis Menten kinetics of GSH transport. We have now established the sigmoidicity of the low-affinity unit (K(m) approximately 9) in SMV, consistent with other models, while the high-affinity unit has been retained intact with Michaelis-Menten kinetics (K(m) approximately 0.13 mM). We capitalized on the negligible cross contributions of the two units to total transport at the low and high ends of GSH concentrations and investigated their characteristics separately, using radiation inactivation, as we did in canalicular GSH transport (Am. J. Physiol. 274 (1998) G923-G930). We studied the functional sizes of the proteins that mediate high- and low-affinity GSH transport in SMV by inactivation of transport at low (trace and 0.02 mM) and high (25 and 50 mM) concentrations of GSH. The low-affinity unit in SMV was much less affected by radiation than in canalicular membrane vesicles (CMV). The target size of the low-affinity sinusoidal GSH transporter appeared to be considerably smaller than both the canalicular low- and high-affinity transporters. The high-affinity unit in SMV was markedly inactivated upon irradiation, revealing a single protein structure with a functional size of approximately 70 kDa. This size is indistinguishable from that of the high affinity GSH transporter in CMV reported earlier. PMID- 10727609 TI - A hyperosmotic stress-induced mRNA of carp cell encodes Na(+)- and Cl(-) dependent high affinity taurine transporter. AB - A cDNA clone encoding a Na(+)- and Cl(-)-dependent high affinity taurine transporter was isolated from a common carp cell line, Epithelioma papulosum cyprini (EPC), as a hyperosmotic stress-inducible gene by RNA arbitrarily primed PCR. The clone contained a 2.5-kb cDNA fragment including an open reading frame of 1878 bp encoding a protein of 625 amino acids. The deduced amino acid sequence of carp taurine transporter shows 78-80% identity to those of cloned mammalian taurine transporters. The functional characteristics of the cloned transporter were analyzed by expression in COS-7 cells. Transfection with the cDNA induced Na(+)- and Cl(-)-dependent taurine transport activity with an apparent K(m) of 56 microM. The Na(+)/Cl(-)hepatopancreas. Taurine transporter mRNA level increased up to 7.5-fold on raising the ambient osmolality from 300 to 450 mosmol/kgH(2)O. These data suggest the significant role of taurine as an osmolyte in carp cells. PMID- 10727610 TI - Variation of phospholamban in slow-twitch muscle sarcoplasmic reticulum between mammalian species and a link to the substrate specificity of endogenous Ca(2+) calmodulin-dependent protein kinase. AB - Systematic immunological and biochemical studies indicate that the level of expression of sarcoplasmic reticulum (SR) Ca(2+)-ATPase regulatory protein phospholamban (PLB) in mammalian slow-twitch fibers varies from zero, in the rat, to significant levels in the rabbit, and even higher in humans. The lack of PLB expression in the rat, at the mRNA level, is shown to be exclusive to slow-twitch skeletal muscle, and not to be shared by the heart, thus suggesting a tissue specific, in addition to a species-specific regulation of PLB. A comparison of sucrose density-purified SR of rat and rabbit slow-twitch muscle, with regard to protein compositional and phosphorylation properties, demonstrates that the biodiversity is two-fold, i.e. (a) in PLB membrane density; and (b) in the ability of membrane-bound Ca(2+)-calmodulin (CaM)-dependent protein kinase II to phosphorylate both PLB and SERCA2a (slow-twitch isoform of Ca(2+)-ATPase). The basal phosphorylation state of PLB at Thr-17 in isolated SR vesicles from rabbit slow-twitch muscle, colocalization of CaM K II with PLB and SERCA2a at the same membrane domain, and the divergent subcellular distribution of PKA, taken together, seem to argue for a differential heterogeneity in the regulation of Ca(2+) transport between such muscle and heart muscle. PMID- 10727611 TI - Biological function of the LH receptor is associated with slow receptor rotational diffusion. AB - The biological activity of luteinizing hormone (LH) receptors can be affected by modifications to the receptor's amino acid sequence or by binding of hormone antagonists such as deglycosylated hCG. Here we have compared rotational diffusion of LH receptors capable of activating adenylate cyclase with that of non-functional hormone-occupied receptors at 4 degrees C and 37 degrees C using time-resolved phosphorescence anisotropy techniques. Binding of hCG to the rat wild-type receptor expressed on 293 cells (LHR-wt cells) or to the LH receptor on MA-10 cells produces functional receptors which exhibit rotational correlation times longer than 1000 micros. However, modification of the LH receptor by substitution of Lys583-->Arg (LHR-K583R) results in a receptor that is non functional and which has a significantly shorter rotational correlation time of 130+/-12 micros following binding of hCG. When these receptors are treated with deglycosylated hCG, an inactive form of hCG, the rotational correlation times for the LH receptors on LHR-wt and MA-10 cells are also shorter, namely 64+/-8 and 76+/-14 micros, respectively. Finally, a biologically active truncated form of the rat LH receptor expressed in 293 cells (LHR-t631) has slow rotational diffusion, greater than 1000 micros, when occupied by hCG and a significantly shorter rotational correlation time of 103+/-12 micros when occupied by deglycosylated hCG. The effects of rat LH binding to LH receptors on these various cell lines were similar to those of hCG although the magnitude of the changes in receptor rotational diffusion were less pronounced. We suggest that functional LH receptors are present in membrane complexes that exhibit slow rotational diffusion or are rotationally immobile. Shorter rotational correlation times for non-functional hormone-receptor complexes may reflect the absence of essential interactions between these complexes and other membrane proteins. PMID- 10727612 TI - Interaction of oligonucleotides with cationic lipids: the relationship between electrostatics, hydration and state of aggregation. AB - Lipoplexes, which are spontaneously formed complexes between oligonucleotide (ODN) and cationic lipid, can be used to deliver ODNs into cells, both in vitro and in vivo. The present study was aimed at characterizing the interactions associated with the formation of lipoplexes, specifically in terms of electrostatics, hydration and particle size. Large unilamellar vesicles (approximately 100 nm diameter), composed of either DOTAP, DOTAP/cholesterol (mole ratio 1:1) or DOTAP/DOPE (mole ratio 1:1) were employed as a model of cationic liposomes. Neutral vesicles ( approximately 100 nm diameter), composed of DOPC/DOPE (mole ratio 1:1), were employed as control liposomes. After ODN addition to vesicles, at different mole ratios, changes in pH and electrical surface potential at the lipid-water interface were analyzed by using the fluorophore heptadecyl-7-hydroxycoumarin. In separate 'mirror image' experiments, liposomes were added at different mole ratios to fluorescein isothiocyanate labeled ODNs, thus yielding data about changes in the pH near the ODN molecules induced by the complexation with the cationic lipid. Particle size distribution and turbidity fluctuations were analyzed by the use of photon correlation spectroscopy and static light-scattering, respectively. In additional fluorescent probe studies, TMADPH was used to quantify membrane defects while laurdan was used to measure the level of hydration at the water-lipid interface. The results indicate that mutual neutralization of cationic lipids by ODNs and vice versa is a spontaneous reaction and that this neutralization is the main driving force for lipoplex generation. When lipid neutralization is partial, induced membrane defects cause the lipoplexes to exhibit increased size instability. PMID- 10727614 TI - The effect of hexadecaprenyl diphosphate on phospholipid membranes. AB - In the present study we investigated phospholipid bilayer membranes and phospholipid vesicles made from dioleoylphosphatidylcholine (DOPC) or its mixture with the phosphate ester derivative of long-chain polyprenol (hexadecaprenyl diphosphate, C(80)-PP) by electrophysiological and transmission electron microscopy (TEM) techniques. The membrane conductance-temperature relationships and the membrane breakdown voltage have been measured for different mixtures of C(80)-PP/DOPC. The current-voltage characteristics, the membrane conductance, the activation energy of ion migration across the membrane and the membrane breakdown voltage were determined. Hexadecaprenyl diphosphate decreases the membrane conductance, increases the activation energy and the membrane breakdown voltage for the various values of C(80)-PP/DOPC mole ratio. The analysis of TEM micrographs shows several characteristic structures, which have been described. The data indicate that hexadecaprenyl diphosphate modulates the surface curvature of the membranes by the formation of aggregates in liquid-crystalline phospholipid membranes. The properties of modified membranes can result from the presence of the negative charges in the hydrophilic part of C(80)-PP molecules and can be modulated by the concentration of this compound in membranes. We suggest that the dynamics and conformation of hexadecaprenyl diphosphate in membranes depend on the transmembrane electrical potential. PMID- 10727613 TI - Effects of lectins on calcification by vesicles isolated from aortas of cholesterol-fed rabbits. AB - Advanced vascular calcification in atherosclerosis weakens arterial walls, thereby imposing a serious rupturing effect. However, the mechanism of dystrophic calcification remains unknown. Although accumulating morphological and biochemical evidence reveals a role for calcifiable vesicles in plaque calcification, the mechanism of vesicle-mediated calcification has not been fully explored. To study whether vesicles' membrane components, such as carbohydrates, may have a role in vesicle-mediated calcification, the effect of sugar-binding lectins on calcification was investigated. Atherosclerosis was developed by feeding rabbits with a diet supplemented with 0.5% cholesterol and 2% peanut oil for 4 months. Calcifiable vesicles were then isolated from thoracic aortas by collagenase digestion. The histological examination of aortas with hematoxylin counter-staining indicated abnormal formation of large plaques enriched with macrophage-derived foam cells. Fourier transform spectroscopy revealed mild calcification in aortas indicating that advanced stages of heavy calcification have yet to be reached. However, vesicles isolated from the aortas were capable of calcification in the presence of physiological levels of Ca(2+), Pi, and ATP. Thus, at this stage of atherosclerosis, aortas may start to produce calcifiable vesicles, but at a level insufficient for substantial formation of mineral in aortas. The assessments by FT-IR analysis and Alizarin red staining indicated that concanavalin A (Con A) substantially increased mineral formation by isolated vesicles. Con A also exerted a marked stimulatory effect on (45)Ca and (32)Pi deposition in a dose-dependent fashion with a half-maximal effect at 6-10 microg/ml. Either alpha-methylmannoside or alpha-methylglucoside, but not mannitol, at 10 mM abolished the stimulation. Con A stimulation was abolished after Con A was removed from calcifying media, suggesting that covalent binding may not be involved in the effect. Galactosides appear to also be implicated in (45)Ca and (32)Pi deposition since Abrus precartorius agglutinin, which specifically binds galactosides, enhanced the deposition. Neither wheat-germ agglutinin that binds N-acetylglucoside nor N-acetylgalactoside-specific Helix pomatia agglutinin was effective, suggesting that the acetylated forms of carbohydrate moieties are either absent in vesicles or may not be involved in calcification. None of these lectins exerted an effect on ATPase. Thus, the effects of lectins appeared to be mediated through interactions with carbohydrate moieties of calcifiable vesicles. Whether stimulation of vesicle-calcification by lectins is of pathological significance in atherosclerotic calcification requires further investigation. PMID- 10727616 TI - Oligonucleotide delivery by a cationic derivative of the polyene antibiotic amphotericin B. I: interaction oligonucleotide/vector as studied by optical spectroscopy and electron microscopy. AB - Antisense strategy requires efficient systems for the delivery of oligodeoxyribonucleotides (ODN) into target cells. Cationic amphiphiles have shown good efficiency in vitro and a lot of attention is currently paid to their interaction with nucleic acids. In the present study, this interaction was, for the first time, analysed at the molecular level, taking advantage of the spectroscopic properties of the positively charged chiral polyene molecule amphotericin B 3-dimethylaminopropyl amide (AMA), the efficiency of which, as delivery system, has been demonstrated [Garcia et al., Pharmacol. Ther. (2000), in press]. By UV-visible absorption and circular dichroism (CD) we studied its self-association properties in pure water, saline and RPMI medium. Drastic changes were observed upon ODN addition, stronger in pure water than in media of high ionic strength. At low AMA concentration (<10(-6) M), the strong increase of the CD signal, characteristic of self-association, indicated condensation of AMA on the ODN molecules. At a higher concentration (10(-4) M), and for a nucleic acid negative charge/AMA positive charge ratio higher than 1, spectra were interpreted as a reorganisation of free self-associated AMA species into smaller ones 'decorating' the nucleic acid molecule. Electron microscopy data were interpreted according to this scheme. PMID- 10727615 TI - Biochemical and biophysical characterization of in vitro folded outer membrane porin PorA of Neisseria meningitidis. AB - Two subtypes of the outer membrane porin PorA of Neisseria meningitidis, P1.6 and P1.7,16, were folded in vitro after overexpression in, and isolation from Escherichia coli. The PorA porins could be folded efficiently by quick dilution in an appropriate buffer containing the detergent n-dodecyl-N, N-dimethyl-1 ammonio-3-propanesulphonate. Although the two PorA porins are highly homologous, they required different acidities for optimal folding, that is, a pH above the pI was needed for efficient folding. Furthermore, whereas trimers of PorA P1.7,16 were almost completely stable in 2% sodium dodecyl sulphate (SDS), those of P1.6 dissociated in the presence of SDS. The higher electrophoretic mobility of the in vitro folded porins could be explained by the stable association of the RmpM protein to the porins in vivo. This association of RmpM contributes to the stability of the porins. The P1.6 pores were moderately cation-selective and displayed a single-channel conductance of 2.8 nS in 1 M KCl. The PorA P1.6 pores, but not the PorA P1.7,16 pores, showed an unusual non-linear dependence of the single-channel conductance on the salt concentration of the subphase. We hypothesize that a cluster of three negatively charged residues in L5 of P1.6 is responsible for the higher conductance at low salt concentrations. PMID- 10727617 TI - Oligonucleotide delivery by a cationic derivative of the polyene antibiotic amphotericin B. II: study of the interactions of the oligonucleotide/cationic vector complexes with lipid monolayers and lipid unilamellar vesicles. AB - We report a study of the behavior of oligodeoxyribonucleotide (ODN)/amphotericin B3-(N'-dimethylamino)propylamide (AMA) complexes, in the presence of lipid monolayers and large unilamellar vesicles. This study follows the recent discovery of the capacity of AMA, as a new cationic vector, to enhance ODN cellular uptake and efficacy. It aims at investigating the internalization mode of a nucleic acid by AMA. A first study at the air-water interface of AMA and AMA/ODN by surface pressure measurement shows that only free AMA would adsorb at the air-water interface. Second, in the presence of zwitterionic phospholipid- and sterol-containing mixture, ODN-AMA interactions in solution would be higher than lipid-AMA interactions at the interface. In monolayer or with large unilamellar vesicles, AMA monomers adsorb mainly at the phospholipid interface. These results favor a crossing mechanism through AMA channel formation, despite the size of ODN. PMID- 10727618 TI - Brain regions where cholecystokinin suppresses feeding in rats. AB - The gut-brain peptide, cholecystokinin (CCK), inhibits food intake when injected either systemically or within the brain. To determine whether CCK's effect in the brain is anatomically specific, CCK-8 (0. 8, 4, 20, 100, 500 pmol) was microinjected into one of 14 different brain sites of rats, and its impact on subsequent food intake was measured. CCK-8 at 500 pmol significantly suppressed intake during the first hour post-injection following administration into six hypothalamic sites (anterior hypothalamus, dorsomedial hypothalamus, lateral hypothalamus, paraventricular nucleus, supraoptic nucleus, ventromedial hypothalamus) and two hindbrain sites (nucleus tractus solitarius, fourth ventricle). Although lower doses were sometimes effective (anterior hypothalamus, dorsomedial hypothalamus, nucleus tractus solitarius), there appeared to be no significant difference in potency among sites. Injections into the medial amygdala, nucleus accumbens, posterior hypothalamus, dorsal raphe, and ventral tegmental area were either ineffective or produced a delayed response. The higher doses required for most sites, as well as the widespread effectiveness of CCK-8 within the hypothalamus, suggest that spread of CCK-8 to adjacent brain sites, and (or) to the periphery, may have been required for anorexia to occur. Findings reported in an accompanying paper provide strong evidence that paraventricular nucleus injection of CCK-8 (500 pmol) did not increase plasma CCK-levels sufficiently to suppress feeding by a peripheral mechanism. Together, these results suggest that CCK may be acting as a neurotransmitter or neuromodulator within two different brain regions to produce satiety - one region which includes the nucleus tractus solitarius in the hindbrain, and another more distributed region within the medial-basal hypothalamus. PMID- 10727619 TI - Effects of paraventricular nucleus injection of CCK-8 on plasma CCK-8 levels in rats. AB - The aim of the study was to determine whether paraventricular nucleus (PVN) injection of an anorexic 500-pmol dose of cholecystokinin (CCK)-8 could increase plasma CCK-8 levels sufficiently to suppress feeding by a peripheral mechanism. Rats received PVN injections of CCK-8 either alone or with 3H-labelled propionylated CCK-8 (3H-pCCK-8) and plasma samples were taken at various times from 3 to 120 min post-injection. Plasma CCK-8 levels were estimated from measurements of both total plasma CCK-like immunoreactivity (CCK-LI) and 3H-pCCK 8 activity. PVN injections of CCK-8 and 3H-pCCK-8 produced estimated peak increases in plasma CCK-8 of 15+/-11 and 22+/-3 pM, respectively. The i.v. infusion of CCK-8 doses (0.2 and 1 nmol/kg h) that bracketed the threshold dose for suppression of feeding, increased plasma CCK-LI from a basal level of 6+/-1 to 49+/-10 and 166+/-36 pM, respectively. The i.v. injections of 600 and 4800 pmol of CCK-8 did not suppress feeding. These results suggest that PVN injection of an anorexic 500-pmol dose of CCK-8 does not increase plasma CCK-8 levels sufficiently to suppress feeding by a peripheral mechanism. PMID- 10727620 TI - Relationship between large conductance calcium-activated potassium channel and bursting activity. AB - To elucidate the role of the large conductance calcium-activated potassium channel (BK(Ca) channel) in the production of bursting activity, which is characteristic of convulsions, effects of iberiotoxin (IbTX), a selective blocker of the BK(Ca) channel, on bursting activity, induced by various procedures were examined using primary cultured neurons from the cerebral cortex of mice. IbTX completely inhibited bursting activity induced by pentylenetetrazol (PTZ), caffeine, 1,4,5-inositol triphosphate (IP3) and direct forced increase of intracellular calcium. Inherent spontaneous bursting activity in the cerebral cortical neurons of the El mouse, which shows a high susceptibility to convulsions was also completely inhibited by IbTX. Apamin, a specific blocker of the small conductance calcium-activated potassium channel (SK(Ca) channel) showed no inhibition of bursting activity. These findings suggest that the BK(Ca) channel is essential for the production of bursting activity, and also suggest the possibility of clinical use of blocking agents of the BK(Ca) channel against intractable epilepsy. PMID- 10727622 TI - Distribution of the GABA(B) receptor subunit gb2 in rat CNS. AB - We have identified and isolated human and rat cDNAs for a novel receptor, gb2, with 38% homology to the GABA(B) receptors gb1a and gb1b. These receptors comprise a new subfamily of seven transmembrane G protein-coupled receptors (GPCRs) that share structure and sequence similarities with the metabotropic glutamate receptors. In situ hybridization histochemistry using an antisense probe to this novel receptor mRNA shows a distribution in rat CNS nearly identical to that for the gb1 receptor, although some regions showed significant differences. Specifically, message levels for gb2 were virtually absent in the caudate/putamen, and significantly lower in the medial basal hypothalamus, septum and brainstem as compared with gb1 message levels. In contrast to gb1, gb2 mRNA was never detected in white matter suggesting that gb2 message is found exclusively in neurons. Finally, in rat brain regions showing significant overlap of message for gb1 and gb2, the transcripts are often found in the same cells. Data from our previous work showing that coexpression of gb2 with gb1 is necessary for expression of a functional receptor together with the detailed anatomical data presented here indicate that native GABA(B) receptors function as heteromeric proteins, the most abundant form being the gb1/gb2 receptor. However, the more limited distribution of gb2 receptor mRNA suggests that there are brain regions where GABA(B) receptors are composed of gb1 and as yet unidentified family members. PMID- 10727621 TI - Phenobarbital administration directed against kindled seizures delays functional recovery following brain insult. AB - Anti-convulsant drug administration or recurrent seizures can impact functional recovery following brain insult. The nature of that impact depends on a variety of factors, including timing of drug administration and drug mechanism of action, as well as seizure number, timing, and severity. The objective of this study was to determine the functional consequences of anti-convulsant administration directed against seizure activity in brain-damaged animals. To this end, phenobarbital was coupled with daily electrical kindling of the amygdala beginning 48 h after a unilateral anteromedial cortex lesion. Recovery from somatosensory deficits was assessed, as was regional atrophy and basic fibroblast growth factor (bFGF) expression. Animals receiving phenobarbital prior to daily kindling failed to recover within 2 months of testing. In contrast, animals receiving saline prior to kindling as well as phenobarbital-treated non-kindled animals recovered within 2 months after the lesion. Though the exact mechanisms underlying these behavioral phenomena remain uncertain, patterns of bFGF expression among the groups provide some insight. Taken together, results from the present study suggest that anti-convulsant drug administration directed against subclinical seizure activity can be more detrimental to functional recovery than seizures alone or anti-convulsant drug treatment after seizure activity has occurred. PMID- 10727623 TI - Differential expression of EGR-1 mRNA in the amygdala following diazepam in contextual fear conditioning. AB - The amygdaloid complex is thought to be a major site of action of anxiolytic benzodiazepine agonists. To investigate whether activity in the amygdaloid complex is altered with anxiolytic effects of diazepam, mRNA expression of the immediate-early gene EGR-1 was examined in the amygdala following blockade of fear conditioning by diazepam. It was previously shown that mRNA expression of EGR-1 (also called, NGFI-A, Zif 268, Krox 24) increases in the lateral nucleus of the amygdala (LA) shortly following contextual fear conditioning. It was therefore hypothesized that diazepam would block both contextual fear and the concomitant increase in EGR-1 mRNA expression in the LA induced by fear conditioning. Rats administered systemic diazepam before fear conditioning displayed both anxiolytic effects during the post-shock period and amnesic effects during a retention test 24 h later. Diazepam blocked the fear conditioning-induced increase in EGR-1 expression in the LA. In addition, diazepam significantly increased EGR-1 mRNA expression in the central nucleus of the amygdala (CeA) in a dose-dependent manner. The results reveal differential regulation of EGR-1 by diazepam in the central and lateral nuclei of the amygdala suggesting that these two amygdala nuclei act in a reciprocal manner during the anxiolytic and amnesic action of the benzodiazepine agonist. PMID- 10727624 TI - A MEG study of sleep. AB - A 64-channel, whole cortex magnetoencephalographic system was employed to obtain sleep data from three healthy subjects. Based upon visual inspection of the signals and the corresponding power spectra, we were able to discern a number of features characterizing the evolution of sleep. These included: (1) the transition from records dominated by the alpha rhythm to records in which alpha is attenuated and slower waves increase; (2) the appearance of sleep spindles, particularly in the parietal channels; and, perhaps most interesting, (3) a slow wave phase whose multichannel spectral signature is a broad rounded maximum in the frequency region around 0.5 Hz. Topographical features of the sleep record were also studied. In two of our subjects, rough lateral symmetry was apparent. As their sleep deepened, the distribution of signal power over the head changed such that the maximum moved in the forward and lateral directions, with parietal and temporal signals strengthening relative to the occipital. The records of the third subject showed a tendency toward right dominance, while topographic changes with sleep depth were minimal. Only one of the subjects was able to sustain the deep, slow-wave stage. Here, characteristic multi-detector outbursts appeared, lasting between 150 and 500 ms. During these intervals, widespread topographic patterns were sustained over the head (often with striking dipolar or quadrupolar forms), while crude source modeling yielded two persisting dipoles, laterally paired. Thus, these outbursts seem to represent large-scale, quasi-static configurations of brain activity perhaps related to the K-complexes, which occur earlier in sleep. Finally, we compare our results with those of previous investigators, including work on human electroencephalographic data and research reported by Steriade et al. from animal studies. PMID- 10727625 TI - Neonatal lesions of the left entorhinal cortex affect dopamine metabolism in the rat brain. AB - The present study was performed to determine the effects of neonatal excitotoxic lesions of the left entorhinal cortex on dopamine (DA) metabolism and release in limbic regions of the rat brain. Quinolinic acid or phosphate buffered saline was infused into the left entorhinal cortex of rat pups on postnatal day 7 (PD7). Concentrations of DA,3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the lateral amygdala, nucleus accumbens, caudate-putamen, and medial prefrontal cortex were determined in the postmortem brains of lesioned and sham operated rats on PD35 and PD56. On PD35, concentrations of DA in the bilateral lateral amygdala and HVA in the left lateral amygdala were significantly increased in lesioned rats compared with sham-operated animals, while no significant change was observed in the other three brain areas. On PD56, in addition to the increased concentration of DA in the left lateral amygdala, those of DA, DOPAC and HVA in the caudate-putamen, and DA in the nucleus accumbens were found to be increased, but DA concentrations in the right medial prefrontal cortex were decreased. The DOPAC/DA concentration ratio was, however, decreased in the amygdala and nucleus accumbens of the lesioned rats. In an in vivo microdialysis study, methamphetamine (MAP: 2 mg/kg, i.p.)-induced DA release in the amygdala of lesioned rats was significantly enhanced compared with sham operated rats on both PD35 and PD56. There were no significant differences in MAP induced DA release in the caudate-putamen between the sham-operated and lesioned rats at any time point. These findings provide evidence that neonatally induced structural abnormalities in the entorhinal cortex affect DA transmission in the limbic regions at the adolescent stage. PMID- 10727626 TI - The human cerebellum and associative learning: dissociation between the acquisition, retention and extinction of conditioned eyeblinks. AB - The present paper is part of a systematic exploration of the neural substrates of conditioned eyeblink responses in humans. Normal subjects and patients with lesions restricted to the cerebellum were examined for their ability to acquire new classically conditioned eyeblinks to an auditory conditioned stimulus and whether they were able to perform and extinguish a previously learned natural anticipatory eyeblink response - the kinesthetic threat eyeblink response (KTER). In classical conditioning to an auditory conditioned stimulus, cerebellar patients failed to acquire new conditioned responses. In contrast to this impairment, in the KTER task both cerebellar patients and control subjects exhibited a high incidence of anticipatory eyeblinks which were initiated before the forehead tap. These results indicate that the cerebellar circuits, which are critical for the acquisition of new conditioned responses, are not essential for the storage and expression of naturally acquired conditioned responses. In the extinction experiment, cerebellar patients failed to extinguish their KTERs. This finding suggests that in humans, the acquisition of new and the extinction of previously learned conditioned responses depends on a similar set of cerebellar circuits. PMID- 10727627 TI - Alterations in hippocampal GAP-43 phosphorylation and protein level following contextual fear conditioning. AB - C57BL/6 (B6) mice display better contextual learning than the DBA/2 (D2) mice. The possibility that GAP-43, is differentially affected as a function of strain and learning was investigated in the present study. No basal difference between C57BL/6J (B6) and DBA/2J (D2) mice in the amount of hippocampal GAP-43 was observed, but naive D2 mice have slightly lower basal levels of GAP-43 phosphorylation than do B6 mice. Interestingly, alterations in hippocampal GAP-43 protein levels and phosphorylation state in response to training for contextual learning were observed only in B6 mice. Immediate-shocked mice, serving as nonlearning controls, showed no GAP-43 alterations, nor did D2 mice subjected to either training condition. These results suggest that modulation of hippocampal GAP-43 may be important for contextual learning and that strain-specific alterations in GAP-43 may be part of a disrupted pathway in D2 mice that is essential for learning. PMID- 10727628 TI - Reduction in excessive muscle tone by selective depletion of serotonin in intercollicularly decerebrated rats. AB - Intercollicular decerebration in animals induces sustained facilitation of muscle tone of the limbs and this animal model has been used to assess centrally acting muscle relaxants. We have examined the involvement of central and spinal cord serotonergic pathways in the onset of excessive muscle tone in an intercollicularly decerebrated rat. Descending serotonergic pathways are known to modulate, directly or indirectly, the excitability of spinal cord motoneurons and it is inferred that serotonin (5-HT) plays an important role in locomotion. Alteration of muscle tone has been investigated in 5-HT-depleted rats with a neurotoxin, 5, 7-dihydroxytryptamine (5,7-DHT) after pretreatment with desipramine. Intracerebroventricular (i.c.v.) administration of 5,7-DHT reduced 5 HT content in the forebrain to 50.5% and that in the spinal cord to 10.5%, while intrathecal (i.t.) administration of 5,7-DHT decreased 5-HT content in the spinal cord to 8.9% without causing any change in the forebrain. In contrast, noradrenaline or dopamine content was not affected by the neurotoxin in both tissues. These treatments significantly attenuated the muscle tone in the animal models. Moreover, the measurement of 5-HT and 5-hydroxyindoleacetic acid content in intact rats after decerebration showed that facilitation of the 5-HT turnover in the spinal cord, but not in the forebrain, was enhanced compared with sham operated rats. These findings suggest that the descending serotonergic pathways are essential to induce excessive muscle tone in the intercollicular decerebrated rats and that 5-HT antagonists might be candidates for centrally acting muscle relaxants. PMID- 10727629 TI - Melatonin reversal of DOI-induced hypophagia in rats; possible mechanism by suppressing 5-HT(2A) receptor-mediated activation of HPA axis. AB - Serotonin type 2A (5-HT(2A)) receptor-mediated neurotransmitter is known to activate hypothalamic-pituitary-adrenal (HPA) axis, regulate sleep-awake cycle, induce anorexia and hyperthermia. Interaction between melatonin and 5-HT(2A) receptors in the regulation of the sleep-awake cycle and head-twitch response in rat have been reported. Previous studies have shown that melatonin has suppressant effect on HPA axis activation, decreases core body temperature and induces hyperphagia in animals. However, melatonin interaction with 5-HT(2A) receptors in mediation of these actions is not yet reported. We have studied the acute effect of melatonin and its antagonist, luzindole on centrally administered (+/-)-1-(2, 5-dimethoxy-4-iodophenyl) 2-amino propane (DOI; a 5-HT(2A/2C) agonist)-induced activation of HPA axis, hypophagia and hyperthermia in 24-h food deprived rats. Like ritanserin [(1 mg/kg, i.p.) 5-HT(2A/2C) antagonist], peripherally administered melatonin (1.5 and 3 mg/kg, i.p.) did not affect the food intake, rectal temperature or basal adrenal ascorbic acid level. However, pretreatment of rats with it significantly reversed DOI (10 microgram, intraventricular)-induced anorexia and activation of HPA axis. But the hyperthermia induced by DOI was not sensitive to reversal by melatonin. Mel(1) receptor subtype antagonist luzindole (5 microgram, intraventricular) did not modulate the DOI effect but antagonized the melatonin (3 mg/kg, i.p.) reversal of 5-HT(2A) agonist response. The present data suggest that melatonin reversal of DOI-induced hypophagia could be due to suppression of 5-HT(2A) mediated activation of HPA axis. PMID- 10727630 TI - Chemosensitivity of non-respiratory rat CNS neurons in tissue culture. AB - Neurons from many brainstem nuclei involved in respiratory control increase their firing rate in response to acidosis in vitro, suggesting that they are central chemoreceptors. This property has been considered to be either unique to neurons involved in respiratory control, or at least very unusual for non-respiratory neurons. However, recordings of intrinsic pH responses of neurons have not been made from enough non-respiratory regions of the CNS to be certain this assumption is true. Here, we have quantified changes in firing rate of neurons cultured from the hippocampus (n=43), neocortex (n=33), and cerebellum (n=29) in response to changes in CO(2) between 3% and 9% (pH approximately 7.6-7.2) after blockade of glutamatergic and GABAergic transmission. The responses of neurons from these three regions were similar, with a subset of neurons (12% of the total 105) inhibited by acidosis, decreasing their firing rate to a mean of 70% of control in response to a decrease in pH of 0.2. Some neurons (5% of total) were stimulated by acidosis, with an increase in firing rate to a mean of 175% of control in response to a decrease in pH of 0.2. We previously quantified chemosensitivity of neurons from the medullary raphe using the same methods [W. Wang, J.H. Pizzonia, G.B. Richerson, Chemosensitivity of rat medullary raphe neurones in primary tissue culture, J. Physiol., 511 (1998) 433-450]. Compared to these non-respiratory neurons, more raphe neurons were stimulated by acidosis (22%), and the average response was greater (to 300% of control) in response to the same stimulus. Thus, over a physiologically relevant pH range, stimulation by acidosis occurs in a significant percentage of neurons not involved in respiratory chemoreception. However, the degree of chemosensitivity of these neurons was less than medullary raphe neurons under the same conditions. Chemosensitivity is not an all-or-none neuronal property, and the degree of chemosensitivity may be relevant to the role neurons play in sensing pH in vivo. PMID- 10727631 TI - Increasing CNS norepinephrine levels by the precursor L-DOPS facilitates beam walking recovery after sensorimotor cortex ablation in rats. AB - The present investigation was conducted to document a role of L-threo-3,4 dihydroxyphenylserine (L-DOPS), precursor of L-norepinephrine (NE), in the functional recovery from beam-walking performance deficits in rats after unilateral sensorimotor cortex ablation. L-DOPS was administered simultaneously with benserazide (BSZ; a peripheral aromatic amino acid decarboxylase inhibitor), and the regional contents of NE in the cerebral cortex, hippocampus, and cerebellum were assayed. Behavioral recovery was demonstrated by the rats treated with L-DOPS and BSZ, and the rate of recovery was significantly different from that of either BSZ-treated or vehicle-treated control rats. The NE tissue levels in the three discrete regions of the rat brain were significantly elevated in the experimental rats receiving both L-DOPS and BSZ. The present studies indicate that increasing NE levels by the precursor L-DOPS may be responsible for facilitating behavioral recovery from beam-walking performance deficits in rats, and further suggest that L-DOPS may become one of the candidate compounds for further clinical human trials promoting functional recovery after injuries to the cerebral cortex. PMID- 10727632 TI - Morphine-induced sensitization of locomotor activity in mice: effect of social isolation on plasma corticosterone levels. AB - This study examined the influence of social isolation on behavioural sensitization to the locomotor effect of morphine and the link between this behaviour and plasma corticosterone concentrations. Four weeks isolation induced an increase in the locomotor effect of morphine. In social and isolated mice, repeated administrations (6) of morphine (one injection every 3 or 4 days) followed by 3 h in an actimeter induced behavioural sensitization to the locomotor effect of morphine. No interaction was observed between social isolation and behavioural sensitization to morphine. Resocializing previously isolated mice for 3 weeks reduced the morphine-induced locomotor effect without altering the behavioural sensitization. Corticosterone plasma levels were more increased (416%) in mice isolated 5 weeks than in mice isolated for 2 weeks (243%) and they return to the control levels following 3 weeks of resocialization. Since there was no interaction between the increase in morphine locomotor effect induced by social isolation and the morphine-induced behavioural sensitization, it is suggested that each of these two events acts independently. Whether or not a common mechanism (plasma corticosterone levels?) partly underlies both effects, the result resembles a simple additive effect. PMID- 10727633 TI - Dose-response and duration effects of acute administrations of cocaine and GBR12909 on dopamine synthesis and transporter in the conscious monkey brain: PET studies combined with microdialysis. AB - The dose-response and duration effects of acute administration of the dopamine transporter (DAT) blocker cocaine and GBR12909 on dopamine synthesis and transporter availability were evaluated in the brains of conscious monkeys using high-resolution positron emission tomography (PET) in combination with microdialysis. Rate of dopamine synthesis and DAT availability were evaluated using L-[beta-11C]DOPA and [11C]beta-CFT (WIN35,428), respectively. Administration of cocaine (0.5, 2 and 5 mg/kg) resulted in dose-dependent elevation of dopamine level in the striatal extracellular fluid (ECF) at 0.5 h after injection, and returned to the baseline level within 1.5 h post-injection. At 0.5 post-injection, cocaine reduced dopamine synthesis rate and DAT availability in a dose-dependent manner. The reduction of DAT availability by cocaine (2 mg/kg) returned to baseline level at 3 h post-injection and thereafter. Interestingly, dopamine synthesis rate was significantly higher at 3 h than baseline level and returned to baseline level 5.5 h post-injection. When GBR12909 (0.5, 2 and 5 mg/kg) was administered 0.5 h before tracer injection, dopamine synthesis rate and DAT availability were significantly decreased in a dose-dependent manner. These reductions induced by GBR12909 (2 mg/kg) lasted at least until 5.5 h post-injection. GBR12909 induced dose-dependent elevation of dopamine level in ECF, and the elevation lasted up to 7 h. The present results indicated that cocaine and GBR12909 affect dopamine synthesis rate and DAT availability in the striatum with difference time courses as measured by PET in the conscious monkey brains. PMID- 10727635 TI - Diphosphorylation of extracellular signal-regulated kinases and c-Jun N-terminal protein kinases in brain ischemic tolerance in rat. AB - Extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal protein kinases (JNKs) activation in brain ischemic tolerance were examined by Western immunoblot. ERK but not JNK diphosphorylation (activation) were increased after preconditioning ischemia. The increased JNK1 but not ERK diphosphorylation after lethal ischemia was eliminated by pretreatment with preconditioning ischemia. The results suggest that the elimination of JNK1 activation after lethal ischemia by preconditioning ischemia may be one of the important protective mechanisms in ischemic tolerance, and ERKs activation may be involved in the induction of the protective responses. PMID- 10727634 TI - Systemically administered D-glucose conjugates of 7-chlorokynurenic acid are centrally available and exert anticonvulsant activity in rodents. AB - We have synthesized D-glucose or D-galactose esters of 7-chlorokynurenic acid (7ClKynA) as prodrugs to facilitate the transport of 7ClKynA across the blood brain barrier. Intraperitoneal (i.p.) administration of either 7ClKynA-D glucopyranos-6'-ylester (7ClKynA/Glu6) or 7ClKynA-D-glucopyranos-3'-yl ester (7ClKynA/Glu3) was protective against seizures induced by N-methyl-D-aspartate (NMDA) in mice, with the former drug showing the highest anticonvulsive activity. Systemic injection of equal amounts of 7ClKynA-D-galactopyranos-6'-yl ester (7ClKynA/Gal6) or free 7ClKynA did not protect against NMDA seizures. Microdialysis in freely moving rats showed the presence of significant amounts of 7ClKynA/Glu6, as well as of 7ClKynA or KynA, in cortical perfusates after i.p. injections of 7ClKynA/Glu6. In contrast, only small amounts of 7ClKynA or KynA were detected after i.p. injection of unconjugated 7ClKynA. Prodrug metabolism has also been examined in mouse cortical cultures containing both neurons and astrocytes. 7ClKynA/Glu6 and 7ClKynA/Gal6 were rapidly metabolized into 7ClKynA and KynA, whereas 7ClKynA/Glu3 was metabolized with a slower kinetics. As a result of its conversion into 7ClKynA and KynA, 7ClKynA/Glu6 protected cortical neurons against NMDA toxicity. We conclude that sugar conjugates of 7ClKynA (and perhaps of other excitatory amino acid receptor antagonists) are prodrugs of potential interest in the experimental therapy of epilepsy and acute or chronic neurodegenerative disorders. PMID- 10727636 TI - Complex hippocampal responses to ATP: fade due to nucleotidase inhibition and P2 receptor-mediated adenosine release. AB - When ATP or the related stable analogue, betagamma-imidoATP, were applied to rat hippocampal slices showing population spikes larger than 5 mV peak-to-peak amplitude, a depression of spike size was obtained, which showed a marked fade during the 10-min period of superfusion. The inhibitory responses were prevented by adenosine deaminase or 8-phenyltheophylline. Adenosine responses showed no fade. alphabeta-MethyleneADP enhanced the fade, while suramin at 50 micrometer prevented the early component of the responses. The results suggest that in slices with large population spikes, inhibitory responses to nucleotides are partly due to their conversion to adenosine, and partly due to the activation of P2 receptors which trigger the release of endogenous adenosine. PMID- 10727637 TI - Protective effect of fluvastatin, a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on MPP(+)-induced hydroxyl radical in the rat striatum. AB - We examined whether fluvastatin, an inhibitor of low-density lipoprotein (LDL) oxidation, can resist 1-methyl-4-phenylpyridine (MPP(+))-induced hydroxyl radical generation (.OH) in the extracellular fluid of rat striatum. Rats were anesthetized and sodium salicylate in Ringer's solution (0.5 nmol/microliter/min) was infused through a microdialysis probe to detect the generation of.OH as reflected by the nonenzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. MPP(+) (5 mM; total dose 75 nmol) clearly produced an increase in.OH formation. However, fluvastatin (100 microM) reduced the.OH formation by the action of MPP(+). These results indicated that fluvastatin, a potent inhibitor of LDL oxidation, may resist the formation of.OH products of MPP(+). PMID- 10727638 TI - Ultrastructural identification of synapses between mitral/tufted cell dendrites. AB - Asymmetrical, type 1 synapses between mitral and/or tufted (M/T) cell dendrites were observed in the glomerular layer (GL) and juxtaglomerular external plexiform layer (EPL) of salamander olfactory bulb sections. The dendrites had electron lucent cytoplasm containing regularly-arrayed microtubules and spherical translucent vesicles. The vesicles were clustered against a thin pre-synaptic density that was aligned with a 17-20 nm-wide synaptic cleft and a thicker post synaptic density. These dendrodendritic synapses could be a source of the delayed, prolonged excitation that originates from the GL/EPL. During spatiotemporal encoding of odor stimuli, they could amplify or synchronize M/T cell responses. PMID- 10727640 TI - The expression and cellular localization of brain/kidney protein in the rat retina. AB - Brain/kidney (B/K) protein is a new protein of 474 amino acids, which contains two C2 domains structurally homologous to those present in synaptotagmins. The expression of B/K protein was identified in the rat retina and B/K protein immunoreactivity was localized to a number of ganglion cells, a few amacrine cells and the radial processes of Muller cells. Thus, B/K protein appears to be important in the homeostasis in these cells of the rat retina. PMID- 10727639 TI - Developmental regulation of membrane type-5 matrix metalloproteinase (MT5-MMP) expression in the rat nervous system. AB - An intricate balance between extracellular matrix (ECM) synthesis and degradation must be maintained during developmental tissue remodeling. Matrix metalloproteinases (MMPs) are the main mediators of ECM degradation. A subset of MMPs, referred to as membrane-type MMPs, contains a transmembrane domain that restricts protease activity at the cell surface. Membrane type-5 MMP is predominantly expressed in the brain. The present report is the first to demonstrate the temporal regulation and spatial distribution of MT5-MMP mRNA during nervous system development. PMID- 10727641 TI - Monoamines and sleep: microdialysis findings in pons and amygdala. AB - This is the first microdialysis report comparing concentrations (pg/microliter) of norepinephrine (NE), serotonin (5-HT) and dopamine (DA) derived from feline locus ceruleus complex (LC) and amygdala. NE and 5-HT declined progressively from waking to slow-wave-sleep (SWS) and then to rapid-eye-movement (REM) sleep. Concentrations of DA did not change at either collection site across the sleep wake cycle. We conclude that release of NE and 5-HT release modulates physiologic components related to the sleep-wake cycle, but DA does not. PMID- 10727642 TI - Effects of the partial glycine agonist D-cycloserine on cognitive functioning in chronic low dose MPTP-treated monkeys. AB - D-Cycloserine, a partial agonist at the glycine recognition site of the N-methyl D-aspartate (NMDA) receptor complex, has been shown to facilitate certain forms of memory formation and to improve visual recognition memory in normal monkeys. In the present study, the effects of D-cycloserine on spatial short-term memory deficits in monkeys induced by chronic low-dose 1-methyl-4-phenyl-1,2,3, 6 tetrahydropyridine (MPTP) administration were examined. Chronic low-dose MPTP administration resulted in deficits in the performance of a variable delayed response task (VDR). Single administration of D-cycloserine (320 or 1000 microgram/kg) significantly improved the performance on this task. High-dose D cycloserine (8000 microgram/kg) or MK-801 (10-32 microgram/kg) administration had no effects on delayed-response performance but impaired performance on a visual discrimination (VD) task that was not adversely affected by MPTP administration. These results show that at low doses, D-cycloserine has cognition-enhancing properties in this model of early Parkinsonism. PMID- 10727643 TI - Neuroprotective effects of creatine administration against NMDA and malonate toxicity. AB - We examined whether creatine administration could exert neuroprotective effects against excitotoxicity mediated by N-methyl-D-aspartate (NMDA), alpha-amino-3 hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainic acid. Oral administration of 1% creatine significantly attenuated striatal excitotoxic lesions produced by NMDA, but had no effect on lesions produced by AMPA or kainic acid. Both creatine and nicotinamide can exert significant protective effects against malonate-induced striatal lesions. We, therefore, examined whether nicotinamide could exert additive neuroprotective effects with creatine against malonate-induced lesions. Nicotinamide with creatine produced significantly better neuroprotection than creatine alone against malonate-induced lesions. Creatine can, therefore, produce significant neuroprotective effects against NMDA mediated excitotoxic lesions in vivo and the combination of nicotinamide with creatine exerts additive neuroprotective effects. PMID- 10727644 TI - Disturbance of rat lever-press learning by hippocampo-prefrontal disconnection. AB - To determine whether the medial prefrontal cortex (PFC), ventral hippocampus and hippocampo-PFC pathway are involved in operant lever-press learning, we conducted lidocaine injections to these brain sites. Rats were injected immediately after lever-press acquisition in the first training, and the second 5-min test the next day. Results showed the response rate of either PFC- or ventral hippocampus inactivated rats to be lower than that of control rats in the test the next day. Rats having lidocaine injected into the unilateral ventral hippocampus combined with contralateral medial PFC also showed lower response rate in their tests. These results suggest that hippocampo-PFC disconnection disturbs operant learning. PMID- 10727645 TI - The effect of neonatal capsaicin on the c-Fos-like immunoreactivity induced in subnucleus oralis neurons by noxious intraoral stimulation. AB - The noxious stimulus-dependent induction of c-Fos-like immunoreactivity (Fos-LI) in neurons in the subnucleus oralis and the medullary dorsal horn (MDH) was significantly suppressed by the selective destruction of unmyelinated primary neurons. The induction of Fos-LI by topical capsaicin application to the lingual mucosal stimulation was almost completely suppressed by neonatal capsaicin treatment. Fos-LI induction by the tooth pulp stimulation and by formalin injection to the lingual mucosa were only partially reduced. These results provide an evidence that the noxious signals from the intraoral structures are transmitted by both unmyelinated and myelinated nociceptors to the subnucleus oralis as well as the MDH. PMID- 10727646 TI - Where do our reviewers come from? PMID- 10727647 TI - What we can learn from individual resuscitated patients. PMID- 10727648 TI - Hypertrophy and dilation: a TOTally new story? PMID- 10727649 TI - Sex and NO--beyond regulation of vasomotor tone. PMID- 10727650 TI - Mitochondria--potential role in cell life and death. PMID- 10727651 TI - The roles of gender, the menopause and hormone replacement on cardiovascular function. PMID- 10727652 TI - Platelets, oxidant stress and erectile dysfunction: an hypothesis. PMID- 10727653 TI - Electrophysiological characterization of SCN5A mutations causing long QT (E1784K) and Brugada (R1512W and R1432G) syndromes. AB - Familial long QT syndrome (LQTS) and Brugada syndrome are two distinct human hereditary cardiac diseases known to cause ventricular tachyarrhythmias (torsade de pointes) and idiopathic ventricular fibrillation, respectively, which can both lead to sudden death. OBJECTIVE: In this study we have identified and electrophysiologically characterized, in patients having either LQTS or Brugada syndrome, three mutations in SCN5A (a cardiac sodium channel gene). METHOD: The mutant channels were expressed in a mammalian expression system and studied by means of the patch clamp technique. RESULTS: The R1512W mutation found in our first patient diagnosed with Brugada syndrome produced a slowing of both inactivation and recovery from inactivation. The R4132G mutation found in our second patient who also presented Brugada syndrome, resulted in no measurable sodium currents. Both Brugada syndrome patients showed ST segment elevation and right bundle-branch block, and had experienced syncopes. The E1784K mutation found in the LQTS showed a persistent inward sodium current, a hyperpolarized shift of the steady-sate inactivation and a faster recovery from inactivation. CONCLUSION: The different clinical manifestations of these three mutations most probably originate from the distinct electrophysiological abnormalities of the mutant cardiac sodium channels reported in this study. PMID- 10727655 TI - Type 2 angiotensin II receptor is downregulated in cardiomyocytes of patients with heart failure. AB - BACKGROUND: The human heart expresses type 2 angiotensin (AT(2)) receptor, but the function is poorly defined. METHODS: In the present study, we investigated (1) the cellular localization of the AT(2) receptor and (2) the relationship between the AT(2) receptor protein expression and the cardiac function of patients with ischemic heart disease. The receptor localization was assessed by immunohistochemistry and the protein expression was quantified by Western blotting in atrial tissues freshly obtained from 22 patients undergoing coronary artery bypass graft surgery (63.0+/-11.0 years old; male ratio, 85%). Prior to the surgery, blood was drawn for determination of atrial-natriuretic hormone level and the left ventricular function was assessed by ultrasound cardiography. RESULTS: The results of immunohistochemistry showed that the AT(2) receptor was localized to cardiomyocytes and was not present in fibroblasts, vascular smooth muscles, or vascular endothelium. Atrial tissues showed various degrees of structural remodeling, but the localization of the AT(2) receptor was not altered in any tissue sections. The amount of the AT(2) receptor was negatively correlated with end-diastolic left ventricular diastolic dimension (r=-0.56, P<0.01), calculated left ventricular mass index (r=-0.51, P<0.02) and the plasma atrial natriuretic peptide (ANP) concentration (r=-0. 62, P<0.01) and positively correlated with left ventricular ejection fraction (r=0.48, P<0.05). CONCLUSIONS: (1) The AT(2) receptor is localized to cardiomyocytes independently of the cardiac function. (2) Left ventricular dysfunction is associated with decreased expression of myocardial AT(2) receptor protein. PMID- 10727654 TI - Angiotensin II receptor blockade attenuates the deleterious effects of exercise training on post-MI ventricular remodelling in rats. AB - OBJECTIVES: The effects of exercise training on LV remodelling following large anterior myocardial infarction (MI) remains controversial. Blockade of the renin angiotensin system has been shown to prevent ventricular dilation and deleterious remodeling. We therefore tested, in a rat model of chronic MI, whether any potentially deleterious effects of exercise on post-MI remodelling could be ameliorated by angiotensin II receptor blockade. METHODS: Male Wistar rats underwent coronary ligation or sham operation. Treatment with losartan (10 mg/kg/day) began 1 week post-MI and moderate treadmill exercise (25 m/min, 60 min/day, 5 days/week) was initiated 2 weeks post-MI. Systolic and diastolic pressure-volume relationships were measured in isolated, red-cell perfused, isovolumically beating hearts 8 weeks post-MI. Morphometric measurements were performed in trichrome stained cross sections of the heart. Five groups of animals were compared: sham (n=13), control MI (MI; n=11), MI plus losartan (MI Los; n=13), MI plus exercise (MI-Ex; n=10) and MI plus exercise and losartan (MI Ex-Los; n=12). RESULTS: Infarct size (% of left ventricle, LV) was similar among the infarcted groups [MI=43+/-4%, MI-Los=49+/-2%, MI-Ex=45+/-1%, MI-Ex-Los=48+/ 2% (NS)]. Exercise, losartan and exercise+losartan treatments all attenuated LV dilation post-MI to a similar degree. Exercise training increased LV developed pressure in both untreated and losartan treated hearts (P<0.05 vs. other MI groups). In addition, exercise resulted in additional scar thinning in untreated hearts, while no additional scar thinning was seen in post-infarct hearts receiving both losartan and exercise. CONCLUSIONS: Following large anterior MI, losartan attenuated LV dilation and scar thinning. In untreated animals, exercise decreased dilation, but also contributed to scar thinning. Therefore, exercise concurrent with blockade of the renin-angiotensin system may provide optimal therapeutic benefit following large anterior MI. PMID- 10727656 TI - Comparison of intravenous and pulmonary artery injections of hypertonic saline for the assessment of conductance catheter parallel conductance. AB - OBJECTIVE: The conductance catheter provides a continuous measure of left ventricular volume. Conversion of raw data to calibrated absolute volume requires assessment of parallel conductance. Conventionally, parallel conductance is determined by injecting a small bolus hypertonic saline into the pulmonary artery and analyzing the signal obtained during passage of the bolus through the left ventricle. However, in some cases, a pulmonary artery catheter is not practicable. Therefore, we investigated whether intravenous hypertonic saline injections yield reliable parallel conductance estimates. METHODS: In 13 anesthetized sheep (33+/-5 kg) parallel conductance was obtained by pulmonary artery and by intravenous injections. Measurements (triplicate) were done at baseline, during dobutamine and pacing, and repeated after embolization of the right coronary artery in order to assess the effects of enlarged right ventricular volumes. We used a multiple linear regression model to determine the relation between parallel conductance obtained by the two methods and to quantify the effects of dobutamine, pacing, and embolization. RESULTS: The two methods show an excellent correlation with a systematic overestimation for intravenous injection. The mean parallel conductance obtained by pulmonary artery injection was 0.690+/-0.009 ohm(-1) whereas intravenous injection yielded 0.739+/-0.015 ohm(-1). Interanimal variability was 0.138 ohm(-1). The difference between the two methods was relatively small, but highly significant (+0.049+/-0.012 ohm(-1), P<0.001). Embolization resulted in significantly higher values (+0.141+/-0.017 ohm(-1), P<0.001), but dobutamine and pacing did not significantly affect parallel conductance (+0.021+/-0.016 ohm(-1), NS). There was no interaction between these interventions and the injection method, indicating that the relation between parallel conductances obtained by the two methods was maintained in all conditions. CONCLUSION: Parallel conductance obtained by intravenous injection was significantly higher (+7%) than by pulmonary artery injection. However, the relation between the two methods is highly linear with an excellent correlation and is not affected by large hemodynamic changes. The systematic difference between the two methods is likely due to increased conductivity of blood in the right ventricle which is present with intravenous injection but not with pulmonary artery injection. Determination of parallel conductance by intravenous injection is a good alternative for conventional pulmonary artery injection and may be applied in studies where pulmonary artery injection is problematic. This may include studies in very small animals or studies in patients prone to arrhythmias or with cardiac anomalies such as pulmonary artery stenosis. In addition, intravenous injection could be used in biventricular studies to obtain right and left ventricular parallel conductances from a single saline injection. PMID- 10727657 TI - Hypertrophic defect unmasked by calcineurin expression in asymptomatic tropomodulin overexpressing transgenic mice. AB - OBJECTIVE: Dilation and hypertrophy often occur concurrently in cardiomyopathy, yet the interaction between these two functionally distinct conditions remains unknown. METHODS: Combinatorial effects of hypertrophy and dilation were investigated by cross-breeding of two cardiomyopathic transgenic mouse lines which develop either hypertrophy (calcineurin-mediated) or dilation (tropomodulin mediated). RESULTS: Altering the intensity of signals driving hypertrophy and dilation in cross-bred litters resulted in novel disease phenotypes different from either parental line. Augmenting the calcineurin-dependent hypertrophic stimulus in tropomodulin overexpressing transgenics elevated heart:body weight ratios, increased ventricular wall thickness, and significantly accelerated mortality. These effects were evident in calcineurin cross-breeding to tropomodulin backgrounds of transgene homozygosity (severe dilation) or heterozygosity (mild dilation to asymptomatic). Molecular analyses indicated that tropomodulin and calcineurin signaling events in the first week after birth were critical for determination of disease outcome, substantiated by demonstration that temporary neonatal inhibition of tropomodulin expression prevents dilation. CONCLUSIONS: This study shows that postnatal timing of altered signaling in cardiomyopathic transgenic mouse models is a pivotal part of determining outcome. In addition, intensifying hypertrophic stimulation exacerbates dilated cardiomyopathy, supporting the concept of shared molecular signaling between hypertrophy and dilation. PMID- 10727658 TI - Cardioprotective effects of the Na(+)/H(+)-exchange inhibitor cariporide in infarct-induced heart failure. AB - OBJECTIVE: We investigated the effect of chronic treatment with the new Na(+)/H(+)-exchange inhibitor, cariporide, on cardiac function and remodelling 6 weeks after myocardial infarction (MI) in rats. METHODS: Treatment with cariporide was commenced either 1 week pre or 30 min, 3 h, 24 h or 7 days after ligation of the left ventricular artery and was continued until haemodynamic parameters were obtained 6 weeks after MI in conscious rats. RESULTS: Compared to sham animals, untreated MI-controls developed pronounced heart failure after 6 weeks. Basal left ventricular end-diastolic pressure (in mmHg) was reduced in the groups in which cariporide was started 1 week pre (16.0+/-1.7) or 30 min (12.5+/ 1.1), 3 h (11.8+/-1.0) and 24 h (13.0+/-2.5) after MI compared to untreated MI controls (22. 4+/-1.5; P<0.01). Basal myocardial contractility (in 1000 mmHg/s) was only increased when treatment was initiated after 30 min (9. 0+/-0.7), 3 h (8.5+/-0.3) and 24 h (8.0+/-0.7) compared to untreated MI-controls (5.8+/-0.7; P<0.05-0.01). Infarct size (in % of left ventricular circumference) was 40.0+/ 2.1 in MI-controls and was decreased when treatment was begun after 30 min (32.6+/-2.7) or 3 h (32.4+/-2.3) (P<0.05). In animals, in which cariporide was started 3 h after induction of MI, heart weight/body weight ratio was significantly decreased, indicating reduced cardiac hypertrophy. When treatment started 7 days after MI, cariporide did not exert any beneficial actions on structural and functional cardiac parameters. CONCLUSION: Our results show for the first time that chronic treatment with the Na(+)/H(+)-exchange inhibitor cariporide engendered marked cardioprotective effects when commenced before and up to 24 h after MI. The optimal time for the start of treatment was between 30 min and 3 h post MI. PMID- 10727659 TI - Activation of Ca(2+)-independent nitric oxide synthase by 17beta-estradiol in post-ischemic rat heart. AB - BACKGROUND: Nitric oxide (NO) donors or facilitation of endogenous NO production is cardioprotective. This study sought to determine whether enhanced myocardial NO production might contribute to estrogen-induced cardioprotection. METHODS: Ca(2+)-dependent and Ca(2+)-independent NOS activities (pmol min(-1) mg(-1) protein), NOS protein expression (quantitative immunoblot), cGMP content (pmol mg(-1) protein) and LV work (Joules) were measured in hearts isolated from ovariectomized rats that were either untreated or treated chronically with 17beta estradiol (0.25 mg, 21 day release formulation). RESULTS: After 14 days, serum levels of 17beta-estradiol were 6+/-1 and 135+/-16 pg ml(-1) in untreated and 17beta-estradiol-treated animals, respectively. After 60 min aerobic working mode perfusion, Ca(2+)-dependent NOS (untreated, 1.47+/-0.36; 17beta-estradiol 1.13+/ 0.25) and Ca(2+)-independent NOS (untreated, 0.45+/-0.24; 17beta-estradiol, 0.41+/-0.21) activities, eNOS and iNOS proteins and cGMP content (untreated, 0.64+/-0.08; 17beta-estradiol, 0.76+/-0.12) were not different in the two groups. After 60 min low-flow (0.5 ml min(-1)) ischemia and 30 min reperfusion, Ca(2+) dependent NOS activities were again similar (untreated, 1.25+/-0.23; 17beta estradiol, 0.78+/-0.27). However, after reperfusion, Ca(2+)-independent NOS activity (untreated, 0. 39+/-0.10; 17beta-estradiol, 1.36+/-0.36) was 3.5-fold higher (P=0. 008) and cGMP content (untreated, 0.30+/-0.03; 17beta-estradiol, 0. 49+/-0.07) was 1.6-fold higher (P=0.017) in hearts from 17beta-estradiol-treated animals. Although pre-ischemic function was similar, recovery of post-ischemic LV work was 2-fold greater (P=0.024) in the 17beta-estradiol group. CONCLUSION: The ability of ischemia and reperfusion in combination with chronic 17beta-estradiol to increase Ca(2+)-independent NOS activity and cGMP content supports a role for enhanced myocardial NO signaling in 17beta-estradiol-induced cardioprotection. PMID- 10727660 TI - Sphingosine 1-phosphate induces sinus tachycardia and coronary vasoconstriction in the canine heart. AB - OBJECTIVE: Sphingosine 1-phosphate is a naturally occurring biologically active lysophospholipid. Recent studies suggested that sphingosine 1-phosphate is released into the blood flow from activated platelets upon stimulation to exert multiple biological phenomenon. The purpose of this study was to assess the effects of sphingosine 1-phosphate on sinus automaticity, ventricular contraction and coronary blood flow. METHODS: The canine isolated, blood-perfused sinoatrial node and papillary muscle preparations were used. RESULTS: Sphingosine 1 phosphate increased the sinoatrial rate, while it decreased the coronary blood flow, which was followed by a weak negative inotropic effect. These positive chronotropic and coronary vasoconstrictor effects were not attenuated by the beta and alpha-adrenoceptor antagonists atenolol and prazosin, respectively. Furthermore, sphingosine 1-phosphate did not affect the adenylate cyclase activity of the membrane preparations made from the canine right atrium and right ventricle, indicating the involvement of a novel signaling pathway in sphingosine 1-phosphate-induced cardiac effects. CONCLUSIONS: These results may provide a clue to better understanding the physiological as well as the pathophysiological regulation of sphingosine 1-phosphate in the heart. PMID- 10727661 TI - Evaluation of fluorescent dyes for the detection of mitochondrial membrane potential changes in cultured cardiomyocytes. AB - OBJECTIVE: Maintenance of the mitochondrial membrane potential (Deltapsim) is fundamental for the normal performance and survival of cells such as cardiomyocytes, that have a high energy requirement. Measurement of Deltapsim is therefore essential in order to develop an understanding of the molecular mechanisms controlling cardiomyocyte function. Here we have evaluated various potentiometric dyes for their ability to detect alterations of Deltapsim, using flow cytometry and confocal microscopy. METHODS: Primary cultures of cardiomyocytes from neonate rats were treated with mitochondrial uncouplers before or after loading with Rho123, DiOC(6)(3), CMXRos or JC-1, and then analysed by flow cytometry. Apoptotic cells were identified by light scatter and Annexin V staining. RESULTS: The four potentiometric dyes tested were able to discriminate between viable and apoptotic cells. However, only JC-1 was able to detect the collapse of Deltapsim induced by uncouplers of mitochondrial respiration. Confocal microscopic analysis confirmed that JC-1 stained mitochondria in a potential-dependent manner. In contrast, CMXRos stained cardiomyocytes irrespective of alterations in Deltapsim. CONCLUSIONS: We conclude that JC-1 is the optimal dye to use when measuring Deltapsim in cardiomyocytes. PMID- 10727663 TI - Effects of ambasilide, quinidine, flecainide and verapamil on ultra-rapid delayed rectifier potassium currents in canine atrial myocytes. AB - OBJECTIVE: A dog atrial ultra-rapid delayed rectifier current (I(Kur. d)) is involved in canine atrial repolarization and shares similarities with the human atrial ultra-rapid delayed rectifier (I(Kur)). Almost no information is available about the actions of antiarrhythmic drugs on I(Kur.d). This study evaluated effects of ambasilide, quinidine, flecainide and verapamil on I(Kur.d) in isolated canine atrial myocytes. METHODS: Standard whole-cell patch clamp techniques were used to study the effects of multiple concentrations of each drug. RESULTS: All drugs produced reversible concentration-, voltage- and time dependent I(Kur.d) inhibition. Significant effects of quinidine, flecainide and ambasilide were noted at atrial-effective antiarrhythmic concentrations in the dog. Upon the onset of a depolarizing pulse, block developed exponentially in relation to time, with the blocking rate-constant increasing with drug concentration, consistent with open-channel blockade and permitting the calculation of forward and reverse rate-constants. For all drugs, the 50% blocking concentration (EC(50)) showed significant voltage-dependence, decreasing at more positive potentials. The magnitude of voltage-dependent block was directly related to the degree of drug-induced shift in the voltage dependence of activation (r=0.97), pointing to open-channel block as a mechanism for voltage dependent action. An additional component of voltage-dependence suggested that blocking sites were subjected to 17-21% of the transmembrane voltage field. CONCLUSIONS: Ambasilide, quinidine, flecainide and verapamil inhibit I(Kur.d), with preferential action on the open state. I(Kur.d) inhibition may play a role in antiarrhythmic effects in canine atrial arrhythmia models. Comparisons between the effects of these drugs on I(Kur.d) and previously studied effects on I(Kur) suggest potential opportunities for investigating the molecular structural determinants of drug-blocking action on atrial-specific ultrarapid delayed rectifiers. PMID- 10727662 TI - Hypoxia differentially regulates stress proteins in cultured cardiomyocytes: role of the p38 stress-activated kinase signaling cascade, and relation to cytoprotection. AB - OBJECTIVE: Stress proteins (heat shock proteins, HSPs) are molecular chaperones that have been shown to enhance the survival of cells exposed to environmental stress. We sought to investigate the effects of hypoxia on the levels of HSP27 and heme oxygenase-1 (HO-1 or HSP32) in an established model of rat neonatal cardiac myocytes in culture. METHODS: Myocytes were subjected to hypoxia (<0.5% O(2) for 16 h). Studies of cell viability and nuclear morphology showed no evidence of cell death under these conditions. RESULTS: Messenger RNA analysis demonstrated constitutive expression of HSP27 and low levels of HO-1. Hypoxia strongly induced HO-1 mRNA without affecting HSP27 mRNA. In parallel to mRNA levels, hypoxia increased HO-1 protein level without affecting HSP27. To further assess the signaling pathways implicated in HO-1 induction, we used inhibition experiments. The tyrosine kinase inhibitor tyrphostin and the mitogen-activated protein kinase inhibitor PD98059 did not prevent HO-1 induction, while the protein kinase C inhibitor chelerythrine partially blocked this response. The p38 stress-activated kinase inhibitor SB203580 was the most potent in suppressing hypoxia-induced HO-1. In vitro kinase assays, cell labeling and immunoprecipitation showed activation of signaling pathways downstream of p38 stress-activated kinase as revealed by an increase in phosphorylation of MAPKAPK 2/3 kinases and HSP27. CONCLUSIONS: These data show a differential pattern of hypoxia-induced HSP expression and implicate the stress kinase in HO-1 induction. Thus, selective regulation of HSP levels may play a role in the cardioprotective mechanisms that participate in the adaptive response to hypoxia-induced stress. PMID- 10727664 TI - Attenuated cardiac contractile responsiveness to insulin-like growth factor I in ventricular myocytes from biobreeding spontaneous diabetic rats. AB - OBJECTIVE: Insulin-like growth factor I (IGF-1) stimulates cardiac growth and contraction, but resistance to its action has been reported in diabetes. This study was to determine if IGF-1-induced cardiac contractile action is altered in rats genetically predisposed to diabetes. METHOD: Ventricular myocytes were isolated from spontaneously biobreeding diabetes-prone (BB/DP) rats and their diabetes-resistant littermates (BB/DR). Mechanical properties were evaluated in cardiomyocytes using a video-based edge-detection system. Myocytes were electrically stimulated at 0.5 Hz. Contractile properties analyzed included peak shortening (PS), time-to-PS (TPS) and time-to-90% relengthening (TR(90)). Intracellular Ca(2+) transients were measured as changes in fura-2 fluorescence intensity (DeltaFFI). RESULTS: Myocytes from BB/DP rats displayed increased PS, prolonged TPS and TR(90,) as well as reduced resting FFI compared to the BB/DR group. IGF-1 (10(-10)-10(-6) M) caused a dose-dependent increase in PS in myocytes from BB/DR but not BB/DP rats. The increase of PS was blunted by IGF-1 antagonist H-1356, phosphatidylinositol-3 (PI-3) kinase inhibitor wortmannin, but not tyrosine kinase inhibitor genistein. None of these agents affected responses to IGF-1 in BB/DP myocytes. Interestingly, IGF-1 elicited a comparable dose dependent increase in Ca(2+) transients in myocytes from both BB/DR and BB/DP rats. CONCLUSION: These results suggest that the attenuation of IGF-1-induced cardiac contractile response in chemically-induced diabetes also exists in diabetes of genetic origin, possibly due to mechanisms involving PI-3 kinase and intracellular Ca(2+) sensitivity. PMID- 10727665 TI - Methylation of the estrogen receptor-alpha gene promoter is selectively increased in proliferating human aortic smooth muscle cells. AB - OBJECTIVE: Atherosclerosis is a multigenic process leading to the progressive occlusion of arteries of mid to large caliber. A key step of the atherogenic process is the proliferation and migration of vascular smooth muscle cells into the intimal layer of the arterial conduit. The phenotype of smooth muscle cells, once within the intima, is known to switch from contractile to de-differentiated, yet the regulation of this switch at the genomic level is unknown. Estrogen has been shown to regulate cell proliferation both for cancer cells and for vascular cells. However, methylation of the estrogen receptor-alpha gene (ERalpha) promoter blocks the expression of ERalpha, and thereby can antagonize the regulatory effect of estrogen on cell proliferation. We sought to determine whether methylation of the ERalpha is differentially and selectively regulated in contractile versus de-differentiated arterial smooth muscle cells. METHODS: We used Southern blot assay, combined bisulfite restriction analysis (Cobra) and restriction landmark genome scanning (RLGS-M) to determine the methylation status of ERalpha in human aortic smooth muscle cells, either in situ (normal aortic tissue, contractile phenotype), or the same cells explanted from the aorta and cultured in vitro (de-differentiated phenotype). RESULTS: We provide evidence that methylation of the ERalpha in smooth muscle cells that display a proliferative phenotype is altered relative to the same cells studied within the media of non-atherosclerotic aortas. Thus, the ERalpha promoter does not appear to be methylated in situ (normal aorta), but becomes methylated in proliferating aortic smooth muscle cells. Using a screening technique, RLGS-M, we show that alteration in methylation associated with the smooth muscle cell phenotypic switch does not seem to require heightened activity of the methyltransferase enzyme, and appears to be selective for the ERalpha and a limited pool of genes whose CpG island becomes either demethylated or de novo methylated. CONCLUSIONS: Our data support the concept that the genome of aortic smooth muscle cells is responsive to environmental conditions, and that DNA methylation, in particular methylation of the ERalpha, could contribute to the switch in phenotype observed in these cells. PMID- 10727666 TI - Nitric oxide mediated endothelium-dependent relaxation induced by glibenclamide in rat isolated aorta. AB - OBJECTIVES: Glibenclamide was found to act as both a selective ATP-sensitive K(+) channel blocker and a vasorelaxant. The exact mechanisms underlying the relaxant effect of glibenclamide are unknown. The present study was designed to examine the role of endothelium/nitric oxide in glibenclamide-induced relaxation in rat isolated aortic rings. METHODS: A combination of experimental approaches including isometric force measurement, cell culture, Ca(2+) fluorescence measurement and radioimmunoassay were used to examine the vascular effect of glibenclamide. RESULTS: Glibenclamide induced a concentration-dependent relaxation more effectively in rings with endothelium (IC(50) of 32+/-4 microM) than those without endothelium (IC(50) of 365+/-29 microM). Incubation with N(G) nitro-L-arginine methyl ester (L-NAME) or methylene blue significantly reduced and L-arginine (3 mM) potentiated the glibenclamide-induced relaxation. L Arginine (3 mM) partially antagonized the effect of L-NAME. Glibenclamide (100 microM) increased the cyclic GMP content of endothelium-intact tissues. Pretreatment with N(G)-nitro-L-arginine (100 microM) or removal of endothelium significantly suppressed the effect of glibenclamide on cyclic GMP production. Glibenclamide elevated the intracellular Ca(2+) levels in cultured rat aortic endothelial cells. Glibenclamide also inhibited the endothelium-independent contractile response to 60 mM K(+) (IC(50) of 137+/-21 microM) and caused a rightward shift in the concentration-contraction curve for CaCl(2). Besides, glibenclamide inhibited phorbol-12,13-diacetate (1 microM)-induced contraction in Ca(2+)-free Krebs solution. CONCLUSION: These results indicate that glibenclamide induced endothelium-dependent relaxation involves nitric oxide release and this effect may be related to its stimulatory effect on endothelial Ca(2+) levels. However, the glibenclamide-induced endothelium-independent relaxation may be associated with its inhibitory effect on Ca(2+) influx through Ca(2+) channels and on the protein kinase C-mediated contractile mechanism. PMID- 10727667 TI - High-glucose-induced nuclear factor kappaB activation in vascular smooth muscle cells. AB - OBJECTIVE: Vascular smooth muscle cell (VSMC) dysfunction plays a role in diabetic macrovasculopathy. This dysfunction may be caused or exacerbated by expression of many of genes potently activated by the transcriptional factor nuclear factor kappaB (NF-kappaB). We have examined whether culture of VSMCs under high glucose conditions to stimulate the diabetic state can lead to the activation of NF-kappaB. METHODS: NF-kappaB activation was assessed in VSMCs stably transfected with a cis-reporter plasmid containing the NF-kappaB binding sites. RESULTS: Within 3-h incubation, high glucose (27.5 or 55 mmol/l) alone induced an increase in NF-kappaB activity in VSMCs; this increase was mimicked by mannitol given to deliver the same osmolar stress to the cells. High glucose or mannitol also enhanced TNFalpha-stimulated NF-kappaB activity. Incubation with high glucose for 48 h followed by stimulation with TNFalpha led to a marked potentiation of NF-kappaB activation compared with normoglycemic (5.5 mmol/l) VSMCs exposed to TNFalpha, while mannitol attenuated this effect. A 48-h incubation with high glucose substantially reduced glutathione (GSH) levels compared with normoglycemic VSMCs, whereas mannitol significantly increased GSH levels. An antioxidant N-acetyl-L-cysteine and a selective protein kinase C (PKC) inhibitor GF109203X significantly suppressed the TNFalpha-induced NF-kappaB activation, and abrogated potentiation of TNFalpha-induced NF-kappaB activity caused by high glucose (27.5 mmol/l). CONCLUSION: These results suggest that acutely high glucose causes alterations in osmolarity leading to activation of NF kappaB, but that exposure to high glucose for more prolonged times causes changes in antioxidant defences and activation of PKC, which potentiates cytokine activation of NF-kappaB. Further definition of these pathways will help to delineate important signals mediating the aberrant behavior of VSMCs under hyperglycemic/diabetic conditions. PMID- 10727669 TI - Magnesium. An update on physiological, clinical and analytical aspects. AB - There is an increased interest in the role of magnesium ions in clinical medicine, nutrition and physiology. The characteristics of the binding of magnesium and calcium ions to various components, macromolecules and biological membranes are described. Magnesium affects many cellular functions, including transport of potassium and calcium ions, and modulates signal transduction, energy metabolism and cell proliferation. The mechanism of cellular uptake and efflux of magnesium, its intracellular transport, intestinal absorption, renal excretion and the effect of hormones on these are reviewed. Magnesium deficiency is not uncommon among the general population: its intake has decreased over the years especially in the western world. The magnesium supplementation or intravenous infusion may be beneficial in various diseased states. Of special interest is the magnesium status in alcoholism, eclampsia, hypertension, atherosclerosis, cardiac diseases, diabetes, and asthma. The development of instrumentation for the assay of ionized magnesium is reviewed, as are the analytical procedures for total magnesium in blood and free magnesium in the cytosol. The improved procedures for the assay of different magnesium states are useful in understanding the role of magnesium in health and disease. PMID- 10727668 TI - Neurally mediated depressor hemodynamic response induced by intracoronary catheter balloon inflation in pigs. AB - OBJECTIVES: To assess whether intracoronary catheter balloon inflation triggers a neurally mediated hemodynamic response that interacts with the ischemia-induced myocardial dysfunction. METHODS: Forty-eight chloralose anesthetized pigs underwent a 60 s intraluminal catheter balloon inflation of the proximal left anterior descending (LAD) coronary artery before and after one of these treatments: disruption of LAD pericoronary nerves with phenol (n=6), bilateral stellectomy (n=8), bilateral cervical vagotomy (n=6), atropine (n=5), and ganglionic blockade with hexamethonium (n=10). In 13 other pigs, we assessed the reproducibility of two balloon inflations spaced 15 min (n=6) or 60 min (n=7). The ECG, left ventricular (LV) pressure, and LV dP/dt were recorded during each intervention. Right ventricular (RV) pressure, RV dP/dt, and aortic blood flow were also measured in a subset of pigs. RESULTS: Balloon inflation induced an early (10 s) and reproducible (ANOVA, P<0.001) drop in systolic pressure and peak dP/dt; a decrease in aortic blood flow; a rise in end-diastolic pressure; and elevation of the ST segment. Pericoronary denervation, stellectomy and ganglionic blockade attenuated (P<0.001) the drop in LV parameters during coronary inflation, but atropine and vagotomy did not. CONCLUSIONS: A depressor hemodynamic response subserved by pericoronary nerves worsens the LV dysfunction induced by brief coronary catheter balloon inflation in anesthetized pigs. Cholinergic fibers do not appear to play a major role. PMID- 10727670 TI - DX-9065a, a specific factor Xa inhibitor, as a universal anticoagulant for blood collection tubes. AB - DX-9065a is a direct and selective factor Xa inhibitor. We evaluated the usefulness of DX-9065a in anticoagulating specimens for routine laboratory tests. Results using blood anticoagulated with DX9065a corresponded well with results with blood treated with ethylendiamine tetraacetic acid (EDTA) in the complete blood count (CBC), including white blood cell (WBC) differential count and morphology of blood cells. Clinical chemistry results from DX-9065a-treated samples were similar to results obtained with serum specimens except for leucine aminopeptidase (LAP) and cholinesterase (Ch-E). Thus, DX-9065a may be a useful universal anticoagulant for laboratory medicine. PMID- 10727671 TI - Lipoprotein abnormalities in patients with asymptomatic acute porphyria. AB - There have been discrepancies in reports of total cholesterol and low density lipoprotein (LDL)-cholesterol levels in patients with acute porphyria. Some studies have found that acute porphyria patients have increased levels while others do not. The aim of this study has been to evaluate the lipid profile in a series of patients with acute porphyria, in order to help clarify these differences. Serum lipoprotein levels were studied in 30 patients (25 women and five men; age:38+/-10 years) with asymptomatic acute porphyria. Controls were 30 healthy volunteers matched for age and gender. For 13 patients and 15 controls, lipoprotein lipase and hepatic lipase activities were determined. Patients exhibited increased levels of total-cholesterol, LDL-cholesterol, high density lipoprotein (HDL)-cholesterol and apolipoprotein (apo)-A1 compared with controls (P4 mmol/l in 15 patients (50%). Levels of total triglycerides, very low density lipoprotein (VLDL)-triglycerides, VLDL-cholesterol, apo-B and lipoprotein(a) were similar in patients and controls. The hepatic lipase activity tended to be lower in patients than controls (33.8+/-17.7 vs. 50.4+/-23.0 pkat/ml; P=0.05). In conclusion, in patients with asymptomatic acute porphyria an increase of total and LDL-cholesterol was found. The cardiovascular risk conferred by this factor may be attenuated by increased HDL-cholesterol and apo-A1. PMID- 10727672 TI - Plasma cholesterol and endogenous cholesterol synthesis during refeeding in anorexia nervosa. AB - Normal or high levels of cholesterol have been measured in patients with anorexia nervosa (AN). Given that cholesterol intake in AN is usually very low, the reasons for this anomaly are not clearly understood. We studied lipid and lipoprotein profiles and endogenous cholesterol synthesis, estimated by serum lathosterol, in a population of 14 girls with AN, before and during a period of 30 days refeeding. The initial body mass index (BMI) of the patients was 13.41+/ 1.62 kg/m(2). No changes were observed during refeeding in endocrine parameters (ACTH, cortisol and estradiol). At Day 0 the lipids data measured here showed normal levels of triglycerides, and total cholesterol at the upper limits of the normal range (5.44+/-1 mmol/l). At this time, total and LDL cholesterol were negatively correlated with transthyretin and BMI. Serum lathosterol (a precursor in cholesterol synthesis pathway) increased significantly (5.99+/-1.75 (Day 0) vs. 8.39+/-2.96 (Day 30); P=0.02) while there was a significant decrease in apo B (0.79+/-0.33 (Day 0) vs. 0. 60+/-0.17 g/l (Day 30), P=0.02) with refeeding. Thus, patients with initial high cholesterol levels have the worst nutritional status and high cholesterol levels are not related to a de novo synthesis. This profile returns to normal with refeeding. An increase of cellular cholesterol uptake may be responsible for this apparently paradoxical evolution with increase of cholesterol synthesis and decrease of apo B during renutrition. PMID- 10727673 TI - Comparison of serum catalytic activity and immunoreactivity of bone alkaline phosphatase in hypophosphatasia. AB - Hypophosphatasia is a rare bone disorder characterised by low levels of tissue non-specific alkaline phosphatase (TNSALP). Although TNSALP is widespread in virtually all tissues the clinical effects, when produced, seem only to affect the mineralizing tissue such as teeth and skeleton. The skeleton is severely affected in the perinatal form of the disease, when death may occur in utero, or may not be affected in the adult type variety of the disease. We therefore compared the catalytic (cBALP) and immunoreactivity (iBALP) of bone alkaline phosphatase isoenzyme in six families with hypophosphatasia. iBALP was measured using an IRMA method. cBALP was measured after electrophoretic separation of serum alkaline phosphatase isoenzymes on lectin containing agarose gel. The percentage of different isoenzymes was calculated using densitometric scanning and cBALP calculated from the known total serum alkaline phosphatase activity. Results showed cBALP=0.796+3. 269iBALP, r=0.9 p<0.01, in cases of hypophosphatasia. In general, the lower the iBALP and cBALP the more severe the skeletal disease. The bone isoenzyme level predicts the clinical severity of bone disease. PMID- 10727674 TI - Comparison of the analytical and clinical performance characteristics of an N-MID versus an intact osteocalcin immunoradiometric assay. AB - Osteocalcin is the most abundant non-collagenous protein in bone, reflecting its formation. It was reported that the instability of intact osteocalcin results from the cleavage of the C-terminal sequence of osteocalcin to produce a large N terminal osteocalcin fragment. A two-site immunoassay for detecting both the N terminal osteocalcin fragment and the intact osteocalcin was developed that were both independent of an unstable C-terminal sequence. The aim of this study is to investigate the performance of an N-MID osteocalcin immunoradiometric assay and to compare it with an intact osteocalcin assay. Ten serum samples were repeatedly frozen and thawed up to seven times. The variability of the values of N-MID osteocalcin was less than that of the intact osteocalcin. For stability of osteocalcin in serum after storage, the mean value of N-MID was 94. 3% of the initial value after 7 days at 4 degrees C, whereas the intact was 73.4%. The reduction of intact values were significantly larger than that of N-MID after 2, 5 and 7 days. At -30 degrees C, the values of N-MID did not change for up to 10 weeks. The concentrations of osteocalcin measured by an N-MID osteocalcin and an intact osteocalcin were investigated in 27 premenopausal subjects, 27 postmenopausal subjects, and 68 osteoporotic patients (23 with vertebral fractures and 45 with hip fractures). The percent mean increase of osteocalcin in postmenopausal subjects over premenopausal subjects was 98% in N-MID versus 42% in the intact assay. The z-scores of N-MID and intact showed similar results in all groups. N-MID osteocalcin significantly correlated with intact osteocalcin (r=0.755), and other biochemical markers for bone formation, such as bone specific alkaline phosphatase (r=0.606) and C-terminal propeptide of type I procollagen (PICP) (r=0.568). An N-MID IRMA had better stability during storage than intact and had the discriminative ability which is similar to the intact assay in postmenopause and osteoporosis. Therefore, an N-MID osteocalcin IRMA could improve the clinical utility and evaluation of osteocalcin. PMID- 10727675 TI - Analytical performance and clinical efficacy of three routine procedures for LDL cholesterol measurement compared with the ultracentrifugation-dextran sulfate Mg(2+) method. AB - The diagnosis and management of adults with hypercholesterolemia in the US are largely based on low-density lipoprotein cholesterol (LDL-C) concentration. In order to classify someone correctly into the National Cholesterol Education Program cut-points, LDL-C must be measured with a total error of /=6 kPa and from May to at least July at pwO(2)>/=2 kPa. Results are discussed in terms of season-related physiological performance, as hypoxic events mainly occur during the hot season. PMID- 10727692 TI - Occurrence of human alpha-synuclein immunoreactive neurons with neurofibrillary tangle formation in the limbic areas of patients with Alzheimer's disease. AB - We examined alpha-synuclein immunoreactivity in the brains from 23 patients with Alzheimer's disease (AD) and two patients with Down's syndrome. In ten of the 23 AD cases and both the two Down's syndrome cases, alpha-synuclein immunoreactivities were observed in the neurons of the limbic areas, predominantly of the amygdala. Nearly all alpha-synuclein-positive neurons had tau-positive neurofibrillary tangles (NFT) in the same neurons, and these consisted of intermingled-type and superimposed-type. By immunoelectron microscopy, the intermingled-type revealed aggregations of alpha-synuclein positive filamentous components, which were in continuity with paired helical filaments (PHF), while the superimposed-type revealed accumulations of alpha synuclein-positive non-filamentous components in PHF bundles. These findings suggest that alpha-synuclein can accumulate in PHF and form filamentous aggregations in neurons of the limbic areas in AD cases. PMID- 10727693 TI - Brain atrophy in relapsing-remitting multiple sclerosis: relationship with 'black holes', disease duration and clinical disability. AB - Recent MRI studies in multiple sclerosis have highlighted the potential role of brain atrophy evaluation as a putative marker of disease progression. In the present study, we evaluated the supratentorial and infratentorial brain volume in patients with relapsing remitting multiple sclerosis (RR MS) and in healthy subjects. Moreover, we determined whether brain volumes of MS patients are associated with different aspects of brain MRI abnormalities and clinical findings. Two-dimensional acquired MRI was performed on 52 relapsing-remitting multiple sclerosis and 30 healthy subjects. The volume of supratentorial and infratentorial structures was measured in selected representative slices. Gd enhancement, T2 hyperintense, T1 hypointense (i.e. 'black holes') total lesion load, as well as the area of corpus callosum was calculated in the MS group and related to brain volume measures. Correlations between MRI parameters and clinical features were also considered. MS patients had significantly lower supratentorial, infratentorial brain volume and corpus callosum area than healthy subjects (P<0.01). Supratentorial brain volume was significantly related to corpus callosum area (r=0.58; P<0.01) and T1 hypointense lesion load (r=0.48; P<0.01), but not with T2 hyperintense lesion load. Infratentorial/supratentorial ratio was significantly associated with disease duration and EDSS score (r=-0.34; P=0.02 and r=-0.49; P<0.01, respectively). This study documents that brain atrophy is an early MRI finding in RR MS and it is closely related to 'black holes' burden. The use of relative values (infratentorial/supratentorial ratio) may increase the conspicuity of correlation between clinical and MRI findings. PMID- 10727694 TI - Neuromuscular disturbance outlasts other symptoms of exercise-induced muscle damage. AB - This study examined the biochemical, immunological, functional, and neuromuscular responses associated with exercise-induced muscle damage in the quadriceps of untrained men. Muscle damage and soreness was elicited with maximal concentric/eccentric muscle actions at 0.53 rads s(-1). Significant (P<0.05) soreness was evident 1, 2, and 3 days following muscle insult, while plasma creatine kinase, a marker of muscle damage, was elevated 3 and 5 days post insult. Plasma interleukin-Ibeta was significantly increased within 5 min, and remained elevated 1, 2, 5, and 7 days post-insult. Maximal isometric quadriceps function was impaired (P<0. 05) for 5 days following muscle challenge. Maximal isokinetic performance at 1.09 rads s(-1) was diminished (P<0.05) for 2 days post insult; no significant decrements at 3.14 rads s(-1) were noted. Average electrical activation (iEMG) of the quadriceps was unaltered, but iEMG activity of the rectus femoris - where soreness was focused - was significantly increased. Neuromuscular efficiency (torque/iEMG) was compromised throughout the 10-day post insult period investigated. While other symptoms of exercise-induced muscle damage dissipate within 7 days, neuromuscular perturbation persists for at least 10 days. PMID- 10727695 TI - Relationship between ataxin-1 nuclear inclusions and Purkinje cell specific proteins in SCA-1 transgenic mice. AB - Spinocerebellar ataxia-1 (SCA-1), like other polyglutamine diseases, is associated with aggregation of mutant protein ataxin-1 in the nuclei of susceptible neurons. The role of ataxin-1 aggregates in the pathogenesis of susceptible neurons, especially cerebellar Purkinje cells, is unknown. The present study was initiated to determine the temporal relationship between ataxin 1 aggregation and the sequence of specific biochemical changes in Purkinje cells in SCA-1 transgenic mice (TM). Earlier, we demonstrated that SCA-1 TM with no Purkinje cell loss and no alterations in home cage behavior show decreased expression of calcium-binding proteins calbindin-D28k (CaB) and parvalbumin (PV) in Purkinje cells. To determine if increased expression of mutant ataxin-1 in TM is also associated with earlier biochemical changes in Purkinje cells, both heterozygous and homozygous (B05 line of SCA-1) TM were used. The age of onset of ataxia in SCA-1 TM was at 12 weeks in heterozygotes and 6 weeks in homozygotes. In 6 week old heterozygous TM, Western blot analysis of growth associated protein 43 (GAP-43) and synaptophysin revealed no significant alterations as compared with the age-matched nontransgenic mice (nTM), whereas CaB was significantly reduced. beta-III-Tubulin was used as a specific Purkinje cell marker protein, immunohistochemical localization showed strong beta-III-tubulin immunoreactivity (IR) in Purkinje cells in 6 week old heterozygous TM, whereas CaB and PV IR were markedly reduced in the same neurons (double immunofluorescence staining). Most Purkinje cells from heterozygous (12 weeks old) and homozygous (6 weeks old) TM contained ataxin-1 nuclear inclusions (NIs). Cells with and without visible NIs revealed reduced PV and CaB IR; however, the changes were overtly more severe in cells with visible NIs. In contrast, the same cells were strongly immunoreactive to beta-III-tubulin. CaB, which is also present in the nucleus, colocalized with ataxin-1 and ubiquitin positive NIs. Further, RT-PCR analysis of CaB mRNA in the cerebellum in 6 week old heterozygous TM demonstrated a significant decrease in mRNA in comparison with the aged-matched nTM. These data suggest that there are selective alterations in the expression of CaB and PV in Purkinje cells which possibly occur earlier than ataxin-1 aggregation. Further, we speculate that ataxin-1 aggregates may not be toxic in general; however, they may deplete specific proteins essential for Purkinje cell viability in SCA-1 TM. PMID- 10727696 TI - Volumetric study of lobar atrophy in Pick complex and Alzheimer's disease. AB - BACKGROUND: Lobar atrophy is an important neuroimaging feature of Pick complex (PiC). However, differences in patterns of focal brain atrophy between PiC and Alzheimer's disease (AD), and among PiC subgroups, have not been studied quantitatively. OBJECTIVE: To compare volumetric measures among primary progressive aphasia (PPA), frontotemporal dementia (FTD) and AD; to assess association between brain atrophy and cognition. PATIENTS: Seventeen patients with PPA, 11 with FTD and 24 with probable AD were studied. METHODS: We measured total and regional volume quantitatively using MRI and computerized volumetry. Contributing factors were controlled statistically or by adopting brain volume ratios. We investigated the classifying power of volumetry and correlated regional brain volume with cognitive and language test scores. RESULTS: The ratio for fronto-temporo-central region was smaller on the left in PPA and on the right in FTD. AD and some PPA patients had smaller parietal lobes. The frontal ratios correctly classified 93% of PPA and FTD patients, but only 50% of the entire PiC and AD patients. Language-dependent examinations correlated with the left fronto temporal volume. CONCLUSIONS: Brain atrophy differs in PPA, FTD and AD, but there is some morphological overlap between PiC and AD in parietal volumes. Focal brain atrophy is most consistently associated with language impairments. PMID- 10727697 TI - Magnetic stimulation study during observation of motor tasks. AB - The aim of the study was to assess if the observation of single or more complex muscle movements activates the premotor cortex in man. We stimulated by transcranial magnetic stimulation the right and left motor cortex recording from the abductor pollicis brevis of eight normal subjects, during observation of different movements performed by the examiner: (1) single movements: thumb abduction, arm elevation; (2) motor sequences: finger opposing movements performed in an ordinate sequence: 1-2, 1-3, 1-4, 1-5, 1-2ellipsis, and in a non consecutive non-repetitive order: 1-3, 1-5, 1-4, 1-2, 1-5, 1-2ellipsis We found an increased excitability of the right cortex during observation of isolated muscle movement regardless of which muscle is moved. At the stimulation of the left cortex, MEPs were significantly increased during observation of complex muscular synergies. PMID- 10727698 TI - Practice as an intervention to improve speeded motor performance and motor learning in Parkinson's disease. AB - Individuals with Parkinson's disease have difficulty initiating and performing complex, sequential movements. Practice generally leads to faster initiation and execution of movements in healthy adults, however, whether practice similarly improves motor performance in patients with Parkinson's disease remains controversial. To assess the effects of practice on motor performance, patients with Parkinson's disease and control subjects practiced two, rapid arm-reaching tasks with different levels of movement complexity for 120 trials each over 2 days. Response programming was studied by analyzing the overall reaction time latency of each movement and its fractionated sub-components, premotor and motor time. Practice effects were investigated by comparing pretest performance to immediate and delayed retention test performances (10-min and 48-h rest intervals, respectively). Both patients with Parkinson's disease and control subjects improved speeded performance of sequential targeting tasks by practice and retained the improvement across both retention test intervals. Finding a learning effect for persons with Parkinson's disease supports practice as an effective rehabilitation strategy to improve motor performance of specific tasks for patients with Parkinson's disease. PMID- 10727700 TI - Quantitative measurement of motor and somatosensory impairments after mild (30 min) and severe (2 h) transient middle cerebral artery occlusion in rats. AB - We tested the hypothesis that mild and severe ischemic cell damage are reflected in neurological and functional recovery after stroke. Rats were subjected to either 30 min or 120 min of middle cerebral artery occlusion or sham operation. Neurological and functional tests including, gross neurological score, and rotarod and adhesive removal tests were performed at various time points up to 21 days after stroke. Significant differences between groups of animals were detected using the rotarod and adhesive removal test. A significant correlation between lesion volume and adhesive removal test was detected in rats subjected to 30 min of ischemia. Our data indicate that quantitative rotarod and adhesive removal tests measure different aspects of functional recovery after stroke, and both are useful in characterizing functional recovery from an ischemic insult. PMID- 10727699 TI - Fragmentation of the Golgi apparatus of the anterior horn cells in patients with familial amyotrophic lateral sclerosis with SOD1 mutations and posterior column involvement. AB - The Golgi apparatus (GA) of the anterior horn cells in the spinal cord was examined by immunohistological methods with an antibody against the MG-160 protein, a conserved intrinsic membrane sialoglycoprotein of the medial cisternae of the GA, in three patients with familial amyotrophic lateral sclerosis (FALS) with posterior column involvement. Large motor neurons in the anterior horns were markedly reduced in number and 10 of total 14 remaining large motor neurons showed fragmentation and a reduction in the number of the elements of the GA. The fragmentation of the GA was identical to that previously reported in motor neurons of the spinal cord and motor cortex from patients with sporadic ALS and in transgenic mice expressing the G93A mutation of the gene encoding the Cu/Zn superoxide dismutase months before the onset of paralysis. This is the first report of fragmented GA of the anterior horn cells in patients with FALS with posterior column involvement. The findings suggest that the GA is a common target in the neuronal degeneration in sporadic and FALS. PMID- 10727701 TI - Acute disseminated encephalomyelitis in a pregnant woman successfully treated with plasmapheresis. PMID- 10727702 TI - Interactions between AMPA receptors and intracellular proteins. AB - alpha-Amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) receptors mediate most fast excitatory synaptic transmission in the mammalian CNS. They play a central role in synapse stabilisation and plasticity and their prolonged activation is potently neurotoxic. Developmental and activity-dependent changes in the functional synaptic expression of these receptors are subject to tight cellular regulation. The molecular and cellular mechanisms which control the postsynaptic insertion and arrangement of individual AMPA receptor variants are therefore the subject of intense investigation and in the last two years there has been significant progress towards elucidating some of the processes involved. Much of the new information has come from the application of the yeast two-hybrid assay, which has led to the discovery of a hitherto unexpected complexity of proteins which selectively interact with individual AMPA receptor subunits. These proteins have been implicated in the regulation of AMPA receptor post-translational modification, targeting and trafficking, surface expression and anchoring. The aim of this article is to present an overview of the major interacting proteins described so far and to place these in the context of how they may participate in the well ordered series of events controlling the cell biology of AMPA receptors. PMID- 10727703 TI - Developmental regulation of AMPA-receptor properties in CA1 pyramidal neurons of rat hippocampus. AB - AMPA-receptor (AMPA-R) currents were recorded from CA1 pyramidal neurons in situ and after acute isolation from the hippocampus of 3- to 45-day-old rats. Membrane currents were analyzed by combining the patch clamp method with fast application techniques. The complete block of receptor currents by GYKI 53655 and the absence of modulation by Concanavalin A indicated that the cells exclusively expressed non-NMDA glutamate receptors of the AMPA subtype while functional kainate receptors could not be detected. The lowest sensitivity to kainate and NBQX was observed at postnatal day (p) 18. These changes might reflect a lower abundance of GluR1 at that developmental stage. A decrease of potentiation of receptor currents by cyclothiazide (CTZ), an acceleration of the recovery from CTZ potentiation and a faster and more complete desensitization of glutamate-evoked currents suggest an up-regulation of flop splice variants with increasing age. These functional data indicate that AMPA-R expression in CA1 pyramidal neurons varies during postnatal development which can be expected to influence the kinetics of synaptic transmission and the excitotoxic vulnerability as well. PMID- 10727704 TI - Input-specific targeting of NMDA receptor subtypes at mouse hippocampal CA3 pyramidal neuron synapses. AB - Hippocampal CA3 pyramidal neurons receive synaptic inputs from commissural and associational fibers on both apical and basal dendrites. NMDA receptors at these synapses were examined in hippocampal slices of wild-type mice and GluRvarepsilon1 (NR2A) subunit knockout mice. Electrical stimulations at the CA3 stratum radiatum or stratum oriens activate both commissural and associational (C/A) synapses, whereas stimulations at ventral fimbria mainly activate commissural synapses. Ro 25-6981 and ifenprodil, the GluRepsilon2 (NR2B) subunit selective NMDA receptor antagonists, suppressed NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) at the commissural-CA3 synapses on basal dendrites more strongly than those at the C/A-CA3 synapses on apical or basal dendrites. However, glutamate-evoked NMDA receptor currents were reduced by the GluRepsilon1 subunit knockout to a similar extent at both apical and basal dendrites. The GluRepsilon1 subunit knockout also reduced NMDA EPSCs at the C/A CA3 synapses on basal dendrites, but did not affect NMDA EPSCs at the commissural CA3 synapses on basal dendrites. These results confirmed our previous findings that NMDA receptors operating at different synapses in CA3 pyramidal cells have different GluRepsilon subunit compositions, and further show that the GluRepsilon subunit composition may be regulated depending on the types of synaptic inputs, even within a single CA3 pyramidal neuron. PMID- 10727705 TI - Lack of expression of long-term potentiation in the dentate gyrus but not in the CA1 region of the hippocampus of mu-opioid receptor-deficient mice. AB - The possible involvement of the mu-opioid receptor subtype in mechanisms of long term potentiation (LTP) of the lateral perforant pathway to the dentate gyrus neurons, as well as of the Schaffer collateral-commissural input of CA1 neurons, was investigated using mu-opioid receptor-deficient mutant mice. In transversal hippocampal slices from mice lacking the mu-opioid receptor (MOR) only a short potentiation in the dentate gyrus after tetanization of the lateral perforant pathway was found. In contrast, the loss of the mu-opioid receptor in the CA1 region did not affect the potentiation of the field potentials induced by tetanization of the Schaffer collaterals. In parallel experiments, the application of 10 microM of the selective MOR-antagonist, funaltrexamine, decreased LTP in the dentate gyrus of wild-type mice but again did not alter the potentiation of the field potentials in the CA1. The loss of MOR-binding in the hippocampus was accompanied by a reduction in D2-binding sites indicating a possible compensatory role of the dopaminergic system. The D1- and glutamate binding was not affected. These observations confirm earlier results with pharmacological blockade of opioid receptors in the dentate gyrus and demonstrate an essential role of MOR activation for the generation of LTP in the dentate gyrus of the mouse but not necessarily in the CA1 region. PMID- 10727706 TI - Intracerebroventricular injection of clonidine releases beta-endorphin to induce mucosal protection in the rat. AB - The possibility that the endogenous opioid system could be involved in the central nervous system (CNS)-mediated gastroprotective effect of clonidine was investigated. Intracerebroventricularly (i.c.v.) injected clonidine (470 pmol/rat) inhibited the gastric mucosal lesions induced by (orally administered) acidified ethanol in a significant manner in the rat. The gastroprotective effect of the centrally administered clonidine was antagonised by i.c.v. or intracisternally (i.c.) administered presynaptic alpha-2 adrenoceptor antagonist, yohimbine; the non-selective opioid receptor antagonist, naloxone; and the delta opioid receptor antagonist naltrindole. These results suggest that an interaction between central alpha-2 adrenoceptors and endogenous opioid systems is involved in mediating the mucosal protective effect. beta-endorphin antiserum (i.c.) also antagonised the gastroprotection induced by intracerebroventricularly injected clonidine indicating that beta-endorphin release is likely to be a key factor in the gastroprotective effect of clonidine. Furthermore, the i.c.v. or i.c. injection of beta-endorphin produced a potent gastroprotection in the picomolar range. The mucosal protective effect of clonidine was abolished after vagotomy indicating that the central effect may be conveyed to the periphery by vagal efferents. Since atropine (1 mg/kg i.v.) failed to modify, but hexamethonium (10 mg/kg i.v.) antagonised the gastroprotective effect of clonidine, it would appear that in the periphery nicotinic, but not muscarinic, cholinergic receptors are likely to be involved in the mucosal protective effect of clonidine. In conclusion, clonidine (i.c.v.) induces gastroprotective action by releasing an endogenous opioid substance - most likely beta-endorphin - in the rat. The clonidine-induced central gastroprotection requires the integrity of vagal pathway; cholinergic nicotinic - but not muscarinic - receptors might mediate the effect in the periphery. PMID- 10727707 TI - Morphine attenuates the effects of juvenile isolation in rats. AB - The acute effects of juvenile isolation on sucrose intake and its long-term consequences on adult social behavior were investigated. Additionally, the role of the endogenous opioid systems was studied. Juvenile rats were housed in one of three conditions: in groups or in isolation with (partial isolation, PI) or without 30 min of daily social contact from 22 to 35 days-of-age. During this period the rats were treated daily with saline or morphine. Juvenile isolated rats showed an increased sucrose intake as compared to non-isolated controls, with PI-rats somewhere in-between, suggesting a negative correlation between the amount of social contact and sucrose consumption. Morphine treatment during the isolation period enhanced the sucrose intake in non-isolated rats, whereas it decreased sucrose consumption in (partial) isolated rats. With regard to the long term effects, (partial) isolated rats decreased social activity as compared to non-isolated controls which was reversed by morphine treatment during the isolation period. In non-isolated rats, morphine treatment caused an opposite effect: it decreased social activity as compared to the saline treated controls. The data suggest that stimulation of endogenous opioid systems in the juvenile phase may have an important modulatory role in the expression of adult social behavior. The results are discussed in relation to a possible function of morphine as a substitute for the release of endogenous opioid peptides during social play. PMID- 10727709 TI - Tonic benzodiazepine-sensitive GABAergic inhibition in cultured rodent cerebellar granule cells. AB - Recent studies have demonstrated that granule cells in rat cerebellar slices exhibit a tonic form of GABAergic inhibition. The presence of a similar constitutive GABAergic conductance was investigated in synaptically coupled cultures of neonatal rat cerebellum. In cells exhibiting spontaneous inhibitory postsynaptic currents (IPSCs), application of the GABA(A) receptor antagonist bicuculline (10 microM) eliminated the IPSCs and also produced a significant decrease in holding current. This latter effect was lacking in cells that did not exhibit IPSCs. Application of TTX (1 microM) and Cd(2+) (100 microM) decreased the IPSC frequency and also produced a change in holding current; these effects were eliminated by the prior application of bicuculline. In the presence of TTX, application of the benzodiazepine (BDZ) Flunitrazepam (1 microM) caused a 85+/ 15% increase in the component of holding current that arose from GABA(A) receptor activity. Noise analysis indicated that the GABA(A) receptors underlying this tonic form of GABAergic inhibition exhibited a mean single channel conductance close to 14 pS, a value similar to that seen for somatic GABA(A) receptors in these cells. Thus, like their counterparts in cerebellar slices, cerebellar granule cells in culture exhibit a background GABAergic conductance. The most likely source of this tonic current is GABA spilt over from active inhibitory synapses. As this conductance was sensitive to benzodiazepine receptor agonists it is unlikely to arise entirely from GABA(A) receptors containing the alpha6 subunit. PMID- 10727708 TI - Different sensitivities of human and rat rho(1) GABA receptors to extracellular pH. AB - We have examined the sensitivity of human and rat homo-oligomeric rho(1) GABA receptors to variations in extracellular pH (pH(o)) using the whole-cell patch clamp technique. The GABA-induced conductance mediated by the rat rho(1) receptor (rho(1)-R) decreased with a decrease in pH(o) between 9.0 to 5.4. Below pH(o) 7.4 the effect of protons on the GABA-induced conductance was apparently competitive, but above pH(o) 7.4 the inhibitory effect of extracellular protons was almost independent on the GABA concentration. Titration of the GABA-induced conductance at 3 microM GABA revealed two protonation sites on rat rho(1)-R with pKa 6.4 and pKa 8.2. At 10 microM GABA the low pKa (6.4) was shifted to a clearly lower value (5.6), but the high pKa was only slightly decreased (from 8.2 to 7.9). Zn(2+) ions were capable of relieving the proton inhibition at low pH(o) indicating that Zn(2+) interacts with the low pKa site. Unlike the rat rho(1)-R, the human rho(1) R was sensitive only to changes in pH(o) at acidic levels. Proton inhibition of human rho(1)-R was apparently competitive, as observed on rat-rho(1) at acidic pH(o). Titration of the human rho(1)-R gave a single H(+) binding site with a pKa of 6.3, similar to the value for the low pKa on rat rho(1)-R. The pKa value of human rho(1)-R was not dependent on the GABA concentration. A chimeric receptor, consisting of the N-terminal part of the rat rho(1)-R and C-terminal part of the human rho(1)-R, displayed pH(o) sensitivity similar to that observed for rat rho(1)-R. This indicates that the high pKa of rat rho(1)-R is attributable to the 11 amino acid differences between the rat and human rho(1)-R extracellular domains. PMID- 10727710 TI - GABAergic involvement in motor effects of an adenosine A(2A) receptor agonist in mice. AB - Adenosine A(2A) agonists are known to induce catalepsy and inhibit dopamine mediated motor hyperactivity. An antagonistic interaction between adenosine A(2A) and dopamine D(2) receptors is known to regulate GABA-mediated neurotransmission in striatopallidal neurons. Stimulation of adenosine A(2A) and dopamine D(2) receptors has been shown to increase and inhibit GABA release respectively in pallidal GABAergic neurons. However, the role of GABAergic neurotransmission in the motor effects of adenosine A(2A) receptors is not yet known. Therefore in the present study the effect of GABAergic agents on adenosine A(2A) receptor agonist (NECA- or CGS 21680) induced catalepsy and inhibition of amphetamine elicited motor hyperactivity was examined. Pretreatment with GABA, the GABA(A) agonist muscimol or the GABA(B) agonist baclofen potentiated whereas the GABA(A) antagonist bicuculline attenuated NECA- or CGS 21680-induced catalepsy. However, the GABA(B) antagonists phaclophen and delta-aminovaleric acid had no effect. Administration of NECA or CGS 21680 not only reduced spontaneous locomotor activity but also antagonized amphetamine elicited motor hyperactivity. These effects of NECA and CGS 21680 were potentiated by GABA or muscimol and antagonized by bicuculline. These findings provide behavioral evidence for the role of GABA in the motor effects of adenosine A(2A) receptor agonists. Activation of adenosine A(2A) receptors increases GABA release which could reduce dopaminergic tone and induce catalepsy or inhibit amphetamine mediated motor hyperactivity. PMID- 10727711 TI - The protective effect of riluzole in the MPTP model of Parkinson's disease in mice is not due to a decrease in MPP(+) accumulation. AB - Riluzole, has previously been shown to be protective in animal models of Parkinson's disease in vivo. In the present study the effects of riluzole on the intrastriatal formation and accumulation of MPP(+), after i.p. injection of MPTP were tested in mice, using two different experimental protocols. In the first protocol, mice were treated with a single dose (15 mg/kg i.p.) of MPTP and MPP(+) accumulation was measured 30 min, 1 h and 3 h after the injection of the toxin. Riluzole (10 mg/kg p.o.), administered 30 min before MPTP, did not modify the accumulation kinetic of MPP(+). Contrarily to riluzole, a single dose of 50 mg/kg p.o. of 7-nitroindazole (7-NI), a non-selective non hypertensive inhibitor of nitric oxide synthase (NOS), significantly decreased MPP(+) levels. In the second protocol, consisting of 3 injections of MPTP (15 mg/kg i.p.), riluzole, administered 4 times at the dose of 5 mg/kg p.o., had no effect on MPP(+) levels. The protective effect of repeated treatments of riluzole and 7-NI against MPTP induced depletion of dopamine (DA) is also reported. Our data obtained with 7-NI (in agreement with previous studies reported by others) suggest that a part of the protection observed with this NOS inhibitor is probably due to in vivo inhibition of monoamine oxidase-B (MAO-B). That riluzole does not modify MPP(+) accumulation demonstrates that its protective effect against MPTP toxicity was not due to an in vivo interference with MPTP metabolism. PMID- 10727712 TI - Pharmacological identification of the K(+) currents mediating the hypoglycemic hyperpolarization of rat midbrain dopaminergic neurones. AB - Hypoglycemia (zero glucose) initially depolarized the membrane and increased the spontaneous firing of rat midbrain dopaminergic neurones (more than 50%) intracellularly recorded in an in vitro slice preparation. Under single-electrode voltage-clamp mode (V(h) -55 mV), this transient phase correlated with an inward current of -18 pA. In all the cells tested (n=30), an inhibition fully developed over 16.9 min of hypoglycemia and was associated with a hyperpolarization of the membrane (7.7 mV) or outward current (95.6 pA). Upon re-application of a control solution (glucose 10 mM) a rebound hyperpolarization/outward current developed. The depression of firing was only seen when the artificial cerebrospinal fluid (ACSF) contained less than 1 mM glucose. In addition, the period of time required to block the spontaneous activity decreased, by diminishing the extracellular concentration of glucose from 1 to 0 mM. The hypoglycemia-induced outward current was associated with an increase in membrane conductance and reversed polarity at 100.4 mV, close to the reversal potential of K(+). The post-hypoglycemic outward current was not associated with an increase in membrane conductance and did not reverse. The K(+)-ATP channel blockers, tolbutamide (300 microM-1 mM) and glibenclamide (3-30 microM) reduced the hypoglycemia-induced inhibition. In addition, the blocker of the Ca(++)-activated K(+)-channels, charybdotoxin (100 400 nM) partially counteracted the hypoglycemic hyperpolarization. Furthermore, barium (100-300 microM) fully antagonized the hypoglycemia-induced inhibition. The post-hypoglycemic hyperpolarization/outward current was not observed in cells treated with the Na(+)/K(+) ATPase pump inhibitor strophanthidin (1-3 microM). Our data suggest that midbrain dopaminergic cells respond to glucose deprivation with a hyperpolarization generated by the opening of several K(+) channels (sulphonylurea-sensitive, charybdotoxin-sensitive and sulphonylurea and charybdotoxin-insensitive) and by the activation of the Na(+)/K(+) ATPase pump after the hypoglycemic period. PMID- 10727713 TI - Inhibition of voltage-gated calcium channels by fluoxetine in rat hippocampal pyramidal cells. AB - Fluoxetine, an antidepressant which is used world-wide, is a prominent member of the class of selective serotonin re-uptake inhibitors. Recently, inhibition of voltage-gated Na(+) and K(+) channels by fluoxetine has also been reported. We examined the effect of fluoxetine on voltage-gated calcium channels using the patch-clamp technique in the whole-cell configuration. In hippocampal pyramidal cells, fluoxetine inhibited the low-voltage-activated (T-type) calcium current with an IC(50) of 6.8 microM. Fluoxetine decreased the high-voltage-activated (HVA) calcium current with an IC(50) between 1 and 2 microM. Nifedipine and omega conotoxin GVIA inhibited the HVA current by 24% and 43%, respectively. Fluoxetine (3 microM), applied in addition to nifedipine or omega-conotoxin, further reduced the current. When fluoxetine (3 microM) was applied first neither nifedipine nor omega-conotoxin attenuated the remaining component of the HVA current. This observation indicates that fluoxetine inhibits both L- and N-type currents. In addition, fluoxetine inhibited the HVA calcium current in carotid body type I chemoreceptor cells and pyramidal neurons prepared from prefrontal cortex. In hippocampal pyramidal cells high K(+)-induced seizure-like activity was inhibited by 1 microM fluoxetine; the mean burst duration was shortened by an average of 44%. These results provide evidence for inhibition of T-, N- and L-type voltage gated calcium channels by fluoxetine at therapeutically relevant concentrations. PMID- 10727714 TI - Roles of 5-HT(1A) receptors in the dorsal raphe and dorsal hippocampus in anxiety assessed by the behavioral effects of 8-OH-DPAT and S 15535 in a modified Geller Seifter conflict model. AB - 8-OH-DPAT [8-hydroxy-2-(di-N-propylamino)tetralin], a 5-HT(1A) receptor agonist, and S 15535 (4-benzodioxan-5-yl)1-(indan-2-yl)piperazine, a partial agonist at 5 HT(1A) receptors, were administered into the dorsal raphe nucleus and dorsal hippocampus and their behavioral effects were assessed in a modified Geller Seifter conflict model. Injected into the dorsal raphe nucleus 8-OH-DPAT, 1 microg but not 0.04 or 0.2 microg 0.5 microl(-1), and S 15535, 2.5 microg but not 0.1 or 0.5 microg 0.5 microl(-1), significantly increased punished responding with no effect on rates of unpunished or time-out responding. WAY 100635, a selective 5-HT(1A) receptor antagonist, injected subcutaneously at 0. 3 mg kg(-1) 30 min before 1 microg 8-OH-DPAT or 2.5 microg S 15535 in the dorsal raphe, completely antagonized their effects on punished responding. At doses ranging from 1 to 10 microg microl(-1) injected into the CA1 region of the dorsal hippocampus neither 8-OH-DPAT nor S 15535 modified punished responding or the rates of time-out. At the highest doses, 8-OH-DPAT significantly reduced unpunished responding whereas S 15535 had the opposite effect. The results suggest that stimulation of 5-HT(1A) receptors in the dorsal raphe nucleus has anxiolytic-like effects whereas stimulation of postsynaptic receptors in the dorsal hippocampus has no anxiolytic or anxiogenic effects, at least judging from changes in rates of punished responding. These results are compatible with the hypothesis that 5-HT(1A) receptor agonists and partial agonists attenuate anxiety by reducing serotonergic transmission in brain areas innervated by the dorsal raphe nucleus. PMID- 10727715 TI - The role of 5-HT(1A) and 5-HT(1B/1D) receptors on the modulation of acute fluoxetine-induced changes in extracellular 5-HT: the mechanism of action of (+/ )pindolol. AB - Some clinical evidence has suggested that (+/-)pindolol can be effective at producing a shortened time to onset of antidepressant activity when co administered with a serotonin specific reuptake inhibitor (SSRI). This effect has been attributed to the antagonist effects of pindolol at the 5-HT(1A) receptor. In the present study, we compared the pharmacology of (+/-)pindolol, WAY-100635 (a 5-HT(1A) antagonist), GR127935 (a 5-HT(1B/1D) antagonist), and isamoltane (a 5 HT(1B) antagonist), when given acutely in combination with fluoxetine, using in vivo microdialysis in the frontal cortex of the freely moving rat. We have determined that the acute fluoxetine-induced increases in extracellular 5-HT can be augmented by (+/-)pindolol, WAY100635, GR127935 and isamoltane with maximum increases of 216+/-32%, 235+/-49%, 240+/-18% and 171+/-47% of preinjection control levels, respectively. Combination of both 5-HT(1A) and 5-HT(1B/1D) autoreceptor antagonists with fluoxetine produced additive increases in extracellular 5-HT (i.e. WAY100635+GR127935+fluoxetine and WAY100635+isamoltane+fluoxetine produced a four- and five-fold potentiation, respectively), suggesting that this strategy may be useful in further augmenting the action of a SSRI in the treatment of depression. In addition, by comparing the combined administration of (+/-)pindolol with either WAY100635, GR127935 or isamoltane, we have determined that (+/-)pindolol produces much of its acute potentiation of fluoxetine-induced increases in extracellular 5-HT via its action at the 5-HT(1B/D) receptor in addition to any activity it has at the presynaptic 5-HT(1A) receptor. PMID- 10727716 TI - The effect of serotonergic agents on haloperidol-induced striatal dopamine release in vivo: opposite role of 5-HT(2A) and 5-HT(2C) receptor subtypes and significance of the haloperidol dose used. AB - This study investigated, using microdialysis in freely-moving rats, the role of serotonin (5-HT) and 5-HT(2) receptor subtypes in the enhancement of striatal dopamine (DA) release induced by various doses of haloperidol. The subcutaneous injection of 0.01, 0.1 or 1 mg/kg haloperidol dose-dependently increased DA outflow (160, 219 and 230% of baseline, respectively). The effect of 0.01 mg/kg haloperidol was, respectively, potentiated by the 5-HT uptake inhibitor citalopram (1 mg/kg, s.c.; +35%) and reduced by the 5-HT(1A) receptor agonist 8 OH-DPAT (0.025 mg/kg, s.c.; -32%). Also, it was reduced by the 5-HT(2A) antagonist SR 46349B (0.5 mg/kg, s.c. ; -40%) or by the 5-HT(2A/2B/2C) antagonist ritanserin (1.25 mg/kg, i.p.; -34%), and potentiated by the 5-HT(2B/2C) antagonist SB 206553 (5 mg/kg, i.p; +78%). Further, only this latter compound significantly modified basal dopamine release by itself (+26%). Dopamine released by 0.1 mg/kg haloperidol was enhanced (+100%) by citalopram, decreased (-61%) by SR 4634B, but unaltered by SB 206553. Finally, none of the compounds used were able to modify the enhancement of dopamine release induced by 1 mg/kg haloperidol. These results show that central 5-HT(2A) and 5-HT(2C) receptors exert an opposite (respectively excitatory and inhibitory) influence on DA release. Moreover, they suggest that the 5-HT(2A)-dependent modulation depends on the degree of central DA receptor blockade. PMID- 10727717 TI - Inhibition of 5-HT3 receptors by propofol: equilibrium and kinetic measurements. AB - Patch-clamp/rapid solution exchange experiments as well as tracer ([14C] guanidinium) influx measurements were applied to investigate effects of propofol on 5-HT3 receptor channels and compare the results with those obtained with pentobarbital. Currents induced by 30 microM 5-HT were recorded in outside-out patches from N1E-115 cells. Application of propofol 45 s before and during 5-HT application inhibited peak-currents and integrated current responses in a concentration-dependent manner (IC50 values=14.5 and 10.5 microM; Hill coefficients -1.5 and -1.3, respectively). The inhibitory effect of propofol in the current measurements was similar to the propofol-induced inhibition in tracer influx experiments in whole N1E-115 cells (Barann et al., 1993. Naunyn Schmiedeberg's Archives of Pharmacology 347, 125-132). Pentobarbital-induced inhibition of 5-HT3 receptors in both patch-clamp (Barann et al., 1997. Neuropharmacology 36, 655-664) and tracer influx measurements indicated a lower potency and lower slope (IC50 values=130 and 55 microM; Hill coefficients -0.8 and -0.7, respectively) compared to propofol. Propofol, in contrast to pentobarbital, showed nearly the full potency when applied to the patches exclusively 45 s before 5-HT. Propofol was least effective when administered exclusively during 5-HT. The onset of inhibition of 5-HT-induced peak currents by propofol had a time constant of 220 ms, similar to the kinetics of 5-HT-induced desensitization. PMID- 10727719 TI - Antagonists of P2 receptor prevent NGF-dependent neuritogenesis in PC12 cells. AB - The pheochromocytoma PC12 cell line that develops neuronal characteristics of sympathetic cells after treatment with nerve growth factor (NGF) represents a well-established cellular model system for studying NGF signalling. Interesting information on the different mechanistic pathways of NGF can be obtained by adopting the pharmacological approach of inhibiting P2 receptors, expressed in naive PC12 cells and recognised as important biological mediators of neurotransmitters and growth factors. We show here that Basilen Blue, an antagonist of P2 receptor, reversibly prevents NGF-dependent neurite outgrowth with an IC(50) in the 5-10 microM range. Suramin, oxidised-ATP and diisothiocyanatostilbene-disulfonic acid, differently from other purinoceptor ligands, are also effective in this regard. NGF-dependent regeneration and stability of neurites, selected NGF-dependent extracellular and intracellular protein phosphorylations, binding of [(3)H] ATP to PC12 cell membranes are also modulated by Basilen Blue. On the contrary, cell adhesion, cellular duplication, 5'-nucleotidase activity, NGF-induced tyrosine autophosphorylation of TrkA receptors are not affected. NGF furthermore directly modulates the extracellular release of ATP and especially the levels of P2X(2) receptor protein in PC12 cells. In addition, extracellular ATP improves the neuritogenic effect of sub optimal concentrations of NGF. Our study identifies P2 receptor ligands, particularly Basilen Blue, as useful tools to dissect different NGF-evoked functions, suggesting a mechanistic role for P2 receptors in the signalling pathways of NGF. PMID- 10727718 TI - Reserpine or chronic paroxetine treatments do not modify the vesicular monoamine transporter 2 expression in serotonin-containing regions of the rat brain. AB - To date, very little information is available about the regulation of vesicular monoamine transporter in central serotonergic regions. The expression of the vesicular monoamine transporter 2 (VMAT2) has been studied in the serotonergic system of the rat brain after an 18 day treatment with the serotonin selective reuptake inhibitor paroxetine (10 mg/kg, i.p., once daily). This treatment, while increasing serotonergic transmission, did not modify either VMAT2 mRNA expression or (3)H-dihydrotetrabenazine binding site density in any of the studied regions. These results suggest that VMAT2 regulation in the central serotonergic system is not involved in the mechanism of action of antidepressants. In addition, a single administration of reserpine (5 mg/kg, s.c.), while blocking the vesicular monoamine uptake function, had no effect on VMAT2 immunoreactivity in the dorsal raphe nucleus 2 or 30 days after injection. It is concluded that neither a reduction (reserpine) nor an enhancement (paroxetine) of the serotonin transmission induces VMAT2 regulation in serotonergic system in the rat brain. PMID- 10727720 TI - Effect of organochlorine insecticides on nicotinic acetylcholine receptor-rich membranes. AB - The so-called generalized polarization (GP) of the fluorescent probe Laurdan and the steady-state fluorescence anisotropy of the probe diphenylhexatriene (DPH) and its phenylpropionic derivative (PA-DPH) were used to study the effects of several organochlorine insecticides of the chlorophenylethane, chlorinated cyclohexane and chlorinated cyclodiene families on the Torpedo nicotinic acetylcholine receptor (AChR)-rich native membrane. All insecticides, with the exception of Lindane, augmented Laurdan GP both in the native membrane and in model lipid systems. Most organochlorine compounds produced a concentration dependent decrease of DPH and PA-DPH anisotropy in the AChR-rich membrane. These compounds exhibited a dual behavior vis-a-vis the native AChR-rich membrane, exerting disordering effects at the bilayer core while ordering and/or excluding water molecules from the lipid-protein interface region, as sensed by DPH anisotropy and Laurdan GP, respectively. Furthermore, all insecticides decreased the efficiency of fluorescence resonance energy transfer between the intrinsic protein and Laurdan, albeit to different extents. On the basis of all these observations, the existence of potential target sites for insecticides in the protein-lipid interface region is postulated. PMID- 10727721 TI - Intracellular calcium releases facilitate induction of long-term depression. AB - In visual cortex layer II/III pyramidal neurons, long-term depression (LTD) of synaptic currents was induced by a combination of the intracellular increase of inositol-1,4,5-trisphosphate (IP(3)), achieved by photolyzing caged-IP(3), and the tetanization to nearby cortex, but not by either of these two procedures alone. A facilitatory role of an IP(3)-induced calcium release in LTD is suggested. PMID- 10727722 TI - [(35)S]-GTPgammaS autoradiography reveals alpha(2) adrenoceptor-mediated G protein activation in amygdala and lateral septum. AB - alpha(2)-adrenoceptor-mediated G-protein activation was examined by [(35)S] GTPgammaS autoradiography. In alpha(2)-adrenoceptor-rich regions (amygdala, lateral septum), noradrenaline stimulated [(35)S]-GTPgammaS binding. These actions were abolished by the selective alpha(2) antagonist, atipamezole. Conversely, in caudate nucleus, which expresses few alpha(2) receptors, noradrenaline-induced stimulation was not inhibited by atipamezole, suggesting that it is not mediated by alpha(2)-adrenoceptors. PMID- 10727723 TI - Citalopram reduces social interaction in rats by activation of serotonin (5 HT)(2C) receptors. AB - The selective serotonin (5-HT) reuptake inhibitor (SSRI), citalopram (2.5 mg/kg, s.c.), reduced social interaction (SI) in rats. This action was abolished by the 5-HT(2B/2C) receptor antagonist, SB206, 553 (0.63 mg/kg, s.c.), and the 5-HT(2C) receptor antagonist, SB242, 084 (0.04 mg/kg, i.p.), but not by the 5-HT(2A) antagonist, MDL100, 907 (0.04 mg/kg, s.c.), the 5-HT(1A) antagonist, WAY100,635 (0.16 mg/kg, s.c.), or the 5-HT(3) antagonist, ondansetron (0.16 mg/kg, s. c.). These data suggest that 5-HT(2C) receptors are involved in the "anxiogenic" actions of citalopram. PMID- 10727724 TI - Ontogeny of a photic response in the retina and suprachiasmatic nucleus in the mouse. AB - The ontogeny of photic responsiveness in the retina and the suprachiasmatic nucleus (SCN) of C57BL/6J mouse was studied using the enhanced expression of the immediate early gene c-fos as a marker of neuronal activation. c-fos expression was assessed by immunocytochemical localisation of its protein product. Light induction of Fos-like protein in the retina and SCN occurred first at postnatal day four (PD 4). At this stage of development, some cells in the inner part of the neuroblastic layer and in the ganglion cell layer showed positive immunoreaction; the number of Fos-like positive cells increased with age until it reached adult levels by PD 15. Induction of Fos-like expression at PD 4 in the SCN mainly occurred in the ventrolateral region, the region that receives the greatest density of retinal innervation. These results indicate that retinal input can activate cells in the SCN even before eyelids open, and the SCN can be stimulated by photic inputs as early as day 4 after birth. PMID- 10727725 TI - Patterns of CYP26 expression in human prenatal cephalic and hepatic tissues indicate an important role during early brain development. AB - CYP26 (P450RAI) catalyzes catabolic retinoic acid (RA) hydroxylation and thereby appears to play a critical role in retinoid signaling pathways during development. In this study, a quantitative competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for evaluation of CYP26 message levels in human prenatal tissues. Statistical analyses of transcription levels in 12 prenatal human brains and six prenatal human livers demonstrated good sensitivity and reproducibility. Quantitative profiles of CYP26 gene expression in early (gestational days 57-110) prenatal cephalic and hepatic tissues and comparisons with adult counterparts are reported for the first time. Prenatal cephalic tissues at days 57-67 exhibited values of 1950+/-420 (CYP26 molecules/10(6) GAPDH molecules) whereas prenatal cephalic tissues at days 105 110 exhibited values of 22300+/-4450 (CYP26 molecules/10(6) GAPDH molecules), indicating a sharp developmental increase (approximately 11-fold). Levels in human adult cephalic tissues were slightly less than the prenatal cephalic levels measured during the earliest stages of gestation and were approximately 3-fold lower than those measured in adult human hepatic tissues. Levels in human prenatal hepatic tissues at days 63-110 gestation were less than 800 (CYP26 molecules/10(6) GAPDH molecules) and did not exhibit developmental increases. Considered together, the data have strong implications for the importance of CYP26 in early development of the human brain. PMID- 10727726 TI - Development of nitric oxide neurons in the chick embryo retina. AB - Nitric oxide (NO) is a gas involved in neurotransmission in the central nervous system (CNS) and in vertebrate retinas. This paper describes five types of nitrergic neurons in developing and adult chick retina using the nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) reaction. Three of them, nitrergic types 1, 2 and 3, were observed in the inner nuclear layer, while nitrergic type 4 was observed in the ganglion cell layer; nitrergic type 5 were the retinal photoreceptors. Cell processes formed four nitrergic networks, which could be observed in the inner plexiform layer (IPL), at sublayers 1, 3a, 3b and 4. Another nitrergic network was observed in the outer plexiform layer (OPL). From hatching, the dendritic branches were completely developed in the IPL and in the OPL, forming the mentioned networks. Current evidence suggests that NO is coexpressed with other neurotransmitters in neurons of the CNS. Double-staining procedures, using NADPHd and 5HT immunohistochemistry in chicken retina, in a sequential or in an alternative manner, did not reveal the coexistence of these two neurotransmitters in the same neurons, but their networks matched in sublayers 1 and 4 of the IPL. PMID- 10727727 TI - Developmental regulation of pituitary adenylate cyclase-activating polypeptide and PAC(1) receptor mRNA expression in the rat central nervous system. AB - As the brain develops, a homogeneous population of mitotically active progenitors generates the molecularly heterogeneous post-mitotic cells of the mature brain. The balance between cell division, growth arrest and differentiation of these progenitors undoubtedly requires the activation of a vast array of genes. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon family. Within the nervous system, PACAP has been shown to stimulate neurite outgrowth, regulate neurotransmitter production and neuronal survival. These diverse biological actions are mediated through interaction with two types of receptors, a PACAP selective receptor (PAC(1)-R) and receptors which interact almost equally with both VIP and PACAP. Since several lines of evidence suggest that PACAP acts as a neurotrophic factor, we sought to characterize PACAP and PAC(1)-R expression in the developing rat nervous system. The PAC(1)-R is expressed at very high levels in ventricular zones throughout the neuraxis. In addition to the embryonic enrichment in proliferative zones, PAC(1)-R expression is maintained in areas of neurogenesis in the adult central nervous system (CNS), namely, the subventricular zone of the olfactory bulb and hippocampal dentate gyrus. In contrast, PACAP is expressed primarily in the post-mitotic parenchyma. This temporal regulation and cellular distribution suggests that PACAP, through its interaction with the PAC(1)-R, may play a role in mammalian neurogenesis. PMID- 10727728 TI - Localization of mRNAs for phospholipase D (PLD) type 1 and 2 in the brain of developing and mature rat. AB - Phospholipase D (PLD) is known as one of the key enzymes in the lipid metabolism which produces several second messengers. We demonstrated the localization of the gene expression for PLD1 and PLD2 using in situ hybridization histochemistry in the brain of developing and mature rats. Whereas PLD1 mRNA expression was detected mainly in presumptive oligodendrocytes, PLD2 mRNA expression was detected mainly in presumptive astrocytes. In addition, the gene expression for PLDs were expressed in neuroepithelial cells of the ventricular/ependymal zones and the gene for PLD2 was expressed transiently in early postnatal gray matters, presumptive neurons. PMID- 10727729 TI - Extracellular matrix proteins increase the expression of pro-TRH and pro-protein convertase PC1 in fetal hypothalamic neurons in vitro. AB - External clues for neuron development include extracellular matrix (ECM) molecules. To explore ECM influence on the early development of peptide phenotype in the CNS, we have compared pro-TRH levels in primary cultures of rat hypothalamic cells plated either on poly-lysine (PL) (control) or on PL plus one of various ECM molecules at 10 microgram/ml. Fetal day 17 cells plated at a density of 1250/mm(2) were grown in a serum free medium made of Neurobasal medium supplemented with B27 (GIBCO). Cultures, consisting mainly of neurons, were analyzed at DIV 2. ECM proteins induced morphological effects in agreement with previously published studies. The amount of pro-TRH per dish, quantified by Western blotting, was increased to 275% for laminin, 191% for fibronectin and 173% for tenascin-C (control=100%); there was no effect of vitronectin. Laminin or fibronectin did not change pro-TRH mRNA or TRH levels but enhanced levels of the pro-protein convertase PC1 suggesting that the ECM molecules did regulate the translational status of pro-TRH. In conclusion, we have shown that some ECM proteins increased pro-TRH level in vitro; this may contribute to the enhancement of pro-TRH levels observed early in vivo in the hypothalamus. PMID- 10727730 TI - Identification of the novel developmentally regulated gene, Bdm2, which is highly expressed in fetal rat brain. AB - Most of the neurogenesis take place during the embryonic stage; the genes expressed predominantly in this stage may play important roles in the control of development of the central nervous system. Using a differential display method, we identified the novel rat gene, brain development-related molecule 2 (Bdm2), that is expressed more abundantly in the embryonic brain than in the adult brain. Full-length Bdm2 cDNA consists of 1842 base pairs (bp) and contains an open reading frame of 1260 bp. Northern blot analysis demonstrated that Bdm2 was strongly expressed in the late embryonic brain and was still detected at lower levels in an early postnatal period; in adults, Bdm2 mRNA was decreased to an undetectable level in brain, though the expression of this mRNA was revealed in other tissues. Level of Bdm2 mRNA was maintained during neuronal differentiation of mouse embryonal carcinoma cell P19, but decreased during the differentiation to glial and unidentified non-neuronal cells. In situ hybridization study demonstrated the wide distribution of Bdm2 mRNA in the embryonic brain; in the adult brain, the hybridization signals became more restricted to the hippocampus, olfactory bulb, cerebellum, and neocortex, almost coinciding with the regions where nascent and immature neurons are present. Thus, it appears likely that Bdm2 encodes a protein that is involved in both the regulation of growth of undifferentiated neural cells and the terminal differentiation of neuronal cells. PMID- 10727731 TI - Histogenesis of the cerebral cortex in rat fetuses with a mutation in the Pax-6 gene. AB - The embryonic development of the cerebral cortex was histologically examined in rat homozygotes with a mutation of the Paired box (Pax)-6 gene, rat Small eye (rSey(2)/rSey(2)). Although the cerebral wall was thinner in rSey(2)/rSey(2) than in the wild type at embryonic day 16 (E16), cortical cells of mutants labeled with 5'-bromodeoxyuridine (BrdU) at E13 migrated as normal, settling in superficial layer at E16. Mitotic activity in the ventricular zone, estimated by immunoreactivity for proliferating cell nuclear antigen (PCNA), was also retained. On the other hand, after E20 cells were clustered in abnormally expanded ventricular and intermediate zones of the rSey(2)/rSey(2) cortex. Birthdating studies using BrdU revealed that most of these clustered cells were generated between E18 and E20. Most of clustered cells were immunoreactive for PCNA and highly polysialylated NCAM, while immunoreaction for neurofilament and microtubule-associated protein-2 (MAP-2) was hardly detected in the clusters. Furthermore, apoptosis detected with terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick end labeling (TUNEL) was rarely seen, suggesting that the clustered cells remain in an undifferentiating state, but not degenerated by the end of the gestational period. Considering that Pax-6 immunoreactivity was exclusively localized in the ventricular zone of the normal rat cortex throughout the fetal period, the present results suggest that Pax-6 is crucial for differentiation and migration of late-generated cortical neurons. PMID- 10727732 TI - Prenatal Delta(9)-tetrahydrocannabinol exposure modifies proenkephalin gene expression in the fetal rat brain: sex-dependent differences. AB - Perinatal Delta(9)-tetrahydrocannabinol (Delta(9)-THC) exposure in rats resulted in enhanced morphine self-administration behavior, naloxone-precipitated withdrawal signs or changes in pain sensitivity, which have been related to changes in micro-opioid receptor binding and/or proenkephalin mRNA levels in several brain regions. However, despite exposure of these animals to Delta(9)-THC from fetal ages, the effects were studied only when animals matured, whereas there is no study on possible changes caused by this cannabinoid during the prenatal ontogeny of opioidergic neurons. The purpose of the present study was to examine the changes in proenkephalin mRNA levels, measured by using in situ hybridization, in several brain nuclei of rat fetuses that had been daily exposed to Delta(9)-THC from the 5th day of gestation. Results were as follows. Prenatal Delta(9)-THC exposure altered proenkephalin mRNA levels in most of the brain areas studied at different fetal ages, but the effects were different between sexes. Thus, proenkephalin mRNA levels increased in females, but decreased in males that had been prenatally exposed to Delta(9)-THC. This was observed in the caudate-putamen, hypothalamic paraventricular and ventromedial nuclei and cerebral cortex. No changes were observed, however, in the subventricular zones of the caudate-putamen, neocortex and nucleus accumbens. In summary, prenatal Delta(9)-THC exposure produced a sex-dependent effect in proenkephalin mRNA levels in several brain structures of rat fetuses. PMID- 10727733 TI - In vivo evidences of early neurosteroid synthesis in the developing rat central nervous system and placenta. AB - The aim of the present study was to determine the developmental pattern of progesterone metabolism in rat brain and spinal cord from embryonic day 13 (E13) to the perinatal period. A marked decrease in the 5alpha-reduction of progesterone in brain cortex was observed between E13 and postnatal day 5 (P5). Isopregnanolone was the predominant isomer in E13 in both cortex and spinal cord and its synthesis diminished gradually, while the concentration of allopregnanolone did not change significantly during development. The placental tissue was able to synthesize the 3alpha and 3beta isomers in E13, E16 and E19 embryos with allopregnanolone being the major metabolite in all the samples. We conclude that embryonic central nervous system tissues are able to synthesize neurosteroids at least from stage E13 and that they are developmentally regulated. PMID- 10727734 TI - Myelination in the splenium of the corpus callosum in adult male and female rats. AB - Previous work reported increases in the number of myelinated axons in the splenium of the rat corpus callosum between 25 and 60 days of age. In the present study, we quantified the area occupied by myelinated axons using a light microscopic point counting technique at 60, 120 and 180 days. Myelinated axons increased across these ages (p=0.001). Thus, myelination of the rat corpus callosum persists well into adulthood. PMID- 10727735 TI - The cortical vibrissae representation is normal in transgenic mice lacking the 5 HT(1B) receptor. AB - Recent studies have suggested that 5-HT may modulate thalamocortical development in somatosensory cortex (S-I) of rats and mice, and that the 5-HT(1B) receptor may play a critical role in this process. Analysis of CO-stained sections through lamina IV of S-I in perinatal and adult 5-HT(1B) knockout mice revealed a normal vibrissae-related pattern, indicating that activation of the 5-HT(1B) receptor is not necessary for the normal development of the vibrissae representation in S-I. PMID- 10727736 TI - Evidence of deprenyl-insensitive apoptosis of nigral dopamine neurons during development. AB - Apoptosis of dopamine neurons occurs naturally in the substantia nigra during development, culminating in approximately 30% loss of these cells during the perinatal period. Deprenyl, independent of its monoamine oxidase (MAO)-B inhibitory properties, can prevent dopamine neuronal apoptosis in models of neurodegeneration. Our current study demonstrate that apoptotic death of dopamine neurons during development is insensitive to daily treatment of pregnant mothers and then newborns with deprenyl (0.1, 1, or 10 mg/kg). This result is not due to poor crossing of the placental and blood-brain barriers, since deprenyl caused a dose-dependent inhibition of brain MAO-B activity in pups at birth. Determining the pathway(s) leading to deprenyl-insensitive apoptosis of nigral dopamine neurons in development may shed light on mechanisms underlying the premature death of dopamine neurons in neurodegenerative disorders. PMID- 10727737 TI - Postnatal development of the basal forebrain cholinergic projections to the medial prefrontal cortex in mice. AB - The postnatal development of basal forebrain cholinergic projections to the medial prefrontal cortex in mice was analyzed by means of the double labeling track-tracing study. The tracer was injected into the medial prefrontal cortex of mice, on the day of birth (P0) to 60 days after birth. The total number of basal forebrain neurons increased from P4 to P8, and began to decrease until P13 (52.9% vs. the maximal average (P8)). After P13, the mean average remains stable up to P60. On the other hand, differential pattern of frontocortical projections of the anterior, intermediate, and posterior regions can be observed. PMID- 10727738 TI - Refinement of the ipsilateral retinocollicular projection is disrupted in double endothelial and neuronal nitric oxide synthase gene knockout mice. AB - Development of retinal connections to the superior colliculus (SC) requires an activity dependent refinement process in which axons gradually become restricted to appropriate retinotopic locations. Nitric oxide has been implicated in this process. We tested this possibility by studying the refinement of the ipsilateral retinocollicular projections (IRP) in normal C57-BL/6 mice and in double knockout mice in which the genes for the edothelial and neuronal isoforms of nitric oxide synthase (e, nNOS) were disrupted. Mice aged between P19 and adulthood were perfused 44-48 h after anterograde injections of WGA-HRP into one eye in order to measure the distribution of the labeled IRP. In normal mice, segregation of the IRP was complete at P21, with the ipsilateral projection restricted to the rostro medial SC. By contrast, the ipsilateral projection was spread over much more of the SC in double e, nNOS knockouts at P21 with patches of label distributed across the entire medio-lateral axis of the rostral 700 microm. Although the distribution of the ipsilateral projection became more restricted in knockout animals at later ages, it was still more extensive than that of normal mice of the same age at P28 and P42. In the adult, the distribution of axons was similar in both normal and double knockout animals. These results show that refinement of the IRP is delayed when expression of eNOS and nNOS is disrupted, presumably to axons with uncorrelated activity because nitric oxide serves as a repellant molecule during normal development. PMID- 10727739 TI - Increased expression of endogenous biotin, but not BDNF, in telencephalic song regions during zebra finch vocal learning. AB - Brain-derived neurotrophic factor (BDNF) is thought to regulate multiple aspects of brain development and neural plasticity in vertebrates. We have examined BDNF expression in two telencephalic nuclei (RA and HVC) in the zebra finch brain that control song learning by juvenile males and the production of already-learned song by adults. Using two different antibody-labeling techniques (avidin-biotin complex and horseradish peroxidase), we were unable to detect BDNF-like immunoreactivity in RA of juvenile or adult birds. BDNF-like immunoreactive labeling of somata was detected in HVC, but the density of labeled cells was not different between juvenile and adult birds. Immunocytochemical findings were confirmed by RT-PCR for BDNF mRNA. Thus, in contrast to a previous report (Akutagawa and Konishi, Proc. Natl. Acad. Sci. USA 95 (1998) 11429-11434), we did not observe elevated levels of BDNF immunoreactivity in RA and HVC of juvenile birds that were learning to sing. However, RA and HVC of juvenile birds were found to express elevated levels of endogenous biotin (as detected by avidin peroxidase), suggesting a possible role for biotin-regulated mechanisms in songbird vocal learning. PMID- 10727740 TI - Bulk-tank milk ELISA antibodies for estimating the prevalence of paratuberculosis in Danish dairy herds. AB - Paratuberculosis (Johne's disease) has been widespread in Danish dairy herds for a long time but the herd-level prevalence has never been determined precisely. To evaluate the prevalence of paratuberculosis in Danish dairy herds in various regions, an ELISA based on a commercially available antigen was adapted for testing bulk-tank milk for the presence of antibodies to Mycobacterium avium subsp. paratuberculosis. Bulk-tank milk samples were collected from six milk collecting centres from six different areas of the country. Samples from 900 herds (about 7.5% of all Danish dairy herds) were examined, and 70% were positive at the statistically optimal cut-off (sensitivity 97.1%; specificity 83.3%). The technical performance of the ELISA was not sufficient to provide a tool for surveillance because even slight changes in optical density for the samples would change the classification of some samples. The infection is more widespread than previous investigations have shown. PMID- 10727741 TI - Risk factors for Salmonella persistence after cleansing and disinfection in French broiler-chicken houses. AB - A prospective survey was carried out in 86 broiler houses in western France to identify risk factors for Salmonella persistence in French broiler houses. The Salmonella status of the house after cleansing and disinfection, was assessed from gauze-swab samples taken from the walls, feeders, ventilation system and bedding (analysed with classical bacteriological methods). Thirty three (38%) houses had at least one contaminated sample and were classified as Salmonella contaminated houses in the logistic regression. The absence of a terminal disinfection and a disinfection procedure performed by the farm staff rather than a contractor were positively related to the Salmonella contamination of the house after cleansing and disinfection. The risk for Salmonella persistence after decontamination was increased if rodents were observed by the farmer, if a large part of the access area to the house was accessible to trucks, and if a disease leading to a treatment occurred in the previous flock. PMID- 10727742 TI - Evaluating control strategies for outbreaks in BHV1-free areas using stochastic and spatial simulation. AB - Several countries within the EU have successfully eradicated bovine herpesvirus type I (BHV1), while others are still making efforts to eradicate the virus. Reintroduction of the virus into BHV1-free areas can lead to major outbreaks - thereby causing severe economic losses. To give decision-makers more insight into the risk and economic consequences of BHV1 reintroduction and into the effectiveness of various control strategies, we developed the simulation model InterIBR. InterIBR is a dynamic model that takes into account risk and uncertainty and the geographic location of individual farms. Simulation of a BHV1 outbreak in the Netherlands starts with introduction of the virus on a predefined farm type, after which both within-farm and between-farm transmission are simulated. Monitoring and control measures are implemented to simulate detection of the infection and subsequent control. Economic consequences included in this study are related to losses due to infection and costs of control. In the simulated basic control strategy, dairy farms are monitored by monthly bulk-milk tests and miscellaneous farms are monitored by half-yearly serological tests. After detection, movement-control measures apply, animal contacts are traced and neighbour farms are put on surveillance. Given current assumptions on transmission dynamics, we conclude that a strategy with either rapid removal or vaccination of infected cattle does not reduce the number of infected farms compared to this basic strategy - but will cost more to control. Farm type with first introduction of BHV1 has a considerable impact on the expected number of secondarily infected farms and total costs. To limit the number of infected farms and total costs due to outbreaks, we suggest intensifying the monitoring program on farms with a high frequency of cattle trade, and monthly bulk-milk testing on dairy farms. PMID- 10727743 TI - The foot-and-mouth disease risk posed by African buffalo within wildlife conservancies to the cattle industry of Zimbabwe. AB - Quantification of the risk that African buffalo (Syncerus caffer) (isolated within wildlife conservancies in Zimbabwe by a double fencing system) would infect cattle outside the conservancies with foot-and-mouth disease (FMD) virus was assessed by scenario-pathway analysis. Of the five scenarios considered, the greatest annual risk (1:5000) for cattle would be from antelope jumping over the outer perimeter fence of the conservancy and infecting cattle on the outside. The other transmission scenarios (including air-borne transmission) had a FMD risk that was low to very low. Risk management would include means to prevent the escape of antelope from the conservancies and restriction of cattle density in the proximity of the perimeter fence. PMID- 10727744 TI - Corrigendum to "A linear programming assessment of the profit from strategies to reduce the prevalence of Staphylococcus aureus mastitis" [Prev. Vet. Med. 33 (1998) 183-193]. AB - We used a linear programming model to estimate the financial returns to a Staphylococcus aureus testing and control program over a 1-year period for a 100 cow herd, with a 8636kg rolling-herd average. Six tests, which vary in sensitivity from 0.80 to 0.98 and specificity of 0.99, were examined in simulated herds with 10, 20, and 30% prevalence of S. aureus infection. Sensitivity of these results to a range of assumptions regarding rolling-herd average, milk price, somatic cell-count premium, and cost and cure rate of dry treatment were examined to determine the profits from the program. The profits of a control program are most dependent upon prevalence and cell-count premium. In our simulation for a 100-cow herd, a testing and control program results in a profit ranging from US$1.50 to US$20 per cow per year, except under the lowest prevalence and most-adverse conditions (low yield or low SCC premium). PMID- 10727745 TI - Community-based active surveillance for rabies in Machakos District, Kenya. AB - The rabies problem in Kenya has been greatest in Machakos District where the disease has persisted endemically for over 40 years. This paper presents the results of a one-year community-based active surveillance for rabies in six randomly selected sublocations in the district for the period 1992-1993. Approximately 860 rabid dogs per 100000 dogs were confirmed in this study, compared to approximately 12 per 100000 confirmed rabid dogs reported by the existing passive-surveillance system. This active surveillance underestimated the true rabies incidence, because only 41% (130/317) of the potential specimens could be diagnosed. Dogs accounted for 92% (179/194) of primary animal-rabies suspects, 80% (66/83) of secondary suspects, and 81% of the confirmed animal rabies cases. The annual incidence of animal-bites of humans was 234 per 100000 people and the point estimate of human-rabies incidence per year was 25 per million people. Almost all (97%) animal-bites of humans were due to dogs.The traditional passive-surveillance system grossly underestimated the importance of rabies as a public-health problem in Machakos District. Community-based active surveillance provides a potential cost-effective strategy for greatly improving estimates of rabies incidence and epidemiology to inform veterinary and policy decision-making. PMID- 10727746 TI - Risk factors for epidemic respiratory disease in Norwegian cattle herds. AB - An epidemic of acute respiratory disease associated with bovine respiratory syncytial virus (BRSV) occurred during the winter and spring of 1995 in two neighbouring veterinary districts in the south-eastern part of Norway. The objective of this study was to describe the time course of the outbreak associated with BRSV in the cattle herds, and to determine the association between selected herd factors and the risk of experiencing a herd outbreak of acute respiratory disease. Data from 431 cattle herds on the dates of disease occurrence, location of the farms, herd size, age profile and production type were collected retrospectively for 1995. The risk of acute respiratory disease occurring in a cattle herd was related to the herd size as well as the type of production, with an expressed interaction between these two variables. From the Cox proportional-hazards model, the risk of a herd outbreak in a mixed herd of 20 animals was estimated to be 1.7-times greater than in a dairy herd and 3.3-times greater than a beef herd (reference category) of a comparable size. On increasing the herd size to 50 animals, the risk increased 1.3-fold for a mixed herd, 3.3 fold for a dairy herd, and 2.1-fold for a beef herd, compared to the risk for a corresponding type of herd of 20 animals. PMID- 10727747 TI - Risk factors for high herd level calf morbidity risk from birth to weaning in beef herds in the USA. AB - We analyzed data from a national survey of beef cow-calf producers in the USA to quantify the effects of hypothesized risk factors on herd-level calf morbidity risk from birth to weaning. The analysis included 2490 herds from 23 states. Two stepwise logistic regressions were fit to identify factors associated with ≥10% morbidity. The first model included all herds dichotomized into high morbidity herds with ≥10% morbidity and low-morbidity herds with <10% morbidity. The second model excluded herds with between 5 and 10% morbidity, and compared >/=10% morbidity with 70% of cows and heifers calves in confinement also increased the odds of being a high-morbidity herd (OR=1.8). The population attributable fractions for dystocia and confined calving for the model including all herds dichotomized at 10% morbidity were 0.41 and 0.11, respectively. The summary population attributable fraction for both factors was 0.46. PMID- 10727748 TI - Mortality in sahelian goats in Nigeria. AB - The cause of mortality in sahelian goats was investigated in three local government areas of Borno State (Kukawa, Maiduguri and Mongonu) that are known for high goat production. A total of 150 selected flocks (50 flocks from each of the local government areas) were administered questionnaires through spot visits and interviews of the flock owners. On the whole, 644 (21.8%) goats died between May 1996 and April 1997 out of the 2956 goats in the 150 flocks. Mortality (41.4%) was higher in kids (<6-month old) than in adults (14.4%). Gastrointestinal disorders (dyspepsia), and respiratory diseases were the most common causes of mortality. Cause-specific mortality risks did not differ between kids and adults. PMID- 10727749 TI - A matched case-control study of factors associated with retention of fetal membranes in dairy ewes in Southern Greece. AB - We defined retention of fetal membranes (RFM) in dairy ewes as failure to expel the placenta within 6h after lambing the last lamb and designed a matched case control study to identify factors that affect the risk of retention. For each ewe with RFM, the next ewe in the flock that lambed and expelled the placenta in <6h after lambing the last lamb was selected as control. Data analyzed included 92 pairs of ewes from 25 flocks comprising a total of 7275 ewes (median flocksize 270 ewes). Factors investigated for associations with RFM were induction of lambing, obstetrical assistance because of dystocia, parity, the number of liveborn lambs, the occurrence of stillbirth(s), the mean weight of the litter on the third day post-lambing and the occurrence of neonatal death in the litter. Conditional logistic regression indicated (1) that the risk of RFM increased linearly with increasing number of liveborn lambs and (2) that the risk of retention was 4-fold higher in ewes that received assistance at lambing than those that lambed normally. PMID- 10727751 TI - Differential impact of nicotine on cellular proliferation and cytokine production by LPS-stimulated murine splenocytes. AB - The immunoregulatory effects of nicotine have not been fully clarified and the reported data are often conflicting. The present study investigated the role of nicotine as an immunomodulator of murine splenocytes stimulated by lipopolysaccharide (LPS), the endotoxin component of gram-negative bacteria. BALB/c female mice of two different ages, young (2-3 months) and old (18-22 months), were used. The cells were incubated with nicotine at two different time points, 3 h pre-incubation and concurrent incubation relevant to LPS stimulation, before further incubation for 48 or 72 h. Treatment of murine splenocytes with nicotine showed an impact on cellular proliferation as well as on the production of the pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). The results indicated that nicotine significantly inhibited cellular proliferation of murine splenocytes in a concentration-related manner (32, 64 and 128 microg/ml). Timing of nicotine exposure prior to LPS stimulation was critical in terms of immunological impact on cytokine production. TNF-alpha and IL-6 production were significantly enhanced by 1 microg/ml of nicotine when cells were pre-incubated with nicotine for 3 h compared to concurrent incubation relative to LPS stimulation. The alteration in cytokine production varied with the age of the mouse. TNF-alpha production was significantly inhibited by nicotine in young mice, while IL-6 production was significantly inhibited by nicotine in old mice. Since any immunomodulation that alters the profile of these cytokines may cause an imbalance in the immune system impinging on health status, these findings may be important when dealing with the concept of nicotine as a therapeutic agent. PMID- 10727752 TI - Inhibitory effect of anaphylactic shock by caffeine in rats. AB - Caffeine is known to reduce evoked histamine secretion, but the effects of caffeine on anaphylactic shock have not been clarified. We have investigated the effects of caffeine on anaphylactic shock in rats. Systemic anaphylactic shock by compound 48/80 injection was monitored for 1 h. An IgE-dependent local anaphylactic shock was generated by sensitizing the skin with anti-dinitrophenyl (DNP) IgE followed 48 h later with an injection of antigen. Caffeine inhibited compound 48/80-induced anaphylatic shock to 40% with a dose of 1 mg/kg. Caffeine (0.1 mg/kg) inhibited to 56.4+/-0.4% passive cutaneous anaphylactic shock activated by anti-DNP IgE. Caffeine (5-20 mM) significantly inhibited histamine release from rat peritoneal mast cells (RPMCs) activated by compound 48/80 or anti-DNP IgE. Especially, caffeine (20 mM) inhibited by 96.7+/-0.5% histamine release activated by compound 48/80. Moreover, caffeine (1-20 mM) had a significant inhibitory effect on anti-DNP IgE-induced tumor necrosis factor-alpha production from RPMCs. The level of cAMP in RPMCs, when caffeine (20 mM) was added, increased significantly after 5-60 min compared with that of a normal control. These results indicate that caffeine inhibits immediate-type allergic reactions by inhibition of mast cell degranulation in vivo and in vitro. PMID- 10727753 TI - Modulation of lung local immune responses by oral administration of a herbal medicine Sho-saiko-to. AB - Sho-saiko-to (SST), a Chinese/Japanese herbal medicine (Kampo medicine) widely used to treat chronic hepatitis in Japan, is known to modulate immune responses, and thus its immunomodulating activity may be responsible for its bi-directional effects on the lungs as therapeutic efficacy in various lung diseases and involvement in development of interstitial pneumonia. We administered SST to BALB/c mice orally and examined the lung tissue levels of pro/anti-inflammatory cytokines, interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), and the effects of SST on acute lung injury induced by instillation of lipopolysaccharide (LPS) or IL-1. Although SST had no effect on lung TNF-alpha or IL-1beta level, it increased IL-6. Investigation of active fractions of SST suggested that multiple ingredients were supposed to be responsible for IL-6-inducing activity. Liquiritigenin, a metabolite of liquiritin which is one of the major ingredients in SST enhanced in vitro IL-6 production in anti-CD3 monoclonal antibody (anti-CD3 mAb)-stimulated lung mononuclear cells in a cell-type specific and dose-dependent manner. SST suppressed LPS-induced lung injury at the later phase when lung leak was evident while being ineffective on initial neutrophil sequestration to the lung in these models. These findings suggest that SST modulates lung inflammation by regulating local immune response. PMID- 10727754 TI - Differential regulation of type IV collagenases and metalloelastase in murine macrophages by the synthetic bacterial lipopeptide JBT 3002. AB - We determined whether the expression of matrix metalloproteinases (MMP) and tissue inhibitors of MMPs (TIMP) in murine macrophages is regulated by the novel synthetic bacterial lipopeptide JBT 3002. Multilamellar liposomes (MLV) encapsulating JBT 3002 (MLV-JBT 3002) stimulated the production of 72-kDa and 92 kDa (gelatinase A and B) type IV collagenase and inhibited the production of murine metalloelastase (MME) in a dose-dependent manner in murine peritoneal macrophages. MLV-JBT 3002 also induced production of TIMP-1. MLV-JBT 3002 did not induce collagenase production in tumor cells. Priming murine macrophages with interferon-gamma (IFN-gamma) inhibited JBT 3002-stimulated production of both MMP 9 and MMP-2 and further inhibited production of MME by a mechanism involving nitric oxide (NO). This conclusion is based on data showing that IFN-gamma failed to inhibit production of MMP in the presence of L-methyl arginine or in macrophages from inducible nitric oxide synthase knockout mice. These data suggest that JBT 3002 differentially regulates the production of various MMPs and TIMP in macrophages. PMID- 10727755 TI - Suppression of tumor necrosis factor secretion from white blood cells by synthetic antisense phosphorothioate oligodeoxynucleotides. AB - In this ex vivo, rather than in vitro, experiment, a synthetic antisense oligodeoxynucleotide was tested to suppress tumor necrosis factor - alpha(TNF) secretion from lipopolysaccharide-stimulated white blood cells. Antisense oligomer showed significant and specific suppressive effect to the secretion of TNF at concentrations of 1.0 and 10 microM. At the concentration of 1 microM, there were 68.4 and 63.9% suppression of TNF secretion at 2 and 24 h after resuspension of blood cells. At the concentration of 10 microM, the suppressions were slightly higher than those at 1 microM, which were 71.8 and 76.2%, respectively. A 50%-matched scrambler showed suppressive effect only at 10 microM concentration, and the suppression only occurred at 2 and 24 h after incubation. Sense oligomer showed no suppressive effects at any of the concentrations. The specificity of this oligomer was documented by dose-effect phenomenon, sequence dependent suppression and absence of effect on the synthesis of another cytokine (interleukin-6). A series of parallel studies was performed and showed that all three oligomers at any concentration tested had no effect on the interleukin-6 secretion after LPS stimulation.In conclusion, properly designed antisense oligodeoxynucleotide can significantly and specifically suppress the secretion of TNF by blood cells in an ex vivo system and it may be a good "information" drug to treat diseases that are caused by over production of TNF. PMID- 10727756 TI - Human chorionic gonadotropin induces nitric oxide synthesis by murine microglia. AB - This study investigated the effects of human chorionic gonadotropin (hCG) on the synthesis of nitric oxide (NO) in murine neonatal microglial cells. When hCG was used in combination with interferon-gamma (IFN-gamma), there was a marked cooperative induction of NO synthesis in a dose-dependent manner. This increase in NO synthesis was reflected as an increased amount of iNOS protein. The increase of NO synthesis by IFN-gamma-plus-hCG was associated with the increase of tumor necrosis factor-alpha (TNF-alpha) secretion and hCG-induced NO production was decreased by the treatment with anti-murine TNF-alpha neutralizing antibody. This study provides evidence that hCG activates expression of iNOS protein in murine microglial cells accompanied by NO accumulation via pathway dependent on L-arginine in the culture medium, and further offers that TNF-alpha acts on the NO synthesis from IFN-gamma-primed murine microglial cells. PMID- 10727757 TI - Inhibition of interleukin 5 production with no influence on interleukin 4 production by an anti-allergic drug, tranilast, in Toxocara canis-infected mice. AB - Tranilast is well-known as a useful drug for allergic diseases. This drug is believed to exhibit its therapeutic effects by inhibiting the release of chemical mediators from mast cells and basophils. Effects of tranilast on T helper type 2 (Th2) cytokine production were investigated in mice infected with Toxocara canis (Tc). Tranilast reduced interleukin (IL)-5 production in a dose-dependent manner but not IL-4 production at all in lung and spleen cells from Tc-infected mice cultured under stimulation with excretory-secretory antigen. Obvious IL-5 mRNA expression was observed at week 1 in the lung alone, and IL-4 mRNA expression was detected at similar levels at weeks 1-6 of infection in both lung and spleen. IL 5 but not IL-4 mRNA expression in the lung was significantly inhibited by daily administration of 100 mg/kg of tranilast for 1 week. This treatment also reduced the serum IL-5 level. Thus, tranilast inhibited IL-5 but not IL-4 production either in vitro or in vivo. The results imply that IL-5 and IL-4 production by Th2 cells may be controlled through different mechanisms. PMID- 10727758 TI - Quantification of liver iron concentration with magnetic resonance imaging by combining T1-, T2-weighted spin echo sequences and a gradient echo sequence. AB - BACKGROUND: The aim of the study was to quantify hepatic iron by MRI for practical use. METHODS: In twenty-three patients with various degrees of iron overload, measurements were carried out with a 1.5 Tesla MR unit. A combination of pulse sequences (T1, T2 and gradient echo) enabled us to quantify smaller amounts of liver iron as accurately as larger amounts of liver iron. The gradient echo sequence provided us with a good correlation when detecting smaller amounts of iron in the liver where the T1 sequence provided a good correlation when larger amounts of iron were present. RESULTS: The combination of the three sequences showed a nice correlation (r=-0. 93, P<0.001) and provided us with an accurate estimate of the liver iron content (LIC). This correlation was achieved with a LIC from the lower range of normal up to LIC of 146 mmol/kg dry weight, which seems the highest measurable liver iron content for a 1.5 Tesla MRI. Measuring in the lower range makes it possible to decide whether further invasive diagnostic investigations by a liver biopsy are indicated. CONCLUSION: MRI is a useful tool to quantify iron overload non-invasively. In cases where a liver biopsy is hazardous MRI can easily be used to obtain reliable, quantitative information about the initial LIC. Quantification by MRI could also be used for follow up of the iron content during depletion treatment by phlebotomy or iron chelation. The stronger the magnet the more sensitive the detection of concentrations up to 150 mmol/kg is. A semi-quantitative judgement will only be possible with severe iron overload over 150 mmol/kg. If such an iron excess is found, a liver biopsy should be performed to exclude cirrhosis. PMID- 10727759 TI - Normohomocysteinaemia and vitamin-treated hyperhomocysteinaemia are associated with similar risks of cardiovascular events in patients with premature atherothrombotic cerebrovascular disease. A prospective cohort study. AB - BACKGROUND: Mild hyperhomocysteinaemia (HHC) is associated with an increased risk of premature atherothrombotic cerebrovascular disease. We investigated the clinical efficacy with regard to the incidence of cardiovascular events of treatment of mild HHC with vitamin B(6) plus folic acid. METHODS: We studied 224 consecutive patients with clinically manifest atherothrombotic cerebrovascular disease with onset before the age of 56. Follow-up was obtained in 203 (90.6%) patients. At baseline, 52 (25.6%) were hyperhomocysteinaemic after methionine loading and started treatment with vitamin B(6) (250 mg) plus folic acid (5 mg); 151 (74.4%) were normohomocysteinaemic (reference group). RESULTS: During follow up (median 57 months), 31 (20.5%) of the normo- and 11 (21.2%) of the hyperhomocysteinaemic patients had a new cardiovascular event. The crude incidence rate per person-year for any cardiovascular event was similar in both groups (0.043 [CI, 0.029-0.057] in the normo- vs. 0.045 [CI, 0.021-0. 069] in the hyperhomocysteinaemic group). Multivariate Cox-regression analyses showed that hypertension and cholesterol levels were associated with an increased risk of new cardiovascular events in the total group [relative risk [RR] (yes vs. no), 7.4 (3. 4-16.0) and RR (per 1 mmol/l), 1.9 (CI, 1.4-2.7)]. The adjusted RR for new cardiovascular events in the hyper- as compared to the normohomocysteinaemic patients was 0.96 (CI, 0.48-1.92). CONCLUSION: These data are consistent with a protective effect of treatment with vitamin B(6) plus folic acid in patients with premature atherothrombotic cerebrovascular disease and post-methionine HHC. PMID- 10727760 TI - Asystole during combination chemotherapy for non-Hodgkin's lymphoma: the acute tumor lysis syndrome. AB - The acute tumor lysis syndrome is a rare condition that has most frequently been documented in patients with rapidly dividing myeloproliferative and lymphoproliferative malignancies. It is characterized by the development of hyperuricemia, hyperkalemia, hyperphosphatemia, hypocalcemia, acute renal failure and metabolic acidosis, as a result of massive tumor cell destruction, usually secondary to effective cytotoxic treatment. We want to present the case history of a 62-year-old woman who died from cardiorespiratory arrest during combination chemotherapy for non-Hodgkin's lymphoma due to acute tumor lysis syndrome. Despite general preventive measures, severe electrolyte abnormalities developed within 18 h of the start of chemotherapy. The general guidelines for the management of this potentially fatal oncologic emergency are discussed, with special emphasis on the pathogenetic mechanisms and risk factors in our patient. PMID- 10727761 TI - Pitfall of the accessory spleen. AB - Two patients, one with insulinoma and one with Cushing's syndrome, are presented. Biochemical evaluation readily suggested the correct diagnosis. During radiologic imaging, the anatomic abnormality giving rise to these diseases, i.e. a pancreatic islet cell tumor, and an adrenal adenoma, at first were mistakenly interpreted as an accessory spleen on the basis of specific computed tomography and magnetic resonance imaging appearances. The insulinoma was identified as such during laparotomy, whereas additional jodo-cholesterol scintigraphy revealed the real nature of the lesion in the patient with Cushing's syndrome. Both patients were operated successfully. PMID- 10727762 TI - Gustatory sweating and diabetes. AB - Gustatory sweating as a feature of autonomic neuropathy is an unusual phenomenon in diabetes mellitus. We describe a patient with type 1 diabetes mellitus complicated by retinopathy, nephropathy and neuropathy. This patient presented with bilateral diffuse facial sweating during eating. She was treated with the antimuscarine agent oxybutynine, which provided a striking relief from the gustatory sweating. PMID- 10727763 TI - Referees for 1999 PMID- 10727765 TI - Bacillus thuringiensis crystal delta-endotoxin: role of proteases in the conversion of protoxin to toxin. AB - The conversion of delta-endoprotoxins of Bacillus thuringiensis to active toxins is mediated by trypsin, insect gut (exogenous) and bacterial (endogenous) proteases. The biochemical aspects of exogenous and endogenous proteases involved in the conversion of protoxin to toxin are reviewed. Perhaps, these proteases also play a role in influencing the host range of toxin and in the development of resistance to toxin. PMID- 10727764 TI - MAPs and POEP of the roads from prokaryotic to eukaryotic kingdoms. AB - Methionine aminopeptidases (MAPs) play important roles in protein processing. MAPs from various organisms, for example E. coli, S. typhimurium, P. furiosus, Saccharomyces cerevisiae, and porcine have been purified to homogeneity and their MAP activities have been tested in vitro and in vivo. The DNA sequence analyses of MAP genes from the above organisms reveal sequence homologies with other prokaryotic MAPs as well as with various eukaryotic homologues of rat p67. The cellular glycoprotein, p67 protects the alpha-subunit of eukaryotic initiation factor 2 (eIF2) from phosphorylation by its kinases. We call this POEP (protection of eIF2alpha phosphorylation) activity of p67. The POEP activity of p67 is observed in different stress-related situations such as during heme deficiency of reticulocytes, serum starvation and heat-shock of mammalian cells, vaccinia virus infection of mammalian cells, baculovirus infection of insect cells, mitosis, apoptosis, and possibly during normal cell growth. The POEP activity of p67 is regulated by an enzyme, called p67-deglycosylase (p67-DG). When active, p67-DG inactivates p67 by removing its carbohydrate moieties. Remarkable amino acid sequence similarities at the C-terminus of rat p67 with its eukaryotic and prokaryotic homologues which have MAP activities, raise several important questions: i) does rat p67 have MAP activity?; and ii) if it does have MAP activity, how the two activities (POEP and MAP) of p67 are used by mammalian cells during their growth and differentiation. In this review, discussions have been made to evaluate both POEP and MAP activities of p67 and their possible involvement during normal growth and cancerous growth of mammalian cells. PMID- 10727766 TI - The selenocysteine insertion sequence binding protein SBP is different from the Y box protein dbpB. AB - In eukaryotes, translation of internal UGA selenocysteine codons requires the SECIS stem-loop structure in the 3'UTR of selenoprotein mRNAs. In an earlier work, we identified SBP as a selenocysteine insertion sequence (SECIS)-binding protein. Here, the yeast three-hybrid screen was employed to capture the cDNA of SBP. One candidate, satisfying the genetic screens, was identified as the already known dbpB protein. Although it was also found by another group, but with a different strategy, to carry SECIS-binding activity, further experiments enabled us to show that dbpB was unable to bind the SECIS element in vitro. Altogether, our findings led us to conclude that, under our conditions, dbpB and SBP are two distinct proteins. PMID- 10727767 TI - Expression of the Trypanosoma brucei phosphoenolpyruvate carboxykinase gene in Saccharomyces cerevisiae. AB - Plasmid pTbp60B (Kueng et al., J. Biol. Chem. 264 (1989) 5203-5209) was employed to obtain, through the polymerase chain reaction, the Trypanosoma brucei gene coding for phosphoenolpyruvate (PEP) carboxykinase, and then cloned into the yeast expression plasmid pYES2. The cloned gene was completely sequenced and the expression plasmid transformed into Saccharomyces cerevisiae PUK-3B (MATalpha pck1 ura3 ade1) competent cells. Gene expression took place upon induction with 2% galactose, and the recombinant T. brucei PEP carboxykinase was purified to near homogeneity. The basic molecular and catalytic characteristics of the recombinant enzyme were determined, and they showed to be essentially similar to those reported for wild type T. brucei PEP carboxykinase (Hunt and Kohler, Biochim. Biophys. Acta 1249 (1995) 15-22). The expression system here described is a reliable non-pathogenic source of T. brucei PEP carboxykinase. PMID- 10727768 TI - The distribution of aminoacylase I among mammalian species and localization of the enzyme in porcine kidney. AB - Aminoacylase I (Acy-1, EC 3.5.1.14) is found in many mammalian tissues, with highest activities occurring in kidney. The enzyme hydrolyzes a variety of N acylated amino acids; however, the physiological role and the exact cellular localization of Acy-1 are still a matter of debate. The comparison of Acy-1 activities in kidney and liver homogenates of 11 mammalian species showed that the enzyme is most abundant in true herbivores such as sheep and cattle as well as in omnivores, while activities were very low in both rodents and the cat. Acy 1 activity was not detected in livers of dogs of five different breeds. Using in situ hybridization of porcine kidney sections with DIG-labeled RNA probes, Acy-1 mRNA was shown to be evenly distributed throughout the tubular system, while glomeruli and the interstitium were free of stain. During subcellular fractionation, porcine Acy-1 behaved like a typical cytosolic enzyme. Commonly, Acy-1 is thought to catalyze hydrolytic reactions, i.e., the formation of free amino acids from acylated derivatives. Based on the present results and literature data, we propose a novel hypothesis, i.e., that Acy-1 catalyzes the synthesis (rather than the hydrolysis) of hippurate that is formed as a detoxification product of aromatic compounds. PMID- 10727769 TI - Dimerization of the Epstein-Barr virus ZEBRA protein in the yeast two-hybrid system. Comparison Of a ZEBRA variant with the B95-8 form. AB - Epstein-Barr virus (EBV) is a herpes virus associated with several human tumors. The EBV protein, ZEBRA, is a transactivator of the basic leucine zipper family (bZip). It binds to specific sequences on DNA and is able to interact with cellular proteins such as p53. The interaction of the ZEBRA protein with its cognate DNA sequences is stable as long as the dimerization domain is functional. Recent work from this laboratory identified a ZEBRA variant (Z206) with a single amino acid change at residue 206. An alanine is substituted for a serine, and this replacement is present in 72% of nasopharyngeal carcinoma from Europe and North Africa. As amino acid 206 lies within the dimerization domain it could be instrumental in interactions with other proteins. The yeast two-hybrid system was used to study ZEBRA-protein interactions. As ZEBRA by itself is a transactivator in yeast, it cannot be used directly in this assay. This paper describes modifications in ZEBRA amino acid sequences, rendering it usable in the yeast two hybrid assay. We compared the dimerization capacity of the Z206 variant to that of ZEBRA from B95-8 (Z95) and observed that reporter gene activity with Z206 was consistently lower than that of Z95 (P < 0.05). Furthermore, no interaction was found to occur between either form of ZEBRA (Z206 or Z95) and the tumor suppressor, p53 in the yeast two-hybrid system. PMID- 10727770 TI - The effect of gallium nitrate on synoviocyte MMP activity. AB - Gallium, a group IIIa metal salt, has been demonstrated to be an effective immunosuppressive agent. Gallium has also been shown to inhibit the production of inflammatory cytokines, such as IL-1beta, produced by macrophage-like cells in vitro. To further characterize the effects of gallium on the inflammatory process, we examined the effects of gallium nitrate on matrix metalloproteinase (MMP) activity utilizing the rabbit synoviocyte cell line HIG-82. HIG-82 cells were incubated with IL-1beta and TPA, with and without increasing concentrations of gallium nitrate. Conditioned medium was collected and assayed for MMP activity using a synthetic substrate and substrate gel zymography. IL-1beta and TPA alone induced MMP activity in HIG-82 cells. A dose-dependent inhibition of IL-1beta and TPA stimulated MMP activity by gallium nitrate at increasing concentrations was observed. This study demonstrates that gallium nitrate can inhibit the activity of MMPs and may be useful as a modulator of inflammation in arthritis. PMID- 10727771 TI - Characterization of turkey pancreatic lipase. AB - Turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Pure TPL (glycerol ester hydrolase, EC 3.1.1.3) was obtained after ammonium sulfate fractionation, Sephacryl S-200 gel filtration, anion exchange chromatography (DEAE-Sepharose) and size exclusion column using high performance liquid chromatography system (HPLC). The pure lipase, which is not a glycoprotein, was presented as a monomer having a molecular mass of about 45 kDa. The lipase activity was maximal at pH 8.5 and 37 degrees C. TPL hydrolyses the long chains triacylglycerols more efficiently than the short ones. A specific activity of 4300 U/mg was measured on triolein as substrate at 37 degrees C and at pH 8.5 in the presence of colipase and 4 mM NaTDC. This enzyme presents the interfacial activation when using tripropionin as substrate. TPL was inactivated when the enzyme was incubated at 65 degrees C or at pH less than 5. Natural detergent (NaTDC), synthetic detergent (Tween-20) or amphipatic protein (beta-lactoglobulin A) act as potent inhibitors of TPL activity. To restore the lipase activity inhibited by NaTDC, colipase should be added to the hydrolysis system. When lipase is inhibited by synthetic detergent or protein, simultaneous addition of colipase and NaTDC was required to restore the TPL activity. The first 22 N terminal amino acid residues were sequenced. This sequence was similar to those of mammal's pancreatic lipases. The biochemical properties of pancreatic lipase isolated from bird are similar to those of mammals. PMID- 10727772 TI - Are there protective enzymatic pathways to regulate high local nitric oxide (NO) concentrations in cells under stress conditions? AB - This paper examines, from a chemical perspective, the hypothesis of the existence of protective enzymes whose role would be to regulate the high local nitric oxide (NO) concentrations that are released in NO-generating cells in situations of response to oxidative stress. These enzymes should play the role, with respect to NO, either of a reductase or of a dismutase. The energetics of the intervening transformations is herein presented, along with a review of pertinent literature. An attempt is made in order to describe the physiognomy of such enzymes, in relation with the literature data. Experimental investigation is needed to further evaluate the validity of such a hypothesis. PMID- 10727773 TI - Initiation of protein synthesis with fluorophore-Met-tRNA(f) and the involvement of IF-2. AB - The complicity of initiation factor 2 (IF-2) in causing the observed low incorporation of N-terminal fluorophore from fluorophore-methionyl-tRNA(f) during protein synthesis in an in vitro coupled transcription/translation system was investigated. The low incorporation in comparison to formyl-methionine was not due to the lack of interaction of fluorophore-Met-tRNA(f) with IF-2. Fluorescence measurements of cascade yellow-, eosin-, pyrene-, or coumarin-Met-tRNA(f) determined that all were capable of binding IF-2 at 4 mM Mg(2+) and 37 degrees C. Filter binding assays conducted in the absence of magnesium ions on fMet-tRNA(f), eosin-Met-tRNA(f), and cascade yellow-Met-tRNA(f) confirmed the previously reported value for the dissociation constant of fMet-tRNA(f) of about 1 microM and placed the binding constants for the two fluorophore derivatives about three fold higher. Binding of the fluorophore-Met-tRNA(f) species to salt-washed ribosomes showed a more significant decrease compared to fMet-tRNA(f). Stimulation in the amount of tRNA bound to the ribosomes upon the addition of IF 2 was observed in each case. All ribosome-bound cascade yellow-Met-tRNA(f) and eosin-Met-tRNA(f) were as puromycin-reactive as fMet-tRNA(f). Cumulatively, the effects observed for the fluorophore-Met-tRNA species in partial reactions of initiation may account for the reduced incorporation of these probes at the N terminus of polypeptides. PMID- 10727774 TI - Fragmentation of human ceruloplasmin induced by hydrogen peroxide. AB - We investigated the fragmentation of human ceruloplasmin induced by H2O2 to study its oxidative damage. When ceruloplasmin was incubated with H2O2, the frequency of the protein fragmentation increased in a proportion to the concentration of H2O2. It also increased in a time-dependent manner and was accompanied by gradual loss of the oxidase activity. Hydroxyl radical scavengers such as azide and mannitol inhibited the fragmentation of ceruloplasmin. The deoxyribose assay showed that hydroxyl radicals were generated in the reaction of ceruloplasmin with H2O2. Incubation of ceruloplasmin with H2O2 resulted in a time-dependent release of copper ions. The released copper ion may participate in a Fenton-like reaction to produce hydroxyl radical, which enhanced the fragmentation. The protection of the fragmentation by copper chelators such as diethylenetriaminepentaacetic acid and bathocuproine indicates a role for copper ion in the reaction. These results suggest that the fragmentation of ceruloplasmin induced by H2O2 is due to hydroxyl radicals formed by a copper dependent Fenton-like reaction. PMID- 10727775 TI - Synaptic transmission in the striatum: from plasticity to neurodegeneration. AB - Striatal neurones receive myriad of synaptic inputs originating from different sources. Massive afferents from all areas of the cortex and the thalamus represent the most important source of excitatory amino acids, whereas the nigrostriatal pathway and intrinsic circuits provide the striatum with dopamine, acetylcholine, GABA, nitric oxide and adenosine. All these neurotransmitter systems interact each other and with voltage-dependent conductances to regulate the efficacy of the synaptic transmission within this nucleus. The integrative action exerted by striatal projection neurones on this converging information dictates the final output of the striatum to the other basal ganglia structures. Recent morphological, immunohistochemical and electrophysiological findings demonstrated that the striatum also contains different interneurones, whose role in physiological and pathological conditions represents an intriguing challenge in these years. The use of the in vitro brain slice preparation has allowed not only the detailed investigation of the direct pre- and postsynaptic electrophysiological actions of several neurotransmitters in striatal neurones, but also the understanding of their role in two different forms of corticostriatal synaptic plasticity, long-term depression and long-term potentiation. These long-lasting changes in the efficacy of excitatory transmission have been proposed to represent the cellular basis of some forms of motor learning and are altered in animal models of human basal ganglia disorders, such as Parkinson's disease. The striatum also expresses high sensitivity to hypoxic-aglycemic insults. During these pathological conditions, striatal synaptic transmission is altered depending on presynaptic inhibition of transmitter release and opposite membrane potential changes occur in projection neurones and in cholinergic interneurones. These ionic mechanisms might partially explain the selective neuronal vulnerability observed in the striatum during global ischemia and Huntington's disease. PMID- 10727776 TI - Myelin synthesis in the peripheral nervous system. AB - By imposing saltatory conduction on the nervous impulse, the principal role of the myelin sheath is to allow the faster propagation of action potentials along the axons which it surrounds. Peripheral nervous system (PNS) myelin is formed by the differentiation of the plasma membrane of Schwann cells. One of the biochemical characteristics that distinguishes myelin from other biological membranes is its high lipid-to-protein ratio. All the major lipid classes are represented in the myelin membrane, while several myelin-specific proteins have been identified. During development, the presence of axons is required for the initiation of myelination, but the nature of the axonal signal is still unknown. The only certainties are that this signal is synthesized by axons whose diameter is greater than 0.7 microm, and that the signal(s) include(s) a diffusible molecule. Morphological studies have provided us with information concerning the timing of myelination, the mechanism by which immature Schwann cells differentiate into a myelinating phenotype and lay down the myelin sheath around the axon, and the accumulation and the structure of the myelin membrane. The last 20 years have seen the identification and the cDNA and gene cloning of the major PNS myelin proteins, which signalled the beginning of the knock-out decade: transgenic null-mutant mice have been created for almost every protein gene. The study of these animals shows that the formation of myelin is considerably less sensitive to molecular alterations than the maintenance of myelin. During the same period, important data has been gathered concerning the synthesis and function of lipids in PNS myelin, although this field has received relatively little attention compared with that of their protein counterparts. PMID- 10727777 TI - Single and multiple transgenic mice as models for Alzheimer's disease. AB - Transgenic mice expressing in brain different mutant forms of the Amyloid Precursor Protein, develop functional, cognitive and pathological defects which resemble or are reminiscent of symptoms observed in Alzheimer's disease (AD) patients. The late development of amyloid plaques in aging transgenic APP mice is needed to warrant that the earlier behavioural and cognitive defects are informative for the human disorder. We describe and discuss our work, the rationale behind the approach and the techniques used to generate these APP transgenic mice, including specific experimental problems. The APP transgenic mouse models are being comprehensively characterized and offer excellent perspectives for the study and definition of early biochemical and pathological aspects that are not accessible in human AD patients. The ongoing combination by breeding with other transgenic mouse strains, i.e. mice overexpressing human Presenilin 1, ApoE 4 and protein tau to generate "multiple" transgenic mice, offer additional potential to define the pathological interactions of these genetic factors, known to be involved, directly or indirectly, in dementia of the Alzheimer type. Finally, it must be the aim to obtain transgenic mice that not only model amyloidogenesis, but also the neurofibrillary tangle pathology and the involvement of protein tau. PMID- 10727778 TI - Fetal tissue transplants in animal models of Huntington's disease: the effects on damaged neuronal circuitry and behavioral deficits. AB - Accumulating evidence indicates that grafts of embryonic neurons achieve the anatomical and functional reconstruction of damaged neuronal circuitry. The restorative capacity of grafted embryonic neural tissue is most illustrated by studies with striatal tissue transplantation in animals with striatal lesions. Striatal neurons implanted into the lesioned striatum receive some of the major striatal afferents such as the nigrostriatal dopaminergic inputs and the gluatmatergic afferents from the neocortex and thalamus. The grafted neurons also send efferents to the primary striatal targets, including the globus pallidus (GP, the rodent homologue of the external segment of the globus pallidus) and the entopeduncular nucleus (EP, the rodent homologue of the internal segment of the globus pallidus). These anatomical connections provide the reversal of the lesion induced alterations in neuronal activities of primary and secondary striatal targets. Furthermore, intrastriatal striatal grafts improve motor and cognitive deficits seen in animals with striatal lesions. Since the grafts affect motor and cognitive behaviors that are critically dependent on the integrity of neuronal circuits of the basal ganglia, the graft-mediated recovery in these behavioral deficits is most likely attributable to the functional reconstruction of the damaged neuronal circuits. The fact that the extent of the behavioral recovery is positively correlated to the amount of grafted neurons surviving in the striatum encourages this view. Based on the animal studies, embryonic striatal tissue grafting could be a viable strategy to alleviate motor and cognitive disorders seen in patients with Huntington's disease where massive degeneration of striatal neurons occurs. PMID- 10727779 TI - The need for a simple animal model to understand sleep. AB - Simple animal models have allowed biologists to apply the tools of modern molecular genetics to such complex behaviors as circadian rhythms and long-term memory consolidation. The mechanisms and molecules discovered in these simple animals are evolutionarily conserved in other species, including mammals. Sleep research lacks a simple animal model because criteria based on the electroencephalogram have been met only in birds and mammals. We argue that straightforward behavioral criteria could allow the identification of a sleep like rest state that might be useful for molecular investigations to understand the regulation and function of sleep. Candidate model systems are discussed, leading to the conclusion that several species have complementary strengths. Specifically, techniques developed for larval zebrafish can be used to visualize neural firing patterns in the living animal, and the fruit fly Drosophila melanogaster has been used successfully for molecular and genetic dissection of complex behaviors. We conclude with a hypothesis that one putative function of sleep, the optimization of neural plasticity, would also have adaptive value in simple organisms and might therefore be evolutionarily conserved. PMID- 10727780 TI - Conventional and new antidepressant drugs in the elderly. AB - Depression in the elderly is nowadays a predominant health care problem, mainly due to the progressive aging of the population. It results from psychosocial stress, polypathology, as well as some biochemical changes which occur in the aged brain and can lead to cognitive impairments, increased symptoms from medical illness, higher utilization of health care services and increased rates of suicide and nonsuicide mortality. Therefore, it is very important to make an early diagnosis and a suitable pharmacological treatment, not only for resolving the acute episode, but also for preventing relapse and enhancing the quality of life. Age-related changes in pharmacokinetics and in pharmacodynamics have to be kept into account before prescribing an antidepressant therapy in an old patient. In this paper some of the most important and tolerated drugs in the elderly are reviewed. Tricyclic antidepressants have to be used carefully for their important side effects. Nortriptyline, amytriptiline, clomipramine and desipramine as well, seem to be the best tolerated tricyclics in old people. Second generation antidepressants are preferred for the elderly and those patients with heart disease as they have milder side effects and are less toxic in overdose and include the so called atypicals, such as selective serotonin reuptake inhibitors, serotonin noradrenalene reuptake inhibitors and noradrenaline reuptake inhibitors. Monoamine oxidase (MAO) inhibitors are useful drugs in resistant forms of depression in which the above mentioned drugs have no efficacy; the last generation drugs (reversible MAO inhibitors), such as meclobemide, seem to be very successful. Mood stabilizing drugs are widely used for preventing recurrences of depression and for preventing and treating bipolar illness. They include lithium, which is sometimes used especially to prevent recurrence of depression, even if its use is limited in old patients for its side effects, the anticonvulsants carbamazepine and valproic acid. Putative last generation mood stabilizing drugs include the dihydropyridine L-type calcium channel blockers and the anticonvulsants phenytoin, lamotrigine, gabapentin and topiramate, which have unique mechanisms of action and also merit further systematic study. Psychotherapy is often used as an adjunct to pharmacotherapy, while electroconvulsant therapy is used only in the elderly patients with severe depression, high risk of suicide or drug resistant forms. PMID- 10727781 TI - Cerebral blood flow and metabolism during exercise. AB - During exercise regional cerebral blood flow (rCBF), as blood velocity in major cerebral arteries and also blood flow in the internal carotid artery increase, suggesting an increase in blood flow to a large part of the brain. Such an increase in CBF is independent of the concomitant increase in blood pressure but is modified by the alteration in arterial carbon dioxide tension (PaCO(2)). Also, the increase in middle cerebral artery mean blood velocity (MCA V(mean)) reported with exercise appears to depend on the ability to increase cardiac output (CO), as demonstrated in response to beta-1 blockade and in patients with cardiac insufficiency or atrial fibrillation.Near-infrared spectroscopy (NIRS) determined cerebral oxygenation supports the alterations in MCA V(mean) during exercise. Equally, the observation that the cerebrovascular CO(2)-reactivity appears to be smaller in the standing than in the sitting and especially in the supine position could relate to the progressively smaller CO. In contrast, during exercise "global" cerebral blood flow (gCBF), as determined by the Kety-Schmidt technique is regarded as being constant. One limitation of the Kety-Schmidt method for measuring CBF is that blood flow in the two internal jugular veins depends on the origin of drainage and it has not been defined which internal jugular venous flow is evaluated. Such a consideration is equally relevant for an evaluation of cerebral metabolism during exercise. While the regional cerebral uptake of oxygen (O(2)) increases during exercise, the global value is regarded as being constant. Yet, during high intensity exercise lactate is taken up by the brain and its O(2) uptake also increases. Furthermore, in the initial minutes of recovery immediately following exercise, brain glucose and O(2) uptake are elevated and lactate uptake remains high.A maintained substrate uptake by the brain after exercise suggests a role for brain glycogen in cerebral activation, but the fate of brain substrate uptake has not yet been determined. PMID- 10727782 TI - The role of 6R-tetrahydrobiopterin in the nervous system. AB - In addition to its cofactor activities for aromatic L-amino acid hydroxylases and nitric oxide synthase (NOS), 6R-tetrahydrobiopterin (6R-BH(4)) shows diverse actions on neurons. Dopamine release from the rat striatum or PC12 cells was stimulated by 6R-BH(4). The action of 6R-BH(4) was independent of its cofactor activities and stereospecific. Ca(2+) channels in rat brain and PC12 cells were activated by 6R-BH(4) via cAMP-protein kinase A pathway. Membrane potential of PC12 cells was deplorized by 6R-BH(4). Thus, it is assumed that 6R-BH(4) acts on its specific action site (possibly outside of the cell membrane) to stimulate dopamine release by activating Ca(2+) channels. Apoptosis induced by depletion of serum and nerve growth factor in PC12 cells was prevented by 6R-BH(4). The cell surviving effect of 6R-BH(4) was also mediated by activation of Ca(2+) channels and cAMP-protein kinase A pathway. However, since 6R-BH(4) did not activate mitogen activated protein kinase, it did not support neuronal differentiation. Nitric oxide (NO)-induced cell death was prevented by 6R-BH(4) in PC12 cells. NOS activity was not changed by exogenous 6R-BH(4), but NO metabolites in culture medium were decreased by 6R-BH(4). When endogenous 6R-BH(4) was reduced by inhibition of biosynthesis, cell death was induced in PC12 cells. Superoxide is observed to be generated during autoxidation of 6R-BH(4). Superoxide producing system mimicked the cell protective action of 6R-BH(4) against NO toxicity. Thus, it is considered that 6R-BH(4) protects PC12 cells against NO toxicity by generating superoxide during its autoxidation. These results raised the possibility that 6R-BH(4) is a self-protective factor against NO toxicity in NO producing neurons. Our findings indicate that 6R-BH(4) regulates neuronal activities in the brain and that 6R-BH(4) can be a promising drug for neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. PMID- 10727783 TI - Mesolimbocortical and nigrostriatal dopamine responses to salient non-reward events. AB - While it has previously been assumed that mesolimbic dopamine neurons carry a reward signal, recent data from single-unit, microdialysis and voltammetry studies suggest that these neurons respond to a large category of salient and arousing events, including appetitive, aversive, high intensity, and novel stimuli. Elevations in dopamine release within mesolimbic, mesocortical and nigrostriatal target sites coincide with arousal, and the increase in dopamine activity within target sites modulates a number of behavioral functions. However, because dopamine neurons respond to a category of salient events that extend beyond that of reward stimuli, dopamine levels are not likely to code for the reward value of encountered events. The paper (i) examines evidence showing that dopamine neurons respond to salient and arousing change in environmental conditions, regardless of the motivational valence of that change, and (ii) asks how this might shape our thinking about the role of dopamine systems in goal directed behavior. PMID- 10727784 TI - Glutamate dependence of GABA levels in neurons of hypoxic and hypoglycemic rat hippocampal slices. AB - Hypoxia may increase GABA levels in neurons by ATP depletion-induced activation of glutamate decarboxylase and by inhibiting GABA transaminase. Hypoglycemia, which also depletes ATP, reduces neuronal levels of GABA and its precursor glutamate. We examined whether differences in glutamate levels may contribute to these altered GABA levels in hippocampal slices. GABA levels were highly correlated with endogenous glutamate levels during both hypoxia and hypoglycemia (R=0.93 for combined data). Hypoxia maximally increased GABA levels (146+/-6.3% of control, S.E.M.) when glutamate remained above 90% of control levels and ATP was at 30% of control levels. Hypoglycemia with similar ATP levels and glutamate levels at 40% of control decreased GABA levels to 55% of control. Effects of inhibitors of glutamate decarboxylase and GABA transaminase suggested that increased synthesis and decreased catabolism may both contribute to increased hypoxic GABA levels. Immunocytochemical studies suggested that hypoxia increased GABA concentrations primarily in neurons and their processes, but not in glial cells. Severe hypoxic ATP depletion increased the release of both GABA and glutamate. Hypoxia increased GABA levels in neurons, while hypoglycemia with a similar severity of ATP depletion decreased GABA levels. Much of the difference may be related to lower levels of precursor glutamate during hypoglycemia. The twofold higher levels of neuroprotective GABA available for release during hypoxia may contribute to differences in the pathophysiology of these metabolic insults. PMID- 10727785 TI - Subcellular distribution and autophosphorylation of calcium/calmodulin-dependent protein kinase II-alpha in rat hippocampus in a model of ischemic tolerance. AB - A brief period of sublethal ischemia induces resistance to a subsequent, otherwise lethal, ischemic insult, a process named ischemic tolerance or preconditioning. A persistently disturbed cell signaling during reperfusion after cerebral ischemia has been proposed to contribute to ischemic cell death. Here, we report on the effect of ischemic preconditioning on the levels of the regulatory alpha-subunit of calcium/calmodulin protein kinase II and its phosphorylation in the hippocampal CA1 region. We found that during and following lethal cerebral ischemia, calcium/calmodulin protein kinase II-alpha is persistently translocated to cell membranes, where it becomes phosphorylated at threonine 286. In contrast, in the preconditioned brains the translocation and phosphorylation are transient and return to preischemic values after one day of reperfusion. At this time of reperfusion, the total level of calcium/calmodulin protein kinase II-alpha is significantly lower in preconditioned animals compared to the sham and non-conditioned animals. After one day of reperfusion, the level of calcium/calmodulin protein kinase II-alpha messenger RNA decreases in the non conditioned brains, whereas it is unchanged in preconditioned brains. We conclude that, during and after ischemia, calcium/calmodulin protein kinase II-alpha is translocated to cell membranes and becomes phosphorylated at threonine 286. This could detrimentally influence cell survival by changing receptor function and ion channel conductance. Ischemic preconditioning prevents the persistent presence of calcium/calmodulin protein kinase II-alpha at cell membranes, presumably by enhancing its degradation, which could be part of a neuroprotective mechanism of ischemic tolerance. PMID- 10727787 TI - Enhanced nucleus accumbens dopamine and plasma corticosterone stress responses in adult rats with neonatal excitotoxic lesions to the medial prefrontal cortex. AB - The medial prefrontal cortex modulates the nucleus accumbens dopamine response to stress and has been implicated in feedback regulation of hypothalamic-pituitary adrenal axis activation by stress. Here we report on the effects of bilateral neonatal (postnatal day 7) ibotenate-induced lesions to the medial prefrontal cortex on nucleus accumbens dopamine and neuroendocrine function in adult rats. Voltammetry was used to monitor the dopamine response to each of five, once-daily exposures to tail-pinch stress whereas alterations in neuroendocrine function were determined from the plasma corticosterone response to a single 20-min episode of restraint stress. Potential lesion-induced deficits in sensory-motor gating were assessed by measuring prepulse inhibition of the acoustic startle response before and after repeated stress. Our data show that each daily stress episode elicited larger and longer-lasting dopamine increases in prefrontal cortex-lesioned animals than in sham-lesioned controls. Furthermore, greater stress-induced elevations in plasma corticosterone were seen in lesioned animals than in their sham-lesioned counterparts. However, while repeated stress potentiated startle responses in animals of both groups, there was no effect of lesion on the amplitude or on prepulse inhibition of the startle response.Together, these findings indicate that neonatal prefrontal cortex damage can lead to changes in mesolimbic dopamine and neuroendocrine function during adulthood. They also add to a growing body of experimental and clinical evidence implicating abnormal prefrontal cortex neuronal development in the pathophysiology of schizophrenia and other disorders linked to central dopamine dysfunction. PMID- 10727786 TI - Systemic and intra-accumbens administration of amphetamine differentially affects cortical acetylcholine release. AB - The present experiments tested the hypothesis that the amphetamine-induced increase in dopamine release in the nucleus accumbens represents a necessary and sufficient component of the ability of systemically administered amphetamine to stimulate cortical acetylcholine release. The effects of systemic or intra accumbens administration of amphetamine on accumbens dopamine release and cortical acetylcholine release were assessed simultaneously in awake animals equipped with dialysis probes inserted into the shell of the nucleus accumbens and the medial prefrontal cortex. Additionally, the ability of intra-accumbens administration of dopamine D(1) and D(2) receptor antagonists to attenuate the effects of systemic amphetamine on cortical acetylcholine was tested. The effects of all treatments were assessed in interaction with a stimulus-induced activation of cortical acetylcholine release to account for the possibility that the demonstration of the trans-synaptic effects of accumbens dopamine requires pre activation of basal forebrain circuits. Systemic amphetamine resulted in increases in basal cortical acetylcholine and accumbens dopamine efflux. Intra accumbens administration of amphetamine substantially increased accumbens dopamine efflux, but did not significantly affect cortical acetylcholine efflux. Furthermore, intra-accumbens administration of sulpiride or SCH 23390 did not attenuate the systemic amphetamine-induced increase in cortical acetylcholine efflux. Collectively, the present data suggest that increases in accumbens dopamine release are neither sufficient nor necessary for the effects of systemically administered amphetamine on cortical acetylcholine release. The systemic amphetamine-induced increase in cortical acetylcholine may be mediated via multiple, parallel pathways and may not be attributable to a single afferent pathway of the basal forebrain. PMID- 10727788 TI - Extracellular dopamine dynamics in rat caudate-putamen during experimenter delivered and intracranial self-stimulation. AB - Intracranial self-stimulation is an operant behavior whereby animals are conditioned to press a lever in order to receive an electrical stimulation of their dopamine neurons. This paradigm is thought to stimulate brain reward pathways and, as such, has been used to clarify the role of dopamine in reward. Striatal extracellular dopamine concentrations were monitored during the acquisition and maintenance of self-stimulation and compared to dopamine release generated by experimenter-delivered and yoked stimulation. Fast-scan cyclic voltammetry in conjunction with carbon-fiber microelectrodes was used to monitor evoked dopamine release in the caudate-putamen during electrical stimulation of the substantia nigra/ventral tegmental area. The sub-second temporal resolution of fast-scan cyclic voltammetry coupled with the micron spatial resolution of the microelectrodes allowed for the measurement of dopamine neurotransmission in real time. Single experimenter-delivered stimulations, identical to those used during self-stimulation, evoked dopamine release in the caudate-putamen both before and after the self-stimulation sessions. Likewise, yoked stimulations of the substantia nigra/ventral tegmental area delivered to animals untrained to perform self-stimulation resulted in an increase in extracellular dopamine levels. During training sessions, experimenter-delivered stimulations evoked dopamine release. However, as the animals began lever-pressing, extracellular dopamine levels subsequently declined. Taken together, these results suggest that dopamine functions as an alerting device, wherein increases in extracellular dopamine are obtained by unpredicted or novel rewarding stimuli, but not by those which can be anticipated. PMID- 10727789 TI - Somatomotor neuron-specific expression of the human cholinergic gene locus in transgenic mice. AB - We examined the expression pattern of the vesicular acetylcholine transporter in the mouse nervous system, using rodent-specific riboprobes and antibodies, prior to comparing it with the distribution of vesicular acetylcholine transporter expressed from a human transgene in the mouse, using riboprobes and antibodies specific for human. Endogenous vesicular acetylcholine transporter expression was high in spinal and brainstem somatomotor neurons, vagal visceromotor neurons, and postganglionic parasympathetic neurons, moderate in basal forebrain and brainstem projection neurons and striatal interneurons, and low in intestinal intrinsic neurons. Vesicular acetylcholine transporter expression in intrinsic cortical neurons was restricted to the entorhinal cortex. The sequence of the mouse cholinergic gene locus to 5.1kb upstream of the start of transcription of the vesicular acetylcholine transporter gene was determined and compared with the corresponding region of the human gene. Cis-regulatory domains implicated previously in human or rat cholinergic gene regulation are highly conserved in mouse, indicating their probable relevance to the regulation of the mammalian cholinergic gene locus in vivo. Mouse lines were established containing a human transgene that included the vesicular acetylcholine transporter gene and sequences spanning 5kb upstream and 1.8kb downstream of the vesicular acetylcholine transporter open reading frame. In this transgene, the intact human vesicular acetylcholine transporter was able to act as its own reporter. This allowed elements within the vesicular acetylcholine transporter open reading frame itself, shown previously to affect transcription in vitro, to be assessed in vivo with antibodies and riboprobes that reliably distinguished between human and mouse vesicular acetylcholine transporters and their messenger RNAs. Expression of the human vesicular acetylcholine transporter was restricted to mouse cholinergic somatomotor neurons in the spinal cord and brainstem, but absent from other central and peripheral cholinergic neurons. The mouse appears to be an appropriate model for the study of the genetic regulation of the cholinergic gene locus, and the physiology and neurochemistry of the mammalian cholinergic nervous system, although differences exist in the distribution of cortical cholinergic neurons between the mouse and other mammals. The somatomotor neuron-specific expression pattern of the transgenic human vesicular acetylcholine transporter suggests a mosaic model for cholinergic gene locus regulation in separate subdivisions of the mammalian cholinergic nervous system. PMID- 10727790 TI - Modulation of presumed cholinergic mesopontine tegmental neurons by acetylcholine and monoamines applied iontophoretically in unanesthetized cats. AB - The mesopontine tegmentum, which contains both cholinergic and non-cholinergic neurons, plays a crucial role in behavioral state control. Using microiontophoresis in unanesthetized cats, we have examined the effect of cholinergic and monoaminergic drugs on two putative cholinergic neurons located mostly in the laterodorsal tegmental nucleus and X area (or the cholinergic part of the nucleus tegmenti pedunculopontinus, pars compacta): one (type I-S) exhibiting slow tonic discharge during both waking and paradoxical sleep, and the other (PGO-on) displaying single spike activity during waking and burst discharges in association with ponto-geniculo-occipital (PGO) waves during paradoxical sleep. We found that: (i) application of carbachol, a potent cholinergic agonist, inhibited single spike activity in both PGO-on and type I-S neurons, but had no effect on the burst activity of PGO-on neurons during paradoxical sleep; the inhibition was associated with either blockade or increased latency of antidromic responses, suggesting membrane hyperpolarization; (ii) application of glutamate, norepinephrine, epinephrine, or histamine resulted in increased tonic discharge in both PGO-on and type I-S neurons; this was state independent and resulted in a change in the firing mode of PGO-on neurons from phasic to tonic; (iii) application of serotonin had only a weak state-dependent inhibitory effect on a few type I-S neurons; and (iv) application of dopamine had no effect on either type of neuron. The present findings suggest that cholinergic, glutamatergic and monoaminergic (especially noradrenergic, adrenergic and histaminergic) inputs have the capacity to strongly modulate the cholinergic neurons, altering both their rate and mode of discharge, such as to shape their state specific activity, and thereby contribute greatly to their role in behavioral state control. PMID- 10727791 TI - The pedunculopontine tegmental nucleus and the role of cholinergic neurons in nicotine self-administration in the rat: a correlative neuroanatomical and behavioral study. AB - The objective of this study was to determine whether the pedunculopontine tegmental nucleus plays a role in the maintenance of nicotine self administration, and whether the ascending cholinergic projection from this nucleus to midbrain dopamine neurons in the ventral tegmental area might be involved. Studies were done with rats trained to self-administer nicotine intravenously. Self-administration was examined before and after the pedunculopontine tegmental nucleus was lesioned with the ethylcholine mustard aziridinium ion, a selective cholinergic toxin. Lesions were assessed qualitatively and quantitatively in histological sections stained for either nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry to identify cholinergic neurons, or for Nissl. Self-administration was also tested after an acute manipulation in which microinfusions of the nicotinic cholinergic antagonist dihydro-beta-erythroidine were made into the pedunculopontine tegmentum. Infusions of neurotoxin into the pedunculopontine tegmentum reduced nicotine self-administration behaviour when tested weeks later. Toxin treatment reduced the number of cholinergic neurons in the tegmentum, while largely sparing the non-cholinergic population in this area. Lesions were limited to the pedunculopontine area and did not extend to the neighboring laterodorsal tegmental nucleus or to the substantia nigra. Acute manipulation of the pedunculopontine tegmental nucleus with microinfusions of dihydro-beta erythroidine also produced an attenuation of nicotine self-administration. Collectively these data show that the pedunculopontine tegmental nucleus is part of the neuronal circuitry mediating nicotine self-administration, and that the population of cholinergic neurons is likely a critical element. PMID- 10727792 TI - Noradrenergic regulation of parvocellular neurons in the rat hypothalamic paraventricular nucleus. AB - Noradrenergic projections to the hypothalamic paraventricular nucleus have been implicated in the secretory regulation of several anterior pituitary hormones, including adrenocorticotropin, thyroid-stimulating hormone, growth hormone and prolactin. In an attempt to elucidate the effects of norepinephrine on the central control of pituitary hormone secretion, we looked at the actions of norepinephrine on the electrical properties of putative parvocellular neurons of the paraventricular nucleus using whole-cell current-clamp recordings in hypothalamic slices. About half (51%) of the putative parvocellular neurons recorded responded to norepinephrine with either a synaptic excitation or a direct inhibition. Norepinephrine (30-300microM) caused a marked increase in the frequency of excitatory postsynaptic potentials in about 36% of the parvocellular neurons recorded. The increase in excitatory postsynaptic potentials was blocked by prazosin (10microM), but not by propranolol (10microM) or timolol (20microM), indicating that it was mediated by alpha(1)-adrenoreceptor activation. It was also blocked by ionotropic glutamate receptor antagonists, suggesting that the excitatory postsynaptic potentials were caused by glutamate release. The increase in excitatory postsynaptic potentials was completely abolished by tetrodotoxin, indicating the spike dependence of the norepinephrine-induced glutamate release. In a separate group comprising 14% of the parvocellular neurons recorded, norepinephrine elicited a hyperpolarization (6.2+/-0.69mV) that was blocked by the beta-adrenoreceptor antagonists, propranolol (10microM) and timolol (20microM), but not by the alpha(1)-receptor antagonist, prazosin (10microM). This response was not blocked by tetrodotoxin (1.5-3microM), suggesting that it was caused by a direct postsynaptic action of norepinephrine. The topographic distribution within the paraventricular nucleus of the norepinephrine-responsive and non-responsive parvocellular neurons was mapped based on intracellular biocytin labeling and neurophysin immunohistochemistry. These data indicate that one parvocellular subpopulation, consisting of about 36% of the paraventricular parvocellular neurons, receives an excitatory input from norepinephrine-sensitive local glutamatergic interneurons, while a second, separate subpopulation, representing about 14% of the parvocellular neurons in the paraventricular nucleus, responds directly to norepinephrine with a beta-adrenoreceptor-mediated inhibition. This suggests that excitatory inputs to parvocellular neurons of the paraventricular nucleus are mediated mainly by an intrahypothalamic glutamatergic relay, and that only a relatively small subset of paraventricular parvocellular neurons receives direct noradrenergic inputs, which are primarily inhibitory. PMID- 10727793 TI - Biophysical and pharmacological characterization of voltage-sensitive calcium currents in neonatal rat inferior colliculus neurons. AB - Calcium conductances have been found in neonatal inferior colliculus neurons, however the biophysical and pharmacological profiles of the underlying calcium currents have not yet been characterized. In this study, we examined which types of voltage-activated calcium currents comprise the whole-cell inward current of neonatal inferior colliculus neurons (10-22microm in diameter). On the basis of their voltage-dependence and pharmacological sensitivities, three major components of barium currents were identified. A low threshold voltage-activated current that activated around -70mV, a mid threshold voltage-activated current that activated near -50mV, and a high threshold voltage-activated current that activated around -40mV. Low and mid threshold voltage-activated currents were present in 33% and 41% of the recordings, respectively, whereas high threshold voltage-activated currents were recorded in all inferior colliculus neurons tested. Nickel chloride (50microM) and U-92032 (1microM), which both block low threshold voltage-activated currents, reduced the amplitude of low threshold voltage-activated peak currents at a test potential of -60mV by 72% and 10%, respectively. In addition, 50microM nickel chloride and 1microM U-92032 reduced the amplitude of mid threshold voltage-activated peak currents measured at -20mV by 55% and 21%, respectively. Further pharmacological analysis indicated the presence of multiple types of high threshold voltage-activated currents in neonatal inferior colliculus neurons. The dihydropyridine nimodipine (1microM), a selective L-type current antagonist, reduced the amplitude of high threshold voltage-activated peak currents by 25%. In addition, FPL 64176 (1microM), a non dihydropyridine L-type current agonist caused a dramatic 534% increase in the amplitude of the slow sustained component of the tail current measured at -40mV. These data indicate that inferior colliculus neurons express L-type channels. omega-Conotoxin GVIA (1microM), a selective blocker of N-type current, inhibited high threshold voltage-activated peak currents by 28% indicating the presence of N-type channels. omega-Agatoxin IVA (300nM), a potent P/Q-type antagonist, reduced high threshold voltage-activated peak currents by 27%, suggesting that inferior colliculus neurons express P/Q-type channels. Concomitant application of nimodipine (1microM), omega-conotoxin GVIA (1microM) and omega-agatoxin IVA (300nM) onto inferior colliculus neurons decreased the control high threshold voltage-activated peak currents only by 62%.Thus, inferior colliculus neurons may express at least one more type of calcium current in addition to low and mid threshold voltage-activated currents and L-type, N-type and P/Q-type high threshold currents. PMID- 10727794 TI - Antinociceptive role of galanin in periaqueductal grey of rats with experimentally induced mononeuropathy. AB - The present study was performed in rats with experimentally induced mononeuropathy after left common sciatic nerve ligation. The hindpaw withdrawal latencies to thermal and mechanical stimulation increased significantly after intra-periaqueductal grey injection of 2 or 3nmol, but not 1nmol of galanin in rats with mononeuropathy. Intraperitoneal administration of 4.5mg/kg morphine induced significant increases in hindpaw withdrawal latencies to both noxious stimulation, which were attenuated by following intra-periaqueductal grey injection of 2nmol of the galanin antagonist galantide. Furthermore, the antinociceptive effect induced by intra-periaqueductal grey injection of 26.6nmol of morphine was attenuated significantly by following intra-periaqueductal gray administration of 2nmol of galantide. The results demonstrated that in periaqueductal grey galanin plays an antinociceptive role in rats with mononeuropathy and galanin is involved in the mechanisms of opioid-induced antinociception. PMID- 10727795 TI - Bidirectional modulation of nociception by GABA neurons in the dorsolateral pontine tegmentum that tonically inhibit spinally projecting noradrenergic A7 neurons. AB - The A7 catecholamine cell group in the dorsolateral pontine tegmentum constitutes an important part of the descending pathways that modulate nociception. Evidence from immunocytochemical studies demonstrate that noradrenergic A7 neurons are densely innervated by GABA terminals arising from GABA neurons that are located in the dorsolateral pontine tegmentum medial to the A7 cell group. GABA(A) receptors are also located on the somata and dendrites of noradrenergic A7 neurons. These findings suggest that noradrenergic neurons in the A7 cell group may be under tonic inhibitory control by GABA neurons. To test this hypothesis, the GABA(A) antagonist bicuculline methiodide in doses of 0.2 or 1.0nmol was microinjected into sites located dorsal to the A7 cell group and the resulting effects on tail flick and nociceptive foot withdrawal responses were measured. Both doses of bicuculline produced significant increases in tail flick latencies and small, but significant, increases in foot withdrawal latencies. Intrathecal injection of the alpha(2)-adrenoceptor antagonist yohimbine, in a dose of 76.7nmol (30microg), attenuated the antinociceptive effect of bicuculline on both the tail and the feet. In contrast, the alpha(1)-adrenoceptor antagonist WB4101, in a nearly equimolar dose of 78.6nmol (30microg), increased the antinociceptive effect of bicuculline on both the tail and the feet. Intrathecal injection of the antagonists alone did not consistently alter nociceptive responses of either the feet or the tail. These findings suggest that noradrenergic neurons in the A7 cell group are tonically inhibited by local GABA neurons. Furthermore, these findings suggest that inhibition of GABA(A) receptors located on spinally projecting A7 noradrenergic neurons disinhibits, or activates, two populations of A7 neurons that have opposing effects on nociception. One of these populations facilitates nociception by an action mediated by alpha(1)-adrenoceptors in the spinal cord dorsal horn and the other population inhibits nociception by an action mediated by alpha(2)-adrenoceptors. PMID- 10727796 TI - Upregulation of an opioid-mediated antinociceptive mechanism in transgenic mice over-expressing substance P in the spinal cord. AB - In transgenic mice expressing ectopic substance P fibres in the spinal white matter, a normally innocuous mechanical stimulus induces hyperalgesia and allodynia which are reversed by substance P and N-methyl-D-aspartate receptor antagonists. This period of enhanced excitation is followed by a rebound overshoot in these animals. As previous evidence indicates opioid mechanisms in a similar rebound in normal animals, the present study was done to determine the effects of systemic administration of morphine and the opiate receptor antagonist, naloxone, on the stimulus-induced responses in the tail withdrawal reflex. Once baseline reaction times had been taken, different combinations of saline, naloxone and morphine were administered intraperitoneally to transgenic and control mice of either sex. A mechanical conditioning stimulus of 450g was then applied to the tip of the tail for 2s. This stimulus was innocuous in control mice given saline or naloxone, but provoked a nociceptive response in transgenic mice given these compounds. In control and transgenic mice, following morphine administration there was an antinociceptive effect. In control mice the subsequent mechanical stimulus had no effect. However, in transgenic mice the mechanical stimulus produced a further antinociception. Naloxone blocked the effect of morphine and the subsequent conditioning stimulus in both control and transgenic mice. The results indicate that while morphine is equally effective on the withdrawal reflex in both types of animal, in the transgenic mice morphine reveals an intrinsic, naloxone-sensitive antinociceptive mechanism. The data are interpreted to suggest that over-expression of substance P or some other factor in the spinal cord of transgenic mice is associated with the up-regulation or facilitation of an opiate-mediated intrinsic antinociceptive mechanism. This is a novel observation because the genetic manipulation in this transgenic mouse results in a transient over-expression of nerve growth factor during development that leads to the formation of ectopic primary afferent fibres in the spinal cord containing substance P. These fibres persist indefinitely after the nerve growth factor levels return to normal. Opioid mechanisms, which are likely of dorsal horn origin, do not fall under the direct influence of nerve growth factor mechanisms and therefore the intriguing possibility is raised that opioid mechanisms in the spinal cord are regulated at least in part by substance P related mechanisms. PMID- 10727797 TI - State-dependent effects of some neuropeptides and neurotransmitters on neuronal activity of the medial septal area in brain slices of the ground squirrel, Citellus undulatus. AB - Neuronal activity of the medial septal area was recorded extracellularly in brain slices taken from hibernating (winter) and waking (summer) ground squirrels. The effects of neuropeptides identified in the brain tissue of hibernators (Thr-Ser Lys-Tyr, Thr-Ser-Lys-Tyr-Arg and Asp-Tyr) on the background activity and responses to electrical stimulation of the median forebrain bundle were analysed. For comparison, the effects of bath application of noradrenaline and serotonin were also tested. Spontaneous activity in half of all neurons (47-56%) was changed under the influence of neuropeptides in hibernating ground squirrels, while in waking ground squirrels the proportion of responsive neurons was significantly lower (25-30%). The tendency for higher efficacy in hibernating ground squirrels was observed for serotonin; only noradrenaline was equally effective in both groups of animals. Electrically evoked responses of the medial septal nucleus-nucleus of the diagonal band neurons were also strongly modulated by neuropeptides; their changes could occur in the absence of shifts in the level and pattern of spontaneous activity. All three neuropeptides had differential action on the level of spontaneous activity, as well as on inhibitory and excitatory components of electrically evoked responses. Thus, the character and distribution of the effects were state dependent and differed greatly in hibernating and waking ground squirrels. The experiments confirmed that medial septal nucleus-nucleus of the diagonal band neurons have higher excitability and responsiveness to some neuropeptides and neurotransmitters in hibernating ground squirrels.The data obtained suggest an increased latent excitability and responsiveness of septal neurons during hibernation and their possible active participation in urgent arousal under the influence of sensory signals. PMID- 10727798 TI - Microglial activation in the developing rat olfactory bulb. AB - The development of the olfactory bulb, the primary central relay of the olfactory system, is characterized by a striking susceptibility to alterations in the amount of afferent input. For example, blocking airflow through one half of the nasal cavity during early life results in a number of dramatic changes in the bulb, including increased cell death. Previous studies reveal high levels of microglia in the olfactory bulb. Microglia function as phagocytes, aid in synaptogenesis, and produce important trophic and cytotoxic factors. In response to a number of tissue perturbations, microglia undergo an activation process that includes, among other changes, the up-regulation of complement receptor 3. Interestingly, a previous study reported that naris closure had no effect on microglia in the bulb; however, the research did not distinguish the functional activation state of microglia. We further examined the role of microglia in the normally developing and olfactory-deprived rat bulb using immunohistochemical detection of complement receptor 3 as a measure of microglial activation. Expression of the receptor in the bulb is relatively high during postnatal development, in particular when compared to levels in cortical regions caudal to the olfactory bulb. In addition, naris closure performed on the day after birth (but not after the first postnatal month) increases levels of the receptor in an age and laminar-dependent fashion. The presence of an inducible pool of activated microglia in the olfactory bulb may be important for normal development and contribute to the plethora of changes seen after early olfactory deprivation. PMID- 10727799 TI - Extracellular guanosine 5' triphosphate enhances nerve growth factor-induced neurite outgrowth via increases in intracellular calcium. AB - Extracellular guanosine 5' triphosphate (GTP) enhances nerve growth factor dependent neurite outgrowth from rat pheochromocytoma (PC12) cells; cultures of PC12 cells exposed to GTP and nerve growth factor together contain significantly more neurite-bearing cells than do those exposed to either nerve growth factor or GTP alone [Gysbers J. W. and Rathbone M. P. (1996) Int. J. devl Neurosci. 14, 19 34]. PC12 cells contain specific cell surface binding sites for extracellular GTP, which do not bind ATP or uridine 5' triphosphate. Exposure of PC12 cells to extracellular GTP (300microM) produced a robust and sustained increase in intracellular Ca(2+) ([Ca(2+)](i)), different from the transient response to the addition of ATP. The GTP-induced [Ca(2+)](i) increase was blocked by the L-type calcium channel inhibitor, nifedipine. The L-type Ca(2+) channel inhibitors, nifedipine or verapamil, also inhibited the enhancement of neurite outgrowth by GTP, but did not affect neurite outgrowth stimulated by nerve growth factor alone. Pre-treatment of PC12 cells with ryanodine (0.5-50microM) depleted calcium from internal stores and prevented the further release of calcium by GTP. Similarly, pre-treatment of PC12 cells with thapsigargin (an inhibitor of internal store Ca(2+)/ATPase) or dantrolene (which blocks Ca(2+) release from some of these stores) also reduced the enhancement of neurite outgrowth by GTP. Therefore, Ca(2+)-induced Ca(2+) release from specific stores, present in PC12 cells, is involved in the enhancement of nerve growth factor-induced neurite outgrowth by GTP, possibly acting at specific binding sites on the cell surface. GTP is proving to be an important extracellular trophic modulator in the central nervous system. These studies show that the neuritogenic actions of GTP involve moderate but sustained increases in intracellular Ca(2+) which are likely due to activation of L-type Ca(2+) channels and Ca(2+)-induced Ca(2+) release from intracellular stores. These effects of extracellular GTP are likely mediated at the cell surface and may be related to specific GTP binding sites which are distinct from G-proteins and from hitherto described purine nucleotide (P2) receptors. These data indicate a mechanism whereby the neuritogenic effects of GTP are mediated and emphasize the importance of considering GTP as a neurotrophic mediator. PMID- 10727801 TI - Effects of the non-pseudoautosomal region of the Y-chromosome on behavior in female offspring of two congenic strains of mice. AB - The learning behavior of female offspring of two strains of mice congenic for the Y-chromosome, BXSX/MpJ-Yaa and BXSB/MpJ-Yaa+, was examined. Significant differences were found in the Morris water maze and the Lashley III maze, demonstrating that the fathers' Y-chromosome can indirectly affect their daughters' behavior. Approximately half the mice had neocortical ectopias, and females from the two paternal groups reacted differently to the presence or absence of ectopias. Since females do not have a Y-chromosome, these effects must be through non-genetic mechanisms. Prenatal factors that could have played a role include possible differences in gonadal growth and the presence of different H-Y antigens. Postnatally, the sires and male siblings of the two strains may not have behaved the same toward the female offspring and/or the dams, creating differences in behavior. In summary, the behavior of female offspring of two groups of males, genetically the same except for their Y-chromosomes, was examined. Since females do not receive a Y-chromosome from their fathers, in theory their behavior should not differ. Significant differences were found, indicating that the Y-chromosome, through some indirect mechanism, can affect females of the next generation. PMID- 10727800 TI - Postnatal decrease in transforming growth factor alpha is associated with enlarged ventricles, deficient amygdaloid vasculature and performance deficits. AB - It is well established that transforming growth factor alpha is involved prenatally in development of the nervous system, but its role in the postnatal brain is less well understood. Here, we document the occurrence of late-onset, morphological and behavioral deficits in the naturally occurring murine mutant, Waved-1 (Wa-1), whose transforming growth factor alpha levels decrease naturally between early postnatal and adolescent ages. Morphological analyses suggest that reduction in the growth factor postnatally is associated temporally with the onset of enlarged lateral ventricles, a reduction in vasculature in the region of the amygdala and a reduction in size of the central nucleus. Onset of the morphological deficits corresponds to the appearance of a performance deficit in contextual fear conditioning. In contrast, the transforming growth factor alpha gene-targeted null mutants exhibit neither morphological nor performance deficits. These data suggest that transforming growth factor alpha during postnatal maturation of the brain may contribute to maintenance of limbic morphology and vasculature, which may in turn affect some behaviors associated with these specific brain structures. PMID- 10727802 TI - Influence of the target on distribution and functioning of the varicosities of Helix pomatia metacerebral cell C1 in dissociated cell culture. AB - The serotonergic metacerebral giant cell (C1) of Helix pomatia was isolated with its bifurcate axon and plated in culture under five conditions: (i) with no target; (ii) with the appropriate target B2 near the stump of the bigger branch (CBC); (iii) with B2 near the stump of the smaller branch (CC); (iv) with a wrong target (C3) near the stump of the CBC branch and (v) with B2 and C3 positioned near the CBC and CC stump, respectively. The counting of anti-serotonin antibody labelled varicosities of the C1 neuron showed that the presence of the appropriate target in either axonal domain both down-regulated the number of varicosities of the contralateral neuritic field, and increased their average size, whereas the wrong target induced an overall reduction of the number of C1 neuron varicosities, and inhibited the evoked transmitter release. The action potential-evoked calcium concentration increase in the neuritic terminals of the C1 neuron cultured alone, or in presence of the appropriate target, reached a value significantly higher than that reached in presence of the wrong target. These results provide evidence that the postsynaptic neuron regulates both morphological and functional development of presynaptic terminals. PMID- 10727803 TI - A new steroidal alkaloid from the seeds of Holarrhena antidysenterica. AB - A new steroidal alkaloid, named antidysentericine, has been isolated from the seeds of Holarrhena antidysenterica and characterized as 3 beta-dimethylaminocon 5-enin-18-one (1). PMID- 10727804 TI - Responses of Spodoptera littoralis larvae to Tunisian plant extracts and to neo clerodane diterpenoids isolated from Ajuga pseudoiva leaves. AB - Thirteen plants growing in Tunisia were tested against Spodoptera littoralis larvae. From the acetone extract of Ajuga pseudoiva leaves, showing interesting antifeedant activity, three new (1-3) and two known (4-5) active clerodane diterpenoids were isolated following a bioassay-guided chromatography. PMID- 10727805 TI - Hypocholesterolemic activity of Marsilea minuta in gerbils. AB - Feeding of a Marsilea minuta leaf extract [Fr. I] reduced serum cholesterol and triglycerides by 31 and 63%, respectively, in athero diet fed gerbils. Liver cholesterol and triglycerides were also lowered by 71 and 27%, respectively, in comparison to athero fed controls. Moreover, treatment with Fr. I prevented the accumulation of cholesterol and triglycerides in liver and aorta and was able to dissolve atheromatous plaques of thoracic and abdominal aorta. Fecal excretions of cholesterol and triglycerides were significantly increased in Fr. I fed gerbils. PMID- 10727807 TI - New bufadienolides from Urginea maritima sensu strictu. AB - Two new bufadienolides, 11 alpha-acetylgamabufotalin 3-O-(4-O-beta-D-glucosyl) alpha-L-rhamnoside (1) and 11 alpha-hydroxyscilliglaucoside (2), were isolated from bulbs of hexaploid Urginea maritima and identified by means of FAB-MS, (1)H NMR and (13)C-NMR studies. PMID- 10727806 TI - Bioactive constituents of the bark of Parkia biglobosa. AB - In the frame of a systematic analysis of African plants used for the 'cure salee', from the bark of Parkia biglobosa, a long-chain ester of trans-ferulic acid (1) has been isolated together with an unseparable mixture of long-chain cis ferulates (2a-e). In addition, lupeol, 4-O-methyl-epi-gallocatechin, epi gallocatechin, epi-catechin 3-O-gallate, and epi-gallocatechin 3-O-gallate were isolated. PMID- 10727808 TI - Two new triterpene lactones from Centaurium pulchellum. AB - Two new triterpene lactones, centauriol (1) and centaurione (2) were isolated from the whole plant of Centaurium pulchellum. Their structures were determined by spectral data. PMID- 10727809 TI - Polycyclic diterpenoids from Euphorbia characias. AB - In addition to widespread flavonoids, a collection of Euphorbia characias from Sardinia afforded 13 oxygenated diterpenoids of the atisane, abietane, pimarane, and kaurane type. Four of these compounds (1, 3a, 7a,b) are new. The accumulation of substantial amounts of biologically active diterpenoids of limited availability, like ent-atisanes endowed with anti-HIV activity and ent abietanolides active on the central nervous system, makes E. characias an interesting source of lead compounds for biomedical research. PMID- 10727810 TI - Pharmacological screening of the aqueous extract of Musanga cecropiodes. AB - The aqueous extract of the leaves of Musanga cecropioides did not affect ache induced contractile response, but produced significant inhibition of the twitch and pendular movement of the rat and rabbit smooth muscles, respectively, and reduced writhing induced by acetic acid in mice. PMID- 10727811 TI - Antipyretic, antinociceptive and anti-inflammatory activity of Premna herbacea roots. AB - The alcoholic extract of the roots of Premna herbacea was investigated for its antipyretic, antinociceptive and anti-inflammatory potential in animal models. The extract, when administered orally to mice has been found to be safe up to a dose of 8.0 g/kg. A significant antipyretic effect has been observed in rabbits while mild antinociceptive effects were evidenced in mice when tested by chemical as well as thermal methods. The extract did not exhibit any anti-inflammatory activity in acute but significantly reduced the chronic inflammation. PMID- 10727812 TI - Variance of common flavonoids by brand of grapefruit juice. AB - Nine commercial brands of grapefruit juice were analyzed for their flavonoid content by HPLC to determine if significant brand-to-brand variance in grapefruit juice flavonoid content exists. Flavonoid glycosides narirutin, naringin, hesperidin, neohesperidin, didymin, and poncirin have been identified in all the grapefruit juices examined. The aglycone quercetin was detected in only two brands. All the juices were free from methoxylated flavonoid aglycones. There was a significant difference in the amounts of total flavonoids and individual flavonoids in the nine brands. The concentration of total flavonoids ranged between 19.44 and 84.28 mg/100 ml juice. Naringin was found to be the major flavonoid followed by narirutin and hesperidin. Their concentrations ranged from 14.56 to 63.8; 2.25 to 12.20; and 0.24 to 3.12 mg/100 ml juice, respectively. PMID- 10727813 TI - Characterization of proanthocyanidins from grape seeds. AB - Leucoselect (grape seed selected proanthocyanidins) was analyzed. HPLC thermospray mass spectrometry (TSP-MS) allowed the detection of monomeric flavan 3-ols and dimeric proanthocyanidins. Fractionation over Sephadex LH-20 resin and analysis of the isolated fractions by gel permeation chromatography (GPC) and electrospray mass spectrometry (ESI-MS) led to the complete characterization of the proanthocyanidin constituents of Leucoselect. The analysis revealed the presence of approximately 15% of (+)-catechin (1) and (-)-epicatechin (2), 80% of (-)-epicatechin 3-O-gallate (3), dimers, trimers, tetramers and their gallates and 5% of pentamers, hexamers, heptamers and their gallates. PMID- 10727814 TI - Antimicrobial properties of Thonningia sanguinea root extracts. AB - The aqueous and methanolic extracts of Thonningia sanguinea root, as well as seven fractions obtained by PTLC from the methanol extract, have been shown to possess varying degrees of antimicrobial activity. PMID- 10727815 TI - Antimicrobial activity of Trichilia heudelotti leaves. AB - Diterpenes and phenolic acids, including the new compounds 12 beta hydroxysandaracopimar-15-ene (4) and 2-propionoxy-beta-resorcylic acid (8) have been isolated from Trichilia heudelotti leaves. The methanol extract showed antimicrobial activity concentrated in the ethyl acetate fraction and some of its constituents. PMID- 10727816 TI - Cytotoxicity and antileishmanial activity of Annona muricata pericarp. AB - Hexane, ethyl acetate and methanol extracts of Annona muricata pericarp were tested in vitro against Leishmania braziliensis and L. panamensis promastigotes, and against cell line U-937. The ethyl acetate extract was more active than the other extracts and even of Glucantime used as reference substance. Its fractionation led to the isolation of three acetogenins--annonacin, annonacin A and annomuricin A. PMID- 10727817 TI - Antimicrobial activity of methanolic extract of Solanum torvum fruit. AB - The methanolic extract of Solanum torvum fruit showed a wide spectrum of antimicrobial activities against human and animal clinical isolates. PMID- 10727818 TI - Preliminary evaluation of cytotoxic properties of Raphia hookeri fruit mesocarp. AB - The aqueous and n-butanol fractions of the methanolic extract of Raphia hookeri fruit mesocarp was evaluated for cytotoxic properties using three different bioassays. The n-butanol fraction exhibited greater cytotoxicity than the aqueous fraction when tested against brine shrimp, mosquito larvae, and 5-day old tadpoles, the cytotoxicity towards the tadpoles being the most pronounced (LC(50)=0.83 ppm). PMID- 10727819 TI - Antimicrobial activity of Bryophyllum pinnatum leaves. AB - Bryophyllum pinnatum leaf 60% methanolic extract was found to inhibit the growth of five out of eight bacteria used, at a concentration of 25 mg/ml. PMID- 10727821 TI - Antibacterial activity of Nicotiana tabacum leaves. AB - Nicotiana tabacum leaf 60% methanolic extract exhibited antibacterial activity against six out of nine bacterial isolates at a concentration of 25 mg/ml. PMID- 10727820 TI - A phytochemical investigation of Alchornea latifolia. AB - A phytochemical investigation of the chloroform leaf extract of Alchornea latifolia has been undertaken. Along with the triterpenoids taraxerone, friedelin, epifriedelinol, and taraxerol, the plant also contains seco-3,4 friedelin (dihydroputranjivic acid) (1) and seco-3,4-taraxerone (2). These A-ring opened triterpenoids show in vitro cytotoxic activity against Hep-G2 and A-431 human cancer cell lines and are potent inhibitors of topoisomerase II. PMID- 10727822 TI - Luteolin 3'-glucuronide, the major flavonoid from Melissa officinalis subsp. officinalis. AB - Luteolin 3'-O-beta-D-glucuronide (1) was isolated from the leaves of Melissa officinalis subsp. officinalis and characterized by spectroscopic analysis. PMID- 10727823 TI - Constituents of Ipomoea fistulosa leaves. AB - The isolation of flavonol glycosides 1-3 from the leaves of Ipomoea fistulosa is reported. PMID- 10727824 TI - Constituents of Caesalpinia pyramidalis. AB - Two new glycosyl phenylpropenoid acids, 4-O-beta-glucopyranosyloxy-(Z)-7 hydroxycinnamic acid (1) and 4-O-beta-glucopyranosyloxy-(Z)-8-hydroxycinnamic acid (2), besides lupeol and aghatisflavone, were isolated from the leaves of Caesalpinia pyramidalis. PMID- 10727825 TI - Uleine and demethoxyaspidospermine from the bark of Plumeria lancifolia. AB - The isolation of (+)-uleine (1) and (+)-demethoxyaspidospermine (2) from the bark of Plumeria lancifolia is reported along with (1)H- and (13)C-NMR data. PMID- 10727826 TI - Constituents of Bowdichia virgilioides. AB - The isolation and (13)C-NMR data of flavone derivatives from Bowdichia virgilioides roots are reported. PMID- 10727827 TI - Multicaulisin, a new Diels-Alder type adduct from Morus multicaulis. AB - The isolation of multicaulisin, a new Diels-Alder type adduct from Morus multicaulis roots is reported. PMID- 10727828 TI - Constituents of aromatic plants: I. Methyleugenol. AB - Methyleugenol (ME) is a natural constituent of the essential oils of a number of plants widely used in foodstuffs as flavouring agents. Its occurrence, routes of intake, and available information on toxicity, kinetics and metabolism are reviewed. In view of the carcinogenic potential of ME, the need to check its presence in food products with effective analytical methods is pointed out. PMID- 10727829 TI - Isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages. AB - This paper describes the isolation of porcine epidemic diarrhea (PED) virus in Vero and porcine cell cultures, and the influence of age on disease in experimental infection. PED virus was isolated from the small intestine of piglets inoculated with PED samples and cultured in Vero, porcine bladder and kidney cells propagated in collagen-coated tissue culture plates in maintenance medium (MM) containing trypsin. In porcine bladder and kidney cell cultures inoculated with isolated PED virus, cytopathic effects (CPE) including cell fusion were detected. Specific brilliant fluorescence was observed in the cytoplasm of these cells. Two- and 7-day old, and 2-, 4-, 8- and 12-week old specific pathogen-free (SPF) pigs were orally inoculated with PED virus isolated from an outbreak. All 2- and 7-day old pigs inoculated developed severe watery diarrhea from post-inoculation day (PID) 1 and died between PID 3 and 4. Although three of five 2-week old pigs developed diarrhea on PID 1-4, they eventually recovered. In the 4-week old group, three of five pigs had mild diarrhea for 1-2 days. None of the 8- and 12-week old pigs showed any clinical signs. Antibodies against PED virus were detected in all surviving pigs by virus neutralization (VN) test and immunofluorescence assay (IFA). Therefore, there is an age dependent resistance to pathogenic PED virus infection in pigs. PMID- 10727830 TI - Capture ELISA systems for the detection of bovine coronavirus-specific IgA and IgM antibodies in milk and serum. AB - Isotype-capture ELISAs for BCV-specific IgA and IgM were developed and tested on milk and serum samples from Swedish cattle. The capture ELISAs showed higher sensitivity than indirect ELISAs for detection of BCV-specific IgA and IgM. In the capture ELISAs the agreement between detection in milk and serum samples was 94% for IgA and 86% for IgM. The correlation between log(10) titres in milk and serum was r=0.82 (P<0.001) for IgA and 0.84 (P<0.001) for IgM. Milk seemed a better target than serum for diagnosing specific IgA at low levels. There was no variation in the isotype-specific BCV antibody titres between healthy quarters of the same udder, but subclinical mastitis was associated with higher levels of IgA antibodies and weak false IgM positive reactions in undiluted milk. Bovine IgA and IgM antibodies in milk and serum showed high stability towards freezing and thawing and storage at room temperature. The antibody responses to BCV were followed in milk and serum from six dairy cows and in serum from four calves for a period of 1 year after an outbreak of winter dysentery (WD). In this outbreak some animals became reinfected with BCV. The IgA and IgM capture ELISAs differentiated between primarily BCV infected and reinfected animals. In the primarily infected cattle, IgM antibodies were first detected in milk and serum four to nine days after the first WD symptoms observed, and were subsequently detected for at least 2-3 weeks. IgM was also detected in the reinfected cows, but mostly at lower levels and for a shorter period of time than in the primarily infected animals. In milk, however, the IgM response of the reinfected cows was detected for a longer period of time than in serum. Six months after the outbreak, IgA was still detected in both serum and milk of all six cows and also in serum of one calf. The reinfected cows showed higher and more long-lasting peak levels of IgA in milk and serum than the primarily infected cows, indicating boosting of the IgA response. PMID- 10727831 TI - Detection of Borna disease virus in a pregnant mare and her fetus. AB - A pregnant mare showing pyrexia, reduced appetite, ataxia and paresis was euthanized and examined for the presence of Borna disease virus (BDV). Her brain, showing multiple neuronal degeneration and necrosis with hemorrhage, and the histologically normal brain of the fetus were both positive for BDV RNA. The BDV nucleotide sequences were identical in the mare and fetus in the second open reading frame (ORF). This is the first report of the possible vertical transmission of BDV in a horse. PMID- 10727832 TI - Serological and demographic evidence for domestic dogs as a source of canine distemper virus infection for Serengeti wildlife. AB - Following an epidemic of canine distemper virus (CDV) in Serengeti lions in 1994, the role of domestic dogs in the epidemiology of the disease was investigated by serological and demographic analyses. From 1992 to 1994, data were collected from two domestic dog populations bordering the Serengeti National Park. Several lines of evidence indicated that patterns of CDV infection differed significantly between higher-density dog populations of Serengeti District to the west of the park and lower-density populations of Ngorongoro District to the south-east: (a) CDV age-seroprevalence patterns differed significantly between years in Ngorongoro District populations but not in Serengeti District populations; (b) CDV seropositive pups (<12 months of age) were detected in Ngorongoro District only in 1994, whereas a proportion of pups in Serengeti District were seropositive in each year of the study; (c) in Ngorongoro District, the proportion of deaths attributed to disease was significantly higher in 1994 than in 1993, whereas in Serengeti District, there was no significant difference in disease-related mortality between years; (d) in Ngorongoro District, significantly more CDV seronegative dogs than seropositive dogs died in 1994, whereas there was no difference in survival of CDV seropositives and seronegatives between years in Serengeti District. We concluded that, between 1992 and 1994, CDV persisted in higher-density dog populations of Serengeti District, but occurred only sporadically in lower-density Ngorongoro District populations. Data from Ngorongoro District are consistent with exposure of dogs to CDV in 1991 and 1994, but not in 1992 and 1993. These findings suggest that higher-density domestic dog populations to the west of the Serengeti National Park were a more likely source of CDV infection for wildlife during 1994 than lower-density pastoralist dogs to the south and east of the park. PMID- 10727833 TI - Aerosol challenge of calves with Haemophilus somnus and Mycoplasma dispar. AB - The aim of the study was to examine the ability of Haemophilus somnus and Mycoplasma dispar to induce pneumonia in healthy calves under conditions closely resembling the supposed natural way of infection, viz. by inhalation of aerosol droplets containing the microorganisms. The infections were investigated by recording clinical data, cytokine expression of peripheral blood cells and pathology. Twelve calves were included in the study: Three animals were exposed to H. somnus only, and two to M. dispar only, whereas five were challenged to M. dispar followed by exposure to H. somnus 11-14 days later. Also, one calf was exposed to M. dispar followed by exposure to a sterile saline solution 11 days later, and one calf was only exposed to a sterile saline solution. Just one animal, only challenged with H. somnus, developed a focal necrotizing pneumonia, from which H. somnus was isolated. Thus, the ability of H. somnus and M. dispar to act as primary pathogens under these conditions were minimal and inconsistent.However, a transient rise in body temperature, a marked granulocytosis and increased levels of interleukin-8 in peripheral blood after inoculation with H. somnus indicated a clear systemic response, probably as a consequence of the natural non-specific local and systemic defence mechanisms acting in healthy calves. PMID- 10727834 TI - Detection of Ureaplasma diversum in cattle using a newly developed PCR-based detection assay. AB - Ureaplasma diversum has been associated with different clinical manifestations including bovine vulvitis, endometritis, salpingitis, spontaneous abortion and infertility. Because the isolation of this ureaplasma from clinical samples is difficult, there is a need for improved detection methods. We developed a PCR assay based on amplification of a region of the gene encoding 16S rRNA. The specificity of the amplification was verified by sequence analysis. Female bovine vaginal swabs (n=168) were collected and the presence of U. diversum evaluated by both culture methods and by the PCR assay. Culture was positive for 60 samples (35.7%), and PCR-specific amplification was obtained for 89 samples (52.9%). These results indicated a high prevalence of U. diversum in the selected animals and the higher sensitivity of this PCR assay as compared to culture. PMID- 10727835 TI - Phylogeny of the Mycoplasma mycoides cluster as shown by sequencing of a putative membrane protein gene. AB - The Mycoplasma mycoides cluster is made of six species that are closely related both genetically and phenotypically. Two are of particular importance, M. mycoides subsp. mycoides SC causing contagious bovine pleuropneumonia and M. capricolum subsp. capripneumoniae causing contagious caprine pleuropneumonia. The sequences of a putative membrane protein gene and partial flanking open reading frames have been obtained from various strains in this cluster, including all reference strains. Sequence analysis showed this locus is present and fully conserved in all strains of M. mycoides subsp. mycoides SC isolated from geographically most distant places worldwide. In M. capricolum subsp. capripneumoniae polymorphism in this locus has been found at seven positions and revealed that they can be used as epidemiological markers. Conserved regions were used to define a primer pair that enables the amplification by PCR of two fragments 302 and 1298bp long, respectively. The 302bp long fragment contains an intergenic sequence that can be used for phylogenetic studies or for identification purposes. Parsimony analysis on an alignment of 49 DNA sequences show a subdivision of the M. mycoides cluster into two subgroups that is in accordance with results obtained by phenotypic methods. Two lineages exist within the capricolum subgroup, one of them clustering strains identified as M. capricolum subsp. capricolum, M. capricolum subsp. capricolum and M. sp Bovine Group 7. However M. capricolum subsp. capripneumoniae strains can readily be identified by three specific nucleotide positions or by sequencing the 1298bp long fragment. There is no clear subdivision within the mycoides subgroup, supporting the idea that M. mycoides subsp. mycoides LC and M. mycoides subsp. capri should not be separated into two subspecies. Mycoplasma mycoides subsp. mycoides SC strains can easily be distinguished as they bear an insertion sequence 15bp downstream from the stop codon of the membrane protein gene. PMID- 10727837 TI - Colonisation of rabbits with Staphylococcus aureus after experimental infection with high and low virulence strains. AB - Four groups of 12 rabbits each were inoculated in the nose with strains with suspected differences in virulence. In the two groups infected with strains from severe outbreaks, belonging to a rabbit-pathogenic biotype-phage type combination, 6-12 rabbits were found positive at the successive bacteriological samplings over a period of 28 days. In the two other groups, infected with strains obtained from rabbitries without a history of staphylococcosis, the number of Staphylococcus aureus positive animals quickly became negative but increased again after 1 week to 1-5 positive animals until the end of the experiment. Two rabbits in each group inoculated with a high virulence strain developed purulent skin lesions, while in the groups inoculated with low virulence strains, all animals remained clinically healthy. Results indicate that colonisation capacity is an important virulence determinant in rabbit staphylococcosis. PMID- 10727836 TI - Toxigenic Escherichia coli isolated from pigs in Argentina. AB - The presence of porcine toxigenic E. coli (ETEC, VTEC) in 28 piggeries (5% of total) of the central and northeast region of Argentina was studied for a better understanding of the epidemiology of porcine strains. Samples were taken by rectal swabs from healthy piglets and from those with diarrhoea, in addition to their dams. Between 5-10 colonies were isolated from each one of 223 animals sampled from 1992 to 1997. By using specific primers each strain was screened by PCR for VT1, VT2all, VT2e, STIa, and LTI toxin genes. Only strains positive for any of the toxins mentioned above were screened for STb. Their O serogroups were determined by agglutination. All of the above enterotoxins and verocytotoxins were found in E. coli isolated from the animals. The STIa gene was detected in E. coli isolated from 27/127 piglets with diarrhoea, in comparison with LTI (4/127 pigs). No toxin gene was amplified from E. coli isolated from either healthy piglets or their dams. When strains isolated from 48 piglets without diarrhoea but showing delayed growth were analysed by PCR, their toxin profile was determined to be VT1 (1/48 piglets), VT2all (5/48), STIa (1/48), LTI (3/48) and VT2e (3/48). Serogroup O64 prevailed among ETEC; O138 prevailed for ETEC/VTEC strains. This is the first extensive study regarding porcine toxigenic E. coli in Argentina and constitutes an important database for the implementation of prevention measures. PMID- 10727838 TI - The detection of Coxiella burnetii from ovine genital swabs, milk and fecal samples by the use of a single touchdown polymerase chain reaction. AB - The polymerase chain reaction (PCR), targeting the repetitive transposon-like region of Coxiella burnetii (Trans-PCR), was evaluated for its ability to detect directly C. burnetii in genital swabs, milk and fecal specimens of ewes. By using a combination of centrifugation step, DNA purification using Qiamp Tissue kit followed by Trans-PCR assay, the efficiency for detection of coxiella in ewes milk samples was further improved and one C. burnetii-cell could be detected in 1ml of milk. In addition, an effective, simple and rapid method to remove PCR inhibitory substances from fecal specimens by simply diluting the DNA template to 1:100 is described, which made the detection of one microorganism per mg of sample then possible. The results obtained from seropositive ewes proved that C. burnetii could also be detected in milk and fecal samples of naturally infected animals. PMID- 10727839 TI - Experimental models of porcine post-weaning colibacillosis and their relationship to post-weaning diarrhoea and digestive disorders as encountered in the field. AB - The aim of this study was to develop a reliable model system of porcine post weaning colibacillosis, and in doing so to assess the primary relationship of enterotoxigenic Escherichia coli to post-weaning diarrhoea and digestive disorders as encountered in the field. Six sequential experiments were carried out using 168 SPF piglets weaned into an optimal controlled environment at 28 days of age. The piglets were allocated to 23 treatment groups, 17 of which were inoculated either orally or intragastrically with enterotoxigenic strains of E. coli (LT+, STI+, STII+) possessing adhesive factors including K88 (F4). The piglets were challenged either once (Day 4 post-weaning) or on several days post weaning, with the challenge load for each inoculation varying from 10(8) to 10(12) CFU. Overall 14.5% of inoculated pigs developed severe illness and died: these had lesions in their digestive tracts typical of colibacillosis. Diarrhoea occurred on at least 1 day in 50% of inoculated pigs, but was transient (1.7 days on average), appeared very soon after challenge (sometimes within half a day), and was accompanied by signs of depression and low weight gain. Generally a prompt recovery then occurred. In the second 2 weeks post-inoculation daily weight gain reached the same level in most inoculated groups of pigs as in the uninoculated controls. Only a small number of pigs developed a chronic enteritis lasting several days, as is typically observed in field cases. Diarrhoea was more common in the piglets that were tested adhesive positive to the K88 fimbriae receptor, but the disorders were no more severe in these animals. The response of all pigs depended primarily on the inoculum used, and especially on the challenge load. Although enterotoxigenic E. coli are clearly important in the aetiology of post-weaning diarrhoea, other factors are also required for the production of the chronic post-weaning digestive disorders and ill-thrift that are commonly encountered in commercial piggeries. PMID- 10727840 TI - Differentiation between high and low virulence Staphylococcus aureus strains from rabbits by randomly amplified polymorphic DNA (RAPD) analysis. AB - Randomly Amplified Polymorphic DNA (RAPD) typing was performed on 53 rabbit Staphylococcus aureus strains. Twenty-three strains isolated in 13 different rabbitries with chronic problems of staphylococcosis, showed the same RAPD banding pattern. Twenty of these strains belonged to the 'mixed CV-C' biotype and to the phage-type 3A/3C/55/71, previously described to be highly virulent in rabbits, and three strains belonged to other biotypes or phage-types. None of the strains isolated from rabbitries without chronic problems of staphylococcosis showed this specific RAPD pattern. RAPD analysis can be used as a rapid and reliable test method to differentiate between the characteristic genotype corresponding to high virulence and other S. aureus strains from rabbits. This is useful for the diagnosis and prevention of the introduction of these highly virulent strains in industrial rabbitries. PMID- 10727841 TI - Antibody response to toxic shock syndrome toxin-1 of Staphylococcus aureus in dairy cows. AB - Antibody response to toxic shock syndrome toxin-1 (TSST-1) of Staphylococcus aureus in dairy cows was examined by enzyme linked immunosorbent assay (ELISA). Serum antibody to TSST-1 was not detected in 39 (76.5%) of 51 calves, which were 1-6 months of age. In contrast, TSST-1 antibody was demonstrated in 1728 (72.6%) of 2380 lactating cows housed on 36 dairy farms. The ELISA values of antibody ranged from 0.2 to 3.0 OD and presented a distribution with the peak at 1.6 OD. The mean ELISA value differed between farms, and it increased slightly along with parturient history. Somatic cell counts of milk from 174 lactating cows was compared with TSST-1 antibody and tst1,000,000 cells per ml. The mean ELISA values in milk were lower than those of sera, but they rose as somatic cells increased. The tst gene of S. aureus detected in 76.0-86.2% of the milk samples containing somatic cells > 500,000 cells per ml, a level which indicates mastitis. The data suggests that many lactating cows may be infected by TSST-1- producing S. aureus. PMID- 10727842 TI - Detection of type III secretion system genes in animal isolates of Bordetella bronchiseptica. AB - A cosmid clone bank of Bordetella bronchiseptica genomic DNA was screened for the presence of type III secretion (TTS) genes using a probe derived from the TTS system genes of Ralstonia solanacearum. A 3.35kb PstI fragment, sub-cloned from a hybridising cosmid clone, was sequenced and found to contain a 97bp overlap with the previously reported B. bronchiseptica bscIJKLNO TTS gene cluster. DNA and predicted protein homology analysis suggests that a bscPQRST cluster lies immediately downstream of bscIJKLNO. A PCR amplification assay indicated that the bscT locus was present in 27 B. bronchiseptica animal isolates tested (100%). Dot blot DNA hybridisation using probes for bscT and bscP confirmed the presence of these loci in six canine isolates associated with a variety of clinical signs. Although TTS has been implicated in the pathogenicity of B. bronchiseptica, it is likely that different clinical manifestations may be due to variations in gene expression or host factors, rather than the absence or presence of TTS genes. PMID- 10727844 TI - Heparan N-sulfatase gene: two novel mutations and transient expression of 15 defects. AB - Sanfilippo syndrome type A or mucopolysaccharidosis IIIA (MPS IIIA) results from the deficiency of the enzyme heparan N-sulfatase (NS, EC 3.10.1.1), required for the degradation of heparan sulfate. Molecular defects of 24 Italian MPS IIIA patients were recently reported by our group. We report here two novel mutations: 1040insT and Q365X and the expression studies on 15 of the identified defects. Transient expression of COS cells by cDNA mutagenized to correspond to heparan N sulfatase mutations Y40N, A44T, 166delG, G122R, P128L, L146P, R150Q, D179N, R182C, R206P, P227R, 1040insT, 1093insG, E369K, R377C did not yield active enzyme, demonstrating the deleterious nature of the mutations. Western blot analysis and metabolic labeling experiments revealed, for cells transfected with wild-type enzyme, a precursor 62-kDa form and a mature 56-kDa form. Western blot resulted, for 11 mutations, in the presence of both forms, indicating a normal maturation of the mutant enzyme. Western blot, metabolic labeling and immunofluorescence experiments suggested, for mutations 166delG, L146P, 1040insT and 1093insG, an increased degradation of the mutant enzymes. PMID- 10727845 TI - The non-oxidative degradation of ascorbic acid at physiological conditions. AB - The degradation of L-ascorbate (AsA) and its primary oxidation products, L dehydroascorbate (DHA) and 2,3-L-diketogulonate (2, 3-DKG) were studied under physiological conditions. Analysis determined that L-erythrulose (ERU) and oxalate were the primary degradation products of ASA regardless of which compound was used as the starting material. The identification of ERU was determined by proton decoupled (13)C-nuclear magnetic resonance spectroscopy, and was quantified by high performance liquid chromatography, and enzymatic analysis. The molar yield of ERU from 2,3-DKG at pH 7.0 37 degrees C and limiting O(2)97%. This novel ketose product of AsA degradation, was additionally qualitatively identified by gas-liquid chromatography, and by thin layer chromatography. ERU is an extremely reactive ketose, which rapidly glycates and crosslinks proteins, and therefore may mediate the AsA-dependent modification of protein (ascorbylation) seen in vitro, and also proposed to occur in vivo in human lens during diabetic and age-onset cataract formation. PMID- 10727846 TI - Generation of rat eosinophils by recombinant rat interleukin-5 in vitro and in vivo. AB - The addition of recombinant rat interleukin-5 (IL-5), which was purified from the hemolymph of silkworm Bombyx mori larvae infected with IL-5-expressing recombinant virus, to cultures of rat bone marrow cells resulted in an increase in the number of Luxol-fast-blue staining eosinophils in a time- and concentration-dependent manner. After 6 days culture with 100 pM recombinant rat IL-5, more than 90% of the bone marrow cells were eosinophil. The contents of major basic protein (MBP) in the bone marrow cells determined by Western blot analysis using a polyclonal antibody to rat MBP were also increased by recombinant rat IL-5 (100 pM). Furthermore, intravenous injections of recombinant rat IL-5 twice a day for six consecutive days increased the population of eosinophils in peripheral blood cells and in bone marrow cells. These findings indicate that rat IL-5 induces terminal differentiation and proliferation of progenitor cells to mature eosinophils in rats. PMID- 10727847 TI - Alpha-fetoprotein-derived antiestrotrophic octapeptide. AB - Alpha-fetoprotein (AFP) is a major serum protein produced during fetal development. Experimental findings suggest that AFP has antiestrotrophic activity and that it can be developed as a therapeutic agent to treat existing estrogen dependent breast cancer or to prevent premalignant foci from developing into breast cancer. The antiestrotrophic activity of AFP was reported to be localized to a peptide consisting of amino acids 447-480, a 34-mer peptide termed P447. A series of parsings and substitutions of amino acids in the P447 sequence was intended to identify the shortest analog which retained antiestrotrophic activity. Peptides related to P447 were generated using solid phase peptide synthesis. Several shorter peptides, including an 8-mer called P472-2 (amino acids 472-479, peptide sequence EMTPVNPG), retained activity, whereas peptides shorter than eight amino acid residues were inactive. The dose-related antiestrotrophic activity of AFP-derived peptides was determined in an immature mouse uterine growth assay that measures their ability to inhibit estradiol stimulated uterine growth. In this assay, the maximal inhibitory activities exhibited by peptide P472-2 (49%), by peptide P447 (45%), and by intact AFP (35 45%) were comparable. The octapeptide P472-2 was also active against estradiol stimulated growth of T47D human breast cancer cells in culture. These data suggest that peptide P472-2 is the minimal sequence in AFP, which retains the antiestrotrophic activity found with the full-length molecule. The synthetic nature and defined structure of this 8-mer peptide suggest that it can be developed into a new drug which opposes the action of estrogen, perhaps including the promotional effects of estradiol in the development of human breast cancer. PMID- 10727848 TI - Free radical induced inactivation of creatine kinase: sites of interaction, protection, and recovery. AB - The study aims at a clarification of the oxidative damage of creatine kinase isoenzymes by X-ray-induced water radiolysis. The radical species generated by this method (under appropriate conditions) are similar to those discussed in the context of mitochondrial energy metabolism. The decay of the enzyme activity is accompanied by a strong decrease of the number of accessible SH groups and by a reduction of the endogenous tryptophan fluorescence. Free radical effects are diminished if irradiation is carried out in the presence of 2-mercaptoethanol. Partial recovery of the activity (repair) is observed if 2-mercaptoethanol is added after irradiation. The experiments suggest a twofold importance of thiol reagents (RSH): to reduce the concentration of free radicals by scavenger reactions and to modify the inactivation mechanism in such a way that efficient repair of enzyme damage may be achieved. Cysteine 282 of MM-CK (Cys-278 in the case of Mi-CK) seems to play a crucial role in this respect. Blockage of the SH group of cysteine 282 by oxidized glutathione effectively protects the enzyme against inactivation by NO(*)(2) radicals. In the absence of nitrogen dioxide and of thiol reagents, however, inactivation seems to proceed via a less specific mechanism involving additional targets of the enzyme. PMID- 10727849 TI - Abnormal phenotype of in vitro dermal fibroblasts from patients with Pseudoxanthoma elasticum (PXE). AB - Pseudoxanthoma elasticum (PXE) is a genetic connective tissue disease, whose gene and pathogenesis are still unknown. Dermal fibroblasts from patients affected by PXE have been compared in vitro with fibroblasts taken from sex and age-matched normal individuals. Cells were grown and investigated in monolayer, into three dimensional collagen gels and in suspension. Compared with normal cells, PXE fibroblasts cultured in monolayer entered more rapidly within the S phase and exhibited an increased proliferation index; on the contrary, similarly to normal fibroblasts, PXE cells did not grow in suspension. Furthermore, compared with normal fibroblasts, PXE cells exhibited lower efficiency in retracting collagen type I lattices and lower adhesion properties to collagen type I and to plasma fibronectin. This behavior was associated with higher expression of integrin subunits alpha2, alpha5, alphav, whereas beta1 subunit as well as alpha2beta1 and alpha5beta1 integrin expression was lower than in controls. Compared to controls, PXE fibroblasts had higher CAM protein expression in accordance with their high tendency to form cellular aggregates, when kept in suspension. The demonstration that PXE fibroblasts have altered cell-cell and cell-matrix interactions, associated with modified proliferation capabilities, is consistent with the hypothesis that the gene responsible for PXE might have a broad regulatory role on the cellular machinery. PMID- 10727850 TI - Identification of a novel human member of the DEAD box protein family. AB - The cDNA library of human pancreatic islets was screened with sera from patients with insulin-dependent diabetes mellitus (IDDM). From the library screening, we isolated a novel cDNA, RNA helicase-like protein (RHELP), which exhibited strong sequence homology to p68 RNA helicase, a prototypic member of the DEAD (Asp-Glu Ala-Asp) box protein family. Sequence analysis of the cDNA revealed that RHELP contained DEAD sequence motif and other conserved motifs of the DEAD box protein family, indicating that RHELP is a new member of this family. DEAD box-containing proteins are involved in the RNA processing, ribosome assembly, spermatogenesis, embryogenesis, and cell growth and division. RHELP showed 42% and 44% amino acid sequence identity to human p68 RNA helicase and yeast DBP2 RNA helicase, respectively, among the DEAD box protein family. Northern blot analysis revealed that RHELP is expressed in most tissues including the liver, lung, tonsil, thymus, and muscle in addition to the pancreatic islets. In vivo or in vitro functions of RHELP as a putative RNA helicase and its potential role as a diabetic autoantigen need to be further investigated. PMID- 10727851 TI - Loss of fibula in mice overexpressing Hoxc11. AB - This study demonstrates severe malformations of the appendicular skeleton in mice overexpressing Hoxc11. Consistent with the endogenous expression pattern, the most conspicuous defect in Hoxc11 overexpressing neonates is aplasia/hypoplasia of the fibula. This is preceded at day 15.5 of embryonic development by marked reduction of chondrocyte proliferation, lack of PTHR expressing prehypertrophic cells, and the absence of hypertrophic and calcifying chondrocytes. Combined with the lack of an overt phenotype in the majority of Hoxc11 overexpressing embryos at day 13.5, the data suggest inhibition of chondrocyte differentiation during the elongation phase of the fibula bone as a primary effect of elevated Hoxc11 expression. This interpretation is further corroborated by Hoxc11 reporter gene expression in the joint areas at embryonic day 15.5, suggesting an involvement of the periarticular perichondrium in generating the mutant phenotype. PMID- 10727852 TI - Enhancer-independent promoter activity of the mouse alphaB-crystallin/small heat shock protein gene in the lens and cornea of transgenic mice. AB - The alphaB-crystallin/small heat shock protein gene is expressed very highly in the mouse eye lens and to a lesser extent in many other nonocular tissues, including the heart, skeletal muscle and brain. Previously we showed in transgenic mice that lens-specific alphaB-crystallin promoter activity is directed by a proximal promoter fragment (-164/+44) and that non-lens promoter activity depends on an upstream enhancer (-427/-259) composed of at least 5 cis control elements. Here we have used truncated alphaB-crystallin promoter-CAT transgenes to test by biphasic CAT assays and/or histochemistry for specific expression in the cornea and lens. Deletion either of 87 bp (-427/-340) from the 5' end of the alphaB-crystallin enhancer or of the whole enhancer (-427/-258) abolished alphaB-crystallin promoter activity in all tissues except the lens and corneal epithelium when examined by the biphasic CAT assay in 4-5-week-old transgenic mice. These truncations also lowered promoter strength in the lens. The -426/+44-CAT, -339/+44-CAT and -164/+44-CAT (previously thought to be lens specific in transgenic mice) transgenes were all expressed in the 4-6-week-old corneal epithelium when examined histochemically. Immunohistochemical staining confirmed the presence of endogenous alphaB-crystallin in the mature corneal epithelial cells. CAT gene expression driven by the alphaB-crystallin promoter with or without the enhancer was evident in the embryonic and 4-6-week-old lens. By contrast, activity of the alphaB-crystallin promoter/enhancer-CAT transgene was not detectable in the corneal epithelium before birth. Taken together, these results indicate that the intact enhancer of the alphaB-crystallin/small heat shock protein gene is required for promoter activity in all tissues tested except the lens and cornea. PMID- 10727853 TI - A late wave of melanoblast differentiation and rostrocaudal migration revealed in patch and rump-white embryos. AB - Melanocytes originate from a small number of precursors localized either side of the dorsal midline. The tyrosine kinase receptor Kit and its ligand Mgf (Steel Factor) are essential for melanoblast survival and proliferation during their migration from the neural crest. Inappropriate Kit expression in the dermatome and dermis of patch and rump-white mouse mutants apparently sequester Mgf, inhibiting melanoblast dispersal. Using a reporter transgene Dct-lacZ, extensive regions of the mutant trunks appear devoid of melanoblasts between E12.5 and E15.5, a much larger area than seen in mutant adults. Melanoblast recolonization of the underpopulated lumbar regions occurs very rapidly by E16.5 giving rise to patterns consistent with those observed in adults. The mutations permit observation of aspects of melanoblast development that are not seen, or are obscured, in normal embryos. PMID- 10727854 TI - Identification of a mouse germ cell-less homologue with conserved activity in Drosophila. AB - Drosophila Germ cell-less (Gcl) has previously been shown to be important in early events during the formation of pole cells, which are the germ cell precursors in the fly. We have isolated a 524 amino acid mouse gene with 32% identity and 49% similarity to Drosophila gcl, termed mgcl-1. Like Drosophila Gcl, mGcl-1 localizes to the nuclear envelope. Ectopic expression of mgcl-1 in Drosophila rescues the gcl-null phenotype, indicating that mGcl-1 is a functional homologue of Gcl. mgcl-1 maps to chromosome 6 at 47.3 cM, and is expressed at low levels at all embryonic stages examined from 8.5 to 18.5 d.p.c. as well as in many adult tissues. Different from Drosophila gcl, mgcl-1 is not highly expressed at the time the primordial germ cells appear in the mouse, but high mgcl-1 expression is found in selected mouse adult male germ cells. The differences in these expression patterns in light of conserved activity between the two genes is discussed. PMID- 10727855 TI - minidiscs encodes a putative amino acid transporter subunit required non autonomously for imaginal cell proliferation. AB - Drosophila minidiscs mutant larvae have smaller imaginal discs than wild-type larvae. However, transplantation experiments have revealed that minidiscs mutant imaginal discs can grow if cultured in non-mutant hosts. These data suggest that minidiscs is required in one or more non-imaginal tissues for synthesis and/or secretion of a diffusible factor that stimulates imaginal cell proliferation. The 2. 3 kb minidiscs transcript accumulates in the larval fat body and encodes a protein containing 12 putative membrane spanning domains that is similar in sequence to amino acid transporter subunits from other eukaryotes, including humans. We propose that in response to amino acid uptake by the transporter encoded by minidiscs, the fat body secretes a diffusible factor required for imaginal disc proliferation. PMID- 10727856 TI - The expression of dopamine beta-hydroxylase, tyrosine hydroxylase, and Phox2 transcription factors in sympathetic neurons: evidence for common regulation during noradrenergic induction and diverging regulation later in development. AB - During differentiation of sympathetic neurons in chick embryos, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) mRNAs become detectable during the same developmental period and are both induced by BMP 4. Later during sympathetic ganglion development, DBH is detectable in TH-positive and -negative cells. Moreover, BMPs reduce DBH mRNA in cultures of sympathetic neurons while leaving TH unaffected. The data provide evidence for a common regulation of TH and DBH early during sympathetic neuron differentiation and indicate that BMPs promote their initial expression but not the maintenance during later development. The time course of Phox2a and 2b expression suggests an evolutionary conserved role in noradrenergic induction. In addition, Phox2a, Phox2b, and c-ret may be involved in the differentiation of cholinergic sympathetic neurons. PMID- 10727857 TI - Analysis of Drosophila salivary gland, epidermis and CNS development suggests an additional function of brinker in anterior-posterior cell fate specification. AB - Salivary glands are simple structured organs which can serve as a model system in the study of organogenesis. Following a large EMS mutagenesis we have identified a number of genes required for normal salivary gland development. Mutations in the locus small salivary glands-1 (ssg-1) lead to a drastic reduction in the size of the salivary glands. The gene ssg-1 was cloned and subsequent sequence and genetic analysis showed identity to the recently published gene brinker. The salivary gland placode in brinker mutants appears reduced along both the anterior posterior and dorso-ventral axis. Analysis of the brinker cuticle phenotype revealed a similar loss of anterior-posterior as well as lateral cell fates. The abdominal ventral denticle belts show a reduced number of setae in the first denticle row. Furthermore, we observed a preferential loss of lateral neuroblasts in the anterior parasegment. Together, these phenotypes suggest that brinker not only plays a role in dorso-ventral but also in anterior-posterior axis patterning. PMID- 10727858 TI - Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4. AB - Differentiation of mouse embryonic stem (ES) cells via embryoid bodies was established as a suitable model to study development in vitro. Here, we show that differentiation of ES cells in vitro into chondrocytes can be modulated by members of the transforming growth factor-beta family (TGF-beta(1), BMP-2 and 4). ES cell differentiation into chondrocytes was characterized by the appearance of Alcian blue-stained areas and the expression of cartilage-associated genes and proteins. Different stages of cartilage differentiation could be distinguished according to the expression pattern of the transcription factor scleraxis, and the cartilage matrix protein collagen II. The number of Alcian-blue-stained areas decreased slightly after application of TGF-beta(1), whereas BMP-2 or -4 induced chondrogenic differentiation. The inducing effect of BMP-2 was found to be dependent on the time of application, consistent with its role to recruit precursor cells to the chondrogenic fate. PMID- 10727859 TI - Activity-regulated, cytoskeleton-associated protein (Arc) is essential for visceral endoderm organization during early embryogenesis. AB - Activity-regulated, cytoskeleton-associated protein (Arc) was first identified as an immediate-early gene regulated by synaptic activity. We have studied its functional role in vivo using a gene-targeting approach. We found that Arc is encoded by a single exon, and Arc mRNA is ubiquitously expressed in early mouse embryos. Homozygous Arc mutants are severely growth-retarded, fail to gastrulate and subsequently die before day 8.5 of embryogenesis. Further analysis revealed severe disorganization of visceral endoderm formation, and total separation and ectopic location of embryonic and extraembryonic structure. These findings demonstrate that Arc function is essential for early embryo development and patterning in mice, and support the hypothesis that signaling from visceral endoderm is essential for normal patterning of the extraembryonic and embryonic structure. PMID- 10727860 TI - Coincidence of otx2 and BMP4 signaling correlates with Xenopus cement gland formation. AB - We previously showed that otx2 activates ectopic formation of the Xenopus cement gland only in ventrolateral ectoderm, defining a region of the embryo permissive for cement gland formation. In this paper, we explore the molecular identity of this permissive area. One candidate permissive factor is BMP4, whose putative graded inhibition by factors such as noggin has been proposed to activate both cement gland and neural fates. Several lines of evidence are presented to suggest that BMP signaling and otx2 work together to activate cement gland formation. First, BMP4 is highly expressed in the cement gland primordium together with otx2. Second, cement gland formation in isolated ectoderm is always accompanied by coexpression of otx2 and BMP4 RNA, whether cement gland is induced by otx2 or by the BMP protein inhibitor noggin. Third, BMP signaling can modulate otx2 activity, such that increasing BMP signaling preferentially inhibits neural induction by otx2, while decreasing BMP signaling prevents cement gland formation. In addition, we show that a hormone-inducible otx2 activates both ectopic neural and cement gland formation within the cement gland permissive region, in a pattern reminiscent of that found in the embryo. We discuss this observation in view of a model that BMP4 and otx2 work together to promote cement gland formation. PMID- 10727861 TI - Xenopus frizzled 7 can act in canonical and non-canonical Wnt signaling pathways: implications on early patterning and morphogenesis. AB - Here we report the cloning of a Xenopus frizzled transmembrane receptor, Xfz7, and describe its expression pattern during early embryogenesis. Xfz7 mRNA is provided maternally and zygotic transcription peaks in gastrula stages. At that time, transcripts are preferentially localized to the marginal zone and become restricted to distinct regions of the tadpoles in tailbud stages. Overexpression of Xfz7 in embryos perturbs the morphogenesis of trunk and tail, blocks convergence-extension movements in animal caps induced with activin and dorsal lip explants and decreases cadherin-mediated cell adhesion. Xfz7 can interact specifically with Xwnt-8b and signal in the canonical, dorsalizing Wnt pathway. Overexpression of Xfz7 does not trigger the Wnt-1-type pathway but acts in a non canonical Wnt or morphogenetic-effector pathway involving the activation of protein kinase C (PKC). Xfz7 seems to be involved in different aspects of Wnt signaling during the course of embryogenesis. PMID- 10727862 TI - Fibronectin matrix composition and organization can regulate cell migration during amphibian development. AB - Fibronectin (FN) is an adhesive extracellular matrix component that is essential for vertebrate development. It forms a fibrillar matrix at the cell surface which controls cell morphology, migration, proliferation, and other important cellular processes. To address specific functions of FN matrix structure during early vertebrate development, we introduced normal and mutant recombinant FNs (recFNs) into the blastocoel cavity of embryos of the amphibian Pleurodeles waltl. Here we show that a native recFN FN(A-B-) as well as recFNs with specific mutations in the cell-binding domain, FN(RGD-) and FN(syn-), or in a FN-binding region, FNDeltaIII(1), are assembled into fibrillar matrix. A recFN (FNDeltaIII(1-7)) that forms a structurally distinct matrix in cultured cells was assembled into aggregates at the cell periphery and was able to inhibit assembly of endogenous amphibian FN matrix in a dose-dependent manner. Cell adhesion, spreading, and migration were perturbed in vitro and in vivo on chimeric matrices containing FN(RGD-), FN(syn-), or FNDeltaIII(1-7) co-assembled with amphibian FN. Developmentally, this perturbation resulted in defects in mesoderm patterning and inhibition of gastrulation. These results indicate that FN matrix fibrillar structure and composition are important determinants of cell adhesion and migration during development. PMID- 10727863 TI - The Drosophila tumor suppressor expanded regulates growth, apoptosis, and patterning during development. AB - The Drosophila expanded (ex) gene encodes a protein thought to play a role in signaling at apical junctions of epithelial cells. Previous studies have characterized this gene as a tumor suppressor involved in regulating the growth of a subset of Drosophila imaginal discs (Boedigheimer, M., Laughon, A., 1993. expanded: a gene involved in the control of cell proliferation in imaginal discs, Development 118, 1291-1301); although ex negatively regulates cell proliferation in the developing wing, it appeared to have a conflicting role in the eye. In contrast, our analysis of the loss-of-function phenotype indicates that ex does, in fact, regulate growth in the eye. We also show that this gene plays a role in patterning of the eye, mainly at the level of planar polarity. Our studies further demonstrate that, contrary to what was expected based on loss-of-function data, the tissue reduction phenotypes resulting from Ex overexpression are attributable to the induction of apoptotic cell death. Taken together, our data suggest that Ex is a versatile molecule that plays a role in most of the processes that govern disc development. PMID- 10727864 TI - Lissencephaly associated mutations suggest a requirement for the PAFAH1B heterotrimeric complex in brain development. AB - Human brain malformations, such as Miller-Dieker syndrome (MDS) or isolated lissencephaly sequence (ILS) may result from abnormal neuronal migration during brain development. MDS and ILS patients have a hemizygous deletion or mutation in the LIS1 gene (PAFAH1B1), therefore, the LIS1 encoded protein (Lis1) may play a role in neuronal migration. Lis1 is a subunit of a brain platelet-activating factor acetylhydrolase (PAFAH1B) where it forms a heterotrimeric complex with two hydrolase subunits, referred to as 29 kDa (PAFAH1B3) and 30 kDa (PAFAH1B2). In order to determine whether this heterotrimer is required for the developmental functions of PAFAH1B, we examined the binding properties of 29 and 30 kDa subunits to mutant Lis1 proteins. The results defined the critical regions of Lis1 for PAFAH1B complex formation and demonstrated that all human LIS1 mutations examined resulted in abolished or reduced capacity of Lis1 to interact with the 29 and 30 kDa subunits, suggesting that the PAFAH1B complex participates in the process of neuronal migration. PMID- 10727865 TI - Cloning and expression pattern of a Xenopus pronephros-specific gene, XSMP-30. AB - The first step in kidney development is the formation of the pronephros which is derived from mesoderm. Xenopus is an appropriate model to study this process since the pronephros can be efficiently induced in animal cap explants by treatment with activin and retinoic acid (RA). Using this in vitro system, we isolated a Xenopus homologue of SMP-30 (Senescence marker protein-30), which is a Ca(2+)-binding protein that is highly conserved in vertebrates. This gene, termed XSMP-30, was found to be selectively expressed in pronephric tubules from the late tadpole stage, by whole mount in situ hybridization. Furthermore XSMP-30 was expressed in animal caps treated with both activin and RA, a condition in which the pronephros is formed in vitro. These data indicate that XSMP-30 is a specific marker for the pronephros. PMID- 10727866 TI - Oracle, a novel PDZ-LIM domain protein expressed in heart and skeletal muscle. AB - In order to identify novel genes enriched in adult heart, we performed a subtractive hybridization for genes expressed in mouse heart but not in skeletal muscle. We identified two alternative splicing variants of a novel PDZ-LIM domain protein, which we named Oracle. Both variants contain a PDZ domain at the amino terminus and three LIM domains at the carboxy-terminus. Highest homology of Oracle was found with the human and rat enigma proteins in the PDZ domain (62 and 61%, respectively) and in the LIM domains (60 and 69%, respectively). By Northern hybridization analysis, we showed that expression is highest in adult mouse heart, low in skeletal muscle and undetectable in other adult mouse tissues. In situ hybridization in mouse embryos confirmed and extended these data by showing high expression of Oracle mRNA in atrial and ventricular myocardial cells from E8.5. From E9.5 low expression of Oracle mRNA was detectable in myotomes. These data suggest a role for Oracle in the early development and function of heart and skeletal muscle. PMID- 10727867 TI - The expression pattern of a novel Deltex homologue during chicken embryogenesis. AB - We isolated a partial cDNA encoding a novel chicken homologue of human Deltex (DTX1), a member of the Notch signaling pathway. The cDtx2 sequence showed higher homology to KIAA0937 protein (92% identical) than to DTX1 (68% identical). cDtx2 is expressed widely in the epiblast at stage 4. Later in development it is expressed in many neural and sensory structures, such as neural tube, migrating neural crest cells, epidermal placodes, cranial ganglia, and the optic and otic vesicles. PMID- 10727868 TI - Expression of CRYP-alpha, LAR, PTP-delta, and PTP-rho in the developing Xenopus visual system. AB - Receptor protein tyrosine phosphatases (RPTPs), are involved in axon outgrowth and guidance not only in the Drosophila visual system (Garrity et al., 1999. Neuron 22, 707-717) but also in the developing vertebrate retina (Ledig et al., 1999a. J. Cell Biol. 147, 375-388). We have cloned a variety of Xenopus RPTPs, including four RPTPs expressed in the developing visual system (LAR, PTP-delta, CRYP-alpha and PTP-rho). These four RPTPs are transcribed in the developing optic vesicle during differentiation and in overlapping but distinct patterns in the developing retina during retinal layer formation. LAR, PTP-delta, and CRYP-alpha are also expressed in retinal ganglion cells during axonogenesis and during axon guidance from the retina to the optic tectum. PMID- 10727869 TI - Differential expression of retinoic acid-inducible (Stra) genes during mouse placentation. AB - Several retinoid binding proteins and nuclear receptors are specifically expressed in murine placenta. However, little is known about molecular events and target genes regulated by retinoids during placentation. Here, we report that several retinoic acid-inducible (Stra) genes, originally isolated by a differential screening procedure, exhibit specific expression patterns in mouse placental tissues. Three Stra genes, including the ephrinB1 receptor tyrosine kinase ligand, are prominently expressed in the regions of exchanges between maternal and embryonic circulations, i.e. the yolk sac and/or the labyrinthine zone of the mature placenta. The Meis2 homeobox gene appears to be specifically expressed in maternally-derived cell populations. Three other Stra genes, including the AP-2-related gene AP-2gamma, are differentially expressed in the trophoblastic cell lineage. Thus, retinoids may regulate various signaling pathways in specific placental cell-types. PMID- 10727870 TI - Expression of PTTG and prc1 genes during telencephalic neurogenesis. AB - We present the first time/space analysis using in situ hybridization for PTTG and prc1 genes during development of the mouse telencephalon. During the stages E11.5 E13.5 PTTG and prc1 are expressed in most tissues of the embryo. Within the telencephalon, PTTG and prc1 are found exclusively inside of the ventricular zone (VZ). The intensity of the expression of both genes in the ventricular zone reaches its peak by E15.5. Expression starts to decrease by E18.5, it is still visible at least up to P2 and not detectable in the adult brains. Expression of the prc1 gene, but not that of the PTTG, is also found in the mitoticaly active cells outside of the VZ within the telencephalon. Most of the cells expressing the PTTG gene were found in the lower part of the ventricular zone suggesting that the level of PTTG mRNA is regulated during different phases of the mitotic cycle. PMID- 10727871 TI - mRNA localization patterns in zebrafish oocytes. AB - In both invertebrate and vertebrate systems, the localization of maternal mRNAs is a common mechanism used to influence developmental processes, including the establishment of the dorsal/ventral axis, anterior/posterior axis, and the germ line (for review, see Bashirullah et al., 1998. Annu. Rev. Biochem. 67, 335-394). While the existence of localized maternal mRNAs has been reported in the zebrafish, Danio rerio, the precise localization patterns of these molecules during oogenesis has not been determined. In this study, in situ hybridization experiments were performed on zebrafish ovaries and activated eggs to examine different mRNA localization patterns. The results establish that while some maternal mRNAs remain ubiquitously distributed throughout the oocyte, other mRNAs follow specific localization patterns, including localization to the animal pole, localization to the vegetal pole, and cortical localization. The animal/vegetal axis is first apparent in stage II oocytes when the earliest mRNA localization is seen. Unique patterns of localization are seen in mature eggs as well. Some mRNAs maintain their oocyte localization patterns, while others localize upon egg activation (fertilization). PMID- 10727872 TI - Sequence, expression, and location of zebrafish frizzled 10. AB - Members of the frizzled gene family encode seven-pass transmembrane proteins that function in the interpretation and reception of Wnt-mediated cell-cell communication events. To investigate frizzled function in early zebrafish development, we isolated the maternally contributed frizzled 10 (fz10) gene and localized it to linkage group 8 using radiation hybrid mapping. The cloned zebrafish fz10 is closely related to the fz10 group from other organisms. Zygotic expression of fz10 is observed in the posterior tail mesenchyme, dorsal neural tube, and different parts of the brain. PMID- 10727873 TI - On the use of partial AUC as an early exposure metric. PMID- 10727874 TI - Influence of lipids on the mannitol flux during transdermal iontophoresis in vitro. AB - The aim of the present study was to evaluate the influence of the lipids EPC (L alpha-phosphatidylcholine, egg lecithin), DSPC (distearoylphosphatidylcholine), and SA (stearylamine) on the iontophoretic mannitol transport through human skin in vitro. The skin was pretreated with 1 mM lipid suspension with ethanol (32%) for 24 h prior to the iontophoretic experiment with mannitol. In addition, the penetration of fluorescent lipids into the epidermis during the pretreatment was studied by confocal laser scanning microscopy (CLSM). The results of the present study show that pretreatment of the skin with zwitterionic EPC increases the iontophoretic transdermal mannitol flux about three-fold compared to iontophoretic control without pretreatment. However, skin pretreatment with another zwitterionic phospholipid, DSPC, did not influence the iontophoretic flux of mannitol. In contrast, pretreatment of the skin with cationic SA decreased the iontophoretic mannitol flow from the anode. It is concluded that EPC works as a penetration enhancer further increasing the transdermal mannitol flux during iontophoresis. In contrast, the cationic stearylamine changes the charge of the skin, thus leading to decreased electroosmosis and decreased mannitol flux. Hence, the effects of stearylamine are assumed to be mediated by the alterations in the charge of the stratum corneum structures, while EPC is suggested to decrease the permeability barrier of the skin. PMID- 10727875 TI - Melt pelletisation of a hygroscopic drug in a high shear mixer. Part 2. Mutual compensation of influence variables. AB - The process of melt pelletisation in a Diosna P10 high shear mixer was examined for sodium valproate and glycerol monostearate. The effects of binder concentration, impeller speed and massing time on mean granule size, size distribution and liquid saturation were investigated. Spherical pellets of almost similar size and size distribution were obtained after 20 min of massing time, with a binder content from 3.1 to 14.1% w/w by adjusting the impeller speed. Granule growth was observed at low levels of binder concentration and liquid saturation (<80%) which is untypical for melt granulation. The liquid saturation seemed to have no major influence on the final pellet size. Additional, mutually compensating effects on granule growth were found to be impeller speed and massing time for a fixed binder concentration. Low levels of both, binder concentration and impeller speed, allowed for good control of the process. The amount of water adsorbed by the hygroscopic drug was found to accelerate granule growth. PMID- 10727876 TI - Development of local patches containing melilot extract and ex vivo-in vivo evaluation of skin permeation. AB - Melilot extract could be effective in treating localised varicose syndrome or capillary fragility. The monolayer patch was selected to obtain a prolonged release of coumarin contained in the phytocomplex. Two types of methacrylic patches (patch 1 based on a blend of Eudragit E100 and Eudragit NE; patch 2 based on Eudragit L100) were prepared. Both patches were equivalent in terms of coumarin release and ex vivo skin permeation profiles. The two patches differed significantly as regards respective adhesive properties. At low peel rate only patch 1 showed adhesive failure as confirmed by the in vivo performance. When comparing the behaviour of the patches containing melilot extract with analogous patches containing synthetic coumarin, no melilot phytocomplex enhancer effect was shown. The data of the ex vivo coumarin skin permeation and those obtained by the in vivo stripping technique showed a good correlation (r(2)=0.9727 for patch 1, r(2)=0.9835 for patch 2). PMID- 10727877 TI - In vitro inhibition properties of a new group of thiobenzanilides in relation to yeasts. AB - The antifungal potency of a series of 2,4-dihydroxythiobenzanilides was tested. MIC assessments were used for the estimation of potential activity in vitro against Candida, Cryptococcus, Geotrichum and Trichosporon species. The strongest fungistatic activity was observed for dichloro derivatives (MIC 7.82-31.21 microg/ml). The action of these compounds depends on lipophilicity, determined by the substitution of N-aryl moiety and the electron properties of molecules. The lipophilicity, expressed by R(Mw) values, was determined in the reversed-phase system. The changes in the nature of the thioamide bond were interpreted on the basis of UV and 1H NMR spectra. PMID- 10727878 TI - Relation between intracellular acidification and camptothecin-induced apoptosis in leukemia cells. AB - Leukemia cells (HL-60 and P388) treated with the topoisomerase I inhibitor camptothecin (CPT) undergo rapid apoptosis as judged from internucleosomal degradation of genomic DNA, morphological changes and flow cytometry analysis. The intracellular free calcium concentration is not affected by the treatment with a high dose of CPT. In contrast, fluorescence measurements of cells loaded with the pH indicator BCECF-AM indicate that the intracellular pH decreases significantly. Incubation of the leukemia cells with a high drug concentration for 5 h or with lower drug concentrations for 15 h results in a pronounced intracellular acidification. Measurements with the whole cell population show a decrease of 0.3-0.4 pH units. The extent of the acidic shift is proportional to the drug concentration and the period of incubation. No such effects were observed with P388CPT5 cells resistant to CPT. The results support the hypothesis that apoptosis induced in leukemia cells by CPT is associated with decreased intracellular pH. Modification of intracellular pH by topoisomerase inhibitors is viewed as an essential event responsible for the induction and/or propagation of apoptosis. The role of CPT-induced cellular acidification in the mechanism of action of the drug is discussed. PMID- 10727879 TI - Improvement of intestinal peptide absorption by a synthetic bile acid derivative, cholylsarcosine. AB - The potential of the nontoxic bile salt derivative, cholylsarcosine, to enhance the intestinal absorption of peptides was investigated in vitro and in situ. The permeation of the two model peptides octreotide and vasopressin-[arg(8)CT>/=CS, whereas ursodeoxycholic acid exhibited no absorption enhancement. Determination of the cytotoxic potential of the bile salts revealed the same rank order. In rats, octreotide and desmopressin were absorbed from the gastrointestinal-tract with moderate absorption efficiency. Coadministration of bile salts resulted in an increased absorption efficiency. The effect of CS was similar to that of CT. In conclusion, CS shows absorption enhancement properties and a relatively low cytotoxicity. It offers an alternative as absorption enhancer as compared to conventional bile acids which may have a potential cocarcinogenic risk. PMID- 10727880 TI - Pharmacokinetic interaction trial between co-artemether and mefloquine. AB - Forty-two healthy subjects were randomized in a parallel three-group design trial to investigate potential electrocardiographic and pharmacokinetic interactions between the new antimalarial co-artemether, a combination of artemether and lumefantrine (both of which are predominantly metabolized through CYP3A4), and mefloquine, another antimalarial described as a substrate (and possible inhibitor) of CYP3A4. Subjects were assigned to one of the three possible treatment groups (i.e., co-artemether alone or mefloquine alone or the combination of both). The dosage was 1000 mg mefloquine (divided into three doses over 12 h) followed 12 h later by six applications of co-artemether (40 mg artemether+480 mg lumefantrine each) over 60 h. The study medications were generally well tolerated after all treatments. Concomitant administration with mefloquine caused statistically significant lower (around 30-40%) plasma concentrations of lumefantrine than when co-artemether was administered alone. Even if important, this decrease in lumefantrine exposure was considered unlikely to impact clinical efficacy given the wide therapeutic index of co-artemether and the usual high variability in lumefantrine plasma levels, mostly and more importantly influenced by food intake. However, patients should be encouraged to eat at dosing times to compensate for this decreased bioavailability. The pharmacokinetics of artemether, DHA or mefloquine were not affected. Artemether concentrations significantly decreased over doses, independently of mefloquine co administration, while DHA concentrations slightly (not significantly) increased. Therefore, no clinically relevant risks due to pharmacokinetic drug-drug interaction are expected at the enzymatic level following co-administration of co artemether with CYP3A4 substrates with similar affinity to that of mefloquine. PMID- 10727881 TI - Proposition of group molar constants for sodium to calculate the partial solubility parameters of sodium salts using the van Krevelen group contribution method. AB - The aim of this study is to propose, for the first time, a set of group molar constants for sodium to calculate the partial solubility parameters of sodium salts. The values were estimated using the few experimental partial solubility parameters of acid/sodium salt series available either from the literature (benzoic acid/Na, ibuprofen acid/Na, diclofenac Na) or determined in this work (salicylic acid/Na, p-aminobenzoic acid/Na, diclofenac), the group contribution method of van Krevelen to calculate the partial parameters of the acids, and three reasonable hypothesis. The experimental method used is a modification of the extended Hansen approach based on a regression analysis of the solubility mole fraction of the drug lnX(2) against models including three- or four-partial solubility parameters of a series of pure solvents ranging from non-polar (heptane) to highly polar (water). The modified method combined with the four parameter model provided the best results for both acids and sodium derivatives. The replacement of the acidic proton by sodium increased the dipolar and basic partial solubility parameters, whereas the dispersion parameter remained unaltered, thus increasing the overall total solubility parameter of the salt. The proposed group molar constants of sodium are consistent with the experimental results as sodium has a relatively low London dispersion molar constant (identical to that of -OH), a very high Keesom dipolar molar constant (identical to that of -NO(2), two times larger than that of -OH), and a very high hydrogen bonding molar constant (identical to that of -OH). The proposed values are: F((Na)d)=270 (J cm(3))(1/2) mol(-1); F((Na)p)=1030 (J cm(3))(1/2) mol(-1); U((Na)h)=17000 J mol(-1). Like the constants for the other groups, the group molar constants proposed for sodium are certainly not the exact values. However, they are believed to be a fair approximation of the impact of sodium on the partial solubility parameters and, therefore, can be used as such in the group contribution method of van Krevelen. PMID- 10727883 TI - Protection against murine tuberculosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the 30-kDa antigen of Mycobacterium bovis BCG. AB - A recombinant (r-) Salmonella typhimurium aroA vaccine that secretes the naturally secreted protein of Mycobacterium bovis strain BCG, Ag85B, by means of the HlyB/HlyD/TolC export machinery (termed p30 in the following) was constructed. In contrast to r-S. typhimurium control, oral vaccination of mice with the r-S. typhimurium p30 construct induced partial protection against an intravenous challenge with the intracellular pathogen Mycobacterium tuberculosis, resulting in similar vaccine efficacy comparable to that of the systemically administered attenuated M. bovis BCG strain. The immune response induced by r-S. typhimurium p30 was accompanied by augmented interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) levels produced by restimulated splenocytes. These data suggest that the HlyB/HlyD/TolC-based antigen delivery system with attenuated r-S. typhimurium as carrier is capable of inducing an immune response against mycobacterial antigens. PMID- 10727882 TI - Kinetics of the mucosal antibody secreting cell response and evidence of specific lymphocyte migration to the lung after oral immunisation with attenuated S. enterica var. typhimurium. AB - The kinetic of mucosal secretory responses elicited by the vaccine vector Salmonella enterica var. typhimurium (S. typhimurium) was examined by enzyme linked immunospot (ELISPOT) and compared with serum responses. Mice immunised orally with BRD509, the aroA, aroD mutant of virulent S. typhimurium SL1344 expressing the C Fragment of tetanus toxin (TT), simultaneously developed an IgA antibody secreting cells (ASC) response in the gastrointestinal lamina propria, the spleen and the lung, against both S. typhimurium lipopolysaccharide (LPS) and TT. The magnitude of the ASC response was greatest in the gut, was boosted by a secondary immunisation at day 25, and the kinetic of the response did not correlate with the appearance of serum antibodies. This study suggests that S. typhimurium can engage the common mucosal immune system to effect mucosal secretory responses at distal sites, however, the magnitude of the responses is both greatest in the gut and antigen-specific. The ASC origin of the serum antibodies specific for S. typhimurium and antigens expressed by the bacterium is yet to be elucidated. PMID- 10727884 TI - Chimeric animal and plant viruses expressing epitopes of outer membrane protein F as a combined vaccine against Pseudomonas aeruginosa lung infection. AB - Outer membrane protein F of Pseudomonas aeruginosa has vaccine efficacy against infection by P. aeruginosa as demonstrated in a variety of animal models. Through the use of synthetic peptides, three surface-exposed epitopes have been identified. These are called peptides 9 (aa 261-274 in the mature F protein, TDAYNQKLSERRAN), 10 (aa 305-318, NATAEGRAINRRVE), and 18 (aa 282-295, NEYGVEGGRVNAVG). Both the peptide 9 and 10 epitopes are protective when administered as a vaccine. In order to develop a vaccine that is suitable for use in humans, including infants with cystic fibrosis, the use of viral vector systems to present the protective epitopes has been investigated. An 11-amino acid portion of epitope 10 (AEGRAINRRVE) was successfully inserted into the antigenic B site of the hemagglutinin on the surface of influenza virus. This chimeric influenza virus protects against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Attempts to derive a chimeric influenza virus carrying epitope 9 have been unsuccessful. A chimeric plant virus, cowpea mosaic virus (CPMV), with epitopes 18 and 10 expressed in tandem on the large coat protein subunit (CPMV-PAE5) was found to elicit antibodies that reacted exclusively with the 10 epitope and not with epitope 18. Use of this chimeric virus as a vaccine afforded protection against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Chimeric CPMVs with a single peptide containing epitopes 9 and 18 expressed on either of the coat proteins are in the process of being evaluated. Epitope 9 was successfully expressed on the coat protein of tobacco mosaic virus (TMV), and this chimeric virus is protective when used as a vaccine in the mouse model of chronic pulmonary infection. However, initial attempts to express epitope 10 on the coat protein of TMV have been unsuccessful. Efforts are continuing to construct chimeric viruses that express both the 9 and 10 epitopes in the same virus vector system. Ideally, the use of a vaccine containing two epitopes of protein F is desirable in order to greatly reduce the likelihood of selecting a variant of P. aeruginosa that escapes protective antibodies in immunized humans via a mutation in a single epitope within protein F. When the chimeric influenza virus containing epitope 10 and the chimeric TMV containing epitope 9 were given together as a combined vaccine, the immunized mice produced antibodies directed toward both epitopes 9 and 10. The combined vaccine afforded protection against challenge with P. aeruginosa in the chronic pulmonary infection model at approximately the same level of efficacy as provided by the individual chimeric virus vaccines. These results prove in principle that a combined chimeric viral vaccine presenting both epitopes 9 and 10 of protein F has vaccine potential warranting continued development into a vaccine for use in humans. PMID- 10727885 TI - Novel bacterial systems for the delivery of recombinant protein or DNA. AB - On the basis of attenuated intracellular bacteria, we have developed two delivery systems for either heterologous proteins or DNA vaccine vectors. The first system utilizes attenuated strains of Gram-negative bacteria which are engineered to secrete heterologous antigens via the alpha-hemolysin secretion system of Escherichia coli. The second system is based on attenuated suicide strains of Listeria monocytogenes, which are used for the direct delivery of eukaryotic antigen expression vectors into professional antigen presenting cells (APC) like macrophages in vitro as well as in vivo. PMID- 10727886 TI - Modulation of immune responses following antigen administration by mucosal route. AB - Most microbial infections are either restricted to the mucosal membranes or the etiologic agents needed to transit the mucosa. Thus, it is desirable to stimulate a mucosal response following vaccination, to block both infection and disease development. Attenuated vaccine carriers mimic natural infections, triggering also mucosal responses. Similar results can be achieved by administering antigens with appropriate adjuvants. However, the delivery of antigens per se is not sufficient to engender a protective response. A successful immunization requires the elicitation of an appropriate type of immune response (e.g. antibodies vs. cell-mediated immunity, Th1 vs. Th2 helper pattern). Therefore, a successful vaccination strategy demands the choice of adequate antigens, and their appropriate delivery and/or formulation to promote the required quality of immune response. Different strategies to optimize the immune responses elicited following vaccine administration by the mucosal route are discussed. PMID- 10727887 TI - Salmonella infection of bone marrow-derived macrophages and dendritic cells: influence on antigen presentation and initiating an immune response. AB - Traditionally macrophages (MPhi) have been considered to be the key type of antigen presenting cells (APC) to combat bacterial infections by phagocytosing and destroying bacteria and presenting bacteria-derived antigens to T cells. However, data in recent years have demonstrated that dendritic cells (DC), at their immature stage of differentiation, are capable of phagocytosing particulate antigens including bacteria. Thus, DC may also be important APC for initiating an immune response to bacterial infections. Our studies focus on studying how DC and MPhi process antigens derived from bacteria with no known mechanism of phagosomal escape (i.e. Salmonella typhimurium) for T cell stimulation as well as what role these APC types have in Salmonella infection in vivo. Using an in vitro antigen processing and presentation assay with bone marrow-derived (BM) APC showed that, in addition to peritoneal elicited MPhi and BMMPhi, BMDC can phagocytose and process Escherichia coli and S. typhimurium for peptide presentation on major histocompatibility complex (MHC) class I (MHC-I) and class II MHC-II. These studies showed that both elicited peritoneal MPhi and BMMPhi use an alternate MHC I presentation pathway that does not require the transporter associated with antigen processing (TAP) or the proteasome and involves peptide loading onto a preformed pool of post-Golgi MHC-I molecules. In contrast, DC process E. coli and S. typhimurium for peptide presentation on MHC-I using the cytosolic MHC-I presentation pathway that requires TAP, the proteasome and uses newly synthesized MHC-I molecules. We further investigated the interaction of Salmonella with BMDC and BMMPhi by analyzing surface molecule expression and cytokine secretion following S. typhimurium infection of BMDC and BMMPhi. These data reveal that Salmonella co-incubation with BMDC as well as BMMPhi results in upregulation of MHC-I and MHC-II as well as several co-stimulatory molecules including CD80 and CD86. Salmonella infection of BMDC or BMMPhi also results in secretion of cytokines including IL-6 and IL-12. Finally, injecting mice with BMDC that have been loaded in vitro with S. typhimurium primes naive CD4(+) and CD8(+) T cells to Salmonella-encoded antigens. Taken together, our data suggest that DC may be an important type of APC that contributes to the immune response to Salmonella. PMID- 10727888 TI - Cell surface presentation of recombinant (poly-) peptides including functional T cell epitopes by the AIDA autotransporter system. AB - For the efficient surface presentation and release of virulence factors especially pathogenic Gram-negative bacteria have developed several distinct secretion mechanisms. An increasing number of pathogens in various species employs a mechanism denoted the 'autotransporter' pathway. This pathway is characterised by an outer membrane translocator module representing the C terminal domain of the transported protein itself. An intriguing potential application of such systems involves the transport and surface expression of recombinant proteins or peptides, like e.g. the presentation of antigens for the generation of live oral vectors as vaccine carriers. Here we report on the incorporation of heterologous (poly-) peptides in permissive sites of the translocator module of the adhesin-involved-in-diffuse-adherence (AIDA) autotransporter system. We demonstrate the presentation of the B subunit of the heat labile enterotoxin of Escherichia coli (LTB) as well as of functional T-cell epitopes of Yersinia enterocolitica heat-shock protein 60 (Y-hsp60) on the surface of E. coli. PMID- 10727889 TI - Molecular analysis of hemolysin-mediated secretion of a human interleukin-6 fusion protein in Salmonella typhimurium. AB - Previously, we reported a plasmid-bearing Salmonella typhimurium strain capable of secreting human interleukin-6 (hIL-6) when genetically fused to the Escherichia coli hemolysin transport signal (HlyA(S)). Stationary phase culture supernatants of this strain revealed three major forms of hIL-6-HlyA(S) fusion protein (apparent molecular masses 32.4, 30.3, 27.0 kDa), at which the largest protein presumably represented full-length hIL-6-HlyA(S). The biological activity of the hIL-6-HlyA(S) protein mixture was similar to that of mature hIL-6. Accumulation of hIL-6-HlyA(S) in the culture supernatant occurred only during the initial growth phase, whereas in stationary phase and under in vitro conditions successive cleavage into the two truncated forms was observed. On the other hand, in whole cell lysates only full-length hIL-6-HlyA(S) could be detected, accounting for more than 50% of the totally synthesized protein. Upon cell fractionation, cellular hIL-6-HlyA(S) was exclusively found in the membrane fraction. These results suggest, that in S. typhimurium production and secretion of hIL-6-HlyA(S) is restricted to growing cells. A specific processing by a Salmonella-derived protease did not affect the biological activity of the fusion protein. PMID- 10727890 TI - Oral delivery of DNA vaccines using attenuated Salmonella typhimurium as carrier. AB - The efficacious delivery of eukaryotic expression plasmids to inductive cells of the immune system constitutes a key prerequisite for the generation of effective DNA vaccines. Here, we have explored the use of bacteria as vehicles to orally deliver expression plasmids. Attenuated Salmonella typhimurium aroA harbouring eukaryotic expression plasmids that encoded virulence factors of Listeria monocytogenes were administered orally to BALB/c mice. Strong cytotoxic and helper T cell responses as well as antibody production were elicited even after a single administration. Mice immunised four times with Salmonella that carried a eukaryotic expression plasmid encoding the secretory listerial protein listeriolysin were protected against a subsequent lethal challenge with this pathogen. A single dose was already partially protective. The efficiency of this vaccination procedure was due to transfer of the expression plasmid from the bacterial carrier to the mammalian host. Evidence for such an event could be obtained in vivo and in vitro. Expression of the desired antigen in various lymphoid tissues was already detectable 1 day after administration of the DNA vaccine and persisted for at least 1 month in spleen and mesenteric lymph nodes. Induction of cytotoxic and helper T cell responses was observed in all mouse strains tested including outbred strains whereas antibodies were mainly detected in BALB/c. Furthermore, we could show that immunogenicity could be improved by increasing the invasiveness of the bacterial carrier. PMID- 10727891 TI - Induction of mucosal immune responses in the human genital tract. PMID- 10727893 TI - Salivary amylase activity of the phlebotomine sand fly, Lutzomyia longipalpis. AB - Both male and female adult stages of the sand fly Lutzomyia longipalpis have detectable amylase activity in their salivary glands, as indicated by formation of p-nitrophenyl-alpha-D-maltoside from p-nitrophenyl-alpha-D-octoside and by hydrolysis of 4-nitrophenyl-alpha-D-maltoheptaoside-4,6,-O-ethylidene. No salivary alpha-glucosidase was detected. Amylase activity was also found in the crop and midgut of female flies, although in a smaller amount. Salivary amylase is significantly reduced from the salivary glands immediately after a blood meal, as is the case with salivary alpha-glucosidases in mosquitoes. Presence of salivary gland amylase in these sand flies, and absence of salivary alpha glucosidase, indicates that in nature these insects may have a significant intake of carbohydrates in the form of starch, as suggested by their plant-feeding behavior, previously demonstrated by Schlein and Warburg (Schlein, Y., Warburg, A., 1986. Phytophagy and the feeding cycle of Phlebotomus papatasi (Diptera: Psychodidae) under experimental conditions. Journal of Medical Entomology 23, 11 15), and Alexander and Usma (Alexander, B., Usma, M.C., 1994. Potential sources of sugar for the phlebotomine sandfly Lutzomyia youngi (Diptera: Psychodidae) in a Columbia coffee plantation. Ann. Trop. Med. Parasitol. 88, 543-549). PMID- 10727892 TI - Recombinant live Salmonella spp. for human vaccination against heterologous pathogens. AB - Live attenuated Salmonella spp. are promising candidates as oral vaccine delivery systems for heterologous antigens. Clinical trials have demonstrated that this approach is feasible for human vaccinations but further optimisation is necessary to obtain a better efficacy. Here, we discuss how existing clinical and pre clinical data can be used to guide such optimisation efforts. PMID- 10727894 TI - The salivary 5'-nucleotidase/phosphodiesterase of the hematophagus sand fly, Lutzomyia longipalpis [corrected]. AB - Salivary gland homogenates from adult female Lutzomyia longipalpis sand flies contain large amounts of 5'-nucleotidase and phosphodiesterase activities. Phosphodiesterase activity was found to be associated with 5'-nucleotidase in several independent experiments: (i) it coelutes with 5'-nucleotidase on a molecular sieving column, (ii) it coelutes with 5'-nucleotidase on a chromatofocusing column, and (iii) it has the same thermal inactivation kinetics as the 5'-nucleotidase activity. Additionally, both activities are independent of divalent cations, and both are decreased following a blood meal, suggesting that they reside in the same molecule. The role of salivary nucleotidases and purine nucleotides in blood-feeding by sand flies is discussed. PMID- 10727895 TI - Identification, characterization and cDNA cloning of two novel proteins secreted into the external space of the regenerating leg of Periplaneta americana. AB - We have isolated two proteins (Rap 60 and Rap 40) that were expressed specifically in regenerating legs of the American cockroach (Periplaneta americana). These proteins appear to be derived from a precursor protein with 443 amino acid residues termed regeneration-associated protein (RAP). Rap 60 and Rap 40 corresponded to Glu 223 to Ser 443 and Glu 223 to Glu 366 of RAP, respectively. Hence, Rap 40 is the N-terminal part of Rap 60. These proteins contained 13 repeats of a novel motif, [Glu/Asp-Glu/Asp-Val/Ala-Lys]. The gene encoding RAP was shown to be expressed during embryogenesis and by newly-formed epidermal cells of regenerating legs. Rap 60 and Rap 40 were secreted into the external space of regenerating leg saccules, where they accumulated, suggesting that they are components of extracellular matrix of regenerating leg saccules. PMID- 10727896 TI - Ferritin acts as the most abundant binding protein for snowdrop lectin in the midgut of rice brown planthoppers (Nilaparvata lugens). AB - The mannose-specific snowdrop lectin [Galanthus nivalis agglutinin (GNA)] displays toxicity to the rice brown planthopper Nilaparvata lugens. A 26kDa GNA binding polypeptide from N. lugens midgut was identified by lectin blotting and affinity chromatography, and characterized by N-terminal sequencing. This polypeptide is the most abundant binding protein for GNA in the N. lugens midgut. A cDNA (fersub2) encoding this protein was isolated from an N. lugens cDNA library. The deduced amino acid sequence shows significant homology to ferritin subunits from Manduca sexta and other arthropods, plants and vertebrates, and contains a putative N-glycosylation site. Native ferritin was purified from whole insects as a protein of more than 400kDa in size and characterized biochemically. Three subunits of 20, 26 and 27kDa were released from the native complex. The 26kDa subunit binds GNA, and its N-terminal sequence was identical to that of fersub2. A second cDNA (fersub1), exhibiting strong homology with dipteran ferritin, was identified as an abundant cDNA in an N. lugens midgut-specific cDNA library, and could encode the larger ferritin subunit. The fersub1 cDNA carries a stem-loop structure (iron-responsive element) upstream from the start codon, similar to structures that have been shown to play a role in the control of ferritin synthesis in other insects. PMID- 10727897 TI - Isolation and characterization of juvenile hormone esterase from hemolymph of Lymantria dispar by affinity- and by anion-exchange chromatography. AB - Juvenile hormone esterase (JHE), which catalyzes the hydrolysis of juvenile hormone, was isolated from the hemolymph of 5(th) instars of Lymantria dispar by two different procedures. One procedure was based on affinity chromatography and the other on anion-exchange chromatography. The material from both purifications showed bands of approximately 50 kDa when analyzed by SDS-PAGE. Isoelectric focusing (IEF) gels in combination with enzyme activity assays indicated two isoelectric forms with the same pI values (pH 5.1. and 5.3) from affinity purification and from anion-exchange chromatography. Amino acid sequencing of several internal peptides from the 50 kDa band following affinity purification and alignment of these sequences with JHEs from previously purified lepidopteran species (Heliothis virescens, Manduca sexta) showed high homology of these enzymes. The isolated JHE, at least in the stage of insect used, was different from the enzyme reported earlier [Valaitis, A.P., 1991. Characterization of hemolymph juvenile hormone esterase from Lymantria dispar. Insect Biochemistry 21, 583-595] to hydrolyze JH in the hemolymph of gypsy moth, based on molecular weight and amino acid sequence. PMID- 10727898 TI - The purine biosynthesis enzyme PRAT detected in proenzyme and mature forms during development of Drosophila melanogaster. AB - Glutamine phosphoribosylpyrophosphate amidotransferase (PRAT; EC 2.4. 2.14) is the first and rate-limiting enzyme of de novo purine biosynthesis. PRAT expression in Drosophila development was examined to determine if it is correlated with cell proliferation and/or nutritional isolation. An antiserum, raised against the 16 carboxyl-terminal amino acids of PRAT, detects two proteins corresponding to a 60 kDa proenzyme and 55 kDa mature enzyme, consistent with a 53 amino acid propeptide predicted from the gene sequence. Mature enzyme is maternally expressed, and proenzyme appears in embryos at 2-8 h, corresponding to the interval during which zygotic transcription is initiated. Upon hatching of first instar larvae, proenzyme levels are reduced and remain low relative to mature enzyme. Adult females have higher levels of both proteins relative to males, consistent with maternal expression. Maternal expression reflects a requirement for the enzyme during embryogenesis, while reduction in expression following hatching reflects a switch to an exogenous source of purines. Prat mRNA levels follow a similar overall pattern in the same developmental stages examined for the protein. Discovery of a second gene encoding PRAT with 78% amino acid identity leads to the possibility that the antiserum raised against the carboxyl terminus detects two enzymes. PMID- 10727899 TI - Absence of protein polymorphism attributable to insecticide-insensitivity of acetylcholinesterase in the green rice leafhopper, Nephotettix cincticeps. AB - The cDNA sequence of acetylcholinesterase (AChE) from the green rice leafhopper, Nephotettix cincticeps, was amplified, based on conserved peptide sequences of AChEs. A 2.3 kb contiguous sequence, containing an ORF encoding an AChE precursor with 677 amino acid residues was obtained. The deduced protein sequence showed the most similarity to that of AChE in the Colorado potato beetle, having common features in the primary AChE structure. cDNA sequences of individual leafhoppers from an insecticide susceptible strain and the resistant strain Nakagawara, whose methylcarbamate-insensitive AChEs show 10(2) or more I(50) ratio for propoxur, were compared. No fixed inter-strain difference was identified in the protein sequence, though amino acid substitution polymorphism was found at one position in the susceptible strain. Insecticide-insensitivity of leafhopper AChE does not result from changes in the protein primary structure that is encoded by the AChE gene sequence isolated in this study. PMID- 10727900 TI - Identification of a polymorphic ryanodine receptor gene from Heliothis virescens (Lepidoptera: noctuidae). AB - cDNAs encoding the C-terminal 1172 amino acids of a ryanodine receptor (RyR) from the lepidopteran pest Heliothis virescens (Hv-RyR) have been cloned and characterised. Sequence comparisons, organisational studies on corresponding genomic regions and a genetic segregation analysis provide evidence for two polymorphic alleles of the Hv-RyR locus. Comparison of the Hv-RyR C-terminal amino acid sequence with equivalent regions of other RyRs reveals a high level of overall amino acid homology (74% identity with D. melanogaster and between 47.9 and 50.1% with vertebrate isoforms). Homologies are however not uniformly distributed, though regions of high and low similarity are consistent with patterns in other RyR isoforms. The structural similarity of Hv-RyR with other RyRs is also indicated by comparison of hydropathy profiles and other previously described functional domains. Such results are consistent with this region of Hv RyR containing the Ca(2+) channel itself and being intimately involved in RyR regulation. Potential uses of the cDNAs described in the discovery and development of novel ryanodine like insecticides are discussed. PMID- 10727901 TI - Induction of premature chromosome condensation by a phosphatase inhibitor and a protein kinase in unstimulated human peripheral blood lymphocytes: a simple and rapid technique to study chromosome aberrations using specific whole-chromosome DNA hybridization probes for biological dosimetry. AB - We developed a simple and rapid method to study chromosome aberrations involving specific chromosomes using unstimulated human peripheral blood lymphocytes (HPBL). Premature chromosome condensation (PCC) was induced by incubating unstimulated HPBL in the presence of okadaic acid (OA, a phosphatase inhibitor), adenosine triphosphate (ATP), and p34(cdc2)/cyclin B kinase [an essential component of mitosis-promoting factor (MPF)], which eliminated the need for fusion with mitotic cells. OA concentration and duration of incubation for PCC induction was optimized using mitogen-stimulated HPBL; a final concentration of 0.75 microM incubated for 3 h was optimum, resulting in approximately 20% PCC yield. In unstimulated HPBL, PCC was induced by the addition of p34(cdc2)/cyclin B kinase at concentrations as low as 5 units/ml to a cell culture medium containing OA. Increases in the concentration of p34(cdc2)/cyclin B kinase from 5 to 50 units/ml resulted in a concentration-dependent increase in PCC yield (30% to 42%). We demonstrate that this technique of inducing PCC in unstimulated HPBL is suitable for studying radiation-induced aberrations involving a specific chromosome (chromosome 1) after 24 h repair using a whole-chromosome in situ hybridization probe and chromosome painting. Cells with aberrant chromosome number 1 are characterized with more than two chromosome spots. The frequency of cells with aberrant chromosome 1 increased with 60Co gamma-radiation doses in the region 0-7.5 Gy. The observed dose-effect relationship for the percentage of cells with aberrant chromosome 1 (Y) was explained by using both a linear [Y=(2.77+/-0.230)D+0.90+/-0.431, r(2)=0.966] and a nonlinear power [Y=(5.70+/ 0.46)D((0.61+/-0.05)), r(2)=0.9901) model. This technique can be applied to biological dosimetry of radiation exposures involving uniform whole-body low linear energy transfer (LET) exposures. PMID- 10727902 TI - Comparison of three different in vitro mutation assays used for the investigation of cytochrome P450-mediated mutagenicity of nitro-polycyclic aromatic hydrocarbons. AB - Three different in vitro mutation assays were used to investigate the involvement of cytochrome P450 enzymes in the activation of the nitro-polycyclic aromatic hydrocarbons (nitroPAHs) 1-nitropyrene and 2-nitrofluorene and their reduced metabolites amino-polycyclic aromatic hydrocarbons (aminoPAHs) 1-aminopyrene and 2-aminofluorene. Mutagenicity was investigated at the HPRT locus in Chinese hamster V79 cells with (V79-NH) or without (V79-MZ) endogenous acetyltransferase activity, stably expressing human cytochrome P450 cDNAs; in NIH/3T3 control or stably expressing human CYP1A2 cells, in combination with a shuttle vector containing a reporter gene; and in Salmonella typhimurium TA98, by inhibition of cytochrome P450 enzymes in rat liver S9 mix. Both the HPRT assay and the Ames test did not show any involvement of CYP3A in the activation of 1-nitropyrene to a mutagenic metabolite. In addition, a clear involvement of CYP1A2 in the activation of the nitroPAH 1-nitropyrene was demonstrated in both mutation assays using eukaryotic cells. However, no activation of 1-nitropyrene was seen in the eukaryotic cell lines when expressing only CYP1A2 (V79-MZ1A2) or acetyltransferase (V79-NH, 3T3-LNCX). The reduced metabolite of 1-nitropyrene, 1 aminopyrene, was also shown to be activated to a mutagenic metabolite by CYP1A2, using 3T3-1A2 cells in combination with a shuttle vector, and the Amestest in combination with the specific CYP1A2 inhibitor furafylline. No clear involvement of cytochrome P450 could be demonstrated for activation of 2-nitrofluorene to a mutagenic metabolite, whereas a role for CYP1A2 in the bioactivation of 2 aminofluorene is suggested. In the present study, we have demonstrated the complementary value of the three in vitro mutation assays in the examination of promutagen activation pathways. PMID- 10727904 TI - Genotoxicity of drinking water from three Korean cities. AB - Organic content of drinking tap water from Seoul, Taejon, and Suwon was extracted with an XAD-2 resin column and organic solvents. Four doses of the extract equivalent to 4, 2, 1, and 0.5 l water were tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 mix. The organic extracts of the water from all three cities were mutagenic in TA 98 without S9 mix and in TA 100 with and without S9 mix. The highest number of revertants per plate was found in the absence of S9 mix. Three doses of the extract (equivalent to 22, 11, and 3.7 l water) were also tested in the bone marrow micronucleus test using BDF1 mice. At the highest dose, a significant increase of the micronucleus frequency was observed. The time required to be on the effect, however, varied with the source of the water. Our results indicate that the drinking tap waters from the three cities were genotoxic clearly in the bacterial test and also in the in vivo assay with mice. As we found no genotoxicity of the source water as seen in a previous study, it is likely that the chlorination process leads to the genotoxicity of the tap water. PMID- 10727903 TI - Evaluation of the SOS/umu-test post-treatment assay for the detection of genotoxic activities of pure compounds and complex environmental mixtures. AB - This study presents an evaluation of the SOS/umu-test after introducing an additional dilution and incubation in the post-treatment assay. This treatment reduces the influence of coloured test compounds that otherwise affect the colorimetric determination of the beta-galactosidase activity and the bacterial growth measurement during the testing of complex environmental samples. The post treatment assay significantly increased the beta-galactosidase activity and consequently the enzyme induction ratios at higher doses of model genotoxins 4 nitroquinoline-N-oxide, N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminoanthracene, benzo(a)pyrene with low or no effect on the sensitivity of the test itself. On the other hand tests of environmental extracts indicated significant increases in sensitivity after additional incubation. 4-Nitroquinoline-N-oxide treatments of bacteria in the test affected cell division and caused filamentous growth. The size of filamentous bacteria and incidence rate of the length categories was positively correlated with the concentrations of genotoxins. Presence of filamentous tester bacteria proved induction of SOS response and genotoxic activity of environment samples in SOS/umu-test. PMID- 10727905 TI - The modifying effect of beta-carotene on gamma radiation-induced elevation of oxidative reactions and genotoxicity in male rats. AB - The aim of the present work was to evaluate the modulatory role of beta-carotene on the radiation-induced changes in certain biochemical and cytogenetic parameters. beta-Carotene was given by gavage at a dose of 5 mg/kg body weight for 7 consecutive days before whole body gamma irradiation with 7 Gy (single dose). The levels of beta-carotene in plasma, thiobarbituric acid-reactive substances (TBARS) in plasma and liver, the activities of superoxide dismutase (SOD) and catalase in blood and liver were the selected parameters. Furthermore, the frequency of micronuclei (MN) of polychromatic erythrocytes (PCEs), normochromatic erythrocytes (NCEs), the ratio of PCEs/NCEs and the mitotic index (MI) of bone marrow cells were also evaluated. The biochemical and cytogenetic determinations were carried out 1, 24, and 72 h after radiation exposure. The results obtained revealed that administration of beta-carotene pre-irradiation significantly inhibited the decrease in plasma beta-carotene, significantly reduced the levels of TBARS in plasma and liver. Significant protection of the radiation-induced changes in the activities of SOD and catalase was also recorded in the blood and liver of beta-carotene-treated and -irradiated rats. beta Carotene resulted in significant inhibition in the frequency of radiation-induced MN, as well as in the ratio of PCEs/NCEs and the MI of bone marrow cells. These results suggest that beta-carotene as a natural product with its antioxidant capacity and capability of quenching singlet oxygen, could play a modulatory role against the cellular damage affected by free radicals induced by whole body irradiation. PMID- 10727906 TI - Study on DNA strand breaks induced by sodium nitroprusside, a nitric oxide donor, in vivo and in vitro. AB - Nitric oxide (NO) as well as its donors has been shown to generate mutation and DNA damage in in vitro assays. The objective of this study was to identify that DNA single-strand breaks (SSBs) could be elicited by NO, not only in vitro but also in vivo. The alkaline single-cell gel electrophoresis (SCGE) was performed to examine the DNA damage in g12 cells and the cells isolated from the organs of mice exposed to sodium nitroprusside (SNP). A modified method, in which neither collagenase nor trypsin was necessary, was used to prepare the single-cell suspension isolated from organs of mice. Results showed that the exposure of g12 cells to 0.13-0.5 micromol/ml SNP with S9 for 1 h induced a concentration dependent increase in DNA SSBs in g12 cells. The significant increase in DNA migration and comet frequency has appeared in the cells isolated from the spleen, thymus, and peritoneal macrophages of mice after injecting i.p. SNP in the dosage range of 0.67-6.0 mg/kg b.wt for 1 h. However, no obvious increase in DNA strand breaks was observed in the cells isolated from the liver, kidney, lung, brain and heart obtained from the same treated mice. These results suggested that DNA SSBs could be induced by NO in some cells both in vivo and in vitro. There were organ differences in sensitivity in the mice exposed to NO. Spleen, thymus, and macrophages might be the important targets of NO. PMID- 10727907 TI - Stimulus-specific oscillatory responses of the brain: a time/frequency-related coding process. AB - OBJECTIVES: To review the coherent, rhythmic oscillations above approximately 20 Hz that occur in response to sensory inputs in the firing rate and membrane or local field potentials of distributed neuron aggregates of CNS layered structures. RESULTS: Oscillatory activity at approximately 20-80 Hz occurs in response to either olfactory, auditory and visual (contrast) stimuli; oscillations at frequencies centered on 100-120 Hz or 600 Hz are recorded, respectively, from the visual system (luminance stimulation) and from the somatosensory cortex. Experimental evidence suggests sources/mechanisms of generation that depend on inhibitory interneurons and pyramidal cells and are partially independent from those of conventional (broadband) evoked responses. In the olfactory and visual systems, the oscillatory responses reflect the global stimulus properties. A time/phase correlation between firing rate, spiking coincidence and oscillatory field responses has been documented. The oscillatory responses are postsynaptic both in cortex and in precortical structures (e.g. retina; LGN). Evidence indicates intracortical and thalamocortical interacting mechanisms of regulation as well as GABAergic and cholinergic modulation. In the visual cortex the oscillatory responses are driven by oscillations in the synaptic input. Oscillatory potentials are dependent on resonance phenomena and produce narrow-band synchronization of activated neurons. They may have a role in the 'binding' of separate neuronal aggregates into sensory units. CONCLUSIONS: Oscillatory responses contribute as a time/frequency coding mechanism to pacing neurons selectively for the physical properties of stimulus and are involved in sensory information processing. PMID- 10727908 TI - Clinical correlates and prognosis in early spindle coma. AB - OBJECTIVE: To determine the prognostic significance of spindle coma (SC) according to etiology and EEG reactivity. METHODS: We reviewed 15 patients with SC due to various causes within 8 days of coma to determine the prognostic significance of this EEG pattern. RESULTS: The outcome among survivors was favorable: among 13 survivors, 9 were independent in all activities of daily living (ADLs) at 6 months; 3 were dependent in all ADLs; and one remained in coma. EEG reactivity to noxious stimuli best predicted outcome: All patients (whatever the coma etiology) with EEG reactivity survived; conversely, not all patients without EEG reactivity died. CONCLUSION: In our patients, EEG reactivity independent of etiology predicted survival, neurological examination did not predict outcome. Most SC survivors had a meaningful recovery achieving all ADLs. From the literature, the cause of SC was predictive of outcome: encephalopathy, seizures and trauma had the best prognosis while hypoxia, CRA and structural lesions carried the worst. Literature review revealed that 23% of patients [56/242] died or remained in a persistent vegetative state (PVS). Best outcomes occurred when SC was due to drugs, encephalopathy or seizures: (0/14 died or were in a PVS). With trauma 15% [25/169] died or were in a PVS). Intermediate outcomes occurred with hypoxia and cardio-respiratory arrest (CRA): 33% [7/21] died or were in a PVS. The gravest outcomes occurred with brain-stem and cerebral infarctions, and tumors: 73% [22/30] died or were in a PVS. PMID- 10727909 TI - Age-related changes of cortical excitability in subjects with sleep-enhanced centrotemporal spikes: a somatosensory evoked potential study. AB - Middle-latency somatosensory evoked potentials (SEPs) of particularly large amplitude (giant) have been reported in subjects with benign childhood epilepsy with centrotemporal spikes (BECT) and in normal children, which usually show significant age-related changes. However, the mechanisms by which age modifies the appearance of centrotemporal spikes and giant SEPs in these children, are not known. The characteristics of SEPs were studied in a group of 18 subjects (10 males and 8 females, aged 7.1-17.2 years) with sleep-enhanced centrotemporal spikes, with or without BECT and the results were compared with those obtained from a group of age-matched normal controls. Giant SEPs were recorded in 6 subjects and, in 3 of these, EEG spikes evoked by hand tapping were obtained also. No subjects with giant SEPs were found in subjects older than 12 years, and an age-related decrease in amplitude of giant SEPs as this age approached was observed. Moreover, at repeated SEP recordings, a clear trend towards a more important reduction in amplitude of giant SEPs over the temporal and parietal than over the central regions was evident. The study of EEG spikes evoked by hand tapping showed a striking similarity between the averaged evoked spikes and the main negative component of giant SEPs. It was also possible to observe that the spike negative peak recorded over the central areas always preceded the same component recorded over the parietal and temporal areas by 5-15 ms. Our study seems to support the idea that giant SEPs in subjects with centrotemporal spikes are generated by a complex mechanism different from that at the basis of the normal N60 component of SEPs; they also show peculiar age-related modifications which can be interpreted in terms of maturational changes of brain excitability/inhibition and probably constitute a tool for monitoring the clinical course of BECT, when present. PMID- 10727910 TI - Continuous bitemporal rhythmic subclinical epileptiform activity in an adult without epileptic seizures. AB - We report the occurrence of an unusually sustained, non-reactive, bilateral, rhythmic, subclinical discharge in a young adult without epileptic seizures PMID- 10727911 TI - Magnetoencephalographic analysis of cortical activity in Alzheimer's disease: a pilot study. AB - OBJECTIVES: In the present study, MEG was used to analyze spectral power and reference-free coherence in patients with probable Alzheimer's disease (AD). METHODS: Sixty-one channel MEG was recorded in 5 AD patients and 5 age-matched controls at rest with eyes open and eyes closed, as well as during the performance of two different mental tasks. Artefact-free epochs were selected for the analysis of power and coherence values in each of 5 4-Hz wide frequency bands ranging from 2 to 22 Hz. RESULTS: In AD patients, the absolute low frequency magnetic power was significantly and rather diffusely increased relative to controls with a fronto-central maximum. High frequency power values were significantly decreased over the occipital and temporal areas. Reactivity to eye opening and mental tasks was reduced in the patient group. Relative to controls, a general decrease of MEG coherence values, including all frequencies analyzed, was found in AD patients. CONCLUSIONS: These observations confirm the pattern of changes in spectral power and reactivity known from EEG studies and suggest that coherence decreases in AD patients are widespread and include frequencies outside the alpha band. PMID- 10727912 TI - The roles of proximal and distal muscles in anticipatory postural adjustments under asymmetrical perturbations and during standing on rollerskates. AB - OBJECTIVES: The study addresses two controversial issues surrounding the nature of anticipatory postural adjustments (APAs). The first deals with the reproducible APA patterns in proximal postural muscles and variable APA patterns reported for the muscles controlling the ankle joint (TA-SOL). We hypothesized that the TA-SOL muscles participate mainly in the compensation of lateral and rotational perturbations, in particular those associated with asymmetrical movements. The second issue deals with decreased APAs reported during both very stable and unstable standing. We hypothesized that APA changes during unstable standing might depend on the actual mechanical nature of instability. METHODS: Eight healthy subjects were recruited who had had no prior experience with rollerskates. They performed series of bilateral and unilateral, flexion and extension movements during regular standing and bilateral movements during standing on rollerskates. EMG changes and shifts of the center of pressure were analyzed within a time window typical of APAs. RESULTS: We found that APAs in proximal muscles were decreased during unilateral shoulder movements as compared to APAs during bilateral movements but did not show right-left differences. In contrast, the distal muscles (TA-SOL) showed little involvement during bilateral movements, while a clear right-left asymmetry was seen during unilateral movements. Bilateral movements performed while standing on rollerskates were accompanied by unchanged APAs in the proximal muscle pairs and increased APAs in the TA-SOL pair. CONCLUSIONS: We conclude that the proximal muscles provide a general pattern counteracting expected perturbations in the anterior-posterior direction while the distal muscles deal with asymmetrical perturbations and the modulation of APAs in unusual conditions such as standing on rollerskates. There seems to be no unambiguous relation between the magnitude of APAs and the stability of standing: Depending on the exact mechanical nature of postural instability, it could be associated with qualitatively different changes in the APAs. PMID- 10727913 TI - Hemispheric asymmetries of cortico-cortical connections in human hand motor areas. AB - OBJECTIVE: To evaluate possible functional asymmetries of the motor cortex on the hand-dominant versus the non-dominant hemisphere. METHODS: We assessed the handedness of 15 consenting volunteers using the Edinburgh Inventory. They were divided in two groups: 9 right-handers and 6 left-handers. We used single- and paired-transcranial magnetic stimulation (TMS) to measure the relaxed and active motor threshold and the ipsilateral cortico-cortical inhibition and facilitation curve for both hand motor areas. We looked for hemispheric asymmetries of variables related to the side of stimulation (dominant versus non-dominant) and to handedness. RESULTS: We found no significant intra- or intergroup hemispheric asymmetry for the relaxed and active thresholds. Among the right-handers, the cortico-cortical inhibition and facilitation curve showed an increased amount of facilitation in the dominant as compared with the non-dominant hand area. No such changes were seen among the left-handers. Both the dominant and the non-dominant hand areas of the right-handers showed more inhibition and less facilitation on the cortico-cortical inhibition and facilitation curve than the corresponding areas of left-handers. CONCLUSION: In the right-handers, paired TMS studies showed a functional asymmetry of the motor cortex between the dominant and the non-dominant hand. The left-handers did not show this lateralization. Under TMS investigation their motor cortex function appeared different from that of right handers. PMID- 10727914 TI - Central motor conduction to lower limb after transcranial magnetic stimulation in spinocerebellar ataxia type 2 (SCA2). AB - OBJECTIVES: To evaluate central motor conduction to lower limbs in spinocerebellar ataxia type 2 (SCA2). METHODS: Transcranial magnetic stimulation was performed to study the corticospinal tracts of 18 patients with SCA2. RESULTS: Central motor conduction time (CMCT) to lower limbs and thresholds were abnormal in 8 patients (44%); CMCT and thresholds were significantly correlated with disease duration and disability. CONCLUSIONS: Corticospinal tract involvement is more frequent than previously reported in SCA2. Prolonged CMCT and increased threshold should not be used to differentiate between various type of autosomal dominant cerebellar ataxia. Similar to that reported in Friedreich's ataxia, we suggest that examining central motor conduction to the lower limbs may assist in evaluating the progressive steps of neurodegeneration in SCA2. PMID- 10727915 TI - Oscillatory cortical activity and movement-related potentials in proximal and distal movements. AB - OBJECTIVES: Event-related desynchronization (ERD) of alpha- and beta-rhythms, the post-movement beta-synchronization and the cortical movement-related potentials were analyzed in distal (finger) and proximal (shoulder) movements. METHODS: EEG was recorded in 7 healthy right-handed men using a 59-channel whole-head EEG system while subjects performed self-paced movements. RESULTS: The amplitude of the Bereitschaftspotential (BP) was greater over the central midline area and smaller over the contralateral sensorimotor hand area in shoulder than in finger movements. The maximal alpha- and beta-ERD was localized at parietal electrodes in shoulder movements and over the left and right sensorimotor hand area in finger movements. The post-movement beta-ERS was greater in shoulder than in finger movements, especially at the electrode located 3.5 cm left of the central midline electrode. A significant correlation between the slope of the terminal portion of the BP (negative slope) and amplitude of the post-movement beta synchronization was observed in shoulder but not in finger movements. CONCLUSIONS: Enhancement of BP over the central midline electrode suggests increased activation of the supplementary motor area in proximal movements. The spatial distribution of the alpha- and beta-ERD and of the post-movement beta-ERS shows topographic differences which may refer to the somatotopic organization of the primary sensorimotor cortex with shoulder representation medial to hand and fingers. The correlation between the negative slope and the post-movement beta ERS in proximal movements supports the view that the brief post-movement inhibition over the motor cortical area is related to the pre-movement activation of that area. PMID- 10727916 TI - Postural and resting tremor in the upper limb. AB - OBJECTIVE: Tremor from multiple segments of the upper limb was recorded under postural and resting conditions. The aims of this study were to examine the nature of tremor within a single limb segment, intra- and inter-limb co ordination of tremor, and the influence of cardiac mechanical events on physiological tremor. METHODS: Tremor was recorded from eight healthy adult subjects during a postural pointing task where the level of support for the upper limb segments was successively increased. The dynamics of tremor within a single segment were examined using power spectral, ApEn and amplitude analyses. Inter segment tremor relations were determined using coherence and Cross-correlation analyses. RESULTS: Single segment analysis demonstrated that each (unsupported) limb segment contained two major frequency peaks (at 1-4 Hz and 8-12 Hz). Both peaks were still evident in the distal segments when the proximal segments were supported. External support of the more proximal limb segments also resulted in decreased finger tremor, but these changes were not simply additive over segments within a limb or equal across fingers. There were significant relations between adjacent proximal and distal limb segment pairs but no correlations between contralateral limb segments or between heart rate and limb tremor. CONCLUSIONS: These findings imply that: the low frequency component (1-4 Hz) of physiological tremor in the hand and finger could not be attributed to passive transmission of oscillations from the upper arm and forearm; and the contribution of proximal segments on tremor in the index finger tremor could not be predicted from mechanical principles alone. The minimization of finger tremor involved compensatory coupling of segments of the upper arm with particular emphasis upon active control of the wrist joint. PMID- 10727917 TI - H-reflex changes under spinal loading and unloading conditions in normal subjects. AB - OBJECTIVE: The soleus H- reflex is usually tested clinically in patients lying prone, with the H-latency always the criterion of choice for detecting abnormality. However, stresses on the spine vary during lying, standing, loading (weight lifting) and unloading. So the objective of this study was to measure changes in the H-reflex under 4 different loading conditions and to investigate whether mechanical loading of the spine would affect the H-reflex parameters. METHODS: Twenty healthy volunteers (22-46 years) with no history of significant low back pain or radiculopathy participated in the study. A Cadwell Excel electromyography unit was used to elicit and record the soleus H-reflex. The tibial nerve was stimulated at the popliteal fossa using 1 ms pulses at 0.2 pps of H-max. Each subject was tested under 4 different conditions: prone lying, free standing, standing while lifting 20% of his or her body weight, and standing while unloaded by 25% of his/or her body weight by a ZUNI II unloading system. For each subject, the peak-to-peak amplitudes of the maximum obtained H- reflex and the onset latencies of 8 separate traces were averaged for both lower extremities. Two-factor, repeated-measures ANOVAs were used to test the effect of the condition and side on the H-reflex amplitude and latency with ( approximately =0.025). RESULTS: The H-reflex was inhibited during standing, loading and unloading as compared with prone lying. The H-reflex was recovered during loading as compared with during standing. There were no significant changes in the H reflex latency under the 4 different conditions. Both lower extremities showed similar pattern of changes in the H-reflexes. CONCLUSIONS: These results imply a significant interplay between peripheral and central mechanisms and their effects on the spinal motoneurons. This in turn suggests that testing of the H-reflex amplitude and latency under functional conditions, such as standing may be useful in detecting subtle changes in root impingement. PMID- 10727918 TI - Motor cortex disinhibition in acute stroke. AB - OBJECTIVES: To test whether a disinhibition occurs in the human motor cortex after stroke. METHODS: Patients with a mild to moderate hemiparesis after an acute unilateral ischemic stroke were compared with age-matched healthy controls. We used paired transcranial magnetic stimuli (TMS) to investigate intracortical inhibition and facilitation. Single TMS were applied to obtain a cortical silent period. RESULTS: Intracortical inhibition was significantly reduced in the affected hemisphere at interstimulus intervals of 2, 3 and 4 ms. The cortical silent period was significantly prolonged when compared to the unaffected hemisphere of the patients and to the control group. Motor cortex disinhibition observed in stroke patients was associated either with minimal impairment at the onset of symptoms or with rapidly improving motor functions. CONCLUSIONS: Motor cortex disinhibition occurs in humans after stroke. We suggest that this disinhibition is indicative of compensatory mechanisms, which are involved in recovery-related reorganization. PMID- 10727919 TI - Leg muscle reflexes mediated by cutaneous A-beta fibres are normal during gait in reflex sympathetic dystrophy. AB - OBJECTIVES: Reflex sympathetic dystrophy (RSD) is, from the onset, characterized by various neurological deficits such as an alteration of sensation and a decrease in muscle strength. We investigated if afferent A-beta fibre-mediated reflexes are changed in lower extremities affected by acute RSD. METHODS: The involvement of these fibres was determined by analyzing reflex responses from the tibialis anterior (TA) and biceps femoris (BF) muscles after electrical stimulation of the sural nerve. The reflexes were studied during walking on a treadmill to investigate whether the abnormalities in gait of the patients were related either to abnormal amplitudes or deficient phase-dependent modulation of reflexes. In 5 patients with acute RSD of the leg and 5 healthy volunteers these reflex responses were determined during the early and late swing phase of the step cycle. RESULTS: No significant difference was found between the RSD and the volunteers. During early swing the mean amplitude of the facilitatory P2 responses in BF and TA increased as a function of stimulus intensity (1.5, 2 and 2.5 times the perception threshold) in both groups. At end swing the same stimuli induced suppressive responses in TA. This phase-dependent reflex reversal from facilitation in early swing to suppression in late swing occurred equally in both groups. CONCLUSIONS: In the acute phase of RSD of the lower extremity there is no evidence for abnormal A-beta fibre-mediated reflexes or for defective regulation of such reflexes. This finding has implications for both the theory on RSD pathophysiology and RSD models, which are based on abnormal functioning of A-beta fibres. PMID- 10727920 TI - Surface EMG mapping of the human trapezius muscle: the topography of monopolar and bipolar surface EMG amplitude and spectrum parameters at varied forces and in fatigue. AB - OBJECTIVES: To investigate the factors affecting the topography of trapezius muscle EMG, multichannel recordings were made at different forces of isometric shoulder elevation and during fatiguing exercise. METHODS: Twenty-eight channels of monopolar EMG were recorded from an array of 4 x 7 electrodes placed on the upper trapezius muscle. From the monopolar EMG and the bipolar derivations the root mean square (RMS(monopolar), RMS(bipolar)) and power spectrum median frequency (MF(monopolar), MF(bipolar)) were calculated. RESULTS: The maximum RMS(monopolar) was located above the middle part of the trapezius muscle, where a minimum was found for RMS(bipolar). The cranial-caudal RMS distribution shifted upwards when the force was increased from 50 to 100% MVC and during fatigue. MF(bipolar) showed a peak above the endplate region, where the MF(monopolar) was low. During fatigue the normalized MF slope was independent of the cranial-caudal electrode position, but MF(monopolar) decreased most strongly at positions above the endplate region, where MF(bipolar) decreased less. CONCLUSIONS: While the changes in MF reflected metabolic properties and volume conduction phenomena in the muscle, changes in RMS reflected a compensation for the fatigue processes within the muscle. The RMS changes in fatigue can be explained by the direction of the fibres involved in shoulder elevation. PMID- 10727921 TI - Event-related brain potentials distinguish processing stages involved in face perception and recognition. AB - OBJECTIVES: An event-related brain potential (ERP) study investigated how different processing stages involved in face identification are reflected by ERP modulations, and how stimulus repetitions and attentional set influence such effects. METHODS: ERPs were recorded in response to photographs of familiar faces, unfamiliar faces, and houses. In Part I, participants had to detect infrequently presented targets (hands), in Part II, attention was either directed towards or away from the pictorial stimuli. RESULTS: The face-specific N170 component elicited maximally at lateral temporal electrodes was not affected by face familiarity. When compared with unfamiliar faces, familiar faces elicited an enhanced negativity between 300 and 500 ms ('N400f') which was followed by an enhanced positivity beyond 500 ms post-stimulus ('P600f'). In contrast to the 'classical' N400, these effects were parietocentrally distributed. They were attenuated, but still reliable, for repeated presentations of familiar faces. When attention was directed to another demanding task, no 'N400f' was elicited, but the 'P600f' effect remained to be present. CONCLUSIONS: While the N170 reflects the pre-categorical structural encoding of faces, the 'N400f' and 'P600f' are likely to indicate subsequent processes involved in face recognition. Impaired structural encoding can result in the disruption of face identification. This is illustrated by a neuropsychological case study, demonstrating the absence of the N170 and later ERP indicators of face recognition in a prosopagnosic patient. PMID- 10727922 TI - EEG brain mapping of phonological and semantic tasks in Italian and German languages. AB - OBJECTIVES: Event-related potential correlates of phonological encoding - as compared with lexical access and semantic categorization - were measured in two studies involving two groups of 14 German and 14 Italian subjects. METHODS: A two stimulus reaction time paradigm was used. Stimulus pairs presented one-by-one with 2 s inter-stimulus intervals (ISI) had to be matched with respect to lexical identity (word-picture) in a word comprehension task or with respect to the phonological representative of objects in a rhyming task. A semantic categorization task was added for the Italian sample. In both studies, the EEG was recorded from 26 scalp electrodes according to the 10-20 system. The slow negative potential during the ISI (CNV) was determined as the electrocortical correlate of preparation for and activation of the specific language-related task. RESULTS: In both samples, phonological encoding (rhyming) evoked a more pronounced CNV over the left- compared with the right-frontal area, while less lateralized central dominance of the CNV was found in the word comprehension task. Semantic categorization was accompanied by the least asymmetry of activity. CONCLUSIONS: Results indicate that the different degree of asymmetry induced by phonological and semantic processing may be determined from the scalp distribution of slow cortical potentials with cross-lingual reliability. PMID- 10727923 TI - After-effect of transcutaneous electrical nerve stimulation (TENS) on pain related evoked potentials and magnetic fields in normal subjects. AB - OBJECTIVES: The after-effect of transcutaneous electrical nerve stimulation (TENS) on pain-related brain responses was investigated using electroencephalography (EEG) and magnetoencephalography (MEG). METHODS: We studied 13 healthy volunteers for the main experiment and 7 for the control experiment. The pain-related evoked cerebral potentials (PREP) at Cz and magnetic cortical fields (PRCF) on both hemispheres following painful electrical finger stimulation were simultaneously recorded before and after TENS on the right forearm of the median nerve territory at 50 Hz for 30 min. PREP and PRCF were similarly recorded without TENS in the control experiment. RESULTS: The PREP components, N150 and P220, were significantly attenuated after TENS, compared to those before TENS (P<0.01, two-way repeated ANOVA). However, there was no consistent change of the PRCF components. Eleven of 13 subjects reported no change of pain sensation after TENS. There was no change of PREP in control experiment without TENS. CONCLUSIONS: The results indicated that TENS reduced PREP following painful electrical stimulation, and that the origin of PREP was, at least partially, different from that of PRCF which was not changed after TENS. An after-effect of TENS significantly affected the generation process of PREP, but it was not enough to relieve the subjective painful feeling. PMID- 10727924 TI - Clinical evaluation criteria for the assessment of impaired pain sensitivity by thulium-laser evoked potentials. AB - OBJECTIVES: Cortical potentials evoked by carbon dioxide laser pulses have been applied in clinical practice to study nociceptive pathways for several years. In this study, we evaluate the properties of an infrared laser (thulium-YAG) with a penetration depth in the skin that matches the intracutaneous depth of nociceptors. METHODS: Temperature measurements and modelling showed that the thulium laser generates painful intracutaneous temperatures with less surface heating than the carbon dioxide laser and with no side effects (up to 600 mJ pulse energy). To develop clinical evaluation criteria, laser-evoked potentials (LEPs) were recorded from 3 midline positions (Fz, Cz, Pz) versus linked earlobes in 23 healthy subjects. Within a session, two skin areas were studied twice in a balanced sequence using randomized interstimulus intervals and two intensities in randomized order. RESULTS: After hand and foot stimulation with 540 mJ pulses, all subjects showed reproducible biphasic vertex potential, consisting of a negativity (hand: 210 ms, foot: 250 ms) and a positivity (hand: 330 ms, foot: 380 ms). Mean habituation of the vertex potential amplitude across runs was 25% (hand) or 16% (foot); due to the balanced sequence it did not affect the other comparisons. Following foot stimulation, peak latencies were significantly longer (by 40-50 ms) and amplitudes were significantly smaller than following hand stimulation (22.5+/-6.7 vs. 30.3+/-10.9 microV, mean+/-SD). Using 2. 5 standard deviations from the mean as a cut-off, absolute normative values were determined for peak latencies and amplitudes. In addition, relative normative values were determined for paired comparisons (hand-hand, foot-foot, hand-foot). CONCLUSIONS: The thulium-YAG laser is a useful tool for assessment of impaired pain sensitivity. Representative case reports illustrate that unlike for early SEP components, the most frequent LEP abnormalities were amplitude differences. PMID- 10727925 TI - Spinally elicited peripheral nerve responses are sensory rather than motor. AB - OBJECTIVES: Spinally elicited peripheral nerve responses, commonly called neurogenic motor evoked potentials (NMEPs), are widely used to monitor spinal cord motor function during surgery. However, numerous evidence suggests that these responses are primarily sensory rather than motor. The collision technique was utilized to address this issue. METHODS: Collision studies were performed in 7 patients during surgery. An ascending volley of sensory (AS) and motor activity (AM) was elicited by posterior tibial nerve stimulation at the popliteal fossa. After a short time delay, high cervical spinal stimulation produced a descending volley of sensory (DS) and motor (DM) activity. The AM volley ascended only to the anterior horn cells whereas the AS and DS volleys collided in the spinal cord. The inter-stimulus delays were varied so as to affect the degree of spinal cord collision. The DS and DM activity which remained after collision was recorded from the posterior tibial nerves at the ankle. RESULTS: Inter-stimulus delays of 18 ms or less resulted in no apparent peripheral descending volleys. These findings were consistent for all the patients studied. CONCLUSIONS: Spinally elicited peripheral nerve responses are primarily sensory rather than motor and are mediated by the same neural pathways as SEPs. PMID- 10727926 TI - Mismatch negativity: a tool for the assessment of stimuli discrimination in cochlear implant subjects. AB - OBJECTIVES: The performance of cochlear implants varies among users. This variability may be due to the ability to process auditory information. The mismatch negativity should provide an index of discrimination in cochlear implantees (Kraus N, McGee T, Carrell T, Sharma A. Neurophysiologic bases of speech discrimination. Ear Hear. 1995;16:19-37). Our aim was to analyze MMN in cochlear implant (Digisonic) subjects to assess electrode discrimination and to study the relationship between MMN and speech performance. METHODS: The mismatch was determined by stimulating three pairs of different electrodes. Two sessions were performed with both standard and deviant stimuli reversed. Speech recognition abilities were evaluated using 4 speech tests. The statistics included the results of 6 subjects. They indicated that MMN may be obtained when stimulating two different electrodes. A difference occurred between standard and deviant stimuli within a session but also when the response to the deviant stimulus was compared to the response of the same stimulus in a standard condition, validating the discrimination process. MMN latency was about 140 ms, and amplitude about -2.8 microV. No differences were shown with respect to electrode spacing. No relationship between MMN and speech performance was found. A clinical application of this method might be to assess the auditory processing of electrical stimuli in congenitally deaf subjects at the pre-implantation stage. PMID- 10727927 TI - 'Slave' metabolites and enzymes. A rapid way of delineating metabolic control. AB - Although control of fluxes and concentrations tends to be distributed rather than confined to a single rate-limiting enzyme, the extent of control can differ widely between enzymes in a metabolic network. In some cases, there are enzymes that lack control completely. This paper identifies one surprising origin of such lack of control: If, in a metabolic system, there is a metabolite that affects the catalytic rate of only one enzyme, the corresponding enzyme cannot control any metabolic variable other than the concentration of that metabolite. We call such enzymes 'slave enzymes', and the corresponding metabolites 'slave metabolites'. Implications of the existence of slave enzymes for the control properties of enzymes further down the metabolic pathway are discussed and examined for the glycolytic pathway of yeast. Inadvertent assumptions in metabolic models may cause the latter incorrectly to calculate absence of metabolic control. The phenomenon of slave enzymes may well be important in enhancing metabolic signal transduction. PMID- 10727928 TI - Maculatin 1.1, an anti-microbial peptide from the Australian tree frog, Litoria genimaculata solution structure and biological activity. AB - The dorsal glands of Australian tree frogs from the Litoria species contain a diversity of antibiotic peptides that forms part of the defence system of the animal. Here, the antibiotic activity and structure of maculatin 1.1, a 21 amino acid peptide from Litoria genimaculata, are compared. The activity data on maculatin 1.1 and a series of its analogues imply that the mechanism of action of maculatin 1.1 involves binding to, and subsequent lysis of, the bacterial cell membrane. The structure of maculatin 1.1 was determined using NMR spectroscopy in a trifluoroethanol/water mixture and when incorporated into dodecylphosphocholine micelles. Under both conditions, the peptide adopts a very similar conformation, i.e. a helical structure with a central kink in the vicinity of Pro15. The kink allows the peptide to adopt a well-defined amphipathic conformation along its entire length. The similar structures determined under both solvent conditions imply that structures of membrane-interacting peptides in trifluoroethanol/water mixtures are representative of those adopted in a membrane environment, e.g. when incorporated into micelles. The synthetic Ala15 analogue of maculatin 1.1 has markedly reduced activity and its NMR-derived structure is a well-defined helix, which lacks the central kink and flexibility of the parent molecule. It is concluded that the kink is important for full biological activity of the peptide, probably because it allows maximum amphipathicity of the peptide to facilitate interaction with the membrane. The structure of maculatin 1.1 is compared with a related peptide, caerin 1.1 [Wong, H., Bowie, J.H. and Carver, J.A. (1997) Eur. J. Biochem. 247, 545-557], which has an additional central proline residue and enhanced central flexibility compared with maculatin 1.1. The role of central flexibility within antibiotic peptides in their interaction with bacterial membranes is discussed. PMID- 10727929 TI - cDNA cloning, genomic cloning, and tissue-specific regulation of mouse cerebroside sulfotransferase. AB - We have isolated a mouse cDNA clone encoding 3'-phosphoadenylylsulfate galactosylceramide 3'-sulfotransferase (cerebroside sulfotransferase; CST; EC 2.8.2.11) from a kidney cDNA library, using a human CST cDNA clone [Honke, K., Tsuda, M., Hirahara, Y., Ishii, A., Makita, A. & Wada, Y. (1997) J. Biol. Chem. 272, 4864-4868] as a probe. A recombinant protein of the cloned cDNA showed CST activity. The deduced protein is composed of the same 423 amino acids as human CST and its sequence exhibits 84% identity with that of the human counterpart. Northern-blot analysis and subquantitative reverse transcription-PCR (RT-PCR) analysis showed that the CST gene is preferentially transcribed in stomach, small intestine, brain, kidney, lung, and testis, in that order. To examine differences in transcripts in various tissues, we isolated CST cDNA clones from stomach, small intestine, brain, kidney, and testis by 5'-RACE analysis. We found seven different nucleotide sequences in the 5'-UTR, while the DNA sequences of all the isolated cDNA clones were identical in the coding region. In addition, we isolated CST genomic DNA clones from a mouse genomic library. The clones covered all the 5'-UTR sequences and coding exons including 3'-UTR. RT-PCR analyses of CST mRNAs from various tissues confirmed that CST transcripts are tissue specifically spliced by alternative use of multiple exons 1. These observations suggest that the tissue-specific expression of the CST gene is explained by alternative usage of multiple 5'-UTR exons flanked with tissue-specific promoters. PMID- 10727930 TI - Functional expression of a locust visual pigment in transgenic Drosophila melanogaster. AB - The cDNA encoding a visual pigment of the locust Schistocerca gregaria has been inserted into the germline of the ninaE mutant of Drosophila melanogaster by P element-mediated transformation. Functional expression has been documented by recording light-regulated electroretinograms in transgenic flies. The spectral properties of the expressed visual pigment were determined with detergent solubilized material, prepared from the eyecups of the transgenic D. melanogaster. The recombinant locust pigment, as well as the genuine pigment of the fruitfly (Rh1) that served as a control for transformation/expression, showed photoreversibility between the pigment and metapigment forms. The absorptions of the difference spectra identify the locust visual pigment as a short wavelength absorbing, blue-light-sensitive photoreceptor. The absorption maxima are similar to those recorded on living locust animals. These results show that, although locust visual pigments contain 11-cis retinal as chromophore, the expressed protein is able to adopt 3-hydroxyretinal that is provided by the transgenic fruitflies. The electrophysiological recordings reveal that the locust visual pigment is able to induce phototransduction in the fruitfly. The reported results have two important consequences: On the one hand, the binding site of the locust opsin is apparently able to interact with the 3-hydroxyretinal from Drosophila in a way that the biological signal generated by the photoisomerization of the chromophore can be used by the protein to adopt a physiologically active conformation. On the other hand, despite the relatively large phylogenetic distance between both insect species, the extent of conservation between the protein domains thought to be involved in G-protein activation is striking. PMID- 10727931 TI - Mouse Hsp25, a small shock protein. The role of its C-terminal extension in oligomerization and chaperone action. AB - Under conditions of cellular stress, small heat shock proteins (sHsps), e.g. Hsp25, stabilize unfolding proteins and prevent their precipitation from solution. 1H NMR spectroscopy has shown that mammalian sHsps possess short, polar and highly flexible C-terminal extensions. A mutant of mouse Hsp25 without this extension has been constructed. CD spectroscopy reveals some differences in secondary and tertiary structure between this mutant and the wild-type protein but analytical ultracentrifugation and electron microscopy show that the proteins have very similar oligomeric masses and quaternary structures. The mutant shows chaperone ability comparable to that of wild-type Hsp25 in a thermal aggregation assay using citrate synthase, but does not stabilize alpha-lactalbumin against precipitation following reduction with dithiothreitol. The accessible hydrophobic surface of the mutant protein is less than that of the wild-type protein and the mutant is also less stable at elevated temperature. 1H NMR spectroscopy reveals that deletion of the C-terminal extension of Hsp25 leads to induction of extra C terminal flexibility in the molecule. Monitoring complex formation between Hsp25 and dithiothreitol-reduced alpha-lactalbumin by 1H NMR spectroscopy indicates that the C-terminal extension of Hsp25 retains its flexibility during this interaction. Overall, these data suggest that a highly flexible C-terminal extension in mammalian sHsps is required for full chaperone activity. PMID- 10727932 TI - Epidermal growth factor receptor is modulated by redox through multiple mechanisms. Effects of reductants and H2O2. AB - The cellular redox state has been shown to play an essential role in cellular signaling systems. Here we investigate the effects of reductants and H2O2 on the signaling of epidermal growth factor (EGF) in cells. H2O2 induced the phosphorylation of the EGF receptor and the formation of a receptor complex comprising Shc, Grb2, Sos, and the EGF receptor. Dimerization or oligomerization of the EGF receptor was not induced by H2O2. Protein tyrosine phosphatase (PTP) assay showed that H2O2 suppressed dephosphorylation of the EGF receptor in cell lysates, suggesting that inactivation of PTP was involved in H2O2-induced activation of the EGF receptor. In contrast, the reductants N-acetyl-L-cysteine [Cys(Ac)] and dithiothreitol markedly suppressed EGF-induced dimerization and activation of the EGF receptor in cells. In accordance with suppression of the EGF receptor, Cys(Ac) suppressed EGF-induced activation of Ras, phosphatidylinositol 3-kinase and mitogen-activated protein kinase. Dithiothreitol completely inhibited EGF binding and kinase activation of the EGF receptor both in vitro and in vivo. In contrast, Cys(Ac) suppressed high-affinity EGF-binding sites on the cells, but had no effect on low-affinity binding sites. Furthermore, Cys(Ac) did not suppress EGF-induced kinase activation or dimerization of the EGF receptor in vitro, indicating that it suppressed the EGF receptor through a redox-sensitive cellular process or processes. Thus, the EGF receptor is regulated by redox through multiple steps including dephosphorylation by PTP, ligand binding, and a Cys(Ac)-sensitive cellular process or processes. PMID- 10727933 TI - Isolation of ERp29, a novel endoplasmic reticulum protein, from rat enamel cells. Evidence for a unique role in secretory-protein synthesis. AB - Recently we cloned and described ERp29, a novel 29-kDa endoplasmic reticulum (ER) protein that is widely expressed in rat tissues. Here we report our original isolation of ERp29 from dental enamel cells, and the comprehensive sequence analysis that correlated ERp29 with its cognate cDNA, both in enamel cells and liver. Fractionation of enamel cells using a new freeze-thaw procedure showed that ERp29 partitioned with known reticuloplasmins, and not with soluble proteins from mitochondria or cytosol. The absence of ERp29 in secreted enamel matrix indicated that the C-terminal tetrapeptide (KEEL motif) confers effective ER retention in enamel cells. ERp29 behaved as a single species (approximately 40 kDa) during size-exclusion chromatography of liver reticuloplasm, suggesting that most ERp29 is not stably associated with other proteins. Immunoblot analysis showed that ERp29 was up-regulated during enamel secretion and expressed most highly in secretory tissues, indicative of a role in secretory-protein synthesis. Unlike other reticuloplasmins, ERp29 was down-regulated during enamel mineralization and thereby dissociated from a calcium-handling role. Tissue specific variations in ERp29 molecular abundance were revealed by quantification of reticuloplasmin mole ratios. IN CONCLUSION: (a) ERp29 is a novel reticuloplasmin of general functional importance; (b) a unique role in protein processing is implicit from the distinctive expression patterns and molecular structure; (c) ERp29 is primarily involved in normal protein secretory events, not the ER stress response; (d) a major role is likely in tissues where ERp29 was equimolar with established molecular chaperones and foldases. This study implicates ERp29 as a new member of the ER protein-processing machinery, and identifies tissues where the physiological role of ERp29 is most likely to be clearly manifested. PMID- 10727934 TI - Expression, stability and performance of the three-component alkane mono oxygenase of Pseudomonas oleovorans in Escherichia coli. AB - We tested the synthesis and in vivo function of the inducible alkane hydroxylase of Pseudomonas oleovorans GPo1 in several Escherichia coli recombinants. The enzyme components (AlkB, AlkG and AlkT) were synthesized at various rates in different E. coli hosts, which after induction produced between twofold and tenfold more of the Alk components than did P. oleovorans. The enzyme components were less stable in recombinant E. coli hosts than in P. oleovorans. In addition, the specific activity of the alkane mono-oxygenase component AlkB was five or six times lower in E. coli than in P. oleovorans. Evidently, optimal functioning of the hydroxylase system requires factors or a molecular environment that are available in Pseudomonas but not in E. coli. These factors are likely to include correct interactions of AlkB with the membrane and incorporation of iron into the AlkG and AlkB apoproteins. PMID- 10727935 TI - Substrate specificity of a maize ribosome-inactivating protein differs across diverse taxa. AB - The superfamily of ribosome-inactivating proteins (RIPs) consists of toxins that catalytically inactivate ribosomes at a universally conserved region of the large ribosomal RNA. RIPs carry out a single N-glycosidation event that alters the binding site of the translational elongational factor eEF1A and causes a cessation of protein synthesis that leads to subsequent cell death. Maize RIP1 is a kernel-specific RIP with the unusual property of being produced as a zymogen, proRIP1. ProRIP1 accumulates during seed development and becomes active during germination when cellular proteases remove acidic residues from a central domain and both termini. These deletions also result in RIP activation in vitro. However, the effectiveness of RIP1 activity against target ribosomes remains species-dependent. To determine the potential efficiency of maize RIP1 as a plant defense protein, we used quantitative RNA gel blots to detect products of RIP activity against intact ribosomal substrates from various species. We determined the enzyme specificity of recombinant maize proRIP1 (rproRIP1), papain-activated rproRIP1 and MOD1 (an active deletion mutant of rproRIP1) against ribosomal substrates with differing levels of RIP sensitivity. The rproRIP1 had no detectable enzymatic activity against ribosomes from any of the species assayed. The papain-activated rproRIP1 was more active than MOD1 against ribosomes from either rabbit or the corn pathogen, Aspergillus flavus, but the difference was much more marked when rabbit ribosomes were used as a substrate. The papain activated rproRIP1 was much more active against rabbit ribosomes than homologous Zea mays ribosomes and had no detectable effect on Escherichia coli ribosomes. PMID- 10727936 TI - Characterization of potato proteinase inhibitor II reactive site mutants. AB - Potato proteinase inhibitor II (PI-2) is composed of two sequence repeats. It contains two reactive site domains. We developed an improved protocol for the production of PI-2 using the yeast Pichia pastoris as the expression host. We then assessed the role of its two reactive sites in the inhibition of trypsin and chymotrypsin by mutating each of the two reactive sites in various ways. From these studies it appears that the second reactive site strongly inhibits both trypsin (Ki = 0.4 nM) and chymotrypsin (Ki = 0.9 nM), and is quite robust towards mutations at positions P2 or P1'. In contrast, the first reactive site inhibits only chymotrypsin (Ki = 2 nM), and this activity is very sensitive to mutations. Remarkably, replacing the reactive site amino acids of domain I with those of domain II did not result in inhibitory activities similar to domain II. The fitness for protein engineering of each domain is discussed. PMID- 10727937 TI - Characterization of L-carnitine transport into rat skeletal muscle plasma membrane vesicles. AB - Transport of L-carnitine into skeletal muscle was investigated using rat sarcolemmal membrane vesicles. In the presence of an inwardly directed sodium chloride gradient, L-carnitine transport showed a clear overshoot. The uptake of L-carnitine was increased, when vesicles were preloaded with potassium. When sodium was replaced by lithium or cesium, and chloride by nitrate or thiocyanate, transport activities were not different from in the presence of sodium chloride. However, L-carnitine transport was clearly lower in the presence of sulfate or gluconate, suggesting potential-dependent transport. An osmolarity plot revealed a positive slope and a significant intercept, indicating transport of L-carnitine into the vesicle lumen and binding to the vesicle membrane. Displacement experiments revealed that approximately 30% of the L-carnitine associated with the vesicles was bound to the outer and 30% to the inner surface of the vesicle membrane, whereas 40% was unbound inside the vesicle. Saturable transport could be described by Michaelis-Menten kinetics with an apparent Km of 13.1 microM and a Vmax of 2.1 pmol.(mg protein-1).s-1. L-Carnitine transport could be trans stimulated by preloading the vesicles with L-carnitine but not with the carnitine precursor butyrobetaine, and was cis-inhibited by L-palmitoylcarnitine, L isovalerylcarnitine, and glycinebetaine. On comparing carnitine transport into rat kidney brush-border membrane vesicles and OCTN2, a sodium-dependent high affinity human carnitine transporter, cloned recently from human kidney also expressed in muscle, the Km values are similar but driving forces, pattern of inhibition and stereospecificity are different. This suggests the existence of more than one carnitine carrier in skeletal muscle. PMID- 10727938 TI - Nickel serves as a substrate recognition motif for the endopeptidase involved in hydrogenase maturation. AB - The interaction of the hydrogenase maturation endopeptidase HycI with its substrate, the precursor of the large subunit, was studied. Replacement of conserved amino-acid residues in HycI, which have been shown to bind a cadmium ion from the crystallization buffer in crystals of HybD (endopeptidase for hydrogenase 2), abolished or strongly reduced processing activity. Atomic absorption spectroscopy of purified HycI and HybD proteins showed the absence of nickel. In vitro processing assays showed that the reaction requires nickel to be bound to the precursor and the protease does not have a function in nickel delivery to the substrate. Radioactive labelling of cells with 63Ni, devoid of endopeptidase, resolved several forms of the precursor which are possibly intermediates in the maturation pathway. It is concluded that the endopeptidase uses the metal in the large subunit of [NiFe]-hydrogenases as a recognition motif. PMID- 10727939 TI - Formation of a new class of oxylipins from N-acyl(ethanol)amines by the lipoxygenase pathway. AB - N-Acylethanolamines (NAEs) constitute a new class of plant lipids and are thought to play a role in plant defense strategies against pathogens. In plant defense systems, oxylipins generated by the lipoxygenase pathway are important actors. To date, it is not known whether plants also use endogeneous oxylipins derived from NAEs in their defense reactions. We tested whether members of the NAE class can be converted by enzymes constituting this pathway, such as (soybean) lipoxygenase 1, (alfalfa) hydroperoxide lyase and (flax seed) allene oxide synthase. We found that both alpha-N-linolenoylethanolamine and gamma-N-linolenoylethanolamine (18:3), as well as alpha-N-linolenoylamine and gamma-N-linolenoylamine were converted into their (13S)-hydroperoxide derivatives by lipoxygenase. Interestingly, only the hydroperoxides of alpha-N-linolenoyl(ethanol)amines and their linoleic acid analogs (18:2) were suitable substrates for hydroperoxide lyase. Hexanal and (3Z)-hexenal were identified as volatile products of the 18:2 and 18:3 fatty acid (ethanol)amides, respectively. 12-Oxo-N-(9Z) dodecenoyl(ethanol)amine was the nonvolatile hydrolysis product. Kinetic studies with lipoxygenase and hydroperoxide lyase revealed that the fatty acid ethanolamides were converted as readily or even better than the corresponding free fatty acids. Allene oxide synthase utilized all substrates, but was most active on (13S)-hydroperoxy-alpha-N-linolenoylethanolamine and the (13S) hydroperoxide of linoleic acid and its ethanolamine derivative. alpha-Ketols and gamma-ketols were characterized as products. In addition, cyclized products, i.e. 12-oxo-N-phytodienoylamines, derived from (13S)-hydroperoxy-alpha-N linolenoylamines were found. The results presented here show that, in principle, hydroperoxide NAEs can be formed in plants and subsequently converted into novel phytooxylipins. PMID- 10727940 TI - Biological activities of lipopolysaccharides are determined by the shape of their lipid A portion. AB - Lipopolysaccharide (LPS) represents a major virulence factor of Gram-negative bacteria ('endotoxin') that can cause septic shock in mammals including man. The lipid anchor of LPS to the outer membrane, lipid A, has a peculiar chemical structure, harbours the 'endotoxic principle' of LPS and is responsible for the expression of pathophysiological effects. Chemically modified lipid A can be endotoxically inactive, but may express strong antagonistic activity against LPS, a property that can be utilized in antisepsis treatment. We show here that these different biological activities are directly correlated with the molecular shape of lipid A. Only (hexaacyl) lipid A with a conical/concave shape, the cross section of the hydrophobic region being larger than that of the hydrophilic region, exhibited strong interleukin-6 (IL-6)-inducing capacity. Most strikingly, a correlation between a cylindrical molecular shape of lipid A and antagonistic activity was established: IL-6 induction by enterobacterial LPS was inhibited by cylindrically shaped lipid A except for compounds with reduced headgroup charge. The antagonistic action is interpreted by assuming that lipid A molecules intercalate into the cytoplasmic membrane of mononuclear cells, and subsequently blocking of the putative signaling protein by the lipid A with cylindrical shape. PMID- 10727941 TI - The structure of the linkage between the O-specific polysaccharide and the core region of the lipopolysaccharide from Salmonella enterica serovar Typhimurium revisited. AB - Salmonella enterica sv. Typhimurium strain 1135 possesses smooth(S)-form lipopolysaccharide (LPS). Although the structures of the core region and the O specific polysaccharide were investigated intensively between the 1960s and the 1980s, the structure of the linkage region between the O-chain and the core was not elucidated unequivocally. By using modern MS and high-field NMR spectroscopy for analysis of the isolated carbohydrate backbone of the LPS, it has been shown that it is a beta-D-Galp residue that links the first repeating unit of the O specific polysaccharide to O-4 of the last D-Glcp residue of the core region. Interestingly, this particular D-Galp residue is alpha-linked in all following repeating units. The data are discussed with regard to the ligation of O-specific polysaccharide and core region during LPS biosynthesis. PMID- 10727942 TI - Gene cloning, nucleotide sequencing, and purification and characterization of the D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3. AB - The gene encoding the D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3 was cloned and sequenced. Analysis of 7.3 kb of genomic DNA revealed the presence of six ORFs, one of which (daaA) encodes the D-amino-acid amidase. This enzyme, DaaA, is composed of 363 amino-acid residues (molecular mass 40 082 Da), and the deduced amino-acid sequence exhibits homology to alkaline D peptidase from Bacillus cereus DF4-B (32% identity), DD-peptidase from Streptomyces R61 (29% identity), and other penicillin-recognizing proteins. The DaaA protein contains the typical SXXK, YXN, and H(K)XG active-site motifs identified in the penicillin-binding proteins and beta-lactamases. The daaA gene modified in the nucleotide sequence upstream from its start codon was overexpressed in Escherichia coli. The activity of the recombinant DaaA enzyme in cell-free extracts of E. coli was 33.6 U. mg-1 with D-phenylalaninamide as substrate, which is about 350-fold higher than in extracts of O. anthropi SV3. This enzyme was purified to electrophoretic homogeneity by ammonium sulfate fractionation and three column chromatography steps. On gel-filtration chromatography, DaaA appeared to be a monomer with a molecular mass of 40 kDa. It had maximal activity at 45 degrees C and pH 9.0, and was completely inactivated in the presence of phenylmethanesulfonyl fluoride or Zn2+. DaaA had hydrolyzing activity toward D-amino-acid amides with aromatic or hydrophobic side chains, but did not act on the substrates for the DD-peptidase and beta-lactamase, despite their sequence similarity to DaaA. The characteristics of the recombinant DaaA are similar to those found for the native enzyme partially purified from O. anthropi SV3. PMID- 10727943 TI - Intracellular site of gamma-secretase cleavage for Abeta42 generation in neuro 2a cells harbouring a presenilin 1 mutation. AB - Previously, we reported that mutations in presenilin 1 (PS1) increased the intracellular levels of amyloid beta-protein (Abeta)42. However, it is still not known at which cellular site or how PS1 mutations exert their effect of enhancing Abeta42-gamma-secretase cleavage. In this study, to clarify the molecular mechanisms underlying this enhancement of Abeta42-gamma-secretase cleavage, we focused on determining the intracellular site of the cleavage. To address this issue, we used APP-C100 encoding the C-terminal beta-amyloid precursor protein (APP) fragment truncated at the N terminus of Abeta (C100); C100 requires only gamma-secretase cleavage to yield Abeta. Mutated PS1 (M146L)-induced Neuro 2a cells showed enhanced Abeta1-42 generation from transiently expressed C100 as well as from full-length APP, whereas the generation of Abeta1-40 was not increased. The intracellular generation of Abeta1-42 from transiently expressed C100 in both mutated PS1-induced and wild-type Neuro 2a cells was inhibited by brefeldin A. Moreover, the generation of Abeta1-42 and Abeta1-40 from a C100 mutant containing a di-lysine endoplasmic reticulum retention signal was greatly decreased, indicating that the major intracellular site of gamma-secretase cleavage is not the endoplasmic reticulum. The intracellular generation of Abeta1 42/40 from C100 was not influenced by monensin treatment, and the level of Abeta1 42/40 generated from C100 carrying a sorting signal for the trans-Golgi network was higher than that generated from wild-type C100. These results using PS1 mutation-harbouring and wild-type Neuro 2a cells suggest that Abeta42/40-gamma secretase cleavages occur in the Golgi compartment and the trans-Golgi network, and that the PS1 mutation does not alter the intracelluar site of Abeta42-gamma secretase cleavage in the normal APP proteolytic processing pathway. PMID- 10727944 TI - Isolation and characterization of novel inducible serine protease inhibitors from larval hemolymph of the greater wax moth Galleria mellonella. AB - Three inducible serine protease inhibitors (ISPI-1, 2, 3) have been purified from larval hemolymph of greater wax moth larvae, Galleria mellonella, and characterized at a molecular level. These inhibitors were synthesized after larvae were injected with a yeast polysaccharide, zymosan preparation. ISPI-1,2,3 were active against various serine proteases including trypsin and toxic proteases released by the entomopathogenic fungus Metarhizium anisopliae. Precipitation by trichloroacetic acid and heat, followed by FPLC and HPLC separation steps were used for purification of the protease inhibitors from cell free hemolymph samples. The molecular masses of purified proteins were determined by MS to be 9.2 kDa (ISPI-1), 6.3 kDa (ISPI-2) and 8.2 kDa (ISPI-3) with isoelectric points ranging between 7.2 and 8.3. The N-terminal amino-acid sequences of ISPI-1 and ISPI-3 are not similar to other known proteins, whereas that of ISPI-2 exhibits extensive similarity to known Kunitz-type protease inhibitors. PMID- 10727945 TI - Aldehyde oxidoreductase activity in Desulfovibrio alaskensis NCIMB 13491 EPR assignment of the proximal [2Fe-2S] cluster to the Mo site. AB - A novel molybdenum iron-sulfur-containing aldehyde oxidoreductase (AOR) belonging to the xanthine oxidase family was isolated and characterized from the sulfate reducing bacterium Desulfovibrio alaskensis NCIMB 13491, a strain isolated from a soured oil reservoir in Purdu Bay, Alaska. D. alaskensis AOR is closely related to other AORs isolated from the Desulfovibrio genus. The protein is a 97-kDa homodimer, with 0.6 +/- 0.1 Mo, 3.6 +/- 0.1 Fe and 0.9 +/- 0.1 pterin cytosine dinucleotides per monomer. The enzyme catalyses the oxidation of aldehydes to their carboxylic acid form, following simple Michaelis-Menten kinetics, with the following parameters (for benzaldehyde): K(app/m)= 6.65 microM; V app = 13.12 microM.min(-1); k(app/cat) = 0.96 s(-1). Three different EPR signals were recorded upon long reduction of the protein with excess dithionite: an almost axial signal split by hyperfine interaction with one proton associated with Mo(V) species and two rhombic signals with EPR parameters and relaxation behavior typical of [2Fe-2S] clusters termed Fe/S I and Fe/S II, respectively. EPR results reveal the existence of magnetic interactions between Mo(V) and one of the Fe/S clusters, as well as between the two Fe/S clusters. Redox titration monitored by EPR yielded midpoint redox potentials of -275 and -325 mV for the Fe/S I and Fe/S II, respectively. The redox potential gap between the two clusters is large enough to obtain differentiated populations of these paramagnetic centers. This fact, together with the observed interactions among paramagnetic centers, was used to assign the EPR-distinguishable Fe/S I and Fe/S II to those seen in the reported crystal structures of homologous enzymes. PMID- 10727946 TI - Functional and molecular identification of novel members of the ubiquitous membrane fusion proteins alpha- and gamma-SNAP (soluble N-ethylmaleimide sensitive factor-attachment proteins) families in Dictyostelium discoideum. AB - The soluble N-ethylmaleimide-sensitive-factor-attachment proteins (SNAP) are eukaryotic soluble proteins required for membrane fusion. Based on their initial identification in bovine brain cytosol, they are divided in alpha/beta and gamma subfamilies. SNAPs act as adapters between N-ethylmaleimide-sensitive factor (NSF), a hexameric ATPase, and membrane SNARE proteins (SNAP receptors). Within the NSF/SNAP/SNARE complex, SNAPs contribute to the catalysis of an ATP-driven conformational change in the SNAREs, resulting in dissociation of the complex. We have constructed a Dictyostelium discoideum strain overexpressing a c-myc-tagged form of D. discoideum NSF (NSF-myc). Its immunoprecipitation from detergent solubilized membrane extracts reveals two associated polypeptides with apparent molecular masses of 33 and 36 kDa (p33 and p36) that are absent in NSF-myc immunoprecipitates from cytosol. Analysis of trypsin-digested peptides by microsequencing and mass spectrometry and comparison with cDNA sequences identify p33 and p36 as the D. discoideum homologues of alpha- and gamma-SNAP, respectively. The alpha-/gamma-SNAP molar ratio is close to 3 in vegetative amoebae from this organism. The molecular identification of gamma-SNAP in plants (Arabidopsis thaliana) and insects (Drosophila melanogaster) documents, for the first time, the wide distribution of the gamma subtype. Altogether, these results suggest a specific role for gamma-SNAP, distinct from that of alpha-SNAP. PMID- 10727947 TI - Effect of streptozotocin-induced diabetes on rat liver Na+/K+-ATPase. AB - Na+/K+-ATPase during diabetes may be regulated by synthesis of its alpha and beta subunits and by changes in membrane fluidity and lipid composition. As these mechanisms were unknown in liver, we studied in rats the effect of streptozotocin induced diabetes on liver Na+/K+-ATPase. We then evaluated whether fish oil treatment prevented the diabetes-induced changes. Diabetes mellitus induced an increased Na+/K+-ATPase activity and an enhanced expression of the beta1 subunit; there was no change in the amount of the alpha1 and beta3 isoenzymes. Biphasic ouabain inhibition curves were obtained for diabetic groups indicating the presence of low and high affinity sites. No alpha2 and alpha3 isoenzymes could be detected. Diabetes mellitus led to a decrease in membrane fluidity and a change in membrane lipid composition. The diabetes-induced changes are not prevented by fish oil treatment. The results suggest that the increase of Na+/K+-ATPase activity can be associated with the enhanced expression of the beta1 subunit in the diabetic state, but cannot be attributed to changes in membrane fluidity as typically this enzyme will increase in response to an enhancement of membrane fluidity. The presence of a high-affinity site for ouabain (IC50 = 10-7 M) could be explained by the presence of (alphabeta)2 diprotomeric structure of Na+/K+ ATPase or an as yet unknown alpha subunit isoform that may exist in diabetes mellitus. These stimulations might be related, in part, to the modification of fatty acid content during diabetes. PMID- 10727948 TI - Requirements for the mitochondrial import and localization of dihydroorotate dehydrogenase. AB - In animals, dihydroorotate dehydrogenase (DHODH) is a mitochondrial protein that carries out the fourth step in de novo pyrimidine biosynthesis. Because this is the only enzyme of this pathway that is localized to mitochondria and because the enzyme is cytosolic in some bacteria and fungi, we carried out studies to understand the mode of targeting of animal DHODH and its submitochondrial localization. Analysis of fractionated rat liver mitochondria revealed that DHODH is an integral membrane protein exposed to the intermembrane space. In vitro synthesized Drosophila, rat and human DHODH proteins were efficiently imported into the intermembrane space of isolated yeast mitochondria. Import did not alter the size of the in vitro synthesized protein, nor was there a detectable size difference when compared to the DHODH protein found in vivo. Thus, there is no apparent proteolytic processing of the protein during import either in vitro or in vivo. Import of rat DHODH into isolated yeast mitochondria required inner membrane potential and was at least partially dependent upon matrix ATP, indicating that its localization uses the well described import machinery of the mitochondrial inner membrane. The DHODH proteins of animals differ from the cytosolic proteins found in some bacteria and fungi by the presence of an N terminal segment that resembles mitochondrial-targeting presequences. Deletion of the cationic portion of this N-terminal sequence from the rat DHODH protein blocked its import into isolated yeast mitochondria, whereas deletion of the adjacent hydrophobic segment resulted in import of the protein into the matrix. Thus, the N-terminus of the DHODH protein contains a bipartite signal that governs import and correct insertion into the mitochondrial inner membrane. PMID- 10727949 TI - Evidence against a direct role of the integrin alpha2beta1 in collagen-induced tyrosine phosphorylation in human platelets. AB - In the present study we have investigated whether the collagen receptor alpha2beta1 (GPIa-IIa; GP, glycoprotein) regulates protein tyrosine phosphorylation in platelets directly through activation of tyrosine kinases or indirectly through modification of the response to GPVI. The interaction of collagen with alpha2beta1 was inhibited in two distinct ways, using the metalloprotease jararhagin, which cleaves the beta1 subunit, or the antibody P1E6 which competes with binding of collagen to the integrin. The two inhibitors caused a shift to the right in the collagen concentration response curves for protein tyrosine phosphorylation and platelet activation consistent with a causal relationship between the two events. There was no change in the overall pattern of tyrosine phosphorylation in response to high concentrations of collagen in the presence of alpha2beta1 blockade demonstrating that the integrin is not required for this event. In contrast, jararhagin and P1E6 had a small, almost negligible inhibitory effect against responses to the GPVI-selective agonist collagen related peptide (CRP) and the G protein-coupled receptor agonist thrombin. Crosslinking of alpha2beta1 in solution or by adhesion to a monolayer using a variety of antibodies to either subunit of the integrin did not induce detectable protein tyrosine phosphorylation in whole cell lysates. The snake venom toxin trimucytin-stimulated a similar pattern of tyrosine phosphorylation to that induced by crosslinking of GPVI which was maintained in the presence of jararhagin. Trimucytin may therefore induce activation via GPVI rather than alpha2beta1 as previously thought. These observations show that the integrin alpha2beta1 is not required for regulation of tyrosine phosphorylation by collagen. PMID- 10727950 TI - Turkey cytochrome c oxidase contains subunit VIa of the liver type associated with low efficiency of energy transduction. AB - Cytochrome c oxidase was isolated from turkey liver, heart and breast skeletal muscle and separated by SDS/PAGE. The N-terminal amino-acid sequence of subunit VIa from all tissues and internal sequences from the skeletal muscle enzyme show homology to the mammalian liver-type subunit VIaL, which was verified by isolation and sequencing of the cDNA of turkey subunit VIa. No cDNA corresponding to subunit VIaH (mammalian heart-type) could be found by RACE-PCR with mRNA from all turkey tissues. Measurement of proton translocation with the reconstituted enzymes from turkey liver and heart revealed H+/e- ratios below 0.5 that were independent of the intraliposomal ATP/ADP ratio, as previously found with the bovine liver enzyme. Under identical conditions, the bovine heart enzyme revealed H+/e- ratios of 0.85 at low and 0.48 at high intraliposomal ATP/ADP ratios. The results suggest that in birds the lower H+/e-ratio of cytochrome c oxidase participates in elevated resting metabolic rate and thermogenesis. PMID- 10727951 TI - Structural characterization of the oligosaccharide chains of human alpha1 microglobulin from urine and amniotic fluid. AB - Human alpha1-microglobulin (alpha1-m; also called protein HC), a glycoprotein belonging to the lipocalin superfamily, was isolated by sequential anion-exchange chromatography and gel filtration from the urine of hemodialized patients and from amniotic fluid collected in the week 16-18 of pregnancy. The carbohydrate chains of the protein purified from the two sources, which are organized in two Asn-linked and one Thr-linked oligosaccharides, were structurally characterized using matrix-assisted laser desorption ionization and electrospray mass spectrometry. The glycans attached to Thr5 are differently truncated NeuHexHexNAc sequences, and O-glycosylation in the amniotic fluid protein is only partial. Asn96 has both diantennary and triantennary structures attached in the case of urinary alpha1-m and only diantennary glycans in the amniotic fluid protein. The main carbohydrate units attached to Asn17 are in both proteins monosialylated and disialylated diantennary glycans. The position of the oligosaccharide chains in a three-dimensional model of the protein, produced using the automated Swiss-Model service, is also discussed. PMID- 10727952 TI - Identification of tudor repeat associator with PCTAIRE 2 (Trap). A novel protein that interacts with the N-terminal domain of PCTAIRE 2 in rat brain. AB - PCTAIRE 2 is a Cdc2-related kinase that is predominantly expressed in the terminally differentiated neuron. To elucidate the function of PCTAIRE 2, proteins that associate with PCTAIRE 2 were screened by the yeast two-hybrid system. A positive clone was found to encode a novel protein that could bind to PCTAIRE 2 in vitro as well as in vivo, and was designated as Trap (tudor repeat associator with PCTAIRE 2). The overall structure of Trap shows no significant homology to any proteins, but contains five repeated domains (the tudor-like domain), conserved in Drosophila tudor protein. Trap associates with the N terminal domain of PCTAIRE 2 through its C-terminal domain, which contains two tudor-like domains. PCTAIRE 1, but not PCTAIRE 3, can also associate with Trap. Trap is predominantly expressed in brain and testis, and gradually increases during brain development throughout life, consistent with the expression pattern of PCTAIRE 2. Immunoreactivities for PCTAIRE 2 and Trap were colocalized to the mitochondria in COS 7 cells. Immunohistochemical analyses showed that PCTAIRE 2 and Trap were distributed in the same cell layer of the cerebral cortex and cerebellum. These findings suggest that Trap is a physiological partner of PCTAIRE 2 in terminally differentiated neurons. PMID- 10727953 TI - Intracellular trafficking and release of intact edible mushroom lectin from HT29 human colon cancer cells. AB - Our previous studies have shown that the Galbeta1-3GalNAcalpha- (Thomsen Friedenreich antigen)-binding lectin from the common edible mushroom Agaricus bisporus (ABL) reversibly inhibits cell proliferation, and this effect is a consequence of inhibition of nuclear localization sequence-dependent nuclear protein import after ABL internalization [Yu, L.G., Fernig, D.G., White, M.R.H., Spiller, D.G., Appleton, P., Evans, R.C., Grierson, I., Smith, J.A., Davies, H., Gerasimenko, O.V., Petersen, O.H., Milton, J.D. & Rhodes, J.M. (1999) J. Biol. Chem. 274, 4890-4899]. Here, we have investigated further the intracellular trafficking and fate of ABL after internalization in HT29 human colon cancer cells. Internalization of 125I-ABL occurred within 30 min of the lectin being bound to the cell surface. Subcellular fractionation after pulse labelling of the cells with 125I-ABL for 2 h at 4 degrees C followed by culture of the cells at 37 degrees C demonstrated a steady increase in radioactivity in a crude nuclear extract. The radioactivity in this extract reached a maximum after 10 h and declined after 20 h. Release of ABL from the cell, after pulse labelling, was assessed using both fluorescein isothiocyanate-labelled ABL and 125I-ABL and was slow, with a t1/2 of 48 h. Most of the 125I-ABL both inside cells and in the medium remained intact, as determined by trichloroacetic acid precipitation and SDS/PAGE, and after 48 h only 22 +/- 2% of ABL in the medium and 14 +/- 2% inside the cells was degraded. This study suggests that the reversibility of the antiproliferative effect of ABL is associated with its release from cells after internalization. The internalization and subsequent slow release, with little degradation of ABL, reflects the tendency of lectins to resist biodegradation and implies that other endogenous or exogenous lectins may be processed in this way by intestinal epithelial cells. PMID- 10727955 TI - Getting started in academic cardiothoracic surgery. AB - Preparing to begin a career in academic cardiothoracic surgery requires forethought and desire. Success mandates honesty, discipline, opportunity, and support. This article will attempt to review some fundamental concepts important in starting such an academic career. The thoughts are somewhat personal and not meant to be inclusive. The article will briefly discuss the following issues: choosing the first job, transitions, effective time management, developing clinical confidence, the continued need for mentorship, developing educational value, developing a philosophy of academic growth, intellectual and emotional honesty, myths, mental and physical health, and keys to success. PMID- 10727954 TI - Compact residual structure in lentil lectin at pH 2. AB - Lentil lectin obtained from Lens culinaris collected in the La Armuna area (Salamanca, Spain) was examined by high-sensitivity differential scanning calorimetry, fluorimetry and measurements of circular dichroism at pH 2.0 and 7.4. At pH 2.0 the lentil lectin is not in the native state; however, at this pH it does show signs of a residual structure that breaks down upon heating. The lentil lectin at pH 2 shares some similarities with what has become known as the molten globule state. The thermal denaturation of intact (pH 7.4) and partially unfolded (pH 2.0) lentil lectin was irreversible and strongly dependent upon the scan rate, suggesting that its denaturation is under kinetic control. The process of lentil lectin denaturation is interpreted in terms of the simple kinetic model, Nk --> D, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state. PMID- 10727956 TI - Clinical science research. AB - Clinical science research incorporates the fields of clinical investigation and health services research. With a focus on the use of either human specimens or subjects, clinical investigation research projects translate knowledge gained from basic science research based on animal models for disease. The goal of clinical investigation is to develop new prevention, intervention, and therapeutic approaches to improve patient clinical outcomes. In contrast, health services research focuses on the improvement of the efficacy, cost-effectiveness, and outcomes of care. Health services research projects examine options to improve the health care delivery system, organization, financing, and reimbursement mechanisms in place today. The purpose of this article is to review common terminology and methodologic approaches that are used in clinical science research. The process of designing a research project is reviewed. Beginning with the development of a research question and hypothesis, the steps for successful completion of the project are discussed. Different study design approaches are presented with their respective strengths and weaknesses. The challenges associated with conducting a clinical research study are discussed, including the development of an appropriate sampling strategy, the designing of data collection, instruments, and the assurance of study data integrity. Possible threats to study validity and generalizability are assessed.One the major advantages of clinical research is that it offers an opportunity to study clinical questions in the clinical setting without the expenses of a basic research laboratory and basic science technology. Thus important clinical questions related to patient care, new technology assessment, clinical practice management, health care administration, or health policy may be addressed. PMID- 10727957 TI - Developing the academic thoracic surgeon: teaching surgery. AB - Teaching surgery can be a very gratifying experience for those of us involved in academic thoracic surgery. Fundamentals of a good residency program require that patients should always be placed in the highest priority. However, the residency program should also be committed to teaching as a priority. Creating the proper operating room environment is essential for optimal conduct of the operation. This environment is similar to that of the airline industry, which is known as crew or cockpit resource management. The design of a teaching program needs to have evaluation as one of its key elements. In addition to resident evaluation, it is also important to have faculty evaluation by the residents. The goal of any residency program should be to foster the development of the future leaders in our specialty. The information contained within this article represents the art and science of teaching thoracic surgery as applied by the faculty in the Division of Cardiac Surgery at The Johns Hopkins Hospital. PMID- 10727958 TI - Getting funded. AB - Getting funded is still possible for cardiothoracic surgeons. You must have a clear hypothesis, have an organized approach, and develop excellent preliminary data. Most important, you need to apply to get funded. PMID- 10727959 TI - The academic surgeon and industry. AB - Academic surgeons should be aware of the tremendous potential that exists to partner with private companies on projects relating either to basic or clinical research. This is particularly timely now because many of these companies are experiencing unprecedented growth and market valuation. The development of these relationships requires knowing who to deal with as well as how to negotiate. Most academic medical centers have individuals who have expertise in developing sponsored research agreements, and young investigators should take advantage of this expertise. PMID- 10727960 TI - Developing administrative skills. PMID- 10727961 TI - Innovation in surgery. PMID- 10727962 TI - Becoming a surgical leader. PMID- 10727963 TI - Mentoring. PMID- 10727964 TI - A view on the science: physical anthropology at the millennium. AB - EDITOR's NOTE The year 2000 marks the onset of the 21st century. Physical anthropologists will provide brief reflections on our discipline, including what attracted them to it, and their views on the directions our discipline may pursue as we enter, in January 2001, the third millennium. PMID- 10727965 TI - On the age of the hominid fossils at the Sima de los Huesos, Sierra de Atapuerca, Spain: paleomagnetic evidence. AB - We report new paleomagnetic data for the Middle Pleistocene hominid-bearing strata in the Sima de los Huesos, North Spain. Sediments (brown muds with human and bear fossils and the underlying sterile clayey and sandy unit) preserve both normal and reversed magnetic components. The sterile unit has exclusively reversed magnetization, dating back to the Matuyama Chron, and thus is Lower Pleistocene in age. The overlying fossiliferous muds have a dominant normal magnetization that overprints a partially resolved reversed magnetization. These data are compatible with one of the reversal events that occurred during the Brunhes Chron. Combined with the existing U-series dates and evidence from the macro- and microfauna, these paleomagnetic results suggest an age of the hominid fossils between 325 to 205 ka, whereas the underlying sand and silts are older than 780 ka. PMID- 10727966 TI - Statistical power comparisons among alternative morphometric methods. AB - This paper compares the statistical power of various tests that have been proposed to test for equality of shape in two populations. Power surfaces are computed with emphasis on the simplest case of three points in the plane (i.e., landmarks at the vertices of a triangle). Goodall's ([1991] J Roy Stat Soc Serb 53:285-339) F-test was found to have the highest power followed by T(2)-tests using Kendall tangent space coordinates. Power for T(2)-tests using Bookstein shape coordinates was good if the baseline was not the shortest side of the triangle. The Rao and Suryawanshi ([1996] Proc Natl Acad Sci 93:12132-12136 and Rao and Suryawanshi [1998] Proc Natl Acad Sci 95:4121-4125) shape variables had much lower power when triangles were not close to being equilateral. Power surfaces for the EDMA-I T statistic revealed very low power for many shape comparisons including those between very different shapes. Power surface for the EDMA-II Z statistic were also complicated and depended strongly on the choice of baseline used for size scaling. The type I error rate was also often not correct for this method. Results for more than three landmarks are also presented. The implications of the results for practical applications of morphometrics are discussed. PMID- 10727967 TI - Principal components analysis of distal humeral shape in Pliocene to recent African hominids: the contribution of geometric morphometrics. AB - The shape of the distal humerus in Homo, Pan (P. paniscus and P. troglodytes), Gorilla, and six australopithecines is compared using a geometric approach (Procrustes superimposition of landmarks). Fourteen landmarks are defined on the humerus in a two-dimensional space. Principal components analysis (PCA) is performed on all superimposed coordinates. I have chosen to discuss the precise place of KNM-KP 271 variously assigned to Australopithecus anamensis, Homo sp., or Praeanthropus africanus, in comparison with a sample of australopithecines. AL 288-1, AL 137-48 (Hadar), STW 431 (Sterkfontein), and TM 1517 (Kromdraai) are commonly attributed to Australopithecus afarensis (the two former), Australopithecus africanus, and Paranthropus robustus, respectively, while the taxonomic place of KNM-ER 739 (Homo or Paranthropus?) is not yet clearly defined. The analysis does not emphasize a particular affinity between KNM-KP 271 and modern Homo, nor with A. afarensis, as previously demonstrated (Lague and Jungers [1996] PMID- 10727968 TI - Phenotypic covariance structure in tamarins (genus Saguinus): a comparison of variation patterns using matrix correlation and common principal component analysis. AB - Constancy of variation/covariation structure among populations is frequently assumed in order to measure the differential selective forces which have caused population differentiation through evolutionary time. Following Steppan ([1997] Evolution 51:571-594), this assumption is examined among closely related tamarin species (genus Saguinus), using two distinct approaches applied to the task of evaluating similarity in patterns of morphological variation: common principal component analysis and matrix correlations. While the results of these analyses may appear contradictory, closer examination reveals them as complementary, highlighting the wisdom of combined methodologies. Overall, the results reveal a close relationship among the morphologically based variance structures of the tamarin species a relationship whose pattern is consistent with the pattern of phylogenetic relatedness as found via a molecular genetic study. More specifically, both methodological approaches provide some support for divergence of S. geoffroyi and S. oedipus (with regards to their patterns of morphological variation) from other tamarin species. This suggests that variance/covariance structure may have diverged through evolutionary time in the tamarin lineage, placing assumptions of constancy in doubt. PMID- 10727969 TI - Spatio-temporal gait characteristics of the hind-limb cycles during voluntary bipedal and quadrupedal walking in bonobos (Pan paniscus). AB - Spatio-temporal gait characteristics (step and stride length, stride frequency, duty factor) were determined for the hind-limb cycles of nine bonobos (Pan paniscus) walking quadrupedally and bipedally at a range of speeds. The data were recalculated to dimensionless quantities according to the principle of dynamic similarity. Lower leg length was used as the reference length. Interindividual variability in speed modulation strategy of bonobos appears to be low. Compared to quadrupedal walking, bipedal bonobos use smaller steps to attain a given speed (differences increase with speed), resulting in shorter strides at a higher frequency. In the context of the ("hybrid") dynamic pattern approach to locomotion (Latach, 1998) we argue that, despite these absolute differences, intended walking speed is the basic control variable which elicits both quadrupedal and bipedal walking kinematics in a similar way. Differences in the initial status of the dynamic system may be responsible for the differences in step length between both gaits. Comparison with data deduced from the literature shows that the effects of walking speed on stride length and frequency are similar in bonobos, common chimpanzees, and humans. This suggests that (at least) within extant homininae, spatio-temporal gait characteristics are highly comparable, and this in spite of obvious differences in mass distribution and bipedal posture. PMID- 10727971 TI - Genetic and environmental influence on the asymmetry of dermatoglyphic traits. AB - Fluctuating asymmetry (FA) is defined as random deviations from bilateral symmetry of the body. Thus, its magnitude is often used to evaluate developmental homeostasis. In this study we evaluate the following hypotheses: 1) FA of dermatoglyphic traits has a significant genetic component; 2) prenatal maternal environment (PME) has a significant effect on the FA of dermatoglyphic traits in developmentally healthy individuals; and 3) genetic or environmental factors affect FA on organismal or systemic levels. Therefore, their effect is better seen in composite scores of FA rather than in FA indices for single traits. We analyzed 15 dermatoglyphic traits from 140 pairs of monozygous twins, 120 pairs of dizygous twins, and 106 pairs of mothers and daughters. All individuals were developmentally healthy. The influence of genetic and environmental factors on FA was evaluated by analysis of variance and regression analysis. For a majority of the traits in our study, FA showed significant but weak heritabilities, with values falling within the 0.20-0.35 range. None of the traits taken separately demonstrated the effect of PME on FA to be significantly greater than zero. The composite score of FA tended to have greater heritability values than individual traits. One of them, obtained in principal components analysis, showed a significant PME effect, supporting the hypothesis that FA is a systemic property. PMID- 10727970 TI - Rib remodeling dynamics in a skeletal population from Kulubnarti, Nubia. AB - Bone remodeling variables in the rib were analyzed for a skeletal population of medieval antiquity (ca. A.D. 550-1450) from Kulubnarti, in Sudanese Nubia. The skeletal remains are naturally mummified and in an excellent state of preservation. The study sample consists of thin sections from the ribs of 80 individuals, ranging in age from 15-50+ years. Ribs were examined using a standard microscope and image analysis software. Numbers of intact osteons, fragmentary osteons, forming osteons, and resorption spaces were counted, osteon and Haversian canal areas were measured, and several variables were calculated to assess morphometric and remodeling status in the rib. Variables calculated included mean annual activation frequency, mean bone formation rate, and net osteonal remodeling. Results indicate that age changes are consistent with those observed for other archaeological and modern samples. High numbers of resorption spaces in young males may reflect slower skeletal development in boys compared to girls. Comparisons of rib data with results of a previous study on patterns of femoral bone remodeling in the same population indicate that ribs have more osteons and higher bone formation rates compared to the femur. Also, sexual differences in osteon size observed in the femur were not observed in the rib. Activation frequency and bone formation rate are low in the Kulubnarti population compared to previously published data for a modern sample, a finding consistent with reported results from other archaeological samples. Genetic factors influencing the minimum effective strain setpoint and duration of skeletal maturation, in addition to repetitive high strains at Kulubnarti, may contribute to observed differences. PMID- 10727972 TI - Secular trend in body height and weight of Australian children and adolescents. AB - Secular changes in growth and maturation have been well documented in various world populations, with secular increase especially noticeable in the developed countries. To assess the trend in both adult size and tempo of growth we compared the data on stature and body weight obtained in 1992-1993 from 1,804 Melbourne school students aged 5 to 17 with historical data collected from white Australians during the last 100 years. We illustrate the age-dependent trend in stature and body weight by means of regression surfaces. These were constructed by fitting local regression models to historical data and by simple plots showing the overall, and per decade, secular increase in both these measures at peripubertal and adult ages. Because of limited information on sample sizes and variability provided by the historical data, statistical comparisons have been performed only between the present 1992-1993 survey and two earlier independent surveys conducted in 1985 and 1970. The results have shown secular increase in adult stature over the last century, with the rate of increase varying between 0.4 and 2.1 cm/decade in males and 0.01 and 1.6 cm/decade in females. While secular increase in stature has significantly slowed down during the last two decades, the increase in body weight is still continuing at a high rate, and this increase is more pronounced in females. The period of strong secular increase, especially in the tempo of growth, coincided both with the shift toward earlier menarche and the improvement of socioeconomic conditions of the Australian population. The need for further studies to identify factors determining the continuing increase in body weight is emphasized, and caution in using the existing national growth standards for stature and weight is recommended. PMID- 10727973 TI - Implications of the distribution of Albumin Naskapi and Albumin Mexico for new world prehistory. AB - The known distributions of two mutational variants of the albumin gene that are restricted to Mexico and/or North America, Albumin Mexico (AL*Mexico) and Albumin Naskapi (AL*Naskapi), were expanded by the electrophoretic analysis of sera collected from more than 3, 500 Native Americans representing several dozen tribal groups. With a few exceptions that could be due to recent, isolated cases of admixture, AL*Naskapi is limited to groups that speak Athapaskan and Algonquian, two widely distributed language families not thought to be related, and to several linguistically unrelated groups geographically proximate to its probable ancestral homeland. Similarly, AL*Mexico is limited to groups that speak Yuman or Uto-Aztecan, two language groups in the American Southwest and Baja California not thought to be closely related to each other, and to several linguistically unrelated groups throughout Mexico. The simultaneous consideration of genetic, historical, linguistic, and archaeological evidence suggests that AL*Naskapi probably originated on the northwestern coast of North America, perhaps in some group ancestral to both Athapaskans and Algonquians, and then spread by migration and admixture to contiguous unrelated, or distantly related, tribal groups. AL*Mexico probably originated in Mexico before 3,000 years BP then spread northward along the Tepiman corridor together with cultural influences to several unrelated groups that participated in the Hohokam culture. PMID- 10727974 TI - Technical note: evaluating mandibular ramus flexure as a morphological indicator of sex. AB - Described as a highly reliable method of sex identification, mandibular ramus flexure is a morphological trait expressed on the posterior border of the ramus at the occlusal plane (Loth and Henneberg [1996] Am. J. Phys. Anthropol. 99:473 485). In a blind test, 158 mandibles were examined for the presence of flexure as defined by Loth and Henneberg, resulting in 79.1% accuracy, which is well below the reported 91-99% accuracy. Twenty-five of these mandibles were assigned the ambiguous score of 0, an outcome of a +1 score for one side, and a -1 score for the other. Seventeen mandibles were examined twice to measure intraobserver error. Only 64.7% of the scores were duplicated in the second session, suggesting difficulty in consistent identification of flexure. Low overall accuracy, an invalid scoring system, and high intraobserver error indicate that mandibular ramus flexure is an unreliable technique for estimation of sex. PMID- 10727975 TI - Brief communication: cutmarks on a plio-pleistocene hominid from Sterkfontein, South Africa. AB - Cutmarks inflicted by a stone tool were observed on the right maxilla of Stw 53, an early hominid partial skull from Sterkfontein "Member 5" (South Africa). The morphology of the marks, their anatomical placement, and the lack of random striae on the specimen all support an interpretation of this linear damage as cutmarks. The location of the marks on the lateral aspect of the zygomatic process of the maxilla is consistent with that expected from slicing through the masseter muscle, presumably to remove the mandible from the cranium. Although radioisotopic dates are not available and relative faunal dating of the deposit from which Stw 53 derives is problematic, the morphology of the hominid skull suggests a Plio-Pleistocene age for the specimen. This therefore constitutes the earliest unambiguous evidence that hominids disarticulated the remains of one another. PMID- 10727976 TI - Biological continuum of benign, atypical, and malignant mesenchymal neoplasms - does it exist? AB - The characterization of specific cytogenetic and molecular abnormalities in benign and malignant soft tissue tumours has increased our understanding and knowledge of the biology of these rare neoplasms in recent years and has led to the modification of a number of traditional classification schemes. Contrary to popular belief, it is reasonable to propose that there exists a molecular, genetic, and morphological continuum of benign, atypical, and malignant mesenchymal neoplasms. The identification of characteristic molecular changes in benign lipomas, lipomas with minimal atypia, and atypical lipomatous tumours, well-differentiated liposarcomas supports the hypothesis of a stepwise process in the pathogenesis of these neoplasms. Not only are these findings important for our understanding of the biology of soft tissue tumours, but they also may increase diagnostic and prognostic accuracy. PMID- 10727977 TI - Aberrant P-cadherin expression is a feature of clonal expansion in the gastrointestinal tract associated with repair and neoplasia. AB - The recognition of key roles for cadherins in the determination of epithelial cell phenotype, migration, differentiation, and tumour dissemination have stimulated much interest in this family of adhesion molecules. In the gastrointestinal tract, alteration of the expression of classical cadherins with aberrant P-cadherin up-regulation, associated with co-expression or loss of E cadherin expression, is seen in neoplastic transformation of oral and oesophageal squamous mucosa and in lesions representing early neoplastic transformation of glandular mucosa, such as aberrant crypt foci and metaplastic and adenomatous polyps. This same phenotype is seen in enterocytes adjacent to foci of ulceration in the intestine in colitis, including inflammatory bowel disease, and in colitis associated dysplasia. In coeliac disease, reversible E-cadherin down-regulation correlates with the degree of villous atrophy, but in contrast with colitis, aberrant P-cadherin expression is not a feature. Aberrant epithelial P-cadherin expression is thus associated with a proliferative phenotype related to ulceration and neoplastic transformation in the gastrointestinal tract, which may confer a survival advantage on these cells, but the relative functional roles of P-cadherin and E-cadherin and the molecular mechanisms underlyingP cadherin/catenin interactions have yet to be elucidated. PMID- 10727978 TI - Coordinated expression and amplification of the MDM2, CDK4, and HMGI-C genes in atypical lipomatous tumours. AB - Atypical lipomatous tumours (ALTs) represent a distinctive subset of mesenchymal neoplasms featuring mature adipocytic differentiation. Most ALTs are characterized cytogenetically by the presence of supernumerary ring and/or long marker chromosomes derived from the chromosomal region 12q13-15. The 12q13-15 chromosome region contains several genes which may play an important role in human tumorigenesis. A series of ALTs was analysed by investigating the MDM2, CDK4, and HMGI-C genes and their proteins. The study was extended to a series of ordinary lipomas, to determine whether the immunohistochemical investigation of these gene products might play any diagnostic role. Cytogenetic analysis revealed the presence of various cytogenetic aberrations involving the 12q13-15 region in 11/18 (61%) lipomas and of ring chromosomes in all ALTs. Overexpression of mdm2 protein was observed in 6/12 (50%) atypical lipomatous tumours. All lipomas were mdm2-negative. cdk4 overexpression was present in 100% of ALTs. Weak cdk4 immunopositivity was detected in 2/18 (11%) ordinary lipomas in a minority of cells. HMGI-C immunopositivity was observed in 10/12 (83%) ALTs. Positive immunoreactivity was also observed in 8/18 (44%) lipomas. Southern blot analysis revealed amplification of the CDK4 and MDM2 genes in 3/5 ALTs analysed. HMGI-C was amplified in 3/5 cases and was deleted in one case. Mutation analysis of the CDK4 gene did not demonstrate any mutation. These data support the hypothesis that ordinary lipomas may form a molecular genetic and morphological continuum with ALT. At one end of the spectrum are lipomas characterized by 12q13-15 rearrangements and HMGI-C activation and at the other end are ALTs with ring chromosomes, 12q13-15 amplification with overrepresentation of the HMGI-C, CDK4 or MDM2 genes, and aberrant cdk4, mdm2, and HMGI-C protein expression. These findings not only provide insights into the molecular pathogenesis of lipomatous tumours, but also indicate that the immunohistochemical analysis of mdm2 and cdk4 may help to increase diagnostic accuracy. PMID- 10727979 TI - The location of pKi67 in the outer dense fibrillary compartment of the nucleolus points to a role in ribosome biogenesis during the cell division cycle. AB - Although widely used as a marker of cell proliferation, the biochemical properties and function of the Ki67 antigen remain poorly understood. Recent data indicate that it can interact with RNA, DNA, and a number of cellular proteins including elements of the ubiquitin proteolytic pathway and a novel kinase. The evidence for its expression only in cycling cells is extensive and it is not regulated by stress, apoptosis or DNA damage. It was reasoned that a detailed characterization of the localization of pKi67 and analysis of its spatial relationship to other nucleolar proteins may provide insights into its function. Using high-resolution laser scanning confocal microscopy with double and triple labelling, pKi67 expression in MCF7 cells has been defined in relation to the distribution of nucleolin, fibrillarin, p130 (human Nopp 140 homologue), p120 (Nol 1), RH-II/Gu helicase, and topoisomerase II beta. All of these molecules are perichromosomal during mitosis and all but fibrillarin and p130 show extra nucleolar distribution in early G1. The majority of p120 (Nol 1) and RH-II/Gu helicase co-localize in the diffuse fibrillar centre (DFC) of nucleoli, while there is only partial overlap with nucleolin and fibrillarin. There is no co localization between p130 and pKi67. These data refine current understanding of the distribution of pKi67 and its physical relationship with functional domains of the nucleolus and place pKi67 in a zone of the DFC associated with late rRNA processing. Taken together with recent biochemical data, these observations allow the proposal of a model of pKi67 function in which it acts as an 'efficiency factor' in ribosome biogenesis during the heavy metabolic demands placed on a cell during the cell division cycle. PMID- 10727980 TI - Decreased expression of Ki-67 in atrophic cervical epithelium of post-menopausal women. AB - Papanicolaou-stained cervical smears (Pap smears) of post-menopausal women often present difficulties in distinguishing atrophic cervical epithelium from high grade cervical intraepithelial neoplasia (CIN2-3). The aim of this study was to disclose differences in proliferative activity in normal cervical epithelium, cervical atrophy, and high-grade CIN lesions, in order to develop specific and sensitive classifiers to discriminate between cervical atrophy and high-grade CIN, both in cervical smears and in tissue sections. A case-control study was done on 83 patients. Proliferative activity was assessed in histological sections using the monoclonal antibody MIB1. An image analysis system was used to characterize different proliferation-associated features. Preceding Pap smears were restained with MIB1 and proliferative activity was measured by a point counting procedure, carried out on a training set of 32 cases and a test set of 51 cases. In cervical atrophy, proliferative activity was significantly lower than in normal epithelium (p<0.001). Proliferative activity measured in both biopsies and cervical smears was considerably higher in high-grade CIN than in normal epithelium (p<0.001). Discriminant analyses resulted in four classifiers, based on proliferation parameters, to discriminate between cervical atrophy and high-grade CIN, and between CIN2 and CIN3, in biopsy specimens and cervical smears, respectively. The two classifiers for biopsy specimens resulted in 100% correct classification. Application of the classifier obtained from the training set of Pap smears resulted in 100% correct classification of the Pap smears in the test set. The classifier to discriminate between CIN2 and CIN3 in Pap smears, obtained from 36 patients, resulted in 87% and 90% correct classification, respectively. PMID- 10727982 TI - 20q13 and cyclin D1 in ovarian carcinomas. Analysis by fluorescence in situ hybridization. AB - In ovarian carcinomas, alterations of the chromosomal region 20q13 and the cyclin D1 gene have been described. This study has sought to determine their prognostic significance. Fluorescence in situ hybridization (FISH) on dissociated nuclei and paraffin sections with DNA probes for 20q13.2 and cyclin D1, as well as immunohistochemistry (cyclin D1), were applied to formalin-fixed tissue of 69 invasive ovarian carcinomas, mainly of serous type. On dissociated nuclei 33/47 cases (70%) and on tissue sections 13/66 cases (20%) demonstrated an increase of 20q13.2 copies. The presence of > or =4 copies per nucleus (isolated nuclei) and > or =3 copies per nucleus (sections) was associated with an adverse prognosis (Kaplan-Meier for FIGO stage III after stratification for residual tumour: p=0.0049 and p=0.03, respectively). Thirty-four out of 47 cases (72%) showed an increase of cyclin D1 copies. Kaplan-Meier analysis for FIGO stage III after stratification for residual tumour>2 cm or < or =2 cm revealed an unfavourable outcome for cases with more than two cyclin D1 copies (p=0.04). No correlation was seen between FISH and immunohistochemistry. Multivariate analysis identified residual tumour (p=0.0002), 20q13.2 gain (p=0.0004) and cyclin D1 gain (p=0.0343) as independent prognostic factors. It is concluded that gains of chromosomal region 20q13.2 and the cyclin D1 gene are frequent and biologically important events, with prognostic relevance, in advanced ovarian carcinomas. PMID- 10727981 TI - Up-regulation of pS2 expression during the development of adenocarcinomas but not squamous cell carcinomas of the uterine cervix, independently of expression of c jun or oestrogen and progesterone receptors. AB - The pS2 gene product was firstly identified as an oestrogen-induced molecule in a breast cancer cell line, while recent studies demonstrate a close association with mucus-secreting epithelia. To assess pS2 expression in uterine cervical adenocarcinomas (C-ACas) and invasive squamous cell carcinomas (C-ISCCs), a series of 94 and 86 cases, respectively, as well as 77 samples of normal cervix, were immunohistochemically investigated and the results compared with data for expression of oestrogen and progesterone receptors (ER and PR) and c-jun. RT-PCR and western blot assays were also applied to 21 cervical carcinomas and 24 normal tissues. With cervical glandular lesions, significant up-regulation of pS2 expression at both the mRNA and the protein levels was observed for adenocarcinomas in situ (AISs) and overt carcinomas, closely linked with mucinous differentiation and tumour grades. pS2 scores were inversely related to ERalpha status for all cervical glandular categories, while there was no association with ERbeta and PR values. In squamous lesions, pS2 values did not differ between normal and malignant lesions, in contrast to the significant down-regulation of ERalpha expression with tumour development. Although c-jun expression significantly correlated with ERalpha values for all squamous categories, it did not relate to pS2 status in either C-ACas or ISCCs. These results indicate that alterations in pS2 expression may occur relatively early in the development of cervical glandular, but not squamous lesions, independently of factors known to promote transcription of the pS2 gene. PMID- 10727983 TI - Micro-anatomy related antigen expression in melanocytic lesions. AB - The in situ expression of antigens associated with melanosomes (gp-100), pigmentation (PAA), tyrosinase (TRP-1), melanoma (MAA-1/MAA-2), and HLA-DR was investigated immunohistochemically in frozen archival specimens of common acquired melanocytic naevi, in dysplastic melanocytic naevi, and in lymph node metastases of melanoma. Expression of these antigens was also studied in established cultured normal human melanocytes, naevus-derived melanocytes and melanoma cell lines of varying metastatic potential, by immunohistochemistry and flow cytometry. Compared with normal melanocytes, melanocytic naevi exhibited increased expression of gp-100, PAA, and TRP-1 in the lesional cells at or very near the dermo-epidermal junction, but with diminishing expression towards the intra-dermal base of the lesions. In contrast, expression of MAA-1 and MAA-2 was observed in melanocytes throughout the dermal part of the naevi. Melanocytes located at the basal layer of the epidermis were positive only for gp-100, PAA, and TRP-1 antigens. Dysplastic melanocytic naevi showed staining of gp-100, PAA, TRP-1, HLA-DR, MAA-1, and MAA-2 of junctional lesional melanocytes, but less intense than that of common acquired naevi. These antigens were not detectable in the dermal part of the dysplastic naevi. Expression of these antigens in lymph node metastases of melanoma was either positive or negative. Similar results regarding antigen expression were observed in all cultured melanocytic cells, both by immunohistochemistry and by flow cytometry. The present data suggest that analysis of these antigens may contribute to the discrimination of common acquired melanocytic naevi from their dysplastic counterparts. Furthermore, variations in the levels of expression in naevi may be consistently related to the micro-anatomy of the lesions, indicating that the micro-environment may have an influence on the expression levels of these antigens in different lesional melanocytes. PMID- 10727984 TI - TGF-beta isoforms are differentially expressed in increasing malignant grades of HaCaT keratinocytes, suggesting separate roles in skin carcinogenesis. AB - The three mammalian isoforms of transforming growth factor-beta (TGF-beta1, beta2, and -beta3) are potent regulators of cell growth, differentiation, and extracellular matrix deposition. To study their role in skin carcinogenesis, normal human keratinocytes, early (31) and late (310) passage immortalized keratinocytes (HaCaT cells), and five HaCaT-ras clones exhibiting benign (A-5, I 7), malignant (II-4, A-5 RT1), and highly aggressive (A-5 RT3) tumourigenic phenotypes were examined for the expression of TGF-beta isoforms, by immunohistochemistry. This was performed under in vivo conditions, in surface transplants and subcutaneously growing tumours in nude mice. Generally, all tissues that formed keratinized epithelia demonstrated an immunostaining pattern similar to normal human skin. TGF-beta1 was localized to the upper differentiated layers, the stratum granulosum and corneum, in a perimembranous pattern, whereas TGF-beta2 and, weaker, TGF-beta3 immunostaining was present in all suprabasal layers of normal keratinizing epithelia. In contrast, non-keratinizing transplants of non-tumourigenic or highly aggressive cells showed little to no immunoreactivity for TGF-beta1. Whereas TGF-beta2 expression was moderate in the upper layers of non-tumourigenic epithelia, large tumour cells of the malignant HaCaT-ras clones, particularly at the invasion front, were strongly positive for TGF-beta2. TGF-beta3 immunostaining was most pronounced in the stroma of malignant tumours, implying its paracrine induction by the malignant tumour transplants. These results suggest differential functions for each TGF-beta isoform in epidermal carcinogenesis, such that TGF-beta1 is associated with the more differentiated state, TGF-beta2 with highly malignant and invading cells, and TGF-beta3 with tumour stroma formation and angiogenesis. Furthermore, the expression of TGF-betas by both early- and late-stage tumours implies that the isoforms may have distinct functions at different stages of malignancy, supporting their dual role in skin carcinogenesis. PMID- 10727985 TI - Lack of insulin-like growth factor 1 (IGF1) in the basal keratinocyte layer of diabetic skin and diabetic foot ulcers. AB - Wound healing, including re-epithelialization, is delayed in diabetes. Growth factors influence the healing process and amongst these, insulin-like growth factor (IGF) has been shown to stimulate keratinocyte proliferation in vitro. Monoclonal antibodies to insulin-like growth factors 1 and 2 (IGF1 and IGF2) were used to investigate their distribution in diabetic foot ulcers and surrounding tissues by immunohistochemistry, compared with diabetic and non-diabetic uninjured skin. IGF2 was found throughout the epidermis (stratum granulosum, spinosum, and basale) in all three groups. Staining for IGF2 was intense in both normal and diabetic skin as well as in diabetic foot ulcers, being greatest at the ulcer edge. IGF1, in comparison, was found throughout the epidermis of non diabetic skin; expression was restricted to the stratum granulosum and spinosum of uninjured diabetic skin and was absent in the basal layer at the ulcer edge. A similar absence of IGF1 in dermal fibroblasts was found in tissue sections from diabetic patients. This lack of expression of IGF1 within the basal layer and fibroblasts may contribute to retarded wound healing in diabetes mellitus. PMID- 10727986 TI - Higher numbers of autologous fibroblasts in an artificial dermal substitute improve tissue regeneration and modulate scar tissue formation. AB - Cultured skin substitutes are increasingly important for the treatment of burns and chronic wounds. The role of fibroblast numbers present in a living-skin equivalent is at present unknown. The quality of dermal tissue regeneration was therefore investigated in relation to the number of autologous fibroblasts seeded in dermal substitutes, transplanted instantaneously or precultured for 10 days in the substitute. A full-thickness porcine wound model was used to compare acellular dermal substitutes (ADS) with dermal substitutes seeded with fibroblasts at two densities, 1x10(5) (0-DS10) and 5x10(5) cells/cm(2) (0-DS50), and with dermal substitutes seeded 10 days before operation at the same densities (10-DS10 and 10-DS50) (n=7 for each group, five pigs). After transplantation of the dermal substitutes, split-skin mesh grafts were applied on top. Wound healing was evaluated blind for 6 weeks. Cosmetic appearance was evaluated and wound contraction was measured by planimetry. The wound biopsies taken after 3 weeks were stained for myofibroblasts (alpha-smooth muscle actin), and after 6 weeks for scar tissue formation (collagen bundles organized in parallel and the absence of elastin staining). Collagen maturation was investigated with polarized light. For wound cosmetic parameters, the 10-DS50 and 0-DS50 treatments scored significantly better than the ADS treatment, as did the 10-DS50 treatment for wound contraction (p<0.05, paired t-test). Three weeks after wounding, the area with myofibroblasts in the granulation tissue, determined by image analysis, was significantly smaller for 0-DS50, 10-DS10, and 10-DS50 than for the ADS treatment (p<0.04, paired t-test). After 6 weeks, the wounds treated with 0-DS50, 0-DS10, and 10-DS50 had significantly less scar tissue and significantly more mature collagen bundles in the regenerated dermis. This improvement of wound healing was correlated with the higher numbers of fibroblasts present in the dermal substitute at the moment of transplantation. In conclusion, dermal regeneration of experimental full-skin defects was significantly improved by treatment with dermal substitutes containing high numbers of (precultured) autologous fibroblasts. PMID- 10727987 TI - Apoptosis of malignant cells in Hodgkin's disease is related to expression of the cdk inhibitor p27KIP1. AB - Previous results from B-cell chronic lymphocytic leukaemia suggest that expression of p27KIP1 might be important in protection from apoptosis. Given the relevance of apoptosis to the pathogenesis of Hodgkin's disease (HD), it was decided to examine the expression of p27KIP1 in relation to apoptosis in these lesions. Paraffin-wax sections from a total of 65 histologically confirmed HD tumours were used to derive apoptotic index (AI) and DNA fragmentation index (DFI) scores, which were compared with the expression of various cell-cycle regulating proteins, including p27KIP1 (p27), p21WAF1/CIP1 (p21) and cyclin D1, and with Epstein-Barr virus (EBV) status. The DFI was measured by TdT-mediated dUTP-FITC nick end-labelling (TUNEL), and the AI by conventional morphology. Cells showing the typical morphology of apoptosis, together with those resembling so-called 'mummified' Hodgkin/Reed-Sternberg (HRS) cells, were included in AI measurements. Increasing numbers of p27-positive HRS cells were associated with lower levels of apoptosis in these cells, as indicated by significantly lower AI and DFI scores. There was a trend towards poorer survival in those patients with the highest numbers of p27-positive HRS cells and with lower AI and DFI scores, but these differences were not statistically significant. p21-positive HRS cells were significantly more frequent in those cases with lower AI scores. A similar trend was observed for p21 and DFI, although this relationship was not statistically significant. There was also a trend towards higher levels of cyclin D1 protein in HD cases with high AI and DFI values. A tendency for increasing numbers of p27-positive and p21-positive HRS cells in EBV-positive cases was noted, but this relationship was not statistically significant. EBV status did not correlate with either AI or DFI scores. The results of this study suggest that p27, and possibly also p21, may be involved in protection from apoptosis in HD. PMID- 10727988 TI - Expression of the CD30 antigen in non-lymphoid tissues and cells. AB - Originally, expression of the CD30 antigen was shown to be typical of the tumour cells of Hodgkin's disease (HD) and of anaplastic large cell lymphomas (ALCLs). In reactive lymphoid tissue, CD30 is expressed only in a small population of activated lymphoid blasts. Since then, several reports have been published describing CD30 expression in non-lymphoid tissues and malignancies, such as embryonal carcinomas (ECs), seminomas, cultivated macrophages, two histiocytic neoplasms, decidual cells, and mesotheliomas. As CD30 detection is important in the differential diagnosis of HD and ALCL, the expression of CD30 in different non-lymphoid tissues was re-evaluated by immunohistology and in situ hybridization. Extra-lymphoid CD30 expression was found in 48/51 cases of EC or EC components of germ cell tumours, in decidual cells of 1/10 cases, in activated mesothelium in 16/28 pleural and peritoneal effusions, and in small foci of tumour cells in 2/8 mesotheliomas. CD30 expression was not confirmed in 27 germ cell tumours of the testis without an EC component nor in cultivated macrophages and 17 histiocytic malignancies. The knowledge of these CD30 expression patterns is important for the immunohistological differential diagnosis of anaplastic tumours. The absence of CD30 expression in reactive and neoplastic macrophages does not favour the concept that HD and ALCL are derived from these cells. PMID- 10727989 TI - Monocyte chemoattractant protein 1, interleukin 8, and chronic airways inflammation in COPD. AB - Chronic obstructive pulmonary disease (COPD) is one of the most common causes of death, with cigarette smoking among the main risk factors. Hallmarks of COPD include chronic airflow obstruction and chronic inflammation in the airway walls or alveolar septa. An earlier study reported elevated numbers of macrophages and mast cells within the bronchiolar epithelium in smokers with COPD, compared with smokers without. Since specific chemokines may be involved in this influx, the in situ protein and mRNA expression of monocyte chemoattractant protein 1 (MCP-1) and of interleukin 8 (IL-8) were studied in tumour-free peripheral lung tissue resected for lung cancer of current or ex-smokers with COPD (FEV(1)<75%; n=14) and without COPD (FEV(1)>84; n=14). MCP-1 was expressed by macrophages, T cells, and endothelial and epithelial cells. Its receptor, CCR2, is expressed by macrophages, mast cells, and epithelial cells. IL-8 was found in neutrophils, epithelial cells, and macrophages. In subjects with COPD, semi-quantitative analysis revealed 1.5-fold higher levels of MCP-1 mRNA and IL-8 mRNA and protein in bronchiolar epithelium (p<0.01) and 1.4-fold higher levels of CCR2 in macrophages (p=0.014) than in subjects without COPD. The bronchiolar epithelial MCP-1 mRNA expression correlated with both CCR2 expression on macrophages and mast cells (p<0.05) and the numbers of intra-epithelial macrophages and mast cells (p<0.04). The epithelial IL-8 expression did not correlate with the numbers of neutrophils, macrophages, CD45RO+, CD8+, or mast cells. These data suggest that MCP-1 and CCR2 are involved in the recruitment of macrophages and mast cells into the airway epithelium in COPD. PMID- 10727990 TI - Heme oxygenase isoform expression in cellular and antibody-mediated models of acute inflammation in the rat. AB - Heme oxygenase (HO) is the rate-limiting enzyme in the catabolism of heme to biliverdin, carbon monoxide (CO), and free iron. The enzyme exists as a constitutive isoform (HO-2) and an inducible isoform (HO-1), which is also a stress protein (HSP32). HO-1 has previously been shown to be associated with the resolution phase of a non-immune model of acute inflammation. In addition, elevation of the enzyme was markedly anti-inflammatory. In the present study, these observations have been extended to two pleural models of immune-driven inflammation in the rat, an immediate type III hypersensitivity (Arthus) reaction and a delayed type IV hypersensitivity reaction. Whilst these models have differing inflammatory mechanisms and time courses, they both showed HO activity to be maximal during the resolution phase. This activity was associated with increases in exudate bilirubin (a breakdown product of biliverdin) and increased expression of HO-1. Immunocytochemical analysis of inflammatory cell smears from the two models showed that HO-1 and HO-2 expression was restricted to mononuclear cells in the type IV hypersensitivity reaction, but included the polymorphonuclear cell population in the type III hypersensitivity reaction. Thus, irrespective of the pathogenesis of the lesion, evidence is accumulating to suggest that HO-1 has a universal role in the resolution of inflammation. PMID- 10727991 TI - Reflection contrast microscopy (RCM): a forgotten technique? AB - Reflection contrast microscopy (RCM), which utilizes the optical phenomena caused by oblique epi-illumination in combination with a specific optical apparatus, provides an approach for exploring biological phenomena in greater detail. The lack of stray reflection makes it superior to other microscopes. It bridges light and electron microscopic capabilities by allowing the analysis of ultrathin sections beyond the usual light microscopic magnification. By using consecutive image analysis, quantitation can be achieved. The wide range of applications of RCM can be combined with most microscopical techniques, so extending the spectrum of information that can be gathered. Twenty-five years after the development of RCM, there is still scope for its application in modern cell biology. PMID- 10727992 TI - Disorders of sexual differentiation PMID- 10727993 TI - Sex determination and the Y chromosome. AB - Although SRY was first identified 10 years ago, we still know remarkably little about its mode of action or downstream target genes. Recently, potential protein partners have been identified and there has been considerable activity to understand the roles of WT1, SF-1, DAX-1 and SOX9 in gonadogenesis. The emerging picture is one of complex interactions, involving both positive and negative regulatory signals that, depending on the cellular and promoter context, drive the expression of male-specific genes. Despite recent advances, however, we are still unable to explain the genetic cause of most cases of 46,XY gonadal dysgenesis or even a single case of Y-chromosome-negative 46,XX maleness. PMID- 10727994 TI - Ovarian differentiation and gonadal failure. AB - Ovarian failure can result from several different genetic mechanisms-X chromosomal abnormalities, autosomal recessive genes causing various types of XX gonadal dysgenesis, and autosomal dominant genes. The number and precise location of loci on the X are still under investigation, but it is clear that, in aggregate, these genes are responsible for ovarian maintenance, given that monosomy X shows germ cells that undergo accelerated atresia. Despite recent hypotheses, at present there is no evidence for a gene directing primary ovarian differentiation; this process may be constitutive. Phenotypic/karyotypic correlation and limited molecular confirmation have long shown that proximal Xp and proximal Xq contain regions of the most importance to ovarian maintenance. Terminal deletions at Xp11 result in 50% primary amenorrhea and 50% premature ovarian failure or fertility. Deletions at Xq13 usually produce primary amenorrhea. Terminal deletions nearer the telomeres on either Xp of Xq bring about premature ovarian failure more often than complete ovarian failure. The X linked zinc finger gene (ZFX) and diaphanous 2 Drosophila homologue (DIAPH2) are the only candidate genes for ovarian maintenance that map to the X chromosome. Additional, as yet unidentified, genes along the X chromosome must be involved. The search for these genes in humans is hampered by the lack of candidate genes that map to the X chromosome, the scarcity of patients with fortuitous autosomal translocations, and small pedigrees, which hinder mapping of the loci. In addition, difficulties with human germ cell research also make it challenging to dissect genes important to ovarian development. Autosomal genes also are involved in ovarian differentiation and gonadal failure. Follicle-stimulating hormone receptor and ataxia telangiectasia are examples of autosomal genes known to cause human ovarian failure. Transgenic mouse models point to many other candidate autosomal genes, and sequencing of the human homologues in affected women should lead to the discovery of new genes responsible for human ovarian failure. Identification, functional analysis, and mapping of novel genes specifically expressed in the ovary of mice and women eventually should lead to fruitful dissection of essential genes in mammalian ovarian development and maintenance. PMID- 10727995 TI - Syndromal (and nonsyndromal) forms of male pseudohermaphroditism. AB - The term sex determination refers to the genetic events that bring about male or female gonadal development; sex differentiation to all subsequent morphogenetic and physiological events that establish functional sexuality, sexual dimorphism and the secondary sexual characteristics. Virtually all of the steps of sex differentiation are under genetic control; consequently each one of them can fail as result of mutation of the corresponding genes. We shall be concerned with those genes and their mutations that cause pseudohermaphroditism in males and more rarely in females (with the exception of congenital adrenal hyperplasia). Special emphasis will be placed on Swyer, Denys-Drash, RSH, GBBB, campomelic and ATR-X syndromes, whose genes were recently identified. PMID- 10727996 TI - Androgen insensitivity. AB - The androgen receptor (AR) protein regulates transcription of certain genes. Usually, this activity depends upon a central DNA-binding domain that permits the binding of androgen-AR complexes to regulatory DNA sequences near or in a target gene. The AR also has a C-terminal androgen-binding domain (ABD) and an N terminal modulatory domain. These domains interact among themselves and with coregulatory, nonreceptor proteins to determine vector control over a gene's transcription rate. The precise roles of these proteins are active research areas. Severe X-linked androgen receptor gene (AR) mutations cause complete androgen insensitivity, mild ones impair virilization with or without infertility, and moderate ones sometimes yield a wide phenotypic spectrum among sibs. Different expressivity may reflect variability of AR-interactive proteins. The family history must identify heterozygous XX females with sparse, delayed, or asymmetric pubic/axillary hair or delayed menarche and infertile XY maternal aunts or uncles. Mutation type and density vary along the length of the AR. N terminal polyglutamine tract expansion limits AR transactivation, causing a form of mild androgen insensitivity. Analysis of ABD mutations that do not impair androgen binding or impair it selectively will illuminate its intradomain properties. For partial androgen insensitivity and mild androgen insensitivity, pharmacotherapy with certain androgens or other steroids may overcome some dysfunction of certain mutant ARs. Experience with this approach is limited; outcomes have been generally disappointing. PMID- 10727997 TI - Persistence of Mullerian derivatives in males. AB - The persistent mullerian duct syndrome is a rare, autosomal recessive disorder, characterized by the persistence of mullerian duct derivatives-uterus and fallopian tubes-in genetic males otherwise normally virilized. We have collected DNA from 69 families with this syndrome. In 45%, a mutation of the anti-mullerian hormone (AMH) gene was detected; 52% were homozygous. The level of circulating AMH was extremely low in the great majority of patients, even before puberty, when AMH levels are normally high. Single-strand conformation polymorphism (SSCP) polymerase chain reaction (PCR) was a very effective screening method. In 39% of families, characterized by an AMH level normal for the age of the patient, a mutation of the type II receptor of AMH was detected by automatic sequencing, because SSCP-PCR was not very effective. Forty-eight percent of the mutations were homozygous. A 27-base-pair deletion in exon 10 was noted in 45% of the families. When this very common mutation is not taken into account, the proportion of recurrent mutations is 42% for the AMH gene and 33% for the AMH receptor type II gene. In 16% of families, no mutation of either the AMH or the AMH receptor gene was detectable; this group may correspond to mutations of unknown genes involved in AMH processing or in downstream AMH transduction. PMID- 10727998 TI - Genetics of the female reproductive ducts. AB - Familial aggregates of the most common disorders of mullerian differentiation in females-Mullerian aplasia, incomplete Mullerian fusion-are best explained on the basis of polygenic/multifactorial inheritance. No information exists on the number and chromosomal location of responsible genes. Single mutant genes (Mendelian) are responsible for the McKusick-Kaufman syndrome (MKS) and the hand foot-genital syndrome. The molecular basis for the latter condition involves HOXA13, but the molecular basis of MKS and other disorders of the female reproductive ducts is unknown. Vaginal atresia, Mullerian aplasia, and incomplete Mullerian fusion are not infrequently observed in malformation syndromes. PMID- 10727999 TI - Genetics of human hypogonadotropic hypogonadism. AB - Humans with hypogonadotropic hypogonadism (HH) manifest irreversible pubertal delay, infertility, and low serum levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Although the genetic basis of this condition is largely unknown, mutations have been identified in approximately 5-10% of HH patients. Mutations in the KAL gene (Kallmann syndrome) and the AHC gene (adrenal hypoplasia congenita/HH) cause X-linked recessive HH. Autosomal recessive HH may be brought about by mutations in the gonadotropin-releasing hormone receptor, leptin, and the leptin receptor genes. Isolated deficiencies of the gonadotropins FSH and LH are due to corresponding beta-subunit genes. PROP1 gene mutations lead to combined pituitary deficiency, and HESX gene mutations result in septo-optic dysplasia, both of which include HH. These identified gene mutations advance our understanding of normal hypothalamic-pituitary-gonadal function. PMID- 10728016 TI - The role of point-of-care anticoagulation monitoring in arterial and venous thromboembolic disorders. PMID- 10728017 TI - Assessing the optimal level of platelet inhibition with GPIIb/IIIa inhibitors in patients undergoing coronary intervention. Rationale and design of the GOLD study. PMID- 10728018 TI - Early and pre-discharge aspirin administration among patients with acute myocardial infarction: current clinical practice and trends in the United States. AB - OBJECTIVES: The purpose of our study was to determine the frequency of aspirin administration among patients with acute myocardial infarction (MI) as dictated by physicians practicing in the United States. BACKGROUND: Aspirin (ASA), a widely available, inexpensive and generally well-tolerated platelet inhibitor, is recommended for patients with acute coronary syndromes, including acute MI. However, there is concern that aspirin is underutilized in daily clinical practice. METHODS: Early (<24 hours) and predischarge ASA administration were determined among 220,171 patients with suspected acute MI enrolled in the Second National Registry of Myocardial Infarction (NRMI 2) between June, 1994 and April 30, 1996. RESULTS: Overall, 165,122 (74.9%) of patients received ASA within 24 hours of hospital admission, whereas 55,049 patients did not. Early ASA recipients were younger, more often male, arrived at the hospital earlier, and were more likely to be classified as Killip Class II or less compared to those who did not receive ASA. Patients who received aspirin were also more likely to have chest pain, electrocardiographic ST segment elevation, and tended to arrive at the hospital earlier than those who did not receive ASA. However, over 20% of patients with ST segment elevation did not receive early ASA therapy. From the total cohort of early ASA recipients, only 69% received ASA at the time of hospital discharge. Trends in early and pre-discharge aspirin administration over a 2 year time period in all patients (72.6 to 75.1% and 71.5 to 74.6%, respectively; p < 0. 001) and in specific patient subsets were encouraging with a gradual but steady increase; however, utilization remained comparatively low in women and the elderly. By multivariable analysis, in-hospital recurrent MI (OR 0.90, 95% CI;.78-1.0, p = 0.04), stroke (OR 0.65, 95% CI,.52-.80, p < 0.001) and death (OR 0.24, 95% CI,.22-.26, p < 0. 001) occurred less frequently when ASA was administered within 24 hours of hospitalization. CONCLUSION: Aspirin is currently underutilized in routine clinical practice as both primary and adjunctive forms of therapy in MI, especially among patients known to be at risk for recurrent cardiothrombotic events. The targeted and timely use of aspirin reduces early cardiovascular events and should remain a priority in national health care efforts. PMID- 10728019 TI - Circulating levels of IL-1beta, a prothrombotic cytokine, are elevated in unstable angina versus stable angina. AB - BACKGROUND: Multiple studies support a role for inflammation in the pathogenesis of coronary atherosclerosis and unstable cardiac syndromes. However, of the known proinflammatory cytokines, only elevated plasma levels of interleukin-6 have been linked to unstable angina. We sought to examine the plasma levels of other major proinflammatory cytokines in similar clinical settings and to determine the extent of the relationship between inflammation and unstable coronary syndromes by measuring the levels of various proinflammatory cytokines in patients with stable and unstable angina. METHODS: We measured plasma levels of interleukin-1 beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and interleukin 6 (IL 6) in 97 patients: 67 with stable angina, 24 with unstable angina, and 15 healthy controls. RESULTS: Mean levels of IL-1beta were significantly higher in patients with unstable angina as compared to patients with stable angina (p =.009). Levels of IL-6 were significantly higher than control patients for both stable angina and unstable angina patients (p =.031 and.006, respectively). No significant differences were found in the levels of TNF-alpha. CONCLUSIONS: Our results suggest that both IL-1beta and IL-6 contribute to the pathogenesis of unstable angina, and that the profile of circulating plasma levels of proinflammatory cytokines differs in unstable angina from that in stable angina. Abbreviated Abstract. Multiple studies support a role for inflammation in the pathogenesis of coronary atherosclerosis and unstable cardiac syndromes. We measured plasma levels of interleukin-1 beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and interleukin 6 (IL-6) in patients with stable and unstable coronary syndromes. Levels of IL-1beta and IL-6 were found to be elevated in patients with unstable coronary syndromes. No significant differences were found in the levels of TNF alpha. Our results suggest that both IL-1beta and IL-6 contribute to the pathogenesis of unstable angina. PMID- 10728020 TI - Enoxaparin, a low molecular weight heparin, inhibits platelet-dependent prothrombinase assembly and activity by factor-Xa neutralization. AB - BACKGROUND: The available evidence suggests strongly that intravascular thrombosis is mediated predominantly by tissue-factor and its activation of factor X, which in the presence of factor Va, calcium, and phospholipid (prothrombinase complex) effectively converts prothrombin to thrombin. In vitro experiments have shown that low molecular weight heparins (LMWHs) have greater anti-Xa activity than unfractionated heparin; however, it remains unclear as to whether their antithrombotic effects in vivo are determined by a similar mechanism. We determined the ability of plasma obtained from patients with either unstable angina or non-ST segment elevation myocardial infarction (MI) receiving the LMWH enoxaparin (anti Xa:IIa ratio 3:1) to inhibit tissue factor-mediated thrombin generation and to inactivate platelet prothrombinase. METHODS: Platelet rich plasma was prepared by suspending washed donor platelets in the plasma of 7 patients participating in the TIMI 11A study. Samples were obtained before, 1 hour after a 30-mg IV bolus of enoxaparin and 6 hours after the third subcutaneous injection (1. 0-1.25 mg/kg given subcutaneously every 12 hrs). Tissue factor (0.1 ng/ml) and 10 mM CaCl(2) were added to initiate extrinsic coagulation. At timed intervals prothrombin activation fragment 1.2 (F1.2) levels (thrombin generation) were measured using an ELISA technique. Inactivation of reformed platelet prothrombinase by samples obtained at the same time points was also determined. RESULTS: Patient plasma obtained 1 hr after treatment initiation and 6 hours after the third subcutaneous injection inhibited tissue factor mediated prothrombinase assembly by 31% and 11%, respectively and platelet prothrombinase activity by 27% and 22%, respectively. CONCLUSION: We conclude that enoxaparin in plasma concentrations achieved routinely in clinical practice is able to: (1) inhibit tissue factor mediated extrinsic coagulation by preventing platelet surface prothrombinase assembly, and (2) inactivate platelet prothrombinase activity and resulting thrombin generation. These observations suggest that a LMWH's anti-Xa activity (and anti-Xa:IIa profile) is important in determining its overall antithrombotic potential. Clinical trials comparing agents with differing anti-Xa:IIa properties will be required, however, to provide proof of concept. PMID- 10728021 TI - Moderate alcohol consumption is associated with decreased platelet activity in patients presenting with acute myocardial infarction. AB - Moderate alcohol consumption (MAC) and platelet inhibition have been independently associated with a reduced risk for the development of acute myocardial infarction (AMI). The effects of MAC on the initial platelet status in patients presenting with AMI are not elucidated. Here we sought to define the effects of MAC on platelet characteristics in AMI patients before applying any reperfusion strategies. The study was designed as an analysis within the cohort study in 23 patients with AMI enrolled in the GUSTO-III. Platelets were investigated by different techniques, including aggregometry, flow cytometry, and ELISA. MAC patients exhibited mild, but consistent, inhibition of platelet aggregability, surface receptor expression, and released substances as compared to non-alcohol consuming patients. These differences were significant for 5 microM ADP (p = 0.04), 10 microM ADP-induced aggregation (p = 0.02); P-selectin (p = 0.01), and PECAM-1 (p = 0.02) platelet-bound expression. Our study confirms that moderate alcohol consumption is associated with diminished platelet activation in patients presenting with AMI. The ability of MAC to favorably modulate the pre-reperfusion platelet status in such patients is of clinical importance, and further investigation in large-scale clinical trials seem warranted. PMID- 10728022 TI - Bridging the gap with new strategies in acute ST elevation myocardial infarction: bolus thrombolysis, glycoprotein IIb/IIIa inhibitors, combination therapy, percutaneous coronary intervention, and "facilitated" PCI. AB - Achieving early reperfusion with thrombolytic agents or primary percutaneous coronary intervention (PCI) in patients with acute myocardial infarction (AMI) is the cornerstone of current therapy. Two advances in pharmacologic therapy are: (1) bolus thrombolysis, which simplifies therapy, reduces door-to-needle time, and reduces the potential for medication errors, and (2) Low-dose fibrinolytic therapy combined with a glycoprotein (GP) IIb/IIIa inhibitor which can achieve higher rates of reperfusion than fibrinolytic therapy alone. In addition, the IIb/IIIa inhibitor as part of the reperfusion regimen will support any acute phase interventions that are performed. The combination of fibrinolytic therapy and GP IIb/IIIa inhibition to "facilitate" PCI is being examined in TIMI-14, SPEED, and GUSTO IV. Early findings in the SPEED trial have shown promising results with "facilitated" PCI when patency is achieved before PCI is attempted. Results of these trials will further define the role of combination therapy in facilitating mechanical interventions. PMID- 10728023 TI - Does ultrasound influence experimentally induced thrombus formation in the central artery of the rabbit ear? AB - BACKGROUND: Thrombosis is one of the most important causes of morbidity in the medical field. Several independent in vitro studies have shown that the fibrinolytic process may be enhanced by ultrasound, but the effect of ultrasound on thrombus formation in vivo is unexplored. The present study was designed to investigate this matter. METHODS: In a blind randomized study, standardized arteriotomies and intimectomies were performed on the central arteries of the ears of 25 rabbits. The rabbits were allocated to two groups, an untreated control group and a group treated with ultrasound (10 pulses of frequency 1 MHz and intensity 1 W/cm(2) per millisecond giving an averaged intensity of 0.01 W/cm(2)). Immediately after reperfusion, patency was confirmed by a manual empty/refill test, after which blood-flow was monitored using ultrasonic flow probes twice a minute for two hours. At two hours, patency was rechecked. RESULTS: All vessels were patent at reperfusion, but only seven vessels (three control, four treated) were patent when flow-rate measurements started. At 2 h, patency-frequencies were 12/23 in the control group and 11/22 in the treated group. Flow-rate curves in patent vessels in both groups were similar. Microscopic investigation at one week showed no difference in thrombus accumulation. CONCLUSIONS: Ultrasound with the above characteristics does not significantly improve patency in vivo. PMID- 10728024 TI - High shear stress marks: the vulnerable target with von Willebrand factor for platelet deployment -letter-. PMID- 10728026 TI - Monitoring therapy with vitamin K antagonists in patients with lupus anticoagulant: effect on different tests for INR determination. AB - BACKGROUND: Lupus anticoagulant (antiphospholipid antibodies) is associated with venous and arterial thrombosis in patients with and without autoimmune disorders. Vitamin K antagonists are the treatment of choice in patients with thrombosis, of which the dose is titrated by INR monitoring. Several recent reports suggest that the presence of the lupus anticoagulant disturbs the INR test and may lead to unreliable results with a large variation in INR values, dependent on the reagents used. METHODS: We studied 11 lupus anticoagulant positive patients and 11 lupus anticoagulant negative patients, all using vitamin K antagonists. The INR value was determined using seven different tests and the variation in INR values was compared between the two groups. The amidolytic Factor X levels were used as an phospholipid independent measure for intensity of warfarin therapy. Factor VII and X activity were measured to assess the stability of warfarin therapy. RESULTS: The variation of the results with different INR tests within one patient was minimal and comparable in the two groups for INR's in the therapeutic range. The coefficient of variation for the cases and control group was 10.43 and 9.35, respectively. Variation in both groups increased at supratherapeutic levels of anticoagulation and when the anticoagulation was unstable (measured with Factor X/Factor VII ratio). The relationship between INR values and Factor X analysis revealed no influence of the lupus anticoagulant. CONCLUSIONS: In this study, lupus anticoagulant antibodies do not disturb INR laboratory tests. Differences in INR measurements are seen in patients with a high intensity of anticoagulation and in patients who either just started or in whom no stable anticoagulation has been achieved. Abbreviated Abstract. This study investigates the influence of lupus anticoagulant on INR determination tests in patients treated with warfarin. Eleven cases and eleven lupus anticoagulant negative control patients, also on warfarin therapy, were included. Seven INR results per patient were obtained using different laboratory tests. A factor X assay was performed to obtain an independent measure for the intensity of warfarin therapy. The variation of INR results between the cases and controls revealed no difference in these groups. In addition, the relationship between INR values and Factor X analysis indicated no influence of the lupus anticoagulant. What was observed was an increased difference in INR values in patients with a high intensity of anticoagulation and in patients who either just started or in whom no stable anticoagulation has been achieved PMID- 10728027 TI - Symptomatic combined homozygous factor XII deficiency and heterozygous factor V Leiden. luscaber@tin.it. AB - A family with a combined deficiency of factor XII and factor V Leiden is presented. The proposita is a 72-year-old who showed a mild to moderate thrombotic tendency characterized by two episodes of deep venous thrombosis and superficial phlebitis between the age of 50 and 71. She was shown to be carrier of homozygous factor XII deficiency and heterozygous FV Leiden mutation. A sister of the proposita showed the same pattern but remained asymptomatic. Other family members showed either isolated heterozygous factor XII deficiency or combined heterozygous factor XII deficiency and heterozygous FV Leiden mutation but were all asymptomatic. These data lend support to those who maintain that FV Leiden is a mild genetic determinant for thrombosis. The role of FXII deficiency as an additional risk factor remains questionable. PMID- 10728025 TI - A guide to venous thromboembolism risk factor assessment. PMID- 10728028 TI - Decrease in protein C inhibitor activity and acquired APC resistance during normal pregnancy. AB - BACKGROUND: Since protein C inhibitor (PCI) inhibits activated protein C (APC) and a number of proteases, one would expect lower concentrations of PCI in a hypercoagulable state due to increased consumption of the inhibitor. Normal pregnancy is associated with a state of activated hemostasis, where response to APC is depressed. We aimed to study whether PCI function varies during normal pregnancy, and assess the relationship between this inhibitor and acquired APC resistance. METHODS: PCI activity in plasma was tested during pregnancy and postpartum in 28 healthy pregnant women without factor V Leiden Arg(506) - Gln mutation and in 14 non-pregnant female controls. The PCI levels determined in the present study was compared to the APC ratio (APC-r), we investigated previously, in the same samples. RESULTS: The levels of PCI in the pregnant group, as compared to that in the control group (4.74 +/- 0.48), gradually decreased from the first to the third trimester, i.e., 3.30 +/- 1.31 microg/mL in week 12 (p < 0.001), 2.66 +/- 1.44 microg/mL in week 20 (p < 0.001), 1.92 +/- 1.18 microg/mL in week 28 (p < 0.001), 1.30 +/- 0.94 microg/mL in week 32 (p < 0.001) and 1.49 +/- 1.12 microg/mL in week 37 (p < 0.001). After delivery, they rose to 5.02 +/- 1.93 microg/mL, similar to that in the controls (p > 0.05). The values of APC-r showed the same tendency during gestation and postpartum. CONCLUSION: With advance of normal pregnancy, decreasing PCI function corresponds to increasing APC resistance, probably due to that activated hemostasis acts as a link connecting the two variables. PMID- 10728029 TI - Relationship between test frequency and outcomes of anticoagulation: a literature review and commentary with implications for the design of randomized trials of patient self-management. AB - BACKGROUND: Patient self-management (PSM) of anticoagulation, which is primarily based upon the premise that more frequent testing will lead to tighter anticoagulation control and thus to improved clinical outcomes, is a promising model of care. The goals of this paper are (1) to describe the strength of evidence correlating more frequent testing with improved outcomes; and (2) to discuss implications of these findings for the design of randomized controlled trials (RCTs) assessing the effectiveness and cost-effectiveness of PSM. METHODS: We performed two literature reviews: one examining the strength of the relationship between time in target range (TTR) and the clinical outcomes of major bleeding and thromboembolism; and the second examining the strength of the relationship between frequency of testing and TTR. RESULTS: We found that (1) the relationship between TTR and clinical outcomes is strong, thus supporting use of TTR as a primary outcome variable; and (2) more frequent testing seems to increase TTR, although the studies supporting this latter conclusion were relatively few and not definitive. Statistical analysis suggested that a study which uses clinical event rates as its primary outcome would need to be much larger than a comparable study which is based upon TTR. CONCLUSIONS: When designing randomized trials of PSM, the design should (1) use as its control group high quality anticoagulation management rather than usual care; (2) include the maximum possible amount of self-management in the intervention group; (3) include different testing intervals in the intervention group; (4) use TTR as the primary outcome variable and event rates as a secondary outcome; and (5) base the sample size calculations upon a 5-10% absolute improvement in TTR. Additional RCTs are needed in order to determine how the promise of PSM can best be fulfilled. PMID- 10728030 TI - Applications of anti-platelet monitoring in catheterization laboratory. PMID- 10728031 TI - Time to treatment and cost of thrombolysis: a multicenter comparison of tPA and rPA. AB - STUDY OBJECTIVE: This study reports a comparison of the time to treatment and cost of administration of alteplase (tPA) and reteplase (rPA) in patients with acute myocardial infarction (MI). DESIGN: Retrospective chart review. SETTING: Hospital emergency department. INTERVENTIONS: A retrospective chart review of 500 MI patients who received alteplase or reteplase was performed. A comparison of time from presentation in the emergency department to start of treatment was performed, and the cost of administration of drugs, including cost of supplies, monitoring time, and IV line complications, was calculated for each drug. RESULTS: The time from presentation to start of treatment was significantly shorter for reteplase than alteplase (51 vs 34 min). This difference resulted from a shorter decision to treat to start of treatment time for reteplase (11 min) compared to alteplase (31 min). The cost of administration of alteplase ranged from $136 to $184 per patient, while the cost of administration of reteplase ranged from $87 to $120 per patient. DISCUSSION: Given the similar safety and efficacy profiles of these thrombolytic agents, the advantages of reteplase in speed of administration and the reduction in cost should be considered when making formulary and drug product selection decisions.). Abbreviated Abstract. Alteplase (tPA) and reteplase (rPA) were compared in a retrospective review of 500 patients. rPA was associated with 17 minute time savings from presentation-to-treatment compared to tPA. rPA was also associated with a per patient cost savings $49 to $64 compared to tPA. The time and cost advantages of rPA should be considered when making drug product selection decisions. PMID- 10728032 TI - Diabetes in immigrant Asian children. PMID- 10728033 TI - Clinico-immunological profile of juvenile rheumatoid arthritis at Chandigarh. AB - OBJECTIVE: To study the clinical and immunological profile of children with juvenile rheumatoid arthritis (JRA). DESIGN: Retrospective hospital based study. SETTING: Tertiary level center of North India. SUBJECTS: 74 patients attending the Pediatric Rheumatology and Immunology Clinic over last 5 years. RESULTS: The patients were aged between 9 months to 12 years with male female ratio of 1.8:1. Eleven (14.9%) patients had systemic onset JRA, 28 (37.8%) had polyarticular onset type and 35(47.3%) had pauciarticular onset type JRA. Uveitis was present only in one patient and rheumatoid nodules were present in 4(5.4%) patients. Rheumatoid factor was positive in 2(2.7%) and antinuclear antibody was present in one patient only. HLA-B27 was positive in 4 children. Two patients developed amyloidosis. CONCLUSION: The clinico-immunological profile of JRA at Chandigarh appears to be some what different from that reported from other centers in India. PMID- 10728034 TI - Effectiveness of BCG vaccination against tuberculous meningitis. AB - OBJECTIVE: To assess the protective effectiveness of BCG vaccination against tuberculous meningitis, while controlling for age, nutrition and socio-economic status, in children 1 month to 12 years of age. DESIGN: Case-control study. SETTING: Secondary care referral and teaching hospital. METHODS: Cases were those conforming to the definition of tuberculous meningitis and controls were patients admitted after every third consecutive case included in the study from September 1995 till the end of August 1997 and who did not suffer from any central nervous system disorder. RESULTS: Among the 192 cases and 70 controls, BCG scar was present in 57.8% and 75.7%, respectively. The crude odd's ratio (OR)for tuberculosis meningitis with a BCG scar was 0.44 (95% CI, .24-0.81; p = 0.008), while the adjusted OR was 0.53 (95% CI, 0.26-1.06; p value = 0.07) after controlling for weight, age, sex and place of residence. Higher weight for age and urban residence were associated with a decreased risk of tuberculous meningitis in the logistic model. CONCLUSIONS: BCG vaccination offers protection against tuberculous meningitis. Since improvement in weight for age was associated with a decreased risk of disease, further studies are needed to evaluate the association, if any, between nutritional status and vaccine efficacy. PMID- 10728035 TI - IAP policy on age of children for pediatric care. PMID- 10728036 TI - Growth of infants and children in China. AB - Growth of China s infants and children has improved since the introduction of economic reforms. After 1979, secular increases in both weight and length have been observed. In certain industrial centers, infant lengths have caught up to international standards. Overall prevalence of underweight and stunting have declined. However, the health benefits from economic growth have not been shared equally among the Chinese population. There remain large disparities in the standard of health delivered to different sectors of the Chinese population. Rural children and children of minority nationalities continue to suffer from malnutrition and thus lower growth as compared to urban children. Economic reform has also changed the Chinese diet. Infant and child obesity is rising. Therefore, although economic reform has stimulated improvements in the growth of China s children, continued development and attention is needed in disadvantaged areas and populations. Public education is also necessary for China s children to avoid the diet-related diseases frequently seen in the west. PMID- 10728037 TI - Medical uncertainties. PMID- 10728038 TI - Hospital induced malnutrition in infants: prevention by relactation. PMID- 10728039 TI - Risk factors and spectrum of neonatal jaundice in a birth cohort in Karachi. PMID- 10728040 TI - Comparison of two methods of taping peripheral intravenous cannulas. PMID- 10728041 TI - Immunogenicity of recombinant hepatitis B vaccine in thalassemic children. PMID- 10728042 TI - Tracheal agenesis. PMID- 10728043 TI - Scurvy in transfusion dependent beta-thalassemia. PMID- 10728044 TI - A child with a DIC (15p; 22p) centric fusion and fetal Valproate syndrome. PMID- 10728045 TI - Morphea: plaque and linear type. PMID- 10728046 TI - Newer vaccines and 15 doses of OPV. PMID- 10728047 TI - Newer vaccines and 15 doses of OPV - reply PMID- 10728049 TI - Hepatitis B vaccine - reply PMID- 10728048 TI - Hepatitis B vaccine. PMID- 10728050 TI - Serological profile of cases of acute flaccid paralysis. PMID- 10728051 TI - Hemoglobin levels and concomitant intestinal parasitoses among children in and around Patna. PMID- 10728052 TI - Lymphangiomatous mesenteric cyst. PMID- 10728053 TI - Published MCQ's also need qualitative reconsideration. PMID- 10728054 TI - Infectious disease control in India: confused priorities? PMID- 10728055 TI - Bronchodilator therapy in bronchiolitis. PMID- 10728056 TI - Exertional rhabdomyolysis. Potentially life-threatening consequence of intense exercise. PMID- 10728057 TI - Don't be afraid to ask. Confronting your patients' sexual problems. PMID- 10728058 TI - Regulation, reimbursement, and PA practice today. PMID- 10728059 TI - Diabetes self-management. PMID- 10728060 TI - Keeping cardiovascular disease in check with calcium channel blockers. PMID- 10728061 TI - A CHF clinic. How aggressive outpatient care can offset hospitalization. AB - Congestive heart failure (CHF) is a serious problem, responsible for one of the highest rates of hospitalization in the United States for any medical condition. This study evaluated the effectiveness of a multidisciplinary, outpatient CHF clinic in decreasing the number of hospitalizations that CHF patients required. The clinic combines intensive patient and family education with aggressive follow up. A major aspect of the research project was developing a computerized database for maintaining the large volume of information generated by the CHF clinic. Once that database was established, records of 20 clinic patients were examined for a 6-month period surrounding each patient's date of enrollment. Characteristics of each patient during the 3-month preclinic period were compared with those of the 3-month postclinic period, including all admissions, all days in the hospital, admissions due to CHF, and days in the hospital due to CHF. There was a mean reduction of 0.733 admissions of all types and a mean reduction of 5.2 inpatient days for all admissions. There was a mean reduction of 0.8 admissions for CHF and a mean reduction of 4.067 inpatient days for CHF. All four reductions were statistically significant (P < .05). Results suggest that the CHF clinic was effective in reducing the number of, and length of stay during, hospitalizations. PMID- 10728062 TI - Atrial fibrillation. Controlling the most common arrhythmia. PMID- 10728063 TI - Acute myocardial infarction. The diagnosis of utmost importance. PMID- 10728064 TI - Valvular heart disease. Astute evaluation to surgical success. PMID- 10728065 TI - Making an accurate diagnosis of hypertension. PMID- 10728066 TI - Making the physical therapy referral. PMID- 10728067 TI - To break the cycle of menstrual migraine. PMID- 10728068 TI - Chasing profits and leaving the elderly behind. PMID- 10728069 TI - Panic disorder. Workup, management, and referral. PMID- 10728070 TI - Vaginal bleeding during adolescent pregnancy. PMID- 10728071 TI - Exploring complementary therapies in conventional practice. PMID- 10728072 TI - Is your patient competent to make medical decisions? PMID- 10728074 TI - Rural health clinics program: change on the horizon. PMID- 10728073 TI - Tailoring treatment for the Parkinson's disease patient. PMID- 10728075 TI - Alcohol addiction. Identifying the patient who drinks. Part 1. PMID- 10728076 TI - Alcohol addiction. Intervention strategies that work. Part 2. PMID- 10728077 TI - A proposal for master's-level competencies in PA education. PMID- 10728078 TI - An approach to parents who oppose childhood vaccination. PMID- 10728079 TI - Racial disparity in infant mortality. What does it mean? What can you do? PMID- 10728080 TI - The burden of comorbidity among the homeless at a drop-in clinic. AB - METHODS: The study was a retrospective chart review based on 174 patients seen during their initial visit. Information was obtained from data reported by each patient during the history and physical exam. RESULTS: Patients most likely to use the clinic were predominantly male (92%), between the ages of 20 and 39 (71%), African-American (64%), and living in an emergency shelter (73%). Sixty eight (39%) patients had some insurance coverage. Comorbidity was significant with 34 (20%) patients reporting all three types of pathology: physical, mental health, and substance abuse. Homeless patients reporting substance abuse were likely to report the coexistence of a medical condition (54%) or a mental health problem (27%). Patients who reported problems of depression, anxiety, or suicidal ideation or who heard voices had a significant concomitant occurrence of all three types of clinical pathology (P < .001). Patients living in an emergency shelter or on the street (as opposed to living with family or friends) were at high risk of medical problems (P < .001), mental health problems (P < .001), and substance abuse (P < .001). CONCLUSION: The prevalence of a medical problem, mental health problem, and substance abuse among the homeless is significant. Homeless patients may be at high risk of two or more comorbid conditions if they live in an emergency shelter or on the street, have a substance abuse problem, or have a mental health problem. Targeted clinical services and preventive medicine programs would be beneficial to these patients. PMID- 10728081 TI - This child has ringworm--or does he? PMID- 10728082 TI - New topicals for mild and moderate psoriasis. PMID- 10728083 TI - Implications for practice and education. Clinical activities of Iowa family practice PAs. AB - A survey of family practice physician assistants (PAs) in Iowa was undertaken to determine the frequency with which they utilized specific clinical skills. A response was received from 55 of 77 (74%) PAs surveyed. Average age of respondents was 42 years (range, 35 to 50). Respondents had, on average, been practicing for 13 years, mostly in family medicine; on average, they saw 25 patients a day. All reported providing patient education, prescribing and dispensing medication, interpreting radiographs, referring patients, and providing a wide range of services similar to their physician counterparts in Iowa. In regard to 62 activities identified, few differences emerged among respondents when population setting was considered--except that PAs who work in communities of fewer than 10,000 residents often perform a wider range of services than those who work in larger communities. Activities that have been ranked as important by Iowa family practice physicians in other surveys were also the clinical skills that PAs in this survey reported performing most often. This study supports mounting evidence that PAs are an important aspect of primary care delivery across society. Their activities in the delivery of care are similar to those that the physicians with whom they are most closely associated consider important. PMID- 10728084 TI - Preventive care and other issues among minority and immigrant patients. PMID- 10728085 TI - Rely on biopsy when skin characteristics overlap. PMID- 10728086 TI - Foreign bodies, ingested and inhaled. PMID- 10728087 TI - Managing type 2 diabetes. A look at the newer agents. PMID- 10728088 TI - Charcot's foot: often overlooked complication of diabetes. PMID- 10728089 TI - Treating serious mental illness in rural practice. PMID- 10728090 TI - Performing cervical polypectomy. PMID- 10728091 TI - The increasingly common problem of middle-ear barotrauma. PMID- 10728092 TI - An appeal to clinicians. Don't rush to prescribe antibiotics for acute URI. PMID- 10728093 TI - Heighten your response to latex allergy. PMID- 10728094 TI - e-merging technology and the care you provide. PMID- 10728095 TI - Ankle injury guidelines for your practice. PMID- 10728096 TI - Hyperpigmented lesions in an 18-year-old woman. PMID- 10728097 TI - Calcium channel blockers for migraine prophylaxis. PMID- 10728098 TI - Obesity: treating an American epidemic. PMID- 10728100 TI - New ways to manage the old problem of osteoporosis. PMID- 10728099 TI - The regionally endemic scourge of histoplasmosis. PMID- 10728102 TI - A theory of practice. PAs, specialists in team medicine. PMID- 10728101 TI - Managing violence in the emergency department (and other settings). PMID- 10728103 TI - Diagnosing vaginitis with a wet preparation. PMID- 10728104 TI - Understanding the function of performance profiles in health care. PMID- 10728105 TI - Regulation of the IgE allergic immune response by humoral and cellular factors. PMID- 10728106 TI - Testosterone in aging men. PMID- 10728107 TI - Clues to patients' explanations and concerns about their illnesses. A call for active listening. AB - Most patients who experience illness symptoms develop an explanatory model. More frequently than physicians realize, these attributions involve serious and potentially life-threatening medical conditions. Only a minority of patients spontaneously disclose or "offer" their ideas, concerns, and expectations. Often patients suggest or imply their ideas through "clues." Active listening is a skill for recognizing and exploring patients' clues. Without this communication skill, patients' real concerns often go unrecognized by health care professionals. Qualitative techniques including videotape analysis, postinterviewing debriefing, and interpersonal process recall were used to identify types of clues. We propose a taxonomy of clues that includes (1) expression of feelings (especially concern or worry), (2) attempts to understand or explain symptoms, (3) speech clues that underscore particular concerns of the patient, (4) personal stories that link the patient with medical conditions or risks, and (5) behaviors suggestive of unresolved concerns or unmet expectations. This clue taxonomy will help physicians recognize patients' clues more readily and thereby improve their active listening skills. A deeper understanding of the true reasons for the visit should result in increased patient satisfaction and improved outcomes. PMID- 10728108 TI - Personal values of family physicians, practice satisfaction, and service to the underserved. AB - BACKGROUND: Personal values are defined as "desirable goals varying in importance that serve as guiding principles in people's lives," and have been shown to influence specialty choice and relate to practice satisfaction. We wished to examine further the relationship of personal values to practice satisfaction and also to a physician's willingness to care for the underserved. We also wished to study associations that might exist among personal values, practice satisfaction, and a variety of practice characteristics. METHODOLOGY: We randomly surveyed a stratified probability sample of 1224 practicing family physicians about their personal values (using the Schwartz values questionnaire), practice satisfaction, practice location, breadth of practice, demographics, board certification status, teaching involvement, and the payor mix of the practice. RESULTS: Family physicians rated the benevolence (motivation to help those close to you) value type highest, and the ratings of the benevolence value type were positively associated with practice satisfaction (correlation coefficient = 0.14, P = .002). Those involved in teaching medical trainees were more satisfied than those who were not involved (P = .009). Some value-type ratings were found to be positively associated with caring for the underserved. Those whose practices consisted of more than 40% underserved (underserved defined as Medicare, Medicaid, and indigent populations) rated the tradition (motivation to maintain customs of traditional culture and religion) value type significantly higher (P = .02). Those whose practices consisted of more than 30% indigent care rated the universalism (motivation to enhance and protect the well-being of all people) value type significantly higher (P = .03). CONCLUSIONS: Family physicians who viewed benevolence as a guiding principle in their lives reported a higher level of professional satisfaction. Likewise, physicians involved in the teaching of medical trainees were more satisfied with their profession. Family physicians who rate the universalism values highly are more likely to provide care to the indigent. PMID- 10728110 TI - Serum vitamin C levels and use of health care resources for wheezing episodes. AB - BACKGROUND: Evidence suggests that the antioxidant vitamin C may play a role in lung function and wheezing, although the data are limited to laboratory evaluation of pulmonary function. OBJECTIVE: To examine the relationship among serum vitamin C levels, wheezing episodes, and use of health care services. METHODS: Analysis of adult subjects (aged > or = 17 years) surveyed in the third National Health and Nutrition Examination Survey (1988-1994) (n = 19,760), including measurements of serum vitamin C levels and self-reports of wheezing episodes, ambulatory health care visits, and overnight hospitalizations for wheezing. RESULTS: A large proportion of individuals (2377/19,760 [12.0%]) have received a diagnosis of asthma, chronic bronchitis, or emphysema. Among 874 individuals having an ambulatory visit for wheezing, 408 (46.7%) did not have a diagnosed respiratory conditions. Similarly, among 159 individuals hospitalized for wheezing, 61 (38.4%) did not have a diagnosed respiratory condition. Serum vitamin C level had no significant relationship with reported diagnosis of respiratory conditions, episodes of wheezing, or use of health care services for wheezing. In a model computed only with individuals with low or high serum vitamin C levels, after adjustment for potential confounders, no statistically significant relationship was found between serum vitamin C levels and ambulatory care (odds ratio, 0.78; 95% confidence interval, 0.58-1.05) or hospitalization for wheezing episodes (odds ratio, 1.21; 95% confidence interval, 0.67-2.21). CONCLUSIONS: Serum vitamin C levels do not appear to be a marker for use of health care services for wheezing. Future investigations of the role of antioxidants in managing respiratory conditions should focus on the clinically important outcomes of health care use. PMID- 10728109 TI - Family dinner and diet quality among older children and adolescents. AB - CONTEXT: The proportion of children eating dinner with their families declines with age and has decreased over time. Few data exist concerning the nutritional effect of eating family dinner. OBJECTIVE: To examine the associations between frequency of eating dinner with family and measures of diet quality. DESIGN: Cross-sectional. SETTING: A national convenience sample. PARTICIPANTS: There were 8677 girls and 7525 boys in the study, aged 9 to 14 years, who were children of the participants in the ongoing Nurses' Health Study II. MAIN OUTCOME MEASURES: We collected data from a self-administered mailed survey, including food and nutrient intakes from a validated semiquantitative food frequency questionnaire. Main outcome measures included servings per day of selected foods and food groups, daily intakes of selected macronutrients and micronutrients, and frequency of multivitamin use. RESULTS: Approximately 17% of participants ate dinner with members of their family never or some days, 40% on most days, and 43% every day. More than half of the 9-year-olds ate family dinner every day, whereas only about one third of 14-year-olds did so. In age- and sex-adjusted logistic regression models, the odds ratios associated with a frequency of family dinner of most days compared with never or some days, or every day compared with most days, were as follows: for eating at least 5 servings per day of fruits and vegetables, 1.45 (95% confidence interval [CI], 1.37-1.53); for eating any fried foods away from home, 0.67 (95% CI, 0.64-0.70); and for drinking any soda, 0.73 (95% CI, 0.66-0.80). Multiple linear regression showed that an increased frequency of family dinner was also associated with substantially higher intake of several nutrients, including fiber, calcium, folate, iron, vitamins B6, B12, C, and E; lower glycemic load; and lower intake of saturated and trans fat as a percentage of energy. We observed little or no effect on intakes of whole dairy products, red meat, or snack foods. Patterns were similar for boys and girls. CONCLUSIONS: Eating family dinner was associated with healthful dietary intake patterns, including more fruits and vegetables, less fried food and soda, less saturated and trans fat, lower glycemic load, more fiber and micronutrients from food, and no material differences in red meat or snack foods. PMID- 10728111 TI - Fluoride and bacterial content of bottled water vs tap water. AB - CONTEXT: Bottled water has become a status symbol and is frequently used in place of tap water. While both waters are considered safe to drink, is either more beneficial in preventing tooth decay and is there a difference in purity? OBJECTIVE: To determine the fluoride level and bacterial content of commercially bottled waters municipal tap water and to compare the results. DESIGN: Comparative study. SETTING: Cleveland, Ohio. SAMPLE: Fifty-seven samples of 5 categories of bottled waters were purchased from local stores. Samples of tap water were collected in sterile containers from the 4 local water processing plants. Fluoride levels were determined by an ion-selective electrode method. Water was cultured quantitatively and levels of bacteria were calculated as colony-forming units (CFUs) per milliliter. MAIN OUTCOME MEASURE: Fluoride levels and bacterial counts. RESULTS: Fluoride levels within the range recommended for drinking water by the Ohio Environmental Protection Agency, Cincinnati, 0.80 to 1.30 mg/L, were found in only 3 samples of bottled water tested. The fluoride levels of tap water samples were within 0.04 mg/L of the optimal fluoride level of 1.00 mg/L. The bacterial counts in the bottled water samples ranged from less than 0.01 CFU/mL to 4900 CFUs/mL, including 6 samples with levels substantially above 1000 CFUs/mL. In contrast, bacterial counts in samples of tap water ranged from 0.2 to 2.7 CFUs/mL. CONCLUSIONS: Five percent of the bottled water purchased in Cleveland fell within the required fluoride range recommended by the state, compared with 100% of the tap water samples, all of which were also within 0.04 mg/L of the optimal fluoride level of 1.00 mg/L. Use of bottled water based on the assumption of purity can be misguided. Recently, the Environmental Protection Agency, Washington, DC, published a final ruling that requires community water systems to regularly report to the public on the quality of local tap water; there are no similar proposals to determine the quality of bottled water through labeling. PMID- 10728112 TI - Supplemental insurance and mortality in elderly Americans. Findings from a national cohort. AB - CONTEXT: As the burden of out-of-pocket health care expenditures for Medicare beneficiaries has grown, the need to assess the relationship between uncovered costs and health outcomes has become more pressing. OBJECTIVE: To assess the relationship between risk for out-of-pocket expenditures and mortality in elderly persons with private supplemental insurance. DESIGN: Retrospective cohort study using proportional hazards survival analyses to assess mortality as a function of health insurance, adjusting for sociodemographic, access, and case mix-health status measures. SETTING: The 1987 National Medical Expenditure Survey, a representative cohort of the US civilian population, linked to the National Death Index. PARTICIPANTS: A total of 3751 persons aged 65 years and older. MAIN OUTCOMES MEASURES: Five-year mortality rate. RESULTS: After 5 years, 18.5% of persons at low risk for out-of-pocket expenditures, 22.5% of those at intermediate risk, and 22.6% of those at high risk had died. After multivariate adjustment, a significant linear trend (P = .02) toward increasing mortality with increasing risk category was observed. Compared with the low-risk group, persons in the intermediate-risk group had an adjusted hazard ratio of 1.2 (95% confidence interval, 0.9-1.6), whereas those in the high-risk group had an adjusted hazard ratio of 1.4 (95% confidence interval, 1.0-1.9). CONCLUSIONS: Increasing risk for out-of-pocket costs is associated with higher subsequent mortality among elderly Americans with supplemental private coverage. Although research is needed to identify which specific components of out-of-pocket expenditures are adversely associated with health outcomes, findings support policies to decrease out-of-pocket health care expenditures to reduce the risk for premature mortality in elderly Americans. PMID- 10728113 TI - Vitamin and mineral supplement use in the United States. Results from the third National Health and Nutrition Examination Survey. PMID- 10728114 TI - Preventive services for rural and urban African American adults. AB - BACKGROUND: Studies have shown that African Americans and rural patients receive fewer preventive services than other patients. OBJECTIVE: To compare the use of preventive services by African Americans in urban and rural settings to determine if race and rural residence were additive risks for not obtaining preventive services. METHODS: Three hundred African American patients seeking care in family practices in South Carolina were surveyed about preventive health care. RESULTS: Rural and urban African Americans were equally likely to know about preventive services and be up-to-date on receiving these services. In both practices, those with lower incomes were less likely to be up-to-date. Patients seen in the urban setting were more likely to receive counseling regarding exercise and smoking than those in the rural practice (87% vs 71%, P = .003). CONCLUSIONS: For both urban and rural African American patients with access to primary care physicians, preventive service use is high. The best predictor of poor compliance with preventive service recommendations was low income, suggesting that a lack of access to care is the primary reason why rural and African American populations do not receive adequate preventive health care. PMID- 10728115 TI - Pharmacotherapy of smoking cessation. AB - Tobacco use is the number one cause of preventable diseases in the United States. Smoking accounts for more than 400,000 deaths yearly and 30% of all cancer deaths. Primary care physicians have access to 70% of smokers, approximately 60% of whom are perceived to be in excellent health. Recent advances in the pharmacotherapy of nicotine addiction, including nicotine nasal spray, nicotine inhaler, bupropion hydrochloride, and over-the-counter transdermal nicotine patches, have increased the treatment options physicians can offer to smokers. Physicians, especially those in primary care specialties, should familiarize themselves with these products to improve efforts to help their patients stop smoking. This article reviews scientific data on the efficacy of approved medications, benefits, adverse effects, and appropriate use of these products. We also discuss nicotine addiction and treatment for special populations, including women, ethnic minorities, light smokers, and patients with cardiovascular and pulmonary diseases. PMID- 10728116 TI - Diagnostic workup before diagnosing colic. PMID- 10728117 TI - An unusual cause of postpartum vomiting. AB - We are presenting this case to emphasize that the symptoms of Addison disease are very similar to those of pregnancy and, although this disease is rare, the consequences can be catastrophic if it is overlooked, particularly in pregnancy. PMID- 10728118 TI - Physician disclosure of healthy personal behaviors improves credibility and ability to motivate. AB - CONTEXT: While some studies have shown that physicians with healthy personal habits are especially likely to discuss prevention with their patients, to our knowledge no one has published information testing whether physician credibility and patient motivation to adopt healthier habits are enhanced by physician's disclosures of their own healthy behaviors. DESIGN: Two brief health education videos about improving diet and exercise were produced and shown to subjects (n1 = 66, n2 = 65) in an Emory University general medical clinic waiting room in Atlanta, Ga. In one video, the physician revealed an additional half minute of information about her personal healthy dietary and exercise practices and had a bike helmet and an apple visible on her desk (physician-disclosure video). In the other video, discussion of personal practices and the apple and bike helmet were not included (control video). RESULTS: Viewers of the physician-disclosure video considered the physician to be generally healthier, some-what more believable, and more motivating than did viewers of the control video. They also rated this physician to be specifically more believable and motivating regarding exercise and diet (P < or = .001). CONCLUSION: Physicians' abilities to motivate patients to adopt healthy habits can be enhanced by conveying their own healthy habits. Educational institutions should consider encouraging health professionals-in training to practice and demonstrate healthy personal lifestyles. PMID- 10728119 TI - Avascular necrosis. A case history and literature review. AB - We describe a patient with avascular necrosis in both shoulders. Confirmatory testing in making the diagnosis included plain radiography, bone scan, and magnetic resonance imaging. The pathogenesis and staging of the disease by radiography are presented in the article. Treatment options include a conservative regimen of shoulder range of motion exercises and nonsteroidal anti inflammatory agents or surgery (arthroplasty or core decompression). The patient's risk factors include long-term corticosteroid use, smoking, and alcohol consumption. Other known risk factors include sickle cell disease, Gaucher disease, chemotherapy, lymphoma, dysbaric conditions, and trauma. This literature search shows that prevention and early diagnosis lend the best outcomes for the diagnosis of avascular necrosis. PMID- 10728120 TI - Age differences in adult personality: findings from the United States and China. AB - This study used samples of adults from the United States (n = 285) and the People's Republic of China (n = 450) to examine age, gender, and culture differences in personality. Participants in both samples ranged from 20 to 87 years of age and responded to the California Psychological Inventory (CPI). Factor analyses of the CPI resulted in 4 personality factors (i.e., extraversion, control/norm orientation, flexibility, femininity/masculinity), with high congruence across cultures after targeted rotation. Multivariate analyses of variance with scale scores revealed significant Age Group 3 Culture and Gender 3 Culture interactions, and significant main effects of age, gender and culture. Specifically, negative age differences were found for scales that loaded on extraversion and flexibility, with older adults having lower scores on these scales. In contrast, positive age differences were found for scales indicative of control/norm orientation. In general, age differences were more pronounced for Chinese adults than for U.S. adults. PMID- 10728121 TI - Personality traits and suicidal behavior and ideation in depressed inpatients 50 years of age and older. AB - Completed suicide may be the most preventable lethal complication of depressive disorders in older adults. Identification of risk factors for suicidal behavior has therefore become a major public health priority. Using data collected on 81 depressed patients 50 years of age and older, we report analyses designed to determine the associations between the personality traits that constitute the Five Factor Model of personality and measures of suicidal behavior and ideation. We hypothesized that low Extraversion would be associated with a lifetime history of attempted suicide, and high Neuroticism would be associated with suicidal ideation. Results were generally consistent with the hypotheses. We also observed a relationship between Openness to Experience and suicidal ideation. These findings suggest that longstanding patterns of behaving, thinking, and feeling contribute to suicidal behavior and thoughts in older adults and highlight the need to consider personality traits in crafting and targeting prevention strategies. PMID- 10728122 TI - Aging and the development of automaticity in conjunction search. AB - In two experiments, younger and older observers carried out feature searches for targets defined by their luminance contrast and orientation. Additionally, they received consistent-mapping (CM) training in luminance contrast by orientation conjunction search, followed by a brief exposure to conjunction search under reversal conditions. In Experiment 1, display size effects on reaction time suggested that both younger and older observers were conducting a parallel search in all conditions and showed equivalent disruption at reversal. Experiment 2 was a substantive replication of the first using more difficult conjunction search displays. In addition to latency, we measured the number, duration, and feature based selectivity of fixations made during conjunction search. Display size effects were larger than in Experiment 2 and were of equivalent magnitude in younger and older people. There were no age differences in improvement in conjunction search and minimal age differences in disruption following reversal. Both age groups demonstrated early in training that they could select items possessing target features (i.e., the color white), and both age groups demonstrated that they could not completely reverse this selectivity when these features no longer defined the target. These experiments have several implications for models of visual attention and age differences therein. PMID- 10728123 TI - Relationships between mood and estradiol (E2) levels in Alzheimer's disease (AD) patients. AB - This study investigates the relationship between mood and estradiol (E2) levels and assesses the prevalence of mood symptoms in Alzheimer's disease (AD) patients compared to healthy elderly controls. Fifty-two AD patients (26 men, 23 estrogen non-using women and three estrogen-using women), mean age 76.2 years, were recruited and assessed with the Geriatric Depression Scale (GDS), a test of mood, and a radioimmunoassay measure of E2 levels at the time of testing. The AD patients were compared to a control group of age and gender-matched healthy elderly men and women estrogen-users and non-users. No differences were found between the AD patients and the controls in overall E2 levels, but, as expected, the women estrogen-users in both the AD and control groups had higher E2 levels than the men and the female estrogen non-users. Both groups of men had higher E2 levels than the estrogen non-using women. There was a significant negative correlation between E2 levels and GDS scores in the full sample, which was particularly strong in the estrogen-using women. This indicates that those subjects with higher E2 levels had less mood symptomatology. Overall, mood scores in the AD patients were higher than in the healthy controls, indicating higher levels of depressive symptomatology; the highest depression scores occurred in the AD women who were estrogen non-users. This suggests that depressive symptoms are common in AD patients, and that women with AD who are not taking estrogen replacement may be especially vulnerable to depression. PMID- 10728124 TI - Effects of chronic insomnia and use of benzodiazepines on daytime performance in older adults. AB - This study evaluated the impact of insomnia and chronic use of benzodiazepines on the cognitive and psychomotor performance of older adults. Three conditions, matched on age, gender, and education, were compared: 20 prolonged users of benzodiazepines for insomnia, 20 unmedicated insomniacs, and 20 good sleepers. The participants completed neuropsychological tests of memory, attention/concentration, psychomotor speed, and executive functions, as well as subjective evaluations of their actual performance. Individuals with insomnia, both medicated and unmedicated, performed worse than good sleepers on the attention/concentration factor. There was no other objective evidence of performance impairments. However, unmedicated insomniacs had lower performance expectancies and subjectively rated their performance more negatively relative to medicated insomniacs and good sleepers. Both insomnia conditions also rated their performance as lower compared with their perceived potential. It is suggested that the attention/concentration difficulties experienced by medicated and unmedicated older adults with insomnia may be linked to a state of hyperarousal. The discrepancies between subjective reports of daytime deficits and objective impairments may reflect a generalized faulty appraisal of sleep and daytime functioning among individuals with insomnia complaints. The implications of those findings for the assessment and treatment of late-life insomnia are discussed. PMID- 10728125 TI - Predicting caregiver burden and depression in Alzheimer's disease. AB - OBJECTIVES: The purpose of this study was to investigate the predictors of caregiver burden and depression, including objective stressors and mediation forces influencing caregiving outcomes. METHODS: This investigation is based on the 1994 Canadian Study of Health and Aging (CSHA) database. Participants were 613 individuals with dementia, living in either the community or an institution, and their informal caregivers. Participants for the CSHA were identified by screening a large random sample of elderly persons across Canada. Structural equation models representing four alternative pathways from caregiving stressors (e.g., functional limitations, disturbing behaviors, patient residence, assistance given to caregiver) to caregiver burden and depression were compared. RESULTS: The data provided the best fit to a model whereby the effects on the caregiver's well-being are mediated by appraisals of burden. A higher frequency of disturbing behavior, caring for a community-dwelling patient, and low informal support were related to higher burden, which in turn led to more depressive symptomatology. Caregivers of patients exhibiting more disturbing behaviors and functional limitations received less help from family and friends, whereas those whose care recipients resided in an institution received more informal support. DISCUSSION: Our findings add to the preexisting literature because we tested alternative models of caregiver burden using an unusually large sample size of participants and after overcoming methodological limitations of past research. Results highlight the importance of the effective management of disturbing behaviors, the provision of formal services for caregivers with highly impaired patients and no informal support, and the improvement of coping skills in burdened caregivers. PMID- 10728126 TI - Pathways to retirement: patterns of labor force participation and labor market exit among the pre-retirement population by race, Hispanic origin, and sex. AB - OBJECTIVES: This study examines the pre-retirement labor force participation behavior of Black, White, and Hispanic men and women to determine how patterns of labor market exit differ among groups. METHODS: We combine data from the first and second waves of the Health and Retirement Study and apply multinomial logit regression techniques to model labor force status in the first wave of the HRS and change over time. RESULTS: Black, Hispanic, and female elderly persons experience more involuntary job separation in the years immediately prior to retirement, and the resulting periods of joblessness often eventuate in "retirement" or labor force withdrawal. Minority disadvantage in human capital, health, and employment characteristics accounts for a large part of racial and ethnic differences in labor force withdrawal. Nevertheless, Black men and Hispanic women experience more involuntary labor market exits than Whites with similar socioeconomic and demographic characteristics. DISCUSSION: Workers most vulnerable to labor market difficulties during their youth confront formidable obstacles maintaining their desired level of labor force attachment as they approach their golden years. This has significant policy implications for the contours of gender and race/ethnic inequality among elderly persons, particularly as life expectancy and the size of the minority elderly population continue to increase. PMID- 10728127 TI - Tracking the majority: households, older workers, and retirement during the Great Depression. AB - OBJECTIVES: Well into the 20th century, elderly people relied on traditional means of support, such as children's financial contributions or continued labor force activity. After the institution of Social Security in the late 1930s, retirement--permanent withdrawal from the labor force with financial arrangements made for support--became an expected part of the life cycle of men 65 years and older in the United States. This research explores the extent of retirement and methods to finance it in the period just before Social Security became available. METHODS: The 1935-1936 Study of Consumer Purchases (SCP) contains information on demographic and economic conditions for 5,975 households. The SCP is a rich but underutilized source of data on household behavior. The data allow two definitions of labor force activity to be constructed; descriptive statistics identify factors associated with retirement. RESULTS: Alternative sources of support, such as pensions and investment income, have been thought to be relatively insignificant before the 1940s. This article shows that retirement in the modern sense appeared before state provision of support for aged persons. SCP data indicate considerable reliance on such financial instruments, a particularly noteworthy result given Depression conditions. Pension and investment income also helps identify persons who might report a gainful occupation but appear to have withdrawn from labor force activity, meeting a modern definition of retirement. DISCUSSION: The SCP, collected just as Social Security was enacted, reveals that nonfamilial sources of income like pensions and investments had begun to underwrite retirement without dependence on family members. Many older persons relied on these instruments for support in old age, and many did not have children present in their households. These results constitute evidence for an independent, nonfamily-based retirement before governmental provision of assistance through Social Security. PMID- 10728128 TI - Deficiency citations for physical restraint use in nursing homes. AB - OBJECTIVES: The average percentage of residents restrained in nursing homes is approximately 20%. Facilities that do not meet Health Care Financing Administration standards for restraint use may be issued a deficiency citation. This article investigates which structure and process factors of nursing homes are associated with a deficiency citation for restraint use. METHODS: Nationally representative data from the 1997 On-line Survey Certification of Automated Records are used, first, to provide descriptive analyses, and second, for logistic regression analyses of structure and process factors associated with a deficiency citation for restraint use. RESULTS: A total of 2,321 facilities were found to have at least one restraint deficiency citation, and 14,703 had none. After controlling for seven other key variables, five structural factors and six process factors are significant. The structural factors--larger bed size, for profit ownership, and hospital based--were significantly associated with a higher likelihood of a deficiency citation for restraint use; whereas higher numbers of full-time equivalent specialists per resident and nurse aide training were significantly associated with a lower likelihood. The process factors--suctioning therapy, pain management, and bladder training--were significantly associated with a lower likelihood of a deficiency citation for restraint use; whereas intravenous therapy, higher use of catheters, and physical restraints were significantly associated with a higher likelihood of a deficiency citation. DISCUSSION: This analysis establishes linkages between structures and processes and the outcome of a deficiency citation for restraint use. The structural results may have some utility for regulators. They could be used to develop a specific program to target facilities most commonly found to have inappropriate restraint use. The process results may have some utility for providers who could use the information to target residents for review of inappropriate restraint use. PMID- 10728129 TI - Sex differences in the prevalence of mobility disability in old age: the dynamics of incidence, recovery, and mortality. AB - OBJECTIVES: This study examined sex differences in the prevalence of mobility disability in older adults according to the influences of three components of prevalence: disability incidence, recovery from disability, and mortality. METHODS: Participants in a population-based study of older adults from three communities in the United States (N = 10,263) were studied for up to 7 years. Life table methods were used to estimate the influence of each of the three components of disability prevalence in women and men. Sex differences in probabilities for transition states were measured by relative risks derived from a single model using a Markov chain approach. RESULTS: The proportion of disabled women increased from 22% of women aged 70 years to 81% of those aged 90 years. In men, comparable figures were 15% and 57%. Incidence had the greatest impact on the sex differences in disability prevalence until age 90 and older when recovery rates had a greater impact on differences in prevalence. Mortality differences in men and women had only a modest impact on sex differences in disability prevalence. These findings initially seemed to contradict striking sex differences observed in the relative risks for mortality in men compared with women. Subsequent graphical analyses showed that incidence rather than recovery or mortality largely accounted for sex differences in disability prevalence in old age. CONCLUSION: Disability incidence, recovery from disability, and mortality dynamically influence the sex differences in the prevalence of mobility disability. However, incidence has the greatest impact overall on the higher prevalence of disability in women compared with men. PMID- 10728130 TI - Out-of-pocket health care costs among older Americans. AB - OBJECTIVES: Despite Medicare, elderly persons are exposed to substantial out-of pocket health care cost burdens. As Medicare reform proposals are considered, it is important to determine the current size, distribution, and burden of these expenditures. METHODS: Data from the 1995 Medicare Current Beneficiary Survey were used to analyze out-of-pocket expenditures and their burden in relation to income; the proportion of total health care expenditures paid out-of-pocket; and the role of pharmacy, hospital, physician, and other services in overall out-of pocket spending. RESULTS: Expenditures averaged 19.0% of income, for full-year Medicare beneficiaries alive during all of 1995. In bivariate analyses, higher burden subgroups included those in poor health (28.5% of income), older than age 85 (22.4%), and with income in the lowest quintile (31.5%, despite Medicaid coverage for some). Those relying on fee-for-service Medicare only (23.0%) or with self-purchased supplemental insurance (25.5%) experienced more burden than those with employer-sponsored coverage or in HMOs. In multivariate analyses, functional impairment, number of medical conditions, self-perceived health and privately-purchased supplemental coverage were each associated with higher out-of pocket burden, while HMO participation was associated with lower burden. Out-of pocket expenditures averaged 15.2% of total health care expenditures with the proportion highest (22.6%) for those with no supplemental coverage. More than half of out-of-pocket payments for health care services were for prescription drugs and dental services. DISCUSSION: Out-of-pocket cost burdens fall most heavily on those with chronic health conditions and without employer-subsidized supplemental coverage or Medicaid. Impact of Medicare reform proposals on these subgroups needs to be carefully evaluated. PMID- 10728131 TI - Genetics in primary care practice. PMID- 10728132 TI - Appropriate abdominal aortic aneurysm screening. PMID- 10728133 TI - Hypertension in patients with diabetes. Overcoming barriers to effective control. AB - The management of diabetic hypertension poses special problems for the medical community. Although patient adherence is often a major barrier to successful management, physicians' beliefs and prejudices also negatively impact treatment. In addition, healthcare organizations need to provide better support to physicians who feel isolated in their efforts to manage diabetic hypertension. Reductions of morbidity and mortality are achievable goals but require aggressive treatment and improved adherence if they are to be reached. PMID- 10728134 TI - Viral pneumonias. Epidemic respiratory viruses. AB - Influenza, respiratory syncytial, and parainfluenza viruses usually cause mild, self-limited illness in adults. However, elderly and immunocompromised persons are at increased risk for development of severe pneumonia. Clinical and radiographic features of epidemic viral pneumonias are often nonspecific. Newer and faster methods of viral culture and viral antigen detection have improved the capability for definitive diagnosis in recent years. Preventive measures for influenza virus pneumonia center on limiting exposure of high-risk patients to active cases of influenza, administering annual vaccinations, and providing chemoprophylaxis. Prophylaxis against RSV is effective in preventing complications. No effective vaccines have been developed against RSV or parainfluenza. Therapy for viral pneumonia is primarily supportive. Amantadine may be beneficial for influenza virus pneumonia, and ribavirin may be useful for RSV and parainfluenza virus disease. However, further definitive studies are necessary to determine their roles in these viral pneumonias. PMID- 10728135 TI - Genetic counseling in primary care. What questions are patients likely to ask, and how should they be answered? AB - Clues to genetic disorders are often first discovered during routine healthcare visits. This can create a dilemma for both the physician and the patient about what to do next. Because human genetics is a rapidly advancing field, many physicians are wondering which elements of a patient's medical history warrant a visit to the genetics clinic and which should be simply watched. In this article, Ms Facher and Dr Robin answer specific questions that the primary care physician is likely to have or to be asked about the evolving science of genetic testing. PMID- 10728136 TI - Topical therapy for acne vulgaris. How do you choose the best drug for each patient? AB - Although management of acne is sometimes difficult, primary care physicians can offer a number of treatment plans to patients with this skin condition. Comedonal acne usually responds to topical keratolytics, such as salicylic acid, benzoyl peroxide, adapalene, and tretinoin. Inflammatory acne is usually treated with topical therapy plus a systemic antibiotic. Nodulocystic acne generally requires an 8-week course of systemic antibiotics. If the nodulocystic acne does not improve, minocycline or isotretinoin may be needed. Topical therapy is often helpful in the long-term management of nodulocystic acne. New products are available that deliver topical agents in novel ways that decrease skin irritation. With the proper tools and instructions in use, most patients have significant improvement in their acne. PMID- 10728137 TI - Progressive scarring of the conjunctiva. Cicatrical pemphigoid. PMID- 10728138 TI - Nonresponders to hepatitis B vaccine. PMID- 10728139 TI - What is causing excessive daytime sleepiness? Evaluation to distinguish sleep deprivation from sleep disorders. AB - Many people have a temporary spell, often in early afternoon, when they feel drowsy. This passing desire for a quick nap is completely different from excessive daytime sleepiness, which is a much more significant problem. Considering the potentially dire personal and economic consequences of falling asleep unintentionally or at inappropriate times, excessive daytime sleepiness must be taken very seriously. A thorough evaluation, as described by Dr. Mahowald, virtually always leads to a specific underlying cause, allowing effective treatment recommendations. PMID- 10728140 TI - Helping patients who say they cannot sleep. Practical ways to evaluate and treat insomnia. AB - Why do some people spend most of the night tossing and turning while others drop off as quickly as a cat in the sun? There are many possible explanations for problems with falling and staying asleep and, sometimes, patients actually get a lot more sleep than they think they do. Each type of insomnia has its own set of symptoms, which can be used, along with appropriate diagnostic tools, to help in identification. In this article, Dr. Attarian describes differential diagnosis and summarizes the best treatment approaches to the common causes of insomnia. PMID- 10728141 TI - Parasomnias. Managing bizarre sleep-related behavior disorders. AB - Sleep can be a troubling experience for persons plagued by nocturnal disorders known as parasomnias. While they are "asleep," such persons may be walking, screaming in terror, rearranging furniture, eating odd food concoctions, or wielding weapons. Or they may be unable to fall asleep because of the unpleasant sensations of restless legs syndrome. Although these disorders are indeed bizarre, effective treatments are available. In this article, Drs Schenck and Mahowald discuss the evaluation and treatment of parasomnias and provide illustrative patient vignettes from their extensive experience at a sleep disorders center. PMID- 10728142 TI - Sleep disorders in children and teens. Helping patients and their families get some rest. AB - Diagnosing sleep disorders in children and adolescents is challenging and rewarding and requires integration of medical, neurodevelopmental, and behavioral histories. Most patients can be successfully treated once a thorough evaluation has been completed and age-appropriate differential diagnosis of common sleep disorders has been considered. With appropriate knowledge and tools, physicians may find that pediatric sleep disorders are some of the most treatable problems in medicine. PMID- 10728144 TI - Irritable bowel syndrome. Definitive diagnostic criteria help focus symptomatic treatment. AB - A diagnosis of IBS can often be established on the basis of the Manning and Rome criteria, which delineate specific features. A limited evaluation to exclude organic disease confirms this clinical impression. Patients should be reassured that no serious organic illness is causing the symptoms and should be educated about the natural history of the disorder. Treatment must be directed at both achieving control of the gastrointestinal complaints and searching for an underlying psychological or social factor that may contribute to the disorder. PMID- 10728143 TI - Herpes simplex virus infections. New treatment approaches make early diagnosis even more important. AB - The herpes simplex viruses HSV-1 and HSV-2 cause problems for millions of people worldwide. Infections range from simple cold sores and fever blisters to severe central nervous system disorders. Development of effective antiviral medications has made prompt recognition important in primary care practice. Appropriate therapy can significantly reduce both medical and psychosocial ramifications of herpes infections and can greatly improve the quality of life for many patients. PMID- 10728145 TI - Finger 'pebbles'. A dermatologic sign of diabetes mellitus. AB - A 35-year-old obese man presented with a chief complaint of hand dryness of 5 years' duration. He was a store manager and denied exposure to chemicals, repetitive trauma, chronic irritation, and hard manual labor. However, he did admit to frequent hand washing. He had no itching or swelling in his hands, but on occasion he had tenderness in the dry areas. He had no personal or family history of diabetes, heart disease, or renal disease, and he stated that at his annual physical examination 6 months earlier, routine blood work was normal. He reported polyuria (every 2 hours), nocturia (five times per night), and polydipsia but no weakness, weight loss, visual changes, or neurosensory changes. Examination revealed xerosis of his hands and "pebbles" on the dorsal aspect of his fingers. The papules were most dense over the knuckles and interphalangeal joints (figures 1 through 3). He also had dozens of acrochordons (i.e., cutaneous papillomas, or skin tags) 1 to 4 mm in diameter on his neck, axilla, and groin. No other cutaneous lesions were noted. Specifically, there was no scleredema adultorum, necrobiosis lipoidica diabeticorum, acanthosis nigricans, bullae, or patchy pretibial pigmentation, although he did have several brown macules 1 to 5 mm in diameter on the sides of his lower legs. The macules had been present for years. Levels of hemoglobin A1c and glycated hemoglobin were 7.5% and 9.5%, respectively (normal, 4.4% to 5.9% and 5.0% to 7.3%). The patient was referred to his family physician, and his diabetes has been well controlled with insulin. PMID- 10728146 TI - Managing endometriosis. Strategies to minimize pain and damage. AB - Endometriosis is a common condition affecting a significant number of women of childbearing age. The diagnosis is clinical and thus can be difficult to make. History taking is generally most helpful, and diagnostic tests have a limited role. Diagnostic laparoscopy remains the "gold standard" for diagnosis of endometriosis. Treatment is geared toward improving fertility and controlling pain and is often not curative. However, both medical and surgical therapies are of value in controlling the disease. Attention to the psychosocial needs of the patient are also critical. Future therapies will be based on a further understanding of the pathogenesis of endometriosis and the effect of hormones on the disease. For the primary care physician who may not be comfortable prescribing GnRH analogs or other medical therapies, referral to a gynecologist or endocrinologist should be considered. PMID- 10728147 TI - Evaluation of fitness to drive. The physician's role in assessing elderly or demented patients. AB - The role of physicians in deciding whether a patient should continue to drive is purely advisory. However, physicians have a moral and, in some states, a legal obligation to report patients who are no longer fit to drive. The most authoritative test to predict safe driving in the elderly is an on-road evaluation conducted by the state driver's licensing authority, which has ultimate responsibility for deciding a patient's fitness to drive. Patients with mild dementia are generally considered safe drivers, although specialized testing, such as an on-road test, may be indicated. Those with moderate dementia can be further evaluated by the on-road test, since psychological testing to distinguish moderate from mild dementia is imprecise. Severe dementia is generally considered a contraindication to driving. When a patient is deemed unfit to drive, the physician can provide counseling and support to help ease the transition away from driving. PMID- 10728148 TI - Otitis media in children. Diagnosis, treatment, and prevention. Institute for Clinical Systems Improvement. PMID- 10728149 TI - A stepwise approach to acid-base disorders. Practical patient evaluation for metabolic acidosis and other conditions. AB - Acid-base disorders can usually be approached by following the steps outlined in the text and doing the calculations shown in the box on page 257. Clues about the underlying disorder can be obtained from history taking and physical examination. Assessment of pH, PaCO2, and HCO3- allows determination of whether a primary metabolic or respiratory disorder is present. Calculation of the predicted compensatory response for simple acid-base disorders might suggest the presence of an additional disease process if compensation is not appropriate. Calculation of the various gaps can be helpful in differential diagnosis (i.e., anion gap for diagnosis of metabolic acidosis, delta anion gap for diagnosis of high-anion-gap metabolic acidosis, and urine anion gap for diagnosis of a non-anion gap metabolic acidosis). Most acid-base problems can be solved with use of the stepwise approach described. PMID- 10728150 TI - Management of cancer pain. Safe, adequate analgesia to improve quality of life. AB - Pain is one of the most common problems for cancer patients, and its management is often hindered by barriers created by patients and physicians alike. By avoiding potential barriers and understanding the principles of pain management and drug selection and titration provided here by Dr Hartmann and colleagues, physicians can safely administer adequate pain relief to their patients in need. PMID- 10728151 TI - Earliest recollections in anorexia and bulimia. AB - Earliest Recollections (ER) are very useful as they reveal information about a person's main aims and lifestyle, according to the Individual Psychology assumption of the inner coherence of the personality. This paper looks at the utility of analyzing ER in psychopathological research, diagnosis and therapy. Its purpose is to compare particular ER in a group of anorectic outpatients, a group of bulimic outpatients and in a control group. The results provide further evidence that ER are a useful instrument to identify various aspects of family dynamics, particular pathological nuclei which are often of the borderline type. They can also help to overcome the initial hostility towards any therapy that is often present in anorectic and assist the therapist early on in drawing a mental picture of the premises for understanding the lifestyle of the subject both as a child, and now as a patient. PMID- 10728152 TI - The activity of GH/IGF-I axis in anorexia nervosa and in obesity: a comparison with normal subjects and patients with hypopituitarism or critical illness. AB - GH/IGF-I axis activity changes have been reported both in anorexia nervosa (AN) and in obesity (OB). AN is characterized by GH hypersecretion and very low IGF-I levels as a result of undernutrition and acquired peripheral GH resistance. On the other hand OB is a GH hyposecretory state but IGF-I levels are generally preserved. The activity of GH/IGF-I axis in AN and OB has never been directly compared with that of other pathophysiological conditions such as hypopituitarism and critical illness in which a reduction of both GH and IGF-I secretion has been demonstrated. To this aim, we evaluated IGF-I levels and both basal and GHRH (1 microgram/kg) IV-induced GH secretion in 20 female patients with anorexia nervosa (mean age: 19.1 +/- 0.8 years) and in 15 female and 5 male patients with simple obesity (mean age: 39.0 +/- 3.0 years). We then compared the results with those of hypopituitaric patients with severe GH deficiency (GHD), including 10 female and 10 patients (mean age: 32.0 +/- 2.1 years), and with 4 female and 7 male patients with critical illness (CRI) following multiple trauma 72 hours after ICU admission (mean age: 59.2 +/- 1.2 years). Twenty-six normal subjects (NS) including 14 female and 12 male patients (mean age: 37.8 +/- 3.7 years) were studied as controls. Basal IGF-I levels in AN patients (93.5 +/- 11 micrograms/L) were lower (p < 0.001) than in the NS (201.7 +/- 13.5 micrograms/L) and OB (194.5 +/- 28.6 micrograms/L), which, in turn, were similar. IGF-I levels in AN patients were lower than in CRI patients (162.8 +/- 17.4 micrograms/L) and higher than in GHD patients (76.7 +/- 13.5 micrograms/L) but these differences did not attain statistical significance. Basal GH levels in AN patients (7.6 +/- 2.5 micrograms/L) were higher (p < 0.001) than in NS (1.8 +/- 0.3 micrograms/L), OB patients (1.1 +/- 0.5 micrograms/L), CRI patients (1.8 +/- 0.5 micrograms/L) and GHD patients (0.3 +/- 0.1 microgram/L), which were the lowest (p < 0.01). The GHRH-induced GH rise in AN patients (AUC: 2032.9 +/- 253.5 micrograms/L/h) was three fold higher (p < 0.001) than in NS (662.1 +/- 80.3 micrograms/L). On the other hand in OB (332.4 +/- 74.7 micrograms/L/h) the GH response to GHRH was similar to that in CRI (199.6 +/- 98.8 micrograms/L/h); both were clearly higher (p < 0.01) than in GHD patients (25.1 +/- 5.2 micrograms/L/h) but lower (p < 0.01) than in NS. These findings demonstrate that in AN patients, in spite of a clear increase of both basal and GHRH-induced GH secretion, IGF-I synthesis and release are as markedly impaired as in patients with panhypopituitarism and severe GHD. On the other hand in OB and in CRI, IGF-I synthesis and release are preserved despite marked impairment to GHRH-induced GH secretion. These results reinforce the major role of nutrition in conditioning the activity of GH/IGF-I axis in different patho-physiological states. PMID- 10728153 TI - D4 dopamine receptor gene exon III polymorphism and obesity risk. AB - Many genes have been identified that may play a role in increasing individual susceptibility to obesity. Reduced dopamine function appears to play a role in dysfunctional eating patterns and may predispose some individuals to obesity. The long version of the D4 dopamine receptor gene (D4DR) has been shown to alter receptor function and reduce intracellular response to dopamine. It also has been associated with novelty-seeking-related personality traits that are found with greater frequency in obese individuals. We examined the association between the long alleles of the D4DR and obesity in a sample of 115 obese patients participating in a weight management program. No direct relationship was found between the D4DR and body mass or novelty-seeking-related personality traits. We constructed four models of increased obesity risk that included combinations of traditional risk factors (i.e., long-term history of obesity, parental obesity, a body mass index > 40) and elevations on the novelty-seeking-related scales of the Karolinska Scales of Personality. There was a significant increase in the frequency of the D4DR long alleles in individuals defined as high risk using the combination of novelty-seeking-related personality traits, severe obesity (i.e., BMI > 40), and any other traditional risk factor, but not with the traditional risk factors alone. These preliminary data suggest a potential role for the D4DR gene in increasing obesity susceptibility. PMID- 10728154 TI - Obesity, psychopathology and eating attitudes: are they related? AB - The aim of this study was to verify the link between psychopathological disorders and eating attitude in a clinical sample of 195 female obese subjects. A battery of psychological tests, including the Italian versions of the MMPI2, ASQ and EAT scales were administered to all the patients. We analyzed the link between psychopathological traits and eating attitudes by using both Multiple Regression analysis and non-parametric Segmentation Modeling. The results showed that psychopathological aspects, and depression in particular, are strongly linked to the eating attitude of clinically obese subjects. This is an important result also for therapeutic purposes, as it highlights the need for psychological support in diet therapy to intervene on the psychological perceptions and experiences of the patient. PMID- 10728155 TI - Randomized trial of measures of body fat versus body weight in the treatment of anorexia nervosa. AB - OBJECTIVE: To determine whether treatment goals based on body weight or body fat assessed by anthropometrics caused a larger reduction in the Eating Disorder Examination (EDE) over 3 months during the treatment of anorexia nervosa (AN). METHODS: Randomized trial comparing the use of body weight versus measures of body fat by anthropometrics, with the EDE as the outcome measure. Of 23 patients enrolled, 11 subjects who met diagnostic criteria for AN (DSM-IV) and received outpatient treatment from the Eating Disorders clinic of a university teaching hospital completed the study. RESULTS: Comparison of the 2 methods by independent t-tests, verified by Mann-Whitney and Sign tests, suggests no difference (p = 0.33). Due to the small sample size, statistical significance could not be assured. A cross-sectional survey of 48 clinic users found the majority of patients preferred to be followed by skinfold measurements and felt they were more helpful than weight. DISCUSSION: Regardless of which measurement is used, if the clinic staff firmly believe that a method is more helpful and more useful, it is likely that the patients will find it acceptable. PMID- 10728156 TI - Frequency of recovery from anorexia nervosa of a cohort patients re-evaluated on a long-term basis following intensive care. AB - The need to treat the acute onset of anorexia nervosa in a specialty unit is well known, but nevertheless, even "clinically recovered" patients show a high rate of relapse. The aim of our study was to re-evaluate various clinical (hemoglobin, transferrin, insulin-like growth factor I, TSH, PRL, gonadotropins and 17 beta estradiol) and psychiatric [semistructured interview, Eating Disorder Inventory (EDI) Test] parameters in 19 female anorexic patients hospitalized in our department from 1983 to 1993 (with a 9-year median prior to the present study) for the treatment of anorexia nervosa, and to compare these results with those of a previous follow-up performed on the same subjects in 1993. In the present evaluation, no significant variation was found among the clinical, nutritional and hormonal parameters when compared to those of the first follow-up in which all parameters had improved with the exception of the PRL levels, which were significantly low. Meanwhile, the percentage of patients with spontaneous menses increased significantly from 50% to 70%, while the number of patients on psychopharmacological therapy decreased significantly throughout the study. Furthermore the percentage of patients with altered (severe or mild) EDI profiles decreased to 50%. This study emphasizes the positive prognostic role of hospitalization and intensive care in a cohort of anorexic patients. The present study, in addition to demonstrating both a general maintenance of body weight acquired over the years, albeit in the lower normal range, and an increase in the percentage of patients with a regular menstrual cycle, also highlights the persistence of psychiatric abnormalities in a large number of patients, even in those diagnosed as "clinically recovered", thus suggesting the need for long-term psychiatric care. PMID- 10728157 TI - Analyzing the relation between self-esteem and eating disorders: differential effects of self-liking and self-competence. AB - Research in the past has demonstrated an association between low self-esteem and eating disorders. Recent research on self-esteem has shown, however, that self esteem is composed of two distinct factors--self-liking and self-competence. The present study examined the relation between these two self-esteem factors and both eating disorders and eating disturbed cognitions. A total of 51 female participants from a high-risk population were clinically interviewed to identify the eating disorder and administered a series of questionnaires that included a measure of self-liking and self-competence. A strong relationship was found between self-liking and eating disorders, but no relationship was found between self-competence and eating disorders. PMID- 10728159 TI - Evaluation of a programme of adapted physical activity for ED patients. AB - The importance of work focused on body experience in the therapy of Eating Disorders is widely acknowledged. Even so, few researches have been devoted to this aspect. This paper describes the employment of adapted physical activity as part of a residential cure programme for 96 eating disorder patients. The chief aims of this programme relate to individual identity, relations with others, the body, and getting patients to build themselves a correct image of physical activity. The wide variety of working tools includes both individual and group activities: movement exercise, adapted sports, dancing, expressive activities, relaxation. The following are used to evaluate results: individual and group talks, specific observation scales, and video recordings of some meetings. As shown by other authors, the observations indicate that controlled physical activities can be usefully integrated into an eating disorders cure project. PMID- 10728158 TI - Differential hemispheric involvement in anorexia nervosa. AB - Hemispheric function was assessed in 22 restricting anorexia nervosa (AN) female subjects and 22 normal female controls. Two verbal and two visuospatial tasks and a set of psychopathological tests were administered. The failure of the "classic" lateralization tests to reveal the expected left hemisphere dominance or a right hemisphere deficit in AN group, as compared to controls, is counterbalanced by the tendency of the psychopathological tests to prove the neuropsychological hypothesis of the present study. Neuropsychological interpretation of psychopathological data, however, does not constitute direct evidence and so further studies and more sophisticated techniques are needed. PMID- 10728160 TI - Hunger sensation: a chronobiometric approach to its within-day and intra-day recursivity in anorexia nervosa restricting type. AB - Hunger sensation (HS) is a perception with a daily (circadian) and within-day (ultradian) recursive pattern. In human beings, circadian repeatability was investigated by means of the Single Cosinor method, while the ultradian recursivity was investigated by means of the spectral analysis, both applied to the 24-h HS profile (orexigram). Orexigrams were provided by each subject investigated, who self-rated her subjective orectic stimulus (OS) (from 1 to 10 hunger units) every half hour. The study was performed in 19 female patients aged 13-52 newly diagnosed as in the first episode of Anorexia Nervosa Restricting Type, with a BMI below 18.7. The control group consisted of 10 clinically healthy women aged 21 to 52 years with a BMI from 23 to 25. Two types of orexigrams were found. The first was characterized by a low profile with negligible ultradian variability, in which the HS circadian rhythm (CR) was still detectable, but the power spectrum (PS) was composed of unusual ultradian components associated with a very diminished amplitude for the circadian harmonics. The second was characterized by an almost regular profile, in which the ultradian variability was clearly detectable, the CR regularly fluctuated, and the PS was almost regularly composed. These findings indicate that anorectic patients (AP) can be recognized by their orexigram as "hyporectic", or "eurectic". Therefore, the term "anorexia" seems to be appropriate for AP who exhibit the first type of orexigram (anorectic aphagia nervosa), whereas the second identifying those who could be defined as suffering from "eurectic aphagia nervosa". PMID- 10728161 TI - Are leptin levels dependent on body fat distribution in obese men and women? AB - Leptin levels are strongly related to total body fat. It is however not yet clear if leptin is also related to visceral fat accumulation or not. In this study, we investigated whether leptin is also associated with body fat distribution and if this association is different in men and women. Leptin was measured in 143 obese subjects (118 women and 25 men) with a body mass index (BMI) greater than 28. Also weight, skinfolds, waist-to-hip ratio (WHR), fat mass by bioimpedance analysis (BIA) were measured, and abdominal visceral and subcutaneous fat were determined by CT scan. Leptin levels were significantly related with BMI, with fat mass (in kg and percentage body fat) as measured by BIA and skinfolds, and with total abdominal fat mass and subcutaneous fat measured by CT scan. No association was found with visceral fat, waist circumference or WHR. In men and women separately, however, a correlation with visceral fat existed. After correction for total body fat, the correlation remained significant only with subcutaneous fat in women. Multiple regression analyses pointed out that percentage body fat was the most important determinant of leptin for all subjects, while for women subcutaneous fat was the most important parameter, and for men alone total abdominal fat. These results suggest that subcutaneous fat seems to be an important factor related to leptin levels. PMID- 10728162 TI - Weight loss maintenance: determinants of long-term success. AB - To analyse determinants of long-term weight maintenance we studied energy expenditure, dietary intake, eating behaviour and psychological symptoms of 9 women (SS, BMI = 26.6) who reached their ideal weight during 1985-86 and were still an average 16.2 kg below their original weight in 1993. Controls were 42 obese, non-reducing females (OC, BMI = 34.8). Dietary intake was calculated from four-day food records. Dietary restraint was assessed by the Three-Factor Eating Questionnaire (TFEQ) and symptoms of bulimia or binge eating by Bulimic Investigatory Test, Edinburgh (BITE). Physical exercise was estimated by open interviews, resting metabolic rate (RMR) by indirect calorimetry and body composition by bioelectrical impedance. Psychological characteristics were studied using the Defense Style Questionnaire (DSQ), Beck Depression Inventory (BDI) and Symptom Checklist (SCL-90). SS had lower RMR (1320 kcal vs. 1540 kcal, p = 0.004), lower daily energy intake (1208 kcal vs. 1525 kcal, p = 0.020) and higher scores for dietary restraint (14.6 vs. 8.4, p = 0.002) than OC. Restraint scores correlated negatively with energy intake and RMR in both groups, especially in SS (r = -0.75, p = 0.021 and r = -0.87, p = 0.002, respectively). Attempts to incorporate habitual physical activities to daily life (89% vs. 39%, p = 0.011) and weekly sports hobbies (67% vs. 13%, p = 0.002) were more often reported by SS. The scores of BITE, DSQ, BDI and SCL-90 were within normal range in both groups but SS had higher scores in most measures, especially for anxiety (16.4 vs. 14.3, p = 0.045). In conclusion, long-term maintenance of weight loss was associated with highly restrained eating, regular physical activity, and perhaps with increased anxiety. PMID- 10728163 TI - Long-term treatment with acarbose for the treatment of reactive hypoglycemia. AB - OBJECTIVE: Acarbose, a potent alpha-glucosidase inhibitor, provides a new concept for the treatment of metabolic disorders, and particularly diabetes mellitus. It reduces the postprandial blood glucose increment and insulin response. For this reason the drug has been successfully used not only in the treatment of type 1 and type 2 diabetes, but also in the management of reactive hypoglycemia and dumping syndrome. The primary aim of the present study is to evaluate the long term effect of acarbose in reducing hypoglycemic symptoms and influencing laboratory measurements in patients with the diagnosis of reactive hypoglycemia. DESIGN AND METHODS: 21 non-obese (BMI < 27 kg/m2) patients (6 males, 15 females) complaining of postprandial symptoms suggesting hypoglycemia and who showed blood glucose values of < 54 mg/dl on one or more occasions during a 5 h oral glucose tolerance test (OGTT) were selected. RESULTS: Before treatment, ingestion of glucose decreased plasma glucose levels at the 3rd and 4th hours, the lowest levels being 39 mg/dl and 45 mg/dl respectively. Eighteen patients had hypoglycemic symptoms during OGTT. Following 3 months of acarbose treatment, the lowest plasma glucose levels at the 3rd and 4th hours increased to 67 mg/dI and 75 mg/dI respectively. Plasma insulin and c-peptide levels were reduced between the 1st and 5th hours, but only the 1st and 2nd hour decrements were statistically significant. The area under the curve (AUC) between 0-300 minutes for insulin was not significant. Plasma glucose levels were significantly increased during the last 3 hours. The AUC for glucose was not significantly changed. Frequency of hypoglycemic attacks was reduced from 4 times a week to 1. C-peptide levels in 24-hour urine collection did not change significantly: 45 micrograms/I and 56 micrograms/I respectively before and after treatment. CONCLUSIONS: These results confirm that acarbose may be of value in preventing reactive hypoglycemia by reducing the early hyperglycemic stimulus to insulin secretion, and in the treatment of reactive hypoglycemia. PMID- 10728164 TI - Experiential cognitive therapy in anorexia nervosa. AB - The paper details the characteristics of Experiential Cognitive Therapy (ECT), a relatively short-term, integrated, patient-oriented approach that focuses on individual discovery. Its novelty is the use of Virtual Reality, a new technology that could play an important role in clinical psychology, since it enables ECT to address both body experience disturbances and motivation for change, two key topics in the treatment of anorexia nervosa. All the phases of ECT are described with reference to an actual clinical case: a 22-year-old female anorectic patient. PMID- 10728165 TI - Epidemiology of eating disordered symptoms in the Korean general population using a Korean version of the Eating Attitudes Test. AB - OBJECTIVE: The purposes of the present study were to estimate the proportion of subjects with a high score on the Korean version of Eating Attitudes Test--26 (KEAT-26), which may provide preliminary data regarding the prevalence rate of eating disorders in the Korean general population, and to further examine the sociocultural hypothesis of eating disorders. METHOD: Using a multi-stage questionnaire sampling method, we surveyed 3062 subjects (1249 males, 1813 females) from 3896 Korean adults in a nationwide area. RESULTS: 8.5% (260/3062) of subjects scored above the cut-off on the KEAT-26. Their demographic correlates, eating traits, and other characteristics relating to general psychopathology were similar to those of patients with eating disorders and female Caucasian controls in Western countries. DISCUSSION: These results suggest that changes in various sociocultural aspects have increased the risk of developing eating disorders in Korea, and support the sociocultural hypothesis of eating disorders. PMID- 10728166 TI - Hyperphagia and self-mutilation in Prader-Willi syndrome: psychopharmacological issues. AB - This study focused in the treatment of two major Prader-Willi symptoms: hyperphagia and self-injurious behavior (SIB). Four patients participated in a four-year study with monthly follow-ups. Patients lived in a behaviorally structured environment, and were treated with selective serotonin reuptake blockers and phenothiazines. Psychopharmacological intervention improved SIB symptoms, but was ineffective to control appetite satiation. PMID- 10728167 TI - Daily hunger sensation and body composition: I. Their relationships in clinically healthy subjects. AB - The human hunger sensation (HS) is a perceptive signal characterized by day-night variability (DNV). This pattern was investigated with respect to its relations with the body compartments in 22 clinically healthy subjects (CHS, 11 males and 11 females, mean age: 24 +/- 2.5 years, mean BMI: 21 +/- 1.7). The DNV was investigated by means of conventional descriptive statistics and the single cosinor method (SCM). Both procedures were applied to the orexigram, i.e., the 24 h profile of the orectic stimulus (OS) provided by each subject, who self-rated his/her HS (from 1 to 10 hunger units) every half hour. Body composition was investigated by Bioelectrical Impedance Analysis (BIA) on the day when the orexigrams were compiled. It was found that the daily HS level correlates positively with the Free Fat Body Mass (FFBM) and negatively with the Fat Body Mass (FBM). These opposite relations indicate that HS is stimulated by the needs of the FFBM, and inhibited by expansion of the FBM, and provide further evidence of the existence of an "adipostat" anorectic mechanism. PMID- 10728168 TI - Binge eating and its relationship to multiple weight control program attendance and alternative methods of weight control. AB - Binge eating and its relationship to multiple program attendance and use of alternative methods of weight control was assessed among 187 former residential weight control participants. Subjects completed a 68-item questionnaire assessing weight control techniques, exercise behaviors, behavior modification techniques, binge eating, and dieting behaviors. Results indicate that respondents who attended other programs after leaving the residential weight control program had significantly higher scores on monitoring food amounts, binge eating characteristics, and post-binge emotions. Similarly, respondents who tried alternative methods of weight control after leaving the program had significantly higher scores on the variables binge eating severity, feelings during a binge, and post-binge emotions. These findings support the binge/diet/obesity cycle and suggest that obese persons with binge problems should discontinue dieting and be directed toward appropriate intervention strategies. Furthermore, policy aimed at screening individuals for binge eating traits prior to weight control program participation is needed. PMID- 10728169 TI - Comparison of total and regional body composition in adolescent patients with anorexia nervosa and pair-matched controls. AB - Body composition in 31 adolescent girls suffering from anorexia nervosa (AN) was measured at the time of hospitalization in order to assess the muscle/bone relationship as a potential source of the development of osteopenia. Differences in lean tissue, fat and bone mass in total body, weight bearing and non weight bearing limbs were estimated in AN and pair-matched controls aged 14.2 +/- 1.8 years (range: 9-17 years). Further, it was investigated if bone mineral density (BMD) better reflects the muscle/bone relationship than bone mineral content (BMC). At the distal radius parameters measured by peripheral quantitative computed tomography (pQCT) were used to estimate the association of volumetric bone density to bone strength and lean body mass. The correspondence to the same and different body regions was assessed. Total lean mass in the controls was closely related to total body bone mineral content (TBBMC), as was lean tissue and bone mass of the limb subregions (r = 0.82 to 0.93). In contrast, the correlation was significantly lower in AN (r = 0.33 to 0.77). In the controls, the pQCT-derived bone strength was correlated with muscle mass of the forearm (r = 0.78, p < 0.001), but only moderately in AN (r = 0.47, n.s.). Regional lean tissue was 11-20% and fat mass was 56-66% lower in AN (p < 0.01). After adjustment for height, TBBMC was different at p = 0.01. Within the limbs subregions, BMC (but not BMD) was different in both groups only in the upper arm and the thigh. BMC reflected the bone/muscle relationship better than BMD. Intra- and between group regressions gave no significant differences between weight bearing and non weight bearing limbs. In conclusion, the assessment of musculoskeletal factors may be a useful tool to develop individual preventive measures for therapy after recovery of our patients. PMID- 10728170 TI - Validation and reliability of the Italian EAT-26. AB - The Italian from of the short, 26-item Eating Attitudes Test (EAT-26) has been administered to 1277 Roman high-school students, mostly females, as a screening device. Ninety-five students with a total score > 20 and 40 students with a low score, were randomly selected, interviewed and diagnosed. The EAT-26 proved to be more sensitive to the presence of an eating disorder than to a specific clinical entity. Item analysis performed on the EAT-26 variables showed satisfactory reliability coefficients. Factor analysis using an oblique rotation was similar to that obtained by Garner et al. (1). Factor analysis with an orthogonal rotation (Cattell's screen test) identified five factors. Results suggested that the EAT-26 isolates cases at risk of clinical spectrum eating disorders. PMID- 10728171 TI - Effects of bariatric surgery on binge eating and related psychopathology. AB - The Beck Depression Inventory (BDI), the Three-Factor Eating Questionnaire (TFEQ), and the Eating Disorder Examination (EDE) were administered to 50 morbidly obese patients before and after gastric bypass surgery. Subjects were classified as non-binge or binge eaters prior to surgery. Though the two groups differed markedly before operation, they were largely indistinguishable 4 months afterward. All binge eating had ceased and mood had improved markedly. TFEQ Restraint scores increased, and Disinhibition and Hunger scores decreased. EDE Eating Concern, Shape Concern, and Weight Concern scores dropped. EDE Restraint scores decreased in non-binge eaters and increased in binge eaters. The overall findings indicate that gastric bypass surgery had a positive short-term impact on non-binge and binge eaters alike. PMID- 10728172 TI - Estimation of food intake: effects of the unit of estimation. AB - While many studies have shown that individuals under-estimate caloric intake, few studies have examined how individuals estimate intake when using other units of measurement (e.g. cups, ounces). Forty-one women (21 obese, 20 normal weight) ate a test meal of Haagen-Dazs chocolate ice cream and were asked to estimate the amount they ate in both calories and cups. As expected, participants under estimated intake when asked to estimate how much they ate in calories, but considerably over-estimated their intake when measured in cups. Thus, individuals can both under- and over-estimate how much of the same food they have eaten, depending on the unit they are asked to use for estimation. Obesity and eating disorders treatment programs should take into account the tendency to over estimate volumetric portions as well as under-estimate caloric intake. PMID- 10728173 TI - Venlafaxine versus fluoxetine in the treatment of atypical anorectic outpatients: a preliminary study. AB - The efficacy of venlafaxine and fluoxetine in the treatment of atypical anorexia nervosa (AN) was compared in a controlled trial. A consecutive series of 24 atypical anorectic females was assigned to either venlafaxine (75 mg/day) or fluoxetine (40 mg/day) plus cognitive-behavioural therapy (CBT). Eating Disorder Examination (EDE12.0D), Beck Depression Inventory (BDI) and State and Trait Anxiety Inventory (STAI) scores were compared before and after 6 months of treatment. Venlafaxine and fluoxetine determined an increase of body mass index (BMI) and a significant reduction of EDE12.0D and BDI scores; venlafaxine alone reduced STAI scores. It would seem that venlafaxine is as effective as fluoxetine when combined with CBT in the treatment of atypical AN. PMID- 10728174 TI - A psychodynamic hypothesis on the night eating syndrome. AB - The Night Eating Syndrome (NES) is usually interpreted in organicistic and physiological terms. This paper looks at it dynamically in terms of the psychic dimension of the patient through an examination of the contrasting tensions (emptiness and fullness; saving and destroying the object, etc.) that are the unconscious cause of his NES. A relationship is suggested between nocturnal reawakenings as a form of eating behaviour and the undreamt or avoided dreams used by the patient as a defence against "perception" of the unconscious. PMID- 10728176 TI - Knowledge of calories and its effect on eating behavior in overweight, normal weight, and underweight individuals. AB - BACKGROUND: To assess changes in the eating behavior of overweight, normal weight, and underweight individuals in response to knowledge about the caloric content of their food. METHODS: Anonymous self-report questionnaires were completed by 1041 staff members attending the Innsbruck University Hospital cafeteria shortly after it started to display the caloric content of all meals. The questions covered the subjects' weight history, body image, eating behavior, and response to knowledge of caloric content. RESULTS: Among the respondents, 141 were classified as overweight, 728 as normal weight, and 118 as underweight. Overweight individuals reported a significantly more frequent change in eating behavior in response to the information, and were also significantly more distressed about weight and eating and dissatisfied with their bodies and their weight than normal and underweight individuals. CONCLUSIONS: Overweight individuals were more likely to report that they had modified their eating in response to information about calories. Whether this reflects an actual change in eating behavior, or merely a wish to give socially desirable responses, remains uncertain. PMID- 10728175 TI - Body weight in bulimia nervosa. AB - OBJECTIVES: The objectives of this study were to determine: 1) factors associated with body mass index (BMI) in women with bulimia nervosa prior to treatment; 2) changes in BMI during cognitive behavioral therapy (CBT) for bulimia nervosa; and 3) predictors of weight change during CBT treatment. METHODS: Participants were 94 women with DSM-III-R bulimia nervosa enrolled in a randomized clinical trial of CBT. Eating disorder symptomatology, past and current psychopathology, and BMI were measured prior to treatment and after twelve weeks of treatment. RESULTS: A significantly lower BMI prior to treatment was independently associated with a history of anorexia nervosa, substance use, cigarette smoking, and a high frequency of vomiting. Reductions in binge eating, purging, and food restriction after treatment did not result in significant changes in BMI. Increasing age, higher maximum lifetime body weight, and a high frequency of binge eating at baseline predicted weight gain during treatment. Weight loss during treatment was predicted by a high frequency of vomiting, and elevated levels of body dissatisfaction prior to treatment. DISCUSSION: Patients hesitant to engage in treatment for fear of weight gain upon cessation of their bulimic behaviors should be reassured that CBT is not usually accompanied by substantial weight gain. Identification of women at risk of significant weight gain or loss during CBT may enable therapists to focus on its aspects that help to prevent excessive body weight changes. PMID- 10728177 TI - Binge eating disorder in males: a population-based investigation. AB - The authors investigated the prevalence of binge eating behavior in a general male Austrian population. A randomly selected sample of 1000 male adults aged 18 to 88 was interviewed by dieticians over the phone. Some screening instruments were used to detect binge eating behavior. The results showed a total prevalence of eating disorders of 14.9%. Eight subjects (0.8%) met the full diagnostic criteria for binge eating disorder (BED), 42 (4.2%) exhibited a partial binge eating syndrome, 94 (9.4%) an Eating Disorder Not Otherwise Specified, and five subjects (0.5%) a bulimia nervosa. Most subjects with a eating disorder were overweight or obese, particularly those with a full BED syndrome. Our findings indicate that full BED syndromes are very rare in a nonclinical male sample, whereas partial syndromes are fairly common in males. PMID- 10728178 TI - The Body Attitude Test: validation of the Spanish version. AB - A Spanish version of the Body Attitude Test (BAT) is presented. It was validated with 165 eating disorder patients (79 anorexia nervosa, 86 bulimia nervosa) and 220 schoolgirls from the general population. Factor analysis allowed the extraction of four factors, as in the original questionnaire, but with different item loadings on each. Taken together, these four factors accounted for 67.1% of the variance. The alpha reliability coefficient was 0.92 in both groups. The test retest reliability with a one-week interval was 0.91 in a subgroup of 34 eating disorder patients and in a subgroup of 43 girls from the general population. The difference between the mean scores of the girls from the general population and the anorexics on one hand and the bulimics on the other was highly significant (p < 0.0001). On the basis of the discriminant validity study, the use of a cut-off point of 41--which gives a sensitivity of 75.1% and a specificity of 72.7%--is recommended. PMID- 10728179 TI - A generalizability study of the Eating Attitudes Test (EAT-12) in non-clinical adolescents. AB - One of the most frequently used measures of eating and weight problems is the Eating Attitudes Test (EAT). The paper discusses conceptual heterogeneity in the EAT in the light of eating disorder continuum theory. Psychometric qualities of the Norwegian 12-item version of the EAT was based on a sample of 224 female and 261 male adolescents aged 14-15 years. Generalizability theory enabled us to study the impact of multiple sources of measurement errors of the EAT-12. The analyses suggest that acceptable generalizability is achieved by the present design on applying 4 items within 3 categories of the EAT. Alternative designs are suggested, and the utility of G-theory is discussed. The results also provide evidence of a gender difference in the structure of the universe score variance. Non-clinical adolescents' ways of organizing information about food and eating are discussed in the light of gender differences. PMID- 10728180 TI - Shame and its psychopathologic correlates in two women's health problems: binge eating disorder and vulvodynia. AB - Shame is thought to be a ubiquitous and destructive psychological process associated with psychiatric and medical conditions. This study examined its nature in two contrasting health problems that influence women's self-evaluations of their bodies and attractiveness, namely an eating disorder (a psychiatric disorder with medical implications) or vulvodynia (i.e., vulvar pain; a newly identified medical condition with psychiatric implications). A community sample of adult women without medical conditions served as an additional comparison group. Participants were 203 adult females: 72 with DSM-IV-defined Binge Eating Disorder (BED), 57 with vulvodynia, and 74 non-patient controls. The three study groups differed significantly: the BED group reported greater shame than the vulvodynia group, and the BED and vulvodynia groups reported greater shame than the control group. Higher levels of shame were associated with higher levels of symptomatic functioning within each group. PMID- 10728181 TI - One-year weight follow-up in anorexia nervosa after inpatient psycho-nutritional rehabilitative treatment. AB - Many inpatient treatments have been proposed for anorexia nervosa (AN) in the past, but few weight outcomes are available today. The criteria for outcome evaluation have always considered body weight. The fifty-three women with AN [26 restrictive (ANR) and 27 bulimic (ANB), studied in this paper were all those discharged in 1996 after an inpatient psycho-nutritional rehabilitation programme. Body weight and BMI were significantly increased between admission and discharge and this increase was maintained at the one-year follow-up. Mean weight in ANR and ANB were similar both at admission and on discharge, but showed significant differences at the follow-up. Follow-up weight was stable (+/- 5%) in 31%, decreased in 15% and increased in 54% of ANR, compared with 44%, 33% and 23% respectively in ANB. There were no differences in age and illness duration. It is very difficult to study presence or absence of menses because many patients were on estro-progestins. Furthermore, only 28% of patients with menses presented BMI higher than 17.5. Only the ANR group showed a significant weight increase at the follow-up. Their BMI was acceptable in 65% compared with 33% in the ANB group. Bulimic behaviour seems to be itself a negative prognostic factor for weight recovery. PMID- 10728182 TI - Failure of acupuncture in the treatment of obesity: a pilot study. AB - Acupuncture has been anecdotally reported to induce weight loss in obese patients. This pilot study examines its efficacy in a randomised, placebo controlled clinical trial. Forty (33 F, 7 M) obese (BMI > 30 kg/m2) outpatients were randomised to either placebo or acupuncture (12 weekly sessions of minimal acupuncture and somatic moxibustion-acupuncture associated with auriculopuncture respectively). BMI, eating attitudes (BES), anxiety (STAI), depression (BDI), and obesity-related quality of life (ORWELL 97) were measured at the beginning and end of the treatment. Six (30%) patients in the treatment group and 12 (60%) in the placebo group dropped out. Intention-to-treat analysis did not show any significant effect of acupuncture on BMI and obesity-related quality of life; eating attitudes improved in both groups, possibly because of the placebo effect. A significant improvement in anxiety and depression was only observed in the acupuncture group. In conclusion, acupuncture does not promote weight loss and is not recommendable in the treatment of obesity. It may, however, improve the psychological status of obese patients. PMID- 10728183 TI - Glycaemic index of different kinds of carbohydrates in type 2 diabetes. AB - An adequate diet provides good metabolic control in diabetics. Since 1981 when Jenkins showed that complex carbohydrates are digested more slowly and raise blood glucose less than simple sugars, many studies have been performed in this field. In this study, seven kinds of carbohydrate-rich food were compared with glucose in 52 Type 2 diabetic patients and 31 normal volunteers. The subject consumed either macaroni, white rice, potatoes, tarhana soup (tarhana includes wheat flour, yoghurt, tomato and green pepper), noodle soup, white or whole wheat bread, or glucose at one-week intervals after an overnight fast. The glycaemic index (GI) of each food was calculated from the area under its glycaemic response curve (AUC) expressed as a percentage of the mean response to glucose. The results showed that the foods ranked from the highest to the lowest GI as follows: white bread; whole wheat bread; macaroni; tarhana soup; white rice; potatoes; noodle soup. PMID- 10728184 TI - The use of citalopram in an integrated approach to the treatment of eating disorders: an open study. AB - This study investigated the efficacy and safety of citalopram in the treatment of eating disorders. Eighteen female patients gave their informed consent to enrollment in the trial: twelve with bulimia nervosa, six with anorexia nervosa according to the DSM IV criteria. They received individual systemic psychotherapy and took 20 mg/day citalopram for 8 weeks. At the beginning and end of the trial, their BMI, body fat and lean mass were checked and they completed the Eating Disorder Inventory and Binge Scale. The results showed that citalopram is effective and safe in the treatment of eating disorders: binge eating episodes and mean scores in three EDI subscales (bulimia, ineffectiveness and interoceptive awareness) significantly decreased in the bulimic patients, and mean scores in the EDI body dissatisfaction subscale significantly decreased in the anorexic patients. PMID- 10728185 TI - Current treatment in acute demyelinating polyneuropathy (Guillain-Barre syndrome). PMID- 10728186 TI - Intravenous immunoglobulin treatment in children with Guillain-Barre syndrome. AB - We have retrospectively reviewed the data of 75 consecutive children diagnosed with Guillain-Barre syndrome (GBS) and hospitalized in two centres. There were 51 children with GBS treated in Ankara, Turkey and 24 patients treated in Toronto, Canada. To evaluate the effect of intravenous immunoglobulin (IVIG) treatment, the patients were divided into three groups. All 24 Canadian patients received IVIG in a dose of 1 g/kg/day for 2 days. In the Ankara group 23 children received 0.4 g/kg/day for 5 days and the remaining 28 patients in that group received supportive treatment only. In all but two patients IVIG was started within the first 2 weeks of neuropathic symptoms. The patients' data, including mean functional grades, frequency of bulbar signs and autonomic dysfunction and age were similar in all three groups. Analysis of the short-term outcome demonstrated that the patients treated with IVIG had a significantly faster rate of recovery. Mean time-lapse until improvement of one functional grade was 17.4 days in the IVIG group from Toronto, and 20.8 days in the IVIG group and 62.4 days in the non IVIG group of patients from Ankara. We conclude that IVIG has considerable efficacy in the treatment of children with GBS. Furthermore, we have also demonstrated a faster rate of recovery in patients who received a total dose of IVIG in 2 days as opposed to 5 days. PMID- 10728187 TI - Does the brain regenerate after perinatal infarction? AB - We have used registered serial magnetic resonance scans to assess the growth of the brain after perinatal infarction in six infants. The initial scans were performed at ages of 4 days to 8 weeks and follow-up studies were performed from 4 days to 21 weeks later. A three-dimensional volume acquisition was performed on each occasion. Rigid body translations and rotations were used to match the images obtained on each occasion. Subtraction of the first image from the second then provided an assessment of the growth of the brain that had occurred between the two examinations. In the early phase of infarction (up to 2 months) low signal areas with clearly defined margins developed at the site of infarction. In the late phase (2 months onwards) growth was seen in the brain at the margins of the infarct in each case, and the size of the infarcted region showed a marked decrease in size. The rate of growth of the brain into the infarcted area exceeded that of the surrounding brain in some cases and was less in others. Growth of undamaged tissue may provide an important mechanism for recovery of the developing brain. PMID- 10728188 TI - The spectrum of acquired cognitive disturbances in children with partial epilepsy and continuous spike-waves during sleep. A 4-year follow-up case study with prolonged reversible learning arrest and dysfluency. AB - We report a longitudinal study (7-11 years) of a previously normal boy (MR) who presented from the age of 5 years with rare partial motor seizures and atypical 'absences'. The history revealed a stagnation in development and speech difficulties a few months before onset of his epilepsy. The first waking electroencephalogram (EEG) showed rare generalized discharges during hyperventilation. Magnetic resonance imaging revealed an arachnoid cyst in the frontotemporal region. Although his epilepsy never became severe, he experienced important learning difficulties. Subsequent EEGs became increasingly active with left focal epileptic activity and continuous spike-waves during sleep (CSWS) present from the first sleep EEG. The first neuropsychological evaluation (7 years) showed a speech dysfluency, word finding and naming problems, inattention and low intelligence quotient. Carbamazepine was changed to clobazam and later ethosuximide was added with a rapid improvement (within 1 month) in linguistic and cognitive performances as well as in behaviour. Furthermore, the patient showed considerable progress in acquisition over the next months whereas learning to read had previously been very difficult. The epileptic activity gradually disappeared and he was able to follow regular school at an age-appropriate level. This case adds a new facet to the already recognized more obvious acquired neuropsychological disturbances known to occur in some partial childhood epilepsy syndromes with CSWS (aphasia, dementia). It manifested as prolonged insidious stagnation in learning and subtle language disability. This study documents rapid specific language improvement with change in anti-epileptic drugs and a restored immediate and long-term learning capacity, suggesting a direct but 'hidden' role of epilepsy. PMID- 10728189 TI - The aetiology of delayed visual maturation: short review and personal findings in relation to magnetic resonance imaging. PMID- 10728190 TI - A family with hemiplegic migraine and focal seizures. AB - Familial hemiplegic migraine is a distinctive form of migraine with autosomal dominant inheritance. The patients undergo attacks of migraine complicated by hemiplegia. Seizures have not been reported as comprising a part of this syndrome. We describe three generations of a family with hemiplegic migraine and focal seizures occurring concurrently with the migrainous attacks. There were five affected family members whose clinical features included unilateral headache and transient hemiplegia. Two family members also had focal seizures during the migrainous attacks. One of the patients was treated with carbamazepine with good results. The only associated neurological finding was ataxia which was found in the oldest patient. The presence of focal seizures during an episode of hemiplegic migraine suggests that the two phenomena of migraine and focal seizures may share the same underlying pathophysiology. PMID- 10728191 TI - Multiple abnormal magnetic resonance imaging signals in a patient with minimal neurological symptoms. PMID- 10728192 TI - Acute encephalopathy with bilateral striatal necrosis: favourable response to corticosteroid therapy. AB - A case of acute encephalopathy with selective bilateral symmetrical striatal lesions is reported. The patient was a previously healthy 4-year-old boy who became obtunded after a febrile illness and fell into a state of delirium with severe pain in the feet. He showed abnormal postures: hyperextension of the neck and upper limbs and extreme flexion of both lower limbs, and abnormal involuntary movements of the limbs: tremor, athetotic movement and right hemiballismus. Analysis of serum antibody titres suggested recent primary infection of herpes simplex type 1 (HSV-1). Cranial T2-weighted magnetic resonance imaging (MRI) demonstrated areas of high-signal intensity involving the whole basal ganglia bilaterally. He showed rapid clinical improvement after the initiation of corticosteroid therapy; complete clinical recovery was noted 3 months after the onset. Serial MRI studies demonstrated a rapid reduction of the lesions, resulting in only slight T2-hyperintense areas in both caudate nuclei. The pathogenesis of the disorder remains unknown, though an autoimmune mechanism has been speculated. The clinical and laboratory findings in this case suggested a possible role of HSV-1 in the pathomechanism of the disorder and a beneficial effect of early corticosteroid therapy. PMID- 10728194 TI - Hydrosyringomyelia associated with Dandy-Walker malformation--is it really rare or undiagnosed? PMID- 10728193 TI - Acute ophthalmoparesis associated with IgG anti-GQ1b antibody subsequent to Streptococcus pyogenes infection. PMID- 10728195 TI - Gene table: the muscular dystrophies. PMID- 10728196 TI - Gene table: epilepsy. PMID- 10728197 TI - Carbohydrate-deficient glycoconjugate (CDG) syndromes: a new chapter of neuropaediatrics. PMID- 10728198 TI - Aromatic L-amino acid decarboxylase deficiency: an extrapyramidal movement disorder with oculogyric crises. AB - Aromatic L-amino acid decarboxylase (AADC) deficiency results in an impaired synthesis of catecholamines and serotonin, and has been reported only in two middle eastern families. We report on a European family with an affected child. The child showed the characteristic clinical picture of an extrapyramidal movement disorder, oculogyric crises and vegetative symptoms seen in the three patients described previously. Treatment with a combination of the AADC cofactor pyridoxine, the monoamine oxidase B inhibitor selegiline and bromocriptine was started during the fifth year of life and showed only a moderate clinical improvement in contrast to patients who have been treated since the first year of life. PMID- 10728199 TI - Bilateral opercular syndrome caused by perinatal difficulties. AB - Four patients with pseudobulbar palsy, mental retardation and various degrees of speech disturbance associated with perinatal difficulties are described as having an acquired type of opercular syndrome. There were two patients with fetal bradycardia and three with subarachnoid haemorrhage and neonatal convulsion. Magnetic resonance imaging revealed cortical atrophy in the bilateral opercula with some signal abnormalities in the underlying white matter in common. Single photon emission computed tomography (SPECT) also confirmed the presence of hypoperfusion in the regions. Although the opercular syndrome is a clinical entity with a multitude of underlying pathologies, perinatal difficulties could be an important cause of the acquired type. PMID- 10728200 TI - Facilitation of ipsilateral motor pathways during recovery from hemiplegia in two adolescent patients. AB - In two hemiplegic patients with acquired cerebral lesions, transcranial magnetic stimulation (TMS) was carried out to examine the contribution of the ipsilateral motor pathways to recovery from hemiplegia. A 13-year-old girl (patient 1) had acute hemiplegia due to a rupture of an arteriovenous malformation, and a 13-year old boy (patient 2) had subacute hemiplegia due to a brain tumour. They showed complete upper limb palsy but recovered after therapy; patient 1 had slightly disabled motor function of the arm, and patient 2 recovered completely. Motor evoked potentials (MEPs) were recorded from the biceps brachii muscles on both sides. The MEPs of the paretic biceps were only elicited by TMS of the intact hemisphere at the beginning of recovery from hemiplegia, but not by TMS of the affected hemisphere. The MEP amplitudes increased and cortical representation areas for the paretic biceps by TMS were enlarged temporarily during recovery. They regressed in patient 1 and MEPs were not evoked at all in patient 2 after recovery. Conversely, MEPs were obtained by TMS of the affected hemisphere after recovery in both patients. These data indicate that ipsilateral motor pathways play a role in recovery from hemiplegia, especially at the beginning, and become inactivated when the contralateral motor pathways recover. PMID- 10728201 TI - Calcifications of the basal ganglia in children with brain tumours. AB - Calcifications in the basal ganglia have been found in nine (5.4%) of all children treated for any kind of brain tumour in our department. This has occurred over a mean period of 2.8 years after diagnosis. The group of patients has been compared with a group of other children, matched for age, sex, histologic diagnosis and tumour treatment, but without calcifications of the basal ganglia. The groups differ from each other with respect to a significantly higher incidence of hypothyroidism and growth hormone deficiency in the group of children with calcifications in the basal ganglia. Moreover the children with calcifications show a larger IQ-loss. Although the pathogenesis of the calcifications of the basal ganglia is not known, an association of damage to the vascular bed of the basal ganglia due to periods of increased intracranial pressure, together with endocrine deficiencies is discussed. We advise an adequate supplementation in cases of endocrine deficiencies in children treated for brain tumours as early as possible. PMID- 10728202 TI - The effect of insulin-like growth factors on brain myelination and their potential therapeutic application in myelination disorders. AB - Degenerative disorders of the cerebral white matter, leukodystrophies and demyelination diseases, are characterized by the faulty formation or excessive breakdown of myelin. Insulin-like growth factors (IGFs) promote the proliferation of oligodendrocytes as well as their myelin synthesis. IGF-I overexpressing mice show a significant increase in brain weight associated with increased myelin content. In contrast, the brains of IGF-binding protein-1 transgenic mice show a dramatic decrease in myelination. Furthermore, IGFs and IGF-binding proteins are among the factors that are induced by brain injury and have neuroprotective effects. IGFs also induce neurite growth and survival, in particular in glial cells of the peripheral nervous system. In demyelinating diseases, IGF-I may be useful for reducing myelin breakdown and promoting myelin regeneration. These observations may lead to new therapeutic applications for IGFs, for example promoting remyelination or limiting damage following brain injury. PMID- 10728203 TI - Keeping the arm in the limelight: advanced visual control of arm movements in neonates. AB - To test whether newborn babies have voluntary control over their limbs, spontaneous arm-waving movements were measured in the dark while the baby lay supine with its head turned to one side. A narrow beam of light was shone over the baby's nose or chest in such a way that the arm the baby was facing was only visible when the hand encountered the, otherwise, invisible beam of light. The results showed the babies were capable of precisely controlling the position, velocity, and deceleration of their arms so as to keep the hand visible in the light. The findings indicate that newborns can purposely control their arm movements to meet external demands and that the development of visual control of arm movement is underway soon after birth. PMID- 10728204 TI - Neuronal ceroid lipofuscinosis in the Czech Republic: analysis of 57 cases. Report of the 'Prague NCL group'. AB - A series of 57 patients (from 51 families) with neuronal ceroid lipofuscinosis (NCL) has been diagnosed during the last 25 years. Using clinical and electrophysiological criteria together with results of ultrastructural, histochemical, immunohistochemical and neuropathological analyses it has been possible to classify the following NCL types. Two cases were of the infantile type (CLN1), one case of the juvenile (CLN3) type and one case of the adult (CLN4) type. The bulk of the series was represented by 26 cases of the late infantile (CLN2) type and by 27 cases of the early juvenile (CLN6) type (also called non-Finnish variant late infantile, or Lake-Cavanagh). Besides the infantile form, microcephaly was a relatively frequent finding (nine cases) in the late infantile and early juvenile NCLs. In more than half of the late infantile and early juvenile cases there was a significant reduction of the nerve conduction velocity. The early juvenile CLN6 type was found to have a relatively high incidence in the Romany population (12 cases in nine families). Incidence of NCL in the Czech republic is estimated to be 1.3:100,000. PMID- 10728205 TI - Epilepsy in Duchenne and Becker muscular dystrophies. AB - Duchenne and Becker muscular dystrophies are X-linked allelic disorders in which the association of central nervous system dysfunction, typically in the form of mental retardation, is a well recognized feature. They are both due to mutations in the dystrophin gene, whose corresponding protein products are expressed both in the muscle and central nervous system. We have observed an increased frequency of epilepsy in children with Duchenne and Becker muscular dystrophy attending our clinic. Out of 254 boys with this condition (201 Duchenne and 53 Becker), eight children, four in the Duchenne and four in the Becker group, had a confirmed diagnosis of epilepsy (cumulative incidence 3.14%, with a subgroup incidence of 1.99% in the Duchenne and 7.54% in the Becker group). Statistical analysis indicated that only the incidence of epilepsy in Becker muscular dystrophy was significant (p < 0.007). Our data suggests that epilepsy may be a rare associated feature in children with muscular dystrophy secondary to dystrophin deficiency. PMID- 10728206 TI - Neuroendoscopic surgery for brain abscess. PMID- 10728208 TI - Merosin-positive congenital muscular dystrophy with mental retardation and cataracts: a new entity in two families. AB - Merosin-positive congenital muscular dystrophy is a heterogenous group of disorders with varying clinical presentations and severity. In general, central nervous system involvement is not present. There is also evidence for still unclassified forms. Here we report three cases in two families with merosin positive congenital muscular dystrophy, mild mental retardation, bilateral cataracts and normal cranial magnetic resonance imaging. To our knowledge, such an association has not been reported previously, and thus is a new entity within congenital muscular dystrophy nosology. PMID- 10728207 TI - Radiofrequency lesions of the dorsal root ganglion in the treatment of hip flexor spasm: a report of two cases. AB - The current popular model of spasticity is that the abnormalities are primarily due to increased sensitivity of the reflex are at the segmental level of the spinal cord. Neurosurgical procedures, such as open selective dorsal rhizotomy for the reduction of spasticity, have been based on this assumption. We describe two patients with hip flexor spasm of different origin treated with radiofrequency lesions of the dorsal root ganglion. PMID- 10728209 TI - Childhood brain tumours: new directions in management. PMID- 10728210 TI - The new anti-epileptic drugs: a review. PMID- 10728211 TI - What's new in the molecular genetics of spinal muscular atrophy? PMID- 10728212 TI - Loss of CO2 reactivity of cerebral blood flow is associated with severe brain damage in mechanically ventilated very low birth weight infants. AB - BACKGROUND: Early detection of pathophysiological factors associated with permanent and severe brain damage in preterm infants requiring intensive care is a major issue in neonatal neurology. The aim of this study was to investigate if an abnormal CO2 reactivity of cerebral blood flow in high risk very low birth weight infants is associated with severe brain injury demonstrated at autopsy or by neurodevelopment examination at 18 months. METHODS: The CO2 reactivity of cerebral blood flow (xenon-133) was measured in 18 mechanically ventilated, severely ill, very low birthweight infants (gestational age 26-32 weeks, birthweight: 630-1360 g) during the first 36 hours of life. Cerebral outcome was assessed on autopsy findings (n = 8) or at the age of 18 months using Bayley developmental scales (n = 10). RESULTS: Eight infants with normal development at 18 months (within mean +/- 2.5 SD of reference group) and two infants with normal cerebral autopsy findings had a median CO2 reactivity of 24.4%/kPa CO2 (interquartile range 14.7-41.2). Two infants with abnormal development (> 2.5 SD below mean) and six infants with hypoxic-ischaemic encephalopathy at autopsy has a median CO2 reactivity of 3.4%/kPa CO2 (interquartile range 8.0-11.7). CONCLUSION: In mechanically ventilated very low birthweight infants low CO2 reactivity of cerebral blood flow (below 10%/kPa CO2) during the first 36 hours of life was associated with poor neurodevelopmental outcome or hypoxic-ischaemic encephalopathy at autopsy. Loss of CO2 reactivity may play a role in the pathogenesis of hypoxic ischaemic encephalopathy. It is a candidate for predicting early severe brain damage in preterm infants requiring intensive care and for controlling the effect of early interventions. PMID- 10728213 TI - Outcome of seizures in the first year of life. AB - AIM: To determine the frequency and natural history of seizures having an onset during the first year of life. METHODS: A retrospective analysis of the files of all patients treated in the paediatric neurology clinic of our medical centre during 1975-1995. RESULTS: Of our 482 patients with seizure onset prior to age 16 years, the first seizure occurred at age 1-12 months in 80 (16.6%). Of those, 38 (48%) had West syndrome and the rest were classified as follows--partial seizures with or without secondary generalization: 19%, generalized tonic seizures: 8.5%, generalized tonic-clonic seizures: 7.5%, myoclonic: 7.5%, unclassified: 7.5%, and mixed-type seizures: 2%. Follow-up was possible in 91% of the patients. Mean follow-up period from seizure onset was 10 years. Of the non-West syndrome patients who were followed, 19 (50%) were still experiencing seizures at follow up. Eleven of the 15 patients (73%) with partial seizures and four of the 13 patients (31%) with generalized tonic or tonic-clonic seizures had symptomatic seizures. Of the 14 followed patients with partial seizures, 10 (71%) still had seizures at follow-up, as did three (25%) of the 12 followed patients with generalized tonic or tonic-clonic seizures (p = 0.023). CONCLUSIONS: The partial seizures were most often symptomatic with unfavourable prognosis, while the generalized seizures were either symptomatic and severe or cryptogenic and rapidly responsive to anti-epileptic drugs with good outcome. PMID- 10728214 TI - Biotinidase deficiency: result of treatment with biotin from age 12 years. AB - A boy with severe symptoms of biotinidase deficiency diagnosed at the age of 12 years showed a remarkable improvement of his neurological picture and normalization of brain magnetic resonance imaging abnormalities when prescribed oral biotin. PMID- 10728215 TI - Chiari I malformation in asymptomatic young children with Williams syndrome: clinical and MRI study. AB - We report clinical and magnetic resonance imaging findings in two young children, aged 2 years 4 months and 3 years, with Williams syndrome. Both showed a mild global delay, although their neurological examination was completely normal. Their magnetic resonance imaging, however, showed Chiari I malformation and some non-specific changes in the centrum semiovale and in the white matter posterior to the lateral ventricles. Cerebellar tonsils were displaced through the foramen magnum 8.5 and 7.5 mm respectively. Our results suggest that Chiari I malformation can also be a frequent feature in subjects with Williams syndrome even in the absence of overt neurological signs suggestive of it. Whether these children might develop acute signs later is not known at present. Further studies are needed not only to evaluate the incidence of these findings in the global population of subjects with Williams syndrome but also to identify the children who are at risk for developing acute neurological signs. PMID- 10728216 TI - Arthrogryposis multiplex congenita and bilateral mid-brain infarction following maternal overdose of co-proxamol. AB - We report a case of arthrogryposis multiplex congenita secondary to fetal hypokinesia in a 41-week gestation infant following antenatal central nervous system injury. The mother's pregnancy was complicated by an episode of attempted self harm, with an overdose of co-proxamol at 22 weeks of gestational age, and by the use of cocaine in combination with excess alcohol intake. Magnetic resonance imaging showed bilateral mid-brain cysts and marked atrophy of the basal ganglia and thalami. PMID- 10728217 TI - Gene table: neuronal ceroid lipofuscinoses (NCL). PMID- 10728218 TI - Gene table: adult and paediatric movement disorders. PMID- 10728219 TI - How primitive is the Moro reflex? PMID- 10728220 TI - Maternal and Child Health Journal: in the beginning... PMID- 10728221 TI - MCH functions framework: a guide to the role of government in maternal and child health in the 21st century. AB - OBJECTIVES: At the close of the 20th century, the government's role in maternal and child health is in a state of transition. What is needed is a framework defining roles and responsibilities and guidance on how to operationalize these functions. This article presents the Maternal and Child Health (MCH) Functions Framework and discusses its value as an advocacy, planning, evaluation, and educational tool. METHODS: The Johns Hopkins Child and Adolescent Health Policy Center developed the Framework in collaboration with leading public health organizations. The process entailed formulating a conceptual approach and facilitating consensus among the relevant organizations. RESULTS: The Framework consists of three main components: (a) a list of ten essential public health services to promote maternal and child health, (b) an outline detailing program functions specific to MCH that apply to all levels of government and to all MCH populations, and (c) selected examples of local, state, and federal activities for implementing MCH program functions. CONCLUSIONS: The MCH Functions Framework can be used in advocacy, policy development, program planning, organizational assessment, education, and training. To date, it has been used by several state and local MCH agencies and in MCH education and training programs. PMID- 10728223 TI - The influence of site of care on the content of prenatal care for low-income women. AB - OBJECTIVE: To assess whether site of prenatal care influences the content of prenatal care for low-income women. DESIGN: Bivariate and logistic analyses of prenatal care content for low-income women provided at five different types of care sites (private offices, HMOs, publicly funded clinics, hospital clinics, and other sites of care), controlling for sociodemographic, behavioral, and maternal health characteristics. PARTICIPANTS: A sample of 3405 low-income women selected from a nationally representative sample of 9953 women surveyed by the National Maternal and Infant Health Survey, who had singleton live births in 1988, had some prenatal care (PNC), Medicaid participation, or a family income less than $12,000/year. OUTCOME MEASURES: Maternal report of seven initial PNC procedures (individually and combined), six areas of PNC advice (individually and combined), and participation in the Women Infant Children (WIC) nutrition program. RESULTS: The content of PNC provided for low-income women does not meet the recommendations of the U.S. Public Health Service, and varies by site of delivery. Low-income women in publicly funded clinics (health departments and community health centers) report receiving more total initial PNC procedures and total PNC advice and have greater participation in the WIC program than similar women receiving PNC in private offices. CONCLUSIONS: Publicly funded sites of care appear to provide more comprehensive prenatal care services than private office settings. Health care systems reforms which assume equality of care across all sites, or which limit services to restricted sites, may foster unequal access to comprehensive PNC. PMID- 10728222 TI - Monitoring health care for children with chronic conditions in a managed care environment. AB - OBJECTIVE: Children with chronic health conditions face special issues in their interactions with managed care. These children often require additional and more varied services than do other children. Managed care plans increasingly include these children, especially with the growth of Medicaid managed care. This article examines the special issues facing children with chronic conditions and develops strategies for monitoring their care in managed care settings. METHODS: The project staff conducted an extensive review of the research and policy literature related to managed care and the special needs of families with children with chronic conditions. The project also reviewed current and proposed plans of federal, state, and private groups for monitoring and, working with parents and other outside groups, identified key issues to consider in developing monitoring plans. RESULTS: The relative rarity of many childhood conditions and the complex interactions among child, family, and community over time make assessment of their care difficult. We describe these child and family characteristics, outline essential features and domains for monitoring systems, and describe population based and plan-based monitoring systems to assess managed care for these children and their families. CONCLUSIONS: Monitoring for children with chronic conditions in managed care arrangements will require public health agencies and health providers to define populations systematically, assess across a variety of conditions, and monitor several domains central to the health of these families. PMID- 10728224 TI - An evaluation of breastfeeding promotion through peer counseling in Mississippi WIC clinics. AB - OBJECTIVE: This study evaluates the effectiveness of a peer counseling program at increasing breastfeeding by participants in the Mississippi Special Supplemental Nutrition Program for Women, Infants and Children (WIC). METHODS: Data from the 1989-1993 Pediatric Nutrition Surveillance System were analyzed to compare breastfeeding rates in clinics with and without peer counseling programs. A questionnaire completed by program staff to describe the program in greater detail helped identify characteristics associated with greater success. RESULTS: The incidence of breastfeeding rose from 12.3% to 19.9% in those clinics with peer counseling programs, but only from 9.2% to 10.7% in clinics without a program. Clinics that started a program earlier showed greater changes in breastfeeding incidence. However, the presence of lactation specialists or consultants in the clinic appeared to be more important than the presence of less trained peer counselors. Peer counselors who spent more than 45 minutes per participant were more effective than those spending less time. CONCLUSIONS: The peer counseling program significantly increased the incidence of breastfeeding, particularly in clinics with lactation specialists and consultants. Success can be enhanced by ensuring that peer counselors spend a great deal of time with the participants. PMID- 10728225 TI - Prenatal smoking in two consecutive pregnancies: Georgia, 1989-1992. AB - OBJECTIVE: To explore the patterns of prenatal smoking among women whose first and second pregnancies ended in live births. METHODS: We used population-based data to explore prenatal smoking among 14,732 white and 8968 black Georgia residents whose first and second pregnancies ended in live births during 1989 1992. Smoking status was obtained from birth certificates linked for individual mothers. Because of demographic differences, we analyzed white and black women separately. RESULTS: Approximately 15% (2253) of white women and 4% (318) of black women smoked during their first pregnancy. Of those smokers, 69% (1551) of white women and 58% (184) of black women also smoked during their second pregnancy. For both white and black nonsmokers during the first pregnancy, low education was the most significant predictor of smoking during the second pregnancy, after adjusting for consistency of the father's name on the birth certificate, prenatal care, birth interval, mother's county of residence, and birth outcome. CONCLUSIONS: The prevalence of smoking in this study may be low because of underreporting of prenatal smoking on birth certificates. The majority of women who smoked during their first pregnancy also smoked during their second, suggesting that these women exposed their first infant to tobacco smoke both in utero and after delivery. Practitioners should offer smoking cessation programs to women during, as well as after, pregnancy. Pediatricians should educate parents on the health risks to young children of exposure to environmental tobacco smoke and refer smoking parents to smoking cessation programs. PMID- 10728226 TI - Promoting the well-being of children: the need to broaden our vision--the 1996 Martha May Eliot Award Lecture. AB - As those in the field of maternal and child health attempt to redefine their role to meet the challenges of the 1990s and beyond, they should review the history of their field. They should examine the legacy of the Children's Bureau which investigated and reported upon all matters pertaining to the welfare of children. They should note the medicalization of MCH and the drift away from broader child welfare issues. In Dr. Eliot's words, they should remember "the inseparability of the health and social aspects of the growth and development of a child." MCHers can advance the well-being of children by defining child health broadly and by promoting it in many ways. PMID- 10728227 TI - Children's health and the environment--the first Herbert L. Needleman Award Lecture. AB - Children today are exposed extensively to toxins in the environment. Prominent among these are exposures to over 70,000 synthetic chemicals, all newly developed in the past 50 years and largely untested for their hazards to children's health. Children are uniquely vulnerable to toxins, and with increasing incidence they are developing chronic, disabling, life-threatening diseases known or suspected to be of environmental origin-asthma, endocrine disruption, cancer, and the diseases caused by tobacco. Pediatricians need to consider toxic etiologies in the differential diagnosis of childhood illness. PMID- 10728228 TI - In search of the MCH professional. AB - One of the goals of the Maternal and Child Health Journal is to encourage professional development. This letter was written to the Editor in light-hearted spirit to convey the true challenge such a goal poses for the journal and the profession. PMID- 10728229 TI - Health insurance coverage of the children of immigrants in the United States. AB - OBJECTIVES: This study assesses the health insurance coverage of children of immigrants in the United States and variations among immigrant groups. METHOD: The study uses data from the March supplements of the 1994 and 1996 Current Population Survey to compare health insurance coverage of children who report foreign parentage. Separate logistic regressions are conducted to estimate the likelihood of being covered by any insurance, public insurance, and private insurance. RESULTS: 27.3% of all children of immigrants are without health insurance, 34.1% are on public insurance, and 44.3% have private insurance. Foreign-born children who have not yet become U.S. citizens are the most likely to be without health insurance (38.0%). Many of these children are not covered because their parents are unable to find jobs that provide coverage and Medicaid fails to enroll as many of them as possible. Overall, the children's chances of being covered by any health insurance vary little according to when their parents came to this country. However, children of recent immigrants are more likely to rely on public health insurance (40.1% vs. 24.8%) and less likely to be covered through private sources (36.8% vs. 60.6%) than those of established immigrants. Among immigrant groups, children of Haitian (48.4%) and Korean (45.3%) immigrants are at the highest risks of being uninsured. Both children of the Dominican Republic (65.9%) and Laos (83.3%) report high rates of public insurance coverage. CONCLUSIONS: Greater disparity in health insurance coverage among children of immigrants is expected once the new welfare reform bills take effect. In particular, noncitizen children, children of recent immigrants, illegal immigrants, and Dominican Republican immigrants will be affected most. Efforts aimed at reducing the harm should target these vulnerable groups. PMID- 10728230 TI - Delayed childbearing by education level in the United States, 1969-1994. AB - OBJECTIVES: Advanced maternal age at first birth, but not at subsequent births, may have detrimental health implications for both mother and child, such as a poor birth outcome and an increased risk of maternal breast cancer. However, positive outcomes may also result such as an improvement in economic measures and offspring's performance on cognitive tests. Research has indicated that women increasingly are delaying their first births beyond the early twenties, but the recent trends in socioeconomic disparity in age at first birth, and the implications for public health, have not been well described. METHOD: This study used national birth certificate data for 1969-1994 to examine age at first birth by maternal education level. Current Population Survey data were also used to examine changes over time in age and educational distribution among women of childbearing age. RESULTS: Age at first birth increased during the time period. Median age at first birth increased from 21.3 to 24.4 between 1969 and 1994, and the proportion of first-time mothers who were age 30 or older increased from 4.1% to 21.2%. Age at first birth increased rapidly among women with 12 or more years of education; nearly half (45.5%) of college graduate women who had their first birth in 1994 were age 30 or older, compared with 10.2% in 1969. However, little change was observed among women with fewer than 12 years of education; among those with 9-11 years of education, only 2.5% of first births in 1994 occurred at age 30 or older. CONCLUSIONS: The trend toward postponed childbearing has occurred primarily among women with at least a high school education. Health services use, such as infertility treatment and cesarean section, may increase as a result of delayed childbearing among higher educated women. Future examinations of the association between maternal age at first birth and health outcomes may need to take greater account of socioeconomic differentials. PMID- 10728231 TI - Characteristics of current hospital-sponsored and nonhospital birth centers. AB - OBJECTIVES: (1) To describe contemporary birth centers in terms of the population served, organizational and financial characteristics, services provided, mission and philosophy, and planning and marketing techniques. (2) To compare hospital sponsored and nonhospital models with regard to the above characteristics. METHOD: Data from the National Survey of Women's Health Centers conducted in 1994 are analyzed using t-tests and chi-square tests. RESULTS: Contemporary birth centers serve a diverse population of women and provide a range of clinical and nonclinical services. Birth centers are both hospital-sponsored and nonhospital, with the former growing at a faster rate. Compared to hospital-sponsored centers, nonhospital centers serve a larger proportion of uninsured women, provide a broader range of clinical services, and are more committed to women-centered care. Centers utilize different marketing methods and are involved in a number of organizational changes to better position themselves in the changing health care environment. CONCLUSIONS: Birth centers offer an attractive option to consumers and are a viable model for delivering women-centered care. Given that all "birth center" facilities do not share the same philosophy and service mix, women need to have some assurance of what a "birth center" will, and will not, provide. PMID- 10728232 TI - Promoting community-based maternal and child health services: a university-health department partnership. AB - OBJECTIVES: Funded by the Federal Maternal and Child Health Bureau, a partnership between the Johns Hopkins University School of Public Health, Department of Maternal and Child Health (JHU), and the Baltimore City Health Department (BCHD) identifies maternal and child health problems, and develops appropriate interventions. This paper presents the organization and activities of the JHU/BCHD Maternal and Child Community Health Science Consortium as a result of overcoming traditional barriers to collaborative efforts, and discusses what role the Consortium has had in its own collaborative success. METHOD: A review of the literature uncovered a number of barriers to productive interaction. A number of factors contributing to overcoming the barriers was also revealed. The organization and activities of the work of the JHU/BCHD Maternal and Child Community Health Science Consortium has been applied to these barriers and associated factors, and discussed in context of implications for future collaborative efforts. RESULTS: The Consortium has developed a fully integrated administrative structure bridging both the BCHD and JHU. The mission of the Consortium has been translated into four categories of work, each one designed to complement, extend, and augment the other. The infrastructure established in Baltimore, as a direct result of this partnership, has served to overcome traditional barriers to productive academic/agency collaboration, while promoting organizational productivity. This outcome is a result of overcoming the recognized barriers to collaboration. CONCLUSIONS: Health agencies and university public health programs must link resources and collaborate to address public health issues. Commitment to a collaborative approach to the public's health will determine its future. PMID- 10728233 TI - Social HMOs and other capitated arrangements for children with special health care needs. AB - OBJECTIVE: Children with special health care needs are increasingly enrolling in managed care arrangements. However, existing managed care organizations, including traditional HMOs, are often poorly suited for caring for this population. In the adult health care area, new managed care entities, called Social HMOs (S/HMO) and Programs for the All-inclusive Care for the Elderly (PACE), have been created to integrate health and health-related services for chronically ill and disabled adults. We describe these models and assess their potential for serving children with special health care needs. METHOD: We reviewed the literature on managed care for children with special health care needs and evaluation findings from the S/HMO and PACE models for the elderly. RESULTS: Evaluations of the S/HMO and PACE models have yielded mixed findings. Some of the more positive accomplishments include lower use and expenditures for long-term care services compared to other demonstration projects, greater integration of primary care physicians in decision making concerning long-term care, and improved management of transitions between care levels. On the negative side, start-up has been slow, prospective members have been hesitant to enroll, intermittent and sometimes frequent operating deficits have emerged, no discernible positive effects on health or social outcomes are apparent, and no significant overall savings have emerged. CONCLUSIONS: With mixed results so far, caution is required in applying these or similar models for vulnerable child populations. However, given the inadequacies of traditional managed care for this population, we believe experimentation with new models of care that integrate health and health-related services is important. Such experimentation should be fostered only to the extent that the models are carefully designed and then implemented in a manner that protects the interests of children with special health care needs. PMID- 10728234 TI - After graduation, what? An analysis of the job placements of graduates of public health maternal and child health training programs. Project of the Association of Teachers of Maternal and Child Health. AB - OBJECTIVES: In 1995, the Association of Teachers of Maternal and Child Health (ATMCH) decided that information about the employment status of program graduates was essential to attempts to improve MCH curricula. METHOD: ATMCH requested information from 13 MCH programs in schools of public health funded by the federal Maternal and Child Health Bureau and 12 provided information about their master's degree graduates in the 1990-1994 period, including the year of graduation, degree, Bureau traineeship support, position held, and employing agency. RESULTS: The total number of graduates was 742. Four programs averaged less than 8 graduates per year (small); six, 10-16 (midsize); and two more than 22 (large). More than 90% of graduates received a M.P.H. In the 10 programs that provided data on Bureau support, 46% received traineeship support from the Bureau. Midsize programs had the largest percentage of graduates receiving traineeship support. Overall, 45% of graduates were in administrative positions, 32% were involved in patient care, 20% were in policy-analytic positions, and 3% in other positions. Forty-seven percent of program graduates entered into or continued in community-based agencies, 18% in government agencies, 17% in academic or research agencies, and 18% in other agencies. Program size was significantly associated with both position and the agency in which the graduate was employed. Bureau traineeship support was associated with employing agency. CONCLUSIONS: The study suggests the need for changes in MCH curricula, enhanced education opportunities in specialty skill areas, and an ongoing survey of graduates of MCH programs. PMID- 10728235 TI - Assessing for violence during pregnancy using a systematic approach. AB - OBJECTIVES: The purpose of this study was to determine whether a systematic, multiple assessment protocol could increase reporting of prenatal violence compared with a one-time routine assessment. METHOD: In 1994, the Maternity Care Coordination (MCC) program in a health department prenatal clinic in North Carolina implemented a concise, systematic assessment protocol on all 384 women who enrolled in the program from April 1994 to April 1995. The protocol assessed for violence at three times during pregnancy using the direct question, "Have you been hit, slapped, kicked, or hurt during this pregnancy?" To determine the effectiveness of the system, we retrospectively examined the 1991-1993 MCC records (n = 1056) in which the care coordinators routinely screened all clients for violence at their first visit only. RESULTS: Compared with the routine assessment approach, the new systematic assessment protocol increased reporting of prenatal violence at the initial prenatal visit from 6.3% to 10.9% (relative risk = 1.7, 95% confidence interval = 1.2, 2.5), and the multiple assessments increased reporting of prenatal violence to 14.1% (relative risk = 2.2, 95% confidence interval = 1.6, 3.1). CONCLUSIONS: Our study suggests that a concise and systematic screening technique using direct questions combined with multiple assessments increased reporting of prenatal violence compared with a single routine assessment. PMID- 10728236 TI - Abstinence education: resources from the National Center for Education in Maternal and Child Health for program design, implementation, and evaluation. PMID- 10728237 TI - An assessment of the use and impact of ancillary prenatal care services to Medicaid women in managed care. AB - OBJECTIVES: Managed care plans under Medicaid are becoming a usual source of care for low-income pregnant women. This study describes an ancillary prenatal care service intervention developed by one managed care organization (MCO) for Medicaid-enrolled women, assesses the extent to which the intervention services were used, and appraises the influence of the intervention on prenatal care participation. METHOD: There were 226 intervention and 258 control women with a single live birth delivered between 28 and 44 weeks gestation who (1) were enrolled in the MCO's Medicaid program, (2) were high-risk based on a prenatal risk assessment, and (3) started prenatal care prior to 26 weeks gestation. Less than adequate and intensive prenatal care utilization were chosen as intervention outcomes measures. RESULTS: Family planning, a 2-month postpartum baby visit, a maternal postpartum visit, and a WIC (Special Supplemental Nutrition Program for Women, Infants, and Children) referral were among the most self-selected intervention services for this population; home health aide and breast-feeding support were the least requested services. Over 90% of those needing family planning or breast-feeding services received the services, while over 20% of the intervention group refused child care, food assistance and family violence referrals, and home health aide and smoking cessation services. The intervention group had a significantly lower risk of less than adequate utilization of prenatal care (OR = .32; 95% CI: 0.17-0.60) and was more likely to have an intensive number of prenatal care visits (OR = 1.61; 95% CI: 1.05-2.48). CONCLUSIONS: The ability of managed care organizations to provide ongoing prenatal care to Medicaid populations in a cost-effective manner depends partly on their development of packages of prenatal services that foster positive preventive health care utilization behaviors and good pregnancy outcomes. The results of this project suggest that the intervention was beneficial in the area of improving utilization of prenatal care. PMID- 10728238 TI - Receipt of home health care after early discharge: results from a national managed care organization. AB - OBJECTIVES: To determine the frequency of home visits during the postpartum period among women discharged within 24 hours after childbirth, to identify characteristics of women who received at least one home visit, and to examine whether a home visit was related to postpartum experiences. METHOD: Women who were enrolled in a Prudential HealthCare plan and had a recent normal vaginal delivery completed a 15-minute telephone survey (N = 5201). Only women who were discharged within 24 hours after delivery were included in this analysis (N = 3121). Selected variables, including maternal characteristics, pregnancy-related and postpartum experiences, and social support factors, were compared for women who received visits and those who did not receive visits within two weeks after delivery. RESULTS: Slightly more than 30% of women participating in the survey received at least one home health care visit within 2 weeks after delivery discharge. Women who received a home visit were more likely to be Black, employed, primaparous, enrolled in a health maintenance organization, or not living in the Southern United States. Compared with women who did not receive a home visit, women who received a visit were more likely to have their newborn receive a phenylketonuria test after discharge, receive a follow-up phone call or housekeeping service, and access to a 24-hour hotline. CONCLUSIONS: Managed care organizations as well as other organized systems of care should be focused on improving the quality of prenatal and postpartum services, and increasing satisfaction of women using these services. PMID- 10728239 TI - Receipt of recommended prenatal interventions and birth weight among African American women: analysis of data from the 1988 National Maternal and Infant Health Survey. AB - OBJECTIVES: While the importance of exploring and better measuring elements of prenatal care have been noted in the public health literature, the components and timing of such services have been poorly examined for the overall pregnant population and specifically for African-Americans, who traditionally have had higher rates of low birth weight and premature delivery. This study explores the association between patient receipt of selected recommended prenatal care interventions and infant birth weight in a nationally representative sample of African-American women, while controlling for the influence of low birth weight risk indicators. METHOD: This is a retrospective case-control analysis using survey data of women who delivered normal birth weight, moderate low birth weight, and very low birth weight newborns in 1988. A sample of 3905 African American women who responded to the 1988 National Maternal and Infant Health Survey is examined based on maternal recall of receipt of six clinical screening procedures and seven health-promotion recommendations. Birth weight measures were obtained from linked 1988 birth certificate data. RESULTS: The initial results indicated that women who do not receive all of the recommended health-promotion advice are more likely to deliver very low birth weight infants than women who receive all of the advice in the content of their prenatal care, after controlling for low birth weight risks (OR = 1.28; 95% CI = 1.01, 1.7). However, when breast-feeding advice is removed from the aggregation of health-promotion advice, the significant effect of advice on very low birth weight is negated. No other significant group variations in the receipt of clinical screening procedures or health-promotion advice for women who gave birth in the remaining birth weight categories are observed. CONCLUSIONS: Nationally recommended initial clinical screening procedures and health-promotion advice in prenatal care content do not appear to be associated with a reduction in low birth weight for African-American women. More research is needed to better assess the impact of other antenatal interventions, particularly those given to women with a higher prevalence of poor birth outcomes. PMID- 10728240 TI - Correlates of drinking during the third trimester of pregnancy in Alaska. AB - OBJECTIVES: To examine characteristics related to drinking during pregnancy among a population-based sample of women. METHOD: We analyzed data related to third trimester drinking collected from the Alaska Pregnancy Risk Assessment Monitoring System (PRAMS). PRAMS used a population-based, stratified sampling design to survey 9733 of the approximately 44,000 live births to Alaska-resident women during 1991-1994. We defined regular drinking as one or more drinks per week on average during the third trimester. Analyses included bivariate and multivariate associations with any and regular drinking. RESULTS: Of women mailed a survey, 6973 responded and answered the questions related to alcohol consumption. Nine percent reported any drinking during the third trimester and 2.5% were regular third-trimester drinkers. The strongest risk factors for both any and regular third-trimester drinking were older age and marijuana or cocaine use. Other risk factors for any third-trimester drinking included prenatal cigarette smoking, greater education, non-Alaska Native race, the experience of significant life stressors, and residence in a community that did not restrict the sale of alcohol. Other risk factors for regular third-trimester drinking included prenatal cigarette smoking and the experience of domestic violence. Prenatal counseling regarding the effects of alcohol consumption during pregnancy and adequacy of prenatal care were not significantly associated with either outcome variable. CONCLUSIONS: Efforts to decrease prenatal alcohol consumption should be directed at older women and should address social determinants of health, such as education, domestic violence, drug use, and the availability of alcohol. In the absence of these efforts, prenatal alcohol education by health care providers may have little impact on pregnancy-related drinking. PMID- 10728241 TI - Prospective recruitment of women receiving prenatal care from diverse provider arrangements: a potential strategy. AB - OBJECTIVES: This study describes the use of a Medicaid managed care list to prospectively recruit into a research project pregnant women receiving care from a variety of providers. METHOD: A list of women enrolled in Medicaid managed care was used to recruit pregnant African-American and Latina women into a study of prenatal care satisfaction. Due to privacy concerns, the researchers were not able to directly access names from the list. Instead, a managed care contract agency sent recruitment letters to 1009 pregnant African-American and Latina Medicaid recipients. Response rates by ethnicity and several other key variables are calculated. The biases associated with this method of recruiting pregnant women from a variety of providers are discussed. RESULTS: Thirty-five percent of the women contacted returned consent forms and agreed to have researchers approach them; the response rate for African-American women was 43% and for Latinas was 29% (p < 0.0001). Respondents were younger and later in their pregnancies than nonrespondents, but did not differ from them by zip code of residence. The women recruited into the study obtained prenatal care from a diverse group of providers. CONCLUSIONS: While the use of a prospectively generated list of pregnant Medicaid recipients to recruit low-income pregnant women into a research study may be associated with some selection bias, the potential cost savings, decreased effort, and diminished recall bias may make their use a feasible sampling alternative, particularly when the researcher desires to recruit women seeking care from a variety of provider arrangements. PMID- 10728243 TI - Where is women's health in maternal and child health? PMID- 10728242 TI - Changing definitions of women's health: implications for health care and policy. AB - OBJECTIVES: To present an overview of how and why normative conceptions of women's health are changing and to discuss some implications of definitional shifts in the context of the changing U.S. health care system. METHOD: The paper describes the historical development of views of women's health and health care, contrasts the biomedical and biopsychosocial perspectives on women's health, and presents some evidence of challenges and opportunities for change in health care and policy. RESULTS: While women's health has generally been equated with reproductive functions, expanded definitions focus on health through the life span and in the context of women's multiple roles and diverse social circumstances. This expanded view highlights the limitations of health services and policy based on narrower conceptions and program mandates and the need for strategies for integrated, continuous care. There is evidence of change in women's health care, including in Title V programs. CONCLUSIONS: New understandings of women's health are particularly relevant to maternal and child health programs, which are positioned to provide model approaches for improving women's health care. PMID- 10728244 TI - Bright futures ahead. PMID- 10728245 TI - Lead in bone and hypertension. AB - In a recent study, Hu et al. showed that patients with elevated bone lead as preschoolers had elevated blood pressure in the absence of elevated blood lead later in life. We found in 486 high school patients with normal blood lead levels that those who had elevated lead as preschoolers had 9.3% prevalence of hypertension versus 3.6% in those of normal lead levels as preschoolers. This finding suggests a possible association between chronic accumulation of lead in bone and later adolescent hypertension. PMID- 10728246 TI - Improved disability population estimates of functional limitation among American children aged 5-17. AB - OBJECTIVES: This paper (a) creates and validates measures for population survey data to assess functional limitation in mobility, self-care, communication, and learning ability for school-age American children; (b) calculates rates of functional limitation using these measures, and provides population estimates of the number of children with limitations; and (c) examines these limitations as a function of socioeconomic factors. METHOD: The study is based on data for children aged 5-17 collected in the 1994 National Health Interview Survey on Disability. Ordinal values are assigned to survey items in the four functional areas and analyzed to produce scales of high reliability. These measures are used to identify within a 95% confidence interval the number of children with these limitations. Ordered logistic regression models measure the effects of functional limitations on disability and societal limitation. Socioeconomic differences are measured with an ordered logistic regression model that predicts severity and comorbidity. RESULTS: Limitations in learning ability (10.6%) and communication (5.5%) are the most common, with mobility (1.3%) and self-care (0.9%) occurring less often. Six percent of children have one serious functional limitation and 2.0% have two or more serious functional limitations. This corresponds to 4.0 million school-age American children with serious functional limitations. Functional limitation is strongly linked to socioeconomic disadvantage and to residence in single-mother households. CONCLUSIONS: Future population research should use multiple-item scales for four distinct areas of functional limitation, and a summary that takes into account both severity and comorbidity. The improved estimates of the number of school-age children with functional limitation in this paper may help contribute to a more informed scientific and policy discussion of functional limitation and disability among American school-age children. Future research on the disability process among children must consider the role of socioeconomic disadvantage and family structure. PMID- 10728247 TI - The effects of race, socioeconomic status, and household structure on injury mortality in children and young adults. AB - OBJECTIVES: Injuries are the leading killer of young persons in the United States, yet significant gaps in our understanding of this cause of death remain. By examining the independent influences of race, education, income, household structure, and residential location on injury mortality in young persons, this study addresses these gaps. METHOD: Using data from the National Longitudinal Mortality Study, survival analysis is used to examine the injury mortality risk faced by 0 to 17 year olds over a nine-year follow-up period. Separate models are estimated for homicide, suicide, unintentional injury deaths, and all injury deaths. RESULTS: Household head's education has an independent effect on youth homicide and unintentional injury mortality risk. By contrast, family income and household structure do not have independent effects on any of the injury outcomes. Finally, much of the excess homicide risk faced by young African Americans is explained by underlying racial differentials in socioeconomic status, household structure, and residential location. CONCLUSIONS: By finding an independent effect of household head's education on youth mortality risk from homicide and unintentional injuries, this study adds to the large body of evidence linking socioeconomic differentials to inequality in life chances. PMID- 10728249 TI - Length of maternal hospital stay for uncomplicated deliveries, 1988-1995: the impact of maternal and hospital characteristics. AB - OBJECTIVES: To determine the independent association of selected maternal and hospital characteristics with length of maternal hospital stay for uncomplicated vaginal deliveries. METHOD: Linear regression analysis using National Hospital Discharge Survey data from 1988 to 1995. Independent variables were year, maternal age and race, method of payment, and hospital ownership, size, and geographic location. The outcome measure was length of maternal hospital stay for uncomplicated vaginal deliveries. RESULTS: Length of stay was independently associated with year, geographic region, payment method, and hospital size. From 1988 to 1995, the mean length of stay fell from 2.1 to 1.5 days. The rate of decrease was similar for all regions, methods of payment, and hospital size. Women in the West had a shorter mean length of stay (1.5 days) than women in the Northeast (2.2 days). The difference by method of payment was smaller. Length of stay was shortest for women without insurance (1.8 days) and longest for women covered by Blue Cross (2.1 days). Maternal age and race and type of hospital ownership were not independently associated with the length of stay. CONCLUSIONS: Significant variations existed in the length of time women are hospitalized for normal childbirth. These variations are primarily associated with where a woman lives and whether she is insured. Given the current public debate on the impact of shortened hospital stays, these variations need to be explored and their effects on maternal and infant well-being clarified. PMID- 10728248 TI - Racial differences in perceived barriers to prenatal care. AB - OBJECTIVES: It is well known that black women are less likely to receive adequate prenatal care than white women. This study examines whether there are differences in barriers to prenatal care reported by black and white mothers. METHOD: Data from the 1988 National Maternal and Infant Health Survey were used to measure relationship between race and reported financial, service, and personal barriers to prenatal care use, adjusting for maternal age, parity, education, poverty level, and insurance coverage during pregnancy. All analyses were stratified by marital status because of substantial effect modification. RESULTS: Among married women, 12.1% of black women reported at least one barrier to prenatal care compared to 9.8% of white women. However, after adjustment for demographic and socioeconomic factors, black women were less likely to report a barrier odds ratio [OR] of 0.8; 0.6-1.0). Unmarried black women were less likely to report any barriers to care than white unmarried women (17.9% vs. 25.6%). After adjustment, the OR was 0.4 (0.3-0.5). CONCLUSIONS: This study suggests that though black women are less likely to receive prenatal care than white women, they are less likely to report barriers to such care. This may be due to differing expectations from the health system. The investigation of perceived barriers to care is important to the consequent understanding of what steps must be taken to assure that pregnant women do not experience obstacles to the receipt of prenatal care. PMID- 10728250 TI - Integrating children's health services: evaluation of a national demonstration project. AB - OBJECTIVES: Increasingly, the public and private sectors are turning to "service integration" efforts to reduce, if not eliminate, barriers to needed care created by categorical programs. In 1991, the Robert Wood Johnson Foundation established a new national demonstration project, called the Child Health Initiative, intended to test the feasibility of developing mechanisms at the community level to coordinate the delivery of health services and to pay for those services through a flexible pool of previously categorical funds. This article presents the findings of an independent evaluation of the Child Health Initiative. METHOD: The evaluation utilized a combination of qualitative methods to assess and describe the experiences of the communities as they developed and implemented integrated health services. It used a repeated measures design involving two site visits and interim telephone interviews, as well as review of documents. RESULTS: Overall, the demonstration project achieved mixed success. Both care coordination and the production of community health report cards were found to be achievable within the relatively short life of the foundation grant. However, many sites experienced significant delays in the production of report cards and implementing care coordination plans because the sites largely did not benefit from the successful models already in existence. Little clear progress was made in implementing the decategorization component of the project. Sites experienced difficulties due to lack of previous experience with this new undertaking, the inability to secure active cooperation from local, state, and federal agencies, the relatively short duration of the project, and other factors. CONCLUSIONS: A number of lessons were learned from this project that may be useful in future decategorization experiments, including (1) a clear understanding of the concept and its applications among all parties is essential, (2) high-level political commitments to the effort are needed between all levels of government, (3) adequate technical assistance should be provided to surmount technical considerations in establishing a workable approach to decategorization, and (4) decategorization and service integration efforts should focus on both the health and social sectors. PMID- 10728251 TI - Women's health in maternal and child health: time for a new tradition? AB - OBJECTIVES: The status quo in maternal and child health (MCH) focuses on obstetric health. An emerging alternative is to broaden the notion to reproductive health. An inclusive perspective encompasses women's health issues in MCH. The purpose of this paper is to further the debate on the relationship of women's health to MCH. Specific aims are (1) to describe activities promoting women's health in MCH and (2) to examine consequences of alternative perspectives for MCH research, services, and training. METHOD: To achieve the first objective, I discuss developments in a state health agency and pertinent documents from the MCH Section of the American Public Health Association. To address the second aim, I follow the Bush Policy Analysis Model of weighing the three paradigms against the following evaluative criteria: equity, efficiency, satisfaction, stigma, indirect effects, feasibility, sensitivity to class and race, and social responsibility. RESULTS: The obstetric approach meets most criteria in a positive fashion; reproductive health satisfies criteria more positively and less equivocally. A women's health perspective bears the most potential for improving reproductive outcomes at this time, since no area of women's general health has been definitively shown to be irrelevant to reproduction (or vice versa). CONCLUSIONS: This analysis suggests that women's health should be incorporated more fully into the MCH field, as well as other areas of public health and medicine. Once research deficits have been addressed and the scope of reproductive health delineated more clearly, the alignment of women's health with MCH may be reevaluated. PMID- 10728253 TI - State of the journal: 1998. PMID- 10728252 TI - Southeastern Title V program staff perceptions of state-level maternal and child health assessment skills. AB - OBJECTIVES: Since the publication of the Future of Public Health, a high priority has been placed on the development of the assessment capacity in public health programs. METHOD: Key informant interviews were conducted by telephone with selected program personnel of state Maternal and Child Health and Children with Special Health Care Needs programs in ten southeastern states to determine perceived deficiencies in skills needed to carry out assessments. RESULTS: We found that professional staff perceived that several fundamental assessment skills were available in their units, including collecting secondary data, and creating interagency groups and frameworks for assessment. In contrast, program staff perceived that their units did not have adequate skills to carry out many other critical assessment tasks, including the ability to analyze data using descriptive statistics. CONCLUSIONS: Our findings support the argument that more funds and staff resources must be devoted to the development of analytic skills, and to ensuring that consistent application of these skills is reinforced. PMID- 10728254 TI - The transition from Medicaid fee-for-service to managed care among private practitioners in New York City: effect on immunization and screening rates. AB - OBJECTIVES: This study examined the association between participation in Medicaid managed care and up-to-date coverage for childhood immunizations and screenings among private practice physicians serving New York City's poorest neighborhoods. METHOD: A random sample of 2174 children 3-35 months of age was drawn from 60 physician practices in 1995, and a cross-sectional analysis was used to compare up-to-date status for immunizations, and lead and anemia screening tests, for children cared for by managed care and nonmanaged care physicians. In 1996, an independent sample of 2380 children from the same practices was used to compare up-to-date status for individual children enrolled in Medicaid managed care and children predominantly enrolled in traditional fee-for-service Medicaid. Information from physician interviews augmented chart review data. Chi-square analysis and logistic regression were used. RESULTS: Physicians who participate in Medicaid managed care and those who do not had equal up-to-date coverage for immunizations (41.0 vs. 36.9%, p = .527), and lead (46.8 vs. 38.7%, p = .199) and anemia screening (63.2 vs. 56.5%, p = .272). Measures of the process of care were also similar for the two groups of physicians. Children themselves enrolled in Medicaid managed care appeared significantly more likely to be up-to-date than their nonmanaged care counterparts for immunizations (OR = 1.53, p = .027) and anemia screening (OR = 2.95, p = .000). CONCLUSIONS: Participation in managed care does not seem to change physicians' overall preventive care practice behavior. Available data did not reveal major differences in demographics or health status between individual children enrolled in managed care and those not enrolled. That children enrolled in managed care were better immunized and screened than those in fee-for-service Medicaid suggests that physicians receiving compensation under two payment systems may treat children differently depending on each child's mode of reimbursement. PMID- 10728255 TI - Perceptions of motivators and barriers to public prenatal care among first-time and follow-up adolescent patients and their providers. AB - OBJECTIVES: To compare perceptions of the motivators and barriers to obtaining public prenatal care from the perspectives of pregnant adolescents coming for first-time and follow-up appointments, as well as among those of their prenatal care providers. METHOD: The patient sample consisted of 250 consecutive, adolescent, public prenatal patients coming to one of the 5 prenatal clinics in one county in Arkansas. Patient responses were analyzed by appointment status (first-time vs. follow-up visitors). Sixteen providers at the same public prenatal clinics were also interviewed using the same survey instrument. RESULTS: We observed striking differences between patients and providers with respect to their perceptions of both the motivators and barriers to prenatal care. Adolescents reported "concern over the health of their baby" as a primary motivation, while providers identified adolescents' "concern over their own health" as the most important reason. With regard to barriers, adolescents were more likely to identify system-related barriers (e.g., lack of finances and transportation, and waiting time for appointments), while providers were more likely to identify personal barriers (e.g., feeling depressed, fear of procedures, and needing time to deal with problems at home). Patients and providers agreed, however, that fear of procedures and not wanting to be pregnant were important barriers to care. CONCLUSIONS: The differences in perceptions between adolescents and their prenatal care providers suggest that poor patient provider communication may represent one of the single most important nonfinancial barriers to care. Possible explanations for inadequate patient provider communication as well as solutions to improve their clinic interactions are discussed. PMID- 10728256 TI - Measuring satisfaction among low-income women: a prenatal care questionnaire. AB - OBJECTIVE: To evaluate the reliability and construct validity of a prenatal care satisfaction scale. METHOD: A prenatal care satisfaction scale that included six dimensions of care based on the literature was tested during a telephone interview with 101 first-time African-American and Mexican-American mothers 18 and over who receive Medicaid. RESULTS: The scale exhibited high reliability (Cronbach's alpha = 0.95), as well as good construct validity. The correlation between the scale and rating of the quality of care overall was 0.74 (p < 0.001); the correlation between the scale and whether the women would recommend this provider to a friend was 0.67 (p < 0.001). Results from correlation and factor analysis suggested a different set of dimensions than those described in the literature. CONCLUSION: The 22-question satisfaction with prenatal care scale has excellent reliability and construct validity and taps six established dimensions of satisfaction, including the art of care, technical quality, access, physical environment, availability, and efficacy. The multidimensional scale allows for alternative groupings of the domains of satisfaction as our understanding of prenatal care satisfaction increases. PMID- 10728257 TI - The maternal and child health sites' practices regarding HIV education, counseling, and testing of women of reproductive age in Chicago: barriers to universal implementation. AB - OBJECTIVES: Women of reproductive age are increasingly at risk for human immunodeficiency virus (HIV) infection. Recent advances in reducing perinatal transmission have resulted in official guidelines on universal HIV education, counseling, and voluntary testing of women of reproductive age, especially pregnant women. This study assesses to what extent the maternal child health (MCH) sites are implementing these guidelines with their female patient population (including pregnant women) and examines the barriers that prevent them from implementing these guidelines. METHOD: The study uses survey data from 92 hospitals and community health centers offering MCH services in Chicago regarding their providers' practices on HIV education, counseling, and testing, implementation of zidovudine (ZDV) therapy to reduce perinatal transmission, and the barriers to implementing these services. In addition, 20 taped in-depth interviews were conducted with experts to examine the barriers to universal implementation. RESULTS: Almost half (45% of perinatal care and 50% of family planning providers) of the institutions are not consistently offering HIV testing. One-third of those institutions that offer testing are not offering pretest counseling. Thirty-nine percent of the perinatal care providers in these institutions are not providing posttest counseling to HIV-negative women. Over one-third (35%) of these institutions reported that they are not set up to implement ZDV therapy during labor and delivery. Almost half (49%) had no protocols for ZDV therapy in place. Barriers to implementation included lack of provider training, limited staff time, physician resistance, unavailability or avoidance to seek perinatal care by high-risk women, cost, absence of a statewide and hospital-specific plan, lack of reproductive choice focus in posttest counseling, lack of provider knowledge about the administration of ZDV or its availability during labor, and lack of consumer education on perinatal risk reduction. CONCLUSIONS: MCH sites and their providers need assistance to overcome many barriers they face to implement universal HIV education, counseling, and testing of women of reproductive age. PMID- 10728258 TI - The role of state maternal and child health programs in the issue of newborn discharge. AB - OBJECTIVES: Since dramatically shortened newborn hospitalization has shifted the focus of care from the hospital, a central policy question has become how to assure a system of care that extends into the home and community. The objective of this study was to examine the role of the state Maternal and Child Health Title V programs in the assessment of the issue of newborn discharge, the development of policies, and the assurance of appropriate care. METHOD: The director or their designee in all 50 states and the District of Columbia participated in a structured telephone interview lasting 30-60 minutes. RESULTS: Twenty-eight states reported new or previously implemented mandates for 48 hours of private insurance coverage for postpartum hospitalization. Only 6 states reported mandates concerning private insurance coverage of inpatient services, but 20 states reported mandates for postdischarge services. In the assessment function, only 18 maternal and child health (MCH) programs reported that they had undertaken specific studies on the effects of discharge timing in their states. In policy development, 18 of the 51 respondents reported that the MCH program initiated newborn discharge discussions, 23 reported that the agency participated in discussions, and 10 indicated that they did not participate. In assurance, 29 programs reported that they had taken action to provide technical assistance to local communities in developing follow-up systems. The relationship between the performance of core functions and the development of specific discharge policies in the state was minimal. CONCLUSIONS: The MCH programs appear to have played varied, but often limited, roles in the development of discharge policies. It is essential for MCH programs to engage actively in the policy-making process. But in the political environment surrounding newborn discharge policy, where laws and regulations consistent with the well-being of infants and mothers were being enacted, the limited MCH program roles may have been appropriate. Arguing against this conclusion, however, is that only 15 programs believed they had been effective and only 19 were satisfied with the discharge policies in their states, which suggests that a more proactive role may be necessary. PMID- 10728259 TI - Child health benefits packages: lessons from Minnesota for State Children's Health Insurance Programs. AB - The State Children's Health Insurance Program (SCHIP) provides state maternal and child health (MCH) programs an opportunity to assure comprehensive health services are available to children through the selection of an appropriate benefits package. Minnesota had this chance in 1994 when the state was considering development of a universal standard benefits set. While the Minnesota MCH program was not at the policy table, they worked through a process that increased their influence on decisions by responding to a legislative call for a definition of "appropriate and necessary" care. This definition was ultimately adopted by policymakers in creating the recommended Universal Standard Benefits Set. This experience may provide an important incentive to other state MCH programs seeking opportunities to participate in the development of state SCHIP plans. PMID- 10728260 TI - District of Columbia Family Policy Seminar: a tool for devolution. PMID- 10728261 TI - Healthy People 2010. PMID- 10728262 TI - Cause-specific trends in neonatal mortality among black and white infants, United States, 1980-1995. AB - OBJECTIVE: Although neonatal mortality has been declining more rapidly than postneonatal mortality in recent decades, neonatal mortality continues to account for close to two-thirds of all infant deaths. This report uses U.S. vital statistics data to describe national trends in the major causes of neonatal mortality among black and white infants from 1980 to 1995. METHODS: Mortality rates were estimated as the number of deaths due to each cause (based on International Classification of Diseases, 9th Revision, codes) divided by the number of live births during the same time period. Linear regression models and smoothed rates were used to describe trends. RESULTS: During the study period, neonatal mortality declined 4.0% per year for white infants and 2.2% per year for black infants, and the black-white gap increased from 2.0 to 2.4. By 1995, disorders relating to short gestation and low birth weight were the number one cause of neonatal death for black infants and the number two cause for white infants, had the highest black-white disparity (4.6, up from 3.3 in 1980), and accounted for almost 40% of excess deaths to black infants (up from 24% in 1980). Congenital anomalies were the number two cause of neonatal death for black infants and the highest ranked cause for white infants in 1995, and it is the only cause for which there was not a substantial excess risk to black infants. CONCLUSIONS: Large declines in neonatal mortality have been achieved in recent years, but not in the black-white gap, which has increased. Declines were slower for black than white infants overall and for almost all causes. Prevention of preterm delivery and low birth weight continue to be a priority for reducing neonatal mortality, particularly among black infants. Although congenital anomalies do not contribute substantially to the black-white gap, their diagnosis, treatment, and prevention is critical to reducing overall neonatal mortality. PMID- 10728263 TI - Quality of measurement of smoking status by self-report and saliva cotinine among pregnant women. AB - OBJECTIVE: The objectives of this paper were to determine the rate of misclassification of smoking and nonsmoking status by self-reports and saliva continine of pregnant women participating in a smoking cessation trial, determine the relationship of the number of cigarettes smoked per day and saliva continine, and examine whether misclassification was due to an inappropriate saliva continine cutoff point. METHODS: End of pregnancy self-reports of smoking status and saliva continine were used to calculate misclassification rates. RESULTS: The findings revealed that 61 of 441 self-reported smokers had biochemical values inconsistent with smoking status for a smoking misclassification rate of 13.8%. The results also revealed that 28 of 107 self-reported quitters had continine values consistent with smoking status for a nonsmoking misclassification rate of 26.2%. Receiver Operating Characteristic (ROC) curves were then plotted to determine whether misclassification resulted from an inappropriate cutoff point. The continine cutoff point that maximized sensitivity and specificity for all women was 24 ng/ml. Racial ROC comparisons indicated a higher cutoff point for blacks than whites. Use of any of the ROC indicated cutoff points would not change the misclassification rates. CONCLUSIONS: These findings suggest that underreporting of smoking status during pregnancy is high and that social desirability of nonsmoking status may have contributed to the lack of precision in saliva continine to distinguish smoking status in this study. PMID- 10728264 TI - Women in a prenatal care/substance abuse treatment program: links between domestic violence and mental health. AB - OBJECTIVES: This study examines the prevalence of violence experienced by patients enrolled in the Step by Step program, a combined prenatal care/substance abuse treatment program at the Wake County Health Department in North Carolina. In addition, potential associations between violence and sociodemographic characteristics, substance use, and mental health are investigated. METHOD: All prenatal care/substance abuse treatment patients who met study eligibility criteria (N = 84) were assessed by health care providers. Descriptive statistics and bivariate analyses were used to compare victims of violence and nonvictims on a wide range of variables. Multiple linear regression analysis estimated the impact of the women's experiences of violence on their levels of mental health symptoms while controlling for confounding factors. RESULTS: Forty-two percent of patients had been victims of both sexual and physical violence, and 30% had been victims of physical violence alone. The combination of sexual and physical violence was significantly less common among African-American women compared with other women. No other significant differences were found between victims and nonvictims in terms of sociodemographics or substance use. Compared with nonvictims, victims of the combination of sexual and physical violence had significantly elevated levels of general psychological distress as well as elevated levels of hostility, depression, anxiety, interpersonal sensitivity, and somatization. However, no significant differences in levels of mental health symptoms were observed among women who had experienced physical violence in the absence of sexual violence. CONCLUSIONS: Questions concerning experiences of violence, including sexual victimization, should be incorporated into the clinical history-taking procedures of professionals working within prenatal care/substance abuse treatment programs so that effective interventions that take experiences of violence into account can be put into place for these high-risk women. PMID- 10728265 TI - Community health monitoring: taking the pulse of America's children. AB - OBJECTIVE: To describe the development, content, enablers/barriers, and impact of child health reports in nine communities participating in the Robert Wood Johnson Foundation funded Child Health Initiative (1991-1996). METHODS: A qualitative, prospective, multiyear, longitudinal evaluation using a multiple case-study methodology. Three waves of structured in-person and telephone interviews of the project staff, community leaders, and key participants tracked the development of child health reports in all nine communities. A mailed survey of project directors was administered to assess accomplishments at the completion of the project. Content analysis of each community health report was conducted using different conceptual frameworks for health measurement and reporting. RESULTS: All communities succeeded in creating a report that contained a broad set of outcome indicators reflecting children's health and well-being. The process of creating these reports, their content, level of analysis, presentation formats, and dissemination varied across sites based on available resources, data and analysis capacity, and other political considerations. While commonly accepted outcome measures were used in most reports (e.g., infant mortality, teen births, immunization rates), process indicators, important for quality monitoring and community health improvement, were notably lacking. In each community the reports were credited with providing a more comprehensive and integrated view of the health needs of children. CONCLUSIONS: Additional conceptual and technical work is needed to improve the ability of community health reports to capture key indicators of interest. Community reports can serve an important role in building the consensus needed to create program and policy changes. Community reports may have additional utility in monitoring the impact of health systems change on population health. Community reports can also facilitate a shared learning process for the participants and the community, and can be a useful tool to advance a children's health policy agenda. PMID- 10728266 TI - Trends in pregnancy weight gain within and outside ranges recommended by the Institute of Medicine in a WIC population. AB - OBJECTIVES: To examine the proportion of women with a pregnancy weight gain below, within, and above ranges recommended by the Institute of Medicine from 1990 to 1996. METHODS: Our study population included women attending Special Supplemental Nutrition Program for Women, Infants and Children (WIC) clinics in five states who delivered a liveborn singleton infant at term (N = 120,531). Pregnancy weight gain was self-reported at the postpartum visit. RESULTS: Only 34% of women gained weight within recommended ranges and there was little change in this proportion from 1990 to 1996. The proportion of women gaining less than their recommended weight decreased from 23.4% to 22.0%, and the proportion gaining more than recommended increased from 41.5% to 43.7% during the study period. Stratified analyses revealed similar trends within all race-ethnicity, age, parity, trimester of WIC initiation, and trimester of prenatal care initiation strata and among women in low, average, and high prepregnancy body mass index categories. There was no change in the weight gain distribution among obese women. Absolute and relative increases in the proportion of women gaining more weight than recommended were greatest among women who were underweight, Asian or Native American, less than 20 years of age, multiparous, and who initiated WIC and prenatal care in the third trimester. CONCLUSIONS: Pregnancy weight gain increased among this population of WIC participants from 1990 to 1996. PMID- 10728267 TI - Knowledge of treatment to reduce perinatal human immunodeficiency virus (HIV) transmission and likelihood of testing for HIV: results from two surveys of women of childbearing age. AB - OBJECTIVE: To determine whether knowledge of zidovudine treatment to reduce the risk of mother-to-child transmission of HIV increases the likelihood of HIV testing among women of childbearing age at increased risk for HIV infection. METHOD: Data from two samples were analyzed. The Young Women Survey was a population-based, door-to-door survey of 2,545 women aged 18 to 29 years who were living in lower-income neighborhoods. For the Perinatal Survey, women receiving prenatal care or who had delivered within the previous six months (N = 850) were recruited at randomly selected sites. Most (71.2%) of these participants were covered by public payment sources for their prenatal care. Data were collected within California counties with high rates of HIV among newborns, high rates of HIV among female clients of alternative test sites, and high rates of AIDS among female injection drug users. Most participants from both surveys were women of color. RESULTS: Participants who knew about zidovudine therapy for HIV-positive pregnant women were more likely to have had an HIV test, regardless of race/ethnicity, age, education, or number of previous births. The majority of women (79.3%) from both samples stated that they were more likely to take a test for HIV knowing about zidovudine treatment. CONCLUSIONS: HIV educators should include information on zidovudine therapy in campaigns designed to promote HIV testing among women. Prenatal care providers should incorporate a brief discussion about the benefits of zidovudine treatment when providing HIV counseling to patients. PMID- 10728268 TI - Maternal reporting of prepregnancy weight and birth outcome: consistency and completeness compared with the clinical record. AB - OBJECTIVES: We examined whether data obtained by maternal report could be used for research in clinical settings in place of abstraction of the clinical record. METHOD: Reported prepregnancy weight, delivery type, and infant's birth date, birth weight, and length were compared to the same information from the clinical record. RESULTS: Reported data, obtained from 198 women, were more complete than data in the 168 clinical records obtained. Prepregnancy weight from the clinical record was highly correlated with weight measured by us in early pregnancy, and with the value reported by the mother (differing significantly only in underweight women, who overreported by 2.4 lbs). There was complete concordance on birth date and method of delivery, and no significant differences in mean birth weight or length, between the reported and recorded information. CONCLUSION: The mother's report is a satisfactory substitute for clinical record data, being consistent with the record, and more complete, yet easier to obtain for clinical studies. PMID- 10728269 TI - Outreach and the State Children's Health Insurance Program. PMID- 10728270 TI - A performance indicator of psychosocial services in enhanced prenatal care of Medicaid-eligible women. AB - OBJECTIVE: Psychosocial services for low-income pregnant women vary widely in practice, and validated indicators of effective performance are lacking. The study presented here aims to determine whether a measure of provider compliance with a psychosocial service delivery guideline is associated with improved birth outcomes and therefore meets an important validity criterion of a performance indicator. METHODS: Data on psychosocial services delivered to 3467 pregnant women came from 27 sites certified by the California Department of Health Services to provide enhanced perinatal services to Medicaid-eligible women. Multivariate regression analyses were used to test the association of adequate service delivery according to a performance guideline with birth outcomes and the dependence of the association on the credentials of the provider and the type of practice setting. RESULTS: Women who received at least one psychosocial assessment each trimester of care according to the guideline were half as likely as women with inadequate services to have a low birthweight (OR = 0.49; CI 0.34, 0.71) or preterm birth (OR = 0.53; CI 0.40, 0.72) outcome. The effect did not depend on the credentials of the provider or the practice setting type. CONCLUSIONS: The indicator of the adequacy of psychosocial services according to a performance guideline appears to meet a fundamental criterion for a performance indicator, an association with improved outcomes. This indicator may be useful in monitoring performance of enhanced perinatal services for continuous quality improvement of services to low-income pregnant women. PMID- 10728272 TI - Pregnancy intentions, pregnancy attitudes, and the use of prenatal care in Missouri. AB - OBJECTIVES: This study uses data from 2378 mothers of live-born infants from the NICHD/Missouri Maternal and Infant Health Survey to examine the relationship between pregnancy intention and adequacy of prenatal care. METHODS: Pregnancy intention was measured using traditional classifications of mistimed and unwanted pregnancies as well as additional measures of women's attitudes about their pregnancies. Odds ratios for inadequate prenatal care and its component parts (initiation of care and receipt of services) were calculated using multiple logistic regression in separate models and in a combined model for the measures of intention and attitude. RESULTS: Women's attitudes about their pregnancies were associated with inadequate prenatal care, including both inadequate initiation of care and inadequate receipt of services. Traditional measures of intendedness were significantly related only to inadequate initiation of care. Women who were unhappy about the pregnancy (OR = 1.44), unsure that they wanted to be pregnant (OR = 2.81), or denied their pregnancies (OR = 4.82) were more likely to have inadequate prenatal care than women who did not have these attitudes. Women who were unhappy about being pregnant (OR = 1.86), unsure that they wanted to be pregnant (OR = 3.44), or who denied the pregnancy (OR = 6.69) were more likely to have inadequate initiation of care. Women who were unsure that they wanted to be pregnant (OR = 1.95) or who denied their pregnancies (OR = 2.47) were more likely to have received inadequate care once they had entered care. CONCLUSIONS: This study suggests that attitudes about pregnancy may be a psychosocial barrier to women obtaining early and continuous prenatal care. Pregnancy attitudes should be assessed and appropriate services provided to improve women's utilization of prenatal care. New measures of pregnancy attitude, beyond the traditional intention measures, can be useful in assessing pregnancy wantedness and identifying women to target for these services. PMID- 10728271 TI - How well do birth certificates describe the pregnancies they report? The Washington State experience with low-risk pregnancies. AB - OBJECTIVES: Birth certificates are a major source of population-based data on maternal and perinatal health, but their value depends on the accuracy of the data. This study assesses the validity of information recorded on the birth certificates for women in Washington State who were considered to be low risk at entry into care. METHODS: Birth certificates were matched to data abstracted from prenatal and intrapartum clinic and hospital records of a sample of 1937 Washington State obstetrical patients who were considered to be low risk at the beginning of their pregnancies. Accuracy of a variety of pregnancy characteristics (e.g., complications, procedures) on the birth certificate was analyzed using percentage agreement and sensitivity with record abstracts as the "gold standard." Next, we weighted the data from each source to produce estimates of pregnancy characteristics in the population. We compared these estimates from the two data sources to see whether they provide similar pictures of this subpopulation. RESULTS: Missing data for specific items on the birth certificates ranged from 0% to 24%. The birth certificate accurately captured gravidity and parity, but was less likely to report prenatal and intrapartum complications. The population estimates of the two data sources were significantly different. CONCLUSIONS: Because birth certificates significantly underestimated the complications of pregnancies, number of interventions, number of procedures, and prenatal visits, use of these data for health policy development or resource allocation should be tempered with caution. PMID- 10728273 TI - Factors associated with very early weaning among primiparas intending to breastfeed. AB - OBJECTIVES: The major objective of this study was to identify predictor variables that accurately differentiated breastfeeding women who weaned during the first 4 weeks, those who weaned between 5 and 26 weeks, and those who weaned after 26 weeks. Predictors were demographic variables, Theory of Planned Behavior (TPB) variables, breastfeeding knowledge, and difficulties experienced during the first month. METHODS: Primiparas who delivered healthy infants in an urban midwestern hospital provided initial data prior to discharge. Follow-up occurred at 1, 3, 6, 9, and 12 months. Following appropriate bivariate analyses, polychotomous logistic regression was used to determine predictors of weaning group. Linear multiple regression was used to predict intended duration. RESULTS: Most of the 84 women who weaned very early had intended to breastfeed considerably longer. According to the multivariate analysis, women who weaned earlier were younger, had completed fewer years of education, had a more positive bottle-feeding attitude and a less positive breastfeeding attitude, intended to breastfeed less time, had lower knowledge scores, had higher perceived insufficient milk scores, and planned to work outside the home. Variables postulated by the TPB to be direct predictors of intention explained 36% of the variance in intended duration. CONCLUSIONS: Women at risk for early weaning can be identified with reasonable accuracy using a TPB-based conceptual framework expanded to include breastfeeding specific variables. Casefinding using empirically derived screening methods and careful postpartum follow-up, along with professional intervention, should be used to avert unintended early weaning. PMID- 10728274 TI - The 1998 Herbert L. Needleman Award Lecture. Adolescent health: priorities for the next millennium. AB - There have been dramatic changes in adolescent health status over the past decade that have resulted from successful interventions. Overall mortality rates are down 14%, and many morbidities have declined. Today we know many of the elements that reduce risk: parental caring and connectedness, parental expectations for school and parent availability all outweigh family structure, ethnicity, and income. Likewise, schools can be extremely protective when young people feel connectedness. Factors associated with successful interventions include: strengthening families; strengthening educational involvement; expanding economic opportunities; and supporting youth development, not just problem reduction. Priorities for the next decade include: establishing resiliency-building interventions; developing positive correlates of negative behaviors; establishing broader multisectorial interdisciplinary teams; and formulating a new, more inclusive framework for adolescent health and development. PMID- 10728275 TI - Live births resulting from unintended pregnancies: an evaluation of synthetic state-based estimates. AB - OBJECTIVES: Most states lack information on the proportion of live births resulting from unintended pregnancies. We evaluated a potential solution to the lack of data, a synthetic state-based estimate of the percentage of live births resulting from unintended pregnancies for the state of Georgia. METHODS: We constructed the synthetic estimate by standardizing the 1995 National Survey of Family Growth data by the race, marital status, and age distribution of Georgia residents ages 15-44 years who delivered a live birth during 1990-1994. Two surveys conducted in Georgia during the same period that collected information on unintended pregnancies were used for comparison: the Georgia Women's Health Survey (GWHS) and the Georgia Pregnancy Risk Assessment Monitoring System (PRAMS). RESULTS: The synthetic estimate (35.2%, 95% CI = 33.5%-36.7%) was not statistically different from the GWHS estimate (39.6%, 95% CI = 35.7%-43.5%), but was significantly lower than the Georgia PRAMS estimate (49.0%, 95% CI = 45.5% 52.5%). When we stratified by race, marital status, and age, the synthetic and GWHS estimates were statistically similar except for married females and females ages 25-34 years, for whom the synthetic estimates were lower. For all groups of females, the synthetic estimates were statistically lower than the Georgia PRAMS estimates. CONCLUSIONS: The synthetic estimate can be a useful method for states that need to know the overall magnitude of the percentage of live births resulting from unintended pregnancy for purposes such as program planning. PMID- 10728276 TI - Reducing preterm and low birthweight rates in the United States: is psychosocial assessment the answer? PMID- 10728277 TI - Adolescent males' combined use of condoms with partners' use of female contraceptive methods. AB - OBJECTIVE: Protection from both sexually transmitted diseases and pregnancy is best obtained by the combined use of male condoms and effective female contraceptive methods. This research examines dual contraceptive method use among teenage men. METHOD: Analyzed data from the 1995 National Survey of Adolescent Males, a nationally representative survey of 15 to 19-year-old males. Used bivariate analyses and logistic regression to examine the correlates of combined use of condoms and female methods. RESULTS: At last intercourse, 17% of sexually active males reported use of a condom and a female method of contraception. Condom use, alone and in combination with a female method, was positively associated with talking with the partner about contraception and condoms, believing that males have a responsibility for contraception, and being in an earlier stage of a relationship. Only high levels of worry about sexually transmitted diseases differentially influenced dual method use, increasing the likelihood of using a condom with a female method, but not using condoms alone. CONCLUSIONS: The results suggest that efforts to increase condom use in general should also influence young men's use of condoms when their partner is using a female method. Providing information to young males about the high prevalence and serious consequences of sexually transmitted diseases may increase dual method use among adolescents. PMID- 10728278 TI - A new method to examine very low birth weight fetal and hebdomadal mortality in a regionalized system of perinatal care. AB - OBJECTIVE: Aggressive maternal transport of very low birth weight (VLBW) live births from community hospitals to regional perinatal centers may artificially increase community fetal death rates. By allocating maternal transports according to the location of antepartum and intrapartum care and separately computing antepartum and intrapartum fetal mortality rates, a more appropriate measure of hospital-based mortality may be determined. METHOD: Delivery charts were reviewed for 568 VLBW deliveries (including 97 fetal deaths and 77 hebdomadal deaths) occurring between 1990 and 1992 in a geographically defined perinatal region. Maternal transports were analyzed with community hospitals for antepartum mortality rates and with the regional center for intrapartum mortality rates. RESULTS: Using traditional methods, the fetal mortality rates for community hospitals and the regional center were antepartum 385.1 vs. 45.2, respectively, and intrapartum 120.9 vs. 24.9, respectively. When regional center live births (maternal transports) are placed with community hospitals for analysis of antepartum mortality, the new antepartum mortality rates were 185.7 vs. 72.8, respectively. The hebdomadal mortality rate for community hospitals was 250.0 as compared to 145.8 for the regional center. CONCLUSION: Maternal transports to a regional center represent successful antepartum management by community care providers. Even though they delivered in the regional center, they should be analyzed with community hospitals for antepartum fetal mortality comparisons. Therefore, antepartum and intrapartum fetal mortality should be examined separately in a functioning regionalized perinatal care program where the location of patient care differs from location of delivery. PMID- 10728279 TI - Characteristics of children having multiple Medicaid-paid asthma hospitalizations. AB - OBJECTIVES: We undertook this population-based study to describe the characteristics of poor children with multiple asthma hospitalizations and to discern if poor minority children have a greater risk for these events than poor white children. METHODS: We conducted a retrospective analysis of 1994 California hospital discharge data for asthma hospitalizations among 1 to 12-year-old Medicaid patients (N = 6844 discharges). Risk factors for multiple Medicaid asthma hospitalizations were calculated by using logistic regression procedures. RESULTS: In 1994, asthma hospitalizations accounted for 11.6% of Medicaid-funded hospitalizations for 1 to 12-year-olds in California. These hospitalizations had a mean length of 2.7 days and a mean hospital charge of $6532. After we controlled for source of admission and length of stay, African American children (OR, 1.93; 95% CI 1.49-2.49) and Latino children (OR, 1.34; 95% CI 1.04-1.72) had a higher risk of multiple Medicaid-paid hospitalizations for asthma than did white children. Adjusted odds ratios for multiple asthma hospitalizations were 1.35 (CI, 1.05-1.74) for children with emergency room admissions, and 1.16 (CI, 0.97-1.39) for children having hospital stays of at least 5 days duration. CONCLUSIONS: Among children with Medicaid-paid hospitalizations for asthma, the risk for multiple asthma hospitalizations within a year was greater among African Americans and Latinos than among whites. Programs attempting to decrease repeat hospitalizations for asthma may benefit by focusing on these populations. PMID- 10728280 TI - Adopting immunization recommendations: a new dissemination model. AB - OBJECTIVE: This paper presents a new approach for understanding factors related to physician adoption of clinical guidelines, using children's vaccine recommendations as a case study. METHODS: The model traces sequential steps, from awareness to agreement to adoption and, finally, adherence to the guideline. Movement through these stages can be catalyzed or retarded by many influences, grouped into two major categories: environmental characteristics of the physician's practice, and information characteristics of the guideline. Environmental characteristics include sociocultural factors, professional characteristics, and practice organization factors. Information characteristics include the guideline's relative advantage, complexity, and compatibility with existing guidelines and protocols, as well as mechanisms of guideline dissemination. IMPLICATIONS: This model can be used to identify characteristics that will likely impede or facilitate guideline adoption, and to focus dissemination efforts on key issues. PMID- 10728281 TI - Model indicators for maternal and child health: an overview of process, product, and applications. AB - OBJECTIVES: Further improvements in the health of mothers and children depend, in part, on collecting, analyzing, and interpreting relevant data correctly. Despite consistent efforts to improve data capacity and use during the past two decades, the need persists for a model set of maternal and child health (MCH) indicators to guide decisions about health conditions to be monitored, elements to be included in data sets, and definitions of measures. This article describes development, key characteristics, and major applications of a set of MCH Model Indicators (MCH MI) created to address these needs. METHODS: A conceptual model with five domains was created to organize and guide development of the indicators. The development process included systematic specification of concepts, formulas, age/gender groups, and data sources, as well as recommendations for frequency of surveillance. Information sources included published reports and expert opinion. RESULTS: There are 217 indicators distributed across domains as follows: 75 health status, 9 contextual characteristics, 16 health systems capacity and adequacy, 49 risk/protective status, and 68 health and related services. Twenty of the indicators, all of them in the health status domain, are recommended for routine surveillance. CONCLUSIONS: The indicators can be used to identify and address MCH problems, to complement and expand other sets of MCH indicators, to serve as standards for consistent definitions, to provide guidance for creation and revision of MCH and related data bases, and to provide a foundation for the development of related sets of indicators. Some of the indicators require further development, but the total MCH MI package constitutes a solid foundation for subsequent work, as well as for ongoing modifications that are essential if the Model Indicators are to remain responsive to MCH needs. PMID- 10728282 TI - Keeping "in touch" with women and children: maternal and child health model indicators. PMID- 10728283 TI - Improving children's access to oral health services: the oral health initiative. PMID- 10728284 TI - State of the journal: 1999. PMID- 10728285 TI - Making U.S. Maternal and Child Health policy: from "early discharge" to "drive through deliveries" to a national law. AB - OBJECTIVES: To examine the national policy-making process in maternal and child health through a case study of the passage of a national law aimed at extending postpartum hospital stays. METHODS: The study is based on a review of official and unofficial documents associated with the bill's legislative progress as well as an examination of scholarly research on early discharge and media coverage of the issue. Interviews were also conducted with legislative and interest group staff. RESULTS: The passage of early discharge legislation occurred in an unusually short time frame and was aided by its minimal public costs, the willingness of its supporters to compromise on key elements, and its perceived appeal to female voters in an election year. Clinical and public health research had little influence on the process. CONCLUSIONS: Maternal and child health advocates, while facing a problem with a politically weak constituency base, can benefit from their important symbolic role in policymakers' minds. They must respect the importance of compromise to legislative institutions and be alert to opportunities to frame their issues in a politically popular way. PMID- 10728286 TI - School performance in a longitudinal cohort of children at risk of maltreatment. AB - OBJECTIVE: Previous research has suggested that child maltreatment is associated with poor school performance. However, previous studies have largely been cross sectional or, if longitudinal, have had small sample sizes, short follow-up periods, or have not adequately controlled for confounders. The objective of this study is to determine the relationship between child maltreatment and school performance in a cohort of children at risk of maltreatment and followed since birth. METHOD: This prospective study followed children born at risk for maltreatment with semi-annual reviews of the North Carolina Central Registry of Child Abuse and Neglect. At ages six and eight years, children's teachers were surveyed using the Achenbach Teacher Report Form and project-developed questions regarding peer status. This information, along with control variables from maternal interviews, was used in logistic regression models to determine the impact of maltreatment on academic performance, peer status, and adaptive functioning. The generalized estimating equations (GEE) method was applied to adjust variance estimates for within-person correlations of school performance measures at two points in time. RESULTS: A substantiated maltreatment report is significantly associated with poorer academic performance (p < 0.01) and poorer adaptive functioning (p < 0.001) but not with peer status. CONCLUSIONS: Understanding the consequences of maltreatment, including poor academic performance and adaptive functioning, is important in planning educational, health, and social service interventions that may help abused or neglected children succeed in school and later in life. Longitudinal analysis is the best way to establish a causal relationship between maltreatment and subsequent school problems. PMID- 10728287 TI - Physician response to prenatal substance exposure. AB - OBJECTIVES: To examine physician responses to suspected prenatal substance exposure and the reasons underlying these responses. METHODS: National mail survey of practicing obstetricians and pediatricians who see neonates. Response rate: 63%. RESULTS: More than 70% of physicians reported having ever suspected prenatal substance exposure. Response rates did not vary by specialty. Twenty seven percent reported that they had never suspected prenatal substance exposure. The most common lifetime pattern (60%) was some response whenever prenatal substance exposure was suspected; next most common was no suspicion (27%). Just over 10% had a discretionary response: acting in some cases of suspected prenatal substance exposure but ignoring others. Two percent consistently ignored their suspicions. Getting help for the patient and protecting the fetus were the most common reasons to act. Among those who had ignored their suspicions, lack of sufficient evidence of substance use was the most often cited reason. There were some important specialty differences in reasons for response and non-response and in specific responses likely to be taken. Obstetricians are far more likely to provide the patient with information and get a substance use history; pediatricians are more inclined to involve outsiders. CONCLUSIONS: Obstetricians and pediatricians seem quite willing to act on their suspicions of prenatal substance exposure, and generally respond by taking positive actions. Specialty differences are few and reflect practice differences. PMID- 10728288 TI - The communicable disease impact of eliminating publicly funded prenatal care for undocumented immigrants. AB - OBJECTIVES: In 1996, California proposed regulations to eliminate publicly funded prenatal care for undocumented immigrants. Prenatal treatment of sexually transmitted infections (STIs) can prevent STI-related adverse outcomes of pregnancy (AOP). The study assessed the STI-related health and economic impact of the proposed regulations in Los Angeles County (LAC). METHODS: We modeled excess STI-related AOPs and associated costs that would occur in LAC as if the regulations were implemented in 1995. Using attributable fractions in the exposed for five STIs and their associated AOPs, we calculated excess STI-related AOPs and their costs that would result from the regulations and the degree to which excess morbidity would offset gross savings. RESULTS: The model indicates that, depending on regulatory level, 74, 110, or 132 excess AOPs would occur subsequent to the regulations, representing lost prevented morbidity. These excess AOPs would cost $5.1, $7.6, or $9.2 million dollars in direct medical expenses, offsetting anticipated savings by 19.2%, 29.0%, or 34.9%. This analysis does not include other costs of these STIs or costs associated with non-STI-related sequelae of diminished prenatal care, all of which could further offset anticipated savings. CONCLUSIONS: The proposed regulations would likely lead to increased STI-related morbidity and costs, thereby offsetting anticipated savings. Health departments are in a special position to promptly respond to policy issues affecting vulnerable populations. The development of a practical and rational framework for local-level policy assessment can be important for encouraging good scientific approaches that respond to calls for reductions in basic preventive health services. PMID- 10728289 TI - Breaking away: advocacy, education, and the relationship between Maternal and Child Health Professionals and the American Public Health Association. AB - Since its founding in 1921, the mission of the Maternal and Child Health (MCH) Section of the American Public Health Association (APHA) has been to develop innovative and creative approaches to addressing the health needs of mothers, children, and families. However, the growth and structure of APHA have impeded the capacity of MCH Section members to accomplish this mission. An independent organization would enhance the effectiveness of advocacy by MCH professionals and facilitate the dissemination of new strategies to address the needs of mothers and children. Consistent with the evolution of the profession of MCH, MCH professionals should withdraw from APHA and create a freestanding organization. PMID- 10728290 TI - Harnessing our energy: a counterpoint to "Breaking away". PMID- 10728291 TI - [What does the internet provide for the physician? Everyone becomes an infonaut]. PMID- 10728292 TI - [The internet will soon be indispensible for physicians]. PMID- 10728294 TI - [Budget stressing test strips. For cystitis diagnosis is often not necessary]. PMID- 10728293 TI - [What is the value of somnoplasty? Facelift from the inside for snorers. Interview by Dr. Christina Berndt]. PMID- 10728295 TI - [2 German myocardial infarct registries indicate: PTCA is more effective than lysis]. PMID- 10728296 TI - [How competence are Germany's carotid surgeons. Evaluation after 15,000 interventions]. AB - With the aim of assuring the quality of surgical treatment of carotid stenoses- also in conformity with legal requirements--the German Vascular Society (DGG) has established the recording of surgical data of patients prior to, during and following carotid reconstructive procedures. Since its inception, the data from 158 centers covering 15,116 interventions are now available. In comparison with data in the literature, the rate of severe post-operative neurological deficits was found to be only 1.9%, and the mortality rate 1%. Statistical analysis has shown that the risks of morbidity and mortality depend on the patient's risk profile and on the nature and morphology of the carotid lesion. PMID- 10728297 TI - [Are you familiar with success rate of your vascular surgeon? Operation permitted only in clinics with quality certificate]. PMID- 10728298 TI - [Carotid bruit: what should be done? Color duplex ultrasound indicates if operation is necessary]. AB - Carotid bruits need to be investigated without delay. The best imaging procedure available is color-coded duplex ultrasonography in the hands of an experienced examiner. Intra-arterial digital subtraction angiography should be used only when a surgical option appears indicated and the surgeon requests it prior to the operation. Determination of the intima/media thickness with the aid of the B scan provides good prognostic information. In the case of higher-grade stenoses with corresponding symptomatology, carotid surgery is indicated. Before carrying out such operations, however, the patient's risk and the surgery-related risk of a stroke or some other event occurring must be carefully weighed, one against the other, with the results achieved by the surgeon in this area being a major factor. PMID- 10728299 TI - [Practice tips for modern insulin therapy. "Bed time insulins without adverse morning aftereffect"]. PMID- 10728300 TI - [Depression and suicidal behavior more effectively controlled by a team approach. Pilot project to optimize diagnosis and therapy]. AB - The aim of the competition (Specialist-field Networks in Medicine "MedNet") announced by the Federal Ministry for Education and Research (BMBF) is to stimulate the creation of supraregional networks for specific diseases, in order, in this way, to improve cooperation and the transfer of knowledge between research institutions and various levels of medical care. The MedNet "Depression, Suicidal tendency" is one of the winners of this BMBF competition". In view of the central role played by the family doctor in the care given to patients with depressive disorders, permanent improvement in cooperation between physicians in private practice and the research institutions is a central aspect of the planned MedNet activities. PMID- 10728302 TI - [Antibiotic therapy in bronchial infections. 1: Acute and chronic bronchitis]. PMID- 10728301 TI - [Wegener's granulomatosis. Formerly poor prognosis, long-term therapy today]. PMID- 10728303 TI - [Diagnostic quiz. Exanthema in liver disease. Purpura in chronic hepatitis C with possible liver cirrhosis]. PMID- 10728304 TI - A brief history of Cardiovascular Research. PMID- 10728305 TI - The most frequently cited papers of Cardiovascular Research (1967-1998): 'the Millennium Minutes'. The Editorial Team. PMID- 10728306 TI - Simultaneous measurement of cardiac output and its distribution with microspheres in the rat. PMID- 10728308 TI - Pulsed Doppler: determination of diameter, blood flow velocity, and volumic flow of brachial artery in man. PMID- 10728307 TI - Blood flow distributions by microsphere deposition methods. AB - The art and science of the use of deposition markers for the estimation of blood flow distributions throughout the body and within organs is reviewed. Development of diffusible tracer techniques started 50 years ago. Twenty years later, radioactive 15 micron microspheres became the standard marker. Early studies on small animals, fetal sheep in 1967 and rats in 1976, provoked much of the technical development. Needs for avoiding the use of radioactivity, for having long lasting labels, and for providing higher spatial resolution, are driving the continuing exploration of newer techniques using colored and fluorescent microspheres and molecular deposition markers. Strengths and weaknesses of the various methods are compared. PMID- 10728309 TI - Noninvasive vascular ultrasound: an asset in vascular medicine. PMID- 10728310 TI - A fourth class of anti-dysrhythmic action? Effect of verapamil and ouabain toxicity, on atrial and ventricular intracellular potentials, and on other features of cardiac function. PMID- 10728311 TI - Voltage-gated calcium-channels and antiarrhythmic drug action. PMID- 10728312 TI - Kinetics of onset of rate-dependent effects of class I antiarrhythmic drugs are important in determining their effects on refractoriness in guinea-pig ventricle, and provide a theoretical basis for their subclassification. PMID- 10728313 TI - Classification of antiarrhythmic agents and the two laws of pharmacology. PMID- 10728314 TI - The context: investigation into hypertension and cardiac hypertrophy. PMID- 10728315 TI - Recent advances in cardiac hypertrophy. PMID- 10728316 TI - Effect of methyldopa, clonidine, and hydralazine on cardiac mass and haemodynamics in Wistar Kyoto and spontaneously hypertensive rats: an historical account and some follow-up. PMID- 10728317 TI - The influence of antihypertensive drug treatment on the prevention and regression of left ventricular hypertrophy. AB - Left ventricular hypertrophy (LVH) has been recognized as an important cardiovascular risk factor. Hypertensive disease is the most frequent background of LVH and it is generally felt that anti-hypertensive treatment should not only lower blood pressure but also cause regression of LVH. In the present survey the patho-physiology of LVH, its measurements and animal models used to study LVH are briefly discussed. Subsequently, the effects of various drugs in animal models and in human hypertensives are reviewed. It has been shown repeatedly that various types of antihypertensive drugs show differential activities on the prevention or regression of LVH. It is not only the lowering of blood pressure which determines the anti-LVH activity, but also the interaction of drugs with neuro-endocrine mechanisms such as the renin-angiotensin-aldosterone system and the sympathetic nervous system. PMID- 10728318 TI - Commentary on "Effects of ischaemia and reperfusion on calcium exchange and mechanical function in isolated rabbit myocardium". PMID- 10728319 TI - Cellular calcium homeostasis during ischemia; a thermodynamic approach. PMID- 10728320 TI - Distribution of myocardial injury and its relation to epicardial ST-segment change after coronary occlusion in the dog. PMID- 10728321 TI - ST-segment elevation in the electrocardiogram: a sign of myocardial ischemia. AB - In 1972 Kjekshus et al. published the seminal article 'Distribution of myocardial injury and its relations to epicardial ST-segment changes after coronary occlusion in the dog' in Cardiovascular Research. In this article it was shown that the ST-segment elevation occurring early after occlusion of the left descending coronary artery was closely related to the depletion of the necrotic cells from creatine kinase and to flow reduction at a later stage (24 h). This correlation was especially prominent if the infarction was transmural. Starting from these phenomenological relationships, this article briefly describes and summarizes the experimental research which was carried out in other laboratories after the publication of Kjekshus et al. Special emphasis is laid on the discussion of the main basic mechanisms which underly the clinically observed ST segment elevation and its evolution after the acute stage of ischemia, i.e. the changes in the transmembrane action potential and the alteration in electrical cell-to-cell coupling. PMID- 10728322 TI - The calcium paradox. PMID- 10728323 TI - Morphological changes of heart muscle caused by successive perfusion with calcium free and calcium-containing solutions (calcium paradox) PMID- 10728324 TI - The calcium paradox revisited: an artefact of great heuristic value. PMID- 10728325 TI - Improved functional recovery by ischaemic preconditioning is not mediated by adenosine in the globally ischaemic isolated rat heart. PMID- 10728326 TI - Adenosine and preconditioning in the rat heart. PMID- 10728327 TI - Nitric oxide synthesised from L-arginine mediates endothelium dependent dilatation in human veins in vivo. PMID- 10728328 TI - Inducible nitric oxide synthase and cardiovascular disease. PMID- 10728329 TI - A historic overview of the paper: an excitatory nociceptive cardiac reflex elicited by bradykinin and potentiated by prostaglandins and myocardial ischaemia (1976) PMID- 10728330 TI - Cardiovascular reflex responses induced by epicardial chemoreceptor stimulation. AB - The cardiac mechano- and chemoreceptors are broadly distributed in the myocardium and coronary vessels. A portion of these receptors extends over the epicardium and pericardium and therefore can be excited by mechanical or chemical stimuli directly applied to the surface of the heart. Excitation of epicardial receptors by topical application of chemical compounds elicits a variety of reflex cardiovascular responses, without the vascular or systemic effects of the drug administered systemically. A considerable number of studies has used the epicardial sensory field as a tool to delineate the functional characteristics of the cardiac afferent neurones in normal as well as in pathological conditions. In this review we analyze the cardiovascular reflex responses induced by epicardial application of a variety of substances like bradykinin, nicotine, muscarine, isoprenaline, adenosine, potassium chloride, capsaicin, prostaglandins or substance P in physiological models and also in models with acute myocardial ischemia or heart failure. The data highlight the contribution of the epicardial sensory neurites to the overall control of the cardiovascular system and, on the other hand, strengthen the need for further investigations directed to better elucidate the reflex cardiovascular responses that may develop in patients with pericardial abnormalities. PMID- 10728331 TI - The normal range and determinants of the intrinsic heart rate in man PMID- 10728332 TI - The normal range and determinants of the intrinsic heart rate in man. AB - Jose and Collison published a study on the normal range and the determinants of intrinsic heart rate in man in Cardiovascular Research in 1970 [Jose AD, Collison D. The normal range and determinants of the intrinsic heart rate in man. Cardiovasc Res 1970; 4: 160-167)]. The intrinsic heart rate is the heart rate under complete pharmacological blockade. They showed that (i) the resting heart rate is lower than the intrinsic heart rate and that (ii) the intrinsic heart rate declines with age. They also established that the variability in intrinsic heart rate between individuals of the same age is of the same order as the effect of ageing at the population level. This update discusses the relevance of these data with emphasis on sinus node function and autonomic balance. The paper of Jose and Collison was cited more than 200 times. The frequency of citation started to increase more than 10 years after publication. PMID- 10728333 TI - Inhibition by beta-blockade of the ST segment elevation after acute myocardial infarction in man. PMID- 10728334 TI - ST segment mapping and infarct size. PMID- 10728335 TI - Sleep revisited. PMID- 10728336 TI - An update on: cardiovascular and respiratory changes during sleep in normal and hypertensive subjects. PMID- 10728337 TI - Species variation in the coronary collateral circulation during regional myocardial ischaemia: a critical determinant of the rate of evolution and extent of myocardial infarction. PMID- 10728338 TI - Quo vadis collateral blood flow? A commentary on a highly cited paper. AB - This text is a commentary on the highly cited paper by Maxwell-Hearse-Yellon describing the amount of collateral blood flow in several species of mammals after coronary artery occlusion. The measurement of collateral blood flow, an academic exercise in previous times because of its invariance and the futility of changing the degree of adaptation under chronic conditions, has reached new importance because collateral vessel growth (presently called arteriogenesis) can now be manipulated with growth factors, their genes or peptides. The early successes and failures are discussed and a plea is made for the rigorous application of goldstandard methods (like in the Maxwell-Hearse-Yellon paper) to avoid disappointments in the new science of 'therapeutic angiogenesis'. PMID- 10728339 TI - Electrocardiogram of the normal mouse, Mus musculus: general considerations and genetic aspects. PMID- 10728340 TI - Mouse electrocardiography: an interval of thirty years. AB - The interest in mouse electrophysiology is expanding fast, despite the marked differences between mouse and man. Different methods have become available to analyse the electrical activity in the mouse heart in vivo. The mouse genome can be altered with relative ease, which allows the molecular dissection of the various components that contribute to de- and repolarisation of the cardiomyocyte and the initiation and propagation of cardiac arrhythmias. Mouse ischaemia reperfusion models have been used recently to study preventive measures against ischaemic myocyte damage. In the present review, the electrophysiological measurements performed in mice to date are discussed and complemented with results from a mouse ischaemia reperfusion model. PMID- 10728341 TI - Messenger RNA expression and immunological quantification of phospholamban and SR Ca(2+)-ATPase in failing and nonfailing human hearts. PMID- 10728342 TI - Regulation of sarcoplasmic reticulum Ca(2+)-ATPase and phospholamban in the failing and nonfailing heart. PMID- 10728343 TI - Regrets or no regrets? No regrets! The fate of rejected manuscripts. PMID- 10728344 TI - Mechano-electrical feedback. PMID- 10728345 TI - Chemokines and cardiovascular diseases. PMID- 10728346 TI - About the role of shear stress in atherogenesis. PMID- 10728347 TI - The role of the cytoskeleton in heart failure. AB - The cytoskeleton of cardiac myocytes consists of actin, the intermediate filament desmin and of alpha- and beta-tubulin that form the microtubules by polymerization. Vinculin, talin, dystrophin and spectrin represent a separate group of membrane-associated proteins. In numerous experimental studies, the role of cytoskeletal alterations especially of microtubules and desmin, in cardiac hypertrophy and failure (CHF) has been described. Microtubules were found to be accumulated thereby posing an increased load on myocytes which impedes sarcomere motion and promotes cardiac dysfunction. Other groups were unable to confirm microtubular densification. The possibility exists that these changes are species, load and chamber dependent. Recently, damage of the dystrophin molecule and MLP (muscle LIM protein) were identified as possible causes of CHF. Our own studies in human hearts with chronic CHF due to dilated cardiomyopathy (DCM) showed that a morphological basis of reduced contractile function exists: the cytoskeletal and membrane-associated proteins are disorganized and increased in amount confirming experimental reports. In contrast, the contractile myofilaments and the proteins of the sarcomeric skeleton including titin, alpha-actinin, and myomesin are significantly decreased. These changes can be assumed to occur in stages and are here presented as a testable hypothesis: (1) The early and reversible stage as present in animal experiments is characterized by accumulation of cytoskeletal proteins to counteract an increased strain without loss of contractile material. (2) Further accumulation of microtubules and desmin to compensate for the increasing loss of myofilaments and titin represents the late clinical and irreversible state. We suggest, based on a structural basis for heart failure, an integrative view which closes the gap between changes within cardiac myocytes and the involvement of the extracellular matrix, including the development of fibrosis. These factors contribute significantly to structural ventricular remodeling and dilatation finally resulting in reduced cardiac function. PMID- 10728348 TI - Cardiac fatty acid uptake and transport in health and disease. AB - Fatty acids are important energy donors for the healthy heart. These substrates are supplied to the myocardium bound to albumin to overcome their low solubility in aqueous solutions such as blood plasma. Transport from the microvascular compartment to the mitochondria inside the cardiomyocytes is most likely a combination of passive and protein-mediated diffusion. Alterations in tissue content of fatty acid-transport proteins may contribute to myocardial diseases such as the diabetic heart, and cardiac hypertrophy and failure. PMID- 10728349 TI - Angiogenic growth factors: potential new treatment for acute myocardial infarction? AB - In models of hind-limb ischemia and progressive coronary artery occlusion, angiogenic growth factor proteins and genes expressing growth factors have been shown to induce the development of collateral vessels and remodeling of existing collaterals. The therapeutic potential of growth factors in the setting of acute myocardial infarction may be related to non-angiogenic properties of growth factors as well, and is the focus of this review. PMID- 10728350 TI - Effect of physiological mechanical perturbations on intact human myocardial repolarization. AB - OBJECTIVE: The objective of this study was to investigate the relationship between acute decreases in right ventricular volume during Valsalva strain (with resultant changes in autonomic neural tone) and measures of local endocardial repolarization time independent of heart rate and autonomic neural tone. METHODS: Patients implanted with a stimulus to T wave (Stim-T) sensing pacemaker specially adapted to output a validate measure of beat to beat local repolarization (n = 9) performed Valsalva manoeuvers (40 mmHg for 15 s) while paced at a cycle length of 500 ms. Stim-T intervals were measured before and after autonomic blockade (Block: 0.03 mg/kg i.v. atropine +/- 0.15 mg/kg propranolol). Right ventricular end diastolic volume was estimated by simultaneous 2D-echocardiography. RESULTS: Without autonomic blockade, compared to baseline, repolarization significantly prolonged during Valsalva strain (1.1 +/- 0.7%) and shortened during release ( 1.4 +/- 1.0%). After block, strain related repolarization prolongation was also observed (1.0 +/- 0.6%), with significantly less release related repolarization shortening (-0.8 +/- 0.8%) compared to pre-block (P < 0.05). Right ventricular end diastolic volume decreased during strain by 11 +/- 10 and 9 +/- 16% from baseline, pre- and post-block respectively (P < 0.05). CONCLUSION: In a chronically instrumented human model, an acute physiologic volume reduction modestly prolongs right ventricular repolarization independent of changes in rate or autonomic tone. PMID- 10728351 TI - Ventricular arrhythmias induced by endothelin-1 or by acute ischemia: a comparative analysis using three-dimensional mapping. AB - OBJECTIVES: To analyze three-dimensional activation patterns of ventricular arrhythmias induced by endothelin-1 in comparison with ischemia-induced tachycardias. METHODS: Following AV node ablation, sixty pin electrodes containing four bipoles each were inserted into both ventricles of ten foxhounds. Using a computerized mapping system, this would allow to simultaneously record 240 endo-, epi- and midmyocardial electrograms for reconstruction of the three dimensional activation pattern. In five dogs, endothelin-1 was infused into the LAD at 60 pmol/min. In another five animals, the LAD was ligated. During the following 40 min, all ventricular arrhythmias were recorded for subsequent analysis. Furthermore, left ventricular conduction times during constant pacing and local effective refractory periods at eight left ventricular sites were determined before and after either intervention. RESULTS: Endothelin-1 had no significant effect on conduction time and refractoriness, whereas ligation prolonged both parameters significantly. Endothelin-1 as well as ligation induced multiple mono- and polymorphic nonsustained ventricular tachycardias. Endothelin 1-induced arrhythmias were exclusively based on focal mechanisms, whereas during ligation, macroreentrant mechanisms were involved in the maintenance of tachycardias in 29% of episodes. CONCLUSION: The differences in the effects of endothelin-1 and LAD ligation on electrophysiologic properties and the difference in the mechanism of induced ventricular tachycardias support the hypothesis that, apart from vasoconstrictive properties, endothelin-1 exerts an intrinsic arrhythmogenic effect. PMID- 10728353 TI - Serial echocardiographic assessment of left ventricular dimensions and function after myocardial infarction in mice. AB - OBJECTIVE: To test the usage of serial echocardiography in mice with induced myocardial infarct (MI) and to characterize the mouse model of MI. METHODS: C57 mice underwent open-chest surgery to induce left coronary artery occlusion or sham-operation (SH). Echocardiography was performed before and at 1, 2.5, 6 and 9 weeks after surgery. Left ventricular end diastolic and end systolic dimensions (LVEDd, LVESd) and fractional shortening (FS) were measured. Haemodynamics was determined at week 9 by LV catheterization and hearts were examined morphologically. RESULTS: Post-infarct mortality was 46% (10/22), of which, 70% died of acute heart failure or LV rupture within the first week. LV dimensions and FS remained stable in SH group (n = 10) during the study period. In surviving MI mice (n = 12), there was modest LV dilatation and fall in FS at week 1. Compared with week 0 values, there were progressive increase in LVEDd (+50( )+66%) and LVESd (+124(-)+171%), and decline in FS (-53(-)-73%) during the 2.5-9 week period. Infarcted mice also had lower LV systolic pressure (LVSP), dP/dtmax and dP/dtmin (all P < 0.01 vs. SH group). Infarct size, LVSP and dP/dt significantly correlated with FS and LV dimensions (r = 0.61-0.80, all P < 0.01). CONCLUSIONS: LV remodeling and dysfunction in mice with MI are time-dependent processes and early remodeling seems associated with high risk of rupture and acute pump failure. Our findings provide a baseline description of this murine model and confirm echocardiography as a reliable means to serially assess changes of cardiac structure and function after MI. PMID- 10728352 TI - Glucose metabolism in reperfused myocardium measured by [2-18F] 2 fluorodeoxyglucose and PET. AB - OBJECTIVE: [2-18F] 2-fluorodeoxyglucose (FDG) is widely used to trace glucose metabolism for cardiac imaging with positron emission tomography. Because the transport and phosphorylation rates differ for glucose and FDG, a lumped constant (LC) is used to correct for these differences. The effects of ischemia and reperfusion on the LC in vivo are unknown. To determine the validity of FDG as a tracer of glucose metabolism in post-ischemic myocardium in vivo, the relationship between glucose uptake (GU) and FDG metabolic rate (FDG-MR) was assessed early post-reperfusion following a transient ischemic event. METHODS: FDG metabolic rate, measured with FDG and PET, was compared to invasive measurements of substrate metabolism in reperfused and global myocardium of dogs subjected to 25 min ischemia and 2 h reperfusion. RESULTS: The FDG metabolic rate was decreased 20 +/- 4% in reperfused relative to remote myocardium. Glucose oxidation and lactate uptake were also decreased in reperfused relative to global myocardium, by 26 +/- 6% and 60 +/- 8% respectively. Glucose uptake did not differ significantly between reperfused and global myocardium. A linear correlation between FDG metabolic rate and glucose uptake was found in both reperfused and remote myocardium. Estimates of the LC from the slopes of the regression lines were similar in reperfused and remote myocardium, 1.25 and 1.44 respectively, and did not differ significantly from the LC determined in control dogs, 1.1. CONCLUSIONS: We conclude that the FDG metabolic rate continues to correlate well with glucose metabolism in reperfused myocardium. While FDG metabolic rate was modestly decreased in the absence of a significant change in glucose uptake, large alterations in the LC are not found 2 h post-reperfusion in vivo. PMID- 10728354 TI - Preconditioning the human myocardium by simulated ischemia: studies on the early and delayed protection. AB - BACKGROUND: There are data supporting the existence of ischemic preconditioning in man. This study investigated the most effective preconditioning protocol for the human myocardium and whether the second window of ischemic preconditioning (24 h) is as protective as the first window (< or = 2 h). METHODS AND RESULTS: Right atrial appendages (n = 6/group) obtained during coronary bypass surgery were prepared and superfused with normoxic and normothermic Krebs-Henseleit solution. After 30 min stabilisation, muscles were subjected to various preconditioning protocols followed by 90 min ischemia and 120 min reperfusion. At the end of each protocol, the leakage of creatinine kinase (CK, U/g wet wt) and the reduction of MTT to insoluble formazan dye (OD/mg wet wt), an index of cell viability, were measured. In study 1, preconditioning was induced by 2, 3, 5 and 10 min of ischemia followed by 5 min reperfusion. In study 2, 1-4 cycles of 2 or 5 min ischemia-5 min reperfusion were applied. In study 3, preconditioning was induced by 5 min ischemia-5 min reperfusion followed by 1, 2, 3 or 4 h reperfusion before the subsequent 90 min ischemia. In study 4, preconditioning with 5 min ischemia followed by 5 min reperfusion either immediately preceded 30 or 90 min ischemia/120 min reperfusion or was applied 24 h before. In study 1 and 2, optimal protection was achieved with 5 min or two cycles of 2 min preconditioning ischemia (CK = 3.06 +/- 0.31 and 2.89 +/- 0.02; MTT = 0.56 +/- 0.05 and 0.47 +/- 0.09, respectively vs. CK = 5.56 +/- 0.52 and MTT = 0.18 +/- 0.04 in ischemia alone group; P < 0.05). In study 3, protection was observed 2 h after preconditioning (CK = 3.43 +/- 0.22 and MTT = 0.46 +/- 0.09; P < 0.01 vs. ischemia alone group) but it was lost beyond 2 h (CK = 6.30 +/- 0.56 and MTT = 0.16 +/- 0.02 after 3 h; P = NS vs. ischemia alone group). In study 4, protection was observed 24 h following preconditioning when the atrial specimens were exposed to 30 min ischemia (CK = 2.96 +/- 0.38 and MTT = 0.61 +/- 0.01 vs. CK = 4.56 +/- 0.26 and MTT = 0.43 +/- 0.02 in ischemia alone group, P < 0.05); however, when the period of ischemia was extended to 90 min the beneficial effect of preconditioning was lost (CK = 10.28 +/- 0.05 and MTT = 0.11 +/- 0.05 vs. CK = 9.56 +/- 0.62 and MTT = 0.104 +/- 0.05 in ischemia alone group, P = NS). CONCLUSIONS: In the isolated human myocardium maximal protection induced by preconditioning is achieved by a total 4-5 min ischemic stimulus, an effect that is lost beyond 2 h of its application. Two windows of protection were identified, the first (< or = 2 h) being more potent than the second (24 h). PMID- 10728355 TI - Urodilatin limits acute reperfusion injury in the isolated rat heart. AB - OBJECTIVES: Hypercontracture is an important mechanism of myocyte death during reperfusion. cGMP modulates the sensitivity of contractile myofilaments to Ca2+, and increasing cGMP concentration during the last minutes of anoxia prevents reoxygenation-induced hypercontracture in isolated cardiomyocytes. The purpose of this study was to determine whether stimulation of particulate guanylyl cyclase with the natriuretic peptide urodilatin, given at the time of reperfusion, reduces myocardial necrosis in the rat heart submitted to transient ischemia. METHODS: Isolated rat hearts (n = 38) were submitted to either 40 or 60 min of no flow ischemia and 2 h of reperfusion, and were allocated to receive or not receive 0.05 microM urodilatin during the first 15 min of reperfusion or non reperfusion treatment. RESULTS: A marked reduction in myocardial cGMP concentration was observed in control hearts during reperfusion after 40 or 60 min of ischemia. Urodilatin significantly attenuated cGMP depletion during initial reperfusion, markedly improved contractile recovery after 40 min of ischemia (P < 0.0309), and reduced reperfusion-induced increase in left ventricular end-diastolic pressure (P = 0.0139), LDH release (P = 0.0263), and contraction band necrosis (P = 0.0179) after 60 min of ischemia. The beneficial effect of urodilatin was reproduced by the membrane permeable cGMP analog 8-Bromo cGMP. CONCLUSIONS: These results indicate that reduced cGMP concentration may impair myocyte survival during reperfusion. Stimulation of particulate guanylyl cyclase may appear as a new strategy to prevent immediate lethal reperfusion injury. PMID- 10728356 TI - Protection of hearts from reperfusion injury by propofol is associated with inhibition of the mitochondrial permeability transition. AB - OBJECTIVE: Diminishing oxidative stress may protect the heart against ischaemia reperfusion injury by preventing opening of the mitochondrial permeability transition (MPT) pore. The general anaesthetic agent propofol, a free radical scavenger, has been investigated for its effect on the MPT and its cardioprotective action following global and cardioplegic ischaemic arrest. METHOD: Isolated perfused Wistar rat hearts were subjected to either warm global ischaemia (Langendorff) or cold St. Thomas' cardioplegia (working heart mode) in the presence or absence of propofol. MPT pore opening was determined using [3H]-2 deoxyglucose-6-phosphate ([3H]-DOG-6P) entrapment. The respiratory function of isolated mitochondria was also determined for evidence of oxidative stress. RESULTS: Propofol (2 micrograms/ml) significantly improved the functional recovery of Langendorff hearts on reperfusion (left ventricular developed pressure from 28.4 +/- 6.2 to 53.3 +/- 7.3 mmHg and left ventricular end diastolic pressure from 52.9 +/- 4.3 to 37.5 +/- 3.9 mmHg). Recovery was also improved in propofol (4 micrograms/ml) treated working hearts following cold cardioplegic arrest. External cardiac work on reperfusion improved from 0.42 +/- 0.05 to 0.60 +/- 0.03 J/s, representing 45-64% of baseline values, when compared to controls (P < 0.05). Propofol inhibited MPT pore opening during reperfusion, [3H]-DOG-6P entrapment being 16.7 vs. 22.5 ratio units in controls (P < 0.05). Mitochondria isolated from non-ischaemic, propofol-treated hearts exhibited increased respiratory chain activity and were less sensitive to calcium-induced MPT pore opening. CONCLUSION: Propofol confers significant protection against global normothermic ischaemia and during cold cardioplegic arrest. This effect is associated with less opening of mitochondrial MPT pores, probably as a result of diminished oxidative stress. Propofol may be a useful adjunct to cardioplegic solutions in heart surgery. PMID- 10728357 TI - Mechanoelectric feedback after left ventricular infarction in rats. AB - BACKGROUND: Myocardial infarction can lead to electrical abnormalities and rhythm disturbances. However, there is limited data on the electrophysiological basis for these events. Since regional contraction abnormalities feature prominently in infarction, we investigated whether stretch of myocardium from the infarction borderzone can modulate the electrophysiological properties of cardiomyocytes via mechanoelectric feedback providing a mechanism for post-infarction arrhythmia. METHODS: Five weeks after experimental myocardial infarction (MI) in rats due to ligation of the left coronary artery (n = 26) or after sham operation (SO, n = 16), action potentials (AP) were measured in left ventricular preparations from the infarction borderzone. Sustained stretch was applied via a micrometer. RESULTS: Preparations from MI generated spontaneous electrical and contractile activity. Cardiomyocytes from MI had a comparable AP amplitude, a more negative resting membrane potential, and a prolonged AP duration (APD) when compared to SO. In SO, stretch of 150 microns increased the APD90. This was associated with stretch activated depolarizations near APD90 (SAD-90). In MI, significantly lower stretch, of only 20 microns, elicited SAD-90s, or SADs near APD50 (SAD-50). Stretch-induced events were suppressed by gadolinium, at a concentration (40 microM) normally used to inhibit stretch-activated channels. CONCLUSION: After MI, SADs are generated in the infarction borderzone at lower degrees of stretch. Increased sensitivity of the membrane potential of cardiac myocytes to mechanical stimuli may contribute to the high risk of arrhythmia after infarction. These SADs may involve the opening of stretch-activated channels. PMID- 10728358 TI - The rate and anisotropy of impulse propagation in the postnatal terminal crest are correlated with remodeling of Cx43 gap junction pattern. AB - BACKGROUND: Disruptions to intermyocyte coupling have been implicated in arrhythmogenesis and development of conduction disturbances. At present, understanding of the relationship between the microscopic organization of intercellular coupling and the macroscopic spread of impulse in the normal and diseased heart is largely confined to theoretical analyses. METHODS AND RESULTS: The abundance and arrangement of gap junctions, as well as conduction properties, were assessed in terminal crest preparations isolated from the atria of neonate, weanling, and adult rabbits. We report that the connexin composition of terminal crest was uncomplicated, with Cx43 being the most prominent isoform detectable by Western blotting and immunostaining. Terminal crest myocytes showed little change in total Cx43-gap junction per cell during postnatal growth as assessed by stereology. However, marked non-uniformities emerged in the sarcolemmal distribution of Cx43-gap junctions. Cx43-gap junction area at myocyte termini increased 3.5-fold from birth to adulthood. Correlated with this change in Cx43, impulse propagation velocity parallel to the myofiber axis, as assessed by multi site optical mapping using voltage-sensitive dye (di-4-ANEPPS), increased 2.4 fold. Conversely, the amount of Cx43-gap junctions on myocyte sides, and the conduction velocity transverse to the myofiber axis, remained relatively invariant during maturation. Hence, the increasing electrical anisotropy of maturing terminal crest was wholly accounted for by increases in conductance velocity along the bundle. This increase in longitudinal conduction velocity was correlated with changes in the sarcolemmal pattern, but not the overall density, of Cx43-gap junctions. CONCLUSIONS: This study provides the first correlative structure/function analysis of the relationship between the macroscopic conduction of impulse and the microscopic cellular organization of gap junctions in a differentiating cardiac bundle. Confirmation is provided for theoretical predictions which emphasize the importance of the cell-to-cell geometry of coupling in determining the spread and pattern of myocardial activation. PMID- 10728359 TI - Long-term treatment of spontaneously hypertensive rats with losartan and electrophysiological remodeling of cardiac myocytes. AB - OBJECTIVE: Cardiac hypertrophy due to pressure overload is associated with several cellular electrophysiological alterations such as prolongation of action potential duration (APD), decrease in transient outward current (Ito) and occurrence of the pacemaker current I(f). These alterations may play a role in sudden arrhythmic death, which is a major risk factor in myocardial hypertrophy and failure. Since angiotensin II is a key signal for myocyte hypertrophy, we tested if an 8-week treatment of old spontaneously hypertensive rats (SHR) with the antagonist of type-1 angiotensin II receptor (AT1), losartan (10 mg/kg/day), was able to influence the cellular electrophysiologic remodeling associated with cardiac hypertrophy. METHODS: Left ventricular myocytes were isolated from control (CTR) or losartan-treated (LOS) 18-month old SHR. Patch-clamped LVM were superfused with a normal Tyrode's solution (to measure action potential) or appropriately modified Tyrode's solution (to measure Ito and I(f)). RESULTS: Heart weight to body weight ratio (HW/BW) was significantly smaller in LOS (5.69 +/- 0.25 mg/g) than in CTR rats (6.67 +/- 0.37 mg/g; P < 0.05). Membrane capacitance, an index of cell size, was significantly reduced in LOS (342 +/- 12, n = 92) vs. CTR (422 +/- 14 pF, n = 96, P < 0.001). APD was significantly shorter in LOS than in CTR (at -60 mV: 197 +/- 23 vs. 277 +/- 19 ms, n = 28, P < 0.001); this effect was paralleled by a larger maximum Ito density in the LOS group (LOS: 15.1 +/- 1.4 pA/pF, CTR: 10.0 +/- 0.8 pA/pF) (n = 27, P < 0.02). I(f), elicited by hyperpolarizing steps (range: -60 to -130 mV), was consistently recorded in SHR cells; however, its maximal specific conductance was significantly lower in LOS than in CTR rats (28.6 +/- 3.6 vs. 54.2 +/- 8.0 pS/pF, n = 55, P < 0.001). Voltage of half-maximal activation (V1/2) of both Ito and I(f) was unchanged by the treatment. CONCLUSIONS: AT1 receptor blockade with losartan prevents the development of myocyte hypertrophy and associated electrophysiological alterations in old SHR. PMID- 10728360 TI - Heterogeneous changes in action potential and intracellular Ca2+ in left ventricular myocyte sub-types from rabbits with heart failure. AB - OBJECTIVE: Myocardial cellular electrophysiology and intracellular Ca2+ regulation are altered in heart failure. The extent of these changes may vary within the layers of the ventricular wall. To examine this, cell size, action potential and intracellular Ca2+ transient characteristics (Fura-2) were measured in single cardiac myocytes from sub-epicardial, mid-myocardial, and sub endocardial regions of the left ventricle of rabbits with heart failure. METHODS: Myocytes were isolated from animals with heart failure induced by chronic coronary artery ligation and from sham operated controls. Trans-membrane potential was measured using high resistance microelectrodes electrodes (30 M omega; 2 M KC1). Fura-2 was loaded into cells by incubation with the AM form. Subsequent fluorescence measurements were used to measure intracellular Ca2+ concentration at a range of stimulus frequencies. RESULTS: Resting cell length was significantly greater in the heart failure group; approximately 115% of control values in sub-epicardial and mid-myocardial cells, and approximately 108% in sub-endocardial cells. Using criteria described by previous studies on other mammalian hearts, functional M cells were identified by a higher maximum rate of depolarisation and longer action potential duration at 90% repolarisation (APD90) compared to the two other myocyte sub-types. In the heart failure group, APD90 and Ca2+ transient duration (CaD50) were prolonged in sub-epicardial and M cells but shortened in sub-endocardial myocytes. These changes were significant at lower stimulus frequencies, but the relative effect diminished at higher frequencies (3 Hz). Peak systolic [Ca2+] was reduced in sub-epicardial and M cells but increased in sub-endocardial cells in the heart failure group compared to controls. At higher stimulus frequencies, end diastolic Ca2+ levels were lower in sub-epicardial cells but higher in sub-endocardial myocytes of the heart failure group compared with controls. In general, changes were greater in heart failure animals with more severe in vivo ventricular dysfunction (ejection fraction < or = 44%). CONCLUSIONS: Heart failure was associated with an increased cell size throughout the left ventricle, but the form of the changes in electrophysiology and Ca2+ transient were dependent on the myocyte sub-type. In particular sub-endocardial cells displayed markedly different changes compared to the other myocyte sub-types. PMID- 10728361 TI - Central role for ornithine decarboxylase in beta-adrenoceptor mediated hypertrophy. AB - OBJECTIVE: TGF-beta stimulation of cardiac myocytes induces a hypertrophic responsiveness to beta-adrenoceptor stimulation. This study investigates whether this beta-adrenoceptor mediated effect depends on induction of ornithine decarboxylase (ODC). METHODS: Isolated adult ventricular cardiomyocytes from rats were used as an experimental model. Cells were either cultured in 20% (v/v) FCS to activate autocrine released TGF-beta or used without pre-treatment. The hypertrophic response was characterized by an increased 14C-phenylalanine incorporation, RNA and protein mass or by an increased expression of atrionatriurectic factor and ODC. The results on cell cultures were compared to those achieved by isoprenaline perfused mice hearts from transgenic mice overexpressing TGF-beta 1. RESULTS: ODC activity and expression increased within 2 h in TGF-beta 1 pre-treated cells under isoprenaline. In the presence of ODC inhibitors (alpha-methylornithine or difluoromethylornithine) this increase remained absent and the increases in 14C-phenylalanine incorporation, protein and RNA mass under isoprenaline were abolished. In cells not exposed to TGF-beta no induction of ODC was observed. Isoprenaline also induced ODC in isolated perfused ventricles from transgenic mice overexpressing TGF-beta 1, but not in ventricles from their nontransgenic counterparts. CONCLUSIONS: This study shows first, a pivotal role for ODC induction in the hypertrophic response of cardiomyocytes to beta-adrenoceptor stimulation and second, that ODC induction in vivo and in vitro requires pre-treatment of cardiomyocytes with TGF-beta. It is concluded that TGF beta induces a hypertrophic responsiveness to beta-adrenoceptor stimulation that is characterized by ODC induction. PMID- 10728362 TI - Participation of prostaglandin E2 in the endothelial protective effect of ischaemic preconditioning in isolated rat heart. AB - OBJECTIVE: To assess whether the protective effect of ischaemic preconditioning (IPC) on endothelial function in coronary arteries of the rat involves prostaglandins. METHODS: Isolated rat hearts perfused under constant flow conditions were exposed to 30 min of partial ischaemia (flow-rate 1 ml/min) followed by 20 min of reperfusion, after which coronaries were precontracted with U-46619 0.1 microM, and the coronary response to the endothelium-dependent vasodilator, serotonin (5-HT, 10 microM), was compared to that of the endothelium independent vasodilator, sodium nitroprusside (SNP, 3 microM). Prostaglandin production was blocked with a perfusion of indomethacin 10 microM started 15 min before IPC or a corresponding sham period and stopped just before the 20-min reperfusion period. RESULTS: In untreated hearts, ischaemia diminished selectively 5-HT-induced vasodilatation, compared to sham hearts. The vasodilatation by SNP was unaffected after ischaemia and reperfusion. IPC (5 min of zero-flow ischaemia followed by 10 min reperfusion before the 30-min partial ischaemia) preserved the vasodilatation produced by 5-HT. Enzymeimmunoassays showed an increased production of PGE2 in the IPC group. Treatment of hearts with indomethacin blocked the protective effect of IPC on the vasodilatation produced by 5-HT and decreased the production of PGE2. A 5-min perfusion with 3 nM PGE2 started 15 min before the partial ischaemia, protected the endothelium. This was blocked by 1 microM chelerythrine, but not by 0.3 microM glibenclamide. CONCLUSIONS: These results suggest that IPC affords protection to endothelial function in coronary arteries of the rat partially via the release of PGE2. Under our experimental conditions, the protective effect of PGE2 is mediated by PKC. PMID- 10728363 TI - CXC-chemokines, a new group of cytokines in congestive heart failure--possible role of platelets and monocytes. AB - OBJECTIVES: The purpose of the present study was to examine the circulating levels of CXC-chemokines in patients with various degree of congestive heart failure (CHF). BACKGROUND: CXC-chemokines may be important mediators in the persistent immune activation observed in CHF patients by activation of circulating neutrophils, T-cells and monocytes and possibly by the recruitment of these cells into the failing myocardium. METHODS: Levels of interleukin (IL)-8, growth-regulated oncogene (GRO) alpha and epithelial neutrophil activating peptide (ENA)-78 were measured both in serum and in platelet-free plasma by enzyme immunoassay in 47 patients with CHF and in 20 healthy controls. RESULTS: (i) CHF patients had significantly elevated levels of all the three CXC chemokines with IL-8 and GRO alpha showing a gradual increase along with increasing NYHA class. (ii) There was an inverse correlation between IL-8 and left ventricular ejection fraction (EF) and cardiac index (CI). (iii) Both unstimulated and lipopolysaccharide (LPS)-stimulated monocytes from CHF patients released markedly elevated amounts of all three CXC-chemokines. (iv) Platelets from patients with severe CHF were characterised by decreased content of GRO alpha and ENA-78 as well as decreased release of these chemokines upon thrombin receptor stimulation. (v) Activated platelets stimulated peripheral blood mononuclear cells in vitro to enhanced release of IL-8, and neutralising antibodies against ENA-78 inhibited this interaction. CONCLUSIONS: This study demonstrates for the first time elevated levels of CXC-chemokines in CHF, which may be of importance for progression of heart failure. Our findings further suggest that activated monocytes and platelets may contribute to enhanced CXC chemokine levels in CHF. PMID- 10728364 TI - Paradoxical inhibition of fibrinogen binding and potentiation of alpha-granule release by specific types of inhibitors of glycoprotein IIb-IIIa. AB - OBJECTIVE: To determine whether glycoprotein (GP) IIb-IIIa inhibitors can paradoxically augment activation of platelets, activation of GP IIb-IIIa, alpha granule degranulation, and lysosome release were induced after exposure of platelets to GP IIb-IIIa inhibitors. METHODS: ADP-induced platelet activation was assessed after exposure of platelets to Abciximab, or to a non-peptide ligand, the free acid of Orbofiban (Orbofiban(a)). Activation of GP IIb-IIIa was detected based on binding of fluorochrome labeled fibrinogen or a labeled monoclonal antibody, PAC-1. alpha-Granule degranulation was detected based on surface expression of P-selectin and lysosome release was detected based on surface expression of CD63. RESULTS: Despite significant inter-individual variability in inhibition of fibrinogen binding in response to each of the GP IIb-IIIa inhibitors used, a concentration dependent decrease in fibrinogen binding was seen with each agent in samples from each subject. Binding of PAC-1 was inhibited in a parallel manner. Abciximab increased ADP-induced P-selectin expression. Orbofiban(a) did not alter ADP-induced P-selectin expression. Neither agent altered ADP-induced CD63 expression. When platelets were exposed to Abciximab and Orbofiban(a), both Abciximab and Orbofiban(a) were found in the alpha-granules (by confocal microscopy), consistent with potentiation of agonist-induced release of alpha-granular products associated with uptake of proteins. CONCLUSIONS: Specific types of GP IIb-IIIa inhibitors can paradoxically augment agonist induced release of alpha-granules despite inhibiting agonist-induced fibrinogen binding. PMID- 10728365 TI - Role of nitric oxide in the vasodilator effect of recombinant human growth hormone in patients with dilated cardiomyopathy. AB - OBJECTIVE: Dilated cardiomyopathy is characterized by elevated arterial vascular resistance and impaired nitric oxide (NO)-dependent vasodilation. Insulin-like growth factor-I (IGF-I) has been shown to stimulate endothelial NO-synthase resulting in endothelium-dependent vasodilation. Growth hormone (GH) substitution therapy leads in GH-deficient patients to significant increases of IGF-I which may alter systemic vascular resistance by stimulating NO production. This study was designed to evaluate the effects of treatment with recombinant human growth hormone (GH) on NO production and NO-dependent vascular effects in patients with dilated cardiomyopathy. METHODS: 50 patients with dilated cardiomyopathy were randomly assigned to double-blind treatment with 2 I.U. of GH or placebo for 3 months. Central hemodynamics were determined by Swan-Ganz catheter and cardiac output was obtained by the thermodilution method. Serum GH and IGF-I levels were measured and systemic NO production was determined from urinary nitrate and cyclic GMP excretion rates in 42 patients. RESULTS: GH treatment caused in comparison to the placebo group a significant increase of IGF-I by 91 ng/ml (P = 0.0001). Urinary excretion rates of nitrate and cyclic GMP increased also significantly by 38 mumol/mmol creatinine (P = 0.027) and 65 nmol/mmol creatinine (P = 0.003), respectively. The parallel increase of both marker molecules indicates increased systemic NO production during GH treatment. CONCLUSION: GH treatment induces a significant, but moderate increase of systemic NO production in patients with dilated cardiomyopathy. This effect may be mediated by IGF-I stimulating endothelial NO synthase. PMID- 10728366 TI - Genistein supplementation and estrogen replacement therapy improve endothelial dysfunction induced by ovariectomy in rats. AB - BACKGROUND: We investigated the effect of genistein, a phytoestrogen derived from a soy diet with a flavonoid chemical structure, on endothelial dysfunction induced by estrogen deficiency in rats. METHODS: Female mature Sprague-Dawley rats were subjected to a bilateral ovariectomy (OVX rats). Sham-operated animals (Sham OVX rats) were used as controls. Three weeks after surgery animals were randomized to the following treatments: genistein (0.2 mg/kg/day, s.c. for 4 weeks), 17 beta-estradiol (20 micrograms/kg/day, s.c. for 4 weeks) or their respective vehicles. Mean arterial blood pressure (MAP), heart rate (HR), total plasma cholesterol, plasma estradiol, plasma genistein levels and uterine weights were studied. Furthermore, we investigated acetylcholine (ACh 10 nM-10 microM) and sodium nitroprusside: (SN 15-30 nM) induced relaxation of aortic rings as well as NG-L-arginine (L-NMA: 10-100 microM) induced vasoconstriction in phenylephrine precontracted aortic segments and calcium-dependent nitric oxide synthase (cNOS) activity in homogenates of lungs taken from both sham OVX and OVX rats. RESULTS: Untreated OVX rats had, compared with sham OVX animals, unchanged body weight, MAP, HR and plasma cholesterol. In contrast ovariectomy impaired endothelial responses, blunted L-NMA induced contraction (L-NMA 100 microM: Sham OVX = 2.1 +/- 0.2 g/mg tissue; OVX = 1.7 +/- 0.4 g/mg tissue) and reduced cNOS activity. Treatment with 17 beta-estradiol increased the hormone plasma levels, reverted the endothelial dysfunction and increased cNOS activity in lung homogenates. Genistein supplementation enhanced the circulating levels of the phytoestrogen and affected NOS activity and endothelial dysfunction to the same extent. CONCLUSIONS: Our data suggest that genistein and 17 beta-estradiol show overlapping effects on experimental endothelial dysfunction. PMID- 10728367 TI - Impaired potentiation by endothelin-1 and vasopressin of sympathetic contraction in tail artery from hypertensive rats. AB - OBJECTIVE: To analyse the effects of endothelin-1 and vasopressin on the sympathetic vasoconstriction during hypertension. METHODS: Electrical field stimulation (4 Hz) was applied to isolated, 2 mm segments of the tail artery from spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats prepared for isometric tension recording. RESULTS: The contraction to electrical stimulation was potentiated by endothelin-1 (10(-10)-10(-8) M) in arteries from WKY but not from SHR, and by vasopressin (10(-12)-10(-10) M) more markedly in arteries from WKY than from SHR. The potentiation by endothelin-1 was reduced more markedly by the antagonist of endothelin ETA receptors BQ-123 (10(-5) M) than by the endothelin ETB receptor antagonist BQ-788 (10(-5) M). The potentiation by vasopressin was reduced by the antagonist of vasopressin V1 receptors d(CH2)5Tyr(Me)AVP (10(-7) M), but not by the vasopressin V2 receptor antagonist d(CH2)5D-Ile2, Ile4AVP (10(-7) M). The blocker of L-type calcium channels verapamil (10(-5) M) reduced the potentiation by both endothelin-1 and vasopressin in arteries from WKY rats, and increased the potentiation by vasopressin in arteries from SHR. Noradrenaline (10(-8)-10(-4) M) contraction was not modified by endothelin-1 (3 x 10(-9) M) or vasopressin (3 x 10(-11) M), and contraction to endothelin-1 (10(-9)-10(-7) M) and vasopressin (10(-10)-10(-7) M) was lower in arteries from SHR than from WKY rats. CONCLUSIONS: (1) The potentiation by endothelin-1 and vasopressin of the sympathetic vasoconstriction, probably due to increased release of noradrenaline, is impaired during hypertension, and (2) this potentiation is mediated mainly by endothelin ETA receptors, and by vasopressin V1 receptors, in both WKY and SHR, and for both peptides it is mediated by L-type calcium channels in arteries from normotensive but not in those from hypertensive animals. PMID- 10728368 TI - Endothelin-dependent effects limit flow-induced dilation of conductance coronary vessels after blockade of nitric oxide formation in conscious dogs. AB - OBJECTIVE: To determine whether endothelin (ET)-dependent effects limit shear stress-induced dilation of large epicardial coronary arteries after blockade of nitric oxide (NO) formation. METHODS: In conscious dogs instrumented for measuring coronary blood flow (CBF) and external diameter (CD) of the circumflex coronary artery, flow-dependent CD dilation was elicited by intracoronary (i.c.) adenosine (500 ng kg-1 min-1). RESULTS: I.c. adenosine increased CBF by 28 +/- 4 from 38 +/- 5 ml min-1 and CD by 0.25 +/- 0.03 from 3.53 +/- 0.07 mm without other hemodynamic effects. After N omega-nitro-L-arginine methyl ester (L-NAME), baseline CD fell (P < 0.01) to 3.35 +/- 0.08 mm but CBF was not significantly altered (36 +/- 5 ml min-1). CBF increases caused by adenosine were smaller (17 +/- 2 ml min-1, P < 0.05) and CD responses were nearly abolished (0.02 +/- 0.01 mm, P < 0.01). I.c. Ro 61-1790, an ETA receptor blocker, given after L-NAME did not significantly influence baseline CBF (36 +/- 5 ml min-1) but increased (P < 0.01) CD to 3.45 +/- 0.09 mm. CBF responses to adenosine were not significantly altered by Ro 61-1790 but CD responses (0.10 +/- 0.01 mm) were partially restored (P < 0.01). In contrast, blockade of ETB receptors with Ro 46-8443 after L-NAME had no further effects on CD and CBF responses to adenosine. CONCLUSION: ETA receptor-mediated effects limit flow-dependent dilation of large epicardial coronary arteries in conscious dogs. Suppression of the L-arginine/NO-dependent pathway with L-NAME reveals significant ET-dependent effects. PMID- 10728369 TI - Reduction in the response to coronary and iliac artery injury with photodynamic therapy using 5-aminolaevulinic acid. AB - OBJECTIVE: Photodynamic therapy (PDT) uses red light (non-thermal, non-ionising) to activate a previously administered photosensitizing drug. This inhibits neointimal hyperplasia in injured arteries in small animals where it appears safe and well tolerated. Our aim was to develop a method for percutaneous application of PDT to iliac and coronary arteries in a large animal model and investigate its influence on the remodeling and intimal hyperplastic response to balloon injury. METHODS: Studies were undertaken on 13 juvenile Large White-Landrace crossbred pigs (15-20 kg). After intravenous administration of the photosensitizing agent 5 amino laevulinic acid (ALA), the arterial tree was accessed via the left common carotid artery and balloon injuries made by over-distension in both common iliacs (thirteen animals) and one or two main coronary arteries (eight animals). Half the injured sites were then illuminated with red laser light transmitted via the catheter. Animals were culled 28 days later and tissue harvested for histomorphometry. RESULTS: Compared with control injured vessels, PDT treated, balloon injured coronary arteries had a larger lumen (1.4 vs. 0.8 mm2, P = 0.002), larger area within the external elastic lamina (2.8 vs. 2.2 mm2, P = 0.006) and smaller area of neointimal hyperplasia (0.4 vs. 0.7 mm2, P = 0.06), 28 days after intervention. Less neointimal hyperplasia and the absence of negative remodeling resulted in the lumen of PDT-treated, injured segments being the same as that of adjacent reference segments (1.5 vs. 1.6 mm2). Similar trends, but with smaller differences, were seen in the iliac vessels. CONCLUSIONS: Intra arterial, trans-catheter PDT favourably influences the arterial response to balloon injury in both the coronary and peripheral circulations. This technique offers a promising new approach to restenosis after endovascular procedures. PMID- 10728370 TI - Shear stress induces angiotensin converting enzyme expression in cultured smooth muscle cells: possible involvement of bFGF. AB - OBJECTIVE: Hemodynamic stresses are considered to be important regulators of gene expression in vascular cells. In this study, we have investigated the role of shear stress on ACE expression in cultured rat vascular cells, and focused on the regulation of ACE expression in smooth muscle cells. METHODS: Rat aortic endothelial cells, smooth muscle cells and fibroblasts isolated from Wistar rats were submitted to shear stress using a laminar shear flow parallel chamber. RESULTS: A 10 dynes/cm2 shear rate for 24 h increased ACE activity in the three vascular cell types (x 2.14 in endothelial cells, x 2.9 in smooth muscle cells, x 3.33 in fibroblasts). This induction was blocked by a 24 h pre-incubation with a translation blocker (10(-4)M cycloheximide) showing the role of protein neosynthesis. Therefore the study was focused on smooth muscle cells and we demonstrated that the increase in ACE activity was due to an elevation in ACE mRNA level in response to a 10 dynes/cm2 shear stress for 24 h. This induction was dependent on the shear intensity (P < 0.0001). Six hours of a 15 dynes/cm2 shear stress showed no effect on ACE activity or mRNA expression. In contrast, the same duration of shear significantly increased bFGF mRNA level (x 3.7). Conversely, bFGF dose dependently increased ACE mRNA expression and activity in smooth muscle cells. This result suggests that bFGF could be one of the potential inductors of ACE expression in the stressed smooth muscle cells. CONCLUSIONS: Mechanical stress increases ACE expression in vascular cells. bFGF could be one of the potential factors involved in this activation. This phenomenon could participate in the role of ACE activity in vascular wall remodeling. PMID- 10728371 TI - Galectin 1 is involved in vascular smooth muscle cell proliferation. AB - OBJECTIVE: Smooth muscle cell (SMC) migration and proliferation are the key steps in the development of atherosclerosis and restenosis. Matricellular proteins have been implicated in cell adhesion, migration and proliferation. Here we investigated the role of the matricellular protein galectin-1 (Gal-1), a beta galactoside-binding lectin, in SMC proliferation in atheroma and DNA synthesis in cell culture. METHODS: Protein expression was visualised by tissue section immunostaining. RNA expression was analysed using Northern blot analysis. DNA synthesis of human vascular SMCs was determined by 3H-thymidine incorporation. Recombinant glutathione S-transferase-galectin-1 fusion protein (Gal FP) binding to extracellular matrix (ECM) proteins was measured by ELISA. Gal-1 binding to cells and ECM was estimated using 125I-labelled Gal FP. RESULTS: Prominent Gal-1 staining coincided with SMC proliferation in human coronary endarterectomy samples in organoid culture. In cell culture, Gal-1 mRNA was upregulated in growing SMCs. Gal FP increased serum-induced DNA synthesis of human SMCs on plastic or endogenous ECM, but not of a rat PAC1 SM cell line. Also, Gal FP slightly increased SMC adhesion to ECM. SMCs exhibited a complex pattern of receptor-ligand interactions with Gal FP. The Gal-1 binding to SMCs was much stronger than to ECM, produced by these SMCs. We identified new ECM proteins: thrombospondin, vitronectin and osteopontin, which bound to Gal FP in a dose- and beta-galactoside-dependent manner in ELISA. CONCLUSIONS: Gal-1 binding to SMCs was stronger than to ECM, although ECM of atherosclerotic blood vessels contained additional ECM proteins which bound to Gal-1. Gal-1 was upregulated during SMC growth and Gal FP enhanced serum-induced DNA synthesis in SMCs. Overall, Gal-1 upregulation is likely to provide a reinforcement of serum-induced events during vascular injury. PMID- 10728372 TI - Heterogeneity in the response of vascular smooth muscle to heparin: altered signaling in heparin-resistant cells. AB - OBJECTIVE: Vascular smooth muscle cells show phenotypic heterogeneity in vivo that affects the extent to which they respond to the antimitogenic effects of heparin. In vitro, heparin-resistant cells are readily selected. This study was undertaken to determine whether differences in the antiproliferative response to heparin involve differences in activity of heparin-sensitive signal transduction pathways. METHODS: Rat thoracic aorta smooth muscle cells (ASMC) at early passage together with two established vascular smooth muscle lines, PAC-1 and A10, were examined before and after selection for growth in the presence of heparin (10 micrograms/ml). Cells were rendered quiescent and then stimulated with serum. RESULTS: The three cell types showed different sensitivities to the antimitogenic effects of heparin. With respect to [3H]thymidine incorporation, A10 cells were insensitive to 1 microgram/ml heparin whereas PAC-1 cells responded down to 0.05 microgram/ml and ASMC were of intermediate sensitivity. ASMC and PAC-1 cells but not A10 showed a decrease in c-fos mRNA in response to 1 microgram/ml heparin, and a decrease in the c-Fos content of AP-1 DNA binding activity. None of the cells had decreased c-jun mRNA in the presence of heparin. Although induction of c-fos by serum is thought to signal through the Erk mitogen activated protein kinase family, Erk activity was decreased more by 1 microgram/ml heparin in A10 cells than in PAC-1 or ASMC. When cells were selected by growth in the presence of 10 micrograms/ml heparin, A10 cells were unaffected but PAC-1 and ASMC showed a blunted effect of heparin on serum stimulation. In contrast to A10 and their controls not exposed to continuous heparin, heparin-selected PAC-1 and ASMC showed a diminished ability to induce c-fos in response to serum. CONCLUSIONS: Smooth muscle cell lines show different responses to the antimitogenic effects of heparin that correlate with the heparin sensitivity of c-Fos/c-Jun expression. Although Erk is implicated in c-fos induction, cells comparatively resistant to heparin still show heparin-dependent inhibition of Erk activation, suggesting that other pathways may be more important for heparin resistance. Furthermore, cells selected for heparin resistance may develop c-fos-independent pathways for proliferation. PMID- 10728373 TI - Involvement of protein kinase C in superoxide anion-induced activation of nuclear factor-kappa B in human endothelial cells. AB - OBJECTIVE: Nuclear factor-kappa B (NF-kappa B) plays an important role in the regulation of redox-sensitive genes which are related to the pathogenesis of various vascular diseases. Although oxygen free-radicals are known to activate NF kappa B, the signaling pathway of oxygen free radical-induced NF-kappa B activation remains largely unclear. Thus, this study was performed to examine the possible involvement of protein kinase C (PKC) in the oxygen free radical-induced NF-kappa B activation in human umbilical vein endothelial cells (HUVECs). METHODS: Superoxide anion was generated by xanthine and xanthine oxidase. An electrophoretic mobility shift assay (EMSA) was performed using a kappa B-motif oligonucleotide and nuclear extracts from HUVECs. Immunoblot analysis using an antibody against I kappa B alpha, phosphorylated by I kappa B alpha kinase, or myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylated by protein kinase C was carried out. An NF-kappa B luciferase reporter gene assay was also performed. RESULTS: The treatment of the cells with superoxide anion for 60 min increased the NF-kappa B/DNA binding activity. Immunoblot analysis showed that superoxide anion induced phosphorylation of I kappa B alpha within 10 min. Furthermore, phosphorylation of MARCKS occurred more rapidly than phosphorylation of I kappa B alpha. Pretreatment of the cells with calphostin C (100-400 nmol/l) and chelerythrine chloride (5-10 mumol/l), inhibitors of PKC, abolished the superoxide anion-induced NF-kappa B activation. Down-regulation of endogenous PKC by long-term exposure to phorbol 12-myristate 13-acetate decreased the superoxide anion-induced NF-kappa B activation to a basal level. Superoxide anion induced the luciferase reporter gene and this induction was completely inhibited by calphostin C (200 nmol/l) and 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron). CONCLUSION: These results suggest that PKC is involved in the activation of NF kappa B by superoxide anion in human endothelial cells. PMID- 10728374 TI - Morphologic and biochemical hallmarks of apoptosis. AB - Apoptosis is characterised by a series of typical morphological features, such as shrinkage of the cell, fragmentation into membrane-bound apoptotic bodies and rapid phagocytosis by neighbouring cells. This paper reviews the current knowledge on the molecular mechanisms of apoptosis as they relate to the morphologic hallmarks and their implications for the detection of apoptosis in cardiac tissue. Activation of cysteine proteases called caspases plays a major role in the execution of apoptosis. These proteases selectively cleave vital cellular substrates, which results in apoptotic morphology and internucleosomal fragmentation of DNA by selectively activated DNases. In response to several pro apoptotic signals, mitochondria release caspase activating factors, that initiate an escalating caspase cascade and commit the cell to die. Members of the Bcl-2 oncoprotein family control mitochondrial events and are able to prevent, or induce, both apoptotic and non-apoptotic types of cell death. This suggests that different types of cell death share common mechanisms in the early phases, whereas activation of caspases determines the phenotype of cell death. Detection of apoptotic cells in tissue samples currently relies on the TUNEL assay. TUNEL positive cardiomyocytes show morphological features of apoptosis and the typical ladder pattern in DNA electrophoresis. Thus, provided that the staining protocol is carefully standardised, this quantitative methodology provides reproducible results of the occurrence of cardiomyocyte apoptosis in cardiac samples. Recently, potentially more specific assays based on analysis of DNA fragmentation or demonstration of caspase activation have been developed. Applicability of these assays to demonstrate cardiomyocyte apoptosis should be tested. PMID- 10728375 TI - Metabolic aspects of programmed cell survival and cell death in the heart. AB - Normal cardiac function requires a tight interaction between metabolism, contractile function and gene expression. The main perturbation challenging this equilibrium in vivo is ischemia, which alters energy flux through the control of key enzymes. The review highlights metabolic imprints and energetic aspects of programmed cell survival, programmed cell death, and of necrosis. When sustained and severe, ischemia leads to a total collapse of energy transfer, to the accumulation of metabolic endproducts, and to the development of myocardial necrosis. When moderate, ischemia results in a coordinated cellular response including enhanced anaerobic glucose metabolism, a modification of cardiac gene expression, and the development of specific mechanisms for programmed cell survival (preconditioning, stunning, hibernation). Repetitive stress results in a decrease of contractile function, a downregulation of gene expression and an impairment of energy transfer, which eventually cause the heart to fail. When the failing heart becomes energy-depleted, the programs of cell survival are no longer operational and programmed cell death ensues. To define the point of departure from programmed cell survival to cell death remains a major challenge. PMID- 10728376 TI - Markers of apoptosis in cardiovascular tissues: focus on Annexin V. AB - In the last decade, apoptosis (or programmed cell death) has become appreciated as an important process in the development of the cardiovascular system. Moreover, apoptosis contributes to the adaptation of the system to the environment. We are at the beginning of understanding its relevance to cardiovascular physiology and pathology. This understanding forms the key to implement apoptosis in diagnosis and therapy of cardiovascular diseases. New avenues for pharmacological intervention are expected to arise from the synergy of our knowledge about the molecular mechanisms of apoptosis, and how apoptosis integrates in the complex environment of the cardiovascular tissue. The latter strongly depends on techniques to measure apoptosis. Currently, we are facing a relative paucity in available techniques, covering both specificity and sensitivity, and furthermore allowing quantitative analysis, preferably in combination with morphology. This field, however, is rapidly evolving and is fed by the expanding knowledge about the molecular mechanisms of apoptosis. In this paper we will briefly review the available techniques to detect and/or quantify apoptosis. These methods are based on the analysis of cellular morphology, either by light- or electron microscopy, DNA fragmentation (TdT-mediated X-dUTP nick end labeling or in situ nick end labeling), or cytoplasmic and membrane changes. Furthermore, the advantages and limitations of these techniques for their use in cardiovascular research will be outlined. In the text we will refer to available reviews and protocols which discuss the techniques in more detail. The main part of this article will, however, focus on a recently introduced technique, the Annexin V-based apoptosis detection assay. The principle, characteristics, pro's and contra's of this new apoptosis detection assay will be discussed. PMID- 10728377 TI - Apoptosis in cardiac diseases: stress- and mitogen-activated signaling pathways. AB - Apoptosis is a form of cell death that involves discrete genetic and molecular programs, de novo protein expression and a unique cellular phenotype. Evidence for the existence of apoptosis in the human heart has been reported in various cardiac diseases, including ischemic and non-ischemic heart failure, myocardial infarction and arrhythmias. Among the most potent stimuli that elicit cardiomyocyte apoptosis are: oxygen radicals (including NO), cytokines (FAS/TNF alpha-receptor signaling), stress conditions (chemical or physical, e.g., radiation), sphingolipid metabolites (ceramide) and autocoids, e.g., angiotensin II. Apoptosis of cardiac myocytes may contribute to progressive pump-failure, arrhythmias and cardiac remodeling. The recognition of numerous molecular targets associated with cardiomyocyte apoptosis may provide novel therapeutic strategies for diverse cardiac ailments, as recently suggested by pharmacologic studies in experimental animals. This review paper is aimed to highlight the role of protein kinase signaling pathways in apoptosis with special attention to the stress activated protein kinases (SAPK) and mitogen-activated protein kinases (MAPK) systems. PMID- 10728379 TI - T cells expressing the gamma delta T cell receptor induce apoptosis in cardiac myocytes. AB - OBJECTIVE: Enterovirus infections are major etiological factors in myocarditis and dilated cardiomyopathy. Using an experimental murine model of this disease, previous studies have shown that myocarditis susceptibility depends upon activation of T lymphocytes expressing the gamma delta T cell receptor (TcR), and that only mouse strains which accumulate gamma delta T cells in the myocardium show apoptosis of myocytes or evidence of dilated cardiomyopathy-like disease. The objective of the present studies is to demonstrate that gamma delta T cells directly induce greater Fas-dependent apoptosis of cultured myocytes than T cells expressing the alpha beta TcR. METHODS: Bl.Tg.E alpha mice were infected for 7 days with coxsackievirus B3 (CVB3). Hearts were removed and were either formalin fixed, sectioned and stained with hematoxylin and eosin for inflammation, and using TdT-TUNEL for apoptosis, or were minced and collagenase digested for isolation of gamma delta+ and alpha beta+ T cells using immunomagnetic bead separation. Neonatal cultures of cardiac myocytes were isolated from mice less than 2 days old by collagenase and pancreatin digestion, and were either untreated or infected with virus. Levels of Fas (CD95) were measured using FITC conjugated hamster anti-mouse Fas monoclonal antibody and flow cytometry. Susceptibility of myocytes to Fas-dependent killing was measured by 51Cr-release by labeled myocytes incubated for 4 h on either 3T3-mock or 3T3-FasL transfected cell monolayers. Killing by T cells was also measured in a 4 h 51Cr-release assay. Fas-dependent and perforin-dependent cytotoxicity was determined by specific blocking using either Fas-Fc or concanamycin A. RESULTS: Virally infected myocyte cultures showed significantly enhanced Fas expression compared to uninfected cells, with maximal upregulation of Fas occurring 18-24 h after virus infection. Both infected and uninfected myocytes were selectively killed by FasL-transfected 3T3 cells but not by mock control cells. Approximately 38% of CD3+ lymphocytes isolated from the heart express the gamma delta TcR with the remainder expressing the alpha beta TcR. Both gamma delta+ and alpha beta+ T cells lysed myocyte targets. Blocking studies indicate that gamma delta+ T cells induced predominantly Fas-mediated killing, while alpha beta+ cell produced more perforin-mediated death, although these effectors were capable of Fas-dependent killing as well. CONCLUSIONS: These studies demonstrate that T cells expressing the gamma delta TcR are more effective mediators of myocyte apoptosis than alpha beta+ T cells in vitro and suggests that these effectors may be primarily responsible for myocardial injury associated with dilated cardiomyopathy-like signs during coxsackievirus B3-induced myocarditis. PMID- 10728378 TI - Adenovirus-encoded hammerhead ribozyme to Bcl-2 inhibits neointimal hyperplasia and induces vascular smooth muscle cell apoptosis. AB - OBJECTIVE: The balance between apoptosis and cell proliferation is vital for cellular homeostasis, yet little is known about the mechanisms that coordinate these two cell fates, particularly in the vessel wall. It is well established that the members of Bcl-2-gene family are regulators of apoptosis, but their role in cellular proliferation is less clear. METHODS: We analyzed the effects of disrupting Bcl-2 expression in vascular smooth muscle cells (VSMCs) by adenoviral mediated delivery of a hammerhead ribozyme against bcl-2 mRNA (Ad-Rbz-Bcl-2). RESULTS: Forced ablation of Bcl-2 in balloon-injured rat carotid arteries reduced cell number and inhibited neointimal hyperplasia. In vitro, VSMCs transduced with the Ad-Rbz-Bcl-2 underwent apoptosis as indicated by a reduction in cell number and DNA fragmentation. Ad-Rbz-Bcl-2-transduced cells also exhibited aberrations in both G1- and S-phases of the cell cycle. However, forced perturbations in cell cycle activity by serum-stimulation or treatment with chemical inhibitors did not affect Ad-Rbz-Bcl-2-induced cell death, indicating that these cell cycle changes are not essential for apoptosis. CONCLUSION: These data show that physiological levels of Bcl-2 are essential for VSMC viability and that ablation of Bcl-2 alters cell cycle activity through the execution of the apoptotic process. PMID- 10728380 TI - Apoptosis induction by nitric oxide in adult cardiomyocytes via cGMP-signaling and its impairment after simulated ischemia. AB - OBJECTIVE: Nitric oxide (NO) has been shown to induce apoptosis in cardiomyocytes under normoxic conditions. The ability of NO to induce apoptosis after ischemia reperfusion, a situation of increased NO release in vivo, has not been investigated. The present study was undertaken to characterize the pathway of induction of apoptosis by NO and the influence of ischemia on this pathway in cardiomyocytes. METHODS: The study was performed on isolated adult cardiomyocytes of the rat. Ischemia was simulated by anoxia in a glucose free medium, pH 6.4. Induction of apoptosis was detected (1) by annexinV-fluorescein isothiocyanate (annexinV-FITC) binding to cells under exclusion of propidium iodide and (2) by laddering of genomic DNA. RESULTS: Incubation of cardiomyocytes with the NO-donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 100 microM) induced apoptosis in 14.1 +/- 1.9% of the cells and necrosis in 24.4 +/- 4.6%. The induction of apoptosis but not necrosis could be blocked by inhibition of soluble guanylyl cyclase or of protein kinase G. Apoptosis induction was mimicked by incubation of cardiomyocytes with 8-pCPT-cGMP (100 microM, 9.6 +/- 0.6% apoptotic cells) or YC 1 (75 microM, 14.6 +/- 2.8% apoptotic cells), a direct activator of soluble guanylyl cyclase. After 3 h of anoxia, cardiomyocytes were transiently protected against apoptosis induced by NO, but not by 8-pCPT-cGMP or YC-1 (8.9 +/- 0.7% or 13.4 +/- 2.4% apoptotic cells). A correlation of the apoptotic response to SNAP or YC-1 with an increased activity of soluble guanylyl cyclase, determined by measurements of intracellular cGMP contents, was found. CONCLUSIONS: NO induces apoptosis in a cGMP dependent manner in isolated adult cardiomyocytes whereas induction of necrosis seems cGMP-independent. After simulated in vitro ischemia the activation of soluble guanylyl cyclase by NO is transiently inhibited resulting in a transient anti-apoptotic protection. PMID- 10728381 TI - Tumor necrosis factor-alpha induces apoptosis via inducible nitric oxide synthase in neonatal mouse cardiomyocytes. AB - OBJECTIVE: It has been demonstrated that tumor necrosis factor-alpha (TNF alpha) induces apoptosis in cardiac myocytes. However, its mechanism of action is still not well understood. In the present study, we hypothesized that TNF alpha induces myocardial apoptosis by induction of inducible nitric oxide synthase (iNOS). METHODS: Neonatal cardiac myocytes were isolated from iNOS (-/-) mutant and C57BL6 wild type mice. Cells were cultured for 3 days before treatment with an NO donor or TNF alpha. Following treatment with S-nitroso-N-acetyl-penicillamine (SNAP) or TNF-alpha, cells were tested for apoptosis by terminal deoxynucleotidyl transfer-mediated end labeling (TUNEL) staining and cell death detection ELISA. NO production was measured by nitrite concentration in the culture medium. Cardiomyocyte expression of iNOS and TNF type 1 receptor (TNFR1) mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: SNAP (0.01-100 microM) induced apoptosis of cardiac myocytes in a concentration dependent manner in the wild type mice (n = 5, P < 0.01). TNFR1 mRNA was expressed in neonatal cardiomyocytes from both wild type and iNOS (-/-) mutant mice. TNF alpha induced a concentration-dependent increase in iNOS mRNA expression and nitrite production as well as significant apoptosis of cardiomyocytes in the wild type mice (n = 4, P < 0.01). However, without iNOS expression, the apoptotic effects of TNF-alpha were significantly attenuated in cardiomyocytes from iNOS (-/-) mutant mice (n = 4, P < 0.05). CONCLUSION: TNF alpha induces apoptosis via iNOS expression and NO production in neonatal mouse cardiomyocytes. PMID- 10728382 TI - Programmed cell death in the developing heart. PMID- 10728383 TI - Apoptosis-related genes expressed in cardiovascular development and disease: an EST approach. AB - Apoptosis (programmed cell death) is an important process which, in conjunction with cell proliferation, maintains cell number homeostasis. Although apoptosis has been more extensively investigated in other tissues [1,2], only recently has this process been suspected as a significant contributor to both disease and normal development of the cardiovascular system [3-6]. Grasping a comprehension of the underlying genetic mechanisms of apoptosis is especially crucial considering that cardiac myocytes irreversibly exit the cell cycle and thus fail to proliferate during pathological conditions. Despite great strides in understanding the molecular pathways of apoptosis, there still remain numerous questions to be answered. Identifying key genes that are involved in the regulatory process of apoptosis in the cardiovascular system will serve as a basis for creating more effective therapeutic treatments in cardiovascular disease and provide an understanding of how cardiac development is modulated. This review provides a brief summary of recent data implicating genes that may be involved in apoptosis in the cardiovascular system. It also outlines the continued usefulness of large-scale generation of expressed sequence tags (ESTs) to establish expression profiles from the cardiovascular system and as a means of identifying potentially significant apoptotic regulators previously characterized in other tissues but not as yet in the cardiovascular system. PMID- 10728384 TI - Apoptosis in relevant clinical situations: contribution of apoptosis in myocardial infarction. AB - Myocardial infarction is associated with increased TUNEL-positivity in cardiac resident and infiltrated cells. Apoptosis of proliferated interstitial myofibroblasts and infiltrated inflammatory cells may have a role in terminating tissue repair processes after infarction. Lateral and endocardial border zones of infarction within the risk area have frequent appearance of TUNEL-positive cardiomyocytes. Although the typical ultrastructural morphology of apoptosis has rarely been detected in ischaemic cardiomyocytes, there are many reports in which the TUNEL method was used for assessment of cardiomyocyte apoptosis. It has become evident that TUNEL-positivity reflects a wide range of cellular conditions; viable cells undergoing DNA repair, apoptosis, and necrosis. Therefore, it is controversial whether TUNEL-positive cardiomyocytes in infarcted myocardium are all apoptotic. Methods which will be more specific for identifying apoptosis are required for future study. TUNEL-positivity can be attenuated by anti-apoptotic interventions such as inhibition of caspases, mitochondrial protection, free radical scavenging, and some conventional pharmacotherapies. However, it remains to be determined whether anti-apoptotic interventions result in satisfactory reduction of infarct size. The injurious impact of myocardial ischaemia comes from a mixture of pro-apoptotic and necrosis-promoting signals, and the target of both signals is mitochondria. Through a common pathway they may cause permeability transition. Interventions which act only at the post mitochondrial stage of apoptosis may fail to reduce infarct size, whereas those acting at pre-mitochondrial and mitochondrial stages may reduce infarct size. Progress in investigating the basic mechanisms of apoptosis and recognition of the modes of cardiomyocytes death will contribute to advances in cardioprotective therapy in myocardial infarction. PMID- 10728385 TI - Effect of caspase inhibitors on myocardial infarct size and myocyte DNA fragmentation in the ischemia-reperfused rat heart. AB - OBJECTIVE: Caspase family proteases are recognized as key mediators of apoptosis. However, the role of caspases in the ischemia-reperfused heart remains uncertain. We evaluated the effect of caspase inhibitors on myocardial infarct size and the myocyte DNA fragmentation in the ischemia-reperfused rat hearts. METHODS: Three groups of Sprague-Dawley rats (n = 7, each) were subjected to 30 min of ischemia followed by 6 h of reperfusion. One of the following drugs: (1) YVAD-aldehyde, a caspase-1-like protease inhibitor (3.5 mg/kg; YVAD), (2) DEVD-aldehyde, a caspase 3-like protease inhibitor (3.5 mg/kg, DEVD), (3) vehicle (140 microliters/kg) was administered intravenously 5 min prior to the ischemia in each group. Myocardial infarct size was defined by triphenyltetrazolium chloride (TTC) staining. Immunohistochemical staining by in situ nick end labeling (TUNEL) of cardiomyocytes and DNA electrophoresis were used for detecting DNA fragmentation. Ultrastructural analysis was done by electron microscopy. The caspase activity was measured in the myocardium of both groups. RESULTS: The percentage of TUNEL positive myocyte nuclei (%AP) was quantified by microscopy. A ladder pattern was detected by electrophoresis of DNA from the risk area and TUNEL-positive myocytes were seen in the risk area. The %AP was significantly reduced from 20 +/- 1% to 12 +/- 3% by YVAD and to 10 +/- 3% by DEVD (both P < 0.01). However, caspase inhibitors did not significantly change the infarct size. Electronmicrograph showed similar salcolemmal and mitochondrial damage in both group. The caspase activity was blocked by DEVD at 4 h after reperfusion. CONCLUSION: Myocyte DNA fragmentation and caspase activation was inhibited by caspase inhibitors without reduction of the infarct size in ischemia-reperfused rat hearts. PMID- 10728386 TI - Reperfusion induces myocardial apoptotic cell death. AB - OBJECTIVE: The purpose of the present study was to investigate whether apoptosis is triggered during ischemia (I) and reperfusion (R) and whether I/R-induced apoptosis is correlated with changes in expression of Bcl-2 and Bax. METHODS: Anesthetized open-chest dogs were divided into two groups. Group I: 7 h of permanent I without R (PI, n = 7); Group II: 60 min I followed by 6 h R (I/R, n = 8). Apoptosis was identified as "DNA ladder" by agarose gel electrophoresis or confirmed histologically using the terminal transferase UTP nick end labeling (TUNEL) assay. RESULTS: Collateral myocardial coronary blood flow during I, confirmed by colored microspheres was comparable in both groups. Although PI caused 72 +/- 5% infarct size, very few TUNEL-positive cells were detected in the necrotic area (0.2 +/- 0.1% of total normal nuclei), consistent with an absence of DNA laddering. In contrast, the appearance of TUNEL-positive cells was significantly displayed after 6 h R in the necrotic area in I/R group (26 +/- 4%, P < 0.001 vs. PI group), and DNA ladder occurred in all experimental animals, suggesting that myocardial apoptosis is primarily elicited by R. Densitometrically, Western blot analysis showed significant reduction in expression of Bcl-2 (16 +/- 1%) and increase in Bax (29 +/- 8%) after 6 h R in the necrotic area compared with normal tissue while expression of these two proteins was not changed in the PI group. Polymorphonuclear neutrophil (PMN) accumulation in the necrotic area determined either by immunohistochemistry with anti-CD18 antibody or by myeloperoxidase activity was significantly increased in the I/R group compared to the PI group (358 +/- 24 vs. 24 +/- 2, mm2 myocardium, P < 0.01) and (2.9 +/- 0.3 vs. 0.4 +/- 0.1, U/100 mg tissue, P < 0.01). There was a significant linear relationship between CD18-positive PMNs and TUNEL-positive cells (P < 0.05) in the I/R group. CONCLUSIONS: These results indicate that (1) PI without R did not induce apoptotic cell death, while two types of cell death, necrosis and apoptosis were found after I/R, (2) the Bcl-2 family may participate in early R-induced myocardial apoptosis, (3) PMN accumulation may play a role in the development of apoptosis. PMID- 10728387 TI - Preconditioning decreases Bax expression, PMN accumulation and apoptosis in reperfused rat heart. AB - OBJECTIVE: Recent studies suggest that ischemic preconditioning (IPC) inhibits myocardial apoptosis after ischemia and reperfusion. This study tested the hypothesis that IPC reduces ischemia/reperfusion-induced myocardial apoptosis by inhibiting neutrophil (PMN) accumulation and altering expression of Bcl-2 and Bax proteins. METHODS: Eighteen rats were subjected to 30 min of left coronary artery occlusion followed by 180 min of reperfusion with IPC (5 min ischemia and 10 min of reperfusion, n = 10) or without IPC (n = 8). Myocardial apoptosis was detected histologically using the terminal transferase UTP nick end labeling (TUNEL) assay and confirmed by DNA ladder on agarose gel electrophoresis. PMN accumulation was detected immunohistochemically with anti-rat CD18 antibody (WT3) and expression of Bcl-2 and Bax proteins was analyzed using Western blot assay. RESULTS: IPC significantly decreased TUNEL positive cells (% total nuclei) in the ischemic zone from 28.6 +/- 2.8 to 3.4 +/- 0.9 (P < 0.05), consistent with the absence of DNA ladders in the IPC group. IPC significantly attenuated PMN accumulation (cells/mm2 myocardium) in the ischemic zone from 243 +/- 19 to 118 +/- 19 (P < 0.05). By regression analysis, there was a significant correlation between TUNEL positive cells and accumulated CD18 positive PMNs in the ischemic zone (r = 0.8, P < 0.001), which was shifted downward by IPC. Densitometrically, IPC significantly attenuated the ischemia/reperfusion-upregulated expression of Bax protein in the ischemic zone from 204 +/- 57% in the control group to 76 +/- 7% (P < 0.05), while the expression of Bcl-2 was not different from the non-ischemic zone in either group. CONCLUSION: These data suggest that ischemic preconditioning may reduce myocardial apoptosis by inhibiting PMN accumulation and down-regulating expression of Bax. PMID- 10728389 TI - Antioxidants attenuate myocyte apoptosis in the remote non-infarcted myocardium following large myocardial infarction. AB - OBJECTIVE: Increased oxidative stress and myocyte apoptosis co-exist in the remote non-infarcted myocardium (RM) following a large myocardial infarction. We proposed that these phenomena are causally related. METHODS AND RESULTS: On day 3 after induction of myocardial infarction, Sprague-Dawley rats were randomized to receive probucol and pyrrolidine dithiocarbamate (MI-T), or vehicle only (MI) for 7 weeks. Control rats (C) received vehicle. At 7 weeks, lipidperoxidation within the RM was assessed by measuring thiobarbituric acid reactive substances, which were significantly increased in MI vs. C, while MI-T was not different from C. There was a significant increase in cardiac myocytes positive for in situ TdT-UTP nick-end labeling within the RM in MI vs. C, which was inhibited in MI-T. Furthermore, internucleosomal DNA fragmentation was clearly demonstrated on agarose gels from RM in the MI group, while it was much less apparent on gels from RM in the C and MI-T groups. Western blot analysis showed a significant increase in p53, Bax and caspase-3 protein expression within the RM of MI vs. C, all of which were inhibited in the MI-T group. Furthermore, there was evidence for an increase in caspase-3 activity within the RM from MI vs. C, which was normalized in the MI-T group. CONCLUSIONS: Long-term treatment with the antioxidants probucol and pyrrolidine dithiocarbamate attenuates oxidative stress, myocyte apoptosis, caspase-3 like activity and the expression of p53, bax and caspase-3 within RM in rats after a large myocardial infarction. PMID- 10728388 TI - Inhibition of endogenous nitric oxide synthase potentiates ischemia-reperfusion induced myocardial apoptosis via a caspase-3 dependent pathway. AB - OBJECTIVE: Apoptosis of cardiomyocytes may contribute to ischemia-reperfusion injury. The role of nitric oxide (NO) in apoptosis is controversial. Therefore, we investigated the effect of NO synthase inhibition on apoptosis of cardiomyocytes during ischemia and reperfusion and elucidated the underlying mechanisms. METHODS AND RESULTS: Isolated perfused rat hearts (n = 6/group) were subjected to ischemia (30 min) and reperfusion (30 min) in the presence or absence of the NO synthase inhibitor NG-mono-methyl-L-arginine. Reperfusion induced cardiomyocyte apoptosis as assessed by immunohistochemistry (TUNEL staining) and the demonstration of the typical DNA laddering. Apoptosis during reperfusion was associated with the cleavage of caspase-3, the final down-stream executioner caspase, whereas the protein levels of the anti-apoptotic protein Bcl 2 and the pro-apoptotic protein Bax were unchanged. Inhibition of the NO synthase drastically increased ischemia and reperfusion-induced apoptosis of cardiomyocytes. Moreover, the NO synthase inhibitor enhanced the activation of caspase-3, suggesting that NO interferes with the activation of caspases in ischemia-reperfusion. CONCLUSION: The results of the present study demonstrate that inhibition of endogenous NO synthesis during ischemia and reperfusion leads to an enhanced induction of apoptosis, suggesting that the endogenous NO synthesis protects against apoptotic cell death. Inhibition of NO synthesis thereby activates the caspase cascade, whereas the Bcl-2/Bax protein levels remained unchanged. PMID- 10728390 TI - Tumor necrosis factor in myocardial hypertrophy and ischaemia--an anti-apoptotic perspective. PMID- 10728391 TI - Apoptosis in chronic hibernating myocardium: sleeping to death? AB - Is the 'smart heart' smart enough? Since the introduction of the term 'hibernating myocardium', this has been referred to as the 'smart heart', however more recently several publications have suggested that cell death accompanies the hibernation process, so that revascularisation of patients with hibernating myocardium should be performed without delay. Other data, however, point to cellular dedifferentiation instead of cellular degeneration, which means that cardiac hibernation is an adaptive mechanism capable of preserving the myocardial viability for a prolonged period. In an attempt to find an answer to the above mentioned question, this review summarises and discusses the findings in this field, also giving attention to possible explanations for the discrepant findings. PMID- 10728392 TI - Apoptotic cell death in heart failure. PMID- 10728393 TI - Adrenergic regulation of myocardial apoptosis. AB - Increased sympathetic nerve activity to the myocardium is a central feature in patients with heart failure. Norepinephrine, the primary transmitter of the sympathetic nervous system, signals via binding to alpha- and beta-adrenergic receptors (AR) that are coupled to G-proteins. Pharmacologic studies of cardiac myocytes in vitro demonstrate that beta-AR can stimulate apoptosis. Likewise, in transgenic mice overexpression of beta 1-AR or G alpha s is associated with myocyte apoptosis and the development of dilated cardiomyopathy. Whereas beta 1 AR stimulate apoptosis in vitro and in vivo, beta 2-AR may either stimulate or inhibit apoptosis and myocardial failure depending on the level of expression. Receptors coupling to Gi and Gq may also be able to mediate or modulate apoptosis and the development of myocardial failure, suggesting the potential for interactions between the beta-AR system and numerous remodeling stimuli that act through Gi or Gq signaling pathways. It appears likely that the mitogen-activated protein kinase superfamily plays a key role in mediating the actions of adrenergic pathways on myocyte apoptosis. These observations suggest that the adrenergic nervous system plays an important role in the regulation of myocyte apoptosis, and may thus contribute to the development of myocardial failure. PMID- 10728394 TI - Expression of secreted frizzled related proteins 3 and 4 in human ventricular myocardium correlates with apoptosis related gene expression. AB - OBJECTIVE: Overload-induced heart failure is associated with myocyte apoptosis induced by unknown mechanisms. Wnt genes encode secreted signaling molecules that bind to frizzled receptors and stabilize cytosolic beta-catenin which is translocated into the nucleus, acts as transcriptional activator and imparts an apoptosis resistant phenotype. This signaling pathway is antagonized by secreted frizzled related proteins (sFRPs) which modulate apoptosis susceptibility in cell culture models. On the basis of these considerations, the present investigation compares myocardial mRNA expression of sFRPs and the level of soluble beta catenin in tissue samples from nonfailing and failing hearts. METHODS: Nonischemic transmural samples from human failing left ventricles and from nonfailing donor ventricles were used in the present study. The mRNA concentration of the Wnt-antagonists sFRP 1-4 were determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). The myocardial localization of sFRP 3 and 4 expression was investigated using in situ RT-PCR. The pool of soluble beta-catenin was quantified by Western blot analysis of protein extracts. RESULTS: The mRNA levels of proapoptotic sFRPs 3 and 4 but not of sFRP 1 and 2 were elevated in failing ventricles compared to donor hearts. There was no significant difference between patients suffering from a dilated cardiomyopathy or a coronary heart disease. sFRPs 3 and 4 were expressed in cardiomyocytes and their expression correlated with the mRNA expression of the proapoptotic Fas/Fas-antagonist ratio, but inversely with the mRNA levels of the antiapoptotic bcl-xL. The size of the pool of 0.1% Triton soluble beta-catenin tended to decrease in myocardial samples with high sFRP 3 and 4 expression levels. CONCLUSIONS: The results support the hypothesis that in failing human myocardium the Wnt/beta-catenin pathway is attenuated by enhanced expression of two endogenous Wnt-antagonists. This might contribute to an apoptosis susceptible phenotype of overloaded human myocardium. PMID- 10728395 TI - Increased apoptosis in the heart of genetic hypertension, associated with increased fibroblasts. AB - OBJECTIVE: The present studies were undertaken to identify apoptosis in cardiomyocytes of genetic hypertension and to study the relationship among apoptosis, aging and blood pressure, and the effect of angiotensin-converting enzyme (ACE) inhibitors on apoptosis. METHODS: Apoptosis in the hearts of spontaneously hypertensive rats (SHR) was identified by electron microscopy (EM) and DNA laddering, and quantified from age 3 weeks to 64 weeks in comparison with normotensive rats (WKY). Fibroblasts and protein products of Bcl-2 and Bax were measured by quantitative immunohistochemistry. SHR were treated with ramipril, an ACE inhibitor. RESULTS: The results showed that: (1) ultrastructural characteristics of apoptosis were observed in cardiomyocytes of SHR, with shrinkage of the cell and condensation of the cytoplasm and chromatin. A DNA ladder was shown; (2) a significant increase in apoptosis in SHR began as early as age 4 weeks and reached a plateau at 16 weeks and maintained at high levels up to 64 weeks. Blood pressure (BP) in SHR started to increase significantly at age 5 weeks; (3) fibroblasts were significantly increased in the heart of SHR; (4) the ratio of Bcl-2/Bax was significantly reduced in SHR; and (6) ramipril effectively reduced apoptosis and fibroblasts, and increased the ratio of Bcl 2/Bax. CONCLUSION: Apoptosis occurs in the cardiomyocytes of genetic hypertension although fibroblasts are increased, and a significant, age-dependent increase in apoptosis is observed. The increase in apoptosis occurs before the difference in blood pressure is detectable. The ACE inhibitor ramipril may be useful for prevention of apoptosis in the heart. PMID- 10728396 TI - Apoptosis in atherosclerosis: beneficial or detrimental? AB - Several groups have demonstrated apoptotic cell death in atherosclerotic plaques. The significance of apoptosis in atherosclerosis depends on the stage of the plaque, localization and the cell types involved. Both macrophages and smooth muscle cells undergo apoptosis in atherosclerotic plaques. Apoptosis of macrophages is mainly present in regions showing signs of DNA synthesis/repair. Smooth muscle cell apoptosis is mainly present in less cellular regions and is not associated with DNA synthesis/repair. Even in early stages of atherosclerosis smooth muscle cells become susceptible to undergoing apoptosis since they increase different pro-apoptotic factors. Moreover, recent data indicate that smooth muscle cells may be killed by activated macrophages. The loss of the smooth muscle cells can be detrimental for plaque stability since most of the interstitial collagen fibers, which are important for the tensile strength of the fibrous cap, are produced by SMC. Apoptosis of macrophages could be beneficial for plaque stability if apoptotic bodies are removed. Apoptotic cells that are not scavenged in the plaque activate thrombin which could further induce intraplaque thrombosis. It can be concluded that apoptosis in the primary atherosclerosis is detrimental since it could lead to plaque rupture and thrombosis. Recent data of our group indicate that apoptosis decreases after lipid lowering which could be important in our understanding of the cell biology of plaque stabilization. PMID- 10728397 TI - The regulation of vascular smooth muscle cell apoptosis. AB - Apoptosis describes the morphological changes that identify a specific form of regulated cell death. Over recent years, the importance of either aberrant onset or suppression of apoptosis within tissues has become apparent and is associated with the development of several terminal diseases. Here we describe the relevance of apoptosis to the maintenance of vascular homeostasis. Specifically, we address the role of vascular smooth muscle cell death, how this may be regulated at the molecular level and whether any of these molecular mediators will provide targets for intervention in diseases such as atherosclerosis. PMID- 10728398 TI - Apoptosis in inflammatory-fibroproliferative disorders of the vessel wall. AB - Apoptotic cell death is a hallmark of inflammatory-fibroproliferative disorders of the vessel wall. Here, we review what is currently known about cell death within atherosclerotic and restenotic lesions. We also examine evidence suggesting that inflammatory cells contribute to the regulation of cell turnover within these lesions, and discuss the molecules expressed by vascular cells that modulate these processes. In toto, these studies suggest that apoptosis is prevalent in vascular lesions, controlling the viability of both inflammatory and vascular cells, and thus determining the cellular composition of the vessel wall. PMID- 10728399 TI - Incidence of intimal proliferation and apoptosis following balloon angioplasty in an atherosclerotic rabbit model. AB - OBJECTIVE: The aim of this study was to determine the occurrence of apoptosis in relation to the proliferative response in the intimal layer after experimental balloon angioplasty of a pre-existing plaque. METHODS: After induction of an intimal plaque in the right carotid artery by electrical stimulation, 26 rabbits underwent balloon angioplasty. Twelve animals served as a control group without performance of angioplasty after plaque induction. To study the time course of intimal apoptosis and cell proliferation the vessels were excised on day 7, 14 and 28 after balloon angioplasty. For in situ detection of apoptosis, the TUNEL technique (TdT-mediated d-UTP fluorescein nick end labeling) was used. In addition, bromodeoxyuridine labeling in all animals allowed the determination of the percentage of cells undergoing DNA synthesis in the neointimal area. Additionally, smooth muscle cells were detected by immunostaining of alpha-actin and macrophages by a specific antibody (RAM 11). RESULTS: Within 28 days of balloon angioplasty, the number of cells undergoing apoptosis remained at a very low level and was not significantly different to the control group without interventional treatment (controls: 0.1 +/- 0.15%; 7 days: 0.44 +/- 0.68%; 14 days: 0.13 +/- 0.11%; 28 days: 0.1 +/- 0.1%). In contrast, the number of cells undergoing DNA synthesis was significantly increased at day 7 after angioplasty (3.72 +/- 2.0% vs. 0.51 +/- 0.29% in controls), resulting in an increase of the total intimal area from 0.088 +/- 0.037 mm2 in the control animals up to 0.256 +/ 0.172 mm2 at day 28 following balloon dilatation. CONCLUSIONS: Our data showed that significant changes in the occurrence of apoptosis are not involved in the regulation of cellular turnover during the examined time period after vessel wall injury. The lacking up-regulation of apoptosis in comparison to the increased cell proliferation in order to maintain the tissue balance is perhaps an important regulatory mechanism leading to intimal hyperplasia after vascular injury in this animal model. Overall, we suggest that there may be a delicate balance between cell proliferation and apoptosis in smooth muscle cells of the vessel wall, and only small shifts in this balance could account for both cellular accumulation in restenotic lesions as well as cell death in mature atheroma. PMID- 10728400 TI - ACE-inhibition promotes apoptosis after balloon injury of rat carotid arteries. AB - OBJECTIVE: Angiotensin II stimulates vascular smooth muscle cell (VSMC) growth, and is considered to be an important mediator of intimal thickening after vascular injury. Recent evidence has indicated that VSMC apoptosis plays a major role in the response to balloon injury, and we therefore examined the effect of angiotensin converting enzyme (ACE)-inhibition on VSMC apoptosis and vascular lesion formation in the rat model of balloon injury. METHODS: Male Sprague-Dawley rats were subjected to carotid artery balloon injury and randomised to a standard diet or a diet supplemented with 1 mg/ml captopril in the drinking water. Animals were sacrificed 2 and 14 days after injury for assessment of apoptosis and proliferation by in situ terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labelling (TUNEL) and proliferating cell nuclear antigen (PCNA) immunohistochemistry, respectively. At 14 days post injury, vessel cross-sections were subjected to microscopic morphometry and total cell numbers were determined. RESULTS: At 2 days after balloon injury, captopril-treated animals displayed a significant increase in the percentage of TUNEL-positive VSMCs in the medial area (12 +/- 4% vs. 1 +/- 1%; P < 0.05) as compared to controls. This increase in early apoptosis was associated with decreased intimal cellularity 14 days post injury (238 +/- 47 cells/cross-section vs. 449 +/- 75 cells/cross-section; P < 0.05), and a reduction of neointimal formation (0.13 +/- 0.02 mm2 vs. 0.23 +/- 0.04 mm2; P < 0.05). The fraction of PCNA-positive VSMCs per cross-section 2 or 14 days after injury was not significantly altered by captopril administration. CONCLUSION: Captopril inhibits neointimal formation in the rat model of arterial injury by mechanisms involving induction of VSMC apoptosis. PMID- 10728401 TI - Antisense Bcl-x oligonucleotide induces apoptosis and prevents arterial neointimal formation in murine cardiac allografts. AB - OBJECTIVE: Cardiac allograft arteriosclerosis, which limits long-term survival of recipients, cannot be prevented by conservative therapies. The arteriopathy is characterized by diffuse intimal thickening comprised of proliferative smooth muscle cells (SMCs). Cell death is a prominent feature of atherosclerosis; Bcl-x is one of the anti-apoptotic mediators. METHODS: To test the hypothesis that antisense bcl-x oligodeoxynucleotide (ODN) is effective in preventing intimal hyperplasia through enhancing apoptosis after cardiac transplantation, we performed single intraluminal delivery of antisense bcl-x ODN into murine cardiac allografts (n = 9). DBA/2 (H-2d) hearts were transplanted into B10.D2 (H-2d) mice. Sense bcl-x ODN (n = 8) and no treatment (n = 8) studies were also performed. RESULTS: Allografts were harvested at 4 weeks after transplantation; all allografts kept beating throughout the period. Coronary intimal thickening had developed in nontreated and sense ODN transfected allografts at 4 weeks after transplantation with enhanced expression of Bcl-x and cell adhesion molecules, and suppressed apoptosis. However, antisense bcl-x ODN prevented neointimal formation through enhanced apoptosis. CONCLUSION: These results indicate that apoptosis of vascular SMCs induced by Bcl-x is associated with initial hyperplasia after heart transplantation. Antisense bcl-x ODN inhibits SMC proliferation by inducing apoptosis in graft coronary arteries. PMID- 10728402 TI - Carvedilol prevents epinephrine-induced apoptosis in human coronary artery endothelial cells: modulation of Fas/Fas ligand and caspase-3 pathway. AB - BACKGROUND: Several studies have shown that carvedilol, a multiple action neurohumoral antagonist, reduces mortality in patients with congestive heart failure (CHF). In addition to being a beta-adrenoceptor antagonist, carvedilol is a potent antioxidant. Since there is evidence for elevation of catecholamine levels in plasma and coronary artery endothelial cell injury in CHF, the present study was designed to test the hypothesis that carvedilol inhibits epinephrine induced apoptosis, and the inhibitory effect is mediated by modulation of Fas, Fas ligand (FasL) and caspase-3 pathway, in cultured human coronary artery endothelial cells (HCAECs). METHODS AND RESULTS: HCAECs were exposed to epinephrine alone, carvedilol + epinephrine, or atenolol + epinephrine for 24 h. Epinephrine increased the number of apoptotic cells, measured by in situ nick end labeling staining (from 4.2 +/- 1.3% to 28.6 +/- 6.0%, P < 0.01, n = 6) and by DNA laddering on agarose gel electrophoresis. Epinephrine also increased Fas and FasL protein expression (P < 0.01 vs. control, n = 6), and activated intracellular protease caspase-3 (P < 0.01 vs. control, n = 6). These effects of epinephrine were completely inhibited by carvedilol. Atenolol in equimolar concentration also attenuated epinephrine-mediated effects, but the effects of atenolol were less marked than those of carvedilol (P < 0.01). To explore the basis of differential effects of carvedilol and atenolol, effects of these agents on epinephrine-induced lipid peroxidation was measured. Lipid peroxidation was completely blocked by carvedilol, whereas equimolar concentration of atenolol had much less (P < 0.05) effect. CONCLUSION: Epinephrine induces apoptosis in HCAECs, and this effect is associated with activation of Fas-FasL and caspase-3 signal transduction pathway. Carvedilol can, more effectively than atenolol, inhibit these effects of epinephrine. The potent antioxidant effect of carvedilol is probably responsible for the superior effect. PMID- 10728403 TI - Future perspectives and potential implications of cardiac myocyte apoptosis. AB - Recent advances in the understanding of the molecular mechanisms of apoptosis has gained increasing interest in the cardiovascular research community. Apoptotic myocyte loss has been detected in different cardiac disease states such as ischemic heart disease and congestive heart failure. In addition, some evidence for the molecular mechanisms in cardiac myocyte apoptosis has been evolving, although at present the implications thereof for clinical cardiac disease are not known in most of the cases. Based on these new insights, it is the intention of this article to highlight some topics in apoptosis research that might be of particular interest to define the future role and potentials of new therapeutic approaches aimed at preventing myocyte apoptosis. PMID- 10728404 TI - Mitochondrial dysfunction in heart failure: potential for therapeutic interventions? PMID- 10728405 TI - Closing the gap in understanding the regulation of intercellular communication. PMID- 10728406 TI - Ischemia-reperfusion associated myocardial contractile dysfunction may depend on Ca(2+)-activated cytoskeleton protein degradation. PMID- 10728407 TI - Diastolic heart failure. AB - Primary diastolic failure is typically seen in patients with hypertensive or valvular heart disease as well as in hypertrophic or restrictive cardiomyopathy but can also occur in a variety of clinical disorders, especially tachycardia and ischemia. Diastolic dysfunction has a particularly high prevalence in elderly patients and is generally associated, with low mortality but high morbidity. The pathophysiology of diastolic dysfunction includes delayed relaxation, impaired LV filling and/or increased stiffness. These conditions result typically in an upward displacement of the diastolic pressure-volume relationship with increased end-diastolic, left atrial and pulmo-capillary wedge pressure leading to symptoms of pulmonary congestion. Diagnosis of diastolic heart failure requires three conditions: (1) presence of signs or symptoms of heart failure; (2) presence of normal or slightly reduced LV ejection fraction (EF > 50%) and (3) presence of increased diastolic filling pressure. Assessment of diastolic function can be performed with several non-invasive (2D- and Doppler-echocardiography, color Doppler M-mode, Doppler tissue imaging, MR-myocardial tagging, radionuclide ventriculography) and invasive techniques (micromanometry, angiography, conductance method). Doppler-echocardiography is the most useful tool to routinely measure diastolic function. Different techniques can be used alone or in combination to assess LV diastolic function, but most of them are dependent on heart rate, pre- and afterload. The transmitral flow pattern remains the starting point, since it is easy to acquire and rapidly categorizes patients into normal (E > A), delayed relaxation (E < A), and restrictive (E >> A) filling patterns. Invasive assessment of diastolic function allows determination of the time constant of relaxation from the exponential pressure decay during isovolumic relaxation, and the evaluation of the passive elastic properties from the slope of the diastolic pressure-volume (= constant of chamber stiffness) and stress strain relationship (= constant of myocardial stiffness). The prognosis of diastolic heart failure is usually better than for systolic dysfunction. Diastolic heart failure is associated with a lower annual mortality rate of approximately 8% as compared to annual mortality of 19% in heart failure with systolic dysfunction, however, morbidity rate can be substantial. Thus, diastolic heart failure is an important clinical disorder mainly seen in the elderly patients with hypertensive heart disease. Early recognition and appropriate therapy of diastolic dysfunction is advisable to prevent further progression to diastolic heart failure and death. There is no specific therapy to improve LV diastolic function directly. Medical therapy of diastolic dysfunction is often empirical and lacks clear-cut pathophysiologic concepts. Nevertheless, there is growing evidence that calcium channel blockers, beta-blockers, ACE-inhibitors and AT2-blockers as well as nitric oxide donors can be beneficial. Treatment of the underlying disease is currently the most important therapeutic approach. PMID- 10728408 TI - Signalling via stress-activated mitogen-activated protein kinases in the cardiovascular system. AB - A number of physiological, pharmacological and pathological stimuli initiate cardiac hypertrophy. The intracellular signalling events activated by these stimuli are equally complex. Our ability to treat the hypertrophic and failing myocardium effectively will require clarification of which signalling events regulate growth, remodelling and failure. Much recent attention has focused on the regulation of the mitogen-activated protein kinase cascades (MAPKs), with the importance of these cascades in the development of cardiovascular diseases being extensively explored. These signalling pathways may provide one link from the diverse stress and pharmacological extracellular stimuli to the regulation of gene expression, contractile protein regulation and protein function. This review focuses on the recent progress made in the understanding of the regulation and function of MAPKs in the cardiovascular system, with particular emphasis being placed on the events in the cardiac ventricular myocyte. PMID- 10728409 TI - Arterial remodeling in atherosclerosis, restenosis and after alteration of blood flow: potential mechanisms and clinical implications. AB - Arterial remodeling is currently being recognized as an important determinant in vascular pathology in which narrowing of the lumen is the predominant feature. Not only expansive remodeling (enlargement), but also constrictive remodeling (shrinkage) is observed in de novo atherosclerosis, in restenosis and in transplant vasculopathy. Expansive remodeling prevents and constrictive remodeling enhances luminal narrowing by plaque formation or intimal hyperplasia. The mechanisms of the opposite remodeling modes is unknown. Insight into the processes that determine the direction of local arterial remodeling may help to develop new strategies to prevent arterial occlusive disease. In the present paper the current status of research in the field of arterial remodeling in cardiovascular disease is reviewed. Mechanisms of arterial remodeling, potential interventions to influence the mode of remodeling as well as the methodological limitations of remodeling studies are discussed. PMID- 10728410 TI - Peri-operative myocardial tissue injury and the release of inflammatory mediators in coronary artery bypass graft patients. AB - OBJECTIVE: This study was conducted to evaluate to what extent the ischemia reperfusion injury resulting from the cardiopulmonary bypass (CPB) and aortic cross-clamping procedures during coronary artery bypass grafting (CABG) contributes to the systemic inflammatory response generally found in these patients. METHODS: Serum levels of enzymes (CK and CK-MB) and non-enzymatic proteins (FABP and myoglobin) as markers of myocardial tissue injury, bactericidal permeability increasing protein (BPI) as an indicator of neutrophil activation, interleukin-6 (IL-6) as inducer of the acute phase response and lipopolysaccharide binding protein (LBP) as parameter of the acute phase response were measured in 15 low-risk CABG patients with cardiopulmonary bypass (CPB), and 17 low-risk CABG patients without CPB. RESULTS: Already 0.5 h after reperfusion significantly increased plasma levels of all markers of myocardial tissue injury were noted in patients having surgery with CPB, but not in non-CPB patients. No significant differences were found between both groups for BPI and IL-6 levels in the early reperfusion period. BPI and IL-6 levels were higher in the non-CPB group on the first post-operative day (P < 0.05). However, no correlations were found for any marker of peri-operative tissue damage with either early neutrophil activation, or acute phase reactants. CONCLUSIONS: Perioperative myocardial injury resulting from CPB and aortic cross-clamping in low-risk CABG patients does not contribute to the release of inflammatory mediators in these patients. PMID- 10728412 TI - Gene expression of Na+/Ca2+ exchanger during development in human heart. AB - OBJECTIVE: In immature animal hearts, lower activity of sarcoplasmic reticulum and lower densities of Ca2+ channels highlight the potentially vital role of the Na+/Ca2+ exchanger (NCX) to excitation-contraction coupling. To date, studies on NCX expression have been restricted to late developmental stages. The distribution and gene expression of NCX during early ontogeny is not known, especially in humans. In the present report, we systematically characterized changes in NCX gene expression in human heart during development, with particular emphasis in early ontogeny. METHODS: Human hearts during early gestation (9- to 20-week gestation), neonatal (1 to 2 days after birth) and adulthood (18-40 years old) were used. NCX mRNA levels were studied using RNase Protection Assay (RPA) and NCX protein levels were assessed by Western blot. Wet weight was also used as the tissue base. Immunolocalization studies using confocal microscopy were performed in isolated fetal cardiac myocytes. RESULTS: Normalization of NCX mRNA derived from ventricles against an early gestational age (10-week gestation) shows that NCX mRNA levels nominally increased from 1 to 1.13 at 19-week gestation then decreased to 0.74 (P < 0.05) at neonate and further decreased to 0.23 (P < 0.05) at adult stages. NCX protein levels increased from 1 at 9-week gestation to 3 (P < 0.05) at 20-week gestation and then decreased to 1.8 (P < 0.05) at neonate and to 1.87 (P < 0.05) at adult stages. Confocal imaging of fetal cardiac myocytes revealed intense homogeneous membrane staining and abundance of NCX protein at this stage. CONCLUSIONS: The data demonstrate changes in NCX transcript and NCX protein levels as well as total RNA and proteins during human heart development. Per wet weight, NCX mRNA was 4.5 times greater at early fetal than adult stages and NCX protein was 2 times greater at adult than the early fetal stage indicating considerable post-transcriptional regulation. These findings provide new insights into the understanding of temporal changes in NCX in the developing heart at the gene level. The functional significance remains to be determined. PMID- 10728411 TI - Mitochondrial function in heart muscle from patients with idiopathic dilated cardiomyopathy. AB - OBJECTIVE: To study the mitochondrial respiratory chain enzyme activities in patients with idiopathic dilated cardiomyopathy (IDC). METHODS: Mitochondrial respiratory chain enzyme activities were assessed spectrophotometrically in left ventricular tissue of 17 patients with IDC undergoing cardiac transplantation, as well as in two groups of controls: a group of six patients suffering from ischemic dilated cardiomyopathy (IC) also undergoing cardiac transplantation, and a group of 17 organ donors considered normal from a cardiac point of view. Cytochrome b gene from three IDC patients whose complex III activity was particularly low and from three controls was also sequenced. RESULTS: We found that complex III enzymatic activity was lower not only in IDC but also in IC patients when compared with normal controls. When analysing cytochrome b gene we only found neutral polymorphisms previously described. CONCLUSIONS: In view of such results, we believe that the decrease of respiratory chain complex III activity found in some cases of IDC is a secondary phenomenon, and not due to a primary mitochondrial disease. PMID- 10728413 TI - Accumulation of oxidized LDL in human semilunar valves correlates with coronary atherosclerosis. AB - OBJECTIVE: Recent data indicate that oxidized low-density lipoprotein (ox-LDL) has several proatherogenic effects, e.g. induction of macrophage chemoattractants, adhesion molecules, cytokines, type-1 plasminogen activator inhibitor and platelet-derived growth factor A-chain by smooth muscle cells. Therefore, ox-LDL has been utilized as a marker of oxidative modification of proteins in atherosclerosis. Because heart valves consist of smooth muscle cells, fibroblasts and endothelial cells, and because valvular disease and coronary atherosclerosis could result from similar biological processes, we investigated ox-LDL accumulation in isolated aortic and pulmonary valves and coronary arteries from patients with angiographically proven coronary heart disease (CHD, n = 19), patients with idiopathic congestive heart failure (IDCM = idiopathic dilated cardiomyopathy, n = 20), and transplant donors. METHODS: Masson-Goldner staining and immunohistochemistry utilizing anti ox-LDL and CD68 were performed on paraffin sections of freshly isolated semilunar valves. Data were analyzed by digital image planimetry and by visual scoring of staining intensity. RESULTS: Ox LDL immunoreactivity was identified in the vascular aspect of the attachment line, in the deep valve stroma, and in the ventricular and vascular endothelium of the semilunar valves, colocalizing with macrophages. Valvular ox-LDL area was significantly increased in CHD-patients (P < 0.03) and IDCM-patients (P < 0.04) compared with controls. More ox-LDL was accumulating in the pulmonary valves than in the aortic valves (P = 0.04) as assessed by area and staining intensity. Valvular ox-LDL area in pulmonary valve and aortic valve was significantly correlated with ox-LDL accumulation in the intimal layer (P < 0.001) and medial layer (P < 0.001) of coronary arteries from the same patients. CONCLUSION: The data suggest that the biological process leading to ox-LDL accumulation in coronary atherosclerosis also involves heart valves. Therefore, accumulation of the oxidative stress marker ox-LDL in heart valves illustrates atherosclerosis as an additional mechanisms accelerating valvular degeneration in these patients. PMID- 10728414 TI - Alterations in Ca2+ cycling proteins and G alpha q signaling after left ventricular assist device support in failing human hearts. AB - OBJECTIVE: Left ventricular assist device support mechanically unloads the failing ventricle with resultant improvement in cardiac geometry and function in patients with end-stage heart failure. Activation of the G alpha q signaling pathway, including protein kinase C, appears to be involved in the progression of heart failure. Similarly down-regulation of Ca2+ cycling proteins may contribute to contractile depression in this clinical syndrome. Thus we examined whether protein kinase C activation and decreased Ca2+ cycling protein levels could be reversed by left ventricular assist device support. METHODS: Left ventricular myocardial specimens were obtained from seven patients during placement of left ventricular assist device and heart transplantation. We examined changes in protein levels of G alpha q, phospholipase C beta 1, regulators of G protein signaling (RGS), sarcoplasmic reticulum Ca2+ ATPase, phospholamban and translocation of protein kinase C isoforms (alpha, beta 1, and beta 2). RESULTS: The paired pre- and post-left ventricular assist device samples revealed that RGS2, a selective inhibitor of G alpha q, was decreased (P < 0.01), while the status of G alpha q, phospholipase C beta 1, RGS3 and RGS4 were unchanged after left ventricular assist device implantation. Translocation of protein kinase C isoforms remained unchanged. Left ventricular assist device support increased sarcoplasmic reticulum Ca2+ ATPase protein level (P < 0.01), while phospholamban abundance was unchanged. CONCLUSIONS: We conclude that altered protein expression and stoichiometry of the major cardiomyocyte Ca2+ cycling proteins rather than reduced phospholipase C beta 1 activation may contribute to improved mechanical function produced by left ventricular assist device support in human heart failure. PMID- 10728415 TI - Early detection of cardiovascular autonomic neuropathy in diabetic pigs using blood pressure and heart rate variability. AB - Cardiac autonomic neuropathy is a common complication in insulin dependent diabetes mellitus. Nevertheless, little is known about when this impairment occurs during the time course of the disease. Analysis of blood pressure (BP) and heart rate (HR) variability could be used to detect early signs of autonomic alteration. To test this proposal, twelve sexually mature male Yucatan miniature pigs were equipped with an arterial catheter for telemetric BP analysis, and with a venous access. BP and HR were recorded together with respiratory movements while the animals were resting in a sling. After the first recording session performed when the pigs were 5 months old, streptozotocin (STZ) was used to induce diabetes in seven pigs, while the five others were controls. BP and HR were measured 3 and 6 months after the onset of diabetes and at a similar age in the controls. BP and HR oscillated at the respiratory range (0.19 Hz). Spectral analysis showed this respiratory component was the main determinant of the short term variability of BP and HR. Atropine increased HR and BP and markedly diminished the respiratory sinus arrhythmia. Propranolol diminished HR and the respiratory peak of HR. A reduced respiratory oscillation of BP paralleled the diminution of the respiratory peak of HR. Baroreceptor-HR reflex was estimated using injections of phenylephrine and nitroprusside, and by cross-spectral analysis between BP and HR. Atropine shifted the curve to higher HR values, while propranolol reduced the level of the upper plateau. Atropine decreased both the coherence and gain of the cross-spectral analysis. STZ injection resulted in a type 1 diabetes. At 3 months, diabetic pigs exhibited low levels of BP and a reduced overall variability of HR and BP. Spectral analysis indicated the respiratory sinus arrhythmia was markedly reduced. In addition, the sensitivity of the baroreceptor-HR reflex was reduced. At a latter stage of diabetes these alterations were marked and the level of the resting HR was increased. These data demonstrate the dual (vagal and sympathetic) control of HR in pigs and the dominant role of respiration in the genesis of HR and BP fluctuations. The spectral and cross-spectral analysis of BP and HR were altered after 3 months of diabetes and could be proposed as early detectors of cardiac autonomic neuropathy. PMID- 10728416 TI - The bradycardic agent zatebradine enhances baroreflex sensitivity and heart rate variability in rats early after myocardial infarction. AB - OBJECTIVE: The bradycardic agent zatebradine (UL-FS 49) reduces heart rate without negative inotropic or proarrhythmic effects. The aim was to experimentally characterize the influence of zatebradine on arterial baroreflex sensitivity (BRS) and heart rate variability (HRV) which are generally considered as estimates of vagal activity and have prognostic value in patients after myocardial infarction (MI). METHODS: Conscious rats were studied 3 days after left coronary artery ligation or sham-operation (SH). BRS was determined by linear regression analysis of RR-interval and mean arterial pressure changes evoked by intravenous (i.v.) injections of methoxamine and nitroprusside. HRV at rest was calculated from high-resolution electrocardiogram-recordings. RESULTS: In MI-rats heart rate was similar to SH-rats, mean arterial pressure was lower and both BRS and HRV were markedly reduced. Zatebradine (0.5 mg/kg i.v.) reduced heart rate in MI-rats from 400 +/- 15 to 350 +/- 19 and in SH-rats from 390 +/- 19 to 324 +/- 6 beats/min without changing mean arterial pressure. Both BRS and HRV were restored in MI- and further increased in SH-rats by the drug. Effects of 0.05, 0.5 and 5 mg/kg zatebradine revealed a dose-dependency of heart rate reduction. The lowest dose enhanced reflex bradycardia despite little effect on heart rate and lack of effect on both reflex tachycardia and HRV. CONCLUSIONS: Both BRS and HRV are reduced in rats early after MI, indicating a depressed reflex and tonic vagal activity. Treatment with zatebradine enhances both BRS and HRV. These data suggest that the drug has both peripheral and central effects, leading to an increase of vagal control of heart rate. PMID- 10728417 TI - Gingerol, isoproterenol and ouabain normalize impaired post-rest behavior but not force-frequency relation in failing human myocardium. AB - OBJECTIVE: Rest- and stimulation frequency-dependent potentiation of contractile force is blunted in failing human myocardium. These alterations have been related to reduced sarcoplasmic reticulum (SR) Ca(2+)-reuptake and enhanced transsarcolemmal Ca(2+)-elimination by Na+/Ca(2+)-exchange. We investigated whether inotropic interventions that enhance SR Ca(2+)-uptake, or reduce Ca(2+) elimination by Na+/Ca(2+)-exchange, normalize impaired post-rest and force frequency behavior in left ventricular muscle strips from failing human hearts. METHODS: We tested the influence of [10]-gingerol which activates SR Ca(2+) ATPase (10 mumol/l; n = 13), and isoproterenol which activates cAMP-dependent pathways (0.01, 0.1, 1 mumol/l; n = 40) on post-rest and force-frequency behavior. Ouabain which blocks Na+/K(+)-ATPase (0.03 mumol/l; n = 16) was used to test the effects of inhibiting Ca(2+)-elimination by Na+/Ca(2+)-exchange. For comparison, the effects of blocking SR Ca(2+)-uptake by thapsigargin (10 mumol/l; n = 14) were tested. In addition, Ca(2+)-uptake in myocardial homogenates was measured for gingerol (10 mumol/l; n = 6). RESULTS: Gingerol, isoproterenol (0.1, 1 mumol/l) and ouabain exerted significant positive inotropic effects under basal experimental conditions and normalized post-rest behavior. In contrast, force frequency relation was only slightly improved by gingerol and isoproterenol (0.01 mumol/l). Ouabain and isoproterenol (1 mumol/l) further deteriorated force frequency relation due to frequency-dependent significant increases in diastolic tension. Thapsigargin exerted negative inotropic effects and significantly deteriorated post-rest and force-frequency behavior. In addition, gingerol increased SR Ca(2+)-uptake significantly in myocardial homogenates. CONCLUSIONS: Inotropic interventions that stimulate SR Ca(2+)-ATPase or inhibit Na+/Ca(2+) exchange normalize impaired post-rest behavior. Force-frequency behavior is only slightly improved by stimulation of SR Ca(2+)-ATPase but not by inhibition of Na+/Ca(2+)-exchange. This dissociation between post-rest and force-frequency behavior results from diastolic dysfunction at high stimulation rates. PMID- 10728418 TI - Biphasic redistribution of muscarinic receptor and the altered receptor phosphorylation and gene transcription are underlying mechanisms in the rat heart during sepsis. AB - OBJECTIVE: The purpose of this study was to investigate intracellular redistribution of muscarinic cholinergic receptor (m2AChR) and the roles of receptor phosphorylation and gene transcription as underlying mechanisms in the rat heart during different phases of sepsis. METHODS: Sepsis was induced by cecal ligation and puncture (CLP). The density of m2AChR in the sarcolemmal and light vesicle fractions was studied using [3H]-quinuclidinyl benzilate ([3H]-QNB). Phosphorylation of m2AChR was studied by labeling of the myocardial ATP pool by perfusing isolated hearts with [32P]H3PO4 followed by identification of the phosphorylated m2AChR with SDS-PAGE. The steady-state level of m2AChR mRNA was determined by RT-PCR and Southern blot analysis. RESULTS: Septic rat hearts exhibit an initial hypercardiodynamic (9 h after CLP, early sepsis) and a subsequent hypocardiodynamic (18 h after CLP, late sepsis) state. During early sepsis, the Bmax for [3H]-QNB binding was increased in sarcolemma (+69%) but decreased in light vesicles (-22%), whereas during late sepsis, the Bmax was decreased in sarcolemma (-20%) but increased in light vesicles (+32%). The sum of Bmax for sarcolemmal and light vesicle fractions was increased during early sepsis (+43%) but decreased during late sepsis (-14%). The phosphorylation of m2AChR was decreased during early sepsis (-73%) but increased during late sepsis (+36% to +90%). The m2AChR mRNA abundance was increased during early sepsis (+52%) but decreased during late sepsis (-28%). CONCLUSIONS: The m2AChR in the rat heart was externalized from light vesicles to sarcolemma (overexpression) during early sepsis but internalized from surface membranes to intracellular sites (underexpression) during late sepsis. Furthermore, changes in the receptor phosphorylation and gene transcription are responsible for the biphasic redistribution and the altered expression of m2AChR in the rat heart during the progression of sepsis. PMID- 10728419 TI - Evidence for mitochondrial K ATP channels as effectors of human myocardial preconditioning. AB - BACKGROUND: Sublethal periods of ischemia preceding a prolonged interval of ischaemia protect the myocardium. This myocardial preconditioning (PC) appears to be effected by KATP channels. These channels occur both in the sarcolemma and the mitochondrial membrane. We investigated whether mitochondrial KATP channels are the end-effector of PC in the human myocardium. METHODS: Right atrium specimens obtained from patients undergoing cardiac surgery were prepared and incubated in buffer solution at 37 degrees C. After 30-min stabilisation, the muscles were made ischemic for 90 min and then reperfused for 120 min. The preparations were randomised into eight experimental groups (n = 6/group): (1) Aerobic control- incubated in oxygenated buffer for 210 min, (2) ischemia alone--90 min ischemia followed by 120 min reperfusion, (3) PC--preconditioned with 5 min ischemia/5 min reperfusion, (4) Glibenclamide (10 microM) in the incubation media for 10 min before PC, (5) 5-hydroxydecanoate (5-HD, MitoKATP blocker, 1 mM) in the incubation media for 10 min before PC, (6) HMR 1883 (SarcKATP blocker, 10 microM) in the incubation media for 10 min before PC, (7) Pinacidil (0.5 mM) in the incubation media for 10 min before ischemia, and (8) Diazoxide (MitoKATP opener, 0.1 mM) in the incubation media for 10 min before ischemia. Creatinine kinase leakage into the medium (CK, IU/g wet wt) and MTT reduction (OD/mg wet wt.), an index of cell viability, were assessed at the end of the experiment. RESULTS: Ischemia alone resulted in a significant increase in CK leakage (8.01 +/- 0.35) and decrease in MTT (0.15 +/- 0.01) from the values seen in the aerobic control (2.24 +/- 0.52 and 0.78 +/- 0.10 respectively, P < 0.05 in both instances). PC fully reversed the effect of ischemia (CK = 2.97 +/- 0.31 and MTT = 0.61 +/- 0.05; P < 0.05 vs. ischemia alone group but P = NS vs. aerobic control group). Both Glibenclamide and 5-HD abolished the protection induced by PC (CK = 6.23 +/- 0.5 and 7.84 +/- 0.64; MTT = 0.18 +/- 0.03 and 0.13 +/- 0.02, respectively, P < 0.05 vs. PC), but interestingly, the protective effect of PC was not abolished by HMR 1883 (CK = 2.85 +/- 0.24 and MTT = 0.58 +/- 0.05, P = NS vs. PC). Diazoxide mimicked the protective effect of PC (CK = 3.56 +/- 0.32 and MTT = 0.58 +/- 0.02, P = NS vs. PC), however pinacidil exhibited less protection than PC (CK = 4.02 +/ 0.16 and MTT = 0.30 +/- 0.02, P < 0.05 vs. PC). CONCLUSIONS: These studies demonstrate that KATP channels are the end-effectors of ischemic preconditioning and that protection is mediated by mitochondrial KATP channels in human right atrial myocardium. PMID- 10728420 TI - Human connexin40 gap junction channels are modulated by cAMP. AB - OBJECTIVE: Gap junction channels provide for direct electrical coupling between cells, and play an important role in homeostasis and electrical coupling. One of the proteins that form gap junctions, Connexin40 (Cx40), shows restricted expression in the body, and is found in blood vessels and in the atrium and conduction system of the heart. We have investigated whether gap junction channels formed of Cx40 are modulated by protein-kinase-A-mediated phosphorylation. METHODS: A communication-deficient human hepatoma cell line (SKHep1) was stably transfected with human Cx40 cDNA and the properties of Cx40 gap junctions channels and their modulation by cAMP were analyzed using immunocytochemistry, Western blotting, dual patch clamp, and dye coupling. RESULTS: Administration of 1 mM 8-Br-cAMP resulted in a mobility shift of Cx40 protein on western blot and increased macroscopic gap junctional conductance between cell pairs by 46.2 +/- 12.0% (mean +/- S.E.M., n = 8). Under control conditions, single channel experiments revealed three single channel conductances around 30, 80 and 120 pS. When cAMP was added, channel conductances of 46 and 120 pS were observed. In monolayers, cAMP also increased the permeability of Cx40 gap junction channels for Lucifer Yellow by 58%. CONCLUSIONS: Macroscopic conductance and permeability of Cx40 gap junctions is strongly increased by cAMP and may play a role in the regulation of intercellular communication in the heart and vasculature. PMID- 10728421 TI - M cells and transmural heterogeneity of action potential configuration in myocytes from the left ventricular wall of the pig heart. AB - OBJECTIVE: Heterogeneity of action potential configuration in the left ventricle (LV), and the contribution of M cells to it, has been observed in the human heart and is important for arrhythmogenesis. Whether the pig heart has similar properties remains a controversial but important issue as the pig heart is currently under study for use in xenotransplantation. METHODS: Single myocytes were enzymatically isolated from the epicardium (EPI, ncells = 29), midmyocardium (MID, ncells = 38), and endocardium (ENDO, ncells = 13) of the free LV wall (npigs = 26, 14-22 weeks old, 55-80 kg), and studied at different stimulation rates during whole-cell recording (normal Tyrode's solution, K(+)-aspartate-based pipette solution, 50 microM K5fluo-3 as [Ca2+]i indicator, 37 degrees C). Standard six-lead ECGs were recorded from anesthetized pigs. RESULTS: The action potential duration (APD) was not significantly different at 0.25 Hz vs. 2 Hz for the majority of cells in all three layers. However, a subpopulation of cells behaved like M cells and had a very steep frequency response (APD90 at 0.25 Hz 538 +/- 30 ms, vs. 337 +/- 9 ms at 2 Hz, P < 0.05, n = 22). These cells were found predominantly in the MID layer (34% of cells), but also (24%) in EPI. M cells had a more pronounced spike-and-dome configuration, with a significantly larger phase 1 magnitude and plateau voltage. The frequency response of these parameters was different from the other cell types. [Ca2+]i transients tended to be larger in M cells. For the in vivo ECG of anesthetized pigs, the QT time was close to the APD90 of M cells, and J waves were seen in 7/12 recordings. CONCLUSIONS: In young adult pigs, M cells can be identified by a steep frequency response of the APD and by a spike-and-dome configuration. These cells are mostly, but not exclusively, found in the midmyocardium, and could contribute to the ECG characteristics. Their properties may however be different from those of other species, including humans. PMID- 10728422 TI - Immunolocalization of annexins IV, V and VI in the failing and non-failing human heart. AB - The failing human heart is characterized by changes in the expression and function of proteins involved in intracellular Ca2+ cycling, resulting in altered Ca2+ transients and impaired contractile properties of cardiac muscle. The role of the cardiac annexins in this process remains unclear. Annexins may play a role in the regulation of Ca2+ pumps and exchangers on the sarcolemma, and have been shown to be altered in some cardiac disease states. OBJECTIVE: The goal of this study was to compare the immunolocalization and expression of annexins IV, V and VI in failing and non-failing human hearts. METHODS: We used immunostaining to identify the subcellular location of annexins IV, V and VI proteins within the myocardial cell, and Western blot analysis to quantify the proteins in the same hearts. RESULTS: Annexin IV showed a cytoplasmic distribution in both failing and non-failing human heart cells. Annexin V was localized at the z-line, around lipofuscin granules, and in the cytosol in the non-failing heart cells. Annexin VI was localized at the sarcolemma and intercalated disc. Protein levels of annexins IV and V were up-regulated in failing human hearts, while the expression of annexin VI was unchanged. CONCLUSIONS: Alterations in the intracellular localization of annexins, along with up-regulation of annexins IV and V in the failing human heart cells, suggests differential regulation of these Ca2+ regulatory proteins during heart failure. PMID- 10728424 TI - Calpain-I induced alterations in the cytoskeletal structure and impaired mechanical properties of single myocytes of rat heart. AB - OBJECTIVE: The involvement of Calpain-I mediated proteolysis has been implicated in myofibrillar dysfunction of reperfused myocardium following ischemia (stunning). This study addresses the question whether ultrastructural alterations might be responsible for the depressed contractility. METHODS: Mechanical properties and protein composition of isolated myocytes after Calpain-I exposure (1.25 U/ml; 10 min; 15 degrees C; pCa 5.0) and of ischemic rat hearts following reperfusion were characterized. RESULTS: Maximal isometric force (44 +/- 5 kN/m2) at pCa 4.5 (pCa = -log[Ca2+]) decreased by 42.5% in Triton permeabilized myocytes (n = 11) after Calpain-I treatment. Force (and consequent myofilament disarrangement) during Calpain-I treatment was prevented by 40 mM BDM. The contractile force of Calpain-I exposed myocytes was significantly higher at submaximal levels of activation (pCa 5.5, 5.4 and 5.3) before maximal force development (pCa 4.5) than after maximal force development. The pCa50 value (5.40 +/- 0.02) determined from these initial test contractures did not differ significantly from that of untreated controls (5.44 +/- 0.03). However, after full activation Ca(2+)-sensitivity of force production in Calpain-I treated myocytes was significantly reduced (pCa50 5.34 +/- 0.02). This change in pCa50 was positively correlated with the reduction in maximal isometric force and was accompanied by sarcomere disorder. These findings imply that at least part of the Calpain-I induced mechanical alterations are dependent on force history. Measurements of the rate of force redevelopment after unloaded shortening suggested that Calpain-I did not affect cross-bridge kinetics. SDS gel electrophoresis and Western immunoblotting of Calpain-I treated myocytes revealed desmin degradation. The desmin content of postischemic myocardium was also reduced. CONCLUSION: Our results indicate that ultrastructural alterations may play an important role in the Calpain-I mediated cardiac dysfunction. PMID- 10728423 TI - Novel mutations in KvLQT1 that affect Iks activation through interactions with Isk. AB - OBJECTIVES: We report the functional expression of four KCNQ1 mutations affecting arginine residues and resulting in Romano-Ward (RW) and the Jervell and Lange Nielsen (JLN) congenital long QT syndromes. RESULTS: The R539W and R190Q mutations were found in typical RW families with an autosomal dominant transmission. The R243H mutation was found in a compound heterozygous JLN patient who presents with deafness and cardiac symptoms. The fourth mutation, R533W, was a new case of recessive form of the RW syndrome since homozygous carriers experienced syncopes but showed no deafness, whereas the heterozygous carriers were asymptomatic. The R190Q mutation failed to produce functional homomeric channels. The R243H, R533W and R539W mutations induced a positive voltage shift of the channel activation but only when co-expressed with IsK, pointing out the critical role of these positively charged residues in the modulation of the gating properties of KvLQT1 by IsK. The positive shift induced by R533W was merely 15%. This small effect was compatible with the recessive character of the RW phenotype transmission. The average QTc was significantly longer (P < 0.01) in patients carrying mutations inducing a total loss of channel function and those patients were also prone to cardiac adverse symptoms (whether syncopes or sudden death) to a greater extent (62 vs. 21%, P < 0.001). CONCLUSIONS: Novel mutations are described that induce a voltage shift of the channel activation only in the presence of IsK. They appear associated with a milder cardiac phenotype. PMID- 10728425 TI - Altered paracrine effect of endothelin in blood vessels of the hyperinsulinemic, insulin resistant obese Zucker rat. AB - OBJECTIVE: Earlier, we reported that high insulin incubation in vitro leads to increased ETA receptor expression in cultured rat aortic smooth muscle cells (Diabetes 1998, 47: 934-944). Our later observation of enhanced endothelin-1 evoked vasoconstriction in aorta from the hyperinsulinemic obese Zucker rat indicated that this interaction might also be relevant in vivo. To further examine the relationship between insulinemia and endothelin, we characterized endothelin receptor expression and endothelin-1 peptide levels in vascular tissues and plasma from young and old obese Zucker rats. METHODS: 12 and 40-week old Zucker obese and lean rats were used. Plasma endothelin-1 levels and endothelin-1 peptide content in the mesenteric artery and in the thoracic aorta were examined by radioimmunoassay. Messenger RNA levels of endothelin-1 peptide and ETA and ETB receptors were examined in the aortic and mesenteric vessels using RT-PCR. RESULTS: Obese rats from both age groups had significantly higher plasma levels of insulin (4-10 fold), total cholesterol (2-3 fold), triglycerides (10-fold), and glucose (approximately 1.5 fold) than their lean counterparts. There was a trend toward worsening lipoproteinemia and glycemia, but improved insulinemia with age in the obese rats. In association with these changes, obese rats exhibited attenuated endothelin-1 peptide and preproET-1 mRNA levels, but conversely elevated ETA and ETB receptor mRNA levels in both aortic and mesenteric vessels. CONCLUSION: These data suggest that vascular tissue from the metabolically dysregulated obese Zucker rat exhibits attenuated endothelin-1 peptide production and elevated endothelin receptor levels. Since elevated insulin levels have been linked to increased endothelin receptor expression, it is plausible that hyperinsulinemia upregulates endothelin receptors contributing to elevated vasoconstrictor responses to endothelin-1 in this model of obesity and hypertension. PMID- 10728426 TI - Reversal of glibenclamide-induced coronary vasoconstriction by enhanced perfusion pulsatility: possible role for nitric oxide. AB - OBJECTIVES: ATP-sensitive potassium channels (K+ATP) prominently contribute to basal coronary tone; however, flow reserve during exercise remains unchanged despite channel blockade with glibenclamide (GLI). We hypothesized that increasing perfusion pulsatility, as accompanies exercise, offsets vasoconstriction from K+ATP-channel blockade, and that this effect is blunted by nitric oxide synthase (NOS) inhibition. METHODS: In 31 anaesthetized dogs the left anterior descending artery was blood-perfused by computer-controlled servo pump, with real-time arterial perfusion pulse pressure (PP) varied from 40 and 100 mm Hg at a constant mean pressure and cardiac workload. RESULTS: At control PP (40 mm Hg), GLI (50 micrograms/min/kg, i.c.) lowered mean regional coronary flow from 37 +/- 5 to 25 +/- 4 ml/min (P < 0.001). However, this was not observed at 100 mm Hg PP (41 +/- 2 vs. 45 +/- 4). NOS inhibition by NG-monomethyl-L arginine (L-NMMA) did not alter basal flow at 40 mm Hg PP, but modestly lowered flow (-5%, P < 0.001) at higher PP (100 mm Hg), reducing PP-flow augmentation by 36%, and acetylcholine (ACh) induced flow elevation by -39%. Co-infusion of L NMMA with GLI resulted in net vasoconstriction at both PP levels (-60% and -40% at 40 and 100 mm Hg PP, respectively). Unlike GLI, vasoconstriction by vasopressin (-43 +/- 3% flow reduction at 40 mm Hg PP) or quinacrine (-23 +/- 7%) was not offset at higher pulsatility (-44 +/- 4 and -23 +/- 6%, respectively). Neither of the latter agents inhibited ACh- or PP-induced flow responses, nor did they modify the effect of L-NMMA on these responses. CONCLUSIONS: Increased coronary flow pulsatility offsets vasoconstriction from K+ATP blockade by likely enhancing NO release. This mechanism may assist exercise-mediated dilation in settings where K+ATP opening is partially compromised. PMID- 10728427 TI - Acute impairment of relaxation by low levels of testosterone in porcine coronary arteries. AB - OBJECTIVES: While there are many suggested reasons for the marked gender bias in cardiovascular events, much of the available data indicate that circulating estrogens are cardioprotective. The possibility that endogenous androgens may be detrimental to the cardiovascular system has received relatively less attention. We investigated the short-term modulatory effects of various concentrations of testosterone on vascular function in isolated porcine coronary artery rings. RESULTS: The higher concentrations (> 1 microM) of testosterone relaxed U46619 contracted coronary artery rings in an endothelium-independent manner. This direct effect was insensitive to the testosterone receptor antagonists, flutamide and cyproterone acetate. Short-term exposure (20 min) to low levels of testosterone (1-100 nM), which were ineffective on their own on vascular function, significantly diminished relaxation to bradykinin and calcium ionophore A23187 but not those produced by levcromakalim and sodium nitroprusside. The inhibitory effect observed with 1 nM testosterone was only partially reversed by flutamide and cyproterone acetate and unaltered in the presence of actinomycin D and cycloheximide. CONCLUSIONS: These results demonstrate that acute treatment with testosterone, at concentrations that have no effect on their own, reduces vasorelaxation. Furthermore, they suggest that this modulatory action may be in part independent of the classical testosterone receptor since it was not completely sensitive to the anti-androgens and was not inhibited by the transcriptional and translational inhibitors. These findings support the postulation that testosterone may have unfavorable influences on vascular function. PMID- 10728428 TI - Chronic non-vascular cytomegalovirus infection: effects on the neointimal response to experimental vascular injury. AB - OBJECTIVE: Epidemiologic and mechanistic evidence implicates a role for cytomegalovirus (CMV) in atherogenesis. Recently, we demonstrated that CMV has the capacity to causally contribute to atherogenesis; acute infection of rats with rat CMV (RCMV) 1 day after carotid artery injury increased neointimal accumulation. Importantly, in the injured vessel infectious virus could not be detected and viral genome was present only transiently, suggesting that additional mechanisms play a role in the virus-induced exacerbation of the vascular injury response other than the changes caused by direct infection of vessel wall cells. The present investigation was designed to determine whether chronic persistent RCMV infection, more relevant to the clinical situation, also exacerbates the response to injury and, if so, whether similar mechanisms are operative. METHODS: Sixty 3-week-old male Spraque-Dawley rats received an i.p. injection of either 10(6) TCID50 RCMV (Priscott strain) or normal saline. The left carotid artery was balloon-injured 3 months after infection. Rats were killed 6 weeks later. This model produces persistent infection, as demonstrated by presence of infectious virus in the salivary glands at time of sacrifice. RESULTS: The neointima to media (N/M) ratio of the injured vessel was 41% greater in the RCMV-infected than in control rats (1.40 +/- 0.48 vs. 0.99 +/- 0.45; P = 0.003). The aorta never contained infectious RCMV, and exhibited RCMV DNA, detected by PCR, only transiently. The persistent infection of non-vascular tissues was associated with increased serum levels of IL-2, IL-4 and IFN-gamma. CONCLUSIONS: CMV infection of young rats causes persistent infection of non vascular tissues and increased cytokine levels. The neointimal response to subsequent vascular injury is increased, despite absence of virus from the vessel wall. These findings, as in acute infection following vascular injury, suggest that inflammatory and immune responses to chronic persistent CMV infection contribute to an exaggerated response to vascular injury. PMID- 10728429 TI - Age-dependent increase in c-fos activity and cyclin A expression in vascular smooth muscle cells. A potential link between aging, smooth muscle cell proliferation and atherosclerosis. AB - OBJECTIVE: Aging can be defined as a progressive deterioration of biological functions after the organism has attained its maximal reproductive competence, which is usually associated with a decrease in proliferative ability in most cell types. However, in certain pathological situations such as atherosclerosis and restenosis, aging has been shown to be associated with a higher level of vascular smooth muscle cell (VSMC) proliferation and neointimal lesion formation after angioplasty. In the present study, we investigated potential mechanisms involved in the age-dependent increase in VSMC proliferation. METHODS AND RESULTS: Primary cultures of VSMCs were isolated from young (6-8-month-old) and old (4-5-year-old) New Zealand rabbits. Results from cell counting assays and FACS analysis were consistent with a shortening of the cell cycle in old VSMCs. Western blot analysis in serum stimulated cells showed a significant increase in the level of cyclin A and cyclin-dependent kinase 2 proteins in the old vs. young VSMCs. In marked contrast, expression of cyclin E in VSMCs was not influenced by aging. Transient transfection assays showed an age-dependent increase in transcription from the human cyclin A promoter. Parallel studies demonstrated that the expression of the AP1 transcription factor c-fos, which interacts with the cyclin A promoter and stimulates VSMC proliferation, was also increased in old VSMCs. Consistent with this notion, electrophoretic mobility shift assays demonstrated an increase in AP1 DNA-binding activity in old VSMCs. CONCLUSIONS: These studies suggest that age-associated increase in c-fos activity contributes to augmented cyclin A expression and VSMC proliferation in old animals. These mechanisms might contribute to the higher prevalence and severity of atherosclerosis in the elderly. PMID- 10728430 TI - Evidence for the stimulatory effect of resveratrol on Ca(2+)-activated K+ current in vascular endothelial cells. AB - OBJECTIVE: Resveratrol, a natural phytoalexin compound, is present in grapes and wine, and it can produce vasorelaxation. However, little is known of its mechanisms of action on ionic currents in endothelial cells. METHODS: The effect of resveratrol on Ca(2+)-activated K+ currents in an endothelial cell line (HUV EC-C) originally derived from human umbilical vein was investigated with the aid of the patch-clamp technique. RESULTS: In the whole-cell configuration, resveratrol reversibly increased the amplitude of K+ outward currents. The increase in outward current caused by resveratrol was greatly inhibited by iberiotoxin (200 nM) or paxilline (1 microM), but not by glibenclamide (10 microM), tamoxifen (10 microM), or beta-bungarotoxin (200 nM). Thus, this outward current is believed to be Ca(2+)-activated K+ current (I K(Ca)). In the inside out configuration, bath application of resveratrol (30 microM) caused no change in the single-channel conductance, but increased the activity of large conductance Ca(2+)-activated K+ (BKCa) channels. Resveratrol enhanced the channel activity in a concentration-dependent manner. The EC50 value for resveratrol induced channel activity was 20 microM. The resveratrol-stimulated increase in the channel activity was independent of internal Ca2+. Resveratrol (30 microM) also shifted the activation curve of BKCa channels to less positive membrane potentials. The change in the kinetic behavior of BKCa channels caused by resveratrol in these cells in due to an increase in mean open time and a decrease in mean closed time. In a pancreatic islet endothelial cell line (MS1), resveratrol (30 microM) also increased the activity of intermediate-conductance KCa channels. CONCLUSIONS: These results provide evidence that in addition to the presence of antioxidative activity, resveratrol can also stimulate KCa channels in endothelial cells. The direct stimulation of these KCa channels by resveratrol may be responsible for its effect on the functional activities of endothelial cells. PMID- 10728431 TI - Subarachnoid hemorrhage induced sympathoexcitation arises due to changes in endothelin and/or nitric oxide activity. AB - OBJECTIVE: The demonstration of the effectiveness of endothelin antagonists and nitric oxide donors in managing vasospasm following subarachnoid hemorrhage is encouraging. Whether such drugs can modify the sympathoexcitation that accompanies this condition remains unknown and was the basis for the present report. METHODS: Subarachnoid hemorrhage was induced in conscious rats by injecting blood via a catheter placed along the surface of the brain and directed towards the circle of Willis. We combined measurements of arterial plasma catecholamines with the spectral analysis of blood pressure variability in order to examine sympathetic nervous activation following subarachnoid hemorrhage. Experiments were performed in untreated animals and in rats following pretreatment with either bosentan or sodium nitroprusside. RESULTS: Indicative of a pronounced sympathoexcitation, the 0.2-0.6 Hz frequency components of blood pressure were markedly elevated following subarachnoid hemorrhage (2.5 +/- 0.5 vs. 8.9 +/- 2.6 mmHg2, P < 0.01). Parallel changes in plasma norepinephrine concentration were observed (1.0 +/- 0.2 vs. 2.4 +/- 0.4 nmol/l, P < 0.01). The subarachnoid injection of saline did not modify blood pressure variability or plasma norepinephrine concentrations. Pretreatment with either bosentan or sodium nitroprusside completely prevented the subarachnoid hemorrhage induced sympathoexcitation. CONCLUSIONS: Experimental subarachnoid hemorrhage is associated with a pronounced activation of the sympathetic nervous system. It would appear that this sympathoexcitation has its roots ensconced in either the release of endothelin or an impairment in nitric oxide mediated vasodilation. PMID- 10728432 TI - Elf-pulsed magnetic fields modulate opioid peptide gene expression in myocardial cells. AB - OBJECTIVES: Magnetic fields have been shown to affect cell proliferation and growth factor expression in cultured cells. Although the activation of endorphin systems is a recurring motif among the biological events elicited by magnetic fields, compelling evidence indicating that magnetic fields may modulate opioid gene expression is still lacking. We therefore investigated whether extremely low frequency (ELF) pulsed magnetic fields (PMF) may affect opioid peptide gene expression and the signaling pathways controlling opioid peptide gene transcription in the adult ventricular myocyte, a cell type behaving both as a target and as a source for opioid peptides. METHODS: Prodynorphin gene expression was investigated in adult rat myocytes exposed to PMF by the aid of RNase protection and nuclear run-off transcription assays. In PMF-exposed nuclei, nuclear protein kinase C (PKC) activity was followed by measuring the phosphorylation rate of the acrylodan-labeled MARCKS peptide. The effect of PMF on the subcellular distribution of different PKC isozymes was assessed by immunoblotting. A radioimmunoassay procedure coupled to reversed-phase high performance liquid chromatography was used to monitor the expression of dynorphin B. RESULTS: Here, we show that PMF enhanced myocardial opioid gene expression and that a direct exposure of isolated myocyte nuclei to PMF markedly enhanced prodynorphin gene transcription, as in the intact cell. The PMF action was mediated by nuclear PKC activation but occurred independently from changes in PKC isozyme expression and enzyme translocation. PMF also led to a marked increase in the synthesis and secretion of dynorphin B. CONCLUSIONS: The present findings demonstrate that an opioid gene is activated by myocyte exposure to PMF and that the cell nucleus and nuclear embedded PKC are a crucial target for the PMF action. Due to the wide ranging importance of opioid peptides in myocardial cell homeostasis, the current data may suggest consideration for potential biological effects of PMF in the cardiovascular system. PMID- 10728433 TI - Nonlinear biphasic relationship between the time constant tau and load. PMID- 10728435 TI - Abnormal sarcoplasmic reticulum Ca2+ release in heart failure PMID- 10728434 TI - Can changes of ryanodine receptor expression affect cardiac contractility? PMID- 10728436 TI - Can fibroblasts determine the late differing outcome between systemic sclerosis and primary hypertrophic osteoarthropathy (pachydermoperiostosis)? PMID- 10728437 TI - The volume of rheumatoid synovial membrane, determined by magnetic resonance, reflects disease activity and predicts joint destruction. PMID- 10728438 TI - Comparison of clinical and immunogenetic features in familial versus sporadic psoriatic arthritis. AB - OBJECTIVE: To compare patients with familial versus sporadic psoriatic arthritis (PsA) with respect to clinical, radiological and immunogenetic features. METHODS: All patients were identified from the University of Toronto Psoriatic Arthritis Clinic. Familial and sporadic PsA were distinguished based on the proband's self reported history. The probands were compared at presentation to clinic with respect to: demographic information, age of onset of psoriasis and inflammatory arthritis, disease activity, disease damage, laboratory variables, functional class and HLA antigens. The two groups were compared using a univariate analysis. RESULTS: In total 407 patients were included. Thirty-six patients (8.8%) were eliminated as they reported a family history of arthritis in the absence of psoriasis. Of the remaining 371 patients, 150 patients reported a positive family of either PsA or psoriasis. 221 patients (54.2%) had no family history of psoriatic arthritis, psoriasis, or "arthritis". The familial group were younger at presentation to clinic (p = 0.003), had an earlier age of onset of psoriasis (p = 0.001) and inflammatory arthritis (p = 0.001) and were more likely to be receiving treatment (p = 0.001). The mean number of actively inflamed joints was higher in the sporadic group (p = 0.035), along with a higher frequency of rheumatoid factor positivity (p = 0.04). Only the age of onset variables and medication use retained significance after correction for multiple comparisons. CONCLUSIONS: In comparing probands with familial versus sporadic PsA, we noted a marked difference in the age of onset of psoriasis and inflammatory arthritis, along with other differences in several clinical variables. These differences may be helpful in identifying PsA patients with a stronger genetic predisposition. PMID- 10728439 TI - Joint cartilage repair with transplantation of embryonic chondrocytes embedded in collagen-fibrin matrices. AB - OBJECTIVE: The objective of this study was to assess the feasibility of transplanting embryonic chondrogenic cells within a collagen-fibrin substrate for the reconstitution of full-thickness cartilage defects in chicken knee joints. METHODS: Full-thickness cartilage defects were created mechanically on the weight bearing surface of the tibial condyle in 45 adult chickens and subsequently filled with chondrocytes embedded in a chondrocyte-collagen-fibrin gel. The transplants were compared to untreated defects and collagen-fibrin transplants without cells. The results were analyzed using histochemical and morphometrical methods after 3, 12 and 24 weeks. A semiquantitative histological grading system was applied to evaluate the transplant integration and the newly formed cartilage architecture. RESULTS: Chondrocyte-gel grafts developed to hyaline-like cartilage without any granulation tissue in the interface after 3 weeks. After 12 weeks the defects in the experimental group were filled completely with hyaline cartilage. The defects in the control groups in all cases healed with fibrous repair tissue. CONCLUSION: Fibrin-collagen gel allowed stable graft fixation and provided an adequate microenvironment for embryonic chondrocytes to generate hyaline-like neocartilage in a full-thickness cartilage defect. PMID- 10728440 TI - Antibodies to neuroblastoma cells in rheumatoid arthritis: a potential marker for neuropathy. AB - OBJECTIVE: To investigate the prevalence of antibodies to neuroblastoma cells in patients with rheumatoid arthritis (RA) complicated by peripheral neuropathy (PN), and to determine whether there is any relationship of these antibodies with the severity of neuropathy. METHODS: The study was carried out on 28 patients with RA complicated by PN, 29 RA patients without PN and 28 healthy volunteers (HV). A cell-based ELISA method was used to test sera for the presence of IgG and IgM anti-neuroblastoma cell antibodies. Localisation and characterisation of neuroblastoma antigens recognised by patients' sera was carried out by immunofluorescent microscopy and Western blotting. RESULTS: Elevated levels of IgG anti-neuroblastoma cell antibodies were found in 10 (36%) neuropathic patients and in 1 (3%) RA control (chi 2 = 9.53, P = 0.002), while significant levels of IgM anti-neuroblastoma cell antibodies were demonstrated in 10 (36%) neuropathic patients and in 2 (7%) RA controls (chi 2 = 7.12, P = 0.008). Overall, the levels of antibodies in healthy volunteers were significantly lower than in RA controls and patients with PN. No significant relationship was found between the level of anti-neuroblastoma cell antibodies and severity of RA or neuropathy. Immunofluorescence staining of neuroblastoma cells with sera from 18 neuropathic patients demonstrated cytoplasmic and/or nuclear patterns. Western blotting demonstrated reactivity with a heterogeneous group of neuroblastoma antigens. Little or no reactivity was seen with RA control or HV sera. CONCLUSION: Antibodies against neuroblastoma cells are more prevalent in RA patients with peripheral neuropathy than in RA patients without peripheral nerve involvement. Such antibodies may be useful diagnostic markers for peripheral neuropathy in RA. PMID- 10728441 TI - Assessment of cardiac function by echocardiography in Paget's disease of bone. AB - OBJECTIVE: To identify the mechanisms which influence the development of cardiac insufficiency in Paget's disease of bone (PD). METHODS: In this hospital-based case-control study 23 consecutive, recently diagnosed and untreated PD patients were compared against 23 controls frequency-matched by sex, age and body index. All subjects underwent non-invasive assessment of cardiac function by two dimensional Doppler echocardiography. Calcium, phosphate, and creatinine were determined in the serum and urine, along with alkaline phosphatase and hydroxyproline excretion, two biochemical parameters of PD activity. RESULTS: Peripheral vascular resistance proved lower (1604.9 +/- 390.1 vs 1801.2 +/- 421.0) and the stroke volume higher in PD patients (67.2 +/- 14.4 vs 56.0 +/- 8.6; p = 0.07) compared with controls. These differences were greater (1504.7 +/- 289.9 and 71.0 +/- 6.2) and attained statistical significance (p = 0.008) when the subgroup with more extensive skeletal disease only was considered. A moderate correlation was observed between hydroxyproline excretion and the E/A ratio (r = 0.45; p = 0.03), peripheral vascular resistance (r = -0.42; p = 0.04), and diastolic arterial pressure (r = -0.42; p = 0.04). The final model obtained via multivariate analysis identified both urinary hydroxyproline and age as predictive variables linked to peripheral vascular resistance. CONCLUSION: In the early phases of PD there is a trend towards a reduction in peripheral vascular resistance. If this persists, it may lead progressively to increased cardiac output, which is mainly influenced by the degree of turnover impairment and the age of the individual. PMID- 10728442 TI - Synovial fluid lymphocyte proliferation in response to crude microbial antigens is not useful as a diagnostic test to specifically indicate a bacterial cause of arthritis. AB - OBJECTIVE: To determine the role of lymphocyte proliferation assay of synovial fluid mononuclear cells (SFMC) with whole fraction bacteria in the diagnosis of reactive arthritis (ReA) or arthritis of unknown origin. METHODS: We stimulated SFMC of 52 unselected patients who consecutively presented in our rheumatology outpatient clinic with the following diagnoses: ReA (n = 8), rheumatoid arthritis (RA) (n = 16), ankylosing spondylitis (AS) (n = 6), osteoarthritis (OA) (n = 5), psoriatic arthritis (PsA) (n = 5) and arthritis of varying origin (AVO) (n = 12) and peripheral blood MC (PBMC) of 10 healthy controls with arthritogenic (Y. entero-colitica, S. enteritidis, C. trachomatis) and non-arthritogenic (E. coli, K. pneumoniae, S. pyogenes, C. albicans) bacteria/mitogens and Tetanus toxoid. T cell proliferation was measured in a standard [3H] Thymidine uptake assay. RESULTS: In all groups of patients tested, SFMC could be stimulated both by arthritogenic and non-arthritogenic bacteria. So-called specific responses were observed in patients with ReA, but also in RA and AS. CONCLUSION: Our findings show that a lymphocyte proliferation assay with SFMC with whole fraction bacteria is not an adequate diagnostic tool to confirm bacterial involvement in inflammatory arthritis. PMID- 10728443 TI - Involvement of cAMP responsive element binding protein (CREB) in the synovial cell hyperfunction in patients with rheumatoid arthritis. AB - OBJECTIVE: To elucidate a possible role of cAMP responsive element binding protein (CREB) in rheumatoid arthritis (RA) synovial cell function, we have studied CREB expression of synovial cells and the effects of an inhibitor of the cAMP/CREB signal pathway on synovial cell function in patients with RA. METHODS: We examined CREB expression by immunohistochemical staining, immunocytochemical staining, and gel shift assays. Effects of cAMP/CREB inhibitor on the proliferation of RA synovial cells were assessed by [3H]-TdR incorporation, and those on proinflammatory cytokine and matrix metalloproteinase (MMP) production by reverse transcription PCR and ELISAs. RESULTS: Immunohistochemical staining of synovial tissue revealed that CREB is expressed mainly in the lining and sublining layers of synovium in patients with RA. DNA binding activity of CREB was ascertained by a gel shift assay. We also confirmed nuclear translocation and phosphorylation of CREB in TNF-alpha stimulated RA fibroblast-like synovial cells by immunocytochemical staining. Modulators of cAMP/CREB signaling pathway, such as Rp-cAMP, had an inhibitory potential on RA synovial cell proliferation in vitro. Rp-cAMP also inhibited the proinflammatory cytokine and MMP production. CONCLUSION: CREB is involved in the synovial cell activity in patients with RA. Inhibition of CREB activity by its inhibitor brings about the correction of aberrant synovial cell functions in patients with RA, thus suggesting a possible clinical application of cAMP/CREB inhibitors. PMID- 10728444 TI - Vascular endothelial growth factor and hypertrophic osteoarthropathy. AB - OBJECTIVE: Hypertrophic osteoarthropathy (HOA) is characterized by the coexistence of digital clubbing and periosteal proliferation of the tubular bones. Localized vascular proliferation associated with platelet/endothelial cell activation are recognized features of this syndrome. Current knowledge suggests that HOA develops from the presence in the systemic circulation of one or more growth factors that are normally inactivated in the lungs. The nature of these purported growth factors has not yet been identified. Vascular endothelial growth factor (VEGF) has several features that may fit in with the pathogenesis of HOA. The objective of our study was to measure serum and plasma levels of VEGF in different groups of patients with HOA. METHODS: We studied 24 patients with HOA; of these, in 12 the HOA was secondary to cyanotic congenital heart disease and in 7 to lung cancer, while 5 represented primary cases. As controls we studied 28 individuals without HOA; of these, 12 were apparently healthy individuals, 7 had cyanosis secondary to chronic obstructive pulmonary disease, and 9 had lung cancer. ELISA was used to measure serum and plasma levels of VEGF. RESULTS: Plasma levels of VEGF were significantly higher in the patients with primary HOA (median 46.2; range 19.4-398.8 pg/ml) and in those with lung cancer-HOA (median 75.5; range 24.6-166.7), compared to healthy controls (median 7.4; range: 0 26.1), p < 0.05. Serum VEGF levels were higher in patients with lung cancer and HOA (median 411.4; range 164.2-959.5 pg/ml) compared with lung cancer patients without HOA (median 74.5; range 13.2-205.4), p < 0.001. CONCLUSIONS: Patients with primary HOA and those with HOA and lung cancer have increased circulating levels of VEGF. This cytokine may play a role in the pathogenesis of HOA. PMID- 10728445 TI - Marked elevation of serum N-acetyl-beta-D-hexosaminidase activity in rheumatoid rheumatoid arthritis. AB - OBJECTIVE: To study N-acetyl-beta-D-hexosaminidase (NAHase) activity in the sera of rheumatoid arthritis (RA) patients and to determine its source. METHODS: NAHase activity in the serum and synovial fluid of RA patients was measured with p-nitrophenyl beta-N-acetylglucosaminide as substrate. The p-nitrophenol released was measured spectrophotometrically in an ELISA reader. Rabbit articular chondrocytes in primary culture were stimulated with interleukin-1 beta (IL-1 beta). RESULTS: Serum NAHase activity was higher in 35% of the RA patients than in healthy patients. The median activity was about twice that of the serum of healthy volunteers. RA patients with high serum NAHase activity also had more joint destruction (85%) than those with normal NAHase activity (57%, p < 0.05), but their inflammatory status was similar. The source of NAHase in RA was investigated by assaying it in RA synovial fluids (SF) and measuring its release from articular chondrocytes in primary culture. NAHase activity was detected in all 23 RA SF, at a median concentration that was 2 times that of the serum. NAHase activity in the medium of articular chondrocytes was stimulated by IL-1 beta (p < 0.005 compared to unstimulated cells), suggesting that cartilage is a source of serum and SF NAHase activity. CONCLUSION: The serum concentration of the matrix hydrolase, NAHase, is higher in destructive RA than in inflammatory RA. PMID- 10728446 TI - A prospective cohort study of the outcome of acute whiplash injury in Greece. AB - OBJECTIVE: An earlier pilot study suggested that the late whiplash syndrome is uncommon in Greece. The purpose of the present study is to extend the evaluation to a larger sample, and include the prevalence of specific symptoms in the evaluation. METHODS: In a prospective, cohort study, a total of 180 accident victims were consecutively recruited following Emergency ward presentation. A standard questionnaire asked about neck pain, headache, shoulder pain, limb numbness or pain, and dizziness. Accident victims were followed for 6 months. RESULTS: In the initial 4 weeks after the accident, accident victims reported neck pain, headache, shoulder pain, arm numbness or pain, and dizziness, but at 4 weeks more than 90% had recovered from these, the remainder of the subjects having minor symptoms (not requiring therapy), and returning to their pre accident state of health (which included minor symptoms). There were no cases of chronic disability. CONCLUSION: In Greece, symptoms after an acute whiplash injury are self-limiting, brief, and do not appear to evolve into the so-called late whiplash syndrome. PMID- 10728447 TI - Pathergy reaction in Behcet's disease: lack of correlation with mucocutaneous manifestations and systemic disease expression. AB - OBJECTIVE: The pathergy reaction is a unique feature of Behcet's disease (BD) and, according to the International Study Group (ISG), is among the major criteria required for the diagnosis. Different positive pathergy reaction rates in BD have been reported worldwide. We evaluated the prevalence of the pathergy reaction in Israeli BD patients, and its relation to mucocutaneous and systemic manifestations of the disease. METHODS: Forty-three patients were studied, all of whom fulfilled the ISG criteria for BD. The mucocutaneous and systemic disease manifestations were analyzed with respect to the presence of the pathergy reaction, and a systemic severity score for BD was calculated according to the potential morbidity and mortality associated with various clinical features. RESULTS: Nineteen patients (44.2%) had a positive pathergy test. The pathergy positive and pathergy-negative BD groups showed a similar male:female ratio, age at disease onset, and mean disease duration. They also exhibited similar HLA-B5 levels and a similar frequency of oral ulcerations in close family members. The mucocutaneous manifestations, systemic disease expression, and severity score were similar in patients with and without the pathergy reaction. CONCLUSION: The presence of a positive pathergy reaction, although common in Israeli BD patients, is not associated with an increased risk for specific mucocutaneous or systemic manifestations of the disease, and probably does not predict a more severe disease course. PMID- 10728448 TI - Survey of Turkish systemic lupus erythematosus patients for a particular mutation of C1Q deficiency. AB - OBJECTIVE: Hereditary C1q deficiency is a rare disease and up to now only 41 cases have been reported. Since all but 3 cases developed SLE or SLE-like disease, C1q deficiency represents the most powerful disease susceptibility gene identified for the development of SLE in humans. A molecular defect in homozygous C1q deficiency has been identified in 13 families. Four of these families are Turkish in origin and they all share the same mutation which is a CAG to TAG change at codon 186 in the A chain. This led us to investigate whether this mutation might be found in Turkish SLE patients and whether it could cause increased disease susceptibility when expressed in the heterozygous form. METHODS: We screened 65 Turkish lupus patients and 49 healthy Turkish individuals by carrying out an amplification of exon 2 of the A chain and restriction enzyme analysis for the C1qA mutation. RESULTS: We found no other example of this mutation in either the homozygous or heterozygous forms. CONCLUSION: C1q deficiency is one of the very strong disease susceptibility genes in lupus and may cause SLE via a critical role in the physiological clearance of apoptotic cells. However, C1q deficiency caused by a particular mutation in the A chain in a heterozygous form is not found in the Turkish SLE population. PMID- 10728449 TI - Lymphocytic hypophysitis in a patient with systemic lupus erythematosus. AB - A case of lymphocytic hypophysitis in a patient with systemic lupus erythematosus is described. A 20-year-old woman was admitted to our hospital with generalized myalgia and facial rash in May 1998. The patient had a medical history, physical examination, and laboratory findings compatible with systemic lupus erythematosus (SLE). Headache and nausea had developed 3 months previously and worsened over the following months. Hormonal investigation showed hypopituitarism except for prolactin. A magnetic resonance image of the brain showed a mass lesion in the pituitary fossa. A trans-sphenoidal surgical procedure was performed which revealed a dark-yellowish hematoma. Microscopic examination showed diffuse infiltration of lymphocytes and plasma cells with fibrosis in the anterior pituitary. Post-operatively the patient's headaches and nausea resolved. This indicates that lymphocytic hypophysitis may be associated with SLE. PMID- 10728450 TI - Immune-mediated pathology following hepatitis B vaccination. Two cases of polyarteritis nodosa and one case of pityriasis rosea-like drug eruption. AB - The association of hepatitis B virus infection and vasculitis or other immune mediated manifestations is well documented. Reports on such manifestations in relation to hepatitis B vaccination are scarce, however. We report 2 patients who developed polyarteritis nodosa following vaccination against hepatitis B. In one patient this resulted in an ischemic and necrotic digital ulcus, necessitating surgical amputation. The other patient presented with typical cutaneous polyarteritis nodosa which responded well to corticosteroid treatment. A third patient developed a severe pityrias rosea-like eruption. He was treated with topical steroids with healing of the lesions, leaving only post-inflammatory hyperpigmentation. The literature on these associations is reviewed. PMID- 10728451 TI - A case of polymyositis associated with hepatitis B infection. AB - This report describes the case of a 47-year old man who developed myositis in association with hepatitis B surface antigen-positive hepatitis. Interestingly, the myositis repeatedly worsened 2 months after the exacerbation of hepatitis in this case, suggesting a close association between hepatitis B infection and myositis. The dose of prednisolone was increased twice in order to treat the exacerbating myositis, resulting in improvement of the muscle symptoms, but the patient eventually died of liver failure. Only 5 other myositis patients with hepatitis B antigenemia have been reported in the literature. We review these cases of the association between hepatitis B infection and myositis. PMID- 10728452 TI - Factitious disorders mimicking systemic lupus erythematosus. AB - Factitious disorders are one of the most difficult challenges to the sagacity of the physician. Self-inflicted injuries and diseases have been recognized since Biblical times. In the Middle Ages, hysterics were known to place leeches in their mouths to simulate hemoptysis and to abrade their skin to reproduce skin conditions. Munchausen syndrome, originally described in 1951, is the term applied to persons who seek medical care by feigning illness in the absence of any organic medical or surgical disease. Since the first report of the syndrome, many case reports have documented the performance of unneeded operations and the administration of dangerous drugs to these patients. Rheumatologic manifestations in this syndrome are rare, and include septic arthritis, osteomyelitis, destructive arthropathy, reflex sympathetic dystrophy and systemic lupus erythematosus (SLE). We reviewed the English literature for the past 29 years and found only 8 patients with Munchausen syndrome who simulated SLE. It is interesting to consider the damage mechanisms (some of the patients satisfied 4 or more of the criteria for the classification of SLE) and how a complex disease with a broad spectrum of manifestations such as lupus can be simulated, the only limits being the patient's knowledge and imagination. PMID- 10728453 TI - Development of a Spanish (Castillian) version of the Childhood Health Assessment Questionnaire. Measurement of health status in children with juvenile chronic arthritis. AB - OBJECTIVE: To demonstrate that the Spanish (Castillian) version of the Childhood Health Assessment Questionnaire (cHAQ-S) is a valid and reliable instrument for measuring the health status of children with juvenile chronic (or rheumatoid) arthritis (JCA) and is sensitive to change. METHODS: A conceptual translation of the original questionnaire into Spanish and two back-translations were performed. The cHAQ-S was completed by the parents of young children (aged 1 to 19 years) affected by JCA, and additionally by those children aged over 9. A second cHAQ-S was administered at least 15 months after the first one. RESULTS: The cHAQ-S was administered to 79 patients of patients affected by JCA. The test-retest reliability was evaluated among 16 patients, and no significant differences between the first and second administration were found (0.88 versus 0.84; p > 0.6; intraclass correlation coefficient R = 0.94). The Cronbach's alpha coefficient was 0.948, indicating an excellent internal reliability with a mean correlation between the different components of the questionnaire varying from 0.3557 to 0.7831. For the between-observer reliability, an intraclass correlation coefficient of 0.96 was obtained. Correlations between DI (Disability Index) and several measures of disease activity were all statistically significant (Spearman's R ranged from 0.42 to 0.87; p < 0.005). Patients who improved showed similar improvement in the DI (p = 0.015), while patients who worsened showed a worsening of the DI (p = 0.1) and patients whose condition was stable showed no change in DI (p = 0.6). CONCLUSION: The cHAQ-S is a feasible, reliable and valid instrument for the determination of the health status of Spanish children suffering from JCA. It is also sensitive to changes in the child's health status. PMID- 10728454 TI - Living with a child with familial Mediterranean fever: does it affect the quality of life of the parents? AB - OBJECTIVE: The aim of the present study was to assess the quality of life (QOL) and the psychological status of parents of children with familial Mediterranean fever (FMF). METHODS: The QOL, anxiety and depression of the parents of 35 children with FMF were evaluated and compared to the parents of 23 healthy children. RESULTS: Mothers of FMF children had lower QOL scores than mothers of healthy children: 5.5 +/- 1.1 versus 6.0 +/- 0.6 (p = 0.048). They also expressed higher levels of anxiety and depression. Within each group, mothers were more anxious and depressed than fathers. Parents with several FMF children were not significantly different from parents with only one FMF child. CONCLUSION: The QOL and psychological well being of parents with FMF children were found to be slightly impaired, especially that of the mothers. PMID- 10728455 TI - Klinefelter's syndrome and systemic lupus erythematosus. PMID- 10728456 TI - Acute arthritis associated with acute Q fever. PMID- 10728457 TI - Calcium pyrophosphate dihydrate crystal deposition disease and primary hyperparathyroidism associated with rheumatoid arthritis: description of 3 cases. PMID- 10728458 TI - Absence of relation between TGF beta 1 serum levels and bone mass in ankylosing spondylitis patients. PMID- 10728459 TI - Intestinal obstruction associated with scleroderma: not always pseudo obstruction. PMID- 10728460 TI - Failure of aggressive anticoagulant therapy in catastrophic antiphospholipid syndrome. PMID- 10728461 TI - Systemic lupus erythematosus in Turkish men. PMID- 10728462 TI - Haematogenous vertebral osteomyelitis caused by Staphylococcus epidermidis: report of 4 cases. PMID- 10728463 TI - Current and novel agents for the treatment of cytomegalovirus retinitis. AB - The incidence of cytomegalovirus (CMV) retinitis appears to be declining with improved treatment protocols for HIV and prophylaxis protocols for other high risk patients. Despite this, CMV retinitis remains a major cause of visual loss in patients who are severely immunocompromised. Treatment options are evolving in the light of viral resistance, new methods of drug delivery, new drugs, and the increased potential for improvement of immune function that modern antiretroviral therapy provides. In this article, we review the changing options available for the management of CMV retinitis and discuss possible therapeutic agents for the future. PMID- 10728464 TI - Agents in development for cytomegalovirus infection. Summary and table. PMID- 10728465 TI - Adefovir dipivoxil. Bis-POM PMEA, GS 0840, GS 840, Piv2PMEA. PMID- 10728466 TI - Benzimidavir. 1263W94, BW 1263W94, GW 1263. PMID- 10728467 TI - Cytomegalovirus glycoprotein vaccine (Chiron). PMID- 10728468 TI - Cytomegalovirus vaccine live (Pasteur Merieux Connaught). PMID- 10728469 TI - GEM 132. Gene expression modulation 132. PMID- 10728470 TI - Sevirumab. Protovir, MSL 109, EV2 7, SDZ MSL 109. PMID- 10728471 TI - Valganciclovir. Cymeval, RO 1079070/194, RS 079070194, RS 79070. PMID- 10728472 TI - The emerging role of fusion inhibitors in HIV infection. AB - Fusion of HIV with its host cell requires the interaction of the viral envelope glycoprotein 120 (gp120) with the chemokine receptor CXCR4 [T cell-tropic (T tropic) or X4 HIV strains] or CCR5 [macrophage-tropic (M-tropic) or R5 HIV strains] followed by a 'spring-loaded' action of the glycoprotein 41 (gp41) that ensures fusion of the viral and cellular lipid membranes and permits the viral nucleocapsid to enter the cell. The overall fusion process can be blocked by a number of compounds. These include siamycin analogues, SPC 3 (a synthetic peptide derived from the V3 domain of gp120), pentafuside (T 20, DP 178) [a synthetic peptide corresponding to amino acid residues 127 to 162 of gp41], the betulinic acid derivative RPR 103611, TAK 779 (a low molecular weight non-peptide CCR5 antagonist) and a number of compounds (T 22, T 134, ALX40-4C, CGP64222 and AMD 3100) that are targeted at the CXCR4 receptor. In particular, the bicyclam AMD 3100 has proved highly potent and selective as a CXCR4 antagonist that blocks the infectivity of X4 HIV strains in the nanomolar concentration range. The proof-of concept that fusion inhibitors should be able to suppress viral replication in vivo has been demonstrated with pentafuside. Pentafuside and AMD 3100 have now proceeded to phase II clinical trials. PMID- 10728473 TI - Fusion inhibitors in HIV infection. Summary and table. PMID- 10728474 TI - AMD 3100. JM 3100, SDZ SID 791. PMID- 10728475 TI - Dextrin-2-sulfate. D2S, Emmelle, Viraldon. PMID- 10728476 TI - FP 21399. PMID- 10728477 TI - ISIS 5320. NSC 665353. PMID- 10728478 TI - Pentafuside. DP 178, T 20. PMID- 10728479 TI - PRO 542. CD4 IgG2. PMID- 10728480 TI - SPC 3. PMID- 10728481 TI - The new millennium: health care evolution in the 21st century. AB - The health care industry has evolved tremendously in recent years. It will experience even greater changes in the 21st century. Evolution will occur in health care delivery, system formation, and the roles played by government, the insurance industry, and organized business. Major focuses will be on management, competition, and quality. PMID- 10728482 TI - The ability of managed care to control health care costs: how much is enough? AB - This article argues that moral hazard is the main source of market failure in the health care sector. Cost sharing and managed care both are designed to control the extra costs of moral hazard. Managed care organizations (MCOs) have the potential to control costs by changing provider incentives away from excessive utilization of resources toward less costly and more effective treatments. However, MCOs have been given the wrong instructions by short-sighted employers who have overemphasized cost control. The solution is to give consumers more information and a choice of plans that emphasize different types and levels of cost control. PMID- 10728483 TI - The Medicare mix: efficient and inefficient combinations of social and private health insurance for U.S. elderly. AB - This paper explores two theories about the reason why the United States provides mixed public (Medicare) and private (Medigap) insurance for the elderly: that this represents an efficient combination of financing methods for a public good, or that it represents public provision of a private good for distributional reasons. It is argued that the first theory has more support than the second, but that the current configuration of Medicare and Medigap is not optimal. Two main problems are identified: the absence of coordination between public and private insurers, and the provision of overly generous coverage to the well off. Recent Medicare reforms have helped with the first problem, but the second remains to be solved. PMID- 10728484 TI - Criteria for the future division of labor between private and social health insurance. AB - This article's point of departure is that the individual has to manage three stochastic assets, namely health, wealth, and wisdom (skills), which tend to be positively correlated. It shows that the unexpected components of insurance payments should be negatively correlated for minimizing total asset volatility. The empirical finding is that in the United States, Japan, and Germany, the lines of social insurance contribute less to diversification than do those of private insurance. The article concludes with suggestions for new, umbrella-type insurance contracts that in the future should help individuals in the efficient management of their assets. PMID- 10728485 TI - Forecasting health expenditures: short, medium, and long (long) term. AB - As the forecasting perspective changes from short to medium to long run, the appropriate measure of health spending and the choice of which variables to hold constant and which to include changes. In the short run, current dollar spending is the best unit and the primary factors affecting spending are contracts (insurance, labor) and inflation. In the medium run, real per capita health expenditures are a better measure and prior growth of income is the primary factor. In the long run, the percentage of gross domestic product is the best measure and the structure of the health system and operation of budget constraints become the most important determinants. Accuracy of +/- 1-2 percent per year is obtainable. A major issue is whether the recent moderation in the rate of growth of spending is a pause or a turning point. PMID- 10728486 TI - Managing the cost of care: a predictive study to identify critical care patients at risk for nosocomial pneumonia. AB - Nosocomial infections represent a major health problem and can have a significant impact on the cost of treating a patient. Hospital-acquired pneumonia (HAP) is the second most common nosocomial infection in the United States and the leading cause of death due to a nosocomial infection. The high prevalence of HAP and its significant impact on increased length of stay and incremental treatment costs identify nosocomial pneumonia (NP) as a key component in managing the total cost of care. The study's objective was to develop a predictive tool for identifying those adult patients in critical care (CC) who are at greatest risk of developing NP to better manage the costs of care. The authors also expected to determine the expected probability of a patient developing NP in CC. A prospective study of longer stay critical care unit (CCU) patients was performed in nine U.S. CCUs. There were no interventions in the study. Development was based on variables common to CC and specific patient profile risk factors. Twelve statistically significant and clinically meaningful risk factors were identified and placed in a sequential cascade fashion. The positive predictive value of the sequential decision process and corresponding tool was 87.03 percent. PMID- 10728487 TI - Health care financing reform and the productivity change in Finnish hospitals. AB - The purpose of this study was to analyze productivity and efficiency changes in the production of hospital care in Finland during the period 1988-1994. A special interest was directed to years 1993-1994 because of health care financing reform that took place in 1993. The analysis is based on the Malmquist index approach using linear programming. Positive productivity changes were found particularly towards the end of the observation period, although a few years did not show significant improvements in productivity and efficiency. There was a significantly higher rate of productivity change in 1992-1993 and 1993-1994, suggesting that the state subsidy reform in 1993 may have strengthened hospitals' efforts to improve performance. PMID- 10728488 TI - Cellular pharmacokinetics: effects of cytoplasmic diffusion and binding on organ transit time distribution. AB - Distribution between well-stirred compartments is the classical paradigm in pharmacokinetics. Also in capillary-issue exchange modeling a barrier-limited approach is mostly adopted. As a consequence of tissue binding, however, drug distribution cannot be regarded as instantaneous even at the cellular level and the distribution process consists of at least two components: transmembrane exchange and cytoplasmic transport. Two concepts have been proposed for the cytoplasmic distribution process of hydrophobic or amphipathic molecules, (i) slowing of diffusion due to instantaneous binding to immobile cellular structures and (ii) slow binding after instantaneous distribution throughout the cytosol. The purpose of this study was to develop a general approach for comparing both models using a stochastic model of intra- and extravascular drug distribution. Criteria for model discrimination are developed using the first three central moments (mean, variance, and skewness) of the cellular residence time and organ transit time distribution, respectively. After matching the models for the relative dispersion the remaining differences in relative skewness are predicted, discussing the relative roles of membrane permeability, cellular binding and cytoplasmic transport. It is shown under which conditions the models are indistinguishable on the basis of venous organ outflow concentration-time curves. The relative dispersion of cellular residence times is introduced as a model independent measure of cytoplasmic equilibration kinetics, which indicates whether diffusion through the cytoplasm is rate limiting. If differences in outflow curve shapes (their relative skewness) cannot be detected, independent information on binding and/or diffusion kinetics is necessary to avoid model misspecification. The method is applied to previously published hepatic outflow data of enalaprilat, triiodothyronine, and diclofenac. It provides a general framework for the modeling of cellular pharmacokinetics. PMID- 10728489 TI - A combined specific target site binding and pharmacokinetic model to explore the non-linear disposition of draflazine. AB - The capacity-limited high-affinity target site binding of draflazine to the nucleoside transporters located on the erythrocytes is a source of nonlinearity in the pharmacokinetics of the drug. An attractive feature of draflazine is that the specific target site binding characteristics can be determined easily by simultaneously measuring plasma and whole blood concentrations of the drug. Measured drug concentrations following various infusion rates and infusion durations were used to develop a model in which the interrelated blood-plasma distribution, elimination, and specific target site binding of draflazine were incorporated simultaneously. The estimated binding (dissociation) constant Kd was 0.57 ng/ml plasma and the maximal specific erythrocyte binding capacity (BmaxRBC) was 163 ng/ml RBC. The maximal specific binding capacity to the tissues (Bmaxtissue) was estimated to be about 1 mg. The estimated volume of the central compartment (Vplasma + tissue fluids) was 12.9 L and the total intrinsic CL was 645 ml/min. After validation, the model was used to further investigate the impact of the specific high-affinity target site binding of draflazine on its disposition in plasma. The time required to reach steady-state plasma concentrations of draflazine decreased with an increasing infusion rate. Time profiles of the plasma concentrations were not always representative for the time profiles of the specific target site (RBC) occupancy of draflazine, but the t1/2,z in plasma paralleled that of the drug at target sites. The apparent Vd and the t1/2,z decreased with increasing single doses whereas the total CL remained constant. The recovery of draflazine was also dose dependent and increased with increasing doses. Finally, the total CL and apparent Vd of the first dose were greater than those of the second dose of draflazine. PMID- 10728490 TI - Analysis of antiplatelet effect of ticlopidine in humans: modeling based on irreversible inhibition of platelet precursors in bone marrow. AB - The relationship between plasma concentration of ticlopidine and its inhibitory effect on platelet aggregation in human was analyzed using a pharmacokinetic/pharmacodynamic (PK/PD) model. The data of plasma concentration and inhibitory effect on platelet aggregation were taken from the literature. A two-compartment open model was fitted to plasma ticlopidine concentrations. Assuming that ticlopidine acts on platelet precursors in the bone marrow, the apparent reaction rate constant of ticlopidine and platelet precursors (K), apparent transformation rate constant of platelet precursors (kr) and apparent elimination rate constant of platelets (ke) were estimated. The estimated values +/- S.D. were 1.01 +/- 1.08 ml micrograms-1 hr-1 for K, 0.265 +/- 0.259 hr-1 for kr and 0.0747 +/- 0.0112 hr-1 for ke. The antiaggregation effects of ticlopidine on platelets after administration of 100, 200, and 300 mg (bid for 8 days) were simulated using the PD parameters of K, kr, and ke. While the antiaggregation effect reached steady state within 3-4 days without dose dependency of the interval, the maximum effect increased with dose. Furthermore, changing the elimination rate constant of ticlopidine from the central compartment in the model significantly changed the duration of inhibitory effect of ticlopidine on platelet aggregation. Therefore, the reported long duration of antiplatelet effect after discontinuation of ticlopidine, which is believed to be irreversible binding to the platelet, might have been partially caused by the delayed plasma elimination after a long therapy of ticlopidine. On the other hand, the mean life span of platelets in the blood estimated by 1/ke after administration of ticlopidine was 14 hr, far below the life-span of platelets in the blood. For a more detailed analysis of the antiplatelet effect of ticlopidine, the possible contribution of reversible binding of the drug to glycoprotein IIb/IIIa should be considered in future PK/PD models. PMID- 10728491 TI - Predicting the time needed to achieve steady state if absorption and elimination constants are equal. AB - A simple table is derived to facilitate the rapid estimation of the number of dose administrations needed to achieve a certain fraction of the steady-state plasma concentration in the case of one-compartment model with uniform multiple oral dosing and equal absorption and elimination constants. PMID- 10728492 TI - Application of a combined "effect compartment/indirect response model" to the central nervous system effects of tiagabine in the rat. AB - Pharmacological inhibition of GABA uptake transporters provides a mechanism for increasing GABAergic transmission, which may be useful in the treatment of various neurological disorders. The purpose of our investigations was to develop an integrated pharmacokinetic-pharmacodynamic (PK/PD) model for the characterization of the pharmacological effect of tiagabine, R-N-(4,4-di-(3 methylthien-2-yl)but-3-enyl)nipecotic acid, in individual rats in vivo. The tiagabine-induced increase in the amplitude of the EEG 11.5-30 Hz frequency band (beta), was used as pharmacodynamic endpoint. Chronically instrumented male Wistar rats were randomly allocated to four groups which received an infusion of 3, 10, or 30 mg kg-1 of tiagabine or vehicle over 10 min. The EEG was continuously recorded in conjunction with frequent arterial blood sampling. The pharmacokinetics of tiagabine could be described by a biexponential equation. The pharmacokinetics of tiagabine were not dose dependent, and the pooled values for clearance, volume of distribution at steady state and terminal half-life were (mean +/- SE, n 23) 96 +/- 9 ml min-1 kg-1, 1.5 +/- 0.1 L kg-1 and 20 +/- 0.2 min. A time delay was observed between the occurrence of maximum plasma drug concentrations and maximal response. A physiological PK/PD model has been used to account for this time delay, in which a biophase was postulated to account for tiagabine available to the GABA uptake carriers in the synaptic cleft and the increase in EEG effect was considered an indirect response due to inhibition of GABA uptake carriers. The population values for the pharmacodynamic parameters characterizing the delay in pharmacological response relative to plasma concentrations were keo = 0.030 min-1 and kout = 81 min-1, respectively. Because of the large difference in these values the PK/PD model was simplified to the effect compartment model. Population estimates (mean +/- SE) were E0 = 155 +/- 6 microV, Emax = 100 +/- 5 microV, EC50 = 287 +/- 7 ng ml-1, Hill factor = 1.8 +/- 0.2 and keo = 0.030 +/- 0.002 min-1. The results of this analysis show that for tiagabine the combined "effect compartment-indirect response" model can be simplified to the classical "effect compartment" model. PMID- 10728493 TI - Rapid attainment of steady-state plasma drug concentrations within precise limits in multicompartment mammillary systems. AB - We have previously described a method of rapidly obtaining a specified steady state plasma concentration of an intravenous drug within precise limits. However the method is limited to drugs whose disposition may be characterized by an open two-compartment system. In this paper, we illustrate how the method can be extended to drugs whose disposition may be characterized by a mammillary model with any number of compartments. Refinements of our previous technique are also described. PMID- 10728494 TI - A compartmental analysis of the pharmacokinetics of propofol in sheep. AB - Conventional compartmental pharmacokinetic analysis may provide inaccurate prediction of drug concentrations after rapid i.v. administration. To examine this, compartment and effect compartment analysis was applied to measured arterial and brain concentrations of propofol in sheep after i.v. administration at a range of doses and dose rates. Although arterial and brain concentrations were reasonably well fitted to compartmental and effect compartment models for individual doses and dose rates, the structure and parameters of all models differed with changes in both dose and rate of administration. There were large discrepancies between predicted and measured arterial and brain concentrations when these models were used to predict drug concentrations across doses and dose rates. These data support the limitations of this type of modeling in the setting of rapid propofol administration. PMID- 10728495 TI - Therapeutic membrane plasmapheresis. 1981. PMID- 10728496 TI - An experimental artificial liver utilizing extracorporeal metabolism with sliced or granulated canine liver. 1963. PMID- 10728497 TI - Microemboli-free blood detoxification utilizing plasma filtration. 1976. PMID- 10728498 TI - New artificial liver support system (plasma perfusion detoxification) for hepatic coma. 1978. PMID- 10728499 TI - Efficient membrane and adsorbent for artificial liver support system. 1978. PMID- 10728500 TI - Double filtration plasmapheresis. 1980. PMID- 10728501 TI - On-line separation of macromolecules by membrane filtration with cryogelation. 1980. PMID- 10728502 TI - Therapeutic cryogel removal in autoimmune disease: what is cryogel. 1982. PMID- 10728503 TI - Clinical thermofiltration: initial application. 1985. PMID- 10728504 TI - Membrane plasma separation. 1983. PMID- 10728505 TI - An evaluation of polymethylmethacrylate plasma separator by animal experiment. 1983. PMID- 10728506 TI - Polyvinylalcohol membranes for plasma separation. 1983. PMID- 10728507 TI - Study of filtration characteristics of EVAL secondary filters in plasma diseases. 1985. PMID- 10728508 TI - Are nucleated red cells significant in patients with thrombotic thrombocytopenic purpura? PMID- 10728509 TI - Pitfalls in the diagnosis and management of systolic hypertension. AB - BACKGROUND: Systolic blood pressure (SBP) is even more important than diastolic blood pressure (DBP) with regard to the risk of cardiovascular complications. METHODS AND RESULTS: Pitfalls in the diagnosis of systolic hypertension include the auscultatory gap, use of the proper size cuff (obese adult size for mid-arm circumference >33 cm and child's cuff for mid-arm circumference <23 cm), a "white coat" effect of about 17 mm Hg, regression toward the mean, and a tendency to focus only on hypertension rather than all of the cardiovascular risk factors. Pitfalls in the pharmacologic management of systolic hypertension include being too aggressive with "acute" therapy, too fast in up-titration, too complacent about adverse effects, too unaware of important drug or food interactions, and too content with the achieved level of SBP. CONCLUSION: In treated hypertensives, SBP is typically less well controlled than DBP. Clinicians must not generally be content with partial control of SBP. PMID- 10728510 TI - Differential diagnosis of gallstone-induced complications. AB - Early recognition and prompt intervention are the most crucial steps in the management of gallstone-induced biliary disease. Many conditions can mimic the presentation of gallstone-induced complications. Therefore, participation of a clinically astute physician is essential in evaluating symptoms and interpreting diagnostic data in patients with symptomatic gallstones. PMID- 10728511 TI - Scalp laceration: an obvious 'occult' cause of shock. AB - Scalp lacerations are often present in patients requiring emergency care for blunt trauma. These injuries are most commonly seen in unrestrained drivers or occupants involved in motor vehicle crashes in which the victim is partially or totally ejected. Patients with scalp lacerations often have associated injuries that redirect the clinician's attention to other injury sites. Some scalp lacerations are severe enough to cause hypovolemic shock and acute anemia. If the patient arrives in shock, the perfusion pressure may be low, and there may be minimal active scalp bleeding. Under such circumstances, the scalp wound may be initially dismissed as trivial and attention appropriately turned to assuring an adequate airway, establishing intravenous lines, initiating volume resuscitation, and searching for more "occult" sources of blood loss. However, as the blood pressure returns toward normal, bleeding from the scalp wound becomes more profuse and presents a hemostatic challenge to the clinician. A case presentation illustrates some of these issues and confirms the effectiveness of an often overlooked but simple technique to control scalp hemorrhage--Raney clip application. PMID- 10728512 TI - The epilepsy of First Lady Ida Saxton McKinley. PMID- 10728513 TI - Pellagra in the United States: a historical perspective. AB - Pellagra was in existence for nearly two centuries in Europe before being recognized in the United States, where it was first reported in 1902. Over the next two decades, pellagra occurred in epidemic proportions in the American South. Poverty and consumption of corn were the most frequently observed risk factors. Since the exact cause and cure of pellagra was not known, a culture of "pellagraphobia" formed among the public. Patients were shunned and ostracized. The medical community implicated spoiled corn as the cause of pellagra, which had economic repercussions for agriculturists. Joseph Goldberger, MD, of the United States Public Health Service eventually solved the secret of the malady: faulty diet. Goldberger was able to prevent and induce pellagra by dietary modification, a landmark event in the annals of medicine, nutrition, and epidemiology. His work and the social history of that period are reviewed. PMID- 10728514 TI - Lipid profiles of obese children and adolescents before and after significant weight loss: differences according to sex. AB - BACKGROUND: Childhood obesity has been associated with elevated serum levels of total cholesterol, triglycerides, and low-density lipoproteins (LDLs). We observed the lipid profiles of obese female subjects versus obese male subjects before and after significant weight loss. METHODS: We studied 29 girls and 21 boys enrolled in a multidisciplinary weight reduction program. RESULTS: Measures were taken at enrollment and at 10 weeks. Significant improvements were observed for changes in percentage of ideal body weight and total cholesterol and triglyceride levels. In addition, LDL decreased significantly in girls but not in boys. CONCLUSIONS: A combination of diet, behavior modification, and exercise, is an effective instrument for lowering total cholesterol and triglyceride levels in obese children. In addition, girls tend to be more susceptible to a decrease in LDL level, which might result in an increased cardiovascular protective effect. PMID- 10728515 TI - Are patients with hyperlipidemia being treated? Investigation of cholesterol treatment practices in an HMO primary care setting. AB - BACKGROUND: The main study objective was to investigate cholesterol treatment practices of primary care physicians in a managed care setting. METHODS: The study was a retrospective review of data with a quasiexperimental design. The National Cholesterol Education Program-Adult Treatment Panel II (NCEP-ATP II) guidelines were used as the reference for conducting a measurement model in the study. Data were randomly selected via a systematic probability sampling method from a health maintenance organization (HMO) capitated risk-based contracting medical clinic in southern Florida. RESULTS: Of the 348 patients selected for the study, 224 (65%) needed either dietary therapy (n = 106) or drug therapy (n = 118). However, only 16 patients (13.6%) had ever had cholesterol-lowering drug regimens prescribed during the 5-year study period. CONCLUSIONS: Our findings indicate that (1) primary care physicians have poorly adopted the cholesterol management practice recommended by NCEP guidelines and need to improve their recognition and treatment of hypercholesterolemia; and (2) the problem of underutilizing prescription medications may be associated with risk-sharing capitation arrangements between physicians and third-party insurers. PMID- 10728516 TI - Initial experience with isradipine for the treatment of hypertension in children. AB - BACKGROUND: Isradipine is a calcium channel blocker of the dihydropyridine class. It has limited effects on myocardial contractility; is available in a powder filled capsule and has a half-life of 6 to 8 hours. METHODS: Pharmacy records were reviewed to identify pediatric patients who had received isradipine. The following demographic data were obtained: age, weight, sex, underlying medical problems, and initial blood pressure values. Information concerning isradipine included the initial dose and its interval, subsequent dose escalations, blood pressure response to the medication, and duration of therapy. RESULTS: The study population comprised 12 patients, ranging in age from 10 days to 11 years. The etiology of the hypertension was renal in 9 cases and nonrenal in 3. Initial dosing with isradipine was 0.1 mg/kg/dose. Six patients had emergent hypertension, and their blood pressure had been controlled with intravenous nicardipine before oral isradipine. Six patients received initial therapy with oral isradipine. Isradipine was monotherapy in 7 patients and in combination with other agents in 5 patients. The dose of isradipine required for blood pressure control was 0.6 +/- 0.3 mg/kg/day (range, 0.3 to 1.2 mg/kg/day). Isradipine failed to provide effective blood pressure control in 2 patients. In 1 of these patients, isradipine was effective after peritoneal dialysis. CONCLUSIONS: Isradipine is an effective, orally administered agent for control of hypertension in children. PMID- 10728517 TI - Caffeine in the treatment of apnea associated with respiratory syncytial virus infection in neonates and infants. AB - BACKGROUND: The xanthines have been shown to be effective in the treatment of apnea of prematurity. Limited reports are available in the literature concerning the use of these agents with apnea related to respiratory syncytial virus (RSV) infections. METHODS: A retrospective review was done to identify infants who received caffeine therapy for RSV-associated apnea. The number of apneic episodes during the 2 hours before the use of caffeine and the number of apneic episodes after the administration of caffeine were compared using a Wilcoxon nonparametric test. RESULTS: The 7 infants ranged in age from 14 to 64 days and in weight from 2.8 to 4.4 kg. The number of apneic episodes per hour for the 2 to 3 hours before the administration of caffeine ranged from 7 to 12, and the number of episodes during the 3 hours after the administration of the first dose of caffeine ranged from 0 to 2. The apneic episodes after caffeine responded to external stimulation. Apnea recurred in 3 infants, 18 to 24 hours after the first dose of caffeine. These infants received a second dose of caffeine (5 mg/kg). CONCLUSIONS: Caffeine should be considered in the treatment of apnea related to RSV infections in neonates and infants. PMID- 10728518 TI - Comparing the effectiveness of video and written material for improving knowledge among sleep disorders clinic patients with limited literacy skills. AB - BACKGROUND: Health care professionals often use written material or video recordings to teach their patients without knowing which is more effective for comprehension of the information. METHODS: Patients watched either an instructional videotape about sleep apnea or read a newly designed brochure, then responded to a structured questionnaire containing 11 knowledge-based questions and 1 open-ended question (requesting suggestions for improvement of the brochure or videotape). RESULTS: Mean reported educational level was grade 12, and mean reading level was between grade 7 and 8. Using video significantly improved only two areas of knowledge for low-level (below grade 8) readers: defining sleep apnea (66% vs 43%) and identifying what continuous positive airway pressure (CPAP) does for the patient (94% vs 78%). Patients requested material with more diverse cultural representation, more information on treatment and outcomes, and fewer polysyllabic words. CONCLUSIONS: Emphasis on diagnosis and treatment, explained using simple words, should be reflected in the content of patient education brochures or videos. Providing information by video alone may have limited benefits. PMID- 10728519 TI - Correlation between increased colloid osmotic pressure and the resolution of refractory ascites after transjugular intrahepatic portosystemic shunt. AB - BACKGROUND: This study compared the changes in serum albumin, globulin, and colloid osmotic pressure (COP) before and after transjugular intrahepatic portosystemic shunt (TIPS) or large volume paracentesis (LVP) in patients with ascites. METHODS: Of 23 patients with refractory ascites, 17 had TIPS and 6 had LVP with infusion of albumin. Colloid osmotic pressure measurements were calculated, using the formula previously proposed by Hoefs: COP = A (1.058G + 0.163A + 3.11) where A = serum albumin and G = serum globulin. RESULTS: After 1 month, ascites resolved in 9 of the 17 patients who had TIPS and in none of the 6 who had LVP. Colloid osmotic pressure increased significantly in patients whose ascites resolved after TIPS. Colloid osmotic pressure did not change in the patients whose ascites did not resolve after TIPS, and COP decreased significantly in the LVP group. A statistically significant difference was found in the pre-TIPS COP measurements between those patients who had resolution of ascites and those who did not. A pre-TIPS COP of < or =20 mm Hg predicted resolution of ascites with an 88% sensitivity and a 78% specificity. CONCLUSIONS: Serum COP increased significantly in patients with resolution of ascites but remained unchanged in patients with persistent ascites after TIPS. Serum COP decreased after LVP. A statistically significant difference in the pre-TIPS COP was found between patients whose ascites resolved and patients having persistent ascites. PMID- 10728520 TI - Suicidal behavior in psychiatric emergency room patients. AB - BACKGROUND: Psychiatric emergency room (ER) patients are thought to be at increased risk of suicide. The prevalence and characteristics of suicidal behavior in a recent sample of patients who came to the ER for psychiatric evaluation were examined. METHODS: Charts of 311 consecutive psychiatric ER patients were reviewed. Suicidal behavior was considered present if current suicidal ideation or attempts within 24 hours of or during the emergency evaluation were noted in the chart. RESULTS: Suicidal behavior was present in 38% of the psychiatric ER patients. Younger age, white race, affective disorders in female patients, and substance abuse disorders in male patients were features of the suicidal group. Sex of the patient was not associated with suicidal behavior. CONCLUSIONS: Suicidal behavior is prevalent in the psychiatric ER. Effective suicide prevention in this setting will hinge on finding more specific risk factors. PMID- 10728521 TI - Life-threatening subcutaneous hematoma caused by an axillary pad in an anticoagulated patient. AB - A large subcutaneous hematoma extending from the left axillary region to the left flank developed in a 70-year-old man receiving anticoagulant therapy. The cause was repeated microtrauma caused by the axillary pad on a walker. Physicians and physiotherapists should be aware that rehabilitation devices causing pressure on the skin increase hemorrhagic risk in patients taking anticoagulants. Accordingly, these patients should systematically be checked for hemorrhagic complications, and the use of such devices should be limited. PMID- 10728522 TI - Primary extra-nodal lymphoma of the urinary bladder. AB - Primary non-Hodgkin's lymphoma of the urinary bladder is rare. Only a few cases have been reported. We report the case of a woman with non-Hodgkin's lymphoma of urinary bladder and review the literature. PMID- 10728524 TI - Atypical presentation of microscopically advanced ovarian carcinoma. AB - Epithelial ovarian cancer (EOC) continues to be an academically challenging and clinically problematic disease. Even with recent advances, the overall 5-year survival is still 31% to 42% in various studies. Deaths from EOC outnumber those due to cervical, vulvar, and endometrial carcinomas combined. Screening for EOC has shown limited success in early detection. The Pap smear is not a dependable tool in EOC screening, though at times it can be the first evidence of ovarian disease. We report a case of EOC that was diagnosed during evaluation of an abnormal Pap smear. On completion of evaluation, stage IIIA endometrioid-type adenocarcinoma of the ovary was diagnosed. Occult EOC should be considered in patients with abnormal findings on cervical cytology after cervical and uterine carcinomas are ruled out. PMID- 10728523 TI - Beware of short-course therapy for Staphylococcus aureus bacteremia without a removable cause. AB - Staphylococcus aureus bacteremia has long been known to cause significant morbidity and mortality. The optimal treatment of this disease has evolved over the years. Recently, criteria have been established for the use of shorter courses of antibiotic therapy in certain patients, most notably those with an easily removed source of the bacteremia. We present the case of a 55-year-old man with S aureus bacteremia unrelated to an intravascular device. He was treated with "short-course" antibiotic therapy, and lumbar diskitis and an epidural abscess developed. PMID- 10728525 TI - Morphology of right ventricular paced beats in posterior myocardial infarction. AB - Premature ventricular beats or paced beats can unmask new or old myocardial infarction when sinus beats fail to exhibit the typical infarct pattern. Knowledge of their morphology is vital, since they may be the earliest or even the only evidence of a myocardial infarction in certain cases. Morphology of premature ventricular beats or paced beats in anterior, anteroseptal, and inferior infarct has been described but not in the setting of a posterior infarct. We describe the morphology of right ventricular paced beats in a patient with posterior infarct and discuss its significance. PMID- 10728526 TI - Successful thrombolytic therapy for massive pulmonary embolism. AB - The use and scope of thrombolytic therapy in the management of pulmonary embolism (PE) continues to evolve. The results of small studies suggest that thrombolytic therapy might have an impact on survival in massive PE with cardiogenic shock; however, no large studies to further this notion exist. Furthermore, the expanded application of thrombolytic therapy to patients with PE and right ventricular dysfunction (RVD) but without overt hemodynamic collapse remains controversial. We report successful use of the thrombolytic agent tissue plasminogen activator (tPA) in the management of life-threatening PE with RVD without overt cardiovascular collapse. We present evidence for the meritorious use of thrombolytic therapy in this category of PE patients. We believe that a broadened application of thrombolytic therapy to patients with PE and RVD but without cardiogenic shock, especially in younger patients, is beneficial and worth the risk. PMID- 10728527 TI - Pyomyositis in the acquired immunodeficiency syndrome. AB - Pyomyositis, a purulent infection of skeletal muscle, is usually caused by Staphylococcus aureus. Many cases of pyomyositis in human immunodeficiency virus (HIV) seronegative patients have been reported in North America and have been reviewed extensively. Moreover, pyomyositis has been reported in association with HIV infection in patients with or without the acquired immunodeficiency syndrome (AIDS). We describe two patients with pyomyositis and HIV and review the available English language literature. Leukocytosis and bacteremia tend to occur less frequently in those with HIV infection and pyomyositis. However, fever, S aureus infection, and bilateral involvement occur more frequently in HIV-positive patients. Antibiotic therapy together with surgical drainage or aspiration is usually sufficient. PMID- 10728528 TI - Improved sexual function in three men taking lamotrigine for epilepsy. AB - Little information exists about the effects of newer antiepileptic drugs (AEDs) on sexual function in men with epilepsy. We report a series of three male veterans whose sexual disorders improved with lamotrigine. All three had partial seizures. One patient was taking phenobarbital and gabapentin and complained of decreased potency and anorgasmia. After lamotrigine was added for better seizure control and the dosage of gabapentin was tapered, anorgasmia improved. The second patient complained of impotence after a rash while taking phenytoin and carbamazepine. Impotence persisted with phenobarbital, valproate, and gabapentin. Eight months after gabapentin was replaced with lamotrigine, impotence improved. The third patient complained of long-standing impotence. Treatment with five AEDs had no effect on the dysfunction. Lamotrigine was added to the carbamazepine regimen; impotence improved with decrease in carbamazepine and increase in lamotrigine. The favorable effect of lamotrigine on sexual disorders in these three patients suggests this drug should be considered under appropriate circumstances for men who have sexual dysfunction while taking other antiepileptic agents. PMID- 10728529 TI - Benign enterogenous cyst of the pancreas. AB - A 26-year-old woman was evaluated for a mass found by computed tomography (CT). Radiographically, the mass resembled a pancreatic cystadenoma. The patient had had left upper quadrant abdominal pain for several years and low grade fevers and indigestion for 5 months. At surgery, a unilocular cystic mass was found anteriorly and caudally to the tail of the pancreas in the lesser sac. The cystic structure was connected to the pancreas by a tubular structure, which was suture ligated and divided. A review of the literature revealed this cyst to be the fifth reported case of enterogenous cyst of the pancreas. The case is unique, differing from previous reports in that ciliated respiratory epithelium, transitional epithelium, gastric mucosa, a bilayered smooth muscle wall, and a serosal surface were present in the cyst wall. PMID- 10728530 TI - Recurrent Merkel cell carcinoma of the upper extremity. AB - We report a case of recurrent Merkel cell carcinoma (MCC) of the upper extremity, treated aggressively with wide local excision, regional lymphadenectomy, and immediate reconstruction. Five years after surgery, there is no clinical or diagnostic evidence of locoregional recurrence or distant disease. The patient's upper extremity and hand remain fully functional, without evidence of median or ulnar nerve dysfunction. No donor site morbidity has been noted. PMID- 10728531 TI - Editorial elaborations. PMID- 10728532 TI - Venting spleen: helmet legislation. PMID- 10728533 TI - Ragnar Berfenstam's legacy. PMID- 10728534 TI - Youth injury data in the Canadian Hospitals Injury Reporting and Prevention Program: do they represent the Canadian experience? AB - OBJECTIVE: Injuries to Canadian youth (11-15 years) identified from a population based health survey (World Health Organization-Health Behaviour in School-Aged Children Survey, or WHO-HBSC) were compared with youth injuries from a national, emergency department based surveillance system. Comparisons focused on external causes of injury, and examined whether similar rankings of injury patterns and hence priorities for intervention were identified by the different systems. SETTING: The Canadian version of the WHO-HBSC was conducted in 1998. The Canadian Hospitals Injury Reporting and Prevention Program (CHIRPP) is the national, emergency room based, surveillance program. Two hospitals involved in CHIRPP collectively provide population based data for Kingston, Ontario. METHOD: Numbers of injuries selected for study varied by data source: WHO-HBSC (n=3673); CHIRPP (n=20,133); Kingston CHIRPP (n=1944). WHO-HBSC and Kingston CHIRPP records were coded according to four variables in the draft International Classification of External Causes of Injury. Existing CHIRPP codes were available to compare Kingston and other CHIRPP data by five variables. Males and females in the three datasets were ranked according to the external causes. Data classified by source and sex were compared using Spearman's rank correlation statistic. RESULTS: Rank orders of four variables describing external causes were remarkably similar between the WHO-HBSC and Kingston CHIRPP (p>0.78; p<0.004) for mechanism, object, location, and activity). The Kingston and other CHIRPP data were also similar (p>0.87; p<0.001) for the variables available to describe external causes of injury (including intent). CONCLUSION: The two subsets of the CHIRPP data and the WHO-HBSC data identified similar priorities for injury prevention among young people. These findings indicate that CHIRPP may be representative of general youth injury patterns in Canada. Our study provides a novel and practical model for the validation of injury surveillance programs. PMID- 10728535 TI - Seasonal variations in incidence of fractures among elderly people. AB - OBJECTIVE: To investigate seasonal variations in the incidence of fall related fractures among people 65 years and older. POPULATION AND METHODS: A prospective, population based cohort study was performed on people aged 65 years and older followed up from 1990 to 1997, a total of 459,904 person years. Cases were identified through a prospective registration system. RESULTS: There were 10,992 (2390 per 100,000 person years) fall related fractures. The risk was higher in the colder seasons (October through March) among people aged 65-79 years (relative risk (RR) = 1.39, 95% confidence interval (CI) 1.32 to 1.47) and in people aged 80 years and older (RR = 1.17, 95% CI 1.09 to 1.22). For arm fractures, the RR was 1.69 (95% CI 1.56 to 1.83) among people aged 65-79 years and 1.30 (95% CI 1.13 to 1.43) among those aged 80 years and older. The RR for hip fractures was 1.27 (95% CI 1.15 to 1.37) among people aged 65-79 years and 1.08 (95% CI 1.00 to 1.15) for people aged 80 years and older. Slipping on ice and snow seems to entirely explain the excessive incidence of hip and arm fractures during winter months. CONCLUSION: Season affects the incidence of all types of fractures in elderly people. Slipping on ice and snow seems to be a causal mechanism behind the seasonal effect. Preventive measures targeting this causal mechanism are likely to reduce the risk of fracture, but the size of the effect is difficult to estimate with certainty. PMID- 10728536 TI - An international comparison of childhood injuries in Hong Kong. AB - OBJECTIVES: This study describes 7813 childhood injuries in Shatin, Hong Kong. Supplementary analyses include developmental specificity of external causes and comparison with international childhood injury data. METHODS: Children aged 0-15 attending the accident and emergency (A&E) department of the Prince of Wales Hospital in Hong Kong were recruited for the study. Attendance records of participants from the A&E department were analyzed. Details concerning the injury, including the International Classification of Diseases, ninth revision, external cause of injury (E code), nature of injury (N code), abbreviated injury scale, and injury severity scale constitute core measurements, along with participants' age, gender, and respective A&E procedural data. RESULTS: Males (65.7%) and fall related injuries (44.2%) predominate, while contusion (34.6%) is the prevailing nature of injury. Two age external cause dimensions are derived from a correspondence analysis. Children 0-1 years old are associated with falls, poisoning, scalds, and machinery related injury. Adolescents aged 12-15 are associated with motor related injury, animal related injury, and cuts/piercings. In comparison with international data, unintentional child injuries in Hong Kong comprised more falls but fewer poisonings and burns. CONCLUSION: A large proportion of falls, along with low proportion of poisonings and burns, are characteristics of childhood injury profile in Hong Kong. From the results of age external cause correspondence analysis, prevention strategies for different external cause should be developmentally specific. PMID- 10728537 TI - Child seating position and restraint use in three states. AB - OBJECTIVE: Because of risks from deploying airbags to children in front seats, extensive publicity has been aimed at getting them restrained and in rear seats. The objective of this study was to assess restraint use and seating positions among children in vehicles with and without airbags. METHOD: Surveys were conducted in cities in Michigan, North Carolina, and Texas 1998. Restraint use and seating position were noted for all children, as well as their estimated age, driver belt use, airbag presence, and vehicle license plate number. RESULTS: Fewer children were observed in the front seats of vehicles with passenger airbags (24%) than in vehicles without them (36%). Most of the children seated in front were ages 7-12 (44%-61%), followed by 3-6 year olds (29%-35%). Very few children ages 0-2 were seated in front (5%-12%). The overwhelming majority of children ages 0-2 were restrained. However, children ages 3-6 seated in the front were least likely to be restrained and most likely to be improperly restrained. Restraint use was higher when the driver was belted, but about 30% of 3-6 year olds were unrestrained even with a belted driver. CONCLUSIONS: Efforts should continue to educate parents about the importance of correct restraint use and rear seating for children, particularly once children move from child safety seats into adult belts. Efforts also should be made to enforce the seat belt laws that exist in every state. PMID- 10728539 TI - Increasing age and experience: are both protective against motorcycle injury? A case-control study. AB - OBJECTIVES: To assess the associations between age, experience, and motorcycle injury. SETTING: Motorcycle riding on non-residential roads between 6 am and midnight over a three year period from February 1993 in Auckland, New Zealand. METHODS: A population based case-control study was conducted. Cases were 490 motorcycle drivers involved in a crash and controls were 1518 drivers identified at random roadside surveys. Crash involvement was defined in terms of a motorcycle crash resulting in either a driver or pillion passenger being killed, hospitalised, or presenting to a public hospital emergency department with an injury severity score > OR =5. RESULTS: There was a strong and consistent relationship between increasing driver age and decreasing risk of moderate to fatal injury. In multivariate analyses, drivers older than 25 years had more than 50% lower risk than those aged from 15-19 years (odds ratio (OR) 0.46; 95% confidence interval (CI) 0.26 to 0.81). In univariate analyses, a protective effect from riding more than five years compared with less than two years was observed. However, this protection was not sustained when driver age and other potential confounding variables were included in the analyses. Familiarity with the specific motorcycle was the only experience measure associated with a strong protective effect (OR (> OR =10,000 km experience) 0.52; 95% Ci 0.35 to 0.79) in multivariate analyses. CONCLUSIONS: Current licensing regulations should continue to emphasise the importance of increased age and might consider restrictions that favour experience with a specific motorcycle. PMID- 10728538 TI - Epidemiology of unintentional drowning and near-drowning in Denmark in 1995. AB - OBJECTIVES: To determine the pattern of accidental drowning and near-drowning in Denmark. DESIGN: Prospective study of all cases of accidental drowning and near drowning during one year. SETTING: Denmark, 1995. SUBJECTS: All patients brought to Danish hospitals after incidents of unintentional near-drowning or cooling in water and all fatal cases. MAIN OUTCOME MEASURES: Number of near-drowned patients reported prospectively by hospital departments supplemented by cases reported after requests based on the National Patient Register. Number of drownings reported by public health medical officers (as medical examiners), institutes of forensic medicine, and hospitals. RESULTS: Sixty three (38%) of 167 unintentionally immersed persons died: eight (17%) of 47 children and 55 (46%) of 120 adults. The annual incidence of serious immersion events in children leading to hospital contact was 5.2/100,000; mortality was 0.7/100,000. For adult males the annual incidence of serious unintentional immersions was 4.3/100,000 and for females 1.2/100,000. For foreigners the risk was three to four times higher than for Danes. CONCLUSIONS: More attention should be paid to the risk of drowning in children, adult males, and foreigners. PMID- 10728540 TI - Effectiveness of toughened glassware in terms of reducing injury in bars: a randomised controlled trial. AB - OBJECTIVE: To evaluate the effectiveness, in terms of injury prevention, of toughened pint glassware in bars. DESIGN: Randomised controlled trial. SETTING: A random sample of 57 bars in South Wales, West Midlands, and West of England. SUBJECTS: A total of 1229 bar workers. INTERVENTION: Complete replacement of pint glasses with annealed (control) or toughened (intervention) glassware. MAIN OUTCOME MEASURES: Bar staff injuries recorded monthly: number, site, and severity (lifestyle impact; treatment need) of injuries. RESULTS: Ninety eight bar staff experienced 115 injuries: 43 in the control group, 72 in the intervention group. Adjusting for people at risk gave a relative risk (RR) of 1.48 (confidence interval (CI) 1.02 to 2.15). Similarly, adjusting for hours worked gave RR 1.57 (CI 1.08 to 2.29). Thus, injury rate was 60% higher in the intervention group (p<0.05), with no significant difference in severity. Most were hand injuries requiring first aid. Injuries tended to occur simultaneously in more than one body part in the intervention group, reportedly caused by spontaneous disintegration of toughened glassware. Impact resistance testing showed the energy required to break annealed glass (1.8 +/- 0.2 J) was greater than that for toughened glass (1.4 +/- 0.2 J), though the difference was not significant. CONCLUSIONS: Glass with lower impact resistance caused more injuries. "Toughened" glassware had lower impact resistance. Standards for toughening need to be developed. PMID- 10728541 TI - International comparisons of child injuries and prevention programs: recommendations for an improved prevention program in Germany. AB - OBJECTIVES: To compare child injury mortality in Germany with that of four neighboring countries, and to examine injury prevention models in these countries with a view to improving prevention programs in Germany. METHODS: Based on official cause of death certificates, child injury mortality rates in Germany are compared with those of Austria, The Netherlands, Sweden, and Switzerland. The main structures and funding of injury prevention programs in these countries are described. RESULTS: In all five countries, mortality is highest among children aged 1-4 years for home and leisure accidents and drownings. Transport accidents are the main cause of death in the 5-14 age group. Mortality in both age groups has fallen significantly since 1980, most markedly in Sweden and The Netherlands. CONCLUSION: Drawing on the injury mortality data and experience of the comparison countries, the following recommendations are proposed to further reduce home and leisure injuries among children in Germany: (1) establish a soundly funded, central institution responsible for child injury surveillance, research, and the coordination of injury prevention activities, (2) improve product control legislation, and (3) disseminate specific safety information to target groups and the general public. PMID- 10728542 TI - Children are not goldfish--mark/recapture techniques and their application to injury data. AB - OBJECTIVES: Mark/recapture (or capture-recapture) is a simple technique commonly applied to estimate the hypothetical total (including undercount) in a register composed of cases from two or more independent and separately incomplete case lists. This paper seeks to illustrate serious drawbacks in the use of the mark/recapture technique when applied to injuries. SETTING AND SUBJECTS: Northumbrian children under 15 years of age who were seriously injured in motor vehicle accidents (MVAs) over a five year period ascertained from two data sources: police reports and hospital inpatient records. METHODS: Individuals (n) appearing in both police (S) and hospital (H) case lists are identified using various matching criteria. The separate and combined influence of age, sex, and casualty class (cyclist, passengers, pedestrians) on the probability of such matching is estimated using multivariate techniques. The hypothetical total incidence of child MVA victims (N) is calculated from N = (S x H)/n. MAIN OUTCOMES: Estimates of the incidences of "serious" injuries in MVAs under various conditions of stratification and matching. The overall procedure is tested for conformity with accepted criteria for valid use of mark/recapture. RESULTS: About one third of the 1009 police and 836 hospital records could be exactly matched. There were significant variations in matching proportions by class of accident (pedestrian v passenger v cyclist). This selective recapture or "heterogeneity" was not affected by sex, but was independently influenced by the age of the child. Further uncertainty was introduced when matching criteria were slightly relaxed. Estimates of the total population of children with serious injuries vary accordingly from 1729 to 2743. A number of plausible reasons why these two data sources might not be unbiased or mutually independent samples of the total target population are proposed as explanations for this heterogeneity. CONCLUSION: This typical example of two sample mark/recapture estimation in an epidemiological setting can be shown to violate virtually all the requirements for valid use of the technique. Very little can be deduced accurately about the scale or characteristics of an unobserved group by the use of mark/recapture applied to two overlapping health event registers. PMID- 10728543 TI - Parental attitudes regarding interviews about injuries to their children. AB - BACKGROUND: Questions about the circumstances of injuries, especially to infants and young children, might be perceived by parents as threatening or intrusive. Some institutional research review committees express concerns that interviews about childhood injuries may be offensive to parents. The perceived value and potential risk of questions about a young child's injury could affect the quality of responses. OBJECTIVES: To assess parents' perceptions of threat and value of interviews about injury to their young children. SETTING: District of Columbia, 1 October 1995 to 30 September 1996. METHODS: Trained research assistants telephoned the parents of children seen in an emergency department or admitted to the hospital after an injury. To be eligible for inclusion the child must have been <3 years of age and a resident of the District of Columbia at the time of the event. After collection of sociodemographic information and circumstances of injury, the respondents were asked if the interview caused them to feel angry, offended or threatened, and if participation in the study was considered worthwhile. RESULTS: Seventy eight per cent of eligible families were contacted. Among those contacted, 93% completed the interview. Eighty two per cent of respondents were mothers and 11% fathers. Ninety per cent (95% confidence interval (CI) 88.4 to 91.6) of the respondents reported that the interview did not make them feel angry, offended, or threatened. Only 13 (1%; 95% CI 0.5 to 1.5) reported being very angry and 7.1% (95% CI 5.8 to 8.5) reported being a little angry. The majority of participants (61.2%, 95% CI 58.6 to 63.8) felt that participation in the study was definitely worthwhile and only 5.5% (95% CI 4.3 to 6.7) felt that it was not at all worthwhile. Parents of children with intentional injuries were more likely to report feelings of anger than parents of children with unintentional injuries (24% v 8%; p=0.02). The per cent of respondents reporting any anger was greater when the interview was conducted within 14 days of the hospital visit compared with later interviews (11% v 7%; p=0.02). CONCLUSIONS: In similar populations most parents of young, injured children are neither upset nor threatened by interviews that probe for details about how their children become injured. In general, collecting data aimed to prevent injuries is perceived as worthwhile, and parents readily cooperate with providing this information. Investigators and review committees should consider that interviews about infant and young child injuries are of no or minimal risk. PMID- 10728544 TI - Adult-worn child carriers: a potential risk for injury. AB - OBJECTIVES: To examine and describe types of injuries associated with adult-worn child carriers and illustrate the need for careful use of these products by parents. METHODS: A literature search for the terms infant carriers, backpack carriers, infant slings, baby carriers, and baby slings was conducted. Information was also obtained and tabulated from the three Consumer Product Safety Commission databases: the National Electronic Injury Surveillance System (NEISS), the In-Depth Investigations File, and the Injury/Potential Injury Incident File. RESULTS: No reports of injuries were found in the medical literature. In the NEISS database, 51 injuries were reported between January 1990 and September 1998. Of these injuries, 38 (74.5%) were head traumas and eight (15.7%) were facial trauma. Of the 51 injuries, 11 (22%) required hospitalization. CONCLUSIONS: Based on the data presented in this paper, injuries associated with the use of adult-worn child carriers appear to come from three general sources: product appropriateness and design, product condition, and product use. It is important for health care providers to assist in the dissemination of information regarding the safe use of these products to parents in an effort to prevent injuries. PMID- 10728545 TI - Equestrian injuries: a five year review of hospital admissions in British Columbia, Canada. AB - AIM: To determine the demographics of hospital admissions and mortality associated with equestrian activities in the 33,000 riders in British Columbia (BC). METHOD: Analysis of admission data from the Ministry of Health for the years 1991-96, review of information obtained from the Office of the Chief Coroner, and comparison of data from Canadian Hospitals Injury Reporting and Prevention Program. RESULTS: The mean number of admissions per year was 390. Head injury was the most common cause of admission to hospital (20%) in BC. Females most often required admission (62%). Teenagers and children have a higher incidence of head injuries than the general population. The injury rate was 0.49/1000 hours of riding. There were three deaths per year, 1/10,000 riders; 60% were caused by head injury and females predominated. CONCLUSION: Head injuries and other serious injuries occur with equestrian activities and it is important for doctors, instructors, and parents to promote the use of appropriate safety equipment, including helmets, especially for children. PMID- 10728546 TI - Mechanical analysis of survival in falls from heights of fifty to one hundred and fifty feet. 1942. PMID- 10728547 TI - Open invitation from the International Poverty and Health Network to all health professionals. PMID- 10728548 TI - Let's emphasize fire sprinklers as an injury prevention technology. PMID- 10728549 TI - Argument for accident and emergency (A&E) collection flawed. PMID- 10728550 TI - Foam party risks. PMID- 10728551 TI - Randomly amplified polymorphic DNA (RAPD) PCR for the identification of yeasts isolated from dairy products. AB - In the present work randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) with primers M13 and RF2 was applied to the identification at species level of yeast strains isolated from cheeses. RAPD-PCR analysis of the type strains of different yeast species gave distinctive band profiles that allowed a clear differentiation of all the considered species. Forty-two of the 48 dairy associated yeasts were clearly assigned to the species Saccharomyces cerevisiae, Kluyveromyces marxianus (anamorph Candida kefyr), Kluyveromyces lactis (anamorph Candida sphaerica), Debaryomyces hansenii (anamorph Candida famata), Yarrowia lipolytica and Torulaspora delbrueckii (anamorph Candida colliculosa). The method, which is rapid and easy to perform, could be a useful tool for the identification of yeasts present in dairy products. PMID- 10728552 TI - The mucus binding of Bifidobacterium lactis Bb12 is enhanced in the presence of Lactobacillus GG and Lact. delbrueckii subsp. bulgaricus. AB - The ability to adhere to mucosal surfaces is related to many probiotic health effects. In the presence of Lactobacillus GG or Lact. bulgaricus, the adhesion of Bifidobacterium lactis Bb12 to a mucus model was more than doubled. Other tested lactobacilli did not affect the adhesion, nor was the adhesion of the lactobacilli influenced by the bifidobacteria. Co-aggregation between Bif. lactis Bb12 and the tested lactobacilli was insignificant and does not explain the observed effect. The results suggest that combinations of probiotics strains may have synergistic adhesion effects. Such specific strain combinations should also be assessed in clinical studies. PMID- 10728553 TI - Analysis of ammonia-oxidizing bacteria populations in acid forest soil during conditions of moisture limitation. AB - Ammonia-oxidizer numbers decreased under conditions of moisture limitation in litter, fermentation and humus layers of forest soil in the field, but the extent of regrowth after rehydration varied between layers. Nitrosospira 16S rRNA genes were amplified from all layers, regardless of moisture content or soil pH which varied between 4.1 and 5.2. Nitrosomonas spp. were detected less often, but appeared to exhibit more rapid recovery than the Nitrosospira spp. when drought conditions were relieved by rainfall. PMID- 10728554 TI - Selection of microbial populations degrading recalcitrant hydrocarbons of gasoline by monitoring of culture-headspace composition. AB - A methodology was devised and was found useful for the selection of populations degrading recalcitrant hydrocarbons. The work was part of a programme aiming at developing knowledge of the intrinsic capacities of autochtonous microflorae of the environment for gasoline biodegradation. The methodology involved monitoring the progress of degradation in enrichment liquid cultures on the selected hydrocarbon by gas chromatographic analysis of CO2 production and O2 consumption. Populations degrading in particular o-xylene, 1,2,4-trimethylbenzene, cyclohexane were obtained. Concerning 2,2,4-trimethylpentane (isooctane), one microflora (and a pure strain derived from it) growing on this hydrocarbon were obtained from gasoline-polluted water. PMID- 10728555 TI - Osmoprotectants and cryoprotectants for Listeria monocytogenes. AB - Listeria monocytogenes is a foodborne pathogen that can grow in high osmotic strength environments and at refrigeration temperatures. Glycine betaine, proline betaine, acetylcarnitine, carnitine, gamma-butyrobetaine and 3 dimethylsulphoniopropionate all acted as osmoprotectants, as evidenced by an increase in growth rate of L. monocytogenes 10403S and Scott A when provided with these compounds, while being stressed in defined medium containing 0.7 M NaCl. These same compounds exhibited cryoprotective activity, as evidenced by increasing the growth rate of L. monocytogenes at 5 degrees C. Ectoine, hydroxy ectoine, pipecolic acid and proline were ineffective as osmoprotectants or cryoprotectants under these conditions. The presence of osmoprotectants and cryoprotectants in foods may provide compounds assisting L. monocytogenes to overcome the barriers of high osmotic strength and low temperature that otherwise control microbial growth. PMID- 10728557 TI - Biodetoxification of silver-cyanide from electroplating industry wastewater. AB - A bacterial consortium capable of utilizing metal-cyanides as a source of nitrogen was used to develop a microbiological process for the detoxification of silver-cyanide from electroplating wastewater. When the treatment was carried out in a 27-l rotating biological contactor (R3C) in continuous mode, the system could achieve > 99.5% removal of 0.1 mmol l(-1) silver-cyanide (approximately 5 mg l(-1) cyanide and 10 mg l(-1) silver) in 10 h with sugarcane molasses (0.1 ml l(-1)) as carbon source. The silver ions set free during biodegradation were efficiently adsorbed by the bacterial biomass. The RBC-treated effluent was found to be safe for discharge into the environment, as confirmed by chemical analysis and fish bioassay studies. PMID- 10728556 TI - Detection of GroEL in activated sludge: a model for detection of system stress. AB - GroEL is a ubiquitous constitutively synthesized protein that is also stress inducible. Activated sludge, which is a standard biological process used in wastewater treatment systems, is made up of a diverse microbial consortium. The synthesis of GroEL in activated sludge was significantly induced after heat (42 degrees C) shock. The increased level of GroEL expression was shown to be due to de novo protein synthesis. We have demonstrated a method which shows that stress proteins can be detected in activated sludge, and propose their use as specific indicators of system stress. PMID- 10728558 TI - Development of simple and efficient protocol for isolation of plasmids from mycobacteria using zirconia beads. AB - A two-step protocol has been developed for isolation of plasmids from recombinant mycobacteria via Escherichia coli. First either mycobacterial primary transformants or propagated cultures were lysed in a mini-bead beater using zirconia beads and the lysate thus obtained was used to transform E. coli recA mutant cells. Secondly, plasmid DNA was isolated from recombinant E. coli cells and analysed. Bead beating times of 2 min for Mycobacterium smegmatis, a rapid grower, and 4 min for M. bovis BCG, a slow grower, were found to be optimal for recovery of plasmid DNA. This protocol was also amenable to other mycobacterial species such as M. avium, M. fortuitum and M. tuberculosis H37Ra. Plasmid recovery from the recombinant M. bovis BCG using this protocol is approximately 300-fold higher than that reported for the electroduction method. PMID- 10728559 TI - Triton X-114 phase partitioning for the isolation of a pediocin-like bacteriocin from Carnobacterium divergens. AB - A new procedure combining Triton X-114 phase partitioning and cation exchange chromatography was developed to purify a bacteriocin from a complex culture medium. This pediocin-like bacteriocin, secreted by Carnobacterium divergens and named divercin V41, was entirely recovered in the lower detergent-rich phase whereas all other substances (compounds from culture medium, bacterial metabolites) remained in the upper detergent-poor phase. Subsequent cation exchange chromatography of the TX-114-rich phase allowed recovery of the pure active bacteriocin and also detergent removing. This new purification method is versatile, fast (only two steps) and can be carried out on whole broth. PMID- 10728560 TI - The development of an ultrasonic apparatus for the noninvasive and repeatable removal of fouling in food processing equipment. AB - A new ultrasonic apparatus operating at a frequency of 40 kHz was developed to dislodge biofilms from food processing equipment in order to assess the effectiveness of cleaning protocols. Sonication conditions to remove biofilms and quantification by ATP-bioluminescence are described. An industrial meat process was developed at the laboratory level to form a biofilm with industrial characteristics. Our results show that the biofilm removal by sonication during 10 s is reproducible and four times greater compared to the swabbing method (83% removal of fouling material against 20%). Unlike the swabbing method, this ultrasonic apparatus permitted the immediately demonstration of the inefficiency (within 1 min) of an industrial meat cleaning protocol. This apparatus is portable, easy to use and can be operated by unskilled users. PMID- 10728561 TI - A simple method for extraction of fungal genomic DNA. AB - We have developed a new, simple and effective method for extraction of fungal genomic DNA. The initial steps involved suspension of freeze-dried mycelium in buffer containing sodium dodecyl sulphate, detachment of DNA from polysaccharides by mild shearing, NaCl precipitation of polysaccharides and protein, chloroform extraction and ethanol precipitation. The ethanol precipitate was then subjected to a second round of mild shearing, NaCl precipitation, chloroform extraction and ethanol precipitation. The procedure required approximately 1 h to perform. The method yielded 8-32 microg of high molecular weight DNA per 30 mg of freeze-dried mycelium when tested on six fungal species: Aspergillus niger, A. flavus, Fusarium graminarum, Neotyphodium lolii, Penicillium citrinum and Rhizopus nigricanes. The DNA was digestible with EcoRI, HindIII, SalI and BamHI. For the slow-growing N. lolii, a modification of the method was developed that removed the agar residue from colonies grown on agar plates by centrifugation at 13 000 rev min(-1) in the presence of CsCl. The modified method yielded 1.5-2 microg of high molecular weight DNA per colony. PMID- 10728562 TI - Growth of Aeromonas species on increasing concentrations of sodium chloride. AB - The growth of 16 strains of Aeromonas, representing 12 species of the genera, were examined at different salt levels (0-1.71 M NaCl). All the strains grew on media with 0.34 M NaCl, and nine on media with 0.68 M. Two strains, Aer. enteropelogenes and Aer. trota, were able to grow on media with 0.85 M and 1.02 M NaCl, respectively. Comparison of the growth curves of Aer. hydrophila ATCC7966 and Aer. trota ATCC 49657 on four concentrations of NaCl (0.08, 0.34, 0.68 and 1.02 M) confirm the high tolerance of Aer. trota, and indicate that high concentrations of salt increase the lag time and decrease the maximum growth rate. However, both strains were able to grow, slowly, in at least 0.68 M NaCl, a sodium chloride concentration currently used as food preservative. PMID- 10728563 TI - Microbial populations and volatile compounds in the 'bone taint' spoilage of dry cured ham. AB - 'Bone taint' is one of the most important causes of spoilage of dry cured ham. This alteration is characterized by a foul-smelling odour. The microbial population and volatile compounds associated with incipient 'bone taint' were evaluated. Enterobacteriaceae species were found at levels of 7.94 x 10(5) cfu g( 1) in spoiled hams and were not detected in unspoiled hams. Serratia sp. and Proteus sp. are the main organisms characterized. The volatile compounds from the spoiled hams give rise to higher levels of ketones, alcohols and esters than unspoiled hams, that could be originated by microbial metabolism of the above bacteria. Thus, volatile and Enterobacteriaceae analyses should be used to determine this incipient spoilage in the meat industry. PMID- 10728564 TI - Hydrocarbon degradation and protease production by Nocardiopsis sp. NCIM 5124. AB - An actinomycete isolated from an oil-contaminated marine environment and identified as Nocardiopsis sp. degraded hydrocarbons and also produced extracellular protease. Conditions for crude oil degradation and simultaneous production of extracellular protease were studied. An alternative approach for bio-augmented clean-up of oil spills using a micro-organism capable of degrading hydrocarbons and recruiting organic nitrogen by producing proteases is reported. PMID- 10728565 TI - A rapid biochemical test to aid identification of Mycoplasma mycoides subsp. mycoides small colony (SC) strains. AB - The ability to utilize maltose, as determined by measurement of oxygen uptake, is used to differentiate Mycoplasma mycoides subsp. mycoides small colony (SC) and M. capricolum subsp. capripneumoniae (all strains negative) from other members of the M. mycoides cluster (M. mycoides subsp. capri, M. mycoides subsp. mycoides large colony (LC), M. capricolum subsp. capricolum; and bovine serogroup 7; 94% of strains positive). Rapid tests for maltose utilizing ability were developed, based on hydrolysis of a chromogenic alpha-glucosidase (maltase) substrate (p nitrophenyl-alpha-D-glucopyranoside, colourless) to give a brightly coloured product (p-nitrophenol, yellow). On agar plates, colonies of maltose-utilizing strains became coloured within 40 min. PMID- 10728566 TI - An RNA transcription-based amplification technique (NASBA) for the detection of viable Salmonella enterica. AB - Possession of mRNA is indicative of cell viability. RTPCR is not appropriate for mRNA detection as it cannot unambiguously detect mRNA in a DNA background. The alternative amplification technique, NASBA, avoids the disadvantages of RTPCR. We have devised a method for detection of viable Salmonella enterica. This involves NASBA amplification of mRNA transcribed from the dnaK gene. Amplification of mRNA extracted from viable and heat-killed cells from the same population produced consistent and highly significant (P > 0.01) differences between the respective signals. The signal obtained from viable cells was completely eradicated by RNase treatment, while PCR amplification of treated and untreated samples was unaffected, indicating that NASBA was unaffected by background DNA. PMID- 10728567 TI - Molecular identification of Dunaliella sp. utilizing the 18S rDNA gene. AB - The utilization of micro-algae for the production of food and fine chemicals is growing in importance. However, confusion of names and species makes comparison of results by different authors very difficult. In this work, five species of Dunaliella were characterized using their 18S ribosomal RNA genes. Conserved oligonucleotides complementary to 5' and 3' termini of the 18S rDNAs were designed and utilized to amplify theme, restriction fragment length polymorphism analysis of the polymerase chain reaction products was developed. Species specific primers were also designed and utilized to corroborate the identification. PMID- 10728568 TI - Quantification of micro-organisms in binary mixed populations by Fourier transform infrared (FT-IR) spectroscopy. AB - Fourier Transform Infrared (FT-IR) spectroscopy was used for the first time to determine the ratios of different microorganisms in mixtures. Exemplarily, systems composed of two food-associated yeast species (Saccharomyces cerevisiae/Hanseniaspora uvarum) and two yoghurt lactic acid bacteria (Lactobacillus acidophilus/Streptococcus salivarius ssp. thermophilus) were investigated. Determination of the cell number ratio in the lactic acid bacteria system was possible with a minimal prediction accuracy of +/- 16 ratio percentage points while the minimum accuracy of prediction in the yeast two-component system was +/- 4% (both at a 95% confidence level). These results show that FT-IR spectroscopy is potentially a rapid method for the quantification of cell ratios in mixtures of two different microorganisms, provided that the cell ratio does not drop below a certain, system-specific threshold. PMID- 10728569 TI - Phagocytosis of mycobacteria by U937 cells: a rapid method for monitoring uptake and separating phagocytosed and free bacteria by magnetic beads. AB - A human-derived monocytic cell line (U937) was induced to phagocytose Mycobacterium phlei by the addition of phorbol myristate acetate (PMA) to the culture medium for 50-60 h. Cells not treated with PMA were unable to phagocytose M. phlei. Magnetic beads enabled a rapid and highly efficient separation of phagocytosed and free bacteria to be achieved, an approach which is particularly useful if colony plating is used to enumerate bacterial survival within phagocytic cells. Fluorescence-activated cell sorting (FACS) analysis showed that 98% of U937 cells contained viable bacteria after 3 h. PMID- 10728570 TI - Motogenic and biosynthetic response of adult skin fibroblasts to TGF-beta isoforms (-1, -2 and -3) determined by 'tissue response unit': role of cell density and substratum. AB - We have recently demonstrated that the three principal mammalian isoforms of transforming growth factor beta (TGF-beta) exert distinct effects upon: (1) the migration of confluent adult fibroblasts into 3D gels of native type I collagen fibres (i.e. TGF-beta-1 and -2 had no apparent motogenic activity, whilst TGF beta-3 induced a dose-dependent stimulation of cell migration); and (2) the synthesis of hyaluronan (HA) by these cells is also affected by the TGF-beta isoforms in a manner which parallels their effect on cell migration. The objective of the present study is to elucidate the manner in which this differential activity of the TGF-beta-1, -2 and -3 may be modulated by experimental parameters. Data presented in this communication indicate that cytokine bioactivity is determined by a combination of cell density and the nature of the macromolecular substratum. Thus, we now report that all three TGF beta isoforms inhibit the migration of subconfluent cells in the collagen gel assay. Our data confirm that the migration of confluent cells is stimulated by TGF-beta-3 and further indicate that this motogenic activity is completely abrogated by either TGF-beta-1 or -2 when these are co-incubated with TGF-beta-3. In contrast to these results obtained using a native type I collagen substratum, all three isoforms stimulated adult fibroblast migration in the transmembrane assay (in which cells are adherent to a 2-D porous polycarbonate substratum). The precise effect of TGF-beta isoforms on HA synthesis was also affected by cell density and the nature of the substratum in a manner which paralleled their diverse effects on cell migration (i.e. stimulation, inhibition or no effect). Streptomyces hyaluronidase completely neutralized the TGF-beta-3-induced stimulation of confluent fibroblast migration, thus suggesting a mechanistic link between the cytokine-induced cell migration and HA synthesis under these conditions. Taken together, these data indicate that: (1) the bioactivity of TGF beta-1, -2 and -3 are determined by cell density, the macromolecular substratum and the presence of other cytokines; and (2) it is therefore necessary to define cytokine bioactivity within the context of a larger 'tissue response unit' which more fully defines the activity state of the target cell and its microenvironment. PMID- 10728571 TI - Ha-ras-transformation alters the metabolism of phosphatidylethanolamine and phosphatidylcholine in NIH 3T3 fibroblasts. AB - Cultured NIH 3T3 fibroblasts were employed to investigate the changes in the phospholipid metabolism induced by Ha-ras transformation. All phospholipid fractions were reduced in ras-transformed fibroblasts except phosphatidylethanolamine (PE). The incorporation of labeled choline and ethanolamine into phosphatidylcholine (PC), PE and their corresponding metabolites were elevated in a similar manner in the transformed cells. The enhanced uptake of choline and ethanolamine correlated with the activation of choline kinase and ethanolamine kinase. Similarly, the uptake of arachidonic, oleic and palmitic acids by PC and PE was higher in ras-cells. Acyl-CoA synthetases, which esterify fatty acid before their incorporation into lysophospholipids, were also activated. However, both CTP:phosphocholine cytidylyltransferase and CTP:phosphoethanolamine-chytidyltransferase were inhibited in the transformed cells. This fact, taken together with the observed activation of choline- and ethanolamine kinases, led to accumulation of phosphocholine and phosphoethanolamine, which have been presumed to participate in the processes of tumor development. PC biosynthesis seemed to be carried out through the CDP-choline pathway, which was stimulated in the oncogenic cells, whereas PE was more likely, a product of phosphatidylserine decarboxylation rather than the CDP-ethanolamine pathway. PMID- 10728572 TI - Apoptosis induced by gamma irradiation in peripheral blood mononuclear cells is not mediated by cytochrome-c release and only partially involves caspase-3-like proteases. AB - Caspase 3 has been shown to be actively involved in the apoptotic process in thymocytes after gamma-irradiation. We examined caspase 3 activation in mature peripheral blood lymphocytes (PBL) after gamma irradiation. Since the activation of caspase 3 is generally prceded by a decrease in mitochondrial membrane potential (delta psi m) and cytochrome c release, these two parameters were also examined. Apoptosis in PBL after a 5-Gy gamma irradiation, is characterized by a decrease in delta psi m, but surprisingly no release of cytochrome-c and only a weak caspase 3 activation was noticed. In contrast, staurosporin treated PBL showed a decrease in delta psi m with cytochrome-c release and a clear caspase 3 activation. We were unable to block the decrease in delta psi m with the caspase inhibitors zVAD-fmk or zDEVD-fmk after gamma irradiation, but DNA fragmentation as measured by the TUNEL assay was partially inhibited. Therefore, in gamma irradiated mature PBL, caspase-dependent and -independent pathways, but not cytochrome c, seem to be involved in the apoptotic process. PMID- 10728573 TI - Cell pairing and methylation in Tetrahymena thermophila are altered by exogenous homocysteine. AB - Homocysteine is causally associated with birth defects such as spina bifida, and with premature vascular disease. We have investigated the effects of homocysteine on a cell-cell interaction in a fundamental eukaryotic system, the free-living ciliate Tetrahymena. Exogenously added homocysteine inhibits cell pairing in a dose-dependent manner. These effects are exacerbated by adenosine, which by itself has little demonstrable influence on pairing. S-adenosylhomocysteine (SAH) is a product of the reaction between adenosine and homocysteine, and is an inhibitor of methyl transferases. We therefore predicted that protein methylation would be significantly inhibited by homocysteine. A direct test of that hypothesis involved a demonstration that incorporation of an isotopically labeled methyl group from methionine into proteins was significantly reduced by homocysteine. The undermethylated proteins are of low molecular weight, and might correspond to known methylatable signaling proteins. We show that vanadate, an inhibitor of protein phosphatase, also inhibits cell pairing, and that the effects of vanadate and homocysteine are additive. This is the first demonstration that methylation and possibly phosphorylation play a regulatory role in cell-cell interactions in ciliates. PMID- 10728574 TI - Annexin V relocates to the periphery of activated platelets following thrombin activation: an ultrastructural immunohistochemical approach. AB - We have previously shown biochemically that the physiological agonist thrombin can cause translocation of endogenous annexin V to a fraction containing all platelet membranes. This paper reports ultrastructural immunohistochemical data revealing that annexin V molecules localize with plasma membranes of blood platelets following thrombin activation. When ultrathin sections of resting platelets were examined by immunogold staining, annexin V was found to be cytosolic, having a generalized distribution throughout the platelet. After thrombin activation, annexin V became peripheral in location and plasmalemma association increased. Morphometric analysis of gold particles shows that annexin V relocates specifically to the plasma membrane and its underlying cytoskeleton following treatment with thrombin. In control platelets 6.1% +/- 0.78 of annexin V is present at the plasma membrane and 15.0% +/- 0.82 in the region corresponding to the membrane cytoskeleton (10-80 nm); after stimulation with 0.5 unit/ml thrombin for 2 min this increased to 16.7% +/- 0.22 and 40.4% +/- 0.53, respectively. PMID- 10728575 TI - Reactive oxygen species induce apoptosis of synoviocytes in vitro. Alpha tocopherol provides no protection. AB - Reactive oxygen species (ROS) are released during the inflammation of the synovial membrane associated with cartilage degradation in osteoarthritis. In this work, we exposed synoviocytes to superoxide anions at concentrations that may cause either apoptosis or necrosis. We studied membrane organization, dehydrogenase mitochondrial activity and nuclear morphology and integrity, to determine the nature of the death process initiated by superoxide anions and tried to counteract ROS effects with alpha-tocopherol. We found that oxidative stress caused synoviocytes to undergo a process of cell death of an apoptotic nature rather than necrotic. Mitochondrial injury occurred at an early stage, and the FITC-annexin-V-positive/propidium iodide-positive cells occurred later than the metabolic changes. DNA strand breaks were evident at 8 h and nuclear condensation at 24 h. No LDH activity was detected in culture supernatants. In our experimental conditions, alpha-tocopherol had little effect on stress damage; the antioxidant properties of this molecule did not affect the apoptosis caused by superoxide anions. PMID- 10728576 TI - Gender-related differences in adhesion, growth and differentiation of vascular smooth muscle cells are enhanced in serum-deprived cultures. AB - In 1-day cultures with 10% serum, the number of rat aortic smooth muscle cells (VSMC) adhering to the growth support was similar in cells from both sexes, whereas in 1% serum, the number of VSMC from male donors was lower. In 10% serum medium, the doubling time was significantly shorter and the number of [3H]thymidine-labelled nuclei was higher in cells of high passage from male rats. In serum-free medium, these differences increased and were also seen in cells of low passage number. Morphologically, the cells in male-derived cultures at higher passage number were mainly spindle-shaped, formed well-developed 'hills and valleys' and possessed longitudinally oriented bundles of alpha-actin-containing microfilaments. Most cells from female rats were flat, polygonal, the multilayered 'hills' were less prominent, with alpha-actin microfilaments forming a mesh-like network. PMID- 10728577 TI - "Is there a need for blood substitutes in the new millennium and what should we expect in the way of safety and efficacy?". PMID- 10728578 TI - "Is there a need for blood substitutes in the new millennium and what should we expect in the way of safety and efficacy?". PMID- 10728579 TI - "Is there a need for blood substitutes in the new millennium and what should we expect in the way of safety and efficacy?". PMID- 10728580 TI - A model of stepwise isovolaemic blood exchange in anaesthetised, spontaneously breathing rats to evaluate the oxygen transport efficiency of artificial oxygen carriers. AB - Our research pursues the production of hypo-oncotic artificial oxygen carriers, based on artificial covalently cross-linked hyperpolymeric mammalian haemoglobins. To evaluate their in vivo efficiency in oxygen delivery to the tissue we developed a small animal model of stepwise isovolaemic blood exchange in anaesthetised, spontaneously breathing rats. With the aid of a two-way respiratory micro valve for small animals the overall oxygen uptake by the tissue of the animal can be determined. Measurements of oxygen contents in arterial and mixed venous blood and of some further blood parameters together with known oxygen-binding characteristics of artificial and native oxygen carriers, permits the determination of the way the two oxygen carriers contribute to the overall oxygen uptake. These so-called partial oxygen net to transport rates (i.e. partial oxygen uptakes), related to the corresponding intravascular mass flow of the transporters, are characteristic measures of the efficiency of the oxygen transporter, the so-called oxygen transport quality. Other biological indicators for an adequate oxygen supply are oxygen-dependent changes of ventilation, cardiac output, heart rate, and systemic vascular resistance. The performance of artificial oxygen carriers is elucidated by a comparison with experimental results from the analogous treatment of rats with non oxygen-transporting plasma expanders. PMID- 10728581 TI - Non-invasive evaluation of the location, the functional integrity and the oxygen supply of implants: 19F nuclear magnetic resonance imaging of perfluorocarbon loaded Ba2+-alginate beads. AB - 19F nuclear magnetic resonance imaging (MRI) can be used as a non-invasive tool to simultaneously determine the location, the integrity and the oxygen supply of Ba2+-alginate implants. This requires that the beads (implants) are pre-loaded with the perfluorocarbon compound F-44E. Implantation of solid 19F-labelled beads into the peritoneum, below the kidney capsule or into the muscle of Wistar WU rats demonstrated that these beads could be detected by 19F-MRI for up to 18 months after implantation. This indicated that F-44E is not considerably released from the beads during implantation. The signal to noise ratio of liquid-core beads was higher by a factor of 4 than the signal to noise ratio of solid beads, but liquid-core beads were more fragile and also too large for implantation under the kidney capsule and into the intramuscular tissue. Quantitative 2-dimensional 19F-T1 maps (resolution 0.5 x 0.5 mm) could be deduced from 19F-MRI measurements. These T1-maps correlated to the local pO2-values. The partial oxygen pressure estimated in F-44E-loaded Ba2+-alginate beads showed that the oxygen supply inside the beads was very poor when they were implanted below the kidney capsule or into the peritoneal cavity. These low pO2-values obtained for the renal subcapsular site and the peritoneum may explain the failure of previous immunoisolated islet transplantation studies using these locations. PMID- 10728582 TI - The collagen-containing alginate/poly(L-lysine)/alginate microcapsules. AB - A method of preparing microcapsules containing collagen fibrous network is reported in this study. This method takes advantage of miscibility of collagen and alginate and the ability of this mixture to form spherical gel beads in the presence of CaCl2. Collagen was then reconstituted within the microcapsules at 37 after alginate was liquefied with citrate. GH3 rat pituitary tumor cell, which can be cultured in both suspended and attached forms, were entrapped within the microcapsules. The cell proliferated faster in the collagen-containing capsule as compared to those in the conventional microcapsules. PMID- 10728583 TI - Attachment of 3T3 and MDBK cells onto poly(EGDMA/HEMA) based microbeads and their biologically modified forms. AB - Poly(EGDMA/HEMA) based microbeads were prepared by suspension polymerization. A comonomer, i.e., 2-hydroxyethylmethacrylate (HEMA) was included in the recipe in order to have functional hydroxyl groups on the microbead surfaces. Toluene was used in the polymerization formulations to introduce porosity into the matrix. Hydroxyl groups were first oxidized with NaIO4, and then two biological molecules, namely collagen and fibronectin were immobilized by using glutaraldehyde. A spacer-arm, i.e., hexamethylene diamine, was also used in some cases. More protein molecules were immobilized onto more swellable microbeads using spacer-arm. Higher amounts of collagen were immobilized, more than fibronectin immobilization. Attachment of two cell lines (i.e., 3T3 and MDBK cell lines) on these microbeads with a wide variety of surface properties was studied in vitro culture media. Attachments of both cells even onto the plain microbeads were significant. More cells did attach to more swellable microbeads. Introducing both fibronectin and collagen onto the microbeads caused significant increase in the cell attachment. More cells attached to the microbeads carrying fibronectin covalently attached onto the microbeads through the spacer-arm molecules. Fibronectine was better than collagen for high attachment values. The mathematical model proposed successfully simulated attachment kinetics. PMID- 10728584 TI - Improved controlled release study of lomustine. AB - Lomustine (CCNU) microcapsules was prepared by improved recoacervation method, then mixed microcapsules with 0.7% collagen swelling solution to prepare the emulsion, spreaded the emulsion on the plate to form membrane and cross-linked it, the membrane would be planted into body and was expected to release at steady speed. The concentration of CCNU and the CCNU content of microcapsules were measured by ultraviolet spectrophotometry to observe the release of CCNU be slow and constant, approach to 0-class release approximately. PMID- 10728585 TI - In vitro pathological model of osteopenia to test orthopaedic biomaterials. AB - The association of in vitro tests and in vivo bone implants, has significantly improved the characterization of biomaterials for orthopaedic devices before their clinical use. However, neither cell cultures nor most animals models used for these tests entirely reflect the clinical conditions in which biomaterials are implanted. Pathological animal models are considered to substantially improve our knowledge of osteointegration of biomaterials; for this reason researchers increasingly use aged, osteopenic or arthritic animals in their experimental tests. The development of "pathological cell cultures" would also be of great importance for the study of biomaterials. It would allow a complete material evaluation, beginning with a biocompatibility test to a more finalized and specific preclinical evaluation. The present study, looks at the possibility of using cell culture methodology for the improvement of in vitro biomaterials characterization in the case of osteopenia. Cultures derived from normal (NB-OST) rats were compared to those of osteopenic (OB-OST) rats, by testing the osteoblasts against common parameters of characterization. Moreover, the reaction of these cultures to two biological glasses of known in vivo behavior (both in normal and osteopenic bone) by means of parameters on biocompatibility and bone formation index, was evaluated. Our results showed that there was no evidence of differences between the NB-OST and OB-OST cultures. After 6 days of culturing, the bioglass that did not osteointegrate in osteopenic animals, did not induce cytotoxicity in NB-OST, but a significative reduction of viability and Osteocalcin level in OB-OST was observed. We think that these data should stimulate researchers to develop further tests in order to improve preliminary in vitro comprehension on biomaterials. PMID- 10728586 TI - Biostimulation of human chondrocytes with Ga-Al-As diode laser: 'in vitro' research. AB - The aim of this study was to verify the effects of laser therapy performed with Ga-Al-As Diode Lasers (780 nm, 2500 mW) on human cartilage cells in vitro. The cartilage sample used for the biostimulation treatment was taken from the right knee of a 19-year-old patient. After the chondrocytes were isolated and suspended for cultivation, the cultures were incubated for 10 days. The cultures were divided into four groups. Groups I, II, III were subject to biostimulation with the following laser parameters: 300 J, 1 W, 100 Hz, 10 min. exposure, pulsating emission; 300 J, 1 W, 300 Hz, 10 min. exposure, pulsating emission; and 300 J, 1 W, 500 Hz, 10 min. exposure, pulsating emission, respectively. Group IV did not receive any treatment. The laser biostimulation was conducted for five consecutive days. At the end of the treatment, the Calcium, Alkaline Phosphate, MTT tests and proteoglycan were performed to assess cell metabolism and toxicity level. The data showed good results in terms of cell viability and levels of Ca and Alkaline Phosphate in the groups treated with laser biostimulation compared to the untreated group. The results obtained confirm our previous positive in vitro results that the Ga-Al-As Laser provides biostimulation without cell damage. PMID- 10728587 TI - Environmental and genetic determinants of aflatoxin-albumin adducts in the Gambia. AB - Aflatoxins together with chronic hepatitis B virus (HBV) infection contribute to the high incidence of hepatocellular carcinoma in developing countries. An understanding of the mechanism of interaction between these factors would provide a strong rationale for developing effective prevention strategies. In this study in The Gambia we examined the effect of environmental (place of residence and timing of sample collection) and host factors (age, sex, HBV status and interindividual variations in carcinogen metabolising enzymes) in determining blood aflatoxin-albumin adduct levels in 357 individuals of whom 181 were chronic HBV carriers. Samples were analysed for aflatoxin-albumin adducts, HBV status and genotypes of glutathione S-transferase (GST) M1, GSTT1, GSTP1 and epoxide hydrolase (EPXH). Urine samples were analysed for 6beta-hydroxycortisol:cortisol ratio as a marker of cytochrome P450 (CYP) 3A4 activity. Adduct levels were significantly higher in subjects resident in rural [geometric mean adduct level 34.9 pg aflatoxin B1-lysine equivalent (28.5-42.8; 95%CI)/mg albumin] than in periurban areas [22.2 pg (14.9-33.4)/mg] and were approximately twice as high in the dry season [mid-February to March; 83.2 pg (53.3-130.8)/mg] than the wet [July to August; 34.9 pg (28.5-42.8)/mg]. In contrast, HBV status, CYP3A4 phenotype, GSTT1, GSTP1 and EPXH genotypes were not associated with aflatoxin albumin adduct level. However, mean adduct levels were significantly higher in non-HBV infected subjects with GSTM1 null genotype. The main factors which affect aflatoxin-albumin adduct levels in this population are environmental, notably place of residence and timing of sample collection. This study further emphasises the priority to reduce aflatoxin exposure in these communities by primary prevention measures. PMID- 10728588 TI - Involvement of genotoxic effects in the initiation of estrogen-induced cellular transformation: studies using Syrian hamster embryo cells treated with 17beta estradiol and eight of its metabolites. AB - To examine a direct involvement of genotoxic effects of estrogens in the initiation of hormonal carcinogenesis, the abilities of 17beta-estradiol (E2) and 8 of its metabolites to induce cellular transformation and genetic effects were studied using the Syrian hamster embryo (SHE) cell model. Treatment with E2, estrone (E1), 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), 2 methoxyestrone (2-MeOE1), 16alpha-hydroxyestrone (16alpha-OHE1), 2 hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2) or estriol (E3) for I to 3 days inhibited SHE cell growth in a concentration-dependent manner. Concentration dependent increases in the frequency of morphological transformation in SHE cells were exhibited by treatment for 48 hr with each of all estrogens examined, except for E3. The transforming activities of the estrogens, determined by the induced transformation frequencies, were ranked as follows: 4-OHE1 > 2-OHE1 > 4-OHE2 > 2 OHE2 > or = E2 or E1 > 2-MeOE1 or 16alpha-OHE1 > E3. Somatic mutations in SHE cells at the Na+/K+ATPase and /or hprt loci were induced only when the cells were treated with 4-OHE1, 2-MeOE1 or 4-OHE2 for 48 hr. Some estrogen metabolites induced chromosome aberrations in SHE cells following treatment for 24 hr. The rank order of the clastogenic activities of the estrogens that induced chromosome aberrations was 4-OHE1 > 2-OHE1 or 4-OHE2 > 2-OHE2 > E1. Significant increases in the percentage of aneuploid cells in the near diploid range were exhibited in SHE cells treated for 48 hr or 72 hr with each of the estrogens, except for 4-OHE1 and E3. Our results indicate that the transforming activities of all estrogens tested correspond to at least one of the genotoxic effects by each estrogen, i.e., chromosome aberrations, aneuploidy or gene mutations, suggesting the possible involvement of genotoxicity in the initiation of estrogen-induced carcinogenesis. PMID- 10728589 TI - Functional activation of integrin alpha V beta 3 in tumor cells expressing membrane-type 1 matrix metalloproteinase. AB - Matrix metalloproteinases (MMPs) and integrins have been implicated in a variety of processes involved in tumor progression. To evaluate the individual roles of integrin alphavbeta3 and membrane-type 1 matrix metalloproteinase (MT1-MMP), as well as the effects of their joint expression on tumor cell functions, MCF7 breast carcinoma cells were transfected stably with either the MT1-MMP, the beta3 integrin subunit or both MT1-MMP and beta3 cDNAs. MT1-MMP expression is accompanied by the functional activation of integrin alphaVbeta3, thereby increasing vitronectin-mediated adhesion and migration of MCF7 cells transfected with MT1-MMP and integrin alphaVbeta3. MT1-MMP-dependent functional activation of alphaVbeta3 correlates with modification(s) of the beta3 subunit, including its higher electrophoretic mobility and affected the LM609-binding site. MCF7 cells jointly expressing MT1-MMP and alphaVbeta3 were the most efficient in adhesion to the recombinant C-terminal domain of MMP-2 as well as in generating soluble and cell surface associated mature MMP-2 enzyme. These findings suggest a mechanism of selective docking of MMP-2 at tumor cell surfaces, specifically at the sites that include MT1-MMP and activated integrin alphaVbeta3. These mechanisms may provide a link between spatial regulation of focal proteolysis by the cell surface associated MMPs and the regulation of integrin-mediated motility of tumor cells. PMID- 10728590 TI - Clinicopathologic significance of urokinase receptor- and MMP-9-positive stromal cells in human colorectal cancer: functional multiplicity of matrix degradation on hematogenous metastasis. AB - Our previous clinicopathologic study revealed an inverse association of liver metastasis of colorectal cancer and stromal expression of matrix metalloproteinase-9 (MMP-9) or urokinase receptor (uPAR). This suggests that host cells, particularly macrophages, expressing matrix-degrading enzymes/factors could be protective for the host against hematogenous metastasis. However, our previous study was unable to differentiate whether our results were causes or effects of widely spread cancer. To solve this point, we designed the present study on colorectal cancers that developed hematogenous metastasis after operation, ie., metachronous hematogenous metastasis. These cancers, being solely micrometastasized at the time of operation, allowed us to eliminate possible systemic effects by widely spread cancer. Sixty-two primary tumors with metachronous metastasis showed a decreased number of MMP-9+ stromal cells and CD68+ macrophages along the invasive margin with unchanged uPAR+ stromal area as compared with those in 72 control cases, which were free from tumor metastasis or recurrence for more than 5 years. Therefore, we judged the decrease of MMP-9+ host cells or macrophages in the primary site is irrelevant of effects of widely spread metastasis but probably related to causes of metastasis. Our data also characterized the metachronous metastasis group by uPAR expression in fibroblasts. The number of uPAR+ cancer cells, although small in number, were also larger in the metachronous metastasis group. Our data revealed that macrophages, a major source of uPAR and one of the sources of MMP-9, could be inhibitory to hematogenous metastasis, while uPAR+ fibroblasts and cancer cells, in turn, facilitate hematogenous metastasis. This suggests the functional multiplicity of matrix degradation processes in cancer tissue. PMID- 10728591 TI - Nitric oxide promotes murine mammary tumour growth and metastasis by stimulating tumour cell migration, invasiveness and angiogenesis. AB - The contributory role of nitric oxide (NO) on tumour growth and metastasis was evaluated in a murine mammary tumour model. NO synthase (NOS) protein expression levels were examined in spontaneously arising C3H/HeJ mammary adenocarcinomas and respective lung metastases. In addition, 2 clonal derivatives of a single spontaneous tumour differing in metastatic phenotype (C3L5 and C10; highly and weakly metastatic, respectively) were utilised to investigate (i) the relationship between NOS expression levels and the biological behaviour of tumour cells (e.g., in vitro migratory and invasive capacities, in vivo tumour growth rate and metastatic and angiogenic capacities) and (ii) whether tumour-derived NO stimulated the invasive, migratory and angiogenic capacities of tumour cells. A heterogeneous pattern of endothelial NOS (eNOS) expression was observed in tumour cells in spontaneous primary tumours, and eNOS expression was higher in undifferentiated relative to differentiated tumour zones. However, tumour cells in lung metastatic sites were always strongly eNOS-positive, suggesting that eNOS expression facilitated metastasis. Findings using clonal derivatives supported this notion; s.c. primary tumour growth rate, efficiency of spontaneous metastasis and eNOS expression were higher for C3L5 relative to C10 cell lines. Nevertheless, lung metastases derived from both tumour cell lines were always strongly and homogeneously eNOS-positive. C3L5 cells were more invasive than C10 cells in vitro, but the migratory capacities of the cell lines did not differ. However, migration and invasiveness of both cell lines were inhibited with L-NAME and restored with excess L-arginine. Tumour-associated angiogenesis, measured in Matrigel implants inclusive of tumour cells, was higher for C3L5 relative to C10 cells, and C3L5-induced angiogenesis was reduced with chronic L-NAME treatment of host animals. These findings suggest that tumour-derived eNOS promoted tumour growth and metastasis by multiple mechanisms: stimulation of tumour cell migration, invasiveness and angiogenesis. PMID- 10728592 TI - Androgen receptor and vitamin D receptor in human ovarian cancer: growth stimulation and inhibition by ligands. AB - The data suggest that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and androgens are essential for regulation of growth and differentiation in, e.g., human reproductive tissues. We investigated the possible cross-talk between 1,25(OH)2D3 and androgens in the human ovarian cancer cell line OVCAR-3. Our data demonstrate that 1,25(OH)2D3 and androgen (dihydrotestosterone, DHT) regulate the growth of OVCAR-3 cells. Nine days' treatment of OVCAR-3 cells with 100 nM DHT resulted in 48% stimulation of growth, whereas growth inhibition (73%) was observed after treatment with 100 nM 1,25(OH)2D3. The combination of 1,25(OH)2D3 and DHT showed that 1,25(OH)2D3 clearly reduces the growth-stimulatory effect of DHT on OVCAR-3 cells. Moreover, Western blot analysis revealed that these cells contain receptors for 1,25(OH)2D3 (VDR) and androgen (AR). Expression of VDR and AR was up-regulated by their cognate ligands. Up-regulation of AR by 1,25(OH)2D3 and of VDR by DHT provides evidence of cross-talk between 2 signaling pathways in OVCAR 3 cells. We also studied the immuno-histochemical distribution of VDRs and ARs in rat ovaries and human ovarian cancer cases. In rat ovaries, VDRs were observed mainly in granulosa and theca cells and ARs in granulosa cells and surface epithelium. In the human ovarian cancer cases studied, 43% were VDR-positive and 64% AR-positive. Combining the results suggests that the growth of ovarian tissue might be regulated by 1,25(OH)2D3 and androgen. PMID- 10728593 TI - VEGFc and VEGFR3 expression in human thyroid pathologies. AB - In vertebrates, vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are major determinants of angiogenesis. In adults, the interaction between VEGFc and VEGFR3 (previously FLT4) is more specifically involved in the biology of lymphatics. Using PCR amplification of reverse-transcribed mRNA, we studied the expression of the VEGFR3 (including its short and long forms) and VEGFc genes in 38 samples of various human thyroid pathologies. VEGFR3 mRNA was detected in all samples of adenomas, nodular goiters and focal goitrogenic alterations; in all samples of thyroid tissue from patients with auto-immune diseases; and in some samples of adenocarcinomas. VEGFc mRNA was detected in most samples. We studied expression of the VEGFR3 and VEGFc proteins in thyroid tumors using appropriate antibodies. Co-expression of VEGFR3 and VEGFc was observed in most samples. PMID- 10728594 TI - Transforming growth factor-B3 protects murine small intestinal crypt stem cells and animal survival after irradiation, possibly by reducing stem-cell cycling. AB - Damage to the normal replacing tissues of the body, specifically the gastro intestinal tract, limits the treatment and hence, cure rate of cancer patients. Here, we investigate the possibility that the sensitivity of the gastro intestinal tract can be manipulated by transforming growth factor beta3 (TGF beta3), making it more resistant to radiation in a murine model. The effects of TGF-beta3 were assessed using the crypt microcolony assay, a test of crypt stem cell functional competence, in animal survival studies examining diarrhoea severity, labelling index and crypt size. Prior treatment with TGF-beta3 can result in a 3- to 4-fold increase (protection factor, PF) in surviving crypts, whilst longer exposure can raise the PF to almost 12. Protection of intestinal clonogenic stem cells results in marked protection of survival with a corresponding reduction in the duration and level of diarrhoea and ultimate restoration of normal histology in surviving mice. Inhibition of proliferation can be demonstrated when sufficient TGF-beta3 exposure is studied. Crypt size is also reduced. In conclusion, TGF-beta3 protects small intestinal clonogenic stem cells from radiation damage, reducing diarrhoea and animal mortality. The mode of action is believed to be specific inhibition of stem-cell proliferation. PMID- 10728595 TI - Microsatellite instability and hMLH1/hMSH2 expression in young endometrial carcinoma patients: associations with family history and histopathology. AB - Endometrial cancer is the second most common malignancy in patients with hereditary nonpolyposis colorectal cancer (HNPCC). The age at diagnosis of HNPCC associated endometrial cancer is approximately 15 years younger than for sporadic endometrial cancer. Our current study was undertaken to determine the frequency of microsatellite instability (MSI) and absence of hMLH1 or hMSH2 protein expression in young patients with endometrial carcinoma and to correlate these findings with histopathologic and clinical features. Endometrial carcinoma from 62 women (23-52 years, median age 46) were assessed for MSI. Twenty-one of the 62 (34%) tumors demonstrated MSI. Of the 21 tumors demonstrating MSI, 12 showed an absence of hMLH1 expression, 4 showed an absence of hMSH2 expression, and 5 demonstrated normal expression of both proteins. All 41 tumors without MSI demonstrated normal hMLH1 and hMSH2 expression. Two patients with MSI tumors fulfilled the Amsterdam criteria for HNPCC, while 2 had histories suggestive of HNPCC. None of the patients with tumors without MSI had a personal or family cancer history suggestive of HNPCC. The MSI phenotype was associated (p < 0.05) with high FIGO stage and grade, cribriform growth pattern, mucinous differentiation and necrosis. Our findings suggest that the frequency of HNPCC in young endometrial cancer patients is relatively low when compared with the frequency of HNPCC in young colorectal cancer patients. Defects of the MMR proteins hMSH2 or hMLH1 account for MSI in most but not all endometrial cancers from young patients. PMID- 10728596 TI - Human hemangiosarcomas have a common polymorphism but no mutations in the connexin37 gene. AB - Gap junctional intercellular communication is often impaired in cancers, and the genes which encode the connexin gap junction proteins are considered to be tumor suppressor genes. In this study, we analyzed the presence of mutations in the connexin 37 (Cx37) gene in 22 human hepatic angiosarcomas, 6 and 4 of which were associated with exposure to vinyl chloride and Thorotrast, respectively. The other 12 samples were from patients with no history of exposure to these 2 agents. In 9 samples, a proline (ACC) to serine (ACT) amino acid change in codon 319 was detected. However, DNA from non-tumorigenic tissue of the same patients also showed this amino acid change, suggesting that this is a polymorphism rather than a mutation. Subsequent analysis of 84 DNA samples from normal donors revealed the frequencies of Pro/Pro, Pro/Ser and Ser/Ser alleles to be 65.5%, 23.8% and 10.7%, respectively, while among the group of angiosarcoma patients the corresponding figures were 59.1%, 31.8% and 9. 1%, respectively. Thus, there was no correlation between the polymorphism at codon 319 and hepatic angiosarcoma occurrence. However, among the 6 cases of vinyl chloride-associated angiosarcoma, the percentages of the polymorphic alleles were 33.3%, 66.7% and 0%, respectively. While the number of samples was too small to allow us to conclude that the Ser319 allele in Cx37 predisposes to this rare type of human cancer, it may be noted that codon 319 is located at the cytoplasmic tail of Cx37, where most regulatory sequences reside, and that it could be a site of phosphorylation for some protein kinases, which may in turn affect the function of Cx37, including intercellular communication. PMID- 10728597 TI - Antibodies to the Epstein-Barr virus transactivator protein (ZEBRA) as a valuable biomarker in young patients with nasopharyngeal carcinoma. AB - Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) generally occurs in adults, especially in high-prevalence populations such as the Chinese and Eskimos. In Maghrebian populations, young patients affected with this malignancy represent 25% of the total NPC cases. In adults with NPC, relatively high titers of IgA antibodies to the EBV viral capsid antigen (VCA) and early antigen (EA) represent important markers. However, nearly 50% of young NPC patients are negative for IgA-anti-VCA and -EA or exhibit very low titers of these antibodies. We report here that 92% of sera from young NPC patients negative for IgA-EA and 89% of those negative for IgA-VCA were positive for IgG antibodies to the EBV transactivator protein (ZEBRA) at very high titers. Our results show that in young patients with NPC these antibodies represent the most reliable marker for diagnosis and prognosis, particularly when compared with conventional NPC markers, i.e., IgA-VCA (58%) and anti-EA (25%). The titers of IgG-ZEBRA antibodies increased along with lymph node involvement only in the young patient group, suggesting a prognostic value of this marker in this patient group. PMID- 10728598 TI - PCR with degenerate primers for highly conserved DNA polymerase gene of the herpesvirus family shows neither human herpesvirus 8 nor a related variant in bone marrow stromal cells from multiple myeloma patients. AB - The possibility has been raised that either a human herpesvirus-8 (HHV-8) variant or a novel, unidentified, gamma-herpesvirus related to HHV-8 is frequently associated with multiple myeloma (MM), which could explain the lack of antibodies to HHV-8 antigens and the discordant results from polymerase chain reaction (PCR) studies of HHV-8-specific sequences in MM patients. Thus, we used a sensitive PCR assay with degenerate primers targeting the highly conserved DNA polymerase gene of the herpesvirus family to examine the long-term cultures of bone marrow stromal cells (BMSCs) from 19 MM, 3 monoclonal gammopathies of undetermined significance and 6 control patients. Both the culture supernatant and the adherent stromal layer were examined from the 2nd until the 8th week of culture to assess the immunophenotype of the various cell types harvested for the molecular analysis. BMSCs consisted of a mixed population of fibroblast, macrophage, dendritic and endothelial cells. An amplified product of the expected size was obtained only in 3 MM cases, both in the adherent and nonadherent fractions. Direct sequencing and alignment of the nucleotide and amino acid sequences showed that the DNA sequences were 100% identical to Epstein-Barr virus (EBV) DNA. The PCR positivity was due to the presence of EBV-infected lymphoblastoid cells with plasmacytoid features, expressing the EBV-encoded latent membrane protein-1 and detectable either in the stromal cells or in the culture supernatant. Our data do not support a causal role of either HHV-8 or a novel herpesviral variant related to HHV-8 in MM. PMID- 10728599 TI - Role of a bacillus Calmette-Guerin fibronectin attachment protein in BCG-induced antitumor activity. AB - Intravesical Mycobacterium bovis bacillus Calmette-Gu*erin (BCG) is the treatment of choice for superficial bladder cancer. Previous studies showed that attachment of BCG to fibronectin within the bladder was necessary for mediation of the antitumor response. Further studies identified a bacterial receptor, fibronectin attachment protein (FAP), as an important mediator of BCG attachment to fibronectin. In vitro studies showed that a stable BCG/fibronectin interaction was dependent on FAP binding to fibronectin; however, no role for FAP in the attachment of BCG in vivo has been characterized. We now report the cloning of the M. bovis BCG FAP (FAP-B) and demonstrate an important role for FAP in the in vivo attachment of BCG to the bladder wall and in the induction of BCG-mediated antitumor activity. The predicted amino acid sequence for FAP-B shows 61% and 71% homology, respectively, with Mycobacterium avium FAP (FAP-A) and Mycobacterium leprae FAP (FAP-L). Rabbit polyclonal antibodies against Mycobacterium vaccae FAP (FAP-V) reacted with all 3 recombinant FAP proteins on Western blots. Functional studies show FAP-B to bind fibronectin via the highly conserved attachment regions previously identified for FAP-A and FAP-L and also to competitively inhibit attachment of BCG to matrix fibronectin. In vivo studies show FAP to be a necessary protein for the stable attachment of BCG to the bladder wall. Moreover, stable binding of BCG via FAP was shown to be necessary for the expression of BCG induced antitumor activity. Our results demonstrate a biological role for FAP in the mediation of BCG-induced antitumor activity. PMID- 10728600 TI - Genetic immunization of mice with human tyrosinase-related protein 2: implications for the immunotherapy of melanoma. AB - The melanosomal protein TRP2 expressed by melanocytes and most melanoma cells is an attractive, clinically relevant model antigen for the experimental development of melanoma immunotherapy in mice. A peptide shared by murine and human TRP2 can be recognized by melanoma-reactive CTL in C57BL/6 mice, as well as in human melanoma patients. Previous experiments demonstrated that gene gun immunization of mice with plasmid DNA encoding autologous murine TRP2 was unable to induce protective immunity against B16 melanoma cells naturally expressing TRP2. In the present study, we investigated whether the use of cDNA encoding xenogeneic human TRP2, which is highly homologous to murine TRP2, would be more effective. Genetic immunization of mice with human TRP2 resulted in coat depigmentation as a sign of autoimmune-mediated destruction of melanocytes and provided significant protection against metastatic growth of B16 melanoma Induction of protective immunity was associated with TRP2-reactive antibodies and CD8+ T cells. Furthermore, immunization with recombinant adenovirus was more effective than immunization with plasmid DNA using the gene gun. Our results provide new insights for the development of antigen-specific immunotherapy of melanoma. PMID- 10728601 TI - Increased expression of the MRP5 gene is associated with exposure to platinum drugs in lung cancer. AB - To investigate the role of the multidrug resistance-associated protein (MRP1) homologue MRP5 in relation to platinum drug resistance, we examined the steady state levels of the mRNAs for MRP5 in both lung cancer cell lines and peripheral mononuclear cells (PMN) after exposure to platinum drug and in normal lung and lung cancer tissue specimens. Firstly, we examined MRP5 gene expression levels in 80 autopsy samples (40 primary tumors and 40 corresponding normal lung tissues) from 40 patients who had died from lung cancer. Next, we monitored MRP5 gene expression levels within 24 hr in both lung cancer cell lines incubated with cisplatin and in PMN from 10 previously untreated lung cancer patients after carboplatin administration alone. The MRP5 gene expression levels were assessed by quantitative reverse transcription polymerase chain reaction or RNase protection assay. The MRP5 expression levels in normal lung tissues and in tumors from patients exposed to platinum drugs during their lifetime were significantly higher than those in tissues from non-exposed patients. On the other hand, the MRP5 expression levels were not rapidly induced by platinum drugs either in lung cancer cell lines or in PMN within 24 hr. Our results suggest that increased expression levels of the MRP5 gene are associated with exposure to platinum drugs in lung cancer in vivo and/or the chronic stress response to xenobiotics. PMID- 10728602 TI - The influence of expression of P-glycoprotein on the penetration of anticancer drugs through multicellular layers. AB - The success of chemotherapy in the treatment of solid tumours may be limited by cellular mechanisms leading to drug resistance and/or by the slow penetration of drugs through tissue, resulting in a steep concentration gradient from tumour blood vessels. One mechanism leading to the development of multidrug resistance is overexpression of the membrane-based export pump P-glycoprotein (P-gp). The relationship between expression of P-gp by constituent cells and the penetration of P-gp substrates through tissue was studied by comparing the penetration of P gp substrates through multicellular layers derived from either wild-type or P-gp overexpressing cell lines. P-gp reversal agents were added to confirm the contribution of P-gp in influencing the penetration of its substrates. Our data indicate: 1) penetration of the P-gp substrates, 99mTc-sestaMIBI and 14C doxorubicin, is greater through multicellular layers formed from P-gp overexpressing cell lines as compared with wild-type cells; 2) the addition of agents that inhibit the function of P-gp results in decreased penetration of these substrates through multicellular layers with P-gp expression. There was no effect of P-gp reversal agents on penetration of 14C-sucrose or of 3H-5 fluorouracil (non-substrate controls). Our data suggest that the administration of agents that inhibit the function of P-gp might have opposing effects on therapeutic index in solid tumours: increased sensitivity of perivascular tumour cells but decreased penetration of P-gp substrates to more distal cells. These effects may explain, in part, the limited therapeutic benefit for solid tumours that has accrued from use of agents that reverse the effects of P-gp. PMID- 10728603 TI - Efficacy of the chemotherapeutic action of HPMA copolymer-bound doxorubicin in a solid tumor model of ovarian carcinoma. AB - Anticancer activity and main mechanisms of action of free doxorubicin (DOX) and HPMA copolymer-bound DOX (P(GFLG)-DOX) were studied in solid tumor mice models of DOX sensitive and resistant human ovarian carcinoma. Free DOX was effective only in sensitive tumors decreasing the tumor size about three times, whereas P(GFLG) DOX decreased the tumor size 28 and 18 times in the sensitive and resistant tumors. An enhanced accumulation of P(GFLG)-DOX in the tumor was observed, whereas only low concentrations of DOX were detected in other organs following P(GFLG)-DOX administration. This effect was dependent on the high permeability of blood vessels in untreated tumors. After treatment with P(GFLG)-DOX the permeability decreased concomitantly with the downregulation of VEGF gene expression. P(GFLG)-DOX effectively killed both types of tumors inducing apoptosis and necrosis through the activation of p53, Apaf-1, caspase 9, c-fos, or c-jun pathways, and the downregulation of the bcl-2 gene. HPMA copolymer-bound DOX preserved its activity inside cells, inhibited detoxification and defensive mechanisms encoded by GST-pi, BUDP, and HSP-70 genes, and limited DNA repair, replication, and biosynthesis by downregulation of Topo-IIalpha,beta, and TK1 genes. P(GFLG)-DOX also produced tumor tissue hypoxia and significantly activated lipid peroxidation in tumors. No damage to other organs after exposure to P(GFLG) DOX was detectable. On the other hand, free DOX activated lipid peroxidation and led to tissue hypoxia in many organs. All data relevant to the mechanism of anticancer action of P(GFLG)-DOX indicated a higher antitumor activity and lower systemic toxicity of HPMA copolymer-bound DOX when compared with free DOX. PMID- 10728604 TI - Case-control study in a subtropical Australian population to assess the relation between non-melanoma skin cancer and epidermodysplasia verruciformis human papillomavirus DNA in plucked eyebrow hairs. The Nambour Skin Cancer Prevention Study Group. AB - Epidermodysplasia verruciformis human papillomavirus (EV-HPV) DNA has been demonstrated in malignant and benign skin lesions and in hairs plucked from renal transplant recipients and immunocompetent patients. We investigated the association between EV-HPV DNA in hairs plucked from eyebrows and the occurrence of non-melanoma skin cancer (NMSC) in a community-based study. Within a cohort of residents of a Queensland township (Nambour), nested case-control studies of recently developed NMSC (64 cases), basal-cell carcinoma (BCC) (51 cases) and squamous-cell carcinoma (SCC) (25 cases) were conducted. EV-HPV DNA in hair and a small number of available tumour samples was detected using a nested PCR specific for EV-HPV types. EV-HPV DNA was detected in hairs from 94 of 143 individuals (66%), and 36 (39%) of the samples contained 2 or more different EV-HPV types. Only known or putatively new EV-HPV types were detectable after sequencing 93 samples. EV-HPV status agreed for 12 of 20 subjects who had both hair and skin tumour samples available. In 4 of 5 pairs of positive samples, the same EV-HPV type was found. There were non-significant negative associations between EV-HPV and NMSC (OR 0.77, 95% CI 0.34-1.8) and BCC (OR 0.58, 95% CI 0.23-1.5) but a non significant positive association with SCC (OR 2.00, 95% CI 0.50-8.0). PMID- 10728605 TI - Selected micronutrients and oral and pharyngeal cancer. AB - The relation between selected micronutrients and oral and pharyngeal cancer risk was investigated using data from a case-control study conducted between January 1992 and November 1997 in Italy and Switzerland. Cases were 754 incident, histologically confirmed oral cancers (344 of the oral cavity and 410 of the pharynx) admitted to the major teaching and general hospitals in the study areas. Controls were 1,775 subjects with no history of cancer admitted to hospitals in the same catchment areas for acute, non-neoplastic diseases. Dietary habits were investigated using a validated food-frequency questionnaire. Odds ratios (ORs) were computed after allowance for age, sex, center, education, occupation, body mass index, smoking and drinking habits and non-alcohol energy intake. Micronutrients were analyzed both as continuous variables and in quintiles. In the former case, the unit was set to 1 SD of the distribution of controls. ORs for the continuous analysis were 0.95 for retinol, 0.61 for carotene, 0.91 for lycopene, 0.83 for vitamin D, 0.74 for vitamin E, 0.63 for vitamin C, 0.82 for thiamine, 0.87 for riboflavin, 0.59 for vitamin B6, 0.61 for folic acid, 0.62 for niacin, 0.91 for calcium, 0.88 for phosphorus, 0.65 for potassium, 0.82 for iron, 0.67 for non-alcohol iron and 0.89 for zinc; the 95% confidence interval excluded one for carotene, vitamin C and E, thiamine, vitamin B6, folic acid, niacin, potassium and iron. ORs were similar for the 2 sexes and in strata of age. When the combined intake of vitamins C and E and carotene was considered, the protective effect of each nutrient was more marked or restricted to subjects with low intake of the other 2. The association with vitamin C and carotene was independent of smoking and drinking habits, while that with vitamin E was less evident in those heavily exposed to alcohol or tobacco. In general, the more a micronutrient was correlated to total vegetable and fruit intake, the stronger was its protective effect against oral cancer. PMID- 10728606 TI - Paan without tobacco: an independent risk factor for oral cancer. AB - Oral cancer is the second most common cancer in women and the third most common in men in Pakistan. Tobacco is smoked and chewed extensively in Pakistan. Paan is a quid of piper betel leaf that contains areca nut, lime, condiment, sweeteners, and sometimes tobacco, which is also used extensively. We did this study to clarify the independent association of paan and oral cancer. Between July 1996 and March 1998, we recruited biopsy-proven, primary cases of oral squamous-cell carcinoma, from 3 tertiary teaching centers in Karachi, Pakistan, and controls pair-matched for age, gender, hospital and time of occurrence, excluding persons with a past or present history of any malignancy. There were 79 cases and 149 controls. Approximately 68% of the cases were men, 49 years old on average, the youngest being 22 years old and the eldest 80. People with oral submucous fibrosis were 19.1 times more likely to develop oral cancer than those without it, after adjusting for other risk factors. People using paan without tobacco were 9.9 times, those using paan with tobacco 8.4 times, more likely to develop oral cancer as compared with non-users, after adjustment for other covariates. This study identifies an independent effect of paan without tobacco in the causation of oral cancer. Its findings may be of significance in South Asian communities where paan is used, and among health-care providers who treat persons from South Asia. PMID- 10728607 TI - Bladder-cancer incidence in relation to vegetable and fruit consumption: a prospective study of atomic-bomb survivors. AB - We examined the relation between consumption of 22 dietary items and subsequent bladder-cancer incidence in a cohort of atomic-bomb survivors in Japan. Subjects were 38,540 people (14,873 men and 23,667 women) who responded to a mail survey carried out between 1979 and 1981 and who had no known cancer diagnosed before the start of follow-up (1 January 1980 for men, 1 February 1981 for women). Consumption frequencies for 22 dietary items were ascertained with pre-coded answers. As of the end of 1993, there were 114 (83 men and 31 women) incident cases of bladder cancer among 450,326 person-years at risk. Statistical analysis was done using Poisson regression for grouped survival data Consumption of green yellow vegetables and fruit were protectively associated with risk. Adjusted for gender, age, radiation exposure, smoking status, educational level, body-mass index and calendar time, the relative risk (RR) for those consuming green-yellow vegetables 2-4 times per week and almost everyday was 0.62 [95% confidence interval (CI) 0.39-0.98] and 0.54 (95% CI 0.30-0.94) respectively, as compared with those consuming once per week or less. The corresponding RR for fruit consumption was 0.50 (0.30-0.81) and 0.62 (0.39-0.99) respectively. Chicken consumption was unexpectedly associated with decreased risk, but additional adjustment for consumption did not change the relation of green-yellow vegetables or of fruit to risk. The consumption of the other dietary items, including meat and green tea, was not related to risk. The findings add to evidence that high consumption of vegetables and fruit are protective against bladder cancer. PMID- 10728608 TI - The role of nutritional habits during gestation and child life in pediatric brain tumor etiology. AB - Our aim was to evaluate the role of maternal nutritional habits during the period of gestation and of children subsequent diet in the etiology of pediatric brain tumors. All cases of incident nervous system tumors under age 18, diagnosed between 1984 and 1993 (n = 300) in Israel were identified. Two matched population controls per case were selected (n = 574). Personal interviews, using a semi quantified three-step food frequency questionnaire, were performed. Univariate analysis showed that increased child consumption of vegetable fat [p trend 0.01; 95% confidence interval (CI) 1.1-3.2], carbohydrates (p trend 0.05; CI 1.0-5.9), and vitamin E (p trend 0.05; CI 1.0-3.3), were significantly associated with brain tumor risk. No associations were found with nitrate, nitrite or vitamin C. A significant positive association with potassium consumption (p trend 0.01; CI 1.1-3.7) was noted during gestation. Results of multivariate analysis showed that the only persisting associations were with vegetable fat (OR = 1.36; CI 1.06 1.73) in the child diet and potassium intake during gestation (OR = 1.44; CI 1.04 1.99). In conclusion, nutritional associations with pediatric brain tumor etiology, remain unsubstantiated. PMID- 10728609 TI - Smoking, type of alcoholic beverage and squamous-cell oesophageal cancer in northern Italy. AB - Between 1992 and 1997, we conducted a case-control study of oesophageal cancer in 3 areas of northern Italy. Cases were 275 men, ages 39-77 years (median age 60), with a first incident squamous-cell carcinoma of the oesophagus. Controls were 593 men, ages 36-77 years (median age 60) admitted for acute illnesses, unrelated to tobacco and alcohol, to major hospitals of the areas under surveillance. Number of daily cigarettes was strongly associated with risk [odds ratio (OR) for > or =25 cigarettes/day = 7.0)]. Long-duration smoking showed particularly elevated ORs (OR = 6.4 for > or =35 years), and excess risk declined after smoking cessation (OR = 1.5 after > or = 10 years). Oesophageal cancer risk steeply rose with increasing level of alcohol consumption. ORs were 6.2 for 35-55 drinks and 24.5 for 84 drinks or more per week. No trend in risk emerged for duration of alcohol drinking or age at start of drinking. The risk in the highest joint level of alcohol drinking and current smoking was increased 130 folds (i.e., compatible with a multiplicative model). Excess risk in drinkers chiefly derived from wine. In conclusion, alcohol drinking and cigarette smoking were both important, but the roles of dose and duration of exposure differed. The association with alcohol was stronger than the one with smoking by exposure intensity, but apparently unaffected by duration or other temporal variables. PMID- 10728610 TI - Effect of proteolytic starter cultures of Staphylococcus spp. on biogenic amine formation during the ripening of dry fermented sausages. AB - The effect of proteolytic starter cultures of Staphylococcus carnosus and Staphylococcus xylosus on biogenic amine production was examined during the fermentation process of dry sausages. Microbial counts (lactic acid bacteria, Micrococcaceae and Enterobactenaceae), pH, moisture and proteolysis-related parameters were also studied. The polyamines spermine and spermidine were the main amines found in the raw material and they only showed slight fluctuations during the fermentation. The four elaborated batches presented a significant (P < 0.001) formation of tyramine and putrescine. The main rate of amine production was during the first three days, when a sharp pH decrease and the development of lactic acid bacteria occurred. Sausages fermented with starters had lower amounts of tyramine than naturally fermented sausages (control), but differences in the Micrococcaceae counts were only significant during the first week of the ripening process. A slight formation of diaminopropane, cadaverine, agmatine, tryptamine and phenylethylamine was observed. The amounts of histamine were constant and remained below 0.5 mg/kg of dry matter, while serotonin, octopamine and dopamine were not detected. The sausages with Staphylococcus as starter culture showed strong proteolysis that was correlated with higher pH values than those of the control sausages. However, no positive correlation was found between the proteolysis index and biogenic amine production. Since proteolysis was stronger during the second half of the ripening process, the release of free amino acids as amine precursors occurred later than the early amine production. PMID- 10728611 TI - Screening of non-Saccharomyces wine yeasts for the production of beta-D xylosidase activity. AB - Fifty-four yeast strains belonging to the genera Candida, Dekkera, Hanseniaspora, Metschnikowia, Pichia, Rhodotorula, Schizosaccharomyces and Zygosaccharomyces, mainly isolated from grapes and wines, were screened for the production of beta-D xylosidase activity. Beta-D-xylosidase activity was only detected in eight yeast strains belonging to the genera Hanseniaspora (H. osmophila and H. uvarum) and Pichia (P. anomala). Beta-D-xylosidase preparations active against p-nitrophenyl beta-D-xyloside were characterised with respect to their optimal pH and temperature conditions. H. uvarum 11105 and 11107 and P. anomala 10320 beta-D xylosidase preparations were active at pH and temperature ranges and at concentrations of glucose and ethanol usually found during winemaking processes. PMID- 10728612 TI - Cryptosporidium parvum studies with dairy products. AB - Cryptosporidium parvum is a protozoan parasite capable of causing massive waterborne outbreaks. This study was conducted to model the transfer of C. parvum oocysts from contaminated water via food contact surfaces into yogurt and ice cream, as well as to examine oocyst survival. Propidium iodide staining, combined with a direct immunofluorescence assay, was used for oocyst viability determination. Oocysts were recovered from milk products by a sucrose flotation based procedure, with average recoveries of 82.3, 60.7, and 62.5% from low (1%) fat milk, 9% fat ice-cream, and 98% fat-free yogurt, respectively. Oocysts were also recovered, by rinsing with tap water, from stainless steel surfaces inoculated with oocyst suspension, with average recoveries of 93.1% when the surface was still wet and 69.0% after the surface had air-dried at room temperature. Viability of oocysts on the surface was significantly affected by desiccation; 5% of the oocysts remained viable after 4 h of air-drying at room temperature, while the proportion of viable oocysts was 81, 69, and 45% after air drying for 10 min, 1 h, and 2 h, respectively. In contrast, oocyst viability only dropped from 82 to 75% after 30 min contact at room temperature with 5% bleach solution (equivalent to 0.26% NaOCl). Transfer of oocysts from milk and stainless steel surfaces into yogurt, and oocyst survival during the process were analyzed. Yogurt was made from pasteurized low fat milk and live yogurt starter by incubating at 37 degrees C for 48 h and then stored at 4 degrees C. Oocyst viability decreased from 83% (80%) to approximately 60% after 48 h at 37 degrees C and to approximately 58% following 8 days of storage, similar to oocyst survival in the controls using pasteurized milk without the addition of live yogurt. Oocyst survival in ice-cream was investigated by inoculating oocysts into ice-cream mix, and mixing and freezing in an ice-cream freezer, and hardening at 20 degrees C. Although approximately 20% (25 and 18%) of oocysts were viable before hardening, none were viable after 24 h at -20 degrees C. Control samples of oocysts suspended in distilled water and stored at -20 degrees C were taken at the same time intervals and 8% of the oocysts were still viable after 24 h. PMID- 10728614 TI - Validation of predictive models describing the growth of Listeria monocytogenes. AB - In this study, predictions for growth rate of Listeria on food products were evaluated by both general applicable models and specific growth models. Literature values, obtained from a large number of publications, for growth rates in/on a variety of foods were compared by graphical and mathematical analysis with predictions given by various models. Apart for the great advantage of being generally applicable, the general models performed best. However, only small differences between the various models were observed. Model predictions were accurate within a factor of about two to four, depending on the type of product. The predictions should therefore not be considered as absolute; it is important to understand the limitations of the performance of models. All results and all assumptions should be criticised, but in many cases the accuracy will be sufficient to use these types of models as a tool in management decisions. PMID- 10728613 TI - Using an electronic nose for determining the spoilage of vacuum-packaged beef. AB - The use of an electronic nose in the quantitative determination of the degree of spoilage of vacuum-packaged beef was evaluated. Beef from four different slaughterhouses was sliced, vacuum-packaged and stored at 4 degrees C for 8 weeks. Samples were withdrawn for bacterial (aerobic bacteria, lactic acid bacteria, Brochothrix thermosphacta, Pseudomonas and Enterobacteriaceae) and sensorial analyses and analysis of the volatile compounds during the storage period. A trained panel was used for the sensorial evaluations. The volatile compounds were analysed using an electronic nose containing a sensory array composed of 10 metal oxide semiconductor field-effect transistors, four Tagushi type sensors and one CO2-sensitive sensor. Four of the 15 sensors were excluded due to lack of response or overloading. Partial least-squares regression was used to define the mathematical relationships between the degree of spoilage of vacuum packaged beef, as determined by the sensory panel, and the signal magnitudes of the sensors of the electronic nose. The mathematical models were validated after 6 months using a new set of samples. The stability of the sensors during this period was examined and it was shown that the sensitivity of five of the 11 sensors used had changed. Using the six remaining sensors, the signal patterns obtained from the meat from the different slaughterhouses did not change over a period of 6 months. It was shown that the degree of spoilage, as calculated using a model based on two Tagushi sensors, correlated well with the degree of spoilage determined by the sensory panel (r2 = 0.94). PMID- 10728615 TI - The effect of homogenization of whole milk, skim milk and milk fat on nisin activity against Listeria innocua. AB - Whole milk, skim milk and an emulsion of milk fat in water, inoculated with approx. 10(5) cfu/ml of Listeria innocua, were treated at 30 degrees C with 100 IU/ml of nisin, homogenization at 200 bar or both procedures. Nisin activity and survival of L. innocua after treatments were determined. Recovery of nisin activity from non-homogenized whole milk treated with 100 IU/ml of nisin was complete, whereas a loss of 18 to 28% of activity was detected in non-homogenized fat-in-water emulsion. Loss in nisin activity due to homogenization represented up to 64% in whole milk and 62% in fat-in-water emulsion. Nisin addition by itself achieved a reduction in L. innocua counts of 3.7-3.8 log units in whole milk and 3.6 log units in fat-in-water emulsion compared to numbers in untreated samples. When nisin-containing whole milk and fat-in-water emulsion were homogenized, L. innocua counts were only reduced by 2.6-2.9 log units and 2.5 log units, respectively, compared to numbers in untreated samples. Homogenization of nisin-containing skim milk resulted in a loss of nisin activity of 20% but achieved a reduction of 3.0 log units in L. innocua counts. PMID- 10728616 TI - The effects of environmental conditions on the lipolytic activity of strains of Penicillium roqueforti. AB - The lipolytic activity of 30 strains of Penicillium roqueforti was investigated by agar diffusion tests on tributyrin (esterase activity) and olive oil agar (lipase activity), by titration of the free fatty acids (FFA) produced and by gas chromatographic analysis of the individual FFA released after growth at 25 or 10 degrees C in butterfat emulsions containing 0, 2 or 7% NaCl. All strains investigated by the agar diffusion tests possessed esterase activity and 23 strains were also able to hydrolyse olive oil, but differences in esterase activity were seen. The agar diffusion tests and the titration of FFA showed that the amount of FFA released by a strain of P. roqueforti is determined by both esterase and lipase activity. A large release of FFA was only seen for strains with the ability to hydrolyse both short- and long-chained fatty acids, while strains with esterase activity produced smaller amounts of FFA. Between 7 and 14 days of incubation a steep increase in the release of FFA was observed both by the titration and by GC analysis, and then a decline from 14 to 21 days, probably caused by conversion of FFA to methyl ketones. Identical FFA profiles were found for two strains with different lipolytic activity. Long-chained fatty acids dominated the profile, while the short-chained fatty acids only were detected in small amounts and mainly in the end of incubation. Both strains were stimulated by NaCl in the emulsions. PMID- 10728617 TI - Behaviour of Listeria spp. in naturally contaminated chorizo (Spanish fermented sausage). AB - Counts of Listeria spp. were determined during the manufacture and drying of 21 lots of five chorizo varieties produced by three different manufacturers. Presumptive Listeria were not isolated from any of the batches produced in a large factory (F3) using starter, sorbate and controlled ripening at high temperatures. Initial levels in factory 1 (F1), with no starter added, but controlled ripening at low temperatures, were ca 3.5 log10 cfu/g while those in factory 2 (F2), with no starter added and ripening under natural climatic conditions, were 1.17 log10 cfu/g. Numbers of listeriae in batches obtained from F1 remained almost constant before decreasing by ca 0.5 log units/g in the finished product (32 days), while the levels in F2 increased by 1.47 log units/g after 11 days of ripening and declined further to levels above the original amount. Manufacturing procedure and smoking significantly affected presumptive listeriae counts. Thirteen strains recovered from F1 batches were identified as: Listeria monocytogenes (three strains of serovar 1/2c), Listeria innocua (eight strains of serovar 6b) and Listeria welshimeri (two strains of serovar 6b). Listeria strains from F2 were assigned to L. innocua and L. welshimeri. PMID- 10728618 TI - Growth of Bacillus cereus in pasteurized milk products. PMID- 10728619 TI - Evaluating GA733-2 mRNA as a marker for the detection of micrometastatic breast cancer in peripheral blood and bone marrow. AB - The GA733-2 gene encodes the epithelial glycoprotein 40, a homophilic cell-cell adhesion molecule, which is expressed on the surface of epithelial cells and associated with a variety of carcinomas, e.g. breast, colorectal and lung carcinomas. To test if it could serve as a tumor marker, we have analysed the expression of GA733-2 in bone marrow (BM) and peripheral blood (PB) from healthy donors, and of patients with haematological malignancies or breast cancer using reverse transcriptase-polymerase chain reaction (RT-PCR). The GA733-2 nested PCR was positive in 100% (8/8) of BM and 40% (16/40) of PB from healthy donors, in 100% (33/33) of BM from patients with breast cancer who had no evidence of distant metastases and also in BM and PB from patients with haematological malignancies. GA733-2 mRNA is not specific as a marker for the detection of breast cancer cells in BM and PB. PMID- 10728620 TI - Pregnancy and perinatal outcomes in migraineurs using sumatriptan: a prospective study. AB - BACKGROUND: Sumatriptan is an acute treatment for migraine which is often used by women in their child-bearing years, and who become unexpectedly pregnant. Within the context of the post-marketing use of sumatriptan injection for the acute treatment of migraine, and in compliance with approved labeling, we wished to compare perinatal pregnancy outcomes in women who did and did not use the drug after conception. METHODS: Open-label, prospective study conducted in 12,339 migraineurs (including 9,861 women) whose demography and consumption pattern of sumatriptan injections were typical, and were predicted to include 150 pregnancies. Outcome of pregnancy was the end-point. RESULTS: There were 168 of 173 pregnancies that were well-documented. Sumatriptan was only used prior to conception in 92 cases. There were 76 first trimester exposures to sumatriptan. There were no differences in pregnancy outcome between the two groups. CONCLUSIONS: Perinatal and pregnancy outcome did not differ between patients who had and had not used sumatriptan after conception, at the resolution of these sample sizes. This study design complements the ongoing pregnancy registry, which is now widened to patients exposed to all formulations of sumatriptan. PMID- 10728621 TI - Changes in uterine size after vaginal delivery and cesarean section determined by vaginal sonography in the puerperium. AB - There have been few reports on postpartum changes in the uterus during the three months after delivery. The aim of this study was to evaluate uterine morphological changes in women after vaginal delivery (n=262-351) and in women after cesarean section (n=64-82) and to evaluate the relation between breast feeding and parity, and uterine involution at 1 and 3 months postpartum measured by vaginal ultrasonography. There were no significant differences in parity between the vaginal delivery group and the cesarean section group. The length of the uterus at one month (7.93+/-1.16 cm, mean+/-SD) and, three months (7.03+/ 1.19 cm) and the width of the uterus at three months (3.83+/-0.94 cm) after delivery in the cesarean section group were greater than in the transvaginal group (7.64+/-1.03 cm, 6.65+/-0.99 cm, 3.57+/-0.62 cm, respectively). Increasing maternal parity was associated slightly with larger uterine size at one month post partum. The length of the uterus of women with a breast-feeding rate of 80% or more per day was 6.35+/-0.85 cm, and shorter than in women with a rate of 20% or less 7.03+/-1.04 cm, at three months after delivery. The width of the uterine body of women with a breast-feeding rate of 80% or more per day was 3.32+/-0.45 cm, and shorter than in women with a rate of 20% or less 3.87+/-0.66 cm, at 3 months after delivery. Stepwise regression and multiple regression analysis among parity, the history of cesarean section, the breast-feeding rate at one and three months after the delivery, and the restoration of the menses at three months after the delivery showed that the uterine size at one month after the delivery was related to the cesarean section and that the uterine size at three months after delivery was mostly related to the rate of breast-feeding. These results indicated that uterine involution was related to delivery mode at one and three months postpartum, feeding mode at three months postpartum, the menses restoration, and parity. The rate of breastfeeding was mostly related to the uterine size at three months postpartum. PMID- 10728622 TI - Contraceptive knowledge and behavior of conventionalists, careerists, idealists, bouncers, desperadoes, and outsiders. AB - The aim of the survey was to determine the contraceptive behavior and knowledge on contraceptive methods of adolescents. We defined six clusters according to personality, leisure activities, and sociodemographic characteristics: conventionalists, careerists, idealists, bouncers, desperadoes, and outsiders. 1010 Austrian adolescents aged 14-24 years participated in the survey. 96%-100% indicated that they were familiar with the condom and the contraceptive pill. The two most popular contraceptive methods cited were the condom (39%) and OC (34%). Whereas 40% of conventionalists reported that OC were their preferred method of contraception, 53% of idealists indicated that they preferred the condom. 66% of the desperadoes indicated that they considered the pill expensive. 85% of conventionalists and careerists believe that OC offer the best protection against pregnancy. Clusters with higher self-esteem and those with a higher educational level were more likely to believe that this method is unreliable. The results obtained indicate that outsiders, desperatoes, and bouncers show deficits in sex education; only careerists and idealists appeared to be better informed. Research on adolescent contraceptive behavior should drop the traditional stratifying criteria and appreciate the diversity of adolescent populations in order to be better able to classify adolescents according to their contraceptive attitudes. PMID- 10728623 TI - Reactive oxygen species in the process of labor. AB - Levels of thiobarbituric acid reactive substances (TBARS), an indicative parameter for oxidative damage, were measured in maternal and cord arterial and venous bloods, and compared between abdominal and vaginal deliveries. Spontaneous labor resulting in either vaginal or emergency abdominal deliveries was associated with a statistically significant higher levels of TBARS in cord artery compared to cord vein and maternal blood (p<0.05). The results support a role of reactive oxygen species in the initiation of labor, possibly through their effect on prostaglandin metabolism. Alternatively, this may be a marker of fetal oxidative stress, secondary to the process of labor. PMID- 10728624 TI - Ovarian abscess and heterotopic triplet pregnancy: two complications after IVF in one patient. AB - A patient is reported, who suffered from ovarian abscess after ovarian puncture of a functional ovarian cyst. The cyst has developed after administration of a GnRH agonist depot preparation in the preceeding luteal phase. She was planned to be stimulated for IVF according to the long luteal protocol. The abscess was removed by laparoscopy. and stimulation started two months later after administration of two further GnRH against depot preparations. The patient got pregnant after embryo transfer of three embryos. and a heterotopic triplet pregnancy, with intrauterine twins and a tubal singleton was established. Bilateral salpingectomy was performed, because of bilateral haematosalpinx and previously described bilateral tubal occlusion. The further pregnancy was uncomplicated. PMID- 10728626 TI - Accuracy of rapid antigen detection test for group B streptococci in the indigenous vaginal bacterial flora. AB - OBJECTIVE: To test the sensitivity of the rapid group B streptococci (GBS) antigen test ICONR and compare its accuracy in women with vaginal enterococci or with non-specific disturbance of the lactobacillary flora. STUDY DESIGN: The ICONR, aerobic culture and a microscopic wet mount evaluation were done on a vaginal sample in 254 unselected women presenting for routine gynecologic care in an academic hospital in Flanders, Belgium and tested by Chi2 [diagnostic odds ratio (DOR) and its 95 percent confidence limits]. RESULTS: Sensitivity of the test was 70%, specificity 99.5%. Prevalence of GBS was 10.6% overall, 23% in the group with abnormal vaginal flora and 7% in the normal group (p=0.002). Accuracy of the ICONR was not affected by abnormal vaginal flora, but was significantly lower in the presence of enterococci: the DOR decreased from 490 to 58, and the positive predictive value from 94 to 80%. CONCLUSION: With a sensitivity of 70% the enzyme immunoassay ICONR does not appear to be suitable as a practical screening tool for detecting GBS carriage in normal or preterm laboring women. In the presence of enterococci the test performed less well, with a DOR falling by 8 to 9 fold. We presume this is due to lower specificity in vivo in the presence of enterococci, as non-specific disturbance of the lactobacillary flora did not interfere with test results. PMID- 10728625 TI - Study of borderline and invasive mucinous ovarian tumors using Ki-67 (MIB 1) antibodies and nucleolar organizer region (NOR) staining. AB - Extensive application of new methods in histopathology has resulted in large quantities of data on molecular markers in different types of human tumors. The main purpose has been to obtain additional parameters for the characterization of various types of malignancy to give more precise information on their biological behavior. In this study we tested AgNOR and Ki-67 (MIB 1) staining in 39 ovarian mucinous tumors to evaluate its diagnostic potential in distinguishing between borderline tumors and well-differentiated carcinomas at stage I of FIGO classification. In general, there was an increase in AgNOR median values from benign (1.83 +/- 0.8) to well-differentiated carcinomas (2.91 +/- 1.3) with an intermediate value in borderline (2.3 +/- 1.6). The lowest Ki-67 labeling index (LI) was found in benign cystadenomas (5.7% +/- 3.4%) and papillary mucinous cystadenomas (7.9 +/- 5.8%). In borderline tumors mean LI was 12.2 +/- 10.9% whereas well-differentiated carcinomas had higher LI (16.8 +/- 10.2%). There were no statistically significant differences between the evaluated tumors and techniques applied. In our opinion the AgNOR count and Ki-67 LI do not appear useful for assessing differences between borderline and well-differentiated mucinous ovarian tumors at stage I of clinical advancement. PMID- 10728627 TI - In vitro effect of bioactive natriuretic peptides on perfusion pressure in placentas from normal and pre-eclamptic pregnancies. AB - The number of placental vascular guanylate-coupled receptors. corresponding to bioactive natriuretic peptide receptors is greater in preeclampsia. but there are no clear data about atrial natriuretic peptide (ANP) concentration in preeclampsia. The influence of various doses of ANP and urodilatin (URO) on placental perfusion pressure in preeclampsia was investigated by perfusing 16 human placentas in vitro. The placental vessels were submaximally preconstricted by continuous infusion of N-omega-nitro-L-arginine (NOLA). Perfusion pressure was measured continuously. Over 180 min various doses of alphaANP or URO were administrated (25, 50, 100, 200 nmol/l, 2 min increments). The effects of pretreatment with the guanylate cyclase inhibitor, LY 83583 was also examined. We found that ANP and URO attenuated NOLA-induced vasoconstriction, that URO given in higher doses produced stronger vasocilation than ANP, and that the mean decrease of perfusion pressure was higher in preeclampsia. The possibility of a non cGMP-mediated pathway of ANP and URO action should be considered. PMID- 10728628 TI - Importance of surgical margins in conization for cervical intraepithelial neoplasia grade III. AB - We conducted a retrospective study on 201 conizations for CIN III done over the last 14 years. Of these, 53 (26.4%) had involvement of the margins, 114 (56.7%) had margins free of neoplasia, and 34 (16.9%) were not evaluated. Of the 53 cases with margin involvement, 28 (52.8%) had complementary surgery and 25 (47.2%) cytological follow-up. Residual CIN was identified in the surgical specimen in 15 cases (53.6%) and microinvasion was present in 2 cases (7.1%). Of the 114 patients with margins free of neoplasia in the cone, 12 had a hysterectomy during follow-up for CIN III diagnosed by cytology and biopsy. Seven of these 12 patients (58.3%) had CIN in the surgical specimen and none had microinvasive cancer. PMID- 10728629 TI - Persistent increase of PRL after oral contraceptive treatment. Alterations in dopaminergic regulation as possible etiology. AB - BACKGROUND: The mechanism involved in estrogen induced hyperprolactinemia is not completely known, although one of the possible theories suggest inhibition of dopaminergic tone. Our objective was to study the mechanism implied in the increment of PRL levels as a consequence of oral contraceptive treatment and possible modifications in TSH levels. MATERIAL AND METHODS: We performed a trial on 21 healthy women, nulliparas. We administered 35 microg of Etinil-Estradiol (EE) and 2 mg of Ciproterone Acetate (CA) for a period of 12 months. Stimulation tests with Metoclopramide and TRH were carried out before treatment, after 3, 6 and 12 months of treatment and finally 6 months after cessation of treatment. RESULTS: Basal levels of PRL (mean=12.62 ng/ml) increased significantly (p<0.05) during the year of treatment (mean12=17.04 ng/ml) and maintained higher levels 6 months after cessation (meanl8=17.53 ng/ml). Maximum values obtained in response to metoclopramide (mean1=154.78) were significantly higher after 12 months (mean12=173.29), persisting 6 months after cessation of treatment (mean18=245.28). We also observed significant differences in the maximum response of TSH to metoclopramide during the same period of study (mean6=2.45), (mean12=2.76) and (mean18=2.07) respectively (p<0.05). We did not find changes in PRL and TSH responses to TRH stimulation after a year of treatment with EE and CA. CONCLUSION: Treatment with EE (35 microg) and CA (2 mg) induces an increase in PRL levels that persist 6 months after cessation of treatment. Our results rule out the possibility that this increase in PRL is due to a decrease in dopaminergic tone or an increase in TRH sensitivity. PMID- 10728630 TI - A study of fetal macrosomia. AB - We describe the maternal characteristics in pregnancy with fetal macrosomia, fetal and maternal complications related to macrosomia, and the risk of impaired glucose tolerance. The study is based on a comparison of maternal and neonatal data in 956 cases of fetal macrosomia (birthweight > or =4000 g) in non-diabetic pregnancy with data in a control group of 6407 mothers with non-macrosomic infants (birthweight 3000-3999 g). The main factors investigated were maternal age, weight, parity, gestosis rate, maternal and fetal birth injuries, maternal oral glucose tolerance test results and umbilical blood insulin levels. Macrosomic infants occurred in 9.1% of all deliveries. Mothers delivering macrosomic infants were significantly older, of higher parity and of greater weight than mothers of the control group. Fetal macrosomia was associated with a higher frequency of gestosis, operative deliveries, birth injuries and postpartum haemorrhages. 26.2% of the mothers had abnormal of oGTT results. The macrosomic infants were more often male and had a significantly higher risk of shoulder dystocia and birth injuries. No essential differences could be observed in the Apgar-scores and umbilical artery pH values. 34% of macrosomic infants had higher insulin levels in umbilical blood. PMID- 10728631 TI - Effects of general anaesthesia vs. sedation on fertilization, cleavage and pregnancy rates in an IVF program. AB - Anaesthetic management for oocyte retrieval may interfere with the results and success rate of an in vitro fertilization (IVF) program. This study was conducted to compare the effects of two different anaesthetic techniques (general anaesthesia versus sedation) used for oocyte retrieval on IVF outcome. For general anaesthesia we used a combination of remifentanil (Ultiva) with either propofol or isoflurane in hypnotic concentrations. For sedation the protocol included midazolam, diazepam or propofol according to clinical needs. In total, 202 women were enrolled in the study. 96 women opted for sedation and 106 for general anaesthesia. The number of collected oocytes was significantly higher with general anaesthesia (10.54+/-5.43 [mean+/-SD]) than with sedation (6.25+/ 3.65, p<0.0001), whereas the number of fertilized oocytes was not different (4.70+/-3.57 vs. 4.23+/-2.90). There were no significant differences in cleavage and pregnancy rates. We therefore believe that remifentanil-based general anaesthesia without nitrous oxide is a suitable alternative to sedation and may be recommended for IVF oocyte retrieval if general anaesthesia is requested. PMID- 10728632 TI - User friendly inquiry and computer program for following patients with ovarian malignancy. AB - The establishing of a correct diagnosis in patients with ovarian malignancy requires the combination of a variety of methods of examination. An inquiry for following of malignoma patients has been compiled, accompanied by an adequate computer program. Beside general information regarding the patient, diagnosis and treatment, the inquiry also includes the case history, data regarding clinical, ultrasonographic and preoperative examinations, surgery, histologic findings and follow-up after termination of treatment. A user-friendly computer program was developed for recording and processing of all available data collected during the diagnostic and treatment procedures. The inquiry and the computer program have been in use for three years and we find they are very useful in follow-up of patients with ovarian malignancy. Data are collected at one site, transparent and always available. Statistical analysis of collected data can be performed by computer. The aim of this uniform inquiry and computer program is a concurrent, radical and simplified collecting, processing and comparing of data at each center and between centers dealing with the treatment of ovarian malignancy. PMID- 10728633 TI - Placenta accreta/increta. Review of 10 cases and a case report. AB - A review of the patients seen at the Department of Obstetrics at Dokkyo University Hospital who had suffered placenta accreta/increta in the past 18 years, was performed. There were 10 such cases out of 9,716 deliveries during this period. This incidence is higher than that which has been reported in other Western countries. Forty percent of the patients in our study had placenta accreta/increta accompanied by placenta previa or low lying; 30% had had a prior cesarean section (C/S); 70% had previously experienced dilatation and curettage (D & C); 80% had previously undergone a C/S and/or D & C: and 40% had a history of miscarriage. Three of the ten patients with placenta accreta/increta required a hysterectomy; 2 patients were successfully treated with hemostatic stitches on the endometrium; and the remaining 5 mild cases were treated with removal of the placenta, either manually or with the use of forceps. There was no case of maternal death. In 2 cases, neonatal asphyxia was noted, but the neonate immediately recovered. PMID- 10728634 TI - Intravenous drug abuse is an indication for antepartum screening for RH alloimmunization. A case report and review of literature. AB - Intravenous drug abuse is a risk for alloimmunization in pregnancy. One case is presented of a nulliparous Rhesus-negative parturient who shared her Rhesus positive partner's needles for the injection of heroin; she subsequently developed elevated antepartum anti-D titers despite routine anti-D prophylaxis. PMID- 10728636 TI - Solitary splenic recurrence of ovarian cancer: case report and review of the literature. AB - We report a rare case of solitary recurrence of ovarian cancer in the spleen which developed 4 years after initial treatment. Only six cases have been reported in the literature and all were serous carcinoma. Our patient had a splenectomy without any complications but developed a liver metastasis 10 months after splenectomy. PMID- 10728637 TI - Changing sonographic features of fetal ovarian cysts during pregnancy and the neonatal period. AB - We present a case in which serial sonographic examinations revealed changing pattern of cystic ovarian masses from hypoechogenic to echogenic appearance. These changes were attributed to suspected torsion of ovarian cyst. Nevertheless, a healthy baby girl was born without clinical and sonographic evidence of ovarian torsion. PMID- 10728635 TI - Interval of 9 h between birth of twins at term: case report and review of the literature. AB - We report the case of a primipara who delivered healthy twins vaginally at term with a time interval between twins of 9 h and 19 min. Neonatal outcome and further development were normal in both twins. PMID- 10728638 TI - Heterotopic pregnancy (triplets) following in vitro fertilisation: case report. AB - We report a patient who had in vitro fertilization for secondary infertility due to tubal disease. Following transfer of three embryos, a twin intrauterine and a tubal ectopic pregnancy resulted (heterotopic pregnancy). PMID- 10728639 TI - Synchronization in plant cells--an introduction. PMID- 10728640 TI - Cell cycle synchronization. AB - The yeast Saccharomyces cerevisiae has been an excellent model system for cell cycle studies. Many such studies require cells synchronized in some particular portion of the cell cycle. Here, methods are described for obtaining and examining synchronized cells as they pass through one or more rounds of the cell cycle. The methods are of two types. First, block-and-release methods, where cells are initially synchronized by blocking them at some particular cell cycle stage, then releasing them from the block under conditions suitable for growth, and taking samples at different times after the release, thereby obtaining samples representing different cell cycle stages. The second type of method is elutriation. Centrifugal elutriation can be used to obtain samples of uniformly sized cells, and because cell size is correlated with cell cycle stage, these cells are synchronized with respect to their position in the cycle. Because elutriation is a very different method from block-and-release, it is ideal as a second method of synchronization to ensure that results achieved by block-and release are not artefactual. Here, block-and-release experiments with the mating pheromone alpha factor, and with the cdc15-2 mutation, are described in detail, as are some elutriation methods. PMID- 10728641 TI - Synchronization of yeast cell populations. AB - The study of synchronous populations of yeast cells has provided a wealth of information into regulatory aspects of the eukaryotic cell division cycle. Synchronized yeast cultures may also have potential benefit when exploiting yeasts in biotechnology. This paper provides an overview of the methods which have been used in the synchronization of cell division in budding and fission yeasts. The relative merits of these methods are outlined and protocols for preferred synchronization methods, based on size selection techniques, are described. In particular, centrifugal elutriation protocols for Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast) are detailed as this method is regarded as one of the best ways of preparing 'unperturbed' synchronous yeast cultures for cell cycle studies. PMID- 10728642 TI - Cell cycle synchronization in plant root meristems. AB - The analysis of structure and metabolism of a cell at a defined phase of cell cycle is often difficult because cell cycle progression in somatic tissues is asynchronous and only a fraction of cells are cycling. An elegant solution to obtain populations of cells enriched for single stage of the cell cycle is to impose the synchrony artificially. Different systems have been used to obtain synchronized populations of plant cells, including suspension-cultured cells, leaf mesophyll protoplasts and root tip meristems. Root tips have been frequently used in a variety of studies ranging from chromosome analysis to cell cycle and its regulation. Seedlings with actively growing roots may be obtained in most plant species, they are easy to handle, the experimental system is well defined, reproducible and can be easily modified for different species. This paper describes a protocol for cell cycle synchronization in root tips of Vicia faba, which is based on the use of DNA synthesis inhibitor hydroxyurea [18]. Modifications of the protocol for Pisum sativum, Medicago sativa, Hordeum vulgare, Secale cereale, Triticum aestivum, and Zea mays are also given. Flow cytometric data indicate that about 90% of root tip cells are synchronized. On average, mitotic indices exceeding 50% are obtained with the method. Synchronized cells may be accumulated at metaphase using a mitotic spindle inhibitor to achieve metaphase indices exceeding 50%. PMID- 10728643 TI - Study of phase-specific gene expression in synchronized tobacco cells. AB - Although the basic mechanisms which control the progression through the cell cycle appear to be conserved in all higher eukaryotes, the unique features of the plant developmental programme must be somehow reflected in a plant-specific regulation of the factors which control cell division. In the last few years, considerable progress has been achieved in identifying the major components of the cell cycle in plants. The question of how these components direct expression of specific genes at specific stages of the cell cycle, and how they are themselves regulated, constitutes a challenge for the present and the next years. This review summarizes our current knowledge at molecular and biochemical levels of cell cycle-regulated expression in the model system, the synchronized tobacco BY2 cell suspension, and discusses the results in comparison to those obtained by different methods and in other plant systems. PMID- 10728644 TI - Plant cell biology through the window of the highly synchronized tobacco BY-2 cell line. AB - Synchronous cell systems are highly desirable for investigating various aspects of plant cell biology. However, to date, the tobacco BY-2 cell line is the only plant cell line which can be synchronized to high levels. A cell synchrony starting from S phase is obtained after release of BY-2 cells from aphidicolin treatment, while that from M phase is available after release from a sequential treatment of aphidicolin followed by propyzamide. A high level of synchrony is only attained by using rapidly growing tobacco BY-2 cells that propagate ca. 100 fold in a week. Reduced levels of synchrony result if the growth rate becomes lower. Technical notes for maintaining the high growth rate of the tobacco BY-2 cell are described. Using this highly synchronous system it has been possible to demonstrate the cell cycle-dependent oscillation of many genes, such as cyclins, and characterize their role during the cell cycle. Furthermore, this system has facilitated the structural and biochemical analysis of cell cycle specific events such as the development of the phragmoplast and the formation of cortical microtubules. Other potential uses of this highly synchronized cells are also described. PMID- 10728645 TI - Synchronization of somatic embryogenesis at high frequency using carrot suspension cultures: model systems and application in plant development. AB - Materials and methods for the high frequency induction and synchronous somatic embryogenesis from cultured cells of higher plants are described, using carrot suspension cultures as a model system of higher plants. The following four synchronous systems of somatic embryogenesis, which were established in our laboratories, are reported: (1) Somatic embryogenesis from single cells. a) Small spherical single cells, obtained from suspension cultures in the presence of 2,4 D, zeatin and mannitol by sieving, density gradient centrifugation in Percoll solutions and manual picking up, form embryogenic cell clusters, which differentiate to embryos at high frequency, when embryogenic cell clusters are transferred to a medium lacking 2,4-D. b) Explants of hypocotyls of regenerated plantlets from somatic embryos were cultured after treatment with 2,4-D for 12-24 h, and then transferred into a fresh medium lacking 2,4-D. Single cells are released from hypocotyl explants and differentiated into embryos at high frequency. In this system, a large number of single cells and embryogenic cells can be collected. (2) Somatic embryogenesis from embryogenic cell clusters, which are obtained from suspension cultures by sieving, density gradient centrifugation in Ficoll solutions, and subsequent centrifugation at a low speed, differentiate synchronously to globular embryos at high frequency. Plantlets are formed from globular embryos. (3) Embryogenic cell clusters obtained according to the procedure described in (2) are cultured at cell densities of 2x10(3) cell clusters ml(-1). Globular embryos differentiate to torpedo-shaped embryos and subsequently to plantlets at high frequency when they are cultured at densities below 150 globular embryos ml(-1). PMID- 10728646 TI - Establishment of synchronization in carrot cell suspension culture and studies on stage specific activation of glyoxalase I. AB - The present report summarizes and compares the effects of three cell cycle inhibitors, viz. aphidicolin, hydroxyurea and mimosine, in inducing synchronization of a rapidly proliferating suspension culture of carrot. These treatments efficiently synchronized the cell cycle as the doubling time of the cell population was roughly equal to the total length of one cell cycle. Protoplasts derived from mimosine treated cell suspension culture were resolved via flow cytometry to get an idea of the temporal organization of the cell cycle events. The biochemical analysis showed a rise in stage specific activity of glyoxalase I, an auxin inducible marker enzyme activated at G2-M. This activity peak could be shifted to an early phase of interphase in response to auxin treatment. PMID- 10728647 TI - Cultured green cells of tobacco as a useful material for the study of chloroplast replication. AB - Chloroplast replication in cultured cells of tobacco (Nicotiana tabacum cv. Samsun NN) was investigated by electron microscopy in comparison with that of green leaves. The structure of chloroplasts in cultured cells changed conspicuously during cell growth especially in photoautotrophic cells. The frequency of dumbbell-shaped chloroplasts (intermediate of chloroplast division) was the maximum (about 23% of total chloroplast) in photoautotrophic cells at 3 days after inoculation, before the cells had started to grow. By contrast, in photomixotrophically cultured cells, the highest frequency of dividing chloroplasts was observed at the early exponential phase (about 7 days after inoculation). The dividing chloroplast was hardly detected in green leaves even at a young stage. The advantages of cultured cells for the study of chloroplast replication and ultrastructural development are discussed. PMID- 10728648 TI - Bivariate flow cytometry DNA/BrdUrd analysis of plant cell cycle. AB - We describe a protocol for flow cytometry analysis of cell cycle in plants using indirect immunolabelling staining and Vicia faba, Pisum sativum and Zea mays root tip cells as model systems. The protocol is based on simultaneous analysis of two fluorescent signals. The first, obtained after staining with propidium iodide, is used to quantify nuclear DNA content. The second, obtained after indirect immunofluorescent staining of bromodeoxyuridine (BrdUrd), is used to quantify the amount of BrdUrd incorporated into nuclear DNA. In an attempt to standardize the procedure, the effects of various conditions for partial DNA denaturation using HCl, as well as of BrdUrd concentration and incorporation time on flow cytometry DNA/BrdUrd content analysis have been studied. Maximum BrdUrd-linked fluorescence was observed after a 30 min pulse with 10 microM BrdUrd and after DNA denaturation with 1.5 N HCl (final concentration) for 30 min at 25 degrees C. Under these conditions, DNA content histograms with relatively small coefficient of variation (< 4%, full peak) could be obtained. To avoid non-specific staining of cytoplasm and cell walls, the protocol involves the use of nuclei isolated from formaldehyde-fixed tissues. Fixed isolated nuclei are stable and may be stored in hexylene glycol 0.75 M at 4 degrees C for prolonged periods prior to actual staining and analysis. PMID- 10728649 TI - Signal transduction events elicited by natural products: role of MAPK and caspase pathways in homeostatic response and induction of apoptosis. AB - Many natural products elicit diverse pharmacological effects. Using two classes of potential chemopreventive compounds, the phenolic compounds and the isothiocyanates, we review the potential utility of two signaling events, the mitogen-activated protein kinases (MAPKs) and the ICE/Ced-3 proteases (caspases) stimulated by these agents in mammalian cell lines. Studies with phenolic antioxidants (BHA, tBHQ), and natural products (flavonoids; EGCG, ECG, and isothiocyanates; PEITC, sulforaphane), provided important insights into the signaling pathways induced by these compounds. At low concentrations, these chemicals may activate the MAPK (ERK2, JNK1, p38) leading to gene expression of survival genes (c-Fos, c-Jun) and defensive genes (Phase II detoxifying enzymes; GST, QR) resulting in survival and protective mechanisms (homeostasis response). Increasing the concentrations of these compounds will additionally activate the caspase pathway, leading to apoptosis (potential cytotoxicity). Further increment to suprapharmacological concentrations will lead to nonspecific necrotic cell death. The wider and narrow concentration ranges between the activation of MAPK/gene induction and caspases/cell death exhibited by phenolic compounds and isothiocyanates, respectively, in mammalian cells, may reflect their respective therapeutic windows in vivo. Consequently, the studies of signaling pathways elicited by natural products will advance our understanding of their efficacy and safety, of which many may become important therapeutic drugs of the future. PMID- 10728650 TI - Synthesis and anti-inflammatory and analgesic activities of pyridyloxy- and phenoxyalkanoic acid derivatives. AB - Synthesis of pyridyloxy-, pyridyloxyphenoxy- and phenoxylphenoxyalkanate derivatives and their anti-inflammatory and analgesic activities were investigated. Analysis of structure-activity relationships showed that in pyridyloxyalkanoic acid derivatives anti-edematous potency was associated with the presence of chlorophenoxypropionic acid moiety and 2-nitrated methyl propionates contributed to the analgesic activity. PMID- 10728651 TI - Naphthazarin derivatives (V): formation of glutathione conjugate and cytotoxic activity of 2-or 6-substituted 5,8-dimethoxy-1,4-napthoquinones in the presence of glutathione-S-transferase, in rat liver S-9 fraction and mouse liver perfusate. AB - Formation of glutathione (GSH) conjugates with 2- or 6-(1-hydroxymethyl)- and 2 (1-hydroxyethyl)-DMNQ derivatives (DMNQ, 5,8-dimethoxy-1,4-naphthoquone) was carried out in phosphate buffer (pH 7.4), in the presence of glutathione-S transferase (GST), in rat liver S-9 fraction and by perfusion, and the rates of conjugates formation were compared and correlated to cytotoxicity. The GSH conjugates of 6-(1-hydroxyalkyl)-DMNQ derivatives were formed faster than 2-(1 hydroxyalkyl)-DMNQ derivatives under all of the media, implying that steric hindrance was the cause of lowering the rate of conjugate formation of 2 substituted derivatives. For both isomers, addition of GST did not improve the reaction rate, compared with that in buffer, while the reaction in the S-9 fraction and the perfusate was accelerated to a great extent. The catalytic effect of the S-9 fraction and the perfusion on 2-isomers was greater than on 6 substituted ones, suggesting that S-9 fraction and the perfusate contain an effective system relaxing the steric hindrance of 2-(1-hydroxyalkyl)-DMNQ derivatives. Furthermore, a good correlation between the formation of the GSH conjugates and the cytotoxic activity of both naphthazarin isomers suggests that the steric hindrance is a cause of lowering the cytotoxicity of 2-isomers. PMID- 10728652 TI - Reversed-phase high performance liquid chromatographic separation of the enantiomers of terbutaline by derivatization with 2,3,4,6-tetra-o-acetyl-beta-D glucopyranosyl isothiocyanate. AB - The enantiomers of the bronchodilator terbutaline were separated by reversed phase high performance liquid chromatography after derivatization with 2,3,4,6 tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate(GITC) reagent. The derivatization proceeded quantitatively within 1 h at room temperature. The corresponding diastereomeric thiourea derivatives were well resolved on an ODS column with acetonitrile-acetate buffer as a mobile phase. Elution orders of the diastereomers were confirmed by derivatization of R-(-)-terbutaline and S-(+) terbutaline which were collected by semi-preparative chiral HPLC using Sumichiral OA-4700 column. The native fluorescence of terbutaline was quenched by derivatization with GITC. The detection limit was 25 ng when monitored at UV 278 nm. PMID- 10728653 TI - Synthesis of 7,8-dichloro-6-nitro-1H-1,5-benzodiazepine-2,4-(3H, 5H)-dione as a potential NMDA receptor glycine site antagonist. AB - An efficient procedure for the preparation of 7,8-dichloro-6-nitro-1H-1,5 benzodiazepine-2,4-(3H, 5H)-dione(7) as a potential lead compound for the NMDA receptor glycine binding site antagonist, starting from readily available 4,5 dichloro-2-nitroaniline(8), is described. The key step in the synthesis involves the cyclization of malonic ester amide 10 to compound 11. PMID- 10728654 TI - Stereochemical requirement at 4-position of 4-phenyl-1 arylsulfonylimidazolidinones for their cytotoxicities. AB - In order to investigate the stereochemical requirements of planar structure at 4 position of 4-phenyl-1-arylsulfonylimidazolidinones (1) for their cytoxicities against human cancer cell lines, the size, the distance from imidazolidinone ring, and the conformation of this moiety were variegated. Replacement of phenyl moiety with naphthyl in compounds 2 and 3 or benzyl moiety in compound 4 sharply reduced activity of 1. Conformational restriction on phenyl ring in compound 5 also resulted in the loss of activity of 1. Therefore, phenyl moiety without any substituents directly attached to imidazolidinone ring of 1 should be considered as an essential pharmacophore for this analog. PMID- 10728655 TI - Cytotoxic activities of 6-arylamino-7-halo-5,8-quinolinediones against human tumor cell lines. AB - 6-Arylamino-7-halo-5,8-quinolinediones (4a-4k, 5a-5b) were tested for in vitro cytotoxicity against human solid tumor cell lines such as A 549 (non-small cell lung), SK-OV-3 (ovarian), SK-MEL-2 (melanoma), HCT-15 (colon) and XF 498 (CNS) by SRB assay. The arylamino-7-chloro-5,8-quinolinediones 4 were also evaluated for cyclin-dependent kinase (CDK2 and CDK4) inhibitory effect. Among them, the 5,8 quinolinediones 4a and 5a with 7-(4-fluorophenyl)amino group were found to be potent cytotoxic against HCT 15, SKOV-3 and XF 498, and the compounds 4f and 4i showed inhibitory activities for the CDK4. PMID- 10728656 TI - Antifungal activity of magnolol and honokiol. AB - Two neolignan compounds, magnolol (5,5'-diallyl-2,2'-dihydroxybiphenyl, 1) and honokiol (5,5'-diallyl-2,4'-dihydroxybiphenyl, 2), were isolated from the stem bark of Magnolia obovata and evaluated for antifungal activity against various human pathogenic fungi. Compound 1 and 2 showed significant inhibitory activities against Trichophyton mentagrophytes, Microsporium gypseum, Epidermophyton floccosum, Aspergillus niger, Cryptococcus neoformans, and Candida albicans with minimum inhibitory concentrations (MIC) in a range of 25-100 microg/ml. Therefore, compound 1 and 2 could be used as lead compounds for the development of novel antifungal agents. PMID- 10728657 TI - Diarylheptanoids from the leaves of Alnus hirsuta Turcz. AB - Diarylheptanoids, (5S)-1,7-bis-(3,4-dihydroxyphenyl)-5-hydroxyheptane-3-one (1, hirsutanonol), (5S)-1,7-bis-(3,4-dihydroxyphenyl)-heptane-3-one-5-O-beta-D xylopyranosi de (2, oregonin), (5R)-1,7-bis-(3,4-dihydroxyphenyl)-heptane-5-O beta-D-xylopyranoside (3), and (5R)-1,7-bis-(3,4-dihydroxyphenyl)-heptane-5-O beta-D-glucopyranoside (4) were isolated from the leaves of Alnus hirsuta Turcz. The structures of these compounds were identified based on the spectral and physicochemical data. PMID- 10728658 TI - Inhibition of LPS-induced NO production and NF-kappaB activation by a sesquiterpene from Saussurea lappa. AB - To elucidate the molecular mechanisms for the suppression of LPS-induced nitric oxide (NO) production by a dehydrocostus lactone (DL) from Saussurea lappa, we examined the preventive effect of this compound on NF-kappaB activation in LPS treated RAW 264.7 macrophages and U937 human monocytic cells. The results suggest that the suppression of NO production is mediated by the inhibitory action on the i-NOS gene expression through the inactivation of NF-kappaB and this sesquiterpene lactone can act as a pharmacological inhibitor of the NF-kappaB activation. PMID- 10728659 TI - Effect of extracellular cations on the chemotherapeutic efficacy of anticancer drugs. AB - Cancer development and the efficiency of chemotherapy relies on the patients calcium-related pathological status such as hyper- or hypocalcemia. In the present study, we investigated the effect of extracellular cations such as calcium and magnesium on the therapeutic efficacy of antitumor drugs. The analytic parameters used were cellular drug uptake/excretion and the chemosensitivity of the human breast cancer cell lines, MCF7 and MCF7/ADR. Both calcium and magnesium ions decreased the membrane permeability of cancer cells, which was determined by cell size analysis. These divalent ions also lowered the drug uptake and the cytoplasmic levels of rhodamine 123 and adriamycin, suggesting that they might interfere with the diffusion of these drugs by modifying the physical properties of the cytoplasmic membrane. The acute cytotoxicity of adriamycin after a short period of incubation correlated with changes in its cytoplasmic level. Our results indicate that these extracellular cations might play an important role in the therapeutic activities of anticancer drugs in cancer patients. These results also provide insight a new aspect of chemotherapy, because they suggest that the therapeutic doses of anti-cancer drugs should be modified in cancer-bearing patients presenting with abnormal blood calcium levels. PMID- 10728660 TI - Application of dry elixir system to oriental traditional medicine: taste masking of peonjahwan by coated dry elixir. AB - Peonjahwan, an oriental traditional medicine composed of crude herbal drugs and animal tissues is bitter and poorly water-soluble. To mask the bitterness of peonjahwan and enhance the release of bilirubin, one of the crude active ingredients of peonjahwan, peonja dry elixir (PDE), was prepared using a spray dryer after extracting the crude materials in ethanol-water solution. Coated peonja dry elixir (CPDE) was then prepared by coating the PDE with Eudragit acrylic resin. Panel assessed bitterness and release test of bilirubin from PDE and CPDE were carried out and compared with peonjahwan alone. PDE was found to have little effect upon the reduction of the bitterness of peonjahwan. However, the bitterness of CPDE was found to reduce to 1/4 of that of peonjahwan due to the encapsulation of crude active ingredients by the dextrin and Eudragit shell (P<0.05). The release rate of bilirubin from PDE and CPDE for 60 min increased about 3.5- and 2.5- fold, respectively, compared to peonjahwan at pH 1.2. It is concluded that CPDE, which masked the bitterness of peonjahwan and enhanced the release of bilirubin, is a preferable delivery system for peonjahwan. PMID- 10728661 TI - General pharmacological properties of YJA20379-2, a new antiulcer agent. AB - The general pharmacological properties of YJA20379-2 2-dimethylamino-4,5 dihydrothiazolo[4,5:3,4]pyridol[1,2-a]++ +benzoimidazole, a novel proton pump inhibitor with antiulcer activities were investigated in mice, rats, guinea pigs and rabbits. YJA20379-2 at oral doses of 50, 100 and 200 mg/kg did not affect the general behaviour, hexobarbital hypnosis and motor coordination in mice. The drug did not have analgesic or anticonvulsant action at 200 mg/kg. Locomotor activity and body temperature were not influenced at 100 mg/kg. At a concentration up to 2 x 10(-4) g/ml, YJA20379-2 did not produce any contraction or relaxation of isolated preparations, such as the rat fundus, the guinea pig ileum and the rat uterus, and did not antagonize the contractile response to several spasmogens, such as histamine, acetylcholine, serotonin and oxytocin. At dosages up to 200mg/kg p.o. YJA20379-2 did not affect the pupil size of mice. Intestinal propulsion of mice was not affected up to 200 mg/kg p.o. and the drug did not affect urinary excretion at 100 mg/kg p.o. These results indicate that at dosages up to 100 mg/kg p.o. YJA20379 was found not to affect this pharmacological profile. However, at 200 mg/kg the drug lowered body temperature and showed decreases in locomotor activity and urine volume. PMID- 10728662 TI - Generation of nuclear hybrids overcoming the natural barrier of incompatibility: transfer of nuclei from Lentinula edodes into protoplasts of Coriolus versicolor. AB - Heterokaryotic nuclear hybrids overcoming the natural barriers of incompatibility have been studied in basidiomycetes. To produce these nuclear hybrids between incompatible mushrooms, which have several potent pharmacological effects, nuclear transfer was performed between Lentinula edodes and Coriolus versicolor. Nuclei from serine auxotrophs of Lentinula edodes, LE207 (Ser-) were transferred into the protoplasts of arginine auxotrophs of Coriolus versicolor, CV17 (Arg-), using 30% polyethylene glycol 4000 in 10 mM CaCl2-glycine solution (pH 8.0). Nuclear transfer progenies were selected by nutritional complementation on minimal media supplemented with 0.6 M sucrose. The progenies were classified based on colony morphology to L. edodes-like, C. versicolor-like and non-parental type. Most of the progenies grew slower than either parent. The number of nuclei per cell was similar but the DNA content varied between progenies. The isozyme patterns of nuclear hybrids resembled either of the parent profiles or showed a mixed profile. PMID- 10728663 TI - Thermo-sensitive self-assembled micelles prepared by cholic acid and poly(N isopropylacrylamide). PMID- 10728664 TI - Chemotherapeutic DNA-damaging drugs activate interferon regulatory factor-7 by the mitogen-activated protein kinase kinase-4-cJun NH2-terminal kinase pathway. AB - Chemotherapeutic drugs and energy-rich radiation cause DNA damage, inducing signaling pathways for apoptotic cell death or cell growth arrest. The tumor suppressor gene p53 plays the critical role in the regulation of these DNA damage responses. Human tumor cells can become resistant to chemotherapy through functional inactivation of p53. Thus, it is important to identify p53-independent DNA damage signaling pathways. Here, treatment of cells with chemotherapeutic drugs or UV irradiation potentiated the transcriptional activity of IFN regulatory factor-7 (IRF7), inducing its phosphorylation and its nuclear translocation. Furthermore, IRF7 was activated by the c-Jun NH2-terminal kinase (JNK) in response to DNA-damaging agents. Activation of JNK by mitogen-activated protein kinase kinase-4 stimulated the transcriptional activity of IRF7 and induced its translocation into the nucleus. Thus, activation of IRF7 through the JNK signaling pathway may play a role in the transcriptional regulation of genes in response to DNA-damaging agents. PMID- 10728665 TI - Antitumor effect of adenovirus-mediated Bax gene transfer on p53-sensitive and p53-resistant cancer lines. AB - Antitumor effects of the proapoptotic Bax gene have been evaluated in vitro and in vivo by a binary adenovirus system expressing the human Bax gene. Overexpression of the Bax gene in cultured cell lines from human lung carcinoma results in caspase activation, apoptosis induction, and cell growth suppression. Intratumoral injection of adenovirus vector expressing the Bar gene suppressed growth of human lung cancer xenografts established in nude mice. Histological examination of tumors from mice treated with the Bax gene demonstrated high levels of Bax expression and extensive apoptosis in tumors. In comparison with the treatment by an adenoviral vector expressing human p53, the Bax gene can effectively suppress tumor growth in both p53-sensitive and p53-resistant human lung carcinoma cell lines. Toxicity was not detected in liver and other systems in animals treated intralesionally with the Bax gene. Therefore, our results suggest that the Bar gene may be useful in cancer treatment. PMID- 10728666 TI - Association of the Bloom syndrome protein with topoisomerase IIIalpha in somatic and meiotic cells. AB - Bloom syndrome (BS) is characterized by genomic instability and cancer susceptibility caused by defects in BLM, a DNA helicase of the RecQ-family (J. German and N. A. Ellis, The Genetic Basis of Human Cancer, pp. 301-316, 1998). RecQ helicases and topoisomerase III proteins interact physically and functionally in yeast (S. Gangloff et al., Mol. Cell. Biol., 14: 8391-8398, 1994) and in Escherichia coli can function together to enable passage of double stranded DNA (F. G. Harmon et al., Mol. Cell, 3: 611-620, 1999). We demonstrate in somatic and meiotic human cells an association between BLM and topoisomerase IIIalpha. These proteins colocalize in promyelocytic leukemia protein nuclear bodies, and this localization is disrupted in BS cells. Thus, mechanisms by which RecQ helicases and topoisomerase III proteins cooperate to maintain genomic stability in model organisms likely apply to humans. PMID- 10728667 TI - Interleukin-13 receptor alpha chain: a novel tumor-associated transmembrane protein in primary explants of human malignant gliomas. AB - Human malignant glioma cell lines express high levels of interleukin-13 receptor (IL-13R). However, the subunit structure of this receptor in primary brain tumor cells is not known. Herein, we examined the subunit composition of IL-13R by analyzing the expression of four different putative subunits of IL-13R complex in 25 primary explants of malignant brain tumors. Reverse transcription-PCR (RT-PCR) of RNA from these tumor cells, normal astrocytes, and normal brain tissue showed that transcripts of IL-13R alpha chain were present in greater abundance in malignant glioma cells compared with normal astrocytes or normal brain tissues. The transcripts for two other chains (e.g., IL-13Ralpha' and IL-4Rbeta), on the other hand, yielded similar PCR positivity in brain tumors as well as in normal samples, whereas transcripts for gammac chain were absent in all brain tumor cells and normal tissues. The specificity of RT-PCR products for these genes was confirmed by oligo liquid hybridization analysis using a radiolabeled sequence specific internal probe. Indirect immunofluorescence studies for different receptor chains confirmed the RT-PCR results and demonstrated a striking difference in the level of expression of IL-13Ralpha protein between normal astrocytes and malignant astrocytoma cells. These studies establish the IL 13Ralpha subunit as a novel tumor-specific protein that may be useful as a tumor marker, a target for cytotoxin/immunotoxin, or alternatively, a tumor-associated antigen for active, specific immunotherapy. PMID- 10728668 TI - Carbonyl reductase: a novel metastasis-modulating function. AB - To explore reasons for differences in the malignancy of tumors, we have compared two cell lines derived from a mouse lung adenocarcinoma cell line that differ 10 fold in their capacity to form lung metastases from s.c. primary tumors or after i.v. injection. One mRNA encoding carbonyl reductase was identified at a relatively high abundance in the subline with low metastatic capacity but was not detectable in the highly metastatic subline. Transfection of the former subline with a plasmid construct expressing antisense carbonyl reductase rendered the cells highly metastatic. Conversely, the capacity of the highly metastatic cells to metastasize was markedly reduced after transfection with a construct expressing carbonyl reductase. We also found that human prostate cancers show loss of carbonyl reductase expression compared with normal prostate epithelia. These data suggest that carbonyl reductase has an important function in modifying the metastatic behavior of malignant tumors. PMID- 10728669 TI - Altered expression of Fhit in carcinoma and precarcinomatous lesions of the esophagus. AB - The FHIT gene, located at chromosome 3p14.2, is a tumor suppressor gene often involved in tumors resulting from exposure to environmental carcinogens. We studied 46 pairs of esophageal primary tumors and corresponding normal squamous mucosa specimens by molecular genetic and immunohistochemical methods to investigate the role of the FHIT gene in esophageal carcinoma. In addition, we studied several different types of lesions, such as carcinoma in situ or dysplasia by immunohistochemistry. Loss of heterozygosity at or around the FHIT gene was observed in 35 (76%) primary tumors. Immunohistochemical detection of Fhit protein in the primary tumors demonstrated that 14 (30%) were positive and 32 (70%) were negative. We observed concordance between loss of Fhit protein and loss of heterozygosity and between loss of Fhit protein and RNA abnormalities. Because the FHIT/FRA3B locus is susceptible to damage by environmental carcinogens, we investigated the correlation between Fhit expression and smoking or alcohol habits. In this relatively small study, the patients who were both heavy users of tobacco and alcohol showed a significantly higher frequency of loss of Fhit expression than those who were light users. Noncarcinomatous squamous epithelium showed positive Fhit reactivity in most cases; however, five showed negative Fhit reactivity. Interestingly, all of these five patients had habits of heavy use of tobacco and alcohol. Eight of 12 carcinomas in situ, 2 of 4 severe dysplasias, 4 of 8 moderate dysplasias, and 3 of 9 mild dysplastic lesions showed negative Fhit reactivity. These findings indicated that loss of Fhit expression may be an early event in the development of human esophageal carcinoma and may occur even in normal-appearing squamous epithelium in some patients heavily exposed to environmental carcinogens. PMID- 10728670 TI - Transforming growth factor beta2 promotes glucose carbon incorporation into nucleic acid ribose through the nonoxidative pentose cycle in lung epithelial carcinoma cells. AB - The invasive transformation of A-459 lung epithelial carcinoma cells has been linked to the autocrine regulation of malignant phenotypic changes by transforming growth factor beta (TGF-beta). Here we demonstrate, using stable 13C glucose isotopes, that the transformed phenotype is characterized by decreased CO2 production via direct glucose oxidation but increased nucleic acid ribose synthesis through the nonoxidative reactions of the pentose cycle. Increased nucleic acid synthesis through the nonoxidative pentose cycle imparts the metabolic adaptation of nontransformed cells to the invasive phenotype that potentially explains the fundamental metabolic disturbance in tumor cells: highly increased nucleic acid synthesis despite hypoxia and decreased glucose oxidation. PMID- 10728671 TI - Further evidence that the radioprotective aminothiol, WR-1065, catalytically inactivates mammalian topoisomerase II. AB - It has recently been proposed that the thiol form of the cytoprotective drug amifostine that is designated WR-1065 [2-((aminopropyl)amino)ethanethiol] exerts its cytoprotective effects in part via a catalytic inhibition of DNA topoisomerase II (topo II)alpha. This in turn leads to the subsequent accumulation of cells in G2 phase and a prolongation of the cell cycle. We have used a Chinese hamster V79 cell-based micronucleus assay to further evaluate this hypothesis. It is demonstrated that WR-1065 strongly inhibits the clastogenesis of the topo II poisons etoposide and clinafloxacin at clinically attained exposure levels while having no effect on clastogenesis induced by topo II noninteractive chemicals. These findings are consistent with the hypothesis that WR-1065 is a catalytic inhibitor of topo II in mammalian cells. These studies also suggest that WR-1065 might be expected to reduce the toxicity and clastogenicity in clinical applications of etoposide or quinolone antibiotics in dose-limiting normal tissues. PMID- 10728672 TI - Characterization of CPT-11 hydrolysis by human liver carboxylesterase isoforms hCE-1 and hCE-2. AB - 7-Ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy-camptothecin (irinotecan; CPT-11) is a prodrug activated by carboxylesterase enzymes. We characterized the hydrolysis of CPT-11 by two recently identified human carboxylesterase (hCE) enzymes, hCE-1 and hCE-2. Km and Vmax for hCE-1 and hCE-2 are 43 microM and 0.53 nmol/min/mg protein and 3.4 microM and 2.5 nmol/min/mg protein, respectively. hCE 2 has a 12.5-fold higher affinity for CPT-11 and a 5-fold higher maximal rate of CPT-11 hydrolysis when compared with hCE-1. In cytotoxicity assays, incubation of 1 microM CPT-11 with hCE-2 (3.6 microg/ml) resulted in a 60% reduction in survival of SQ20b cells. No significant reduction in cell survival was observed after incubation of CPT-11 with hCE-1. These data indicate that hCE-2 is a high affinity, high-velocity enzyme with respect to CPT-11. hCE-2 likely plays a substantial role in CPT-11 activation in human liver at relevant pharmacological concentrations. PMID- 10728673 TI - In vivo antitumor activity of ONYX-015 is influenced by p53 status and is augmented by radiotherapy. AB - The E1B-deleted, replication-competent ONYX-015 (dl1520) adenovirus was originally described as being able to selectively kill p53-deficient cells due to a requirement of p53 inactivation for efficient viral replication. This hypothesis has become controversial because subsequent in vitro studies have demonstrated that the host range specificity of ONYX-015 is independent of p53 gene status. Using a pair of isogenic cell lines that differ only in their p53 status, we demonstrate here that although ONYX-015 can replicate in both p53 wild type and mutant cells in vitro, the virus demonstrates significantly greater antitumor activity against mutant p53 tumors in vivo. Moreover, ONYX-015 viral therapy can be combined with radiation to improve tumor control beyond that of either monotherapy. The results demonstrate that ONYX-015 can discern in vivo between tumors having a different p53 status and that it may be an effective neoadjuvant to radiation therapy. PMID- 10728674 TI - A new temperature-sensitive liposome for use with mild hyperthermia: characterization and testing in a human tumor xenograft model. AB - The single biggest challenge now facing drug delivery (for liposomes and indeed other carriers) is to initiate and produce release of the encapsulated drug only at the diseased site and at controllable rates. Our efforts have focused on developing a new thermal-sensitive drug delivery system, specifically for the local control of solid tumors. We describe here a new lipid formulation containing doxorubicin that has been optimized for both mild hyperthermic temperatures (39 degrees C to 40 degrees C) that are readily achievable in the clinic and rapid release times of drug (tens of seconds). This new liposome, in combination with mild hyperthermia, was found to be significantly more effective than free drug or current liposome formulations at reducing tumor growth in a human squamous cell carcinoma xenograft line (FaDu), producing 11 of 11 complete regressions lasting up to 60 days posttreatment. PMID- 10728675 TI - Nuclear factor-kappaB/Rel is apoptogenic in cytokine withdrawal-induced programmed cell death. AB - In the complex microenvironment where they evolve, developing cells undergo rapid programmed cell death (PCD) when cytokines that support them become limiting. The transcriptional mechanisms of cytokine-withdrawal apoptosis are poorly understood. In this report, we used early B-lymphocyte tissue culture and transgenic cells to demonstrate that nuclear factor-kappaB (NF-kappaB) promotes apoptosis during cytokine withdrawal-induced PCD. In the progenitor B lymphocyte model FL5.12, whereas NF-kappaB has an antiapoptotic function in response to tumor necrosis factor-alpha, cytokine withdrawal causes nuclear translocation of NF-kappaB/cRel, where it is apoptogenic. Inhibition of NF-kappaB activation delays cytokine withdrawal-induced PCD in both FL5.12 and transgenic early B cells. Additionally, reconstituting a bone marrow microenvironment ex vivo abrogates the differential apoptotic pattern between control and transgenic early B cells. PMID- 10728676 TI - Major improvement in the efficacy of BRCA1 mutation screening using morphoclinical features of breast cancer. AB - A family history of breast and/or ovarian cancer is the main criterion used in screening BRCA1 gene carriers. However, ascertaining a patient's family history is a difficult task, which significantly restricts the use of this parameter in clinical practice. Alternative individual criteria that can be used to identity BRCA1 gene carriers would, therefore, be of great value. In this context, it was recently established that BRCA1-associated breast cancers (BRCA1-BCs) show a specific morphoclinical pattern. In multivariate analyses, the two most discriminant morphoclinical parameters available for establishing the BRCA1 status, in addition to an early age at onset, are estrogen receptor negativity (ER-) and poor tumor differentiation (TD3). Here we tested the efficacy of these two morphological parameters as BRCA1 mutation indicators and investigated their economic impact, in a population-based survey on a series of women who developed invasive breast cancer by the age of 35 years, regardless of their family history. A high rate of 28.6% of BRCA1 mutations was found to have occurred in the group of tumors with both ER- and TD3 versus only 3.6% in tumors with other profiles (P = 0.007; odds ratio, 10.8). When the sole criterion used was early onset by the age of 35 years, the mutation rate was found to be 8.6%. The resulting cost of testing only women with ER- and TD3 tumors worked out at 30% that of testing the whole population of women with cancer by the age of 35 years, and the sensitivity was found to be of 66%. Lastly, the family history of ER- and TD3 cases with a BRCA1 mutation was investigated retrospectively, and none of these cases was found to have a particularly extensive family history of breast and/or ovarian cancer. The use of these morphological features of BRCA1-BCs that are currently typed in clinical practice, therefore, provides a helpful and cost effective tool for those making decisions about genetic screening. This strategy makes it possible to identify gene carriers who would be overlooked using current criteria. PMID- 10728677 TI - Loss of pRb expression in pituitary adenomas is associated with methylation of the RB1 CpG island. AB - We recently showed loss of pRb in a proportion of pituitary tumors that was not associated with loss of heterozygosity of an RB1 intragenic marker. To further define the mechanism responsible for loss of retinoblastoma protein (pRb) expression, we have investigated the methylation status of the CpG island contained within the promoter region of the RB1 gene, together with sequence analysis of the essential promoter region and exons coding for the protein binding pocket domain. Methylation of the CpG island within the RB1 promoter region was detected in 6 of 10 tumors that failed to express pRb. In contrast, 18 of 20 tumors and all six histologically normal postmortem pituitaries that expressed pRb were unmethylated. No inactivating mutations were found within the RB1 promoter region in the four unmethylated tumors that failed to express pRB. However, one or more exons comprising the coding region for the protein-binding pocket domain were shown to be homozygously deleted in three of four tumors available for analysis. This study describes an additional tumor type, in addition to retinoblastoma, in which methylation of the RB1 promoter is associated with loss of pRb expression. Furthermore, we show that in addition to methylation of the RB1 promoter region, deletion within the protein-binding pocket domain is associated with a loss of detectable pRb expression. The reactivation of tumor suppressor genes, silenced through methylation, represents a promising therapeutic target in sporadic pituitary adenomas. PMID- 10728678 TI - CD95 (Fas/APO-1) and p53 signal apoptosis independently in diverse cell types. AB - The tumor suppressor p53 exerts its antioncogenic effects in cells chiefly by regulating their progression through the cell cycle and by inducing cell death. It has been claimed that p53-transduced apoptosis involves the death receptor CD95 (Fas/APO-1). We report that thymocytes from mice lacking functional Fas ligand (gld) show normal sensitivity to apoptosis transduced by p53, and that hepatocytes fromp53-/- mice have normal sensitivity to apoptosis triggered through ligation of CD95. p53 and CD95, therefore, function in independent pathways to cell death in these diverse cell types. PMID- 10728679 TI - Fibroblast growth factor receptor-1 alpha-exon exclusion and polypyrimidine tract binding protein in glioblastoma multiforme tumors. AB - Neoplastic transformation of glial cells alters inclusion of the alpha exon in human fibroblast growth factor receptor-1 (FGFR-1) mRNA transcripts. Although normal cells predominantly include the alpha exon, this exon is excluded in most glioblastoma cell transcripts, creating a high-affinity receptor form. In this study, we identified polypyrimidine tract-binding protein (PTB) as a regulator of FGFR-1 splicing. PTB interacted in a sequence-specific manner with the ISS-1 regulatory element in the intron upstream of the a exon. PTB expression was also strongly increased in seven malignant glioblastoma multiforme tumors relative to adjacent normal tissue, but not in a low-grade astrocytoma. These results suggest that increased expression of PTB may contribute to glial cell malignancy. PMID- 10728680 TI - Inhibition of constitutively activated Stat3 signaling pathway suppresses growth of prostate cancer cells. AB - Overexpression of interleukin 6, a downstream target of the GBX2 homeobox gene, has been linked to the progression of prostate cancer. The Janus kinase-signal transducers and activators of transcription signaling pathway transmits interleukin 6-mediated signals from cell surface receptors to the target genes in the nucleus and is critical in mediating cellular growth and differentiation. We demonstrate that cells derived from both rat and human prostate cancers have constitutively activated Stat3, with Stat3 activation being correlated with malignant potential. Blockade of activated Stat3 by ectopic expression of a dominant-negative Stat3 in human prostate cancer cells significantly suppresses their growth in vitro and their tumorigenicity in vivo. Furthermore, the Janus kinase inhibitor, tyrphostin AG490, inhibited the constitutive activation of Stat3 and suppressed the growth of human prostate cancer cells. These results indicate that activation of Stat3 signaling is essential in the progression of prostate cancer cells and suggest that targeting Stat3 signaling may yield a potential therapeutic intervention for prostate cancer. PMID- 10728681 TI - Tetrocarcin A inhibits mitochondrial functions of Bcl-2 and suppresses its anti apoptotic activity. AB - Bcl-2 is an integral, intracellular membrane protein that prevents cells from undergoing apoptosis in response to a variety of cell death signals. It negatively regulates the activation of Caspase-3, which functions as effector of mammalian cell death pathways. Overexpression of Bcl-2 inhibits the caspase activities and apoptosis. A microbial secondary metabolite, Tetrocarcin A (TC-A), was identified as an inhibitor of the anti-apoptotic function of Bcl-2. Apoptosis could be induced in cell lines that overexpressed Bcl-2 or Bcl-XL when the cells were treated with anti-Fas antibody, tumor necrosis factor alpha, staurosporine, or Bax, in addition to TC-A. TC-A showed selectivity against the pro-apoptotic Bcl-2 family members, in that cells overexpressing CrmA or dominant-negative FADD could not undergo apoptosis with TC-A treatment. In Bcl-2-overexpressing cell lines, TC-A inhibited mitochondrial functions regulated by Bcl-2, resulting in Fas-triggered mitochondrial transmembrane potential loss and cytochrome c release. Inhibition of the mitochondrial functions of Bcl-2 and, thereby, its anti-apoptotic effect could serve as useful pharmacological targets. Thus, TC-A should serve as an archetype for specific inhibitors of Bcl-2 functions. PMID- 10728682 TI - The tumor growth-inhibiting cell adhesion molecule CEACAM1 (C-CAM) is differently expressed in proliferating and quiescent epithelial cells and regulates cell proliferation. AB - The homophilic cell adhesion molecule CEACAM1 (C-CAM, BGP, CD66a) occurs as two coexpressed isoforms, CEACAM1-L and CEACAM1-S, in epithelia, endothelia, and leukocytes. CEACAM1-L can inhibit tumor growth; this effect is influenced by CEACAM1-S. To characterize the growth regulatory properties of CEACAM1, we analyzed the expression patterns of the isoforms, and here we demonstrate that both the expression levels and the S:L isoform ratios differ in proliferating and quiescent rat epithelial cells. Quiescent prostate NbE cells expressed more CEACAM1 than quiescent bladder NBT-II cells, a pattern that correlated with the expression levels in the parental tissues. In contrast, both the expression levels and the isoform ratios were strikingly similar in proliferating NbE and NBT-II cells, showing that a particular CEACAM1 expression pattern is compatible with cell proliferation. However, in confluent cells, CEACAM1 seemed to exert inhibitory effects on cell proliferation. Addition of anti-CEACAM1 antibodies to quiescent, confluent cells caused decreased expression of the cyclin-dependent kinase inhibitor, p27Klp1, stimulated growth factor-dependent DNA synthesis, and altered the S:L isoform ratio toward the ratio characteristic of proliferating cells. Taken together, our data suggest that CEACAM1 contributes to contact inhibition of cell proliferation in confluent cells but allows proliferation when expressed at different isoform ratios. PMID- 10728683 TI - DNA-dependent protein kinase stimulates an independently active, nonhomologous, end-joining apparatus. AB - Double-strand breaks (DSBs) can be efficiently removed from the DNA of higher eukaryotes by nonhomologous end-joining (NHEJ). Genetic studies implicate the DNA dependent protein kinase (DNA-PK) in NHEJ, but the exact function of this protein complex in the rejoining reaction remains to be elucidated. We compared rejoining of DNA DSBs in a human glioma cell line, M059-J, lacking the catalytic subunit of DNA-PK (DNA-PKcs), and their isogenic but DNA-PK-proficient counterpart, M059-K. In both cell lines, rejoining of DNA DSBs was biphasic, with a fast and a slow component operating with a half-life of approximately 22 min and 12 h, respectively. Deficiency in DNA-PK activity did not alter the half-times of either of these components of rejoining but increased from 17 to 72% the proportion of DNA DSB rejoining with slow kinetics. DNA DSB rejoining was nearly complete in both cell lines, and there was only a small increase in the number of unrejoined breaks in M059-J as compared with M059-K cells after 30 h of incubation. Wortmannin radiosensitized to killing M059-K cells and strongly inhibited DNA DSB rejoining. Wortmannin did not affect the radiosensitivity to killing and produced only a modest inhibition in DNA DSB rejoining in M059-J cells, suggesting that, for these end points, DNA-PK is the principal target of the drug. These observations demonstrate that DNA-PK deficiency profoundly decreases the proportion of DNA DSB rejoining with fast kinetics but has only a small effect on the fraction remaining unrejoined. We propose that in higher eukaryotes, an evolutionarily conserved, independently active, but inherently slow NHEJ pathway is stimulated 30-fold by DNA-PKcs to rapidly remove DNA DSBs from the genome. The stimulation is expected to be of local nature and the presence of DNA-PKcs in the vicinity of the DNA DSB determines whether rejoining will follow fast or slow kinetics. Structural and regulatory functions of DNA PKcs may mediate this impressive acceleration of DNA DSB rejoining, and regions of chromatin within a certain range from this large protein may benefit from these activities. We propose the term DNA-PK surveillance domains to describe these regions. PMID- 10728684 TI - Irradiated mammary gland stroma promotes the expression of tumorigenic potential by unirradiated epithelial cells. AB - We have shown that ionizing radiation, a known carcinogen of human breast, elicits rapid, persistent, and global changes in the mammary microenvironment as evidenced by altered extracellular matrix composition and growth factor activities. To address whether these events contribute to radiogenic carcinogenesis, we evaluated the effect of irradiated mammary stroma on the neoplastic potential of COMMA-D mammary epithelial cells. Although COMMA-D cells harbor mutations in both alleles of p53, they are nontumorigenic when injected s.c. into syngeneic hosts. Unirradiated COMMA-D cells transplanted to mammary fat pads cleared previously of epithelia preferentially formed tumors in irradiated hosts. Tumor incidence at 6 weeks was 81% +/- 12 SE when animals were irradiated with 4 Gy, 3 days prior to transplantation, compared with 19% +/- 2 SE (P < 0.005) in sham-irradiated hosts. This effect was evident when cells were transplanted 1 to 14 days after irradiation. Furthermore, tumors were significantly larger (243.1 +/- 61.3 mm3 versus 30.8 +/- 8.7 mm3) and arose more quickly (100% by 6 weeks versus 39% over 10 weeks in sham hosts) in fat pads in irradiated hosts. The contribution of local versus systemic effects was evaluated using hemibody (left versus right) irradiation; tumors formed only in fat pads on the irradiated side. These data indicate that radiation-induced changes in the stromal microenvironment can contribute to neoplastic progression in vivo. Disruption of solid tissue interactions is a heretofore unrecognized activity of ionizing radiation as a carcinogen. PMID- 10728685 TI - Overexpression of human aspartyl (asparaginyl) beta-hydroxylase is associated with malignant transformation. AB - The human aspartyl (asparaginyl) beta-hydroxylase (HAAH) is a highly conserved enzyme that hydroxylates epidermal growth factor-like domains in transformation associated proteins. We previously reported overexpression of the HAAH gene in human hepatocellular carcinomas and cholangiocarcinomas (L. Lavaissiere et al., J. Clin. Investig., 98: 1313-1323, 1996). In the present study, we determined whether HAAH protein overexpression was linked to cellular proliferation or malignant transformation of bile ducts by using a human disease and rat model of bile duct proliferation. In addition, the transforming properties of the AAH genes were assessed by transient and stable transfection of NIH-3T3 cells with human and murine wild-type as well as mutant cDNA constructs that lacked hydroxylation activity. Cellular characteristics of the malignant phenotype were assessed by formation of transformed foci, growth in soft agar, and tumor development in nude mice. We found that HAAH gene expression was undetectable during bile duct proliferation in both human disease and rat models as compared with cholangiocarcinoma. Overexpression of HAAH in NIH-3T3 cells was associated with generation of a malignant phenotype, and enzymatic activity was required for cellular transformation. These findings suggest that overexpression of HAAH is linked to cellular transformation of biliary epithelial cells. PMID- 10728686 TI - Sensitivity of the cervical transformation zone to estrogen-induced squamous carcinogenesis. AB - Regions where one type of epithelium replaces another (metaplasia) have a predilection for cancer formation. Environmental factors are closely linked to metaplastic carcinogenesis. In particular, cervical cancers associated with human papillomavirus (HPV) infection develop primarily at the transformation zone, a region where metaplastic squamous cells are detected in otherwise columnar epithelial-lined endocervical glands. Previously, we reported estrogen-induced multistage vaginal and cervical carcinogenesis in transgenic mice expressing HPV16 oncogenes in basal squamous epithelial cells. In the present study to investigate the threshold neoplastic response to exogenous estrogen, we treated groups of transgenic mice with lower hormone doses. A 5-fold reduction in estrogen dose induced squamous carcinogenesis solely at the cervical transformation zone compared with other reproductive tract sites. Further study delineated stages of transformation zone carcinogenesis, including formation of hyperplastic lower uterine glands and emergence of multiple foci of squamous metaplasia from individual stem-like glandular reserve cells, followed by neoplastic progression of metaplasia to dysplasia and squamous cancer. We propose that a combination of low-dose estrogen and low-level HPV oncogene expression biases transformation zone glandular reserve cells toward squamous rather than columnar epithelial fate decisions. Synergistic activation of proliferation by viral oncoprotein cell cycle dysregulation and estrogen receptor signaling, together with altered paracrine stromal-epithelial interactions, may conspire to support and promote neoplastic progression and cancer formation. PMID- 10728687 TI - Detection of DNA alkylphosphotriesters by 32P postlabeling: evidence for the nonrandom manifestation of phosphotriester lesions in vivo. AB - Many genotoxic carcinogens react with the sugar-phosphate backbone in DNA to form phosphotriester (PTE) adducts. These lesions are relatively abundant and persistent for some alkylating carcinogens and may therefore serve as useful biomarkers with which to assess genotoxic exposure and potential mutagenic risk. In the present study, we have developed a 32p postlabeling method that permits analysis of total methyl and/or ethyl PTE in DNA at the femtomole level. The technique is based on the inability of all known nucleolytic enzymes to cleave the internucleotide PTE bond. Consequently, complete digestion of alkylated DNA with these nucleases in the presence of an alkaline phosphatase yields PTE dinucleoside phosphates. These species are then converted to the corresponding dinucleoside phosphates (dNpdNs) by treatment with alkali to permit subsequent 32p labeling. The resulting labeled dinucleotides (32pd-NpdN) are then analyzed by PAGE. Validation of this method has been carried out using a polydeoxythymidylic acid oligonucleotide containing a site-specific methyl PTE. The method has been applied to the in vitro analysis of calf thymus (CT) DNA treated with dimethylsulfate (DMS) or diethylsulfate (DES) and to the analysis of liver DNA from mice treated in vivo with nitrosodiethylamine. In each case, autoradiograms of the polyacrylamide gels showed the anticipated five bands representing the sixteen labeled dinucleotides, with proportional increases observed as the concentrations of DMS or DES used in the in vitro treatment of CT DNA were increased. The identity and frequency of the nucleosides located 5' to the PTE lesions were obtained by nuclease P1 digestion of the gel-isolated 32pdNpdN species and by analysis of the released labeled mononucleotides, 32pdN, by high-performance liquid chromatography with radioactivity detection. Results obtained from CT DNA treated with DMS or DES showed that the frequency of the four detected nucleotides reflected the normal nucleoside content of CT DNA, indicating the random formation of methyl and ethyl PTE adducts in the in vitro modified DNA. However, studies using liver DNA from three strains of mice treated in vivo with nitrosodiethylamine indicated that the frequency of the thymidine and the 2'-deoxyguanosine 5' to the ethyl PTE was significantly different from the corresponding normal nucleoside content. These results are indicative of (a) the nonrandom formation of ethyl PTE in vivo and/or (b) base sequence-specific ethyl PTE repair. PMID- 10728688 TI - The food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine activates S-phase checkpoint and apoptosis, and induces gene mutation in human lymphoblastoid TK6 cells. AB - The mutagenic heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-blpyridine (PhIP) is formed at parts per billion levels when meat is cooked. It is efficiently absorbed from cooked food and extensively activated to its genotoxic N-hydroxy derivative by human cytochrome P4501A enzymes. It is also a rodent carcinogen. To better understand the genetic toxicity of PhIP, we have examined its effect on the cell cycle and gene mutation frequency using human lymphoblastoid cells (TK6) as a model. Because TK6 cells are unable to activate PhIP, we have cultured the cells in the presence of irradiated Chinese hamster XEMh1A2-MZ cells that have been genetically engineered to express human CYP1A2. Asynchronized TK6 cells were harvested at various times after treatment with PhIP (1.25-10 microg/ml), fixed and stained with propidium iodide for the examination of cell cycle by fluorescence-activated flow cytometry. After 20 h of PhIP treatment, a slight S-phase delay of the cell cycle was observed. Normal cell cycle recovered after the cells were washed and further cultured in the absence of PhIP for 5 days. However, PhIP treatment for 40 h induced a more pronounced S phase arrest that was accompanied by a decrease in the level of cyclin A, an S phase cyclin. This was followed by the appearance of a sub-G1 population (indicative of apoptotic cell death), range from 13 to 54% with PhIP concentrations from 1.25 to 10 microg/ml, compared with 5% in the vehicle control. A concomitant increase of mutation frequency at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus, assessed by colony formation assay in the presence of 6-thioguanine, was detected after 40 h-range, 16 to 45 x 10(-6) compared with 12 x 10(-6) in cultures without PhIP. In G1-enriched cell populations (synchronized culture), although PhIP induced S-phase delay, the induction of sub-G1 cells was substantially decreased. Our studies show that in TK6 cells, PhIP activates S-phase checkpoint, yet eludes G1 and G2-M checkpoints, and is accompanied by increased apoptosis and gene mutation. If treatment with PhIP induces similar cellular reactions in vivo, then activation of S-phase checkpoint with avoidance of G1 and G2-M checkpoints could be important factors in PhIP-induced genetic damage and neoplastic disease. PMID- 10728689 TI - Factors underlying the cell growth-related bystander responses to alpha particles. AB - Increases in cell proliferation are widely viewed as being of importance in carcinogenesis. We report that exposure of normal human lung fibroblasts to a low dose of alpha particles like those emitted by radon/radon progeny stimulates their proliferation in vitro, and this response also occurs when unirradiated cells are treated with supernatants from alpha-irradiated cells. We attribute the promitogenic response to superoxide dismutase- and catalase-inhibitable a particle-induced increases in the concentrations of transforming growth factor beta1 (TGF-beta1) in cell supernatants. TGF-beta1 at concentrations commensurate with those in the supernatants capably induces increases in intracellular reactive oxygen species (ROS) in unirradiated cells. Furthermore, the addition of supernatants from alpha-irradiated cells to unirradiated cells decreases cellular levels of TP53 and CDKN1A and increases CDC2 and proliferating cell nuclear antigen in the latter. Like the increased intracellular ROS bystander effect, this "decreased TP53/CDKN1A response" can be mimicked in otherwise untreated cells by the addition of low concentrations of TGF-beta1. Our results indicate that alpha particle-associated increases in cell growth correlate with intracellular increases in ROS along with decreases in TP53 and CDKN1A, and that these cellular responses are mechanistically coupled. As well, the proliferating cell nuclear antigen and CDC2 increases that occur along with the decreased TP53/CDKN1A bystander effect also would expectedly favor enhanced cell growth. Such processes may account for cell hyperplastic responses in the conducting airways of the lower respiratory track that occur after inhalation exposure to radon/ radon progeny, as well as, perhaps, other ROS-associated environmental stresses. PMID- 10728690 TI - Increased urinary excretion of 2-hydroxyestrone but not 16alpha-hydroxyestrone in premenopausal women during a soya diet containing isoflavones. AB - Asian diets high in soy are associated with lower risk for breast cancer compared with Western diets. Moreover, higher levels of two putative carcinogenic metabolites of 17beta-estradiol, 4- and 16alpha-hydroxyestrogen, and lower amounts of anticarcinogenic metabolites, 2-hydroxyestrogens, have been associated with greater breast cancer risk. In this study, we tested the hypothesis that consumption of a soya diet containing the weakly estrogenic isoflavones genistein and daidzein may alter the metabolism of 17beta-estradiol to 2- and 16alpha hydroxylated products. Eight pre-menopausal women were placed on a soya containing, constant diet in a metabolic unit. The diet provided 400 kilocalories from soymilk and 113-202 mg/day (158 +/- 26 mg/day, mean +/- SD) isoflavones daily for a complete menstrual cycle. After a washout period of 4 months, the subjects consumed the same diet, but with soymilk that contained <4.5 mg/day isoflavones ("isoflavone-free"). Urine samples were collected for 24 h daily for the entire cycle during each soya diet period for the analysis of daidzein, genistein, and 2- and 16alpha-hydroxyestrone. Subjects excreted measurable amounts of daidzein (11.6-39.2 mg/day) and genistein (2.9-18.2 mg/day) during the isoflavone-rich soya diet but not during the isoflavone-free soya diet. The diet rich in isoflavones increased the cycle mean daily urinary excretion of 2 hydroxyestrone (averaged over the entire cycle) from 11.6 +/- 2.06 to 17.0 +/- 2.96 nmol/12-h (P = 0.03), a 47% increase. However, the mean daily excretion of 16alpha-hydroxyestrone did not change (7.0 +/- 1.14 nmol/12-h during the isoflavone-free and 7.7 +/- 1.25 nmol/12-h during the isoflavone-rich diet; P = 0.36). The ratio of 2-hydroxyestrone to 16alpha-hydroxyestrone was higher during the isoflavone-rich soya diet (2.6 +/- 0.34) than during the isoflavone-free diet (2.0 +/- 0.32; P = 0.01), a 27% increase. These results suggest that soya isoflavones increase the metabolism of endogenous estrogens to the protective 2 hydroxylated estrogens in women, and this may play an important role in lowering 17beta-estradiol levels and the long-term risk for breast cancer. PMID- 10728691 TI - Antiangiogenic and antitumor activities of cyclooxygenase-2 inhibitors. AB - We provide evidence that cyclooxygenase (COX)-2-derived prostaglandins contribute to tumor growth by inducing newly formed blood vessels (neoangiogenesis) that sustain tumor cell viability and growth. COX-2 is expressed within human tumor neovasculature as well as in neoplastic cells present in human colon, breast, prostate, and lung cancer biopsy tissue. COX-1 is broadly distributed in normal, as well as in neoplastic, tissues. The contribution of COX-2 to human tumor growth was indicated by the ability of celecoxib, an agent that inhibits the COX 2 enzyme, to suppress growth of lung and colon tumors implanted into recipient mice. Mechanistically, celecoxib demonstrated a potent antiangiogenic activity. In a rat model of angiogenesis, we observe that corneal blood vessel formation is suppressed by celecoxib, but not by a COX-1 inhibitor. These and other data indicate that COX-2 and COX-2-derived prostaglandins may play a major role in development of cancer through numerous biochemical mechanisms, including stimulation of tumor cell growth and neovascularization. The ability of celecoxib to block angiogenesis and suppress tumor growth suggests a novel application of this anti-inflammatory drug in the treatment of human cancer. PMID- 10728692 TI - Copper-transporting P-type adenosine triphosphatase (ATP7B) is associated with cisplatin resistance. AB - The accumulation of cisplatin is decreased in many cisplatin-resistant cell lines, and an active efflux pump for cisplatin exists in some of them, but it has not yet been identified. In this study, we transfected the copper-transporting P type ATPase cDNA (ATP7B) into human epidermoid carcinoma KB-3-1 cells. The transfectant, KB/WD cell line, which overexpressed the P-type ATPase, ATP7B, was resistant to both cisplatin (8.9-fold) and copper (2.0-fold). The accumulation of cisplatin in KB/WD cells was lower than in mock-transfected KB/CV cells, and the efflux of cisplatin from KB/WD cells was enhanced compared with KB/CV cells. KB/WD cells were sensitive to other heavy metals, such as antimony, arsenate, arsenite, cadmium, and cobalt. ATP7B was overexpressed in cisplatin-resistant prostate carcinoma PC-5 cells but not in the parental PC-3 cells and the revertant PC-5R cells. ATP7B may be involved in cisplatin resistance in some tumors. PMID- 10728693 TI - Cdc25 inhibition and cell cycle arrest by a synthetic thioalkyl vitamin K analogue. AB - A synthetic vitamin K analogue, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or compound 5 (Cpd 5), was found previously to be a potent inhibitor of tumor cell growth. We now demonstrate that Cpd 5 arrested cell cycle progression at both G1 and G2-M. Because of the potential arylating activity of Cpd 5, it might inhibit Cdc25 phosphatases, which contain a cysteine in the catalytic site. To test this hypothesis, we examined the inhibitory activity of Cpd 5 against several cell cycle-relevant protein tyrosine phosphatases and found that Cpd 5 was a potent, selective, and partially competitive inhibitor of Cdc25 phosphatases. Furthermore, Cpd 5 caused time-dependent, irreversible enzyme inhibition, consistent with arylation of the catalytic cysteine in Cdc25. Treatment of cells with Cpd 5 blocked dephosphorylation of the Cdc25C substrate, Cdc2, and its kinase activity. Cpd 5 enhanced tyrosine phosphorylation of both potent regulators of G1 transition, ie., Cdk2 and Cdk4, and decreased the phosphorylation of Rb, an endogenous substrate for Cdk4 kinase. Furthermore, close chemical analogues that lacked in vitro Cdc25 inhibitory activity failed to block cell cycle progression and Cdc2 kinase activity. Cpd 5 did not alter the levels of p53 or the endogenous cyclin-dependent kinase inhibitors, p21 and p16. Our results support the hypothesis that the disruption in cell cycle transition caused by Cpd 5 was attributable to intracellular Cdc25 inhibition. This novel thioalkyl K vitamin analogue could be useful for cell cycle control studies and may provide a valuable pharmacophore for the design of future therapeutics. PMID- 10728694 TI - Preferential enhancement of tumor radioresponse by a cyclooxygenase-2 inhibitor. AB - Cyclooxygenase-2 (COX-2), an inducible isoform of cyclooxygenase, is overexpressed in many types of malignant tumors, where it mediates production of prostaglandins (PGs), which in turn may stimulate tumor growth and protect against damage by cytotoxic agents. This study investigated whether SC-'236, a selective inhibitor of COX-2, potentiates antitumor efficacy of radiation without increasing radiation injury to normal tissue. Mice bearing the sarcoma FSA in the hind legs were treated daily for 10 days with SC-'236 (6 mg/kg given in the drinking water) when tumors were 6 mm in diameter. When tumors reached 8 mm in diameter, the mice were given 11- to 50-Gy single-dose local tumor irradiation with or without SC-'236. SC-'236 inhibited tumor growth on its own, and it greatly enhanced the effect of tumor irradiation. The growth delay was increased from 14.8 days after 25-Gy single dose to 28.4 days after the combined treatment (P = 0.01). SC-'236 reduced TCD50 (radiation dose yielding 50% tumor cure) from 39.2 Gy to 20.9 Gy (enhancement factor = 1.87). SC-'236 did not appreciably alter radiation damage to jejunal crypt cells and tissue involved in the development of radiation-induced leg contractures. The SC-'236-induced enhancement of tumor radioresponse was associated with a decrease in PGE2 levels in FSA tumors. The drug had no effect on radiation-induced apoptosis. Neoangiogenesis was inhibited by SC-'236, which could account for some of the increase in tumor radioresponse. Overall, our findings demonstrated that treatment with a selective inhibitor of COX-2 greatly enhanced tumor radioresponse without markedly affecting normal tissue radioresponse. Thus, COX-2 inhibitors have a high potential for increasing the therapeutic ratio of radiotherapy. PMID- 10728695 TI - A role for Id-1 in the aggressive phenotype and steroid hormone response of human breast cancer cells. AB - The helix-loop-helix protein Id-1 inhibits the activity of basic helix-loop-helix transcription factors, and is an important regulator of cell growth and tissue specific differentiation. We have shown (P. Y. Desprez et al., Mol. Cell. Biol., 18: 4577-4588, 1998) that ectopic expression of Id-1 inhibits differentiation and stimulates the proliferation and invasiveness of mouse mammary epithelial cells, and that there is a correlation between the levels of Id-1 protein and the aggressiveness of several human breast cancer cell lines. Here, we show that aggressive and metastatic breast cancer cells express high levels of Id-1 mRNA because of a loss of serum-dependent regulation that is mediated by a 2.2-kb region of the human Id-1 promoter. Three lines of evidence suggest that unregulated Id-1 expression may be an important regulator of the aggressive phenotype of a subset of human breast cancer cells: (a) a constitutively expressed Id-1 cDNA, when introduced into a nonaggressive breast cancer cell line (T47D), conferred a more aggressive phenotype, as measured by growth and invasiveness; (b) Id-1 was an important mediator of the effects of sex steroid hormones on T47D cell proliferation. Estrogen stimulated proliferation and induced Id-1 expression, whereas progesterone inhibited proliferation and repressed Id-1 expression. Progesterone repressed Id-1 expression, at least in part by repressing transcription. Most importantly, an antisense oligonucleotide that reduced Id-1 protein levels reduced the ability of estrogen to stimulate cell proliferation, whereas constitutive Id-1 expression rendered cells refractory to growth inhibition by progesterone; and (c) using a limited number of breast cancer biopsies, we showed that Id-1 was more frequently expressed in infiltrating carcinomas compared with ductal carcinomas in situ. Our results suggest that Id-1 can control the malignant progression of breast cancer cells, particularly that mediated by sex steroid hormones. Moreover, Id-1 has the potential to serve as a marker for aggressive breast tumors. PMID- 10728696 TI - A minimal critical region of the 8p22-23 amplicon in esophageal adenocarcinomas defined using sequence tagged site-amplification mapping and quantitative polymerase chain reaction includes the GATA-4 gene. AB - The incidence of esophageal adenocarcinomas has increased greatly over the past 20 years. The genetic alterations associated with this disease, however, remain largely unknown. We identified recently a novel amplicon at 8p22-23 in esophageal adenocarcinomas using the restriction landmark genomic scanning two-dimensional gel technique. Four known genes within or near this amplicon were initially characterized. The cathepsin B (CTSB) gene was found to be amplified in 13% of esophageal tumors. CTSB was shown previously to be overexpressed without amplification in many other human cancers. An approach termed sequence tagged site-amplification mapping has been implemented in the present study, allowing the 8p22-23 amplicon to be narrowed from 12 cM to a <2-cM minimal amplified area located between markers D8S552 and D8S1759. The CTSB gene maps within this region. To identify other cancer-related candidate genes in this region, a positional candidate gene approach was subsequently applied to characterize this minimal critical region. An expressed sequence tag (EST), which was included in the minimal critical region, demonstrated both amplification and overexpression. This EST and the extended sequence from the EST were determined to be a novel sequence in the 3' untranslated region of the human GATA-4 gene. GATA-4, a member of a zinc finger transcription factor family, was confirmed to be amplified and overexpressed in esophageal adenocarcinomas and was localized within <0.5 kb from CTSB. Furthermore, amplification of 8p22-23 was detected in one of eight gastric cardia adenocarcinomas but was not observed in either human lung adenocarcinomas (n = 39) or in esophageal squamous cell carcinomas (n = 24). The relatively high frequency of the 8p22-23 amplification in esophageal (13.6%) and gastric cardia (12.5%) adenocarcinomas may indicate a specificity of this amplicon for tumors of gastroesophageal origin. PMID- 10728697 TI - Prostate-specific transcription factor hPSE is translated only in normal prostate epithelial cells. AB - We recently cloned a novel transcription factor gene, hPSE, which belongs to the Ets gene family. hPSE mRNA was expressed specifically in prostate glandular epithelial cells and also in the human prostate carcinoma cell lines PC-3 and LNCaP. On the other hand, on immunoblot analysis with anti-hPSE antiserum, hPSE protein was detected only in human prostate tissue samples and not in PC-3 or LNCaP culture cells. Immunohistochemistry and in situ hybridization analysis revealed that hPSE protein was translated in normal prostate glandular epithelial cells, but not in carcinoma cells with hPSE transcripts. These findings suggest that expression of hPSE is regulated translationally in prostate epithelial cells and that hPSE protein is a candidate for a marker distinguishing normal cells from cancer cells in the prostate. It appeared that the 5'- and 3'-untranslated regions of hPSE transcripts might be necessary for translational control of hPSE, on the basis of results of transfection analysis in non-prostate lineage cells (HEK-293) using some deletion mutants of hPSE cDNA. PMID- 10728698 TI - Identification of a human glioma-associated growth factor gene, granulin, using differential immuno-absorption. AB - Identification of the genes that are differentially expressed in brain tumor cells but not in normal brain cells is important for understanding the molecular basis of these neurological cancers and for defining possible targets for therapeutic intervention. In an effort to discover potentially antigenic proteins that may be involved in the malignant transformation and progression of human glioblastomas, a novel antibody-based approach was developed to identify and isolate gene products that are expressed in brain tumors versus normal brain tissue. Using this method, whereby tumor-specific antibodies were isolated and used to screen a glioblastoma cDNA expression library, 28 gene products were identified. Nine of these clones had homology to known gene products, and 19 were novel. The expression of these genes in multiple different human gliomas was then evaluated by cDNA microarray hybridization. One of the isolated clones had consistently higher levels of expression (3-30-fold) in brain tumors compared with normal brain. Northern blot analysis and in situ hybridization confirmed this differential overexpression. cDNA sequence analysis revealed that this gene was identical to a relatively new class of growth regulators known as granulins, which have tertiary structures resembling the epidermal growth factor-like proteins. The 2.1-kb granulin mRNA was expressed predominantly in glial tumors, with lower levels in spleen, kidney, and testes, whereas expression was not detected in non-tumor brain tissues. Functional assays using [3H]thymidine incorporation indicated that granulin may be a glial mitogen, as addition of synthetic granulin peptide to primary rat astrocytes and three different early passage human glioblastoma cultures increased cell proliferation in vitro, whereas increasing concentrations of granulin antibody inhibited cell growth in a dose-dependent manner. The differential expression pattern, tissue distribution, and implication of this glioma-associated molecule in growth regulation suggest a potentially important role for granulin in the pathogenesis and/or malignant progression of primary brain neoplasms. PMID- 10728699 TI - BRCA1-related papillary serous carcinoma of the peritoneum has a unique molecular pathogenesis. AB - Papillary serous carcinoma of the peritoneum (PSCP) is believed to develop de novo from the peritoneal lining of the pelvis and abdomen. Although it is histologically indistinguishable from serous ovarian carcinoma, PSCP exhibits minimal or absent ovarian involvement and may even develop in a woman years after prophylactic oophorectomy. We have shown previously that patients with germ-line BRCA1 mutations who develop PSCP are more likely to have disease originating from multiple peritoneal sites compared with patients with wild-type BRCA1. In this study, we tested the hypothesis that BRCA1-related PSCP has a unique molecular pathogenesis. DNA was extracted from normal tissue and multiple tumor sites in patients with PSCP. BRCA1 and p53 gene mutations were screened for using single strand conformation polymorphism. Loss of heterozygosity was determined at the BRCA1 and p53 loci. Immunohistochemical analyses of p53, epidermal growth factor receptor, erbB-2, erbB-3, erbB-4, and Bcl-2 expression were performed. We detected germ-line BRCA1 mutations in 11 (26%) of 43 PSCP patients. BRCA1 mutation carriers had a higher overall incidence of p53 mutations (89% versus 47%; P = 0.052), were more likely to exhibit multifocal or null p53 mutations (63% versus 7%; P = 0.014), and were less likely to exhibit erbB-2 overexpression (P = 0.013) than wild-type BRCA1 case subjects. We propose that the unique molecular pathogenesis of BRCA1-related PSCP may affect the ability of current methods to reliably prevent or detect this disease prior to metastasis. PMID- 10728700 TI - High levels of tyrosine phosphorylated proto-ret in sporadic phenochromocytomas. AB - Pheochromocytomas are tumors originating from chromaffin cells, the large majority of which are sporadic neoplasms. The genetic and molecular events determining their tumorigenesis continue to remain unknown. On the other hand, RET germ-line mutations cause the inheritance of familial tumors in multiple endocrine neoplasia (MEN)-2 diseases, which account for a minority of pheochromocytomas. We investigated the expression of the RET gene in 14 sporadic tumors harboring no activating mutations. A subset of highly RET-expressing tumors (50%) could be distinguished. They showed RET transcript, protein amounts as well as Ret-associated phosphotyrosine levels similar to those measured in MEN 2A-associated pheochromocytomas. We also determined the GDNF and GDNF family receptor alpha (GFRalpha)-1 transcript levels in tumors and in normal tissues. Whereas the GFRalpha-1 transcripts were detected at similar levels in normal tissues and in tumors, GDNF was frequently found expressed in sporadic tumors at levels several times higher than in controls. These results led us to propose the existence of an autocrine or paracrine loop leading to chronic stimulation of the Ret signaling pathway, which could participate in the pathogenesis of a number of sporadic pheochromocytomas. PMID- 10728701 TI - BRCA1 and BRCA2 have a limited role in familial prostate cancer. AB - Epidemiological studies have suggested that the breast cancer susceptibility genes, BRCA1 and BRCA2, may be involved in the development of prostate cancer. Several studies have screened prostate cancer populations for the presence of BRCA1 and BRCA2 mutations, with few mutations identified. In this study, 22 high risk prostate cancer families (at least three cases of prostate cancer) were screened by conformation-sensitive gel electrophoresis (CSGE) for mutations in BRCA1 and BRCA2. To maximize the chance of finding mutations in these two genes, families were also selected for the presence of at least two cases of breast and/or ovarian cancer. We identified one previously reported BRCA2 missense mutation and two previously unreported BRCA2 intron polymorphisms. No BRCA1 or BRCA2 truncating mutations were detected. Thus, BRCA1 and BRCA2 appear to have a limited role in familial prostate cancer, and families with both prostate and breast cancer may result from mutations in other predisposition genes. PMID- 10728702 TI - Class A helix-loop-helix proteins are positive regulators of several cyclin dependent kinase inhibitors' promoter activity and negatively affect cell growth. AB - The class A of basic helix-loop-helix (bHLH) proteins are ubiquitously expressed transcription factors playing a pivotal role in the regulation of cell growth and differentiation. We determined that enforced expression of all four different mammalian members of this family, E12, E47, E2-2, and HEB, suppresses the cell colony-forming efficiency of several cell lines. To gain insights into the mechanisms by which class A bHLH factors affect cell growth, we have investigated their role in the transcriptional regulation of cyclin-dependent kinase inhibitors. We found that p21CIP1/ WAF1, p15INK4B, and p16INK4B promoter sequences contain E-boxes that render these genes competent for class A bHLH mediated transcriptional activation and Id-mediated repression. The mechanism underlying the class A bHLH-mediated inhibition of cell growth does not involve an arrest of G1 progression in 293T cells. In fact, contrary to what has been found in 3T3 NIH fibroblasts, we found that enhanced expression of class A bHLH proteins led to a decreased proliferation rate by promoting cell death associated with the induction of apoptosis. These findings highlight the role of the class A bHLH proteins as general negative regulators of cell proliferation through a mechanism(s) that involves both enhancement of several cyclin-dependent kinase inhibitor genes expression and promotion of cell death. PMID- 10728703 TI - Diversity and frequency of epidermal growth factor receptor mutations in human glioblastomas. AB - Several types of epidermal growth factor receptor (EGFR) gene mutations have been reported in glioblastomas, and in nearly all cases the alterations have been reported in tumors with EGFR amplification. The objectives of this study were to determine the frequency and diversity of EGFR mutations in glioblastomas and to determine whether gene mutation is inevitably associated with increased EGFR gene dosage. To accomplish these aims, we sequenced cDNA products representing the entire EGFR coding region in 44 glioblastomas, half of which had EGFR amplification. Coding sequence alterations were identified in 17 of the tumors, and each of these cases had amplified EGFR. No mutations were identified in the 22 tumors without EGFR amplification. An additional 26 glioblastomas with EGFR amplification were then examined to establish more reliable frequencies for each type of mutation identified in the tumors for which the entire gene was sequenced. Transcripts associated with the most common mutation lacked coding sequence for amino acids 6-273 (67%). This mutation has been described extensively in the literature. Transcripts encoding receptors that would truncate at amino acid 958 and transcripts encoding receptors that would lack amino acids 521-603 were the next most common types of alteration. Each of these were observed in 15% of the tumors with EGFR amplification. Other mutations were observed at lower frequencies, but among these were three cases with missense mutations. Sixteen of the 48 tumors with EGFR amplification showed multiple types of EGFR mutations (33%), and in one case it was determined that multiple alterations had occurred in the same transcript. In total, these data are consistent with EGFR mutation being exclusively and frequently associated with EGFR amplification. Furthermore, the determination of multiple EGFR mutations within individual tumors suggests that glioblastomas with EGFR amplification have the capacity to produce a variety of functionally distinct EGFRs. PMID- 10728704 TI - Heterogeneity of angiogenesis and blood vessel maturation in human tumors: implications for antiangiogenic tumor therapies. AB - Microvessel density (MVD) counting techniques have been widely used to assess the vasculature in tumors. MVD counts assess the presence of blood vessels but do not give an indication of the degree of angiogenesis and the functional status of the tumor neovasculature. To analyze angiogenesis and the functional status of the tumor vascular bed, we have quantitated endothelial cell proliferation and the recruitment of pericytes in human tumors [glioblastomas (n = 30), renal cell carcinomas (n = 22), colon carcinomas (n = 18), mammary carcinomas (n = 24), lung carcinomas (n = 15), and prostate carcinomas (n = 19)]. These findings were compared to the physiological angiogenesis in the cyclic bovine ovarian corpus luteum. Tissue sections were examined applying double-labeling immunohistochemical techniques to detect proliferating endothelial cells and to colocalize endothelial cells and pericytes. The following parameters were quantitated: (a) MVD count; (b) proliferating capillary index (PCI); (c) proliferating tumor versus endothelial cell index; and (d) microvessel pericyte coverage index (MPI). Based on endothelial cell proliferation, angiogenesis was found to be present in all tumors with characteristic and significant differences between the tumor types (glioblastomas, PCI = 9.6 +/- 6.1%; renal cell carcinomas, PCI = 9.4 +/- 5.2%; colon carcinomas, PCI = 7.8 +/- 5.2%; mammary carcinomas, PCI = 5.0 +/- 4.8%; lung carcinomas, PCI = 2.6 +/- 2.5%; prostate carcinomas, PCI = 2.0 +/- 1.4%). There was a considerable degree of heterogeneity in the intensity of angiogenesis within each tumor group, as indicated by large standard deviations. Even in the most angiogenic tumors, angiogenesis was found to be 4 to 20 times less intense as compared with the physiological angiogenesis in the growing ovarian corpus rubrum (PCI = 40.6 +/- 6.2%). Varying degrees of pericyte recruitment to the tumor microvasculature were determined in the different tumor types (glioblastomas, MPI = 12.7 +/- 7.9%; renal cell carcinomas, MPI = 17.9 +/- 7.8%; colon carcinomas, MPI = 65.4 +/- 10.5%; mammary carcinomas, MPI = 67.3 +/- 14.2%; lung carcinomas, MPI = 40.8 +/- 14.5%; prostate carcinomas, MPI = 29.6 +/- 9.5%). The data demonstrate distinct quantitative variations in the intensity of angiogenesis in malignant human tumors. Furthermore, the varying degrees of pericyte recruitment indicate differences in the functional status of the tumor vasculature in different tumors that may reflect varying degrees of maturation of the tumor vascular bed. PMID- 10728705 TI - Autocrine stimulatory mechanism by transforming growth factor beta in human hepatocellular carcinoma. AB - The serum concentration of transforming growth factor beta (TGF-beta) is elevated as tumors progress in hepatocellular carcinoma (HCC) patients. In this study, we examined whether modulation of tumor-derived TGF-beta signal transduction contributes to malignant progression. We investigated the production of TGF beta1, the biological effects of TGF-beta and neutralizing antibody on HCC cells, activation of Smad 2, Smad 3, and Smad 4, induction of antagonistic Smads (Smad 6 and Smad 7), and promoter activities of two target genes, plasminogen activator inhibitor type 1 (PAI-1) and p15INK4B. In human cell lines HCC-M and HCC-T, TGF beta accelerates their proliferation. Smad 2 was activated constitutively by an autocrine mechanism, because in the absence of exogenous TGF-beta, a high level of Smad 2 phosphorylation, induction of PAI-1 transcripts, and nuclear localization of Smad 2 were observed. This constitutive activation of Smad 2 was, at least in part, attributable to the lack of induction of antagonistic Smads by TGF-beta. However, Smads activated by tumor-derived TGF-beta constantly suppressed p151NK4B expression. In addition, 3 of 10 human HCC tissues showed nuclear localization of Smad 2 and low mRNA levels of p15INK4B and antagonistic Smads but a high level of PAI-1. Our observations suggest that this constant suppression of the p15INK4B gene could be involved in the malignant progression of HCC. PMID- 10728706 TI - Identification of the putative brain tumor antigen BF7/GE2 as the (de)toxifying enzyme microsomal epoxide hydrolase. AB - Malignant gliomas are the main cause of death from primary brain tumors. Despite surgery, radiation, and chemotherapy, patients have a median survival of less than a few years; therefore, it is clearly imperative to investigate new ways of treatment. The development of new therapeutic strategies for brain tumors is dependent on a better understanding of the differences between normal and tumoral brain cells. Our group had described previously a Mr 48,000 antigen defined by reactivity with two monoclonal antibodies (GE2 and BF7) obtained by immunization of mice with human glioblastoma cells. Here, we describe the identification of the GE2/BF7 antigen as microsomal epoxide hydrolase (mEH), a drug-metabolizing enzyme that is involved both in toxification and detoxification of carcinogens. We initially used immunoaffinity purification using GE2 and BF7 and analyzed the purified proteins by microsequencing. Edman degradation identified 15 amino acids of the NH2-terminal sequence that were 100% identical to mEH. To further confirm the identity of the BF7/GE2 antigen as mEH, we showed that the protein immunopurified with GE2 and BF7 was recognized by an anti-mEH antibody and that in vitro and in vivo synthesized human mEH is recognized by BF7 and GE2 antibodies. Furthermore, anti-mEH antibody recognizes an antigen expressed both in gliomas and reactive astrocytes, as do BF7 and GE2. Finally, we demonstrate that in contrast to what has been reported in rat embryo fibroblasts, p53 does not regulate mEH mRNA expression in glioma cells. PMID- 10728707 TI - Expression of sialyl 6-sulfo Lewis X is inversely correlated with conventional sialyl Lewis X expression in human colorectal cancer. AB - Sialyl 6-sulfo Lewis X determinant has been described recently as a major ligand for L-selectin on high endothelial venules of human peripheral lymph nodes. From our investigation of its distribution in human colorectal cancer tissues and cultured colon cancer cells, the sialyl 6-sulfo Lewis X determinant was preferentially expressed in the nonmalignant colonic epithelia rather than cancer cells (P < 0.001; n = 23). This was in contrast to the distribution of conventional sialyl Lewis X, which was preferentially expressed in cancer tissues rather than nonmalignant epithelia (P = 0.007; n = 23), indicating that 6 sulfation predominantly occurs in nonmalignant tissues and is suppressed upon malignant transformation. In confirmation of this, a nonsialylated determinant 6 sulfo Lewis X was also found to be preferentially localized in the nonmalignant epithelia. Significant expression of sialyl 6-sulfo Lewis X was observed in only 2 lines, whereas 8 were positive for conventional sialyl Lewis X, among 13 cultured colon cancer cell lines. Transfection of cells with fucosyltransferase (Fuc-T) VI induced expression of sialyl 6-sulfo Lewis X, whereas transfection of Fuc-T III did not, suggesting that the determinant was synthesized mainly by Fuc T VI in colonic epithelia. Members of the sialic acid cyclase pathway, the de-N acetyl sialyl 6-sulfo Lewis X and cyclic sialyl 6-sulfo Lewis X determinants, were also preferentially expressed in the nonmalignant epithelia rather than colonic cancer cells (P < 0.001; n = 23). Stimulation of the sialyl 6-sulfo Lewis X-positive colon cancer cell line with a calcium ionophore ionomycin markedly reduced sialyl 6-sulfo Lewis X and induced cyclic sialyl 6-sulfo Lewis X expression. These results suggested that the metabolic conversion of sialyl 6 sulfo Lewis X into cyclic sialyl 6-sulfo Lewis X by a calcium-dependent enzyme, sialic acid cyclase, as we hypothesized for human leukocytes previously (C. Mitsuoka et al., Proc. Natl. Acad. Sci. USA, 96: 1597-1602, 1999), also occurs in nonmalignant colonic epithelia. PMID- 10728708 TI - A mechanistic, predictive model of dose-response curves for cell cycle phase specific and -nonspecific drugs. AB - In vitro dose-response curves for anticancer agents are useful for predicting the clinical response to chemotherapy, and models to capture the time-dependency of dose-response curves are necessary for potential clinical extrapolation. Usually, the modified Hill model is used (see Levasseur et al., Cancer Res., 58: 5749 5761, 1998), although this model is neither mechanistic nor predictive for understanding how drug and tumor cell characteristics affect the shape of the dose-response curve. A new exponential kill (EK) model is proposed to predict the shape of dose-response curves based on the cell cycle phase specificity of a drug, the cell cycle time, the duration and concentration of drug exposure at the site of action, and a scaling factor for the level of drug resistance. Explicit analytical equations are presented for predicting the ICx (the concentration required to reduce cell growth by x%), the maximum cell kill achievable at high doses after a given duration of drug exposure, and the slope of the survival fraction versus log (concentration) plot at the ICx. Numerical solutions illustrate that there may be an optimal, finite duration of drug exposure that maximizes cell kill for a given area under the concentration versus time curve, and an analytical equation is given to calculate when such an optimal, finite duration exists. The EK model generates sigmoidal dose-response curves, like those seen empirically and previously described by the Hill model, which eventually plateau with increasing drug concentration at levels that depend on the cell cycle specificity of the drug, the cell cycle time, and the duration of exposure to the drug. This study includes no original data. Instead, empirical results in the literature are used to test the model. Because data by Levasseur et al. (1998) was fit to the Hill model assuming the plateau in the effect versus concentration curve to be independent of exposure duration, a full test of the model is not possible using their published data. Some tests of the EK model were possible, however, showing that EK model predictions yield good fits to in vitro data published in that and in another study. In addition, combining the EK model with a pharmacokinetic model resulted in predictions that were consistent with results of clinical studies comparing etoposide given in different schedules. Further tests of the model are necessary. PMID- 10728709 TI - Sulforaphane, a naturally occurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer cells. AB - Sulforaphane is an isothiocyanate that is present naturally in widely consumed vegetables and has a particularly high concentration in broccoli. This compound has been shown to block the formation of tumors initiated by chemicals in the rat. Although sulforaphane has been proposed to modulate the metabolism of carcinogens, its mechanism of action remains poorly understood. We have previously demonstrated that sulforaphane inhibits the reinitiation of growth and decreases the cellular viability of quiescent human colon carcinoma cells (HT29). Moreover, the weak effect observed on differentiated CaCo2 cells suggests a specific anticancer activity for this compound. Here we investigated the effect of sulforaphane on the growth and viability of HT29 cells during their exponentially growing phase. We observed that sulforaphane induced a cell cycle arrest in a dose-dependent manner, followed by cell death. This sulforaphane induced cell cycle arrest was correlated with an increased expression of cyclins A and B1. Moreover, we clearly demonstrated that sulforaphane induced cell death via an apoptotic process. Indeed, a large proportion of treated cells display the following: (a) translocation of phosphatidylserine from the inner layer to the outer layer of the plasma membrane; (b) typical chromatin condensation; and (c) ultrastructural modifications related to apoptotic cell death. We also showed that the expression of p53 was not changed in sulforaphane-treated cells. In contrast, whereas bcl-2 was not detected, we observed increased expression of the proapoptotic protein bax, the release of cytochrome c from the mitochondria to the cytosol, and the proteolytic cleavage of poly(ADP-ribose) polymerase. In conclusion, our results strongly suggest that in addition to the activation of detoxifying enzymes, induction of apoptosis is also involved in the sulforaphane associated chemoprevention of cancer. PMID- 10728710 TI - Estrogen receptors in human myeloma cells. AB - It has recently been reported that the human myeloma cell line U266 proceeds to undergo apoptosis after cultivation with the antiestrogen tamoxifen, thus raising the possibility that antiestrogens may be candidates for use in myeloma therapy. To obtain basic information on the effects of antiestrogens on myeloma cells, we investigated the mRNA expression levels of estrogen receptor (ER)-alpha, ER-beta, and coactivators and corepressors in nine human myeloma cell lines and compared them with those of seven human breast cancer cell lines including four ER positive and three ER-negative lines. The alterations in cell growth and mRNA expression of the target genes of ER or those of cytokines in the myeloma lines by estradiol or antiestrogens (tamoxifen and toremifene) were also investigated. In addition, effects on membrane Fas expression, appearance of apoptosis, and cell cycle perturbation were analyzed. It was revealed that ER-beta and corepressors were dominantly expressed in myeloma cells, and antiestrogens induced growth inhibition through apoptosis mediated by a Fas-related pathway and G1 arrest of the cell cycle in myeloma cell lines. PMID- 10728711 TI - Local imbalance of proangiogenic and antiangiogenic factors: a potential mechanism of focal necrosis and dormancy in tumors. AB - Solid tumors produce both stimulators and inhibitors of angiogenesis. The suppression of metastases by some primary tumors has been attributed to the longer circulatory half-lives of the inhibitors. We propose that intrinsic differences in the physicochemical properties of these regulators may also explain focal suppression of angiogenesis within the primary tumor. We present a mathematical framework that describes production, diffusion, and degradation of these factors in tumor and host tissue and their effect on angiogenesis at local and distal sites. Results show focal suppression of angiogenesis, provide an explanation for tumor dormancy and focal necrosis, and predict a suppressive influence of primary tumors on angiogenesis at metastatic sites. They suggest generally that diffusible factors produced by tumors can stimulate responses in adjacent host tissue, preparing it for further tumor invasion. This study presents a new paradigm for the development of tumor necrosis and offers new insight into angiogenesis regulation and therapy. The framework established for modeling the competing effects of diffusible stimulators and inhibitors can be applied more generally to growth factors/inhibitors and other opposing factors produced in the tumor environment. PMID- 10728712 TI - Restoration of alpha(1,2) fucosyltransferase activity decreases adhesive and metastatic properties of human pancreatic cancer cells. AB - The expression of alpha(1,2) fucosyltransferases that catalyze the fucose transfer to galactose of the N-acetyl(iso)lactosamine chain is decreased in human metastatic pancreatic cancer cells. alpha(2,3) Sialyltransferases catalyze the transfer of sialic acid to the same substrate to form, with alpha(1,3/1,4) fucosyltransferases, sialyl-Lewis a and sialyl-Lewis x determinants on cell surface that are involved in pancreatic metastatic invasion. The aim of this study was to determine whether this decrease of alpha(1,2) fucosyltransferase expression can favor the alpha(2,3) sialyltransferase activity to form metastatic sialyl-Lewis antigens. Restoration of alpha(1,2) fucosyltransferase activity in the human pancreatic cancer cell line BxPC-3 was obtained by selecting stable transfectants expressing FUT1. Overexpression of FUTI in BxPC-3 cells resulted in a substantial reduction of sialyl-Lewis antigen expression that correlated with an increase of expression of Lewis y and H-type antigens on cell surface. The modified oligosaccharide structures were preferentially restricted to three major glycoproteins, which could in part be related to mucin-type glycoproteins. The reduction of sialyl-Lewis antigen expression was associated with an inhibition of adhesive properties to E-selectin and a decrease of gastrointestinal metastatic power of BxPC-3 cells after xenograft transplantation into nude mice. This study provides evidence that the expression level of alpha(1,2) fucosyltransferase may regulate the expression of sialyl-Lewis a and sialyl-Lewis x antigens and consequently could play an important role in metastatic properties of human pancreatic cancer cells. PMID- 10728713 TI - Overexpression of PTEN/MMAC1 and decreased activation of Akt in human papillomavirus-infected laryngeal papillomas. AB - Laryngeal papillomas are benign, human papillomavirus-induced hyperplastic tumors of the respiratory tract. They are characterized by overexpression of the epidermal growth factor receptor, constitutive activation of mitogen-activated protein kinase, a low proliferative rate, and defects in differentiation. We have now found that phosphoinositol 3-kinase (PI 3-K) activity is significantly increased in papilloma tissue. However, phosphorylated Akt (also known as protein kinase B), a downstream effector of PI 3-K, is reduced when compared with normal tissue. The ratio of activated Akt to total Akt is much lower in papillomas than in normal laryngeal tissue, suggesting decreased Akt activation. PTEN/ MMAC1 is a tumor suppressor that dephosphorylates phosphatidylinositol 3,4,5-triphosphate, an intermediate in the PI 3-K/Akt signaling pathway. We have found that PTEN protein is overexpressed in laryngeal papillomas when compared with normal laryngeal tissues. On the basis of reverse transcription-PCR analysis, PTEN mRNA is more abundant in papillomas, suggesting transcriptional up-regulation. We postulate that negative regulation of the PI 3-K/Akt pathway by PTEN may modulate the effects of the hyperactive epidermal growth factor receptor/mitogen-activated protein kinase pathway, contributing to the low proliferation and dysfunctional differentiation of laryngeal papillomas. PMID- 10728714 TI - Correspondence re: Q-R. Chen et al., Liposomes complexed to plasmids encoding angiostatin and endostatin inhibit breast cancer in nude mice. Cancer Res., 59: 3308-3312, 1999. PMID- 10728715 TI - Summary of the International Cancer Microenvironment from meeting in Pittsburgh, Pennsylvania on October 3-6, 1999. PMID- 10728716 TI - Effects of age, medication, and illness duration on the N-acetyl aspartate signal of the anterior cingulate region in schizophrenia. AB - The authors performed a MRSI study of the anterior cingulate gyrus in 19 schizophrenic patients under stable medication and 16 controls in order to corroborate previous findings of reduced NAA in the anterior cingulate region in schizophrenia. Furthermore, correlations between NAA in the anterior cingulate gyrus and age or illness duration have been determined. A decreased NAA signal was found in the anterior cingulate gyrus of patients compared to controls. Subdividing the patient group into two groups depending on medication revealed that the group of patients receiving a typical neuroleptic medication showed a lower mean NAA in comparison to the group of patients receiving atypical antipsychotic drugs. No significant group differences in the creatine and phosphocreatine signal or the signal from choline-containing compounds were found. The NAA signal significantly correlated with age, and therefore, individual NAA values were corrected for the age effect found in the control group. The age-corrected NAA signal in schizophrenia correlated significantly with the duration of illness. The detected correlations of NAA decrease with age and illness duration are consistent with recent imaging studies where progressing cortical atrophy in schizophrenia was found. Further studies will be needed to corroborate a possible favorable effect of atypical antipsychotics on the NAA signal. PMID- 10728717 TI - A magnetic resonance imaging study of corpus callosum size in familial schizophrenic subjects, their relatives, and normal controls. AB - The corpus callosum is one of several brain regions thought to be abnormal in schizophrenia. We sought to investigate whether the size of the corpus callosum would be abnormally small in schizophrenic subjects from families with familial schizophrenia and their healthy relatives. We wished to determine whether an abnormal corpus callosum size is found in healthy relatives who are genetically at a greater risk than normal of developing or transmitting the disorder. Twenty seven familial schizophrenics, 53 of their healthy first-degree relatives, and 35 normal volunteers underwent MRI brain scans. We defined 11 of the relatives as presumed 'obligate carriers', i.e. an individual who appears to be transmitting the schizophrenic gene(s). The mid-sagittal slice of the corpus callosum and the whole brain volume were measured blind to diagnostic and family group. We found no difference between schizophrenics, their relatives, and normal controls in the mid-sagittal area of the corpus callosum. There remained no difference when the relatives were divided into two groups comprising presumed 'obligate carriers' and 'non-obligate carriers'. Adjusting for age and whole brain area made no difference to the results. Families with several schizophrenic members are not associated with abnormality in the size of the corpus callosum. PMID- 10728718 TI - Apoptosis and schizophrenia: is the tumour suppressor gene, p53, a candidate susceptibility gene? AB - This paper reviews the six published incidence studies of the relative risk of cancer in patients with schizophrenia compared with the general population. These studies used: incidence data, register case ascertainment, and controlled for age and sex. It is concluded that schizophrenia is associated with a lower risk of developing cancer. The role of apoptosis (programmed cell death) in cancer and brain development is briefly described. The possibility is explored that increased apoptosis may account for neurodevelopmental abnormalities as well as tumour resistance associated with schizophrenia. The authors propose that p53, a tumour suppressor gene central to regulation of apoptosis, should be investigated as a candidate susceptibility gene in schizophrenia. PMID- 10728719 TI - Premorbid IQ in patients with functional psychosis and their first-degree relatives. AB - Numerous studies have found deficits in premorbid IQ in schizophrenic patients, but it is not clear whether this deficit is shared by (a) patients with other functional psychoses, and (b) relatives of these patients. Ninety-one schizophrenic patients, 66 affective psychotic patients (29 schizoaffective and 37 manic or depressed), and 50 normal control subjects were administered the National Adult Reading Test (NART) which provides an estimate of premorbid IQ. The NART was also completed by 85 first-degree relatives of schizophrenic patients and by 65 first-degree relatives of affective psychotic patients. After adjustments were made for sex, social class, ethnicity and years of education, schizophrenic patients had significantly lower premorbid IQ than their relatives, the affective psychotic patients and controls. Manic and depressed patients had significantly lower NART scores than their first-degree relatives, but schizoaffective patients did not, and neither group differed significantly from controls. There was no significant difference in premorbid IQ between patients who had experienced obstetric complications (OC+) and those who had not (OC-). Both OC+ and OC- schizophrenic patients differed significantly from their relatives, but the disparity was greatest between OC+ patients and their relatives. Relatives of OC+ schizophrenic patients had significantly higher IQ than relatives of OC- schizophrenic patients. PMID- 10728720 TI - Memory and organizational strategies in chronic and acute schizophrenic patients. AB - The memory profile of acute and chronic schizophrenic patients was examined according to the information processing model, with which encoding, retention and retrieval processes of these patients were compared. The effects of an external organizational strategy on their verbal learning and memory were also examined. Twenty chronic and 20 acute schizophrenic patients were tested with a list learning task consisting of a random (i.e., words presented randomly) and a blocked (i.e., words presented in clusters) word-list. The schizophrenic patients, as compared with age- and education-matched normal control subjects, demonstrated impaired learning, and the duration of their illness was not a significant factor in the severity of their learning impairment. However, the acute and chronic schizophrenic patients seem able to retain most of the newly acquired materials, regardless of the presentation format, after 30 min. In addition, the learning and subjective organizational strategy of the chronic patients, but not that of the acute patients, improved significantly by the blocked presentation. However, semantic organization could facilitate both the chronic and acute schizophrenic patients to retain more newly learned items. PMID- 10728721 TI - Visuospatial working memory in schizotypal personality disorder patients. AB - BACKGROUND: Cognitive processing deficits have been identified as an abnormality that schizotypal personality disorder (SPD) individuals share with schizophrenic patients. It has been hypothesized that impaired working memory may be a critical component of several of the more complex cognitive deficits found in schizophrenia spectrum patients. METHOD: 18 DSM-III-R SPD patients, and 17 normal comparison subjects were compared on a pen and paper visuospatial working memory task. Moreover, we identified a second psychiatric comparison group comprised of nine patients with other, non-odd cluster personality disorder diagnoses who met no more than one of the SPD criteria and were also tested on the same task. Each person was given 14 immediate recall trials and 10 trials using a 10 s delay. RESULTS: SPD patients performed significantly worse than normal control subjects on the working memory task. SPD patients also performed significantly worse compared to the non-schizophrenia-related personality disorder psychiatric comparison group. CONCLUSIONS: Like schizophrenic patients, SPD patients demonstrate working memory impairment compared to normal controls. This impairment may be specific to the schizophrenia-related personality disorders. PMID- 10728722 TI - Seasonality of first admissions for schizophrenia in the Southern Hemisphere. AB - Northern Hemisphere studies of first admissions for schizophrenia have shown an excess of summer admissions (June, July and August) compared to other times of the year. The within-year fluctuations in first admissions could be related to meteorological factors that fluctuate between seasons (e.g. temperature, photoperiod) and/or social factors (e.g. holidays, religious events). If meteorological factors were primarily responsible for the fluctuation, then Southern Hemisphere studies should find excess first admissions in December, January and February. This paper presents the first season of first admission study of schizophrenia in the Southern Hemisphere. The month and year of first admission for schizophrenia (ICD 8/9) for 4487 male and 3252 female, Australian born individuals were extracted from a Queensland mental health register. Spectral analysis showed a strong annual periodicity of first admissions for males with the peak in August (Southern Hemisphere winter) and a trough in the summer months (December to February). The pattern for females also displayed annual periodicity. These results correspond to the Northern Hemisphere reports of excess of schizophrenia first admissions in terms of the month of the year but not the season of excess. Further work is needed in order to clarify the impact of latitude and meteorological factors on the month of first admission for schizophrenia. PMID- 10728723 TI - Interactive ERP recording increases the amplitude of the endogenous P300 peak in schizophrenia. AB - The P300 is a long-latency cognitive event-related potential (ERP) elicited by the presentation of relevant target stimuli. Unfortunately, the standard ERP recording technique takes no account of the background EEG during stimulus presentation. However, a recently developed technique (interactive recording) controls for variability in the EEG by applying stimuli only when the background EEG is in a predetermined state. The use of the interactive technique has led to significant changes in the P300 amplitude in control studies. Since P300 amplitude data are commonly used in schizophrenia research, and have previously been associated with cognitive deficits in schizophrenia, we studied the effects of interactive recording in a schizophrenia population. The same ERP paradigm was implemented twice for each subject, using both standard ERP and the interactive recording techniques. There was a significant increase in the amplitude of the P300 peak with interactive recording, although no significant change in latency. The results indicate that the effect of background EEG may need to be taken into account when conducting general ERP-based studies in schizophrenia. The results also indicate that the method may be used to investigate the effects of EEG variation on ERP values and, by implication, on cognitive processes. In particular, the recognition of EEG states that lead to a higher amplitude ERP value may aid in the further development of EEG investigations of schizophrenia. A similar development is indicated between cognitive research in schizophrenia and the sub-second brain-states in which cognitive function may be reflected. PMID- 10728724 TI - Verbal memory and negative symptoms of schizophrenia revisited. PMID- 10728725 TI - The interaction of neutrophils with respiratory epithelial cells in viral infection. AB - Viral respiratory infection is very common. Respiratory syncytial virus (RSV) infects almost all children during the first 2 years of life. Respiratory syncytial virus is the most frequent cause of bronchiolitis, which is strongly linked with asthma. However, the pathophysiology of RSV bronchiolitis is unclear. Neutrophils are the predominant airway leucocytes in RSV bronchiolitis and other viral infections. Neutrophils and their products are likely to play an important role in viral infection. Current evidence indicates that: (i) viral infection of epithelial cells increases the production of neutrophil chemoattractants or chemokines, which induce neutrophil migration into the inflammatory sites; (ii) the expression of adhesion molecules on neutrophils and epithelial cells is up regulated in viral infection, and neutrophil-epithelial adhesion is increased; (iii) neutrophils augment epithelial damage and detachment induced by viral infection and contribute to the pathophysiology of viral disease; (iv) neutrophil apoptosis is up-regulated in RSV infection, which may be an in vivo mechanism to limit neutrophil-induced epithelial damage; (v) inhibitors of chemokines, adhesion molecules or neutrophil proteases may be useful in prevention of neutrophil-induced epithelial damage. In conclusion, neutrophils play an important role in viral infection, and intervention to prevent neutrophil-induced epithelial damage may be a potential clinical therapy. PMID- 10728726 TI - Flow-volume curve changes in patients with obstructive sleep apnoea and brief upper airway dysfunction. AB - OBJECTIVE: Patients with obstructive sleep apnoea (OSA) and those with brief upper airway dysfunction (BUAD) have been reported to have abnormalities of maximal flow-volume curves. This study was designed to assess the ability of flow volume curves to predict the presence of OSA or BUAD. METHODOLOGY: Four maximal flow-volume manoeuvres performed by 33 OSA patients and 16 BUAD patients were compared with those of 36 normal subjects. Flow-volume indices, their variability, saw-toothing in the curve and an algorithm based on the flow ratios and shape of the curves were assessed. RESULTS: When the confounding factors, body mass index (BMI), age, gender and smoking status were taken into account, there was no significant difference in a variety of indices derived from the flow volume curves between OSA and normal subjects. No BUAD patient had normal flow volume curves as determined with the algorithm. After BMI, age, gender and smoking status were accounted for, decreased forced expiratory volume in 1 s (FEV1), and increased variability of peak expiratory flow (PEF)/peak inspiratory flow (PIF) and FEV1/PEF remained significantly associated with BUAD. CONCLUSIONS: These findings suggest that flow-volume curve indices have no value in predicting OSA. Some abnormalities are found in patients with BUAD; a normal flow-volume curve makes the diagnosis of BUAD unlikely. PMID- 10728727 TI - A dynamic analysis of chest wall motions with MRI in healthy young subjects. AB - OBJECTIVE: The objective of this study was to analyse respiratory-related motion of the chest wall with non-invasive method. METHODOLOGY: Using magnetic resonance image (MRI), 30 sequential images (scanning time, 0.4 s per image) on sagittal, axial and coronal planes were obtained in nine healthy young subjects during quiet breathing (QB) and maximal deep breathing (MDB). The coronal planes were obtained in five of nine subjects during MDB. Ventilation was simultaneously measured with pneumotachometer. RESULTS: There was a linear correlation between instantaneous lung volume and lung cross-sectional area. Motion of the diaphragm and rib cage was also linearly related to instantaneous lung volume. The exception was lower anteroposterior (AP) diameter of the rib cage. The contribution of individual part of the chest wall motion to a unit lung volume change was assessed by slope (S) of the linear regression line. The S at the anterior diaphragm was significantly smaller than those at middle and posterior parts during MDB. The S of middle and posterior diaphragmatic motion was approximately five times that of AP motion of upper rib cage. The S of AP motion of upper rib cage was twice that of transverse motion during either QB or MDB. CONCLUSION: We concluded that dynamic MRI study with concurrent ventilation measurement is a simple and reliable method for evaluation of local chest wall motion, and that neither diaphragm nor rib cage works as a single functional unit during active ventilation. PMID- 10728728 TI - Suppressive effect of saprogrelate hydrochloride on Raynaud's phenomenon and respiratory failure in patients with systemic sclerosis. AB - OBJECTIVE: In seven patients with systemic sclerosis (SS), we evaluated the clinical effectiveness of oral administration of saprogrelate hydrochloride (SH: 300 mg/day) for 2 months on Raynaud's phenomenon (RP) and respiratory failure estimated by Hugh-Jones classification. METHODOLOGY: We evaluated laboratory data (arterial blood gas (pH, PaO2 and PaCO2), pulmonary function tests (%VC, FEV1/FVC and %DL(CO)), mean pulmonary arterial pressure (mPAP), white blood cell count, C reactive protein and the plasma levels of fibrinopeptide A (FPA), beta thrombogloblin (beta-TG), platelet factor 4 (PF4) and thrombomodulin (TM)) before and after SH administration. RESULTS: The frequency and duration of RP, as well as the coldness, numbness and pain of RP were significantly decreased after SH administration (P < 0.05, P < 0.01 and P < 0.001). Respiratory failure estimated by Hugh-Jones classification was also significantly decreased after SH administration (P < 0.05), and the %DL(CO) was significantly increased (P < 0.01). The mPAP decreased significantly after SH administration (P < 0.05). Plasma FPA, beta-TG and PF4 significantly decreased after administration (P < 0.05 and P < 0.01). CONCLUSIONS: SH therapy could prevent RP and respiratory failure in patients with SS. PMID- 10728729 TI - Causes of pleural exudates in a region with a high incidence of tuberculosis. AB - OBJECTIVE: To define the causes of exudative pleural effusions in our region. METHODOLOGY: A retrospective study was performed on consecutive patients with exudative pleural effusion seen in our hospital during a 4-year period. RESULTS: Of 186 patients with a mean age (+/- SD) of 51.2 (+/- 19.2) years with exudative pleural effusions, 131 (70.4%) were males and 55 (29.6%) were females. The most frequent cause of exudative pleural effusions was tuberculosis (44.1%), followed by malignancy (29.6%). The majority (94.5%) of malignant pleural effusions were due to lung cancer. Apart from a patient with bilateral pleural effusions due to cryptococcosis, patients with tuberculous pleural effusion (mean age (+/- SD), 39.7 (+/- 17.5)) were significantly younger than the rest (P < 0.05). Tuberculous effusions were most frequent in the first five decades (60/82, 73.2%) and were the most common type of pleural effusion, accounting for 60 (69.8%) of 86 cases, in this age range. Malignant effusions were more frequent among the older age groups, 74.5% (41/55) of patients with malignant effusions being older than 50 years. Most types of pleural effusions showed a preference for the right side. Of the 44 cases of large effusions, 28 (63.6%) were caused by malignancy. CONCLUSIONS: In our region with a high incidence of tuberculosis, the most frequent cause of pleural exudates is tuberculosis followed by malignancy, particularly lung cancer. PMID- 10728730 TI - Utility of near patient testing and home care analysis using a portable critical care analyser. AB - OBJECTIVE: This study was conducted in order to determine the usefulness of the application of home patient care using a portable critical care analyser (OPTI-C) after the precision of the OPTI-C was examined. METHODOLOGY: The OPTI-C, which measures sodium (Na+) and potassium (K+) as well as pH, PCO2 and PO2 in whole blood, was used. In order to confirm the linearity of the OPTI-C, analysis using reference materials was performed (n=81). Correlation testing with whole blood samples from patients was also performed using other analysers in the laboratory (n=108). Home patient testing was performed with 15 patients. RESULTS: The slope linearity and correlation coefficients were as follows: pH, 1.0174+/-0.0091, 0.99972; PCO2, 0.9681+/-0.0094, 0.99967; PO2, 0.9844+/-0.0085, 0.99974; NaST, 0.9788+/-0.0115, 0.99911 and KST 0.9964+/-0.0128, 0.99893. The equivalent results from other analysers showed pH, 1.071+/-0.009, 0.9963; PCO2 0.985+/-0.016, 0.9870; PO2 0.989+/-0.007, 0.9976; Na+, 1.075+/-0.278, 0.9662 and K+ 1.120+/ 0.020, 0.9842. The correlation coefficients and P values were as follows: pH, 0.9790, P>0.1; PCO2, 0.9990, P>0.1; PO2, 0.9980, P>0.1; Na+, 0.9438, P>0.05, and K+, 0.9174, P>0.1. CONCLUSION: The use of portable critical care analysers for near patient testing for home-care patients is recommended in order to increase the efficiency of visits as well as to improve the quality of patient care. PMID- 10728731 TI - Prehospital management of exacerbations of asthma: relation to patient and disease characteristics. AB - OBJECTIVE: Admission to hospital due to an exacerbation of asthma may represent a failure of prehospital management to prevent an attack or bring about its remission. We aim to describe the steps taken prior to hospital attendance in patients with asthma and to identify patient or disease characteristics that were associated with failure to take appropriate actions. METHODOLOGY: Patients aged over 15 years who were discharged from hospital or the Emergency Department with a primary diagnosis of asthma completed a questionnaire about the impact of asthma and its management prior to the onset of the recent exacerbation. The impact of asthma on quality of life was measured using our Asthma Quality of Life Questionnaire (AQLQ). RESULTS: Seventy-eight subjects completed the questionnaire including 49 who had been inpatients and 29 who had been discharged from the Emergency Department. They included many subjects with severe asthma: 41% had nocturnal symptoms three or more times per week and 41% had been admitted to hospital with asthma in the 12 months prior to this episode. Their AQLQ scores were high (severe) (mean 4.5+/-2.2). Most subjects described their presenting exacerbation as slow in onset and moderate or severe in intensity. During this exacerbation and prior to hospital attendance, only 27% of subjects had measured their peak expiratory flow rate, 19% had commenced or increased the dose of inhaled steroids, and 22% had commenced oral steroids. These actions were not related to the self-perceived speed of onset of the attack. Patients with lower levels of asthma-related concerns for health and more severe mood disturbance associated with asthma were less likely to take these appropriate self-management steps. CONCLUSIONS: This study shows that the failure of prehospital management to prevent the necessity of hospital attendance in most cases stems from a failure to implement currently recommended actions or treatments for exacerbations. Addressing this problem should result in a reduction in rates of hospitalization for asthma. PMID- 10728732 TI - Induced sputum eosinophils in the assessment of asthma and chronic cough. AB - OBJECTIVE: To evaluate induced sputum eosinophils in asthma and chronic cough. DESIGN: This was an analytical, cross-sectional study set in an ambulatory respiratory clinic. SUBJECTS: Subjects (n=75) referred for evaluation of symptomatic asthma or episodic respiratory symptoms had a clinical assessment, spirometry, hypertonic saline challenge and induced sputum. Two diagnostic groups were identified. The first group comprised subjects with symptomatic asthma and variable airway obstruction (VAO) (n=32). The second group included subjects with episodic respiratory symptoms and no VAO (n=43). RESULTS: The prevalence of eosinophilic bronchitis (eosinophils >2.75%) was greatest in asthma (n=14, 44%), compared to the episodic respiratory symptoms group (n=9, 21%, P = 0.02). Clinical variables did not predict increased eosinophils (P > 0.05). Sputum eosinophils were highest in asthmatics not using inhaled corticosteroids (6.5% vs 0.5%, P = 0.02). Sputum neutrophils were higher in subjects using inhaled corticosteroid (53% vs 25%, P = 0.04). CONCLUSION: Airway inflammation with eosinophilia was common among patients presenting to a respiratory clinic, especially those with asthma who were not using inhaled corticosteroids. Induced sputum also identified eosinophilic bronchitis in those without asthma. It was not possible to detect the presence or absence of airway eosinophilia by routine clinical assessment. The results in this study imply that the assessment of induced sputum eosinophils may be a useful guide to therapy, especially in the assessment of persistent symptoms in asthmatics on corticosteroids, and in the assessment of non-asthmatic subjects with symptoms. PMID- 10728733 TI - A comparative clinical study of pneumonia by penicillin-resistant and -sensitive Streptococcus pneumoniae in a community hospital. AB - OBJECTIVE: This study aimed to determine the clinical difference of pneumonia between penicillin-resistant and penicillin-sensitive Streptococcus pneumoniae. METHODOLOGY: Forty-nine cases in 46 patients of pneumococcal pneumonia were studied from December 1992 to May 1997. There were 24 cases (in 22 patients) of penicillin-resistant pneumococci (PRSP) pneumonia which were compared with 25 cases (in 24 patients) with penicillin-sensitive pneumococci (PSSP). RESULTS: Both the mean age and the underlying disease states did not differ between the two groups. However, hospital-acquired pneumonia and previous use of antibiotics were observed in eight (33.3%) and 12 (50.0%) patients in PRSP compared with three (12.0%) and two (8.0%) in PSSP, respectively. The clinical efficacy rate and bacteriological eradication rates were 87.5 and 87.5% in PRSP compared with 87.5 and 87.0% in PSSP, respectively. Minimum inhibitory concentration (MIC) of antibiotics against 30 pneumococcal isolates was examined, and 10 strains ranged from 0.10-0.78 microg/mL and five strains were more than 1.56 microg/mL against penicillin G, while the MIC showed higher resistance to other antibiotics except for the carbapenems. Serotyping of the isolates by antiserum revealed differences in the predominant types PRSP (19F) and PSSP (6A,9V) [corrected]. CONCLUSIONS: We must care for not only community-acquired infection but also nosocomial transmission of PRSP pneumonia. Most patients with infections due to PRSP tended to have a milder illness with a good outcome (no patient died). As such it appears that empiric therapy for pneumococcal pneumonia does not require modification from what is recommended at present. However, in patients with infection due to highly resistant strains, and who are not responding to conventional therapy should have their treatment modified according to subsequent susceptibility testing. PMID- 10728734 TI - The effect of intratracheal pulmonary ventilation on the decrease of dead space in rabbits with acute respiratory failure. AB - OBJECTIVE: A technique that improves the efficiency of alveolar ventilation should decrease the pressure required and reduce the potential for lung injury during mechanical ventilation. High partial pressure of carbon dioxide (PaCO2) can be tolerated if associated with a lower airway pressure as in permissive hypercapnia (PH). Intratracheal pulmonary ventilation (ITPV) was developed to allow a decrease in physiological dead space during mechanical ventilation. We compared the effect of hybrid ventilation (HV) as a modification of ITPV with PH on the decrease of tidal volume and airway pressures in rabbits with acute respiratory failure. METHODOLOGY: Tracheostomy was performed in seven rabbits ventilated under volume-controlled mode in the supine position. Arterial blood gas analysis, airway pressures, and dead space ventilation were measured at respiratory rates of 20/min as control values. Oleic acid (OA) of 0.06 mL/kg was injected to induce acute respiratory failure. Tidal volume (V(T)) was elevated to maintain PaCO2 in the normal range. These same parameters were measured as OA values. Then V(T) was reduced to the control level to allow PH. Hybrid ventilation was initiated by inserting a reverse thrust catheter (RTC) into the endotracheal tube. Hybrid ventilation consists of a pressure-controlled mode of mechanical ventilation and ITPV while flushing fresh gas continuously via the RTC. Respiratory parameters were compared under control, OA, PH and HV conditions. RESULTS: Oleic acid injection decreased partial pressure of oxygen (PaCO2) from 401+/-35 mmHg to 129+/-39 mmHg, increased V(T) from 42+/-5 mL to 52+/-10 mL, and increased dead space (V(D))/V(T) ratio from 0.65+/-0.07 to 0.71+/ 0.07. During PH, the increase in PaCO2 was accompanied by an increase in V(D)/V(T) ratio from 0.71+/-0.07 to 0.79+/-0.03 and by a decrease of peak inspiratory pressure (PIP) from 19.4+/-4.0 cmH2O to 16.8+/-3.1 cmH2O. PaCO2 was lowered from 50+/-5 mmHg in PH to 39+/-5 mmHg in HV with a lower V(T). V(D)/V(T) ratio in HV which was as low as that in control. CONCLUSIONS: Hybrid ventilation is an effective and easy-to-use ventilatory modality to reduce PaCO2 and airway pressures by the reduction in V(D)/V(T) ratio in acute respiratory failure model. PMID- 10728735 TI - Intravascular ultrasound imaging of the pulmonary arteries in primary pulmonary hypertension. AB - OBJECTIVE: Intravascular ultrasound has the unique ability to provide cross sectional images of the arterial wall. This study examined intravascular ultrasound (IVUS) images of the proximal pulmonary arteries in primary pulmonary hypertension (PPH). METHODOLOGY: Study 1: Specimens from four patients who had died of PPH (in vitro PPH group) were compared with those of three patients who had died of subarachnoid haemorrhage but had no evidence of cardiopulmonary disease (in vitro control group). Three-centimetre segments of the following levels were examined by IVUS: pulmonary trunk, eight secondary branch arteries of the upper, middle, and lower lobes of both lungs, and the thoracic descending aorta. Study 2: Four patients with PPH (in vivo PPH group) and five patients without pulmonary hypertension and no evidence of cardiopulmonary disease (in vivo control group) were examined. The IVUS images of the apical segmental artery of the right upper lobe and the descending branch of the right pulmonary artery were studied. RESULTS: Echographic examination of formalin-fixed preparations of secondary branch sections of the pulmonary artery failed to show a clear three layer structure in the in vitro control group (24 preparations), but a distinct three-layer structure and increased vessel wall thickness were observed in the in vitro PPH group (32 preparations). Similar findings were obtained in the in vivo study. The mean echo density of the proximal pulmonary arterial wall correlated well with the mean pulmonary arterial pressure (mPA) in the in vitro PPH, and also correlated with the mPA in the in vivo study (r = 0.960, P < 0.0001). The echo intensity of secondary branch sections of the pulmonary artery was higher in the in vitro PPH group than in the in vitro control group (180.5 +/- 27.0 vs 132.5 +/- 26.7 counts, P < 0.001); similar results were obtained in the in vivo study (144.7 +/- 23.4 vs 85.0 +/- 14.3 counts, P < 0.01). CONCLUSIONS: These results suggest that the histological changes detected in the pulmonary artery walls in the PPH group were responsible for the increased echo intensity. PMID- 10728736 TI - Pleural amyloidosis in a patient with intractable pleural effusion and multiple myeloma. AB - Pleural involvement of systemic amyloidosis has been rarely reported. We report a case with multiple myeloma presenting an intractable right pleural effusion, in which pleural amyloidosis was diagnosed through pleural biopsy using a Cope needle. The diagnosis of pleural amyloidosis is important, because its refractory pleural effusion should be treated with pleurodesis. Since closed pleural biopsy using a Cope needle is much less invasive than thoracoscopy, the former should be attempted first whenever pleural amyloidosis is suspected. PMID- 10728737 TI - Bronchiolitis obliterans organizing pneumonia (BOOP) in a case of smouldering adult T-cell leukaemia. AB - Although various respiratory diseases have been reported in human T lymphotropic virus type-1 (HTLV-1) carriers or patients with adult T-cell leukaemia (ATL), there appears to be no report of the development of bronchiolitis obliterans organizing pneumonia (BOOP) in ATL or HTLV-1-related disorders. We describe a 51 year-old male with smouldering ATL who developed BOOP during a long-term follow up. Bronchoalveolar lavage (BAL) and transbronchial lung biopsy (TBLB) were performed in the right lower lobe B6 with infiltrative shadows. As a result of flow cytometric analysis of peripheral lymphocytes and BAL lymphocytes, histological examination of the biopsied lung specimen, and the clinical course, we excluded the pulmonary infiltration of ATL cells and bacterial infection. Thus, he was diagnosed as having BOOP and successfully treated with corticosteroid therapy. This is probably the first report of BOOP developing in ATL. Bronchiolitis obliterans organizing pneumonia should be considered in the differential diagnosis of pulmonary complications in HTLV-1 carriers or ATL patients since BOOP can be successfully treated by corticosteroids. PMID- 10728738 TI - Multi-organ failure caused by reactivated coccidioidomycosis without dissemination in a patient with renal transplantation. AB - OBJECTIVE: The acute respiratory failure caused by pulmonary coccidioidomycosis without dissemination is an extremely unusual event. CASE REPORT: We report a 47 year-old renal transplanted man with a reactivated pulmonary coccidioidomycosis, whose clinical course presented as fulminant respiratory failure, disseminated intravascular coagulation and profound hypotension mimicking bacterial pneumonia and septic shock. Lung biopsy showed conglomerated necrotizing granulomas containing many spherules filled with endospores of Coccidioides immitis. CONCLUSION: Coccidioidomycosis should be included in the differential diagnosis of acute sepsis, particularly in an immunocompromised host who has travelled in an endemic area. PMID- 10728739 TI - When does rheumatoid arthritis begin and why do we need to know? PMID- 10728740 TI - Genetic factors predisposing to spondylarthropathies. PMID- 10728741 TI - Treatment with leflunomide slows radiographic progression of rheumatoid arthritis: results from three randomized controlled trials of leflunomide in patients with active rheumatoid arthritis. Leflunomide Rheumatoid Arthritis Investigators Group. AB - OBJECTIVE: To determine whether treatment with leflunomide (LEF), methotrexate (MTX), or sulfasalazine (SSZ) for 6-12 months retards progression of radiographic damage and to identify clinical variables that correlate with radiographic progression. METHODS: Radiographs of the hands and feet were performed at baseline and at the end of study or early exit in 3 randomized controlled trials. Protocol US301 was a 12-month controlled trial of LEF or MTX treatment compared with placebo in 482 patients randomized in a 3:3:2 ratio. Protocol MN301 compared 6 months of LEF or SSZ treatment with placebo in 358 patients, randomized in a 3:3:2 ratio, with continued blinded treatment in the active control arms for 12 months. Protocol MN302 compared 12 months of LEF treatment with MTX in 999 patients. Radiographs were blinded for sequence and treatment and were scored for erosions and joint space narrowing. All analyses were by intent-to-treat. Sensitivity analyses were performed to account for missing data. RESULTS: LEF, MTX, and SSZ treatment resulted in statistically significantly less radiographic progression compared with placebo at 6 and 12 months: for protocol US301, LEF versus placebo P = 0.0007 and MTX versus placebo P = 0.0196; for protocol MN301, LEF versus placebo P = 0.0004 and SSZ versus placebo P = 0.0484. The effect of LEF treatment was similar to that of MTX and SSZ. CONCLUSION: These are the first 6- and 12-month randomized placebo- and active drug-controlled trials to demonstrate retardation of radiographic progression by a new disease-modifying antirheumatic drug (DMARD), LEF, as well as 2 commonly used DMARDs, MTX and SSZ. PMID- 10728742 TI - Clinical improvement as reflected in measures of function and health-related quality of life following treatment with leflunomide compared with methotrexate in patients with rheumatoid arthritis: sensitivity and relative efficiency to detect a treatment effect in a twelve-month, placebo-controlled trial. Leflunomide Rheumatoid Arthritis Investigators Group. AB - OBJECTIVE: To examine correlations between clinical improvement as defined by the American College of Rheumatology (ACR) responder analysis and clinical improvement as determined by 4 function and/or health-related quality of life measures, and to estimate the sensitivity and relative efficiency of these measures compared with changes in the tender joint count in patients with rheumatoid arthritis (RA). METHODS: A 52-week, multicenter, double-blind controlled trial was conducted to compare treatment with leflunomide (n = 182), methotrexate (n = 180), or placebo (n = 118) in patients with active RA. ACR response rates and improvement in scores on the Health Assessment Questionnaire (HAQ), Problem Elicitation Technique (PET), and Medical Outcomes Survey Short Form 36 (SF-36) were compared in 438 of the patients. RESULTS: In comparing leflunomide with placebo, the patient global assessment, HAQ disability index, and SF-36 bodily pain scale were most responsive to treatment group differences. The modified HAQ (M-HAQ), PET Top 5, SF-36 physical component score, physician global assessment, pain intensity scale, and SF-36 physical functioning scale were more responsive to treatment group differences than was the tender joint count. In comparing methotrexate with placebo, the patient and physician global assessments were most responsive. These 2 measures, as well as the pain intensity scale and the C-reactive protein level, were more responsive to treatment group differences than was the tender joint count, while the SF-36 mental health component score was least responsive. A close correlation between changes in the M-HAQ and HAQ scores indicated that the M-HAQ was similarly responsive to change over time. Improvements in the PET, SF-36 physical component score, bodily pain, and physical functioning scales correlated with the ACR responder status. CONCLUSION: Both disease-specific and generic measures of function and health related quality of life detect improvements in RA patients. Using both types of measures for evaluating therapies will identify discernible changes that are important to patients, and will facilitate comparisons across different disease states. PMID- 10728743 TI - The association of variant mannose-binding lectin genotypes with radiographic outcome in rheumatoid arthritis. AB - OBJECTIVE: To investigate the possible association of mannose-binding lectin (MBL) genotypes with the outcome of rheumatoid arthritis (RA). METHODS: MBL genotypes and plasma concentrations were retrospectively determined in 140 RA patients who were selected from a major cohort followed up prospectively for up to 32 years. RESULTS: MBL-insufficient patients (those with 2 defective structural MBL alleles or with 1 defective allele combined with a low-expression variant of the normal allele) had unfavorable outcomes. The relative risk of a severe radiographic outcome event (30% of maximum radiographic destruction, or an RE30) was 3.1 (95% confidence interval 1.8-5.1) in the MBL-insufficient group versus the MBL-competent group (P < 0.0001). An RE30 occurred in 50% of MBL competent patients within 17 years, while such an event occurred 9 years earlier in MBL-insufficient patients (i.e., within 8 years) (P < 0.0001). During the first 15 years, there was a significant trend toward lower hemoglobin levels (P < 0.04), higher erythrocyte sedimentation rates (P < 0.02), and a higher number of swollen joints (P < 0.05) in the MBL-insufficient group. CONCLUSION: MBL genotypes giving rise to MBL insufficiency are highly significant risk factors for fast progression of radiographic joint destruction. PMID- 10728744 TI - Bone mineral density and frequency of osteoporosis in female patients with rheumatoid arthritis: results from 394 patients in the Oslo County Rheumatoid Arthritis register. AB - OBJECTIVE: To examine the bone mineral density (BMD), frequency of osteoporosis, and risk factors for BMD reduction in a representative population of female rheumatoid arthritis (RA) patients ages 20-70 years. METHODS: BMD in the femoral neck, total hip, and spine L2-4 (anterior-posterior view) was measured in 394 RA patients recruited from a validated county RA register (completeness 85%) comprising 721 women ages 20-70 years. BMD was measured with dual-energy x-ray absorptiometry, and age-specific values were compared with pooled values from a European/US population of healthy subjects free from earlier fractures, chronic diseases, and medications influencing bone metabolism. A multiple linear regression model was used to determine individual predictors of BMD. RESULTS: No statistically significant differences were found in demographic, disease activity, disease severity, or health status parameters between the RA register patients in whom BMD was measured and the remaining register patients. Femoral neck BMD was significantly reduced by 4.2% in the age group 50-59 years, and by 5.0% in those ages 60-70 years. For BMD in the total hip, the significant reductions were 3.7%, 6.0%, and 8.5% in the age groups 40-49 years, 50-59 years, and 60-70 years, respectively. No significant reduction in spine L2-4 BMD was found. A 2-fold increased frequency of osteoporosis was observed in all 4 age groups of RA patients compared with the reference population, ranging from 0% to 28.6% in the femoral neck, 0% to 29.9% in the total hip, and 1.8% to 31.5% in the spine. Predictors of reduced BMD were as follows: at the femoral neck, older age, low body weight, current use of corticosteroids, greater physical disability (as measured by the modified Health Assessment Questionnaire [M-HAQ]), and presence of rheumatoid factor; at the total hip, older age, low weight, current use of corticosteroids, and higher M-HAQ disability score; and at the lumbar spine, older age, low weight, and current use of corticosteroids. CONCLUSION: Register based prevalence data on BMD reduction in female RA patients ages 20-70 years are presented for the first time in this report, which demonstrates a 2-fold increase in osteoporosis in this representative population. PMID- 10728745 TI - Total-body bone mineral content in non-corticosteroid-treated postpubertal females with juvenile rheumatoid arthritis: frequency of osteopenia and contributing factors. AB - OBJECTIVE: To determine the extent of low total-body bone mineral content (BMC) in non-corticosteroid-treated white postpubertal females with juvenile rheumatoid arthritis (JRA) compared with healthy age- and race-matched female controls, and to identify variables that significantly contribute to total-body BMC. METHODS: Thirty-six females with definite JRA who had never received corticosteroids and 51 healthy female controls were evaluated. All subjects had had their first menstrual period at least 2 years prior to enrollment. Total-body BMC, lumbar spine bone mineral density, and body composition were determined by dual x-ray absorptiometry. Total-body BMC Z-scores were calculated for JRA patients using data from controls. JRA patients were dichotomized into those with "normal" bone mass (total-body BMC at or above the mean or no more than 1 SD below the mean) and those with "low" bone mass (total-body BMC more than 1 SD below the mean). Comparisons of anthropometric measurements, laboratory measurements of bone metabolism, disease activity, dietary intake, and physical activity were performed. Stepwise logistic regression was utilized to determine the presence or absence of low total-body BMC and to identify associated contributing factors. RESULTS: Total-body BMC was 4.5% lower in JRA patients than in controls (mean +/- SD 2,050 +/- 379 gm versus 2,143 +/- 308 gm; P = 0.21). Twenty-five of 36 patients (69.4%) had "normal" and 11 of 36 (30.6%) had "low" total-body BMC. Comparison of JRA patients with "normal" versus those with "low" total-body BMC revealed significant differences in disease characteristics, anthropometric and physical development characteristics, laboratory measures of bone mineralization, and dietary intake. The final regression model contained only lean mass (P = 0.01), which accounted for 76.3% of the variance in total-body BMC. The odds ratio for lean mass was 0.4451 (95% confidence interval 0.2374-0.8348). CONCLUSION: In this study, approximately 30% of the subjects in a sample of postpubertal female patients with mild-to-moderate, non-corticosteroid-treated JRA had low bone mass. The predictor variable that significantly contributed to total-body BMC was lean mass, which demonstrated a protective effect of 0.56 risk reduction for low total-body BMC. PMID- 10728746 TI - Medium- and long-term functional outcomes in a multicenter cohort of children with juvenile dermatomyositis. AB - OBJECTIVE: To evaluate functional outcomes in a cohort of patients with juvenile dermatomyositis (DM). METHODS: A retrospective inception cohort of patients diagnosed as having juvenile DM between January 1, 1984 and January 1, 1995 was established at 4 Canadian tertiary care pediatric centers. Informed consent was obtained. Each subject was interviewed by telephone or in person. The primary outcome was physical function, as measured by the Childhood Health Assessment Questionnaire (CHAQ). Additional outcomes were educational and vocational achievement, growth, development of calcinosis, patient satisfaction with outcome, and development of other illnesses. Data regarding illness presentation, treatment, and disease course were obtained through chart review. RESULTS: Sixty five of 80 patients (81%; 46 females and 19 males) could be contacted. The median followup time was 7.2 years (range 3.2-13.9 years), with a median age at followup of 13 years (range 7-26 years). Twenty-four patients (37%) had a monocyclic course, while the remaining 41 (63%) had a chronic continuous or polycyclic course. Sixty-two patients (95%) were treated with corticosteroids, while 41 (63%) received a second-line agent. Physical function was excellent, with a median CHAQ score of 0 (range 0-2.50). Eighteen patients had scores >0, and only 5 had moderate-to-severe disability, as defined by a CHAQ score >1.0. Females had higher CHAQ scores, and all but 1 of the patients with scores >0 were female (range 0-2.50; P = 0.015). Patients with a chronic continuous course also had higher CHAQ scores. Sixteen patients in the chronic continuous group had CHAQ scores >0 (range 0-2.50; P = 0.0009). Calcinosis developed in 22 patients (34%) and persisted to followup in 14. Development of calcinosis was not related to initial therapy, sex, or disease course, but was significantly associated with higher CHAQ scores (range 0-1.0 versus 0-2.5; P = 0.01). At the time of followup, 26 patients (40%) still had rash, 15 (23%) still reported weakness, and 23 (35%) continued taking medications, despite the fact that all were at least 3 years postdiagnosis. There was 1 death. CONCLUSION: In general, patients in this cohort had favorable outcomes. Most had CHAQ scores of 0, and only 8% met our definition of moderate-to-severe disability. However, many patients continued to have chronic disease, persistent rash, and continued taking medications >3 years after diagnosis. Further research is needed to improve outcomes for patients with juvenile DM. PMID- 10728747 TI - Ovulation induction and in vitro fertilization in systemic lupus erythematosus and antiphospholipid syndrome. AB - OBJECTIVE: During ovulation induction (OI), ovarian stimulation is accomplished by hormonal manipulation, which includes administration of gonadotropins, gonadotropin-releasing hormone agonists, follicle-stimulating hormone, and luteinizing hormone. In in vitro fertilization (IVF), progesterone is often added. Because of the possibility of hormone-associated flare or thrombosis, patients with systemic lupus erythematosus (SLE) and primary antiphospholipid syndrome (primary APS) undergoing OI/IVF are potentially at increased risk. The present study was conducted in order to assess this risk. METHODS: Nineteen women who underwent 68 cycles of OI/IVF were studied by interview and retrospective chart review. RESULTS: Four OI/IVF cycles (25%) in SLE patients resulted in increased lupus activity and 2 (13%) in ovarian hyperstimulation syndrome. One patient with primary APS who was given heparin during multiple cycles developed osteopenia. No thrombosis occurred. Pregnancy complications included toxemia, lupus flare, gastrointestinal hemorrhage due to Mallory-Weiss tear, polygestation, and diabetes. Postpartum complications included nephritis flare, costochondritis, and suicidal depression. Lupus flares occurred at expected rates. Five of 16 cycles (31%) in 7 SLE patients, 5 of 48 cycles (10%) in 10 primary APS patients, and 0 of 5 cycles in 2 women with antiphospholipid antibody (without SLE or primary APS) resulted in liveborn children, including multiple gestations (3 twin sets with 4 surviving infants and 2 triplet sets with 3 surviving infants). Seven of 14 living children (50%) were premature, 3 had neonatal lupus, and 1 had pulmonic stenosis. Five surviving infants (38%) had complications unrelated to prematurity. CONCLUSION: Although OI/IVF can be successful in SLE and primary APS patients, rates of fetal and maternal complications are high. PMID- 10728748 TI - Prevalence of anti-protein S antibodies in patients with systemic lupus erythematosus. AB - OBJECTIVE: To determine the prevalence of circulating anti-protein S (anti-PS) antibodies in association with PS deficiency in patients with systemic lupus erythematosus (SLE). METHODS: Plasma was obtained from blood samples collected from 27 patients with SLE (5 men, 22 women; mean +/- SD age 32 +/- 10 years). Anti-PS antibodies were detected by immunoblotting according to a previously described method. Levels of free and total PS antigen were measured by enzyme linked immunosorbent assay. PS activity was assayed by a clotting method. Levels of C4b binding protein (C4bBP) were measured by latex immunoassay. RESULTS: Among 27 patients with SLE, 19 (70.4%) had PS activity, and 12 (44.4%) had PS free antigen below the lower limit established for a normal population (mean -2SD). There was good correlation between PS activity and levels of PS free antigen (r = 0.851, P < 0.001), PS total antigen (r = 0.743, P < 0.001), and C4bBP (r = 0.597, P = 0.001). Circulating anti-PS antibodies and antiphospholipid antibodies (aPL) (IgG type) were detected in 7 (25.9%) and 14 (51.9%) of the 27 patients with SLE, respectively. Only 1 patient (3.7%) had both anti-PS antibodies and aPL. PS activities in patients who were positive for anti-PS antibodies (mean 38.7%) tended to be lower than those in patients who were negative for anti-PS antibodies (mean 54.9%), but the difference was not statistically significant. CONCLUSION: Anti-PS antibodies, independently of aPL, may play a role in the occurrence of PS deficiency in some patients with SLE, with possible effects on the function of PS that do not change the levels of PS antigens. PMID- 10728749 TI - The association between chronic widespread pain and mental disorder: a population based study. AB - OBJECTIVE: Patients with chronic widespread pain (CWP) have been reported to have a greater prevalence of mental disorders and somatization than that found in the general population, but the true association between CWP and mental disorders is unknown. In this study, we investigated whether there is an increased prevalence of mental disorder in people with CWP from the general population. We also describe the psychiatric diagnoses associated with CWP. METHODS: In a population based case-control study, 1,953 subjects (75% of a random sample of individuals age 18-65 years) completed a questionnaire that included a pain assessment and the 12-item General Health Questionnaire (GHQ-12). Of 710 subjects scoring >1 on the GHQ-12, 301 were assessed further using a structured psychiatric interview and detailed assessment of medical records to identify cases of mental disorder, in accordance with criteria of the 10th edition of the International Classification of Diseases. The association between CWP and mental disorder was modeled using logistic regression, adjusting for possible confounders including age, sex, and nonresponders. RESULTS: We estimated the overall population prevalence of mental illness to be 11.9%. The odds of having a mental disorder for subjects with versus those without CWP were 3.18 (95% confidence interval 1.97-5.11). Most subjects with mental disorders were diagnosed as having mood and anxiety disorders. Only 3 cases of somatoform disorders were identified, and all were associated with pain. CONCLUSION: This study, although unable to demonstrate a cause-and-effect relationship, showed that 16.9% of those with CWP were estimated to have a psychiatric diagnosis, suggesting that these disorders should be identified and treated. PMID- 10728750 TI - The mechanism of the effect of obesity in knee osteoarthritis: the mediating role of malalignment. AB - OBJECTIVE: Obesity is most strongly linked to osteoarthritis (OA) at the knee. Varus malalignment was examined as a possible local mediator that may increase the impact of body weight at the knee, versus the hip or ankle. Compartment load distribution is more equitable in valgus than in varus knees, and valgus knees may better tolerate obesity. We therefore tested whether 1) body mass index (BMI) is correlated with OA severity in varus knees, 2) the BMI-OA severity correlation is weaker in valgus than in varus knees, 3) BMI is correlated with the severity of varus malalignment, and 4) the BMI-medial tibiofemoral OA severity relationship is reduced after controlling for varus malalignment. METHODS: In 300 community-recruited patients with knee OA, 2 groups (varus and valgus) were identified based on dominant knee alignment on a full-limb radiograph, i.e., the angle formed by the intersection of the femoral and tibial mechanical axes. Severity of knee OA was assessed by measurement of the narrowest joint space width on radiographs of knees in a fluoroscopy-confirmed semiflexed position. RESULTS: Alignment direction was symmetric (or neutral in 1 limb) in 87% of patients. One hundred fifty-four patients had varus knees and 115 had valgus knees. BMI correlated with OA severity in the varus group (r = -0.29, P = 0.0009) but not in the valgus group (r = -0.13, P = 0.17). BMI correlated with malalignment in those with varus knees (r = 0.26) but not in those with valgus knees (r = 0.16). The partial correlation of BMI and OA severity, controlling for sex, was reduced from 0.24 (P = 0.002) to 0.04 (P = 0.42) when varus malalignment was added to the model. CONCLUSION: BMI was related to OA severity in those with varus knees but not in those with valgus knees. Much of the effect of BMI on the severity of medial tibiofemoral OA was explained by varus malalignment, after controlling for sex. Whether it precedes or follows the onset of disease, varus malalignment is one local factor that may contribute to rendering the knee most vulnerable to the effects of obesity. PMID- 10728751 TI - The mechanism of formation of bony spurs (enthesophytes) in the achilles tendon. AB - OBJECTIVE: To investigate the early stages in the formation of bony spurs in relation to normal enthesis development. METHODS: Histologic sections of rat Achilles tendons, stained with toluidine blue or Masson's trichrome, were examined in animals ranging from 2 weeks to 1 year of age. Further material prepared for immunohistochemistry was labeled with monoclonal antibodies for laminin and type IV collagen to highlight the presence of small blood vessels at the enthesis. Sections of small spurs from the Achilles tendons of elderly humans were also examined for comparison. RESULTS: As a part of normal development, bone grows into the Achilles tendon as the calcaneus enlarges. Ossification is preceded by vascular invasion, which occurs along rows of enthesis fibrocartilage cells. Small bony spurs develop when ossification at one point on the enthesis outstrips that on either side. CONCLUSION: Bony spurs can develop in the Achilles tendon without the need for preceding microtears or any inflammatory reaction, and they form by endochondral ossification of enthesis fibrocartilage. The increased surface area created at the tendon-bone junction may be an adaptive mechanism to ensure the integrity of the interface in response to increased mechanical loads. PMID- 10728752 TI - Erythema nodosum: etiologic and predictive factors in a defined population. AB - OBJECTIVE: To examine the frequency and features of erythema nodosum (EN), establish disease associations, and identify the optimal set of predictors for the occurrence of secondary EN. METHODS: We performed a retrospective study of an unselected population of patients 14 years and older with biopsy-proven EN diagnosed at a referral hospital between 1988 and 1997. Patients were classified as having either idiopathic EN or EN secondary to other diseases if the skin nodules occurred in the context of a well-defined disease, or if there was a precipitating event in close temporal proximity to the onset of EN. RESULTS: One hundred six patients (82 women) were diagnosed as having biopsy-proven EN. At the time of diagnosis, no precipitating events or underlying diseases were identified in 36.8% of patients. Sarcoidosis and nonstreptococcal upper respiratory tract infections (URI) were the most common conditions associated with secondary EN. Only 1 of 35 patients with an initial diagnosis of idiopathic EN and a followup of at least 1 year was finally diagnosed as having secondary EN. The best predictive model of secondary EN included an abnormal results on a chest radiograph, a previous history of nonstreptococcal URI, and a significant change in antistreptolysin O (ASO) titer in 2 consecutive determinations performed in a 2-4-week interval. Also, the presence of peripheral synovitis, a positive tuberculin skin test, and a history of diarrhea suggested the presence of secondary EN. This model showed high sensitivity and specificity. CONCLUSION: Idiopathic EN is common. A basic procedure including careful medical history taking, a physical examination for peripheral synovitis, 2 consecutive ASO determinations, a tuberculin skin test, and chest radiography may be sufficient to diagnose EN. PMID- 10728753 TI - Presence of bacterial DNA and bacterial peptidoglycans in joints of patients with rheumatoid arthritis and other arthritides. AB - OBJECTIVE: The continuous presence of bacteria or their degraded antigens in the synovium may be involved in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to determine the presence of bacterial nucleic acids and bacterial cell wall constituents in the joints of patients with RA and other forms of arthritis. METHODS: Joint samples were obtained from patients with RA (n = 26), septic arthritis (n = 2), inflammatory osteoarthritis (n = 5), and gout (n = 6), and joint trauma (n = 1). Universal 16S-ribosomal RNA primers were used to detect the presence of bacterial DNA in these samples, using stringent regimens for sample collection and molecular microbiologic analysis. Automated sequencing and comparative data analysis were performed to identify the species. The presence of bacterial peptidoglycan-polysaccharide complexes in synovial tissue was detected by immunohistologic analysis with a specific antibody. RESULTS: The bacterial species cultured from the synovium could be identified in both of the patients with septic arthritis. DNA amplicons were also detected in the synovial fluid and/or tissue samples from 5 patients with RA and 2 patients with crystal induced arthritis; these originated from multiple bacterial species. Staining for peptidoglycan-polysaccharide complexes was positive in the synovial tissue of both patients with septic arthritis, 16 with RA, 4 with inflammatory osteoarthritis, 4 with crystal-induced arthropathy, and 1 with joint trauma. The staining was mainly found in cells in the synovial sublining, including macrophages. CONCLUSION: The results indicate that bacterial DNA and bacterial cell wall constituents are retained in the joints of some patients with arthritis, where they might enhance synovial inflammation. PMID- 10728754 TI - Expression of sentrin, a novel antiapoptotic molecule, at sites of synovial invasion in rheumatoid arthritis. AB - OBJECTIVE: Sentrin, a novel antiapoptotic molecule, has been shown to interact with the signal-competent form of Fas/APO-1 and tumor necrosis factor receptor I (TNFRI), and thereby, to protect cells against anti-Fas/APO-1- and TNF-induced cell death. Since reduced apoptosis in the synovial lining is supposed to contribute to synovial hyperplasia in rheumatoid arthritis (RA), we searched for the expression of sentrin-1 messenger RNA (mRNA) in synovium from patients with RA. METHODS: The expression of sentrin-1 mRNA was examined by in situ hybridization on snap-frozen sections of normal and RA synovial tissues as well as on paraffin-embedded RA synovial specimens, including the interface of cartilage-bone and invading synovium. Immunohistochemical double labeling after in situ hybridization was performed to further characterize sentrin-1 mRNA expressing cells. In addition, quantitative analysis of sentrin-1 mRNA expression in RA synovial fibroblasts (RASF), osteoarthritis synovial fibroblasts (OASF), and normal fibroblasts was performed by quantitative real-time polymerase chain reaction. Expression levels were standardized to the expression of GAPDH. The in vivo maintenance of sentrin expression in RASF aggressively invading human cartilage was explored in the SCID mouse model of RA. RESULTS: A marked expression of sentrin-1 mRNA could be seen in all RA synovial specimens, predominantly in SF of the lining layer and at sites of invasion of RA synovium into cartilage. In normal synovial tissues, no sentrin-1 mRNA was detectable. RASF showed a maximum 32.5-fold (mean +/- SD 14.9 +/- 11.6) increase of sentrin-1 mRNA expression compared with normal fibroblasts and a maximum 31.4-fold (mean +/ SD 14.3 +/- 10.9) increase compared with OASF. When coimplanted with normal human cartilage in the SCID mouse model, invading RASF maintained their sentrin-1 mRNA expression for at least 60 days in vivo. CONCLUSION: The marked expression of sentrin in rheumatoid synovial tissue, but not in normal or OA synovial tissue, may contribute to the modulation of Fas- and TNFR-mediated apoptosis in RA synovium, and thereby extend the lifespan of invasive, cartilage-destructive SF. PMID- 10728755 TI - Induction of tumor necrosis factor alpha production by adhered human monocytes: a key role for Fcgamma receptor type IIIa in rheumatoid arthritis. AB - OBJECTIVE: Small IgG rheumatoid factor immune complexes may provide the trigger for macrophage-derived tumor necrosis factor alpha (TNFalpha) production in rheumatoid arthritis. Immune complexes may bind to any of 3 IgG Fc receptors (FcgammaR). Therefore, the ability of monocyte-derived macrophages to produce TNFalpha was examined following ligation of each of the 3 human FcgammaR, using murine monoclonal antibodies (mAb) to each receptor as a model for small immune complexes. METHODS: Adhered human monocytes expressing all 3 FcgammaR were incubated with murine anti-FcgammaR mAb directed against FcgammaRI, FcgammaRII, or FcgammaRIII. Supernatants were collected at various time points and tested for the presence of TNFalpha and interleukin-1alpha (IL-1alpha) by enzyme-linked immunosorbent assay. RESULTS: The anti-FcgammaRIII mAb induced adhered human monocytes to release TNFalpha. However, F(ab)2 and Fab fragments of the anti FcgammaRIII mAb failed to induce TNFalpha production. TNFalpha was undetectable following incubation with the anti-FcgammaRI or anti-FcgammaRII mAb. Furthermore, blocking FcgammaRI or FcgammaRII had no effect on the levels of TNFalpha released in response to the anti-FcgammaRIII mAb. Of the 3 anti-FcgammaR mAb, only anti FcgammaRIII induced IL-1alpha production from adhered human monocytes, and this was inhibited by the presence of a neutralizing anti-TNFalpha mAb. CONCLUSION: This study suggests a dominant role for FcgammaRIIIA in the induction of both TNFalpha and IL-1alpha production by human macrophages in rheumatoid arthritis following receptor ligation by small immune complexes. The signaling of TNFalpha production may require the ligation of either 3 FcgammaRIIIA receptors or only 2 FcgammaRIIIA receptors, where one interaction must involve binding via an Fc domain. In addition, IL-1alpha production following FcgammaRIIIA ligation appears to be dependent on the presence of TNFalpha. PMID- 10728756 TI - Reduced expression of the regulatory CD4+ T cell subset is related to Th1/Th2 balance and disease severity in rheumatoid arthritis. AB - OBJECTIVE: To elucidate the involvement of the regulatory CD4+ T cells that produce high levels of interleukin-10 (IL-10) and low levels of IL-4 and IL-2 in the pathogenesis of rheumatoid arthritis (RA), we investigated whether the frequency of this type of CD4+ T cell subset in peripheral blood lymphocytes (PBL) or synovial lymphocyte infiltrates of patients with RA correlated with disease severity and histologic features in rheumatoid synovium. METHODS: PBL and synovial lymphocyte infiltrates were isolated from peripheral blood samples and synovial tissues obtained from 25 patients with RA. Control specimens were obtained from 18 patients with osteoarthritis (OA) and 10 patients with traumatic injuries of the knee joint. CD4+ T cell subsets were categorized as Th1 (production of interferon-gamma [IFNgamma], but not IL-4), Th2 (production of IL 4, but not IFNgamma), or CD4+ T cell subsets producing IL-10, IL-2, or IL-4. The percentages of these T helper subsets among PBL and among synovial infiltrating lymphocytes were determined by an intracellular staining assay with flow cytometric analysis. RESULTS: The level of expression of CD4+ T cells producing IL-10 but not IL-2 and IL-4 in the peripheral blood and synovial tissue was significantly lower in RA patients than in OA patients and trauma patients. In RA patients, the frequency of this type of CD4+ T cell subset among synovial infiltrating CD4+ T cells was inversely correlated with the frequency of Th1 cells and the Th1/Th2 balance in synovial lymphocytes, serum C-reactive protein value, disease activity score, and the degree of synovial lining hyperplasia and lymphocyte infiltration in rheumatoid synovium. There was a reciprocal relationship between the frequency of Thl cells and CD4+ T cells producing IL-10 but not IL-2 and IL-4 in the peripheral blood of RA patients. CONCLUSION: In RA, reduced expression of the CD4+ T cell subset producing IL-10 but not IL-2 and IL 4 may be responsible for the dominance of Th1 over Th2 cells at sites of inflamed synovium and in the peripheral blood. Decreases in this type of CD4+ T cell subset may induce the down-regulation of T cell tolerance and exacerbate the inflammatory process in RA. PMID- 10728757 TI - Myeloid-related proteins 8 and 14 are specifically secreted during interaction of phagocytes and activated endothelium and are useful markers for monitoring disease activity in pauciarticular-onset juvenile rheumatoid arthritis. AB - OBJECTIVE: To analyze which physiologic stimuli induce secretion of myeloid related protein 8 (MRP8) and MRP14, two S100 proteins expressed in neutrophils and monocytes, and to determine whether serum concentrations of these proteins are reliable parameters for monitoring inflammatory activity in pauciarticular juvenile rheumatoid arthritis (JRA). METHODS: Secretion of MRP8 and MRP14 was analyzed using a coculture system of endothelial cells and monocytes. Concentrations of MRP8/MRP14 in the serum and synovial fluid of JRA patients or culture medium were determined by enzyme-linked immunosorbent assay. The expression of MRP8 and MRP14 by leukocytes in synovial tissue or fluid was investigated using immunohistochemistry. RESULTS: MRP8 and MRP14 were specifically released during interaction of activated monocytes with tumor necrosis factor-stimulated endothelial cells. Secretion was mediated via an increase in intracellular calcium levels in monocytes. In contrast, contact with resting endothelium inhibited protein kinase C-induced secretion of the proteins by monocytes. In JRA patients, MRP8 and MRP14 were strongly expressed in infiltrating neutrophils and monocytes within the inflamed joints and could be found in significantly higher concentrations in synovial fluid (mean 42,800 ng/ml) compared with serum (2,060 ng/ml). Concentrations of MRP8/MRP14 in serum correlated well with those in synovial fluid (r = 0.78) and showed a strong correlation with disease activity (r = 0.62). After intraarticular triamcinolone therapy, the serum concentrations of MRP8/MRP14 decreased significantly in therapy responders, whereas no differences were found in patients who showed no clinical benefit. CONCLUSION: MRP8 and MRP14 are specifically released during the interaction of monocytes with inflammatory activated endothelium, probably at sites of local inflammation. Their serum concentrations represent a useful marker for monitoring local inflammation in JRA. PMID- 10728758 TI - A comparative study into the mechanisms of action of anti-tumor necrosis factor alpha, anti-CD4, and combined anti-tumor necrosis factor alpha/anti-CD4 treatment in early collagen-induced arthritis. AB - OBJECTIVE: Anti-tumor necrosis factor alpha (anti-TNFalpha) therapy is very effective in rheumatoid arthritis (RA), whereas depleting anti-CD4 therapy is relatively ineffective. To explain the differences in efficacy between these 2 therapies, we used an animal model of RA to compare their effects on different aspects of the disease process. METHODS: Mice with collagen-induced arthritis were treated with depleting anti-CD4 monoclonal antibodies (mAb), anti-TNFalpha mAb, or phosphate buffered saline. Another group was given a combination of anti TNFalpha plus anti-CD4. The treatments were compared for their ability to down regulate the expression of proinflammatory cytokines and adhesion molecules, reduce the cellularity of the joint, and inhibit Th1 activity. RESULTS: Anti TNFalpha significantly reduced the numbers of cells expressing TNFalpha, interleukin-1beta (IL-1beta), very late activation antigen 4 (VLA-4), vascular cell adhesion molecule 1 (VCAM-1), and numbers of CD4+ T cells and macrophages in the joint. Anti-CD4 treatment led to a small reduction in the expression of TNFalpha, IL-1beta, VLA-4, and VCAM-1, but this did not reach statistical significance. Depleting anti-CD4 was also surprisingly ineffective in eliminating CD4+ T cells from the joint. Anti-TNFalpha therapy was also more effective than anti-CD4 in reducing Thl activity, as assessed by the production of interferon gamma in lymph node cell cultures. There was a synergistic relationship between anti-TNFalpha and anti-CD4 in the reduction of histologic score and inhibition of TNFalpha/IL-1beta expression in the joints. CONCLUSION: The efficacy of the 3 treatments correlated with their ability to modulate the expression of inflammatory cytokines and adhesion molecules in the joint, reduce the cellularity of the joint, and inhibit Th1 activity. This kind of analysis may prove useful in the testing of novel therapies for RA. PMID- 10728759 TI - Induction of tolerance with intranasal administration of human cartilage gp-39 in DBA/1 mice: amelioration of clinical, histologic, and radiologic signs of type II collagen-induced arthritis. AB - OBJECTIVE: Human cartilage glycoprotein 39 (HC gp-39) was recently identified as a candidate autoantigen in the pathogenesis of rheumatoid arthritis. In the present studies, we investigated the capacity of HC gp-39 to interfere in clinical disease induced by an unrelated autoantigen, type II collagen (CII), by the induction of cross-tolerance. METHODS: DBA-1j/Bom mice were immunized with bovine CII/complete Freund's adjuvant and were given intraperitoneal booster injections of CII on day 21. Tolerance was induced via the intranasal pathway with either the disease-inducing antigen (CII), a control antigen (ovalbumin), or HC gp-39 either before priming with CII or near the day of the booster injection. Arthritis was monitored visually, and joint pathology was examined histologically and radiologically. In addition, CII antibody levels in serum were analyzed by enzyme-linked immunosorbent assay. RESULTS: In contrast to treatment before priming, intranasal application of HC gp-39 after immunization markedly suppressed disease activity and prevented joint destruction, whereas application of ovalbumin or CII was ineffective. Interference of HC gp-39 with the immune response to CII was demonstrated by decreased anti-CII antibody levels. The combined data indicate that intranasal treatment with HC gp-39 may trigger modulatory or regulatory mechanisms that interfere with the expression of disease in murine collagen-induced arthritis. CONCLUSION: HC gp-39 is the first cross tolerance-inducing protein in arthritis that down-modulates a spectrum of disease features when given in a semitherapeutic protocol. PMID- 10728760 TI - Reversal of the antiinflammatory effects of methotrexate by the nonselective adenosine receptor antagonists theophylline and caffeine: evidence that the antiinflammatory effects of methotrexate are mediated via multiple adenosine receptors in rat adjuvant arthritis. AB - OBJECTIVE: Weekly low-dose methotrexate (MTX) remains the mainstay of second-line therapy for rheumatoid arthritis (RA). We have previously reported that adenosine, acting at specific receptors on inflammatory cells, mediates the antiinflammatory effects of MTX in both in vitro and in vivo models of acute inflammation, but the mechanism by which MTX suppresses the chronic inflammation of arthritis remains controversial. The present study was undertaken to further investigate the means by which adenosine mediates the antiinflammatory effects of MTX. METHODS: The effects of 2 nonselective adenosine receptor antagonists, theophylline and caffeine, were examined, using the rat adjuvant arthritis model of RA. These agents were given alone and in conjunction with MTX, and arthritis severity was assessed clinically, radiologically, and histologically. Since rodent adenosine A3 receptors are not blocked by theophylline, selective A1, A2A, and A2B receptor antagonists were tested as well. RESULTS: Control animals developed severe arthritis, which was markedly attenuated by weekly treatment with MTX (0.75 mg/kg/week). Neither theophylline alone nor caffeine alone (each at 10 mg/kg/day) significantly affected the severity of the arthritis, but both agents markedly reversed the effect of MTX as measured by a severity index, hindpaw swelling, and hindpaw ankylosis. Radiographic and histologic analyses confirmed these observations. Neither A1, A2A, nor A2B receptor antagonists affected the capacity of MTX to ameliorate inflammation in adjuvant arthritis. CONCLUSION: These results provide strong evidence that adenosine mediates the antiinflammatory effects of MTX in this model of RA. Moreover, the findings suggest that abstinence from caffeine, a ubiquitous food additive and medication, may enhance the therapeutic effects of MTX in RA. PMID- 10728761 TI - Comparison of the degradation of type II collagen and proteoglycan in nasal and articular cartilages induced by interleukin-1 and the selective inhibition of type II collagen cleavage by collagenase. AB - OBJECTIVE: To compare interleukin-1alpha (IL-1alpha)-induced degradation of nasal and articular cartilages in terms of proteoglycan loss and type II collagen cleavage, denaturation, and release; to examine the temporal relationship of these changes; and to investigate the effects of an inhibitor of collagenase 2 and collagenase 3 on these catabolic processes. METHODS: Discs of mature bovine nasal and articular cartilages were cultured with or without human IL-1alpha (5 ng/ml) with or without RS102,481, a selective synthetic inhibitor of collagenase 2 and collagenase 3 (matrix metalloproteinase 8 [MMP-8] and MMP-13, respectively) but not of collagenase 1 (MMP-1). Immunoassays were used to measure collagenase generated type II collagen cleavage neoepitope (antibody COL2-3/4C(short)) and denaturation (antibody COL2-3/4m), as well as total type II collagen content (antibody COL2-3/4m) in articular cartilage and culture media. A colorimetric assay was used to measure total proteoglycan concentration (principally of aggrecan) as sulfated glycosaminoglycans (sGAG). RESULTS: IL-1alpha initially induced a decrease in tissue proteoglycan content in nasal cartilage. A progressive loss of proteoglycan was noted during culture in articular cartilages, irrespective of the presence of IL-1alpha. In both cartilages, proteoglycan loss was followed by IL-1alpha-induced cleavage of type II collagen by collagenase, which was often reflected by increased denaturation. The inhibitor RS102,481 had no clear effect on the reduction in proteoglycan content (measured by sGAG) and collagen denaturation in either cartilage, but at 10 nM it inhibited the enhanced cleavage of type II collagen, partially in nasal cartilage and completely in articular cartilage. CONCLUSION: IL-1alpha-induced cleavage and denaturation of type II collagen is observed in both hyaline cartilages and is secondary to proteoglycan loss. It probably involves different collagenases, since there is no evidence of a rate-limiting role for collagenase 1 in articular cartilage, unlike the case for nasal cartilage. Inhibitors of this kind may be of value in the treatment of cartilage damage in arthritis. Also, the ability to detect the release of type II collagen collagenase-generated fragments from degraded cartilage offers the potential to monitor cartilage collagen damage and its control in vivo. PMID- 10728762 TI - Selective enhancement of collagenase-mediated cleavage of resident type II collagen in cultured osteoarthritic cartilage and arrest with a synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1). AB - OBJECTIVE: To examine whether type II collagen cleavage by collagenase and loss of proteoglycan are excessive in human osteoarthritic (OA) articular cartilage compared with nonarthritic articular cartilage, and whether this can be inhibited by a selective synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1 [MMP-1]). METHODS: Articular cartilage samples were obtained during surgery from 11 patients with OA and at autopsy from 5 adults without arthritis. The articular cartilage samples were cultured in serum-free medium. A collagenase-generated neoepitope, which reflects cleavage of type II collagen, and proteoglycan glycosaminoglycan (GAG), which predominantly reflects aggrecan release, were assayed in culture media. In addition, cultures were performed using either of 2 synthetic MMP inhibitors, both of which inhibited collagenase 2 (MMP-8) and collagenase 3 (MMP-13), but one of which spared collagenase 1. Cultures were also biolabeled with 3H-proline in the presence and absence of these inhibitors to measure collagen synthesis (as tritiated hydroxyproline) and incorporation in articular cartilage. RESULTS: As a group, cleavage of type II collagen by collagenase was significantly increased in OA cartilage samples. In contrast, proteoglycan (GAG) release was not increased. This release of a collagenase-generated epitope was inhibited by both MMP inhibitors in 2 of 5 nonarthritic samples and in 9 of 11 OA cartilage samples. The inhibitor that spared collagenase 1 was generally more effective and inhibited release from 4 of 5 nonarthritic cartilage samples and the same OA cartilage samples. Group analyses revealed that the inhibition of collagenase neoepitope release by both inhibitors was significant in the OA patient cartilage, but not in the nonarthritic cartilage. Proteoglycan loss was unaffected by either inhibitor. Newly synthesized collagen (predominantly, type II) exhibited increased incorporation in OA cartilage, but only in the presence of the inhibitor that arrested collagenase 1 activity. CONCLUSION: These results further indicate that the digestion of type II collagen by collagenase is selectively increased in OA cartilage, and that this can be inhibited in the majority of cases by a synthetic inhibitor that can inhibit collagenases 2 and 3, but not collagenase 1. The results also suggest that in OA, newly synthesized collagen is digested, but in a different manner than that of resident molecules. Proteoglycan release was not increased in OA cartilage and was unaffected by these inhibitors. Inhibitors of this kind may be of value in preventing damage to type II collagen in human arthritic articular cartilage. PMID- 10728763 TI - Association of antiphosphatidylserine/prothrombin autoantibodies with HLA class II genes. AB - OBJECTIVE: To investigate the associations between HLA class II genes and antiphosphatidylserine/prothrombin antibodies (aPS/PT) in a group of British caucasoid patients with antiphospholipid antibodies (aPL). METHODS: This study included 82 patients with aPL. IgG aPS/PT were detected in sera using enzyme linked immunosorbent assays. HLA-DQB1, DQA1, and DRB1 genotypes were determined by polymerase chain reaction using sequence-specific primers. All results were compared with 177 matched healthy control subjects. RESULTS: IgG aPS/PT were present in 41 of 82 patients (50%). The frequencies of DQB1*0301/4, DQB1*0604/5/6/7/9, and DRB1*1302 alleles were increased in patients with aPS/PT compared with controls. To minimize the interference of the association between anti-beta2-glycoprotein I (anti-beta2GPI) and HLA, patients with anti-beta2GPI were excluded from further analyses, and only HLA-DQB1*0301/4 remained significant compared with controls (odds ratio [OR] 2.75, 95% confidence interval [95% CI] 1.2-6.5, P < 0.03). In the haplotype analysis, HLA-DQB1*0301/4;DQA1* 0301/2;DRB1*04 was significantly increased in patients with IgG aPS/PT compared with controls (OR 4.75, 95% CI 1.72-13.10, P = 0.0063). CONCLUSION: The HLA DQB1*0301/4;DQA1*0301/ 2;DRB1*04 haplotype and its components may influence the production of aPS/PT in the antiphospholipid syndrome, which partly explains the correlation between the lupus anticoagulant and DQB1*03. PMID- 10728764 TI - Quantitative radioligand assays using de novo-synthesized recombinant autoantigens in connective tissue diseases: new tools to approach the pathogenic significance of anti-RNP antibodies in rheumatic diseases. AB - OBJECTIVE: To describe new assays for the detection and quantification of antibodies to RNPs in rheumatic diseases, using soluble nuclear antigens synthesized de novo in reticulocyte lysates. METHODS: Sera from 381 patients with various rheumatic diseases, including 212 patients with systemic lupus erythematosus (SLE), were analyzed in order to evaluate the sensitivity and specificity of serum autoantibody reactivities to several recombinant soluble autoantigens: U1-A RNP, Sm-B, SSA/Ro 52 and SSA/Ro 60, SSB/La, and Ku. Radioligand assays (RLAs) were performed following the in vitro transcription and translation of each autoantigen from the corresponding complementary DNA, labeled with 35S-methionine. The radiolabeled protein was then bound by the specific serum autoantibody, forming immune complexes that were captured by protein A Sepharose beads and quantified by counting the radioactivity. RESULTS: Among the SLE patients, 44% were positive for anti-U1-A RNP activity, 34% for anti-Sm-B, 44% for anti-SSA (32% for Ro 52 and 46% for Ro 60), 32% for anti-SSB/La, and 11% for anti-Ku reactivities. SSA antibodies had a high frequency in patients with mixed connective tissue disease (MCTD) (80%); 65% of these patient sera reacted with Ro 52, 45% with Ro 60, and 45% with U1-A RNP. Twenty percent of the MCTD patients also exhibited antibodies to Sm-B and Ku. In patients with Sjogren's syndrome, anti-SSA was the main anti-RNP antibody (63%), together with SSB/La antibodies (44%). Among patients with inflammatory myopathy, only antibodies against Ro 52 (36%) and Ro 60 (36%) were present. These new RLA allowed observation of a strong correlation (P < 0.0001) between Sm-B antibody levels and the severity of SLE (as measured by the SLE Disease Activity Index), and establishment of a correlation between anti-U1-A RNP antibodies and the occurrence of SLE nephritis (P < 0.02). All RLAs were highly specific for the antigen tested and displayed, in the disease groups studied, a higher sensitivity than conventional immunodiffusion assays. CONCLUSION: These highly sensitive, specific, and quantitative RLAs represent new tools for the detection of autoantibodies to RNP antigens in rheumatic diseases, and may be useful for (differential) diagnosis in clinical practice. PMID- 10728765 TI - Clinical Images: High-resolution computed tomography in the diagnosis and management of pulmonary Wegener's granulomatosis in a patient with normal chest radiography findings. PMID- 10728766 TI - Pulmonary edema complicating prostacyclin therapy in pulmonary hypertension associated with scleroderma: a case of pulmonary capillary hemangiomatosis. AB - Continuous intravenous infusion of prostacyclin is an effective treatment for primary pulmonary hypertension (PPH), and has recently been shown to be of benefit in PH associated with scleroderma (SSc). Pulmonary capillary hemangiomatosis (PCH) is a rare cause of PPH. Prostacyclin therapy has been complicated by pulmonary edema in cases of PCH. We describe a case of PH associated with limited SSc, where treatment with prostacyclin was complicated by pulmonary edema. Autopsy revealed PCH as the pathologic basis for the PH. There were no clinical features of PCH prior to initiation of vasodilator therapy, illustrating the potential difficulty in establishing the diagnosis. This is the first reported case of PCH in SSc. PMID- 10728767 TI - Mixed connective tissue disease presenting with pneumonitis and pneumatosis intestinalis. PMID- 10728768 TI - Clinical Image : Carpal tunnel syndrome from accessory lumbrical muscles. PMID- 10728769 TI - Fibromyalgia in Frida Kahlo's life and art. PMID- 10728770 TI - Clinical Images: Tuberculous arthritis. PMID- 10728771 TI - Bone mineral density and juvenile chronic arthritis: comment on the article by Zak et al. PMID- 10728772 TI - Cytokine-producing T cells in systemic lupus erythematosus: comment on the article by Akahoshi et al. PMID- 10728773 TI - Psoriatic arthritis--an enthesopathy-based disorder? Comment on the article by McGonagle et al. PMID- 10728774 TI - Clinical Images: Ehlers-Danlos syndrome presenting as olecranon bursitis. PMID- 10728775 TI - Diallyl sulfide enhances azoxymethane-induced preneoplasia in Fischer 344 rat colon. AB - Azoxymethane (AOM) is an indirect-acting colon carcinogen that produces a high incidence of precancerous lesions, referred to as aberrant crypt foci (ACF), in rats. This study was undertaken to determine whether high dose gavage administration of the cytochrome P-450 2E1 (CYP2E1) inhibitor and chemopreventive agent, diallyl sulfide, would reduce the incidence and severity of ACF formation in the distal colons of AOM-treated Fischer 344 rats. Seven-week-old male rats received 150 or 50 mg/kg diallyl sulfide by gavage 24 and 2 h prior to two weekly i.p. injections of AOM (20 mg/kg). Ten weeks after the last injection of AOM the rats were sacrificed and the colons removed and stained with 0.2% methylene blue. ACF were visualized using stereomicroscopy. Rats pretreated with diallyl sulfide exhibited a significant increase in the number of ACF/cm in the distal colon compared with rats receiving AOM alone. This increase in ACF number was seen in ACF of all sizes. To examine the effects of diallyl sulfide on the initiation stage of AOM-induced carcinogenesis, mutations in the K-ras proto-oncogene were also investigated. ACF and normal appearing colonic mucosa (0.2-0.5 mm3) were microdissected for subsequent PCR-RFLP analysis of a codon 12 (GGT-GGA) activating mutation in the K-ras gene. Greater than 90% of ACF from AOM-treated animals, regardless of diallyl sulfide treatment, exhibited activating K-ras mutations. K-ras mutations were also detected in normal appearing mucosa of AOM treated animals, although at a lesser frequency (15-35%). These studies demonstrate that diallyl sulfide given in large gavage doses enhances AOM-induced preneoplasia in rats and suggests that diallyl sulfide may alter the disposition of AOM intermediates and/or enhance colonic promotional activity in the rat. PMID- 10728776 TI - Familial dysalbuminemic byperthyroxinemia may result in altered warfarin pharmacokinetics. AB - Two distinct genotypes that result in the amino acid substitutions R218P and R218H in subdomain 2A of human serum albumin (HSA) have been identified as the cause of familial dysalbuminemic hyperthyroxinemia (FDH). These substitutions increase the affinity of subdomain 2A for thyroxine by approximately 10-fold elevating plasma thyroxine levels in affected individuals. While many studies have examined the binding of thyroxine to FDH HSA, the binding of FDH HSA to drugs has not been widely investigated. The widely administered drug warfarin was selected as a model compound to study FDH HSA/drug interactions since it binds to subdomain 2A and its pharmacokinetics are dramatically influenced by HSA binding. Using two independent methods, fluorescence spectroscopy and equilibrium dialysis with radioactive warfarin, the binding of recombinant R218P, R218H, R218M and wild type HSA to warfarin was measured. Both methods showed an approximately 5 fold decrease in the affinity of R218P, R218H and R218M HSA for warfarin relative to wild type HSA. The Kd values determined by fluorescence spectroscopy for wild type, R218H, R218P and R218M HSA binding to warfarin were 1.35, 5.38, 5.61, and 8.34 microM, respectively. The values determined by equilibrium dialysis were 5.36, 29.5, 14.5, and 23.4 microM, respectively. Based on the above findings one would expect the free serum warfarin concentration in homozygous R218P and R218H FDH patients to be elevated about 5-fold, resulting in about a 5-fold reduction in the serum half-life of the drug. PMID- 10728778 TI - Lipoxygenase-mediated hydrogen peroxide-dependent N-demethylation of N,N dimethylaniline and related compounds. AB - To date, studies of xenobiotic N-demethylation have focused on heme-proteins such as P450 and peroxidases. In this study we investigated the ability of non-heme iron proteins, namely soybean lipoxygenase (SLO) and human term placental lipoxygenase (HTPLO) to mediate N-demethylation of N,N-dimethylaniline (DMA) and related compounds in the presence of hydrogen peroxide. In addition to being hydrogen peroxide dependent, the reaction was also dependent on incubation time, concentration of enzyme and DMA and the pH of the medium. Using Nash reagent to estimate formaldehyde production, we determined the specific activity for SLO mediated N-demethylation of DMA to be 200 + 18 nmol HCHO/min per mg protein or 23 +/- 2 nmol/min per nmol of enzyme, while that of HTPLO was 33 +/- 4 nmol HCHO/min per mg protein. Nordihydroguaiaretic acid (NDGA), a classical inhibitor of lipoxygenase (LO), as well as antioxidants and free radical reducing agents, caused a marked reduction in the rate of production of formaldehyde from DMA by SLO. Besides N,N-dimethylaniline, N-methylaniline, N,N,N',N' tetramethylbenzidine, N,N-dimethyl-p-phenylenediamine, N,N-dimethyl-3 nitroaniline and N,N-dimethyl-p-toluidine were also demethylated by SLO. The formation of a DMA N-oxide was not detected. Preliminary experiments suggested SLO-mediated hydrogen peroxide-dependent S-dealkylation of methiocarb or O dealkylation of 4-nitroanisole does not occur. PMID- 10728777 TI - 2,3,7,8 Tetrachlorodibenzo-p-dioxin induction of cytochrome P4501A in cultured rat and human hepatocytes. AB - We report here a novel observation that 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) induced predominantly cytochrome P4501A1 (CYP1A1) in rat hepatocytes and predominantly CYP1A2 in human hepatocytes. As part of our research program to evaluate species-differences in response to CYP inducers, we studied the effects of TCDD on CYP1A activity, protein, and gene expression in primary cultures of rat and human hepatocytes. TCDD was found to induce CYP1A activity, measured as ethoxyresorufin-O-deethylase (EROD) activity, in both rat and human hepatocytes. TCDD induction of EROD activity in human hepatocytes (2-5 fold of concurrent solvent control), was significantly lower than that found in rat hepatocytes ( 20 fold of concurrent solvent control). Two structural analogs of TCDD, 2,3,7,8 tetrachlorodibenzofuran (TCDF) and 6-nitro-1,3,8-trichlorodibenzofuran (6-NCDF), were also evaluated. As observed for TCDD, human hepatocytes consistently showed a lower response than rat hepatocytes. As most TCDD-related effects are believed to be mediated via binding of the TCDD-Ah receptor (AhR) complex to DNA, nuclear AhR levels were measured in rat and human hepatocytes after TCDD treatment. We found that the nuclear AhR levels in TCDD-treated rat hepatocytes were approximately 4 times higher than found in TCDD-treated human hepatocytes. However, the estimated binding affinity of [3H]TCDD to nuclear AhR from rat hepatocytes was similar. The species difference in response to TCDD was further evaluated by analysis of CYP1A1 and CYP1A2 mRNA levels using Northern analysis, and P4501A1 and 1A2 protein levels using Western immunoblotting. Results showed that, at both gene expression and protein levels, TCDD induced predominantly CYP1A1 in rat hepatocytes and CYP1A2 in human hepatocytes. PMID- 10728779 TI - Estrous cycle-dependent changes in the expression of aromatic hydrocarbon receptor (AHR) and AHR-nuclear translocator (ARNT) mRNAs in the rat ovary and liver. AB - The aromatic hydrocarbon receptor (AHR) and AHR nuclear translocator protein (ARNT) mediate the toxic effects of a wide variety of halogenated and polycyclic aromatic hydrocarbons. While it can be assumed that AHR has an endogenous function, its role in reproduction is currently undefined. The present study seeks to examine the regulation of AHR and ARNT mRNAs in liver and ovarian tissues across the rat estrous cycle. Message for hepatic AHR was increased significantly on the morning of proestrus, and decreased dramatically by the evening of proestrus; while hepatic ARNT mRNA was significantly decreased between diestrus and the morning of proestrus, and between the evening of proestrus and the morning of estrus. Ovarian AHR mRNA was unchanged from diestrus to proestrus, and was decreased on the evening of proestrus. Changes in the expression of ARNT mRNA mirrored changes in the liver. To assess interaction between the AHR- and estrogen-receptor (ER)-signaling pathways and to test the hypothesis that estrogen regulates AHR mRNA, 25-day-old female rats were injected with either 17beta-estradiol, the ER antagonist ICI 182 780, or with vehicle, and hepatic AHR mRNA was measured. Treatment with estrogen or the estrogen antagonist did not alter the abundance of AHR mRNA in the liver. These data suggest that while estrogen may not be the key regulator of AHR mRNA expression, a factor associated with the rat reproductive cycle may be important in regulating the expression of both the AHR and ARNT genes in the ovary and liver. PMID- 10728780 TI - Excretion of copper complexed with thiomolybdate into the bile and blood in LEC rats. AB - Copper (Cu) accumulating in a form bound to metallothionein (MT) in the liver of Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease, was removed with ammonium tetrathiomolybdate (TTM), and the fate of the Cu complexed with TTM and mobilized from the liver was determined. TTM was injected intravenously as a single dose of 2, 10 or 50 mg TTM/kg body weight into LEC and Wistar (normal Cu metabolism) rats, and then the concentrations of Cu and molybdenum (Mo) in the bile and plasma were monitored with time after the injection. In Wistar rats, most of the Mo was excreted into the urine, only a small quantity being excreted into the bile, while Cu excreted into the urine decreased. However, in LEC rats, Cu and Mo were excreted into the bile and blood, and the bile is recognized for the first time as the major route of excretion. The Cu excreted into both the bile and plasma was accompanied by an equimolar amount of Mo. The relative ratio of the amounts of Cu excreted into the bile and plasma was 40/60 for the low and high dose groups, and 70/30 for the medium dose group. The systemic dispositions of the Cu mobilized from the liver and the Mo complexed with the Cu were also determined for the kidneys, spleen and brain together with their urinal excretion. Although Mo in the three organs and Cu in the kidneys and spleen were increased or showed a tendency to increase, Cu in the brain was not increased at all doses of TTM. PMID- 10728781 TI - 'Know thy cell!' A comment on the UKCCCR guidelines for the use of cell lines. U.K. Co-ordinating Committee on Cancer Research. PMID- 10728782 TI - Interaction between protein kinase C and sphingomyelin/cholesterol. AB - Physical characteristics of binding of protein kinase C with sphingomyelin/cholesterol lipid bilayers were analysed using three complementary approaches: acrylodan fluorescence, fluorescence energy transfer and quenching of tryptophan fluorescence. It was demonstrated that sphingomyelin/cholesterol lipid membranes were available for protein kinase C binding. The intensity of the binding was dependent on the sphingomyelin content. The results of quenching of intrinsic tryptophan fluorescence showed that the enzyme molecule penetrated the sphingomyelin/cholesterol lipid bilayer to the C-16 position of labeled fatty acid probes. Our results also showed sphingomyelin itself restrains protein kinase C activity. A possible explanation for our results is that caveolae function as signaling storage devices. PMID- 10728783 TI - Transcriptional activation of heme oxygenase-1 gene in mouse spleen, liver and kidney cells after treatment with lipopolysaccharide or hemoglobin. AB - Heme oxygenase (HO)-1 catalyzes the conversion of heme to biliverdin, iron and carbon monoxide. HO-1 is induced by many reagents including heme, Hb and lipopolysaccharide (LPS). LPS is known to activate the HO-1 gene in cultured mouse liver and macrophage cells through oxidative activation of NF-kappaB. But little is known about the effect of LPS and Hb on the HO-1 gene in living organisms. To study this issue, we examined the HO-1 and its mRNA levels in mouse liver, spleen and kidney after intravenous administration of LPS and Hb. On LPS treatment, the amount of HO-1 and its mRNA increased markedly mainly in mouse spleen, but on Hb treatment the amounts of HO-1 and its mRNA increased slightly only in liver. Run-off transcription assay supported the above results and band shift assays also revealed that LPS significantly activates an NF-kappaB-like factor in spleen cells, while Hb slightly activates it in liver cells. According to our previous study, a small amount of Hb injected to mouse is selectively taken up by liver as Hb-haptoglobin complex. These results suggest different pathways for the HO gene activation in mouse organs; one by LPS in spleen cells and the other by Hb in liver cells. PMID- 10728784 TI - Epidermal growth factor receptor (EGF-R) localization in the apical membrane of the enterocytes of rat duodenum. AB - The maintenance of gastrointestinal epithelium integrity requires a fine balance between proliferation and differentiation as well as protection against gastric acid secretion. Some growth factors, such as epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha), bind to epidermal growth factor receptor (EGF-R) to exert these functions. The exact location of EGF-R within the duodenal area is controversial and still not completely known. Immunohistochemical and Western blot techniques using a monoclonal anti-EGF-R antibody were performed on the adult rat duodenum. The duodenal enterocytes expressed EGF-R in the apical membrane and in the supranuclear area along the length of the villuos. The Lieberkhum crypts and Brunner's glands also showed a positive immunostaining. By Western blot analysis we identified in the duodenal scrape a band with an apparent molecular weight of 175 kDa. Our results suggest a functional role for the luminal EGF and/or TGF-alpha in the establishment and maintenance of the epithelial renewal, probably by stimulation of cell proliferation, differentiation and migration. PMID- 10728786 TI - D,L-alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, induces differentiation in MEL cells. AB - In the present study, the effect of D,L-alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), on Friend's murine erythroleukemia (MEL) cell differentiation is investigated. DFMO was able to induce differentiation of MEL cells in culture as determined by haemoglobin (Hb) content and percentage of cells synthesizing Hb detected by benzidine staining. DFMO at a concentration of 2 mM resulted in about 70% benzidine-positive cells on the fifth day. There was a time-dependent increase in the percentage of benzidine positive cells starting from day three. However, only a 24 h presence of DFMO in the medium was required to induce differentiation suggesting that DFMO switches on a pathway during this period leading to terminal differentiation of MEL cells. DFMO induced differentiation of MEL cells was sensitive to dexamethasone and 5 bromo-2'-deoxyuridine. PMID- 10728785 TI - Effects of 1alpha,25-dihydroxycholecalciferol and cortisol on the growth and differentiation of primary cultures of mouse mammary epithelial cells in collagen gel. AB - We examined the effects of 1alpha,25-dihydroxycholecalciferol (1,25-DHCC) and the glucocorticoid, cortisol, on primary mouse mammary epithelial cells in collagen gel cell culture systems. Physiological low concentrations (10(-11)-10(-9) M) of 1,25-DHCC stimulated growth of the cells in a collagen gel matrix culture in serum-free DMEM+Ham's F12 (1:1) medium containing BSA, EGF and cholera toxin, and the cell number reached 1.8-fold the control after 6 d in culture. In contrast, supraphysiological concentrations (10(-8)-10(-7) M) of 1,25-DHCC suppressed cell growth. Cortisol produced similar, but smaller, dose-dependent effects. The addition of serum to the culture medium masked the stimulatory effect of 1,25 DHCC and both the stimulatory and inhibitory effects of cortisol. 1,25-DHCC also affected casein synthesis by cells cultured in a serum-free floating collagen gel culture containing prolactin, insulin and cortisol, enhancing synthesis at low concentrations (10(-11)-10(-9) M) and inhibiting it above 10(-8) M. In the absence of cortisol, no detectable change in casein synthesis was induced by 1,25 DHCC. These results suggest a physiological role for 1,25-DHCC in stimulating both growth and differentiation of mouse mammary epithelial cells, though 1,25 DHCC does not substitute for glucocorticoids in the differentiation of the cells. PMID- 10728787 TI - Changes in plasma membrane fluidity of immortal rodent cells induced by anticancer drugs doxorubicin, aclarubicin and mitoxantrone. AB - The aim of this study was to examine the effect of three structurally different anticancer drugs-the pro-oxidative anthracyclines doxorubicin (DOX) and aclarubicin (ACL), and antioxidative anthraquinone mitoxantrone (MTX) on the fluidity of plasma membrane of immortalized rodent fibroblasts using fluorescence spectroscopy and electron spin resonance (ESR) techniques. Two kinds of fluorescent probes (TMA-DPH and 12-AS) and spin labels (5-DS and methyl-12-DS) were used to monitor fluidity in the hydrophobic core and in the polar headgroup region of the lipid bilayer. Immortalized hamster B14 and NIH 3T3 mouse fibroblasts were exposed to DOX, ACL and MTX. We demonstrate that these drugs influence predominantly the hydrophobic core of the lipid bilayer, inducing significant decrease in its fluidity at low concentrations (2-5 microM). A decreased membrane fluidity at the surface of the lipid bilayer was observed only at a higher concentration (20 microM) of the drugs, which indicates that DOX, ACL and MTX intercalate mainly into the hydrophobic core of the membrane, thereby perturbing its structure. PMID- 10728788 TI - Reorganization of myosin and focal adhesion proteins in Swiss 3T3 fibroblasts induced by transforming growth factor beta. AB - Certain types of cells show a dramatic change in cell morphology cultured in the presence of transforming growth factor beta (TGF-beta). To identify cellular components or factors leading to morphological changes, we investigated if any members of cytoskeletal proteins and cell-adhesion molecules were redistributed in TGF-beta-treated Swiss 3T3 fibroblasts by indirect immunofluorescence and Western-blot analysis. Changes in cell morphology became apparent within 12 h of the addition of TGF-beta and new RNA and protein synthesis was necessitated by the changes. While TGF-beta induced reorganization of microfilaments as reported in earlier studies, one of the actin isoforms, alpha actin of smooth muscle, was induced to form stress fibers in Swiss 3T3 cells. It was observed that myosin light chain was relocated from cell periphery to cytoplasmic filamentous structures by TGF-beta treatment, with an increased amount. In addition, the cell shape change was accompanied by an increase in the level of vinculin and tyrosine phosphorylation at focal adhesions. These results suggest that new protein synthesis is required for the cell-shape change, and acto-myosin filaments and focal adhesion proteins are involved in the alteration of cell morphology induced by TGF-beta in Swiss 3T3 fibroblasts. PMID- 10728789 TI - Modulation of A2A adenosine receptor(s) by K+(ATP) channels in bovine brain striatal membranes. AB - The modulation of adenosine receptor with K+(ATP) channel blocker, glibenclamide, was investigated using the radiolabeled A2A-receptor selective agonist [3H]CGS 21680. Radioligand binding studies in bovine brain striatal membranes (BBM) indicated that unlabeled CGS 21680 displaced the bound [3H]CGS 21680 in a concentration-dependent manner with a maximum displacement being approximately 65% at 10(-4) M. In the presence of 10(-5) M glibenclamide, unlabeled CGS 21680 increased the displacement of bound [3H]CGS 21860 by approximately 28% at 10(-4) M. [3H]CGS 21680 bound to BBM in a saturable manner to a single binding site (Kd = 10.6+/-1.71 nM; Bmax = 221.4+/-6.43 fmol/mg of protein). In contrast, [3H]CGS 21680 showed saturable binding to two sites in the presence of 10(-5) M glibenclamide; (Kd = 1.3+/-0.22 nM; Bmax = 74.3+/-2.14 fmol/mg protein; and Kd = 8.9+/-0.64 nM; Bmax = 243.2+/-5.71 fmol/mg protein), indicating modulation of adenosine A2A receptors by glibenclamide. These studies suggest that the K+(ATP) channel blocker, glibenclamide, modulated the adenosine A2A receptor in such a manner that [3H]CGS 21680 alone recognizes a single affinity adenosine receptor, but that the interactions between K+(ATP) channels and adenosine receptors. PMID- 10728790 TI - Hepatobiliary transport. AB - The alterations of hepatobiliary transport that occur in cholestasis can be divided into primary defects, such as mutations of transporter genes or acquired dysfunctions of transport systems that cause defective canalicular or cholangiocellular secretion, and secondary defects, which result from biliary obstruction. The dysfunction of distinct biliary transport systems as a primary cause of cholestasis is exemplified by the genetic defects in progressive familial intrahepatic cholestasis or by the direct inhibition of transporter gene expression by cytokines. In both, the hepatocellular accumulation of toxic cholephilic compounds causes multiple alterations of hepatocellular transporter expression. In addition, lack of specific components of bile caused by a defective transporter, as in the case of mdr2/MDR3 deficiency, unmasks the toxic potential of other components. The production of bile is critically dependent upon the coordinated regulation and function of sinusoidal and canalicular transporters, for instance of Na+-taurocholate cotransporting polypeptide (NTCP) and bile salt export pump (BSEP). Whereas the downregulation of the unidirectional sinusoidal uptake system NTCP protects the hepatocyte from further intracellular accumulation of bile salts, the relative preservation of canalicular BSEP expression serves to uphold bile salt secretion, even in complete biliary obstruction. Conversely, the strong downregulation of canalicular MRP2 (MRP, multidrug resistance protein) in cholestasis forces the hepatocyte to upregulate basolateral efflux systems such as MRP3 and MRP1, indicating an inverse regulation of basolateral and apical transporters The regulation of hepatocellular transporters in cholestasis adheres to the law of parsimony, since many of the cellular mechanisms are pivotally governed by the effect of bile salts. The discovery that bile salts are the natural ligand of the farnesoid X receptor has shown us how the major bile component is able to regulate its own enterohepatic circulation by affecting transcription of the genes critically involved in transport and metabolism. PMID- 10728791 TI - Liver regeneration. AB - The liver can precisely regulate its growth and mass. Surgical resection of hepatic lobes or hepatocyte loss caused by viral or chemical injury triggers hepatocyte replication while enlarged liver mass is corrected by apoptosis. Hepatocytes have a great replicative capacity and are capable of repopulating the liver. However, "stem-like" cells proliferate when hepatocyte replication is blocked or delayed. Detailed studies of the mechanisms that regulate liver growth have been done in animals subjected to partial hepatectomy or chemical injury. Substantial progress has been achieved using appropriate transgenic and knockout mouse models for this work. Gene expression in the regenerating liver can be divided into several phases, starting with expression of a large number of immediate early genes. Hepatocytes need to be primed before they can fully respond to the growth factors HGF (Hepatocyte Growth Factor), TGFalpha (Transforming Growth Factor Alpha), and EGF (Epidermal Growth Factor) in vitro. Priming requires the cytokines TNF and IL-6 in addition to other agents that prevent cytotoxicity. Reactive Oxygen Species and glutathione content can determine whether the TNF effect on hepatocytes is proliferative or apoptotic. At least four transcription factors, NFkappaB, STAT3 (which are strongly induced by TNF), AP-1 and C/EBPbeta play major roles in the initiation of liver regeneration. In addition, extensive remodeling of the hepatic extracellular matrix occurs shortly after partial hepatectomy. Progression through the cell cycle beyond the initiation phase requires growth factors. The expression of Cyclin D1 probably establishes the stage at which replication becomes growth factor-independent and autonomous. Knowledge about the mechanisms of liver regeneration can now be applied to correct clinical problems caused by deficient liver growth. PMID- 10728792 TI - New aspects of hepatic fibrosis. AB - Hepatic stellate cells are the major source of extracellular matrix proteins in hepatic fibrosis, including Type I collagen. In response to liver injury, the hepatic stellate cells change from a quiescent to an activated phenotype. This activation process includes a phenotypic change to a myofibroblast-like cell, increased proliferation rate, loss of retinoid stores, increased production of extracellular matrix proteins, chemokines, and cytokines, and contractility. Ongoing studies are characterizing the genes that are differentially expressed in the quiescent and activated hepatic stellate cells. We have also investigated the regulation of Type I collagen expression, the cleavage of collagen propeptides, and the formation of collagen cross-links. Understanding these pathways may provide new insights into the molecular pathogenesis of hepatic fibrosis. PMID- 10728793 TI - Mechanisms of liver cell injury. AB - Liver cell death is triggered by a number of insults arising from the external environment or from within the cell. These insults may engage cell surface receptors with death domaines leading to a proteolytic cascade involving initiator and executioner caspases and an apoptotic demise. Alternatively, the insults may profoundly disrupt mitochondrial function and result in loss of homeostasis accompanied by activation of hydrolases and a necrotic or lytic demise. The distinction between apoptotic and necrotic cell death has become blurred recently by the recognition that the same stimuli can induce either form of cell death as well as caspase independent apoptosis. Mitochondria play a key role in the shape of cell death; selective release of mediators amplifies the apoptosis program and profound loss of mitochondrial function leads to necrosis. Reactive oxygen metabolites and nitric oxide participate as initiating factors and modulators. The extensive knowledge gained in recent years about the mechanisms of cell death will undoubtedly lead to new and exciting advances in the prevention and treatment of liver diseases. Important targets include death receptors, death signaling mechanisms, the mitochondrial permeability transition and approaches which selectively inhibit or activate cell death in parenchymal versus nonparenchymal cells. PMID- 10728794 TI - Haemochromatosis in the new millennium. AB - Hereditary haemochromatosis (HHC) is a common inherited disorder of iron metabolism characterised by progressive iron loading of parenchymal cells of the liver, pancreas, heart and other organs ultimately leading to cirrhosis and organ failure. Despite HLA studies which localised the defective gene to the short arm of chromosome 6, the haemochromatosis gene remained elusive until 1996, when the gene was identified by a massive positional cloning effort. The haemochromatosis gene (HFE) encodes a novel nonclassical MHC class-1-like molecule. Two missense mutations have been identified in patients with HHC, a G to A at nucleotide 845, resulting in a substitution of tyrosine for cysteine at amino acid 282 (referred to as the C282Y mutation) and a C to G at nucleotide 187, resulting in a substitution of aspartate for histidine at amino acid 63 (H63D). An average of 85 90% of patients with typical clinical features of HHC are homozygous for the C282Y mutation. H63D is not associated with the same degree of iron loading as C282Y. Clinical expression is variable depending on environmental (dietary) iron, physiological and pathological blood loss and as yet unidentified modifying genetic factors. One recent Australian study indicates that only about 50% of homozygous subjects are fully expressing and symptomatic and that about 30% show no clinical or biochemical expression. Genetic tests for identifying mutations in the HFE gene provide precise means for diagnosis, family testing and population screening and have led to re-evaluation of the indications for liver biopsy in this disease. At the present time, however, the most practical and cost-effective method of screening is for phenotypic expression by transferrin saturation or unsaturated iron binding capacity measurement. In the future, population screening by genotype should be feasible once the relevant technical, legal and ethical issues are resolved. PMID- 10728795 TI - Acute liver failure: targeted artificial and hepatocyte-based support of liver regeneration and reversal of multiorgan failure. AB - Acute liver failure (ALF) still represents a major therapeutic challenge for hepatologists due to its high mortality rate as a result of multiorgan failure. Although emergency orthotopic liver transplantation represents a major advance in the management of selected patients, it is not applicable to all candidates due to limited organ availability. Therefore, new therapeutic options should be developed to bridge selected patients to transplantation or to treat patients not candidates for liver transplantation. Although new techniques for cell culture and perfusion have resulted in a number of promising devices for the provision of temporary liver support in acute liver failure, their clinical efficacy is as yet uncertain. Controlled trials on a multi-centre basis in well-defined patient groups and with standardised outcome measures, including the extent to which treatment influences cell damage and regeneration and prevents or reverses multiorgan failure, will be essential to properly evaluate the clinical value of current and evolving artificial and bioartificial devices. The same considerations must also apply to the assessment of therapeutic efficacy of hepatocyte transplantation. A better understanding of mechanisms responsible for the development of liver cell death, along with cellular and molecular mechanisms allowing surviving cells to proliferate in a hostile environment, will be required if a more targeted therapeutic approach to decreasing hepatocellular injury and enhancing liver regeneration is to be achieved. Whether extracorporeal devices or the transplantation of primary hepatocytes, stem cells or cells genetically engineered to over-express key metabolic functions, a proliferative phenotype and/or cytoprotective pathways will be best suited to meeting these demanding challenges remains to be determined. PMID- 10728796 TI - Drug-induced liver diseases. AB - Drug-induced liver injuries make up a persisting and challenging problem for physicians, health agencies and pharmaceutical firms. The clinical expression is polymorphous, acute hepatitis being predominant. The diagnosis is frequently difficult because of the absence of specific signs in most cases and mainly relies on the exclusion of other causes. The diagnosis should be particularly evoked in patients over 50 yr who are taking many drugs, after viral infections have been ruled out. Acute hepatocellular hepatitis is particularly severe because of the risk of fulminant hepatitis or of a more insidious course leading to cirrhosis. Cross hepatotoxicity can sometimes occur. One should avoid re administration of not only the causative agents but also of other drugs belonging to the same family or having a related chemical structure. The prediction of the hepatotoxicity of new drugs must be improved. Investigations would be particularly useful for drugs having critical chemical structures and belonging to families with an established history of hepatotoxicity. PMID- 10728797 TI - Hepatitis B infection: pathogenesis and management. AB - Although hepatitis B is an ancient disease, most of the advances in our knowledge of its epidemiology, prevention, pathogenesis, natural history and treatment were made in the last 30 years. The prospect of global eradication of HBV infection within the next 50 years is technologically possible but implementation of worldwide vaccination against hepatitis B will require significantly more time to overcome the social and economic hurdles. While there is reasonable optimism that HBV infection will be eradicated, there are currently 300 million HBV carriers worldwide who are at risk of dying from liver failure or hepatocellular carcinoma, and there will continue to be new cases of HBV infection for many more years. Thus, HBV infection cannot be considered to be a health problem of the past. The focus of hepatitis B research at the turn of the millenium will be the development of more effective therapies that can be applied to all patients with chronic HBV infection. These treatments need to be effective in inhibiting HBV DNA synthesis and in eliminating ccc DNA. They may involve monotherapy with more potent antiviral agents that do not induce resistance, but are more likely to require a combination of antiviral agents or antiviral and immunomodulatory agents. These treatments must be safe, convenient to administer, and affordable. It is likely that new therapies with increasing efficacy will be available in the next one to two decades and combination therapy will be used widely by 2010. These treatments will induce sustained remission in the majority of patients who can afford them but provision of treatment to all those who need them will be more difficult. Other areas of hepatitis B that need to be addressed are the prevalence of occult HBV infection, the changing epidemiology and clinical significance of HBV variants, in particular the A1896 mutant, and the mechanisms of immune clearance and pathogenesis. PMID- 10728798 TI - Pathogenesis, diagnosis and management of hepatitis C. AB - The hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide. It is estimated that about 170 million people are chronically infected with HCV. Chronic hepatitis C is a major cause of cirrhosis and hepatocellular carcinoma and HCV-related end-stage liver disease is, in many countries, the first cause of liver transplantation. HCV infection is characterized by its propensity to chronicity. Because of its high genetic variability, HCV has the capability to escape the immune response of the host. HCV is not directly cytopathic and liver lesions are mainly related to immune-mediated mechanisms, which are characterized by a predominant type 1 helper cell response. Co-factors influencing the outcome of the disease including age, gender and alcohol consumption are poorly understood and other factors such as immunologic and genetic factors may play an important role. Recent studies have shown that the combination therapy with alpha interferon and ribavirin induces a sustained virological response in about 40% of patients with chronic hepatitis C. The sustained response rates are mainly dependent on the viral genotype (roughly 60% in genotype non-1 and 30% in genotype 1). Reliable diagnostic tools are now available and useful for detecting HCV infection, to quantify viral load and to determine the viral type. The assessment of the viral quasispecies and the characterization of viral sequences might be clinically relevant but standardized and simple techniques are needed. The lack of animal models and of in vitro culture systems hampers the understanding of the pathogenesis of chronic hepatitis C and the development of new antivirals. New therapeutic schedules with higher and/or daily doses of alpha interferon do not seem to improve the efficacy greatly. The conjugation with polyethylene glycol (PEG) improved the pharmacodynamics and the efficacy of alpha interferon. Emerging new therapies include inhibitors of viral enzymes (protease, helicase and polymerase), cytokines (IL-12 and IL-10), antisense oligonucleotides and ribozymes. The first candidate compounds should be available in the next few years. The development of an effective vaccine remains the most difficult and pressing challenge. Because of the high protein variability of HCV, protective vaccines could be extremely difficult to produce and therapeutic vaccines seem more realistic. Considerable progress has been made in the field of HCV since its discovery 10 years ago but a major effort needs to be made in the next decade to control HCV-related liver disease. PMID- 10728799 TI - Alcoholic liver disease: new insights in pathogenesis lead to new treatments. AB - Much progress has been made in the understanding of the pathogenesis of alcoholic liver disease, resulting in improvement of prevention and therapy, with promising prospects for even more effective treatments. The most successful approaches that one can expect to evolve are those that deal with the fundamental cellular disturbances resulting from excessive alcohol consumption. Two pathologic concepts are emerging as particularly useful therapeutically. Whereas it continues to be important to replenish nutritional deficiencies, when present, it is crucial to recognize that because of the alcohol-induced disease process, some of the nutritional requirements change. This is exemplified by methionine, which normally is one of the essential amino acids for humans, but needs to be activated to S-adenosylmethionine (SAMe), a process impaired by the disease. Thus, SAMe rather than methionine is the compound that must be supplemented in the presence of significant liver disease. Indeed, SAMe was found to attenuate mitochondrial lesions in baboons, replenish glutathione, and significantly reduce mortality in patients with Child A or B cirrhosis. Similarly, polyenylphosphatidylcholine (PPC) corrects the ethanol-induced hepatic phospholipid depletion as well as the decreased phosphatidylethanolamine methyltransferase activity and opposes oxidative stress. It also deactivates hepatic stellate cells, whereas its dilinoleoyl species (DLPC) increases collagenase activity, resulting in prevention of ethanol-induced septal fibrosis and cirrhosis in the baboon. Clinical trials with PPC are ongoing in patients with alcoholic liver disease. Furthermore, enzymes useful for detoxification, such as CYP2E1, when excessively induced, become harmful and should be downregulated. PPC is one of the substances with anti-CYP2E1 properties that is now emerging. Another important aspect is the association of alcoholic liver disease with hepatitis C: a quarter of all patients with alcoholic liver disease also have markers of HCV infection, with an even higher incidence in some urban areas but, at present, no specific therapy is available since interferon is contraindicated in that population. However, in addition to antiviral medications, agents that oppose oxidative stress and fibrosis should also be tested for hepatitis C treatment since these two processes contribute much to the pathology and mortality associated with the virus. In addition to antioxidants (such as PPC, silymarin, alpha-tocopherol and selenium), anti-inflammatory medications (corticosteroids, colchicine, anticytokines) are also being tested as antifibrotics. Transplantation is now accepted treatment in alcoholics who have brought their alcoholism under control and who benefit from adequate social support but organ availability is still the major limiting factor and should be expanded more aggressively. Finally, abstinence from excessive drinking is always indicated; it is difficult to achieve but agents that oppose alcohol craving are becoming available and they should be used more extensively. PMID- 10728800 TI - Chronic cholestatic diseases. AB - Chronic cholestatic diseases, whether occurring in infancy, childhood or adulthood, are characterized by defective bile acid transport from the liver to the intestine, which is caused by primary damage to the biliary epithelium in most cases. In this article, approaches to diagnosis and management of the main specific disorders are provided and some of the recent developments in this field are discussed. Major advances in the understanding of the cellular and molecular physiology of bile secretion have led to identification of genetic defects responsible for the different types of progressive familial intrahepatic cholestasis (PFIC). The potential role of the genes involved in PFIC in some adult cholestatic disorders remains to be determined. The majority of adult patients with chronic cholestasis have primary biliary cirrhosis (PBC) or primary sclerosing cholangitis (PSC). Recently, variant forms of PBC have been described. The term autoimmune cholangitis is used to describe patients having chronic non suppurative cholangitis with negative antimitochondrial antibodies (AMA) but positive antinuclear and/or antismooth muscle antibodies. Autoimmune cholangitis and AMA-positive PBC are quite similar in terms of clinical presentation, survival and response to ursodeoxycholic acid (UDCA) therapy. In contrast, autoimmune cholangitis must be distinguished from PBC-autoimmune hepatitis (AIH) overlap syndrome in which biochemical and histological characteristics of both PBC and AIH coexist. Combination of UDCA and corticosteroids is required in most patients with overlap syndrome to obtain a complete clinical and biochemical response. Long-term UDCA treatment improves survival without liver transplantation in PBC patients. Among the putative mechanisms of the beneficial effects of UDCA, description of anti-apoptotic properties and effect on endotoxin disposal in biliary cells have provided new insights. In patients with incomplete response to UDCA, combination of UDCA with antiinflammatory or immunosuppressive drugs is under evaluation. Variant forms of PSC have also been described, including PSC-AIH overlap syndrome, especially in children or young adults, and small-duct PSC, which is characterized by normal cholangiogram in patients having chronic cholestasis, histologic features compatible with PSC and inflammatory bowel disease. Development of cholangiocarcinoma (CC) is a major feature of PSC, occurring in 10-15% of patients. Early diagnosis of CC is a difficult challenge, although positron emission tomography seems a promising tool. Unlike PBC, effective medical therapy is not yet available in PSC, reflecting the lack of knowledge about the exact pathogenesis of the disease. Currently, liver transplantation is the only effective therapy for patients with advanced disease, although recurrence of PSC in the graft may occur. PMID- 10728801 TI - Complications of cirrhosis. I. Portal hypertension. AB - Increased resistance to portal blood flow is the primary factor in the pathophysiology of portal hypertension, and is mainly determined by the morphological changes occurring in chronic liver diseases. This is aggravated by a dynamic component, due to the active-reversible- contraction of different elements of the porto-hepatic bed. A decreased synthesis of NO in the intrahepatic circulation is the main determinant of this dynamic component. This provides a rationale for the use of vasodilators to reduce intrahepatic resistance and portal pressure. Another factor contributing to aggravate the portal hypertension is a significant increase in portal blood flow, caused by arteriolar splanchnic vasodilation and hyperkinetic circulation. Splanchnic arteriolar vasodilation is a multifactorial phenomenon, which may involve local (endothelial) mechanisms as well as neurogenic and humoral pathways. Most pharmacological treatments have been aimed at correcting the increased portal blood inflow by the use of splanchnic vasoconstrictors, such as beta-blockers, vasopressin derivatives and somatostatin. Several studies have demonstrated that changes in the hepatic venous pressure gradient (HVPG) during maintenance therapy are useful to identify those patients who are going to have a variceal bleeding or rebleeding. The wide individual variation in the HVPG response to pharmacological treatment makes it desirable to schedule follow-up measurements of HVPG during pharmacological therapy. A priority for research in the forthcoming years is to develop accurate non-invasive methods to assess prognosis, which can be used to substitute or as surrogate indicators of the HVPG response. In the clinical management of portal hypertension, beta-blockers are at present the only accepted treatment for the prevention of variceal bleeding. Whether the association of isosorbide-5-mononitrate will improve the high efficacy of beta-blockers is questionable. The efficacy of more aggressive techniques, such as endoscopic band ligation, should be further tested against beta-blockers in patients with a high risk of bleeding. In the treatment of acute variceal bleeding, administration of somatostatin or terlipressin is an established therapy. It may be used alone or, preferably, as an initial treatment before sclerotherapy or endoscopic band ligation. No more than two sessions of endoscopic treatment should be used to control the bleeding. If the bleeding is not easily controlled, other alternatives such as transjugular intrahepatic portosystemic shunts (TIPS) or derivative surgery should be considered, the former being the best in patients with poor liver function. Recent studies suggest that early measurement of HVPG during variceal bleeding may be used as a guide for therapeutic decisions in the treatment of patients with acute variceal bleeding. Those patients with a high HVPG have a high risk of poor evolution, and may be candidates for more intensive and aggressive therapy, such as surgery or TIPS. Those with lower HVPG have a very high probability of an uneventful evolution, and may thus be managed more conservatively using medical and endoscopic treatments. Pharmacological agents (propranolol or nadolol), endoscopic treatment (preferably banding ligation) or surgery can be used to prevent rebleeding. A pending task for the new millennium is to assess whether the early treatment of asymptomatic, compensated cirrhotic patients with portal pressure reducing agents can prevent the development of esophageal varices and of other complications of portal hypertension. PMID- 10728802 TI - Complications of cirrhosis. II. Renal and circulatory dysfunction. Lights and shadows in an important clinical problem. AB - The pathophysiology of circulatory and renal dysfunction in cirrhosis and the treatment of ascites and related conditions (hepatorenal syndrome and spontaneous bacterial peritonitis) have been research topics of major interest during the last two decades. However, many aspects of these problem remain unclear and will constitute major areas of investigation in the next millennium. The pathogenesis of sodium retention, the most prevalent renal function abnormality of cirrhosis, is only partially known. In approximately one third of patients with ascites, sodium retention occurs despite normal activity of the renin-aldosterone and sympathetic nervous systems and increased circulating plasma levels of natriuretic peptides and activity of the so-called natriuretic hormone. These patients present an impairment in circulatory function which, although less intense, is similar to that of patients with increased activity of the renin aldosterone and sympathetic nervous systems, suggesting that antinatriuretic factors more sensitive to changes in circulatory function that these systems may be important in the pathogenesis of sodium retention in cirrhosis. The development of drugs that inhibit the tubular effect of antidiuretic hormone and increase renal water excretion without affecting urine solute excretion has opened a field of great interest for the management of water retention and dilutional hyponatremia in cirrhosis. Two families of drugs, the V2 vasopressin receptor antagonists and the kappa-opioid agonists, have been shown to improve free water clearance and correct dilutional hyponatremia in human and experimental cirrhosis with ascites. The first type of drugs blocks the tubular effect of antidiuretic hormone and the second inhibits antidiuretic hormone secretion by the neurohypophysis. On the other hand, two new treatments have also been proved to reverse hepatorenal syndrome in cirrhosis. The most interesting one is that based on the simultaneous administration of plasma volume expansion and vasoconstrictors. The second is transjugular intrahepatic porto-systemic shunt. The long-term administration (1-3 weeks) of analogs of vasopressin (ornipressin or terlipressin) or other vasoconstrictors together with plasma volume expansion with albumin is associated with a dramatic improvement in circulatory function and normalization of serum creatinine concentration in patients with severe hepatorenal syndrome. Of interest is the observation that in many of these patients, hepatorenal syndrome does not recur following discontinuation of the treatment, thus raising important questions about the mechanism by which hepatorenal syndrome follows a progressive course in most untreated cases. The pathogenesis of circulatory dysfunction in cirrhosis and the role of local mechanisms in the development of the splanchnic arteriolar vasodilation associated with portal hypertension will continue as important topics in clinical and basic research in Hepatology. Of special interest is the study of the mechanism by which circulatory function further deteriorates following complications such as severe bacterial infection or therapeutic interventions such as therapeutic paracentesis, and the adverse consequences of the impairment in circulatory function on renal and hepatic hemodynamics. Finally, although major advances have been made concerning the treatment and secondary prophylaxis of spontaneous bacterial peritonitis in cirrhosis, many aspects of the pathogenesis of this infection remain unclear. The mechanism of bacterial translocation and of the colonization of bacteria in the ascitic fluid are particularly important to design adequate measures for primary prophylaxis of this severe bacterial infection. PMID- 10728803 TI - Complications of cirrhosis III. Hepatic encephalopathy. AB - Hepatic encephalopathy (HE) is a major neuropsychiatric complication of cirrhosis. HE develops slowly in cirrhotic patients, starting with altered sleep patterns and eventually progressing through asterixis to stupor and coma. Precipitating factors are common and include an oral protein load, gastrointestinal bleeding and the use of sedatives. HE is common following transjugular intrahepatic portosystemic stent shunts (TIPS). Neuropathologically, HE in cirrhotic patients is characterized by astrocytic (rather than neuronal) changes known as Alzheimer type II astrocytosis and in altered expression of key astrocytic proteins. Magnetic resonance imaging in cirrhotic patients reveals bilateral signal hyperintensities particularly in globus pallidus on T1-weighted imaging, a phenomenon which may result from manganese deposition. Proton (1H) magnetic resonance spectroscopy shows increases in the glutamine resonance in brain, a finding which confirms previous biochemical studies and results no doubt from increased brain ammonia removal (glutamine synthesis). Additional evidence for increased brain ammonia uptake and removal in cirrhotic patients is provided by studies using positron emission tomography and 13NH3. Recent molecular biological studies demonstrate increased expression of genes coding for neurotransmitter-related proteins in chronic liver failure. Such genes include monoamine oxidase (MAO-A isoform), the peripheral-type benzodiazepine receptor and nitric oxide synthase (nNOS isoform). Activation of these systems has the potential to lead to alterations of monoamine and amino acid neurotransmitter function as well as modified cerebral perfusion in chronic liver failure. Prevention and treatment of HE in cirrhotic patients continues to rely on ammonia lowering strategies which include assessment of dietary protein intake and the use of lactulose, neomycin, sodium benzoate and L-ornithine-aspartate. The benzodiazepine receptor antagonist flumazenil may be effective in certain cases. A more widespread use of central nervous system-acting drugs awaits a more complete understanding of the precise neurotransmitter systems involved in the pathogenesis of HE in chronic liver failure. PMID- 10728804 TI - Autoimmune hepatitis. AB - Autoimmune hepatitis (AIH) is a rare disease, characterized by female predominance, hypergammaglobulinemia, autoantibodies, association with HLA DR3 and HLA DR4 and a good response to immunosuppression. Different subtypes of AIH may be distinguished, based on differences in the autoantibody patterns. AIH type 1 is characterized by anti-nuclear (ANA) and/or anti-smooth muscular (SMA) autoantibodies. AIH type 2 is characterized by liver/kidney microsomal autoantibodies (LKM). AIH type 3 may be distinguished by autoantibodies to soluble liver proteins (SLA) or the liver pancreas antigen (LP). AIH-2 affects predominantly pediatric patients and is characterized by a more severe clinical course, a higher frequency of relapse under immunosuppressive treatment and a more frequent progression to cirrhosis. In contrast, AIH types 1 and 3 show a higher age of onset and a better long-term response to immunosuppressive treatment. At present, the treatment of choice is prednisone alone or a combination with prednisone and azathioprine. Both treatment protocols show high survival rates. However, a rate of 13% of treatment failures and the failure to induce permanent remission in most patients underlines the urgent need to develop additional treatment regimens. A yet unknown genetic predisposition is believed to act as the underlying etiological factor in AIH. This genetic predisposition includes a few known risk factors such as the presence of HLA DR3 or HLA DR4, deletions of C4A alleles and female gender. Furthermore, it has to be postulated that defects in immunoregulatory genes exist. A model for such defects may be the autoimmune polyglandular syndrome type 1 (APS1), which results from the defects in a single gene, the autoimmune regulator type 1 (AIRE-1). Patients with APS1 suffer from mucocutaneous candidiasis and a number of organ-specific autoimmune diseases. Characteristic is a high variability in the number and character of the disease components in APS1, indicating that other genetic and environmental factors may strongly modulate the outcome of disease. Environmental factors may comprise chemical influences, such as nutritional compounds and drugs, or virus infections. Several drugs or chemicals were shown to induce hepatitis with autoimmune involvement, e.g. tienilic acid, dihydralazine and halothane. Adduct formation of an activated metabolite is believed to act as a trigger and to induce a specific immune response. Similarly, viruses were repeatedly shown to trigger autoimmune hepatitis. In virus infections, sequence similarities between viral and self-proteins may trigger autoimmune processes and the simultaneous presence of inflammatory cytokines during virus infection may further increase the risk of developing self-perpetuating autoimmune reactions which overshoot. PMID- 10728805 TI - Liver transplantation. AB - Since the first human liver transplant done in 1963, the procedure has become a routine procedure with an excellent outcome in terms both of quality and of length of survival. The development and introduction into clinical practice of a variety of immunosuppressive agents has given the clinician a bewildering array of therapeutic options but with a lack of evidence on which to select for optimal immunosuppression. Tolerance can be reliably achieved in some animal transplants but remains to be achieved in humans. One of the major challenges facing the transplant community is the shortage of donor organs: imaginative approaches to overcome this problem include more effective use of marginal donor livers, splitting livers and development of living related transplants. While advances have been made in the field of xenotransplantation, there remain many hurdles to be overcome before this approach can be introduced into human transplantation. In the meanwhile, there are difficulties in determining the optimal criteria for listing patients for transplantation and for treating some of the complications arising after transplantation such as recurrence of disease and complications of immunosuppression, e.g. renal failure, malignancy and vascular disease. PMID- 10728806 TI - Hepatobiliary surgery. AB - The transformation of liver and biliary tract surgery into a full speciality began with the application of functional anatomy to segmental surgery in the 1950's, reinforced by ultrasound and new imaging techniques. The spectrum of gall stone disease encountered by the hepatobiliary surgeon has changed with the laparoscopic approach to cholecystectomy. There is increased need for conservation techniques to repair the bile duct injuries that arise more often in the laparoscopic approach to cholecystectomy. These and other surgical interventions on the bile ducts should be selected as a function of risk versus benefit in relation to the patient's requirements and the institutional expertise. Bile duct cancers, including hilar cholangiocarcinoma, and gallbladder cancers have a dismal reputation, but evidence is accumulating for better survivals from aggressive approaches performed by specialist hepatobiliary surgeons. Hepatic surgery has increased in safety and effectiveness, largely due to the segmental approach, but also to experience with techniques for vascular control and exclusion used in liver transplantation. Techniques such as portal vein embolisation, which induces hypertrophy of the future remnant liver, percutaneous local tumour destruction using cryotherapy or radiofrequency tumour coagulation and more effective chemotherapy are beginning to increase the number of patients who can undergo curative resection. In liver transplantation, segmental surgery has been applied to graft reduction and to split liver grafts, and is opening new perspectives for living donor transplantation. Today the limitation to survival in primary and metastatic liver cancer lies not in the surgical technique but in the difficulty of dealing with microscopic and extrahepatic disease. Progress in these fields will enable the hepatobiliary surgeon to further extend the possibilities for proposing curative resections. PMID- 10728807 TI - Hepatocellular carcinoma. AB - Hepatocellular carcinoma (HCC) is increasing in many countries as a result of an increase in hepatitis C virus (HCV) infection since World War II. The epidemiology of HCC varies with the global region. There have been conflicting observations from different parts of the world concerning the frequency of HCC in patients who in the distant past had post-transfusion non-A, non-B hepatitis. The genetic basis of hepatocarcinogenesis is still poorly understood. In hepatitis B virus (HVB) associated HCC, codon 249 mutation in the p 53 gene seems more related to exposure to aflatoxin B1 than to hepatocarcinogenesis itself. HCC that occurs in children in high HBV endemic regions could be associated with germ-line mutations, but little information is available; not much is known about chemical hepatocarcinogens in the environment other than aflatoxins. The X gene of HBV seems to play an important role in HBV-associated hepatocarcinogenesis. There are preliminary observations on the molecular mechanism of HCV-associated HCC, such as HCV core protein inducing HCC in transgenic mice and the NS3 genome transforming NIH 3T3 cells. Pathological distinction between preneoplastic and very early transformed lesions still depends on classical morphology, and a more genetically oriented differential diagnosis is required. Clinical diagnosis based on modern imaging has improved greatly, but is still unsatisfactory in the differential diagnosis of preneoplastic and early transformed nodules, because the vasculature changes that occur within the nodule are not accurately discerned with the current imaging. Use of sensitive des-gamma-carboxy prothrombin (PIVKA II) assay, and lectin affinity chromatography separating HCC specific subspecies of AFP molecules with a more practical biochemical technique will further improve diagnosis. Early diagnosis and transplantation are the best treatment at the moment, but transplantation is not widely available because of the donor shortage. Despite successful resection, the remnant cirrhotic liver frequently develops new HCC lesions, seriously curtailing long-term survival. All-out efforts should be directed to the prevention of HCC, through prevention of viral hepatitis, prevention of acute hepatitis from becoming chronic, prevention of chronic hepatitis from progressing to cirrhosis, and prevention of the cirrhotic liver from developing HCC (chemoprevention). At the moment, very few such studies exist. PMID- 10728808 TI - Liver-directed gene therapy: promises, problems and prospects at the turn of the century. AB - Although liver-directed gene therapy arrived later than gene therapy directed at bone marrow cells, intrinsic advantages of the liver as a target organ make it likely that gene therapy for liver diseases will be among the first therapeutically relevant applications of this treatment modality at the onset of the 21st century. Vectorology for gene transfer to the liver is advancing rapidly, and it is safe to predict that gene therapy vehicles that will be in clinical use a decade from now, have not yet been developed. None of the currently available modes of gene transfer to the liver is optimal for all types of applications. Nonetheless, the concerted effort of many investigators has provided a wide choice of non-viral and viral vectors for gene transfer to the liver for use in specific situations. Original strategies for liver-directed gene therapy included substitution of missing gene products, overexpression of intrinsic or extrinsic genes and inhibition of expression of specific genes. To the list is now added the possibility of site-specific correction or generation of mutations within specific genes in somatic cells of living adult animals. Thus, despite some initial faux pas, liver-directed gene therapy is poised to make an important impact on health care in the year 2000 and beyond. PMID- 10728809 TI - Lectin-binding pattern of primary malignant melanomas and melanocytic nevi. AB - A panel of six biotinylated lectins was applied in order to study the composition and distribution of plasma membrane carbohydrate residues in 83 primary cutaneous melanomas (MMs) and in 85 melanocytic nevi (MN) with the avidin-biotin peroxidase technique. No clear-cut differences between MN and MMs were observed with regard to the staining with lectins. In MN and MMs derived from different patients, the lectin-binding pattern was variable and heterogeneous even within the individual nevi or melanomas. It seems reasonable, therefore, to assume that the lectin binding pattern cannot be regarded as a reliable histochemical marker for the differentiation of MN from MMs. Moreover, because the pattern reveals no statistically significant correlation with the thickness or the depth of invasion of MM, it seems to lack prognostic significance. PMID- 10728810 TI - Expression of the basal cell adhesion molecule (B-CAM) in normal and diseased human skin. AB - The basal cell adhesion molecule (B-CAM) is a 90-kD cell surface glycoprotein with a characteristic immunoglobulin domain structure. The pattern of B-CAM expression in cultured cells suggests that the molecule is associated with a substrate-adherent growth pattern in some lineages. We investigated the expression of B-CAM in normal and diseased human epidermis by means of immunohistochemistry employing a single batch of high-titer mouse monoclonal antibody G253. Snap-frozen biopsy material from normal skin (n = 8), psoriasis (n = 5), contact dermatitis (n = 6), basal cell carcinoma (n = 5) and fetal skin (n = 6) was studied. In normal human skin, B-CAM was found in varying degrees throughout the epidermis with a preference for suprabasal expression, hair follicles were regularly of a B-CAM-positive phenotype. There were no qualitative differences with regard to the B-CAM expression pattern in normal skin in comparison to psoriasis and contact dermatitis. In contrast, fetal skin (15th to 18th week of gestation) was characterized by B-CAM-positive cells in the basal layer of the epidermis as well as in the outer root sheath of hair follicles. Basal cell carcinomas also regularly expressed high levels of B-CAM. A strong B CAM-positive phenotype can be found in the outer root sheath of hair follicles of adult and fetal human skin as well as in fetal basal keratinocytes. PMID- 10728811 TI - Lichenoid, erosive and ulcerated dermatofibromas. Three additional clinico pathologic variants. AB - On the occasion of a case of dermatofibroma with histological lichenoid features, we reviewed from our files all the cases in which the epidermis, usually hyperplastic in dermatofibroma, was, in some way, partially or completely destroyed. Among a total of 484 dermatofibromas, we found three lichenoid, six erosive and two ulcerated cases. In the three lichenoid cases, the columnar epidermal basal cells were lacking (squamotization of the basal layer) and in two of them there was a cleft between the epidermis and the dermatofibroma. Three of the six eroded cases were large pedunculated dermatofibromas with inflammatory phenomena of variable intensity. One case was in the center of a plaque of lichen simplex chronicus with some eroded area. In the other two cases, as well as in the two ulcerated lesions, neither inflammation nor epidermal changes usually attributed to rubbing or scratching were seen. Only in three of the eleven cases dermatofibroma was proposed (with question mark) as a clinical diagnosis. Both follow-up and histopathology supported the benign nature of these cases. We may conclude that: i) Lichenoid, erosive and ulcerated changes in dermatofibroma are infrequent phenomena which may make a clinical diagnosis difficult; and ii) in the presence of an otherwise histopathologically typical dermatofibroma, erosion and ulceration should not be considered as suspicious of malignancy. PMID- 10728812 TI - Analysis of thyroid transcription factor-1 and cytokeratin 20 separates merkel cell carcinoma from small cell carcinoma of lung. AB - Merkel cell carcinoma needs to be separated from small cell carcinoma metastatic from visceral sites to skin. Pulmonary small cell carcinoma is the most common primary site of small cell carcinoma. We evaluated the immunophenotypic characteristics of 21 Merkel cell carcinomas and 33 small cell carcinomas of lung using thyroid transcription factor-1 and cytokeratin 20. Thyroid transcription factor-1 was 100% specific for the diagnosis of small cell carcinoma of lung associated with a diagnostic sensitivity of 85%. Cytokeratin 20 was present in 95% of Merkel cell carcinomas; however, 33% of small cell carcinoma of lung were also positive. Both antibodies typically demonstrate diffuse and intense staining of their respective tumor cells. We conclude that thyroid transcription factor-1 is a sensitive and specific marker for small cell carcinomas of lung and that a combination of thyroid transcription factor-1 and cytokeratin 20 is indicated to assist in the differentiation of metastatic small cell carcinoma of lung from merkel cell carcinoma. PMID- 10728813 TI - Apoptosis in the areas of squamous differentiation of irritated seborrheic keratosis. AB - Seborrheic keratosis (SK) consists of a localized proliferation of basaloid keratinocytes, often accompanied by hyperkeratosis and hyperpigmentation. In irritated SK, these features are associated with areas of squamous differentiation with larger keratinocytes and squamous cell eddies. This work is concerned with the evaluation of apoptosis, as demonstrated by the TUNEL method, in the different varieties of SK. Apoptosis was highly expressed in the areas of squamous differentiation of irritated SK, but only mildly increased in the other varieties of SK. These data support the hypothesis that apoptosis has a role in the squamous differentiation of irritated SK. In consideration also of previous data showing that irritated SK is associated with downregulation of EGF-R expression and 125I-EGF binding, we postulate that the morphologic features of irritated SK could correspond to an involution phase of the disease, characterized by altered cell balance with inadequate cell renewal and increased cell loss. PMID- 10728814 TI - Cutaneous infections due to nontuberculous mycobacteria: histopathological review of 28 cases. Comparative study between lesions observed in immunosuppressed patients and normal hosts. AB - To evaluate the histopathological features observed in patients with cutaneous infections due to nontuberculous mycobacteria (NTM) and to compare the histopathological patterns observed in immunosuppressed patients and normal hosts. Twenty-eight biopsy specimens corresponding to 27 patients with cutaneous infections due to NTM were reviewed. Eighteen biopsies corresponded to normal hosts (14 Mycobacterium marinum, 2 Mycobacterium chelonae, 1 Mycobacterium terrae and 1 Mycobacterium gordonae) and 10 biopsy specimens were obtained from 9 immunosuppressed patients (3 Mycobacterium chelonae, one of which had two biopsies, 1 Mycobacterium abscessus, 2 Mycobacterium kansasii, 1 Mycobacterium marinum, 1 Mycobacterium avium complex and 1 Mycobacterium simiae). A panel of histopathological features was evaluated by two independent observers in each biopsy specimen. Epidermal changes (acanthosis, pseudoepitheliomatous hyperplasia, exocytosis) were mainly observed in M. marinum infections. In immunosuppressed patients the infiltrate tended to be deeper, involving the subcutaneous tissue (100%) with a more diffuse distribution and constant abscess formation. A marked granulomatous inflammatory reaction was observed in 83% of immunocompetent and in 60% of immunosuppressed patients. In immunosuppressed patients a relationship between the chronic evolution of the disease and granuloma formation was demonstrated. A diffuse infiltrate of histiocytes with occasionally foamy appearance was noted in three biopsy specimens from three patients with AIDS. Acute and chronic panniculitis was detected in 8 biopsy specimens. In one biopsy (M. chelonae) an acute suppurative folliculitis was observed. Different histopathological patterns can be noted in biopsy specimens from cutaneous nontuberculous mycobacterial infections. The evolution of the disease and the immunologic status of the host may explain this spectrum of morphological changes. Tuberculoid, palisading and sarcoid-like granulomas, a diffuse infiltrate of histiocytic foamy cells, acute and chronic panniculitis, non-specific chronic inflammation, cutaneous abscesses, suppurative granulomas and necrotizing folliculitis can be detected. Suppurative granulomas are the most characteristic feature in skin biopsy specimens from cutaneous NTM infections. Some histopathological patterns seem more prevalent in immunosuppressed patients. PMID- 10728815 TI - An immunohistopathologic study in cutaneous necrotizing vasculitis. AB - In order to investigate the importance of timing in the immunophenotypical characteristics of the inflammatory infiltrate and in the adhesion molecules expression in cutaneous necrotizing vasculitis (CNV) we carried on an immunohistopathologic study. An avidin-biotin-streptavidin peroxidase technique was performed on 21 lesional skin biopsy specimens obtained sequentially at 0 to 24, 72 and 120 hours from seven patients with a CNV presenting as palpable purpura. A panel of monoclonal antibodies specific for inflammatory cells (T lymphocytes, polymorphonuclear leukocytes, macrophages, dendritic cells) and different adhesion molecules (E-selectin, ICAM-1, VCAM-1, LFA-1, VLA-4) was used. Moreover, HECA-450 monoclonal antibody was used to identify cutaneous lymphocyte antigen (CLA) in the inflammatory infiltrate. In all cases, polymorphonuclear leukocytes predominated in the early phase of CNV and their number decreased significantly with time (p = 0.0001). The T lymphocytes were present from the beginning and their number remained stable or increased slightly in time (p = 0.1), thus becoming predominant in the perivascular infiltrate in older lesions. Macrophages were scattered on interstitium since the early phase and they showed a time-dependent increase (p = 0.0003). E-selectin (ELAM-1) expression was detected at the first biopsy and it decreased depending on the age of the evolving vasculitis (p = 0.0033). The expression of CLA decreased also with time in 5 of the 7 cases (p = 0.0001). Our study supports the existence of an unique histopathologic pattern in CNV, in which the inflammatory infiltrate varies with time at the expense of the number of polymorphonuclear cells and macrophages. PMID- 10728816 TI - Factor XIIIa+ dermal dendrocytes in erythema elevatum diutinum and ordinary cutaneous leukocytoclastic vasculitis lesions. AB - Factor XIIIa+ dermal dendrocytes belong to the dermal microvascular unit and are related to wound healing, angiogenic and fibrogenic processes. Erythema elevatum diutinum (EED) is a leukocytoclastic vasculitis followed by repair and fibrosis. In order to verify the involvement of fXIIIa+DD in the pathogenesis of EED and ordinary leukocytoclastic vasculitis (OLV) these cells were immune labeled with anti-factor XIIIa antibody and quantified in 15 biopsies of EED, 18 of OLV and compared with 11 fragments of normal skin (NS). The number of vessels was evaluated by endothelial cell staining with anti CD34 antibody. FXIIIa+DD appeared in both groups of vasculitis with hyperthophic dendrites, with no difference in their number at any level of the dermis. The number of fXIIIa+DD in the superficial dermis was higher in OLV than in NS (p<0.001). The number of dermal vessels in the EED group was higher at all dermis depths evaluated when compared with NS (p<0.05) and in the middle and deep dermis when compared with OLV (p<0.05). The results suggest the participation of fXIIIa+DD in the immunopathological mechanisms of both groups of vasculitis studied. However, there was no correlation between the number of fXIIIa+DD and angiogenesis and fibrogenesis in the EED lesions. PMID- 10728817 TI - Expression of the intermediate filament peripherin in extraskeletal myxoid chondrosarcoma. AB - The embryologic histogenesis of cartilage is not well characterized. While cranial cartilage is believed to be derived from pluripotential precursor cells of the neural crest, chondrocytes found elsewhere in the body are thought to be derived from mesoderm. As such, soft tissue tumors with cartilaginous differentiation may be related to neural crest or mesoderm. Peripherin is an intermediate filament encoded on chromosome 12, involved in growth and development of the peripheral nervous system. Peripherin is apparently expressed exclusively in cells derived from the neural crest and neural tube. A group of six soft tissue tumor types was selected because they are either of controversial differentiation or cytogenetically related to chromosome 12. A total of 41 cases was evaluated with antibodies against the intermediate filament peripherin. A panel of neural and neuroendocrine differentiation markers was used in selected cases. Three of five extraskeletal myxoid chondrosarcomas showed strong cytoplasmic reactivity with anti-peripherin. No peripherin expression was noted in any of eleven epithelioid sarcomas, eight liposarcomas, seven conventional chondrosarcomas, four neurothekeomas, three alveolar soft part sarcomas, or three clear cell sarcomas. The finding of peripherin expression in some extraskeletal myxoid chondrosarcomas may suggest the ability of some tumors to demonstrate both neural and chondroid differentiation. PMID- 10728818 TI - Elastic tissue in scars and alopecia. AB - A recent report suggests that elastic fibers appear in scars in a time-dependent fashion. This observation prompted our investigation, because we have found elastic tissue stains helpful in determining the pattern of scarring in cases of permanent alopecia. We carried out this investigation to determine if the Verhoeff-Van Gieson (VVG) elastic stain can reliably differentiate scarred from non-scarred dermis and to test our hypothesis that elastic stained sections are helpful in distinguishing lichen planopilaris (LPP) from lupus erythematosus (LE), central progressive alopecia in black females ("follicular degeneration syndrome" and "hot comb alopecia" are other terms used to describe this condition) and classic ivory white idiopathic pseudopelade. We studied histological sections from surgical scars of known duration, stained with the VVG elastic stain and VVG-stained sections of scalp biopsies from patients with established lesions of permanent alopecia. In most cases, both vertical and transverse sections were examined. In every case, the VVG stain clearly differentiated scar from the normal surrounding dermis. Distinct patterns of elastic tissue allowed for correct classification in most of the well-established cases of permanent alopecia studied. We determined that the Verhoeff-Van Gieson stain is an excellent stain to evaluate the pattern of scarring in cases of permanent alopecia and elastic tissue stains may be helpful in the histological evaluation of alopecia. PMID- 10728819 TI - Cutaneous malignant melanoma associated with extensive pseudoepitheliomatous hyperplasia. Report of a case and discussion of the origin of pseudoepitheliomatous hyperplasia. AB - We report a case of cutaneous malignant melanoma associated with extensive pseudoepitheliomatous hyperplasia. Pseudoepitheliomatous hyperplasia may mimic squamous cell carcinoma and may complicate the diagnosis of cutaneous melanoma. This diagnostic pitfall is important to both recognize and be cognizant of, so as to avoid diagnostic errors. The observation of the pseudoepitheliomatous hyperplasia, in this case with an extensive proliferation of eccrine ducts, provides further evidence that cutaneous pseudoepitheliomatous hyperplasia arises within the eccrine apparatus. PMID- 10728820 TI - Simple PCR amplification of the entire glucocerebrosidase gene (GBA) coding region for diagnostic sequence analysis. AB - Mutations in the human glucocerebrosidase gene (GBA) may lead to Gaucher disease an autosomal recessive, lysosomal storage disease. In about 15-25% of Caucasian patients with Gaucher disease yet the disease-causing mutations remain to be identified. There exists 16kb downstream from the functional GBA gene (chromosome 1q21) a highly homologous transcribed pseudogene (GBAP) with some sequence differences to GBA. These sequence differences might erroneously imitate a true mutation in the functional gene if an unintentional co-investigation of the pseudogene occurred. We describe a protocol which allows the selective analysis of a PCR-amplified 7.1 kb genomic GBA-fragment encompassing the entire GBA coding region. Direct, nonradioactive double stranded cycle-sequencing procedure of nested PCR fragments from this long range GBA-specific product allowed the sequencing of the coding exons including the flanking splice sites. Several, so far unknown coding mutation were identified in non-Jewish families with Gaucher disease. This protocol allows the rapid detection of new GBA mutations. PMID- 10728821 TI - Structure of an ovine CYP11B1 gene. AB - Glucocorticoids play an important role in the normal development and proliferation of cells, and are also involved in inflammatory responses. The level of active glucocorticoids in the body is controlled in part by the enzyme CYP11B1, which catalyses the final step of its biosynthesis. In this report, we have completely characterised the ovine CYP11B1 gene using two overlapping clones isolated from an lambdaEMBL3 sheep liver genomic library. The gene comprised 9 exons and 8 introns, spanning over a region of 8.0 kb. Two ovine CYP11B1 transcripts, with molecular sizes of 1.9 and 4.0 kb, have also been isolated from the adrenal zona fasciculata region, which showed that they arose from the usage of the two polyadenylation sites situated 2.1 kb apart in exon 9. The transcriptional start sites of the gene has been mapped using primer extension analysis. Three major start sites were identified at positions -5, -6 and -77 from the first ATG codon (Met), with two minor sites located at positions -306 and -413. When examined in context with the ovine CYP11B1 5' regulatory region, the results suggested that the ovine CYP11B1 gene contained two additional core promoters located further upstream of a proximal TATA box which could be utilised to produce mRNAs with alternative transcriptional start sites. PMID- 10728822 TI - Tracts of adenosine and cytidine residues in the genomes of prokaryotes and eukaryotes. AB - Large segments of the S. cerevisiae, C. elegans, D. melanogaster, mouse, and human genomes, as well as the genomes of four bacterial species, have been analyzed for the occurrence of tracts of separated, alternating, and mixed adenosine and cytidine residues. Several surprising features have been observed. Although both yeast and nematode DNA are rich in AT base pairs, the genomes of these organisms have widely different biases for long homonucleotide tracts. Yeast has many long tracts of oligoadenosine, while C. elegans has an extraordinary abundance of oligocytidine tracts. Tracts of alternating A-C residues are overrepresented in most eukaryotic organisms examined. Tracts of mixed adenosine and cytidine residues, however, are especially frequent in the human genome. PMID- 10728823 TI - Characterization of pig connective tissue growth factor (CTGF) cDNA, mRNA and protein from uterine tissue. AB - Connective tissue growth factor (CTGF) is a 38kDa mitogen and chemotactic factor for fibroblasts that is transcriptionally activated by serum or transforming growth factor-beta and may play a role in wound healing and various skin diseases. In these studies, pig endometrium was shown to contain a single CTGF transcript of 2.4kb and to produce a 38kDa CTGF-immunoreactive protein. Cloning and sequencing of a 1.5kb pig uterine CTGF cDNA revealed that the predicted pCTGF primary translation product displayed 92% identity to human CTGF and 93% identity to mouse CTGF. The pCTGF cDNA encoded a 26 amino acid signal peptide followed by a 323-residue sequence containing 38 highly conserved cysteine residues. In common with mouse and human CTGF proteins, pCTGF is predicted to resemble a multi functional mosaic protein that contains four distinct modules. PMID- 10728824 TI - DNA sequence of the fowl adenovirus serotype 10 short fiber gene. AB - The complete nucleotide sequence and map location of the short fiber gene of fowl adenovirus serotype 10 (FAV-10) strain CFA20 was determined. The coding sequence of the short fiber gene was found to be 1383 bp, encoding a putative polypeptide of 461 amino acids. The gene was located between 69.2 to 71.7 map units on the FAV-10 genome. A consensus splice acceptor site was located 30 bp upstream of the putative translation start codon and a polyA recognition sequence was located 24 bp downstream of the translation stop codon, signaling the end of late transcription unit five. PMID- 10728825 TI - Cloning and sequence determination of the chloroplast psbA gene in Magnolia pyramidata (Magnoliales; Magnoliaceae). AB - The full length gene encoding the D1 protein of photosynthesis (psbA) has been cloned and sequenced from Magnolia pyramidata (Magnoliaceae). Despite considerable investigation into psbA structure and function in many algal lineages and a few agricultural plants, there has been little effort invested toward characterizing a broader range of plant psbA genes. This is the first report of a psbA gene sequence from a primitive angiosperm. The DNA and deduced amino acid sequences maintain high overall conservation with other taxa, suggesting a role for psbA in broad based angiosperm phylogenetic reconstruction. PMID- 10728826 TI - Comparison of the nucleotide sequence and secondary structure of the 5.8S ribosomal RNA gene of Chlamydomonas tetragama with those of green algae. AB - We have determined the nucleotide sequence of a PCR product corresponding to the 5.8S ribosomal RNA gene in Chlamydomonas tetragama and compared the obtained sequence with those of one red and eleven green algae. A phylogenetic tree based on the 5.8S rDNAs and comparison of their predicted secondary RNA structure, with attention to a region which has been proposed to base-pair with an internal guide sequence of an intron in the large subunit chloroplast rRNA, suggested that C. tetragama was not closely related with Chlamydomonas reinhardtii. These results support the previous idea that the genus Chlamydomonas is heterogenous. PMID- 10728827 TI - Structure of the human D1F15S1A locus: a chromosome 1 locus with 97% identity to the chromosome 3 gene coding for hepatocyte growth factor-like protein. AB - The human chromosome 3 locus coding for hepatocyte growth factor-like protein/macrophage stimulating protein (HGFL/MSP) is homologous to two sets of amplified loci on human chromosome 1 at 1p36. One copy of one of the amplified loci (D1F15S1A) has been further characterized by restriction enzyme and DNA sequence analysis. A total of 8331 bp of continuous sequence was determined for this locus. The first 6878 bp of sequence is 96.1% identical to the HGFL/MSP gene, while there is no homology between the two genes following nucleotide 6878. Based on the presence of a 5 bp deletion in putative exon 2 and several downstream stop codons it is very likely that this gene is a pseudogene. Screening of a human liver cDNA library with a chromosome 1-specific probe indicates that at least several other members of the chromosome 1 loci are transcribed. PMID- 10728828 TI - Cloning and characterization of the cDNA sequences of two venom peptides from Chinese scorpion Buthus martensii Karsch (BmK). AB - From a cDNA library made from venom glands of Chinese scorpions of Buthus martensii Karsch, full-length cDNAs encoding precursors of two venom peptides have been isolated using a cDNA probe synthesized by polymerase chain reaction. Sequence analysis of the cDNAs revealed that one encoded precursor was 85 amino acid residues long including a signal peptide of 19 residues and a mature peptide (named BmK T) of 66 residues, and another encoded precursor was 84 residues long containing the same length signal peptide and a mature peptide (BmK M4 isoform, named BmK M4') of 64 residues. The analysis of amino acid sequence similarity indicated that the BmK T was homologous with both mammalian and insect toxins from BmK scorpion or other scorpions, and the BmK M4' was highly homologous with the members of the mammalian neurotoxin family of BmK, having two point mutations in amino acid residue sequence compared to BmK M4, a natural toxin from BmK. PMID- 10728829 TI - Structure of basic phospholipase A2 from Agkistrodon halys Pallas: implications for its association, hemolytic and anticoagulant activities. AB - The basic phospholipase A2 (PLA2) from the venom of Agkistrodon halys Pallas is a potent hemolytic toxin and anticoagulant. Crystal structure of the enzyme complexed with detergent n-octyl beta-D-glucopyranoside (beta-OG) in monoclinic crystal form has been determined to 2.6 A resolution. Beta-OG molecules were found in the hydrophobic channels of the enzyme. SDS-PAGE and dynamic light scattering measurements showed that the enzyme had a strong tendency to dimerise in aqueous solution. In the crystal structure the enzyme molecules associate into a tetramer with pseudo 222 symmetry, and the interfacial recognition site linked dimers constituting the tetramer have intensive interface interactions, and may be stable in solution. The structure reveals a unique positively charged face at the C-terminal region and a characteristic non-cationic 'anticoagulant' region (53-77). The face is supposed to be the hemolytic site, and based on sequence and structure comparison residues Trp70 and Glu53 instead of the basic residues in 'anticoagulant' region might play an important role in the anticoagulant activity. PMID- 10728831 TI - Enzymatic characterization of the major phospholipase A2 component of sea anemone (Aiptasia pallida) nematocyst venom. AB - The purified beta phospholipase A2 (PLA2; EC 3.1.1.4) (PLA2) from sea anemone (Aiptasia pallida) nematocysts is larger and more labile than other known venom PLA2s. In common with all other known venoms and most secretory PLA2s, the beta PLA2 requires mM Ca2+ for optimal activity and is surface-activated by aggregated lipids such as mixed micelles of detergent and phospholipid. The beta PLA2 exhibits an unusually steep and narrow pH optimum of activity at pH 7.7. The effects of changes in pH on the activity of the enzyme suggest that the active site contains functional groups having a pKs of about 7.0 and 8.0. The effects of temperature on beta PLA2 activity show a marked decrease in the energy of activation above the pre-transition temperature, suggesting that the enzyme "melts" both fatty chains in order for catalysis to occur. PMID- 10728830 TI - Multiple organ damage caused by a new toxin azaspiracid, isolated from mussels produced in Ireland. AB - A new type of food poisoning resulting from ingestion of mussels produced in Ireland occurred in the Netherlands in 1995 and then reoccurred in Ireland in 1997. As the causative agent, azaspiracid, was isolated in pure form and revealed to have a structure entirely unlike other known algal toxins, in vivo studies with mice were carried out to elucidate the pathological injuries caused by the toxin. By per os administration, the toxin caused necrosis in the lamina propria of the small intestine and in lymphoid tissues such as thymus, spleen and the Peyer's patches. Both T and B lymphocytes were injured. Additionally a fatty change was observed in the liver. These injuries distinctly differed from those caused by the representative diarrhetic shellfish toxin, okadaic acid. PMID- 10728832 TI - Effects of trachynilysin, a protein isolated from stonefish (Synanceia trachynis) venom, on frog atrial heart muscle. AB - The effects of trachynilysin (TLY), a protein toxin isolated from stonefish (Synanceia trachynis) venom, were studied on the electrical and mechanical activities of frog atrial fibres. TLY (1 microg/ml) hyperpolarized the membrane, shortened the action potential (AP) duration (APD), exerted a negative inotropic effect and elicited contracture. These effects did not develop in the presence of atropine. TLY shortened the APD of fibres isolated from a frog completely paralyzed with botulinum type A toxin, in the presence of Ca2+ but not when Ca2+ was replaced by Sr2+. TLY increased the basal and the peak of the fluorescence ratio of stimulated fibres loaded with fura-2. Confocal laser scanning microscopy revealed the existence of a diffuse innervation in atrial tissue. Our results suggest that TLY enhances the release of acetylcholine from atrial cholinergic nerve terminals and activates indirectly muscarinic receptors leading to a shortening of APD. They also show that the mechanical effects induced by TLY are due to an increase of the Ca2+ influx and to a rise in intracellular Ca2+ levels which leads to (i) a slowing of the Na+/Ca2+ exchange activity, which accounts for the contracture and (ii) the activation of a Ca2+-dependent K+ current involved in the APD shortening. PMID- 10728833 TI - Myotoxic activity of an acidic phospholipase A2 isolated from Lachesis muta (Bushmaster) snake venom. AB - An acidic phospholipase A2 isolated from Lachesis muta snake venom denoted LM PLA2, showed neither toxic nor anticoagulant activities in contrast to a potent inhibitory effect of collagen-induced platelet aggregation [Fuly, A.L., Machado. O.L.T., Alves, E.W. and Carlini, C.R., 1997. Thromb. Haemost 78, 1372-1380.]. Now, the myotoxicity induced by LM-PLA2 was investigated by using both in vivo and in vitro experiments. LM-PLA2 induced in vitro a dose- and time-dependent release of creatine-kinase (CK) from mouse Extensor Digitorium Longus (EDL) muscles and also increased the plasma CK activity in treated animals. Histopathological studies confirm myonecrosis of mouse skeletal muscles as a major effect. Edema could also be seen in muscle tissue. The amino-terminal sequence of LM-PLA2 (previously reported) indicates an aspartic acid residue located at position 49, together with other conserved amino acids present in the Asp-49 phospholipases, such as Tyr-28, Gly-30, Gly-32, His-48. Chemical modification of the protein moiety was also performed. Histidine alkylation with p-bromophenacyl bromide and lysine acetylation with acetic anhydride, abolished both indirect hemolytic and myotoxic activities of LM-PLA2. On the other hand, contrarily to what has been observed with several basic myotoxic phospholipases, the myotoxic effect induced by LM-PLA2 was not abolished by heparin. PMID- 10728834 TI - Antigenic cross reactivity among the venoms and toxins from unrelated diverse sources. AB - Numerous investigators have studied and reported the antigenic reactivity of venoms from the species of snakes belonging to a genus or a family. However, there is very little published data on the inter-family antigenic cross reactivity among the venoms of snakes and absolutely no data on venoms from other sources such as honey bee, scorpion and toad. This report describes the antigenic and immunological cross reactivity among the venoms of snakes from major families: Crotalidae, Elapidae, Viperidae, Hydrophiidae and venoms from honey bee, scorpion and toad. The homologous polyclonal antisera versus snake venoms showed high reactivity to the respective venoms and varying degree to other venoms revealing the inter-family antigenic cross-reactivity. Surprisingly, venoms from bee, scorpion and toad showed antigenic cross reactivity to snake venoms. Antisera against snake venoms reacted immunologically to venoms from bee and scorpion but toad venom reacted only to anti C. atrox venom. The immunological cross reactivity among singular toxins, cobratoxin, ricin A, botulinum A and cholera was studied by using respective polyclonal antibodies. Immunological high cross reactivity was observed between bee venom and anti ricin, similarly between anti botulinum and cobratoxin. Bee venom reacted immunologically to all anti-toxins. PMID- 10728835 TI - Neurotoxic shellfish poisoning and brevetoxin metabolites: a case study from Florida. AB - In June of 1996, three family members were diagnosed as suffering from neurotoxic shellfish poisoning (NSP) as a result of eating shellfish harvested from Sarasota Bay, Florida. Urine from two of these patients and extracts of shellfish collected from the same location were analyzed by radioimmunoassay (RIA) and by receptor binding assay. Activity consistent with brevetoxins was present in both urine and shellfish extracts. High performance liquid chromatographic (HPLC) analysis of shellfish extracts demonstrated multiple fractions recognized by specific anti-brevetoxin antibodies, suggesting metabolic conversion of parent brevetoxins. Affinity-purification of these extracts yielded four major peaks of activity. One peak was identified by HPLC-mass spectroscopy (HPLC-MS) to be PbTx 3, which was likely produced metabolically from the dominant parent toxin PbTx-2. No PbTx-2, however, was detected. Other peaks of activity were determined to consist of compounds of apparent masses of [M + H]+ of 1018, 1034, and 1005. These higher masses are suggestive of conjugated metabolites, but their structures have yet to be determined. The material associated with these latter three peaks were recognized by both RIA and receptor binding assay, but they quantitated differently. This finding suggests that these metabolites react differently in the two assays, and this result may have important implications for seafood safety and regulation. We suggest these metabolites to be the true cause of NSP, and they should be taken into account during regulatory testing. PMID- 10728836 TI - Efficacy of bothropic antivenom and its IgG(T) fraction in restoring fibrinogen levels of Bothrops jararaca envenomed mice. AB - Bothropic antivenom and its IgG(T) fraction, administered 4 h after experimental envenoming by Bothrops jararaca in Swiss mice, were compared for their abilities to restore fibrinogen 24 or 48 h after treatment. IgG(T) was able to normalise fibrinogen levels as efficiently as conventional antivenom. As IgG(T) also neutralises most anti-toxic activities of Bothrops venom, our results suggest that IgG(T) could be a better alternative treatment for envenoming due to the reduced amount of extraneous proteins, which may facilitate the induction of early adverse reactions. PMID- 10728837 TI - Bibliography of toxinology. PMID- 10728838 TI - More than just "interesting!" Anthropology, health and human rights. PMID- 10728839 TI - Physical access to primary health care in Andean Bolivia. AB - Limited physical access to primary health care is a major factor contributing to the poor health of populations in developing countries, particularly in mountain areas with rugged topography, harsh climates and extensive socioeconomic barriers. Assessing physical access to primary health care is an important exercise for health care planners and policy makers. The development of geographic information system (GIS) technology has greatly improved this assessment process in industrialized countries where digital cartographic data are widely available. In developing countries particularly in mountain areas, however, detailed cartographic data, even in hardcopy form, are nonexistent, inaccurate or severely lacking. This paper uses GIS technology to assess physical access to primary health care in a remote and impoverished region of Andean Bolivia. In addition, it proposes an alternative model of health personnel distribution to maximize physical accessibility. Methods involved extensive fieldwork in the region, utilizing GPS (global positioning system) technology in the development of the GIS and gathering other pertinent health data for the study. Satellite imagery also contributed to the development of the GIS and in the modeling process. The results indicate significant variation in physical access to primary health care across the three study sites. More importantly, this paper highlights the use of GIS technology as a powerful tool in improving physical accessibility in mountain areas of developing countries. PMID- 10728840 TI - The influence of maternal intergenerational education on health behaviors of women in peri-urban Bolivia. AB - The influence of maternal education on infant mortality has been demonstrated repeatedly in health and social science literature. Less explored is the influence of the education level of the mother's mother. In the present paper the authors examine the possible effect of grandmother's education on maternal behaviors. The relationship between intergenerational education and selected health behaviors, including utilization of health services for prenatal care, breast-feeding and family planning, are reported. The data were collected in peri urban Santa Cruz, Bolivia among mothers of infants between 0 and 18 months of age. It appears that grandmother's education does exert an effect on health behaviors above and beyond the effect of maternal education. This effect is more pronounced for health services which fall clearly in the domain of the formal health care system. While the results are exploratory, the results suggest the worth of further study and consideration of the influence of mothers' mothers in the design of culturally sensitive quality health services. PMID- 10728841 TI - Distance and health care utilization among the rural elderly. AB - This paper explores the relationship between distance and the utilization of health care by a group of elderly residents in rural Vermont. By drawing on recent work on the geography of health we frame the decision to visit a primary care physician in the context of the experience of place. The paper devises a test of this broader reading of the role of distance for utilization, and operationalizes this test using a custom designed survey. Using a randomized mail survey of elderly residents of Vermont's North East Kingdom we explore how grocery shopping, travel to work, home location relative to local services, access to private transportation, and living arrangements are associated with the number of doctor visits made to primary health care providers. Although the results confirm the idea that increased distance from provider does reduce utilization, they strongly suggest that distance to provider is a surrogate for location in a richer web of relations between residents and their local communities. We conclude by calling for further research that establishes links between place and the use of health facilities. PMID- 10728842 TI - Socioeconomic status and infant mortality in Australia: a national study of small urban areas, 1985-89. AB - This study uses small-area data for the period 1985-89 to examine the relationship between socioeconomic status and infant mortality in each of the mainland State capital cities of Australia. An unweighted OLS regression analysis based on 195 Statistical Local Areas (SLAs) that recorded five or more deaths over the reference period shows that standardised infant mortality ratios were significantly higher in areas with greater concentrations of low income families. This relationship was independent of the effects of low birthweight, Aboriginality, ethnicity and variability between each of the capital cities. To test for the robustness of this result a sensitivity analysis was undertaken. This involved (a) performing a Principal Components Analysis on a wide range of sociodemographic variables to derive factor scales that were subsequently included in a regression analysis, (b) using weighted least-squares regression and a Poisson generalised linear model and (c) including in the analysis all SLAs irrespective of the number of infant deaths. The sensitivity analysis supported the results of this study, thus validating the observed association between the socioeconomic characteristics of urban areas and their rate of infant mortality. Despite marked reductions in overall rates of infant mortality over the last century in Australia. socioeconomic disparities were still evident during the mid to-late 1980s. Whether and to what extent this situation persisted during the early-to-mid 1990s will be known in the near future when the next collection of area-based data are publicly released. The results of this study, therefore, represent an important baseline against which more contemporary national trends can be monitored. PMID- 10728843 TI - Women's perceptions of caesarean section: reflections from a Turkish teaching hospital. AB - Caesarean section as a contentious topic has attracted attention world-wide and different dimensions of the issue has been investigated. The primary reason behind these initiatives have been the upsurge of caesarean sections both in the developed and developing world and the realisation that the operation may not always contribute positively to the mother's and baby's health. By contrast, several studies have demonstrated both the short and long term negative effects. Research has also revealed that factors other than medical necessity play an important role in the decision to perform a caesarean section. Turkey, although reliable data does not exist, can be classified among the countries experiencing the caesarean epidemic, at least among highly educated and wealthy mothers. This research, exploring the perceptions of mothers in a teaching hospital with a high caesarean rate, is a rare example of its kind in Turkey. The main finding is the dissatisfaction of the mothers undergoing caesareans during their stay in the hospital. PMID- 10728844 TI - Career preferences and the work-family balance in medicine: gender differences among medical specialists. AB - In this article career preferences of medical specialists in the Netherlands are analysed, based on a survey among the members of medical associations of five specialties. Four different career preferences were offered, each of which implied a possible variation in working hours. A questionnaire was sent to a random selected group of working specialists in general practice, internal medicine, anaesthesiology, ophthalmology and psychiatry. Logistic regressions were used to predict career preferences. Besides individual characteristics, work and home domain characteristics were taken into the analysis. Not surprisingly, the preference for career change in respect of working hours is higher among full time MDs, especially women, than among part-time workers. In contradiction to what was expected, home domain characteristics did not predict a part-time preference for female, but for male MDs. One home domain characteristic, children's age, did predict the male part-time preference. Further gender differences were found in respect of the fit between actual and preferred working hours (A/P-fit). The majority of male MDs with a full-time preference had achieved an A/P-fit, whereas significantly less female MDs achieved their preferences. It was found that hospital-bound specialists are less positive towards part-time careers than other specialists. Furthermore, the change of working hours would imply a reduction in FTE for all specialties, if all preferences were met. Especially in hospital-bound specialisms it was not confirmed that the reduction in FTE would be low; this was found only in respect of interns. It may be concluded that individual preferences in career paths are very diverse. Personnel policy in medical specialties, especially in hospitals, will have to cope with changes in traditional vertical and age-related career paths. Flexible careers related to home domain determinants or other activities will reinforce a life cycle approach, in which the centrality of work is decreasing. PMID- 10728845 TI - Interpretation of nonvocal behavior and the meaning of voicelessness in critical care. AB - This paper presents two interrelated psychosocial constructs, voicelessness and interpretation, which were derived from a participant observation study of critically ill older adults in the USA. Voicelessness occurs when physiological, psychosocial and/or technological barriers limit the abilities of critically ill patients to represent their thoughts, feelings, desires and needs fully to others. Voicelessness influences not only the responses of critically ill patients to their condition, environment and caregivers, but also profoundly effects the responses of family members and clinicians. Thus, communicative interactions as well as certain clinical and treatment decisions in ICU hinge on clinician and family member interpretation of patients' nonvocal behaviors. Conditions and factors contributing to interpretation are described and a hypothesis proposed, that interpretation mitigates the detrimental effects of voicelessness. PMID- 10728846 TI - Reinventing fatherhood in Japan and Canada. AB - This article summarizes the findings of a transnational study of Japanese and Canadian families. Fathers' perceptions are presented for a critical developmental phase of life: pregnancy, labor, birth, the early and late postpartum periods. Using qualitative and quantitative research methods, 33 fathers and 194 mothers were interviewed: Japanese in Tokyo and Montreal, and English and French Canadians in Montreal. In addition, the study examines the impact of pregnancy and birth on the relationship of marriage. The results suggest that the social meaning of fatherhood has been transformed, legitimizing the presence of the father in the domestic sphere as a consequence of shifting extended family household structures, economic conditions and the empowerment of women. Other significant (p < 0.05) patterns were identified. (1) Canadian fathers participate in labor and delivery to a higher degree than Japanese fathers in Montreal and Tokyo do (mothers in Tokyo felt more isolated). (2) Canadian men (60%) are more likely to take time off from work during this phase than the Japanese. (3) Overall, the majority of fathers, in all four groups, cared for the child to a higher degree than expected and (4). Birth and early child rearing appears to have a significant impact on marriage in all four groups. Public policy issues are addressed and potential responses provided. PMID- 10728847 TI - AIDS, risk and social governance. AB - This paper considers the discursive properties of public health literature produced around AIDS in the 1980s and early 1990s. Attention is focused upon the role of health promotion in the UK government's response to the epidemic and on the language used in the educational campaigns conducted by the Health Education Council and its replacement the Health Education Authority. Using an analytical approach influenced by the work of Michel Foucault, the paper argues that the knowledges of AIDS produced by these various public health institutions constructed discursive boundaries between the idea of 'normal' and 'abnormal' behavioural practices. The notion of risk, produced as it is from epidemiological knowledge, is a central mechanism in this process. It is through the production, articulation and normalisation of 'at risk' groups that society is fragmented and hence subject to the governance strategies of late-modern liberal economies. PMID- 10728848 TI - Life course accumulation of disadvantage: childhood health and hazard exposure during adulthood. AB - The present paper examines the association between physical and social disadvantage during childhood and lifetime exposure to health-damaging environments. Study members were participants of Boyd Orr's clinical, social and dietary survey conducted between 1937 and 1939 and were aged between 5 and 14 years at clinical examination. Study participants were traced and between 1997 and 1998 a random sample of 294 were interviewed. The lifegrid interview method was used to collect full occupational, residential and household histories, from which accumulated lifetime exposures to a range of environmental hazards were estimated. Age-adjusted height during childhood was found to be inversely related to subsequent exposure to all hazards combined (males p = 0.002; females p = 0.001). This relationship was found in males with manual fathers (p = 0.044) and females with non-manual fathers (p = 0.035). Chronic disease during childhood was also associated with greater subsequent hazard exposure in males with manual fathers (p = 0.008). Among females with non-manual fathers, in contrast, chronic disease during childhood was associated with reduced subsequent hazard exposure (p = 0.05). These findings suggest that exposure to health-damaging environments during adulthood may accumulate on top of health disadvantage during childhood and that this process of life course accumulation of disadvantage may vary by gender and childhood social class. PMID- 10728849 TI - Racial differences in Norplant use in the United States. AB - The introduction of the contraceptive implant Norplant has focused attention on how social factors may affect contraceptive use. In the United States, race is a central category of social organization which may impact Norplant use. I use data from the 1995 National Survey of Family Growth to answer three main questions. (1) Are women of color more likely to use Norplant? (2) To what extent can racial differences in Norplant use be explained by a structural bias in the provision of medical care? (3) To what extent can racial differences in Norplant use be explained by life circumstances which may affect individual women's contraceptive decisions? I find that African American and Native American women are more likely than white or Asian American women to be recent Norplant users. There are no differences in recent use by Hispanic origin. Both a structural bias in the provision of care and differences in life circumstances account for the disparity in Norplant use between African Americans and whites. However, none of the factors examined here explain Native American women's high rate of use. Concerns about health risks for Norplant use are also discussed. These findings point out the importance of examining structural, individual and health status factors in studies of the use of health services. PMID- 10728850 TI - Treatment seeking for malaria in Morong, Bataan, the Philippines. AB - Early diagnosis and treatment for malaria has a significant impact on the severity of the disease and contributes to the interruption of its transmission. Fourteen high-risk families and nine locality-matched families, with no recent history of malaria, participated in an ethnographic study which aimed to document malaria episodes and to examine treatment paths for fevers locally termed malaria and perceived to be malaria. The study, conducted in Morong, Bataan, a low malaria endemic area in the Philippines, used a combination of qualitative and quantitative methods over a period of 12 months. Six treatment categories were identified; self-treatment with Western medicines and clinic consultations had almost equal frequencies. Twenty-six treatment paths were recognised which consisted of the six categories singly or in combination. More than 80% of the undiagnosed malarya illnesses were treated with antimalarials of inappropriate dosages. More adult men than women self-treated, but there was no significant difference by gender in terms of clinic consultation for illness. The majority of clinic consultations were made for young children. The implications for control are discussed. PMID- 10728851 TI - Job strain, effort-reward imbalance and employee well-being: a large-scale cross sectional study. AB - This study investigated the effects of the Job Demand-Control (JD-C) Model and the Effort-Reward Imbalance (ERI) Model on employee well-being. A cross-sectional survey was conducted comprising a large representative sample of 11,636 employed Dutch men and women. Logistic regression analyses were used. Controlling for job sector, demographic characteristics (including educational level) and managerial position, employees reporting high job demands (i.e. psychological and physical demands) and low job control had elevated risks of emotional exhaustion, psychosomatic and physical health complaints and job dissatisfaction (odds ratios ranged from 2.89 to 10.94). Odds ratios were generally higher in employees reporting both high (psychological and physical) efforts and low rewards (i.e. poor salary, job insecurity and low work support): they ranged from 3.23 to 15.43. Furthermore, overcommitted people had higher risks of poor well-being due to a high effort-low reward mismatch (ORs: 3.57-20.81) than their less committed counterparts (ORs: 3.01-12.71). Finally, high efforts and low occupational rewards were stronger predictors of poor well-being than low job control when both job stress models were simultaneously adjusted. In conclusion, our findings show independent cumulative effects of both the JD-C Model and the ERI Model on employee well-being and are not significantly different in men and women as well as in young and old people. In particular, high (psychological and physical) efforts and low rewards adversely affected employee well-being. Preliminary findings also indicate excess risks of poor well-being in overcommitted persons suffering from high cost--low gain conditions at work. PMID- 10728852 TI - Ochre-differentiation through micro-Raman and micro-FTIR spectroscopies: application on wall paintings at Meteora and Mount Athos, Greece. AB - The most widely-used inorganic pigments of Byzantine and post-Byzantine hagiography are earth pigments called ochres such as, red and yellow ochres, limonite, goethite, raw and burnt sienna, caput mortuum and hematite. The present experimental work proposes a technique of differentiation that allows one to distinguish among all the different kinds of iron oxides, thereby providing a better understanding of the painting technique used on portable icons and wall paintings. The ratios between the main spectroscopic peaks, attributable to the major components usually present in ochres, were calculated and compared, one against the another, from the spectra obtained through micro-Raman spectroscopy. Elementary composition is also revealed through a scanning electron microscopy (SEM) analysis. The possibility for detailed study on a particular Byzantine ochre palette can thus be performed based on the small differences in its nature and composition. These differences can first be observed and then measured among all of the natural earth pigments, through microRaman and microFTIR spectroscopies. PMID- 10728853 TI - High resolution spectral analysis of 13CH3OH in the excited torsional states. AB - In this work, we have extended our previous analysis of the Hamiltonian of 13C substituted methanol to include a large number of spectral lines involving the second excited torsional state using an improved model. The data set consisted of 2529 Fourier transform and microwave transitions with the rotational angular momentum J < or = 10, K < or = 6 and n < or = 2 (with 336 MW lines). The data set was fitted with the new Hamiltonian model to derive the molecular parameters. The results indicate that the model developed for the other methanol species (CH3OH, CH3(18)OH and CH3OD) is also valid for the C-13 substituted species. The results will allow the energy levels of the molecule to be calculated for higher torsional levels above the internal rotational barrier with improved precision and allow the analysis to be carried out for more excited torsional states. PMID- 10728854 TI - Conformational stability of CH3CH2PH2BH3 from temperature dependent FT-IR spectra of xenon solutions and r0 structural parameters. AB - Variable temperature (-55--100 degrees C) studies of the infrared spectra (3500 400 cm(-1)) of ethylphosphine-borane, CH3CH2PH2BH3, and ethylphosphine-borane-d5 dissolved in liquid xenon have been recorded. From these data, the enthalpy difference has been determined to be 86 +/- 8 cm(-1) (1.03 +/- 0.10 kJ/mol), with the trans conformer the more stable rotamer. Complete vibrational assignments are presented for both conformers, which are consistent with the predicted frequencies obtained from the ab initio MP2/6-31G(d) calculations. The optimized geometries, conformational stabilities, harmonic force fields, infrared intensities, Raman activities, and depolarization ratios have been obtained from RHF/6-31G(d) and/or MP2/6-31G(d) ab initio calculations. These quantities are compared to the corresponding experimental quantities when appropriate as well as with some corresponding results for some similar molecules. The r0 structural parameters have been obtained from a combination of the previously reported microwave rotational constants and ab initio predicted parameters. PMID- 10728855 TI - The (2 + 2) REMPI study of methylamine in the 430-485-nm region. AB - The mass resolved (2 + 2) resonance enhanced multiphoton ionization (REMPI) spectra of methylamine (MA) via the (nN,3s) Rydberg state were obtained in the 430-485-nm region using a time-of-flight (TOF) mass spectrometer. They have the same vibrational structure mainly due to NH2-wagging mode excitation. The parent ion relative intensity increases at longer wavelengths. The multiphoton ionization mechanism is discussed. PMID- 10728856 TI - 13C nuclear magnetic resonance data of bile acid derivatives. AB - The 13C-NMR spectra of well over 100 bile acid derivatives have been analyzed and summarized. A diagnostic gamma-oxygen shielding effect has been identified. PMID- 10728857 TI - The resonance Raman effect of uranyl chloride in dimethyl sulfoxide. AB - A study was carried out of the resonance Raman scattering spectra of uranyl chloride (UO2Cl2) in dimethyl sulfoxide ((CH3)2SO) (DMSO) under laser excitation of the UO2(2+) ion in resonance with the 1sigma(g)+ --> 1phi(g) Laport-forbidden f-f electronic transitions span from 530 to 450 nm by using ten output lines of the argon-ion laser at room temperature. The resonance Raman excitation profile of the totally symmetric stretching vibrational mode of uranyl observed at 832 cm(-1) is presented and analyzed in terms of transform theory within the non Condon model to give relatively good agreement with experimental results. The disagreement between the experimental data and the calculated resonance Raman excitation profile, at the long-wave part of the the 1sigma(g)+ --> 1phi(g) electronic transitions, may be referred to interference between the weak scattering from the neighboring forbidden electronic states (1delta(g)) and strong preresonance scattering from allowed electronic states at higher levels. An amount of change in the experimental resonance Raman excitation profile of the uranyl-DMSO system depends considerably upon the ligands (L) bound to the uranyl group. Elongation of the U-O equilibrium bond length resulting from the 1sigma(g)+ --> 1phi(g) electronic transitions is related to the magnitude of the change in the excitation profile of UO2L2 (L = NO3, CH3COO, Cl) type uranyl compounds in (DMSO). PMID- 10728858 TI - NMR studies on derivatives of heteroaromatic compounds: exceptionally small carbon-carbon couplings, 1J(CC), in 2-lithiothiophene, 2-lithio-N-methylpyrrole and 2-lithiofuran. AB - Spin-spin carbon-carbon coupling constants across one bond and carbon proton coupling constants across one, two and three bonds have been measured for a large series of derivatives of five-membered heterocyclic compounds. This included 2 methyl and 2-lithio derivatives of furan, N-methyl pyrrole and thiophene and a series of 2-R-substituted thiophenes where R = O-t-Bu, Cl, Br, I, Si(CH3)3, MgBr and MgTh. For the long-range C-H couplings their signs have been determined in several compounds by the use of modern 2D NMR techniques, and in all the cases they have been found to be positive. A good linear dependence upon electronegativity of the substituent has been observed for 1J(CC), 2J(C2H3), and 3J(C2H4). Very small 1J(CC) couplings of ca. 30 Hz only have been found in all three lithio compounds; they belong to the smallest couplings of this type reported up to now. They are accompanied by very large and positive two-bond couplings, 2J(C2H3) of ca. +20 Hz, which in turn are the largest couplings of this type reported so far. In both cases the changes observed are interpreted in terms of the changes in the Fermi contact contribution. PMID- 10728859 TI - Temperature dependent luminescence characteristics of Sm3+-doped silicate glass. AB - We report here on the optical characterisation of Sm3+ (5 wt%): SiO2 + Al2O3 + Li2O + Na2O + MgO glass from the measurements of optical absorption spectra (at 300 K), total luminescence spectra (10-300 K) and fluorescence lifetimes (10-300 K) of the prominent emission transitions of the Sm3+ ions. Besides its spectral properties, physical and nonlinearity characterising property parameters have also been computed to understand the optical dispersive power of this glass. By the application of Judd-Ofelt parameters (omega(lambda)) of the measured absorption spectrum, the radiative transition probability factors (A) and stimulated emission cross-section (sigma(p)E) of the observed fluorescent levels have been analysed. Both emission intensity and measured lifetimes of the prominent luminescent transition (4G(5/2) --> 6H(7/2)) concerning Sm3+-glass has been showing a descending trend with the rise in temperature with N2-laser (337.1 nm) as the source of excitation. PMID- 10728861 TI - Vibrational spectra and structure of benzophenone and its (18)O and d10 labelled derivatives: an ab initio and experimental study. AB - Infrared (4000-100 cm(-1)) and Raman (4000-10 cm(-1)) spectra of benzophenone, benzophenone-d10 and benzophenone-(18)O have been studied in the solid state and in solution and their fundamental frequencies have been assigned using isotopic frequency shifts and differential infrared linear dichroic spectra of oriented polycrystalline layers (4000-400 cm(-1)). Ab initio MO calculations have been carried out for the three benzophenone isotopomers at the HF/3-21G, 6-31G and 6 31G** levels and the computed vibrational frequencies have been compared with the experimental ones. Best agreement is achieved with the 6-31G data, the mean deviation being 25.4 cm(-1). The calculated isotopic frequency shifts induced by the (18)O and d10 labelling, are also in a good accordance with the measured ones. All geometry parameters calculated for the isolated molecule are in good agreement with the X-ray data for the benzophenone single crystal. PMID- 10728860 TI - Triplet-state energies and substituent effects of excited aroyl compounds in the gas phase. AB - Triplet-state energy values obtained from the gas phase are still scarce. In this study, the triplet-state energies of 58 aroyl compounds, introduced as gas chromatographic peaks at atmospheric pressure and typically 473 K, have been determined from the 0-0 bands of their n --> pi* type phosphorescence spectra in excited nitrogen. Correlations of those gas-phase triplet-state energies with Hammett constants could be observed for substituted acetophenones, benzaldehydes and benzophenones. PMID- 10728862 TI - Photo-degradation and emission characteristics of benzidine in halomethane solvents. AB - Benzidine is an aromatic base of importance in industry. It represents a serious pollutant in many industrial effluents and its photodegradation is of great interest. The mechanism of photo-chemical decomposition of benzidine in different halomethane solvents in addition to the corresponding UV absorption spectra and fluorescence emission spectra are discussed. The photochemical quantum yields (phiC lambda ex = 254 nm) of benzidine in halomethanes is dependent upon the halogen content in the solvent. This effect may be explained by the assumption that free radicals are formed during photolysis of these solvents followed by the abstraction of electrons from a benzidine molecule forming macroradicals of the latter. Both mono- and di-radical benzidine cations have been detected by different techniques. The well known electron absorption peak at 283 nm is characteristic of neutral benzidine while those observed at ca. 370 and 565 nm are assigned to the blue-monoradical cation and yellow-diradical cation, respectively. The blue-monoradical cation has been isolated after irradiation as a blue precipitate. A comparison between neutral benzidine and the blue monoradical cation are made using both IR and differential thermal analysis (DTA) techniques. The fluorescence quenching of solutions of benzidine in inert solvents using halomethanes have been studied. The results obtained are interpreted in terms of a diffusional quenching mechanism. PMID- 10728863 TI - Piezoreflectance spectra of 1s Z12 exciton in CuBr. AB - We have investigated the piezoreflectance spectra (deltaR/R) of the 1s Z12 exciton in single crystals of copper bromide CuBr at the temperature of 95 K with linearly polarized light. The spectra were successively studied with the pressure p along the [001] and [111] axis and the wave vector k of the incident light parallel to the [110] direction. The shear deformation potentials b and d of the 1s Z12 exciton were deduced from the stress-induced shift and splitting. The results are compared with those obtained in other works. PMID- 10728864 TI - Cooperative effect in hydrogen bonding of N-methylacetamide in carbon tetrachloride solution confirmed by NMR and IR spectroscopies. AB - Recently we carried out ab initio molecular orbital calculations of the hydrogen bond energies in the dimers and trimers of N-methylacetamide (NMA), and found the existence of a cooperative effect in the hydrogen bonding, by which formation of one hydrogen bond in a hydrogen-bonded chain enhances the strength of another hydrogen bond. In order to confirm the existence of such a cooperative effect, we have determined experimentally the enthalpy changes occurring upon hydrogen bonding of NMA in CCl4 solution. First, following the method proposed by us, the population fractions of free (non-bonded) NH protons are obtained from the observed amide proton NMR chemical shifts and the IR intensities of the free NH stretching bands. Next, the enthalpy changes are evaluated by analyzing the equilibrium between the free and bonded states of an NH proton. In this analysis, the existence of the CCl4 solvent is taken into account. The stabilization energy of hydrogen bonds in a trimer, as compared with twice the hydrogen bond energy in a dimer, is 5.4 kJ mol(-1), in good agreement with the calculated value (5.9 kJ mol(-1)). This result provides experimental confirmation of the existence of a cooperative effect in hydrogen bonding. PMID- 10728865 TI - A resonance Raman investigation on the interaction of styrene and 4-methyl styrene oligomers on sulphated titanium oxide. AB - In order to understand the nature of the interaction that gives rise to the yellow-orange colour observed when styrene or 4-methyl styrene are put in contact with sulphated TiO2, the resonance Raman spectra of such systems, including deuterated styrene (ring-deuterated d5 and perdeuterated d8) and allylbenzene were investigated. In all cases a substantial enhancement of the ring v(CC) stretching mode was observed. A charge transfer process involving a transition from the ring pi-electrons to the empty d-pi orbitals of titanium was ascribed responsible for the absorption in the visible. Two types of resonance Raman spectra were observed depending on the excitation wavelength, which can be explained by the presence of two kinds of oligomers, saturated and unsaturated, on the surface of the oxide with the former giving rise to a Raman enhancement at a higher excitation energy. PMID- 10728866 TI - UV-laser photoisomerization of fumaryl chloride: the first identification of maleoyl chloride: matrix isolation infrared and ab initio studies. AB - Fourier transform infrared spectra of fumaryl chloride 1 isolated in an argon matrix at 10 K have been analyzed. The comparison between the ab initio HF/6-31G calculated infrared spectra with the experimental ones reveals the existence of three planar conformers, the cis cis 1a, the cis trans 1b and the trans trans 1c. Laser UV irradiation of 1 at lambda = 340 nm yields maleoyl chloride 2 by a carbon carbon double bond photoisomerization process. The first identification of this compound was performed by comparison of the experimental infrared spectra with the calculated ones at the MP2/6-1G** level. AM1 semiempirical and ab initio calculations were used to calculate the structure and the relative stability of the three non planar maleoyl chloride conformers. PMID- 10728867 TI - Resonance Raman study of the polyiodide complex formed in the reaction of iodine with the polysulphur cyclic base 1,4,7,10,13,16-hexathiacyclooctadecane. AB - Resonance Raman spectroscopy has been used to study the reaction of iodine with the interesting polysulphur cyclic base, 1,4,7,10,13,16,-haxathiacyclootadecane (HTCOD). The results indicate that the complex [(HTCOD)2]+ x I5- is formed. The I5- unit exists in the form of distorted I2 linked to I3- unit which has two unequivalent I-I bonds. The v(I-I) for I2 occurs at 194 cm(-1) while for I-I, inner and outer bonds in I3- at 143 and 160 cm(-1), respectively. PMID- 10728868 TI - Optical transient absorption spectroscopy of LaO produced by pulsed laser ablation of La2O3. AB - Temporally and spatially resolved absorption spectrometry has been used to study molecular LaO absorption in laser ablated plume from an La2O3 target The absorption time-of-flight (TOF) spectra of ground-state LaO molecules were measured The TOF spectra indicate that only one component is observed in vacuum and in an Ar ambient, while there are two component, a fast and a broad slow component, observed at higher O2 pressure. The absorbance of LaO molecules decreases gradually with increasing ambient gas pressure. The dependence of the absorbance of LaO on the detection distance can be fitted by an exponential decay equation. The shock wave model is used to describe the behavior of ground state LaO molecules in high O2 pressure region. PMID- 10728869 TI - NMR, UV-vis and CD study on the interaction of pradimicin BMY-28864 with divalent cations of alkaline earth metal. AB - In order to clarify the mechanism of the calcium-activated anti-fungal action of pradimicin derivatives, we investigated the compatibility of four divalent metal cations, Ca2+, Mg2+, Sr2+ and Ba2+, when combined with pradimicin BMY-28864 in an aqueous solution. The 1H NMR studies suggest that all the tested cations can induce a chemical exchange between two types of aggregation of BMY-28864 in an aqueous solution. The exchange rate, however, varies according to the cation species. In descending order they are: Ba2+ > Sr2+ > Ca2+ > Mg2+. The differences in the binding ability of BMY-28864 to the cations and the solubility of the chloride salts are explained by the electronegativity of these cations. The UV Vis and CD spectra of these solutions show isosbestic points that correspond to an exchange process in the cations' binding to BMY-28864. The results provide experimental support for the hypothesis that there is a common mechanism underlying their cations interactions with the antibiotic. The CD study also provided evidence about the stoichiometric relation of the divalent metal ions to the antibiotic, 1:2, which was obtained by NMR analyses. PMID- 10728870 TI - Vibrational raman spectroscopic study of scytonemin, the UV-protective cyanobacterial pigment. AB - The Raman spectrum of the photoprotective pigment scytonemin found in cyanobacterial sheaths has been obtained for the first time. Its skeletal structure is extensively conjugated and unique in nature. Detailed molecular vibrational assignments are proposed and a distinctive group of four corroborative vibrational bands have been identified as unique indicators for the compound. These bands, especially a prominent feature at wavenumber 1590 cm(-1), are sufficiently conspicuous to be detectable in the mixed biomolecular pools of undisturbed natural microbial communities. This has been confirmed by demonstrating the Raman spectral bands for scytonemin in a sample of an intact intertidal cyanobacterial mat. PMID- 10728871 TI - Coupling of intramolecular hydrogen bonding to the cis-to-trans isomerization of a proline imide bond of small model peptides. AB - A relationship between intramolecular hydrogen bonding and the cis-trans isomerization of a proline imide bond for proline-containing short peptides were studied by proton NMR and infrared spectroscopy using DMSO-d6/CDCl3 mixed solvents. The percentage of the trans form increases with increasing fraction of CDCl3 in the mixed solvents except for compounds without possibility of intramolecular hydrogen bonding. Chemical shift variations of amide protons with solvent mixing ratios were found to be useful for judging whether the amide protons take part in the intramolecular hydrogen bonding to a considerable degree or not. These results and infrared spectra were used to specify intramolecularly hydrogen bonded structures of the peptides. Formation of the 10-membered or 13 membered hydrogen bonded ring which includes the carbonyl group precedent to the prolyl residue facilitates the cis-to-trans isomerization and these hydrogen bonded rings are strong enough to restrict the proline imide bond to the trans form in CDCl3 solution. On the other hand, a 7-membered hydrogen bonded ring is not so effective in restricting the proline imide bond. PMID- 10728872 TI - Reinvestigation of the triplet-minus-singlet spectrum of chloroplasts. AB - A comparison of the triplet-minus-singlet (TmS) absorption spectrum of spinach chloroplasts, recorded some thirty years ago, with the more recently published TmS spectrum of isolated Chla/b LHCII (light-harvesting complexes associated with photosystem II of higher plants) shows that the two spectra are very similar, which is to be expected, since only the carotenoid pigments contribute to each spectrum. Be that as it may, the comparison also reveals a dissimilarity: photoexcitation of the sample does, or does not, affect the absorbance in the Qy region (650-700 nm), depending on whether the sample is a suspension of chloroplasts or of isolated LHCII. The Qy-signal in the TmS spectrum of LHCII decays, it should be noted, at the same rate as the rest of the difference spectrum, and its most prominent feature is a negative peak. As the carotenoids do not absorb in the Qy region, the presence of a signal in this region calls for an explanation: van der Vos, Carbonera and Hoff, the first to find as well as fathom the phenomenon, attributed the Qy-signal to a change, in the absorption spectrum of a chlorophyll a (Chla) molecule, brought about by the presence of triplet excitation on a neighbouring carotenoid (Car). The difference in the behaviours of chloroplasts and LHCII, if reproducible, would imply that the Car triplets which give rise to the TmS spectrum of chloroplasts do not influence the absorption spectra of their Chla neighbours. With a view to reaching a firm conclusion about this vexed issue, spinach chloroplasts and thylakoids have been examined with the aid of the same kinetic spectrometer as that used for investigating LHCII; the TmS spectra of both chloroplasts and thylakoids contain prominent bleaching signals centred at 680 nm, and the triplet decay time in each case is comparable to that of the Chla/b LHCII triplets. Results pertaining to other closely related systems are recalled, and it is concluded that, so far as the overall appearance of the TmS spectrum is concerned, spinach chloroplasts are by no means abnormal. PMID- 10728873 TI - Effect of chain length on the conformation and T cell recognition of synthetic hemagglutinin fragments. AB - Circular dichroism and Fourier-transform infrared spectroscopies were used to compare the conformational mobility of 13-mer peptides covering the 317-329 region of the envelope protein hemagglutinin of human influenza A virus subtypes H1, H2 and H3 with that of their truncated deca- and nonapeptide analogs. These peptides were demonstrated to bind to the murine I-Ed major histocompatibility complex encoded class II and human HLA-B*2705 class I molecules. Despite the amino acid substitutions in the three 13-mer subtype sequences, no significant differences in the conformational properties could be shown. Deletion of the N terminal three residues resulted in a shift to an increased alpha-helical conformer population in the 317-329 H1 peptide and the breakage of the 3(10) or weakly H-bonded (nascent) alpha-helix in the H2 and H3 peptides. The conformational change observed upon deletion did not influence the efficiency of I-Ed peptide interaction, however, the C-terminal Arg had a beneficial effect both on MHC class II and class I binding without causing any remarkable change in solution conformation. PMID- 10728874 TI - The concept of transmitter receptors: 100 years on. AB - It is nearly one hundred years since John Langley of Cambridge developed the idea of the 'receptive substance' or 'receptors' as we now call them. This historical review traces the background to his introduction of this concept of the transmitter receptor and of how succeeding generations built on his ideas to generalise the applicability of this concept to synapses in general. It starts with a consideration of the discovery by Bernard (1844) that curare could paralyse rabbits without affecting their hearts because, as Vulpian (1866) suggested, curare acts on some intermediate zone between nerve and muscle. No further progress could be made without establishing the idea of chemical transmission, which Elliott (1904) then achieved, building on observations concerning sympathetic transmission to smooth muscle made previously by his mentor Langley (1901). Then between 1905 and 1907 Langley, in a wonderful act of creative ability, carried out a series of experiments on the somatic neuromuscular junction which established the idea of transmitter receptors. This review gives details of the experiments which persuaded both Langley and a recalcitrant Ehrlich that pharmacological substances could possess the necessary structure for them to combine with appropriate molecules on cells. The subsequent identification by Dale and his colleagues (1936) of acetylcholine as the transmitter acting on the receptors first discovered by Langley at the somatic neuromuscular junction as well as of acetylcholine on receptors in the heart by Loewi (1921) is then detailed. The review concludes with the triumph of the first recordings of the electrical signs of single channel openings by Neher and Sakmann (1976) at the receptors which Langley had first described. PMID- 10728875 TI - The 5-HT1A antagonist, WAY 100 635, alleviates cognitive impairments induced by dizocilpine (MK-801) in monkeys. AB - Central glutamate neurotransmission is modulated by an upregulatory cholinergic influence and an inhibitory serotonergic influence. In Alzheimer's disease, cognitive decline is associated with loss of both glutamatergic and cholinergic neurones (Francis et al., 1992, Progress in Neurobiology 39, 517-545). While therapeutic strategies for alleviating this cognitive decline have concentrated on restoring cholinergic tone, we suggest that 5-HT1A antagonists also have the potential to alleviate the cognitive symptoms of Alzheimer's disease. Previous studies have shown that dizocilpine (MK-801), a glutamatergic antagonist acting at the NMDA receptor, produces learning impairments in the common marmoset, a non human primate. Specifically, it impairs the acquisition of shape discrimination and visuospatial conditional tasks, at doses that do not affect locomotor behaviour or coordination (Harder et al., 1998, Society for Neuroscience Abstracts 23(1), 219). In the present study we investigated the effects of WAY 100 635, a 5-HT1A antagonist, on the cognitive deficits induced by dizocilpine. The number of trials required to learn each type of task under combined treatment with dizocilpine and WAY 100 635 was significantly lower than under dizocilpine treatment alone, and did not differ significantly from the number of trials required under saline, demonstrating that the cognitive effects of glutamatergic blockade can be overcome by treatment with a 5-HT1A antagonist. PMID- 10728876 TI - Enhanced 5-HT metabolism and synthesis rate by the new selective r5-HT1B receptor antagonist, NAS-181 in the rat brain. AB - NAS-181 ((R)-(+)-2-(3-morpholinomethyl-2H-chromen-8-yl) oxymethyl-morpholine methanesulfonate) is a novel rat 5-hydroxytryptamine1B, (r5-HT1B) receptor antagonist with high selectivity. The in vivo effects of NAS-181 on 5-HT metabolism and synthesis in the rat brain were examined. 5-HT metabolism, measured as the ratio 5-hydroxyindoleacetic acid (5-HIAA)/5-HT, was dose dependently increased in all four brain regions analysed (hypothalamus, hippocampus, frontal cortex and striatum) at doses 0.1 to 20 mg/kg s.c. NAS-181. The enhancement of 5-HT metabolism at the dose 20 mg/kg s.c. was maximal one hour after the injection and was still significant eight hours but not 24 hours after the injection. 5-HT synthesis rate measured as the accumulation of 5 hydroxytryptophan (5-HTP) after inhibition of the aromatic amino acid decarboxylase activity was also elevated by NAS-181 at doses 0.3 to 20 mg/kg s.c. NAS-181 competitively antagonised the decrease in 5-HT metabolism evoked by the r5-HT1B receptor agonist, anpirtoline, in hypothalamus, hippocampus and frontal cortex. Anpirtoline had no effect on 5-HT metabolism in striatum. However, anpirtoline antagonised the enhancement of 5-HT metabolism induced by NAS-181 in striatum. Combined treatment of rats with NAS-181 and the 5-HT1A receptor antagonist, WAY-100635, increased 5'-HT metabolism considerably more than when the compounds were given alone. PMID- 10728877 TI - Ethanol and trichloroethanol alter gating of 5-HT3 receptor-channels in NCB-20 neuroblastoma cells. AB - Alcohol potentiation of 5-HT3 receptors was examined in NCB-20 neuroblastoma cells using whole-cell patch-clamp electrophysiological techniques. Activation of the receptor with the weak partial agonist dopamine (DA) was used to examine alcohol effects under conditions of full agonist occupancy, but low probability of channel opening. Dopamine activation of the receptor increased in a concentration-dependent manner (EC50=0.28 mM), and on average maximal responses to DA were 8.0+/-0.8% of the maximal response to 5-HT. Ethanol (EtOH) and trichloroethanol (TCEt) potentiated DA-activated ion current mediated by 5-HT3 receptors. Potentiation of responses to a maximally effective dopamine concentration averaged 52.0+/-8.0% for EtOH and 567+/-43% for TCEt, which was comparable to the potentiation observed when receptors were activated by a low concentration of 5-HT. The alcohols increased both the potency and efficacy with which dopamine activated the receptor. The observation that alcohols increase the maximal efficacy of dopamine activation of the receptor indicates that one action of alcohols on the 5-HT3 receptor is to increase the probability of channel opening independent of any effect on agonist affinity. PMID- 10728878 TI - Nitric oxide-mediated regulation of dopamine release in the hippocampus in vivo. AB - Infusion of N-methyl-D-aspartate (NMDA) into the hippocampus of freely moving rats produced a concentration-dependent decrease in the extracellular levels of dopamine, an effect which was reversed by D-2-amino-5-phosphonovaleric acid (D AP5). To determine the involvement of nitric oxide (NO) in this response, two nitric oxide synthase (NOS) inhibitors, N-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI), were examined for their ability to modify both basal and NMDA-inhibited dopamine release. When infused alone both NOS inhibitors elicited an increase in extracellular dopamine concentration, moreover, when administered prior to the application of NMDA, the agonist failed to elicit a decrease in dopamine levels. Infusion of the NO donor S-nitroso-N acetylpenicillamine (SNAP) over a 30 min period caused either an increase or a decrease in dopamine release depending upon the concentration used. At the lower concentration (0.5 mM) SNAP promoted dopamine release whilst at the higher concentration (5 mM), the donor elicited a long lasting reduction in basal dopamine levels. The effect of the lower concentration of SNAP was reversed by the prior application of D-AP5, but that of the higher concentration was unaffected by the antagonist. PMID- 10728879 TI - Dopamine transporter synthesis and degradation rate in rat striatum and nucleus accumbens using RTI-76. AB - Intracerebroventricular injections of the irreversible dopamine transporter (DAT) inhibitor, RTI-76 [3beta-(3-p-chlorophenyl) tropan-2beta-carboxylic acid p isothiocyanatophenylethyl ester hydrochloride], decreased DAT binding in both the striatum and nucleus accumbens as measured by both [3H]GBR12935 and by [3H]WIN35,428. This decrease was dose-related, with 100 nmol RTI-76 producing approximately a 50% decrease in both regions. The maximal inhibition of DAT binding was observed 24 h after RTI-76 injection, and binding was fully restored 7 days after injection. The DAT protein half-life determined under these conditions was about 2 days. [3H]Nisoxetine binding at norepinephrine transporters in the cortex was not altered by RTI-76 administration at any time point or dose examined. PMID- 10728880 TI - Discriminative stimulus properties of the dopamine D3 receptor agonists, PD128,907 and 7-OH-DPAT: a comparative characterization with novel ligands at D3 versus D2 receptors. AB - Rats were trained to recognize a discriminative stimulus (DS) elicited by the preferential dopamine D3 receptor agonists, PD128,907 (0.16 mg/kg, i.p.) and 7-OH DPAT (0.16 mg/kg, i.p.). PD128,907 and 7-OH-DPAT showed "full" (> or = 80%) and mutual generalization. Chemically-diverse, preferential D3 versus D2 agonists, quinelorane, CGS15855A, pramipexole, ropinirole and piribedil, generalized to PD128,907 (and 7-OH-DPAT) in this order of potency, which correlated more strongly with affinity/activity at cloned human (h)D3 (r=0.68/0.81, n=7) than hD2 (0.27/0.64, n=7) receptors. Further, generalization potency strongly correlated with potency for suppression of response rates (0.86), induction of hypothermia (0.92), reduction of striatal dopamine turnover (0.92) and diminution of immobility in a forced-swim procedure (0.97). Nafadotride, UH232 and AJ76, which show a mild preference for D3 versus D2 sites, blocked the PD128,907 DS, and the modestly-selective D3 antagonist, U99194A, was partially effective. Both nafadotride and U99194A blocked the 7-OH-DPAT DS. However, antagonist potency (n=4) versus PD128,907 correlated better with affinity at D2 (0.89) versus D3 (0.27) sites. Further, whereas the preferential D2 versus D3 antagonist, L741,626, antagonized the PD128,907 DS, the selective D3 antagonists, S11566, S14297 (its eutomer) and GR218,231 were ineffective against PD128907 and 7-OH DPAT DS. S11566 and GR218,231 likewise did not generalize to PD128,907. In conclusion, under the present conditions, D2 receptors are principally implicated in the DS properties of PD128,907 and 7-OH-DPAT. PMID- 10728881 TI - Prenatal cocaine exposure alters behavioral and neurochemical sensitization to amphetamine in adult rats. AB - This study examined the neurochemical correlates of amphetamine (AMPH)-induced behavioral effects in prenatally saline (PSAL)-exposed or cocaine (PCOC)-exposed male rats. Pregnant Long-Evans rats received saline or saline containing cocaine hydrochloride (20 mg/kg s.c., b.i.d.) from gestational days 15-21. Animals were left with their biological mothers. Adult offspring were exposed to daily saline or AMPH (0.5, 1.5, or 5 mg/kg, i.p.) injections for 7 days. Behaviors were recorded in an open field during the first hour post-injection. PCOC rats did not exhibit behavioral anomalies during habituation to injection-stress or placement in the open field. PCOC rats displayed significant alterations in stereotyped responses to acute or intermittent exposure to various doses of AMPH. Within 48 h of the final testing day, striatal tissue was obtained from these animals and electrically-evoked [3H]acetylcholine (ACh) release was measured from striatal slices. Superfusion of tissue slices with various concentrations of AMPH (1-1000 nM) produced dose-dependent inhibition of ACh release in both PSAL and PCOC rats repeatedly injected with saline as adults. However, AMPH-induced inhibition of ACh release was decreased in PCOC rats repeatedly injected with AMPH as adults. At 5 mg/kg AMPH, PCOC rats exhibited increased mortality compared to PSAL rats. These data suggest that PCOC exposure produces long-lasting alterations in nigrostriatal transmission and behaviors mediated by this system. PMID- 10728882 TI - The influence of an endogenous beta3 subunit on recombinant GABA(A) receptor assembly and pharmacology in WSS-1 cells and transiently transfected HEK293 cells. AB - Cell lines are commonly used for studying recombinant heterooligomeric ion channels with defined subunit composition. Such studies often ignore the contribution of endogenous proteins in the assembly of mature channels. We examined whether an endogenous subunit was required for the functional expression of gamma-aminobutyric acid type A (GABA(A)) receptors in WSS-1 cells, HEK293 cells stably expressing recombinant alpha1 and gamma2 subunits. Our pharmacological and RT-PCR analyses of GABA(A) receptors and their mRNAs in WSS-1 cells confirm the presence of alpha1 and gamma2 subunits and suggest the existence of an endogenous beta3 subunit. Whole-cell GABA-evoked currents recorded from untransfected WSS-1 cells were blocked by bicuculline methiodide and enhanced by anesthetics and anticonvulsants including the subunit-selective compounds diazepam and loreclezole. These data suggest that, in addition to the gamma2 subunit, WSS-1 cell receptors also contain beta2/3 subunits. RT-PCR revealed that WSS-1 cells and parental HEK293 cells contain beta3 mRNA. We examined the contribution of the beta3 subunit in the function of receptors formed by expression of alpha1 and gamma2S subunits. Untransfected HEK293 cells were unresponsive to GABA. Cells transfected with alpha1 and gamma2S cDNAs displayed small diazepam and loreclezole responsive GABA-activated currents. By contrast, the expression of alpha1 and gamma2S cDNAs in the neuroblastoma NB41A3 cell line, that lacks beta subunit mRNAs, failed to produce functional receptors. These data reaffirm that alpha1 and gamma2S subunits alone do not form functional GABA(A) receptors and that receptors of WSS-1 cells contain alpha1, beta3 and gamma2S subunits. PMID- 10728883 TI - Mobilisation of intracellular Ca2+ by mGluR5 metabotropic glutamate receptor activation in neonatal rat cultured dorsal root ganglia neurones. AB - The ability of metabotropic glutamate receptor activation to mobilise intracellular calcium was investigated in cultured dorsal root ganglion (DRG) neurones from neonatal rats using the calcium sensitive fluorescent dye Fura-2. L glutamate (10 microM) caused sustained and oscillatory increases in intracellular calcium concentration ([Ca2+]i) in a subpopulation of cultured DRG neurones. The oscillatory responses were not blocked by combined application of the ionotropic glutamate receptor antagonists MK 801 (2 microM) and CNQX (20 microM). Oscillations in [Ca2+]i were also observed following application of the nonselective metabotropic glutamate receptor (mGluR) agonist, trans-(1S,3R)-1 aminocyclopentane-1S, 3R-dicarboxylic acid (1S,3R)-ACPD, 20 microM) and the mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 500 microM). These responses were blocked by the selective Group I mGluR antagonist (RS)-1 aminoindan-1,5-dicarboxylic acid (AIDA) (100 microM) and Ca2+ release channel inhibitors ryanodine (100 microM) and dantrolene (10 microM). The predominantly Group II agonist (2S,2'R,3'R)-2-(2'3'-dicarboxy-cyclopropyl)glycine (DCG-IV, 100 microM) failed to produce Ca2+ transients alone but suppressed responses to CHPG. Reverse transcriptase PCR techniques, using primers specific to Group I mGluRs, revealed the presence of mGluR5 but not mGluR1 mRNA in these cells. Therefore, glutamate can cause a slowly activating and reversible mobilisation of [Ca2+]i in sensory neurones by activation of ionotropic receptors, and can induce oscillatory calcium transients by selectively activating metabotropic glutamate receptors that are likely to be of the mGluR5 subtype. PMID- 10728884 TI - Differential effects of NMDA-receptor antagonists on long-term potentiation and hypoxic/hypoglycaemic excitotoxicity in hippocampal slices. AB - Whole-cell patch clamp recording from cultured hippocampal neurones was used to investigate the NMDA antagonistic effects of the glycineB antagonist 5,7-DCKA and the competitive antagonist CGP 37849. Extracellular field potential recording from area CA1 of hippocampal slices was used to investigate their effects on the induction of LTP and hypoxia/hypoglycaemia-induced suppression of fEPSPs. Additionally, memantine and (+)MK-801 were tested in the later model. 5,7-DCKA inhibited NMDA-induced plateau currents (IC50=0.24+/-0.02 microM) with around nine times higher potency than against peak (IC50=2.14+/-0.17 microM). In contrast, CGP 37849 slowed the onset of NMDA-induced currents considerably and antagonized currents at the time point when the peak component occurred in control responses (IC50=0.18+/-0.01 microM) with around seven times higher potency than against plateau (IC50=1.26+/-0.19 microM). Both 5,7-DCKA and CGP 37849 inhibited the induction of LTP (IC50s=2.53+/-0.13 and 0.37+/-0.04 microM respectively) with potencies close to those inhibiting peak currents in patch clamp studies. 5,7-DCKA and CGP 37849 also blocked the hypoxia/hypoglycaemia induced suppression of fEPSPs but CGP 37849 (EC50=4.3+/-0.33 microM) was far less potent than against the induction of LTP whilst 5,7-DCKA (EC50=1.47+/-0.04 microM) had similar potency in these two models. Memantine and (+)MK-801 also blocked hypoxia/hypoglycaemia-induced suppression of fEPSPs with EC50s of 14.1+/ 0.52 and 0.53+/-0.02 microM respectively. Whereas memantine blocked this effect with similar potency as we previously reported for LTP, (+)MK-801 was four time less potent in this model. The calculated relative therapeutic indices (IC50 LTP over EC50 hypoxia/hypoglycaemia) for 5,7-DCKA, CGP 37849, memantine and (+)MK-801 were 1.72, 0.09, 0.82 and 0.24 respectively. These results show that even in a severe model of hypoxia/hypoglycaemia, glycineB site antagonists and moderate affinity channel blockers exhibit a better therapeutic index than competitive antagonists and high affinity channel blockers. It is likely that in milder forms of pathology the observed differences in therapeutic indices remain the same but the absolute values are expected to be higher. PMID- 10728885 TI - Long-term potentiation in dentate gyrus of the rat is inhibited by the phosphoinositide 3-kinase inhibitor, wortmannin. AB - The pivotal role of inositol phospholipids in cell signalling has been placed centre-stage again with the recognition that phosphoinositide (PI) 3-kinase is implicated in several cellular processes. Stimulation of PI-3 kinase requires activation of the 85 kD regulatory subunit which relies on tyrosine phosphorylation, one consequence of which is activation of the 110 kD catalytic subunit. In this study, we have investigated the role of PI 3-kinase in the expression of long-term potentiation (LTP) in perforant path-granule cell synapses of the rat. We report that intracerebroventricular injection of wortmannin inhibited expression of LTP, though it did not affect the early change in the synaptic response. Activation of PI 3-kinase was enhanced in tetanized tissue prepared from dentate gyrus, compared with untetanized tissue, but this effect was inhibited in tissue prepared from wortmannin-pretreated rats. LTP was associated with increased glutamate release, as previously described, but this effect was also inhibited in tissue prepared from wortmannin-pretreated rats. The results presented demonstrate that wortmannin also exerted an inhibitory effect on KCl-stimulated glutamate release and calcium influx in hippocampal synaptosomes in vitro. The evidence presented is consistent with the hypothesis that PI 3-kinase activation, possibly by NGF, plays a role in expression of LTP in dentate gyrus. PMID- 10728886 TI - The novel NK1 receptor antagonist MK-0869 (L-754,030) and its water soluble phosphoryl prodrug, L-758,298, inhibit acute and delayed cisplatin-induced emesis in ferrets. AB - The anti-emetic profile of the novel brain penetrant tachykinin NK1 receptor antagonist MK-0869 (L-754,030) 2-(R)-(1-(R)-(3,5 bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluor o)phenyl-4-(3-oxo-1,2,4-triazol 5-yl)methylmorpholine and its water soluble prodrug, L-758,298, has been examined against emesis induced by cisplatin in ferrets. In a 4 h observation period, MK 0869 and L-758,298 (3 mg/kg i.v. or p.o.) inhibited the emetic response to cisplatin (10 mg/kg i.v.). The anti-emetic protection afforded by MK-0869 (0.1 mg/kg i.v.) was enhanced by combined treatment with either dexamethasone (20 mg/kg i.v.) or the 5-HT3 receptor antagonist ondansetron (0.1 mg/kg i.v.). In a model of acute and delayed emesis, ferrets were dosed with cisplatin (5 mg/kg i.p.) and the retching and vomiting response recorded for 72 h. Pretreatment with MK-0869 (4-16 mg/kg p.o.) dose-dependently inhibited the emetic response to cisplatin. Once daily treatment with MK-0869 (2 and 4 mg/kg p.o.) completely prevented retching and vomiting in all ferrets tested. Further when daily dosing began at 24 h after cisplatin injection, when the acute phase of emesis had already become established, MK-0869 (4 mg/kg p.o. at 24 and 48 h after cisplatin) prevented retching and vomiting in three out of four ferrets. These data show that MK-0869 and its prodrug, L-758,298, have good activity against cisplatin induced emesis in ferrets and provided a basis for the clinical testing of these agents for the treatment of emesis associated with cancer chemotherapy. PMID- 10728887 TI - Brain uptake and receptor binding of two [11C]labelled selective high affinity NK1-antagonists, GR203040 and GR205171--PET studies in rhesus monkey. AB - Two high affinity and selective NK1-receptor antagonists, GR203040 and GR205171, were labelled with 11C and used in a series of experiments in rhesus monkeys. The purpose of these studies was to evaluate the brain uptake pattern and to explore the potential use of these compounds as PET ligands to characterise NK1-receptor binding. Seventeen studies were carried out with [11C]GR205171 and five experiments with [11C]GR203040, including baseline studies and studies performed after a 5 min infusion of cold compound at doses between 0.05 and 1 mg/kg. Both compounds demonstrated a significant and rapid uptake in the brain, but the uptake of [11C]GR205171 was more than double the uptake of [11C]GR203040. At tracer doses of [11C]GR205171 and all doses of [11C]GR203040 the uptake reached a plateau with no washout during the examination time, whereas [11C]GR205171 after pre-treatment with cold GR205171 showed a significant washout. Using a model with the cerebellum as reference, a method for quantitation was applied to the studies with [11C]GR205171 and the results indicated that the highest specific binding was in the striatum. The pre-treatment dose of cold GR205171 needed for 50% inhibition of binding was less than 0.04 mg/kg. The studies indicated that [11C]GR205171 could be used for the in vivo characterisation of NK1-receptor binding. PMID- 10728888 TI - The influence of nicotinic receptor subunit composition upon agonist, alpha bungarotoxin and insecticide (imidacloprid) binding affinity. AB - A series of cell lines stably expressing recombinant nicotinic acetylcholine receptors (nAChRs) has been established by transfection of mammalian (rat) and insect (Drosophila) nicotinic subunit cDNAs. By equilibrium radioligand binding, we have examined the influence of individual subunits upon the affinity of two nicotinic agonists (epibatidine and methylcarbamylcholine), an antagonist (the snake neurotoxin, alpha-bungarotoxin) and a recently developed chloronicotinyl insecticide (imidacloprid). Imidacloprid bound with very low affinity to the rat alpha4/beta2 nAChR but did so with high affinity to hybrid nAChRs containing Drosophila alpha subunits co-assembled with rat beta2. Of the subunit combinations examined, imidacloprid showed highest affinity binding to nAChRs containing the recently identified Drosophila alpha subunit, D alpha3, co assembled with beta2. In contrast, no specific binding of imidacloprid was detected when D alpha3 was co-expressed with the mammalian neuronal beta4 subunit, or with the muscle-type (gamma or delta) subunits. However, despite the absence of imidacloprid binding to D alpha3/beta4, D alpha3/gamma or D alpha3/delta, these subunit combinations all exhibited high affinity binding of other nicotinic radioligands. Epibatidine showed substantially higher affinity binding to subunit combinations containing neuronal (beta2 or beta4) subunits than it did to combinations containing muscle-type (gamma or delta) subunits. In contrast, alpha-bungarotoxin bound with higher affinity to combinations containing muscle-type subunits. Our results demonstrate that both alpha and non alpha subunits exert a profound influence upon the affinity of nicotinic ligands for recombinant nAChRs. PMID- 10728889 TI - Differential expression, activity and regulation of the sodium/myo-inositol cotransporter in astrocyte cultures from different regions of the rat brain. AB - The high-affinity sodium/myo-inositol cotransporter (SMIT) is involved in osmoregulation in several cells and tissues. In the CNS the activity of SMIT also determines the individual susceptibility of neural cells to the inositol depleting effect of lithium, which is considered to be important in lithium's therapeutic effects in manic-depressive illness. Among neural cells SMIT is particularly active in astrocytes. In the present work we have cloned the cDNA of SMIT of the rat and assessed its activity, expression and regulation in primary astroglia cultures derived from five different rat brain regions: cerebellum, cortex, diencephalon, hippocampus and tegmentum. After an incubation period of 24 h in medium containing 3[H]labeled myo-inositol different steady-state concentrations were detected which were dependent on the brain region from which the astrocytes were cultured. In addition, myo-inositol uptake in astrocytes from different areas was characterized by two different Km values (27 microM for cerebellum and diencephalon, 50 microM for cortex, hippocampus and tegmentum) and by three different v(max) values (approx. 200 pmol/mg protein/min for astrocytes from cerebellum and tegmentum, 298 for hippocampus and 465 for cortex), indicating that the active myo-inositol uptake into astroglial cells is distinct in the various brain regions. The efficacy of uptake as determined by v(max) values of 3[H]myo-inositol uptake correlated with the level of mRNA of SMIT in the astrocyte cultures from the various brain regions as determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Both 3[H]myo-inositol uptake and SMIT mRNA content was upregulated by incubation of astrocytes in medium of increased osmolarity. In astrocytes from cerebellum, cortex, hippocampus and tegmentum 3[H]myo-inositol uptake was downregulated by chronic incubation with 400 microM inositol. This effect was not observed in astrocytes from diencephalon. Furthermore, in astrocytes from cortex and hippocampus but not from cerebellum, diencephalon and tegmentum incubation with corticosterone for three days upregulated 3[H]myo-inositol uptake. It is concluded that SMIT is differentially expressed and regulated in astrocytes from distinct brain regions. These regional differences suggest particular consideration of localized effects in investigations of the role of myo-inositol in the mechanism of action of antibipolar drugs. PMID- 10728890 TI - Diffusion-weighted MRI of infarct growth in a rat photochemical stroke model: effect of lubeluzole. AB - We studied the neuroprotective effect of lubeluzole, a NOS (nitric oxide synthase) pathway modulator, on the development of ischemic damage within the first six hours after a photochemically induced neocortical infarct in rats using diffusion-weighted MRI and Apparent Diffusion Coefficient (ADC) maps. A unilateral photochemical infarct was induced in the hindlimb sensorimotor neocortex of Wistar rats. One hour after infarction, rats received either vehicle (n=10) or lubeluzole (n=11; a 0.31 mg/kg i.v. bolus followed by a one-hour 0.31 mg/kg i.v. infusion). During the first six hours after infarct induction, multislice T2- and Diffusion-Weighted magnetic resonance images (MRI) were obtained to measure percent change of volume of ischemic damage, whereas regional ADC maps were used to measure time-dependent density of ischemic damage. Lubeluzole reduced the percent increase of volume of ischemic damage relative to baseline (at 1 h after infarct induction just before drug treatment), by 18% at 5 and 6 hrs after infarct induction. Lubeluzole attenuated the ADC decreases in the peripheral rim of the infarct, but left the ADC values in the core unaffected. In conclusion, the neuroprotectant lubeluzole attenuates growth of ischemic damage as well as its density in the periphery of a photochemically induced neocortical infarct in rats. PMID- 10728891 TI - The activation of B-Raf and Raf-1 after electroconvulsive shock in the rat hippocampus. AB - We demonstrated that ECS activates the kinase activity of B-Raf and Raf-1 in the rat hippocampus. The activity was maximal at one minute after ECS and temporally coincided with the increased membrane translocation of Rafs and the reported activity of MAPK, but not with the phosphorylation of Rafs. PMID- 10728892 TI - Role of regional neurotransmitter receptors in corticotropin-releasing factor (CRF)-mediated modulation of fear conditioning. AB - We have demonstrated previously that stimulation of hippocampal corticotropin releasing factor (CRF) receptors enhances, whereas stimulation of CRF receptors in the lateral intermediate septum impairs learning, as indicated by fear conditioning. Here, we report that the action of CRF within the hippocampus and lateral septum require muscarinic and D2 receptors, respectively. PMID- 10728893 TI - Intermanual coordination in relation to handedness, familial sinistrality and lateral preferences. AB - Intermanual coordination assessed by alternating finger tapping and finger tapping asymmetry were investigated in 105 healthy right- and 105 left-handers and related to handedness, familial sinistrality and lateral preferences (in hand clasping, arm-folding and eyedness). Compared to right-handers, left-handers with less pronounced left-hand preferences (Subgroup B) showed higher values in intermanual coordination and lower values in finger-tapping asymmetry. Moreover, familial sinistrality and eyedness interacted with handedness effects. While in right-handers intermanual coordination was significantly higher in subjects with dominant left eye, in left-handers of the Subgroup B it was somewhat higher in those with dominant right eye. Higher values in intermanual coordination and reduced asymmetry in finger tapping may be associated with a greater bihemispheric control and better performance in fast bimanual movements. PMID- 10728894 TI - The effects of selective response preparation on corticospinal excitability. AB - OBJECTIVE: Both motor imagery and selective motor cues enhance performance. Motor cortex is activated during motor imagery. We wanted to learn if selective motor cueing also activates motor cortex. METHODS: We gave normal right-handed subjects information about which hand to use to respond to an imperative stimulus (selective intention) or where in space an imperative stimulus would occur (selective attention). To minimize anticipatory responses, warning stimulus validity was 80%. During this choice reaction time task, we recorded magnetic motor evoked potentials. Imperative stimuli and transcranial magnetic stimulation were presented randomly to assess the effect of warning cues on reaction times and corticospinal excitability. RESULTS: Selective intentional and attentional warning cues reduced reaction times, but neither stimulus altered motor evoked potentials. CONCLUSIONS: These results suggest that unlike motor imagery, selective intention to respond to an imperative stimulus and shifting spatial attention to an imperative stimulus do not alter corticospinal excitability. PMID- 10728895 TI - Plasticity and reorganization during language development in children with early brain injury. AB - Although some studies have reported subtle language deficits following early focal brain lesions (EFBL), most studies find no evidence for differential language outcomes as a function of lesion side or lesion type in children with congenital injuries to one side of the brain. However, recent prospective studies of the first stages of language development in English-speaking children with EFBL have reported greater delays in expressive vocabulary in children with left hemisphere damage, particularly if the lesion involves left temporal cortex. In the present study, first stages in the development of word production were studied in 43 Italian children with congenital EFBL, between 13 and 46 months of age. As a group, the EFBL children were markedly delayed in expressive vocabulary. Among children who were in the first stage of language learning, delays were significantly greater with left-hemisphere injury. However, this left right difference was not evident in children who had moved on to the next stage of language development, producing at least some sentences. Discussion centers on the role of developmental plasticity in determining the outcomes of early focal brain injury, suggesting that recovery from initial delays may take place in the early stages of language development, at least for some children. PMID- 10728896 TI - Memory for object locations in Korsakoff's amnesia. AB - Deficits in spatial context memory are an important characteristic of Korsakoff's amnesia. In memory for spatial context information, there is evidence for a functional dissociation of three separate processes: (1) binding of object information to locations (i.e. binding complex memories), (2) exact, metric processing of Euclidean co-ordinates, and (3) an integration mechanism. In the present study, these sub-mechanisms were assessed experimentally in a group of Korsakoff patients (N = 20) and compared to healthy controls (N = 20) to see whether selective deficits can be demonstrated. It was found that Korsakoff patients display deficits on all three spatial-memory conditions, which are not the primary result of deficits in visuo-spatial construction and memory for object identity. No evidence for selective impairments could be observed. These impairments can be linked to damage of diencephalic regions and perhaps the parietal cortex. PMID- 10728897 TI - The neural basis of perceptual and conceptual word priming--a PET study. AB - Positron emission tomography scans were obtained in 13 normal subjects during perceptual and conceptual word priming tasks with the aim to investigate the neural system specific to the two priming conditions. In the prescan phase, subjects were primed perceptually or conceptually with two separate procedures, while in the scan phase, they performed the same stem completion task. Therefore we could compare the results of the two priming tasks in a direct manner. A fixation control task and a baseline task (completion of stems that did not correspond to previously seen words) were also given. A specific blood flow decrease was found in the left inferior temporal cortex in the perceptual word priming condition and in the left superior temporal / inferior parietal cortex in the conceptual word priming condition. Each blood flow change may reflect transient changes in the cortical areas subserving the processing of the perceptual and conceptual components of word priming. PMID- 10728898 TI - "Deaf hearing": unacknowledged detection of auditory stimuli in a patient with cerebral deafness. AB - We describe a patient with the rare disorder of total deafness caused by a bilateral lesion in the temporal lobes and lesions in the central pontine area. Although she displayed no voluntary ability to detect or localize or identify sounds and denied hearing them when asked in writing, she retained some ability to respond reflexively to sounds. When attempts were made to restore awareness of sounds and/or voluntary responses to sounds by drawing her attention to her appropriate orienting head movements her performance improved and she began to respond successfully in a "forced-choice" paradigm. However, even when she became confident at detecting and localizing sounds she remained densely agnosic to their meaning. Her condition of deaf-hearing bears many similarities to that of blindsight. PMID- 10728899 TI - Sensory and response interference by ipsilesional stimuli in tactile extinction. AB - Extinction is thought to be due to a pathologically limited attentional capacity in which multiple stimuli cannot be processed simultaneously to conscious awareness. Patients with tactile extinction are aware of being touched on a contralesional limb, but seem unaware of similar contralesional touch if touched simultaneously on their ipsilesional limb. The ipsilesional stimulus interferes and competes with the processing of the contralesional stimulus. Most theorists assume that the ipsilesional stimulus affects the sensory processing of the contralesional stimulus, although the precise functional level at which this interference occurs is not clear. We report a series of experiments using signal detection analyses to investigate tactile extinction in one patient (DC). These analyses revealed that ipsilesional stimuli, in addition to interfering with processing of contralateral sensations, also interfere with verbal reports of those sensations. This influence on responses suggests that interference in tactile extinction can occur at a post-perceptual level, further 'downstream' than previously thought. PMID- 10728900 TI - Patterns of attentional impairment following closed head injury: a collaborative European study. AB - A comprehensive assessment of both selective (focused attention, divided attention) and intensive (alertness and vigilance) attentional processes was performed on 106 patients with closed head injury using a computerised battery for the evaluation of attention. All patients were tested at least five months after their accident. A high percentage of patients were pathological in tests mapping the selective components of attention while only a minority were impaired on tests mapping the intensive components of attention. Three different subgroups of patients with consistent performance patterns were evidenced. The psychometric characteristics of the battery and its possible clinical usefulness are discussed. PMID- 10728901 TI - Visual-spatial attention in developmental dyslexia. AB - Orienting and focusing of visual attention are two processes strictly involved in reading. They were studied in a group of dyslexic children and normal readers. Shifting of attention by both peripheral and central visual cues was studied by means of the covert orienting paradigm. Focusing, consisting in the ability to control the size of the attentional focus, was investigated using simple reaction times in central vision. Results showed that dyslexics had a specific disability in the shifting of attention caused by a peripheral cue at short SOAs, and were also able to maintain attention focused for short periods of time only, presumably not long enough for efficient visual processing. Our results support the suggestion that visual selective attention deficits in disabled readers may be due to a specific difficulty in orienting and focusing. PMID- 10728902 TI - A case of prosopagnosia following moderate closed head injury with left hemisphere focal lesion. AB - This study describes a left handed woman with prosopagnosia following traumatic brain injury with a focal lesion confined to the left-posterior hemisphere. Few cases of prosopagnosia following unilateral left hemisphere lesions have previously been reported in the literature. Corrected visual acuity was 20/70 (binocular), color vision was intact on screening, and shape detection was borderline. Impairments in higher order visual perception were evident to varying degrees on nonfacial tasks. Matching of unfamiliar faces was very slow but accurate. A marked impairment in the ability to recognize familiar faces and learn new face-name associations was evident on experimental tasks relative to the performance of healthy control subjects. In contrast, identification of characteristics of faces (gender, age) and identification and matching of facial expressions were relatively preserved. We discuss the cognitive processing stages that appear to be disrupted using Bruce and Young's (1986) model of facial recognition and perception as a framework. PMID- 10728903 TI - Naming famous faces and buildings. AB - Reports of a dissociation between impaired retrieval of people's names and spared retrieval of other categories of proper names have led to the suggestion that there are separate brain mechanisms for processing different categories of proper names. An alternative explanation states that geographical proper names (e.g. cities, countries) may be spared in patients with proper name anomia because they are easier to retrieve than people's names. Unlike people's names, geographical names are also used as adjectives. However, this explanation can not explain why retrieval of proper names that are never used as adjectives (e.g. buildings) was spared in some patients. This study investigated whether retrieving people's names is equally difficult as retrieving the names of buildings. Results from normal subjects and closed-head injured patients revealed no differences between retrieval of people's names and buildings' names, and suggested that both categories of proper names are equally vulnerable to brain injury. PMID- 10728904 TI - Mitogenesis in glioblastoma multiforme cell lines: a role for NGF and its TrkA receptors. AB - Neurotrophins have definitive roles in the growth/maintenance of neuronal populations, but their function in malignant gliomas is unknown. The ability for nerve growth factor (NGF) to serve as a mitogenic agent was investigated in several human glioblastoma multiforme (GBM) cell lines, including U251, U87, and U373. In a serum-free medium, the addition of NGF (200 ng/ml) to these cell lines increased cell counts over controls, after 3 days in culture by 9%, 16%, and 33%, respectively. Dose-dependent increases in cell counts and [3H]thymidine uptake were found in the more rigorously investigated U373 cell line. Proteins for both the high affinity NGF-specific tyrosine kinase binding site (p140TrkA; TrkA) and the low affinity neurotrophin (p75NTR) receptor were present in all three GBM cell lines. TrkA mRNA was identified in U373 (only cell line studied). NGF stimulated proliferation was inhibited in a dose-dependent fashion by K252a, a blocker of Trk-induced receptor kinases. NGF, measured by ELISA, was detectable in all GBM cell lines even after 7 days of growth in serum-free medium. These data suggest that GBM cell growth can be enhanced by NGF acting via Trk receptor phosphorylation. Future studies of antiproliferative therapies should consider agents directed against intracellular Trk signaling cascades. PMID- 10728905 TI - ACNU, MTX and 5-FU penetration of rat brain tissue and tumors. AB - The distribution of radio-labeled ACNU, MTX and 5-FU in brain and tumor tissue was studied in female Wistar rats by macroautoradiography after intrathecal administration. In normal rats, ACNU and 5-FU, administered intracisternally, distributed rapidly in the subarachnoid space, ventricular system and cerebrospinal fluid (CSF). 5-FU and MTX penetrated the brain deeply; the diffusional transport of ACNU was limited to a depth of 1 or 2 mm from the CSF surface of the brain. MTX and 5-FU clearance into the blood circulation was rather slow while ACNU cleared relatively quickly. The half time of ACNU, 5-FU and MTX radioactivity at the ventricular surface was 10, 21, and 110 min, respectively, at their maximal concentration after intracisternal administration. In rats with leptomeningeal tumor induced by intracisternal inoculation of Walker 256 cells, the distribution patterns of ACNU, 5-FU, and MTX were essentially the same as in normal rats despite 10-20 cell layers of tumor growing in the subarachnoid space. 5-FU and MTX were able to penetrate tumor masses in the subarachnoid space; MTX penetration was slower than that of 5-FU and ACNU failed to penetrate to more than a depth of 1 or 2 mm from the tumor surface. PMID- 10728906 TI - Differential effect of cycloheximide on neuronal and glioma cells treated with chemotherapy and radiation. AB - Dividing cells and non-dividing cells are distinct in their cell cycle kinetics, and react differently when facing cytotoxic stimuli. A protein synthesis inhibitor, cycloheximide (CHX), has recently been found to protect neuronal cells from oxidative stress. We investigated whether CHX exerts differential effects on dividing and non-dividing cells in the brain under cytotoxic stimuli. Mitotic C6 rat glioma cells and postmitotic neuronal cells were studied with a cytotoxic regimen combining gamma-irradiation (RT) and 1,3-bis,2-chloroethyl-1-nitrosourea (BCNU). Cells were exposed to BCNU (1 g/ml) for 15 h before gamma-irradiation and incubated with CHX (1 g/ml) from 30 min before and until 5 h after irradiation. Clonogenic assay was used to assess cytotoxic effects on C6 glioma cells. LDH assay was used for the viability of H19-7 postmitotic neuronal cells. A 2.27-3.75 fold enhancement of cytotoxicity was noticed with the addition of CHX to BCNU and 2-10 Gy of radiation. Our data demonstrated that CHX enhanced cytotoxicity of RT plus BCNU, while no additional toxicity was incurred to the postmitotic neuronal cells when CHX was added. We further studied whether the inhibition of DNA repair, assayed by single-cell DNA electrophoresis (comet assay), is a contributing factor for the enhanced cytotoxicity on C6 glioma cells. Interestingly, the initial DNA damage after RT plus BCNU was equivalent; whereas DNA repair was significantly less at 5 h after radiation in CHX-treated C6 glioma cells. Protecting non-dividing neuronal cells to avoid excessive functional deficit is an integral part of a successful brain tumor treatment regimen. Taking advantage of the differential effect of CHX on glioma and neuronal cells may improve tumor control without excessive neural toxicity. PMID- 10728907 TI - Brain derived neurotrophic factor protects human neuroblastoma cells from DNA damaging agents. AB - Neurotrophins are required for survival of neurons during development and may act as survival factors to cells undergoing stress. We tested whether brain derived neurotrophic factor (BDNF) protects neuroblastoma (NB) cells from cytotoxic agents using a model NB cell line, NB 1643, which expresses functional tropomyosin related kinase B (TRKB) following treatment with all-trans-retinoic acid. TRKB is the receptor for BDNF. BDNF increases the EC50 values in survival assays for cisplatin, doxorubicin, and topotecan by two to three fold. Thus, BDNF does indeed protect cells drugs that damage DNA. Cisplatin and doxorubicin are used to treat NB. Topotecan is in clinical studies for the treatment of NB. Since these drugs induce DNA damage, we also investigated whether BDNF might afford protection from gamma irradiation. BDNF also induces more than a two fold resistance to gamma irradiation. Since BDNF protects cells from agents with different mechanisms of damaging DNA and resistance, it seems likely that BDNF may alter a common signaling pathway required for cell death initiation by DNA damaging agents. PMID- 10728908 TI - Increased phosphorylation of DNA topoisomerase II in etoposide resistant mutants of human glioma cell line. AB - The efficacy of the epipodophyllotoxins VP-16 and VM-26 is limited by the occurrence of drug resistance in the tumor cell population. Cellular insensitivity to drugs that stabilize the cleavable complex is frequently expressed as multidrug resistance (MDR). In some cell lines, overexpression of MDR-1/P-glycoprotein or the multidrug resistance associated protein (MRP) has been demonstrated and implicated as the mechanism of resistance. Typically, these cells have reduced drug accumulation, secondary to increased drug efflux. In other cell lines, an atypical MDR phenotype has been identified, with the predominant mechanism of resistance shown to be qualitative and/or quantitative changes in the levels and activity of topoisomerase II. For VP-16, increased expression of MDR-1 or MRP and alterations in topoisomerase II have been shown to confer tolerance. To further understand resistance to VP-16, T98G-VP(1000) was initially isolated as a single clone from parental cell, T98G, by exposure to VP 16. Subsequently, a population of cells from this subline was exposed to three fold higher drug concentration allowing stable sublines to be established at higher extracellular drug concentration. Characterization of the resistant sublines demonstrates the adaptation that occurs with advancing drug concentrations during in vitro selections. Reduced topoisomerase II mRNA and protein levels were observed in the initial isolate. This reduction was accompanied by a decrease in topoisomerase II activity and cellular growth rate and was associated with 47-fold resistance to topoisomerase II poisons. With advancing resistance, MRP expression increased, with increased VP-16 efflux and reduced accumulation. This adaptation allowed for partial restoration of topoisomerase II activity secondary to increased expression and hyperphosphorylation, with a resultant increase in growth rate. In this cell line, hyperphosphorylation coincided with increased casein kinase II mRNA protein levels, without increased PKC protein levels, suggesting a role for this kinase in the acquired hyperphosphorylation. In this cell line, hyperphosphorylation mediated the increased activity despite a fall in topoisomerase II protein levels secondary to an acquired 615 bp deletion in one topoisomerase II allele, which resulted in reduced protein levels. In this subline, high levels of resistance were attained as a result of synergism between the reduced topoisomerase II levels and MRP overexpression. These studies demonstrate how cellular adaptation to increasing drug pressure occurs and how more than one mechanism can contribute to the resistant phenotype when increasing selecting pressure is applied. Reduced expression of topoisomerase II is sufficient to confer substantial resistance early in the selection process, with synergy from additional mechanisms helping to confer high levels of resistance. PMID- 10728909 TI - Etoposide with or without mannitol for the treatment of recurrent or primarily unresponsive brain tumors: a Children's Cancer Group Study, CCG-9881. AB - PURPOSE: This study was undertaken to evaluate the response of recurrent brain tumors to intravenous etoposide and to evaluate the efficacy of mannitol in augmenting etoposide's tumoricidal effect. PATIENTS AND METHODS: Ninety-nine children between one and 21 years of age with recurrent brain tumors were randomly assigned to treatment with intravenous etoposide 150 mg/M2, with or without mannitol 15 gm/M2, daily for five days every three weeks for one year or until disease progression or death. Computerized tomographic (CT) or magnetic resonance image (MRI) scans, obtained after three cycles of therapy, were compared with pre-therapy scans. Scans were centrally reviewed. RESULTS: Of 87 evaluable patients, 12 (13.8%) were determined to have had an objective response by the institutional radiologist. On central review, 7/66 (10.6%) responses were documented. Responses in centrally reviewed patients were observed in 2/12 (16.7%) low grade astrocytomas, 4/26 (15.4%) medulloblastoma or primitive neuroectodermal tumors (PNET), 1/13 (7.7%) high grade astrocytomas and 0/15 (0%) brain stem gliomas. Survival at one year was 53% (SE 12%) for low grade astrocytomas, 38% (SE 7%) for medulloblastoma or PNET, 28% (SE 10%) for high grade astrocytomas and 9% (SE 5%) for brain stem gliomas. An effect of mannitol was not observed. CONCLUSION: Intravenous etoposide has a low level of activity in the treatment of recurrent low grade astrocytomas and medulloblastoma or PNET. The efficacy of this agent was not enhanced by the coincident intravenous administration of mannitol. PMID- 10728910 TI - Metastatic medulloblastoma in 10-year-old girl treated successfully with chemotherapy without radiotherapy. AB - We report a case of high risk medulloblastoma with leptomeningeal intracranial and spinal metastasis in a 10-year-old girl treated successfully with conventional prolonged chemotherapy without radiotherapy. This is a particular case of medulloblastoma that at onset did not receive standard therapy for medulloblastoma i.e. neither surgery nor craniospinal irradiation. This 10-year old Chinese girl affected with localized medulloblastoma was previously treated at a medical department in China only with radiotherapy on the posterior fossa. When the child arrived in Italy with progressed metastatic medulloblastoma, she was treated with carboplatin/etoposide association i.v. followed by oral etoposide and partial surgery of the primitive mass. The schedule of chemotherapy was etoposide 300 mg/sqm followed by carboplatin 1000 mg/sqm in one day every 21 28 days for the first six courses, then etoposide 200 mg/sqm and carboplatin 600 mg/sqm in one day every 28-35 days for further 11 courses and oral etoposide 50 mg/sqm/day for ten consecutive days and one week interval between two cycles for one year. At present the girl is alive and disease-free, and has been off-therapy for 31 months. Interestingly, in this case a long-lasting complete remission was obtained without radiotherapy and without myeloablative chemotherapy. Oral etoposide played an important role in achieving a complete remission. PMID- 10728911 TI - Chemotherapy for advanced CNS ependymoma. AB - BACKGROUND: The role of chemotherapy in recurrent ependymoma is poorly defined. This study was performed to help clarify the benefits of chemotherapy in this setting. PATIENTS AND METHODS: We retrospectively reviewed the charts of patients with advanced ependymoma of the CNS who received chemotherapy in our institution between 1974 and 1993, inclusive. Sixteen consecutive patients were treated with regimens containing either nitrosourea, platinum, or other combinations exclusive of nitrosourea or platinum. No patient received nitrosourea and platinum concurrently. Two methods were used to define response. The first was a direct comparison of radiographic images before and after chemotherapy more than one month apart. A second broader definition of response that employed four other criteria in addition to imaging studies (symptoms, signs, performance status, and neurologic functional status) was also used. RESULTS: Results were as follows (response rate by imaging studies followed by response rate by scoring in parenthesis): Platinum-based chemotherapy resulted in a 67% (83%) response rate with 33% (0%) remaining stable. Nitrosourea-based regimens resulted in a 25% (60%) response rate with 50% (10%) remaining stable. When combinations other than platinum or nitrosourea were used, 11% (22%) responded and 56% (44%) remained stable. Relative differences in response rates between chemotherapy regimens persisted when the data were analyzed by grade. Median time to progression was 6, 10, and 3 months, respectively. CONCLUSION: Platinum-based chemotherapy regimens appear to result in higher response rates with lower rates of progression than nitrosourea-based regimens. Other regimens that do not include cisplatinum or nitrosourea appear to be even less effective. PMID- 10728912 TI - Efficacy of proton magnetic resonance spectroscopy in clinical decision making for patients with suspected malignant brain tumors. AB - We wished to determine the utility of single voxel proton (1H) magnetic resonance spectroscopy (MRS) when used as an alternative or adjunct to brain biopsy in patients harboring lesions suggestive of brain tumors identified by MRI scan. Fifteen patients (age 7-58 years) with MRI scans and clinical histories suggestive of primary brain tumors underwent single voxel 1H-MRS. MRS (16 regions of interest in 15 patients) was used to aid in differentiation between tumor and other pathologies such as stroke or demyelinating plaque (n = 6), radiation necrosis (n = 5), or edema (n = 5). Spectra were quantified to determine absolute molar values of N-acetyl aspartate (NAA), choline (Cho), creatine (Cr), lactate (LAC), and myo-inositol (mI), metabolite ratios relative to Cr were calculated, and spectra were interpreted based on metabolite ratios. Subsequent clinical management was based on MRS interpretation, and patients were then followed to determine if MRS interpretation accurately predicted clinical outcome or surgical findings. Mean follow-up was 12.5 months (range 3-28 months). MRS suggested the presence of recurrent tumor in 7 cases, all of which were subsequently 'confirmed' by tumor resection (n = 4) or disease progression (n = 3). MRS suggested the presence of new tumor in 1 case, subsequently confirmed by surgical resection. MRS suggested the presence of necrosis in 3 patients; all 3 remained radiographically stable during the follow-up period, and one was confirmed by stereotactic biopsy. MRS suggested non-neoplastic lesions in 4 cases, 3 of whom were followed until radiographic resolution of lesions and one of which was confirmed as a pyogenic abscess via stereotactic aspiration. Overall, MRS accurately predicted the pathological nature and clinical outcome of lesions in 15/16 (96%) situations, influenced clinical decision making in 12 cases, and altered surgery planning in 7 patients. In appropriate circumstances MRS can reduce the need for biopsy, and provide an important guide for clinical decision making in difficult cases. PMID- 10728914 TI - Cyclic adenosine monophosphate inhibits ursolic acid-induced apoptosis via activation of protein kinase A in human leukaemic HL-60 cells. AB - This study was designed to investigate the effect of cAMP on ursolic acid-induced apoptosis of HL-60 cells. Ursolic acid decreased the viability of the cells in a dose-dependent manner, which was revealed as an apototic process characterized by ladder-pattern DNA fragmentation in agarose gel electrophoresis and segmented nuclei in DAPI-sulpharhodamin 101 staining. Ursolic acid-induced apoptosis of the cells was markedly inhibited by the addition of cAMP-elevating agents including DB-cAMP, CPT-cAMP, 8-Br-cAMP and forskolin. These results were further evidenced by the fact that inhibitors of cAMP-dependent protein kinase including H89 and KT5720 completely inhibited the cAMP-mediated rescue of HL-60 cells from ursolic acid-induced apoptosis. In addition, differentiating agents of the cells such as dimethyl sulfoxide and retinoic acid did not affect the ursolic acid-induced apoptosis of HL-60 cells. These results suggest that signaling pathway of cAMP dependent activation of protein kinase A may affect the responsiveness of tumor cells upon ursolic acid. PMID- 10728915 TI - Effects of anticonvulsants on local anaesthetic-induced neurotoxicity in rats. AB - The effects of various anticonvulsants on local anaesthetics procaine- and lidocaine-induced convulsions were investigated in rats. Pretreatment with diazepam (2.5-5 mg/kg, intraperitoneally) and clonazepam (5-10 mg/kg, intraperitoneally) completely protected the rats against both local anaesthetic induced convulsions. Phenobarbital (12.5-50 mg/kg, subcutaneously) also significantly decreased the incidence of both convulsions and prolonged their latencies. Carbabazepine (10-40 mg/kg, intraperitoneally) did not completely repress both convulsions, but it prolonged their latencies. Phenytoin (5-20 mg/kg, intraperitoneally) and primidone (30-60 mg/kg, intraperitoneally) markedly enhanced both local anaesthetic-induced convulsions, as shown by shortening of latency and increase in mortality. Valproate (100-200 mg/kg, intraperitoneally) produced a protective effect against procaine-induced convulsions, while it strongly enhanced lidocaine-induced convulsions. These results suggest that the benzodiazepines are effective drugs to prevent neurotoxicity induced by local anaesthetics, while phenytoin and primidone potentiate them. PMID- 10728913 TI - Molecular basis of pituitary oncogenesis. AB - Recent advances in the molecular biology has served to unveil the underlying genetic and epigenetic alterations in pituitary adenomas. Three nuclear transcriptional factors, AP-1, CREB, and Pit-1, which are targets of protein kinase C and A, appear to play critical roles in both neoplastic growth and hormone secretion in hormone-producing adenomas. The alteration of G proteins such as Gs and Gi2 is a direct cause of the activation of such transcriptional factors. Autocrine growth factor/cytokine loops also contribute to the augmented signal transductions. Bromocriptine and somatostatin analogs have effects to lower cellular cAMP level through inhibitory G proteins, although the mechanism leading to cellular apoptosis is unknown. On the other hand, most non-functioning adenomas may not have PKC- or PKA-mediated oncogenic mechanisms. Although the loss of Rb and p27Kip1 genes has been demonstrated as a cause of murine pituitary adenomas, the role of tumor suppressor genes for human pituitary adenomas remains elusive. However, potential candidates for the suppressor genes are now emerging. The recently cloned multiple endocrine neoplasia type I gene is one example. Alterations of c-myc/bcl-2, and ras, although rare, appear to be an important cause of the process by which adenoma cells acquire aggressive phenotypes. Further studies on the links between abnormal signal transductions and aberrant tumor suppressor genes will be needed to clarify the whole picture of pituitary oncogenesis. PMID- 10728916 TI - The influence of the nitric oxide synthase inhibitor L-NOARG on the effects of ethanol in rats after acute ethanol administration. AB - The aim of this work was to study the effects of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine (L-NOARG) on the sedative and toxic effects of ethanol in rats. Ethanol at a dose of 3 g/kg, intraperitoneally induced sleep in rats (sleep time: 111.2+/-10.3 min.). Administration of the nitric oxide synthase inhibitor L-NOARG (20 and 40 mg/kg, intraperitoneally) 30 min. before ethanol significantly increased the duration of ethanol-induced sleep. L-NOARG at doses of 20 and 40 mg/kg reduced the exploratory activity of rats in the open-field test and significantly enhanced the sedative effect of ethanol in this test. It is possible that this effect is not caused by the interaction of ethanol with nitric oxide pathways but by synergistic CNS depression caused by ethanol and L NOARG. L-NOARG (20 and 40 mg/kg) had no effect on ethanol concentrations in blood after acute ethanol administration (2 and 3 g/kg). Moreover, the combined administration of ethanol (2 g/kg) and L-NOARG (20 and 40 mg/kg) caused a decrease in the body weight of animals, observed for 14 days. Also, livers of these rats were studied for necrosis and connective tissue reaction. In histological studies L-NOARG at a dose of 40 mg/kg had no effect on hepatic necrosis caused by the acute administration of ethanol but strengthened connective tissue reaction. L-NOARG is widely used in pharmacological studies, including those concerning the effects of ethanol. However, on the basis of our data the possibility of toxic interactions with ethanol should be considered. PMID- 10728917 TI - Beta,gamma-methylene ATP but not alpha,beta-methylene ATP mimics the inhibitory effect of ATP on ventricular automaticity. AB - The effects of alpha,beta- and beta,gamma-methylene ATP on ventricular automaticity induced by local injury in the right ventricle of rats pretreated with reserpine, were investigated and compared with the effects of ATP and adenosine. Beta,gamma-methylene ATP but not alpha,beta-methylene ATP mimicked the inhibitory effect of both ATP and adenosine on the spontaneous automaticity In low concentrations, adenosine was more effective than ATP. Alpha,beta-methylene ATP caused little or no effect on ventricular automaticity. The results suggest that the effect of ATP might depend on its hydrolysis into AMP/adenosine. PMID- 10728918 TI - Differential activities of CYP1A isozymes in hepatic and intestinal microsomes of control and 3-methylcholanthrene-induced rats. AB - Differences in expression of CYP1A isoforms (CYP1A1 and CYP1A2) in liver and small intestine of male Wistar rats and their inducibility by 3 methylcholanthrene as well as the effect of different CYP1A1/1A2 expression on caffeine metabolism were investigated. In rat liver, CYP1A2 is the predominant isoform and CYP1A1 protein expression in liver is significantly increased after treatment by 3-methylcholanthrene. In contrast, only CYP1A1 was detected in control and 3-methylcholanthrene induced small intestine microsomes. Treatment with 3-methylcholanthrene (40 mg/kg intraperitoneally daily during 1, 2, 3 or 4 days) demonstrated that liver CYP1A1 is more sensitive for the induction effects than CYP1A2 and also that significant induction of CYP1A1 in rat small intestine only occurred after 3 to 4 days pretreatment. Caffeine metabolism and inhibition studies by furafylline, CYP1A1 antiserum and ketoconazole revealed that the differences in the expression of CYP1A1 and CYP1A2 in the two tissues led to significant changes in the contribution of the various isoenzymes involved in the biotransformation of caffeine. Whereas in liver paraxanthine formation was almost exclusively catalyzed by CYP1A2, in rat proximal intestine it was formed by CYP1A1. In addition, other CYP enzymes (most probably CYP3A) play a significant role in theobromine and theophylline formation from caffeine in rat intestine. Overall, this study shows different expression and inducibility of CYP1A1/1A2 by 3-methylcholanthrene in rat liver and small intestine. Furthermore in rat intestine cytochrome P450 isozymes such as CYP1A1 and CYP3A replace CYP1A2 in the caffeine metabolism. PMID- 10728919 TI - Effect of loperamide on mucosal guanylyl cyclase activity in rat jejunum following Escherichia coli heat-stable toxin-induced fluid accumulation. AB - Loperamide has antidiarrhoeal activities against secretagogues with different mechanisms of action. Besides its opioid-like effect on intestinal motility and secretion it might exhibit additional antisecretory properties which may not be completely elucidated yet. Direct effects of loperamide on mucosal guanylyl cyclase have never been observed. We therefore investigated the effect of loperamide on intestinal fluid transport altered by heat-stable Escherichia coli enterotoxin which acts by stimulating mucosal guanylyl cyclase. Net fluid movement was determined during a 1 hr incubation period in ligated jejunal loops of anaesthetised female Wistar rats. Transport rates of net fluid movement were calculated from the loop contents measured gravimetrically at the beginning and the end of the experiments. Addition of heat-stable Escherichia coli enterotoxin to the luminal solution resulted in a net secretion of water which was significantly reversed into net absorption by loperamide. The specific activity of the particulate guanylyl cyclase was determined in mucosal scrapings of the jejunum without and with the addition of heat-stable Escherichia coli enterotoxin and/or loperamide. Additions of loperamide of up to 10 micromol/l did not change guanylyl cyclase activity. We conclude that the effect of loperamide counteracting heat-stable Escherichia coli enterotoxin induced changes of intestinal fluid transport does not involve a direct effect on guanylyl cyclase. PMID- 10728920 TI - Induction of apoptosis by lovastatin through activation of caspase-3 and DNase II in leukaemia HL-60 cells. AB - Lovastatin, an HMG-CoA reductase inhibitor, was found to suppress growth and induce apoptosis in culture human promyelocytic leukaemic cell, HL-60. However, the mechanisms of lovastatin-induced apoptosis are still unclear. In this study, we attempted to elucidate the signal transduction pathway for lovastatin-induced apoptosis in HL-60 cells in a dose- and time-dependent manner. The features of this apoptosis were attenuated by the presence of mevalonate, a metabolic intermediate of cholesterol synthesis. Treatment of lovastatin caused a rapid release of mitochondrial cytochrome c into cytosol and subsequent induction of caspase-3, but not caspase-1 activity. Lovastatin also stimulated proteolytic cleavage of poly-(ADP-ribose) polymerase (PARP), and followed by the appearance of caspase activity and DNA fragmentation. Pretreatment with caspase-3 inhibitors, Ac-DEVD-CHO and Z-VAD-FMK, inhibited lovastatin induced caspase-3 activity and DNA fragmentation. Furthermore, we demonstrated that DNase II was involved in the DNA fragmentation induced by lovastatin. These results suggested that the mechanism of lovastatin induced HL-60 cells apoptosis through activation of caspase-3 and DNase II activities. PMID- 10728921 TI - Toxicity study of di(2-ethylhexyl)phthalate (DEHP) in combination with acetone in rats. AB - In two separate studies with exposure duration 9 weeks or 4 weeks, male Wistar rats were dosed with di(2-ethylhexyl)phthalate (DEHP) by gavage and exposed to drinking water with or without acetone (0.5% wt/v in the 9-week study, 1.0% wt/v in the 4-week study). In the 9-week study the doses of DEHP were 0, 125, 250, 500 or 1000 mg/kg b.wt. In the 4-week study the doses of DEHP were increased to 1000, 5000 and 10,000 mg/kg b.wt. In the 9-week study, the relative liver weight was increased in the rats exposed to 500 and 1000 mg/kg b.wt. No interaction of DEHP and acetone was observed in any of the measured parameters. In the 4-week study DEHP, at the highest dose level, resulted in severe general toxicity. The group exposed to DEHP in combination with acetone was more affected. Male fertility was decreased. Body weight was decreased, and the relative weight of the liver, kidney, heart, brain and adrenals increased. The relative weight of the testes decreased in the 5000 and 10,000 mg/kg b.wt. groups. The weight of seminal vesicles and epididymals decreased at 10,000 mg/kg b.wt. In animals exposed to 5000 and 10,000 mg DEHP/kg b.wt. a severe atrophy of the seminiferous tubules and a slight diffuse Leydig's cell hyperplasia was observed. The cellular debris and conglomerates of desquamated cells found in the lumen of the seminiferous tubules were immunostained positive for vimentin. This indicates that Sertoli cell cytoplasm is included in the conglomerates an interesting finding not previously described. No specific interaction of DEHP and acetone was observed in any of the measured parameters. PMID- 10728922 TI - Absence of mRNA encoding estrogen receptor in the rat cochlea. AB - Based on changes in hearing thresholds and tinnitus that are co-related with the menstrual cycle, it has been suggested that the cochlea may respond directly to estrogen. For this to occur, the cochlea should express estrogen receptors. In situ mRNA hybridization was performed on normal female rat cochleas, using radiolabeled RNA probes complementary to mRNA encoding estrogen receptor, to determine whether estrogen receptors are present in the cochlea. Strong hybridization of the riboprobes to sections of uterus and hypothalamus indicated that the technique detected estrogen receptor mRNA. No hybridization to any cochlear tissues was observed. The results indicate that estrogen receptors are not expressed on cochlear cells, at least in rats. This in turn suggests that variation in cochlear responses during the estrus cycle are not the result of the direct effect of estrogen on the cochlea. Such variation may, however, be caused by systemic changes in fluid regulation induced by estrogen receptors at a distant site, or by other hormone receptors. PMID- 10728923 TI - A temporal bone study of posterior semicircular canal resection for exposure of the internal auditory canal (PSCC resection). AB - Partial resection of the labyrinth is becoming accepted as a means of improving access to the internal auditory canal and central skull base neoplasms. In this investigation, an infralabyrinthine approach was performed on 20 temporal bones. The dissection was extended by transection of the endolymphatic duct, then excision of the posterior semicircular canal. The maximal lateral exposure of the internal auditory canal (IAC) was measured after each manoeuvre. Resection of the posterior semicircular canal increased lateral exposure in 7/20 specimens to an average 61% of the length of the IAC. Posterior canal resection improved superior exposure and increased the circumference of exposure in all specimens. PMID- 10728924 TI - Recording of mechano-electrical transduction currents by a nystatin perforated patch method. AB - Mechano-electrical transduction (MET) currents in isolated cochlear hair cells of chicks were recorded by use of a nystatin perforated-patch method. The membrane of a cell-attached patch was permeabilized by nystatin in the patch pipette, thus providing electrical continuity between the pipette and the cytoplasm of the cell without loss of cytoplasmic compounds. The current-voltage relationship was linear for the inward-going MET current at negative membrane potentials, but outward currents were reduced at positive membrane potentials, evidence of inward going rectification. Elevation of the intracellular concentration of calcium at positive membrane potential, mediated via a voltage-dependent Ca2+ channel, may suppress the outward-going MET current by acting from within the cell. PMID- 10728925 TI - Cow's milk allergy is associated with recurrent otitis media during childhood. AB - To determine whether cow's milk allergy (CMA) in infancy is associated with recurrent otitis media (ROM) or other chronic ear infections, we conducted a cohort study by enrolling 56 milk-allergic and 204 control schoolchildren. We also studied the association between ear problems and different atopic manifestations. A higher proportion of children with CMA had had ROM. defined as at least 15 acute otitis media episodes by the age of 10 years (27%, vs 12%, p = 0.009), and had undergone adenoidectomy and or tympanostomy compared with the controls (48%, vs 28%, p = 0.005). However, this was only true of the children who had developed respiratory atopy. Asthma and/or allergic rhinitis, but not atopic dermatitis, posed a significant risk for ROM, while all the three atopic manifestations enhanced the risk for secretory otitis media. Positive skin prick tests with food, but not with inhaled allergens, tended to be associated with ear problems. In conclusion, we found that children with CMA in infancy, even when properly treated, had experienced significantly more ROM, the risk associating with concomitant development of respiratory atopy. PMID- 10728926 TI - Immunohistochemical study of cell proliferation using BrdU labelling on tympanic membrane, external auditory canal and induced cholesteatoma in Mongolian gerbils. AB - The Mongolian gerbil is a well-known animal model for induction of aural cholesteatomas. This animal model is useful for studying changes in the keratinizing epithelium. It is not known whether keratin accumulation can increase the proliferative activity of the keratinizing epithelium in tympanic membrane and meatal skin. In this study, we investigated the proliferative activity of the epidermis in induced aural cholesteatoma at various stages and in different areas of the tympanic membrane and meatal skin in normal gerbils. Anti 5-bromo-2- deoxyuridine (BrdU) was injected intraperitoneally to detect the proliferative activity of keratinizing epithelium. Immunohistochemistry with monoclonal BrdU antibody in the normal gerbil showed intense immunolabelled keratinocytes at the handle of malleus, and the superior parts of pars tensa and pars flaccida. Also, mitotic activity in the deep meatal skin was more active than in the lateral meatal skin. The induced aural cholesteatoma showed more active proliferation centre of the epithelial cell than eardrum and external ear canal of the normal gerbil. These observations suggest that the accumulation of the keratin debris might induce changes of the cellular proliferation in the external auditory meatus. PMID- 10728927 TI - Middle ear pressure: effect of body position and sleep. AB - The aim of this study was to investigate whether a middle ear (ME) pressure rise noted during sleep is an effect of the recumbent position, the state of sleep or Eustachian tube (ET) function, respectively. Eleven subjects with no history of ear disease were subjected to continuous, 24-h, direct ME pressure measurements and tubal function tests, respectively. At the start of the measurements the subjects were in the erect position. This position was maintained for at least 2 h (during which they were permitted to conduct normal everyday activities). The subjects then rested in the recumbent position for at least 2 h, after which they again resumed the erect position with normal activities for a period of at least 2 h. Then the subjects went to sleep in the recumbent position. A mean pressure rise in the ME of 36.4 daPa was seen during sleep compared with being awake in the same position. The rise was not caused by tubal opening and active insufflation of air via the ET. The number of tubal openings was significantly fewer during sleep compared with resting awake and the erect position, respectively. The ME pressure was not significantly different in the erect position compared with the recumbent position while resting awake. Tubal function tests demonstrated results in accordance with normal tubal function for all subjects. In conclusion, the state of sleep induced a ME pressure rise, not the recumbent position per se. These findings support the significance of a two directional gas diffusion for regulation of the ME pressure. PMID- 10728928 TI - Vestibulo-ocular reflex in patients with Meniere's disease between attacks. AB - In Meniere's disease, spontaneous nystagmus beating toward the affected ear (ipsilateral nystagmus) is frequently observed especially during vertiginous periods. VOR against horizontal rotation was recorded in 19 patients with Meniere's disease exhibiting ispilateral beating nystagmus, and the dynamic aspect of vestibular function during vertiginous periods was examined by determining VOR gain and directional preponderance (DP). The patients sat on a rotation chair and were passively rotated at about 0.3-0.6 Hz. The maximal head velocity ranged from 80 to 120 deg/s and the duration of rotation from 20 to 30 s. In most patients exhibiting ipsilateral nystagmus, VOR gain toward the affected side was higher than that toward the intact side, indicating that the peripheral vestibular system on the affected side could still respond to head movements and that its dynamic function was increased. This asymmetry disappeared along with disappearance of ipsilateral nystagmus. During the period with contralateral nystagmus, VOR gain toward the affected side became lower than that toward the intact side. These findings indicate that alterations in the dynamic property of the peripheral vestibular system may correlate with the directional change of spontaneous nystagmus. However, the degree of VOR DP (VOR DP%) did not correlate with the slow phase velocity of spontaneous nystagmus, indicating that observation of spontaneous nystagmus alone cannot determine with precision the degree of imbalance in the dynamic aspect of vestibular function. PMID- 10728929 TI - Influence of head position on the vestibulo-ocular reflex during rotational testing. AB - The influence of two static head positions on the horizontal nystagmus induced by sinusoidal rotational stimulation in a group of 24 healthy subjects was investigated. The rotation test was performed with the subject's head in the primary position (upright) and with the head 30 degree pitched down. Computerized electronystagmography was used to measure the maximum slow component velocity of the horizontal nystagmus and to calculate gain, phase and asymmetry. The vestibulo-ocular reflex (VOR) gain obtained during rotation with the head in the primary position was significantly higher than with the head bent 30 degree forward. No other significant differences were found. PMID- 10728930 TI - Vestibular neuritis: a follow-up study. AB - Seven to eight years after a disease period of vestibular neuritis 19 patients previously examined at our department were given a questionnaire about whether they had experienced any audiovestibular symptoms since they first fell ill. All 19 patients responded and 18 consented to participate in the long-term follow-up study. The patients were subjected to a clinical evaluation, a bithermal caloric test and audiological examinations with stapedius reflex measurements with use of the same technique as at the first examinations. Ten of the 18 patients had experienced recurrent vestibular symptoms with sensation of dysequilibrium and vertigo. Six of these 10 patients also had periods of positional vertigo. The remaining nine patients were free of symptoms. None of the patients had noticed any change in their hearing ability. The mean caloric side difference at the 7- or 8-year follow-up was not significantly higher in the group of patients with symptoms than in the group without symptoms. Neither did the group of patients with pathologically elevated stapedius reflex thresholds at onset display a larger mean caloric side difference than the group of patients with normal thresholds. However, a small caloric side difference at onset of the disease served as a predictor for residual vestibular symptoms. It is speculated whether the group of patients with residual symptoms had a different pathophysiological localization of their disease within the vestibular system than the group of patients with no symptoms at follow-up. PMID- 10728931 TI - Spatial and temporal patterns of evoked neural activity from auditory nuclei in chick brainstem detected by optical recording. AB - In order to detect the spatial patterning of the auditory projection of the embryonic chick brainstem, anatomical methods such as orthograde transport of horseradish peroxidase have been used. However, these methods do not provide the continuous information required about the absolute value and time-course of varying neural excitement. Furthermore, the use of conventional electrophysiological methods makes it difficult or impossible to detect the transmembrane voltage change because of the small size and fragility of the cells of the young chick brainstem. We thus believe that optical measurement of membrane potential might be beneficial in circumstances where electrodes are difficult to use for reasons of cell size, complexity, or membrane topology. In the present work, we therefore examined the feasibility of an optical method for delineating the synaptic transmission of afferent input in the auditory nuclei in the chick brainstem. We used embryonic chick brainstem slice preparations featuring an intact eighth nerve, and loaded depolarizing square current pulses from tungsten microelectrodes into the auditory nerve for stimulation of these preparations. In this approach, we used a multiple-site optical recording system comprising a 16 x 16-element photodiode array and a voltage sensitive dye (NK 2761). Neural excitation evoked by stimulation to the left auditory nerve was propagated to the dorsal side of the brainstem. This area in which the optical signal was detected is located on the auditory nuclei. Since the physiological spatial patterning of the auditory nerve projection could be roughly estimated by the optical technique, the technique is considered useful for examining the electrical activity generated from auditory nuclei in the brainstem. This is the first report of spatial patterning of auditory neurons in the embryonic chick brainstem generated through optical recording. PMID- 10728932 TI - Effects of TNF-alpha and IL-1 beta on mucin, lysozyme, IL-6 and IL-8 in passage-2 normal human nasal epithelial cells. AB - Little is known about the regulatory effects of cytokines on various nasal secretions in normal human nasal epithelial cells. The aim of this study was to examine whether TNF-alpha, IL-1beta or their combination can increase the secretion of mucin as an indicator of mucous secretion, the secretion of lysozyme as an indicator of serous secretion and the secretion of IL-6 and IL-8 as important cytokines. In addition, we wanted to examine their message levels in normal human nasal epithelium. On day 12 of culture, passage-2 normal human nasal epithelial cells were treated with 10 ng/ml TNF-alpha, 10 ng/ml IL-1beta and combinations of both. Twenty-four hours later, the apical secretions were collected. A mixture of TNF-alpha and IL-1beta synergistically increased secretion of mucin, IL-6 and IL-8, but did not increase secretion of lysozyme. A combination of TNF-alpha and IL-1beta showed a questionable increase of MUC2 mRNA levels. TNF-alpha, IL-1beta and a combination of both all significantly increased MUC8 mRNA levels. Neither TNF-alpha, IL-1beta nor a combination of both increased MUC5AC, MUC5B and lysozyme mRNA levels. IL-1beta alone or a combination of TNF alpha and IL-1beta comparably increased IL-6 and IL-8 mRNA levels slightly. In conclusion, a mixture of inflammatory mediators can synergistically increase secretion of mucin, IL-6 and IL-8 in human nasal epithelium. Accordingly, nasal secretions may be under the control of an inflammatory mediator network. PMID- 10728933 TI - Effect of submucosal diathermy to the inferior turbinates on unilateral and total nasal airflow in patients with rhinitis. AB - The efficacy of the controversial treatment of submucosal diathermy to the inferior turbinates (SMDIT) was evaluated objectively. Twenty-seven patients with chronic rhinitis were investigated by hourly posterior rhinomanometry to assess changes in total and minimum (Fmin) and maximum (Fmax) unilateral nasal airflow over 5 h, before and 2-3 months after standardized SMDIT treatment. Nasal airflow was recorded at a sample pressure of 75 Pa and the results are reported as medians with interquartile range. Whilst SMDIT caused a significant 51% increase (p < 0.0001) in total nasal airflow from 246 cm3/s (131) to 371 cm3/s (133) the changes in unilateral airflow provided further evidence which strongly supported the benefits of this operation. Unilateral Fmin significantly increased by 136% (p < 0.0001) from 69 cm3/s (82) to 163 cm3/s (74) and Fmax significantly increased by 23% (p < 0.0001) from 171 cm3/s (74) to 211 cm3/s (59). The effect of surgery was to "splint" to the turbinate in a state of relative vasoconstriction. Our findings therefore provide functional evidence of submucosal fibrosis following SMDIT. The greater percentage change in unilateral Fmin suggests that this parameter is a more sensitive index of the effect of nasal surgery than total nasal airflow measurements. The importance of considering the nose as two separate airways in the evaluation of nasal treatments is emphasized. PMID- 10728934 TI - A-mode ultrasound in the diagnosis of chronic polypous sinusitis. AB - A-mode ultrasound (A-US) is a simple, non-invasive and non-ionizing method for detecting fluid or even mucosal swelling in inflamed maxillary and frontal sinuses. A-US has been shown to be a quite reliable tool in the diagnosis of acute maxillary sinusitis. However, controversy still exists over the reliability of A-US in detecting fluid retention or mucosal swelling in patients suffering from chronic polypous rhinosinusitis or in transantrally operated maxillary sinuses. We have compared the results of maxillary sinus A-US with computed tomography (CT) images in a selected series of chronic polypous rhinosinusitis comprising 40 patients. Fluid retention was seen in 20 of 79 maxillary sinuses on CT scanning. Only 6 of these 20 retentions were detected with A-US. There were 11 false positive findings. In six of these cases a back-wall echo was received through polypoid masses in the sinus. Mucosal swelling was also difficult to diagnose. The results of A-US were not easily reproduced; only in 50% of cases were identical results obtained by two investigators. We do not recommend the use of A-US to diagnose fluid retention or mucosal swelling in a patient with chronic mucosal changes in the maxillary sinus or if surgery has been performed on the anterior wall of the maxillary sinus. PMID- 10728935 TI - Evaluation of rhinostereometry compared with acoustic rhinometry. AB - Measurement of the nasal mucosa is a challenging task. There are many different methods; each with advantages and disadvantages. In the last decade two new methods have been used extensively: acoustic rhinometry and rhinostereometry. Many studies with rhinostereometry have shown interesting results. However, there have been doubts about this method, since only a few investigators have used it, and it has never been compared with other methods. On the other hand, the acoustic rhinometer has been compared with many other methods. In this study, we compare the results of measurements with acoustic rhinometry and rhinostereometry. Thirty patients with vasomotor rhinitis participated in the study. They were challenged with three histamine concentrations on two occasions. resulting in 180 observations with each method. The results were compared with each other using the linear correlation test, and showed a poor but significant correlation (p < 0.01. r = 0.25). We conclude that acoustic rhinometry and rhinostereometry are sensitive methods for studying nasal mucosal swelling, but that there is poor correlation between the two methods. PMID- 10728936 TI - Heavy snorer's disease: a progressive local neuropathy. AB - "Heavy snorer's disease" is defined as progression from heavy snoring to obstructive sleep apnoea syndrome (OSAS). Apart from significant weight gain, the aetiology underlying progression to a collapse of the upper airways during inspiration and sleep remains unclear. Previous studies have shown that nocturnal respiratory disturbances became worse, even in some OSAS patients who did not gain weight. The patency of the upper airways depends on the balance between the negative intrapharyngeal pressure developed during inspiration and its counteraction by dilating muscles. The reflexogenic dilation is probably mediated by afferent nerve endings in the pharyngeal mucosa. Chronic vibration of a tissue may cause neuronal damage. Therefore, the hypothesis that snoring per se might cause progressive pharyngeal nerve lesion has been tested in a series of studies from the Karolinska Institute, Stockholm, which, along with other studies, will be reviewed here. In these studies it was found that a majority of patients with heavy snoring and different degrees of respiratory disturbance had signs of pharyngeal afferent and efferent (motor) nerve lesions. These lesions may cause the collapse of upper airways in OSAS. Since it is not known which "heavy snorer" will develop OSAS, early effective prevention and or treatment of snoring is called for. PMID- 10728937 TI - Internal thermistors in differentiating between oral and nasal breathing during sleep. AB - To select patients with sleep apnoea hypopnoea syndrome (SAHS) who will benefit from surgery, we use information from four different pressure sensors in the upper airways and oesophagus during polysomnography (PSG). These pressure sensors also have the ability to act as internal thermistors and can hence indirectly measure flow as well as pressure. This new method for measuring flow has proven to be very accurate for scoring hypopnoeas as well as apnoeas. The aim of this study was to determine whether the flow and pressure sensors located in the epipharynx, oropharynx and hypopharynx could differentiate between nasal and oral breathing. The design was a prospective cross-over study in 124 patients referred to the hospital for SAHS diagnosis. The awake patients were asked to breathe first through the nose and then through the mouth while the nose was blocked with a clip. A standard nocturnal PSG with pressure and flow measurement in the upper airways was performed in all patients. The procedure was repeated in a lateral position, and again the next morning in 32 of the patients. Reduction in flow signals from the nose was calculated, and a paired t-test was performed for statistical analysis. The difference between nasal and oral breathing was quite distinct in the flow tracings from the internal thermistors. The mean reduction in nasal flow signals when changing from nasal to oral breathing was 83.7% (SD 14.5, p < 0.0001). The same was seen in a lateral position, 82.2% (SD 16.4, p < 0.0001). Testing for changes in properties of the internal thermistors revealed no significant difference between the reduction in flow when the test started and after the patients had slept the whole night (p > 0.1). It is possible to differentiate between nasal and oral breathing using internal thermistors. PMID- 10728938 TI - Immunoglobulin-secreting cells in the surface secretion on the pharyngeal tonsils. AB - As B-lymphocytes on the pharyngeal tonsils constitute a considerable part of the leukocytes in the surface secretion, and their biological role is obscure, we explored their possible function with respect to immunoglobulin production. Twenty children scheduled for routine adenoidectomy participated. Surface secretion from 10 children was analysed for presence of plasma cells and cells from the secretions of the other 10 children were tested in enzyme-linked immunosorbent spot assays (ELISPOT-assays) for their capacity to secrete and produce IgA, IgM and IgG. Plasma cells and cells that secreted IgA, IgM and IgG respectively were present in the secretions of all tested children. In eight of ten children the IgG immunocytes, Ig-producing blasts and plasma cells. outnumbered the IgA immunocytes. The number of immunoglobulin secreting cells (ISCs) was reduced by half or more in cell suspensions exposed to the reversible protein synthesis inhibitor cycloheximide. It is concluded that immunocytes that produce and secrete immunoglobulin are present in the surface secretion on the pharyngeal tonsils. The production represents an addition to the immunoglobulins transported to the secretion by the poly-Ig receptor and by passive diffusion. The results shed new light on the pathogenesis of mucosal infections in the upper airways. PMID- 10728939 TI - Tolerance and efficacy of interfering alpha-streptococci in recurrence of streptococcal pharyngotonsillitis: a placebo-controlled study. AB - A total of 342 patients with clinical signs of tonsillitis and suspected group A beta-haemolytic streptococci (GAS) aetiology, verified with rapid test and GAS culture, were enrolled in a randomized, placebo-controlled, double-blind, multicentre study. They received antibiotic treatment for 10 days, followed by 10 days of alpha-streptococcal or placebo spray treatment in the ratio of 2 : 1. Pharyngeal status, throat culture and adverse events were investigated up to 75 days after treatment. The frequency of bacteriologically verified clinical recurrence was 13% in the alpha-streptococcal group and 15% in the placebo group at the follow-up on day 22. The corresponding figures at the last valid visit after 45-75 days were 19% and 30%, respectively, a statistically significant difference (p = 0.037). Furthermore, at the last valid visit 5% of subjects in the alpha-streptococcal and 12% in the placebo group were healthy carriers, bacteriological treatment failures, of GAS (p = 0.029). Treatment with alpha streptococci and placebo spray were equally well tolerated. Thus, re-colonization with alpha-streptococci seem to hinder late recurrences of GAS pharyngotonsillitis. PMID- 10728940 TI - Increased levels of urokinase receptor in plasma of head and neck squamous cell carcinoma patients. AB - Urokinase-type plasminogen activator (uPA) is important for matrix degradation and motility of cancer cells. The binding of uPA to its cell surface receptor on cancer cells is essential for effective invasion. A soluble form of urokinase receptor (suPAR) has been described in serum and ascites of ovarian cancer patients and in plasma samples of non-small cell lung cancer patients. Plasma samples from 36 head and neck squamous cell carcinoma patients and 24 healthy control persons were analysed for the presence of suPAR using enzyme-linked immunosorbent assay (ELISA) and the expression levels were correlated with clinical and histopathological data. Significantly elevated levels of suPAR in blood plasma from head and neck cancer patients were observed (p = 0.000), and the suPAR plasma levels decreased after resection of the carcinoma in 8 of 11 patients. suPAR plasma levels of cancer patients showed no significant correlations with T staging, metastasis, recurrence or differentiation stage of the tumours. The significance of suPAR plasma levels in head and neck squamous cell carcinoma patients for prognosis of the disease is discussed. PMID- 10728941 TI - Relation between myocardial viability and abnormalities on the signal-averaged electrocardiogram in patients with low (<40%) ejection fraction and coronary artery disease. AB - To determine a possible mechanism for the previously observed improved outcome after bypass surgery in patients with poor ventricular function and viable myocardium, we sought to examine the relation between the extent of viability and the frequency of an abnormal signal-averaged electrocardiogram (SAECG) in patients with reduced ejection fraction and coronary artery disease. Fifty-two patients with coronary disease and ventricular dysfunction underwent quantitative redistribution thallium-201 (Tl-201) scintigraphy at rest to determine the extent of viability. The presence of late potentials was assessed by SAECG. Long-term, cardiac event-free survival was determined. Patients with greater viability (group 1, n = 23) were similar to patients with less viability (group 2, n = 29) with respect to age, gender, ejection fraction, and incidence of arrhythmia. Fewer group 1 patients had late potentials (33% vs. 65%, p = 0.05) and individual parameters were significantly more abnormal in the group 2 patients. Patients with late potentials had less viability than patients without late potentials (viability index 0.61+/-0.15 vs. 0.69+/-0.14, respectively, p = 0.05). By multivariate analysis, only the extent of viable myocardium and the left ventricular end-diastolic pressure were independent predictors of late potentials. Survival free of cardiac death or transplantation was better in patients with a normal SAECG (p<0.04) and in patients with predominantly viable myocardium (p<0.005). Thus, patients with low ejection fraction, coronary disease, and viable myocardium have a lower frequency of late potentials, suggesting reduced susceptibility to ventricular arrhythmia. PMID- 10728942 TI - Usefulness of the severity and extent of wall motion abnormalities as prognostic markers of an adverse outcome after a first myocardial infarction treated with thrombolytic therapy. AB - The prognostic value of wall motion score index (WMSI), assessed at predischarge after a first acute myocardial infarction (AMI) in the thrombolytic era, is still not well known. One-hundred forty-four consecutive patients with a first AMI treated with thrombolytic therapy underwent exercise testing and echocardiography at rest before discharge and were followed-up for a mean period of 18 months. During follow-up, there were 32 cardiac events (12 patients had cardiac deaths, 8 had unstable angina pectoris, 1 had nonfatal reinfarction, and 11 patients had congestive heart failure). The patients who experienced any cardiac event had a higher WMSI (1.67+/-0.15 vs. 1.30+/-0.16, p<0.0001), a higher end-systolic volume (75.1+/-34 vs. 59.5+/-22 ml, p<0.01), and a lower ejection fraction (47+/-16% vs. 55+/-10%, p<0.001) at predischarge than patients without events. The incidence of a positive predischarge exercise testing did not differ between patients with and without cardiac events (22% vs. 24%, p = NS). Multivariate Cox regression analysis, including clinical, exercise results, and echocardiographic parameters, showed that the most powerful predictor of a subsequent event was a resting WMSI > or =1.50 before discharge (chi-square 17.8, p<0.0001). Thus, in patients with a first AMI who underwent thrombolysis, the severity and extent of echocardiographically detected wall motion abnormalities are important independent predictors of cardiac events. PMID- 10728943 TI - Maximal exercise systolic pressure, exercise training, and mortality in myocardial infarction patients. AB - The relation of maximal exercise systolic pressure to physical conditioning and to mortality was determined in 641 men with > or =1 myocardial infarctions. Each performed a standardized multistage exercise test before randomized assignment either to an exercise group or a control group and at scheduled periodic intervals over 3 years. This study compares 123 men with maximal exercise systolic pressures (MESP) of < or =140 mm Hg with 518 men whose maximal exercise systolic pressure was > or =140 mm Hg. At baseline, the 2 groups were comparable for age, entry time since the occurrence of the qualifying cardiac event, and reported use of antihypertensive medications. Men with low MESP used more beta blockers, had lower systolic pressure measurements at rest and by definition at maximal exercise, and lower work capacity than men with higher levels of MESP. Men with low MESP experienced: (1) no reduction in mortality with exercise conditioning (p<0.86), and (2) a significantly higher mortality rate over 3 years (p<0.003) compared with men with higher levels of MESP. The relation of a low MESP to mortality persisted: (1) whether MESP or work capacity increased from the baseline exercise test to the last performed exercise test, and (2) whether it was measured at low (<6 METs) or high (> or =6 METs) levels of work capacity. We conclude that low maximal exercise systolic blood pressure is a predictor of mortality and is associated with an ineffective training response in men with myocardial infarction. PMID- 10728944 TI - Comparison of troponin T versus creatine kinase-MB in suspected acute coronary syndromes. AB - Limitations of creatine kinase-MB (CK-MB) have led to alternative biochemical markers, including troponin T (TnT), to detect myocardial necrosis. Limited data are available regarding the predictive value of this new marker in patients with chest pain of uncertain etiology. Therefore, we prospectively compared CK-MB and TnT in a broad population with suspected acute coronary syndromes, including those admitted to a short-stay chest pain unit. CK-MB, quantitative TnT levels, and a rapid bedside assay were performed at 0, 4, 8, and 16 hours. Adverse events, including infarction, recurrent ischemia, coronary surgery, need for catheterization and/or intervention, stroke, congestive heart failure, or death, were identified by chart review and by follow-up phone call at 6 months. Of 707 patients, 104 were excluded for creatinine >2 mg/dl or incomplete data, leaving a total cohort of 603 patients. Coronary Care Unit admissions were 18%, intermediate care admissions were 14%, telemetry admissions is 21%, and admissions to 24-hour short-stay area were 47%. TnT (at 0.1 ng/ml) and CK-MB were positive in a similar proportion of patients (20.4% and 19.7%, respectively); however, the patients identified by TnT and CK-MB were not identical. In-hospital adverse events occurred in 37.1% with no differences in positive predictive value for the markers (p = NS). If CK-MB and TnT were negative, the early adverse event rate was 27%. No cardiac marker was positive by 16 hours in 54.9% of patients with an adverse event. Six-month follow-up was obtained in 576 of the 603 patients (95.5%). One hundred fifty-five late adverse events occurred in 134 patients (23.3%) at an average of 3.3+/-2.5 months after discharge. If both markers were negative, the late event rate was 20.2% and did not increase in patients with positive CK-MB or TnT >0.2 ng/ml. However, the late event rate was substantially higher (52.9%) in those with intermediate TnT levels of 0.1 to 0.2 ng/ml (p = 0.002). Thus, TnT is a suitable alternative to CK-MB in patients with suspected acute coronary syndromes. The rapid bedside assay is comparable to quantitative TnT and may enable early diagnosis and triage. A negative cardiac marker value (TnT or CK-MB) does not necessarily confer a low risk of complication in patients presenting with acute chest pain to an emergency department. PMID- 10728945 TI - Timing of and risk factors for myocardial ischemic events after percutaneous coronary intervention (IMPACT-II). Integrilin to Minimize Platelet Aggregation and Coronary Thrombosis. AB - We studied both the time course and risk factors for adverse clinical events after percutaneous coronary intervention (PCI). Such information is critical to clinical decision-making, but scant quantitative data exist to describe the time course of these adverse outcomes. Patients enrolled in the Integrilin to Minimize Platelet Aggregation and Coronary Thrombosis-II (IMPACT-II) trial were analyzed. Patients undergoing elective, urgent, or emergency PCI (n = 4,010) were randomized to receive either placebo or 1 of 2 eptifibatide regimens during intervention. We evaluated the time to the primary end point of the trial, the 30 day composite of death, myocardial infarction, repeat nonelective PCI, nonelective bypass surgery, or stenting for abrupt closure. Adverse events occurred in 407 patients (10.1%). Because the risk of events declined substantially between 6 and 9 hours (66% occurred within 6 hours), events were classified as occurring before or after 6 hours. Independent predictors of "early" events included dissection, pre- and postprocedural coronary blood flow, side-branch occlusion, procedural thrombolytic use, previous bypass, presentation with unstable angina, absence of diabetes, and hyperlipidemia. The predictors of "late" events included lower weight, increased baseline heart rate, coronary dissection, and procedural thrombolytic use. The risk of ischemic events were greatest immediately after PCI and rapidly declined, so that by 9 hours the hazard function plot was flat; 66% of events occurred within 6 hours of PCI. Knowledge of the risk factors for early and late events help risk-stratify patients before and after intervention for myocardial ischemic events. PMID- 10728946 TI - Efficacy of abciximab readministration in coronary intervention. AB - Abciximab, an Fab monoclonal antibody fragment that blocks the platelet glycoprotein IIb/IIIa receptor, is increasingly used as an adjunct to coronary intervention. Little is known, however, about the efficacy and safety of readministration of abciximab. This study examined and characterized outcomes of patients receiving abciximab for a second time. From April 1995 to June 1997, 164 consecutive patients were readministered abciximab at our 3 institutions. We retrospectively examined and analyzed in-hospital outcomes in this cohort. The median time to readministration was 95 days. The angiographic success rate of percutaneous intervention was 99.5%. Rates and 95% confidence intervals of in hospital events were death 2% (0.7% to 6.1%), myocardial infarction 3% (1% to 7%), coronary bypass surgery 0% (0% to 2.2%), and intracranial hemorrhage 2% (0.4% to 5.3%). Severe thrombocytopenia was observed in 4% of patients (1.4% to 7.8%) after readministration. Allergic or anaphylactic reactions were not observed. Major bleeding was associated with excessive concomitant antithrombotic therapy. Patients undergoing readministration of abciximab within 2 weeks of first administration experienced a higher incidence of severe thrombocytopenia (12% vs. 2%, p = 0.046). Thus, abciximab remains clinically efficacious when readministered as an adjunct to percutaneous coronary intervention. However, concomitant heparin administration must be carefully monitored and warfarin therapy should be avoided. Vigilant surveillance for thrombocytopenia should be employed. Reduced dosing may be necessary when abciximab is readministered within days of the initial administration. PMID- 10728947 TI - Intravascular ultrasonic predictors of angiographic restenosis after long coronary stenting. AB - The intravascular ultrasound (IVUS) criteria for stent optimization have not been determined in stenting long lesions. We evaluated the predictors of angiographic restenosis and compared it with stent lumen cross-sectional area (CSA) and stent length between short (stent length <20 mm) and long (> or =20 mm) coronary stenting. IVUS-guided coronary stenting was successfully performed in 285 consecutive patients with 304 native coronary lesions. Six-month follow-up angiogram was performed in 236 patients (82.8%) with 246 lesions (80.9%). Results were evaluated using conventional (clinical, angiographic, and IVUS) methods. The overall angiographic restenosis rate was 22.8% (56 of 246 lesions) (short stent 17.6% vs. long stent 32.2%, p = 0.009). Using multivariate logistic regression analysis, the independent predictors of angiographic restenosis were the IVUS stent lumen CSA (odds ratio 1.51, 95% confidence intervals 1.18 to 1.92, p = 0.001) and stent length (odds ratio 0.95, 95% confidence intervals 0.91 to 1.00, p = 0.039). The angiographic restenosis rate was 54.8% for stent lumen CSA of <5.0 mm2 (short stent 37.5% vs. long stent 73.3%, p = 0.049), 27.4% for CSA between 5.0 and 7.0 mm2 (short stent 24.1% vs. long stent 31.7%, p = 0.409), 10.5% for CSA between 7.0 and 9.0 mm2 (short stent 10.0% vs. long stent 12.5%, p = 0.772), and 11.4% for stent lumen CSA of > or =9.0 mm2 (short stent 10.4% vs. long stent 13.3%, p = 0.767) (p = 0.001). Compared with short coronary stenting, long coronary stenting is effective treatment modality to cover long lesions with comparable long-term clinical outcomes in cases of stent lumen CSA of > or =7.0 mm2. Regardless of the stent length, the most important factor determining angiographic restenosis was the IVUS stent lumen CSA in relatively large coronary artery lesions. PMID- 10728948 TI - Outcomes following interventions in small coronary arteries with the use of hand crimped Palmaz-Schatz stents. AB - Although coronary stenting has been shown to be effective, retrospective studies have suggested that stents do not provide better results than angioplasty in small coronary arteries. We sought to examine procedural, in-hospital, and long term outcomes of patients undergoing small-vessel stenting with Palmaz-Schatz stents hand-crimped on a balloon catheter <3 mm in diameter. We retrospectively analyzed the outcomes of 117 patients who underwent this type of coronary stent implantation at Duke University Medical Center between January 1, 1997 and May 30, 1998. The clinical indications for percutaneous revascularization included unstable angina in 67.5% of patients, acute myocardial infarction in 4.3%, postinfarct angina in 3.4%, silent ischemia in 3.4%, and stable angina in 1% of patients. Quantitative angiographic analysis was performed immediately before angioplasty and after stent implantation. Stents were used for elective indications in 24%, for suboptimal angiographic result in 61.5%, and for abrupt and/or threatened closure in 14.5% of patients. Reference vessel diameter was similar before and after the procedure. Minimum luminal diameter increased from 0.63 to 2.35 mm, an acute gain of 1.72+/-0.43 mm. Percent stenosis decreased from 74.2% to 4.7%. The clinical composite of death (n = 1, 1%), nonfatal myocardial infarction (n = 6, 5.1%), and revascularization (n = 1, 1%) occurred in-hospital in only 8 patients (6.8%), resulting in clinical procedure success in 109 patients (93%). Our data suggest that stents designed for vessels >3.0 mm can be deployed in small vessels, with a low in-hospital event rate. However, target lesion revascularization in small vessels remains high. Development of antiproliferative strategies could improve long-term outcomes for small-vessel interventions. PMID- 10728949 TI - Association between elevated plasma fibrinogen and the small, dense low-density lipoprotein phenotype among postmenopausal women. AB - A predominance of small, dense low-density lipoprotein (LDL) particles (subclass pattern B) has been associated with a 2- to threefold increase in coronary heart disease risk. Recently, it has been reported that LDL subclass pattern B is associated with hyperfibrinogenemia, which is also a coronary heart disease risk factor. The present study examined the relation between hyperfibrinogenemia and LDL subclass pattern in 258 postmenopausal women. A significant univariate correlation was observed between the concentration of cholesterol carried in small, dense LDL particles and plasma fibrinogen concentration (r = 0.17, p = 0.01). The prevalence of LDL subclass pattern B was 41.9% in the highest fibrinogen tertile, compared with 27.9% and 24.4% in the first and second tertiles, respectively (global chi-square 6.8, p = 0.03). The crude odds ratio (OR) for LDL subclass pattern B among women in the highest fibrinogen tertile, compared with the lower tertiles, was 2.03 (95% confidence interval [CI] 1.18 to 3.51, p = 0.01). After adjustment for age and plasma lipids (log(e) triglycerides, LDL cholesterol, and high-density lipoprotein cholesterol), the OR was 2.14 (95% CI 1.17 to 3.96, p = 0.01). Further adjustment for hematocrit, indicators of carbohydrate homeostasis, body mass index, waist circumference, and several variables related to lifestyle did not attenuate this association (OR 2.56, 95% CI 1.27 to 5.27, p = 0.01). These data suggest that hyperfibrinogenemia and LDL subclass pattern B may be 2 components of a common syndrome and suggest that hyperfibrinogenemia may contribute to the increased coronary heart disease risk associated with LDL subclass pattern B. PMID- 10728950 TI - Clinical and genetic variables associated with acute arousal and nonarousal related cardiac events among subjects with long QT syndrome. AB - In patients with the long QT syndrome (LQTS), the occurrence of cardiac events (syncope or cardiac arrest) is frequently associated with acute arousal caused by exercise, swimming, emotion, or noise. However, cardiac events may also occur during sleep or with ordinary daily activities. The purpose of this study was to determine whether there are differential clinical, electrocardiographic, and genetic features among LQTS patients who experienced cardiac events with and without acute arousal. We identified 1,325 patients with cardiac events from the International LQTS Registry. Based on the precipitating conditions of the first event, 427 patients were classified as arousal, 345 as nonarousal, and the remaining 553 were unknown (not classifiable). Gene linkage was known in 78 of the 772 patients with classifiable first events. The age at first cardiac event was significantly younger in the arousal than the nonarousal group (11.7 vs. 15.5 years, respectively; p<0.001). The arousal-type patients had a higher rate of subsequent cardiac events during follow-up after the index event than the nonarousal-type patients (p = 0.02). Arousal-related cardiac events occurred in 85% of LQT1, 67% of LQT2, and 33% of LQT3 patients (p = 0.008). This study provides evidence that the genotype is an important determinant of the LQTS phenotype in terms of arousal and nonarousal-related cardiac events. PMID- 10728951 TI - Effectiveness of amiodarone as a single oral dose for recent-onset atrial fibrillation. AB - The efficacy of amiodarone has been proved in long-term maintenance of sinus rhythm (SR) in patients with paroxysmal atrial fibrillation (AF). The present study evaluates the efficacy and safety of a single oral dose of amiodarone in patients with recent-onset AF (<48 hours). Seventy-two patients were randomized to receive 30 mg/kg of either amiodarone or placebo. Conversion to SR was verified by 24-hour Holter monitoring. Ten patients were excluded because of SR in the beginning of monitoring or technical failure during Holter monitoring. The remaining study groups were comparable (n = 31 for each), except that in the placebo group beta blockers were more common. The patients receiving amiodarone converted to SR more effectively than those receiving placebo (p<0.0001). At 8 hours, approximately 50% of patients in the amiodarone group and 20% in the placebo group (Holter successful) had converted to SR, whereas after 24 hours the corresponding figures were 87% and 35%, respectively. The median time for conversion (8.7 hours for amiodarone and 7.9 hours for placebo) did not differ in the groups. Amiodarone was hemodynamically well tolerated, and the number of adverse events in the study groups was similar. Amiodarone as a single oral dose of 30 mg/kg appears to be effective and safe in patients with recent-onset AF. PMID- 10728952 TI - Left ventricular filling patterns in patients with systemic hypertension and left ventricular hypertrophy (the LIFE study). Losartan Intervention For Endpoint. AB - Abnormal left ventricular (LV) filling may exist in early stages of hypertension. Whether this finding is related to LV hypertrophy is currently controversial. This study was undertaken to assess relations between abnormal diastolic LV filling and LV geometry in a large series of hypertensive patients with electrocardiographic LV hypertrophy. M-mode, 2-dimensional, and pulsed Doppler echocardiographic recordings of mitral inflow velocity and isovolumetric relaxation time (IVRT) were obtained in 750 patients with stage I to III hypertension and LV hypertrophy determined by electrocardiography (sex-adjusted Cornell voltage duration criteria or Sokolow-Lyon voltage criteria) after 14 days of placebo treatment. The patients' mean age was 67+/-7 years and 44% were women. One hundred forty patients (19%) had normal LV geometric pattern, 79 (11%) had concentric remodeling, 342 (45%) had eccentric LV hypertrophy, and 189 (25%) had concentric LV hypertrophy. A normal LV filling pattern was found in 116 patients (16%), abnormal relaxation in 519 (69%), "pseudonormal" filling was found in 83 (11%), and a restrictive filling pattern in 32 (4%). Prolonged IVRT was associated with LV hypertrophy (p<0.01) as well as elevated relative wall thickness (p<0.05). A stronger difference (p<0.01) in IVRT was found between groups with and without LV hypertrophy. Multiple regression analysis revealed that increased LV mass correlated with prolonged IVRT, whereas LV mass and geometry were not associated with peak early LV filling velocity (E), peak atrial filling velocity (A) ratio or mitral valve E-peak deceleration time, although IVRT was found to be an independent correlate of E/A ratio and deceleration time. Thus, abnormal IVRT was highly prevalent in all LV geometric subgroups among hypertensive patients with electrocardiographic LV hypertrophy, even in those with normal LV geometry determined by echocardiography. We found that IVRT differed significantly among patient groups with different LV geometric patterns, primarily because of the association of IVRT to LV mass. PMID- 10728953 TI - Impact of left ventricular diastolic dysfunction on maximal treadmill performance in normotensive subjects with well-controlled type 2 diabetes mellitus. AB - Patients with type 2 diabetes often have impaired exercise capacity compared with nondiabetic subjects. Left ventricular (LV) diastolic dysfunction has been shown to limit exercise performance in nondiabetic subjects. Men with well-controlled type 2 diabetes were divided into 2 groups: normal LV diastolic function (group 1, n = 9) or LV diastolic dysfunction (group 2, n = 10) based on standard echocardiographic criteria using pulmonary veins and transmitral flow recordings. They were matched for age and had no evidence of systemic hypertension, macroalbuminuria, coronary artery disease, congestive heart failure, clinical diabetic complications, and thyroid disease. Good metabolic control was demonstrated by glycated hemoglobin levels of 6.7+/-1.6% and 6.6+/-2.5% (means +/ SD) in patients with LV diastolic dysfunction and in controls, respectively. Each subject performed a symptom-limited modified Bruce protocol treadmill exercise test. Maximal treadmill performance was higher in subjects with normal diastolic function compared with subjects with LV diastolic dysfunction when expressed in time (803+/-29 vs. 662+/-44 seconds, respectively, p<0.02) or in METs (11.4+/-1.2 vs. 9.5+/-1.9 METs, respectively, p<0.02). Moreover, there was a correlation between E/A ratio and exercise duration (r = 0.64, p = 0.004) or E/A ratio and METs (r = 0.658, p = 0.003). There were no significant differences in maximal heart rate, maximal systolic and diastolic blood pressure, or maximal rate-pressure product attained during the exercise test. In conclusion, this study demonstrated that LV diastolic dysfunction influences maximal treadmill performance and could explain lower maximal performance observed in patients with type 2 diabetes. PMID- 10728954 TI - Usefulness of hypotension during dobutamine echocardiography in predicting perioperative cardiac events. AB - This study was undertaken to determine the prognostic significance of hypotension induced during preoperative dobutamine stress echocardiography (DSE) before vascular and noncardiac thoracic surgery. Wall motion abnormality during DSE predicts perioperative risk. Although hypotension during DSE has not been shown to correlate with the presence or severity of coronary artery disease, its significance in perioperative risk assessment is unknown. We retrospectively studied 300 patients who had DSE within 6 months of noncardiac surgery. Perioperative events including death, myocardial infarction, ischemia, and arrhythmias were recorded. Odds ratios with 95% confidence intervals were used to examine the association between clinical and echocardiographic variables and perioperative events. A hypotensive response during DSE was seen in 85 patients (28%). Forty-eight patients (16%) had 54 perioperative complications including 4 cardiac-related deaths, 10 myocardial infarctions, 12 myocardial ischemic events, and 28 arrhythmias. Hypotension during DSE was predictive of the combined end point of perioperative cardiac mortality, myocardial infarction, and ischemia (odds ratio 4.04, 95% confidence interval 1.72 to 9.51). In a multivariate logistic regression model, hypotension during DSE remained a significant predictor (odds ratio 4.10, p<0.01). DSE-related hypotension was predictive of perioperative cardiac events and therefore may have a role in risk stratification before vascular or noncardiac thoracic surgery. PMID- 10728955 TI - Treating isolated low high-density lipoprotein cholesterol: prescient or premature? PMID- 10728956 TI - Prediction of the site of total occlusion in the left anterior descending coronary artery using admission electrocardiogram in anterior wall acute myocardial infarction. AB - In anterior acute myocardial infarction, ST elevation in aVL and ST depression in II, III, and aVF predict a culprit lesion in the left anterior descending artery proximal to the origin of the first diagonal branch, with good specificity and positive predictive value. Inferior ST depression is not related to remote ischemia but represents an electrocardiographic phenomenon reciprocal to ST elevation in aVL. PMID- 10728957 TI - Increased platelet reactivity in patients given orbofiban after an acute coronary syndrome: an OPUS-TIMI 16 substudy. Orbofiban in Patients with Unstable coronary syndromes. Thrombolysis In Myocardial Infarction. AB - Patients receiving the oral glycoprotein IIb-IIIa inhibitor orbofiban in the OPUS -TIMI 16 trial had a paradoxical increase in platelet reactivity with respect to both fibrinogen binding and alpha-granule degranulation, suggesting a mechanism for the lack of efficacy of orbofiban in the trial. Thus, sensitive assays of platelet reactivity may be helpful in the design of further clinical studies and implementation of antiplatelet therapy in patients. PMID- 10728959 TI - Frequency of collateral blood flow in the infarct-related coronary artery in rupture of the ventricular septum after acute myocardial infarction. AB - Patients with postinfarction ventricular septal rupture have poor residual or collateral blood flow in the infarct artery and do not benefit from ischemic preconditioning. This suggests that rupture of the ventricular septum occurs on an unprotected and unprepared myocardium. PMID- 10728958 TI - Formation of platelet aggregates after attacks of coronary spastic angina pectoris. AB - Using a novel laser-light scattering method, we examined platelet aggregability, especially small-sized platelet aggregates, at baseline and after spontaneous coronary spastic attacks in 14 patients with coronary spastic angina, and before and after anginal attacks during an exercise test in 11 patients with stable exertional angina. The number of small-sized platelet aggregates after coronary spastic anginal attacks increased significantly, but not in patients with stable exertional angina. These results imply that an increase in the number of small sized platelet aggregates from coronary spasm may be a trigger for coronary thrombosis via medium- and large-sized platelet aggregates. PMID- 10728960 TI - Serum cholesterol level, cigarette smoking, and vasomotor responses to L-arginine in narrowed epicardial coronary arteries. AB - We examined the impact of serum cholesterol and cigarette smoking on the coronary vasomotor effects of L-arginine in patients with atherosclerotic coronary artery disease. The dilation of proximal and distal segments in response to low-dose L arginine was greater in patients with a serum cholesterol level < or =200 mg/dl than in patients with a level >200 mg/dl, whereas the response was the same in smokers and nonsmokers. PMID- 10728961 TI - Clinical and cost comparison of ibutilide and direct-current cardioversion for atrial fibrillation and flutter. AB - The clinical effectiveness and cost to convert recent-onset atrial fibrillation or flutter to sinus after 3 to 4 weeks of anticoagulation with intravenous ibutilide was compared with direct-current cardioversion. The low success rate with ibutilide made direct-current cardioversion the more clinical and cost effective method to restore sinus rhythm. PMID- 10728962 TI - Vasopressin in the treatment of milrinone-induced hypotension in severe heart failure. AB - The use of phosphodiesterase inhibitors such as milrinone in the treatment of severe heart failure is frequently restricted because they cause vasodilation and hypotension. In patients with decompensated heart failure with hypotension after treatment with milrinone, low doses of vasopressin restored blood pressure without inhibiting the inotropic effect of milrinone. PMID- 10728963 TI - Beta-blocker titration failure is independent of severity of heart failure. AB - In the present study, predictors of complicated initiation of beta blockade in patients with idiopathic dilated cardiomyopathy was studied. We found that generally accepted measures of severity of heart failure are not predictable, whereas low systolic blood pressure (< or =120 mm Hg) was the strongest predictor for problematic (up)titration. PMID- 10728964 TI - Pulmonary capillary wedge pressure in mitral stenosis accurately reflects mean left atrial pressure but overestimates transmitral gradient. AB - Current opinion varies as to whether pulmonary capillary wedge pressure assessment of transmitral gradient in mitral stenosis is accurate; we therefore compared transmitral gradient in 36 patients awaiting balloon valvuloplasty using both pulmonary capillary wedge pressure and direct left atrial pressure. Mean pulmonary capillary wedge pressure correlated well with mean left atrial pressure (limits of agreement -1.5 to +3.7 mm Hg), but mean diastolic mitral gradient calculated using pulmonary capillary wedge pressure differed significantly from that calculated using left atrial pressure (limits of agreement -1.2 to +9.8 mm Hg): wedge pressure-assessed transmitral gradient is therefore misleading, routinely overestimating stenosis severity. PMID- 10728966 TI - Comparison of central venous and inferior vena caval pressures. AB - Inferior vena caval pressures were measured in 60 patients undergoing cardiac catheterization and compared with central venous pressure from within the right atrium. Mean pressures within the abdominal inferior vena cava were essentially the same as mean right atrial pressure, suggesting that the inferior vena cava provides a useful safe alternative for measuring central venous pressure. PMID- 10728965 TI - Single-photon emission computed tomographic myocardial perfusion imaging in patients with mitral valve prolapse. AB - The possibility of exercise-induced myocardial ischemia due to mitral valve prolapse (MVP) in the absence of coronary artery disease was evaluated with single-photon emission computed tomographic analysis using thallium-201 and technetium-99m sestamibi in 72 patients with MVP. Exercise electrocardiography was positive in 5 patients (8%), but single-photon emission computed tomography was found to be normal in all patients, and exercise-induced chest pain, electrocardiographic changes, and arrhythmias were found not to be related to myocardial ischemia in patients with MVP. PMID- 10728967 TI - Life-saving needle aspiration of a cardiac-compressing pericardial cyst. PMID- 10728968 TI - Case 3: A patient with systemic hypertension and left ventricular hypertrophy. AB - Hypertension is often referred to as the "silent killer" because most hypertensive patients are asymptomatic until cardiovascular sequelae such as stroke, myocardial infarction, heart failure, or renal failure occur. LVH is a common finding in patients with hypertension, especially African-Americans. Data from the Framingham Heart Study indicate that LVH is an independent risk factor for major cardiovascular events. In the Amlodipine Cardiovascular Community Trial, 37% of 124 hypertensive patients screened by means of echocardiography had LVH at baseline. Although there was no difference in the prevalence of LVH by gender or age, African-American patients were nearly twice as likely to have LVH than white patients (64% vs. 34%, p<0.05). Hence, aggressive therapy to reach target goals outlined in the Sixth Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC VI) is especially important in this group of patients. Even lifestyle modifications such as weight reduction and limitation of salt intake, if sufficiently aggressive, can lead to regression of LVH, as demonstrated by results of the Treatment of Mild Hypertension Study (TOMHS). Most classes of antihypertensive drugs are effective in causing regression of LVH. Vasodilators, such as minoxidil and hydralazine, do not have an effect on regression, possibly because reflex tachycardia and stimulation of catecholamines and the renin-angiotensin system associated with these agents may negate the benefit of reduced afterload. There is some controversy regarding the ability of the angiotensin receptor blockers to reduce LVH. In some studies, these agents were associated with regression, whereas in others they were not. Whether targeting LVH as the primary treatment goal in hypertensive patients will have long-term benefits on outcome above and beyond simply reducing blood pressure is not clear. PMID- 10728969 TI - Prion protein is not detectable in dental pulp from patients with Creutzfeldt Jakob disease. PMID- 10728970 TI - Adenoviral-mediated gene transfer to mouse salivary glands. AB - Adenoviral vectors effectively transfer genes to rat salivary glands. However, potent immune responses limit their use in vivo. Mice offer more opportunities than rats for the study of these immune processes. We first established conditions for infection of mouse salivary glands, with an adenoviral vector. The effects of time, viral dose, viral diluent buffer volume, and dexamethasone on expression of a transgene, luciferase, were determined by means of the recombinant vector AdCMVluc. Optimal luciferase expression was observed when the vector was suspended in 50 microL of buffer. This volume completely filled the gland parenchyma and slightly distended the capsule. Dexamethasone increased immediate transgene expression and reduced the acute inflammation one day following viral administration, but did not alter subsequent mononuclear inflammation or transgene expression 14 or 28 days later. An adenoviral vector encoding either anti-inflammatory cytokine IL-4 or IL-10 was co-administered with AdCMVluc to increase transgene expression at 14 and 28 days. While this strategy did not extend the duration of luciferase expression, co-administration of AdCMVIL-10 with AdCMVluc almost completely eliminated the chronic inflammatory infiltrate in the glands after 28 days. This study demonstrates that adenoviral mediated gene transfer to mouse submandibular glands is possible by intraductal cannulation and that reduction of either the acute or chronic inflammatory infiltrates was insufficient to increase long-term transgene expression in this tissue. PMID- 10728971 TI - Evidence of chemical bonding at biomaterial-hard tissue interfaces. AB - For many years, glass-polyalkenoate cements have been described as possessing the unique properties of self-adherence to human hard tissues, such as bones or teeth. However, direct experimental evidence to prove the existence of chemical bonding has not been advanced. X-ray Photoelectron Spectroscopy (XPS) was used to analyze the chemical interaction of a synthesized polyalkenoic acid with enamel and synthetic hydroxyapatite. For both enamel and hydroxyapatite, the peak representing the carboxyl groups of the polyalkenoic acid was detected to have significantly shifted to a lower binding energy. De-convolution of this shifted peak disclosed two components with a peak representing unreacted carboxyl groups and a peak suggesting chemical bonding to hydroxyapatite. On average, 67.5% of the carboxyl groups of the polyalkenoic acid were measured to have bonded to hydroxyapatite. XPS of hydroxyapatite also disclosed its surface to be enriched in calcium and decreased in phosphorus, indicating that phosphorus was extracted at a relatively higher rate than calcium. Analysis of these data supports the mechanism in which carboxylic groups replace phosphate ions (PO4(3-)) of the substrate and make ionic bonds with calcium ions of hydroxyapatite. It is concluded that an ultrathin layer of a polyalkenoic acid can be prepared on a hydroxyapatite-based substrate by careful removal of non-bonded molecules. With this specimen-processing method, XPS not only provided direct evidence of chemical bonding, but also enabled us to quantify the percentages of functional groups of the polyalkenoic acids that bonded to calcium of hydroxyapatite. PMID- 10728972 TI - Design and wear testing of a temporomandibular joint prosthesis articulation. AB - As part of the development of a total temporomandibular joint prosthesis, a prosthesis articulation was designed. The articulation consists of a spherical head (ball) of the mandibular part, rotating against an enveloping ultra-high molecular-weight polyethylene (UHMWPE) disc with a flat cranial side, which slides along the opposing skull part. The aim of this study was to determine the in vitro wear rate of the articulation, and to predict the in vivo wear rate from the results. Based on a disc thickness of 5 mm and a ball diameter of 8 mm, the stresses within the disc were calculated by means of a finite element computer model. The wear rate of the ball-disc articulation was determined by in vitro wear tests, with a stainless-steel ball rotating against a UHMWPE disc in a serum based lubricant. Eight discs were tested for seven million cycles each. The in vitro wear rate of the disc-skull part articulation was calculated from the test results of the ball-disc articulation. The maximum Von Mises' stress was less than the yield strength of UHMWPE and, therefore, was sufficiently low. The in vitro wear rate of the ball-disc articulation was 0.47 mm3 per million cycles. The in vivo expected total wear rate is 0.65 mm3 per year, corresponding with a yearly decrease of disc thickness of 0.0094 mm. Although it is difficult to judge whether this wear rate is sufficiently low, because the influence of UHMWPE wear particles in the TMJ region is unknown, both the expected wear rate and the decrease of thickness appear to be acceptable. PMID- 10728973 TI - Lifetime-limiting strength degradation from contact fatigue in dental ceramics. AB - The hypothesis under examination in this paper is that the lifetimes of dental restorations are limited by the accumulation of contact damage during oral function; and, moreover, that strengths of dental ceramics are significantly lower after multi-cycle loading than after single-cycle loading. Accordingly, indentation damage and associated strength degradation from multi-cycle contacts with spherical indenters in water are evaluated in four dental ceramics: "aesthetic" ceramics-porcelain and micaceous glass-ceramic (MGC), and "structural" ceramics-glass-infiltrated alumina and yttria-stabilized tetragonal zirconia polycrystal (Y-TZP). At large numbers of contact cycles, all materials show an abrupt transition in damage mode, consisting of strongly enhanced damage inside the contact area and attendant initiation of radial cracks outside. This transition in damage mode is not observed in comparative static loading tests, attesting to a strong mechanical component in the fatigue mechanism. Radial cracks, once formed, lead to rapid degradation in strength properties, signaling the end of the useful lifetime of the material. Strength degradation from multi cycle contacts is examined in the test materials, after indentation at loads from 200 to 3000 N up to 10(6) cycles. Degradation occurs in the porcelain and MGC after approximately 10(4) cycles at loads as low as 200 N; comparable degradation in the alumina and Y-TZP requires loads higher than 500 N, well above the clinically significant range. PMID- 10728974 TI - Molecular mapping of statherin- and histatin-binding domains in human salivary mucin MG1 (MUC5B) by the yeast two-hybrid system. AB - MGI is a high-molecular-weight mucin secreted by mucous acinar cells in human submandibular and sublingual glands. We have recently shown that the tracheobronchial mucin MUC5B is a major component of MG1. MUC5B is organized into cysteine-rich N- and C-terminal regions that flank a central tandem-repeat region containing cysteine-rich subdomains and imperfect 29-residue tandem repeats. In earlier work, we have shown that this mucin selectively forms heterotypic complexes with amylase, proline-rich proteins, statherin, and histatins in salivary secretions, and the aim of this study was to identify specific binding domains within MUC5B using the yeast two-hybrid system. Interactions of cysteine rich domains in the tandem-repeat region (Cys1-Cys4) and C-terminal region (Cys8a, Cys8b, Cys8c) of MUC5B with statherin and histatins were investigated. These studies indicated that histatin 1 selectively bound to Cysl and Cys2, whereas statherin and histatin 1, 3, and 5 selectively bound to Cys8a. Analysis of the primary sequences of the identified binding domains suggests that these domains most probably can fold into globular-like structures in the native mucin. A ProDom blast search revealed that sequences in Cys1, Cys2, and Cys8a exhibit similarity to domains in evolutionarily diverse extracellular proteins known to participate in a wide variety of protein-protein interactions. PMID- 10728975 TI - Human gingival crevicular fluid contains MRP8 (S100A8) and MRP14 (S100A9), two calcium-binding proteins of the S100 family. AB - Human gingival crevicular fluid contains unidentified proteins which might play a role as markers in periodontal diseases. Therefore, low-molecular-weight proteins found in human gingival crevicular fluid (GCF), but absent from serum, were identified in the present study by means of two-dimensional electrophoresis (2-D PAGE) analysis. GCF, serum, and whole saliva were collected from periodontitis and healthy subjects, as well as from edentulous and newborn subjects. Protein samples were separated by two-dimensional polyacrylamide gel electrophoresis, stained with silver, and compared with reference protein maps in the SWISS-2D PAGE database. In GCF and saliva from periodontitis patients and healthy subjects, four dominant low-molecular-mass (from 8 to 14 kDa) acidic spots were observed. They were not found in serum and were less visible in saliva from edentulous and newborn subjects. From N-terminal amino acid sequencing, the two 2 D protein spots of 8 kDa and isoelectric points between 6.5 and 7.0 were both identified as protein MRP8 (SI00A8), a member of the S100 family of calcium binding proteins. Using peptide mass fingerprinting and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), we identified the other two protein spots, with mass of 14 kDa and isoelectric points between 5.5 and 6.0, as protein MRP14 (S100A9), also belonging to the S100 family. The presence of MRP8 and MRP14 in GCF was confirmed by Western blot, with monoclonal antibodies. The two polypeptides, MRP8 and MRP14, identified in GCF represent the major difference between the 2-D PAGE patterns of serum and GCF, and we hypothesize that they may play an important role in the gingival sulcus and could represent possible markers for periodontal diseases. PMID- 10728976 TI - Prostaglandin E2 predominantly induces production of hepatocyte growth factor/scatter factor in human dental pulp in acute inflammation. AB - Hepatocyte growth factor (HGF), which is also known as the scatter factor, is a broad-spectrum and multifunctional cytokine, mediates epithelial-mesenchyme interaction, and is shown to be involved in the development and regeneration of various tissues, including tooth. Here, we report that HGF was present in adult human dental pulps, and its levels increased during acute inflammation of the tissue. Levels of HGF mRNA in dental pulps also increased with inflammation, as determined by reverse-transcription/polymerase chain-reaction. The production of HGF in fibroblasts from dental pulps in culture was dose-dependently stimulated by inflammatory cytokines such as interleukin (IL)-1alpha and tumor necrosis factor (TNF)-alpha, and by prostaglandin (PG) E2, as determined by an enzyme linked immunosorbent assay. We also showed that indomethacin did not affect the increase in HGF production by the cells with IL-1alpha, TNF-alpha, and PGE2. The levels of HGF mRNA in the cells were simultaneously increased by these stimulants, as determined by Northern blotting. Since the production of PGs is known to increase at the beginning of inflammation, PGE2 may be involved in the regeneration of dental pulps by the induction of HGF expression after inflammation. PMID- 10728977 TI - Age, dental infections, and coronary heart disease. AB - Epidemiological and intervention studies have suggested that infections are risk factors for coronary heart disease (CHD). Dental infections have appeared as cardiovascular risk factors in cross-sectional and in follow-up studies, and the association has been independent of the "classic" coronary risk factors. This case-control study aimed at detailed assessment of the dental pathology found in various CHD categories (including elderly patients). Altogether, 85 patients with proven coronary heart disease and 53 random controls, matched for sex, age, geographic area, and socio-economic status, were compared with regard to dental status, assessed blindly with four separate scores, and to the "classic" coronary risk factors (seven of the controls had CHD, and they were not included in the analyses). The dental indices were higher among CHD patients than in the controls, but, contrary to previous studies, the differences were not significant (between the CHD patients and their matched controls or among the different CHD categories). This result could not be explained by potential confounding factors. The participants in the present study were older and had more often undergone recent dental treatment in comparison with subjects in our earlier studies. Age correlated with the severity of dental infections only in the random controls but not in the coronary patients who, although young, already had high dental scores. We believe that the higher age of the participants in the present study is the most likely reason for the results. Other possible explanations include an age related selection bias among older CHD patients, and the fact that those participating in studies like this may have better general health and thus also less severe dental infections. Thus, the role of dental infections as a coronary risk factor varies according to the characteristics of the population studied. PMID- 10728978 TI - The effects of a break in water fluoridation on the development of dental caries and fluorosis. AB - Durham, NC, fluoridated since 1962, had an 11-month cessation of fluoridation between September, 1990, and August, 1991. The purpose of this study was to assess the effects of this break on the development of caries and fluorosis in children. Study participants were continuously-resident children in Kindergarten through Grade 5 in Durham's elementary schools. There were 1696 children, 81.4% of those eligible, for whom a questionnaire was completed and clinical data recorded. Age cohorts were defined by a child's age at the time that fluoridation ceased. Caries was recorded in children in the Birth Cohort through Cohort 3, and fluorosis for children in Cohorts 1 through 5. Caries was assessed in the primary first and second molars according to the decayed-filled index; fluorosis on the labial surfaces of the upper permanent central and lateral incisors was assessed by the Thylstrup-Fejerskov (TF) index. Mother's education was associated with caries; higher education of the mother had an odds ratio of 0.53 (95% CI 0.40, 0.76) for caries in the child. No cohort effects could be discerned for caries. Overall prevalence of fluorosis was 44%. Prevalence in Cohorts 1, 2, 3, 4, and 5 was 39.8%, 32.3%, 33.0%, 62.3%, and 57.1%, respectively. These cohort differences remained statistically significant in regression analysis. It was concluded that while the break had little effect on caries, dental fluorosis is sensitive to even small changes in fluoride exposure from drinking water, and this sensitivity is greater at 1 to 3 years of age than at 4 or 5 years. PMID- 10728979 TI - The pH of dental plaque in its relation to early enamel caries and dental plaque flora in humans. AB - Dental caries appears to result from the action of multiple, interrelated factors. A companion study dealt with the plaque-flora/caries relationship (van Ruyven et al., 2000). The plaque-pH/caries relationship is the subject of this study. Since both studies involve the same subjects, plaques, and tooth surfaces, data on the examined factors have also been integrated. In vivo plaque pH determinations (microelectrode) were done on buccal sound (s) and "white-spot" (ws) caries surfaces in a selected dentition area in a low-caries (no ws) and higher-caries subject group. The pH response to sugar was evaluated before and after a sugar rinse, a local sugar application, or sucking on a sugary lozenge. pH profiles with sugar rinsing and normal or limited salivary flow conditions, showed progressively decreasing plaque pH values at various time points in the order of: low-caries subjects (s sites), higher-caries subjects (s sites), higher caries subjects (s + ws sites), and higher-caries subjects (ws sites). The minimum pH values showed the same trend. Analyses of all data indicated only a statistical difference for minimum values for s sites in low-caries subjects vs. ws sites in higher-caries subjects, and for s and ws sites in the latter. Local sugar application and sucking on a sugary lozenge induced smaller pH drops than sugar rinsing; such suboptimal sugar exposure caused a disappearance of the difference between the minimum pH values for s and ws sites observed with sugar rinsing in the higher-caries subjects. Initial plaque pH values were similar regardless of subject or tooth caries status. The values were also not correlated with the plaque levels of strongly iodophilic polysaccharide-storing bacteria. Collectively, both studies indicate that increasing subject caries status is characterized by increasing plaque levels of highly-acid-tolerant, acidogenic bacteria and an increasing plaque-pH-lowering potential and support the dynamic relationship between these parameters. PMID- 10728980 TI - Relationship among mutans streptococci, "low-pH" bacteria, and lodophilic polysaccharide-producing bacteria in dental plaque and early enamel caries in humans. AB - Multiple interactions occur among major determinants of dental caries. We have studied the bacterial flora and pH-lowering capacity of the same dental plaques in relation to caries. The findings on the plaque flora are reported here. The buccal surfaces of upper teeth in each subject were selected for study. A low caries group had no "white spot" caries (ws) in the selected dentition area; a higher-caries group averaged 4.1 ws in this area. The latter group was divided into subjects with 2, 3, or 4 ws and subjects with 5, 6, or 7 ws. Enumerated organisms in plaque samples (sound and ws sites) from all subjects were: (1) mutans streptococci (MS) on mitis-salivarius-bacitracin and mitis-salivarius agar; (2) non-mutans streptococci (non-MS) on mitis-salivarius agar; (3) organisms that were categorized according to their minimum pH in sugar broth, i.e., the predominant undifferentiated total flora on blood agar or the predominant non-MS flora on mitis-salivarius agar; and (4) iodophilic polysaccharide-storing organisms on trypticase-yeast extract-salts agar. Plaques covering ws lesions contained generally only low proportions (< 0.1%) of MS. The plaque proportions of all the above 4 bacterial groups were increased in the higher-caries group but were similar for s and ws sites in this group. Over half of the total plaque flora in subjects with 5, 6, or 7 ws consisted of "low-pH" type organisms (minimum pH < 4.4). Many of these were neither MS nor "low-pH" non MS. The numerical emergence of MS in plaque appeared to be preceded often by other types of "low-pH" bacteria, including the non-MS. Caries development in the absence or presence of MS as well as different bacterial successions in plaque can be explained readily by the dynamic and positive relationship among the factors carbohydrate consumption, plaque flora composition, plaque acidogenic potential, and caries activity. PMID- 10728981 TI - Campylobacter species in health, gingivitis, and periodontitis. AB - At least seven Campylobacter species have been identified from subgingival sites. Campylobacter rectus has been implicated as a periodontal pathogen; however, association with periodontal infections of other Campylobacter species, especially the newly described Campylobacter showae, is unclear. This study examined which Campylobacter species were associated with periodontal health and disease. Subgingival Campylobacter species from initial and established periodontitis were compared with species from periodontally healthy subjects, including subjects with gingivitis. Campylobacter species were isolated on selective media and identified by whole-cell protein profiles (SDS-PAGE). Except for C. rectus, Campylobacter levels were frequently below the detection limit (2 5% of the microbiota) of non-selective culture methods. C. rectus and C. showae, including Campylobacter X, were found more frequently and in higher levels from diseased than from healthy periodontal sites. C. gracilis was the dominant Campylobacter species found in relatively shallow pockets; however, its presence was unrelated to periodontal health or disease. C. concisus was isolated in higher proportions from relatively shallow and healthy sites, compared with deeper pockets. C. curvus was unrelated to periodontal health or disease. Analysis of the study data confirmed the relationship of C. rectus with diseased subgingival sites and indicated that C. showae may also be associated with periodontal disease. PMID- 10728982 TI - The impact of developmental biology on cancer research: an overview. AB - In recent years developmental biology has contributed a great deal to cancer research. This is in part because both fields address the question of how genes control the three-dimensional organisation of tissues, and how mutation of genes alters this. But also in recent years, the discovery that signalling pathways are conserved from worms to man, combined with the power of developmental biology's model organisms, principally Drosophila and C. elegans, to reveal signalling pathways that control tissue growth and organisation, has had a huge impact. Examples of this are the subject of the reviews in this issue, including the EGF receptor, Wnt/APC/catenin, TGF-beta/Smad and hedgehog/patched/smoothened pathways, all of which were discovered and/or pieced together in model organisms, and all of which are disrupted by mutation in human cancer. Other topics considered are the control and execution of apoptosis; the search for tumour suppressor-like genes in Drosophila; and genes of the Polycomb and Trithorax Groups that regulate the commitment of cells to patterns of differentiation, and that are among the targets for chromosome translocations. These stories illustrate how developmental biology has shown that there are many more signalling pathways relevant to neoplasia than the receptor tyrosine kinase pathways that first dominated the field; and that the signalling is more than just mitogenic or anti-mitogenic, and should be viewed as providing cells with information about their position and neighbours, that determines their role, differentiation and behaviour. PMID- 10728983 TI - Control of EGF receptor signalling: lessons from fruitflies. AB - The EGF receptor, and the related ErbB family of receptor tyrosine kinases, have been much implicated in human cancer. Hyperactive receptor signalling promotes deregulated growth control and the onset of malignancy, as well as the disruption of developmental programmes. Very little, however, is known about ErbB physiological regulation in humans. The fruitfly, Drosophila melanogaster, has a single receptor homologous to the four ErbB receptors and in this review we discuss how a genetic approach has led to significant insights into how the fly receptor is regulated. As signalling mechanisms have been well conserved between flies and mammals, these results of experiments in flies are relevant to the study of the human receptors in development and disease. Two areas of recent progress are emphasised. First, a number of signal modulators have been identified, including three EGF receptor inhibitors, several of which have human homologues. Second, we describe how the signalling molecules are integrated into regulatory networks that specify the elaborate activation profiles needed in development--positive and negative feedback control of EGF receptor signalling emerges as a central theme. Although the study of the Drosophila EGF receptor has no direct clinical application, the mechanistic insight it provides suggests new avenues of more applied research, including potential therapeutic targets. PMID- 10728984 TI - C. elegans vulval development as a model system to study the cancer biology of EGFR signaling. AB - Molecular genetic studies of C. elegans vulval development have helped to define an evolutionarily conserved signaling pathway from an EGF-like ligand through EGF receptor, Ras and MAP kinase to the nucleus. Further studies have identified novel positive regulators such as KSR-1 and SUR-8/SOC-2 and negative regulators such as cbl/SLI-1. The many negative regulatory proteins might serve to prevent inappropriate signaling, and thus are analogous to tumor suppressor genes. PMID- 10728985 TI - Wnt signalling in mammalian development and cancer. AB - Wnt signalling is involved in a variety of mammalian developmental processes, including cell proliferation, differentiation and epithelial-mesenchymal interactions, through which they contribute to the development of tissues and organs such as the limbs, the brain, the reproductive tract and the kidney. Wnts are secreted ligands that control cell processes via at least two pathways, one of which, the 'canonical' Wnt signalling pathway, operates through the cytosolic stabilisation of a transcriptional co-factor, beta-catenin. This is achieved by downregulating the activity of a beta-catenin turnover complex. Evidence from tumour expression studies, transgenic animals and in vitro experiments suggests that inappropriate activation of the canonical Wnt signalling pathway is a major feature in human neoplasia and that oncogenic activation of this pathway can occur at many levels. Inappropriate expression of the Wnt ligand and Wnt binding proteins have been found in a variety of human tumours. Further downstream, dysregulation of the beta-catenin turnover complex, by loss of the Adenomatous Polyposis Coli or Protein Phosphatase 2A proteins, or by activating mutations of beta-catenin, has been found in several tumour types, and is believed to be a key step in neoplastic progression. Transcriptional targets of the Wnt pathway include the cellular oncogenes cyclin D1 and c-myc. Activation of the Wnt signalling pathway by various means can therefore be a primary cause in oncogenesis, affecting cell proliferation, morphology and contact inhibition, as well as co-operating with other oncogenes in multistep tumour progression. PMID- 10728986 TI - The control of beta-catenin and TCF during embryonic development and cancer. AB - The Wnt signaling pathway functions reiteratively during animal development to control cell fate decisions. Inappropriate deregulation of this pathway leads to cancer in a number of tissues. The components that transduce the Wnt signal from the cell membrane to the cell nucleus are well conserved between vertebrates and Drosophila. A pivotal Wnt effector is the protein beta-catenin/Armadillo whose stability in the cytoplasm is low in unstimulated cells. Beta-catenin/Armadillo is targetted for proteasome-mediated degradation by a protein complex to which it binds. This complex consists of Axin, a putative scaffold protein which also binds to the tumor suppressor Adenomatous polyposis coli (APC) and glycogen synthase kinase 3 (GSK3)/Shaggy. Wnt signaling somehow inhibits the kinase activity of the quaternary complex. As a consequence, beta-catenin/Armadillo accumulates in the cytoplasm, translocates to the nucleus and becomes a transcriptional co-activator of T cell factor (TCF), the ultimate nuclear target of Wnt signaling. TCF is an architectural protein, mediating the assembly of multi-protein enhancer complexes. It cooperates with other enhancer-binding proteins and, together with beta-catenin/Armadillo, stimulates the transcription of Wnt target genes. Recently, repressors have been identified that prevent TCF from being active in the absence of Wnt signaling. PMID- 10728987 TI - TGFbeta signaling pathways and human diseases. AB - Recent progress in deciphering the TGFbeta pathway has uncovered a new signaling molecule, the Smads, and with this finding now gives us insights into how TGFbeta like signals are transmitted from outside the cell to the nucleus. As we learn more about how TGFbeta regulates normal development, we also are gaining insights into diseases that are caused by mis-regulation or mutation of various components of the signaling pathways. PMID- 10728988 TI - The hedgehog signalling pathway and its role in basal cell carcinoma. AB - The hedgehog signalling pathway plays a vital role in Drosophila embryonic patterning and development. Hedgehog is a secreted protein, unrelated to classical growth factors, which seems to form concentration gradients across those tissues involved in pattern formation. Cloning of vertebrate homologues of hedgehog and other genes has illustrated the remarkable conservation of function of this pathway throughout evolution. The human homologue of patched, a receptor for the hedgehog protein, was cloned as the gene responsible for naevoid basal cell carcinoma syndrome (NBCCS/'Gorlin Syndrome'), an autosomal dominant condition in which patients suffer from multiple basal cell carcinomas and a wide spectrum of developmental abnormalities. Its role as a tumour suppressor gene in both NBCCS and sporadic basal cell carcinoma led to the suggestion that mutation or inactivation of human patched may be an essential step in development of basal cell carcinomas and other skin tumours. This review describes our current understanding of hedgehog signalling in Drosophila and vertebrates and its relation to the development of human basal cell carcinoma and other skin tumours, together with a discussion of future avenues of research into this critical and intriguing pathway. PMID- 10728989 TI - Programmed cell death in C. elegans. AB - Genetic screens in the hermaphrodite nematode worm Caenorhabditis elegans resulted in the identification of the basal conserved machinery of apoptosis, arguably the single most important finding for our understanding of cell death. The last two years have seen enormous progress in the elucidation of the molecular interactions that lie at the heart of this conserved machinery, along with major insights both into how cell death is activated in the worm and into the mechanism of recognition and engulfment of the cell corpses. In this review, I set out the current models of cell death regulation and execution in C. elegans, focussing in particular on the similarities between cell death in C. elegans and vertebrates. Finally, I attempt to highlight key areas for future progress in cell death research in C. elegans and explore additional ways in which the worm can be used to understand the regulation of cell death in mammals. PMID- 10728990 TI - What is Drosophila telling us about cancer? AB - In Drosophila, genetic loss of the tumour suppressor protein Dlg (in dlg mutants) or p127 (in lgl mutants) leads to loss of epithelial structure and excess proliferation in the imaginal discs and brain of the developing larva. These phenotypes show most of the characteristic features of human neoplasia, so study of the gene products may contribute to our understanding of cancer. Both proteins occur in high molecular-mass complexes in the membrane-associated cytoskeleton, and they both appear to play dual roles as structural proteins and active partners in signal transduction. Dlg is a membrane-associated guanylate kinase homolog (MAGUK) found at septate junctions between epithelial cells, as well as at neuromuscular junctions. Specific domains of the protein are required for membrane targeting and for localisation injunctions, and for epithelial cell proliferation control; all of these functions are probably mediated through binding to other proteins. Loss of Dlg results in the absence of septate junctions, delocalisation of several proteins including Fasciclin III, Coracle, actin and tubulin, and loss of cell polarity. p127, although mostly associated with the plasma membrane, is in most cell types also present in the cytoplasm. It shows a dynamic subcellular distribution, and its cytosolic and membrane associated forms play distinctive roles by interacting with different binding partners, in particular the non-muscle myosin II heavy chain. Defects associated with the lgl temperature-sensitive allele include loss of the columnar organisation of epithelial cells, indicating that p127 contributes to cell structure, presumably by stabilising the plasma membrane. In addition to their organising functions, both Dlg and p127 appear to be involved in signal transduction pathways. Study of these genes shows that some proteins play both structural and functional roles, and that cancer can involve changes in the organisation of signalling pathways in addition to changes in individual pathway components. PMID- 10728991 TI - Cell memory and cancer--the story of the trithorax and Polycomb group genes. AB - Most of the cells in an adult organism possess the same DNA content and the same complement of genes, yet we can recognise many different cell types in the adult. Development is the process by which the cells of the embryo progressively acquire their adult fate and position. The genetic network which controls these processes is beginning to be unravelled at an increasing pace, yet one key area is still somewhat neglected--namely, developmental cell memory, the process by which cells record their ontogeny. A significant component of the phenotype of cancer cells may be explained by deregulation of genes whose normal role is to control the division, differentiation and migration of embryonic cells during development. It should therefore not be surprising that genes implicated in cell memory processes during development are also implicated in disease. In this review we outline what is known about the Polycomb and trithorax group of proteins as candidate genes for the memory process, both in terms of basic functions and the roles of abnormalities in these genes resulting in cancer. PMID- 10728992 TI - Enhancing effect of acute fasting on ethanol suppression of pulsatile luteinizing hormone release via an estrogen-dependent mechanism in Holstein heifers. AB - This study was conducted to determine whether acute fasting in Holstein heifers enhances the suppressive effect of an intravenous injection of ethanol on pulsatile LH release (LH pulse) and, additionally, to establish whether or not the mechanism is estrogen-dependent. After estrus synchronization (Day 0 = estrus), 29 heifers were either fasted (fasting group; n = 14) or fully fed as a control (control group; n = 15) from Days 1 to 4. On Day 4, blood samples were taken at 10-min intervals for 4 h before (pre-injection period) and after (post injection period) an intravenous injection of 1.5 mL of saline, 1.5 mL of ethanol , or 35 mg of tamoxifen dissolved in 1.5 mL of ethanol . We analyzed the mean LH level, the number of LH peaks per 4 h, and the amplitude of LH peaks. No differences were observed in the LH pulse in the pre-injection period between the control and the fasting group. However, in the post-injection period, compared with the saline injected control heifers, ethanol suppression of the LH pulse was observed in the number of LH peaks of the ethanol injected control heifers and in all pulse parameters of the ethanol injected fasting heifers. Furthermore, tamoxifen inhibited suppression of ethanol on LH pulse was observed in the control and fasting heifers injected with tamoxifen dissolved in ethanol. It was concluded that acute fasting in Holstein heifers has an enhancing effect on ethanol inhibition of the LH pulse and that the mechanism may be estrogen dependent. PMID- 10728993 TI - Influences of retrieval stages and glutathione addition on post-thaw viability of quick frozen mouse morula during in vitro culture. AB - Effects of the embryo retrieval stages and addition of glutathione (GSH) on post thaw development of mouse morula were evaluated in 2 consecutive experiments. In the first experiment, 1-, 2-, 3- to 4- and 5- to 8-cell stage embryos were collected and cultured to the morula stage in Whitten's medium containing 0.1 mM ethylenediaminetetraacetic acid (EDTA). The development rate of 1-cell embryos to the morula stage was lower than that of the other stages (P<0.01). The post-thaw development rate of the morulae obtained from in vitro culture of 1-, 2-, 3- to 4 , and 5- to 8-cell embryos and from in vivo embryos (control) to the blastocyst stage was 55.5, 84.9, 87.4, 90.1 and 90.8%, respectively. The post-thaw development rate of morula obtained from in vitro produced 1-cell embryos was significantly lower than from the other stages or from the in vivo counterparts (P<0.0001). In Experiment 2, the impact of GSH supplementation of the culture medium in the presence or absence of EDTA was evaluated for embryo development to the morula stage and post-thaw survival, using in the 2 x 2 factorial design. Although EDTA supplementation increased development rates to the morulae (P<0.01) stage, GSH did not have an influence on morula development. However, the presence of either GSH or EDTA in the culture medium supported development to the blastocyst stage (P<0.01) of in vitro produced morulae. These data demonstrate that 1-cell embryos from a blocking-strain mouse cultured in vitro to the morula stage have a lower development rate following freezing and thawing than embryos collected at the 2-cell or later stages. Addition of EDTA or GSH, individually or in combination, to the culture medium may improve the development rate of morula to blastocyst stage following cryopreservation. PMID- 10728994 TI - Failure of antibiotics gentamycin, tylosin, lincomycin and spectinomycin to eliminate Mycoplasma bovis in artificially infected frozen bovine semen. AB - To study the effect of antibiotics upon Mycoplasma bovis in fresh bovine semen just before freezing, specimens of bovine semen were artificially infected with 1 of 9 different strains of M. bovis. Inocula of each strain were prepared to contain 10(5) to 10(6)/mL colony-forming units of M. bovis at 3 different stages of the growth phase. The infected semen was diluted with a Tris extender by a 3 step procedure using an antibiotic mixture of gentamicin, tylosin, lincomycin and spectinomycin (GTLS). This semen-antibiotic mixture was placed into French straws that were stored at -196 degrees C. The control semen specimens contained no antibiotics Mycoplasmas were counted after 8 d of storage in 3 decimal dilutions of the frozen semen. No evident effect was noticed upon the 9 tested strains of mycoplasmas in the semen frozen with the antibiotics, compared with that of the untreated control samples. It was further shown that this lack of effect was irrespective of the stage of the growth phase of the mycoplasmas. It was concluded that the antibiotic mixture (GTLS) in semen specimens is not capable of total elimination of mycoplasmas in frozen bovine semen. PMID- 10728995 TI - The time interval between FSH administration and ovarian aspiration influences the development of cattle oocytes. AB - Depriving the ovary of exogenous FSH for 1, 2 or 3 d following a bolus injection of FSH was shown to influence the quality of the recovered oocytes. Thus, we compared the developmental competence of oocytes from heifers which had been stimulated for 3 d with FSH (Folltropin-V) and, after an interval of 36, 48 or 60 h, underwent blind transvaginal aspiration. The ovaries of heifers with a palpable or functional corpus luteum were aspirated to remove all large follicles 2 d prior to being injected with either 6 doses of saline (S), 6 doses (20 mg/mL) of FSH (F), or in 6 decreasing doses of FSH (3, 3, 2, 2, 1, 1 mL; Fd). Follicles were counted and classified (medium: 5 to 10 mm, large: >10 mm) with ultrasonography before each aspiration. The oocytes recovered were classified, matured, fertilized, and developed in vitro. On a per animal basis, 1.5, 5.2 and 4.7 large and 1.5, 10.7 and 10.7 medium follicles were counted for S, F and Fd, respectively. A mean of 3.3, 9.1 and 7.7 oocytes was recovered for treatments S, F and Fd, respectively and 58, 94 and 82% were enclosed in a nonexpanded cumulus or a corona layer. Oocyte development rates were based on counts of embryos with 32 or more nuclei at Day 6.5. When oocytes were recovered 36 h after the last injection, an average of 1, 2.7 and 2 embryos per animal was obtained with S, F and Fd, respectively; at 48 h, 0.75, 4.25 and 1 embryo; and at 60 h, 0, 2.5 and 2.7 embryos. Variance analysis was performed, and the protected LSD test indicated that treatment F at 48 h resulted in a significantly higher embryo rate than Fd at 48 h (P<0.05) or S (all times; P<0.05). The reduced effect of the Fd regimen could be due to the decreasing FSH support during follicular growth or to the lower total amount of FSH given. In conclusion, these results indicate an advantage of using moderate (3 d) follicle stimulation followed by a period of FSH starvation to obtain optimal embryo production. PMID- 10728996 TI - Studies on a chemically defined medium for in vitro culture of in vitro matured and fertilized porcine oocytes. AB - The present study evaluated the effects of various components in a chemically defined medium on the development of IVM/IVF porcine embryos. The investigated components included energy substrates (lactate, pyruvate or glucose, alone or in various combinations), amino acids (glutamine, glycine or alanine), PVP and HEPES buffer. The effects of each energy substrate were the same as the control. However, a mixture of lactate with either of the other energy substrates increased the development rate. Glutamine tended to decrease rate of the development more than other amino acids, and this inhibition was dose dependent. Both PVP and HEPES buffer did not affect development rate. However, more than 35 mM HEPES buffer induced fragmentation From the above results, a new culture medium was designed (supplemented with 0.276 mM glycine, 0.176 mM alanine, 15 mM HEPES buffer and 1% (wt/vol) PVP in BSA-free Whitten's medium with or without glucose). The new medium resulted in a higher embryo development rate (20.4 and 16.3%) than that obtained with the control medium (10.0%). PMID- 10728997 TI - Hypoosmotic test in equine spermatozoa. AB - The aim of the study was to evaluate equine sperm membrane integrity using the hypoosmotic swelling (HOS) test and to correlate this test with different sperm parameters in raw and frozen thawed semen. The HOS solutions were made with fructose, sucrose, lactose and sodium citrate each at 300, 150, 100, 50 and 25 mosm. Maximum numbers of swollen spermatozoa were observed in solutions of fructose, sucrose and lactose each at 100, 50 and 25 mosm. Correlations between progressive motility, morphologically normal spermatozoa and the HOS test were r = 0.75 and r = 0.51 in raw semen and r = 0.26 and r = -0.22 in frozen-thawed semen. The correlation between HOS and percentage of intact membranes with the fluorescent stain was r = 0.32 in frozen-thawed semen. The HOS test is a simple and accessible method which could be used as a complement to routine equine semen analysis. It has the added advantages of being less susceptible to the immediate effects of cold shock and of evaluating individual spermatozoa rather than the population as a whole, as does progressive motility. PMID- 10728998 TI - Immunohistochemical detection of estrogen receptors in the canine uterus and their relation to sex steroid hormone levels. AB - Cyclic changes in estrogen receptor expression in the uterine tissue of 60 female dogs were evaluated, using an immunohistochemical technique on formalin-fixed paraffin-embedded sections. The expression of estrogen receptors in the uterine horns, body and cervix was quantified by means of an immunohistochemical score. A negative correlation was found between staining scores in the uterine horns and serum progesterone levels. Generally, staining scores in the uterine horns were highest during proestrus, declined during estrus and were lowest during early metestrus. During anestrus high staining scores for estrogen receptors were observed, indicating sensitivity for estrogens in a sexual quiescence stage. Compared with the uterine horns, high staining scores were found in the uterine body and cervix during estrus and metestrus. No positive staining for estrogen receptors was detected in 1 pregnant uterus. Fluctuations in estrogen receptors were more pronounced in endometrial stroma cells than in epithelial cells of the uterine horns. The importance of stromal cells in the sexual cyclicity of the canine uterus should not be underestimated when studying uterine endocrinology and pathology. PMID- 10728999 TI - In vitro development of individually matured bovine oocytes in relation to follicular wall atresia. AB - Morphologically good-quality cumulus oocyte complexes (COCs) can originate from slightly atretic follicles. Biochemical and ultrastructural investigations reveal that a very high percentage of bovine antral follicles express some degree of atresia. The aim of the present study was to determine the developmental competence of good quality COCs in relation to their biochemically estimated follicular wall apoptosis. For experimental design a single oocyte maturation system was established, followed by group culture processing oocytes together according to their level of follicular wall atresia estimated by an ELISA for apoptotic cell death. Single oocyte culture during maturation reduced the developmental capacity of oocytes significantly (P < 0.01), with 5% blastocysts versus 25% after common group culture. Blastocyst formation for single oocyte maturation was found exclusively in oocytes isolated from luteal stage ovaries with low degree of apoptosis. The level of follicular wall apoptosis in luteal stage follicles (0.79 +/- 0.05 units/mg protein, n = 198) was lower than in follicular stage follicles (1.14 +/- 0.05 units/mg protein, n = 208). This was caused by significant higher levels in small (< 3.5 mm diameter) and large (> 5.5 mm diameter) follicles of the latter group. In conclusion, despite reduced developmental capacity after single oocyte maturation, we were able to reveal some functional relationship between oocyte origin and quality. It was shown that morphologically good quality COCs isolated from follicles with higher degree of apoptosis lose their developmental capacity. PMID- 10729000 TI - Ultrasound-guided follicle aspiration: the collection of bovine cumulus-oocyte complexes from ovaries of slaughtered or live cows. AB - To increase the collection efficiency of bovine cumulus-oocyte-complexes (COCs) by transvaginal aspiration, the effects of aspiration pressure and needle diameter on bovine follicular oocyte collection were assessed. Oocytes were aspirated from ovaries of slaughtered cows using 2 different diameter needles (18 or 21-gauge) with 4 different aspiration pressures (40, 80, 120 or 160 mmHg) and of live cows using 18-gauge needles with 40 or 80 mmHg, or using 21-gauge needles with 80 or 120 mmHg. The recovered oocytes were divided into 4 categories according to the surrounding cumulus cells and quality of oocytes: 1) 4 or more layers, 2) between 1 and 3 layers, 3) completely or partially denuded and 4) all others, including expanded cumulus cells and degenerated oocytes. The highest oocyte recovery rates from Categories 1 and 2 were obtained using 18-gauge needles with 40 mmHg pressure and 21-gauge needles with 120 mmHg pressure, respectively, from the ovaries of slaughtered cows. When oocytes were collected from live cows, the highest recovery rates for Categories 1 and 2 were obtained using an 18-gauge needle and 40 mmHg pressure, and 21-gauge needle and 80 mmHg, respectively. In addition, the proportion of oocytes in each category were compared between ovaries from slaughtered and live cows. The proportion of Category 1 oocytes collected from live cows was lower than from slaughtered cows when 18-gauge needles at 80 mmHg (P<0.05). The results show that the combination of aspiration pressure and needle diameter is crucial for COC collection, and they suggest that optimal aspiration conditions for ovaries of slaughtered cows are not necessarily applicable to live cows. PMID- 10729001 TI - Bull sperm surface "craters" and other aspects of semen quality. AB - Semen from 200 Holstein bulls in an artificial insemination center was examined for the frequency of craters on the surface of sperm heads, as visualized with the aid of differential interference contrast microscopy. Semen from 100 of these bulls was examined in more detail in 2 experiments by staining with eosin-aniline blue to determine the relationship of unstained spermatozoa, and spermatozoa with normal acrosomes with apical ridges to the incidence of craters and fertility. Only 3 of 100 bulls had a substantial incidence of craters (15 to 23%), whereas the average of the other 97 bulls in 2 experiments was 1 to 3%. The percentage of sperm cells with craters was correlated (P < 0.05) with the percentage of unstained spermatozoa (r = -0.29 and sperm cells with normal acrosomes (r = 0.52) but was not significantly correlated (r = -0.24) with the nonreturn rate. One bull with many sperm cells with craters was slaughtered, and the epididymal spermatozoa were examined. The high incidence of sperm cells with craters was limited to one side, with the testis on that side having 2 Sertoli cell tumors. The remaining 2 bulls as well as one other that produced 16% of sperm cells with craters did so only temporarily. Within a few months crater sperm production had decreased and semen quality increased. The condition usually appears to be transitory, presumably due to temporary stress. PMID- 10729002 TI - Effect of hyaluronic acid on the development of porcine 1-cell embryos produced by a conventional or new in vitro maturation/fertilization system. AB - In pigs, it is difficult to produce normal fertilized embryos from immature oocytes in vitro. However, a new maturation/fertilization system in which the percentage of normal fertilized embryos is comparatively high has been developed recently. In the present study, porcine 1-cell embryos were produced both by a conventional and a new system and then cultured in NCSU-23 supplemented with hyaluronic acid at various concentrations. In the conventional system, the percentage of oocytes with monospermic penetration and 1 male pronucleus and 1 female pronucleus was only 6%. At 144 h after insemination, the percentage (5%) of embryos developing to the blastocyst stage in medium supplemented with 0.5 mg/mL hyaluronic acid was significantly (P<0.05) higher than that (2%) in medium without hyaluronic acid. When oocytes were matured and inseminated using the new system, monospermic penetration and the formation of 1 male and 1 female pronucleus were observed in 69% of the penetrated oocytes. However, blastocyst formation (8 to 14%) at 144 h after insemination was not affected by the concentration (0 to 1.0 mg/mL) of hyaluronic acid. These results indicate that the effect of hyaluronic acid on the development of in vitro-produced porcine embryos varies with the conditions of oocyte maturation and fertilization. PMID- 10729003 TI - Cloning and sequencing of buffalo male-specific repetitive DNA: sexing of in vitro developed buffalo embryos using multiplex and nested polymerase chain reaction. AB - Buffalo Y-chromosome specific repetitive DNA (BuRY.I) was cloned and sequenced in order to develop a sensitive method for sexing of buffalo preimplantation stage embryos using polymerase chain reaction (PCR). A highly sensitive and reliable sex determination assay using a primary (BRY.I), nested (BuRYN.I) and multiplex (BuRYN.I, ZFX/ZFY) PCR was developed. The BRY.I and BuRYN.I primers are targeted to amplify Y-specific sequences, while the ZFX/ZFY loci was amplified to serve as a positive control for both male and female samples. Accuracy of the sex determination assay was initially verified with genomic DNA obtained from blood of known gender. Further sensitivity and reproducibility of the assay was examined using DNA obtained from 1 or 2 blastomeres to demi embryos. Altogether, 80 IVF-derived embryos ranging from the 2 to 4 cell to the blastocyst stage were used for sex determination. Definite and clear signals following PCR amplification were obtained from all embryo samples. Accuracy of assays was determined by comparing results from a single cell with those of blastocyst stage embryos, thereby indicating that 1 or 2 blastomeres from a preimplantation buffalo embryo is sufficient for sex determination by PCR. No misidentification was observed within the embryo samples using nested (BuRY.I), primary (BRY.I) and multiplex (BuRYN.I; ZFX/ZFY) PCR, suggesting that this technique is a highly reliable method for sexing buffalo embryos. PMID- 10729004 TI - Maturation, fertilization and complete development of porcine oocytes matured under different systems. AB - This study was designed 1) to determine the effectiveness of 2 in vitro maturation systems commonly employed to produce nuclear and cytoplasmically mature pig oocytes, 2) to assess the effects of boar, sperm concentration and maturation system on oocyte penetrability and male pronucleus formation and 3) to determine the ability of the in vitro matured oocytes to be fertilized in vivo by artificial insemination (AI) of sows. The differences examined between the 2 maturation systems included the culture medium (Waymouth vs TCM199), hormones, additives, culture conditions (static vs gentle agitation) presence or absence of porcine follicular fluid (PFF) and presence or absence of follicular shells. The results showed that nuclear maturation rate was similar in both systems (83.3 +/- 3.5 vs 86.4 +/- 2.5%), and intracellular content of glutathione was 5.21 +/- 0.73 vs 3.5 +/- 0.39 pmol/oocyte, although no correlation between these parameters was observed. The penetration rate and number of sperm cells per oocyte were dependent on the boar, maturation system and sperm concentration, but the rate of male pronuclear formation seemed to be influenced only by the boar and the maturation system but not by sperm concentration. In vivo fertilization of in vitro matured oocytes showed that both maturation systems could yield viable oocytes since 3 of 4 gilts and 2 of 4 gilts, respectively, became pregnant. Failure to become pregnant was not associated with inadequate oocyte maturation since control gilts, which received their own ovulated oocytes rather than in vitro matured oocytes at transfer, also did not become pregnant. We conclude that polyspermy may be an inherent problem in the IVF but not in the IVM systems. PMID- 10729005 TI - The relationship between vaginal mucous impedance and serum concentrations of estradiol and progesterone throughout the sheep estrous cycle. AB - The objective of this experiment was to assess the relationship between electrical resistance of the vaginal mucosa and serum concentrations of estradiol (E2) and progesterone (P4) during the estrous cycle in ewes. Vaginal impedance was recorded daily using a 2-electrode impedometer in 10 nonprolific Western white-faced and 7 prolific Finn ewes, during the mid-breeding season (October to December). Transrectal ultrasonography of ovaries was performed once a day to confirm ovulation and monitor follicle growth (follicles > or =3 mm in diameter) and development of corpora lutea (CL). Jugular blood samples were collected daily for radioimmunoassay (RIA) of estradiol and progesterone. In all ewes, a decline in vaginal impedance (to <40 ohms) was closely associated with the onset of behavioral estrus. In both breeds of sheep, there was no significant correlation between daily serum concentrations of estradiol and vaginal impedance throughout the estrous cycle. Daily serum concentrations of progesterone and the E2:P4 ratio were correlated with vaginal impedance during the period of luteolysis and follicular phase in both breeds (Western white-faced ewes: r = 0.62, P = 0.0002 and r = -0.56, P = 0.0002; Finn ewes: r = 0.61, P = 0.001 and r = -0.45, P = 0.03, respectively) and early in the cycle (Days 0 to 2, Day 0 = day of ovulation) in white-faced ewes (r = 0.61, P = 0.0003 and r = -0.36, P = 0.052, respectively) but not during the remaining portion of the luteal phase in either breed. In conclusion, vaginal mucous impedance appears to be primarily controlled by progesterone, but it also changes in response to shifts in the E2:P4 ratio when progesterone concentrations are low. Impedometric characteristics of the vaginal mucosa in cyclic ewes are an indicator of serum concentrations of progesterone and E2:P4 ratios during the terminal stage of the estrous cycle. PMID- 10729006 TI - Pregnancy diagnosis in sows: direct ELISA for estrone in feces and its prospects for an on-farm test, in comparison to ultrasonography. AB - The usefulness of fecal estrone (E1) measurement as a tool for pregnancy diagnosis was investigated. Concentrations of E1 were measured in feces from pregnant and nonpregnant sows by a direct ELISA without extraction. Highly significant differences in E1 concentrations were found in feces from nonpregnant and pregnant sows (P = 0.016 to < 0.001). Pregnancy diagnosis on Days 26 to 32 after insemination, based both on fecal E1 concentrations as measured by ELISA and ultrasonography using a 5.0 MHz linear-array transducer, was performed in a group of 496 gilts and sows. The fecal E1 test had a sensitivity (correct diagnosis of pregnancy) of 96.5% and a specificity (correct diagnosis of nonpregnancy) of 93.6%, using 3.65 ng E1/g feces as a cut-off value. For ultrasonographic pregnancy diagnosis the test sensitivity and specificity were 99.3 and 92.5%, respectively. Although an increase of fecal E1 concentrations was noticed for increasing litter sizes, the results indicated that these concentrations could not be used to predict litter size. It is concluded that the distribution of fecal E1 concentrations in both nonpregnant and pregnant sows offers a suitable basis for the development of a simple, sow-side pregnancy test. PMID- 10729007 TI - Determining the sex of bovine embryos using polymerase chain reaction results: a six-year retrospective study. AB - Knowing the sex of embryos produced for use in an embryo transfer program can assist the dairy producer in managing his resources more effectively. A reliable procedure for accomplishing this goal is to apply PCR technology to the biopsy of an embryo. A description is provided of how the technique has been applied on a large scale in a commercial setting in western Canada between 1992 and 1997. A total of 4,183 embryos was biopsied over a 6-yr period. The sex was determined with more than 90% of the embryos. The results showed that there was a seasonal variation in the sex ratio, with more females being recorded in the period of least light (October to March), than in April to September. While both sire and embryo quality affected the sex ratio, the differences were too small to be of value in most breeding programs. Pregnancy rates with fresh sexed embryos (58 to 71%) were comparable to those with fresh unsexed embryos. The results following freezing and thawing of sexed embryos were low (37 to 66%) but sufficient to be viable commercially. When the sex assigned by PCR was verified by fetal sexing at 60 d of gestation, the error rate was 7%. This study demonstrates that sexing of embryos can be carried out on a large scale. Demand for quick, reliable determination of sex can be met in a cost effective manner. The pregnancy rates achieved with embryos after biopsy are suitable for use in a commercial setting. PMID- 10729008 TI - Oxytocin in the semen and gonads of the stallion. AB - It has been suggested that oxytocin is involved in sperm transport and motility in domestic animals. Immunoreactive oxytocin was measured in seminal fractions (pre-ejaculatory fluid, seminal plasma, gel and sperm) and in extracts of testis and epididymis from stallions. In addition, sections of gonadal tissue from stallions were immunostained for the presence of oxytocin and its neurophysin. Oxytocin was detected in all of the seminal fractions, being highest in the gel. It was also present in washed, lysed sperm and in extracts from the testis and epididymis. Immunostaining for oxytocin was present in occasional interstitial cells in the testis and in the epididymal epithelium and smooth muscle. However, immunostaining for neurophysin was detected in a few interstitial cells in the testis of only 1 of 8 stallions and was absent from all areas of the epididymis. These data demonstrate for the first time the presence of oxytocin in stallion semen and gonadal tissue; however, lack of immunostaining for neurophysin indicated that it was unlikely that there was local synthesis within the gonads. PMID- 10729009 TI - Acyloxyacyl hydrolase activity of neutrophil leukocytes in normal early postpartum dairy cows and in cows with retained placenta. AB - Acyloxyacyl hydrolase (AOAH) is an enzyme of bovine polymorphonuclear neutrophil leukocytes (PMN) that is capable of detoxifying endotoxin (25). The activity of AOAH in PMN isolated from the blood was investigated in dairy cows that expelled the fetal membranes normally (Group NFM) and in cows with retained fetal membranes (Group RFM) to obtain better insight into the role of the AOAH enzyme of neutrophils in endotoxin-related diseases, which occur frequently in dairy cows during the early postpartum period, especially in RFM cows. Twenty early postpartum dairy cows were used in the study: 13 NFM cows and 7 RFM cows. In the RFM cows, the percentage of PMN in blood (29+/-4%) was significantly (P<0.05) lower than in NFM cows (43+/-4%). The average AOAH activity in RFM cows (mean +/- SEM = 89+/-13 pmol fatty acid/10(7) PMN/h) was lower than in NFM cows (107+/-6 pmol fatty acid/10(7) PMN/h), but the difference in neutrophil AOAH activity between the 2 groups was not significant. There was also a higher percentage of immature neutrophils in isolated leukocyte suspensions from RFM cows (22+/-8%) than from NFM cows (15+/-4%), so that impairment of AOAH activity in early postpartum cows could be explained, in part, by immaturity of the neutrophils. These results suggest that the decreased AOAH activity of PMN could play a role in the pathogenesis of endotoxin-related diseases in dairy cows during the early postpartum period. PMID- 10729010 TI - Prevalence of antibodies to IBR and BVD viruses in dairy cows with reproductive disorders. AB - We determined the prevalence of antibodies to infectious bovine rhinotracheitis virus (IBRV) and bovine viral diarrhea virus (BVDV) in sera of dairy cows on 4 different farms in the Republic of Croatia. A high percentage (60.8%) of cows had various reproductive disorders. The results showed that seroprevalence of infectious bovine rhinotracheitis (IBR) was 85.8% and that of bovine viral diarrhea (BVD) was 79.2% in tested cows. Antibodies to both viruses were found in 80.8% of cows with reproductive disorders but in only 46.8% of cows without reproductive disorders. This difference was statistically significant (P<0.01), and indicated a connection between reproductive disorders and simultaneous infections with IBR and BVD viruses in dairy cows. PMID- 10729011 TI - Effect of a deep uterine insemination on spermatozoal accessibility to the ovum in cattle: a competitive insemination study. AB - A competitive insemination study was conducted to determine the effect of a deep uterine insemination on accessory sperm number per embryo in cattle. Cryopreserved semen of a fertile bull characterized by spermatozoa with a semi flattened region of the anterior sperm head (marked bull) was matched with cryopreserved semen from an unmarked bull having spermatozoa with a conventional head shape. Using 0.25-mL French straws and a side delivery embryo transfer device, deep uterine insemination (0.125 mL deposited in each horn) was performed 2 cm from the uterotubal junction. Immediately after, the uterine body was artificially inseminated using semen (0.25 mL) from an alternate bull and a conventional insemination device. The complete dose (both inseminations) was 50x10(6) total sperm cells consisting of an equal number of spermatozoa from each bull. Single ovulating cows (n = 95) were inseminated at random with either the unmarked semen in the uterine body and marked semen in the uterine horn, or the unmarked semen in the uterine horn and marked semen in the uterine body. Sixty one embryos(ova) were recovered nonsurgically 6 d post insemination, of which 40 were fertilized and contained accessory spermatozoa. The ratio and total number of accessory spermatozoa recovered was different among treatments: 62:38 (326) for the unmarked semen in the uterine body and marked semen in the uterine horn, and 72:28 (454) for the unmarked semen in the uterine horn and marked semen in the uterine body (P<0.05). Deep uterine insemination using this semen in a split dose and a side delivery device favors accessibility of spermatozoa to the ovum compared with conventional uterine body insemination. PMID- 10729012 TI - Successful in vitro fertilization of in vivo matured oocytes aspirated laparoscopically from red deer hinds (Cervus elaphus). AB - Most current protocols of in vitro fertilization in ruminants are based on in vitro maturation of oocytes derived from abattoir material. For application of IVF technology to captive endangered species, however, noninvasive techniques are required which allow repeated collection of oocytes from live females. The aim of this study was to develop a method for embryo production from mature oocytes collected laparoscopically from red deer hinds. Follicular development was synchronized in red deer hinds by the insertion of intravaginal progesterone releasing devices for 10 d, and ovarian stimulation was induced with 1000 IU, i.m. PMSG 48 h before progesterone device removal. Oocytes were harvested by laparoscopy under xylazine/ketamine sedation 24 h after progesterone device removal and then co-incubated with frozen-thawed red deer spermatozoa for 24 h. In Experiment 1, oocytes and embryos were fixed and stained at different developmental timepoints. Their external morphological changes (cumulus expansion, extrusion of the second polar body and cytokinesis) paralleled their nuclear developmental changes (formation of the 2nd metaphase spindle of meiosis, pronuclear formation and nuclear division, respectively). In Experiment 2, embryos were maintained in vitro until they ceased to undergo cell division. A total of 39 aspiration procedures was carried out on 14 red deer hinds. Forty four cumulus-oocyte complexes (COC) were aspirated from 95 large Graafian follicles; of these, 27 were classed as mature/nondegenerated on the basis of cumulus/cytoplasmic morphology. Seventeen oocytes cleaved following in vitro fertilization, yielding six 2-cell embryos, six 4-cell embryos, four 8-cell embryos and one 16-cell embryo. The results indicate that laparoscopic aspiration of mature oocytes from hormone-treated females offers a valuable source of genetic material for assisted deer breeding programs. PMID- 10729014 TI - Development and application of competitive ELISA assays for rat LH and FSH. AB - Rat LH (rLH) and FSH (rFSH) were measured by sensitive and specific competition ELISAs. The rat LH ELISA used rLH-I-9 coated plates, an antiserum against rLH and an antibody against rabbit IgG labeled with peroxidase. Using rLH-RP-3 as a standard, rat LH was determined by binding of the anti-LH antibody to rLH-I-9 coated plates. The sensitivity of the assay was 0.8 ng/mL. Similarly, the rat FSH ELISA used rFSH-I-8 coated plates, an antiserum against rFSH and an antibody against rabbit IgG labeled with peroxidase. Using rFSH-RP-3 as a standard, the FSH-ELISA was also determined by binding of the anti-FSH antibody to rFSH-I-8 coated plates. The sensitivity of this assay was 1.25 ng/mL. Both rat LH and FSH ELISA assays are highly specific and provide accurate determination of gonadotrophins in buffers, sera, cell culture media, and anterior pituitary extracts. These assays were used for monitoring the gonadotrophin surge attenuating factor (GnSAF) and inhibin activities present in human follicular fluid (hFF). The 2 new ELISA procedures have practical advantages (safety, convenience, economy) over the RIA methods, and they perform as well as the RIA techniques at the same range of concentrations. PMID- 10729013 TI - The inhibitory effect of hCG on counter current transfer of GnRH and the presence of LH/hCG receptors in the perihypophyseal cavernous sinus--carotid rete vascular complex of ewes. AB - The existence of the hormone passage from venous blood into arterial blood in the area of the perihypophyseal vascular complex has been demonstrated in some mammals, but its mechanism has not been defined. To study the regulatory mechanism we infused hCG into perihypophyseal cavernous sinus of ovariectomized, conscious ewes to test if the hCG would affect putative LH/hCG receptors and inhibit counter-current transfer of GnRH from the venous cavernous sinus to the arterial carotid rete. The latter study was done on an isolated head model. Ewes were ovariectomized in mid-anestrus and, after 4 to 5 wk were used in the experiments. On the day of experiment ewes were treated intramuscularly with estradiol benzoate or oil vehicle, and 18 to 20 h later were infused either with a multielectrolyte solution or hCG for 2 h via the venae angularis oculi. Immediately thereafter the ewes were anesthetized and exanguinated, and subsequently decapitated. The isolated head was perfused with Dextran in multielectrolyte. The 125I-GnRH was infused into the cavernous sinus via the venae angularis oculi for 5 min; contemporaneous samples were taken from the carotid rete and both jugular veins at 1-min intervals. Transfer of 125I-GnRH from the cavernous sinus to the carotid rete was inhibited by hCG in ewes pretreated with estradiol benzoate but not with oil (P<0.005). We collected tissue samples from the vascular complex of the cavernous sinus and carotid rete of cyclic ewes to determine the presence of LH/hCG receptors. In situ hybridization showed the presence of LH/hCG receptor mRNA transcripts in the walls of both arterial and venous compartments of the cavernous sinus-carotid rete complex, and immunohistochemistry revealed the presence of receptor proteins. These novel findings confirm previously obtained data suggesting that LH is a modulatory factor for the counter-current transfer of neuropeptides from the venous blood of the cavernous sinus to the arterial blood supplying the brain and hypophysis. The LH could modulate 125I-GnRH transfer acting directly on the vascular smooth muscle. PMID- 10729015 TI - Effect of semen preparation on IVF of prepubertal goat oocytes. AB - The aim of these experiments was to study the effects of different methods of washing and selection of spermatozoa on the IVF of IVM oocytes from prepubertal goats. Fresh ejaculates from 3 males of proven fertility were processed according to the following treatments: 1) centrifugation in TALP, 2) centrifugation in sucrose-based Ficoll medium, 3) centrifugation in Percoll gradients at 40 and 80%, 4) by swim-up and 5) by dilution of spermatozoa (1:40) in (1:1) TALP. In all 5 treatments spermatozoa were incubated for 45 min with 100 microg/mL of heparin and then added to Fert-TALP. Oocytes were matured for 27 h in TCM-199 supplemented with 20% estrous goat serum (EGS), FSH, LH and estradiol-17beta. Spermatozoa (4x10(6) cells/mL) were coincubated with oocytes in 100 microL of Fert-TALP with hypotaurine for 24 h, after which the oocytes were transferred to a granulosa cells monolayer in TCM-199 plus 10% of EGS for 24 h (48 h post insemination). At 17 h post insemination a sample of sperm-exposed oocytes was taken and stained in lacmoid to observe sperm penetration and the formation of pronuclei. At 48 h post insemination the cleavage rate of oocytes was evaluated. Motility, viability and acrosome status of the spermatozoa were evaluated immediately after the mixing of the ejaculates, after washing and selection treatments, and after incubation with heparin and at 17 h post insemination. The different ejaculate treatments did not affect the penetration and cleavage rates of oocytes. At 48 h post insemination the cleavage rate was 46.9, 36.6 and 29.0% for dilution, Ficoll and swim-up preparations, respectively. Only the swim-up protocol improved sperm motility and viability compared with that of the initial semen sample and with the other sample treatments. At 17 h post insemination the semen parameters were the same for all sperm sample treatments. PMID- 10729016 TI - Development of bovine IVF oocytes cultured in medium supplemented with a nitric oxide scavenger or inhibitor in a co-culture system. AB - Bovine IVF oocytes were cultured in modified bovine embryo culture medium (mBECM) supplemented with either a nitric oxide (NO) scavenger, hemoglobin (Hb, 1 microg/mL) and/or a NO synthesis inhibitor, L(omega)-nitro-L-arginine methyl ester (L-NAME, 1 or 1000 nM) in a cumulus-granulosa cell co-culture system. In Experiment 1, a total of 1,675 cumulus-oocytes complexes was collected for 7 mo and cultured to the blastocyst stage in mBECM with or without Hb after IVM and IVF. There were significant (P<0.0024) model effects of Hb addition and month of oocyte collection on embryo development. A significant (P<0.0023) monthly variation was detected in all developmental stages. However, addition of Hb to mBECM consistently enhanced embryo development to the blastocyst stage over all months. No statistical differences were found in the interaction between Hb addition and month except for the cleavage rate. Overall, a greater percentage of oocytes developed to the 8-cell (P<0.0459), 16-cell (P<0.001), morula (P<0.0013) and blastocyst (P<0.0024) stages after the addition of Hb. In Experiment 2, addition of L-NAME to mBECM supplemented with Hb did not further stimulate prehatched development. In conclusion, the promoting effect of Hb on in vitro development of embryos is highly repeatable over an extended period of time. PMID- 10729018 TI - Electroactivation of rabbit oocytes in an hypotonic pulsing medium and parthenogenetic in vitro development without cytochalasin B-diploidizing pretreatment. AB - We investigated the electroactivation frequencies, type of activation and in vitro development of rabbit oocytes. In Experiment 1, activation (8 pulses, 12 min apart, 60 microsec, 0.6 kVcm(-1)) was performed by altering osmolarity (190 vs. 320 mOsm kg(-1)) and Ca++ concentration (10, 60 or 100 microM) in mannitol pulsing media. More oocytes were activated in hypotonic pulsing medium, regardless of Ca++ concentration (96 to 100%). Both haploid and diploid parthenogenetic embryos developed to compacted morulae (57 to 92% and 63 to 100%, respectively) regardless of the activation treatment; however, the blastocyst rates were more variable (0 to 74% and 0 to 73%, respectively). In Experiment 2, the effects of pulse duration (30 or 60 microsec) and number of applied pulses (4, 8 or 12) under hypotonic conditions were studied. Activation frequencies were the lowest after four 30 microsec-pulses (58 vs. 88 to 100%, respectively). A lower haploid frequency was obtained when more than four 30 or 60 microsec-pulses were applied (from 67 to 25% and 83 to 0%, respectively). Increasing the number of 60-microsec pulses improved the compacted morula rate of haploid and diploid oocytes (47 to 83% and 57 to 96%, respectively). Overall, haploid development to morulae and blastocysts was lower than diploid development to these stages (69 and 25% vs. 74 and 44%, respectively). PMID- 10729017 TI - In vivo oocyte recovery and in vitro embryo production from bovine donors aspirated at different frequencies or following FSH treatment. AB - The effects of frequency of follicular aspiration and treatment of donor cattle with FSH on in vivo oocyte recovery and in vitro embryo production were studied. Simmental heifers (n = 24) formed 8 replicates of 3 treatments in which oocyte donors were aspirated 1) once a week, 2) twice a week, or 3) once a week following treatment with FSH for 3 d prior to aspiration. Oocytes were graded, washed, matured for 20 to 24 h and then inseminated with frozen/thawed semen from a single sire, followed by co-culture on granulosa cell layers. Embryo development was observed until Day 7 after insemination. Significantly fewer follicles per heifer per week were counted (14.7+/-2.3 vs. 27.4+/-3.1 vs. 23.1+/ 2.8) and aspirated (12.0+/-2.0 vs. 21.8+/-2.7 vs. 20.1+/-2.6) in heifers on the once-weekly than twice-weekly aspiration treatment (P<0.01) or on the once-weekly aspiration after FSH treatment (P<0.05). There were no significant differences between treatments in the total number of oocytes recovered per week (5.6+/-1.2 vs. 8.9+/-1.5 vs. 6.1+/-1.2), but significantly more oocytes per heifer per week recovered from animals treated with FSH were graded Category 1 (2.8+/-0.4), i.e., >4 layers good cumulus with a clear, even cytoplasm, than from animals aspirated once (0.9+/-0.2; P<0.01) or twice a week (1.5+/-0.3; P<0.05). The number of transferable morulae plus blastocysts produced per heifer per week was higher from animals aspirated twice a week (2.4+/-0.4; P<0.05) or once a week following FSH treatment (2.1+/-0.4; P<0.05) than from animals aspirated once a week without FSH treatment (1.0+/-0.3). In conclusion, FSH treatment of bovine oocyte donors aspirated once a week enabled a similar number of transferable embryos to be produced per donor week as aspiration twice a week without FSH treatment. These 2 treatments produced twice as many transferable embryos per donor week as aspiration once a week without FSH treatment. PMID- 10729019 TI - Effect of growth hormone and induced IGF-I release on germ cell population and apoptosis in the bovine testis. AB - Bovine growth hormone has been used in dairy cattle to increase milk production,but it also increases the twin parturition rate. This effect is mediated by insulin-like growth factor-I (IGF-I), which prevents follicular atresia by hindering apoptosis of granulosa cells. The action of GH and IGF-I on testicular function remains unclear. The goal of this study, therefore, was to verify the effects of short-term administration of GH and induced IGF-I release on the number of testicular germ cells, testicular morphology, and apoptosis in the bovine testis. Twenty Zebu bulls were split into 2 groups. The bulls in Group 1 (n = 10) were treated with 2 subcutaneous injections of bovine GH (500 mg/bull) 7 d apart. Group 2 bulls (n = 10) received placebos under the same protocol. All of the bulls were slaughtered 14 d after the start of treatment. Fragments of the testis were collected, fixed in Bouin's solution, embedded in paraffin, and the sections stained with hematoxilin and eosin. The paraffin-embedded sections were also used for in situ detection of apoptotic cells. Blood samples were collected at slaughter to measure serum levels of IGF-I, FSH and LH. Neither the number of Stage I seminiferous epithelium germ cells and the morphometric parameters (tubular diameter, seminiferous epithelium height, and volumetric proportions of structural components) nor the blood levels of FSH and LH showed a significant difference between the 2 groups. However, the treated animals showed an increase in serum IGF-I (P<0.01). Apoptotic germ cells were detected in the testis of both groups, showing the same pattern and a stage-specific apoptosis pattern. Most of the labeled cells were spermatocytes. The localization of apoptotic germ cells did not differ between groups. These results suggest that short-term administration of GH does not affect bovine spermatogenesis in adult bulls. PMID- 10729020 TI - Estrus synchronization and artificial insemination of hair sheep ewes in the tropics. AB - Hair sheep ewes (St. Croix White and Barbados Blackbelly) were used to evaluate 3 methods of estrus synchronization for use with transcervical artificial insemination (TAI). To synchronize estrus, ewes (n = 18) were treated with PGF2alpha (15 mg, im) 10 d apart, with controlled internal drug release (CIDR) devices containing 300 mg progesterone for 12 d (n = 18), or with intravaginal sponges containing 500 mg progesterone for 12 d (n = 18). On the day of the second PGF2alpha injection or at CIDR or sponge removal, sterile rams were placed with the ewes. Jugular blood samples were collected from the ewes at 6-h intervals until the time of ovulation, and daily for 16 d after estrus (Day 0). Plasma was harvested and stored at -20 degrees C until LH, and progesterone concentrations were determined by RIA. There was no difference (P>0.10) in time to estrus among the CIDR-, PGF2alpha- or sponge-treated ewes. All of the ewes in the CIDR group and 94.4% of the sponge treated ewes exhibited estrus by 36 h after ram introduction, while only 72.2% of PGF2alpha-treated ewes showed signs of estrus by this time (P<0.06). The time from ram introduction to ovulation was not different (P>0.10) among the CIDR-, PGF2alpha- or sponge-treated ewes. The time to the preovulatory LH surge was similar (P>0.10) among CIDR, PGF2alpha and sponge treated ewes. Progesterone levels through Day 16 after the synchronized estrus were not different (P>0.10) among treatment groups. Hair sheep ewes (n = 23) were synchronized using PGF2alpha and bred by TAI using frozen-thawed semen 48 h after the second injection. The conception rate to TAI was 2/23 (8.7%) and produced 3 ram lambs. In a subsequent trial, 17 ewes were synchronized with CIDR devices and bred by TAI using frozen-thawed semen 48 h after CIDR removal, resulting in a conception rate of 52.9% (9/17). It is possible to synchronize estrus in hair sheep using either CIDRs, sponges or PGF2alpha. Even though there were no significant differences in the timing of ovulation or the LH surge among the treatment groups, a higher conception rate was achieved in ewes synchronized with CIDR devices during the second trial. This may reflect an increase in the skill level of the TAI technician. PMID- 10729021 TI - Full-term development of nuclear transfer calves produced from open-pulled straw (OPS) vitrified cytoplasts: work in progress. AB - Cryopreservation of cytoplasts would help to resolve the logistics of matching the availability of oocytes with embryo donors in nuclear transfer. Therefore, the developmental potential of nuclear transfer bovine embryos reconstructed using vitrified cytoplasts was investigated. In vitro matured oocytes were denuded, enucleated, activated with calcium ionophore (10 microM, 5 min) and cycloheximide (10 microg/mL, 6 h) and then vitrified by the open pulled straw (OPS) method. After immediate warming, the nuclear transfer embryos were reconstructed using blastomeres from nonvitrified,in vitro-produced embryo donors. Compared with control nuclear transfer embryos that were reconstructed using nonvitrified cytoplasts, fusion rates (% +/- SEM) were not affected (83.7+/ 9.2 vs. 79.8+/-4.6; P>0.05), but cleavage (55.7+/-2.9 vs. 92.8+/-3.9; P = 0.0002) and blastocyst rates (7.2+/-5.0 vs. 32.6+/-7.8; P = 0.0025, vitrified vs. nonvitrified cytoplasts, respectively) per successful fusion were reduced. One nuclear transfer blastocyst reconstructed from a vitrified cytoplast was transferred to a synchronized recipient. After a normal length gestation (265 d), twin calves (21 and 26 kg) were delivered. Microsatellite analysis confirmed that the calves were homozygotic (the embryo split in utero), and were derived from the in vitro-produced embryo donor. The twins were dead at birth, but post-mortem analysis of the calves indicated no abnormalities or infections, suggesting that their death was related to the twin pregnancy and the known fragility of nuclear transfer calves. These data demonstrate that open pulled straw-vitrified cytoplasts are capable of supporting full-term development of nuclear transfer embryos. PMID- 10729022 TI - Substantial decrease of heat-shock protein 90 precedes the decline of sperm motility during cooling of boar spermatozoa. AB - The decline in boar semen quality after cryopreservation may be attributed to changes in intracellular proteins. Thus, the aim of the present study was to evaluate the change of protein profiles in boar spermatozoa during the process of cooling and after cryopreservation. A total of 9 sexually mature boars (mean age = 25.5+/-12.3 mo) was used. Samples for protein analysis were collected before chilling, after cooling to 15 degrees C, after cooling to 5 degrees C, following thawing after freezing to -100 degrees C, and following thawing after 1 wk of cryopreservation at -196 degrees C. Semen characteristics evaluated included progressive motility and the percentage of morphologically normal spermatozoa. Total proteins from 5x10(6) spermatozoa were separated and analyzed by SDS-PAGE. The results revealed that there was a substantial decrease of a 90 kDa protein in the frozen-thawed spermatozoa. Western blot analysis demonstrated that this protein was 90 kDa heat-shock protein (HSP90). Time course study showed that the decrease of HSP90 in spermatozoa initially occurred in the first hour during cooling to 5 degrees C. When compared with the fresh spermatozoa before chilling, there was a 64% decrease of HSP90 in spermatozoa after cooling to 5 degrees C. However, the motility and percentage of normal spermatozoa did not significantly decrease during this period of treatment. Both declined substantially as the semen was thawed after freezing from -100 degrees C. The results indicated that the decrease of HSP90 precedes the decline of semen characteristics. The length of time between a decrease of HSP90 and the decline in sperm motility was estimated to be 2 to 3 h. Taken together, the above results suggested that a substantial decrease of HSP90 might be associated with a decline in sperm motility during cooling of boar spermatozoa. PMID- 10729023 TI - Changes in intrauterine pressure after oxytocin administration in reproductively normal mares and in those with a delay in uterine clearance. AB - Intrauterine pressure was measured in 4 reproductively normal mares and 4 mares with delay in uterine clearance after administration of oxytocin to determine if intrauterine pressure varied between dosage and group. Changes in intrauterine pressure were measured during estrus, when a follicle was > or =35 mm, using a Millar "Mikro-tip" catheter that had 3 discrete pressure sensors/channels. Mares received 4 different treatments of 10, 5, 2.5 or 0 IU (vehicle) of oxytocin. The protocol for each treatment consisted of a 10-min baseline recording, administration of treatment and measurement of changes in intrauterine pressure for 65 min. After administration of the first two treatments, mares were rested for 2 h and the protocol repeated for the remaining 2 treatments. Changes in intrauterine pressure were measured on a physiograph and stored in a computer. The results were analyzed by 4x4 Latin Square Design analysis of variance (ANOVA) using the GLM procedure of the Statistical Analysis System. The ANOVA detected a main effect of treatment (P<0.01) and mare (nested within group; P<0.01) but no effect of channels, group or treatment-by-group interaction. There was a dose dependent increase in uterine activity in both normal mares and those with delayed uterine clearance. A dose of 10 IU of oxytocin induced a larger number of uterine contractions (5.67+/-0.06) for a longer time (24.09+/-1.18 min) than the 5 IU (4.16+/-0.06 contractions and 16.31+/-1.18; P<0.01 min) or 2.5 IU dose (4.08+/-0.06 contractions and 17.61+/-1.18 min). The first intrauterine wave occurred most often near the tip of the horn in 10 of 12 recordings in normal mares and in 8 of 12 recordings in mares with delayed uterine clearance. It was then propagated from the middle of the horn to the uterine body just cranial to the cervix. There was no pattern of propagation for subsequent intrauterine pressure waves. We conclude that the difference in spontaneous clearance of the uterus between the 2 groups is not reflected in their response to exogenous oxytocin as determined by changes in intrauterine pressure. PMID- 10729024 TI - Glucocorticoid influence on porcine granulosa cell IGF-I and steroid hormone production in vitro. AB - The effect of cortisol on granulosa cell (GC) insulin-like growth factor I (IGF I) synthesis, and IGF-mediated steroid production was examined at various stages of follicle maturation. Granulosa cells were recovered from gilts on Days 14, 18, and 20 of the estrous cycle, while luteinizing GC were recovered on Day 21, just prior to ovulation. The cells were cultured in serum-free medium with increasing concentrations of cortisol (0, 1, 10, and 100 microg/mL) for 5 d with or without IGF-I stimulation (10 ng/mL). During culture all cells were supplemented with FSH and androstenedione (A4). Cellular IGF-I, progesterone (P4) and estradiol-17beta (E2) production was determined by specific radioimmunoassays (RIA), and cell proliferation was assessed. Granulosa cell IGF-I and steroid hormone synthesis increased (P<0.05) with follicle maturation. Direct exposure to high cortisol concentrations, however, altered both IGF-I synthesis and action. Cortisol treatment lowered (P<0.05) IGF-I production by GC recovered on Days 18, 20, and 21. Furthermore, it reduced (P<0.05) IGF-stimulated P4 synthesis at all stages and decreased (P<0.05) IGF-stimulated E2 synthesis by cells recovered on Day 14. In contrast, cortisol enhanced (P<0.05) FSH-stimulated P4 production by GC collected on Days 14 and 18. The opposing effects on FSH and IGF-I action indicate that cortisol did not promote an overall suppressive effect on cell function, nor did it impair cell proliferation. Hence, these results demonstrate that elevated cortisol concentrations can disrupt both IGF-I synthesis and IGF mediated actions by porcine GC under in vitro conditions, and that specific disruptions are dependent on the stage of follicle maturation. PMID- 10729025 TI - Fertility after vaginal or uterine deposition of dog semen frozen in a tris extender with or without Equex STM paste. AB - Twenty-five bitches were artificially inseminated with semen that was frozen thawed using an egg yolk-Tris-glucose-citrate extender containing 5% glycerol with, or without the addition of 0.5% Equex STM Paste. Semen was collected on 2 occasions from 11 dogs, pooled, and evaluated for sperm motility, morphology and plasma membrane integrity. Each pool was then divided in 2 parts, diluted with 1 of the 2 extenders, and frozen in 0.5-mL straws. In the bitches, plasma progesterone was assayed daily during late proestrus and estrus. Artificial insemination (AI) was performed twice on Days 3 and 5 after the estimated LH peak. For each insemination, 200x10(6) spermatozoa were used. Ten bitches were inseminated with semen frozen without Equex: In 5 females, semen was deposited transcervically into the uterus with the aid of a fiberoptic endoscope and a urethral catheter, while the remaining 5 bitches were inseminated in the cranial vagina using a Norwegian catheter. Fifteen bitches were inseminated with semen frozen-thawed with Equex: Two groups of 5 bitches were inseminated according to the techniques described above, while 5 bitches were inseminated vaginally using the Osiris catheter. Pregnancy was diagnosed and the number of fetuses counted by ultrasound examination. Post-thaw, spermatozoa frozen with Equex tended to have higher total and progressive motility and to survive longer in vitro than when the extender without Equex was used. Spermatozoal concentration, age of the bitches, duration of heat and estrus, and progesterone concentration at LH peak and at the first and second AI did not differ among the 5 groups. The overall pregnancy rate of 84% (21/25) was close to what can be expected from well controlled natural matings. For both freezing extenders tested, 5/5 bitches were pregnant after uterine deposition of semen and 4/5 were pregnant when semen was deposited in the anterior vagina using the Norwegian catheter. With the Osiris catheter, 3/5 inseminations resulted in a pregnancy. No significant differences in pregnancy rate or number of fetuses were found between groups, site of deposition or freezing extender. PMID- 10729026 TI - Agreement between ultrasonographic classification of the CL and plasma progesterone concentration in dairy cows. AB - We evaluated the agreement between ultrasonographic characteristics of the corpus luteum (CL) and plasma progesterone (P4) concentration in dairy cows. In Phase I of the study, the ovaries of 8 cows were ultrasonographically examined, and P4 was analyzed daily from estrus (Day 0) to Day 4, then at Day 7 and Day 10, and again daily from Day 17 to the onset of next estrus. In Phase 2, the ovaries of 157 randomly selected Friesian cows were examined once by ultrasonography, and blood samples collected concurrently were analyzed for plasma P4. On the basis of the P4 values, the function of CLs was classified as follows: 1) non-secretory CL when plasma P4 was lower than 1 ng/mL (n=41); 2) evolving CL when plasma P4 was between 1 and 4 ng/mL (n=55); and 3) mid-cycle CL when plasma P4 was more than 4 ng/mL (n=61). On the basis of ultrasonographic examination, 3 additional groups were established (absence of CL, evolving CL, midcycle CL). Ultrasonographic characteristics and size of Day 3 to 4 CLs and their respective plasma P4 concentrations were not distinguishable from those of CLs observed 3 to 4 d before the subsequent estrus. The degree of agreement between the two classification was 72%. The data indicate that the functional classification of CLs is difficult to determine based on ultrasonography alone. PMID- 10729027 TI - Acute-phase response in dairy cows with acute postpartum metritis. AB - The diagnostic value of 2 plasma acute-phase proteins, haptoglobin and alpha1 acid glycoprotein, and plasma N-acetyl-beta-D-glucosaminidase enzyme activity were studied in 29 newly calved dairy cows. Nineteen had developed acute metritis with putrid vaginal discharge within 2 wk after calving; 10 were clinically healthy controls. Plasma haptoglobin concentration remained low in most cows with acute postpartum metritis. Only the 3 most severely affected cows exhibited a strong haptoglobin response. These were later culled due to poor condition and reduced fertility. This suggests that in acute uterine infection a highly increased haptoglobin concentration indicates poor prognosis for repeat conception. Plasma alpha1-acid glycoprotein concentration increased in acute postpartum metritis, the response pattern being less prominent than that for haptoglobin. The alpha1-acid glycoprotein concentrations did not correlate with severity of disease, and, consequently, the capacity of alpha1-acid glycoprotein in differentiating genital infections was relatively poor. The highest alpha1 acid glycoprotein concentrations were detected in cows with retained placenta and/or dystocia. Plasma N-acetyl-beta-D-glucosaminidase activity levels did not differ between the cows with acute postpartum metritis and healthy control cows. PMID- 10729028 TI - Relationship between bull field fertility and in vitro embryo production using sperm preparation methods with and without somatic cell co-culture. AB - Experiments were designed to compare rates of embryonic development following oocyte exposure to cryopreserved spermatozoa from bulls of varying proven fertility, utilizing 3 different sperm preparation methods prior to oocyte introduction. These included 1) sperm co-culture with bovine oviductal epithelial cells (BOEC); 2) sperm co-culture with buffalo rat liver cells (BRLC); or 3) control culture in a routine, cell-free culture system. Semen from 9 bulls was classified by lifetime 60- to 90-d nonreturn rates as having either (mean +/- SEM) high (n=3) 73.2 +/- 3a, medium (n=3) 70.3 +/- 2b or low (n=3) 65.8 +/- 3c field fertility ((ac)p< 0.01; (bc)p< 0.05). There was no difference in embryo cleavage rates for spermatozoa from the high (58 +/- 18%), medium (57 +/-23%) or low (57 +/- 18%) fertility groups. Development to morula or beyond of oocytes fertilized with high (53 +/- 30%) or low (58 +/- 27%) fertility semen tended (P<0.10) to be higher than of those fertilized with medium fertility (33 +/- 28%) semen. This lack of relationship between in vivo fertility and in vitro embryo outcome was consistent across all sperm preparation methods. Therefore, pooled data were used to evaluate the effect of sperm preparation on embryo outcome. There was no difference in embryo cleavage rates between BOEC monolayers (51 +/- 22%), BRLC monolayers (60 +/- 20%) and the cell-free controls (60 +/- 17%). Subsequent embryonic development to compact morula and beyond was higher (P<0.01) with the BRLC monolayer treatment (61 +/- 28%) than with the BOEC monolayers (42 +/- 33%) or control culture (39 +/- 24%). In conclusion, these studies suggest that there is no predictive relationship between bull field fertility (in the ranges evaluated here) and in vitro embryo cleavage or development rates. However, oocytes inseminated with sperm cells co-cultured on BRLC monolayers develop to the morula stage or beyond at a higher rate than oocytes inseminated with spermatozoa from the BOEC or cell-free system. PMID- 10729029 TI - Transvaginal ultrasound-guided oocyte retrieval following FSH stimulation of domestic goats. AB - The objectives of this study were to evaluate different ovarian stimulation protocols on donor goats and to develop a safe, repeatable method for harvesting oocytes from FSH-treated does (Experiment I). Based on the preliminary findings of the first experiment, 32 crossbred does were used in a second experiment (Experiment II), 16 that had not been previously aspirated and 16 that had undergone one previous aspiration, were used to fine tune the procedure. Females were randomly subjected to 1 of the 2 ovarian stimulation protocols: Treatment (A) does were implanted with a norgestomet ear implant. Starting 10 d post implantation, does were administered FSH daily for 4 d. Does in Treatment (B) were treated similarly to those in (A) but were implanted for only 3 d before starting the FSH injections and implants were not removed prior to aspiration. Using a 2 x 2 factorial arrangement, fresh does (n=16), not previously aspirated, were then further randomly assigned to either a laparoscopic aspiration procedure (LAP) or a transvaginal ultrasound-guided aspiration procedure (TUGA). The LAP procedure was performed using a fiber optics. For the TUGA, the doe was placed in dorsal recumbency, and a 5 MHz human transvaginal transducer, attached to the ultrasound unit, was positioned vaginally for oocyte aspiration. In summary, there was no significant difference among treatment groups for parameters evaluated, with the exception of methods for oocyte collection. The number of follicles detected and oocytes harvested using TUGA (9.5 and 4.3, respectively) was less than for females obtained by LAP (17.4 and 14.4, respectfully). The percentage of oocytes recovered from does subjected to the TUGA (68%), however, was similar to those subjected to the LAP (69%). Unlike donor does subjected to a repeated LAP, there was no evidence of adhesions in donor does from the repeated TUGA group. The TUGA approach to oocyte collection should not be overlooked in an effort to decrease the chances of adhesions in valuable donor goats. PMID- 10729030 TI - Assessment of estrus detection by visual observation and electronic detection methods and characterization of factors associated with estrus and pregnancy in beef heifers. AB - One hundred and sixty-four beef heifers representing Angus, Brahman and their crosses were subjected to estrus synchronization treatment following evaluation of weight, body condition score and reproductive tract. Heifers were assigned to 1 of 2 methods of estrus detection, either visual observation for signs of standing estrus or a rump-mounted pressure-sensitive detection device. All heifers were artificially inseminated during a 25d insemination period and then bred by a bull. The effectiveness of estrus detection and timely insemination were evaluated by the type of detection method, breed and breeding event resulting in a pregnancy. Although there was not a significant difference in first service conception for method of detection, at the end of a 25-d insemination period, 60.5% of the visually observed heifers were pregnant, while only 45.8% of the heifers detected by the mount detection device were pregnant (P = 0.05). The reduced 25-d conception rate in the pressure-sensitive detection group suggested that insemination of detected heifers may not have been optimal for pregnancy. The mean time to estrus after implant removal and the time of insemination were evaluated. Heifers pregnant at the first service had a shorter time to estrus (32.11 +/- 1.6 h, P=0.13), a longer mean interval from the start of estrus to insemination (12.10 +/- 1.2 h, P = 0.16) and a positive mean interval between the end of standing estrus and the time of insemination (3.17 +/ 1.7 h, P=0.19) than heifers not conceiving at the synchronized estrus (38.5 +/- 2.1, 7.42 +/- 1.9 h and -2.04 +/- 2.1 h, respectively). Breed differences were observed in estrus durations (Angus 8.52 +/- 1.2 h, Brahman 6.65 +/- 1.2 h, crossbred 11.90 +/- 1.2 h; P = 0.03), number of mounts (19 +/- 3.6, 25 +/- 5.4, 37 +/- 5.5, respectively; P=0.02) and gestation length (281 +/- 1.2, 291 +/- 1.8, 286 +/- 1.1 d, respectively; P = 0.001). PMID- 10729031 TI - Relationship between triacylglycerol concentration in the liver and first ovulation in postpartum dairy cows. AB - The relationship between accumulation of triacylglycerols in the liver and first ovulation post partum was studied in an experiment with 32 dairy cows. During the dry period, 16 treated cows were fed a high energy diet to induce overconditioning, while 16 control cows were fed a recommended diet. After parturition, the treated cows entered deeper negative energy balance state, required more days to reach the nadir of negative energy balance, required more days to regain positive value of energy balance, and lost more body weight than the control cows. The treated cows also accumulated greater amounts of triacylglycerols in the liver and had higher concentrations of nonesterified fatty acids and 3-hydroxybutyrate post partum than the control cows. Although the number of days to first ovulation did not differ significantly between the 2 groups, the results of pooled data demonstrated that the liver triacylglycerol concentration was positively correlated with the number of days to first ovulation. In conclusion, negative energy balance caused the body fat of cows to mobilize. As a consequence, the cows accumulated greater amounts of triacylglycerols in the liver, and this accumulation was associated with an increase in the interval from parturition to first ovulation. PMID- 10729032 TI - Administration of oxytocin immediately after insemination does not improve pregnancy rates in mares bred by fertile or subfertile stallions. AB - It is probable that reduced pregnancy rates in mares bred to subfertile stallions is attributable, in part, to the reduced number of normal spermatozoa that colonize the oviduct. Administration of oxytocin stimulates both uterine and oviductal contractility. The hypothesis that oxytocin may enhance sperm transport to/into the oviducts, and thereby increase pregnancy rates, was tested in 2 trials. For both trials, fertile estrous mares with follicles > or = 35 mm in diameter were inseminated once at 24 h after administration of 1500 to 2000 U hCG. The inseminate dose was limited to 100 million spermatozoa in order to lower pregnancy rates and thus increase the chance of detecting a treatment effect. Pregnancy status was determined by transrectal ultrasound examination 14 to 16 d after insemination. In Trial 1, 49 mares were inseminated with 4 mL extended semen from 1 of 3 stallions (1 fertile and 2 subfertile males). Immediately after insemination, the mares were administered either 20 U oxytocin or 1 mL saline intravenously. In Trial 2, 51 mares were inseminated with 4 mL extended semen from 1 of 4 stallions (1 fertile and 1 subfertile male used in Trial 1, and 2 additional fertile males). Immediately after insemination, and again 30 min later, mares were administered either 5 U oxytocin or 0.25 mL saline intramuscularly. To test for effects of treatment with oxytocin and for the interaction between semen quality and treatment, a generalized linear mixed regression model was used that accounted for the split-plot design (treatment within stallions), the random effect of stallion, the fixed effect of semen quality, the binary outcome of a single breeding trial, and the varying number of trials per stallion/treatment groups. Three treatment protocols or regimens were used: placebo, 5 U oxytocin injected twice intramuscularly, and 20 units oxytocin injected twice intravenously. Semen was classified as high (fertile stallions) or low (subfertile stallions) quality. No interaction between semen quality and treatment was detected (P > 0.10). The pregnancy rate of mares treated with oxytocin immediately after insemination was 30% (15/50) compared with 50% (25/50) for mares treated with saline immediately after breeding. Administration of oxytocin did not affect pregnancy rates (P > 0.10). PMID- 10729033 TI - Repeatability and heritability of response to superovulation in Holstein cows. AB - The objective of this study was to estimate the relative effects of genetic and phenotypic factors on the efficacy and efficiency of superovulation for Holstein Friesian cows reared in Brazil. A database, established by the Associacao Brasileira de Criadores de Bovinos da Raca Holandesa, consisting of a total of 5387 superovulations of 2941 cows distributed over 473 herds and sired by 690 bulls was used for the analysis. The records were analyzed by MTDFREML (Multiple Trait Derivative-Free Restricted Maximum Likelihood), using a repeatability animal model. The fixed effects included in the model were contemporaneous group (veterinarian, herd, year and season of the superovulation); number of semen doses; cow age; and superovulation order. The estimated repeatability of the number of the transferable embryos was low (0.13), and the estimated heritability was 0.03. These results indicate that environmental factors play a critical role in the response of a cow to a superovulation treatment. There is little evidence that future responses to superovulation by individual females can be predicted by previous treatment(s) or that superovulation response is an heritable trait. PMID- 10729034 TI - Quality of stallion semen obtained by a new semen collection phantom (Equidame) versus a Missouri artificial vagina. AB - A study was performed to test a new semen collection device (Equidame phantom) that fractionates the ejaculate by comparing the quality of semen obtained by the Equidame phantom with that obtained by a Missouri artificial vagina. Semen from 4 Finnhorse stallions was collected 4 times per stallion by both methods. Half of the ejaculate was frozen and the other half extended and loaded into 2 Equitainer transport containers (24- and 48-h samples). Motility parameters were determined by a Hamilton-Thorn motility analyzer after cooled storage for 24 and 48 h and again after freezing/thawing. Raw and chilled semen samples were cultured and the number of bacterial colonies counted after incubations of 24 and 48 h. After a 24 h incubation the number of colony-forming units (CFU) in raw semen was significantly higher (P<0.01) when collected by the Missouri artificial vagina than by the Equidame phantom. After cooled storage, 75% of the semen samples contained no bacteria after an incubation of 24 h, and 69% yielded no growth after 48 h. The sperm-rich fractions (Cup 2) collected by the Equidame phantom had lower mean volumes (22.1 +/- 2.3 mL [+/- SEM] versus 101.6 +/- 9.3 mL) and significantly higher mean sperm concentrations (218.0 +/- 25.8 x 10(6) vs 86.2 +/ 8.1 x 10(6) cells/mL; P<0.05) than the total ejaculates collected by the Missouri device. The total and progressive motility of chilled and frozen-thawed semen did not differ significantly between collection methods. The Equidame phantom yielded semen that was of a lower bacteriological colony counts, but had sperm motility similar to that of semen collected with the traditional method by the Missouri artificial vagina. PMID- 10729035 TI - Effect of equine chorionic gonadotropin on weaning-to-first service interval and litter size of female swine. AB - We evaluated the effect of PMSG on the weaning-to-first service interval, total litter size and born alive litter size in swine. Four doses of PMSG (0, 500, 750 and 1,000 IU) were administered intramuscularly after weaning to sows at 3 different farms, grouped by parities (1, 2 and 3 or higher) and 2 distinct time periods. The associations among main effects and response variables were assessed by analysis of variance. Polynomial orthogonal terms were used to adjust the estimates of weaning-to-first service interval, total litter size and born alive litter size for the interaction effect of parity and PMSG treatment. The weaning to-first service interval did not differ across periods and farms (P>0.05), although the interval was shorter (P<0.05) for Parity 3+ sows (4.97 d) than for Parity 1 sows (5.29 d), with no other differences in intervals observed across parities (P>0.05). Time period did not influence litter size (P>0.05), but there were differences in litter size across farms (P<0.05). Both litter size traits were lower for Parity 1 sows than for higher parity sows (P<0.05), but there were no differences in litter size between Parity 2 and 3+ sows (P>0.05). Litter size increased with PMSG dose in both Parities 1 and 2 (P<0.05), but not in Parity 3+ (P>0.05). A significant quadratic effect (P<0.05) of PMSG treatment in weaning-to first service interval was observed for both Parity 1 and 2 sows, with the shortest intervals occurring with the 750 IU dose for Parity 1 sows. Administration of PMSG after weaning was associated with a shortened weaning-to first service interval in Parity 1 sows and increased litter size in Parity 1 and 2 sows. PMID- 10729036 TI - Isolation of immunologically active uterine luminal proteins associated with follicular and luteal phases of the ovary in buffalo (Bubalus bubalus). AB - Uterine luminal proteins (ULP) collected from the genital tract of buffalo during the follicular (Group F) and luteal (Group L) phases of the estrous cycle were chromatographed using sephacryl S-200 gel. Five peaks were detected in each group. Different protein concentrations (10 to 200 microg) from Peaks I and V in each group were examined for immunological activity on polymorph nuclear leukocytic cells (PMNL) in vitro. All concentrations except 10 microg of ULP Peak I (< or = 250 kDa) in Group F enhanced phagocytic activity of PMNL. Peak V (56 kDa) in the same group enhanced phagocytic activity of PMNL only at low protein concentrations (10, 20 and 40 microg protein), while at greater concentrations (80, 150 and 200 microg protein) PMNL activity was suppressed. On the other hand, all protein concentrations from Peak 1 (> or = 250 kDa) in Group L suppressed PMNL activity in a dose-dependent manner. Proteins from Peak V (31 kDa) in Group L suppressed PMNL phagocytic activity at all concentrations but not to the same extent as in Peak I. Electrophoretic analysis of Peaks I and V in both groups revealed only 3 detectable protein bands (subunits) in Peak I and 1 detectable subunit in Peak V. Several additional proteins were probably not detected. The molecular weights of the detected subunits in Peaks I and V in Group F were greater than those in Group L as indicated by SDS-PAGE analysis. The results of this study show that ULP collected from buffalo possessed proteins that modulated phagocytic activity of PMNL in vitro. Proteins collected during the follicular phase, especially Peak I, enhanced phagocytic activity of the PMNL, whereas those collected during the luteal phase (Peaks I and V) suppressed activity. Changes in the molecular weights of ULP detected in this experiment may be related to the changes in phagocytic activity of PMNL tested in vitro. PMID- 10729037 TI - Relationships among seminal culture, seminal white blood cells, and the percentage of primary sperm abnormalities in bulls evaluated prior to the breeding season. AB - Semen samples from 100 beef breed bulls were evaluated for sperm morphology (phased contrast microscopy), seminal white blood cells, and the presence of potential reproductive pathogens. Eligibility required visualization of the glans penis throughout semen collection. Based on clinical spermiograms, bulls were grouped into normal, marginal, or unsatisfactory morphology classifications. The 3 experimental groups were similar in age and scrotal circumference and differed significantly in the percentage of primary sperm abnormalities. Most semen samples (94%) contained one or more potential reproductive pathogens (Hemophilus somnus. Mycoplasma bovigenitalium, Arcanobacterium pyogenes and Ureaplasma diversum). No significant relationship could be demonstrated between primary abnormalities and the assigned culture score. Our experimental results suggest that clinicians should interpret clinical semen culture results with great care. No significant relationship could be demonstrated between primary abnormalities and assigned white blood cell (WBC) score, although, only 1% of the samples was scored >5 WBC per high power field. The use of seminal WBC score may be valid adjunct to routine semen evaluation when that threshold is the basis for clinical decisions. PMID- 10729038 TI - Live birth of a bear cub following nonsurgical embryo collection. AB - In the near future, 6 of 8 bear species will face extinction mainly because of loss of their natural habitat. This loss of habitat will ultimately require some of these bears to be maintained in zoos and wildlife preserves in the hope of conserving genetic diversity. If the giant panda is representative of other bear species, reproductive performance will be inhibited in such an environment. In this study, we used the nonendangered American black bear (Ursus americanus) as the model for developing appropriate embryo transfer procedures. The donor bear mated numerous times between late May and early June. In late July we anesthetized her and used a series of telescoping sheaths to gain access to the uterus Then we passed a catheter through the largest sheath, inflated the balloon, and, using a 20-mL syringe, repeatedly infused into and then aspirated from the uterus PBS + BSA. We emptied the syringe into Petri dishes and observed 2 embryos. We rinsed the embryos, placed them in human tubal fluid + HSA + HEPES and then held them at 35 degrees C for 5 h. The recipient mated during mid-June; in late July we anesthetized her and, with the aid of laparoscopy, transferred an embryo into the cranial portion of the uterine horn ipsilateral to the ovary containing a CL. The recipient delivered 2 cubs in January. Necropsy results indicated that the neonates lived for 6 to 8 wk before succumbing to flooding in the den. The DNA from hair samples belonging to the neonates indicated that the male cub belonged to the donor, the female cub to the recipient. The delayed implantation mechanism in bears probably allowed for the successful development of the embryo in the presence of a substantial asynchrony between the donor and the recipient (13 d). We conclude that embryo transfer is possible in the American black bear and can lead to the birth of live cubs. PMID- 10729039 TI - Effect of monensin and progesterone priming on ram-induced reproductive performance of boutsiko mountain breed ewes. AB - The effects of monensin and progesterone priming on reproductive performance (estrous response, lambing rate and prolificacy) of grazing Boutsiko mountain breed adult and 18-mo.-old ewes at the end of seasonal anestrus were investigated. In Experiment 1 the feed supplement with or without monensin was offered for 21 d after introduction of vasectomized rams (Day 0). Progesterone was administered to the ewes in the respective groups as a single injection at Day -3. Ewes of both age groups were assigned randomly to 1 of 4 treatments: C, C+P, C+M and C+M+P. In Experiment 2 the supplement C or M was offered from Day 26 to Day 21. The treatments consisted of C, C+P and C+M+P. Blood samples were taken 50 h after ram introduction for determination of plasma concentrations of P and insulin-like growth factor-I (IGF-I). There was a greater increase in estrous response at Days 17 to 19 and at Days 0 to 19 when supplementation was offered before rather than after ram introduction in both age groups. In the adult group ewes synchronization of estrus at Days 17 to 19 was significantly increased by administration of monensin (P<0.05) and progesterone (P<0.01) compared with the control group in the first but not the second experiment. The incidence of estrus at Days 17 to 19 or at Days 0 to 19 was highest in the adult groups treated with monensin and progesterone in both experiments. In 18-mo.-old ewes progesterone was effective in synchronizing estrus only in Experiment 2. Mean plasma IGF-I concentrations were increased by monensin treatment (P<0.05) in adult ewes that were at the periovulatory stage at blood sampling time. Correlation coefficients between IGF-I and progesterone concentrations in monensin plus progesterone group adults were -0.715 (P<0.02) and -0.516 (P<0.01), respectively across all treatments. The results suggest that monensin and progesterone priming improved reproductive performance, and the monensin-induced increase in plasma IGF-I levels at the periovulatory stage may be causally related to the ability of ovulatory follicles to develop into functional corpora lutea (CL). PMID- 10729040 TI - Effect of lactate dehydrogenase activity and isoenzyme localization in bovine oocytes and utilization of oxidative substrates on in vitro maturation. AB - Oocyte nutritional metabolism changes during maturation in order to increase the energy available to support metabolic requirements. The aim of this work was to study pyruvate and lactate utilization as oxidative substrates on IVM and lactate dehydrogenase (LDH) activity and localization of their isoenzymes in bovine oocytes. Immature cumulus-oocyte complexes (COCs) were recovered by aspiration of antral follicles in ovaries obtained from slaughtered cows. The COCs and denuded oocytes were separately cultured in TCM-199 with steer serum (controls) and were supplemented with pyruvate, lactate or lactate plus NAD for 24 h at 39 degrees C in 5% CO2:95% humidified air. No significant differences were found in IVM rates of COCs matured according to the various treatments (P>0.05). The IVM rate in denuded oocytes without supplementation was 47.8%. The presence of pyruvate in the culture medium resulted in an increased number of matured denuded oocytes (59.4%; P<0.05), but the addition of lactate failed to improve the IVM rate of matured denuded oocytes (47.6%, P>0.05). When the medium was supplemented with lactate plus NAD, the IVM rate of denuded oocytes likewise failed to differ from that obtained with the addition of pyruvate (59.9%, P>0.05). The LDH activity in immature and matured COCs and denuded oocytes was (3.1+/-1.6) 10(-3), (3.3+/-1.6) 10(-3) U/COC, (5.2+/-2.0) 10(-5), (5.4+/-3.5) 10(-5) U/oocyte with pyruvate as substrate, and (1.2+/-0.5) 10(-3), (1.0+/-0.5) 10(-3) U/COC, (2.2+/-0.1) 10(-5), (2.5+/-1.4) 10(-5) U/oocyte respectively, with lactate; no significant differences due to maturation status were observed (P>0.05; n = 9 for each LDH activity). Electrophoresis disclosed that the principal band corresponded to the LDH-1 isoenzyme in oocytes, while there was no predominance of any isoenzyme in cumulus cells. Due to the fact that LDH-1 is the main oocyte isoenzyme, the pyruvate used during oocyte maturation could be partly produced from lactate when the NAD supply is adequate. Cumulus cells would be responsible for providing pyruvate and/or lactate as oxidative substrates to be used by the bovine oocyte and this supply would be regulated by the LDH activity in these cells. PMID- 10729041 TI - Improvement of in vitro co-culture systems for bovine embryos using a low concentration of carbon dioxide and medium supplemented with beta mercaptoethanol. AB - Two experiments were conducted to investigate the effect of carbon dioxide (CO2) gas atmosphere and beta-mercaptoethanol on the development of bovine embryos in an in vitro co-culture system. In Experiment 1, in vitro-matured bovine oocytes were inseminated and then co-cultured with cumulus cells in culture medium (CM; 25 mM HEPES buffered TCM-199 supplemented with 5% superovulated cow serum and 0.5 mM sodium pyruvate). Oocytes matured and fertilized in 2 or 5% CO2 in air exhibited similar cleavage rates, but the proportion of embryos that developed to the blastocyst stage was higher for embryos co-cultured in 2 versus 5% CO2 in air. In Experiment two, 4- to 8-cell embryos produced under the condition of 2% CO2 in air were co-cultured with cumulus cells in CM supplemented with various levels of beta-mercaptoethanol (0, 5, 10, 50 microM). The percentage of embryos that developed to the blastocyst stage in CM with 10 microM beta-mercaptoethanol was higher (P<0.05) than that of embryos co-cultured with 0 or 50 microM beta mercaptoethanol. These results indicate that cumulus cell co-culture in an atmosphere of 2% CO2 in air has a marked stimulatory effect on in vitro development of bovine embryos and that addition of beta-mercaptoethanol to the co culture medium 2 d after insemination improved the in vitro development of bovine 4- to 8-cell embryos to the blastocyst stage. PMID- 10729042 TI - Effects of season and phase of the estrous cycle on steroidogenesis and LH-FSH sensitivity of large ovine follicles perfused in vitro. AB - The aims of this study were to compare steroidogenesis (progesterone, androstenedione and estradiol production) and response to LH and FSH challenge by whole perifused follicles 4 to 5.5 mm in diameter, obtained at different periods of the breeding season (onset, middle, end), during anestrus and the luteal phase. We have observed that all follicles do not have the same steroidogenetic potential and do not respond with the same intensity to LH and FSH. At the middle of the breeding season, LH and FSH supplementation was ineffective in increasing progesterone secretion by follicles (0.19+/-0.05 vs. 0.20+/-0.03 ng/mL). In contrast, gonadotrophin challenge elicited significant (P<0.05) increases in androstenedione (0.94+/-0.34 vs. 0.35+/-0.09 ng/mL) and estradiol (120+/-11 vs. 49+/-10 pg/mL) production immediately after its administration. At the onset of the breeding season, steroidogenesis was identical under both basal and gonadotrophin-stimulated conditions unlike that in middle of the breeding season. However follicles were more sensitive to the gonadotrophin challenge in terms of estradiol production than those collected at the middle of the breeding season (220+/-45 vs. 120+/-11 pg/mL). Follicles obtained at the end of the breeding season featured higher progesterone (2.61+/-0.81 vs. 0.19+/-0.05 ng/mL; P<0.05) and lower estradiol production (10+/-3 vs. 49+/-10 pg/mL; P<0.05) that was not influenced by LH and FSH. Basal androstenedione secretion was comparable to that observed at the middle of the breeding season (0.42+/-0.10 vs. 0.35+/-0.09 ng/mL), but the response to stimulation was significantly higher (1.82+/-0.61 vs. 0.94+/-0.34 ng/mL; P<0.05). In anoestrus and the luteal phase, follicles presented higher progesterone and androstenedione and lower estradiol concentrations (P<0.05) compared with those obtained during the follicular phase at the middle of the breeding season. In the luteal phase, follicles remained capable of responding to LH-FSH challenge by increasing estradiol secretion (9+/ 1 before and 21+/-6 pg/mL after LH-FSH; P<0.05). In contrast, in the luteal phase, estradiol production was not increased by LH-FSH challenge (7+/-2 vs. 12+/ 4 pg/mL). PMID- 10729043 TI - Cryopreservation of sperm from the endangered formosan landlocked salmon (Oncorhynchus masou formosanus). AB - The Formosan landlocked salmon (Oncorhynchus masou formosanus) are at a high risk of extinction, and the sustained maintenance of the population will soon depend on aquaculture systems, which use cryopreservation of spermatozoa to increase genetic diversity. We investigated the effectiveness of dimethyl sulfoxide (DMSO), dimethyl-acetamide (DMA), and methanol as cryoprotectants in combination with 300 mM glucose as extender on the freezing of Formosan landlocked salmon spermatozoa. We also evaluated the morphological changes of Formosan landlocked salmon spermatozoa after their immediate dilution in the 300 mM glucose-DMSO extender and after freeze-thawing. The spermatozoa frozen with DMSO as a cryoprotectant showed significantly higher post-thaw motility and fertility than spermatozoa frozen with DMA or methanol. The fertilization capacity of frozen thawed Formosan landlocked salmon was comparable to that of fresh spermatozoa. Intersubspecies fertilization trials between cryopreserved Formosan landlocked salmon spermatozoa and Amago salmon eggs showed high fertilization rates. Based on the findings, the potential value of using sperm bank to safeguard this endangered species is discussed. PMID- 10729044 TI - Effects of bovine oviductal proteins on bull spermatozoal function. AB - The effects of bovine oviductal proteins on bull sperm viability, acrosome reaction and motility were studied. Motile frozen/thawed spermatozoa from Percoll gradients were incubated with 1.0 mg/mL oviductal proteins (>8 kDa) extracted by ammonium sulphate precipitation from oviductal extract (OE) or serum-free oviductal epithelial cell-conditioned media (CM), treated in the presence (CM+) or absence (CM-) of 1 microg/mL 17beta-estradiol. Inclusion of oviductal proteins had a significant beneficial effect on sperm viability (76.3 to 80.6%+/-5.3) compared with the control (without oviductal proteins; 57.8%+/-5.3) immediately after the commencement of incubation. After 5 h, viability was significantly higher for CM- and OE treatments than for the control, although no differences were observed at 24 h. Acrosomal status only differed among treatments after 24 h, when higher percentages of acrosome- reacted spermatozoa were found in the control (46.0%+/-2.5) than in the oviductal protein treatments (33.1 to 38.2%+/ 2.5). No differences in percentages of motile spermatozoa occurred within the first hour of incubation, although inclusion of CM proteins decreased sperm velocities, beat cross frequency, linearity, and straightness but increased values for mean angular displacement. These findings suggest that proteins secreted by oviductal epithelium promote viability, delay the acrosome reaction and suppress the motion of spermatozoa. PMID- 10729045 TI - Effect of stage of anestrus on the induction of estrus by the dopamine agonist cabergoline in dogs. AB - Beagle bitches were administered the dopamine D2 receptor agonist cabergoline in 3 groups of 5 animals each, starting on known days of the estrous cycle. Cabergoline treatment was started in either early anestrus (Days 93 to 108), mid anestrus (Days 123 to 156), or late anestrus (Days 161 to 192) at doses of 5 ug/kg/d, per os, and was continued until the confirmation of induced proestrus or for 40 d. Reproductive parameters were compared with those in 5 control anestrous bitches (Days 90 to 150). In control bitches, the mean (+/- SEM) interval to the next proestrus (73+/-11 d) resulted in an interestrus interval (192+/-9 d) similar to that of the previous cycles (196+/-11 d). In 14 of the 15 cabergoline treated bitches, the next proestrus occurred within 4 to 30 d, was premature in early and mid-anestrous bitches and developed with low variability within groups. The resulting intervals to proestrus in bitches treated with cabergoline in early anestrus (20+/-2 d), mid-anestrus (14+/-3 d) and late anestrus (6+/-1 d) resulted in interestrus intervals in those groups of 131+/-5, 166+/-7 and 196+/-2 d, respectively. In response to treatment, interestrus intervals were reduced (P<0.05) and more synchronous (P<0.05) in early and mid-anestrus bitches, and were more synchronous (P<0.05) in late-anestrous bitches compared with those of control bitches or those of the previous cycle. Periovulatory estradiol and progesterone profiles of induced cycles in treated bitches were similar to those of spontaneous cycles in control bitches. Four of 5 control bitches and 12 of the 14 responding cabergoline-treated bitches became pregnant and produced normal litters. Plasma prolactin concentrations at Days 2 and 5 of treatment (0.3+/-0.1 ng/mL) and at the onset of proestrus shortly before the end of treatment (0.4+/ 0.1 ng/mL) were lower (P<0.05) than those present in anestrus prior to treatment (1.7+/-0.6 ng/mL) or in control bitches. Prolactin was also low at the onset of proestrus in control bitches (0.5+/-0.2 ng/mL). The results demonstrate that prolactin-lowering doses of the dopamine agonist cabergoline can terminate the normal obligate anestrus in dogs, and that the effect occurs more slowly in early anestrus than in mid or late anestrus. PMID- 10729046 TI - Allopurinol, an inhibitor of xanthine oxidase, improves the development of IVM/IVF bovine embryos (>4 cell) in vitro under certain culture conditions. AB - To determine the origin of free oxygen radicals in the culture medium of bovine embryos, the effect of allopurinol, an inhibitor of xanthine oxidase, on the development of embryos (>4 cell) in modified synthetic oviduct fluid (m-SOF) medium was examined. When embryos were cultured in the presence of 0.2 mM allopurinol under high oxygen tension (5% CO2 in air), the blastocyst rate significantly (P<0.05) increased compared with the absence of allopurinol (allopurinol (+) 42 vs. (-) 25%; Day 6, 63 vs. 51%; Day 7, 69 vs. 58%; Day 8). However, allopurinol had no effect on embryo development under low oxygen tension (5% CO2, 5% O2, 90% N2). Moreover, it was found that the developmental rate and the total cell number of blastocysts decreased (development rate: 60 vs. 28%, cell number: 132 vs. 74) when the embryos were cultured in medium containing 0.01 U/mL xanthine oxidase (XOD) and 0.1 mM hypoxanthine (HXT), and the damaging effect of XOD and HXT was removed by the addition of 0.2 mM allopurinol. The beneficial effect of allopurinol was also observed when the glucose concentration was increased to 4.5 mM from 1.5 mM (control: 22% vs. allopurinol: 34%; Day 8), but no beneficial effects were observed in the media without glucose (control: 55% vs. allopurinol: 59%). Taken together, these results suggested that a portion of the free oxygen radicals are generated from the XOD and HXT reactions under culture conditions, and this generation is enhanced by high oxygen tension in the gas atmosphere or by high glucose concentrations in the medium. PMID- 10729047 TI - Sperm-lectin agglutination combined with swim-up leads to an efficient selection of highly motile, viable and heterogeneous ram spermatozoa. AB - Lectins, high molecular weight glycoproteins with different sugar-binding specificity, are able to agglutinate different cell types. The recovery of high quality spermatozoa can be facilitated by the agglutination induced by the lectin binding. The objective of this study was to combine sperm-lectin agglutination with a dextran/swim-up procedure for developing a new selection technique for ram spermatozoa. To study sperm quality, cell viability (plasma membrane integrity), the HOS-test response and progressive individual motility were assessed. Simultaneously, centrifugal countercurrent distribution (CCCD) in an aqueous two phase system was carried out to analyze sperm surface heterogeneity. Semen from 3 mature Saltz rams was pooled, and 0.5-mL aliquots were incubated with 4 fluorescein-labelled lectins (ECL, JAC, PSA, RCA). Then, a dextran solution was gently added and overlaid with medium. The top layer of the medium containing the spermatozoa was collected and replaced by careful addition of fresh medium. The incubation sequence was repeated 3 times at 10-min intervals. The consecutive 4 top layers obtained were pooled to give the swim-up combined sample. The highest rate of improvement in sperm quality was obtained after incubation with RCA, with a 50% increase in progressive individual motility, 21.6% in HOS value and 39.5% in viability. Total cell recovery was 64% (1.56x10(9) cells), with a viable cell recovery rate of 86%. The obtained sample showed 82% motility, 80% HOS score and 77% viability, up from the pre-swim-up values of 51, 60 and 57 %, respectively. Comparative CCCD analysis revealed a very high heterogeneous population in the RCA/swim-up sample obtained, while a much more homogeneous population was obtained in the sample after the dextran/swim-up procedure previously developed byus With this simple method, a large proportion of highly-motile spermatozoa with preserved plasma membrane and high heterogeneity can be obtained. These results strongly suggest that this selection procedure could result in a high fertility rate. PMID- 10729048 TI - Comparison of the longevity of motility of stallion spermatozoa incubated at 38 degrees C in different capacitating media and containers. AB - This study was designed to compare the effects of different media and containers on longevity of motility of spermatozoa during in vitro incubation at 38 degrees C in either air or 5% CO2 atmosphere. Three ejaculates were collected from each of 4 stallions. The media tested were skim milk-glucose, modified Krebs/Ringer and Hank's salts solution for incubation in an air atmosphere, and modified Krebs/Ringer and Brackett and Oliphant (BO) defined medium for incubation in a 5% CO2 atmosphere. All samples were incubated in 5-mL borosilicate glass tubes filled with 3 mL of extended spermatozoa, 5-mL borosilicate tubes filled with 6 mL (topped) of extended spermatozoa, 35-mm Petri dishes filled with 3 mL of extended spermatozoa, and 35-mm Petri dishes with 200-microL microdroplets of extended spermatozoa under sterile mineral oil. For all treatments, individual samples were removed at 2, 4, 6 and 12 h of incubation to determine the percentage of motile cells. Overall, spermatozoa incubated in Petri dishes in both 3-mL and microdroplet treatments had significantly higher motility than those incubated in glass tubes (P<0.01). At 6 and 12 h of incubation in Petri dishes, progressive motility was significantly higher for spermatozoa extended in the Hank's salts solution than in the other media. Both the medium and container used significantly affected the longevity of motility of spermatozoa incubated at 38 degrees C. PMID- 10729049 TI - Synchronization of estrus and fertility in zebu beef heifers treated with three estrus synchronization protocols. AB - The effects on estrus and fertility of 3 estrus synchronization protocols were studied in Brahman beef heifers. In Treatment 1 (PGF protocol; n=234), heifers received 7.5 mg, i.m. prostianol on Day 0 and were inseminated after observed estrus until Day 5. Treatment 2 (10-d NOR protocol; n = 220) consisted of norgestomet (NOR; 3 mg, s.c. implant and 3 mg, i.m.) and estradiol valerate (5 mg, i.m.) treatment on Day -10, NOR implant removal and 400 IU, i.m. PMSG on Day 0, and AI after observed estrus through to Day 5. Treatment 3 (14-d NOR+PGF protocol; n = 168) constituted a NOR implant (3 mg, sc) on Day -14, NOR implant removal on Day 0, PGF on Day 16, and AI after observed estrus through to Day 21. All heifers were examined for return to estrus at the next cycle and inseminated after observed estrus. The heifers were then exposed to bulls for at least 21 d. During the period of estrus observation (5 d) after treatment, those heifers treated with the PGF protocol had a lower (P<0.01) rate of estrual response (58%) than heifers treated with the 10-d NOR (87%) or 14-d NOR+PGF (88%) protocol. Heifers treated with the 10-d NOR protocol displayed estrus earlier and had a closer synchrony of estrus than heifers treated with either the PGF or the 14-d NOR+PGF protocol. Heifers treated with the 14-d NOR+PGF protocol had higher (P<0.05) conception and calving rates (51 and 46%) to AI at the induced estrus than heifers treated with the PGF (45 and 27%) or the 10-d NOR (38 and 33%) protocol. Calving rate to 2 rounds of AI was greater (P<0.05) for heifers treated with the 14-d NOR-PGF (50%) protocol than heifers treated with the 10-d NOR (38%) but not the PGF (43%) protocol. Breeding season calving rates were similar among the 3 protocols. The results show that the 14-d NOR+PGF estrus synchronization protocol induced a high incidence of estrus with comparatively high fertility in Brahman heifers. PMID- 10729051 TI - Influence of follicle size, medium, temperature and time on the incidence of diploid bovine oocytes matured in vitro. AB - The present work describes a cytogenetic study of in vitro-matured bovine oocytes designed to analyze the incidence of diploid oocytes induced by concentration of serum in the culture medium, follicle size, culture temperature and incubation time. In Experiment 1, immature follicular oocytes from follicles of the same size were cultured for 24 h in TCM-199 supplemented with increasing concentrations 0, 10, 20 and 50% of estrous cow serum (ECS). In Experiment 2, immature oocytes harvested from follicles of different sizes were cultured for 24 h in TCM-199 supplemented with 20% ECS at 39 degrees C in 5% CO2. In Experiment 3, immature follicular oocytes were matured in TCM-199 supplemented with 20% ECS at 2 different temperatures (37 degrees C or 39 degrees C) in 5% CO2. In Experiment 4, immature oocytes were matured over 4 different incubation times (24, 36 and 48 h) in TCM-199 supplemented with 20% ECS in 5% CO2. The highest concentration (50%) of ECS supplement in the culture medium induced the highest incidence of diploid oocytes. This incidence of diploid oocytes matured in vitro was higher in oocytes from follicles with a diameter between 11 and 15 mm. Finally, lower culture temperature (37 degrees C) and prolonged incubation time (48 h) also significantly (P<0.01) increased the percentage of diploid oocytes. PMID- 10729050 TI - Preparation of young preactivated oocytes with high enucleation efficiency for bovine nuclear transfer. AB - To improve the enucleation rate in newly matured bovine oocytes, we investigated the position of cytoplasmic chromatin in relation to the polar body and the consequent enucleation efficiency before and after sequential activation with calcium ionophore A23187 and cycloheximide. Oocytes aspirated from the follicles of slaughterhouse-collected ovaries were cultured for 18 to 20 h. With Hoechst staining, only 40.7% of the chromatin material was found adjacent to the first polar body in metaphase II oocytes, while 100% was located adjacent to the second polar body in oocytes after the activation. Enucleation trials after activation showed a higher enucleation rate (91.5%) than that before activation (59.9%). The following experiment determined the effect of using both kinds of cytoplast on the in vitro development of nuclear transfer embryos. Blastomeres of the 32-cell stage in vitro-produced embryos were transferred, fused to the activated cytoplasts and cultured in vitro. No significant difference was detected in fusion, cleavage or development to blastocysts obtained 7 d (174 h) post fusion. In conclusion, this study showed that young in vitro-matured bovine oocytes sequentially activated with calcium ionophore and cycloheximide have cytoplasmic chromatin material adjacent to the second polar body, leading to a high enucleation rate. PMID- 10729052 TI - R.M. Moor, recipient of the 1999 Pioneer Award. PMID- 10729053 TI - Commercialization of academic research (patent, publish and flourish). PMID- 10729054 TI - Patenting reproductive technologies: underlying principals and emerging issues. PMID- 10729055 TI - Commercializing reproductive biotechnology--the approach used by XY, Inc. PMID- 10729056 TI - Production of commercial valuable products using transgenic technology. PMID- 10729057 TI - Taking the best in high-tech to market. PMID- 10729058 TI - Reproduction as a marketplace. PMID- 10729059 TI - Factors that influence follicle recruitment, growth and ovulation during ovarian superstimulation in heifers: opportunities to increase ovulation rate and embryo recovery by delaying the exposure of follicles to LH. AB - The outcome of ovarian follicular superstimulation protocols in heifers is influenced by the number of follicles that are stimulated to grow and the number induced to ovulate. At present, only a proportion of the follicles that are stimulated to grow progress to ovulation. The argument is developed in this review that failure of some of these follicles to ovulate may be due not to an intrinsic deficiency but rather to their relatively small size when the FSH treatment is initiated. Consequently, these follicles do not have the opportunity to undergo full maturation within the time frame of a conventional superstimulation protocol Based on this argument, we propose that delaying the LH surge would allow for completion of maturation by a greater number of follicles, resulting in an increased ovulation rate and in recovery of a greater number of viable embryos. PMID- 10729060 TI - Effects of superovulation on endogenous LH secretion in cattle, and consequences for embryo production. AB - Stimulation of follicular growth during superovulation is achieved by the injection of FSH or compounds with high FSH-bioactivities. However, some LH activity is required for follicle maturation. It is of relevance to evaluate, therefore, the effect of superovulatory treatments on endogenous LH secretion. Luteinizing hormone is secreted in discrete pulses, and the pattern of pulsatile LH secretion during superovulation is reviewed. Four of five published studies have shown that LH pulse frequency is significantly reduced by injection of eCG or FSH preparations. This suppression appears within 8 h of treatment Effects of superovulation on LH pulse amplitude are less consistent. The reasons for the decrease in pulse frequency have been investigated, and although the answer is not definitive, it would seem that increased follicular estradiol, acting perhaps in synergism with progesterone, may play a role. Changes in plasma progesterone concentrations are not related to changes in LH pulse frequency. What is the significance of decreased LH pulse frequency? We attempted to investigate this by inducing LH pulses during superovulation, but the result was a major reduction in ovulation rate. More research is required to determine if modification of endogenous LH secretion can improve superovulatory responses. PMID- 10729061 TI - Remote assessment of ovarian response and follicular status using visual analysis of ultrasound images. AB - Computer assisted evaluation of ultrasonographic image attributes indicative of viability and atresia of ovarian follicles has the potential to become an integral part of ovarian superstimulation protocols. However, in many cases, animal handling facilities, laboratories providing image analysis services and the individual making clinical decisions are geographically separated. The feasibility of remote assessment of follicular status and ovarian response to superstimulation is demonstrated using internet and video conferencing techniques. A cohort of heifers (n = 6) was subjected to ovarian superstimulation. Ultrasound images of the ovarian responses were digitally acquired and transmitted to a distant laboratory for quantitative assessment. Images from follicles which ovulated in response to luteolysis and GnRH treatments were visually and quantitatively different from follicles committed to atresia. Two types of atresia were observed; images of non-ovulatory follicles were characterized as being reflective of the potential to develop into either follicular or luteal cysts. It is probable that the response of individual follicles to the pharmacologic agents used for ovarian superstimulation and ovulation induction may be as important as the total number of follicles recruited. Thus, assessment of the progress and fates of individual follicles may be used to tailor ovarian stimulation to individual donor animals to increase the probability of successful ovarian stimulation and embryo production. The use of the internet for data transfer, image analysis and clinical evaluation places the prospects of providing useful information within the grasp of practitioners who wish to have access to the biological information, but do not wish to invest in the equipment required to make quantitative assessments of visual data. PMID- 10729062 TI - In vitro embryo production in the cow: an effective alternative to the conventional embryo production approach. AB - Development of new technology related to in vitro embryo production has allowed for the commercial use of this method of reproduction. In the present work, we evaluate the efficiency of this technology compared with conventional embryo production based on results obtained with a standard procedure, including the sexing of embryos. The donor animals were mature nonlactating dairy cows (n = 92) kept under a constant environment and feeding program in an ET center. Ultrasound guided transvaginal ovum pick-up following 48 h pre-treatment with FSH has been used for the IVF-IVC protocol. A total of 437 oocyte recovery sessions performed on 92 cows yielded 4145 oocytes, which were used in an IVF-IVC protocol. Using the conventional approach, 156 embryo collections on 49 cows yielded 1652 ova and embryos. All Quality 1 and 2 embryos were sexed by a PCR procedure, and embryos of the desired sex were transferred to synchronized recipients located at the center. The results obtained in the IVF protocol showed that 4 oocyte collections per cow performed within 60 d, yielded 38 oocytes, which resulted in 18.8 viable embryos, of which 7.05 were female. After transfer of the female embryos, an average of 3.8 recipients were pregnant at 60 d. One embryo collection under the conventional approach yielded an average of 1.2 female pregnancies, which was confirmed during the same 60-d time period. These results indicate that IVF procedures can effectively replace conventional embryo production methods when a predetermined number of pregnancies of known sex are needed within a short period of time. PMID- 10729063 TI - The prospect for international regulatory interventions in embryo transfer and reproductive technologies in the next century. AB - Historically, international regulatory interventions in the area of animal reproductive technologies have focused on the need for mitigation against the dissemination of diseases with the movement of genetics and germplasm across international borders. The continued globalization of agriculture under the Sanitary/Phytosanitary (SPS) Agreement of the World Trade Organization (WTO) ensures that disease considerations arising from third and fourth generation reproductive technologies such as in vitro fertilized embryos, transgenics and xenotransplantation will continue to give rise to animal health regulatory measures. Furthermore, in the aftermath of the raising of the public consciousness and the ensuing consumer confidence crisis concerning animal husbandry and livestock production practices following the Bovine Spongiform Encephalopathy outbreak, evolving societal values are expected to expand regulatory considerations to address veterinary public health and ethical concerns. Consequently, it is expected that the role of the International Embryo Transfer Society in fostering meaningful dialogue and profiling of the research necessary to provide for appropriate science based regulation development will increase in importance. PMID- 10729064 TI - State of the art in pig embryo transfer. AB - Embryo transfer in pigs has required surgical procedures in both donors and recipients. Over the last decade, procedures have been developed for transferring embryos by endoscopic or nonsurgical (transcervical) procedures. The feasibility of these procedures for practical application and the latest results of these new approaches are compared here. Factors affecting the current results and obstacles to be overcome in the near future are also discussed. Finally, some relevant embryo collection procedures and applications are briefly summarized. PMID- 10729065 TI - The current status of equine embryo transfer. AB - The use of embryo transfer in the horse has increased steadily over the past two decades. However, several unique biological features as well as technical problems have limited its widespread use in the horse as compared with that in the cattle industry. Factors that affect embryo recovery include the day of recovery, number of ovulations, age of the donor and the quality of sire's semen. Generally, embryo recoveries are performed 7 or 8 d after ovulation unless the embryos are to be frozen, in which case recovery is performed 6 d after ovulation. Most embryos are recovered from single-ovulating mares. Because there is no commercially available hormonal preparation for inducing multiple ovulation in the horse, equine pituitary extract has been used to increase the number of ovulations in treated mares, but FSH of ovine or porcine origin is relatively ineffective in inducing multiple ovulation in the mare. Factors shown to affect pregnancy rates after embryo transfer include method of transfer, synchrony of the donor and recipient, embryo quality, and management of the recipient. One of the major improvements in equine embryo transfer over the last several years is the ability to store embryos at 5 degrees C and thus ship them to a centralized station for transfer into recipient mares. Embryos are collected by practitioners on the farm, cooled to 5 degrees C in a passive cooling unit and shipped to an embryo transfer station without a major decrease in fertility. However, progress in developing techniques for freezing equine embryos has been slow. Currently, only small, Day-6 equine embryos can be frozen with reasonable success. Additional studies are needed to refine the techniques for freezing embryos collected from mares 7 or 8 d after ovulation. Demand for the development of assisted reproductive techniques in the horse has increased dramatically. Collection of equine oocytes by transvaginal, ultrasound-guided puncture and the transfer of these oocytes into recipients is now being used to produce pregnancies from donors that had previously been unable to provide embryos. In vitro fertilization, however, has been essentially unsuccessful in the horse. One alternative to in vitro fertilization that has shown promise is intracytoplasmic sperm injection. However, culture conditions for in vitro-produced embryos appear to be inadequate. The continued demand for assisted reproductive technology will likely result in the further development of techniques that are suitable for use in the horse. PMID- 10729066 TI - State of the art in sheep-goat embryo transfer. AB - Considerable advances have been made in the last 25 yr in sheep and goat embryo production and transfer technology. This presentation covers the procedures used to overcome the variability of ovarian response after treatment with exogeneous gonadotropins, the asynchrony of ovulations, failure of fertilization in females showing a high ovulatory response, and the side-effects of repeated treatments (surgical trauma, gonadotropins and their antibodies). In the ewe, prior antigonadotrophic pretreatment results in a significant gain in ovulation rate due to the elimination of nonresponses and in a two-fold increase in embryo yield. A better comprehension of the relationships between oocyte quality and follicular characteristics after superovulation can be gained using in vitro techniques. This knowledge will subsequently be used for the optimization of embryo production needed for the genetic improvement of livestock and the development of new biotechnologies. PMID- 10729067 TI - Gene expression regulating blastocyst formation. AB - Development of embryos to the blastocyst stage is a critical event in the early lives of all eutherian mammalian species. Blastocyst formation is essential for implantation and is the principal morphological determinant of embryo quality prior to embryo transfer. The physiological events and roles of specific gene families that regulate blastocyst formation are subjects of intense research Recent findings have demonstrated that bovine embryos express multiple members of the Na/K-ATPase ion transporter gene family. Two members of this family have been co-localized to bovine trophectoderm, but each becomes largely confined to opposing cell membrane margins. Bovine blastocysts display a greater sensitivity to ouabain (potent inhibitor of the Na/K-ATPase) than murine blastocysts, and enzyme activity (ouabain sensitive 86Rb+ uptake) undergoes a 9-fold increase from the bovine morula to the blastocyst stage. Disruption of Na/K-ATPase gene expression by antisense oligodeoxynucleotide inhibition abolishes blastocyst formation. These results have implicated the Na/K-ATPase as a key regulator of bovine blastocyst formation and have provided insights necessary for the production of healthy bovine embryos by the application of in vitro maturation, in vitro fertilization and in vitro culture methods. PMID- 10729068 TI - Utilization of gene mapping information in livestock animals. AB - A number of recent advances in genomic research will change and improve livestock production in the near future. Genetic linkage maps have been developed for a number of livestock species including cattle, sheep, and pigs. These maps allow scientists to identify chromosomal regions that influence traits of economic importance. This information will lead to improved genetic selection practices by identifying animals with superior copies of the chromosomal regions that affect the selected trait. This mapping information will also be used to identify the genes controlling the trait. A number of genomic regions or loci have already been reported that affect production, carcass or disease traits, and in a few cases, a specific gene has been identified. Production of transgenic animals with sequence changes in these genes may be beneficial for evaluating the effect of the gene upon the selected trait and more specifically the effect of certain polymorphisms (mutations) within the gene. PMID- 10729069 TI - Genetic modification of animals in the next century. AB - Since the initial demonstration in 1982 of profound phenotypic effects stemming from the expression of a single transgene, genetic engineering has revolutionized fundamental biological and biomedical research. The application of transgenic technology to farm animals has held the promise of being able to improve animal agriculture significantly and has resulted in a new industry, i.e., the successful expression of foreign proteins in the mammary gland for the pharmaceutical industry. Work over the last few years in model species (e.g., the mouse) and new technical developments such as cloning have now set the stage for the initial application of transgenic technology for the improvement of farm animals. Major limitations that remain are the lack understanding of which genes we should transfer in order to alter quantitative production traits usefully and the low efficiency of producting transgenic founders. Furthermore, more research is needed concerning the consequences and potential problems arising from the integration of transgenes into populations with varying genetic backgrounds. Recent advances suggest that within the first decade of the 21 st century the first transgenic animals will become available to the livestock industry, with acceptance depending upon their cost versus their potential economic benefit to the producers. PMID- 10729070 TI - Interspecies pregnancy of Spanish ibex (Capra pyrenaica) fetus in domestic goat (Capra hircus) recipients induces abnormally high plasmatic levels of pregnancy associated glycoprotein. AB - Interspecies embryo transfer could be a valuable tool in preservation programs of endangered species. In this work the results of both interspecific-monospecific (ibex-in-goat) and interspecific-bispecific (mixed-species; ibex+goat-in-goat) embryo transfers in the capra genus are reported. The aim of this work was to compare the PAG plasmatic profiles occurring in these interspecific gestations to those encountered in normal (i.e. intraspecies) pregnancies of Spanish ibex and domestic goat. Spanish Ibex females were superovulated with 9 mg NIADDK-oFSH-17 and embryos were surgically collected 5.5 d after estrus. Two embryos were transferred per recipient. Domestic goat recipients were previously mated either to vasectomized domestic bucks (n=17 females; interspecific-monospecific gestations) or to fertile ones (n=9 females; interspecific-bispecific gestations). Intraspecific pregnancies were obtained by natural mating between males and females of the same species (Spanish ibex: n=6; domestic goat: n=1). Pregnancy rate diagnosed by progesterone was low in both interspecific monospecific (7/17) and interspecific-bispecific (3/9) transfers. None of the monospecific (0/7) and 2 (2/3) of the bispecific established pregnancies developed to term. Ibex-in-ibex PAG profile showed 2 similar peaks of 60 to 70 ng/mL on Days 34 and 153 of pregnancy, while goat-in-goat had the maximum value (60 to 70 ng/mL) at Day 50, decreasing slightly afterwards until parturition. Mixed-species gestations (ibex+goat in goat) showed a first peak of 500 to 1000 ng/mL on Day 70 and a second one (200 to 500 ng/mL) on Day 140 of pregnancy. Four ibex-in-goat gestations that terminated with the expulsion of dead fetuses at Days 110 to 170 had their maximum PAG values (100 to 700 ng/mL) on Days 60 to 90. We conclude that it is possible to achieve pregnancies after transfer of ibex embryos into domestic goats, but this requires a great change of the PAG profiles, which increase significantly. Live ibex kids can be produced when embryos from both species share the uterus. This is the first report of successful interspecific pregnancies in the capra genus. PMID- 10729071 TI - Ovine endometrial cells fail to lyse K-562 target cells: a preliminary investigation. AB - The ability of unfractionated and fractionated endometrial cells to lyse K-562 target cells was investigated within ewes during the late luteal phase of the estrous cycle (Days 12 to 14) and pregnancy (Days 16 and 19). In separate experiments, lymphokine activated killer (LAK) cells and endometrial cells, both designated as effector cells, were co-cultured with chromium-51 (51Cr)-labeled K 562 target cells in varying effector: target cell ratios. At 22 h, lytic activity was assessed by the release of 51Cr into the culture medium. The LAK cells exhibited lytic activity in a ratio-dependent manner, whereas the unfractionated and fractionated endometrial cells failed to lyse the target cells. For ratios combined, the rate of cytotoxicity for unfractionated endometrial cells recovered from ewes in the cyclic and pregnant (Days 16 and 19 combined) groups was 13.9 and 5.4%, respectively. Although the findings are preliminary, they indicate that ovine endometrial cells recovered during the late luteal phase and early pregnancy failed to exhibit natural killer activity upon K-562 target cells. PMID- 10729072 TI - Fertilization and in vitro development of porcine oocytes following intracytoplasmic injection of round spermatid or round spermatid nuclei. AB - The objective of this study was to determine fertilization rates and developmental ability of porcine oocytes following injection of round spermatid and round spermatid nucleus with artificial activation either 2 h before or immediately after injection. Electrical stimulation at 2 h before spermatid injection significantly increased the incidence of normal fertilization compared with that following injection without stimulation or with stimulation immediately after injection. Incidences of formation of 2 pronuclei and of apposition were not different in oocytes following intracytoplasmic spermatid and spermatid nucleus injection. Chromosome analysis revealed that most oocytes were diploid either following round spermatid or round spermatid nucleus injection. There was no diploid set of chromatin in oocytes at 20 h following sham injection. At 6 d following injection blastocoele formation was seen in the oocytes following round spermatid (25%) and round spermatid nucleus injection (27%). However, none of the oocytes developed to the blastocyst stage 6 d following sham injection. The average cell numbers of blastocysts 8 d after injection of spermatid and spermatid nucleus were 99 and 87, respectively. These results suggest that electrical stimulation before injection enhances the incidence of fertilization following round spermatid injection in the pig. Our study also indicates that either the round spermatid or it's nucleus can be used to produce viable embryos by injection into unfertilized porcine oocytes. PMID- 10729074 TI - A technique for the evaluation of sperm penetrating ability and quality of bovine semen processed in an extender made with Brackett-Oliphant medium and egg yolk. AB - Egg yolk-sodium citrate (EYC) semen extender was compared with an extender made of Brackett-Oliphant medium and egg yolk (BOEY). Ejaculates were divided into equal portions, processed and frozen. Semen was thawed and evaluated for quality. Additional semen was thawed, stained with Hoechst 33342 and the spermatozoa capacitated, after which they were co-incubated with zona-free hamster oocytes to determine their penetrating ability. Sperm penetration of non-compressed, unfixed oocytes was evaluated using an optical sectioning technique on a standard research microscope. Sperm penetration was considered successful if a fluorescing sperm head was observed within the living oocyte in a hanging drop of fertilization medium. There were small differences in percentage of secondary abnormalities and percentage of progressive motility immediately after thawing between spermatozoa extended in EYC or BOEY diluent. There were no differences due to by extender composition in percentage of spermatozoa with intact acrosomes or percent of progressively motile after a 3 h incubation at 37 degrees C, nor the percentage of spermatozoa with head abnormalities. While there were significant correlations between all seminal quality characteristics, no quality measurements were correlated to percentage of oocyte penetration. The new penetration evaluation method allowed for examination of the fertilized oocytes using fluorescent microscopy initially and again after re-incubation for further development. PMID- 10729073 TI - Clinical and pathologic features of cloned transgenic calves and fetuses (13 case studies). AB - The neonatal abnormalities, treatments and outcomes in a group of 13 cloned transgenic calves and fetuses that progressed into the third trimester of pregnancy are described. From these 13 fetuses, 8 calves were born live, 4 stillborn fetuses were recovered from 3 cows that died 7 d to 2 mo before term, and 1 aborted fetus was recovered at 8 mo gestation. All fetuses and calves were derived from the same male fetal Holstein fibroblast cell line transfected with a beta-galactosidase marker gene. Six calves were delivered by Cesarian section and two by vaginal delivery between 278 and 288 d of gestation. Birth weights ranged from 44 to 58.6 kg. Five of the 8 live born calves were judged to be normal within 4 h of birth based on clinical signs and blood gas measurements. One of these 5 calves died at 6 wk of age from a suspected dilated cardiomyopathy. Three of the 8 calves were diagnosed with neonatal respiratory distress immediately following birth, one of which died (at 4 d of age) as a result of pulmonary surfactant deficiency coupled with pulmonary hypertension and elevated systemic venous pressures. Similar findings of chronic pulmonary hypertension were also observed in 2 of 5 fetuses. Placental edema was present in both calves that later died and in the 2 fetuses with cardiopulmonary abnormalities. Hydrallantois occurred with or without placental edema in 6 cows, and only 1 calf from this group survived. The 6 cows without hydrallantois or placental edema produced 5 live calves and 1 aborted fetus. The cardiopulmonary abnormalities observed in the calves and fetuses occurred in utero in conjunction with placental abnormalities, and it is likely that the cloning technique and/or in vitro embryo culture conditions contributed to these abnormalities, although the mechanism remains to be determined. PMID- 10729075 TI - Risk factors for stillbirth in Holstein heifers under field conditions in France: a prospective survey. AB - Risk factors for stillbirth, defined as birth of a dead calf or a calf dead within 24 h after parturition, were studied in Holstein heifers. Data came from an overall 4-yr prospective survey conducted in French dairy herds. Only heifers that delivered a single calf were included in the study. The stillbirth incidence was 6.9%. The predictive indicators of stillbirth risk were: gestation length, prepartum body condition and dirtiness scores, biochemical and hematological blood parameters measured during the last 2 mo of gestation, and calving conditions. Multiple logistic regressions were run using herd, calving year, calving season, blood sampling-to-parturition interval, and body scoring-to parturition interval as the fixed effects. The results, expressed as the ratio of the odds of disease occurrence in the exposed and non-exposed subgroups (OR), indicated that dystocia and a body condition score (BCS) higher than 4 before calving were significant risk factors for stillbirth (OR=14.6, P<0.0001 and OR=2.98, P<0.05, respectively). Prepartum circulating neutrophil counts higher than 1950/mm3 (OR=0.50, P<0.05) were associated with a lower risk of stillbirth. A higher occurrence of placental retention, lower fertility and a lower 305-d milk yield were significant consequences of stillbirth. PMID- 10729076 TI - Effects of ultrasound-guided transvaginal follicular aspiration on oocyte recovery and hormonal profiles before and after GnRH treatment. AB - Endocrine changes and recovered oocytes were evaluated during 16 wk of ultrasound guided transvaginal follicular aspiration (TVFA) and prior to and following administration of GnRH at the cessation of aspiration. Nonlactating previously aspirated (PAC, n = 4) and non-aspirated, (AC, n = 4) Holstein cows were subjected to 16 wk of twice-weekly aspiration. Four control cows (OAC) were aspirated 1 time only at the final TVFA session (wk 16). Jugular blood samples were collected from all cows during aspiration, before and after the final TVFA session, and during an 18-d period following cessation of aspiration. Ovarian activity was monitored in all cows after cessation of aspiration for 18 d. The PAC and AC cows averaged 3.4 +/- 1.2 (+/- SE) and 6.8 +/- 1.2 oocytes per session, respectively. Progesterone concentrations during TVFA did not differ between the PAC and AC (0.8 +/- 0.1 and 0.9 +/- 0.1 ng/mL, respectively). Progesterone concentration in OAC was 4.5 +/- 0.2 ng/mL before TVFA, while the PAC and AC averaged 0.5 +/- 0.2 and 0.3 +/- 0.2 ng/mL, respectively, at 16 wk. At Week 16 LH was 1.0 +/- 0.2 ng/mL and it increased to 7.5 +/- 0.1 ng/mL after GnRH treatment. The LH concentration before the final aspiration session was higher at peak amplitude in PAC than in AC groups and peak length was longer in OAC than in AC cows (P < 0.07). Between 18 and 24 h after the last aspiration there were more LH peaks and greater peak frequencies in PAC than in OAC cows (P < 0.07), and the interval between peaks was longer in PAC and AC cows (P < 0.10) than in OAC cows. Mean FSH concentrations were lower (P < 0.01) for OAC than for PAC and AC groups at 20 and 24 h after the last aspiration. Follicle numbers after GnRH varied most among treatment groups for follicles < 9 mm, with the PAC, AC and OAC averaging 5.1 +/- 1.0, 5.1 +/- 1.0, and 3.8 +/- 1.0 follicles/d, respectively. Progesterone concentrations increased to 1.1 +/- 0.3 ng/mL in PAC cows and 2.5 +/- 0.3 and 3.4 +/- 0.3 ng/mL in AC and OAC groups, respectively, during the 18-d period. These results suggest that long-term TVFA affects progesterone, LH and FSH profiles and ovarian dynamics in cows. PMID- 10729077 TI - Progestin implants can rescue demi-embryo pregnancies in goats: a case study. AB - Survival after transfer of demi-embryos (i.e., half-embryos produced by embryo splitting) to recipients usually is lower than survival after transfer of intact embryos. Reduced survival after demi-embryo transfer could be due to loss of viability after splitting, failure of a viable demi-embryo to prevent corpus luteum (CL) regression in the recipient female, or a combination of factors. From a retrospective analysis of pregnancy and embryo survival rates after demi-embryo transfer in sheep and goats, we report the rescue of caprine demi-embryo pregnancies in which CL regression occurred at the end of diestrus despite the presence of a viable conceptus in the uterus with progestin implants. Day 5 or 6 morulae and blastocysts were flushed from superovulated ewes and does and split into demi-embryos of approximately equal halves. Demi-embryos were either transferred fresh to synchronized recipients of the homologous species or frozen in liquid nitrogen. Approximately half of the recipient does and ewes were treated with norgestomet implants on Day 10 of the embryo transfer cycle and again 2 wk later. Serum collected on Day 25 from recipients with implants was assayed for progesterone to determine if a CL of pregnancy had been maintained. Pregnancy was diagnosed by ultrasonography on Day 35 of gestation. Corpus luteum regression occurred despite the presence of a viable conceptus in the uterus in 6 of 55 progestin-treated caprine demi-embryo recipients and in 0 of 66 ovine demi embryo recipients. Five of the caprine pregnancies were maintained to term with norgestomet implants and produced 5 live kids. The sixth fetus, which was carried by a progestin implant-treated 8-mo-old doeling, died at approximately 50 d of gestation. These results suggest that, at least in goats, some demi-embryos may provide inadequate signaling for maternal recognition of pregnancy, and such pregnancies can be rescued with progestin treatment to the doe. PMID- 10729078 TI - Viability assessment of honey bee, Apis mellifera, sperm using dual fluorescent staining. AB - Since the development of instrumental insemination of honey bee (Apis mellifera) queens in the 1930s, there has been interest in the evaluation and in vitro storage of semen. Several fluorescent stains, when used in combination, have been effectively used to assess sperm viability in mammalian and avian species. Our objectives were to test two combinations of living:dead fluorescent stains, SYBR 14 with propidium iodide (PI), or Calcein-AM with PI, and validate the use of these probes with honey bee sperm. SYBR-14 is a nuclear stain producing green fluorescence of the DNA in living sperm, Calcein-AM is a membrane-permeant esterase substrate staining entire sperm green, and PI is a traditional dead cell stain giving a contrasting red color. Both living stains fluoresced bee sperm, but the SYBR-14:PI produced a clearer distinction between the living and dead sperm. A graduated series of known living:dead sperm proportions was used to validate the accuracy of the stains for determining sperm viability in honey bees. PMID- 10729079 TI - Assessment of sows mating efficacy during the low productive period after early weaning: a field study. AB - Data on sows bred after weaning (n = 9,540) and their lactation feed intake records (average lactation length <20 d) were obtained from 16 commercial farms. Weaning-to-first-mating intervals (WMI) at 6 to 12 d and 0 to 6 d after weaning were defined as the low and high productive periods, respectively. Of the 9,192 sows mated, 80.5 and 19.5% were mated at 0 to 6 d and 7 to 12 d, respectively. In logistic regression analysis, lower parity, shorter lactation length, lower average daily feed intake (ADFI) during lactation, and a greater number of weaned pigs were associated with mating at 7 to 12 d after weaning (P < or = 0.045). Exponentiating the coefficients in logistic regression analysis, the odds ratios were 0.79 for parity, 0.84 for ADFI during lactation, 0.85 for lactation length, and 1.05 for weaned pigs, respectively. A sow with a 14-d lactation length is 2.3 (1/0.85(5)) times as likely to mate within a 6- to 12-d WMI as a sow with a 19-d lactation length. Thus, the early weaned sows are more likely to mate during the low productive period than the later weaned sows. The odds for party 0.79 imply that Parity 1 sows were 1.6 (1/0.79(2)) times as likely to mate within a WMI 6 to 12 d as Parity 3 sows. For each 1-kg increase in ADFI, a mating occurrence during the low productive period decreased by 0.84 times. Sows are mated during the low productive period because this period is a part of the distribution of WMI in a herd. However, our research suggests that increasing feed intake during lactation and maintaining parity proportion appropriate to the herd can decrease the proportion of sows mated during the low productivity period. PMID- 10729080 TI - Bovine viral diarrhea virus: its effects on estradiol, progesterone and prostaglandin secretion in the cow. AB - Bovine viral diarrhea virus (BVDV) is a major cattle pathogen responsible for a spectrum of symptoms, including reproductive failure. This study was designed to establish the effects of BVDV infection on estradiol, progesterone and PGF2alpha secretion in the cow. Seven BVDV-free cows were challenged with non cytopathogenic BVDV (strain Pe 515: 5x10(6) tissue culture infected dose50) so that peak viremia occurred during the initial phase of luteal development in a synchronized estrous cycle. Ovulation was also synchronized in 7 sham-infected animals. Within 2 wk of inoculation, viremia, leukopenia and serum neutralizing antibodies were recorded in all of the BVDV-infected cows but not the sham infected animals. Between Day 4 and Day 9 post estrus the BVDV-infected cows had significantly (P<0.01) lower plasma estradiol levels than the sham-infected animals. However, the BVDV infection did not alter rectal temperatures, plasma progesterone concentrations or PGF2alpha secretion 17, 18 and 19 d post estrus. These data highlight a potential causal link between BVDV viremia, endocrine dysfunction and poor fertility in the cow. PMID- 10729081 TI - Reproductive and safety assessment of vaccination with Gavac against the cattle tick (Boophilus microplus). AB - Recent developments in cattle tick control have incorporated the use of recombinant Bm86 vaccines against this ectoparasite. The vaccine developed by our group (Gavac) contains an antigen expressed in Pichia pastoris, and has been successfully employed for the control of tick infestations and transmission of tick-borne diseases. Here, we examined the safety and effect of the Gavac vaccine on reproductive parameters in cattle. Toxicity tests in mice and guinea pigs demonstrated the safety of Gavac. To study the adverse effects of vaccination on reproduction, a field trial involving 9,500 animals in Cuba was conducted. The cattle at 3 farms were vaccinated while those on a fourth farm were left unvaccinated and served as the control. Following vaccination, the control of tick infestation and the transmission of babesiosis were used to demonstrate the efficacy of the vaccine. No adverse effects were observed in any of the reproductive parameters studied when comparing the data before and after vaccination with Gavac and between the vaccinated farms and the control farm. These results demonstrate that under the conditions of our study vaccination with Gavac is safe for use on cattle. PMID- 10729082 TI - Gynecological examination and pregnancy diagnosis in small ruminants using bimanual palpation technique: a review. AB - A palpation technique using both hands was developed by the author and used for examination of internal reproductive organs, pregnancy diagnosis, and assessment of approximate stages of gestation in small ruminants. The study was conducted in 4 stages on 87 female goats and 30 slaughterhouse source ewes First, 20 doelings and 15 does with no recent history of insemination were examined by two hand method for palpability and structural integrity of internal reproductive organs. Next 52 goats (28 doelings and 24 does) were examined at 28 to 30 d post insemination for pregnancy. Among those diagnosed pregnant, 20 goats were examined at 15 d intervals to assess the palpable changes of pregnancy. Preslaughter palpation followed by post-slaughter verification of the findings were performed in 30 ewes. The two hand method was effective for palpating the reproductive tract in the nonpregnant goats irrespective of parity status. Gross palpation of the ovaries was possible in 83% of the goats. Of the 52 goats examined for pregnancy at 28 to 30 d, 29 (56%) had clear distension of uterus while 23 (44%) did not. The results were confirmed by kidding date or returning to service. Changes of the reproductive tract were monitored in 20 goats at biweekly intervals and the findings categorized by the stage of gestation. Post slaughter verification of preslaughter findings based on two handed palpation indicates that this technique can be used for early pregnancy diagnosis and assessment of the stage of gestation in goats and ewes. PMID- 10729083 TI - Effect of protein supplementation and presence of an antioxidant on the development of bovine zygotes in synthetic oviduct fluid medium under high or low oxygen tension. AB - The aim of this study was to investigate the effect of protein supplementation of culture medium and the presence of a putative antioxidant on bovine zygote development under 5% (low) and 20% (high) O2. In Experiment 1, presumptive zygotes (n=992) were cultured in synthetic oviduct fluid (SOF) alone or supplemented with 3 mg/mL PVP, 3 mg/mL BSA (SOFB), and/or 10% FCS (SOFBF) in 5% CO2, 5% O2, 90% N2. In Experiment 2, zygotes (n=1916) were cultured in SOF, SOFB or SOFBF with or without taurine under high and low O2. In Experiment 1, presence of BSA or BSA plus FCS significantly increased the speed of development compared to SOF or SOF+PVP. Blastocyst quality was also improved, as evidenced by increased hatching rate and cell numbers. In Experiments 2, taurine had no effect on development irrespective of oxygen concentration or protein supplementation. In conclusion, the presence of protein in the culture medium and culture under reduced O2 significantly improved embryo development. Taurine had no effect on development. PMID- 10729084 TI - Transuterine embryo migration in recipient cattle. AB - Transuterine migration of bovine embryos following fertilization in vivo is apparently rare, but little is known about migration following embryo transfer. We studied heifers receiving either 1 or 2 in vitro produced embryos to determine 1) the incidence of transuterine migration, 2) the timing of migration and 3) the random or systematic occurrence of the event. In 4 experiments, 436 heifers received embryos and 218 of these were pregnant at necroscopy on either Day 14, Day 18, Day 26 or Day 60 of pregnancy. Overall, 43/218 (20%) of the heifers had embryos that had migrated. The frequency of migration was higher in twin (30/68) than in single (13/150) embryo transfers of pregnant recipients (44 vs 9%; P<0.001), and in contralateral (9/15) than in ipsilateral (33/170) transfers (60 vs 19%; P<0.001). Among the heifers that received embryos by ipsilateral transfer, the migration rate was similar to that in heifers pregnant with a singleton after the transfer of either 1 (2/48) or 2 (4/60) embryos (4 vs 7%, NS). The migration rate was highest at Day 26 (12/37) in heifers receiving twin embryos by ipsilateral transfer but was similar at all other stages of pregnancy (15/111, 32 vs 14%; P<0.01). Migration was first observed by Day 14, and it appears that either further migration occurred over the next 12 d or that migration was associated with a higher survival rate from Day 14 to Day 26. The low migration rate evident at Day 60 suggests that migration by Day 26 was associated with increased embryo or fetal death by Day 60. The data suggest that embryo migration is probably independent for each of a pair of surviving embryos. We conclude that in cattle embryo migration is embryo-dependent, but this capability is dormant unless more than 1 embryo is present in a uterine horn or the embryos are transferred to the contralateral uterine horn. The relationship between migration and embryo survival remains unclear. PMID- 10729085 TI - Pregnancy outcomes in two commercial dairy herds following hormonal scheduling programs. AB - There are a number of options for hormonal management of post partum dairy cows; however, only a few studies have made direct comparisons of these programs in commercial herd settings. We compared reproductive management programs of 2 commercial dairy herds to evaluate the efficacy of prostaglandin-based treatment regimens on reproductive outcomes. Cows in Herd A were left untreated and served as the negative controls. Cows in Herd B were given PGF2alpha every 14 d until first insemination beginning 45 d post partum and served as the positive controls. Treatment 1 (Ovsynch), initiated randomly during the estrous cycle, consisted of sequential injections of GnRH, PGF2alpha, GnRH again and insemination 16 to 20 h later. Treatment 2 consisted of an Ovsynch protocol, as described above, which was begun 7 d post estrus (Ovsynch + 7). In Herd A, the number of days from parturition to conception (days open) for controls, for Ovsynch and for Ovsynch + 7 were 126, 112 and 102, respectively. In Herd B, respective days open were 102, 100 and 93 for controls, Ovsynch and Ovsynch + 7. Hormonal intervention reduced the number of days open in both herds. PMID- 10729086 TI - Determination of corpora lutea numbers in Booroola--Texel ewes using transrectal ultrasound. AB - Ovulation rates in Booroola--Texel ewes are currently determined by laparoscopic examination of the ovaries during the luteal phase allowing for carrier status of the ewe to be diagnosed. However, this is an invasive procedure and the aim of this work was to determine whether or not transrectal ultrasound would be an acceptable and accurate alternative to laparoscopy for this purpose in ewes. Transrectal ultrasound was performed using a 5.0 MHz transducer in 18 ewes restrained in the standing position. They were then euthanized and laparoscopy was performed, following which the reproductive tracts were recovered. An additional 22 ewes were subjected to scanning in dorsal recumbency, using a 7.5 MHz transducer, at various stages of the luteal phase before being euthanized and the reproductive tracts recovered. The ovarian structures identified using each method were then compared. The results demonstrated that laparoscopy is an accurate method of determining ovulation rates in ewes. Transrectal ultrasound examination appeared to be an acceptable and non-invasive procedure. The use of a 7.5 MHz transducer with the ewes in dorsal recumbency produced the best results and clearest images of the ovaries, which allowed for the determination of the number of corpora lutea (CL) and the presence of other structures. Accuracy improved with experience in the technique, the stage of the luteal phase at which the examination was performed, the number of CL present in the ovary and the age of the ewe. Further work is needed to confirm this finding due to the small numbers of animals in the present study. This ultrasound technique, however, has the potential to provide an alternative to laparoscopy for the determination of numbers of CL in ewes. PMID- 10729087 TI - Influence of the timing of blastomere isolation after the removal of nocodazole in bovine nuclear transfer. AB - This study was conducted to investigate the influence of the timing of blastomere isolation after the removal of nocodazole on the subsequent division of blastomeres and developmental ability of reconstituted bovine embryos. The division rate of isolated blastomeres was examined at 3, 5 and 24 h of culture after nocodazole removal. Furthermore, isolated blastomeres and those of whole embryos were used as donors in nuclear transfer to determine the development of reconstituted embryos. The division rate of isolated blastomeres at 3 h was significantly lower than the presumptive division rate of blastomeres from whole embryos (P<0.05). When these blastomeres were used as donor nuclei, the dividing blastomeres yielded a significantly higher development rate than blastomeres from whole embryos (P<0.05). These results confirm that the timing of blastomere isolation influences the subsequent division of blastomeres and the developmental ability of the reconstituted embryos. PMID- 10729088 TI - Chromosomal diagnosis in each individual blastomere of 5- to 10-cell bovine embryos derived from in vitro fertilization. AB - Chromosomal normality and sex were diagnosed in each blastomere of bovine embryos derived from in vitro fertilization (IVF). Bovine embryos developing to the 5- to 10-cell stage were separated into individual blastomeres with 0.5% protease. After treatment with 100 ng/mL vinblastine sulfate for 8 to 10 h, they were prepared for chromosome samples. In total, 33 bovine embryos and 185 blastomeres were examined. Chromosomal normality was analyzed in 43.8% (81/185) of the blastomeres and 60.6% (20/33) of the embryos; while chromosomal anomalies were found in 16 (80%, 16/20) of the embryos, 5 haploid embryos and 11 mosaic (n/2n) embryos. Mosaicism characteristic of the opposite sex in X-and Y-chromosomes was found in 2 haploid embryos, and that of a Y-chromosome and of XX chromosomes in 1 n/2n embryo. Various sex-chromosome compositions were also observed in the other 10 chromosomal mosaic n/2n embryos. PMID- 10729089 TI - Birth of a foal after oocyte transfer to a nonovulating, hormone-treated recipient mare. AB - A nonovulating, hormone-treated mare was used successfully as an oocyte recipient. The mare's ovarian activity was suppressed using progesterone and estrogen treatment. This treatment was stopped, then estrogen was administered for 3 d prior to the transfer. An oocyte was recovered from the follicle of a donor mare and was transferred via flank laparotomy into the recipient's oviduct. The recipient mare was inseminated 7 h before transfer. The recipient was treated with intramuscular progesterone from the day after transfer until 47 d after transfer, and then with oral altrenogest until 150 d gestation. A normal colt was born at 321 d gestation, and was shown by DNA analysis to be the progeny of the donor mare. This is the first report of fertilization and embryo development to term after transfer of oocytes to a nonovulating mare, and, to our knowledge, the first of its kind in any domestic species. PMID- 10729090 TI - Effect of type and quantity of concentrates on superovulation and embryo yield in beef heifers. AB - Embryo yield and quality can be decreased following superovulation of cattle on high levels of concentrates. Concentrate type can alter rumen fermentation patterns and so affect energy availability and thus embryo quality. The objectives of this experiment were to examine the effect of 2 levels and 2 types of concentrate on superovulatory response and embryo quality in beef heifers. Beef heifers received grass silage as a basal diet and barley at 3 kg (n = 20) or ad-libitum (n = 19), or citrus/beet pulp at 3 kg (n = 18) or ad-libitum (n = 19) as the source of concentrate supplement. Silage was available ad-libitum for heifers offered 3 kg but was restricted to 1 kg DM/day for heifers on ad-libitum concentrate intake. Both concentrates contained 14% crude protein. After 100 d, heifers were treated with an intravaginal progesterone releasing device (CIDR) for 7 d, and superovulation was initiated 60 h before CIDR withdrawal. Heifers received pFSH (a total of 265 mg NIH-FSH-P1 equivalent) administered over 8 injections at 12 h intervals with the last 2 injections administered at 12 and 24 h after CIDR withdrawal; they were inseminated at 56 and 72 h after CIDR withdrawal without reference to estrus. Heifers were slaughtered 6, 7 or 8 d after the first insemination, and embryos were flushed from the uterus with PBS and were graded morphologically. Statistical analyses were performed using analysis of variance. There was no interaction between level and type of diet, and thus data for the main effects are presented separately. Final live weights at the end of the experiment and carcass weights for heifers offered ad-libitum concentrate (643.8+/-6.9 kg; 354+/-8 kg, respectively) were higher (P<0.01) than those of heifers offered 3 kg concentrate (613.1+/-7.5 kg; 338.4+/-3.2 kg, respectively) while live weights and carcass weights of heifers offered barley (625.6+/-8.1 and 345.4+/-4.1 kg, respectively) or citrus/beet pulp concentrate (631.4+/-7.0 and 348.0+/-3.1 kg, respectively) were not different (P>0.05). Superovulatory responses (number of corpora lutea at slaughter) was greater (P<0.06) when heifers were offered 3 kg (15.5+/-1.6) than when offered ad-libitum concentrates (12.3+/-1.4). However, the superovulatory response for both citrus/beet pulp (14.4+/-1.5) and barley (13.4+/-1.5) diets were not different (P>0.05). Heifers offered 3 kg concentrates produced greater (P<0.05) numbers of transferable embryos (4.8+/-0.7) compared with heifers fed ad-libitum concentrates (2.8+/-0.4). Heifers offered citrus/beet pulp produced greater (P<0.05) numbers of transferable embryos (4.8+/-0.7) than heifers offered barley (2.9+/-0.5). These data indicate that high concentrate intake has a negative effect on embryo yield and quality and that a barley compared with citrus/beet pulp based concentrate has a negative effect on embryo quality following superovulation in beef heifers. PMID- 10729091 TI - Use of AI technician scores for body condition, uterine tone and uterine discharge in a model with disease and milk production parameters to predict pregnancy risk at first AI in Holstein dairy cows. AB - Technicians recorded body condition score (BCS) and several parameters related to estrus and/or metritis for 1694 first insemination cows on 23 farms. Additional variables for modeling the adjusted odds ratios (OR) for pregnancy were data on disease prior to or within 21 days of AI and test day milk yields. Significant predictors for pregnancy were farm, year and season, BCS, uterine tone, contaminated insemination gun after AI, fat-protein corrected kilograms milk (FPCM), days in milk (DIM), and diseases. Vaginal mucus, ease of cervical passage, and lameness were not significant predictors for pregnancy. Pregnancy risk at AI increased with increasing DIM, reaching a near optimum after 82 days. Lack of uterine tone was associated with a lowered pregnancy risk (OR = 0.69) as was contaminated insemination gun (OR = 0.67), first-parity lactation, FPCM >33 kg (OR = 0.71), BCS 2.5 at AI (OR = 0.65), clinical mastitis (OR = 0.53), cystic ovarian disease (OR = 0.53), and metritis (OR = 0.74). It was concluded that data on BCS and uterine findings, as collected by AI technicians, are significant predictors of AI outcome. Dairy producers and veterinarians should jointly examine the potential costs and value of such AI technician-based data to improve herd fertility. PMID- 10729092 TI - Plasma GH, IGF-I, and conception rate in cattle treated with low doses of recombinant bovine GH. AB - Blood and uterine concentrations of GH and insulin-like growth factor (IGF)-I are correlated with improved fertility in cattle. We tested incremental doses of a 14 d sustained release recombinant bovine GH (rbGH) to increase blood GH and IGF-I (Experiments 1 and 2). Conception rate after administration of an optimized rbGH dose was also tested (Experiment 3). In Experiment 1, lactating Holstein cows (n = 18) were randomly assigned to receive 0 (n = 5), 100 (n = 5), 200 (n = 5), or 500 (n = 3) mg sc rbGH. Increasing the doses of rbGH was associated with increased serum concentrations of GH and IGF-I. The 100- and 200-mg doses caused an IGF-I release that was below and above, respectively, the perceived optimum response. Therefore, Experiment 2 was designed to test a rbGH dose (167 mg), which was intermediate to the doses tested in Experiment 1. Lactating and nonlactating postpartum beef cows were treated with 0 (n = 9) or 167 (n = 9) mg rbGH at insemination. Plasma concentrations of GH and IGF-I were greater in rbGH treated cows than in controls. Lactating cows had initial IGF-I concentrations that were lower than nonlactating cows. The 167-mg dose of rbGH increased plasma IGF-I concentrations in lactating cows to the levels of those of nonlactating cows. In Experiment 3, cows and heifers were administered either 0 or 167 mg rbGH at insemination. The conception rate for rbGH-treated and control cows was 54.4 and 49.5% (n = 617), and 46.0 and 46.3% for heifers (n = 1123), respectively. Herd (P<0.01) and parity (P<0.01) affected conception rate, but conception rates for rbGH and control cattle were similar. In summary, low doses of rbGH increased blood GH and restored blood IGF-I concentrations in lactating cows to those of nonlactating cows, but the conception rate in cows and heifers was not affected by administration of 14-d sustained-release rbGH at insemination. PMID- 10729093 TI - In vivo development of microinjected embryos from superovulated prepuberal slaughter lambs. AB - This study was performed to investigate the developmental potential of microinjected embryos recovered from superovulated prepuberal lambs. Fifty-nine mixed-bred lambs (about 3 mo old) were superovulated either with 18 mg FSH-P with (Group FSH/+S) or without (Group FSH/-S) progestagen treatment, or with 10 mL Ovagene following progestagen treatment (Group OVA/+S). All animals received hCG to induce ovulation. Ovulation rates and the number of ova recovered per animal for the different groups were 8.7 and 4.7 (55%, FSH/+S); 7.3 and 3.2 (42%, FSH/ S); and 6.4 and 4.0 (65%, OVA/+S), respectively. No significant differences were seen in the ovulation and the recovery rates, but animals without progestagen treatment showed a significantly lower fertilization rate (44%) when compared with progestagen-treated groups (87%; P<0.001). Foreign DNA was microinjected into the pronuclei of fertilized ova (n = 155), which were transferred (n = 98) into synchronized recipient ewes (n = 21). Two animals were detected pregnant and both gave birth to a single lamb. Results of superovulation and embryo recovery from prepuberal lambs were promising, but the low rate of development to term indicates that ova recovered from prepuberal lambs have reduced developmental competence in vivo. Although 2 lambs were born, it seems that this is not a successful method for use in future gene transfer programs. PMID- 10729094 TI - Development of in vitro matured bovine oocytes after cryopreservation with different cryoprotectants. AB - In vitro matured bovine oocytes at the metaphase-II stage were slowly frozen in phosphate buffered saline (PBS) containing 1.0 M glycerol, 1.0 M dimethylsulfoxide (DMSO) or 1.0 M propylene glycol (PROH). When thawed rapidly, more (P<0.05) oocytes were morphologically normal after being frozen with DMSO (86%) or PROH (83%) than with glycerol (62%). When inseminated in vitro with frozen-thawed bull spermatozoa, higher (P<0.05) penetration rates were observed in DMSO (79%) or PROH (76%) than in glycerol (48%). The percentages of oocytes developing to the 2-cell stage at 48 h postinsemination were also significantly (P<0.05) higher in DMSO (51%) and PROH (54%) than in glycerol (33%). However, a significant increase in the proportions of 8-cell embryos (46 vs 21 to 26%; P<0.05) at 72 h postinsemination and morulae (14 vs. 6 to 8%; P<0.05) was derived from oocytes frozen with PROH than with DMSO or glycerol. In conclusion, the type of cryoprotectant used is one of the critical factors affecting developmental competence of bovine oocytes frozen at the metaphase-II stage. For this stage of oocytes, PROH was the most effective, yielding a large number of 8-cell embryos and morulae than either glycerol or DMSO in a slow freezing method combined with a 3-step thawing protocol. PMID- 10729095 TI - Effect of adding reduced glutathione during insemination on the development of porcine embryos in vitro. AB - This study evaluated the effect of adding reduced glutathione (GSH) during sperm washing and insemination on the subsequent fertilization dynamics and development of IVM porcine oocytes. Follicular oocytes were matured in vitro in NCSU 23 medium with porcine follicular fluid, cysteine and hormone supplements for 22 h. They were then matured in the same medium but without hormones for another 22 h. Matured oocytes were stripped of cumulus cells and co-incubated with frozen thawed spermatozoa for 5 h. Putative embryos were cultured in NCSU 23 with BSA for either 7 h to examine fertilization parameters or 6 d to evaluate cleavage (2 d) and blastocyst rates. In Experiment 1, GSH was added to the insemination medium at 0, 0.125, 0.25 or 0.5 mM. The presence of GSH during insemination did not affect (P>0.05) rates of penetration, polyspermy, male pronuclear formation or cleavage, but did increase (P<0.05) blastocyst formation rates when added at concentrations of 0.125 (36%) and 0.25 mM (34%) compared with that of the control (0 mM; 19%). However, the numbers of inner cell mass and trophectoderm cells of blastocysts were unaffected by GSH treatment (P>0.05). The presence of GSH during insemination was found not to significantly increase intracellular glutathione concentrations of oocytes (P>0.05). In Experiment 2, addition of GSH (0.25 mM) during sperm washing did not affect cleavage or blastocyst formation rates or cell numbers (P>0.05). In conclusion, the presence of GSH during insemination improves the developmental competence of IVM pig oocytes in a dose-dependent manner. PMID- 10729096 TI - Determination of pregnancy-associated glycoprotein concentrations in goats (Capra hircus) with unsuccessful pregnancies: a retrospective study. AB - Presented here are the profiles of pregnancy-associated glycoprotein (PAG) concentrations in blood collected weekly from goats experiencing maintained and unsuccessful pregnancies. The analysis of these profiles clearly indicated 4 different situations: the pseudopregnancy syndrome, abortion between Days 89 and 137, parturition of 1 dead and 1 live fetus, and expulsion of macerated or mummified fetuses after full term. A marked reduction in PAG concentration at any time during pregnancy was followed by an event such as abortion or the expulsion of a dead fetus at term or later. PMID- 10729097 TI - Stag exposure advances the LH surge and behavioral estrus in Eld's deer hinds after CIDR device synchronization of estrus. AB - The impact of male presence or absence on the timing of the preovulatory LH surge and estrus was studied in 3 experimental groups (n = 6/group) of Eld's deer hinds pretreated with intravaginal progesterone-releasing devices (CIDR-type G) as follows: Group 1 = indirect male contact barn; Group 2 = direct male contact barn; and Group 3 = male isolation barn. For all hinds, the duration of the preovulatory LH surge averaged 2.5+/-0.5 h, whereas mean peak preovulatory and basal LH concentrations were 2.9+/-0.2 ng mL(-1) and 0.27+/-0.03 ng mL(-1), respectively. Nine of 12 male-exposed hinds exhibited a preovulatory LH surge within 24 to 32 h postCIDR device withdrawal, whereas 0 of 6 male-isolated hinds exhibited a preovulatory LH surge during the same time period. Onset of behavioral estrus (45.2+/-2.3, 52.7+/-5.7 and 66.3+/-1.8 h, respectively) was significantly advanced (P<0.05) after CIDR device withdrawal in male exposed hinds (Groups 1 and 2) compared with male isolated hinds (Group 3). These data suggest that stag exposure is important for modulating the timing of the preovulatory LH surge and behavioral estrus after synchronization of estrus with exogenous progestagens. PMID- 10729098 TI - Reproductive parameters of miniature stallions. AB - Breeding soundness evaluation (BSE) of stallions is a routine component of stud farm practice. Guidelines for assessing satisfactory breeding potential have been developed using data derived from stallions of full-size breeds. In view of the increasing popularity of miniature stallions, knowledge of normal semen parameters of these stallions is important. Therefore, testicular measurements and semen parameters from 216 sexually rested miniature stallions were obtained. Semen was collected twice, 1.5 to 3 h apart, using an artificial vagina. Values were averaged over the 2 collections because of the sexual inexperience of the stallions. The smaller stallions (Group A, 72 to 86 cm; Group B, 87 to 96 cm) had smaller testicles (P<0.05), and Group A stallions had the lowest ejaculate volume (P<0.05) compared with Group C (97 to 104 cm) stallions. Thus, although there was no difference in the concentration of spermatozoa per milliliter between groups of stallions, Group A stallions had fewer total spermatozoa in their ejaculate than Group C stallions (4.31+/-0.47x10(9) vs. 5.41+/-0.30x10(9), P<0.05). Moreover, miniature stallions had smaller testicles and fewer total spermatozoa in their ejaculate than is commonly accepted as normal in full-size stallions. Average total scrotal width of miniature stallions was found to be 7.13, 7.38 and 7.95 cm for Groups A, B and C, respectively. The average total number of spermatozoa in the ejaculates of miniature stallions in this study was 4.94+/ 0.22x10(9) cells, with 1.75+/-0.09x10(9) total normal, motile spermatozoa. When only stallions <96.5 cm in height were considered (conforming to requirements of the American Miniature Horse Association Registry), the average total number of spermatozoa in the ejaculates was 4.59+/-0.30x10(9) cells, with 1.70+/-0.11x 10(9) total normal, motile spermatozoa. Based on these findings, different criteria should be used to evaluate the potential breeding soundness of miniature stallions than are commonly applied to full-size stallions. PMID- 10729099 TI - The effect of subluteal levels of exogenous progesterone on follicular dynamics and endocrine patterns during early luteal phase of the ewe. AB - Nineteen Corriedale ewes were treated with an im dose of a PGF2alpha during the luteal phase to synchronize estrus. After ovulation had been detected by using ultrasonography (Day 0); the ewes were randomly assigned to 2 different groups. In 11 ewes a CIDR, which had previously been used for 10 d, was inserted on the fourth day after ovulation. The ewes then received a dose of PGF2alpha on Day 5 to induce luteolysis. The CIDR remained in place until the end of the experiment (Day 9). Control ewes (n = 8) received no treatment. Blood samples were taken daily for estradiol, progesterone and FSH determinations. In the untreated ewes, 2 follicular waves were detected in all of the animals throughout the monitoring period, with a mean wave interval of 4.5 d. The total number of follicles which were > or =2 mm decreased from Day 0 to Day 4 (8.8+/-1.0 to 5.3+/-0.6; P< or =0.05) and then increased at Day 7 (7.5+/-0.9; P< or =0.05). The growth profiles of both the largest and the second largest follicles of Wave 1 showed significant divergence, while no divergence was observed in Wave 2. Serum estradiol concentrations decreased significantly from the day before to the day of ovulation and then increased again during the growing phase of the largest follicle of Wave 1. Concentrations of FSH were high on the day of emergence of both waves, but while a significant decline was observed after emergence in Wave 1, the levels remained high in Wave 2. In 8 of the 11 treated ewes, the largest follicle of Wave 1 was still present on the ninth day after ovulation (persistent follicle). In the other 3 ewes, the largest follicle of Wave 1 was already regressing on the day that the treatment was administered, and the largest follicle that was present on Day 9 originated from Wave 2 (nonpersistent follicle). In persistent follicle ewes, the largest follicle of Wave 1 prolonged its lifespan significantly, attaining the maximum diameter (Day 8.1+/-0.8) later than in untreated (Day 3.0+/-0.4) and nonpersisted follicle ewes (Day 2.0+/-0.6). The total number of follicles decreased in persistent follicle ewes between Day 0 and Day 4 (7.9+/-1.5 to 4.5+/-0.5, respectively; P< or =0.05) and remained low until the end of the experiment. Progesterone concentrations (nmol/L) between Days 6 and 9 were significantly different between untreated and persistent follicle ewes (12.8+/-1.0 vs. 9.4+/-1.0, P< or =0.02). The present study confirms that the largest follicle of Wave 1 is dominant in the ewe and that subluteal progesterone concentrations can prolong its lifespan and extend this dominance. PMID- 10729100 TI - Effects of prolactin on intracellular stored calcium in the course of bovine oocyte maturation in vitro. AB - At present there are divergent opinions as to the role of prolactin (PRL) in the mechanisms of meiotic regulation in mammals. We investigated the effects of bovine PRL (bPRL) on bovine oocyte maturation in different culture systems and varying levels of intracellular stored calcium ([Ca2+]is) in the oocytes. Cumulus oocyte complexes (COC) were incubated in TCM 199 containing either 10% fetal calf serum (FCS) in the absence (System 1) or presence (System 2) of FSH and estradiol, or 6 mg/mL bovine serum albumin (BSA) in the presence of FSH and estradiol (System 3). Levels of [Ca2+]is in oocytes were determined by using the fluorophore chlortetracycline. The addition of 50 ng/mL bPRL to different culture media increased the percentage of oocytes at telophase I and metaphase II stages (Systems 1 and 2) and/or decreased the percentage of oocytes with degenerated chromosomes (Systems 1 and 3). Compared with the control, lower levels of [Ca2+]is were observed in oocytes cultured for 2.5 h in those systems in which bPRL decreased the rate of oocytes with degenerated chromosomes (1.27+/-0.11 vs. 1.67+/-0.09 arbitrary units (AU) in System 1, P<0.001 and 1.27+/-0.12 vs. 1.52+/ 0.04 AU in System 3, P<0.001). These findings show that the effects of bPRL on bovine oocyte maturation depend on the composition of the culture system and that the decline in the rate of oocytes with degenerated chromosomes in response to bPRL may be the result of the decrease in [Ca2+ ]is levels at early stages of oocyte maturation. PMID- 10729101 TI - In vitro production of pig embryos: comparisons of culture media and boars. AB - The utilization of in vitro produced pig embryos for commercial production or research is dependent upon the development of improved methodology. Our objective was to establish a consistent in vitro embryo production (IVP) system and subsequently utilize the procedures to evaluate culture system components and boar effects. To summarize the IVP system, 403 inseminated oocytes from a total of 2243 were analyzed across 17 replicates for maturation and fertilization efficiency, while 1838 zygotes were cultured in 26 replicates for developmental data. Penetration, cleavage and blastocyst development rates were determined at 18, 44 and either 144 or 168 h post insemination, respectively. Monospermic penetration averaged 31.8+/-7.3% while polyspermy was 30.8+/-17.2%. Cleavage rate was 44.9+/-16.1%, with 21.8+/-7.5% of fertilized oocytes and 51.9+/-15.9% of cleaved embryos developing to blastocysts. For culture medium comparison, fertilized oocytes were cultured in either BECM-6, BECM-7, NCSU-23 or NCSU-23aa and supplemented on Day 5 post insemination (pi) with 10% FCS. These treatments resulted in 4.0, 4.9, 19.8 and 13.6% (+/-3.2%) blastocysts by Day 7 pi, with an average cell number of 44.4+/-9.0, 65.1+/-8.2, 61.3+/-4.5 and 64.4+/-4.8, respectively. These IVP procedures consistently produced zygotes from semen of several different boars, capable of forming blastocysts in vitro. Comparison of developmental rates among the boars indicated that this system is variable among boars but not strictly boar-dependent. Culture media comparisons suggest that NCSU-23 yielded a higher percentage of blastocysts than the other media in this IVP system. PMID- 10729102 TI - Sry-negative XX true hermaphrodite in a Basset hound. AB - A true hermaphrodite was diagnosed in a 7-mo.-old Basset hound. The diagnosis was based on the clinical signs, the histology of the gonads and the karyogram. Additionally, the dog was tested for the Y-linked gene Sry, which was negative. The Basset hound presented here is compared to other XX sex reversed animals described in the literature. In man, XX sex reversal is a heterogenous condition. The pathogenesis in Sry-negative individuals is not understood. Thus Sry-negative animals could serve as an animal model of the human disease. PMID- 10729103 TI - Serum cholesterol and triglycerides in postpartum beef cows and their relationship to the resumption of ovulation. AB - The variations in lipid metabolism according to the physiological stage and their relationship to the resumption of postpartum ovarian cyclicity were assessed in Limousine beef cows fed a grass diet over 3 yr. Weekly blood samples were collected from 59 cows beginning 10 wk before to 20 wk after calving to evaluate serum cholesterol and triglyceride concentrations and electrophoretic lipoprotein fractions. After parturition, progesterone concentrations were also measured at weekly intervals to determine time of resumption of ovulation. Cows were categorized by resumption of postpartum ovarian cyclicity into 3 groups: early (4 to 6 wk post partum, n = 36); mid (7 to 10 wk post partum, n = 46) and late (after 11 wk post partum, n = 38). Higher serum triglyceride values (P<0.05) were observed during the last 10 wk of pregnancy (0.36+/-0.15 g/L) than during the first 20 wk of suckling (0.29+/-0.09 g/L). Cholesterol values decreased significantly (P<0.05) at the end of pregnancy, were minimal (1.01+/-0.03 g/L) at parturition, and increased again up to 9 wk post calving. Increased cholesterolemia and low serum triglyceride values after calving could be linked to the increased bovine alpha-lipoprotein fraction and decreased beta fraction. Serum triglyceride concentrations were not related to the resumption of postpartum ovarian cyclicity. Higher serum cholesterol values were observed from 2 wk before to 4 wk after calving in cows with early rather than mid and late resumption of ovarian cyclicity. Therefore, modifications in lipid metabolism during the puerperium seem to be related to resumption of cyclicity during the early postpartum period. PMID- 10729104 TI - Influence of the dominant follicle on oocytes from subordinate follicles. AB - As the oocyte grows within the follicle, a number of factors influence its health and developmental competence. These factors include follicle size, day of estrous cycle, level of atresia and influence of other follicles such as the dominant follicle. Follicles were dissected from ovaries of synchronized dairy cows on four days during the estrous cycle, and the oocyte from each follicle collected, matured, fertilized and cultured singly until Day 8. Development to blastocyst was greater in oocytes collected during phases of follicular growth than those collected during phases of follicular dominance (P<0.001) over all follicle size categories. Oocyte competence tended to increase with increasing follicle size (P<0.1). Follicular cells analyzed by flow cytometry showed an increase in proportion of apoptotic cells in subordinate follicles during the dominant phase compared to growth phase (P<0.05). Thus, the dominant follicle on both oocyte competence and levels of atresia. Further studies on the effect of dominance has shown that lactate production in cumulus-oocyte-complexes (COCs) from medium sized follicles collected during a dominance phase and small follicles collected during a growth phase are no different from other follicles, despite having significantly lower uptake of glucose (P<0.1). Thus, COCs from different follicle subclasses differ in their nutrient requirements, and current IVM technology needs further improvement to better assist those oocytes that are developmentally challenged. PMID- 10729105 TI - In vitro capacitation of bovine spermatozoa: role of intracellular calcium. AB - The development of successful methods of in vitro fertilization for bovine oocytes has advanced the bovine as a model for reproductive technology. The discovery of heparin as a capacitating agent has made it possible for investigators to have an inexpensive, readily available supply of bovine gametes for experimentation in reproductive biotechnologies such as gene transfer and cloning. The central event that mammalian sperm must undergo before being able to fertilize an oocyte is capacitation. Although we have methods which lead to efficient in vitro fertilization, we still lack understanding about the molecular mechanisms of capacitation. While numerous events occur during capacitation, it appears that regulation of intracellular Ca2+ (Ca(i)) is one of the most important. We found that the influx of Ca2+ into sperm during the first 2 hours of incubation is critical to heparin-induced capacitation. This is a period during capacitation when Ca(i) has not yet increased. We propose that during capacitation, the initial influx of Ca2+ into sperm is used to fill an intracellular Ca2+ store located in the acrosome. We found that thapsigargin, an inhibitor of an acrosomal Ca2+-ATPase, can stimulate capacitated sperm to acrosome react, trigger the opening of a store-operated calcium channel in the plasma membrane and has greater effects on capacitated sperm compared to noncapacitated sperm. An increase in intracellular Ca2+ was also detected in the anterior sperm head during capacitation, suggesting the loading of the acrosome with Ca2+. These observations may be important in the development of new methods for capacitation and understanding the death of sperm after cryopreservation. PMID- 10729106 TI - Factors determining competence of in vitro produced cattle embryos. AB - Concerns have developed in regard to problems associated with pregnancies and calves produced after use of cattle blastocysts made in the laboratory for embryo transfer. For both empirical studies and commercial purposes, there is a need for assurance that the product of these biotechnologies results in a normally functioning entity of its kind. Ability to use more genetic material from a donor female and in producing blastocysts needs to be improved to increase the efficiency of utilizing in vitro biotechnologies in animal production agriculture and for biomedical purposes. The role of gametes used as raw materials for laboratory production cattle embryos and adequacy of culture systems in supporting development of embryos are discussed in relation to competency of embryos produced in vitro. PMID- 10729107 TI - Development of the techniques for nuclear transfer in pigs. AB - Nuclear transfer in pigs was developed in the late 1980's. The techniques were based on previous studies in frogs, mice and cattle. Within stage nuclear transfer, pronuclear exchange, was followed by the transfer of nuclei from cleavage stage embryos. While these have resulted in term development, many problems remain. Recently progress on the problem of inadequate oocyte activation has been made and now there can be a refocus on the other aspects of the nuclear transfer procedure. The emphasis in developing the cloning/transgenic technology is easily justified, not so much by the ability to produce genetically identical animals for production agriculture, but for the potential to use a cell line that can be genetically engineered prior to the nuclear transfer. Pigs with specific genetic modifications will have a great impact on production agriculture as well as human medicine. PMID- 10729108 TI - Development and application of technology for large scale cloning of cattle. AB - Mammalian cloning technologies originally developed as methods of testing hypotheses about the mechanisms of cell differentiation. Embryo splitting procedures demonstrated that each of the cells in the early embryo are capable of developing into a complete new individual. Nuclear transplantation technologies have shown that loss of genetic sequences or even irreversible repression of gene function are also not mechanisms of cell differentiation. Therefore, both of these methods can be used for producing genetically identical animals. Nuclear transplantation has the advantage of being able to produce unlimited numbers of identical offspring. Highly efficient procedures have been developed for nuclear transplantation in mammals and several important characteristics of donor cells have been described. Unfortunately, the efficiency of producing cloned offspring is still low and many factors affecting the development of nuclear transfer embryos to term remain to be investigated. The tremendous potential of the technology for use in agriculture and medicine, however, will ensure that these problems are addressed and solved. PMID- 10729109 TI - Production and breeding of transgenic cattle using in vitro embryo production technology. AB - Transgenic technology permits major modifications of phenotype by introducing subtle changes in genotype. For domestic farm species, genetic modification may be used to enhance agricultural production or to generate novel genotypes capable of producing heterologous proteins for biomedical applications. The advent of in vitro embryo production techniques has facilitated the large-scale, commercial use of transgenic technology in cattle. Accordingly, we employed in vitro produced zygotes and embryos in an effort to generate transgenic cattle. Overall, pronuclei in 36,530 in vitro matured and fertilized zygotes were microinjected with a construct designed to express human alpha-lactalbumin in the mammary gland. Of these, 1,472 developed and were transferred to recipients, including 148 twin transfers. Initial pregnancy rate on Day 30 of gestation was 28% (374/1,324). Subsequent calving rate was 17% (226/1,324). Eighteen calves (8%) were transgenic. In vitro produced embryos were used to facilitate breeding of transgenic bulls. Frequency of transgene transmission varied from 3 to 54% between bulls, indicating varying degrees mosaicism. Embryos produced in vitro by these bulls were biopsied and screened for transgenesis prior to transfer to recipients; so far all (6/6) calves born from screened, transgenic embryos were themselves transgenic. PMID- 10729110 TI - Renal artery aneurysm: endovascular treatment by coil embolisation with preservation of renal blood flow. PMID- 10729111 TI - Multilocus linkage tests based on affected relative pairs. AB - For complex diseases, recent interest has focused on methods that take into account joint effects at interacting loci. Conditioning on effects of disease loci at known locations can lead to increased power to detect effects at other loci. Moreover, use of joint models allows investigation of the etiologic mechanisms that may be involved in the disease. Here we present a method for simultaneous analysis of the joint genetic effects at several loci that uses affected relative pairs. The method is a generalization of the two-locus LOD score analysis for affected sib pairs proposed by Cordell et al. We derive expressions for the relative risk, lambdaR, to a relative of an affected individual, in terms of the additive and epistatic components of variance at an arbitrary number of disease loci, and we show how these can be used to fit a likelihood model to the identity-by-descent sharing among pairs of affected relatives in extended pedigrees. We implement the method by use of a stepwise strategy in which, given evidence of linkage to disease at m-1 locations on the genome, we calculate the conditional likelihood curve across the genome for an mth disease locus, using multipoint methods similar to those proposed by Kruglyak et al. We evaluate the properties of our method by use of simulated data and present an application to real data from families with insulin-dependent diabetes mellitus. PMID- 10729112 TI - A major susceptibility locus influencing plasma triglyceride concentrations is located on chromosome 15q in Mexican Americans. AB - Although several genetic forms of rare or syndromic hypertriglyceridemia have been reported, little is known about the specific chromosomal regions across the genome harboring susceptibility genes for common forms of hypertriglyceridemia. Therefore, we conducted a genomewide scan for susceptibility genes influencing plasma triglyceride (TG) levels in a Mexican American population. We used both phenotypic and genotypic data from 418 individuals distributed across 27 low income, extended Mexican American families. For the analyses, TG values were log transformed (ln TG). We used a variance-components technique to conduct multipoint linkage analyses for localizing susceptibility genes that determine variation in TG levels. We used an approximately 10-15-cM map, which was made on the basis of information from 295 microsatellite markers. After accounting for the effects of sex and sex-specific age terms, we found significant evidence for linkage (LOD = 3.88) of ln TG levels to a genetic location between the markers GABRB3 and D15S165 on chromosome 15q. This putative locus explains 39.7+/-7% (P=.000012) of total phenotypic variation in ln TG levels. Suggestive evidence was found for linkage of ln TG levels to two different locations on chromosome 7, which are approximately 85 cM apart from each other. Also, there is some evidence for linkage of high-density lipoprotein cholesterol concentrations to a genetic location near one of the regions on chromosome 7. In conclusion, we found strong evidence for linkage of ln TG levels to a genetic location on chromosome 15q in a Mexican American population, which is prone to disease conditions such as type 2 diabetes and the insulin-resistance syndrome that are associated with hypertriglyceridemia. This putative locus appears to have a major influence on ln TG variation. PMID- 10729113 TI - Genomic structure and identification of novel mutations in usherin, the gene responsible for Usher syndrome type IIa. AB - Usher syndrome type IIa (USHIIa) is an autosomal recessive disorder characterized by moderate to severe sensorineural hearing loss and progressive retinitis pigmentosa. This disorder maps to human chromosome 1q41. Recently, mutations in USHIIa patients were identified in a novel gene isolated from this chromosomal region. The USH2A gene encodes a protein with a predicted molecular weight of 171.5 kD and possesses laminin epidermal growth factor as well as fibronectin type III domains. These domains are observed in other protein components of the basal lamina and extracellular matrixes; they may also be observed in cell adhesion molecules. The intron/exon organization of the gene whose protein we name "Usherin" was determined by direct sequencing of PCR products and cloned genomic DNA with cDNA-specific primers. The gene is encoded by 21 exons and spans a minimum of 105 kb. A mutation search of 57 independent USHIIa probands was performed with a combination of direct sequencing and heteroduplex analysis of PCR-amplified exons. Fifteen new mutations were found. Of 114 independent USH2A alleles, 58 harbored probable pathologic mutations. Ten cases of USHIIa were true homozygotes and 10 were compound heterozygotes; 18 heterozygotes with only one identifiable mutation were observed. Sixty-five percent (38/58) of cases had at least one mutation, and 51% (58/114) of the total number of possible mutations were identified. The allele 2299delG (previously reported as 2314delG) was the most frequent mutant allele observed (16%; 31/192). Three new missense mutations (C319Y, N346H, and C419F) were discovered; all were restricted to the previously unreported laminin domain VI region of Usherin. The possible significance of this domain, known to be necessary for laminin network assembly, is discussed in the context of domain VI mutations from other proteins. PMID- 10729114 TI - Pyogenic arthritis, pyoderma gangrenosum, and acne syndrome maps to chromosome 15q. AB - Pyoderma gangrenosum, cystic acne, and aseptic arthritis are clinically distinct disorders within the broad class of inflammatory diseases. Although this triad of symptoms is rarely observed in a single patient, a three-generation kindred with autosomal-dominant transmission of these three disorders has been reported as "PAPA syndrome" (MIM 604416). We report mapping of a disease locus for familial pyoderma gangrenosum-acne-arthritis to the long arm of chromosome 15 (maximum two point LOD score, 5.83; recombination fraction [straight theta] 0 at locus D15S206). Under the assumption of complete penetrance, haplotype analysis of recombination events defined a disease interval of 10 cM, between D15S1023 and D15S979. Successful identification of a single disease locus for this syndrome suggests that these clinically distinct disorders may share a genetic etiology. These data further indicate the role of genes outside the major histocompatibility locus in inflammatory disease. PMID- 10729115 TI - A juvenile-onset, progressive cataract locus on chromosome 3q21-q22 is associated with a missense mutation in the beaded filament structural protein-2. AB - Juvenile-onset cataracts are distinguished from congenital cataracts by the initial clarity of the lens at birth and the gradual development of lens opacity in the second and third decades of life. Genomewide linkage analysis in a multigenerational pedigree, segregating for autosomal dominant juvenile-onset cataracts, identified a locus in chromosome region 3q21.2-q22.3. Because of the proximity of the gene coding for lens beaded filament structural protein-2 (BFSP2) to this locus, we screened for mutations in the coding sequence of BFSP2. We observed a unique C-->T transition, one that was not observed in 200 normal chromosomes. We predicted that this led to a nonconservative R287W substitution in exon 4 that cosegregated with cataracts. This mutation alters an evolutionarily conserved arginine residue in the central rod domain of the intermediate filament. On consideration of the proposed function of BFSP2 in the lens cytoskeleton, it is likely that this alteration is the cause of cataracts in the members of the family we studied. This is the first example of a mutation in a noncrystallin structural gene that leads to a juvenile-onset, progressive cataract. PMID- 10729116 TI - Synaptic pathology in Borna disease virus persistent infection. AB - Borna disease virus (BDV) infection of newborn rats leads to a persistent infection of the brain, which is associated with behavioral and neuroanatonomical abnormalities. These disorders occur in the absence of lymphoid cell infiltrates, and BDV-induced cell damage is restricted to defined brain areas. To investigate if damage to synaptic structures anteceded neuronal loss in BDV neonatally infected rats, we analyzed at different times postinfection the expression levels of growth-associated protein 43 and synaptophysin, two molecules involved in neuroplasticity processes. We found that BDV induced a progressive and marked decrease in the expression of these synaptic markers, which was followed by a significant loss of cortical neurons. Our findings suggest that BDV persistent infection interferes with neuroplasticity processes in specific cell populations. This, in turn, could affect the proper supply of growth factors and other molecules required for survival of selective neuronal populations within the cortex and limbic system structures. PMID- 10729117 TI - Virus load and sequence variation in simian retrovirus type 2 infection. AB - The natural history of type D simian retrovirus (SRV) infection is poorly characterized in terms of viral load, antibody status, and sequence variation. To investigate this, blood samples were taken from a small cohort of mostly asymptomatic cynomolgus macaques (Macaca fascicularis), naturally infected with SRV type 2 (SRV-2), some of which were followed over an 8-month period with blood taken every 2 months. Provirus and RNA virus loads were obtained, the samples were screened for presence of antibodies to SRV-2 and neutralizing antibody titers to SRV-2 were assayed. env sequences were aligned to determine intra- and intermonkey variation over time. Virus loads varied greatly among cohort individuals but, conversely, remained steady for each macaque over the 8-month period, regardless of their initial levels. No significant sequence variation was found within an individual over time. No clear picture emerged from these results, which indicate that the variables of SRV-2 infection are complex, differ from those for lentivirus infection, and are not distinctly related to disease outcome. PMID- 10729118 TI - CD4(+) T-cell-mediated antiviral protection of the upper respiratory tract in BALB/c mice following parenteral immunization with a recombinant respiratory syncytial virus G protein fragment. AB - We analyzed the protective mechanisms induced against respiratory syncytial virus subgroup A (RSV-A) infection in the lower and upper respiratory tracts (LRT and URT) of BALB/c mice after intraperitoneal immunization with a recombinant fusion protein incorporating residues 130 to 230 of RSV-A G protein (BBG2Na). Mother-to offspring antibody (Ab) transfer and adoptive transfer of BBG2Na-primed B cells into SCID mice demonstrated that Abs are important for LRT protection but have no effect on URT infection. In contrast, RSV-A clearance in the URT was achieved in a dose-dependent fashion after adoptive transfer of BBG2Na-primed T cells, while it was abolished in BBG2Na-immunized mice upon in vivo depletion of CD4(+), but not CD8(+), T cells. Furthermore, the conserved RSV-A G protein cysteines and residues 193 and 194, overlapping the recently identified T helper cell epitope on the G protein (P. W. Tebbey et al., J. Exp. Med. 188:1967-1972, 1998), were found to be essential for URT but not LRT protection. Taken together, these results demonstrate for the first time that CD4(+) T cells induced upon parenteral immunization with an RSV G protein fragment play a critical role in URT protection of normal mice against RSV infection. PMID- 10729119 TI - Molecular characterization of a bacteriophage (Chp2) from Chlamydia psittaci. AB - Comparisons of the proteome of abortifacient Chlamydia psittaci isolates from sheep by two-dimensional gel electrophoresis identified a novel abundant protein with a molecular mass of 61.4 kDa and an isoelectric point of 6.41. C-terminal sequence analysis of this protein yielded a short peptide sequence that had an identical match to the viral coat protein (VP1) of the avian chlamydiaphage Chp1. Electron microscope studies revealed the presence of a 25-nm-diameter bacteriophage (Chp2) with no apparent spike structures. Thin sections of chlamydia-infected cells showed that Chp2 particles were located to membranous structures surrounding reticulate bodies (RBs), suggesting that Chp2 is cytopathic for ovine C. psittaci RBs. Chp2 double-stranded circular replicative form DNA was purified and used as a template for DNA sequence analysis. The Chp2 genome is 4,567 bp and encodes up to eight open reading frames (ORFs); it is similar in overall organization to the Chp1 genome. Seven of the ORFs (1 to 5, 7, and 8) have sequence homologies with Chp1. However, ORF 6 has a different spatial location and no cognate partner within the Chp1 genome. Chlamydiaphages have three viral structural proteins, VP1, VP2, and VP3, encoded by ORFs 1 to 3, respectively. Amino acid residues in the phiX174 procapsid known to mediate interactions between the viral coat protein and internal scaffolding proteins are conserved in the Chp2 VP1 and VP3 proteins. We suggest that VP3 performs a scaffolding-like function but has evolved into a structural protein. PMID- 10729120 TI - NP and L proteins of lymphocytic choriomeningitis virus (LCMV) are sufficient for efficient transcription and replication of LCMV genomic RNA analogs. AB - The genome of lymphocytic choriomeningitis virus (LCMV) consists of two negative sense single-stranded RNA segments, designated L and S. Both segments contain two viral genes in an ambisense coding strategy, with the genes being separated by an intergenic region (IGR). We have developed a reverse genetic system that allows the investigation of cis-acting signals and trans-acting factors involved in transcription and replication of LCMV. To this end, we constructed an LCMV S minigenome consisting of a negative-sense copy of the chloramphenicol acetyltransferase (CAT) reporter gene flanked upstream by the S 5' untranslated region (UTR) and IGR and downstream by the S 3' UTR. CAT expression was detected in LCMV-infected cells transfected with the minigenome RNA. Intracellular coexpression of the LCMV minigenome and LCMV L and NP proteins supplied from cotransfected plasmids driven by the T7 RNA polymerase provided by the recombinant vaccinia virus vTF7-3 resulted in high levels of CAT activity and synthesis of subgenomic CAT mRNA and antiminigenome RNA species. Thus, L and NP represent the minimal viral trans-acting factors required for efficient RNA synthesis mediated by LCMV polymerase. PMID- 10729121 TI - Identification of antigenic proteins encoded by human herpesvirus 8 and seroprevalence in the general population and among patients with and without Kaposi's sarcoma. AB - To establish a sensitive and specific antibody assay, potent antigenic proteins encoded by human herpesvirus 8 (HHV8) were studied. Fifteen recombinant HHV8 encoded proteins were produced as glutathione S-transferase fusion proteins. The sera from AIDS-associated Kaposi's sarcoma (KS) patients reacted with four proteins encoded by open reading frames (ORFs) K8.1, 59, 65, and 73 in a Western blot assay. An enzyme-linked immunosorbent assay (ELISA) using these four proteins as antigens (mixed-antigen ELISA) revealed that all 26 sera derived from KS patients (24 with and 2 without human immunodeficiency virus infection) became positive for anti-HHV8 antibodies. The presence of HHV8 was demonstrated in 14 (1. 4%) of 1,004 sera from the Japanese general population and 10 (1.9%) of 527 sera from patients without HHV8-associated diseases. The presence of immunoglobulin G (IgG) and IgM antibodies against HHV8 examined further by the mixed-antigen ELISA and Western blotting revealed IgG antibody in all ELISA positive sera, while IgM antibody against ORF K8.1 was absent. These data suggest that the ORF 73 and 65 proteins are potent antigens for a sensitive serological assay. PMID- 10729122 TI - A previously unrecognized H-2D(b)-restricted peptide prominent in the primary influenza A virus-specific CD8(+) T-cell response is much less apparent following secondary challenge. AB - Respiratory challenge of H-2(b) mice with an H3N2 influenza A virus causes an acute, transient pneumonitis characterized by the massive infiltration of CD8(+) T lymphocytes. The inflammatory process monitored by quantitative analysis of lymphocyte populations recovered by bronchoalveolar lavage is greatly enhanced by prior exposure to an H1N1 virus, with the recall of cross-reactive CD8(+)-T-cell memory leading to more rapid clearance of the infection from the lungs. The predominant epitope recognized by the influenza virus-specific CD8(+) set has long been thought to be a nucleoprotein (NP(366-374)) presented by H-2D(b) (D(b)NP(366)). This continues to be true for the secondary H3N2-->H1N1 challenge but can no longer be considered the case for the primary response to either virus. Quantitative analysis based on intracellular staining for gamma interferon has shown that the polymerase 2 protein (PA(224-233)) provides a previously undetected epitope (D(b)PA(224)) that is at least as prominent as D(b)NP(366) during the first 10 days following primary exposure to either the H3N2 or H1N1 virus. The response to D(b)NP(366) seems to continue for longer, even when infectious virus can no longer be detected, but there is no obvious difference in the prevalence of memory T cells specific for D(b)NP(366) and D(b)PA(224). The generalization that the magnitude of the functional memory T-cell pool is a direct consequence of the clonal burst size during the primary response may no longer be useful. Previous CD8(+)-T-cell immunodominance heirarchies defined largely by cytotoxic T-lymphocyte assays may need to be revised. PMID- 10729123 TI - Productive replication of adeno-associated virus can occur in human papillomavirus type 16 (HPV-16) episome-containing keratinocytes and is augmented by the HPV-16 E2 protein. AB - We used a sensitive assay to test whether an adeno-associated virus (AAV) productive replication cycle can occur in immortalized human keratinocytes carrying episomal human papillomavirus type 16 (HPV-16) DNA. Following transfection with cloned AAV DNA, infectious AAV was produced, and the infectivity was blocked by anti-AAV antiserum. The HPV-16 E2 protein substantially increased the yield of AAV. Other HPV early proteins did not, in our experiments, show this ability. E2 has been shown to be able to affect p53 levels and to block cell cycle progression at mitosis. We tested the effect of changes in p53 expression on AAV replication and found that large differences in the level of p53 did not alter AAV DNA replication. In extension of this, we found that cellular help for AAV in response to stress was also independent of p53. To test if a mitotic block could trigger AAV DNA replication, we treated the cells with the mitotic inhibitor nocodazole. AAV DNA replication was stimulated by the presence of nocodazole in these and a number of other cell types tested. Yields of infectious virus, however, were not increased by this treatment. We conclude that the HPV-16 E2 protein stimulates AAV multiplication in these cells and propose that this occurs independently of the effects of E2 on p53 and cell cycle progression. Since the effect of E2 was not seen in keratinocytes lacking the HPV-16 episome, we suggest that E2 can help AAV by working in concert with other HPV-16 proteins. PMID- 10729124 TI - Role of the pseudorabies virus gI cytoplasmic domain in neuroinvasion, virulence, and posttranslational N-linked glycosylation. AB - The glycoproteins I and E of pseudorabies virus are important mediators of cell to-cell spread and virulence in all animal models tested. Although these two proteins form a complex with one another, ascribing any function to the individual proteins has been difficult. We have shown previously, using nonsense mutations, that the N-terminal ectodomain of the gE protein is sufficient for gE mediated transsynaptic spread whereas the cytoplasmic domain of the protein is required for full expression of virulence. These same studies demonstrated that the cytoplasmic domain of gE is also required for endocytosis of the protein. In this report, we describe the construction of viruses with nonsense mutations in gI that allowed us to determine the contributions of the gI cytoplasmic domain to protein expression as well as virus neuroinvasion and virulence after infection of the rat eye. We also constructed double mutants with nonsense mutations in both gE and gI so that the contributions of both the gE and gI cytoplasmic domains could be determined. We observed that the gI cytoplasmic domain is required for efficient posttranslational modification of the gI protein. The gE cytoplasmic domain has no effect on gE posttranslational glycosylation. In addition, we found that infection of all gE-gI-dependent anterograde circuits projecting from the rat retina requires both ectodomains and at least one of the cytoplasmic domains of the proteins. The gI cytoplasmic domain promotes transsynaptic spread of virus better than the gE cytoplasmic domain. Interestingly, both gE and gI cytoplasmic tails are required for virulence; lack of either one or both results in an attenuated infection. These data suggest that gE and gI play differential roles in mediating directional neuroinvasion of the rat; however, the gE and gI cytoplasmic domains most likely function together to promote virulence. PMID- 10729125 TI - Pathogenesis of herpes simplex virus-induced ocular immunoinflammatory lesions in B-cell-deficient mice. AB - The role of B cells and humoral immunity in herpes simplex virus (HSV) ocular infections was studied in immunoglobulin mu chain gene-targeted B-cell-deficient mice (muK/O). At doses of virus well tolerated by immunocompetent mice, heightened susceptibility of muK/O mice to herpetic encephalitis as well as to herpetic stromal keratitis (HSK) was observed. An explanation was sought for the increased severity of HSK in the muK/O mice. First, the lack of antibody responses in muK/O mice resulted in longer viral persistence and dissemination to the corneal stroma, the site of inflammation. Prolonged virus expression in the corneal stroma was suggested to cause bystander activation of Th1-type CD4(+) T cells, further contributing to the severity of HSK lesion expression in muK/O mice. Second, muK/O mice generated minimal Th2 cytokine responses compared to wild-type mice. Such responses might serve to downregulate the severity of Th1 mediated HSK lesions. PMID- 10729126 TI - Characterization of vaccinia virus intracellular cores: implications for viral uncoating and core structure. AB - The entry of vaccinia virus (VV) into the host cell results in the delivery of the double-stranded DNA genome-containing core into the cytoplasm. The core is disassembled, releasing the viral DNA in order to initiate VV cytoplasmic transcription and DNA replication. Core disassembly can be prevented using the VV early transcription inhibitor actinomycin D (actD), since early VV protein synthesis is required for core uncoating. In this study, VV intracellular cores were accumulated in the presence of actD and isolated from infected cells. The content of these cores was analyzed by negative staining EM and by Western blotting using a collection of antibodies to VV core and membrane proteins. By Western blot analyses, intracellular actD cores, as well as cores prepared by NP 40-dithiothreitol treatment of purified virions (NP-40/DTT cores), contained the core proteins p25 (encoded by L4R), 4a (A10L), 4b (A3L), and p39 (A4L) as well as small amounts of the VV membrane proteins p32 (D8L) and p35 (H3L). While NP 40/DTT cores contained the major putative DNA-binding protein p11 (F17R), actD cores entirely lacked this protein. Labeled cryosections of cells infected for different periods of time in the presence or absence of actD were subsequently used to follow the fate of VV core proteins by EM. These EM images confirmed that p11 was lost at the plasma membrane upon core penetration. The cores that accumulated in the presence of actD were labeled with antibodies to 4a, p39, p25, and DNA at all times examined. In the absence of the drug the cores gradually lost their electron-dense inner part, concomitant with the loss of p25 and DNA labeling. The remaining core shell still labeled with antibodies to p39 and 4a/4b, implying that these proteins are part of this structure. These combined data are discussed with respect to the structure of VV as well as core disassembly. PMID- 10729127 TI - Evidence for recombination of live, attenuated immunodeficiency virus vaccine with challenge virus to a more virulent strain. AB - Live, attenuated immunodeficiency virus vaccines, such as nef deletion mutants, are the most effective vaccines tested in the simian immunodeficiency virus (SIV) macaque model. In two independent studies designed to determine the breadth of protection induced by live, attenuated SIV vaccines, we noticed that three of the vaccinated macaques developed higher set point viral load levels than unvaccinated control monkeys. Two of these vaccinated monkeys developed AIDS, while the control monkeys infected in parallel remained asymptomatic. Concomitant with an increase in viral load, a recombinant of the vaccine virus and the challenge virus could be detected. Therefore, the emergence of more-virulent recombinants of live, attenuated immunodeficiency viruses and less-aggressive wild-type viruses seems to be an additional risk of live, attenuated immunodeficiency virus vaccines. PMID- 10729128 TI - Memory in viral quasispecies. AB - Biological adaptive systems share some common features: variation among their constituent elements and continuity of core information. Some of them, such as the immune system, are endowed with memory of past events. In this study we provide direct evidence that evolving viral quasispecies possess a molecular memory in the form of minority components that populate their mutant spectra. The experiments have involved foot-and-mouth disease virus populations with known evolutionary histories. The composition and behavior of the viral population in response to a selective constraint were influenced by past evolutionary history in a way that could not be predicted from examination of consensus nucleotide sequences of the viral populations. The molecular memory of the viral quasispecies influenced both the nature and the intensity of the response of the virus to a selective constraint. PMID- 10729129 TI - Genetic evidence for an interaction between human immunodeficiency virus type 1 matrix and alpha-helix 2 of the gp41 cytoplasmic tail. AB - The incorporation of envelope (Env) glycoproteins into virions is an essential step in the retroviral replication cycle. Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), encode Env glycoproteins with unusually long cytoplasmic tails, the functions of which have not been fully elucidated. In this study, we examine the effects on virus replication of a number of mutations in a helical motif (alpha-helix 2) located near the center of the HIV-1 gp41 cytoplasmic tail. We find that, in T-cell lines, small deletions in this domain disrupt the incorporation of Env glycoproteins into virions and markedly impair virus infectivity. Through the analysis of viral revertants, we demonstrate that a single amino acid change (34VI) in the matrix domain of Gag reverses the Env incorporation and infectivity defect imposed by a small deletion near the C terminus of alpha-helix 2. These results provide genetic evidence, in the context of infected T cells, for an interaction between HIV-1 matrix and the gp41 cytoplasmic tail and identify domains of both proteins involved in this putative interaction. PMID- 10729130 TI - Kinetics of recombinant adeno-associated virus-mediated gene transfer. AB - Recombinant adeno-associated virus (rAAV) vectors have been shown to be useful for efficient gene delivery to a variety of dividing and nondividing cells. Mechanisms responsible for the long-term, persistent expression of the rAAV transgene are not well understood. In this study we investigated the kinetics of rAAV-mediated human factor IX (hFIX) gene transfer into human primary myoblasts and myotubes. Transduction of both myoblasts and myotubes occured with a similar and high efficiency. After 3 to 4 weeks of transduction, rAAV with a cytomegalovirus (CMV) promoter showed 10- to 15-fold higher expression than that with a muscle-specific creatine kinase enhancer linked to beta-actin promoter. Factor IX expression from transduced myoblasts as well as myotubes reached levels as high as approximately 2 microgram of hFIX/10(6) cells/day. Southern blot analyses of high-molecular-weight (HMW) cellular genomic and Hirt DNAs isolated from rAAV/CMVhFIXm1-transduced cells showed that the conversion of single stranded vector genomes to double-stranded DNA forms, but not the level of the integrated forms in HMW DNA, correlated with increasing expression of the transgene. Together, these results indicate that rAAV can transduce both proliferating and terminally differentiated muscle cells at about the same efficiency, that expression of transgenes increases linearly over their lifetime with no initial lag phase, and that increasing expression correlates with the appearance of double-stranded episomal rAAV genomes. Evidence showing that the rAAV virions can copackage hFIX, presumably nonspecifically, was also obtained. PMID- 10729131 TI - Diminishing returns of population size in the rate of RNA virus adaptation. AB - Whenever an asexual viral population evolves by adapting to new environmental conditions, beneficial mutations, the ultimate cause of adaptation, are randomly produced and then fixed in the population. The larger the population size and the higher the mutation rate, the more beneficial mutations can be produced per unit time. With the usually high mutation rate of RNA viruses and in a large enough population, several beneficial mutations could arise at the same time but in different genetic backgrounds, and if the virus is asexual, they will never be brought together through recombination. Thus, the best of these genotypes must outcompete each other on their way to fixation. This competition among beneficial mutations has the effect of slowing the overall rate of adaptation. This phenomenon is known as clonal interference. Clonal interference predicts a speed limit for adaptation as the population size increases. In the present report, by varying the size of evolving vesicular stomatitis virus populations, we found evidence clearly demonstrating this speed limit and thus indicating that clonal interference might be an important factor modulating the rate of adaptation to an in vitro cell system. Several evolutionary and epidemiological implications of the clonal interference model applied to RNA viruses are discussed. PMID- 10729132 TI - Identification and characterization of a shared TNFR-related receptor for subgroup B, D, and E avian leukosis viruses reveal cysteine residues required specifically for subgroup E viral entry. AB - Genetic and receptor interference data have indicated the presence of one or more cellular receptors for subgroup B, D, and E avian leukosis viruses (ALV) encoded by the s1 allele of the chicken tvb locus. Despite the prediction that these viruses use the same receptor, they exhibit a nonreciprocal receptor interference pattern: ALV-B and ALV-D can interfere with infection by all three viral subgroups, but ALV-E only interferes with infection by subgroup E viruses. We identified a tvb(s1) cDNA clone which encodes a tumor necrosis factor receptor related receptor for ALV-B, -D, and -E. The nonreciprocal receptor interference pattern was reconstituted in transfected human 293 cells by coexpressing the cloned receptor with the envelope (Env) proteins of either ALV-B or ALV-E. This pattern of interference was also observed when soluble ALV surface (SU) immunoglobulin fusion proteins were bound to this cellular receptor before viral challenge. These data demonstrate that viral Env-receptor interactions can account for the nonreciprocal interference between ALV subgroups B, D, and E. Furthermore, they indicate that a single chicken gene located at tvb(s1) encodes receptors for these three viral subgroups. The TVB(S1) protein differs exclusively at residue 62 from the published subgroup B- and D-specific receptor, encoded by the s3 allele of tvb. Residue 62 is a cysteine in TVB(S1) but is a serine in TVB(S3), giving TVB(S1) an even number of cysteines in the extracellular domain. We present evidence for a disulfide bond requirement in TVB(S1) for ALV-E infection but not for ALV-B infection. Thus, ALV-B and ALV-E interact in fundamentally different ways with this shared receptor, a finding that may account for the observed biological differences between these two ALV subgroups. PMID- 10729133 TI - The M184V mutation in the reverse transcriptase of human immunodeficiency virus type 1 impairs rescue of chain-terminated DNA synthesis. AB - Nucleoside analog chain terminators such as 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxy-3'-thiacytidine (3TC) represent an important class of drugs that are used in the clinic to inhibit the reverse transcriptase (RT) of human immunodeficiency virus type 1. Recent data have suggested that mutant enzymes associated with AZT resistance are capable of removing the chain-terminating residue with much greater efficiency than wild-type RT and this may, in turn, facilitate rescue of DNA synthesis; these experiments were performed using physiological concentrations of pyrophosphate or nucleoside triphosphates, respectively. The present study demonstrates that the M184V mutation, which confers high-level resistance to 3TC, can severely compromise the removal of chain-terminating nucleotides. Pyrophosphorolysis on 3TC-terminated primer strands was not detectable with M184V-containing, as opposed to wild-type, RT, and rescue of AZT-terminated DNA synthesis was significantly decreased with the former enzyme. Thus, mutated RTs associated with resistance to AZT and 3TC possess opposing, and therefore incompatible, phenotypes in this regard. These results are consistent with tissue culture and clinical data showing sustained antiviral effects of AZT in the context of viruses that contain the M184V mutation in the RT-encoding gene. PMID- 10729134 TI - Kaposi's sarcoma-associated herpesvirus open reading frame 57 encodes a posttranscriptional regulator with multiple distinct activities. AB - Open reading frame (ORF) 57 of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a homolog of known posttranscriptional regulators that are essential for replication in other herpesviruses. Here, we examined the expression of this gene and the function(s) of its product. KSHV ORF 57 is expressed very early in infection from a 1.6-kb spliced RNA bearing several in-frame initiation codons. Its product is a nuclear protein that, in transient assays, has little effect on the expression of luciferase reporter genes driven by a variety of KSHV and heterologous promoters. However, ORF 57 protein enhances the accumulation of several viral transcripts, in a manner suggesting posttranscriptional regulation. These transcripts include not only known cytoplasmic mRNAs (e.g., ORF 59) but also a nuclear RNA (nut-1) that lacks coding potential. Finally, ORF 57 protein can also modulate the effects of the ORF 50 gene product, a classical transactivator known to be required for lytic induction. The expression from some (e.g., nut-1) but not all (e.g., tk) ORF 50-responsive promoters can be synergistically enhanced by coexpression of ORF 50 and ORF 57. This effect is not due to upregulation of ORF 50 expression but rather to a posttranslational enhancement of the transcriptional activity of ORF 50. These data indicate that ORF 57 is a powerful pleiotropic effector that can act on several posttranscriptional levels to modulate the expression of viral genes in infected cells. PMID- 10729135 TI - Herpes simplex virus virion host shutoff (vhs) activity alters periocular disease in mice. AB - During lytic infection, the virion host shutoff (vhs) protein of herpes simplex virus (HSV) mediates the rapid degradation of RNA and shutoff of host protein synthesis. In mice, HSV type 1 (HSV-1) mutants lacking vhs activity are profoundly attenuated. HSV-2 has significantly higher vhs activity than HSV-1, eliciting a faster and more complete shutoff. To examine further the role of vhs activity in pathogenesis, we generated an intertypic recombinant virus (KOSV2) in which the vhs open reading frame of HSV-1 strain KOS was replaced with that of HSV-2 strain 333. KOSV2 and a marker-rescued virus, KOSV2R, were characterized in cell culture and tested in an in vivo mouse eye model of latency and pathogenesis. The RNA degradation kinetics of KOSV2 was identical to that of HSV 2 333, and both showed vhs activity significantly higher than that of KOS. This demonstrated that the fast vhs-mediated degradation phenotype of 333 had been conferred upon KOS. The growth of KOSV2 was comparable to that of KOS, 333, and KOSV2R in cell culture, murine corneas, and trigeminal ganglia and had a reactivation frequency similar to those of KOS and KOSV2R from explanted latently infected trigeminal ganglia. There was, however, significantly reduced blepharitis and viral replication within the periocular skin of KOSV2-infected mice compared to mice infected with either KOS or KOSV2R. Taken together, these data demonstrate that heightened vhs activity, in the context of HSV-1 infection, leads to increased viral clearance from the skin of mice and that the replication of virus in the skin is a determining factor for blepharitis. These data also suggest a role for vhs in modulating host responses to HSV infection. PMID- 10729136 TI - Inhibitory effects of nitric oxide and gamma interferon on in vitro and in vivo replication of Marek's disease virus. AB - The replication of Marek's disease herpesvirus (MDV) and herpesvirus of turkeys (HVT) in chicken embryo fibroblast (CEF) cultures was inhibited by the addition of S-nitroso-N-acetylpenicillamine, a nitric oxide (NO)-generating compound, in a dose-dependent manner. Treatment of CEF culture, prepared from 11-day-old embryos, with recombinant chicken gamma interferon (rChIFN-gamma) and lipopolysaccharide (LPS) resulted in production of NO which was suppressed by the addition of N(G)-monomethyl L-arginine (NMMA), an inhibitor of inducible NO synthase (iNOS). Incubation of CEF cultures for 72 h prior to treatment with rChIFN-gamma plus LPS was required for optimal NO production. Significant differences in NO production were observed in CEF derived from MDV-resistant N2a (major histocompatibility complex [MHC], B(21)B(21)) and MDV-susceptible S(13) (MHC, B(13)B(13)) and P2a (MHC, B(19)B(19)) chickens. N2a-derived CEF produced NO earlier and at higher levels than CEF from the other two lines. The lowest production of NO was detected in P2a-derived CEF. NO production in chicken splenocyte cultures followed a similar pattern, with the highest levels of NO produced in cultures from N2a chickens and the lowest levels produced in cultures from P2a chickens. Replication of MDV and HVT was significantly inhibited in CEF cultures treated with rChIFN-gamma plus LPS and producing NO. The addition of NMMA to CEF treated with rChIFN-gamma plus LPS reduced the inhibition. MDV infection of chickens treated with S-methylisothiourea, an inhibitor of iNOS, resulted in increased virus load compared to nontreated chickens. These results suggest that NO may play an important role in control of MDV replication in vivo. PMID- 10729137 TI - Evidence for a bidirectional element located downstream from the herpes simplex virus type 1 latency-associated promoter that increases its activity during latency. AB - Herpes simplex virus type 1 (HSV-1) latent infection in vivo is characterized by the constitutive expression of the latency-associated transcripts (LAT), which originate from the LAT promoter (LAP). In an attempt to determine the functional parts of LAP, we previously demonstrated that viruses harboring a DNA fragment 3' of the LAT promoter itself were able to maintain detectable promoter expression throughout latency whereas viruses not containing this element could not (J. R. Lokensgard, H. Berthomme, and L. T. Feldman, J. Virol. 71:6714-6719, 1997). This element was therefore called a long-term expression element (LTE). To further study the role of the LTE, we constructed plasmids containing a DNA fragment encompassing the LTE inserted into a synthetic intron between the reporter lacZ gene and either the LAT or the HSV-1 thymidine kinase promoter. Transient expression experiments with both neuronal and nonneuronal cell lines showed that the LTE locus has an enhancer activity that does not activate the cytomegalovirus enhancer but does activate the promoters such as the LAT promoter and the thymidine kinase promoter. The enhancement of these two promoters occurs in both neuronal and nonneuronal cell lines. Recombinant viruses containing enhancer constructs were constructed, and these demonstrated that the enhancer functioned when present in the context of the viral DNA, both for in vitro infections of cells in culture and for in vivo infections of neurons in mouse dorsal root ganglia. In the infections of mouse dorsal root ganglia, there was a very high level of promoter activity in neurons infected with viruses bearing the LAT promoter-enhancer, but this decreased after the first 2 or 3 weeks. By 18 days postinfection, neurons harboring latent virus without the enhancer showed no beta galactosidase (beta-gal) staining whereas those harboring latent virus containing the enhancer continued to show beta-gal staining for long periods, extending to at least 6 months postinfection, the longest time examined. PMID- 10729138 TI - Charged residues in the transmembrane domains of hepatitis C virus glycoproteins play a major role in the processing, subcellular localization, and assembly of these envelope proteins. AB - For most membrane proteins, the transmembrane domain (TMD) is more than just an anchor to the membrane. The TMDs of hepatitis C virus (HCV) envelope proteins E1 and E2 are extreme examples of the multifunctionality of such membrane-spanning sequences. Indeed, they possess a signal sequence function in their C-terminal half, play a major role in endoplasmic reticulum localization of E1 and E2, and are potentially involved in the assembly of these envelope proteins. These multiple functions are supposed to be essential for the formation of the viral envelope. As for the other viruses of the family Flaviviridae, these anchor domains are composed of two stretches of hydrophobic residues separated by a short segment containing at least one fully conserved charged residue. Replacement of these charged residues by an alanine in HCV envelope proteins led to an alteration of all of the functions performed by their TMDs, indicating that these functions are tightly linked together. These data suggest that the charged residues of the TMDs of HCV glycoproteins play a key role in the formation of the viral envelope. PMID- 10729139 TI - Specific phosphorylated forms of glyceraldehyde 3-phosphate dehydrogenase associate with human parainfluenza virus type 3 and inhibit viral transcription in vitro. AB - We previously reported specific interaction of cellular glyceraldehyde 3 phosphate dehydrogenase (GAPDH), the key glycolytic enzyme, and La protein, the RNA polymerase III transcription factor, with the cis-acting RNAs of human parainfluenza virus type 3 (HPIV3) and packaging of these proteins within purified virions (B. P. De, S. Gupta, H. Zhao, J. Z. Drazba, and A. K. Banerjee, J. Biol. Chem. 271:24728-24735, 1996). To gain further insight into these molecular interactions, we analyzed the virion-associated GAPDH and La protein using two-dimensional gel electrophoresis and immunoblotting. The GAPDH was resolved into two major and one minor molecular species migrating in the pI range of 7.6 to 8.3, while the La protein was resolved into five molecular species in the pI range of 6.8 to 7.5. The GAPDH isoforms present in the virions were also detected in the cytoplasmic fraction of CV-1 cell extract, albeit as minor species. On the other hand, the multiple molecular forms of La protein as seen within the virions were readily detected in the total CV-1 cell extract. Further analysis of virion-associated GAPDH by in vivo labeling with [(32)P]orthophosphate revealed the presence of multiple phosphorylated species. The phosphorylated species were able to bind specifically to the viral cis-acting 3' genome sense RNA but failed to bind to the leader sense RNA, as determined by gel mobility shift assay. In contrast, the La protein isoforms present within the virions were not phosphorylated and bound to the viral cis-acting RNAs in a phosphorylation-independent manner. The GAPDH isoforms purified from the CV-1 cell cytoplasmic fraction inhibited viral transcription in vitro. Consistent with this, flag-tagged recombinant GAPDH synthesized by using the vaccinia virus expression system also inhibited viral transcription. Together, these data indicate that specific phosphorylated forms of GAPDH associate with HPIV3 and are involved in the regulation of virus gene expression. PMID- 10729140 TI - Identification of amino acid residues in CD81 critical for interaction with hepatitis C virus envelope glycoprotein E2. AB - Human CD81 has been previously identified as the putative receptor for the hepatitis C virus envelope glycoprotein E2. The large extracellular loop (LEL) of human CD81 differs in four amino acid residues from that of the African green monkey (AGM), which does not bind E2. We mutated each of the four positions in human CD81 to the corresponding AGM residues and expressed them as soluble fusion LEL proteins in bacteria or as complete membrane proteins in mammalian cells. We found human amino acid 186 to be critical for the interaction with the viral envelope glycoprotein. This residue was also important for binding of certain anti-CD81 monoclonal antibodies. Mutating residues 188 and 196 did not affect E2 or antibody binding. Interestingly, mutation of residue 163 increased both E2 and antibody binding, suggesting that this amino acid contributes to the tertiary structure of CD81 and its ligand-binding ability. These observations have implications for the design of soluble high-affinity molecules that could target the CD81-E2 interaction site(s). PMID- 10729141 TI - Bystander sensitization to activation-induced cell death as a mechanism of virus induced immune suppression. AB - Viral infections which induce strong T-cell responses are often characterized by a period of transient immunodeficiency associated with the failure of host T cells to proliferate in response to mitogens or to mount memory recall responses to other antigens. During acute infections, most of the activated, proliferating virus-specific T cells are sensitized to undergo apoptosis on strong T-cell receptor (TCR) stimulation, but it has not been known why memory T cells not specific for the virus fail to proliferate on exposure to their cognate antigen. Using a lymphocytic choriomeningitis virus (LCMV) infection model in which LCMV immune Thy 1.1(+) splenocytes are adoptively transferred into Thy 1.2(+) LCMV carrier mice, we demonstrate here that T cells clearly defined as not specific for the virus are sensitized to undergo activation-induced cell death on TCR stimulation in vitro. This bystander sensitization was in part dependent on the expression of Fas ligand (FasL) on the activated virus-specific cells and gamma interferon (IFN-gamma) receptor expression on the bystander T cells. We propose that FasL from highly activated antiviral T cells may sensitize IFN-gamma conditioned T cells not specific for the virus to undergo apoptosis rather than to proliferate on encountering antigen. This may in part explain the failure of memory T cells to respond to recall antigens during acute and persistent viral infections. PMID- 10729142 TI - Rta of murine gammaherpesvirus 68 reactivates the complete lytic cycle from latency. AB - Herpesviruses are characterized as having two distinct life cycle phases: lytic replication and latency. The mechanisms of latency establishment and maintenance, as well as the switch from latency to lytic replication, are poorly understood. Human gammaherpesviruses, including Epstein-Barr virus (EBV) and human herpesvirus-8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), are associated with lymphoproliferative diseases and several human tumors. Unfortunately, the lack of cell lines to support efficient de novo productive infection and restricted host ranges of EBV and HHV-8 make it difficult to explore certain important biological questions. Murine gammaherpesvirus 68 (MHV-68, or gammaHV68) can establish de novo lytic infection in a variety of cell lines and is also able to infect laboratory mice, offering an ideal model with which to study various aspects of gammaherpesvirus infection. Here we describe in vitro studies of the mechanisms of the switch from latency to lytic replication of MHV-68. An MHV-68 gene, rta (replication and transcription activator), encoded primarily by open reading frame 50 (ORF50), is homologous to the rta genes of other gammaherpesviruses, including HHV-8 and EBV. HHV-8 and EBV Rta have been shown to play central roles in viral reactivation from latency. We first studied the kinetics of MHV-68 rta gene transcription during de novo lytic infection. MHV-68 rta was predominantly expressed as a 2-kb immediate-early transcript. Sequence analysis of MHV-68 rta cDNA revealed that an 866-nucleotide intron 5' of ORF50 was removed to create the Rta ORF of 583 amino acids. To test the functions of MHV-68 Rta in reactivation, a plasmid expressing Rta was transfected into a latently infected cell line, S11E, which was established from a B-cell lymphoma in an MHV-68-infected mouse. Rta induced expression of viral early and late genes, lytic replication of viral DNA, and production of infectious viral particles. We conclude that Rta alone is able to disrupt latency, activate viral lytic replication, and drive the lytic cycle to completion. This study indicates that MHV-68 provides a valuable model for investigating regulation of the balance between latency and lytic replication in vitro and in vivo. PMID- 10729143 TI - A murine leukemia virus (MuLV) long terminal repeat derived from rhesus macaques in the context of a lentivirus vector and MuLV gag sequence results in high-level gene expression in human T lymphocytes. AB - We constructed human immunodeficiency virus type 1 (HIV-1) vectors that will allow higher levels of gene expression in T cells. Gene expression under the control of an internal cytomegalovirus (CMV) immediate-early promoter in a self inactivating lentiviral vector (CSCG) is 4- to 15-fold lower in T-cell lines (SUPT1 and CEMX174) than in non-lymphoid-cell lines (HeLa and 293T). This is in contrast to a Moloney murine leukemia virus (MoMLV)-based retrovirus vector (SRalphaLEGFP). We therefore replaced the internal CMV promoter of CSCG with three different murine oncoretroviral long terminal repeat (LTR) promoters-murine sarcoma virus (MSV), MoMLV (MLV), and the LTR (termed Rh-MLV) that is derived from the ampho-mink cell focus-forming (AMP/MCF) retrovirus in the serum of one rhesus macaque monkey that developed T-cell lymphoma following autologous transplantation of enriched bone marrow stem cells transduced with a retrovirus vector preparation containing replication-competent viruses (E. F. Vanin, M. Kaloss, C. Broscius, and A. W. Nienhuis, J. Virol. 68:4241-4250, 1994). We found that the combination of Rh-MLV LTR and a partial gag sequence of MoMLV (Deltagag(871-1612)) in CS-Rh-MLV-E gave the highest level of enhanced green fluorescent protein (EGFP) gene expression compared with MLV, MSV LTR, phosphoglycerate kinase, and CMV promoters in T-cell lines, as well as activated primary T cells. Interestingly, there was a further two- to threefold increase in EGFP expression (thus, 10-fold-higher expression than with CMV) when the Rh-MLV promoter and Deltagag(871-1612) were used in a self-inactivating-vector setting that has a further deletion in the U3 region of the HIV-1 LTR. These hybrid vectors should prove useful in gene therapy applications for T cells. PMID- 10729144 TI - Elucidating the essential role of the A14 phosphoprotein in vaccinia virus morphogenesis: construction and characterization of a tetracycline-inducible recombinant. AB - We have previously reported the construction and characterization of vindH1, an inducible recombinant in which expression of the vaccinia virus H1 phosphatase is regulated experimentally by IPTG (isopropyl-beta-D-thiogalactopyranoside) (35). In the absence of H1 expression, the transcriptional competence and infectivity of nascent virions are severely compromised. We have sought to identify H1 substrates by characterizing proteins that are hyperphosphorylated in H1 deficient virions. Here, we demonstrate that the A14 protein, a component of the virion membrane, is indeed an H1 phosphatase substrate in vivo and in vitro. A14 is hyperphosphorylated on serine residues in the absence of H1 expression. To enable a genetic analysis of A14's function during the viral life cycle, we have adopted the regulatory components of the tetracycline (TET) operon and created new reagents for the construction of TET-inducible vaccinia virus recombinants. In the context of a virus expressing the TET repressor (tetR), insertion of the TET operator between the transcriptional and translational start sites of a late viral gene enables its expression to be tightly regulated by TET. We constructed a TET-inducible recombinant for the A14 gene, vindA14. In the absence of TET, vindA14 fails to form plaques and the 24-h yield of infectious progeny is reduced by 3 orders of magnitude. The infection arrests early during viral morphogenesis, with the accumulation of large numbers of vesicles and the appearance of "empty" crescents that appear to adhere only loosely to virosomes. This phenotype corresponds closely to that observed for an IPTG-inducible A14 recombinant whose construction and characterization were reported while our work was ongoing (47). The consistency in the phenotypes seen for the IPTG- and TET-inducible recombinants confirms the efficacy of the TET-inducible system and reinforces the value of having a second, independent system available for generating inducible recombinants. PMID- 10729145 TI - Suppression of murine cytomegalovirus (MCMV) replication with a DNA vaccine encoding MCMV M84 (a homolog of human cytomegalovirus pp65). AB - The cytotoxic T-lymphocyte (CTL) response against the murine cytomegalovirus (MCMV) immediate-early gene 1 (IE1) 89-kDa phosphoprotein pp89 plays a major role in protecting BALB/c mice against the lethal effects of the viral infection. CTL populations specific to MCMV early-phase and structural antigens are also generated during infection, but the identities of these antigens and their relative contributions to overall immunity against MCMV are not known. We previously demonstrated that DNA vaccination with a pp89-expressing plasmid effectively generated a CTL response and conferred protection against infection (J. C. Gonzalez Armas, C. S. Morello, L. D. Cranmer, and D. H. Spector, J. Virol. 70:7921-7928, 1996). In this report, we have sought (i) to identify other viral antigens that contribute to immunity against MCMV and (ii) to determine whether the protective response is haplotype specific. DNA immunization was used to test the protective efficacies of plasmids encoding MCMV homologs of human cytomegalovirus (HCMV) tegument (M32, M48, M56, M82, M83, M69, and M99), capsid (M85 and M86), and nonstructural antigens (IE1-pp89 and M84). BALB/c (H-2(d)) and C3H/HeN (H-2(k)) mice were immunized by intradermal injection of either single plasmids or cocktails of up to four expression plasmids and then challenged with sublethal doses of virulent MCMV administered intraperitoneally. In this way, we identified a new viral gene product, M84, that conferred protection against viral replication in the spleens of BALB/c mice. M84 is expressed early in the infection and encodes a nonstructural protein that shares significant amino acid homology with the HCMV UL83-pp65 tegument protein, a major target of protective CTLs in humans. Specificity of the immune response to the M84 protein was confirmed by showing that immunization with pp89 DNA, but not M84 DNA, protected mice against subsequent infection with an MCMV deletion mutant lacking the M84 gene. The other MCMV genes tested did not generate a protective response even when mice were immunized with vaccinia viruses expressing the viral proteins. However, the M84 plasmid was protective when injected in combination with nonprotective plasmids, and coimmunization of BALB/c mice with pp89 and M84 provided a synergistic level of protection in the spleen. Viral titers in the salivary glands were also reduced, but not to the same extent as observed in the spleen, and the decrease was seen only when the BALB/c mice were immunized with pp89 plus M84 or with pp89 alone. The experiments with the C3H/HeN mice showed that the immunity conferred by DNA vaccination was haplotype dependent. In this strain of mice, only pp89 elicited a protective response as measured by a reduction in spleen titer. These results suggest that DNA immunization with the appropriate combination of CMV genes may provide a strategy for improving vaccine efficacy. PMID- 10729146 TI - A novel subgenomic murine leukemia virus RNA transcript results from alternative splicing. AB - Here we show the existence of a novel subgenomic 4.4-kb RNA in cells infected with the prototypic replication-competent Friend or Moloney murine leukemia viruses (MuLV). This RNA derives by splicing from an alternative donor site (SD') within the capsid-coding region to the canonical envelope splice acceptor site. The position and the sequence of SD' was highly conserved among mammalian type C and D oncoviruses. Point mutations used to inactivate SD' without changing the capsid-coding ability affected viral RNA splicing and reduced viral replication in infected cells. PMID- 10729147 TI - Identification and characterization of novel human endogenous retrovirus families by phylogenetic screening of the human genome mapping project database. AB - Human endogenous retroviruses (HERVs) were first identified almost 20 years ago, and since then numerous families have been described. It has, however, been difficult to obtain a good estimate of both the total number of independently derived families and their relationship to each other as well as to other members of the family Retroviridae. In this study, I used sequence data derived from over 150 novel HERVs, obtained from the Human Genome Mapping Project database, and a variety of recently identified nonhuman retroviruses to classify the HERVs into 22 independently acquired families. Of these, 17 families were loosely assigned to the class I HERVs, 3 to the class II HERVs and 2 to the class III HERVs. Many of these families have been identified previously, but six are described here for the first time and another four, for which only partial sequence information was previously available, were further characterized. Members of each of the 10 families are defective, and calculation of their integration dates suggested that most of them are likely to have been present within the human lineage since it diverged from the Old World monkeys more than 25 million years ago. PMID- 10729148 TI - Mutational analysis of the subgroup A avian sarcoma and leukosis virus putative fusion peptide domain. AB - Short hydrophobic regions referred to as fusion peptide domains (FPDs) at or near the amino terminus of the membrane-anchoring subunit of viral glycoproteins are believed to insert into the host membrane during the initial stage of enveloped viral entry. Avian sarcoma and leukosis viruses (ASLV) are unusual among retroviruses in that the region in the envelope glycoprotein (EnvA) proposed to be the FPD is internal and contains a centrally located proline residue. To begin analyzing the function of this region of EnvA, 20 substitution mutations were introduced into the putative FPD. The mutant envelope glycoproteins were evaluated for effects on virion incorporation, receptor binding, and infection. Interestingly, most of the single-substitution mutations had little effect on any of these processes. In contrast, a bulky hydrophobic substitution for the central proline reduced viral titers 15-fold without affecting virion incorporation or receptor binding, whereas substitution of glycine for the proline had only a nominal effect on EnvA function. Similar to other viral FPDs, the putative ASLV FPD has been modeled as an amphipathic helix where most of the bulky hydrophobic residues form a patch on one face of the helix. A series of alanine insertion mutations designed to interrupt the hydrophobic patch on the helix had differential effects on infectivity, and the results of that analysis together with the results observed with the substitution mutations suggest no correlation between maintenance of the hydrophobic patch and glycoprotein function. PMID- 10729149 TI - Functional differences between the long terminal repeat transcriptional promoters of human immunodeficiency virus type 1 subtypes A through G. AB - The current human immunodeficiency virus type 1 (HIV-1) shows an increasing number of distinct viral subtypes, as well as viruses that are recombinants of at least two subtypes. Although no biological differences have been described so far for viruses that belong to different subtypes, there is considerable sequence variation between the different HIV-1 subtypes. The HIV-1 long terminal repeat (LTR) encodes the transcriptional promoter, and the LTR of subtypes A through G was cloned and analyzed to test if there are subtype-specific differences in gene expression. Sequence analysis demonstrated a unique LTR enhancer-promoter configuration for each subtype. Transcription assays with luciferase reporter constructs showed that all subtype LTRs are functional promoters with a low basal transcriptional activity and a high activity in the presence of the viral Tat transcriptional activator protein. All subtype LTRs responded equally well to the Tat trans activator protein of subtype B. This result suggests that there are no major differences in the mechanism of Tat-mediated trans activation among the subtypes. Nevertheless, subtype-specific differences in the activity of the basal LTR promoter were measured in different cell types. Furthermore, we measured a differential response to tumor necrosis factor alpha treatment, and the induction level correlated with the number of NF-kappaB sites in the respective LTRs, which varies from one (subtype E) to three (subtype C). In general, subtype E was found to encode the most potent LTR, and we therefore inserted the core promoter elements of subtype E in the infectious molecular clone of the LAI isolate (subtype B). This recombinant LAI-E virus exhibited a profound replication advantage compared with the original LAI virus in the SupT1 T-cell line, indicating that subtle differences in LTR promoter activity can have a significant impact on viral replication kinetics. These results suggest that there may be considerable biological differences among the HIV-1 subtypes. PMID- 10729150 TI - Mechanisms of human papillomavirus E2-mediated repression of viral oncogene expression and cervical cancer cell growth inhibition. AB - The papillomavirus E2 gene product plays a pivotal role in viral replication. E2 has multiple functions, including (i) transcriptional activation and repression of viral promoters and (ii) the enhancement of viral DNA replication. It was previously reported that E2 suppressed the growth of papillomavirus-positive cervical carcinoma cell lines. In the present study, we investigated the mechanisms of E2 growth inhibition. We found that the transcriptional activation function of E2 is required for inhibition of the growth of HeLa cells as well as for transcriptional repression of the viral E6/E7 promoter. It had been previously postulated that transcriptional repression of the E6/E7 promoter results from E2 binding its cognate sites proximal to the E6/E7 promoter and displacing other cellular transcriptional factors. In this study, we report a requirement for the transcription activation function for the binding of E2 to transcriptionally active templates. PMID- 10729151 TI - The hinge of the human papillomavirus type 11 E2 protein contains major determinants for nuclear localization and nuclear matrix association. AB - The E2 protein of papillomaviruses is a site-specific DNA binding nuclear protein. It functions as the primary replication origin recognition protein and assists in the assembly of the preinitiation complex. It also helps regulate transcription from the native viral promoter. The E2 protein consists of an amino terminal (N) trans-acting domain, a central hinge (H) domain, and a carboxyl terminal (C) protein dimerization and DNA binding domain. The hinge is highly divergent among papillomaviruses, and little is known about its functions. We fused the enhanced green fluorescent protein (GFP) with the full-length human papillomavirus type 11 (HPV-11) E2 protein and showed that the resultant fusion, called gfpE2, maintained transcription and replication functions of the wild-type protein and formed similar subnuclear foci. Using a series of GFP fusion proteins, we showed that the hinge conferred strong nuclear localization, whereas the N or C domain was present in both cytoplasm and nucleus. Biochemical fractionation demonstrated that the N domain and hinge, but not the C domain, independently associated with the nuclear matrix. Mutational analyses showed that a cluster of basic amino acid residues, which is conserved among many mucosotropic papillomaviruses, was required for efficient nuclear localization and nuclear matrix association. This mutation no longer repressed the HPV-11 upstream regulatory region-controlled reporter expression. However, a very small fraction of this mutant colocalized with E1 in the nucleus, perhaps by a piggyback mechanism, and was able to support transient replication. We propose that the hinge is critical for the diverse regulatory functions of the HPV-11 E2 protein during mRNA transcription and viral DNA replication. PMID- 10729152 TI - Golgi network targeting and plasma membrane internalization signals in vaccinia virus B5R envelope protein. AB - The vaccinia virus B5R type I integral membrane protein accumulates in the Golgi network, from where it becomes incorporated into the envelope of extracellular virions. Our objective was to determine the domains of B5R responsible for Golgi membrane targeting in the absence of other viral components. Fusion of an enhanced green fluorescent protein to the C terminus of B5R allowed imaging of the chimeric protein without altering intracellular trafficking and Golgi network localization in transfected cells. Deletion or swapping of B5R domains with corresponding regions of the vesicular stomatitis virus G protein, which is targeted to the plasma membrane, indicated that (i) the N-terminal extracellular domain of B5R had no specific role in Golgi apparatus localization, (ii) the transmembrane domain of B5R was sufficient for exiting the endoplasmic reticulum, and (iii) removal of the cytoplasmic tail impaired Golgi network localization and increased the accumulation of B5R in the plasma membrane. Further experiments demonstrated that the cytoplasmic tail mediated internalization of B5R from the plasma membrane, suggesting a retrieval mechanism. Mutagenesis revealed residues required for Golgi membrane localization and efficient plasma membrane retrieval of the B5R protein: a tyrosine at residue 310 and two adjacent leucines at residues 315 and 316. PMID- 10729153 TI - The IRF-3 transcription factor mediates Sendai virus-induced apoptosis. AB - Virus infection of target cells can result in different biological outcomes: lytic infection, cellular transformation, or cell death by apoptosis. Cells respond to virus infection by the activation of specific transcription factors involved in cytokine gene regulation and cell growth control. The ubiquitously expressed interferon regulatory factor 3 (IRF-3) transcription factor is directly activated following virus infection through posttranslational modification. Phosphorylation of specific C-terminal serine residues results in IRF-3 dimerization, nuclear translocation, and activation of DNA-binding and transactivation potential. Once activated, IRF-3 transcriptionally up regulates alpha/beta interferon genes, the chemokine RANTES, and potentially other genes that inhibit viral infection. We previously generated constitutively active [IRF 3(5D)] and dominant negative (IRF-3 DeltaN) forms of IRF-3 that control target gene expression. In an effort to characterize the growth regulatory properties of IRF-3, we observed that IRF-3 is a mediator of paramyxovirus-induced apoptosis. Expression of the constitutively active form of IRF-3 is toxic, preventing the establishment of stably transfected cells. By using a tetracycline-inducible system, we show that induction of IRF-3(5D) alone is sufficient to induce apoptosis in human embryonic kidney 293 and human Jurkat T cells as measured by DNA laddering, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay, and analysis of DNA content by flow cytometry. Wild-type IRF-3 expression augments paramyxovirus-induced apoptosis, while expression of IRF-3 DeltaN blocks virus-induced apoptosis. In addition, we demonstrate an important role of caspases 8, 9, and 3 in IRF-3-induced apoptosis. These results suggest that IRF-3, in addition to potently activating cytokine genes, regulates apoptotic signalling following virus infection. PMID- 10729154 TI - Nonrandom transduction of recombinant adeno-associated virus vectors in mouse hepatocytes in vivo: cell cycling does not influence hepatocyte transduction. AB - Recombinant adeno-associated virus vectors (rAAV) show promise in preclinical trials for the treatment of genetic diseases including hemophilia. Liver-directed gene transfer results in a slow rise in transgene expression, reaching steady state levels over a period of 5 weeks concomitant with the conversion of the single-stranded rAAV molecules into high-molecular-weight concatemers in about 5% of hepatocytes. Immunohistochemistry and RNA in situ hybridization show that the transgene product is made in about approximately 5% of hepatocytes, suggesting that most rAAV-mediated gene expression occurs in hepatocytes containing the double-stranded concatemers. In this study, the mechanism(s) involved in stable transduction in vivo was evaluated. While only approximately 5% of hepatocytes are stably transduced, in situ hybridization experiments demonstrated that the vast majority of the hepatocytes take up AAV-DNA genomes after portal vein infusion of the vector. Two different vectors were infused together or staggered by 1, 3, or 5 weeks, and two-color fluorescent in situ hybridization and molecular analyses were performed 5 weeks after the infusion of the second vector. These experiments revealed that a small but changing subpopulation of hepatocytes were permissive to stable transduction. Furthermore, in animals that received a single infusion of two vectors, about one-third of the transduced cells contained heteroconcatemers, suggesting that dimer formation was a critical event in the process of concatemer formation. To determine if the progression through the cell cycle was important for rAAV transduction, animals were continuously infused with 5'-bromo-2'-deoxyuridine (BrdU), starting at the time of administration of a rAAV vector that expressed cytoplasmic beta-galactosidase. Colabeling for beta-galactosidase and BrdU revealed that there was no preference for transduction of cycling cells. This was further confirmed by demonstrating no increase in rAAV transduction efficiencies in animals whose livers were induced to cycle at the time of or after vector administration. Taken together, our studies suggest that while virtually all hepatocytes take up vector, unknown cellular factors are required for stable transduction, and that dimer formation is a critical event in the transduction pathway. These studies have important implications for understanding the mechanism of integration and may be useful for improving liver gene transfer in vivo. PMID- 10729155 TI - A beta-stranded motif drives capsid protein oligomers of the parvovirus minute virus of mice into the nucleus for viral assembly. AB - The determinants of nuclear import in the VP-1 and VP-2 capsid proteins of the parvovirus minute virus of mice strain i (MVMi) synthesized in human fibroblasts were sought by genetic analysis in an infectious plasmid. Immunofluorescence of transfected cells revealed that the two proteins were involved in cooperative cytoplasmic interactions for nuclear cotransport. However, while VP-1 translocated regardless of extension of deletions and did not form capsid epitopes by itself, VP-2 seemed to require cytoplasmic folding and the overall conformation for nuclear transport. The sequence (528)KGKLTMRAKLR(538) was found necessary for nuclear uptake of VP-2, even though it was not sufficient to confer a nuclear localization capacity on a heterologous protein. In the icosahaedral MVMi capsid, this sequence forms the carboxy end of the amphipathic beta-strand I (betaI), and all its basic residues are contiguously positioned at the face that in the unassembled subunit would be exposed to solvent. Mutations in singly expressed VP-2 that either decrease the net basic charge of the exposed face (K530N-R534T), perturb the hydrophobicity of the opposite face (L531E), or distort the betaI conformation (G529P) produced cytoplasmic subviral oligomers. Particle formation by betaI mutants indicated that the basic residues clustered at one face of betaI drive VP oligomers into the nucleus preceding and uncoupled to assembly and that the nuclear environment is required for MVMi capsid formation in the infected cell. The degree of VP-1/VP-2 transport cooperativity suggests that VP trimers are the morphogenetic intermediates translocating through the nuclear pore. The results support a model in which nuclear transport signaling preserves the VP-1/VP-2 stoichiometry necessary for efficient intranuclear assembly and in which the beta-stranded VP-2 nuclear localization motif contributes to the quality control of viral morphogenesis. PMID- 10729157 TI - Attenuated, replication-competent herpes simplex virus type 1 mutant G207: safety evaluation in mice. AB - Herpes simplex virus type 1 (HSV-1) mutants that are attenuated for neurovirulence are being used for the treatment of cancer. We have examined the safety of G207, a multimutated replication-competent HSV-1 vector, in mice. BALB/c mice inoculated intracerebrally or intracerebroventricularly with 10(7) PFU of G207 survived for over 20 weeks with no apparent symptoms of disease. In contrast, over 80% of animals inoculated intracerebrally with 1.5 x 10(3) PFU of HSV-1 wild-type strain KOS and 50% of animals inoculated intracerebroventricularly with 10(4) PFU of wild-type strain F died within 10 days. Similarly, after intrahepatic inoculation of G207 (3 x 10(7) PFU) all animals survived for over 10 weeks, whereas no animals survived for even 1 week after inoculation with 10(6) PFU of KOS. After intracerebroventricular inoculation, LacZ expression was initially observed in the cells lining the ventricles and subarachnoid space; expression decreased until almost absent within 5 days postinfection, with no apparent loss of ependymal cells. G207 DNA could be detected by PCR in the brains of mice 8 weeks after intracerebral inoculation; however, no infectious virus could be detected after 2 days. As a model for latent HSV in the brain, we used survivors of an intracerebral inoculation of HSV-1 KOS at the 50% lethal dose. Inoculation of a high dose of G207 at the same stereotactic coordinates did not result in reactivation of detectable infectious virus or symptoms of disease. We conclude that G207 is safe at or above doses that were efficacious in mouse tumor studies. PMID- 10729156 TI - The genome of fowlpox virus. AB - Here we present the genomic sequence, with analysis, of a pathogenic fowlpox virus (FPV). The 288-kbp FPV genome consists of a central coding region bounded by identical 9.5-kbp inverted terminal repeats and contains 260 open reading frames, of which 101 exhibit similarity to genes of known function. Comparison of the FPV genome with those of other chordopoxviruses (ChPVs) revealed 65 conserved gene homologues, encoding proteins involved in transcription and mRNA biogenesis, nucleotide metabolism, DNA replication and repair, protein processing, and virion structure. Comparison of the FPV genome with those of other ChPVs revealed extensive genome colinearity which is interrupted in FPV by a translocation and a major inversion, the presence of multiple and in some cases large gene families, and novel cellular homologues. Large numbers of cellular homologues together with 10 multigene families largely account for the marked size difference between the FPV genome (260 to 309 kbp) and other known ChPV genomes (178 to 191 kbp). Predicted proteins with putative functions involving immune evasion included eight natural killer cell receptors, four CC chemokines, three G-protein-coupled receptors, two beta nerve growth factors, transforming growth factor beta, interleukin-18-binding protein, semaphorin, and five serine proteinase inhibitors (serpins). Other potential FPV host range proteins included homologues of those involved in apoptosis (e.g., Bcl-2 protein), cell growth (e.g., epidermal growth factor domain protein), tissue tropism (e.g., ankyrin repeat-containing gene family, N1R/p28 gene family, and a T10 homologue), and avian host range (e.g., a protein present in both fowl adenovirus and Marek's disease virus). The presence of homologues of genes encoding proteins involved in steroid biogenesis (e.g., hydroxysteroid dehydrogenase), antioxidant functions (e.g., glutathione peroxidase), vesicle trafficking (e.g., two alpha-type soluble NSF attachment proteins), and other, unknown conserved cellular processes (e.g., Hal3 domain protein and GSN1/SUR4) suggests that significant modification of host cell function occurs upon viral infection. The presence of a cyclobutane pyrimidine dimer photolyase homologue in FPV suggests the presence of a photoreactivation DNA repair pathway. This diverse complement of genes with likely host range functions in FPV suggests significant viral adaptation to the avian host. PMID- 10729158 TI - Human cytomegalovirus pp28 (UL99) localizes to a cytoplasmic compartment which overlaps the endoplasmic reticulum-golgi-intermediate compartment. AB - Although the assembly of herpesviruses has remained an active area of investigation, considerable controversy continues to surround the cellular location of tegument and envelope acquisition. This controversy is particularly evident when the proposed pathways for alpha- and beta-herpesvirus assembly are compared. We have approached this aspect of human cytomegalovirus (HCMV) assembly, specifically, envelopment, by investigating the intracellular trafficking of viral tegument proteins which localize in the cytoplasms of infected cells. In this study we have demonstrated that the virion tegument protein pp28 (UL99), a true late protein, was membrane associated as a result of myristoylation. A mutation in this protein which prevented incorporation of [(3)H]myristic acid also altered the detergent solubility and intracellular distribution of the protein when it was expressed in transfected cells. Using a panel of markers for intracellular compartments, we could localize the expression of wild-type pp28 to an intracellular compartment which colocalized with the endoplasmic reticulum-Golgi-intermediate compartment (ERGIC), a dynamic compartment of the secretory pathway which interfaces with both the ER and Golgi apparatus. The localization of this viral tegument protein within an early secretory compartment of the cell provided further evidence that the assembly of the HCMV tegument likely includes a cytoplasmic phase. Because pp28 has been shown to be localized to a cytoplasmic assembly compartment in HCMV-infected cells, our findings also suggested that viral tegument protein interactions within the secretory pathway may have an important role in the assembly of the virion. PMID- 10729159 TI - Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer. AB - In the genetic disease cystic fibrosis, recombinant adeno-associated virus type 2 (AAV2) is being investigated as a vector to transfer CFTR cDNA to airway epithelia. However, earlier work has shown that the apical surface of human airway epithelia is resistant to infection by AAV2, presumably as a result of a lack of heparan sulfate proteoglycans on the apical surface. This inefficiency can be overcome by increasing the amount of vector or by increasing the incubation time. However, these interventions are not very practical for translation into a therapeutic airway-directed vector. Therefore, we examined the efficiency of other AAV serotypes at infecting human airway epithelia. When applied at low multiplicity of infection to the apical surface of differentiated airway epithelia we found that a recombinant AAV5 bound and mediated gene transfer 50-fold more efficiently than AAV2. Furthermore, in contrast to AAV2, AAV5-mediated gene transfer was not inhibited by soluble heparin. Recombinant AAV5 was also more efficient than AAV2 in transferring beta-galactosidase cDNA to murine airway and alveolar epithelia in vivo. These data suggest that AAV5 derived vectors bind and mediate gene transfer to human and murine airway epithelia, and the tropism of AAV5 may be useful to target cells that are not permissive for AAV2. PMID- 10729160 TI - A block to human immunodeficiency virus type 1 assembly in murine cells. AB - Human immunodeficiency virus type 1 (HIV-1) does not replicate in murine cells. We investigated the basis of this block by infecting a murine NIH 3T3 reporter cell line that stably expressed human CD4, CCR5, and cyclin T1 and contained a transactivatable HIV-1 long terminal repeat (LTR)-green fluorescent protein (GFP) cassette. Although the virus entered efficiently, formed provirus, and was expressed at a level close to that in a highly permissive human cell line, the murine cells did not support M-tropic HIV-1 replication. To determine why the virus failed to replicate, the efficiency of each postentry step in the virus replication cycle was analyzed using vesicular stomatitis virus G pseudotypes. The murine cells supported reverse transcription and integration at levels comparable to those in the human osteosarcoma-derived cell line GHOST.R5, and human cyclin T1 restored provirus expression, consistent with earlier findings of others. The infected murine cells contained nearly as much virion protein as did the human cells but released less than 1/500 the amount of p24(gag) into the culture medium. A small amount of p24(gag) was released and was in the form of fully infectious virus. Electron microscopy suggested that aberrantly assembled virion protein had accumulated in cytoplasmic vesicular structures. Virions assembling at the cell membrane were observed but were rare. The entry of M tropic JR.FL-pseudotyped reporter virus was moderately reduced in the murine cells, suggesting a minor reduction in coreceptor function. A small reduction in the abundance of full-length viral mRNA transcripts was also noted; however, the major block was at virion assembly. This could have been due to a failure of Gag to target to the cell membrane. This block must be overcome before a murine model for HIV-1 replication can be developed. PMID- 10729161 TI - Identification of additional coat-scaffolding interactions in a bacteriophage P22 mutant defective in maturation. AB - Scaffolding proteins play a critical role in the assembly of certain viruses by directing the formation and maturation of a precursor capsid. Using electron cryomicroscopy difference mapping, we have identified an altered arrangement of a mutant scaffolding within the bacteriophage P22 procapsid. This mutant scaffolding allows us to directly visualize scaffolding density within the P22 procapsid. Based on these observations we propose a model for why the mutant prevents scaffolding release and capsid maturation. PMID- 10729162 TI - Measles virus-induced disruption of the glial-fibrillary-acidic protein cytoskeleton in an astrocytoma cell line (U-251). AB - A recombinant measles virus which expresses enhanced green fluorescent protein (MVeGFP) has been used to infect two astrocytoma cell lines (GCCM and U-251) to study the effect of virus infection on the cytoskeleton. Indirect immunocytochemistry was used to demonstrate the cellular localization of the cytoskeletal components. Enhanced green fluorescent protein autofluorescence was used to identify measles virus-infected cells. No alteration of the actin, tubulin, or vimentin components of the cytoskeleton was observed in either cell type, whereas a disruption of the glial-fibrillary-acidic protein filament (GFAP) network was noted in MVeGFP-infected U-251 cells. The relative amounts of GFAP present in infected and uninfected U-251 cells were quantified by image analysis of data sets obtained by confocal microscopy by using vimentin, another intermediate filament on which MVeGFP has no effect, as a control. PMID- 10729163 TI - Induction of a novel cellular homolog of interleukin-10, AK155, by transformation of T lymphocytes with herpesvirus saimiri. AB - Although herpesvirus saimiri-transformed T lymphocytes retain multiple normal T cell functions, only a few changes have been described. By subtractive hybridization, we have isolated a novel cellular gene, ak155, a sequence homolog of the interleukin-10 gene. Specifically herpesvirus saimiri-transformed T cells overexpress ak155 and secrete the protein into the supernatant. In other T-cell lines and in native peripheral blood cells, but not in B cells, ak155 is transcribed at low levels. AK155 forms homodimers similarly to interleukin-10. As a lymphokine, AK155 may contribute to the transformed phenotype of human T cells after infection by herpesvirus saimiri. PMID- 10729164 TI - Mutation analysis of the GDD sequence motif of a calicivirus RNA-dependent RNA polymerase. AB - The RNA-dependent RNA polymerase from rabbit hemorrhagic disease virus, a calicivirus, is known to have a conserved GDD amino acid motif and several additional regions of sequence homology with all types of polymerases. To test whether both aspartic acid residues are in fact involved in the catalytic activity and metal ion coordination of the enzyme, several defined mutations have been made in order to replace them by glutamate, asparagine, or glycine. All six mutant enzymes were produced in Escherichia coli, and their in vitro poly(U) polymerase activity was characterized. The results demonstrated that the first aspartate residue was absolutely required for enzyme function and that some flexibility existed with respect to the second, which could be replaced by glutamate. PMID- 10729165 TI - Patterns of genomic sequence diversity among their simian immunodeficiency viruses suggest that L'Hoest monkeys (Cercopithecus lhoesti) are a natural lentivirus reservoir. AB - Recently, we described a novel simian immunodeficiency virus (SIVlhoest) from a wild-caught L'Hoest monkey (Cercopithecus lhoesti) from a North American zoo. To investigate whether L'Hoest monkeys are the natural host for these viruses, we have screened blood samples from 14 wild animals from the Democratic Republic of Congo. Eight (57%) were found to be seropositive for SIV. Nearly full-length genome sequences were obtained for SIV isolates from three of these monkeys and compared to the original isolate and to other SIVs. The four samples of SIVlhoest formed a distinct cluster in phylogenetic trees. Two of these isolates differed on average at only about 5% of nucleotides, suggesting that they were epidemiologically linked; otherwise, the SIVlhoest isolates differed on average by 18%. Both the level of diversity and the pattern of its variation along the genome were very similar to those seen among isolates of SIVagm from vervet monkeys, pointing to similarities in the nature of, and constraints on, SIV evolution in these two species. Discordant phylogenetic relationships among the SIVlhoest isolates for different genomic regions indicated that mosaic viruses have been generated by recombination, implying that individual monkeys have been coinfected by more than one strain of SIV. Taken together, these observations provide strong evidence that L'Hoest monkeys constitute a natural reservoir for SIV. PMID- 10729166 TI - Ultrastructural organization of recombinant Marburg virus nucleoprotein: comparison with Marburg virus inclusions. AB - HeLa cells expressing the recombinant Marburg virus (MBGV) nucleoprotein (NP) have been studied by immunoelectron microscopy. It was found that MBGV NPs assembled into large aggregates which were in close association with membranes of the rough endoplasmic reticulum. Further analysis of these aggregates revealed that NPs formed tubule-like structures which were arranged in a hexagonal pattern. A similar pattern of preformed nucleocapsids was detected in intracellular inclusions induced by MBGV infection. Our data indicated that MBGV NP is able to form nucleocapsid-like structures in the absence of the authentic viral genome and other nucleocapsid-associated proteins. PMID- 10729167 TI - Control of Sindbis virus infection by antibody in interferon-deficient mice. AB - Antibodies clear Sindbis virus from infected animals through an unknown mechanism. To determine whether interferon-induced pathways are required for this clearance, we examined mice which are unable to respond to alpha/beta interferon or gamma interferon. Although extremely susceptible to infection, such mice survived and completely cleared virus if antibodies against Sindbis virus were given. PMID- 10729168 TI - Cell-to-cell spread of wild-type herpes simplex virus type 1, but not of syncytial strains, is mediated by the immunoglobulin-like receptors that mediate virion entry, nectin1 (PRR1/HveC/HIgR) and nectin2 (PRR2/HveB). AB - The immunoglobulin-like receptors that mediate entry of herpes simplex virus type 1 (HSV-1) into human cells were found to mediate the direct cell-to-cell spread of wild-type virus. The receptors here designated Nectin1alpha and -delta and Nectin2alpha were originally designated HIgR, PRR1/HveC, and PRR2alpha/HveB, respectively. We report the following. (i) Wild-type HSV-1 spreads from cell to cell in J cells expressing nectin1alpha or nectin1delta but not in parental J cells that are devoid of entry receptors. A monoclonal antibody to nectin1, which blocks entry, also blocked cell-to-cell spread in nectin1-expressing J cells. Moreover, wild-type virus did not spread from a receptor-positive to a receptor negative cell. (ii) The antibody to nectin1 blocked transmission of wild-type virus in a number of human cell lines, with varying efficiencies, suggesting that nectin1 is the principal mediator of wild-type virus spread in a variety of human cell lines. (iii) Nectin1 did not mediate cell fusion induced by the syncytial strains HSV-1(MP) and HFEM-syn. (iv) Nectin2alpha could serve as a receptor for spread of a mutant virus carrying the L25P substitution in glycoprotein D, but not of wild-type virus, in agreement with its ability to mediate entry of the mutant but not of wild-type virus. PMID- 10729169 TI - Efficient incorporation of HLA class II onto human immunodeficiency virus type 1 requires envelope glycoprotein packaging. AB - HLA class II DR is one of the most abundant cell surface proteins incorporated onto human immunodeficiency virus type 1 (HIV-1) during budding. The mechanism for HLA class II protein incorporation is not known and may involve a viral protein. To determine whether Env affects HLA class II protein incorporation, HIV 1 virions, either with or without Env on their surface, were produced from HLA class II-expressing cells and analyzed by whole-virus immunoprecipitation with antisera against HLA class II proteins. HLA class II proteins were detected on virions only when wild-type Env was incorporated, while similar experiments showed that HLA class I proteins were incorporated independent of Env packaging. Therefore, the packaging of HIV-1 Env protein is required for the efficient incorporation of HLA class II but not class I proteins into the virion. Analysis of two Env mutants revealed that the presence of a 43-amino-acid sequence between amino acids 708 and 750 in the gp41(TM) cytoplasmic tail was required for efficient incorporation of HLA class II proteins. These data show that HIV-1 actively incorporates HLA class II proteins in a process that, either directly or indirectly, requires Env. PMID- 10729171 TI - An antiviral compound that blocks structural transitions of poliovirus prevents receptor binding at low temperatures. AB - Drugs such as WIN51711 that inhibit picornavirus replication are thought to block poliovirus infectivity by binding to the capsid and preventing structural transitions required for uncoating. We examined the activity of WIN51711 at temperatures where capsid flexibility is thought to be decreased. Below 37 degrees C, WIN51711 inhibits the binding of wild-type poliovirus to cells but does not affect the binding of a poliovirus mutant which is believed to undergo structural transitions more readily. These results suggest that the poliovirus capsid must undergo structural changes to bind to its cellular receptor. PMID- 10729170 TI - Naturally occurring TAP-dependent specific T-cell tolerance for a variant of an immunodominant retroviral cytotoxic T-lymphocyte epitope. AB - Upon immunization and restimulation with tumors induced by the endogenous AKR/Gross murine leukemia virus (MuLV), C57BL/6 mice generate vigorous H-2K(b) restricted cytotoxic T-lymphocyte (CTL) responses to a determinant (KSPWFTTL) derived from the p15E transmembrane portion of the viral envelope glycoprotein. By contrast, the highly homologous determinant RSPWFTTL, expressed by tumor cells induced by Friend/Moloney/Rauscher (FMR) MuLV, is not immunogenic, even when presented to the immune system as vaccinia virus-encoded cytosolic or endoplasmic reticulum (ER)-targeted minigene products. Such minigene products are usually highly immunogenic since they bypass the need for cells to liberate the peptide or transport the peptide into the ER by the transporter associated with antigen processing (TAP). Using KSPWFTTL-specific CTLs that cross-react with RSPWFTTL, we previously demonstrated that presentation of RSPWFTTL from its natural viral gene product is TAP dependent. Here, we show first that C57BL/6 mice express mRNA encoding RSPWFTTL but not KSPWFTTL and second that the ER-targeted RSPWFTTL minigene product is highly immunogenic in C57BL/6 mice with a targeted deletion in TAP1. These findings provide the initial demonstration of TAP-dependent tolerance induction to a specific self peptide and demonstrate that this contributes to the differential recognition of RSPWFTTL and KSPWFTTL by C57BL/6 mice. PMID- 10729172 TI - Personal and professional connections: the power of relationships. PMID- 10729173 TI - The good neighbor. PMID- 10729175 TI - Descriptive designs: the case for case studies and case series in research. PMID- 10729174 TI - What my daughter taught me about becoming a WOC nurse. PMID- 10729176 TI - Thriving and surviving in home care and skilled nursing facilities under the Balanced Budget Act of 1997. AB - The Balanced Budget Act of 1997 (BBA 97) contains the most dramatic changes to the Medicare program since its genesis nearly 35 years ago. To remain financially viable under the cost-cutting measures mandated in this Act, hospitals, home health agencies, skilled nursing facilities, and their employees must have a working knowledge of its contents. In addition, the patients served by these health care providers must have well documented and positive health outcomes, and they must be satisfied with the care and service they receive. Nevertheless, merely understanding the changes mandated by BBA 97 is not sufficient for success; clinicians also must develop innovative solutions to the hurdles the Act erects and quickly integrate them into daily practice. Issues of payment and reimbursement have everything to do with the delivery of today's patient care, regardless of the setting where it is delivered. BBA 97 offers special opportunities to wound, ostomy, and continence care clinicians. PMID- 10729177 TI - Non-elastic compression: an alternative in management of chronic venous insufficiency. AB - Non-elastic compression has been used to treat venous insufficiency of the lower extremities for more than 150 years. The best and most recognized example is the Unna boot. When compared with other dressings, the Unna boot has performed as well as or better than other forms of compression. While the Unna boot is used worldwide, a 3- or 4-layer dressing has emerged as the dressing of choice in treating severe chronic venous insufficiency in the United States and English speaking European countries. In the United States, non-elastic compression can also be applied as a CircAid legging. This semirigid support has been compared with heavyweight class 3 below-knee medical stockings. At 2 and 6 hours after application, inelastic compression maintained limb size and reduced venous volume better than did stockings. At 6 hours, the ejection fraction of the calf muscle pump was increased and venous filling index significantly improved with inelastic compression compared with stockings. Comparison of elastic stockings with short stretch bandages has also been completed. The short-stretch bandage was found to be similar but not identical to the semirigid inelastic support device. The studies have shown that venous filling index improved by short-stretch bandaging and that venous reflux time was prolonged more by the short-stretch bandages than by stockings. The findings of these studies demonstrate that the inelastic support dressing mimics the action of the Unna boot in providing counter-pressure to perforating vein outflow. This may improve cutaneous and subcutaneous microcirculation in a manner similar to perforating vein surgery, which has been shown to accelerate leg ulcer healing. PMID- 10729178 TI - Comparison of the nutritional composition of diets of persons with fecal incontinence and that of age- and gender-matched controls. AB - PURPOSE: In our clinical and research experience, persons with fecal incontinence anecdotally report altered diet intake to avoid incontinence. The purpose of this study was to compare the dietary intake of 39 persons with fecal incontinence living in the community with that of age- and gender-matched control subjects who had normal bowel function. The diets of both groups were compared with recommended dietary allowances (RDAs) for their constituent nutrients. METHODS: Subjects prospectively recorded the type, amount, and method of preparing all foods and beverages ingested for 8 consecutive days. Diet records were analyzed using the Nutritionist IV software program. MAIN OUTCOME MEASURES: The main outcome measures were the amounts and percentage of the RDAs of macronutrients and micronutrients in the subjects' diets. RESULTS: There were no significant differences in the intake of total kilocalories, protein, fat, dietary fiber, caffeine, or lactose by the fecal incontinence and control groups. The fecal incontinence group had a greater intake of carbohydrates, manganese, and vitamin B(1) compared with the control group. Diets of both groups exceeded 100% of the RDA for protein, phosphorus, iron, sodium, potassium, Vitamins B(1), B(2), B(3), B(12), and C and folate. Diets of both groups had less than 50% of the RDA for biotin, chromium, copper, and manganese but did not differ significantly. The percentages of the RDA for calcium and vitamin D were 84% +/- 6% and 56% +/- 8% for the fecal incontinence group and 90 +/- 8 (P =.6) and 69 +/- 11 for the control group (P =.4). CONCLUSIONS: The diets of persons with fecal incontinence were similar to those of control subjects with normal bowel function. Both the fecal incontinence and control groups may improve their nutritional patterns by lowering sodium and protein intake and increasing dietary fiber and monounsaturated fat intake. Calcium and vitamin D supplementation may improve dietary deficiencies and lower disease risks. Including a nutritional assessment and consultation in the care of persons with fecal incontinence to improve their general health and prevent disease is recommended, but consideration must be given to altered diet patterns perceived by the patient to prevent fecal incontinence. PMID- 10729179 TI - Diaries as a data collection strategy. PMID- 10729180 TI - The RU-3 project: research utilization in practice. PMID- 10729181 TI - Continence for women: evaluation of AWHONN's third research utilization project. AB - OBJECTIVE: To develop an evidence-based protocol for initial evaluation and treatment of urinary incontinence and to design procedures that would facilitate the protocol's implementation into clinical practice. DESIGN: Descriptive report of the Association of Women's Health, Obstetric and Neonatal Nurses (AWHONN) Continence for Women Project. SETTING: Twenty-one public, private, and other women's health sites. Participants: Women in ambulatory care settings (N = 1474) provided demographic statistics. METHODS: The protocol was developed, sites were selected, site coordinator training was provided, data collection was facilitated by project-specific teleforms, and the overall process was evaluated by the science team. MAIN OUTCOME MEASURES: Site representation, patient representation, site coordinator feedback on the training program, and site coordinator experience during project implementation. RESULTS: The process yielded a representative mix of site and patient diversity appropriate for testing of the protocol. Site coordinators felt well-prepared to implement the protocol and experienced increased professional satisfaction because of therapeutic benefits achieved for patients and positive collaboration with physicians. CONCLUSIONS: The Continence for Women Project demonstrated the potential for developing and testing evidence-based protocols for clinical practice when the resources of an organization such as AWHONN and the research community are combined. PMID- 10729182 TI - Continence for women: a test of AWHONN's evidence-based protocol in clinical practice. AB - OBJECTIVE: To test the effectiveness of an evidence-based protocol for urinary incontinence in increasing identification of women with the condition and improving their outcomes. DESIGN: Prospective formative evaluation study. SETTING: Twenty-one public, private, and other women's health care sites. PARTICIPANTS: Women in ambulatory care settings (N = 1474) provided descriptive statistics. Clinical outcomes were tested in 132 cases for whom pre- and posttreatment data were available. INTERVENTIONS: Standardized screening and baseline follow-up forms were used to minimize time burden on clinicians; bladder and pelvic floor muscle training materials were provided to clinicians for distribution. MAIN OUTCOME MEASURES: Self-reported frequency, volume, and quality of life related to incontinence and cost of self-management were used to assess protocol effectiveness. RESULTS: Frequency of incontinence episodes, estimated volume lost per episode, and the cost of self-management decreased. Quality of life improved, as reflected in decreased bother attributed to incontinence and in the number of women avoiding activities such as shopping, exercising, or travel because of incontinence. CONCLUSIONS: This simple program of pelvic floor muscle and bladder training, as it has been systematically implemented in a variety of ambulatory women's health care settings, has benefitted women's continence status. The results of this project strongly support widespread application. PMID- 10729183 TI - Options for managing an open wound with draining enterocutaneous fistula. PMID- 10729184 TI - Clinician's desk reference: assessment, treatment and management of urinary incontinence. PMID- 10729185 TI - Redox regulatory mechanisms of cellular signal transduction. PMID- 10729186 TI - Effects of ionizing radiation in targeted and nontargeted cells. PMID- 10729187 TI - Detection of oxidative base DNA damage by a new biochemical assay. AB - Reactive oxygen species (ROS) damage DNA which appears to represent the major target involved in mutagenesis, carcinogenesis, and aging cell responses. Various DNA modifications are generated by ROS, but 8-hydroxy-2'-deoxyguanosine (8-oxoG) has retained a lot of attention in the last few years. Therefore, numerous methods have been developed to detect and quantify the extent of 8-oxoG in DNA, most of them requiring a significant amount of DNA that might be limiting in the case of biological samples. 8-oxoG is repaired in Escherichia coli by a specific glycosylase, the Fpg (formamidopyrimidine DNA glycosylase) protein, in a reaction that requires a covalent intermediate favored under reducing conditions. We set up a new assay based on the capture of plasmid DNA into sensitized microplate wells. DNA damaged by photoactivation of methylene blue was adsorbed on a polylysine-treated plastic well. Then the Fpg protein was added, allowed to fix on the damage by taking advantage of minimized glycosylase activity at low temperature and the reductive trapping of the covalent intermediate, yielding to a stable DNA-protein interaction. The trapped protein was subsequently recognized by a specific antibody. A secondary antibody coupled with horseradish peroxidase was used to detect the complex and the measurement was carried out by chemiluminescence. This new assay offers various potentialities, specifically in the field of technology of ROS producers. PMID- 10729188 TI - Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: high-yield expression and purification of human P-glycoprotein. AB - Utilizing human P-glycoprotein (P-gp), we investigated methods to enhance the heterologous expression of ATP-binding cassette transporters in Saccharomyces cerevisiae. Human multidrug resistance gene MDR1 cDNA was placed in a high-copy 2 mu yeast expression plasmid under the control of the inducible GAL1 promoter or the strong constitutive PMA1 promoter from which P-gp was expressed in functional form. Yeast cells expressing P-gp were valinomycin resistant. Basal ATPase activity of P-gp in yeast membranes was 0. 4-0.7 micromol/mg/min indicating excellent functionality. P-glycoprotein expressed in the protease-deficient strain BJ5457 was found in the plasma membrane and was not N-glycosylated. By use of the PMA1 promoter, P-gp could be expressed at 3% of total membrane protein. The expression level could be further enhanced to 8% when cells were grown in the presence of 10% glycerol as a chemical chaperone. Similarly, glycerol enhanced protein levels of P-gp expressed under control of the GAL1 promoter. Glycerol was demonstrated to enhance posttranslational stability of P-gp. Polyhistidine-tagged P-gp was purified by metal affinity chromatography and reconstituted into proteoliposomes in milligram quantities and its ATPase activity was characterized. Turnover numbers as high as 12 s(-1) were observed. The kinetic parameters K(MgATP)(M), V(max), and drug activation were dependent on the lipid composition of proteoliposomes and pH of the assay and were similar to P-gp purified from mammalian sources. In conclusion, we developed a system for cost effective, high-yield, heterologous expression of functional P-gp useful in producing large quantities of normal and mutant P-gp forms for structural and mechanistic studies. PMID- 10729189 TI - Cloning and sequencing of glycogen metabolism genes from Rhodobacter sphaeroides 2.4.1. Expression and characterization of recombinant ADP-glucose pyrophosphorylase. AB - A 6-kb DNA fragment of the Rhodobacter sphaeroides 2.4.1 glg operon was cloned from a genomic library using a polymerase chain reaction probe coding for part of the ADP-glucose pyrophosphorylase (glgC) gene. The DNA fragment was sequenced and found to harbor complete open reading frames for the glgC and glgA (glycogen synthase) genes and partial sequences corresponding to glgP (glycogen phosphorylase) and glgX (glucan hydrolase/transferase) genes. The genomic fragment also contained an apparent truncated sequence corresponding to the C terminus of the glgB gene (branching enzyme). The presence of active branching enzyme activity in crude sonicates of Rb. sphaeroides cells indicates that the genome contains a full-length glgB at another location. The structure of this operon in relation to other glg operons is further discussed. The deduced sequence of the ADP-glucose pyrophosphorylase enzyme is compared to other known ADP-glucose pyrophosphorylase sequences and discussed in relation to the allosteric regulation of this enzyme family. The glgC gene was subcloned in the vector pSE420 (Invitrogen) for high-level expression in E. coli. The successful overexpression of the recombinant enzyme allowed for the purification of over 35 mg of protein from 10 g of cells, representing a dramatic improvement over enzyme isolation from the native strain. The recombinant enzyme was purified to near homogeneity and found to be physically, immunologically, and kinetically identical to the native enzyme, verifying the fidelity of the cloning step. PMID- 10729190 TI - Adaptation of protein carbonyl detection to the requirements of proteome analysis demonstrated for hypoxia/reoxygenation in isolated rat liver mitochondria. AB - The key technique in proteome analysis is high-resolution two-dimensional (2D) electrophoretic separation of proteins from biological samples. This method combines isoelectric focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Derivatization of protein carbonyls with 2, 4 dinitrophenylhydrazine (DNPH) and subsequent anti-dinitrophenyl (DNP) immunoblotting is widely used for the detection of oxidatively modified proteins. In previous studies on adapting this method to 2D electrophoresis the derivatization step was carried out before and after the 2D procedure, resulting in an altered spot pattern and high background staining, respectively. The aim of the present experiments was to develop a method for protein derivatization with DNPH between the IEF and the SDS-PAGE steps. Mitochondria were exposed to 10 min hypoxia and 5 min reoxygenation. After IEF using immobilized pH gradients the gel strips were incubated in DNPH/trifluoroacetic acid/SDS for 20 min and neutralized, and SDS-PAGE was performed. Proteins were either stained with Coomassie dye or subjected to Western blotting using anti-DNP IgG. Gels and blots were scanned and matched to a master gel, and the relative carbonyl content of each spot was calculated and compared for five experiments. Importantly, the spot patterns in DNPH-treated and untreated gels were not different. Protein carbonyls could be detected in 59 of the 125 matched spots. Although there was no significant increase in the total protein carbonyl content after hypoxia/reoxygenation, eighteen 2D spots exhibited an increase in carbonyl content. However, most protein spots did not show a change or even a decline (4 spots) in protein carbonyls. PMID- 10729191 TI - Development of a toxicological gene array and quantitative assessment of this technology. AB - High-density arrays of DNA bound to solid substrates offer a powerful approach to identifying changes in gene expression in response to toxicants. While DNA arrays have been used to explore qualitative changes in gene regulation, less attention has focused on the quantitative aspects of this technology. Arrays containing expressed sequence tags for xenobiotic metabolizing enzymes, proteins associated with glutathione regulation, DNA repair enzymes, heat shock proteins, and housekeeping genes were used to examine gene expression in response to beta naphthoflavone (beta-NF). Upregulation of cytochrome P4501a1 (Cyp1a1) and 1a2 in mouse liver was maximal 8 h after beta-NF administration. Significant upregulation of Cyp1a2 was noted at beta-NF doses as low as 0.62 and 1.2 mg/kg when gene expression was measured by microarray or Northern blotting, respectively. Maximal Cyp1a2 induction is 5-fold by Northern analysis and 10-fold by microarray. Induction of Cyp1a1 was 15- and 20-fold by Northern and microarray analysis, respectively. The coefficient of variation for spot to spot and slide to slide comparisons was <15%; this variability was smaller than interanimal variability (18-60%). Comparison of mRNA expression in control animals indicated that there are differences in labeling/detection associated with Cy3/Cy5 dyes; accordingly, experiments must include methods for establishing baseline signals for all genes. We conclude that the dynamic range and sensitivity of DNA microarrays on glass slides is comparable to Northern blotting analysis and that variability of the data introduced during spotting and hybridization is less than the interanimal variability. PMID- 10729192 TI - Determination of the chelatable iron pool of single intact cells by laser scanning microscopy. AB - We have previously established a method of detecting intracellular chelatable iron in viable cells based on digital fluorescence microscopy. To quantify cellular chelatable iron, it was crucial to determine the intracellular indicator concentration. In the present study, we therefore adapted the method to confocal laser scanning microscopy, which should allow the determination of the indicator concentration on the single-cell level. The fluorescent heavy-metal indicator phen green SK (PG SK), the fluorescence of which is quenched by iron, was loaded into cultured rat hepatocytes. The hepatocellular fluorescence increased when cellular chelatable iron available to PG SK was removed from the probe by an excess of the membrane-permeable transition metal chelator 2,2'-dipyridyl (2, 2' DPD, 5 mM). We optimized the scanning parameters for quantitatively recording changes in fluorescence and determined individual intracellular PG SK concentrations from the unquenched cellular fluorescence (after 2,2'-DPD) compared with PG SK standards in a "cytosolic" medium. An ex situ calibration method based on laser scanning microscopy was set up to determine the concentration of cellular chelatable iron from the increase of PG SK fluorescence after addition of 2,2'-DPD (5 mM). As the stoichiometry of the PG SK:Fe(2+) complex was 3:1 as long as PG SK was not limiting, cellular chelatable iron was calculated directly from absolute changes in cellular fluorescence. Using this method, we found 2.5 +/- 2.2 microM chelatable iron in hepatocytes. This method makes it possible to determine the pool of chelatable iron in single vital cells independently of cellular differences (e.g., dye loading, cell volume) in heterogeneous cell populations. PMID- 10729193 TI - Molecular characterization and physiological role of ascorbate peroxidase from halotolerant Chlamydomonas sp. W80 strain. AB - A cDNA clone encoding an ascorbate peroxidase was isolated from the cDNA library from halotolerant Chlamydomonas W80 by a simple screening method based on the bacterial expression system. The cDNA clone contained an open reading frame encoding a mature protein of 282 amino acids with a calculated molecular mass of 30,031 Da, preceded by the chloroplast transit peptide consisting of 37 amino acids. In fact, ascorbate peroxidase was localized in the chloroplasts of Chlamydomonas W80 cells; the activity was detected in the stromal fraction but not in the thylakoid membrane. The deduced amino acid sequence of the cDNA showed 54 and 49% homology to chloroplastic and cytosolic ascorbate peroxidase isoenzymes of spinach leaves, respectively. The enzyme from Chlamydomonas W80 cells was purified to electrophoretic homogeneity. The molecular properties of the purified enzyme were similar to those of the other algal ascorbate peroxidases rather than those of ascorbate peroxidases from higher plants. The enzyme was relatively stable in ascorbate-depleted medium compared with the chloroplastic ascorbate peroxidase isoenzymes of higher plants. The presence of NaCl (3%) as well as of beta-d-thiogalactopyranoside was needed for the expression of Chlamydomonas W80 ascorbate peroxidase in Escherichia coli. PMID- 10729195 TI - Bradyrhizobium japonicum isocitrate dehydrogenase exhibits calcium-dependent hysteresis. AB - Bradyrhizobium japonicum NADP(+)-dependent isocitrate dehydrogenase was purified both from cultured cells and from the symbiotic form of the bacteria and was found to be identical in terms of N-terminal amino acid sequence, kinetics, and physicochemical properties. Magnesium and glycerol were absolute requirements for maintaining enzyme activity. The N-terminal amino acid sequence of the enzyme was more similar to the sequences from soybean and yeast than to other bacterial sequences. There was no immunological cross-reaction of antibodies from B. japonicum isocitrate dehydrogenase to extracts of soybean, pea, or Escherichia coli, but there was detectable, although weak, cross-reaction of antibodies from E. coli with the B. japonicum enzyme. B. japonicum isocitrate dehydrogenase displayed strong inhibition by NADH, indicating that during symbiotic nitrogen fixation the enzyme activity would be markedly reduced in planta. The enzyme displayed a calcium-dependent hysteresis, with a pronounced lag lasting as long as 2 min. Hysteresis was evident at concentrations of magnesium less than 0.5 mM and calcium greater than 1 microM. The hysteresis could be alleviated by excess magnesium or by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. The results suggest two roles for magnesium during catalysis; one magnesium may be needed to convert the enzyme into the steady-state form and the second needed for chelation of isocitrate for catalysis. The calcium-dependent hysteretic behavior of B. japonicum NADP(+)-isocitrate dehydrogenase suggested that this metal could serve as an intracellular regulator during symbiosis. PMID- 10729194 TI - Enzymes of adenylate metabolism and their role in hibernation of the white-tailed prairie dog, Cynomys leucurus. AB - AMP deaminase (AMPD) and adenylate kinase (AK) were purified from skeletal muscle of the white-tailed prairie dog, Cynomus leucurus, and enzyme properties were assayed at temperatures characteristic of euthermia (37 degrees C) and hibernation (5 degrees C) to analyze their role in adenylate metabolism during hibernation. Total adenylates decreased in muscle of torpid individuals from 6.97 +/- 0. 31 to 4.66 +/- 0.58 micromol/g of wet weight due to a significant drop in ATP but ADP, AMP, IMP, and energy charge were unchanged. The affinity of prairie dog AMPD for AMP was not affected by temperature and did not differ from that of rabbit muscle AMPD, used for comparison. However, both prairie dog and rabbit AMPD showed much stronger inhibition by ions and GTP at 5 degrees C, versus 37 degrees C, and inhibition by inorganic phosphate, NH(4)Cl, and (NH(4))(2)SO(4) was much stronger at 5 degrees C for the prairie dog enzyme. Furthermore, ATP and ADP, which activated AMPD at 37 degrees C, were strong inhibitors of prairie dog AMPD at 5 degrees C, with I(50) values of 1 and 14 microM, respectively. ATP also inhibited rabbit AMPD at 5 degrees C (I(50) = 103 microM). Strong inhibition of AMPD at 5 degrees C by several effectors suggests that enzyme function is specifically suppressed in muscle of hibernating animals. By contrast, AK showed properties that would maintain or even enhance its function at low temperature. K(m) values for substrates (ATP, ADP, AMP) decreased with decreasing temperature, the change in K(m) ATP paralleling the decrease in muscle ATP concentration. AK inhibition by ions was also reduced at 5 degrees C. The data suggest that adenylate degradation via AMPD is blocked during hibernation but that AK maintains its function in stabilizing energy charge. PMID- 10729196 TI - Quantitative RT-PCR measurement of human cytochrome P-450s: application to drug induction studies. AB - A quantitative RT-PCR assay has been developed that is able to measure the mRNA content of the major human CYPs (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5). The technique is highly specific, reproducible, rapid, and sensitive enough to quantitate low and high abundant mRNAs. The PCR primers were selected to specifically match each CYP mRNA, to have a very close annealing temperature, and to render PCR products of similar sizes. The PCR conditions were designed to allow the simultaneous measurement of the various human liver CYPs in a single run. To achieve precise and reproducible quantitation of each cytochrome mRNA, a external standard (luciferase mRNA) is added to the probes to monitor the efficiency of the RT step. The degree of amplification is estimated using appropriate cDNA standards and quantitation of the amplified products by fluorescent measurement. This assay can be used to quantify the most relevant CYPs in human liver and cultured human hepatocytes. CYPs 3A4 and 2E1 were the most abundant mRNAs in human liver (2.5 and 1.7 x 10(8) molecules/microgram of total RNA respectively), whereas 1A1 and 2D6 were the least abundant isoforms (1.2 and 2.1 x 10(6) molecules/microgram of total RNA). A similar pattern was also found in short-term cultured human hepatocytes. This technique is also suitable for assessing CYP mRNA induction by xenobiotics. Cells exposed to 3 methylcholanthrene showed a characteristic increased expression of CYP1A2 and 1A1 mRNAs. Upon incubation with phenobarbital and rifampin (rifampicin), human hepatocytes increased CYP 2B6, 3A4, and 3A5 among others. PMID- 10729197 TI - Sandwich configuration of type I collagen suppresses progesterone production in primary cultured porcine granulosa cells by reducing gene expression of cytochrome P450 cholesterol side-chain cleavage enzyme. AB - When porcine granulosa cells were cultured on type I collagen (TIC)-coated dishes, progesterone was continuously secreted in the culture medium. However, when cells were overlaid with a TIC gel, progesterone production was decreased to 34% (day 3) and 16% (day 4) of the value measured for cells without the overlay. The effect of TIC gel overlay on cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), which catalyzes the conversion of cholesterol to pregnenolone and is thought to be the rate-limiting enzyme in the conversion of cholesterol to progesterone, was examined. P450scc gene expression in cells overlaid with a TIC gel was decreased to 62% (day 3) and 36% (day 4) of the value measured for cells without the overlay. Amounts of P450scc were also reduced in the cells overlaid with a TIC gel. When pregnenolone, the direct precursor of progesterone, was added to the culture medium, the increase in progesterone production by cells overlaid with a TIC gel was much greater than that for cells without a TIC gel and a statistical difference in progesterone production was no longer observed between the two groups of cells. Treatment of the cells with human FSH (hFSH) enhanced progesterone production in a dose-dependent manner, irrespective of the presence of a TIC gel overlay. Moreover, hFSH induced P450scc gene expression in cells with and without a TIC gel overlay. These results indicate that a TIC gel overlay reduces progesterone production in granulosa cells via the suppression of P450scc gene expression. This supports the possibility that the existence of a TIC gel on the apical side of granulosa cells prevents the spontaneous luteinization of granulosa cells cultured on TIC-coated dishes. The fact that hFSH overcomes the suppressive effect of the TIC gel overlay on progesterone production may explain the mechanism for the subtle rise in serum progesterone concentration in the late follicle phase of the "in vitro fertilization" program. PMID- 10729198 TI - Fluorescence and excitation Escherichia coli RecA protein spectra analyzed separately for tyrosine and tryptophan residues. AB - The method for separation of emission (EM) and excitation (EX) spectra of a protein into EM and EX spectra of its tyrosine (Tyr) and tryptophan (Trp) residues was described. The method was applied to analysis of Escherichia coli RecA protein and its complexes with Mg(2+), ATPgammaS or ADP, and single-stranded DNA (ssDNA). RecA consists of a C-terminal domain containing two Trp and two Tyr residues, a major domain with five Tyr residues, and an N-terminal domain without these residues (R. M. Story, I. T. Weber, and T. A. Steitz (1992) Nature (London) 355, 374-376). Because the fluorescence of Tyr residues in the C-terminal domain was shown to be quenched by energy transfer to Trp residues, Trp and Tyr fluorescence of RecA was provided by the C-terminal and the major domains, respectively. Spectral analysis of Trp and Tyr constituents revealed that a relative spatial location of the C-terminal and the major domains in RecA monomers was different for their complexes with either ATPgammaS or ADP, whereas this location did not change upon additional interaction of these complexes with ssDNA. Homogeneous (that is, independent of EX wavelength) and nonhomogeneous (dependent on EX wavelength) types of Tyr and Trp fluorescence quenching were analyzed for RecA and its complexes with nucleotide cofactors and ssDNA. The former was expected to result from singlet-singlet energy transfer from these residues to adenine of ATPgammaS or ADP. By analogy, the latter was suggested to proceed through energy transfer from high vibrational levels of the excited state of Trp and Tyr residues to the adenine. In this case, for correct calculation of the overlap integral, Trp and Tyr donor emission spectra were substituted by the spectral function of convolution of emission and excitation spectra that resulted in a significant increase of the overlap integral and gave an explanation of the nonhomogeneous quenching of Trp residues in the C-terminal domain. PMID- 10729199 TI - Characterization of nucleotide binding sites of the isolated H(+)-ATPase from spinach chloroplasts, CF(0)F(1). AB - Soluble purified CF(0)F(1) from chloroplasts was either oxidized or reduced and then incubated with [alpha-(32)P]ATP in the presence or in the absence of Mg(2+). Depending on the conditions of incubation, the enzyme showed different tight nucleotide binding sites. In the presence of EDTA, two sites bind [alpha (32)P]ATP from the reaction medium at different rates. Both sites promote ATP hydrolysis, since equimolar amounts of [alpha-(32)P]ATP and [alpha-(32)P]ADP are bound to the enzyme. In the presence of Mg(2+), only one site appears during the first hour of incubation, with characteristics similar to those described in the absence of Mg(2+). However, after this time a third site appears also permitting binding of ATP from the reaction medium, but in this case the bound ATP is not hydrolyzed. Covalent derivatization by 2-azido-[alpha-(32)P]ATP was used to distinguish between catalytic and noncatalytic sites. In the presence of Mg(2+), there are at least three distinct nucleotide binding sites that bind nucleotide tightly from the reaction medium: two of them are catalytic and one is noncatalytic. PMID- 10729200 TI - Rapid reactions of peroxynitrite with heme-thiolate proteins as the basis for protection of prostacyclin synthase from inactivation by nitration. AB - Prostacyclin (PGI(2)) synthase is a heme-thiolate (P450) protein which reacts with low levels of peroxynitrite (PN) under tyrosine nitration and inactivation. Studying heme proteins as models, we have found the heme-thiolate protein NADH-NO reductase (P450(NOR)) to be highly efficient in decomposing PN under concomitant nitration of phenol. The present study investigates two other P450 proteins, P450(BM-3) and chloroperoxidase, in order to test for the specific role of the thiolate ligand in the reaction with PN. A comparison with horseradish peroxidase and microperoxidase gives evidence of kinetic differences that classify heme thiolate proteins, but not other heme proteins, as effective inhibitors of PGI(2) synthase nitration and inactivation. P450(BM-3) with PN catalyzes phenol nitration and nitration of its own tyrosine below 10 microM PN, whereas chloroperoxidase and P450(NOR) at such concentrations also nitrate phenol but not enzyme-bound tyrosine residues. We conclude that heme-thiolate proteins in general exhibit high reactivity with PN and turnover, probably due to the special electronic structure of the presumed thiolate-ferryl intermediate. PMID- 10729202 TI - Redistribution of cyclic GMP in response to sodium butyrate in colon cells. AB - The effect of butyrate on the response to guanylin and Escherichia coli heat stable enterotoxin, STa, was assessed in T84 cells and Caco-2 cells, cultured colon cell lines possessing the guanylyl cyclase C which is the receptor for these peptides. Butyrate treatment of these cells resulted in an apparent increase in cyclic GMP (cGMP) accumulation when the cGMP content of cells and the supernatant medium was measured. Butyrate treatment did not change the guanylyl cyclase activity or (125)I-STa binding parameters in T84 cells, but the butyrate effect was completely blocked by cycloheximide. Butyrate did not have any effect on STa-stimulated cGMP accumulation in COS cells transfected with the human or porcine GC-C. Further experiments showed that butyrate treatment caused a large increase in the cGMP released into the culture medium, and in cells grown in polarized fashion in Transwell inserts, cGMP efflux was predominantly from the basolateral surface of the cell; intracellular cGMP was actually lowered by butyrate treatment. Exposure of T84 cells to butyrate had no effect on the disposition of cyclic AMP generated in response to forskolin. The effects of butyrate on cGMP were reversible within 24 h of butyrate withdrawal. In colon cells, butyrate treatment induced a previously undescribed, cGMP-specific efflux mechanism which lowered intracellular cGMP and elevated extracellular cGMP in response to peptide agonists such as guanylin and STa. PMID- 10729201 TI - DNA as a possible target for antitumor ruthenium(III) complexes. AB - The interaction of two experimental ruthenium(III)-containing antitumor complexes Na[trans-RuCl(4)(DMSO)(Im)] (NAMI) and dichloro(1,2 propylendiaminetetraacetate)ruthenium(III) (RAP)-with DNA was investigated through a number of spectroscopic and molecular biology techniques, including spectrophotometry, circular dichroism, gel shift analysis, and restriction enzyme inhibition. It was found that both complexes slightly alter DNA conformation, modify its electrophoretic mobility, and inhibit DNA recognition and cleavage by some restriction enzymes, though they were less effective than cisplatin in producing such effects. Notably, the effects produced by NAMI on DNA were much larger than those induced by RAP. Implications of these results for the mechanism of action of ruthenium(III) antitumor complexes are discussed. PMID- 10729203 TI - Manganese peroxidase isoenzymes produced by Pleurotus ostreatus grown on wood sawdust. AB - The white rot basidiomycete Pleurotus ostreatus produces two manganese peroxidase (MnP) isoenzymes when grown in solid stationary conditions on poplar sawdust, whereas a lower production of these same enzymes is observed on fir sawdust. Addition of Mn(2+) to poplar culture resulted in a threefold increase of MnP activity; the same addition to fir culture was able to increase tenfold the MnP production. The two MnP isoenzymes (MnP2 and MnP3) were purified from P. ostreatus poplar culture. The isoenzymes differ in their pI values, molecular masses, and N-terminal sequences. MnP3 has the same N-terminal sequence as that of a P. ostreatus MnP previously reported. Both isoenzymes exhibit Mn(2+) dependent and Mn(2+)-independent peroxidase activities when tested on phenolic substrates. The gene coding for the new isoenzyme MnP2 was cloned and sequenced and the promoter region analyzed. Furthermore, the chromosomal localization of all known P. ostreatus genes was determined. PMID- 10729204 TI - Post-transcriptional regulation of rat CYP2E1 expression: role of CYP2E1 mRNA untranslated regions in control of translational efficiency and message stability. AB - Altered expression of hepatic CYP2E1 by xenobiotic or physiological stimuli is largely mediated through post-transcriptional mechanisms that may include altered CYP2E1 mRNA translation and/or protein degradation. Examination of the polyribosomal distribution of rat hepatic P450 mRNAs indicated that, whereas nearly all of the CYP2B, CYP3A, and CYP4A mRNAs were recovered in the polysomal fractions, indicating active translation, approximately 30-40% of CYP2E1 mRNA was not associated with polysomes and therefore not actively engaged in protein synthesis. To examine the CYP2E1 mRNA molecule for sequences that might affect its translational efficiency, a series of CYP2E1 recombinant RNAs (rcRNAs) with modified 5' or 3' untranslated regions (UTRs) was translated in vitro using the rabbit reticulocyte lysate system. Deletion of most of the CYP2E1 5' UTR, which was predicted to contain secondary structure, increased in vitro CYP2E1 protein synthesis. Polysomal distribution analyses of 5'-modified rcRNAs demonstrated that, as seen for hepatic CYP2E1 mRNA, a substantial fraction of each CYP2E1 rcRNA was not associated with polysomes. The polysomal distribution analyses of the CYP2E1 rcRNAs also confirmed that the observed changes in CYP2E1 protein synthesis were associated with altered ribosomal loading. Deletion of the poly(A) tail, and partial or complete deletion of the 3' UTR, decreased CYP2E1 protein synthesis. These changes in protein synthesis were accompanied by increased degradation of the CYP2E1 rcRNAs. Incubation with translational inhibitors, but not increased levels of RNase inhibitor, decreased the degradation of the rcRNAs during in vitro translation. In conclusion, these studies suggest that secondary structure in the 5' UTR of CYP2E1 mRNA is at least partially responsible for the inefficient translation of this mRNA. The poly(A) tail and sequences contained within the 3' UTR appear to be important for protecting CYP2E1 mRNA from RNase activity associated with the translation machinery. PMID- 10729205 TI - Effect of retinoic acid on the enhancing effect of acetaldehyde on mouse type I collagen expression. AB - Acetaldehyde alone and retinoic acid alone have been shown to increase and decrease, respectively, collagen production by stellate cells in culture. In this study the effects of retinoic acid on alpha(1)(I) and alpha(2)(I) collagen expression and its influence on the enhancing effects of acetaldehyde were determined. Retinoic acid decreased the activation of the alpha(2)(I) collagen promoter and decreased the message of alpha(2)(I) collagen in cultured stellate cells, but had no effect on either the activation of the alpha(1)(I) collagen promoter or on the alpha(1)(I) collagen message. This depressant effect of retinoic acid was also evident in the transfected alpha(2)(I) collagen promoter mutated at the retinoic acid response element (RARE). The activation of the alpha(2)(I) collagen promoter by acetaldehyde was not decreased significantly by retinoic acid, but was suppressed by the retinoic acid receptor (RAR) selective retinoid SRI-6751-84. Retinoic acid, however, decreased the acetaldehyde-induced enhancement of the alpha(1)(I) and alpha(2)(I) collagen messages. Acetaldehyde also resulted in a decrease in RAR beta message and RARbeta protein. This study shows that retinoic acid depresses alpha(2)(I) collagen gene expression but that this effect is less pronounced when the expression of this collagen is enhanced by acetaldehyde, which also decreases RARbeta message and protein. Furthermore, the action of retinoic acid in inhibiting alpha(2)(I) collagen gene expression occurs at sites other than the RARE site. PMID- 10729206 TI - Identification of CYP2C9 as a human liver microsomal linoleic acid epoxygenase. AB - Leukotoxin (9,10-epoxy-12-octadecanoate) and isoleukotoxin (12, 13-epoxy-9 octadecenoate) are monoepoxides of linoleic acid, synthesized by a cytochrome P450 monooxygenase and possibly by an oxidative burst of inflammatory cells. Recent experiments in this laboratory have indicated that the toxicity of leukotoxin and isoleukotoxin is not due to these epoxides, but to the 9,10- and 12, 13-diol metabolites. Leukotoxin and isoleukotoxin are metabolized primarily by the soluble epoxide hydrolase to form leukotoxin diol. Investigations with recombinant cytochrome P450 enzymes have demonstrated that leukotoxin and isoleukotoxin can be formed by these enzymes. This study used a combination of experimental approaches to identify the major cytochrome P450 enzyme in human liver involved in linoleic acid epoxidation. The kinetic paramenters were determined; the K(m) of linoleic acid epoxidation by pooled human liver microsomes was 170 microM and the V(max) was 58 pmol/mg/min. Correlation analysis was performed using individual samples of human liver microsomes, and the best correlation of linoleic acid epoxidation activity was with tolbutamide hydroxylase activity, CYP2C9. Recombinant CYP2C9 was the most active in linoleic acid epoxygenation, and antibody and chemical inhibition also indicated the importance of CYP2C9. This enzyme, therefore, may serve as a therapeutic target in the treatment of inflammation in order to reduce the amount of circulating leukotoxin/isoleukotoxin and their related diols. PMID- 10729207 TI - Expression of human cytochrome P450 2B6 in Escherichia coli: characterization of catalytic activity and expression levels in human liver. AB - Expression of human cytochrome P450 (P450) 2B6 in Escherichia coli was achieved following supplementation of the expression medium with chloramphenicol. The recombinant protein was purified using Ni(2+)-nitrilotriacetate chromatography and was characterized with regard to its spectral properties and catalytic activities toward typical P450 substrates. The purified recombinant protein was also used to raise polyclonal antibodies in rabbits. Examination of a panel of human liver microsomal preparations revealed expression of P450 2B6 in most samples, with levels of <1 to 30 pmol 2B6/mg microsomal protein. Examination of purified P450 2B6 preparations revealed the presence of a protease-sensitive site located 126 residues away from the N-terminus. The identity of the cleavage boundary was verified by protein sequence analysis. Cleavage of P450 2B6 at that site results in the presence of a lower molecular weight fragment of approximately 35 kDa in purified preparations. An immunoreactive peptide of a similar molecular weight was consistently observed in some but not all human liver microsomal preparations suggesting cleavage at the same site. Examination of catalytic activities of the purified reconstituted protein indicated the potential utility of (S)-mephenytoin N-demethylation and testosterone 16beta hydroxylation as markers for P450 2B6. PMID- 10729208 TI - Oxidative dimerization of proteins: role of tyrosine accessibility. AB - PURPOSE: To investigate the importance of two possible mechanisms of tyrosine oxidation on the yield of protein dimerization. The model chosen is hen and turkey egg-white lysozymes, which differ by seven amino acids, among which one tyrosine is in the 3 position. MATERIALS AND METHODS: Aqueous solutions of proteins were oxidized by OH(*) or N(*)(3) free radicals produced by gamma or pulse irradiation in an atmosphere of N(2)O. Protein dimers were quantified by SDS-PAGE and reverse-phase HPLC. Dityrosines were identified by absorption and fluorescence. RESULTS: Using N(*)(3) free radicals, the initial yields of dimerization are equal to (8.6 +/- 0.7) x 10(-9) mol J(-1) for both proteins. Using OH(*) free radicals, they become equal to (1.23 +/- 0.1) x 10(-8) and (4.42 +/- 0.1) x 10(-8) mol J(-1) for hen and turkey egg-white lysozymes, respectively (gamma radiolysis). DISCUSSION. N(*)(3) radicals react primarily with tryptophan residues only. Tyrosine gets oxidized by intramolecular long-range electron migration, whereas OH(*) may react directly with tyrosines. We propose a low participation of Tyr3 in turkey protein in the intramolecular process, because Tyr3 is far from all tryptophans. On the other hand, Tyr3 is very accessible to solvent and in a flexible area; thus collisions with OH(*) could easily be followed by intermolecular dimerization. PMID- 10729209 TI - Synthesis and use of 3'-(azidoiodosalicyl) derivatives of 2', 5'-dideoxyadenosine as photoaffinity ligands for adenylyl cyclase. AB - 3'-[(4-Azidosalicyl)glycyl]-2',5'-dideoxyadenosine (1), 3'- [(4-azidosalicyl) gamma-aminobutyryl]-2',5'-dideoxyadenosine (2), and the (125)I-labeled mono- and diiodinated analogs of 1 were synthesized and tested as photoaffinity probes for adenylyl cyclases. Kinetics for inhibition of purified type I enzyme by 1 was noncompetitive with respect to Mn(*)5'-ATP in the absence of light, implying a P site mechanism of inhibition. In a UV-dependent manner both 1 and 2 and the iodinated derivative of 1 irreversibly inactivated membrane-bound and purified forms of recombinant type I bovine adenylyl cyclase expressed in ovarian cells of either the fall armyworm (Sf9) or Trichoplasia ni (High Five). Irreversible inactivation was independent of 5'-ATP and was prevented by 2', 5' dideoxyadenosine. Adenylyl cyclase, whether purified from bovine brain or in membranes from High Five cells expressing type I enzyme, when subjected to UV irradiation in the presence of (125)I-labeled 1 resulted in radioactive incorporation into protein migrating at approximately 116 kDa. The cross-linking of 1 and its iodinated derivative with adenylyl cyclase suggests potential for such compounds to be useful in structural studies of adenylyl cyclases or of other proteins for which adenine nucleosides are substrates or allosteric regulators. PMID- 10729212 TI - Editorial. PMID- 10729210 TI - Topoisomerase II poisoning and antineoplastic action by DNA-nonbinding diacetyl and dicarboxaldoxime derivatives of ferrocene. AB - Topoisomerase II is a major molecular target for a number of DNA-binding anticancer drugs. In the present study, we report topoisomerase II inhibition and anticancer activity by four substituted ferrocene derivatives which do not bind to DNA. The first derivative, acetyl-substituted ferrocene (monoacetylferrocene), showed a minor inhibition of topoisomerase II activity along with a consequent inhibition of cancer cell proliferation. The second derivative (diacetylferrocene) showed a higher potency of action compared to the monosubstituted derivative. The third and fourth derivatives, with mono- and disubstituted carboxaldoxime groups (ferrocenecarboxaldoxime and ferrocenedicarboxaldoxime), showed a higher anticancer action and stronger topoisomerase II inhibition. To understand their molecular mechanism of action, cleavage assays were carried out to monitor the drug-induced, topoisomerase II mediated DNA cleavage. The results show that diacetylferrocene and ferrocenedicarboxaldoxime could form an enzyme-drug-DNA ternary complex, called a "cleavage complex," resulting in DNA cleavage. These results along with those of an immunoprecipitation assay indicate that the two compounds interact with topoisomerase II alone and poison its activity by trapping the enzyme and enzyme cleaved DNA in the covalently closed cleavage complex. The formation of such a complex has numerous genetic implications, which ultimately results in neoplastic cell death. PMID- 10729211 TI - Superoxide dismutase reduces susceptibility to metronidazole of the pathogenic protozoan Entamoeba histolytica under microaerophilic but not under anaerobic conditions. PMID- 10729213 TI - Who believes in "communication"? The Norman Cousins Lecture, 1999. PMID- 10729214 TI - Increased natural killer-cell mobilization and cytotoxicity during marital conflict. AB - Natural killer (NK) cells are reproducibly mobilized into the circulation in response to intense physical exercise or acute psychological stress, and altered expression of adhesion molecules potentially contributes to NK-cell mobilization. Studies of leukocyte mobilization during acute stress have used psychological stressors which facilitate tight experimental control but have limited applicability to everyday life. We therefore used a laboratory model of marital conflict as an experientially meaningful acute stressor to elucidate relationships among conflict, cardiovascular reactivity, and altered leukocyte phenotype and function. Forty-one ethnically diverse, nondistressed, healthy married couples were asked to discuss a specific problem in their marriage for 15 min. Blood pressure and heart rate were measured before, during, and after the discussion, and blood was remotely drawn at the same time points to quantify numbers of specific leukocyte subsets, NK-cell adhesion molecule expression, and NK cytotoxicity. Couples responded to the conflict task with cardiovascular reactivity; increases in the percentages of circulating NK cells and CD8(+) T cells and decreases in the percentage of circulating CD4(+) T cells; decreases in the percentage of NK cells that express L-selectin; and increases in NK-cell cytotoxicity without a commensurate increase in per-cell cytotoxicity. Rapid downregulation or shedding of L-selectin (CD62L) from NK cells did not contribute to their mobilization during conflict. Instead, CD62L(-) NK cells were mobilized while CD62L(+) NK cells were selectively retained in the vascular marginating pool and/or in extravascular tissue. From a broader perspective, the data support the hypothesis that altered trafficking of specific leukocyte subsets is an integral component of the fight-or-flight response to an acute stressor. PMID- 10729215 TI - Involvement of granzyme B expression in the enhancement of natural killer activity by beta-endorphin. AB - Beta-Endorphin has been reported to enhance natural killer (NK) activity in vitro. However, few studies have examined the precise regulation of the cytolytic stage of NK cells. We therefore investigated the regulation by beta-endorphin of cytotoxicity-associated molecules such as granzyme B, perforin, and Fas ligand (FasL) in human CD16(+) NK cells. On semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay, the granzyme B mRNA level apparently increased in CD16(+) NK cells from high responding subjects having ratios >1.5 for the LU(30) ratio. An increase in intracellular granzyme B molecules was also detected in CD16(+) NK cells by flow cytometry. On the other hand, perforin and FasL appeared not to be involved in regulation by beta-endorphin. These findings suggest that up-regulation of granzyme B expression may be involved in the enhancement of NK activity by beta-endorphin. PMID- 10729216 TI - Social class, sex, and age differences in mucosal immunity in a large community sample. AB - There have been very few reports addressing levels and distribution of commonly used PNI measures in large community samples. In this study, we report such data for secretion rates of secretory immunoglobulin A (sIgA), as determined from saliva samples taken from 1971 subjects interviewed as part of the West of Scotland Twenty-07 survey of health in West Central Scotland. Univariate analyses of demographic variables found lower sIgA and salivary flow to be significantly related to poorer social class, increased age, and being female. Smokers also had lower sIgA but not lower salivary flow. Multivariate analysis showed that demographic variables were significant predictors of sIgA independently of each other and assay variation. Adding smoking status to the equation confirmed it as an independent predictor and also indicated that social class differences in sIgA are partly explicable in terms of smoking status. In view of reported associations between sIgA levels and stress, its role as a first line of mucosal defense, and its relevance to health, these first results from a large survey are of interest. Further work is now needed to explore which factors, including psychosocial ones, may be contributing to subgroup differences. PMID- 10729217 TI - Modulation of IL-6 production during the menstrual cycle in vivo and in vitro. AB - During the menstrual cycle (MC), premenopausal women experience changes in basal temperature and their physical condition and well-being. Premenopausal female patients with chronic inflammatory diseases demonstrate changes in disease activity during the MC. The study was initiated to explore reasons for these phenomena. The sex hormone-modulated lipopolysaccharide (LPS)-induced interleukin 6 (IL-6) secretion in a whole blood assay, serum IL-6 concentration, and serum sex hormone concentrations were studied throughout the MC in five healthy female subjects (median, 28 years; mean, 31.2+/-2.2 years, 26-38 years). Serum IL-6 concentration demonstrated a significant increase in the luteal phase of the MC and was elevated when serum dehydroepiandrosterone (DHEA) was low and vice versa. DHEA decreased LPS-induced IL-6 secretion at six of seven time points during the MC (DHEA, p = .047). In contrast, beta-estradiol and testosterone increased LPS induced IL-6 secretion in six of seven time points during the MC (significant for testosterone, p = .005). The study demonstrates oscillation of serum IL-6 concentration during the MC and the marked MC-dependent modulation of IL-6 secretion by sex hormones. These mechanisms may be involved in the changes in the basal temperature, the general condition, and, in patients with chronic inflammatory diseases, of disease activity during the MC. PMID- 10729218 TI - In vitro airway responsiveness of Flinders sensitive and resistant line rats. AB - Recently, we reported that freely moving Flinders sensitive line rats (FSL, selectively bred for their cholinergic hyperresponsiveness) are more susceptible to allergen-induced airway hyperresponsiveness than their control counterparts Flinders resistant line (FRL) rats. In this study the two Flinders lines were compared for responsiveness of excised tracheal and primary bronchial smooth muscle in vitro. FSL tissues were slightly but significantly more sensitive to cholinergic stimulation than FRL tissues (slightly lower EC(50) value for carbachol) but the FRL tissues were more responsive, exhibiting larger amplitude of response. Surprisingly, previous exposure to allergen challenge was accompanied by reduced in vitro responses to spasmogens in both rat lines. We conclude that FSL and FRL airways do not differ greatly with respect to sensitivity to cholinergic stimulation in vitro and that inflamed airways show reduced in vitro responses to spasmogens. The discrepancy between the in vivo and in vitro findings suggests that responsiveness of airway smooth muscle involves regulation from both proximal and distal sites. PMID- 10729219 TI - Dual renin gene targeting by Cre-mediated interchromosomal recombination. AB - This study describes a new approach to targeting clustered genes. Our study began with the establishment of two lines of mice carrying different mutations in either Ren1 or Ren2. These two genes, both encoding renin, span over 40 kb in tandem on chromosome 1. Each gene was mutated by gene targeting to contain loxP sites. These two mutants and Cre transgenic mice were mated to produce offspring carrying the mutant Ren1 and Ren2 genes, as well as the Cre transgene concurrently. Initially, two mutant Ren genes were located on separate chromosomes. Southern analysis of mice from the second generation revealed that the mutant Ren1 and Ren2 were interchromosomally recombined at the loxP sites to produce a new dually mutated allele on the chromosome at the rate of 9.6% (7/73). Thus, interchromosomal recombination can be efficiently programmed by mating as designed using the Cre-loxP system. PMID- 10729220 TI - Symmetric and asymmetric DNA methylation in the human IGF2-H19 imprinted region. AB - The two contiguous IGF2 (human insulin-like growth factor II) and H19 genes are reciprocally imprinted in both human and mouse. In most tissues, IGF2 is transcribed only from the paternal chromosome while H19 is transcribed only from the maternal allele. The presence of a differential methylation region (DMR) on the two parental alleles at the 5' flanking region of H19 has been proposed to constitute the gametic imprint, which controls the reciprocal allelic expression of the two genes. Using bisulfite genomic sequencing, we have assessed the methylation status of cytosine (including 154 CpG sites) in six CpG-rich regions of the human IGF2-H19 genes. In a CpG island near promoter P3 of the IGF2 gene, more than 99.8% of all cytosines were converted to thymidine by sodium bisulfite mutagenesis, indicating that none of the CpGs was methylated. In the IGF2 exon 8 9 region, mosaic methylation of 56 CpG sites was observed in fetal tissues and in adult blood DNA. In contrast to the mosaic methylation of IGF2, the allelic methylation of the human H19 DMR was uniform. In the CpG region located 2 kb upstream (-2362 to -1911) of the H19 transcription site, all 25 CpG sites were completely methylated on only one parental allele. Uniform allele-specific methylation was also observed in the CpG island proximal to the H19 promoter ( 711 to -290) with complete methylation of all 25 CpG sites in one parental allele. In contrast, the CpG region in the H19 promoter (-292 to +15) was mosaically methylated in all tissues. In addition, cytosine was methylated at three CpNpG and GpNpC sites on the top DNA strand and one CpNpG site on the bottom DNA strand from the fetal brain. The cytosines at CpG sites were methylated on both DNA strands (symmetric methylation) while cytosines at the CpNpG and GpNpC sites were methylated on only one DNA strand (asymmetric methylation). The asymmetric methylation was associated with tissue-specific disruption of H19 genomic imprinting in fetal brain. PMID- 10729221 TI - Gene structures and expression profiles of three human KCND (Kv4) potassium channels mediating A-type currents I(TO) and I(SA). AB - The four known members of the KCND/Kv4 channel family encode voltage-gated potassium channels. Recent studies provide evidence that members of the Kv4 channel family are responsible for native, rapidly inactivating (A-type) currents described in heart (I(TO)) and neurons (I(SA)). In this study, we cloned the human KCND1 cDNA, localized the KCND1 gene to chromosome Xp11.23-p11.3, and determined the genomic structure and tissue-specific expression of the KCND1, KCND2, and KCND3 genes, respectively. The open reading frame of Kv4. 1 is 1941 nucleotides long, predicting a protein of 647 amino acids. The deduced protein sequence of Kv4.1 shows an overall identity of 60% with Kv4.2 and Kv4.3L and corresponds to the common structure of voltage-gated potassium channels. KCND1 specific transcripts were detectable in human brain, heart, liver, kidney, thyroid gland, and pancreas, as revealed by Northern blot and RT-PCR experiments. The comparison of the expression patterns of the known Kv4 family members shows subtype specificity with significant overlaps. The KCND gene structures exhibit an evolutionarily conserved exon pattern with a large first exon containing the intracellular N-terminus and the putative membrane-spanning regions S1 to S5, as well as part of the pore region. The KCND3 gene contains an additional exon of 57 bp, which is not present in the other two KCND genes and gives rise to the C terminal splice KCND3L variant with an insertion of 19 amino acids. PMID- 10729222 TI - Genomic characterization of the lysophosphatidic acid receptor gene, lp(A2)/Edg4, and identification of a frameshift mutation in a previously characterized cDNA. AB - To understand the regulation, evolution, and genetics of lp(A2)/Edg4, a second lysophosphatidic acid receptor gene, we characterized its complete cDNA sequence, genomic structure, and chromosomal location. The full-length mouse transcript sequence was determined using rapid amplification of cDNA ends. Southern blot and restriction fragment length polymorphism segregation analyses revealed that the mouse gene was present as a single copy and located at the middle of Chromosome 8 near the mutations for myodystrophy (myd) and "kidney-anemia-testes" (kat). This region is syntenic with human chromosome 19p12, where the human genomic clone containing the lp(A2) gene (EDG4) was mapped. Sequence analysis of genomic clones demonstrated that both mouse and human transcripts were encoded by three exons, with an intron separating the coding region for transmembrane domain VI. Reverse transcriptase-PCR demonstrated that the three exons were spliced in all mouse tissues shown to express the transcript. Finally, in a comparison of all human lp(A2) sequences present in the database, we identified several sequence variants in multiple tumors. One such variant (a G deletion) in the initially characterized Edg4 cDNA clone (derived from an ovarian tumor) results in a frameshift mutation near the 3' end of the coding region. In addition to increasing our understanding of the mechanisms underlying lysophosphatidic acid signaling and lysophospholipid receptor gene evolution, these results have important implications regarding the genomic targeting and oncogenic potential of lp(A2). PMID- 10729223 TI - The region on 9p associated with 46,XY sex reversal contains several transcripts expressed in the urogenital system and a novel doublesex-related domain. AB - Deletions of 9p have been associated with 46,XY gonadal dysgenesis, and the smallest region of overlap has been mapped to the tip of chromosome 9. Two candidate genes (DMRT1 and 2) have been found in the region. Despite intensive mutation searches, no mutations have been detected in these genes. To gain insights into the genomics of the region and to isolate other candidate genes for the phenotype, we have constructed a P1 artificial chromosome (PAC)/bacterial artificial chromosome (BAC) contig spanning over 500 kb and covering the consensus critical region. We have analyzed the expression pattern of several ESTs mapped or sublocalized within the framework of the contig. In addition, a sample shotgun sequencing of a PAC containing the mentioned DM genes led to the detection of novel transcripts displaying an expression pattern specific to testis and kidney, consistent with a role in the development of the urogenital system. One of them, expressed in adult testis and human embryos aged 4-5 weeks, encodes a potential polypeptide and is located immediately downstream of a sequence capable of encoding a novel DM domain. The region was partially screened for mutations in sex-reversed patients by Southern blot, sequencing, and FISH. No mutations were found. Our results suggest that the critical region on 9p involved in male-to-female sex reversal displays greater gene density and genomic complexity than previously anticipated. Future investigations will include functional and mutational studies of the novel transcripts mapped or sublocalized within the critical region by this study as well as cloning efforts to isolate additional candidate genes. PMID- 10729224 TI - The human doublesex-related gene, DMRT2, is homologous to a gene involved in somitogenesis and encodes a potential bicistronic transcript. AB - Intense efforts are currently being pursued to identify autosomal genes associated with 46,XY male-to-female sex reversal. The genes DMRT1 and 2 are located on distal 9p, a region deleted in 46,XY sex-reversed patients. They are considered excellent candidates because of their homology to regulators of sex development in invertebrates. We present the genomic structure of DMRT2, showing that it generates several transcripts with distinct coding potential. In addition to the previously reported 226-amino-acid protein-encoding transcript, we describe other mRNA isoforms that are potentially bicistronic and are predicted to encode an additional 328-amino-acid polypeptide. Finally, a stop codon containing exon (exon 4) can be skipped by alternative splicing and can generate a transcript that is predicted to encode a fusion protein. The latter shares 58% amino acid identity with a gene recently described in fish, termed terra. Differences in expression pattern exist for DMRT2 mRNA isoforms among the human adult tissues tested, between adult tissues and human embryos, and between DMRT2 and DMRT1 during embryonic development. We failed to detect mutations by sequencing of DMRT2 in a sample of 46,XY female patients. The interesting structure of DMRT2 coupled to preliminary functional studies in fish showing that terra is involved in somitogenesis suggests that validation or exclusion of this gene as a cause of sex reversal will require more in-depth investigations. PMID- 10729225 TI - A novel murine PKA-related protein kinase involved in neuronal differentiation. AB - Members of the cAMP-dependent second-messenger pathway have been described as regulators of cellular growth and differentiation and were consequently implicated in a variety of embryogenic processes including brain development. Moreover, recent data suggest an indispensable role for cAMP-dependent protein kinases (PKAs) in neuronal differentiation and synaptic plasticity. Using a degenerate primer-based approach, we have identified a novel murine gene closely related to the human cAMP-dependent protein kinase PRKX on Xp22.3. This gene (Pkare) was mapped to the region near the centromere of the murine X chromosome and is expressed in a variety of adult organs including kidney, liver, spleen, testis, ovary, lung, heart, and brain. Antisense in situ hybridization on staged mouse embryos revealed a highly distinctive expression pattern during neuronal development, with elevated Pkare expression observed only in differentiating neurons within the first ganglion, the dorsal root ganglia, and the mantle layer of the telencephalon. Based on the close relationship with the catalytic PKA subunits and its distinct expression in differentiating neuronal cells, Pkare might represent a novel component of the cAMP-regulated pathways involved in brain development and function. PMID- 10729226 TI - Identification and characterization of novel genes located at the t(1;15)(p36.2;q24) translocation breakpoint in the neuroblastoma cell line NGP. AB - The distal portion of chromosome 1p is frequently deleted in several human cancers, suggesting the presence of one or more putative tumor suppressor genes on this chromosomal arm. In human neuroblastoma, a consistently deleted region at 1p36.1-p36.2 has been defined by comparison of molecular loss of heterozygosity (LOH) analyses. Recently we described the identification of a yeast artificial chromosome, YAC 927G4, that spans a translocation/duplication breakpoint within the minimally defined LOH region at 1p36.1-p36.2 in the neuroblastoma cell line NGP. Here we describe the identification of two overlapping P1 artificial chromosomes comprising 220 kb at the distal end of YAC 927G4, which we have used as hybridization probes under modified conditions to screen a composite, normalized cDNA library (IMAGE cDNA library). Hybridization screening resulted in the rapid and comprehensive identification of partial cDNAs of which a portion comprised two novel candidate genes, termed DNB1/ARPh and DNB5, which encode putative proteins of 1011 and 447 amino acids, respectively. The DNB1/ARPh gene, which was found to be ubiquitously expressed in human adult and fetal tissues, is highly related to the DRPLA gene, in which expansion of a CAG triplet appears to be causal in the dentatorubral and pallidolysian atrophy disease phenotype. The DNB5 sequence, in contrast, which is predominantly expressed in brain tissues and fetal kidney, failed to show any similarity to sequences in the public domain. A preliminary assessment of transcription and sequence of both genes in several neuroblastoma cell lines does not, thus far, support a causal role in neuroblastoma. However, further analyses are required to confirm these results. PMID- 10729227 TI - Characterization of a novel gene, C21orf6, mapping to a critical region of chromosome 21q22.1 involved in the monosomy 21 phenotype and of its murine ortholog, orf5. AB - Phenotypic and molecular analyses of patients with partial chromosome 21 monosomy enabled us to define a region, spanning 2.4 Mb between D21S190 and D21S226, associated with arthrogryposis, mental retardation, hypertonia, and several facial anomalies. The markers of the region were used to screen a total human PAC library (Ioannou, RZPD). We isolated 57 PACs, which formed primary contigs. EST clusters (UNIGENE collection) located in a 6-Mb interval, between D21S260 and D21S263, were mapped in individual bacterial clones. We mapped the WI-17843 cluster to the PAC clone J12100, which contains the two anchor markers LB10T and LA329. The open reading frame extends over 960 bp, with three putative start codons. The 1695-bp cDNA containing a polyadenylation signal should correspond to the full-length cDNA. From the genomic sequence, we deduced that the gene contained five exons and that there was a putative promoter sequence upstream from exon 1. In silico screening of DNA databases revealed similarity with a murine EST. The corresponding cDNA (1757 bp) sequence was very similar (>85%) to the human cDNA and had an open reading frame of 876 nucleotides. Somatic hybrid mapping localized the cDNA to mouse chromosome 16. EST analyses and RT-PCR indicated that the third exon in the human gene (exon 2 in the mouse) undergoes alternative splicing. Northern blot hybridization showed that the gene was ubiquitously expressed in humans and mice. The longest mouse clone was used to generate riboprobes, which were hybridized to murine embryos at stages E-9.5, E 10.5, E-12.5, E-13.5, and E-14.5-15, to study the pattern of expression during development. Ubiquitous labeling was observed, with strong signals restricted to limited areas of the telencephalon, the mesencephalon, and the interrhombomeric regions in the central nervous system, and other regions of the body such as the limb buds, branchial arches, and somites. PMID- 10729228 TI - CFTR intron 1 increases luciferase expression driven by CFTR 5'-flanking DNA in a yeast artificial chromosome. AB - The DNA elements that account for the highly regulated expression of the cystic fibrosis transmembrane conductance regulator gene (CFTR) are poorly understood. The goal of this study was to assess the feasibility of using a yeast artificial chromosome (YAC)-based reporter gene construct to define these elements further. An approximately 350-kb YAC (y5'luc) was constructed by replacing CFTR with a luciferase reporter gene (luc). A second YAC (y5'lucI) was similarly constructed but included a putative positive regulatory element from CFTR intron 1. Stable Chinese hamster ovary (CHO-K1) cell clones were derived using each YAC to assess the role that luc copy number and the presence of intron 1 played in luc expression. The CHO-K1 clonal cell lines demonstrated a wide range of luciferase activity. On average, this activity was significantly higher in clones derived from y5'lucI. After correcting for luc copy number, the presence of intron 1 was still associated with an increase in luciferase activity (P < 0.05), despite the fact that luciferase activity did not correlate with luc copy number in y5'luc derived clones (r = -0.12). In contrast, the luciferase activity correlated well with luc copy number in the clones derived from y5'luc (r = 0. 75). These data are consistent with a positive role for intron 1 in regulating CFTR expression, but suggest that copy number is not the only factor that determines expression levels, particularly when this element is present. This YAC-based reporter system will provide a unique strategy for further assessment of the cis-acting elements that control CFTR expression. PMID- 10729230 TI - Foreword PMID- 10729229 TI - Molecular characterization of a novel gene family (PHTF) conserved from Drosophila to mammals. AB - PHTF1 (putative homeodomain transcriptional factor; HGMW-approved symbol PHTF1) is a putative homeobox gene located at band 1p11-p13 of the human genome. We report here the cloning and sequencing of its mouse and Drosophila orthologs. The conservation between mouse and human proteins extends over the entire protein and is localized at the putative homeodomain and at the N- and C-terminal regions of Drosophila protein sequence. Blast searches allowed us to identify another member of the PHTF family, PHTF2, located at 7q11.23-q21 of the human genome. The strongest homologies between human PHTF1 and PHTF2 are localized to the domains that we already described in Drosophila, i.e., the putative homeodomain and the N and C-terminal regions. The human and mouse genes display 98% similarity to one another, 56% similarity with the Drosophila gene, and 67% similarity with PHTF2, suggesting that phtf might define a novel gene family of highly divergent homeobox genes. Finally, Northern blot analysis showed that while PHTF1 is expressed mainly in testis, PHTF2 is predominantly expressed in muscle, suggesting that these two genes may have acquired different functions after their duplication and divergence. PMID- 10729231 TI - The thymus and central tolerance. AB - T cell tolerance to self-components occurs largely in the thymus during early differentiation and leads to death (negative selection) of T cells with overt autoreactivity. In this article we review the evidence that negative selection in mice occurs mainly in the medulla at the level of a population of semimature T cells. The role of Fas and several costimulatory molecules on negative selection and the inhibitory role of certain cytokines are discussed. PMID- 10729232 TI - Balancing signals for negative selection and activation of developing B lymphocytes. PMID- 10729233 TI - The origin and functions of natural killer cells. PMID- 10729234 TI - Immunostimulatory sequence oligodeoxynucleotide: A novel mucosal adjuvant. AB - The majority of infectious agents enter the body via mucosal surfaces. Therefore, there is great interest in developing vaccination strategies that lead to mucosal immunity. However, to generate strong mucosal immune responses to nonreplicating immunogens, mucosal delivery with an adjuvant is required, and no safe and effective mucosal adjuvants are presently available for use in humans. Recently, immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) has been shown to be a potent mucosal adjuvant. Intranasal (i.n.) delivery of antigen with ISS-ODN elicits a secretory IgA response both locally in respiratory secretions and distally in intestinal and vaginal secretions. In this regard, ISS-ODN has been shown to be as effective as cholera toxin (CT), but unlike CT, ISS-ODN has not been shown to have significant toxicity. Furthermore, i.n. antigen/ISS-ODN coimmunization induces a strong Th(1)-biased systemic immune response with cytotoxic T lymphocyte activity. These observations suggest that ISS-ODN would be an ideal mucosal adjuvant for development of vaccines against mucosal pathogens. PMID- 10729235 TI - A protective role for innate immunity in autoimmune disease. AB - Systemic lupus erythematosus is an incurable autoimmune disease characterized by autoantibodies directed against highly conserved nuclear proteins and DNA. While the cause of lupus is not known, B-lymphocytes are essential for disease. Most striking is that deficiency in components of the innate immune system such as complement is a major risk factor in disease. One explanation for the involvement of complement and innate immunity is that they are important in the induction and maintenance of B cell tolerance to lupus antigens. PMID- 10729236 TI - Gene therapy for autoimmune disease. AB - Autoantigen-specific CD4(+) T lymphocytes have been implicated in the pathogenesis of autoimmune diseases. Tissue-specific homing properties of autoantigen-specific CD4(+) T cells suggested that these cells might be ideal vehicles for delivery of retroviral-encoded regulatory proteins in a site specific manner as a therapy for autoimmune diseases. Application of retroviral transduction of autoantigen-reactive CD4(+) T cells in gene therapy of autoimmunity must include systems capable of targeting these rare populations of antigen-activated T cells. Studies discussed below suggest that retroviral transduction of autoantigen-specific murine CD4(+) T cells may provide a method to target and isolate nontransformed autoantigen-specific murine CD4(+) T cells and provide a rational approach to gene therapy in animal models of autoimmunity. PMID- 10729237 TI - Therapeutic vaccination for cancer. PMID- 10729238 TI - Mixed chimerism as an approach to transplantation tolerance. AB - The induction of tolerance to transplanted organs could make transplantation safer and more uniformly successful. One of the most promising approaches currently being investigated involves the induction of deletional tolerance through the establishment of "mixed chimerism." In this laboratory, we first studied mixed chimerism as an approach to transplantation tolerance in mice, using a nonmyeloablative preparative regimen consisting of 300 R whole-body irradiation, 700 R thymic irradiation, and treatment with monoclonal antibodies to CD4 and CD8. This approach has subsequently been extended successfully to the induction of tolerance to renal transplants in fully mismatched cynomolgus monkeys. In addition, the same approach, with minor modifications, has been found effective in producing mixed chimerism and transplantation tolerance in the concordant xenogeneic baboon to cynomolgus monkey species combination. Because pigs have many advantages as a potential xenograft donor for humans, we are also trying to extend our nonmyeloablative regimen for production of mixed chimerism to the discordant pig --> primate combination. We have used absorption of natural antibodies to prevent hyperacute rejection and then proceeded with a mixed chimerism approach. Administration of pig hematopoietic stem cells along with pig recombinant cytokines (SCF and IL-3) to primates has enabled the pig bone marrow to survive in these xenogeneic hosts for over 6 months. This chimerism has apparently been sufficient to markedly diminish T cell immunity and the induction of new T-cell-dependent responses. However, to date we have not succeeded in preventing the return of natural antibodies, which appear to be the cause of eventual loss of organ transplants and are the subject of further intensive investigations. PMID- 10729239 TI - The prophylactic and therapeutic effects of glutamine- and arginine-enriched diets on radiation-induced enteritis in rats. AB - BACKGROUND: Recent studies indicated that glutamine and arginine support the mucosal barrier in several ways. This experimental study hypothesized that administration of glutamine- and arginine-enriched diets before abdominal radiation therapy would provide a radioprotective effect on intestinal mucosa, and this would augment the therapeutic effectiveness provided by postirradiation administration. MATERIALS AND METHODS: A rat model of radiation enteritis was designed with a single dose of 1100 cGy to the abdomen. Thirty-five rats were randomized into five groups of seven. A 7-day glutamine-enriched diet for Group I and a 7-day arginine-enriched diet for Group II were administered both pre- and postradiation. For Groups III and IV, the same glutamine and arginine diets were given, respectively, postradiation only. Group V was fed a glutamine- and arginine-free diet and was the control group. The rats underwent laparotomy for culture of mesenteric lymph nodes and removal of segments of ileum, jejenum, and colon for microscopic examination. RESULTS: Bacterial translocation was significantly higher in Group V (P < 0.05), while intestinal villus count and villus height were significantly higher in all of the groups fed glutamine and arginine when compared with the control group (P < 0.0001 and P < 0.05, respectively). CONCLUSION: Both arginine- and glutamine-enriched diets have protective effects on gut mucosa in the postirradiation state; however, pre- and postirradiation administration together does not provide superior protection versus postradiation administration alone. PMID- 10729240 TI - Effects of portal-systemic shunt following 90% partial hepatectomy in rats. AB - We studied the effects of portal-systemic shunt after massive hepatectomy. Male Wistar rats were divided into two groups: one group underwent laparotomy alone (C group) and in the other group a portal-systemic shunt was placed through laparotomy (S group). After 90% hepatectomy was performed, 3-day and 1-week survival rates and histopathology were examined, and hepatic hemodynamics during the early stage after hepatectomy were measured using dye-containing microspheres. The 3-day survival rate in the S group was significantly higher, and the 1-week survival rate was slightly higher, than those in C group. Sinusoidal dilation 7 days after hepatectomy in the S group was significantly milder than that in the C group. Fatty degeneration of hepatocytes in the S group was significantly milder than that in the C group. With respect to hepatic hemodynamics during the early stage after hepatectomy, the rate of shunt (26.3%) in the S group was significantly higher than that (9.5%) in the C group. Portal pressure, total hepatic blood flow, and total hepatic blood flow per gram of liver in the S group were significantly lower than those in the C group. These results suggest that approximately 26% shunt after 90% hepatectomy in rats increases the early survival rate and improves histological changes in surviving rats 7 days after resection. PMID- 10729241 TI - Aggregation substance promotes colonic mucosal invasion of Enterococcus faecalis in an ex vivo model. AB - BACKGROUND: Bacterial translocation through the gastrointestinal tract is the crucial step in the pathogenesis of intraabdominal infections. We assessed whether aggregation substance (AS), a bacterial adhesin and virulence factor of Enterococcus faecalis, promotes bacterial translocation and colonic mucosal invasion in an ex vivo experiment. METHODS: Colonic mucosa of male Wistar rats was placed in a modified Ussing system. The mucosal side of the chamber was filled with a suspension of E. faecalis OG1X:pAM721 (AS-positive) or E. faecalis OG1X (AS-negative). The serosal side was filled with sterile Dulbecco's modified Eagle's medium. For assessment of colonic mucosal invasion the mucosal side was incubated for 2.5 h with a suspension of AS-positive or AS-negative enterococci. After being washed, a solution of gentamicin and penicillin G in tissue culture medium was added on both sides in order to kill extracellular bacteria. Subsequently, the mucosa was removed from the system, washed, lysed with Triton X 100, and homogenized. Viable intramural bacteria were quantified by plating serial dilutions of the homogenate on Todd-Hewitt broth agar plates. To quantify bacterial translocation samples which were taken at various time points from the serosal side were plated on Todd-Hewitt broth agar plates and colony forming units (CFU) were determined. RESULTS: Invasion of the AS-positive E. faecalis strain OG1X:pAM721 into the colonic mucosa was significantly higher than invasion rates of the AS-negative strain OG1X (2.88 log(10) CFU/ml vs 1.73 log(10) CFU/ml; P = 0.02). However, none of the tested strains was found to translocate from the mucosal to the serosal side within the incubation time of 4 h. CONCLUSIONS: The aggregation substance promotes invasion of E. faecalis into colonic mucosa. PMID- 10729242 TI - Platelet-activating factor contributes to postischemic vasospasm. AB - BACKGROUND: The purpose of the present study was to determine if platelet activating factor is an important mediator that produces vasospasm during reperfusion after ischemia in skeletal muscle. MATERIALS AND METHODS: A vascular isolated cremaster muscle in male Sprague-Dawley rats was coupled with local intraarterial drug infusion as a model to study microcirculation responses to ischemia/reperfusion injury. Arteriole diameters and capillary perfusion were measured using intravital microscopy. Group 1: platelet-activating factor dose response. Group 2: Effects of a cyclooxygenase inhibitor; indomethacin, and a thromboxane synthetase inhibitor, imidazole, on the response to platelet activating factor. Group 3: Effects of nitric oxide synthesis inhibitor; N(omega) nitro-L-arginine methyl ester, on the response to platelet-activating factor. Group 4: Effects of a platelet-activating factor receptor antagonist, CV-3988, indomethacin, and imidazole after 4 h of warm ischemia and reperfusion. RESULTS: Intraarterial infusion of platelet-activating factor produced a dose-related but mild vasoconstriction. Pretreatment with indomethacin or imidazole resulted in significant vasodilation actually emanating from platelet-activating factor infusion. Nitric oxide inhibition (with N(omega)-nitro-L-arginine methyl ester) enhanced the vasoconstriction produced by platelet-activating factor. Pretreatment with CV-3988, indomethacin, or imidazole significantly attenuated ischemia/reperfusion-induced vasospasm and capillary no-reflow in the cremaster muscles. CONCLUSIONS: Ischemia/reperfusion-induced vasoconstriction is at least in part mediated by platelet-activating factor and thromboxane A(2). PMID- 10729243 TI - Endothelial and smooth muscle changes of the thoracic and abdominal aorta with various types of cryopreservation. AB - BACKGROUND: There is a need for cryopreserved arterial allografts in vascular surgery. Vitrification was examined as an alternative to slow cooling. Function of endothelial and adjacent cells was evaluated in the thoracic and abdominal regions of the porcine aorta. MATERIAL AND METHODS: Aortas from young pigs were harvested and cryopreserved with several treatments: slow cooling at 1 degrees C/min with 10% dimethyl sulfoxide (DMSO) and vitrification with a vitrification solution (VS), performed either in the presence (medium) or in the absence (air) of the cryoprotective medium surrounding the sample. Tetrazolium salt reduction and oxygen consumption were used to assess the mitochondrial activity of the endothelial cells and smooth muscle cells from the aorta. RESULTS: Fresh aorta showed an increased function from below the aortic arch to the iliac branch. Exposure to cryoprotective solutions reduced significantly the oxygen consumption of the abdominal sections. Samples from the thoracic region had a better response to cryopreservation than those from the abdominal region. There was a significant reduction in tetrazolium salt reduction and oxygen consumption when the samples were cryopreserved immersed in the medium. Significant flaking of the endothelium was observed after vitrification and contributes generally to lower the function. CONCLUSION: The thoracic region of the aortic wall was more tolerant to the preservation injury, and freezing with DMSO in air offered a better choice of preservation. PMID- 10729244 TI - Gene therapy enhances the antiproliferative effect of radiation in intimal hyperplasia. AB - BACKGROUND: Although ionizing radiation (IR) has been demonstrated to attenuate vessel wall restenosis and intimal hyperplasia (IH), dose-related mural injury and atrophy are possible deleterious side effects. We tested the hypothesis that a radiosensitizing strategy may improve IR-induced inhibition of in vivo vascular smooth muscle cells (VSMCs) without influencing apoptotic cell death. METHODS: In 28 New Zealand White rabbits, the right common carotid artery (CCA) was injured and subjected to low-flow conditions to promote IH. The CCA was transfected with an adenoviral vector incorporating the cytosine deaminase (CD) gene (1 x 10(9) PFU/ml). 5-Fluorocytosine (5-FC), a prodrug that is converted to the radiosensitizing agent 5-fluorouracil (5-FU) by CD, was thereafter administered intravenously. The CCA was exposed to 5 Gy IR at 24 h. Intimal/medial (I/M) area and thickness ratios were determined in the harvested CCAs at 14 days. VSMC proliferative and apoptotic indices were assessed with immunohistochemistry. RESULTS: A 50% reduction in I/M area was found in rabbits treated with IR and IR + CD/5-FC (0.19 +/- 0.03 and 0.18 +/- 0.02) when compared with untreated controls (UC) (0.37 +/- 0.06) (P = 0.005). This finding was substantiated by attenuation of I/M thickness in the IR groups [0.47 +/- 0.13 (IR), 0.41 +/- 0.11 (IR + CD/5 FC), 0.61 +/- 0.17 (UC)] (P = 0.007). The number of proliferating VSMCs was notably smaller when IR was combined with CD/5-FC (4.17 +/- 1.16 vs 2.97 +/- 1.09 log transformed cells/mm(2), P < 0.07). Apoptosis was similar in all groups. CONCLUSIONS: Both IR alone and IR combined with a radiosensitizing agent are effective in attenuating experimental IH. However, combination therapy is synergistic and achieves greater inhibition of VSMC proliferation and may involve selective killing of radioresistant S-phase VSMCs. IR + CD/5-FC represents a novel therapeutic strategy that offers potential for long-term control of IH. PMID- 10729245 TI - Mechanisms of myocardial protection by adenosine-supplemented cardioplegia: differential response of calcium-independent protein kinase C isozymes. AB - BACKGROUND: Adenosine-supplemented cardioplegia improves myocardial function after cardioplegic arrest. However, the underlying cellular mechanism(s) responsible for adenosine's protective actions remains unclear. We tested the hypothesis that protection by adenosine-supplemented cardioplegia would be associated with selective activation of protein kinase C (PKC) isozymes delta and epsilon. MATERIALS AND METHODS: Isolated rat hearts were perfused (37 degrees C, Krebs-Ringer bicarbonate buffer) for 30 min, after which baseline functional measurements were made. This was followed by 120 min of cold cardioplegic arrest at 4 degrees C with either St. Thomas No. 2 (ST#2), ST#2 + adenosine (100 microM, ADO) or ST#2 + ADO + 8-sulfophenyltheophylline (50 microM, SPT). Hearts were reperfused for 60 min and functional measurements made. Distribution of PKC isoforms was determined (immunoblotting) after 30 min of warm perfusion (No-CDPL) or after 30 min of perfusion followed by 15 min of cardioplegic arrest. RESULTS: ADO prevented myocardial dysfunction after cardioplegic arrest. PKC-delta did not differ in the cytosolic fraction among groups. However, ADO prevented increases in particulate fraction PKC-delta, but elicited a significant increase in the particulate fraction PKC-epsilon, while ST#2 or SPT significantly decreased the cytosolic fraction PKC-epsilon. Both functional and cellular changes associated with ADO were receptor mediated. CONCLUSION: This novel, dual action of adenosine supplemented cardioplegia on PKC isoforms may be responsible for the associated functional improvements. PMID- 10729246 TI - Inhibition of intestinal tumors by curcumin is associated with changes in the intestinal immune cell profile. AB - BACKGROUND: The C57BL/6J-Min/+ (Min/+) mouse bears a germline mutation in Apc and is therefore a model for familial adenomatous polyposis and sporadic colorectal cancer. Min/+ intestinal mucosa exhibits a marked tendency for spontaneous adenoma formation. Curcumin is a phenolic antioxidant known for its antitumor and immune modulatory functions in vitro. Curcumin prevents adenoma formation in Min/+ mice, through a mechanism that may be related to its immunomodulatory properties. MATERIALS AND METHODS: To study the relationship between intestinal immunity and curcumin-induced antitumor response, we used immunohistochemistry to characterize the effect of curcumin treatment on resident intestinal immune effector cells in Min/+ mice. RESULTS/CONCLUSION: These results show that mucosal CD4(+) T cells and B cells increase in animals treated with curcumin, suggesting that curcumin modulates lymphocyte-mediated immune functions. PMID- 10729247 TI - Vascularized bone marrow transplantation: A new surgical approach using isolated femoral bone/bone marrow. AB - BACKGROUND: Orthotopic composite tissue (limb) transplantation in rats is a unique model for vascularized bone marrow transplantation because bone marrow cells and bone marrow stroma are transplanted by microsurgical means, thus creating immediate bone marrow space and engraftment. However, it contains a skin component and other musculoskeletal tissues that complicate issues related to tolerance induction. MATERIALS AND METHODS: To study only aspects of vascularized bone marrow transplantation, we created a new isolated vascularized bone marrow transplant model in rats. The common iliac (or femoral) artery and vein were microsurgically anastomosed to the recipient abdominal aorta and inferior vena cava in an end-to-side fashion, respectively. Syngeneic male Lewis (RT1(1), n = 20) and allogeneic male BN (RT1(n), n = 10) donors were transplanted to female Lewis recipients. To establish rejection criteria, we examined histopathology and used the polymerase chain reaction (PCR) to assess microchimerism of donor male bone marrow cells in the peripheral blood of female recipients using rat Y chromosome (sex-determining region Y)-specific primers. RESULTS: All recipients were healthy and remained stable without major complications for up to 300 days posttransplant. Morphologically, syngeneic male Lewis bone marrow showed a near normal appearance. Allogeneic male BN bone marrow was clearly rejected. Male bone marrow cells were detected by PCR in the peripheral blood of all syngeneic recipients, but not in allogeneic blood specimens. CONCLUSIONS: A new surgical approach to bone marrow transplantation was established. This consisted of the vascularized femoral bone/bone marrow transplant. Further analyses regarding the ability of vascularized femoral bone marrow transplants to induce systemic transplantation tolerance in adult rats will provide insights into not only various issues of immunology but also the potential clinical application of vascularized bone marrow transplantation. PMID- 10729248 TI - Regenerative capacities of normal and cirrhotic livers following 70% hepatectomy in rats and the effect of alpha-tocopherol on cirrhotic regeneration. AB - BACKGROUND: The regeneration of normal and cirrhotic liver has been very well demonstrated after partial hepatectomy; although the tissue regenerated by cirrhotic liver is also cirrhotic. The structural differences of the regenerated tissues between normal and cirrhotic livers may also indicate different regeneration capacities. The objective of this study was to compare the regeneration capacities of normal and cirrhotic livers by bromodeoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) labeling indices in replicating nuclei and mitotic figures in cells in partially hepatectomized normal and cirrhotic rats and to study the effect of alpha tocopherol on cirrhotic liver regeneration. METHODS: Five groups of adult Wistar rats comprised normal livers, cirrhotic livers, regenerated normal livers, regenerated cirrhotic livers, and alpha-tocopherol-treated regenerated cirrhotic livers. Cirrhosis was induced by intragastric administration of carbon tetrachloride and phenobarbital in the drinking water of the rats. Liver regeneration capacities in normal and cirrhotic rats and following partial hepatectomy in normal and cirrhotic rats and cirrhotic rats that were administered alpha-tocopherol were evaluated through BrdU incorporation, PCNA labeling, and mitotic indices. RESULTS: BrdU and PCNA labeling and mitotic indices were zero for normal rats and 4.3 +/- 3.5, 6.5 +/- 5, and 2.5 +/- 1.5 for cirrhotic rats, respectively. The values after partial hepatectomy in normal and cirrhotic rats were 46.2 +/- 8.7 and 27.8 +/- 7.5 for BrdU labeling, 83.7 +/- 6.5 and 51.3 +/- 6.8 for PCNA labeling, and 31.8 +/- 4.2 and 18.6 +/- 3.4 for mitotic index, respectively. For the fifth group comprising cirrhotic rats that were administered alpha-tocopherol and had undergone partial hepatectomy, BrdU incorporation, PCNA labeling, and mitotic indices were 37.5 +/- 6.3, 76.5 +/- 6.2, and 27.2 +/- 4.2, respectively. When the cirrhotic liver regeneration group was compared with the normal liver regeneration group, rates of liver regeneration in the cirrhotic group were significantly depressed (P < 0.01). Although the BrdU incorporation and PCNA labeling indices of the alpha-tocopherol administered cirrhotic liver regeneration group indicated significantly lower rates of liver regeneration when compared with the normal liver regeneration group (P < 0.05), the liver regeneration rates of the alpha-tocopherol administered cirrhotic group were also significantly higher than those of the cirrhotic liver regeneration group that was not administered alpha-tocopherol (P < 0.01). CONCLUSIONS: Cirrhotic livers revealed a significantly depressed capacity for regeneration following partial hepatectomy. alpha-Tocopherol administration seemed to improve the rates of regeneration in cirrhotic rats with respect to the BrdU incorporation, PCNA labeling, and mitotic indices. PMID- 10729250 TI - Coherent cross-polarization theory for a spin-12 coupled to a general object AB - Zero-order average-Hamiltonian theory is used to extend the product-operator description of coherent spin-spin cross-polarization to the case of a spin-12 coupled to a general object, like a molecular rotor or a quantum oscillator. The object, which is not necessarily in a Boltzmann equilibrium state, is assumed to have no interaction with the lattice and no internal relaxation capacity. The Bloch-Wangsness-Redfield (BWR) theory for incoherent processes like spin-lattice relaxation does not apply for such an isolated spin-object pair. Nevertheless spectral density at the Larmor frequency, of key importance in BWR theory, also plays a central role in object-induced spin polarization. Spectral density in our theory is represented by quantum operators J(-) and J(+). If J(-) and J(+) do not commute, the spin-object coupling may cause spin polarization in an initially saturated spin system. This represents a coherent mechanism for spin cooling, which in specific cases may lead to enhanced spin polarization above the thermal equilibrium value. A master equation is derived for general spin-object cross polarization, and applied to the case of a spin pair inside a uniaxial rotor, and a spin coupled to a microelectronic LC circuit. Copyright 2000 Academic Press. PMID- 10729249 TI - Enhancement of angiogenesis by the implantation of self bone marrow cells in a rat ischemic heart model. AB - Background. Bone marrow contains various kinds of primitive cells which differentiate into endothelial cells and could secrete several growth factors. Therefore, we attempted to induce therapeutic angiogenesis using self bone marrow cells in a rat model. Materials and methods. Quantitative angiogenesis was examined using a sponge implantation assay that indicated whether the rat bone marrow cells had induced angiogenesis or not. Employing a rat ischemic heart model, bone marrow cells were injected directly into the ischemic area and the number of vessels was examined immunohistochemically using the anti-CD31 monoclonal antibody. The contributed growth factors revealed the levels present in the ischemic myocardium by an enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction. Results. The sponge implantation assay showed that bone marrow cells induced angiogenesis. Light microscopic analysis of the vessel count positively stained by anti-CD31 in the ischemic area showed that angiogenesis had been induced to a significantly greater degree in the group implanted with bone marrow cells (BMI group) than in the group injected with phosphate-buffered saline (PBS group) 1 week after BMI. Levels of the inflammatory cytokines interleukin-1 (IL-1beta) and cytokine-induced neutrophil chemoattractant (CINC) in the BMI group were significantly elevated compared with those in the PBS group. Conclusions. Self bone marrow cell implantation induced angiogenesis in a rat ischemic heart model as a result of elevation of the levels of IL-1beta and CINC. Thus, bone marrow implantation could be a novel and simple method to induce therapeutic angiogenesis. PMID- 10729251 TI - Construction of phase cycles of minimum cycle length: MakeCycle AB - An algorithm for the generation of a phase cycle of minimum length for a pulse sequence is developed from the basic requirement that only specified coherence transfer pathways will be accumulated. The efficacy of the algorithm is shown by determining the phase cycles of minimum length for DQFCOSY, GHMBC, and INEPT pulse sequences. Copyright 2000 Academic Press. PMID- 10729252 TI - Separation of quadrupolar and magnetic contributions to spin-lattice relaxation in the case of a single isotope AB - We present a NMR pulse double-irradiation method which allows one to separate magnetic from quadrupolar contributions in the spin-lattice relaxation. The pulse sequence fully saturates one transition while another is observed. In the presence of a ||Deltam || = 2 quadrupolar contribution, the intensity of the observed line is altered compared to a standard spin-echo experiment. We calculated analytically this intensity change for spins I = 1, 32, 52, thus providing a quantitative analysis of the experimental results. Since the pulse sequence we used takes care of the absorbed radiofrequency power, no problems due to heating arise. The method is especially suited when only one NMR sensitive isotope is available. Different cross-checks were performed to prove the reliability of the results obtained. The applicability of this method is demonstrated by a study of the plane oxygen (17)O (I = 52) in the high temperature superconductor YBa(2)Cu(4)O(8): the (17)O spin-lattice relaxation rate consists of magnetic as well as quadrupolar contributions. Copyright 2000 Academic Press. PMID- 10729253 TI - Electron paramagnetic resonance imaging using magnetic-field-gradient spinning AB - A novel X-band CW EPR imaging has been developed using magnetic-field-gradient (MFG) spinning to obtain spatial distributions of electron paramagnetic species. Spinning MFG EPR imaging for 65 projection spectra required just 55 s while conventional imaging took 11 min 40 s, that is, the acquisition time for the new system is one order of magnitude shorter than that for conventional EPR imaging. Spinning MFG EPR imaging allows one to measure reconstructed images in an interactive manner where resolution and condition can be changed quickly. Copyright 2000 Academic Press. PMID- 10729254 TI - Refocused primary echo: A zero dead time detection of the electron spin echo envelope modulation AB - We report on the two-dimensional (2D) implementation of the refocused primary electron spin echo envelope modulation (ESEEM) technique, its theory and experimental application to a model system and a system of biological interest. This technique is virtually free of dead time along one time coordinate. The ESEEM obtained by integration of the 2D time-domain data of the refocused primary ESEEM over one of the time coordinates shows the intensity of the sum combination harmonics proportional to k(2) for k << 1 and proportional to k for k approximately 1 (k is a usual notation for the modulation amplitude factor). This feature, in combination with the adjustment of k by means of variation of the operational frequency of the spectrometer, was found to be very useful for detection of protons with distributed hyperfine interaction parameters situated close to the electron spin. Copyright 2000 Academic Press. PMID- 10729255 TI - Novel peak assignments of in vivo (13)C MRS in human brain at 1.5 T. AB - (13)C MRS studies at natural abundance and after intravenous 1-(13)C glucose infusion were performed on a 1.5-T clinical scanner in four subjects. Localization to the occipital cortex was achieved by a surface coil. In natural abundance spectra glucose C(3beta,5beta), myo-inositol, glutamate C(1,2,5), glutamine C(1,2,5), N-acetyl-aspartate C(1-4,C=O), creatine CH(2), CH(3), and C(C=N), taurine C(2,3), bicarbonate HCO(-)(3) were identified. After glucose infusion (13)C enrichment of glucose C(1alpha,1beta), glutamate C(1-4), glutamine C(1-4), aspartate C(2,3), N-acetyl-aspartate C(2,3), lactate C(3), alanine C(3), and HCO(-)(3) were observed. The observation of (13)C enrichment of resonances resonating at >150 ppm is an extension of previously published studies and will provide a more precise determination of metabolic rates and substrate decarboxylation in human brain. PMID- 10729256 TI - Broadband echo sequence using a pi composite pulse for the pure NQR of a spin I = 32 powder sample AB - This work presents a numerical approach to optimizing sequences with composite pulses for the pure NQR of a spin I = 32 powder sample. The calculations are based on a formalism developed in a previous paper, which allows a fast powder averaging procedure to be implemented. The framework of the Cayley-Klein matrices to describe space rotations by 2 x 2 unitary and unimodular complex matrices is used to calculate the pulse propagators. The object of such a study is to design a high-performance echo sequence composed of a single preparation pulse and a three-pulse composite transfer pulse. We mean a sequence leading to a large excitation bandwidth with a good signal-to-noise ratio, a flat excitation profile near the irradiation frequency, and a good linearity of the phase as a function of frequency offset. Such a composite echo sequence is intended to give a better excitation profile than the classical Hahn (θ)-tau-(2θ) echo sequence. It is argued that in pure NQR of a powder sample, the sequence must be optimized as a whole since both the excitation and the reception of the signal depend on the relative orientation of the crystallites with respect to the coil axis. To our knowledge, this is the first time such a global approach is presented. An extensive numerical study of the composite echo sequence described above is performed in this article. The key of the discrimination between the sequences lies in using the first five reduced moments of the excitation profile as well as an estimator of the phase linearity. Based on such information, we suggest that the echo sequence that best fulfills our criterion is (1)(0)-tau (0.35)(0)(2.1)(pi)(0.35)(0), the pulse angles omega(RF)t(p) being in radians. The subscripts are the relative pulse phases. We outlined the way to implement the spin echo mapping method to reconstruct large spectra with this sequence, and it is shown that it reduces the acquisition time by a factor of 1.7 if compared to the classical Hahn echo. Some other broadband echo sequences are also briefly discussed. We also study the effect of a small delay between the pulses of the composite pulse to take into account the experimental constraint imposed by the spectrometer. To be complete, we give an estimation of the performances of the Hahn echo, the stimulated echo, and the composite echo sequence optimized for a powder sample when applied to a monocrystal. Experiments performed on chloranil at ambient temperature confirm the predicted excitation profiles. Copyright 2000 Academic Press. PMID- 10729257 TI - Cramer-Rao bound expressions for parametric estimation of overlapping peaks: influence of prior knowledge AB - We have derived analytical expressions of the Cramer-Rao lower bounds on spectral parameters for singlet, doublet, and triplet peaks in noise. We considered exponential damping (Lorentzian lineshape) and white Gaussian noise. The expressions, valid if a sufficiently large number of samples is used, were derived in the time domain for algebraic convenience. They enable one to judge the precision of any unbiased estimator as a function of the spectral and experimental parameters, which is useful for quantitation objectives and experimental design. The influence of constraints (chemical prior knowledge) on parameters of the peaks of doublets and triplets is demonstrated both analytically and numerically and the inherent benefits for quantitation are shown. Our expressions also enable analysis of spectra comprising many peaks. Copyright 2000 Academic Press. PMID- 10729258 TI - Heteronuclear local field NMR spectroscopy under fast magic-angle sample spinning conditions AB - The acquisition of bidimensional heteronuclear nuclear magnetic resonance local field spectra under moderately fast magic-angle spinning (MAS) conditions is discussed. It is shown both experimentally and with the aid of numerical simulations on multispin systems that when sufficiently fast MAS rates are employed, quantitative dipolar sideband patterns from directly bonded spin pairs can be acquired in the absence of (1)H-(1)H multiple-pulse homonuclear decoupling even for "real" organic solids. The MAS speeds involved are well within the range of commercially available systems (10-14 kHz) and provide sidebands with sufficient intensity to enable a reliable quantification of heteronuclear dipolar couplings from methine groups. Simulations and experiments show that useful information can be extracted in this manner even from more tightly coupled -CH(2) moieties, although the agreement with the patterns simulated solely on the basis of heteronuclear interactions is not in this case as satisfactory as for methines. Preliminary applications of this simple approach to the analysis of molecular motions in solids are presented; characteristics and potential extensions of the method are also discussed. Copyright 2000 Academic Press. PMID- 10729259 TI - Comparison and use of vector and quantum representations of J-coupled spin evolution in an IS spin system during RF irradiation of one spin AB - A comprehensive survey is provided of the analytical expressions for the orthogonal product operator states arising from any initial state of an IS J coupled spin system during arbitrary RF irradiation of one spin. These equations exactly characterize the effect of J coupling during the application of the RF field. The survey differentiates two kinds of spin rotation, classical and nonclassical, where the second kind comprises any interconversion that includes the transverse two-spin coherence states, 2S(y)I(x) or 2S(y)I(y), as initial, transient, or final states, and the first kind comprises all other rotations. Classical rotations are defined as linear rotations of the nuclear spin magnetization vectors around effective fields and there is an exact correspondence between the resulting vector model and the quantum mechanical (QM) equations at all RF field strengths. The effect of scalar coupling can be neglected for B(1) > 5J. Nonclassical rotations are nonlinear in time for a constant RF field. At high field (B(1) > 50J), the effect of J modulation is negligible, and the rotation of magnetizations is classical to a very good approximation. At intermediate strengths (5J < B(1) < 50J), a semi-classical vector model of I-spin irradiation is applicable in which the J-coupled precession of the S spins is determined from a reduced coupling constant, but the effect of the S spins on the I spins is ignored (this model has previously been used to determine the effect of coupling during adiabatic pulses). At lower powers, the exact QM-derived equations must be used for nonclassical rotations, but continuous pictorial descriptions of the rotation of magnetizations determined from the vector sum of the product operator states are helpful in designing novel NMR applications. At all powers it is proven that the instantaneous reduced coupling constant acting on the S spins is J cosφ, where φ is the polar angle of the I-spin magnetizations, thus establishing the central tenet of the semi-classical model applicable at moderate power. Several spinstate transformations that combine the effects of RF and scalar coupling to produce the overall rotation can be generated in 100% yield using low power irradiation. Analogous transformations are also available using classical rotations and, in combination with their nonclassical counterparts, form a general class of frequency-selective pulses that we call J pulses. Any combination of a 90 degrees pulse and a consecutive (2J)(-1) delay period can be replaced with a J pulse, and some initial approaches to designing shaped J pulses with improved offset profiles are explored. Copyright 2000 Academic Press. PMID- 10729260 TI - Improvement of spectral editing in solids: A sequence for obtaining (13)CH + (13)CH(2)-only (13)C spectra AB - An improved spectral editing method for solids is described which allows one to obtain a set of subspectra in roughly two-thirds the amount of time as our original CPPI editing method for the same signal to noise. This improvement is afforded by a new pulse sequence that is used to acquire a (13)CH + (13)CH(2) spectrum which has very little (13)CH(3) or nonprotonated carbon contamination. By using this new sequence the (13)CH-only subspectrum is obtained much more efficiently. Criteria for optimizing the signal to noise in the edited subspectra are discussed. Copyright 2000 Academic Press. PMID- 10729261 TI - Effects of off-resonance irradiation, cross-relaxation, and chemical exchange on steady-state magnetization and effective spin-lattice relaxation times AB - In the presence of an off-resonance radiofrequency field, recovery of longitudinal magnetization to a steady state is not purely monoexponential. Under reasonable conditions with zero initial magnetization, recovery is nearly exponential and an effective relaxation rate constant R(1eff) = 1/T(1eff) can be obtained. Exact and approximate formulas for R(1eff) and steady-state magnetization are derived from the Bloch equations for spins undergoing cross relaxation and chemical exchange between two sites in the presence of an off resonance radiofrequency field. The relaxation formulas require that the magnetization of one spin is constant, but not necessarily zero, while the other spin relaxes. Extension to three sites with one radiofrequency field is explained. The special cases of off-resonance effects alone and with cross relaxation or chemical exchange, cross-relaxation alone, and chemical exchange alone are compared. The inaccuracy in saturation transfer measurements of exchange rate constants by published formulas is discussed for the creatine kinase reaction. Copyright 2000 Academic Press. PMID- 10729262 TI - NMR characterization of the pore structure and anisotropic self-diffusion in salt water Ice AB - NMR imaging and one- and two-dimensional self-diffusion propagator measurements of the liquid phase in salt water ice are presented. The properties of the network of brine-filled pores are found to depend on the growth conditions of the ice. Two types of samples are compared: (a) shock-frozen ice produced in the probe in situ and (b) ice grown over several hours under controlled conditions. By shock-freezing, an ice structure could be produced which featured streak-like porous channels of diameters of up to 300 &mgr;m allowing almost unrestricted self-diffusion along one preferential axis but reduced diffusivities in the remaining directions. In ice grown under controlled conditions, the pore sizes are near the resolution limit of the imaging experiment of typically 50 &mgr;m. For this type of samples, strongly non-Gaussian self-diffusion propagators are obtained, indicating restricted self-diffusion on rms scales of 30 &mgr;m. Common to all samples was the observation of highly anisotropic self-diffusion. One- and two-dimensional propagators are compared in order to estimate the degree of anisotropy and the size of the restrictions. Copyright 2000 Academic Press. PMID- 10729263 TI - Enhanced suppression of residual water in a "270" WET sequence. AB - In certain water suppression experiments, the residual water, which comes from a region away from the center of the RF coil and experiences a much smaller flip angle than the designed one, may appear. The residual water in the WET sequence can be reduced significantly by using a composite 90(x)( degrees )90(y)( degrees )90(-x)( degrees )90(-y)( degrees ) pulse, which de-excites molecules experiencing a small flip angle. The composite pulse, however, has two null excitation points near on resonance, causing a severe loss of spectrum intensity and baseline distortion toward the null points. Since the residual water experiences a very small flip angle, it can be treated as a linear spin system; i.e., the intensity of the residual water is proportional to the pulse strength and width. Based on this principle, the residual water can be reduced dramatically by replacing the 90 degrees pulse in the "270" WET sequence with a 270 degrees pulse for one out of every four scans, without noticeable loss of intensity and baseline distortion. PMID- 10729264 TI - New techniques for the measurement of C'N and C'H(N) J coupling constants across hydrogen bonds in proteins. AB - Two new two- or three-dimensional NMR methods for measuring (3h)J(C'N) and (2h)J(C'H) coupling constants across hydrogen bonds in proteins are presented. They are tailored to suit the size of the TROSY effect, i.e., the degree of interference between dipolar and chemical shift anisotropy relaxation mechanisms. The methods edit 2D or 3D spectra into two separate subspectra corresponding to the two possible spin states of the (1)H(N) spin during evolution of (13)CO coherences. This allows (2h)J(C'H) to be measured in an E.COSY-type way while (3h)J(C'N) can be measured in the so-called quantitative way provided a reference spectrum is also recorded. A demonstration of the new methods is shown for the (15)N,(13)C-labeled protein chymotrypsin inhibitor 2. PMID- 10729265 TI - Semi-constant-time HMSQC (SCT-HMSQC-HA) for the measurement of (3)J(H(N))(H(alpha)) couplings in (15)N-labeled proteins. AB - A simple method for accurately measuring (3)J(H(N))(H(alpha)) coupling constants in (15)N-labeled proteins is described. This semi-constant-time HMSQC-HA experiment combines the rapidity and convenience of the recently introduced CT HMQC-HA scheme (Postingl and Otting, J. Biomol. NMR 12, 319-324 (1998)) with the high resolution and robustness of the HSQC experiment. The proposed method is demonstrated for the 76-residue human ubiquitin and Saccharopolyspora erythraea calerythrin (176 residues). Our results imply that the SCT-HMSQC-HA experiment is suitable also for proteins with less favorable NMR properties due to its good resolution and sensitivity. PMID- 10729266 TI - Determining pore sizes using an internal magnetic field AB - A concept is proposed to measure the pore size length scale by the internal magnetic field (B(i)) in porous materials. The spatial distribution of the magnetic field inhomogeneity, a result of the magnetic susceptibility contrast between the porous material and the fluid, reflects the underlying pore geometry. Diffusion in B(i) causes the initial decay of magnetization. At long times, the effect of B(i) saturates when the diffusion length reaches a characteristic pore size. This method is independent of surface spin relaxation in determining pore sizes. Nuclear magnetic resonance experiments on packed glass beads and sedimentary rock samples will be presented. Copyright 2000 Academic Press. PMID- 10729267 TI - Variation of molecular alignment as a means of resolving orientational ambiguities in protein structures from dipolar couplings. AB - Residual dipolar couplings for pairs of proximate magnetic nuclei in macromolecules can easily be measured using high-resolution NMR methods when the molecules are dissolved in dilute liquid crystalline media. The resulting couplings can in principle be used to constrain the relative orientation of molecular fragments in macromolecular systems to build a complete structure. However, determination of relative fragment orientations based on a single set of residual dipolar couplings is inherently hindered by the multi-valued nature of the angular dependence of the dipolar interaction. Even with unlimited dipolar data, this gives rise to a fourfold degeneracy in fragment orientations. In this Communication, we demonstrate a procedure based on an order tensor analysis that completely removes this degeneracy by combining residual dipolar coupling measurements from two alignment media. Application is demonstrated on (15)N-(1)H residual dipolar coupling data acquired on the protein zinc rubredoxin from Clostridium pasteurianum dissolved in two different bicelle media. PMID- 10729268 TI - Sensitivity enhancement of HCACO by using an HMQC magnetization transfer scheme. AB - Previous theoretical calculations have demonstrated that the multiquantum relaxation rate of (1)H(alpha)-(13)C(alpha)(R(MQ)) is, on average, 1.3 +/- 0.4 or 1.7 +/- 0.6 times slower than the single-quantum relaxation rate of (13)C(alpha)(R(C)) for a sample with or without, respectively, amide protons. By taking advantage of this fact and by using the PEP sensitivity enhancement scheme, an HMQC version of the HCACO experiment has been developed. We demonstrate that this new experiment is 23 and 55% more sensitive than the original HSQC version of the HCACO experiment, at constant times of 7 and 27 ms, respectively, for a sample of the BC domain of the ciliary neurotrophic factor receptor protein dissolved in D(2)O at 20 degrees C. PMID- 10729269 TI - Sample optimization and identification of signal patterns of amino acid side chains in 2D RFDR spectra of the alpha-spectrin SH3 domain. AB - Future structural investigations of proteins by solid-state CPMAS NMR will rely on uniformly labeled protein samples showing spectra with an excellent resolution. NMR samples of the solid alpha-spectrin SH3 domain were generated in four different ways, and their (13)C CPMAS spectra were compared. The spectrum of a [u-(13)C, (15)N]-labeled sample generated by precipitation shows very narrow (13)C signals and resolved scalar carbon-carbon couplings. Linewidths of 16-19 Hz were found for the three alanine C(beta )signals of a selectively labeled [70% 3 (13)C]alanine-enriched SH3 sample. The signal pattern of the isoleucine, of all prolines, valines, alanines, and serines, and of three of the four threonines were identified in 2D (13)C-(13)C RFDR spectra of the [u-(13)C, (15)N]-labeled SH3 sample. A comparison of the (13)C chemical shifts of the found signal patterns with the (13)C assignment obtained in solution shows an intriguing match. PMID- 10729270 TI - Simple suppression of spurious peaks in TROSY experiments. AB - In (1)H-(15)N TROSY experiments of proteins and nucleic acids, where the second coherence transfer delay time tau' has been fixed as 5.6 ms, 1/(2(1)J(NH)), in order to achieve complete spin-state selection, spurious negative peaks are observed along the (15)N axes. These peaks are often annoyingly large, especially for nucleic acids. A simple product operator calculation, however, indicated that the shortening of the second delay time tau', which is next to the t1 period, would efficiently suppress these spurious peaks, without sacrificing the sensitivities of the TROSY peaks too much. We have shown for three systems, two 11- and 17-kDa proteins and one 8-kDa DNA duplex, that these spurious peaks can be effectively suppressed with delay times of 3.3 ms for the two proteins and 2.3 ms for the DNA. These delay times, optimized by trial and error, for the spurious peak suppression did not depend on the magnetic field strength and the temperature very much. Although the shortened tau' delay times attenuate the TROSY peak intensities by about 10 and 20% for the two proteins and the DNA, respectively, this simple modification will be useful for the quantitative uses of TROSY peaks and will result in cleaner spectra for various TROSY-based multiple resonance experiments. PMID- 10729272 TI - Anisotropic diffusion in a nematic liquid crystal- An electric field PFG NMR approach AB - The access to self-diffusion coefficients in anisotropic systems such as thermotropic liquid crystals by means of PFG NMR is complicated by strong dipolar interactions. Additionally, problems arise due to the immediate orientation of low-molar-mass nematic liquid crystals in an external field. The director orientation can be changed by the application of an additional electric field. This can be exploited in order to reduce the dipolar interaction to such an extent that the NMR linewidths change from a solid-state to a liquid-like situation enabling PFG NMR experiments. Copyright 2000 Academic Press. PMID- 10729271 TI - Protein dynamics measurements by TROSY-based NMR experiments. AB - The described TROSY-based experiments for investigating backbone dynamics of proteins make it possible to elucidate internal motions in large proteins via measurements of T(1), T(2), and NOE of backbone (15)N nuclei. In our proposed sequences, the INEPT sequence is eliminated and the PEP sequence is replaced by the ST2-PT sequence from the HSQC-based experiments. This has the benefit of shortening the pulse sequences by 5.4 ms (=1/2J) and results in an increase in the intrinsic sensitivity of the proposed TROSY-based experiments. The TROSY based experiments are on average of 13% more sensitive than the corresponding HSQC-based experiments on a uniformly (15)N-labeled Xenopus laevis calcium-bound calmodulin sample on a 750-MHz spectrometer at 5 degrees C. The amide proton linewidths of the TROSY-based experiments are 2-13 Hz narrower than those of the HSQC experiments. More sensitivity gain and higher resolution are expected if the protein sample is deuterated. PMID- 10729273 TI - 3hJ coupling between C(alpha) and H(N) across hydrogen bonds in proteins. AB - J couplings between (13)C(alpha) and (1)H(N) across hydrogen bonds in proteins are reported for the first time, and a two- or three-dimensional NMR technique for their measurement is presented. The technique exploits the TROSY effect, i.e., the degree of interference between dipolar and chemical shift anisotropy relaxation mechanisms, for sensitivity enhancement. The 2D or 3D spectra exhibit E.COSY patterns where the splittings in the (13)CO and (1)H(N) dimensions are (1)J((13)C(alpha), (13)CO) and the desired (3h)J((13)C(alpha), (1)H(N)), respectively. A demonstration of the new method is shown for the (15)N,(13)C labeled protein chymotrypsin inhibitor 2 where 17 (3h)J((13)C(alpha), (1)H(N)) coupling constants ranging from 0 to 1.4 Hz where identified and all of positive sign. PMID- 10729274 TI - Magnetization transfer via residual dipolar couplings: application to proton proton correlations in partially aligned proteins. AB - A novel three-dimensional NMR experiment is reported that allows the observation of correlations between amide and other protons via residual dipolar couplings in partially oriented proteins. The experiment is designed to permit quantitative measurement of the magnitude of proton-proton residual dipolar couplings in larger molecules and at higher degree of alignments. The observed couplings contain data valuable for protein resonance assignment, local protein structure refinement, and determination of low-resolution protein folds. PMID- 10729275 TI - Mammalian oxygen sensing, signalling and gene regulation. AB - Oxygen is essential to the life of all aerobic organisms. Virtually every cell type is able to sense a limited oxygen supply (hypoxia) and specifically to induce a set of oxygen-regulated genes. This review summarizes current concepts of mammalian oxygen-sensing and signal-transduction pathways. Since the discovery of the hypoxia-inducible factors (HIFs), a great deal of progress has been made in our comprehension of how hypoxia induces the expression of oxygen-regulated genes. The alpha subunit of the heterodimeric transcription factors HIF-1, 2 and 3 is unstable under normoxia but is rapidly stabilized upon exposure to hypoxic conditions. Following heterodimerization with the constitutively expressed beta subunit, HIFs activate the transcription of an increasing number of genes involved in maintaining oxygen homeostasis at the cellular, local and systemic levels. PMID- 10729276 TI - Spray mechanism of the most primitive bombardier beetle (Metrius contractus). AB - The bombardier beetle Metrius contractus discharges its defensive secretion as a froth that clings to its body. When attacked from the rear, it allows the froth to build up over the gland openings near the abdominal tip; when attacked from the front, it conveys the secretion forwards along special elytral tracks. M. contractus has two-chambered defensive glands typical of bombardier beetles, and its secretion, like that of other bombardiers, is quinonoid and hot. Its frothing mechanism, however, is unique for bombardiers and possibly illustrative of the ancestral glandular discharge mechanism of these beetles. M. contractus, thus, could be the least derived of extant bombardiers. PMID- 10729277 TI - Concentrations of myoglobin and myoglobin mRNA in heart ventricles from Antarctic fishes. AB - We used a combined immunochemical and molecular approach to ascertain the presence and concentrations of both the intracellular oxygen-binding hemoprotein myoglobin (Mb) and its messenger RNA (mRNA) in 13 of 15 known species of Antarctic channichthyid icefishes. Mb protein is present in the hearts of eight species of icefishes: Chionodraco rastrospinosus, Chionodraco hamatus, Chionodraco myersi, Chaenodraco wilsoni, Pseudochaenichthys georgianus, Cryodraco antarcticus, Chionobathyscus dewitti and Neopagetopsis ionah. Five icefish species lack detectable Mb protein: Chaenocephalus aceratus, Pagetopsis macropterus, Pagetopsis maculatus, Champsocephalus gunnari and Dacodraco hunteri. Mb concentrations range from 0.44+/-0.02 to 0.71+/-0.08 mg Mb g(-)(1 )wet mass in heart ventricle of species expressing the protein. A Mb-mRNA-specific cDNA probe was used to quantify mRNA in five Mb-expressing icefishes. Mb mRNA was found in low but detectable amounts in Champsocephalus gunnari, one of the species lacking detectable Mb. Mb mRNA concentrations in heart ventricle from Mb-expressing species ranged from 0.78+/-0.02 to 16.22+/-2.17 pg Mb mRNA microg(-)(1 )total RNA). Mb protein and Mb mRNA are absent from the oxidative skeletal muscle of all icefishes. Steady-state concentrations of Mb protein do not parallel steady-state concentrations of Mb mRNA within and among icefishes, indicating that the concentration of Mb protein is not determined by the size of its mRNA pool. PMID- 10729278 TI - The interplay among cardiac ultrastructure, metabolism and the expression of oxygen-binding proteins in Antarctic fishes. AB - We examined heart ventricle from three species of Antarctic fishes that vary in their expression of oxygen-binding proteins to investigate how some of these fishes maintain cardiac function despite the loss of hemoglobin (Hb) and/or myoglobin (Mb). We quantified ultrastructural features and enzymatic indices of metabolic capacity in cardiac muscle from Gobionotothen gibberifrons, which expresses both Hb and Mb, Chionodraco rastrospinosus, which lacks Hb but expresses Mb, and Chaenocephalus aceratus, which lacks both Hb and Mb. The most striking difference in cellular architecture of the heart among these species is the percentage of cell volume occupied by mitochondria, V(v)(mit,f), which is greatest in Chaenocephalus aceratus (36.53+/-2.07), intermediate in Chionodraco rastrospinosus (20.10+/-0.74) and lowest in G. gibberifrons (15.87+/-0.74). There are also differences in mitochondrial morphologies among the three species. The surface area of inner mitochondrial membrane per volume of mitochondria, S(v)(imm, mit), varies inversely with mitochondrial volume density so that S(v)(imm,mit) is greatest in G. gibberifrons (29.63+/-1.62 microm(-)(1)), lower in Chionodraco rastrospinosus (21.52+/-0.69 microm(-)(1)) and smallest in Chaenocephalus aceratus (20.04+/-0.79 microm(-)(1)). The surface area of mitochondrial cristae per gram of tissue, however, is greater in Chaenocephalus aceratus than in G. gibberifrons and Chionodraco rastrospinosus, whose surface areas are similar. Despite significant ultrastructural differences, oxidative capacities, estimated from measurements of maximal activities per gram of tissue of enzymes from aerobic metabolic pathways, are similar among the three species. The combination of ultrastructural and enzymatic data indicates that there are differences in the density of electron transport chain proteins within the inner mitochondrial membrane; proteins are less densely packed within the cristae of hearts from Chaenocephalus aceratus than in the other two species. High mitochondrial densities within hearts from species that lack oxygen-binding proteins may help maintain oxygen flux by decreasing the diffusion distance between the ventricular lumen and mitochondrial membrane. Also, high mitochondrial densities result in a high intracellular lipid content, which may enhance oxygen diffusion because of the higher solubility of oxygen in lipid compared with cytoplasm. These results indicate that features of cardiac myocyte architecture in species lacking oxygen-binding proteins may maintain oxygen flux, ensuring that aerobic metabolic capacity is not diminished and that cardiac function is maintained. PMID- 10729279 TI - How do inositol and glucose modulate feeding in Manduca sexta caterpillars? AB - Many species of caterpillar possess taste cells that respond exceptionally vigorously to the sugar alcohol myo-inositol. We examined the functional significance of these inositol-sensitive taste cells in Manduca sexta caterpillars through an integrated series of electrophysiological and behavioral studies. Neural recordings from all the gustatory chemosensilla revealed that M. sexta have only two pairs of inositol-sensitive taste cells, which respond strongly and selectively to myo-inositol, and two pairs of sugar-sensitive taste cells, which respond relatively weakly to sugars (glucose and sucrose). Behavioral studies established that myo-inositol incites feeding and counteracts the inhibitory effects of aversive taste stimuli (e.g. caffeine) on feeding, but does not promote increased consumption once feeding has been initiated. In contrast, glucose and sucrose did not produce any robust effects on feeding. We failed to obtain any evidence of sensory inhibition between taste cells that responded to myo-inositol and caffeine, indicating that myo-inositol counteracts the inhibitory effects of caffeine on feeding through a central gustatory mechanism. We conclude that sensory input from the inositol-sensitive taste cells, but not the sugar-sensitive taste cells, plays an important role in regulating feeding in M. sexta. PMID- 10729280 TI - Fatigue quality of mammalian tendons. AB - When excised tendons are subjected to a prolonged load, whether constant or oscillatory, fatigue damage accumulates, leading eventually to rupture. 'Fatigue quality', assessed by the time-to-rupture under a given stress, was found to vary hugely among the tendons of a wallaby hind limb. This material property correlates with the varied stresses to which tendons from different anatomical sites are exposed in life. The correlation was demonstrated by subjecting each excised tendon to a load equal to the maximum isometric force that its muscle could have developed. The time-to-rupture was then approximately the same for each tendon, on average 4.2 h. A model is introduced in which damage is proposed as the trigger for adaptation of fatigue quality. The model aims, in particular, to explain why low-stressed tendons are not made of a 'better' material, although this clearly exists since it is used in high-stressed tendons. The principle of design to a minimum quality is viable in biology because of the availability of self-repair to balance routine damage. Clinical symptoms, to be included under the general heading of 'overuse injuries', will only arise when this balance fails. PMID- 10729281 TI - Excitatory and inhibitory roles of central ganglia in initiation of the insect ecdysis behavioural sequence. AB - Insects shed their old cuticle by performing the ecdysis behavioural sequence. To activate each subunit of this set of programmed behaviours in Manduca sexta, specific central ganglia are targeted by pre-ecdysis-triggering (PETH) and ecdysis-triggering (ETH) hormones secreted from Inka cells. PETH and ETH act on each abdominal ganglion to initiate, within a few minutes, pre-ecdysis I and II, respectively. Shortly thereafter, ETH targets the tritocerebrum and suboesophageal ganglion to activate the ecdysis neural network in abdominal ganglia through the elevation of cyclic GMP (cGMP) levels. However, the onset of ecdysis behaviour is delayed by inhibitory factor(s) from the cephalic and thoracic ganglia. The switch from pre-ecdysis to ecdysis is controlled by an independent clock in each abdominal ganglion and is considerably accelerated after removal of the head and thorax. Eclosion hormone (EH) appears to be one of the central signals inducing elevation of cGMP levels and ecdysis, but these actions are quite variable and usually restricted to anterior ganglia. EH treatment of desheathed ganglia also elicits strong production of cGMP in intact ganglia, suggesting that this induction occurs via the release of additional downstream factors. Our data suggest that the initiation of pre-ecdysis and the transition to ecdysis are regulated by stimulatory and inhibitory factors released within the central nervous system after the initial actions of PETH and ETH. PMID- 10729282 TI - Ocellar pigmentation and phototaxis in the nematode Mermis nigrescens: changes during development. AB - After 1 or 2 years of dormancy in the soil, Mermis nigrescens females emerge to lay eggs on vegetation where their grasshopper hosts are likely to feed. Females collected at this life stage exhibit a strong positive phototaxis and have a tubular region of pigmentation near the anterior tip consisting of concentrated oxyhaemoglobin. A previous investigation of the scanning motion of the 'head' and orientation of the 'neck' has implicated the shadowing of a photoreceptor inside the tube as the mechanism for identifying the direction of light during phototaxis. Here, we describe the development of the pigment in young adult females and investigate phototaxis in early developmental stages that lack the pigment. The orientation of the neck to a horizontal 420 nm stimulus (intensity 10(13 )photons s(-)(1 )cm(-)(2)) was measured for unpigmented fourth-stage larvae and immature adult females as well as mature females with pigmented ocelli. The orientation of the larvae and immature adults was weakly negative, whereas that of the mature adults was strongly positive. Head and neck movements were otherwise the same in the three stages. Thus, the pigmentation appears to be required for positive phototaxis, and the results provide further support for the shadowing role of ocellar haemoglobin. PMID- 10729283 TI - Sensory preconditioning in honeybees. AB - Sensory preconditioning means that reinforcement of stimulus A after unreinforced exposure to a compound AB also leads to responses to stimulus B. Here, we describe and analyze sensory preconditioning in an insect, the honeybee Apis mellifera. Using two-element odorant compounds in classical conditioning of the proboscis extension reflex, we found (i) that sensory preconditioning is not due to stimulus generalization, (ii) that paired, but not unpaired, presentation of elements supports sensory preconditioning, (iii) that simultaneous, but not sequential, exposure to the elements of the compound supports sensory preconditioning and (iv) that a single presentation of the compound yields maximal sensory preconditioning. The results are discussed with respect to configural and chain-like associative explanations for sensory preconditioning. We suggest an experience-dependent step of compound processing, establishing configural units, as an additional explanation for sensory preconditioning. PMID- 10729284 TI - Energy metabolism of the Virginia opossum during fasting and exercise. AB - Rates of oxygen consumption and CO(2) production were measured in Virginia opossums (Didelphis virginiana) during fasting and prolonged exercise to quantify changes in total energy expenditure and oxidative fuel selection. We hypothesized that fasting would cause metabolic depression and a progressive shift towards lipid utilization to spare alternative substrates. It was also predicted that prolonged exercise would cause the same relative changes in fuel preference as fasting, but on a compressed time scale. The results show that hypometabolism is not used by the Virginia opossum to cope with food deprivation. However, a rapid exhaustion of limited carbohydrate reserves is prevented through a sixfold reduction in the percentage contribution of carbohydrates to total energy expenditure made possible by an increase in lipid utilization. No protein sparing is observed in this species. Prolonged low-intensity exercise elicits a potent mobilization of lipids that allows maximal running time to be extended by delaying the depletion of limited carbohydrate reserves. We conclude that fasting and prolonged low-intensity exercise cause similar changes in the relative use of lipids and carbohydrates, but on a different time scale, supporting the idea that endurance exercise is the metabolic equivalent of 'accelerated fasting'. The absence of metabolic depression and protein sparing during fasting shows that such physiological strategies have not been necessary for the rapid range expansion towards the North recently shown by this species. PMID- 10729285 TI - Active NaCl absorption across split lamellae of posterior gills of the chinese crab Eriocheir sinensis: stimulation by eyestalk extract. AB - Split lamellae of the posterior gills of freshwater-adapted Chinese crabs (Eriocheir sinensis) were mounted in a modified Ussing-type chamber, and active and electrogenic absorption of Na(+) and Cl(-) were measured as positive (I(Na)) or negative (I(Cl)) short-circuit currents. Haemolymph-side addition of eyestalk extract stimulated I(Cl) by increasing both the transcellular Cl(-) conductance and the electromotive force for Cl(-) absorption. The effect was dose-dependent. Boiling the eyestalk extract did not change its effectiveness. The stimulating factor passed through dialysis tubing, indicating that it has a molecular mass of less than 2 kDa. R(p)cAMPS, a blocker of protein kinase A, reduced the stimulated I(Cl). Eyestalk extract stimulated I(Na) by increasing the transcellular Na(+) conductance at constant electromotive force. Amiloride-induced current-noise analysis revealed that stimulation of I(Na) was accompanied by an increase in the apparent number of open apical Na(+) channels at a slightly reduced single channel current. In addition to the electrophysiological experiments, whole gills were perfused in the presence and in the absence of putative transport stimulators, and the specific activities of the V-ATPase and the Na(+)/K(+) ATPase were measured. Eyestalk extract, theophylline or dibutyryl-cyclic AMP stimulated the activity of the V-ATPase, whereas the activity of the Na(+)/K(+) ATPase was unaffected. The simultaneous presence of R(p)cAMPS prevented the stimulation of V-ATPase by eyestalk extract or theophylline. PMID- 10729286 TI - Haemolymph Mg(2+) regulation in decapod crustaceans: physiological correlates and ecological consequences in polar areas. AB - Reptant decapod crustaceans are almost absent from the Southern Ocean south of the Antarctic Convergence. We tested the hypothesis that this may be due to the reduced ability of this group to regulate Mg(2+) levels in the haemolymph ([Mg(2+)](HL)). Mg(2+) acts as an anaesthetic in marine invertebrates and its level is higher in Reptantia (crabs such as Cancer spp., Chionoecetes spp., Maja spp., 30-50 mmol l(-)(1)) than in Natantia (prawns such as Pandalus spp., Palaemon spp., Crangon spp., 5-12 mmol l(-)(1)). We varied [Mg(2+)](HL) in three species of reptant decapod crustaceans, Carcinus maenas, Hyas araneus and Eurypodius latreillei, and investigated heart rate, the rate of oxygen consumption and levels of spontaneous and forced activity at different temperatures. The rate of oxygen consumption and heart rate increased significantly with reduction in [Mg(2+)](HL) over the entire temperature range investigated in E. latreillei. In H. araneus, an increase in metabolic and heart rates compared with control values was found only at temperatures below 2 degrees C. Forced and spontaneous activity levels increased significantly in the group of [Mg(2+)](HL)-reduced animals below 0 degrees C, at which control animals were mostly inactive. At a reduced [Mg(2+)](HL) of 5-12 mmol l(-)(1), which is the [Mg(2+)](HL) of caridean shrimps in the Southern Ocean, Q(10) and activation energy were reduced for all these variables and extended the temperature range over which physiological functions were maintained. We suggest that the high [Mg(2+)](HL) in Reptantia causes relaxation of the animals and reduces their scope for activity, especially at temperatures below 0 degrees C. The hypothesis that the synergistic effects of high [Mg(2+)](HL) and low temperature probably prevented the Reptantia from recolonizing the permanently cold water of polar areas is discussed. PMID- 10729287 TI - Disease and "dis-ease" in patients with uveal melanoma. PMID- 10729288 TI - Foveal translocation for exudative age related macular degeneration. PMID- 10729289 TI - Development and validation of a patient based measure of outcome in ocular melanoma. AB - BACKGROUND: Patients with uveal melanoma can be treated by a number of modalities. As none of the different treatments offer a survival advantage, a key factor in choosing among treatments is their differential impact on patients' quality of life. A short, patient based questionnaire was developed and validated for evaluating outcomes following treatment for uveal melanoma. METHODS: The 21 item measure of outcome in ocular disease (MOOD) assesses the patient's view of outcome in terms of visual function and the impact of treatment. The reliability and validity of the three MOOD scores (total, vision, impact) were evaluated in 176 patients who had been treated for uveal melanoma (75 brachytherapy, 78 proton beam radiotherapy, 23 enucleation). Of these, 165 patients also completed the SF 36. RESULTS: All three MOOD scales met standard criteria for acceptability, reliability, and validity. The proportion of missing data was low, and responses to all items were well distributed across response categories. Internal consistency, assessed by Cronbach's alpha coefficients, exceeded the standard criterion of 0. 70 for all three summary scores. Item total correlations ranged from 0.22 to 0.77 (mean item total correlation 0.58), indicating good homogeneity. Test-retest correlations for all three summary scores exceeded 0.85. Scaling assumptions, assessed by item convergent and discriminant validity correlations, were met for the vision and impact scores. The MOOD showed good content validity, as assessed by review by ophthalmologists and patients. Construct validity was demonstrated by high intercorrelations between the vision and impact scores and the total scale; higher scores for patients who reported being very satisfied compared with those who were not very satisfied and for those who reported persistent red eye compared with those who did not have this complication (known group differences/hypothesis testing); moderate correlations between the MOOD and the SF-36 and visual acuity (convergent validity); and low correlations between the MOOD and age and sex (discriminant validity). CONCLUSIONS: The MOOD is a practical and scientifically sound patient based measure which can be used in research and audit to evaluate outcomes following treatment for uveal melanoma. It takes 5 minutes to complete and meets standard psychometric criteria for reliability and validity. PMID- 10729290 TI - Foveal relocation by redistribution of the neurosensory retina. AB - AIM: To describe a new surgical technique for foveal relocation, and to report the outcome in nine patients treated with this procedure. METHODS: Nine consecutive patients with subfoveal choroidal neovascular membranes (CNVMs) secondary to age related macular degeneration underwent foveal relocation surgery by redistribution of the neurosensory retina (RNR). The technique involved induction of a retinal detachment via a single retinotomy, relocation of the fovea by "sweeping" the retinal tissue with a retinal brush, and stabilisation of the retina in its new location using perfluorocarbon liquid peroperatively and silicone oil postoperatively. RESULTS: In eight of nine eyes successful relocation of the fovea was achieved; in one eye the CNVM remained in a subfoveal location postoperatively. Visual acuity improved in two eyes, remained unchanged in three, and decreased in four eyes after a median follow up of 4 months (range 2.5-6 months). Complications included rupture of a foveal cyst with the development of a macular hole in one eye and epimacular membrane formation in another eye. In two eyes, macular retinal vessel closure occurred at the time of laser photocoagulation; one of these eyes later developed cystoid macular oedema and the other an epiretinal membrane. Recurrence of the CNVM was observed in one eye, but was controlled with further laser treatment. CONCLUSIONS: Foveal relocation by RNR appears to be feasible, obviating the need for extensive retinotomies or scleral shortening. PMID- 10729291 TI - Genetic heterogeneity in familial exudative vitreoretinopathy; exclusion of the EVR1 locus on chromosome 11q in a large autosomal dominant pedigree. AB - BACKGROUND/AIMS: Familial exudative vitreoretinopathy (FEVR) is associated with mutations in the Norrie disease gene in X linked pedigrees and with linkage to the EVR1 locus at 11q13 in autosomal dominant cases. A large autosomal dominant FEVR family was studied, both clinically and by linkage analysis, to determine whether it differed from the known forms of FEVR. METHODS: Affected members and obligate gene carriers from this family were examined by slit lamp biomicroscopy, indirect ophthalmoscopy, and in some cases fluorescein angiography. Patient DNAs were genotyped for markers at the EVR1 locus on chromosome 11q13. RESULTS: The clinical evaluation in this family is consistent with previous descriptions of FEVR pedigrees, but linkage analysis proves that it has a form of FEVR genetically distinct from the EVR1 locus on 11q. CONCLUSION: This proves that there are at least three different loci associated with comparable FEVR phenotypes, a situation similar to that existing for many forms of retinal degeneration. PMID- 10729292 TI - COL2A1 exon 2 mutations: relevance to the Stickler and Wagner syndromes. AB - AIMS: To compare the clinical and molecular genetic features of two phenotypically distinct subgroups of families with type 1 Stickler syndrome. BACKGROUND: Stickler syndrome (hereditary arthro-ophthalmopathy, McKusick Nos 108300 and 184840) is a dominantly inherited disorder of collagen connective tissue, resulting in an abnormal vitreous, myopia, and a variable degree of orofacial abnormality, deafness, and arthropathy. Stickler syndrome is the commonest inherited cause of rhegmatogenous retinal detachment in childhood with a risk of giant retinal tear (GRT) which is commonly bilateral and a frequent cause of blindness. METHOD: Pedigrees were identified from the vitreoretinal service database and subclassified according to vitreoretinal phenotype. Ophthalmic, skeletal, auditory, and orofacial features were assessed. Linkage analysis was carried out with markers for the candidate genes COL2A1, COL11A1, and COL11A2. The COL2A1 gene was amplified as five overlapping PCR products. Direct sequencing of individual exons identified mutations. RESULTS: Eight families exhibiting the type 1 vitreous phenotype were studied. Seven were consistent for linkage to COL2A1, with lod scores ranging from 2.1 to 0.3. In most instances linkage to COL11A1 and COL11A2 could be excluded. One family was analysed without prior linkage analysis. Three of the families exhibited a predominantly ocular phenotype with minimal or absent systemic involvement and were found to have mutations in exon 2 of COL2A1. Five other pedigrees with an identical ocular phenotype plus orofacial, auditory, and articular involvement had mutations in others regions of the COL2A1 gene. None of the pedigrees exhibited the characteristic lenticular, retinal pigment epithelial, or choroidal changes seen in Wagner syndrome. CONCLUSIONS: These data confirm that type 1 Stickler syndrome is caused by mutations in the gene encoding type II collagen (COL2A1). In addition, data are submitted showing that mutations involving exon 2 of COL2A1 are characterised by a predominantly ocular variant of this disorder, consistent with the major form of type II procollagen in non-ocular tissues having exon 2 spliced out. Such patients are all at high risk of retinal detachment. This has important implications for counselling patients with regard to the development of systemic complications. It also emphasises the importance and reliability of the ophthalmic examination in the differential diagnosis of this predominantly ocular form of Stickler syndrome from Wagner's vitreoretinopathy. PMID- 10729293 TI - Prevention of visual field defects after macular hole surgery. AB - BACKGROUND/AIM: The pathogenesis of visual field loss associated with macular hole surgery is uncertain but a number of explanations have been proposed, the most convincing of which is the effect of peeling of the posterior hyaloid, causing either direct damage to the nerve fibre layer or to its blood supply at the optic nerve head. The purpose of this preliminary prospective study was to determine the incidence of visual field defects following macular hole surgery in cases in which peeling of the posterior hyaloid was confined only to the area of the macula. METHODS: 102 consecutive eyes that had macular hole surgery had preoperative and postoperative visual field examination using a Humphrey's perimeter. A comparison was made between two groups: I, those treated with vitrectomy with complete posterior cortical vitreous peeling; and II, those treated with a vitrectomy with peeling of the posterior hyaloid in the area of the macula but without attempting a complete posterior vitreous detachment. Specifically, no attempt was made to separate the posterior hyaloid from the optic nerve head. Eyes with stage II or III macular holes were operated. Autologous platelet concentrate and non-expansile gas tamponade was used. Patients were postured prone for 1 week. RESULTS: In group I, 22% of patients were found to have visual field defects. In group II, it was possible to separate the posterior hyaloid from the macula without stripping it from the optic nerve head and in these eyes no pattern of postoperative visual field loss emerged. There were no significant vision threatening complications in this group. The difference in the incidence of visual field loss between group I and group II was significant (p=0.02). The anatomical and visual success rates were comparable between both groups. CONCLUSION: The results from this preliminary study suggest that the complication of visual field loss after macular surgery may be reduced if peeling of the posterior hyaloid is confined to the area of the macula so that the hyaloid remains attached at the optic nerve head. The postoperative clinical course does not appear to differ from eyes in which a complete posterior vitreous detachment has been effected during surgery. PMID- 10729294 TI - Fluoroquinolone and fortified antibiotics for treating bacterial corneal ulcers. AB - AIM: To compare the clinical efficacy of commercially available fluoroquinolone drops with the use of combined fortified antibiotics (tobramycin 1.3%-cefazolin 5%) in treatment of bacterial corneal ulcer. METHODS: The medical records of 140 patients with a diagnosis of bacterial corneal ulcer who were admitted to the Royal Victorian Eye and Ear Hospital, Melbourne, Australia between January 1993 and December 1997 were reviewed retrospectively. Final outcome and results of 138 ulcer episodes were compared between those treated initially with fluoroquinolone and those who received fortified antibiotics. Two patients had been treated with chloramphenicol. RESULTS: No significant treatment difference was found between fluoroquinolone and fortified therapy in terms of final visual outcome. However, serious complications such as corneal perforation, evisceration, or enucleation of the affected eye were more common with fluoroquinolone therapy (16.7%) compared with the fortified therapy (2.4%, p= 0.02). The duration of intensive therapy was less with fluoroquinolone especially in those over 60 years of age (4 days v 6 days, p=0.01). Hospital stay was also less in the fluoroquinolone group compared with the fortified group for all patients and was significantly less with fluoroquinolone treatment (7 days v 10 days, p=0.02) in patients in the age group over 60 years old. CONCLUSIONS: Monotherapy with fluoroquinolone eye drops for the treatment of bacterial corneal ulcers led to shorter duration of intensive therapy and shorter hospital stay compared with combined fortified therapy (tobramycin-cefazolin). This finding may have resulted from quicker clinical response of healing as a result of less toxicity found in the patients treated with fluoroquinolone. However, as some serious complications were encountered more commonly in the fluoroquinolone group, caution should be exercised in using fluoroquinolones in large, deep ulcers in the elderly. PMID- 10729295 TI - Analysis of variation in success rates in conjunctival autografting for primary and recurrent pterygium. AB - AIMS: To evaluate the success rates of conjunctival autografting for primary and recurrent pterygium performed in a tertiary ophthalmic centre. METHODS: The outcome of 139 cases with primary pterygia and 64 cases with recurrent pterygia who underwent excision with conjunctival autografting was retrospectively reviewed. Outcome was evaluated in terms of recurrence of pterygia onto the cornea. The recurrence rates were determined using Weibull survival functions, in a mixture model that included a component allowing for cure. The suitability of this model was verified using Turnbull's non-parametric method for interval censored data (1974). Estimated recurrence free probabilities were based on the fitted Weibull survival curves. RESULTS: Mean follow up was 8.4 months in the primary group, and 9.5 months for the recurrent group. 29 out of 139 cases of primary pterygia recurred (20.8%) while 20 out of 64 cases in the recurrent group (31.2%) recurred. Recurrence rates varied widely among surgeons, ranging from 5% to 82%. Recurrence rates were inversely related to previous experience in performing conjunctival grafting. The recurrence free probability was 84% at 3 months, 73% at 1 year for primary pterygia, and 80% at 3 months, 67% at 1 year for recurrent pterygia. There was no statistical difference in recurrence rates between primary and recurrent groups (p= 0.80). CONCLUSION: The success of conjunctival autografting for pterygium in this series varies widely, and may be related to a significant learning curve or differing surgical techniques for this procedure. This may account for the wide variation in reported success of this procedure in the ophthalmic literature. PMID- 10729297 TI - No strong association between alleles of tumour necrosis factor alpha and interleukin-1 receptor antagonist and corneal melting associated with systemic vasculitis. AB - AIMS: To investigate polymorphism within the tumour necrosis factor alpha (TNF alpha) promoter region and within the interleukin-1 receptor antagonist (IL-1Ra) gene in a group of patients with vasculitis associated corneal melting. METHODS: The polymorphic regions at position -308 on the TNF-alpha promoter region and in intron 2 of the IL-1Ra gene were amplified by the polymerase chain reaction (PCR). The resultant products were separated by electrophoresis on agarose gels and visualised by ethidium bromide staining. Genotype and allele frequencies for the 20 patients were compared with healthy controls from the same geographical area. RESULTS: The allele frequencies in the patient and control groups respectively for the TNF-alpha and IL-1Ra sites studied were as follows: TNF1, 82.5% and 80.2%; TNF2, 17.5% and 19.8%; IL-1Ra*1, 82. 5% and 78.3%; IL-1Ra*2, 15% and 20%; IL-1Ra*3 2.5% and 1.5%. Although there was a trend for the IL1Ra*2 allele to be more common in the control group, no allele was found to have a statistically significantly association with the patient group: TNF1 p = 0.89; TNF2 p = 0.89; IL-1Ra*1 p = 0.65; IL-1Ra*2 p = 0.68; IL-1Ra*3 p= 0. 50. CONCLUSIONS: The results suggest that the polymorphic alleles of TNF-alpha and IL 1Ra studied play little or no part in the susceptibility to corneal melting among these patients with systemic vasculitis. PMID- 10729296 TI - Late onset lattice corneal dystrophy with systemic familial amyloidosis, amyloidosis V, in an English family. AB - AIMS: To establish a clinical and molecular diagnosis in a family with late onset lattice corneal dystrophy. METHODS: Linkage analysis, single strand conformation polymorphism (SSCP) analysis, and direct sequencing of genomic DNA were performed. A review of the patients' clinical symptoms and signs was undertaken. RESULTS: Linkage to chromosome 9q34 was established and a mutation in the gelsolin gene was found in affected individuals. Numerous symptoms experienced by the patients were attributable to this mutation. CONCLUSION: A diagnosis of amyloidosis type V (familial amyloidosis, Finnish type, FAF/Meretoja syndrome/gelsolin related amyloidosis) was made. This is the first case of amyloidosis type V described in the UK. This emphasises the importance of recognition of the extraocular manifestations of eye disease both in the diagnosis and management of the patient. In addition, these findings can help molecular geneticists in their search for disease-causing mutations. PMID- 10729298 TI - Oxygen application by a nasal probe prevents hypoxia but not rebreathing of carbon dioxide in patients undergoing eye surgery under local anaesthesia. AB - BACKGROUND/AIM: Hypoxia and carbon dioxide rebreathing are potential problems during eye surgery in spontaneously breathing patients. The aim of the present study was to determine effectiveness of nasal application of oxygen to prevent hypoxia and carbon dioxide accumulation in spontaneously breathing patients undergoing cataract surgery. METHODS: Oxygenation and carbon dioxide rebreathing were examined in 40 elderly patients using two different methods of oxygen supply nasal v ambient air-with a constant flow of 2 l/min. Partial pressure of carbon dioxide under ophthalmic drapes, transcutaneous pressure of carbon dioxide, and the respiratory rate were measured during 25 minutes while oxygen was supplied via a nasal cannula or into the ambient air under the drapes. RESULTS: In both groups carbon dioxide accumulation under the drapes, carbon dioxide rebreathing, tachypnoea, and an increase in peripheral oxygen saturation occurred. No significant differences were found between the two methods. CONCLUSION: Nasal application of oxygen prevented hypoxia but did not prevent carbon dioxide accumulation in patients undergoing eye surgery under retrobulbar anaesthesia. Additionally, as a side effect when using nasal probes, irritation of the nose was described in half of the patients investigated. PMID- 10729299 TI - A new digital optic disc stereo camera: intraobserver and interobserver repeatability of optic disc measurements. AB - AIMS: To investigate the intraobserver and interobserver repeatability of optic disc measurement using a new digital optic disc stereo camera. METHODS: 112 consecutive new patients presenting to a glaucoma service had dilated optic disc photography performed using a new digital stereo camera (Discam, Marcher Enterprises Ltd, Hereford). The images were analysed by two masked observers using a stereo viewer and computer simulated stereopsis. Vertical and horizontal cup:disc ratios (CDR), cup area:disc area, and cup circumference:disc circumference were computed. Intraobserver and interobserver repeatability analyses were performed. Intraclass correlation coefficients (ICC) and 95% tolerance for change (TC) were computed. RESULTS: 220 optic discs were photographed, of which 196 were suitable for analysis (10 were of poor image quality and 14 had anomalous discs). Mean age of patients was 65 years, 60 were male and 48 female. For intraobserver measurements of: horizontal CDR, ICC = 0.94, TC = 0.11 (15% of range); vertical CDR, ICC = 0.92, TC = 0.14 (16% of range); cup area:disc area, ICC = 0.95, TC = 0.10 (13% of range), and cup circumference:disc circumference, ICC = 0.95, TC = 0.09 (14% of range). For interobserver measurements of: horizontal CDR, ICC = 0.89, TC = 0.14 (19% of range); vertical CDR, ICC = 0.90, TC = 0.14 (16% of range); cup area:disc area, ICC = 0.92, TC = 0.13 (16% of range), and cup circumference:disc circumference, ICC = 0.90, TC = 0.12 (17% of range). Systematic bias between observers was within acceptable limits. CONCLUSIONS: Digital stereo disc photography and analysis provide repeatable measures of optic disc variables. The results compare favourably with ophthalmoscopic and stereophotographic methods of assessment of the optic disc. PMID- 10729300 TI - Both CD4+ and CD8+ T cells are involved in protection against HSV-1 induced corneal scarring. AB - AIM: To determine the relative impact of CD4+ T cells and CD8+ T cells in protecting mice against ocular HSV-1 challenge. METHODS: CD4+ T cell knockout mice (CD4-/- mice), CD8+ T cell knockout mice (CD8-/- mice), and mice depleted for CD4+ or CD8+ T cells by antibody (CD4+ depleted and CD8+ depleted mice), were examined for their ability to withstand HSV-1 ocular challenge. The parental mice for both knockout mice were C57BL/6J. RESULTS: These results suggest that: (1) both CD4+ deficient mice (CD4-/- and CD4+ depleted mice) and CD8+ deficient mice (CD8-/-, and CD8+ depleted mice) developed significantly more corneal scarring than their C57BL/6J parental strain; (2) the duration of virus clearance from the eyes of the CD4+ deficient mice was 4 days longer than that of the CD8+ deficient mice; and (3) the severity of corneal scarring in the CD4+ deficient mice was approximately twice that of the CD8+ deficient mice. CONCLUSIONS: It was reported here that: (1) CD4+ and CD8+ T cells were both involved in protection against lethal ocular HSV-1 infection; and (2) CD4+ and CD8+ T cells were both involved in protection against HSV-1 induced corneal scarring. PMID- 10729301 TI - HLA-B27 typing in the categorisation of uveitis in a HLA-B27 rich population. AB - AIMS: To determine whether HLA-B27 typing helps the clinician in the diagnostic examination of uveitis in a HLA-B27 rich population and also whether the clinical picture of HLA-B27 positive unilateral acute or recurrent anterior uveitis (AAU) is distinguishable from the idiopathic negative form. METHODS: During a 3 year period 220 consecutive patients with undetermined uveitis at onset were examined in the Helsinki University Eye Clinic. HLA-B27 antigen was tested for 85% of the patients. Other laboratory or x ray examinations were performed on the basis of the anatomical classification of uveitis and the biomicroscopic features characteristic of uveitis associated systemic diseases. RESULTS: HLA-B27 antigen was found significantly more often in patients with anterior (71%) and acute/recurrent unilateral (79%) uveitis than in patients with intermediate, posterior panuveitis (7%), and chronic (7%) or bilateral (12%) forms. Of the 16 cases of HLA-B27 negative unilateral AAU, five showed biomicroscopic features representing uveitis entities. The remaining 11 cases did not differ in any respect from the cases of HLA-B27 positive unilateral AAU. CONCLUSION: HLA-B27 antigen helps the clinician in the diagnostic examination of unilateral AAU. Positive test results serve as a clue to search for spondyloarthropathies, and negative results indicate the need to look for specific uveitis entities and other systemic diseases. The occurrence of HLA-B27 positivity in conjunction with uveitis entities other than unilateral AAU is of the same level or less than in the population of Finland in general. PMID- 10729302 TI - Regulation of plasminogen activation by TGF-beta in cultured human retinal endothelial cells. AB - BACKGROUND/AIMS: Regulation of plasmin mediated extracellular matrix degradation by vascular endothelial cells is important in the development of angiogenesis. The aim was to determine whether transforming growth factor beta (TGF-beta) affected the regulation of components of the plasminogen system by human retinal endothelial cells, in order to define more clearly the role of TGF-beta in retinal angiogenesis in the context of diabetes mellitus. METHODS: Human retinal endothelial cells (HREC) were isolated from donor eyes and used between passages 4-8. The cells were cultured in medium supplemented with 2, 5, 15, or 25 mM glucose, plus or minus TGF-beta (1 ng/ml). The concentrations of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and plasminogen activator inhibitor type 1 (PAI-1) in cell conditioned medium were determined by ELISA and the level of PAI-1 mRNA was determined using northern hybridisation. Cell associated plasminogen activity was determined using a clot lysis assay and a chromogenic assay. RESULTS: Under basal conditions (5 mM glucose), HREC produced PAI-1, t-PA, and trace amounts of u-PA. Cell surface plasminogen activation observed by lysis of fibrin or by cleavage of chromogenic substrate, was mediated by t-PA. Glucose at varying concentrations (2-25 mM) had no significant effect on t-PA mediated clot lysis. In contrast, treatment with TGF-beta resulted in increased synthesis of PAI-1 protein and mRNA. The increased expression of the PAI-1 mRNAs by TGF-beta did not occur uniformly, the 2.3 kb mRNA transcript was preferentially increased in comparison with the 3.2 kb mRNA (p<0.05). CONCLUSIONS: These data demonstrate that TGF-beta increases PAI-1 and decreases cell associated lysis. This is sufficient to decrease the normal lytic potential of HREC. PMID- 10729303 TI - Responses of different cell lines from ocular tissues to elevated hydrostatic pressure. AB - BACKGROUND/AIMS: Mechanical forces are thought to induce cellular responses through activation of signalling pathways. Cells within the intraocular environment are exposed to constant changes in the levels of intraocular pressure. In this study, an attempt was made to determine the acute effects of elevated hydrostatic pressure on different intraocular cells grown in culture. METHODS: Different cell lines derived from ocular tissues including non-pigmented and pigmented ciliary epithelium, trabecular meshwork, retina, and lamina cribrosa were incubated in a pressurised chamber at 50 mm Hg in a culture incubator at 37 degrees C for up to 6 hours. Control cells were incubated at atmospheric pressure. The viability of the cells was examined using their intracellular esterase activity. The morphology and cytoskeleton of the cells were investigated using microscopy and phalloidin staining. Adenylyl cyclase activity was assessed by measuring the conversion of [(3)H]-cAMP from [(3)H]-ATP in response to elevated hydrostatic pressure for 1-6 hours. In addition, at the end of incubation period under elevated hydrostatic pressure the recovery of adenylyl cyclase activity to control levels was examined. RESULTS: Cell viability did not change following exposure to elevated hydrostatic pressure for 6 hours. Cells subjected to elevated hydrostatic pressure demonstrated morphological differences characterised by a more rounded shape and a redistribution of actin stress fibres that was most prominent in lamina cribrosa astrocytes. A time dependent increase in basal adenylyl cyclase activity, and a decrease in maximum forskolin stimulated activity were observed in all cell lines following exposure to elevated hydrostatic pressure. CONCLUSION: These observations demonstrate that cell lines from different ocular tissues are sensitive to changes in external pressure in vitro. They exhibit morphological and cytoskeletal changes as well as significant alterations of intracellular adenylyl cyclase activity following exposure to acute and sustained levels of elevated hydrostatic pressure of up to 6 hours' duration. PMID- 10729304 TI - What factors influence cataract waiting list time? AB - AIMS: To determine whether there were any specific factors that influenced waiting list time (WLT) for patients undergoing cataract surgery. METHODS: 70 preoperative cataract patients were interviewed by one of the authors using a questionnaire to score visual acuity, coexisting ocular pathology and disabilities, threat to independent living/employment, and perceived visual handicap for detailed, gross, and driving vision. Individuals were analysed separately according to whether it was their first or second cataract operation. RESULTS: The median WLT for first eye surgery was 9 months (n = 31) and 13 months for second eye surgery (n = 36). The WLT ranged from 2 to 25 months for first eyes and 0.25-18 months for second eyes. Where there was a perceived threat to independent living or employment the WLT was found to be significantly shorter than the median. A high overall score correlated with a shorter WLT. Surgical priority was also given to individuals with anisometropia >3 dioptres. CONCLUSION: This study has demonstrated that there are specific factors that influence clinicians when prioritising patients for cataract surgery. PMID- 10729305 TI - Use of vision tests in clinical decision making about cataract surgery: results of a national survey. AB - AIM: To provide information on the use of vision tests in clinical decision making about cataract surgery in the UK. METHOD: A questionnaire survey was mailed to 703 consultant ophthalmologists. RESULTS: A response rate of 70% was obtained. Monocular distance visual acuity was the only visual function that was tested routinely by all surgeons. Supplementary use of contrast sensitivity and glare testing was low. Many surgeons (35%) were willing to consider surgery at acuity levels better than 6/9 and a small but substantial number (12%) indicated that they did not use an acuity criterion. Being prepared to consider surgery at relatively good levels of acuity was not associated with more common use of other tests of vision. CONCLUSION: Many UK surgeons are prepared to consider cataract extraction at relatively good levels of visual acuity and use other vision tests infrequently. PMID- 10729306 TI - Sub-Tenon's local anaesthesia: the effect of hyaluronidase. AB - AIMS: A prospective, randomised, double blind study was used to investigate the effect of hyaluronidase on the quality of block achieved with sub-Tenon's local anaesthesia. METHODS: 150 patients scheduled for elective cataract surgery were randomly allocated to either sub-Tenon's block with 3 ml lignocaine 2%/adrenaline 1:200 000 alone or with the addition of 30 IU/ml of hyaluronidase. The blocks were assessed for degree of akinesia and reduction of eyelid movement, and also post-injection and postoperative pain scores. RESULTS: Akinesia and reduction of eyelid movement measured 10 minutes after injection were significantly better in the group with hyaluronidase added to the anaesthetic solution. Postoperative pain scores were not significantly different between the two groups but the post injection pain score was greater (marginally significant) in the group with hyaluronidase added. CONCLUSION: The addition of hyaluronidase significantly improves the quality of the motor blockade achieved with sub-Tenon's local anaesthesia, but has no effect on the sensory blockade. PMID- 10729308 TI - Norman henry ashton CBE, DSC (LOND), FRCP, FRCS, FRCPATH, FRCOPHTH, FRS, KSTJ, b 11 september 1913, d 4 january 2000 PMID- 10729307 TI - Painful blind eye: efficacy of enucleation and evisceration in resolving ocular pain. AB - AIMS: To assess the effectiveness of enucleation or evisceration in relieving pain from painful blind eyes. METHODS: 24 patients with intractable ocular pain underwent enucleation or evisceration with or without an orbital implant. RESULTS: Complete pain relief was achieved in all patients at an average time of 3 months (range 1-15 months). Seven patients required further medical or surgical treatment in addition to removal of the globe. CONCLUSION: Enucleation and evisceration were effective in relieving ocular pain in all patients with a painful blind eye in our study. However, complications of surgery and orbital implants can cause recurrent pain. PMID- 10729309 TI - Validation of a simple Yq deletion screening programme in an ICSI candidate population. AB - This study reports on the validation of a diagnostic screening programme for Yq deletions in a population of infertile men. First, an unselected group of 402 intracytoplasmic sperm injection (ICSI) candidate patients was screened prospectively by means of three polymerase chain reactions (PCR) each with one marker in the region AZFa, AZFb or AZFc. With this screening strategy, eight males (2.2%) were found to carry a deletion in Yq11. Secondly, a subgroup of males were further analysed by multiplex PCR with 27 sequence-tagged sites. In this group of 229 cytogenetically normal males with azoospermia, cryptozoospermia or extreme oligozoospermia, including some patients with varicocele or a history of cryptorchidism, only one additional microdeleted patient was found with the multiplex PCR. Hence we obtained a frequency of 2.2% (9/402) or 4% (9/229) in the unselected and selected patient groups respectively. We conclude that in a diagnostic programme for Yq deletions in ICSI candidates it might be sufficient to use only four markers representing the three AZF regions and a more distal region in AZFc. In this way, it is possible to detect most, if not all, Yq deletions which might be the causal factor in the patient's infertility. PMID- 10729310 TI - Absence of mutations involving the INSL3 gene in human idiopathic cryptorchidism. AB - The aetiology of cryptorchidism is for the most part unknown and appears to be multifactorial. Recently, a product of Leydig cells termed Leydig insulin-like hormone (INSL3) has been proposed as a putative trophic hormone of the first part of descent. Absence of Insl3 in male mice results in bilateral cryptorchidism and mutations involving this gene may be a cause of cryptorchidism in man. We sequenced both exons of the human INSL3 gene in 31 men who presented with idiopathic unilateral or bilateral cryptorchidism. The only sequence variant was an amino acid substitution in the C-peptide of the molecule. This change was also found in a control group of normal fertile men indicating that it is a polymorphism unrelated to the phenotype. These results suggest that mutations involving the human INSL3 gene are not a common cause of cryptorchidism in man. PMID- 10729311 TI - Molecular characterization of a voltage-gated potassium channel expressed in rat testis. AB - Potassium (K(+)) channels are present in both mammalian testis and spermatozoa. Immunofluorescent detection of sperm-bound biotinylated charybdotoxin, an inhibitor of Ca(2+)-activated and of delayed rectifier K(+) channels, indicated that these ion channels are uniformly distributed over the surface of both heads and tails of unfixed rat epididymal spermatozoa. Reverse transcription-polymerase chain reaction (RT-PCR) analysis on rat testis RNA with PCR primers, based on known nucleotide sequences of different classes of K(+) channels, amplified sequences homologous to delayed rectifier K(+) channels. In-situ RT-PCR on rat testis sections showed that these K(+) channel transcripts are present in the cytoplasm of primary spermatocytes and post-meiotic elongating spermatids. Northern blot analysis of various rat tissues identified multiple K(+) channel transcripts, some of which were observed only in testis. An attempt to obtain a full length rat testis K(+) channel cDNA sequence gave an assembled sequence of 2693 base pairs with >90% homology to a delayed rectifier K(+) channel, Kv1.3. A method for rapid amplification of cDNA ends was employed to amplify the 5' sequences of the rat testis cDNA but a unique sequence could not be obtained. DNA sequencer traces suggest that multiple related K(+) channels which differed at their 5' ends were amplified in rat testis. PMID- 10729312 TI - Mapping of the POF1 locus and identification of putative genes for premature ovarian failure. AB - We have identified a breakpoint on the X chromosome which is associated with premature ovarian failure (POF). Using polymerase chain reaction (PCR) probes of polymorphic microsatellites and fluorescent in-situ hybridization (FISH), this breakpoint has been narrowed to a region of 300 kb spanned by two P1 artificial chromosomes (PAC). Computer exon prediction and gene homology programs revealed three genes in this area. Our results suggest that two of these genes, HS6ST and E2F, and LINE 1 elements may be involved in ovarian development. Interruption of these genes could be the cause of POF. This study demonstrates how various molecular techniques and bioinformatic searches can complement each other in order to solve a clinical problem. PMID- 10729314 TI - Luteinized human granulosa cells are associated with endogenous basement membrane like components in culture. AB - Human granulosa cells (GC), prepared from follicular aspirates using a non enzymic method, were maintained in culture on chamber slides in a defined medium without additional attachment factors or extracellular matrix (ECM). In this system, GC clustered to a limited extent and attached only loosely to the substratum necessitating medium replacement through repeated partial changes to avoid cell loss. Using this new culture system, cell size and progesterone production per cell increased, consistent with continuing luteinization. These processes were associated with maintenance and deposition of endogenous ECM components. Thus, pericellular heparan sulphate proteoglycan (HSPG) was clearly visible by immunocytochemistry around the luteinized GC after culture. Also progressive accumulation of laminin (particularly alpha(2)-, beta(1)- and gamma(1)-subunits) during culture was shown by Western blotting of GC extracts. Small patches of collagen IV, shown to be already present between freshly prepared GC, were maintained in culture. A clear effect of gonadotrophin on the maintenance of progesterone production in culture was paralleled by an apparent increased pericellular deposition of HSPG. To conclude, luteinization and maintenance of the GC-derived layer of the corpus luteum is likely to involve deposition and conservation of pericellular ECM components. PMID- 10729313 TI - Expression of TP and TIE2 genes in normal ovary with corpus luteum and in ovarian cancer: correlation with ultrasound-derived peak systolic velocity. AB - Transvaginal colour and pulsed Doppler ultrasonography analyses of blood flow velocity have indicated that intra-tumoral peak systolic velocity (PSV) is a good indicator of ovarian malignancy. Therefore, we examined whether there was an association between the expression of angiogenic genes, e.g. thymidine phosphorylase (TP) and TIE2 and the PSV of blood flow in normal and cancerous ovaries. Initially, 40 patients were examined by transvaginal ultrasonography and 23 ovaries were surgically removed; 14 were normal with corpora lutea (CL) and nine showed ovarian cancer. The ovarian tissue was dissected according to areas of high blood velocity and gene expression was examined using the reverse transcriptase-polymerase chain reaction (RT-PCR). No significant differences were found between PSV in the normal ovary with CL and ovarian cancer (P = 0.95). TP gene expression was significantly higher in ovarian cancer than in normal ovary with CL (P = 0.02), while TIE2 gene expression was not significantly different (P = 0.186). There was a significant correlation between TIE2 gene expression and PSV in the normal ovary with CL (r = 0.633, P = 0.015), while TP expression was significantly correlated with the PSV in ovarian cancer (r = 0.757, P = 0.018). These results indicate that there is a biological difference between physiological and pathological angiogenesis, TIE2 having a physiological role and TP being involved in pathological angiogenesis. PMID- 10729315 TI - Localization of proteasomes in human oocytes and preimplantation embryos. AB - In the present study we describe the localization of proteasomes in human oocytes, apoptotic preimplantation embryos, and triploid preimplantation embryos by means of immunolabelling with the MCP21 monoclonal antibody detected by confocal microscopy. While in the oocytes proteasomes are scattered throughout the cytoplasm, in the pronuclear zygote they appear to concentrate at the periphery of the cytoplasm and do not enter the pronuclei. During early cleavage stages, proteasome immunolabelling is concentrated in the nuclei, while the examination of triploid blastocysts showed that proteasomes had a similar cellular distribution to somatic cell lines, i.e. in the nuclei but not in the nucleoli or the cytoplasm. It appears that the distribution of proteasomes dramatically changes during human preimplantation embryo development. PMID- 10729316 TI - Leukaemia inhibitory factor in the endometrium of the common marmoset Callithrix jacchus: localization, expression and hormonal regulation. AB - In the present study, changes in the immunohistochemical localization of leukaemia inhibitory factor (LIF) in the endometrium during various phases of ovarian cyclicity of the common marmoset have been reported. LIF was absent during the early and late follicular phases. LIF was observed mainly in the cytoplasm of the endometrial glands during the early luteal phase, reached maximum intensity during the mid-luteal phase and declined again during late luteal phase. In-situ hybridization also showed a similar cyclic pattern in the expression of LIF. Stromal cells only showed signals for LIF during the mid luteal phase. In ovariectomized marmosets, graded dosages of oestradiol alone failed to induce the appearance of LIF protein. Progesterone treatment following oestradiol priming, however, induced distinct glandular localization of LIF, indicating that LIF is a progesterone-dependent protein. Thus endometrial LIF is under maternal control and is secreted in response to the increased progesterone concentrations in circulation. It is possible that high concentrations of LIF during mid-luteal phase may prepare the endometrium for blastocyst implantation in marmosets. PMID- 10729317 TI - Corticotrophin-releasing hormone (CRH) interacts with inflammatory prostaglandins and interleukins and affects the decidualization of human endometrial stroma. AB - The hypothalamic neuropeptide, corticotrophin-releasing hormone (CRH), which is also produced by human endometrium, has been shown to induce its decidualization in vitro. This process, induced mainly by progesterone, has characteristics of an aseptic inflammatory reaction, and is modulated by locally produced pro inflammatory factors. In humans, prostaglandin E(2) (PGE(2)) enhances while interleukin (IL)-1 inhibits the decidualizing effect of progesterone. The aim of the present work was to test the hypothesis that CRH might affect the decidualization of human endometrium interacting with these factors. Therefore, we studied its effects on the production of pro-inflammatory interleukins IL-1, IL-6 and of PGE(2) from human endometrial stromal cells in primary culture. The results strongly suggest that CRH decidualizes stromal cells, as judged by the appearance of cytokeratins and the production of prolactin, two established markers of decidualization. In parallel to its effect on decidualization, CRH also decreased the production of PGE(2), while it increased the production of IL 1 and IL-6. Exposure of endometrial stromal cells to IL-6 also caused decidualization. The data presented here suggest that endometrial CRH regulates the production of local modulators of decidualization, i.e. PGE(2), IL-1 and IL 6. We postulate that, through the regulation of these factors, CRH acts as a local fine-tuner of decidualization initiated by progesterone. PMID- 10729318 TI - Pregnancy is associated with altered response to neuropeptide Y in uterine artery. AB - Pregnancy is associated with a significant increase in uterine blood flow which contributes to optimal fetal development. Although neuropeptide Y (NPY) is considered to be an important regulator of uterine blood flow, it is not known whether: (i) products from the vascular endothelium modulate NPY action in the uterine artery; (ii) pregnancy changes the responsiveness of the uterine artery to NPY, or (iii) NPY interacts with noradrenaline and acetylcholine on the uterine artery, with pregnancy regulating this possible interaction. In the present study, NPY induced a concentration-dependent contraction of guinea pig uterine arterial rings both intact and denuded of endothelium. Pregnancy significantly decreased the potency of NPY to contract uterine artery with and without endothelium. In all preparations, addition of N(G)-monomethyl-l-arginine acetate (l-NMMA), indomethacin and diethylcarbamazine did not modify the effect of NPY. In the presence of NPY concentration-response curves for acetylcholine and noradrenaline were significantly shifted to the right and left respectively. This effect of NPY was independent of endothelial condition or pregnancy status. The receptor reserve (K(A)/EC(50)) for acetylcholine was decreased and for noradrenaline was increased in the presence of NPY, although no changes in the dissociation constants of the neurotransmitter-receptor complexes were observed. Thus, this study has shown that: (i) NPY induces contraction of guinea pig uterine arteries acting on receptors localized in smooth muscle; (ii) pregnancy alters the response of guinea pig uterine arteries to NPY in such a way as to promote vasorelaxation, and (iii) NPY modulates the effect of neurotransmitters on guinea pig uterine arteries, but pregnancy is not associated with the changes at the level of NPY-neurotransmitter interaction. PMID- 10729319 TI - Pregnancy does not alter the response of uterine arteries to vasoactive intestinal polypeptide. AB - In order to provide sufficient nutrients for fetal development, pregnancy is associated with a significant increase in uterine blood flow. Although vasoactive intestinal polypeptide (VIP) is considered to be an important regulator of uterine blood flow it is not known whether endothelium-derived relaxing factors contribute to VIP action in the uterine artery, whether pregnancy alters the effect of VIP in the uterine artery and/or whether VIP interacts with noradrenaline and acetylcholine on the uterine artery and whether pregnancy regulates this possible interaction. In the present study, VIP induced a concentration-dependent relaxation of guinea pig uterine arterial rings, both intact and denuded of endothelium. Pregnancy did not alter the relaxation of uterine artery in response to VIP. In all preparations, addition of N(G) monomethyl-L-arginine (L-NMMA), indomethacin and diethylcarbamazine did not modify the effect of VIP in uterine arteries. The VIP-receptor complex dissociation constant did not differ significantly between studied vessels, and in all experimental groups the relationship between receptor occupancy and the response was linear, with the receptor reserve (K(A)/EC(50)) close to unity. VIP did not modulate acetylcholine-induced relaxation or noradrenaline-induced contraction in both non-pregnant and pregnant guinea pig uterine arteries. This study has shown that: (i) VIP induces relaxation of guinea pig uterine artery acting as a partial agonist on receptors localized in smooth muscle; (ii) pregnancy does not alter the response of guinea pig uterine arteries to VIP and does not change the receptor affinity for VIP, the efficiency of the receptor coupling or the VIP receptor density; and (iii) VIP does not modulate effects of neurotransmitters on guinea pig uterine arteries and pregnancy is not associated with the changes of VIP-neurotransmitter interaction. PMID- 10729320 TI - Modulation of indoleamine 2,3-dioxygenase by interferon-gamma in human placental chorionic villi. AB - The effect of interferon-gamma on indoleamine 2,3-dioxygenase, a tryptophan catabolizing enzyme, was studied in cultured human placental chorionic villi. The activity of indoleamine 2,3-dioxygenase was markedly stimulated by interferon gamma in a time- and concentration-dependent manner. Interferon-alpha and interferon-beta also showed a slight stimulatory effect on indoleamine 2,3 dioxygenase activity. The level of indoleamine 2,3-dioxygenase mRNA expression (determined by reverse transcription-polymerase chain reaction) was also enhanced by interferon-gamma. Interleukin-4 showed a dose-dependent inhibitory effect on interferon-gamma-induced stimulation of indoleamine 2,3-dioxygenase activity and mRNA expression. PMID- 10729321 TI - Human cervical ripening, an inflammatory process mediated by cytokines. AB - An extensive remodelling process, referred to as cervical ripening, takes place in the cervical tissue during pregnancy and labour. It is recognized as softening and dilation of the cervical canal, and starts as a slow process during pregnancy, becoming rapid close to partum. In this study we focus on cytokines as possible mediators of this final remodelling. mRNA levels for interleukin (IL)-8, IL-6 and granulocyte colony-stimulating factor (G-CSF) were upregulated in the ripe postpartum cervical tissue (n = 8) compared to the unripe state (n = 9). Likewise, released cytokine concentrations increased from non-pregnant (n = 11) to the term-pregnant group (n = 13) with a further increase at partum (n = 16). IL-8 concentrations increased 4-fold from non-pregnant to term-pregnant (P<0.01), and a further 10-fold to postpartum state (P<0.0001). Concentrations of IL-6 and G-CSF were similarly increased. Specific IL-8 immunostaining was identified in the epithelia of pregnant cervical tissue (n = 7) and was most pronounced in the epithelia and stroma of postpartum tissue (n = 4). In conclusion, IL-8, IL-6 and G-CSF increase in the human cervix during the ripening process, indicating their important role in the cervical remodelling. These data demonstrate that cervical ripening is similar to an inflammatory process. PMID- 10729322 TI - Studies of cervical ripening in pregnant rats: effects of various treatments. AB - The exact mechanisms that regulate cervical softening or ripening during pregnancy are not completely understood. The aim of this study was to estimate the effects of various agents on cervical softening during pregnancy in rats. Cervical resistance was examined after treatment with nitric oxide (NO) donors and inhibitors and different hormonal agents. Cervical resistance was significantly reduced (P< 0.05) in rats treated with the NO donors: sodium nitroprusside, molsidomine and prostaglandin E(2). However, treatments with the NO synthase (NOS) inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME) and L-N(6)-1-iminoethyl-lysine (L-NIL), or the prostaglandin synthesis inhibitor, indomethacin, significantly increased resistance (P<0.05). The antiprogesterone, onapristone, reduced cervical resistance and its effects were only partially blocked by the progesterone agonist, promegestone. Relaxin reduced cervical resistance and NOS inhibitors partially blocked the effect of relaxin. These studies demonstrate that NO regulates cervical ripening. Relaxin also softens the cervix and may act by stimulating NO synthesis. Progesterone seems important in the control of cervical ripening, but its role appears complex. NO and prostaglandin pathways may independently control ripening by acting in parallel or synergistically. PMID- 10729323 TI - Neurocan is upregulated in injured brain and in cytokine-treated astrocytes. AB - Injury to the CNS results in the formation of the glial scar, a primarily astrocytic structure that represents an obstacle to regrowing axons. Chondroitin sulfate proteoglycans (CSPG) are greatly upregulated in the glial scar, and a large body of evidence suggests that these molecules are inhibitory to axon regeneration. We show that the CSPG neurocan, which is expressed in the CNS, exerts a repulsive effect on growing cerebellar axons. Expression of neurocan was examined in the normal and damaged CNS. Frozen sections labeled with anti neurocan monoclonal antibodies 7 d after a unilateral knife lesion to the cerebral cortex revealed an upregulation of neurocan around the lesion. Western blot analysis of extracts prepared from injured and uninjured tissue also revealed substantially more neurocan in the injured CNS. Western blot analysis revealed neurocan and the processed forms neurocan-C and neurocan-130 to be present in the conditioned medium of highly purified rat astrocytes. The amount detected was increased by transforming growth factor beta and to a greater extent by epidermal growth factor and was decreased by platelet-derived growth factor and, to a lesser extent, by interferon gamma. O-2A lineage cells were also capable of synthesizing and processing neurocan. Immunocytochemistry revealed neurocan to be deposited on the substrate around and under astrocytes but not on the cells. Astrocytes therefore lack the means to retain neurocan at the cell surface. These findings raise the possibility that neurocan interferes with axonal regeneration after CNS injury. PMID- 10729324 TI - Hippocampal abnormalities and enhanced excitability in a murine model of human lissencephaly. AB - Human cortical heterotopia and neuronal migration disorders result in epilepsy; however, the precise mechanisms remain elusive. Here we demonstrate severe neuronal dysplasia and heterotopia throughout the granule cell and pyramidal cell layers of mice containing a heterozygous deletion of Lis1, a mouse model of human 17p13.3-linked lissencephaly. Birth-dating analysis using bromodeoxyuridine revealed that neurons in Lis1+/- murine hippocampus are born at the appropriate time but fail in migration to form a defined cell layer. Heterotopic pyramidal neurons in Lis1+/- mice were stunted and possessed fewer dendritic branches, whereas dentate granule cells were hypertrophic and formed spiny basilar dendrites from which the principal axon emerged. Both somatostatin- and parvalbumin-containing inhibitory neurons were heterotopic and displaced into both stratum radiatum and stratum lacunosum-moleculare. Mechanisms of synaptic transmission were severely disrupted, revealing hyperexcitability at Schaffer collateral-CA1 synapses and depression of mossy fiber-CA3 transmission. In addition, the dynamic range of frequency-dependent facilitation of Lis1+/- mossy fiber transmission was less than that of wild type. Consequently, Lis1+/- hippocampi are prone to interictal electrographic seizure activity in an elevated [K(+)](o) model of epilepsy. In Lis1+/- hippocampus, intense interictal bursting was observed on elevation of extracellular potassium to 6.5 mM, a condition that resulted in only minimal bursting in wild type. These anatomical and physiological hippocampal defects may provide a neuronal basis for seizures associated with lissencephaly. PMID- 10729325 TI - Synaptic activity modulates the induction of bidirectional synaptic changes in adult mouse hippocampus. AB - Activity-dependent synaptic plasticity is critical for learning and memory. Considerable attention has been paid to mechanisms that increase or decrease synaptic efficacy, referred to as long-term potentiation (LTP) and long-term depression (LTD), respectively. It is becoming apparent that synaptic activity also modulates the ability to elicit subsequent synaptic changes. We provide direct experimental evidence that this modulation is attributable, at least in part, to variations in the level of postsynaptic depolarization required for inducing plasticity. In slices from adult hippocampal CA1, a brief pairing protocol known to produce LTP can also induce LTD. The voltage-response function for the induction of LTD and LTP in naive synapses exhibits three parts: at a postsynaptic membrane potential during pairing (V(m)) -20 mV, LTP is generated. This function varies with initial synaptic efficacy. In depressed synapses, Theta(-), the V(m) above which LTD is generated, is shifted toward more depolarized V(ms) and Theta(+), the LTD-LTP crossover point or, equivalently, the V(m) above which LTP is induced, toward more polarized V(ms). Conversely in potentiated synapses, Theta(-) is shifted toward more polarized V(ms). Therefore synaptic activity changes synaptic efficacy and accordingly adjusts the voltages for eliciting subsequent synaptic modifications. The concomitant shifts in the voltages for inducing LTD and LTP in opposite directions promote synaptic potentiation and inhibit synaptic depression in depressed synapses and vice versa in potentiated synapses. PMID- 10729326 TI - Increase in neurite outgrowth mediated by overexpression of actin depolymerizing factor. AB - Growth cone motility is regulated by changes in actin dynamics. Actin depolymerizing factor (ADF) is an important regulator of actin dynamics, and extracellular signal-induced changes in ADF activity may influence growth cone motility and neurite extension. To determine this directly, we overexpressed ADF in primary neurons and analyzed neurite lengths. Recombinant adenoviruses were constructed that express wild-type Xenopus ADF/cofilin [XAC(wt)], as well as two mutant forms of XAC, the active but nonphosphorylatable XAC(A3) and the less active, pseudophosphorylated XAC(E3). XAC expression was detectable on Western blots 24 hr after infection and peaked at 3 d in cultured rat cortical neurons. Peak expression was approximately 75% that of endogenous ADF. XAC(wt) expression caused a slight increase in growth cone area and filopodia but decreased filopodia numbers on neurite shafts. At maximal XAC levels, neurite lengths increased >50% compared with controls infected with a green fluorescent protein expressing adenovirus. Increased neurite extension was directly related to the expression of active XAC. Expression of the XAC(E3) mutant did not increase neurite extension, whereas expression of the XAC(A3) mutant increased neurite extension but to a lesser extent than XAC(wt), which was partially phosphorylated. XAC expression had minimal, if any, impact on F-actin levels and did not result in compensatory changes in the expression of endogenous ADF or actin. However, F-actin turnover appeared to increase based on F-actin loss after treatment with drugs that block actin polymerization. These results provide direct evidence that increased ADF activity promotes process extension and neurite outgrowth. PMID- 10729327 TI - Mechanisms of cannabinoid inhibition of GABA(A) synaptic transmission in the hippocampus. AB - The localization of cannabinoid (CB) receptors to GABAergic interneurons in the hippocampus indicates that CBs may modulate GABAergic function and thereby mediate some of the disruptive effects of marijuana on spatial memory and sensory processing. To investigate the possible mechanisms through which CB receptors may modulate GABAergic neurotransmission in the hippocampus, whole-cell voltage-clamp recordings were performed on CA1 pyramidal neurons in rat brain slices. Stimulus evoked GABA(A) receptor-mediated IPSCs were reduced in a concentration-dependent manner by the CB receptor agonist WIN 55,212-2 (EC(50) of 138 nM). This effect was blocked by the CB1 receptor antagonist SR141716A (1 microM) but not by the opioid antagonist naloxone. In contrast, evoked GABA(B)-mediated IPSCs were insensitive to the CB agonist. WIN 55,212-2 also reduced the frequency of spontaneous, action potential-dependent IPSCs (sIPSCs), without altering action potential-independent miniature IPSCs (mIPSCs), measured while sodium channels were blocked by tetrodotoxin (TTX). Blockade of voltage-dependent calcium channels (VDCCs) by cadmium also eliminated the effect of WIN 55,212-2 on sIPSCs. Depolarization of inhibitory terminals with elevated extracellular potassium caused a large increase in the frequency of mIPSCs that was inhibited by both cadmium and WIN 55,212-2. The presynaptic effect of WIN 55,212-2 was also investigated using the potassium channel blockers barium and 4-aminopyridine. Neither of these agents significantly altered the effect of WIN 55,212-2 on evoked IPSCs. Together, these data suggest that presynaptic CB1 receptors reduce GABA(A)- but not GABA(B)-mediated synaptic inhibition of CA1 pyramidal neurons by inhibiting VDCCs located on inhibitory nerve terminals. PMID- 10729328 TI - Release-independent short-term synaptic depression in cultured hippocampal neurons. AB - Short-term synaptic plasticity may dramatically influence neuronal information transfer, yet the underlying mechanisms remain incompletely understood. In autapses (self-synapses) formed by cultured hippocampal neurons, short-term synaptic depression (STD) had several unusual features. (1) Reduction of neurotransmitter release probability with Cd(2+), a blocker of voltage-gated calcium channels, did not change depression. (2) Lowering [Ca(2+)](o) and/or raising [Mg(2+)](o) had little effect on STD in cells with strong baseline depression, but in cells with more modest baseline depression, it reduced the depression. (3) Random variations in the size of initial EPSCs did not influence successive EPSC sizes. These findings were inconsistent with release-dependent mechanisms, such as vesicle depletion, post-synaptic receptor desensitization, and autoreceptor inhibition. Instead, other results suggested that changes in action potentials (APs) contributed to depression. The somatic APs declined in amplitude with repetitive stimulation, and modest reduction of AP amplitudes with tetrodotoxin inhibited EPSCs. Notably, tetrodotoxin also increased depression. Similar changes in axonal APs could produce STD in at least two ways. First, decreasing presynaptic spike amplitudes could reduce calcium entry and release probability. Alternatively, APs could fail to propagate through some axonal branches, reducing the number of active synapses. To explore these possibilities, we derived the expected variance of EPSCs for the two scenarios. Experimentally, the variance increased and then decreased on average with successive responses during trains of APs, confirming a unique prediction from the conduction failure scenario. Thus, STD had surprising properties, incompatible with commonly postulated mechanisms but consistent with AP conduction failure at axonal branches. PMID- 10729329 TI - Enhancement of the dense-core vesicle secretory cycle by glucocorticoid differentiation of PC12 cells: characteristics of rapid exocytosis and endocytosis. AB - The secretory cycle of dense-core vesicles (DCVs) in physiologically stimulated patch-clamped PC12 cells was analyzed using both amperometry and capacitance measurements. Untreated cells had low or undetectable Ca currents and sparse secretory responses to short depolarizations. Dexamethasone (5 microM) treatment for 5-7 d tripled Ca current magnitude and dramatically increased quantal secretion in response to depolarization with action potentials. Such cells expressed L-, N-, and P-type Ca channels, and depolarization evoked rapid catecholamine secretion recorded as amperometric spikes; the average latency was approximately 50 msec. These spikes were much smaller and shorter than those of primary adrenal chromaffin cells, reflecting the smaller size of DCVs in PC12 cells. Depolarizing pulse trains also elicited a rapid increase in membrane capacitance corresponding to exocytosis in differentiated but not in naive cells. On termination of stimulation, membrane capacitance declined within 20 sec to baseline indicative of rapid endocytosis (RE). RE did not take place when secretion was stimulated in the presence of Ba or Sr, indicating that RE is Ca specific. RE was blocked when either anti-dynamin antibodies or the pleckstrin homology domain of dynamin-1 was loaded into the cell via the patch pipette. These studies indicate that neuroendocrine differentiation of PC12 cells with glucocorticoids enhances the development of the excitable membrane and increases the coupling between Ca channels and vesicle release sites, leading to rapid exocytosis and endocytosis. Slow catecholamine secretion in undifferentiated cells may be caused in part by a lack of localized secretory machinery rather than being an intrinsic property of dense-core vesicles. PMID- 10729330 TI - Regulation of long-term potentiation by H-Ras through NMDA receptor phosphorylation. AB - The proto-oncogene ras plays a critical role in cell proliferation and differentiation. However, ras genes are abundantly expressed in the adult CNS, although neuronal cells normally do not proliferate. Recently, several lines of evidence implicated the involvement of Ras signaling pathway in synaptic plasticity. To explore the role of the Ras proteins in the CNS, we generated knock-out mice lacking the H-ras gene and then used them to study the roles of Ras in synaptic transmission and plasticity. An investigation of protein phosphorylation and synaptic transmission in H-ras null mutant mice has shown that the NMDA receptor is a final target molecule of the Ras protein pathway in the CNS. In the H-ras null mutant hippocampus, the tyrosine phosphorylation of NR2A (epsilon1) and NR2B (epsilon2) subunits of NMDA receptors is increased, and, correspondingly, NMDA synaptic responses are selectively enhanced. In addition, long-term potentiation is markedly enhanced in mutant mice, most likely because of a selective enhancement of NMDA synaptic responses. Therefore, although Ras proteins have been implicated in cell proliferation and differentiation, the regulation of activity-dependent synaptic plasticity in the adult animals by downregulation of the phosphorylation of the NMDA receptor may be another major and pivotal role for H-Ras protein. PMID- 10729331 TI - NMDA receptor content of synapses in stratum radiatum of the hippocampal CA1 area. AB - Glutamate receptors activated by NMDA (NMDARs) or AMPA (AMPARs) are clustered on dendritic spines of pyramidal cells. Both the AMPAR-mediated postsynaptic responses and the synaptic AMPAR immunoreactivity show a large intersynapse variability. Postsynaptic responses mediated by NMDARs show less variability. To assess the variability in NMDAR content and the extent of their coexistence with AMPARs in Schaffer collateral-commissural synapses of adult rat CA1 pyramidal cells, electron microscopic immunogold localization of receptors has been used. Immunoreactivity of NMDARs was detected in virtually all synapses on spines, but AMPARs were undetectable, on average, in 12% of synapses. A proportion of synapses had a very high AMPAR content relative to the mean content, resulting in a distribution more skewed toward larger values than that of NMDARs. The variability of synaptic NMDAR content [coefficient of variation (CV), 0.64-0.70] was much lower than that of the AMPAR content (CV, 1.17-1.45). Unlike the AMPAR content, the NMDAR content showed only a weak correlation with synapse size. As reported previously for AMPARs, the immunoreactivity of NMDARs was also associated with the spine apparatus within spines. The results demonstrate that the majority of the synapses made by CA3 pyramidal cells onto spines of CA1 pyramids express both NMDARs and AMPARs, but with variable ratios. A less variable NMDAR content is accompanied by a wide variability of AMPAR content, indicating that the regulation of expression of the two receptors is not closely linked. These findings support reports that fast excitatory transmission at some of these synapses is mediated by activation mainly of NMDARs. PMID- 10729332 TI - Highly localized Ca(2+) accumulation revealed by multiphoton microscopy in an identified motoneuron and its modulation by dopamine. AB - Calcium is essential for synaptic transmission and the control of the intrinsic firing properties of neurons; this makes Ca(2+) channels a prime target for neuromodulators. A combination of multiphoton microscopy and voltage-clamp recording was used to determine the localization of voltage-dependent Ca(2+) accumulation in the two pyloric dilator (PD) neurons of the pyloric network in the spiny lobster, Panulirus interruptus, and its modulation by dopamine. We monitored [Ca(2+)](i) in fine distal branches in the neuropil >350 microm below the surface of the ganglion during controlled voltage steps in voltage clamp. Ca(2+) accumulation originated mostly from small, fairly rare, spatially restricted varicosities on distal neuritic arborizations. Ca(2+) diffused from these point sources into adjacent regions. Varicosities with similar morphology in the PD neuron have been shown previously to be sites of synaptic contacts. We have demonstrated in earlier studies that dopamine inhibits activity and greatly reduces synaptic transmission from the PD neuron. In approximately 60% of the varicosities, the voltage-activated Ca(2+) accumulation was reduced by exogenous dopamine (DA) (10(-4) M). DA decreased the peak amplitude of Ca(2+) accumulation but had no effect on the rise and decay time. We conclude that DA reduces chemical synaptic transmission from the PD neurons at least in part by decreasing Ca(2+) entry at neurotransmitter release sites. PMID- 10729333 TI - Early and selective loss of neuromuscular synapse subtypes with low sprouting competence in motoneuron diseases. AB - The addition or loss of synapses in response to changes in activity, disease, or aging is a major aspect of nervous system plasticity in the adult. The mechanisms that affect the turnover and maintenance of synapses in the adult are poorly understood and are difficult to investigate in the brain. Here, we exploited a unique anatomical arrangement in the neuromuscular system to determine whether subtypes of synapses can differ in anatomical plasticity and vulnerability. In three genetic mouse models of motoneuron disease of diverse origin and severity, we observed a gradual and selective loss of synaptic connections that begun long before the onset of clinical deficits and correlated with the timing of disease progression. A subgroup of fast-type (fast-fatiguable) neuromuscular synapses was highly vulnerable and was lost very early on. In contrast, slow-type synapses resisted up to the terminal phase of the disease. Muscle-specific differences were also evident. Similar selective losses were detected in aged mice. These selective vulnerability properties of synapses coincided with hitherto unrecognized major differences in stimulus-induced anatomical plasticity that could also be revealed in healthy mice. Using paralysis and/or growth-associated protein 43 overexpression to induce synaptic sprouting, we found that slow-type, disease-resistant synapses were particularly plastic. In contrast, fast-type synapses with the highest vulnerability failed to exhibit any stimulus-induced change. The results reveal pronounced subtype specificity in the anatomical plasticity and susceptibility to loss of neuromuscular synapses and suggest that degenerative motoneuron diseases involve a common early pathway of selective and progressive synaptic weakening also associated with aging. PMID- 10729334 TI - Thrombomodulin as a new marker of lesion-induced astrogliosis: involvement of thrombin through the G-protein-coupled protease-activated receptor-1. AB - Because injury of the CNS causes an astrogliosis, characterized by cell swelling and proliferation, similar to the effects of the serine protease thrombin on astrocytes, we hypothesized that a high level of thrombin at the site of injury might initially induce an astrocyte reaction and later increase the expression of its specific inhibitor, thrombomodulin. Thrombomodulin could then stabilize the astroglial scar through its adhesive properties. Here, we studied the in vivo injury response of astrocytes in the anterior medullary velum of adult rat by immunostaining and in situ hybridization of thrombomodulin. Thrombomodulin was poorly expressed on astrocytes in normal tissue, increased up to 2 d after injury, and was still highly expressed at 6 d. To check that thrombin had a direct effect on thrombomodulin expression by astrocytes, we used brain cortical astrocyte primary cultures treated with either thrombin or the agonist peptide thrombin receptor-activating peptide-6, known to activate directly the thrombin G protein-coupled receptor (GPCR) protease-activated receptor-1 (PAR-1). Modification of thrombomodulin expression was studied by Western blotting and quantitative reverse transcription-PCR. There was a dose-dependent increase in thrombomodulin after 48 hr of treatment, with gene expression peaking at 24 hr but falling to control levels by 48 hr. Together, these results show the following: (1) injury increases astrocyte thrombomodulin expression; (2) thrombin might mediate thrombomodulin expression via the specific receptor PAR-1; and (3) serine proteases, their inhibitors, and the new family of GPCR, PARs, are active on astrogliosis. PMID- 10729335 TI - Morphofunctional plasticity in the adult hypothalamus induces regulation of polysialic acid-neural cell adhesion molecule through changing activity and expression levels of polysialyltransferases. AB - Polysialic acid-neural cell adhesion molecule (PSA-NCAM) expression in the adult nervous system is restricted to regions retaining a capacity for morphological plasticity. For the female rat hypothalamoneurohypophysial system (HNS), we have previously shown that lactation induces a dramatic decrease in PSA-NCAM, while leaving the level of total NCAM protein unchanged. Here, we wanted to elucidate the molecular mechanisms leading to a downregulation of PSA, thereby stabilizing newly established synapses and neurohemal contacts that accompany the increased activity of oxytocinergic neurons. First, we show that the overall specific activity of polysialyltransferases present in tissue extracts from supraoptic nuclei decreases by approximately 50% during lactation. So far, two polysialyltransferase enzymes, STX and PST, have been characterized for their capacity to transfer PSA onto NCAM in vitro. Using a competitive RT-PCR on RNA extracts from the HNS, we demonstrate furthermore a significant decrease in the expression levels of both STX and PST mRNAs in lactating versus virgin animals. Interestingly, this downregulation of NCAM polysialylation is not correlated with the post-transcriptional regulation of variable alternative spliced exon splicing, in contrast to neural development. The control of polysialylation via a regulation of both enzyme activity and expression underlines the important role of this post-translational modification of NCAM in morphofunctional plasticity in adult brain. PMID- 10729336 TI - Dysfunctions in mice by NMDA receptor point mutations NR1(N598Q) and NR1(N598R). AB - NMDA receptors in mice were mutated by gene targeting to substitute asparagine (N) in position 598 of the NR1 subunit to glutamine (Q) or arginine (R). Animals expressing exclusively the mutated NR1 alleles, NR1(Q/Q) and NR1(-/R) mice, developed a perinatally lethal phenotype mainly characterized by respiratory failure. The dysfunctions were partially rescued in heterozygous mice by the presence of pure wild-type receptors. Thus, NR1(+/Q) mice exhibited reduced life expectancy, with females being impaired in nurturing; NR1(+/R) mice displayed signs of underdevelopment such as growth retardation and impaired righting reflex, and died before weaning. We analyzed the key properties of NMDA receptors, high Ca(2+) permeability, and voltage-dependent Mg(2+) block, in the mutant mice. Comparison of the complex physiological and phenotypical changes observed in the different mutants indicates that properties controlled by NR1 subunit residue N598 are important for autonomic brain functions at birth and during postnatal development. We conclude that disturbed NMDA receptor signaling mediates a variety of neurological phenotypes. PMID- 10729337 TI - Role of glycogen synthase kinase-3beta in neuronal apoptosis induced by trophic withdrawal. AB - Glycogen synthase kinase-3beta (GSK3beta) activity is negatively regulated by several signal transduction cascades that protect neurons against apoptosis, including the phosphatidylinositol-3 kinase (PI-3 kinase) pathway. This suggests the interesting possibility that activation of GSK3beta may contribute to neuronal apoptosis. Consequently, we evaluated the role of GSK3beta in apoptosis in cultured cortical neurons induced by trophic factor withdrawal or by PI-3 kinase inhibition. Neurons were subjected to several apoptotic paradigms, including serum deprivation, serum deprivation combined with exposure to NMDA receptor antagonists, or treatment with PI-3 kinase inhibitors. These treatments all led to stimulation of GSK3beta activity in cortical neurons, which preceded the induction of apoptosis. Expression of an inhibitory GSK3beta binding protein or a dominant interfering form of GSK3beta reduced neuronal apoptosis, suggesting that GSK3beta contributes to trophic factor withdrawal-induced apoptosis. Furthermore, overexpression of GSK3beta in neurons increased apoptosis, indicating that activation of this enzyme is sufficient to trigger programmed cell death. Although destabilization of beta-catenin is an important physiological effect of GSK3beta activation, expression of a mutant beta-catenin that is not destabilized by GSK3beta did not protect against apoptosis. We conclude that inhibition of GSK3beta is one of the mechanisms by which PI-3 kinase activation protects neurons from programmed cell death. PMID- 10729338 TI - In vivo structure-function analyses of Caenorhabditis elegans MEC-4, a candidate mechanosensory ion channel subunit. AB - Mechanosensory signaling mediated by mechanically gated ion channels constitutes the basis for the senses of touch and hearing and contributes fundamentally to the development and homeostasis of all organisms. Despite this profound importance in biology, little is known of the molecular identities or functional requirements of mechanically gated ion channels. We report a genetically based structure-function analysis of the candidate mechanotransducing channel subunit MEC-4, a core component of a touch-sensing complex in Caenorhabditis elegans and a member of the DEG/ENaC superfamily. We identify molecular lesions in 40 EMS induced mec-4 alleles and further probe residue and domain function using site directed approaches. Our analysis highlights residues and subdomains critical for MEC-4 activity and suggests possible roles of these in channel assembly and/or function. We describe a class of substitutions that disrupt normal channel activity in touch transduction but remain permissive for neurotoxic channel hyperactivation, and we show that expression of an N-terminal MEC-4 fragment interferes with in vivo channel function. These data advance working models for the MEC-4 mechanotransducing channel and identify residues, unique to MEC-4 or the MEC-4 degenerin subfamily, that might be specifically required for mechanotransducing function. Because many other substitutions identified by our study affect residues conserved within the DEG/ENaC channel superfamily, this work also provides a broad view of structure-function relations in the superfamily as a whole. Because the C. elegans genome encodes representatives of a large number of eukaryotic channel classes, we suggest that similar genetic based structure-activity studies might be generally applied to generate insight into the in vivo function of diverse channel types. PMID- 10729339 TI - Phenotypic knockout of nerve growth factor in adult transgenic mice reveals severe deficits in basal forebrain cholinergic neurons, cell death in the spleen, and skeletal muscle dystrophy. AB - The disruption of the nerve growth factor (NGF) gene in transgenic mice leads to a lethal phenotype (Crowley et al., 1994) and hinders the study of NGF functions in the adult. In this study the phenotypic knockout of NGF in adult mice was achieved by expressing transgenic anti-NGF antibodies, under the control of the human cytomegalovirus promoter. In adult mice, antibody levels are 2000-fold higher than in newborns. Classical NGF targets, including sympathetic and sensory neurons, are severely affected. In the CNS, basal forebrain and hippocampal cholinergic neurons are not affected in the early postnatal period, whereas they are greatly reduced in the adult (55 and 62% reduction, respectively). Adult mice show a reduced ability in spatial learning behavioral tasks. Adult, but not neonatal, transgenic mice further show a new phenotype at the level of peripheral tissues, such as apoptosis in the spleen and dystrophy of skeletal muscles. The analysis of this novel comprehensive transgenic model settles the controversial issue regarding the NGF dependence of cholinergic neurons in adult animals and reveals new NGF functions in adult non-neuronal tissues. The results demonstrate that the decreased availability of NGF in the adult causes phenotypic effects via processes that are at least partially distinct from early developmental effects of NGF deprivation. PMID- 10729340 TI - Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration. AB - Functional recovery is often poor despite the capacity for axonal regeneration in the peripheral nervous system and advances in microsurgical technique. Regeneration of axons in mixed nerve into inappropriate pathways is a major contributing factor to this failure. In this study, we use the rat femoral nerve model of transection and surgical repair to evaluate (1) the effect of nerve transection on the speed of regeneration and the generation of motor-sensory specificity, (2) the efficacy of electrical stimulation in accelerating axonal regeneration and promoting the reinnervation of appropriate muscle pathways by femoral motor nerves, and (3) the mechanism of action of electrical stimulation. Using the retrograde neurotracers fluorogold and fluororuby to backlabel motoneurons that regenerate axons into muscle and cutaneous pathways, we found the following. (1) There is a very protracted period (10 weeks) of axonal outgrowth that adds substantially to the delay in axonal regeneration (staggered regeneration). This process of staggered regeneration is associated with preferential motor reinnervation (PMR). (2) One hour to 2 weeks of 20 Hz continuous electrical stimulation of the parent axons proximal to the repair site dramatically reduces this period (to 3 weeks) and accelerates PMR. (3) The positive effect of short-term electrical stimulation is mediated via the cell body, implicating an enhanced growth program. The effectiveness of such a short period low-frequency electrical stimulation suggests a new therapeutic approach to accelerate nerve regeneration after injury and, in turn, improve functional recovery. PMID- 10729341 TI - Elevated levels of the chemokine GRO-1 correlate with elevated oligodendrocyte progenitor proliferation in the jimpy mutant. AB - The dysmyelinating mutant jimpy (jp) arises from a point mutation in the mouse gene encoding proteolipid protein and is characterized by severe dysmyelination attributable to oligodendrocyte death. This mutant was used to investigate the regulation of oligodendrocyte progenitor proliferation in the postnatal spinal cord. At postnatal day 18, jp spinal cord contained a three- to eightfold greater number of proliferating oligodendrocyte progenitor cells than did wild-type (wt) spinal cord. Increased proliferation in jp spinal cord was accompanied by a twofold increase in the number of progenitor cells. Semiquantitative reverse transcriptase-PCR revealed no change in the level of mRNA encoding the platelet derived growth factor A, transforming growth factor-beta, or insulin-like growth factor-I, all of which have been implicated as regulators of proliferation and differentiation of oligodendrocyte progenitor cells. There was, however, a 17 fold increase in the level of mRNA encoding the chemokine GRO-1 and a 5- to 6 fold increase in GRO-1 protein in the jp spinal cord. Double immunofluorescence labeling revealed elevated levels of GRO-1 in reactive astrocytes in jp spinal cord white matter. In vitro studies indicated that extracts from jp spinal cord stimulated oligodendrocyte progenitor proliferation. Furthermore, removal of GRO 1 from jp extracts by immunoprecipitation reduced the proliferation of progenitor cells to a level similar to that achieved by wt extracts. These findings suggest a novel mechanism by which proliferation of oligodendrocyte progenitor cells is regulated in the postnatal spinal cord in response to insult. PMID- 10729342 TI - Emx2 is required for growth of the hippocampus but not for hippocampal field specification. AB - The vertebrate Emx genes are expressed in a nested pattern in early embryonic cerebral cortex, such that a medial strip of cortex expresses Emx2 but not Emx1. This pattern suggests that Emx genes could play a role in specifying different areas or fields of the cortex along the mediolateral axis. Such a role has been supported by the observation that in mice lacking functional Emx2 the hippocampus is shrunken and the most medial field of the cortex, the hippocampal dentate gyrus, appears by cytoarchitecture to be missing (Pellegrini et al., 1996; Yoshida et al., 1997). Use of region-specific molecular markers shows, however, that hippocampal fields are specified and correctly positioned in the Emx2 mutant. In particular, a dentate cell population is generated, although it fails to form a morphological gyrus. This failure may be part of a more widespread medial cortical defect in the mutant. Examination of cortical cell proliferation and differentiation indicates a disruption of the maturation of the medial cortex in the absence of Emx2. Thus, Emx2 is required for normal growth and maturation of the hippocampus but not for the specification of cells to particular hippocampal field identities. PMID- 10729343 TI - Spontaneous acetylcholine secretion from developing growth cones of Drosophila central neurons in culture: effects of cAMP-pathway mutations. AB - We describe a novel bioassay system that uses Xenopus embryonic myocytes (myoballs) to detect the release of acetylcholine from Drosophila CNS neurons. When a voltage-clamped Xenopus myoball was manipulated into contact with cultured Drosophila "giant" neurons, spontaneous synaptic current-like events were registered. These events were observed within seconds after contact and were blocked by curare and alpha-bungarotoxin, but not by TTX and Cd(2+), suggesting that they are caused by the spontaneous quantal release of acetylcholine (ACh). The secretion occurred not only at the growth cone, but also along the neurite and at the soma, with significantly different release parameters among various regions. The amplitude of these currents displayed a skewed distribution. These features are distinct from synaptic transmission at more mature synapses or autapses formed in this culture system and are reminiscent of the transmitter release process during early development in other preparations. The usefulness of this coculture system in studying presynaptic secretion mechanisms is illustrated by a series of studies on the cAMP pathway mutations, dunce (dnc) and PKA-RI, which disrupt a cAMP-specific phosphodiesterase and the regulatory subunit of cAMP-dependent protein kinase A, respectively. We found that these mutations affected the ACh current kinetics, but not the quantal ACh packet, and that the release frequency was greatly enhanced by repetitive neuronal activity in dnc, but not wild-type, growth cones. These results suggest that the cAMP pathway plays an important role in the activity-dependent regulation of transmitter release not only in mature synapses as previously shown, but also in developing nerve terminals before synaptogenesis. PMID- 10729344 TI - The transmembrane protein semaphorin 6A repels embryonic sympathetic axons. AB - Semaphorin 6A (Sema6A) (previously named Semaphorin VIa) is the originally described member of the vertebrate semaphorin class 6, a group of transmembrane semaphorins homologous to the insect semaphorin class 1. Although Sema-1a (previously named semaphorin I) has been implicated in axon guidance in insects, the function of Sema6A is currently unknown. We have expressed the extracellular domain of Sema6A in mammalian cells as either a monomeric or a dimeric fusion protein and tested for potential axon guidance effects on two populations of embryonic neurons in growth cone collapse and collagen matrix chemorepulsion assays. Sema6A was observed to induce growth cone collapse of sympathetic neurons with an EC50 of approximately 200 pM, although a 10-fold higher (EC50 of approximately 2 nM) concentration was necessary to induce growth cone collapse of dorsal root ganglion neurons. The activity of Sema6A is likely to depend on protein dimerization or oligomerization. Although Sema6A mRNA is expressed in complex patterns during embryonic development, it is strikingly absent from sympathetic ganglia. Sema6A is, however, expressed in areas avoided by sympathetic axons and in areas innervated by sympathetics, but before their arrival. Our results demonstrate that transmembrane semaphorins, like the secreted ones, can act as repulsive axon guidance cues. Our findings are consistent with a role for Sema6A in channeling sympathetic axons into the sympathetic chains and controlling the temporal sequence of sympathetic target innervation. PMID- 10729345 TI - Impaired preference conditioning after anterior temporal lobe resection in humans. AB - Research with animals suggests that structures within the amygdaloid nuclear complex (ANC) are critical for acquiring associations between rewarding events and neutral stimuli, a form of conditioning often manifested in a subsequent preference for those (conditioned) stimuli. In this study, we investigated the relationship between the ANC and preference learning in humans. Three abstract monochrome patterns were presented to each subject over 180 trials in the context of a counting task requiring working memory. One pattern was paired with food reward on 90% of the trials in which it was presented and with no food reward on the other 10% of trials. The other patterns were similarly reinforced, but at ratios of 50:50% and 10:90% with reward and nonreward, respectively. Subsequently, a group of 21 normal participants preferred the pattern paired most often with reward to that paired least often with reward, and they did not explicitly relate their preferences to the conditioning procedure, but instead attributed them to the characteristics of the patterns themselves. Unlike the normal controls, a group of patients with unilateral surgical lesions that included the ANC (15 left, 18 right) did not show conditioned preferences, but performed normally on a measure of working memory. In contrast, 13 patients with unilateral damage confined to frontal cortex exhibited normal conditioned preferences but were impaired on the working memory task. This double dissociation provides clear evidence that, in humans as in other animals, reward related learning (conditioned reward) critically depends on a circuit involving inferotemporal cortex and the ANC. PMID- 10729346 TI - Cortical processing of human somatic and visceral sensation. AB - Somatic sensation can be localized precisely, whereas localization of visceral sensation is vague, possibly reflecting differences in the pattern of somatic and visceral input to the cerebral cortex. We used functional magnetic resonance imaging to study the cortical processing of sensation arising from the proximal (somatic) and distal (visceral) esophagus in six healthy male subjects. Esophageal stimulation was performed by phasic distension of a 2 cm balloon at 0.5 Hz. For each esophageal region, five separate 30 sec periods of nonpainful distension were alternated with five periods of similar duration without distension. Gradient echoplanar images depicting bold contrast were acquired using a 1.5 T GE scanner. Distension of the proximal esophagus was localized precisely to the upper chest and was represented in the trunk region of the left primary somatosensory cortex. In contrast, distension of the distal esophagus was perceived diffusely over the lower chest and was represented bilaterally at the junction of the primary and secondary somatosensory cortices. Different activation patterns were also observed in the anterior cingulate gyrus with the proximal esophagus being represented in the right midanterior cingulate cortex (BA 24) and the distal esophagus in the perigenual area (BA32). Differences in the activation of the dorsolateral prefrontal cortex and cerebellum were also observed for the two esophageal regions. These findings suggest that cortical specialization in the sensory-discriminative, affective, and cognitive areas of the cortex accounts for the perceptual differences observed between the two sensory modalities. PMID- 10729347 TI - A positron emission tomographic study of auditory localization in the congenitally blind. AB - We have used positron emission tomography (PET) to measure regional cerebral blood flow (rCBF) in sighted and congenitally blind subjects performing auditory localization tasks. During scanning, the spectral and binaural cues of localized sound were reproduced by a sound system and delivered via headphones. During tasks that required auditory localization both the sighted and blind subjects strongly activated posterior parietal areas. In addition, the blind subjects activated association areas in the right occipital cortex, the foci of which were similar to areas previously identified in visual location and motion detection experiments in sighted subjects. The blind subjects, therefore, demonstrated visual to auditory cross-modal plasticity with auditory localization activating occipital association areas originally intended for dorsal-stream visual processing. To determine the functional connectivity of pre-selected brain regions in primary and non-primary auditory and posterior parietal cortex in the two cohorts, we performed an inter-regional correlation analysis on the rCBF data set. During auditory localization in the blind subjects, rCBF activity in the right posterior parietal cortex was positively correlated with that in the right occipital region, whereas in sighted subjects correlations were generally negative. There were no significant positive occipital correlations in either cohort when reference regions in temporal or left parietal cortex were chosen. This indicates that in congenitally blind subjects the right occipital cortex participates in a functional network for auditory localization and that occipital activity is more likely to arise from connections with posterior parietal cortex. PMID- 10729348 TI - Distinct ionotropic GABA receptors mediate presynaptic and postsynaptic inhibition in retinal bipolar cells. AB - Ionotropic GABA receptors can mediate presynaptic and postsynaptic inhibition. We assessed the contributions of GABA(A) and GABA(C) receptors to inhibition at the dendrites and axon terminals of ferret retinal bipolar cells by recording currents evoked by focal application of GABA in the retinal slice. Currents elicited at the dendrites were mediated predominantly by GABA(A) receptors, whereas responses evoked at the terminals had GABA(A) and GABA(C) components. The ratio of GABA(C) to GABA(A) (GABA(C):GABA(A)) was highest in rod bipolar cell terminals and variable among cone bipolars, but generally was lower in OFF than in ON classes. Our results also suggest that the GABA(C):GABA(A) could influence the time course of responses. Currents evoked at the terminals decayed slowly in cell types for which the GABA(C):GABA(A) was high, but decayed relatively rapidly in cells for which this ratio was low. Immunohistochemical studies corroborated our physiological results. GABA(A) beta2/3 subunit immunoreactivity was intense in the outer and inner plexiform layers (OPL and IPL, respectively). GABA(C) rho subunit labeling was weak in the OPL but strong in the IPL in which puncta colocalized with terminals of rod bipolars immunoreactive for protein kinase C and of cone bipolars immunoreactive for calbindin or recoverin. These data demonstrate that GABA(A) receptors mediate GABAergic inhibition on bipolar cell dendrites in the OPL, that GABA(A) and GABA(C) receptors mediate inhibition on axon terminals in the IPL, and that the GABA(C):GABA(A) on the terminals may tune the response characteristics of the bipolar cell. PMID- 10729349 TI - A role for somatosensory cortices in the visual recognition of emotion as revealed by three-dimensional lesion mapping. AB - Although lesion and functional imaging studies have broadly implicated the right hemisphere in the recognition of emotion, neither the underlying processes nor the precise anatomical correlates are well understood. We addressed these two issues in a quantitative study of 108 subjects with focal brain lesions, using three different tasks that assessed the recognition and naming of six basic emotions from facial expressions. Lesions were analyzed as a function of task performance by coregistration in a common brain space, and statistical analyses of their joint volumetric density revealed specific regions in which damage was significantly associated with impairment. We show that recognizing emotions from visually presented facial expressions requires right somatosensory-related cortices. The findings are consistent with the idea that we recognize another individual's emotional state by internally generating somatosensory representations that simulate how the other individual would feel when displaying a certain facial expression. Follow-up experiments revealed that conceptual knowledge and knowledge of the name of the emotion draw on neuroanatomically separable systems. Right somatosensory-related cortices thus constitute an additional critical component that functions together with structures such as the amygdala and right visual cortices in retrieving socially relevant information from faces. PMID- 10729350 TI - What and when: parallel and convergent processing in motor control. AB - Successful motor behavior requires making appropriate response (response selection) at the right time (timing adjustment). Earlier psychological studies have suggested that the response selection and timing adjustment processes are performed serially in separate stages. We tested this hypothesis using functional magnetic resonance imaging. The subjects performed a choice reaction time task in four conditions: two (on-line response selection required or not) by two (on-line timing adjustment required or not). We found that the neural correlates for the two processes were indeed separate: the anterior medial premotor cortex (presupplementary motor area) was selectively active in response selection, whereas the cerebellar posterior lobe was selectively active in timing adjustment. However, the functional separation was only partial in that the lateral premotor cortex and the intraparietal sulcus were active equally for response selection and timing adjustment. The lateral premotor cortex was most active when both processes were required, suggesting that it integrates the information on response selection and the information on timing adjustment; alternatively, it might contribute to the allocation of attentional resources during dual information processing. The intraparietal sulcus was equally active when either response selection or timing adjustment was required, suggesting that it modifies, rather than integrates, these processes. Furthermore, our results suggest that these activations related to response selection and timing adjustment were distinct from sensory or motor processes. PMID- 10729351 TI - Neuronal correlates of fear in the lateral amygdala: multiple extracellular recordings in conscious cats. AB - Much data implicates the amygdala in the expression and learning of fear. Yet, few studies have examined the neuronal correlates of fear in the amygdala. This study aimed to determine whether fear is correlated to particular activity patterns in the lateral amygdaloid (LA) nucleus. Cats, chronically implanted with multiple microelectrodes in the LA and a catheter in the femoral artery, learned that a series of tones interrupted by a period of silence (5 sec) preceded the administration of a footshock. During the silent period, their blood pressure increased, indicating that they anticipated the noxious stimulus. In parallel, the firing rate of LA neurons doubled, and the discharges of simultaneously recorded cells became more synchronized. Moreover, cross-correlation of focal LA waves revealed a significant increase in synchrony restricted to the theta band. In keeping with this, perievent histograms of neuronal discharges revealed rhythmic changes in the firing probability of LA neurons in relation to focal theta waves. Finally, the responsiveness of LA cells to the stimuli predicting the footshock (the tones) increased during the trials, whereas responses to unrelated stimuli (perirhinal shocks) remained stable. Thus, during the anticipation of noxious stimuli, a state here defined anthropomorphically as fear, the firing rate of LA neurons increases, and their discharges become more synchronized through a modulation at the theta frequency. The presence of theta oscillations in the LA might facilitate cooperative interactions between the amygdala and cortical areas involved in memory. PMID- 10729352 TI - Fos imaging reveals differential patterns of hippocampal and parahippocampal subfield activation in rats in response to different spatial memory tests. AB - We compared neuronal activation, as measured by Fos staining, during different spatial tasks in two experiments. The counts of Fos-stained neurons in the hippocampus increased as the spatial demands of the tasks increased, the tasks having been carefully matched for other factors. In Experiment 1, matched groups of rats either ran a standard eight-arm radial maze task or were trained to run up and down just one arm of the maze; the number of runs and rewards was identical in both conditions. In Experiment 2, rats were trained on the eight-arm maze but in different rooms. On the critical test day, both groups were run in the same room so that one group now performed with novel landmarks. All hippocampal subfields (dentate gyrus, CA3, CA1, dorsal, ventral, and caudal subiculum) showed a relative increases in c-fos activation in the eight-arm (Experiment 1) and novel room (Experiment 2) conditions, the sole exception being the ventral subiculum in Experiment 2. Although increased c-fos activation was found in both dorsal and ventral hippocampus, in Experiment 2 the relative increase was significantly greater in the dorsal hippocampus. Parahippocampal cortices responded heterogeneously: the perirhinal cortex failed to show increased activation in both experiments, in contrast to the entorhinal and postrhinal cortices. Subsequent comparisons confirmed that the perirhinal and postrhinal cortices responded in qualitatively different ways, the perirhinal cortex differing from the rest of the hippocampal formation. These experiments, which provide the first analysis of hippocampal Fos production during tests of allocentric spatial working memory, reveal that all components of the hippocampus are activated, but that under certain conditions the dorsal hippocampus is disproportionately involved. PMID- 10729353 TI - Task-dependent constraints in motor control: pinhole goggles make the head move like an eye. AB - In the 19th century, Donders observed that only one three-dimensional eye orientation is used for each gaze direction. Listing's law further specifies that the full set of eye orientation vectors forms a plane, whereas the equivalent Donders' law for the head, the Fick strategy, specifies a twisted two-dimensional range. Surprisingly, despite considerable research and speculation, the biological reasons for choosing one such range over another remain obscure. In the current study, human subjects performed head-free gaze shifts between visual targets while wearing pinhole goggles. During fixations, the head orientation range still obeyed Donders' law, but in most subjects, it immediately changed from the twisted Fick-like range to a flattened Listing-like range. Further controls showed that this was not attributable to loss of binocular vision or increased range of head motion, nor was it attributable to blocked peripheral vision; when subjects pointed a helmet-mounted laser toward targets (a task with goggle-like motor demands but normal vision), the head followed Listing's law even more closely. Donders' law of the head only broke down (in favor of a "minimum-rotation strategy") when head motion was dissociated from gaze. These behaviors could not be modeled using current "Donders' operators" but were readily simulated nonholonomically, i.e., by modulating head velocity commands as a function of position and task. We conclude that the gaze control system uses such velocity rules to shape Donders' law on a moment-to-moment basis, not primarily to satisfy perceptual or anatomic demands, but rather for motor optimization; the Fick strategy optimizes the role of the head as a platform for eye movement, whereas Listing's law optimizes rapid control of the eye (or head) as a gaze pointer. PMID- 10729354 TI - Progressive postnatal assembly of limbic-autonomic circuits revealed by central transneuronal transport of pseudorabies virus. AB - The development of neuronal projections to a target and the establishment of synaptic connections with that target can be temporally distinct events, which typically are distinguished by functional assessments. We have applied a novel neuroanatomical approach to characterize the development of limbic forebrain synaptic inputs to autonomic neurons in neonatal rats. Transneuronal labeling of preautonomic forebrain neurons was achieved by inoculating the ventral stomach wall with pseudorabies virus (PRV) on postnatal day 1 (P1), P4, or P8. In each age group, PRV-positive neurons were present in autonomic and preautonomic regions of the spinal cord and brainstem 62-64 hr after inoculation. Transneuronal forebrain labeling in rats injected on P8 was similar to the transneuronal labeling reported previously in adult rats and included neurons in the medial and lateral hypothalamus, amygdala, bed nucleus of the stria terminalis, and visceral cortices. However, no cortex labeling and only modest amygdala and bed nucleus labeling were observed in rats injected with PRV on P4, and only medial hypothalamic labeling was observed in rats injected on P1. Additional tracing experiments involving central injections of PRV or cholera toxin beta indicated that lateral hypothalamic and telencephalic regions projected to the medullary dorsal vagal complex several days before establishing synaptic connections with gastric-related autonomic neurons. These results demonstrate a novel strategy for evaluating synaptic connectivity in developing neural circuits and show a temporally segregated postnatal emergence of medial hypothalamic, lateral hypothalamic, and telencephalic synaptic inputs to central autonomic neurons. PMID- 10729355 TI - Axotomized and intact muscle afferents but no skin afferents develop ongoing discharges of dorsal root ganglion origin after peripheral nerve lesion. AB - After peripheral nerve lesions, some axotomized afferent neurons develop ongoing discharges that originate in the dorsal root ganglion (DRG). We investigated in vivo which functional types of afferent neurons contributed to this ectopic activity. Six to twelve days after the gastrocnemius soleus (GS) nerve supplying skeletal muscle and the sural (SU) nerve supplying skin had been transected (experimental group E1), 20.4% of afferent neurons with myelinated axons projecting into the GS nerve produced ongoing discharges of irregular or bursting pattern. In contrast, all SU neurons were silent. Additional transection of peroneal and tibial nerves (group E2) induced ongoing activity in a similar percentage of GS neurons (22.1%), but their mean discharge frequency was higher (6.0 vs 2.7 Hz), and more of them exhibited bursting discharges (63 vs 17%). When the GS nerve had been left intact while tibial, peroneal, and SU nerve had been transected (group E3), 18.8% of unlesioned GS neurons developed ongoing discharges at a mean frequency of 6.1 Hz; most of them exhibited a bursting pattern. Without a preceding nerve lesion, almost no GS neuron (1.1%) fired spontaneously. Most afferent neurons with ongoing activity had an axonal conduction velocity of 5-30 m/sec indicating that some of these neurons may have had nociceptive function. These findings provide the first evidence that after peripheral nerve injury both axotomized as well as intact afferent neurons supplying skeletal muscle but not skin afferents generate ongoing activity within the DRG, probably because of a yet unknown signal in the DRG triggered by axotomy. PMID- 10729356 TI - Developmental changes in the subcellular localization of calretinin. AB - Brainstem auditory neurons in the chick nucleus magnocellularis (NM) express high levels of the neuron-specific calcium-binding protein calretinin (CR). CR has heretofore been considered a diffusible calcium buffer that is dispersed uniformly throughout the cytosol. Using high-resolution confocal microscopy and complementary biochemical analyses, we have found that during the development of NM neurons, CR changes from being expressed diffusely at low concentrations to being highly concentrated beneath the plasma membrane. This shift in CR localization occurs at the same time as the onset of spontaneous activity, synaptic transmission, and synapse refinement in NM. In the chick brainstem auditory pathway, this subcellular localization appears to occur only in NM neurons and only with respect to CR, because calmodulin remains diffusely expressed in NM. Biochemical analyses show the association of calretinin with the membrane is detergent-soluble and calcium-independent. Because these are highly active neurons with a large number of Ca2+-permeable synaptic AMPA receptors, we hypothesize that localization of CR beneath the plasma membrane is an adaptation to spatially restrict the calcium influxes. PMID- 10729357 TI - Input-specific long-term depression in the lateral amygdala evoked by theta frequency stimulation. AB - Although conditioned fear has been shown to involve mechanisms of synaptic plasticity in the amygdala, the association with afferent input systems is not yet clear. Here we report on homosynaptic long-term depression (LTD) of excitatory responses after stimulation of putative thalamic input fibers, but not of cortical inputs, to the rat lateral amygdala in vitro. LTD is induced by theta frequency stimulation and involves postsynaptic calcium-dependent mechanisms and group II metabotropic glutamate receptors. These input-specific changes in synaptic strength represent potential cellular sources, which regulate the balance between sensory thalamic and cortical input signals to the amygdala. This regulation would function to reduce the influence of relatively undiscriminated stimulus information carried by thalamic afferents in favor of discriminated sensory information mediated by the cortex during fear responses. PMID- 10729358 TI - Activation of D2-like dopamine receptors reduces synaptic inputs to striatal cholinergic interneurons. AB - Dopamine (DA) plays a crucial role in the modulation of striatal function. Striatal cholinergic interneurons represent an important synaptic target of dopaminergic fibers arising from the substantia nigra and cortical glutamatergic inputs. By means of an electrophysiological approach from corticostriatal slices, we isolated three distinct synaptic inputs to cholinergic interneurons: glutamate mediated EPSPs, GABAA-mediated potentials, and Acetylcholine (ACh)-mediated IPSPs. We therefore explored whether DA controls the striatal cholinergic activity through the modulation of these synaptic potentials. We found that SKF38393, a D1-like receptor agonist, induced a membrane depolarization (also see Aosaki et al., 1998) but had no effects on glutamatergic, GABAergic, and cholinergic synaptic potentials. Conversely, D2-like DA receptor activation by quinpirole inhibited both GABAA and cholinergic synaptic potentials. These effects of quinpirole were mimicked by omega-conotoxin GVIA, blocker of N-type calcium channels. The lack of effect both on the intrinsic membrane properties and on exogenously applied GABA and ACh by quinpirole supports a presynaptic site of action for the D2-like receptor-mediated inhibition. Moreover, the quinpirole induced decrease in amplitude was accompanied by an increase in paired pulse facilitation ratio (EPSP2/EPSP1), an index of a decrease in transmitter release. Our findings demonstrate that DA modulates the excitability of cholinergic interneurons through either an excitatory D1-like-mediated postsynaptic mechanism or a presynaptic inhibition of the GABAergic and cholinergic inhibitory synaptic potentials. PMID- 10729359 TI - Cytochrome P450-dependent N-dealkylation of L-deprenyl in C57BL mouse liver microsomes: effects of in vivo pretreatment with ethanol, phenobarbital, beta naphthoflavone and L-deprenyl. AB - The monoamine oxidase inhibitor L-deprenyl [(-)-deprenyl, selegiline] is an effective therapeutic agent for improving early symptoms of idiopathic Parkinson's disease. It appears to exert this action independently of its inhibition of monoamine oxidase B (MAO-B) and some of its metabolites are thought to contribute. Cytochrome P450 (CYP) activities are known to give rise to L deprenyl metabolites that may affect the dopaminergic system. In order to clarify the interactions of L-deprenyl with these enzymes, C57BL mice were treated with L deprenyl, ethanol, phenobarbital or beta-naphthoflavone to induce different CYP isozymes. After preincubation of L-deprenyl with liver microsomes from control or treated mice, the metabolites were analysed by a GLC method. L-deprenyl (10 mg/kg i.p. for 3 days) caused a significant decrease in total CYP levels (0.315+/ 0.019, L-deprenyl; 0.786+/-0.124, control, nmol/mg protein) and CYP2E1-associated p-nitrophenol hydroxylase activity (0.92+/-0.04 vs. 1.17+/-0.06 nmol/min/mg). Both phenobarbital and ethanol increased the N-depropynylation activity towards L deprenyl that leads to the formation of methamphetamine (4. 11+/-0.64, phenobarbital; 4.77+/-1.15, ethanol; 1.77+/-0.34, control, nmol/min/mg). Ethanol alone increased the N-demethylation rate of L-deprenyl, that results in formation of nordeprenyl (3.99+/-0.68, ethanol; 1.41+/-0.31, control, nmol/min/mg). Moreover, the N-dealkylation pathways of deprenyl are inhibited by 4 methylpyrazole and disulfiram, two CYP2E1 inhibitors. None of the other treatments modified L-deprenyl metabolism. These findings indicate that mainly CYP2E1 and to a lesser extent CYP2B isozymes are involved in L-deprenyl metabolism. They also suggest that, by reducing CYP content, L-deprenyl treatment may impair the metabolic disposition of other drugs given in combination regimens. PMID- 10729360 TI - Inhibition of the P-glycoprotein- and multidrug resistance protein-mediated efflux of anthracyclines and calceinacetoxymethyl ester by PAK-104P. AB - Multidrug resistance phenotype in mammalian cells is often correlated with overexpression of P-glycoprotein or Multidrug Resistance-Associated protein (MRP(1)). Both proteins are energy-dependent drug efflux pumps that efficiently reduce the intracellular accumulation and hence the cytotoxicity of many natural cytotoxins. Overexpression of these transporters by tumor cells is thought to be a significant factor in both intrinsic and acquired resistance to anticancer drugs. Consequently a great deal of interest is focused on identifying chemical agents that can either antagonise drug transport by these proteins or that can inhibit the proliferation of tumors cells despite the expression of these transporters. P-glycoprotein-mediated multidrug resistance is reversed by a variety of compounds, but surprisingly, few agents reverse the MRP(1)-mediated multidrug resistance. However, it has recently been shown that 2-[4 (diphenylmethyl)-1-piperazinyl]ethyl-5-(trans-4,6-dimethyl-1, 3, 2 dioxaphosphorinan-2-yl)-2, 6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P oxide (PAK-104P) was able to inhibit the P-glycoprotein and MRP(1)-mediated efflux of several compounds. Understanding of the interactions between transporters and multidrug resistance reversing agents is important in the design of more effective multidrug resistance modulators. We now examined the effect of PAK-104P on Pgp-and MRP1-mediated efflux of three anthracyclines, daunorubicin, pirarubicin, hydroxydoxorubicin and of calcein acetoxymethyl ester and calcein. Our data show that PAK-104P non-competitively inhibits the P-glycoprotein mediated efflux of anthracycline derivatives and calcein acetoxymethyl ester with an inhibitory constant K(I)=0. 25+/-0.05 microM. PAK-104P also non-competitively inhibits the MRP(1)-mediated efflux of daunorubicin, pirarubicin, hydroxyrubicin, calcein acetoxymethyl ester and calcein. However, surprisingly, in this case the K(I) values obtained were very different ranging from 0.06 for hydroxyrubicin to 10 microM for calcein. These data strongly suggested the existence of two different mechanisms for the inhibition by PAK-104P of the MRP(1)-mediated efflux of molecules: a first mechanism, involving a low-affinity site for PAK-104P, and which would concern molecules such as calcein, cysteinyl leukotriene LCT(4) etc. whose efflux do not depend on glutathione. A second mechanism involving a high affinity site for PAK-104P and which would concern molecules such as anthracyclines, calcein acetoxymethyl ester whose efflux depends on the presence of glutathione. PMID- 10729361 TI - Electrophysiological effect of l-cis-diltiazem, the stereoisomer of d-cis diltiazem, on isolated guinea-pig left ventricular myocytes. AB - l-cis-Diltiazem, the stereoisomer of the L-type Ca(2+) channel blocker d-cis diltiazem, protects cardiac myocytes from ischemia and reperfusion injury in the perfused heart and from veratridine-induced Ca(2+) overload. We determined the effect of l-cis-diltiazem on the voltage-dependent Na(+) current (I(Na)) and lysophosphatidylcholine-induced currents in isolated guinea-pig left ventricular myocytes by a whole-cell patch-clamp technique. l-cis-Diltiazem inhibited I(Na) in a dose-dependent manner without altering the current-voltage relationship for I(Na) (K(d) values : 729 and 9 microM at holding potentials of -140 and -80 mV, respectively). A use-dependent block of I(Na), the leftward shift of the steady state inactivation curve and the delay of recovery from inactivation suggest that l-cis-diltiazem has a higher affinity for the inactivated state of Na(+) channels. In addition to I(Na), the lysophosphatidylcholine-induced currents were inhibited by l-cis-diltiazem in a similar concentration range. It is suggested that inhibition of both Na(+) channels and lysophosphatidylcholine-activated non selective cation channels contributes to the cardioprotective effect of l-cis diltiazem. PMID- 10729362 TI - Nitric oxide accelerates the ascorbic acid-induced osteoblastic differentiation of mouse stromal ST2 cells by stimulating the production of prostaglandin E(2). AB - Nitric oxide (NO) promoted the differentiation of clonal stromal cells (ST2 cells) derived from mouse bone marrow to osteoblast-like cells. The level of expression of mRNA for osteocalcin, a marker of osteoblastic differentiation, and the formation of mineralized nodules, increased in ST2 cells treated with a donor of NO. We used the reverse transcriptase-polymerase chain reaction (RT-PCR) to identify the subtypes of NO synthase that were expressed in the ST2 cells and we detected the expression of an inducible NO synthase gene in response to tumor necrosis factor-alpha (TNF-alpha). In various types of cell, NO induces the synthesis of prostaglandin E(2) and cGMP, which are known as regulators of osteoblastic differentiation, by activating cyclooxygenases and soluble guanylate cyclase, respectively. Prostaglandin E(2) was generated in response to NO in ST2 cells, however, no synthesis of cGMP in response to NO was detected. Two inhibitors of cyclooxygenase-2, N-[4-nitro-2-phenoxyphenyl]-methanesulfonamide (nimesulide) and 1-(4-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetic acid (indomethacin), inhibited the formation of mineralized nodules by ST2 cells. Our observations suggest that NO might promote osteoblastic differentiation of ST2 cells by stimulating the production of prostaglandin E(2). PMID- 10729363 TI - Receptor binding characteristics of the novel NMDA receptor glycine site antagonist [3H]GV150526A in rat cerebral cortical membranes. AB - Binding of the glycine site antagonist 3-[2-(Phenylamino-carbonyl)ethenyl]-4,6 dichloro-indole-2-carboxylic acid sodium salt ([3H]GV150526A) was characterised in rat cerebral cortical membranes. Saturation experiments indicated the existence of a high affinity binding site, with a pK(d) value of 9.08 (K(d)=0. 8 nM) and a B(max) of 3.4 pmol/mg of protein. A strong linear correlation was observed between the displacement potencies for [3H]GV150526A and [3H]glycine of 13 glycine site ligands (r=0.991). The association kinetics of [3H]GV150526A binding was monophasic, with a k(on) value of 0.047 (nM)(-1) min(-1). Dissociation was induced by the addition of an excess of glycine, GV150526A, or 5,7-dichlorokynurenic acid (DCKA), another glycine antagonist. With GV150526A and DCKA, the dissociation curves presented similar k(off) values (0.068 and 0.069 min(-1), respectively), as expected from ligands binding to the same site. Conversely, a significantly lower k(off) value (0.027 min(-1)) was found with glycine. Although these data may suggest that glycine agonists and antagonists bind to discrete sites with an allosteric linkage (rather than interacting competitively), the reason for this difference remains to be elucidated. It is concluded that [3H]GV150526A can be considered a new valuable tool to further investigate the properties of the glycine site of the NMDA receptor. PMID- 10729364 TI - Bicuculline antagonizes 5-HT(3A) and alpha2 glycine receptors expressed in Xenopus oocytes. AB - The present study examined the effects of bicuculline on the mouse 5 hydroxytryptamine(3A) receptor (5-HT(3A) receptor and the human alpha2 subunit of the glycine receptor. Bicuculline antagonized both the 5-HT(3A) receptor (IC(50)=20.12+/-0.39 microM) and the alpha2 glycine receptor (IC(50)=169.40+/ 1.73 microM). A competitive form of antagonism by bicuculline was suggested by experiments in which the EC(50)s for 5-HT and glycine were increased in the 5 HT(3A) and alpha2 glycine receptors, respectively, as bicuculline concentrations were increased. A competitive nature of antagonism by bicuculline at the 5-HT(3A) receptor was also suggested by displacement of the competitive antagonist, [3H]GR65630 in SF21 insect cells expressing the 5-HT(3A) receptor (K(i)=19.01+/ 0.71 microM). Our data and that of others reveal that bicuculline, a purported selective antagonist of the GABA(A) receptor, antagonizes at least one receptor subclass in every member of the superfamily of ligand-gated ion channels. PMID- 10729365 TI - Piracetam and levetiracetam, two pyrrolidone derivatives, exert antidystonic activity in a hamster model of paroxysmal dystonia. AB - The effects of the nootropic drug piracetam and its analogue, the antiepileptic drug levetiracetam (ucb L059) on severity of dystonic attacks were studied in a mutant hamster model of idiopathic generalized dystonia. Both drugs significantly decreased the severity of dystonia. In contrast to seizure models, in which levetiracetam is much more potent as an anticonvulsant than piracetam, the antidystonic potency of levetiracetam was only moderately higher than that of piracetam. The antidystonic activity of piracetam and levetiracetam was not associated with any behavioral side effects. The data indicate that piracetam and levetiracetam are interesting novel treatments for idiopathic dystonia. PMID- 10729366 TI - gamma-Aminobutyrate, alpha-carboxy-2-nitrobenzyl ester selectively blocks inhibitory synaptic transmission in rat dentate gyrus. AB - gamma-Aminobutyrate, alpha-carboxy-2-nitrobenzyl ester (cGABA) is a stable photoactivatable probe used to study gamma-aminobutyrate (GABA) receptors. GABA is released from this compound when it is exposed to ultraviolet light, but little is known about the electrophysiological effects of the compound itself. Whole cell patch clamp recordings on rat hippocampal slices demonstrated that cGABA blocked polysynaptic inhibitory postsynaptic currents (IPSCs) evoked in dentate granule cells by antidromic stimulation of the mossy fibers. It also reduced monosynaptically evoked IPSCs with an IC(50) of 28 microM. In contrast, cGABA had no effect on excitatory postsynaptic currents (EPSCs) evoked by perforant path stimulation. The effect of cGABA was not mediated by depression of GABA release through activation of presynaptic GABA(B) receptors. cGABA inhibited muscimol-evoked currents by only 15% at a concentration of 40 microM. At this same concentration, it reduced the mean frequency of miniature inhibitory postsynaptic potentials by 71%, their mean peak amplitude by 44%, their mean decay time constant by 26% and the mean charge transfer per event by 52%. These effects may be explained by a phenothiazine-like modification of GABA(A) receptor kinetics and/or a selective block of somatic GABA synapses. PMID- 10729367 TI - Desipramine binding to noradrenaline reuptake sites in cardiac sympathetic neurons in man in vivo. AB - Noradrenergic reuptake blockade is a recognised mechanism of antidepressant action, but the extent of the blockade necessary for therapeutic effect is not known and plasma levels do not provide a guide to therapy. We report a method to assess noradrenaline reuptake blockade in vivo in man using [11C]meta hydroxyephedrine and the multiple organs' coincidences counter. Eight healthy volunteers had two scans, one with tracer alone and one after preloading with desipramine 50-75 mg p.o. In all subjects, there was an increased washout rate of the radioligand from the heart following preloading (t=4.38; P<0.003) as well as a decrease of the area under the [11C]meta-hydroxyephedrine time activity curve (t=7. 4; P=0.001). In one subject who had three doses of desipramine, the increase in washout rate was dose-dependent. In conclusion, [11C]meta hydroxyephedrine in the multiple organs' coincidences counter gives a valid, low radiation method to assess noradrenergic reuptake blockade in the clinic. PMID- 10729368 TI - Activational role of cannabinoids on movement. AB - Cannabinoid's major effect on movement is hypoactivity. Nevertheless, a biphasic excitatory/inhibitory effect of cannabinoids on movement has been repeatedly acknowledged. However, the literature is lacking a detailed description of such an effect. In this study, we performed a dose-response study of the effects of Delta(9)-tetrahydrocannabinol on movement. Immediately after the administration of vehicle or a dose of Delta(9)-tetrahydrocannabinol (0.2, 0.5, 1, 1.5, 2, 2.5, 3, 4, or 5 mg/kg), the animal was placed in an activity monitor and observed for 1 h. Several parameters were recorded. The horizontal and vertical activities were measured as the number of photobeams broken between the photocells on the walls of an activity monitor. The number of wet dog shakes, scratches with hindpaw, mouth movements, forepaw flutters were also recorded, as was the amount of time in minutes that each subject spent grooming. The number of fecal boluses was recorded as an index of autonomic activity. Each animal was subsequently tested for catalepsy in the bar test. A triphasic effect was observed: low doses of the cannabinoid receptor agonist Delta(9)-tetrahydrocannabinol (0.2 mg/kg) decreased locomotor activity while higher doses (1-2 mg/kg) dose-dependently stimulated movement until catalepsy emerged (2.5 mg/kg) accompanied by decreases in activity. PMID- 10729369 TI - Effects of the cannabinoid receptor agonist, HU 210, on ingestive behaviour and body weight of rats. AB - The effect of the synthetic cannabinoid receptor agonist, (-)11-hydroxy-Delta(8) tetrahydrocannabinol-dymethylheptyl (HU 210), on rat body weight and eating and drinking behaviour was examined. In Experiment 1, the drug (25, 50 or 100 microg/kg), sub-chronically administered for 4 days, produced a dose- and time dependent loss of body weight that, at the highest dose, was not regained by 7 days after the drug was stopped, and remained markedly below that of vehicle treated animals. In Experiment 2, food and water intakes, which were evaluated in fasted rats, tested as in Experiment 1, were significantly inhibited only by the dose of 100 microgram/kg, and this effect was still present 7 days after the last injection of the drug. The possibility that the effects observed are not directly dependent on the control of appetite and might be ascribable to stress-related phenomena is discussed. PMID- 10729370 TI - Angiotensin II mediates pressure loading-induced mitogen-activated protein kinase activation in isolated rat aorta. AB - Vascular hypertrophy occurs during chronic hypertension and contributes to the elevation of peripheral vascular resistance in hypertension. In this study, we examined whether acute pressure overloading of the vascular wall produces activation of mitogen-activated protein (MAP) kinases, enzymes believed to be involved in the pathway for cell proliferation, in isolated perfused rat aortae, and examined whether the mechanical overloading-induced MAP kinase activation is mediated via the vascular angiotensin system. Aortae were perfused with Tyrode solution. Increases in perfusion pressure caused a pressure-dependent increase in MAP kinase activity in endothelium-intact aortae and in endothelium-denuded aortae. The increase in MAP kinase activity induced by pressure loading was inhibited by the angiotensin receptor antagonist, losartan, the renin inhibitor, pepstatin A, and the angiotensin-converting enzyme inhibitor, captopril. Ca(2+) depletion and the Ca(2+) channel antagonist, nifedipine, did not affect the pressure loading-induced MAP kinase activation. The results of the present study suggest that pressure loading of the vascular wall per se can activate MAP kinases in the vasculature and that the MAP kinase activation is mediated at least partly via the vascular angiotensin system. It seems unlikely that the pressure loading-induced increase in MAP kinase activity is mainly mediated via increases in Ca(2+) influx in vascular cells. PMID- 10729371 TI - Buprenorphine and methoclocinnamox: agonist and antagonist effects on respiratory function in rhesus monkeys. AB - Buprenorphine and methoclocinnamox are partial micro-opioid receptor agonists with potential use in the treatment of opioid abuse. The ability of these drugs to suppress respiration as well as their ability to antagonize the respiratory suppressant effects of morphine and heroin were tested in rhesus monkeys. Frequency (f), minute volume (V(e)) tidal volume (V(t)) in monkeys breathing air or 5% CO(2) in air were recorded using a pressure-displacement plethysmograph. Buprenorphine (0.001-10 mg/kg) produced a dose-dependent decrease in respiratory parameters that plateaued at a dose of 1 mg/kg in both air and 5% CO(2). Methoclocinnamox (0. 032-1 mg/kg) also produced dose-dependent respiratory depression that plateaued at a dose of 0.3 mg/kg in air, and was directly related to dose in 5% CO(2). Respiratory suppression produced by buprenorphine 1 and 10 mg/kg lasted for 3 and 7 days, respectively, whereas the suppression produced by the largest dose of methoclocinnamox (1 mg/kg, the solubility limit) lasted less than 24 h. Buprenorphine and methoclocinnamox antagonized morphine- and heroin induced respiratory depression, and this antagonist effect was observed concomitantly with, as well as following, the mu-opioid receptor agonist effects of buprenorphine and methoclocinnamox. The mu-opioid receptor antagonist effects of buprenorphine (10 mg/kg) and methoclocinnamox (1 mg/kg) lasted for 2 weeks. These results suggest that buprenorphine and methoclocinnamox have a wide margin of safety in clinical use and that these two compounds have a prolonged, insurmountable, mu-opioid receptor antagonist effect after the disappearance of their agonist effects. PMID- 10729372 TI - Mechanisms involved in UTP-induced contraction in isolated rat aorta. AB - The mechanisms of UTP-induced contractions in the rat aorta strips were studied. These were only partially inhibited in a Ca(2+)-free medium or by incubation with verapamil or nifedipine. Successive challenges did not decrease the magnitude of the contraction in the absence of external Ca(2+). Quin 2(acetoxymethyl) ester (Quin 2AM), 8-(N,N-diethylamino)octyl 3,4,5-trimetoxybenzoate (TMB-8), thapsigargin and ryanodine inhibited these contractions. The participation of protein kinase C is also very likely, since downregulation by the phorbol 12,13 dibutyrate (PDB) decreased UTP-induced contraction, and staurosporine and 1-(5 isoquinolinesulfonyl)-2-methylpiperazine (H-7) antagonized UTP-induced contractions and relaxed UTP-induced tonic contractions. Therefore, different pools of intracellular Ca(2+) and protein kinase C seem to participate in UTP induced contraction and in the mechanisms of maintenance in a Ca(2+)-free medium. PMID- 10729373 TI - Involvement of vanilloid receptors and purinoceptors in the Phoneutria nigriventer spider venom-induced plasma extravasation in rat skin. AB - Phoneutria nigriventer venom causes stimulation of capsaicin-sensitive primary afferent neurons in the rat dorsal skin, leading to neurogenic plasma protein extravasation due to the release of tachykinin NK(1) receptor agonist. In this study we further investigated the mechanisms involved in the venom-induced activation of capsaicin-sensitive primary afferent neurons. The plasma extravasation in response to venom intradermally injected was measured in Wistar rats as the local accumulation of i.v. injected 125I-labelled human serum albumin into skin sites. The tachykinin NK(1) receptor agonist, D-Ala-[L-Pro(9),Me Leu(8)]substance P-(7-11) (GR73632; 10-100 pmol/site), induced a significant plasma leakage that was abolished by the selective tachykinin NK(1) receptor antagonist, (S)-1-[2-[3-(3,4-dichlorphenyl)-1 (3-isopropoxyphenylacetyl) piperidin-3-yl] ethyl]-4-phenyl-1 azaniabicyclo [2.2.2]octane chloride (SR140333; 1 nmol/site), whereas the leakage after venom (1-10 microgram/site) was significantly inhibited (but not abolished) by SR140333. The calcitonin gene related peptide (CGRP) receptor antagonist, CGRP-(8-37), failed to further reduce the residual plasma extravasation induced by venom plus SR140333. The mu-opioid receptor agonist, [D-Ala(2), Me-Phe(4),Gly-ol(5)]enkephalin (DAMGO), and the local anaesthetic, lignocaine, had no effect on the venom-induced plasma extravasation. Similarly, the L-, N- and P/Q-type voltage-sensitive Ca(2+) channel blockers (verapamil, omega-conotoxin MVIIA and MVIIC, respectively) as well as the Na(+) channel blockers, tetrodotoxin and carbamazepine, had no effect on the venom-induced effect. Neither the systemic treatment nor the local injection of ruthenium red prevented the venom-induced plasma extravasation. However, the vanilloid receptor antagonist, N-[2-(4-chlorophenyl) ethyl]-1,3,4, 5 tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide (capsazepine; 120 micromol/kg, i.v.), reduced by 48% (P<0.05) the venom (10 microgram/site)-induced plasma extravasation. A significant inhibitory effect was also observed with the P(2) purinoceptor agonists, adenosine 5'-triphosphate (ATP; 10 and 30 nmol/site) and adenosine 5'-diphosphate (ADP; 10 nmol/site). The involvement of histamine and/or 5-hydroxytryptamine (5-HT) in the venom-induced plasma extravasation was ruled out since neither histamine and 5-HT receptor antagonists nor depletion of mast cells by compound 48/80 affected the venom response. This was further supported by the failure of venom to degranulate in vitro peritoneal mast cells. In conclusion, only vanilloid receptors and P(2) prejunctional purinoceptors had an inhibitory effect on the neurogenic plasma extravasation evoked by P. nigriventer venom in rat dorsal skin. PMID- 10729374 TI - A novel dual regulator of tumor necrosis factor-alpha and interleukin-10 protects mice from endotoxin-induced shock. AB - A pyrimidylpiperazine derivative, N-[1-(4-?[4-(pyrimidin-2-yl)piperazin-1 yl]methyl?phenyl)cycloprop yl] acetamide (Y-39041), is a dual cytokine regulator of tumor necrosis factor (TNF)-alpha and interleukin-10 production. Lipopolysaccharide-induced TNF-alpha release in BALB/c mice was inhibited by the oral treatment with the compound at 10-100 mg/kg (about 80% suppression) while interleukin-10 release was augmented (about 10-fold increase at 30 mg/kg). In addition, Y-39041 (30 mg/kg, p.o.) completely protected mice from lipopolysaccharide-induced death by the treatment before and after lipopolysaccharide injection. The finding that Y-39041 suppresses TNF-alpha production and stimulates interleukin-10 production at the same time provides new insights for the treatment of septic shock, rheumatoid arthritis and Crohn's diseases. PMID- 10729375 TI - The role of plaque rupture and thrombosis in coronary artery disease. AB - Atherosclerosis and its thrombotic complications are the major cause of morbidity and mortality in the industrialized world. The progression of atherosclerotic plaques in the coronary circulation is dependent on several risk factors. It is now clear that plaque composition is a major determinant of the risk of subsequent plaque rupture and superimposed thrombosis. The vulnerability of plaques to rupture is further determined by extrinsic triggering factors. Following rupture, the fatty core of the plaque and its high content of tissue factor provide a powerful substrate for the activation of the coagulation cascade. Plaque rupture can be clinically silent or cause symptoms of ischaemia depending on thrombus burden and the degree of vessel occlusion. In addition, plaque rupture and subsequent healing is recognized to be a major cause of further rapid plaque progression. This review looks at the mechanisms underlying the development and progression of atherosclerotic plaques, factors leading to plaque rupture and subsequent thrombosis and their clinical consequences. Finally, we speculate on targets for future research. PMID- 10729376 TI - Effects of reserpine on expression of the LDL receptor in liver and on plasma and tissue lipids, low density lipoprotein and fibrinogen in rabbits in vivo. AB - The effects of administering reserpine (0.1 mg/kg) or 17alpha-ethinyloestradiol (2.5 mg/kg) to New Zealand White rabbits on low density lipoprotein receptors in liver, on plasma low density lipoprotein and fibrinogen and on plasma and tissue lipids were determined. Blood pressure and heart rate were also followed. The drugs were injected subcutaneously into conscious unrestrained rabbits for 5 days. On the 6th day homologous 125I-tyramine cellobiose labelled low density lipoprotein (125I-TC-LDL) was injected intravenously and 24 h later the animals were killed. Compared to controls, reserpine significantly increased LDL receptor expression in the liver by about threefold, and reduced total cholesterol in plasma, aorta and heart, without affecting plasma triglycerides. The reductions in plasma cholesterol and heart were due to decreases in both unesterified and esterified cholesterol. Similar effects were observed with oestrogen, except that there was no change in esterified cholesterol in aorta. In liver, a decrease of 24% in total cholesterol was due mainly to decreased esterified cholesterol. In adrenal glands total cholesterol increased by 25%. Reserpine significantly accelerated the plasma clearance of intravenously injected homologous 125I-TC-LDL and reduced its accumulation in aortic wall. Neither reserpine nor oestradiol affected blood pressure, haematocrit or plasma fibrinogen. The results suggest that reserpine is an affective anti-atherogenic drug capable of decreasing cholesterol in plasma, arteries and heart by increasing high affinity LDL receptors in the liver. PMID- 10729377 TI - The effect of vitamin E, probucol, and lovastatin on oxidative status and aortic fatty lesions in hyperlipidemic-diabetic hamsters. AB - Diabetes mellitus is associated with an increased risk of premature atherosclerosis, which may be due in part to an increased rate of low density lipoprotein (LDL) oxidation. Previous studies have shown that vitamin E, probucol, and lovastatin can reduce the oxidative susceptibility of LDL in normoglycemic animal models; however, few studies have investigated this in conjunction with aortic fatty streak lesion formation in diabetic hyperlipidemic models. Forty-eight Syrian hamsters were made diabetic by intraperitoneal injection of low dose streptozotocin. Diabetic animals (12 animals/groups) received a high saturated fat and cholesterol diet for 12.5 weeks. At 2.5 week of dietary treatments, the diet was supplemented with either: (1) 500 IU/day vitamin E (D+E); (2) 1% probucol w/w of the diet (D+P); (3) 25 mg/kg lovastatin (D+L); or (4) diabetic control (D). An age-matched group of hamsters (n=6) receiving the same diet but not made diabetic (ND) was used as control. At the end of the study, aortic arch foam cell-rich fatty streak lesion, plasma glucose, total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), non-HDL-C, triglycerides (TG), phospholipids, alpha-tocopherol, plasma lipid peroxide and the susceptibility of LDL to copper-catalyzed oxidation were determined. Diabetes increased plasma glucose, and when combined with an atherogenic diet resulted in a further increase of plasma lipids. Vitamin E, probucol, and lovastatin significantly reduced plasma TG in the diabetic hamsters fed the atherogenic diet. Vitamin E treatment increased TC, probucol reduced HDL-C without affecting TC; whereas lovastatin reduced TC and selectively decreased non-HDL-C, and significantly reduced fatty streak lesion formation in the aortic arch. While vitamin E and probucol were effective in reducing several indices of oxidative stress including plasma lipid peroxides, cholesterol oxidation products and in vitro LDL oxidation, they had no effect on fatty streak lesion formation. Our results indicate that the LDL in diabetic animals is more susceptible to oxidation than in non-diabetic hamsters and that not only vitamin E and probucol but also lovastatin provide antioxidant protection. It appears that in this combined model of diabetes and hypercholesterolemia, progression of fatty streak lesion formation is mainly associated with changes in TC and non-HDL-C as affected by lovastatin, and is less dependent on the extent of LDL oxidation, changes in plasma TG level and oxidative stress status. PMID- 10729378 TI - Differential effects of long-term renin-angiotensin system blockade on limitation of infarct size in cholesterol-fed rabbits. AB - We evaluated the effects of chronic inhibition of angiotensin-converting enzyme (ACE) or receptor blockade of angiotensin II type I on the size of myocardial infarcts induced by coronary occlusion-reperfusion in rabbits fed a high cholesterol or normal diet for 10 weeks. In treated rabbits, myocardial infarction occurred 24 h after the last dose of enalapril or L-158809, an angiotensin II type I receptor antagonist, because of the drugs' waning effects on hemodynamic parameters. The size of the infarct was significantly larger in cholesterol-fed rabbits than in rabbits fed a normal diet. This augmentation of infarct size in cholesterol-fed rabbits was reversed by long-term treatment with enalapril, but not L-158809. The favorable effects of enalapril treatment disappeared after pretreatment with the bradykinin B(2) receptor blocker HOE 140. Long-term enalapril or L-158809 administration did not reduce the size of the infarct in rabbits fed a normal diet. ACE activity in ischemic myocardium significantly exceeded that in nonischemic myocardium and was further increased in cholesterol-fed rabbits, but was significantly reduced by long-term enalapril, but not L-158809. Moreover, treatment with enalapril, but not L-158809, restored acetylcholine-induced endothelium-dependent relaxation of aortic rings from cholesterol-fed rabbits. These results demonstrate that long-term ACE inhibition, but not angiotensin II type I receptor blockade, effectively reduces the size of myocardial infarcts in cholesterol-fed rabbits. The favorable effects of enalapril treatment may involve primarily a bradykinin B(2) receptor-mediated pathway. PMID- 10729379 TI - Small oxidative changes in atherogenic LDL concentrations irreversibly regulate adhesiveness of human endothelial cells: effect of the lazaroid U74500A. AB - The adherence of monocytes to the endothelium is an early event in atherogenesis which is modulated by low density lipoproteins (LDL). We analyzed the effect of atherogenic LDL levels (180 mg cholesterol/dl, for 24 h) with minimal oxidative modifications (thiobarbituric-acid-reactive-substances (TBARS) concentration between 1.2+/-0.1 and 2.5+/-0.3 nmol of malonaldehyde bis-diethyl acetal (MDA) per mg protein) on human umbilical vein endothelial cell (HUVEC) adhesive properties. We used native LDL (n-LDL), and LDL exposed to spontaneous oxidation without antioxidants (mox-LDL) or with 20 micromol/l of the antioxidant butylated hydroxytoluene (BHT-LDL) or 10 micromol/l U74500A (U74500A-LDL), a scavenger of free radicals. Thiobarbituric-acid-reactive-substances (TBARS) levels were significantly higher in mox-LDL (2.5+/-0.3 nmol MDA/mg protein) than in BHT-LDL (1.6+/-0.2), U74500A-LDL (1.2+/-0.1) or in n-LDL (1.3+/-0.1). mox-LDL induced the greatest adhesion of U937 cells to HUVEC (103+/-9% over controls) followed by BHT LDL (75+/-10%), U74500A-LDL (36+/-9%) and n-LDL (35+/-3%). The lazaroid U74500A efficiently protected U74500A-LDL against oxidative damage and prevented endothelial adhesiveness associated with this LDL modification, inducing adhesion effects similar to those of n-LDL. However, U74500A could not reverse the adhesion induced by previously oxidized LDL (mox-LDL). LDL did not induce the expression of the intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) or E-selectin, but it produced a downregulation of endothelial nitric oxide synthase (NOS III) mRNA levels. Thus, adhesiveness of human endothelial cells (EC) exposed to atherogenic concentrations of LDL is closely modulated by minimal changes in LDL oxidative state, and could be related to a downregulation of NOS III. PMID- 10729381 TI - Arterial intimal retention of pro-atherogenic lipoproteins in insulin deficient rabbits and rats. AB - Recent observations that remnants of triglyceride rich lipoproteins become trapped within the subendothelial of arterial vessels gives rise to the possibility that these particles could initiate the atherogenic cascade. Increased frequency and progression of atherosclerosis in diabetes might in part be a consequence of raised concentrations in plasma of remnant lipoproteins. In addition, diabetes may lead to changes in the arterial vasculature which exacerbate arterial retention of pro-atherogenic lipoproteins. To explore these possibilities, in this study we determined aortic retention of chylomicron remnants, which are of intestinal origin, and of hepatically derived low-density lipoproteins (LDL) in insulin deficient rabbits and rats. The two species were selected because of their disparate susceptibility to develop atherosclerosis in the presence of diabetes induced hyperlipidemia. Chylomicron remnants and LDL were differentially radiolabelled with a residual marker and injected simultaneously into conscious rabbits or rats. Arterial retention was determined 2 h after injection, and relative retention was expressed as a percentage of mean arterial exposure. We found in insulin deficient rabbits and rats that intimal and medial retention of chylomicron remnants was positively related to the degree of hyperglycemia and was significantly greater than in non-diabetic control groups. In contrast, insulin deficiency did not influence arterial retention of LDL. Rabbits which are susceptible to diabetes induced atherogenesis had significantly greater intimal retention of chylomicron remnants compared to rats. Results from this study support the hypothesis that chronic hyperglycemia promotes arterial retention of triglyceride rich remnant lipoproteins and that atherosclerotic susceptibility might be related to degree of remnant entrapment within the subendothelial space. Greater retention of remnant lipoproteins could in part explain the increased prevalence of atherogenesis in diabetes. PMID- 10729380 TI - Regression of poloxamer 407-induced atherosclerotic lesions in C57BL/6 mice using atorvastatin. AB - HMG-CoA reductase inhibitor drugs or 'statins' have been shown to effectively reduce plasma total cholesterol (CHOL), CHOL associated with low-density lipoprotein (LDL), and triglycerides (TG). In addition, slight elevations in HDL CHOL are also typically observed. Poloxamer 407 (P-407), a nonionic surfactant, effectively elevates both plasma CHOL and especially TG in a dose-controlled fashion and results in formation of atherosclerotic lesions in the aortas of C57BL/6 mice without the requirement of dietary cholic acid [1,2]. The purpose of the present study was to assess whether a typical statin, namely atorvastatin (Lipitor(R)) would significantly reduce P-407-induced hypercholesterolemia and hypertriglyceridemia as well as cause regression of atherosclerotic lesions resulting from administration of P-407 to C57BL/6 mice. C57BL/6 mice in the present study were treated with either normal saline (C, controls), 0.5 g/kg of P 407 (P), or a high-fat, high-cholesterol, cholate-containing diet (HF) for 120 days. Mice in all groups were then equally and randomly divided and treated with either atorvastatin or saline for an additional 120 days. Beginning at Day 121 and using mice in groups P and HF as an example, one-fourth of the mice in each group received 20 mg/kg per day of atorvastatin with either concomitant HF feeding or P-407 administration ('progression' treatment groups), one-fourth received 20 mg/kg per day of atorvastatin following cessation of HF feeding or P 407 administration, one-fourth received saline (placebo) with either simultaneous HF feeding or P-407 administration ('progression' placebo groups), and one-fourth received saline (placebo) following cessation of HF feeding or P-407 administration. Total plasma CHOL was significantly (P<0.01) lower for mice in groups P and HF when administered atorvastatin relative to saline, but remained significantly (P<0.05) elevated compared to total plasma CHOL of C mice. With discontinuation of either P-407 administration or HF feeding, total plasma CHOL declined rapidly in both P and HF mice with atorvastatin-treated mice generally demonstrating lower plasma CHOL concentrations relative to saline-treated mice. Total plasma TG was significantly (P<0.01) lower for mice in group P administered atorvastatin relative to saline, but remained significantly (P<0.05) elevated compared to plasma TG of C mice. With discontinuation of P-407 administration, total plasma TG declined rapidly in P mice with atorvastatin-treated mice typically demonstrating lower plasma TG concentrations relative to saline-treated P mice. Aortas of mice treated with 20 mg/kg per day of atorvastatin in both groups P and HF, whether maintained on the HF-diet or treated with P-407 from Day 120 to 240 or whether each treatment was terminated at Day 120, revealed no presence of atherosclerotic lesions relative to saline-treated mice and were indistinguishable from aortas retrieved from C mice. Atorvastatin at a dose of 20 mg/kg per day not only significantly reduced the plasma CHOL and TG concentrations, but also resulted in regression of atherosclerotic lesions induced in C57BL/6 mice by administration of P-407 or ingestion of a HF-diet containing cholic acid. PMID- 10729382 TI - Characterization of atherosclerosis in LDL receptor knockout mice: macrophage accumulation correlates with rapid and sustained expression of aortic MCP-1/JE. AB - Atherosclerosis and the expression of monocyte chemoattractant protein-1 (MCP-1) were quantified in low density lipoprotein receptor knockout (LDLR KO) mice fed 1.25% cholesterol (study #1) or 0.2% cholesterol (study #2). In study #1 plasma total cholesterols leveled-off at 1800 mg/dl whereas plasma triglycerides remained low. In en face specimens of the aortic root and arch, intimal foam cells plus extracellular lipid particles accumulated and by 8 weeks the fatty streak surface area had rapidly expanded at both sites. In study #2, total cholesterols averaged 400 mg/dl and fatty streaks were 2-3-fold smaller compared to those in study #1. In study #3, LDLR KO mice were fed chow or 1.25% cholesterol, and immunostaining demonstrated a few Mac-2-positive intimal macrophages in mice fed chow, and during the first 10 weeks of hypercholesterolemia the number of intimal macrophages increased continuously. In chow-fed mice (0 weeks) there was little MCP-1 in the aorta. After 2 days of hypercholesterolemia intimal macrophages stained for MCP-1, and during the next 10 weeks recently recruited arterial macrophages also expressed MCP-1. Macrophage accumulation was highly correlated with MCP-1 expression. In study #4, feeding LDLR KO mice 1.25% cholesterol for 6 months produced atherosclerotic plaques at both sites and they contained a fibrous cap of smooth muscle cells, macrophage foam cells, connective tissue and cholesterol crystals. In summary, LDLR KO mice fed cholesterol develop fatty streaks that transform into fibrous plaques. Hypercholesterolemia rapidly triggers MCP-1 expression in resident intimal macrophages, which is followed by the accumulation of more macrophages that also express MCP-1, suggesting that this chemokine may both initiate and amplify monocyte recruitment to the artery wall during early atherogenesis. PMID- 10729383 TI - Aortic endothelial cell von Willebrand factor content, and circulating plasminogen activator inhibitor-1 are increased, but expression of endothelial leukocyte adhesion molecules is unchanged in insulin-dependent diabetic BB rats. AB - Endothelial cell injury has been implicated in the increased incidence of vascular disease associated with diabetes mellitus. In diabetic humans, elevated plasma von Willebrand Factor (vWF) has been interpreted as an indication of endothelial damage. In contrast, in an animal model of inherited insulin dependent diabetes, the bio-breeding (BB) rat, plasma vWF levels did not differ from those in age-matched control rats during the first 7 months of diabetes although morphological evidence of mild aortic endothelial alteration or injury was observed. In the present study efforts have been made to define the endothelial alterations in BB diabetic rats compared to controls more precisely over this time period. Thus, adhesion molecules: intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule-1(VCAM-1) were evaluated by in situ immunohistochemistry, vWF content was determined by biochemical analysis of aortic extracts and by quantitative immunohistochemistry, plasma vWF levels were measured by ELISA and vWF mRNA by RNAse protection assay. Neither age nor diabetic state significantly affected either the expression of adhesion molecules, or the levels of circulating vWF. Endothelial vWF content was significantly increased in the diabetic vessels, as observed by both approaches but the vWF mRNA content was not different from that in control vessels. Plasma plasminogen activator inhibitor (PAI-1) activity was significantly increased in diabetic animals. In conclusion, endothelial alterations in BB rats associated with diabetes, together with the raised plasma PAI-1 levels, promote the thrombogenic potential of the vessel wall, and are consistent with an increased risk for vascular disease. PMID- 10729384 TI - Low level expression of hormone-sensitive lipase in arterial macrophage-derived foam cells: potential explanation for low rates of cholesteryl ester hydrolysis. AB - Conversion of arterial macrophages into foam cells is a key process involved in both the initiation and progression of atherosclerotic lesions. Foam cell formation involves the progressive accumulation and storage of lipoprotein derived cholesteryl esters. The resulting imbalance in cholesterol metabolism in arterial foam cells may be due in part to an inadequately low level of cytoplasmic neutral cholesteryl ester hydrolase (NCEH) activity. In this study, we have demonstrated that hormone-sensitive lipase (HSL) mRNA is expressed at very low levels in macrophage-derived foam cells, using the unique approach of extracting mRNA from macrophage-derived foam cells purified from human and rabbit atherosclerotic plaques coupled with reverse transcriptase polymerase chain reaction (RT-PCR). We also demonstrate that macrophage-derived foam cells isolated from rabbit atherosclerotic lesions exhibit a resistance to high density lipoprotein (HDL)-mediated cholesterol efflux along with reduced levels of NCEH activity compared to lipid-loaded mouse peritoneal macrophages. Thus, low level expression of HSL may partially account for the reduced NCEH activity observed in arterial foam cells isolated from atherosclerosis-susceptible species. PMID- 10729385 TI - Increased atherogenesis in Otsuka Long-Evans Tokushima fatty rats before the onset of diabetes mellitus: association with overexpression of PDGF beta receptors in aortic smooth muscle cells. AB - The mechanism of diabetic macroangiopathy was studied from the view point of phenotypic change of arterial smooth muscle cells (SMC). Otsuka Long-Evans Tokushima fatty (OLETF) rat, an animal model of non-insulin dependent diabetes mellitus (NIDDM), develops spontaneous persistent hyperglycemia after the age of 18 weeks. Medial SMC in OLETF rats expressed more platelet-derived growth factor (PDGF) beta-receptor and fibronectin at the protein level than those from control, Long-Evans Tokushima Otsuka (LETO) rats, not only after but also before the onset of diabetes mellitus. Cultured SMC from OLETF rats more strongly responded specifically to the mitogenic stimuli of PDGF-AB and PDGF-BB and also expressed PDGF beta-receptor more intensely compared with those from LETO rats. PDGF is known to be the main contributor to the intimal thickening induced by balloon catheter injury, which is one of several forms of arterial injuries. Intimal thickening of carotid arteries in OLETF rats after balloon catheter injury increased compared with that in LETO rats before the onset of diabetes mellitus. In in vitro culture system, fibronectin synthesis was stimulated by transforming growth factor-beta1(TGF-beta1) in SMC from OLETF rats, but not in those from LETO rats, suggesting that SMC from OLETF rats respond to TGF-beta1. These results indicate that overexpression of PDGF beta-receptor and fibronectin in medial SMC plays an important role in the accelerated intimal thickening before the onset of diabetes mellitus in OLETF rats. PMID- 10729386 TI - Apolipoprotein binding to protruding membrane domains during removal of excess cellular cholesterol. AB - High-density lipoproteins (HDL) are believed to protect against cardiovascular disease by removing excess cholesterol from cells. Lipid-free HDL apolipoproteins remove cellular cholesterol and phospholipids by an active, Golgi-dependent process that is still poorly understood. Here we characterized the morphology of apolipoprotein binding sites on cultured cells by immunogold electron microscopy. After 6 h incubations with lipid-free apoA-I or apoE, immunogold-labeled apolipoproteins were distributed sparsely along the planar surface of human fibroblasts and THP-1 macrophages. Overloading these cells with cholesterol led to a several-fold increase in the concentration of immunogold-labeled apoA-I and apoE on the cell surface, and over 80% of these gold particles were associated with novel electron-opaque structures protruding from the plasma membrane. Protrusions binding apoE were larger (100-200 nm) than those binding apoA-I (10 60 nm), and similar apoA-I-binding structures appeared when cells were incubated with either purified apoA-I or HDL particles. These structures were formed and enlarged by a time-dependent process inhibited by the Golgi disruptor brefledin A, the energy poison NaF, and low temperature. Moreover, formation of these structures was nearly absent in fibroblasts from a subject with Tangier disease, cells that lack a functioning apolipoprotein-mediated lipid removal pathway. Thus, formation of novel apolipoprotein binding structures protruding from the cell surface is an intermediate step in the cellular pathway by which apolipoproteins remove excess cholesterol. PMID- 10729387 TI - Paraoxonase gene polymorphisms are associated with carotid arterial wall thickness in subjects with familial hypercholesterolemia. AB - Human serum paraoxonase (PON) is a high density lipoprotein (HDL) associated enzyme capable of hydrolyzing lipid peroxides in vitro. PON has recently attracted attention as a protective factor against oxidative modification of LDL and may therefore play an important role in the prevention of the atherosclerotic process. Two frequent mutations at the paraoxonase gene locus (PON1) are the leucine (L allele)-->methionine (M allele) and the glutamine (Q allele)- >arginine (R allele) substitutions at residues 55 and 192, respectively. We have examined the influence of these two polymorphisms on carotid atherosclerosis in familial hypercholesterolemia (FH) patients. The allele frequencies of these two polymorphisms were determined by PCR and restriction fragment analysis, for both the FH population and healthy controls. High resolution B-mode ultrasound was used to assess intima-media wall thickness (IMT) of the carotid artery. No differences were found in allele frequencies between the FH and the control population. In FH patients, the LL, LM and MM genotypes at position 55 occurred in 86 (46.0%), 78 (41.7%) and 23 (12.3%) subjects, respectively, whereas the QQ, QR and RR genotypes at position 192 were found in 90 (48.1%), 79 (42.2%) and 18 (9.6%) individuals. When both polymorphisms were considered separately, no different carotid IMTs were found between the genotype groups. However, our data did show a significant association between the various genotypes of the combined polymorphisms at position 55 and 192 of PON1 and the carotid artery IMT in FH subjects. Subjects with the homozygous wildtype LL/QQ for paraoxonase had the highest mean carotid IMTs when compared to other genotypes, combined. Multiple regression analysis demonstrated age (beta=0.34, P<0.0001), total plasma cholesterol (beta=0.17, P=0. 0109) and the LL/QQ genotype of the PON1 gene (beta=0.22, P=0.0018) to be significant risk factors for carotid atherosclerosis in subjects with FH. The LL/QQ genotype could explain 5.3% of total variance of carotid IMT. In conclusion, this is the first study to report an independent association between the combined PON1 polymorphism genotypes and carotid wall thickness. The homozygous wildtype LL/QQ for PON1 may represent an additional risk factor for carotid atherosclerosis in subjects with FH. PMID- 10729388 TI - Infection with virulent strains of Helicobacter pylori is not associated with ischaemic heart disease: evidence from a population-based case-control study of myocardial infarction. AB - BACKGROUND: Although the majority of evidence does not support association between Helicobacter pylori infection and ischaemic heart disease, the nature of this relationship may differ when virulence of the infecting strains are examined. METHODS AND RESULTS: The prevalence of IgG antibody evidence of infection with CagA positive stains of H. pylori was investigated in stored plasma samples from 259 cases of myocardial infarction (aged 25-70 years, 74 males) and 259 population based controls from the same area in Northern Ireland. Two-hundred and seventy (52.1%) subjects were seropositive for anti-CagA IgG. CagA seropositivity was more common in cases than in controls: 56.4 vs 47.9%, odds ratio for seropositivity in cases (95% CI) 1.41 (1.00, 1.99). Substantial attenuation of this relationship occurred on adjustment for age, sex, number of siblings, smoking and measures of socio-economic status: odds ratio (95% CI) 1.16 (0.79, 1.70). A similar pattern was seen for seropositivity for all H. pylori strains. CONCLUSION: Infection with the more virulent strains of H. pylori, as with all strains, is not associated with myocardial infarction. PMID- 10729389 TI - Genetic factors associated with response of LDL subfractions to change in the nature of dietary fat. AB - A preponderance of dense low density lipoprotein (LDL) particles is associated with an increased risk of coronary heart disease. It has been shown that dense LDL levels can be modified by diet. We investigated the contribution of polymorphisms in the genes for apolipoprotein (apo) B, apo AIV, lipoprotein lipase (LPL) and cholesterol ester transfer protein (CETP) to variation in the changes in plasma concentrations of dense LDL between a high saturated and a high polyunsaturated fatty acid diet. A total of 46 freeliving individuals (19 men and 27 women) completed a crossover trial with two dietary interventions of 4 weeks each, a high saturated fat diet (providing 21% energy from saturated fat and 3% energy from polyunsaturated fat) and a high polyunsaturated fat diet (providing 11% energy as saturated fat and 10% energy as polyunsaturated fat). Overall, the change in dense LDL between the saturated and polyunsaturated fat period was 0.17+/-0.33 mmol/L and this change was similar in men and women. Of the polymorphisms studied only variation in the apo AIV gene causing the substitution of histidine for glutamine at position 360 (Q360H) was associated with significant differences in the change in dense LDL concentration. Apo AIV Q/H individuals (n=6) showed a three-fold greater change in dense LDL cholesterol unadjusted for Lp(a) levels than Q/Q individuals (0.46+/-0.27 versus 0.12+/-0.31 mmol/L, p=0.02). The greater decrease in dense LDL cholesterol with an increase in polyunsaturated fat seen in those with the apo AIV H360 variant, who represent roughly 10% of the general population, suggests that they may benefit most from a PUFA rich lipid lowering diet. PMID- 10729390 TI - Detection of missense mutations in the genes for lipoprotein lipase and hepatic triglyceride lipase in patients with dyslipidemia undergoing coronary angiography. AB - Coronary events have a close association with a low HDL/hypertriglyceridemia (LHDL/HTG) phenotype. As enzymes that hydrolyze triglyceride-rich lipoproteins are associated with a modulation of both HDL cholesterol and triglycerides, we have tested the hypothesis that mutations in the genes encoding lipoprotein lipase (LPL) or hepatic lipase (HTGL) may contribute to the formation of coronary atherosclerosis and, thus, of coronary heart disease (CHD). The entire coding and boundary regions of LPL and HTGL genes were analyzed by direct sequencing in 20 patients with both LHDL/HTG and diagnosed CHD. In the LPL gene six different polymorphisms were identified with same frequencies observed in the general population. In the HTGL gene, besides several polymorphisms, we identified three missense mutations: Asn37His, Val73Met, and Ser267Phe. Population screening using allele specific PCR identified Val73Met as a polymorphism while the two others were absent from 100 control individuals. One of the mutations (Ser267Phe) is known to cause HTGL deficiency and is associated with type III hyperlipoproteinemia. Since this dyslipoproteinemia meets the criteria of LHDL/HTG, it is intriguing to speculate that missense mutations in HTGL may play a role in the pathogenesis of this atherogenic phenotype. PMID- 10729391 TI - Relation of inflammation to vascular function in patients with coronary heart disease. AB - Endothelium plays a pivotal role in the regulation of vascular relaxation. Inflammation may in turn induce endothelial dysfunction and thus increase the risk of atherothrombosis. We investigated 31 men with angiographically verified coronary heart disease, aged 57. 7+/-5.3 years, in regard to endothelium dependent, acetylcholine-induced, and to endothelium-independent, sodium nitroprusside-induced vasodilatation in the forearm vasculature by strain-gauge plethysmography. Logistic regression analysis served to determine the relation between forearm vascular function and the inflammatory factors measured, concentration of C-reactive protein, subtypes of peripheral blood T-lymphocytes, and other factors potentially affecting endothelial function (lipoprotein and glucose levels). Concentration of C-reactive protein was an independent determinant of endothelium-dependent vascular function (P<0.001 for low dose acetylcholine, P=0.01 for high dose acetylcholine). Other determinants of endothelium-dependent vascular dysfunction were CD8-lymphocytes expressing ICAM-1 (P=0.001), antibodies to oxidized low-density lipoprotein (P<0.001), and body weight (P=0.007). The present data showed an association between inflammatory risk factors linked to atherothrombosis and endothelial dysfunction in coronary heart disease patients. It is possible that endothelial dysfunction in coronary heart disease patients is related to the chronic inflammation or infection coexisting with atherosclerosis. PMID- 10729392 TI - Influence of fatty acid chain length and cis/trans isomerization on postprandial lipemia and factor VII in healthy subjects (postprandial lipids and factor VII). AB - Exaggerated postprandial lipemia is believed to be atherogenic and to influence risk of thrombosis. The postprandial effects on plasma triacylglycerol concentration, factor VII coagulant activity (FVII(c)) and activated FVII concentration (FVII(a)) of five high fat meals (5.2 MJ, 90 g fat) enriched with medium triacylglycerols (MCT, 8:0+10:0), palmitate(16:0), stearate (18:0), elaidate(18:1 trans) and oleate(18:1 cis) were compared with those following a low fat meal (5.2 MJ,10 g fat) in 16 healthy subjects using a randomized crossover design. Postprandial lipemia measured as the area under the curve (AUC arbitrary units) for plasma triacylglycerol concentration (mean+/-SE) was greater following the oleate (5.8+/-1. 05), elaidate (4.3+/-0.79) and palmitate (4.1+/ 0.64) meals compared with stearate (2.0+/-0.45) and MCT (1.1+/-0.47) meals. Fatty acid analyses of the chylomicron lipids suggested that approximately one fifth of the dietary stearate was not absorbed. FVII(c) increased following the oleate, elaidate and palmitate meals and fell following the low fat meal; the increase in FVII(c) was correlated with the AUC for plasma TAG (r=0.34; P=0.001). FVII(a) concentration increased following all high fat meals but not following the low fat meal. The increase in FVII(a) at 7 h was greater after the oleate meal than after the stearate and MCT meals. These results do not support the hypothesis that dietary stearate and elaidate are responsible for the postprandial increases in FVII associated with high fat intakes. PMID- 10729393 TI - Additional risk factors influence excess mortality in heterozygous familial hypercholesterolaemia. AB - Life expectancy of patients with familial hypercholesterolaemia is decreased. Some untreated patients reach a normal life span and, therefore, additional risk factors and the type of mutation in the low-density lipoprotein (LDL) receptor gene are likely to influence the clinical outcome. We determined all cause mortality in kindreds with the disorder, who were untreated, in order to study (a) additional risk factors for coronary artery disease (CAD) and (b) the types of LDL receptor gene mutations that may contribute to a poor prognosis. The mortality in all 855 first-degree relatives of 113 unrelated patients was compared to the Dutch population after standardisation for age, gender, and calendar period. Analyses restricted to affected relatives could have underestimated the mortality risk due to lack of information about severe cases, who died prematurely. Therefore, all first-degree relatives were analysed and as a result the standardised mortality ratios (SMRs) exhibit only 50% of the excess mortality from familial hypercholesterolaemia. We observed 190 deaths in 32048 person-years leading to an overall SMR of 1.34 (95% confidence interval (CI) 1. 16-1.55, P=0.001). High excess mortality occurred in males between age 40 and 54 (SMR 2.34, 95% CI 1.60-3.31, P<0.001). The excess mortality decreased during the last decades. This change of mortality over calendar time shows that additional risk factors modulate the mortality from the disorder. The SMR of 62 families referred with premature CAD was 1.62 (95% CI 1.32-1.93, P<0.001) and the SMR was 1.10 (95% CI 0.86-1.34, P=0.4) in 51 families without premature CAD. The mortality risk of kindreds with null alleles was similar to that of kindreds with other mutations. In conclusion, the burden of the untreated disorder occurred mainly among middle-aged males and was not influenced by the type of mutation. Additional risk factors increased excess mortality significantly and are highlighted by the presence of premature CAD among first-degree relatives. This underscores the need for active identification of all hypercholesterolaemic relatives of such patients. PMID- 10729394 TI - Postprandial concentrations and distribution of apo C-III in type 2 diabetic patients. Effect Of bezafibrate treatment. AB - Apo C-III plays a key role in the metabolism of triglyceride-rich lipoproteins. It has recently been implicated as a potential determinant of the triglyceride (TG) lowering effect of fibrates, which down-regulate its expression. This hypothesis has been explored in ten moderately hypertriglyceridemic (TG 4.50+/ 2.40 mmol/l) male type 2 diabetic patients tested with a lipid load before and after 4 weeks of treatment with 400 mg bezafibrate daily. Treatment lowered apo C III concentrations by 20%, mainly in VLDL. Postprandially, apo C-III was transferred to chylomicrons in proportion to their TG content exclusively from HDL. VLDL retained their apo C-III and the apo C-III:TG ratio decreased as TG contents increased. At the end of the absorptive period (8 h) HDL did not recover the totality of their apo C-III (net loss 19 and 28% respectively before and after treatment, P<0.0001 for time effect). Bezafibrate lowered apo E by 33% (P<0.03). The apo C-III:apo E ratio did not vary significantly under treatment but underwent a postprandial decrease: 13% before and 18% (P=0.01) after treatment. These results indicate that repression of apo C-III expression and lowering of the apo C-III:E ratio are not likely mechanisms for the lipid lowering effects of fibrates in type 2 diabetic patients. The potent effects on postprandial lipemia are suggestive of an apo C-III-independent stimulation of lipolysis. PMID- 10729395 TI - Evidence for association between paraoxonase gene polymorphisms and atherosclerotic diseases. AB - Paraoxonase 1 (PON1) is proposed to have an anti-atherogenic action. Two polymorphisms at the PON1 (M/L55 and Q/R192) have been shown to be associated with coronary artery disease (CAD). This conclusion is not drawn universally, however, and specific ethnic characteristics may be important determinants in this association. Recently two homologues of PON1 - PON2 and PON3 - were identified and Sanghera et al. demonstrated C/S311 polymorphism at PON2 was associated with the risk of CAD. Within that context, we investigated the association between the aforementioned three polymorphisms and CAD and ischemic stroke in a Japanese population. The study population included 431 control subjects, 210 CAD patients, and 235 ischemic stroke patients. Genotype distributions and allele frequencies of M/L55 and C/S311 were similar among the control and patient groups, whereas the R192 allele frequency was significantly higher (P<0.001) in CAD (75%) and ischemic stroke (76%) patients than in control subjects (65%). When confounding influences of other risk factors were controlled for by multivariate analysis, R192 remained an independent risk determinant (additive model: OR (95% CI), P value CAD: 2.01 (1.45-2.79), 0.0001; ischemic stroke: 1.84 (1.34-2.52), 0.0002 (three genotypes into calculation)). Taken together, our data indicate that the Q/R192 is principally associated with both CAD and ischemic stroke in Japanese. PMID- 10729396 TI - Fibrinogen, angina and coronary heart disease in a Chinese population. AB - Although fibrinogen is an established risk factor of coronary heart disease (CHD), whether fibrinogen is associated with CHD in Chinese is not clear. This population-based cross-sectional study aimed to analyse this relationship in Hong Kong Chinese. Fibrinogen was measured by the Clauss method in 1348 men and 1385 women aged 25-74 years. Severity of CHD was defined as most serious if the subjects had medically diagnosed CHD, as less serious if they had angina only, and as normal if they had neither. The prevalence of angina and CHD was respectively 2.4% and 2.2% in men and 3.2% and 2.7% in women. In men the age adjusted mean fibrinogen concentration was 2.47 (95% confidence interval (CI) 2.43-2.51) g/l in the normal group, 2.65 (95% CI 2.45-2.85) g/l in the angina group, and 2.78 (95% CI 2.56-3. 00) g/l in the CHD cases (P<0.01); in women it was respectively 2.61 (95% CI 2.59-2.63), 2.66 (95% CI 2.50-2.82), 2.90 (95% CI 2.72-3.08) g/l (P<0.01). The differences were significant after adjustment of other significant risk factors. We conclude that fibrinogen should be considered as a risk factor in Chinese. PMID- 10729397 TI - Glutathione S-transferase genotype as a susceptibility factor in smoking-related coronary heart disease. AB - Cancer studies suggest that the null polymorphisms of glutathione S-transferase M1 or T1 (GSTM1/GSTT1) may affect the ability to detoxify or activate chemicals in cigarette smoke. The potential modification of the association between smoking and coronary heart disease (CHD) by GSTM1 and GSTT1 has not been studied in humans. A case-cohort study was conducted to test the hypotheses that specific genotypes of GSTM1 or GSTT1 affect susceptibility to smoking-related CHD. CHD cases (n=400) accrued during 1987-1993 and a cohort-representative sample (n=924) were selected from a biracial cohort of 15792 middle-aged men and women in four US communities. A significantly higher frequency of GSTM1-0 and a lower frequency of GSTT1-0 were found in whites (GSTM1-0=47.1%, GSTT1-0=16.4%) than in African Americans (AAs) (GSTM1-0=17.5%, GSTT1-0=25.9%). A smoking-GSTM1-0 interaction for the risk of CHD was statistically significant on an additive scale, with ever smokers with GSTM1-0 at a approximately 1.5-fold higher risk relative to ever smokers with GSTM1-1 and a approximately 2-fold higher risk relative to never smokers with GSTM1-0, after adjustment for other CHD risk factors. The interaction between having smoked >/=20 pack-years and GSTT1-1 was statistically significant on both multiplicative and additive scales. The risk of CHD given both GSTT1-1 and >/=20 pack-years of smoking was approximately three times greater than the risk given exposure to >/=20 pack-years of smoking alone, and approximately four times greater than the risk given exposure to GSTT1-1 alone. The modification of the smoking-CHD association by GSTM1 or GSTT1 suggests that chemicals in cigarette smoke that are substrates for glutathione S-transferases may be involved in the etiology of CHD. PMID- 10729604 TI - Liposomology PMID- 10729605 TI - IRIV-adjuvanted hepatitis A vaccine: in vivo absorption and biophysical characterization. AB - Immunopotentiating reconstituted influenza virosomes (IRIV) are 150-nm proteoliposomes composed of influenza surface glycoproteins and a mixture of natural and synthetic phospholipids. Due to size, structure and composition of the IRIVs, they serve as an antigen carrier system for efficacious vaccination, as was demonstrated for hepatitis A and influenza. This paper reviews the unique properties of IRIVs and describes the in vivo biodistribution of model antigens using 14C-labeled IRIVs and 125I-labeled streptavidin. IRIV formulated streptavidin induced a strong depot effect after intra muscular (i.m.) vaccination of mice, whereas soluble streptavidin was soon eliminated via the kidney of the animals. A mixture of antigen and IRIVs yielded higher antibody titers after i.m. inoculation than streptavidin alone. The highest immunostimulation was achieved by the binding of the antigen to the investigated adjuvant. The potential penetration of inactivated hepatitis A virions into lipid membranes was assessed by measuring the area increase of a lipid monolayer kept at a constant surface pressure corresponding to that of lipid bilayer vesicles. The monolayers were composed of phosphatidylcholine (POPC) and phosphatidylethanolamine (POPE) (75/25 mol/mol), thus resembling the lipid composition of the IRIV. The results suggested that the hepatitis A antigen may spontaneously bind to the reconstituted IRIV membranes. PMID- 10729606 TI - Lipid-linked proteins of plants. AB - Increasing numbers of plant proteins are being shown to have posttranslationally attached lipids. The modifications include N-myristoylation, S-palmitoylation, prenylation by farnesyl or geranylgeranyl moieties, or attachment of glycosylphosphatidylinositol anchors. This report summarizes recent findings regarding the structure, metabolism and physiological functions of these important protein-linked lipids. PMID- 10729607 TI - Platelet-activating factor (PAF) receptor and genetically engineered PAF receptor mutant mice. AB - Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active phospholipid mediator. Although PAF was initially recognized for its potential to induce platelet aggregation and secretion, intense investigations have elucidated potent biological actions of PAF in a broad range of cell types and tissues, many of which also produce the molecule. PAF acts by binding to a unique G-protein-coupled seven transmembrane receptor. PAF receptor is linked to intracellular signal transduction pathways, including turnover of phosphatidylinositol, elevation in intracellular calcium concentration, and activation of kinases, resulting in versatile bioactions. On the basis of numerous pharmacological reports, PAF is thought to have many pathophysiological and physiological functions. Recently advanced molecular technics enable us not only to clone PAF receptor cDNAs and genes, but also generate PAF receptor mutant animals, i.e., PAF receptor-overexpressing mouse and PAF receptor-deficient mouse. These mutant mice gave us a novel and specific approach for identifying the pathophysiological and physiological functions of PAF. This review also describes the phenotypes of these mutant mice and discusses them by referring to previously reported pharmacological and genetical data. PMID- 10729608 TI - Regulation of fatty acid transporters in mammalian cells. PMID- 10729609 TI - Structural and kinetic characterization of NADP-dependent, non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from celery leaves. AB - NADP-dependent, non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9) from celery leaves was purified over 1200-fold to a specific activity of 35 units/mg protein, and its kinetic, regulatory and structural properties were characterized. The purified enzyme exhibited a homotetrameric structure with a subunit molecular mass of 54 kDa. A high specificity of the enzyme for the substrates NADP(+) (K(m)=7 uM) and D-glyceraldehyde-3-phosphate (K(m)=127 uM) was observed. Maximal activity was determined at pH 8.5. The purified enzyme was highly unstable, requiring the addition of NADP(+) or conditions of high ionic strength in the medium. A hysteretic behavior, with a lag phase of minutes, was observed during activity measurement of the enzyme preincubated in the absence of substrates. The lag was inversely proportional to the protein concentration during preincubation. The hysteretic parameters were affected by the substrates, KCl and mannitol among other compounds. Distinctively, incubation with NADP(+) produced a near twofold activation of the enzyme. Results suggest that in alditol producing plants the enzyme plays a key role in the synthesis and partitioning of photoassimilates. PMID- 10729610 TI - Xyloglucan mobilisation in cotyledons of developing plantlets of Hymenaea courbaril L. (Leguminosae-Caesalpinoideae). AB - Many seeds contain storage compounds that are used by the embryo/plantlet as a source of nutrients after germination. In seeds of Hymenaea courbaril, a leguminous tree, the main reserve consists of a structurally unusual xyloglucan stored in thickened walls of the cotyledon cells. The present work aimed to study H. courbaril xyloglucan metabolism during and after germination in order to compare its degrading system with the other known xyloglucan containing seeds. Polysaccharide degradation occurred after germination between 35 and 55 days after planting. The activities of alpha-xylosidase, beta-glucosidase, beta galactosidase and XET rose during the period of xyloglucan disassembling but a low level of endo-beta-glucanase activity was detected, suggesting that this XET has high affinity for the oligosaccharides. The pH optimum of beta-galactosidase was different from the alpha-xylosidase, beta-glucosidase and XET optima suggesting that the former may be important in the control of the mobilisation process. A tentative model for xyloglucan disassembling in vivo is proposed, where beta-galactosidase allows the free oligosaccharides to bypass a transglycosylation cycle and be disassembled by the other exo-enzymes. Some ecophysiological comparisons among H. courbaril and other xyloglucan storing seeds are discussed. PMID- 10729611 TI - Effect of singlet oxygen generating substances on the ascorbic acid and glutathione content in pea leaves. AB - Ascorbate and glutathione levels were investigated in pea leaf discs exposed to various singlet oxygen generating compounds: eosin, rose bengal, monuron, acifluorfen and 5-amino-levulinic acid (ALA). The cellular level of the major antioxidant ascorbate was markedly decreased by the herbicides monuron, acifluorfen and ALA (in light-dependent reactions), as well as by the xanthene dyes eosin and rose bengal (independently of light). No significant accumulation of dehydroascorbate could be observed in any treatments. In contrast to ascorbate, the foliar glutathione levels were considerably increased by subtoxic or slightly toxic concentrations of eosin, rose bengal, acifluorfen and ALA in a light-dependent manner. Monuron treatments led to unchanged or decreasing glutathione contents. The activities of three antioxidative enzymes (ascorbate peroxidase, glutathione reductase and glutathione S-transferase) were also induced by eosin in light-dependent reactions. PMID- 10729612 TI - Effects of pH on the induction of phosphoenolpyruvate carboxylase kinase in Kalanchoe fedtschenkoi. AB - Previous work has shown that an increase in cytosolic pH plays an important role in the induction of phosphoenolpyruvate carboxylase (PEPc) kinase by light in C(4) plants. The potential involvement of a similar effect in the induction of PEPc kinase in the Crassulacean acid metabolism (CAM) plant Kalanchoe fedtschenkoi was assessed using leaf disks. Treatment of disks with the weak base NH(4)Cl did not affect induction of the kinase. Prolonged treatment of disks with weak acids prevented both decarboxylation of malate during the day and induction of the kinase in the following night, but short treatments had no effect. The data are consistent with the view that a high cytosolic malate content can prevent induction of phosphoenolpyruvate carboxylase kinase. Changes in cytosolic pH may affect induction of the kinase in CAM plants by a secondary effect rather than as part of a primary signalling pathway. PMID- 10729613 TI - Differences in the contents of total sugars, reducing sugars, starch and sucrose in embryogenic and non-embryogenic calli from Medicago arborea L. AB - The total sugars, reducing sugars, starch and sucrose in embryogenic and non embryogenic calli from explants (cotyledons, petioles, hypocotyls and leaves) obtained from Medicago arborea L. seedlings were evaluated. Total sugars were the major components in the calli and no significant differences between embryogenic and non-embryogenic calli were observed. In contrast, important differences between the embryogenic and non-embryogenic calli were observed for reducing sugars, the highest levels being observed in embryogenic calli. The highest starch levels were found in non-embryogenic calli developed in MS medium. During the development of somatic embryogenesis very low starch levels in the callus were found. During the first months of culture, no significant differences in the sucrose content were found between calli that produced embryos and those that did not. The most important differences in sucrose were seen between calli transferred to medium F0, which had the greatest embryogenic capacity, and those transferred to medium F6, which inhibited embryogenesis. In the latter case, an increase in sucrose was observed. PMID- 10729614 TI - A mesocarp-and species-specific cDNA clone from oil palm encodes for sesquiterpene synthase. AB - The differential display method was used to isolate cDNAs corresponding to transcripts that accumulate during the period of lipid synthesis, 12-20 weeks after anthesis (WAA) in the mesocarp of two oil palms, Elaeis oleifera and Elaeis guineensis, Tenera. DNA-free total RNA from mesocarp and kernel of E. guineensis, Tenera and E. oleifera (15 WAA) were used to obtain differential gene expression patterns between these tissues from the two species. In this report, we describe the isolation and characterization of a specific cDNA clone, MO1 (434 bp) which was shown to be mesocarp-specific as well as species-specific for E. oleifera Sequencing of this fragment showed homology to the enzyme sesquiterpene synthase. Its longer cDNA clone, pMO1 (1072 bp), isolated from a 15-week E. oleifera mesocarp cDNA library confirmed that it encodes for sesquiterpene synthase. The complete sequence of 1976 bp was obtained using 5'RACE method. Northern hybridization showed that MO1 and pMO1 mRNA transcripts are highly expressed only in the mesocarp of E. oleifera from 5 to 20 WAA. No expression was detected in the kernel (12-17 WAA) and vegetative tissues of both species nor in the mesocarp of E. guineensis. This is the first communication to document on the isolation and characterisation of a mesocarp-and species-specific cDNA clone from oil palm. PMID- 10729615 TI - Analysis of a ferric leghemoglobin reductase from cowpea (Vigna unguiculata) root nodules. AB - Ferric leghemoglobin reductase (FLbR), an enzyme reducing ferric leghemoglobin (Lb) to ferrous Lb, was purified from cowpea (Vigna unguiculata) root nodules by sequential chromatography on hydroxylapatite followed by Mono-Q HR5/5 FPLC and Sephacryl S-200 gel filtration. The purified cowpea FLbR had a specific activity of 216 nmol Lb(2+)O(2) formed min(-1) mg(-1) of enzyme for cowpea Lb(3+) and a specific activity of 184 nmol Lb(2+)O(2) formed min(-1) mg(-1) of enzyme for soybean Lb(3+). A cDNA clone of cowpea FLbR was obtained by screening a cowpea root nodule cDNA library. The nucleotide sequence of cowpea FLbR cDNA exhibited about 88% similarity with soybean (Glycine max) FLbR and 85% with pea (Pisum sativum) dihydrolipoamide dehydrogenase (DLDH, EC 1.8.1.4) cDNAs. Conserved regions for the FAD-binding site, NAD(P)H-binding site, and disulfide active site were identified among the deduced amino acid sequences of cowpea FLbR, soybean FLbR, pea DLDH and other enzymes in the family of the pyridine nucleotide disulfide oxido-reductases. PMID- 10729616 TI - Transgenic expression of a gene encoding a synthetic antimicrobial peptide results in inhibition of fungal growth in vitro and in planta. AB - Transgenic tobacco plants producing the synthetic antimicrobial peptide D4E1, encoded by a gene under the control of an enhanced cauliflower mosaic virus 35S RNA promoter, were obtained by Agrobacterium-mediated transformation. Successful transformation was demonstrated by PCR and Southern hybridization analysis of tobacco DNAs. Expression of the synthetic D4E1 gene was shown by RT-PCR of tobacco mRNA. Crude protein extracts from leaf tissue of transformed plants significantly reduced the number of fungal colonies arising from germinating conidia of Aspergillus flavus and Verticillium dahliae by up to 75 and 99%, respectively, compared to extracts from plants transformed with pBI121. Compared to negative controls, tobacco plants expressing the D4E1 gene showed greater levels of disease resistance in planta to the fungal pathogen, Colletotrichum destructivum, which causes anthracnose. PMID- 10729617 TI - Half-life of ubiquinone and plastoquinone in spinach cells. AB - The half-life of plastoquinone (PQ), ubiquinone-9 (UQ-9) and ubiquinone-10 (UQ 10) in spinach tissue was determined. This was achieved by monitoring the decay of radioactivity incorporated into these lipids from a labeled precursor. The half-life of PQ was 15 h while for UQ-9 and UQ-10 it was longer, i.e. 30 h. The values of half-lives of PQ and UQ suggest a high rate of turnover of these lipids in spinach cells. PMID- 10729618 TI - Site-directed mutagenesis of Cys-92 from the alpha-polypeptide of Phaseolus vulgaris glutamine synthetase reveals that this highly conserved residue is not essential for enzyme activity but it is involved in thermal stability. AB - The residue Cys-92 from the alpha-polypeptide of Phaseolus vulgaris glutamine synthetase is a highly conserved residue in prokaryotic and eukaryotic glutamine synthetase genes. This cysteine residue was previously proposed as a good candidate for being essential for enzyme activity. We have examined through heterologous expression in Escherichia coli and site-directed mutagenesis the functional importance of this residue. We have found that the thiol group of Cys 92 is not essential either for glutamine synthetase biosynthetic or transferase enzyme activities. The characteristic inhibition by p-hydroxymercuribenzoate (a specific sulphydryl reagent) was not substantially altered as a consequence of replacement of Cys-92 by Ala. Immunoreactivity of the glutamine synthetase mutant protein, examined both under native and denaturing conditions, was similar to the wild-type, indicating that no significant conformational changes were produced as a consequence of the introduced mutation. However, the mutant enzyme C92A was considerably less stable than the wild-type. These results indicate that Cys-92 is not an essential residue for enzyme activity but it is important for stability of the glutamine synthetase protein. PMID- 10729619 TI - Differential effects of melanocortin peptides on ingestive behaviour in rats: evidence against the involvement of MC(3) receptor in the regulation of food intake. AB - The pro-opiomelanocortin-derived peptides decrease food intake possibly via MC4 receptor. In this study we compared the effects of alpha-melanocyte-stimulating hormone (MSH), beta-MSH and gamma(1)-MSH (0.2, 1.0 and 5.0 microg, i.c.v.) on food intake. alpha-MSH and beta-MSH inhibited spontaneous food intake in a dose dependent manner, whereas the gamma(1)-MSH did not. alpha-MSH and beta-MSH but not gamma(1)-MSH (all 5.0 microg, i.c.v.) inhibited fasting-induced food intake about 50%. None of the three peptides inhibited fluid consumption in water deprived (24 h) rats. It is suggested that MC(3) receptor, activated selectively by gamma(1)-MSH, is not involved in the regulation of food intake. PMID- 10729620 TI - A melanocortin agonist reduces neuronal firing rate in rat hypothalamic slices. AB - Bath application of alpha-melanocyte stimulating hormone (alpha-MSH) to rat hypothalamic slices inhibited the spontaneous firing rate of continuously firing neurons in the ventromedial hypothalamic nucleus or paraventricular nucleus. This inhibitory effect is most likely direct and independent of synaptic transmission. The alpha-MSH-responsive neurons tested did not respond to neuropeptide Y (NPY) application. On the other hand, alpha-MSH did not inhibit the intraburst firing rate of phasic bursting neurons, although these bursting neurons were highly responsive to a serotonin 5HT2a/2b/2c agonist with a change of firing pattern to continuous firing and an increase in firing rate which was reversed by NPY. These results suggest that a change of neuronal firing rate may represent a neural correlate of satiety induced by anorexic agents. PMID- 10729622 TI - Cerebral trypsinogen expression in human and rat cerebrospinal fluid. AB - Trypsinogen was identified in cerebrospinal fluid (CSF), where it has not previously been reported and its activation state in experimental subarachnoid haemorrhage (SAH) in rats and in neurosurgical patients was determined. Trypsinogen activation peptide (TAP) release provided an equimolar marker for trypsinogen. Total TAP was significantly reduced to 26% of the baseline level (P<0.02) following experimental SAH in 15 rats but not in ten sham operated controls (P=0.3). TAP was also measured in patients with ruptured (n=11) and unruptured (n=9) aneurysms who underwent craniotomy to clip an aneurysm. Postoperatively there was a significant fall in TAP concentration (P<0.005) in both groups. Trypsinogen, as identified by CSF levels of TAP, is activated by SAH in rats and by craniotomy for aneurysmal clipping in patients. PMID- 10729621 TI - Cholinergic deafferentation of the rabbit cortex: a new animal model of Abeta deposition. AB - Brain deposition of the amyloid beta-peptide (Abeta) is a critical step in the pathogenesis of Alzheimer's disease (AD) and human cerebral amyloid angiopathy (CAA). A small fraction of AD and CAA cases are caused by gene mutations leading to increased production and deposition of Abeta, but for the majority, there is no known direct genetic cause. We have hypothesized that Abeta deposition in these sporadic cases occurs as a result of cortical cholinergic deafferentation. Here we show that cortical cholinergic deafferentation, induced in rabbits by a selective immunotoxin, leads to Abeta deposition in cerebral blood vessels and perivascular neuropil. Biochemical measurements confirmed that lesioned animals had 2.5- and 8-fold elevations of cortical Abeta40 and Abeta42, respectively. Cholinergic deafferentation may be one factor that can contribute to Abeta deposition. PMID- 10729623 TI - During pain-avoidance neurons activated in the macaque anterior cingulate and caudate. AB - Lesions in either the anterior cingulate cortex (ACC) or the caudate nucleus (CN) impair avoidance behavior from noxious somatic stimuli, so these two areas may play a similar role in pain-avoidance behavior. To test this hypothesis, we recorded single neuronal activities in the ACC and in the CN of a monkey while it was performing a pain-avoidance task. Ten of 136 ACC and eleven of 160 CN neurons responded selectively during pain-avoidance behavior. We found little difference in the population distribution or in the response latency and duration. Our present findings are in accordance with previous lesion and anatomical studies which suggest that these two regions could function as one module in pain avoidance behavior. PMID- 10729624 TI - The source generator of respiratory-related anxiety potential in the human brain. AB - In this study, we used the dipole tracing method of a scalp-skull-brain head model to determine the location of the electric current source which correlates with the increased respiratory rate due to anxiety in humans. Anticipatory anxiety was produced by giving electrical stimulation to the left forefinger after the warning red light. While administering anticipatory anxiety, subjects' anxiety state and respiratory rates increased. In averaged electroencephalogram, which was triggered by onset of inspiration, positive waves were observed approximately 350 ms after the onset of inspiration. In this period of time, dipoles were concentrated in the right temporal pole, and the temporal pole and the amygdala in the most anxious subject. This data suggests that there are respiratory-related neural activities in limbic and paralimbic areas which may correlate with anxiety in humans. PMID- 10729625 TI - Choline plus cytidine stimulate phospholipid production, and the expression and secretion of amyloid precursor protein in rat PC12 cells. AB - The amyloid precursor protein (APP) is a transmembrane protein anchored in the membrane lipid bilayer. Choline and cytidine are major precursors of cell membranes, and are regulatory elements in membrane biosynthesis. We examined the levels of cellular APP holoprotein and secreted APPs when rat PC12 cells are stimulated to undergo increase in membrane phospholipids by choline+cytidine (2+2, 5+5, 10+10 or 50+50 microM) treatment. We now show that as phospholipids levels are increased by supplemental choline and cytidine treatment, the levels of cell-associated APP also rise stoichiometrically; these treatments also caused major (up to 6. 8-fold) increases in the amounts of secreted APP released into the cell medium, and also stimulated increased process formation. These results show that choline plus cytidine increase both phospholipid levels, and the expression and secretion in PC12 cells. It appears that agents that stimulate cellular membrane biosynthesis may be used to stimulate the secretion of neurotrophic APPs and neurite formation in neurodegenerative disorders such as Alzheimer's disease. PMID- 10729626 TI - Phosphorylated cAMP response element binding protein increases in neurokinin-1 receptor-immunoreactive neurons in rat spinal cord in response to formalin induced nociception. AB - The rat neurokinin-1 (NK1) receptor gene contains a cyclic adenosine monophosphate (cAMP) response element, and gene transcription may be activated upon binding of phosphorylated cAMP response element binding protein (pCREB). If pCREB contributes to increased expression of NK1 receptors, pCREB should increase in neurons that express NK1 receptors under conditions that increase NK1 receptor mRNA. Evidence for this relationship was found following injection of formalin into one hindpaw of rats. Immunohistochemistry was employed to visualize NK1 receptors and pCREB in spinal cord sections. Formalin injection produced an increase in pCREB-immunofluorescence within NK1 receptor-immunoreactive neurons from segments L4 and L5. No change occurred in pCREB-immunofluorescence within NK1 receptor-immunoreactive neurons from segment T11. These data support the hypothesis that transcription factor pCREB contributes to increased expression of spinal NK1 receptors during persistent pain. PMID- 10729627 TI - GABAergic presynaptic inhibition of rat neostriatal afferents is mediated by Q type Ca(2+) channels. AB - Population spikes associated with the paired pulse facilitation paradigm have been successfully used to measure presynaptic inhibition in several systems. In the present work, this paradigm was used to evaluate the action of baclofen on neostriatal glutamatergic transmission. Baclofen enhanced synaptic facilitation with an EC(50)=0.57 microM and a maximal effect of 457%. Selective antagonists for N-, P- and Q-type Ca(2+)-channels enhanced paired pulse facilitation; suggesting that these channel types participate in the release of transmitter. Nevertheless, neither 1 microM omega-conotoxin GVIA, nor 20 nM omega-agatoxinTK occluded the action of baclofen. Baclofen's action was occluded only by 400 nM omega-agatoxinTK. These data suggest that Q-type Ca(2+)-channels mediate gamma aminobutyric acid(B) presynaptic inhibition of neostriatal afferents. PMID- 10729628 TI - Anxiolytic-like effects in rats produced by ventral pallidal injection of both N- and C-terminal fragments of substance P. AB - Prior studies have shown that the neurokinin substance P (SP) has anxiolytic-like effects when administered into the nucleus basalis (NB) area of the rat ventral pallidum. The present work was performed to examine whether the anxiolytic effects of SP in the nucleus basalis can be assigned its amino (N)- or carboxy (C)-terminal moiety. Using the elevated plus-maze model of anxiety in combination with unilateral injection of N-terminal SP(1-7) or C-terminal SP(7-11) into the NB region, we found that the treatment with either SP-fragment increased the number of entries into and time spent on the open arms as well as excursions into the end of the open arms, indicative of an anxiolytic-like profile. Furthermore, the effective doses of SP(1-7) (0.67 ng) and SP(7-11) (0.45 ng) were equimolar to the dosage of the whole SP molecule (1 ng), which was effective to reduce anxiety. Thus, the results support earlier findings that ventral pallidal injection of SP has anxiolytic-like effects and provide new evidence that fragments of SP, representing the N- and C-terminal domain of the peptide can reduce fear-parameters at a concentration similar to that of the parent peptide. PMID- 10729629 TI - Inhibition of Na(+),K(+)-ATPase activity in cultured rat cerebellar granule cells prevents the onset of apoptosis induced by low potassium. AB - In cerebellar granule cells in culture, lowering of extracellular [K(+)] results in apoptotic death (D'Mello, S.R., Galli, C., Ciotti, T. and Calissano, P., Induction of apoptosis in cerebellar granule neurons by low potassium: inhibition of death by insulin-like growth factor I and cAMP, Proc. Natl. Acad. Sci. USA, 90 (1993) 10989-10993). In this model, we studied the influence of Na(+), K(+) ATPase inhibition on apoptosis. We demonstrate that cell death (93+/-2 vs. 46+/ 1.6%) as well as fragmentation of nuclear DNA induced by low extracellular potassium were prevented by addition of ouabain (0.1 mM), a specific inhibitor of the Na(+),K(+)-ATPase. Blockade of glutamatergic N-methyl-D-aspartate and alpha amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors by 5-methyl-10,11 dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine hydrogen maleate (MK-801; 20 microM) and 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 microM) did not inhibit the protective effect of ouabain. 24 h treatment with ouabain also decreased cell death induced by Fe(2+)/ascorbic acid (74+/-2% to 49+/-3%). We speculate that ouabain pretreatment enhances the resistance against low [K(+)] induced apoptosis independent of glutamate-receptor activation. Since this effect can be mimicked by a free-radical generating system, we suggest an antioxidative effect underlying ouabain-induced neuroprotection. PMID- 10729630 TI - Vascular endothelial growth factor expression under ischemic stress in human meningiomas. AB - Vascular endothelial growth factor (VEGF) is an endothelial cell-specific antigen and angiogenic factor that plays a role in angiogenesis. We analyzed the expression of four VEGF mRNA isoforms in meningiomas. Among 35 meningiomas, 11 came from patients who underwent complete (n=4) or partial (n=7206=189 in all samples. However, the VEGF121 and 165 isoforms were significantly upregulated in samples from patients who underwent partial preoperative embolization. The diffusible VEGF121 isoform may be important for vascularity and edema formation in meningiomas. PMID- 10729631 TI - The echidna manifests typical characteristics of rapid eye movement sleep. AB - The failure to identify rapid eye movement sleep (REMS) in an early study of the echidna at an unmeasured ambient temperature (T(a)) was unexpected, as its brain stem structures resemble those that generate REMS in other mammals. However, typical mammalian REMS was evident in echidnas exposed to several T(a)s. The parallel presence of REMS in birds points to its reptilian origin. PMID- 10729632 TI - An alternative pathway of nitric oxide production by rat astrocytes requires specific antigen and T cell contact. AB - In the present study, we observed an alternative pathway in which nitric oxide (NO) production by rat astrocytes requires specific antigen and cell-cell contact. NO production by astrocytes was significantly inhibited by antibodies against CD40L, B7-1 or B7-2. Astrocyte-derived NO inhibited T cell proliferation and induced T cell apoptosis. In contrast, augmented astrocyte proliferation was correlated to the levels of NO production by astrocytes, implicating a role of NO in regulating local immune responses in the central nervous system. These results suggest that T cell-astrocyte interactions may regulate local immune responses via the NO pathway and influence the fate of infiltrating T cells. PMID- 10729633 TI - Peroxiredoxin I (macrophage 23 kDa stress protein) is highly and widely expressed in the rat nervous system. AB - Expression pattern of peroxiredoxin I (Prx I), a novel stress-inducible 23 kDa protein, initially found in macrophages, was investigated in the normal rat nervous system by western blot and immunohistochemistry. The protein was widely expressed in the central and peripheral nervous system, particularly in oligodendroglia and Schwann cells. Weak staining of axons and neuropil suggests neuronal processes also may contain Prx I. Immunostaining of neuronal cell bodies were not evident. Since Prx I reduces hydrogen peroxide, the widespread glial expression of Prx I indicates that it may play an important protective role against oxidative damage in the nervous system. PMID- 10729634 TI - Co-expression of calcium signaling components in vertebrate taste bud cells. AB - In order to investigate the molecular mechanism of calcium signaling pathways common to the vertebrate gustatory systems, we have analyzed the expression of their molecular components. We first identified a phospholipase C (PLC) beta subtype expressed in the taste buds of pond loach (Misgurnus anguillicaudatus), designated DPLCbeta2, which is closely related to mammalian PLCbeta2 shown recently to be expressed in rat taste buds. The taste bud-specific expression of PLCbeta2 in a fish species as well as rat strongly suggests that PLCbeta2 mediates the tastant-induced second messenger response in taste buds, which is common to vertebrates. Next, we examined the correlation of gene expression of the candidate components leading to PLCbeta2 activation in rat circumvallate papillae, including G proteins, G(i2) and gustducin, and a G protein-coupled receptor, TR2. As a result, it was shown that the mRNAs for PLCbeta2 and G(i2) co exist in the same cells, and PLCbeta2- and G(i2)-positive cells include both gustducin-positive cells and TR2-positive cells. However, no correlation was found between the expressions of TR2 and gustducin as reported previously. Our results thus indicate that a taste transduction pathway comprising TR2, G(i2) and PLCbeta2 occurs in a subset of taste cells. PMID- 10729635 TI - Multi-muscle control of head movements in monkeys: the referent configuration hypothesis. AB - It is suggested that the nervous system may specify a referent configuration (R) of the body determined by the set of the threshold joint angles at which all skeletal muscles may be silent. At the same time, electromyographic (EMG) activity and forces are generated to resist deflections of the body from this configuration. The R configuration may thus be considered an internal geometric image with which the actual body configuration (Q) is compared. Thereby the difference between the R and Q is a major factor determining the recruitment and gradation of the activity of each skeletal muscle. Control systems may produce movements by changing the R configuration according to task demands. The referent hypothesis predicts that when the R and Q configurations match each other, a global minimum in the EMG activity of all muscles involved should occur, an event most likely observed in movements with reversal in direction. To test the validity of the R hypothesis for head movements, three-dimensional kinematics and EMG activity of 14 functionally diverse neck muscles were analysed in monkeys during head rotations to and from fruit targets placed beyond the oculomotor range. Despite the functional and anatomical diversity of the neck muscles, the activity of all muscles was minimised at a reversal point of the movement trajectory, as predicted by the R hypothesis. This study thus illustrates the notion that a change in the internal geometric image of a biomechanical system may underlie movement production. PMID- 10729636 TI - Decline of the monkey's limbic and prefrontal activity during task repetition. AB - In order to investigate the neural mechanism underlying the motivation, we measured the regional cerebral blood flow (rCBF) by positron emission tomography in a monkey that was repeatedly engaged in cognito-behavioural tasks. The rCBF of the anterior cingulate area, medial parts of prefrontal area, and hippocampal region decreased significantly with the succession of tasks within a day. The activity was restored abruptly when the reward was then increased, but decreased again on further repetition of the tasks. These findings suggest that the limbic and prefrontal areas are the regions involved in motivational function in the brain. PMID- 10729637 TI - Postnatal naltrindole treatments induce behavioural modifications in preweanling rats. AB - To investigate the physiological role of the delta-opioid receptor during the preweanling period, we have studied the effects of chronic (daily injections from birth to postnatal day 19) and acute treatments with the selective delta antagonist naltrindole (1 mg/kg), on behavioural and nociceptive responses in 20 day old male rats. Behavioural testing was performed using an open field paradigm. Acute naltrindole induced significant decreases in external and total ambulation (horizontal activity) and rearing behaviour (vertical activity), as well as a significant increase in grooming frequency. In animals chronically treated with naltrindole there was an increase in total ambulation one day after the discontinuation of the treatment. In a test of nociception (tail immersion) no significant effect of chronic naltrindole treatment on baseline latencies or of acute naltrindole on latency quotients (post-treatment latency/pre-treatment latency) were found. However, chronic naltrindole administration significantly decreased the latency quotients. The results show that the delta-opioid receptor participates in the tonic regulation of motor activity during the preweanling period and might be involved in certain aspects of stress responsiveness. PMID- 10729638 TI - Myelin basic protein induces cell death of mature pig oligodendrocytes in vitro and produces demyelination in vivo. AB - Two methods prevail at present in producing demyelinated areas in the central nervous system. One uses the detergent-like effect of lysolecithin, the other is based on a cell killing effect of ethidium bromide plus x-irradiation. Unwanted side-effects are inherent in both methods. Based on the fact that myelin basic protein (MBP) kills adult pig oligodendrocytes but almost no astrocytes in vitro, we have used MBP for creating demyelinated areas in the centrum semiovale of the pig brain. These lesions are characterized by a loss of oligodendrocytes and myelin, a preservation of axons and astrocytes, and by the presence of macrophages. Thus, this type of lesion might present an alternative option for studying the fate of transplanted myelinating cells. PMID- 10729639 TI - Statistical probability mapping reveals high-frequency magnetoencephalographic activity in supplementary motor area during self-paced finger movements. AB - Investigations of both haemodynamic and electroencephalographic measures of brain activity have demonstrated supplementary motor area (SMA) involvement in self paced finger movements. In contrast, analysis of magnetoencephalographic (MEG) signals in the time domain has usually failed to detect SMA activity in healthy individuals. We investigated oscillatory MEG activity in 12 normal adults during (a) a self-paced, complex sequence of finger movements and (b) a simple finger opposition task paced externally by tactile stimuli presented to the contralateral thumb. Statistical probability mapping revealed enhanced non-phase locked spectral amplitudes in the 22-28 Hz range over bilateral frontal cortex during self-paced as compared to externally cued finger movements. This activity may reflect recruitment of cell assemblies in SMA during self-paced, complex movements. PMID- 10729640 TI - Prevalence of Cryptosporidium infection in calves in France. AB - Two multicentre surveys were conducted in France to estimate the prevalence of Cryptosporidium infection in calves using qualitative ELISA for detection of Cryptosporidium coproantigens and oocysts. The first survey involved 4-12-day-old calves in six dairy-calf distribution centres, collecting calves from seven Administrative Regions (Aquitaine, Bretagne, Franche-Comte, Lorraine, Normandie, Nord, Pays de Loire). For each region, 20 calves were selected every month for 12 consecutive months (October 1995-September 1996). Prevalence of Cryptosporidium infection was 17.9% (Confidence Intervals (C.I.) 95%=[16.1%; 19.8%]) among the 1628 selected calves, of which only 5.3% had diarrhoea. The second survey conducted between November 1995 and May 1996 involved 4-21-day-old calves examined by veterinary practitioners who selected 189 livestock farms of dairy- or suckler-type in ten Administrative Departments (Allier, Cantal, Creuse, Doubs, Ille-et-Vilaine, Maine-et-Loire, Manche, Pas-de-Calais, Saone-et-Loire, Vendee). Cryptosporidia were detected in 105 (55.6%) of the farms. Among the 440 calves examined, of which 398 (90.5%) presented diarrhoea, cryptosporidia were found in 191 animals, i.e. a prevalence of 43.4% (C.I. 95%=[38. 8%; 48.0%]). Breed of calves and type of housing had very little impact on prevalence in this survey. Some regional variations could be noticed, even if cryptosporidia infection is widespread. Monthly variations could be related to seasonal peaks in calving with a lower infection rate during summer. PMID- 10729641 TI - Epidemiologic study of Giardia sp. infection in dairy cattle in southeastern New York State. AB - To identify animal and management factors associated with the risk of Giardia sp. infection in dairy cattle in southeastern New York State, an observational analytical epidemiologic study was conducted. A random sample of 2943 animals in 109 dairy herds located in five counties of southeastern New York was selected from the target population. Fecal samples were collected from animals in the study population and examined for the presence of Giardia sp. using a quantitative centrifugation concentration flotation technique. Data on each animal, and on general management, maternity, preweaning, and postweaning practices were collected by personal interview with the farmer or farm manager. The significance of association of these factors, within each management practice group, with the risk of infection was evaluated using the logistic regression analysis. Weighted indices for each of these four groups were developed, and were evaluated together with the age of the animal and season of sampling for their significance of association with the risk of infection using mixed effect logistic regression analysis. Only the maternity management practices, age of the animal, and the season of sampling were significantly associated with the risk of infection with Giardia sp. Summer housing of bred heifers, on pasture or in tie stalls, was associated with increased risk of infection. Calves that were fed fresh colostrum or separated immediately after birth from the dam were at decreased risk of infection. The risk of infection decreased with the age of the animal and varied by the season of sampling. There was no significant extra binomial variation in the risk of infection in this data. If consideration is to be given to reducing the risk of infection with this parasite, management must be a top priority, especially in younger animals. PMID- 10729642 TI - Partial purification and characterization of Gastrothylax crumenifer somatic antigens. AB - The soluble extracts of Gastrothylax crumenifer isolated from the rumen of buffalo (Bubalus bubalis) were fractionated on a Sephadex G-200 column. A total of eight major fractions (F1, F2, F3, F4, F5, F6, F7, and F8) were separated from the whole homogenate of G. crumenifer, and each of these fractions was tested for their antigenicity by ELISA against rabbit hyperimmune sera. It was observed that F1, F2, F3 and F4 were highly antigenic, F6 and F7 were moderately antigenic and F5 and F8 were poorly antigenic. The individual fractions analysed after SDS-PAGE and Western blotting indicated that the antigenic fractions of G. crumenifer are of low molecular weight, in the range of <14-50kDa, and predominant antigenic components which were evident in most of the Sephadex profiles were of Mr 15, 18, 19, 23-24 and 28-32kDa. PMID- 10729643 TI - The influence of age on the variation among sheep in susceptibility to natural nematode infection. AB - A longitudinal study of faecal nematode egg counts was made in naturally infected Scottish Blackface sheep over two grazing seasons to 75 weeks of age. Although egg counts were lower in the second grazing season the variation among animals was greater. Egg counts were repeatable from 3 months of age. The repeatability of faecal egg counts within and between grazing seasons was about 0.3. Animals with lower than average egg counts in the first grazing season tended to have lower than average egg counts in the second grazing season. Therefore lambs with relatively low faecal egg counts after 3 months of age are likely to retain their advantage in the following year. PMID- 10729644 TI - Resilience of second year grazing cattle to parasitic gastroenteritis following negligible to moderate exposure to gastrointestinal nematode infections in their first year. AB - The influence of gastrointestinal nematode infections on performance of four groups of female Holstein Friesian calves was monitored until the end of the second grazing season (SGS). In the first year three groups were grazed and one group (G4) was permanently housed. General and grazing management during the first grazing season (FGS) was arranged such that G1 acquired moderate infections, G2 low infections and G3 very low infections with gastrointestinal nematodes. These infections were monitored through faecal egg counts, differentiation of faecal larval cultures, pasture larval counts, serum pepsinogen values, ELISA with a recombinant Cooperia oncophora protein, weight gain, tracer worm counts and sentinel worm counts. In 1998 all four groups were grazed together as one herd from 23 April to 26 October and infections were monitored with the same techniques with the exception of sentinel calves. In the FGS weight gain was higher in G4 than in the other groups and higher in G3 (28. 6kg) than in G1. Weight gain of G2 was intermediate to G1 and G3 but did not significantly differ from either group. In the SGS weight gain in G4 was far less than in any other group and the mean weight at the end of the experiment was 41.9, 38.6 and 50.9kg lower than G3, G1 and G2, respectively. Though no significant differences were observed between G1, G2 and G3 at the end of the experiment it was obvious that the weight gain advantage of G3 over G1 at the end of the FGS had disappeared. Parasitological and serological findings in the SGS indicated that G3 and G4 had build up less immunity during the FGS compared to G1 and G2. The conclusion of the experiment is that resilience to parasitic gastroenteritis in the SGS depends on the level of exposure to nematodes in the FGS. However, problems with poor weight gain only will be expected when exposure is very low in the FGS and high in the SGS. PMID- 10729645 TI - Differences in susceptibility to gastrointestinal nematode infection between Angus and Brangus cattle in south Louisiana. AB - Breed susceptibility to nematode infection was evaluated in Angus (Bos taurus) and Brangus (B. indicus crossbreed) cattle. A cow-calf herd and a yearling replacement heifer herd were monitored during one grazing season. Calves were born in March and were weaned in October. Individual rectal fecal samples were collected monthly from the two herds and processed for fecal egg counts (FEC) and coprocultures. Cow and calf FEC increased from April, reaching maximum values during the summer. Angus cows and calves had significantly (p<0.05) greater FEC than Brangus cows and calves, and Haemonchus and Cooperia were the predominant genera. Replacement heifer FEC showed a similar pattern with maximum levels during late summer/fall, and Haemonchus was the predominant genus. No significant differences were seen between breeds, however, infection levels were consistently lower in Brangus heifers. Ostertagia was present in cows and heifers only in fall/winter, which is consistent with summer inhibition. The data suggested that cows were an important source of pasture contamination for their susceptible calves and that the Brangus breed was relatively more resistant to infection. The use of B. indicus crossbreeds may help in alleviating reliance on chemical control by reducing the rate of pasture contamination and subsequent infection losses. PMID- 10729646 TI - Evaluation of the persistent efficacy of doramectin and ivermectin injectable against Ostertagia ostertagi and Cooperia oncophora in cattle. AB - The persistent efficacy of doramectin and ivermectin injectable against moderate and high infection levels of Ostertagia ostertagi and Cooperia oncophora were evaluated in cattle. Calves were allocated to six groups of six animals. On Day 0 animals of Groups I1/I2 and D1/D2 were treated with 0.2mg/kg ivermectin and doramectin injectable, respectively. Animals of the C1, I1 and D1 groups received a daily (moderate) infection of 1000 L3 of O. ostertagi and 1000 L3 of C. oncophora, and animals of the C2, I2 and D2 groups received a daily (high) infection of 10,000 L3 of each species. The animals were infected for 21 days with both species, the infections with C. oncophora and O. ostertagi started from Days 8 and 15 post treatment, respectively. Animals were necropsied on Day 40. The calculation of the persistent activity of ivermectin and doramectin was based on the efficacy against the different developmental and adult stages of both parasites. The present study confirmed that infection levels may influence the duration of persistent efficacy of an anthelmintic. Doramectin had at the moderate infection level a persistent efficacy of at least 35 days against O. ostertagi and at least 28 days against C. oncophora; at the high infection dose persistent efficacy was somewhat shorter i.e. up to 33 days and approximately 28 days, respectively. The duration of persistent efficacy of ivermectin against O. ostertagi at the moderate infection level was between 14 and 25 days, at the high dose level up to 25 days. Persistent efficacy of ivermectin against C. oncophora could, at both infection doses, not be measured, with the present experimental design. PMID- 10729647 TI - Heritability of gastrointestinal nematode faecal egg counts in West African village N'Dama cattle and its relation to age. AB - Offspring-dam regression was used to estimate the heritability of strongyle faecal egg counts (FEC) of traditionally raised West African N'Dama cattle in the Central River Division in The Gambia. Faecal samples were taken monthly from June October 1992, and again from July-October 1993, including 179-463 dams and their calves sampled on each occasion. The only proven genetic relationship was the dam offspring relationship. Gastrointestinal strongyle FEC was expressed as epg (eggs per gram faeces). Regression of offspring FEC on dam FEC, showed a heritability (h(2)) of 0.18 (95% Confidence Limits 0.10, 0.25). Heritabilities were higher at the beginning and end of the rainy season than during the months of the peak rainy season. This is in line with earlier suggestions that genetic control of faecal egg counts is most effective during periods of low parasite transmission. There was a significant (p<0.001) increase in heritability of 0.086+/-0.018 with each year of age of the corresponding offspring. In view of the virtual absence of national cattle breeding systems in West Africa, which are a precondition for exploitation of heritable traits in cattle, integrated control using improved management and strategic prophylaxis remain the methods of choice to control gastrointestinal nematodes in the given conditions. PMID- 10729648 TI - A sequential study of the pathology associated with the infection of sheep with adult and larval Ostertagia circumcincta. AB - Disturbances in the physiology of the abomasa of sheep infected with either adult Ostertagia circumcincta given via abomasal cannulae, or larvae (L3) given intraruminally were matched by pathological changes in tissues collected by repeated mucosal biopsy. Within 2-3 days of the transplant of adult worms, abomasal pH had increased markedly in five out of six animals, and there also had been rapid increases in serum gastrin and pepsinogen concentrations in all animals. Reductions in parietal cell number were recorded as early as 1 day after the transplant of adults and were associated with the rapid accumulation of many neutrophils and eosinophils. Mucosal hyperplasia, with increased numbers of cells closer in appearance to mucous/mucous neck cells, was a relatively late development, being most pronounced in the latter part of the infection. In sheep given larvae, changes in secretory physiology were again matched by a concurrent fall in parietal cell number and by the accumulation of inflammatory cells. Changes became maximal when most worms could be expected to be present as adults, confirming the role of adults in the natural disease. Some abnormalities were detected in biopsies collected from animals maintained free of parasites and, although milder in degree, there were similarities to those observed in parasitised tissues, there being fewer parietal cells, a modest degree of mucous cell hyperplasia and inflammatory infiltrates of predominantly neutrophils. These changes were the likely result of trauma to the tissues in the immediate vicinity of the cannula, due either to the presence of the cannula itself or to the frequent collection of biopsy material from areas close to it. PMID- 10729649 TI - Validation of ELISAs for the detection of antibodies to Sarcoptes scabiei in pigs. AB - An Enzyme-linked ImmunoSorbent Assay (ELISA) was developed for the detection of antibodies to Sarcoptes scabiei. This 'Animal Health Service'-ELISA (AHS-ELISA) was compared with a commercial test (Checkit(R) Sarcoptest) using experimental and field sera. The experimental study was a contact infestation experiment. Eighty piglets were randomly divided between the experimental and control group. After introduction of three Sarcoptes scabiei var. suis infested pigs in the experimental group, both groups were monitored by determining scratching indices, taking ear scrapings and blood samples in Weeks 0, 2, 4, 6, 8, 12 and 16. Four pigs in the control group were immunised with either Dermatophagoides pteronyssinus (Dp) antigens (n=2), or Acarus siro (As) antigens (n=2). In the control group all (non-immunised) pigs were negative in all tests. In the experimental group only slightly elevated scratching indices were observed, with a maximum in Week 8. After 2 weeks for the first time an ear scraping was positive (2.5%). In Week 8 the highest number of positive ear scrapings were found (25.0%). Positive results in the Sarcoptest were first obtained in Week 12 (10.5% positive), while eventually 29.0% of the finishing pigs were positive after 16 weeks. The AHS-ELISA first detected a serological response after 6 weeks (5. 0% positives), increasing until after 16 weeks a large proportion (74.2%) of the finishing pigs were seropositive, making the AHS-ELISA the most sensitive test. In the AHS-ELISA one As-immunised pig remained seronegative, but the other hyper-immunised pigs crossreacted. In the Sarcoptest, only Dp-immunised pigs had elevated Optical Densities (OD's) albeit below the cut-off level. Although hyper immunisation is not a representation of field conditions, it cannot be excluded that the AHS-ELISA is not 100% specific.Field samples were taken from 20 sows in 30 herds, classified as mange-free, suspect, or infested. On a herd level there was high agreement among the ELISAs. Both serological tests were suitable to distinguish mange-free herds from infested herds. In one infested herd the decline of maternal antibody in piglets was studied by sampling 40 piglets from 20 different litters. The lowest average OD using the AHS-ELISA was found at 5 weeks of age, followed by a significant increase at 7 weeks. The average OD with the Sarcoptest was at a minimum level at 3 weeks, but no increase was found later. For screening of herds, interference of maternal antibodies is avoided by sampling at an age of 7 weeks or older. PMID- 10729650 TI - The life cycle of Rhipicephalus bursa Canestrini and Fanzago, 1877 (Acarina: ixodidae) under laboratory conditions. AB - The biology of Rhipicephalus bursa has not been thoroughly studied. In the present study R. bursa was bred in the laboratory and its biology worked out.Larvae, nymphs and adult Rhipicephalus bursa ticks were fed on sheep and were held at 28+/-1 degrees C and 89+/-1% relative humidity (RH) in a darkened incubator. The average weight of engorged females was 0.676g (range 0.353-1.128). The average weight of eggs laid by females was 0.344g (range 0.146-0. 554g). The average number of eggs laid was 7527 (range 6263-8860) and this was significantly correlated with the weight of the females. The index of Reproduction Efficiency (number of eggs per g weight of female) was 8469.6. The index of Conversion Efficiency (weight of eggs per weight of female) was 0.5037. Peak daily oviposition, at an average of 840 eggs per female, occurred on the 6th day of oviposition. After Day 6, the rate declined steadily. The average duration of the preoviposition period was 5.1 days (range 4-7 days). The oviposition period averaged 16.5 days (range 12-21 days), and was significantly related to both the weight of the female and the number of eggs laid. The average pre-hatching period was 31.2 days (range 24-45 days). The average feeding time of larvae and nymphs of this two host tick was 15.2 days (range 14-18 days). Female ticks fed for 5.9 days on the average. Heavier nymphs produced females and a shorter premoult period was noted for lighter male nymphs. The males/females ratio was 1.38:1. The time taken to complete the life cycle was on average 142.45 days (range 99-254 days). PMID- 10729651 TI - Increased control of the sheep biting louse Bovicola (Damalinia) ovis with deltamethrin formulated in a fractionated wool grease carrier. AB - The synthetic pyrethroid deltamethrin (DM) containing a trace of [(14)C]-DM was formulated with non-oxidised sterol and wax ester fractions (F1) of wool grease and as the commercial preparation 'Clout-S'. These were applied as a 'backline' strip to sheep immediately after shearing and the concentration of [(14)C]-DM at meridians adjacent to the application strip and at 1/4 and 3/4 of the dorsal ventral distance was determined. The F1 formulation resulted in significantly greater lateral spread of DM with less remaining at the application site (66+/-8% of dose) 98 days after treatment compared to 'Clout-S' (94+/-3% dose). Autoradiographic examination of treated wool demonstrated that there was more DM in the lower half of the wool staple when formulated in F1 compared to 'Clout-S'. Greater mortality occurred when sheep biting lice Bovicola (Damalinia) ovis were exposed in vitro to wool containing DM from F1 compared to 'Clout-S' treated sheep. In field trials there was increased efficacy against synthetic pyrethroid resistant B. ovis with F1 formulation than with 'Clout-S'. The study has demonstrated that synthetic pyrethroid availability, and therefore efficacy, can be significantly increased when the insecticide is formulated in a 'carrier' with the physicochemical characteristics of wool grease. PMID- 10729652 TI - Comparative in vitro isolation of Trypanosoma theileri from cattle in Belgium. AB - Ten blood samples randomly collected from cows on a farm nearby Antwerp, Belgium, were inoculated into KIVI culture medium (Kit for In Vitro Isolation of trypanosomes) and RPMI 10%+feeder medium. Within 3 weeks of incubation all KIVI cultures and four RPMI 10%+feeder revealed presence of Trypanosoma theileri. Some practical implications regarding the use of KIVI for isolation of pathogenic African trypanosomes from cattle and other Bovidae are discussed. PMID- 10729653 TI - Comparative study of strongyle infections of cattle and buffaloes in Mindanao, the Philippines. AB - Faecal samples were collected from 500 cattle and 385 buffaloes belonging to backyard farmers from 24 villages in the province of Bukidnon, Mindanao, the Philippines. Strongyle nematode infections were studied on these samples by faecal egg counts and infective larvae differentiation. The effects of animal species and age on the prevalence and intensity of infections were studied. The overall prevalence of strongyle infection was 53% for cattle and 28% for buffaloes. The prevalence decreased with increasing age in both animal species. Faecal egg counts were significantly lower in buffaloes than in cattle. Adult animals had lower egg counts than calves. This age effect was most pronounced in buffaloes. Six genera of strongyle nematodes were identified from the faecal cultures in both animal species, Mecistocirrus, Haemonchus, Trichostrongylus, Cooperia, Bunostomum and Oesophagostomum. The distribution of larvae did not vary much with age. PMID- 10729654 TI - Efficacy of doramectin against naturally acquired nematode infection in Iberian swine. AB - Studies were carried out to determine the therapeutic efficacy of doramectin, administered intramuscularly at a dose of 300 microg/kg live weight, against naturally acquired helminths of extensively farmed Iberian pigs. The first study (slaughter study) evaluated, through necropsy of the study animals, the product's efficacy against gastrointestinal and pulmonary nematodes (Ascaris suum, Oesophagostomum dentatum and Metastrongylus sp.) whilst the second, faecal egg count reduction study, (FECR study) evaluated the drug's efficacy only against gastrointestinal helminths (A. suum, Trichuris suis and Oesophagostomum sp.). The first study used 20 animals divided into two equal groups of 10 on the basis of body weight and faecal egg count. One group constituted saline treated controls and the other was doramectin treated. On Day 14 post treatment half of the animals in each group were necropsied and the number of parasites present counted. On Day 15 the remaining half of each group underwent the same procedure. The second study was carried out with 40 animals divided equally into two groups of 20. This study determined the effect of doramectin treatment on faecal egg counts as an indicator of parasite burden. The first study demonstrated an efficacy of 100% against adult Metastrongylus sp. and A. suum, whilst the efficacy against O.dentatum was 96.3%. The second study indicated that at Day 21 post treatment there was a 100% reduction in egg counts in faeces in comparison to untreated controls. PMID- 10729655 TI - Kinetics of antibody-based antigen detection in serum and faeces of sheep experimentally infected with Fasciola hepatica. AB - The monoclonal antibody ES78 was used in a sandwich immunosorbent assay (Sandwich ELISA) for the detection of antigens in sera and faeces in the course of Fasciola hepatica infection in 10 experimentally infected sheep. All infected sheep had circulating antigens in the first week post-infection (WPI). Antigenemia was detectable until WPI 3 in four infected sheep, WPI 4 in five infected sheep and in only one sheep by WPI 5. The detection of coproantigens (Fa(g)) was possible in five infected sheep at WPI-4, in four sheep at WPI-5 and in one sheep only at WPI-6. This technique was compared to an indirect ELISA for the detection of antibodies using excretory secretory antigens of F. hepatica. A significant correlation was found between Fa(g) and egg output and also with adult worm numbers. Our method demonstrated that the diagnosis of active fasciolosis in sheep is possible during all periods of infection. PMID- 10729656 TI - A nationwide, multicenter, case-control study comparing risk factors, treatment, and outcome for vancomycin-resistant and -susceptible enterococcal bacteremia. AB - National Nosocomial Resistance Surveillance Group participants from 22 hospitals across the United States reviewed medical records for hospitalized patients with vancomycin-resistant enterococcal (VRE) or vancomycin-susceptible enterococcal (VSE) bacteremia to identify risk factors associated with the acquisition of VRE bacteremia, describe genetic traits of VRE strains, and identify factors predictive of clinical outcome. VRE cases were matched to VSE controls within each institution. Multiple logistic regression (LR) and classification and regression tree (CART) analysis were used to probe for factors associated with VRE bacteremia and clinical outcome. A total of 150 matched-pairs of VRE cases and VSE controls were collected from 1995 to 1997. Using LR, the following were found to be highly associated with VRE bacteremia: history of AIDS, positive HIV status, or drug abuse (OR 9.58); prior exposure with parenteral vancomycin (OR 8.37); and liver transplant history (OR 6. 75). CART analysis revealed that isolation of Enterococcus faecium, prior vancomycin exposure, and serum creatinine values > or = 1.1 mg/dl were predictors of VRE bacteremia. Greater proportions of clinical failure (60% versus 40%, P < 0.001) and all-cause mortality (52% versus 27%, P < 0.001) were seen in patients with VRE versus VSE bacteremia. Results from both LR and CART indicated that patients with persisting enterococcal bacteremia, intubation at baseline, higher APACHE II scores, and VRE bacteremia were at greater risk for poor outcome. PMID- 10729657 TI - Comparison of gen-probe AccuProbe group B streptococcus culture identification test with conventional culture for the detection of group B streptococci in broth cultures of vaginal-anorectal specimens from pregnant women. AB - The performance of the AccuProbe Group B Streptococcus Culture Identification Test (Gen-Probe Incorporated, San Diego, CA, USA) for the detection of group B streptococci (GBS) directly from LIM broth cultures of vaginal-anorectal swab specimens from pregnant women (two swabs per patient in most cases) was evaluated by comparing results to those of conventional GBS culture. Of 411 specimens analyzed, 82 were positive and 312 were negative for GBS by both methods. After initial testing, the percent agreement was 95.9%. The initial sensitivity, specificity, and positive and negative predictive values for the AccuProbe test were 90.1%, 97.5%, 91.1%, and 97.2%, respectively. Results were discrepant for 17 specimens: eight were GBS positive by probe and negative by culture; nine were negative by probe and positive by culture. To resolve discrepancies, culture plates were re-examined for GBS colonies, AccuProbe testing was repeated on the initial LIM broth cultures, and the second swab (if received) was inoculated to LIM broth for AccuProbe testing after overnight incubation. After discrepant resolution testing, the percent agreement between the two test methods was 97.8%. The final sensitivity, specificity, and positive and negative predictive values for the AccuProbe test were 95.6%, 98.4%, 94.6%, and 98.7%, respectively. These data suggest that the AccuProbe test is a reliable method for detecting GBS in vaginal-anorectal specimens, providing results more rapidly than conventional culture. However, strict adherence to the manufacturer's test protocol is necessary to limit technical errors. PMID- 10729658 TI - Evaluation of four commercial transport media for the survival of Neisseria gonorrhoeae. AB - We evaluated four commercial transport systems with a standardized inoculum of clinical isolates of N. gonorrhoeae (NG), and assessed survival after holding for up to 48 hours at both ambient and refrigeration temperatures. Suspensions of clinical isolates of NG were standardized and adsorbed onto four transport swab types: Culturette EZ (Becton Dickinson [BD], Cockeysville, MD, USA); Cultureswab (Difco Laboratories, Detroit, MI, USA); Venturi Transystem (Copan Italia, Bovezzo, Italy); and a recently modified Starswab (Starplex Scientific, Etobicoke, ON). Swabs were plated to chocolate agar at 0, 6, 24, and 48 hours, and colonies counted. Each swab type was tested in quadruplicate with each NG strain for all time and temperature variables. There was a marked reduction in NG CFUs after only 6 hours incubation with each of the swabs tested. Survival was best using Venturi Transystem and Cultureswab transports (colony counts were reduced to 15.3% and 13.0%, respectively, at 6 hours) when compared with the Culturette EZ and Starswab (colony counts were reduced to 2.2% and 4.3%, respectively, at 6 hours). After the 24-hour holding period, 94% of the cultures from the Venturi Transystem were positive, 82% from the Cultureswab, 24% from the Starswab; and 17% from the Culturette EZ. After 48 hours, recovery dropped to 72%, 43%, 14%, and 0.04%, respectively. All of the systems tested had at least an 80% decrease in recovered colonies after only 6 hours. Further studies are required to determine how poor transport conditions influence the number of positive cultures and what the public health implications are. Of the swabs tested, Cultureswab and Venturi Transystem were most acceptable. PMID- 10729659 TI - Evaluation of the clinical usefulness of C. difficile toxin testing in hospitalized patients with diarrhea. AB - Although numerous studies have evaluated the sensitivity and specificity of different assays for Clostridium difficile toxin, none has evaluated how physicians utilize these tests or respond to test results. Therefore, we assessed patient characteristics, clinical findings, and physician responses to positive and negative assay results at two university-affiliated hospitals, one of which used a cell cytotoxicity assay to test for C. difficile toxin and the other of which used an enzyme immunoassay. Two hundred one patient samples at Hospital A and 199 samples at Hospital B were assessed. Positive toxin assays were more frequent at Hospital A than at Hospital B (p < 0.001), at least in part due to the fact that patients tested at Hospital A were more likely to have fever (p < 0.001), an abnormal abdominal exam (p < 0.001), an abnormal leukocyte count (p < 0.001), and a history of prior antibiotic use (p < 0.001). Empiric therapy for C. difficile before results of the toxin assay was more common (p < 0.001) at Hospital A (83/201, 41. 3%) than at Hospital B (25/199, 12.5%). Once empiric therapy was started, most physicians continued therapy despite negative test results (Hospital A, 76%; Hospital B, 69%). Patients who were treated empirically were more likely than patients not treated empirically to have positive toxin assay results and to have fever (p < 0.001), an abnormal abdominal exam (p = 0.003), or an abnormal leukocyte count (p < 0.05). Physicians seldom ordered repeat toxin assays (Hospital A, 14%; Hospital B, 10%) if the initial assay result was negative. In logistic regression analysis, predictors of a positive toxin assay were prior antibiotic therapy, an abnormal abdominal exam, residence at Hospital A, and age >/= 60 years. Predictors of empiric therapy were residence at Hospital A and prior antibiotic therapy. Because physicians electing to empirically treat inpatients with diarrhea rarely alter therapy based on C. difficile toxin assay results, a more cost-effective management strategy may be not to obtain a toxin assay at all in such situations. Testing should be limited to patients who have received antibiotics within the prior month and who have significant diarrhea and/or abdominal pain. PMID- 10729660 TI - Clinical features and in vitro susceptibilities of two varieties of Cryptococcus neoformans in Taiwan. AB - One hundred years after introduction of the Eucalyptus tree to Taiwan, a predominantly subtropical island, we analyzed clinical and microbiological data of 59 patients with Cryptococcus neoformans infection hospitalized at National Taiwan University Hospital during 1982 to 1997. There were 38 (64.4%) cases of cryptococcosis caused by the var. neoformans and 21 (35.6%) caused by the var. gattii. Thirty-three patients (55.9%) had impaired T cell function, which included 12 patients with acquired immunodeficiency syndrome (AIDS). Eleven of the 12 patients with AIDS were diagnosed after 1995, and 11 cases were caused by var. neoformans. Minimum inhibitory concentrations (MICs) determined by the NCCLS broth microdilution method using antibiotic medium 3 improved the discrimination of in vitro susceptibility against amphotericin B and demonstrated that var. gattii isolates were less susceptible (geometric means 0.25 microg/ml versus 0.64 microg/ml, P < 0.001). In addition, a higher proportion of var. gattii were less susceptible to flucytosine as compared with var. neoformans (35.0% versus 64.9%, P = 0.030). There was no seasonal clustering for isolation of var. neoformans, though infections caused by var. gattii peaked in July and August. Compared with the first study of cryptococcosis (1957-1972) at NTUH, this study demonstrated the increase in immunocompromised and elderly patients, as well as a higher proportion of Cryptococcus isolated from blood or bone marrow. Facing the increasing adaptive plantation of Eucalyptus in Taiwan, the importance of field study regarding the role of Eucalyptus plantations in Taiwan and occurrence of cryptococcosis in human beings cannot be over-emphasized. PMID- 10729661 TI - In-vitro activity of FK 037 (Cefoselis), a novel 4(th) generation cephalosporin, compared to cefepime and cefpirome on nosocomial staphylococci and gram-negative isolates. AB - The novel 4(th) generation cephalosporin FK037 was in vitro compared to cefepime and cefpirome on 563 multiresistant nosocomial isolates including methicillin susceptible (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA). Their time-kill effect was studied on MSSA, Escherichia coli, Klebsiella pneumoniae, and isolates of Enterobacter cross-resistant to cefotaxime, ceftriaxone, and to ceftazidime, their interaction with amikacin being also evaluated on the latter isolates. Results revealed that FK037 possessed a superior antistaphylococcal activity on MSSA isolates to both other compounds being however equal active to cefepime and cefpirome on multiresistant enterobacteriaceae. Synergy was documented between 4(th) generation cephalosporins and amikacin on K. pneumoniae and on Enterobacter spp. cross-resistant to 3(rd) generation cephalosporins. In the latter species 4(th) generation cephalosporins remained inactive. The presented results support the need of clinical studies with FK037 as monotherapy for nosocomial infections based on the local surveillance data of the level of antimicrobial resistance of each hospital. PMID- 10729662 TI - Modeling approach to diameter breakpoint determination. AB - In determining zone diameter breakpoints, the error-rate bounded method focuses directly on the observed discrepancy percentages (very major, major, and minor). These percentages, however, are quite variable due to the number of isolates investigated, the drug-specific relationship between MIC and zone diameter, the location of the isolates relative to the MIC intermediate zone, and the inherent variability of each test. To overcome potential sampling problems, a hierarchical model is proposed which explicitly accounts for each of these factors and probabilities from this model are used to determine diameter breakpoints. A simulation study is performed to demonstrate the improved consistency of this model-based procedure. Application to three published scatterplots demonstrate its interpretability advantages. PMID- 10729663 TI - Antibiotic resistance and prevalence of beta-lactamase in Haemophilus influenzae isolates-a surveillance study of patients with respiratory infection in Saudi Arabia. AB - Haemophilus influenzae was isolated from patients with respiratory tract infections in five centers in Saudi Arabia. All of the 129 isolates tested by MIC agar dilution were fully susceptible to ceftazidime and ciprofloxacin but 13.2% were resistant to ampicillin, 7% to tetracycline, 5.4% to chloramphenicol, 3.9% to roxithromycin, and 1.6% to amoxicillin/clavulanic acid. Seventeen (13.2%) of all isolates produced TEM-1 type beta-lactamase, the majority (82%) characterized as biotype I or II with 4 (23.5%) encapsulated and belonging to serotype b. There was a clear distinction between the prevalence of beta-lactamase production in hospital patients (26.3% of 19 isolates) compared with community based patients (10.9% of 110 isolates). In addition, we report an increase in the prevalence of beta-lactamase negative, ampicillin intermediate strains (BLNAI) compared to previous studies in this defined geographical region. Changes in the frequency and nature of antimicrobial resistance in common respiratory pathogens confirms the need to maintain surveillance. PMID- 10729664 TI - Infection by human parvovirus B19: "gloves and socks" papular purpuric syndrome. AB - Human parvovirus B19 is the cause of erythema infectiosum a benign and self limited infection, but sometimes the virus causes an acute and self-limiting dermatosis. It consists of a edema and erythema of the hands and feet in a gloves and sock distribution and is associated with oral lesions and fever. We report a case of a "gloves and socks" infection by human parvovirus B19. PMID- 10729665 TI - Emergency department use by the rural elderly. AB - This study uses Medicare data to compare emergency department (ED) use by rural and urban elderly beneficiaries. The U.S. Health Care Financing Administration's National Claims File was used to identify services provided to Medicare beneficiaries in Washington State in 1994. Patients were classified by urban, adjacent rural, or remote rural residence. We identified ED visits and associated diagnostic codes, assigned severity levels for presenting conditions, and determined the specialties of physicians providing ED services. The rural elderly living in remote areas are 13% less likely to visit the ED than their urban counterparts. Causes of ED use by the elderly do not vary meaningfully by location. Most ED visits by this group are for conditions that seem appropriate for this setting. Given the similarity of diagnostic conditions associated with ED visits, rural EDs must be capable of dealing with the same range of emergency conditions as urban EDs. PMID- 10729666 TI - Emergency staff survey on their role in pediatric injury prevention education-a pilot study. AB - A pilot study to survey the attitudes of emergency department (ED) personnel regarding their role in injury prevention education in children and parents was conducted at a tertiary care trauma center. The survey consisted of 14 statements, asking staff members their level of agreement (from strongly agree to strongly disagree) on a forced-choice four-point scale. These were followed by two questions asking staff members to rank schools, physician's offices, emergency departments, and public health units on their value in providing injury prevention information to children and parents. Before the intervention (an in service training program on the importance of documenting the circumstances of injury on a patient's chart), a 50% randomly selected sample of ED staff members was asked in May 1997 to complete the survey. After the data collection prepilot (4 months later), the remaining 50% was asked in September 1997 to complete the same questionnaire. Administration of the preintervention survey resulted in 53 of 62 surveys being returned (85%). The postintervention survey was completed by 35 of the 41 staff members still eligible (85%), those who were employed in the ED during the entire pilot project. There was no statistically significant difference between the pre- and post-pilot groups on any demographic characteristics. Staff members agreed least with the statement that ED physicians and staff members could impact the severity of injuries to children by providing counseling to parents (68.1% preintervention and 64.5% postintervention agreement). Of most significance was the fact that a lower percentage of staff members agreed postintervention that almost all injuries to children were avoidable. The emergency department was the lowest ranked information dissemination venue for both parents and children. PMID- 10729667 TI - The effects of lead sulfate on new sealed lead acid batteries. AB - Emergency Medical Services (EMS) rely on batteries to power external cardiac defibrillators. While maintenance protocols should be followed to ensure that batteries possess adequate capacity to power their defibrillator, they are not often applied to new batteries. This study examines the effects of prolonged storage on sealed lead acid (SLA) batteries, the number of batteries that are affected by lead sulfate, and the ability of a protocol to restore the capacity in SLA batteries. A prospective cohort of new batteries was subjected to testing and discharge protocols. Initial battery capacities were measured using a battery analyzer. An "over-discharge" protocol fully discharged the battery over a 24-h period, and batteries were recharged and reanalyzed. Capacity measurements were repeated twice. Sulfate buildup was defined a priori as final capacity measurements greater than predischarge measurements. There were 126 batteries studied, a mean of 14 months after manufacture. Overall, 47 batteries (36.5%) had measured capacity that was insufficient (< 65% capacity). Batteries possessing very low initial capacities (< 55%) responded with a significant improvement on average of 54.7% compared with batteries within a normal capacity range (> 65%) whose average improvement was 9.3%. After discharge, there was an average of 17% improvement in the measured capacity, with no differences in the final capacity readings in each battery type. In conclusion, sealed lead acid batteries are affected by prolonged storage. The loss of capacity created by accumulation of lead sulfate can be reversed if battery maintenance protocols are used as part of EMS quality assurance programs. PMID- 10729668 TI - A prospective study of i.v. magnesium and i.v. prochlorperazine in the treatment of headaches. AB - Previous uncontrolled, nonrandomized trials suggest that magnesium sulfate (MgSO(4)) is effective in the treatment of headache. The objective of this study was to determine the efficacy of MgSO(4) vs. prochlorperazine in emergency department (ED) patients with acute headache. Patients presenting to the ED with a chief complaint of headache who met study criteria were enrolled. Each patient rated pain on a visual analog scale before and 30 min after a randomized study drug infusion. Thirty-six similar patients were enrolled. There was complete or partial pain relief in 90% of the prochlorperazine group and 56% of the MgSO(4) group, a statistically significant difference. Prochlorperazine caused significantly fewer side effects, and none of the patients required additional medication during the study period. It was determined that intravenous prochlorperazine is highly effective in the treatment of headache and magnesium is moderately effective. Response to MgSO(4) was unrelated to serum Mg level. PMID- 10729669 TI - Massive esophageal variceal hemorrhage triggered by complicated endotracheal intubation. AB - Esophageal variceal hemorrhage is frequently a catastrophic event. The specific events that trigger variceal rupture are not well understood. Acute elevations in systemic blood pressure and increased splanchnic blood flow, however, may lead to increased intravariceal pressure followed by variceal rupture and hemorrhage. This report describes a strong temporal association between complicated endotracheal intubation and abrupt onset of life-threatening variceal hemorrhage. A 52-year-old man with a history of portal hypertension was intubated emergently for airway protection because of respiratory insufficiency due to sepsis. Intubation was complicated by initial inadvertent esophageal intubation and by a peak mean arterial blood pressure of 155 mmHg. At the conclusion of the procedure, the patient sustained large volume hematemesis due to esophageal variceal rupture. This case suggests a risk of triggering variceal hemorrhage as a result of intubation-induced increase in blood pressure. A number of agents, including fentanyl, have been shown to be effective in attenuating the cardiovascular response to intubation. This case report provides strong evidence in support of administering fentanyl, or a suitable alternative adjunctive medication, before intubation of patients with documented portal hypertension and a history of esophageal variceal hemorrhage. PMID- 10729670 TI - Denture esophageal impaction refractory to endoscopic removal in a psychiatric patient. AB - Impaction of dental prostheses is frequently encountered in psychiatric patients. These patients may present an especially challenging problem because the diagnosis may be delayed, resulting in increased morbidity and mortality. Delay in diagnosis in such patients has been attributed to their inability to give a reliable clinical history. In addition, radiolucent dentures cannot be easily detected by radiographic examination. The purpose of this report is to describe a psychiatric patient with an impacted radiopaque dental prosthesis that was refractory to endoscopic intervention. An esophagotomy was needed to successfully remove the foreign body. PMID- 10729671 TI - Emergency medicine in Belarus. AB - Belarus is a small eastern European nation between Poland and Russia. Except for the lingering effects of Chernobyl radiation contamination, its system of health care and, in particular, emergency care has received little attention in the world literature. Emergency medical care is organized similarly to that provided in Russia and other European countries. A well-coordinated Emergency Medical Services (EMS) system exists, and many ambulance teams include a physician. Most emergency care is provided in an area of the hospital designated as the reception area, the equivalent of a U. S. emergency department (ED). Unlike the practice in U.S. EDs, many patients have a minimal evaluation after admission to the actual ward or hospital ICU. Emergency Medicine has not been officially organized as a specialty. Opportunities exist for interchange and discussion on the delivery of emergency care. PMID- 10729672 TI - The limping child. AB - The limping child often presents to the emergency department with a nonspecific history and physical examination. The components of gait, the pathophysiology of specific abnormalities, and the conditions that may produce long-term morbidity must be identified expeditiously to assure return to normal function. Ancillary data and imaging may be essential for appropriate management. PMID- 10729673 TI - Acebutolol overdose resulting in fatalities. AB - Two fatal cases of Acebutolol intoxication are presented that demonstrate its clinical characteristics and potential lethality. A review of the literature suggests that acebutolol is one of the most toxic beta blockers when taken as an overdose. In addition to demonstrating characteristics of membrane-stabilizing activity, both fatal cases demonstrate significant QTc prolongation and ventricular tachycardia. The latter findings suggest an impact on ventricular repolarization not seen with Propranolol intoxication. Clarification of these findings has important implications regarding identification and treatment of this potentially fatal intoxication. PMID- 10729674 TI - Drug storage temperatures in rescue vehicles. AB - This study was conducted to determine storage temperatures of drugs carried on rescue vehicles. Recording thermometers were placed inside drug boxes carried on rescue vehicles. Those temperatures were compared with ambient air temperatures, temperatures inside mechanically cooled compartments of the rescue vehicles, and USP-recommended drug storage temperatures. The results indicate that drug storage temperatures in some prehospital rescue vehicles exceed USP guidelines. Mechanical cooling of the storage compartment results in drug storage temperatures within the USP guidelines. Mechanical cooling of drug storage compartments on vehicles is technologically and financially possible. PMID- 10729675 TI - Scene disposition and mode of transport following rural trauma: a prospective cohort study comparing patient costs. AB - This prospective cohort study was performed from 1994 to 1996 to compare the impact of scene disposition on prehospital and hospital costs incurred by rural trauma patients transported to a trauma center by helicopter or ground ambulance. The study included all rural adult injury victims who arrived at the tertiary trauma center by ambulance within 24 h of injury. Inclusion criteria consisted of inpatient admission or death in the emergency department, and any traumatic injury except burns. Data collected included mortality, mode of transport, Injury Severity Score (ISS), and costs from impact to discharge or death. Of 105 study patients, 52 initially went to a rural hospital, while 53 went directly to the trauma center. There was no significant difference in survival in the two groups. The ISS was significantly higher for patients taken directly to the trauma center from the scene. The ISS of trauma patients transported from the rural hospital was highest for patients sent by ground transport. The prehospital transport costs were significantly more for patients transported to a rural hospital first. The costs incurred at the trauma center were highest for those patients transported directly from the scene. Many severely injured patients were initially transported to a rural hospital rather than directly to the trauma center. At both the scene and rural hospital, consistent use of triage criteria appeared to be lacking in determining the severity of injury, appropriate destination, and mode of transport for trauma patients. Since no significant difference in prehospital helicopter and ground transport costs was demonstrated, the decision on mode of transport should be in the best interest of patient care. PMID- 10729676 TI - Delayed cardiac tamponade after pacemaker insertion. AB - Cardiogenic shock is one of the most dramatic presentations in Emergency Medicine and requires rapid and accurate assessment, evaluation, and treatment. The cardiovascular disasters that present with shock include acute myocardial infarction with pump failure, aortic dissection, massive pulmonary emboli, and cardiac tamponade. We report a patient who presented to our Emergency Department (ED) in cardiogenic shock 10 days after insertion of a permanent cardiac pacemaker. The patient had developed pericardial tamponade secondary to the insertion. In reviewing the literature, we found many reports relating to complications of pacemakers and even more information regarding the various etiologies of cardiac tamponade, but cardiac tamponade as a consequence of pacemaker insertion rarely has been reported. Cardiac tamponade can occur secondary to perforation of the right ventricle during pacemaker electrode insertion and manipulation. Perforation is generally believed to be benign and self-limiting and only rarely causes tamponade and hemodynamic compromise; however, that was not the case for our patient. PMID- 10729677 TI - Syphilis testing. AB - The organism that causes syphilis, Treponema pallidum, is impossible to culture in the clinical laboratory. Direct visualization of the pathogen is one laboratory technique used in the diagnosis of syphilis. Currently, serologic testing is the most widely used laboratory technique in diagnosing syphilis and monitoring its course after treatment. Serologic tests are divided into two categories, the nontreponemal and treponemal antibody tests. Newer techniques such as enzyme immunoassays have shown excellent results. PMID- 10729678 TI - Cyanosis. PMID- 10729679 TI - Pseudogout. PMID- 10729680 TI - The other olive associated with vomiting. PMID- 10729681 TI - An uncharacteristically helpful white count. PMID- 10729682 TI - Can one size fit all? PMID- 10729683 TI - Revision of the International Classification of Impairments, Disabilities, and Handicaps: developmental issues. AB - Variability in approaches to define and classify disability has constituted persistent problems in documenting the epidemiology of disability and providing appropriate services. The major institutions of health care, mental health, and welfare often have separate systems of classification and terminology related to defining eligibility for programs and funding for services. In 1980, the International Classification of Impairments, Disabilities and Handicaps-ICIDH was published by the World Health Organization as a companion document of the International Classification of Disease to document the consequences of illness or injury. Current problems concerning the classification of childhood disability in health, education, and related services have resulted in growing interest in the revision of the ICIDH as a classification tool. The strengths and limitations of the ICIDH are examined in general, as well as with specific reference to its ability to document the nature and epidemiology of childhood disability. This paper (1) describes the ICIDH taxonomy and representative contributions; (2) reviews issues and concerns contributing to its revision; (3) summarizes changes in the revised ICIDH2 draft document, and (4) identifies issues of particular relevance to children and public health applications. PMID- 10729684 TI - Measurement properties of the activities scale for kids. AB - The Activities Scale for Kids (ASK) is a self-report measure of childhood physical disability, that has excellent reliability (ICC = 0.97). The purpose of this study was to assess further the ASK's measurement properties. ASK questionnaires were completed by 200 children with musculoskeletal limitations (mean age, 10.1 years). Rasch analyses confirmed that all items measured the same construct and supported aggregation of a summary score. Validity of the ASK was demonstrated by a correlation of 0.81 (P<0.0001) with parent-reported Childhood Health Assessment Questionnaire scores; a significant difference in scores according to clinicians' global ratings of disability (P<0.0001), and a correlation of 0.92 (P<0.0001) with clinician-observation. The ASK showed minimal ceiling effects, no floor effects, and changed by 1.73 standard deviation units after clinically important change. The ASK is a valid and responsive outcome measure that permits 5- to 15-year-old children physical functioning in the community to be assessed accurately by mail. The quality of this measure will enable clinical studies to measure outcome not only in a way that is relevant to patients, but also in a way that is sensitive to small amounts of change and is practical and inexpensive. PMID- 10729685 TI - Clinimetric properties of a parent report on their offspring's quality of life. AB - The objective of this study was to assess the reproducibility, construct validity, and responsiveness of the parent report version of the "How are you" (HAY), a quality of life questionnaire for children with a chronic disease. The reproducibility of the HAY was assessed by comparing the scores of two measurement cycles of children with stable asthma. Construct validity was evaluated by testing the differences between scores of the HAY of parents of children with and without asthma and by investigating the correlations between the HAY and instruments supposed to correlate with the HAY. Responsiveness was investigated in parents of children whose clinical status changed clinically relevant between two measurements. The HAY showed acceptable reproducibility. Also the HAY detected anticipated differences in quality of life between parents of asthmatic and healthy children. It furthermore showed acceptable ability to measure change in health status of children with asthma. The parent report version of the HAY shows acceptable reproducibility, supportive evidence for construct validity, and good responsiveness. The measurement of quality of life of asthmatic children and changes therein can truly rely on parental report. PMID- 10729686 TI - Stillbirth risk with social class and deprivation: no evidence for increasing inequality. AB - The objective of this study was to determine whether inequality in stillbirth risk between social strata has changed over time. Subjects were all 288,869 births in Cumbria, northwest England, 1950-1993 and all 8,039,269 births in England and Wales, 1981-1992. Social class of Cumbrian babies was ascertained from birth registrations. Community deprivation scores were calculated from census data for (i) enumeration districts in Cumbria and (ii) county districts in England and Wales. The relative index of inequality was used to measure inequality of stillbirth risk between social strata. Results indicate inequality in stillbirth risk in Cumbria has fallen significantly since 1966 (P< or =0.02) and was not evident in more recent time periods. In England and Wales, there was significant inequality in stillbirth risk in all time periods and no evidence that this has changed over time. Inequality in stillbirth risk has not increased and in some areas has attenuated in recent years. PMID- 10729687 TI - Why use placebos in clinical trials? A narrative review of the methodological literature. AB - Researchers have a number of different options for their choice of control or comparison intervention in randomized trials. We surveyed the methodological literature looking for reasons why a researcher might choose to administer a placebo (i.e., a mimic intervention) to control subjects. Two implicit assumptions were that the issue of placebo is pertinent only to drug trials and that the nonplacebo effect of a treatment is the "real" or "true" effect. Explicit reasons given in the literature for the use of placebos were facilitating blinding and controlling for the placebo effect. The importance of the latter was often inadequately argued. Reasons to avoid placebos in controlled trials, other than ethics and feasibility, are that placebos do not inform real decisions, and may interfere with accurate estimation of effect size and with nonspecific aspects of treatment. Placebo-controlled trials have high internal validity but may be difficult to apply to clinical practice; the situation is reversed for trials without placebo control. PMID- 10729688 TI - Incorporating patient preferences into randomized trials. AB - When designing trials, an important ethical and practical concern is ensuring that proper consideration is given to patient preferences. A number of design variants on the traditional randomized trial can take account of such preferences in different ways. The partially randomized patient preference (PRPP) design is often advocated for taking these preferences into account. In studies of this type, all eligible patients are followed up, but the primary comparison is still between effects observed in patients randomly allocated to treatment arms. This design and other variants on conventional trials each have different scientific, ethical, and practical implications. We consider these diverse aspects of several modifications to the conventional method for obtaining consent and then, in more detail, the relative merits of the PRPP design. We conclude that none of these adjustments is ideal. Those designing trials should therefore always think carefully about the consent process, for its implications on participation and on how the results will be interpreted. PMID- 10729689 TI - Randomized trials versus observational studies in adolescent pregnancy prevention. AB - The objective of this study is to compare the results of randomized trials and observational studies of interventions to prevent adolescent pregnancy. We identified published and unpublished reports through computerized searches of CATLINE, CINAHL, CONFERENCE PAPERS INDEX, DISSERTATION ABSTRACTS ONLINE, EMBASE, ERIC, MEDLINE, NTIS, POPLINE, PsycINFO, and SOCIOLOGICAL ABSTRACTS; manual searches of eight relevant journals; reference lists from primary articles; and contact with content experts. We included randomized trials and observational studies that evaluated the impact of primary prevention interventions including sex education classes, school-based clinics, free-standing clinics, physician/nurse practitioner practice-based service, improved access, and community-based programs on four outcomes: sexual intercourse, birth control use, responsible sexual behavior, or pregnancy in adolescents. One investigator abstracted the data and a second conducted a detailed review of the abstraction. We identified 13 randomized trials and 17 observational studies. We generated estimates of the impact of the interventions separately for males and females for all four outcomes for both observational studies and randomized trials. For six of the eight outcomes the summary odds ratios for the observational studies showed a significant intervention benefit (P<0.05) while the randomized trials did not show a benefit for any outcome in either females or males. The difference between the results of the observational studies and randomized trials was statistically significant in two of the eight outcomes (P<0.05 for initiation of intercourse and pregnancy in females). Observational studies yield systematically greater estimates of treatment effects than randomized trials of adolescent pregnancy prevention interventions. Public policy or individual patient treatment decisions should be based on observational studies only when randomized trials are unavailable and only with careful consideration of possible biases. PMID- 10729690 TI - An investigation into factors associated with Helicobacter pylori infection. AB - This study aims to estimate the prevalence of Helicobacter pylori in Glasgow, and to provide a systematic analysis of factors associated with this prevalence. The data used are from a random population sample of 793 men and 838 women aged 25-64 years conducted in 1995. The prevalence is estimated to be 66% (95% confidence interval: 63-68%); a level that is more typical of developing countries. Prevalence increases with age and social deprivation (P<0.0001) and is slightly higher in men than women (P = 0.07). After adjustment for age, social class, and sex group, H. pylori prevalence increases with increased cotinine (tobacco consumption) (P = 0.0005), increased number of siblings (P<0.0001), and decreased height (P = 0.03). Prevalence of coronary heart disease, hypertension, diabetes and intermittent claudication, alcohol consumption, fibrinogen, total serum cholesterol, HDL cholesterol, triglycerides, marital status, systolic and diastolic blood pressure had no independent association. The infection seems to be spread more readily in deprived, relatively crowded living conditions in childhood. The independent relationship with smoking suggests a possible second source of spread of infection in later years. The high degree of social deprivation in Glasgow is suggested as a major explanation of the high H. pylori prevalence found there. PMID- 10729691 TI - Using medical services claims to assess injuries in the elderly: sensitivity of diagnostic and procedure codes for injury ascertainment. AB - The sensitivity of using physician claims data for injury ascertainment was examined in a cohort of 1,181 elderly who were treated in the emergency department of one of 10 hospitals for injuries in 1993-1994. The clinical record of the type and date of injury was compared with diagnostic and procedure codes in the Quebec health insurance agency records of physician billing claims for the same patients. The proportion of patients correctly classified by claims data was determined for the exact date of injury and for a time window around the date of injury. The most common injuries were fractures (55.4%) and lacerations (19.3%), and 78.9% of injuries were fall related. Overall, the combination of treatment procedure codes and diagnostic codes provided the most sensitive measure of injury occurrence; a sensitivity of 67.3% for the exact date and 81.3% for an expanded data window (95.6% of injuries were within -1 day to +3 days of the injury date). Sensitivity varied by injury type form a low of 14% for abrasions to a high of 97.2% for hip fractures. The combination of diagnostic and procedure codes in physician claims is a sensitive indicator of some common injuries that would not be documented in hospitalization databases. PMID- 10729692 TI - Use of grade membership analysis to profile the practice styles of individual physicians in the management of acute low back pain. AB - The objective of this study was to investigate Grade of Membership analysis (GoM) as a means of profiling the practice styles of individual physicians. GoM uses maximum likelihood techniques to estimate occurrences of management items that define unique practice styles. It also provides statistical estimates (grade of membership coefficients) of how well each individual physician's practice fits the identified styles. Data sources were responses to a mailed survey asking 814 physicians to select from predetermined management options in three scenarios depicting patients with acute low back pain. Four distinct types of practice style were identified: watchful waiting; investigative; guideline based; and physiotherapy focused. Most physicians were characterized by high grades of membership in the two profiles that most closely matched existing guidelines for the management of acute low back pain. GoM successfully identified characteristic practice styles, and the GoM coefficients obtained provided descriptions of individual management approaches. GoM is data driven and less subject to judgment than opinion-based measures of performance. It has several potential applications in identifying physicians for whom some form of intervention would be beneficial. PMID- 10729693 TI - Publication bias in meta-analysis: its causes and consequences. AB - Publication bias is a widespread problem that may seriously distort attempts to estimate the effect under investigation. The literature is reviewed to determine features of the design and execution of both single studies and meta-analyses leading to publication bias, and the role the author, journal editor, and reviewer play in selecting studies for publication. Methods of detecting, correcting for, and preventing publication bias are reviewed. The design of the meta-analysis itself, and the studies included in it, are shown to be important among a number of sources of publication bias. Various factors influence an author's decision to submit results for publication. Journal editors and reviewers are crucial in deciding which studies to publish. Various methods proposed for detecting and correcting for publication bias, though useful, all have limitations. However, prevention of publication bias by registering every trial undertaken or publishing all studies is an ideal that is hard to achieve. PMID- 10729694 TI - Delaying decay: mummies and smoked salmon. PMID- 10729695 TI - A topographic electrophysiologic study of mental rotation. AB - Mental rotation is a task performed when subjects are requested to determine whether two stimuli presented in turn have the same shape (congruency) or a mirror-image shape (incongruency) regardless of any difference in orientation. We compared event-related potentials during mental rotation tasks with narrow and wide angular disparities between the two stimuli to identify electrophysiologic correlates of mental rotation. When angular disparity was wide, a prominent negative component arose 438 ms after the second stimulus. A statistically significant difference detected between amplitudes of the negative components under narrow- and wide-angle conditions was maximal in the right parietal region, suggesting that processing of mental rotation is a right parietal dominant function. PMID- 10729696 TI - Recognising a hand by grasp. AB - The present study aimed to demonstrate that motor representations are used to recognise biological stimuli. In three experiments subjects were required to judge laterality of hands and forearms presented by pictures. The postures of the hands were those assumed when holding a small, medium and large sphere. In experiment 1, the sphere held in hand was presented, whereas in experiment 2 it was absent. In experiment 3, the same images, showing holding-a-sphere hands, as in experiment 1 were presented, but without forearm. In all experiments one finger of each hand could be absent. In experiment 1 recognition time was longer for those hand postures for which the corresponding grasping motor acts required more accuracy. This was confirmed by a control experiment (experiment 4), in which subjects actually grasped the spheres. Absence of fingers did not influence right-left hand recognition. However, the absence of target object in experiment 2, and of forearm in experiment 3 reduced the effects of the type of holding on hand laterality recognition. The results of the present study indicate that grasp representations are used to recognise hand laterality. In particular, the visual description of how hand and object interact in space (the opposition space [M.A. Arbib, Programs, schemas and neural networks for control of hand movement: beyond the RS frameworks, in: M. Jeannerod (Ed.), Attention and Performance XIII: Motor Representation and Control, Lawrence Erlbaum, Hillsdale, NJ, 1990, 111-138; M.A. Arbib, T. Iberall, D. Lyons, Coordinated control programs for movements of the hand, in: A.W. Goodman, I. Darian-Smith (Eds.), Hand function and the neocortex, Springer, Berlin, 1985, pp. 135-170]) and the anchoring of the hand to the agent are the features of the grasp representations used in hand-recognition processes. The data are discussed according to the more general notion that motor representations are automatically extracted in the process of intuiting situations, or people's intentions. These motor representations, which are compared with those of other people, contain concrete information on the actions (the motor program) by which a situation is created and on the aim of the agents executing those actions. PMID- 10729697 TI - Event-related potential indices of semantic priming using masked and unmasked words: evidence that the N400 does not reflect a post-lexical process. AB - Several authors have contended that the N400 is a reflection of a post-lexical event such as that proposed by Neely and Keefe [J.H. Neely, D.E. Keefe, Semantic context effects on visual word processing: a hybrid prospective/retrospective processing theory, in: G.H. Bower (Ed.), The Psychology of Learning and Motivation: Advances in Research and Theory, Vol. 23, Academic Press, New York, 1989, pp. 207-248.], whereby the subject compares the word on the current trial to the "context" provided by the word on the preceding trial [M. Besson, M. Kutas, The many facets of repetition: A cued-recall and event-related potential analysis of repeating words in same versus different sentence contexts, Journal of Experimental Psychology: Learning, Memory and Cognition, 19 (5) (1993), 1115 1133; C. Brown, P. Hagoort, The processing nature of the N400: Evidence from masked priming. Journal of Cognitive Neuroscience, 5(1) (1993), 34-44; P.J. Holcomb, Semantic priming and stimulus degradation: Implications for the role of the N400 in language processing, Psychophysiology 30 (1993), 47-61; M.D. Rugg, M.C. Doyle, Event-related potentials and stimulus repetition in indirect and direct tests of memory, in: H. Heinze, T. Munte, G.R. Mangun (Eds), Cognitive Electrophysiology, Birkhauser Boston, Cambridge, MA, 1994]. A study which used masked primes to directly test this possibility has been reported by Brown and Hagoort [C. Brown, P. Hagoort, The processing nature of the N400: evidence from masked priming. Journal of Cognitive Neuroscience, 5(1) (1993), 34-44]. When the primes were masked, no priming effect was observed on the N400. When behavioral data were collected in the same paradigm, from another group of subjects, the usual priming effect on RT was obtained. Considered together, the data from the two groups of subjects indicated that activation of semantic representations had occurred without conscious awareness. As no N400 priming effect was observed, it was suggested that N400 must reflect a post-lexical process. This interpretation, however, is at odds with the findings of other studies which have reported N400 priming effects under conditions where post-lexical processes would not be thought to operate[J. Anderson, P. Holcomb, Auditory and visual semantic priming using different stimulus onset asynchronies: an event-related brain potential study. Psychophysiology 32 (1995), 177-190; J. Boddy, Event-related potentials in chronometric analysis of primed word recognition with different stimulus onset asynchronies, Psychophysiology 23 (1986), 232-245; D. Deacon, T. Uhm, W. Ritter, S. Hewitt, The lifetime of automatic priming effects may exceed two seconds, Cognitive Brain Research 7 (1999), 465-472; P.J. Holcomb, Automatic and attentional process: an event-related brain potential analysis of semantic priming. Brain and Language 35 (1998) 66-85]. The present study replicated Brown and Hagoort using a repeated measures design, a shorter SOA (stimulus onset asynchrony), and a slightly different threshold setting procedure. Significant priming effects were obtained on the mean amplitude of the N400 regardless of whether the words were masked or unmasked. The findings imply that the processing subserving the N400 is not postlexical, since the N400 was manipulated without the subjects being aware of the identity of the words. PMID- 10729698 TI - Event-related brain potentials dissociate visual working memory processes under categorial and identical comparison conditions. AB - Event-related potentials (ERPs) have been successfully employed to examine the functional and neuronal characteristics of working memory processes. In the present study, we examined the ERP waveforms in a delayed matching task to examine the cognitive processes underlying category and identity comparison and the effects of stimulus complexity. Subjects had to decide whether two visual stimuli are (a) physically identical (identical comparison condition, IC) or (b) identical, irrespective of their orientation (categorial comparison condition, CC). The stimuli were structured five-point patterns, which varied in complexity. For the ERPs elicited during the 1500 ms retention interval, the following pattern of results was obtained: Stimuli in the CC-condition elicited larger P300 components than in the IC-condition. In the IC-condition, the P300 was followed by a broadly distributed negative slow wave. Moreover, complex patterns elicited a posteriorily distributed negativity at 350 ms (N350), whereas the less complex patterns gave rise to a fronto-centrally distributed slow wave that started around 500 ms. These results suggest that S1 was more elaborately processed in the CC-condition, while the more complex figures were associated with an early classification process during the retention interval. PMID- 10729699 TI - Spatiotemporal source analysis of vocalization-associated magnetic fields. AB - The vocalization-related cortical fields (VRCF) following vowel vocalization were studied by magnetoencephalography (MEG) in eight normal subjects. A multiple source model, BESA (Brain Electric Source Analysis), was applied to elucidate the generating mechanism of VRCF in the period from 150 ms before to 150 ms after the onset of vocalization. Six sources provided satisfactory solutions for VRCF activities during that period. Sources 1 and 2, which were activated from approximately 150 ms before the vocalization onset, were located in laryngeal motor areas of the left and right hemispheres, respectively. Sources 5 and 6 were located in the truncal motor area in each hemisphere, and they were very similar to sources 1 and 2 in terms of temporal change of activities. Sources 3 and 4 were located in the auditory cortices of the left and right hemispheres, respectively, and they appeared to be activated just after the vocalization onset. However, all six sources were temporally overlapped in the period approximately 0-100 ms after the vocalization onset. The present results suggested that the bilateral motor cortices, probably laryngeal and truncal areas, were activated just before the vocalization. We considered that the activities of the bilateral auditory areas after the vocalization were the response of the subject's central auditory system to his/her own voice. The motor and auditory activities were temporally overlapped, and BESA was very useful to separate the activities of each source. PMID- 10729700 TI - Effects of distraction on pain-related somatosensory evoked magnetic fields and potentials following painful electrical stimulation. AB - We aimed to compare the effects of distraction on pain-related somatosensory evoked magnetic fields (pain SEF) following painful electrical stimulation with simultaneous recordings of evoked potentials (pain SEP). Painful electrical stimuli were applied to the right index finger of eleven healthy subjects. A table with 25 random two-digit numbers was shown to the subjects, who were asked to add 5 numbers of each line in their mind (calculation task) or to memorize the numbers (memorization task) during the recording. In the SEF recording, 3 short latency components within 50 ms of the stimulation were generated in the primary sensory cortex (SI) of the hemisphere contralateral to the stimulated finger. Middle-latency components between 100 and 250 ms after the stimuli were recorded from the secondary somatosensory cortex (SII) in the bilateral hemispheres or the cingulate cortex. No SEF components were significantly affected by either task. In the SEP recording, the middle-latency components (N140 and P230) were identified as being maximal around the vertex. Amplitudes of the N140 and P230 were not affected by each task, but the peak-to-peak amplitude (N140-P230) was significantly decreased by both the calculation and memorization tasks, particularly by the former. Subjective pain rating was decreased in both the calculation and memorization tasks, particularly in the former. We concluded that distraction tasks reduced activities in the limbic system, in which the middle latency EEG component probably generated, while neither the short-latency SEF components generated in SI nor the primary pain-related SEF components generated in SII-insula are affected. PMID- 10729701 TI - Human oscillatory brain activity within gamma band (30-50 Hz) induced by visual recognition of non-stable postures. AB - Our principal finding from this study is that there were changes at the level of brain electrical activity (EEG) during cognitive tasks while subjects were instructed to visually recognize non-stable postures of a computer animated human body model. In particular, there was clear enhancement of the amplitude within the gamma band (30-50 Hz) activity associated with visual recognition of non stable postures at fronto-central and parietal areas in all subjects. The Morlet's wavelet transform was applied to examine the change of time-frequency (TF) energy within a range of 1-70 Hz frequencies range as a function of experimental tasks. There was a high energy burst within the 35-45 Hz TF cluster at fronto-central and parietal areas when subjects visually recognized non-stable postures. Experimental evidences were provided demonstrating that EEG activity recorded during visual recognition of non-stable postures was related to specific judgement of postural instability. In a series of control experiments, additional evidences were provided to justify the specific sensitivity of EEG 40-Hz activity to the act of visual recognition of postural instability. The contamination of muscle activity in the reported EEG results during perceptual tasks was also ruled out. Our findings are consistent with the notion of existence of specialized neural detectors (predictors) for specific postures and goal-oriented behavior. However, the functional significance and precise cognitive and neurophysiological mechanisms predicting the existence of these detectors remain to be explored. PMID- 10729702 TI - Contextual fear conditioning is associated with an increase of acetylcholine release in the hippocampus of rat. AB - The effects of contextual fear conditioning on the release of acetylcholine (ACh) in the hippocampus of freely moving rats was assessed using microdialysis. Measures were carried out during both acquisition and retention testing (re exposure to the conditioning chamber) and compared between animals that either received foot-shocks as unconditioned stimulus (conditioned group) or no foot shocks (control group) during acquisition. Results showed that during acquisition, hippocampal ACh extracellular level was increased with respect to baseline but that this increase was of similar magnitude in both groups. By contrast, re-exposure to the conditioning chamber the day after (retention testing) produced a significantly greater increase in ACh extracellular level in the conditioned (that, otherwise, displayed conditioned freezing behavior to contextual cues), than in the control group (which displayed virtually no freezing). This enhanced hippocampal ACh release seems to result from the greater hippocampal processing of contextual stimuli in conditioned animals with respect to controls. PMID- 10729703 TI - Temporal cost of switching between kinds of visual stimuli in a memory task. AB - These experiments measured the extra time required to respond when the type of stimulus was changed from one trial to the next. Heretofore, switching costs have been measured for a change in task, but we wished to isolate the cost of changing the sensorial component per se and its necessary analytical processing, as distinct from changing the question being posed or the type of response to be given. Thus, the task was identical throughout the experiments: continuous recognition in a single, 240-trial session, in which the subject was required to distinguish initial from repeat appearances of a stimulus, the single repetition of each stimulus occurring after 1-31 intervening trials. There were two categories of 200-ms stimuli, linguistic (words and non-words) and images (multiple-colored or gray scale panels, human faces, or butterflies); and two conditions of switching, predictable (alternating on each trial) or unpredictable, in which the switch occurred after three to eight trials of one kind. In the majority of cases, there was a robust switching cost, from 24 to 92 ms. The similarity of costs in the predictable and unpredictable modes suggests that this cost is derived at least as much from terminating the modus operandi for the preceding type of stimulus as from a reconfiguration of processing for the new type of stimulus. In the switch between words and images, the costs were "paradoxical" (asymmetrical), in that the switching from an image to a word was more costly than the reverse, that is, changing from the more to the less difficult required the greater time. This, too, is compatible with the idea that termination of the previous mode of processing is a major component of the cost. Thus, in contemplating the neuronal/cognitive events underlying visual memory, consideration must be given to the inertia of pre-existing linkages. PMID- 10729704 TI - Idiosyncratic orientation strategies influence self-controlled whole-body rotations in the dark. AB - The present experiment examined the influence of spatial orientation strategies on human subjects' accuracy in a self-controlled whole-body rotation task in the dark. Subjects were seated on a robotic chair and had to perform 360 degrees rotations with or without the presentation of a space-fixed target. Performance was compared between subjects who preferably used an "egocentric" or an "allocentric" strategy. Results suggest that orientation strategies might be tightly linked to sensory integration processes. PMID- 10729705 TI - Neural correlates of memory retrieval and evaluation. AB - Results from recent neuroimaging studies have led to a controversy as to whether right or left prefrontal regions are relatively more important for episodic retrieval. To address this issue, we recorded event-related brain potentials during two recognition tests with identical stimuli but differing retrieval demands. In both tests, participants viewed a sequence of object drawings, half of which were identical to ones viewed earlier except for a change in size and half of which were new. Instructions were to discriminate between old and new objects (general test) or to additionally decide whether old objects were larger or smaller at study (specific test). Frontal brain potentials that were more positive during the specific than during the general test for both old and new objects were interpreted as neural correlates of the process by which specific attributes of test cues are compared with information retrieved from memory. Another ERP difference between the specific and general tests, which was observed for old objects only, had a left posterior scalp topography and was interpreted to reflect the reactivation of memories for studied objects. Frontal and posterior potentials thus reflected two memory processes important for accurate episodic retrieval. Furthermore, our findings suggest that both left and right prefrontal regions were engaged when demands to retrieve and evaluate perceptual information increased. PMID- 10729706 TI - Localization, sequence analysis, and ethnic distribution of a 96-bp insertion in the promoter of the human CYP2E1 gene. PMID- 10729707 TI - Use of archival tissue in epidemiologic studies: collection procedures and assessment of potential sources of bias. AB - Collection and analyses of archival tumor tissue as a means to increase our understanding of disease pathways is becoming an important avenue of epidemiologic research. In this paper, we present methods of collection and processing of archival tissue and assess the population characteristics of those for whom we were able to and unable to obtain tumor DNA. Cases of colon cancer diagnosed between September, 1991 and October, 1994 living in Utah, Northern California, or the Twin Cities Metropolitan area of Minnesota were targeted for this study. Of the 2477 people for whom we had permission to obtain tumor blocks, we were able to collect blocks and extract DNA for 2117 (85.5%). There were no differences in age, tumor site, or diet and lifestyle characteristics between those with and without DNA extracted. However, we were less likely to be able to extract DNA if the case was diagnosed at a more advanced disease stage or at the earliest disease. Potential bias from exclusion of those with the most advanced disease stage is discussed. PMID- 10729708 TI - Predicted changes in pre-mRNA secondary structure vary in their association with exon skipping for mutations in exons 2, 4, and 8 of the Hprt gene and exon 51 of the fibrillin gene. AB - Exon skipping that accompanies exonic mutation might be caused by an effect of the mutation on pre-mRNA secondary structure. Previous attempts to associate predicted secondary structure of pre-mRNA with exon skipping have been hindered by either a small number of available mutations, sub-optimal structures, or weak effects on exon skipping. This report identifies more extensive sets of mutations from the human and hamster Hprt gene whose association with exon skipping is clear. Optimal secondary structures of the wild-type and mutant pre-mRNA surrounding each exon were predicted by energy minimization and were compared by energy dot plots. A significant association was found between the occurrence of exon skipping and the disruption of a stem containing the acceptor site consensus sequences of exon 8 of the human Hprt gene. However, no change in secondary structure was associated with skipping of exon 4 of the hamster Hprt gene. Using updated energy parameters we found a different structure than that previously reported for exon 2 of the hamster Hprt gene. In contrast to the previously reported structure, no significant association was found between predicted structural changes and skipping of exon 2. For all three Hprt exons studied, there was a significantly greater number of deoxythymidine substitutions among mutations accompanied by exon skipping than among mutations without exon skipping. For exon 8, deoxythymidine substitution was also associated with structural changes in the stem containing the acceptor site consensus sequences. For exon 51 of the human fibrillin gene, structural differences from wild type were predicted for all four mutations accompanied by exon skipping that were not were predicted for a single mutation without exon skipping. Our results suggest that both primary and secondary pre-mRNA structure contribute to definition of Hprt exons, which may involve exonic splicing enhancers. PMID- 10729709 TI - A nonsense codon of exon 14 reduces lysyl hydroxylase mRNA and leads to aberrant RNA splicing in a patient with Ehlers-Danlos syndrome type VI. AB - Ehlers-Danlos syndrome type VI (EDSVI) is an autosomal recessively inherited connective tissue disease, characterized by kyphoscoliosis, muscular hypotonia and ocular manifestations. The cause of the syndrome is a deficiency in the activity of lysyl hydroxylase (LH), one of the enzymes involved in the post translational modification of collagens. We describe here an unusual compound heterozygote British patient with EDSVI. Our investigations indicate that a maternally inherited nonsense mutation (Y511X) in exon 14 of the LH gene (PLOD1) results in a reduction of the mRNA level as well as a skipping of exon 14 sequences in the mRNA that produces a protein shortened by 38 amino acids. The transcription of the other allele of the LH gene is considerably reduced from the normal for reasons that are not yet known. As a consequence, the LH activity of the skin fibroblasts of the patient is markedly reduced. PMID- 10729710 TI - Novel mutations of the PKD1 gene in Korean patients with autosomal dominant polycystic kidney disease. AB - The gene for the most common form of autosomal dominant polycystic kidney disease (ADPKD), PKD1, has recently been characterized and shown to encode an integral membrane protein, polycystin-1, which is involved in cell-cell and cell-matrix interactions. Until now, approximately 30 mutations of the 3' single copy region of the PKD1 gene have been reported in European and American populations. However, there is no report of mutations in Asian populations. Using the polymerase chain reaction and single-strand conformation polymorphism (SSCP) analysis, 91 Korean patients with ADPKD were screened for mutation in the 3' single copy region of the PKD1 gene. As a result, we have identified and characterized six mutations: three frameshift mutations (11548del8bp, 11674insG and 12722delT), a nonsense mutation (Q4010X), and two missense mutations (R3752W and D3814N). Five mutations except for Q4010X are reported here for the first time. Our findings also indicate that many different mutations are likely to be responsible for ADPKD in the Korean population. The detection of additional disease-causing PKD1 mutations will help in identifying the location of the important functional regions of polycystin-1 and help us to better understand the pathophysiology of ADPKD. PMID- 10729711 TI - Surface ultrastructure of Stictodora tridactyla (Trematoda: Heterophyidae) from Kuwait Bay. AB - The surface morphology of Stictodora tridactyla recovered from a kitten that was fed the killifish, Aphanius dispar, naturally infected with the metacercariae was studied using scanning electron microscopy. The body comprised a rounded head, elongate neck, and widely pyriform hind-body. The head bore a circular oral sucker on the ventral side, and concentric rows of peg-like cephalic spines on the dorsal side. The oral sucker was armed with pre-oral spines similar in shape and size to the cephalic spines. The neck and hind-body were densely covered with scale-like multipointed spines, the size and density of which decreased from anterior to posterior parts of the body. Ciliated dome-shaped papillae were found solitarily or as conjugated groups on the head apex and lips of the oral sucker. Non-ciliated dome-shaped papillae were restricted to the lower lip of the oral sucker. The body was devoid of a ventral sucker. The genital opening appeared as a round depression of the tegument at about the anterior third of the body. This is the first record of the occurrence of S. tridactyla in the Arabian Gulf region and A. dispar is a new second intermediate host. PMID- 10729712 TI - Ecological factors in schistosome transmission, and an environmentally benign method for controlling snails in a recreational lake with a record of schistosome dermatitis. AB - The avian schistosomes, Trichobilharzia stagnicolae, T. physellae and Gigantobilharzia sp., that cause Schistosome Dermatitis (Swimmers' Itch) in humans were studied in the laboratory and at Cultus Lake, British Columbia, Canada in relation to the biology and behavior of their intermediate snail hosts, Stagnicola catascopium, Physa sp. and Gyraulus parvus, respectively, and their definite bird hosts. Wind-driven, surface currents were measured. Populations of snails, close to host-bird roosting logs had a very high prevalence of schistosome infections. An experiment that mechanically disturbed the epilithic habitat of the snails using a boat-mounted rototiller or a tractor and rake, eliminated almost all of the snails if the disturbance was done in areas of high snail concentration in shallow areas of the lake during the breeding and early development phase of the snail. It is proposed that the incorporation of snail habitat disturbance into management programs is an effective way to control Schistosome Dermatitis. PMID- 10729713 TI - Preventive effect of artemether in experimental animals infected with Schistosoma mansoni. AB - The effect of artemether, an antimalarial drug developed from the plant Artemisia annua, has been tested against the larval stages of Schistosoma mansoni covering the time from skin penetration to the early adult liver-stage. The results show that the experimental animals used (hamster and mice) do not develop schistosomiasis mansoni if treated with artemether during the first month after infection. The parasite was found to be especially susceptible between the 3rd and 4th week after infection, resulting in worm reductions of 75.3-82.0% compared to non-treated controls. This level was boosted to 97.2-100% when the animals were subjected to various schedules of repeated treatment. Almost complete protection was also reached in parallel experiments with repeated infections carried out to mirror more closely the real situation of trickle infection. PMID- 10729714 TI - Effect of praziquantel together with artemether on Schistosoma japonicum parasites of different ages in rabbits. AB - The efficacy of combined treatment with praziquantel and artemether against infection with Schistosoma japonicum was tested on infected rabbits, in which 7 to 14-day-old schistosomules and 42-day-old adult schistosomes were simultaneously present. Rabbits were treated orally with praziquantel and artemether using various dosages and schedules. The therapeutic effects were evaluated by estimating the mean total worm burden (TWB) and female worm burden (FWB) and comparing them with the worm burdens in control animals treated with praziquantel or artemether alone. When the rabbits received praziquantel in a single dose (50 mg/kg), or daily for 2-6 days (30-60 mg/kg), the TWB was reduced by 28-66% and the FWB by 26-65%. In rabbits treated with artemether the reductions were 44-56% and 35-54%, respectively. Treatment with praziquantel in combination with artemether resulted in a significantly greater reduction of worm burden than was found for the groups treated with praziquantel or artemether alone, using the same dosages and schedules. TWB was reduced by 79-92%, and FWB by 80-93%. The results demonstrated that when rabbits infected simultaneously with schistosomules and adult schistosomes were treated with praziquantel in combination with artemether, the effects of the individual drugs could be increased significantly. PMID- 10729715 TI - Asymptomatic cyst passers of Entamoeba histolytica but not Entamoeba dispar in institutions for the mentally retarded in Japan. AB - Entamoeba histolytica/Entamoeba dispar was isolated from 50 asymptomatic amebic cyst passers in three institutions for the mentally retarded in Kanagawa Prefecture, Japan. To distinguish between E. histolytica and E. dispar, the isolates were analyzed by PCR, reactivity to monoclonal antibodies, and zymodemes. All isolates were identified as E. histolytica. The results lead us to conceive that, in Japan, E. histolytica is predominant even in asymptomatic cyst passers. PMID- 10729716 TI - Treatment of gram-negative bacterial meningitis in term neonates with third generation cephalosporins plus amikacin. AB - The aim of this retrospective study was to evaluate the clinical efficacy in terms of mortality and long-term morbidity of third generation cephalosporins and amikacin in combination for the treatment of gram-negative bacterial meningitis in a homogeneous group of neonates. A 15-year experience (1983-1997) with 72 term neonates without central nervous system anomalies and with gram-negative organisms grown in their cerebrospinal fluid treated with the above combination of antibiotics is presented. All isolated organisms were sensitive to cefotaxime or ceftazidime and to amikacin but 80% were resistant to ampicillin. The predominant infecting organism was Escherichia coli (68.0%) which was sensitive to both cefotaxime and amikacin in all cases but resistant to ampicillin in 48% of cases. Survival at discharge was 97.2% but ultimate survival was reduced to 94.4%, as 2 patients died a few months following discharge of conditions unrelated to meningitis. Ventriculitis was diagnosed in 10 neonates (13.8%). Among survivors, 1 neonate (1.3%) developed hydrocephalus needing shunting and 1 neonate (1.3%) with Proteus mirabilis developed a brain abscess with relapse of meningitis which was successfully treated with a 6-week course of chloramphenicol. At follow-up at an age greater than 6 months, 91.1% of the surviving infants were normal, while 92.3% of survivors at an age greater than 6 years were normal and attended normal school. These results, despite any reservations due to the nature of the study (retrospective, uncontrolled study), strongly support the use of third generation cephalosporins and amikacin in combination for the treatment of neonatal gram-negative bacterial meningitis. PMID- 10729718 TI - Effect of human milk fortifier on the immunodetection and molecular mass profile of transforming growth factor-alpha. AB - OBJECTIVE: To determine if the addition of human milk fortifier (HMF) affects the distribution, immunoreactivity, or molecular mass profile of transforming growth factor-alpha (TGF-alpha) within the compartments of human milk. METHODS: Fifteen milk samples were obtained. Each sample was divided into two aliquots; a powdered HMF was added to the first aliquot. TGF-alpha concentration was measured via radioimmunoassay in whole milk and its aqueous and fat fractions +/- HMF. TGF alpha molecular mass profiles of the samples (v/v) were measured via Western blotting. RESULTS: TGF-alpha concentration (mean +/- SD) in fortified whole milk (15.7 +/- 7.1 pg/100 microl) vs. nonfortified whole milk (14.8 +/- 8.0 pg/100 microl) and in the aqueous fraction of fortified (14.0 +/- 2.7 pg/100 microl) vs. nonfortified (14.0 +/- 3.5 pg/100 microl) did not differ statistically. There was, however, a marked decrease in the concentration of TGF-alpha in the fat fraction of fortified (30.6 +/- 2.8 pg/100 microl) vs. nonfortified (98.0 +/- 6.9 pg/100 microl) milk samples. Western blot for TGF-alpha in whole milk and its separated fractions revealed characteristic bands at 6.5, 12-16, 22, 26-30 and 46 kD. HMF alone and HMF with sodium taurocholate had a prominent band at 18 kD and fainter bands at 6.5, 26-30, and 46 kD. While whole and aqueous milk samples with HMF also consistently showed the 18-kD band, in 8/15 fat fraction samples with HMF the 18-kD band was nondetectable and was only faintly detectable in the remaining 7/15 samples. CONCLUSIONS: It appears that HMF differentially alters the biochemical profile of human milk with regard to TGF-alpha concentration and molecular mass profile. What effect this alteration in human milk biochemistry has on neonatal gut function remains unknown. PMID- 10729717 TI - Heart rate variability in preterm brain-injured and very-low-birth-weight infants. AB - Heart rate variability (HRV) reflects the complex interplay of the sympathetic and parasympathetic innervation of the heart. Developmental maturation of the fetus and newborn results in predictable alterations in the neural cardiac control of heart rate. Furthermore, patterns of HRV are closely correlated to clinical outcome in several pathologic situations. The first aim of this study was to characterize the maturational patterns of HRV in a group of developmentally at-risk newborns (those with severe hemorrhagic or ischemic brain injury and extremely immature, low-birth-weight infants). Secondly, we sought to determine whether a correlation exists between HRV and length of hospital stay, diagnosis of cerebral palsy, and neurodevelopmental test scores at 1-year corrected age. Time domain indices of HRV were computed longitudinally from 32 to 37 weeks of corrected gestational age in 19 very low birth weight, preterm infants. Among the 19 infants studied, 7 infants had no evidence of brain injury, 7 infants had periventricular leukomalacia (PVL), 3 infants had grade III/IV intraventricular hemorrhage (IVH), and 2 infants had both IVH and PVL. Neurologic injuries were documented using ultrasound and neurodevelopmental progress was followed through 1 year of corrected gestational age. A multivariate repeated measures analysis was performed to determine the relationship between the type of perinatal brain injury and neurodevelopmental status at 1 year of corrected gestational age. The type of perinatal brain injury was highly correlated to specific patterns of HRV with multivariate regression models producing adjusted r(2) values ranging from 0.63 to 0.99. The type of perinatal brain injury was highly correlated to the developmental outcome measures (p < 0.0000) with PVL patients having the lowest neurodevelopmental scores, IVH patients having the highest scores, and noninjured infants having midrange, grossly normal values. Using ANOVA, HRV was correlated to outcome, but individual comparisons revealed statistical significance only for the noninjured group (p < 0.04). However, multivariate models, which characterized outcome within each brain injury group, were highly significant (adjusted r (2) ranged from 0.23 to 0.89). In summary, the type of perinatal brain injury determined the pattern of HRV and HRV was highly correlated to length of hospital stay and neurodevelopmental function assessed at 1 year of corrected gestational age. PMID- 10729719 TI - Nonnutritive sucking during heelstick procedures decreases behavioral distress in the newborn infant. AB - We investigated if nonnutritive sucking (NNS) during heelstick procedures alleviates behavioral distress in neonates. In our NICU, 26 neonates without severe complications (mean Minde score 0.8, range 0-3), undergoing heelstick procedures at least twice a day, in the first 2 weeks of life, were enrolled in the trial (mean gestational age 33.9 weeks, range 26-39 weeks, mean birth weight 1, 988.5 g, range 1,200-4,010 g, mean Apgar score at the first minute 6. 7, range 4-10, at the fifth minute 8.5, range 6-10). Two heelpricks were performed in each neonate with NNS randomly assigned. Behavioral states, transcutaneous oxygen tension (TcPO(2)), heart rate, and respiratory rate were monitored before, during and after the heelstick procedures. Heelstick procedures lasted for a mean of 109 s (range 50-230 s) with NNS, and a mean of 128.8 s (range 20-420 s) without NNS. Compared with baseline, heart rate and behavioral distress increased and respiratory rate decreased during heelstick and after heelstick. Oxygen tension did not change. Nonnutritive sucking had no effect on respiratory rate or transcutaneous oxygen tension, but reduced the time of crying and the heart rate increase during the procedure. In conclusion, NNS can be recommended to reduce distress in newborns undergoing invasive routine procedures. Further studies are needed to evaluate the effects of NNS on respiratory rate and blood gas levels. PMID- 10729720 TI - Decreased mRNA expression of G-CSF receptor in cord blood neutrophils of term newborns: regulation of expression by G-CSF and TNF-alpha. AB - Granulocyte colony-stimulating factor (G-CSF) promotes neutrophil production and enhances neutrophil function. The effects of G-CSF are mediated by binding to its receptor. Since neutrophils are an essential part of the neonatal host defense system, we studied G-CSF receptor expression in neonatal neutrophils. We determined protein and mRNA expression of G-CSF receptor in freshly isolated neutrophils from cord blood of healthy term newborns (n = 16) and of adults (n = 6) as well as the in vitro effect of supplemented recombinant human G-CSF (rhG CSF) and tumor necrosis factor-alpha (TNF-alpha) on G-CSF receptor expression of neutrophils. Expression of G-CSF receptor on the surface of neutrophils of cord blood was significantly lower compared to adults (61 +/- 6 vs. 89 +/- 2%). G-CSF receptor mRNA transcripts of neutrophils from newborns compared to adults was lower, too (77 +/- 14 vs. 152 +/- 33%). Neutrophils isolated from cord blood showed a decrease of G-CSF receptor expression within 24 h of culture. Moreover, we were able to show that supplemented rhG-CSF is necessary for maintenance of G CSF receptor expression. TNF-alpha, however, down-regulated G-CSF receptor expression. We conclude that low protein and mRNA expression of G-CSF receptor in neutrophils of neonates compared to adults may adversely affect granulopoiesis and neutrophil functions by decreased responsiveness to G-CSF. Furthermore, G-CSF receptor expression on neutrophils was modified not only by G-CSF itself, but also by TNF-alpha. PMID- 10729721 TI - The effect of cold stimulation to the face on the metabolic rate of the febrile piglet. AB - Sudden infant death syndrome has been associated with winter climates, infection, and overwrapping of babies. The hypothesis has been tested in this laboratory that two different causes of increased metabolic rate, high core temperature (via the van't Hoff or 'Q10' effect) and face-cooling, might synergistically induce hyperthermia. This proved not to be the case. We now report on a 'febrile' state adding Salmonella abortus equi pyrogens. The combination of face-cooling and pyrogen administration to 14 already hot piglets produced an increase in oxygen consumption of 47% in 6 of the animals (19% overall). Face-cooling alone caused a 6.5% fall in oxygen consumption, and injection of pyrogens alone had no effect on oxygen consumption. We conclude that there may be a danger of life-threatening hyperthermia in the combination of a cold face and febrile state. PMID- 10729722 TI - Dihydrotestosterone prevents glucocorticoid-negative effects on fetal rat metatarsal bone in vitro. AB - The effects of dihydrotestosterone (DHT) on glucocorticoid-pretreated fetal rat long bone were studied in an in vitro culture system. First, dose-response curves of corticosterone, hydrocortisone, and dexamethasone were studied at several concentrations. Then, hydrocortisone (H) at 10(-5) M was selected for the second part of the study, as it slackened rudiment mineralization (104 +/- 16% of the initial dark zone vs. 141 +/- 9% in control bones), as well as its lengthening (140 +/- 4% of the harvesting day length vs. 160 +/- 1% in control bones), by both inhibition of cell proliferation and stimulation of resorption. On the contrary, in H-pretreated metatarsal bones, DHT (10(-7) M) partly limited slackening of mineralization (124 +/- 5%) and lengthening (153 +/- 2%). Moreover, a control-like cell proliferation was re-established and resorption holes were filled in. Thus, in this study, DHT partly limited hydrocortisone-induced impairment of fetal rat metatarsal bone development. PMID- 10729723 TI - Prophylactic treatment of endotoxic shock with monophosphoryl lipid A in newborn rats. AB - Mortality due to gram-negative septic shock remains high despite advances in medical care. Induction of endotoxin tolerance might be a new treatment strategy to prevent septic shock in the newborn. The present study was performed to show that an injection in pregnant rats of monophosphoryl lipid A (MPL), a nontoxic derivative of lipopolysaccharide (LPS), induces tolerance to Salmonella enteritidis LPS and tumor necrosis factor alpha (TNF-alpha) in their offspring. MPL at a dose of 2 mg/kg was injected into pregnant rats on the 19th day of gestation. Their 0-day-old offspring later received an intraperitoneal injection of S. enteritidis LPS or TNF-alpha. Newborn rats of MPL-treated dams exhibited a higher survival rate, absence of lactacidemia and lower plasma TNF-alpha concentration in response to S. enteritidis LPS when compared to the newborn rats of saline-treated dams. Newborn rats of MPL-treated dams were more tolerant to TNF-alpha than those of saline-treated dams. MPL injection into pregnant rats did not increase plasma endotoxin concentration in the fetuses, suggesting no placental passage took place, but it did increase plasma TNF-alpha concentration. We concluded that an injection of MPL into pregnant rats induced tolerance to LPS in their offspring, which might be due to TNF-alpha-induced TNF-alpha tolerance. PMID- 10729724 TI - Timing of surfactant administration determines its physiologic response in a rabbit model of airway lavage. AB - Surfactant response depends on timing of surfactant administration in infants with respiratory distress syndrome (RDS). We performed lung function studies in an animal model to describe the underlying differences in response related to gas exchange, lung volume and lung mechanics comparing early and late surfactant administration protocols. Young New Zealand rabbits were made surfactant deficient by repeated airway lavage with warm saline until the a/A O(2) ratio decreased to ==65 years) women, 66 tissue specimens obtained from patients with stage Ib-IIb cervical carcinoma among two groups of women, 64 years of age or younger and 65 years of age and older, were analyzed for human papillomavirus (HPV) typing via polymerase chain reaction, the expression of p53 via immunohistochemical study, and clinical behavior. The prevalence of HPV DNA was higher in the younger group than in the older group (84.0 vs. 50.0%) as was the detection rate of HPV 16 (44.0 vs. 6.3%). In contrast, HPV 18, 33, 52, 58, and X were frequently detected in older patients. The positive rate of p53 overexpression in the older group was similar to that in the younger group (46.7 vs. 48.8%). There was no significant difference in the incidence of lymph node metastasis, histology, and the distribution of clinical stage between the two groups. Thus, in elderly Japanese women with stage Ib-IIb, the association of HPV of types other than HPV 16 is suggested to influence the progression of cervical cancer. PMID- 10729762 TI - Development and therapeutic effect of adoptively transferred T cells primed by tumor lysate-pulsed autologous dendritic cells in a patient with metastatic endometrial cancer. AB - We describe a 65-year-old woman with a large surgically unresectable and chemoresistant liver metastasis of endometrial carcinoma who was treated by infusion with peripheral blood T cells stimulated with tumor lysate-pulsed autologous dendritic cells (DC). Extensive in vitro characterization of the DC activated T cells included phenotypic analysis, cytotoxicity, and intracellular cytokine secretion. High cytotoxicity was observed against autologous tumor cells, but not against NK-sensitive K562 cells, autologous Con-A lymphoblasts, or autologous Epstein-Barr virus-transformed lymphoblastoid cells. Blocking studies demonstrated that lytic activity was HLA class I restricted. Two-color flow cytometric analysis revealed that a significant proportion of CD8+ T cells was also CD56+, and analysis of intracellular IFN-gamma and IL-4 expression suggested a type 1 cytokine bias. The patient was treated by three infusions of tumor specific T cells at 3- to 4-week intervals, and in vivo distribution of the T cells was followed by (111)In oxine labeling and serial gamma camera imaging. Tumor localization and accumulation of labeled lymphocytes was consistently detected at serial time points following each injection. However, deep infiltration of the large tumor mass by activated T cells was minimal, as evaluated in 3 dimensions by single photon emission computerized tomography (SPECT) imaging. Transient serum increases of the tumor marker lactate dehydrogenase (LDH), were detectable after each injection. Similar posttreatment elevations were seen for serum uric acid and potassium. Clinically, stabilization of the large liver metastasis was obtained during treatment. Collectively, these results indicate that tumor-specific CD8+ cytotoxic T-cell responses can be generated in patients with endometrial cancer, and suggest that T-cell immunotherapy may be of therapeutic value in patients harboring metastatic disease. PMID- 10729763 TI - Alteration of telomerase activity in ovarian cancer after chemotherapy. AB - To elucidate the relationship between telomerase activity and chemosensitivity in epithelial ovarian cancer, telomerase activity and telomere length (TRF) were examined before and after chemotherapy. Of 21 patients, 9 patients responded to chemotherapy and 12 did not. The positivity of telomerase activity did not significantly differ between responders and nonresponders. There were no differences in the mean length and the distribution of TRF between the two groups. Those distributions became narrow after chemotherapy in both groups. Seven nonresponders (58.3%) exhibited an increase in telomerase activity after chemotherapy but none of the responders showed an increase in activity. Telomerase activity may relate to chemosensitivity in epithelial ovarian cancer. PMID- 10729764 TI - Small bowel herniation through subumbilical port site following laparoscopic surgery at the time of reversal of anesthesia. AB - Incisional hernias through laparoscopic trocar sites are unusual complications of laparoscopy. Two cases of small bowel herniation at subumbilical port site occurred at the time of withdrawal of the trocar sheath at the end of the laparoscopic procedure are reported. The herniations were precipitated by the coughing movements of the patients as a result of too early reversal of the general anesthesia. Awareness of the complication, precaution at time of sheath withdrawal and a well-timed reversal of the general anesthesia are important in avoiding such a complication. PMID- 10729765 TI - Ovarian dysgerminoma with massive metastases to para-aortic lymph nodes. AB - We report on a 15-year-old female with left ovarian dysgerminoma accompanied by massive swelling of the para-aortic lymph nodes which was clearly demonstrated by preoperative magnetic resonance imaging (MRI). Metastasis to the para-aortic lymph nodes from dysgerminoma was confirmed by biopsies obtained during surgery. No study has previously reported dysgerminoma with massive para-aortic lymph node metastases clearly demonstrated by MRI. These preoperative MRI findings are presented here. The patient received six cycles of cisplatin-based combination chemotherapy with the BEP regimen (bleomycin, etoposide and cisplatin) after conservative surgery, and no residual para-aortic lymph nodes were detected by MRI or CT after the chemotherapy. PMID- 10729766 TI - Excellent results of postoperative radiotherapy for endometrial stromal sarcoma of low-grade malignancy. AB - Endometrial stromal sarcoma of low-grade malignancy (ESSL) is a rare neoplasm, and neither preoperative diagnostic procedures nor standard therapy have yet been established. We treated 3 cases of ESSL in the past 27 years, and we report here one of these cases that was classified as stage III (according to the FIGO classification of endometrial carcinoma). Postoperative radiotherapy was used to treat a residual tumor, and the patient showed a complete response. PMID- 10729767 TI - Establishment of high and low metastasis cell lines derived from a human tongue squamous cell carcinoma. AB - Malignant tumors are composed of cells with different phenotypic properties and only certain cell subpopulations present metastatic potential. The establishment of cell lines with high or low metastatic potential is necessary to investigate the molecular mechanisms of the metastatic process. However, human oral squamous cell carcinoma (SCC) cell lines that are suitable for the above investigation are scarce. High and low metastatic cells were obtained from a primary lesion of a patient with tongue carcinoma who had not received any therapy. Two distinct cell lines were selected, UM1 with a scattered growth pattern and loose cell-cell adhesion, and UM2 with a colony-formed growth pattern and firm cell-cell adhesion. The expression of E-cadherin in UM1 was clearly lower than that in UM2. UM1 exhibited a higher motility, invasive and metastatic activity than UM2 in vivo and in vitro. A low invasive and a metastatic oral SCC cell line, useful to investigate invasion and metastasis mechanisms, have been established. PMID- 10729768 TI - Effects of all-trans-retinoic acid incorporated into low-density lipoprotein on invasive properties of multidrug-resistant MCF-7 spheroids. AB - Cultured cells grown as spheroids provide an in vitro model that is closer to an in vivo tumour than conventional monolayer techniques. Previous work from our laboratory has demonstrated that spheroids formed from multidrug-resistant MCF-7 cells exhibit invasive characteristics which were not present in their sensitive counterparts. The treatment of these spheroids by all-trans-retinoic acid (ATRA), a potent inducer of in vitro and in vivo differentiation, decreases their proteolytic activity and ability to invade Matrigel-coated filters. The efficiency of ATRA is enhanced by its incorporation into low-density lipoprotein (LDL) (LDL-ATRA). Indeed, invasion through a reconstituted basement membrane was reduced by 73% with 10(-6) M ATRA and 3 x 10(-8) M LDL-ATRA. Furthermore, inhibition of invasion was correlated with a decrease in several factors: (1) secreted matrix metalloproteinase-9 and enzymes degrading type IV collagen and Matrigel films, and (2) tissue plasminogen activator. The results observed were found with a concentration of LDL-ATRA 30 times lower than that of ATRA. This could be due to the protective effect of LDL and to a better targeting of cancer cells through their LDL receptors. LDL-ATRA may therefore represent a new and potent inhibitor of invasion that could be developed for clinical trials. PMID- 10729769 TI - In vitro invasiveness of human breast cancer cells is promoted by low density lipoprotein receptor-related protein. AB - Low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor (LRP) is a surface membrane endocytic receptor, one of whose many functions is the regulation of plasminogen activator-mediated cell migration. LRP is known to have a role in migration and invasion, but its direct involvement has been demonstrated only in non-tumour cells. We investigated six breast cancer cell lines and a normal mammary epithelial cell clone for surface and total cellular LRP expression, and confirmed that its presence corresponds to the ability to invade and migrate in vitro. We showed that LRP in the tumour cell lines is expressed at a wide range of levels: from approximately 300 to approximately 6,300 sites per cell. Four of the breast cancer cell lines expressed LRP at over 1,000 sites/cell and were markedly invasive in our assay, the remainder of the cell lines and the normal clone having far fewer LRP sites and lacking invasive ability. We further showed that the migratory and invasive abilities of a highly invasive breast cancer cell line are both inhibited by receptor-associated protein, a unique LRP ligand which normally has a solely intracellular distribution but which, when added to culture medium, can inhibit all other ligand interactions with this receptor. PMID- 10729770 TI - Maximum effect of urokinase plasminogen activator inhibitors in the control of invasion and metastasis of rat mammary cancer. AB - Experimentally induced pulmonary metastases of mammary cancer in the Fisher 344 rat can be suppressed by the inhibition of urokinase plasminogen activator (uPA). The inhibition of uPA with amiloride or B428 has been shown to be dose dependent. Increased dosage levels of inhibitors might be expected to enhance levels of suppression of metastases. The use of each of these inhibitors at equipotent concentrations that exceeded the doses administered in previous studies failed to eliminate pulmonary metastases. These results demonstrate that a maximum limit is attained for the inhibitory capacities on cells during in vitro invasion or in vivo metastasis. At increased levels, uPA inhibitors continue to suppress, but do not eradicate, experimental pulmonary metastases of MATB cell rat mammary cancer. PMID- 10729771 TI - c-K-ras overexpression is characteristic for metastases derived from a methylcholanthrene-induced fibrosarcoma. AB - We investigated the relationship between the activation of the c-myc and c-K-ras proto-oncogenes and the acquisition of metastatic potential in a methylcholanthrene-induced BALB/c fibrosarcoma. The murine fibrosarcoma GR9 was originally induced in BALB/c mice following exposure to the carcinogenic chemical 3-methylcholanthrene. To induce spontaneous metastasis, we used two tumor cell clones (B9 and G2) known to differ in their metastatic potential, local tumor growth, H-2 class I expression and sensitivity to natural killer (NK) cells. The metastatic nodes were obtained from the lung, liver and kidney. The results showed: (1) amplification of the c-myc proto-oncogene in original tumor clones as well as in all metastatic nodes; (2) mRNA overexpression without amplification of the K-ras proto-oncogene in the metastatic cells, regardless of their anatomical location; (3) no c-K-ras point mutations at codons 12 and 61, and (4) in general, a statistically significantly reduced in vitro sensitivity of metastatic tumor cells to NK cells as compared with the tumor clones used to induce them (p<0.05). These results therefore suggest that overexpressed c-K-ras mRNA is important during tumor progression, perhaps rendering metastatic tumor cells more resistant to lysis by NK cells. PMID- 10729772 TI - Expression of adhesion factors and degrading proteins in primary and secondary glioblastomas and their precursor tumors. AB - In tumor tissue specimens of 27 primary and 17 secondary glioblastomas and the precursor lesions, the immunohistochemical expression patterns of the membrane protein CD44s, the basal lamina proteins laminin, collagen IV, and fibronectin, the lectin galectin-3 recognizing tenascin and N-CAM as well as of the matrix degrading enzymes matrix metalloproteinase MMP-2 and MMP-9, and cathepsin D were studied. Besides expression of basal lamina proteins in vessels, all glioblastomas and the precursor lesions showed strong immunoreactivity of CD44s, tenascin, galectin-3, and N-CAM which were restricted to solid tumor masses. Present in solid tumor areas, MMP-2, MMP-9 and cathepsin D were also strongly expressed by single tumors cells invading adjacent brain tissue at the infiltrative margin. Neither the expression pattern in primary and secondary glioblastomas nor in the precursor tumors revealed significant differences. There was also no intraindividual constant expression pattern during glioma progression or correlation with malignancy. Restricted expression of CD44s, galectin-3, tenascin and N-CAM in solid tumor masses seems to contribute to homotypic tumor cell adhesion while single tumor cells abolish this expression profile and acquire invasive activities by expression of cathepsin D, MMP-2 and MMP-9. PMID- 10729773 TI - Clustering of murine lung metastases reflects fractal nonuniformity in regional lung blood flow. AB - In the experimental metastasis assay certain animals, from groups of similarly treated animals, develop more lung metastases than expected from random chance alone. This clustering of metastases is characterized by a power function relationship, sigma(2) = amu(b), between the variance, sigma(2), and mean, mu, of the numbers of lung metastases per animal (a and b are constants). To determine whether this clustering could be an artifact of experimental metastasis, whether it could be influenced by different experimental conditions, and to attempt to clarify its cause, 22 published data sets from experimental metastasis utilizing 2,145 mice, as well as 8 data sets from spontaneous metastasis utilizing 1,020 mice were analyzed. In these experiments cell cloning, cell-cell fusion, treatment with a protein kinase C inhibitor, treatment with cell adhesion compounds, and transfection with either the ras oncogene, the sialidase gene, or the urokinase sense and antisense genes were used to influence metastasis. They employed 14 different cell lines and 6 different strains of inbred mice. Clustering of metastasis was evident in animals from the spontaneous metastasis assays as well as from the experimental metastasis assays. It was apparent whether mice were injected with tumor cells derived from clones or from cell lines. Clustering was demonstrated within each data set, regardless of the experimental conditions employed. A single variance to mean power function (with a = 2.2 and b = 1.51) characterized the clustering in the 30 data sets. The regional distribution of blood flow through lungs and other organs is nonuniform, exhibiting a fractal symmetry on change of scale. This symmetry implies that the variance of a region's blood flow is related to its mean by the same power function as was observed with metastasis. Indeed, measurements of blood flow from isolated canine lungs yield b = 1.56, similar to the corresponding figure from murine lung metastasis. These findings lend support to the hypothesis that the observed clustering of metastases is a consequence of fractal variations in lung blood flow. PMID- 10729774 TI - Human metastatic prostate PC3 cell lines degrade bone using matrix metalloproteinases. AB - Bone metastases are often associated with osteolysis and subsequent pathological fractures. To determine if metastatic human cancer cells can directly degrade non mineralized and mineralized bone, we used prostate PC3 adenocarcinoma cell lines, which were originally established from skeletal metastases. We show that PC3 cells and their conditioned medium degraded non-mineralized, osteoid-like radiolabelled extracellular matrices from human Saos2 and U2OS osteoblast-like cells. These cells also directly degraded mineralized bone by inducing (45)Ca release from rat fetal calvariae and forming resorption pits on bone slices, an effect increased by transforming growth factor-beta(1). A role for matrix metalloproteinases in degradation was shown by: (1) stimulation by the phorbol ester TPA of PC3-induced matrix degradation and release of matrix metalloproteinase activity; (2) abrogation of matrix degradation by 1,10 phenanthroline, a metalloproteinase inhibitor, and (3) degradation of purified type I collagen by PC3 cells and their conditioned medium. We demonstrate that human prostate cancer cells can directly degrade bone-related matrices and that matrix metalloproteinases have a role in this process. PMID- 10729775 TI - Molecular breeding of allergy vaccines and antiallergic cytokines. AB - Molecular breeding, also called DNA shuffling, is a technology that enables the generation of large libraries of novel genes and vectors, from which improved variants can be selected based on functional properties. In a common format, it involves recursive recombination and mutation, performed by random fragmentation of related DNA sequences, followed by reassembly of the fragments in a self priming polymerase chain reaction. As in natural evolution, the technique takes advantage of crossovers, deletions, insertions, inversions and point mutations of genes to generate large pools of related sequences. Molecular breeding can be used to generate improved variants of proteins used as therapeutics, such as vaccine antigens, growth factors and immunomodulatory molecules. Moreover, the technology can be applied to evolve entire viruses or vectors, including DNA vaccines. Cytokines downregulating allergic immune responses and allergens are attractive targets for evolution by molecular breeding. This review describes approaches to generate chimeric allergens with T cell epitopes from multiple allergen homologues, while reducing the recognition by preexisting IgE. In addition, the results and applications of molecular breeding in the evolution of improved antiallergic cytokines are discussed. PMID- 10729776 TI - Differential regulation of activation-induced cell death in individual human T cell clones. AB - BACKGROUND: Restimulation of T lymphocytes via the TCR/CD3 complex can result in CD95/CD95L-dependent activation-induced cell death (AICD). Although the correlation of AICD sensitivity to the T helper 1 phenotype was confirmed in different studies, the underlying mechanism is still debated. Thus, it has been suggested that in Th2 cells, AICD resistance is controlled by a TCR-induced upregulation of the CD95-associated inhibitory phosphatase, FAP-1. We and others demonstrated that AICD resistance is associated with a reduced surface expression of CD95L upon restimulation. METHODS: Utilizing RT-PCR, Western blotting and flow cytometry, we analyzed time-dependent changes in levels of CD95L mRNA, cytosolic protein and surface expression in five long-term human T cell clones and polarized helper populations. RESULTS: We confirm that the inducible CD95L surface expression is lower or absent in all tested AICD-resistant clones as compared to sensitive cells. It is of interest that striking differences with respect to the activation-dependent inducibility of CD95L mRNA expression in individual resistant clones were observed. In addition, alterations in the expression of the inhibitory phosphatase FAP-1 or TCR-dependent changes in CD95 sensitivity in AICD-resistant clones could be ruled out as a mechanism for AICD resistance of human T cell clones. CONCLUSIONS: (1) The data presented strongly support the previous notion that AICD resistance of human T cell clones is mainly regulated by a differential expression of CD95L. (2) Differential expression of CD95L on individual resistant clones results from a lack of mRNA induction in one set and from a markedly decreased surface expression of translated protein in another set of clones. PMID- 10729777 TI - Cleavage of human IgE mediated by Schistosoma mansoni. AB - BACKGROUND: IgE-mediated mechanisms are important in protection against helminth parasites. However, schistosomes are long-lived in mammalian hosts, presumably as a result of immune evasion strategies. We sought evidence for one such strategy, namely specific cleavage of host IgE. METHODS: Human IgE, IgA and IgG were incubated with extracts from cercarial and schistosomular stages of Schistosoma mansoni or with schistosomular culture supernatants. The resulting products were analysed by Western blotting with Ig-specific antibodies. Numerous protease inhibitors were assessed for ability to inhibit the observed cleavage of IgE by the extracts. Partial purification of the IgE-proteolytic activity from cercarial extract was achieved by gel filtration. To test IgE function, we compared the abilities of untreated and schistosomular-treated IgE to mediate rosette formation through interaction with Fcepsilon receptors. RESULTS: Cercarial and schistosomular extracts were found to cleave human, mouse and rat IgE but not human IgA1, IgA2 or IgG1. Schistosomular culture supernatants displayed similar proteolytic activity towards IgE. Immunoblotting suggested that cleavage occurred close to the Cepsilon2/Cepsilon3 domain interface of the IgE heavy chain. PMSF and elastatinal inhibited cleavage, suggesting that the protease involved is an elastase-like serine protease, particularly since porcine pancreatic elastase also cleaved IgE to give similar-sized products. Further, the chloromethyl ketone derivatized peptide MeO-Suc-Ala-Ala-Pro-Leu- CMK, known to specifically inhibit the schistosome elastase, prevented IgE cleavage. Cleavage of human IgE rendered the antibody molecule unable to interact with U937 cells expressing FcepsilonRII. CONCLUSIONS: An elastase-like protease in S. mansoni is able to render IgE non functional. PMID- 10729778 TI - Purification of group 2 Dermatophagoides pteronyssinus allergen and prevalence of its specific IgE in asthmatics. AB - Group 2 allergens are a major cause of sensitization in patients allergic to house dust mites. This study was performed to determine the prevalence of hypersensitivity to group 2 allergens (Der p 2) of Dermatophagoides pteronyssinus (Dp) in asthmatic patients in Taiwan. To facilitate the analysis of Der p 2 specific IgE, we raised a panel of monoclonal antibodies (MoAbs) to Der p 2 antigens. Purified Der p 2 was obtained after MoAb affinity column purification. There were 82 asthmatic patients (41 adults and 41 children) with hypersensitivity to Dp who were analyzed for hypersensitivity to Der p 2. All of them were both skin test- and serology test-reactive to Dp. Using purified Der p 2, 87.8% (72/82) of patients had a skin-test-positive reaction. Six adults (6/41) and 4 children (4/41) had negative skin tests for Der p 2. Ten families (both parents and children were asthmatics) of the 82 patients were selected for Der p 2 skin testing and Der p 2-specific IgE determination using immunoblot analysis. Results showed that 90% (18/20) of patients' skin reactions to Der p 2 and serum contained specific IgE to Der p 2. Because 87.8% (85.4% of adults and 90.2% of children) of the asthmatic patients with Dp hypersensitivity were allergic to Der p 2, its role in the pathogenesis of asthma in Taiwan appears to be important. Purified Der p 2 allergens can be further used for allergen skin testing and immunotherapy. PMID- 10729779 TI - Identification of Hev b1 in natural latex mattresses. AB - BACKGROUND: Type 1 hypersensitivity to natural rubber latex proteins is a well recognized health problem. Recent data have shown that allergens can be extracted from natural latex mattresses. As Hev b 1 (rubber elongation factor) and Hev b 6.02 (hevein) were described as major allergens, the present work was carried out to evaluate their presence in latex mattresses as well as in latex gloves. METHODS: Extracted proteins from latex mattresses and gloves were separated by SDS-PAGE or two-dimensional gel electrophoresis, transferred onto nitrocellulose and detected with monoclonal antibodies specific for Hev b 1 and Hev b 6.02. RESULTS: The results showed that various forms of Hev b 1, as well as degradation products of Hev b 1 were detected in latex mattresses and gloves, whereas Hev b 6.02 was not detected either in mattresses or in gloves. In a standardized latex extract, Hev b 1 and Hev b 6.01 (prohevein) were identified by the monoclonal antibodies. CONCLUSION: The fact that only Hev b 1 was detected by immunoblot in latex articles indicates that Hev b 1 may be the last protein to be washed out of latex products and that the Hev b 1 content may be used as a criterion for the estimation of the allergenicity of the latex products. PMID- 10729780 TI - Superantigen-induced T cell death by apoptosis: analysis on a single cell level and effect of IFN-gamma and IL-4 treatment. AB - BACKGROUND: A role of bacterial superantigens in several chronic inflammatory diseases has previously been proposed. Many of these diseases are associated with an imbalance of the T helper cell subsets and their cytokine production. METHODS: Peripheral blood mononuclear cells from healthy donors were incubated with various concentrations of staphylococcal enterotoxin B (SEB) with or without IL-4 or IFN-gamma. After different time points cell activation, proliferation, Fas expression, cytokine release and cell death via apoptosis were detected. RESULTS: SEB treatment resulted in sequential T cell activation, proliferation, Fas expression, cytokine release, subsequently followed by cell death via apoptosis in a dose- and time-dependent manner. This biphasic effect occurred preferentially in SEB-responsive cells represented by the expression of Vbeta3 and Vbeta12 on T cells. A strong relationship between T cell activation and apoptosis was observed. The amplitude between these events increased with the dose of SEB. The highest rate of apoptotic T cells was observed at a dose of 1,000 ng/ml SEB. Addition of IFN-gamma to SEB-treated cells significantly reduced the rate of apoptotic cells, whereas IL-4 prevented apoptosis only in SEB untreated cells. CONCLUSION: These results support the concept that the dose of superantigen exposure determines the rate of T cell proliferation and subsequent cell death. This T cell immune response is modulated by the presence and the type of cytokines. PMID- 10729781 TI - Cutaneous CD30+ cells in children with atopic dermatitis. AB - BACKGROUND: CD30 expression can be considered a marker of Th2 cells. We investigated the presence of CD30+ cells in the lesional skin of children with atopic dermatitis (AD). We also analyzed the possible relationship between CD30+ cells and serum soluble CD 30 (sCD30) levels, and IgE, soluble interleukin-2 (IL 2) receptor (sIL-2R) or soluble CD23 (sCD23) levels in the blood, and clinical score. METHODS: Ten eczematous children (4 males, 6 females; median age: 4 years and 5 months; range: 11 months to 14 years), 9 sex- and age-matched control children and an adult control group were studied. A clinical score (SCORAD index), was given to eczematous lesions. Blood was taken for the determination of IgE, sCD30, sIL-2R and sCD23 levels. Punch biopsies of lesional skin were stained with hematoxylin and eosin or incubated with anti-CD30 monoclonal antibodies. Skin prick tests (SPTs) were also performed. RESULTS: In the biopsy specimens, CD30 expression was observed in high proportions of infiltrating cells. In children with AD, total serum IgE, sCD30, sIL-2R, sCD23 and eosinophils were significantly elevated compared to controls. CD30+ cells were not associated with serum IgE, sCD30, sIL-2R, sCD23, or SPT results, score of inflammatory cells in lesional skin or clinical score. Children with AD who had high total IgE and specific IgE antibodies did not differ from those with normal total IgE and negative specific IgE in respect of age, clinical score, number of CD30+ cells, sCD30, sIL-2R and sCD23 levels, score of inflammatory cells in skin or clinical score. CONCLUSION: Our results showed remarkable numbers of CD30+ cells in the lesional skin and high sCD30 in the serum of children with AD. CD30+ cells did not correlate with systemic markers of IgE reaction. PMID- 10729782 TI - Safety of sublingual-swallow immunotherapy in children and adults. AB - BACKGROUND: Immunotherapy is an established treatment of allergic diseases. The safety of this treatment, particularly when administered without direct medical surveillance, as in the case of the sublingual-swallow route needs to be established. The aim of this paper is to review the safety of the sublingual swallow immunotherapy as reported in eight double-blind, placebo-controlled trials carried out in France, Italy and Greece. METHODS: Six hundred and ninety subjects, 472 adults and 218 children, took part in trials of specific immunotherapy (SIT) for the treatment of rhinoconjunctivitis and/or asthma. Three hundred and forty-seven patients received SIT and 343 patients received placebo. Treatment with specific immunotherapy with allergen extracts or placebo was administered using the sublingual-swallow technique. The allergens administered were grass, ambrosia, parietaria and olive pollens, and mites. The daily dose taken during maintenance therapy ranged from 100 to 300 IR (index of reactivity) and cumulative doses ranged from 4,500 to 104,000 IR. Treatment duration ranged from 4 months to 2 years. Adverse events reported either spontaneously by the patient or on direct questioning by the investigator were analysed. RESULTS: One hundred and forty-five unusual events were reported in the subjects receiving active SIT and 79 in those receiving placebo (p < 0.001). Of these 85 were children aged 15 years or less (50 received active SIT, 35 placebo) and 139 were adults (95 received SIT, 44 placebo). Unusual events involving the buccal cavity (61 SIT, 13 placebo) and the gastro-intestinal tract (47 SIT, 15 placebo) were significantly more frequent in the SIT-treated patients (p < 0.001). Wheezing (9 SIT, 21 placebo) was more frequent in the placebo-treated patients (p < 0.05). There were no differences in the frequency of unusual events between adults and children and in the frequency of events involving other body systems. No event was reported as serious. Two events reported as laryngeal oedema were not considered to be accurate descriptions. CONCLUSIONS: No serious adverse event was reported in the studies monitored, confirming the good safety profile of the sublingual-swallow method both in children and adults with rhinitis or moderate asthma. PMID- 10729783 TI - Mediation of the immunomodulatory effect of beta-estradiol on inflammatory responses by inhibition of recruitment and activation of inflammatory cells and their gene expression of TNF-alpha and IFN-gamma. AB - BACKGROUND: Estrogen has long been reported to show immunomodulatory effects on immune responses, yet, its specific anti-inflammatory mechanism is not clear. METHODS: In this study, we analyzed the effects of beta-estradiol (E2), at its contraceptive dose, on both T cell-independent and T cell-dependent inflammations, and the associated immune mechanism, in female mice. The T cell independent inflammation was locally induced either with an intradermal injection of olive oil in the footpad, or by an intraperitoneal injection of proteose peptone (PP). The T cell-dependent inflammation was induced by an intraperitoneal injection of the purified protein derivatives (PPD). RESULTS: While E2 inhibited olive oil-induced inflammation as monitored by the decrease in footpad swelling, it did not affect the gene expression of monocyte chemoattractant protein-1 and IL-6 by cells at the inflammatory locus. E2 also inhibited PP-induced inflammation as monitored by the decrease in the number of inflammatory peritoneal exudate cells (PEC) coinciding with a marked decrease in the number of macrophages and granulocytes (Gr. 1+). While E2 did not affect the gene expression of monocyte chemoattractant protein-1 and IL-6 by PP-elicited PEC, it decreased both gene expression and production of TNF-alpha. E2 also decreased the number of cells expressing the lymphocyte function-activated protein-1 in PP elicited PEC, but not for CD62L. In purified protein derivative-induced T cell dependent inflammation, E2 decreased the total cellularity of PEC and the relative numbers of CD3+ and CD4+ T cells, and the number of cells expressing the lymphocyte activation markers CD40, CD44, CD69 and IL-2Ralpha in PEC. Furthermore, while E2 did not affect the gene expression of the early T lymphocyte activation protein-1 by PEC, it decreased the gene expression of INF gamma. CONCLUSION: Collectively, the results suggest that E2-mediated inhibition of inflammatory responses may be due to a combination of suppression of homing and activation of inflammatory cells and their production of TNF-alpha and IFN gamma. PMID- 10729784 TI - Athymic nude rats develop severe pulmonary hypertension following monocrotaline administration. AB - OBJECTIVE: To investigate the significance of the presence or absence of the thymus in the development of pulmonary hypertension (PH) following monocrotaline (MCT) administration, the degree of MCT-induced PH (MCT-PH) in athymic nude (F344/N Jcl-rnu) rats was compared with that in their euthymic (rnu/+) littermates. METHODS: Histopathological studies of the lung in terms of interstitial edema, congestion, thickening of the alveolar wall, inflammatory cell infiltration and degeneration of arteries were performed by staining with hematoxylin-eosin (HE), elastin van Gieson and Masson's trichrome. The medial wall thickness of the small pulmonary arteries and the weight ratio of the right ventricular free wall (RV) to that of the left ventricle plus septum [RV/(LV + S) weight ratio] were used as indices of the degree of PH. Toluidine blue staining was performed to estimate the number of the mast cells in the lung interstitium. RESULTS: Interstitial edema was significantly severer in MCT- injected euthymic rats than in MCT-injected athymic nude rats (p < 0.01); in contrast, the thickening of the alveolar wall was severer in MCT-injected athymic nude rats than in MCT-injected euthymic rats (p < 0.05). The degree of MCT-PH, as determined by the medial wall thickness of the small pulmonary arteries and RV/(LV + S) weight ratio in MCT-injected athymic nude rats, was significantly severer than in MCT-injected euthymic rats (p < 0.05 and p < 0.01, respectively). The number of mast cells was significantly greater in MCT-injected athymic nude rats than in MCT-injected euthymic rats (p < 0.01). The degree of the medial wall thickness of the small pulmonary arteries was significantly correlated with RV/(LV + S) weight ratio (p < 0.05) as well as with the number of mast cells in MCT-injected rats. CONCLUSIONS: Athymic nude rats developed severer PH than euthymic rats along with a greater number of mast cells and severer histopathological changes, such as thickening of the alveolar wall. Mast cell proliferation was considered to play a pivotal role in the development of PH in MCT-PH. PMID- 10729785 TI - HIV-1 gp41 by N-domain binds the potential receptor protein P45. AB - Recent crystal structure analysis of HIV-1 gp41 revealed that two domains (N- and C-domains) on gp41 play an important role in mediating membrane fusion and HIV-1 entry. The experimental evidence that gp41 by N-domain bound the potential receptor protein P45 could help to understand the mechanism of HIV entry. A recombinant soluble gp41 (rsgp41: Env aa539-684) could bind to P45 in the affinity capillary electrophoresis analysis and the surface plasmon resonance assay. In a blockade assay, peptide P1 (Env aa583-599) could inhibit interaction between rsgp41 and P45, while a control peptide could not. Direct binding of rsgp41, rgp41DP (aa567-648), P1 peptide and (P1)(2) peptide [(aa586-596)(2)] to P45 was examined in an ELISA assay. Rsgp41 bound the potential receptor protein P45 strongly, while rgp41DP and P1 as well as (P1)(2) could all weakly bind to P45, indicating that gp41 by N-domain weakly binds P45 and the region RILAVERYLKD located in the N-domain is defined as the binding site for P45 binding. PMID- 10729786 TI - Identification of epidermal growth factor mRNA-expressing cells in the mouse anterior pituitary. AB - Epidermal growth factor (EGF) produced within the pituitary gland is associated with the growth of pituitary cells in rats. The aim of the present study was to localize EGF- and EGF receptor-expressing cells, and to clarify the involvement of EGF in DNA replication in 2-month-old male mouse pituitary cells. In situ hybridization of the pituitaries of these mice demonstrated that EGF mRNA was expressed in the anterior and intermediate lobes. Within the anterior pituitary, EGF mRNA-expressing cells were medium-sized and round, and made up 40% of the total number of secretory cells. EGF receptor mRNA was only detected in anterior pituitary cells. Forty-seven percent anterior pituitary cells expressed EGF receptor mRNA. An immunocytochemical study showed that most somatotropes and some mammotropes expressed EGF mRNA. When anterior pituitary cells were enzymatically dissociated and cultured in serum-free medium, RT-PCR demonstrated both EGF mRNA and EGF receptor mRNA expression. Treatment with EGF (1 and 10 ng/ml) for 5 days stimulated DNA replication in mammotropes and corticotropes. These results indicate that the DNA replication in mammotropes and corticotropes is regulated by the paracrine and/or autocrine activity of EGF produced at least in part by these cell types themselves. PMID- 10729787 TI - Melatonin regulates the mRNA expression of the mt(1) melatonin receptor in the rat Pars tuberalis. AB - The pars tuberalis (PT) of the pituitary is a major neuroendocrine target site for melatonin as it contains a large number of high-affinity melatonin receptors. We have previously shown that melatonin autoregulates the density of its own receptors in the PT. However, whether melatonin regulation includes mRNA expression in vivo is unclear. In the present study we have used quantitative in situ hybridization to (1) follow the daily profile of mt(1) mRNA expression in the rat PT and (2) investigate whether mt(1) mRNA expression could be regulated in vivo by melatonin. We found clear diurnal variations of mt(1) mRNA expression that persist in constant darkness. We also showed, on pinealectomized animals, that the rhythmic pineal melatonin secretion is necessary for the expression of these daily variations. In a second step, we studied the effect of an acute suppression of endogenous melatonin synthesis on mt(1) melatonin receptors by applying a 1-hour light pulse during the night. We found that light induced a dramatic increase in mt(1) mRNA which was totally prevented by a melatonin injection showing that the acute effect of melatonin on the receptor mRNA is strongly inhibitory. A light pulse applied to animals with a chronic absence of melatonin was ineffective showing that light only affects melatonin receptors via the light-induced plasma melatonin suppression. Altogether our results show that melatonin regulates mt(1) melatonin receptor mRNA expression. However, this regulation seems to be complex: acute changes in plasma melatonin concentration regulate negatively the gene transcription, even if the daily endogenous nocturnal melatonin peak seems a prerequisite for variations in its receptor expression. PMID- 10729788 TI - Dynorphin stimulates corticotropin release from mouse anterior pituitary AtT-20 cells through nonopioid mechanisms. AB - Dynorphin (Dyn) peptides were previously shown to increase plasma corticotropin (ACTH) in the ovine fetus, but the site of its action remains unclear. In the present study, Dyn A(1-17) was found to stimulate ACTH release from mouse anterior pituitary tumor AtT-20 cells in a dose-dependent manner. Naloxone did not block the effect of Dyn A(1-17) and the selective kappa-opioid receptor agonist U50488H did not stimulate ACTH release. Dyn A(2-17), a degradative peptide fragment that does not bind to opioid receptors, also stimulated ACTH release from AtT-20 cells. Although the nonopioid effects of Dyn have previously been attributed to N-methyl-D-aspartate (NMDA) receptors, the ACTH-releasing effects of Dyn A(1-17) in AtT-20 cells were not affected by co-administration of NMDA receptor antagonist LY235959. The ACTH response to Dyn A(1-17) could not be blocked by alpha-helical CRH (CRH antagonist) and was additive with a maximal stimulatory dose of CRH, suggesting different mechanisms of action. These results show that the release of ACTH by Dyn A(1-17) in AtT-20 cells is not mediated by kappa-opioid receptors or by the NMDA receptor. PMID- 10729789 TI - Neuroendocrine secretory protein 55 (NESP55): alternative splicing onto transcripts of the GNAS gene and posttranslational processing of a maternally expressed protein. AB - Recent studies established a novel genomically imprinted gene located 45 kb upstream of the human GNAS1 locus. This locus encoded for the Neuroendocrine Secretory Protein with an apparent molecular weight of 55,000 (NESP55), which is transcribed exclusively from the maternal allele. We sequenced rat and human NESP55 and investigated tissue-specific splicing of its mRNA and posttranslational modifications of the protein in various tissues. Alternative mRNA splicing of NESP55 was analyzed by sequencing of cDNA clones, RT-PCR and Northern blotting. Two main splice variants, which were generated in a tissue specific manner, were identified: The open reading frame encoding NESP55 was spliced onto exons 2-13 of Gsalpha in the adrenal medulla, pituitary and the brain. In addition, in the pituitary a second shorter, prominent mRNA transcript was generated by splicing of NESP55 onto exons 2, 3 and N1 of Gsalpha. Several of the cDNA clones isolated contained inverted repeats of 50-150 bp at their 5' or 3' termini, which might form hairpin stems and thus alter mRNA stability. The NESP55 open reading frame encoded a hydrophilic protein of 28,018 Da (human) and 29,218 Da (rat), respectively, which resembled the class of acidic, neuroendocrine secretory proteins collectively called chromogranins. NESP55 is highly conserved among mammalian species. It is posttranslationally acidified by the addition of keratan sulfate glycosaminoglycan chains and differentially processed by endopeptidases in various endocrine and neuronal tissues. PMID- 10729790 TI - The long form of the leptin receptor (OB-Rb) is widely expressed in the human brain. AB - Leptin exerts important effects on the regulation of food intake and energy expenditure by acting in the brain. Leptin action is mediated by the interaction with a receptor that is alternatively spliced, resulting in at least five different isoforms. The long form (OB-Rb) has a long intracellular domain that is essential for intracellular signal transduction. The specific aim of this study was to further investigate the role that the brain may play in the pathogenesis of obesity in humans. We studied the expression of OB-R mRNA (both short or common and long isoforms) in the brains of obese, lean and diabetic subjects, by in situ hybridization, semiquantitative RT-PCR and Northern blots analysis. We used two alternative probes: one that recognizes all known splice variants (OB Ra) and a second that recognizes only the long form, OB-Rb. Several brain regions, including hypothalamus, cerebellum, neocortex, entorrhinal cortex, amygdala, and rostral medulla, were evaluated. In situ hybridization studies revealed that both OB-Ra and OB-Rb mRNAs are widely distributed in the human brain. The specific hybridization signal with both probes was detected exclusively in the cytoplasm of the cell body, dendrites and proximal axons of neurons. Hypothalamic nuclei, Purkinje cells and dentate nuclei of the cerebellum, inferior olivary and cranial nerves nuclei in the medulla, amygdala and neurons from both neocortex and entorrhinal cortex demonstrated positive signals. The hybridization signal obtained in ependyma was lower than that in neurons and no specific hybridization was detected in glial cells. No significant differences were identified among the regions or among the three groups studied. These results match those previously obtained by us [Couce et al.: Neuroendocrinology 1997;66:145] in which the distribution of the OB-R protein in the human brain was first described. RT-PCR indicated that the OB-Rb was highly expressed in the hypothalamus and cerebellum. No significant differences of OB-Ra or OB-Rb mRNA expression were identified in lean or obese individuals in these two cerebral regions. The levels of OB-Rb were significantly higher in cerebellum compared to hypothalamus in lean and obese individuals. The original hypothesis that OB-Rb is present only in the hypothalamus needs to be reconsidered. This OB Rb isoform seems to be widely expressed in the human brain with highest levels in the cerebellum. Obesity and hyperleptinemia appears not to be associated with down-regulation of the OB-Rb in the human brain. PMID- 10729791 TI - Neuroendocrine consequences of fasting in adult male macaques: effects of recombinant rhesus macaque leptin infusion. AB - Fasting inhibits the gonadotropic axis and stimulates the corticotropic and somatotropic axes. Since leptin is a product of fat cells that has been implicated in the control of both reproduction and metabolism, we hypothesized that the decrease in leptin observed during fasting was responsible for these effects on reproductive and metabolic hormones. Recombinant rhesus leptin (rrhLep) produced in our laboratory was infused (100 microgram/h) into fasted adult male rhesus macaques (6-9 kg) beginning at midnight after the first missed meal and continuing until the end of the study. Bioactive luteinizing hormone (LH), testosterone, cortisol and growth hormone (GH) were measured in plasma from samples collected at 15-min intervals for the last 15 h (42-57 h) of the fast. We analyzed pulsatile LH and GH secretion by deconvolution analysis and the orderliness of pulsatile LH and GH release by the approximate entropy (ApEn) statistic. There was no difference in LH pulse frequency between control and fasted groups, but there was a significant decrease in the mean concentration of LH released (7.6 +/- 1.4 ng/ml control vs. 2.7 +/- 0.65 ng/ml fasted) that was not relieved with rrhLep infusions (2.8 +/- 0.83 ng/ml). Model-free Cluster analysis confirmed these inferences and also indicated that the peak height was lower in the fasted (4.6 +/- 1.0 ng/ml) and the fasted + rrhLep (2.85 +/- 1.0 ng/ml) groups compared to controls (16. 3 +/- 1.4 ng/ml). Testosterone levels reflected those of LH. Fasting resulted in an increase in GH secretory pulse frequency (5.3 +/- 0. 95 pulses/15 h control vs. 12.8 +/- 1.4 pulses/15 h fasted) and this increase was not affected by rrhLep infusion (12.5 +/- 1.4 pulses/15 h). In addition, fasting also increased the ApEn (decreased the orderliness) of pulsatile GH secretion, and this characteristic was not relieved with rrhLep infusions. Cortisol levels in fasted animals were 2- to 3-fold higher than those observed in control studies, and this increase was particularly pronounced at the time when the animals expected their first meal of the day. The increase in circulating cortisol observed in fasted animals was not affected by rrhLep infusion. Glucose levels at the end of the sampling period were 80 mg/dl in controls, 48 mg/dl in fasted animals and 58 mg/dl in the fasted + rrhLep group. Circulating leptin levels averaged 1.2 +/- 0.37 ng/ml in control animals, 0.7 +/- 0.2 ng/ml in fasted animals and 10.1 +/- 5.6 ng/ml in fasted animals infused with rrhLep. These studies suggest that intravenous replacement with homologous leptin does not reverse the acute changes in GH, LH and cortisol secretion observed with fasting in the adult male macaque. PMID- 10729792 TI - Oxytocin/alpha(2)-Adrenoceptor interactions in feeding responses. AB - The modulation of alpha(2)-adrenoceptor-induced food intake by oxytocin has been evaluated in studies on food intake and by quantitative receptor autoradiography in the hypothalamus and the amygdala of the rat. The effects of lateral intracerebroventricular administration of clonidine and oxytocin were evaluated on food intake in satiated animals. Food consumption was measured at 30, 90, 240 min and 22 h (1,320 min) after injection. The coinjection of oxytocin and clonidine was found to counteract the increase in food intake produced by clonidine (p < 0.001) in satiated rats. Receptor autoradiographic experiments showed that oxytocin significantly increased the K(d) values of [(3)H]p aminoclonidine alpha(2)-agonist-binding sites in the hypothalamus. Effective oxytocin concentrations ranged between 0.3 and 1 nM (p < 0.05) with a maximal action of 250% at 1 nM. The B(max) value was significantly increased (p < 0.05) for all concentrations of oxytocin. In the amygdala, oxytocin also increased both the K(d) of [(3)H]p-aminoclonidine-binding sites by about 190% at 1 nM and the B(max) values at 1 and 3 nM (p < 0.05). Oxytocin (1 nM) also significantly and substantially (p < 0.01) increased the K(d) and B(max) values of the [(3)H]UK 14.304 alpha(2)-agonist-binding sites in the hypothalamus and amygdala in agreement with the results obtained with the other agonist of the alpha(2) adrenoceptor [(3)H]p-aminoclonidine. This effect was partially blocked by the presence of the specific oxytocin receptor antagonist, CAP. These findings suggest the existence of an antagonistic oxytocin/alpha(2)-receptor interaction in the hypothalamus and amygdala that may be of relevance for the demonstrated modulation of alpha(2)-adrenoceptor-induced feeding responses by oxytocin. PMID- 10729793 TI - Effectiveness and utility of chemical labyrinthectomy with streptomycin using osmotic pump. AB - To investigate the utility of osmotic pumps, labyrinthectomy was performed surgically (surgical group) or chemically with 30% streptomycin sulfate (SM) using osmotic pumps (SM group) in guinea pigs. After treatment, no statistical difference was observed in the frequency of spontaneous nystagmus and the degree of yaw head tilt between the groups. These data indicate the reliability and efficacy of osmotic pumps, and it might be possible to make guinea pig models using osmotic pumps to predict various grades of damage in the vestibular periphery of humans. PMID- 10729794 TI - Relationship between neurological asymptomatic vertigo and the vertebrobasilar system as revealed by magnetic resonance angiography. AB - Many patients suffering from vertigo have no neurological symptoms except for a positional nystagmus. Vertigo without any neurological findings has not been thought to be a vertebrobasilar (VB) attack. The purpose of this study is to clarify the relationship between vertigo without any neurological symptoms and the VB system using magnetic resonance angiography (MRA). MRAs of the VB system were examined in 31 patients with positional nystagmus of unknown origin (PNU) and in 14 patients with benign paroxysmal positional vertigo (BPPV) as control. MRA was performed with a 1.5-tesla system using the two-dimensional time-of flight technique. Abnormalities such as elongation, bending, narrowing or obstruction of the artery were detected in 27 patients of the PNU group and 8 patients of the BPPV group. A significant difference in the abnormalities was noticed between the two groups (p < 0.05; chi(2) test). This result is almost similar to those of previous studies using conventional vertebral angiography and digital subtraction angiography. Thus, MRA is useful in examination of the VB system. Moreover, our study suggested that the positional nystagmus might result from VB ischemia, even if there were no other neurological signs. PMID- 10729795 TI - Epithelial changes in seasonal allergic rhinitis throughout the year: evidence of coexistent air pollution and local secretory IgA deficiency? AB - To determine possible epithelial changes in seasonal allergic rhinitis, we examined epithelial cells in cytology swab preparations of 38 non-smoking patients, exclusively sensitized to grass pollen, throughout the year, and surgical material of 8 patients. Cytologically, we found a marked goblet cell hyperplasia during the period of grass pollen exposure, while meta- and dysplasia were found throughout the year. Immunohistochemically, a lack of secretroy IgA in the altered epithelium was detected. These results were not obtained in the control group of 60 healthy non-smokers. Since we have recently found the same epithelial changes of meta- and dysplasia in smokers, these findings may point to non-specific toxic damage of the nasal epithelium possibly caused by other air pollutants. The altered epithelium may lead to an impairment of the local secretory IgA defence system and thereby to an increased allergen uptake. PMID- 10729796 TI - Side effects after surgical treatment of snoring. AB - In a questionnaire study detailed side effects after snoring surgery were examined in 415 individuals 2-8 years after surgery. Three years later a new questionnaire was answered by those with side effects affecting taste, smell and voice (n = 74). At the first follow-up 255 had side effects of globus, regurgitation, taste, smell and voice. The globus was the most common (40%). In all spheres except the globus, a significant improvement was seen 3 years later. However, pharyngeal dryness and phlegm had a reported frequency of nearly 60%. No significant differences were seen between the uvulopalatopharyngoplasty and laser uvulopalatoplasty methods. Taste disturbances might be due to surgical damage to the nerves or oral dryness. The olfactory impairment present in 7 patients still needs to be explained. PMID- 10729797 TI - The effects of chronic electrical stimulation on laryngeal muscle physiology and histochemistry. AB - The present study examined the effects of functional neuromuscular stimulation (FNS) on posterior cricoarytenoid (PCA) muscle physiology and histochemistry. In 4 canines, 10 cm of the recurrent laryngeal nerve was resected. A patch electrode array was implanted for PCA stimulation. FNS was applied to 2 canines for a period of 4 weeks with 2 additional animals serving as nonstimulated controls. Results indicated that FNS increased PCA muscle contractility over the period of intervention but had no effect on contraction speed. FNS also protected the muscle from atrophy by preventing muscle weight loss and type 2 fiber deterioration. Finally, it rescued muscle fibers from ensuing fibrosis. PMID- 10729798 TI - The effects of chronic electrical stimulation on laryngeal muscle reinnervation. AB - The present study examined the effects of functional neuromuscular stimulation (FNS) on reinnervation of the posterior cricoarytenoid (PCA) muscle. In 4 canines, the recurrent laryngeal nerve (RLN) was sectioned and anastomosed and a patch electrode array implanted for stimulation and recording at multiple PCA sites. Following implantation, FNS was applied to 2 canines for a period of 6 weeks. Two additional animals served as nonstimulated controls. In each animal, histomorphometric analysis of the RLN was used to assess the quality of nerve regeneration and the potential for muscle reconnection. The magnitude of reinnervation was monitored by electromyographic (EMG) potentials evoked by RLN stimulation. The appropriateness of reconnection was determined by the pattern of spontaneous EMG activity and recovery of vocal fold abduction. Results of this preliminary study indicated that FNS caused an overall repression of reinnervation. However, the repression preferentially inhibited reconnection by foreign nerve fibers, promoting selective reinnervation and preventing synkinesis. PMID- 10729799 TI - Motor potentials of lower-lip mimetic muscles and distal arm muscles to cortical transcranial magnetic stimulation: the possibility of one-dimensional separation of two cortical representation areas. AB - The cortical representation of the abductor pollicis brevis muscle (APB) was investigated by transcranial magnetic stimulation (TMS) along the interaural line on the head surface in 17 healthy subjects. Comparing amplitudes of motor evoked potentials (MEPs) with those obtained by cortical TMS of lower-lip muscles, we found a considerable overlap between the areas from which reproducible MEPs of APB and lower-lip muscles could be elicited. However, there was a statistically significant separation of the two areas, the APB area being placed more medially. With regard to short examination procedures it can be concluded that a separation between distal arm and facial muscles is possible not only by two-dimensional cortical mapping procedures, but also by one-dimensional cortical TMS. PMID- 10729800 TI - Hereditary sensorineural hearing loss of unknown cause involving mitochondrial DNA 1555 mutation. AB - We report on a family with maternally inherited sensorineural hearing loss, in which no history of aminoglycoside injection and no other specific etiology could be identified in any member. A 1555 A-to-G mutation of mitochondrial DNA was found in all members demonstrating hearing loss. The hearing in the propositus and his sister was severely impaired at a younger age than that in the mother. This case suggests that the 1555 point mutation of mitochondrial DNA has potential to promote inherited nonsyndromic hearing loss without any known etiology. PMID- 10729801 TI - Malignant oncocytoma of a minor salivary gland: an unusual presentation at the base of the tongue. AB - In contrast to malignant oncocytomas of the parotid gland, malignant oncocytomas arising from minor extraparotid glandular tissue are rare. The latter may display a locally aggressive behaviour as well as a variable tendency to develop secondary cervical lymph nodes. We report a new case of malignant oncocytoma in a 43-year-old man presenting with greatly enlarged cervical lymph nodes accompanied by a poorly defined swelling at the base of the tongue microscopically identified as a malignant oncocytoma. The diagnosis was strongly supported by the ultrastructural features of the cells almost completely filled with mitochondria. The cervical mass was excised, and the patient has suffered no recurrence for nearly 2 years after surgical treatment. PMID- 10729802 TI - Saccular laryngeal cysts. Three case studies and review of the literature. AB - Supraglottic laryngeal cysts are benign lesions that usually are easily recognized and simply managed. Among these lesions saccular cysts are uncommon, usually large, anomalies of the larynx and can cause severe airway obstruction and even death. Three patients with a saccular cyst were successfully treated by endoscopic CO(2)-laser excision of their lesions. Before microlaryngoscopy and laser procedure, an urgent tracheostomy was required in 1 of our cases. PMID- 10729803 TI - False negative conventional histology of lymph nodes in patients with head and neck cancer. AB - Lymph node micrometastases in patients with head and neck cancer found node negative by clinical examination and conventional histology may be discovered with additional sectioning of the lymph nodes, by immunohistochemistry and by molecular biology. PMID- 10729804 TI - Evaluation of the safety of Rh immunoglobulin by monitoring viral markers among Rh-negative female blood donors. AB - BACKGROUND AND OBJECTIVES: Although immunoglobulin (Ig) preparations including RhIg have been noted for their record of safety, recent reports of hepatitis C virus (HCV) transmission by some Ig preparations have raised concern. This analysis examined the safety of RhIg manufactured in the US by comparing the prevalence and incidence of viral markers in Rh-negative and Rh-positive female blood donors. MATERIALS AND METHODS: Demographic and viral marker data were analyzed for allogeneic donations collected from female donors of childbearing age (17-49 years of age) from April 1992 to May 1996. Prevalence and incidence rates were calculated for HCV, human immunodeficiency virus (HIV), and hepatitis B surface antigen (HBsAg). RESULTS: Of the 624,939 female donors included in the study, 96,355 (15.4%) were Rh-negative and 528,584 (84.6%) Rh-positive. There were no significant differences in the prevalence of HCV and HIV between Rh negative and Rh-positive female donors. HBsAg prevalence was significantly higher among non-white compared to white donors. Following implementation of the more sensitive EIA 2.0 screening test for HCV in April 1992, prevalence and incidence rates declined over time at similar rates for Rh-negative and Rh-positive female donors. CONCLUSIONS: Rh-negative female donors had similar prevalence and incidence rates for most viral markers compared to Rh-positive female donors. This analysis supports the historical safety of RhIg. PMID- 10729805 TI - Large-scale screening for human parvovirus B19 DNA by PCR: application to the quality control of plasma for fractionation. AB - BACKGROUND AND OBJECTIVES: Because human parvovirus B19 (B19) has been transmitted by various plasma-derived medicinal products, the 'Laboratoire francais du Fractionnement et des Biotechnologies' (LFB) implemented PCR screening of plasma pool samples for B19 DNA as part of the quality control of plasma source material. MATERIALS AND METHODS: Plasma pool samples (average of 46.5 donations) were tested for B19 DNA by PCR and by immunological detection of PCR products. The viral DNA content was determined by means of a TaqMantrade mark based, quantitative PCR. RESULTS: From plasma corresponding to 2-year collections in France, and representing 4.26 million donations from approximately 1.25 million voluntary unpaid donors, the average frequency of positive donations was 1/5,950 and reached 1/1,420 during an epidemic. Levels of B19 DNA in positive pools ranged from <10(2) to 10(11) copies/ml. CONCLUSION: A large-scale PCR plasma screening increased the safety of LFB's wide range of products with respect to B19, a virus particularly resistant to physicochemical inactivation procedures. PMID- 10729806 TI - Allogeneic red blood cell transfusion is an independent risk factor for the development of postoperative bacterial infection. AB - BACKGROUND AND OBJECTIVES: Allogeneic red blood cell transfusions may exert immunomodulatory effects in recipients including an increased rate of postoperative bacterial infection. It is controversial whether allogeneic transfusion is an independent predictor for the development of postoperative bacterial infection. METHODS: We analysed a prospectively collected database of 1,349 patients undergoing colorectal surgery in 11 centres across Canada. The primary outcome was the development of either a postoperative wound infection or intra-abdominal sepsis in transfused and nontransfused patients. The effect of allogeneic transfusion on postoperative infection was evaluated with adjustment for all the confounding factors in a multiple regression analysis. RESULTS: The 282 patients who received a total of 832 allogeneic units had a significantly higher frequency of wound infections and intra-abdominal sepsis than the patients who were not transfused (25. 9 vs. 14.2%, p = 0.001). A significant dose-response relationship between transfusion and infection rate was demonstrated. Multiple regression analysis identified allogeneic transfusion as a statistically significant independent predictor for postoperative bacterial infection (OR 1.18, 95% CI 1.05-1.33, p = 0.007). Other independent predictors were anastomotic leak, repeat operation, patient age and preoperative haemoglobin level. The mortality rate was also significantly higher in the transfused group. CONCLUSION: These data support the hypothesis that allogeneic red cell transfusion is an independent risk factor for the development of postoperative bacterial infection in patients undergoing colorectal surgery. This association provides further reason to minimise exposure to allogeneic transfusions in the perioperative setting. PMID- 10729807 TI - Leukocyte depletion of red cell components prevents exposure of transfusion recipients to neutrophil elastase. AB - BACKGROUND: Polymorphonuclear leukocytes contain a large number of enzymes and bactericidal proteins stored in granules. Neutrophil activation induces degranulation and immediate release of these bioactive substances, including human neutrophil elastase (HNE) also known as elastase-2 (ELA2), which may contaminate whole blood units and blood components. MATERIALS AND METHODS: The HNE concentration was determined in the supernatants of blood components with a commercial enzyme-linked immunosorbent assay (ELISA). The effect of leukocyte depletion and storage was evaluated by testing whole blood, buffy-coat-reduced, and leukocyte-depleted red cell units. Buffy-coat-derived platelets and plasma were also tested. RESULTS: HNE concentrations at day 1 were about 50 microg/l in all types of red cell components with the exception of leukocyte-depleted red cells (<0.26 microg/l). In leukocyte-depleted red cells, platelets and plasma, no significant increase was observed during storage. In whole-blood units and buffy coat-reduced red cells, the HNE concentrations increased steadily and often exceeded 1,000 microg/l when the units expired. CONCLUSION: Leukocyte depletion may limit the inadvertent infusion of bioactive substances derived from polymorphonuclear leukocytes, of which HNE is but one example. The accumulation of HNE in buffy-coat-reduced red cells may be greater than that of whole blood units. HNE accumulates during storage and its quantity may have pathophysiologic significance. Platelets and plasma derived from buffy coats contain some HNE, but leukocyte-depleted red cells virtually none. However, we consider the accumulation of HNE in these components not to be clinically important. The quantities, kinetics, and occurrence in various blood components of HNE contamination differ from those observed with cytokines. PMID- 10729808 TI - Retention of coagulation factors in plasma frozen after extended holding at 1-6 degrees C. AB - BACKGROUND AND OBJECTIVES: The ability to use plasma, isolated from units of whole blood and frozen within 24 h of phlebotomy, as a substitute for plasma frozen within 8 h of phlebotomy would have several advantages for blood centers. It should provide increased flexibility pertaining to the freezing of plasma for clinical use. We have conducted studies to assess the influence of an extended holding time for separated plasma, prior to freezing, on the retention of coagulation factor activity. STUDY DESIGN AND METHODS: Freshly harvested plasma from each of 10 units of CPD-whole blood was divided into four equal aliquots. These aliquots were held in plastic packs at 1-6 degrees C for a total of 0, 8, 15 and 24 h. Subsequently, the plasma aliquots were frozen rapidly and stored at 20 degrees C for 4 months. The thawed plasma was tested for coagulant factors V and IX, factor VIII coagulant activity (factor VIII:C), von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor of von Willebrand factor. RESULTS: The levels of factor V, factor vWF:Ag, factor IX and ristocetin cofactor were not influenced by holding the plasma for up to 24 h prior to freezing. Factor VIII:C activity was reduced with extended holding at 1-6 degrees C; the percentage at time zero activity was 75.9+/-2.4% for samples frozen immediately after a 24-hour period. CONCLUSIONS: The data indicate that coagulation factor properties of harvested plasma are retained except for factor VIII for at least 24 h prior to freezing. PMID- 10729809 TI - Plasma levels of eicosanoids after transfusion of intraoperatively salvaged blood. AB - BACKGROUND: The interest in autologous red blood cell salvage is increasing rapidly, but it is well known that platelets and white cells are activated during cell washing. Therefore, we aimed to determine whether or not eicosanoids transfused together with the salvaged blood are responsible for nonhemolytic immunological transfusion reactions. MATERIALS AND METHODS: In 11 patients who underwent radical prostatectomy we studied the release of eicosanoids, prostaglandin E(2) (PGE(2)) and thromboxane B(2) (TxB(2)), in association with reinfusion of intraoperatively salvaged blood. Blood samples were taken before operation, on days 1 and 3 after operation and before and after cell washing. RESULTS: There was a twofold increase in PGE(2) and a twentyfold increase in TxB(2) in the unwashed salvaged blood. Washing caused a reduction in eicosanoid plasma levels of up to 95%, but a significantly lower level in relation to its corresponding preoperative value was only found for PGE(2). After transfusion of an average of 420 ml RBC sediment from washed blood, the patients' plasma concentrations of PGE(2) and TxB(2) on the 1st and 3rd postoperative day did not significantly differ compared to baseline values. CONCLUSION: Our data support the notion that RBC sediment from 'salvaged' and washed autologous blood contains increased amounts of PGE(2) and TxB(2). It remains, however, questionable if these passively infused eicosanoids become biologically active. According to our study, the PGE(2) and TxB(2) transfused are apparently not responsible for nonhemolytic immunological transfusion reactions. PMID- 10729810 TI - Errors in interpreting the pretransfusion bedside compatibility test: an experimental study. AB - BACKGROUND AND OBJECTIVE: Analysis of reports of incidents, involving ABO incompatibility suggests that the main problem is poor interpretation of the pretransfusion bedside compatibility test (PBCT). We studied sources of error as experienced by nurses as to the blood groups of donor blood and of the recipient. MATERIALS AND METHODS: According to their seniority in the profession and on the ward, 48 nurses were randomly selected from four transfusion sectors of the University Hospital of Grenoble, France. Each nurse interpreted 24 photos of PBCTs, including some with procedural irregularities, and was asked to assess the compatibility of the blood types of the donor and the recipient. At random, half the nurses were provided with a diagram to facilitate interpretation. RESULTS: The overall frequency of errors was 39.8%. Errors were fewer when the tests were interpreted as compatible (7.3%) or incompatible (6.3%), and when the nurse had been in the profession between 3 and 5 years and in the ward less than 3 years (25.5%), or worked in hematology (34.7%) or anesthesia (36.5%). Use of the diagram limited the number of errors, provided the test was interpretable (22.2%). CONCLUSION: PBCTs cannot be considered a valid safety procedure. We need other, more effective methods to reduce the risk of incompatibility accidents. PMID- 10729811 TI - Evans antigen: a new hybrid structure occurring on background of D.. and D-- Rh complexes. AB - BACKGROUND AND OBJECTIVES: Previously, the Evans antigen (RH37) of the Rh blood group system was shown to be specified by a novel CE-D-CE hybrid gene. We studied further the heterogeneity of Evans and report here its new molecular type resulting from a novel intraexon fusion event on the background of D. and D-- complexes. MATERIALS AND METHODS: A white family with 2 Evans+ and 2 Evans- members was analyzed by serological methods and molecular techniques. RESULTS: The Evans+ proband (JD) typed D+C-c-E-e- and showed a partial loss of RHCE but an increased dose of RHD on DNA blots. On sequencing of Rh cDNAs, a normal D and 3 hybrid transcripts were detected. The D-CE hybrid is characterized by a single breakpoint located in exon 6. The CE-D hybrid derived its exon 1 (or 1 and 2) from RHCE and exons 2-10 (or 3-10) from RHD. The CE-D-CE hybrid had its internal exons 2-7 (or 3-7) from RHD. Family studies showed that the D-CE and CE-D hybrids were linked and cotransmitted from JD to his son, whereas RHD and CE-D-CE hybrid were transmitted from JD to his daughter. CONCLUSION: In this family, Evans is specified by the novel D-CE intraexon fusion gene which occurs in cis to CE-D and trans to CE-D-CE interexon fusion genes. The observed large duplication of RHD reflects a convergent mosaicism underlying the enhancement of D and ablation of CcEe antigens. PMID- 10729812 TI - The MNS blood group antigens, Vr (MNS12) and Mt(a) (MNS14), each arise from an amino acid substitution on glycophorin A. AB - BACKGROUND AND OBJECTIVES: The antigens, Vr (MNS12) and Mt(a) (MNS14), are low incidence antigens of the MNS blood group system. The Vr antigen has been found only on the red blood cells (RBCs) of persons of Dutch ancestry whereas the Mt(a) antigen has been found on the RBCs of persons from a wide geographic distribution. The objective of this study was to determine the molecular basis of Vr and Mt(a). MATERIALS AND METHODS: Following RT-PCR amplification of total RNA isolated from one Vr+ person (G488) and one Mt(a+) person (GH), the genes encoding glycophorin A (GYPA) and glycophorin B (GYPB) were cloned and sequenced. To confirm the point mutation observed in the cDNA from G488 (Vr+), GYPA exon 3 was cloned and sequenced from the genomic DNA of G488 and a second unrelated Vr+ person (MU). A restriction fragment length polymorphism (RFLP) assay was used to analyze genomic DNA from 11 Mt(a+) persons (10 unrelated) following PCR amplification of GYPA exon 3. RESULTS: The coding sequence of GYPB was normal in both G488 (Vr+) and GH (Mt(a+)). Sequencing data from GYPA clones derived from G488 showed to full length GYPA sequences: A normal GYPA M allele and a GYPA M allele with a point mutation 197C-->A. Sequencing of GYPA exon 3 from G488 and MU confirmed the point mutation. Sequencing data drom GYPA clones derived from GH showed two full length GYPA sequences: a normal GYPA M allele and a GYPA N allele with a point mutation 230C-->T. RFLP analysis based on the point mutation showed that DNA from 11 Mt(a+) samples were heterozygous for the point mutation. CONCLUSION: The Vr antigen arises from a point mutation 197C-->A on GYPA which is predicted to change serine at position 47 to tyrosine. This change introduces a new alpha-chymotrypsin cleavage site. The Mt(a) antigen arises from a point mutation 230C-->T which is predicted to change threonine at position 58 to isoleucine. PMID- 10729813 TI - Screening blood donations for hepatitis C virus by polymerase chain reaction. PMID- 10729814 TI - Bacterial contamination of blood components. PMID- 10729816 TI - Lipid imbalance might contribute to cystic fibrosis. PMID- 10729815 TI - Painting a brighter future for dogs and humans. PMID- 10729817 TI - CREB: New approach for treating obesity? PMID- 10729819 TI - Using bioinformatics in gene and drug discovery. AB - Bioinformatics has, out of necessity, become a key aspect of drug discovery in the genomic revolution, contributing to both target discovery and target validation. The author describes the role that bioinformatics has played and will continue to play in response to the waves of genome-wide data sources that have become available to the industry, including expressed sequence tags, microbial genome sequences, model organism sequences, polymorphisms, gene expression data and proteomics. However, these knowledge sources must be intelligently integrated. PMID- 10729820 TI - Chemical ligands, genomics and drug discovery. AB - The sequencing of the human genome and numerous pathogen genomes has resulted in an explosion of potential drug targets. These targets represent both an unprecedented opportunity and a technological challenge for the pharmaceutical industry. A new strategy is required to initiate small-molecule drug discovery with sets of incompletely characterized, disease-associated proteins. One such strategy is the early application of combinatorial chemistry and other technologies to the discovery of bioactive small-molecule ligands that act on candidate drug targets. Therapeutically active ligands serve to concurrently validate a target and provide lead structures for downstream drug development, thereby accelerating the drug discovery process. PMID- 10729821 TI - Balancing the risks and the benefits. AB - Pharmaceutical research organizations can benefit from outsourcing discovery activities that are not core competencies of the organization. The core competencies for a discovery operation are the expertise and systems that give the organization an advantage over its competition. Successful outsourcing ventures result in cost reduction, increased operation efficiency and optimization of resource allocation. While there are pitfalls to outsourcing, including poor partner selection and inadequate implementation, outsourcing can be a powerful tool for enhancing drug discovery operations. PMID- 10729822 TI - The 'science' of headhunting. AB - Recruitment Consultants use Executive Searches - headhunting - to fill key vacancies in client companies. They cultivate a thorough understanding of the ideal candidate profile and the client's culture/strategy, and undertake a focussed search to identify competent individuals. A well-informed Consultant - sensitive to the requirements of both the client and the candidates throughout - will ultimately introduce the correct candidate to the correct job, to the benefit of all concerned. PMID- 10729823 TI - Monitor: molecules and profiles. AB - Monitor provides an insight into the latest developments in drug discovery through brief synopses of recent presentations and publications together with expert commentaries on the latest technologies. There are two sections: Molecules summarizes the chemistry and the pharmacological significance and biological relevance of new molecules reported in the literature and on the conference scene; Profiles offers commentary on promising lines of research, emerging molecular targets, novel technology, advances in synthetic and separation techniques and legislative issues. PMID- 10729824 TI - Combinatorial chemistry. PMID- 10729825 TI - Radiation-produced chromosome aberrations: colourful clues. AB - Ionizing radiation produces many chromosome aberrations. A rich variety of aberration types can now be seen with the technique of chromosome painting. Apart from being important in medicine and public health, radiation-produced aberrations act as colorful molecular clues to damage-processing mechanisms and, because juxtaposition of different parts of the genome is involved, to interphase nuclear organization. Recent studies using chromosome painting have helped to identify DNA double-strand-break repair and misrepair pathways, to determine the extent of chromosome territories and motions, and to characterize different aberration patterns left behind by different kinds of radiation. PMID- 10729826 TI - Flipping the oncogene switch: illumination of tumor maintenance and regression. AB - The genetic construction of cancer-prone mice, combined with the capacity to control transgene expression in vivo, provides new opportunities to study the role of oncogenes in the maintenance of fully formed tumors. These inducible cancer models provide a means to dissect how specific oncogenic signals influence host-tumor symbiosis, to validate the importance of a given oncogenic lesion in established advanced tumors, and to predict the biological response and adaptations to therapies targeted to that cancer-causing genetic alteration. PMID- 10729827 TI - Retroviruses in plants? PMID- 10729828 TI - Reverse gyrase from hyperthermophiles: probable transfer of a thermoadaptation trait from archaea to bacteria. PMID- 10729829 TI - Expanding the definition of informational suppression. PMID- 10729830 TI - Opsin evolution: out of wild green yonder? PMID- 10729831 TI - Modeling human neurodegenerative diseases in Drosophila: on a wing and a prayer. AB - The ability of Drosophila genetics to reveal new insights into human neurodegenerative disease is highlighted not only by mutants in flies that show neuronal cell loss, but also by targeted expression of human disease genes in the fly. Moreover, study of Drosophila homologs of various human disease genes provides new insight into fundamental aspects of protein function. These recent findings confirm the remarkable homology of gene function in flies when compared with humans. With the advent of complete genomic sequencing on the horizon, Drosophila will continue to be an outstanding model system in which to unravel the complexities, causes and treatments for human neural degeneration. PMID- 10729832 TI - DNA hypermethylation in tumorigenesis: epigenetics joins genetics. AB - Recently, the concept that epigenetic, as well as genetic, events might be central to the evolution of human cancer is re-emerging. Cancers often exhibit an aberrant methylation of gene promoter regions that is associated with loss of gene function. This DNA change constitutes a heritable state, not mediated by altered nucleotide sequence, that appears to be tightly linked to the formation of transcriptionally repressive chromatin. This epigenetic process acts as an alternative to mutations to disrupt tumor-suppressor gene function and can predispose to genetic alterations through inactivating DNA-repair genes. Dissecting the molecular processes that mediate these methylation changes will enhance our understanding of chromatin modeling and gene regulation and might present novel possibilities for cancer therapy. Methylation changes constitute potentially sensitive molecular markers to define risk states, monitor prevention strategies, achieve early diagnosis, and track the prognosis of cancer. PMID- 10729833 TI - Gene therapy: designer promoters for tumour targeting. AB - One of the biggest challenges facing cancer therapy is to generate tumour specific treatment strategies. Gene therapy hopes to achieve this by directing the activity of therapeutic genes specifically to the sites of disease. Of paramount importance for the success of this approach is the availability of tumour-specific delivery systems: both the transductional targeting of the vector vehicle and the restriction of transgene expression to the tumour are promising strategies towards this goal. This review will focus on the recent achievements in the field of transcriptional targeting and the different strategies to improve or design promoters with the desired specificities. PMID- 10729834 TI - Pairing SOX off: with partners in the regulation of embryonic development. AB - The SOX family of high-mobility group (HMG) domain proteins has recently been recognized as a key player in the regulation of embryonic development and in the determination of the cell fate. In the case of certain SOX proteins, they regulate the target genes by being paired off with specific partner factors. This partnering might allow SOX proteins to act in a cell-specific manner, which is key to their role in cell differentiation. The focus of this article is the mechanism of action of SOX proteins, in particular, how SOX proteins specifically pair off with respective partner factors and, as a consequence, select distinct sets of genes as their regulatory targets. PMID- 10729835 TI - GXD: integrated access to gene expression data for the laboratory mouse. PMID- 10729836 TI - Vigilance and the technologic promise of the third millennium. PMID- 10729837 TI - Endovascular neural stimulation via a novel basket electrode catheter: comparison of electrode configurations. AB - We previously showed that parasympathetic stimulation by a basket electrode catheter (BEC) positioned in the superior vena cava (SVC) can slow sinus rate (SR) or ventricular response (VR) during atrial fibrillation (AF). In 11 dogs, anesthetized with Na-pentobarbital, standard ECG leads II and aVR, blood pressure and right atrial electrograms were continuously monitored. Two different BEC configurations (B1, B2) were tested in the SVC. B1 consisted of five metal splines, each 3 cm in length. Stimulation was applied between adjacent splines. B2 consisted of 2 electrodes at opposite ends of each of 5 splines and a larger electrode at the middle of each spline. Stimulation was delivered between the two end electrodes and the middle electrode on the same arm. Stimulation consisted of square wave stimuli, each 0.1 msec duration, frequency 20 Hz at voltages from 1 40 V. Six dogs were studied with B1 and five were studied with the B2 configuration. The average voltage required to produce a 50% decrease in heart rate was 22+/- 12 V when stimulating between adjacent splines (B1) compared to 10+/- 5 V when stimulating along a single spline (B2), a 55% decrease (p60 seconds) and a spontaneously terminating (>/=10 beats and 30 beats/min) escape rates (there were no 12-hour data in 2). The escape rhythm was stable in 17 of the other 18 while 1 had no escape beats at 12 hours. Patients developing right bundle branch block had a greater chance of having an inadequate escape rhythm at 12 hours but this difference was not seen at 24 hours. We conclude that an adequate escape rhythm is usually present immediately after radiofrequency ablation of the AV node and tends to remain stable up to 24 hours. The absence of an escape rhythm immediately after ablation is of limited prognostic value since reliable escape rhythms may emerge subsequently. PMID- 10729852 TI - The effect of ablation sequence and duration on lesion shape using rapidly pulsed radiofrequency energy through multiple electrodes. AB - Sequences of energy application to multiple electrodes and a study of ablation duration with distal tip and multi-electrode ablations were explored with a radiofrequency controller that distributes energy from a generator to up to 4 electrodes with various duty cycles. In vitro ablations were performed on bovine left ventricle in circulating blood and lesions in goats were performed to verify the in vitro results. All of the ablation sequences with simultaneous electrode activation of contiguous electrodes resulted in deeper lesions than those created in sequence. There was also no scalloping of the lesion if contiguous electrodes were activated simultaneously. During all distal tip ablations, lesion volume and depth was greater after 3 minutes of energy delivery than after 1 minute, but did not increase from 3 minutes to 5 minutes. There was a significant increase in multi-electrode ablation lesion depth with each additional minute in the ablation cycle. The in vivo ablations verified these results at 120 and 300 second ablations. Pulsed energy distal tip ablations resulted in deeper lesions than continuous only if power amplitudes over 50 W were employed. In conclusion, contiguous electrodes in simultaneous use create lesions that resemble one large lesion rather than two lesions positioned next to each other. Multi-electrode ablation lesions continue to grow at ablation durations of up to 5 minutes compared to distal tip lesions which reach steady-state between 1 and 3 minutes. Pulsed energy delivery to distal tips may result in deeper lesions than conventional if high powers are employed. PMID- 10729853 TI - Does Genetic Predisposition Influence Surgical Results of Operations for Obesity? AB - BACKGROUND: There is a familial predisposition to obesity. We wished to document the incidence of obesity (BMI > 40 kg m(2)) in the immediate relatives (parents and siblings) of obese patients who were candidates for gastric restrictive surgery. We determined if a familial predisposition to obesity would influence the surgical results. METHODS: The height, weight and BMI were obtained in 1841 relatives of obese patients and in 1059 relatives of normal weight controls. The results of gastric surgery after 52.9 +/- 23.1 months were obtained in 44 patients with a familial history of obesity and in 34 patients without a familial history. RESULTS: Patients presenting with a BMI > 40 kg m(2) were 24.541 times more likely to have a first degree relative with morbid or super obesity than individuals in the control group. Mothers were twice as likely to be severely obese as fathers. A successful result (BMI < 35 kg m(2) or less than 50% excess weight) occurred 52.9 +/- 23.1 months in 77% of patients with a family history of obesity.and in 73% of patients without a familial predisposition (p = 0.79). CONCLUSIONS: There is a strong familial predisposition to obesity but over one half of the immediate family members of obese patients have a BMI < 30 kg m(2). Gastric restrictive surgery induces satiety and produces a successful outcome regardless of familial predisposition. Patients who undergo surgery have a remarkably stable weight over the year prior to operation, suggesting they are defending a markedly elevated BMI. PMID- 10729854 TI - Familial Incidence and Influence on the Development of Obesity. AB - BACKGROUND: The prevalence of obesity in families of patients seeking surgical treatment for their severe obesity was investigated. METHODS: Patients listed for surgery (vertical gastroplasty) for severe obesity (BMI > 35) were interviewed and asked for details of the numbers of members of the immediate family who were at least 6.5 kg overweight. The data collected on 87 morbidly obese patients were compared to data from a group of 50 patients, all of whom had a BMI of less than 25 and were not overweight, who were acting as controls. RESULTS: Obese patients had significantly more overweight family members than normal weight controls,(p = 0.004). CONCLUSION: The prevalence of overweight in the families of obese patients may be genetically and environmentally influenced. PMID- 10729855 TI - The Gastric Bypass for Failed Bariatric Surgical Procedures. AB - METHODS: Evaluated are surgical difficulties, management problems and weight loss in patients with distal gastric bypass as a revisionary procedure. Eighty patients were followed up to 3 years; four were lost to follow-up. Mean age was 43; mean prebariatric surgery weight 134 kg; height 1.65 meters; body mass index 40.1; ideal body weight 62.7 kg; excess weight 70.5 kg; per cent excess weight 214%. A 250 cm stomach-to-ileocecal valve segment of small bowel was used, and the biliopancreatic secretions were brought into the terminal ileum 100 6 in from the ileocecal valve. Mean pouch size was 63 cc; length of hospital stay 5 days; operative blood loss 616 cc; operative time 130 min. RESULTS: Intraoperative complications included three splenic injuries (without splenectomy). Early complications included one deep vein thrombosis, two marginal ulcers, one GI hemorrhage, one wound dehiscence, one pouch outlet obstruction and one pancreatitis. Late complications included: one death from protein malnutrition/ ARDS; 21 hypoproteinemia; six protein malnutrition, and of these, three had hyperalimentation; three cholecystitis; 27 anemia; 22 incisional hernia; two staple-line disruption (reoperated); 26 low serum iron; 11 prolonged (>6 months) diarrhea; three prolonged frequent vomiting; and two unrelated deaths (chronic myelogenous leukemia and amyotrophic lateral sclerosis). Mean excess weight loss was 83% at 12 months; 89% at 24 months; and 94% at 36 months. CONCLUSION: The distal gastric bypass is fraught with the operative and immediate post-operative complications experienced in any revisionary bariatric surgery. Distal gastric bypass is very effective in producing long-term weight loss. Nutritional problems are common but usually easily corrected. The most serious nutritional complication is protein malnutrition, which must be identified and corrected early. Success of this procedure is dependent upon patient compliance with proper nutrition and supplements, and regular office follow-up with monitoring of laboratory data. Patients who are noncompliant are at significant risk for complications. PMID- 10729856 TI - Does Bilio-intestinal Bypass Still have a Role in the Treatment of Super-obesity? AB - BACKGROUND: Super-obesity (BMI > 50) is life-threatening. The jejunoileal bypass had Potential long-term problems, and weight loss with gastroplasty May be inadequate. METHODS: From 1988 to 1995, 19 Patients with morbid obesity had jejunolleal bypass with anastomosis of the fundus of the gallbladder to the proximal end of the bypassed jejunum. Of these patients, 11 were superobese, with ages 19-49 years, weight 125-172 kg, mean excess weight 97 kg (73-119) and BMI 56 (50-67). RESULTS: Mean weight loss at 3 years was 60 kg. There was no mortality and no major complications. Patients had 5-6 stools per day and some flatulence. There have been no hepatic, renal, calcium or electrolyte problems. Diseases secondary to obesity resolved. CONCLUSION: Billo-intestinal bypass has been effective and safe thus far. PMID- 10729857 TI - Six Cases of Barrett's Esophagus after Gastric Restrictive Surgery for Massive Obesity: An Extended Case Report. AB - OBJECTIVE: The purpose of this study is to report and characterize six patients who have developed Barrett's esophagus after; a gastric restrictive procedure for massive obesity. METHOD: Retrospective analysis of patients operated with gastric banding (GB) and vertical banded gastroplasty (VBG) between 1981 and 1994. RESULTS: Four patients (4/92) initially operated with GB have developed Barrett's esophagus a mean of 9 years post-operatively. Two patients (2/198) operated with VBG developed Barrett's esophagus 18 and 47 months postoperatively. The histopathological type of Barrett's esophagus was cardia-like in three cases, gastric-like in two cases and intestinal-like columnar epithelium in one case. None of the biopsies showed signs of dysplasia. CONCLUSION: Gastric banding is again gaining popularity with the development of adjustable bands that can be placed laparoscopically. The development of Barrett's esophagus after GB and VBG, a premalignant lesion, is cause for some concern. Prospective long-term studies are needed to further address this complication. PMID- 10729858 TI - Effect of Abdominal Lipectomy on Lipid Profile, Glucose Handling and Blood Pressure in Patients with Truncal Obesity. AB - BACKGROUND: Previous studies have found that people with apple (android)-shaped body fat distribution are at risk of developing cardiovascular disease, impaired glucose tolerance and hypertension. METHODS: To investigate the effects of lipectomy, we measured lipid and lipoprotein levels, indices associated with glucose tolerance and blood-pressure. The tests were performed pre-operatively and 3 and 12 months post-operatively in 34 subjects (obese lipectomy patients with android-shaped body fat distribution) and 23 controls (obese breast reduction patients). RESULTS: In subjects, total cholesterol, LDL and blood pressure were significantly lower at 3 months follow-up, but returned to pre operative levels at 12 months follow-up. Plasma insulin decreased significantly at 3 months follow-up, and continued to decrease at 12 months follow-up. Triglycerides, HDL, fasting blood sugar, glycosylated hemoglobin and C-peptide did not change at 3 and 12 months follow-up. There were no changes in controls. CONCLUSIONS: Lipectomy in patients with truncal obesity may reduce plasma insulin levels, but had no lasting effect on plasma lipids. PMID- 10729859 TI - Therapeutic Megalipoextraction or Megaliposculture. AB - BACKGROUND: For a long time, surgeons hesitated to extract more than 2.5 liters of adipose tissue from a patient. Through the years, liposuction has advanced, now allowing megaliposculptures to be performed so that more than 10 liters can be extracted without posing any greater risk to the patient than a conventional esthetic liposculpture. METHODS: This article traces the progressive evolution of the technique, discussing complications and the concept of megaliposculpturing. RESULTS: The syringe-assisted extraction of large quantities of adipose tissue without transfusion, hypovolemic shock, or any major complication opens new horizons in the symptomatic treatment of certain types of osteoarthritis of the lower extremities and the potential complications of obesity. CONCLUSIONS: Large quantities of adipose tissue may safely, quickly and efficiently be extracted. Numerous investigations in various pathological domains can now begin after this first step (megalipotherapy). Therapeutic megalipoextraction can be done either as a preliminary indication or as a treatment of the residual adiposities that occur after bariatric operations. PMID- 10729860 TI - Ulcerogenesis in Surgery for Obesity. PMID- 10729861 TI - The Development of General Surgical Operations and Weight-loss Operations. PMID- 10729862 TI - Acceptance of Surgery for Obesity by Academic Surgeons in North America. AB - BACKGROUND: Have surgeons in North America accepted operations for the treatment of severe obesity? This question was posed by organizers of the Ninth International Symposium on Obesity Surgery that met in Stockholm in September, 1995. Study design: In order to obtain opinions which might help to answer the question posed, a 1-page questionnaire was sent to 151 chairmen of academic departments of surgery in North America in December 1994 asking about the acceptance and use of surgical operations for the treatment of severe obesity. RESULTS: Answers, received from 112 or 74%, indicated that operative treatment should be used (74%), was effective (77%) and safe (71 %). However, only 65% of departments provided such operations. Operative treatment for obesity was available outside the department in 53%. The operations most frequently used were Roux-en-Y gastric bypass (RGB) and vertical banded gastroplasty (VBG). A single operation was offered by 44 departments and a choice of two or more operations in 30 departments. RGB and VBG were equally in use. Gastric banding was used as an alternative to VBG in six departments. Other operations were in use in six departments either alone (three) or as alternatives to RGB and VBG (three). CONCLUSIONS: It appears that surgical treatment of obesity is accepted and available in the majority of academic departments of surgery. PMID- 10729864 TI - Vertical Silastic Ring Gastroplasty with Nissen Fundoplication Prevents Reflux. AB - Optimal management of coexisting morbid obesity and gastroesophageal reflux is not known. Silastic ring vertical gastroplasty is an effective treatment for morbid obesity, and Nissen fundoplication is effective in treating gastroesophageal reflux. We combined these two procedures in an animal model and found protection against gastroesophageal reflux without any deleterious effects on pouch emptying. PMID- 10729863 TI - Pay Status as a Predictor of Outcome in Surgical Treatment of Obesity. AB - BACKGROUND: Higher complication rates and lower success in surgery for severe obesity have been reported for patients with government pay status. We examined the effect of pay status upon outcome in surgical treatment of obesity. METHODS: This was an observational study from an aggregate data set of individual patient information. Government pay status (G) was defined as full or partial medical care payment through Medicare, Medicaid, or Veterans Administration. Payment entirely by private insurance was defined as private (P). Operations were classified as either simple (S, gastric restriction) or complex (C, gastric restriction with small bowel bypass). Two measures of outcome, perioperative complication rate and weight loss success ( Obesity surgery, morbid obesity, outcomes, surgical results, survey PMID- 10729882 TI - Believe It or Not, Sometimes Lawyers Are the Good Guys! PMID- 10729883 TI - Beyond Bariatric Surgery.Complications We Fail to Address. AB - As we now progress in the field of bariatric surgery, improving methods and fine tuning operative procedures, we witness many benefits. Fewer complications, shorter hospital stays, and more evidence of successful weight loss consistently appear in the statistics that have been accumulated through years of research. The information accumulated through years of research. The information provided, continues to verify that surgery is the most viable treatment for morbid obesity. There is minimal literature, however, that addresses the emotional issues faced by the patients who have undergone bariatric procedures. Postoperatively we may find at intervals of 3 months, 6 months, and even 1 or more years, a patient struggling to deal with their identity. The resolution or improvements of various medical anomalies, may seem insignificant as the patient now begins to focus on emotional and physical changes. Their bodies' new shape may create distressing personal concerns. Family members may also struggle with these psychological and physical changes. The surgery has forced the patient to deal with the loss of a love that is not easily replaced, the love of food. These, all too frequently, are the actual postoperative complications, co-morbidity changes, and weight loss, most importantly, we must also understand our responsibility to address the patients' emotional well-being. While indicating our concerns for the psychological as well as physiological recovery, the patient may then realize a healthier transition into life beyond bariatric surgery. PMID- 10729884 TI - Progress in the Treatment of Obesity. PMID- 10729885 TI - Adjustments and Leak Detection of the Adjustable Silicone Gastric Band (ASGB) and Lap-Bandtrade mark Adjustable Gastric Band (LAGB) System. AB - BACKGROUND: The detection of a leakage in the system of the adjustable silicone gastric band (ASGB) may be difficult. Gastrografin injection into the port should be avoided because it acts like a glue and blocks the system. METHODS: A syringe containing saline and a syringe containing Thallium-201 chloride is connected to the 4-way stopcock which is connected to the needle. The needle is pushed into the port. The position is confirmed by injection and aspiration of saline. 2 ml of TL-201 chloride (74 MBp) is injected to locate the leakage in the system with planar images with a gamma camera (Elscint SP 6), 30 min, 2, 3, and 24 h after injection. RESULTS: The original ASGB was provided with an injection reservoir which, in our series, was found to be leaking in four cases (3%). CONCLUSIONS: Our technique for adjustment and leak detection appears to be simple and effective. Band-related problems such as reservoir leak should disappear with improvement of the material. PMID- 10729886 TI - Adjunctive Use of Appetite Suppressant Medications for Improved Weight Management in Bariatric Surgical Patients. AB - BACKGROUND: Patients who undergo bariatric surgery sometimes experience late onset or weight gain, when they lapse into negative eating patterns, which adversely affect weight management. Long-term weight management is a process, with a surgical foundation, and requiring adjunctive strategies for best results. We sought to determine if appetite suppressant medications could be safely incorporated into a comprehensive program of weight management. METHODS: Subjects were at least 18 months postoperative, were accessible for weekly follow-up, and weighed at least 9 kg more than their ideal body weight. Phentermine and fenfluramine were prescribed in combination, at the lowest dose necessary to achieve comfortable appetite suppression. RESULTS: Weight losses ranged from 4.5 to 22.7 kg, over a 12-week course of treatment, corresponding to 8-65% of excess body weight. Most side-effects were minor, and did not require cessation of treatment. Two patients discontinued treatment due to side-effects which were unacceptable to them. CONCLUSION: Phentermine and fenfluramine are a safe and useful adjunct to a comprehensive program of weight management. PMID- 10729887 TI - Complications of Biliopancreatic Diversion Surgery as Proposed by Scopinaro in the Treatment of Morbid Obesity. AB - BACKGROUND: This study concerns 33 patients treated for morbid obesity with the procedure proposed by Scopinaro. Results are reviewed retrospectively in terms of complication rates. METHODS: The group consisted of ten men and 23 women with a mean age of 34 years (range 20-51 years), and a mean BMI of 49.5 kg/m(2) (range 37-77). Adequate attempts at medical management had failed repeatedly. The operative procedure involved a 2/3 partial gastrectomy and biliopancreatic diversion by Roux-en-Y reconstruction 50 cm before the ileocecal valve. In one patient, a cholecystectomy was added. RESULTS: The mean weight loss after 6 months was 18.9% of the initial weight, with mean BMI 41 kg/m(2) (range 29-60). Early complications included four wound infections (15%), while two patients complained of an early dumping syndrome (6%), treated by dietary measures. There were no respiratory infections and no pulmonary embolism, likely as a result of the thoracic epidural anesthesia and high doses of prophylactic heparin used. There was no mortality. As to late complications, nine patients complained of diarrhea due to bacterial overgrowth (27%) and were treated with antibiotic therapy. There were five incisional hernias (15%). Five patients had a peptic ulcer (15%) and required medical treatment. Two patients had acute cholecystitis (6%). One patient had an afferent loop obstruction (3%), requiring reoperation. CONCLUSIONS: Overall, this series of intestinal diversion procedures by the method of Scopinaro had a larger complication rate than generally accepted. PMID- 10729888 TI - Vertical Banded Gastroplasty and Distal Gastric Bypass as Primary Procedures: A Comparison. AB - BACKGROUND: Comparing primary vertical banded gastroplasty (VBG) and distal gastric bypass (DGBP) patients might assist decision-making based on patient profiles and desired outcomes. METHODS: A prospective study of 81 vertical banded gastroplasty and 60 distal gastric bypass patients. Technical aspects, complications, weight loss, post-op compliance and satisfaction are reported. Length of follow-up is 48 months (VBG) and 36 (DGBP). Lost-to-follow-up 41% (VBG) and 22% (DGBP). Ten per cent of VBGs were revised, with 1% takedown. Three percent DGBPs were converted to proximal GBPs. Demographics are comparable. RESULTS: Operative time was 40 min VBG and 88 DGBP; blood loss 187 cc vs 335 cc; and hospital stay 3 versus 4 days. Exclusive VBG complications include: 1% staple line leak, 4% intra-abdominal abscess, 1% respiratory failure, 5% pneumonia, 1% intra-abdominal bleed, 1% small bowel obstruction, 2% infected incision, 2% fistula, 2% stenotic or obstructed obstructed stoma, and 1% bezoar. Exclusive DGBP complications include: 2% GI bleed, 12% marginal ulcer, 5% reflux esophagitis, 13% hypocalcemia, 23% hypovitaminosis A and D (12% requiring B12 therapy). Shared complications include hypoproteinemia 6% VBG versus 40% DGBP; excess vomiting (>6 months post-op), 7% versus 10%, excess diarrhea 2% versus 20%, dehydration 1% versus 8%, re-hospitalization 4% versus 15% (hyperalimentation), post-op cholecystectomy 1% versus 5%, weight regain 48% versus 1%. VBG experienced an average of 64% excess weight loss at 36 months versus DGBP 89% excess weight loss. VBG follow-up compliance is generally poor but good for DGBP. Compliance with diet and supplements is equivalent (50%). Satisfaction is 85% and 93% respectively. CONCLUSION: The DGBP provides better long-term weight loss, but nutritional deficiencies occur more often and require close follow-up. The surgery is more complex, but as a primary procedure there are few major complications. PMID- 10729889 TI - The Post-surgical Treatment of the Bariatric Patient: Helping the Patient Succeed. AB - BACKGROUND: A psychological profile of the average bariatric patient demonstrates psychopathology that may contribute to patient noncompliance with post-surgical treatment guidelines. METHODS AND RESULTS: Patient psychopathology is analyzed with regard to noncompliance and its contribution to poor surgical outcome. The interpersonal process approach is reviewed as a psychotherapeutic framework that provides interventions to patient psychopathology. CONCLUSIONS: Treatment of patient psychopathology with the interpersonal process approach encourages post surgical compliance and helps patients succeed. PMID- 10729890 TI - Obesity: The Last Bastion of Prejudice. AB - BACKGROUND: Most people do not realize how prejudicially damaging they behave, particularly towards the obese. Their discrimination has been deemed, unconsciously perhaps, as acceptable by society. METHODS: This paper describes a high school senior's exploration of prejudice and discrimination towards the obese. RESULTS: Through interviews with bariatric surgeons, bariatric patients, an obese victim of prejudice in her high school, attendance at support group meetings, statements from others experiencing similar bias in their workplace as well as a review of the relevant literature, the author developed a new understanding of the extent and depth of prejudice against the obese in North American society. She realized how this prejudice limits social opportunities and access of all sorts, interferes with employment opportunities, and even how deeply it penetrates the medical community. CONCLUSION: The intent of this paper is to educate those in society who continue to discriminate against the obese, and to open our eyes to our own behavior, as the author's have been opened. PMID- 10729891 TI - Impact of Vertical Banded Gastroplasty on Respiratory Insufficiency of Severe Obesity. AB - BACKGROUND: Respiratory insufficiency associated with morbid obesity can include sleep apnea syndrome (SAS), obesity hypoventilation syndrome (OHS), or a combination of both. The aim of our study was to determine the safety and effectiveness of vertical banded gastroplasty (VBG) in the treatment of severely obese patients with respiratory insufficiency. METHODS: From 1983 to 1994, 35 patients (25 males, ten females) who met the criteria for either SAS and OHS (1 9 patients) or SAS alone (1 6 patients) underwent VBG. RESULTS: Six patients (17%) died of subsequent pulmonary-cardiac disease despite significant weight loss. Need for nasal continuous positive airway pressure (CPAP) decreased after VBG from 68% of patients preoperatively to 22% postoperatively. Of the ten patients with sleep studies, the apnea/hyponea index decreased from 45 +/- 11 events per h preoperatively to 12 +/- 6 events per h postoperatively, while per cent ideal body weight (%IBW) also decreased (pre-VBG: 268 +/- 12, post-VBG: 204 +/- 12). Of the seven patients with arterial blood gases, PaCO&inf2; decreased from 55 +/- 4 torr preoperatively to 41 +/- 3 torr postoperatively, and PaO&inf2; increased from 50 +/- 4 torr preoperatively to 73 +/- 6 torr postoperatively, while %IBW decreased (pre-VBG: 263 +/- 16, post-VBG: 193 +/- 14). CONCLUSION: Respiratory insufficiency is a life-threatening complication of morbid obesity. In morbidly obese patients with respiratory insufficiency, VBG offers improvement in both SAS and OHS. Respiratory insufficiency due to obesity should be considered a strong indication for VBG. PMID- 10729892 TI - Gastric Emptying Half-time Following Silastic Ring Vertical Gastroplasty: A Scintigraphic Study (Preliminary Results). AB - BACKGROUND: The mechanism for weight loss following gastroplasty surgery is unknown. A few studies have checked gastric emptying patterns following gastroplasty, but none have analyzed their pattern over time. METHODS: Gastric emptying half-time of Tc-99 phytate-labeled semi-solid meal was studied in 23 obese non-diabetic and one diabetic patient (BMI 34.6-55.7). Gastric emptying was measured by gamma scintigraphy before Silastic Ring Vertical Gastroplasty (SRVG) and 1, 3 and 6 months following surgery. RESULTS: Gastric emptying half-times were maximal in all areas of the stomach 1 month after operation, declined to base-line 3 months following the procedure, and showed statistically significant delay in the upper area of the stomach 6 months after the operation. No correlation was found between the delay in food passage following surgery and loss of weight. CONCLUSIONS: Prolongation of gastric emptying half-time following surgery may be a result of two separate mechanisms: asymptomatic or early inflammatory processes in the ring area and pouch distention as a result of partial denervation. PMID- 10729893 TI - Validation of Pouch Size Measurement Following the Swedish Adjustable Gastric Banding Using Endoscopy, MRI and Barium Swallow. AB - BACKGROUND: Pouch volume appears to be of major importance for subsequent weight loss following any gastric restriction type of surgery for morbid obesity. In order to be able to evaluate pouch volume following Swedish Adjustable Gastric Banding (SAGB), an endoscopic pouch volume classification system was designed in which pouch volume is classified in five categories. The aim of this study was to validate the endoscopic classification system using MRI and barium swallow as reference methods for pouch volume measurement. METHODS: Twenty patients (13 women and seven men) were operated for obesity with SAGB. They were investigated a mean of 3 years (6 weeks-5.5 years) after surgery and had at that time lost a mean of 60 (12-112) kg. During the same afternoon they sequentially underwent endoscopy, MRI and barium swallow with an empty stomach. RESULTS: The mean pouch volume measured with MRI was 70 ml (0-1 80 ml) and with barium swallow was 72 ml (0-1 95 ml). In 17/20 patients the volume as measured by MRI and barium swallow was in the same volume category as with endoscopy. The correlation measured according to Pearson was significant between endoscopy on one hand and MRV barium swallow both independently and together (p < 0.001). CONCLUSION: Based on these results we are confident in using our endoscopic classification system for postoperative follow-up of pouch volume. PMID- 10729894 TI - Pouch Volume, Stoma Diameter and Weight Loss in Swedish Adjustable Gastric Banding (SAGB). AB - BACKGROUND: Weight loss appears to be inversely related to pouch volume following gastric restriction procedures for morbid obesity. The aim of this study was to investigate the changes in pouch volume with time and the relationship between pouch volume and stoma diameter and subsequent weight loss following the Swedish Adjustable Gastric Banding (SAGB). METHODS: During 1990 50 patients were operated upon. Their mean BMI at surgery was 46 and at 2 years 28. We followed these patients with endoscopy at 6 weeks and 3, 18 and 24 months after surgery. During endoscopy pouch volume was estimated according to a standardized classification system and measured stoma diameter using balloon catheters. RESULTS: The results indicate that the pouch dilates during the first few months after surgery but that the size thereafter is fairly stable. There is also a relationship between pouch volume and subsequent weight loss. Pouch volume seems to be the primary determinator for weight loss. CONCLUSION: The smaller the pouch the greater the weight loss. Reduction of the stoma diameter is a good instrument for regulating the degree and speed of weight loss in patients with small pouches, but much less powerful in patients with large pouches. PMID- 10729895 TI - The Influence of Dumping on Weight Loss After Gastric Restrictive Surgery for Morbid Obesity. AB - BACKGROUND: The dumping syndrome that follows Roux-en-Y gastric bypass for morbid obesity is considered to, be the primary mechanism of improved weight loss as compared with the purely restrictive vertical banded gastroplasty. To evaluate the influence of dumping on post-operative weight loss, severity of dumping was determined using Sigstad's clinical diagnostic index. METHODS: One hundred and thirty seven gastric bypass and 19 gastroplasty patients were assessed 18-24 months following surgery. Sigstad's criteria for the dumping syndrome were met by 75.9% of gastric bypass and no gastroplasty patients. Among gastric bypass patients, no relationships were found between severity of dumping and weight loss, as measured by per cent of excess body weight loss or change in body mass index. Weight loss was significantly greater with gastric bypass than gastroplasty patients (72.5 compared to 47.9% of excess body weight loss). All gastroplasty and 24.1% of gastric bypass patients were classified as non-dumpers. The difference in weight loss between surgical procedures was not related to dumping: gastric bypass non-dumpers lost significantly more weight (69.1% excess body weight loss) than gastroplasty patients. CONCLUSIONS: This study fails to demonstrate a significant relationship between dumping severity and weight loss. It is inferred that the superior weight loss of gastric bypass compared to gastroplasty has some other etiology. PMID- 10729896 TI - Effect of Revisional Bariatric Surgery on Weight Loss and Frequency of Complications. AB - BACKGROUND: The relative risks and effectiveness of primary and revision operations done to produce weight loss are of interest both from a patient care and an economic perspective. The possibility that patients requiring revision surgery comprise a treatment resistant subgroup who are more likely to have post operative complications is a valid concern. METHODS: The records of all patients having bariatric procedures since January of 1970 were evaluated for weight loss and complications. RESULTS: Most revisions were from jejunoileal bypass or a gastric restrictive procedure. Early complications were significantly more common following revision surgery (19%) than after primary procedures (6%), although late and combined early and late complication rates were similar. Operative mortality was lower following primary procedures (2/382) than revisions (1/75). Cholecystectomy was a common sequela following primary procedures but did not occur after revision procedures. Regardless of surgical category, weight loss after revision was equivalent to weight loss after primary procedures. CONCLUSIONS: Weight loss following revisional bariatric surgery is equivalent to weight loss following a primary operation of the same type. Although mortality and early complications are more common after revisional bariatric surgery, the frequency of late complications is not different. In all groups wound infections and hernias were relatively common; complications and cholecystectomies are rare after revisional bariatric operations. PMID- 10729897 TI - Biliopancreatic Limb Obstruction in Gastric Bypass at or Proximal to the Jejunojejunostomy: A Potentially Deadly, Catastrophic Event. AB - BACKGROUND: Although unusual, but not rare, obstruction in the vicinity of the jejunojejunostomy in Roux-Y gastric bypass (RYGBP) can progress in a very short period of time to a life-threatening situation. METHODS: Over a 10-year period in 1,174 RYGBPs, we have seen seven instances of acute and subacute partial to complete small bowel obstructions in the vicinity of the jejunojejunostomy, which can lead to acute gastric dilatation due to obstruction of the bilio-pancreatic limb. Signs and symptoms of the obstruction may include tachycardia, oliguria, hypotension, severe epigastric pain with or without a palpable mass in the epigastrium, chronic bile regurgitation and bilious vomiting, and a possible increase in serum amylase. Laboratory data otherwise has not been helpful, and although a palpable abdominal mass may be diagnostic, the best tools have been radiologic, i.e. the acute abdomen series, limited upper GI series in the patients that appear to be only partially obstructed, abdominal ultrasound and probably most importantly, CT of the abdomen. RESULTS: In the seven cases presented, diagnoses included internal hernia, adhesions, an idiopathic spontaneous hematoma of the bowel wall and retrograde intussusception at the jejunojejunostomy. CONCLUSIONS: Since many surgeons who perform bariatric surgery are alone in their community, they should train their non-bariatric surgical colleagues and associates to be aware of these potential deadly problems. PMID- 10729898 TI - Double Application of TA-90 B Four-Row AutoSuture(c) Stapling Instrument: A Safe, Effective Method of Staple-line Production Indicated by Follow-up GI Series. AB - BACKGROUND: Controversy continues, concerning the best method of isolating upper and lower gastric pouches in Roux-Y gastric bypass. This paper reports a technique used from August 1991 through May 1996, in which there was a double application of the TA-90 B Four-Row AutoSuture Stapling Instrument to form the proximal gastric pouch. Because of a significant staple-line failure rate historically, many are separating the pouches. However, due to recent reports of gastro-gastric fistula formation when pouch separation is done, a second look has been taken at stapling the division without separation of the pouches. METHODS: There were 650 patients in this series, and 160 asymptomatic patients 1-4 years postoperatively agreed to have limited upper series. RESULTS: Only one of this group had staple-line failure. Failures were seen in four of 19 symptomatic patients with dyspepsia or rapid weight regain. Putting these two groups together, our staple-line failure rate has been less than 1% using this technique. CONCLUSION: Whether stapling in continuity or dividing the stomach, the two methods appear to be equally effective with comparable morbidity. Patients with staple-line failure or gastro-gastric fistulae will ultimately be forced by their symptoms of rapid weight regain and/or peptic ulcer disease and reflux esophagitis to return for follow-up. Asymptomatic patients very rarely have a connection between upper and lower pouches, and routine follow-up upper GI series are not indicated. PMID- 10729899 TI - Laparoscopic Gastric Bypass, Roux-en-Y: Technique and Results in 75 Patients With 3-30 Months Follow-up. AB - BACKGROUND: Laparoscopic Roux en-Y Gastric Bypass (RYGB) has been performed in 100 patients, in our series. METHODS: The results of surgery, including 3-30 months follow-up, are described with 100% follow-up, for the first 75 patients. Weight loss, operative morbidity and relief of co-morbidities have been thoroughly studied in a prospective fashion. RESULTS: Diabetes mellitus was normalized in 22 of 24 patients and gastroesophageal reflux was relieved in all patients. Length of stay, recovery time and cosmetic results are superior to the 'open' technique, and the operative times are competitive. CONCLUSION: Laparoscopic RYGB deserves a place in the operative repertoire of bariatric surgeons. PMID- 10729900 TI - Comparative study of reductive amination reaction on 5-(4-formyl-3,5 dimethoxyphenoxy)valeric acid and its monomethoxy analog using the Multipin approach. AB - The 5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid (Barany) linker and its monomethoxy analog were applied to the Multipin method of solid phase synthesis. A comparative assessment of reductive amination and cleavage of these linkers under conditions of multiple synthesis indicated that both were applicable to a broad range of primary amines including aniline and 4-nitroaniline. Apart from the greater lability of the dimethoxy version under TFA cleavage, there was no observable advantage of one linker over the other within the described experiment. PMID- 10729901 TI - Solid phase synthesis of hydantoins by thermal cyclization and screening of reaction conditions using APOS 1200. AB - A novel strategy for solid-phase synthesis of hydantoins with high optical purity is described using a thermal pH-neutral cyclization and simultaneous release from resin. Hereby even hydantoins bearing a pH-sensitive side chain (protection) are available. The reaction conditions are well screened applying the parallel organic synthesizer APOS 1200. PMID- 10729902 TI - Use of combinatorial library screening to identify inhibitors of a bacterial two component signal transduction kinase. AB - Bacterial resistance to antibiotics is emerging as a major concern to the medical community. The appearance of several antibiotic-resistant strains, including multidrug-resistant Staphylococcus aureus, raises the prospect that infections by these bacteria could soon become untreatable with currently available antibiotics. In order to address this problem, increased emphasis is being placed on the discovery of novel classes of antibacterial agents that inhibit novel molecular targets using sources of compounds not yet exploited for antibiotic drug discovery. Novel classes of compounds can now be rapidly investigated using combinatorial chemistry approaches. This report describes the identification of novel antibacterial compounds from a combinatorial library of N-acetylated, C amidated D-amino acid hexapeptides. This library of compounds was screened for inhibitors of CheA, a member of the bacterial two-component signal transduction kinase family. Several peptides with apparent IC50 values in the low micromolar range were identified. In addition to inhibiting CheA, these peptides inhibited mammalian protein kinase C (from rat brain) with comparable potency. Finally, these peptides were also found to have significant antibacterial properties, although the true mechanism by which they exhibited inhibition of bacterial growth remains uncertain. PMID- 10729903 TI - Parallel solution synthesis of pyridinethiones, pyridinones and thienopyridines. AB - The parallel solution synthesis of three classes of heterocycles is described. Arrays of pyridinethiones, pyridinones and thienopyridines were prepared using one-step chemistry starting from readily accessible building blocks. The latter class of compounds was accessed by utilising a library-from-library approach. PMID- 10729904 TI - Y-ligation: an efficient method for ligating single-stranded DNAs and RNAs with T4 RNA ligase. AB - Very efficient ligation of oligodeoxyribonucleotides was attained through a simple molecular construct, which is composed of one stem and two branches (Y shape), with use of T4 RNA ligase. Single-stranded DNAs (naturally, RNAs also) of more than 100 nucleotides (even 800 nts) were considerably ligated, approximately as theoretically expected. Owing to the molecular construct adopted, such a tiny amount of ligation products could be amplified to a sufficient amount by PCR and then recovered as single-stranded DNAs. This advantage of being amplifiable is shown to be useful for both combinatorial chemistry and evolutionary molecular engineering, which deal with a pool of diversity molecules. PMID- 10729905 TI - Synthesis of peptidomimetics using a polymer-bound Boc-linker. AB - Boc-resin-bound alpha-hydroxy-beta-amino-aldehydes are accessible starting from N terminally bound amino acid esters by using Dondoni's C1-homologation reaction sequence. The conversion of these synthons to two different peptide mimetics--2 hydroxy-1,3-ethyl-diamines and gamma-hydroxy-delta-amino-vinyl sulfones--has been investigated. The successful transfer of the complex alpha-amino acid homologation reaction sequence into solid-phase chemistry demonstrates the potentials of the Boc-resin for synthesis of peptidomimetics. PMID- 10729906 TI - Comparative conformational analysis of peptide libraries. AB - Six computer-based combinatorial libraries, including tetrapeptide sequences (generated with five amino acids) and conformations (generated with five main chain and three side chain rotamers), were obtained and sequence-conformation probabilities were calculated with a molecular and statistical mechanics procedure. The structural motifs alpha-helix, beta-sheet, 3(10)-helix, reverse turn I and gamma-turn were focused in these calculations. It is shown that sequence-conformation-probability surfaces provide a broad view of structural changes accompanying changes in sequence. Numerical indices are defined to enable comparisons between frequencies of occurrence of these structural motifs in peptide libraries and in a database of low sequence identity protein structures. Fine details of sequence-conformation-probability surfaces show the effect of point mutations. Broad comparisons between different regions of these surfaces indicate how to select the occurrence of structural motifs in the combinatorial synthesis of peptide chains. PMID- 10729907 TI - [Multiple sclerosis: a stake in knowledge for researchers and a therapeutic challenge for physicians]. PMID- 10729908 TI - [Is multiple sclerosis a disease of viral origin?]. AB - MS is a multifactorial disease in which host genetic factors as well as environmental factors may interact. Among them, the role of viruses is still debated since none has been directly or indirectly involved in the disease up to now. The latest candidates are HHV6 and MSRV. Recently, HHV6 antigens have been found in MS brain, but these results require further investigation. MSRV is a retroviral sequence isolated from MS tissue and it belongs to the already known ERV9 family of endogenous retroviruses. Its role in MS is only putative. To conclude, "MS virus" is still unidentified. Nevertheless, the search for a virus in MS should be pursued. In that perspective, animal models illustrate how host immune system interacts with viruses to lead to demyelination, a common feature of a multi-step process of diverse origins. PMID- 10729909 TI - New perspectives in multiple sclerosis: retroviral involvement and glial cell death. AB - Retroviral involvement in the pathogenic cascade in multiple sclerosis (MS) and a cytotoxic activity with narrow specificity towards glial cells have been recently considered as credible working hypotheses to explain some of the complex pathophysiological and neuropathological features of MS. The partial characterization of exogenous retroviral sequences, thought to be associated with MS, has led us to the identification of new human endogenous retroviruses closely related to the extracellular multiple sclerosis associated retrovirus (MSRV). These endogenous retroviruses (HERV-TcR and HERV-7q) have the potential to be transcribed into RNA and proteins. Interestingly, the env domain of HERV-7q could code for a 59.8 kDa secreted glycoprotein (called enverin) with an immunoregulatory region. The presence in various MS biological fluids of a cytotoxic activity able to induce programmed cell death for oligodendrocytes and astrocytes suggests the possibility of a demyelination phenomenon as part of direct glial cell damage. Moreover, both retroviral expression and cytotoxic factor production have been evidenced in MS monocyte/macrophage cultures and MS cerebrospinal fluid. It is now crucial to better characterize the endo/exo retroviruses possibly involved in MS and their pathogenic potential, and to identify the contributing factor(s) to the gliotoxicity found in the MS cerebrospinal fluid or serum, as well as to elucidate the mechanism of induction of the observed programmed glial cell death. PMID- 10729910 TI - [Role of autoimmunity in multiple sclerosis]. AB - A number of evidences indicate that an antigen-specific immune reaction is responsible for the formation and/or the maintenance of multiple sclerosis (MS) lesions. This disease is characterized by the following: 1), an infiltration of the white matter of the brain and spinal cord by inflammatory cells; 2), the T and B lymphocytes, present in the lesions or in the cerebrospinal fluid of patients, show signs of activation; i.e., the classic IgG oligoclonal bands of the cerebrospinal fluid (activation of B lymphocytes) and the presence of activation markers on the surface of the T lymphocytes; 3), the presence of an association, and a linkage between the disease and the genes of the HLA complex. The HLA molecules are implicated in the presentation of the antigen to the T lymphocytes; 4), finally, it should be noted that the therapeutic approaches aimed at reducing (immunosuppressants) or at modulating (beta-interferon, copolymer 1) the immune responses have a positive effect on this disease, whereas those treatments which activate the immune system (gamma-interferon) have a negative effect. PMID- 10729911 TI - Experimental allergic encephalomyelitis animal models for analyzing features of multiple sclerosis. AB - Various animal models with experimental allergic encephalomyelitis (EAE) have been developed applying immunologic, virologic, toxic and traumatic parameters in order to understand features of multiple sclerosis (MS). The main simulating aspects of the EAE models and the precautions for their interpretation in determining differences and common features between EAE and MS are presented. In view of an early diagnosis of CNS lesions in human, we present with particular interest the application of human-related technologies, such as MR imaging techniques, and the development of new markers to follow the dynamic of CNS lesions in vivo in EAE animal models. PMID- 10729912 TI - [Embryonic origin of oligodendrocytes]. AB - Over the past ten years, knowledge regarding the origin of the oligodendrocyte lineage during development has increased considerably. In this review, we present the major findings as a result of which it was determined that only restricted regions of the neural tube have an oligodendrogenetic potential. These findings are based on the study of molecular markers permitting the detection, among the multipotent neutricular cells of the neural tube, of those with a potential to differentiate into oligodendrocytes. At the present time, it appears that these oligodendrocyte precursors can be distinguished in the brain either by the expression of the plp/dm-20 transcript, or by that of the alpha-receptor of PDGF. These two markers allow two oligodendroglial subpopulations to be differentiated, suggesting a multiple origin of oligodendrocytes. An assessment has also been made of the current state of knowledge, still incomplete, regarding the intrinsic and extrinsic factors which cause a multipotent cell strain to follow an oligodendroglial differentiation. A better knowledge of the oligodendrogenesis during embryonic development should provide insight into the regeneration mechanisms, and later to the development of therapeutic strategies aimed at increasing the endogenous potential of remyelinization. PMID- 10729913 TI - [Role of axonal signals in myelination of the central nervous system]. AB - The myelination of the axons of the central nervous system (CNS) is assumed by the oligodendrocytes, which depend at least in part on signals of axonal origin. The axonal influence on myelination seems to consist of the sum of positive and negative factors, which can either act on the axon or on the oligodendrocyte, allowing the neuron to decide when and where myelinization is initiated. The induction factors appear to be mediated, in some cases, by electrical activity. Among the negative factors, certain factors such as the adhesion molecule PSA NCAM seem to act by inhibiting the adhesion between the axon and the oligodendrocytic extension. Others, such as the inhibitory signalling pathway, jagged1/Notch1, appear to trigger an inhibitory oligodendroglial signalling, therapy preventing maturation and myelination. The recent determination of the role of these axonal signals has provided a new approach to the mechanisms of normal myelination. These results could be extrapolated to the process of remyelination in human demyelinating pathologies such as multiple sclerosis, and open up new therapeutic research possibilities aimed at neuronal protection. PMID- 10729914 TI - Do central nervous system axons remyelinate? AB - In multiple sclerosis (MS), one of the most frequent demyelinating diseases in man, remyelination of demyelinating lesions exists but is often incomplete. Also reported in experimental models of demyelination, this phenomenom confirms the regenerating potential of the demyelinated central nervous system (CNS) and, in particular, the existence of an endogenous mechanism of oligodendrocyte renewal. Failure in efficient remyelination could result from exhaustion of the pool of remyelinating cells, loss of axons and absence of a permissive environment for remyelination. Identifying the nature and the origin of the cells capable of generating new oligodendrocytes for remyelination could contribute to strategies to activate these cells, and thereby enhance their potential for myelin repair. Within the adult CNS, several cell types are capable of generating new oligodendrocytes following myelin damage: post-mitotic oligodendrocytes frequently found at the lesion site, oligodendrocyte progenitors whose existence has been confirmed both in vitro and in vivo, and multipotent cells localized in the germinative areas of the brain and the spinal cord. Although restricted to particular sites of the CNS, these multipotent cells, which maintain the capacity to self-renew and to migrate throughout adulthood, could constitute a powerful source of remyelinating cells. The study of the mechanisms of proliferation, migration and differentiation of these cells in response to demyelination should allow the definition of new strategies to promote endogenous remyelination and develop therapeutic approaches for demyelinating diseases such as MS. This goal is an appealing alternative to the transplantation of myelin-forming cells and should efficiently complement strategies aimed at reducing neuronal loss and inflammation. PMID- 10729915 TI - Why are growth factors important in oligodendrocyte physiology? AB - Recent studies in chicken, rodents and transgenic mice have provided new insight on the nature of factors essential to oligodendrocyte development. Here we first review how sonic hedgehog (shh) graded signalling induces emergence of oligodendrocytes in the embryonic spinal cord from birds to man. We then discuss the way in which thyroid hormone successively signals different thyroid receptors to control fate determination, growth and differentiation in the oligodendrocyte lineage. Platelet-derived growth factor (PDGF) is a potent regulator of oligodendrocyte progenitor (OP) migration and proliferation, while insulin-like growth factor 1 (IGF-1) acts both on neurons and myelin-forming cells to promote myelination. The balance between OP proliferation and differentiation appears to be controlled by different sets of growth factors locally synthesized in the central nervous system (CNS) as well as glutamate. In experimental models of multiple sclerosis (MS), the neuregulin isoform glial growth factor 2, IGF-1 and some neurotrophins can promote remyelination after an episode of inflammatory demyelination. A future challenge is to determine how to induce multipotential neural precursors to generate migratory OP and enhance the remyelination process in the adult CNS. PMID- 10729916 TI - [Genetic predisposition for multiple sclerosis]. AB - Although the clinical manifestations and neuropathological signs of multiple sclerosis (MS) have been known for a century, the cause of this disease has not yet been determined. The epidemiological studies indicate that MS is of multifactorial etiology, including both environmental and inherited (genetic predisposition) factors. The role of the HLA system in genetic predisposition to MS has been known since the 1970s. As a result of the progress made in human genetics, it is now possible to study genetic predisposition to MS. PMID- 10729917 TI - Membranous glomerulonephritis: a morphometric study. AB - Archival material from 45 renal biopsies with a diagnosis of idiopathic membranous glomerulonephritis (MGN) were studied by computer-aided image analysis in order to evaluate the prognostic significance of glomerular and interstitial morphometry in MGN. The control group consisted of thirty seven normal renal biopsy specimens. The surface area, the perimeter, the major axis length and the shape factor of renal glomeruli as well as the percentage of the interstitial fibrosis were measured. All the morphometric parameters related to the size of glomeruli had significantly higher values in the patient group (p = 0.000 for all the parameters). However, no significant difference of the glomerular size between different stages of MGN was observed. In contrast, the percentage of interstitial fibrosis increased as the MGN stage rose (median values: 10.3% in stage 1, 14.2% in stage II, 26.9% in stage III, 28.9% in stage IV and 34.2% in stage V, Kruskal-Wallis ANOVA H = 37.645, p = 0.000). In the multivariate analysis the percentage of interstitial fibrosis was the only independent prognostic factor (p = 0.013). Our findings suggest that, in membraneous glomerulonephritis, the interstitial fibrosis increases as the MGN stage progresses, while the size of renal glomeruli has increased at a very early stage of the disease. This fact may indicate that interstitial fibrosis, not glomerular lesions, is mainly responsible for the reduction of renal function. PMID- 10729918 TI - Use of crypt isolation to determine loss of heterozygosity of multiple tumor suppressor genes in colorectal carcinoma. AB - Analysis of loss of heterozygosity (LOH) is very important in the study of tumor suppressor genes. However, accurate LOH analysis of tumor suppressor genes is difficult because of dilution by contaminating non-tumor DNA. Thus, enrichment of tumor DNA is required to accurately determine LOH of the tumor. We developed a new application of the fluorescent polymerase chain reaction by coupling it with crypt isolation to accurately assess the incidence of LOH of tumor suppressor genes in 45 colorectal carcinomas. LOH was observed at p53 in 26 of 37 tumors (70.3%), at APC in 13 of 35 (37.1%), at DCC in 16 of 25 (64.0%), at NF-2 in 5 of 23 (21.7%), and at nm23 H-1 in 7 of 30 (23.3%). We could clearly determine LOH of these genes because the crypt isolation technique was used. Although the incidence of LOH at each of these loci, as determined by using this technique, was similar to that obtained in previous studies using conventional methods, this method provides a simpler, more accurate way to assess LOH. In addition, the morphology of the samples can be analyzed before genetic analysis. PMID- 10729919 TI - Intracranial meningeal tumours in childhood: a clinicopathologic study including MIB-1 immunohistochemistry. AB - Primary tumours of the meninges with a relatively high tendency for malignant behaviour are uncommon in childhood. This study concerns 18 cases of meningeal tumours in children under the age of 16, of which 13 were meningiomas and five were other tumours arising in the meninges. Meningiomas showed a preponderance in females as in adult series, and the majority were supratentorial in localisation. The percentage of meningeal tumours and meningiomas among all brain tumours in our centre were 3.72% and 2.69%, respectively. Four out of 13 meningiomas were fibroblastic, four were transitional, one was meningothelial, two were psammomatous and two were papillary meningiomas. Seven (38.8%) out of 18 tumours showed anaplastic features, including two papillary meningiomas, two hemangiopericytomas, one mesenchymal chondrosarcoma, one pleomorphic sarcoma and one anaplastic meningeal tumour. Papillary meningiomas with hemangiopericytoma like solid areas were seen frequently in our cases (15.3%). Meningoangiomatosis was associated with two meningeal tumours. MIB1 (Ki-67) labelling indices (LIs) ranged between 0% and 13.6% (mean 1.83%) in benign, and between 1% and 20% (mean 7.2%) in malignant tumour, including papillary meningiomas. Mean MIB-1 LIs were 5.61% and 1.14% in non-recurrent and recurrent cases, respectively. MIB-1 LIs showed significant differences between benign and malignant meningeal tumours but no significant correlation either with prognosis or recurrence. Despite the fact that brain tumours are among the most common neoplasms of childhood, meningeal tumours are rare lesions, accounting for less than 2% of published series of intracranial neoplasms in childhood [5, 8, 18, 24, 30, 32]. It has been suggested that the clinical and pathological characteristics of meningiomas in this age group differ from those of adults [14, 18, 24, 45]. Besides meningiomas, there are a few reports of other meningeal tumours in childhood and difficulties in differential diagnosis may arise within this group, especially in anaplastic tumours [11, 13, 32, 44, 46]. One of the major problems in meningiomas and some tumours arising in the meninges is the discordance that arises between the histologic appearance of the tumour and behaviour [4]. Several studies have attempted to determine the proliferation potential of meningiomas, including immunohistochemical labelling with monoclonal antibodies to Ki-67, proliferating cell nuclear antigen (PCNA), and bromodeoxyuridine (BUdR); flow cytometric DNA analysis; or argyrophilic nucleolar organizer regions (AgNORs) counting [9, 10, 15, 19, 22, 26, 31, 35, 53]. The studies concerning proliferation markers have contradictory results [9, 10, 15, 26, 31, 42, 53]. MIB-1 detects the same or a similar epitope as the original antibody Ki-67 and reacts with a proliferation associated antigen expressed in all active parts of the cell cycle, G1, S, G2 and M (mitosis), but not in the G0 or quiescent phases [7]. In this study we examined the clinicopathological characteristics and MIB1 values of 18 meningeal tumours in children under the age of 16 years within the last 25 years (from 1970 to 1995). PMID- 10729921 TI - Apoptosis in breast carcinoma. AB - Apoptosis may play a major role in determining tumor growth and aggressiveness. The aim of this study was to examine the relationship between apoptosis, expression of bcl-2 and p53 proteins, proliferation index, and other clinicopathological features of breast carcinoma. Sixty-five formalin-fixed paraffin-embedded tissue sections from invasive ductal breast carcinomas were studied for the presence of apoptosis by the terminaldeoxynucleotidyl-transferase mediated dUTP-FITC nick end-labeling (TUNEL) method. Immunohistochemical methods were also used to determine the expression of estrogen receptor, Ki67, bcl-2 and p53 proteins. The number of apoptotic cells ranged from 2.0 to 236.0/10HPF (mean 36.26, median 28.0). The observation of 30 apoptotic cells/10HPF was more common in tumors > 3 cm, of histological grade III, with a high mitotic index, Ki67 index > or = 300, and p53 positivity; however, statistical significance was found only for the histological grade. Grade I and III tumors displayed an inverse association between the apoptotic index and bcl-2 and p53 protein expressions; grade I tumors frequently expressed bcl-2 (19/28), lacked p53 (20/28), and presented a low number of apoptotic cells (18/28), whereas grade III tumors tended to express p53 (12/17), lacked bcl-2 (13/17), and displayed a high number of apoptotic cells/10HPF (12/17). Multivariate analysis for survival revealed that estrogen receptors and apoptosis were independent variables. These data suggest that apoptosis, rather than proliferation index or expression of bcl-2 or p53 proteins, is an independent factor for the prognosis of survival. PMID- 10729920 TI - Colonic mucin-carbohydrate components in colorectal tumors and their possible relationship to MUC2, p53 and DCC immunoreactivities. AB - To clarify changes in mucus components during colorectal tumorigenesis, we developed novel monoclonal antibodies (Abs) against carbohydrate chains of human colorectal mucin (HCM) obtained from normal sigmoid and rectal mucosae. A hundred and ninety-nine cases of colorectal carcinoma and 67 cases of tubular adenoma, along with 250 normal colonic tissue samples, were investigated immunohistochemically. The results were compared with clinical stage, survival and MUC2 (core protein of the intestinal type mucin) expression, as well as with the status of the p53 and DCC (deleted in colorectal carcinomas) genes. In the normal colonic epithelium, HCM14 Ab reacted with the cytoplasmic regions of the goblet cells and enterocytes, while HCM21 Ab bound to mucous droplets in the former, suggesting a more mature carbohydrate structure. Both HCM14 and 21 scores were significantly decreased in adenomas and carcinomas. This is in line with an altered PAS-Alcian blue staining, indicating accumulation of mucins with incomplete or abnormal glycosylation in tumors. Levels of HCM14 and 21 binding tended to show a positive correlation with expression of MUC2 and DCC, and a negative association with p53 protein accumulation in carcinomas, although there was no apparent link to Duke's stage or the prognostic outcome. These findings suggest a possible involvement of alterations in mucin carbohydrate in colorectal tumor development. The observed changes may be associated with loss of MUC2 and DCC expression, as well as with p53 protein accumulation. PMID- 10729922 TI - Histopathological study of experimental poststreptococcal pneumonia in mice. Group A, type 50, streptococcal infection of murine nares controls with Staphylococcus aureus and E. coli. AB - Microscopic methods (light and electron microscopy, histochemistry, immunohistochemistry) have been used to assess previously unknown pulmonary inflammatory responses of specific pathogen-free (SPF) mice secondary to infection via the nares by group A, type 50, streptococci suspended in saline ("strep group mice"). As controls for the strep group mice, the animals were either injected with saline alone via nares (no lesions were seen), or with Staphylococcus aureus in saline ("staph group mice") or with E. coli ("E. coli group mice"). The three different bacterial species caused clearly different histological changes in the lung. In the strep group mice, the microscopic findings were consistent with the diagnosis of lymphocytic interstitial pneumonia of bronchiolovascular bundles, secondary to exaggerated pulmonary recirculation of lymphocytes, concomitant with vasoconstrictive angiopathy of encased pulmonary artery branches and nodular inflammatory cell aggregates in lung parenchyma. These aggregates either consisted predominantly of lymphocytes, or of mixed cells (neutrophils, lymphocytes, macrophages) or of activated macrophages only. In 18 of 22 inflamed lungs of strep group mice, no bacteria could be cultured from lung tissue. In staph group mice the microscopic findings are consistent with the diagnosis of lymphocytic interstitial pneumonia of bronchiolovascular bundles, secondary to exaggerated pulmonary recirculation of lymphocytes only. In 12 of 17 inflamed lungs of staph group mice, no bacteria could be cultured from lung tissue. In E. coli group mice the microscopic findings were consistent with the diagnosis of distal terminal bronchiolitis and early pleural-based pneumonitis, in which lymphocytes and neutrophils mingled with macrophages. In 10 of 11 inflamed lungs of E. coli group mice, no bacteria could be cultured from lung tissue. The morphologic approaches described here may have potential for unravelling the complex inflammatory processes underlying different forms of interstitial and parenchymal pneumonia. PMID- 10729923 TI - Hemorrhages of dorsal root ganglia and spinal cord in congenitally hydrocephalic HTX rat. AB - The effects on the brain caused by hydrocephalus have been examined in detail. However, only little attention has been paid to the possibility that hydrocephalus may affect the spinal cord and the spinal ganglia via the spinal canal. Therefore, the present study focused on the pathological changes seen in the spinal cord and the dorsal root ganglia. A total of 651 congenitally hydrocephalic HTX rats were used in this study. The age ranged from postnatal day 0 to postnatal day 520. All of the HTX rats were from littermates raised in our laboratory. Macroscopic and microscopic investigations demonstrated hemorrhages of the dorsal root ganglia in 134 rats among the 235 affected HTX rats. The hemorrhages of the dorsal root ganglia were observed most frequently in the lumbar ganglia and, less frequently, in the cervical ganglia. Of the 134 rats with hemorrhages in the dorsal root ganglia, 34 rats had hemorrhages both in the spinal cord and in the dorsal root ganglia. The spinal cord hemorrhages were distributed mainly around the central canal and in the ventral parts of the posterior funiculus at the lower thoracic and upper lumbar cords. These hemorrhages were seen only in those rats having progressive hydrocephalus. These findings suggest that increased cerebrospinal fluid pressure can cause congestion of the radicular veins, leading to hemorrhages of the spinal cord and the dorsal root ganglia. PMID- 10729924 TI - Formaldehyde neurotoxicity in animal experiments. AB - The aim of this study was to determine whether the inhalation of formaldehyde has a neurotoxicological impact. Forty Wistar rats (Lew.1/K) were trained to find food in a maze within a particular time. When all animals were at an equal level, 13 rats inhaled 2.6 ppm and 13 others inhaled 4.6 ppm formaldehyde 10 min/d, 7 d/week for 90 d. The control group comprised 14 animals inhaling water steam according to the same exposure pattern. During the exposure period and the post trial observation stage (30 d), the time required to find the food and the number of mistakes made on the way were recorded. Between the animals exposed to formaldehyde and the control group a statistically significant difference for both parameters was observed (p < 0.05). The animals exposed to formaldehyde needed more time and made more mistakes than the animals of the control group while going through the maze. The results underline the necessity for a systematic observance of precautions in case of occupational or dwelling-related formaldehyde exposure, and allow us to classify formaldehyde as "probably neurotoxic". Further investigations are required to assess the neurotoxicologic impact of subchronic formaldehyde exposure. PMID- 10729925 TI - Congenital mesoblastic nephroma: report of a Case with review of the most significant literature. AB - AIMS AND BACKGROUND: Congenital mesoblastic nephroma (CMN) is a rare pediatric tumor of the kidney with the highest peak of incidence during the first 3 postnatal months. It has previously been confused with Wilms' tumor (which, on the contrary, is rare during the first six months of age and is still considered a histogenetic congener). CMN almost always has a favourable prognosis. Therefore, CMN needs to be correctly diagnosed and differentiated from other pediatric renal neoplasms. Two morphological subtypes are currently distinguished histologically: the classical or leiomyomatous type and the atypical or cellular type. Mixed forms with a combination of the two patterns are also on record. Recurrence and even tumor-related death have been described in the literature and always related to the atypical form or to the mixed form, particularly in patients aged more than 3 months and in those cases in which the surgical removal was not complete. Opinions concerning post-surgical clinical management, especially in regard to adjuvant therapy, are not unanimous. METHODS: A case of CMN, predominantly of the classical histological subtype diagnosed in a baby with a follow-up of 6 years, is herein presented. The tumor was discovered at birth and surgically removed after one month. Since the tumor showed a high mitotic index (one of the characteristics of the cellular subtype) and the perirenal fat was focally involved with the tumor, the possibility of giving adjuvant chemotherapy was considered. Flow cytometric analysis was also performed which showed a diploid DNA content of neoplastic cells. RESULTS: The tumor was completely removed, surgical margins were free histologically, and no clear-cut histological features of the atypical subtype were noted. Flow cytometrically, it showed the euploid DNA content. Consequently no additional therapy was given. Six years after surgery the patient is developing well and is free of disease. He has regular follow-up examinations. CONCLUSIONS: CMN almost always pursues a benign clinical course if diagnosed under three months of age and if totally surgically excised independent of histological type. Criteria for management of atypical cases are not unanimous in regard to the benefit of additional therapy after surgery. PMID- 10729926 TI - Elastofibroma of the sigmoid colon. AB - A case of elastofibroma occurring in the sigmoid colon of a 69 year-old woman is reported. The woman presented for survey of her gastrointestinal tract. Colonoscopy disclosed two polyps in the sigmoid colon, one of which was clinically considered to be recurrent adenoma. Histologically, the lesion had characteristic eosinophilic fibers and globules, termed elastofibroma fibers with hematoxylin and eosin stain. In addition, these elastinophilic materials were digested by elastase. Histological evaluation confirmed the diagnosis of elastofibroma. Our case might suggest that it is the result of long-term fibrosis after previous endoscopic resection of a sigmoid colonic adenoma. PMID- 10729927 TI - Genetics of patients with borderline personality disorder. AB - An overview of the existing literature suggests that traits similar to BPD are influenced by genes. It is too early to say to what extent BPD is also influenced by genes, but because personality traits generally show a strong genetic influence, this should also be true for BPD. Nonetheless, if the equal environment assumption were to be violated for MZ and DZ pairs, twin studies may be overestimating genetic effects and hiding the effect of common family environment. The less than-ideal reliability of measurements used in this research may also reduce the effects of genes and common environment while increasing the effects of unique or nonshared environment. The effect of genes on the development of BPD is likely substantial. The effect of common family environment may be close to zero. More studies, large and small, are needed to reach firmer conclusions about the influence of genetics on BPD. PMID- 10729928 TI - The biology of impulsivity and suicidality. AB - Abnormalities of 5-HT and noradrenergic functioning have been implicated in aggressive impulsivity, SIB, and suicidal behavior. The role of DA and GABA in human studies of these behaviors requires further investigation. Most studies suggest that impulsive aggression is related to lower levels of CNS 5-HT. Some studies demonstrate that increasing NE correlates to impulsive aggression, whereas other studies demonstrate an opposite relationship. The role of NE in impulsive aggressive behavior is still unclear. Self-injurious behavior is similar to impulsive aggression in that it seems to be mediated by the neurotransmitter systems previously mentioned. For example, the presence of lower levels of 5-HT and abnormalities in the DA system are related to SIB in patients with BPD and depression. SIB severity also seems to be influenced by neglect (e.g., severe isolation during rearing). As animal studies suggest, increasing the amount of isolation and an earlier onset of isolation increase the severity of SIB. Suicidal behaviors and the lethality of suicide attempts may also be linked to the abnormalities in neurotransmitter systems similar to those found in patients with impulsive aggression and SIB, namely, lowered 5-HT transmission and enhanced DA and NE functioning. Understanding the biological triggers of impulsive aggression or SIB may allow for the evaluation of suicidal attempts and completion from a different perspective and, in conjunction with genetic predictors, may eventually help with the early prediction and prevention of suicidal behaviors. Additional studies of live subjects and postmortem brains will assist in clarifying the neurobiology of suicidal behaviors that are common to many disorders and are clinically relevant to BPD. PMID- 10729929 TI - Neurotransmitter dysfunction in patients with borderline personality disorder. AB - Neurotransmitter system dysfunction may contribute to the borderline personality disorder traits of impulsive aggression and affective instability. This article reviews evidence from neurochemical assays, receptor-density studies, neuroendocrine-challenge paradigms, functional neuroimaging studies, and candidate-gene research, which converge to identify particular neurotransmitter systems that seem to be dysregulated in patients with borderline personality disorder. PMID- 10729930 TI - Borderline personality disorder. Neuropsychological testing results. AB - Earlier clinical hypotheses regarding borderline personality disorder, based on projective psychological testing and intelligence tests, are examined. Recent findings of comparative deficits in this population in the domains of verbal and visual memory as well as visual perception are reviewed, together with clinical relevance. Efforts to examine the way individuals with this disorder process emotion-laden material are discussed, with suggestions for further research. PMID- 10729931 TI - Borderline personality disorder. Overview of biologic factors. AB - Exploring the biology of neurotransmitters and polymorphic genes that influence behavior most certainly will increase understanding of the complexity of personality disorders and the people who suffer with them. Continuing biologic exploration of the personality disorders will also provide new and valuable information that will inform and direct ever more sophisticated and specific psychiatric treatments. Further, it will supply knowledge that might begin to erode the strong biases and negative labels that have for too long been applied to these patients. These labels may have been applied not because these patients are bad people, but because of ignorance in understanding their complexity and limited ability to improve their condition. Nonetheless, clinicians must refrain from concluding that the answers to all problems in psychiatric treatment can be answered by the molecular geneticist or biologic researcher. As Reiss et al have written when speaking about nonshared environmental effects and its role in the development of psychopathology: "Psychiatry has been forced into the chronically uncomfortable position of straddling biomedicine and the social sciences and seems always to hunger for relief.... [Yet] the data simply do not permit a conception of the future centered on a straightforward biomedical answer to the fundamental question of the pathogenesis of major disorders. Indeed, a balanced image of the future contains a growing and equal partnership of the social sciences and molecular biology." PMID- 10729932 TI - Childhood precursors of borderline personality disorder. AB - The childhood precursors of borderline personality disorder are largely unknown. High-risk studies of "borderline children," as well as studies of the children of adult borderline patients, can shed light on the issue. Prospective research is needed to address these problems substantively. PMID- 10729933 TI - Childhood experiences associated with the development of borderline personality disorder. AB - In time, mental health professionals will understand the etiology of BPD more fully. Although enormous strides have been made in the past decade, research into the multifactorial basis of BPD is still in its infancy. In particular, studies of children at high risk for developing BPD are needed. For now, the author suggests that one can admire patients with BPD for the integrity with which they have dealt with their pain. After all, not many people remain so loyal to and so respectful of such disheartening childhood experiences. PMID- 10729934 TI - Attachment and borderline personality disorder. A theory and some evidence. AB - An attachment theory approach to severe personality disorder is described. Evidence is presented that suggests that representations of attachment relationships and attachment behaviors of patients with this diagnosis are commonly disorganized in character. It is argued that the capacity to develop mental representations of mental states in self and other (reflective function) develops in the context of attachment relationships and that disorganization of attachment undermines this process. Such disorganization can be associated with trauma but may also be linked to other biological and psychosocial deficits. Many of the clinical characteristics of patients with borderline personality disorder may be seen as consequences of disordered self-organization and a limited rudimentary capacity to think about behavior in mental state terms. The relevance of this model for the practice of psychotherapy with this group of patients is discussed. PMID- 10729935 TI - Sociocultural conceptions of the borderline personality. AB - Although sympathetic to most analytic conceptions of the origins of borderline personality disorder, an addendum reflecting the recent and divisive impact of rapid social and cultural changes is proposed. Specifically noted are social customs that exacerbate rather than remediate early errant parent-child relationships and the diminished power of once reparative institutions that no longer compensate for these ubiquitous early problems. PMID- 10729936 TI - Prospective studies of outcome. Understanding mechanisms of change in patients with borderline personality disorder. AB - The authors propose a conceptual model of recovery from BPD that takes into account empiric findings regarding the dynamic relationships between severity of BPD symptoms, age, impulsivity, and duration of a healing relationship. The model assumes that each factor related to the course of BPD essentially lies on a continuum and interacts with the remaining important factors. The model also preserves the relative relationships, with age and impulsivity being primarily important and the duration of a healing relationship being independent but overall of less importance in modifying the course of illness. (Having said this, however, modifying the presence of a healing relationship may be more accessible for therapists.) By 7 to 10 years' follow-up, half of patients with BPD will be characterized as remitted. Efforts to keep these patients alive through the early course of the disorder will see many patients through to the resolution of the disorder. Impulsivity is a primary element of the disorder that predicts the course of BPD, with impulsivity becoming less as patients age. Efforts to modify the levels of impulsivity, such as rehabilitation for substance-abuse disorder, pharmacologic interventions, and cognitive-behavioral therapies to reduce impulsivity, may affect the course of the disorder. Exposure to an intimate relationship, such as a successful marriage, also may impact the course by allowing the person to be involved in a healing relationship. Additional studies are needed on the role of couple and marital interventions with BPD patients because the development of successful relationships may modify the course of the disorder. This model of change in BPD is conceptually heuristic and accounts for the complex interplay of factors that modify the course of BPD but accounts for the empiric findings to date. This model is also presented to assist clinicians in understanding and conceptualizing the primary features affecting recovery for these patients. As new findings become available, this model should be modified to reflect the nature of those findings and extend the understanding of the course of BPD and the factors that modify that course of illness. PMID- 10729937 TI - Research on dialectical behavior therapy for patients with borderline personality disorder. AB - Research evidence to date indicates that, although DBT was developed for the treatment of patients with suicidal behavior, it can be adapted to treat BPD patients with comorbid substance-abuse disorder and be extended to other patient populations and the treatment of other disorders. Across studies, DBT seems to reduce severe dysfunctional behaviors that are targeted for intervention (e.g., parasuicide, substance abuse, and binge eating), enhance treatment retention, and reduce psychiatric hospitalization. Evidence suggests that additional research is warranted to examine which components of DBT contribute to outcomes. Although preliminary, skills coaching seems to be a crucial ingredient in producing reductions in parasuicidal behavior, and specific strategies (e.g., validation, balance of change, and acceptance interventions) may play an important role in positive behavioral change. Several investigators are evaluating the efficacy of DBT. For example, Asberg et al at the Karolinska Institute in Sweden have begun a pilot study comparing DBT for women who have made multiple suicide attempts to transference focus psychotherapy, a psychodynamic therapy developed by Kernberg. They have planned a randomized clinical trial to compare DBT and transference focus psychotherapy with TAU in the community. van den Bosch has completed a randomized clinical trial for women who met criteria for BPD and substance abuse comparing DBT-S with TAU. Lynch is conducting a randomized clinical trial examining the efficacy of DBT skills training plus medication versus medication only for the treatment of moderate to severe depression in the elderly. Results from these studies should become available over the next several years, providing further empiric evidence by which to evaluate the efficacy of DBT. Additional development of DBT seems warranted to improve its efficacy, and additional investigation is needed to establish its effectiveness in public health settings. Analyses from existing data sets of factors that predict treatment response and elements of the treatment that contribute to outcome are needed. Also, longitudinal follow-up studies to determine suicide rates and maintenance of treatment gains are needed. Because DBT has been adopted in a variety of clinical settings, effectiveness studies are needed. Given the difficulty of conducting treatment research with chronically suicidal individuals, perhaps the largest challenge to further treatment development is recruiting young investigators who are willing to conduct research in this area. Nevertheless, in the 6 years since the treatment manuals were published, DBT seems to be a step toward more effective treatment for severely multidisordered patients. PMID- 10729938 TI - Psychopharmacology of borderline personality disorder. AB - Pharmacotherapy for patients with borderline personality disorder is directed against the psychobiology of cognitive-perceptual, affective, and impulsive behavioral symptoms. A symptom-specific method using current empiric evidence for drug efficacy in each symptom domain is proposed. Drugs in each medication class have some potential utility against specific symptoms in patients with borderline personality disorder. PMID- 10729939 TI - Clinical guidelines for psychotherapy for patients with borderline personality disorder. AB - In planning a course of psychotherapy for borderline patients, clinicians must take into account the heterogeneity of the clinical presentation in the borderline domain. Borderline personality disorder is usually accompanied, for example, by one or several "symptom disorders," such as an eating disorder, depression, posttraumatic stress disorder, premenstrual tension, dissociative disorder, or anxiety disorder--not to mention one or more other personality disorders. The nature of the "comorbidity" in each patient will determine which medications, if any, are applicable. The accompanying personality disorders will have an impact on amenability to psychotherapy. The main forms of therapy currently in use are supportive, cognitive-behavioral (including dialectical behavioral therapy) and psychodynamic (including transference-focused psychotherapy). Group therapy is often used adjunctively with any of these approaches. The main question is no longer, Which of these approaches is best, overall? but rather, Which approach is best for which type of borderline patient? Contemporary research is addressed to this latter question. PMID- 10729940 TI - A practical approach to the treatment of patients with borderline personality disorder. AB - This approach to the treatment of BPD is part of a general framework for treating all forms of personality disorder. The therapeutic model has two components: (1) general strategies to manage core self and interpersonal pathology that characterizes all cases of personality disorder and (2) a specific or tailored component required to treat the problems of individual cases. The general strategies, derived from generic models of therapeutic change and self psychology, emphasize the therapeutic relationship and the importance of a consistent treatment process, validation, and building motivation. A combination of specific interventions, including medication and psychotherapeutic strategies drawn from treatment approaches, is incorporated into this framework as required. The overall model is not an eclectic approach to treatment but an integrated framework based on an understanding of borderline pathology that emerges from recent research. PMID- 10729941 TI - The intrapsychic and intersubjective in psychoanalysis. AB - Both the intrapsychic and the intersubjective take part in the analytic process. A pointless struggle for supremacy may await those who support either point of view exclusively. If the "objectal" perspective is well known, the "subjectal" one is less theoretically defined: it includes the series of the ego, the self, the subject, the I, etc. The drive is the matrix of the subject. An examination of the relationships between perception and representation raises the question of the connections between drive and object. The object is the revealer of the drive. A revision of Freud's theory must underline the role of the object, which is unduly neglected. The new paradigm should consider the indissociable couple, drive-object. The construction of the object leads retroactively to the hypothesis of the drive, which reciprocally constructs the object. The function of the similar other (autre semblable) is defined as a fundamental link (desire and identification). The intersubjective relationship connects two intrapsychic subjects. Force and meaning are intertwined and combine their effects. Psychic causality is at the crossroads of the biological (metabiological) and the cultural. This paper examines the transition from the first topographic model to the second. PMID- 10729942 TI - The unsolved puzzle of trauma. AB - This paper covers the inherent difference between psychoanalysis, which deals with unconscious fantasies, and trauma, which emphasizes the psychic repercussions caused by events in external reality. This distinction has led to variations in the treatment for victims of trauma, which also reflects the duality commonly observed in this group of patients. I suggest that trauma leads to unconscious guilt and the need for exoneration because individuals identify with their fate, and that this guilt can and indeed should be analyzed. Further studies might show that dividedness predates the experience of trauma and that this mode of defense mitigates the impact of trauma. PMID- 10729943 TI - Intersubjectivity and interaction in the analytic relationship: a mainstream view. AB - The authors conceptualize intersubjectivity as a meta-theory that reflects the inherent nature of human relatedness and is conceptually independent of any particular theory of mind or school of psychoanalysis. Their view of intersubjectivity joins the emotional life of the analyst to that of the patient and places the analytic relationship at the center of the analytic process. They contrast intersubjectivity with traditional classical conflict theory so as to clarify the relevance of intersubjectivity for psychoanalytic clinical theory and therapeutic practice. In so doing, they hope to direct analysts more firmly toward the study of the unconscious dyadic contributions to the affective, inactive, and interactive dimensions of the analytic situation and their impact upon the patient's actions within and experience of the analytic relationship. To illustrate their thesis, two hours from an analysis are presented in detail. PMID- 10729944 TI - From countertransference to "passion". AB - Bion's ideas may be extended to describe an emotional phenomenology of the analyst's subjectivity and a methodology which helps differentiate countertransference enactments from fuller emotional participation. Bion called the process of integrating and utilizing one's most basic and important emotions to make meaning, "passion." The analyst's primal feelings--of love, hate, and curiosity--serve as a central organizer of meaning in the analytic interaction. These feelings involve pain, and to the extent the analyst unconsciously decides to evade or foreclose the evolution of the feelings, such that they remain unintegrated in the thinking process, the analyst is liable to become mired in repetitive transference-countertransference experiences without establishing fresh meaning. A case example illustrates the relevance of "passion" to contemporary relational theory and practice. PMID- 10729945 TI - Idealization and mourning in love relationships: normal and pathological spectra. AB - Prior to the last two decades, psychoanalytic literature focused on the psychopathology of sexual life, rather than on an integrated overview of love relationships. Only in the last twenty-five years has its scope been expanded to include the psychodynamics and phenomenology of love relationships per se. Nevertheless, a selective, critical review of the literature indicates that little attention has been paid to a) the interrelation of narcissism, self esteem, and love relationships; b) the role of the ego-ideal and idealizations in the capacity for falling in love and sustaining love relationships; and c) the faculty for, and/or impediments to, transcending intrapsychic self-boundaries in mature love relationships. In this paper, a brief exposition of the ego-ideal developmental sequences and their integration into the superego as a differentiated structure serves as an introduction to the proposal of a developmental continuum of mechanisms of idealization and their respective nodal transmutations throughout the life cycle. This developmental continuum may contribute to the ongoing elucidation of the aforementioned problems. This referential frame is ultimately applied to the exploration of categorical and dimensional pathological variations of idealization and mourning in love relationships and in different levels of personality organization: neurotic, borderline, and narcissistic structures. A clinical vignette illustrates some of the correspondence criteria between this frame of reference and its clinical applications. PMID- 10729946 TI - On discovering otherness of mind. PMID- 10729947 TI - [Legal and structural improvements in rehabilitation--positions of legal social insurance and recommendations for policy]. PMID- 10729948 TI - [Considerations for a "general model of psychosomatic rehabilitation" with departments for appropriate treatment intensity and required length of stay]. AB - Studies undertaken in the scientific area of general systems theory generally seek to elucidate central interrelationships. This paper its no exception in that it presents a "general model of psychosomatic rehabilitation". This new Y-shaped model was inspired firstly by the "general model of psychotherapy" and secondly by general systematic considerations on medical rehabilitation. It was first used to assess the progress of patients undergoing inpatient psychosomatic rehabilitation and to determine the relationship between treatment intensity and length of time-sensitive psychotherapy. PMID- 10729949 TI - [Analysis of the need for clinical neuropsychology in inpatient neurological rehabilitation (phases C and D)--an empirical study of 788 patients]. AB - Recently, several studies concerning the need for neuropsychologists in early or in out-patient rehabilitation settings have been submitted; however, an analysis of classical in-patient neurological rehabilitation which is most important by virtue of its numerical impact has not yet been performed. An empirical study focussing on the need for neuropsychologists during in-patient rehabilitation (phases C and D) is discussed. The two-year study records deficits observed among all patients with damage to the central nervous system (n = 788) admitted to a neurological rehabilitation clinic in north-western Germany. The deficits were divided into the following groups: memory, visual-constructional, deficits involving the planning process, attention deficits and emotional disturbances. Depending on the deficits displayed, patients were prescribed either individual or group therapy, or a combination of both. An empirical pattern of job distribution for neuropsychologists is calculated for the first time based upon findings of the study, which concern the time required for neuropsychological diagnostics, neuropsychological therapy, consultation of relatives and a fixed time-budget established for team supervision and documentation. PMID- 10729950 TI - [Catamnestic results and questions in neurologic rehabilitation--in relation to encephalomyelitis disseminata (multiple sclerosis)]. AB - Owing to the changes in the laws governing the health care system, medical rehabilitation for patients treated as in-patients is becoming increasingly a subject of discussion, especially for chronic illnesses. Using multiple sclerosis (encephalomyelitis disseminata) as an example, a special database is used to examine the courses, contents, improvements or deteriorations seen in in-patient rehabilitation as experienced by 901 patients who were in rehabilitation clinic in the years 1995-1998. This period was selected to allow the ascertainment of possible effects of far-reaching changes in the law taking effect from autumn 1996 onward. The EDSS (Expanded Disability Status Scale) was applied, and patients financed by statutory health insurances were compared with those financed by pension insurance funds for length of stay, outcome and therapeutic content. In the case of 256 patients being financed by pension funds it was possible to use the classification of therapeutic services as a basic tool and the connection with degree of disability was recognizable: different degrees of severity of the illness led to different treatment programmes. Health insurance patients were always more severely ill than those financed by pension insurance. Interestingly, the legal changes had no effect on the treatment outcomes of the patients. The data retrieved confirm the efficacy of the rehabilitation programmes. PMID- 10729951 TI - [Stage-oriented nursing care rates in neurologic rehabilitation]. AB - The aim of this pilot study done in a neurological rehabilitation center for children and adolescents was to increase the transparency of costs in relation to work and to gain differentiated daily rates according to the kind and amount of nursing and multidisciplinary treatment of patients in different phases (B, C, D) of neurorehabilitation. Presented and discussed are methods and instruments for performance registration and connecting them to costs. As the study shows quite different daily rates are necessary to assure adequate treatment for young patients in different phases--early to late--of neurological rehabilitation. PMID- 10729952 TI - [The BARMER AR procedure as a demand-oriented management system--intermediate term evaluation and improvements]. AB - The BARMER Ersatzkasse health fund has introduced its own procedure to make medical rehabilitation available immediately after acute care (Anschlussrehabilitation, AR). Regional cooperation among hospitals and rehabilitation clinics, a differentiated service scheme as well as extensive quality management are the main elements of this case management system. Results of the basis documentation after conclusion of the test period are presented and aspects of specific demand are discussed. PMID- 10729953 TI - [Comment on the contribution "New research data and considerations in managing wakeful coma patients" by A. Zieger]. PMID- 10729954 TI - [Profile comparison systems and performance diagnostic, electronic data processing-supported technology--their use in improving responses to social medicine questions and expert assessments and planning of rehabilitation measures]. AB - Systems for comparing ability and requirement profiles as well as instruments for evaluating functional capacity are current topics in rehabilitation. Only few of them however are related to vocational rehab. This article describes the present state of affairs in the development in procedures, instruments and methods to measure work-related human functional capacity with the aim of vocational rehabilitation and integration, helping to obtain objective results to decide about further steps. They are an addition to sociomedical advice in assessment and vocational centres, and serve to expand the common basis of the WHO International Classification of impairments, disabilities and handicaps (ICIDH). The purpose is intervention. Abilities are contrasted with requirements, as the basis for defining a need for intervention and realizing appropriate action. This is the thinking integrated in the German profiling assessment and documentation system IMBA--Integration von Menschen mit Behinderungen in die Arbeitswelt (Integration of people with disabilities into worklife). Diagnostic, computer based technology and work simulation and measurement to evaluate functional capacity as developed and used in the U.S. is complementary to IMBA. Work simulation is based on databases, such as the D.O.T., and includes standardized work requirement profiles. Such systems have been imported from the United States of America to Switzerland and the Netherlands and now to Germany. There are numerous connections with German assessment systems, and the fundamentals of ICIDH are included. Common application in the field of rehabilitation is dealt with, also in view of pensioning issues. The need for user training and quality management are reported in this article as well. PMID- 10729955 TI - Attachment and coercive sexual behavior. AB - This study examined the relationships between childhood attachment and coercive sexual behavior. One hundred sixty-two male undergraduate students completed self report measures of childhood maternal attachment, childhood paternal attachment, adult attachment, antisociality, aggression, and coercive sexual behavior. As predicted, insecure childhood attachment, especially insecure paternal attachment, was associated with antisociality, aggression, and coercive sexual behavior. Moreover, childhood attachment independently predicted coercive sexual behavior after antisociality and aggression were statistically controlled. The hypothesis that paternal avoidant attachment would predict coercive sexual behavior independently of its relationship with aggression and antisociality was also supported. Posthoc analysis indicated that maternal anxious attachment was associated with antisociality and that paternal avoidant attachment was associated with both antisociality and coercive sexual behavior. These results are consistent with criminological and psychological research linking adverse early family experiences with offending and lend support to an attachment theoretical framework for understanding offending behavior in general and sexual offending behavior in particular. PMID- 10729956 TI - Childhood attachments, sexual abuse, and their relationship to adult coping in child molesters. AB - The present study examined the parent-child attachments, typical coping styles, and childhood sexual abuse among 30 child molesters, 24 nonsexual offenders, and 29 nonoffenders. The results indicated that all subjects reported greater security in their attachments to their mothers than to their fathers and the insecure patterns of childhood attachments were related to ineffective adult coping. The only difference observed in characteristic coping showed that child molesters were more likely to engage in emotion focused strategies. Child molesters reported having experienced high levels of childhood sexual abuse and these experiences appear to have been more distressing to them than to other subjects. The results are discussed in terms of their implications for theory and treatment. PMID- 10729957 TI - The public health approach to the prevention of sexual violence. AB - This paper presents the public health approach to the prevention of sexual violence. The public health approach addresses primary, secondary, and tertiary levels of prevention. An explanation of each level of prevention is given as it relates to the area of sexual violence. Additionally, the public health approach attempts to shift the focus of prevention from potential victims to potential perpetrators. The four steps of the public health model are presented with examples of how the Centers for Disease Control and Prevention are applying this model to sexual violence prevention. PMID- 10729958 TI - Were adolescent sexual offenders children with sexual behavior problems? AB - This article compares responses of three groups of incarcerated adolescents who admitted to sexual offending in an anonymous survey project on measures of trauma, sexual offending, the relationship between trauma and perpetration, and adjudication status. The first group admitted to sexual offending before the age of 12 only (n = 48), the second after the age of 12 only (n = 130), and the third before and after the age of 12 (n = 65). More than 46% of the sexually aggressive adolescents began their deviant behaviors before the age of 12. Level and complexity of perpetration acts were more severe for the continuous offenders than for the other groups. Victimization and perpetration were significantly correlated for all three groups. This study supports a social learning hypothesis for the development of sexual offending by adolescents. Implications for research and clinical practice are drawn. PMID- 10729959 TI - A theory of mind perspective on cognitive, affective, and intimacy deficits in child sexual offenders. AB - Previous work in the area of sexual offending has suggested that factors such as intimacy deficits, problems empathizing with victims, and cognitive distortions have all been associated with the genesis and maintenance of sexual abuse. While researchers have constructed theories to account for the role of these variables in sexual offending, a framework that unites their study is lacking. Recently Ward, Keenan, and Hudson have proposed that sexual offenders may suffer from a deficit in their ability to understand and attribute mental states to others. Their review of the literature on the etiology of sexual offending suggested that intimacy deficits, empathy deficits, and cognitive distortions all point to a lack of awareness of other peoples' beliefs, desires, perspectives, and needs, what is commonly referred to in the developmental literature as a theory of mind. In this paper, we expand on this argument, illustrating some of the developmental pathways by which deficits in one's theory of mind can explain the pattern of deficits exhibited by many sexual offenders. PMID- 10729960 TI - An exploration of child sexual abusers' sexual fantasies before and after treatment. AB - Although there is a substantial literature looking at the relationship between deviant sexual fantasies and child sexual abuse, there is scant previous work that focuses upon the actual content of such fantasies. The present study looks at child sexual abusers' deviant fantasies both pre- and postintervention. Using both qualitative and quantitative methodologies, a description of the frequency and content of, and triggers for, child sexual abusers' deviant fantasies is reported both pre- and postintervention. The implications of this information for subsequent intervention programs is explored. PMID- 10729961 TI - [The Vienna Medical School: on the road to the 3d Vienna Medical School or break with the tradition?]. PMID- 10729962 TI - [Glomus caroticum chemodectoma. Review on current diagnosis and therapy]. AB - Carotid body tumors are rare neoplasms arising from the small chemoreceptor organ in the adventitia of the common carotid bifurcation. Patients with carotid body tumours usually present with a gradually enlarging non-tender anterolateral neck mass. Differential diagnosis includes metastatic lymph nodes, carotid artery aneurysm, salivary gland tumour, branchial cleft cyst, and neurogenic or thyroid tumours. When such a lesion is suspected, a non-invasive Doppler colour flow ultrasonography enables the clinician to arrive at a definite diagnosis. Subsequent arteriography is mandatory, because the finding of an intensely blushing hypervascular mass spreading into the carotid bifurcation further supports the diagnosis and provides accurate preoperative information concerning arterial blood supply. Computed tomography scanning is appropriate to delineate the relation of the tumour to adherent structures, while magnetic resonance tomography demonstrates the relation of the tumour to the adjacent internal jugular vein and the carotid artery. Selective embolization should be performed for safe surgical removal with less bleeding. Early surgery is the treatment of choice and is recommended in order to minimize major risks. Subadventitial resection is the most established technique. Radical resection prevents local recurrence and has the best long-term results. Removal of the internal or common carotid arteries can become mandatory in selected cases of extensive disease. Surgical treatment by an experienced team is associated with considerably low mortality and morbidity. PMID- 10729963 TI - Urinary apo(a) excretion is not altered by changes in glomerular filtration rate and renal plasma flow in healthy males. AB - Lipoprotein(a) (Lp(a)) is an independent risk factor for atherosclerotic disease. However, information concerning the site of Lp(a) catabolism and breakdown is scarce. Several studies have shown that, in renal insufficiency, plasma Lp(a) levels are elevated, and that after normalisation of kidney function they return to normal. We have recently shown that fragments of apo(a) are found in the urine of healthy individuals. Despite this evidence that apo (a) is excreted into the urine, the mode of excretion of apo(a) remains unclear. Since it has been reported that intravenous infusion of somatostatin can reduce glomerular filtration rate (GFR) and renal plasma flow (RPF), we analysed urinary apo(a) excretion in ten healthy volunteers receiving somatostatin infusions. The infusion of somatostatin led to reversible changes in GFR and RPF. Apo(a) excretion was constant in all 10 individuals over the entire time course when normalised for creatinine. There was a highly significant correlation between plasma Lp(a) levels and urinary apo(a) values. Changes in renal plasma flow and glomerular filtration rate did not alter urinary apo(a) excretion. We conclude that a constant amount of apo(a) is excreted into urine, depending on plasma Lp(a) levels, and that urinary apo(a) excretion is not altered by changes in GFR and RPF in healthy males. PMID- 10729964 TI - [The effect of inhalable dust particles (PN10) on lung function and respiratory symptoms of school children in Lower Austria]. AB - The effect of PM10 (particulate matter less than 10 microns in diameter) on respiratory symptoms and lung function was evaluated in 881 children (aged 8 to 11 years) in 8 communities in Lower Austria. In each community, air pollution data (PM10, SO2, NO2, O3) were collected. The examination of each child included a questionnaire (spring 1996), and two lung function tests (autumn 1995, spring 1996). Statistically significant relationships were observed between PM10 levels (annual mean, 15.8-26.9 micrograms/m3) and parameters of lung function (adjusted for sex, height, atopy, passive smoking, altitude, temperature). A 10 micrograms/m3 increase in the last two weeks' mean PM10 in spring 1996 was associated with a 0.05% decrease in FVC, a 0.05% decrease in FEV1, a 0.15% decrease in MEF50, and a 0.13% decrease in MEF75-25. Furthermore, a 10 micrograms/m3 increase in last year's mean PM10 was associated with a 0.07% decrease in FVC. No association between the prevalence of respiratory symptoms and the last year's mean PM10-exposure was found. Our study demonstrates a small effect of low-level particulate air pollution on lung function of healthy school children. PMID- 10729965 TI - [Comparison of manual lymph drainage with physical therapy in complex regional pain syndrome, type I. A comparative randomized controlled therapy study]. AB - Sympathetic blockage and physiotherapy are among the most effective treatment approaches for the complex regional pain syndrome (CRPS). It is important to institute the treatment as early as possible in order to avoid major functional limitations of the affected limb. Unfortunately, there is a paucity of vigorously applied randomised or placebo-controlled trials for these therapeutic approaches. A prospective randomised study of 35 outpatient clinic patients with type I complex regional pain syndrome of the lower extremities lasting less than 6 months is described. One of two treatments, exercise alone or exercise in combination with manual lymph drainage, was applied for six weeks, three times a week, to the affected limb. Clinical and subjective parameters for pain, swelling, temperature, and range of motion were evaluated. Manual lymph drainage was chosen as adequate therapy for oedema reduction, whereas exercise was applied as standard therapy for contracture prophylaxis in reflex sympathetic dystrophy. Both groups were asked not to use analgesics but received extensive instructions for avoiding pain. Significant improvements in clinical parameters were observed in both groups, but no significant effect between treatment groups was found. Pain measurement alone with a verbal rating scale showed a tendency towards greater pain reduction in the group receiving lymph drainage. The results indicate that, during the first 6 months of complex regional pain syndrome type I, manual lymph drainage provides no additional benefit when applied in conjunction with an intensive exercise program. PMID- 10729966 TI - A case of pseudothrombocytopenia after infusion of abciximab in vivo and anticoagulant-independent platelet clumping after rechallenge with abciximab in vitro. AB - A 45-year old man was treated for unstable angina pectoris with percutaneous transluminal angioplasty and stenting of his left anterior descending coronary artery. The procedure was followed by infusion of abciximab. The patient's automated platelet count in an EDTA-anticoagulated blood sample at admission to the hospital was normal, but dropped to 5 x 10(9)/l three hours after the procedure. The infusion of abciximab was stopped and the patient received platelet transfusions although there were no signs of bleeding. Two days later his platelet count was still low (37 x 10(9)/l) in an EDTA-anticoagulated blood sample, but normal (193 x 10(9)/l) in a heparin-anticoagulated sample. Platelet clumps were present only in the sample anticoagulated with EDTA, and pseudothrombocytopenia was diagnosed. The patient's recovery was uneventful. At follow-up visits four months and one year after discharge from hospital, the patient's blood samples were anticoagulated with three different anticoagulants: EDTA, citrate and heparin. The platelet count was normal in all three samples but after mixing with abciximab in vitro it dropped profoundly due to platelet clumping, regardless of the choice of the anticoagulant. Our report raises two points: (a) one needs to consider the possibility of pseudothrombocytopenia in a patient with a low automated platelet count after infusion of abciximab but without signs of bleeding, and (b) the in vitro results suggest that our patient who had initially responded to abciximab with pseudothrombocytopenia could develop true thrombocytopenia after repeated exposure. PMID- 10729967 TI - [Comments on the Special Issue of the Wiener klinische Wochenschrift on the Vienna Anatomical Science, issue 18, 1999]. PMID- 10729968 TI - TENS, electroacupuncture and ice massage: comparison of treatment for osteoarthritis of the knee. AB - The purpose of this study was to compare the effectiveness of transcutaneous nerve stimulation (TENS), electroacupuncture (EA), and ice massage with placebo treatment for the treatment of pain. Subjects (n = 100) diagnosed with osteoarthritis (OA) of the knee were treated with these modalities. The parameters for evaluating the effectiveness of treatment include pain at rest, stiffness, 50 foot walking time, quadriceps muscle strength, and knee flexion degree. The results showed (a) that all three methods could be effective in decreasing not only pain but also the objective parameters in a short period of time; and (b) that the treatment results in TENS, EA and ice massage were superior to placebo. PMID- 10729969 TI - Understanding acute disharmonies of the channel sinews. AB - A wide variety of symptom presentations have been identified in association with exogenous pathogenic attack of the "channel sinews" (CS). In accordance with the Eight Principles of Diagnosis and with reference to the classical literature, the author explores the outcomes of attack by exogenous pathogenic wind, cold, heat, and dampness at the acute stage. PMID- 10729970 TI - Psychology and Chinese medicine: exploring the mental, emotional and spiritual disharmonies of the Kidney energy system. AB - Drawing on his perspective as psychiatrist and longtime practitioner of Chinese medicine (CM), the author introduces his interpretation of the energy concepts of CM as they pertain to the psychology of man. This discussion focuses on the Water Element and Kidney energy system which are associated with supervision the genetic and intrauterine developmental phases of evolution. A faltering of these energies is associated with both pervasive and subtle neurological disorders and a predisposition to the most severe psychological disturbances. The manifestations and consequences are examined with case studies. PMID- 10729971 TI - The paradox of the unity and duality of the kidneys according to Chinese medicine: kidney essence, yin, yang, qi, the mingmen--their origins, relationships, functions and manifestations. AB - This paper explores the relationship between Kidney Essence, Yang, Yin and Qi, and the Mingmen, their origins, the variety of functions they provide to the human organism, mentally, physically and spiritually, and the fundamental paradox they represent in terms of unity and polarity. This exercise is part of an ongoing personal exploration in the service of a more complete understanding of the cosmic and material functions of the Kidney in Chinese medicine and culture, also illuminated by Chinese pulse diagnosis. It is not meant to convey a final statement or as a repository of absolutes, but as an attempt to stimulate and ultimately share new ideas. PMID- 10729972 TI - Energetics and transformation: insights on the paradoxical opportunity presented by chronic illness and pain--Part IV. AB - Following the author's own experiences with chronic pain due to a motorcycle accident during medical school, and the pain's unresponsiveness to conventional medicine, he sought other solutions. His journey led him to a new understanding of health and illness. With these insights and through the use of acupuncture, bodywork and various breathing techniques, he began to see phenomena--emotional release, myoclonic shaking, and regression--and healing that could not be explained in terms of a rational or structural framework. He posits that such phenomena represent different forms of de-stressing which together serve to release "blocked feeling," which he suggests is the "energy block" described by acupuncture theory as "stagnant Qi." The fourth in a series, this installment explores the role of repressed anger and its relationship to chronic illness. PMID- 10729973 TI - The systems, holograms and theory of micro-acupuncture. AB - Although acupuncture has been practiced for more than 5,000 years, micro acupuncture is a recent concept. In the mid-1950s, Nogier discovered that the ear may be utilized as a diagnostic and therapeutic system for the entire body. He explored the topology of acupoints on the ear, observing that it is reiterative of the anatomy. Micro-acupuncture extends these principles to other parts of the body, for example, the foot, hand, nose, and even the orbit of the eye. The development, systems, theory, clinical uses and holograms of micro-acupuncture are reviewed. It is suggested that the holographic paradigm may even have implications for the evolution of a new scientific, social and personal consciousness. PMID- 10729974 TI - Point-counterpoint: is O-ring testing a reliable method? PMID- 10729975 TI - Clinical research at the crossroads. PMID- 10729976 TI - Tongue between tooth significant. PMID- 10729977 TI - Reports promote understanding. PMID- 10729978 TI - Transfer of radiographs. AB - Dental radiographs constitute 25% of all exposures made in the UK and therefore in terms of radiation protection the population risk is not insignificant. Paragraph 11 of The Core of Knowledge in the Schedule to the Protection of persons undergoing medical examination or treatment (POPUMET) regulations specifies the importance of using existing radiological information, whether films or reports, about a patient. The Guidance Notes recommends that in order to reduce unnecessary radiographic examinations, there should be ready availability of previous radiographs. Access to previous radiographs is also commended by the National Radiological Practice Board (NRPB) and the Royal College of Radiologists (PCR) in their report on Patient Dose Reduction in Diagnostic Radiology. In addition to dose limitation, patient care is improved. PMID- 10729979 TI - Biting off more than you can chew: a forensic case report. AB - A case is reported where a forefinger is 'amputated' by a human bite. This type of extreme biting injury is uncommon and probably represents tearing by the premolar teeth rather than a clean bite by incisor teeth. PMID- 10729980 TI - Primary non-Hodgkin's lymphoma of the mandible presenting following tooth extraction. AB - A case of oral non-Hodgkin's lymphoma presenting following a tooth extraction is reported. The clinical presentation was that of a periodontal lesion and an extraction socket that failed to heal, which may have led to initial confusion in diagnosis and management. PMID- 10729981 TI - Dental implants. 7. Basic restorative techniques. AB - Some restorative techniques for implant supported restorations will be familiar to dentists used to providing conventional crown and bridgework. The differences and principles involved when using implants are identified. PMID- 10729982 TI - Measuring oral health: does your treatment really make a difference. AB - An understanding of a broader concept of health is increasingly important for all health professionals, including dentists, and has recently been incorporated as a key principle in the Government White Paper, The New NHS. This aims to deliver a dependable, high quality, egalitarian health service. In the past, performance measurements in the UK have often relied simply on those areas which are most easily quantified. For example, within the hospital service, performance was measured in terms of the cost and the number of finished consultant episodes, from which the 'purchaser efficiency index' was calculated. This tended to produce a driving force rewarding those doing more rather than those doing more better. It is analogous to the system which has been the backbone of NHS dental practice for many years, 'fee per item of service', where throughout is rewarded rather than outcome. However, the White Paper has signalled a move away from simply counting activity. From April 1999 within the hospital service the purchaser efficiency index has been replaced with more rounded measures, reflecting the changing concepts of health, in a new broader performance framework to determine what really counts for patients. It will focus on measuring health improvement, fairer access, better quality and outcome, including the views of patients. PMID- 10729983 TI - Factors influencing the diagnosis and management of teeth with pulpal and periradicular disease by general dental practitioners. Part 1. AB - OBJECTIVE: To identify factors influencing the diagnosis and management of periradicular disease by general dental practitioners. DESIGN: The study was conducted in two stages. 1. Analysis of recent returns to the Scottish Dental Practice Board. 2. Data collection via a postal questionnaire distributed to 617 general dental practitioners in Scotland (33% of practitioners registered with the Dental Practice Board). RESULTS: 417 (69%) questionnaires were completed and returned. The majority of respondents undertook root canal treatment and the number of cases treated had increased in the last five years. The vast majority of the respondents (89.3%) were confident in their diagnosis of periradicular disease and 77.1% were confident of their treatment of the disease. A referral system for treatment was used by 31% of respondents of whom the majority used a specialist in a hospital. Over 50% of the respondents undertook surgical root canal treatment. Only 40% of respondents followed up their completed cases for longer than six months. Constraints on the provision of treatment included the time available and the low level of fees. CONCLUSIONS: Current arrangements for the treatment of periradicular disease in general dental practice are less than optimal. The fiscal arrangements for the provision of these treatments must be developed to encourage a high standard of treatment to be performed thereby maximising the likelihood of success. In addition, efforts to inform patients of the benefits of the treatment of periradicular disease should be increased. PMID- 10729984 TI - Provision of dental general anaesthesia for extractions in child patients at two centres. AB - AIM: To two contrasting centres, to describe the provision of dental general anaesthesia (DGA) for simple non-surgical extractions in terms of the type of treatment provided, including the number of primary and permanent teeth extracted, and the characteristics of child patients attending in terms of their age group and gender. DESIGN: Retrospective analysis of hospital records. METHOD: Data were drawn from records of services over a 12-month period in 1996/97 at: a) a London dental hospital (Centre 1), and b) in the community dental services in Rochdale, Lancashire (Centre 2). Information was collated and analysed using the SPSS statistical software package. RESULTS: The majority of patients at both centres were aged less than 9 years. Almost one third (31%) of those seen at Centre 1 were below 5 years of age, but fewer of this age group were treated at Centre 2. Children aged 9 years or less had an average of 5.4 (SD = 3.0) primary teeth extracted at Centre 1 and 3.0 (SD = 2.0) at Centre 2. For permanent teeth, an average of 3.2 (SD = 1.2) and 2.7 (SD = 1.4) were extracted at Centres 1 and 2 respectively. CONCLUSIONS: Both services were used primarily for the extraction of primary teeth although the services differed in the ages of patients who used them and in the numbers of teeth extracted. Numbers of patients attending the service at Centre 1 had declined over time but numbers of teeth extracted per child had increased. PMID- 10729985 TI - A survey of general dental practitioners' postgraduate education activity and demand for extended modular postgraduate programmes. AB - The aim of this paper is to assess the level of involvement of general dental practitioners in postgraduate education and training, identify their speciality interests, preferred format of course presentation and assess the potential demand for modular postgraduate programmes. A questionnaire analysis of a one in three random sample of general dental practitioners on Health Authority lists in the North West of England and North Wales was designed and carried out. Out of a random sample of 799, 552 GDPs completed an anonymous questionnaire in 1998 which investigated their current postgraduate activity, the areas of specialist interest and their interest in extended modular postgraduate education courses with associated summative assessment. Fifty three percent of respondents attended more than four postgraduate sessions per year of whom the majority (59%) were in 35-44 year old age group. 79% identified Section 63 courses as their first preference and 63% preferred mixed didactic and 'hands on' courses. Seventy five percent of respondents expressed an interest in attending modular programmes which might lead to a postgraduate qualification. In conclusion, an enthusiasm to attend existing postgraduate courses was identified but a demand was also perceived for courses of longer duration possibly leading to additional postgraduate qualifications. PMID- 10729986 TI - Treatment and quality standards for swimming pool water. PMID- 10729987 TI - Genotyping of human isolates of cryptosporidium. PMID- 10729988 TI - Hematologic aspects of the porphyrias. AB - The porphyrias are disorders that can be inherited and acquired, in which the activities of the enzymes of the heme biosynthetic pathway are partially or almost totally deficient. There are 8 enzymes involved in the synthesis of heme, and, with the exception of the first enzyme, an enzymatic defect at every step leads to tissue accumulation and excessive excretion of porphyrins and/or their precursors, such as delta-aminolevulinic acid and porphobilinogen. Whereas heme, the final product of the biosynthetic pathway, is biologically important, porphyrins and their precursors are not only useless but also toxic. Porphyrias can be classified as either photosensitive or neurologic, depending on the type of symptoms, but some porphyrias cause both photosensitive and neurologic symptoms. Alternatively, they can be classified either hepatic or erythropoietic, depending on the principal site of expression of the specific enzymatic defect. The tissue-specific expression of porphyrias is largely due to the tissue specific control of heme pathway gene expression, particularly at the level of delta-aminolevulinate synthase, the first and the rate-limiting enzyme of heme biosynthesis. In this chapter, hematologic aspects of the erythropoietic porphyrias will be described. The 3 major erythropoietic porphyrias are congenital erythropoietic porphyria (CEP), hepatoerythropoietic porphyria (HEP) and erythropoietic protoporphyria (EPP). PMID- 10729989 TI - Pathogenesis and management of chronic idiopathic thrombocytopenic purpura: an update. AB - Idiopathic thrombocytopenic purpura (ITP) is a disorder characterized by platelet destruction caused by an antiplatelet autoantibody that results in platelet phagocytosis via the reticuloendothelial system. The condition will become chronic in 10% to 20% of children with ITP and almost all adult ITP patients. The expert panel established in 1994 by the American Society of Hematology extensively reviewed ITP-related articles before 1995 and published practice guidelines. This publication is of great help in regard to research and management of ITP. However, the pathogenesis of chronic ITP has not been fully elucidated, and its management is primarily empirical. This review focuses mainly on the articles published after 1995, including some experiences in China. PMID- 10729990 TI - Oxidative stress response and signaling in hematological malignancies and HIV infection. AB - Hematopoietic cells can be exposed to a wide spectrum of oxidative stresses. Excessive oxidative stress damages biomolecules such as DNA, proteins, and lipids, leading to cellular dysfunction and cell death. Accumulation of such damage provokes noxious effects on individuals, resulting in diseases such as hematopoietic malignancies. On the other hand, cells have multiple mechanisms to protect themselves from stress. These mechanisms include apoptosis, DNA repair, cell cycle regulation, and induction of antioxidant and detoxifying enzymes. Reactive oxygen species (ROS) may act as intracellular signaling mediators in physiological signal transduction. ROS activate cascades of events, such as activation of tyrosine kinases, small Ras proteins, and the mitogen-activated protein kinase system, followed by the activation of some subsets of transcription factors. Antioxidants are induced by oxidative stress to act not simply as scavengers of ROS but also as important regulators of oxidative stress response. Meanwhile, oxidative stress often causes apoptosis, in which mitochondrial control has been known to play an essential role. The dysregulation of antioxidants and apoptosis is deeply involved in the pathogenesis and pathophysiology of virus-associated hematopoietic disorders, including acquired immunodeficiency syndrome. PMID- 10729991 TI - Thrombopoietin stimulates murine lineage negative, Sca-1+, C-Kit+, CD34- cells: comparative study with stem cell factor or interleukin-3. AB - It has recently been reported that human thrombopoietin (TPO) acts on early hematopoietic progenitor cells. Consequently, we investigated the effects of TPO on murine hematopoietic progenitor cells using lineage negative (Lin-), Sca-1+, c Kit+ marrow cells from 5-fluorouracil-treated mice. One hundred enriched cells were cultured in suspension with various single cytokines for 5 days. When cultured with the single cytokines as stem cell factor (SCF), TPO, or interleukin (IL)-3, these cells were maintained or had increased by day 5, whereas only a few cells survived in cultures with granulocyte colony stimulating factor, IL-11, or IL-6. We extended the study in serum-free or serum-containing suspension cultures with SCF or TPO. Anti-TPO antibodies did not inhibit the effects of SCF on enriched cells but did inhibit the effects of TPO on those cells. We further examined the effects of TPO, SCF, and IL-3 on other populations of murine hematopoietic progenitor cells. Either TPO or SCF as a single cytokine could maintain murine Lin-, Sca-1+, c-Kit+, CD34- marrow cells, which are the most dormant cells. In addition, IL-3 increased Lin-, Sca-1-, c-Kit+ cells more than did SCF and TPO but did not stimulate Lin-, Sca-1+, c-Kit+, CD34- cells more. These results indicate that TPO as well as SCF may be key regulators in the proliferation of murine hematopoietic early progenitor cells. PMID- 10729992 TI - Adult onset cyclic hematopoiesis in a patient with myelodysplastic syndrome. AB - A 75-year-old woman presenting with myelodysplastic syndrome showed cyclic oscillations in her white blood cell and platelet counts. Each cycle lasted for 5 to 6 months, with 4 cycles occurring over the course of a 2-year period. During successive cycles, the white blood cell count fluctuated from 10.1 to 2.6; 13.8 to 1.8; 11.0 to 1.6, and 8.6 to 1.3 x 10(9)/L. The platelet count fluctuated from 242 to 38, 199 to 11, 110 to 5, and 75 to 3 x 10(9)/L. The patient underwent red blood cell transfusions because of red blood cell aplasia; the frequency of the transfusions and the erythropoietin concentration in serum were inversely correlated. The number of circulating granulocyte-macrophage colony-forming units and CD34-positive cells in peripheral blood oscillated in phase with the white blood cell and platelet counts. These patterns suggested a periodic influx of progenitor cells from hematopoietic stem cells. The ratio of neutrophils to mononuclear cells remained essentially constant throughout the clinical course. Lymphocyte subset assessments using monoclonal antibodies showed an inverse CD4/CD8 ratio (less than 1) and extreme B cell lymphopenia throughout the fourth cycle. The percentage of CD3-positive cells oscillated inversely, suggesting that the cyclic cytopenia had an immune mechanism involving T lymphocytes. PMID- 10729993 TI - Constitutively active STAT5A and STAT5B in vitro and in vivo: mutation of STAT5 is not a frequent cause of leukemogenesis. AB - We recently identified several constitutively active forms of signal transducers and activators of transcription 5 (STAT5) using polymerase chain reaction-driven random mutagenesis followed by retrovirus-mediated expression screening. All constitutively active STAT5 showed constitutive phosphorylation on their tyrosine residues and induced factor-independent growth in a mouse interleukin-3-dependent cell line, Ba/F3. Sequence analysis of these active STAT5 revealed two important mutations: S710F and N642H. The N642H mutation localized in the SH2 domain was able to induce autonomous growth of Ba/F3 cells by itself, whereas S710F in the effector domain was able to induce autonomous growth of Ba/F3 cells in concert with a second mutation including H298R and E150G. Recently, constitutive activation of STAT5 has been reported in patients' leukemic cells and is implicated in leukemogenesis. We attempted to clarify whether leukemic cells harbored activating mutations primarily in STAT5 proteins, and analyzed the sequence of STAT5 derived from 49 leukemic patients. No mutations were found, however, in the regions surrounding S710 and N642 of STAT5A and corresponding residues of STAT5B. We also cloned full-length cDNAs for STAT5s from three patients whose leukemic cells exhibited constitutive tyrosine phosphorylation of the STAT5 protein and expressed the derived STAT5 proteins in Ba/F3 cells. However, none of these clones exhibited constitutive tyrosine phosphorylation or gave rise to FI proliferation of Ba/F3 cells. These results indicate that constitutive activation of STAT5 is a secondary event in most leukemias. PMID- 10729994 TI - XYY male with essential thrombocythemia in childhood. AB - We describe a boy with XYY male accompanied with essential thrombocythemia. This is, to our knowledge, the first complete case report of the kind in the pediatric literature. The patient was asymptomatic, but at age 5 his platelet count had increased to 145.5 x 10(4)/microL, and he was diagnosed as having essential thrombocythemia based on the diagnostic criteria of the Polycythemia Vera Study Group. At that time, it was discovered by chromosome analysis of both bone marrow and peripheral blood cells that he was XYY male. At times during the clinical course when his platelet count was 94.1 x 10(4)/microL, his serum thrombopoietin (measured by enzyme-linked immunosorbent assay) was 1.09 fmol/mL, which was normal for his age. Aspirin was administered, and he remained asymptomatic throughout the course. After 2 years, he underwent a spontaneous remission. Because of the small number of reported cases, we have been unable to determine the relation between XYY males and essential thrombocythemia. PMID- 10729995 TI - Effects of cyclosporin A on refractory B-cell chronic lymphocytic leukemia. AB - This report details the effects of cyclosporin A (Cs-A) on the clinical course of B-cell chronic lymphocytic leukemia (B-CLL) with an increase in circulating T cells that express CD3, CD8, CD25, CD45RO, and T-cell receptor alpha beta chain. Cs-A was used for treatment of a female patient with refractory B-CLL and was shown to increase dramatically the number of leukemic B cells in association with a simultaneous increase in T cells in vivo. To study this effect more precisely, CD19+ (B cells) and CD3+ (T cells) cells were isolated from the patient and cultured in the presence or absence of Cs-A. When Cs-A was added to the culture, no cell stimulation was seen. However, CD3+ cells from the patient underwent cell death, relative to those from healthy donors. On the other hand, CD19+ cells from the patient were more resistant to Cs-A than those from healthy donors. Because in vitro experiments cannot explain this phenomenon of Cs-A increasing leukemic cells (not only B-CLL cells but also T cells), care should be exercised in the clinical use of Cs-A in B-CLL patients. PMID- 10729996 TI - Adult T-cell leukemia/lymphoma with initial deafness. AB - In July 1995, a 43-year-old Japanese man presented with deafness in the right ear. On hospital admission, he had deafness in both ears and right facial palsy. Variously sized lymphoid cells with convoluted nuclei were observed in the cerebrospinal fluid. Surface marker analysis revealed monoclonality of T lymphocytes in the spinal fluid. Similar abnormal cells were observed in peripheral blood and bone marrow. Biopsy specimens of the stomach and prostate showed tight proliferation of large lymphoid cells in the interstitium and epithelium. Antibody against human T-lymphotrophic virus type 1 (HTLV-1) was present. The diagnosis of non-Hodgkin's lymphoma, diffuse type, was made. Seven months later, the patient died of sepsis. Autopsy revealed multiple lymphadenopathy in the abdomen and the infiltration of atypical lymphocytes to the pancreas, kidneys, and other organs. A monoclonal band of HTLV-1 provirus was detected by Southern blot analysis. To our knowledge, this is the first report of adult T-cell leukemia/lymphoma with auditory nerve abnormalities as the initial symptom. PMID- 10729997 TI - Differences between lymphocyte subsets, after allogeneic bone marrow transplantation, in patients who received tacrolimus and patients who received cyclosporin A. AB - The subsets of peripheral blood lymphocytes after allogeneic bone marrow transplantation were compared in 20 patients who received tacrolimus and 34 patients who received cyclosporin A (CsA) prophylactically. The phenotypes of CD3, CD4, CD8, and D8/CD57 were analyzed by flow cytometry. The percentage of CD3+ cells in the tacrolimus group (58.8% +/- 21.6%) was significantly lower than in the CsA group (77.2% +/- 12.8%) (P = .0239). The percentage of CD8+CD57+ cells in the patients receiving tacrolimus and developing acute graft-versus-host disease (GVHD) (grade I, 20.1% +/- 10.6%; grade II-IV, 13.2% +/- 6.3%) was significantly higher than in the patients receiving CsA and developing acute GVHD (grade I, 10.7% +/- 5.2%; grade II-IV, 7.7% +/- 4.0%) (grade I, P = .0036; grade II-IV, P = .0255). The absolute number of CD8+CD57+ cells in the patients with grade II-IV acute GVHD was also significantly higher in the tacrolimus group compared with the CsA group. There was no difference in the incidence of acute GVHD in the 2 groups. Recovery from acute GVHD in the tacrolimus group (16.6 +/- 13.6 days) was more rapid than in the CsA group (30.8 +/- 24.8 days) (P = .0124). These results suggest that, compared with CsA, tacrolimus administered prophylactically induces more CD8+CD57+ lymphocytes when acute GVHD occurs and accelerates the recovery from acute GVHD more rapidly. PMID- 10729999 TI - Mutations of the WASP gene in 10 Japanese patients with Wiskott-Aldrich syndrome and X-linked thrombocytopenia. AB - Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, immunodeficiency, and eczema. X-linked thrombocytopenia (XLT) is a mild form of WAS with isolated thrombocytopenia. Both phenotypes are caused by mutation of the Wiskott-Aldrich syndrome protein (WASP) gene. In this study, we identified mutations of the WASP gene in 10 Japanese patients from 9 unrelated families with WAS/XLT. All XLT patients (n = 3) and one WAS patient had a missense mutation at the PH domain of WASP. Two WAS patients had nonsense mutations. One WAS patient had exon 8 skipping caused by one nucleotide deletion at the acceptor site of intron 7. Three WAS patients had genomic deletions; one of the three had a large genomic deletion involving exons 3 to 7. Codons 45 and 86 seem to be the hot spots of the WASP mutation, because missense mutations in these codons have been reported previously in several WAS/XLT patients in addition to the patients in this report, and patients with the same mutation show a similar clinical phenotype. All other mutations are novel, indicating that the mutations of WASP are heterogeneous. EB virus-transformed cell lines from XLT patients expressed nearly normal amounts of WASP, whereas those from typical WAS patients expressed almost undetectable amounts of WASP. We conclude that the analysis of gene mutation and protein expression of WASP are useful together in assessing the severity of WAS. PMID- 10729998 TI - Successful treatment of disseminated nasal NK/T-cell lymphoma using double autologous peripheral blood stem cell transplantation. AB - Nasal natural killer (NK)/T-cell lymphoma is a rare disease with an aggressive clinical course. Prognosis is generally poor and the disease is invariably fatal after systemic dissemination. We report a patient with aggressive nasal NK/T-cell lymphoma who was resistant to therapy and developed systemic dissemination involving the intestine, skin, and stomach. Epstein-Barr virus (EBV) was detected by Southern blotting with EBV-terminal repeat probe and by in situ EBV-encoded small nuclear early region-1 hybridization. The patient was treated using double high-dose chemotherapy and autologous peripheral blood stem cell transplantation (auto-PBSCT). Pretransplant conditioning for the first auto-PBSCT was MCVC (high dose ranimustine, carboplatin, etoposide [VP16], and cyclophosphamide), and for the second auto-PBSCT, modified ICE (high-dose ifosfamide, VP16, and carboplatin). The patient obtained a complete remission and has been free of disease for 3.0 years since the second PBSCT. These observations suggest that double high-dose chemotherapy with PBSCT support may be effective in resistant nasal NK/T-cell lymphoma. PMID- 10730000 TI - A novel type of factor XI deficiency showing compound genetic abnormalities: a nonsense mutation and an impaired transcription. AB - We studied a 29-year-old Japanese male patient with factor XI deficiency; we also studied his parents and one sibling. Factor XI coagulation activity and antigen levels were extremely low (less than 1% of normal level) in both the patient and his brother, and they were half the normal levels in both parents. Sequence analysis of all 15 exons and the exon-intron boundaries of the factor XI gene amplified by polymerase chain reaction revealed a nonsense mutation in exon 8 (Gln263-->Stop). Although the parents are first cousins, the mutation was unexpectedly heterozygous in all the family members except the father, who showed the homozygous wild type, indicating that this mutation alone was not sufficient to account for the factor XI deficiency. To explore the genetic abnormality in the father, we analyzed allele-specific expression of the platelet factor XI gene using reverse transcription-polymerase chain reaction and subsequent restriction enzyme digestion. As a result, gene expression from only one allele was severely impaired in the father. This result implies an additional mutation in some regulatory element of the factor XI gene from paternal inheritance. We concluded that the factor XI deficiency of the patient was caused by compound heterozygous genetic abnormalities. PMID- 10730002 TI - Profile: George Weber. Man of many worlds. Interview by Teresa Amoroso. PMID- 10730001 TI - Dental education. PMID- 10730003 TI - The significance of co-payments. PMID- 10730004 TI - Gingival hyperplasia complicating acute myelomonocytic leukemia. AB - Many systemic illnesses manifest clinical signs in the oral cavity. A remarkable case of gingival hyperplasia heralding the presence of acute myelomonocytic leukemia (AML FAB-M4) is described. The oral manifestations of acute leukemia are reviewed. PMID- 10730005 TI - Unusual palatal presentation of oral psoriasis. AB - Oral lesions of psoriasis are rare clinical observations. Report on oral psoriasis that are well documented show no consistent lesion pattern. The oral lesions range in type from red plaques to white plaques to ulcers. In this report, an unusual palatal presentation of oral psoriasis with red serpiginous concentric arcs is described. The diagnosis of intraoral psoriasis is supported by clinical and microscopic findings. PMID- 10730006 TI - Cosmetic dentistry: is it really health care? PMID- 10730007 TI - Cosmetic dentistry is still dentistry. PMID- 10730008 TI - Comparing characteristics of Canadians who visited dentists and physicians during 1993/94: a secondary analysis. AB - Unlike medical care, dental services are not included in Canada's universal system of health care. Using the data from the 1994 National Population Health Survey, we estimate the proportion of the population aged 12 and older visiting dentists and physicians in 1993/94 and compare the factors that influence the use of dentists' and physicians' services. Overall, 52.4% of Canadians made one or more visits to a dentist and 78.4% visited a physician. Logistic regression analysis indicates that whereas visiting a family physician is more likely to occur for people who are ill (generally, on medications or needing help with daily living) or pregnant, visiting a dentist is more likely to occur for young, healthy, wealthy and highly educated people. Future dental health policy needs better information on health status linked to use of services. PMID- 10730009 TI - Nutrition: its role in dental training and practice. PMID- 10730010 TI - Intimate partner violence. Bridging the gap between law and science. PMID- 10730011 TI - Regulating reproductive technologies. PMID- 10730012 TI - Assisted reproductive technology law. Obtaining informed consent for the commercial cryopreservation of embryos. PMID- 10730013 TI - Access to and cost of reproduction of patient medical records. A comparison of state laws. PMID- 10730014 TI - Mistaken baby switches. An analysis of hospital liability and resulting custody issues. PMID- 10730015 TI - A physician's liability for mistakes of a physician assistant. PMID- 10730016 TI - Pharmacy organizations 1852-1902. PMID- 10730017 TI - Lipid management by pharmacists: evidence of benefits. PMID- 10730018 TI - Establishing a pharmacy-based laboratory service. PMID- 10730019 TI - Pharmaceutical care services and results in project ImPACT: hyperlipidemia. AB - OBJECTIVE: To demonstrate that pharmacists, working collaboratively with patients and physicians and having immediate access to objective point-of-care patient data, promote patient persistence and compliance with prescribed dyslipidemic therapy that enables patients to achieve their National Cholesterol Education Program (NCEP) goals. DESIGN: Observational study. PARTICIPANTS: 26 community based ambulatory care pharmacies: independent, chain-professional, chain-grocery store, home health/home infusion, clinic, health maintenance organization/managed care. MAIN OUTCOME MEASURES: Rates of patient persistence and compliance with medication therapy and achievement of target therapeutic goals. RESULTS: In a population of 397 patients over an average period of 24.6 months, observed rates for persistence and compliance with medication therapy were 93.6% and 90.1%, respectively, and 62.5% of patients had reached and were maintained at their NCEP lipid goal at the end of the project. CONCLUSION: Working collaboratively with patients, physicians, and other health care providers, pharmacists who have ready access to objective clinical data, and who have the necessary knowledge, skills, and resources, can provide an advanced level of care that results in successful management of dyslipidemia. PMID- 10730020 TI - Clinical and humanistic outcomes of a lipid management program in the community pharmacy setting. AB - OBJECTIVES: To identify patients at risk for coronary artery disease (CAD) through a search of a community pharmacy's prescription database, to screen and identify patients with elevated cholesterol and at risk for CAD, to enroll patients in a pharmacist-directed lipid management program, and to evaluate selected clinical and humanistic outcomes. DESIGN: Randomized, pretest-posttest control groups. SETTING: Independent community pharmacy in a suburban metropolitan area. PATIENTS: 51 patients who were not at National Cholesterol Education Program low-density lipoprotein cholesterol (LDL-C) or defined triglyceride goals and who met inclusion criteria. INTERVENTION: Pharmacist directed lipid management program. MAIN OUTCOME MEASURES: Clinical outcome measures included total cholesterol, LDL-C, high-density lipoprotein cholesterol (HDL-C), and triglyceride levels; achievement of LDL-C goal; and risk factor prediction scores. Humanistic outcome measures included patient satisfaction with pharmaceutical care and patient knowledge of hyperlipidemia. RESULTS: LDL-C was decreased in the pharmacist intervention group (n = 25), compared with an increase in the control group at study end. HDL-C levels increased and triglyceride levels decreased in both groups. Of treatment group patients, 32% achieved their cholesterol goals, compared with 15% of control group patients. Risk factor prediction scores improved in the treatment group and worsened in the control group. The treatment group's hyperlipidemia knowledge scores improved significantly from pretest to posttest. Both treatment and control group patient satisfaction scores for the pharmacist investigator were favorable at study end. CONCLUSION: Both treatment and control patients benefited from participating in this study. Patients enrolled in the lipid management program made greater improvements in their knowledge of hyperlipidemia, risk factor scores, and cholesterol levels. PMID- 10730021 TI - Pharmaceutical care for patients with chronic conditions. AB - OBJECTIVE: To assess factors associated with participation in pharmaceutical care and the benefits of participation--in terms of amount of information about medications, administration of medications, and awareness of side effects. DESIGN: Quasi-experimental design, with a control group. Medication Survey, administered 6 months after pharmaceutical care intervention to participants, refusers, and controls. Logistic regression analyses. SETTING: Three staff clinic pharmacies and three contract clinic pharmacies affiliated with a health maintenance organization (HMO). PATIENTS AND OTHER PARTICIPANTS: Patients with chronic health conditions (asthma, chronic obstructive pulmonary disease, or heart disease) enrolled at six intervention sites, identified through the HMO's electronic pharmacy database. Control sample with the same chronic health conditions, without access to pharmaceutical care (n = 210 participants, 162 refusers, and 368 controls; overall adjusted response rate = 72%). INTERVENTION: Pharmaceutical care, in the form of a comprehensive drug therapy management program. MAIN OUTCOME MEASURES: Predictors of participation, amount of information about medications, use of reminder methods, and awareness of side effects. RESULTS: The following variables were significantly associated with the probability of participating in pharmaceutical care (P < .05): number of medications, employment, income, health status, education, and living situation. Participants were more likely than controls to say they received "a lot of information" from their pharmacist about all aspects of medications (odds ratio [OR], 1.75 to 2.68). Participants were more likely to report leaving their medication container in a visible place and using two or more reminder methods (OR, 1.87 to 1.48). There were no significant differences in the probability of missing doses. Participants were more likely to report experiencing "symptoms or problems" associated with prescription medications (OR, 1.81). CONCLUSION: Pharmaceutical care appears to increase the information given to patients about medications, promote more effective self-administration of medications by encouraging patients to use systematic reminders, and increase awareness of medication side effects. PMID- 10730022 TI - New drugs of 1999. AB - OBJECTIVE: To provide information regarding the most important properties of the new therapeutic agents marketed in 1999. DATA SOURCES: Published studies, drug information reference sources, and product labeling. DATA SYNTHESIS: In 1999, 28 new therapeutic agents were marketed. The indications and information on dosage and administration for each new agent are reviewed, as are the most important pharmacokinetic properties, adverse events, drug interactions, and other precautions. Practical considerations for the use of the new agents are also discussed. Where possible, the properties of the new drugs are compared with those of older drugs marketed for the same indications. CONCLUSIONS: A number of the new therapeutic agents marketed in 1999 have important advantages over older medications. An understanding of the properties of these agents is important for the pharmacist to effectively counsel patients about their use and to serve as a valuable source of information for other health care professionals regarding these drugs. PMID- 10730023 TI - New OTC drugs and devices 1999: a selective review. AB - OBJECTIVES: To educate pharmacists about newly introduced nonprescription products/devices/diagnostic products, and to create a heightened awareness of the dilemma of dietary supplements and the problems their use poses for consumers and pharmacists. DATA SOURCES: Recent clinical and pharmaceutical industry literature. DATA SYNTHESIS: It is critical for the pharmacist to be knowledgeable about dietary supplements to be able to appropriately counsel patients on benefits and risks associated with their use. In addition, numerous other nonprescription products, including home diagnostic products and accessories, will continue to become available on the U.S. market. It is important that the pharmacist become as knowledgeable as possible about these and be capable of educating the consuming public about their appropriate use. CONCLUSION: Dietary supplements will continue to flood the self-care market. Pharmacists need a balanced understanding of these products to assist the consumer. The new nonprescription medications cited in this review represent valuable additions to the growing array of self-care products for consumer use. PMID- 10730024 TI - Herbs of special interest to women. AB - OBJECTIVE: To review the efficacy and safety of specific herbal medications that have been used traditionally to treat common conditions in women. DATA SOURCES: Current literature, with emphasis on more rigorously controlled studies. DATA SYNTHESIS: Herbal medicines have long been used in traditional healing systems to treat conditions of particular interest to women, such as premenstrual syndrome (PMS) and menopausal symptoms. For a select number of phytomedicines, including evening primrose oil, black cohosh root extract, dong quai, and chaste tree berry, scientific investigation is elucidating the pharmacologically active constituents, mechanism of action, and clinical value. CONCLUSION: Based on the available evidence, evening primrose oil and chaste tree berry may be reasonable treatment alternatives for some patients with PMS. Dong quai may have some efficacy for PMS when used in traditional Chinese multiple-herb formulas. For relief of menopausal symptoms, black cohosh root extract and dong quai have good safety profiles, but only black cohosh has demonstrated efficacy for this indication. Safety data, especially during pregnancy and lactation, are still largely lacking for many herbal medications, and recommendations for usage and dosage vary. Pharmacists who wish to recommend herbal products for women's health conditions need to evaluate the scientific literature in order to form their own opinions about appropriate use and safety. PMID- 10730025 TI - Postcard and telephone reminders for unclaimed prescriptions: a comparative evaluation using survival analysis. AB - OBJECTIVE: This study investigated unclaimed prescription reminders with the goal of developing practical and useful recommendations for pharmacies interested in reminding patients to pick up unclaimed prescriptions. Based on the recipient and mode of the reminder notification, this study measured differences in unclaimed prescription pickup time. DESIGN: This study was conducted using a convenience sample of three independent pharmacies in a large Midwestern city. A total of 120 subjects with prescriptions remaining unclaimed after 3 or 4 working days were included in the study. Once identified as unclaimed, these prescriptions were randomly assigned into a control group or one of the following four intervention groups: (1) a telephone reminder to the patient, (2) telephone notification to the prescribing physician, (3) a postcard reminder to the patient, and (4) postcard notification to the prescribing physician. RESULTS: The results suggest that different methods of pharmacist-initiated reminder systems may affect time to prescription pickup in community pharmacy practice. Marginally significant differences were found among the five study groups and the time to prescription pickup (P = .09). Compared with the control group, neither telephone nor postcard reminders--to patients or physicians--significantly decreased the mean number of days to pickup of potentially abandoned prescriptions. CONCLUSION: The actual value of an unclaimed prescription reminder program may reside in improved relationships with customers and with the medical community. The effort and expense of implementing and maintaining an unclaimed prescription reminder system should be balanced against the opportunity to establish and improve pharmacist patient and pharmacist-physician relationships. Further research in different pharmacy settings should investigate the effectiveness of (1) postcard versus telephone reminders, (2) physician versus patient notification, and (3) the effects of reminders on patient outcomes. PMID- 10730026 TI - Implementation of a pharmacy-based immunization program in a supermarket chain. AB - OBJECTIVE: To describe procedures for implementing a pharmacy-based immunization program in a supermarket chain. SETTING: Supermarket chain pharmacy. PRACTICE DESCRIPTION: Ukrop's is a local supermarket chain with 27 stores in the greater area of Richmond, Fredericksburg, and Williamsburg, Virginia, 19 of which have pharmacies. Ukrop's offers enhanced patient care services including immunizations, diabetes, asthma, hypertension, hyperlipidemia monitoring, and smoking cessation. All pharmacies offer adult immunizations and host periodic diabetes, hypertension, and hyperlipidemia screening events. PRACTICE INNOVATION: Adult immunization program. INTERVENTIONS: Each pharmacy offered influenza and pneumococcal vaccinations on a walk-up basis during pharmacy hours and during clinics held at least 3 days per week. Immunizations were also offered periodically at off-site locations. Distribution of letters and chart stickers to patients' physicians, and even partnership with a physician to establish the immunization protocol, helped increase awareness of the pharmacy immunization services. This service involved a core group of immunizing pharmacists who developed a policies and procedures manual, distributed the vaccine, and handled additional staffing requirements. MAIN OUTCOME MEASURES: Number of adult influenza and pneumococcal vaccinations administered by pharmacists. RESULTS: Between September and December 1998, Ukrop's pharmacists administered 5,137 influenza vaccinations and 613 pneumococcal vaccinations. Between September 1999 and January 2000, Ukrop's pharmacists administered 18,000 influenza vaccinations and 1,200 pneumococcal vaccinations. CONCLUSION: In addition to immunizing thousands of people in its first year, the program served as a successful marketing tool to increase awareness of enhanced pharmacy services in the community and among local physicians. Administration of vaccines increased pharmacists involvement with and enthusiasm for enhanced patient care services and generated a revenue stream for the pharmacies. PMID- 10730027 TI - Stability of mexiletine in two extemporaneous liquid formulations stored under refrigeration and at room temperature. PMID- 10730028 TI - Homeopathy: attitudes and opinions of members of the American Pharmaceutical Association. PMID- 10730029 TI - A call to action: pharmacists on the front line against insulin resistance. PMID- 10730030 TI - How hard is it to learn to vaccinate? PMID- 10730031 TI - 1999 a disappointing year for biotech approvals. PMID- 10730032 TI - Primary health care in the twenty-first century. PMID- 10730033 TI - The unexplored research potentials of public health in Africa. PMID- 10730034 TI - Bacterial, viral and parasitic aetiology of paediatric diarrhoea in the highlands of Papua New Guinea. AB - Enteropathogens and clinical features associated with diarrhoea were investigated in 1526 children admitted over a 5-year period to the paediatric ward of a hospital in the highlands of Papua New Guinea. Overall, a recognized pathogen was isolated from 39 per cent of the children admitted with diarrhoea. The most commonly isolated agents were rotavirus (23 per cent), Shigella spp. (13 per cent), Campylobacter spp. (12 per cent), Cryptosporidium parvum (10 per cent) and enteropathogenic Escherichia coli (8 per cent). The clearest clinical associations were rotavirus with vomiting, and Shigella with blood and pus in the stool. A control series of children admitted with other complaints was also included, and the odds ratios for diarrhoea for the above five pathogens were 18.2, 9.6, 3.7, 2.2, and 1.6, respectively. PMID- 10730035 TI - The role of expressed breastmilk and continuous positive airway pressure as predictors of survival in extremely low birthweight infants. AB - A case-control study was carried out on 152 extremely low birthweight (ELBW, < 1000 g) infants born consecutively in a large Malaysian maternity hospital during a 21-month period to determine the significant predictors associated with survival at discharge. Forty-nine (32.2 per cent) of these infants survived and 103 (67.8 per cent) died. The survivors weighed significantly heavier (mean = 888 g, SD = 99) than infants who died (mean = 763 g, SD = 131; p < 0.0001). They were also of higher gestational age (mean = 28.7 weeks, SD = 2.2) than those who died (mean = 26.7 weeks, SD = 2.5; p < 0.0001). Logistic regression analysis showed that, after controlling for various confounders, only three factors were significantly associated with the survival of these infants. These were: (1) increasing birthweight of the infants (with every gram increase in birthweight, adjusted odds ratio of survival was: 1.009; 95 per cent CI 1.004, 1.015; p = 0.0006); (2) given nasal continuous positive airway pressure for treatment of respiratory distress syndrome (adjusted odds ratio of survival: 4.2; 95 per cent CI 1.2, 14.0; p = 0.02); and (3) given expressed breastmilk (adjusted odds ratio of survival: 57.5; 95 per cent CI: 7, 474; p = 0.0002). Maternal illness, intrapartum problems, ethnicity, gestational age, use of antenatal steroid, modes of delivery, Apgar scores, congenital anomalies, respiratory distress syndrome, persistent ductus arteriosus, septicemia, necrotising enterocolitis, chronic lung disease, oxygen therapy, intermittent positive pressure ventilation, surfactant therapy, and blood transfusion were not significant factors associated with increased survival. PMID- 10730036 TI - Viral isolates during febrile neutropaenia in children with cancer. AB - We prospectively studied South African children with cancer for viral isolates during episodes of febrile neutropaenia. Viruses were found in seven (31.8 per cent) and bacteria in five (22.7 per cent) of 22 episodes. The most common isolate was the herpes simplex virus and the most common source was from nasopharyngeal aspirates. There was no dual detection of viral and bacterial isolates. This study emphasizes the important contribution of viruses to febrile neutropaenia. PMID- 10730037 TI - Xmn I polymorphic site in Yemeni sickle cell disease patients. AB - To investigate the molecular basis of severe clinical presentation in sickle cell disease (SCD) patients in Yemen, this study was conducted on 30 Yemeni SCD patients living in Riyadh and attending King Khalid University Hospital. Seven individuals without SCD were used as controls. Haematological parameters, red cell indices, Hb A2 and Hb F levels were estimated and haemoglobin variant were identified on electrophoresis profiling. DNA was extracted from the buffy coat separated from fresh blood samples and was treated with the restriction endonuclease: Xmn I. The fragments generated were separated on electrophoresis, transferred to nitrocellulose and hybridized to a 32P-labelled probe of gamma globin gene. After extensive washing, two bands, 8.1 kb and 7.0 kb in size, were obtained. The frequency of occurrence of the presence of Xmn I polymorphic site (7.0 kb fragment) and its absence (8.1 kb fragment) were documented. The results in Yemeni SCD patients were compared with the results obtained previously in Saudi Arabs. Of the 30 SCD patients from Yemen 29 had only the 8.1 kb fragment and one had only the 7.0 kb fragment. This gave the frequency of 0.966 for the absence (-) and 0.033 (+) for the presence of Xmn I polymorphic site. This is the same result as that reported earlier for SCD patient from southwestern Saudi Arabia [(-) = 0.966; (+) = 0.033] but is significantly different from that reported in the eastern province [(-) = 0.068; (+) 0.932)] of Saudi Arabia. This paper presents the nature of molecular linkage in SCD patients from Yemen. PMID- 10730038 TI - The Night Vision Threshold Test (NVTT): a simple instrument for testing dark adaptation in young children. AB - It is estimated that 41 per cent of the population aged under 5 in the developing world has an inadequate vitamin A dietary intake resulting in increased morbidity and mortality. Half a million children go blind each year as a result of vitamin A deficiency. Thirteen and a half million have night blindness, the first sign of vitamin A deficiency. Unfortunately, there is no simple, sensitive and inexpensive means to identify the child who has marginal levels of vitamin A and thus institute means to prevent their development of severe deficiency. A low cost, simple, easy-to-use instrument designed to detect a young child's ability to adapt to darkness was tested in children admitted to the Mwanamugimu Nutrition Unit at Makerere Medical School in Kampala, Uganda. Despite the severe degree of malnutrition found in these children, Night Vision Threshold Test results and serum retinol levels were related (r = 0.41, p < 0.05). Further efficacy trials for this instrument are planned at community sites in Nepal. PMID- 10730039 TI - Double versus single phototherapy in term newborns with significant hyperbilirubinemia. AB - The efficacy of double phototherapy, in the form of conventional phototherapy with special blue light plus fiberoptic phototherapy, was compared with conventional phototherapy consisting of special blue lamps alone in a relatively larger series of term newborns with significant hyperbilirubinemia. During the study period the sum of the average spectral irradiances in the double phototherapy group was significantly higher than that of the single phototherapy group (p < 0.05). Phototherapy was effective in decreasing bilirubin levels in both groups, but the response was greater in the double phototherapy group; the duration of exposure to phototherapy was significantly shorter (31.2 +/- 8.5 vs. 38.98 +/- 14.7 h, p < 0.05), and the overall bilirubin decline rate as mumol/l/h and per cent/h was significantly greater in the double phototherapy group (4.1 +/ 1.37 vs. 3.3 +/- 0.86 mumol/l/h, and 1.29 +/- 0.38 vs. 1.02 +/- 0.44 per cent/h, p < 0.05). In phototherapy treatment of term newborns with significant hyperbilirubinemia, double phototherapy provided more rapid and effective bilirubin reduction than conventional phototherapy alone due to higher spectral irradiance and larger body surface area exposed to phototherapy. The value of double phototherapy in the treatment of newborns with hemolytic hyperbilirubinemia remains to be determined. PMID- 10730040 TI - Hepatic dysfunction in childhood dengue infection. AB - Hepatic functions of 61 children, diagnosed to have dengue infection (DI), aged 2 months to 12 years comprising 37 cases of dengue fever (DF), 16 with dengue hemorrhagic fever (DHF), and eight with dengue shock syndrome (DSS) were prospectively studied during the acute attack. Hepatomegaly (74 per cent), epistaxis (26 per cent), jaundice (25 per cent), and petechial rashes (18 per cent) were the common clinical manifestations of DI. On admission, levels of serum aspartate transaminase (AST), serum alanine transaminase (ALT) and serum alkaline phosphatase (AP) were raised in 80-87 per cent of children with hepatomegaly (group I) and 81 per cent of cases without hepatomegaly (group II). During the second week of hospitalization the proportion of cases with raised levels of AST, ALT, AP and serum bilirubin increased and the mean levels were significantly higher (p < 0.05) in both the groups. These levels gradually declined over the next 2-3 weeks. All the cases with DSS and DHF had raised AST, ALT and AP levels and the mean levels of these enzymes were significantly higher (p < 0.05) as compared to DF. Our results suggest a transient derangement of liver functions in childhood DI, more so in DSS and DHF, with or without hepatomegaly. PMID- 10730041 TI - Perinatal mortality in rural Punjab--a population-based study. AB - The results of a population-based case-control study are reported to examine the factors affecting perinatal mortality in rural Punjab, during the period 1991 1996. There were 91 perinatal deaths in 2424 of the pregnant women registered. The perinatal mortality rate was 34.57/1000 and the stillbirth rate was 30.94/1000. Odds ratio, 95 per cent confidence interval, prevalence and population attributable risk percent were calculated for the various risk factors; of the risk factors studied, material weight less than 40 kg, height less than 152 cm, body mass index < 20, illiteracy, a birth to conception interval less than 100 weeks, prematurity, late registration and home delivery were found to be significant on univariate analysis. When subjected to multiple logistic regression, the full model identified the significance of all the risk factors except late registration. However on the final model, only prematurity and short birth-interval were found to be significant. The highest population attributable risk, 35.16 per cent, was observed for prematurity. PMID- 10730042 TI - Risk factors associated with neonatal hypothermia during cleaning of newborn infants in labour rooms. AB - Cleaning newborn infants with coconut oil shortly after birth is a common practice in Malaysian labour rooms. This study aimed: (1) to determine whether this practice was associated with a significant decrease in the core temperature of infants; and (2) to identify significant risk factors associated with neonatal hypothermia. The core temperature of 227 randomly selected normal-term infants immediately before and after cleaning in labour rooms was measured with an infrared tympanic thermometer inserted into their left ears. Their mean post cleaning body temperature (36.6 degrees C, SD = 1.0) was significantly lower than their mean pre-cleaning temperature (37.1 degrees C, SD = 1.0; p < 0.001). Logistic regression analysis showed that the risk factors significantly associated with pre-cleaning hypothermia (< 36.5 degrees C) were: (1) not being placed under radiant warmer before cleaning p = 0.03); and (2) lower labour room temperature (p < 0.001). Logistic regression analysis also showed that the risk factors significantly associated with post-cleaning hypothermia were: (1) lower labour room temperature (p < 0.001); (2) lower pre-cleaning body temperature (p < 0.001); and (3) longer duration of cleaning (p = 0.002). In conclusion, to prevent neonatal hypothermia, labour room temperature should be set at a higher level and cleaning infants in the labour room should be discouraged. PMID- 10730043 TI - Tuberculin reactivity in young children following neonatal BCG vaccination. PMID- 10730044 TI - Magnesium sulphate for management of pulmonary hypertension in non-ventilated newborns. PMID- 10730045 TI - Pulmonary tuberculosis as a cause of recurrent wheezy chest: the value of serological diagnosis using IgG antibodies to mycobacterium 38 kDa antigen. PMID- 10730046 TI - Indirect assessment of peak expiratory flow rate in healthy Indian children. PMID- 10730047 TI - Azithromycin in the treatment of plasmodium falciparum gametocytes: a preliminary observation. PMID- 10730048 TI - Outcome of neonatal care in Port Moresby, Papua New Guinea: a 19-year review. AB - The effectiveness of changes in practice can only be determined by comparing outcome before and after their introduction. This report presents and discusses the neonatal statistics from Port Moresby General Hospital (PMGH) over the last 19 years and attempts to determine the effects of changes in practice introduced during the last year of the study period. Neonatal mortality rates have been very constant. However, the changes in practice--having all but the sickest babies nursed with their mothers and encouraging kangaroo care--together with strict adherence to breastfeeding policies, including the use of expressed breastmilk wherever possible, and the close involvement of the local breastfeeding support group in follow-up, appeared to have beneficial effects in reducing the length of stay and increasing the rate of weight gain in the very low birthweight babies. PMID- 10730049 TI - No brainer. PMID- 10730050 TI - A wondrous thing, a ray of hope. PMID- 10730051 TI - Arrhythmias in athletes. PMID- 10730052 TI - Diagnosis and nonoperative treatment of common athletic shoulder injuries. PMID- 10730053 TI - Sports-related ocular injuries. PMID- 10730054 TI - Exercise-induced asthma. PMID- 10730055 TI - HIV, infectious diseases, and competitive athletics. PMID- 10730056 TI - Get with the guidelines. PMID- 10730057 TI - Head injuries sustained during sports and recreation. PMID- 10730058 TI - Bone mineral density assessment. PMID- 10730059 TI - [Sleep apneas and cerebral vascular accident: an important association in clinical practice. Are we neurologists looking in another direction?]. PMID- 10730060 TI - [Sleep apnea syndrome and cerebral infarction]. AB - OBJECTIVES: To analyze the relationship between snoring and sleep apnea with brain infarction. METHODS: We studied 79 consecutive patients of both sexes with cerebral infarction and 248 age and sex matched controls. We obtained data reflecting arterial hypertension, diabetes mellitus, hypercholesterolemia, smoking and drinking habits, coronary heart disease, cardiopathy, snoring, respiratory pauses during sleep and daytime sleepiness, by using a standard questionnaire to interview every subject and his/her spouse. RESULTS: 53% of patients and 46% of controls snored often or always (p = 0.27). Snoring was significantly more frequent in men. Thirty four percent of patients and 27% of controls were snorers and suffered apnea during sleep (p = 0.19). Nineteen per cent of patients and 11% of controls presented snoring, respiratory pauses during sleep and daytime sleepiness simultaneously, suggesting obstructive sleep apnea syndrome (OSAS) (p = 0.06). However, by separately analyzing people younger than 65 years, the frequency of OSAS was significantly higher in patients (29%) than in controls (7%) (p = 0.006). Finally, 10% of patients and 3% of controls presented snoring, respiratory pauses during sleep and moderate or severe daytime sleepiness simultaneously, suggesting moderate-severe OSAS (p = 0.01). A multiple logistic regression analysis confirmed the independent contribution of moderate severe OSAS as a risk factor for ischemic stroke, with an adjusted odds ratio of 4.54. In people younger than 65 years, OSAS, regardless of its severity, was also an independent risk factor for ischemic stroke, with an adjusted odds ratio of 5.78. CONCLUSIONS: Clinically diagnosed obstructive sleep apnea syndrome is an independent risk factor for ischemic stroke, especially in people younger than 65 years. PMID- 10730061 TI - [Psychotic symptoms and Alzheimer's disease]. AB - BACKGROUND: Psychotic symptoms appear during the course of Alzheimer's disease, but their frequency and intensity vary according to different studies and their nature remains unsettled. OBJECTIVES: To study the frequency and intensity of psychotic symptoms in two transversal series of patients with Alzheimer's disease and analyze its relationship with the duration of the disease and severity of cognitive impairment. PATIENTS AND METHODS: This study has been carried out in patients suffering from probable Alzheimer's disease (NINDS-ADRDA criteria). The stage of the disease was determined according to FAST, and the intensity of cognitive impairment in Mini Mental State Examination was classified as mild, moderate or severe. Frequency and intensity of psychotic symptoms (delusions, hallucinations and misidentifications) were determined by means of semistructured interviews (BEHAVE-AD 78 patients and CUSPAD 69 patients). The results obtained in these three groups of patients were compared through ANOVA variance analysis and mean contrast. Variance and covariance analysis were done to determine the relationship between psychotic symptoms and other variables (degree of cognitive impairment, length of evolution and stage of the disease). For this purpose, the patients with Alzheimer's disease but without psychotic symptoms were considered as control and compared to patients with psychotic symptoms. RESULTS: Nearly half the patients had psychotic symptoms. Delusions appeared earlier and were more frequent than hallucinations and misinterpretations. The more severe was the cognitive impairment, the more frequent and intense were psychotic symptoms, but the difference was significant only in cases with severe cognitive impairment. Hallucinations appeared mainly in patients with advanced dementia and were related firstly with the intensity of functional and cognitive impairment and secondly with the duration of the disease. CONCLUSIONS: Mild psychotic symptoms, especially delusions, appear early during the course of Alzheimer's disease. The frequency and intensity of these symptoms increase in parallel with the functional and cognitive impairments caused by the disease. Hallucinations, which appear mainly when the dementia is severe, can be considered as an evolutive marker of the process. Psychotic symptoms differ from those occurring in other disorders, either neurologic or psychiatric in nature. PMID- 10730062 TI - [Dementia with Lewy bodies]. AB - Dementia with Lewy bodies (DLB) is the second most frequent cause of primary degenerative dementias, following Alzheimer's disease (AD). The nosologic situation of this disease has fragile limits. There is controversy as to whether Parkinson's disease (PD) and DLB are two different entities or whether they make up part of the same spectrum. The terms diffuse Lewy bodies disease and the variant of Lewy bodies in senile dementia or AD have been used to describe pathologic changes with clinical manifestations of dementia and parkinsonism. At present, DLB should be understood as an entity with the essential feature being the presence of Lewy bodies in the brain stem and cerebral cortex. From the point of view of clinical examination, DLB is characterized by the presence of subcortical or progressive cortical dementia, at times without severe memory disorders, with great fluctuations and well detailed recurrent visual hallucinations. These cognitive alterations are associated with parkinsonism. Other frequent disorders are falls, syncopes, transitory alterations in consciousness, great sensitivity to neuroleptic drugs and visual illusions with pseudoperception. The correct diagnosis of this entity is important to administer adequate treatment, to avoid classical neuroleptic drugs and to establish precise prognosis. From a therapeutic point of view, cholinesterase inhibitors show some efficacy in the treatment of cognitive alterations. PMID- 10730063 TI - [Rizatriptan]. PMID- 10730064 TI - [Isolated paralysis of the hypoglossal nerve due to metastasis at the base of the cranium]. PMID- 10730065 TI - [Intracranial hypertension as the first clinical manifestation of gliomatosis cerebri]. AB - A case of gliomatosis cerebri which clinically presented with a syndrome of intracranial hypertension (ICH), involvement of bilateral sixth cranial nerves, and oppressive holocranial headache of one week of evolution. Cranial MR and CT were performed demonstrating diffuse hypodense cortical-subcortical lesions on tomography and in T1 sequences and hyperdense lesions in T2 sequences with irregular contrast enhancement. Intracranial pressure was measured by ventricular catheter with the appearance of high, maintained pressure waves (Lundberg A waves). Ventricular LCR study and cerebral angiography did not provide additional data. Meningeal and cerebral biopsies showed infiltration by pleomorphous glioma leading to the diagnosis of gliomatosis cerebri. The patient was treated with steroids, hyperosmolar agents, external LCR derivation and tumoral radiotherapy. Nonetheless, the patient dies at six months of initiation of the symptoms. Gliomatosis cerebri should be taken into account in the differential diagnosis of clinical pictures presenting with ICH. PMID- 10730066 TI - [Supranuclear gaze palsy following extracorporeal surgery with induced hypothermia. Report of two cases]. AB - Although up to 5-20% of the patients who underwent surgery with hypothermia and cardiac arrest may present neurological complications, just a few cases of ocularmotor disorders have been described. Acquired supranuclear ocular motor paresis (ASOMP) is a rare disorder characterized by impairment in volitional and reflex saccades and smooth pursuit in one or more directions of gaze with intact extraocular movements in response to vestibular estimulation. We present two cases of acquired supranuclear ocular motor paresis associated with a peculiar gait disorder. Although a partial improvement was observed, both patients continue with ocular motor paresis in vertical direction after one year of evolution. A selective vulnerability of certain brainstem neuronal groups would explain the pathophysiology of these symptoms. PMID- 10730067 TI - [Granulocytic sarcoma of neural localization]. PMID- 10730068 TI - Neuromuscular syndromes in an oncologic center. PMID- 10730069 TI - [Verbal fluency in revisited Alzheimer's disease]. PMID- 10730070 TI - [Total mesorectal excision with ultrasonic coagulation knife ("UltraCision") in surgery of rectal cancer]. AB - The authors report a total of 62 middle and low third rectal cancer cases operated on by total mesorectal excision by the method of Heald. The oncological basis of this procedure is the horizontal regional metastatization of rectal cancer. The total mesorectal excision facilitates, the low anterior resections and preservation of sphincter with an ultra-low colorectal, or coloanal anastomosis using the double stapling technique. In the authors' experience, the "UltraCision" cutting-coagulating device permits an atraumatic, bloodless and oncologically correct dissection. Using the double stapling technique, we succeeded in 60% of our middle- and low-third rectal cancer patients to perform a sphincter preserving low anterior resection. In 9 (28%) of the low third rectal cancer patients, preservation of the sphincter was possible with oncologically correct anterior resection and an ultra-low colo-anal anastomosis. Three anastomotic insufficiencies occurred, two of them healed on lotion-suction drainage, and one on the application of transient protective ileostomy. The literature data suggest a lower local recurrency rate after radical rectal cancer surgery, if total mesorectal excision is performed. PMID- 10730071 TI - [Transtubular potassium gradient in the diagnosis of potassium metabolism disorders]. AB - The transtubular potassium gradient (TTKG) is a simple physiologically based clinical test to study the renal excretion of potassium. This article reviews the most important physiological changes influencing TTKG, the hypokalaemia and hyperkalaemia, the effect of mineralocorticoids, alkalosis, action of diuretics among other drugs etc. The authors studied the abnormalities of TTKG occurring in clinical conditions (renal patients with nephrotic edema, "dry" patients with renal diseases, liver cirrhosis associated with ascites, and primary hyperaldosteronism) and compare them to the results obtained in healthy people. They consider the test to be useful in the recognition of conditions with hypoaldosteronism (including the various types of pseudohypoaldosteronisms and aldosterone resistance) and hyperaldosteronism as well as renal diseases, in accordance with the data published in the literature. On the basis of their own results, they found the method of determination of TTKG informative and helpful also when investigating the site of actions and the effect mechanisms of the diuretics. PMID- 10730072 TI - [Comparing aboral pouch construction with Roux-en-Y reconstruction after total gastrectomy]. AB - The aim of this study was the introduction of a new type of gastric substitute, the aboral pouch after total gastrectomy and comparing the motility, nutritional and quality of life parameters of the patients with aboral pouch to the simple Roux-en-Y group in a prospective, randomized, controlled trial. Between September of 1997 and February of 1999 25 patients entered the study. In the pouch group after Roux-en-Y reconstruction aboral pouch was created as a side to side anastomosis approximately 50 cms distal to the esophagojejunostomy, 15 cms in length. In the control group simple Roux-en-Y reconstruction was performed. During the follow up examinations nutritional measurements, scintigraphic evaluation of the gastrointestinal passage, absorption tests and quality of life interviews were carried out. In the first year of the study 15 patients underwent pouch construction, while 10 patients served as controls. Preliminary results suggest that there is no marked difference in the postoperative adaptation between the two groups. However serum immunoglobulin and serum iron measurements, lipid absorption test and quality of life questionnaires predict a slight superiority of the patients with aboral pouch over the simple Roux-en Y group. PMID- 10730073 TI - [Significance of nuchal edema in fetuses of pregnant women under 35 years of age]. AB - The aim of this study was to survey the connection between the fetal nuchal oedema and the increased risk of the aneuploidies. According to the prenatal care the thickness of the nuchal fold was measured on the 11-12th gestational week by 6.5 MHz transvaginal transducer (ATL-HDI and Hitachi EUB-450). Cases were underlined where the presence of nuchal oedema (> or = 3 mm) made the indication for chromosomal analysis among the 280 single pregnancies and fetal karyotyping was carried out. Nuchal oedema was observed in 72/280 cases of pregnant women under 35 years old and in 20/280 cases of that above 35 years old, the nuchal oedema was another indication beside the age. Abnormal karyotype was observed in 9/72 (12.5%) cases among the pregnant women under 35 years old and in 10/92 (10.87%) cases among the pregnancies with fetal nuchal oedema. It was also observed abnormal karyotype in 6/188 (3.19%) cases of pregnancies without nuchal oedema and in 16/280 (5.71%) cases of chromosomal analyses with or without nuchal oedema. This can be an effective method for screening the fetal aneuploidies and other fetal anomalies, both in the high risk and the normal pregnant population, especially in cases of pregnant women under 35 years old belonging to the normal genetical risk population. PMID- 10730074 TI - [The beginnings of medical education in Russia]. PMID- 10730075 TI - [Women physicians with the Nobel Prize]. PMID- 10730076 TI - [Enuresis. 1925]. PMID- 10730078 TI - Upward migration of the L-P shunt catheter into the cranial base. AB - A rare complication of upward migration of the L-P shunt catheter into the cranial base is reported. A 59-year-old female with hydrocephalus underwent L-P shunt with a one-piece catheter. The catheter was secured with a clip but migrataed up to the cranial base without the clip becoming detached from the catheter. We assume that the catheter slipped at the position of the clip as the result of a strong force produced by lumbar movements, and that the clip may have acted like an one-way valve to push the catheter into the spinal canal. To prevent such a complication, we should tighten the clip as firmly as possible and use more clips, or use another type of clip which prevents slipping of a one piece catheter such as the one described by Imaizumi et al. PMID- 10730077 TI - Induction of human gamma delta T cells in myasthenia gravis thymus transplanted SCID mice. AB - BACKGROUND: TCR-gamma delta cells develop by extrathymic differentiation and might play an important role in thymectomy-resistant myasthenia gravis (MG) patients. In this study, we show development of human TCR-gamma delta cells in periphery of SCID mice by transplantation of human MG thymus or thymoma. METHODS: Three pieces of thymoma tissue and four of non-thymoma thymic tissues were obtained from MG patients by thymectomy. Each tissue was transplanted into 5-6 weeks old female SCID mice by intraperitoneal injection or surgical implantation. Rate of human TCR-positive cell development and its subtypes (TCR-alpha beta, TCR gamma delta) were measured in the mice blood at one, three, and six weeks after the transplantation. RESULTS: Human TCR-positive cells were detected three weeks after transplantation. Rate of TCR-gamma delta T cell development got higher in thymoma transplanted group than in non-thymoma transplanted group. CONCLUSIONS: We could successfully develop human mature T cells in SCID mice by transplantation of human MG thymus or thymoma. PMID- 10730079 TI - Preoperative epidural morphine using double-catheter technique for esophagectomy. AB - This study was undertaken to determine whether preoperative epidural morphine using double-catheter technique would improve postoperative analgesia in patients undergoing esophagectomy with or without continuous intraoperative epidural lidocaine in a randomized double-blind and controlled manner. Thirty patients undergoing esophagectomy for esophageal cancer received preoperative epidural morphine 2 mg at T 6-7 and 2 mg at L 3-4 through the catheters, respectively. Thereafter, continuous thoracic epidural infusion of either 1% lidocaine (lidocaine group, n = 15) or normal saline solution (control group, n = 15) at 6 ml/h was initiated. After surgery, mean verbal rating scale of pain (0 to 10) at rest was maintained below 4 and pain on deep breathing was maintained mild in all patients in both groups. There was no significant difference in these values between the groups. In conclusion, preoperative epidural morphine using double catheter technique provided adequate analgesia following esophagectomy. The addition of intraoperative continuous epidural lidocaine did not improve analgesia. PMID- 10730080 TI - An effective method for recovering CD34 positive progenitor cells from peripheral blood stem cell apheresis products cryopreserved with simplified method. AB - The use of small volume collection chamber (SVCC) during peripheral blood stem cell (PBSC) apheresis, combined with simplified cryopreservation without rate controlled freezing, have successfully been applied to clinical PBSC transplantation following myeloablative chemotherapies. However, the method to effectively select CD34+ progenitor cells from frozen apheresis products obtained with these simplified methods has not been reported. For this goal, after washing the thawed apheresis products with medium containing Dnase I, two different approaches to purify CD34+ progenitor cells from washed WBCs were compared. In method I, CD34+ cells were purified on the same day using immunomagnetic method. In method II, the cells after wash were cultured for overnight in RPMI-1640/10% FCS containing SCF and IL-3, followed by enrichment of CD34+ cells as in method I on the next day. In both methods, CD34+ cells were recovered with high viability. However, subsequent liquid culture revealed that the cells obtained from method II have clearly higher growth potential compared with the cells from method I. In addition, these CD34+ cells from method II well-tolerated to further refreeze and thaw. Thus, allowing to "rest" overnight after thaw may be critical for processing of the simply cryopreserved apheresis products as in method II. PMID- 10730081 TI - Heparin-coated circuits prevent renal dysfunction after open heart surgery. AB - We examined whether heparin-coated (HC) circuits can improve the operative results in patients with chronic renal insufficiency. SUBJECTS AND METHODS: Elective cardiac surgery was performed in 24 patients with a serum creatinine (Cr) level of at least 1.5 mg/dl. Uncoated circuits were used in 12 patients, and HC circuits were used in 12 patients. The results of surgery were compared between the two groups. RESULTS: The mean preoperative Cr concentration was significantly higher in the HC group (2.3 vs 1.8 mg/dl). The heparin doses, protamine dose, and activated clotting times were significantly lower in the HC group. The mean blood loss was also less, although this difference was not significant. The postoperative increase in the Cr level was small in the HC group (2.3-->2.5 mg/dl) compared to that in the noncoated group (1.8-->2.3 mg/dl). Postoperative dialysis was not required in the 5 patients in the HC group with a preoperative Cr clearance less than 20 ml/min. CONCLUSION: HC circuits and low dose heparin can be used to maintain renal function after cardiopulmonary bypass, even in patients with severe kidney disease. PMID- 10730082 TI - Complement activation by mycoloyl glycolipids from Mycobacterium tuberculosis and Rhodococcus ruber. AB - In this study, we examined complement activation by mycoloyl glycolipids (MGL) such as trehalose-6,6'-dimycolate (TDM), often termed cord factor, and trehalose 6-monomycolate from Mycobacterium tuberculosis and Rhodococcus ruber, and also examined the effect of complement binding to MGL on phagocytosis by human monocytes. TDM and TMM, but not glucose mycolate, mannose mycolate or fructose mycolate which differ from TMM only in carbohydrate moiety, exhibited complement activation. TDM and TMM of M.tuberculosis exhibited stronger complement activation than those of R.ruber, the mycolic acids of which are much shorter than those of M.tuberculosis. Neither mycolic acids nor trehalose, which are products of TDM and TMM by hydrolytic cleavage, exhibited no complement activation. TDM activated complement through the alternative pathway, since supplementation with C4-deficient serum completely restored classical pathway mediated hemolytic activity of complement which had been previously consumed by TDM. Next, we examined the effects of TDM and TMM on phagocytosis by human monocytes. Coating of heat-killed Staphylococcus aureus cells with TDM or TMM did not enhance their phagocytosis by monocytes, while, in the presence of complement, phagocytosis of these cells increased significantly. These findings suggest that TDM and TMM act as virulence factors that enhance the entry of mycobacteria into phagocytes via binding of C3 through activation of the alternative complement pathway. PMID- 10730084 TI - [Anesthesia in the first decades of the 21st century]. PMID- 10730083 TI - Clinical results of total cystectomy for 92 patients with bladder cancer. AB - The survival rate of 92 patients with primary bladder cancer who had undergone total cystectomy during a 13-year period from 1984 to 1996 was examined. The mean follow-up period was 1,886 days. The 5-year survival rate was 67.9% and the 10 year survival rate was 55.1%. When survival rates were compared pathohistologically, with 81 patients with transitional cell carcinoma divided into two groups, a high-stage group including T3 and T4 patients and a low-stage group with all other patients, the cancer-specific 5-year survival rate of the low-stage group was 88.9% while that of the high-stage group was 45.4%; this difference was significant (p = 0.0002). There were also significant differences in survival rate between those with and those without regional lymph node metastasis, those with and those without lymphatic infiltration, and those with and those without vascular infiltration. However, there was no significant difference in survival rate for the 34 patients with T3 or T4 disease when those with or without chemotherapy and/or radiation therapy were compared. PMID- 10730085 TI - [Effectiveness of isobaric 0.5% bupivacaine and 5% lidocaine in continuous subarachnoid anesthesia with microcatheters]. AB - OBJECTIVES: To study the clinical effect of two isobaric local anesthetics infused through microcatheters for continuous subarachnoid anesthesia. MATERIAL AND METHODS: Patients undergoing surgery under continuous subarachnoid anesthesia were enrolled prospectively over 12 months. Twenty-seven-gage catheters were inserted through 22 G Sprotte (Intralong) needles. The two isobaric anesthetics (0.5% bupivacaine and 5% lidocaine) were studied in two successive six-month periods. One milliliter of local anesthetic was administered, followed by incremental doses of 0.5 ml until the required anesthetic level was reached. Hemodynamic variables were recorded, as were levels of anesthetic and motor blockade and complications developing during the surgical and postoperative periods. RESULTS: Thirty-one patients were anesthetized with isobaric 0.5% bupivacaine and 40 with isobaric 5% lidocaine. A high blockade was observed in three patients in the bupivacaine group and in 15 in the bupivacaine group (p < 0.05). The highest anesthetic level reached was T4. Hypotension occurred in one patient in the bupivacaine group and in 10 in the lidocaine group (p < 0.05). Blockade was difficult to increase to the appropriate level in 11 lidocaine patients and in one bupivacaine patient, whereas blockade of distal roots was difficult in 13 bupivacaine patients and in 7 lidocaine patients (p < 0.005). The total doses infused were 11.0 +/- 3.0 mg of 0.5% bupivacaine and 95.6 +/- 24.6 mg of 5% lidocaine. CONCLUSIONS: Isobaric 0.5% bupivacaine provides a more predictable anesthetic blockade with greater hemodynamic stability and a lower rate of difficulty in raising the level of blockade than does 5% lidocaine when administered through microcatheters for continuous subarachnoid anesthesia. PMID- 10730086 TI - [Usefulness of an anesthesia information sheet before the preoperative visit]. AB - OBJECTIVE: The information patients receive about anesthesia in the preoperative period must be clear in order to assure their understanding and involvement in making decisions about proposed techniques. A sheet providing basic general information about anesthesia can prepare the patient for consultation with the anesthesiologist. PATIENTS AND METHODS: A questionnaire with 14 yes/no questions was designed for this prospective, descriptive study. We surveyed 184 consecutive patients referred for preanesthetic assessment before elective surgery in a general hospital on the outskirts of Barcelona. RESULTS: The information sheet was judged good and necessary for obtaining informed consent by between 96 and 97% of the patients. A similar percentage judged the comprehensibility of the sheet to be good, although 15% did not correctly answer questions about matters that were covered by the sheet. Anxiety increased after reading the sheet for 12%, and 10% even began to tremble. Additional clarification was requested by 9%. CONCLUSIONS: An information sheet improves comprehension of anesthetic procedures and facilitates informed consent. However, a need for further clarification is generated in a small proportion of patients and a substantial proportion believes the sheet caused greater fear. We therefore believe that information should be adapted to the particular requests and needs of individual patients. PMID- 10730087 TI - [Craniocerebral injuries: initial treatment and at resuscitation units]. AB - Head injury is the main cause of death or disability among under-45-year-olds. This review covers the main pathophysiological aspects of head injury as well as initial treatment and management in the intensive care recovery ward. The chief therapeutic aim is to maintain adequate cerebral perfusion pressure rather than to maintain normal levels of intracranial pressure. An important challenge is to avoid development of secondary lesions, and in this context arterial hypotensive events that affect prognosis and the survival of such patients merit special attention. We reject treatment based on indiscriminate use of hyperventilation and mannitol and underline the importance of providing adequate sedation and analgesia while maintaining normal flow and pressure and adequate monitoring of such patients. PMID- 10730088 TI - [Heart arrest in cemented hip arthroplasty]. AB - Hip arthroplasty is a common surgical intervention in our hospital practice, involving high perioperative risk related to patients age and multiple concomitant diseases. Hemodynamic complications described vary from slight hypotension during surgery to heart failure and sudden death, particularly if the operation involves a cemented femoral component. Because of the type of patients undergoing such operations (elderly patients, with osteoporosis and scarce cardiopulmonary reserve), the unclear origin of complications and the lack of consensus on what constitutes adequate monitoring during surgery, hip arthroplasty is problematic for the specialists involved. We report on five deaths during cemented hip arthroplasty; after reviewing the case history and autopsy report of one, we believe the events leading to death were triggered by massive pulmonary embolism. PMID- 10730089 TI - [Locoregional anesthesia for cesarean section in full-term pregnancy with single ventricle]. AB - A parturient at full-term diagnosed of a single ventricle and with the fetus in podalic presentation underwent scheduled delivery by cesarean section under epidural anesthesia without complications. The number of women with this cyanogenic congenital heart disease who reach childbearing age is high. The rate of death among parturients with mild or moderate heart disease is low; however, mortality is higher for high-risk women for whom conception is considered contraindicated due to heart disease. Few cases of single-ventricle congenital heart disease have been reported in the literature. PMID- 10730090 TI - [Constraints on the use of a nerve stimulator in peripheral conduction blocks when there is disturbance in the neuromuscular junction]. AB - An 81-year-old woman with right lower limb ischemia was scheduled for revascularization of a femoropopliteal bypass grafted 10 years earlier. A popliteal blockade, attempted as part of regional anesthesia with the aid of a nerve stimulator, was not achieved because the posterior tibial nerve could not be located. After surgery, the patient mentioned symptoms in the region of the right knee consistent with complex regional pain syndrome (Ducke's stage 3); the symptoms appeared after the first operation and would explain the absence of response to the nerve stimulator. Using a nerve stimulator to facilitate location of the various nerve trunks for anesthesia involves obtaining a motor response to electrical stimulation. The procedure is becoming more and more frequent because of its many advantages over other more traditional methods. However, it may be impossible to locate a nerve for a variety of reasons. PMID- 10730091 TI - [Septic meningitis following subarachnoid anesthesia]. PMID- 10730092 TI - [Remifentanil in major ambulatory surgery]. PMID- 10730093 TI - [When is the best time to use fracture osteosynthesis in multiple trauma patients with associated craniocerebral injury?]. PMID- 10730094 TI - [Disseminated intravascular coagulation, massive hemothorax, and shock in the immediate postoperative period of uterine curettage]. PMID- 10730095 TI - [Bispectral index (BIS) and heart arrest]. PMID- 10730096 TI - [Depression is by far the most frequent psychiatric disorder]. PMID- 10730097 TI - [Epidemiology and comorbidity of depressive disorders]. AB - Recent epidemiological surveys in general populations of different countries of the world found lifetime prevalence rates of major depressions between 3.3% and 17%. For dysthymia (depressed mood over a period of at least two years with at least two concomitant depressive symptoms) the prevalence rate was found to be between 2% and 7%. The prevalence rates of major depressions and dysthymia are usually higher for females than for males. Bipolar disorders can be observed in about 1% of a general population over lifetime, and they seem to be somewhat more common among males than females. Divorced and separated persons have a higher risk of suffering from major depressions than married persons. Major depressions are thought to be more common among members of the lowest social class than among people belonging to the upper classes. Major depressions usually start between the age of 25 and 30 years, and the age of onset of bipolar disorders is between the age of 18 and 30 years. For western industrial nations a secular trend towards an increase in the prevalence of major depressions may be presumed. However, such a secular trend has not yet been confirmed, owing to biases associated with methodological problems. A notable comorbidity of major depressions can be observed with all anxiety disorders, obsessive-compulsive disorders, eating disorders, post-traumatic stress disorder, disorders of impulse control, abuse and dependence of alcohol and of other legal and illegal drugs, pathological gambling, migraine, fibromyalgia and irritable bowel syndrome. This observation has led to the concept of an "affective spectrum". This phenomenon has to be kept in mind during the diagnostic process and treatment. PMID- 10730098 TI - [Diagnosis of depression]. AB - The importance of the identification of depressions in clinical practice is emphasised. The author suggests the use of modern operationalized diagnostic criteria. In atypical depressions and depressions with comorbid psychiatric disorders the diagnosis is sometimes missed. Occasionally, it is impossible to decide if a depression is present or not. In such cases it is in general indicated to try antidepressive treatment. PMID- 10730099 TI - [Review of several different forms of psychotherapy in treatment of depression]. AB - The present article endeavours to introduce, in a concise form, selected forms of psychotherapy for the treatment of depressions. In particular behaviour therapy (BT), cognitive therapy (CT), interpersonal psychotherapy (IPT) and a psychoanalytically oriented and inspired therapy, subsequently named psychodynamic therapy (PT), are described. The attempt to offer a brief presentation of these treatment methods follows essentially the descriptions of Elizabeth Schramm in her book "Interpersonelle Psychotherapie". We thank the author for giving her kind consent to our publication. Our main concern is to enable the reader to recognize the essential characteristics and differences between these treatment forms, so that he can give the depressive patient advice on the choice of an adequate treatment. The first three brief psychotherapies have been developed specifically for the treatment of depressions. The psychodynamically (psychoanalytically) oriented approach is the most frequently used. CT, BT and IPT in particular have in common that they offer a temporally limited, clearly structured treatment programme in the sense of a manual, and are thus teachable and conveyable and efficient with respect to costs. We would point out, however, that combinations between the different treatment forms are possible and that a strict distinction between the separate methods is chiefly of didactic value. Essential to all treatment methods is the empathic and supporting relation to the patient. The therapist should play an active role and should convey the hope that the patient can be helped. All the described treatment forms are suited for the treatment of light to moderately severe depressions, with or without concomitant psychopharmacological therapy. In psychodynamic therapy the tendency is to dispense with medication, whereas CB, CT and IPT tend to integrate the use of psychoactive drugs in their treatment schedules. In our opinion psychopharmacological treatment should on no account be renounced to in severe depressions and where there is risk of suicide. We would also point out that IPT and CT offer specific group programmes for the in-patient treatment of depressive patients. The four treatment forms are briefly reviewed (theoretic background, treatment indications, monitoring, effectiveness), followed by a short overview of the most essential characteristics and differences between the separate treatment forms and by contact addresses. PMID- 10730100 TI - [Phototherapy]. AB - Recommendations for a light therapy--PREVALENCE OF SEASONAL AFFECTIVE DISORDER (SAD): Ca. 2% of the adult population in Europe; female to male ratio 4:1. DIAGNOSIS: Recurrent major depressive episodes in winter with full remission in summer. DURATION OF LIGHT THERAPY: For 10,000 lux light boxes 30 minutes/day, for 2500 lux light boxes 1-2 hours/day. TIME OF DAY: If possible in the morning after awakening, otherwise at a time of day when a regular daily light therapy is practicable. ONSET OF LIGHT THERAPY EFFECT: Often already after a week (2-4 weeks treatment produces a stable antidepressant effect). SIDE EFFECTS: Rare, can be avoided by reducing the light dosis. OPHTHALMOLOGICAL RISKS: None under normal conditions. Patients with ocular risk factors (e.g. photosensitizing medications) or preexisting retinal diseases should undergo ophthalmologic examination. LIGHT OR ANTIDEPRESSANT DRUGS: Light therapy is the first line treatment for SAD. For partial or non-response, combination of light with SSRIs is recommended. LIGHT THERAPY MAY BE AN ADJUVANT TREATMENT FOR OTHER PSYCHIATRIC ILLNESSES WITH A SEASONAL TIME COURSE: Bulimia nervosa, obsessive-compulsive disorder, panic disorder; there is some preliminary evidence for usefulness in premenstrual dysphoric disorder and also for non-seasonal major depression. PMID- 10730101 TI - [Antidepressive therapy by modifying sleep]. AB - Depressive symptoms are unspecific and occur in several psychiatric disorders. Sleep disturbances are also frequently present in depressed patients. As a consequence, it has been established that a number of modulations of the sleep wake cycle can have an antidepressive effect. Total sleep deprivation or deprivation in the second half of the night have proven successful. The main limitation of the otherwise well tolerated treatment is the short duration of the antidepressive effect, which is mostly reversed in nearly all patients after the following night's sleep. New approaches are to shift the timing of sleep to earlier to ensure a possible longer-lasting effect. In clinical praxis the following manipulations should not be used: sleep deprivation in the first half of the night (not successful), REM-sleep deprivation (experimental setting), induced sleep prolongation (negative risk-benefit-ratio). In addition to patients with affective disorders sleep deprivation has proved relevant in patients with schizophrenia (depressed and/or with predominantly negative symptoms) and premenstrual dysphoric disorder. Very few side effects have been reported. Although many hypotheses have been tested, the mechanism of action underlying the antidepressive effect of sleep deprivation is still unknown. PMID- 10730102 TI - [Pharmacologic treatment of depression]. AB - The pharmacological treatment of depression is focussed on antidepressants. Different substance groups with different biochemical mechanisms and side effects have antidepressant effects. Based on these different substances the pharmacological treatment of depression has become more and more differentiated. The combination of antidepressants and other drugs (lithium, carbamazepine, benzodiazepines, stimulants, antipsychotics, hormones) offers additional possibilities. The combination with other biological treatments of depression enlarges the spectrum of treatments. The inclusion of non-biological therapies makes the therapeutic possibilities with proven antidepressant efficacy rich and powerful. PMID- 10730103 TI - [Electroconvulsive therapy of depressive disorders]. AB - Electroconvulsive therapy (ECT) is the most effective treatment in all types of major depression. On the other side ECT has long suffered from controversial public image, a reputation that has effectively removed it as treatment option for many patients. Today ECT is an effective and safe treatment for those with severe mental illness. Electroconvulsive therapy has undergone fundamental changes since its introduction 65 years ago. It is no longer a memory-modifying, fearsome treatment pictured in films. Anesthesia, controlled oxygenation, and muscle relaxation make the ECT so safe that the risks are less as those which accompany the use of several psychotropic drugs. Indeed, for the elderly, the systematic ill, and pregnant women, electroconvulsive therapy is a safer treatment for mental illness than any alternative. PMID- 10730104 TI - [Diagnosis and therapy of depression in advanced age]. AB - Depressive symptoms and dementia are the most frequent psychiatric disorders in late life. Somatic diseases, social isolation, and functional disability, interfering with the activity of daily life and social participation are among the relevant risk factors. The elderly are particularly prone to subsyndromal depression because of their increased tendency to alexithymia and somatisation, which masks the depression. There is a strong association between comorbid physical illnesses and depressive symptoms for a number of common medical disorders. Antidepressive therapy is proven to be as effective as in younger age groups. Medical treatment should consider the special pharmacological features of old age and should be combined with psychotherapy. When the notion that depression in older people is "justified" is no longer common sense, then successful treatment will be more likely. PMID- 10730105 TI - [Case report. Tailored therapy of depression]. AB - Tailored therapy means an integrative strategy, including all therapeutic possibilities important for the patient, usually: psychotherapy, psychopharmacotherapy, help of the general physician and active support for social problems. Many patients need far more therapies, for example physiotherapy, art therapy, help with homework, dance lessons. This integrative strategy is not focussed on a single therapeutic method but on the patient himself. It is very important, to choose for every method the right moment. PMID- 10730106 TI - The general physician--extinction or evolution? PMID- 10730107 TI - Telemedicine in the National Health Service. PMID- 10730108 TI - Diabetes epidemic in newly westernized populations: is it due to thrifty genes or to genetically unknown foods? AB - Until a few decades ago, certain 'new-world' populations that kept to traditional dietary habits were virtually free from diabetes; then, after they began eating some foods that are common in Europe, the disease reached epidemic proportions. Europeans, by contrast, have a low rate of diabetes. To account for this paradox, it has been suggested that those new-world populations have a thrifty genotype, which would have conferred a selective advantage during the frequent famines of the past, while today it would be detrimental because the recently adopted foods are constantly available. Here it is proposed that thrifty genes are unlikely to exist. Both the diabetes epidemics that occur in newly westernized populations and the low rate of diabetes in Europeans can be explained by the hypothesis that Europeans, through millenary natural selection, have become adapted, albeit incompletely, to some diabetogenic foods for which humankind is genetically unequipped. PMID- 10730109 TI - Problem-based learning as an alternative to lecture-based continuing medical education. PMID- 10730110 TI - Effect of beer consumption on plasma magnesium: randomized comparison with mineral water. AB - Moderate consumption of ethanol lowers mortality from coronary artery disease, and one of the possible mechanisms is an antiarrhythmic action. We therefore investigated the effect of a small daily dose of beer on plasma electrolytes. 52 men who seldom drank alcohol, clinically stable more than one year after coronary bypass surgery, were randomized to drink either 330 mL beer (containing about 20 g ethanol) or mineral water with similar potassium, magnesium, calcium and sodium content daily for 30 days. Plasma electrolytes and liver function indices, and also heart rate, blood pressure and weight, were measured before and after the trial period. The only significant before-and-after difference was in the group consuming beer, whose plasma magnesium rose from 0.89 (SD 0.01) to 0.98 (SD 0.02) mmol/L (P < 0.0025). This level of beer consumption did no obvious harm to liver function and its possibly beneficial effect on plasma magnesium deserves further investigation. PMID- 10730112 TI - Emergency small-bowel resection in a district general hospital. AB - Small-bowel resection has been identified as a core surgical skill that all general surgical trainees must acquire. Most of these resections are performed by the unsupervised higher surgical trainee on call. Reviewing 51 small-bowel resections performed over a five-year period in a district general hospital we found that, although the operation carried a high mortality rate (18%) and a high morbidity rate (21%), these had less to do with the operative technique than with the nature of the underlying disease and the hazards of emergency surgery in general. We conclude that small-bowel resection per se is relatively safe and remains a good training procedure. PMID- 10730111 TI - Management and morbidity of cellulitis of the leg. AB - Ascending cellulitis of the leg is a common emergency. An audit was conducted in two district general hospitals to determine how it is managed and the long-term morbidity, and to formulate a treatment strategy. Case notes were reviewed for 92 patients admitted to hospital under adult specialties. Mean duration of inpatient therapy was 10 days. A likely portal of entry was identified in 51/92 cases, of which the commonest were minor injuries and tinea pedis. Pathogens were rarely identified, group G streptococci being the single most frequent organism. Benzylpenicillin was administered in only 43 cases. Long-term morbidity, identified in 8 of 70 patients with over six months' follow-up, included persistent oedema (6) and leg ulceration (2); an additional 19 patients had either suffered previous episodes or experienced a further episode subsequently. Ascending cellulitis of the leg has substantial short-term and long-term morbidity. Important but often neglected therapeutic suggestions are the inclusion of benzylpenicillin in all cases without a contraindication, assessment and treatment of tinea pedis, use of support hosiery, and serological testing for streptococci to confirm the diagnosis in retrospect. The high frequency of recurrent episodes suggests that longer courses of penicillin, or penicillin prophylaxis, might be useful. PMID- 10730113 TI - Sudden-onset ageusia in the antiphospholipid syndrome. PMID- 10730114 TI - Evaluating the return of prostatic adenocarcinoma. PMID- 10730115 TI - Non-Hodgkin lymphoma diagnosed from a tracheal window biopsy. PMID- 10730116 TI - Renal carcinoma with acute mastoiditis. PMID- 10730117 TI - Ruptured aortic aneurysm with aortocaval fistula. PMID- 10730118 TI - Inguinal hernia presenting as iron deficiency anaemia. PMID- 10730119 TI - Pseudocyst formation after mesh repair of incisional hernia. PMID- 10730120 TI - Long-term morbidity from Pope Ear Wicks. PMID- 10730121 TI - Anal sphincter disruption from a snowboarding injury. PMID- 10730122 TI - Studs, the Ordernet and peer review. PMID- 10730123 TI - This month in history. PMID- 10730124 TI - Consent obtained by the junior house officer. PMID- 10730125 TI - Consent obtained by the junior house officer. PMID- 10730126 TI - Consent obtained by the junior house officer. PMID- 10730127 TI - The NHS at fifty. PMID- 10730128 TI - Alcohol and the heart. PMID- 10730129 TI - Training of surgeons. PMID- 10730130 TI - Nipple trauma. PMID- 10730131 TI - Frey's syndrome without hyperhidrosis. PMID- 10730132 TI - Carbon monoxide poisoning and death following the use of explosives. PMID- 10730133 TI - A new approach to needlestick injuries among health care workers. PMID- 10730134 TI - Bloodborne exposures at a United States Army Medical Center. AB - With the ultimate goal of minimizing exposures, we conducted a hazard analysis on bloodborne disease transmission at our hospital to identify appropriate control interventions. We utilized basic principles of occupational epidemiology to gather information on the severity and extent of exposures. Because we suspected inadequate reporting of needlestick injuries, we collected 339 reported exposures of health care workers; we conducted a survey of all workers requiring universal precautions training for bloodborne pathogens. The annual incidence of exposures reported was 93.7 per 1000 workers who required this training. Sharps accounted for 83.5 percent of these exposures. Exposure sources demonstrated 4.3 percent positive for HIV, 4.4 percent positive for hepatitis B, and 7.1 percent positive for hepatitis C. The survey indicated that blood/body fluid exposures were underreported by at least fourfold. House officers were most at risk. At-risk behaviors were identified by the significant differences in knowledge of HIV transmission and work practices between groups reporting no or single exposures versus groups reporting multiple exposures. Increased emphasis should be placed on education, reporting exposures, and training house officers in procedures requiring the use of hollow bore needles. This study shows how the use of occupational epidemiology principles and methods were utilized in conducting a thorough hazard analysis and in identifying appropriate control methods. PMID- 10730135 TI - Working conditions and health in hairdressing salons. AB - The purpose of the study was to assess the working conditions in hairdressing salons and the influence of work factors on the workers' health. Twenty randomly sampled hairdressing salons in the Helsinki, Finland, metropolitan area were selected for the study. The study was performed during winter 1994-1995; it included a survey of the hairdressing chemicals in use, the measurement of physical and chemical working conditions, and a self-administered questionnaire of the work environment and health of the workers. The air temperature varied between 16-25 degrees C, air velocity 0.02-0.3 m/s, and relative humidity, 18-42 percent. The total dust concentration varied between 66-133 micrograms/m3. The concentration of volatile organic compounds was 84-465 micrograms/m3 and the peaks rose to 25-45 mg/m3. The highest concentration of ammonia detected was 3.5 mg/m3. The long-term concentrations of thioglycolates and persulfates were at their lowest below the detection limit, and at their highest 1.8 micrograms/m3 for thioglycolates and 4.7 micrograms/m3 for persulfates, respectively, and the peaks of persulfates, 30 micrograms/m3. Hairdressing chemicals, awkward work postures, and repetitive movements were the most frequent causes of discomfort and for some had caused a work-related disease. Good general ventilation decreased the health complaints caused by hairdressing chemicals, but caused discomfort as a result of drafts. On average, the physical and chemical working conditions in the hairdressing salons were satisfactory compared with the Finnish criterion for indoor climate. However, the frequent high peak concentrations of chemicals during dyeing, bleaching, permanenting, and aerosol spraying still pose a significant health problem. Although effective general ventilation alleviated the effects of the air pollutants, it could not completely solve the problem. Therefore, local exhaust ventilation is recommended at the mixing places for hairdressing chemicals and wherever they are applied to the hair. According to our results, already increasing the air exchange rate up to 5 to 7 times per hour during the high exposure jobs would improve the situation. PMID- 10730136 TI - Silo gas exposure in New York state following the dry growing season of 1995. AB - Exposure to silo gas is a recognized agricultural hazard. Silo gas produced from corn fermentation may consist of oxides of nitrogen and carbon dioxide. The presence of potentially lethal concentrations of nitrogen dioxide (NO2) within vertical silos has been well documented. The risk of silo gas exposure from other silage storage methodologies--including horizontal "ag-bags" and concrete bunkers -has been less well characterized. A dry growing season is known to be a factor for elevating nitrate levels in corn plants and can result in increased NO2 production. Farms in the northeastern United States faced drought conditions during the 1995 growing season. The New York State (NYS) Department of Health (DOH) and the New York Center for Agricultural Medicine and Health (NYCAMH) investigated four exposure incidents involving six farmworkers during September/October 1995. Four of these workers were hospitalized for multiple days, with two workers receiving treatment in intensive care units. The remaining two workers were treated in hospital emergency departments; one refused admission and left against medical advice. We monitored NO2 levels from "ag-bags" at several New York farms. For four days, outdoor concentrations of NO2 at one site remained in excess of the National Institute for Occupational Safety and Health's (NIOSH) immediately dangerous to life and health value (IDLH) of 20 ppm. As a result of the clinical and industrial hygiene data, and the growing season's abnormal weather conditions, DOH and NYCAMH issued statewide health hazard alerts and conducted educational activities to warn farmers and their families. The findings of this study reinforce the potential hazards associated with silo gas exposure and identify the use of ag-bags as a relatively new avenue for significant worker exposure. PMID- 10730137 TI - Exposures of geotechnical laboratory workers to respirable crystalline silica. AB - Geotechnical laboratory testing involves the determination of the physical properties of soil, rock, and other building materials for engineering purposes. Individuals working in these laboratories are exposed to airborne soil, rock, and other dusts during the preparation and testing of these materials. Crystalline silica as quartz is a common constituent of these materials and represents a potential hazard to geotechnical laboratory workers when airborne as a respirable dust. The authors conducted an examination of the potential for geotechnical laboratory workers to be exposed to respirable dust and respirable quartz during the performance of three routine laboratory tasks. A task-based exposure assessment strategy was used. Although respirable dust was generated during the performance of each of these tasks, its impact on exposures was generally overridden by the presence of respirable quartz in the dust. Quartz content in the respirable dust ranged from below the detection limit to greater than 50 percent. Mean exposure to respirable quartz, based on the duration of the task and assuming no other exposures for the rest of the 8-hour day, exceeded the National Institute for Occupational Safety and Health (NIOSH) "action level" (the exposure level at which certain actions must be taken) of 0.025 mg/m3. If exposure was assumed to continue for the rest of the 8-hour day at the measured concentration, mean exposure to respirable quartz exceeded the American Conference of Governmental Industrial Hygienists (ACGIH) Threshold Limit Value (TLV) time-weighted average (TWA), the Occupational Safety and Health Administration (OSHA) PEL, and the NIOSH REL. Seven percent of 57 individual task exposure measurements exceeded the TLV-TWA and the PEL, 18 percent exceeded the REL, and another 12 percent exceeded excursion limits as defined by ACGIH. The results of this study support the conclusion that geotechnical laboratory workers are potentially exposed to respirable crystalline silica as quartz at levels that may be harmful. Because the quartz content of the materials being tested in these laboratories is highly variable and is almost never determined prior to testing, all materials being tested in the geotechnical laboratory should be assumed to contain quartz. Appropriate controls should be used to protect workers from inhaling dusts generated from these materials. PMID- 10730138 TI - Mortality among North Carolina construction workers, 1988-1994. AB - This study evaluated proportionate mortality patterns among all male construction workers in North Carolina who resided and died in North Carolina during the period 1988-1994. Proportionate Mortality Ratios (PMRs) and Proportionate Cancer Mortality Ratios (PCMRs) compared the number of deaths among male construction workers with the number of deaths expected based on the gender, race, and cause specific mortality experience of the entire North Carolina population by five year age groups for the same years of study. PMRs based on United States death rates also were calculated. Among all male construction workers, significantly elevated mortality was observed for several causes possibly related to work including malignant neoplasms of buccal cavity (PMR = 143), pharynx (PMR = 134), and lung (PMR = 113), pneumoconiosis (PMR = 111), transportation accidents (PMR = 106), and accidental falls (PMR = 132). Elevated mortality also was observed for causes more related to lifestyle and non-occupational factors including alcoholism (PMR = 145), cirrhosis of the liver (PMR = 129), accidental poisoning (PMR = 136), and homicide (PMR = 141). Patterns of elevated mortality for Whites and Black men were similar and PCMR mortality patterns for Blacks and Whites combined were similar to PMRs. Construction workers were at significantly increased risk for deaths resulting from falls from ladders or scaffolds, falls from or out of buildings or structures, and electrocutions. Construction trades found to have statistically elevated cancer risks include laborers and roofers (buccal cavity), painters (pharynx), laborers (peritoneum), and carpenters, painters, brick masons, and operating engineers (lung). These data are consistent with other reports demonstrating excess mortality from asbestos-related diseases (pneumoconiosis, lung cancer, and mesothelioma) among construction workers. Dry wall workers and laborers were found to have a statistically elevated risk of death as a result of respiratory tuberculosis. PMID- 10730139 TI - [Human platelet antigens and their clinical implications (First part)]. PMID- 10730140 TI - [Hand injuries in the ready-made clothing industry]. AB - We collect during 1997, 30 work accidents in the ready-wade clothe sector. These accidents have concerned 28 females and 2 males with a mean age of 21 years. Injury affect predominatly the left hand (17 patients) and the finger of pince. Severity of the lesions was variable according to the traumatic cause. In the distal fingers trauma, skin cicatrisation have been observed after 21 days and bone consolidation within 45 days. PMID- 10730141 TI - [Retrospective study of 167 ovarian tumors]. AB - OBJECTIVE: Our purpose was to assess clinical particularities, diagnosis and therapeutic problems of ovarian tumors. STUDY DESIGN: A retrospective study of 156 patients having undergone an operation for ovarian tumor not suspected to be malignant between january 1991 and june 1998. The clinical, ultrasonographic and tumor marker data were analysed. RESULTS: Mean age of our patients was 34 years. 19 patients were menopausal. All ovarian masses were considered as benign at ultrasonographic scan. Serum CA125 level was measured in 42 cases. It was abnormal in 2 cases. 102 patients underwent laparotomy. 54 patients underwent laparoscopy, of these, 19 were converted to laparotomy due to a suspect macroscopic diagnosis (2 cases) or to technical difficulties (13 cases). Histological findings were 150 benign ovarian tumors, 12 fonctional cysts and 5 malignant ovarian tumors. Therefore, preoperative assessment had failed diagnosis of cancer in 5 cases. CONCLUSION: The clinical, ultrasonographic and tumor markers triad remains the best preoperative approach currently available for ovarian tumors. However, the final diagnosis remains histological. PMID- 10730142 TI - [Study of anemia in giardiasis intestinalis in Tunisian preschool children]. AB - The present work is based upon a prospective in study done in a semi-urban area of suburbs of Tunis, from february to November 1997. A total of 302 children aged between 6 month to 5 years were enrolled in the survey. The study aimed at assessing the extention of parasitoses in preschool aged children. The relationship between the Giardiasis intestinalis and ferropenic anemia. The prevalence of anemia is 31.78% (n = 302). The parasitologic analysis has shown that 113 children out of 302 are infected; 37.41%. We observed an obvious predominance of Giardia Lamblia: 62% (n = 113) pathogenic protozoon. The rate of anemia parasited children is amounted to 19.78%. During the Giardiasis, anemia is present in 23.17% of the cases. The Polyparasitism concerns 16% of the infested children. This anemia could be caused by a global bad absorption syndrome or by a ferro-elective bad absorption. A proper sanitary education, a purification action and also a curative treatment of the beaners carriers will be the only guarantees to decrease its morbidity. PMID- 10730143 TI - [Diagnostic imaging in neuro-Behcet's disease. Report of 5 cases]. AB - The authors report five patients with neuro-Behcet's disease. Computed tomography showed low-density lesions with or without mass effect and contrast enhancement. Magnetic resonance imaging revealed scattered areas of high signal intensity on T2-weighted images, and iso or low signal intensity on T1-weighted images with enhancement after gadolinium injection. These lesions affected all the central nervous system structures with predilection to the brain stem, basal brain ganglia, internal capsula, subcortical and deep cerebral white matter. By its availability, computed tomography remains a precious tool for diagnosis and assessment of severity of neurological involvement. Brain magnetic resonance imaging reveals silent lesions on CT, specifies their exact topography, identifies lesional association reminiscent of neuro-Behcet and contributes to the differential diagnosis with multiple sclerosis. CT and MRI permit the follow up of lesions under treatment and evaluate prognosis. PMID- 10730144 TI - [Testicular non-Hodgkin's lymphoma. Report of 2 cases]. AB - We report two cases of non Hodgkin malignant lymphoma of the testis observed in two patients 36 and 32 years old. Diagnosis was made on pathological examination after orchiectomy. One patient had an involvement of the lung and Waldeyer's ring and the other presented cerebral involvement. Despite combination chemotherapy and external beam radiation therapy, the disease progression was rapid and fatal in the first patient (19 months) while the second had multiple cerebral relapses and died by disease progression 21 months after orchiectomy. PMID- 10730145 TI - [Neuroendocrine carcinoma in Barrett's esophagus. Report of a case]. AB - The neuroendocrine tumours of the oesophagus are exceptional. They are a spectrum of heterogeneous tumours often not clearly defined in the literature. We report a case of moderately differentiated neuroendocrine tumour of the oesophagus arising from Barrett's mucosa in 51-year-old man treated surgically and who was free of disease 28 months after excision. We discuss about this case, the classification, the histogenesis and therapeutic approaches of these tumours. PMID- 10730146 TI - [Congenital colic diverticulum and colo-duodenal fistula]. PMID- 10730147 TI - [Periarteritis nodosa associated with lung cancer. A new observation]. AB - The cases of vasculitis associated to malignant tumour are uncommon, their course which is often parallel suggests a direct link between them. We report the observation of periarteritis nodosa associated with a lung cancer discovered in the following of vasculitis; whereas the course is favorable in 83 years old man without particular history. The frequency of neoplasia with vasculitis is estimated between 3 and 8%, and periarteritis nodosa can't be a paraneoplastic syndrome. Lung and colic tumours are the most frequent reported of the solid tumours. The delay of appearance of vasculitis varies from 25 months before to 9 months after cancer diagnosis. There are many factors, either some treatment, the neoantigens involve the formation of immune complexes, lymphokines and some vasoactive substances. PMID- 10730148 TI - [The 5q(-) syndrome. Report of 2 cases]. AB - A rare and primitive myelodysplastic syndrome 5q(-) is characterised first, by the persistence of the cytogenetic anomaly 5q(-), and second, by its feminine predominance. Among 13 cases of myelodysplastic syndromes, the subject of a substantial and systematic cytogenetic medullar study (1996-1998), this paper is a case study of 2 syndromes 5q(-) diagnosed in two male patients, respectively, aged 41 and 68. The following diagnosis was made on the basis of an aregenerative macrocytic anaemia, a high platelet count, and a megakaryocytic hyperplasia, along with dysmegakaryocytopoiesis. The diagnosis of the 5q(-) syndrome was verified by cytogenetic analysis showing in one of the patients a deletion 5q( )(q13, q33) and 5q(-)(q14, q34) with trisomy in the second one. Treatment was only limited to a blood transfusion. Subsequently one of the patients developed an advanced case of leukaemia. This paper suggests that a systematic medullar cytogenetic study must be conducted in the case of any refractory anaemia in order to identify the syndrome 5q(-) in individual cases. PMID- 10730149 TI - [Tubo-ovarian xanthogranulomatous inflammation. Report of a case]. AB - Xanthogranulomatous inflammation in the female genital tract is rare and characterized by a massive infiltration of the tissue by lipid containing histiocytic cells, together with lymphocytes and plasma cells. We report a new case of Xanthogranulomatous inflammation of the female genital tract. PMID- 10730150 TI - [Polyneuropathy due to glue-sniffing intoxication. Report of a case]. AB - We report a case of a 24 years old female who presented a glue sniffing neuropathy. Symptoms began with progressive weakness which initially involves the lower extremities. Neurological examination noted a decreased Achille reflexes. Nerve conduction studies showed a slowing nerve conduction velocities, a delayed F waves and distal latencies and diffuse nerve conduction blocks. The disease continued to worsen beyond two weeks after stopping intoxication with persistent evidence of ongoing demyelination. 40 days later we have noted an improvement in nerve conduction velocities and in conduction blocks, neuropathy is the most frequent glue sniffing complications, there is a clinic and electric improvement of the polyneuropathy after stopping intoxication. PMID- 10730151 TI - [Alzheimer's disease]. PMID- 10730152 TI - [Results of a prospective protocol for the treatment of adult Hodgkin's disease]. AB - To report the results of an adapted protocol of treatment of Hodgkin disease in Tunisian patients. 70 patients (47 males and 23 females, sex-ratio = 2.04) with a mean age of 38.5 years (15 to 75) are enrolled in a therapeutic protocol to the prognostic factors and based on chemotherapy with MOPP/ABV or hybrid associated to radiotherapy. We perform an evaluation of response to chemotherapy after the 4th cycle, after the 6th cycle and then at the end of the protocol. Our population is characterized by the frequency of young patients(34% between 30 and 40 years), histologic types 2 and 3 (45 and 48%) and advanced disease with 60% of stages III and IV. After the 4th cycle, 32 patients(45%) are in complete response and 31(44%) in partial response, while 6 patients(9%) progress under chemotherapy. After 6 cycles, we observe 44 in complete response(72%) including 46% of the bad responders after 4 cycles. At the end of the protocol and on the 58 evaluable patients, 50 remain in complete response(86%). We observe 5 deaths occurred in 3 progressing patients and in 2 patients by infection after chemotherapy. 5-year actuarial and disease-free survival is 60% and 56% and median survival is 83 months. In the univariate analysis, response to chemotherapy represent the unic significant prognostic factor. PMID- 10730153 TI - [Colon cancer. Study of 153 cases and comparison with a series of 140 historic cases]. AB - The authors study the epidemiological, diagnostic and therapeutic features of 153 consecutive cases of colon cancers collected between 1985 and 1998. They compare the results with those of a previous study performed in the same hospital service (140 cases between 1966 and 1984). It is a series of 79 men and 74 women (sex ratio = 1.07). The mean age was 56.6 +/- 14 years old with 13.7% of the patients who were less than 40 years old. The operability and the resectability were respectively 98.7% and 83.7% with a global operative mortality of 7.9%. According to the Dukes classification, they were 2 A stages, 73 B stages, 32 C stages and 45 D stages. Starting from 1993, 53% of the C stages an 48% of the B stages had an adjuvant chemotherapy. The comparison between our present results and those of our previous series does not show any modification in what regards the mean age, the cancer frequency in young people, the complicated forms proportion and time needed for the diagnosis by comparison to the beginning of symptomatology. The colic cancer prognostic improvement is attained through an earlier diagnosis, making it possible to reduce the developed forms and the complicated forms proportion, the consequence of which is the operative mortality decrease and the log-range survival increase. PMID- 10730154 TI - [Cerebral stereotactic biopsy and surgery: Report of 100 cases]. AB - OBJECTIVE: To report the results of the use of the stereotactic techniques in the management of intra cranial lesions. MATERIALS AND METHODS: Between july 1994 and march 1998, we carried out 117 stereotactic procedures of whom only 100 cases were analyzed. All the procedures were achieved after a CT scan. Patients were separated in two groups: (A) stereotactic biopsy (91 patients), (B) surgery with laser guidance (9 patients). The mean age in group A was 38 years (2-75 years) versus 27 years (11-66 years) in group B. The sex-ratio was 1.3. RESULTS: In the group A, the correct pathological diagnosis was obtained in 91.2% of cases. Glial tumors was the frequent histopathological variety of tumor (67.47%). Only one patient was operated after biopsy for a bilateral meningioma of the anterior 1/3 of the falx. There was only one death (1.09%) and 3.29% of transitory complications. In the group B, all patients were operated with laser guidance. Thirty seven per cent of patients underwent radiotherapy after the stereotactic biopsy or surgery. CONCLUSION: Stereotactic biopsy is a reliable method for the histopathological diagnosis of deep-seated brain lesions. Surgery with laser guidance is a useful alternative for the management of small deep-seated lesions or lesions located in functional areas. PMID- 10730155 TI - [Role of cryptosporidia and microsporidia in diarrhea in immunocompromised patients]. AB - Cryptosporidium and Microsporidian play an important part in the diarrhoeic pathology of the immunocompromised patients. The study of 35 cases of cryptosporidiosis and 4 cases of intestinal microsporidiosis diagnosed in the parasitology laboratory of Rabta hospital of Tunis shows that cryptosporidiosis prevalence is 17.24% for AIDS patients, 3.45% for immunocompromised patients VIH (-), and microsporidiosis prevalence is 5.7% for patients with acquired immunodeficiency syndrome. Common points for these two parasitosis are: Clinical syndromes dominated by an acute diarrhea A diagnosis based on specific techniques showing the significance of the clinical orientation. Lack of an effective specific therapy. PMID- 10730156 TI - Prognostic factors in congenital hemiplegia in full-term and preterm children. AB - OBJECTIVES: To determine whether the cerebral CT scan patterns, the type of E.E.G recording, the gestational age, the etiological factors and the side of hemiplegia would influence the functional prognosis in congenital hemiplegia. MATERIAL AND METHODS: 53 children (35 males, 18 females) suffering from congenital hemiplegia were included in the study. They were divided into prematures (16 cases) and full-term children (37 cases). All of them were evaluated for I.Q level, motor performance and language ability. CT scan was performed in all cases and E.E.G recording in the majority of them. RESULTS: Full term gestational age, cortical and subcortical lesions in term children, irritative E.E.G recording and hemispheric atrophy in both gestational age groups were found to be bad prognostic indicators for functional status in congenital cerebral hemiplegia. PMID- 10730157 TI - [Syndrome of inappropriate macrophage activation associated with infantile visceral leishmaniasis]. AB - Hemophagocytosis has already been in cases of visceral leishmaniasis and thus may complicate search for diagnosis. We report a case of hemophagocytosis in a 20 month-old boy presenting with fever, hepatosplenomegaly, pancytopenia and coagulopathy. An initial diagnosis of kala-azar was refuted because of absence of biological inflammatory syndrome and negativity of bone-marrow aspiration. Specific serology for visceral leishmaniasis become positive. The boy was given stibogluconate for 21 days; he improves gradually with complete remission. PMID- 10730158 TI - [Role of lactate dehydrogenase (LDH) level in the aqueous humor in the diagnosis of retinoblastoma (RB)]. AB - In spite of the progress of the imagery in ophthalmology, the retinoblastoma remains in some particular cases, difficult to make. Moreover, the therapy sanction is often heavy. The lactate dehydrogenase (LDH) dosage in aqueous humor is an invasive technique conceivable when there are diagnosis problems particularly in retino-blastoma of old children and in affections simulating retino-blastoma. The purpose of this work is to lay out three observations where the LDH dosage enabled to keep the retinoblastoma diagnosis in one 8-year old child and eliminate it in two children having pseudoglioma with spontaneous evolution allowing to confirm the non-tumoral nature of the affection. Thus, the technique of LDH dosage has been reliable for the 3 cases which we have introduced. The authors compare the reliability of the different cytochemical techniques during the retinoblastoma diagnosis. PMID- 10730159 TI - [Vermian agenesis: Report of a family and review of the literature]. AB - Vermian agenesis constitute an heterogeneous group of clinical and neuroradiological entities with different prognosis. Authors report a kindred with vermian agenesis associated to characteristic facial dysmorphy and to mental retardation. After review of the literature, we found no previous description of such an association. We believe this is a new entity of familial vermian agenesis with autosomal recessive transmission. PMID- 10730160 TI - When is an examination complete? Lessons to be learned from cost-effectiveness analysis. PMID- 10730161 TI - Optimal management strategy for use of compression US for deep venous thrombosis in symptomatic patients: a cost-effectiveness analysis. AB - RATIONALE AND OBJECTIVES: The authors' purpose was to identify the optimal strategy for using compression ultrasonography (US) in patients suspected of having deep venous thrombosis (DVT). MATERIALS AND METHODS: The authors developed a decision-analytic model representing the natural history of DVT and the benefits and risks of anticoagulation therapy. They evaluated six initial imaging strategies: (a) unilateral examination of the common femoral and popliteal veins; (b) unilateral examination of the common femoral, popliteal, and femoral veins; (c) bilateral examination of the common femoral and popliteal veins; (d) bilateral examination of the common femoral, popliteal, and femoral veins; (e) complete unilateral examination of the symptomatic leg (including calf veins); and (f) complete bilateral examination of both legs. RESULTS: For 65-year-old men with unilateral symptoms of DVT, the most effective strategy was bilateral examination of the common femoral and popliteal veins with anticoagulation therapy in patients with proximal DVT and follow-up bilateral examination of the common femoral and popliteal veins in patients without an initial diagnosis of DVT with an incremental cost-effectiveness ratio of $39,000 per quality-adjusted life year gained. CONCLUSION: These results suggest that bilateral examination limited to the common femoral and popliteal veins, with follow-up bilateral examination limited to the common femoral and popliteal veins, was as cost effective as other well-accepted medical interventions. The results were sensitive to the distribution of clot, diagnostic accuracy of compression US, and probability of bleeding with long-term morbidity. PMID- 10730162 TI - Perceived features reported as nodules: interpretation of spiral chest CT scans. AB - RATIONALE AND OBJECTIVES: The purpose of this study was to evaluate nontarget locations identified in a study of lung nodule detection with spiral computed tomographic (CT) scans that compared cine and film presentations. MATERIALS AND METHODS: In a previous study of lung nodule detection, eight observers were asked to identify 10 nodule locations in each of five CT scans containing eight simulated nodules. In the current study, each nontarget location that was reported more than once in the previous study was inspected with a stack-mode display in both cine and static modes. The nontarget locations were evaluated for probable identity, shape, and distance from the peripheral lung surface. RESULTS: Fifty-two nontarget locations included clinically undetected pulmonary nodules (n = 12), lymph nodes (n = 2), unclassifiable structures (n = 2), pleural scars (n = 8), and vascular structures (n = 28). Five nontarget locations contained vessels with complex courses apparent only with cine mode. As a group, nontarget locations were significantly closer to the periphery than would be expected by chance (for all locations, P < .0001; for locations not touching the pleural surface, P = .013). CONCLUSION: The lower reporting threshold caused by the observer instructions to find 10 targets resulted in increased reporting of structure with a nodular appearance. The locations of these reports in the lung periphery can be attributed to the relationship between frequent disease and a nearly featureless background in the lung periphery. PMID- 10730163 TI - Nontraumatic osteonecrosis: MR perfusion imaging evaluation in an experimental model. AB - RATIONALE AND OBJECTIVES: Because the nature and time course of changes in early, nontraumatic osteonecrosis at perfusion and magnetic resonance (MR) imaging are unknown, the authors evaluated this technique in the assessment of early osteonecrosis with a nontraumatic model. MATERIALS AND METHODS: Five rabbits underwent intravenous injection of lipopolysaccharide endotoxin followed by intramuscular injection of methylprednisolone. MR imaging of the femora was performed before and at weekly intervals after endotoxin injection. Histologic findings from the areas of osteonecrosis were correlated with the findings of MR imaging and MR perfusion studies. RESULTS: Histologic evaluation showed osteonecrosis in six femora of four animals 2-4 weeks after endotoxin injection. Findings on T1-weighted images of the femur were normal in all animals; T2 weighted images of one femur showed equivocal changes. On MR perfusion images, the baseline mean peak percentage of enhancement was 52.7% +/- 12.6. In the six areas without osteonecrosis, the mean percentage of enhancement was similar to the baseline percentage of enhancement at 1 week (62.2% +/- 31.2). In the four areas with diffuse osteonecrosis, there was essentially no contrast enhancement 1 4 weeks after endotoxin injection. CONCLUSION: T1- and T2-weighted MR imaging is insensitive to the presence of early nontraumatic osteonecrosis. MR perfusion imaging might be useful to detect early nontraumatic osteonecrosis. PMID- 10730164 TI - Accuracy and precision of spiral CT in the assessment of neoplastic lesions associated with the mandible. AB - RATIONALE AND OBJECTIVES: The purpose of this study was to determine the accuracy (validity) and precision (reliability) of spiral computed tomographic (CT) images by using film- and computer graphics-based measurements of simulated neoplastic lesions associated with the mandible. MATERIALS AND METHODS: Four cadaver heads, each with two simulated tumors containing contrast medium positioned medial to the mandibles, were examined by using a subsecond spiral CT scanner. Data were transferred to film and to a computer workstation. With computer graphics, data were analyzed by using multiplanar reconstructed images. Linear measurements of the length, width, and depth of simulated tumors were made by two observers, twice each, on the film scans by using manual calipers and on the multiplanar reconstructed images by using computerized measurements. The soft tissues were then removed from the cadavers and the same measurements made by using the same calipers. RESULTS: No statistically significant differences between computer graphics- or film-based measurements and physical measurements (P > .05) or between inter- and intraobserver measurements (P > .05) were found. CONCLUSION: The authors found high reproducibility of measurements for all dimensions. Spiral CT allows accurate computer graphics- and film-based measurements of neoplastic lesions associated with the mandible. PMID- 10730165 TI - Through-transmission US applied to breast imaging. AB - RATIONALE AND OBJECTIVES: The purpose of this study was to evaluate the application of a new method of through-transmission ultrasonography (US) to breast imaging. MATERIALS AND METHODS: Through-transmission US was used to image breast tissue in 18 women; 12 had no known lesions, and six had masses, five of which were palpable. Of those with no known lesions, two had implants, and two had mammary duct ectasia. Lesion sizes and locations on the through-transmission sonograms were correlated with findings from conventional imaging to determine successful imaging. Histopathologic findings and lesion size were recorded from pathology reports. Detailed comparative analyses of through-transmission US, conventional x-ray mammography, and conventional US were performed. The following lesion variables were compared: maximum diameter, shape, margins, and internal architecture. RESULTS: Through-transmission US produced images of all masses, some of which were mammographically occult. Comparisons among through transmission US, mammography, and conventional US showed high correlation in lesion characteristics such as maximum diameter, shape, margins, and internal architecture. For two malignant masses, through-transmission US appeared to be more accurate in assessing tumor extent, compared with mammography and conventional US. CONCLUSION: This new method of through-transmission US produces images of breast tissue in women with a variety of breast types and can be used to detect and characterize a variety of lesions, some of which are mammographically occult. PMID- 10730166 TI - Curriculum in radiology for residents: what, why, how, when, and where. AB - Developing a curriculum in chest radiology should follow the same general principles that are used when developing a curriculum in any subspecialty area of radiology. A curriculum is more than a "list of topics" with which a resident should be familiar after 4 years of training. It includes objectives and goals, content, faculty, methods, and evaluation. Numerous resources are available for those who are charged with developing a curriculum in chest radiology. In addition to faculty members in the department, whose input during development can ensure successful implementation of the curriculum, organizations (i.e., ACR, APDR, STR) already have begun to develop "model" curricula. Attending the annual meeting of the Association of American Medical Colleges is a way to meet and hear from professionals who develop and oversee curriculum development at their medical schools, and another important resource available at some medical schools is the Office of Medical Education. The faculty within such offices are uniquely qualified to assist with curriculum and faculty development, especially for those areas in which radiology faculty traditionally are less experienced, such as development of valid and reliable assessment forms and construction of behaviorally based objectives. PMID- 10730167 TI - Personal diary: on being an interim chairman. PMID- 10730168 TI - Imaging of congenital and acquired gastric abnormalities in children. PMID- 10730169 TI - [Auditing of drug use practices in anesthesia]. AB - OBJECTIVES: Internal pharmaceutical practice guidelines were produced in a department of anaesthesia of a University hospital in 1995, after a preliminary evaluation showing controversial and expensive practices. After approval, these recommendations were circulated to all members of the department. Phase I of this study was started 18 months later, to evaluate the compliance of anaesthetists with these guidelines. STUDY DESIGN: Prospective survey. METHOD: An audit was performed by a research assistant pharmacist, previously trained, who compared anaesthetic agents and fresh gas flows used during anaesthetics with those recommended in the internal guidelines. RESULTS: Implementation of guidelines was observed in more than 90% of cases for all agents studied, except for non depolarizing neuromuscular relaxants. Relaxants of intermediate duration were used in 52% of cases with an expected surgery duration of more than 90 min, in opposition to the guidelines stating that pancuronium should have been administered in such circumstances. These results were presented and discussed. Slight changes in the guidelines (especially concerning monitoring of neuromuscular blockade) were made and evaluated again, using the same method (Phase II). A small but significant improvement was seen with induction agents (use of propofol in non-approved situations: 5 versus 0%, P = 0.03), while a non significant trend toward better implementation of guidelines was seen with non depolarizing agents (use of pancuronium in surgery of expected duration > 90 min: phase I vs phase II = 47 vs 52%, NS; use of pancuronium in surgery of expected duration < 90 min: phase I vs phase II = 3.5 vs 0%, NS). CONCLUSION: This study suggests that implementation of internal guidelines is easy when clinical indications of the agents are not controversial. PMID- 10730170 TI - [Staphylococcus nasal carriage and infection of central venous ports in oncology]. AB - OBJECTIVE: To assess the risk of infection of either subcutaneously implanted central venous access devices or percutaneous central venous catheters inserted via a subcutaneous tunnel in cancer patients with a positive staphylococcal nasal carriage. STUDY DESIGN: Prospective study. PATIENTS: The study included 266 patients undergoing cancer chemotherapy. METHOD: A nasal swab was taken prior to insertion of the venous access device and the patients were followed over 30 days for the occurrence of a staphylococcal infection (hemoculture and device or site of insertion). RESULTS: A nasal staphylococcal nasal carriage was found in 227 patients. Out of the 15 developing a device infection, a staphylococcal nasal colonization was existing in nine patients. Bacteriological screening a sensitivity of 60% and a specificity of 13%. CONCLUSION: Bacteriological screening at the time of device insertion of a central venous access device is of no value for the detection of patients at risk of staphylococcal infection of the device. PMID- 10730171 TI - [Allergic risk of aprotinin]. AB - OBJECTIVE: To analyse the risk of anaphylactic reaction with the administration of aprotinin, either by i.v. route or as a biological sealant application and to propose updated guidelines in accordance with current data of the literature. DATA SOURCES: Search in the Medline data base of articles in French, English and German, published since 1960, using following key words: aprotinin, allergy, anaphylaxis. STUDY SELECTION: All categories of articles on this topic have been selected. DATA EXTRACTION: Articles have been analysed for history, incidence and mechanisms of anaphylactic reactions, symptomatology, factors of risk, diagnosis and precautions of use. DATA SYNTHESIS: Aprotinin is widely used for decreasing preoperative bleeding, especially in cardiac and orthopaedic surgery. This heterologue protein can cause anaphylactic reactions in 0.5 to 5.8% of patients, depending of the inclusion criteria. They are mediated by IgG and IgE antibodies. Aprotinin has also a direct, non specific, histaminoliberation effect. The clinical presentation includes various degrees of severity, up to cardiac arrest. Documented factors of risk are a previous parotinin administration, 15 days to 6 months before, and intolerance to beef meat, white of egg, cheese and milk. The immediate biological diagnosis is obtained on assessing the degranulation of basophiles (histamine) and mastocytes (tryptase), as well as the concentration of anti-aprotinin antibodies (RAST IgE), with a test of inhibition. The secondary assessment, six weeks later, includes prick-tests and intradermoreactions if the former are negative. The mean precaution consists to search factors of risk at preanaesthetic assessment. The predictive value of systematic prick-tests has not yet been validated. Anti H1 and anti H2 premedication is inefficient. A test dose can trigger a severe reaction. CONCLUSION: Considering a significant anaphylactic risk, aprotinin administration becomes only licit after a careful evaluation of the benefit-risk ratio. PMID- 10730172 TI - [Myocardial rhabdomyolysis following paraphenylene diamine poisoning]. AB - Acute intoxication with paraphenylene diamine, a mineral compound used as hair dye, associated asphyxia due to cervical oedema and rhabdomyolysis. We report the case of a patient with lethal cardiogenic shock secondary to myocardial rhabdomyolysis confirmed by a postmortem biopsy. PMID- 10730173 TI - [Iatrogenic gas embolism following surgical lavage of a wound with hydrogen peroxide]. AB - The use of hydrogen peroxide (H2O2) in surgery for its antiseptic properties has been associated with life-threatening complications. We report a case of severe oxygen embolism after wound irrigation with H2O2 in a 17-year-old boy undergoing surgical dressing of a large thigh trauma under general anaesthesia. During muscle lavage with 400 mL of H2O2 3%, severe shock suddenly occurred. On the basis of clinical presentation, the diagnosis of pulmonary gas embolism was strongly suspected. Symptomatic treatment initiated immediately, restaured a normal haemodynamic state within a few minutes and the patient recovered without sequelae. The degradation of H2O2 results in considerable amounts of gaseous oxygen. One mL of H2O2 can produce in the tissues 10 mL of oxygen. This gas can enter the circulation and determine severe embolism. The treatment should be initiated without delay. The administration of H2O2 under pressure is contraindicated during surgery. PMID- 10730174 TI - [Monitoring jugular venous oxygen saturation in severe cerebral vasospasm after subarachnoid hemorrhage]. AB - Cerebral arterial vasospasm is a major complication of aneurysmal subarachnoid haemorrhage. The conventional treatment of this complication includes haemodilution, hypervolaemia, arterial hypertension and nimodipine. Some patients do not respond to this therapy and require an intraarterial infusion of papaverine and/or a cerebral angioplasty. Transcranial Doppler detects cerebral vasospasm. However it does not provide an accurate metabolic information on the ischaemic status of the cerebral tissue. This article describes the monitoring of jugular venous bulb oxygen saturation to obtain a real time information on the metabolic effect of cerebral vasospasm and its variations after intra-arterial infusion of papaverine. PMID- 10730175 TI - [Tracheal gas insufflation associated with mechanical ventilation for CO2 removal]. AB - OBJECTIVE: Tracheal gas insufflation (TGI) either continuously, or at inspiration, or at expiration, is a technique associated with mechanical ventilation aimed to enhance CO2 elimination in favouring washout of anatomical dead space. This article analyses the mechanism of action, the techniques and the effects of TGI in presence of hypercapnia, especially in the fame of ARDS in adults. DATA SOURCES: In addition to some historical or major references, the articles on TGI published over the past five years have been searched in the Medline data base. STUDY SELECTION: Articles with data on TGI associated with mechanical ventilation were selected. DATA EXTRACTION: Data on mechanisms of action, technical and practical aspects of TGI were extracted. DATA SYNTHESIS: CO2 elimination is increased when the TGI catheter tip is close to the carina, when the gas jet is directed towards the latter, by a continuous gas jet, by a high washing gas volume. The effect on oxygenation is minor. The work of breathing is decreased. An increased intracranial pressure is decreased. Circulatory effects are minor. The major risk is dynamic pulmonary over distension. Local complications include dessiccation and lesion of bronchial mucosa by the gas jet. CONCLUSION: In mechanically ventilated patients, additional TGI is a valuable technique for decreasing anatomical dead space. TGI decreases hypercapnia during mechanical ventilation with limited tidal volumes in permissive hypercapnia. Further clinical studies with large series of patients are required to assess the benefits and the effect of TGI on outcome. PMID- 10730176 TI - [Comparison of two measurement methods: the Bland and Altman assessment]. AB - Bland-Altman analysis for comparison of two methods of clinical measurement is frequently used in scientific publications. This article is more appropriate than the conventional linear regression analysis. This paper gives an overview of the principles for the use of Bland-Altman analysis as well as the specific terminology attached to it. The Bland-Altman comparison analysis is mainly a tool for clinical interpretation. The bias and the agreement limits provide the variation of the values of the technique compared to the other. The difference between the two methods of measurement is plotted against the average obtained with each of the two techniques. Bland-Altman analysis can also be used to check the repeatability of a measurement technique within the same subject and to determine a repeatability coefficient. With an adaptation of the calculation of the agreement limits, the average of multiple measurements for each subject with two measurement techniques can be used for the Bland-Altman analysis. PMID- 10730177 TI - [Intravenous locoregional anesthesia and severe arterial hypertension]. PMID- 10730178 TI - [Hypopharyngeal injuries secondary to nasograstric tube]. PMID- 10730179 TI - [Iconography of a bent endotracheal tube]. PMID- 10730180 TI - [Anesthesia and sickle cell disease. Analysis of 54 cases]. PMID- 10730181 TI - [Three potentially serious problems, occurred during the use of the anesthesia device Kion (Siemens)]. PMID- 10730182 TI - [Blood transfusions against the will of the patients...]. PMID- 10730183 TI - [Treatment refusal and life emergencies: a conflict between the aim of medical practice and the duty to respect the will of the person]. PMID- 10730184 TI - [Letter DGS/DH dated November 18, 1999 on the treatment of paranesthetic malignant hyperthermia (MH)]. PMID- 10730185 TI - The role of efflux in bacterial resistance to soft metals and metalloids. AB - Bacteria have evolved various types of resistance mechanism to toxic soft metals and metalloids, including cadmium/zinc, copper/silver and arsenic/antimony. Active efflux of the metal is a frequently utilized stratagem to produce resistance by lowering the intracellular concentration to subtoxic levels. Reduction to a less-toxic form or to a form recognized by an efflux system also occurs. Pumps utilized for resistance may have evolved from normal cellular systems. For example, plasmid-mediated cadmium resistances may have evolved from a common ancestor of the pump involved in zinc homoeostasis. Pumps are more efficient than carriers and may have evolved by developing carriers that associate with ATPase subunits. PMID- 10730186 TI - Bacterial detoxification of Hg(II) and organomercurials. AB - The most common bacterial mechanism for resistance to mercuric-ion species involves intracellular reduction of Hg(II) to Hg(0). Key proteins of the pathway typically include: MerR, which regulates pathway expression; MerP, which protects the external environment; MerT or MerC, which transport Hg(II) species across the inner membrane; MerA, which catalyses reduction of Hg(II); and sometimes MerB, which catalyses cleavage of C-Hg bonds in organomercurials. Cysteine residues of varying number are arranged in each of the key proteins to optimize their unique roles in sensing (high affinity), transporting (exchangeability), and reducing (redox accessibility) Hg(II). Nature's regulator of this pathway, MerR, is an exquisitely sensitive, Hg(II)-binding, DNA-binding protein that holds the system primed for immediate transcription at the slightest influx of Hg(II). PMID- 10730187 TI - Non-haem iron-containing oxygenases involved in the microbial biodegradation of aromatic hydrocarbons. AB - A wide variety of aromatic hydrocarbons can be degraded aerobically by micro organisms. A large fraction of the metabolic pathways are initiated by oxygenases containing Fe(II) at the active sites, which participates in the oxygenation and activation of the hydrocarbons. Mono-oxygenations and dioxygenations are found in these pathways. Some of these enzymes can catalyse either or both reactions, depending on the nature of the substrate. Two general themes are found: mononuclear Fe(II) centres that must be reduced by one electron at a time, or di iron centres that can be reduced by two electrons. The electrons from NAD(P)H can be delivered by either an electron-transfer chain consisting of a flavin and one or more [2Fe-2S] centres, or a pterin. Proposed mechanisms generally involve higher oxidation states of the iron (Fe = O), analogous to those for P450, and peroxidase systems. These strong oxidants are necessary to oxidize aromatic and aliphatic compounds. Mechanisms currently considered viable for these reactions require significant changes in ligation during catalysis. The structures of the non-haem iron centres may be particularly well-suited for such transformations. PMID- 10730188 TI - Haem iron-containing peroxidases. AB - Peroxidases are enzymes that utilize hydrogen peroxide to oxidize substrates. A histidine residue on the proximal side of the haem iron ligates most peroxidases. The various oxidation states and ligand complexes have been spectroscopically characterized. HRP-I is two oxidation states above ferric HRP. It contains an oxoferryl (= oxyferryl) iron with a pi-radical cation that resides on the haem. HRP-II is one oxidation state above ferric HRP and contains an oxoferryl iron. HRP-III is equivalent to the oxyferrous state. Only compounds I and II are part of the peroxidase reaction cycle. CCP-ES contains an oxoferryl iron but the radical cation resides on the Trp-191 residue and not on the haem. CPO is the only known peroxidase that is ligated by a cysteine residue rather than a histidine residue, on the proximal side of the haem iron. CPO is a more versatile enzyme, catalysing numerous types of reaction: peroxidase, catalase and halogenation reactions. The various CPO species are less stable than other peroxidase species and more elusive, thus needing further characterization. The roles of the amino acid residues on the proximal and distal sides of the haem need more investigation to further decipher their specific roles. Haem proteins, especially peroxidases, are structure-function-specific. PMID- 10730189 TI - Nature's universal oxygenases: the cytochromes P450. AB - Cytochromes P450 are utilized in an enormous diversity of biological reactions, including degradation of xenobiotics, generation of hormones and biosynthesis of a variety of important biological compounds. The cytochrome P450 family is a major participant in nearly all metabolism of pharmaceutical reagents. The presence of different P450 enzymes in various quantities in individuals makes the prediction of drug responses in patients highly complex. A large literature describing mechanistic studies has characterized several intermediates in the oxygenation pathway. It has recently been shown that two or more possible oxygenated forms of the P450 haem can participate in various oxygenations, with some intermediates being highly electrophilic and others being nucleophilic. PMID- 10730190 TI - Oxygen-carrying proteins: three solutions to a common problem. AB - Nature has used transition-metal ions with unpaired d-electrons to overcome the kinetic inertness of O2 and to control its thermodynamic tendency towards reduction. High-resolution X-ray crystal structures of O2-carrying proteins show that Nature has devised three distinct solutions to the problem of reversible O2 binding. The three types can be classified according to their active sites: Hb (haem iron); Hr (non-haem di-iron); and Hcy (dicopper). The reversible O2 binding to the three types of active site are formally oxidative additions: Fe(II) to Fe(III)-O2- for Hb; [Fe(II),Fe(II)] to [Fe(III),Fe(III)O(2)2-] for Hr; and [Cu(I),Cu(I)] to [Cu(II)(mu-O(2)2-) Cu(II)] for Hcy. In all cases the O-O bond is weakened, but not cleaved, upon binding. The 'textbook' explanation for discrimination against CO and O2 binding to Hb has been revised: steric constraints to the preferred linear Fe-C-O geometry imposed by the 'distal' histidine are no longer thought to play a major role. Instead, recent experimental evidence indicates that the polarity of the binding pocket favours the polar Fe-O-O unit over the relatively non-polar Fe-C-O unit, and that a C-O binding pocket near the haem also inhibits the preferred linear Fe-C-O geometry. Reversible O2 binding to the di-iron site of Hr involves an internal proton transfer as well as electron transfer to O2, but the elementary steps governing the rates of O2 binding and release, especially the effects of the surrounding protein, remain to be delineated. An unusual side-on-bonded O2 that bridges the two copper ions explains both the unusually low O-O stretching frequency and the diamagnetism of oxyHcy. O2-activating-enzyme counterparts exist for each of the three known types of O2-carrying protein. Detailed comparisons of these protein/enzyme pairs are likely to clarify the factors that tune the delicate balance between reversible O2 binding and controlled O-O bond cleavage. PMID- 10730191 TI - Biological electron-transfer reactions. AB - A wide range of biological processes makes extensive use of electron-transfer reactions. Rigorous characterization of a biological electron-transfer reaction requires a combination of kinetic, thermodynamic, structural and theoretical methods. The rate of electron transfer from an electron donor to an electron acceptor through a protein is dependent on the difference in reduction potential of the electron acceptor and electron donor and the distance over which electron transfer occurs. The manner in which the rate of electron transfer also depends on the structure of the protein located between the electron donor and acceptor sites remains an active topic of investigation. Diverse protein-engineering strategies are providing new insights into fundamental mechanistic considerations regarding electron-transfer properties of biological molecules, and they can provide novel means by which insights concerning biological electron-transfer reactions can be employed to develop new and useful types of chemistry. PMID- 10730192 TI - Molybdenum enzymes. AB - There are many molybdenum-containing enzymes distributed throughout the biosphere. The availability of molybdenum to biological systems is due to the high water solubility of oxidized forms of the metal. Molybdenum enzymes can be grouped on the basis of the structure of the metal centre. Three principal families of enzyme exist, with active sites consisting of (ppt)MoOS(OH) (the molybdenum hydroxylases), (ppt)MoO2(S-Cys) (the eukaryotic oxotransferases) and (ppt)2MoOX (the bacterial oxotransferases). Here, ppt represents a unique ppt cofactor (pyranopterin) that co-ordinates to the metal, and X is a metalliganded serine, cysteine or selenocysteine. The molybdenum hydroxylases catalyse their reactions differently to other hydroxylase enzymes, with water rather than molecular oxygen as the ultimate source of the oxygen atom incorporated into product, and with the generation rather than consumption of reducing equivalents. The active sites possess a catalytically labile Mo-OH (or possibly Mo-OH2) group that is transferred to substrate in the course of the hydroxylation reaction. These enzymes invariably have other redoxactive centres. The eukaryotic oxotransferases consist of the sulphite oxidases and plant nitrate reductases. They catalyse the transfer of an oxygen atom to or from their substrate (to and from nitrate) in a manner that involves formal oxidation-state changes of the molybdenum. As with the molybdenum hydroxylases, the ultimate source of oxygen is water rather than molecular oxygen. The bacterial oxotransferases and related enzymes differ from the other two groups of molybdenum enzymes in having two equivalents of the ppt cofactor co-ordinated to the metal. This family is quite diverse, as reflected in the fact that serine, cysteine or selenocysteine may be found co-ordinated to the molybdenum, depending on the enzyme. As in the case of the molybdenum hydroxylases, both eukaryotic and bacterial oxotransferases utilize water (rather than molecular oxygen) as the source of the oxygen atom incorporated into product, although for these enzymes, the catalytically labile oxygen in the active site is an Mo = O group rather than an Mo-OH. PMID- 10730193 TI - Coenzyme B12 (cobalamin)-dependent enzymes. AB - The B12 or cobalamin coenzymes are complex macrocycles whose reactivity is associated with a unique cobalt-carbon bond. The two biologically active forms are MeCbl and AdoCbl and their closely related cobamide forms. MeCbl participates as the intermediate carrier of activated methyl groups. During the catalytic cycle the coenzyme shuttles between MeCbl and the highly nucleophilic cob(I)alamin form. Examples of MeCbl-dependent enzymes include methionine synthase and Me-H4-MPT: coenzyme M methyl transferase. AdoCbl functions as a source of carbon-based free radicals that are unmasked by homolysis of the coenzyme's cobalt-carbon bond. The free radicals are subsequently used to remove non-acid hydrogen atoms from substrates to facilitate a variety of reactions involving cleavage of carbon-carbon, carbon-oxygen and carbon-nitrogen bonds. Most reactions involve 1,2 migrations of hydroxy-, amino- and carbon-containing groups, but there is also one class of ribonucleotide reductases that uses AdoCbl. The structures of two cobalamin-dependent enzymes, methionine synthase and methylmalonyl-CoA mutase, have been solved. In both cases the cobalt is co ordinated by a histidine ligand from the protein. The significance of this binding motif is presently unclear since in other cobalamin-dependent enzymes spectroscopic evidence suggests that the coenzyme's nucleotide 'tail' remains co ordinated to cobalt when bound to the protein. PMID- 10730194 TI - Oxygen reactions of the copper oxidases. AB - The copper oxidases are a remarkable family of metalloenzymes that have evolved specialized mechanisms to accomplish the controlled reduction of dioxygen, delivering the equivalent of H2 from organic substrates to O2 to form hydrogen peroxide, a ubiquitous oxygen metabolite that is involved in a wide range of biological interactions. These enzymes display their virtuosity in dioxygen chemistry by harnessing the oxidizing power of that molecule not only during catalytic turnover, but also in transforming themselves in the biogenesis of their catalytic redox cofactor. PMID- 10730195 TI - Catechol dioxygenases. AB - Catechol dioxygenases are key enzymes in the metabolism of aromatic rings by soil bacteria. Catechol dioxygenases have been found that participate in the metabolism of halogenated aromatic compounds and, in doing so, play a key role in bioremediation of halogenated pollutants. The catechol dioxygenases can be divided into two major groups: those that cleave the aromatic ring between the vicinal diols (the intradiol enzymes) and those that cleave the ring to one side of the vicinal diols (the extradiol enzymes). Whereas both types of catechol dioxygenase contain an active-site iron that is required absolutely for enzymic activity, the intradiol enzymes contain Fe(III), while the extradiol enzymes contain Fe(II). The nature of the protein ligands determines this specificity. The differences in oxidation state of the active-site iron appear to result in mechanistic differences that lead to the differing regioselectivity of the two groups of catechol dioxygenase. Mechanistic proposals based on available evidence suggest a substrate-activation mechanism for the intradiol enzymes and an oxygen activation mechanism for the extradiol enzymes. PMID- 10730196 TI - Cisplatin. AB - Cisplatin is a widely used anti-cancer drug that is exceptionally effective against testicular cancer. trans-DDP, the geometric isomer of cisplatin, is ineffective as a chemotherapeutic agent. The anti-tumour activity of cisplatin is generally attributed to its formation of DNA adducts, both intrastrand and interstrand crosslinks, which induce structural distortions in DNA. The DNA adducts of cisplatin are thought to mediate its cytotoxic effects by inhibiting DNA replication and transcription and, ultimately, by inducing programmed cell death, or apoptosis. The adducts of both cis- and trans-DDP are removed from DNA by the nucleotide-excision-repair pathway. Cellular proteins possessing certain DNA-binding motifs, including the HMG domain, bind selectively to DNA modified by cisplatin, but not to DNA adducts of trans-DDP; evidence suggests a possible role for these proteins in modulating cisplatin cytotoxicity. Both intrinsic and drug induced resistance often limit the success of cisplatin; several specific mechanisms of cisplatin resistance have been identified. PMID- 10730197 TI - Comparative studies on carbohydrates of several myxosporean parasites of fish using lectin histochemical methods. AB - A histochemical study using lectin methods was performed on myxosporean parasites from vastly different fish hosts from marine and fresh waters. Six biotinylated lectins were used (WGA, SBA, BS-I, Con-A, UEA-I and SNA). The binding pattern of Con-A and WGA revealed the presence of mannose and/or glucose, and N-acetyl-D glucosamine respectively, in polar capsules and valves of most of the myxosporea assayed. Thus, chitin may be present in polar capsules and/or valves of myxosporean spores. The BS-I binding pattern showed the presence of alpha-D galactose and/or N-acetyl-D-galactosamine residues in polar capsules of Kudoa sp., Zschokkella mugilis Sitja-Bobadilla et Alvarez-Pellitero, 1993 and Leptotheca sp., and in the valves of the latter. Scarce amounts of N-acetyl-D galactosamine and/or alpha-D-galactose were demonstrated by SBA binding in Sphaerospora dicentrarchi Sitja-Bobadilla et Alvarez-Pellitero 1992, Leptotheca sp. and Kudoa sp. valves, and in Leptotheca sp. polar capsules. The UEA-I staining indicated the absence of alpha-L-fucose in all the myxosporea assayed except in Leptotheca sp. N-acetylneuraminic acid was detected with SNA in the polar capsules and sporoplasms of Polysporoplasma sparis Sitja-Bobadilla et Alvarez-Pellitero, 1995 and in the polar capsules and valves of Kudoa sp. These results indicate that, although Myxosporea may have conserved carbohydrate structures, some of them can show significantly different binding patterns, which may be useful in diagnostic and functional studies. PMID- 10730198 TI - Hemibranch preference by freshwater monogeneans a function of gill area, water current, or both? AB - Differences in the occurrence of monogeneans on lamellae of fish gill arches were observed in this study. These differences were attributed to variations in water current on the gill surfaces or to greater area of certain arches. Two computer simulation programs based on gill area and water current were written to generate parasite metapopulations with clumped patterns. The results obtained were compared with true distributions of selected freshwater monogenean taxa. The combination of both theoretical models (gill area and water current) had greater explanatory power than either of the models alone. PMID- 10730199 TI - Monogeneans of freshwater fishes from cenotes (sinkholes) of the Yucatan Peninsula, Mexico. AB - During a survey of the parasites of freshwater fishes from cenotes (sinkholes) of the Yucatan Peninsula the following species of monogeneans were found on cichlid, pimelodid, characid and poeciliid fishes: Sciadicleithrum mexicanum Kritsky, Vidal-Martinez et Rodriguez-Canul, 1994 from Cichlasoma urophthalmus (Gunther) (type host), Cichlasoma friedrichsthali (Heckel), Cichlasoma octofasciatum (Regan), and Cichlasoma synspilum Hubbs, all new host records; Sciadicleithrum meekii Mendoza-Franco, Scholz et Vidal-Martinez, 1997 from Cichlasoma meeki (Brind); Urocleidoides chavarriai (Price, 1938) and Urocleidoides travassosi (Price, 1938) from Rhamdia guatemalensis (Gunther); Urocleidoides costaricensis (Price et Bussing, 1967), Urocleidoides heteroancistrium (Price et Bussing, 1968), Urocleidoides anops Kritsky et Thatcher, 1974, Anacanthocotyle anacanthocotyle Kritsky et Fritts, 1970, and Gyrodactylus neotropicalis Kritsky et Fritts, 1970 from Astyanax fasciatus; and Gyrodactylus sp. from Gambusia yucatana Regan. Urocleidoides chavarriai, U. travassosi, U. costaricensis, U. heteroancistrium, U. anops, Anacanthocotyle anacanthocotyle and Gyrodactylus neotropicalis are reported from North America (Mexico) for the first time. These findings support the idea about the dispersion of freshwater fishes and their monogenean parasites from South America through Central America to southeastern Mexico, following the emergence of the Panamanian isthmus between 2 and 5 million years ago. PMID- 10730200 TI - Redescription of Eucoleus schvalovoj (Nematoda: Capillariidae), an oesophageal parasite of the Eurasian otter, Lutra lutra, in Spain. AB - Eucoleus schvalovoj Kontrimavichus, 1963 (Nematoda: Capillariidae) is redescribed. The original description of this species was brief and inadequate in that it was based on just a few specimens removed from the Eurasian otter, Lutra lutra (Linnaeus, 1758) in the Khabarovsk region, USSR. Detailed morphological study of several specimens of E. schvalovoj from the oesophagus of L. lutra from Spain revealed new characters, above all in males, and allows for a better characterisation of this species. Since its original description E. schvalovoj has only been recorded in Spain. PMID- 10730201 TI - Observations on cucullanid nematodes from freshwater fishes in Mexico, including Dichelyne mexicanus sp. n. AB - A new cucullanid nematode, Dichelyne mexicanus sp. n., is described from the intestine of three species of fishes, Agonostomus monticola (Bancroft) (Mugilidae, Perciformes) (type host), Ictalurus balsanus (Jordan et Snyder) (Ictaluridae, Siluriformes) and Cichlasoma beani (Jordan) (Cichlidae, Perciformes), from three rivers (La Maquina River, Veracruz; Chontalcoatlan River, Guerrero and Santiago River, Nayarit) in central Mexico. This species is characterised by the absence of a ventral sucker in the male (subgenus Dichelyne) and it differs from its congeners mainly in possessing very unequal and dissimilar spicules (left 0.465-0.768 mm and right 293-548 mm long), an asymmetrical gubernaculum, and two intestinal caeca. Another cucullanid nematode, Cucullanus caballeroi Petter, 1977, is reported from Dormitator maculatus (Bloch) (Eleotridae, Perciformes) from the La Palma and La Maquina Rivers and Balzapote stream, Veracruz, being briefly described and illustrated; this represents a new host record. Findings of D. mexicanus and C. caballeroi represent a new record of cucullanid nematodes from fishes in Mexican fresh waters. PMID- 10730203 TI - Hypoechinorhynchus robustus sp. n. from Notolabrus parilus (Labridae) from Western Australia with a discussion on the validity of the Hypoechinorhynchidae (Acanthocephala: Palaeacanthocephala). AB - Hypoechinorhynchus robustus sp. n. is described from Notolabrus parilus (Richardson) (Labridae) from Pt Peron, Western Australia. It has a proboscis with 30 hooks arranged in ten longitudinal rows: 5 rows of a small apical spine, a large anterior hook and a small posterior spine, 5 rows of a large anterior hook, a middle spine and a posterior spine. The new species is distinguished from other species of the genus by having a set of 5 small apical spines anterior to the large hooks on the proboscis, by having lemnisci that barely extend beyond the proboscis receptacle and testes which are more adjacent than tandem. H. robustus also has robust trunk spines anteriorly. Re-examination of Hypoechinorhynchus alaeopis Yamaguti, 1939 (type species) revealed trunk spines that had been overlooked previously. The Hypoechinorhynchidae is made a junior synonym of Arhythmacanthidae because there is considerable overlap between the two family diagnoses, particularly in that both families have a proboscis armature that changes abruptly from small basal spines to large apical (or subapical if present) hooks. The genus Hypoechinorhynchus is placed in the subfamily Arhythmacanthinae because it has trunk spines and a spherical proboscis with few hooks (relative to other arhythmacanthid genera). It is also proposed that Heterosentis magellanicus (Szidat, 1950) be returned to the genus Hypoechinorhynchus since it was transferred to Heterosentis primarily because it had trunk spines. The other hypoechinorhynchid genus contained only Bolborhynchoides exiguus (Achmerov et Dombrowskaja-Achmerova, 1941) Achmerov, 1959 and is relegated to incertae sedis. PMID- 10730202 TI - Some nematodes and acanthocephalans from exotic ornamental freshwater fishes imported into Germany. AB - Five species of adult nematodes, unidentifiable nematode larvae, and three species of acanthocephalans, were found in freshwater ornamental fishes newly imported into Germany from Brazil, Colombia, Indonesia, Malaysia, Nigeria, Peru, Sri Lanka and Thailand. The following species were identified: Adult Nematoda: Pseudocapillaria tomentosa, Capillariidae gen. sp., Dichelyne hartwichi sp. n., Procamallanus (Spirocamallanus) pintoi and Spinitectus allaeri; Acanthocephala: Pseudogorgorhynchus arii gen. et sp. n., Neoechinorhynchus sp. and Pallisentis sp. The nematode Dichelyne hartwichi sp. n. (male only) from the intestine of Chelonodon fluviatilis (Hamilton) from Thailand is characterised mainly by the presence of minute cuticular spines on the tail tip, length of spicules (510 microns) and arrangement of caudal papillae. The acanthocephalan Pseudogorgorhynchus arii sp. n. from the intestine of Ariopsis seemanni (Gunther) from Colombia represents a new genus Pseudogorgorhynchus gen. n., differing from other genera of the Rhadinorhynchidae mainly in possessing a small proboscis armed with markedly few (18) hooks arranged in six spiral rows. Spinitectus macheirus Boomker et Puylaert, 1994 and Spinitectus moraveci Boomker et Puylaert, 1994 are considered junior synonyms of Spinitectus allaeri Campana-Rouget, 1961. PMID- 10730204 TI - Trichoecius calomysci sp. n. (Acari: Myocoptidae), a new mite species from Iran. AB - A new species of myocoptid mite, Trichoecius calomysci sp. n. (Acari: Myocoptidae), from Calomyscus sp. (Rodentia: Cricetidae) from Iran is described. PMID- 10730205 TI - Structures of confinement in 19th-century asylums. A comparative study using England and Ontario. PMID- 10730206 TI - Rethinking risk assessment for incest offenders. PMID- 10730207 TI - The death of the subject. Human rights, due process, and psychiatry. PMID- 10730208 TI - The "pass-through" model of psychiatric emergency room assessment. PMID- 10730209 TI - The nature and diurnal variation of criminal acts committed by patients with mood disorders. PMID- 10730210 TI - The emergence of assisted (supported) decision-making in the Canadian law of adult guardianship and substitute decision-making. PMID- 10730211 TI - Impact of first contacts with the criminal justice or mental health systems on the subsequent orientation of mentally disordered persons toward either system. PMID- 10730212 TI - Involvement of gap junctional communication in myogenesis. AB - Cell-to-cell communication plays important roles in development and in tissue morphogenesis. Gap junctional intercellular communication (GJIC) has been implicated in embryonic development of various tissues and provides a pathway to exchange ions, secondary messengers, and metabolites through the intercellular gap junction channels. Although GJIC is absent in adult skeletal muscles, the formation of skeletal muscles involves a sequence of complex events including cell-cell interaction processes where myogenic cells closely adhere to each other. Much experimental evidence has shown that myogenic precursors and developing muscle fibers can directly communicate through junctional channels. This review summarizes current knowledge on the GJIC and developmental events involved in the formation of skeletal muscle fibers and describes recent progress in the investigation of the role of GJIC in myogenesis: evidence of gap junctions in somitic and myotomal tissue as well as in developing muscle fibers in situ, GJIC between perfusion myoblasts in culture, and involvement of GJIC in cytodifferentiation of skeletal muscle cells and in myoblast fusion. A model of intercellular signaling is proposed where GJIC participates to coordinate a multicellular population of interacting myogenic precursors to allow commitment to the skeletal muscle fate. PMID- 10730213 TI - Genetic analysis of plant morphogenesis in vitro. AB - Plant morphogenesis in vitro such as somatic embryogenesis and adventitious organogenesis has provided useful systems for physiological, biochemical, and molecular biological studies on plant development. Advantages of the in vitro systems are currently being combined with genetic techniques to generate new insights into fundamental mechanisms of plant development. This article presents an overview of genetic analysis of plant morphogenesis in vitro including genetic variation of tissue culture responses, mutational analysis of somatic embryogenesis, and mutational analysis of adventitious organogenesis. PMID- 10730214 TI - The cell and developmental biology of tendons and ligaments. AB - We have sought to create, for the first time in a single comprehensive review, a modern synthesis of opinion on the cell, developmental, and molecular biology of tendons, ligaments, and their associated structures (tendon sheaths, vinculi, and retinacula). Particular attention has been paid to highlighting new data on the early development of tendons, the signaling molecules involved in their patterning, and the diversity of specialized regions (entheses, wrap-around regions, and myotendinous junctions) that characterize fully formed tendons and ligaments. We have emphasized the complexities of adult tendon and ligament cell shape and related these to their early development. The importance of gap junctions in allowing cell communication throughout an extensive extracellular matrix (ECM) has also been highlighted, particularly in relation to understanding how tendon and ligament cells respond to changes in mechanical load. Finally, we have considered the influence of growth factors and related molecules on cell proliferation and ECM synthesis. PMID- 10730215 TI - Some aspects of plant karyology and karyosystematics. AB - The significance of the 4C value (where C is the amount of DNA in the unreplicated haploid genome) in angiosperm plants is discussed. The DNA amount is a stable feature used in biosystematics. Although this parameter varies even in closely related taxa, there is no correlation between the DNA amount and the structural and functional organization of plants. The role of DNA amount, including "excess" DNA, in plant evolution is considered. Some rules governing the distribution of DNA amount among different plant taxa are postulated, together with the possibility of using the data in systematics, phylogeny, and solutions of problems of genetic apparatus organization and evolution. The decrease in DNA value per genome during plant evolution and the high level of species formation in taxa with large DNA values have been shown. Plant taxa with a small DNA value per genome have a high percentage and higher degree of polyploidy. The nature of the differential staining of euchromatin and heterochromatin bands of prophase and metaphase chromosomes is also discussed. Data that could explain the mechanism of heterochromatin visualization under cold pretreatment of cells are reviewed. Phenomena involved in the arrangement of chromocenters in interphase nuclei and chromosomes in metaphase during consecutive cell generations are discussed. PMID- 10730216 TI - Eph receptors and ephrins: regulators of guidance and assembly. AB - Recent advances have started to elucidate the developmental functions and biochemistry of Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins. Interactions between these molecules are promiscuous, but they largely fall into two groups: EphA receptors bind to GPI-anchored ephrin-A ligands, while EphB receptors bind to ephrin-B proteins that have a transmembrane and cytoplasmic domain. Remarkably, ephrin-B proteins transduce signals, such that bidirectional signaling can occur upon interaction with Eph receptor. In many tissues, specific Eph receptors and ephrins have complementary domains, whereas other family members may overlap in their expression. An important role of Eph receptors and ephrins is to mediate cell-contact-dependent repulsion. Complementary and overlapping gradients of expression underlie establishment of a topographic map of neuronal projections in the retinotectal system. Eph receptors and ephrins also act at boundaries to channel neuronal growth cones along specific pathways, restrict the migration of neural crest cells, and via bidirectional signaling prevent intermingling between hindbrain segments. Intriguingly, Eph receptors and ephrins can also trigger an adhesive response of endothelial cells and are required for the remodeling of blood vessels. Biochemical studies suggest that the extent of multimerization of Eph receptors modulates the cellular response and that the actin cytoskeleton is one major target of the intracellular pathways activated by Eph receptors. Eph receptors and ephrins have thus emerged as key regulators of the repulsion and adhesion of cells that underlie the establishment, maintenance, and remodeling of patterns of cellular organization. PMID- 10730218 TI - [Announcement--a stressful winter for ophthalmologists]. PMID- 10730217 TI - Renewal of photoreceptor outer segments and their phagocytosis by the retinal pigment epithelium. AB - The discovery of disc protein renewal in rod outer segments, in 1960s, was followed by the observation that old discs were ingested by the retinal pigment epithelium. This process occurs in both rods and cones and is crucial for their survival. Photoreceptors completely degenerate in the Royal College of Surgeons mutant rat, whose pigment epithelium cannot ingest old discs. The complete renewal process includes the following sequential steps involving both photoreceptor and pigment epithelium activity: new disc assembly and old disc shedding by photoreceptor cells; recognition and binding to pigment epithelium membranes; then ingestion, digestion, and segregation of residual bodies in pigment epithelium cytoplasm. Regulating factors are involved at each step. While disc assembly is mostly genetically controlled, disc shedding and the subsequent pigment epithelium phagocytosis appear regulated by environmental factors (light and temperature). Disc shedding is rhythmically controlled by an eye intrinsic circadian oscillator using endogenous dopamine and melatonin as light and dark signal, respectively. Of special interest is the regulation of phagocytosis by multiple receptors, including specific phagocytosis receptors and receptors for neuroactive substances released from the neuroretina. The candidates for phagocytosis receptors are presented, but it is acknowledged that they are not completely known. The main neuromodulators are adenosine, dopamine, glutamate, serotonin, and melatonin. Although the transduction mechanisms are not fully understood, attention was brought to cyclic AMP, phosphoinositides, and calcium. The chapter points to the multiplicity of regulating factors and the complexity of their intermingling modes of action. Promising areas for future research still exist in this field. PMID- 10730219 TI - [Anterior segment involvement in HIV-related eye disease after the commencement of highly active antiretroviral therapy (HAART)]. AB - The clinical picture of HIV-associated eye disease has changed dramatically since the introduction of highly active antiretroviral chemotherapy (HAART). As a consequence of the marked reconstitution of immune function and the control of retroviral replication, thereby effected, the clinical manifestations of infectious eye disease are not so patent. Although direct infectious destruction of tissue is less severe, inflammatory infiltration is augmented, and this gives rise to a situation that is open to misinterpretation. Furthermore, several completely new disease entities have been described. One of these is the so called immune-recovery uveitis, which involves mainly the anterior uvea and vitreous, and is not uncommonly associated with a marked disturbance of visual function. Another group of new diseases has been attributed to the toxic effects of drugs, i.e. of Cidofovir and Rifabutin. In both instances, a principally anterior form of uveitis develops, which is characterized by a discrepancy between clinical symptoms and morphological changes; the former condition is distinguished by severe pain, and the latter by marked inflammation. This article describes the clinical pictures characterizing these new affections of the anterior segment, postulates on the possible causes of the seemingly paradoxical clinical, morphological and immunological situations sometimes presented by them, and comments on recommended treatment strategies. The information furnished is designed to help the clinical practitioner in making his/her diagnostic and therapeutic decisions. PMID- 10730220 TI - [Posterior segment involvement in HIV-related eye disease after commencement of highly-active antiretroviral therapy (HAART)]. AB - The panel of therapeutic options available for the treatment of retroviral disorders has expanded explosively in recent years with the result that it has now become possible to reconstitute the immune function of individuals with advanced HIV-disease to a remarkable degree. The ophthalmologist is thus confronted with completely new clinical situations, courses of treatment and decisions. For example, a CMV-retinitis developing within six weeks of the onset of HAART does not necessarily require specific treatment, and reactivation of a retinitis within the same time span is often barely distinguishable from an inflammatory reaction induced by the reconstituted lymphocytes. Marked cellular infiltration of the vitreous--in the absence of an active retinal or chorioretinal lesion--is the hallmark of this immune-recovery vitreitis and responds nicely to systemic steroid treatment. In the course of retinitis, macular edema and epiretinal membrane formation are not uncommonly responsible for visual impairment. If, on the other hand, immunological parameters remain stable during the course of three months, i.e., if the CD4 cell count is more than 150-250 microliters and the plasma load of HIV-1 is less than 5-10,000 copies/microliter, then what has until now been deemed to be a lifelong therapy for CMV-retinitis may be interrupted, provided the clinical (i.e., retinal) situation can be controlled on a regular basis. In this article, an attempt is made to discuss and interpret clinical findings pertaining to posterior segment involvement in ocular HIV disease, with a view to guiding the clinical practitioner in his/her course of action. PMID- 10730221 TI - [Protein-condensation diseases--molecular basis of cataract formation]. AB - There are reasons to classify a number of apparently disparate diseases as "condensation" (or molecular aggregation) diseases. Examples of such condensation diseases include the late phase of diabetes mellitus, Alzheimer's disease and others. With an expanding knowledge, the list of these diseases is likely to increase. We shall describe the underlying common mechanisms, the aim being to find anticataractogenic drugs based on this insight. The common, most important denominator of various clinically differing condensation diseases derives from the interaction of the macromolecules which is in part attractive and in part repulsive. Aggregation resp. clumping of the macromolecules of the crystalline lens, the reasons for light scattering, may be prevented by introducing a number of molecules of various designs into the original macromolecular complex which reduce the tendency of aggregation. Cataract inhibitors of this category may be regarded as effective if they are able to increase the time constant of the normal aging process (i.e. the increment of scatter) by about 20%. PMID- 10730223 TI - [Therapeutic strategies in RP (retinitis pigmentosa): light at the end of the tunnel?]. AB - Retinitis pigmentosa (RP) is a hereditary retinal dystrophy which leads to severe visual impairment or blindness and affects about 3.5/1000 of individuals in the industrial world. During the past decades, numerous animal models carrying mutations analogous to mutations in human RP have been studied to elucidate the molecular mechanisms leading to apoptotic photoreceptor cell death in this disease. Up to date, there is no effective treatment to influence the fatal outcome of RP. Recent progress in basic research promotes the development of new therapeutic strategies. In order to restore visual function in blind individuals, the development of electronic photoreceptor prosthesis is being investigated by several researchgroups. Other promising approaches are somatic gene therapy, the application of growth factors and/or pharmacological agents and the inhibition of photoreceptor cell death by interfering with the apoptotic pathway. However, a better understanding of the molecular events leading to cell loss due to photoreceptor apoptosis will be essential for the development of effective treatment. PMID- 10730222 TI - [Strabismus surgery under subconjunctival anesthesia: technique and evaluation by patient and his surgeon]. AB - BACKGROUND: In adults, eye muscle surgery can be performed under local anesthesia without retrobulbar injection. We wanted to know how patients and surgeons perceive this procedure. PATIENTS AND METHODS: 50 patients and 3 surgeons were interviewed with a questionnaire after operation on one or two straight eye muscles, including revisions in 11 cases. The patients were sedated with benzodiazepines. The conjunctiva was anesthetized with tetracaine eye drops. During the operation, mepivacaine was irrigated under the muscle insertion using a blunt needle. RESULTS: The patients perceived little or no pain during the operation. This corresponded to the surgeon's judgement. 96% of the patients would prefer local over general anesthesia in case of a second operation. CONCLUSION: The technique can be recommended for surgery of rectus muscles in adults, even in the case of revision. PMID- 10730224 TI - [Rehabilitation of homonymous hemianopsia]. AB - BACKGROUND: Homonymous hemianopia, most often caused by cerebral arterial infarction, represents for the patient a severe handicap, whereby beside bumping into persons and objects disturbance of reading and writing are predominant. In the beginning the patients often are not aware of the hemianopia (anosognosia) or even completely deny it (hemi-neglect). For rehabilitation of homonymous hemianopia different successful methods are at disposal. METHODS: 1. Improvement of the patient's attention for the blind hemifield, 2. Enlargement of the saccadic search field. 3. Systematization of the search strategies, 4. Treatment of the hemianopic reading and writing disturbance, 5. Auxiliary optical measures. CONCLUSIONS: The rehabilitation of homonymous hemianopia with the described different methods is most successful and improves for the patient not only elementary and higher visual functions (visual exploration, reading and writing ability, parafoveal visual field, recognition of forms and colours), but may under favorable circumstances even restore partial or total working capacity. PMID- 10730225 TI - [Intraocular pressure after phacoemulsification and implantation of silicone plate haptic intraocular lenses without viscoelastics]. AB - BACKGROUND: A rise in intraocular pressure (IOP) after cataract operation is a well known problem. Avoidance of the use of viscoelastics seems to solve the problem. PATIENTS AND METHODS: The IOP was measured in a group of 33 eyes of 33 patients one day before, as well as 6 hours, 24 hours, and 7 days after phacoemulsification and implantation of a foldable silicone plate haptic intraocular lens without viscoelastics and by means of an injector and of the anterior chamber maintainer. RESULTS: Mean preoperative IOP was 16.1 +/- 3.9 mm Hg (range 10 to 28 mm Hg). Postoperatively mean IOP was 12.3 +/- 3.3 mm Hg (range 5 to 18 mm Hg) after 6 hours (p < 0.0001), 13.8 +/- 2.8 mm Hg (range 7 to 19 mm Hg) after 24 hours (p = 0.032), and 15.5 +/- 3.4 (range 10 to 24) after one week (p = 0.39). In none of the eyes was the pressure in the first 24 hours higher than 18 mm Hg. When excluding the 4 patients with glaucoma and PEX or when taking them as a separate group, the results were similar. CONCLUSIONS: Pressure elevation after cataract operation without the use of viscoelastic substances can be avoided, thus contributing not only to lower costs but also to a higher safety. PMID- 10730226 TI - [Ocular hemoglobinometry--physiological variations]. AB - BACKGROUND: To explore the relationship between ocular (fundus) hemoglobin and that sampled and measured conventionally. To look for differences in hemoglobin density determined by both methods when the body hemoglobin is acutely (blood donation) or pathologically e.g. anemia altered. PATIENTS AND METHODS: Conventional (capillary and antecubital) and ocular fundus (papillary and choroidal) determinations of hemoglobin density in 14 females and 23 males, aged 25 to 30 years were compared. Application of the ocular method before and after blood donation in 21 females and 12 males, aged 20 to 68 years was performed. All these subjects were ophthalmologically and systemically healthy. Five male and 5 female anemia patients, aged 27 to 90 years, were also measured as above. RESULTS: Good correlation between fundus hemoglobin density and capillary (r = 0.81) and venous (r = 0.61) hemoglobin was observed in healthy persons. Differences in hemoglobin density according to gender were obvious at all fundus sites measured. Following blood donation, papillary hemoglobin density in males moreover increased, while that in females decreased (F = 7.53), suggesting a gender-specific difference in the ocular blood regulation, an effect also noted in the anemia patients. CONCLUSIONS: Comparison of conventional and ocular determination of hemoglobin reveals good correlation in healthy people. However, in acute or chronic blood loss the papillary hemoglobin level differs from that measured peripherally. A gender-related regulatory capacity of the ocular tissues under low-level conditions can be shown: Male persons maintain ocular hemoglobin at a normal level even when peripheral hemoglobin falls to low values, whereas female persons show a decrease in ocular hemoglobin parallel to the venous levels. Hence, under such extreme conditions,--and only in males--the ocular method yields values other than those from the conventional method, because ocular regulatory mechanisms, otherwise undetected, are exquisitely revealed. PMID- 10730227 TI - [Glaucoma and cataract: combined operation or trabeculectomy first and cataract extraction later?]. AB - BACKGROUND: The aim of this retrospective study is to evaluate the difference in postoperative intraocular pressure (IOP) control, number of antiglaucoma medications and visual acuity (VA) between combined phacoemulsification and trabeculectomy (Glaucoma triple) surgery and two-stage procedures with trabeculectomy first and cataract extraction later. PATIENTS AND METHODS: Two groups of glaucoma patients were assessed: In group 150 consecutive patients undergoing combined phacoemulsification and trabeculectomy were enrolled. In group two, 51 consecutive patients were included, that had undergone trabeculectomy first and cataract surgery later. The mean interval between the two procedures was 3.2 years, 35/51 patients (71%) underwent conventional extracapsular cataract extraction, the other 16 patients (29%) had phacoemusification via clear cornea incision. RESULTS: One year postoperative both groups revealed a significant IOP reduction. IOP-reduction was significantly better in the two-stage group one year after trabeculectomy (12.8 +/- 4.0 mm Hg) and one year after cataract extraction (14.0 +/- 2.8 mm Hg) as compared to the combined surgery group (15.3 +/- 4.0 mm Hg). Kaplan-Meier survival analysis revealed a 90% success-rate 12 months after trabeculectomy only as compared to 65% success-rate in the combined surgery group. The number of antiglaucoma medications was significantly reduced in both groups. One year after cataract surgery no difference in VA could be seen between the two groups. CONCLUSIONS: If medically uncontrolled glaucoma with advanced optic nerve head changes in patients with coexisting glaucoma and cataracts is the main indication for surgery, a two-stage procedure (i.e. trabeculectomy first, cataract extraction later) yields better long-term IOP control. If glaucoma is medically controlled with a simple regimen, conjunctiva-sparing cataract surgery may be the treatment of choice. In all other cases of coexisting glaucoma and cataract combined phacoemulsification and trabeculectomy (Glaucoma triple) is preferrable, either performed through a single incision or as two separate procedures in the same session (i.e. trabeculectomy from above, phacoemulsifiaction via clear cornea from temporal). PMID- 10730228 TI - [Radiotherapy for choroidal neovascularization (CNV) in age-related macular degeneration (AMD)]. AB - AIM OF THE STUDY: Several pilot studies have indicated, that radiation therapy might have a beneficial effect on the course of CNV in AMD. This controlled study was aimed at the question, whether such treatment might halt progression of neovascular AMD and whether a low or a high radiation dose should be applied. METHODS AND PATIENTS: Enclosed were patients aged > or = 60 and < or = 85 and eyes with a VA of > or = 0.1 and < or = 0.6, revealing a juxta/subfoveal CNV either of the occult or the classic type. Treatment was performed with a linear accelerator at fractions of 2 Gy up to a total dose of 10 Gy or 36 Gy. 95 eyes had completed follow-up of > or = 12 < or = 24 months. RESULTS: Among eyes with occult CNV 8 received 36 Gy, 16 were treated with 10 Gy and 21 were in the control group. Mean visual loss was 3.5 lines after 12 months and 5 lines after 24 months with no difference between irradiated eyes and those in the control group. In the groups with classic CNV 8 eyes were treated with 36 Gy, 27 eyes received 10 Gy and 15 eyes were in the control group. Mean visual loss after 6 months was 2.2 lines in eyes of both groups treated with radiation and 5.7 lines in the control group. This was statistically significant (p < 0.05). VA was < 0.1 after 12 (24) months of follow-up in 50 (75)% of the cases with 36 Gy, in 48 (83)% with 10 Gy and in 60 (83)% of the controls. These results were statistically significant after 12 months only. DISCUSSION AND CONCLUSIONS: The natural course of occult CNV could not be improved by irradiation with 10 or 36 Gy. In eyes with classic CNV a VA of > or = 0.1 was maintained significantly more often in irradiated eyes than in those of the control group. Treatment with 36 Gy however was associated with an unacceptable incidence of radiation retinopathy. PMID- 10730229 TI - [Bilateral cystoid macular edema in an older female patient with tubulointerstitial nephritis and uveitis (TINU syndrome)]. AB - BACKGROUND: TINU-Syndrome (Acute tubulointerstitial nephritis and uveitis) is a rare immune-mediated disease, primarily occurring in children and young women. Uveitis may precede, follow or occur concomitantly with nephritis. Corticosteroids are the mainstay of therapy. PATIENT: A 73-year old female patient with tubulointerstitial nephritis and recurrences of anterior uveitis was diagnosed having TINU syndrome. During follow-up she developed bilateral cystoid macular edema with accompanying visual loss. Nephritis and uveitis responded well to oral corticosteroids. CONCLUSIONS: Uveitis screening should exclude renal disease independent of the age of the patient. Diagnosis can often be inferred from anamnesis, physical examination, or laboratory tests. Renal biopsy as the most definite method of diagnosis may be necessary. Ophthalmologic follow-up on a regular basis is mandatory for a successful management of long-term complications. PMID- 10730230 TI - [Pronounced physiological pupillary hippus]. AB - BACKGROUND: The term "hippus" describes pupillary oscillations of variable amplitude and frequency and may be physiological or part of a pathological entity. Thus, pupillary hippus is often the target of extensive clinical investigation. CASE REPORT: A 9-year-old boy was seen in our emergency room in March 1998. Examination showed distinct pupillary oscillations in the absence of any other pathological findings. We presented the patient to a pediatric neurologist. Apart from a history of enuresis and of motor tics in the neck region, the present neurological examination was normal. CONCLUSIONS: Even a pronounced hippus may be physiological and does not necessarily require extensive diagnostic investigations. PMID- 10730231 TI - [Simultaneous ICG- and fluorescein-angiography for fundus examination]. AB - BACKGROUND: An indocyanine-green (ICG) angiography is rarely used as the only diagnostic procedure. Almost always it is performed additionally to a fluorescein angiography. The use of simultaneous ICG and fluorescein (SIF) angiography therefore makes sense. Several examples for the application of SIF-angiography are presented. MATERIALS AND METHODS: SIF-angiography was performed using a 2 wavelength scanning laser ophthalmoscope (SLO). Images were digitally recorded in real time with a graphics workstation. The following cases will be presented: choroidal neovascularization in age-related macular degeneration, choroidal hemangioma, inflammatory fundus disease (APMPPE) and idiopathic polypoidal choroidal vasculopathy. RESULTS: ICG and fluorescein angiography can be simultaneously recorded with the 2-wavelenghth SLO. The quality of the combined pictures is comparable to single-channel recordings of separate ICG and fluorescein images. We show results of the above mentioned cases. CONCLUSIONS: SIF angiography is time efficient and allows precise comparison and analysis of the transit of both dyes through retinal and choroidal circulation. The topographic relation of pathologic findings in ICG angiograms with the critical retinal vascular landmarks is facilitated. PMID- 10730232 TI - MR imaging of the thoracic aorta. AB - MR imaging is an effective modality for noninvasive morphologic and functional assessment of the thoracic aorta. MR imaging provides several advantages for vascular imaging, including intrinsic contrast between the blood pool and vascular structures, multiplanar imaging capability, and the absence of ionizing radiation. By combining imaging and flow-sensitive techniques, MR imaging can be used to delineate morphology and to quantify bloodflow volume and velocity. In patients who need sequential examinations over time to monitor disease severity, a noninvasive technique such as MR imaging is desirable. PMID- 10730233 TI - MR imaging of thoracic veins. AB - Anomalies of thoracic veins are a diverse but important group of abnormalities. MR imaging is useful in delineating the venous anomalies, provides physiologic information, and is a valuable complementary technique to echocardiography, angiography, and CT scan. PMID- 10730234 TI - MR imaging of thoracic lymph nodes. A comparison of computed tomography and positron emission tomography. AB - CT scanning has traditionally been the preferred imaging modality for the assessment of patients with non-small-cell lung cancer. The low sensitivity and specificity of CT scanning, however, has limited its usefulness in assessing nodal status. Despite this limitation, CT scanning still plays an important role by aiding the selection of the most appropriate procedure for staging purposes, guiding biopsy procedures, and providing anatomic information for visual correlation with FDG-PET images. Anatomic imaging with MR imaging has been shown to have accuracy comparable with CT scanning in assessing mediastinal lymph nodes. MR imaging, however, is more accurate than CT in the assessment of hilar lymph nodes. At present, anatomic imaging of lymph nodes with MR imaging should be considered a secondary, problem-solving tool for cases in which CT scanning is inconclusive. Advances in physiologic imaging of mediastinal lymph nodes with FDG PET imaging have resulted in better diagnostic accuracy than obtained with anatomic imaging with CT scans or MR imaging. At present, an imaging strategy that employs both FDG-PET imaging and CT scanning appears to be the most accurate, noninvasive, and cost-effective means available for assessing nodal status in patients with non-small-cell lung cancer. Physiologic MR imaging with iron oxide is currently being assessed in clinical trials. Future studies are necessary to determine the clinical efficacy, accuracy, and cost effectiveness of this technique. PMID- 10730235 TI - MR imaging of lung cancer. AB - Computed tomography of the chest is the present mainstay of cross-sectional imaging in patients with lung cancer. MR imaging, however, plays a complementary role in evaluating apical tumor and is often used as a problem-solving tool when CT findings are equivocal. New developments such as contrast-enhanced MR imaging and whole-body MR imaging may broaden the indications for this technique in the future. PMID- 10730236 TI - MR imaging of mediastinal masses. AB - Although ongoing developments in MR imaging have resulted in improved image quality and decreased acquisition time, MR imaging is largely used as an adjunct to CT scanning in the evaluation of mediastinal abnormalities. In this role, MR imaging often provides additional information about the nature, location, and extent of disease. MR imaging is useful in confirming the cystic nature of mediastinal lesions that appear solid on CT and, by revealing small amounts of intralesional fat, can suggest the diagnosis of hemangioma, teratoma, or extramedullary hematopoesis. MR imaging is the preferred modality for imaging neurogenic tumors, because its multiplanar capability and high contrast resolution can best demonstrate the number and nature of the lesions (differentiating cysts from neoplasms), intraspinal extension, and craniocaudad extent. MR imaging is also especially useful for evaluating the mediastinum of patients for whom the administration of iodinated contrast material is contraindicated. PMID- 10730237 TI - MR imaging of thoracic trauma. AB - Although MR imaging and MR angiography should not be considered a primary study for patients sustaining blunt or penetrating thoracic trauma, it has achieved importance as an ancillary problem-solving modality. In general, the authors have found MR imaging to be a useful ancillary study to confirm or exclude diaphragm injury, if spiral CT scan with reformations are equivocal; to help assess the aorta and mediastinum in cases of potential injury that are not clarified by CT scan or thoracic angiography; and to identify the true origin of trauma-related masses that may simulate thoracic neoplasms. PMID- 10730238 TI - MR imaging of pulmonary parenchyma. AB - Poor image quality has severely limited the clinical effectiveness of MR imaging of lung parenchyma. Recent improvements in imaging gradients and the introduction of phased-array receiver coils have improved image quality and should allow the diagnostic advantages inherent in MR imaging to be applied for images of lung parenchyma. The recent introduction of MR ventilation imaging should also provide further insights into airways diseases, including chronic obstructive lung disease (COLD), asthma, and bronchiolitis obliterans. The continuing research in MR imaging of lung parenchyma attests to the difficulty of obtaining good images and to the potential power of this technology. PMID- 10730239 TI - MR imaging of the pleura and chest wall. AB - Evaluation of pleural and chest-wall disease begins with a chest radiograph. In many cases, further evaluation of the chest wall and pleura requires cross sectional imaging with CT scan or MR imaging. MR imaging is reserved for specific cases of tumors, infections, pleural effusions, and masses. The superior soft tissue contrast of MR imaging is useful in evaluating the extent of infections and tumors involving the pleura and chest wall. The multiplanar capability of MR imaging aids in the evaluation of chest wall and pleural abnormalities, particularly in the apical regions. PMID- 10730240 TI - MR imaging of the thoracoabdominal junction. AB - Recent advances in MR imaging techniques and its multiplanar capability allow for improved detection and characterization of benign and malignant processes occurring in the thoracoabdominal region. It is important to the staging process of patients with known malignancy to correctly diagnose incidental masses in this region, including hepatic or adrenal lesions, which initially may be identified by other imaging modalities. MR imaging provides a complimentary role in lesion detection and diagnosis that may ultimately affect therapeutic management and outcome. PMID- 10730241 TI - MR imaging of the thyroid and parathyroid glands. AB - Whereas scintigraphy and ultrasonography are the primary imaging modalities used for the investigation of thyroid disorders, MR imaging is generally used for specific indications, including evaluating the extent of substernal goiters, assessing the effect on adjacent structures, imaging the local extent of thyroid carcinomas, and localizing recurrent sites of thyroid neoplasia. MR imaging also has been used for the investigation of congenital disorders of the thyroid gland and the evaluation of diffuse thyroid disease, such as Grave's disease, Hashimoto's and Riedel thyroiditis, and hemochromatosis. MR imaging of the parathyroid glands is generally used for patients with recurrent or persistent hyperparathyroidism following neck exploration. MR imaging provides useful information about morphologic abnormalities of the thyroid and parathyroid glands. MR imaging has good sensitivity and positive predictive value for the identification of non-ectopic and ectopic abnormal parathyroid glands. The detailed anatomic information provided by MR imaging is useful in planning a surgical approach and is complementary to other imaging methods used in the investigation of recurrent or persistent hyperparathyroidism. PMID- 10730242 TI - MR imaging of the thoracic inlet. AB - The borders of the thoracic inlet define an oblique plane that angles downward from the spine anteriorly to the first ribs. It is therefore best to consider the thoracic inlet as a region or "zone" which extends a short distance above and below this plane to include the lower portion of the infrahyoid neck and the upper portion of the superior mediastinum. MRs multiplanar imaging capability allows the thoracic inlet to be subdivided into four distinct zones; visceral, neurovascular, pulmonary, and spinal. The first section of this article reviews the normal anatomy of each zone and the sequence protocol for imaging of the brachial plexus. The second section discusses differential diagnoses relevant to each zone. Pathologic processes are also presented. PMID- 10730243 TI - Novel techniques for MR imaging of pulmonary airspaces. AB - Hyperpolarized gas- and molecular oxygen-enhanced MR imaging are two new techniques for high-resolution MR imaging of pulmonary airspaces. Both techniques produce excellent images in a safe, reproducible, and technically feasible manner. Because morphologic and functional information is obtained, and radiation is not used, these techniques may prove ideal for serially evaluating patients with a variety of lung diseases that affect pulmonary ventilation, such as cystic fibrosis, emphysema, asthma, or bronchiolitis obliterans syndrome in lung transplant recipients. At present, the greatest clinical experience is with hyperpolarized He-3-enhanced MR imaging. This technique is limited, however, by the limited availability of He-3, by its polarization requirements, and by the need to tune the MR system to the resonant frequency of the gas. There is less clinical experience with oxygen-enhanced MR imaging. Although this technique produces images with more inherent noise than hyperpolarized He-3 imaging, this problem can be overcome by signal averaging. Oxygen-enhanced imaging has the major advantages of lower cost and ready availability. For oxygen-enhanced imaging, the MR imaging system does not need to be readjusted; imaging is performed at the conventional hydrogen proton frequency. PMID- 10730244 TI - Insights into T-cell development from studies using transgenic and knockout mice. PMID- 10730245 TI - Isolation and characterization of murine early intrathymic precursor populations. PMID- 10730246 TI - Differentiation of mouse thymocytes in fetal thymus organ culture. PMID- 10730247 TI - Purification of immature CD4+CD8+ thymocytes by panning with anti-CD8 antibody. PMID- 10730248 TI - Transfection and transcription of genes in developing thymocytes. PMID- 10730249 TI - Analysis of CD4/CD8 lineage commitment by pronase treatment and reexpression assay. PMID- 10730250 TI - Intrathymic delivery of MHC genes using recombinant adenoviruses. PMID- 10730251 TI - Quantitative analysis of the usage of human T-cell receptor alpha and beta chain variable regions by reverse dot-blot hybridization. PMID- 10730252 TI - Flow cytometric detection of cytoplasmic and surface CD3-epsilon expression in developing T cells. PMID- 10730253 TI - Assessing apoptosis of developing T cells by flow cytometry. PMID- 10730254 TI - Isolation of T-cell subsets by magnetic cell sorting (MACS). PMID- 10730255 TI - Isolation of T-cell antigens by retrovirus-mediated expression cloning. PMID- 10730256 TI - Flow cytometric analysis of murine T lymphocytes. A practical guide. PMID- 10730257 TI - Production and characterization of immature murine T-lymphoma cell lines. PMID- 10730258 TI - Production and characterization of T cell hybridomas. PMID- 10730259 TI - Something happens; a brief history of TCR signal transduction. PMID- 10730260 TI - Measurement of phosphatidylinositol (PI) hydrolysis in activated T lymphocytes. PMID- 10730261 TI - Fluorescence polarization as an early measure of T-lymphocyte stimulation. PMID- 10730262 TI - Measurement of lymphoproliferation at the single-cell level by flow cytometry. PMID- 10730263 TI - Digital image analysis of lymphocyte activation. PMID- 10730264 TI - Induction of T-cell signaling by immobilized integrin ligands. PMID- 10730265 TI - Cytotoxic T-cell adherence assay (CAA). PMID- 10730266 TI - Detecting ubiquitinated T-cell antigen receptor subunits by immunoblotting. PMID- 10730267 TI - Determination of CD45 tyrosine phosphatase activity in T lymphocytes. PMID- 10730268 TI - Measurement of protein tyrosine phosphorylation in T-cell subsets by flow cytometry. PMID- 10730269 TI - Biochemical analysis of activated T lymphocytes. Protein phosphorylation and Ras, ERK, and JNK activation. PMID- 10730270 TI - Activation of heterotrimeric GTP-binding proteins upon TCR/CD3 engagement. PMID- 10730271 TI - Generation of human T helper 1 and T helper 2 subsets from peripheral blood derived naive CD4+ T cells. PMID- 10730272 TI - Transcriptional expression analysis of T-cell activation by multiplex messenger assay (MMA). PMID- 10730273 TI - [About nephrology vocabulary]. PMID- 10730274 TI - Alport syndrome and diffuse leiomyomatosis. Clinical aspects, pathology, molecular biology and extracellular matrix studies. A synthesis. AB - The Alport syndrome-diffuse leiomyomatosis association can be defined as a hereditary disease of type IV collagen combining features of Alport syndrome (hematuric nephropathy, deafness and ocular abnormalities: anterior lenticonus, maculopathy) and leiomyomatosis involving oesophagus (diffuse type), tracheobronchial tree, and genitals (only in women). This entity is transmitted as an X-linked dominant trait. Mutations of both the COL4A5 and COL4A6 genes, located head to head in Xq22 encoding the alpha 5 and alpha 6(IV) chains are responsible for the abnormalities. Molecular studies have shown deletions of the 5' end of both COL4A5 and COL4A6 including the intergenic region. The breakpoint in COL4A6 is always located within intron 2. Immunohistochemistry has shown significant alterations of basement membranes in the kidney and esophageal leiomyomas. Leiomyomas lack alpha 5 and alpha 6(IV) chains, fibronectin and laminin beta 1 chains in the muscle basement membranes where they are normally expressed. The tumors also show myocyte anomalies: irregular expression of the alpha 5 integrin subunits, and disorganization of actin and desmin filaments. It is hypothesized that a third as yet unknown gene, situated within the large intron 2 in a critical 90 kb region, is responsible for the smooth muscle proliferation. Abnormalities of the basement membranes could destabilize interactions between muscular cells and the extracellular matrix. PMID- 10730275 TI - [Cystinosis from childhood to adulthood]. AB - Nephropathic cystinosis is a metabolic disease related to lysosomal cystine accumulation in almost all tissues of the body. The first symptoms set up from 5 or 6 months of age including anorexia vomiting polyurodipsia and failure to thrive associated with a proximal tubulopathy (glycosuria, tubular proteinuria, loss of bicarbonate, potassium, phosphorus, etc.) Treatment by cysteamine dramatically changed the prognostic. If started early this treatment allows to delay and possibly to prevent the spontaneous evolution towards end stage renal disease between 6 and 12 years of age and to avoid the growth failure. On the long term the disease involves other organs: eyes, thyroid endocrine pancreas, muscle and central nervous system. The diagnosis of cystinosis is based on the cystine leukocyte assay allowing also the follow up and the adjustment of the treatment. Prenatal diagnosis is possible on chorionic sample. The gene of this recessive disease, mapping on chromosome 17 was recently identified. This gene encodes a protein of the lysosomal membrane involved in the transport of cystine out of the lysosome. There is a juvenile, late onset, form of cystinosis its main symptom is proteinuria with variable tubular alterations. The so called adult form is asymptomatic its only symptom is corneal deposits most often found by chance examination. PMID- 10730276 TI - [Prevalence of subcutaneous, labial and rectal amyloid lesions in patients wi histologically confirmed renal amyloidosis]. AB - Our work is to evaluate the biopsies rectal (RB), cutaneous and one about the accessory salivary glands (ASGB) in the diagnosis of amyloidosis renal through 20 cases of renal amyloidosis confirmed by renal biopsy in unit of nephrology and hemodialysis in UHC lbn Rochd from February 1996 to January 1998. The mean age of our patients was 39 years old (15-80) with a male predominance of (4/1). The infectious pathology has been the most frequent cause (70%) which consisted essentially in the tuberculosis and the surinfection on dilatation of the bronchi. All the patients had nephrologic symptomatology dominated by the nephrotic syndrome. The 4 biopsies were practised in the 20 patients. Wright's reaction practised in all renal biopsy confirmed the AA nature of the amyloidosis. The amyloid deposits were noticed in 100% of renal needle biopsy, 80% of accessory gland biopsy, 75% of rectal biopsy and in 35% of cutaneous biopsy. The association of RB and ASGB was positive in 90% of the cases. If the renal biopsy gave more positivity than the other biopsies, it caused complications that were severe sometimes. Also, the biopsy of the ASG, the simple technique is very reliable in the recognition of the amylosis and seems to be the best diagnostic test for this affection. PMID- 10730277 TI - [The Society of Renal Pathology (1948-1949)]. AB - Founded in 1948 under the leadership J. Hamburger, the Societe de pathologie renale played an important role for the development of what will become within 10 years a new discipline, Nephrology. Our discipline is unique since it does not come from internal medicine but from fundamental biology which is needed to analyze and understand the normal and diseased kidney. In the Societe, where European and Francophone investigators met, several aspects of Nephrology were presented, discussed and sometimes solved such as acute renal failure and its treatment by peritoneal dialysis or hemodialysis, electrolytic disorders and their correction sin intensive care units, renal biopsies in primitive and secondary renal diseases. In 1959, this society became Societe de nephrologie, preceding the first international congress of Nephrology, another success of J. Hamburger. PMID- 10730278 TI - [Validation of 24-hour urine sampling by estimation of the 24-hour creatinuria according to the Cockroft and Gault formula]. PMID- 10730279 TI - Local anesthesia toxicity review revisited. PMID- 10730280 TI - Comparison of direct digital and conventional radiography for the detection of proximal surface caries in the mixed dentition. AB - PURPOSE: The aim of this study was to compare the performance of direct digital radiography and traditional dental radiography for the detection of proximal surface dental caries in the mixed dentition. METHODS: 15 quadrants of extracted teeth, arranged from the primary canine to permanent first molar, were imaged using direct digital (Schick Technologies, Long Island City, NY, USA) and conventional films (D-speed and E-speed Plus; Eastman Kodak Co., Rochester, NY, USA). Five pediatric dentists viewed the images and scored the 270 proximal surfaces for presence of caries on a 5 point scale and extent of caries on a 4 point scale. The teeth were sectioned and viewed microscopically to determine the gold standard. Receiver operating characteristic (ROC) analysis and analysis of variance (ANOVA) were used to evaluate the viewer's performance for detecting proximal caries using the 3 different image receptor types. RESULTS: Experienced examiners were significantly more accurate in diagnosis of proximal surface caries using either D-speed or E-speed Plus films than they were using the direct digital receptor. The mean areas under the ROC curve (Az) for the viewers were 0.7595 for D-speed film, 0.7557 for E-speed Plus film, and 0.5928 for the direct digital receptor. The results also indicated that selected viewers' accuracy increased when viewing the direct digital images a second time. CONCLUSION: CCD based direct digital radiography was not as accurate as conventional film images for the purpose of diagnosing proximal surface caries in the mixed dentition. However, the results imply that with increased experience, direct digital images may be as accurate as conventional film based images for diagnosis. PMID- 10730281 TI - Pediatric dental visits during 1996: an analysis of the federal Medical Expenditure Panel Survey. AB - Pediatric health policy articulated by the government, health professions, and child advocates is concerned with eliminating health disparities among children and increasing access to essential pediatric health services. National data are widely available on dental health status and associated disparities by income and race, but little data have been reported on dental service utilization by children. Data from the 1996 federal Medical Expenditure Panel Survey were analyzed to determine the percentage of children who obtained a dental visit and the number of visits children experienced by age, sex, ethnic/racial background, family income, and parental education. Overall, 43% of all children ages birth through 18 obtained at least one dental visit in 1996. Among children who see a dentist, the average number of visits during 1996 was 2.7. Low income, low education, and minority status are all associated with both lower odds of having a dental visit and lower number of visits per utilizer. Children under 6 had less than half the dental visit rate of older children and had fewer visits per person among utilizers. PMID- 10730282 TI - Hospital charges for dental caries related emergency admissions. AB - PURPOSE: The purpose of this investigation was to develop and test a model for identifying hospital charges resulting from patient admissions through the emergency room of a children's hospital to manage pediatric nontraumatic dental disease. METHOD: Model development involved data identification and collection at Children's Medical Center of Dallas, Texas. Its utility was tested in 4 children's hospitals across the United States. RESULTS: The model proved effective in determining hospital charges for pediatric caries-related admissions. Diagnosis codes assigned at the time of admission were not specific enough to limit identification to nontraumatic dental admissions. Extensive review of patient records determined that only one-third of admitted patients identified by the model were caries-related admissions. Fifty-two children were identified who were admitted to the 5 children's hospitals in 1997 due to dental caries or its complications. Median hospital charge per admission was $3,223 and the total hospital charges for these 52 children was $250,000. CONCLUSIONS: More specific ICD-9 diagnosis codes should be developed to identify these patients. PMID- 10730283 TI - A cost analysis of treating pediatric dental patients using general anesthesia versus conscious sedation. AB - PURPOSE: The purpose of this pilot study is to report a cost comparison of general anesthesia (GA) versus oral conscious sedation (CS) for the treatment of pediatric dental patients. METHODS: The study sample included 22 children whose parents/guardian selected GA care for their child. Selection criteria limited inclusion to healthy children (ASA I) ages 24-60 m.o. The subjects acted as their own comparison group to an estimation CS model. Models were developed to assess societal costs for treatment under GA and CS. Treatment rendered was equalized using the dental Relative Based Value Units Scale (RBVU). RESULTS: Ordinary Least Squares Regression analysis techniques showed the association of RBVU to the total societal costs of GA and CS to be significant (P < 0.01) with an adjusted R2 of .64 and .78 respectively. When regression lines were plotted, the intersection represented RBVU level at which societal costs of GA and CS were the same. CONCLUSION: Under the conditions of this pilot study, it is concluded that CS costs exceed GA costs at a RBVU level of 66.4, which would equate to more than three CS appointments. PMID- 10730284 TI - A comparison of restorations for children with early childhood caries treated under general anesthesia or conscious sedation. AB - PURPOSE: There is no data in the dental literature concerning the quality of the restorations performed in young children with early childhood caries (ECC) under sedation as compared with those treated under general anesthesia (GA). The aim of this study was to compare the quality of restorations and recurrent caries in 65 children with ECC who had dental treatment under GA or sedation. METHODS: Thirty four children, mean age 34.4 months were treated under GA and 31 children with a mean age of 37.2 months were treated under sedation and re-examined 6-24 months after completion of treatment. The quality of the restorations was evaluated using a modified Cvar & Ryge index. RESULTS: Fifty-nine percent of children treated under GA required further dental treatment compared to 74% of children treated under sedation. The majority of the required treatment was due to new caries: 57% in the GA group and 60% in the sedation group. A total of 248 restorations were evaluated for the GA group, with a 94% success rate for marginal adaptation, 92% success for anatomic form, and 97% had no secondary caries. In the sedation group, out of 224 restorations, 78% demonstrated perfect marginal adaptation, 79% showed adequate anatomic form, and 90% had no secondary caries. Successful marginal adaptation was found in 90% of strip crowns placed under GA, compared to 63% of those placed under sedation. CONCLUSION: It is concluded that the outcome of treatments related to quality of the restorations performed under GA is better for all parameters examined. PMID- 10730286 TI - Smoking interventions delivered by pediatric dentists: special recommendations for pediatric cancer patients. AB - Child and adolescent cancer patients who use tobacco present challenging management problems for the pediatric dentist. An approach to counseling patients about their oral health risks secondary to their cancer treatment, which can be adversely impacted by tobacco use, is discussed. Strategies for conducting dental examinations with pediatric cancer patients with attention to potential tobacco related complications are also provided. PMID- 10730285 TI - Microleakage of Compoglass-F and Dyract-AP compomers in Class V preparations after salivary contamination. AB - PURPOSE: The purpose of this study was to investigate the effect of salivary contamination on the microleakage within Class V preparations of teeth restored with either Compoglass-F/Syntac SC and Dyract-AP/Prime and Bond 2.1. METHODS: Class V cavity preparations with occlusal margins in enamel and gingival margins in cementum were prepared on the buccal and lingual surfaces of extracted human permanent molar teeth. Preparations were distributed randomly into 4 treatment groups (N = 16) consisting of the two compomers and their respective bonding agents with and without salivary contamination. After treatment, the samples were stored in tap water for 24 hours, thermocycled, stained with dye, sectioned, and scored for microleakage. RESULTS: Salivary contamination had no significant effect on enamel microleakage but significantly increased both linear and penetrating microleakage versus non-contaminated for both compomer/dentin bonding systems. CONCLUSION: These data indicate salivary contamination adversely effects gingival but not enamel microleakage when Class V restorations are restored with either Compoglass F/Syntac SC or Dyract-AP/Prime and Bond 2.1. PMID- 10730287 TI - Anodontia with hypohidrotic ectodermal dysplasia in a young female: a case report. AB - A five-year-old Japanese girl was referred to our clinic for non-eruption of the teeth. Panoramic radiographs revealed absence of all teeth of both primary and permanent dentitions. She showed hypotricosis, hypohidrosis, and anodontia. In this article, the reported cases of anodontia were reviewed and the fabricating procedure of full dentures for a young child was described. PMID- 10730288 TI - Odontogenic keratocysts in a 5-year-old: initial manifestations of nevoid basal cell carcinoma syndrome. AB - The purpose of this paper is to report the occurrence of odontogenic keratocysts in a young child. Odontogenic keratocysts are one of the principal features of nevoid basal cell carcinoma syndrome. Their occurrence in this syndrome is usually during the second or third decades of life. This report describes the occurrence of odontogenic keratocysts in a 5-year-old, which proved to be the initial presentation of nevoid basal cell carcinoma syndrome and highlights the need to consider this syndrome as a possible diagnosis in all cases of odontogenic keratocysts. PMID- 10730289 TI - Long-term oral and craniofacial complications following pediatric bone marrow transplantation. AB - Bone marrow transplantation (BMT) has become a common form of treatment for childhood diseases and disorders that directly or indirectly affect the production of stem cells which give origin to blood and immune system elements. The pre-transplant protocols include, but are not limited to, chemotherapy and/or radiotherapy, which can cause considerable acute and long-term undesired effects in the oral cavity and the craniofacial complex. This manuscript discusses the sequelae that a pediatric dentist may encounter when treating a BMT survivor. PMID- 10730290 TI - Ectopic erythema migrans in an adolescent with a skin disorder. AB - Red and white circular lesions of the buccal and labial mucosa were observed in an adolescent. Periodically, these nontender patches would resolve and move to other oral sites. Detection of ectopic erythema migrans aided in the diagnosis of a bothersome skin condition. PMID- 10730291 TI - Transitional anterior esthetic restorations for patients with enamel defects. PMID- 10730292 TI - Early treatment mechanics of the Class II division 2 malocclusion. AB - A most common type of early malocclusion that the pediatric dentist comes in contact with daily is the developing Class II Division 2 malocclusion (Fig 1 a,b). It is the malocclusion that the parents of the children we serve bring to our attention. Parental concern is the early crowding that develops in the anterior of the lower arch with risk of periodontal involvement. This malocclusion is readily amenable to interception at age 7 or 8 and can proceed with a protocol of defined objectives and predictable outcomes (Fig 2). With efficient and effective utility arch wire (UAW) mechanics a state of normalcy can be achieved within six to eight months of treatment. PMID- 10730293 TI - Orthodontic alignment of permanent incisors following previous trauma of a primary tooth. PMID- 10730295 TI - Making child patient cooperation a choice. "Do you want to do it again?". PMID- 10730294 TI - Rubber dam clamp placement on partially erupted molars without anesthesia. PMID- 10730296 TI - What parents should know about estrogen-like compounds in dental materials. AB - The use of pit and fissure sealants has been reported to increase exposure to xenoestrogens. Because these estrogen-mimics are suspected of having many deleterious effects in animals, and perhaps humans, several types of studies were undertaken by our Biocompatibility Group. We confirmed that bisphenol A (BPA) and bisphenol A dimethacrylate (BPA-DM) have proliferative effects in cells with high levels of estrogen receptors. However, BPA was not detected by our group in American-made sealants, and BPA-DM was detectable in only a few. In addition, the surface layer of the sealant can be treated to reduce the possibility of unpolymerized BPA-DM being left on the tooth. We believe it is important to reassure parents that their children are less likely to be exposed to BPA from sealants than from the ingestion of soft drinks or canned food. PMID- 10730297 TI - Use of nickel-titanium rotary files for root canal preparation in primary teeth. AB - This article reviews the use of nickel-titanium rotary files for root canal instrumentation in primary teeth. The pulpectomy technique is described and the advantages and disadvantages of using rotary files are discussed. Specific recommendations for the selection of materials and devices are made. PMID- 10730298 TI - Why family planning matters. PMID- 10730299 TI - Determining the incidence of dengue hemorrhagic fever in Puerto Rico. PMID- 10730300 TI - Surveillance for an emerging disease: dengue hemorrhagic fever in Puerto Rico, 1988-1997. Puerto Rico Association of Epidemiologists. AB - Surveillance for emerging diseases is critically dependent on four factors: reporting methods, case definition, laboratory diagnosis, and knowledge of the disease among health-care professionals. The dengue hemorrhagic fever (DHF) surveillance system in Puerto Rico collects patient data from three sources: dengue case investigation (DCI) forms sent with diagnostic samples, clinical reports from hospital infection control nurses (ICNs), and hospital records. Recruitment of ICN reporting produced a marked increase in notifications (67 to 294). Hospital records of possible DHF cases showed that tests for ascertaining diagnosis (e.g., blood in stool, serum albumin) were frequently not performed. DCI and ICN reports underestimated severity. After hospital record review, the ratio of total DHF cases to cases detected by surveillance was approximately 3:1, whether using clinical criteria or using clinical and dengue laboratory diagnosis. An important determinant for the low sensitivity (28.4%) and high specificity (96.5%) of the surveillance system was the World Health Organization (WHO) clinical definition for DHF. In spite of such limitations, DHF surveillance data in Puerto Rico provide abundant, reliable information for monitoring disease trends. These methods may be applied to other situations to define the characteristics and incidence trends of emerging infections. PMID- 10730301 TI - An evaluation of modified case definitions for the detection of dengue hemorrhagic fever. Puerto Rico Association of Epidemiologists. AB - The case definition for dengue hemorrhagic fever (DHF) requires fever, platelets < or = 100,000/mm3, any hemorrhagic manifestation, and plasma leakage evidenced by hemoconcentration > or = 20%, pleural or abdominal effusions, hypoproteinemia or hypoalbuminemia. We evaluated the specificity and yield of modified DHF case definitions and the recently proposed World Health Organization criteria for a provisional diagnosis of DHF, using a data base of laboratory-positive and laboratory-negative reports of hospitalizations for suspected dengue in Puerto Rico, 1994 to 1996. By design, all modifications had 100% sensitivity. More liberal criteria for plasma leakage were examined: 1) adding as evidence a single hematocrit > or = 50% (specificity 97.4%); 2) accepting hemoconcentration > or = 10% (specificity 90.1%); and 3) accepting either hematocrit > or = 50% or hemoconcentration > or = 10% (specificity 88.8%). The new DHF cases identified by these definitions (and percent laboratory positive) were 25 (100.0%), 95 (90.5%), and 107 (91.6%), respectively. In contrast, the provisional diagnosis of DHF (fever and hemorrhage, and one or more of platelets < or = 100,000/mm3, or hemoconcentration > or = 20%, or at least a rising hematocrit [redefined quantitatively as a 5% or greater relative change]) showed a specificity of 66.8%, and identified 318 new DHF cases, of which 282 (88.7%) were laboratory positive. Very small changes in the criteria may result in a large number of new cases. The modification that accepted either hematocrit > or = 50% or hemoconcentration > or = 10% had acceptable specificity, while doubling the detection of DHF-compatible, laboratory-positive severe cases, but "provisional diagnosis" showed even lower specificity, and may produce inflated DHF incidence figures. Modified case definitions should be prospectively evaluated with patients in a health-care facility before they are recommended for widespread use. PMID- 10730302 TI - [Obstetricians and pediatricians: experience with breastfeeding in their residency programs]. AB - A prior study had the objective of identifying knowledge and experience on breastfeeding of obstetricians and pediatricians in two private hospitals of the Metropolitan Area of San Juan, Puerto Rico. This study presents the experience of those physicians regarding breastfeeding education during their residency training. An exploratory research design was used, the sample was non probabilistic and was selected according to availability for participation among the physicians who visited those hospitals during June, 1997. The studied sample included 29 pediatricians and 26 obstetricians. Descriptive statistics and crosstab tables were used to describe the results. It was found that participating physicians had little or no exposition to breastfeeding related topics during their respective residency programs. They are aware of the importance of their role in the achievement of successful breastfeeding. They perceive themselves as effective in stimulating breastfeeding although they recognize that they should have received better training on the subject. This attitude is worrisome in face of the fact that this same group presented significant deficiencies in a previous study with regards to knowledge in the management of breastfeeding. PMID- 10730303 TI - [Analysis of the role of caregivers in human lactation and breastfeeding]. AB - The provision of care and support in breastfeeding and human lactation is a function of health professionals as well as of the members of the community. These functions must be appropriately defined so that the importance and degree of responsibility of health professionals and of the community be understood. The legal aspects of the practice of Lactation Consultant are discussed. The training course for Lactation Educator offered by the Maternal and Child Health Program in the Graduate School of Public Health is presented as an alternative in basic training and service. The scope of the practice of the Lactation Educator should be determined by the basic role of the graduate within the system of care for the breastfeeding mother and her infant. PMID- 10730304 TI - Suppressor mutations derived from the most severe protein folding mutation known. AB - This brief report describes the isolation and initial characterization of revertants to the most severe temperature sensitive folding mutant known. The revertants or suppressors may describe amino acid interactions that occur during the folding of the P22 tailspike polypeptide chain. Results indicate that several different types of suppressors may have been obtained. PMID- 10730305 TI - [Description of health status and functional capacity of the 65-year-old and older population residing in the University Region of Health of Puerto Rico]. AB - This study intended to describe the health and functional status of the population 65 years and over resident in the Puerto Rico University Health Region (municipalities of Canovanas, Loiza, Carolina and Trujillo Alto, Puerto Rico). Four ninety one hundred elderly subjects selected from a random sample of households, were interviewed. A questionnaire was designed to gather data of the following variables: health conditions, functional capacity, health services utilization, social support and preventive measures. Descriptive measures and chi square were utilized for the statistical analysis. Findings revealed a population composed mostly of women with a higher prevalence of health conditions than their male counterparts. More than half of the sample report visual problems, arthritis or hypertension. Almost a quarter had diabetes mellitus. The prevalence of these conditions was higher in the age group over 75, with the exception of diabetes. Thirty percent of the sample was classified as functionally dependent, condition that increased with the subject's age. Findings evidence the need of an early assessment in this population in order to intervene with potentially modifiable factors to prevent future disability and improve quality of life of the aged. PMID- 10730306 TI - [Health status and functional limitations in the elderly population of the University Region of Health of Puerto Rico: methodologic aspects and prevalence estimates]. AB - In order to plan the health services for the elderly population, it is necessary to quantify their health status and their functional capacity. In Puerto Rico, few epidemiological studies have been conducted regarding functional capacity and chronic diseases in the elderly population. One of the difficulties to undertake these studies is the high cost and risks to move this population for clinical exams, in addition to the methodology limitation of self-report in the elderly population. This study shows the use of logistic regression to estimate the prevalence of chronic diseases and functional capacity, when the observed data are not consisted with the planed sampling scheme. Four hundred and eighty-seven elderly persons (65 yr. and over) were interviewed in the municipalities of Canovanas, Carolina, Loiza and Trujillo Alto in Puerto Rico, using a cross sectional design. More than half of the elderly had visual problems (IC 95%: 54.8%, 63.8%), arthritis (IC 95%: 52.7%, 61.5%) and hypertension (IC 95%: 47.3%, 56.3%). In the case of hypertension, significant differences (p < 0.05) by sex were observed, where women reported a higher prevalence than men. One of the higher prevalences in the functional capacity status was with urine accidents. More than one-third of the population is estimated to have this problem (IC 95%: 34.7%, 43.4%). One-fourth of the population had limitations with going out for shopping (IC 95%: 23.9%, 31.9%) and using public and private transportation (IC 95%: 19.5%, 26.9%). We conclude that the applied methodology was consistent with the estimation presented in the literature and statistics from the Puerto Rico Health Department. However, it is necessary to continue assessing the design and analytical methodology, in order to undertake consistent and periodic evaluations of the elderly population. PMID- 10730307 TI - Variations in the survival probabilities of the PVC-protected red mangrove propagules: testing the encased replanting technique. AB - The EcoElectrica Mangrove Planting Project, a five-year voluntary effort, has the purpose of testing a recently developed mangrove planting technique at the EcoElectrica site in Penuelas, Puerto Rico. The goal of the project is to provide empirical validation to promote or improve the technique to be used in recovering mangrove ecosystems in Puerto Rico and United States. The research presented herein analyzed the information collected on the first two years of the project. The proportions of remaining casings and seeds per study zone were compared using the chi-square distribution. Zone 1 had the least pipes lost while Zone 4 had the most (p < 0.05). Forty-three percent of the seeds in Zone 1 remained in the casing, while 26% remained in Zone 2 (p = 0.03). Median growth rates of seeds per study zone showed that Zone 1 had the highest median growth rates. Survival analysis described the survival experience of the seeds, and differences in survival probabilities were compared with the log-rank test. Zone 1 seeds had a better survival experience compared to Zones 2, 3 and 4 (p < 0.0001). Survival probabilities for being free of spots were over 60% during the whole study period. No significant differences were observed in the survival experience with the use-or-no use of casing extensions (p = 0.40), and the use-or-no use of nursed seeds (p = 0.26). Differences in survival probabilities might be attributed to variations in wave energy, depth or substrate conditions. This hypothesis will be evaluated in the second phase of the study. PMID- 10730308 TI - [Women and HIV/AIDS: barriers and new challenges]. AB - Women have been placed at a vulnerable situation regarding the HIV epidemic. Recent advances in antiretroviral therapies have placed in evidence the gender disparities and the new challenges to overcome them. The mortality of AIDS has decreased dramatically in the United States and Puerto Rico as a consequence of new combination therapies. Still, women constitute the fastest growing group of AIDS cases. There are gender differences in access to treatment and care, economic income and social and personal power. Among women's barriers to care are the lack of knowledge about AIDS in women by health providers, the family responsibilities and the burden and fear of disclosure. The authors suggest the need for empowerment as strategy for attaining better health and improving the quality of life in women living with HIV. PMID- 10730309 TI - Adoption of a pharmacist conscience clause by Professional Associations and Boards of Pharmacy in the United States. AB - OBJECTIVE: To determine the number of professional associations and boards of pharmacy in the United States that have adopted or are considering to adopt a conscience clause as part of their codes of ethics, rules, laws or regulations. BACKGROUND: Pharmacists are often exposed to ethical dilemmas in their day-to-day practice and their response depends on a number of factors, including the personal beliefs and values of those involved. This has lead some professional associations to address whether their members have the right to refuse to participate in procedures which are contrary to their conscience or moral convictions. The outcome of these discussions is usually the development and adoption of a conscience clause. METHODS: A one-page self-administered questionnaire was sent by Fax to the highest ranking officer of 108 pharmacy organizations in the United States. RESULTS: Thirty-five completed questionnaires were received for a 32.4% response rate. In general, it was found that there is a lack of knowledge as to what a conscience clause is. Only two state associations and one board of pharmacy responded that they have a conscience clause as part of their code of ethics or regulations. Reasons given for not having a conscience clause included lack of interest and low priority. Nevertheless, four state associations, one national association and one board of pharmacy responded that they are considering developing a pharmacist conscience clause. More professional associations than boards of pharmacy expressed interest to obtain information about conscience clauses. CONCLUSION: Although many state associations and boards of pharmacy that responded do not have a conscience clause as part of their codes of ethics, laws or regulations, this survey shows an increasing interest to learn about it. This is expected as the pharmacist assumes increasing responsibility in patient care. PMID- 10730310 TI - Substituted judgement: should life-support decisions be made by a surrogate? AB - OBJECTIVE: To evaluate the utility of the substituted judgement standard in terminally ill patients by determining agreement between patients, family proxies and physicians. BACKGROUND: Several studies have addressed the utility of substituted judgement, showing conflicting data whether surrogates are accurate predicting patient's wishes. METHODS: Patients with acquired immunodeficiency syndrome, congestive heart failure, chronic liver disease admitted to the San Juan Veterans Affairs Medical Center in Puerto Rico from November 1997 to February 1998 were evaluated. A questionnaire presented three hypothetical situations on withholding and withdrawal of life-support and CPR. The percent agreement was used as a measure of concordance between choices made by physician, surrogate and patients. RESULTS: Twenty patients met inclusion criteria (5 chronic liver disease, 9 heart failure, 6 AIDS). Relatives had a higher percent of agreement as compared to physicians in all vignettes. Even though, none did better than chance in predicting patient's wishes (k < 0.4). There was a tendency for relatives not to provide a wanted life-support measure, and for physicians to provide an unwanted life-support measure. CONCLUSION: The poor agreement between patients and surrogates suggests that substituted judgement is not an accurate tool to make end-of-life decisions. These findings, although similar to previous published studies, are unique because the direction of discrepant responses is opposite to the findings of studies published elsewhere. These results could reflect religious, cultural and socioeconomic differences. PMID- 10730311 TI - Academic and ethical implications of health reforms based on managed care: some critical reflections. PMID- 10730312 TI - [Clinical relevance of periodic limb movements during sleep in obstructive sleep apnea patients]. AB - INTRODUCTION: The periodic limb movements disorder (PLMD) is frequently associated with the obstructive sleep apnea syndrome (OSAS), but the prevalence and clinical relevance of this association have not been studied in detail. OBJECTIVE: The objectives were to make a prospective study on the prevalence of PLMD in patients with OSAS, and correlate this association with clinical and respiratory parameters. PATIENTS AND METHODS: Forty-two patients diagnosed with OSAS, without clinical suspicion of PLMD, underwent a polysomnographic study. Clinical symptoms and signs were evaluated with an structured questionnaire, and respiratory parameters were obtained from the nocturnal study. RESULTS: Periodic limb movements were found in 10 patients (24%). There were no differences in clinical parameters between both groups (with and without periodical limb movements). However, respiratory parameters were significantly worse in patients without PLMD. CONCLUSIONS: PLMD is very frequent in patients with OSAS, and can contribute to worsen clinical signs and symptoms in these patients independently from respiratory parameters. PMID- 10730313 TI - [Economic costs of childhood epilepsy in Spain]. AB - INTRODUCTION: Health expenditure increases continuously in all developed countries. Common disorders, including epilepsy are responsible for most of the health budget. OBJECTIVE: To quantify the economic effect of childhood epilepsy in Spain by analyzing its major components. PATIENTS AND METHODS: We asked a number of neuropediatricians to complete to questionnaire including data from which to calculate, from a social aspect, the direct and indirect costs (medical and non-medical) of the last 12 months follow-up of children under 14 years old with controlled and uncontrolled epilepsy. RESULTS: During 1998, the average annual cost of children with controlled epilepsy was 308,352 pesetas and of children with uncontrolled epilepsy 823,635 pesetas, or 2.7 times more. The cost of the prevalence of epilepsy in Spain during 1998 was nearly 11,300 million pesetas with direct costs making up most of the total cost. CONCLUSIONS: The size of the economic aspect increases the impact of the disease on the child and his family. The different cost distribution in children and in adults with epilepsy makes early treatment necessary so as to permit lower indirect costs of the disease when the patient becomes adult. Pharmaco-economic assessment is also necessary to justify the use of newer anti-epileptic drugs which are considerably more expensive than the classical anti-epileptic drugs. PMID- 10730315 TI - [Causes of dementia in Maracaibo, Venezuela: a re-evaluation]. AB - INTRODUCTION AND OBJECTIVES: In a previous report we analyzed the causes of dementia in an autopsy population from four hospitals of Maracaibo, Venezuela. Our results showed that vascular dementia (VD) accounted for 86.7% of all clinically diagnosed demented patients, whereas no cases of Alzheimer's disease (AD) were neuropathologically demonstrated. Six years later, we undertook a similar study, in order to re-evaluate the frequency of the different causes of dementia in our country. MATERIAL AND METHODS: We studied 611 adult brains obtained from autopsies performed during the period 1992-1998; 39 of these came from clinically demented patients. RESULTS: Vascular dementia was disclosed in 33 cases (84.6%), five cases (12.82%) showed evidence of specific causes for dementia other than VD or AD, and in one of the cases (2.56%) the neuropathological changes were specific for AD. CONCLUSIONS: Our current results confirm the conclusions of our previous study and contrast with the vast majority of reports in the medical literature regarding the frequency of the different causes of dementia, except for studies from China, Japan and Russia that yielded similar findings as ours. At present, further investigations are being undertaken in order to come up with possible explanations for these differences. PMID- 10730314 TI - [Neurophysiological clinical study of 70 patients with type 2 spinocerebellar ataxia]. AB - INTRODUCTION: The prevalence of hereditary ataxia in Holguin, Cuba, is 43 per 100,000 inhabitants, but in some regions reaches 503 per 100,000 inhabitants, a figure never previously recorded in the international literature. OBJECTIVES: To evaluate the part played by non-cerebellar structures and vias in causing some of the clinical signs of the disorder by means of clinico-electro physiological correlation. PATIENTS AND METHODS: A neurological examination, peripheral nerve conduction studies and somato-sensorial evoked potentials of the median and posterior tibial nerves were done in 70 patients with type 2 spinocerebellar ataxia. RESULTS: The commonest clinical signs were ataxia on walking, dysarthria, dysmetria and adiadochokinesia. Correlation analysis showed that the variable most significantly correlated with the degree of ataxia and Romberg's sign was latency of the P40 component, whilst tendon reflexes were best correlated with H reflex latency. However, the duration of the disease was not found to have statistical correlation. CONCLUSIONS: These results suggest that degeneration of long peripheral nerves and sensory paths contributes to the degree of ataxia and other clinical signs. At present no correlation has been found between the duration of the illness and increasing electrophysiological changes, and therefore seems not to have a major effect on neurodegenerative mechanisms. PMID- 10730316 TI - [Neurology and the reform of the health care system in Colombia]. AB - INTRODUCTION AND OBJECTIVES: Despite the magnitude of the Colombian health system reform, introduced in 1993, changes in specialized medical practice have not been evaluated. This paper is the follow-up of a similar study on the practice of neurology done by the authors before the reform. MATERIAL AND METHODS: Of the 62 members of the Colombian Association of Neurology living in Bogota, 47 (76%) of them responded an anonymous survey inquiring on the characteristics of their medical practice and registering all the medical encounters during one week in October 1998. RESULTS: Two thirds of the total working time is devoted by neurologists to clinical work. Half of the neurologists in the sample have invested in diagnostic equipment. There was no significant change in the total number of patients attended during the week, as compared with the 1993 study, and the diagnostic profile was similar. The proportion of private patients was significantly lower, while patients from prepaid medical schemes increased. Patients belonging to the Obligatory Health Plan, designed to cover lowest income population, and to prepaid medicine are not distributed homogeneously throughout all ages. The first year of life is particularly uncovered by the Obligatory Health Plan, while prepaid schemes do not address the problems of individuals age 60 or more. CONCLUSIONS: There has been a significant reduction in private practice compensated by an increase in prepaid medicine. There is no evidence of increased coverage for neurological disorders. PMID- 10730317 TI - [Early diagnosis and the course of Alzheimer's disease]. AB - INTRODUCTION: The limits of normal aging are poorly defined and this is also true of Alzheimer's disease (AD), where the diagnostic criteria are quantitative and quite arbitrary. We ought also to mention the condition currently know as 'mild cognitive deterioration' considering this to be an illness with a high annual rate of conversion to dementia. AD is considered to be the main cause of dementia at the present time. It is defined as a neurodegenerative disorder of the central nervous system characterized by progressive deterioration of memory and other cognitive functions. OBJECTIVE: Firstly to differentiate a sick from a healthy person of the same age; determine the functions involved and to what degree and finally to discover its possible development by determining the factors which may predict its course, and determine the treatment, bearing in mind the values which will be useful in effectively controlling the drug. PATIENTS AND METHODS: The sample used was made up of 20 persons, 10 of whom complained of loss of memory whilst the remainder had no illness of any kind when they were examined. RESULTS: The results show statistically significant differences between the two groups. CONCLUSIONS: Neurophysiological tests give objective effective methodology for the diagnosis of AD in its early stages and for evaluation of the progress of the illness. This efficacy is comparable to that of other techniques of examination which are regularly used in our setting. PMID- 10730319 TI - [Malignant catatonia]. AB - INTRODUCTION: Malignant catatonia is thought to be due to a severely decreased central dopaminergic activity of a medical, psychiatric, toxic-pharmacologic or idiopathic origin. CLINICAL CASE: A 24 year-old man presented with a generalized tonic-clonic seizure of focal onset. Within the following 48 hours, he developed agitation and paranoid ideation, which evolved into a severe catatonic syndrome complicated by aspiration pneumonia requiring transfer to Intensive Care Unit (ICU). Complementary tests (CT scan MRI, CSF, blood and urine studies) were negative or could not provide an etiological diagnosis. Initial EEG showed theta and delta waves, originating in the right frontal regions which later generalized. He stayed in ICU for the following three months in a state of catatonic stupor with frequent medical complications. During this time, he was given 27 sessions of electroconvulsive therapy (ECT) that eventually led to a remission of the catatonic syndrome. He was discharged from hospital one month later, fully asymptomatic except for a residuary amnesia, with a final diagnosis of idiopathic malignant catatonia. CONCLUSIONS: The authors have reviewed the management of this syndrome, which can be challenging for the physician because of the wide array of possible etiologies and its potentially lethal nature. A favorable outcome can be expected when ECT is started early, provided the underlying pathological process is treatable and there are no structural lesions in the central nervous system. PMID- 10730318 TI - [In vitro study of the effect of ethanol on pyroglutamyl aminopeptidase activity in mouse synaptosomes under basal and stimulated conditions]. AB - INTRODUCTION AND OBJECTIVE: Pyroglutamyl aminopeptidase (pGluAP) is an omega peptidase which removes pyroglutamyl N-terminals residues from peptides and arylamidase derivatives. This activity is thought to be involved in the regulation of several physiological mechanisms on the central nervous system. pGluAP can modulate various susceptible endogenous substrates such as thyrotrophin-releasing hormone (TRH). It is well known that TRH plays an important role in the modulation of the behavioral changes induced by ethanol and others drugs. The aim of this work was to study the in vitro effects of ethanol (25, 50 and 100 mM) on the pGluAP activity and its ability for modulating the TRH. MATERIAL AND METHODS: pGluAP activity was measured in synaptosomes from cerebral cortex of mouse, using pyroglutamyl-beta-naphthylamide as substrate in basal and stimulated (K+ 25 mM) conditions, and in presence or absence of calcium on the buffer. RESULTS: In basal conditions, ethanol produced an inhibition of the pGluAP activity in presence or absence of calcium, being this inhibition non dose-related. However, the stimulation with K+ 25 mM did not produce a modification of pGluAP activity in presence of calcium, but produced a light increase in absence of it. Depolarization in presence or absence of calcium and ethanol produced an inhibition of pGluAP activity, which changed in function of the ethanol concentration used. CONCLUSIONS: Ethanol modifies pGluAP activity in basal conditions by a mechanism independent of calcium, but the changes observed after the stimulation with high K+ may be due to a calcium-dependent mechanism. These variations of pGluAP activity induced by ethanol, and their effects on their endogenous substrates, specially TRH, may be responsible for the behavioral changes associated to the alcoholism and mediated by TRH. PMID- 10730320 TI - [Intrathecal perfusion pump with baclofen in generalized dystonia]. AB - OBJECTIVES: We show the effectiveness of treatment with continuous intrathecal baclofen infusion in a case of hereditary generalized dystonia refractory to anticholinergics, tetrabenazine, pimozide, L-dopa, benzodiazepines and thalamotomy. CLINICAL CASE: A 26 years old female patient, when she was 11 years old began with torsion dystonia in her left feet, that progressively worsened to involve her entire body. She had painful spasms. She had four brothers, three of them with dystonia and one healthy. Her uncle grandfather had similar symptoms. Complementary explorations to reject secondary origin was negatives. She was treated with high and progressive dosages of anticholinergics, pimozide, tetrabenazine, benzodiazepines, L-dopa and thalamotomy without improvement. Underwent intrathecal baclofen test dosing, we used 25, 50, 100 micrograms/day, the last one with improvement during 10 hours. A pump was inserted with an initial dose of 220 micrograms/day. After pump insertion, baclofen dosage was gradually increased to 450 micrograms/day. She had a great improvement in right part of her body and less in her left body. Painful spasms had disappeared. CONCLUSION: We propose continuous baclofen intrathecal infusion pump for patients with severe torsion dystonia that not response to ordinal treatment. PMID- 10730321 TI - [The evaluation of cell proliferation in gliomas]. AB - INTRODUCTION: The homeostasis of tissues depends on a strict control of cell growth, differentiation and death. Several proteins, which are involved on the regulation of the cell cycle, can suffer diverse alterations and produce an uncontrolled cell proliferation and the genesis of a neoplastic process. The assessment of cell proliferation is an useful method applied to Neuro-oncology in order to know the behavior of gliomas. DEVELOPMENT: This work is focussed on the analysis of different methods, all of them employed to study the cell proliferation: immunostaining of proliferating cell nuclear antigen (PCNA) and Ki 67, DNA content and ploidy by flow cytometry, in vitro incorporation of bromodeoxyuridine (BrdU) and the identification of apoptotic cells. The study of the DNA by flow cytometry establishes a relationship between ploidy and the prognostic of gliomas. The assessment of PCNA provides us with objective data about the proliferative activity of gliomas. Both Ki-67 expression and BrdU incorporation are also useful methods in the study of gliomas. CONCLUSIONS: In short, the most malignant gliomas are characterized by a high frequency of aneuploidies and high PCNA, Ki-67 and BrdU labelling indexes. All of these described methods can be used as prognostic markers complementary to the classic criteria employed nowadays. PMID- 10730322 TI - [Bio-equivalence and generic drugs. Studies of bio-equivalence. II. Special situations. Reflections on problems which may arise with drugs habitually used in neurology]. AB - OBJECTIVE: To discuss some controversial aspects and special situations which should be considered when assessing the use of generic drugs, especially those frequently used in neurology. DEVELOPMENT: We discuss the difference between the concept of average bio-equivalence and individual bio-equivalence, and the implications the selection of one or other criterion may have in the treatment of individual patients with carbamazepine. We also discuss the peculiarities and problems concerning bio-equivalence in the case of drugs with non-linear pharmacokinetics, controlled-release formulations, racemic drugs with two or more stereo-isomers and when there are differences in the excipients of different formulations of the same drug. CONCLUSIONS: There are drugs in which it may be very difficult to show the bio-equivalence of a generic formulation because of its pharmacokinetic characteristics as occurs in the case of controlled-release drugs, the presence of active metabolites or differences in their enantiomers or even due to problems with the excipients used, although if the therapeutic range is wide and these variables are controlled in the studies done, the conclusions regarding bio-equivalence may be valid. PMID- 10730323 TI - [Application of magnetic resonance spectroscopy in the study of brain disease]. AB - INTRODUCTION AND OBJECTIVE: Magnetic resonance spectroscopy (MRS) is a non invasive technique used for the neurochemical study of the brain in vivo. The aim of this work is to review the main investigations that have focused on the study of cerebral pathology. DEVELOPMENT: Cerebral MRS studies started in 1983 but the maximum development has been in the 90's. The pathologies more investigated were Alzheimer's disease, multiple sclerosis, schizophrenia, and depression. Although several nuclei can be observed the most investigated are phosphor (P31) and hydrogen (H1). Nowadays proton MRS is the more frequently used technique. The low levels of N-acetyl-aspartate are a good indicator of neuronal loss. Their determination is complementary to the volumetric structural studies from magnetic resonance imaging. The peak of myo-inositol seems to be a neurochemical marker for the Alzheimer's disease. CONCLUSIONS: MRS have contributed to the increase of knowledge about the physiopathology of normal aging, degenerative processes, demyelinating and psychiatric diseases. Potentially can contribute to differential diagnose in Alzheimer's and Parkinson's diseases. Recently it has also been opened a new research potential in the field of the pharmacological treatment effects in discrete cerebral regions. PMID- 10730324 TI - [Genetics and Alzheimer's disease]. AB - INTRODUCTION: Alzheimer's disease is one of the 'plagues' of modern society. The number of people affected, directly or indirectly, and the huge cost in health resources are frequently discussed in the press. DEVELOPMENT: In recent years we have seen a 'scientific mini-revolution' involving this condition. Most of this 'revolution' is due to the 'success' of the genetic strategy for study of the condition and began with the description of mutations in the gene for amyloid protein, the main component of one of the lesions which shows the disease. Following this, many laboratories have contributed to our knowledge of this protein and the mechanisms which appear to be involved in the disorder. Some years later other genes were also found to be involved in the disorder and once again, many groups have managed to discover part of the process leading to this disease. Even so, the original aims have still not been achieved. The disease still cannot be detected early on, at the preclinical stage preferably and there is still no therapeutic strategy to detain or ideally to reverse the advance of the condition. CONCLUSION: It is to be hoped that the contribution of molecular genetics, which has been so striking to date, continues in the future with the application of new molecular and statistical techniques. PMID- 10730325 TI - [Multiple sclerosis: epidemiological-genetic studies in the population of Antioquia, Colombia. Disequilibrium of HLA DQ alpha]. AB - INTRODUCTION AND OBJECTIVE: Discrimination and quantification of the environmental and genetic components involved in developing multiple sclerosis (MS) have not been made. In order to discriminate these components we have ascertained affected individuals by MS belonging to the Paisa community from Antioquia, Colombia, a state localized in the tropical area of South America, to detect eventual linkage disequilibrium to HLA, locus DQ alpha, which could demonstrate the relevance of the genetic component. DEVELOPMENT: A contingence analysis among case-control HLA DQ alpha genotype distributions, by using Monte Carlo resampling method to solve small number sample, showed that there are significant differences between the two groups. We observe that HLA DQ alpha 1.1, 1.2 allele frequencies were higher in the cases than in the controls. Also, there was significant HLA DQ alpha 3 allele lower frequency (p < 0.05) in the cases than in the controls. CONCLUSIONS: Similar results have been described in other Caucasian populations living in non tropical areas. Before results could indicate that the Caucasoid populations genetic component implied in the susceptibility to MS have remained in Paisa community, whether the environmental component, being meaningful to develop MS. PMID- 10730326 TI - [Molecular genetic aspects of the susceptibility to develop idiopathic epilepsy]. AB - INTRODUCTION: There are more than 40 clinical types of epilepsy classified according to aetiology and/or mode of clinical onset. The term idiopathic epilepsy is reserved for cases with convulsions but no detectable structural lesions of the brain or neurological anomalies. DEVELOPMENT AND CONCLUSIONS: In spite of many studies confirming the importance of genetic factors in the occurrence of idiopathic epilepsy, these appear to be complex and probably involve a locus of variable expression or several loci with similar phenotype expression (epistaxis). Also, environmental factors have variable effects. In recent years the principal genes involved in susceptibility to develop epilepsy have been mapped. In this way one mitochondrial and three autosomic genes have been cloned as responsible for the development of certain forms of this disorder. Also several studies of genetic linkage have given evidence, sometimes inconsistent, regarding the influence of another five loci in the susceptibility to develop epilepsy (6p21.2, 6q23-25, 8q24, 8p, 10q). On occasions the same locus has been linked with different forms of epilepsy, and on other occasions one form of epilepsy has been shown to be linked to several loci. PMID- 10730327 TI - [Disorders of sleep in children: a vision from the primary care stand point]. AB - INTRODUCTION AND OBJECTIVE: Problems with sleep are common in paediatrics. Approximately 20-25% of the child population have some type of sleep disorder. The objective of this study was to study its course, and the commonest changes seen in childhood and to suggest methodology for its study at a Primary Care level. METHOD: We review the specific characteristics of sleep and its alterations in the paediatric age group, observing the variations in different parameters such as reduction in REM sleep. We define the concept of sleep disorder, differentiating it from a problem with sleep. We also review the different disorders, particularly sleep due to bad habits and anomalous conduct during sleep, including night terrors, nocturnal rhythms, static phenomena and respiratory changes during sleep. We assess the different studies which show a high incidence of such disorders in childhood and introduce a methodology of study in Primary Care based mainly on the clinical history and study of the circadian rhythm of the binomial child-family, suggesting two algorithms for clinical diagnosis and the indications of remission of a Paediatric Sleep Unit. CONCLUSION: The presence of certain problems with sleep is a normal phenomenon at a specific time of life. It is important to associate the different problems with the age groups of presentation, and to assess the rapid changes of maturation when studying a child with a sleep disorder. PMID- 10730328 TI - [Idiopathic hypersomnia of the central nervous system]. AB - Idiopathic hypersomnia of the central nervous system is a cause of excessive diurnal somnolence which affects 5-10% of the patients who attend sleep clinics for this reason. We describe three male patients who consulted for excessive diurnal somnolence. Nocturnal polysomnographic studies followed by tests for multiple latencies of sleep were done. In all cases there was confirmation of lengthening of the time of nocturnal sleep with normal phases of sleep and an increase in the number of sleep spindles in phase II. Similarly there was an average latency of sleep of less than 10 minutes and fewer than two phases of REM in the multiple latencies test. All patients improved with drugs stimulating vigil, two of them with centramine and the third with methilphenidate. We consider the clinical data the polysomnographic criteria which help to establish the diagnosis. PMID- 10730329 TI - [Childhood insomnia due to disorderly habits]. AB - A common disorder in children aged between 6 months and 5 years is childhood insomnia due to disorderly habits. The clinical characteristics of this condition are difficulty in going to sleep on their own and multiple nocturnal wakenings. It occurs in perfectly normal children who are seen to have difficulty in the normalization of their sleep-waking rhythm due to absence or weak application of external synchronizers. The only point on which we can act to enforce synchronization of the sleep-waking circadian rhythm is on the habits related to getting to sleep. To initiate this synchronization of sleep-waking rhythm, it is essential to create a ritual around the action of going to bed. The possibility of the child getting back to sleep during the many physiological wakenings he has during the night depends on this ritual. It is essential that the child be awake when he leaves the bedroom. The child must learn to go to sleep with external elements which are associated with sleep, and during the physiological wakenings during the night, he will reclaim the circumstances which he associates with sleep. If the child goes to sleep on his own, he will go back to sleep on his own when he wakes at night; but if he has gone to sleep in someone's arms or 'has been put to sleep' by rocking, he will want the arms or rocking again. Once the routine is completed, the parents will leave the room and should follow a pattern of waiting time, increasing his progressively, following techniques for the modification of conduct until the child manages to get to sleep on his own. PMID- 10730330 TI - [Monoaminergic selectivity of the antidepressive drugs and sleep: neurophysiological implications of depression]. AB - OBJECTIVES: To obtain better understanding of the neurophysiology of sleep and of depression, together with any relationship there might be between them. DEVELOPMENT: We review the mechanisms of action of the neurotransmitters involved in the sleep-waking cycle, and also the antidepressive drugs, concentrating on those with more selective mechanisms of action, and the selective serotonin re uptake inhibitors. CONCLUSIONS: It is well know that the disorders associated with sleep play a predominant part in the symptoms of depressed patients, and these may revert with antidepressant treatment. Antidepressants cause, often markedly, changes in the characteristics of sleep at the same time as they improve the depressive symptoms. Thus, knowing the basis of the mode of action of antidepressive drugs, not only does the understanding of the neurophysiology of sleep progress, but also the understanding of sleep. PMID- 10730331 TI - [Brief history of dreams]. AB - Throughout history dreams have played a crucial role. Dreams have inspired great works of art, solved scientific problems and, because of the premonitory value attached to them, have influenced transcendental decisions. This paper reviews some of the dreams that have been a part of the world's literature and historical tradition. PMID- 10730332 TI - [Cerebral amyloid angiopathy: forms of presentation]. PMID- 10730333 TI - Intervention trials. PMID- 10730334 TI - The role of statisticians in intervention trials. AB - The planning, conduct, analysis and reporting of randomized trials to reliably evaluate an intervention create a complex intellectual and logistical endeavour. Success generally requires active collaboration of a well-trained, experienced and committed statistician. A good intervention trial asks an important question, gets a reliable answer and is honestly reported. Many trials fail on one of these components and some of these failures could have been avoided with better statistical involvement. Many subject matter investigators do not understand how to effectively collaborate with statisticians. The objective here is to outline some of the important responsibilities of the statistician in intervention trials. PMID- 10730335 TI - Individually randomized intervention trials for disease prevention and control. AB - It is argued that randomized, controlled trials should fulfil a critical role in the identification of practical approaches to the prevention and control of chronic diseases. Because of the great public health potential of chemopreventive and behavioural approaches to chronic disease prevention there is need for a major interdisciplinary scientific effort aimed at intervention development. Because of the cost and duration of controlled trials to evaluate specific interventions there is a need for well-conducted feasibility, pilot and intermediate outcome trials, to inform and to justify corresponding full-scale trials having clinical disease outcomes. Compared to therapeutic trials, prevention trials need to have a greater emphasis on overall benefit versus risk assessment. Such trials need to be large enough, and of sufficient duration, to yield powerful tests of key hypotheses, and informative benefit versus risk summary statements. These requirements have a range of implications for intervention trial design, conduct, monitoring and reporting, which are reviewed and discussed. The clinical trial component of the ongoing Women's Health Initiative provides illustration throughout this discussion. PMID- 10730336 TI - Estimating the population impact of an intervention: a decision-analytic approach. AB - The aim of this paper is to highlight the role for decision analysis in assessing outcomes of medical interventions at a population level. The basic steps of decision analysis are introduced and an illustrative hypothetical preventive intervention is examined. Specific modelling challenges that arise when estimating the population impact of an intervention are described and each is accompanied by an example. Decision analysis can provide useful information for health policy decision makers by identifying the intervention(s) with the largest beneficial impact on health over a wide range of assumptions. In addition, by focusing attention on the parameters with the greatest influence on projected outcomes, decision analysis can aid in identifying critical areas for future research. PMID- 10730337 TI - StatView for Windows, Version 5.0 PMID- 10730338 TI - [Preliminary results of antibiotic resistance monitoring in the Netherlands]. AB - Qualitative tests are used to monitor antimicrobial resistance in bacteria of animal origin in the Netherlands. Quantitative information on trends in resistance is thus not obtained. Moreover, in general a limited panel of antibiotics is tested. The present study describes resistance in zoonotic food borne pathogens Salmonella, Campylobacter, and Escherichia coli O157 isolated from human clinical cases and from faeces of healthy food animals in 1998 and 1999, as determined with quantitative susceptibility tests. The resistance of the indicator organisms E. coli and Enterococcus faecium isolated from faecal samples of broilers and pigs randomly sampled at slaughterhouses was also determined. For this end, faecal samples from veal calves were sampled in 1996 and 1997 at the three main Dutch veal calf slaughterhouses. In 1998 only a limited number of faecal samples of veal calves were taken at farms. For E. coli and Salmonella the following antibiotics were tested: amoxicillin, amoxicillin-clavulanic acid, piperacillin, cefotaxime, ceftazidime, imipenem, gentamicin, doxycycline, trimethoprim, trimethoprim/sulphamethoxazole, ciprofloxacin, chloramphenicol, florfenicol, carbadox, and flumequine. For E. faecium the following antibiotics were tested: amoxicillin, amoxicillin-clavulanic acid, chloramphenicol, doxycycline, erythromycin, vancomycin, teicoplanin, streptomycin ('high level' > 2000 mg/ml), gentamicin ('high level' > 500 mg/ml), ciprofloxacin, bacitracin, flavofosfolipol, salinomycin, quinupristin-dalfopristin, virginiamycin, tilmicosin, avilamycin, and everninomycin. For Campylobacter the following antibiotics were tested: erythromycin, doxycycline, gentamicin, carbadox, flavofosfolipol, ciprofloxacin, trimethoprim/sulphamethoxazole, amoxicillin, and metronidazole. PMID- 10730339 TI - [In vitro activity of 12 antibiotics used in veterinary medicine against Mannheimia haemolytica and Pasteurella multocida isolated from calves in the Netherlands]. AB - Results of susceptibility tests of clinical isolates of animal pathogens are periodically summarized and reported by the Animal Health Service. However, these results are based upon qualitative test methods. In the present paper results of quantitative susceptibility tests of twelve antibacterial agents against Mannheimia haemolytica (MHA) and Pasteurella multocida (PMU) isolated from Dutch calves in 1996 and 1997 are presented. Minimum inhibitory concentrations of amoxicillin, ceftiofur, tetracycline, trimethoprim-sulphamethoxazole, tilmicosin, neomycin, gentamicin, spectinomycin, flumequine, enrofloxacin, chloramphenicol and florfenicol were determined. No resistance was detected for ceftiofur and florfenicol. Three strains had an intermediate susceptibility to tilmicosin. The resistance percentages of MHA and PMU for neomycin, gentamicin, spectinomycin, flumequine, enrofloxacin, and chloramphenicol varied from 2% to 16%. Higher resistance percentages (16%-53%) were observed for amoxicillin, tetracycline, and trimethoprim-sulphamethoxazole. The MIC breakpoints used to determine whether a strain is susceptible, intermediate, or resistant are arbitrary and discussed in this paper. PMID- 10730340 TI - [Blood surveillance in Europe]. AB - The organisation of haemovigilance in Europe is quite heterogeneous between the different member states of the European Union, which requires an exchange of information between the different systems through the creation of the European Haemovigilance Network. This article presents, firstly, the various regulatory and administrative levels where the European policy in this matter is decided, and secondly, the different situations of haemovigilance organisation in the main European countries. PMID- 10730341 TI - [Immunologic risk analysis of blood transfusion: 1991-1998]. AB - The immunologic risk associated to erythrocyte transfusions is bound to the polymorphism of blood group systems and to the respect of blood transfusion regulations. The results of three studies are presented, which were carried out respectively by the French Society of Blood Transfusion, the National Institute of Blood Transfusion and the National Haemovigilance Network. Two hundred and twenty-seven cases of immunologic accidents are analysed using the Kaplan's interpretation model and the traditional method of process analysis. The results show three critical factors in the occurrence of this type of incident: the relevance of the clinical examinations prescribed, the way in which the biological results are taken into account, and the relationship/exchange of information between private and public hospitals, and blood transfusion centers. PMID- 10730342 TI - [Blood transfusion and bacterial risk]. AB - Initial hemovigilance data confirm the incidence and severity of transfusion reactions due to the bacterial contamination of blood components (TRBCs). With 18 deaths reported through the French hemovigilance network over the past five years, bacterial risks represent one of the major immediate complications of BC transfusion. BC contamination may lead to more or less severe TRBCs, depending on their origin: bacteria growth, the BC itself or unknown origin. Although the rate of donated blood or BC contamination is known (0.5% and 0.05%, respectively), it is still difficult to assess the actual incidence of TRBCs, as it is difficult to identify and relate them to transfusion. Likewise, a better knowledge of bacteria, symptoms, and outcome is required to improve prevention methods. Better prevention can reduce BC contamination and proliferation of bacteria at each stage of blood transfusion. Methods of detecting BC contamination are still under investigation. Through continuous education of hemovigilance participants in identifying and dealing with TRBCs, as well as drawing up procedures to perform inquiries and specific bacterial analyses, case reporting can be further improved, in order to achieve more efficient prevention. PMID- 10730343 TI - [Controlled viral risks]. AB - The prevalence of virological markers in blood donors has been continuously decreasing since the implementation, as soon as they were available, of serological screening methods. In 1998, the prevalences (per 10,000 donations) were 0.17 for antibody to HIV, 0.08 for antibody to HTLV, 2.23 for HBs Ag and 2.52 for antibody to HCV. The values are of course higher in new donors when compared to regular donors: approximately five-fold for HIV, 50-fold for HCV and 300-fold for HBs Ag. The remaining major questions concern the residual risk due to infectious donations which could escape the preventive measures. It seems evident that the major risk is imputable mainly to donations collected during the window period. During the 1996-1998 period, the residual risk for HIV was one out of 1,350,000 donations, 0 for HTLV, one out of 375,000 for HCV, and one out of 220,000 for HBV. A few cases of 'immuno-silent' patients have been reported. They remain exceptional. The first data collected after implementation of Nucleic Acid Technology (NAT) confirm the very low residual risk. The molecular epidemiology of the concerned viruses applied to the evaluation of screening assays highlighted the impact of the genetic diversity on the efficiency of these assays. This is particularly evident for HIV and HBV. The recent introduction of leucodepletion probably brought an important contribution in diminishing the risk of transmission of leucotropic viruses such as cytomegalovirus, Epstein-Barr viruses, human herpesviruses 6 and 7, and HTLV. If the purification process of plasma derived medicinal products including inactivation procedures permits confidence in the elimination of infectivity due to enveloped viruses, the detection of nucleic acid sequences derived from naked viruses in plasma pools may greatly contribute to their safety. PMID- 10730344 TI - [Transfusion safety: emergent or hypothetical risks]. AB - Three categories of emerging risks are studied: 1) A new variant of Creutzfeld Jakob disease, different from its sporadic form; limited to the British isles (48 of 51 cases), it affects younger patients, and has a higher duration with a predominance of psychiatric symptoms. Environmental risk factors include a previous stay in the British isles and oral transmission via contaminated food. No link has been made evident between blood component (BC) transfusion and occurrence of the disease. A potential risk exists if its agent is found in blood and peripheral lymphoid tissues and if buffy coat from infected animals has been inoculated intracerebrally. Since 1993, prevention measures have been taken: exclusion of donors with a potential risk as well as transfused donors, systematic leukocyte reduction and implementation of disease surveillance. Excluding donors after a several month-stay in the British Isles is being discussed. 2) Novel hepatitis viruses. Hepatitis G virus (HGV) has been detected in 2-4% of blood donors. Ten percent of patients with chronic non-A-E hepatitis are HGV RNA positive. The incidence of HGV infection is higher than expected from PCR studies. HGV has a high prevalence in the world. Novel DNA non-enveloped virus (TTV) has a normal distribution. Its prevalence varies from 2 to 80%, depending on the country. Although it has not been shown to be aggressive for the liver, prolonged follow-up is required. 3) Human herpes virus 8 (HHV8) is associated with Kaposi's sarcoma in 80% of cases. Its prevalence (0-20%) varies depending on the country. Kaposi's sarcoma has never been reported after BC transfusion. PCR-based viral DNA searches have yielded negative results in 19 poly-transfused subjects. Continuous monitoring is required for recipients at risk (e.g., immunosuppressed). In response to a possible health risk, emerging risks govern the "Precaution Principle", so difficult to implement. PMID- 10730345 TI - [Agent liability: a law in flux]. AB - This communication aims to show the impact of the recent reform of the French sanitary organization, and of the evolutions of the French law of liability. Contrary to what it seems, the important reform of the safety system has less immediate consequences than the change in laws on the way in which transfusion professionals perceive their juridical situation. PMID- 10730346 TI - Development and validation of a genotyping kit for the eight principal human platelet alloantigen systems. AB - Thrombocytopenia in newborns is often due to maternal alloimmunization against platelet alloantigens of the foetus which have been inherited from the father and are absent in the mother. Our aim was to develop a "ready-to-use" typing kit based on polymerase chain reactions, using sequence-specific primers for rapid and simultaneous genotyping of the eight principal human platelet alloantigens. The typing technique uses two specific primer pairs for each bi-allelic system and a monomorphic primer pair as amplification control, with a single temperature cycle programme and identical PCR stringency conditions for all pairs of primers. This kit allows typing of blood samples of small volume within three hours after reception. Validation criteria are essential to check the reliability and specificity of the test, and DNA controls carrying the targeted HPA alleles must be obtained from typed individuals or created in vitro by PCR. PMID- 10730347 TI - [Antibodies against hepatitis B virus core antigen among blood donors]. AB - The prevalence of antibodies to the hepatitis B virus (HBV) core antigen (anti HBc) and the risk factors are evaluated in different blood donor groups. In 1998, on 12,456 first donations, 163 (1.31%) were positive with the two anti-HBc screening tests. Samples were from 69 women (42.3%) and 94 men (57.7%). Three subjects had no anti-HBs but were anti-HBc IgM negative. Forty (24.5%) donors were born in another country than France, and the majority (25 donors, 62.5%) were from North Africa. HBV vaccine had been previously given in 14 subjects (8.6%). Eight (4.9%) had hepatitis before the first donation. Trips in endemic areas (Africa and Asia) were taken by 26 subjects out of 76 donors with follow up. Two (2.6%) had been previously transfused and six (7.9%) had contact with HBV infected people. Among 78,033 repeat donations, 26 were positive with the two Anti-HBc screening tests (0.033%). Sixteen were negative after a second test and were probably false positive. Among the ten last donors, nine were Anti-HBs positive. One had anti-HBc IgM and had been recently infected by HBV. The prevalence of Anti-HBc in first-donation persons remains low. Trips in endemic areas and contact with an HBV-infected subject are the most frequent risk factors. Lastly, HBV seroconversion in repeat blood donors is a rare event. Anti HBc screening in transfusion remains limited. PMID- 10730348 TI - [The platelet/leukocyte ratio in red blood cell concentrates is an essential indicator of leukocyte removal filter efficiency which limits their use]. AB - Performances of filter systems for leucocyte removal (Pall BPF4 and Sepacell RZ 200 B) have been studied following the preparation of red blood cell concentrates (RBCCs) obtained either by a soft-spin centrifugation at 2967xg (n = 438) or by a hard-spin centrifugation at 5275xg (n = 187). After deleucocytation, 43, i.e., 9.8% of RBCCs obtained following a soft-spin centrifugation contained a number of residual leucocytes higher than 1 x 10(6) leucocytes/RBCC which represents the legal upper limit in France, whereas RBCCs obtained following a hard-spin centrifugation always contained a number of residual leucocytes lower than this limit. Our results strongly suggest that the quality of deleucocytation depends on an adequate ratio of platelets to leucocytes, which favours the capture of leucocytes by filters. In fact, this ratio was higher in RBCCs obtained following a hard-spin centrifugation. Our results demonstrate that the use of these filters does not allow the removal of leucocytes complying with the legal limit of leucodepletion for all preparations of RBCCs. PMID- 10730349 TI - [The virus isolated from patient TT (TTV): still an orphan 2 years after its discovery]. AB - TTV is the acronym for a virus isolated two years ago from a patient whose initials were T.T. It is a naked virus probably belonging to the Circoviridae family. TTV has a particle size of 30-50 nm and possesses a single-strand circular DNA. Epidemiologic data are derived from studies looking for the viral DNA by the polymerase chain reaction (PCR). Important differences between early and recent studies appear to be due to the use of PCR assays based on primers located in different regions of the genome. Based on the most recent studies, the prevalence of TTV infections seems very high in the general population. TTV is present in the feces and would be transmitted through the fecal-oral route. It appears to be a ubiquitous virus, also present in various animal species, from chickens to chimpanzees. No association to any pathology has been identified so far, and TTV infection does not have a significant effect on liver disease. PMID- 10730350 TI - [Problem-solving in immunohematology: interpretation of ABO typing and its difficulties]. AB - Practice in immunohematology is replete with complex problems that require practitioners' problem-solving performance. In immunohematology, the acquisition of the reasoning process and necessary skills for making clinical decisions is based on teaching problem-solving strategies which potentially reduce errors and improve patient outcome. We discuss the recognition and resolution of the common causes of discrepancies in ABO typing results using problem-solving strategies. PMID- 10730351 TI - [Materials surveillance, a surveillance performed on the use of medical devices]. AB - One of the missions of the Agence Francaise due Sang was to set up a haemovigilance system based on a national network of surveillance and alert, for the whole blood chain from blood collection to receivers' follow-up. This system is now operational and contributes with efficacy to the safety of blood transfusions (see: A quoi sert l'hemovigilance? Transfus Clin Biol 1998; 5: 415 21.). The French law No. 98-535 dated July 1st 1998, whose enforcement orders were published in March 1999, is the legal foundation of the system. In this article the national and regional structures of the national system of sanitary safety are described, and the main guidelines of material vigilance, the last element of this system, is presented. PMID- 10730352 TI - [Chronic lumbago-ischialgia syndrome from the psychosomatic viewpoint. Farewell to the stress reaction concept]. PMID- 10730353 TI - [Predictors for complications and unsatisfactory outcome in lumbar intervertebral disk operation]. PMID- 10730354 TI - [Ehlers-Danlos syndrome]. PMID- 10730355 TI - [The Journal of Orthopedics at the beginning of year 2000]. PMID- 10730356 TI - [Reliability of manual medical examination techniques of the cervical spine. Study of quality assurance in manual diagnosis]. AB - OBJECTIVE: Chiropractic techniques are of particular importance for the examination of the cervical spine. The aim of this study was to assess interexaminer reliability of examination techniques of the cervical spine in subjects with and without musculoskeletal distortions of the neck. The interrater method was used with five independent examiners. METHOD: Twenty patients suffering from neck diseases and 20 asymptomatic subjects of similar age and gender were randomized and assessed by five examiners blind to patient histories. Statistical analysis was carried out using multiple logistic regression and the calculation of kappa. RESULTS: Compared to asymptomatic subjects, patients experienced pain significantly often when pressure was applied to the cervical zygapophysial joints and the superficial neck muscles (p < or = 0.05 and p < or = 0.01). In addition, segmental function tests induced kinesialgia significantly more often in patients than in asymptomatic subjects (p < or = 0.05 and p < or = 0.01). A significant relationship was not found between the patient's health status and the findings from muscle palpation and functional examination of the motion segments. The assessment of agreement within examiners beyond chance had to be calculated, aside from for few exceptions, little to moderate (0.2 < kappa < or = 0.6). CONCLUSIONS: Chiropractic techniques are an essential part of every examination of the cervical spine. The clinical impact has not been scientifically established until up to now. Based on our findings and literature, we conclude that interexaminer reliability of manual diagnosis in the examination of the cervical spine should be improved by standardizing the examination process and setting guidelines for documentation and evaluation criteria. Controlled and frequently repeated training sessions also contribute to the reproducibility of findings from manual examinations. PMID- 10730357 TI - [Effects of elastic lumbar belts on the effect of a muscle training program for patients with chronic back pain]. AB - QUESTION: Aim of the study was to evaluate the influence of elastic lumbar belts on the effect of muscle training for patients with low back pain. METHODS: 97 male subjects aged from 23 to 42 years with and without low back pain participated in the investigation. The low back pain patients (n = 63) were randomized into a training group without and a training group with elastic lumbar belts and a control group. The subjects with healthy backs (n = 34) were divided age-matched into a training group with elastic lumbar belts and a control group. The three training groups took part in a muscle strenghtening program over 8 weeks. The control groups did not receive any alternative physiotherapeutic treatment. All groups were tested at the beginning, after 8 weeks and further 6 months later. RESULTS: The data obtained for the control groups remained virtually unchanged over the period of investigation. However, a significant increase of the muscle flexibility of the lower limbs could be proved for the training groups. Furthermore the coordination between the lumbar spine and pelvis when flexing the trunk deeply forward was more leveled out for the training groups with patients suffered low back pain. The results confirmed a reduction for pain severity and for limitations in activities of daily living as well. The modifications for the criteria investigated were significant stronger for the training group with patients using the elastic lumbar belt. CONCLUSIONS: The effectiveness of the muscle strengthening program for patients with low back pain could be improved significantly by means of the elastic lumbar belt as an applicable therapy instrument in the functional rehabilitation of spinal injuries. PMID- 10730358 TI - [Effect of spongiosa density on load bearing of the lumbar spine.A finite element analysis]. AB - OBJECTIVE: Different parts of the human spine have to accomplish different functions. But little is known about the exact distribution of forces within the spine and whether this is influenced by bone quality. The purpose of this study was to predict fields and extent of greatest load in compression in a human lumbar spine motion segment using a finite element model. METHODS: A three dimensional isotrophic finite element model was generated using the software ANSYS 5.4. Spinal loading was performed in axial compression (600 N). The model was validated by biomechanical analysis using 12 human spinal segments that were loaded with the same forces. Prediction was done with different E-modulus for cancellous bone, representing a wide range of bone quality between osteoporotic and strong bone quality. RESULTS: Load-sharing was influenced by bone quality: the weaker bone quality is, the higher is the extent of load that is passed through the posterior part of the spine. CONCLUSION: This finite element model predicts that load-sharing in a lumbar spine segment with decreased bone mineral density is different from that in healthy segments. A decrease of bone mineral density is resulting in an increase of load that is passed through the posterior part of the lumbar spine. Keeping in mind the simplifications of this model, the results may influence surgical treatment of patients suffering from osteoporosis or osteolytic destructions of the lumbar spine. PMID- 10730359 TI - [Halm-Zielke instrumentation in idiopathic scoliosis. Results in 25 consecutive patients with a minimum follow-up of 2 years]. AB - AIM OF THE STUDY: Halm-Zielke Instrumentation (HZI) was developed to eliminate the disadvantage of VDS-Zielke in terms of lack of primary stability and in order to simplify sagittal plane control. Since 1993 we study within a prospective clinical trial, whether HZI fulfills these demands. METHODS: HZI is an anterior double-rod system with a two screw per vertebral body fixation. The longitudinal components consist of a threaded VDS-rod and a solid rod, which are attached to a hinge-conducted lid plate. 25 consecutive patients with idiopathic scoliosis and curves ranging from 36 degrees to 92 degrees were treated with HZI. The follow-up period ranges from 2 to 4 years. RESULTS: Correction of the frontal plane within the instrumented levels averaged 71.4% and 70.4% postoperatively and at follow up, respectively. Derotation averaged 51.7% and mean correction of the tilt of the lowest instrumented vertebra was 69.5% at final follow-up. Thoracolumbar kyphosis was present in 7 patients and always completely corrected. One implant related complication, a screw breakage 12 months postoperatively without adverse effects was noted. There was no case of pseudarthrosis. All patients were mobilized without any additional external immobilization in terms of a brace or cast and were allowed to go swimming for physio-therapeutical purposes immediately after wound healing. CONCLUSION: This study proves that HZI is a primary stable implant to perform the Ventral Derotation Spondylodesis. The VDS typical implant related disadvantages are eliminated. With this the period of rehabilitation is shortened by many months due to avoidance of cast and brace treatment. PMID- 10730360 TI - [Effect of extracorporeal shockwave administration on biological behavior of bone cells in vitro]. AB - AIM: Osteo-destructive effects as well as stimulation of bone growth are often described after extracorporeal shock-wave application (ESWA). A correlation between the applicated energy and outcome is assumed. The purpose of this study was to analyze, whether ESWA has an influence on growth and proliferation of bone cells in vitro. METHODS: Human cancellous bone was cultivated until a confluent cell layer had grown. 5 x 10(5) bone cells were transferred into U-formed tubes, centrifuged and covered with cultivation medium. Thereafter ESWA was applicated in a standardized manner. Number and intensity of ESWA were systematically combined (500, 1000, 2000 and 0.15, 0.26, 0.51 mJ/mm2 energy flux density--EFD, respectively). Ten samples per combination were analyzed. In addition, we examined an untreated control group. Survival, metabolism (alkaline phosphatase activity), type I collagene-synthesis as well as proliferation were determined. RESULTS: There is a decrease of survival after ESWA depending on the number of impulses and intensity (dose-dependent survival). Cell survival was significantly reduced to 40% after 2000 impulses with high energy rates. Metabolism of surviving cells is not altered by ESWA in comparison with controls. Depending on the number but not on the energy of impulses the type I collagene-synthesis of surviving cells decreased. Between the 3rd and the 8th day after ESWA proliferation increased significantly in cell cultures treated with 2000 impulses of medium or high energy rates. CONCLUSIONS: There is a direct relation between dose and effect for ESWA: A minimum number of impulses and EFD is needed to cause effects on bone-cells. This mainly depends on the numbers of impulses. Destruction of cells is a short-time effect of high shock-wave-doses, a medium term effect is a cell-stimulation. PMID- 10730361 TI - [Analgesic effect of low energy extracorporeal shock waves in tendinosis calcarea, epicondylitis humeri radialis and plantar fasciitis]. AB - PURPOSE OF THE STUDY: Is there a pain reduction at the application site after extracorporeal shockwave application for tendinitis calcarea, epicondylitis radialis and plantar fasziitis? METHODS: In a prospective study 85 patients were observed. Shockwave application was performed three or five times using low energies (0.09-0.18 ml/mm2). Before and after shockwave application pain was evaluated using SF-36 score and Visual Analog Scale (VAS). RESULTS: After 5 months for all three indications a significant improvement of the pain situation could be reached. Patients with plantar fasziitis demonstrated the highest decrease of pain, followed by tendinosis calcarea and epicondylitis radialis. The number of applications had no influence to the clinical result of the ESWT. RELEVANCE: In the present study the analgetic effect of ESWT after repeated low energy application was described for the standard indications. PMID- 10730362 TI - [Total endoprosthetic management of the hip joint after failed osteosynthesis of para-articular hip fracture]. AB - OBJECTIVE: Treatment of fractures of the proximal femur by open reduction and internal fixation is prone to complications and frequently requiring secondary joint replacement. The aim of the present study was to examine the results of total hip arthroplasty as a salvage procedure for failed internal fixation of femoral or acetabular fractures. PATIENTS AND METHODS: We retrospectively studied 145 patients who had undergone 146 total hip arthroplasties for failed internal fixation of femoral (n = 135) or acetabular fractures (n = 11). Mean follow-up time after insertion of the hip endoprosthesis was 7.1 years (1.5-14.7 years). Patient evaluation included a history, clinical examination, and standard radiographs. RESULTS: Twenty-eight patients had died, and 18 patients were lost to follow-up. Kaplan-Meier analysis with revision of the implants as the end point demonstrated 85 percent survival after ten years. Using the Merle d'Aubigne rating system, we found good or excellent results in 85 percent of the cases. However, only 73.9 percent of the patients were satisfied with their result, and 40.9 percent still showed a positive Trendelenburg gait at follow-up. The perioperative mortality was 2.7 percent. Surgery-related femoral fractures or fissures were observed in 18 cases, and deep infections in four. CONCLUSIONS: In comparison with data of patients who had undergone primary total hip arthroplasty for osteoarthritis in our department, the results reported here after secondary hip replacement are clearly inferior. Nonetheless, alloarthroplasty of the hip still is the most effective procedure after failed internal fixation for acetabular or proximal femoral fractures. PMID- 10730363 TI - [Migration analysis of cemented Muller polyethylene acetabular cups versus cement free Zweymuller screw-attached acetabular cups]. AB - OBJECTIVE: Is the cementless Zweymuller hip cup superior to the cemented Muller cup? METHOD: This article presents a radiographic analysis of 25 cemented Muller acetabular cups versus 22 cementless Zweymuller cups using the Einbildrontgenanalyse (EBRA), a software tool for radiographic measurement of acetabular cup migration. In addition, we determined the effects of the cup anteversion and inclination, the polyethylene wear, the lateral bone coverage of the acetabular cup, the position of the center of rotation, and individual factors on the incidence of cup migration. RESULTS: The incidence of cup migration was 64% in the cementless group and 48% in the cemented group after a mean follow-up of 6 years. The average migration rate was 0.33 mm/a for cementless Zweymuller cups and 0.38 mm/a for cemented Muller cups. Cup anteversion and inclination showed no effect on the incidence of cup migration. The combination metal-polyethylene (0.17 mm per year) demonstrated a significantly higher wear rate in comparison to the ceramic-polyethylene combination (0.11 mm per year). Incompletely lateral covered cups demonstrated a significantly higher incidence of cup migration. Cranial or medial deviations of the center of rotation up to 5 mm are tolerable, in contrast to caudal or lateral deviations that lead to a significantly higher incidence of cup migration. CONCLUSION: The superiority of the cementless Zweymuller cup was not observed. We recommend a complete lateral bone coverage of the hip cup. Cranial and medial deviations of the center of rotation up to 5 mm are tolerable. In the present study the polyethylene wear of the ceramic-polyethylene combination was significantly less as compared with the metal-polyethylene combination. PMID- 10730364 TI - [10 year results with a cemented fine-grit-blasted titanium-aluminum-vanadium hip endoprosthesis shaft]. AB - PURPOSE: Are the results of cemented titanium hip arthroplasty as bad as described in literature? We present the 10-year results of a cemented mild grid blasted Titanium-Aluminium-Vanadium Stem in Total-Hip-Arthroplasty. METHODS: The clinical and radiological reexamination was documented in standardized questionnaire in which several scores were integrated. All X-rays were stored and analysed by a special hardware and software computer system. RESULTS: 35% of all patients could be reexamined both clinically and roentgenologically. 71 patients were male (mean age 56.1 years) and 130 female (mean age 59.3 years). The average follow-up period was 11 years and 4 months. The mean Merle d'Aubigne hip score increased from 10.5 to 16.0 points at the follow-up evaluation. Radiolucent lines were according to the zones of Gruen seen in 1% to 22% depending on the zone and the size of the femoral component. 18 reoperations of the femoral component (2.9%) had to be performed. The survivorship analysis (Kaplan-Meier) showed a 95.4% survival of the femoral component after 10 years. CONCLUSION: Our findings with a cemented mild grid blasted Titanium-Aluminum-Vanadium stem demonstrate excellent results unlike it was reported for cemented femoral components of similar alloy but different design and cementation philosophy. PMID- 10730365 TI - [Complete dislocation of the polyethylene inlay after internal hemipelvectomy and individually fitted tumor endoprosthesis]. AB - We report about an exceptional complication after internal hemipelvectomy and replacement of the defect with a custom-made endoprosthesis. A complete luxation of the PE-inlay out of the metal cup occurred. Radiologically we assumed a luxation of the femoral head in dorsal direction. Revision after a failed attempt of closed reposition showed a complete luxation of the inlay, which was caused by deficient fixation in the acetabular component. The absence of a contrast wire in the PE-inlay delayed the right diagnosis and made it difficult to find the dislocated inlay in the large wound. PMID- 10730366 TI - [Effect of a rasp surface on cement penetration in paired cadaver femurs]. AB - AIM: The purpose of this investigation was to study two different broach surface designs with regard to cement penetration into human cancellous bone. METHODS: In a cadaver study 15 paired human cadaver femora were prepared using broaches of identical geometry but different surface characteristics. All left femora were prepared using chipped toothed broaches, all right femora using diamond shaped broaches. Cancellous bone was irrigated with 1 liter pulsed lavage. The specimens were imbedded in specially designed pots. Bone cement was applied in a retrograde manner and subjected to a standard pressure protocol with a constant force of 3000 N. Radiographs were taken and horizontal sections were obtained at predefined levels using a diamond saw. Microradiographs were taken and analyzed using image analysis to assess cement penetration into cancellous bone. RESULTS: Pressure curves recorded during cement pressurisation were comparable. The microradiographic evaluation revealed no significant morphometric differences in the different groups with regard to cement penetration into cancellous bone. These findings were similar in all sections obtained. CONCLUSIONS: A standardized model was developed allowing comparison of cement penetration into cancellous bone depending on bone preparation. In the presence of pulsed lavage there is no significant influence of broach surface characteristics on cement penetration into cancellous bone of the upper end of the femur. PMID- 10730367 TI - [Knee flexion after rotational knee endoprosthesis]. AB - In the presented study the question was evaluated if--after rotational knee arthroplasty--range of motion must be restored at discharge or if range of motion can also be restored with delay during the first postoperative year. METHOD: A retrospective study was performed on 45 patients. These had been rehabilitated with a specific therapeutical concept which did not include early restoration at discharge of complete range of motion as the key principle. RESULTS: The mean range of motion was 60 degrees +/- 21 degrees at discharge. At the date of presentation 12 to 18 months postoperatively the mean range of motion had improved to 110 degrees +/- 19 degrees. CONCLUSIONS: The presented study indicates that complete restoration of range of motion after rotational knee arthroplasty may not necessarily be restored at discharge. PMID- 10730368 TI - [Gonarthrosis and empyema in geriatric patients. Combined synovectomy and KTEP implantation procedure]. AB - ISSUE: Is a one stage procedures with open synovectomy and implantation of cemented knee endoprostheses in patients with empyemas of the knee and concomitant osteoarthritis suitable to reduce morbidity and increase early mobility? METHOD: Three female patients (age [symbol: see text] 88 yrs.) with acute empyema of arthritic knee joints (Pathogen: Staph. aureus) were treated with open synovectomies and implantations of cemented TKA's in a one stage procedure at mean 3 days (1-6) after admission. After 4.6 months (3-6) we carried out the last clinical and radiographic examination and after 15 months (11-21) a telephone questionnaire. RESULTS: The mean operation time was 2.4 hrs. (2-3), the perioperative blood loss at mean 1040 cc (800-1180 cc) and patients received 4 units blood (4-5). Parenteral antibiotics were administered for at mean 21 days (18-25 d). 33 days (27-39) after surgery the patients could be discharged, all of them ambulated with a walker, the operated joints had no signs of infection and blood parameters i.e. leucocyte count, crp and ESR dropped. At the time of the latest follow up examination there were neither clinical nor radiographic any signs that infections recurred. Two patients ambulated with crutches, one still with a walker. The HSS-Score increased from preop. 54 pts. up to 80 pts. at the follow up. CONCLUSION: Due to our results, a one stage procedure with open synovectomy and implantation of cemented total knee endoprostheses seems to be a considerable alternative for the treatment of geriatric patients suffering from knee empyema and severe osteoarthritis. PMID- 10730369 TI - [Inpatient and follow-up nosocomial wound infection in orthopedics]. AB - SUBJECT: This study was designed to determine if direct documentation (in patients) and questionnaire (out-patients) are reliable follow-up methods to record nosocomial wound infections (NWI) after orthopedic surgery. Furthermore if postdischarge NWI were also detected without this project. MATERIAL AND METHODS: 1664 patients after orthopedic surgery were evaluated over 20 month. In-hospital infections were directly documented. Later infections were recorded by questionnaire 3 months following patient's discharge. Data of all cases with NWI were retrospectively evaluated as a control. RESULTS: A total of 18 NWI (1.1%) were recorded. 2 of these were deep wound infections after hip or knee arthroplasty, no re-operation was required. 6 NWI (33.3%) occurred after discharge. All of these post-discharge NWI were detected in our out-patient clinics. CONCLUSION: This study demonstrate, that postoperative wound infections surveillance must be continued after discharge. 33.3% NWI occurred after discharge and all cases were detected at our out-patient clinics. We conclude that questionnaires to record post-discharge NWI are not necessary in hospitals with routinely follow-up in out-patient clinics. PMID- 10730370 TI - [Disruption of the arteria nutricia tibiae by reamed and unreamed intramedullary nailing. Study of the vascular architecture of the human tibial intramedullary cavity]. AB - AIM: By reason of the pseudarthrotic healing of fractures due to vascular complications after reamed and unreamed intramedullary nailing, the intraosseous course and branching of the tibial nutrient artery and its impairment by nailing procedures needs an actual analysis. METHOD: The nutrient vessel of 24 tibiae taken from fresh corpses were prepared by injection of Technovit and lead oxide. After this procedure the medullary cavities of 12 bones were opened by a frontal cut. These specimens were subjected to routine maceration. The other 12 tibiae were naed with the unreamed (6) and the reamed nailing (6) techniques. X-rays were also taken routinely. RESULTS: After penetration of the tibial compact bone the main trunk of the nutrient vessel runs through a perforated osseous tunnel (pars tecta arteriae nutriciae tibiae). At its end the vessel divides into a descending branch (obligate) and two ascending branches (facultative). The descending branch lies near to the centromedial region of the medullary cavity which is termed as the pars liberal arteriae nutriciae tibiae. All branches pass through supporting horizontal osseous lamellas. Due to this topography the reamed nailing technique destroyed the nutrient vessel completely in all specimens. In contrast to this observation the unreamed nailing destroyed the vessel completely only in 1 (16.7%) and partially in 3 (50%) bones; 2 (33.3%) specimens exhibited no destruction of the vessel. CONCLUSION: The unreamed as well as the reamed nailing technique can destroy the intramedullary course of the tibial nutrient artery. Fractures as well as displacement osteotomies or corticotomies are able to diminish the medullary blood supply seriously, if they are localised within the area of the tactic an of the tibial nutrient artery. PMID- 10730372 TI - [The problems and outcome with newborns who are extremely low birth weight, appropriate for gestational age and small for gestational age]. AB - The aim of this study is to evaluate the consequences of being extremely low birth weight (ELBW) under 1000 g at birth, small (SGA) or appropriate for gestational age (AGA). METHODOLOGY: A retrospective comparison of two cohorts of ELBW AGA (n = 47) and SGA (n = 38) infants, admitted to the Intensive Care Unite of State Maternity Hospital "Maichin Dom" from 01, 1995 to 06, 1998 is carried out. Infants with major congenital anomalies and those, who died in the first two hours of life, being extremely premature and unviable, are excluded. RESULTS AND CONCLUSIONS: The mean birth weight of the AGA group is 863 g, their mean gestational age is 26.6 weeks of gestation and of the SGA group--795 g and 29.6 weeks respectively. In the Sga C-section is preferred in 85%, vs only 34% in the AGA group. SGA babies are with two times greater survival rate (61%) then AGA (32%). The causes of death are similar. The AGA survivors have a greater rate of stage III-IV intraventricular hemorrhage, more prolonged need for ventilatory support (19 vs 12 days for SGA) and oxygen supplementation (37.5 vs 30.7 for the SGA), more difficulties with enteral feeding and their median hospital stay is somewhat longer (81.9 days), compared with those of the SGA (76.6 days). The two groups show a similar rate of respiratory distress syndrome, chronic lung disease, infections and neurological complications among the survivors. PMID- 10730371 TI - [Ambulatory diagnosis and therapy of gonarthrosis]. AB - AIM OF STUDY: To develop recommendations for appropriate diagnostic procedures and conservative treatment of knee osteoarthritis in outpatients. MATERIAL AND METHODS: Following a consensus conference and expert reviews basic recommendations were developed. RESULTS: While standardized radiographic assessment is mandatory, MRI investigation should be restricted to problems apart from osteoarthritis. Indications for physical therapy, bracing and pharmaco treatment depend on the severity of the disease. Guidelines for intraarticular injections are presented. CONCLUSION: Effective treatment of knee osteoarthritis must be based on available recommendations and guidelines. PMID- 10730373 TI - [Myomectomy during cesarean section]. AB - The purpose of this prospective study is to assess the possibility of myomectomy during CS as a routine method. The material includes 21 cases of myomectomy during CS. 162 consecutive CS without myomectomy are control group for hemorrhage during the operation. The myomectomy is accomplish according classical technique. Our data show that the myomas and pregnancy is found mainly in women after 30 years of age and is prevalent in nulliparas (70%). Single myomas were found in 85% of cases, 63% located in the corpus uteri and 46% of all myomas are confined to the myometrium. Related to the isthmus uteri are 23% of myomas. Myomectomy as a separate operation during CS increases the hemorrhage by 10%. The analysis of the cases with severe hemorrhage point to the placental disorders (abruptio placentae and placenta praevia) as a main cause of overall increased blood lost. Our limited experience with myomectomy as a protocol during CS irrespective of number and magnitude of myomas shows that this is possible. The postoperative period is without complications. PMID- 10730374 TI - [The dry period in preterm birth and premature rupture of the fetal membranes]. AB - It is widely believed that premature rupture of membranes accelerates fetal pulmonary maturity. The purpose of our study was to determine the duration of the latent phase, in the cases of premature rupture of membranes, required to achieve this effect. Retrospective analysis of our database yielded a group of 42 patients, who were delivered between 26 and 34 weeks gestation. The results of this study suggest that pulmonary maturation continues but is not accelerated after premature rupture of membranes. Delaying delivery for more than 72 hours after rupture of membranes is more likely to result in chorioamnionitis than accelerated pulmonary maturation. PMID- 10730375 TI - [Transvaginal sonography in the study of the cervix uteri in pregnancy]. AB - The purpose of this prospective and cross sectional study is to evaluate the changes in the cervical length and wide of the cervical canal throughout normal pregnancy and the effect of parity on this process. The material consists of 215 unselected and non complicated pregnancies between 8th and 42nd weeks of gestation. 119 were nulliparous and 96 multiparous women. The cases are divided in subgroups through 5 weeks. The cervical length is measured between OICC and OECC by 5 MHz transvaginal transducer and the wide of the cervical canal at the middle of the cervix. In 6 cases there was a funneling and the pregnancies terminated in abortions or preterm labors, so they were excluded. Unlike other studies we find increase in the cervical length with advancing gestational age until 31 week and a progressive shortening to the end of pregnancy. The wide of the cervical canal is increasing to the end of pregnancy. The wide of the cervical is increasing to the end of pregnancy is between 2 to 6 mm. The parity has insignificant influence on the ultrasound measured cervical length and wide of cervical canal. PMID- 10730376 TI - [The Apgar score, acid-base equilibrium of the umbilical cord vessels and early postnatal adaptation in healthy term newborns]. AB - The aim of the study is to establish a correlation between Apgar score, acid-base status (ABS) and blood gases (bg) from cord blood and the early postnatal adaptation in healthy term newborns. The study is prospective and includes 52 babies at term born at the State University Hospital Maichin Dom, Sofia during a three month period--03-05, 1998 without evidence of asphyxia before and during delivery. All babies are monitored for Apgar score at minute 1 and 5, ABS and bg from umbilical artery (u.a.) and vein (u.v.) examined at birth, as well as capillary ABS and bg 1 hour after birth. Early postnatal adaptation is judged by a neonatologist in the course of two hours. RESULTS: A significant difference is found between all the examined points in the ABS and the blood gases in samples from umbilical artery and vein (p < 0.05), most significantly differ pH, pO2 and O2 Sat (p < 0.001). There is a correlation between 1 minute Apgar score and ABS and bg from umbilical vessels, babies with 1 minute Apgar score 7 having significantly lower pH from u.a. requiring wider range of resuscitation. Babies with 1 minute Apgar score 9/8 and 5 minute Apgar score 10 have definitely less early adaptational problems. CONCLUSION: The use of a combination of evaluation criteria for the condition of the newborn after birth (Apgar score, ABS and bg from cord blood and strict monitoring of early cardio-pulmonary adaptation) guarantees adequate resuscitation in term babies. PMID- 10730377 TI - [The duration and extent of intensive therapy for newborns]. AB - The aim of the study is to evaluate the duration, the extent and the complications of the intensive care of neonates, treated in the Neonatal Intensive Care Unit (NICU) of DUB "Maichin Dom" Sofia. Included retrospectively are all 383 neonates, admitted in the NICU for 1995, classified as mature--131, premature stage I--70, stage II--75, stage III--71 and stage IV--36 babies. The duration of the stay is increasing progressively according their weight: from 4 days for the mature children to 15.6 and 18.4 respectively for the newborns with very low and extremely low birth weight. The latter two groups have considerably higher need of ventilatory support (respectively 52.1% and 86.1% of them are ventilated) and higher mean duration of the ventilation--242 and 271 hours. Although they represent 28% of the patients in the NICU, the babies weighting below 1500 g accumulate 74% (17,347 hours) of the whole sum of ventilatory hours in the unit. They also develop more complications due to the immaturity and the assisted ventilation--pneumothorax, bronchopulmonary dysplasia etc. A conclusion is to be made, that the charge of a NICU is due mainly to the premature infants with very low and extremely low birth weight. These babies and the amount of ventilatory support in the unit (as total number of ventilatory hours and ventilated children) can serve as criteria for the extent of loading of the NICU. PMID- 10730378 TI - [Streptococcus group B isolated in 3 microscopic displays from the vaginal secretions of pregnant women]. AB - Over a period of 20 months we investigated 1366 vaginal specimens from pregnant women for GBS (group B streptococci) carrier in three microscopic patterns, evaluated by Nugent score system. More frequently we isolated GBS in group I intermedia (score 4-6)--20.8%, (when Lactobacillus spp. is missing--67.4%, without or associated with other nonanaerobic microorganisms (60.5%). All isolated GBS strains showed sensitivity to ampicillin and carbenicillin. Good sensitivity was found to cefazolin (92.4%) ant to cefuroxim (94.9%). The strains showed 13.7% resistance to erythromycin and 4.1% to clindamycin. PMID- 10730379 TI - [The etiology of infectious cervicitis in women]. AB - Infectious cervicitis is a common disease in women of reproductive age. A prospective study was carried out including 70 women with clinical manifestations of cervicitis during gynecological and colposcopic examination. In 19 women (27%), Chlamydia trachomatis was demonstrated by direct immunofluorescence in cervical smears. Neisseria gonorrhoeae was isolated in 2 women and 1 had a concomitant infection with Neisseria gonorrhoeae and Chlamydia trachomatis. Mycoplasma hominis and Ureaplasma urealyticum were isolated in 7 patients and 4 had cytological smears consistent with Human Papilloma Virus infection. In the rest 47 patients no etiological agent was found. The results of the present study indicate that the most common etiological cause for infectious cervicitis can be Chlamydia trachomatis and an examination for this organism should me mandatory in women with infectious cervicitis for guiding the correct diagnosis and treatment. PMID- 10730380 TI - [The correlation between the study data on the Papanicolaou cytological smear and the data on direct immunofluorescence for C. trachomatis]. AB - A study was carried out to examine the relationship between the clinical data, the data from the Papanicolaou smear and the direct immunofluorescence for C. trachomatis in 70 women with infectious cervicitis. The prevalence of Chlamydial infection in the study population was 27%. The study showed that one third of the women in whom the Papanicolaou smear revealed inflammatory changes might be expected to be infected with C. trachomatis. The probability of not having infection when no inflammatory changes were observed in the Papanicolaou smear was 0.74--negative predictive value which means that more than two thirds of the women without inflammatory changes in cytology smear will not be infected with C. trachomatis. PMID- 10730382 TI - [The progesterone test and transvaginal sonography as methods for the early discovery and screening of endometrial cancer in women in the postmenopause from some risk groups]. AB - We wanted to assess the effect of tamoxifen treated women with breast cancer, as well as to examine patients with diabetes blood hypertension, and obesity. The influence of Tamoxifen was searched over the endometrium in patients treated with it for 3 years. In this research work is assessed the effect of this medicament from this risk group. The second risk group were the women with high blood pressure, obesity and diabetes mellitus. We used transvaginal sonography and the progesterone test to find endometrial pathology and especially endometrial cancer in asymptomatic women-measuring the depth of the endometrium, and using abrasio probatoria separata in order to find endometrial cancer. With these tests we found polyps, hyperplasia, and early endometrial cancer. We wanted with progesteron test and vaginal sonography in these risk groups to find endometrial pathology and especially early endometrial cancer in asymptomatic women. PMID- 10730381 TI - [Changes in the concentration of individual proteins in women using current peroral contraceptives]. AB - BUT: The aim of the present study was to establish the influence of the moderate combined oral contraceptives on the concentration of the individual proteins in serum. MATERIAL AND METHODS: The study included 70 women at the age from 16 to 30, separated into two groups: control group, including 30 women who have not used OC basic group, including 40 women using Cilest and Marvelon in the period of one to three years. There have been examined the following individual proteins in serum: prealbumin; orosomucoid; haptoglobin; TRA; C3; C4; CRP; IgA; IgG and IgM. Their level in serum was defined with immunoturbidimetric method with monoclonal antibodies of the firm Kone (Fin). RESULTS: The data showed reduced concentrations of Orosomucoid and increased concentrations of TRA in the basic group. CONCLUSIONS: The continuous use of Cilest and Marvelon do not influence the concentrations of the examined individual proteins in the serum. They lead to reduced concentrations of orosomucoid and increased concentrations of TRA. PMID- 10730383 TI - [The incidence of sperm antibodies in patients included in a program of assisted reproduction]. AB - The aim of the present investigation was to establish the sperm antibody incidence among patients included in a program for assisted reproduction by the means of the classical and new methods for sperm antibody detection; to analyze the correlation between the results demonstrated by the different techniques; to evaluate the obtained data in the context of the application of new assisted reproduction technologies (ART). In the Laboratory of Reproductive Immunology, Department of Biology, Medical University of Sofia, 73 sera from patients (35 couples and 3 men) with primary and secondary infertility, aged 23-46 years, grouped according to the diagnosis and included in the program for assisted reproduction "Technobioassistance" were tested. Our results demonstrated the highest incidence of sperm antibodies amongst patients with primary unexplained infertility. The percentage of positive reacting couples for at least one of the applied tests was 31%. The highest relative share of those reacting positively, was observed with ELISA and the tray agglutination test (TAT) of Friberg. For ELISA 31.48% of the sera reacted positively, while clinically relevant liters of sperm antibodies were found with TAT in 21.92% of the tested sera. We also found that 17.46% and 13.7% of the tested sera were positive, respectively in the sperm immobilization test (SIT) of Isojima and the gelatin agglutination test (GAT) of Kibrick. The high degree of correlation (P < 0.0001) between the tests of Kibrick and Friberg showed the appropriateness of their application for patients in an ART program. At the other hand, the lack of correlation between the other applied tests (the Isojima test and ELISA) confirmed the assumption that immunity against spermatozoa should be sought with at least two diagnostic tools. Five of the tested families with clinically relevant TAT titers were included in our ART program for "in vitro" fertilization and embryo transfer, after preliminary absorption of the seminal fluid with autologous semen, or underwent the ICSI technique. For these patients, one ICSI pregnancy finished with the successful birth of a healthy child and one "in vitro" pregnancy is developing at the moment. PMID- 10730384 TI - [Carcinoma of the vagina]. AB - In our study we present the cases with vaginal cancer treated in the gynaecological clinic of the National Oncological Centre in Sofia for the period from 1989 till 1998. Most of the women were with squamous cell carcinoma of the vagina. Twenty seven patients were examined--19 of them were operated, 18 patients received radiotherapy and 2 chemotherapy. The tumors that were localized in the upper 2/3 of the vagina were operated by radical hysterectomy a modo Wertheim-Meigs and extirpation of the vagina. When the tumor was localized in the lower third of the vagina we performed additionally radical vulvectomy and bilateral inguinofemoral lymph-node dissection. For more advanced cancers we did exenteration anterior and posterior. When there was no possibility for surgical approach or for radiotherapy we did chemotherapy with 5-Fu, methotrexate, cyclophosphamide and vincristin. The prognosis for the vaginal cancers is not good, in 25-30% of the patients survive 5 years. As a whole we assess that we are having the same results, as well as survival rate like other western clinics. PMID- 10730385 TI - [The hysteroscopic diagnosis of endometrial carcinoma]. AB - Panoramic hysteroscopy as a testing method was introduced into the Department of Obstetrics and Gynaecology at the Medical University--Plovdiv in 1995. For four years the authors have performed 308 panoramic hysteroscopies. With 19 of these cases (6.17%) the diagnosis cancer of the endometrium has been confirmed histologically, too. Using the advantages of this method within the complex of modem screening testing methods for early diagnosing of this kind of cancer, the authors have described its typical hysteroscopic image. PMID- 10730386 TI - [The TUNEL technic for demonstrating apoptosis on paraffin sections]. AB - TUNEL technique, i.e. terminal deoxynucleotidil transferase-mediated dUTP nick end-labeling, has become a widely used staining method to assist in detection of apoptotic cells in tissue section for routine pathology and scientific investigation. Terminal deoxyribonucleotidyl transferase catalyzed the addition of biotinylated dUTP to free 3'OH ends of DNA fragments, with the synthesis of a polydeoxynucleodide polymer. The signal is amplified by avidin-(biotin) peroxidase and diaminobenzidine is used as chromogen. We have used this technique to study apoptosis in a group of 9 patients with primary breast carcinoma, 4 of them treated with primary chemotherapy. In situ labeling technique identify apoptotic cells, including those with early nuclear chromatin margination. Necrotic cells have multiple random DNA breaks and stain less intensely. Using these technique will allow for the comparison of the quantitative rate of apoptosis in specific target cell types before and after therapy. Such comparison should enhance knowledge as to the role that apoptosis plays in process of cancer therapy. PMID- 10730387 TI - [Bulgarian research on immune infertility]. PMID- 10730388 TI - [Polycystic ovarian disease. 1. The current data on its etiology and pathogenesis]. PMID- 10730389 TI - [The use of GnRH agonists in severe uterine hemorrhages in adolescence caused by congenital aplastic and hypoplastic anemias]. AB - GnRH agonists, applied for a long period of time or as depot forms, lead to blocking of gonadotropin and ovarian steroid synthesis. We used their property to induce amenorrhea for treating menorrhagic and menometrorrhagic bleeding in adolescence caused by inborn aplastic and hypoplastic anaemia. In patients with uterine bleeding during the first regular menstrual period or recurring during menstrual periods Zoladex (depot GnRH agonist) has been applied, resulting in stop of bleeding in 2 to 4 days. The drug-induced amenorrhea provides the possibility for physiologic restoring of hemoglobin levels, improvement of therapeutic results from adjuvant antianaemic therapy. Thus a more favourable basis for treating of the main disorder is being created. When surgery is indicated, GnRH agonists provide the opportunity for planning of operating management or for laser ablation of endometrium. PMID- 10730390 TI - [The potentials for combined estrogen-androgen therapy in the postmenopause]. PMID- 10730391 TI - [Myoma uteri permagna in a 21-year-old woman]. AB - The authors inform about very rarely case of myoma uteri permagna of yang person 21 years old. They had diagnostically and operative difficulties. PMID- 10730392 TI - [The results of a single-stage sex-change operation from male to female]. AB - The authors report a result of single-act operation for sex changing from man to woman. PMID- 10730393 TI - [Our 3- to 5-year experience with Anteovin]. AB - Anteovin was used in the treatment of dysmenorrhoea, climax precox, dysfunctional uterine bleeding, bleeding with IUD and oral contraceptive. 247 women and adolescents for 6761 cycles were treated with Anteovin for period of 3-5 years. Results were excellent. 21 women--8.17% had side effects. PMID- 10730394 TI - [Modern noninvasive methods for terminating pregnancy in the 2nd trimester]. AB - Pregnancy interruption upon medical indications between the 13th and 21st gestational weeks is carried out mainly by two-phase invasive techniques including intraamnial application of NaCl and vaginally prostaglandins, laminarie or solutio rivanoli, followed by a surgical treatment (instrumental revision). Because of its invasive character these techniques can cause severe complications. The application of prostaglandin analogues (P.G.) is safer but it is connected with gastrointestinal side effects and their action is often protrachirated especially "primigravida" with a undeveloped infantile P.V.C.U. the author proposes a highly effective non-invasive technique implementing the following combination: sensibilization with an antiprogestin (synthetic norsteroid--Ru 486), applied perorally: 1 tablet 200 mg; 24 hours before the intracervical (vaginal) insertion of the PGO2 tablet or gel. Results are extremely effective and good: the time for pregnancy interruption is reduced to more than 50% (average 5 to 10 hours), side effects and complications are minimized and the hospital stay is significantly reduced. PMID- 10730395 TI - [In Process Citation] PMID- 10730396 TI - [Thyroid dysfunction in the aged]. PMID- 10730397 TI - [Thyroid function in the aged admitted to a nursing home]. AB - BACKGROUND: Intrinsic thyroid diseases and so-called sick euthyroid syndrome are frequent among the elderly. Therefore, we set out to assess the usefulness of the measurement of thyroid hormones and TSH on admission to a public nursing home. METHODS: A medical history, physical examination, geriatric assessment and a venous sample were taken from 201 elderly subjects on admission to a nursing home. Thyroid hormones and TSH were measured in all cases. All subjects were classified as valid, mentally impaired, or physically impaired, according to the Spanish Red Cross Scales. RESULTS: Sixty non-disabled and 141 disabled elders were studied. Sixteen (7.9%) cases of primary hypothyroidism were found, of whom 7 (3.5%) were deemed deserving treatment with L-thyroxine, their mean daily dose being 114.3 mcg. Sick euthyroid syndrome was considered to be present in 28 (13.9%) cases, of whom 25 had a normal T4, three had a low T4, and none had a high T4. No cases of hyperthyroidism were detected. Thyroid hormone abnormalities were not statistically associated with age, gender, or physical or mental disability. CONCLUSIONS: On admission to the nursing home, nearly 8% of the elders have hypothyroidism, and an additional 14% have the sick euthyroid syndrome. Routine measurement of T4 and TSH in elders on admission to a nursing home has a favorable cost-utility ratio. PMID- 10730398 TI - [Primary hemochromatosis in asymptomatic young patients]. AB - OBJECTIVES: In order to increase our knowledge of adult haemochromatosis epidemiology and its clinical behaviour in young patients, we studied 4 patients from 3 pedigrees with idiopathic haemochromatosis. METHODS: Diagnostic criteria were: 1) Discarding the presence of secondary haemosiderosis. 2) Histological and histochemical confirmation of hepatic iron overload. Mean age at time of diagnosis was 25.2 years. Male/female ratio was 1. RESULTS: All of them were clinically asymptomatic, what shows the main difference with juvenile haemochromatosis, presenting earlier and more aggressively. Physical examination could not find abnormalities in any case. Regarding analytical studies, the main results were as follows: mean serum iron level was 209.5 mg/dl (s = 37.8), transferrin 206 mg/dl (s = 24.5), transferrin saturation was 77.4% (s = 9); ferritin, ASAT & ALAT were abnormal exclusively in the 2 males), the other measurements being between normal limits in all cases. Abdominal MRI showed an impaired hepatic signal in 2 cases. Liver biopsy was accomplished in 3 of them, showing massive deposits of hemosiderin in the hepatocytes in the 2 male cases and chronic hepatitis in one of them. There was no evidence of damage to other organs. Genetic studies showed that the 2 siblings were homozygous for the HFE 845GAEA (C282Y) mutation and in the other 2 cases HLA-A3,B7 was found to be associated. At present, after a year or more, all of them remain asymptomatic, liver function tests and mean serum ferritin levels are within normal limits, IST is < 65%, while Hb continues being > 11 g/dl. CONCLUSIONS: To conclude, we would like to emphasize the extraordinary importance of early diagnosis and careful treatment in this common and remediable illness (otherwise lethal), to reach a normal and symptoms-free lifespan. PMID- 10730399 TI - [Tuberculosis and human immunodeficiency virus infection: clinical manifestations and performance of diagnostic procedures according to distinct forms of the localization of disease]. AB - OBJECTIVES: To analyse the clinical manifestations and performance of the diagnosis methods in tuberculosis (TB) diagnosed in patients with human immunodeficiency virus infection (HIV), according to the location forms of disease (LF). MATERIAL AND METHODS: We revised 80 cases of TB diagnosed in patients with infection by HIV. The data were gathered in relation to TB location, the clinical data and the microbiological and histological diagnosed studies. In the statistic analysis the values p < 0.05 were taken into account. RESULTS: The prevalence of LF was: 1) pulmonary forms (PF) 32 (40%), of which 12 were typical pulmonary (TP) and 20 atypical pulmonary (AP); 2) mixed forms (MF) 21 (26%); 3) extrapulmonary forms (EF) 19 (24%), of which 17 were lymphadenitis; 4) miliary tuberculosis (MT) 8 cases (10%). The prevailing symptoms were: fever (71%) mainly in MF and MT, cough (69%), with less frequency in AP, EF and MT (p < 0.05) and adenomegalies especially in EF and MF. The diagnosis was based on the study of the sputum smears and lymph node. The bacilloscope of the spontaneous sputum was 53% with a minor performance in the AP (p < 0.05), while in lymph node the smear for AFB was positive in 78% cases and caseous granulomas were observed in 87%. CONCLUSIONS: In our study the major confirmed locations were pulmonary and lymph node, the most important clinical symptoms were fever, cough and adenomegalies. The diagnosis was based, in the most of cases, on the microbiologic and histologic examination of bronchial secretion and lymph node samples. PMID- 10730400 TI - [Etiology, clinical features, precipitating factors, type of ventricular dysfunction, length of stay and mortality of 305 patients admitted to hospital because of heart failure]. AB - OBJECTIVE: To study the etiology, precipitating factors, clinic features, patterns of ventricular disfunction, treatment, hospital length of stay and mortality of patients admitted to hospital because of heart failure. PATIENTS AND METHODS: It's a prospective study of 305 patients with heart failure admitted consecutively in a short stay unit. RESULTS: The mean age was 73.6 +/- 10.8 years. 50.5% were women and 49.5% men. Women (76.5 +/- 9.7 years) were older than men (70.5 +/- 11.0 years). The etiology was valvular heart disease 30.8%, hypertensive heart disease 26.2%, ischemic heart disease 21.6%, idiopathic dilated cardiomyopathy 8.2% and alcoholic cardiomyopathy 5.6%. The more frequent precipitating factors were respiratory infection (37.3%) and arrhythmias (28.7%). No precipitating factor was detected in 13.9%. 46.1% had systolic disfunction (erection fraction < 50%). 96.7% were treated with diuretics, 79.3% with ACE inhibitors and 51.9% with digoxin. 5.3% needed cardiac surgery. Mean length of stay was 5.64 days. In hospital mortality was 5.6%. PMID- 10730401 TI - [Cardiac tamponade as clinical manifestation of neoplastic process: presentation of 11 cases and review of the literature]. AB - We analyse the clinical presentation of pericardial effusion in patients with malignancy. The diagnostic sensibility of cytology of the pericardial effusion, current management strategies, recurrences and survival are considered. Retrospective single centre study (Fundacion Jimenez Diaz). Eleven patients with malignant pericardial effusion (period: 1992-1996). The most frequent symptom was dyspnea (100%); echocardiogram accuracy was 100% and the sensitivity of pericardial cytology 64%. 73% of all effusions were adenocarcinomas (87.5% of the lung). Pericardiocentesis was practised in 73% patients. Survival did not improve when adding radiotherapy, using local sclerotherapy or surgical pericardial windows (pericardiotomy (medium survival of 109 days). Those patients who died did not develop symptoms of hemodynamic clinical emergency. Prognostic of malignant cardiac tamponade is closely related to the extent of disease and its sensitivity to treatment. PMID- 10730402 TI - [Osteopoikilosis: report of 3 cases and review of the literature]. AB - Osteopoikilosis, osteopathia condensans disseminata, is a rare hereditary autosomal dominant sclerosing bone dysplasia, more common in males. The diagnosis is usually made incidentally from radiographs which show multiple, small, well defined, variably shaped and widely distributed (over the skeleton) sclerotic areas. The involvement is symmetrical, and the predilected locations are the phalanges of the hand, carpal bones, metacarpals, foot phalanges, metatarsals, tarsal bones, ilium, femur, radio and sacrum. It must be distinguished from melorheostosis, osteopathia striata and fundamentally from osteoblastic bone metastases, on the basis of the clinical, radiological (roentgenographs, computed tomography and magnetic resonance) and radionuclide scanning characteristics. Histologically, there are focal condensations of compact lamellar bone within the spongiosa. We report three cases of osteopoikilosis and review the literature. Two cases didn't have affectation in phalanges of the hand, which had not been previously reported, to our knowledge. PMID- 10730403 TI - [Intra-abdominal abscess as presentation of colonic cancer]. AB - Splenic abscess and retroperitoneal abscess are uncommon, although severe diseases, with a high mortality rate that has been attributed to delayed diagnosis, due to the unspecificity of clinical symptoms. We report two patients with a splenic and a retroperitoneal abscess, respectively, in both cases as an onset of colon cancer. The two patients complained of abdominal pain and fever as onset symptoms. Abdominal ultrasonography was normal in the case of retroperitoneal abscess and abnormal in the case of splenic abscess. CT Scan was diagnostic in both cases. In the patient with splenic abscess. CT Scan established a further diagnostic suspect of colon cancer, which was confirmed by colonoscopy. In the patient with retroperitoneal abscess, diagnosis of colon carcinoma was made during the surgical act. In spite of an adequate, combined medical and surgical therapy, both patients died within a short time after surgery. PMID- 10730404 TI - [Hydrops of the gallbladder associated with Epstein-Barr virus infection]. AB - A 50-year-old male developed Hydrops of Gallbladder during the course of Epstein Barr virus infection. The patient had a history of acute encephalitis one month prior to admission. Physical examination revealed jaundice and hepatomegaly. Liver function tests were abnormal and the white blood count was normal with 15% of atypical lymphocytes. Ultrasonography revealed a distended gall-bladder without wall thickening or cholelithiasis. The diagnoses of primary Epstein-Barr infection was made by positivity from EBV VCA IgM serological study. Two weeks later, total clinical, biochemical and ultrasonography resolution were observed. We comment this exceptionally presentation of EBV infection. The great variability of clinical pictures of Infectious Mononucleosis was emphasized. PMID- 10730405 TI - [Psychological variables and adherence to antiretroviral treatment]. AB - The aim of this study is to discuss the implications of a good adherence to antiretroviral therapy, the factors affecting adherence, and the different methods used today to evaluate it. As conclusion, the authors emphasize the convenience of an interdisciplinary collaboration between professionals in order to improve patient's adherence. The possibility to use a multifactorial approach to assess adherence, taking into account psychological variables, is also underlined. PMID- 10730406 TI - [Rheumatic polymyalgia]. AB - Polymyalgia rheumatica (PMR) is a common clinical syndrome that is characterized by pain and stiffness in neck, shoulder girdle and pelvic girdle. The aetiology is unknown. However, recent studies have documented an association with HLA antigens and infectious agents. It occurs mostly after the age of 50 years and is often accompanied by systemic features such as fever, asthenia, hyporexia and weight loss. An erythrocyte sedimentation rate (ESR) of at least 40 mm/hour has been considered diagnostic criterion. Nevertheless, a normal ESR accounted for up to 20% of cases of PMR. A dramatic and prompt response to corticosteroid treatment is characteristic. Giant cell arteritis/temporal arteritis (GCA) has been found in 0-80% of cases of PMR. Temporal biopsy could initially be deferred in patients with PMR younger than 70 years with no cranial symptoms, in which the risk of GCA is very low. PMID- 10730407 TI - [Inappropriate admissions to the Department of Internal Medicine evaluated by the AEP (Appropriateness Evaluation Protocol)]. PMID- 10730408 TI - [Liver cirrhosis caused by hepatitis C virus and septic polyarthritis due to Streptococcus agalactiae]. PMID- 10730409 TI - [Coumarin-induced retroperitoneal hematoma: 2 case reports]. PMID- 10730410 TI - [Yersinia enterocolitica sepsis in a patient with liver cirrhosis and hypersideremia]. PMID- 10730411 TI - [Pneumococcal pericarditis treated successfully by pericardiocentesis]. PMID- 10730412 TI - [Weekly variation of the incidence of acute myocardial infarction (IMI) in a local hospital]. PMID- 10730413 TI - [Improvement of severe psoriasis in a HIV(+) patient]. PMID- 10730414 TI - [Liver abscess caused by enteric Staphylococcus aureus]. PMID- 10730415 TI - [Giant duodenal leiomyoma: a case report]. PMID- 10730416 TI - [Nephrotic syndrome and acute pancreatitis related to glucantime administration]. PMID- 10730417 TI - [Isolated ACTH deficiency: a rare cause of chronic adrenal failure]. PMID- 10730418 TI - [Quantification of bladder tumor antigen (BTA trak) and its correlation with bladder cancer grade and stage]. AB - OBJECTIVE: To analyze the efficacy of the BTA Trak test as a diagnostic marker for bladder cancer, determine its correlation with tumor grade and stage, compare its sensitivity with urinary cytology and its utility in urological conditions other than bladder cancer. METHODS: 77 patients comprised the study; 33 with bladder cancer and 44 without. A urine sample was obtained from all patients for the BTA Trak test and another three samples for urinary cytology from each of the 33 patients with bladder cancer. Of the 44 patients without bladder cancer, 36 had conditions involving the urinary tract and 8 presented conditions without urothelial involvement. The BTA Trak test was repeated three months after treatment in 11 of the patients with bladder cancer and three months after the first test in 13 patients without bladder cancer. RESULTS: Using BTA Trak threshold values of 14 U/ml as first reference and 50 U/ml as the value indicating clinical warning, these were found to be higher in 24 of 33 patients with bladder tumor (sensitivity 72.7%). By tumor stage, BTA Trak was positive in 12 of 21 pT1 (sensitivity 57%) and in 11 of 11 pT2 (sensitivity 100%). Concerning tumor grade, values were higher than threshold in 13 of 22 GI-II (sensitivity 59.9%) and 10 of 10 GIII (sensitivity 100%). Urinary cytology was positive in only 8 cases (sensitivity 24.4%). In the patients without bladder cancer, values are higher than threshold in 3 of 8 patients with urological conditions without urinary tract involvement and 14 of 36 with a probable urothelial involvement without tumor, accounting for an overall sensitivity of BTA Trak of 61.4%. The BTA Trak test three months after treatment showed lower value in 7 of 8 patients with bladder tumor and pretreatment values higher than threshold. CONCLUSIONS: The BTA Trak test is a simple quantitative method with a high sensitivity for the diagnosis of bladder tumors, especially those in the advanced stages and grades. It is superior to cytology, although it has a lower specificity, particularly in conditions with urinary tract involvement. PMID- 10730419 TI - [T.V.T. (tension-free vaginal tape). New surgical technique in the treatment of stress urinary incontinence]. AB - OBJECTIVES: To describe the tension-free vaginal tape procedure (TVT) for treatment of female stress urinary incontinence and the short-term results. METHODS/RESULTS: From November 1998 to May 1999, the TVT procedure was performed in 20 patients with genuine stress urinary incontinence. All patients had a bladder catheter for 24 hours after surgery. Only one patient required intermittent catheterization for one month due to high quantities of post-void residual urine. There were no intraoperative complications. Early postoperatively one patient presented a hematoma in the posterior rectal aspect which was managed conservatively. At 4.5 months mean follow-up, 95% of the patients are continent and one is incontinent due to incorrect positioning of the prolene band. CONCLUSIONS: Although the initial results appear to be encouraging, further studies are warranted to determine the long-term efficacy of this technique. PMID- 10730420 TI - [Cystic pyeloureteritis and infection. Presentation forms and review of the literature]. AB - OBJECTIVE: To present three illustrative cases of pyeloureteritis cystica and review the literature. METHODS: Three illustrative cases diagnosed at our department are described. Patient history, clinical features, diagnostic procedures and treatment are analyzed and the literature is reviewed. RESULTS: Our patients had no specific symptoms. All three patients had urinary tract infection with pyeloureteral involvement, which was bilateral in two cases. One of these patients had a long-indwelling catheter. CONCLUSIONS: Pyeloureteritis cystica is a benign and uncommon condition whose etiology is not well-known. It is generally associated with chronic infection and inflammation, and may be difficult to distinguish from other filling defects of the urinary tract. Due to its benign nature, treatment must always be conservative and close follow-up is recommended. PMID- 10730421 TI - [Comparative study of BTA stat test, NMP-22, and cytology in the diagnosis of bladder cancer]. AB - OBJECTIVE: To compare the sensitivity and specificity of the BTA stat test, NMP 22 and voided urine cytology in the diagnosis of bladder cancer. METHODS: The study comprised 100 patients or follow-up or with a suspicion of bladder cancer. A voided urine sample was obtained and alliquoted in three samples for the BTA stat test, NMP-22 and cytology. The patients were subsequently evaluated by cystoscopy and TUR was performed when cancer was suspected. The bladder cancer was classified according to TNM stage and WHO grade. The McNemar test was utilized to compare the results. The cut-off level used for NMP-22 was 10 U/ml. ROC curves were plotted to determine the NMP-22 values for optimal sensitivity and specificity in our seires. RESULTS: Two patients were excluded from the study. The overall sensitivity was 76.47% for cytology, 78.43% for the BTA stat test and 84.31% for NMP-22 (p = n.s.). The specificity was 91.49%, 87.23% and 87.23% respectively (p = n.s.). By grade and stage, NMP-22 showed the best results followed by the BTA stat test and lastly cytology, although the differences were not significant. The ideal cut-off for NMP-22 in our series was 6 U/ml and not the generally recognized 10 U/ml. CONCLUSIONS: NMP-22 is superior to the BTA stat test and cytology in the diagnosis of bladder cancer, although the differences were not significant. The ideal cut-off in our series was 6 U/ml. The BTA stat test has the advantage of being easy to perform and provides the results in 5 minutes. In our view, NMP-22 and BTA stat test can replace cytology in the diagnosis of bladder cancer. PMID- 10730422 TI - [DNA ploidy and cell cycle phase analysis with flow cytometry in bladder wash. Preliminary experience]. AB - OBJECTIVE: To analyze the prognostic significance in bladder carcinoma of DNA ploidy and cell phase fractions measured by bladder wash flow cytometry. METHODS: Samples were obtained from 25 patients by bladder irrigation; 16 before surgery and 9 during follow-up cystoscopic examination. Cells were stained with propidium iodide and analyzed with the FacScan flow cytometer and Cellfit 2.01 (Becton Dickinson). RESULTS: The number of cells obtained was sufficient for flow cytometric analysis in all cases. In 13 tumor samples (8 superficial and 5 invasive tumors), aneuploidy cells were detected in 3 cases that had a worse outcome; the only superficial tumor in which aneuploidy was detected presented a recurrent bladder carcinoma 15 months later. Of the 5 patients with invasive tumors, two patients with aneuploidy died within 6 months from tumor metastases. Of the patients without macroscopic tumor, only one showed an increase in the percentage of the S phase fraction (19.5% of cells in S phase). A recurrent bladder carcinoma was detected in this patient 6 months after the analysis. In patients with macroscopic tumor, analysis of the S phase fraction was not relevant for prognosis. CONCLUSIONS: Analysis of DNA ploidy and cell phase fractions by flow cytometry of bladder washings can increase the prognostic information in bladder carcinoma. Aneuploidy was associated with a worse prognosis and an increase in the S phase fraction predicted a recurrent bladder carcinoma months before it manifested clinically. PMID- 10730423 TI - [TESE-ICSI in the treatment of male infertility secondary to tuberculosis. Report of a case]. AB - OBJECTIVE: To report a case of infertility treated by TESE-ICSI in a patient with tuberculosis of the seminal duct. METHODS: A 32-year-old patient consulted for primary infertility with a history of 7 years. The fertility studies demonstrated azoospermia, hypospermia and genitourinary tuberculosis involving the entire seminal duct. RESULTS: After the tuberculosis had been treated satisfactorily, TESE, IVF and ICSI were performed with success. CONCLUSIONS: Tuberculosis of the seminal duct can cause primary obstructive infertility. TRA is an alternative when spermatogenesis is preserved. In this patient TESE-ICSI was performed with success. PMID- 10730424 TI - [Renal vein partial thrombosis in 3 recipients of kidney transplantation]. AB - OBJECTIVE: To report on three patients with partial graft renal vein thrombosis diagnosed by ultrasound. METHODS: The vascular complications diagnosed by ultrasound in 400 transplants performed from 1995-1998 were reviewed. RESULTS: Three patients with partial graft renal vein thrombosis were found. Two patients were treated with anticoagulant therapy. No thrombosis could be detected in all three patients on subsequent US control evaluation. CONCLUSIONS: Unlike complete thrombosis, partial graft renal vein thrombosis presents late, without significant clinical features and can be managed conservatively. PMID- 10730426 TI - [Spermatic cord liposarcoma: differential diagnostic criteria and treatment]. AB - OBJECTIVE: To report a case of liposarcoma of the spermatic cord in a young male, with special reference to the difficulties encountered in making the clinical and histopathological differential diagnosis. METHODS/RESULTS: A 43-year-old patient underwent surgery for a tumor (8 x 7 cms) in the left spermatic cord. Pathological analysis demonstrated a well-differentiated liposarcoma with myxoid areas. Radical orchidectomy was performed. The patient is well, with no tumor recurrence or metastasis. RESULTS/CONCLUSIONS: Paratesticular liposarcoma is rare. It is therefore difficult to establish the guidelines for treatment, prognosis and differential diagnosis. Similarly, difficulty is encountered when making the preoperative diagnosis, although US and CT can be useful. As in liposarcomas localized to other sites, the histological type and grade of the lesion are useful for the prognosis. Radical inguinal orchidectomy is the treatment of choice. PMID- 10730425 TI - [HLA compatibility and antigenic re-exposure. Impact on the clinical course of cadaver kidney transplantation]. AB - OBJECTIVES: A number of prediction factors on the outcome of cadaver renal transplants have been recognized, particularly the influence of recipient response to the first graft. However, the influence of HLA compatibility and of repeated recipient and donor antigen mismatches remain a controversy. The importance of the foregoing on the survival of the second renal graft is analyzed in this study. METHODS: The clinical records of 80 patients who had undergone a second cadaver renal transplantation from 1985 to 1995 at the Hospital 12 de Octubre (Madrid, Spain) were reviewed. Data on the recipient, donor and graft characteristics and outcome of the first and second transplants were collected for multivariate analysis of graft survival using the Cox regression method. The importance of the overall HLA compatibility and of each HLA locus, and the effect of antigenic re-exposure on the survival of the second renal graft were analyzed. RESULTS: The multivariate analysis showed a significant increase of the survival of the second graft when there were at least three compatible antigens between donor and recipient (p = 0.02). Compatibility of the HLA-A and DR loci appeared to improve the outcome, although the differences were not statistically significant. However, a significant improvement in the survival of the second cadaver renal allograft was found for repeated HLA mismatches between the recipient and donors (p = 0.006). CONCLUSIONS: Our data corroborate the benefits of assigning renal grafts according to the degree of HLA compatibility also for the second renal transplant. Furthermore, our data show that re-exposure to previous mismatched antigens need not be hazardous for the second renal transplant in the cyclosporine era. PMID- 10730427 TI - [Oat cell bladder carcinoma. Report of a case]. AB - OBJECTIVE: To report an additional case of small cell (oat cell) carcinoma of the bladder. METHODS/RESULTS: A 78-year-old patient who consulted for hematuria is described. A TUR biopsy demonstrated small cell undifferentiated carcinoma. The patient received neoadjuvant chemotherapy and subsequently underwent surgery, but died 6 months postoperatively. CONCLUSIONS: Regardless of the treatment utilized, this tumor type maintains its aggressive behaviour and can be associated with other carcinomatous components (papillary transitional cell carcinoma and carcinoma in situ). However, after treatment with CDDP + VP-16 and partial cystectomy, our patient was asymptomatic for 5 months. PMID- 10730428 TI - [Pigmented renal carcinoma, rich in hemosiderin]. AB - OBJECTIVE: To report a case of hemosiderin-rich, pigmented renal cell carcinoma. METHODS: A 70-year-old patient referred several episodes of gross hematuria during the last few weeks, without any other symptoms. CT demonstrated a renal mass compatible with carcinoma. A right renal nephrectomy was performed. RESULTS: Anatomopathological analysis revealed a hemosiderin-rich, pigmented, papillary renal cell carcinoma localized to the renal capsule that stained blue with the iron staining method. The postoperative period was unremarkable and the patient is asymptomatic one year postoperatively. CONCLUSION: Papillary renal cell carcinoma can present as a pigmented lesion due to the accumulation of iron in the cytoplasm. This peculiar form must be distinguished from neuromelanin pigmented lesions. PMID- 10730429 TI - [A new case of entero-vesical fistula, secondary to Crohn's disease]. AB - OBJECTIVE: To emphasize that urological symptoms and signs can be the presenting features of Crohn's disease. METHODS: A case of enterovesical fistula in a patient with Crohn's disease is described. The patient presented with hematuria and mild voiding symptoms, but there were no GI or other symptoms. The clinical aspects, diagnostic methods (ultrasound, cystoscopy, rectosigmoidoscopy) and treatment are discussed. RESULTS/CONCLUSIONS: Although Crohn's disease is rare, it should be considered when making the differential diagnosis in patients who present with voiding symptoms and hematuria. PMID- 10730430 TI - [Giant bladder diverticulum, inside inguinal hernia. Clinical case and review of the literature]. AB - OBJECTIVE: To report a case of giant diverticulum of the bladder associated with inguinal hernia. METHODS/RESULTS: An uncommon case of giant diverticulum associated with inguinal hernia is presented and the literature reviewed. The enormous size of the diverticulum and the associated lower urinary tract obstruction arising from hyperplasia of the prostate required resolution of both conditions. This was achieved by diverticulectomy with herniorrhaphy and TURP. CONCLUSIONS: Diverticula of the bladder are associated with lower urinary tract obstruction that are generally caused by a prostatic condition. Hernia with bladder diverticulum is uncommon. The symptoms are usually scanty and cannot be distinguished from the voiding symptoms arising from prostatic involvement. Apart form the clinical findings, US, cystography or IVP are useful for diagnosis. Treatment is usually by surgical correction of both the bladder diverticulum and voiding obstruction from prostatic hyperplasia. PMID- 10730431 TI - [Gas-containing balano-preputial abscess; diagnostic and therapeutic approach]. AB - OBJECTIVE: A case of balanopreputial abscess containing gas in a patient with adhesion of the glans penis to preputial mucosa is presented. METHODS: The clinical features, diagnostic methods (penile ultrasound) and treatment (debridement + step (staged?) circumcision) are described. RESULTS/CONCLUSIONS: Severe phimosis and balanopreputial adhesions can cause urological complications as shown in the case described. Ultrasound demonstrated the presence of abscess and gas. PMID- 10730432 TI - [Renal carcinoma with intranuclear inclusions associated with colon carcinoma]. AB - OBJECTIVE: To report a case of renal cell carcinoma associated with carcinoma of the colon. METHODS: A right renal mass was detected during US control evaluation of a patient that had undergone surgery for rectosigmoid carcinoma 4 months earlier. A previous abdominal CT scan revealed a renal mass with characteristics of malignancy. Fine-needle aspiration biopsy showed clear cell renal carcinoma. This case was considered as two different and synchronous primaries, and the patient was submitted to surgery. RESULTS: Intestinal adhesions secondary to radiotherapy were found intraoperatively, but there was no evidence of local intestinal recurrence, and therefore nephrectomy was performed. The histopathological study demonstrated clear cell renal carcinoma with intranuclear inclusions. CONCLUSIONS: Although the patient may have a previous history of carcinoma at another site, the finding of a renal mass excludes synchronous tumors and nephrectomy should be the treatment of choice. The finding of clear cells in the cytological study supports the interpretation that these lesions were different primaries. Renal carcinoma should be included among the neoplasms with intranuclear inclusions. PMID- 10730434 TI - [The relationship with the pharmaceutical industry: an ethical dilemma?]. PMID- 10730433 TI - Early cystic relapse of embryonal carcinoma of testis in obturator fossa. AB - OBJECTIVE: We report a case of embryonal carcinoma stage IIB arising from the right testis that subsequently underwent chemotherapy and retroperitoneal lymph node dissection and presented with an early cystic recurrence in the obturator fossa. METHODS: This case is reanalyzed retrospectively and literature is reevaluated for the early recurrences of testicular tumors at atypical locations. We discuss the rarity of obturator fossa as a location for early recurrences of testis tumors. RESULTS: Only one case of recurrence in obturator fossa has been reported. CONCLUSIONS: This case provides an example of the possibility of recurrence in an unpredictable short interval subsequent to proper therapies and underscores the importance of close follow-up. PMID- 10730435 TI - [The diagnostic situation with prostatic cancer in primary care]. AB - OBJECTIVES: To evaluate the quality of diagnosis in primary care (PC) of prostate cancer (CP) and to analyse the factors linked to late diagnosis. DESIGN: Retrospective, cross-sectional study. SETTING: Five PC centres and a hospital (covering about 130,000 inhabitants). PATIENTS: All CP diagnosed in PC between April 1989 and October 1996. MEASUREMENTS AND MAIN RESULTS: PC clinical history, hospital records and request for specialist consultation in 41 cases were reviewed. In cases coming from PC (71%), the most frequent clinical picture was mixed prostate syndrome (44.8%). PSA (20.7%) and urine sediment (17.2%) were the most commonly requested investigations. Rectal touch (RT) occurred in one case (3.4%) and four cases were diagnosed as CP. In the urology service, RT occurred in 68% of cases, with PSA determined in 59% of those who had not had it. After the first visit, 44% were diagnosed as CP. Two time intervals in common between PC and hospital were studied: referral-specialist visit and specialist visit diagnosis. In PC there were mean delays of 50 days (SD, 53; percentile [P] 25 = 14; P50 = 35; P75 = 75) and 420 days (SD, 595; P25 = 72; P50 = 194; P75 = 490), respectively. In the hospital the times were 6 days for the first (SD, 6; P25 = 2; P50 = 5; P75 = 8.5), and 168 for the second (SD, 176; P25 = 34; P50 = 130; P75 = 271). The differences were statistically significant: p = 0.0006 and p = 0.05. CONCLUSIONS: Doing RT and PSA determination in primary care would favour diagnosis in the cases of CP. The creation of training programmes and rapid referral routes to hospital could reduce the delays looked at. PMID- 10730436 TI - [Television and children: is television responsible for all the evils attributed to it?]. AB - OBJECTIVE: The purpose of this study was to analyze children's television viewing habits and their parents attitudes towards such viewing. DESIGN: Cross-sectional descriptive study. SETTING: Primary care. PARTICIPANTS: A survey was undertaken with 317 three to fourteen year old children and their parents as part of the primary care check-up program for healthy children. MEASUREMENTS AND MAIN RESULTS: Time devoted to television viewing was 106 +/- 50 minutes on weekdays and 141 +/- 80 minutes weekends. Despite this, 49.2% of parents thought their children saw little television, especially those with children under six (57.6%). Children of parents in highly qualified positions and of parents in the uppermost socioeconomic group saw television the least, on non-working days (70 +/- 61 minutes and 144 +/- 78 minutes respectively, p < or = 0.0001). Some 71.9% of children watched television alone and 34% did so at meal-times. Altogether 48.3% of parents were unaware as to what their children watched and some 61.5% encouraged television viewing, above all those having children of under six (76%). The youngest children preferred to watch cartoons which were generally of a violent nature. For those aged from 11 to 14, 19.5% chose as their favorite programs those having a high level of violence. CONCLUSION: Television habits are an educational problem for parents, an important shake-up in their attitudes being called for, in which pediatricians should be involved in developing health programs aimed at proper use of the television. PMID- 10730437 TI - [An epidemic outbreak of mumps. A study of vaccinal efficacy]. AB - OBJECTIVE: Description of an outbreak of mumps in an urban area. Study of the effectiveness of the mumps component of the triple virus (TV) vaccination. DESIGN: Cross sectional descriptive study of the outbreak. The cases were obtained from the Andalusian Register of Infectious Diseases (SVEA in Spanish). The effectiveness of the vaccine was studied through a retrospective cohort design, with the cohorts defined according to their vaccination history. SETTING: Urban area of low social class and income and young population pyramid. The outbreak occurred between March and November 1997. The effectiveness of vaccination at one school was studied. PATIENTS AND OTHER PARTICIPANTS, RESIDENTS IN THE AREA: The source of the population was the 1996 municipal census. INTERVENTIONS: The declared cases of mumps were taken from the SVEA. The state of vaccination was documented through a review of the school registration records, vaccination cards and the health centre register of names. MEASUREMENTS AND MAIN RESULTS: A total of 283 cases of mumps were declared. There were more cases in the 0-10 age group than at older ages. 79% of cases were vaccinated (95% CI, 74.3 73.7). Effectiveness of a dose of the mumps component of the vaccine was low (46%; CI, 0-84), though the effectiveness of a second dose was higher (87%; CI, 27-99). CONCLUSIONS: The low effectiveness of the mumps component of the TV vaccination was confirmed. It is proposed that the age for the second dose should be brought forward to school-starting age (primary, first year). Ensuring school vaccination is recommended. PMID- 10730438 TI - [A pharmacoeconomic study of the antibiotic treatment of the acute manifestations of chronic bronchitis in primary care. The DAFNE Group]. AB - OBJECTIVE: To evaluate the effectiveness and the cost of antibiotic treatment of chronic bronchitis (CB) crises and COPD in PC. DESIGN: Prospective cohort study of patients with CB and COPD monitored in health districts. Information was requested on the first ten patients, without any selection, who attended for consultation with the diagnosis of a crisis in their CB or COPD. They were followed for 30 days and the direct costs of their care were evaluated. The cost effectiveness of the various types of antibiotic treatment was analysed. RESULTS: 268 doctors took part and provided 2354 patients who were valid for the study. 20.8% (490/2354) came back within 30 days because of poor evolution: 79 (3.3%) of these needed hospital admission. The average cost of the failure was 57,687 pesetas. The cost of the use of cephixime was 14,388 pesetas per crisis, with 82.5% effectiveness. The cost for the alternative of "other antibiotics" was 19,775 pesetas, with 73.4% effectiveness. For the alternative amoxycillin clavulanic acid, the cost was 18,647 pesetas with 74.4% effectiveness. CONCLUSIONS: A fifth of the patients evolved poorly, which led to the admission of 3.3%. Cephixime treatment was the dominant treatment, as it is more effective and cheaper than all the other antibiotics and than amoxycillin-clavulanic acid treatment. The cost of the therapeutic failure of crises is more than three times greater than the cost of antibiotic treatment. The rate of failure, which means considerable morbidity at very high cost, must be reduced. PMID- 10730439 TI - [The QUALI Project: a quality study of the structure of the primary care teams (PCT) of Catalonia. The Quality Improvement Commission of the Societat Catalana de Medicina Familiar i Comunitaria]. AB - OBJECTIVE: To analyse the compliance with the primary health care quality criteria of the Committee for Quality Improvement of the Catalan Society of Family and Community Medicine. DESIGN: Cross-sectional descriptive study. SETTING: Reformed primary care centres (PCC) in Catalonia (186), of which 73 (39.2%) filled out the disk with the results. MEASUREMENTS AND MAIN RESULTS: A total of 202 criteria, divided into five sections (structure and material resources, human resources, work organisation, systems of recording and access, and continuity, confidentiality and participation), were self-evaluated in each PCC. These criteria were defined as basic or optimal and compliance with them was graded from 1 to 4. 72.8% of the criteria were fully complied with. In 7.4% there was partial compliance, and in 19.7% non-compliance. The basic criteria had 74% total compliance, whereas the optimal criteria had 51.9%. The groups with least compliance (sum of the total plus the partial) were records systems (68.6%) and human resources (73.5%). The organisation group had the best compliance (85.5%). By sub-groups, the greatest compliance was found in privacy (93.7%), direct care (89.2%) and recording actions (89.1%). Sub-groups with least compliance were long term treatments (61.8%), vaccinations (46.4%) and storage (42%). Eight of the 18 criteria not complied with by 50% or more were considered responsibility of the team. There was 37.8% total compliance and 24% partial in the criteria referring to the specific training of primary care professionals. Up to 80% non-compliance was found in quality of long-term prescription records, vaccinations and storage. CONCLUSIONS: The high level of compliance in the sections which depend most on professionals should be noted (accessibility-continuity and organisation). This study is a first approach towards understanding the quality of the structure of PC in Catalonia, and enables proposals for improvement to be set up. PMID- 10730440 TI - [The prevalence of diabetes mellitus and its association with other cardiovascular risk factors in the province of Albacete. The Vascular Disease Group of Albacete (GEVA)]. AB - OBJECTIVE: To find the prevalence of DM in the population of Albacete, and their relationship with other risk factors. DESIGN: A descriptive cross-sectional population study. SETTING: Community setting in the province of Albacete. PARTICIPANTS: A sample of 2121 people+ over 18 (843 from the capital and 1278 from the rest of the province) was selected through stratified random sampling. MEASUREMENTS AND MAIN RESULTS: Appointments were made by mail. All those attending underwent anamnesis, a physical examination, EKG and basic analytic test. 1263 people older than 18 years were studied. The prevalence of DM observed with WHO criteria was 6.7% (95% CI, 5.9%-7.4%), of which 6.5% corresponded to known DM and only 0.2% to unknown. If ADA criteria were used, the prevalence was 9.8%. We found association between DM and age, obesity, hypertension, hyperlipaemia, low HDL-c, high levels of fibrinogen, left ventricular hypertrophy, arrhythmias and personal history of ischemic cardiopathy and peripheric arteriopathy. CONCLUSIONS: DM is a highly prevalent pathology in Albacete. We consider necessary to standard epidemiological methods that allow us to know the real situation of DM in our country. Other cardiovascular risk factors are more prevalent in diabetic patients, increasing their coronary risk. The control of modifiable factors is fundamental to avoid as possible the chronic complications of the disease. PMID- 10730441 TI - [Why do our patients go to hospital emergency departments?]. AB - OBJECTIVE: To analyse the causes of overuse of hospital emergency services (HES). DESIGN: Cross sectional, descriptive study. SETTING: Emergency service at a general hospital. PATIENTS: Patients who attended the HES on their own initiative. MEASUREMENTS AND MAIN RESULTS: Telephone poll to a representative sample of patients attending on their own initiative the emergency department of the 12 de Octubre Hospital in Madrid between October 5th and 12th. Average age: 46.95 (SD, 20.81); 52.2% women and 47.8% men, 50% were ignorant of the existence of primary care emergencies. 77% were ignorant of the existence of ongoing care points. Main motives for attendance at HES were: ignorance of non-hospital emergency services (32%), better technical means (25.6%), quicker care (21%), sensation of vital urgency (11.4%), poor quality of care in PC (8%). The care received at HES was evaluated as positive in 90% of cases, though 33% thought the information provided insufficient, and 34% the waiting-time excessive. Although 40% believed afterwards that their problem could have been resolved in PC, as many as 75% would return to the hospital. CONCLUSIONS: Widespread ignorance of the existence of non-hospital emergency services affects the over-use of HES. Most users use the HES as a service of immediate PC, a rapid way of obtaining health care. Users have a very favourable opinion of HES care, which is not stated in the case of non-hospital emergency services. In order to improve use of the HES, the population needs to receive better health education. PMID- 10730442 TI - [The mortality at 2 years in chronic patients confined to home]. AB - OBJECTIVE: To analyse the relationship between the mortality of the chronically ill confined to their homes and the risk of death predicted by the doctor and other variables. DESIGN: Longitudinal descriptive study. PATIENTS: 223 patients were studied over 2 years. MEASUREMENTS AND MAIN RESULTS: Survival was related to the variables measured at the beginning of the follow-up: age, sex, social class, time of confinement, number of diagnoses, hours in bed, evaluation with the Katz index and the Nottingham health profile, number of medicines, family help available, and doctor's opinion on the risk of death. 67% of the patients considered at very serious risk of death died within 6 months, as against 6.91 who died in the same period but had a different prognosis. 40.6% at serious risk died within a year, against 12.4% with a different prognosis. The variables independently associated with higher mortality are: the doctor considering that the risk of death is serious/very serious, OR and 95% CI; 2.57 (1.64-4.03); remaining over 16 hours a day in bed, OR 2.39 (1.31-4.36); being over 80, OR 3.41 (1.74-6.66) and being male, OR 1.61 (1.11-2.33). CONCLUSIONS: The prognostic judgement of the general doctor behaved as a predictor of mortality, and may be an indirect indicator of his/her capacity to foresee the need for health services. PMID- 10730443 TI - [The focus group as a qualitative research technic in health: its design and practical application]. PMID- 10730444 TI - [On bioethics and family medicine (II). The Working Group of the semFYC (Sociedad Espanola de Medicina Familiar y Comunitaria)]. PMID- 10730445 TI - [Antibiotics in urinary infections: a problem of choice]. PMID- 10730446 TI - [On palliative care]. PMID- 10730448 TI - [Nursing: a key part for controlling osteoporosis from the primary care viewpoint]. PMID- 10730447 TI - [Do management teams and primary care professionals assess complaints differently?]. PMID- 10730449 TI - [Vertigo of central origin in a hypertensive patient]. PMID- 10730450 TI - [The family doctor and well-being medications]. PMID- 10730451 TI - [Social and family characteristics in children born to women infected by the human immunodeficiency virus]. AB - OBJECTIVES: To evaluate the social and family characteristics of children born to women infected by the human immunodeficiency virus (HIV). As secondary objectives, to analyse their schooling and the number of hospital admissions and lengths of stay that these children required. DESIGN: A prospective observation study. SETTING: HIV unit in a children's hospital. PARTICIPANTS: INCLUSION CRITERIA: all the children recruited from the HIV unit who had their infection status defined during the study period, understood as between the first known case in 1985 and April 1994. The sample included 177 children (62 HIV-infected and 115 not infected). MEASUREMENTS AND MAIN RESULTS: Through an interview the social, family and school variables were collected. On comparison between infected and non-infected children, there were no important differences as to the lack of protection of the new-born (8.1% vs 13%), scant mother-child relationship (31.2% vs 36.5%) or people responsible for the custody of these children. Less schooling and greater problems of school integration were detected in infected cases, with odds ratios of 2.68 (p = 0.004) and 11.36 (p = 0.004), respectively. Children infected also needed more admissions (4.3 +/- 5.7) than the non-infected (1.7 +/- 0.9) (p = 0.001), and more days of hospital stay (75.1 +/- 110.3 vs 23.3 +/- 19.6) (p = 0.0003). CONCLUSIONS: Infected children and non-infected children had similar social and family characteristics. However, less schooling, problems of school integration, and more and longer hospital admissions were related to HIV infection in children, and not so much to their status as children of seropositive mothers. PMID- 10730452 TI - [A proposal for capitation payment, based on age, chronicity, and gender, using management databases]. AB - OBJECTIVES: To propose a case-mix methodology for primary care, based on chronicity, type and age. To describe the explanatory value of these variables in the variability of the medical case-load. DESIGN: Observation, descriptive and retrospective study. SETTING: Primary care. Rochapea Health Centre, Pamplona. MATERIAL AND METHODS: Computer records of all the consultations between January 1996 and June 1997. Dependent variable: case-load. INDEPENDENT VARIABLES: age, type, chronic pathologies (diabetes, lipaemia, chronic neurological diseases, COPD-asthma, chronic psychiatric illnesses, cardiopathy, hypertension, alcohol and other drug abuse). The Kruskal-Wallis test was used to compare work-loads by age groups; and multiple linear regression analysis to calculate the predictive power of the independent variables. RESULTS: Significant differences were observed for age groups. In the multivariate model used for general practitioners, all the variables could be included. They explained 24.2% of the variability in work load (R2). For paediatricians, age and asthma, explaining 23.48%, could also be included. CONCLUSIONS: Age, type and chronicity are useful variables for predicting case load from administrative data bases. They can be used in adjustments for case load applicable to capitation payment systems. PMID- 10730453 TI - [Professionals and managers on internal communication in the Program of Mother Child Health of Andalucia. Evaluation Team of the PSMI]. AB - OBJECTIVE: To find the views of primary health area directors and of professionals in health centres (HC) in Andalusia on internal communication within the Mother-Child Health Programme introduced in this autonomous community. DESIGN: Descriptive study that uses qualitative and quantitative methodology. SETTING: Primary care (PC) areas and HC in Andalusia, between July 1994 and February 1995. PARTICIPANTS: 51 PC area directors and 91 HC professionals. MEASUREMENTS AND MAIN RESULTS: Self-administered questionnaire (directors) and focus groups (professionals) were used. Analysis by non-parametric tests compared averages and analysed discourse contents. Internal communication was an important weakness in the Mother-Child Health Programme and in PC in general. There were important differences between the perceptions of directors and professionals in relation to the guidelines of the Mother-Child Programme. As such, in all the area administrations it was affirmed that there were programme objectives agreed with the professionals, and transmission channels for these objectives between the area and the centres, although these were transmitted in writing in under 25% of the areas. However, professionals thought that the objectives and guidelines of the programme were unclear and ill-defined. In addition, the view that the annual objectives are not negotiated was quite common. CONCLUSIONS: Internal communication in PC is a considerable weakness in HC management. The professionals perceive considerable conflicts of interest between them and the organisation's directors. PMID- 10730454 TI - [Adoption of new medications by prescribing physicians. The innovator physician]. AB - OBJECTIVES: To determine how new medicines are adopted by primary care doctors, identifying innovators or first adopters. Also, to define the variables determining the characteristics of innovative doctors. DESIGN: Retrospective, longitudinal, observational and descriptive study. SETTING: Primary care doctors from the Bahia-Vejer Area in Cadiz (78 general practitioners and 22 paediatricians). MEASUREMENTS AND MAIN RESULTS: All the prescriptions dispensed in pharmacy offices between 1/1/94 and 31/12/96 and prescribed by doctors with over three years experience (74 doctors) were analysed. The active principles sold for the first time between 1/10/93 and 31/12/96 and mainly used in primary care were chosen. Medicines coming on prescription were grouped by three-month periods (28 medicines) and studied for a year. The number of containers of each group prescribed by each doctor was calculated. The doctors were classified in decreasing order according to the number of containers used and given scores through weighting more the initial periods of the prescription of each medicine. The characteristics of the innovative doctor were found with the following variables: age, gender, training, type of care network, type of contract, drugs expenditure and prescription quality through logistic regression. The OR of prevalence for each variable analysed was calculated, and 90% confidence intervals were also determined. 33% of the total (25 doctors) were identified as innovative doctors or first adopters. The statistically significant variables correlating with this group of doctors were: doctors aged > 45, male, non-MIR training, temporary contract, not their sole job, with drug expenditure over the area's 50 percentile, from non-reformed centres. CONCLUSIONS: The identification of this group of doctors will enable specific programmes to be set up in an attempt to alter their attitude to the marketing of new medicines (objective information, evaluation comparing new medicines with existing ones, and increasing doctors' awareness of perceived risk of the use of medicines). PMID- 10730455 TI - [Continuing education in a primary care team: analysis of the 1996-1998 teaching sessions]. AB - OBJECTIVES: To describe the teaching sessions of a primary care team in the three year period 1996-1998. To identify the professionals who ran them and study the areas of knowledge tackled. DESIGN: A retrospective, cross-over, descriptive study. SETTING: Teaching health centre belonging to a rural health district. PARTICIPANTS: All the teaching sessions that took place during the three-year period (n = 249). INTERVENTIONS: The following variables were extracted from the monthly register sheet of the ongoing training programme of our management: date of activity, duration, number attending, type of session, teaching professional and contents of activity (classified by pathology according to organs and systems for bibliographic, clinical and expert sessions; portfolio of 1996 Primary Care INSALUD services for session on programme; computer studies). MEASUREMENTS AND MAIN RESULTS: Mean sessions per month: 6.9 (SD: 4.8). Mean attendance: 9.3 persons (SD: 3.01). Mean length: 36.5 minutes (SD: 11.0). Type of session: bibliographic 65.2%, on programme 18%, session with expert 7.2%, computer studies 5.6%, clinical 4%. Responsible for teaching: intern 39.4%; family doctor tutor 34.9%; family doctor not a tutor 7.2%; nurse 6.4%; hospital doctor 4%; locum family doctor 3.6%; pharmacist 2.8%; paediatrician 1.2%; physiotherapist 0.4%. Most common contents: non-specific general pathology (16.1%), skin diseases (8.8%), diseases of the endocrine system (7.6%). CONCLUSIONS: Low frequency of clinical sessions. The teachers in charge were mainly family doctor tutors and interns, with the rest of the staff participating little. PMID- 10730456 TI - [Value of brief questionnaires in predicting smoking cessation in primary care]. AB - OBJECTIVES: 1. To study the factors predicting the giving-up of tobacco and to correlate the categories in the Smoker's Aid Programme (SAP) with levels of motivation and dependency, using brief questionnaires. 2. To find the annual percentage in the "winners" category, using the SAP methodology. DESIGN: Prospective, observational, intervention study without a control group of a random sample of smokers attending primary care clinics. SETTING: Four urban health centres in Zaragoza and Barcelona. INTERVENTIONS: Systematic intervention on smokers in the change phase (SAP category). Use of questionnaires on motivation/dependency and of biochemical checks that they had actually given up. PATIENTS: 362 patients of whom 288 smokers (79.5%) took part. Average age was 40.85 (SD: 15.7). 60.8% were males. MEASUREMENTS AND MAIN RESULTS: 21.4% of the smokers had tobacco-related pathology. The number of previous attempts to give up smoking was 1.6 (SD: 2.9) Patients smoked an average of 13.4 cigarettes for 22.2 years (SD: 15.7). Average age at start of consumption was 17 (SD: 4.7). Initial motivation on the Richmond Test was 4.78 (SD: 3.2); and average dependency according to the modified Fagerstrom Test (TFm) was 0.6 (SD: 0.7). The number of SAP interventions was 1.9 (SD: 2.9). By the end 15.6% of the patients had achieved "winner" status, which was confirmed in 95.6% of them by the biochemical test. The logistic regression model confirmed that the factors predicting giving up tobacco were age, the initial levels of motivation and the initial number of cigarettes. CONCLUSIONS: The short tests of motivation (Richmond) and, to a lesser extent, dependency (TFm) were good predictors of giving up tobacco and can be recommended in primary care. PMID- 10730458 TI - [Qualitative study analysis]. PMID- 10730457 TI - [Length of temporal disability caused by tuberculosis]. AB - OBJECTIVE: To determine the average length of time off work in working persons ill with tuberculosis in a health district. DESIGN: Descriptive and retrospective. SETTING: Community level, within a health district. PARTICIPANTS: Persons diagnosed between 1989 and 1998 with tuberculosis which caused time off work. MEASUREMENTS AND MAIN RESULTS: An epidemiological data base of people with tuberculosis was crossed with the time-off-work records of the district's medical inspectorate, producing 58 patients. Average time off work was 220.3 days. Mean age was 37, almost all men (93.1%). 36.2% of the workers were miners, with their time off work greater than other workers (p = 0.003). No significant differences in the length of time off work were found for the variables of age, sex, health district, type of tuberculosis or year of diagnosis. CONCLUSIONS: We found that time off work lasted much longer than is customary, and was related to the hardship of the physical work. Return to work should be posed when there is clinical improvement in the patient and a definitive response to the tuberculosis treatment. PMID- 10730459 TI - [The model of primary health care: balance and perspectives]. PMID- 10730460 TI - [Family practice as knowledge area]. PMID- 10730461 TI - [To publish in all the languages of the Spanish State]. PMID- 10730462 TI - [Pharmacologic prescription in 3 geriatric homes in Barcelona]. PMID- 10730463 TI - [Prevalence of hypodontia in permanent teeth]. PMID- 10730464 TI - [Diet, hormones, genes, and breast cancer]. AB - The review describes on-going studies at the Unit of cancer epidemiology of the National Cancer Institute in Milan-Breast carcinogenesis is reviewed addressing: 1) Hormones and breast cancer 2) Diet and breast cancer 3) Diet and hormones 4) Potentiality of dietary prevention in women at high genetic risk. PMID- 10730465 TI - [Confounding and causality in epidemiology]. AB - Since the beginning of the discipline epidemiologists developed a specific methodology to assess causal relationships, even opposed to statistics. A special effort to assure unbiasedness explains the importance of the concept of confounding in epidemiology. However, there is an intrinsic conflict in the formal definition of confounding based on association criteria. This conflict is solved in practice by gathering information on the subject specific matter. Causal criteria for confounding generalize with regards to identifiability of causal relationships in observational studies. PMID- 10730466 TI - [Causality in epidemiology: populations, individuals, and alternative explanations]. AB - The purpose of the present paper is to review issues concerning causal reasoning in epidemiology, with special emphasis on the relationship between observation at the individual and population level. The sequence of the three sections of the paper, causality in epidemiology, individual and population, alternative explanations, reflects the need to deal with issues of research methodology mainly derived from discussions in the frame of risk assessment, decision making, regulation and legal setting. In discussing the aforementioned topics, special attention is given to the attribution of causality to a single exposure in the presence of a plurality of potential causes. As a case study, data concerning the occurrence of hepatic and pulmonary tumors among subjects exposed to vinyl chloride are considered making reference to hearings on this topic which took place in Court. PMID- 10730468 TI - [Occupational experience, epidemiologic observations, and scientific evidence in an illustrative case: adverse effects of the production and use of aromatic amines in Italy]. AB - Economical, social and technical interrelations between synthetic dyes' production and bladder cancer in workmen engaged in the manufacture are reviewed. International situation is the background of Italian situation, mainly of the Lombardy region and Turin area. For decades social actors, individuals and institutions, acted as separate bodies, contributed to delay and omit the protection of workers' health. The situation changed when scientific knowledge, technical developments and workers' commitment prioritized workers' health as a main goal. The epidemic of bladder cancers among dyestuff workers was a cornerstone in the development of a different scenario in the production responsibility, workers' participation and eventually scientific community. The authors hypothesise that in our country these events stimulated the growth of epidemiology and epidemiologists; furthermore since then institutions promulgated a new and up-to-date wave of protective legislation in the workers' H&S field. PMID- 10730467 TI - [Air pollution and daily mortality in Turin, 1991-1996]. AB - A time series study was carried out to assess the relationship between the air pollutants measured by the air quality monitoring network and daily mortality in Turin, Italy. We used TSP, SO2, NO2, O3, and CO concentrations measured from 1-1 1991 to 31-12-1996 at three stations of the city network, chosen to represent different, typical circumstances of exposure to air pollution in the town. The analysis was performed by robust Poisson regression model including loss smoothing functions to allow for long-time trend, seasons, temperature and relative humidity. Dummy variables for the days of the week and holidays were also included. The relative risk of death for a unit increase in the pollutant concentration either during the same day (lag 0) in the previous ones (lag up to 5) was computed. The unit increase was 50 micrograms/m3 for TSP, SO2, NO2, O3, and 2 mg/m3 for CO. The average daily number of deaths for natural causes was 21. The relative risks for total mortality were 1.04 for TSP (lag 1), 1.10 for SO2 (lag 2), 1.06 for NO2 (lag 1), 1.01 for O3 (lag 0), 1.03 for CO (lag 1). The relative risks were 1.05 for TSP (lag 0), 1.12 for SO2 (lag 2), 1.07 for NO2 (lag 1), 1.03 for O3 (lag 0), 1.03 for CO (lag 1) for cardiovascular mortality, and 1.08 for TSP (lag 2), 1.20 for SO2 (lag 2), 1.12 for NO2 (lag 2), 1.03 for O3 (lag 2), 1.05 for CO (lag 2) for respiratory mortality. PMID- 10730469 TI - [Quality of data or quality of care? Comparison of diverse standarization methods by clinical severity, based on the discharge form, in the analysis of hospital mortality]. AB - Using discharge abstract data, we analysed hospital mortality comparing four different methods of risk adjustment. All patients discharged from the S. Giovanni Battista (Molinette) hospital in Turin (Italy) between January 1996 and June 1999 (n = 169,746) were classified with All Patient Refined--Diagnosis Related Groups (APR-DRG). A first analysis evaluated the time trend of hospital mortality by semester. A second analysis compared hospital mortality during the last 12 months among eight units of internal medicine (n = 5592). All comparisons were made through logistic regression models. As the quality of discharge abstracts increased during time and showed variation among units with similar patients, all comparisons were repeated using four models, characterised by increasing predictivity and sensitivity to quality of data. In addition to crude comparisons (A), the other models included as risk factors: B) age and emergency admission; C) same as 'B' plus expected mortality by APR-DRG; D) same as 'B' plus expected mortality by APR-DRG and risk of death subclass. If no risk factors were considered (A), hospital mortality showed an increasing trend, with an odds ratio (OR) of 1.02 by semester, with a 95% confidence interval (CI) between 1.01 and 1.03. The association was weakened when age and mode of admission were taken into account (B) and disappeared when the APR-DRG expected mortality was also considered (C) (OR = 1.00; CI = 0.98-1.01). Finally, if the comparisons were adjusted also for the expected mortality by APR-DRG and risk of death subclass (D) a reversed trend appeared (OR = 0.95; CI = 0.94-0.97). The comparison among the units of internal medicine gave discordant results according to the method used to adjust for confounders. The most striking variations were detected for those units with the best and the worst clinical data. The unit with the poorer clinical data (average number of diagnoses per patient = 2.9) showed a crude OR of 1.38 (CI = 0.99-1.93) and an adjusted OR (D) of 1.71 (CI = 1.10-2.66); the unit with the best quality of data (average number of diagnoses per patient = 4.4) changed the OR from 1.55 (CI = 1.06-2.26) (A) to 0.66 (CI = 0.37-1.17) (D). In conclusion, these results confirm the high sensitivity of the APR-DRG classification to the quality of data and, more in general, suggest to be prudent when using powerful instruments like this to assess quality of care, especially if the quality of data among the units compared is less than optimal or not homogeneous. PMID- 10730470 TI - [Social differences in cancer incidence, fatality, and mortality in Turin]. AB - INTRODUCTION: The study of social differences in health is today focused on determinants. The knowledge of determinants is useful for the definition of policies to tackle inequities. The aim of this paper is to analyse the social distribution of cancer incidence, fatality and mortality in the city of Turin during the 80's and to estimate the amount of inequality produced within the health system. SOURCES OF DATA: The social distribution of the risk of death in Turin during 1981-1989, estimated from the Turin Longitudinal Study, is compared with incidence and fatality during approximately the same time period. RESULTS: Among men, the relative risks for all cancer sites, among social classes, appear to confirm the hypothesis of a multiplicative model of incidence and fatality. In particular, colo-rectal, prostatic, bladder cancers and lymphoma and leukaemia show relative risks similar to the expected ones. Cancers of larynx, lung and stomach seem to be less coherent. Among women, colon-rectum, lung, breast and lymphoma and leukaemia show coherent results, whereas relative risks for uterus are far from the expected. DISCUSSION: The coherence with the expected values of the social distribution of mortality suggests the adequacy of the multiplicative model for the provision of mortality from incidence and fatality. Therefore it is possible to state that the portion of inequality in health originated within the health system is big enough to suggest the need to put the equity in the access to health services in the agenda of interventions. PMID- 10730471 TI - [Occupational cancer in criminal law]. AB - This paper addresses the issue of occupational cancer and criminal law with respect to: 1) causal links between occupation and cancer; 2) certainty and probability in causal links; 3) occupational and non occupational causes of cancer; 4) cancer predictability and employer behaviour. PMID- 10730472 TI - [Malignant Mesothelioma Registry from Piedmont. Incidence in 1990-1995]. AB - This paper describes methods and results of the Piedmont Malignant Mesothelioma Registry. The Registry is active since 1990 and collects all histologically confirmed incident cases of malignant mesothelioma (m.m.) occurring in the residents of Piedmont. In the period 1990-95, 346 cases of pleural m.m. (211 males and 135 females) and 41 (28 males and 13 females) of peritoneal m.m. have been observed. Amongst the inhabitants of the Local Health Authority of Casale Monferrato, where manufacturing of cement asbestos has determined serious asbestos exposures both in the work place and general environment, there have been 105 pleural m.m. and 17 peritoneal m.m. (incidence rate were 15.6 for men and 13.0 for women and 3.6 for men and 0.6 for women respectively). Leaving out the Local Health Authority (LHA) of Casale Monferrato, the annual incidence rate in Piedmont (for 10(5) person-years, age standardised on the 1981 Italian population), has been 1.0 in men and 0.6 in women for the pleural m.m. (respectively 154 and 87 cases) and 0.09 and 0.06 for peritoneal m.m. (14 and 10 cases). Possible cases of m.m. (cytological and/or x-ray diagnosis) have been searched in the file of hospital admission and discharges (SDO) in 1994-95: 46 additional cases were found, with a 25% increase in incidence rates. The analysis of incidence according to geographical aggregations (defined according to the LHA borders) has identified, besides some already known important sources of exposures, as Casale Monferrato and the LHA of Lanzo (Balangero mine), other areas with excess of incidence as the LHA's of Galliate and Caluso which show an increased incidence of pleural m.m. in men or Vercelli and Chieri with increased incidence of pleural m.m. in women. These observation deserves further analysis. PMID- 10730473 TI - [Trends in mortality from primary pleural tumor and incidence of pleural mesothelioma in Italy: a particularly serious situation]. AB - We present: a) an analysis of the past mortality from Primary Pleural Tumors (PPT) occurred in Italy between 1968 and 1992 by an age-cohort-period model, using a Poisson regression model, estimating the risk of dying by birth cohort, the Lifetime Cumulative risk (25-84 years) by birth cohort, the risk by calendar period and testing the full model (age-cohort-period effects); b) a summary of the incidence of mesothelioma as recorded in Italy by Cancer Registries and Mesothelioma Registries. The highest Lifetime Cumulative Risk of dying from TTP is recorded for the birth cohort 1946-'50 (6.2 per thousand among males, 1.64 among females). Whereas the risk by birth cohort becomes flat among females born after 1936, among males the risk is increasing up to the youngest birth cohorts. By calendar period, the highest risk of dying is observed in the last period (1991-'92). The inclusion in the full model of the calendar period term increases significantly the goodness-of-fit of the model among females, but not among males. The highest incidence of mesothelioma in both genders registered by 150 Cancer Registries all over the world is currently recorded among the population of Genoa and Trieste, where large ship-building plants are located. Even higher incidence mesothelioma rates have been recently recorded in other areas of Italy. The trend in PPT mortality in Italy could have been influenced, but not explained, by the increased awareness over time of the disease, but it fits well with the pattern occurring in most industrialized countries of western Europe, with the unprotected industrial use of asbestos which occurred in Italy, and also with the gender characteristics of the work-force employed in asbestos-exposing activities. A ban of asbestos use has been introduced in Italy in 1992. However, considering that asbestos seems to act as an initiator for mesothelioma, the trend in male mortality for PPT will not peak until two or three decades. PMID- 10730474 TI - [Proportion of lung tumors attributable to occupation]. AB - This paper reviews estimates of the proportion of lung cancer attributable to occupational exposures and presents estimates for the City of Turin where a population-based case-control study on lung cancer has been conducted in 1991 1992 among the residents. An analysis of six hundred and five cases (505 males and 100 females) and 859 controls (684 males and 175 females) with histological/cytological confirmed diagnosis of lung cancer and occupational and smoking complete histories has been performed to estimate the proportion of lung cancers attributable to occupation in the city of Turin. The occupational exposure has been assessed with two different classifications: a) classification of a checklist of occupations as obtained from the study questionnaire according to industries and occupations known (List A) and suspected (List B) to be associated with lung cancer; b) translation of ISCO-ISIC codes for industries and occupations according to the same List A and List B. The population attributable risks are comparable when estimated with the two different classifications. In the city of Turin, at the beginning of the Nineties, about 10%-15% of lung cancers in males and 2%-5% in females could be attributed to occupational exposure. PMID- 10730475 TI - [Quality of life at the end of life. Analysis of the quality of life of oncologic patients treated with palliative care. Results of a multicenter observational study (staging)]. AB - Outcome in palliative care can be defined as patients' quality of life, quality of death and satisfaction with care. In an Italian multicentre prospectic study ('Staging') the quality of life of 571 palliative care patients with advanced cancer disease was assessed since the beginning of palliative care till the end of the study. We analyzed the tissue of quality of life missing data and the possibility to input the missing quality of life evaluation through the quality of life evaluation made by a proxy (doctor, nurse). The greatest functional impairment and an increasing level of some symptoms (fatigue, general malaise, emotional status) were observed during the last two weeks of life, whereas for other symptoms (gastro-intestinal, pain) some degree of control was possible. The quality of life analysis for palliative care patients should consider the different response of different quality of life components to the palliative care intervention. PMID- 10730476 TI - [Estimated number of workers exposed to carcinogens in Italy, within the context of the European study CAREX]. AB - CAREX is an international information system on occupational exposure to known or suspected carcinogens, built within the framework of the Europe Against Cancer Programme of the European Union. It provides estimates of the number of exposed workers by country, industry, and agent, including data about 139 agents evaluated by the International Agency for Research on Cancer (group 1 and 2A agents, plus selected group 2B ones), across 55 industries classified according to UN-ISIC revision 2. The 1990-3 occupational exposures to these agents were estimated for the fifteen countries of the European Union in two steps. At first, estimates were generated on the basis of the national workforce data, and of the exposure prevalence estimates from two reference countries: Finland and the USA. These estimates are adjusted by economic structure, but they do not take into account country specific patterns of exposure to carcinogens. For selected countries, among which Italy, it was possible to correct these estimates by national experts, who were invited to allow for the exposure patterns that they considered specific to their countries. According to the estimates for Italy, there were about 4.2 million workers, i.e. 24% of the work force, exposed to the agents included in CAREX, with some 5.5 million exposures. The most common exposures were: environmental tobacco smoke (770,000 exposed workers), solar radiation (550,000), diesel engine exhaust (550,000), asbestos (350,000), wood dust (300,000), crystalline silica (260,000), lead and inorganic lead compounds (220,000), benzene (180,000), hexavalent chromium and compounds (130,000) and PAHs (130,000). PMID- 10730477 TI - [Pediatric Tumor Registry of Piedmont. Descriptive epidemiology of malignant tumors in children in Piedmont, 1976-1994]. AB - Since 1967, the Childhood Cancer Registry of Piedmont measures cancer incidence and lethality among children aged 0-14 residents in the Region. Two thousand seven hundred twenty cases were recorded in the period 1967-94. Males were 55.4%. The highest frequency was observed in the age class 0-4 including 41.3% of cases. The most frequent malignancies were: Acute Lymphocytic Leukemias, CNS Tumours and Lymphomas. Incidence rates showed limited variation, both for total neoplasm and for the largest diagnostic categories. On the contrary, lethality decreased markedly: rate (per million children years) was 77.2 in 1967-69 and 59.4 in 1988 94. This trend as more evident for acute leukemias and CNS tumours. Better diagnostic techniques, anticancer and support therapies are the likely explanation for the improved prognosis. Prevalence increased, as a consequence of improved survival and curability: in the Province of Torino it increased from 62 cases per 100,000 children (age 0-14) in 1980 to 98 cases in 1994. PMID- 10730478 TI - [Generic screening for breast cancer]. AB - Following the cloning of BRCA1 and BRCA2 it has been suggested that genetic screening may be of benefit for women at high familial risk. To be able to assess whether this is the case more research is needed on the penetrance of the different mutations, their prevalence in the population, the biological and environmental factors affecting the development of cancer in mutation carriers and the natural history of genetically determined breast cancer. Furthermore, studies on the efficacy of preventive measures, on counselling and on psychological effects of testing are also needed. PMID- 10730479 TI - [Use of molecular tests of human papilloma virus (HPV) as screening test for cervix cancer: a review]. AB - Infection from some types of human papillomavirus has been recognised as carcinogenic for the uterine cervix. With reliable techniques (PCR and Hybrid Capture II) "high risk" HPV types are found in a very high proportion of women with invasive cancer and high-grade pre-invasive lesions. On the other hand prevalence is low in cytologically normal women, except in young women, who seem to have a high frequency of transient infections in the years following the beginning of sexual activity. Some studies found a role of the presence and persistence of "high-risk" HPV types in the progression of low-grade pre-invasive lesions vs. high-grade rather then vs. spontaneous regression. For these reasons HPV testing has been suggested as a possible tool for primary screening. A few studies suggest that it could allow increasing sensitivity, although problems of extrapolability of results exist. It must, however, be considered that traditional cytological screening is already very protective and that simply adding a further test would lead to an unfavourable cost-benefit ratio. An appealing possibility is applying HPV testing with long intervals between screening rounds. This would reduce the burden for women and plausibly allow reaching higher coverage at each round. A key element is the duration of infection before progression to pre-invasive lesion. A long duration would allow selecting women at low risk of developing a lesion for years (those HPV negative), who could have long-interval test, and others (those HPV-positive) at high risk, to be followed more strictly. Published data from longitudinal studies are limited and don't allow an adequately precise estimate. Therefore the use of HPV testing for primary screening is very promising but to the moment must be considered as a research object. PMID- 10730480 TI - [Strategies of epidemiologic research applied to BSE]. AB - Published epidemiological literature relating BSE was reviewed to point out strategies and methods used by the veterinary epidemiologists involved. Classification keys were used to identify descriptive, analytical and theoretical epidemiological studies used to formulate etiologic hypotheses, to assess the relevance of several risk factors and to monitor and predict the future of the epidemic. Case series, incidence and ecological studies, case-control studies, a cohort study and modelling epidemiology studies have been considered. PMID- 10730481 TI - [Epidemiology between ethics and politics]. AB - Over the last two decades ethics in epidemiology has been the subject of a substantial number of conferences, books, articles, guidelines as well as training courses. The relevance of ethics for epidemiology does not make the ethical approach sufficient to exhaust the analysis of the relationship between epidemiology and society: on one side the ethical assessment of an epidemiological investigation is not self-sufficient but depends on the evaluation of the scientific worth of the study (a planned study involving humans is automatically unethical if scientifically unsound) and, on the other side, to be of practical value it needs expanding as to its policy implications. A few remarks on two issues, ethics committees and human reproductive cloning, support the general contention that translating knowledge into effective public health actions may be grounded in ethical considerations but necessitates a substantial development at political level. PMID- 10730482 TI - [Compliance, conscious participation, and informed consent in tumor screening programs]. AB - Compliance and coverage are supposed to be indicators of effectiveness in screening activities. Yet effectiveness is a necessary but not sufficient condition for a screening programme: adverse effects are intrinsic to screening practice; to persuade to comply to screening programme should imply that the advantages overwhelm the disadvantages; if at community level the balance is in favour of the screening, it is not possible to predict the weight for each individual of any potential harms. To accomplish individual values is fundamental from ethical point of view: we must avoid non responsible participation or uninformed refusal, through a reliable information on screening benefits and harms to the invited population. Therefore the concept of compliance should be abandon: "participation" should be used instead. The consequence of a different approach to participation in screening evaluation should be appreciated: new indicators and standards have to be defined. The following ones are proposed: prevalence of informed target population; prevalence of consent to invitation within the informed population; ratio consent/refusal to invitation; prevalence of participation in the invited group. It is unrealistic to expect an informed participation, to screening programme, from all population but incorporation of patient values and preferences is seen as the next frontier in attempts to devise valid practice guidelines. Research is needed to develop instrument on risk communication and informed consent, taking into account the current organisation of screening programs. PMID- 10730483 TI - [Urinary cotinine as indicator of exposure to passive smoking]. AB - We studied the association between self-reported exposure to passive smoking (ETS) and urinary cotinine among 95 ex-smokers, who had quit in the context of a smoking cessation trial in general practice, and among 33 ex-smokers who had been contacted in the context of a study aimed at estimating the spontaneous cessation rate among smokers, listed in the roosters of the General Practitioners participating in the trial, who had not been offered recruitment. The results of multivariate linear regression analysis indicated that duration of exposure at home was strongly related to cotinine levels (increase of 1 ng/mg every 3 hours of exposure at home), quantified by radioimmunoassay. Duration of exposure in public places showed a similar effect, but the association was significant only among subjects who reported having been exposed in a heavily ETS polluted environment (increase of 1 ng/mg every 45 minutes of exposure). Among subjects exposed at work cotinine concentration was associated to subjects' judgement of the concentration of ETS, rather than to duration of exposure. Standardized questionnaires may provide a reasonably accurate description of ETS exposure and they can be used to assess exposure in etiologic studies. Non-smokers non exposed to ETS at home may not represent an appropriate control group in such studies. Indeed, they may be exposed to other sources, as indicated by the relatively high levels of urinary cotinine among ex-smokers non exposed at home in our study. Misclassification of these subjects may result in a dilution of the effect of the exposure. PMID- 10730484 TI - [Development of registries]. AB - Cancer registration took place in Italy in the first half of the Seventies with only one Cancer Registry in the province of Varese. More recently several cancer registries started their activity attaining population coverage of approximately 18% of the national population. The contribution from a network of cancer registries in Italy to the activity of cancer research and control is discussed in view of an improved exploitation of this tool. PMID- 10730485 TI - [Commitment and ambiguous complexity in scientific research]. AB - The manicheistic temptation of concluding that the good, but also the best science, is the science made by scientists morally and ideologically committed to alleviate both physical illness and health and social inequalities, would take us far away from the painful complexity of the mixture of good and bad, right and wrong, bright and obscure, and from the ambiguities and ambivalent situations that may originate from it, in which not only scientific research but our entire existence is soaked. The scientist should see very clearly his obligations toward society and his fellow humans in order to resist the ambiguous complexity where the interest for a scientific result can be exploited for ignoble purposes or, as Robert N. Proctor reminds us in his recent book "The Nazi War on Cancer", where "the routine practice of science can so easily coexist with the routine exercise of cruelty." Research committed to prevention and to the struggle for health and social equity was for quite a while successfully hampered by closing the financial channels and depriving it of the necessary means. The block by lack of funds has been replaced in recent times by a block by plenty through the attraction exerted on many scientists by availability of conspicuous and sure funds for research on themes chosen by the economic power. PMID- 10730486 TI - [Short-term effects of exposure to urban air pollution on human health in Europe. The APHEA Projects (Air Pollution and Health: a European Approach]. AB - During the '80s, evidence was collected that air pollutants concentrations close to, or lower than, air quality standards could negatively influence public health at short term, i.e. within a few days. The European Union financed, between 1993 and 1995, the study "Short term effects of Air Pollution on Health: a European Approach using epidemiological time-series data" (APHEA-1 project), involving more than 25 millions inhabitants in 15 cities, investigated between 1977 and 1991. In this paper, the main results, already published in various scientific journals, are reported. The health effects were studied as mortality for natural causes, cardiovascular and respiratory diseases, and as emergency hospital admissions for all respiratory diseases, bronchial asthma, and chronic obstructive pulmonary disease (COPD). The pollutants whose measures were available for the analysis are sulphur dioxide (SO2), nitrogen dioxide (NO2), particulate matter (PM) measured either as black smoke or total suspended particulate, and ozone (O3). The analysis was carried out by each participating group following a detailed protocol, defined during various workshops, through the construction of Poisson regression models, adjusted for autocorrelation and overdispersion, accounting for variables influencing the daily count of deaths, such as long time trends, season, temperature, day of the week. This paper reports the results of the meta-analysis, performed using the values of each city, as relative risk of dying or being admitted to hospital associated with increases of 50 micrograms/m3 in the 24 hours average concentrations of each pollutant. The daily number of natural deaths was associated with increases in the levels of PM, SO2, O3, and NO2. Cardiovascular and respiratory deaths were associated with increases of the levels of PM, SO2, and O3; cardiovascular deaths were associated also with increases of NO2 concentrations. Emergency hospital admissions for the whole group of respiratory disorders are less consistently associated with PM, SO2, and NO2, whereas there is evidence of association with O3. COPD admissions are related to the air pollutant levels, especially those of O3. Lastly, NO2 levels may play a role in exhacerbating asthma, and SO2 levels can have an effect on asthma in children. A second study is currently going on, involving over 40 millions residents in 34 cities. The aims of this project are: to evaluate the dose-response relationship, to investigate on synergy among pollutants, on the "harvesting" phenomenon, on the geographical differences inside Europe and with the U.S. through the collaboration with a similar American study now in progress. The existence of an association between daily variations in the levels of urban air pollution and adverse health effects was confirmed in Europe. This association is weak, but it involves the whole resident population, so it is a major cause of concern from the public health point of view. PMID- 10730487 TI - [Estimate of the proportion of skin tumors attributable to sun exposure in 3 Italian populations]. AB - Translation of recommendations on skin cancer prevention formulated by many national and international institutions into efficient steps requires measuring the potential impact in terms of preventable cases. This study analyses the proportion of skin cancers attributable to sun exposure in Turin, Trento and Ragusa. Estimates have been reached by applying the risk models calculated from the case-control study on melanoma in Turin and the Helios multi-centric study on skin carcinomas. The proportion of cases attributable to sun exposure has been subsequently calculated by correcting for the different proportion of skin characteristics among the populations taken into consideration. Attributable risks so calculated show medium to high values for melanoma in Turin (38%) and Trento (29%), while values are lower in Ragusa (17%). As for basal-cell carcinoma, attributable risk values are slightly inferior to those of melanoma. The risk of squamous-cell carcinoma attributable to sun exposure during outdoor work is the highest in Ragusa, with a value of 25%. Taking an annual incidence rate for melanoma of 9 cases per 100,000 inhabitants, and a preventive intervention with 50% of effectiveness, preventable cases should be 2 cases per 100,000 each year. This figure compared with 8 preventable cases of cervix cancer, in a similar population suggests an extremely cautious attitude in taking on programmes aiming to change life styles. PMID- 10730488 TI - Distribution and seasonal occurrence of Forcipomyia taiwana (Diptera: Ceratopogonidae) in the Nantou area in Taiwan. AB - We studied the distribution of Forcipomyia taiwana (Shiraki) in Taiwan, and found this species almost island-wide. Midge seasonality was studied for 4 yr at 3 sites in Nantou, central Taiwan, to identify the extent and causes of midge population outbreaks. The midge population in 1995 was significantly lower than in 3 other years because several typhoons inundated breeding sites. Maximum populations of F. taiwana occurred in June, July, and August. There was a highly significant correlation between the monthly abundance of F. taiwana and temperature and rainfall. A step-up multiple regression indicated that temperature was the most important factor leading to the outbreaks of F. taiwana. Temperature increases from 15 degrees C to near 30 degrees C will increase the midge abundance. PMID- 10730489 TI - Experimental bovine hypodermosis in Spain. AB - An experimental survey was carried out in western Spain to investigate both the chronobiology of Hypoderma spp. and the immunoresponse of their bovine hosts. This study was initiated with a new system of obtaining Hypoderma spp. larvae directly from their host, including the eclosion of adults from their pupae, infestation under natural but controlled conditions, and confirmation of the resulting infection. This survey was carried out over 2 cattle grub seasons; it was possible to infest and reinfest the experimental animals and to monitor them by both parasitological methods and by an enzyme-linked immunosorbent assay. This method permitted the evaluation of the development of anti-Hypoderma antibodies during the experiment. The experimental design also enabled us to establish the period of detectable H. lineatum infection to be from December until the end of April with the largest number of warbles observed during March and April. After a pupal period of < 30 d, adults were seen in April and May. Hypoderma bovis (de Geer) showed a delay of 2 m.o. relative to H. lineatum (de Villiers). This study reports a completed biological life cycle of Hypoderma spp. under controlled conditions in both natural and experimental environments. PMID- 10730490 TI - Temporal and spatial variation of phlebotomine (Diptera: Psychodidae) community diversity in a cutaneous leishmaniasis endemic area of Costa Rica. AB - Phlebotomine sand fly species composition and diversity were studied in Acosta, an area of Costa Rica endemic for cutaneous leishmaniasis. Two CDC miniature light traps per house were set overnight in 72 houses distributed in 6 localities, once a month, during the rainy seasons of 1994 and 1995. Of the 22 species collected, the most common were Lutzomyia serrana (Damasceno & Arouck), L. youngi Feliciangelli & Murillo, L. ylephiletor (Fairchild & Hertig), L. shannoni (Dyar), L. cruciata (Coquillett), L. gomezi (Nitzulescu), L. sanguinaria (Fairchild & Hertig), and Warileya rotundipennis (Fairchild & Hertig). The Czekanowsky index and the Sorensen index as modified by Bray and Curtis to consider abundance were used to compare species composition and beta-diversity. Comparisons were made among localities within years, between years, and localities each year. The data strongly indicated that phlebotomine species composition is more stable than diversity or abundance. PMID- 10730491 TI - Repellent and antifeedant activity of salicylic acid and related compounds against the biting midge, Culicoides impunctatus (Diptera: Ceratopogonidae). AB - An artificial membrane, blood-feeding method was used to assess the repellent and antifeedant effects of derivatives of salicylic acid on the biting midge Culicoides impunctatus Goetghebeur. Trans-2-hydroxycyclohexanecarboxylic acid, 6 methylsalicylic acid, 2,6-dihydroxy-4-methylbenzoic acid, and salicyluric acid caused highly significant inhibition of feeding, with salicyluric acid performing significantly greater than the other compounds. Investigating the effects of alkyl substitution of salicylic acid, o-thymotic and o-cresotic acids were also effective. A pilot clinical trial using salicyluric acid indicated that it provided a protective effect and it was hypothesized that this may have resulted primarily from contact, because no significant repellent effects were shown for this compound in a microscope slide test, designed to demonstrate repellency with volatile compounds. PMID- 10730492 TI - Inhibition of immature Ctenocephalides felis felis (Siphonaptera: Pulicidae) development in the immediate environment of cats treated with imidacloprid. AB - The hypothesis that dermally applied imidacloprid may transfer from treated cats, Felis catus L., to their immediate environment in quantities sufficient to have a significant effect on developing immature cat fleas, Ctenocephalides felis (Bouche), was tested in a controlled experiment. Flea eggs harvested from untreated donor cats were incubated on replicated samples from blankets used by treated or untreated cats under standardized conditions. As compared with controls, the percentage of adult flea emergence on blankets used by treated animals was reduced by 100% in the 1st wk after treatment and by 84, 60, and 74% in subsequent weeks (P < 0.001). PMID- 10730493 TI - Factors associated with distribution of Anopheles aquasalis and Anopheles oswaldoi (Diptera: Culicidae) in a malarious area, northeastern Venezuela. AB - Spatial and temporal abundance patterns of anopheline larvae and their relationships with wetland conditions were studied in an endemic malaria area in northeastern Venezuela, where Anopheles aquasalis Curry is the main vector. Larvae were sampled over a 2-yr period in 7 wetland types (brackish and freshwater herbaceous swamps, mangrove swamps, freshwater ponds, clear-cut marsh forests, small irrigation canals, and swamp forests), covering 3 environmental gradients (salinity, aquatic vegetation, and habitat permanence). Twelve variable were quantified to describe each habitat. Two species of anophelines were collected. An. aquasalis was the species with the widest distribution, and its highest abundance was in the seasonal brackish mangrove habitat during the rainy season. An. oswaldoi Peryassu was rarely encountered, but was mainly associated with the dry season and with the permanent fresh water wetlands (such as ponds). Principal components and correlation analyses revealed that the physicochemical (salinity, dissolved oxygen) variables of the wetland were associated most strongly with the spatial distribution of both species. Variations in salinity were strongly associated with the abundance of An. aquasalis. Both the occurrence and abundance of An. oswaldoi were most closely correlated with dissolved oxygen. Changes in seasonal abundance of both species were associated with rainfall. The relevance of these results to vector control in northern Venezuela is discussed. PMID- 10730494 TI - Intra-puparial development in Oestrus ovis (Diptera: Oestridae). AB - The phenology of intrapuparial development in Oestrus ovis L. is described, based on 302 specimens collected from the head cavities of goats and reared in the laboratory at a photoperiod of 12:12 (L:D) h and 32 and 16 degrees C. Dissection and histology of puparia at pupariation and at 3, 6, 9, 12, 15, 18, 21, 24, 30, 36, 42, 48, 66, and 72 h after pupariation and every day of the intra-puparial period showed that pupariation was achieved in approximately 12 h in heavily pigmented larvae (range, 2-46 h in postfeeding period). Larval-pupal apolysis began immediately after pupariation and was completed by 18-36 h after pupariation (prepupal period). The cryptocephalic pupa was found from this time to the 5th d, when head eversion occurred. Pupal-adult apolysis was initiated before head eversion and completed by day 7. The pharate adult presented progressive coloration in compound eyes (transparent, white, yellow, orange, red, brown, silver) while integumental pigmentation and sclerotization were in progress. Adult emergence occurred at 22 and 23 d in males and females, respectively. Changes in the weekly puparial weight of specimens reared under both field and laboratory conditions was described. It was concluded that although the intra-puparial development of O. ovis displayed some unique characteristics, it was essentially similar to other cyclorraphous flies. The actual pupal period of O. ovis lasted from the 2-7 d post-pupariation, whereas approximately two-thirds of the intra-puparial period was used for the maturation of the pharate adult. PMID- 10730495 TI - Bactericidal effects of sugar-fed antibiotics on resident midgut bacteria of newly emerged anopheline mosquitoes (Diptera: Culicidae). AB - A protocol was developed for significantly reducing resident midgut bacteria in newly emerged anopheline mosquitoes using a combination of antibiotics. Pupa harvested from colony-reared Anopheles gambiae s.l. Giles and Anopheles stephensi (Liston) were placed in cages wiped previously with 70% alcohol and kept under UV light for 24 h. Emerging adult mosquitoes were fed for 3 consecutive days on antibiotic solution, consisting of 0.4% gentamicin sulfate and 1% penicillin streptomycin solution in a 10% sterile sucrose solution. Bacterial suspensions of Escherichia coli, Klebsiella pneumoniae (Schroeter, 1886), and Pseudomonas stutzeri (Lehmann & Neumann, 1896) isolated from wild-caught anophelines were fed to antibiotic-treated mosquitoes starved for 24 h via either sugar or membrane feeding. Mosquitoes dissected 1 and 24 h after blood-feeding or sugar-feeding, and plated on trypticase soy agar plates, yielded the same type of bacteria fed originally without evidence of contaminants. There was no residual effect of the antibiotics on introduced single bacteria strains as judged by the presence of bacteria in antibiotic-treated mosquitoes. This experimental reduction of resident midgut bacteria and their replacement with single strains in newly emerged anopheline mosquitoes should facilitate further investigations of the interactions between malaria parasites and bacteria found in the midguts of mosquitoes. PMID- 10730496 TI - Method of infection does not alter response of chicks and house finches to western equine encephalomyelitis and St. Louis encephalitis viruses. AB - The effects of method of infection and virus dose on the viremia and antibody responses of 1-wk-old chicks and after-hatching-year house finches to infection with western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) viruses were studied under laboratory conditions. Using a capillary tube technique, females from 2 strains of Culex tarsalis Coquillett mosquitoes were estimated to expectorate from 1.0 to 1.7 log10 plaque forming units (PFU) of WEE and from 1.9 to 2.2 log10 PFU of SLE. Based on the proportion of parenterally infected females that transmitted and the number that blood fed during each experiment, virus doses per bird were estimated to be 1.0-1.9 log10 PFU for WEE and 1.4-2.3 log10 PFU for SLE. When infected with comparable doses of WEE by subcutaneous inoculation, there was no significant difference in the duration or magnitude of the viremia response between birds infected by mosquito bite or syringe; few birds developed a viremia response after infection with SLE, precluding analysis. In chickens, increasing the syringe dose of WEE from 0.3 to 1.7 log10 PFU/0.1 ml shortened the time when viremia first appeared from 3 to 1 d postinfection and increased the duration of the viremia period from 1 to 3 d, but did not alter the maximum viremia titer. In house finches, increasing the syringe dose of WEE from 2.6 to 3.3 log10 PFU/0.1 ml did not alter markedly the viremia response. Most birds developed antibody detected by enzyme immunoassay (EIA) or plaque reduction neutralization test (PRNT). In chickens, WEE EIA levels and PRNT titers were higher for birds infected by syringe than by mosquito bite, whereas in house finches the pattern was reversed. For birds infected with SLE, there was overlap among groups infected by mosquito bite or syringe. These results indicate that subcutaneous syringe inoculation provides a biologically sound mode of infection that did not alter viremia and antibody responses when compared with infection by mosquito bite. PMID- 10730497 TI - Response of house finches to infection with sympatric and allopatric strains of western equine encephalomyelitis and St. Louis encephalitis viruses from California. AB - Adult house finches from Kern County were inoculated subcutaneously with recent sympatric and allopatric isolates of western equine encephalomyelitis and St. Louis encephalitis (SLE) viruses made from Culex tarsalis Coquillett collected in Kern County and Coachella Valley, CA, respectively. Virulence, as measured by the amplitude of the viremia response during days 1 and 2 postinfection, varied significantly among strains, but independently of geographic origin. The intensity of the immune response, as measured by an enzyme immunoassay and a plaque reduction neutralization test, seemed to be independent of virulence, especially for SLE where some strains failed to produce a detectable viremia but elicited a strong antibody response. Our preliminary data indicated that strain virulence may be associated with the level of enzootic activity during the year of isolation. PMID- 10730498 TI - Response of the tick Dermacentor variabilis (Acari: Ixodidae) to hemocoelic inoculation of Borrelia burgdorferi (Spirochetales). AB - When Borrelia burgdorferi B31 low passage strain spirochetes were directly injected into the hemocoel of Dermacentor variabilis (Say) females, the bacteria were cleared from the hemocoel within < 24 h. Viable spirochetes were not found in hemolymph, salivary gland, or ovary tissues by subculturing or by IFA. The hemocyte population increased approximately 6 times within the first 6 h after inoculation compared with the uninoculated controls. In contrast, the soluble total hemolymph protein content decreased inversely with the increase in hemocytes. Borreliacidal activity was demonstrated with cell-free hemolymph from D. variabilis. In vitro antimicrobial assays using hemolymph from borrelia challenged and nonchallenged ticks resulted in 72% spirochete reductions compared with only 11.5%, respectively, within 1 h. Additional evidence of induced antimicrobial hemolymph protein activity was demonstrated by SDS-PAGE, which revealed upregulation of a lysozyme-like peptide (approximately 15 kDa) (22% increase) and the induction of a approximately 5.8 kDa peptide in the B. burgdorferi-challenged ticks. In contrast with the nonvector borne Bacillus subtilis, D. variabilis presented a rapid and robust response to challenge with cultured B. burgdorferi spirochetes and lead to their early elimination. The role of the tick immune system, including possible differences between vector and nonvector ticks, in determining the success of invasive bacteria is discussed. PMID- 10730499 TI - Horizontal transmission of triatoma virus through the fecal-oral route in Triatoma infestans (Hemiptera: Triatomidae). AB - Feces from Triatoma infestans (Klug) infected with TrV showed a large number of well-preserved viral particles when examined by electron microscopy. No viral particles were observed in suspensions of feces uninfected insects. Fecal suspensions inoculated parenterally into uninfected triatomines killed the insects within 36 h, showing that infective TrV is present in the feces of infected insects. It also is demonstrated that T. infestans becomes infected with TrV while feeding on contaminated chickens, and all the chickens used to feed a colony of triatomines infected with TrV showed high anti-TrV titer in their sera, although no TrV replication could be demonstrated in chickens. Oral infection of T. infestans by contaminated feces probably contributes to virus dispersal in nature. This observation provides the rationale for the potential use of TrV as a biological control agent. PMID- 10730500 TI - Prevalence of Ehrlichia chaffeensis (Rickettsiales: Rickettsiaceae) in Amblyomma americanum (Acari: Ixodidae) from the Georgia coast and Barrier Islands. AB - Human monocytic ehrlichiosis is an emerging zoonosis caused by infection with Ehrlichia chaffeensis Anderson, Dawson, Jones & Wilson, which is transmitted to mammals by ixodid ticks. Prevalence of infected ticks and distribution of infection foci indicate relative risk of human exposure to ehrlichiosis and may be influenced by factors such as geographic isolation, human disturbance, and the availability of suitable mammalian reservoir hosts. To test, this, individual and pooled lone star ticks, Amblyomma americanum (L.), were collected from three populations from the Georgia coast (1 mainland site and two barrier islands) and screened for E. chaffeensis. A species-specific, nested polymerase chain reaction (PCR) assay was used to amplify a 572 bp fragment of the E. chaffeensis 16S rRNA gene from infected ticks. PCR product specificity was confirmed by nucleotide sequencing. Our results showed the prevalence of infected ticks to be 0.0% (n = 151), 0.9% (n = 111), and 9.3% (n = 129) for Sapelo Island, St. Catherine's Island, and Fort McAllister, respectively. PMID- 10730501 TI - Laboratory and field evaluation of metallic copper on Aedes albopictus (Diptera: Culicidae) larval development. AB - Laboratory bioassays and field trials were carried out to study the effect of metallic copper on the development of Aedes albopictus (Skuse). Multiwire electric cable was used as a source of metallic copper. Three different doses were used in laboratory tests (5, 10, and 20 g/liter) and two in field tests (20 and 40 g/liter). In the laboratory, 10 g/liter induced high mortality and a lack of development in Ae. albopictus larvae and doses of 20 g/liter completely inhibited development. Larval mortality was higher in earlier instars than in third through fourth instars and pupae. No effects were reported on egg hatching. Copper ion concentration in water increased up to 574 ppb for 5 g/liter dose, 710 ppb for 10 g/liter dose, and 1,210 ppb for 20 g/liter dose, within week 6. The increasing concentration of copper in water was correlated positively with the decreasing production of adults. Copper ions concentration < 500 ppb did not or only slightly affected larval development and mortality of Ae. albopictus in laboratory tests. Copper concentrations between 500 and 1,000 ppb delayed larval development and caused high mortality. Copper concentrations > 1,000 ppb inhibited larval development completely killing all the larvae. This last result has been achieved by the use of a 20 g/liter dose of metallic copper in water. Copper also affected adult weight. In field trials, 20 g/liter reduced the number of larvae in treated pots by 90%, and 40 g/liter completely prevented oviposition. Moreover, the persistence of the toxic action of metallic copper in the field lasted for several months. PMID- 10730502 TI - Absence of insecticide resistance in Anopheles gambiae s.l. (Diptera: Culicidae) after four decades of residual house spraying in Gokwe District, Zimbabwe. AB - Field tests showed that Anopheles gambiae s.l. from 4 villages in Gokwe District, Midlands Province, Zimbabwe, were susceptible to 4% DDT, 0.025% deltamethrin, 0.025% alpha-cypermethrin, and 0.1% lambdacyhalothrin. In the laboratory, F1 progeny of field-caught females that were identified as Anopheles arabiensis Patton were completely susceptible to deltamethrin, alpha-cypermethrin, lambdacyhathrin, and DDT. The use of insecticides in Gokwe district has not yet induced detectable resistance in An gambiae s.l. PMID- 10730503 TI - Electrophoretic and morphological differentiation between two sympatric species of Adlerius: Phlebotomus brevis and Phlebotomus simici (Diptera: Psychodidae). AB - Phlebotomus simici Theodor and P. brevis Artemiev & Dergacheva are closely related species in the subgenus Adlerius that only may be identified by the male terminalia. Females only may be differentiated by collecting them in copula. Because females of P. simici may be involved in Leishmania transmission, their identity should be verified. Our study separated individual from 2 sympatric populations in Lebanon by means of agarose enzyme electrophoresis. Differential patterns were found at the PGM and HK loci. Results were confirmed by morphological reexamination of female spermathecae and pharyngeal armature. PMID- 10730504 TI - Vector competence of two species of mosquitoes (Diptera: Culicidae) from southern California for Dirofilaria immitis (Filariidea: Onchocercidae). AB - Two species of mosquitoes reared to adults from field-collected larvae or egg rafts in or near Los Angeles County, CA, were exposed to dogs with known levels of Dirofilaria immitis (Leidy) microfilariae per 20 cubic millimeters of blood. Aedes taeniorhynchus (Wiedemann) readily blood fed on an infected dog ingested microfilariae but did not become infected. Culiseta incidens (Thomson) also fed on an infected dog became infected, and 10% of the microfilariae developed to the L3 stage. Cs. incidens is a peridomestic species broadly distributed in Los Angeles County and abundant from February through December. The wide distribution of this species as well as its long annual period of reproductive activity in Los Angeles County may contribute to its potential as a vector of D. immitis. PMID- 10730505 TI - Alzheimer's disease diagnosis. AB - This section reviews some of the more prominent alternate causes of dementia that need to be excluded when pursuing a diagnosis of Alzheimer's disease. In practice, this exclusion cannot be done with 100% precision. Consequently, the accuracy of the clinical diagnosis of Alzheimer's disease is approximately 85%. Brain biopsy, which is seldom performed in practice, is the only method of moving diagnostic accuracy to near 100%. There is yet no intervention that halts or reverses the underlying pathophysiology of Alzheimer's disease, so the invasive brain biopsy is rarely justified to confirm a diagnosis. PMID- 10730506 TI - Treatment options for Alzheimer's disease. AB - An understanding of the basic pathophysiology and molecular mechanisms of Alzheimer's disease is essential to effective treatment of the disease. Despite multiple hypotheses related to the development and progression of Alzheimer's disease, no unifying theory is currently available. Inflammation, oxidation stress, estrogen hormone status, pathways for production of beta-amyloid42, apolipoprotein E state, cholinergic neuron depletion, and head injury are all possible contributors and therefore provide points of intervention or potential intervention in the development and progression of Alzheimer's disease. Thus, this article reviews current therapeutic modalities, including estrogen replacement therapy, Ginkgo biloba, and the two cholinesterase inhibitors approved in the United States, tacrine and donepezil. PMID- 10730507 TI - Management of behavioral problems in elderly patients with dementia. AB - A major challenge confronting healthcare providers in geriatrics involves the treatment of behavior problems in the elderly patient with dementia. The agitation affects the patient and the caregiver and their quality of life. A definitive diagnosis is difficult, and treatment often involves the injudicious use of medications. The purpose of this article is to identify the disorders involved, the assessment strategies, and pharmacologic and nonpharmacologic modes of therapy. PMID- 10730508 TI - Psychiatric disorders associated with Alzheimer's disease. AB - Alzheimer's disease is a cortical dementia with an insidious onset and relatively slow progression. In the early stages and throughout most of the disease, memory impairment is the primary problem. Any manifestation of psychiatric symptoms is generally secondary to the amnesia, the paramount symptom of early Alzheimer's disease. The psychiatric symptoms emanate from the memory impairment. Therefore, testing memory is essential. The first stage of Alzheimer's disease commonly is marked by anxiety and depression secondary to memory impairment, and delusions. In the second stage, delusions often become more bizarre. Impairment of visuospatial memory, improper advances, and obscene language begin to replace disinhibited behavior, often to the point of violence directed at others. Increasing agitation requires restraints. In the third and final stage, screaming, banging, and cursing are common features. Verbal and behavioral perseverations are very common. Fecal and urinary incontinence and gait apraxia are other features of the final stage, again with restraints often necessary. In addition to outlining the progression of Alzheimer's disease through these stages, this article summarizes the available pharmacotherapy for the various psychiatric manifestations of the illness prevalent at each stage. PMID- 10730509 TI - Caring for the caregivers. AB - Although direct care of the patient with Alzheimer's disease remains the focus of the physician's concern, the importance of evaluating and responding to the needs of that patient's caregivers cannot be ignored. Osteopathic physicians provide a truly holistic approach to treatment of the patient with Alzheimer's disease when that treatment includes the care of the entire family. This article provides a brief overview of the major issues affecting caregiving for the patient with Alzheimer's disease. It describes the warning signs of caregiver burnout; and it provides guidelines for accessing resources available to assist the caregivers. PMID- 10730510 TI - Residual patent ductus arteriosus shunting after single Gianturco coil occluding. AB - Transcatheter occlusion with Gianturco coils has become the treatment of choice for small patent ductus arteriosus (PDA). Coil occlusion was attempted in 20 patients with ductus diameter less than 4 mm who did not require other cardiac surgery. Sixteen of 20 patients had successful implantation. The mean age was 4.2 years. Their mean weight was 14.1 +/- 5.9 kg. The mean ductus diameter was 2.21 +/- 0.91 mm (range 1-3.7 mm). Nine patients had complete occlusion but 7 had residual shunting immediately after the procedure. However, 4 patients had spontaneous resolution of residual shunts at 6 months after the procedure. The other 3 who had diameter of ductus greater than 3 mm still had significant residual shunt at 6 months and 1 year after the procedure. The second coil was successfully implanted in one of these 3 patients and the closure of PDA was accomplished. We concluded that the second coil should be implanted if the ductus diameter is greater than 3 mm and significant residual shunt is still demonstrated angiographically after the first coil implantation. PMID- 10730511 TI - Factors affecting mortality in Ebstein's anomaly of the tricuspid valve. AB - Ebstein's anomaly of the tricuspid valve is a relatively uncommon congenital heart defect. Twenty-one patients (11 boys and 10 girls) with Ebstein's anomaly were reviewed regarding clinical characteristics and factors related to the outcome of this lesion. Their ages at presentation ranged from 1 day to 13 years (median = 11 months). Eleven patients presented in infancy period, 6 of them were neonates. Common clinical findings were systolic murmur (85.7%) and cyanosis (57.1%). Laboratory findings included cardiomegaly on chest X-rays (95.2%), right bundle branch block pattern (76.2%) and right atrial enlargement (61.9%) on electrocardiography. Diagnosis and grading of severity were established by echocardiography. Among 21 patients, 4 were lost during follow-up. Seventeen patients were followed for 3-72 months. Six patients (28.6%) required surgery, 5 of whom died following surgery. Two patients died during the medical follow-up. Factors affecting cardiac death were the younger age at presentation, onset of cyanosis in infancy period, associated PS or PA, the lower insertion of the septal leaflet of the tricuspid valve and the higher ratio of the combined area of right atrium and atrialized right ventricle to that of functional right ventricle and left heart chambers. PMID- 10730512 TI - The role of prostacyclin (PGI2) and thromboxane A2 (TXA2) in pathogenesis of dengue hemorrhagic fever (DHF). AB - In previous studies it has been demonstrated that the levels of plasma 6 keto prostaglandin F1 alpha (6-K-PGF1), the stable metabolite of PGI2 were elevated in DHF patients during shock. In this study it is hypothesized that excessive PGI2 production plays a very important role in developing serious clinical manifestations of dengue shock syndrome (DSS) patients. In addition, an attempt was made to determine whether TXA2 has any significant role in such patients. Plasma 6-K-PGF1 and thromboxane B2 (TXB2), the stable metabolites of TXA2 were determined in 43 normal healthy children (NC) and 54 DHF patients without shock (DHF-N) and 33 DHF patients with shock (DHF-S). Subjects aged between 2 and 14 years. Plasma 6-K-PGF1 and TXB2 were measured by radioimmunoassay and the ratio of TXB2/6-K-PGF1 were also calculated. In 43 NC the values of plasma TXB2, 6-K PGF1 and TXB2/6-K-PGF1 ratio were (mean +/- SE) 372.3 +/- 17.1, 150.1 +/- 2.4 and 2.52 +/- 0.12 pg/ml, respectively. In 54 DHF-N patients the corresponding values were 409.1 +/- 16.0, 278.4 +/- 11.6 and 1.54 +/- 0.06 pg/ml; whereas those in 33 DHF-S patients were 254.3 +/- 26.2, 349.1 +/- 20.5 and 0.757 +/- 0.073 pg/ml, respectively. Plasma 6-K-PGF1 levels of DHF-N and DHF-S patients were significantly greater than those in normal children (p < 0.001, p < 0.01 respectively). The plasma 6-K-PGF1 levels seem to be greater in DHF-S patients than in the DHF-N patients, however the difference in values were not statistically significant (p > 0.05). These findings indicate that plasma PGI2 level is significantly increased in DHF particularly during shock. Plasma TXB2 levels of DHF-N had no significant statistical difference from those of NC (p > 0.05); however, those in DHF-S patients were significantly lowered (p < 0.001) than those of NC and DHF-N patients. The findings suggest the important role of TXA2 to compensate for excessive PGI2 secretion in DHF patients. The failure or inadequate TXA2 production may eventually lead to shock. The ratios were significantly reduced in both DHF-N and DHF-S patients when compared to those of NC (p < 0.001 both). The ratio in DHF-S patients was also significantly lowered than that in DHF-N patients (p < 0.001). It is suggested that the imbalance between TXA2 and PGI2 production exists during DHF infection. The more reduction of plasma TXA2/PGI2 ratio leads to more overt and serious clinical manifestations of the disease. PMID- 10730513 TI - Thyroid function in healthy Thai neonates. AB - OBJECTIVES: To construct a normative data for serum thyroxine (T4), free T4 (FT4), triiodothyronine (T3) and thyrotropin (TSH) in Thai neonates. STUDY DESIGN: A cross-sectional study of 275 healthy full-term neonates was conducted. Blood samples were obtained from umbilical cords of the neonates and from heel pads of infants aged 1-30 days. Hormone measurements included serum T4, FT4, T3 and TSH. RESULTS: Mean serum T4 and FT4 levels rapidly increased after delivery to the maximum level at 1-3 days of age. Thereafter, they declined to a steady state level within 2-4 weeks. Mean serum T3 level was very low at birth. The concentration increased 3-5 times and reached a steady state levels within 1 week. In contrast, mean serum TSH declined from birth and the level at 1-3 days of age was slightly less than that of the cord blood. It changed little after 3 days of age. Previous studies have shown a transient TSH surge in the first 24-48 hour of life. TSH surge was not apparent in our study because samples were not obtained from infants < 24 hours old. Therefore, if TSH is measured for screening of congenital hypothyroidism, samples should be obtained from umbilical cord or infants aged > 48 hours. CONCLUSIONS: This study provides the normative data for thyroid function tests in Thai full-term neonates. These data are useful for detection and verification of hypothyroidism in a screening program for congenital hypothyroidism. PMID- 10730514 TI - Five-year thyrotropin screening for congenital hypothyroidism in Ramathibodi Hospital. AB - OBJECTIVES: To detect newborns with congenital hypothyroidism (CH) and to treat the affected infants as early as possible. STUDY DESIGN: Cord blood thyrotropin (TSH) screening for CH in Ramathibodi Hospital began in 1993. From October 1993 to December 1998, 35,390 neonates were screened. The infants with elevated TSH level of greater than 30 mU/L were recalled for verification of CH. Confirmation tests included total thyroxine, free thyroxine and TSH level. Thyroid scan and uptake were performed in some affected infants. RESULTS: Twelve infants with CH were detected resulting in an incidence of one in 2,949 live-births. All affected infants were asymptomatic at birth. Of 12 infants with CH, one premature neonate had a delayed TSH elevation and was diagnosed as having primary hypothyroidism at 2 months of age. The recall rate for validation of CH based on a cut-off value at serum TSH level of greater than 30 mU/L is 1.1 per cent. If the cut-off value of serum TSH level was raised to greater than 40 mU/L, the recall rate would decrease to 0.43 per cent. None of the affected infants had cord blood TSH level of less than 50 mU/L except one premature patient. Therefore, beginning in January 1997, the cut-off value of TSH was raised to 40 mU/L or greater. Pitfalls in this program include incomplete blood-specimen collection and incomplete follow-up. To strengthen the program, improvements were made in the follow-up system from 1996 onward. Therefore, the coverage for blood-specimen collection progressively increased from 84 per cent in 1994 to 96 per cent in 1998. Simultaneously, the patients' return after recalls also increased from 38 per cent to 100 per cent. CONCLUSIONS: The incidence of CH in Ramathibodi Hospital is approximately 1:3,000 live-births. The optimal cord blood TSH level for recall is 40 mU/L or greater. The intensification of follow-up strategy resulted in better response to recall and earlier treatment in the affected infants. PMID- 10730515 TI - Gonadotropin-releasing hormone testing in premature thelarche. AB - Premature thelarche (PT) is characterized by isolated breast development in girls prior to 8 years of age. In addition, there is neither growth spurt nor advanced bone age. It has been suggested that luteinizing hormone (LH) response to gonadotropin-releasing hormone (GnRH) alone is adequate to distinguish central precocious puberty from PT. However, LH response to GnRH is greater in infancy than that in childhood. Therefore, gonadotropin response to GnRH in girls with isolated premature breast development in different age group was studied. Thirty six girls with isolated PT (aged 0.25-8 years) were evaluated. They were classified into 2 groups; aged < 4 years (group A: mean age 1.57 +/- 0.87 years, n = 13) and > or = 4 years (group B: mean age 6.97 +/- 0.94 years, n = 23). Initial evaluation included X-ray bone age, pelvic sonography and GnRH testing. Patients were followed for at least 1 year to confirm that no patient had progression into puberty. Bone ages in both groups were within mean +/- 2 SD in all patients. Pelvic sonography was performed in all patients which revealed no abnormality of ovaries and uterus. Pubertal response to GnRH stimulation is characterized by peak LH of > 20 IU/L or delta LH of > 15 IU/L which is generally greater than peak follicle stimulating hormone (FSH) or delta FSH, respectively. Mean peak LH and delta LH in group A were 13.0 +/- 6.06 and 11.4 +/- 5.92 IU/L whereas those in the group B were 8.5 +/- 4.10 and 6.3 +/- 3.49 IU/L. Therefore, LH response to GnRH in group A was significantly higher than that in group B (p < 0.05). In addition, the mean peak FSH and delta FSH in group A were 120.5 +/- 45.87 and 109.9 +/- 42.09 IU/L whereas those in the group B were 48.7 +/- 24.05 and 39.9 +/- 23.69 IU/L. Therefore, FSH response to GnRH in group A was significantly greater than that in group B (p < 0.001). LH response to GnRH alone can distinguish prepuberty from puberty in girls > 4 years of age. However, in prepubertal young girls with PT aged < 4 years, pubertal LH response can occur, i.e. peak LH > 20 IU/L. Hence, the greater FSH response to GnRH than that of LH would confirm the diagnosis of premature thelarche in this group. Therefore, the evaluation of FSH response to GnRH is beneficial to distinguish puberty from prepuberty in young girls. PMID- 10730516 TI - Combined raw cornstarch and nifedipine as an additional treatment in persistent hyperinsulinemic hypoglycemia of infancy. AB - This study reports the result of treatment with the combination of raw cornstarch and nifedipine in two infants affected with hyperinsulinemic hypoglycemia of variable severity. The first infant developed hypoglycemia during early neonatal period and required subtotal pancreatectomy. She still developed hypoglycemia after her second operation. The second infant developed hypoglycemia at the age of 7 months. Raw cornstarch and nifedipine efficiently normalized both infants' blood glucose levels. Although they still need frequent feedings, no hypoglycemic episode was reported except when they were sick. Their growth and development were markedly improved after initiation of treatment. PMID- 10730517 TI - Reduction of rotavirus infection in children receiving bifidobacteria supplemented formula. AB - This study was conducted at Pakkred Babies Home, Bangkok, Thailand; with the hypothesis that children receiving probiotic-supplemented milk-based formula may be protected from developing diarrheal diseases. Salivary rotavirus-specific IgA antibody was used as an indicator of rotavirus infection. One hundred and seventy five children, aged 6-36 months, were enrolled in the study. They were divided into 3 groups according to the type of formula given. There were 81 episodes of diarrhea during an 8-month study period, most of which were caused by bacterial enteropathogens. Ninety-seven pairs of salivary samples were adequate for the analysis of rotavirus antibody. Among 23 children receiving milk-based follow-up formula and serving as control group, 30.4 per cent of them had > or = 4-fold increase in the antibody titre, indicating subclinical rotavirus infection. The majority of children in the other 2 study groups, receiving the same formula supplemented with either Bifidobacterium Bb12 alone or together with Streptococcus thermophilus, had no significant change in the antibody titres between the two time points. The results of this study support our hypothesis that children receiving bifidobacteria-supplemented milk-based formula may be protected against symptomatic rotavirus infection. PMID- 10730518 TI - Low cost locally prepared fibrin glue for clinical applications: reported of 145 cases. Committee of Bangkok International Hemophilia Training Center. AB - Fibrin glue (FG) is one of the blood products known to be very useful for local hemostatic measure and as a medically valuable tool for adhesion, sealing, anastomosis, repair microvascular and nerve grafts in medical and surgical procedures. Before 1996, FG was used to a limited extent in Thailand due to the high cost. Technology for locally prepared FG was transferred to Bangkok International Hemophilia Training Center of the World Federation of Hemophilia (IHTC-WFH) in July 1996 by Prof. Uri Martinowitz and the late Prof. Henri Horoszowski. Since then FG has been widely used and proved to be very useful in Thailand. This paper reports 145 cases using low cost locally prepared FG at Ramathibodi Hospital during November 1996 to December 1997. A total of 145 cases with age range from 5 months to 73 years, which included 55 pediatrics and 90 adults, 100 males and 45 females. The amount of FG used was 1-80 ml per case. Clinical procedures included dental surgery (46), open heart surgery (35), ENT (28), orthopedic (13) including 2-3 joint correction in one session in 2 hemophiliacs, neurology (11), plastic repair (7), liver (2) and severe bleeding in dengue hemorrhagic fever (3). Forty-seven cases had hemostatic disorders. The result of local hemostatic, adhesive and sealant effect of FG was satisfactory with no complications. In open heart surgery, the amount of content in chest drain decreased and none required reopen-surgery to stop bleeding. Dental surgery was performed in 43 patients with bleeding disorders i.e. hemophilia, idiopathic thrombocytopenic purpura, leukemia, severe thrombocytopenia, patients on anticoagulant, etc. Only 3 cases (7%) required blood component compared to all of the 50 no-FG controlled cases (100%) that required blood component therapy. FG has proved to be very useful in many aspects i.e. minimizing blood product usage, decreasing medical workload, reducing medical cost and increasing patients' convenience and satisfaction in particular. PMID- 10730519 TI - Home treatment for patients with congenital bleeding disorders in a developing country. AB - Ninety-six patients with congenital bleeding disorders were enrolled in a home treatment program from 1979 to 1997. The diagnosis included severe and moderate hemophilia A and B (n = 63), mild hemophilia A (n = 18), von Willebrand disease (vWD) (n = 12) and congenital factor VII deficiency (n = 3). The median age was 9 years and the median duration of follow-up was 4 3/12 years. The home treatment was modified 4 ways: (1) Using locally-prepared single units of fresh dry plasma in the majority of the patients while mild hemophilia A and vWD patients received 1-deamino 8D-arginine vasopressin. (2) Recruiting local health personnel as the primary care providers. (3) Teaching and training patients and parents intensively. (4) Maintaining an effective control system. The heartful effort of the health personnel was not in vain; the patients learned to take good care of themselves. Twenty patients and 20 parents or relatives were able to perform venipuncture properly and no adverse effect was observed. Since the hemorrhage was treated very early, the severity and sequelae of bleeding were decreased. The utilized blood components and days of hospitalization were reduced. Impressively, the absenteeism from work or school was minimized so that the patients could enjoy a near normal life in their family, school and society. PMID- 10730520 TI - Disseminated intravascular coagulation findings in 100 patients. AB - A retrospective study of 100 patients with disseminated intravascular coagulation from 1993 to 1997 is reported. Forty-five patients were neonates with a mean age of 12.6 days and 55 patients were infants, children and adolescents with a mean age of 6 years and 3 months. Most of them (91.5%) had complicated underlying conditions which included congenital anomalies, prematurity, malignancy, hematological and various diseases. Additionally, every patient had triggering conditions commonly identified as gram-negative septicemia. Bleeding and thromboembolic manifestations were found in 59.4 per cent and 19.8 per cent, respectively. The laboratory findings revealed red blood cell fragmentation, 89.6 per cent and thrombocytopenia, 85.8 per cent. Natural anticoagulants were studied in a few cases and revealed low levels of antithrombin III and protein C. The prompt effective management included treatment of underlying diseases, identification and relief of triggering conditions, correction of thrombocytopenia and coagulopathy, and fully supportive care. The overall case fatality rate was 41.6 per cent which was not correlated with age, underlying diseases, triggering conditions, manifestation of bleeding, thromboembolism or shock, and exchange transfusion. However, a significant lower case-fatality rate was found in patients with positive culture (25%) as compared to those with sepsis and negative culture (51.7%) (p = 0.044). In addition, the febrile neutropenic patients, who showed good response to the administrated granulocyte colony stimulating factor (G-CSF), survived from the DIC. PMID- 10730521 TI - The efficacy and safety of lyophilized cryoprecipitate in hemophilia A. AB - This prospective study of assessing the efficacy and safety of lyophilized cryoprecipitate (LC), which was heat-treated at 60 degrees C for 25 hours, was conducted in 23 patients with hemophilia A (severe 13, moderate 9, mild 1) at the International Hemophilia Training Center, Bangkok from 1997 to 1998. A total of 223 infusions of LC were given. The status of the patients could be classified into 4 groups: group I, non-bleeding (n = 13); group II, severe bleeding requiring hospitalization (n = 9); group III, appendectomy (n = 1) and group IV, early bleeding controlled by modified home treatment (n = 200). Pharmacokinetic studies were conducted in groups I and II. The mean in vivo half-life of factor VIII clotting activity (F VIII:C) was 12.6 hours and the mean in vivo incremental recovery at baseline was 2.1 per cent/unit/kg. The mean clearance was 3.22 ml/kg/h. There was no statistically significant difference in these parameters between groups I and II (p > 0.05). The hemostasis was successfully achieved and 1 to 2 small urticarial wheals were observed in only 2 infusions. In addition, 9 out of 23 patients received LC exclusively for 1 year. None of them developed inhibitor to F VIII:C nor did any contract additional transfusion-transmitted infection except one who developed anti-hepatitis C virus seroconversion after receiving 16 bottles of LC in 4 months. Therefore, the more efficient virus inactivation in the preparation of LC should be established. PMID- 10730522 TI - The correlation of transferrin saturation and ferritin in non-splenectomized thalassemic children. AB - Thalassemia is a public health problem in Thailand. Progressive iron overload is the life-limiting complication commonly found in thalassemic patients. The assessment of body iron stores is essential for determining the need and efficacy of iron chelation. The parameters of serum iron, total iron binding capacity (TIBC), and serum ferritin were studied in 79 children with thalassemia diseases. The ages ranged from 1 to 17 years with a mean of 7 years and 10 months. Neither of them had clinical symptoms of hepatitis. The correlation between transferrin saturation (TS = serum iron/TIBC x 100) and serum ferritin was shown in the equation of TS = 10.253 In (ferritin) (r = 0.956, p = 0.000). For example, TS = 70.83 per cent indicates serum ferritin of 1,000 ng/ml. Thus, where serum ferritin is not feasible but serum iron and TIBC are available, TS can be used to estimate the level of serum ferritin. Therefore, the assessment of iron stores and monitoring of iron chelation in thalassemic patients can be effectively achieved. PMID- 10730523 TI - Subcutaneous portacath utilization in pediatric oncology patients: Ramathibodi Hospital experience. AB - Subcutaneous portacaths (SQP) placement in 19 pediatric oncology patients were studied. Complications of SQP were evaluated. Two patients had premature SQP removal due to fungal infection and breakage, 1 for each. Two patients had catheter-related bacteremia which was resolved by antibiotic administration. Only 1 patient had occasional difficult blood drawing episodes, because the tip of catheter was inserted through external jugular vein instead of subclavian vein. There were no other serious complications except that some of them had clotted formations, which were resolved easily by urokinase administration. Long-term SQP utilization was possible in 17 of 19 patients, with the average time of 7.5 months. Few complications occurred in the group of patients studied. SQP improved quality of medical care and significantly lessened the anxiety of patients who need long-term chemotherapy treatment. Therefore, placement of the intravenous access device is feasible for pediatric oncology patients in Thailand. The patients are no longer suffering from repeated venipunctures. Although it is expensive, it is convenient and useful for some patients with relatively high socioeconomic status. It should be considered for every pediatric cancer patient who needs prolonged chemotherapy and who has affordable means. PMID- 10730524 TI - Neonatal polycythemia: effects of partial exchange transfusion using fresh frozen plasma, Haemaccel and normal saline. AB - BACKGROUND: Neonatal polycythemia remains a significant clinical problem in Thailand. Partial exchanges transfusion (PET) with fresh frozen plasma (FFP) has been the mainstay of management for this condition in Thailand. Since FFP is difficult to find in certain areas and can cause concerns of transfusion related diseases, this study was undertaken to investigate the possibility of using plasma substitute and normal saline (NSS) for PET in the newborn infant with polycythemia. OBJECTIVE: 1. To compare the rate and duration of decrease of venous hematocrit (Hct) before and after PET with FFP, Haemaccel and NSS. 2. To compare any complications from using FFP, Haemaccel and NSS such as coagulation defect, electrolytes change, etc. in PET. METHODS AND SUBJECTS: A randomized prospective trial was conducted in Neonatal Unit, Department of Pediatrics, Ramathibodi Hospital. The first phase of study: July 1, 1993 to June 30, 1994: randomized prospective trial using FFP or Haemaccel for PET in 26 newborn infants with polycythemia. The second phase of study: July 1, 1994 to June 30, 1995: consecutive enrollment trial using NSS for PET in 38 consecutive newborn infants with polycythemia. RESULTS: There was significant decrease in Hct in both groups after PET but there was no statistically significant difference in the rate of decrease of Hct. There was no significant difference in biochemical profiles in both groups of infants 24 hours after PET. In the NSS group, there was significant decrease of Hct level after PET. There was no significant change of biochemical profiles and coagulation activity in these patients 24 hours after exchange transfusion. There were 2 patients with complications related to umbilical venous catheter and PET. CONCLUSION: Haemaccel and NSS can be safely used for PET to treat neonatal polycythemia. However, the attending physician should be aware of possible complications related to umbilical venous catheterization and PET. PMID- 10730525 TI - Indomethacin prophylaxis for patent ductus arteriosus (PDA) in infants with a birth weight of less than 1250 grams. AB - BACKGROUND: Very low birth weight (VLBW, less than 1500 g) and extremely low birth weight infants (ELBW, less than 1000 g) are the premature infants that are most likely to develop symptomatic PDA. Intravenous indomethacin has proven effective in prevention of PDA in many prospective trials. This strategy will be a useful adjunctive therapy for premature infants in Thailand. OBJECTIVE: To answer the following questions: 1. Will multiple doses of intravenous indomethacin, given to VLBW infants within the first day of life, effectively prevent the occurrence of symptomatic PDA? Are there any side effects or complications? 2. Will this strategy be more beneficial in ELBW? METHODS AND SUBJECTS: The study included thirty VLBW infants born at Ramathibodi Hospital, with birth weights ranging from 630 to 1230 g. They were randomized into 2 groups of 15 infants each. One group received 3 doses of intravenous indomethacin at the dosage of 0.2 mg/kg initially and then 0.1 mg/kg every 12 hours for 2 more doses; the second group received a placebo. The study was performed by a double blind control. RESULTS: Sixteen infants developed symptomatic PDA, 4 in the indomethacin group and 12 in the placebo group. The decrease in incidence of PDA is statistically significant. But when the data was analyzed separately for the VLBW and ELBW groups. The effects were only significantly different in ELBW but not yet significant in the VLBW group. There was a statistically significant difference in the incidence of severe intraventricular hemorrhage (IVH) (grade 3 or higher) in the ELBW infants. CONCLUSION: Intravenous indomethacin therapy given to VLBW infants with a birth weight of less than 1250 g decreased incidence of symptomatic PDA with no significant permanent side effects. The effect was markedly noticeable in ELBW infants. Incidence of severe IVH was also markedly decreased in the ELBW infants who received indomethacin. PMID- 10730526 TI - Single daily dose of gentamicin in the treatment of pediatric urinary tract infection. AB - Fourty-nine patients aged 6 months to 12 years old with suspected urinary tract infection (UTI) were evaluated in this open randomized study. Twenty-seven patients received gentamicin 4.5 mg/kg/d once daily (OD group) and 22 patients received the same daily dose in three divided doses (TID group) for 3 days before being switched to amoxy-clavulanic acid. Ninety-six per cent (26/27) of the OD group had peak gentamicin within therapeutic level while 40 per cent (9/22) of the TID group had peak gentamicin within therapeutic level. One in OD group had high gentamicin level due to technical error in obtaining blood sample. None in neither group had trough level in toxic level. Only 24 patients had confirmed UTI and were evaluated for clinical efficacy and toxicity. Demographic data were the same in both groups except there were more males in OD group (8:3 vs 4:9). Patients in OD group became afebrile earlier than TID group (8.69 vs 15.31 hours) but no statistically significant difference. All patients had negative urine culture results within 48 hours. None had clinical nephrotoxicity in both groups. More patients in TID group had laboratory nephrotoxicity (5/11 vs 2/13) but no statistically significant difference. We conclude that gentamicin can be given safely and efficiently as single daily dose or thrice daily but more cost effective and less time consuming in once daily dose. PMID- 10730527 TI - Comparison of urine anion gap, urine osmolal gap and modified urine osmolal gap in assessing the urine ammonium in metabolic acidosis. AB - Twenty-four hour urine and spot urine samples from 29 patients with metabolic acidosis were collected for evaluation of urine ammonium in relation to urine anion gap, urine osmolal gap (OG) and modified urine osmolal gap (MOG). Their underlying diseases included SLE in 8, RTA in 7, CRF in 6, RPGN in 2 (one with SLE), Lowe syndrome in 2, on acetazolamide in 2, gastroenteritis in 2, and CAH in one. Twenty-three patients had normal serum anion gap (< 14 mmol/L). Their mean CO2 was 13.77 (9.4-17.9) mmol/L, net acid excretion (NAE) was 33.18 +/- 35.36 mmol/24 hour, NH+4 excretion was 29.16 +/- 31.97 mmol/24 hour. Neither the 24 hour urine nor spot urine anion gap correlated with corresponding urine NH+4 with or without adding urine HCO-3 in the calculation. Spot urine NH+4 correlated well with urine OG (r2 = 0.82, p < 0.001) and less with MOG (r2 = 0.339, p < 0.006). The urine osmolality was well correlated with the sum of 2 (Na+ + K+ + NH+4) + urea for both spot (r2 = 0.990, p < 0.001) and 24 hour urine (r2 = 0.907, p < 0.001) collection. Twenty-four hour urine NH+4 did not correlate with the OG or the MOG. There was no correlation between spot urine NH4/Cr ratio and 24 hour urine NH4/Cr ratio (r2 = 0.243, p = 0.53) nor between spot NAE/Cr ratio and 24 hour urine NAE/Cr ratio (r2 = 0.380, p = 0.014). Therefore in the presence of low urine NH+4 (< 100 mmol/L), urine osmolal gap may be used to determine urine NH+4 indirectly with good correlation. Twenty-four hour urine collection is still necessary to assess renal acidification. PMID- 10730528 TI - The efficacy of intravenous pulse cyclophosphamide in the treatment of severe lupus nephritis in children. AB - BACKGROUND: The intermittent intravenous cyclophosphamide in the treatment of lupus nephritis in adults is well tolerated and associated with clinical improvement and long term stability of renal function. However, there are few reports about the efficacy of intravenous pulse cyclophosphamide (IPC) of severe lupus nephritis in children. OBJECTIVE: To evaluate the clinical efficacy, renal function, renal outcome and complications of IPC therapy in children with severe lupus nephritis. METHOD: Prospective study. PATIENTS: Children with severe lupus nephritis have been followed-up for at least 6 months. Treatment regimen: Intravenous pulse cyclophosphamide 0.5-0.75 g/m2 given monthly for 6 months with subsequent doses given at 2-3 months interval up to 3 years and combined with low dose oral prednisolone therapy. RESULTS: Thirty-one children (mean age: 12.31 +/- 2.03 years; female:male = 24:7) with severe lupus nephritis received IPC therapy. 24 out of 28 patients (85.7%) had diffuse proliferative glomerulonephritis. After 3 months of treatment, most patients were clinically improved as evidenced by significant improvements in 24-hour urine protein, creatinine clearance, serum creatinine, BUN, serum albumin and C3 level. These improvements were sustained up to 18 months and were accompanied by a significant reduction in prednisolone dosage. Renal outcome at the last follow-up (range = 6-76 months) demonstrated that twelve patients (38.7%) had complete remission, 18 patients (58.0%) still had significant proteinuria and only one had serum creatinine of 1.6 mg/dl at 42 months. One child progressed to end stage renal diseases during IPC therapy. Five patients had severe infections during the treatment resulting in one death. Hemorrhagic cystitis and malignancies were not found. CONCLUSION: Treatment of severe lupus nephritis in children with intravenous pulse cyclophosphamide is associated with favorable short term results. Severe infections are the major complications. PMID- 10730529 TI - Seizures after intracranial surgery in pediatric patients. AB - We retrospectively reviewed the occurrence of seizure after performing intracranial operations in children aged less than 15 years. During a 5-year period, there were 18 patients out of a total of 155 who developed one or more seizures within 1 year after operation. The majority of patients (55.6%) had the onset of seizures within 24 hours. Seventy-two per cent of the patients had partial seizures. Two patients who developed immediate postoperative seizures had sodium derangement. Eight of 10 patients who had early onset seizures had had an operation for supratentorial lesions. Among the 6 patients who developed seizures after infratentorial tumor removal, the cause of seizure was not known in 4 patients. We emphasize that prompt investigations to exclude any structural lesions and other possible causes of seizure, especially electrolyte disturbance along with appropriate antiepileptic drug administration, are important in patients who develop seizures after the operation. The recommendation for routine administration of antiepileptic drug prior to the operation has yet to be concluded. PMID- 10730530 TI - Elevated serum cholesterol levels in Bangkok children and adolescents. AB - Hypercholesterolemia is a major cardiovascular risk factor. This study aimed to assess serum total cholesterol (TC) levels of children and adolescents living in Bangkok, Thailand. During 1995-1997, nonfasting blood samples were obtained from 570 healthy school children and adolescents aged 9-18 years. The mean TC levels ranged from 143-180 mg/dl in males and from 145-202 mg/dl in females. The prevalences of hypercholesterolemia (TC > or = 200 mg/dl) were 12.2 per cent and 20.3 per cent in males and females, respectively. Twenty-eight per cent of males and 26.9 per cent of females had borderline values (TC 170-199 mg/dl). TC inversely correlated with age (r = -0.16, P < 0.01) in males. The findings indicate that notable percentage of these children had elevated cholesterol levels and warrant additional study concerning risk factors and tracking of lipoprotein levels from childhood into adulthood. PMID- 10730531 TI - Effects of tuna fish oil on hyperlipidemia and proteinuria in childhood nephrotic syndrome. AB - This is a randomized, double-blind, placebo-controlled, cross-over study to determine whether tuna fish oil decreased hyperlipidemia and proteinuria in children with steroid-resistant nephrotic syndrome. Five boys were supplemented with both 4 grams of tuna fish oil and placebo in a randomized order for 8 weeks of each treatment separated by 6-week washout period. The results showed no statistically significant difference in serum creatinine, triglyceride, cholesterol, urine protein and creatinine clearance between fish oil supplemented group and placebo group. Small sample size, low dosage, short duration of supplementation and wash-out period are among the important limitations in this study. Further study should be performed to identify the effects of fish oil on this entity in nephrotic syndrome. PMID- 10730532 TI - Plasma free amino acid contents in healthy Thai subjects. AB - This preliminary investigation determined the plasma amino acid concentrations in 136 healthy subjects. The subjects were divided into four groups according to their ages: gr 1; 1-3 years, gr 2; 4-11 years, gr 3; 12-19 years and gr 4; 20-45 years. Comparing among the groups, the results showed that in younger children (age 1-3 years) the essential amino acids were slightly lower than the other groups. A wide range for most amino acids were observed. The ratio of essential (EAA): non-essential amino acid (NEAA) in very young children (1-3 years) was lower than children, adolescents and adults. These ratios were normal (> 0.5). The data have been compared with those from several studies performed in normal healthy subjects of different age groups, races and dietary habits. The results showed that most of the mean values of individual amino acid were higher than others except cystine. The EAA:NEAA ratio of each age group was comparable with other reports. The mean values of plasma amino acids and EAA:NEAA ratio in healthy subjects were determined for diagnosis, follow-up and prognosis of malnourished condition, abnormal amino acid metabolism and as a reference for other purposes. PMID- 10730533 TI - Peak expiratory flow rate values of students in Bangkok. AB - BACKGROUND: As there are considerable variations in normal values of peak expiratory flow rate (PEFR) shown by studies from various population, a study is required to obtain normal values of PEFR in Thai children. OBJECTIVE: To determine the values of PEFR of students in Bangkok. METHODS: In a cross sectional study of PEFR measured with standard Wright peak flow meter, 501 normal students, aged 5 to 15 years, from five public schools in Bangkok were investigated. In the selection process of subjects, strict criteria of "normality" were applied and included history of medical illnesses, physical examination and nutritional status. RESULTS: The relationship between PEFR and height was approximately linear in both male and female children. Prediction equations for each sex were: Male children: PEFR (L/min) = [3.52 x Height (cm)] - 186.80 Female children: PEFR (L/min) = [3.48 x Height (cm)] - 204.11 The PEFR values of students in this study were different from the predicted values of PEFR in those of previous reports in Thai children. These discrepancies might be explained by a variety of study population and environmental factors. CONCLUSIONS: The relationship between PEFR and height of students in Bangkok is best described by a regression equation. The prediction graphs for each sex may be used to monitor PEFR values of children with obstructive airway diseases and to compare an individual's PEFR with those of others of the same height and sex. PMID- 10730534 TI - Pulmonary involvement in childhood systemic lupus erythematosus. AB - Twenty-four children (aged 6-15 years, M:F = 1:11) with systemic lupus erythematosus (SLE), who had respiratory symptoms, were retrospectively reviewed. Chest radiographs obtained from all patients revealed pleural effusion in 13, alveolar infiltration in 9, pericardial effusion and cardiomegaly in 6, interstitial infiltration in 4, hilar adenopathy in 3, lung abscess in 2 and pneumatocele with pneumothorax in 1. Etiologic organisms were identified in 7 cases; (3 cases of nocardia isolated from pleural effusion and sputum, 2 cases of tuberculosis, 1 case with staphylococcus aureus septicemia and 1 case with salmonella septicemia). All except one patient improved with medical treatment. One patient died from pneumonitis. Although pulmonary involvement is increasingly recognized in children with SLE, neither roentgenogram nor clinical findings were specific. The differentiation of pulmonary infiltrates caused by lupus lung disease from pulmonary infection should be carefully evaluated. PMID- 10730535 TI - Pulmonary function and exercise challenge test in Thai children: 10 years post respiratory syncytial virus infection. AB - OBJECTIVE: To report the occurrence of asthma, pulmonary function and exercise challenge test abnormalities found in Thai children, who 10 years earlier had lower respiratory tract infection caused by respiratory syncytial virus (RSV LRI). PATIENTS AND METHODS: 37 children, with RSV-LRI admitted at Ramathibodi Hospital during January 1986 through December 1987, were contacted, of whom 13 patients responded and completed history-review-questionnaires. Pulmonary function measurements at pre- and post-exercise and testing for bronchodilator response were performed. RESULTS: The occurrence of physician-diagnosed asthma in the patients with and without family history of allergic diseases was 75 per cent and 40 per cent, respectively. Of 11 patients who performed spirometry, 45 per cent had concave flow-volume curves, 36 per cent showed decreased ratio of forced expiratory flow in the first second (FEV1) to forced vital capacity (FVC) and 27 per cent showed either decreased forced expiratory flow between 25-75 per cent of FVC (FEF25-75%) or peak expiratory flow rate (PEFR). Of 8 patients who completed exercise challenge tests, 87.5 per cent showed markedly decreased PEFR at post exercise challenge tests and 37.5 per cent had markedly decreased FEV1. 62.5 per cent and 25 per cent showed markedly increased PEFR and FEV, respectively, after inhalation of bronchodilators. CONCLUSION: This study suggests that the occurrence of asthma in children, who had previous RSV-LRI, is relatively high compared with that reported in general Thai children. Baseline pulmonary function tests showed evidence of airway obstruction as demonstrated by concave-shaped flow-volume curves, decreased FEV1/FVC ratio, FEF25-75% and PEFR. The presence of bronchial hyperreactivity to exercise challenge test and positive response to bronchodilators confirms the evidence of exercise-induced bronchospasm in children with history of RSV-LRI. PMID- 10730536 TI - Blood lead level in Bangkok children. AB - Lead poisoning is one of the most harmful pollutant in children since it permanently effects the growth and intelligence. OBJECTIVE: To evaluate the lead problem in Bangkok children and identify risk factors and impact associated with high lead levels (> 10 micrograms/dL). MATERIALS AND METHODS: The longitudinal study (N = 84) followed blood lead levels at birth, 6, 12, 18 and 24 months of age. Also multiple cross-sectional studies comprising of children under 15 years of age attending the outpatient clinic, Ramathibodi Hospital (N = 511), kindergartens (N = 60) and secondary school students (N = 377) in Bangkok were conducted. The control for under 2 year-old children (N = 188) were those attending Metapracharak Hospital, Nakornprathom Province. Physical examinations were performed by pediatricians. Blood lead levels were assessed by Atomic Absorption Spectrometry. Questionnaires to identify risk factors were completed by parents of under 2-year-old children. Standford Binet tests were performed by psychologists for assessing the IQ in the longitudinal group at 2 years of age. RESULTS: The mean blood lead levels were increasing with age from 5.57 +/- 2.31 micrograms/dL at birth, to 4.75 +/- 3.25 micrograms/dL at 2 years of age, 6.74 +/ 2.02 micrograms/dL in kindergartens, and 9.03 +/- 3.65 micrograms/dL in secondary school students. They were in the acceptable range. However, the proportion of blood lead higher than 10 micrograms/dL were increasing from 1 to 6, 10 per cent at birth to 6 per cent at 2 years of age, 10 per cent in kindergartens and 35 per cent in secondary school students. The mean lead level in Bangkok children under 2 years of age was higher than those of the control group in Nakornprathom province, but not statistically significant. In addition, there was no identified significant risk factor except that the high lead group had a higher mean age and larger family size than those in the low lead group. In the kindergartens and secondary school, males had higher lead levels than females in the same age group. CONCLUSION: The blood lead levels in Bangkok children were not as high as expected. On the contrary, they tended to decrease following the reduction of ambient lead levels due to unleaded gasoline usage. PMID- 10730537 TI - Cyanide poisoning, 2 cases report and treatment review. AB - Two patients, a 4-year-old girl and her brother 1 1/2 year-old, with cyanide poisoning are reported. They vomited and became comatose 9 hours after ingestion of boiled cassava. At a community hospital, they were intubated and given ventilatory support. The girl was transferred to Ramathibodi Intensive Care Unit. At 19 hours after ingestion, sodium nitrite and sodium thiosulfate were given as well as other supportive treatment. She recovered with normal breathing on the next day. The boy was referred to Ramathibodi 4 hours later. On arrival, he appeared normal except for the bitter almond breathe. Only supportive treatment was given. Their blood cyanide levels on arrival were 0.56 and 0.32 microgram/ml (normal value < 0.3 microgram/ml) respectively confirming the diagnosis of cyanide poisoning. Other abnormal laboratory findings included metabolic acidosis and lactic acidemia. The pathogenesis and management of cyanide poisoning are reviewed. PMID- 10730538 TI - Pediatric injuries in emergency room, Ramathibodi Hospital. AB - The purposes of this study were to analyze the epidemiological data of child injuries from the computerized data system of the emergency room at Ramathibodi Hospital from June 1995 to May 1996 and assess the accuracy of triage system for child injury cases. Among 14,427 pediatric patients, 1,023 patients (7%) were injured. Most were under 5 years of age (48%), and most were male. Twenty-five per cent of cases were triaged as true emergency cases. Twelve per cent were disposed on an admission or referral by physicians. The negative predictive value of triage system to classify as a non-urgent was 91 per cent. Common causes of injury included falls (38%), inanimate force (19%), transportation (14%), and animal bite (12%). Older age group had significantly more severe injuries than younger (p = 0.002). However, only the transportation injuries were significantly more severe than other types (p = 0.003). The present ER-based injury surveillance system is useful to describe the basic epidemiology of pediatric injuries and to evaluate the triage system. However, for injury prevention purposes, the injury surveillance system should include more data of injury circumstances, associated environment and products. PMID- 10730539 TI - Williams syndrome and the elastin gene in Thai patients. AB - Williams syndrome (WS) has long been known as a complex disorder of dysmorphic facial features, described as elfin face, mental retardation or learning disability, loquacious personality, and supravalvular aortic stenosis. The etiology is now known to be due to deletion of the elastin gene (ELN) on long arm of chromosome 7. Thai patients were previously reported by clinical diagnosis. This study reports the first two cases of WS with ELN deletion diagnosed by fluorescent in situ hybridization (FISH) technique. Clinically, hyperacusis is a common finding in WS associated with otitis media. Neither of the patients had hyperacusis, but one of them had bilateral sensorineural hearing loss, which to our knowledge, has never been reported. PMID- 10730540 TI - Chromosome 22q11 deletion syndrome: the first three cases reported in Thailand. AB - The DiGeorge, velocardiofacial, and conotruncal anomaly face syndromes were originally described as separate disorders due to different concerns regarding phenotypes. However, all these disorders have some common clinical manifestations, including congenital heart defect, facial anomaly, and developmental delay. It is now clear that most cases of these syndromes have a common cause resulting from microdeletion of chromosome 22q11. This study reports the first three cases of Thai children presented with developmental delays. All are females who were known cases of congenital heart diseases. Their minor facial anomalies were subtle and not previously recognized as of any syndromes. The chromosome study by fluorescent in situ hybridization technique yielded microdeletion of chromosome 22q11. Without known prevalence in Asian populations, except in Japanese children, further study for chromosome 22q11 deletion syndrome in Asian children with conotruncal heart defects, who also have minor facial anomalies or developmental delays, should be undertaken. PMID- 10730541 TI - DNA extraction and amplification of 10-day, room-temperature blood samples. AB - DNA was serially studied in 20 samples of buffy coat stored at room temperature. Each sample was divided into 5 equal volumes, namely D0, D3, D5, D7 and D10. DNA extraction was performed on days 0, 3, 5, 7 and 10 after blood collection. The mean ratio of OD260/OD280 of the DNA obtained from D0 to D10 ranged from 1.77 to 1.79, and the mean amounts of the DNA obtained from D0 to D10 ranged from 602 to 740 ng/ul. There were no significant differences in the mean ratio and amounts of DNA obtained among these samples (p > 0.05). Subsequently, amplification was successfully performed from this template DNA to yield products of 1.4 kb and 142 bp at the sites associated with beta globin and factor VIII genes, respectively. These findings suggest the possibility of sending blood samples for DNA analysis by mail, or no ice is required during transportation. PMID- 10730543 TI - Drug-resistant tuberculosis: what do we do now? AB - Drug-resistant tuberculosis (TB) represents a threat to TB control programmes. Erratic and inappropriate use of currently available medications, HIV-TB co infection, and concern about transmission of drug-resistant strains in the general population all contribute to a worrying picture. What do we do now? In the last few years, there has been considerable progress in the understanding of mechanisms of action and resistance to antituberculosis agents, and in establishing the value of directly observed therapy in preventing treatment failure. However, a limited effort has been devoted to the development of new active compounds or of rapid diagnostic tests, and their relevance to global tuberculosis control has been questioned. PMID- 10730542 TI - Inflammation, infection and atherosclerosis: do antibacterials have a role in the therapy of coronary artery disease? AB - Since the recent publication of 3 studies on the use of antibacterials in patients with coronary artery disease (CAD), there has been a phenomenal interest in the role of infection in the genesis of CAD. It is now generally accepted that inflammation accompanies atherosclerosis from its initiation to the evolution of end-events. Inflammation may occur in response to traditional risk factors, such as hyperlipidaemia, smoking and diabetes mellitus. There is a recent resurgence of the concept that inflammation may have an infectious basis. This concept is based on the identification of microorganisms in the atherosclerotic plaque and seropositivity. The data on eradication of the offending organism with antibiotics and prevention of atherosclerosis-related events have, however, been inconsistent. This may reflect lack of precise understanding of steps leading to atherosclerosis and the evolution of acute ischaemic events. Further work in this area may help identify subsets of patient populations within which infection may play a causative role in the genesis of CAD. Targeted therapy then may be considered logical. PMID- 10730544 TI - Important drug interactions in dermatology. AB - Drug interactions can occur at any step from absorption to elimination of a drug, and can induce adverse as well as beneficial effects. Since systemic drugs are increasingly available and important in the treatment of dermatological diseases, a variety of possible interactions between concomitantly administered drugs have to be considered by dermatologists. The xenobiotic-metabolising enzyme system cytochrome P450 (CYP) is involved in the metabolism of many drugs, regulating their plasma concentrations and activities. Furthermore, the adverse effects of many drugs depend on the basal activity and inducibility of particular CYP isoenzymes in an individual patient. Since drug therapy in dermatological practice is of increasing complexity, and an increasing number of potent systemic drugs have become commonly used therapeutic agents, this review focuses on the following topics with the aim of optimising dermatological drug therapy. In the first section, all the different types of drug interactions that can occur through pharmacokinetic and pharmacodynamic mechanisms are introduced briefly, and then discussed systematically with special reference to drugs important for dermatologists. Then, the network of drug interactions that may occur from absorption to elimination is presented. The most important drug interactions mediated by CYP isoenzymes are listed. Finally, the importance of pharmacogenetics for the development of new drugs and its potential impact on the optimisation of individual therapy regimens is discussed. PMID- 10730547 TI - Lidocaine patch 5%. AB - Lidocaine patch 5% comprises a soft, stretchy adhesive patch (l0 by 14 cm) containing 5% lidocaine (700 mg) for the topical treatment of pain associated with postherpetic neuralgia (PHN). Lidocaine provides analgesic relief by blocking neuronal sodium channels. In clinical trials (conducted over 12 hours to 24 days) involving patients with allodynia associated with PHN, treatment with lidocaine patch 5% resulted in a significant reduction in pain intensity and increased pain relief compared with vehicle patch. Lidocaine patch 5% was associated with few adverse events, the most frequent being mild skin redness or irritation at the application site which occurred with a similar incidence with lidocaine and vehicle patch. PMID- 10730548 TI - Insulin glargine. AB - Insulin glargine is an extended-action biosynthetic human insulin. It precipitates in the neutral environment of subcutaneous tissue and is thus gradually absorbed into the bloodstream. The addition of small amounts of zinc to the formulation further delays absorption. In small euglycaemic clamp studies, the onset of action of insulin glargine was shown to be later, the duration of action longer and the time-action profile flatter than that of Neutral Protamine Hagedorn (NPH) insulin in patients with type 1 diabetes mellitus and healthy volunteers. Four large clinical trials of up to 28 weeks' duration have shown that a single bedtime dose of insulin glargine, in combination with preprandial short-acting insulin, is as effective or more effective than once or twice daily NPH plus short-acting insulin in improving glycaemic control in patients with type 1 diabetes mellitus. In 3 large comparative trials, insulin glargine decreased glycosylated haemoglobin and/or fasting blood glucose levels to a similar extent to that seen with NPH insulin in patients with insulin-dependent or non-insulin-dependent type 2 diabetes mellitus, either as monotherapy or in combination with oral hypoglycaemic agents. Insulin glargine appears to be well tolerated. A lower incidence of hypoglycaemia, especially at night, was reported in most trials with insulin glargine when compared with NPH insulin. PMID- 10730545 TI - Phosphodiesterase 4 inhibitors and the treatment of asthma: where are we now and where do we go from here? AB - Research conducted over the last 20 years has established that inflammation of the airways is central to the airway dysfunction that characterises asthma. Typically, the airway wall is infiltrated by a variety of cells including mast cells, eosinophils and T lymphocytes, which have deviated towards a T(H)2 phenotype. Together, these cells release a plethora of mediators including interleukin (IL)-4, IL-5, granulocyte/macrophage colony-stimulating factor and eotaxin which ultimately cause the histopathology and symptoms of asthma. Glucocorticosteroids are the only drugs currently available that effectively impact upon this inflammation and resolve, to a greater or lesser extent, compromised lung function. However, steroids are nonselective and generally unsuitable for paediatric use. New drugs are clearly required. One group of potential therapeutic agents for asthma are inhibitors of cyclic AMP-specific phosphodiesterase (PDE), of which theophylline may be considered a prototype. It is now known that PDE is a generic term which refers to at least 11 distinct enzyme families that hydrolyse cAMP and/or cGMP. Over the last decade, inhibitors of PDE4 (a cAMP-specific family that negatively regulates the function of almost all pro-inflammatory and immune cells, and exerts widespread anti-inflammatory activity in animal models of asthma) have been developed with the view to reducing the adverse effects profile associated with non-selective inhibitors such as theophylline. Such is the optimism regarding PDE4 as a viable therapeutic target that more than 100 PDE4 inhibitor patent applications have been filed since 1996 by 13 major pharmaceutical companies. This article reviews the progress of PDE4 inhibitors as anti-inflammatory agents, and identifies problems that have been encountered by the pharmaceutical industry in the clinical development of these drugs and what strategies are being considered to overcome them. PMID- 10730549 TI - Dexmedetomidine. AB - Dexmedetomidine is a potent alpha2-adrenoceptor agonist with 8 times higher affinity for the alpha2-adrenoceptor than clonidine. Dexmedetomidine has shown sedative, analgesic and anxiolytic effects after intravenous administration to healthy volunteers or postsurgical patients in the intensive care unit. Dexmedetomidine produced a predictable haemodynamic decline (dose-dependently decreased arterial blood pressure and heart rate) in postsurgical patients coinciding with reductions in plasma catecholamines. In phase III clinical trials, dexmedetomidine 0.2 to 0.7 microg/kg/h produced clinically effective sedation and significantly reduced the analgesic requirements of postsurgical ventilated intensive care unit patients. There was no clinically apparent respiratory depression after cessation of assisted ventilation. Dexmedetomidine produced rapid and stable sedation in postsurgical ventilated patients while maintaining a high degree of patient rousability and anxiety reduction. Dexmedetomidine was well tolerated in phase III studies. The most frequently observed adverse events were hypotension, bradycardia and nausea. PMID- 10730546 TI - Prevention and treatment of postoperative nausea and vomiting. AB - Pain, nausea and vomiting are frequently listed by patients as their most important perioperative concerns. With the change in emphasis from an inpatient to outpatient hospital and office-based medical/surgical environment, there has been increased interest in the 'big little problem' of postoperative nausea and vomiting (PONV). Currently, the overall incidence of PONV is estimated to be 25 to 30%, with severe, intractable PONV estimated to occur in approximately 0.18% of all patients undergoing surgery. PONV can lead to delayed postanaesthesia care unit (PACU) recovery room discharge and unanticipated hospital admission, thereby increasing medical costs. The aetiology and consequences of PONV are complex and multifactorial, with patient-, medical- and surgery-related factors. A thorough understanding of these factors, as well as the neuropharmacology of multiple emetic receptors [dopaminergic, muscarinic, cholinergic, opioid, histamine, serotonin (5-hydroxy-tryptamine; 5-HT)] and physiology [cranial nerves VIII (acoustic-vestibular), IX (glossopharyngeal) and X (vagus), gastrointestinal reflex] relating to PONV are necessary to most effectively manage PONV. Commonly used older, traditional antiemetics for PONV include the anticholinergics (scopolamine), phenothiazines (promethazine), antihistamines (diphenhydramine), butyrophenones (droperidol) and benzamides (metoclopramide). These antiemetics have adverse effects such as dry mouth, sedation, hypotension, extrapyramidal symptoms, dystonic effects and restlessness. The newest class of antiemetics used for the prevention and treatment of PONV are the serotonin receptor antagonists (ondansetron, granisetron, tropisetron, dolasetron). These antiemetics do not have the adverse effects of the older, traditional antiemetics. Headache and dizziness are the main adverse effects of the serotonin receptor antagonists in the dosages used for PONV. The serotonin receptor antagonists have improved antiemetic effectiveness but are not as completely efficacious for PONV as they are for chemotherapy-induced nausea and vomiting. Older, traditional antiemetics (such as droperidol) compare favourably with the serotonin receptor antagonists regarding efficacy for PONV prevention. Combination antiemetic therapy improves efficacy for PONV prevention and treatment. In the difficult-to-treat PONV patient (as in the chemotherapy patient), suppression of numerous emetogenic peripheral stimuli and central neuroemetic receptors may be necessary. This multimodal PONV management approach includes use of: (i) multiple different antiemetic medications (double or triple combination antiemetic therapy acting at different neuroreceptor sites); (ii) less emetogenic anaesthesia techniques; (iii) adequate intravenous hydration; and (iv) adequate pain control. PMID- 10730550 TI - Azimilide. AB - Azimilide is a potassium channel antagonist that, in contrast to existing class III antiarrhythmic agents, blocks both the rapidly (I(Kr)) and slowly (I(Ks)) activating components of the delayed rectifier potassium current. In animal and clinical studies, azimilide prolonged repolarisation by increasing the action potential duration and effective refractory period. In animal models, azimilide was effective in terminating both atrial and ventricular arrhythmias. Azimilide also demonstrated antifibrillatory efficacy in a canine model of sudden cardiac death. In patients with a history of atrial fibrillation/flutter, oral azimilide controlled arrhythmias more effectively than placebo in a 6-month randomised double-blind study. At a dosage of 125 mg once daily, azimilide significantly increased the time to first symptomatic recurrence of atrial fibrillation/flutter. However, no significant difference between placebo and azimilide was found in another study. Oral azimilide 100 mg once daily demonstrated clinically significant treatment effects in patients with paroxysmal supraventricular tachycardia. In clinical trials, azimilide was generally well tolerated and headache was the most commonly occurring adverse event. Azimilide is associated with a low incidence of proarrhythmic events, such as torsades de pointes, and few serious adverse events have been reported. PMID- 10730551 TI - Adjuvanted Lyme disease vaccine: a review of its use in the management of Lyme disease. AB - Lyme disease is a potentially serious infection which is caused by the spirochaete Borrelia burgdorferi and is endemic in certain areas of North America, Europe and Asia. Lyme disease vaccine (LYMErix) is an adjuvanted formulation of the outer surface protein A (OspA) of the causative spirochaete. It acts by inducing high titres of anti-OspA antibodies (anti-OspA), which must be present in vaccinated individuals before exposure to B. burgdorferi to provide protection against Lyme disease. Lyme disease vaccine efficacy against Lyme disease was 80% for definite and asymptomatic cases and 76% for definite cases at year 2 using the recommended dosage regimen [30 microg at months 0, 1 and 12 (0, 1, 12 schedule)] in a randomised, double-blind, multicentre trial in 10,936 enrolled adult volunteers who resided in areas of the US endemic for Lyme disease. On the basis of an anti-OspA correlate of protection, Lyme disease vaccine 30 microg was equally effective when administered by a shorter schedule (0, 1, 6 schedule); > or = 90% of adult volunteers developed protective anti-OspA titres with this or the 0, 1, 12 schedule. Although published data are fewer, a 0, 1, 2 schedule has also shown promise in adults. In addition, virtually all children (aged 2 to 15 years) given Lyme disease vaccine 30 microg developed protective anti-OspA titres, but published data are also limited and results of a large paediatric trial are awaited with interest. Long term protection against Lyme disease appears to be possible with Lyme disease vaccine. Although anti-OspA titres decline rapidly after completion of the recommended schedule, booster doses of 30 microg of the vaccine induced protective anti-OspA titres in > or = 96% of adult volunteers when administered 12 and/or 24 months later. Lyme disease vaccine 30 microg is well tolerated: most vaccination-related adverse events were transient and mild or moderate in severity in clinical trials. The most common spontaneously reported adverse event was pain at the injection site in 24% of vaccine recipients (vs 7.6% of the placebo group). The incidence of spontaneously reported, early nonspecific systemic adverse events was <4% but was higher with the vaccine than with placebo for some events (e.g. myalgias, fever and chills but not arthralgia). There appeared to be no association between the vaccine and the incidence of arthritis or any late systemic adverse events. The tolerability profile of Lyme disease vaccine did not appear to vary with the schedule of administration, nor to differ between adults and children. CONCLUSIONS: Lyme disease vaccine, an adjuvanted formulation of OspA, protects most adults against Lyme disease when administered by the recommended 0, 1, 12 schedule before disease exposure, and is well tolerated. The optimal schedule(s) of administration, duration of protection against Lyme disease, long term tolerability in adults and potential role in children are not fully defined for this vaccine. Lyme disease vaccine is indicated in North America for active immunisation of adults at moderate to high risk of contracting Lyme disease. PMID- 10730552 TI - Fexofenadine: a review of its use in the management of seasonal allergic rhinitis and chronic idiopathic urticaria. AB - Fexofenadine, the active metabolite of terfenadine, is a selective histamine H1 receptor antagonist that does not cross the blood brain barrier and appears to display some anti-inflammatory properties. Fexofenadine is rapidly absorbed (onset of relief < or = 2 hours) and has a long duration of action, making it suitable for once daily administration. Clinical trials (< or = 2 weeks' duration) have shown fexofenadine 60 mg twice daily and 120 mg once daily to be as effective as loratadine 10 mg once daily, and fexofenadine 120 mg once daily to be as effective as cetirizine 10 mg once daily in the overall reduction of symptoms of seasonal allergic rhinitis. When given in combination, fexofenadine and extended release pseudoephedrine had complementary activity. Fexofenadine was effective in relieving the symptoms of sneezing, rhinorrhoea, itchy nose palate or throat, and itchy, watery, red eyes in patients with seasonal allergic rhinitis. There were often small improvements in nasal congestion that were further improved by pseudoephedrine. Fexofenadine produced greater improvements in quality of life than loratadine to an extent considered to be clinically meaningful, and enhanced patients' quality of life when added to pseudoephedrine treatment. Although no comparative data with other H1 antagonists exist, fexofenadine 180 mg once daily was effective in reducing the symptoms of chronic idiopathic urticaria for up to 6 weeks. Fexofenadine was well tolerated in clinical trials in adults and adolescents and the adverse event profile was similar to placebo in all studies. The most frequently reported adverse event during fexofenadine treatment was headache, which occurred with a similar incidence to that seen in placebo recipients. Fexofenadine does not inhibit cardiac K+ channels and is not associated with prolongation of the corrected QT interval. When given alone or in combination with erythromycin or ketoconazole, it was not associated with any adverse cardiac events in clinical trials. As it does not cross the blood brain barrier, fexofenadine is free of the sedative effects associated with first generation antihistamines, even at dosages of up to 240 mg/day. CONCLUSIONS: fexofenadine is clinically effective in the treatment of seasonal allergic rhinitis and chronic idiopathic urticaria for which it is a suitable option for first-line therapy. Comparative data suggest that fexofenadine is as effective as loratadine or cetirizine in the treatment of seasonal allergic rhinitis. In those with excessive nasal congestion the combination of fexofenadine plus pseudoephedrine may be useful. In clinical trials fexofenadine is not associated with adverse cardiac or cognitive/psychomotor effects. PMID- 10730554 TI - "Nonspecific" inflammatory bowel disease (ulcerative colitis and Crohn's disease) after 100 years--what next? PMID- 10730555 TI - Secrecy and openness in peer review--time for a change of culture? PMID- 10730556 TI - Comparison between McCoy cell line and HeLa cell line for detecting Helicobacter pylori cytotoxicity: clinical and pathological relevance. AB - BACKGROUND: Cell culture assay is an accurate test for detecting Helicobacter pylori cytotoxicity. AIMS: To evaluate McCoy cells for detecting Helicobacter pylori cytotoxicity by comparing with HeLa cells, and determine the association of cytotoxic strains with endoscopic and histological findings. METHODS: Helicobacter pylori isolates from 68 dyspeptic patients and 11 asymptomatic volunteers were tested. 180 microl (1.8 x 10(4) cells) of grown McCoy or HeLa cell suspension was seeded into each well of a 96-well microtitre tray and the medium was replaced once after 24 hours. Sonicate (20 microl) of each isolate was then added to the wells, in duplicate. After 24 and 48 hours incubation, intracellular vacuolation was assessed by inverted light microscopy. RESULTS: Using McCoy cells 57% of isolates showed cytotoxicity (23% weak and 34% strong), while using HeLa cells 30% of isolates showed strong cytotoxicity. All isolates toxic in HeLa cells were also toxic in McCoy cells. The prevalence of cytotoxic strains was not significantly different between the endoscopic findings; 50% in normal endoscopy, 60% in non-ulcer dyspepsia and 59% in peptic ulcer disease. However, cytotoxic strains were more common in subjects with severe histological gastritis than in those with normal mucosa or mild gastritis (66% vs 30%, p<0.01). Similarly, the prevalence of cytotoxic strains was also higher in subjects with active gastritis than in those without (64% vs 23%, p<0.01). CONCLUSIONS: McCoy cells are more sensitive than HeLa cells for detecting Helicobacter pylori cytotoxicity in vitro. There is an association between cytotoxic strains and the severity and activity of histological gastritis. PMID- 10730557 TI - Immunohistochemical detection of Helicobacter pylori vacuolating cytotoxin in the hepatocytes of patients with isolated hypertransaminasaemia. AB - BACKGROUND AND AIM: Even if different Helicobacter species can colonise animal livers and induce hepatitis, there is no evidence that Helicobacter pylori can damage this organ and only a potential capacity of cytotoxic strains to increase transaminases in humans has been suggested. We have, therefore, carried out an immunohistochemical study on vacuolating cytotoxin in the hepatocytes of subjects with isolated hypertransaminasaemia. PATIENTS, METHODS AND RESULTS: Five male patients with isolated hypertransaminasaemia without signs of known causes of liver diseases were studied. Endoscopy demonstrated diffuse mucosal hyperaemia in 3 patients and duodenal ulcer in one. Histology revealed active chronic pangastritis in all. Helicobacter pylori was assessed by histology and culture and its cytotoxity, demonstrated by positive immunoblotting for both anti-CagA and VacA. Percutaneous liver biopsy showed minimal changes. Hepatic and gastric sections were tested either with autologous serum and rabbit antibody to VacA toxin. Immune reaction was revealed by immunoperoxidase. Both autologous sera and anti-VacA toxin antibody showed a reaction with a similar pattern which involved 60% of hepatocytes. Anti-VacA toxin showed a reaction to gastric epithelial cells and autologous sera to parietal cells in 4/5 patients. All patients received triple therapy and eradication of Helicobacter pylori was assessed by urea breath test. Serum transaminase levels 3 months after eradication, are still abnormal. CONCLUSIONS: Our immunohistochemical findings suggest that vacuolating cytotoxin could reach the hepatocytes of patients suffering from both isolated hypertransaminasaemia and infection by cytotoxic strains of Helicobacter pylori. Nevertheless, a clear relationship between these two condition remains uncertain. PMID- 10730558 TI - Helicobacters everywhere? Role of Helicobacter spp. in hypertransaminasaemia. PMID- 10730553 TI - Tacrolimus: a further update of its pharmacology and therapeutic use in the management of organ transplantation. AB - Tacrolimus (FK-506) is an immunosuppressant agent that acts by a variety of different mechanisms which include inhibition of calcineurin. It is used as a therapeutic alternative to cyclosporin, and therefore represents a cornerstone of immunosuppressive therapy in organ transplant recipients. Tacrolimus is now well established for primary immunosuppression in liver and kidney transplantation, and experience with its use in other types of solid organ transplantation, including heart, lung, pancreas and intestinal, as well as its use for the prevention of graft-versus-host disease in allogeneic bone marrow transplantation (BMT), is rapidly accumulating. Large randomised nonblind multicentre studies conducted in the US and Europe in both liver and kidney transplantation showed similar patient and graft survival rates between treatment groups (although rates were numerically higher with tacrolimus- versus cyclosporin-based immunosuppression in adults with liver transplants), and a consistent statistically significant advantage for tacrolimus with respect to acute rejection rate. Chronic rejection rates were also significantly lower with tacrolimus in a large randomised liver transplantation trial, and a trend towards a lower rate of chronic rejection was noted with tacrolimus in a large multicentre renal transplantation study. In general, a similar trend in overall efficacy has been demonstrated in a number of additional clinical trials comparing tacrolimus- with cyclosporin-based immunosuppression in various types of transplantation. One notable exception is in BMT, where a large randomised trial showed significantly better 2-year patient survival with cyclosporin over tacrolimus, which was primarily attributed to patients with advanced haematological malignancies at the time of (matched sibling donor) BMT. These survival results in BMT require further elucidation. Tacrolimus has also demonstrated efficacy in various types of transplantation as rescue therapy in patients who experience persistent acute rejection (or significant adverse effect's) with cyclosporin-based therapy, whereas cyclosporin has not demonstrated a similar capacity to reverse refractory acute rejection. A corticosteroid-sparing effect has been demonstrated in several studies with tacrolimus, which may be a particularly useful consideration in children receiving transplants. The differences in the tolerability profiles of tacrolimus and cyclosporin may well be an influential factor in selecting the optimal treatment for patients undergoing organ transplantation. Although both drugs have a similar degree of nephrotoxicity, cyclosporin has a higher incidence of significant hypertension, hypercholesterolaemia, hirsutism and gingival hyperplasia, while tacrolimus has a higher incidence of diabetes mellitus, some types of neurotoxicity (e.g. tremor, paraesthesia), diarrhoea and alopecia. CONCLUSION: Tacrolimus is an important therapeutic option for the optimal individualisation of immunosuppressive therapy in transplant recipients. PMID- 10730559 TI - Mesalazine foam (Salofalk foam) in the treatment of active distal ulcerative colitis. A comparative trial vs Salofalk enema. The SAF-3 study group. AB - BACKGROUND: Mesalazine enemas are of well proven efficacy for the topical treatment of distal ulcerative colitis. Although new rectal formulations of mesalazine are not expected to be superior in efficacy and tolerability to standard formulations, they may offer secondary advantages in terms of overall acceptability. AIM: To compare the efficacy, tolerability and overall acceptability of a new mesalazine rectal foam (Salofalk foam) with mesalazine enema in the treatment of active distal ulcerative colitis. PATIENTS AND METHODS: A multicentre study was carried out in patients with active proctitis, proctosigmoiditis and left-sided ulcerative colitis as evaluated by the Clinical Activity Index (CAI > or =4) and Endoscopic Index (EI > or =6). Patients were randomly assigned to receive, in open-label fashion, either mesalazine foam 2 g twice a day or mesalazine enema (2 g/60 ml twice a day) for 3 weeks. Patients who did not achieve remission (defined as CAI <4 and EI <6) after 3 weeks continued the study receiving the alternative galenic formulation for a further 3 weeks. RESULTS: A total of 195 patients were enrolled. Characteristics at baseline were similar except for concomitant therapy with oral 5-ASA products: during the 1st treatment phase, 41% of patients on enema received such treatment vs only 29% of those on foam. Patients with at least one post-treatment efficacy evaluation were included in the intent-to-treat analysis (n=89 foam, n=96 enema). After 3 weeks of treatment, 112 patients were in remission and only 59 patients entered the 2nd treatment phase thus providing data on acceptability. Remission was achieved after 3 weeks in 54% of patients treated with foam and in 67% of those treated with enema. The 90% confidence interval for the difference in remission rates was 0 to 24 and thus within the clinically acceptable range of therapeutic equivalence. At the end of the 2nd phase, 70% of patients switched to foam were in remission vs 65% to the enema. Two patients discontinued treatment with foam prematurely due to anal burning. No clinically important changes were seen in the laboratory tests. CONCLUSIONS: Salofalk foam and enema are equally effective for the treatment of proctitis, proctosigmoiditis and left-sided ulcerative colitis. The new foam preparation is as well tolerated and accepted as enemas and can be used as a therapeutic alternative to conventional mesalazine enema formulations. PMID- 10730560 TI - Treatment of distal ulcerative colitis: now the patients have the choice. PMID- 10730561 TI - Interferon alpha plus ketoprofen or interferon alpha plus ribavirin in chronic hepatitis C non-responder to interferon alpha alone: results of a pilot study. AB - BACKGROUND: Recently, in vitro and in vivo studies demonstrated that non steroidal anti-inflammatory drugs are able to enhance the activity of interferon alpha. AIM: To evaluate the efficacy and tolerability of ketoprofen (a non steroidal anti-inflammatory drug) plus interferon alpha (group B) compared to interferon alpha plus ribavirin (group C) and interferon alpha alone (group A) in chronic hepatitis C non-responders after a 5-month course with interferon alpha. PATIENTS AND METHODS: Without stopping interferon alpha, 49 patients were randomized to receive one of the three treatment regimens for 4 months. RESULTS: Three patients discontinued the therapy. One out of 16 patients in group A, 6/16 in group B and 5/14 in group C, alanine aminotransferase returned to normal at the end of the therapies (B vs A: p=0.04); serum hepatitis C virus-RNA became negative in 1 patient in group A and in 4 patients in both group B and group C. Six months after treatment, normal alanine transferase and negative hepatitis C virus-RNA were observed in 3 patients in group B and 2 in group C. In these patients, liver histology significantly improved. CONCLUSIONS: These results indicate that a certain number of non-responder patients to interferon alpha can benefit from a combination therapy of interferon alpha plus ketoprofen that is at least as effective as the combination interferon alpha plus ribavirin. PMID- 10730562 TI - Beyond white light endoscopy--the prospect for endoscopic optical biopsy. AB - The most important factor in the successful treatment of cancer is early detection. This will be more likely to facilitate eradication of abnormal cells prior to systemic invasion. White light endoscopy has been an essential tool in medical diagnosis for a number of years. Direct endoscopic inspection of gastrointestinal organs has revolutionised diagnostic techniques, improving the targeting of biopsies of macroscopic morphological abnormalities. Recent technological developments are threatening a further revolution enabling the instantaneous and non-invasive diagnosis of microscopic tissue abnormalities in vivo. This is made possible by improving the level of information that can be obtained from the tissue. As well as the two-dimensional surface morphology image, which the traditional endoscope can view, new techniques enable structure at depth, i.e., the third-dimension, to be imaged in high resolution. Other advances enable the detection of biochemical changes in tissue that precede any changes in morphology, thus enabling earlier diagnosis of tissue abnormalities. This review details recent advances that have the greatest potential, for use in partnership with endoscopy, for the diagnosis of malignancy and pre-malignancy. PMID- 10730563 TI - Relationship of Helicobacter pylori infection with gastrointestinal motility. AB - The interest of gastroenterologists in the relationship between Helicobacter pylori and gastrointestinal motility emerges from the observation that Helicobacter pylori may be involved in the pathogenesis of functional dyspepsia and that a relatively large percentage of patients with dyspepsia may show impaired gastrointestinal motility. A number of studies have been published on the interaction between Helicobacter pylori infection and gastrointestinal motility with controversial results, and, therefore, there are no definite conclusions, as yet, as to whether Helicobacter pylori is able, at all, or in which degree, to influence the motility of the upper gastrointestinal tract. Motility of the upper gastrointestinal tract has been studied in Helicobacter pylori positive and negative individuals by means of manometry, scintigraphy, radio-opaque markers or by other, recently developed, procedures such as breath tests, ultrasonography, and barostat. The vast majority of studies do not support the hypothesis that Helicobacter pylori may influence gastrointestinal motility. Nearly all these studies are, however, affected by methodological problems related to the small numbers of patients, different methodological approaches, and to the well-known difficulties in studying both gastrointestinal motility and functional dyspepsia. PMID- 10730564 TI - Virtual colonoscopy--technique and applications. AB - Virtual colonoscopy is a new method for evaluating the colon which uses thin section computed tomography of the clean air distended colon. The acquired computed tomography data is then subjected to computer manipulation to demonstrate the colonic mucosa. It is a safe, non-invasive, well-tolerated method that has potential as a method of colorectal cancer screening. This review will describe the technique, review preliminary results, and discuss the present and future applications of this technique. PMID- 10730565 TI - From Roma88 to Roma99: back to the future. AB - Some objectives of the Rome International Congress of Gastroenterology and Digestive Endoscopy (1988) and their development during the past eleven years are described in the perspective of Roma99, the Seventh United European Gastroenterology Week. PMID- 10730566 TI - Meta-analysis as a source of evidence in gastroenterology: a critical approach. AB - Meta-analysis is increasingly used in hepatogastroenterology. Meta-analysis is of value to provide a systematic review of related trials and to display their results in an objective, easily understandable manner. When the trials are sufficiently homogeneous, meta-analysis can document the superiority, (a), or the lack of superiority (b) of a treatment with respect to another (e.g., (a) Interferon plus ribavirin vs Interferon for chronic hepatitis; (b) 5-ASA vs sulfasalazine for maintaining remission in ulcerative colitis). However the interpretation of meta-analysis requires caution. Meta-analysis can be unreliable or unstable if based on a few, small trials (e.g., Tamoxifen vs non-active treatment for hepatocellular carcinoma), or if distorted by confounding variables and publication bias (e.g., glucocorticoids vs standard treatment in alcoholic hepatitis). Eventually, qualitative heterogeneity makes the pooled results of meta-analysis meaningless or questionable (e.g., endoscopic sclerotherapy for prevention of first variceal bleeding in cirrhosis) and should prompt the search for its sources to plan future studies. Finally, meta-analysis of trials measuring the treatment effect of a drug vs a placebo when an active drug is available for comparison provides the limited informative content for the physician of the individual trials (e.g. 5-ASA vs placebo for maintaining remission in ulcerative colitis). PMID- 10730567 TI - Basic research in endoscopy. AB - Endoscopic research includes Technologic Development, Facilitated Research in which endoscopy may simply be a means to an end (such as to gauge a response to a pharmacologic agent or obtain tissue which is used for genetic analysis); Translational Research which can be defined as the clinical evaluation of endoscopic techniques or technology as well as the delivery of basic science innovations to patients endoscopically; and Outcomes Research. Defining the barriers to endoscopic research is much more important than the distinction of whether the research is basic or clinical. These barriers are multifactorial and include inadequate infrastructure and training and lack of both funding and protected time at the divisional level. Governmental, GI Societal, and industry financial support will all be required as will training programme revisions. The latter may include training in areas such as outcomes, physics, or biomedical engineering; and should also entail a formal mentoring process, "seed money" early in the investigator's career and protected time. PMID- 10730568 TI - Cost-effectiveness of gastroscopy. AB - In a health economics perspective, no other decision area in gastroenterology has been more debated than the role of gastroscopy in dyspepsia. Cost-effectiveness analyses correlate costs with health outcomes. The cost-effectiveness of a diagnostic strategy must be compared to that of a competing strategy to produce useful information. This review examines the cost-effectiveness of gastroscopy in the management of dyspeptic patients, in particular in relation to the competing strategies of deferred referral guided by the outcome of either empirical antisecretory medication or of a non-invasive Helicobacter pylori test. Estimates of costs of gastroscopy differ widely. Cost estimates are usually much lower in European countries compared to the US. This is probably mainly due to differences in the calculations and in the payer perspective used. Cost-effectiveness analyses in dyspepsia management and the role of gastroscopy are difficult to interpret and impossible to compare due to a lack of uniformity in designing, measuring and reporting costs and health-care related outcome. Compared to empirical acid inhibition and to a test-and-scope strategy initial endoscopy is probably cost-effective--at least in Europe. Based on preliminary findings from clinical trials and from decision analyses a test-and-eradicate management strategy in young dyspeptic patients without warning symptoms seems to be cost effective compared to early endoscopy. Implementing a test-and-eradicate strategy in primary care will probably save endoscopies without harmful effects. In view of the rapidly changing epidemiology of Helicobacter pylori infection in the Western world the long-term effects of such a policy should be monitored closely. PMID- 10730569 TI - Digestive endoscopy: relevance of negative findings. AB - In patients with digestive complaints, endoscopy is the gold standard and is often the primary exploration. Positive findings in upper gastrointestinal endoscopy occur in 30 to 40% in relation to a benign or malignant structural disease. Negative findings occur therefore in 60 to 70% of cases: dyspepsia and/or reflux are functional diseases. Colonoscopy aims essentially at screening of colorectal cancer. Digestive cancer is very rare under 50 years, and most cases detected with symptoms are advanced; asymptomatic early and curable cancer is detected in screening. A negative finding at endoscopy is reliable in the hands of an experienced endoscopist controlling the risk of false negative and false positive findings. A reliable negative endoscopy is beneficial for the quality of life when it relieves anxiousness of the patient. It may be cost effective if drugs and consultations are decreased. Overuse of negative endoscopy is not so much through large indications, that in repetition of negative procedures. The relevance of negative endoscopy in screening for cancer in the upper and lower digestive tract, is whether is should be repeated. There is tendency to increased intervals in surveillance protocols. In the very near future the first role of endoscopy in the primary diagnosis could be challenged by concurrent techniques. Future progress is based upon a Quality Assurance program for quality, experience of the operator a control of the cost and a research programme on tolerance with a trend to reduced sedation. PMID- 10730570 TI - Coeliac disease in the year 2000. AB - Coeliac disease is a permanent intolerance to wheat gliadins and related prolamines. Patients who have an obvious malabsorption syndrome form only a small minority of the total number of people with coeliac disease. There are, in fact, no pathognomonic clinical features, and the condition is defined and diagnosed by the presence of pathological changes in the small bowel mucosa related to the presence of toxic prolamines. Susceptibility to coeliac disease is determined to a significant extent by genetic factors. A large part of the genetic susceptibility maps to the HLA region on chromosome 6, as approximately 95% of coeliac disease patients carry an almost identical HLA DQ2/heterodimer; a role of non-HLA genes has also been postulated. From a pathogenetic point of view, most evidence supports the notion of a DQ-restricted gluten-specific Th1 response in the lamina propria; nonetheless, it is possible that, in coeliac subjects, gluten, prior to T cell activation, could exert a direct toxic effect leading to the production of proinflammatory signals. PMID- 10730571 TI - Therapy of chronic viral hepatitis: a critical view. AB - Many oral nucleoside analogues that are potent inhibitors of hepatitis B virus have recently been developed for the treatment of hepatitis B. The problems with these drugs are bioavailability, toxicity and the time-dependent emergence of resistant hepatitis B virus mutants. Lamivudine appears to be the most useful in terms of clinical benefit, safety and tolerance. It is active on wild type hepatitis B virus as well as on HBeAg-minus variants of the virus. However, although hepatitis B virus is consistently repressed while on therapy, only a minority of patients are cured or remain in remission after Lamivudine withdrawal. Maintenance therapy would appear to be in order, but the long-term use of Lamivudine is precluded by the emergence of polymerase gene-mutants which may rekindle disease. Combination with other antivirals (Adefovir?) active also against Lamivudine escape mutants opens promising new prospects. There is, as yet, no valid therapy for chronic hepatitis D virus hepatitis. Attempts to improve the results of alpha-interferon therapy in chronic hepatitis C with new interferons, or the manipulation of interferon monotherapy so as to obtain the maximum results compatible with tolerance, have not produced significantly better results than the classic protocols of alpha-interferon monotherapy. A more concrete improvement has been achieved by the combination of interferon with Ribavirin, with the overall rate of response increasing three times compared to interferon monotherapy. Anaemia, however, is a common additional side-effect induced by Ribavirin. Combination therapy has become the treatment of choice for interferon naive patients as well as for interferon relapses; it is not efficacious in patients who have not responded to interferon. PMID- 10730572 TI - New and future drugs in nerve-gut dysfunction. AB - There is increasing evidence that modifications in brain-gut communications are responsible for the occurrence of Functional Bowel Disorders. Based on various experimental models of modified gut sensitivity and the emergence of new pharmacological tools, it is now possible to identify new targets for the corrections of altered brain-gut communications and to improve our understanding of functional gastrointestinal disorders. Both local inflammatory related components and central nervous system acting factors are associated to trigger dysfunctioning and neuropeptides such as tachykinins, bradykinin and calcitonin gene-related peptide are involved in peripheral and spinal sensitization of afferent neurons. Serotonin released from entero chromaffin cells, mast cells, platelets or nerves also play a role, through different receptor subtypes, in initiating gut hypersensitivity. Brain modulation of impaired ascending messages also appears to be an important approach for the correction of symptoms related to gut hyper-responsiveness. PMID- 10730573 TI - Immunodeficiency and the gut: clues to the role of the immune system in gastrointestinal disease. AB - Gastrointestinal disease occurs in a high proportion of patients with primary immunodeficiency. While enteric infections are responsible for the gastrointestinal pathology in many individuals, it is now clear that infection alone is not sufficient to explain the gut pathology found in many immunodeficiencies. This article focuses on recent developments in this field and highlights the insights gained in understanding the complex relationship between immunodeficiency and the gut. PMID- 10730574 TI - Spectrum of acute self-limiting colitis: role of the clinician and pathologist. AB - Acute self-limited colitis encompasses several diagnostic possibilities such as infectious colitis, post-antibiotic colitis, drug-induced colitis and should be differentiated from acute forms of inflammatory bowel disease. Diverticular disease in the elderly patient with colonic ischaemia may also give symptoms of acute bloody mucoid rectal discharge and should be recognised, although the clinical picture is usually completely different. Recognition of the causative agent--if possible--is particularly important in the patient with a foudroyant colitis (e.g. toxic megacolon), when the clinician has to decide, whether antibiotics or corticosteroids should be given or even a resection should be performed. A short history usually indicates towards infection, but a long standing history of inflammatory bowel disease may be complicated by a superinfection. Faecal cultures, endoscopy with colonic biopsy should be performed and results be discussed. New techniques for the assessment and follow up of difficult cases are: white cell scintigraphy, computerized tomography scanning and magnetic resonance imaging scanning. Acute self-limited colitis can usually be classified properly and treated accordingly. This review discusses the role to be played by the clinician, microbiologist and pathologist and is illustrated by several clinical examples, in which patients presented with unusual forms of acute self-limited colitis. PMID- 10730575 TI - Treatment of rectal cancer--a critical update. AB - The treatment of rectal cancer still remains primarily surgical in nature. Techniques have evolved from complete extirpation of the rectum and anal canal which left the patient with a permanent colostomy, to sphincter-saving procedures. Effort is now concentrated on reducing local recurrence by combining total mesorectal excision with adjuvant radiotherapy and there is good evidence that both are effective. However, it is likely that only adjuvant chemotherapy will improve survival, and numerous studies are currently in progress to determine the best combination of drugs. In addition, attempts are being made to improve the functional results of sphincter-saving surgery, most notably by the addition of a colonic pouch to the procedure. PMID- 10730576 TI - Kinetic studies on the effects of ADP and ionic strength on the interaction between myosin subfragment-1 and actin: implications for load-sensitivity and regulation of the crossbridge cycle. AB - The dynamics of the interaction of fast skeletal muscle myosin subfragment-1 with pyrene-labelled actin were examined using both stopped-flow and pressure relaxation methods. The data suggest a four-step model i.e.: A + M.(N)(K0)<-->A approximately M.(N)(K1)<-->A - M.(N)(K2)<-->A.M.(N)(K3)<-->A.M.(N)#. ADP weakens the acto-S1 affinity via a reduction in Ko, with no apparent effect on K1 and no effect on K2, whilst k(+2) and k(-2) are both markedly reduced. Increased ionic strength reduces both K0 and k(+2) with no major effect on k(+1). Step 3 represents an extension to previous models and is ADP-dependent. The present work is discussed in relation to earlier studies which led to somewhat different conclusions (Taylor EW (1991) J Biol Chem 266: 294-302; Geeves MA (1989) Biochemistry 28: 5864-5871). It is likely that the interaction proceeds via formation of a disordered complex stabilised by ionic interactions (corresponding to step 0), followed by a disordered-to-ordered transition involving additional hydrophobic contacts (step 1) after which further contacts of both types are made coupled to internal conformational changes (steps 2 and 3). Step 3 could have a role in extending the lifetime of force-generating crossbridges and limiting ATP turnover during contraction against a load, and may be equivalent to a structural change observed in recent cryo-EM studies on the smooth muscle system (Whittaker M, Wilsonkubalek EM, Smith JE, Faust L, Milligan RA and Sweeney HL (1995) Nature 378: 748-751). Cooperative interactions between the two myosin heads also appear to have a role in this putative latch mechanism. PMID- 10730577 TI - Synthesis of a spin-labeled photoaffinity ATP analogue, and its use to specifically photolabel myosin cross-bridges in skeletal muscle fibers. AB - A spin-labeled photoaffinity ATP analogue 3'(2')-O-[4-[4-oxo-(4-amido-2,2,6,6 tetramethyl-piperidino-1-oxyl)]-benz oyl]benzoyl adenosine 5'-triphosphate (SL Bz2ATP) was synthesized and used to photolabel myosin in muscle fibers. Previous work has shown that 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz2ATP) photolabeled Ser-324 of the 50 kDa tryptic fragment of skeletal S1 heavy chain. In this work, [alpha-32P]SL-Bz2ATP was hydrolyzed and trapped as the diphosphate analogue with Co2+ and orthovanadate at the active site of myosin in rabbit psoas muscle fibers. After UV irradiation, the myosin heavy chain was the only protein band found to be significantly photolabeled as assayed by gel electrophoresis and radioactivity counting. The labeling was localized after brief trypsin digestion by SDS-PAGE to be on the 50 kDa tryptic fragment of the S1 heavy chain. Ca. 35% of the myosin in fibers was covalently photolabeled. The fibers photolabeled with SL-Bz2ATP had the same active tension and maximum shortening velocity as the control fibers. The resulting spin label on myosin was too mobile to report the orientation of the heads in fibers. Nonetheless, this is the first work to show the feasibility of utilizing active site binding and photoaffinity labeling to place covalent spectroscopic probes at the myosin active site in fibers with high specificity and yield without affecting mechanical function. PMID- 10730578 TI - Thin-filament-binding domains of cardiac and fast skeletal muscle troponin I isoforms as studied by epitope tagging. AB - We examined the binding domains of cardiac and fast skeletal muscle troponin I (CTnI and FTnI, respectively) to myofibrils (MFs). Deletion mutants containing CTnI amino acid residues 1-79, 43-207 and 80-207 (CTnI-head, CTnI-tail-I and CTnI tail-2, respectively) and FTnI amino acid residues 1-54 and 55-182 (FTnI-head and FTnI-tail, respectively) were transiently expressed in cardiac and fast skeletal muscle cells. To monitor the intracellular localization of these exogenously introduced truncated TnIs, epitope tagging was used. CTnI-tail-1 was incorporated into cardiac MFs specifically, but CTnI-tail-2 was not assembled onto any MFs examined. This suggests that there is no potent actin filament-binding site in CTnI-tail-2. Since CTnI-tail-1 has an amino acid extension (CTnI residues 43-79) whose sequence is longer than that of CTnI-head-2; it appears that this sequence extension is important in binding to cardiac MFs. FTnI-tail, containing the inhibitory domain of actomyosin ATPase, showed intensive and specific incorporation into fast MFs. FTnI-tail was a homologous fragment of CTnI-tail-2, but the binding patterns of these two domains differed greatly from each other. It is possible that the absence of potent binding affinity of CTnI-tail-2 corresponding to the inhibitory domain of actomyosin ATPase is advantageous for continuous cardiac muscle contraction, since a potent inhibitory activity is a serious obstacle to cardiac muscle contraction. It can be assumed that distinctive binding ability of functional domains of TnI-tails reflect unique adaptations to muscles with different physiological properties. PMID- 10730579 TI - Contraction of epithelial (MDCK) cells in response to low extracellular calcium is dependent on extracellular sodium. AB - Like other cells of epithelial origin, MDCK cells respond with a reversible structural transformation to a diminution in the concentration of extracellular Ca2+. Upon deprivation of Ca2+ in the medium the cells undergo an active contraction mediated by the actin-myosin cytoskeleton, in parallel to detachment of the intercellular contacts and appearance of free spaces in the epithelium or monolayer (Castillo et al., 1998). We now present results indicating that the decrease of external Ca2+ plays an indirect and non-specific role in activating contraction, probably by allowing an influx of Na+. The omission of external Ca2+ had no effect when it was replaced by Mg2+, Ba2+ or Hg2+, and the addition of any of these divalent cations induced relaxation of cells previously contracted by exposure to low Ca2+. A null or weak response was observed also when Ca2+ was lowered in a solution where Na+ was replaced by choline or in the presence of amiloride (30 microM), which reduces the permeability of the plasma membrane to Na+. Restitution of Na+ or removal of amiloride were followed by contraction in the same cultures. Li+ proved an able substitute of Na+ as requisite for cell contraction in response to Ca2+ depletion. Monensin (0.1 mM) -an ionophore selective for Na+- and to a lesser extent ouabain (0.1 mM) -an inhibitor of Na+ extrusion across the plasma membrane- , both stimulated contraction in the presence of the normal level of external Ca2+. Decreasing by half the normal concentration of external K+ facilitated cell contraction, but typical responses were observed when K+ was increased to 40 mM by partial substitution for Na+. These findings attest that cell contraction in response to low Ca2+ is likely due to an increase in the permeability of the plasma membrane to Na+, though not to membrane depolarization as such. Evidences from other motile systems suggest that Na+ influx might in turn cause an elevation of cytoplasmic Ca2+, which activates the actin-myosin cytoskeleton. PMID- 10730580 TI - Complex three-dimensional patterns of myosin isoform expression: differences between and within specific extraocular muscles. AB - Because complex structural differences in adult extraocular muscles may have physiological and pathophysiological significance, the three-dimensional pattern of myosin heavy chain (MHC) isoform expression within the orbital and global layers of the muscle bellies compared with the distal tendon ends was quantitatively assessed. Three of the six extraocular muscles of adult rabbits were examined for immunohistologic expression of all fast, fast IIA/X, slow, neonatal and developmental MHC isoforms. The percentages of myofibers positive for each of these 5 myosin isoforms were determined in the orbital and global layers. There were relatively similar patterns of fast and slow MHC expression in the orbital and global layers of each of the three muscles examined. There were high levels of developmental MHC in the orbital layers, but significantly fewer developmental MHC positive myofibers in the global layer. The most variable expression was found with the neonatal MHC. There were significant differences between the longitudinal expression of the various isoforms in the middle of each muscle compared with the tendon end. In the orbital layer of all three muscles examined, the large numbers of fibers positive for fast MHC in the middle of the muscle dramatically decreased at the tendon end, with a concomitant increase in expression of slow myosin. There was a greater number of developmental MHC positive myofibers at the tendon end than in the middle of the muscle in all three muscles examined. In the global layer, the IIA/X-positive myofibers comprised only half of the total number of fast-positive myofibers whereas in the orbital layer they comprised all or almost all of the fast positive myofibers. The configuration of the extraocular muscles is more complex than might be indicated by previous studies. The lateral rectus muscle had the most individual pattern of MHC expression when compared with the inferior rectus and inferior oblique muscles. Together with dramatic cross-sectional MHC fiber type differences between the orbital and global layers of the muscles, there are pronounced longitudinal differences in the proportions of myofibers expressing these five MHC isoforms in the middle region of the muscles and those in the distal tendon ends. This longitudinal progression appears to occur both within single myofibers, as well as within the series of myofibers that comprise the length of the muscle. We also confirm that the number of myofibers is reduced at the tendonous end while the cross-sectional area of each of the remaining myofibers is proportionally increased with regard to those in the muscle belly. Future studies may yet require two additional schemes for anatomic classification of the named extraocular muscles. One will be based on immunohistochemical features of their constituent myofibers as a supplement to classifications based on their electron microscopic appearance, innervation patterns or relative position with regard to the globe and orbit. Another will be based on the proportional length and longitudinal position of individual myofibers within an individual extraocular muscle. PMID- 10730581 TI - A single-fiber in vitro motility assay. In vitro sliding velocity of F-actin vs. unloaded shortening velocity in skinned muscle fibers. AB - We describe an approach that allows us to form a micro in vitro motility assay with as little myosin as can be retrieved from a short (approximately 10 mm) segment of a single skinned skeletal muscle fiber (diameter some 100 microm). Myosin is directly extracted from the single fiber segment by a high ionic strength solution in the presence of MgATP, and the extracted myosin is immediately applied to a miniaturized flow cell that has been pretreated with BSA. The observed sliding velocities of fluorescently labeled F-actin are essentially identical with those reported in the literature. Since at the single fiber level most muscle fibers contain only a single myosin heavy chain isoform this approach allows us to determine without additional purification steps, the sliding velocity driven by myosins with different heavy chain isoforms. In addition, this approach can be used to directly correlate under identical experimental conditions unloaded shortening velocity measured in segments of skinned muscle fibers with the in vitro sliding velocity of fluorescently labeled F-actin by extraction of myosin from the same skinned fibers. Such direct correlation was performed with different myosin heavy chain isoforms as well as at different temperatures and ionic strengths. Under all conditions studied, unloaded shortening velocity was 4- to 8-fold faster than sliding velocity in the motility assay even at high temperature (22 degrees C) and ionic strengths >50 mM. This suggests that sliding velocity in the motility assay is limited by additional factors beyond those thought to limit velocity of unloaded shortening in muscle fibers. One such factor might be unspecific ionic interactions between F-actin and the substrate in the motility assay resulting in somewhat higher sensitivity for ionic strength of sliding velocity in the motility assay. This might become of special relevance when using in vitro sliding velocity in assessing functional consequences of mutations involving charged residues of actin or myosin. PMID- 10730583 TI - The XXVIII European Muscle conference. York, UK, 4-7 September 1999. PMID- 10730582 TI - The Drosophila projectin mutant, bentD, has reduced stretch activation and altered indirect flight muscle kinetics. AB - Projectin is a ca. 900 kDa protein that is a member of the titin protein superfamily. In skeletal muscle titins are involved in the longitudinal reinforcement of the sarcomere by connecting the Z-band to the M-line. In insect indirect flight muscle (IFM), projectin is believed to form the connecting filaments that link the Z-band to the thick filaments and is responsible for the high relaxed stiffness found in this muscle type. The Drosophila mutant bentD (btD) has been shown to have a breakpoint close to the carboxy-terminal kinase domain of the projectin sequence. Homozygotes for btD are embryonic lethal but heterozygotes (btD/+) are viable. Here we show that btD/+ flies have normal flight ability and a slightly elevated wing beat frequency (btD/+ 223+/-13 Hz; +/+ 203+/-5 Hz, mean +/- SD; P < 0.01). Electron microscopy of btD/+ IFM show normal ultrastructure but skinned fiber mechanics show reduced stretch activation and oscillatory work. Although btD/+ IFM power output was at wild-type levels, maximum power was achieved at a higher frequency of applied length perturbation (btD/+ 151+/-6 Hz; +/+ 102+/-14 Hz; P < 0.01). Results were interpreted in the context of a viscoelastic model of the sarcomere and indicate altered cross bridge kinetics of the power-producing step. These results show that the btD mutation reduces oscillatory work in a way consistent with the proposed role of the connecting filaments in the stretch activation response of IFM. PMID- 10730585 TI - Meiosis in holocentric chromosomes: orientation and segregation of an autosome and sex chromosomes in Triatoma infestans (Heteroptera). AB - The meiotic behaviour of the X chromosome and one autosomal pair of the heteropteran Triatoma infestans was analysed by means of C-banding plus DAPI staining. At first metaphase, the X univalent is oriented with its long axis parallel to the equatorial plate, which suggests a holocentric interaction with the spindle fibres. After this initial orientation, kinetic activity is restricted to one of both chromatid ends. The election of the active chromatid end is random and it is independent of the end selected in the sister chromatid. At second metaphase, the X and Y chromatids associate side by side forming a pseudobivalent. After that, the kinetic activity is again restricted to either of both chromosomal ends in a random fashion. At first metaphase, the fourth autosomal bivalent shows two alternative random orientations depending on the chromosome end showing kinetic activity (DAPI positive or opposite). At second metaphase, half bivalents are oriented with their long axis parallel to the equatorial plate. Three different segregation patterns are observed. The kinetic activity can be localised: (i) in the end with the DAPI signal (46.9%), (ii) in the opposite end (44.6%) or (iii) in one DAPI-positive end in one chromatid and in the opposite end in the other one (8.5%). The existence of the last pattern indicates that the same end can show kinetic activity during both meiotic divisions. Our results provide new information on the comparative meiotic behaviour of autosomes and sex chromosomes in holocentric systems. PMID- 10730584 TI - Structure and organization of the rDNA intergenic spacer in lake trout (Salvelinus namaycush). AB - A total-genomic cosmid library was created to isolate complete copies of the rDNA cistron of lake trout (Salvelinus namaycush) in order to study the structure and organization of the intergenic spacer (IGS) in this species. A total of 60 rDNA positive clones (average inserts > 25 kb) was recovered by screening the library with a rDNA-specific probe. Positive clones were assayed for the presence of the two internal rDNA spacers (ITS-1 and ITS-2) and the entire IGS fragment was successfully amplified from 42 clones by PCR. Length of the IGS fragments ranged from 9.4 to 17.8 kb. Comparative restriction mapping of the IGS-PCR products of several clones indicated two regions of extensive length variation surrounding a central region with sequence conservation. DNA sequence analysis was used to investigate the molecular basis of the IGS length variation and focused on identifying the region responsible for this variation. Over 9 kb of DNA sequence was obtained for one clone (A1) with a total IGS length of approximately 12.4 kb. Sequence of a conserved central region contained two open reading frames and a number of short direct repeats. Length variation in the IGS was determined by RFLP to result from differences in the number of copies of repetitive DNA sequences. These included an 89-bp tandem repeat (alpha repeats), an 82-bp element (beta repeats), a 168-177-bp element (chi repeats), and a 179-201-bp element (delta repeats). Overall nucleotide composition of the IGS was biased towards A and T (%GC = 47.4). Maintenance of discrete rDNA-length variants in lake trout suggests that the rate of gene conversion is insufficient to produce homogeneous copies across the genome. PMID- 10730586 TI - Localization of repetitive DNAs to zebrafish (Danio rerio) chromosomes by fluorescence in situ hybridization (FISH). AB - The genome of the zebrafish, Danio rerio, contains two major classes of tandem repetitive elements (AT-rich and GC-rich). The AT-rich repeats can be further subdivided into two subgroups which differ by about 10% of 185 bp in the repeating unit. The chromosomal location of these sequences and the moderately repetitive 5S rDNA sequences was determined in two diploid zebrafish cell lines using in-situ hybridization with fluorochrome-labeled probes. The AT-rich sequences were found at the centromeres of all chromosome pairs and the GC-rich sequences were found in paracentromeric location on over half of the chromosomal pairs. Different patterns of hybridization were found for the two subgroups of the AT-rich family. One type hybridized primarily to centromeres of one half to two thirds of the chromosomal pairs and the other type to centromeres of about three fourths of the chromosomal pairs. The pattern of hybridization with the GC rich sequences varied somewhat between the cell lines consistent with interindividual variation in the location of paracentromeric heterochromatin. The 5S rRNA genes are found on the long arm of chromosome 3. Most of this chromosome arm is late replicating, but apparently does not contain either the AT-rich or GC rich repetitive sequences. PMID- 10730587 TI - Early phase karyotype analysis of chromosome segregation after formation of mouse mouse hybridomas with chromosome painting probes. AB - FISH analysis with chromosome painting probes allows, better than karyotyping after Giemsa banding, the study of chromosome segregation after hybridoma formation. FISH is particularly useful for intraspecies hybrids and allows visualization of small chromosome fragments. Cell hybrids were constructed between P3 x 63Ag8.653 mouse myeloma cells and lymphocytes from BALB/c mice by PEG fusion and by selection in hypoxanthine azaserine medium. Three hybridomas (A4, D8, F10) were selected and, after cloning, the cells were cultivated in vitro over a period of 28 days. During this time in culture, air-dried metaphase spreads were prepared by standard methods. For FISH chromosome painting, digoxigenin- and biotin-labeled mouse chromosome painting probes and rhodamine antidigoxigenin antibodies and fluorescein-avidin were used for dual color detection. Total chromosome numbers and the numbers of mouse chromosomes 1, X, 6 and 12 were estimated as function of days in culture. Mean chromosome numbers of 78 (D8), 82 (F10) and 150 (A4) were observed. The major rearrangements of chromosome numbers occured in the first 28 days in culture and did not change significantly between day 28 and day 56. Mouse chromosome #12, which had the largest chromosome fragments in the parent myeloma, remained stable while the number of X chromosomes, which were significantly fragmented already in the parent myeloma, decreased by approximately 50%. PMID- 10730588 TI - Synteny and regional marker order assignment of 26 type I and microsatellite markers to the horse X- and Y-chromosomes. AB - The hypothesis that the conservation of sex-chromosome-linked genes among placental mammals could be extended to the horse genome was tested using the UCDavis horse-mouse somatic cell hybrid (SCH) panel. By exploiting the fluorescence in-situ hybridization (FISH) technique to localize an anchor locus, X-inactivation-specific transcript (XIST) on the horse X chromosome, together with the fragmentation and translocation of the X- and Y-chromosome fragments in a somatic cell hybrid panel, we regionally assigned 13 type I and 13 type II (microsatellite) markers to the horse X- and Y-chromosomes. The synteny groups that correspond to horse X- and Y-chromosomes were identified by synteny mapping of sex-specific loci zinc finger protein X-linked (ZFX), zinc finger protein Y linked (ZFY) and sex-determining region Y (SRY) on the SCH panel. A non pseudoautosomal gene in the human steroid sulfatase (STS) was identified in both X- and Y-chromosome-containing clones. The regional order of the X-linked type I markers examined in this study, from Xp- to Xq-distal, was [STS-X, the voltage gated chloride channel 4 (CLCN4)], [ZFX, delta-aminolevulinate synthase 2 (ALAS2)], XIST, coagulation factor IX (F9) and [biglycan (BGN), equine F18, glucose-6-phosphate dehydrogenase (G6PD)] (precise marker order could not be determined for genes within the same brackets). The order of the Y-linked type I markers was STS-Y, SRY and ZFY These orders are the same arrangements as reported for the human X- and Y-chromosomes, supporting the conservation of genomic organization between the human and the horse sex chromosomes. Regional ordering of X-linked type I and microsatellite markers provides the first integration of type I and type II markers in the horse X chromosome. PMID- 10730589 TI - PRINS analysis of the telomeric sequence in seven lemurs. AB - We examined the chromosomal localization of the telomeric sequence, (TTAGGG)n, in seven species of the lemurs and one greater galago, as an outgroup, using the primed in-situ labeling (PRINS) technique. As expected, the telomeric sequence was identified at both ends of all chromosomes of the eight prosimians. However, six species showed a signal at some pericentromeric regions involving constitutive heterochromatin as well. The pericentromeric region of chromosome 1 of Verreaux's sifaka (Propithecus verreauxi verreauxi) was labeled with a large and intense signal. The range of the signal considerably exceeded the area of DAPI positive heterochromatin. On the other hand, in the five lemurs, a large signal was detected also in the short arm of acrocentric chromosomes. Acquisition of the large block of the telomeric sequence into the acrocentric short arm might be interpretable in terms of the tandem growth of the heterochromatic short arm and the reciprocal translocation between heterochromatic short arms involving the telomeric sequence. Subsequently, it was postulated that meta- or submetacentric chromosomes possessing the telomeric sequence at the pericentromeric region could be formed by centric fusion between such acrocentric chromosomes. PMID- 10730590 TI - Organization of repetitive DNA sequences at pachytene chromosomes of gilthead seabream Sparus aurata (Pisces, Perciformes). AB - A method of preparing two-dimensional surface spreads of fish synaptonemal complexes (SCs) associated with fluorescent in-situ hybridization is described. This technique permits a novel approach to the analysis of chromatin organization and the construction of physical maps at meiosis, since surface-spread pachytene chromosomes are several times the length of metaphase chromosomes and the decondensed chromatin loops are attached to the lateral elements of the SC. We have applied this technique to analyze the location and organization of three different repetitive DNA sequences, rDNA, an EcoRI satellite DNA of the Sparidae family and telomere DNA in the gilthead seabream Sparus aurata. Our observations indicate that, depending on the type of sequence, the chromatin has different properties with regard to anchorage to the SC. PMID- 10730591 TI - Ty1-copia-retrotransposon behavior in a polyploid cotton. AB - Retrotransposons constitute a ubiquitous and dynamic component of plant genomes. Intragenomic and intergenomic comparisons of related genomes offer potential insights into retrotransposon behavior and genomic effects. Here, we have used fluorescent in-situ hybridization to determine the chromosomal distributions of a Ty1-copia-like retrotransposon in the cotton AD-genome tetraploid Gossypium hirsutum and closely related putative A- and D-genome diploid ancestors. Retrotransposon clone A108 hybridized to all G. hirsutum chromosomes, approximately equal in intensity in the A- and D-subgenomes. Similar results were obtained by hybridization of A108 to the A-genome diploid G. arboreum, whereas no signal was detected on chromosomes of the D-genome diploid G. raimondii. The significance and potential causes of these observations are discussed. PMID- 10730592 TI - CIS--cloning of identical sequences between two complex genomes. AB - Development of the methods permitting cloning of identical sequences between two sources of DNA can be very useful for many purposes, including isolation of disease genes. Here we describe a new method called CIS (cloning of identical sequences). A combination of digestion with MvnI, treatment with mung bean nuclease, UDG (uracil-DNA glycosylase) and PCR with 5'-methyl-dCTP and dUTP was used to isolate identical sequences between two micro-cell hybrid lines (MCH). In a control experiment, mouse MCH903.1 and MCH939.2 containing human chromosome 3 from different individuals, were compared using the CIS procedure. Only background fluorescence in-situ hybridization (FISH) was achieved. In another experiment, mouse MCH903.1, containing complete human chromosome 3, and rat MCH429.11, containing a part of human 3q from the same chromosome were compared. The experiment showed that the original MCH429.11 and the DNA purified using the CIS procedure had identical FISH patterns to human metaphase chromosomes, thus demonstrating the efficiency of CIS. PMID- 10730593 TI - Localization of 5S RNA genes on tobacco chromosomes. PMID- 10730594 TI - Managing change: an overview. AB - As increasingly powerful informatics systems are designed, developed, and implemented, they inevitably affect larger, more heterogeneous groups of people and more organizational areas. In turn, the major challenges to system success are often more behavioral than technical. Successfully introducing such systems into complex health care organizations requires an effective blend of good technical and good organizational skills. People who have low psychological ownership in a system and who vigorously resist its implementation can bring a "technically best" system to its knees. However, effective leadership can sharply reduce the behavioral resistance to change-including to new technologies-to achieve a more rapid and productive introduction of informatics technology. This paper looks at four major areas-why information system failures occur, the core theories supporting change management, the practical applications of change management, and the change management efforts in informatics. PMID- 10730595 TI - Managing change: analysis of a hypothetical case. PMID- 10730597 TI - Integration and beyond: panel discussion. PMID- 10730596 TI - Integration and beyond: linking information from disparate sources and into workflow. AB - The vision of integrating information-from a variety of sources, into the way people work, to improve decisions and process-is one of the cornerstones of biomedical informatics. Thoughts on how this vision might be realized have evolved as improvements in information and communication technologies, together with discoveries in biomedical informatics, and have changed the art of the possible. This review identified three distinct generations of "integration" projects. First-generation projects create a database and use it for multiple purposes. Second-generation projects integrate by bringing information from various sources together through enterprise information architecture. Third generation projects inter-relate disparate but accessible information sources to provide the appearance of integration. The review suggests that the ideas developed in the earlier generations have not been supplanted by ideas from subsequent generations. Instead, the ideas represent a continuum of progress along the three dimensions of workflow, structure, and extraction. PMID- 10730598 TI - The shadow uniform resource locator: standardizing citations of electronically published materials. AB - Citation of scientific materials published on the Internet is often cumbersome because of unwieldy uniform resource locators (URLs). The authors describe a format for URLs that simplifies citation of scholarly materials. Its use depends on a simple HTML device, the "refresh page." Uniform citation would follow this format: [Author I. Title of article. http:// domain/year/month-day(e#).html]. The HTML code for such a page is: (HTML) (head) (meta HTTP-EQUIV="Refresh" CONTENT="0; URL= http://Actual-URL/ for-article/ referred-to/ incitation.html") (/head) (/HTML). The code instructs the browser to suppress the content of the refresh page and bring up the title page of the cited article instead. Citations would be succinct and predictable. An electronic journal would not need to alter its existing file hierarchy but would need to establish a distinct domain name and maintain a file of refresh pages. Utilization of the "shadow" URL would bring us one step closer to truly universal resource locators. PMID- 10730599 TI - Temporal expressiveness in querying a time-stamp--based clinical database. AB - Most health care databases include time-stamped instant data as the only temporal representation of patient information. Many previous efforts have attempted to provide frameworks in which medical databases could be queried in relation to time. These, however, have required either a sophisticated database representation of time, including time intervals, or a time-stamp-based database coupled with a nonstandard temporal query language. In this work, the authors demonstrate how their previously described data retrieval application, DXtractor, can be used as a database querying application with expressive power close to that of temporal databases and temporal query languages, using only standard SQL and existing time-stamp-based repositories. DXtractor provides the ability to compose temporal queries through an interface that is understood by nonprogramming medical personnel. Not all temporal constructs are easily implemented using this framework; nonetheless, DXtractor's temporal capabilities provide a significant improvement in the temporal expressivity accessible to clinicians using standard time-stamped clinical databases. PMID- 10730600 TI - Comparing response time, errors, and satisfaction between text-based and graphical user interfaces during nursing order tasks. AB - Despite the general adoption of graphical users interfaces (GUIs) in health care, few empirical data document the impact of this move on system users. This study compares two distinctly different user interfaces, a legacy text-based interface and a prototype graphical interface, for differences in nurses' response time (RT), errors, and satisfaction when the interfaces are used in the performance of computerized nursing order tasks. In a medical center on the East Coast of the United States, 98 randomly selected male and female nurses completed 40 tasks using each interface. Nurses completed four different types of order tasks (create, activate, modify, and discontinue). Using a repeated-measures and Latin square design, the study was counterbalanced for tasks, interface types, and blocks of trials. Overall, nurses had significantly faster response times (P < 0.0001) and fewer errors (P < 0.0001) using the prototype GUI than the text-based interface. The GUI was also rated significantly higher for satisfaction than the text system, and the GUI was faster to leam (P < 0.0001). Therefore, the results indicated that the use of a prototype GUI for nursing orders significantly enhances user performance and satisfaction. Consideration should be given to redesigning older user interfaces to create more modern ones by using human factors principles and input from user-centered focus groups. Future work should examine prospective nursing interfaces for highly complex interactions in computer-based patient records, detail the severity of errors made on line, and explore designs to optimize interactions in life-critical systems. PMID- 10730601 TI - The risks of multimedia methods: effects of actor's race and gender on preferences for health states. AB - OBJECTIVE: While the use of multimedia methods in medical education and decision support can facilitate learning, it also has certain hazards. One potential hazard is the inadvertent triggering of racial and gender bias by the appearance of actors or patients in presentations. The authors hypothesized that race and gender affect preferences. To explore this issue they studied the effects of actors' race and gender on preference ratings for health states that include symptoms of schizophrenia. DESIGN: A convenience sample of patients with schizophrenia, family members of patients, and health professionals was used. Participants were randomly assigned to rate two health states, one portrayed by either a man of mixed race (Hispanic-black) or a white man and the second portrayed by either a white woman or a white man. MEASUREMENTS: Visual analog scale (VAS) and standard gamble ratings of health state preferences for health states that include symptoms of mild and moderate schizophrenia. RESULTS: Studies of the effects of the race of the actor (n = 114) revealed that racial mismatch between the actor and the participant affected the participant's preferences for health states. Ratings were lower when racial groups differed (mean difference, 0.098 for visual analog scale ratings and 0.053 lower in standard gamble, P = 0.006 for interactions between the race of the subject and the actor). In studies of the effects of a female actress on ratings (n = 117), we found no evidence of a corresponding interaction between the gender of the actor and the study participant. Rather, an interaction between actor's gender and method of assessment was observed. Standard gamble ratings (difference between means, 0.151), but not visual analog scale ratings (difference, 0.005), were markedly higher when the state was portrayed by the actress (P = 0.003 for interactions between actor's gender and method of preference assessment). Differential effects on standard gamble ratings suggest that an actor's gender may influence the willingness of viewers to gamble to gain health benefits (or risk attitude). CONCLUSIONS: Educators and researchers considering the use of multimedia methods for decision support need to be aware of the potential for the race and gender of patients or actors to influence preferences for health states and thus, potentially, medical decisions. PMID- 10730602 TI - Implementation of clinical guidelines via a computer charting system: effect on the care of febrile children less than three years of age. AB - OBJECTIVE: The authors have shown that clinical guidelines embedded in an electronic medical record improved the quality, while lowering the cost, of care for health care workers who incurred occupational exposures to body fluid. They seek to determine whether this system has similar effects on the emergency department care of young children with febrile illness. DESIGN: Off-on-off, interrupted time series with intent-to-treat analysis. SETTING: University hospital emergency department. SUBJECTS: 830 febrile children less than 3 years of age and the physicians who treated them. INTERVENTIONS: Implementation of an electronic medical record that provides real-time advice regarding the content of the history and physical examination and recommendations regarding laboratory testing, treatment, diagnosis, and disposition. MEASUREMENTS: Documentation of essential items in the medical record and after-care instructions; compliance with guidelines regarding testing, treatment, and diagnosis; charges. RESULTS: The computer was used in 64 percent of eligible cases. Mean percentage documentation of 21 essential history and physical examination items increased from 80 percent during the baseline period to 92 percent in the intervention phase (13 percent increase; 95 percent CI, 10-15 percent). Mean percentage documentation of ten items in the after-care instructions increased from 48 percent at baseline to 81 percent during the intervention phase (33 percent increase; 95 percent confidence interval, 28-38 percent). All documentation decreased to baseline when the computer system was removed. There were no demonstrable improvements in appropriateness of care, nor was there evidence that appropriateness worsened. Mean charges were not changed by the intervention. CONCLUSION: The intervention markedly improved documentation, had little effect on the appropriateness of the process of care, and had no effect on charges. Results for the febrile child module differ from those for the module for occupational blood and body fluid exposure (a more focused and straightforward medical condition), underscoring the need for implementation methods to be tailored to specific clinical complaints. PMID- 10730603 TI - A comparison of the effects of computer and manual reminders on compliance with a mental health clinical practice guideline. AB - OBJECTIVE: To evaluate the relative effectiveness of computer and manual reminder systems on the implementation of a clinical practice guideline. DESIGN: Seventy eight outpatients in a mental health clinic were randomly assigned within clinician to one of the two reminder systems. The computer system, called CaseWalker, reminded clinicians when guideline-recommended screening for mood disorder was due, ensured the fidelity of the diagnosis of major depressive disorder to criteria of the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV), and generated a progress note. The manual system was a checklist inserted in the paper medical record. MEASURES: Screening rates for mood disorder and the completeness of the documentation of which DSM-IV criteria were met by patients who were said to have major depressive disorder were compared. RESULTS: The CaseWalker, compared with the paper checklist, resulted in a higher screening rate for mood disorder (86.5 vs. 61 percent, P = 0.008) and a higher rate of complete documentation of DSM-IV criteria (100 vs. 5.6 percent, P < 0.001). CONCLUSIONS: In an outpatient mental health clinic, computer reminders were shown to be superior to manual reminders in improving adherence to a clinical practice guideline for depression. PMID- 10730604 TI - The cornerstones of medical informatics. PMID- 10730605 TI - An AMIA perspective on proposed regulation of privacy of health information. PMID- 10730606 TI - Graduate education in medical informatics. PMID- 10730607 TI - Plasticity of the pathologic heart. AB - This review addresses two relevant issues concerning the adaptation of the failing heart: myocyte growth and myocyte death. Recent results are summarized to support the notion that adult ventricular myocytes are not terminally differentiated cells and myocyte replication occurs in the normal heart and is potentiated by overloads. On this basis, myocyte hypertrophy and proliferation both contribute to the remodeling of the pathologic heart in animals and humans. Additionally, the controversy regarding the activation of apoptosis in the stressed myocardium is emphasized and published results are discussed. Available information demonstrates unequivocally that cell death by this mechanism takes place in the diseased heart and may have significant implications in the progression of ventricular dysfunction to end-stage failure. The importance of recognizing that electron microscopy is inappropriate for the identification and quantification of myocyte apoptosis is strongly indicated. Moreover, myocyte necrosis is presented as a relevant component of the decompensated heart. In summary, the dogma that myocytes cannot reenter the cell cycle and undergo mitotic division is proven to be obsolete and invalid. Similarly, the dogma that myocytes can die only by necrosis is contrary to any objective interpretation of published findings. Myocyte necrosis and apoptosis, and myocyte hypertrophy and proliferation are major elements of the plasticity of the heart. PMID- 10730608 TI - Angiotensin II AT2 subtype receptors: an emerging target for cardiovascular therapy. AB - This article reviews the impressive amount of knowledge accumulated in the last few years on the angiotensin II AT2 subtype receptor. Although still elusive, a large body of experimental evidence strongly suggests that it may play an important role in the adaptive changes of the cardiovascular structures in response to pathological conditions such as myocardial infarction, congestive heart failure or hypertension. The most intriguing aspects of the biology of this receptor, however, appear to be: 1) the regulation of its transcription, which plays an important role in the expression of the protein in adults or in injured tissues; 2) its interaction or "cross-talk" with the predominant angiotensin II receptor, the AT1 subtype, or with the receptors of other growth factors or cytokines; and 3) its connections with the bradykinin/nitric oxide pathways. These aspects may be relevant for the therapeutical use of drugs which antagonize the renin-angiotensin system, such as angiotensin-converting enzyme inhibitors or angiotensin receptor antagonists, as well as for new therapeutic approaches to the treatment of cardiovascular diseases. PMID- 10730609 TI - Towards a molecular understanding of the atheroprotective effects of estrogens: a review of estrogen effects on endothelial activation. AB - Early phases of atherosclerosis are characterized by an increased adhesion of leukocytes to vascular endothelium, leading to a recruitment of white blood cells into the intima. Leukocyte adhesion is mediated by the expression of specific adhesion molecules on endothelial cells, dependent on a dysfunctional status of vascular endothelium (endothelial activation), potentially caused by the exposure to diverse atherogenic stimuli. Female sex steroid hormones regulate vascular function acting directly on vascular cells, and producing a net anti-inflammatory effect. Among the various mechanisms mediating the anti-atherogenic effects of estrogens, the inhibition of endothelial activation process and of leukocyte adhesion molecule expression are particularly important, for the strategic pathophysiological role played by these processes during atherogenesis. This brief review discusses recent discoveries on the molecular mechanisms of estrogens on endothelial activation, as well as pathophysiological and clinical implications of these effects. PMID- 10730611 TI - Angiographic follow-up after coronary implantation of the Multilink stent: a prospective observation. AB - BACKGROUND: A growing variety of coronary stents is becoming available on the market. Results of randomized trials may be difficult to apply to less selected patients, and experience with every device cannot be obtained in every center. Detailed information about the immediate and long-term results achieved with one device can be a helpful reference for interventional cardiologists. The aim of this study was to test the applicability and the clinical and angiographic results, both immediate and at 6 months, of the Multilink coronary stent in a cohort of unselected patients undergoing coronary angioplasty. METHODS: From March 1997 to June 1998 coronary angioplasty was performed in 391 patients in our center, with the use of stents in 339 patients. RESULTS: Three hundred and seventeen Multilink stents were successfully implanted in 295 lesions in 277 patients; an acute coronary syndrome was present in 209 cases (75%), and lesion types B2 and C accounted for 30% of lesions. In 7 cases (2.4%) the Multilink stent did not cross the lesion, and another device was implanted. Subacute stent occlusion occurred in 1 patient (0.36%) after primary angioplasty. After 6 months from the procedure, clinical follow-up data were available for 252 out of 254 patients: none had died, and angina or myocardial ischemia occurred in 25 patients (9.9%). A control angiogram was performed in 239 out of 254 patients (94%) at 178 +/- 34 days. Restenosis occurred in 44/239 patients (18.4%) and in 48/247 lesions (19.4%). In patients with vs without restenosis the original lesion was longer (p = 0.009), and diabetes mellitus was more frequent (p = 0.002), as was the use of multiple stents (p = 0.005). In single 15, 25 and 35 mm long stents restenosis occurred in 13.9, 15.5 and 46.2% of cases, respectively (p = NS). CONCLUSIONS: The Multilink stent showed a low rate of subacute occlusion (0.36%) and could be used safely also in patients with acute coronary syndromes. The use of a single, 15 or 25 mm long Multilink stent was associated with a low angiographic recurrence rate (14-16%). PMID- 10730610 TI - Microvascular damage during myocardial ischemia-reperfusion: pathophysiology, clinical implications and potential therapeutic approach evaluated by myocardial contrast echocardiography. AB - During myocardial ischemia produced by coronary occlusion, coronary microvessels and cardiac myocytes undergo progressive functional and structural changes. The prompt reopening of the epicardial vessel is the main therapeutic strategy to limit the vascular and cellular damage. However, the full benefit of reperfusion can be limited by progressive microvascular obstruction and cell death occurring after the reestablishment of flow. During ischemia-reperfusion, preservation of the integrity of the coronary microvasculature is a fundamental prerequisite to ensuring myocardial viability. Therefore, therapeutic approaches should be developed to prevent and treat microvascular impairment resulting from ischemia reperfusion. Also, given the importance of the assessment and treatment of post reperfusion disorders of coronary microvasculature, a diagnostic tool able to evaluate the structural and functional status of the microcirculation in vivo is needed. Myocardial contrast echocardiography has been demonstrated to be extremely useful in this setting. In this review, the anatomic and functional characteristics of the coronary microcirculation are described during normal conditions, as well as in the presence of ischemia-reperfusion injury. The role of myocardial contrast echocardiography in the assessment of microvascular dysfunction and specific potential therapeutic approaches to the treatment of microvascular damage during ischemia and after reperfusion are also discussed. PMID- 10730612 TI - Left ventricular performance in chronic mitral regurgitation: temporal response to valve repair and prognostic value of early postoperative echocardiographic parameters. AB - BACKGROUND: The temporal response of the left ventricle due to the relief of volume loading after mitral valve repair, and the prognostic value of early changes in left ventricular size and function, are not fully documented. The purpose of this study was to analyze the evolution of left ventricular performance after surgery, and to evaluate how early postoperative echocardiographic parameters compare with late ventricular function. METHODS: We studied 58 patients with chronic degenerative mitral regurgitation using echocardiography, before, and 9 +/- 3 days and 38 +/- 6 months after mitral valve repair. RESULTS: Between the preoperative and early postoperative study, left ventricular end-diastolic and left atrial size, and ejection fraction decreased, whereas left ventricular end-systolic dimension did not change. Between the early and late postoperative study left ventricular end-systolic size decreased significantly, there was a further decrease in left ventricular end-diastolic dimension and a significant increase in ejection fraction; left atrial size did not change. Multivariate analysis showed that preoperative and early postoperative ejection fraction, and the early postoperative reduction in diastolic dimension were the best predictors of late left ventricular function. CONCLUSIONS: In patients with chronic degenerative mitral regurgitation, the greatest reduction in end-diastolic dimension occurs within 2 weeks of the reversal of volume overload; a significant reduction in end-systolic dimension with an increase in ejection fraction occurs later. In our experience, early postoperative echocardiographic measurements of left ventricular size and function can provide important prognostic information. PMID- 10730613 TI - Left atrial and appendage mechanical function after pharmacological or electrical cardioversion in patients with chronic atrial fibrillation: a multicenter, randomized study. AB - BACKGROUND: Transient atrial and appendage dysfunction occurs after cardioversion of atrial fibrillation. It has been suggested that one component of early dysfunction is related to the method of restoration of sinus rhythm and it is less severe in patients undergoing pharmacological than electrical cardioversion. The aim of this study was to compare left atrial chamber and left atrial appendage mechanical function before and after 48 hours from electrical or pharmacological cardioversion in patients with chronic atrial fibrillation. METHODS: We studied the effects of the mode of cardioversion on Doppler left atrial and appendage function in 19 patients with persistent atrial fibrillation (> or = 4 weeks), who were randomized to pharmacological (quinidine) or electrical cardioversion (protocol: 200, 300, 360 J) after pre-treatment with verapamil. Transthoracic and transesophageal echocardiography were performed before and 48 hours after the restoration of sinus rhythm. To determine left atrial and appendage mechanical dysfunction, the peak A wave velocities were obtained from transmitral flow velocity profiles recorded in the apical 4-chamber view, and peak emptying and filling appendage velocities were measured by the transesophageal approach with the sample volume placed at the orifice of the left atrial appendage. All the patients were pre-treated with verapamil before cardioversion in order to achieve a satisfactory control of heart rate. RESULTS: Mean peak A wave velocities were 0.52 +/- 0.12 m/s in the patients treated electrically and 0.54 +/- 0.08 m/s in those treated pharmacologically (p = NS). Before and after electrical cardioversion, the peak filling velocities of the left atrial appendage were 0.42 +/- 0.17 and 0.43 +/- 0.17 m/s respectively, and the peak emptying velocities 0.30 +/- 0.14 and 0.36 +/- 0.17 m/s respectively; before and after pharmacological treatment, the peak filling velocities were 0.38 +/- 0.1 and 0.43 +/- 0.1 m/s respectively, and the peak emptying velocities were 0.30 +/- 0.13 and 0.43 +/- 0.24 m/s respectively (p = 0.08). CONCLUSIONS: Even a long period of atrial fibrillation does not lead to a marked depression of global left atrial and left atrial appendage function 48 hours after the restoration of sinus rhythm by means of electrical or pharmacological cardioversion. There is no evidence that electrical cardioversion causes greater post-cardioversion atrial and/or appendage dysfunction than pharmacological treatment after 48 hours. Pre treatment with verapamil may have reduced the dysfunction (probably because of a reduction in mechanical remodeling during atrial fibrillation). PMID- 10730615 TI - Low energy transvenous atrial defibrillation. When and how to use it. PMID- 10730614 TI - Low energy intracardiac cardioversion of chronic atrial fibrillation by single femoral approach: safety and effectiveness of the procedure. AB - BACKGROUND: Low energy intracardiac cardioversion has recently been introduced into clinical practice to treat both acute and chronic atrial fibrillation. It has also been suggested that low energy intracardiac cardioversion has a higher efficacy rate in restoring sinus rhythm than conventional external cardioversion. METHODS: A prospective study was started in 41 patients (mean age 64.5 years) with chronic atrial fibrillation (mean duration 6.5 months), in order to obtain more data on low energy intracardiac cardioversion concerning: 1) time required to perform low energy intracardiac cardioversion by single venous femoral approach; 2) acute efficacy; 3) incidence of complications; 4) persistence of sinus rhythm after 1 month. RESULTS: Twenty patients had right atrium-coronary sinus (Group A) and 20 right atrium-left pulmonary artery (Group B) electrode configuration for defibrillation. In 1 patient the configuration was not available. In all patients (100%) sinus rhythm was acutely restored. No statistically significant differences were found between the two groups concerning mean energy and impedance required to obtain cardioversion. With mild sedation the discomfort induced by the electrical shock was minimal or mild. Only 44% of patients were in sinus rhythm 1 month after low energy intracardiac cardioversion, in spite of adequate pharmacological therapy. CONCLUSIONS: Low energy intracardiac cardioversion by single venous femoral approach may be considered a very effective and not time consuming procedure in acutely restoring sinus rhythm, with low complication rate; in addition the procedure was well accepted by all patients. PMID- 10730616 TI - Myocardial protection by the nitroderivative of aspirin, NCX 4016: in vitro and in vivo experiments in the rabbit. AB - BACKGROUND: A new family of nitroderivatives of conventional non-steroidal anti inflammatory drugs capable of releasing nitric oxide has been synthesized. Among these compounds, a nitroderivative of aspirin (NCX 4016), which displays antiplatelet and vasodilating activities, appears to have clinical potential in cardiac pathology related to coronary insufficiency. METHODS: In this study the beneficial effects of NCX 4016 and aspirin were evaluated in vitro in a model of myocardial ischemia-reperfusion of the rabbit and in vivo in a model of acute myocardial infarction of the same animal species. RESULTS: The NCX 4016 (from 1 x 10(-5) M to 3 x 10(-4) M) caused dose-dependent cardiac protection in isolated rabbit hearts subjected to low flow ischemia-reperfusion. Inhibition of 6-keto prostaglandin F1alpha (6-keto-PGF1alpha) generation and proportional reduction of creatine kinase (CK) activity at reperfusion was observed. Aspirin (1 x 10(-4)M) markedly worsened the post-ischemic ventricular dysfunction and this event was paralleled by a 63% increase in CK activity and abolition of 6-keto-PGF1alpha formation. Perfusion of the hearts with NG-monomethyl-L-arginine (1 x 10(-5) M) worsened the ischemia-reperfusion damage in perfused hearts. This event was prevented by prior treatment with NCX 4016 (1 x 10(-4) M) but not with aspirin (1 x 10(-4) M). Ligation of the first antero-lateral branch of the left coronary artery in rabbits resulted in acute myocardial infarction with a mortality rate of 60% at 24 hours. NCX 4016 (0.5 mg/kg/min for 2 hours) significantly reduced the mortality rate by 10%, protected the rabbits against electrocardiogram derangement and almost abolished CK activity in plasma and myeloperoxidase activity in cardiac tissue. Aspirin was devoid of any protective activity. CONCLUSIONS: In the rabbit NCX 4016 appears to exert a relevant cardioprotection likely mediated by nitric oxide donation. These results suggest that this nitroderivative of aspirin may lead to innovative therapy in myocardial ischemia and infarction. PMID- 10730617 TI - Are you feeding the energy vampire? PMID- 10730618 TI - Diagnosis and treatment of canine separation anxiety and the use of clomipramine hydrochloride (clomicalm). PMID- 10730620 TI - Intrapericardial cyst causing cardiac tamponade in a cat. AB - A two-year-old cat with episodic dyspnea was diagnosed with an intrapericardial cyst via two-dimensional echocardiography. The cyst directly compressed the right ventricle, resulting in cardiac tamponade. Centesis of the cyst was performed to reduce tamponade prior to surgery. At surgery, a large, fluid-filled cystic structure was found within the pericardium. The cystic structure was continuous with a pedicle of liver that passed through a small peritoneopericardial diaphragmatic hernia. Surgical resolution was achieved by median sternotomy, midline pericardotomy, resection of the cyst, and diaphragmatic herniorrhaphy. PMID- 10730619 TI - The effects of sodium ampicillin, sodium cefazolin, and sodium cefoxitin on blood pressures and heart rates in healthy, anesthetized dogs. AB - This study determined the effects of intravenous ampicillin, cefazolin, and cefoxitin on blood pressures and heart rates in healthy, anesthetized dogs. Forty dogs were each randomly assigned to a control, ampicillin, cefazolin, or cefoxitin group. Antibiotics or saline was delivered by intravenous bolus prior to surgical stimulation. Heart rate; systolic, mean, and diastolic arterial pressures; oxygen saturation; end-tidal halothane; and end-tidal carbon dioxide (CO2) were recorded before and every minute for 10 minutes after the test drug was administered. No significant differences were recorded between the antibiotic and control groups. The prophylactic use of these antibiotics should be considered safe in healthy, anesthetized dogs. PMID- 10730621 TI - Pericardial effusion associated with metastatic disease from an unknown primary tumor in a dog. AB - A 6.5-year-old, spayed female Siberian husky presented with signs of cardiac tamponade and weakness. Pleural, pericardial, and abdominal effusion were identified with radiographs and ultrasound. Pericardiocentesis relieved signs of tamponade, and the dog was clinically improved. Pericardial effusion recurred, and pericardiectomy was performed. Histopathological examination of excised tissues failed to reveal evidence of infectious or neoplastic disease. After pericardiectomy, clinically apparent thoracic effusion persisted. The dog was euthanized, and postmortem histopathological examination revealed emboli of metastatic carcinoma cells in the epicardium. The location of intrathoracic disease in this dog made antemortem diagnosis difficult, if not impossible. PMID- 10730622 TI - The prevalence of cardiomyopathy in the Irish wolfhound: a clinical study of 500 dogs. AB - The prevalence of cardiomyopathy in Irish wolfhounds was evaluated by retrospective review of the results of cardiovascular examinations carried out in 500 dogs presented for veterinary services at the author's practice. Abnormalities were found in 209 (41.8%) of the dogs examined. Dilated cardiomyopathy (DCM) was diagnosed in 121 (24.2%) of the dogs and was accompanied by atrial fibrillation in 106 dogs. Seventeen dogs were suffering from advanced congestive heart failure (CHF), and 55 dogs were suffering from mild to moderate CHF as a result of DCM. Congestive heart failure was most commonly characterized by mild to severe pleural effusion due to right-sided heart failure in addition to pulmonary edema. Rhythm disturbances without evidence of DCM were detected in 48 dogs. Forty dogs had echocardiographic abnormalities without signs of DCM. Soft to moderate mitral regurgitations were diagnosed in 13 (2.6%) of these 40 dogs examined. In 39 dogs that died as a result of DCM, the median survival time from the time of diagnosis was 5.1 months, and in 59 dogs with DCM that are still alive, the median survival time is 15.7 months. PMID- 10730623 TI - Left ventricular outflow tract to left atrial fistula associated with endocarditis in a dog. AB - A one-year-old, intact male, 28-kg, mixed-breed dog developed neurological episodes consistent with emboli. An acquired III/VI holosystolic heart murmur was ausculted in the mitral area, and valvular endocarditis with pulmonic and aortic insufficiency were noted at echocardiographic examination. An abnormal communication (i.e., fistula) between the left ventricular outflow tract (LVOT) and the left atrium adjacent to the mitral valve annulus was noted with Doppler imaging and confirmed with angiography. Infective valvular endocarditis was confirmed based on two of three blood cultures being positive for Staphylococcus intermedius. In humans, a sequela to infective endocarditis of the aortic or mitral valve, or both, is rupture of the mitral-aortic intervalvular fibrosa, resulting in a communication between the LVOT and the left atrium. This is the first report of this sequela in the dog. PMID- 10730624 TI - Seroconversion of puppies to canine parvovirus and canine distemper virus: a comparison of two combination vaccines. AB - Sixty puppies were randomly assigned to receive one of two commercially available combination vaccines, and responses to the canine parvovirus and canine distemper virus components of the vaccines were determined by measuring serum antibody titers. The percentage of puppies that seroconverted to canine parvovirus was significantly higher and the mean time for seroconversion was significantly shorter for puppies that received one of the vaccines than for puppies that received the other vaccine. Percentages of puppies that seroconverted to canine distemper virus were not significantly different. PMID- 10730625 TI - Thrombosis of the caudal vena cava presenting as an unusual cause of an abdominal mass and thrombocytopenia in a dog. AB - Thrombosis of the caudal vena cava in a dog secondary to metastatic neoplasia is described. The dog had a palpable abdominal mass and persistent thrombocytopenia due to a thrombosed caudal vena cava that was surgically removed. A few days after its removal, the dog died and neoplastic cells of neural crest origin were identified at the edge of the thrombus. Massive thrombosis can be an unusual cause of platelet consumption, leading to thrombocytopenia and disseminated intravascular coagulation. Deep vein thrombosis of the vena cava can occur in dogs and may mimic an abdominal mass. Multiple mechanisms may be involved in the development of venous thrombosis, including endothelial damage by neoplastic cells and the presence of a hypercoagulable state secondary to neoplasia. Extensive collateral circulation may allow removal of diseased vena cava. PMID- 10730626 TI - Comparison of needle-core (Trucut) biopsy and surgical biopsy for the diagnosis of cutaneous and subcutaneous masses: a prospective study of 51 cases (November 1997-August 1998). AB - Cutaneous or subcutaneous masses in 51 dogs and cats were examined by needle-core (i.e., Trucut) biopsy, and results were compared to results of surgical biopsy to assess the accuracy of the former. Needle-core specimens obtained before surgical biopsy were submitted to a single pathologist who evaluated both samples and was blinded to the results of surgical biopsy when evaluating the Trucut specimen. The results indicate that needle-core biopsy can accurately predict surgical biopsy. Thus, needle-core biopsy performed before surgical excision of masses can facilitate planning and reduce the need for numerous surgical procedures. Needle core biopsy can direct appropriate treatment of nonmalignant masses. PMID- 10730627 TI - Blastomycosis granuloma involving the cranial vena cava associated with chylothorax and cranial vena caval syndrome in a dog. AB - A four-year-old, sexually intact, male dachshund was diagnosed with pulmonary blastomycosis. Itraconazole was administered for 60 days, and the dog was considered to be disease-free at three- and 12-month reevaluations. Two years following discontinuation of itraconazole, the dog developed a granuloma of the cranial vena cava resulting in chylothorax and cranial vena caval obstruction. To the authors' knowledge, this is the first case of a blastomycotic granuloma involving the vena cava reported in the dog. Blastomycosis should be considered as a differential diagnosis for both chylothorax and cranial vena caval syndrome in the dog. PMID- 10730628 TI - Primary hemangiosarcoma of the iliopsoas muscle eliciting a peripheral neuropathy. AB - An eight-year-old, male castrated bullmastiff presented to the Kansas State University Veterinary Medical Teaching Hospital with left hind-limb paralysis. A mass was identified in the left paralumbar soft tissue adjacent to the fourth (L4) to sixth (L6) lumbar vertebrae by magnetic resonance imaging. The iliopsoas muscle contained the mass which was identified as a hemangiosarcoma on histopathological examination. Hemangiosarcoma is rarely reported as a primary tumor arising from muscle vascular endothelium. PMID- 10730629 TI - Diffuse annular fusiform adenocarcinoma in a dog. AB - Canine colonic intestinal adenocarcinoma typically presents as rectal polypoid or annular stenotic masses causing clinical signs consistent with large bowel disease. This report discusses an unusual case of intestinal adenocarcinoma in an 11-year-old, neutered male German shepherd dog presented for evaluation of anorexia, profuse watery diarrhea, and weight loss. In this dog, colonic adenocarcinoma diffusely infiltrated the entire large bowel and caused an annular fusiform lesion, as confirmed by endoscopic biopsies and postmortem examination. Other unique features included a paucity of desmoplasia associated with the neoplastic lesion and widespread metastasis to regional lymph nodes, lung, and prostate. PMID- 10730630 TI - Luxation of the superficial digital flexor tendon in a cat. AB - Superficial digital flexor luxation has been described in dogs, horses, and cattle. To the authors' knowledge, it has not been reported in cats. In the case of this report, monofilament nonabsorbable suture material was used to repair a laterally luxating superficial digital flexor tendon in a cat. The repair was similar to that which has been described in dogs. Whereas many etiologies of superficial tendon luxation have been proposed in dogs, trauma was believed to have contributed to the tendon luxation in this cat. PMID- 10730631 TI - A careful neurologic examination should precede neuroimaging studies in HIV infected patients with headache. PMID- 10730632 TI - Funded research and neuroradiology. PMID- 10730633 TI - Treatment of atherosclerotic disease at the cervical carotid bifurcation: current status and review of the literature. PMID- 10730634 TI - Screening CT of the brain determined by CD4 count in HIV-positive patients presenting with headache. AB - BACKGROUND AND PURPOSE: Few studies have examined HIV-positive patients presenting with uncomplicated headache for clinical variables that might be predictive of those patients who would most benefit from CT. Because of the value of CD4 counts in predicting the relative risk of developing opportunistic infections and neoplasms, we assessed the diagnostic yield of screening CT in HIV positive patients presenting with headache as sorted by CD4 count. METHODS: We reviewed CT scan results and CD4 counts in patients presenting with headache uncomplicated by altered mental status, meningeal signs, neurologic findings, or symptoms of subarachnoid hemorrhage. For analysis, scans were considered positive or negative and were grouped according to CD4 counts of less than 200 cells/microL, 200 to 499 cells/microL, and equal to or greater than 500 cells/microL. The results were then analyzed using the chi2 test. RESULTS: One hundred seventy-eight HIV-positive patients underwent a total of 204 unenhanced and contrast-enhanced CT examinations. One hundred twenty-eight (62.7%) of the scans were negative, and 76 (37.3%) were positive. Of the positive scans, 58 (76.3%) showed atrophy only and 18 (23.7%) showed mass lesions or white matter lesions. All cases that were positive for mass lesions or white matter lesions occurred in patients with CD4 counts less than 200 cells/microL (P = .04). CONCLUSION: A recent CD4 count provides an important predictor variable when considering performing CT in HIV-positive patients presenting with uncomplicated headache. Performing CT of the head for patients with CD4 counts equal to or greater than 200 cells/microL is of questionable value considering the low prevalence of positive CT findings. For this select group of patients, MR imaging may be more appropriate than CT. Patients with CD4 counts less than 200 cells/microL should undergo CT because of the high prevalence of positive scans. PMID- 10730635 TI - Nipah viral encephalitis or Japanese encephalitis? MR findings in a new zoonotic disease. AB - BACKGROUND AND PURPOSE: An epidemic of suspected Japanese encephalitis occurred in Malaysia in 1998-1999 among pig farmers. In neighboring Singapore, an outbreak occurred among pig slaughterhouse workers. It was subsequently established that the causative agent in the outbreak was not the Japanese encephalitis virus but a previously unknown Hendra-like paramyxovirus named Nipah virus. METHODS: The brain MR images of eight patients with Nipah virus infection were reviewed. All patients tested negative for acute Japanese encephalitis virus. Seven patients had contrast-enhanced studies and six had diffusion-weighted examinations. RESULTS: All patients had multiple small bilateral foci of T2 prolongation within the subcortical and deep white matter. The periventricular region and corpus callosum were also involved. In addition to white matter disease, five patients had cortical lesions, three had brain stem involvement, and a single thalamic lesion was detected in one patient. All lesions were less than 1 cm in maximum diameter. In five patients, diffusion-weighted images showed increased signal. Four patients had leptomeningeal enhancement and four had enhancement of parenchymal lesions. CONCLUSION: The brain MR findings in patients infected with the newly discovered Nipah paramyxovirus are different from those of patients with Japanese encephalitis. In a zoonotic epidemic, this striking difference in the appearance and distribution of lesions is useful in differentiating these diseases. Diffusion-weighted imaging was advantageous in increasing lesion conspicuity. PMID- 10730636 TI - A CT method to measure hemodynamics in brain tumors: validation and application of cerebral blood flow maps. AB - BACKGROUND AND PURPOSE: CT is an imaging technique that is routinely used for evaluating brain tumors. Nonetheless, imaging often cannot show the distinction between radiation necrosis and neoplastic growth among patients with recurrent symptoms after radiation therapy. In such cases, a diagnostic tool that provides perfusion measurements with high anatomic detail would show the separation between necrotic areas, which are characterized by low perfusion, from neoplastic areas, which are characterized by elevated CBF. We attempted to validate a dynamic contrast-enhanced CT method for the measurement of regional CBF in brain tumors, and to apply this method by creating CBF maps. METHODS: We studied nine New Zealand White rabbits with implanted brain tumors. We obtained dynamic CT measurements of CBF, cerebral blood volume (CBV), and permeability surface (PS) from the tumor, peritumor, and contralateral normal tissue regions. In all nine rabbits (two studies per rabbit), we compared CT-derived CBF values with those simultaneously obtained by the standard of reference ex vivo microsphere technique. Using CT, we examined three rabbits to assess the variability of repeated CBF and CBV measurements; we examined the other six to evaluate regional CBF reactivity to arterial carbon dioxide tensions. Finally, CT CBF maps were obtained from a rabbit with a brain tumor during normocapnia and hypocapnia. RESULTS: We found a significant linear correlation (r = 0.847) between the regional CT-and microsphere-derived CBF values, with a slope not significantly different from unity (0.99+/-0.03, P>.01). The mean difference between regional CBF measurements obtained using both methods did not significantly deviate from zero (P>.10). During normocapnia, tumor had significantly higher CBF, CBV, and PS values (P<.05) than did peritumor and normal tissues. The variability in CT derived CBF and CBV measurements in the repeated studies was 13% and 7%, respectively. CT revealed no significantly different CBF CO2 reactivity from that determined by the microsphere method (P>.10). The CBF map of tumor regions during normocapnia showed much higher flow than normal regions manifested, and this difference was reduced on the hypocapnia CBF map. CONCLUSION: The dynamic CT method presented herein provides absolute CBF measurements in brain tumors that are accurate and precise. Preliminary CBF maps derived with this method demonstrate their potential for depicting areas of different blood flow within tumors and surrounding tissue, indicating its possible use in the clinical setting. PMID- 10730637 TI - Prognostic factors in recurrent glioblastoma multiforme and anaplastic astrocytoma treated with selective intra-arterial chemotherapy. AB - BACKGROUND AND PURPOSE: Factors predictive of primary brain tumor outcome have been studied extensively, although the prognostic value of radiologic data, such as MR imaging and angiographic characteristics, has not been studied in depth. The purpose of this study was to determine whether radiologic data were prognostic factors among patients with recurrent glioblastoma multiforme and anaplastic astrocytoma treated with selective intra-arterial chemotherapy. METHODS: Forty-six patients were enrolled in a Phase II study of intra-arterial chemotherapy with carboplatin and Cereport (Alkermes Inc.; Cambridge, MA), a bradykinin analog that selectively increases permeability of the blood-tumor barrier. MR imaging volumes of enhancing tumor, resection cavity, and T2 signal abnormality were measured with T1-weighted and T2-weighted sequences. Volumes were analyzed individually and in various combinations. Tumor vascularity was graded on angiograms. Outcome was measured by time to tumor progression and survival. RESULTS: Of 46 patients included in this study, 41 underwent evaluation. Thirty were male and 11 were female; mean age was 48.5 years. Karnofsky scores ranged from 70 to 100. Thirty-two patients had glioblastoma multiforme, whereas nine had anaplastic astrocytoma. Twenty-eight patients had tumor progression and 13 had stable disease. Twenty-three patients died after an average of 205 days; 18 were surviving at an average of 324 days from the start of intra-arterial chemotherapy. In multivariate analysis, time from diagnosis to intra-arterial chemotherapy was predictive both of time to tumor progression and survival. Net tumor volume and vascularity also were significant for survival. Age, Karnofsky performance status, histologic findings, gender, MR imaging area, resection cavity volume, T2 signal abnormality volume, and various combined volumes were not significant. CONCLUSION: If confirmed by further studies, radiologic factors such as tumor volume and angiographic vascularity should be considered in design and stratification of future chemotherapy trials. PMID- 10730638 TI - Cortical laminar necrosis caused by immunosuppressive therapy and chemotherapy. AB - We report three patients in whom neurologic symptoms and cortical laminar necrosis developed after immunosuppressive treatment (cyclosporin A and FK 506) and polychemotherapy (vincristine and methotrexate). Initial neuroradiologic studies showed cortical and white matter involvement. Follow-up studies showed cortical hyper-intense lesions on T1-weighted MR images, consistent with cortical laminar necrosis. The clinical and radiologic data indicate that a transient hypoxic-ischemic process could have been responsible for the encephalic lesions in these three patients. PMID- 10730639 TI - MR imaging findings of Rathke's cleft cysts: significance of intracystic nodules. AB - BACKGROUND AND PURPOSE: Rathke's cleft cysts often may be difficult to differentiate from other intrasellar or suprasellar masses on radiologic studies. The purpose of this study was to describe the significance of intracystic nodules, a diagnostic characteristic found in Rathke's cleft cysts, on MR images. METHODS: A retrospective review of MR studies was conducted for 13 patients who, after pathologic analysis, were diagnosed as having Rathke's cleft cyst. These patients underwent unenhanced and contrast-enhanced T1- and T2-weighted axial and coronal spin-echo sequential imaging. The signal intensity and incidence of the intracystic nodules on T1- and T2-weighted images were analyzed. The signal intensity of the nodule was compared with that of white matter and surrounding cyst fluid. The signal intensity of cyst fluid was compared with the intraoperative appearance of the cyst fluid. Biochemical and pathologic analyses of the intracystic nodules were conducted in two cases. RESULTS: An intracystic nodule having high signal intensity on T1-weighted images and low signal intensity on T2-weighted images was observed in 10 (77%) of the cases. At surgery, intracystic nodules were yellow, waxy, solid masses. Pathologic analysis showed this nodule to be a mucin clump. Biochemical analysis of the intracystic nodules showed cholesterol and proteins as the main constituents. In the Rathke's cleft cyst with intracystic nodules, cyst fluid revealed low signal intensity to isointensity relative to the intensity of the nodules on T1-weighted images, and isointensity to high signal intensity on T2-weighted images. Intracystic nodules were clearly visible on T2-weighted images. CONCLUSION: Because cyst fluid of Rathke's cleft cysts shows variable intensities on MR images, the specific diagnosis is often difficult when based on MR signal intensity values alone. The presence of an intracystic nodule with characteristic signal intensities on MR images may be indicative of the diagnosis of Rathke's cleft cyst. PMID- 10730640 TI - Colloid cysts of the third ventricle: are MR imaging patterns predictive of difficulty with percutaneous treatment? AB - BACKGROUND AND PURPOSE: Colloid cysts of the third ventricle are rare benign brain tumors. The purpose of this study was to correlate their patterns on MR images with the probability of success of percutaneous treatment. METHODS: Nineteen patients underwent endoscopic treatment for colloid cysts of the third ventricle. The cases were divided into two groups based on difficulty of the aspiration procedure. We reviewed CT scans and MR images and divided cysts into groups based on their signal intensity on the MR images and their density on CT scans. Intensity and density were correlated with difficulty of aspiration during the endoscopic procedure. RESULTS: The aspiration procedure was difficult in 63% of the cases. Eighty-nine percent of hyperdense cysts on unenhanced axial CT scans were categorized as difficult, and 75% of hypodense cysts were categorized as easy. On T2-weighted MR sequences, 100% of low-signal cyst contents were difficult and nearly 63% of high-signal lesions were easy. There was a significant correlation between the T2-weighted sequences and the CT scans regarding the difficulty of the aspiration procedure. CONCLUSION: T2-weighted MR sequences are useful for predicting difficulty of aspiration during stereotactic or endoscopic procedures. A T2-weighted low-signal cyst is correlated with high viscosity intracystic contents. PMID- 10730641 TI - CSF flow studies of intracranial cysts and cyst-like lesions achieved using reversed fast imaging with steady-state precession MR sequences. AB - BACKGROUND AND PURPOSE: Differentiating between intracranial cysts or cyst-like structures and communicating or noncommunicating cysts is often not possible with cranial CT or nonfunctional MR imaging. We evaluated a retrospective ECG-gated fast imaging with steady-state precession (PSIF) MR sequence with optional cine mode to differentiate cystic masses from enlarged CSF spaces and to determine the accuracy of detecting communication between cysts and neighboring CSF spaces. METHODS: Fourteen patients with intracranial cystic masses underwent CSF flow studies with an ungated and a retrospective ECG-gated cine-mode PSIF sequence in addition to spin-echo imaging. Findings were evaluated retrospectively by using a five-point rating scale and without knowledge of clinical or other imaging findings. Results were compared with intraoperative findings or with results of intrathecal contrast studies. RESULTS: Eighteen arachnoid cysts and one enlarged cisterna magna were diagnosed. Improved differentiation between cysts and enlarged CSF spaces was obtained with cine-mode PSIF imaging in six lesions (six patients). Increased diagnostic certainty as to communication between cysts and CSF spaces was obtained in 18 cysts (13 patients). Diagnoses were verified by membranectomy in five lesions, by CT cisternography in five lesions, and indirectly by shunting in one cystic lesion. In one case, MR diagnosis was not confirmed by CT cisternography. CONCLUSION: Cine-mode MR imaging with a retrospective ECG-gated flow-sensitive PSIF sequence contributed to the certainty of communication between arachnoid cysts and neighboring CSF spaces with an accuracy of 90%, using surgical findings or intrathecal contrast studies as reference. Differentiation between intracranial cysts and enlargement of CSF spaces and other cystic masses was improved in 25% of cases. PMID- 10730642 TI - Intraventricular CSF pulsation artifact on fast fluid-attenuated inversion recovery MR images: analysis of 100 consecutive normal studies. AB - BACKGROUND AND PURPOSE: CSF pulsation artifact is a pitfall of fast fluid attenuated inversion-recovery (FLAIR) brain MR imaging. We studied ventricular CSF pulsation artifact (VCSFA) on axial FLAIR images and its relationship to age and ventricular size. METHODS: Fast FLAIR axial images were obtained on a 1.5-T unit (8000/150/2 [TR/TE/ excitations], inversion time = 2200, field of view = 24 cm, matrix = 189x256, and 5-mm interleaved sections). Two observers rated VCSFA (hyperintensity on FLAIR images) in the lateral, third, and fourth ventricles by using a three-point ordinal scale in 100 consecutive subjects (ages 20-86 years) with normal brain MR studies. Left-to-right third ventricular width was also measured. RESULTS: Seventy-two subjects had VCSFA in at least one ventricular cavity. The fourth ventricle was the most common site of VCSFA (n = 58), followed by the third ventricle (n = 47) and the lateral ventricles (n = 13). VCSFA was usually severe in the third and fourth ventricles and less severe in the lateral ventricles. Fourth ventricular VCSFA was significantly associated with third ventricular VCSFA. Increasing third ventricular size and, to a lesser extent, increasing age was significantly associated with VCSFA. Ghost pulsation of VCSFA occurred across the brain parenchyma in the phase-encoding direction. VCSFA seen in the fourth ventricle on axial FLAIR images disappeared on sagittal FLAIR images in one subject. CONCLUSION: VCSFA on axial FLAIR images represents inflow artifact caused by inversion delay and ghosting effects. VCSFA might obscure or mimic intraventricular lesions, especially in the third and fourth ventricles. Although common in adults of all ages, VCSFA is associated with advancing age and increasing ventricular size. Thus, altered CSF flow dynamics that occur with ventriculomegaly and aging contribute to VCSFA on axial FLAIR MR images. PMID- 10730643 TI - Contrast-enhanced transcranial color-coded duplexsonography in stroke patients with limited bone windows. AB - BACKGROUND AND PURPOSE: Thickening of the temporal bone in stroke-age patients may obviate sonographic evaluation of the circle of Willis in 20% to 30% of patients. We assessed the diagnostic efficacy of contrast-enhanced transcranial color-coded duplexsonography (TCCD) for noninvasive evaluation of the circle of Willis in stroke patients with limited bone windows. METHODS: Of 171 consecutive patients who presented with ischemic symptoms in the middle cerebral artery (MCA) territory, 49 patients (32 female, 17 male; age range, 70.5+/-10.6 years) had no detectable colorflow signals from the circle of Willis by TCCD because of limited acoustic windows. These 49 patients received an IV injection of a sonographic contrast-enhancing agent, Levovist (Schering; Berlin, Germany), and were re examined. Correlative imaging studies of the circle of Willis were obtained in 42 of 49 of these patients. RESULTS: In 38 of 49 patients, contrast-enhanced TCCD enabled full visualization of the circle of Willis bilaterally; in an additional five patients, contrast-enhanced TCCD revealed only the portion of the circle of Willis ipsilateral to the probe through one temporal bone. In six of these 43 patients, contrast-enhanced TCCD showed MCA stenosis and MCA occlusion in three; three of the six cases of MCA stenosis and all three cases of the MCA occlusion were found on the symptomatic side. In six of 49 patients, no colorflow signals were obtained after contrast enhancement. All contrast-enhanced TCCD findings were confirmed by CT angiography, transfemoral digital subtraction angiography, MR angiography, or a combination of all three correlative studies. Levovist produced no serious adverse events. CONCLUSION: In stroke-age patients with limited acoustic windows, contrast-enhancement with Levovist can markedly increase the sensitivity of TCCD and increase the detection of clinically relevant intracranial arterial disease. PMID- 10730644 TI - Pure sensory stroke caused by a cerebral hemorrhage: clinical-radiologic correlations in seven patients. AB - BACKGROUND AND PURPOSE: Pure sensory stroke (PSS) usually is caused by a lacunar infarct; reports of PSS caused by cerebral hemorrhage have been rare. We correlated clinical and neuroradiologic findings in patients with PSS caused by cerebral hemorrhage. METHODS: We retrospectively studied seven patients with appropriate clinical findings and lesions revealed by X-ray CT and MR imaging (five men, two women; age range, 46-64 years; mean age, 55.9 years). RESULTS: Hemorrhages involved the thalamus, pons, internal capsule, or cerebral cortex. MR imaging revealed thalamic PSS was located in the ventral posterior lateral (VPL) or ventral posterior medial (VPM) nucleus; a lesion producing a thalamic cheiro oral syndrome was situated on the border between the VPL and VPM. Pontine PSS involved the medial lemniscus together with the ventral trigeminothalamic tract, sparing the anterior and lateral spinothalamic tracts. Accordingly, pontine PSS, but not thalamic PSS, selectively affected vibration and position sense while leaving pinprick and temperature perception intact, and oral sensory involvement was bilateral when cheiro-oral syndrome had a pontine origin. MR imaging revealed hemorrhage in the postcentral gyrus in the cortical variety of PSS and in the posterior part of the posterior limb (thalamocortical sensory pathway) in PSS of internal capsular origin. The postcentral gyral lesion impaired stereognosis and graphesthesia. CONCLUSION: Focal hemorrhages can lead to purely sensory stroke syndromes, and the clinical deficits are fairly well linked with the locations of the bleeds. PMID- 10730645 TI - Diffuse vasospasm after pretruncal nonaneurysmal subarachnoid hemorrhage. AB - Pretruncal (perimesencephalic) nonaneurysmal hemorrhage is a benign form of subarachnoid hemorrhage (SAH). Angiographic changes of vasospasm are uncommon in patients with this type of hemorrhage, and if vasospasm is present, it is mild and focal. We report two patients with pretruncal nonaneurysmal SAH who developed severe and diffuse vasospasm, expanding the clinical spectrum of this type of SAH. The first patient was a 40-year-old woman who suffered pretruncal nonaneurysmal SAH. Angiography performed on the seventh day post hemorrhage showed diffuse and severe vasospasm affecting both the anterior and the posterior circulation. The patient was treated with hypervolemia, and she remained asymptomatic. Follow-up angiography showed resolution of the vasospasm. The second patient was a 67-year-old woman who suffered pretruncal nonaneurysmal SAH. The results of the initial angiography were normal. Repeat angiography on the ninth day post hemorrhage showed severe vasospasm in the anterior circulation and moderate vasospasm in the posterior circulation. Nine hours later, the patient developed transient dysphasia, and she was treated with hypervolemia. Three days later, a transcranial Doppler examination showed normalization of blood velocities. The presence of diffuse and severe vasospasm does not exclude a diagnosis of pretruncal nonaneurysmal SAH. PMID- 10730646 TI - Functional MR imaging using a visually guided saccade paradigm for comparing activation patterns in patients with probable Alzheimer's disease and in cognitively able elderly volunteers. AB - BACKGROUND AND PURPOSE: Alzheimer's disease is associated with progressive visuospatial dysfunction. This study used functional MR (fMR) imaging with an eye movement paradigm to investigate differences in visuospatial cognition between patients with probable Alzheimer's disease (pAD) and cognitively able elderly volunteers. METHODS: Using established, although imperfect, clinical criteria, patients with pAD (n = 18) and cognitively able elderly volunteers (n = 10) were selected for study. All patients underwent echo-planar fMR imaging at 1.5 T. The visually guided saccade paradigm consisted of alternating periods (30 s) of central fixation and visually guided saccades to a target appearing randomly along the horizontal meridian. Activation maps were derived using a voxelwise t test, comparing the signal intensities between the two steady-state conditions. The activation patterns were characterized by Talairach coordinates, activation volumes, and laterality ratios (LRs). RESULTS: Statistically significant differences existed between the activation patterns of the patients with pAD and those of the volunteers. In contrast to the control group, a left-dominant parietal activation pattern and enhanced prefrontal cortical activation were observed in most patients with pAD. CONCLUSION: Within the limitations of the imperfect clinical standard of reference, the reduction in right parietal activation producing the left-dominant LR for the intraparietal sulcus may reflect the progressive dysfunction in spatial attention associated with Alzheimer's disease, considering the known parietal lobe involvement in this function and the disease. The high specificity of a positive intraparietal sulcal LR measured by fMR imaging may have a role in detecting and monitoring Alzheimer's disease. PMID- 10730647 TI - Endovascular problem solving with intravascular stents. AB - BACKGROUND AND PURPOSE: Intravascular stents are being used with increasing frequency in interventional neuroradiology. They provide the potential to expand the therapeutic capabilities of the endovascular therapist and stand to revolutionize endovascular intervention within both the intracranial and extracranial vessels. We present our application of stent technology to further the understanding of endovascular rescue from procedural complications and the solving of complex clinical problems. METHODS: Three patients underwent unplanned placement of intravascular stents. In two patients a stent was used to provide stabilization of an irretrievable intravascular device; in the third patient a stent was used to provide a scaffolding for proximal external carotid sacrifice. RESULTS: Stent deployment was successful in all patients. The intravascular devices stabilized by stent placement included unraveled fragments of a Guglielmi detachable coil (GDC) and a partially deployed coronary stent. Proximal external carotid sacrifice was achieved with the aid of a stent in one patient to control hemorrhage from recurrence of laryngeal cancer. No periprocedural neurologic complications were encountered. Six-month follow-up angiography in one patient showed only minimal myointimal hyperplasia induced by stent-stabilized GDC fragments adjacent to the internal carotid vessel wall. CONCLUSION: Stents can be used to provide stabilization of irretrievable intravascular devices or as a scaffolding for proximal vessel sacrifice. These applications may allow endovascular rescue of procedural complications and solve unique clinical problems. PMID- 10730648 TI - Arterial dissections complicating cerebral angiography and cerebrovascular interventions. AB - BACKGROUND AND PURPOSE: Iatrogenic dissections are an uncommon complication of cerebral angiography. We retrospectively reviewed 12 cases of arterial dissections complicating cerebral angiography and cerebrovascular interventions to evaluate the clinical course of these dissections. METHODS: Cases from a large tertiary center performing a large number of neurovascular procedures were collected retrospectively. The patients' medical records and imaging studies were reviewed, with particular attention given to the cause of the dissection, the development of ischemic events resulting from the dissection, and the treatment used. RESULTS: Each of nine dissections affected a vertebral artery, each of two affected an internal carotid artery, and one affected a common carotid artery. The prevalence of iatrogenic dissections was 0.4%. Seven of the dissections were noted at the time of contrast material injection for the filming of cerebral angiograms. The other five dissections occurred during catheter or wire manipulations for interventional neuroradiologic procedures. Five of the patients in our series were treated with IV administered heparin for 24 to 48 hours. The other seven patients had recently suffered acute intracranial hemorrhage or undergone neurosurgery and could not undergo anticoagulant therapy. None of the patients developed symptoms of ischemia, but one was later found to have an asymptomatic infarct in the territory supplied by the dissected artery. CONCLUSION: Arterial dissections are an uncommon complication of cerebral angiography and cerebrovascular interventions and usually have a benign clinical course. PMID- 10730649 TI - Internal carotid artery aneurysm visualized during successful endovascular treatment of carotid embolism. AB - We herein present a case of an internal carotid artery embolism associated with a hidden internal carotid artery aneurysm. The aneurysm was visualized during successful endovascular treatment of the carotid embolism. In retrospect, the aneurysm was at risk of rupture during the procedure. In the endovascular treatment of cerebral embolism, the possibility that aneurysms are hidden by emboli must be borne in mind. Care should be taken not to injure unidentified arterial walls while advancing a catheter blindly. PMID- 10730650 TI - Idiopathic ischemic cerebral infarction in childhood: depiction of arterial abnormalities by MR angiography and catheter angiography. AB - BACKGROUND AND PURPOSE: We report our experience with MR imaging, MR angiography, and catheter angiography in children with acute idiopathic cerebral infarction and suggest that catheter angiography may still play an important role in this setting. METHODS: During the past 8 years, 18 children with idiopathic cerebral infarction underwent MR imaging and catheter angiography; 17 were also studied with MR angiography. MR imaging was done within 34 hours after onset of hemiplegia or seizures or both. Sixteen patients underwent catheter angiography within 36 hours of MR imaging; 12 studies were performed within 22 hours. Two patients underwent catheter angiography, in both cases within 72 hours. Infarcts were compared with arterial abnormalities seen at catheter angiography, and the results of MR angiography were compared with those seen at catheter angiography. RESULTS: Comparing MR angiography with catheter angiography, we found the positive predictive value of MR angiography for arteriopathy was 100%, with a negative predictive value of 88%. MR angiography was equivalent to catheter angiography in the detection and depiction of proximal middle cerebral artery disease; however, depiction of disease in the internal carotid artery (ICA) and detection of peripheral embolic disease were better with catheter angiography than MR angiography. CONCLUSION: Basal ganglia lesions associated with ICA disease by MR angiography should probably be studied with digital subtraction angiography, as MR angiography did not depict the length and severity of ICA disease as well as catheter angiography did. Hemispheric infarcts should be studied with catheter angiography, as emboli may occur in the absence of heart disease; the circle of Willis may be uninvolved with embolic disease, and MR angiography is not sensitive to emboli in small peripheral intracranial arteries. PMID- 10730651 TI - Tuberous sclerosis: differences between cerebral and cerebellar cortical tubers in a pediatric population. AB - BACKGROUND AND PURPOSE: Histologic evidence of cortical tubers is pathognomonic of tuberous sclerosis (TS) disease. Cerebellar tubers, however, are uncommonly found. Our objective was to evaluate the prevalence and characteristics of cerebellar tubers in a large series of pediatric patients with TS studied with MR imaging. In particular, their relationship with volume loss and age will be analyzed. METHODS: MR images of 34 children with TS and cortical tubers were reviewed. There were 17 female and 17 male patients. The mean age was 8.9+/-4.5 years (mean +/- SD), with a range from 2 to 14 years. The number and location (supratentorial, infratentorial) of tubers and volume loss of the underlying parenchyma were recorded. The relationship between tuber location, patient age, and number of tubers was studied using the Student's t test for independent samples. The relationship between tuber location and presence of volume loss was established using the Pearson chi2 test. RESULTS: The mean number of cortical tubers was 14.3+/-8.5. Fifteen (44.1%) patients had cerebellar tubers associated with cerebral lesions. Patients with cerebellar and cerebral tubers had significantly more global cortical lesions than did patients with isolated cerebral tubers (17.9+/-8.9 versus 11.4+/-7.2 tubers, P = .026). Patients with cerebellar tubers were significantly older than those with isolated supratentorial tubers (11.3+/-3.4 versus 7.1+/-4.4 years, P = .005). In only four (11.8%) patients were tubers associated with focal parenchymal volume loss. The tubers in all of these cases were located in the cerebellum-indicating a significant relationship with volume loss (Pearson chi2 test, P = .017). CONCLUSION: In this series, cerebellar tubers were frequent and were always present in association with cerebral cortical tubers. They were seen in older children with a larger total number of tubers. Cerebellar tubers may be associated with focal volume loss. PMID- 10730652 TI - Multivariate feature analysis of sonographic findings of metastatic cervical lymph nodes: contribution of blood flow features revealed by power Doppler sonography for predicting metastasis. AB - BACKGROUND AND PURPOSE: Sonographic criteria of the lymph node have been found to be good indicators for metastatic lymph nodes. We determined which sonographic features are most predictive of metastasis in cervical lymph nodes among patients with head and neck cancer. METHODS: Gray-scale and power Doppler sonograms were retrospectively analyzed in 133 cervical lymph nodes (57 metastatic and 76 reactive nodes) from 52 patients with head and neck cancer. The gray-scale sonographic features of the presence or absence of hilar echoes, parenchymal echogenicity, and short and long axis lengths as well as the power Doppler features of normal hilar flow and abnormal parenchymal flow were evaluated. Univariate and multivariate logistic regression analyses were conducted to determine the relative value of each sonographic feature. RESULTS: At univariate analysis, all sonographic features assessed were found to be important. Multivariate analysis, however, suggested that the presence or absence of hilar echoes, increases in short axis length, and the presence of normal hilar flow were the only sonographic features that were predictive of reactive (presence of hilar echoes and hilar flow) and metastatic (increases in short axis length) lymph nodes. Although multivariate analysis did not indicate any significant contribution of the color-flow criteria for predicting metastatic nodes, the color-flow criteria appeared to improve the overall diagnostic accuracy for the less experienced observer. CONCLUSION: The sonographic criteria most predictive of metastatic cervical lymph nodes were absent hilar echoes and increases in short axis length, as assessed by logistic regression analysis. Compared with these gray-scale criteria, color-flow criteria had fewer predictive advantages. PMID- 10730653 TI - Great auricular nerve: anatomy and imaging in a case of perineural tumor spread. AB - We present the imaging and clinical findings of a case of recurrent cutaneous squamous cell carcinoma of the face in which CT and MR imaging revealed perineural tumor spread along the great auricular nerve. The great auricular nerve is a superficial cutaneous branch of the cervical plexus, providing sensory innervation to the skin of the parotid and periauricular region. Our purpose was to familiarize the reader with the anatomy of this nerve and imaging's potential role in the diagnosis of perineural tumor spread along this seldom seen structure. PMID- 10730654 TI - The persistent stapedial artery. AB - The persistent stapedial artery is a rare congenital vascular anomaly that may present as a pulsatile middle ear mass or that may appear as an incidental finding. Five cases of persistent stapedial artery are presented. The CT findings include the absence of the ipsilateral foramen spinosum and a soft-tissue prominence in the region of the tympanic segment of the facial nerve. Three cases were associated with an aberrant internal carotid artery. Imaging identification of this variant may obviate unnecessary surgery and may help in planning surgical or endovascular interventions. PMID- 10730655 TI - Recurrence of clival chordoma along the surgical pathway. AB - Chordomas are locally aggressive malignant tumors of notochordal origin whose metastatic potential is increasingly recognized. Surgical pathway recurrence has been noted only rarely in the literature. We present three patients with clival chordomas whose sole or initial recurrence was along the pathway of prior surgical access. A characteristic mass found along the pathway of prior surgical access for resection of a chordoma should suggest recurrent chordoma. PMID- 10730656 TI - Dedifferentiated chondrosarcoma of the larynx. AB - A 74-year-old man with a history of a chondroid lesion of the larynx noted an enlarging neck mass. Axial CT showed a large expansile lesion arising from the left thyroid cartilage. Multiple rings and arcs with relatively intact cortex indicated a chondroid lesion. Irregularity of the anterolateral margin abutted a prominent soft-tissue component. The specimen obtained from fine needle aspiration was suggestive of a malignant fibrous histiocytoma. After further resection, the final diagnosis was dedifferentiated chondrosarcoma. A new soft tissue component or rapid growth of the mass can be indicative of a diagnosis of dedifferentiated chondrosarcoma. PMID- 10730657 TI - Diffusion-weighted MR imaging of the spinal cord. AB - Diffusion-weighted MR imaging may increase the sensitivity and specificity of MR imaging for certain pathologic conditions of the spinal cord but is rarely performed because of several technical issues. We therefore tested a novel phase navigated spin-echo diffusion-weighted interleaved echo-planar imaging sequence in seven healthy volunteers and six patients with intramedullary lesions. We performed diffusion-weighted MR imaging of the spinal cord with high spatial resolution. Different patterns of diffusion abnormalities observed in patient studies support the possible diagnostic impact of diffusion-weighted MR imaging for diseases of the spinal cord. PMID- 10730658 TI - Treatment of an aneurysmal bone cyst of the spine by radionuclide ablation. AB - We report the nonoperative treatment of a recurrent, multilevel spinal aneurysmal bone cyst by injection of 32P chromic phosphate colloid into the cyst. The patient was then followed up with serial CT examinations, which showed stabilization and progressive ossification within the lesion. The rationale, alternatives, and possible contraindications to radionuclide ablation of spinal aneurysmal bone cysts are discussed. PMID- 10730659 TI - Combined anomaly of intramedullary arteriovenous malformation and lipomyelomeningocele. AB - We report a rare situation in which a lipomyelomeningocele and an intramedullary arteriovenous malformation (AVM) occurred together at the T11-L1 level in a 44 year-old man. MR images showed a hypervascular lesion intradurally and a fatty component extradurally. Spinal angiography revealed this lesion to be an intramedullary AVM with multiple feeding arteries from the right T12 and left T10 intercostal artery and the left L1 lumbar artery, drained by tortuous, dilated, perimedullary veins. PMID- 10730660 TI - Primary amyloidoma of the cervical spine. AB - Primary solitary amyloidoma of the spine is a disease characterized by localized deposits of amyloid. We describe and illustrate the radiologic appearance of primary solitary amyloidoma of the spine on plain radiographs, CT scans, and MR images. The imaging findings revealed features of a nonspecific soft-tissue mass with calcifications. Epidural extension of the amyloidoma caused spinal cord compression. PMID- 10730661 TI - Neuroradiology classics. PMID- 10730662 TI - In re: radiologic and histopathologic evaluation of canine artery occlusion after collagen-coated platinum microcoil delivery. PMID- 10730663 TI - Intraventricular melanoma. PMID- 10730664 TI - Is an early angiogram needed? PMID- 10730665 TI - A case of cleidocranial dysplasia confirmed by 3D CT of the cranium. PMID- 10730666 TI - Spinal cord herniation--which one is really traumatic? PMID- 10730667 TI - Spinal cord decompression sickness. PMID- 10730668 TI - The evolutionary and embryologic basis for the development and anatomy of the cavum veli interpositi. PMID- 10730669 TI - The esthetic impact of extraction and nonextraction treatments on Caucasian patients. AB - This investigation was designed to compare the esthetic effects of extraction and nonextraction treatments. Panels of 58 laypersons and 42 dentists evaluated randomly presented pre- and posttreatment profiles of 70 extraction and 50 nonextraction Class I and II Caucasian patients. The samples were similar at the outset; however, at the end of treatment, the extraction patients' faces were, on average, 1.8 mm "flatter" than the faces of nonextraction subjects. The flatter faces were preferred by both panels, dentists more so than laypersons. In general, nonextraction treatment was seen as having little effect on the profile, whereas the perceived effect of extraction treatment was a statistically significant function of initial soft tissue protrusion-the greater the initial protrusion, the greater the benefit. The point at which a reduction in protrusion produces a perceived improvement was explored by way of regression analysis. Both panels saw extraction as being potentially beneficial when the lips were more protrusive than 2 to 3 mm behind Ricketts' E-plane. It is concluded that extraction treatment can produce improved facial esthetics for many patients who present with some combination of crowding and protrusion. PMID- 10730670 TI - Investigation of bacteremia following orthodontic debanding. AB - The aim of this study was to investigate the prevalence of bacteremia after orthodontic debanding and debonding. The study group comprised 30 patients (10 men, 20 women). All were treated using the Edgewise technique. Patients with acceptable oral hygiene and fixed appliances in both jaws were included in the study group. Blood samples were obtained using a strict aseptic technique before and after removal of bands and brackets. A 6.6% bacteremia prevalence was observed in both preoperative and postoperative blood samples. PMID- 10730671 TI - Hypodontia in children with various types of clefts. AB - The prevalence of hypodontia in children with clefts, both inside and outside the cleft region, and the possible association between the side of the cleft and the side of the missing teeth were studied using radiographs of 278 patients with cleft lip, cleft palate, or both (158 boys and 120 girls), age 5 to 18 years (mean age 10.4 years). A hypodontia prevalence of 77% (excluding third molars) was found for the total cleft sample. This was significantly higher, both statistically and clinically, than the incidence of hypodontia reported for noncleft populations, and considerably higher than the prevalence reported in other studies of children with clefts. The maxillary permanent lateral incisors were the teeth most frequently missing on the cleft side (259 teeth) followed by the maxillary (47 teeth) and mandibular (23 teeth) second premolars, in both boys and girls. The teeth that were most often missing on the noncleft side were the maxillary second premolars (12 teeth), followed by the maxillary lateral incisors (10 teeth) and mandibular second premolars (6 teeth). Hypodontia of both the maxillary lateral incisors and second premolars was found more frequently on the left side, which also has a higher frequency of clefting. PMID- 10730672 TI - Effects of a nonrinse conditioner and 17% ethylenediaminetetraacetic acid on the etch pattern of intact human permanent enamel. AB - The purpose of this study was to evaluate the effects of 2 new acid-etching solutions, nonrinse conditioner (NRC) and 17% ethylenediaminetetraacetic acid (EDTA), on enamel surface morphology, and to compare the new solutions with traditional 37% phosphoric acid. The effect of prolonged etching time was also investigated. The buccal surfaces of 80 extracted third molars were etched with one of the 3 acids for 15, 30, or 60 seconds. The central regions of the specimens were examined with a scanning electron microscope. Shorter etching time with phosphoric acid resulted in a relatively smooth enamel surface compared with longer treatments. Irrespective of treatment time, NRC produced an aprismatic etch pattern, which suggested a potentially retentive morphological character. EDTA treatment had the least effect of all etchants tested. PMID- 10730673 TI - Polymerization with the argon laser: curing time and shear bond strength. AB - The objective of this study was to determine the efficiency of an argon laser in polymerizing a light-cured orthodontic adhesive. Metal brackets were bonded to 185 premolars, divided into 5 different protocol groups of 37 each as follows: light 40-second buccal, light 40-second lingual, laser 5-second lingual, laser 10 second lingual, and laser 15-second lingual. All bonded specimens were placed in distilled water for 30 days at 37 degrees C followed by thermal cycling for 24 hours. Brackets were detached using a shearpeel load delivered by an Instron machine. The site of bond failure was examined under 10x magnification. The difference in the shear-peel bond strength between the light 40-second buccal (13.31 MPa) and the light 40-second lingual (11.95 MPa) groups was not statistically significant. The mean shear-peel bond strengths for the laser cured groups were quite similar for the 5-, 10- and 15-second laser groups (10.86, 11.32, and 10.80 MPa). The difference in mean lingual bond strength between the light 40-second and laser 5-second groups was not statistically significant (t = 1.26; P = .212). The adhesive remnant index analysis revealed principally cohesive bond failures. An increased frequency of enamel fractures at debond was noted in the lingual light-cured and 10-second laser-cured groups, at 35.1% (13/37) and 21.6% (8/ 37), respectively. All other groups displayed enamel fractures of 16.2% (6/37). A 5-second cure using an argon laser produced bond failure loads comparable to those obtained after 40 seconds of conventional light cure, with less than half the frequency of enamel fracture at debond. PMID- 10730674 TI - Sliding mechanics of coated composite wires and the development of an engineering model for binding. AB - A tribological (friction and wear) study, which was designed to simulate clinical sliding mechanics, was conducted as part of an effort to determine the suitability of poly(chloro-p-xylylene) coatings for composite orthodontic archwires. Prototype composite wires, having stiffnesses similar to those of current initial and intermediate alignment wires, were tested against stainless steel and ceramic brackets in the passive and active configurations (with and without angulation). Kinetic coefficient of friction values, which were determined to quantify sliding resistances as functions of the normal forces of ligation, had a mean that was 72% greater than uncoated wire couples at 0.43. To improve analysis of the active configuration, a mathematical model was developed that related bracket angulation, bracket width, interbracket distance, wire geometry, and wire elastic modulus to sliding resistance. From this model, kinetic coefficients of binding were determined to quantify sliding resistances as functions of the normal forces of binding. The mean binding coefficient was the same as that of uncoated wire couples at 0.42. Although penetrations through the coating were observed on many specimens, the glass-fiber reinforcement within the composite wires was undamaged for all conditions tested. This finding implies that the risk of glass fiber release during clinical use would be eliminated by the coating. In addition, the frictional and binding coefficients were still within the limits outlined by conventional orthodontic wire-bracket couples. Consequently, the coatings were regarded as an improvement to the clinical acceptability of composite orthodontic archwires. PMID- 10730675 TI - Effects of T-loop geometry on its forces and moments. AB - The moments and forces produced by various orthodontic T-loop spring designs were measured. The effects of dimension changes (within clinically used ranges) and the addition of gable bends with heat treatment were assessed. Increasing the vertical or horizontal dimension reduced the spring's load-deflection rate and its moment-to-force ratio. Gable preactivation with heat treatment had the opposite effects. PMID- 10730676 TI - Specialized spring design in segmented edgewise orthodontics: further verification of dedicated software. AB - A software program for use in the investigation of specialized springs used in the segmented arch technique has previously been developed and an example of its clinical application has been shown. The purpose of this study was to further assess the reliability of the software in 4 tests using T-loop springs. A numerical simulation of a determined experimental condition relative to a T-loop spring was carried out in each test. Numerical and experimental results were compared, and the precision of the analytical tool was assessed relative to a variety of parameters of the spring. Since the comparison between numerical and experimental results showed good agreement along the entire range of activation of the spring, the reliability of the software is sufficient for the clinical purposes of the segmented arch technique. PMID- 10730677 TI - Respiratory-related genioglossus electromyographic activity in response to head rotation and changes in body position. AB - The purpose of this study was to assess the effect of changes in body and head positions on respiratory-related activity of the genioglossus muscle in normal subjects in 8 body and head positions: (1) upright body with head straight, (2) upright body with head rotated to the right, (3) upright body with head rotated to the left, (4) supine body with head straight, (5) supine body with head rotated to the right, (6) supine body with head rotated to the left, (7) lateral recumbent body to the right, and (8) lateral recumbent body to the left. Phasic activity of the genioglossus muscle decreased significantly when subjects rotated their heads and moved from the supine to the lateral recumbent position. It is therefore concluded that genioglossus muscle activity is modulated in response to head rotation and changes in body position. PMID- 10730678 TI - Osseous morphology and spatial relationships of the temporomandibular joint: comparisons of normal and anterior disc positions. AB - The objective of this study was to determine differences in spatial relationships and osseous morphology between temporomandibular joints with normal and anterior disc positions. Magnetic resonance imaging was employed to determine disc position in 335 temporomandibular joints in 175 subjects (106 female and 69 male) between the ages of 7.27 years and 20.0 years (mean age: 13.08 years). Twelve tomographic variables were measured from preorthodontic tomograms of the same individuals. Tomographic data were cross-referenced with MRI data for those with normal and full anterior disc displacement. Independent sample t-tests revealed significant differences for all measures of joint space, condylar position, and morphology of the articular eminence (P < .05) between joints with normal disc position and with full anterior disc displacement. This study indicated that measures of joint space and eminence morphology might provide diagnostic information for the assessment of joint status in the adolescent population. PMID- 10730679 TI - Craniofacial asymmetry and temporomandibular joint internal derangement in female adolescents: a posteroanterior cephalometric study. AB - Unilateral or bilateral pathology of the osseous components of the temporomandibular joint (TMJ) can result in pronounced facial asymmetry because of dissimilar size and shape of the right and left sides of the mandible. To date, it is unknown whether abnormalities of the soft tissues of the TMJ are associated with greater than normal craniofacial asymmetry. In this study, we investigated the amount of craniofacial asymmetry in female orthodontic patients with unilateral or bilateral TMJ internal derangement (TMJ ID) relative to the amount in female patients without TMJ ID. The total sample consisted of 80 female adolescents. Bilateral TMJ magnetic resonance images were used as a database for objectively scoring the severity of TMJ ID. Craniofacial asymmetry was measured from posteroanterior cephalograms. Females with bilateral TMJ ID had significantly greater asymmetry in the vertical position of the antegonion. If the TMJ ID was more advanced on the right side, the ipsilateral ramus was shorter, resulting in significant asymmetry in this region. In all other craniofacial regions, the amount of asymmetry was not significant between females with normal TMJs and those with TMJ ID. The results indicate that a female orthodontic patient with bilateral TMJ ID or unilateral right TMJ ID may present with or develop a vertical mandibular discrepancy. PMID- 10730680 TI - Case report: Long-term outcome of class II division 1 malocclusion treated with rapid palatal expansion and cervical traction. AB - A case of a Class II Division 1 malocclusion with reduced transpalatal width and unfavorable axial inclinations of the posterior teeth is reported. Rapid palatal expansion (RPE) was used for maxillary enhancement and molar distalization therapy to correct the anteroposterior dental discrepancy. This case report illustrates the results of the method of treatment used with a long-term (16-year posttreatment) follow-up. PMID- 10730681 TI - Moxifloxacin, a new antibiotic designed to treat community-acquired respiratory tract infections: a review of microbiologic and pharmacokinetic-pharmacodynamic characteristics. AB - Moxifloxacin (BAY 12-8039) is a new 8-methoxy-fluoroquinolone antibacterial agent. The minimum inhibitory concentration for 90% of organisms (MIC90) is less than 0.25 mg/L for commonly isolated community-acquired respiratory tract pathogens including penicillin-susceptible and -resistant Streptococcus pneumoniae, Haemophilus sp, and Moraxella catarrhalis, and less than 1.0 mg/L for atypical pathogens such as Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella pneumophila. To date, emergence of resistance to moxifloxacin has been uncommon, including selection of resistance under experimental conditions (methicillin-sensitive Staphylococcus aureus, S. pneumoniae). A postantibiotic effect is observed for both gram-positive and gram-negative bacteria. Human pharmacokinetics in healthy volunteers after a single 400-mg oral dose were mean maximum concentration (Cmax) 3.2 mg/L, area under the curve (AUC) 37 mg x hour/L, and terminal elimination half-life 12.0 hours. At steady-state, Cmax and AUC were approximately 4.5 mg/L and 48 mg x hour/L, respectively. Because of a balanced system of excretion, no dosage adjustments are required in patients with renal or hepatic impairment. Moxifloxacin also has excellent penetration into upper and lower respiratory tissues. Laboratory pharmacodynamic models suggest that MIC and AUC values predict therapeutic response. Notably, the drug can be administered once/day and is not associated with drug interactions secondary to altered hepatic metabolism. In addition, since its metabolism does not involve the cytochrome P450 system, many common drug interactions are absent. The agent is being investigated in clinical trials and shows promise as a safe and effective once-daily treatment of respiratory infections. In addition, its chemical structure and pharmacokinetic and pharmacodynamic properties indicate that it has enhanced potential to minimize emergence of bacterial resistance, which should make it an excellent choice for treating respiratory tract infections now and in the future. PMID- 10730682 TI - Safety issues with herbal medicine. AB - As herbal medicines are used by increasing numbers of people, pharmacists must be knowledgeable about their safety. This requires appreciation of the magnitude of use, as well as regulations under which the products are marketed that may affect their safety. Unexpected effects of many popular herbal products are described in the literature. Thus the pharmacist's role in ensuring protection of the consumer continues to grow. PMID- 10730684 TI - Altering the estrogenic milieu of breast cancer with a focus on the new aromatase inhibitors. AB - Aromatase is a dual-enzyme complex that catalyzes the synthesis of estrogen from androgenic precursors. Although evidence implicates estrogens in the pathogenesis of breast cancer, recent findings suggest that deregulation of aromatase may be a crucial link between these hormones and this neoplasm. Whereas tamoxifen is the endocrine therapy of choice, selective inhibition of aromatase may be equally effective, and possibly less toxic, in the management of patients with hormone responsive breast tumors. PMID- 10730683 TI - Orlistat, a new lipase inhibitor for the management of obesity. AB - Orlistat, a weight-loss agent with a novel mechanism of action, recently was approved by the Food and Drug Administration for the treatment of obesity. It inhibits gastric and pancreatic lipases in the lumen of the gastrointestinal tract to decrease systemic absorption of dietary fat. In several trials lasting up to 2 years, orlistat was more effective than diet alone for weight reduction and maintenance of lost weight. Orlistat treatment also results in modest improvements in total cholesterol, low-density lipoprotein, blood pressure, and fasting glucose and insulin concentrations. The major adverse effects are gastrointestinal, usually occur early in therapy, and tend to decrease with continued treatment. Because orlistat may decrease the absorption of fat-soluble vitamins, a standard multiple-vitamin supplement is recommended daily during therapy to prevent abnormalities in vitamin serum concentrations. The potential for severe gastrointestinal discomfort and the modest degree of weight loss may limit the agent's clinical utility. Its long-term safety and effectiveness for weight maintenance, cost-effectiveness of treatment, and overall reduction in obesity-related morbidity and mortality remain to be determined. PMID- 10730685 TI - Evaluation and management of drug-induced thrombocytopenia in the acutely ill patient. AB - The numerous drugs to which the acutely ill are exposed place these patients at a significant risk of developing drug-induced thrombocytopenia. Such patients tend to have preexisting hemostatic defects that place them at additional risk of complications as a result of the drug-induced thrombocytopenia. The clinical challenge is to provide rapid identification and removal of the offending agent before clinically significant bleeding or, in the case of heparin, thrombosis results. Drug-induced thrombocytopenic disorders can be classified into three mechanisms: bone marrow suppression, immune-mediated destruction, and platelet aggregation. Clinical characteristics, preliminary laboratory findings, and drug history specific to the mechanisms can assist clinicians in rapidly isolating the causative drug. PMID- 10730686 TI - Psychotropic drugs and falls: new evidence pertaining to serotonin reuptake inhibitors. AB - Falls are a significant cause of fatal and nonfatal injuries in older persons. Risk factors include previous falls, several disease states, and certain drugs such as tricyclic antidepressants and antihypertensives. We conducted a MEDLINE search from January 1966-March 1999 to identify studies and review articles on the association of neuroleptics, benzodiazepines, and antidepressants with fall risk in older people. The focus was on the risk associated with serotonin reuptake inhibitors, biologic plausibility, and limitations of these studies. It was thought that the agents did not increase the risk of falls, although recent evidence suggests that this is not the case. PMID- 10730687 TI - The pharmacokinetics and pharmacodynamics of argatroban: effects of age, gender, and hepatic or renal dysfunction. AB - STUDY OBJECTIVE: To determine the pharmacokinetics and pharmacodynamics of argatroban in healthy volunteers and patients with hepatic or renal dysfunction. DESIGN: Prospective, open-label study (studies 1 and 3); prospective, open-label, parallel-group study (study 2). SETTINGS: Two research centers and an inpatient clinic. SUBJECTS: Study 1, healthy volunteers; study 2, healthy volunteers and volunteers with hepatic disease; study 3, volunteers with normal to severely impaired renal function assigned to one of four groups based on creatinine clearance. INTERVENTION: Study 1, argatroban 125-microg/kg bolus followed by 4 hour continuous infusion of 2.5 microg/kg/minute; study 2, 4-hour infusion of 2.5 microg/kg/minute (1.25 microg/kg/minute in one patient with hepatic impairment); study 3, 5-microg/kg/minute continuous infusion over 4 hours. MEASUREMENTS AND MAIN RESULTS: Blood samples were obtained to assess plasma argatroban concentration, plasma activated partial thromboplastin time (aPTT), and whole blood activated clotting time (ACT). Study 1: the pharmacokinetic profile was well described by a two-compartment model with first-order elimination; effect response and plasma argatroban concentrations were well correlated. Mean +/- SD clearance, steady-state volume of distribution, and half-life values (40 healthy volunteers) were 4.7 +/- 1.1 ml/minute/kg, 179.5 +/- 33.0 ml/kg, and 46.2 +/- 10.2 minutes, respectively. The only effect of age or gender was the approximately 20% lower clearance in elderly men versus elderly women, which did not translate to clinically or statistically significant differences in pharmacodynamic response. Study 2: in patients with hepatic impairment, area under the concentration versus time curve (AUC) from time zero (t0) to last measurable concentration, AUC from t0 to infinity, maximum concentration, and half-life of argatroban were increased approximately 2- to 3-fold; clearance was one-fourth that of healthy volunteers. For aPTT and ACT, AUC over time for mean effect and mean maximum effect was higher in these volunteers. Study 3: no significant differences were detected. All four groups had predictable response profiles over time. CONCLUSION: Argatroban should be easy to monitor and control, with little potential for underdosing or overdosing, regardless of age, gender, or renal function. Dosing precautions are recommended, however, in patients with hepatic dysfunction. PMID- 10730688 TI - Open-label, nonrandomized study of the effects of gatifloxacin on the pharmacokinetics of midazolam in healthy male volunteers. AB - STUDY OBJECTIVE: To confirm findings from an in vitro study that showed gatifloxacin did not substantially inhibit cytochrome P450 (CYP) 3A4 model substrate metabolism. DESIGN: Open-label, nonrandomized trial. SETTING: Clinical pharmacology unit. SUBJECTS: Fourteen healthy adult men. INTERVENTION: Using midazolam probe methodology, the clearance of midazolam in the presence of multiple-dose gatifloxacin was evaluated. MEASUREMENTS AND MAIN RESULTS: Typical steady-state concentrations of gatifloxacin 400 mg once/day had no effect on midazolam clearance, and gatifloxacin pharmacokinetics were unaffected by midazolam. All doses of both agents were well tolerated. CONCLUSION: Data from this in vivo trial support in vitro experience with gatifloxacin and suggest that interactions are unlikely between gatifloxacin and drugs that are metabolized by CYP3A. PMID- 10730689 TI - The effects of anger management groups in a day school for emotionally disturbed adolescents. AB - Drawn from a day school for emotionally disturbed adolescents, seven students who scored in the clinical range on the Conduct subscale of the Conners Teacher Rating Scale took part in an anger management program. The program included psychoeducation, anger discrimination training, logging incidents of anger, and training in prosocial responses to anger. Pre-post assessments provided evidence of positive effects. The adolescents showed significant improvement on both the teacher (p<.03) and the parent (p<.04) versions of the Conduct subscale. They also exhibited a trend toward fewer incidents of physical aggression (p<.06). The implications of these findings for future research are discussed. PMID- 10730690 TI - Teachers' and students' work-culture variables associated with positive school outcome. AB - Little is known about the relationship between teachers' family-of-origin variables, impacting their work attitudes and interpersonal skills, and students' academic outcome. This study investigated whether goodness of fit between teachers' and students' backgrounds is associated with subjective grading and objective achievement at school. One hundred one seventh graders and twenty of their teachers completed the Self-Report Family Inventory. Similarity between teachers' and students' work-culture variables was associated with the subjective grading practices of teachers. The self-report data also revealed effective teacher and successful student profiles. PMID- 10730691 TI - Factors associated with parenting among incarcerated juvenile offenders. AB - In regard to the injured offender, research indicates that violent victimization represents only one facet of a constellation of associated risks and consequences, including promiscuity and adolescent parenthood. A relationship between firearm injuries and self-reported promiscuity among incarcerated juvenile offenders has previously been noted. The present study was an attempt to gain additional insight into the larger consequences of violent injuries. Information pertaining to the fathering of children was collected from 258 incarcerated male adolescents from the Richmond, Virginia, metropolitan area during a two-year period. It was hypothesized that adolescent parenting would be associated with firearm injuries. The results indicated that 20% of the juvenile offenders fathered at least one child. Analyses revealed a significant relationship between firearm injuries and increased prevalence of adolescent parenting. Continued involvement in illegal activities, as indicated by a second commitment to a juvenile correctional center, also was associated with increased prevalence of adolescent parenting, while race and involvement in drug selling or violent offending were not. The social and economic implications of these findings, particularly in terms of the health care and social service delivery systems, are discussed. PMID- 10730692 TI - Onset of disordered eating attitudes and behaviors in early adolescence: interplay of pubertal status, gender, weight, and age. AB - The interplay of puberty, gender, weight, and age in regard to body image and disordered eating behaviors and attitudes was investigated in a sample of early adolescents. It was found that, after menarche, females had increased personal expectations and were dissatisfied with weight/shape changes. They wanted to reduce their weight and between-meal eating (which was associated with increased feelings of inadequacy, loss of control, and decreased self-esteem). Young postmenarcheal females who were higher achievers and had higher trait anxiety were more likely to have lower weight. Young males at puberty desired to build up their bodies, believing that appearance was important to their sexual appeal. They were also more willing to form close relationships. Postpubertal males felt most in control of their lives, had the highest self-esteem, and were more trusting of their body cues. Overall, high achieving, more anxious postmenarcheal females were at greatest risk for eating disturbances. These results indicate that the interaction of pubertal status and gender must be considered when developing intervention programs. PMID- 10730693 TI - Academic dishonesty among high school students. AB - Research on academic dishonesty has generally relied on survey techniques, which may fail to capture students' true feelings about cheating. The present investigation used focus group discussions to gain a fuller understanding of students' beliefs about academic dishonesty. The results suggest that, in regard to their cheating, students generally place the blame on others. PMID- 10730694 TI - "She looked at me crazy": escalation of conflict through telegraphed emotion. AB - This exploratory study investigated a feature of adolescent social interaction that can quickly initiate or escalate conflict: telegraphed emotion (a specific emotion conveyed through a subset of its expressive components or through a brief, extreme display of the complete expression; Shields & Maybury, manuscript in preparation). In the form of "the look," telegraphed emotion conveys intensely felt, yet controlled, affect. Using focus groups, teenage females were asked to define the look and to discuss its use, such as who employs it, when it is used, and the consequence of using the look. They reported that the look has different meanings depending on whether it is directed at a friend or a nonfriend, and whether it is used by females or males. With friends, the purpose is to communicate anger, sadness, or disgust; with nonfriends, it indicates disgust or anger, but within a framework of asserting power or status. The look often results in escalation of conflict, especially a verbal or physical fight. These findings are discussed, and the implications for investigating and preventing adolescent interpersonal conflict are explored. PMID- 10730695 TI - A developmental-contextual perspective on youth covictimization by community violence. AB - Witnessing violence within the community-covictimization-has been recognized as a threat to the optimal development of youth. Following a discussion of methodological issues that plague covictimization research, the literature on covictimization is reviewed, beginning with findings on its prevalence. Correlates of covictimization are then examined within the Shakoor and Chalmers framework, comprising emotional, cognitive, and behavioral domains. Recent lines of research investigating interactions among these three domains are also explored. Further, a developmental-contextual approach toward studying covictimization is outlined and, from this perspective, the relationship of covictimization to social cognition, health-related behaviors, parenting, and social support is discussed. Finally, recommendations for further research are presented. PMID- 10730696 TI - Stress-buffering factors related to adolescent coping: a path analysis. AB - To uncover those factors that buffer the impact of stressful negative experiences on adolescent adjustment, a theoretical model of adolescent stress and coping, with social support and social problem solving proposed as moderators, was investigated using path analysis. The study was conducted with 122 ninth-and tenth-grade nonreferred high school students. Using the LISREL statistical package (Joreskog & Sorbom, 1986), it was found that a recursive loop leading from stress outcomes back to negative stressors did not allow for a successful solution to the model. However, the effects of stressful events on adjustment were mediated by coping resources, which included a combination of problem solving abilities and social support. Overall, the findings replicated previous investigations that have demonstrated direct relationships among stressful life events, social support, problem solving, and adolescent adjustment. While a successful fit to the theoretical model was not attained, it was concluded that a refined model may provide a more acceptable solution. PMID- 10730697 TI - Suicidal behavior in chemically dependent adolescents. AB - Among 250 adolescents in a short-term residential treatment program for chemical dependency, 20% had attempted suicide in the two years prior to admission. Females, however, were found to have a higher attempt rate than did males. Suicide attempters were compared with a group of 50 nonsuicidal adolescents from the same treatment program and 50 non-chemically dependent, nonsuicidal high school students. Each adolescent was administered the Symptom Checklist 90 Revised (SCL-90-R), and a biographical history was obtained. The suicidal group was found to be more psychologically distressed than were the other two groups. Post hoc analysis revealed that there were significant differences between the chemically dependent groups (suicidal, nonsuicidal) on the Global Severity Index of the SCL-90-R, as well as the following subscales: Somatization, Depression, Anxiety, and Phobic Anxiety. It was also found that the majority of suicidal gestures or attempts had gone untreated beyond medical management. It was found that only 28% of the suicide attempters had received crisis intervention or emergency room treatment, and only 27% had received some type of follow-up treatment or counseling. These results are discussed, particularly in regard to the issue of "covert suicide." PMID- 10730698 TI - The resiliency of street children in Brazil. AB - This study examined the relationship between social support and the ability of Brazilian adolescents to adapt to life on the streets. Participants included thirty male street children in Rio de Janeiro. These youths, and a comparison group, were 13 to 18 years old. It was hypothesized that street children with more social support would adapt better to life on the streets. It was also hypothesized that street children with higher quality support would adapt better. Findings indicated that neither quantity nor quality of social support was related to adaptation. The implications of these findings with regard to the resiliency of street children are discussed. PMID- 10730699 TI - American adolescents touch each other less and are more aggressive toward their peers as compared with French adolescents. AB - Forty adolescents were observed at McDonald's restaurants in Paris and Miami to assess the amount of touching and aggression during their peer interactions. The American adolescents spent less time leaning against, stroking, kissing, and hugging their peers than did the French adolescents. Instead, they showed more self-touching and more aggressive verbal and physical behavior. PMID- 10730700 TI - Music training and mathematics achievement. AB - Iowa Tests of Basic Skills (ITBS) mathematics scores of eighth graders who had received music instruction were compared according to whether the students were given private lessons. Comparisons also were made between students whose lessons were on the keyboard versus other music lessons. Analyses indicated that students who had private lessons for two or more years performed significantly better on the composite mathematics portion of the ITBS than did students who did not have private lessons. In addition, students who received lessons on the keyboard had significantly higher ITBS mathematics scores than did students whose lessons did not involve the keyboard. These results are discussed in relation to previous research on music training and mathematics achievement. PMID- 10730701 TI - Migrant labor in South Africa: a comparative analysis of the academic achievement of father-present and father-absent adolescents. AB - This study was designed to determine whether there are significant differences in academic achievement between father-present and father-absent (due to migrant labor) adolescents. Data were collected from 276 high school students in South Africa. Academic achievement was measured by the Human Sciences Research Council's (HSRC) Scholastic Achievement Test, covering biology, English (second language), and mathematics. Father-present students were found to score significantly higher than father-absent students. The findings suggest that a father's absence due to work conditions has deleterious effects on the scholastic performance of young people. PMID- 10730702 TI - Mentoring and the spiritual well-being of late adolescents. AB - The purpose of this study was to investigate the possible impact of faculty mentoring on the spiritual well-being of late adolescents. The sample consisted of randomly chosen students in their first year at a Christian liberal arts college in New England. Students in the experimental group participated in the freshman seminar program (small classes with seminar leaders/mentors), while those in the control group did not have the freshman seminar experience. Students in both groups were administered a self-assessment survey in September of their freshman year and again in May to determine if there was any change in their spiritual well-being and to explore their perceptions of mentor-student interactions. The findings revealed significant differences between the two groups. In addition, the three aspects of mentoring were positively correlated with the two components of spiritual well-being. PMID- 10730703 TI - Object relations and referential activity in physically abused adolescents. AB - This study compared the object relations and language functions of 15 physically abused and 15 nonabused adolescents. The adolescents provided a 5-minute narrative about their mothers, which was scored for referential activity and object relations. As predicted, the abused adolescents tended to have lower levels of affect tone, indicating more malevolent relationship paradigms. Contrary to what was predicted, however, there were no significant differences between groups on overall measures of referential activity and object relations. Further, the abused adolescents tended to have higher levels of two elements of referential activity (concreteness, imagery), indicating increased verbal ability to express emotional experience. While predicted correlations were found between object relations and referential activity for the nonabused group, the abused group showed higher symbolizing and verbalizing capacity, associated with more malevolent representations of relationships. The findings do not support the view that physically abused adolescents experience developmental lags, instead suggesting that they organize and use emotional and symbolizing processes differently from nonabused adolescents. The implications for treatment are discussed. PMID- 10730704 TI - Perception of classroom environment in Hong Kong: differences between students in junior and senior forms. AB - In Hong Kong secondary schools, students in the senior forms take competitive examinations to proceed to the next level, whereas those in the junior forms have to attend school regardless of their ability and motivation. It was hypothesized that this produces different classroom climates. To test this hypothesis, 602 Chinese secondary school students were administered the short version of the Classroom Environment Scale. Significant differences were found, varying by type of school, on the dimensions of Involvement, Task Orientation, Teacher Support, Competition, Order and Organization, Affiliation, Innovation, Rule Clarity, and Teacher Control. These findings are discussed. PMID- 10730705 TI - The effects of perioperative hypothermia and the duration of anesthesia on postoperative wound infection rate in clean wounds: a retrospective study. AB - OBJECTIVE: To determine the effects of hypothermia and duration of anesthesia on the infection rate in clean wounds in dogs and cats. STUDY DESIGN: Retrospective clinical study. SAMPLE POPULATION: Seven hundred seventy-seven dogs and cats undergoing clean surgical procedures. METHODS: Records of animals prospectively evaluated for postoperative wound infection were retrospectively evaluated for the prevalence of perioperative hypothermia. Body temperatures during the perioperative period and the duration of surgery and anesthesia were recorded. Data were analyzed to identify differences between animals with infected and uninfected wounds and multiple logistic regression modeling was used to evaluate the independent contribution of risk factors to the incidence of postoperative wound infection. RESULTS: No statistically significant differences were found in the analysis of temperature data between animals with infected and uninfected wounds. Duration of anesthesia was significantly greater in animals with wound infections (P = .01). Multiple logistic regression modeling identified duration of anesthesia as a risk factor for postoperative wound infection independent of the duration of surgery. CONCLUSIONS: In animals with clean surgical wounds, mild perioperative hypothermia is not a significant risk factor for postoperative wound infection. The duration of anesthesia, however, is a significant risk factor independent of the duration of surgery. CLINICAL RELEVANCE: To decrease the incidence of postoperative wound infection, the duration of anesthesia should be minimized. Surgical time, as well as the time required for ancillary diagnostic tests while under anesthesia should, therefore, be kept to a reasonable minimum. PMID- 10730706 TI - Evaluation of a free vascularized medial tibial bone graft in dogs. AB - OBJECTIVE: To develop a free vascularized tibial bone graft based on the periosteal saphenous blood supply. STUDY DESIGN: Preliminary anatomic study of medial tibial blood supply. In vivo comparison of a vascularized and avascular tibial bone graft. ANIMALS: Nine canine cadavers; 14 healthy adult dogs that weighed 25 to 32 kg. METHODS: An anatomic study of the vascular supply of the medial aspect of the tibia was performed using the Spalteholz technique. A bone graft consisting of the medial aspect of the tibia was transferred to a mandibular defect as a vascularized graft in 7 dogs and as an avascular graft in 7 dogs. Bone scans were performed to evaluate graft perfusion. Radiographic evaluation of the mandibles and tibias was performed. The dogs were killed after 60 days, five mandibles from each group were examined histologically, and two from each group were evaluated using the Spalteholz technique. RESULTS: The saphenous vascular pedicle provides vascular perfusion to the medial tibial cortex. Bone scans and radiographic evaluations were consistent with viable bone in the vascularized grafts, and nonviable bone in the avascular grafts. Histological examination revealed live, healing bone in vascular grafts and necrotic bone in avascular grafts. Spalteholz evaluation revealed many small arborizing vessels in the vascular grafts and no organized vasculature in the avascular grafts. CONCLUSIONS: The vascularized medial tibial cortical bone graft survived and proceeded to bony union in the mandibular body defect more readily than the avascular graft in this experimental model. CLINICAL RELEVANCE: A vascularized medial tibial bone graft is a suitable free graft for use in reconstructing bone defects in dogs. PMID- 10730707 TI - Tissue strength and wound morphology of the equine linea alba after ventral median celiotomy. AB - OBJECTIVE: To evaluate the tensile strength and wound morphology of the equine linea alba at intervals over 6 months after ventral median celiotomy. STUDY DESIGN: Linea alba tensile strength and wound morphology were determined at 2, 4, 8, 16, and 24 weeks postoperatively. Linea alba samples from 3 unoperated horses were used as controls. ANIMALS: Eighteen adult horses, weighing 400 to 500 kg, 4 to 15 years old. METHODS: Tensile strength and thickness of incised linea alba samples collected at 2, 4, 8, 16, and 24 weeks after ventral median celiotomy were compared with control linea alba samples. Additional samples were subjectively evaluated for wound morphology, notably collagen morphology. RESULTS: Control linea alba had a mean (+/-SEM) tensile strength of 484.9 +/- 58.3 N and was significantly (P < or = .05) stronger than at 2 weeks (87.7 +/- 61.4 N) after surgery. The tensile strength of 4-week (305.8 +/- 61.7 N), 8-week (465.4 +/- 56.5 N), and 16-week (477.8 +/- 57.2 N) samples were not significantly different from control linea alba. At 24 weeks, the tensile strength (721.0 +/- 57.9 N) was significantly stronger than control. The 2-, 4-, and 8-week samples were significantly thicker than controls, whereas the 16- and 24-week samples were not different from controls. On microscopy, control samples were characterized by dense mature collagen bundles. At 2 weeks, samples consisted primarily of granulation tissue, whereas at 4 weeks, samples had immature collagen fibers that were not formed into bundles, and at 8, 16, and 24 weeks, all samples had abundant mature collagen fibers formed into bundles. CONCLUSIONS: At 8 weeks, incised and sutured equine linea alba had a tensile strength comparable with non-incised linea alba and was characterized by mature collagen. CLINICAL RELEVANCE: Based on tensile strength and wound morphology, horses that have had an uncomplicated recovery after ventral median celiotomy should be able to return to controlled exercise as early as 60 days after surgery. PMID- 10730708 TI - A biomechanical comparison of double-plate and Y-plate fixation for comminuted equine second phalangeal fractures. AB - OBJECTIVES: To compare the biomechanical properties, in full limb preparations, of intact second phalanx and a simulated comminuted second phalangeal fracture stabilized with either two bone plates or a custom Y-plate. STUDY DESIGN: In vitro biomechanical assessment of intact limbs and of paired limbs with a simulated second phalangeal fracture stabilized by one of two fixation methods. Animal Population-Thirteen pairs of equine cadaveric forelimbs. METHODS: A comminuted second phalangeal fracture was created in six paired cadaveric limbs. For each limb pair, the fracture was stabilized with two plates in one limb, and with a Y-plate in the contralateral limb. These limbs and seven pairs of intact limbs were subjected to axial compression in a single cycle until failure. Mechanical properties were compared with a mixed-model ANOVA and post hoc contrasts. Joint contact pressure, screw insertion torque, and final screw torque remaining after mechanical testing were also evaluated for constructs. RESULTS: No significant differences in mechanical testing variables were detected between construct types. However, the Y-Plate construct had significantly greater yield load, yield displacement and yield energy, and failure load and stiffness values than those for intact specimens, whereas the double-plate construct only had greater stiffness than intact specimens. There were no significant differences in joint contact pressures for both constructs. The final screw torque for proximal phalangeal screws was significantly greater for the Y-plate constructs than for double-plate constructs. CONCLUSIONS: The Y-plate was as effective as the double plate technique for stabilization of simulated comminuted second phalangeal fractures in monotonically tested equine cadaveric forelimbs. CLINICAL RELEVANCE: This investigation supports evaluation of the Y-plate for repair of comminuted second phalangeal fractures in equine patients. Its specific design may facilitate repair of second phalangeal fractures, and may provide increased stability by allowing the proximal fragments of the second phalanx to be fixed with three screws placed through the plate. PMID- 10730709 TI - In vitro cyclic biomechanical properties of an interlocking equine tibial nail. AB - OBJECTIVE: To determine cyclic biomechanical properties of gap osteotomized adult equine tibiae stabilized with an equine interlocking nail (EIN). STUDY DESIGN: In vitro experimental biomechanical investigation. SAMPLE POPULATION: Thirteen adult equine cadaveric tibiae. METHODS: Adult equine tibiae with transverse, midshaft, 1-cm gap osteotomies, stabilized with an equine interlocking nail, underwent cyclic biomechanical testing in vitro under axial compression, 4-point bending, and torsion. Different specimens were subjected to different load levels that represented estimated in vivo loads at 2 Hz for 740,000 cycles. Fatigue life and gap strain were calculated. RESULTS: Compression and bending, but not torsional, fatigue life were longer than time necessary for bone healing. Compressive, but not bending or torsional, gap strains were small enough to be compatible with fracture healing by primary bone formation. Gap strains for compressive, bending, and torsional loads were compatible with indirect, or secondary, bone formation. CONCLUSIONS: Further modification should be made to the equine interlocking nail to increase bending stiffness and torsional fatigue life. CLINICAL RELEVANCE: The stainless steel equine intramedullary interlocking nail is unlikely to provide appropriate long-term stability for fracture healing in adult equine tibiae without modifications in the nail design and material. PMID- 10730710 TI - Paranasal sinus surgery through a frontonasal flap in sedated, standing horses. AB - OBJECTIVE: To report experience with paranasal sinus surgery through a frontonasal flap in sedated, standing horses. STUDY DESIGN: Treatment of 10 horses with naturally occurring paranasal sinus disease through a frontonasal bone flap created with the horses standing. ANIMALS: Ten adult horses. METHODS: After restraint and sedation, local anesthetic was injected subcutaneously along the proposed incision line over the conchofrontal sinus and was instilled into the sinuses through a small hole created in the frontal bone. A 3-sided, rectangular, cutaneous incision that extended through the periosteum was created over the frontal and nasal bones. The incision was extended into the conchofrontal sinus using a bone saw, and the base of the flap, on the midline of the face, was fractured. The sinuses were explored, and the horse was treated for the disease encountered. The flap was repositioned; subcutaneous tissue and skin were sutured separately. RESULTS: The horses had few signs of discomfort during creation of the bone flap and during disease treatment. Diseases encountered included inspissated exudate in the ventral conchal sinus (five horses), feed and exudate throughout the sinuses (one horse), occlusion of the nasomaxillary aperature (one horse), polyp (one horse), osteoma (one horse), and progressive ethmoidal hematoma (one horse). CONCLUSION: In selected cases, surgery of the paranasal sinuses can be performed safely on sedated and standing horses through a frontonasal bone flap. CLINICAL RELEVANCE: Performing surgery through a frontonasal bone flap with the horse standing and sedated, rather than anesthetized, eliminates risks and expense of general anesthesia. PMID- 10730711 TI - Effect of public symphysiodesis on pelvic development in the skeletally immature greyhound. AB - OBJECTIVE: To evaluate the effect of pubic symphysiodesis (PS) on pelvic development in skeletally immature dogs. STUDY DESIGN: Prospective randomized clinical trial. ANIMALS: Eight 4 month-old, sexually intact female Greyhounds. METHODS: Initial PS was performed at 4 months of age using a powered stapling device. Because of failure of the initial surgery, a second PS was performed 1 month later by resecting the pubic symphysis with a rongeur followed by placement of handmade bone staples in four dogs. Sham PS was performed in four control dogs at 4 months of age. Pubic growth rate and pelvic development were evaluated using standard plane radiography and computed tomography. Specific measurements included acetabular ventroversion, Norberg angle, lateral center-edge angle, and pelvic inlet dimensions. Hip distraction indices were determined as well. RESULTS: PS at 4 months of age using a stapling device failed. Pubic symphysiodesis using hand made staples was successful at 5 months of age and did not result in any clinically significant intraoperative or postoperative complications. Pubic symphysiodesis markedly decreased pubic symphysis growth in the treatment group. Hip distraction indices and pelvic inlet circumference, area, and width significantly decreased in treated dogs compared to those in the control group. Acetabular ventroversion was significantly increased in treated dogs compared to those in the control group. CONCLUSIONS: PS decreases pelvic canal size, increases acetabular ventroversion, and does not appear to have any clinically significant complications. CLINICAL RELEVANCE: PS performed in skeletally immature dogs with hip dysplasia may provide an effect similar to a triple pelvic osteotomy and warrants further investigation. PMID- 10730712 TI - Samarium 153-labeled hydroxyapatite microspheres for radiation synovectomy in the horse: a study of the biokinetics, dosimetry, clinical, and morphologic response in normal metacarpophalangeal and metatarsophalangeal joints. AB - OBJECTIVE: To determine the effects of Samarium-153 bound to hydroxyapatite microspheres (153SmM) when injected into the metacarpophalangeal and metatarsophalangeal joints of horses. STUDY DESIGN: - Horses were injected with 153SmM in metacarpophalangeal and metatarsophalangeal joints with the diagonal contralateral joints used as untreated controls. ANIMALS OR SAMPLE POPULATION: Twelve adult horses without pre-existing disease involving the metacarpo/metatarsophalangeal joints. METHODS: Horses were divided into three groups: high-dose Samarium-153 (12.5 to 17.0 millicurie [mCi]), intermediate dose (6.5 to 12.0 mCi), and low dose (3.5 to 6.0 mCi). Horses were examined daily for 7 days postinjection for clinical abnormalities, lameness, and surface and systemic radiation levels. One horse from each group was euthanatized at 14, 30, and 60 days postinjection and the effects of the 153SmM examined microscopically in the cartilage and synovial membrane. RESULTS: Intraarticular(153)SmM caused inflammation characterized by lameness, effusion, and regional edema for 48 to 72 hours. Minimal levels of active 153SmM were identified in the blood or urine and were well below the maximal tolerance of 1 mCi. Microscopically the radiation caused no effects on the articular cartilage. The synovectomy created was good but not ideal in that some areas did have necrosis into the subintimal regions and a few islands of intact intimal cells persisted. CONCLUSIONS: The use of 153SmM is an effective means of targeting the synovial intimal cells with minimal extrasynovial leakage of radiation. CLINICAL RELEVANCE: The metacarpophalangeal and metatarsophalangeal joints of the horse can be safely treated with 153SmM without damage to the cartilage or significant extracapsular leakage. PMID- 10730714 TI - Production frontier analyses: comments on the methodology. PMID- 10730713 TI - The effect of changing the mode of ventilation on the arterial-to-end-tidal CO2 difference and physiological dead space in laterally and dorsally recumbent horses during halothane anesthesia. AB - OBJECTIVE: To evaluate the effect of changing the mode of ventilation from spontaneous to controlled on the arterial-to-end-tidal CO2 difference [P(a ET)CO2] and physiological dead space (VD(phys)/VT) in laterally and dorsally recumbent halothane-anesthetized horses. STUDY DESIGN; Prospective, experimental, nonrandomized trial. ANIMALS: Seven mixed breed adult horses (1 male and 6 female) weighing 320 +/- 11 kg. METHODS: Horses were anesthetized in 2 positions right lateral and dorsal recumbency-with a minimum interval of 1 month. Anesthesia was maintained with halothane in oxygen for 180 minutes. Spontaneous ventilation (SV) was used for 90 minutes followed by 90 minutes of controlled ventilation (CV). The same ventilator settings were used for both laterally and dorsally recumbent horses. Arterial blood gas analysis was performed every 30 minutes during anesthesia. End-tidal CO2 (PETCO2) was measured continuously. P(a ET)CO2 and VD(phys)NT were calculated. Statistical analysis included analysis of variance for repeated measures over time, followed by Student-Newman-Keuls test. Comparison between groups was performed using a paired t test; P < .05 was considered significant. RESULTS: P(a-ET)CO2 and VD(phys)/VT increased during SV, whereas CV reduced these variables. The variables did not change significantly throughout mechanical ventilation in either group. Dorsally recumbent horses showed greater P(a-ET)CO2 and VD(phys)/VT values throughout. PaCO2 was greater during CV in dorsally positioned horses. CONCLUSIONS AND CLINICAL RELEVANCE: Changing the mode of ventilation from spontaneous to controlled was effective in reducing P(a-ET)CO2 and physiological dead space in both laterally and dorsally recumbent halothane-anesthetized horses. Dorsal recumbency resulted in greater impairment of effective ventilation. Capnometry has a limited value for accurate estimation of PaCO2 in anesthetized horses, although it may be used to evaluate pulmonary function when paired with arterial blood gas analysis. PMID- 10730715 TI - Caries prevalence and distribution in 3-20-year-olds in Jonkoping, Sweden, in 1973, 1978, 1983, and 1993. AB - Four cross-sectional studies were carried out in 1973, 1978, 1983, and 1993 to collect clinical and radiographic epidemiological data on the dental health status of the inhabitants of Jonkoping, Sweden. The aim of the present paper was to use these data to analyze trends in the development of caries among children and adolescents between 1973 and 1993. Approximately 500 randomly selected individuals evenly distributed among the age groups 3, 5, 10, 15, and 20 years participated in each study. The main results show that the numbers of caries-free individuals increased in all age groups. In 1993, the mean number of decayed and filled tooth surfaces in the primary (dfs) and the permanent (DFS) dentition in all age groups was less than half of that found in 1973. Most of this decrease took place during the first 5 years, i.e., between 1973 and 1978. Between 1978 and 1983, only minor changes were observed. There was a further reduction of approximately 30%-50% in dfs/DFS between 1983 and 1993 in 3-, 5-, 10-, and 20 year-olds. The frequency distributions of dfs/DFS for 5- and 15-year-olds revealed an increasing skewness over time: in 1993, a large majority of the children and adolescents had a low or moderate caries severity while only a small group had high scores of dfs/DFS. PMID- 10730716 TI - Caries prevalence and distribution in 20-80-year-olds in Jonkoping, Sweden, in 1973, 1983, and 1993. AB - In 1973, a cross-sectional study on oral health status was performed on 1000 individuals in the age groups 3-70 years in Jonkoping, Sweden. In 1983 and 1993, new cross-sectional studies were carried out in the age groups 3-80 years. The aim of the present study was to analyze caries prevalence and distribution in the three investigations 1973, 1983, and 1993 in the age groups 20-80 years. In the younger age groups (20-40 years), a larger proportion of individuals with good oral health was found in 1993 than in 1973 or 1983. A steady increase in the number of teeth in the age groups 40-80 years could be found, which was also reflected in the increasing number of decayed and filled tooth surfaces (DFS) in the same age groups. A marked decrease in proximal DFS in 20-50-year-olds in 1993 compared to 1973 and 1983 was found. However, a rather large and unchanging group of individuals suffering from severe caries was also observed. This situation demands an individualized caries treatment strategy based on risk assessment. PMID- 10730717 TI - Children's acceptance of xylitol-based foods. AB - Daily consumption of xylitol (5-10 g/day) added to chewing gum and confectionary foods has been previously shown to prevent dental caries in children. METHODS: Snack foods containing xylitol were developed and tested for acceptability in a convenience sample of 31 children ages 3 to 6 years. In order to mimic an after meal snack, all children were tested during mid-morning, approximately 1 h after eating. Preference testing was based on the methodology of Birch et al. (J Nutr Educ, 1979; 11: 77-80). In the first phase, each child was presented with a tray of six xylitol-based foods (popsicles, pudding, gum drops, gelatin dessert, cookies, popcorn) and asked to taste each item in any desired order. Immediately after tasting a food, the child was asked to place it in front of one of three cartoon faces (smile, frown or neutral) representing the child's response to the taste of that particular food. In the second phase, the child was asked to rank order the foods in each face category (smile, frown or neutral). Ranks within categories were then combined to obtain a rank ordering for all of the foods. RESULTS: Non-parametric data analysis indicated significant differences in ranking between the foods when they were compared to each other (Friedman ANOVA by ranks, P<0.01). Pudding was significantly less preferred than the other foods (sign tests, P<0.04). At least 84% of the children found five of the six foods very good or satisfactory, when considered individually. CONCLUSIONS: These results suggest that snack foods developed with xylitol are generally well accepted by children. PMID- 10730718 TI - Oral health changes in an adult Norwegian population: a cohort analytical approach. AB - OBJECTIVES: The purpose of this analysis was to study temporal variation in oral health in an adult population. The cohort analysis comprised 1) estimation of longitudinal, cross-sectional, and time-lag differences in caries and treatment experience of the adult population in Trondelag from 1973 to 1983 and to 1994; 2) assessment of which of the effects of age, period and cohort was the most plausible explanation for the observed differences and 3) discussion of causal and other factors underlying the effects of age, period and cohort. METHODS: Cohort analysis was used to study changes in oral health over time. Study participants were selected by random sampling in 1973, 1983, and 1994 (n = 1959, n = 3195, n = 2341 respectively). The methods of data collection comprised standardised clinical measurements and a self-administered questionnaire. Cohort tables were established to analyse changes in DFT from 1973 to 1983 and DFT and DFS from 1983 to 1994. RESULTS: Four patterns of changes in oral health were identified from adolescence to older adulthood. They point in different directions which indicate that complex processes are present. There were two and three significant changes in the subsets of analysis. Assumptions and conditions that allow identification of separate cohort, period, and age effects appeared not to be present. The pattern of changes was consistent with main effects of both age, period, and cohort. The effects were discussed from the perspective of the chosen frame of explanatory factors. CONCLUSIONS: The changes in oral health between populations in 1973, 1983 and 1994 were the combined consequences of simultaneously occurring factors that account for both age, period and cohort effects. PMID- 10730719 TI - Trends in periodontal status and oral hygiene habits in Dutch adults between 1983 and 1995. AB - In 1995, a dental survey, similar to one carried out in 1983, was performed among persons aged 25-54 in the city of 's-Hertogenbosch, the Netherlands. OBJECTIVE: To describe trends in adults periodontal health between 1983 and 1995. METHODS: The participants were interviewed at home and clinically examined in a dental van. RESULTS: Average scores for plaque, calculus, bleeding after probing and number of buccal root surfaces exposed to the oral cavity were lower in 1995 than in 1983. There was no significant change in the percentage of teeth with pocket depth of 3.5 mm or more between 1983 and 1995. No difference was found between frequency of toothbrushing in 1983 and 1995. CONCLUSION: The results suggest that the periodontal health of Dutch adults aged 25-54 years has improved since 1983. PMID- 10730720 TI - Determinants of patients' treatment preferences in a clinical trial. AB - Several researchers have suggested that patients' preferences for a particular form of treatment should be taken into account in clinical trials. Preferences may influence the outcome of treatment, especially in trials when patients cannot be blinded to the type of treatment received and the outcome is based on patients' evaluations of therapy. Participants in this study were 136 edentulous patients who took part in a randomised controlled clinical trial comparing two types of treatments for edentulism: conventional dentures and implant-supported prostheses. Prior to receiving treatment, subjects were required to complete a questionnaire regarding their satisfaction with their present prostheses. In addition, they were asked to indicate which treatment they would prefer if given a choice. The objective of this study was to determine whether there are important differences among study participants between patients who have a treatment preference and those who do not. The effects of satisfaction with pre treatment prostheses, age, gender and level of education on preferences were examined. Level of satisfaction with the original dentures and level of education were significant predictors of preference. Compared to subjects who rated their satisfaction with their current condition as 'low', the odds ratios associated with having a preference for implant treatment were 0.31 (95% CI: 0.09 to 0.96) for subjects who rated their prostheses in the 'medium' range and 0.11 (95% CI: 0.03 to 0.41) for those who rated in the 'high' range. In addition, subjects with high levels of education were significantly less likely to have a preference for either conventional or implant treatments (OR = 0.18, 95% CI: 0.02 to 0.77 and OR = 0.20, 95% CI: 0.05 to 0.76, respectively) compared to those with low education. Neither age nor gender was a significant predictor of preference. We suggest that study designs which incorporate patients' preferences must take into account possible differences between preference groups that might confound the relationship between preference and the outcome of interest. PMID- 10730721 TI - Reasons for the caries decline in Slovenia. AB - In 1987, 1993 and 1998, nationwide surveys on the prevalence of dental caries were carried out in Slovenia. Sampling and examinations were performed according to WHO standards. The age groups 6, 12, 15, 18, 35-44 and 65 years or older were studied. In each age group, 200 persons or more were examined on each occasion. The results showed that the proportion of caries-free children and adolescents increased considerably over the 12-year period between the first and the last survey (from 6% to 40% for 12-year-olds). In the same period, the mean DMFT values decreased as follows: from 5.1 to 1.8 for 12-year-olds, from 10.2 to 4.3 for 15-year-olds, from 12.9 to 7.0 for 18-years-olds, from 20.5 to 14.7 for 35-44 year-olds, and from 27.0 to 22.5 for subjects aged 65 years or more. The notable improvement of dental health can be explained by the preventive programmes in operation in various periods. The most recent decline was most likely due to supervised brushing (with concentrated fluoride gel) taking place some 16-18 times a year in primary schools attended by children aged 7-15 years, improved oral hygiene, and a comprehensive programme of applying fissure sealants, particularly on first molars. PMID- 10730722 TI - An investigation of factors influencing patients' use of new complete dentures using structural equation modelling techniques. AB - A review of the literature demonstrated that research had not identified a set of prognostic indicators for prospective complete denture patients, with different authors publishing contradictory results. OBJECTIVE: To investigate possible relationships between patient and clinical factors and denture wear and use of dentures for eating. METHODS: Patients attending Guy's Dental Hospital, London, for provision of complete dentures were recruited. For each patient relevant history was recorded and an examination of the edentulous mouth and existing dentures, where present, was undertaken at the beginning of treatment. At the first post-insertion appointment the edentulous mouth, previous and new dentures were examined. Three months after the review appointment patients were sent a postal questionnaire to assess patient use of dentures and patient satisfaction. Data were analysed using structural equation modelling techniques. RESULTS: 723 patients were recruited. Significant relationships were found between quality of complete dentures and patients' use of complete dentures and between quality of residual edentulous ridges and patients' use of complete dentures. These results contrast with most previous research into prognostic indicators for success in complete denture treatments. CONCLUSIONS: Quality of residual edentulous ridges and quality of new complete dentures predict patients' use of new complete dentures. PMID- 10730723 TI - Effects of an oral health program on the occurrence of oral candidosis in a long term care facility. AB - OBJECTIVES: The aim was to evaluate the effectiveness of a preventive oral health program on the prevalence of oral candidosis in 237 frail or dependent residents in a long-term care facility. Half of the residents were included in an experimental group which benefited from a preventive oral hygiene program including instruction of the carers and implementation of a recall program for professional oral hygiene care. METHODS: Intraoral examinations and yeast cultures from the oral mucosa and the fitting denture surface were carried out at baseline and 18 months later. The outgrowth of yeast was estimated on Oricult-N dip slides using the scale: no growth; 1-20 colonies; 21-100 colonies; >100 colonies. RESULTS: At baseline (n = 237) and at 18 months (n = 159) the experimental and the control groups were similar with regard to the residents' distribution by age, sex, dental and prosthetic status and prevalence of denture stomatitis. The 78 residents lost had the same baseline characteristics as the survivors, except for being older. In the experimental group the severity of the inflammation of the palate decreased (P = 0.005) as well as the prevalence of glossitis (P = 0.005). At baseline high yeast scores from the mucosa (>20) were observed in about 50% of the residents in the experimental as well as the control group. At 18 months this figure was 23.4% for the experimental and 48.7% for the control group (P = 0.001). There was also a reduction of the number of residents with positive cultures and the denture yeast scores at 18 months in the experimental group (P = 0.05). CONCLUSIONS: This study has shown that the preventive program was effective in reducing the colonization of the oral mucosa and dentures by Candida and thereby improving the health of the oral mucosa. PMID- 10730724 TI - Factors related to betel chewing among junior high school students in Taiwan. AB - A cross-sectional study was conducted to explore the betel-chewing behavior of junior high school students in Taichung County in 1998. The sample consisted of 2244 students in the general area and 84 students in the aboriginal area. Fifty percent of the students in the aboriginal area had had the experience of chewing betel, and this figure includes 30.1% of the habitual chewers. The prevalence of habitual betel chewers among the students in the general area is 4.3% to 5.0%. The major source of betel quid is friends or classmates, and curiosity accounts for most students' first experience of betel chewing. Sixty percent of the students started betel chewing in elementary school. An effective health education program for students in the aboriginal area and younger students in the general areas as well as an evaluation procedure for the effectiveness of the program are needed to prevent the increase of betel chewing among Taiwan's youth. PMID- 10730725 TI - Evidence for dental caries decline among children in an East European country (Hungary). AB - In Central and Eastern Europe oral health systems are in transition due to the economic and political changes. The aim of the present study was to highlight the long-term trend in dental caries prevalence of Hungarian children. A WHO National Pathfinder Survey of oral health status was conducted in 1996 which included 6 year-olds (n = 900) and 12-year-olds (n = 900). Similar surveys were performed in 1985 (age 6-7, n = 895; age 12, n = 893) and in 1991 (age 5-6, n = 898; age 12, n = 898) and the surveys were based on the same sites and examination criteria. Clinical data were collected by trained dental examiners according to the WHO Basic Methods. In 1996, 73% of 5-6-year-olds and 84.5% of 12-year-olds were affected by dental caries. At age 5-6 the mean dmft was 4.5 and at age 12 the DMFT was 3.8. In both groups, the d/D-components were high (5-6 years: dt/dmft = 89%; 12 years: DT/DMFT = 45%). For children aged 5-6, the mean caries experience increased from 1991 (3.7 dmft) to 1996 (4.5 dmft) (P<0.01) whereas DMFT of 12 year-olds declined from 5.0 in 1985 and 4.3 in 1991 (P<0.01). The differences in caries experience by urbanization level were reduced over time. In 1996, 72% of 12-year-olds had gingival bleeding (CPI scores 1+2), and 23% had moderate/severe malocclusion. In conclusion, in order to meet the need for dental care in schoolchildren in Hungary health authorities are encouraged to revitalize the school dental services. Implementation of community-based health promotion is needed to control oral disease in Hungarian children. PMID- 10730726 TI - Trained nurses can provide safe and effective sedation of MRI in pediatric patients. PMID- 10730727 TI - The new anesthesia machines. PMID- 10730728 TI - Trained nurses can provide safe and effective sedation for MRI in pediatric patients. AB - PURPOSE: To determine the success rate, safety and complications using a standard protocol and trained nurses to provide sedation for MRI under the supervision of a radiologist. MATERIALS AND METHODS: Nurses were trained to provide sedation via a standard protocol for pediatric patients undergoing diagnostic MRI. Oral chloral hydrate (80-100 mg x kg(-1)) was used for children less than 18 mo of age. Older children received either 1-6 mg x kg(-1) pentobarbital i.v., with or without 1-2 microg x kg x hr(-1) fentanyl, or 25 mg x kg(-1) thiopental pr. Sedation was defined as successful if it allowed completion of the MRI without image distorting patient movement. The records of 572 MRIs performed on 488 pediatric patients (mean age 5+/-4 yr; age 2 mo-14 yr) from 1991 to July 1995 were reviewed to determine the success rate and complications using the sedation program. RESULTS: Most, 91.8% (525/572), of the MRIs were successfully completed in 445 patients. The reasons for failure were inadequate sedation (45, 95.7%) and coughing (2, 4.2%). The failure rate was much higher before 1994 (38/272, 14%) than after (9/300, 3%; P<0.0001). Failure was more common if rectal thiopental was used (23/172, 14%) than intravenous pentobarbital (19/256, 7.4%; P<0.05). The failure rate was also high in patients with a history of a behavioural disorder (10/59, 17%). There were no deaths or unexpected admissions as a result of the sedation program. CONCLUSION: A high success rate can be achieved as experience is gained using a standard protocol and trained nurses to sedate children for MRI. PMID- 10730730 TI - Advancement of the mandible facilitates nasal breathing in human subjects sedated with midazolam. AB - PURPOSE: To determine how anterior advancement of the mandible (ADM) affects spontaneous breathing through the nasal route in healthy human volunteers sedated with intravenous midazolam. METHODS: In four subjects who exhibited nasal breathing during midazolam sedation (intravenous dose: 0.09+/-0.02 mg x kg(-1), mean +/- SD), we measured respiratory rate (RR), peak nasal inspiratory airflow rate (V(nIpeak)) peak nasal expiratory airflow rate (V(nEpeak), duty ratio (Ti/Ttot) and nasal resistance (Rn) before and after ADM. Nasal resistance was calculated by dividing the difference between nasal mask and oropharyngeal pressure by airflow rate at peak nasal inspiratory airflow. RESULTS: The RR, V(nIpeak), and V(nEpeak) increased following ADM (P<0.001, respectively). On the contrary, Ti/Ttot decreased after ADM (P<0.001). Consequently, ADM decreased Rn from 30.4+/-40.8 to 5.0+/-5.6 (cm H2O x l(-1) x sec(-1)) (mean +/- SD) (P<0.001). In these four subjects, no respiratory airflow was observed through the oral route before and after ADM. CONCLUSION: Advancement of the mandible decreases nasal resistance, thereby facilitating spontaneous breathing through the nasal route in normal humans sedated with midazolam. PMID- 10730729 TI - A comparison of midazolam, alfentanil and propofol for sedation in outpatient intraocular surgery. AB - PURPOSE: To determine the ideal sedative regimen for intraocular surgery under peribulbar or retrobulbar block. The addition of alfentanil and or propofol to midazolam was evaluated with regard to hemodynamic variables, respiratory rate, pain, anxiety, sedation, postoperative recovery and patient satisfaction. METHODS: Eighty two patients aged between 50 and 85 were recruited into this prospective, randomised, double blind study. Patients, in four groups, received 0.015 mg x kg(-1) midazolam, 5 microg x kg(-1) alfentanil and 0.15 mg x kg(-1) propofol; 0.015 mg x kg(-1) midazolam and 0.15 mg x kg(-1) propofol; 0.015 mg x kg(-1) midazolam and 5 microg x kg(-1) alfentanil or 0.015 mg x kg(-1) midazolam alone. Blood pressure, heart rate, respiratory rate, pain, anxiety and sedation scores were measured. Times to discharge from the Post Anesthesia Care Unit (PACU) and Day Surgery Unit (DSU) were documented. A 24 hr telephone interview was carried out to determine patient satisfaction. RESULT: Systolic blood pressure of patients in groups that had received alfentanil was 6% lower than that of patients who had not (P<0.05) at the time of insertion of intraocular block. Patients in the alfentanil groups also had lower respiratory rates during the first 15 min after drug administration, but all patients were given supplemental oxygen therefore oxygen saturation was unaffected. Pain scores of patients who had been given alfentanil were lower during the first postoperative hour than those who had not. CONCLUSION: The addition of alfentanil to midazolam is advantageous in providing sedation for insertion of intraocular block. PMID- 10730731 TI - Diclofenac premedication but not intra-articular ropivacaine alleviates pain following day-case knee arthroscopy. AB - PURPOSE: To compare the postoperative analgesic effects of 50 mg diclofenac p.o. before surgery and intra-articular ropivacaine injected after diagnostic day-case knee arthroscopy performed under spinal anesthesia. METHODS: In a randomized, double-blind investigation, 200 ASA physical status 1-2 outpatients, age 18-60 yr, received either 50 mg diclofenac p.o. or placebo one hour before operation (100 patients per group), and intraarticular injections of either 20 ml of ropivacaine 0.5% or 20 ml of saline 0.9% (50 patients in each premedication groups). Patients received 50 mg diclofenac p.o. prn and, if needed, 0.1 mg x kg( 1) oxycodone im for postoperative pain relief. Patients were discharged home with a supply of 50 mg diclofenac tablets and were given a sheet of paper with knee pain VAS scales and a questionnaire of analgesics taken. Patients rated their VAS scores eight hours after surgery and in the moming and at the end of the first and the second postoperative days, respectively. RESULTS: The only statistically significant difference was found when the diclofenac groups were combined and compared with the combined placebo premedication groups. The VAS scores of knee pain at eight hours after the operation were 19+/-22 in the two diclofenac premedication groups and 32+/-28 in the two placebo groups (P = 0.001). CONCLUSIONS: Diclofenac premedication p.o. reduced the VAS scores at eight hours postoperatively while intra-articular ropivacaine did not. PMID- 10730732 TI - Intubating conditions and blockade after mivacurium, rocuronium and their combination in young and elderly adults. AB - PURPOSE: Mivacurium-rocuronium combinations have been demonstrated to be more potent than either drug given alone. Combinations were compared with mivacurium and rocuronium, with respect to onset, intubating conditions, and duration of action in young and elderly adults. METHODS: Fentanyl-propofol-N2O-isoflurane anesthesia was given to ASA I and II adults aged 18-65 yr (45 patients) and over 66 yr (45 patients). In this blinded randomized study, we compared accelerographic adductor pollicis response and visual assessment of response to facial nerve stimulation after 0.25 mg x kg(-1) mivacurium, 0.6 mg x kg(-1) rocuronium, and a combination of 0.08 mg kg(-1) mivacurium plus 0.2 mg x kg(-1) rocuronium. Intubating conditions at 2.5 min were rated as excellent, good, fair or poor. RESULTS: Onset times were similar for all drugs regimens and for both age groups (204-276 sec at the thumb; 142-196 sec at the eye) (P<0.05 between muscles). Intubating conditions were similar in all groups, and rated good or excellent, except in two subjects. In young patients duration to 25% recovery was longer (P<0.05) for rocuronium (mean +/- SD) (39+/-11 min) than for either mivacurium (23+/-6 min), or the combination (27+/-7 min). Duration was prolonged in the elderly for rocuronium (54+/-17 min), and the combination (35+/-11 min), but not for mivacurium (24+/-6 min). CONCLUSIONS: Mivacurium-rocuronium combinations yield onset times and intubating conditions similar to either parent agent with only two thirds as much total drug. Duration for such a mixture is similar to that of mivacurium in young adults and slightly prolonged in the elderly. PMID- 10730734 TI - Ephedrine, dopamine, or dobutamine to treat hypotension with propofol during epidural anesthesia. AB - PURPOSE: To compare the efficacy of ephedrine, dopamine and dobutamine for circulatory support during thoracic epidural anesthesia after anesthetic induction with propofol. METHODS: Forty patients undergoing lobectomy or mastectomy were divided into four groups of 10: a control group received no vasopressor; an ephedrine group received 5 mg ephedrine when the mean arterial pressure (MAP), measured every 2.5 min, decreased by 10% from baseline; dopamine and dobutamine groups received 5 microg x kg(-1) x min(-1) dopamine or 3 microg x kg(-1) x min(-1) dobutamine from five minutes after epidural injection of local anesthetic to the end of tracheal intubation. Anesthesia was induced with 2 mg x kg(-1) propofol. The MAP and heart rate (HR) were measured at baseline, 20 min after epidural injection, three minutes after propofol, and one minute after tracheal intubation. RESULTS: In the control group, MAP and HR decreased from 86+/-9 mmHg, 74+/-8 bpm to 62+/-9 mm Hg; P<0.0001, 60+/-8 bpm; P = 0.0003 after propofol. After tracheal intubation, MAP was restored to (81+/-13 mmHg, 70+/-13 bpm). In the ephedrine, dopamine, and dobutamine groups, MAP and HR remained unchanged during epidural anesthesia and propofol induction. However, after tracheal intubation, MAP and HR increased in the ephedrine (104+/-11 mm Hg; P = 0.004, 87+/-11 bpm; P<0.0001) and dobutamine (117+/-13 mm Hg; P = 0.0005, 100+/ 11 bpm; P<0.0001) groups, but not in the dopamine group compared with baseline. CONCLUSION: Dopamine is preferable to ephedrine and dobutamine in providing hemodynamic stability during propofol induction and tracheal intubation following epidural anesthesia. PMID- 10730733 TI - Comparison of combined spinal-epidural and low dose epidural for labour analgesia. AB - PURPOSE: To compare the combined spinal-epidural (CSE) technique with the epidural technique with regard to time to initiate and manage, motor block, onset of analgesia and satisfaction during labour. METHODS: Upon requesting analgesia, 50 healthy term parturients were randomized in a prospective, double-blind fashion to receive either CSE analgesia or lumbar epidural analgesia in the labour floor of a university hospital at an academic medical centre. The epidural group (n = 24) received bupivacaine 0.0625%-fentanyl 0.0002% with 0.05 ml in 10 ml local anesthetic sodium bicarbonate 8.4% and epinephrine 1:200,000. The CSE group (n = 26) received intrathecal 25 microg fentanyl and 2.5 mg bupivacaine. Additional analgesia was provided upon maternal request. RESULTS: There were no differences (P>0.05) in time to perform either technique, motor blockade, or parturient satisfaction or in the number of times that the anesthesiologist was called to perform any intervention. Although the first sign of analgesia was not different between the two groups, the onset of complete analgesia was more rapid with the CSE technique (Visual Analogue Pain Score (VAPS) at five minutes < three: 26/26 vs. 17/24, P+/-0.001). CONCLUSION: Although epidural analgesia with a low concentration of local anesthetic and opioid mixture takes longer to produce complete analgesia, it is a satisfactory alternative to CSE. PMID- 10730735 TI - Sevoflurane mask anesthesia for urgent tracheostomy in an uncooperative trauma patient with a difficult airway. AB - PURPOSE: Proper care of the trauma patient often includes tracheal intubation to insure adequate ventilation and oxygenation, protect the airway from aspiration, and facilitate surgery. Airway management can be particularly complex when there are facial bone fractures, head injury and cervical spine instability. CLINICAL FEATURES: A 29-yr-old intoxicated woman suffered a motor vehicle accident. Injuries consisted of multiple abrasions to her head, forehead, and face, right temporal lobe hemorrhage, and complex mandibular fractures with displacement. Mouth opening was <10 mm. Blood pressure was 106/71 mm Hg, pulse 109, respirations 18, temperature 37.3 degrees C, SpO2 100%. Chest and pelvic radiographs were normal and the there was increased anterior angulation of C4-C5 on the cervical spine film. Drug screen was positive for cocaine and alcohol. The initial plan was to perform awake tracheostomy with local anesthesia. However, the patient was uncooperative despite sedation and infiltration of local anesthesia. Sevoflurane, 1%, inspired in oxygen 100%, was administered via face mask. The concentration of sevoflurane was gradually increased to 4%, and loss of consciousness occurred within one minute. The patient breathed spontaneously and required gentle chin lift and jaw thrust. A cuffed tracheostomy tube was surgically inserted without complication. Blood gas showed pH 7.40, PCO2 35 mm Hg, PO2 396 mm Hg, hematocrit 33.6%. Diagnostic peritoneal lavage was negative. Pulmonary aspiration did not occur. Oxygenation and ventilation were maintained throughout the procedure. CONCLUSION: Continuous mask ventilation with sevoflurane is an appropriate technique when confronted with an uncooperative trauma patient with a difficult airway. PMID- 10730736 TI - [Serotonin syndrome during fluoxetine poisoning in a patient taking moclobemide]. AB - PURPOSE: To present a case of delayed serotonin syndome (SS), a less well-known adverse effect of fluoxetine intoxication. CLINICAL PRESENTATION: A 21-yr-old woman was admitted following voluntary intoxication with fluoxetine and benzodiazepines. At the time of admission, she was slightly drowsy and hypotonic but, eight hours later, she developed severe hypertonic coma despite blood concentrations of fluoxetine within the therapeutic range. Repeated toxicological analyses revealed the presence of moclobemide at non-measurable concentrations, suggesting earlier ingestion of this monoamine oxydase inhibitor. Having excluded all other likely causes of the neurological syndrome observed, a SS was postulated. Treatment was symptomatic with mechanical ventilation, sedation with thiopental and fentanyl, and neuromuscular block with pancuronium bromide. The patient recovered spontaneously 20 hr later. CONCLUSION: Physicians managing patients presenting with fluoxetine intoxication must be aware of the potential risk of SS. Treatment is symptomatic, but SS may be severe and require vital support in the intensive care environment. Review of published reports does not allow the authors to recommend a specific anesthetic management. PMID- 10730737 TI - Torsades de pointes secondary to intravenous haloperidol after coronary bypass grafting surgery. AB - PURPOSE: Postoperative delirium occurs in about 2% of patients undergoing major cardiac surgery including coronary artery bypass grafting surgery (CABG). Haloperidol (Sabex, Boucherville, Canada) is a drug commonly used in the intensive care unit for the treatment of delirium and is usually considered safe even at high doses and is rarely implicated in the development of malignant ventricular arrhythmias such as torsades de pointes. The purpose of this study is to report such a complication of use of haloperidol after myocardial revascularization. CLINICAL FEATURES: The patient reported underwent uneventful triple bypass surgery. Administration of large intravenous doses of haloperidol was necessary for control of psychomotor agitation due to delirium. Torsades de pointes occurred in the absence of QT prolongation on the third postoperative day following use of the drug with no other obvious etiological factor. CONCLUSION: Awareness of this rare complication is key to judicious use of this drug in the post CABG patient in whom such an arrhythmia may have very deleterious consequences because of the underlying cardiac condition. PMID- 10730738 TI - Recombinant hirudin anticoagulation for aortic valve replacement in heparin induced thrombocytopenia. AB - PURPOSE: To report the case of a patient with HIT that received a prolonged infusion of r-hirudin (lepirudin; Refludan; Hoechst, France) before, during and after cardiopulmonary bypass (CPB) for aortic surgery. Although administration of r-hirudin for CPB anticoagulation has previously been reported, many questions persist concerning the best therapeutic regimen for CPB anticoagulation as well as the time of onset and the doses for postoperative anticoagulation. CLINICAL FEATURES: A 65-yr-old man was admitted for surgery of aortic stenosis after an episode of acute pulmonary edema complicated by deep venous thrombosis in the context of documented HIT. The patient received r-hirudin for 13 dy before surgery at doses (0.4 mg x kg(-1) bolus followed by 0.15 mg x kg(-1) x hr(-1) continuous infusion) that maintained activated partial thromboplastin time (aPTT) ratios between 2 and 2.5. Anticoagulation for CPB was performed with r-hirudin given as 0.1 mg x kg(-1) i.v. bolus and 0.2 mg kg(-1) in the CPB priming volume. Anticoagulation during CPB was monitored with the whole blood activated coagulation time and ecarin clotting time (ECT) performed in the operating room with values corresponding to r-hirudin concentrations >5 microg x ml(-1) during CPB. Anticoagulation during CPB was uneventful. Two bleeding episodes, related to the r-hirudin regimen and necessitating allogeneic blood transfusion, occurred after surgery. CONCLUSION: This case report confirms previous experience of the use of r-hirudin for anticoagulation during CPB and provides additional information in the context of prolonged r-hirudin infusion before and after CPB. PMID- 10730739 TI - An unusual cause of tracheal stenosis. AB - PURPOSE: To report a large chronic tracheal foreign body, causing tracheal stenosis in an 11-yr-old girl. CLINICAL FEATURES: The history was suggestive of obstructive airways disease with secondary bronchiectasis. Physical findings were crepitations and rhonchi all over the chest. Blood gases were normal. Chest X-ray showed bronchiectasis and a ventilation perfusion scan identified a tracheo esophageal fistula. During anesthesia to confirm this, intubation and ventilation were difficult because of tracheal stenosis. The hypoventilation resulted in severe hypercarbia and acidosis. A subsequent CT scan showed a stenosis of 2 mm diameter and 1 cm length in the middle third of trachea, bronchiectasis, and an air filled pocket between the trachea and esophagus. PFT showed a severe obstruction. Antitubercular treatment which was started on the presumptive diagnosis of tuberculous stenosis and tracheoesophageal fistula caused a delay with deterioration of patient from intermittent dyspnea to orthopnea with severe hypecarbia and acidosis. The anesthetic management of the tracheal reconstruction was difficult due to her moribund condition even after medical treatment, the short length of the trachea above the obstruction, its severity and lack of resources for alternative techniques. A large foreign body was found lying obliquely in the trachea dividing it into an anterior narrow airway mimicking a stenosed trachea, and a wider posterior blind passage. CONCLUSION: The anesthetic consequences were peculiar to the unexpected etiology of the stenosis and poor general condition of the patient. Minor details like the tracheal tube bevel and ventilatory pattern became vitally important. PMID- 10730740 TI - Modifications of the hemodynamic consequences of theophylline intoxication with landiolol in halothane-anesthetized dogs. AB - PURPOSE: To examine the effect of landiolol (ONO-1101), a new ultra-short acting, highly selective beta1 blocker, on hemodynamic response to acute theophylline intoxication in anesthetized dogs. METHODS: Thirty-four dogs were studied during halothane anesthesia. Aminophylline (50 mg x kg(-1) over 20 min followed by infusion at 1.75 mg x kg(-1) x hr(-1)) was administered as a model of acute theophylline intoxication. Dogs were randomly enrolled into four landiolol groups (0, 1, 10, 100 microg x kg(-1) x min(-1)) to treat tachyarrhythmias. Hemodynamic variables, heart rate (HR), systemic blood pressure (SBP), pulmonary artery pressure, pulmonary artery occlusion pressure, and cardiac output (CO) were measured along with plasma concentrations of theophylline, epinephrine, and norepinephrine. RESULTS: After 60 min, plasma concentration of theophylline reached 46.6+/-4.0 (mean +/- SD) microg x ml(-1), HR increased from 129+/-21 to 193+/-27 bpm (P<0.0001) and CO increased from 1.6+/-0.5 l x min(-1) to 2.1+/-0.4 l x min(-1) (P<0.0001), whereas SBP decreased from 139+/-25 to 121+/-25 mm Hg (P<0.0001), with decreasing systemic vascular resistance. After intoxication, plasma epinephrine concentration increased from 125 +/-112 to 325+/-239 pg x ml( 1) (P<0.0001), and norepinephrine concentration from 103+/-61 to 133+/-61 pg x ml(-1) (P<0.0011). Landiolol 10 microg x kg(-1) x min(-1) decreased HR to pre intoxication level, whereas HR returned to the intoxication baseline by 30 min after cessation of landiolol infusion. CONCLUSIONS: Landiolol controlled tachyarrhythmias associated with theophylline toxicity. The optimal effective dose of landiolol was 10 microg x kg(-1) x min(-1). PMID- 10730741 TI - Propofol directly depresses lumbar dorsal horn neuronal responses to noxious stimulation in goats. AB - PURPOSE: We tested the hypothesis that propofol, acting in the brain, would either enhance, or have no effect, on lumbar dorsal horn neuronal responses to a noxious mechanical stimulus applied to the hindlimb. We recorded the response of lumbar dorsal horn neurons during differential delivery of propofol to the brain and torso of goats. METHODS: Goats were anesthetized with isoflurane and neck dissections performed which permitted cranial bypass. A laminectomy was made to allow microelectrode recording of lumbar dorsal horn neuronal activity. Isoflurane was maintained at 0.8+/-0.1% to both head and torso throughout the study. During cranial bypass propofol was separately administered to the torso (1 mg x kg(-1), n = 7; 3.75 mg x kg(-1), n = 8) or cranial (0.04 mg x kg(-1), n = 7; 0.14 mg kg(-1), n = 8) circulations. RESULTS: Propofol administered to the torso depressed dorsal horn neuronal responses to noxious stimulation: low dose: 500+/ 243 to 174+/-240 impulses x min(-1) at one minute post-injection, P<0.001; high dose: 478+/-204 to 91+/-138 impulses x min(-1) at one minute post-injection, P<0.05). Propofol administered to the cranial circulation had no effect: low dose: 315+/-150 to 410+/-272 impulses x min(-1), P>0.05; high dose: 462+/-261 to 371+/-196 impulses x min(-1), P>0.05. CONCLUSIONS: These data indicate that propofol has a direct depressant effect on dorsal horn neuronal responses to noxious stimulation, with little or no indirect supraspinal effect. PMID- 10730742 TI - Difficult airway management: comparison of the Bullard laryngoscope with the video-optical intubation stylet. AB - PURPOSE: To evaluate, whether the video-optical intubation stylet (VOIS) was more successful for difficult tracheal intubation than the Bullard laryngoscope (BL). METHODS: An intubation mannequin head was modified so that, using a Macintosh blade size 3, only the epiglottis was visible at direct laryngoscopy, representing a grade III laryngoscopic view. Forty anesthesiologists attempted tracheal intubation using each technique. Tracheal intubation with the Bullard laryngoscope was performed using the attached non-malleable intubating stylet preloaded with an endotracheal tube. The video-optical intubation stylet inserted into an endotracheal tube was used with direct laryngoscopy. During conventional laryngoscopy, the video-view from the stylet tip allowed the tracheal tube to be guided behind the epiglottis into the trachea. Ten attempts with each technique were performed by each anesthesiologist in randomized order. Intubation time, and failed intubation (> 60 sec/esophageal intubation) were recorded. The operators assessed the degree of difficulty of each method using a Likert-scale. RESULTS: Mean intubation time (19.2+/-4.5 sec for the BL and 18.8+/-4.6 sec for the VOIS) was almost the identical. The video-optical intubation stylet was associated with fewer failed intubations (8 vs. 41; P<0.005) and had a lower degree of difficulty (1.7+/-0.65 for the VOIS and 2.6+/-0.74 for the BL; P<0.0001). No correlation was found between the anesthesiologist's experience and mean intubation time, estimated degree of difficulty or number of unsuccessful intubation. CONCLUSION: The video-optical intubation stylet was a more effective and simpler intubation device to facilitate difficult tracheal intubation than the Bullard laryngoscope. PMID- 10730743 TI - New media. PMID- 10730744 TI - Physician resources. PMID- 10730745 TI - Epidural blood patch (EBP) and septic complication. PMID- 10730746 TI - High frequency ventilation. PMID- 10730747 TI - [Painful injection of propofol into a central vein]. PMID- 10730748 TI - Fragrance allergy. PMID- 10730749 TI - What are some of the lessons learnt from in vitro studies of severe unpredictable drug reactions? PMID- 10730750 TI - Understanding of the pathophysiology of pemphigus suggests innovative therapeutic approaches. PMID- 10730751 TI - Characterization of an in vitro model of human melanoma invasion based on reconstructed human skin. AB - The purpose of this study was to compare the invasive properties of normal human cutaneous melanocytes and of a cutaneous melanoma cell line (HBL) in a three dimensional model of reconstructed human skin. Specifically, we asked to what extent the pigmentary and invasive behaviour of both cells is influenced by their interaction with adjacent skin cells (keratinocytes and fibroblasts) and the basement membrane (BM). In the presence of a BM, normal human melanocytes within this model remained within the basal layer of keratinocytes and did not pigment spontaneously. When the BM was removed, melanocytes were found suprabasally and pigmented extensively. No significant invasion of melanocytes into the dermis was detected in the presence or absence of the BM. HBL melanoma cells showed no significant ability to invade into the dermis in the absence of other cells, irrespective of the presence or absence of the BM. However, when added to keratinocytes and fibroblasts, HBL cells showed a capacity to invade into the dermis, both in the presence and absence of the BM. Associated with HBL invasion into the dermis, we noted significant keratinocyte entry into the dermis. On their own, keratinocytes entered the dermis in the absence of the BM but showed no significant penetration into the dermis when the BM was present. In summary, this model demonstrates clear differences between melanocytes and a melanoma cell line with respect to their invasive properties. It also allows demonstration of interactions between cells, and between cells and the BM. The study also provides evidence for a synergistic interaction between this melanoma cell line and keratinocytes in penetrating the BM. PMID- 10730752 TI - Expression of transglutaminase activity in developing human epidermis. AB - Epidermal transglutaminase (TGase) is known to catalyse cross-linking of several precursor proteins in the formation of cornified cell envelope at the terminal differentiation of keratinocytes. Expression of TGase activity was studied using an in situ TGase activity assay in human fetal skin samples of 49-163 days estimated gestational age (EGA). In the early two-layered epidermis (49-56 days EGA), in situ TGase activity was not observed in the periderm cells or the basal cells. In the late two-layered epidermis (57-65 days EGA), in situ TGase activity became weakly positive in the periderm cells, but not in the basal cells. In the three-layered (66-95 days EGA) and in four- or more layered (96-135 days EGA) stratified epidermis, in situ TGase activity was still restricted only to the periderm cells. After keratinization occurred in the interfollicular epidermis (163 days EGA), in situ TGase activity was expressed in the granular and cornified layers. This unique localization of TGase activity further support the hypothesis that periderm cells form cornified cell envelope during their regression process in human fetal skin development. PMID- 10730753 TI - Topical hyaluronidase decreases hyaluronic acid and CD44 in human skin and in reconstituted human epidermis: evidence that hyaluronidase can permeate the stratum corneum. AB - Hyaluronic acid (HA), a high molecular weight glycosaminoglycan of the extracellular matrix involved in growth, inflammation and wound healing, also contributes to the hydration and plastic properties of skin. Several drug and cosmetic formulations contain HA. We have initiated investigations that explore whether it is possible, by topical application, to modulate endogenous HA levels in skin. We developed a model epidermal culture system that exhibited a differentiated stratum corneum, and expressed HA and the HA receptor CD44, in a pattern similar to that observed in intact skin. Such in vitro skin equivalents are useful models for investigating the effect of topical drugs. HA and bacterial hyaluronidase were applied to the in vitro skin equivalent and to human skin. Their effects on endogenous HA and CD44 expression were examined using histochemical analysis. Topical HA treatment had no significant effect on HA or CD44 expression in either system. However, hyaluronidase decreased HA and CD44 expression in a dose-dependent manner in both the epidermal culture system and in skin. Apparently, HA is not able to permeate the epidermal culture system or human skin to a significant degree, but bacterial hyaluronidase does permeate both human skin and the culture system, depleting HA and decreasing CD44 expression. These effects were more prominent in the dermal than in the epidermal layers, suggesting that marked differences in HA metabolism exist in these two skin compartments. The ability of hyaluronidase to permeate the stratum corneum suggests that topical application may, additionally, be useful as a clinical modality. PMID- 10730754 TI - The distribution of choline acetyltransferase- and acetylcholinesterase-like immunoreactivity in the palmar skin of patients with palmoplantar pustulosis. AB - The distribution of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in involved skin in patients with palmoplantar pustulosis (PPP) and in normal palmar skin in healthy non-smokers and smokers has been studied by immunohistochemistry, especially in relation to the sweat gland apparatus. The sweat gland and its duct showed ChAT- and AChE-like immunoreactivity (LI) of varying intensity in all three groups and with stronger reactivity than in the epidermis. ChAT-LI was present in the coil and in the duct except in the corneal layer. Smokers and patients with PPP displayed significantly fewer ChAT+ acrosyringia than non-smokers. In the patients with PPP, the granulocytes in the pustules and in the papillary dermis displayed ChAT-LI. Western blot analysis of granulocytes from peripheral blood from healthy donors confirmed the presence of ChAT-like proteins in large amounts in neutrophils and small amounts in eosinophils. AChE-LI of varying intensity was found in all parts of the sweat gland apparatus in all three groups. The strongest AChE-LI in the acrosyringia was seen in the lowest part of the stratum corneum, where the PPP pustules are located. No significant differences in staining pattern or intensity were found between the coils, nerve fibres surrounding the coils or ducts. The number of mast cells in the papillary dermis was about four times larger in the patients with PPP than in the control subjects. AChE-LI was observed in about 25% of the mast cells in non-smoking control subjects and in patients with PPP, but only in 10% of those in the smoking control subjects. Our findings indicate that the (non neuronal) cholinergic system may be involved in cutaneous inflammatory processes. PMID- 10730755 TI - The use of cytokeratins 7 and 20 in the diagnosis of primary and secondary extramammary Paget's disease. AB - Despite the similarity in clinical appearance, there is a significant difference in the prognosis between primary extramammary Paget's disease (EPD) and the pagetoid spread of underlying regional internal malignancy (secondary EPD, pagetoid phenomenon). Fifteen cases of primary EPD (11 carcinoma in situ and four invasive carcinoma), seven cases of secondary EPD (five colorectal adenocarcinoma and two urothelial carcinoma), and six cases of anal canal carcinoma were retrieved and analysed immunohistochemically using six kinds of monoclonal anticytokeratin antibodies. No expression of cytokeratins 1, 5, 10, 13 and 14 was observed in any cases examined in this study. All 15 cases of primary EPD had the immunophenotype cytokeratin (CK)7+/CK20-. CK20 expression was diffusely positive in six cases of secondary pagetoid spread (two urothelial carcinoma and four colorectal adenocarcinoma), and focally in one case (a colorectal adenocarcinoma). In anal canal carcinoma, three of six cases showed CK20 diffuse expression and the remaining three cases expressed CK20 focally. CK7 expression was observed in three of six cases of anal canal carcinoma and in two of five cases of secondary EPD associated with colorectal adenocarcinoma. The combination of CK7 and CK20 demonstrates these to be useful markers in distinguishing 'primary' EPD from a pagetoid spread of extracutaneous malignancies. Namely, immunophenotypes other than CK7+/CK20- in Paget cells suggest underlying regional internal malignancy. PMID- 10730756 TI - Refined localization of the gene for Clouston syndrome (hidrotic ectodermal dysplasia) in a large French family. AB - Hidrotic ectodermal dysplasia (HED) or Clouston syndrome is a rare autosomal dominant disorder characterized by nail dystrophy, alopecia and palmoplantar hyperkeratosis, which maps to chromosome 13q11-q12.1. We confirmed linkage of HED to this region in a large French family. To define the critical region for HED, detailed haplotypes were constructed with new pericentromeric polymorphic markers. A recombination event in the family indicates that the HED locus maps centromeric to D13S1832. Our French family does not share a common haplotype with other pedigrees previously published (particularly French-Canadian), indicating that the mutations in these families are likely to be of different origin. PMID- 10730757 TI - Sequential assessment of an antidrug antibody response in a patient with a systemic delayed-onset sulphonamide hypersensitivity syndrome reaction. AB - A 19-year-old man was treated with trimethoprim-sulphamethoxazole intermittently over 4 weeks. He developed a rash and fever. Despite treatment with low-dose methylprednisolone, his condition worsened. He developed a confluent erythematous macular eruption, elevated liver enzymes, lymphadenopathy, polyserositis and eosinophilia. A tentative diagnosis of sulphonamide hypersensitivity syndrome reaction (SHSR) was made and a serum sample (acute) was obtained to screen for antibodies associated with SHSR. Intravenous methylprednisolone sodium succinate (250 mg every 6 h for 48 h) was administered. The patient's condition improved, and he was discharged with oral prednisone. A convalescent serum sample was obtained 14 weeks later. By Western blotting and enzyme-linked immunosorbent assay (ELISA), antisulphamethoxazole IgG antibodies were detected in the acute serum sample, supporting the clinical diagnosis of SHSR. Contrary to expectations, antibodies were not detected in the convalescent serum sample by immunoblotting. Antisulphamethoxazole antibodies were detected by ELISA in the convalescent serum, but the titre was decreased approximately 45-fold. One possible explanation for the decrease in antibody concentration in the convalescent sample was the administration of high-dose glucocorticoids to the patient following collection of the acute serum sample. PMID- 10730758 TI - VML 295 (LY-293111), a novel LTB4 antagonist, is not effective in the prevention of relapse in psoriasis. AB - Leukotriene B4 (LTB4) receptor antagonists have been the subject of several studies in the treatment of inflammatory diseases, including psoriasis. A novel oral LTB4 antagonist, VML 295 (LY-293111) has recently been developed and has a pronounced effect on epidermal inflammatory parameters. However, oral treatment of psoriasis for 4 weeks did not result in a decrease in disease severity. The present study was performed in order to investigate whether prolonged treatment with VML 295 up to 8 weeks has a beneficial effect on the overall severity of psoriasis. Moreover, we studied to what extent VML 295 is able to prevent relapse in psoriasis. In the present study, 35 patients with stable chronic plaque psoriasis were included. A representative plaque of at least 16 cm2 was initially treated with clobetasol-17-propionate lotion under hydrocolloid occlusion in all patients. Clearance was achieved within 6 weeks in 31 patients. After clearance, the patients were randomized to treatment and received oral VML 295 capsules 200 mg twice daily or placebo for 8 weeks. Twenty-five patients completed the study. The psoriasis area and severity index (PASI) was assessed before treatment, at clearance, and on days 15, 29, 43 and 5 7 of the treatment period. Biopsies were taken from the treated lesion before treatment, after clearance and at relapse, and cells were analysed by flow cytometry with markers for differentiation (keratin 10), inflammation (vimentin), and proliferation (DNA content). After 8 weeks of treatment, 14 of 15 VML 295-treated patients had relapsed and 11 of 16 placebo-treated patients had relapsed. A total of six patients were withdrawn. The time to relapse and the number of relapsed patients was not significantly different comparing the treatment groups. There was no significant difference in PASI scores between VML 295-treated patients and placebo-treated patients after 8 weeks of treatment. Flow cytometric parameters for differentiation, inflammation and proliferation did not show significant differences between VML 295- and placebo-treated patients. We conclude that oral VML 295 (LY-293111) is not effective in preventing relapse in psoriasis, either clinically or at the cellular level, and that in our group of patients VML 295 had no beneficial effect on overall psoriasis severity. Moreover, we conclude that further development of LTB4 modulating drugs for the treatment of psoriasis is not indicated. PMID- 10730759 TI - Histological increase in inflammatory infiltrate in sun-exposed skin of female subjects: the possible involvement of matrix metalloproteinase-1 produced by inflammatory infiltrate on collagen degradation. AB - To investigate morphological changes occurring during cutaneous photoageing, a correlation between the number of infiltrating cells in the dermis and the degree of collagen damage was examined using sections from clinically normal chronically sun-exposed and sun-protected skin of Japanese female subjects. Haematoxylin and eosin-stained sections from 134 sun-exposed (subjects aged 3-82 years) and 73 sun protected (subjects aged 1-86 years) areas demonstrated a predominant lymphoid cell and to a lesser extent histiocyte infiltration. The mean +/- SD number of lymphoid cells and histiocytes in the sun-exposed skin sections (427.0+/-192.2 and 147.8+/-83.3 cells/mm2, respectively) was significantly higher than in the sun-protected skin sections (292.6+/-98.3 and 125.9+/-59.0 cells/mm2, respectively) (P < 0.001 and P < 0.05, respectively), and the number of lymphoid cells in the sun-exposed skin sections increased significantly with age up to 50 years (r = 0.400, P < 0.001). Sun-exposed skin sections with severe collagen degeneration had a significantly higher number of lymphoid cells than those with slightly degenerated collagen (mean 626.3 vs. 482.4 cells/mm2, P < 0.01). The mean count of mast cells in sun-exposed skin was 202.0 cells/mm2; this did not vary with the age of the subjects or the level of collagen damage. Immunohistochemical studies using 24 frozen sections identified most of the lymphoid cells infiltrating sun-exposed skin as memory T lymphocytes (CD3+, CD4+ and CD45RO+). The number of cells which displayed immunoreactivity to matrix metalloproteinase (MMP)-1 in the sun-exposed skin sections was significantly higher than in the sun-protected skin sections (mean 170.2 vs. 113.6 cells/mm2, P < 0.05). Among these cells were observed CD3 and MMP-1 double-stained T lymphocytes, and T lymphocytes contacting MMP-1-positive cells. These morphological observations suggest that T lymphocytes infiltrating photodamaged skin may play a part in the degeneration and reduction of collagen through MMP-1 activity. PMID- 10730760 TI - The British Association of Dermatologists audit of atopic eczema management in secondary care. Phase 2: audit of service process. AB - An audit of atopic eczema management, conducted on behalf of the British Association of Dermatologists, examined service structure (phase 1), process (phase 2) and outcome (phase 3). In phase 2, an on-site case-note audit was conducted in 19 hospital dermatology departments randomly selected from the original sample of 187 centres across the U.K. In total, 630 sets of notes were examined for completeness of: (i) information given to general practitioners (GPs) in clinic letters and (ii) facts relevant to the management of atopic eczema recorded in the patients' notes. In general, the information given to GPs in the clinic letters was good, with the recording of diagnosis, treatment and follow-up approaching the 100% working standard. Factual information such as site and severity of eczema (83% and 74%), and presence or absence of asthma (53%) were better recorded than quality of life issues such as sleep loss secondary to itching (21%) and effect on school, work or social life (6%). On average, only 51% of all audit measures were recorded across all centres, with slight variation between centres (41-61%). The centre with the best recording had a purpose designed data sheet for doctors to complete when seeing new patients with atopic eczema. Such data sheets may help improve case-note recording. Similar data sheets for patients to complete may be more time-efficient. PMID- 10730766 TI - Lichen planus with lesions on the palms and/or soles: prevalence and clinicopathological study of 36 patients. AB - According to the literature, palmoplantar lesions in lichen planus (LP) are uncommon, and do not usually have the classically described clinical morphology. The aim of this study was to evaluate the prevalence of palmoplantar lesions in LP and to describe the characteristic clinicopathological appearances of LP affecting palms and soles. Palmoplantar LP with accompanying skin involvement accounted for 26% of our cases. The presence of very pruriginous erythematous scaly and/or hyperkeratotic plaques, with well-defined edges, located on the internal plantar arch, without involvement of the fingertips, and which usually disappear in a few months, is characteristic of palmoplantar LP. Histopathological examination shows the characteristic features of LP. PMID- 10730763 TI - The Nottingham Eczema Severity Score: preliminary refinement of the Rajka and Langeland grading. AB - A method for assessing disease severity of atopic dermatitis (AD) in children has been developed for population-based research. Based on an index first described by Rajka and Langeland in 1989, disease severity is determined by evaluating the three elements of clinical course, disease intensity and extent of examined AD. This paper describes development of the index for use in epidemiological studies based on a community-based study of 290 pre-school children (aged 1-5 years). Construct validity of the index was evaluated with respect to clinical severity assessment according to a dermatologist, parental severity assessment, use of topical corticosteroids and impairment of quality of life. The severity distribution of AD in this community-based sample of children was: mild 82% (n = 237), moderate 12% (n = 36) and severe 6% (n = 17) according to this new index. In this sample 24% of children had suffered from AD of more than 9 months duration in the preceding 12 months, 4.5% had experienced significant sleep loss (6 or more nights of average sleep loss per week over 12 months) and 11% had experienced widespread extent of involvement (more than 10 body sites involved). Construct validity of the index was demonstrated for clinical and patient-derived severity assessment. This included a comparison between the new index and a global severity assessment by a dermatologist in which exact agreement was achieved in 88% of the cases. A small subgroup of children suffering from persistent localized forms of AD (discoid pattern, hand/ foot dermatitis, perioral dermatitis), who reported considerable morbidity, was identified using quality of life measures of severity; they would otherwise have been misclassified by the dermatologist or new index. Preliminary use of the Nottingham Eczema Severity Score would support further development as a research tool for a simple assessment of disease severity that could be used in epidemiological studies. Further validation is required with respect to use in older children, administration by researchers/health professionals and development as a wholly questionnaire-based assessment. PMID- 10730761 TI - The frequency of fragrance allergy in a patch-test population over a 17-year period. AB - Fragrances are widely encountered in our daily environment and are known to be a common cause of allergic contact dermatitis. We have reviewed our patch test data from 1980 to 1996 to establish whether the pattern of fragrance allergy has changed with time. During this period, 25,545 patients (10,450 male, 15,005 female) were patch tested with the European standard series. The mean annual frequency of positive reactions to the fragrance mix was 8.5% in females (range 6.1-10.9) and 6.7% in males (range 5.1-12.9). Females were 1.3 times more likely to be allergic to fragrance (P < 0.001, 95% confidence interval, CI 1.17-1.41). Males with fragrance allergy were older than females by 5.6 years (mean age 48.2 vs. 42.6 years; P < 0.001, 95% CI 3.9-7.3). The incidence of a concomitant positive patch test to balsam of Peru in fragrance-sensitive patients showed wide variation, suggesting that it is not a reliable marker of fragrance allergy. There was a positive correlation between the isomers isoeugenol and eugenol. Oak moss remained the most common overall allergen throughout the study, positive in 38.3% of females and 35.6% of males who were tested to the constituents of the fragrance mix. During the period of the study the incidence of positive tests to oak moss increased by 5% yearly (P = 0.001, 95% CI 2.2-8.7). The frequency of allergic reactions to eugenol and geraniol remained relatively constant. Isoeugenol and alpha-amyl cinnamic aldehyde sensitivity increased and hydroxycitronellal showed a slow decline. There was a striking reduction in the frequency of sensitivity to cinnamic aldehyde (by 18% yearly; P < 0.001, 95% CI 14.3-21.0) and cinnamic alcohol (by 9% yearly; P < 0.001, 95% CI 5.2-12.9); these are now uncommon fragrance allergens. These data show temporal trends which may reflect the frequency of population exposure to individual fragrances. PMID- 10730764 TI - Hand eczema in twins: a questionnaire investigation. AB - Hand eczema in a population-based twin cohort of 6666 persons aged 20-44 years was investigated by means of a questionnaire regarding skin symptoms on the hands and self- or physician-diagnosed hand eczema. Genetic influence was observed by significant differences between identical and fraternal twins regarding casewise concordance rate and correlations in liability under the threshold model. The casewise concordance rates were almost twice as high in identical compared with fraternal twins in both sexes. By extension of the threshold model a joint analysis could be performed, resulting in a heritability estimate of 0.65. Tendencies towards stronger genetic influence for men and for younger individuals were present, but neither was statistically significant. No particular symptom pattern expressed especially strong or weak genetic influence. Hereditary factors for hand eczema were observed despite a presumably substantial individual specific environmental variation. The hypothesis that hereditary risk factors may play a significant part in the development of hand eczema in the general population, when no extreme environmental exposure exists, seems justifiable. The relevance of known individual risk factors such as atopic dermatitis or contact allergy in relation to heredity remains to be analysed. The possible importance of age and temporal change also needs further consideration. PMID- 10730767 TI - Laryngeal involvement in the Dowling-Meara variant of epidermolysis bullosa simplex with keratin mutations of severely disruptive potential. AB - The clinical features of the Dowling-Meara variant of epidermolysis bullosa simplex (EBS-DM) can, in an infant, be indistinguishable from other severe forms of epidermolysis bullosa (EB). Two unrelated infants with no family history of skin disease are described who, within hours of birth, developed extensive blistering of skin and oral mucosae and who both subsequently developed hoarse cries. Despite this superficial resemblance to other forms of EB, electron microscopy revealed a basal cell rupture and keratin aggregates characteristic of EBS-DM in the skin of both infants and in the vocal cord epithelium of one. Molecular analysis confirmed the diagnosis by identification of mis-sense point mutations in basal cell keratin genes in both cases. One patient carries a point mutation in keratin 14 (converting arginine at position 125 to histidine) and the other has a novel point mutation in keratin 5 (converting serine at position 181 to proline). Hoarseness is not a well documented feature of EBS-DM and is usually associated with junctional EB. These two patients demonstrate that the presence of a hoarse cry in an infant affected by severe EB does not necessarily indicate a poor prognosis. PMID- 10730765 TI - Atopic dermatitis and melanocytic naevi. AB - A study was performed to test the clinical impression that adults with severe atopic dermatitis (AD) have a low number of common naevi (CN). The number of CN > or = 2 mm was investigated in 51 Caucasian patients aged 20-63 years with severe AD since early childhood. The control group consisted of 379 randomly selected subjects, aged 30-50 years, investigated in an earlier study. Patients with AD had a significantly (P < 0.0001) lower total body count of CN (mean 9, median 5) compared with the control group (mean 67, median 53). It was also found that in the AD group there was a significant (P < 0.001) negative correlation between serum IgE and number of CN [r(s) = -0.50, 95% CI (-0.69; -0.24)]. The explanation for the low number of naevi that we have found in this highly selected subgroup of AD patients is not known. The atopic inflammation in the skin, genetics and treatment used for eczema are possible factors that may influence the formation of melanocytic naevi. PMID- 10730762 TI - Descriptive epidemiology of hand dermatitis at the St John's contact dermatitis clinic 1983-97. AB - For 15 years, hand dermatitis accounted for a quarter of patients seen in this clinic. The highest proportion occurred in women aged 17-30 years. Overall, the male to female ratio was 0.8, which contrasts with population-based studies. Catering was most frequently associated with occupational hand dermatitis. Other frequent occupational associations included metalworking, hairdressing, healthcare and mechanical work. PMID- 10730768 TI - Development of chimeric molecules for recognition and targeting of antigen specific B cells in pemphigus vulgaris. AB - Pemphigus vulgaris (PV) is an autoimmune blistering disease characterized by circulating pathogenic IgG antibodies against desmoglein 3 (Dsg3). The purpose of this study was to develop chimeric molecules for specific recognition and elimination of autoimmune B cells in PV. Mouse hybridoma cell lines producing anti-Dsg3 antibody (5H10, 12A2) were developed as an in vitro model system for targeting B cells. Dsg3-GFP, a baculoprotein containing the entire extracellular domain of Dsg3 fused with green fluorescence protein, recognized and targeted the hybridoma cells through their surface immunoglobulin receptors in an antigen specific way. The epitopes of these monoclonal antibodies were mapped on the amino terminal EC1 and part of EC2, a region considered functionally important in cadherins. Chimeric toxin molecules containing the mapped region (Dsg3deltaN1) and modified Pseudomonas exotoxin were produced in bacteria (Dsg3deltaN1-PE40 KDEL, PE3 7-Dsg3deltaN1-KDEL) and tested in vitro on hybridoma cell lines. The chimeric toxins, but not Dsg3deltaN1 alone, showed dose-dependent toxic activity with a reduction in hybridoma cell number to 40-60% of toxin-negative control cultures, compared with little or no effect on anti-Dsg3-negative hybridoma cells. Furthermore, these toxins showed toxic effects on anti-Dsg3 IgG-producing B cells from Dsg3deltaN1-immunized mice, with a 60% reduction in cell number compared with Dsg3deltaN1 alone. Thus, specific recognition and targeting of antigen-specific B cells in PV was demonstrated; this strategy may hold promise as a future therapeutic option for PV and other autoimmune diseases. PMID- 10730769 TI - Teaching non-specialist health care professionals how to identify the atypical mole syndrome phenotype: a multinational study. AB - The atypical mole syndrome (AMS) phenotype is the strongest known risk factor for cutaneous melanoma but recognition of the phenotype has been claimed to be problematic and to require specialist assessment. This study determined the ability of previously unskilled doctors and nurses in five countries to recognize the phenotype after brief training. The system used was the AMS scoring system. This incorporates melanocytic naevus counts, clinical atypia of naevi and distribution of naevi. The agreement in scoring between the dermatologist and trained personnel was determined in 986 patients; overall agreement in diagnosis was 94.5% (kappa 0.70, P < 0.0001). The kappa scores in different countries ranged from 0.65 to 0.77 for individual naevus characteristics, indicative of good agreement. Accurate diagnosis of the atypical mole syndrome phenotype is possible by non-specialists. This has implications for collaborative studies of naevi, for screening and for both primary and secondary prevention of melanoma. PMID- 10730770 TI - Photodynamic therapy with delta-aminolaevulinic acid for nodular basal cell carcinomas using a prior debulking technique. AB - The incidence of basal cell carcinomas (BCCs) is still increasing, and there is a demand for an easy, effective and selective non-invasive treatment such as topical photodynamic therapy (PDT). Twenty-three patients with 24 nodular BCCs were treated once with delta-aminolaevulinic acid (delta-ALA) PDT (100 mW cm(-2), 120 J/cm2) 3 weeks after prior debulking of the BCCs. Three months after PDT, all lesions were surgically excised and histopathologically evaluated for residual tumour. Twenty-two (92%) of the 24 nodular BCCs showed a complete response on clinical and histopathological examination. PDT for superficially abraded nodular BCCs with topically applied delta-ALA and the VersaLight as light source is an easy, effective and safe therapy, with excellent cosmetic results and no serious side-effects, in cases where non-surgical treatment of BCCs is indicated. PMID- 10730771 TI - Flegel's disease treated with psoralen ultraviolet A. AB - Flegel's disease is an uncommon condition which causes asymptomatic keratotic papules on the limbs. It usually develops in the fourth or fifth decade. Therapeutic options are limited to emollients, topical 5-fluorouracil and retinoids, but none of these treatments is consistently helpful. We report a patient with Flegel's disease who responded to psoralen ultraviolet A treatment. PMID- 10730772 TI - Redefining Rowell's syndrome. AB - Rowell's syndrome is believed to be a distinct and rare clinical entity originally described as lupus erythematosus associated with erythema multiforme like lesions with immunological findings of speckled antinuclear antibodies, anti La antibodies and a positive test for rheumatoid factor. We report two additional patients with Rowell's syndrome and review all the diagnostic criteria found in the literature. In view of the inconsistent findings of some of the diagnostic features, we propose that major and minor criteria be used to diagnose Rowell's syndrome. PMID- 10730773 TI - Pityriasis lichenoides-like mycosis fungoides in children. AB - We report three children with clinical features of pityriasis lichenoides (scaly red to brown papules and macules) in whom there were histopathological findings of mycosis fungoides (disproportionate epidermotropism, Pautrier's microabscesses, and wiry and coarse collagen bundles). Immunohistochemical staining revealed a prevalence of T lymphocytes in the infiltrate. T-cell receptor gene rearrangement analysis in lesional skin demonstrated rearrangement of the gamma chain in all cases. Human T-cell lymphotropic virus type 1 serology was negative in the two patients in whom this test was performed. Thus, lesions resembling pityriasis lichenoides can be an unusual and potentially misleading presentation of mycosis fungoides. PMID- 10730774 TI - Granulomatous slack skin: successful treatment with recombinant interferon-gamma. AB - Granulomatous slack skin is a rare variant of indolent cutaneous lymphoma, characterized by a cutis laxa-like clinical appearance and widespread granulomatous infiltration. A 25-year-old man had suffered from slowly progressive infiltrative, scaly and atrophic plaques and flaccid nodules with deep induration in the axillae, trunk and thighs. Histopathologically, dense lymphoid cell infiltration with numerous multinucleated giant cells (MGC) throughout the dermis to the subcutaneous tissue was observed. T-cell receptor gene rearrangement was detected in the skin lesions. Granulomatous infiltration with MGC was found in enlarged lymph nodes and the liver. Acute exacerbation was successfully treated with systemic recombinant interferon-gamma. PMID- 10730775 TI - Phacomatosis pigmentovascularis type IIa successfully treated with two types of laser therapy. AB - We describe a 28-year-old Japanese woman with phacomatosis pigmentovascularis type IIa who was treated by 27 sessions of Q-switched ruby laser irradiation to the site of dermal melanosis on her face and three sessions of dye laser irradiation to the port-wine stain on her left cheek. This is the first report of the successful treatment of a cutaneous lesion in a patient with phacomatosis pigmentovascularis. As the outcome of the treatments was excellent, we conclude that phacomatosis pigmentovascularis type IIa can be treated successfully by the combination of the two types of laser therapy. PMID- 10730776 TI - Acquired digital arteriovenous malformation: a report of six cases. AB - Arteriovenous malformation (AVM) is usually congenital, but an acquired type is also known, of which most are due to a previous injury. We report six patients with acquired digital AVM, all having a small patch of AVM localized to the tip of one finger, and therefore quite different from ordinary AVM which consists of a large pulsatile mass. Histologically, dilated venous and arterial vessels were present in the dermis. Tumour-like growth was absent; thus, these six cases could be differentiated from arteriovenous haemangioma. Because of the characteristic anatomical site, unique clinical manifestations and histology, this appears to be a new and distinctive entity. PMID- 10730778 TI - The beta subunit of the high-affinity IgE receptor, a candidate for atopic dermatitis, is not imprinted. PMID- 10730777 TI - Cutis marmorata telangiectatica congenita: report of 18 cases. AB - We report 18 patients with cutis marmorata telangiectatica congenita (CMTC) who visited the University Hospital Nijmegen between 1982 and 1999. In 11 patients (61%), associated abnormalities of varying severity were observed. Three of these patients had a previously described distinct syndrome comprising CMTC, a naevus flammeus at the upper lip/philtrum, macrocephaly and syndactyly, among other abnormalities. Findings in the other eight patients with associated abnormalities included hydrocephalus, hyperplasia/hypoplasia (body asymmetry), anal atresia, hearing loss, cardiovascular abnormalities, strabismus hypothyroidism, haemangioma, naevus anaemicus, cafe-au-lait spot, lipoma and an elevation of the right diaphragm. All cases were sporadic. PMID- 10730779 TI - Macular amyloidosis with an incontinentia pigmenti-like pattern. PMID- 10730780 TI - Pustular vasculitis secondary to achalasia of the cardia. PMID- 10730781 TI - Sneddon's syndrome in a child. PMID- 10730782 TI - Pityriasis rubra pilaris treated with acitretin and narrow-band ultraviolet B (Re TL-01) PMID- 10730783 TI - Lichen planus with xanthomatous change in a patient with primary biliary cirrhosis. PMID- 10730784 TI - Cerebrotendinous xanthomatosis: report of a case. PMID- 10730785 TI - Keratoacanthoma arising in hypertrophic lichen planus. PMID- 10730786 TI - Naevoid hyperkeratosis of the nipple and areola in a man. PMID- 10730787 TI - Hepatocellular adenomatosis presenting as prurigo. PMID- 10730788 TI - Fulminating septicaemia caused by Vibrio vulnificus. PMID- 10730789 TI - A case of cold abscess due to disseminated tuberculosis in an AIDS patient. PMID- 10730790 TI - Exacerbation of asthma by isotretinoin. PMID- 10730791 TI - Photosensitizing drugs may lower the narrow-band ultraviolet B (TL-01) minimal erythema dose. PMID- 10730792 TI - Reactive perforating collagenosis associated with underlying malignancy. PMID- 10730793 TI - Vascular endothelial growth factor and basic fibroblast growth factor in melanoma. PMID- 10730794 TI - Television as a venue for cancer education: the general hospital experience. PMID- 10730795 TI - A multidimensional approach to breast cancer education. AB - BACKGROUND: This study was undertaken to present a multidimensional breast cancer education package (BCEP) to medical students in an effort to improve breast cancer education. METHODS: The students were exposed to a four-part BCEP consisting of a hands-on structured clinical instruction module (SCIM), a lecture, a problem-based learning (PBL) small-group discussion, and a written manual. Each component was evaluated with a questionnaire. Students responded to the items using a five-point Likert scale ranging from 1 (strongly disagree) to 5 (strongly agree). RESULTS: The mean overall evaluations for the BCEP components were: SCIM, 4.6; lecture, 4.0; manual 3.8, and PBL discussion, 3.6. Highly rated qualities of the SCIM included organization (4.7), faculty preparedness (4.8), and opportunity to practice skills (4.5). The students agreed that the lecture (4.1), manual (3.8), and PBL discussion (4.2) had prepared them for the SCIM. CONCLUSION: This innovative BCEP effectively improved students' understanding of breast cancer. PMID- 10730796 TI - Training in clinical breast examination as part of a general surgery core curriculum. AB - BACKGROUND: Early detection of breast cancer relies on a multidisciplinary approach that includes patient breast self-examination, radiographic studies, and clinical breast examination (CBE). This study was undertaken to assess the value of formal CBE instruction by the surgery department using solicone breast models. METHODS: Thirty students were randomized in their first week of junior surgical clerkship to undergo or forgo a one-hour CBE retraining session. They were subsequently evaluated on technical competence and the ability to detect masses in an opaque silicone breast model. These skills were reassessed one month later. RESULTS: The students who underwent the teaching session performed significantly better than the control group in both the early (scores 1.23 vs 2.67, p < 0.05) and the late (scores 0.15 vs 2.14, p < 0.05) testing sessions. CONCLUSIONS: These data suggest significant improvement in CBE in students receiving formalized instruction. Further evaluation is needed prior to incorporation of this technique into the surgery core curriculum. PMID- 10730797 TI - Changing physicians' attitudes toward self-help groups: an educational intervention. AB - BACKGROUND: Members of self-help groups (SHGs) for support of cancer patients are concerned that physicians are skeptical about these groups and see them as potentially harmful. The purpose of this study was to assess family physicians' attitudes towards self-help groups and see whether these could be changed through an educational intervention. METHODS: A questionnaire assessing attitudes toward SHGs was mailed to 1,422 eligible Ontario family physicians, to which 911 responded (64% response rate). Responders were sent an educational package consisting of an article about self-help groups, a list of local cancer self-help groups, and a follow-up questionnaire. RESULTS: The study was completed by 584/911 family physicians (64%). After being exposed to educational material, the physicians were more positive about the helpfulness of SHGs (p = 0.021), and less concerned about SHGs' being harmful (p = 0.003). They were more positive about the potential for SHGs to provide participants with opportunities for: sharing information (p = 0.004), bonding with other patients (p < 0.001), feeling understood (p = 0.004), sharing common experiences (p = 0.004), providing hope (p < 0.001), sharing laughter (p = 0.001), becoming more assertive (p < 0.001), communicating with health professionals (p = 0.04), dealing with issues related to death and dying (p = 0.005), advocacy (p = 0.01), and overcoming isolation (p = 0.002). They were less concerned with the potential for SHGs to provide misinformation (p = 0.003), the negative effects of associating with the very ill (p = 0.002), dwelling on illness (p = 0.002), or cultivating false hope (p = 0.001). CONCLUSION: Having family physicians complete a questionnaire, followed by educational material specific to their concerns, changed their attitudes toward self-help groups. Further study is needed to see whether behavioral changes resulted. PMID- 10730798 TI - Sources of information used by patients to learn about chemotherapy side effects. AB - BACKGROUND: Little is known about the relative importance of specific information sources patients use to obtain knowledge about treatment side effects. The authors examined information sources used to learn about side effects, why patients believe they will experience some but not others, and the meanings side effects have in terms of treatment efficacy. METHODS: Before treatment, 31 ovarian cancer patients and 81 men and women with a variety of cancer diagnoses completed a questionnaire assessing their expectations about experiencing specific side effects of chemotherapy and information sources used. RESULTS: The doctor or nurse was the most frequently cited source of side-effect information, with readings second. While most thought they would get certain side effects because the doctor or nurse had said so, most instinctively believed they would not get others. CONCLUSIONS: Patients relied on medical and non-medical information sources. Further research could examine other sources for their influences on information-seeking activities. PMID- 10730799 TI - An analysis of printed breast cancer information for African American women. AB - BACKGROUND: The 1995-1998 Delta Project was designed to increase breast cancer screening among disadvantaged African American women with limited literacy skills by educating their health care professionals about breast health. The research team intended to provide onsite training and appropriate educational materials; however, they found no suitable materials. This article presents the results of an assessment of available materials and defines the need for suitable materials. METHODS: Nineteen organizations that develop cancer-related publications submitted materials intended for African American audiences. Sixty-one documents were examined for readability and cultural sensitivity. The Flesch Reading Ease (FRE), Flesch-Kincaid (F-K), and Cultural Sensitivity Assessment Tools (CSAT) were used in testing. RESULTS: The mean FRE score of 65 yielded a F-K mean grade level of 7.5 (desired level: 3.5). Using CSAT, 16 documents (26%) were eliminated because they had no visuals. Twenty-two publications (37%) were culturally sensitive for all audiences and 19 (31%) were for white audiences. Four (6%) pieces specifically addressed African American women. CONCLUSIONS: Printed educational materials on breast cancer do not adequately provide information to undereducated, economically disadvantaged African American women. PMID- 10730800 TI - Native American recruitment into breast cancer screening: the NAWWA project. AB - BACKGROUND: American Indian women's historically low breast cancer incidence and mortality rates have gradually increased such that in many parts of the United States they equal "U.S. All Races" rates. Thus, American Indian women need screening to maintain their low rates. METHODS: In an outreach program, local American Indian women were trained as lay health advisers, "Native Sisters," to locate and contact American Indian women in the Denver metropolitan area and provide education and encouragement to increase participation in mammography screening. Participation was monitored and interviews collected descriptive information and information about risk factors for breast cancer. An interrupted time-series design was used to assess changes in mammography participation. RESULTS: The NAWWA program increased recruitment of American Indian women (p < 0.05). Women recruited by the Native Sisters were more likely to be currently on hormone replacement therapy and to be menopausal. CONCLUSIONS: The lay health adviser program was effective in recruiting American Indian women to have screening mammography. Barriers to participation were complex and often involved cultural values and beliefs. PMID- 10730801 TI - Black cosmetologists promoting health program: pilot study outcomes. AB - BACKGROUND: African Americans suffer a disproportionate burden of illness and premature mortality. METHODS: A health education program delivered via cosmetologists was pilot tested as a supplement to other programs seeking to reach this community with information designed to remedy this inequality. Eight cosmetologists were randomized to either an active or a passive educational intervention arm, with the active arm (experimental arm) focused on breast cancer early detection. RESULTS: Both cosmetologists and clients found this an acceptable intervention. Nearly all women in the study demonstrated that they had heard the mainstream messages about the value of breast cancer early detection, but a considerable proportion appeared not to realize breast cancer's high level of morbidity and mortality within their own community. CONCLUSION: The results suggest this approach is worthy of further evaluation. PMID- 10730803 TI - Rural Mexican American men's attitudes and beliefs about cancer screening. AB - INTRODUCTION: Mexican American men are thought to receive fewer cancer screening services for prostate and colon cancer than the general male population; however, little is known about this group. METHODS: A population-based cross-sectional study was conducted to examine the knowledge, beliefs, attitudes, and use of cancer screening services among a sample of high-acculturated Mexican American, low-acculturated Mexican American, and non-Mexican American white men in Washington State. A total of 158 men (127 Mexican American; 31 non-Mexican American white) completed an in-person interview. RESULTS: The Mexican American men were more likely to report employment in agriculture and had completed fewer years of formal education compared with the non-Mexican American men, and were more likely than their non-Mexican American counterparts to have little knowledge about the causes of cancer and the need for cancer prevention practices. Further, the Mexican American men were more likely to report avoidant and fatalistic attitudes about cancer. CONCLUSION: Future research should address variables that are related to the low level of cancer screening among Mexican American men. PMID- 10730802 TI - A pilot study using nurse education as an intervention to increase skin self examination for melanoma. AB - BACKGROUND: A pilot study of an intervention using nurse education for skin self examination (SSE) was conducted in order to determine specific factors that would be important for the design of a larger intervention. METHODS: Seventy-five subjects completed a pretest, a posttest, a thorough skin examination by a dermatologist, and an educational session by a nurse. RESULTS: Results showed that the subjects increased the frequency of SSE to a predetermined optimal frequency of once every one to two months. Increases in optimal SSE were associated with increases in knowledge about melanoma (p = 0.006). CONCLUSIONS: Before intensive campaigns are launched to improve SSE, models incorporating theories of behavioral change should be tested. PMID- 10730804 TI - Screening to the converted: an educational intervention in African American churches. AB - BACKGROUND: African American women have higher incidences of breast and cervical cancers and African American men present with more advanced stages of colon and prostate cancers than do their non-African American counterparts. Since the church is central to the organization of the African American community, the authors set out to determine whether a church-directed educational project could influence parishioners to obtain cancer screening. METHODS: Three African American churches having memberships of 250, 500, and 1,500, respectively, were selected for their different socioeconomic strata: one congregation was composed mostly of working poor, the second was more affluent, and the third consisted primarily of retirees. During a five-week summer period, appropriate literature, health fairs, testimonials by cancer survivors, and visits by representatives of the medical community were used to increase awareness of cancer screening. Surveys regarding cancer-screening behaviors were distributed at the end of church services. Using the guidelines established by the American Cancer Society, individual recommendations for screening examinations were developed and sent to parishioners based on their survey responses. RESULTS: Of 437 parishioners surveyed (73% female, 27% male), 75% were 40 years old or older. Many reported up to-date screening for breast (84%), cervical (78%), colon (62%), and prostate (89%) cancers. The results were remarkably similar in all three churches. Telephone follow-up seven months after the survey directed at the 120 parishioners identified as noncompliant for at least one cancer screening revealed that 49% had obtained the appropriate screenings. CONCLUSIONS: These African American churchgoers were well screened compared with estimated national averages, possibly due to previous efforts of the activist ministers in the churches selected. The message for cancer screening is heeded when delivered through the African American church. PMID- 10730805 TI - Hepatitis B knowledge among Vietnamese immigrants: implications for prevention of hepatocellular carcinoma. AB - BACKGROUND: Vietnamese have higher liver cancer rates than any other racial/ethnic group in the United States. Approximately 80% of liver cancers are etiologically associated with hepatitis B virus (HBV) infection, which is endemic in Southeast Asia. METHODS: A telephone survey of randomly selected Vietnamese households (n = 75) was conducted during 1998 to examine HBV knowledge among Seattle's Vietnamese community. The questionnaire included items related to the transmission of HBV, the possible sequelae of infection, and disease prevention. RESULTS: The response rate was 70% among reachable and eligible households. Prior to being provided with a description of the disease, two thirds of our respondents had heard of HBV infection. Less than 60% knew that asymptomatic individuals can transmit the disease to others. Most thought that HBV infection can cause liver cancer (63%) and death (80%). However, only a minority knew that infection can be lifelong (38%) and incurable (22%). Finally, 28% had never heard of the HBV vaccine. There were significant associations between knowledge and educational level as well as home ownership. CONCLUSIONS: The findings suggest that Vietnamese immigrants have low levels of knowledge about HBV infection, and indicate a need for targeted educational interventions aimed at reducing HBV related liver cancer mortality. PMID- 10730806 TI - Small good things. PMID- 10730807 TI - Evaluation of the quality of self-education mammography material available for patients on the Internet. AB - RATIONALE AND OBJECTIVES: The Internet offers many advantages for educating patients but has no standards for publication. This limitation could negatively affect patient care. The purpose of this study was to evaluate the quality of information on mammography that a patient could find on the Internet. MATERIALS AND METHODS: Three search utilities were used to research the term "mammography." For each utility, the first 50 addresses for Web pages were evaluated (or all the addresses, if fewer than 50 were returned). Web sites selected contained information that could guide an asymptomatic woman in deciding whether to undergo screening mammography. These sites were then evaluated for whether they indicated their sponsorship, authorship, the currency of information, and references. Sites were also noted if they advised women older than 50 years to undergo screening mammography at intervals of longer than 1 year or otherwise severely diminished the role of mammography. RESULTS: Thirty-eight Web sites were identified. Ten indicated authorship, 29 indicated the currency of the information, and 27 provided references. All of the Web sites indicated sponsorship. Three sites recommended screening mammography at intervals of longer than 1 year for women 50 years of age or older. Two sites suggested that mammography is not substantially more sensitive than physical examination. CONCLUSION: Many Web sites do not meet the standards for disseminating information required in professional peer reviewed journals. Some Web sites contain statements that might lead asymptomatic women over age 50 years to delay screening mammography or to undergo screening at intervals of longer than 1 year. PMID- 10730808 TI - Fast algorithm for soft straightening of the colon. AB - RATIONALE AND OBJECTIVES: In this study, the authors developed a fast algorithm for soft straightening of the colon with computed tomographic data that greatly accelerates the unraveling process based on the interpolation of representative electric force lines. MATERIALS AND METHODS: Each curved cross section of the colon is defined by electric force lines of a common origin on an electrically charged central path and is constructed by interpolating most of these force lines from a limited number of representative force lines that are traced directly. Both a synthetic colon phantom and a colon in a living patient were used to demonstrate the feasibility of the fast interpolation algorithm compared with direct implementation for soft straightening of the colon. RESULTS: The interpolation-based soft-straightening algorithm ran approximately 40 times faster than the direct implementation of the electric field-based soft straightening algorithm. CONCLUSION: The fast algorithm for soft straightening of the colon has potential for use in computed tomographic colonography. PMID- 10730809 TI - Improving the quality of care through routine teleradiology consultation. AB - RATIONALE AND OBJECTIVES: The hypotheses of this study were as follows: (a) University subspecialty radiologists can provide consultations effectively to general radiologists as part of routine clinical operations; (b) these consultations will improve the quality of the final radiologic report; and (c) the consultations will improve the care process and may save money, as well. MATERIALS AND METHODS: For 2,012 consecutive computed tomographic or magnetic resonance (MR) imaging studies, the initial interpretations provided by radiology generalists were subsequently reviewed by specialists, with a final consensus report available. "Truth" was established by final consensus reports. To control for potential bias, 150 adult MR imaging and 250 pediatric radiologic studies were interpreted initially by specialists and then by generalists. Again, truth was established by final consensus reports. RESULTS: There was disagreement between generalist and specialist radiologist interpretations in 427 (21.2%) of the cases reviewed. These disagreements were stratified further by independent specialists, who graded them as important, very important, or unimportant. Differences were considered important or very important in 99% of the cases reviewed. CONCLUSION: Consultations by subspecialty radiologists improved the quality of the radiology reports studied and, at least in some cases, improved the process of care by eliminating unnecessary procedures or suggesting more specific follow-up examinations. The consultation services can be provided cost effectively from the payer's perspective and may save additional costs when unnecessary procedures can be eliminated. PMID- 10730811 TI - Emergency department coverage by academic departments of radiology. AB - RATIONALE AND OBJECTIVES: The purpose of this study was to survey academic radiology departments to determine how emergency radiology coverage is handled and whether there are any prerequisites for those individuals providing this coverage. MATERIALS AND METHODS: The authors developed a simple two-page survey and sent it to a total of 608 program directors, chiefs of diagnostic radiology, chairpersons, and chief residents at academic departments of radiology. RESULTS: Of the 608 surveys sent, 278 (46%) were returned. More than half of the departments have an emergency radiology section that provides "wet read" coverage during the day, and most academic departments cover the emergency department during the night and on weekends. Nighttime and weekend coverage is handled mostly by residents. Most departments give time off for lunch, with few other prerequisites for faculty who provide emergency coverage. Sixty percent of the departments have teleradiology capability, and many use it for emergency department coverage. CONCLUSION: These results can serve as the basis for discussion and comparison with other institutions regarding a variety of aspects of emergency department coverage. PMID- 10730810 TI - Characteristics and pitfalls of contrast-enhanced, T1-weighted magnetization transfer images of the brain. AB - RATIONALE AND OBJECTIVES: This study was undertaken to clarify the difference in signal pattern on contrast material-enhanced T1-weighted magnetic resonance (MR) magnetization transfer (MT) images between enhancing and nonenhancing lesions in various intracranial diseases and to determine the necessity of nonenhanced MT images for evaluating lesional contrast enhancement. MATERIALS AND METHODS: MR images of 116 patients who underwent nonenhanced T1-weighted imaging, nonenhanced MT imaging, and contrast-enhanced MT imaging were reviewed. The increase in signal intensity of lesions relative to normal brain was compared between nonenhanced T1-weighted images and contrast-enhanced MT images. Signal intensity of lesions was compared with that of the striate nucleus and white matter on contrast-enhanced MT images. True enhancement was determined by comparison with nonenhanced MT images. RESULTS: In all, 143 lesions, including 86 enhancing and 57 nonenhancing lesions, were identified among 63 patients. Almost all (99%) of the enhancing lesions were hyperintense to striate nucleus on contrast-enhanced MT images, and most (>87%) showed moderate to marked signal intensity increase from nonenhanced T1-weighted images to contrast-enhanced MT images. Most (>95%) of the nonenhancing lesions showed mild or no increase in relative signal intensity, and most (75%) were iso- or hypointense to striate nucleus on contrast enhanced MT images. A few nonenhancing lesions (4%-6%), however, showed increase in signal intensity that was indistinguishable from true enhancement without comparison to non-enhanced MT images. CONCLUSION: Nonenhanced MT images should be obtained to assess pathologic enhancement accurately. PMID- 10730812 TI - Evaluation of a mandatory radiology resident research rotation. AB - RATIONALE AND OBJECTIVES: This purpose of this study was to determine if a 1 month, mandatory research rotation results in resident interest and productivity in research. MATERIALS AND METHODS: A survey composed primarily of closed-end questions was constructed to evaluate the opinions and experience of 24 residents who completed the research rotation during a 3 1/2-year period. RESULTS: All 24 residents filled out the survey. Seventeen residents did clinical projects, two did bench research, and two did projects that were socioeconomic or legal in nature. Thirteen residents presented data at a meeting, and three of these presentations won awards. Resident research projects resulted in 18 manuscripts at the time of the survey (seven published, eight in preparation, and three in revision or review). Seventeen residents thought the experience was worthwhile and the research month should be continued; two residents did not. Ten residents reported that in retrospect, they would have done the rotation even if it had not been required, and eight reported the experience increased their appreciation of the value of research in medicine. CONCLUSION: A 1-month, mandatory resident research rotation produces resident interest in research and enhances departmental research productivity. PMID- 10730813 TI - Report of the 1997 SCAROP survey on resident training. Society of Chairmen of Academic Radiation Oncology Programs. AB - RATIONALE AND OBJECTIVES: Members of the Society of Chairmen of Academic Radiation Oncology Programs (SCAROP) were surveyed in November 1997 to evaluate the current status of radiation oncology training in the United States and to help determine how it should be carried out in the coming decade. MATERIALS AND METHODS: A detailed questionnaire was sent to all members of SCAROP; 68 of 82 questionnaires were returned, for a response rate of 83%. RESULTS: The responses to the survey show a serious shortage of radiation oncologists in university settings, despite an apparent surplus in private practice. Although recent changes in health care have added additional clinical responsibilities for radiation oncologists in university practices, approximately 75% of the chairpersons answering the survey continue to give their faculty protected time for research. Even with additional research and teaching responsibilities, the average radiation oncologist in university practice saw 206 patients per year in 1997, a number similar to that reported by the Patterns of Care Study for radiation oncologists overall. Approximately two-thirds of respondents believe that academic chairs should strive to have all clinical faculty members participating in research. Nevertheless, most think that basic research is better performed by dedicated researchers with PhD degrees rather than radiologists with MD degrees. Most respondents believe that the training programs adequately prepare radiation oncologists for a career in academic medicine but do not provide good training in research. Eighty-four percent agreed that resident performance on the American Board of Radiology examination should be considered in the accreditation of residency programs in radiation oncology but should not be the major criterion. CONCLUSION: There is a shortage of academic radiation oncologists in the United States despite the large number of radiation oncologists completing training. This probably is due to a variety of factors, including a relatively small pool of candidates for academic positions, increasing demands for performance from academic physicians (to see more patients, perform research, publish, write grants, and teach), and competition from the private sector for recruitment of these individuals. PMID- 10730814 TI - Quality, access, and change: proceedings of the 110th annual meeting of the Association of American Medical Colleges. PMID- 10730815 TI - Imaging findings of chronic pulmonary embolism. PMID- 10730816 TI - Empowering the online educator. PMID- 10730817 TI - Advancing toward an institute for imaging: a status report from the Academy of Radiology Research. PMID- 10730818 TI - Mitochondria, oxidative stress and aging. AB - In the eighties, Miquel and Fleming suggested that mitochondria play a key role in cellular aging. Mitochondria, and specially mitochondrial DNA (mtDNA), are major targets of free radical attack. At present, it is well established that mitochondrial deficits accumulate upon aging due to oxidative damage. Thus, oxidative lesions to mtDNA accumulate with age in human and rodent tissues. Furthermore, levels of oxidative damage to mtDNA are several times higher than those of nuclear DNA. Mitochondrial size increases whereas mitochondrial membrane potential decreases with age in brain and liver. Recently, we have shown that treatment with certain antioxidants, such as sulphur-containing antioxidants, vitamins C and E or the Ginkgo biloba extract EGb 761, protects against the age associated oxidative damage to mtDNA and oxidation of mitochondrial glutathione. Moreover, the extract EGb 761 also prevents changes in mitochondrial morphology and function associated with aging of the brain and liver. Thus, mitochondrial aging may be prevented by antioxidants. Furthermore, late onset administration of certain antioxidants is also able to prevent the impairment in physiological performance, particularly motor co-ordination, that occurs upon aging. PMID- 10730819 TI - Free radical scavenging and copper chelation: a potentially beneficial action of captopril. AB - Captopril (CpSH), an angiotensin converting enzyme (ACE) inhibitor, is reported to provide protection against free-radical mediated damage. The purpose of this study was to investigate, by means of pulse radiolysis technique, the behaviour of CpSH towards radiation-induced radicals in the absence and in the presence of copper(II) ions, which can play a relevant role in the metal catalysed generation of reactive oxygen species. The results indicate that the -SH group is crucial in determining the radical scavenging action of CpSH and the nature of the resulting CpSH transient products in the absence or in the presence of oxygen. In the presence of Cu(II), the -SH group is still involved in the biological action of the molecule participating both in the one-electron reduction of Cu(II) with formation of CpSSCp, and in Cu(I) chelation. This conclusion is supported by the Raman spectroscopic data which allow to identify the CpSH sites involved in the copper complex at different pH. These results suggest that CpSH may potentially inhibit oxidative damage both through free radical scavenging and metal chelation. Considering the low CpSH concentration in vivo, the metal chelation mechanism, more than the direct radical scavenging, could play the major role in moderating the toxicological effects of free radicals. PMID- 10730820 TI - Inhibitory effects of post low dose gamma-ray irradiation on ferric nitrilotriacetate-induced mice liver damage. AB - We studied the effects of a single post whole-body low-dose irradiation (50 cGy of gamma-ray) on mice with ferric nitrilotriacetate (Fe3+-NTA)-induced transient hepatopathy. As a result, low-dose irradiation accelerated the rate of recovery. Based on the changes in glutamic oxaloacetic transaminase (GOT) activities, glutamic pyruvic transaminase (GPT) activities and lipid peroxide levels, it was shown that hepatopathy was improved by low-dose irradiation 3 h after Fe3+-NTA administration. This may be because of the enhancement of antioxidant agents such as total glutathione (GSH + GSSG), glutathione peroxidase (GPX), glutathione reductase (GR) and gamma-glutamylcysteine synthetase (gamma-GCS) by low-dose irradiation. These findings suggest that low-dose irradiation relieved functional disorders at least in the livers of mice with active oxygen species related diseases. PMID- 10730821 TI - Regulation of iNOS mRNA levels in endothelial cells by glutathione, a double edged sword. AB - Both inducible nitric oxide synthase (iNOS) and glutathione are important mediators in various physiological and pathological conditions in humans. In human endothelial cells the intracellular glutathione levels were modulated by N acetyl-L-cysteine (NAC), a precursor of glutathione and 1,3-bis(chloroethyl)-1 nitrosourea (BCNU), an inhibitor of glutathione reductase. BCNU significantly decreased reduced glutathione (GSH) but increased oxidized glutathione (GSSG) whereas NAC markedly elevated GSH with a relatively small increase in GSSG. Appropriate concentrations of GSH and GSSG increase the expression of iNOS gene. However, either GSH or GSSG at a too high concentration inhibits its expression, indicating that iNOS gene is fine tuned by the metabolites of glutathione cycle. The changes of iNOS mRNA steady state levels by the glutathione metabolites were associated with a similar alteration in its gene transcription and NF-kappaB activity. BCNU at high concentrations also shortens the half-life of iNOS mRNA, suggesting a role of GSSG in the stability of the iNOS gene. Thus, the change of glutathione levels in vitro can regulate iNOS mRNA steady state levels in a bi phasic manner in human endothelial cells. PMID- 10730822 TI - Is allantoin in serum and urine a useful indicator of exercise-induced oxidative stress in humans? AB - To assess whether allantoin levels in serum and urine are influenced by exhaustive and moderate exercise and whether allantoin is a useful indicator of exercise-induced oxidative stress in humans, we made subjects perform exhaustive and moderate (100% and 40% VO2max) cycling exercise and examined the levels of allantoin, thiobarbituric acid reactive substances (TBARS) and urate in serum and urine. Immediately after exercise at 100% VO2max, the serum allantoin/urate ratio was significantly elevated compared with the resting levels while the serum urate levels was significantly elevated 30 min after exercise. The serum TBARS levels did not increase significantly compared with the resting levels. Urinary allantoin excretion significantly increased during 60 min of recovery after exercise, however, urinary urate excretion decreased significantly during the same period. The urinary allantoin/urate ratio also rapidly increased during 60 min of recovery after exercise. Urinary TBARS excretion decreased during the first 60 min of the recovery period and thereafter significantly increased during the latter half of the recovery period. On the contrary, after 40% VO2max of exercise, no significant changes in the levels of urate, allantoin and TBARS in serum or urine were observed. These findings suggest that allantoin levels in serum and urine may reflect the extent of oxidative stress in vivo and that the allantoin which appeared following exercise may have originated not from urate formed as a result of exercise but from urate that previously existed in the body. Furthermore, these findings support the view that allantoin in serum and urine is a more sensitive and reliable indicator of in vivo oxidative stress than lipid peroxidation products measured as TBARS. PMID- 10730823 TI - Autoxidation of naphthohydroquinones: effects of pH, naphthoquinones and superoxide dismutase. AB - The rates of autoxidation of a number of pure naphthohydroquinones have been determined, and the effects of pH, superoxide dismutase (SOD) and of the parent naphthoquinone on the oxidation rates have been investigated. Most compounds were slowly oxidised in acid solution with the rates increasing with increasing pH, although 2-hydroxy-, 2-hydroxy-3-methyl- and 2-amino-1,4-naphthohydroquinone were rapidly oxidised at pH 5 and the rates of oxidation of these substances were comparatively unresponsive to changes in pH. At pH 7.4, autoxidation rates decreased in the order 2,3-dichloro-1,4-naphthohydroquinone > 5-hydroxy > 2-bromo > 2-hydroxy-3-methyl > 2-amino > 2-hydroxy > 2-methoxy > 2,3-dimethoxy > 2,3 dimethyl > 2-methyl > unsubstituted hydroquinone. The autoxidation rates of the alkyl, alkoxy, hydroxy and amino derivatives were decreased in the presence of SOD, but this enzyme had no effect on the rate of autoxidation of the 2,3 dichloro and 2-bromo derivatives while that of the 5-hydroxy derivative was increased. The rates of autoxidation of all compounds except the halogen derivatives and 5-hydroxy-1,4-naphthohydroquinone were increased by addition of the parent naphthoquinone, and quinone addition partially or completely overcame the inhibitory effect of SOD. There is evidence that the reduction of quinones to hydroquinones in vivo may lead either to detoxification or to activation. This may be due to differences in the rate or mechanism of autoxidation of the hydroquinones that are formed, and the data gained in this study will provide a framework for testing this possibility. PMID- 10730824 TI - Antioxidative property of T-0970, a new ureidophenol derivative. AB - We investigated the antioxidative property of T-0970, a newly synthesized ureidophenol derivative. The inhibitory effect of T-0970 on spontaneous lipid peroxidation in rat brain was 10 times greater than those of well-known antioxidants such as butylhydroxytoluene (BHT), probucol and alpha-tocopherol. T 0970 also showed dose-dependent free radical scavenging activities in vitro for both superoxide anions and hydroxyl radicals. The radical-scavenging potencies of T-0970 were about 10-30 times stronger than those of BHT. We evaluated the in vivo antioxidative ability of T-0970 in the animal model of acute oxidative tissue injury in rats. Intraperitoneal injection of ferric nitrilotriacetate (Fe/NTA) caused an acute and remarkable increase in the level of thiobarbituric acid-reactive substances (TBARS) in both plasma and the liver, and also resulted in a considerable elevation of the plasma levels of GOT and GPT indicative of hepatic injury. Both oral and intravenous administration of T-0970 dose dependently depressed these diagnostic parameters. These results indicate that T 0970 may have a therapeutic potential in various diseases associated with oxidative tissue injury. PMID- 10730825 TI - Pitfalls of using lucigenin in endothelial cells: implications for NAD(P)H dependent superoxide formation. AB - Since an increased endothelial superoxide formation plays an important role in the pathogenesis of endothelial dysfunction its specific detection is of particular interest. The widely used superoxide probe lucigenin, however, has been reported to induce superoxide under certain conditions, especially in the presence of NADH. This raises questions as to the conclusion of a NAD(P)H oxidase as the major source of endothelial superoxide. Using independent methods, we showed that lucigenin in the presence of NADH leads to the production of substantial amount of superoxide (approximately 15-fold of control) in endothelial cell homogenates. On the other hand, these independent methods revealed that endothelial cells without lucigenin still produce superoxide in a NAD(P)H-dependent manner. This was blocked by inhibitors of the neutrophil NADPH oxidase diphenyleniodonium and phenylarsine oxide. Our results demonstrate that a NAD(P)H-dependent oxidase is an important source for endothelial superoxide but the latter, however, cannot be measured reliably by lucigenin. PMID- 10730826 TI - Absorption of ferulic acid from low-alcohol beer. AB - Flavonoids and monophenolic compounds have been well-described over recent years for their properties as antioxidants and scavengers of reactive oxygen and nitrogen species. A number of epidemiological studies implicate a role for flavonoids in reducing the risk of coronary heart disease. In particular, the focus has been on flavonol-rich fruit and vegetables and flavonoid-rich beverages, especially tea and red wine. Mechanisms of protection are unclear since the absorption, distribution, metabolism and elimination of dietary phenolics have not yet been extensively investigated. Here we report the bioavailability of ferulic acid, 4-hydroxy-3-methoxy-cinnamic acid, the major hydroxycinnamate in beer. Studies of the pharmacokinetics of urinary excretion of ferulic acid from low alcohol beer consumption in humans have been undertaken. The results show that ferulic acid is absorbed with a peak time for maximal excretion of ca. 8 h and the mean cumulative amount excreted is 5.8 +/- 3.2 mg. These findings are consistent with the uptake of ferulic acid from dietary sources, such as tomatoes, and suggest that ferulic acid is more bioavailable than individual dietary flavonoids and phenolics so far studied. PMID- 10730827 TI - The effects of graded doses of endothelin-1 on coronary perfusion pressure and vital organ blood flow during cardiac arrest. AB - BACKGROUND: Endothelin-1 (ET-1) is a potent vasoconstrictor and has been shown to improve coronary perfusion pressure (CPP) during arrest. The effects of ET-1 on organ blood flow during arrest have not been extensively studied. OBJECTIVE: To investigate the effects of ET-1 on myocardial and cerebral blood flow during cardiac arrest. METHODS: Sixty immature swine were anesthetized and instrumented. The animals were randomized to receive one of three doses of ET-1 (50, 150, or 300 microg) or placebo with/without standard-dose epinephrine (SDE) during cardiac arrest. After a 10-minute period of no-flow ventricular fibrillation (VF), cardiopulmonary resuscitation (CPR) was performed for 3 minutes, followed by drug administration. Placebo or SDE was given every 3 minutes. Myocardial and cerebral blood flow was measured using a fluorescent microsphere technique. RESULTS: Prearrest and CPR variables were not different between groups. Beginning 4 minutes after giving 300 microg ET-1 with or without SDE, CPP was significantly increased compared with SDE alone. Total myocardial blood flow following ET-1 administration was no different than myocardial blood flow following SDE alone. Cerebral blood flow increased 3.5 minutes after administration of 300 microg ET-1 with SDE and reached significance 9.5 minutes after drug administration when compared with SDE alone [92.5 (48.8-117.9) vs 15.6 (7.7-23) mL/min/100 g]. CONCLUSIONS: Three hundred microg ET-1 with SDE increases CPP and improves cerebral blood flow but does not improve myocardial blood flow during cardiac arrest. The peripheral effects of ET-1 significantly improve CPP and cerebral blood flow, but myocardial blood flow is not increased due to coronary vasoconstriction. PMID- 10730828 TI - Octylcyanoacrylate for the treatment of contaminated partial-thickness burns in swine: a randomized controlled experiment. AB - OBJECTIVE: To compare infection and reepithelialization rates of contaminated second-degree burns treated with octylcyanoacrylate (OCA), silver sulfadiazine (SSD), polyurethane (PU) film, and dry gauze (control; C) in swine. METHODS: Eighty standardized burns were created by applying an aluminum bar preheated to 80 degrees C to the backs and flanks of young pigs for 20 seconds. All burns were immediately contaminated with 0.1 mL of Staphylococcus aureus 10(5)/mL and randomly treated with OCA spray, SSD, PU, or dry gauze (C). Full-thickness biopsies were taken at 3, 7, and 14 days for blinded histopathologic evaluation using hematoxylin and eosin (H&E)-stained slides. Burns were considered infected in the presence of interstitial reticular dermal neutrophils containing bacteria (intraobserver agreement, kappa = 1.00). Quantitative wound cultures were performed on a second day-3 specimen. RESULTS: At day 3, wound infection rates were 30% (OCA), 50% (SSD), 55% (PU), and 50% (C); p = 0.40 (Kruskal-Wallis test). At day 7, infection rates were 35% (OCA), 85% (SSD), 70% (PU), and 65% (C); p = 0.01. Median bacterial counts at day 3 were 6,500 (OCA), 20,000 (SSD), 1,000,000 (PU), and 650,000 (C); p = 0.29. The proportion of completely reepithelialized wounds at day 14 were 75% (OCA), 90% (SSD), 85% (PU), and 90% (C); p = 0.50. CONCLUSIONS: Treatment of contaminated partial-thickness burns with OCA spray resulted in fewer infections at one week than with the other three treatments. PMID- 10730829 TI - A combination of midazolam and ketamine for procedural sedation and analgesia in adult emergency department patients. AB - OBJECTIVE: To describe the clinical characteristics of a combination of midazolam and ketamine for procedural sedation and analgesia in adult emergency department (ED) patients. METHODS: This was a prospective, observational trial, conducted in the ED of an urban level II trauma center. Patients > or = 18 years of age requiring procedural sedation and analgesia were eligible, and enrolled patients received 0.07 mg/kg of intravenous midazolam followed by 2 mg/kg of intravenous ketamine. Vital signs were recorded at regular intervals. The adequacy of sedation, adverse effects, patient satisfaction, and time to reach discharge alertness were determined. Descriptive statistics were calculated using statistical analysis software. RESULTS: Seventy-seven patients were enrolled. Three were excluded due to protocol violations, three due to lack of documentation, and one due to subcutaneous infiltration of ketamine, leaving 70 patients for analysis. The average age was 31 years, and 41 (59%) were female. Indications for procedural sedation and analgesia included abscess incision and drainage (66%), fracture/joint reduction (26%), and other (8%). The mean dose of midazolam was 5.6 +/- 1.4 mg and the mean dose of ketamine was 159 +/- 42 mg. The mean time to achieve discharge criteria was 64 +/- 24 minutes. Five patients experienced mild emergence reactions, but there were no episodes of hallucinations, delirium, or other serious emergence reactions. Eighteen (25%) patients recalled dreaming while sedated; twelve (17%) were described as pleasant, two (3%) unpleasant, three (4%) both pleasant and unpleasant, and one (1%) neither pleasant nor unpleasant. There were four (6%) cases of respiratory compromise, two (3%) episodes of emesis, and one (1%) case of myoclonia. All of these were transient and did not result in a change in the patient's disposition. Only one (1%) patient indicated that she was not satisfied with the sedation regimen. CONCLUSIONS: The combination of midazolam and ketamine provides effective procedural sedation and analgesia in adult ED patients, and appears to be safe. PMID- 10730830 TI - Reliability and validity of a new five-level triage instrument. AB - OBJECTIVES: Triage is the initial clinical sorting process in hospital emergency departments (EDs). Because of poor reproducibility and validity of three-level triage, the authors developed and validated a new five-level triage instrument, the Emergency Severity Index (ESI). The study objectives were: 1) to validate the triage instrument against ED patients' clinical resource and hospitalization needs, and 2) to measure the interrater reliability (reproducibility) of the instrument. METHODS: This was a prospective, observational cohort study of a population-based convenience sample of adult patients triaged during 100 hours at two urban referral hospitals. Validation by resource use and hospitalization (criterion standards) and reproducibility by blinded paired triage assignments compared with weighted kappa analysis were assessed. RESULTS: Five hundred thirty eight patients were enrolled; 45 were excluded due to incomplete evaluations. The resulting cohort of 493 patients was 52% female, was 26% nonwhite, and had a median age of 40 years (range 16-95); overall, 159 (32%) patients were hospitalized. Weighted kappa for triage assignment was 0.80 (95% CI = 0.76 to 0.84). Resource use and hospitalization rates were strongly associated with triage level. For patients in category 5, only one-fourth (17/67) required any diagnostic test or procedure, and none were hospitalized (upper confidence limit, 5%). Conversely, in category 1, one of twelve patients was discharged (upper confidence limit, 25%), and none required fewer than two resources. CONCLUSIONS: This five-level triage instrument was shown to be both valid and reliable in the authors' practice settings. It reproducibly triages patients into five distinct strata, from very high hospitalization/resource intensity to very low hospitalization/resource intensity. PMID- 10730831 TI - Domestic violence and out-of-hospital providers: a potential resource to protect battered women. AB - OBJECTIVE: The primary objective was to determine the prevalence of domestic violence (DV) in a subset of women presenting to the Boston emergency medical services (EMS) system and to evaluate documentation. A secondary objective was to determine the rate of refusal of transport to the hospital for DV-positive patients, compared with the general population. METHODS: A retrospective chart review of ambulance run sheets from a nonconsecutive, convenience sample between July and December 1995 was performed. Women presenting with injury, obstetric/ gynecologic complaints, or psychiatric complaints were included. Records were reviewed, and labeled as positive, probable, suggestive, or negative for DV, based on a previously used classification system. A weighted kappa test was performed, and data were analyzed using chi-square and t-test. RESULTS: Among 1,251 charts reviewed, 876 met criteria for inclusion. The percentage of positive cases was 5.4% (95% CI = 3.9% to 6.9%), probable 10.8% (8.8% to 12.9%), suggestive 2.6% (1.6% to 3.7%), and negative 81.2% (78.6% to 83.6%). Among DV positive patients, the refusal to transport rate was 23.4% (11.3% to 35.5%), compared with a 7.1% (5.8% to 9.3%) rate for the entire study population (n = 876), and 4.7% for the general Boston EMS population during the same year. More DV patients presented during the night shift compared with other shifts. CONCLUSIONS: Domestic violence is common in this high-risk population. A substantial proportion of women in this population refuse transport to the hospital. Out-of-hospital personnel should be trained with the tools to identify and document DV, assess patient safety, offer timely resources, and empower victims to make choices. PMID- 10730832 TI - Return to the emergency department among elders: patterns and predictors. AB - OBJECTIVES: 1) To describe the pattern of return visits to the emergency department (ED) among elders over the six months following an index visit; 2) to identify the predictors of early return (within 30 days) and frequent return (three or more return visits in six months); and 3) to evaluate a newly developed screening tool for functional decline, Identification of Seniors At Risk (ISAR), with regard to its ability to predict return visits. METHODS: Subjects were patients aged 65 years or more who visited the EDs of four Canadian hospitals during the weekday shift over a three-month recruitment period. Excluded were patients who: could not be interviewed, due either to their medical conditions or to cognitive impairment, and no other informant was available; refused linkage of study data; or were admitted to hospital at the initial (index) visit. Measures made at the index ED visit included: 27 self-report screening questions on social, physical, and mental risk factors, medical history, use of hospital services, medications, and alcohol. Six of these questions comprised the ISAR scale. Return visits and diagnoses during the six months after the index visit were abstracted from the utilization database. RESULTS: Among 1,122 patients released from the ED, 492 (43.9%) made one or more return visits; 216 (19.3%) returned early and 84 (7.5%) returned frequently. Earlier returns were more likely than later returns to be for the same diagnosis (p = 0.003). Using logistic regression, hospitalization during the previous six months, feeling depressed, and certain diagnoses predicted both early and frequent returns. Also, a history of heart disease, having ever been married, and not drinking alcohol daily predicted early return; a history of diabetes, a recent ED visit, and lack of support predicted frequent use. CONCLUSIONS: In the first month after an ED visit, return rates are highest and are more likely to be for the same diagnosis. Both medical and social factors predict early and frequent returns to the ED; patients at increased risk of return can be quickly identified with a short, self report questionnaire. The ISAR screening tool, developed to identify patients at increased risk of functional decline, can also identify patients who are more likely to return to the ED. PMID- 10730833 TI - Prospective confirmation of low arrest rates among intoxicated drivers in motor vehicle crashes. AB - OBJECTIVE: Several states have legally sanctioned or mandated physician reporting of drivers who were driving while intoxicated (DWI). Valid prospective evidence demonstrating extremely poor performance of the criminal justice system seems ethically and scientifically essential if overriding public health considerations are to abrogate the fundamental principles of patient-physician confidentiality. No such evidence is available. The authors reasoned that poor performance of the judicial system would be most evident if drivers who were DWI were not arrested under conditions selected to optimize legal intervention. The authors therefore wished to estimate the unbiased proportion of DWI drivers brought to an emergency department (ED) under these optimized conditions who escape detection by law enforcement officials. METHODS: Prospective, consecutive cohort of drivers transported to an urban ED following a motor vehicle crash (MVC). Conditions selected to optimize legal intervention included: police at scene; inebriation of driver clinically evident to out-of-hospital personnel; and confirmatory blood ethanol level > or =100 mg/dL (> or =22 mmol/L). Main outcome measure was arrest for DWI. RESULTS: Of 294 drivers in MVCs, 270 had ethanol levels, of whom 18 met criteria for optimum likelihood of legal intervention. Of these, 22% were arrested for DWI (95% CI = 6% to 48%). Adjustment for missing data, under assumptions designed to maximize arrest frequency for DWI, did not materially alter these findings. No patients were lost to follow-up. CONCLUSION: These findings prospectively confirm that, even under conditions selected to optimize detection by law enforcement officials, only about one of every five drivers who were DWI and were brought to an ED following an MVC-and almost certainly no more than a minority-comes to the attention of the criminal justice system. PMID- 10730834 TI - Do diabetic patients have higher in-hospital complication rates when admitted from the emergency department for possible myocardial ischemia? AB - OBJECTIVE: To compare in-hospital complication rates for diabetic and nondiabetic patients admitted from the emergency department (ED) for possible myocardial ischemia. METHODS: This was a prospective, observational study of consecutive consenting patients presenting to a suburban university hospital ED during study hours with typical and atypical symptoms consistent with cardiac ischemia. Demographic, historical, and clinical data were recorded by trained research assistants using a standardized, closed-question, data collection instrument. Inpatient records were reviewed by trained data abstractors to ascertain hospital course and occurrence of complications. Final discharge diagnosis of acute myocardial infarction (AMI) was assigned by World Health Organization criteria. Categorical and continuous data were analyzed by chi-square and t-tests, respectively. All tests were two-tailed with alpha set at 0.05. RESULTS: There were 1,543 patients enrolled who did not have complications at initial presentation; 283 were diabetic. The rule-in rate for AMI was 13.8% for nondiabetic patients and 17.7% for diabetic patients (p = 0.09). Times to presentation were similar for nondiabetic vs diabetic patients [248 minutes (95% CI = 231 to 266) vs 235 minutes (95% CI = 202 to 269); p = 0.32]. Nondiabetic patients tended to be younger [56.6 years (95% CI = 55.8 to 57.4) vs 61.6 years (95% CI = 60.2 to 63.1); p = 0.001] and were less likely to be female (34.3% vs 48.1%; p = 0.001). The two groups had similar prevalences for initial electrocardiograms diagnostic for AMI (5.5% vs 7.4%; p = 0.21). There was no significant difference between nondiabetic and diabetic patients for the occurrence of the following complications after admission to the hospital: congestive heart failure (1.3% vs 1.1%, p = 0.77); nonsustained ventricular tachycardia (VT) (1.3% vs 1.2%, p = 0.93); sustained VT (1.2% vs 1.1%, p = 0.85); supraventricular tachycardia (1.7% vs 3.2%, p = 0.12); bradydysrhythmias (1.9% vs 1.1%, p = 0.33); hypotension necessitating the use of pressors (0.9% vs 1.1%, p = 0.76); cardiopulmonary resuscitation (0.2% vs 0.7%, p = 0.10); and death (0.3% vs 0.7%, p = 0.34). One or more complications occurred with similar frequencies for patients in the two groups (6.3% vs 5.7%; p = 0.70). CONCLUSIONS: No statistically significant difference was found in the postadmission complication rates for initially stable diabetic vs nondiabetic patients admitted for possible myocardial ischemia. Based on these results, the presence or absence of diabetes as a comorbid condition does not indicate a need to alter admitting decisions with respect to risk for inpatient complications. PMID- 10730835 TI - A continuous quality improvement approach to IL-372 documentation compliance in an academic emergency department, and its impact on dictation costs, billing practices, and average patient length of stay. AB - OBJECTIVE: To determine whether continuous quality improvement (CQI) methodology could improve and maintain IL-372 documentation compliance in an academic emergency department (ED). The impact on transcription costs, billing practices, and average patient length of stay was also analyzed. METHODS: Baseline IL-372 compliance data were collected and shared with staff during a multidisciplinary educational session. Faculty dictation became mandatory. Pocket-sized dictation templates were provided. A Documentation Improvement Committee monitored outcomes. Each month of the study period, a compliance officer reviewed approximately 100 records. The following indicators were monitored: IL-372 compliance rates, dictation rates, transcription costs, down-coding rates, percentage of billable records, and average patient length of stay. Individualized results were provided to faculty. RESULTS: During the ten-month study period, compliance rates increased from 60% to 100% (p-trend < 0.001), while dictation rates increased from 69% to 100% (p < 0.001). Rates of down coding adjustments improved from 54% to 2% (p-trend < 0.001). The percentage of billable records increased from 65% to 100% (p-trend < 0.001). Transcription costs increased a modest 16%. The average patient length of stay remained unchanged. CONCLUSION: The application of CQI methodology, combined with the availability of dictation, resulted in sustained improvement in IL-372 compliance. This was associated with a parallel increase in dictation rates, although concurrent transcription costs increased only modestly. The percentage of billable records increased, while the number of charts requiring down-coding decreased, both beneficial outcomes. Average length of stay was not adversely impacted by this added documentation requirement. PMID- 10730836 TI - The uninsured: emergency medicine's challenge to our political leaders. PMID- 10730837 TI - Ketamine in adults: what emergency physicians need to know about patient selection and emergence reactions. PMID- 10730838 TI - Developing a "change-ready" emergency department. PMID- 10730839 TI - The emergency physician as integrative physiologist: inauguration of the academic emergency medicine "bench to bedside" series. PMID- 10730840 TI - Bench to bedside: nitric oxide in emergency medicine. PMID- 10730841 TI - The utility of an alcohol oxidase reaction test to expedite the detection of toxic alcohol exposures. PMID- 10730842 TI - Cerebrospinal fluid glucose and protein in disposition and treatment decisions. PMID- 10730843 TI - Development of rhabdomyolysis after rapid opioid detoxification with subcutaneous naltrexone maintenance therapy. PMID- 10730844 TI - The ASE position statement on echocardiography in the emergency department. PMID- 10730845 TI - Medical application of the Dremel Multipro rotary tool. PMID- 10730846 TI - High-dose intravenous benzodiazepine. PMID- 10730847 TI - My personal recollections of Radslav Kinsky's immunological career on the occasion of his 70th birthday. PMID- 10730848 TI - A personal short history of some concepts of the foeto-maternal relationship. PMID- 10730849 TI - Antibody against 28-kDa intra-acrosomal sperm protein as a tool for evaluation of acrosomal integrity in bull spermatozoa. AB - Monoclonal antibody ACR.4 recognizing specifically the 28-kDa intra-acrosomal protein was prepared by immunization of mice with acetic acid extract of boar spermatozoa, but cross-reacted also with bull intra-acrosomal protein. This monoclonal antibody was used for immunostaining analysis of bull spermatozoa before and during capacitation and ionophore-induced acrosome reaction. Immunostaining analysis showed changes of 28-kDa protein in the acrosome during capacitation and loss of this protein after induced acrosome reaction by ionophore A23187. Therefore, this monoclonal antibody can be used in the bull spermatozoa as an immunological test for detection of the acrosome state after manipulation with spermatozoa or after freezing/thawing. This test could be useful (apart from morphology and motility) for the selection of suitable spermatozoa for insemination or in vitro fertilization. PMID- 10730850 TI - A possible cross-talk of retinoic acid- and TPA-driven myeloid differentiation pathways. AB - Retinoic acid (RA) and phorbol esters are important regulators of cellular proliferation and differentiation processes which are capable of arresting growth and inducing differentiation of various leukemic cells. We have studied and compared the effects of these agents on the line of v-myb oncogene-transformed chicken monoblasts BM2. We found that although there are differences in the differentiation kinetics and cell cycle stop points, the differentiation pathways induced by RA and phorbol esters seem to be significantly interconnected. PMID- 10730851 TI - Linkage mapping of the interleukin 1beta converting enzyme (Il1bc) and the glutamate receptor subunit KA1 (Grik4) genes to rat chromosome 8. AB - Genes for interleukin 1beta converting enzyme (Il1bc) and the glutamate receptor subunit KA1 (Grik4) have been mapped to a centromeric region of rat chromosome 8 using linkage analysis of HXB and BXH recombinant inbred strains. The current results demonstrate that rat chromosome 8 is largely homologous to mouse chromosome 9. PMID- 10730852 TI - Effect of pH on proteinase secretion by transformed fibroblast populations. AB - Effect of pH on secretion of proteolytic enzymes in cell cultures of three clonal lines of transformed fibroblasts (K2, T15 and K4) was studied by using 14C labelled denatured proteins as substrate. One line of malignant macrophages derived from mouse reticulum cell sarcoma (J774.1) was used for comparison. The relative motility index of all cell lines was derived by computer analysis of quantitative estimations of cell dispersion in single-cell-derived colonies. Cultivation at pH 6.5 decreased the growth rate in most experiments as compared with that at pH 7.4, and stimulated cell motility to a different extent. The population of mouse malignant macrophages produced several-fold higher extracellular proteolytic activity than the fibroblast lines. Secretion of proteinases by the malignant macrophages was significantly stimulated by the lower pH. Enzyme secretion by two of the three fibroblast derivatives was also stimulated by acidic pH but to a lesser extent than the secretion of the malignant macrophages. The assessment of motility done by measurement of dispersion of cells in colony proved a positive correlation between motility and proteinase secretion in J774.1 cells and one transformed fibroblast clone (T15) but not in the two other clonal lines. PMID- 10730853 TI - Cellular activity of murine phagocytes isolated from peripheral blood by a discontinuous gradient. AB - A mixture of Ficoll 400 and sodium diatrizoate (Hypaque) at a density of 1.077 g/ml has been used to isolate the mononuclear cells from the remaining haematic cells. A simple, inexpensive and classical method was established to obtain substantially erythrocyte-free polymorphonuclear cell preparations from mouse peripheral blood, using a mixture of the same substances but at a density of 1.119 g/ml. This method along with that at a density of 1.077 g/ml allows two cellular bands to appear which contain mononuclear and polymorphonuclear (PMN) cells, respectively. Using this method, the counts of monocytes isolated from peripheral blood are significantly greater than those obtained by a one-step Ficoll-Hypaque procedure. On the contrary, the counts of PMN cells are significantly smaller than when sedimentation in dextran (6% solution) is used after gradient centrifugation. In this paper, chemiluminescence assay has been used to analyze the possible variations in phagocytic activity of cells isolated by both procedures, since it appears to be one of the most sensitive methods available for this purpose. The results obtained show a slightly greater activation in monocytes and PMN cells isolated by one-step Ficoll-Hypaque procedure, in comparison with another method which uses both Ficoll-Hypaque 1077 and Ficoll-Hypaque 1119, although statistical differences were not significant. PMID- 10730854 TI - Transplantation tolerance and cytokines: is suppressor cell activity mediated by Th2 cells? PMID- 10730855 TI - Linkage analysis for blood pressure with rat chromosome 15 markers. AB - A series of markers located across rat chromosome (Chr.) 15 was mapped and then systematically genotyped for cosegregation with blood pressure in an F2 population that originated from crosses of the Dahl salt-sensitive (S) rats with rats of the Wistar-Kyoto (WKY) strain, i.e., F2(S x WKY). Alleles for the markers centered around the locus for endothelin-receptor B (Ednrb) weakly cosegregated with blood pressure in the F2(S x WKY) population. Therefore, it appears highly unlikely that there is major blood pressure quantitative trait locus (QTL) on rat Chr. 15, which possesses a strong effect on blood pressure in the S rat. The Chr. 15 map will be useful for mapping genes and searching for QTL of other biological traits. PMID- 10730856 TI - The influence of calcium on thyroid follicular cells FRTL-5 in vitro. AB - The present work is based on the results of in vivo experiments on rats, which had shown that hypercalcemia had led to morphological and biochemical hyperfunction of thyroid follicular cells. The regulation of the activity of follicular cells should directly, or indirectly via paracrine action of serotonin secreted from parafollicular cells, depend on the presence of calcium ions. The effect of calcium was studied on a cell line of rat follicular cells FRTL-5 (Fischer Rat Thyroid cells in Low serum) using three methods: measuring the quantity of produced cAMP (cyclic adenosine 3',5'-monophosphate), measuring [3H]thymidine incorporation into cell DNA and transmission electron microscopy. Results show that calcium has no effect on cAMP production. Calcium at 1.3 mM, 3 mM, 10 mM, 20 mM and 30 mM concentrations increases [3H]thymidine incorporation into cell DNA when compared with controls without calcium. Calcium at the concentration of 30 mM has no effect on FRTL-5 cell morphology. TSH (thyrotropin) stimulates follicular cells; at higher extracellular concentrations (3 mM, 10 mM, 20 mM, 30 mM), calcium diminishes its effect, presumably by activation of a cAMP phosphodiesterase which disintegrates cAMP and/or by inhibition of adenyl cyclase. PMID- 10730857 TI - A dose-dependent transformation of embryotoxicity manifestations in the population of chick embryos. AB - The world-wide and long-lasting stagnation in general incidence of inborn defects does not agree with the increasing impact of the environment. However, inborn defects alone are definitely a poor indicator of the reproductive risk being accompanied by much more frequent embryotoxicity manifestations -- death of the conceptus and growth retardation. Dose-dependent transformations of the embryotoxicity manifestations may explain both the stagnation in the incidence of inborn defects and the observed shifts in malformation spectra. An attempt was made to design a reliable experimental model to produce individual transformations resulting from the specific dose-response relationships inherited in teratogenesis. A population of chick embryos in different stages of development was exposed to increasing doses of cyclophosphamide, a model teratogen. All types of the transformations were recorded. Prenatal extinction of defective embryos, transition of single moderate defects to more severe and multiple malformations, as well as the changes in the incidence of certain types of defects exhibit a clear-cut positive dose dependence. Introducing this experimental system we will be able to study mechanisms underlying the particular transformations. PMID- 10730858 TI - Characterization of the differentiated phenotype of an organotypic model of skin derived from human keratinocytes and dried porcine dermis. AB - A number of skin models have been developed, but a simple method for rapidly producing large quantities of differentiated epidermis has been missing. We show the differentiated phenotype of human keratinocytes in organotypic culture arising in vitro by air-exposure of keratinocytes cultured with feeders on dried pig dermis. Keratinocytes were seeded at low density on the dermis covered with irradiated NIH-3T3 feeder cells and after reaching confluence lifted to the air medium interface. A well differentiated epidermis with distinct basal, spinous, granular and stratum corneum layers was formed within 1 week. In this way, 100 cm2 of the differentiated recombined human/pig skin (D-RHPS) can be obtained from 106 secondary keratinocytes in 14 days. The entire keratinocyte life cycle takes place on the dermal substrate - from single cells to stratified epidermis. The differentiation was characterized using a panel of monoclonal antibodies. Similarly as in the normal skin, keratin 14 was expressed in all cell layers, keratin 10 in suprabasal layers, beta1-integrin and epitopes to antibody LH8 in the basal layer, involucrin and transglutaminase in the granular and horny layer of the epidermis. Keratins 16 and 7/17, which are absent in the normal epidermis, but present suprabasally in the psoriatic one, were expressed strongly in all suprabasal layers and in a subpopulation of basal cells. The keratinocytes can be combined with two other cell types cultured either on the dermal side of the dermis and/or on the bottom of the dish. It appears that this simple skin model can be used in studies of epithelial/mesenchymal interactions and interactions between epidermal cells and infectious agents. It may be particularly useful for the study of human papilloma viruses. PMID- 10730859 TI - Clonal expansion of epithelial cells from primary human breast carcinoma with 3T3 feeder layer technique. AB - 3T3 feeder layer technique provided support for clonal growth and serial propagation of two apparently single epithelial cells isolated from a peroperative biopsy of a primary ductal breast carcinoma. The total culture lifetime was estimated to be more than 30 doublings, 21 of which took place during the primary culture. The two cells were the only survivors of two-week exposure to stressing conditions that resembled the microenvironment in a tumour (low pH, depleted nutrition and accumulation of metabolic waste). The epithelial character of the cells was proved by positive immunostaining for keratins 7/17. The majority of growing cells did not express keratin 19. Only quiescent cells in some colonies, which appeared to reach a more advanced stage of differentiation, expressed keratin 19. These features correspond with the characteristics of mammary luminal cells which in vivo undergo differentiation from the stem K19- to secretory K19+ cells. The luminal cells are supposed to be the target of malignant transformation in the mammary gland. The described technique opens a regular way for the in vitro clonal growth of individual primary cells from breast tumours. Such an approach can improve our understanding of the biology of breast cancer cell populations and also simplify the predictive chemosensitivity assay on breast cancer cells from individual patients. PMID- 10730860 TI - A new monoclonal antibody against Rpg1p. PMID- 10730861 TI - Fifty years of immunological tolerance. PMID- 10730862 TI - Infection-activated T lymphocytes resist nitric oxide-mediated immunosuppression in the course of Francisella tularensis 15L experimental infection. AB - Study of the inhibition of splenocyte proliferation stimulated by concanavalin A (Con A), induced by Francisella tularensis 15L infection, showed that immunosuppression is mediated by nitric oxide (NO). A certain fraction of cells, however, resist the antiproliferative activities of NO and these were characterized as Thy-1.2 positive infection-activated T lymphocytes. The importance of this phenomenon for the development of specific anti-infectious immunity was studied further in naturally resistant and susceptible mouse strains. The naturally resistant mouse strain (C57BL/10) was characterized by early production of NO and depressed splenocyte responsiveness to the mitogen. Early production of NO prevented activation of certain fractions of T lymphocytes. Hence the antibodies of these animals were only directed against three main F. tularensis antigens. Late or reduced release of NO from activated macrophages of susceptible strains (C3H/He and BALB/c) on the other hand was accompanied by late or reduced immunosuppression. This resulted in polyclonal activation of the immune system because the antibodies of these mice reacted with 6-12 compounds of the tularaemic antigen. The difference in heterogeneity of specific antibodies was not caused by a defect in the clonal network, as both the susceptible and resistant strains responded similarly to inactivated F. tularensis antigen. PMID- 10730863 TI - Transferrin receptor expression is controlled differently by transferrin-bound and non-transferrin iron in human cells. AB - We studied the effects of iron supplied as transferrin-bound iron and iron supplied as non-transferrin iron on transferrin receptor expression by human cell lines. Defined conditions of iron supply were represented by (i) 5 microg/ml of iron-saturated transferrin (transferrin medium) and by (ii) 500 microM ferric citrate (ferric citrate medium). Transferrin receptor expression of studied cell lines (HeLa, K562, Jiyoye) grown as long-term cultures in transferrin medium was somewhat higher (up to 137% of the mean fluorescence intensity) than in ferric citrate medium. The receptor expression corresponded with cellular iron regulatory protein (IRP) activity (ratio activated/total), which was also higher in transferrin medium (0.69-0.84) than in ferric citrate medium (0.33-0.60). However, unexpectedly much higher (about 65-135-fold) cellular iron levels were found in ferric citrate medium (13.9-14.9 nmol/10(6) cells) than in transferrin medium (0.11-0.21 nmol/10(6) cells). In contrast to the iron levels, cellular ferritin levels of the cells in ferric citrate medium (38.3-130 ng/10(6) cells) were only about 2-7-fold higher than in transferrin medium (6.8-61.5 ng/10(6) cells). We suggest that iron supplied as non-transferrin iron (ferric citrate) is apparently less available for the control of transferrin receptor expression via IRP activity than iron supplied as transferrin. PMID- 10730864 TI - Effects of ethinylestradiol-norethisterone acetate combinations on the ultrastructure of liver, kidney, ovary and endometrium cells. AB - Sexually mature female Wistar rats were given 0.05 mg ethinylestradiol (EE) + 0.5 mg norethisterone acetate (NET), or 0.1 mg EE + 1.0 mg NET for 6 or 12 sexual cycles, i.e., for 30 or 60 days. Rat hepatocytes, renal proximal tubule cells, ovarian granulosa cells and endometrial gland cells showed ultrastructural changes that correlated with the dose and duration of EE/NET treatment. The most common ultrastructural features were as follows: reduced RER, mitochondrial damage, and an increased number of lysosomes. The study has shown that EE/NET combinations used in oral contraception produce ultrastructural lesions, which may impair protein biosynthesis and energy production processes, and may simultaneously enhance cellular catabolism. PMID- 10730865 TI - Influence of dietary supplementation with olive oil on pyroglutamyl-beta naphthylamide hydrolysing activity in serum and different tissues of mice. AB - Pyroglutamyl aminopeptidase is an omega peptidase which removes pyroglutamyl N terminal residues from peptides and arylamide derivatives. To date, three distinct types of this enzyme have been described and called serum thyroliberinase, cytosolic pyroglutamyl aminopeptidase type I and membrane-bound pyroglutamyl aminopeptidase type II. The activity of all of them is thought to be involved in the regulation, more or less restricted in their substrate specificity, of various susceptible endogenous substrates such as TRH, GnRH, neurotensin, bombesin and anorexogenic peptide. It is well known that the type and amount of fat in the diet not only modify blood lipid concentrations, including cholesterol levels, but change the cell membrane lipid composition. Modifications in the composition and physical properties of the membrane lead to alterations in the activities of membrane-bound enzymes and carriers. The aim of this work was to compare the effect of a standard diet and a high fat diet (olive oil, 20% wt/wt) on pyroglutamyl-beta-naphthylamide hydrolysing activity, in serum and in soluble and membrane-bound fractions from different tissues of male mice. After ten weeks of feeding, pyroglutamyl-beta-naphthylamide hydrolysing activity was measured fluorometrically using pyroglutamyl-beta-naphthylamide as substrate. Mice fed the high fat diet had higher rates of body weight than controls starting from the second week of feeding. Serum total cholesterol concentrations were higher after feeding the high fat diet than after feeding the control diet. In serum, no changes were observed in the high fat group. In selected tissues, only pyroglutamyl-beta-naphthylamide hydrolysing activity was modified significantly in the soluble fraction, but not in the membrane-bound one, decreasing in the adrenal gland of high fat fed animals. The results may reflect functional modifications in susceptible endogenous substrates. PMID- 10730866 TI - Prednisolone-induced alteration in hepatic and muscular protein and glycogen level: its correlation with blood glucose level in mice. AB - Prednisolone, a potent glucocorticoid, is well known for its immunosuppressive effects on animals. A single dose of 100 microg, 200 microg and 300 microg prednisolone/kg body weight was administered in Swiss albino male mice. Seven days after treatment, the animals were sacrificed to investigate the effect of prednisolone on hepatic and muscular protein contents and glycogen levels along with the level of blood glucose. A catabolic effect on the protein level was evidenced but at the same time the mice became hypoglycemic. PMID- 10730867 TI - Cells in the spotlight of cancer research. PMID- 10730868 TI - Cyclosporin A-induced autoimmunity: the result of defective de novo T-cell development. AB - Cyclosporin A-induced autoimmunity (CsA-AI) is an autoimmune disease, caused by the combinatory treatment with irradiation and cyclosporin A (CsA). CsA-AI is the result of defective T-cell maturation leading to disturbed T-cell balances in the periphery. Increases in Th1 cells and reduction of autoregulatory cells eventually enable the enumerated autoreactive CD4 and CD8 T cells to disturb the homeostasis in the target organs. In unravelling the effect of CsA on T-cell maturation and the role of T cells in CsA-AI many pieces have been put in their places; nevertheless, some remain the topic of debate. The identity of the autoantigen(s) remains elusive, the working mechanism of the autoregulatory cells still is to be determined, and the interplay between CD4 and CD8 T-cell subsets in relation to type-1 and type-2 responses is a matter of interest. In spite of all these unknowns, the CsA-AI autoimmune model is, in contrast to many autoimmune models induced by immunization with a foreign protein in adjuvant, an interesting physiological model based on defective T-cell development including aberrant selection in the thymus and disturbed T-cell balances in the periphery. PMID- 10730869 TI - A structure-activity study of a C-terminal endothelin analogue. AB - We report a structure-activity study of an endothelin (ET) analogue, obtained by introduction of a non-aminoacidic portion on the C-terminal ET pentapeptide. The peptidic moiety was modified with systematic replacement of each residue by alanine (Ala scan); further modifications were performed at the C-terminus. The biological activity was analyzed at both ET(A) and ET(B) receptor subtypes, showing that the two C-terminal residues (Ile-Trp) are very important for the activity. On the contrary, the aminoacidic central portion of the molecule appears to be much more tolerant toward modifications. PMID- 10730870 TI - Saccharide-binding properties of boar AQN spermadhesins and DQH sperm surface protein. AB - Heparin-binding proteins BHB 2-BHB 5 were purified from boar seminal plasma by affinity chromatography on a heparin-polyacrylamide column and reversed phase HPLC. Three of the proteins, BHB 3-BHB 5, were found to be identical to spermadhesins AQN 1-AQN 3 isolated from boar spermatozoa. The lectin-like properties of the isolated proteins BHB 2-BHB 5 were studied using double diffusion in agarose gel, enzyme-linked binding assay, and inhibition assays of erythroagglutinating activity. It was found that proteins BHB 3-BHB 5 (spermadhesins AQN 1-AQN 3) interacted with glycoproteins containing O glycosidically bound oligosaccharide chains, but not with those containing only N linked carbohydrate chains. The strongest interaction was observed between BHB 3 (AQN 1) and desialyzed bovine submaxillary gland mucin, the glycoprotein containing only O-glycosidically linked saccharides. No interaction of BHB 3-BHB 5 proteins with simple saccharides, their derivatives or acidic polysaccharides was observed. Both the hemagglutinating activity and saccharide-binding properties of BHB 2 protein were quite different. Agglutinating activity of human erythrocytes by BHB 2 protein was significantly higher than that by BHB 3-BHB 5 proteins (AQN spermadhesins). In contrast to AQN spermadhesins, BHB 2 protein (DQH sperm surface protein) interacted strongly with acidic polysaccharides and sialyzed glycoproteins, but no binding of desialyzed glycoproteins as well as N acetyl-alpha-D-galactosaminyl-O-serine,simple monosaccharides and amino sugars was observed. PMID- 10730871 TI - Seroprevalence of human T cell leukaemia/lymphoma virus type I (HTLV-I) in pregnant women, patients attending venereological outpatient services and intravenous drug users from Slovenia. AB - To establish current seroprevalence of human T cell leukaemia/lymphoma virus type I (HTLV-I) infection in some low- and high-risk populations from Slovenia, 10,369 and 869 serum samples collected during Slovenian 1994 unlinked surveys of human immunodeficiency viruses seroprevalence in pregnant women and patients attending venereological outpatient services, respectively, and 219 serum samples collected from Slovenian intravenous drug abusers during 1995 and 1996, were screened for the presence of anti-HTLV-I antibodies using commercial particle agglutination test Serodia HTLV-I (Fujirebio, Tokyo, Japan). Only one sample obtained from a pregnant woman was found repeatedly positive in the screening test. Presence of anti-HTLV-I antibodies in the reactive sample was undoubtedly confirmed with supplemental Western blot test. The prevalence of antibodies to HTLV-I in the Slovenian population might be somewhere between one in 10,000 (0.01%) and one in 15,000 (0.0066%), which is similar or even higher to prevalence rates in other European countries. PMID- 10730872 TI - Fine specificity of anti-Le(X) monoclonal antibody TEC-01. AB - TEC-01 monoclonal antibody recognizes the oligosaccharide structure Galbeta1- >4[Fucalpha1-->3] GlcNAc (Le(X) hapten). To determine its fine specificity, reactivity of TEC-01 antibody with Le(X) glycosphingolipids, isolated from human liver metastasis of adenocarcinoma, and Le(X) neoglycolipid were analysed. Immunostaining of Le(X) glycosphingolipids, fractionated by thin-layer chromatography, showed that TEC-01 reacted with difucosyl and trifucosyl Le(X) glycosphingolipids but not with Le(X) pentasaccharide ceramide. Interestingly, TEC-01 also reacted with Le(X) neoglycolipid prepared by reductive amination from Le(X) pentasaccharide, lacto-N-fucopentaose III, and L-1,2-dipalmitoyl-sn glycerophosphoethanolamine. The combined data suggest that TEC-01 recognizes Le(X) hapten in glycolipids with longer oligosaccharide chain moiety. PMID- 10730873 TI - A new monoclonal antibody against p34 CDC28. PMID- 10730874 TI - Novel monoclonal antibodies TU-08 and TU-16 specific for tubulin subunits. PMID- 10730875 TI - New resources of functional genomics: unique vertebrate models from Prague. PMID- 10730876 TI - Depression in the level of cadherin and alpha-, beta-, gamma-catenins in transgenic Xenopus laevis highly expressing c-Src. AB - Aberrant morphogenesis of transgenic Xenopus laevis 5-day embryos carrying Rous sarcoma virus LTR in their DNA and expressing a high level of c-Src protein kinase was found to be accompanied with a profound depression in the level of cadherins and alpha-, beta-, and gamma-(plakoglobin) catenins in their tissues, as revealed by immunohistochemical analysis. Simultaneously, an increased level of phosphotyrosine staining was detected. However, an analogous increase in the level of phosphotyrosine immunostaining and a slightly higher level of Src were also detected in tissues of originally defective but later spontaneously repaired frog embryos that displayed essentially normal patterns of staining for cadherins and catenins. Our results provide evidence that the defective morphogenesis of frog embryos expressing a high level of c-Src is characterized by the loss of the cadherin-catenin complexes. It appears that to induce frog morphogenetic malformations, the c-Src overproduction and the loss of cadherins-catenins are simultaneously required. Phosphorylation is not likely to be the cause of cadherin and catenin disappearance from the tissues of strongly aberrant frog embryos. PMID- 10730877 TI - Irradiation of genetically modified plasmacytoma vaccines results in upregulation of CD80 molecule expression, IL-2 production and higher therapeutic efficacy of the vaccines. AB - It has been found previously that irradiated, IL-2 gene-modified plasmacytoma (X63-m-IL-2) vaccines are more efficient in the therapy of the parental (X63 Ag8.653) plasmacytoma than live plasmacytoma vaccines. In this communication, we have demonstrated that irradiation of murine IL-2-producing plasmacytoma vaccines resulted in upregulation of CD80 molecule expression and IL-2 production. The expression of MHC class I antigens was not altered. The upregulation of the CD80 membrane molecule expression in X63-m-IL-2 cells was higher after irradiation with 150 Gy than after irradiation with 50 Gy. Comparable upregulation of the CD80 molecule expression has also been demonstrated after irradiation of the parental murine X63-Ag8.653 plasmacytoma cells. The results indicate that upregulation of the CD80 molecule expression and enhanced IL-2 production in irradiated X63-m-IL-2 cells was responsible for the higher therapeutic effectiveness of the irradiated plasmacytoma vaccine. PMID- 10730878 TI - The chicken--a laboratory animal of the class Aves. AB - Prague inbred lines of chickens represent a unique system of MHC(B) congenic partners differing in the immune-based resistance/susceptibility to v-src-induced oncogenesis. Mapping in chickens can be facilitated by the availability of inbred lines, since many well described differences in disease susceptibility and MHC(B) haplotypes exist among the defined lines. Long-term intensive research on human, mouse, and rat MHC has established a canonical picture of this multigene complex. The chicken MHC(B) is clearly the best characterized outside the mammals and it was the first MHC clearly different from the paradigmatic structure of the above mentioned mammalian species. Chickens were in many aspects the poor relatives of mice, and they had to wait for introduction of molecular biology methods. But, when it happened, the newly gained data could be easily reconciled with classical genetic studies using available congenic chicken lines. We have established permanent tumor cell lines from ex vivo tumors induced by the LTR, v-src, LTR provirus in inbred chickens. These cells express a high level of the v-src oncogene and are of defined MHC(B) genotype. We witness a dramatic acceleration of the development of chicken (avian) genomics. The chicken is not only a good comparative model for basic science, but it is also an object of the poultry industry, which is threatened by several avian diseases. The reason for genome mapping in chickens is thus more than academic. PMID- 10730879 TI - SHR.BN-congenic strains for genetic analysis of multifactorially determined traits. AB - The laboratory rat is an important laboratory animal with multiple well-defined inbred strains, including some of the most widely used animal models of human diseases. Recent advances in the development of rat genetic resources will enable the exploitation of the full potential of rat models of human diseases and, in addition, the rat can provide useful information for comparative genomics of humans and mice. In the current review, we describe the development of congenics strains by introgression of differential chromosome segments from the Brown Norway (BN) rat to the genetic background of the spontaneously hypertensive rat (SHR). These SHR.BN-congenic strains and recombinant sublines derived from them were developed as a model system for genetic analysis of multifactorially determined pathophysiological and morphological conditions. PMID- 10730880 TI - PWD/Ph and PWK/Ph inbred mouse strains of Mus m. musculus subspecies--a valuable resource of phenotypic variations and genomic polymorphisms. AB - PWD/Ph and PWK/Ph (abbreviated PW*) are highly inbred mouse strains (F66 and F70) derived from wild mice of Mus musculus musculus subspecies. When compared with laboratory inbred strains, they display a plethora of differences in many complex phenotypes such as body weight, fat distribution pattern, blood levels of intermediary metabolites, sensitivity to type-1 diabetes or behaviour patterns. The PWD/Ph genes can rescue the lethal effect of lack of the Igf2 receptor. The male-limited hybrid sterility of (PWD/Ph x laboratory strain)F1 hybrids is a specific phenotype controlled by three or four unlinked loci. These complex phenotypic traits can be genetically dissected by QTL analysis using microsatellite markers of known genetic location. The PW strains are particularly useful for such genome-wide scans since 70-80% of randomly chosen microsatellite markers are polymorphic in (PW x laboratory strain) crosses compared to 35-45% in crosses between two laboratory strains. The list of polymorphic microsatellite loci is included in this report. The high degree of sequence polymorphism allows easier distinction between paternal and maternal mRNA transcripts in PW hybrids, which makes the PW* strains a useful tool also in molecular studies of genomic imprinting. The high frequency of phenotypic differences together with the high degree of sequence polymorphism and the relatively easy breeding of PW strains make them a valuable mammalian model organism for the functional genomics of the traits of biomedical importance. PMID- 10730881 TI - Colon mucosal cells after high-dose fractional irradiation. AB - The aim of this study was to investigate histological and stereological changes in cryptal enterocytes, mucosal lymphocytes and mast cells 10 days after irradiation. For experimental model, 24 Beagle dogs 1-2 years old were used. Twelve dogs were irradiated 20 days with 32 Gy over the whole pelvis and tail. Another 12 dogs represented a control group. For the detection of apoptosis, the TUNEL technique was used. Histological and stereological analyses were performed using a Wild sampling microscope M 1000. In the irradiated group, volume density (P < 0.01), numerical density (P < 0.05) and average volume of lymphocytes (P < 0.001) were significantly lower than in the nonirradiated group. Numerical areal density of mast cells in the irradiated group was also significantly lower (P < 0.05). Volume density (P < 0.001) and average volume of mast cells (P < 0.001) were significantly higher in the irradiated group. The results of our experiments show that irradiation causes injury and loss of lymphocytes and mast cells in the colon mucosa. Apoptosis was detected in enterocytes and lymphocytes in the irradiated group and in nonirradiated group in equal numbers (2.5+/-0.3 vs. 2.3+/ 0.3; ns.), suggesting that 10 days after high-dose irradiation, the cell loss is not due to apoptosis. PMID- 10730882 TI - Development of the ectopia cordis induced by hydrocortisone administration. AB - Our previous study on the development of thorax in chick embryos revealed that mechanical disturbance of the so-called membrana reuniens causes the development of the ectopia cordis (EC). To assess whether membrana reuniens disturbance was really essential for EC development, we employed hydrocortisone, a teratogen known to produce a high incidence of EC. The incidence of EC after the hydrocortisone intraamniotic application on the 4th embryonic day reached 84,8%. It was found that although in the whole course of EC development the membrana reuniens appeared very thin, it nevertheless remained continuous. The morphology of the membrana reuniens in embryos with fully developed EC, studied in classical serial histological sections, was similar to that of the amniotic membrane. Flow cytometry analysis of the cell cycle revealed that EC induced by hydrocortisone administration was associated with a significantly lowered proliferation activity of the prospective body-wall mesenchyme involved in the closure of the anterior wall of thorax. The probable mechanism of EC development is suggested. PMID- 10730883 TI - Monoclonal antibody to human sperm acrosomal protein. AB - A new monoclonal antibody designated Hs-14 was generated after immunization of BALB/c mice with the acid extract of human sperm. In indirect immunofluorescence Hs-14 mAb binds to the acrosome of permeabilized sperm cells and consequently recognizes some intra-acrosomal protein. Western blotting analysis revealed that under non-reducing conditions the Hs-14 mAb detects a protein with a molecular mass of 220 kDa. Under reducing conditions the Hs-14 recognizes several peptide bands within the range from 55 kDa to 110 kDa. Beside human sperm the antibody positively reacts also with sperm of some other mammalian species. Using Hs-14 mAb it is possible to evaluate the acrosomal integrity of spermatozoa and to reveal sperm pathology. PMID- 10730884 TI - Genomics on the eve of the 21st century. PMID- 10730885 TI - Granulocyte-macrophage colony-stimulating factor-producing tumour vaccines. AB - Murine sarcoma MC12 cells were transfected with the gene coding for murine granulocyte-macrophage colony-stimulating factor (GM-CSF). Tumorigenicity of a variety of cell clones with different expression of the inserted gene was assessed. All of the genetically manipulated MC12 cell clones examined were found to be less tumorigenic than the parental MC12 cell population. No correlation was observed between the production of GM-CSF by the clones and their tumorigenicity. It has been found that irradiation of the GM-CSF-producing cells with the dose of 150 Gy did not significantly inhibit the GM-CSF production during the period of 5 days after irradiation. These findings provided us with the rationale for using the irradiated GM-CSF-producing MC12 sarcoma vaccine for therapy. It has further been found than immunosensitivity of the genetically manipulated, GM-CSF producing tumour targets to the IL-2-activated killer (LAK) cell-mediated cytolysis was significantly increased, as compared to the parental target cell population. Irradiated, GM-CSF-producing tumour vaccines were used for therapy of 3-day-old MC12 sarcoma transplants in syngeneic mice and for therapy of surgically induced minimal residual tumour disease. Neither small tumour transplants, nor tumour residua after surgery were significantly sensitive to the therapy with GM-CSF-producing tumour vaccines. PMID- 10730886 TI - Monitoring of residual disease in non-Hodgkin's lymphomas by quantitative PCR (preliminary report). AB - Highly sensitive PCR techniques are often used in molecular monitoring of hematological malignancies, and a quantification of residual disease is important for further prognosis. Here, the limiting dilution methodology and the multiplex IgH/ras PCR are proposed as approaches to molecular monitoring of NHLs. Applying the limiting dilution methodology as a simple dose-response assay for the translocation t(14,18) and CDR3 clonal rearrangement of IgH, critical amounts of total cells determined with stored consecutive diagnostic samples in the same PCR run are compared. Assuming that specific targets are diluted proportionally in dilution of total genomic DNA, the samples showing lower critical concentrations of total DNA are considered as containing higher portion of cells possessing the specific disease marker and vice versa. So far, the correlation of results with the disease outcome confirmed that this simple semi-quantitative approach may in some cases substitute laborious precisely quantifying techniques in the monitoring of the disease. In optimized multiplex IgH/ras PCR co-amplifying clonal CDR3 rearrangement of IgH and the codon 61 of Hras 1 gene, the amount of CDR3 product as the disease marker is related to the ras product as a standard marker of all cells, and quantitative results are obtained by software analyses of detecting gels. Presumably, both approaches may provide clinically useful information on the disease activity and treatment outcome. PMID- 10730888 TI - Aggregated forms of heparin-binding and non-heparin-binding proteins of boar seminal plasma and their binding properties. AB - Boar seminal plasma proteins were separated by affinity chromatography on immobilized heparin into two portions: heparin-binding (H+) and non-heparin binding (H-) proteins. Gel chromatography of the H+ portion yielded four main protein fractions of >150, 45, 30 and 20 kDa, while that of the H- portion resulted in the separation into three main protein fractions of >150, 30 and 20 kDa. HPLC analysis and N-terminal sequencing used to characterize the composition of the protein fractions obtained by gel chromatography revealed that all consisted of low (12-16 kDa) molecular weight components: the H+ fraction consisted of DQH sperm protein, AQN and AWN spermadhesins whereas the H- fraction consisted of PSPI and PSPII spermadhesins. The high molecular weight values of fractions obtained by gel chromatography thus suggest that the proteins are present in boar seminal plasma in the form of aggregates. Interactions of individual boar seminal plasma proteins and their aggregates present in the H+ and H- fractions with acid polysaccharides were estimated. PMID- 10730887 TI - Selective cytotoxic activity of a novel ribonucleoside diphosphate reductase inhibitor MDL-101,731 against thyroid cancer in vitro. AB - The dramatically increased number of new cases of disseminated children thyroid cancer in Belarus after the Chernobyl accident requires development of novel strategies to treat this disease. In addition to conventional therapy using radioiodine, chemotherapy with new effective drugs can be an alternative kind of treatment. We tested the antitumor activity of (E)-2'-fluoromethylene-2' deoxycytidine (MDL-101,731), a ribonucleoside diphosphate reductase inhibitor against three thyroid carcinoma cell lines established from anaplastic (8505C), papillary (B-CPAP) and poorly differentiated papillary (BHT-101) cancer. MDL 101,371 decreased both cell growth and DNA synthesis of tumor cells at concentrations lower than 100 nM, while the concentrations higher than 5000 nM showed only moderate effects on growth of normal human fibroblasts. The effects of MDL-101, 371 on tumor cells were associated with induction of apoptosis, as demonstrated by DNA fragmentation assay. Flow cytometric analysis of the expression of apoptosis-related genes revealed the increased levels of Fas antigen, whereas the levels of Bcl-2 were not significantly influenced in thyroid cancer cells treated with MDL-101,731. These results demonstrated that MDL 101,731 is a potent antitumor agent against cultured thyroid cancer cells due to its ability to induce apoptosis in association with increased Fas expression. PMID- 10730889 TI - HXB/Ipcv and BXH/Cub recombinant inbred strains of the rat: strain distribution patterns of 632 alleles. AB - The HXB/Ipcv and BXH/Cub sets of recombinant inbred (RI) strains were derived from the spontaneously hypertensive rats (SHR/OlaIpcv) and normotensive Brown Norway (BN-Lx/Cub) rats. The RI strains were produced as a model system for genetic and correlation analysis of spontaneous hypertension and other risk factors of cardiovascular disease such as insulin resistance and dyslipidemia. The RI strains were phenotyped in multiple hemodynamic and metabolic traits. In the current study, we describe strain distribution patterns of 632 genetic markers. PMID- 10730890 TI - Local hypertrophic/hyperplastic changes of keratinizing squamous epithelium of pinna induced by concanavalin A and other immunomodulators in mice. AB - Intradermal administration of concanavalin A, a potent T-cell mitogen, into an ear lap resulted in activation of chondrogenesis and stimulation of epidermis proliferation. This proliferation is sometimes invasive in character (pearls and epidermal nests form in the underlying connective tissue) but never turns into true cancerous lesions. This reaction can be delayed, but not prevented, by the prostaglandin inhibitor indomethacin. Stimulation of epidermis proliferation was also caused by administration of other immunomodulators, such as carrageenan type IV, Moloney sarcoma development, and rarely in the course of GvHr, but to much lesser degree than with concanavalin A. It is suggested that the same growth factors, which are mediators of local chondrocyte stimulation, are also mediators of keratinocyte activation. PMID- 10730892 TI - Dendritic-cell-based cancer vaccines. PMID- 10730891 TI - A set of monoclonal antibodies specific for bovine cell surface molecule CD9. PMID- 10730893 TI - Baculovirus expression system cells expressing v-myb oncogene: the distribution of RNA and DNA in specific nuclear compartments with respect to structures interacting with anti-v-Myb antibody. AB - The distribution of RNA, total DNA and newly synthesized DNA within nucleoli-like structures in insect cells overexpressing v-myb oncogene was investigated. Three types of these structures which revealed interaction with anti-v-Myb oncoprotein antibody were found at the ultrastructural level. Specific staining by toluidine blue at pH 5.2 showed the presence of RNA in these nucleoli-like structures. To detect total DNA, the in situ terminal deoxynucleotidyl transferase-immunogold technique was used. In addition to an expected labeling of host condensed chromatin, the labeling of the three types of nucleoli-like structures differed from each other. While the compact (type I) and ring-shaped (type II) nucleoli like structures were labeled only on their periphery and in the proximity of baculovirus particles that interacted with them, the structures with an appearance of nucleolonemas (type III) were labeled strongly. Incorporation of 5 bromo-2-deoxyuridine, in spite of a poor labeling of newly synthesized DNA, confirmed these results. We suggest that the nucleoli-like structures of type I and II are of nucleolar origin. The type III more likely represents virogenic stroma or viral DNA storage site. PMID- 10730894 TI - Genetic analysis of the rat hypodactylous mutation. AB - Autosomal recessive rat hypodactylous mutation Hd leads in homozygous condition to reductive changes of the digital arch of all feet. There is a variable preaxial reduction of the number of fingers in both sexes. Moreover, homozygous males are sterile. Testes of homozygous Hd/Hd and +/Hd adult rats were examined in the light and electron microscopes. Spatial organization of stages of the spermatogenetic cycle was not confirmed in Hd/Hd testes comparing with +/Hd males. Significant decrease in the number of germ cells in seminiferous tubules of Hd/Hd testes was accompanied with loosening and vacuolization of the seminiferous epithelium. The assignment of the Hd locus to RNO10 excluded the suspected homology between rat and mouse Hd mutations. More precise mapping using microsatellite markers revealed close linkage of the Hd locus with the D1OMit8 marker defining Syb2 gene coding for synaptobrevin 2. A chromosomal segment of RNO10 carrying Hd and Syb genes is being incrossed onto BN and SHR inbred strains in order to examine the modifying influences of different genetic backgrounds. PMID- 10730895 TI - Experimental lesion of medial frontal cortex mediates inhibition of expression of carbohydrate-binding sites in the spleen macrophages in rat. AB - The fundamental concept of a neuroimmunological network is well appreciated although detailed description of the individual mechanisms has not yet been attained. In an effort to close this gap, the effect of electrolesion of the frontal medial cortex, a structure with the known "immunoreactivity", on selected phenotypic features of spleen macrophages was studied. Since sugar receptors (lectins) are pivotal for recognition, custom-made tools termed neoglycoproteins were employed to delineate any injury-induced changes of their expression. The total number of macrophages in the spleen red pulp was assessed using the ED-1 monoclonal antibody. The results showed that after lesion of the medial frontal cortex, the extent of expression of carbohydrate-binding sites in red pulp spleen macrophages significantly decreased without affecting the total number of these cells. These data intimate that distinct brain regions are involved in the control of the phenotype of macrophages in the central lymphoid organs by currently elusive biochemical mechanisms. PMID- 10730896 TI - Mechanisms of horizontal gene transfer (review). PMID- 10730897 TI - Free-Tree--freeware program for construction of phylogenetic trees on the basis of distance data and bootstrap/jackknife analysis of the tree robustness. Application in the RAPD analysis of genus Frenkelia. AB - The Win95 program for computation of distance matrixes and construction of phylogenetic or phenetic trees on the basis of RAPD, RFLP and allozyme data was presented. In contrast with other presently available software, the program FreeTree can also assess the robustness of the tree topology by bootstrap, jackknife or OTU-jackknife analysis. Moreover, the program can be used also for an analysis of data obtained in several independent experiments performed with nonidentical subsets of taxa. The function of the program was demonstrated by an analysis of RAPD data from 22 strains of Frenkelia. The program is available as an autoextractive archive containing the installation files of FreeTree and TreeView, manual in MS Word format and a sample of the input file at http://www.natur.cuni.cz/flegr/programs/+ ++freetree. PMID- 10730898 TI - A signalling accessory molecule revealed by a new anti-fibroblastoid L cell monoclonal antibody. AB - Fibroblastoid mouse L-cells are widely used in immunological models because when transfected with class II-coding genes they become efficient antigen presenting cells. Little is known, however, about the cell surface markers borne by L-cells and their putative involvement/Interference with the experimental models studied. Rats were immunized against DAP.3 cells (subclone of L-cells) and monoclonal antibodies (mAbs) were prepared. One of them, 4D4, was studied in detail. It recognizes an epitope which is neither cell lineage- nor strain- nor species restricted since, in addition to DAP.3 cells, it binds, as determined by flow cytometry and immunohistochemistry, to various cells such as CD8+ T cells from thymus, spleen, lymph node or intestinal epithelium, mouse peritoneal B cells and various tissues such as renal, pulmonary or intestinal epithelia. 4D4 mAb immunoprecipitates an undescribed 68 kDa protein. Functionally, this mAb inhibits the IL-2 secretion of a T cell clone in response to its peptide presented by appropriate class II-transfected L-cells and induces a negative selection of double positive CD4+CD8+ thymocytes. Since the 4D4 ligand is found on cells which are submitted to selection (T cells) and on cells which mediate selection (epithelial and antigen presenting cells), we conclude that 4D4 mAb defines a cell surface antigen involved, as an accessory molecule, in a cell selection process. PMID- 10730899 TI - Methylation mechanisms in pituitary tumorigenesis. AB - Methylation is essential for embryonic development, however aberrant methylation of CpG islands associated with the tumour suppressor genes (TSGs) and leading to gene silencing is found in numerous tumour types. The TSG p16/CDKN2A is involved in the genesis of many tumour types and frequent methylation of the CpG island of the p16/CDKN2A gene is associated with loss of protein expression in pituitary tumours. In addition, CpG sites are mutational hotspots and abnormal methylation patterns have been shown to lead to genetic instability, predisposing to, and preceding allelic loss. Although several studies of pituitary tumours have shown loss of genetic material at known and putative TSGs loci, studies of the retained alleles have revealed infrequent mutation. Equally, for several other TSGs no mechanisms have been described for their reduced expression. Methylation may represent a unifying theme, responsible in some cases for an absence or reduced expression and in other cases predisposing to allelic loss that may or may not encompass a TSG. In several tumour types treatment of tumours or their cognate cell lines with demethylating agents induces expression of previously methylated genes. Using the mouse corticotroph cell line AtT20 as a model system, transfection studies showed restoration of growth control through induction of ectopically expressed p16/CDKN2A. These effects were reversed by prior in vitro methylation of the constructs' CpG sites within the coding region of this gene. Methylation of an otherwise unmethylated CpG island renders a gene transcriptionally incompetent and clinically these genes represent attractive therapeutic targets since the gene is neither lost nor mutated, but may be reactivated. Future studies will no doubt describe more efficacious pharmacological interventions and identify the mechanisms responsible for the abnormal methylation patterns seen in tumours including those of pituitary origin. PMID- 10730900 TI - Multiple endocrine neoplasia type 1. AB - Combined clinical and laboratory investigations of multiple endocrine neoplasia type 1 (MEN1) have resulted in an increased understanding of this disorder which may be inherited as an autosomal dominant condition. Defining the features of each disease manifestation in MEN1 has improved patient management and treatment, and has also facilitated a screening protocol to be instituted. The application of the techniques of molecular biology has enabled the identification of the gene causing MEN1 and the detection of mutations in patients. The function of the protein encoded by the MEN1 gene has been shown to be in the regulation of JunD mediated transcription but much still remains to be elucidated. However, these recent advances provide for the identification of mutant MEN1 gene carriers who are at a high risk of developing this disorder and thus require regular and biochemical screening to detect the development of endocrine tumours. PMID- 10730901 TI - Gene therapy for pituitary tumours. PMID- 10730902 TI - Hormonal approaches to the chemoprevention of endocrine-dependent tumors. AB - The estrogen dependency of human breast cancer has been successfully exploited in the treatment of early and advanced diseases and provides a unique opportunity for chemoprevention of this common malignancy. Preliminary results with the antiestrogens Tamoxifen and Raloxifene show an encouraging reduction in the incidence of breast cancer. Alternative approaches include the use of highly selective and non-toxic aromatase inhibitors and, in premenopausal women, the use of LHRH agonists in conjunction with the administration of small doses of estrogen and progesterone. The rationale for these chemopreventive strategies and their possible limitations are briefly discussed. PMID- 10730903 TI - Prostate cancer: molecular biology of early progression to androgen independence. AB - To improve the therapy for prostate cancer, it will be necessary to address the problems of progression to androgen independence and the process of metastatic spread of tumour. The complexity of the latter condition is likely to mitigate against the immediate development of relevant therapeutic approaches. However, the basis of androgen independence appears to be a problem of simpler dimensions and more amenable to treatment with current therapeutic technology. Since early tumour progression can be detected by an incomplete prostate-specific antigen (PSA) response to androgen withdrawal therapy, a study of the molecular biology of PSA gene regulation may well provide insight into new methods for preventing or delaying this problem. Mounting evidence suggests that ligand-independent activation of the androgen receptor may be one underlying mechanism of androgen independence. In the absence of androgen, a compensatory increase in the activity of cAMP-dependent protein kinase (PKA) enhances the ability of the androgen receptor to bind to the response elements regulating PSA gene expression. The activation of the androgen receptor through up-regulation of the PKA signal transduction pathway involves the amino-terminus of the androgen receptor, the function of which may be altered either by modifications such as phosphorylation, or through interactions with co-regulators or other proteins. Of therapeutic interest is the fact that this effect can be counteracted experimentally by the anti-androgen, bicalutamide, and clinically by several other similar agents. We speculate that the inhibition of PKA-activated androgen receptor might also be accomplished by decoy molecules that can bind to the relevant activated site on the amino-terminus or competitively interact with proteins recruited by the PKA pathway that are responsible for activating the receptor in the absence of androgen. Such molecules might include small mimetic substances or agents that can gain access to the nucleus of the cell. PMID- 10730904 TI - Neuroendocrine cells in tumour growth of the prostate. AB - The prognostic significance of neuroendocrine differentiation in prostatic malignancy is controversial, but the results of recent studies with markers such as chromogranin A and neurone-specific enolase suggest that neuroendocrine differentiation, as reflected by increased tissue expression or blood concentrations of these neuroendocrine secretory products, is associated with a poor prognosis, tumour progression, and androgen independence. As all malignant neuroendocrine cells are devoid of androgen receptors and the expression of neuroendocrine cells is not suppressed by androgen ablation, clonal propagation of androgen receptor-negative neuroendocrine cells may have an important role in the development of androgen-independent prostatic carcinoma. This has significant implications for the treatment of prostate cancer, because several of the hormones that are secreted by neuroendocrine differentiated, malignant prostatic cells are potential candidates for use in drug treatment. A limited number of hormones have been tested in this context, in particular somatostatin, bombesin, and serotonin. As there is currently no successful treatment for differentiated prostate cancer, new therapeutic procedures and trials need to be developed to test drugs based on neuroendocrine hormones or their antagonists. PMID- 10730905 TI - BRCA1/2 carriers and endocrine risk modifiers. PMID- 10730906 TI - Changes in neoplastic cell features and sensitivity to mitotane during mitotane induced remission in a patient with recurrent, metastatic adrenocortical carcinoma. AB - A 58-year-old man had adrenocortical carcinoma in the right adrenal gland. The tumour secreted excessive cortisol and dehydroepiandrosterone-sulphate (DHEA-S), and had invaded the right hepatic lobe and vena cava. Eleven months after surgical tumour resection, the serum DHEA-S levels again increased. Local tumour recurrence and a metastasis was found in the lung. Eleven months after surgery chemotherapy with mitotane (o,p'-DDD) was initiated. Twelve weeks of mitotane reduced serum DHEA-S levels and caused these tumours to disappear. The patient was then treated with low-dose mitotane (1.5-2.0 g/day) for 2 years. Serum levels of mitotane remained at less than 10 microg/ml. Although such low serum levels of mitotane and delayed initiation of mitotane after surgery have been proposed to weaken the antineoplastic effect of mitotane, the patient had a remission for 2 years. However, there was then local re-recurrence with an increase in serum DHEA S and death 4 months later. The histological features of neoplastic cells were quite different comparing tumour resected at surgery and tumour at autopsy. The latter had more frequent mitotic nuclei. This tumour was initially sensitive to mitotane, but later became insensitive. PMID- 10730907 TI - Onychomycosis: current treatment and future challenges. AB - Onychomycosis is a fungal infection of the nails, more often of the toenails. It is a common condition, with an estimated overall prevalence of 3-10% in European populations. Dermatophytes, especially Trichophyton rubrum and Trichophyton mentagrophytes, are the usual pathogens. Some 50% of infected patients fail to seek medical advice. Medically confirmed onychomycosis should be treated. This recommendation is based on several disease-specific considerations: cosmetic and functional disability, lack of spontaneous remission, impairment of health and wellbeing in elderly patients and the need to reduce contamination in communal bathing places. Current treatments for onychomycosis include oral antifungal agents such as terbinafine (Lamisil) and itraconazole (Sporanox). They offer significantly improved rates of cure, shorter treatment regimens and a lower level of adverse events than was previously the case. Comparative studies have shown that terbinafine is more effective than griseofulvin, fluconazole or itraconazole in the treatment of this condition, providing a cure rate of 70-80% and an excellent tolerability profile. Terbinafine is also the most cost effective agent. However, several problems remain that will provide future challenges in the treatment of onychomycosis, not least the consistent treatment failure rate of 20%. In many of these cases, surgery may need to precede drug therapy in order to maximise the prospects of clinical and mycological cure. In addition, duration of treatment also needs to be more closely adjusted to the individual case by prior identification of severity and extent of toenail infection, and combined oral and topical therapy also requires further investigation. PMID- 10730909 TI - Long-term outcomes in the treatment of toenail onychomycosis. AB - Most clinical studies in subjects with toenail onychomycosis end with a final assessment at 48-52 weeks. This fails to take full account of the physiology of toenail growth, as toenails can take up to 12-18 months to grow out fully. Accurate assessment of long-term outcomes therefore requires follow-up of at least 2 years after completion of the study. We have evaluated long-term outcomes of treatment in the patients whom we contributed to two multicentre studies of oral therapy for toenail onychomycosis caused by dermatophyte infection. In the first, a dose-finding study for terbinafine (Lamisil), the high rates of mycological and clinical cure achieved by terbinafine at week 48 were maintained more than 2 years after completion of the study. In the second, a comparative study between terbinafine and itraconazole (Sporanox), the excellent mycological and clinical cure rates achieved by terbinafine at week 48 were again maintained more than 2 years after completion of the study. By contrast, the failure and relapse rates seen with itraconazole were much higher. Other studies undertaken in recent years have confirmed these positive findings with respect to terbinafine, and have demonstrated its superiority over itraconazole in maintaining mycological and clinical cure over long periods. These long-term benefits of terbinafine probably relate to its primarily fungicidal action against dermatophytes, compared to the fungistatic action of itraconazole and other triazole agents. Future clinical studies should therefore incorporate at least 2 years' follow-up. PMID- 10730908 TI - L.I.ON. Study: efficacy and tolerability of continuous terbinafine (Lamisil) compared to intermittent itraconazole in the treatment of toenail onychomycosis. Lamisil vs. Itraconazole in Onychomycosis. AB - We undertook a prospective, randomised, double-blind, double-dummy, multicentre, parallel-group study to compare the efficacy and tolerability of continuous terbinafine (Lamisil) with intermittent itraconazole (Sporanox) in the treatment of toenail onychomycosis. A total of 496 patients (age range 18-75 years) with a clinical diagnosis of dermatophyte toenail onychomycosis, confirmed by positive mycological culture and microscopy (KOH), were recruited from 35 centres in six European countries. They were randomly divided into four parallel groups to receive either terbinafine 250 mg/day for 12 or 16 weeks (groups T12 and T16), or itraconazole 400 mg/day for 1 week in every 4 weeks for 12 or 16 weeks (groups I3 and I4). The primary efficacy measurement at week 72 was mycological cure, defined as negative microscopy and negative culture of samples from the target toenail. At week 72, the mycological cure rates were 75.5% (81/107) in the T12 group and 80.8% (80/99) in the T16 group, compared with 38.3% (41/107) in the I3 group and 49.1% (53/108) in the I4 group. All comparisons (T12 vs. I3, T12 vs. I4, T16 vs. I3, T16 vs. I4) showed significantly higher cure rates in the terbinafine groups (all P<0.0001). In addition, all secondary clinical outcome measures were significantly in favour of terbinafine at week 72. Both treatments were well tolerated, with no significant differences in the number or type of adverse events reported. We conclude that continuous terbinafine is significantly more effective than intermittent itraconazole in the treatment of toenail dermatophyte onychomycosis. PMID- 10730910 TI - Terbinafine: tolerability in general medical practice. AB - Four open, prospective, postmarketing studies with consistent protocols were undertaken, involving over 25,000 patients and nearly 3600 physicians in four European countries (UK, Netherlands, Germany and Austria). The purpose was to determine the tolerability of terbinafine when prescribed to a large, unselected patient population in general medical practice. Patients were recruited from dermatology departments and from general and family practitioners. All received at least one dose of terbinafine. No specific exclusion criteria were applied. The only treatment instructions provided were those contained in the manufacturer's product information. Patients were monitored for adverse events at baseline, during treatment, and at the end of treatment. Of the 25,884 patients entering the study, 38.6% (9991) had concomitant disease, 42.8% (11,078) were taking other medications, and 22.7% (5876) were older than 60 years. The predominant indication for prescribing terbinafine was onychomycosis. Mean duration of treatment was 13.2 weeks. No adverse events were reported in 89.5% (23,167) of patients. The remaining 10.5% (2717) reported one or more adverse events, primarily gastrointestinal (4.9%) or dermatological (2.3%). A total of 115 serious adverse events were recorded, but of these only four were probably and eight possibly related to terbinafine. There were no reported drug-drug interactions, even in patients receiving concomitant medications metabolized by cytochrome P-450 enzymes. There were also no clinically significant drug-disease interactions. In conclusion, terbinafine is well tolerated in an unselected general practice population. This confirms its good tolerability previously established during controlled clinical trials. PMID- 10730911 TI - Drug interactions of the newer oral antifungal agents. AB - The newer oral antifungal agents, such as fluconazole, itraconazole and terbinafine, are generally both effective and well tolerated in the management of widespread or resistant dermatomycoses such as onychomycosis. However, these agents differ markedly in their potential to cause clinically significant drug interactions. Triazoles such as fluconazole and itraconazole have been responsible for a greater number of clinically significant drug interactions than terbinafine. For example, itraconazole, and to a lesser extent fluconazole (in high doses) are inhibitors of CYP3A4. Therefore certain agents that are substrates of this enzyme, such as some of the new generation of H1 antihistamines, several HMG-CoA reductase inhibitors and certain benzodiazepines, are contraindicated. Other drugs like cyclosporine and quinidine need careful monitoring if administered concurrently with these triazoles. In contrast, there are no drug-drug contraindications with terbinafine. Indeed, in a postmarketing surveillance study, in which 42.8% of the 25,884 participating patients were taking a variety of concomitant therapies, no new drug-drug interactions were revealed. Physicians should be aware of the potential for interaction of the medications that they prescribe, in order to prevent or reduce the burden of adverse events. Terbinafine may be the most rational choice of oral antifungal agent in patients receiving concomitant medications that may adversely affect or be affected by either fluconazole or itraconazole. PMID- 10730912 TI - Resistance of Candida species to antifungal agents used in the treatment of onychomycosis: a review of current problems. AB - Treatment of Candida infections with fluconazole has resulted in the emergence of drug resistance, a problem particularly apparent in HIV-infected patients. Frequently, the yeast is also cross-resistant to itraconazole and other azoles. In neutropenic patients fluconazole therapy or prophylaxis has caused overgrowth and infection by inherently less susceptible species of Candida, principally C. glabrata and C. krusei. Consequently, the use of intermittent long-term azole therapy to treat onychomycosis could result in changes in the commensal yeast flora of patients--either resistance or pathogen shift. An 'off-study' investigation undertaken in patients receiving either continuous terbinafine or intermittent itraconazole for toenail onychomycosis (L.I.ON. study) showed no evidence of changes in the yeast species present, nor in their sensitivity to itraconazole or fluconazole. Although intermittent itraconazole seems unlikely to cause problems in this respect, the situation with regard to intermittent fluconazole therapy of onychomycosis needs further study. PMID- 10730913 TI - Therapeutic potential of terbinafine in subcutaneous and systemic mycoses. AB - Mycoses vary widely in severity, and may present as superficial, subcutaneous and/or systemic infection. Effective treatments for most superficial mycoses now exist, but new agents with convenient dosing regimens and a low level of adverse events are still needed to reduce morbidity and mortality from serious subcutaneous and systemic fungal infections. In vitro, terbinafine exhibits a broad spectrum of activity against the pathogenic fungi responsible for deep mycoses. Clinical data, while not abundant, suggest that this in vitro activity of terbinafine is reflected in its in vivo efficacy. The limited data show that terbinafine is a useful first-line treatment in chromoblastomycosis patients and has efficacy in pulmonary aspergillosis. There are also data to suggest that terbinafine may be effective in treating histoplasmosis, Pneumocystis carinii infection, fungal mycetoma, and cutaneous leishmaniasis. Moreover, there is some evidence of terbinafine having synergistic activity with amphotericin B, itraconazole, and fluconazole against clinical isolates of Candida species. Thus, the therapeutic potential of terbinafine extends well beyond its current use in acute and chronic dermatophytoses to include a wide range of subcutaneous and systemic mycoses. Studies are needed to determine the optimum dose in each disease, and whether combination therapy would have advantages in certain circumstances. PMID- 10730914 TI - Regnier de Graaf: Paris, purging, and the pancreas. PMID- 10730915 TI - Foods and gastric motor function. PMID- 10730916 TI - Biofeedback therapy for dyssynergic (obstructive) defecation. PMID- 10730917 TI - The diagnosis and work-up of the patient with gastroparesis. AB - Gastroparesis-or delayed gastric emptying--is associated with upper gastrointestinal symptoms that include early satiety, nausea, vomiting, regurgitation, fullness, and bloating. Gastroparesis should be considered in the diagnosis of a patient with these symptoms after mechanical and structural lesions have been ruled out. This review briefly summarizes gastric motor physiology and discusses the etiology and diagnostic approach to the treatment of a patient with possible gastroparesis. We highlight the methods available to measure gastric motility and describe their relative advantages and disadvantages. PMID- 10730918 TI - Hepatitis C: an update on the silent epidemic. AB - Hepatitis C virus (HCV) currently infects an estimated 2-3 million people in the United States and 175 million people globally. Over 80% of infected patients go on to develop chronic disease. Most patients remain asymptomatic despite silent, insidious progression of the disease. The sequelae of HCV-induced chronic liver disease accounts for 8,000-10,000 deaths annually in the United States and is currently the leading indication for liver transplantation. The cost of this epidemic to the United States was estimated in 1991 at $600 million in terms of medical expenses (excluding costs related to liver transplantation) and work lost. Over the last decade, since the viral genome of HCV was first sequenced in 1989, there has been a great increase in understanding of this infection. This review summarizes current knowledge about the hepatitis C epidemic with particular reference to epidemiology of infection, viral characteristics, risk factors for disease, diagnostic testing, clinical manifestations, and current, as well as potential, therapeutic options. PMID- 10730919 TI - Gastrointestinal manifestations of Behcet's disease. AB - Behcet's disease (BD) is a multisystem, chronic, relapsing vasculitis of unknown origin that affects nearly all organs and systems. While recurrent oral ulcerations are a "sine qua non" of BD, the frequency of extra-oral parts of the gastrointestinal involvement varies widely in different countries. The most frequent extra-oral sites of gastrointestinal involvement are the ileocecal region and the colon. The liver (except with Budd-Chiari syndrome), pancreas, and spleen are rarely involved. The symptoms associated with these extra-oral manifestations of BD are abdominal pain, nausea, vomiting, diarrhea with or without blood, and constipation. The lesions typically are resistant to medical treatment and frequently recur with surgical treatment. We review the literature regarding the gastrointestinal and hepatobiliary systems in BD. Also, we present a patient who had BD complicated with radiologically-proven hepatic veins involvement (Budd-Chiari syndrome) and complete occlusion of hepatic portion of inferior vena cava and who had a good response to colchicine and penicillin treatment. PMID- 10730920 TI - Treating Budd-Chiari Syndrome: making rational choices from a myriad of options. AB - Budd-Chiari syndrome represents a spectrum of disorders characterized by obstruction to hepatic venous drainage. Originally described as an "obliterating endophlebitis of the hepatic veins," this condition has come to refer to the manifestations of hepatic venous outflow obstruction anywhere above the level of the hepatic venulae regardless of the etiology, position, or severity of the obstruction or of the clinical course. Depending on the nature and anatomy of the obstruction, the disease presents acutely, with a rapidly progressive course, or insidiously, with gradual development of symptoms. The optimal management strategy for a given patient with Budd-Chiari syndrome depends on the anatomy of the obstruction, its physiologic consequences, and the natural history of the specific lesion. The specific treatments available and their use in the treatment of Budd-Chiari syndrome are reviewed. PMID- 10730921 TI - Effect of gastric myoelectric activity on carbohydrate absorption of fruit juice in children. AB - Juices have a different rate of gastric emptying than other foods. This may alter the rate of delivery of carbohydrates to the small bowel for absorption. The aim of the study is to demonstrate that faster gastric emptying is associated with greater production of hydrogen through a randomized, crossover study of 39 healthy children. The electrogastrography (indicator of the gastric myoelectric activities) and breath hydrogen tests (indicator of carbohydrate malabsorption) were performed at baseline and after ingestion of 240 to 330 mL of grape or pear juice given in a random order. The cutaneous electrogastrogram was analyzed by running spectral analysis to compute pre- and postprandial period dominant power (PDP) and running spectrum total power (RSTP). Postprandial PDP and RSTP were higher (p < 0.02) in the pear juice group than in the grape juice group, suggesting higher antral myoelectric activities. Twenty three percent of the subjects had significant movement artifacts that suggested discomfort after drinking pear juice compared to 5% after grape juice (p < 0.03). Breath hydrogen test was more frequently positive (increase >20 part per million [ppm] above baseline) after pear juice (52.2%; mean, 36 +/- 33 ppm) than after grape juice (4.3%, 6 +/- 6 ppm). In a multiple regression analysis, the most predictive independent variable of hydrogen concentration was found to be either postprandial PDP (r2 = 0.24; p < 0.002), or RSTP (r2 = 0.37; p < 0.001). Juices affect gastric myoelectric activity. Grape juice induces lower antral myoelectric activities and is better absorbed. The malabsorption of carbohydrates of juices is in part related to their effect on the gastric physiology. PMID- 10730922 TI - Secretion and tissue content of eosinophil cationic protein in Crohn's disease. AB - To define further the role of eosinophils in the pathogenesis of Crohn's disease, the secretion rate and tissue content of eosinophilic cationic protein (ECP) were measured in colorectal samples. Mucosal biopsies were obtained from 22 controls and 20 patients with Crohn's disease during colonoscopy. After measurement of the initial basal ECP, release samples were incubated using a mucosa oxygenation system. Spontaneous and antiimmunoglobulin E (anti-IgE)-induced secretions of ECP were measured at different time points. For detection of the remaining tissue, ECP amount samples were mechanically homogenized after the incubation period. ECP measurement was performed by radioimmunoassay. Spontaneous ECP secretion rates during the incubation period were similar in all patient groups, whereas the initial basal ECP secretion was significantly increased in tissue affected by Crohn's disease. After stimulation with anti-IgE, ECP secretion was increased two fold in controls and three-fold in patients with Crohn's disease. In tissue affected by Crohn's disease, ECP levels were found to be significantly increased in most segments of the lower gastrointestinal tract with the highest ECP concentrations in affected mucosa. This functional study demonstrates an enhanced immunologically mediated ECP secretion and an accumulation of ECP in the intestinal mucosa of Crohn's disease, indicating a local upregulation of eosinophils. PMID- 10730923 TI - Pelvic floor study in patients with obstructive defecation: influence of biofeedback. AB - The aim of this study was to evaluate the pathophysiologic abnormalities in patients with obstructive defecation or dyssynergia and to assess the role of biofeedback treatment. Three groups were studied. Group A had 24 patients with obstructive defecation; B, 25 patients with constipation; and C, 22 healthy volunteers. Rectosigmoid segmental transit time of group A was 28.5 hours (SD +/- 13.4); B, 17.2 hours (SD +/- 11.5); and C, 8.5 hours (SD +/- 6.3) (p < 0.05). There was no statistical difference in resting and squeezing anal pressure among the three groups. Anorectal angle at rest revealed no difference among the three groups. At strain, a statistically significant difference between groups A and C (p < 0.05) and a marginal difference between groups A and B was noted. Rectocele of the anterior rectal wall was present at strain in 17/24 patients of group A and 7/22 patients of group C (p < 0.05). Electromyography during strain revealed abnormal contractions of puborectalis muscle and external anal sphincter, in 13 and 14 patients of group A, respectively, which differed from that observed in groups B and C (p < 0.001). Biofeedback treatment was applied with good results in 7 of 11 patients of group A. At six months, constipation relapsed in only one of treated patients. Patients suffering from obstructive defecation seem to have slower rectosigmoid transit time than the others. Defecography shows smaller anorectal angle at strain and rectocele of the anterior rectal wall more often. Abnormal pelvic floor contraction at strain is often noted in anal electromyography. Some of these patients seem to respond favorably to biofeedback treatment. PMID- 10730924 TI - Adenosine deaminase isoenzymes and neopterin in liver cirrhosis. AB - The aim of this study was to define the pattern of neopterin and ADA isoenzymes in liver cirrhosis. A total of 117 patients with liver cirrhosis were included. Serum levels of ADA were assayed in the presence and absence of a specific inhibitor for ADA1. Serum neopterin was measured using a competitive enzyme linked immunosorbent assay. The grade of liver insufficiency was assessed according to the Child-Pugh classification and the monoethylglycinexylidide test. Serum ADA, ADA1, ADA2 and neopterin were higher in cirrhotic patients than in control subjects. A stepwise increase in serum ADA level was observed with increasing severity of liver cirrhosis. The probability of ADA2 being greater than the mean was approximately 2.5 times higher (2.48, CI 95%: 1.36-4.52) in patients with liver cirrhosis due to hepatitis C virus (HCV) infection than in those patients with cirrhosis of a different etiology. No correlation was found between ADA2 and neopterin. Our data show that liver insufficiency and HCV infection increase the serum levels of ADA and its major isoenzyme ADA2. Furthermore, ADA isoenzyme determination adds no value to total ADA value. The absence of a correlation between ADA2 and neopterin suggests that different physiologic processes are involved in their increase. PMID- 10730925 TI - Reappraisal of biochemical hepatitis C activity in hemodialysis patients. AB - We reappraised biochemical hepatitis C activity in hemodialysis patients in comparison with normal controls. A total of 111 hemodialysis patients and 66 healthy volunteer blood donors with hepatitis C virus (HCV) infection were consecutively enrolled. Serum alanine aminotransferase (ALT) levels were normal (< or =45 U/L) in 103 (93%) hemodialysis patients and 34 (52%) donors (p < 0.001). HCV viremic levels were lower in the hemodialysis group (p = 0.044), with no difference in the HCV genotype prevalence. During two-year follow-up, 60 (67%) of 90 hemodialysis patients and 13 (26%) of 50 donors showed persistently normal ALT levels (p < 0.001). For hemodialysis patients, however, the upper normal limit of ALT activity was reset at 25 U/L corresponding to the mean + 2 x SD for the normalized ALT distribution in 400 control patients. The adjusted ALT levels were initially normal in 73 (66%) hemodialysis patients and persistently normal in 19 (21%). Thus, ALT levels were the same for the two groups. GB virus C (GBV C)/hepatitis G virus (HGV) coinfection found only in the hemodialysis group (10/111) had no influence on the disease. A relationship was noted between low disease activity and female gender in both groups. These findings indicate that biochemical hepatitis C activity in hemodialysis patients is similar to that in normal controls and should be monitored based on adjusted ALT levels. PMID- 10730926 TI - The beneficial effect of mesalazine on esophageal ulcers in intestinal Behcet's disease. AB - Intestinal Behcet's disease in a 19-year-old girl was diagnosed because of the history of recurrent oral aphthous ulcers and typical endoscopic findings of esophageal and ileal ulcers. Her symptoms (e.g., dysphagia and retrosternal pain) were gradually relieved by treatment with prednisolone and total parenteral nutrition. However, about one month later, oral and esophageal ulcers appeared again. Mesalazine was added. Oral and esophageal ulcers healed promptly, and have not relapsed for about one year. Although mesalazine appears to act locally in the small intestine and colon, the therapeutic effect of mesalazine in this case may be explained by the systemic antiinflammatory effect. This case suggests that mesalazine is an effective drug and is a good candidate in the treatment of intestinal Behcet's disease, especially accompanied with esophageal involvement. PMID- 10730927 TI - Granular cell tumors of the colon: report of a case and review of the literature. AB - Granular cell tumors are uncommon, usually benign tumors that can be located anywhere in the body. They commonly occur in the oral cavity and in subcutaneous tissue. In the gastrointestinal tract, granular cell tumors are uncommon and are quite rare in the colon. To date, 55 patients diagnosed with granular cell tumors of the colon have been reported in the literature, only 15 had multiple tumors. We describe the case of a 38-year-old man with a family history of colon cancer who was diagnosed with multiple colonic granular cell tumors after a screening colonoscopy. This seems to be the first report of this type. However, in our patient, the diagnosis of colonic granular cell tumors was incidental and there is no data that correlates adenomas or colorectal cancer with granular cell tumors of the colon. Finally, since granular cell tumors are usually benign, we suggest a conservative approach to patients with multiple granular cell tumors of the colon by means of endoscopic resection and a strict endoscopic follow-up. PMID- 10730928 TI - Oral tacrolimus (FK 506) in Crohn's disease complicated by fistulae of the perineum. AB - We describe the cases of two patients with Crohn's disease affected by severe perineal fistulae resistant to conventional therapies, successfully treated with FK 506, a new immunomodulatory drug. It is well absorbed from diseased bowel and preliminary experiences have indicated its short-term use in complicated Crohn's disease. The first patient was a 24-year-old male with perineal fistula and severe skin ulceration (8 cm of external opening diameter). He had undergone colectomy and ileostomy because of severe pancolitis refractory to medical treatment and had been treated with azathioprine and metronidazole. Two months after starting FK 506, a dramatic improvement made further surgical operation unnecessary. Local and general benefit was observed during the following 26 months, until FK 506 was withdrawn. The second patient was a 28-year-old male with a diagnosis of ulcerative pancolitis changed to Crohn's disease two months after the onset of a perineal fistula, recurring despite drainage procedures, steroid therapy, and total parenteral nutrition. FK 506 was administered for two months with a complete healing of fistula. Successively, it was stopped and corticosteroids (associated to enteral nutrition) were given because of recurrent rectal bleeding. Our experience encourages the use of oral FK 506 in complicated Crohn's disease and suggests the possibility of a long-term primary therapy other than the use as a "bridge" to other treatments. PMID- 10730929 TI - Budd-Chiari syndrome in a patient with ulcerative colitis: association with anticardiolipin antibodies. AB - This is the first reported case of a young patient with ulcerative colitis who developed acute Budd-Chiari syndrome and was found positive for anticardiolipin antibodies. Although an association between ulcerative colitis and Budd-Chiari syndrome, and between the latter and antiphospholipid antibodies, has been shown in the literature, the coexistence of these three conditions has never been reported. The patient we describe did poorly and was considered as a candidate for liver transplantation in spite of prompt heparin therapy, probably because of the presence of multiple risk factors. PMID- 10730930 TI - A case of sclerosing cholangitis managed by a percutaneous approach. AB - In 1992, a 61-year-old man who complained of recurrent episodes of fever and jaundice was diagnosed as having sclerosing cholangitis. In the three years that followed, the clinical picture progressively worsened; and, in 1995, the patient was hospitalized again for biliary obstruction. A liver transplantation was excluded because of concomitant severe coronary heart disease. A percutaneous transhepatic cholangiogram showed several critical strictures of the intrahepatic biliary tree and a temporary internal-external biliary drainage was placed to relieve the obstruction. After 40 days, a two-step percutaneous biliary balloon dilation was performed followed by topical steroid treatment through the catheter. After 45 days, the catheter was removed and steroid treatment tapered orally. In the three years that followed, the patient was well. He experienced only about 1-2 episodes of ascending cholangitis per year requiring antimicrobial therapy. Laboratory analysis showed a gradual improvement in hepatic chemistry, serum bilirubin, and erythrocyte sedimentation rate (ESR). In our patient, the association of percutaneous balloon dilation and topical steroid treatment improved both the clinical and radiological picture, without significant side effects. This approach should be considered a valuable and cost-effective option in primary sclerosing cholangitis, mainly for patients not eligible for liver transplantation. PMID- 10730931 TI - Idiopathic adulthood ductopenia: a diagnosis: two clinicopathologic courses. AB - Idiopathic adulthood ductopenia is a chronic cholestatic entity of unknown origin that was discovered in the past decade. Although it is classically a progressive and severe disease, a benign and responsive-to-therapy form has been reported recently. We present two middle-aged females that represent the extremes of the clinicopathologic spectrum: One with a severe form of the disease for whom liver transplantation was considered and the other with a benign form. PMID- 10730932 TI - Anti-HBc in hepatocellular carcinoma without HBsAg, HBV DNA, anti-HCV, and HCV RNA. PMID- 10730934 TI - Dormia basket fracture: an unusual complication of mechanical lithotripsy. PMID- 10730933 TI - Benign course of congestive cirrhosis associated with tricuspid regurgitation: does pulsatility protect against complications of venous hypertension? PMID- 10730935 TI - Acute appendicitis with an unusual complication. PMID- 10730936 TI - HCV hepatitis and polyarthritis: a therapeutic approach. PMID- 10730937 TI - A perspective on copper and liver disease in the dog. AB - Copper is a ubiquitous trace metal necessary for normal function of a variety of cellular proteins. Intracellular copper metabolism is complex, and only a few of the proteins/genes involved are known. Copper deficiency does not appear to be a clinical problem in dogs. Excess copper accumulation in the liver as a cause of hepatitis and cirrhosis was first demonstrated among Bedlington terriers. Subsequently, copper accumulation in the liver has been shown to occur in several other breeds of dogs. Excess hepatic copper has been found in dogs with normal liver histology, dogs with hepatitis, and dogs with end stage cirrhosis. Evidence is accumulating that copper is a cause of liver disease in breeds of dogs other than Bedlington terriers. Moreover, as more data are accumulated, the copper storage disease appears to have characteristics that are very similar among all of the affected breeds. PMID- 10730939 TI - A statistical model for assessing sample size for bacterial colony selection: a case study of Escherichia coli and avian cellulitis. AB - A general problem for microbiologists is determining the number of phenotypically similar colonies growing on an agar plate that must be analyzed in order to be confident of identifying all of the different strains present in the sample. If a specified number of colonies is picked from a plate on which the number of unique strains of bacteria is unknown, assigning a probability of correctly identifying all of the strains present on the plate is not a simple task. With Escherichia coli of avian cellulitis origin as a case study, a statistical model was designed that would delineate sample sizes for efficient and consistent identification of all the strains of phenotypically similar bacteria in a clinical sample. This model enables the microbiologist to calculate the probability that all of the strains contained within the sample are correctly identified and to generate probability-based sample sizes for colony identification. The probability of cellulitis lesions containing a single strain of E. coli was 95.4%. If one E. coli strain is observed out of three colonies randomly selected from a future agar plate, the probability is 98.8% that only one strain is on the plate. These results are specific for this cellulitis E. coli scenario. For systems in which the number of bacterial strains per sample is variable, this model provides a quantitative means by which sample sizes can be determined. PMID- 10730938 TI - Apoptotic and proliferation indexes in canine lymphoma. AB - Proliferative and apoptotic fractions of tumors were evaluated in 41 dogs with lymphoma for prediction of response to chemotherapy. All dogs had advanced clinical stage tumors, were untreated prior to study, and received identical induction-remission chemotherapy. Tumor cell proliferation was determined in all pretreatment biopsy specimens and in 18 specimens collected at the time of clinical relapse from remission. Quantitative measures included mitotic index and immunoreactivities for proliferating cell nuclear antigen (PCNA) and Ki-67. Apoptotic index was evaluated from 40 dogs pretreatment and from 16 dogs at the time of first relapse. Pretreatment tumor values for Ki-67, PCNA, and apoptosis were compared with posttreatment values. The median first relapse-free interval (RFI) and overall survival (OS) time were 174 days and 445 days, respectively. Of the proliferation markers, only the results of the Ki-67 analysis were predictive for duration of the first RFI but not OS. Pretreatment apoptotic index was also predictive of the duration of first RFI but not OS. No significant predictive value for comparison of the pretreatment and postrelapse values was demonstrated. Ki-67 labeling index and apoptotic indexes were combined to form both a proliferation/apoptotic ratio (PAR) and a sum, or turnover index. Only the PAR was predictive for duration of first RFI on multivariate analysis. Other variables that were evaluated for their influence on treatment outcome included patient age, weight, gender, clinical stage, clinical substage, and tumor immunophenotype. Of these variables, only immunophenotype was found to be of value for predicting duration of first RFI and OS. PMID- 10730940 TI - Canines as sentinels for Lyme disease in San Diego County, California. AB - Prevalence of Lyme borreliosis in canine sentinels has been shown to correlate with infection in humans. One thousand canine sera (917 dogs, 83 coyotes) obtained from animal control authorities and area veterinarians were screened by ELISA for antibodies to Borrelia burgdorferi. Results were validated by Western blot and indirect fluorescent antibody (IFA) tests at referee laboratories. Criterion for a positive Western blot was presence of 5 of 10 of the most common antigen IgG bands; for IFA, >1:128 or the equivalent when correcting for interlaboratory variability. Twenty-two of 1,000 canines were confirmed serologically positive (21 dogs and 1 coyote; seroprevalence 2.3% and 1.2%, respectively). Lifestyle, breed size, gender, and age were not statistically predictive of seropositive status. No regional clustering of seropositive animals was detected. The low prevalence of seropositivity in sentinel canines suggests the Lyme borreliosis hazard in San Diego County is minimal. PMID- 10730941 TI - Evaluation of a novel enzyme-linked immunosorbent assay for detection of antibodies against Salmonella, employing a stable coating of lipopolysaccharide derived antigens covalently attached to polystyrene microwells. AB - Polysaccharides derived from Salmonella typhimurium lipopolysaccharide (LPS) representing the O-antigen factors 1, 4, 5, and 12 and the O-antigen factors 6 and 7 from Salmonella choleraesuis LPS were derivatized with the photoreactive compound anthraquinone and subsequently covalently coupled to microtiter polystyrene plates by ultraviolet irradiation. Both polysaccharide antigens could be coupled simultaneously to the same microtiter plate. The coated surface was used in indirect ELISA for the determination of serum antibodies from pigs infected with bacteria of the two Salmonella groups and from uninfected pigs. This ELISA proved itself by having a good long-term durability and a high degree of reproducibility, including low day-to-day variations and low interplate variations. Furthermore, the ELISA showed good specificity and sensitivity when data were compared with the optical density levels of a panel of pig sera as determined by a conventional ELISA on the basis of passive coating of the two Salmonella LPS antigens (the mix-ELISA). The covalent anthraquinone mix-ELISA shows promise as a stable and durable alternative to the existing conventional ELISA for serological surveillance of Salmonella infections in pigs. PMID- 10730942 TI - Evaluation of solid-phase chemiluminescent enzyme immunoassay, enzyme-linked immunosorbent assay, and latex agglutination tests for screening toxoplasma IgG in samples obtained from cats and pigs. AB - Serum samples from cats and pigs were analyzed by the solid-phase chemiluminescent enzyme immunoassay (SPCEI), enzyme-linked immunosorbent assay (ELISA), and indirect latex agglutination (ILA) methods. The SPCEI and ILA methods accurately analyzed Toxoplasma IgG (T-IgG) in both clinical and spiked samples from pigs and cats. The ELISA method accurately analyzed T-IgG in spiked samples from cats and pigs or clinical samples from pigs, but it did not accurately analyze T-IgG in clinical samples from cats. The antibody used in the ELISA kit did not cross-react with cat T-IgG. The SPCEI method that uses a stand alone automated analyzer provided quantitative analysis, whereas the ELISA and ILA methods provided qualitative or, at best, semiquantitative analysis of T-IgG. The SPCEI and ELISA methods were rapid (60-90 minutes for 30 samples), whereas the ILA method required 13-15 hours for 30 samples. Although the three methods accurately distinguished positive from negative samples, the ILA method yielded many weakly positive results that were not confirmed by either the ELISA or SPCEI method. Thus, the indirect agglutination tests may give nonspecific responses at lower T-IgG concentrations. PMID- 10730943 TI - Development of an enzyme-linked immunosorbent assay for the detection of antibody to epizootic hemorrhagic disease of deer virus. AB - An enzyme-linked immunosorbent assay has been developed to detect antibodies to epizootic hemorrhagic disease of deer virus (EHDV). The assay incorporates a monoclonal antibody to EHDV serotype 2 (EHDV-2) that demonstrates specificity for the viral structural protein, VP7. The assay was evaluated with sequential sera collected from cattle experimentally infected with EHDV serotype 1 (EHDV-1) and EHDV-2, as well as the four serotypes of bluetongue virus (BTV), BTV-10, BTV-11, BTV-13, and BTV-17, that currently circulate in the US. A competitive and a blocking format as well as the use of antigen produced from both EHDV-1- and EHDV 2-infected cells were evaluated. The assay was able to detect specific antibody as early as 7 days after infection and could differentiate animals experimentally infected with EHDV from those experimentally infected with BTV. The diagnostic potential of this assay was demonstrated with field-collected serum samples from cattle, deer, and buffalo. PMID- 10730944 TI - Lesions associated with postweaning multisystemic wasting syndrome in pigs from Prince Edward Island, Canada. PMID- 10730945 TI - Porcine postweaning multisystemic wasting syndrome in Korean pig: detection of porcine circovirus 2 infection by immunohistochemistry and polymerase chain reaction. AB - This report describes the first diagnosis of porcine circovirus (PCV) infection in weaned pigs with postweaning multisystemic wasting syndrome in Korea by immunohistochemistry and polymerase chain reaction. The most unique lesions were multifocal granulomatous inflammation affecting lymph nodes, liver, and spleen, characterized by infiltrates of epithelioid macrophages and multinucleated giant cells. Circoviral antigen was detected in formalin-fixed sections and was usually present in large, round, dendritic cells in the white pulp of spleen and remnants of follicles in lymph nodes. Lymphoid follicles in the tonsils also contained PCV antigen. A 530-bp DNA fragment of circovirus was successfully amplified from all tested lymph nodes, liver, and spleen. PMID- 10730946 TI - Neonatal enterocolitis associated with coronavirus infection in a foal: a case report. PMID- 10730947 TI - Isolation and genome characterization of porcine reproductive and respiratory syndrome virus in P.R. China. PMID- 10730948 TI - Nonsuppurative meningoencephalitis associated with Aleutian mink disease parvovirus infection in ranch mink. AB - Severe nonsuppurative meningoencephalitis associated with Aleutian mink disease parvovirus (ADV) infection was observed in adult ranch mink. Brain lesions included severe, locally extensive to coalescing lymphoplasmacytic meningoencephalitis with accompanying gliosis, satellitosis, and mild extension of inflammation into the leptomeninges. ADV was identified in mesenteric lymph node, spleen, brain, and liver of affected mink by polymerase chain reaction techniques. Sequences of the ADV isolate (TH5) revealed 2 unique residues in the region of the viral genome that determines pathogenicity. These findings suggest that certain strains of ADV may preferentially cause disease in the nervous system. ADV infection should be considered in the differential diagnosis of neurologic disorders in mink. PMID- 10730950 TI - Isolation of avian pneumovirus from an outbreak of respiratory illness in Minnesota turkeys. AB - Antibodies to avian pneumovirus (APV) were first detected in Minnesota turkeys in 1997. Virus isolation was attempted on 32 samples (28 tracheal swabs, 4 pools of trachea and turbinates) that were positive for APV by reverse transcriptase polymerase chain reaction (RT-PCR). The cell cultures used were chicken embryo fibroblast (CEF), Vero cells, and QT-35 cells. Five virus isolates were obtained from these samples, and the identity of the isolates was confirmed by RT-PCR. Four isolates were obtained by inoculation of CEF cells, and 1 isolate was obtained in QT-35 cells after 3-7 blind passages in cell cultures. Vero cells did not yield any isolate on primary isolation; however, all 5 isolates could be adapted to grow in Vero cells following primary isolation in CEF or QT-35 cells. This is the first report of isolation of APV in Minnesota and also the first report of primary isolation of APV in QT-35 cells. PMID- 10730949 TI - Halicephalobus gingivalis (H. deletrix) infection in two horses in southern California. AB - Two horses, a 16-year-old male Holsteiner and a 5-year-old male miniature horse, were diagnosed with halicephalobiasis at the California Veterinary Diagnostic Laboratory System, San Bernardino Branch, in April and June of 1998. Over a period of 4 weeks, the Holsteiner horse developed renal dysfunction, blepharospasm, and blindness in the right eye. A 15-cm-diameter mass was detected on ultrasound examination in the right kidney. Terminally, the animal developed seizures and was euthanized. The miniature horse had a 6-week-long illness characterized by testicular enlargement and uveitis. This animal developed ataxia and died. Necropsy examination revealed bilateral enlargement of the kidneys in both horses, petechial hemorrhages of the optic nerve (Holsteiner), and a diffusely firm and enlarged left testicle (miniature horse). Microscopic evaluation of tissues revealed granulomatous nephritis, optic neuritis, retinitis, and encephalitis in both horses and orchitis in only the miniature horse with intralesional rhabditiform nematodes. Halicephalobus gingivalis was found in the urine sediment of both animals and in semen of the Holsteiner horse. PMID- 10730951 TI - An outbreak of Klebsiella pneumoniae infection in dogs with severe enteritis and septicemia. PMID- 10730952 TI - Listeria monocytogenes septicemia in a Thoroughbred foal. AB - Listeria monocytogenes septicemia was diagnosed in a 6-day-old Thoroughbred foal. Primary clinical signs included fever, depression, diarrhea, and respiratory distress. Hematologic abnormalities included leukopenia, neutropenia, degenerative left shift, and hyperfibrinogenemia. Clinical chemistry and blood gas abnormalities included metabolic acidosis, hypoxemia, hypocapnia, hypoglycemia, and hyponatremia. Despite aggressive therapeutic intervention and intensive care, the foal died within 12 hours of admission. A postmortem examination was performed, and the primary gross lesion was bilaterally severe, focally extensive bronchopneumonia. Histopathology revealed severe subacute multifocal suppurative bronchopneumonia with necrotizing vasculitis and intralesional coccobacilli. Cultures of blood collected at admission and immediately prior to death were positive for L. monocytogenes, as were cultures obtained from lung and liver at necropsy. Immunohistochemical examination of formalin-fixed tissues revealed abundant intra- and extracellular L. monocytogenes antigen within the lung and intravascularly in multiple organs. PMID- 10730953 TI - Chromobacteriosis in a Chinese red panda (Ailurus fulgens styani). AB - An adult Chinese red panda (Ailurus fulgens styani) transported by airplane from Florida to a North Dakota zoo died 1 week after arrival. Grossly, an interscapular abscess, subcutaneous inflammation, lymphadenitis, and pulmonary abscesses were observed. Microscopic findings included necrotizing inflammation in liver, lung, lymph node, and spleen. Chromobacterium violaceum was cultured from the interscapular abscess, liver, lung, and spleen and was injected into Swiss Webster mice. These mice died 18 hours postinoculation, and C. violaceum was cultured from liver, lung, and spleen. Chromobacterium violaceum is a sporadically reported but highly virulent pathogenic bacterium of both animals and humans typically found as a soil and water inhabitant of tropical and subtropical regions. PMID- 10730954 TI - Diagnosis of sporotrichosis in a donkey using direct fluorescein-labeled antibody testing. AB - A 4-year-old female donkey residing in an open field in Indiana was admitted for evaluation of facial lesions of 2 years duration. Cytologic and histologic examination of exudate and tissue from the lesions revealed a pyogranulomatous inflammatory reaction with numerous yeasts. Sporothrix schenckii was suspected to be the infectious agent; however, multiple culture attempts did not provide positive identification of the organism. Serologic examination supported infection with S. schenckii. A specific direct immunofluorescent antibody test performed on paraffin-embedded tissue sections confirmed the organism as S. schenckii. Clinical signs resolved after appropriate iodide therapy. PMID- 10730955 TI - Comparison of virus isolation and reverse transcription polymerase chain reaction assay for detection of bovine viral diarrhea virus in bulk milk tank samples. AB - The use of a reverse transcriptase polymerase chain reaction (RT-PCR) assay to screen bulk milk tank samples for bovine viral diarrhea virus (BVDV) has proven to be a sensitive and economical means to evaluate the lactating animals in a herd. The assay is capable of detecting the presence of a single persistently infected animal within a group of several hundred cows. Over a 3-year period, 144 samples from 97 farms were tested for BVDV using an RT-PCR assay in conjunction with a classical virus isolation (VI) procedure to measure the relative effectiveness of the techniques. Virus could be detected with both methods when the milk from a single persistently infected animal was diluted 1:600 with the milk from a herd of BVDV-negative animals. Based on individual farms, there was an overall prevalence of 12.4% BVDV infection, and the correlation between the 2 assays was 95.9%. In terms of sensitivity, specificity, and turnaround time, RT PCR was superior to VI. However, of the 17 samples that were VI positive, 4 were RT-PCR negative. RT-PCR may not detect all naturally occurring BVDV isolates because they may contain minor sequence variations in the primer regions. VI and RT-PCR are both suitable for detection of BVDV in bulk milk samples when used independently, but to increase the probability of successful detection and to provide cross-checks against assay contamination, it is desirable to utilize both methods in parallel. PMID- 10730956 TI - Diagnosis of feline herpesvirus infection by immunohistochemistry, polymerase chain reaction, and in situ hybridization. AB - An adult domestic shorthair cat had severe chemosis due to purulent and necrotizing blepharitis and conjunctivitis. Purulent rhinitis, necrotizing glossitis, and dermatitis were also diagnosed. The cat was positive for feline immunodeficiency virus and feline leukemia virus. Histologically, intranuclear Cowdry type A inclusions were found within numerous epithelial cells adjacent to the lesions in skin, conjunctiva, and tongue. Electron microscopic examination revealed herpesviral particles within the lesions. Paraffin-embedded skin and tongue tissues were processed in a polymerase chain reaction, using primers to amplify a 306-bp region of the thymidine kinase gene of feline herpesvirus type 1, resulting in a distinct amplification product of the predicted size. The distribution of feline herpesvirus was demonstrated by immunohistochemistry and nonradioactive in situ hybridization. Positive immunostaining was found in nuclei and cytoplasm of numerous epithelial cells within and next to the lesions, whereas in situ hybridization, performed with a digoxigenin-labeled double stranded DNA probe, revealed hybridization signal only in nuclei of intact epithelial cells. Neither immunohistochemistry nor in situ hybridization showed feline herpesvirus type 1 in tissues of lungs, liver, spleen, intestine, or brain. PMID- 10730957 TI - Determination of absorbed dose to water in reference conditions for radiotherapy kilovoltage x-rays between 10 and 300 kV: a comparison of the data in the IAEA, IPEMB, DIN and NCS dosimetry protocols. AB - A comparison of four of the most commonly used dosimetry protocols for the determination of absorbed dose to water in therapeutic kilovoltage x-rays using an ionization chamber (IAEA TRS-277, IPEMB, DIN and NCS) has been carried out. Owing to the different energy ranges and HVLs recommended by each protocol, backscatter factors, water-to-air mass energy absorption coefficient ratios and perturbation correction factors have been recast to a common quality range that all protocols satisfy individually to make a comparison possible. The results of the comparison show that in the sometimes reduced quality range originally included by the different protocols, determinations of absorbed dose to water at all beam qualities agree to within +/-1.0% with that obtained using the second edition of the IAEA TRS-277 code of practice (1997). The extrapolation of data to a common beam quality range practically preserves the agreement for all the protocols except for that issued by the NCS at the extremes of the range, where differences of up to 1.8% and 1.4% have been found for low and medium energies respectively. In all cases the DIN protocol yields very good agreement with TRS 277. PMID- 10730958 TI - Experimental partial and total kerma coefficients for carbon deduced from microscopic cross sections at incident neutron energies below 75 MeV. AB - The double-differential cross sections for proton, deuteron, triton and alpha particle production in fast neutron induced reactions on carbon have been measured at several neutron energies from 25 to 75 MeV in the angular range from 20 degrees to 160 degrees. Experimental energy-differential, angle-differential and total cross sections are deduced, and consequently the partial and total kerma coefficients. The obtained experimental partial kerma coefficients are extrapolated down to the threshold energy of each measured reaction channel. Results on the experimental double differential, energy-differential and total cross sections are briefly presented. The deduced partial and total kerma coefficients of the present work are compared with previous measurements and theoretical predictions. PMID- 10730960 TI - A method of improving the spatial resolution of treatments that involve a multileaf collimator. AB - In this paper we present a novel method of reducing the dosimetric effects of the finite leaf width of a multileaf collimator (MLC) in conformal and intensity modulated radiotherapy (IMRT). This is achieved by decomposing the required high resolution fluence distribution into two orthogonal components, which are delivered with two leaf sweeps with head-twists differing by 90 degrees. Before the decomposition stage, a filter is applied to the required beam to force it to have a constant gradient in the two delivery directions. The component deliveries were found to be very spiky in nature, resulting in very inefficient delivery with the scanning leaves of our MLC. This method was evaluated using film dosimetry of four idealized beams: a 45 degree edge, a circle, a hemispherical intensity modulated beam (IMB) and a sine-like IMB. The measurements showed that this method had significantly reduced the effects of the 1 cm leaf width of our MLC at the 50% isodose level, but there was significant overdosage at the edge of the field and immediately inside the held edge. This method shows promise but further work is required before it may find clinical utility. PMID- 10730959 TI - Effective point of measurement of cylindrical ionization chambers for heavy charged particles. AB - Cylindrical ionization chambers are used for the determination of absorbed dose in beams of heavy charged particles, where the effective point of measurement, Peff (the point in depth to which the measured dose refers), is a priori not known. A measurement of Peff for a Farmer-type chamber in a carbon ion beam is presented. It is based on a comparison of relative depth dose curves measured with a cylindrical chamber and a plane-parallel Markus chamber. Both measurements were compared against another high-precision relative depth dose measurement using large-area plane-parallel chambers. For Peff, a value of 72 +/- 7% of the inner radius of the chamber is obtained. The relative depth dose curve for the cylindrical chamber is calculated using an averaging of the depth dose values over the curved inner surface of the active volume while taking account of the different depths of points on the inner surface. Within the measurement uncertainty of 0.2 mm the measurements agree well with the calculated Bragg curve for the Farmer chamber. The result for Peff is in correspondence with the value suggested in a new code of practice by the IAEA for protons and ions, and somewhat less than that suggested by Palmans for protons. The measurements show that cylindrical chambers are in general well suited for depth dose measurements in fields of heavy charged particles if the correct Peff is used. PMID- 10730961 TI - A solution to the long-object problem in helical cone-beam tomography. AB - This paper presents a new algorithm for the long-object problem in helical cone beam (CB) computerized tomography (CT). This problem consists in reconstructing a region-of-interest (ROI) bounded by two given transaxial slices, using axially truncated CB projections corresponding to a helix segment long enough to cover the ROI, but not long enough to cover the whole axial extent of the object. The new algorithm is based on a previously published method, referred to as CB-FBP (Kudo et al 1998 Phys. Med. Biol. 43 2885-909), which is suitable for quasi-exact reconstruction when the helix extends well beyond the support of the object. We first show that the CB-FBP algorithm simplifies dramatically, and furthermore constitutes a solution to the long-object problem, when the object under study has line integrals which vanish along all PI-lines. (A PI line is a line which connects two points of the helix separated by less than one pitch.) Exploiting a geometric property of the helix, we then show how the image can be expressed as the sum of two images, where the first image can be reconstructed from the measured CB projections by a simple backprojection procedure, and the second image has zero PI-line integrals and hence can be reconstructed using the simplified CB-FBP algorithm. The resulting method is a quasi-exact solution to the long-object problem, called the ZB method. We present its implementation and illustrate its performance using simulated CB data of the 3D Shepp phantom and of a more challenging head-like phantom. PMID- 10730962 TI - Exact analytical solution of the convolution integral equation for a general profile fitting function and Gaussian detector kernel. AB - One of the most important aspects in the metrology of radiation fields is the problem of the measurement of dose profiles in regions where the dose gradient is large. In such zones, the 'detector size effect' may produce experimental measurements that do not correspond to reality. Mathematically it can be proved, under some general assumptions of spatial linearity, that the disturbance induced in the measurement by the effect of the finite size of the detector is equal to the convolution of the real profile with a representative kernel of the detector. In this work the exact relation between the measured profile and the real profile is shown, through the analytical resolution of the integral equation for a general type of profile fitting function using Gaussian convolution kernels. PMID- 10730963 TI - Experimental kerma coefficients and dose distributions of C, N, O, Mg, Al, Si, Fe, Zr, A-150 plastic, Al203, AlN, SiO2 and ZrO2 for neutron energies up to 66 MeV. AB - Low-pressure proportional counters (LPPCs) with walls made from the elements C, Mg, Al, Si, Fe and Zr and from the chemical compounds A-150 plastic, AlN, Al2O3, SiO2 and ZrO2 were used to measure neutron fluence-to-kerma conversion coefficients at energies up to 66 MeV. The LPPCs served to measure the absorbed dose deposited in the gas of a cavity surrounded by the counter walls that could be converted to the absorbed dose to the wall on the basis of the Bragg-Gray cavity theory. Numerically the absorbed doses to the walls were almost equal to the corresponding kerma values of the wall materials. The neutron fluence was determined by various experimental methods based on the reference cross sections of the 1H(n, p) scattering and/or the 238U(n, f) reactions. The measurements were performed in monoenergetic neutron fields of energies of 5 MeV, 8 MeV, 15 MeV and 17 MeV and in polyenergetic neutron beams with prominent peaks of energies of 34 MeV, 44 MeV and 66 MeV. For the measurements in the polyenergetic neutron beams, significant corrections for the contributions of the non peak energy neutrons were applied. The fluence-to kerma conversion coefficients of N and O were determined using the difference technique applied with matched pairs of LPPCs made from a chemical compound and a pure element. This paper reports experimental fluence-to-kerma conversion coefficient values of eight elements and four compounds measured for seven neutron energies, and presents a comparison with data from previous measurements and theoretical predictions. The distributions of the absorbed dose as a function of the lineal energy were measured for monoenergetic neutrons or, for polyenergetic neutron fields, deduced by applying iterative unfolding procedures in order to subtract the contributions from non peak energy neutrons. The dose distributions provide insight into the neutron interaction processes. PMID- 10730964 TI - Comparative dosimetry in narrow high-energy photon beams. AB - A comparison of the response of different dosimeters in narrow photon beams (phi > or = 4 mm) of 6 and 18 MV bremsstrahlung has been performed. The detectors used were a natural diamond detector, a liquid ionization chamber, a plastic scintillator and two dedicated silicon diodes. The diodes had a very small detection volume and one was a specially designed double diode using two parallel opposed active volumes with compensating interface perturbations. The characteristics of the detectors were investigated both for dose distribution measurements, such as depth-dose curves and lateral beam profiles, and for output factors. The dose rate and angular dependence of the diamond and the two diodes were also studied separately. The depth-dose distributions for small fields agree well for the diamond, the scintillator and the single diode, while the measured dose maximum for the double diode is about 1% higher and for the liquid chamber about 1% lower than the mean of the others when normalized at a depth of 10 cm. The plastic scintillator and the liquid ionization chamber detect a penumbra width that is slightly broadened due to the influence of their finite size, while the double diode may even underestimate the penumbra width due to its small size and high density. When corrected for the extension of the detector volume a good agreement with Monte Carlo calculated beam profiles was obtained for the plastic scintillator and the liquid ionization chamber. Profiles measured with the diamond show an asymmetry when positioned with the smallest dimension facing the beam, while the double diode, the scintillator and the liquid chamber measure symmetric profiles irrespective of positioning. Significant differences in the output factors were obtained with the different detectors. The natural diamond detector measures output factors close to those with an ionization chamber (less than 1% difference) for field sizes between 3 x 3 and 15 x 15 cm2, but overestimates the output factors for large fields and underestimates the output factors for the smallest field sizes. The single and double diodes overestimated the output factor for large field sizes by up to 7 and 12% respectively due to the high content of low-energy photons. The double diode, and to some extent the single diode, also showed a relative increase in response compared with the more water equivalent liquid chamber and plastic scintillator at the smallest fields where there is a lack of lateral electron equilibrium. Both the plastic scintillator and the liquid chamber also show responses that deviate from the ionization chamber for larger field sizes. The major deviations can be explained based on the characteristics of the sensitive materials and the construction of the detectors. PMID- 10730965 TI - Radial dose functions for 103Pd, 125I, 169Yb and 192Ir brachytherapy sources: an EGS4 Monte Carlo study. AB - Radial dose functions g(r) in water around 103Pd, 125I, 169Yb and 192Ir brachytherapy sources were estimated by means of the EGS4 simulation system and extensively compared with experimental as well as with theoretical results. The DLC-136/PHOTX cross section library, water molecular form factors, bound Compton scattering and Doppler broadening of the Compton-scattered photon energy were considered in the calculations. Use of the point source approach produces reasonably accurate values of the radial dose function only at distances beyond 0.5 cm for 103Pd sources. It is shown that binding corrections for Compton scattering have a negligible effect on radial dose function for 169Yb and 192Ir seeds and for 103Pd seeds under 5.0 cm from the source centre and for the 125I seed model 6702 under 8.0 cm. Beyond those limits there is an increasing influence of binding corrections on radial dose function for 103Pd and 125I sources. Results in solid water medium underestimate radial dose function for low energy sources by as much as 6% for 103Pd and 2.5% for 125I already at 2 cm from source centre resulting in a direct underestimation of absolute dose rate values. It was found necessary to consider medium boundaries when comparing results for the radial dose function of 169Yb and 192Ir sources to avoid discrepancies due to the backscattering contribution in the phantom medium. Values of g(r) for all source types studied are presented. Uncertainties lie under 1% within one standard deviation. PMID- 10730966 TI - Linear programming based on neural networks for radiotherapy treatment planning. AB - In this paper, we propose a neural network model for linear programming that is designed to optimize radiotherapy treatment planning (RTP). This kind of neural network can be easily implemented by using a kind of 'neural' electronic system in order to obtain an optimization solution in real time. We first give an introduction to the RTP problem and construct a non-constraint objective function for the neural network model. We adopt a gradient algorithm to minimize the objective function and design the structure of the neural network for RTP. Compared to traditional linear programming methods, this neural network model can reduce the time needed for convergence, the size of problems (i.e., the number of variables to be searched) and the number of extra slack and surplus variables needed. We obtained a set of optimized beam weights that result in a better dose distribution as compared to that obtained using the simplex algorithm under the same initial condition. The example presented in this paper shows that this model is feasible in three-dimensional RTP. PMID- 10730967 TI - An efficient method of measuring the 4 mm helmet output factor for the Gamma knife. AB - It is essential to have accurate measurements of the 4 mm helmet output factor in the treatment of trigeminal neuralgia patients using the Gamma Knife. Because of the small collimator size and the sharp dose gradient at the beam focus, this measurement is generally tedious and difficult. We have developed an efficient method of measuring the 4 mm helmet output factor using regular radiographic films. The helmet output factor was measured by exposing a single Kodak XV film in the standard Leksell spherical phantom using the 18 mm helmet with 30-40 of its plug collimators replaced by the 4 mm plug collimators. The 4 mm helmet output factor was measured to be 0.876 +/- 0.009. This is in excellent agreement with our EGS4 Monte Carlo simulated value of 0.876 +/- 0.005. This helmet output factor value also agrees with more tedious TLD, diode and radiochromic film measurements that were each obtained using two separate measurements with the 18 mm helmet and the 4 mm helmet respectively. The 4 mm helmet output factor measured by the diode was 0.884 +/- 0.016, and the TLD measurement was 0.890 +/- 0.020. The radiochromic film measured value was 0.870 +/- 0.018. Because a single exposure measurement was performed instead of a double exposure measurement, most of the systematic errors that appeared in the double-exposure measurements due to experimental setup variations were cancelled out. Consequently, the 4 mm helmet output factor is more precisely determined by the single-exposure approach. Therefore, routine measurement and quality assurance of the 4 mm helmet output factor of the Gamma Knife could be efficiently carried out using the proposed single-exposure technique. PMID- 10730968 TI - Spectrophotometric assessment of pigmented skin lesions: methods and feature selection for evaluation of diagnostic performance. AB - This study documents the optical reflectance characteristics of pigmented skin lesions and evaluates their potential for improving the differential diagnosis of malignant melanoma from benign pigmented skin lesions. Optical reflectance spectra in the wavelength range 320-1100 nm were obtained from 121 lesions already selected by expert dermatologists as suspicious of malignancy. Characteristic differences in spectra from benign and malignant lesions were studied. Feature extraction showed significant differences between lesion groups classified by histology. Seven of the most relevant features were used in the discriminant analysis of reflectance spectra from 15 melanoma and 32 compound naevi which resulted in a sensitivity of 100% and specificity of 84.4% when compared with histology. This simple objective technique appears to perform as well as the expert dermatologist and may improve the diagnostic accuracy of non specialists such as trainees and GPs. Further prospective clinical study of reflectance spectrophotometry in a larger patient group is now required. PMID- 10730969 TI - Cryogen spray cooling during laser tissue welding. AB - Cryogen cooling during laser tissue welding was explored as a means of reducing lateral thermal damage near the tissue surface and shortening operative time. Two centimetre long full-thickness incisions were made on the epilated backs of guinea pigs, in vivo. India ink was applied to the incision edges then clamps were used to appose the edges. A 4 mm diameter beam of 16 W, continuous-wave, 1.06 microm, Nd:YAG laser radiation was scanned over the incisions, producing approximately 100 ms pulses. There was a delay of 2 s between scans. The total irradiation time was varied from 1-2 min. Cryogen was delivered to the weld site through a solenoid valve in spurt durations of 20, 60 and 100 ms. The time between spurts was either 2 or 4 s, corresponding to one spurt every one or two laser scans. Histology and tensile strength measurements were used to evaluate laser welds. Total irradiation times were reduced from 10 min without surface cooling to under 1 min with surface cooling. The thermal denaturation profile showed less denaturation in the papillary dermis than in the mid-dermis. Welds created using optimized irradiation and cooling parameters had significantly higher tensile strengths (1.7 +/- 0.4 kg cm(-2)) than measured in the control studies without cryogen cooling (1.0 +/- 0.2 kg cm(-2)) (p < 0.05). Cryogen cooling of the tissue surface during laser welding results in increased weld strengths while reducing thermal damage and operative times. Long-term studies will be necessary to determine weld strengths and the amount of scarring during wound healing. PMID- 10730970 TI - Modelling individual temperature profiles from an isolated perfused bovine tongue. AB - To predict the temperature distribution during hyperthermia treatments a thermal model that accounts for the thermal effect of blood flow is mandatory. The DIscrete VAsculature (DIVA) thermal model developed at our department is able to do so; geometrically described vessels are handled individually and the remaining vasculature is modelled collectively. The goal of this paper is to experimentally validate the DIVA model by comparing measured with modelled temperature profiles on an individual basis. Temperature profiles in an isolated bovine tongue heated with three hot water tubes were measured at three controlled perfusion levels, 0, 6 and 24 ml (100 g)(-1) min(-1). The geometries of the tongue, the hot water tubes, thermocouples and discrete vasculature down to 0.5 mm diameter were reconstructed by using cryo-microtome slices at 0.1 mm cubic resolution. This reconstruction of the experimental set-up is used for the modelling of individual profiles. In a no-flow agar-agar phantom, DIVA showed nearly perfect correspondence between measurements and simulations. In the isolated bovine tongue the correspondence at no flow was slightly disturbed due to geometrical distortion in the reconstruction of the experimental set-up. Measurements showed decreasing temperature profiles with increasing perfusion. DIVA correctly predicted this decrease in temperature as well as the thermal impact of a large vessel close to a thermocouple. Blood flow was modelled using discrete vasculature and using a heat sink model. Although at 24 ml (100 g)(-1) min(-1) correspondence between heat sink simulations and measurements was reasonable, modelling discrete vasculature yielded the best correspondence at both 6 and 24 ml (100 g)(-1) min(-1). The results strongly suggest that with accurate data acquisition DIVA can predict temperature profiles on an individual basis. For this kind of patient-specific treatment planning in the clinic, geometrical reconstruction of the anatomy, vasculature and the heating implant is necessary. MRI is capable of providing these data. Further research will be done on thermal simulations of actual clinical hyperthermia treatments. PMID- 10730971 TI - Performance of a PSPMT based detector for scintimammography. AB - In breast scintigraphy, compact detectors with high intrinsic spatial resolution and small inactive peripheries can provide improvements in extrinsic spatial resolution, efficiency and contrast for small lesions relative to larger conventional cameras. We are developing a pixelated small field-of-view gamma camera for scintimammography. Extensive measurements of the imaging properties of a prototype system have been made, including spatial resolution, sensitivity, uniformity of response, geometric linearity and energy resolution. An anthropomorphic torso phantom providing a realistic breast exit gamma spectrum has been used in a qualitative study of lesion detectability. A new type of breast imaging system that combines scintimammography and digital mammography in a single upright unit has also been developed. The system provides automatic co registration between the scintigram and the digital mammogram, obtained with the breast in a single configuration. Intrinsic spatial resolution was evaluated via calculation of the phase-dependent modulation transfer function (MTF). Measurements of extrinsic spatial resolution, sensitivity and uniformity of response were made for two types of parallel hole collimator using NEMA (National Electrical Manufacturers Association) protocols. Geometric linearity was quantified using a line input and least squares analysis of the measured line shape. Energy resolution was measured for seven different crystal types, and the effectiveness of optical grease coupling was assessed. Exit gamma spectra were obtained using a cadmium zinc telluride based spectrometer. These were used to identify appropriate radioisotope concentrations for the various regions of an anthropomorphic torso phantom, such that realistic scatter conditions could be obtained during phantom measurements. For prone scintimammography, a special imaging table was constructed that permits simultaneous imaging of both breasts, as well as craniocaudal views. A dedicated breast imaging system was also developed that permits simultaneous acquisition and superposition of planar gamma images and digital x-ray images. The intrinsic MTF is nonstationary, and is dependent on the phase relationship between the signal and the crystal array matrix. Averaged over all phases, the MTF is approximately 0.75, 0.57 and 0.40 at spatial frequencies of 1.0, 1.5 and 2.0 cycles per cm, respectively. The phase averaged line spread function (LSF) has a FWHM value of 2.6 mm. Following uniformity corrections, the RMS deviations in flood images are only slightly greater than is predicted from counting statistics. Across an 80 mm section of the active area, the differential linearity is 0.83 mm and the absolute linearity 2.0 mm. Using an anthropomorphic torso phantom with detachable breasts, scatter radiation similar to that observed exiting the breast of scintimammography patients was observed. It was observed that scattered gamma rays can constitute the majority of the radiation incident on the detector, but that the scatter-to primary ratio varies significantly across the field of view, being greatest in the caudal portion of the breast, where scatter from the liver is high. Using a lesion-to-breast concentration ratio of 6:1, a 1.0 cm3 simulated breast lesion was detectable in lateral images obtained with both the developmental camera and with a clinical camera, while a 0.35 cm3 lesion was detectable in neither. Utilization of the dual x-ray transmission, gamma emission breast imaging system greatly increases the conspicuity of scintimammographic lesions relative to prone imaging, as well as greatly facilitating the localization and identification of structures in the gamma image. The prototype imaging gamma detector exhibits spatial resolution superior to that of conventional cameras, and comparable uniformity of response and geometric linearity. Because of light losses in the crystals, the energy resolution is inferior to that of single crystal NaI(Tl) came PMID- 10730972 TI - Dosimetric implications of age related glandular changes in screening mammography. AB - The UK National Health Service Breast Screening Programme is currently organized to routinely screen women between the ages of 50 and 64, with screening for older women available on request. The lower end of this age range closely matches the median age for the menopause (51 years), during which significant changes in the composition of the breast are known to occur. In order to quantify the dosimetric effect of these changes, radiographic factors and compressed breast thickness data for a cohort of 1258 women aged between 35 and 79 undergoing breast screening mammography have been used to derive estimates of breast glandularity and mean glandular dose (MGD), and examine their variation with age. The variation of mean radiographic exposure factors with age is also investigated. The presence of a significant number of age trial women within the cohort allowed an extended age range to be studied. Estimates of MGD including corrections for breast glandularity based on compressed breast thickness only, compressed breast thickness and age and for each individual woman are compared with the MGD based on the conventional assumption of a 50:50 adipose/glandular composition. It has been found that the use of the conventional 50:50 assumption leads to overestimates of MGD of up to 13% over the age range considered. By using compressed breast thickness to estimate breast glandularity, this error range can be reduced to 8%, whilst age and compressed breast thickness based glandularity estimates result in an error range of 1%. PMID- 10730973 TI - Monitor unit calculation for an intensity modulated photon field by a simple scatter-summation algorithm. AB - An important issue in intensity modulated radiation therapy (IMRT) is the verification of the monitor unit (MU) calculation of the planning system using an independent procedure. Because of the intensity modulation and the dynamic nature of the delivery process, the problem becomes much more involved than that in conventional radiation therapy. In this work, a closed formula for MU calculation is derived. The approach is independent of the specific form of leaf sequence algorithms. It is straightforward to implement the procedure using a simple computer program. The approach is illustrated by a simplified example and is demonstrated by a few CORVUS (NOMOS Corporation, Sewickley, PA) treatment plans. The results indicate that it is robust and suitable for IMRT MU verification. PMID- 10730974 TI - Risk factors for post partum ovarian dysfunction in high producing dairy cows in Belgium: a field study. AB - An epidemiological study of risk factors for postpartal ovarian disturbances was carried out on 334 high-yielding dairy cows in 6 well-managed Belgian herds. Ovarian activity was closely monitored using progesterone profiles, based on twice weekly RIA-analysis for progesterone in milk fat, starting at 10 d after calving and continuing until the confirmation of a new pregnancy. Attention was focused on abnormal cyclicity during the preservice, postpartum period; cows were divided into 6 different categories. Three of these categories (normal profile, delayed cyclicity, and prolonged luteal phase) were of major importance and were analyzed using a multiple variable logistic regression model. Season of calving (stable vs pasture, odds ratio (OR)=5.7), an extended length of the previous dry period (> 77 vs < or = 63 d, OR=2.9), problem calvings (OR=3.6), abnormal vaginal discharge (OR=4.5), health problems during the first month of lactation (clinical disease, OR=5.4; ketosis, OR=11.3), and clinical parameters illustrating the appearance of a severe negative energy balance significantly increased the risk for delayed cyclicity before service. Parity (> or = 4 vs 1, OR=2.5), problem calvings (OR=2.9), occurrence of puerperal disturbances (OR ranged from 3.5 to 11.0), health problems during the first month of lactation (OR=3.1), and an early resumption of ovarian cyclicity after calving (< 19 d vs > 32 d, OR=2.8) increased the risk for prolonged luteal cycles before service. PMID- 10730975 TI - Assessment of sperm characteristics post-thaw and response to calcium ionophore in relation to fertility in Swedish dairy AI bulls. AB - The present study examined the relationship between bull sperm characteristics post-thawing, after swim-up, and after challenge to calcium ionophore in relation to fertility (56-d nonreturn rates) after artificial insemination (AI). Spermatozoa from 25 semen batches derived from 15 Swedish Red and White AI bulls were evaluated with regard to post-thaw motility, membrane integrity, and migration through a swim-up procedure. The swim-up separated spermatozoa were assessed in terms of sperm concentration, viability and capacitation status as well as their response to exogenous calcium ionophore (A23187). Acrosome reactions were evaluated by fluorescence microscopy and flow cytometry. Sperm motility and viability post-thawing were significantly correlated with fertility. For the swim-up separated semen, significant correlations to nonreturn rates were found for concentration, viability, number of viable spermatozoa and sperm capacitation status (Pattern F and Pattern B). The only parameter significantly correlated to fertility after the ionophore challenge was the percentage of acrosome-reacted spermatozoa with remaining equatorial fluorescence, as assessed by fluorescence microscopy, but not by flow cytometry. The regression analysis showed that combining the results of sperm membrane integrity assessment post thawing with those of capacitation status after swim-up provided the best prediction of fertility. The accuracy of prediction did not improve when these parameters were combined with the percentage of spermatozoa in which acrosome reaction was induced by ionophore challenge. PMID- 10730976 TI - Placental lactogen as a regulator of luteal cells function during pregnancy in sheep. AB - The luteotropic activity of ovine placental lactogen (oPL) on different days of gestation in ewes was assessed using in vitro methods. Corpora lutea (CL) harvested on Days 45, 70, 95, 120 and 135 of gestation and during parturition were enzymatically dispersed and plated on multiwell plates. After 48 h of incubation, all cultures were terminated and media were frozen for further steroid analysis. Cells were cultured in control medium, with addition of oPL alone, or in combination with PGE2 or PGF2alpha. Supplementation of culture media with oPL increased basal progesterone secretion by cells isolated on Days 45 and 70 of gestation. There was no effect on progesterone secretion by cells isolated on other days of gestation; PGE2 added to the culture media increased progesterone production only by cells isolated on Day 70 of pregnancy. Simultaneous oPL treatment with PGE2 had a statistically significant and stimulatory effect on progesterone production by luteal cells collected on Days 70 and 95 of pregnancy. In contrast, PGF2alpha alone in culture media decreased progesterone secretion by cells isolated on Days 45, 70 and 95 of gestation, while oPL plus PGF2alpha on Days 70 and 95 of gestation protected against luteolytic action of PGF2alpha. The results showed 1) a direct effect of the oPL on luteal cells isolated on Days 45 and 70 of gestation; 2) synergism between PL and PGE2 in progesterone production; by cells isolated on Day 70; 3) and a luteoprotective effect of oPL against the luteolytic action of prostaglandin F (PGF2alpha) observed on Days 70 and 95 of gestation. PMID- 10730977 TI - Evaluation of sperm acrosome reaction in the Asiatic elephant. AB - This study focuses on the effect of chemicals on acrosome reaction in elephant spermatozoa. Semen was collected at the Washington Park Zoo in Portland, Oregon, from an 11-yr-old Asian elephant by artificial vagina (7 ejaculates) and transported to Mahidol University in Bangkok in extender at 4 to 5 degrees C within 24 to 28 h. A total of 500 x 10(6) sperm/mL was used for the control and for each of the 4 treatment groups: 1) cAMP (0.1 mM); 2) caffeine (0.1 mM); 3) Penicillamine hypotaurine and epinephine, PHE (penicillamine 2 mM, hypotaurine 1 mM, epinephine 1 mM); and 4) heparin (10 microg/mL) at 39 degrees C for 2 h. Aliquots were removed and the sperm viability, abnormal morphology, and acrosome status were evaluated by triple stain technique. Transmission electron microscopy (TEM) was used to observe changes of the sperm head membrane in all treatment groups. Trypan blue reliably stained dead spermatozoa, while rose Bengal stained only the spermatozoa with intact acrosomes. The concentration of dead sperm cells was similar in the 4 groups. The percentages of live acrosome-reacted spermatozoa in the control and in groups treated with caffeine, PHE, cAMP and heparin were 19.5 +/- 4.3, 38.1 +/- 4.0, 34.8 +/- 3.7, 29.8 +/- 0.8 and 28.0 +/- 4.2, respectively. The acrosome reaction rate was higher in the treatment groups than in the control (P<0.05). Caffeine and PHE caused significantly higher acrosome reaction of the sperm head than cAMP or heparin (P<0.05). The electron micrographs showed that the acrosome reaction occurred by the presence of apical vesiculation. The results indicated that 1) the triple stain technique allowed for evaluation of both viability and acrosome reaction simultaneously in elephant spermatozoa,2) acrosome reaction occurred at a high rate in all 3 treatment groups. 3) the effects of caffeine and PHE were significantly higher (P<0.05) than of cAMP and heparin, and 4) the data obtained from the triple stain technique corresponded to those from TEM. PMID- 10730978 TI - Localization of apoptotic cells in the cystic ovarian follicles of cows: a DNA end labeling histochemical study. AB - We examined the frequency of apoptosis in cystic follicular cells to investigate the cause of the delay in regression of cystic follicles. Paraffin sections of healthy antral follicles, early and late atretic ones, and early and late cystic ones were stained using the terminal deoxynucleotidyl transferase (Tdt)-mediated biotinylated deoxyuridine triphosphates (dUTP) nick end-labeling (TUNEL) method to detect apoptotic cells. In the granulosa layer of early cystic and atretic follicles, TUNEL-positive cells were evident. In the theca interna of both early and late atresia, high frequencies of TUNEL-positive cells were observed. In the theca interna, a high frequency of TUNEL-positive cells was noted in the early cystic follicles, whereas their frequency decreased in late cystic follicles. These results suggest that apoptosis occurs in the granulosa and theca interna cells of cystic as well as atretic follicles, but the frequency of apoptosis in theca interna cells decreases in late cystic follicles, which may be responsible for the delay of follicular regression. PMID- 10730979 TI - The timing of parturition in the pig is altered by intravenous naloxone. AB - This experiment tested the hypothesis that opioid antagonists could influence the timing of the onset and progress of parturition in the pig. Primiparous pigs (gilts) received a jugular catheter on Days 104 to 106 of pregnancy. At 1400 h on Day 112 the gilts received 10 mg PGF2alpha, i.m. to induce parturition. At 1000 h on Day 113 (i.e., 20 h later) gilts received either saline (n=6), 1 mg/kg, i.v. naltrexone (n=4) or 1 mg/kg, i.v. naloxone (n=5). Blood samples were taken daily from Days 108 to 116. On Day 113, blood samples were taken hourly from 0500 to 0900 h and then every 30 min until 2400 h, or until the birth of the last piglet (BLP) (whichever was sooner) and assayed for progesterone, oxytocin (OT), cortisol and PRL. Additional blood samples for OT and cortisol assay were taken every minute from 0930 to 1100 h on Day 113 and for 30 min during parturition. Naloxone, but not naltrexone, delayed the onset of parturition relative to saline controls (by 14 h 21 min; P<0.05). Duration of parturition and rate of births were not significantly affected by treatment. Mean plasma OT increased in the 4 h following naloxone but not saline treatment, during which time OT plasma pulse amplitude was reduced in naloxone and naltrexone-treated animals relative to saline treated controls. The PRL secretion rose following treatment in saline treated animals, consistent with approaching parturition, but failed to rise in opioid antagonist treated animals. Progesterone concentrations remained elevated in naloxone-treated animals for longer than in the other groups. These data suggest that a rapid change in overall effect of parenteral administration of naloxone to parturient pigs occurs from delaying its onset when administered as in these experiments, to facilitating its progress when given during parturition (earlier experiments). The delay of onset of parturition may be mediated by interference with hypothalamic control of OT or PRL release. PMID- 10730980 TI - Temporal interrelationships among luteolysis, FSH and LH concentrations and follicle deviation in mares. AB - The effect of altered LH concentrations on the deviation in growth rates between the 2 largest follicles was studied in pony mares. The progestational phase was shortened by administration of PGF2alpha on Day 10 (Day 0=ovulation; n=9) or lengthened by daily administration of 100 mg of progesterone on Days 10 to 30 (n=11; controls, n=10). All follicles > or = 5 mm were ablated on Day 10 in all groups to initiate a new follicular wave. The interovulatory interval was not altered by the PGF2alpha treatment despite a 4-day earlier decrease in progesterone concentrations. Time required for growth of the follicles of the new wave apparently delayed the interval to ovulation after luteolysis. The FSH concentrations of the first post-ablation FSH surge were not different among groups. A second FSH surge with an associated follicular wave began by Day 22 in 7 of 11 mares in the progesterone group and in 0 of 19 mares in the other groups, indicating reduced functional competence of the largest follicle. A prolonged elevation in LH concentrations began on the mean day of wave emergence (Day 11) in the prostaglandin group (19.2 +/- 2.2 vs 9.0 +/- 0.7 ng/mL in controls; P<0.05), an average of 4 d before an increase in the controls. Concentrations of LH in the progesterone group initially increased until Day 14 and then decreased so that by Day 18 the concentrations were lower (P<0.05) than in the control group (12.9 +/- 1.6 vs 20.2 +/- 2.6 ng/mL). Neither the early and prolonged increase nor the early decrease in LH concentrations altered the growth profile of the second-largest follicle, suggesting that LH was not involved in the initiation of deviation. However, the early decrease in LH concentrations in the progesterone group was followed by a smaller (P<0.05) diameter of the largest follicle by Day 20 (26.9 +/- 1.7 mm) than the controls (30.3 +/- 1.7 mm), suggesting that LH was necessary for continued growth of the largest follicle after deviation. PMID- 10730981 TI - Termination of mid-gestation pregnancy in bitches with aglepristone, a progesterone receptor antagonist. AB - Six pregnancies were terminated in mid-gestation with aglepristone, a progesterone receptor antagonist, in 5 beagle bitches in order to determine the effects of aglepristone on plasma concentrations of prolactin and progesterone, the duration of the luteal phase, and the interestrous interval. In addition, the effects of aglepristone on the condition of the uterus and fetuses were examined by ultrasonography. After confirmation of pregnancy by ultrasonography, the dogs received 10 mg, s.c. aglepristone per kg body weight on 2 consecutive days at about 30 d post ovulation. Before, during and after treatment with aglepristone, plasma samples were collected for determination of the concentrations of prolactin and progesterone. The condition of the uterus and fetuses was assessed by ultrasonography the day before and at least 3 times a week for at least 2 wk after aglepristone administration. Termination of pregnancy occurred within 4 to 7 d after the start of aglepristone treatment, which was well tolerated, with no side-effects except slight vaginal discharge. The results of ultrasonographic examination indicated that aglepristone leads to abortion but not to fetal resorption. Elevated plasma concentrations of prolactin were observed during aglepristone treatment, while plasma progesterone levels remained unchanged. Pregnancy termination with aglepristone resulted in premature cessation of luteal function. In addition, the interestrous interval was shortened. The latter effects may be the consequence of actions of the progesterone receptor antagonist at the hypothalamus-pituitary level. In conclusion, aglepristone proved to be a safe and effective abortifacient in mid-gestation in the bitch. The results of the present study also indicated that aglepristone directly or indirectly influences pituitary function. PMID- 10730982 TI - Effects of oxytocin on follicular development and duration of the estrous cycle in heifers. AB - Holstein heifers were used to study effects of exogenous administration of oxytocin on luteal function and ovarian follicular development. Twelve heifers were monitored for 1 estrous cycle to confirm normal ovarian function. At the subsequent estrus, these animals were randomly assigned to 1 of 3 treatments: saline control, (Group 1, n=4), oxytocin (Group 2, n=4) and saline pregnant (Group 3, n=4). Group 2 received continuous infusion of oxytocin (1.9 mg/d) from Days 14 to 26 after estrus, while Groups 1 and 3 received saline infusion during the same period. Group 3 were artificially inseminated at estrus. Daily blood samples were collected for oxytocin and progesterone assay. Ovarian follicles and corpus luteum (CL) development were monitored daily by transrectal ultrasonography until Day 32 after estrus. Plasma progesterone (P4) concentrations prior to initiation of infusion were 7.6+/-1.3 ng/mL on Day 14. They then decreased to <1 ng/mL on Day 19 for Group 1 and on Day 28 for Group 2. The interestrous interval was longer (P <0.05) for heifers that received oxytocin infusion. During the infusion period P4 concentrations were not different (P >0.05) between Group 2 and 3 but declined gradually from Day 20 in Group 2 despite the presence of high plasma oxytocin concentrations. Control heifers had 2 waves of follicular growth, with the second dominant follicle ovulating. Three of the 4 oxytocin-infused animals had an additional wave, with the third dominant follicle ovulating. Oxytocin infusion had no effect on size of the ovulating follicle (P >0.05) and the number of Class 1 follicles (3 to 5 mm, P >0.1). Differences in the number of Class 2 follicles (6 to 9 mm) among treatments on Days 15 to 22 after estrus were not detected (P >0.1) except on Days 23 to 26, when Group 2 had fewer follicles than Group 3 (P <0.05). The results show that continuous infusion of oxytocin during normal luteolysis delays luteal regression without inhibiting follicular development. PMID- 10730983 TI - Effects of oxytocin on cloprostenol-induced luteolysis, follicular growth, ovulation and corpus luteum function in heifers. AB - Twenty-five normally cyclic Holstein heifers were used to examine the effects of oxytocin on cloprostenol-induced luteolysis, subsequent ovulation, and early luteal and follicular development. The heifers were randomly assigned to 1 of 4 treatments: Group SC-SC (n=6), Group SC-OT (n=6), Group OT-SC (n=6) and Group OT OT (n=7). The SC-SC and SC-OT groups received continuous saline infusion, while Groups OT-SC and OT-OT received continuous oxytocin infusion (1:9 mg/d) on Days 14 to 26 after estrus. All animals received 500 microg, i.m. cloprostenol 2 d after initiation of infusion (Day 16) to induce luteolysis. Groups SC-OT and OT OT received oxytocin twice daily (12 h apart) (0.33 USP units/kg body weight, s.c.) on Days 3 to 6 of the estrous cycle following cloprostenol-induced luteolysis, while Groups SC-SC and OT-SC received an equivalent volume of saline. Daily plasma progesterone (P4) concentrations prior to cloprostenol-induced luteolysis and rates of decline in P4 following the induced luteolysis did not differ between oxytocin-infused (OT-OT and OT-SC) and saline-infused (SC-SC and SC-OT) groups (P >0.1). Duration of the estrous cycle was shortened in saline infused heifers receiving oxytocin daily during the first week of the estrous cycle. In contrast, oxytocin injections did not result in premature inhibition of luteal function and return to estrus in heifers that received oxytocin infusion (OT-OT). Day of ovulation, size of ovulating follicle and time of peak LH after cloprostenol administration for oxytocin and saline-treated control heifers did not differ (P >0.1). During the first 3 d of the estrous cycle following luteal regression, fewer (P <0.01) follicles of all classes were observed in the oxytocin-infused animals. Day of emergence of the first follicular wave in heifers treated with oxytocin was delayed (P <0.05). The results show that continuous infusion of oxytocin during the mid-luteal stage of the estrous cycle has no effect on cloprostenol-induced luteal regression, timing of preovulatory LH peak or ovulation. Further, the finding support that an episodic rather than continuous administration of oxytocin during the first week of the estrous cycle results in premature loss of luteal function. The data suggest minor inhibitory effects of oxytocin on follicular growth during the first 3 d of the estrous cycle following cloprostenol-induced luteolysis. PMID- 10730984 TI - Effect of genetic merit, milk yield, body condition and lactation number on in vitro oocyte development in dairy cows. AB - The effects of milk yield, body condition score (BCS) and lactation number on the number of oocytes recovered and blastocysts formed were studied following in vitro maturation, fertilization and culture of bovine oocytes collected from 48 high and 46 medium genetic merit dairy cows in their first and third lactation. The cows were slaughtered between 125 and 229 d post partum. Ovaries were recovered, and 2- to 10-mm follicles were aspirated. Cleavage rate and number of blastocysts were determined at 44 h and 7 d after insemination, respectively. Oocytes from high genetic merit cows formed fewer blastocysts and had lower cleavage and blastocyst formation rates than those from medium genetic merit cows (0.36 +/- 0.19, 70.4 and 6.8% vs 0.85 +/- 0.22, 77.4 and 11.4%, respectively). The effect of milk production was tested by grouping cows in their third lactation into high and low groups. There was no difference in number of oocytes recovered and subsequent development into blastocysts between the cows in the high milk production group (4559 to 5114 kg, n = 20) and cows in the low yield (3162 to 3972 kg, n = 20) group (6.9 +/- 1.34 vs 8.9 +/- 1.32, respectively). The effect of BCS was tested by grouping cows in their first or third lactation into high and low groups. Cleavage and blastocyst formation rates were greater for oocytes from cows with a high BCS (3.3 to 4.0, n = 20) than a low BCS (1.5 to 2.5, n = 20) (75.7 vs 61.9% and 9.9 vs 3.0%, respectively). Cows in the first lactation yielded fewer oocytes (5.7 +/- 1.24) than cows in the third lactation (7.8 +/- 0.79). Thus, the quality of oocytes probably contributes to reduced fertility, often evident in high genetic merit dairy cows. PMID- 10730985 TI - Sexual behavior and pregnancy rate of Bos indicus bulls. AB - To study sexual behavior and pregnancy rate of Zebu bulls, 9 Brahman and 6 Nelore bulls were first examined for libido and serving capacity, semen characteristics and scrotal circumference, a week before being placed, individually, into a group of 16 estrus-syncronized heifers to observe mating activity. Scores for libido (0 to 10) and for serving capacity (based on the number of services per period) were assigned for each bull over a 15-min period. Estrus was synchronized with Norgestomet (3 mg, s.c. implant and 3 mg, i.m.) and estradiol valerate (5 mg, i.m.). The implants remained in place for 10 d, then 40 h after their removal a bull was placed with the heifers. Four Brahman and 6 Nelore bulls were monitored for 2 h while the remaining 5 Brahman bulls were observed for 30 h. Number of services and mounts per bull, the hour at which each event took place, and the identification of the heifers serviced were recorded. Pregnancy was diagnosed by transrectal palpation 50 to 60 d after mating. Mean libido score (+/- SEM) of the 15 bulls was 6.4 +/- 0.5, and mean serving capacity was 0.53 +/- 0.19; both of these measures were correlated with age (r = 0.78, P < 0.01, and r = 0.56, P < 0.05, respectively). In the 2-h trials, the number of mounts was correlated with the libido score (r = 0.73, P < 0.05), and the number of services was correlated with age (r = 0.76, P < 0.05) and with libido and serving capacity score (r = 0.91 and 0.92, P < 0.01). In the 30-h trials, the mean (+/- SEM) number of mounts and of services per bull was 74.4 +/- 21.1 and 14.6 +/- 0.5, respectively. The mount-to-service ratio and percentage of serving efficiency were 5.1 +/- 1.4, and 19.8 +/- 4%, respectively. Libido score was correlated with the mount-to-service ratio (r = -0.86, P < 0.10) and with serving efficiency (r = 0.84, P < 0.10). The percentage of heifers serviced, of those serviced 2 or more times and of those pregnant was 55 +/- 5.3; 47.1 +/- 6.4, and 59.2 +/- 5.8, respectively. Pregnancy rate was not correlated with any trait (P > 0.10). It was concluded that libido and the serving capacity test seem to indicate the mating potential of Zebu bulls over a relatively short period of time (e.g. 30 hours), in which resultant pregnancy rates are comparable to those of European bulls. PMID- 10730986 TI - Timing of insemination relative to ovulation in pigs: effects on sex ratio of offspring. AB - The objective of the present study was to identify effects of the interval between insemination and ovulation in pigs on the sex ratio and sex ratio dispersion of offspring. Crossbred sows that had farrowed 2 to 9 litters were weaned (Day 0) and came into estrus between Days 3 and 7 after weaning. Ultrasonography was performed every 6 h, from 12 h after the onset of estrus until ovulation had been observed. The sows were inseminated once at various intervals from the onset of estrus. At farrowing, the numbers of viable piglets and dead piglets were recorded per sow. In four 12-h intervals between insemination and ovulation (36 to 24 h before ovulation, 24 to 12 h before ovulation, 12 to 0 h before ovulation and 0 to 12 h after ovulation), the total number of piglets was (mean+/-SEM) 10.8+/-1.2 (n=15); 13.4+/-0.7 (n=23); 13.2+/ 0.9 (n=21); and 12.1+/-1.0 (n=16), respectively (P>0.05). The percentage of male piglets per litter in the four 12-h intervals was 52.1+/-3.6, 50.5+/-2.7, 54.9+/ 2.8 and 47.8+/-4.5, respectively (P>0.05). Sex ratio was not influenced by litter size (P>0.05), and its distribution was normally dispersed (i.e., as expected under a binomial distribution) in all 4 intervals between insemination and ovulation (P>0.05). PMID- 10730987 TI - Induction of superovulation and recovery of fertilized oocytes in prepubertal miniature pigs after treatment with PG600. AB - The purpose of this study was to examine whether superovulation can be induced by hormonal treatment with PG600 (400 IU eCG and 200 IU hCG) at the prepubertal stage in miniature pigs. In Experiment 1, 14 prepubertal miniature pigs received 1, 1/2 or 1/4 vial of PG600, im on Day 0 (the first day of treatment). Presentation of estrus was monitored thereafter. On Days 10 to 13 (i.e., 6 to 8 d after estrus), the number of corpora lutea (CL) and residual follicles was counted by an exploratory laparotomy. Injection of 1/2 vial of PG600 effectively induced estrus and ovulation in the pigs. In Experiment 2, 15 prepubertal miniature pigs that received 1/2 vial of PG600 were artificially inseminated into the uterus by an exploratory laparotomy at 100 to 104 h after PG600 injection. Oocytes were recovered from the oviducts at 121 to 145 h after PG600 administration. The oocyte recovery rate was 66% (15 oocytes/pig, average), and 84% of these were at the 1-cell stage. In Experiment 3, 5 prepubertal miniature pigs that received 1/2 vial of PG600, followed by 100 IU hCG 70 h later, were artificially inseminated into the uterus. Oocytes were recovered synchronously at 120 to 122 h after PG600 treatment. The recovery rate was 80% (17 oocytes/pig, average) and 90% of the oocytes recovered were at the 1-cell stage. These results suggest that superovulation of prepubertal miniature pigs can be induced by 1/2 vial of PG600 injection, and by the combined treatment with PG600 and hCG injection, the fertilized ova can be synchronously recovered at around 120 h after PG600 injection. This procedure may provide a useful system for biomedical research using the miniature pigs, especially for producing transgenic animals for use in human disease models. PMID- 10730988 TI - Exercise-related lower leg pain: an overview. PMID- 10730989 TI - A review of chronic exertional compartment syndrome in the lower leg. AB - Chronic exertional compartment syndrome (CECS) in the lower leg has been described as early as 1956. This review describes the five relevant anatomical compartments and the important clinical features on presentation used in diagnosis. Compartment pressure testing using various types of catheter is useful as a confirmatory investigation. Although the pathophysiology of this condition is poorly understood, current hypotheses are discussed. Surgical treatment is usually definitive and techniques for decompression of the five compartments are presented with comparison to available conservative treatments. PMID- 10730990 TI - Exercise-related leg pain: neurological perspective. PMID- 10730991 TI - Exercise-related lower leg pain: bone. PMID- 10730992 TI - Medial tibial stress syndrome. PMID- 10730993 TI - Exercise-related lower leg pain: vascular. PMID- 10730994 TI - Long-term exercise adherence after intensive rehabilitation for chronic low back pain. AB - PURPOSE: The purpose of this study was to examine exercise compliance in patients with chronic low back pain (CLBP) after participation in an intensive spine rehabilitation program. METHODS: Exercise behaviors in 122 consecutive subjects with CLBP who completed a program of quota based exercise were examined. Frequency per week of performance of four exercise activities, Oswestry disability scores, and visual analog scale (VAS) scores were assessed at evaluation, 3-month, and 12-month follow-up by patient-completed questionnaires. RESULTS: Percentage of patients responding to initial, 3-month, and 12-month questionnaires were 100%, 86%, and 71%, respectively. Frequencies of exercise behaviors were compared by Wilcoxon signed-rank test and were found to increase significantly between evaluation and 3 months (P < 0.000), and evaluation and 12 month follow-up (P < 0.000). The percentages of patients reporting three or more times weekly performance of the following activities at evaluation and at three month follow-up, respectively, were: 1) stretching for the back and legs, 35% and 93%; 2) aerobic exercise, 44% and 87%; 3) back-strengthening exercises, 15% and 82%; and, 4) weight training, 6% and 71%. Evaluation and follow-up Oswestry disability and visual analog scale (VAS) scores for back pain were compared using Student's t-test. Significant improvements (P < 0.000) were noted for each of these scales at 3-month follow-up that were maintained at 12-month follow-up. CONCLUSION: It is concluded that exercise behaviors can be increased and maintained in CLBP patients without adversely affecting pain or function. PMID- 10730995 TI - Hyperbaric oxygen therapy does not affect recovery from delayed onset muscle soreness. AB - PURPOSE: This study investigated whether hyperbaric oxygen therapy (HBOT) improves recovery after exercise-induced muscle injury. METHODS: Healthy male subjects (N = 24) were randomly assigned to either a placebo group or a HBOT group. Subjects were tested for maximal isometric strength (preexercise) of their right elbow flexors. Each subject then completed a high-force eccentric workout of the elbow flexor muscle group to induce delayed onset muscle soreness (DOMS). On the seven successive days after this workout, the subjects were exposed to a hyperbaric environment of 2.5 ATA for 60 min, inspiring either a normoxic mixture (P(I)O2 = 0.2 ATA; placebo group) or a hyperoxic gas mixture (P(I)O2 = 2.5 ATA; HBOT group). Before the eccentric workout and daily for the next 10 d, measurements were obtained regarding: maximal isometric muscle strength of the elbow flexor muscles, right upper arm circumferences, and rating of the perceived muscle soreness. RESULTS: Isometric strength decreased significantly from preexercise levels of 25.1 +/- 3.8 kp to postexercise levels of 12.0 +/- 4.6 kp, for the HBOT group, and from 24.6 +/- 3.4 kp to 12.5 +/- 3.7 kp, respectively, for the placebo group. Over the 10-d recovery period, there was no difference in the rate of recovery of muscle strength between the two groups. Perceived soreness peaked at about 48 h after exercise with no difference between groups. Also, the exercise-induced increases in arm circumference were similar in the two groups. CONCLUSIONS: These results indicate that HBOT is not an effective therapy for the treatment of DOMS. PMID- 10730996 TI - Intensity effect of active recovery from glycolytic exercise on decreasing blood lactate concentration in prepubertal children. AB - PURPOSE: Children's performance after intense exercise is known to recover faster than that of adults. However, very little is known about the physiological processes that differentiate children from adults in their recovery. The purpose of this study was to compare, in children, the decrease in blood lactate concentration ([La]) during various intensities of active recovery from highly intense exercise with that during passive recovery. METHODS: Subjects were 15 healthy, physically active, prepubertal, 9- to 11-yr-old boys (N = 8) and girls (N = 7). Subjects performed three 40-s cycling bouts at 150% peak oxygen consumption (VO2peak), with two 50-s rest intervals, followed by 2 min of passive recovery and 23 min of one of four randomly-assigned recovery levels: passive and 40%, 50%, and 60% VO2peak (RP, R40, R50, and R60, respectively). RESULTS: Mean values of peak [La] (by treatment) ranged between 9.9 +/- 1.5 and 10.8 +/- 2.0. Whereas HR and VO2 remained relatively higher, [La] decreased faster during all active recoveries compared with the passive mode. [La] during R60 was higher compared with [La] during R40. [La] was slightly higher in the first 10 min of R40 compared with R50, whereas from the 15th min onward, this difference was reversed. A similar pattern was seen in the boys and girls, separately. The calculated half-life of [La] was significantly higher during the passive compared with all three active recoveries, with no differences among the latter (22.0 +/- 5.0, 10.3 +/- 1.9, 10.5 +/- 2.2, and 11.5 +/- 2.1 min during RP, R40, R50, and R60, respectively). CONCLUSIONS: In summary, similar to the case in adults, the decrease in [La] after intense exercise in children is faster during active recovery compared with the passive mode. Further research is required to determine whether performance recovery parallels that of [La] in children and adults of both genders. PMID- 10730997 TI - Correlated neurocardiologic and fitness changes in athletes interrupting training. AB - PURPOSE: We studied nine male Dutch top marathon skaters during a 1-month interruption of their training schedules after their last contest in the winter to investigate a possible decline in baroreflex sensitivity. METHODS: Before and after this period, a maximal exercise test was done, and at days 0, 4, 7, 14, and 28 neurocardiologic measurement sessions--heart rate and noninvasive baroreflex sensitivity, recumbent and tilt--were performed. RESULTS: Interruption of training resulted in a significant and relevant decrease in the maximal oxygen uptake (from 65.7 +/- 5.8 to 61.6 +/- 4.7 mL O2 x kg(-1) x min(-1); P = 0.03), most likely associated with decreased competitive possibilities. Resting heart rate modestly increased (from 54.6 +/- 7.2 to 58.8 +/- 7.5 bpm), however, not significantly. Heart rate during 60 degrees tilt increased considerably (from 70.1 +/- 6.1 to 80.1 +/- 9.1 bpm; P = 0.01), possibly due to a decrease in blood volume and an increase in cardiopulmonary baroreflex gain. Arterial baroreflex sensitivity decreased significantly in the recumbent (from 13.3 +/- 5.4 to 9.8 +/ 3.8 ms x mm Hg(-1), P = 0.04), but not in the 60 degrees tilt position (from 6.7 +/- 2.0 to 6.0 +/- 2.5 ms x mm Hg(-1)). The relative decrease in baroreflex sensitivity and maximal oxygen uptake correlated significantly (r = 0.71, P = 0.02). CONCLUSIONS: In summary, our data show that correlated detrimental changes in fitness and baroreflex sensitivity are measurable in these athletes after a month of interruption of training. PMID- 10730998 TI - Safety of chronic exercise in a rat model of kidney disease. AB - PURPOSE: The objective of this study was to determine the effect of treadmill running on polycystic kidney disease (PKD) progression and bone mineral density in Han:SPRD-cy rats, an animal model of PKD. METHODS: Using a 2 x 2 design, normal and polycystic male rats were divided randomly into exercise and sedentary groups at 4 wk of age. The exercising group performed treadmill exercise (14 m x min(-1)) for 30 min 3 d x wk(-1) for 6 wk, whereas the control group remained sedentary. This 6-wk period represents the period of greatest cyst growth in this model. RESULTS: Both exercised and sedentary polycystic animals had significantly greater kidney weights, as well as greater concentrations of serum urea nitrogen and serum creatinine than control animals. Exercise did not alter these parameters in either normal or polycystic animals. In addition, bone mineral density and bone mineral content, determined by dual-energy x-ray absorptiometry, were not altered by exercise in these animals. Bone mineral content, however, was marginally lower in polycystic animals. CONCLUSIONS: These results support the safety of moderate exercise in PKD. Additional research in this area is needed since there may be other benefits that are derived from exercise in this population. PMID- 10730999 TI - Vagal and cardiac reactivity to psychological stressors in trained and untrained men. AB - PURPOSE: The aim of this study was to determine whether higher aerobic fitness is associated with enhanced vagal influences on the myocardium, resulting in moderation of chronotropic cardiac activity during psychological stress and recovery. METHOD: Heart period (HP) and respiratory sinus arrhythmia (RSA) were obtained from 10 aerobically trained (AT) and 10 untrained (UT) college-aged men at rest and during three contiguous psychological challenges and 3 min of recovery. Ratings of perceived stress were obtained at the end of the rest period, at the midpoint of each stressor, and at 30 s into recovery. Time series methods were used to quantify RSA from the beat-to-beat HP series. Responsivity was assessed both in terms of absolute levels of activity and phasic changes in activity (task or recovery minus baseline). RESULTS: Both groups reported similar levels of subjective stress throughout the experiment. The AT exhibited longer HP at rest and during psychological stress and recovery than did the UT. However, the groups did not differ on RSA at rest or during psychological stress and recovery, nor did they differ on phasic changes in RSA or HP during stress or recovery. Additionally, aerobic capacity was not correlated with absolute levels or phasic changes in RSA during psychological challenge for either group and, except in Min 2 for the UT, similar results were obtained for recovery. CONCLUSIONS: The results supported the hypothesis that, among young men, higher aerobic fitness is associated with longer HP at rest and during psychological stress and recovery. However, the lower cardiac chronotropic activation observed among the AT relative to the UT was not paralleled by a group difference in the amplitude of RSA. These results suggest that the group difference in HP was not mediated directly by the vagal mechanisms manifested in the amplitude of RSA. PMID- 10731000 TI - HSP expression in human leukocytes is modulated by endurance exercise. AB - PURPOSE: Temperature increase, oxidative stress, and inflammatory reactions after endurance exercise were expected to stimulate the synthesis of heat shock proteins (HSP) in peripheral blood leukocytes. Furthermore, it was of interest whether regular endurance training influences HSP expression. METHODS: The expression of HSP27, HSP60, HSP70, constitutive HSC70, and HSP90 in the cytoplasma and surface of lymphocytes, monocytes, and granulocytes of 12 trained athletes was analyzed by flow cytometry before and after (0, 3, and 24 h) a half marathon. Twelve untrained persons at rest were included as control. RESULTS: After the race, there was a significantly greater percentage of leukocytes expressing cytoplasmic HSP27, HSP60, and HSP70 (P < 0.01), whereas HSC70 and HSP90 remained unchanged. The fluorescence intensity increased significantly in monocytes for HSP27 (0 and 3 h) and HSP70 (0, 3, and 24 h) and in granulocytes, only 24 h postexercise for HSP70. The percent values of trained athletes at rest were significantly lower compared with untrained persons (P < 0,01). CONCLUSIONS: Strenuous exercise increased HSP expression in blood immediately after the run, indicating a protective function of HSP in leukocytes of athletes to maintain function after heavy exercise. The downregulation of HSP-positive cells in trained athletes at rest seems to be a result of adaptation mechanisms to regular endurance training. PMID- 10731001 TI - Vitamin E regulates changes in tissue antioxidants induced by fish oil and acute exercise. AB - PURPOSE: Prooxidant effects of fish oil supplementation could unfavorably affect the cardiovascular benefits of fish oil. We tested the effects of 8 wk vitamin E cosupplementation with fish oil on antioxidant defenses at rest and in response to exhaustive exercise in rats. METHODS: Rats (N = 80) were divided into fish oil, fish oil and vitamin E (FOVE), soy oil, and soy oil and vitamin E (SOVE) supplemented groups. For the vitamin E supplemented rats, corresponding groups (FOVE-Ex and SOVE-Ex) performed an acute bout of exhaustive exercise after the supplementation period. RESULTS: Fish oil supplementation increased the activity of catalase, glutathione peroxidase, and glutathione-S-transferase in the liver and red gastrocnemius (RG) muscle. Fish oil decreased liver total glutathione (TGSH) levels. Vitamin E supplementation decreased antioxidant enzyme activities to levels at or near those in SOVE in a tissue specific pattern. Vitamin E increased TGSH in liver, heart, and RG. Regression analysis showed TGSH to be a negative determinant of protein oxidative damage as measured by protein carbonyl levels in both liver and RG. Catalase activity was associated with liver lipid peroxidation as measured by thiobarbituric acid-reacting substances. The exercise induced decrease in hepatic TGSH tended to be less in FOVE versus SOVE. Exhaustive exercise also modulated tissue antioxidant enzymes. CONCLUSIONS: Vitamin E supplementation markedly decreased fish oil induced antioxidant enzyme activities in all tissues. Sparing of glutathione may be an important mechanism by which vitamin E decreased tissue protein oxidative damage. PMID- 10731002 TI - Plasma catecholamine and blood lactate responses to incremental arm and leg exercise. AB - PURPOSE AND METHODS: The present study was conducted to examine the pattern of plasma catecholamine and blood lactate responses to incremental arm and leg exercise. Seven untrained male subjects performed two incremental exercise tests on separate days in random order. One test consisted of 1-arm cranking (5W x 2 min(-1)), whereas the other exercise test was 2-leg cycling (20-25W x 2 min(-1)). Blood samples were obtained from the nonexercising arm during 1-arm cranking and from the same arm and vein during 2-leg cycling. Thresholds for blood lactate (T(La)), epinephrine (T(Epi)) and norepinephrine (T(NE)) were determined for each subject under both exercise conditions and defined as breakpoints when plotted as a function of power output. RESULTS: When the two modes of exercise were compared, T(La), T(Epi), and T(NE) were all significantly lower for 1-arm cranking than for 2-leg cycling (P < 0.01). During 1-arm cranking, T(La) (0.96 +/ 0.10 L x min(-1)), T(Epi) (1.02 +/- 0.07 L x min(-1)), and T(NE) (1.07 +/- 0.09 L x min(-1)) occurred simultaneously. During 2-leg cycling, T(La) (1.77 +/- 0.20 L x min(-1)), T(Epi) (1.74 +/- 0.17 L x min(-1)), and T(NE) (1.98 +/- 0.17 L x min(-1)) occurred at similar levels of VO2 and were not significantly different. The correlation observed between the VO2 measured at the T(La) and T(Epi) was 0.917 for arm and 0.929 for leg exercise (P < 0.001). The epinephrine concentration ([Epi]) obtained at the T(La) was not significantly different for arm (0.144 ng x mL(-1)) and leg (0.152 ng x mL(-1)) exercise. CONCLUSIONS: The breakpoint in plasma [Epi] shifted in an identical manner and occurred simultaneously with that of T(La) regardless of the mode of exercise (arm or leg). The Epi concentrations observed at the T(La) agree with those previously reported to produce a rise in blood lactate during Epi infusion at rest. These results support the hypothesis that a rise in plasma [Epi] may contribute to the breakpoint in blood lactate that occurs during incremental exercise. PMID- 10731003 TI - Familial risk ratios for high and low physical fitness levels in the Canadian population. AB - PURPOSE: To estimate the familial risk of being physically fit or unfit in Canada. METHODS: The sample consisted of 11,680 participants from 4144 nuclear families of the 1981 Canada Fitness Survey. Indicators of physical fitness included estimated physical working capacity at a heart rate of 150 beats x min( 1) derived from a step test (PWC150), hand grip strength, sit-ups, and trunk flexibility. Probands were defined as reference individuals who were physically fit (95th, 85th, and 75th percentiles) or physically unfit (25th, 15th, and 5th percentiles) for each fitness indicator, respectively. RESULTS: Standardized risk ratios, adjusted for age and sex (SRR), for spouses and first-degree relatives of probands exceeding the 95th percentile are 1.63 and 1.81 for PWC150, 2.38 and 3.16 for grip strength, 2.63 and 3.98 for sit-ups, and 2.59 and 3.56 for trunk flexibility, respectively, whereas the SRR for spouses and first-degree relatives of probands below the 5th percentile are 1.54 and 1.34 for PWC150, 1.83 and 1.85 for grip strength, 1.13 and 1.53 for sit-ups, and 1.42 and 1.84 for trunk flexibility, respectively. The familial risks tend to be greatest at the extremes (95th and 5th percentiles) and the risks for first-degree relatives of physically fit probands are generally greater than those for spouses for grip strength, sit ups and trunk flexibility, whereas those for PWC150 show no clear pattern. CONCLUSION: There is significant familial risk for being physically fit or unfit in the Canadian population. The pattern of SRR suggests that the familial risk for indicators of strength and flexibility may be due, in part, to genetic factors, whereas the shared family environment is largely contributing to the familial risk for PWC150. PMID- 10731004 TI - Leisure time physical activity, cardiorespiratory fitness, and plasma fibrinogen concentrations in nonsmoking middle-aged men. AB - PURPOSE: The relationship of both leisure time physical activity and predicted maximum oxygen consumption (VO2max) with plasma fibrinogen concentration was examined within a cohort of employed middle-aged men. METHODS: Analyses were performed on a subsample of 635 nonsmoking men (46.7 +/- 7.7 yr) who completed a preventive medical assessment between 1992 and 1996. RESULTS: Among nonsmokers, mean age-adjusted fibrinogen concentration decreased significantly with higher physical activity index (PAL) categories and quartiles of predicted VO2max (mL x kg(-1) x min(-1)) (both P = 0.001). Mean age-adjusted plasma fibrinogen concentrations were significantly different (P < 0.05) between inactive and vigorous PAI groups and extreme quartiles of predicted VO2max (mL x kg(-1) x min( 1)). These relationships were no longer significant after adjustment for the confounding effect of other ischemic heart disease risk factors. Stepwise multiple regression analyses showed that age, sum of skinfolds, and blood leukocyte count were the strongest predictors of plasma fibrinogen concentration. CONCLUSION: These data do not confirm a significant independent association of both physical activity and predicted VO2max (mL x kg(-1) x min(-1)) with fibrinogen concentrations among nonsmoking middle-aged men of similar high social class. PMID- 10731006 TI - Push-off mechanics in speed skating with conventional skates and klapskates. AB - PURPOSE: Personal and world records in speed skating improved tremendously after the introduction of the klapskate, which allows the foot to plantar flex at the end of the push-off while the full blade continues to glide on the ice. The purpose of this study was to gain insight into the differences in skating technique with conventional versus klapskates and to unveil the source of power enhancement using klapskates. METHODS: Ten elite speed skaters skated four 400-m laps at maximal effort with both conventional and klapskates. On the straight high-speed film, push-off force and EMG data were collected. An inverse dynamics analysis was performed in the moving reference plane through hip, knee, and ankle. RESULTS: Skating velocity increased 5% as a result of an increase in mean power output of 25 W when klapskates were used instead of conventional skates. The increase in mean power output was achieved through an 11-J increase in work per stroke and an increase in stroke frequency from 1.30 to 1.36 strokes x s(-1). The difference in work per stroke occurs during the final 50 ms of the push-off. This is the result of the ineffective way in which push-off forces are generated with conventional skates when the foot rotates about the long front end of the blade. No differences in muscle coordination were observed from EMG. CONCLUSION: A hinge under the ball of the foot enhances the effectiveness of plantar flexion during the final 50 ms of the push off with klapskates and increases work per stroke and mean power output. PMID- 10731005 TI - Calcaneal loading during walking and running. AB - PURPOSE: This study of the foot uses experimentally measured kinematic and kinetic data with a numerical model to evaluate in vivo calcaneal stresses during walking and running. METHODS: External ground reaction forces (GRF) and kinematic data were measured during walking and running using cineradiography and force plate measurements. A contact-coupled finite element model of the foot was developed to assess the forces acting on the calcaneus during gait. RESULTS: We found that the calculated force-time profiles of the joint contact, ligament, and Achilles tendon forces varied with the time-history curve of the moment about the ankle joint. The model predicted peak talocalcaneal and calcaneocuboid joint loads of 5.4 and 4.2 body weights (BW) during walking and 11.1 and 7.9 BW during running. The maximum predicted Achilles tendon forces were 3.9 and 7.7 BW for walking and running. CONCLUSIONS: Large magnitude forces and calcaneal stresses are generated late in the stance phase, with maximum loads occurring at approximately 70% of the stance phase during walking and at approximately 60% of the stance phase during running, for the gait velocities analyzed. The trajectories of the principal stresses, during both walking and running, corresponded to each other and qualitatively to the calcaneal trabecular architecture. PMID- 10731007 TI - Assessment of speed of human locomotion using a differential satellite global positioning system. AB - PURPOSE: The objective was to explore whether a satellite-based navigation system, global positioning system used in differential mode (DGPS), could accurately assess the speed of running in humans. METHODS: A subject was equipped with a portable GPS receptor coupled to a receiver for differential corrections, while running outdoors on a straight asphalt road at 27 different speeds. Actual speed (reference method) was assessed by chronometry. RESULTS: The accuracy of speed prediction had a standard deviation (SD) of 0.08 km x h(-1) for walking, 0.11 km x h(-1) for running, yielding a coefficient of variation (SD/mean) of 1.38% and 0.82%, respectively. There was a highly significant linear relationship between actual and DGPS speed assessment (r2 = 0.999) with little bias in the prediction equation, because the slope of the regression line was close to unity (0.997). CONCLUSION: the DGPS technique appears to be a valid and inconspicuous tool for "on line" monitoring of the speed of displacement of individuals located on any field on earth, for prolonged periods of time and unlimited distance, but only in specific environmental conditions ("open sky"). Furthermore, the accuracy of speed assessment using the differential GPS mode was improved by a factor of 10 as compared to non-differential GPS. PMID- 10731008 TI - Does fatigue induced by repeated dynamic efforts affect hamstring muscle function? AB - PURPOSE: The purpose of this study was to determine the effects of hamstring fatigue induced by repeated maximal efforts on hamstring muscle function during maximal sprint running. METHODS: Twelve subjects performed three maximal 40-m sprints during which time high-speed film of the subjects' sprint action and EMG of five lower extremity muscles were recorded (nonfatigued condition, NFC). Subjects then performed specific and general hamstring fatigue tasks followed by three final 40-m sprints (fatigued condition, FC) during which time high-speed film and EMG of the same muscles were again recorded. RESULTS: Statistical analysis of the kinematic data indicated the following significant (P < 0.05) changes in the subjects' running action from the NFC to the FC: decreased hip and knee flexion at maximum knee extension in the swing phase of the sprint cycle, decreased leg angular velocity immediately before foot-ground contact (FGC), and decreased angular displacement of the trunk, thigh, and leg segments during the late swing phase. Statistical analysis of the EMG data indicated a significant increase in the duration of hamstring activity and earlier cessation of rectus femoris activity during the swing phase of the sprint stride. CONCLUSIONS: It was concluded that these changes in the kinematic and EMG parameters of sprint running primarily served as protective mechanisms to reduce stress placed on the hamstring muscles at critical phases of the stride cycle. PMID- 10731009 TI - Effects of oral creatine supplementation on muscular strength and body composition. AB - PURPOSE: The purpose of this investigation was to examine the effects of 6 wk of oral creatine supplementation during a periodized program of arm flexor strength training on arm flexor IRM, upper arm muscle area, and body composition. METHODS: Twenty-three male volunteers with at least 1 yr of weight training experience were assigned in a double blind fashion to two groups (Cr, N = 10; Placebo, N = 13) with no significant mean pretest one repetition maximum (IRM) differences in arm flexor strength. Cr ingested 5 g of creatine monohydrate in a flavored, sucrose drink four times per day for 5 d. After 5 d, supplementation was reduced to 2 g x d(-1). Placebo ingested a flavored, sucrose drink. Both drinks were 500 mL and made with 32 g of sucrose. IRM strength of the arm flexors, body composition, and anthropometric upper arm muscle area (UAMA) were measured before and after a 6-wk resistance training program. Subjects trained twice per week with training loads that began at 6RM and progressed to 2RM. RESULTS: IRM for Cr increased (P < 0.01) from (mean +/- SD) 42.8 +/- 17.7 kg to 54.7 +/- 14.1 kg, while IRM for Placebo increased (P < 0.01) from 42.5 +/- 15.9 kg to 49.3 +/- 15.7 kg. At post-test IRM was significantly (P < 0.01) greater for Cr than for Placebo. Body mass for Cr increased (P < 0.01) from 86.7 +/- 14.7 kg to 88.7 +/- 13.8 kg. Fat-free mass for Cr increased (P < 0.01) from 71.2 +/- 10.0 kg to 72.8 +/- 10.1 kg. No changes in body mass or fat-free mass were found for Placebo. There were no changes in fat mass and percent body fat for either group. UAMA increased (P < 0.01) 7.9 cm2 for Cr and did not change for Placebo. CONCLUSION: Creatine supplementation during arm flexor strength training lead to greater increases in arm flexor muscular strength, upper arm muscle area, and fat-free mass than strength training alone. PMID- 10731010 TI - Relationship between energy deficits and body composition in elite female gymnasts and runners. AB - PURPOSE: The purpose of this study was to evaluate energy balance and body composition in 42 gymnasts (mean age = 15.5 yr) and 20 runners (mean age = 26.6 yr), all of whom were on national teams or were nationally ranked. METHODS: Athletes were assessed for body composition using DEXA and skinfolds, and energy balance was determined with a Computerized Time-Line Energy Analysis (CTLEA) procedure. RESULTS: Results from the CTLEA were assessed as the number of within day energy deficits (largest and frequency) and within-day energy surpluses (largest and frequency). There was a significant difference (P = 0.000) in the mean number of hourly energy deficits > 300 kcal experienced by gymnasts (9.45 +/ 6.00) and runners (3.70 +/- 5.34). There was also a significant difference (P = 0.001) in the mean number of hourly energy surpluses > 300 kcal experienced by gymnasts (1.40 +/- 3.04) and runners (6.20 +/- 5.50). The mean largest daily energy deficit was 743 (+/- 392) kcal for gymnasts and 435 (+/- 340) kcal for runners. The mean largest daily energy surplus was 239 (+/- 219) kcal for gymnasts, and 536 (+/- 340) kcal for runners. There was a significant relationship between the number of daily energy deficits > 300 kcal and DEXA derived body fat percent for gymnasts (r = 0.508; P = 0.001) and for runners (r = 0.461; P = 0.041). There was also a negative relationship between the largest daily energy surplus and DEXA-derived body fat percentage for gymnasts (r = 0.418; P = 0.003). Using the energy balance variables, age, and athlete type (artistic gymnast, rhythmic gymnast, middle-distance runner, long-distance runner) as independent variables in a forward stepwise regression analysis, a small but significant amount of variance was explained in DEXA-derived (P = 0.000; R2 = 0.309) and skinfold-derived (P = 0.000; R2 = 0.298) body fat percent by the number of energy deficits > 300 kcal and age. CONCLUSIONS: These data suggest that within-day energy deficits (measured by frequency and/or magnitude of deficit) are associated with higher body fat percentage in both anaerobic and aerobic elite athletes, possibly from an adaptive reduction in the REE. These data should discourage athletes from following restrained or delayed eating patterns to achieve a desired body composition. PMID- 10731011 TI - Effect of oral sodium loading on high-intensity arm ergometry in college wrestlers. AB - PURPOSE: The aim of this study was to examine the effect of 0.3 g x kg(-1) of NaHCO3, 0.21 g x kg(-1) of NaCl, and a low-calorie placebo control (PC) on high intensity arm ergometry in eight college wrestlers (aged 20.6 +/- 0.8 yr, body mass 70.4 +/- 2.1 kg). METHODS: Subjects performed eight 15-s intervals of maximal effort arm ergometry separated by 20 s of recovery cranking. Treatments were administered in a randomized, double-blind manner in two equal doses at 90 and 60 min before testing. Venous blood samples were withdrawn at baseline, preexercise, and postexercise intervals. RESULTS: Preexercise pH (7.33 +/- 0.01, 7.31 +/- 0.01, and 7.40 +/- 0.01) and base excess (2.41 +/- 0.35, 0.93 +/- 0.39, and 8.45 +/- 0.51) after PC and NaCl ingestion, respectively, were similar, whereas ingestion of NaHCO3 resulted in significantly higher values (P < or = 0.05). Postexercise pH (7.02 +/- 0.01, 7.02 +/- 0.03, and 7.09 +/- 0.03) and base excess (-13.29 +/- 0.96, -14.49 +/- 1.01, and -8.83 +/- 1.38) were significantly lower after both PC and NaCl ingestion compared with NaHCO3 ingestion. Postexercise plasma [lactate] was also greater in both PC and NaHCO3 trials (21.42 +/- 1.52, 20.07 +/- 1.39, and 22.65 +/- 1.77 mmol x L(-1)). However, peak power (370.7 +/- 26.0, 346.3 +/- 13.6, and 354.3 +/- 18.9 W) and total work accomplished in eight intervals (30.2 +/- 1.5, 29.6 +/- 1.1, and 29.9 +/- 1.1 kJ), and percent fatigue (31.0 +/- 2.7, 29.0 +/- 3.2, and 29.2 +/- 4.0%) were similar. CONCLUSIONS: These data contradict previous reports of ergogenic benefits NaHCO3 and NaCl administration before exercise and further suggest that performance in this type of activity may not be enhanced by exogenously induced metabolic alkalosis or sodium ingestion. PMID- 10731012 TI - Changes in stress and recovery in elite rowers during preparation for the Olympic Games. AB - PURPOSE: The purpose was to investigate changes in stress and recovery during preparation for the 1996 Atlanta Olympic Games. METHODS: Eleven elite rowers of the German National Rowing Team completed four times the Recovery-Stress Questionnaire for Athletes (RESTQ-Sport). The eight rowers who competed at the Olympic Games filled out the RESTQ-Sport a fifth time, 2 d before the preliminaries. RESULTS: Trend parameters revealed significant alterations of somatic components of stress (Lack of Engery, Somatic Complaints, Fitness/Injury) and recovery factors (Fitness/Being in Shape) over time that mirrored the average length of daily extensive endurance training sessions. Significant changes in the scales Conflicts/Pressure and Social Relaxation reflected interpersonal processes within the team. CONCLUSIONS: The importance of balancing training stress and recovery for an optimal performance development is highlighted as well as the potential of the RESTQ-Sport for training monitoring. PMID- 10731013 TI - Changes in glutamine and glutamate concentrations for tracking training tolerance. AB - PURPOSE: The purpose was to monitor high-performance athletes throughout training macrocycles and competitions and examine the changes in plasma glutamine (Gm) and glutamate (Ga) concentrations in order to develop a model of tolerance to training. METHODS: Plasma glutamine and glutamate concentrations of 52 National team athletes (31 male and 21 female) divided into male and female groups of speed skating, swimming, and cross-country skiing were measured in an early season rested condition to determine highest Gm and lowest Ga concentrations and over 2-4 macrocycles, which included heavy training to establish lowest Gm and highest Ga concentrations. RESULTS: In the rested condition, there were no differences within and between the male and female groups, excluding five athletes (OTA) who became overtrained in heavy training. The mean (+/-SD) Gm concentration was 585 +/- 54 micromol x L(-1), Ga concentration 101 +/- 16 micromol x L(-1), and Gm/Ga ratio 5.88 +/- 0.84 micromol x L(-1). The OTA had a significantly higher Ga concentration of 128 +/- 16 micromol x L(-1) and lower Gm/Ga ratio of 4.43 +/- 0.49 micromol x L(-1) than all the other groups. In heavy training, there was a significant decrease (P < 0.05) in Gm concentration to 522 +/- 53 micromol x L(-1), significant increase in Ga concentration to 128 +/- 19 micromol x L(-1) and significant decrease in Gm/Ga ratio to 4.15 +/- 0.57 micromol x L(-1). The OTA Gm concentration of 488 +/- 31 micromol x L(-1) was significant lower than only the male speed skating and swimming groups. However, the Ga concentration of 171 +/- 17 micromol x L(-1) and Gm/Ga ratio of 2.88 +/- 0.27 micromol x L(-1) were significantly higher and lower respectively than all other groups. CONCLUSIONS: Based on the changes in Gm and Ga concentration under different training conditions, we propose an athlete tolerance to training model where glutamine concentration reflects tolerance to volume of work and glutamate concentration reflects tolerance to high intensity training. We suggest that the Gm/Ga ratio may globally represent overall tolerance to training. PMID- 10731014 TI - High-carbohydrate versus high-fat diets: effect on body composition in trained cyclists. AB - PURPOSE: Recent research suggesting the benefits of high-fat diets for endurance athletes have been viewed with caution because of the potential negative health consequences, including increased adiposity. METHODS: We have examined the effects of a high-fat (HF, 50% of total energy from fat and 37% from carbohydrate) versus a high-carbohydrate (HC, 15% of total energy from fat and 69% from carbohydrate) diet on body fat, lean body mass, and bone mineral density (BMD) in 32 endurance-trained cyclists over a period of 3 months. Body composition was assessed by dual x-ray absorptiometry at baseline and after 3 months. RESULTS: Total and percent body fat, lean tissue mass, and body weight were not significantly different within and between each diet group from baseline to week 12. Total body BMD increased significantly within HF (P = 0.02), but nonsignificantly in HC, over the 12-wk period. CONCLUSIONS: In conclusion, during periods of endurance training when energy requirements are high, increasing the percentage of fat in the diet to approximately 50% of total energy does not increase body weight or adiposity because athletes are able to maintain energy balance. PMID- 10731015 TI - Field evaluation of the Computer Science and Applications, Inc. physical activity monitor. AB - PURPOSE: The purpose of this study was to evaluate the Computer Science and Applications, Inc. (CSA) activity monitor to quantify physical activity in free living subjects using an activity diary as the criterion measure. METHODS: Subjects also completed a 7-d physical activity recall at the end of the monitoring period. Nine male and 10 female subjects (mean, SD) (25.0, 3.6 yr) wore the CSA monitor for 7 consecutive days. On 3 of those days, subjects completed an activity diary (2 weekdays and 1 weekend day). Total kcal per day (Dkcal(tot)) was calculated from the self-reported diary classifications of the subject's activities. For the 3 days that coincided with the diary, total number of counts accumulated per day (cnt(tot)) was obtained from the monitor. RESULTS: The amount of activity per day recorded by the CSA monitor followed the same pattern of change as the activity diary. The cnt(tot) and Dkcal(tot) were significantly (P < or = 0.05) correlated on day 1 (r = 0.65), day 2 (r = 0.49), day 3 (r = 0.55), and for the 3 days pooled (r = 0.51). Subjects were classified as low, moderate, or highly active based on tertiles of kcal from the diary and counts from the CSA monitor. The percentage agreement between the CSA and the activity diary was 68.4% (Kappa = 0.53, P < 0.01). The number of minutes spent in the various activity categories were compared between instruments using an ANOVA model. The results of these analyses suggest that the CSA overestimated light activity and underestimated vigorous activity compared with the diary. CONCLUSION: In conclusion, the CSA monitor may be useful in a field situation where total physical activity and patterns of physical activity are the desired outcomes. PMID- 10731016 TI - Prediction of peak oxygen uptake in men using pulmonary and hemodynamic variables during exercise. AB - PURPOSE: Many attempts have been made to predict peak VO2 from data obtained at rest or submaximal exercise. Predictive submaximal tests using the heart rate (HR) response have limited accuracy. Some tests incorporate submaximal gas exchange data, but a predictive test without gas exchange measurements would be of benefit. Addition of stroke volume and pulmonary function (PF) measurements might increase the predictability of a submaximal exercise test. METHODS: In this study, an incremental exercise test (10 W x min(-1)) was performed in 30 healthy men of various habitual activity levels. Step-wise multiple regression analysis was used to isolate the most important predictor variables of peak VO2 from a set of measurements of PF: lung volumes, diffusion capacity, airway resistance, and maximum inspiratory and expiratory pressures; gas exchange; minute ventilation (V(E)), tidal volume (V(T)), respiratory exchange ratio (RER = carbon dioxide output divided by VO2); and hemodynamics (HR, stroke index (SI) = stroke volume/body surface area, and mean arterial pressure). These measurements were made at rest and during submaximal exercise. RESULTS: Using the set of PF variables (expressed as percentages of predicted), FEV1 explained 30% of the variance of peak VO2. No other PF variables were predictive. After addition of resting hemodynamic data, SI was included in the prediction equation, raising the predictability to 40%. At the 60-W exercise level, 48% of the variance in peak VO2 could be explained by SI and FEV1. At 150 W, the prediction increased to 81%. At this level VCO2/O2 (RER) also entered the prediction equation of peak VO2: 6.44 x FEV1(%) + 13.0 x SI - 1921 x RER + 2380 (SE = 142 mL x min(-1) x m(-2), P < 0.0001). Leaving out the gas exchange variable RER, maximally 64% of the variance in peak VO2 could be explained. CONCLUSION: In conclusion, inclusion of pulmonary function and hemodynamic measurements could improve the prediction accuracy of a submaximal exercise test. The submaximal exercise test should be performed until a level of 150 W is reached. Noninvasive stroke volume measurements by means of EIC have additional value to measurement of HR alone. Finally, measurement of gas exchange significantly improves the predictability of peak VO2. PMID- 10731017 TI - American College of Sports Medicine roundtable. The physiological and health effects of oral creatine supplementation. AB - Creatine (Cr) supplementation has become a common practice among professional, elite, collegiate, amateur, and recreational athletes with the expectation of enhancing exercise performance. Research indicates that Cr supplementation can increase muscle phosphocreatine (PCr) content, but not in all individuals. A high dose of 20 g x d(-1) that is common to many research studies is not necessary, as 3 g x d(-1) will achieve the same increase in PCr given time. Coincident ingestion of carbohydrate with Cr may increase muscle uptake; however, the procedure requires a large amount of carbohydrate. Exercise performance involving short periods of extremely powerful activity can be enhanced, especially during repeated bouts of activity. This is in keeping with the theoretical importance of an elevated PCr content in skeletal muscle. Cr supplementation does not increase maximal isometric strength, the rate of maximal force production, nor aerobic exercise performance. Most of the evidence has been obtained from healthy young adult male subjects with mixed athletic ability and training status. Less research information is available related to the alterations due to age and gender. Cr supplementation leads to weight gain within the first few days, likely due to water retention related to Cr uptake in the muscle. Cr supplementation is associated with an enhanced accrual of strength in strength-training programs, a response not independent from the initial weight gain, but may be related to a greater volume and intensity of training that can be achieved. There is no definitive evidence that Cr supplementation causes gastrointestinal, renal, and/or muscle cramping complications. The potential acute effects of high-dose Cr supplementation on body fluid balance has not been fully investigated, and ingestion of Cr before or during exercise is not recommended. There is evidence that medical use of Cr supplementation is warranted in certain patients (e.g.. neuromuscular disease); future research may establish its potential usefulness in other medical applications. Although Cr supplementation exhibits small but significant physiological and performance changes, the increases in performance are realized during very specific exercise conditions. This suggests that the apparent high expectations for performance enhancement, evident by the extensive use of Cr supplementation, are inordinate. PMID- 10731018 TI - Xanthone 6-hydroxylase from cell cultures of Centaurium erythraea RAFN and Hypericum androsaemium L. AB - Xanthone 6-hydroxylase activity was detected in the microsomal fractions from two plant cell cultures. The enzyme from cultured cells of Centaurium erythraea (Gentianaceae) exhibited absolute specificity for 1,3,5-trihydroxyxanthone as substrate, whereas xanthone 6-hydroxylase from cell cultures of Hypericum androsaemum (Hypericacaea) preferred the isomeric 1,3,7-trihydroxyxanthone but used 1,3,5-trihydroxyxanthone also to a small extent. Both xanthones were regioselectively hydroxylated in position 6. The xanthone 6-hydoxylases are cytochrome P450 monooxygenases, as shown by their dependence on NADPH and molecular oxygen and their inhibition by carbon monoxide and typical P450 inhibitors. In both cell cultures, xanthone accumulation was preceded by an increase in xanthone 6-hydroxylase activity. PMID- 10731020 TI - Vacuolar transport of the glutathione conjugate of trans-cinnamic acid. AB - Red beet (Beta vulgaris L.) tonoplast membrane vesicles and [14C]trans-cinnamic acid-glutatione were used to study the vacuolar transport of phynylpropanoid glutathione conjugates which are formed in peroxidase-mediated reactions. It was determined that the uptake of [14C]trans-cinnamic acid-glutathione into the tonoplast membrane vesicles was MgATP dependent and was 10-fold faster than the uptake of non-conjugated [14C]trans-cinnamic acid. Uptake of the conjugate in the presence of MgATP was not dependent on a trans-tonoblast H+-electrochemical gradient, because uptake was not affected by the addition of NH4Cl (1 mM; 0% inhibition) and was only slightly affected by gramicidin-D (5 microM; 14% inhibition). Uptake of the conjugate was inhibited 92% by the addition of vanadate (1 mM) and 71% by the addition of the model substrate S-(2,4 dinitrophenyl) glutathione (500 microM). Uptake did not occur when a nonhydrolyzable analog of ATP was used in place of MgATP. The calculated Km and Vmax values for uptake were 142 microM amd 5.95 nmol mg(-1) min(-1), respectively. Based on these results, phenylpropanoid-glutation conjugates formed in peroxidase-mediated reactions appear to be transported into the vacuole by the glutathione S-conjugate pump(s) located in the tonoplast membrane. PMID- 10731019 TI - L-Ascorbic acid and L-galactose are sources for oxalic acid and calcium oxalate in Pistia stratiotes. AB - Axenic Pistia stratiotes L. plants were pulse-chase labeled with [14C]oxalic acid, L[1-14C]ascorbic acid, L-6-14C]ascorbic acid, D-[1-14C]erythorbic acid, L [1-14C]galactose, or [1-14C]glycolate. Specific radioactivities of L-ascorbic acid (AsA), free oxalic acid (OxA) and calcium oxalate (CaOx) in labeled plants were compared. Samples of leaf tissue were fixed for microautoradiography and examined by confocal microscopy. Results demonstrate a biosynthetic role for AsA as precursor of OxA and its crystalline deposition product, CaOx, in idioblast cells of P. stratiotes and support the recent discovery of Wheeler, Jones and Smirnoff (Wheeler, G.L., Jones M.A., & Smirnoff, N. (1998). The biosynthetic pathway of vitamin C in higher plants. Nature, 393, 365-369) that L-galactose is a key intermediate in the conversion of D-glucose to AsA in plants. D-[1 14C]erythorbic acid (a diastereomeric analog of AsA) is utilized also by P. stratiotes as a precursor of OxA and its calcium salt deposition product in idioblasts. Labeled OxA is rapidly incorporated into CaOx in idioblasts, but microautoradiography shows there is also significant incorporation of carbon from OxA into other components of growing cells, contrary to the dogma that OxA is a relatively stable end product of metabolism. Glycolate is a poor substrate for synthesis of OxA and CaOx formation, further establishing AsA as th immediate precursor in the synthesis of OxA used for calcium precipitation in crystal idioblasts. PMID- 10731021 TI - Biotransformation of the Trichoderma metabolite 6-n-pentyl-2H-pyran-2-one by cell suspension cultures of Pinus radiata. AB - Cell suspension cultures of Pinus radiata metabolize the antifungal Trichoderma secondary metabolite 6-n-pentyl-2H-pyran-2-one (6PAP) (1) via hydroxylation of the pentyl side chain. Examination of the culture medium following dosing studies with 1 revealed that 79-85% of this bioactive compound had been metabolised after 144 h. At that time, 34-40% of the metabolized dose was recovered as a series of monohydroxylated isomers of 1, the principal metabolite being 5-(2-pyron-6 yl)pentan-5-ol (7). PMID- 10731022 TI - Occurrence and characterization of oils rich in gamma-linolenic acid Part I: Echium seeds from Macaronesia. AB - Nineteen species of the genus Echium (Fam. Boraginaceae) collected in Macaronesia were surveyed in a search for new sources of gamma-linolenic acid (GLA, 18:3omega6). High amounts of this acid were found in all of them, ranging from 9.15% (E. plantagineum) to 26.31% (E. callithyrsum) of total seed fatty acids. The amounts of GLA related to total seed weight were also significant, ranging from 1.77% (E. sventenii) to 5.02% (E. nervosum). In addition, considerable amounts of stearidonic acid (SA, 18:4omega3) were detected, ranging from 3.03% (E. auberianum) to 12.94% (E. plantagineum) of total fatty acids. These data allow us to consider tile members of the genus Echium from Macaronesia as one of the richest sources of gamma-linolenic acid found so far in nature. The results obtained from multivariable data analysis and the taxonomic relationships among the species is discussed. PMID- 10731023 TI - Anti-HIV-1 phorbol esters from the seeds of Croton tiglium. AB - Five phorbol diesters, together with three known ones, were isolated from a MeOH extract of the seeds of Croton tiglium, and their structures were determined by spectroscopic methods and selective hydrolysis of acyl groups. These compounds were assessed for their abilities to inhibit an HIV-induced cytopathic effect (CPE) on MT-4 cells and to activate protein kinase C (PKC) associated with tumor promoting action. 12-O-Acetylphorbol-13-decanoate and 12-O-decanoylphorbol-13-(2 methylbutyrate) effectively inhibited the cytopathic effect of HIV-1 [complete inhibitory concentration (IC100) values of 7.6 ng/ml and 7.81 microg/ml, and minimum cytotoxic concentration (CC0) value of 62.5 and 31.3 microg/ml, respectively]; however, 12-O-acetylphorbol-13-decanoate showed no activation of PKC at concentrations of 10 and 100 ng/ml. 12-O-Tetradecanoylphorbol-13-acetate (TPA) was found to be not only the most potent inhibitor of HIV-1-induced CPE (IC100 value of 0.48 ng/ml), but also the most potent activator of PKC (100% activation at 10 ng/ml). PMID- 10731024 TI - Delevoyin C, a tetranortriterpenoid from Entandrophragma delevoyi. AB - The hexane extract of the bark of Entandrophragma delevoyi has yielded a novel tetranortriterpenoid, delevoyin C. This compound contains a cyclobutanyl ring incorporating C-19 and a cycloheptanyl ring C including C-30. Gedunin and 11beta Acetoxygedunin were isolated from the hexane extract of the wood. PMID- 10731025 TI - Flavonoid glycosides and saponins from Astragalus shikokianus. AB - A new flavonol glycoside, kaempferol 3-O-alpha-L-rhamnopyranosyl -(1-->6)-[alpha L-rhamnopyranosyl-(1-->2)]-beta-D-galactopyranosyl-7-O-a lpha-L-rhamnopyranoside, named astrasikokioside I, was isolated from aerial part of Astragalus shikokianus, together with two flavonol glycosides, kaempferol 3-O-alpha-L rhamnopyranosyl-(1-->2)-beta-D-galactopyranosyl-7-O-alpha-L- rhamnopyranoside, robinin, and three triterpenoid glycosides, soyasaponin I, sophoraflavoside II and robinioside E. PMID- 10731026 TI - Sesquiterpene lactone glucosides from Sonchus asper. AB - From the methanolic extract of the roots of Sonchus asper, two new sesquiterpene glucosides, together with two known sesquiterpene glucosides and a known aglycone were isolated. Structures were identified as 11beta,13-dihydrourospermal A, 15-O beta-D-glucopyranosyl-11beta,13-dihydrourospermal A, 15-O-beta-D glucopyranosylurospermal A, 15-O-[6'-(p-hydroxyphenylacetyl)]-beta-D glucopyranosylurosperm al A and 14-O-methylacetal-15-O-[6'-(p hydroxyphenylacetyl)]-beta-D-+ ++glucopyranosylurospermal A, by spectroscopic means. PMID- 10731027 TI - Eudesmane derivatives from Tessaria integrifolia. AB - Five eudesmane-type sesquiterpenoids were isolated from the methanol extract of the aerial part of Tessaria integrifolia Ruiz. et Pavon (Compositae). Their structures were elucidated on the basis of spectroscopic analysis is well as chemical evidence. PMID- 10731028 TI - Steroidal glycosides from the aerial part of Asclepias incarnata. AB - The aerial part of Asclepias incarnata afforded 34 pregnane glycosides. These were confirmed to have lineolon, isolineolon, ikemagenin, 12-O nicotinoyllineolon, deacylmetaplexigenin, metaplexigenin, rostratamine, 12-O acetyllineolon, 15beta-hydroxylineolon and 15beta-hydroxyisolineolon moieties as their aglycones, and 2.6-dideoxyhexopyranose, glucopyranose and allopyranose as the corresponding sugar constituents. Their structures were determined using both spectroscopic and chemical methods. PMID- 10731029 TI - Iridoid glycosides from Lonicera quinquelocularis. AB - A new iridoid glycoside 6'-O-beta-apiofuranosylsweroside was isolated from the ethanolic extract of the roots of Lonicera quinquelocularis along with the known compounds loganin and sweroside. PMID- 10731030 TI - Four alkaloids, lucidine B, oxolucidine A, lucidine A, and lucidulinone from Lycopodium lucidulum. AB - The structures of four alkaloids extracted from Lycopodium lucidulum (Lycopodiaceae) were established by X-ray and 2D NMR spectroscopic analyses. The dihydro-derivative of oxolucidine A, which was obtained by NaBH4 reduction of oxolucidine A, was treated with p-bromobenzoyl chloride to afford crystals, whose X-ray crystallographic analysis established the stereostructure, including the absolute configuration. The 2D NMR spectra of tetrahydrodeoxylucidine B were fully analyzed to establish the full structure of lucidine B, and the hitherto unknown stereochemistry at the C-14 position was established as beta-H. The structure of a new alkaloid, lucidulinone, was determined by spectroscopic analysis to be luciduline lactam. PMID- 10731031 TI - A chalcone and a dihydrochalcone from Uvaria dulcis. AB - 2',3'-Dihydroxy-4',6'-dimethoxchalcone and the corresponding dihydrochalcone were isolated from the leaves of Uvaria dulcis and characterized by chemical and spectral methods. PMID- 10731032 TI - A biflavanone from Cycas beddomei. AB - A new biflavanone, 7,7"-di-O-methyltetrahydrohinokiflavone together with tetrahydrohinokiflavone were isolated from the stems of Cycas beddomei. The structures were established on the basis of spectral and chemical studies. PMID- 10731033 TI - Gibberellins in seedlings and flowering trees of Prunus avium L. AB - Extracts of acids from mature seeds, germinating seeds, first, second and third year seedlings as well as mature, flowering trees of sweet cherry (Prunus avium L. cv. Stella) were analysed by gas chromatography-mass spectrometry. The presence of the known gibberellins (GAs) GA1 (1), GA3 (4), GA5 (7), GA8 (11), GA19 (14), GA20 (12), GA29 (13), GA32 (5), GA85 (2), GA86 (3) and GA87 (6) was confirmed by comparison of their mass spectra and Kovats retention indices with those of standards or literature values. In addition, 16alpha,17-dihydrodihydroxy GA25 (16) was identified and its stereochemistry confirmed by rational synthesis. The 12alpha,13-dihydroxy GAs, GA32 (5), GA86 (2), GA86 (3) and GA87 (6), were detected in mature seeds, germinating seeds and young seedlings, but not in flowering plants. The 13-hydroxy GAs, GA1 (1) and GA3 (4), were present in germinating seeds and, in addition to these, GA5 (7), GA8 (11), GA19 (14), GA20 (12) and GA29 (13) were detected in seedlings and mature flowering plants. In germinating seeds and seedlings (while the plants were growing actively), concentrations of the 12alpha,13-dihydroxy GAs, measured by bioassay, declined and those of the 13-hydroxy GAs increased. The results are discussed with reference to the known and predicted effects of the GAs on the vegetative growth and flowering of P. avium plants. PMID- 10731034 TI - Activation of the A3 adenosine receptor affects cell cycle progression and cell growth. AB - The A3 adenosine receptor has been implicated in modulation of cell growth. As a first step to the characterization of the underlying mechanisms, we exposed Chinese hamster ovary (CHO) cells transfected with the human A3 receptor (A3R CHO) to selective A3 receptor ligands. At micromolar concentrations, the A3 agonists N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) and its 2 chloro derivative Cl-IB-MECA reduced cell number, with no effects on either parental CHO cells (not expressing any adenosine receptor), or CHO cells transfected with the human A1 receptor. Cl-IB-MECA also reduced cell number in the human HEK293 cell line transfected with the human A3 receptor cDNA as opposed to the respective untransfected wild-type cells. In A3R-CHO, agonist-induced effects were antagonized by nanomolar concentrations of A3 antagonists, including the triazoloquinazoline derivative MRS 1220, the dihydropyridine derivative MRS 1191, and the triazolonaphthyridine derivative L-249,313. A3 agonist-induced effects were not due to modulation of cell adhesion, nor to necrosis or apoptosis. Growth curves revealed highly impeded growth, and flow-cytometric analysis showed markedly reduced bromodeoxyuridine incorporation into nuclei. The effect on cell cycle was completely antagonized by MRS1191. Hence, activation of the human A3 receptor in A3R-CHO results in markedly impaired cell cycle progression, suggesting an important role for this adenosine receptor subtype in cell cycle regulation and cell growth. PMID- 10731035 TI - Long chain diamines inhibit growth of C6 glioma cells according to their hydrophobicity. An in vitro and molecular modeling study. AB - A series of diamines with the general structure NH2(CH2)xNH2, x=2-12, was tested for their potential effects on cell proliferation of cultured rat C6 glioma cells in comparison to natural polyamines. Long chain diamines reduced cell number after 48 h in culture with a sequence of 1,12-diaminododecane (1,12-DD) >1,10 diaminodecane >1,9-diaminononane. Polyamines (putrescine, spermidine and spermine) as well as diamines up to a CH2-chain length of x=8 were found to be ineffective. The spermine analogue 1,12-DD was the most effective molecule in reducing cell number in an irreversible, dose-dependent manner (EC50=3 microM under serum-free conditions). In further experiments we investigated the mechanisms of action of 1,12-DD. The compound had only a minor effect on cell cycle and did not affect free internal calcium concentration. Under physiological conditions 1,12-DD interacts with triplex DNA but not with duplex DNA. Ornithine decarboxylase activity as well as the concentration of internal polyamines were found to be reduced by 1,12-DD. Polyamine application, however, was not able to reverse the effect of 1,12-DD, indicating a polyamine-independent or non competitive mechanism of action. 1,12-DD reduced cell number by induction of apoptosis as well as necrosis. In molecular modeling studies it was found that a minimal hydrophobic intersegment of at least 4 A was required to make a diamine an effective drug in respect to cellular growth. A hydrophobic gap of this size fits the minimum requirement expected from molecular modeling to provide space for hydrophobic interactions with parts of proteins like a CH3-group. Our results show that 1,12-DD acts as a potent drug, reducing the number of C6 glioma cells, and suggest that its spatial and hydrophobic properties are responsible for its mechanism of action. PMID- 10731036 TI - PLA2 phosphorylation and cyclooxygenase-2 induction, through p38 MAP kinase pathway, is involved in the IL-1beta-induced bradykinin B2 receptor gene transcription. AB - We hypothesized that inflammatory mediators such as interleukin-1beta (IL-1beta) might be responsible for the hyperreactivity to bradykinin observed in asthmatic patients. We reported previously that IL-1beta induced a prostanoid-dependent increase in the density of bradykinin B2 receptors in cultured human bronchial smooth muscle cells. Our experiments demonstrate that the rapid prostaglandin E2 (PGE2) synthesis induced by IL-1beta is abolished by cycloheximide, suggesting the involvement of protein synthesis. The formation of PGE2 is preceded by the phosphorylation of cPLA2 and the expression of cyclooxygenase-2 (Cox-2). The inhibition of p38 MAP kinase inhibited PGE2 synthesis, cPLA2 phosphorylation and abolished Cox-2 expression. The inhibition of Cox-2 expression correlated with a decrease of bradykinin B2 receptor expression. These data demonstrate that the activation of p38 MAP kinase elicited by IL-1beta leads to the phosphorylation of cPLA2 and Cox-2 overexpression, allowing rapid synthesis of PGE2 as a prerequisite for bradykinin B2 gene expression in human bronchial smooth muscle cells which could explain the hyperresponsiveness of asthmatic patients to bradykinin. PMID- 10731037 TI - Further characterisation of the interaction of haloperidol metabolites with neurotransmitter transporters in rat neuronal cultures and in transfected COS-7 cells. AB - It has been proposed that the extrapyramidal symptoms such as tardive dyskinesia developed by patients on long-term haloperidol treatment may be the result of uptake of haloperidol metabolites into neurons via the monoamine neurotransmitter transporters followed by neurotoxic events, as occurs for MPP+, the pyridinium metabolite of MPTP. We recently showed that haloperidol and its metabolites are inhibitors of the human noradrenaline transporter (NAT), dopamine transporter (DAT) and serotonin transporter (SERT), and determined their Ki values for inhibition of the three transporters expressed in transfected COS-7 cells. In this study, we extended the investigation of these compounds to their inhibitory effects on DAT, SERT and the high affinity choline uptake (HACU) in neuronal cultures from embryonic rat brain, and investigated whether the compounds are substrates or non-transported inhibitors of the NAT, DAT and SERT in transfected COS-7 cells and DAT and SERT in the neuronal cultures. Haloperidol and its metabolites inhibited DAT, SERT and HACU in the neuronal cultures, indicating that they are not specific inhibitors of the monoamine neurotransmitter transporters. The ratio of the Ki values of the least and most potent inhibitors were found to be 2.8 for DAT, 24 for SERT and 7.6 for HACU. The compounds were more potent inhibitors of DAT and SERT in neuronal cultures than we found previously in transfected COS-7 cells. The question of whether the compounds are substrates or non-transported inhibitors of the monoamine transporters was investigated by determining whether they caused an increase in efflux of [3H]amine in transfected COS-7 cells or neuronal cultures preloaded with [3H]amine. Haloperidol metabolites were weak substrates for SERT, but not for NAT or DAT, in transporter-transfected COS-7 cells. In neuronal cultures, the metabolites appeared to be non-transported inhibitors or very weak substrates of DAT and SERT. Despite inhibition of the monoamine transporters by haloperidol and its metabolites, there is little evidence to support the proposal that these compounds are likely to cause neurotoxic effects via neuronal uptake using the monoamine transporters. The mechanisms of the side effects of haloperidol therapy, such as tardive dyskinesia, are still unclear, but are unlikely to depend on interactions of the drug or its metabolites with NAT, DAT or SERT. PMID- 10731038 TI - Effects of 2-arachidonylglycerol, an endogenous cannabinoid, on neuronal activity in rat hippocampal slices. AB - The monoacylglycerol 2-arachidonylglycerol is an endogenous ligand of cannabinoid receptors. We examined whether 2-arachidonylglycerol can influence excessive neuronal activity by investigating stimulation-induced population spikes and epileptiform activity in rat hippocampal slices. For this purpose, the effects of 2-arachidonylglycerol were compared with those of the synthetic cannabinoid agonist WIN 55,212-2. At concentrations of 10-50 microM, 2-arachidonylglycerol attenuated the amplitude of the orthodromic population spike and the slope of the field excitatory postsynaptic potential (field EPSP). However, the effect of the synthetic cannabinoid WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3 [(morpholinyl)methyl]pyrrolol[ 1,2,3-de]-1,4-benzoxazinyl]-(1 naphthalenyl)methanone; 0.1 microM and 1 microM) was significantly higher than that of the endogenous ligand. At a concentration of 1 microM, WIN 55,212-2 completely suppressed the field EPSP. However, none of the investigated compounds did affect the presynaptic fiber spike of the afferents. The CB1 receptor antagonist SR 141716 (N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorphenyl)-4 methyl-3- pyrazole-carboxamide) blocked the inhibition evoked by the cannabinoids. Both 2-arachidonylglycerol (30 microM) and WIN 55,212-2 (100 nM) shifted the input-output curve of the postsynaptic spike and the field EPSP to the right and increased the magnitude of paired-pulse facilitation, indicating a presynaptic mechanism of action. 2-Arachidonylglycerol and WIN 55,212-2 attenuated the frequency of spontaneously occurring epileptiform burst discharges in CA3 elicited by omission of Mg2+ and elevation of K+ to 8 mM. The antiepileptiform effect of these cannabinoids was blocked by SR 141716. In conclusion, 2-arachidonylglycerol seems to limit neuronal excitability via cannabinoid receptors of the CB1 type. By acting predominantly at a presynaptic site, it is capable of reducing excitatory neurotransmission, a mechanism which might be involved in the prevention of excessive excitability leading to epileptiform activity. PMID- 10731039 TI - Effects of peptidase inhibitors on anti-nociceptive action of dynorphin-(1-8) in rats. AB - Previous in vitro studies showed that the degradation of dynorphin-(1-8) [dyn-(1 8)] by cerebral membrane preparations is almost completely prevented by a mixture of three peptidase inhibitors (PIs), amastatin, captopril and phosphoramidon. In the present investigations, effects of the three PIs on the anti-nociception induced by the intra-third-ventricular (i.t.v.) administration of dyn-(1-8) were examined. The inhibitory effect of dyn-(1-8) on the tail-flick response was increased more than 100-fold by the i.t.v. pretreatment of rats with the three PIs. The inhibition produced by dyn-(1-8) in rats pretreated with any combination of two PIs was significantly smaller than that in rats pretreated with three PIs, indicating that any residual single peptidase could inactivate significant amounts of dyn-(1-8). The antagonistic effectiveness of naloxone, a relatively selective mu-opioid antagonist, indicates that dyn-(1-8)-induced inhibition of tail-flick response in rats pretreated with three PIs is mediated by mu-opioid receptors. Furthermore, mu-receptor-mediated inhibition induced by dyn-(1-8) was significantly greater than that produced by [Met5]-enkephalin in rats pretreated with three PIs. The data obtained in the present investigations together with those obtained in previous studies strongly indicate that dyn-(1-8) not only has well-known kappa-agonist activity but also has high mu-agonist activity. PMID- 10731040 TI - Short-acting NMDA receptor antagonist MRZ 2/576 produces prolonged suppression of morphine withdrawal in mice. AB - The present study sought to evaluate the time-course of the effects of a short acting glycine site NMDA receptor antagonist, MRZ 2/576 (half-life of about 20 min), on the expression of morphine withdrawal syndrome in mice. Morphine-naive and morphine-dependent mice (10-100 mg/kg, b.i.d., s.c., 9 days) were injected with a combination of naltrexone (vehicle or 1 mg/kg, s.c.) and MRZ 2/576 (vehicle, 0.3-10 mg/kg, i.p.) 24 h after the last morphine injection. MRZ 2/576 suppressed expression of several signs of morphine withdrawal (jumping, shaking, forelimb tremor). Effects of MRZ 2/576 were equally expressed throughout 1-h observation test of both spontaneous and naltrexone-facilitated withdrawal. These results suggest that despite its short half-life, MRZ 2/576 produces prolonged suppression of morphine withdrawal syndrome and this effect cannot be attributed to repeated morphine-induced increase in sensitivity to naltrexone. PMID- 10731041 TI - Roles of group II metabotropic glutamate receptors in modulation of seizure activity. AB - Evidence suggests that metabotropic glutamate receptors (mGluR) are involved in mediating seizures and epileptogenesis. In the present experiments, the selective, group II mGluR agonist (+)-2-aminobicyclo-[3.1.0]hexane-2,6 dicarboxylic acid (LY354740, 0.1-1.0 microM) inhibited spontaneous epileptiform discharges which developed in rat cortical slices in Mg2+-free medium. LY354740 (4-16 mg/kg) administered prior to an injection of pentylenetetrazol (80 mg/kg) or picrotoxin (3.2 mg/kg) produced a dose-dependent decrease in the number of mice exhibiting clonic convulsions, but had no effect on N-methyl-D-aspartate (NMDA, 150 mg/kg)-induced convulsions. LY354740 (4-16 mg/kg) did not affect lethality induced in mice by pentylenetetrazol, picrotoxin or NMDA. LY354740 potentiated the anticonvulsant activity of the conventional antiepileptic drug diazepam, significantly decreasing the ED50 for that drug's effect on pentylenetetrazol-induced convulsions by 30%, but had no influence on anticonvulsant activity of ethosuximide and valproic acid. A pharmacokinetic interaction between LY354740 and diazepam, leading to the lowering of the plasma level of free diazepam, was also demonstrated. Our data suggest that the group II mGluR agonist LY354740 possesses anti-seizure activity and may modify the effects of some conventional antiepileptic drugs. PMID- 10731042 TI - Activation of striatal group II metabotropic glutamate receptors has a differential effect on dopamine-D1 and -D2 receptor antagonist-induced hypokinesia in the rat. AB - Motor function of group II metabotropic glutamate receptors was investigated by quantifying motor effects of bilateral infusions of the preferential group II metabotropic glutamate receptor agonist (2S,3S,4S)-alpha-carboxycyclopropyl glycine (15, 30, 60 nmol/0.5 microl) into the striatum of conscious rats. (2S,3S,4S)-alpha-carboxycyclopropyl-glycine reduced spontaneous sniffing activity in an experimental chamber, but did not affect spontaneous locomotor (line crossings) or exploratory behaviour (rearings, hole visits) in an open field equipped with a hole-board. Intrastriatal infusion of the selective group III metabotropic glutamate receptor agonist L-2-amino-4-phosphobutyric acid (15, 30, 60, 120 nmol/0.5 microl) did not influence spontaneous motor behaviour. Intrastriatal infusion of (2S,3S,4S)-alpha-carboxycyclopropyl-glycine (15 nmol/0.5 microl and 30 nmol/0.5 microl) further depressed spontaneous motor behaviour in rats pretreated with the dopamine-D1 receptor antagonist (-)-trans 6,7,7a,8,9,13b-hexahydro-3-chloro-2-hydroxy-N-methyl-5H- benzo[d]naphtho-(2,1 b)azepine, but not if rats were pretreated with the preferential dopamine-D2 receptor antagonist haloperidol. It appears likely that the depression of spontaneous motor behaviour evoked by the preferential group II agonist (2S,3S,4S)-alpha-carboxycyclopropyl-glycine is mediated by activation of group II metabotropic glutamate receptors, since activation of group I metabotropic glutamate receptors has been shown to stimulate motor behaviour, and activation of group III metabotropic glutamate receptors had no effect, as shown in this study. Therefore, it is reasonable to speculate that the striatum may contribute to the motor-depressant effects of systemically applied group II metabotropic glutamate receptor agonists, as reported by us recently. The present findings further suggest that a functional dopamine-D1 antagonism contributes to the motor effects of group II metabotropic glutamate receptor agonists. PMID- 10731043 TI - JL13, a pyridobenzoxazepine compound with potential atypical antipsychotic activity, increases extracellular dopamine in the prefrontal cortex, but not in the striatum and the nucleus accumbens of rats. AB - In behavioral and receptor binding studies, 5-(4-methylpiperazin-1-yl)-8-chloro pyridol[2,3b] [1,5]benzoxazepine (JL13) shows an atypical antipsychotic profile. We used microdialysis in awake rats to study the effects of various intraperitoneal doses of JL13 on extracellular concentrations of dopamine in the prefrontal cortex, nucleus accumbens and striatum. JL13 at 20 mg/kg and 40 mg/kg dose-dependently raised extracellular dopamine (234% and 434% of basal levels at peak, respectively) in the prefrontal cortex whereas lower doses (5 mg/kg and 10 mg/kg) had no effect. Extracellular concentrations of dihydroxyphenylacetic acid and homovanillic acid were also significantly increased in the prefrontal cortex of rats given 40 mg/kg JL13 (310% and 230% of basal levels, respectively). At 20 mg/kg and 40 mg/kg JL13 did not affect the extracellular concentrations of dopamine and its metabolites in the striatum and nucleus accumbens. The mechanisms by which JL13 increases cortical dopamine release and the significance for potential antipsychotic efficacy are discussed. PMID- 10731045 TI - Chloroquine blocks the background potassium current in guinea pig atrial myocytes. AB - In this study we examined the effects of chloroquine on the muscarinic potassium current, I(K-ACh), and the inward rectifying potassium current, I(K1). We utilized three ways to induce I(K-ACh): activating the M2-muscarinic receptors with carbachol, activating the purinergic A1-receptors with adenosine and directly activating the G(K)-protein coupled with these receptors in an irreversible way with GTPgammaS. In experiments using the whole-cell configuration of the patch-clamp technique, we found that chloroquine, independently from the manner of activation of I(K-ACh), was able to block this current with similar potency. These results strongly suggest that chloroquine may be acting directly on the muscarinic potassium channel. Chloroquine also blocked I(K1) with similar potency, in both guinea pig atrial and ventricular myocytes. PMID- 10731044 TI - Electrophysiological effects of bimoclomol in canine ventricular myocytes. AB - Concentration-dependent effects of bimoclomol, a novel heat shock protein (HSP) coinducer, were studied on the parameters of action potential and transmembrane ionic currents in enzymatically dispersed canine ventricular cardiomyocytes using conventional microelectrode and whole cell voltage clamp techniques. Bimoclomol (10-100 microM) decreased the maximum velocity of depolarization (Vmax) and amplitude of action potentials in a concentration-dependent manner. These effects were fully reversible after a 5-min period of washout in drug-free medium. Action potential duration measured at 50% or 90% level of repolarization (APD-50 and APD 90, respectively) was markedly shortened by bimoclomol. Both APD-50 and APD-90 were decreased, but the reduction in APD-50 was more pronounced. The APD shortening effect of bimoclomol was significantly reduced in the presence of 20 nM charybdotoxin (inhibitor of the Ca-dependent K current) or 0.5 mM anthracene-9 carboxylic acid (inhibitor of the Ca-dependent Cl current) or 1 microM glibenclamide (inhibitor of the ATP-sensitive K current). In the presence of anthracene-9-carboxylic acid, APD-90 was lengthened by bimoclomol. The APD shortening effect of bimoclomol was also partially antagonized by chelation of intracellular Ca2+ by application of the cell permeant form of BAPTA, or when using 10 mM EGTA-containing patch pipettes to record action potentials. The Vmax depressant effect of bimoclomol was not affected by charybdotoxin, anthracene-9 carboxylic acid, glibenclamide, or BAPTA load. In voltage clamped cardiomyocytes bimoclomol (100 microM) had no effect on the amplitude of I(Ca), but decreased significantly the inactivation time constant of I(Ca) (from 19.8+/-1.6 ms to 16.8+/-1.2 ms at 0 mV). Bimoclomol also decreased significantly the amplitude of I(K1) (from -20.5+/-1.1 pA/pF to -16.6+/-0.8 pA/pF at -135 mV), causing reduction in slope of the negative branch of the I-V curve. At positive potentials, however, bimoclomol increased outward current. The bimoclomol-induced current, therefore, was studied in the presence of BaCl2, when I(K1) current was blocked. The bimoclomol-induced current had a reversal potential close to -90 mV. Bimoclomol (100 microM) had no effect on the amplitude or kinetic properties of the transient outward K current (I(to)) and the delayed rectifier K current (I(K)). It is concluded that bimoclomol exerts both Ca-independent (inhibition of I(Na) and I(K1), activation of the ATP-sensitive K current) and Ca-dependent effects (mediated by Ca-activated Cl and probably K currents) in canine ventricular myocytes. PMID- 10731046 TI - Nitric oxide, but not vasopressin V2 receptor-mediated vasodilation, modulates vasopressin-induced renal vasoconstriction in rats. AB - The renal vascular response to vasopressin and its modulation were evaluated in vivo by infusing the peptide directly into the renal artery of anaesthetized rats. The intra-renal artery (i.r.a) infusion of vasopressin induced a dose dependent decrease in renal blood flow. Vasoconstriction was obvious at a dose of 3 ng/kg per min and reached a maximum at 100 ng/kg per min. The dose required for a half-maximal response (ED50) was 24+/-4 ng/kg per min (mean+/-SEM, n=8), corresponding to an estimated concentration in renal arterial blood required for a half-maximal response (EC50) of 1.9+/-0.6 nM. Thiobutabarbitone anaesthesia markedly increased plasma vasopressin concentration. This increase was prevented partially by hypotonic hydration of the rats without any change in the renal vascular response to exogenous vasopressin. Vasopressin-induced vasoconstriction dose/response curves were similar in homozygous and heterozygous Brattleboro rats. Infusion of desmopressin (1-1000 ng/kg per min, i.r.a.), a vasopressin V2 receptor-selective agonist, failed to induce renal vasodilation or vasoconstriction. In the presence of SR 49059 (1 mg/kg i.v.), a vasopressin V1A receptor antagonist that completely abolished the vasopressin-induced renal vasoconstriction, desmopressin again failed to induce vasodilation. Inhibition of nitric oxide synthase by N(omega)-nitro-L-arginine (L-NNA, 100 microg/kg for 10 min and 7.5 microg/kg per min, i.r.a.) enhanced vasopressin-induced renal vasoconstriction (EC50 0.6+/-0.1 nM, P<0.05). In contrast, cyclooxygenase blockade by indomethacin (5 mg/kg, i.v.) neither modified the vasopressin-induced decrease in renal blood flow nor altered the potentiation of vasoconstriction by L-NNA. These results show that the constrictor response of the rat renal vascular bed in vivo is observed only with high local concentrations of vasopressin. This hyporeactivity in vivo was not explained by an anaesthesia-elicited increase in endogenous vasopressin, nor by a modulatory effect linked to V2 receptor activation or prostanoid release. In contrast, NO release contributed to the attenuation of vasopressin-induced renal vasoconstriction. PMID- 10731047 TI - Pharmacological studies on the inhibitory action of melatonin and putative melatonin analogues on porcine vascular smooth muscle. AB - The effect of high concentrations of melatonin, and related indole-based and naphthalene-based derivatives, has been examined in the porcine coronary artery, pulmonary artery and the marginal artery of the colon. In addition, we have pharmacologically examined the role of cyclic GMP in the relaxatory action of these agents. Cumulative addition of melatonin (3-300 microM) caused a slowly developing relaxation in all three vascular preparations pre-contracted with 9,11 dideoxy-9a,11a-methanoepoxy prostaglandin F2alpha (U46619), a thromboxane mimetic agent. The estimated pIC50 values were 4.10-3.70 (coronary artery), 3.89 (pulmonary artery) and 3.96 (marginal artery). All melatonin analogues examined also produced concentration-dependent inhibition of U46619-induced contractions of the coronary and marginal arteries in a qualitatively similar manner to melatonin. The rank order of potency (based on the pIC50 values) of these compounds in both vascular tissues was N-[2-(3-ethyl-7-methoxynaphthyl) ethyl] acetamide (S21634) >2-iodomelatonin = N-[2-(7-methoxynaphth-1-yl)-ethyl] acetamide (S20098) = N-[2-naphth-1-yl-ethyl]-cyclobutyl carboxamide (S20928) >melatonin >N-acetyl-5-HT. Finally, the pharmacological characteristics of melatonin and S21634 as phosphodiesterase inhibitors were compared to those of zaprinast, a known cyclic GMP-specific phosphodiesterase inhibitor. Zaprinast also caused concentration-dependent inhibition of U46619-induced tone. All three compounds, zaprinast (10 microM), melatonin (300 microM) and S21634 (30 microM), significantly enhanced sodium nitroprusside-induced relaxations. The inhibitory action of zaprinast per se was greater in the presence of the endothelium and significantly attenuated by 3 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a selective inhibitor of guanylyl cyclase. In marked contrast, the vasorelaxant action of melatonin and S21634 was not affected by the removal of the endothelium or the addition of ODQ. In summary, we have shown that porcine arterial smooth muscle relaxes in response to high concentrations of melatonin and other related melatonin receptor ligands. However, it appears that the receptive site is pharmacologically different from the melatonin receptors currently known and does not involve inhibition of cyclic GMP-specific phosphodiesterase. PMID- 10731048 TI - Role of NK1 receptors on cisplatin-induced nephrotoxicity in the rat. AB - The role of NK1 receptors on the nephrotoxicity associated with cisplatin treatment was evaluated. Adult Sprague-Dawley male rats (300-400 g) were treated with saline (0.1 ml/100 g, i.p., every 8 h for 72 h) or the selective NK1 receptor antagonist (GR205171; 2 mg/kg, i.p., every 8 h for 72 h). Treatments were started 5 min before cisplatin (7.5 mg/kg, i.p., single dose). All evaluations were made from 72 h to 96 h after cisplatin. An oral load of 10 ml/kg of water was given at time 0 (72 h after cisplatin). Cisplatin reduced the urinary volume (-45%), creatinine clearance (>90%), lithium clearance (-76%) and urinary potassium excretion (-54%). Protein and sodium excretion was not affected by cisplatin. GR205171 prevented the reduction in urine volume induced by cisplatin. In addition, the decreases in creatinine and lithium clearances induced by cisplatin were also attenuated by the NK1 receptor antagonist. The clearance of creatinine averaged 0.57+/-0.2 ml/min in controls, 0.004+/-0.01 ml/min after cisplatin, and 0.09+/-0.02 ml/min after cisplatin + GR205171. The lithium clearance was 0.09+/-0.04 ml/min in controls, 0.02+/-0.01 ml/min after cisplatin and 0.06+/-0.01 ml/min after cisplatin + GR205171. Cisplatin induced marked necrosis, vacuolation and edema of proximal renal tubules; these changes were considerably reduced in GR205171-treated animals. These results indicate that treatment with a selective NK1 receptor antagonist ameliorated cisplatin induced renal toxicity in rats, as evidenced by improvements in renal function and histology. PMID- 10731049 TI - Differential alteration of cisplatin cytotoxicity and myelotoxicity by the paclitaxel vehicle cremophor EL. AB - Cremophor EL (CR), the paclitaxel vehicle, has previously been reported to alter the pharmacokinetics and/or pharmacodynamics of some anticancer drugs including paclitaxel. Several experimental and clinical studies suggested that cisplatin (CDDP) in combination with paclitaxel results in less hematological toxicity than anticipated. To reveal the role of CR in this important pharmacological interaction, we evaluated the interaction of CR with CDDP in vitro and in vivo using experimental Ehrlich ascites carcinoma (EAC) tumor. CR (1 microg/ml) significantly enhanced the in vitro cytotoxicity of CDDP in cultured EAC cells. This enhancement was not associated with a parallel increase in CDDP cellular uptake. In tumor-bearing mice, CR (2.5 ml/kg, i.v.) given in combination with CDDP (7 mg/kg, i.v.) did not significantly change CDDP pharmacokinetics, antitumor activity or nephrotoxicity. On the other hand, CDDP-induced hematological toxicity was significantly reduced by CR. This protective effect was related to CR-induced inhibition of cellular CDDP accumulation in bone marrow. This study presents evidence that CR may play an important role in the pharmacological interaction between CDDP and paclitaxel. The present data may suggest formulation of CDDP with CR for systemic treatment. Further studies are yet necessary to establish the clinical value of CR as a modifier for CDDP therapeutic index. PMID- 10731050 TI - Asymptomatic inferior vena cava abnormalities in three children with end-stage renal disease: risk factors and screening guidelines for pretransplant diagnosis. PMID- 10731051 TI - Therapeutic drug monitoring of cyclosporin A: should we use the area under the concentration-time curve and forget trough levels? PMID- 10731052 TI - Evaluation of anti-human leukocyte antigen allo-immunization in pediatric cadaveric kidney transplantation. AB - Forty-eight pediatric cadaveric renal transplantations, performed between May 1986 and February 1997, were retrospectively screened, pre- and post-transplant, for antibodies to human leukocyte antigen (anti-HLA) using complement-dependent cytotoxicity (CDC) assay and enzyme immunoassay (EIA). The correlation between anti-HLA immunization and graft outcome was investigated. The combined analysis of CDC and EIA enabled the differentiation between complement-fixing and non complement-fixing, anti-HLA class I and anti-HLA class II antibodies. The median post-transplant follow-up for all patients with a functioning graft was 86 months (range 10-138 months). In the whole population, 16 grafts were lost: six following a non-immunologic complication; and 10 as a result of rejection. Of these 10 grafts lost, eight were in patients with pre- and/or post-transplant donor antigen specific (DAS) anti-HLA class I or class I + II antibodies; and two were in patients with DAS anti-HLA class II antibodies only. Three of these grafts were lost in patients with weak pre-existing DAS anti-HLA class I antibodies. Immunological graft loss appeared at a median post-transplant time of 38 months (range 2-68 months). All patients without DAS anti-HLA antibodies had a good graft outcome. The presence of pre- and post-transplant DAS anti-HLA antibodies, especially if directed against HLA class I, were associated with a poor graft outcome. A systematic search for, and identification of, anti-HLA antibodies should therefore be part of a pretransplant evaluation to allow the identification of 'unacceptable' donor HLA antigens, following which the impact of the HLA-cross-match on graft outcome will improve. Screening for DAS anti-HLA antibodies post-transplant could be helpful for detecting patients with an increased risk for graft loss following rejection episodes. PMID- 10731053 TI - Impact of FK506 and steroids on adaptation after intestinal resection or segmental transplantation. AB - Segmental small intestinal transplantation (SIT) using living related donors (LRD) is being evaluated as a therapy, clinically. Advantages of this technique include an increase in the donor pool, optimization of the timing of transplants, and potential immunologic benefits. However, the ability of a short segment of intestine to function after transplantation has not been investigated in large animal models. This study evaluates the impact of immunosuppression on the adaptive process and the ability of a transplanted segment of intestine to adapt. A pig model of segmental SIT was used. Animals were resected, leaving 150 cm of distal ileum (n = 5), resected and treated with FK506 (n = 4), or steroids (n = 4), or with FK506 + steroids (n = 7), or transplanted using a similar segment of ileum and treated with FK506 + steroid immunosuppression (n = 9). Animals undergoing resection, or resection plus steroid treatment, did well, gaining weight post-operatively (37% and 15% of preoperative weight, respectively). However, animals undergoing resection and treated with FK506 or FK506 + steroids did poorly, losing weight (-14% and -22% of preoperative weight, respectively) and showing significant impairment of intestinal adaptation, morphologically and functionally. Furthermore, FK506-treated animals developed inflammatory changes in the intestinal mucosa, mimicking rejection. Segmental SIT animals had a high rate of rejection (66%) and showed a similar impairment in adaptation. Hence, segmental SIT is a stringent physiological test of intestinal adaptation. FK506 appears to impair gut function after resection, either directly, or by interfering with the adaptive process. In this model of segmental SIT, FK506 and steroids at the doses tested did not provide adequate immunosuppression to prevent rejection and the graft did not function adequately to allow growth. Further studies are required to evaluate the mechanisms underlying these findings, and to determine if similar effects occur in humans. PMID- 10731054 TI - Use of anti-hypertensive medications and post-transplant renal allograft function in children. AB - Post-transplant hypertension is a common occurrence in children. The relative effect of this hypertension on renal allograft function is uncertain. Examining the accumulated data for pediatric renal transplant recipients at our institution from monthly visits for up to three years, we determined whether the use of anti hypertensive medications (anti-HTN medications) was associated with allograft dysfunction. Monthly clinical data included height, weight, serum creatinine, cyclosporin A (CsA) trough levels, number of acute rejection episodes, and number of anti-HTN medications. Estimated glomerular filtration rate (eGFR) was calculated monthly for each patient using the Schwartz formula. Time post transplant was grouped into 6-month intervals. One thousand three hundred and sixty-three monthly data sets from 6 months (n = 76 patients) to 3 yr post transplant (n = 47 patients) were analyzed. Overall mean eGFR was 75 mL/min/1.73 m2 at 6 months and 54 mL/min/1.73 m2 at 3 yr. A lower eGFR was found at all post transplant time intervals for patients receiving anti-HTN medications compared with those who were not (p < 0.01). This lower eGFR was found at some but not all times post-transplant when patients were grouped by donor type or history of acute rejection episodes and analyzed separately. Mean CsA trough levels were higher at all post-transplant time intervals in patients receiving anti-HTN medications (p < 0.05). While a causal relationship between post-transplant hypertension and graft dysfunction cannot be established from this study, we conclude that the need for anti-HTN medications is associated with worse allograft function. PMID- 10731055 TI - Asymptomatic inferior vena cava abnormalities in three children with end-stage renal disease: risk factors and screening guidelines for pretransplant diagnosis. AB - We report two children with end-stage renal disease (ESRD) found to have inferior vena cava (IVC) thrombosis at the time of renal transplantation. The children suffered from renal diseases that included congenital hepatic fibrosis and portal hypertension as part of their pathophysiology. Neither child had evidence of hypercoaguability or clinical symptoms of IVC thrombosis. Prior to transplantation, the renal replacement therapy consisted primarily of peritoneal dialysis. During their hospital courses, these children had central venous catheters placed for temporary hemodialysis, episodes of peritonitis and numerous abdominal surgeries. The medical literature to date has not identified a link between IVC thrombosis and portal hypertension, nor has an association between the patients' primary renal disease and IVC thrombosis been found. We also report the finding of asymptomatic IVC narrowing in a third patient with obstructive uropathy, colonic dysmotility and numerous abdominal surgeries. IVC narrowing was diagnosed by CT scan during his pretransplant evaluation. In this paper, we consider similarities between these three patients that may have predisposed each of them to asymptomatic IVC pathology, including large-bore central venous access as young children and/or recurrent scarring abdominal processes. A discussion regarding appropriate screening of the 'high-risk patient' for IVC pathology prior to kidney transplantation and surgical options for children with this rare complication are presented. PMID- 10731056 TI - Pulmonary capillary leak syndrome with intravenous cyclosporin A in pediatric renal transplantation. AB - Despite frequent use of intravenous (i.v.) cyclosporin A (CsA) in the early post operative course of transplant recipients, allergic reactions have been infrequently described. Of 134 transplants, we report four pediatric renal transplant recipients with severe reaction to i.v. CsA with pulmonary capillary leak syndrome. Pulmonary edema developed at a mean time of 3.5 h after commencement of i.v. CsA, with two patients requiring mechanical ventilation. Discontinuation of i.v. CsA and conversion to oral CsA was followed by rapid resolution of pulmonary edema, suggesting that cremaphor, the solubilizing agent in the i.v. formulation, is likely to be responsible for this adverse response. Skin prick testing with cremaphor was negative in all patients and alternative mechanisms for the cremaphor response are proposed. It is likely that inadequate mixing of the i.v. CsA solution triggered this reaction, by delivering a higher concentration of cremaphor at the start of the CsA infusion. Pulmonary edema in the early post-transplant course in the absence of obvious fluid overload should prompt the diagnosis of an i.v. CsA reaction. This life-threatening reaction is easily reversible if recognized, and can be managed easily without compromise to the allograft, by discontinuing i.v. CsA and switching early to an oral CsA formulation. PMID- 10731057 TI - Pulmonary complications following liver transplantation in pediatric patients. AB - The purpose of this study was to define the incidence and type of pulmonary complications experienced by children after orthotopic liver transplantation (OLT). The radiological records of all patients receiving OLT during a 3-yr period were reviewed to identify evidence of a pulmonary abnormality. Medical records were then reviewed to determine type, duration, therapy and outcome of pulmonary disorders. Potential risk factors for the development of persistent pleural effusions were also analyzed. One hundred and fifty-one pediatric liver transplantations were performed on 113 patients during this period. Pneumonia developed in 27 patients, including 11 proven bacterial, six presumed bacterial, six viral and four fungal cases. All three deaths related to pulmonary complications were in this group. Three patients developed mild pulmonary hemorrhages, and three developed pulmonary calcifications, which did not impair lung function. Sixteen patients developed paralysis of the right hemidiaphragm, four required diaphragm plication. Pleural effusions developed in 86 patients, 38 persisted longer than 7 days. Patients with persistent effusions were more likely to develop allograft rejection than patients with no persistent effusion (p < 0.01) by chi2 analysis. Seven patients required tracheostomy placement. Of these, four were successfully decannulated, two died from non-pulmonary complications and one is receiving home ventilator support. In conclusion, the majority of children experience at least one pulmonary complication after OLT, but mortality due to pulmonary disease is low in this population. Persistent pleural effusions may be a heralding sign of allograft rejection. Viral, bacterial and fungal pneumonia were the only pulmonary causes of death and only one patient in this series has had significant chronic lung disease. PMID- 10731058 TI - Sirolimus in pediatric gastrointestinal transplantation: the use of sirolimus for pediatric transplant patients with tacrolimus-related cardiomyopathy. AB - Hypertrophic obstructive cardiomyopathy (HOCM) associated with the use of tacrolimus is a rare complication of liver and intestinal transplantation seen almost exclusively among pediatric patients. Reduction of tacrolimus dosage or conversion to cyclosporin A (CsA) has been used as an effective treatment in reviewed cases. We present three pediatric transplant recipients who developed hypertrophic obstructive cardiomyopathy while under tacrolimus immunosuppression and were treated with conversion to sirolimus (Rapamycin). The patients (ages 6 yr, 12 yr and 11 months) were transplant recipients (liver, n = 2; liver and intestine, n = 1) who developed significant cardiomyopathy 15 and 96 months post transplant. One patient died of post-transplant lymphoproliferative disorder 21 days after starting sirolimus. One patient had received two liver transplants and had been on CsA for 12 yr before conversion to tacrolimus at 60 months post transplant for acute and chronic rejection. The surviving patients were receiving mycophenolate mofetil, tacrolimus and steroids at the time of diagnosis. Dose reduction of tacrolimus and treatment with beta blockers failed to alleviate the hemodynamic changes. The patients were converted to sirolimus 1.6, 37 and 148 months post-transplant and maintained a whole-blood trough level of 15-20 ng/mL 21 days after starting sirolimus. Repeat echocardiograms in the surviving patients showed improvement in cardiomyopathy. One patient had one rejection episode (intestinal biopsy, mild acute cellular rejection) after starting sirolimus that responded to a transient increase in steroids. The early demise of the third patient after sirolimus conversion prevented an adequate assessment of cardiomyopathy. Conversion to sirolimus was associated with a reduction in the cardiomyopathy of the two surviving patients while still providing effective immunosuppression. To our knowledge this observation has not been previously reported. PMID- 10731059 TI - Cryptosporidial infections after solid organ transplantation in children. AB - The diagnosis and treatment of moderate-to-severe diarrhea in solid organ transplant recipients is often a challenge because of the variety of infectious and non-infectious causes. The morbidity associated with this clinical condition is of particular significance in the pediatric population where malnutrition may lead to poor growth and development. Rarely, Cryptosporidium has been identified as the cause of clinically significant diarrhea in pediatric solid organ transplant patients. A retrospective review identified cases of cryptosporidiosis among the 1160 non-renal, abdominal organ transplant recipients cared for at the Children's Hospital of Pittsburgh between 1981 and June 1998. Four cases of clinically significant diarrhea were identified in three liver transplant recipients and one small bowel transplant recipient. Endoscopy and biopsy with histologic confirmation diagnosed three cases; ova and parasitic examination of stool specimens identified the fourth case. Therapy varied among the patients depending on when they had been diagnosed as, over the years, different and newer agents have been indicated for the treatment of cryptosporidiosis. All four patients resolved their infections. Hence, endoscopy and biopsy is recommended for pediatric transplant patients who present with chronic diarrhea of unknown etiology. The patients who may be at a higher risk for cryptosporidial infections include those with an increased immunosuppressive state (i.e. pre-existing immunodeficiency, malignancy, re-transplantation, and those receiving higher doses of immunosuppressive therapy). While cryptosporidiosis is a non-lethal complication, it allows the clinician to gain further insight into the degree of immunosuppression of their patient. PMID- 10731060 TI - Successful transplantation of a cadaveric kidney with post-infectious glomerulonephritis. AB - This report describes a successful renal Tx in a patient with chronic renal failure, caused by dysplastic kidneys, who received a cadaveric kidney with post infectious glomerulonephritis. Sequential renal biopsies were performed at 12 h before Tx, and at 1 h and on days 8 and 58 post-Tx. Post-operative hematuria disappeared on day 9 and proteinuria on day 13. Normal graft function was observed within 1 month, with histologic resolution. Our study suggests that while the donor kidney facilitates deposition of certain immune reactants, this is a host (environmental) problem and when transplanted into a new host (new environment), the problem is no longer sustained. PMID- 10731061 TI - Complete necrosis of the renal pelvis and ureter after cadaveric renal transplantation. AB - Urological complications of transplantation, in particular, necrosis of the transplanted ureter, are rare. We report on a 20-yr-old boy who received a well matched cadaveric kidney transplant that was complicated by poor graft function and subsequent exploratory laparotomy for complete necrosis of the renal pelvis and ureter. PMID- 10731062 TI - Increased tacrolimus levels in a pediatric renal transplant patient attributed to chronic diarrhea. AB - Tacrolimus is an immunosuppressant used to prevent rejection of transplanted organs. It is metabolized in both the gut and the liver by the cytochrome P450 (CYP) 3A4 enzyme system and is a substrate for the P-glycoprotein (P-gp) drug efflux pump. As CYP3A4 enzymes and P-gp are present at differing concentrations throughout the gastrointestinal tract, the bioavailability of tacrolimus may be influenced by changes in gastrointestinal transit time in addition to changes in hepatic metabolism. We report the case of a pediatric renal transplant patient who experienced a three-fold increase in serum tacrolimus concentrations during an episode of gastroenteritis with chronic diarrhea. PMID- 10731063 TI - Inappropriate liver transplantation in a child with Alpers-Huttenlocher syndrome misdiagnosed as valproate-induced acute liver failure. AB - A 3-yr-old boy received valproic acid (VPA) for recurrent seizures. He developed coma and acute liver failure that were attributed to VPA toxicity, and underwent emergency orthotopic liver transplantation (OLTx). Despite good graft function, his neurological state worsened and led to death a few months later. The diagnosis of Alpers-Huttenlocher Syndrome (AHS) was suspected, subsequently to liver Tx, in view of ongoing neurologic deterioration and magnetic resonance imaging (MRI) findings. The syndrome, recessively inherited, associates brain degeneration with liver failure, and is now considered a mitochondrial disease. Enzyme activity deficiencies of the respiratory chain were identified in muscle mitochondria, as well as morphologic abnormalities of mitochondria in the explanted liver. Guidelines for diagnosis are presented, in order to differentiate the liver failure in AHS from that induced by genuine VPA toxicity. It is recommended to avoid liver Tx in patients with AHS given the fatal neurological course of the disease. PMID- 10731064 TI - Pediatric renal transplantation in the Netherlands. AB - In the Netherlands, pediatric kidney transplantation programs are available in four centers. We retrospectively analyzed the results obtained over the past decade. Between 1985 and 1995, 231 patients (139 boys) received 269 transplants, including 61 repeat. The recipients were aged 1.9-21.8 yrs (mean 10.9), the donors 0.3-63.3 yrs (median 11.4, mean 19.7). Immunosuppression consisted of corticosteroids, cyclosporin A and azathioprine, in various combinations and dosages. The patient survival during follow-up was 97%. The overall graft survival was 73% at 1 yr and 60% at 5 yrs after transplantation. Major causes of graft loss were acute rejection (21%), thrombosis (12%) and chronic rejection (28%). Acute rejection episodes were noted in 74% of all grafts. First acute rejection episodes had a moderate predictive value for graft loss (relative risk (RR), compared to rejection-free grafts, 5.9). First rejection episodes occurring later than 3 months after transplantation were considerably more predictive (RR 18.3) than early ones. Grafts from living related donors (n = 35) yielded a superior 5-yr graft survival (77%) and remained free of rejection more often than grafts from adult cadaveric donors(43% vs. 25%). The results of pre-emptive transplants were excellent (n = 13, 5-yr survival 100%). Repeat transplants had the same results as primary transplants. Recipients younger than 4 yrs showed a poor 5-yr graft survival of 38% (n = 13). Single kidney grafts from donors younger than 4 yrs (n = 35) had a 5-yr graft survival of 44%. In contrast, kidneys from these young donors did well if transplanted en bloc (n = 10, 5-yr graft survival 89%). These overall results are in line with those of others. The results may be improved by expansion of immunosuppressive therapy in the first year and by thrombosis prophylaxis in high-risk patient-donor combinations. Better results may be expected from more extensive use of living related donations, pre-emptive transplantation and en bloc transplantation instead of single kidneys of young donors. PMID- 10731065 TI - Bovine TB: working towards a long-term solution. PMID- 10731066 TI - 'Radical moves' to cut red tape in the meat industry. PMID- 10731067 TI - Virulent strain of IBR virus causes problems for Scottish cattle. PMID- 10731069 TI - Epidemiological and diagnostic observations following the elimination of porcine reproductive and respiratory syndrome virus from a breeding herd of pigs by the test and removal protocol. AB - The protocol of test and removal for the elimination of porcine reproductive and respiratory syndrome (PRRS) virus was applied to an 825-sow breeding herd. All the adult animals were tested and serum samples analysed by ELISA and polymerase chain reaction (PCR). Eighty-eight animals (10 x 7 per cent) were removed from the herd and, of these, three were ELISA-pOSitive and PCR-positive, and 85 were ELISA-positive and PCR-negative. They tended to be either individual sows, or groups of four to six animals housed in adjacent gestation stalls. Four of the ELISA-positive, PCR-negative sows were slaughtered and PRRS virus nucleic acid was detected in a sample of sternal lymph node from one of them. After the completion of the test and removal protocol, the breeding and finishing populations were monitored for 12 consecutive months by ELISA. The 960 samples taken were negative for PRRS virus antibodies. PMID- 10731068 TI - Bovine tuberculosis: towards a future control strategy. AB - The Independent Scientific Group on Cattle TB was appointed by the Government in 1998 to implement and develop the research recommendations of the 1997 Krebs report on bovine tuberculosis in cattle and badgers. In this article, members of the group discuss the approach they are adopting in attempting to ensure that future control strategies are scientifically based. In a second article, to be published in next week's Veterinary Record, the group will consider the extent to which efforts to control the disease in cattle may be constrained by limitations in current testing procedures. PMID- 10731070 TI - Dermatomyositis in five Shetland sheepdogs in the United Kingdom. AB - Five cases of dermatomyositis in four Shetland sheepdog puppies and one adult bitch are described. The dogs all had well-defined patches of scaling, crusting and alopecia over the muzzle, periorbital skin and distal limbs, and the tail, perineum and pinnae were affected in some of them. The affected puppies were all sired by the same stud dog. The affected adult bitch was unrelated to the puppies. Three of the four dogs tested had high serum creatine kinase concentrations and electromyographic abnormalities were detected in three of the four dogs tested. The histological changes observed in the skin of four of the dogs strongly supported the diagnosis of dermatomyositis, and in the fifth dog they were compatible with this diagnosis. Two of the puppies were euthanised shortly after being diagnosed. In the other two puppies and the adult the disease remains stable and non-progressive 15 to 18 months after diagnosis. The sire of the four affected puppies has been used extensively because it was considered to be genetically clear of collie eye anomaly. PMID- 10731071 TI - Sarcocystis infections in gazelles at the King Khalid Wildlife Research Centre, Saudi Arabia. AB - The prevalence of Sarcocystis species in muscle samples from gazelles kept as breeding groups at the King Khalid Wildlife Research Centre, Saudi Arabia, was determined by fibreoptic examination, pepsin digestion and histological techniques. No macroscopic sarcocysts were detected by fibreoptic examination, and the overall prevalence of Sarcocystis was 66 x 7 per cent by pepsin digestion, and 39 x 9 per cent by histological examination. By digestion, the tongue contained the highest density of bradyzoites in Gazella dorcas, and Gazella gazella erlangeri, the oesophagus in Gazella subgutturosa marica and skeletal muscle in Gazella gazella and Gazella thomsoni. Skeletal muscle was least affected in G dorcas, the oesophagus in G gazella, and the diaphragm in G g erlangeri, G s marica and G thomsoni. By histology, the heart contained most microcysts, except in G g erlangeri, in which the tongue was most affected. No single tissue type was therefore suitable for the diagnosis of sarcocystosis in this multispecies collection, although digestion was more sensitive in detecting infection than histology. The level of Sarcocystis infection was significantly higher in free-ranging gazelles kept in a main enclosure than in gazellas kept in breeding pens, and higher in adult gazelles than in juveniles. PMID- 10731072 TI - Gasterophilus nasalis infection: prevalence and pathological changes in equids in south-west England. PMID- 10731073 TI - Bilateral testicular interstitial cell tumour in an aged boar. PMID- 10731074 TI - Unusual outbreak of sporozoan encephalomyelitis in Bluefaced Leicester ram lambs. PMID- 10731075 TI - Thrombosis of the splenic vein in llamas (Lama glama). PMID- 10731076 TI - Intestinal candidiasis in a dog. PMID- 10731077 TI - Intraspecific aggression and suspected infanticide in otters. PMID- 10731078 TI - Canine distemper virus and metaphyseal osteopathy. PMID- 10731079 TI - RCVS awarding body audit-visits to practices. PMID- 10731080 TI - Influence of exercise on plasma and muscle free amino acids in trained rainbow trout. AB - Variations in the concentration of free amino acids in the muscle and plasma of trout submitted to 5 minutes of intense exercise have been studied. The responses of untrained fish and those trained performing the same type of exercise twice daily for 28 days are compared. Total amino acid concentrations in muscle tend to diminish after intense exercise. Significant decreases are observed in muscle content of alanine, beta-alanine, isoleucine and ornithine. Plasma amino acids tend to increase after exercise with significant differences in glutamate, GABA, methionine and NH4+. The small variations due to intense exercise suggest that the amino acids are mobilised. Training led to a decrease in total amino acid concentration in plasma but not in muscle, where levels of aspartate and ornithine increased. This suggests a metabolic adaptation to exercise, with amino acid level retention in the muscle. PMID- 10731081 TI - High hemoglobin affinity to oxygen and its relationships with lipid peroxidation during fever. AB - The effects of high hemoglobin-oxygen affinity (HOA) on rectal temperature and lipid free radical oxidation were investigated in red blood cells, heart, liver and kidneys of male rats during fever. Fever was induced by intraperitoneal injection of Salmonella typhi lipopolysaccharide (LPS; 5.0 mg kg(-1)). HOA was increased by addition of 0.5% sodium cyanate to drinking water for eight weeks. HOA modification (actual half-saturation oxygen pressure, P50act, decreased to 23.3+/-0.7 vs. 31.6+/-0.7 Torr in control; p < 0.001) weakened a febrile response: rise of temperature after 4 hours was 0.79+/-0.2 degrees C vs. 1.38+/ 0.1 degrees C in rats with normal HOA (p < 0.05). In red cells and tissues of rats with normal HOA, concentrations of conjugated dienes and Schiff bases increased during fever, and alpha-tocopherol level and catalase activity decreased. Rats with increased HOA had an inverse pattern of such changes. Changes in rectal temperature and markers of free radical oxidation correlated with a shift of oxyhemoglobin dissociation curve leftwards. The present results indicate that the intentional increment of HOA may substantially diminish lipid peroxidation activity, increase the body antioxidant content during fever and decrease the febrile response on LPS. PMID- 10731082 TI - Plasma estradiol-17beta levels and gonadosomatic index in tench (Tinca tinca L.) reared in natural and controlled conditions. AB - The plasma estradiol-17beta levels (E2-17beta) and gonadosomatic index (GSI) of female tench (Tinca tinca L.) reared outdoors and indoors were studied from December 1996 to April 1997. Water temperature and daylight were gradually increased (from 10 to 25 degrees C and from 9 to 16 hours per day, respectively) in the indoor group. Animals reared in natural conditions had similar plasma levels throughout the experiment but animals under controlled conditions experienced an increase in these levels in March in relation to previous months (4.81+/-0.73 ng/ml in March vs. 0.29+/-0.03 ng/ml and 0.99+/-0.31 ng/ml in January and February, respectively; P < 0.05) and in relation to animals reared outdoors (4.81+/-0.73 ng/ml vs. 0.47+/-0.19 ng/ml, respectively; P < 0.05). In April, a sharp decrease in estradiol-17beta in these animals was observed, probably associated with the end of vitellogenesis. GSI increased in both groups through the experiment and indoor animals showed greater values in March vs outdoor fishes (7.57+/-2.18 vs. 4.05+/-0.63; P < 0.05). No significant correlation was found between E2-17beta and GSI, (r = 0.35). It is concluded that manipulation of the photoperiod and temperature can modify gonadal development in tench, and this is similar to the situation in other Teleostei species. This research is the first step towards achieving, out of season, spawning in tench. PMID- 10731083 TI - Effect of long-term GHRH and somatostatin administration on GH release and body weight in prepubertal female rats. AB - In order to find a chronic GHRH administration capable of stimulating growth rate without depleting pituitary GH content, prepubertal female rats were subcutaneously (sc) treated with GHRH (1-29)-NH2 and somatostatin (SS). In experiment 1, the rats received sc injections of GHRH and cyclic natural SS for 19 days. In the second study, female rats were continuously treated during 21 days with GHRH, using a slow release pellet, alone or combined with one daily injection of long acting SS (octreotide). In experiment 1, body weight was significantly increased when GHRH was administered at the highest daily dosage (1200 microg/day), accompanied by an slight increment in pituitary GH content. Hypothalamic SS concentrations decreased when GHRH or SS were administered alone whereas the combined treatment with both peptides did not modify this parameter, which suggests the existence of a balance between the chronic actions of both peptides on hypothalamus. In experiment 2, the continuous infusion of GHRH increased plasma GH levels and tended to enhance pituitary GH content. Nevertheless, GHRH effect was not effective enough to increase body weight. By adding one daily injection of SS both GHRH effects on the pituitary gland were abolished. Our study indicates that female rats retain responsiveness to chronic GHRH and SS treatments at both pituitary and hypothalamic levels. PMID- 10731084 TI - Effect of MPTP on brain mitochondrial H2O2 and ATP production and on dopamine and DOPAC in the striatum. AB - An experimental rat model of Parkinson's disease was established by injecting rats directly in the striatum with the neurotoxic agent 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP). In order to study the action mechanism of this neurotoxic agent, MPTP and its main metabolite 1-methyl-4-phenylpyridinium (MPP+) were also added to suspensions of pyruvate/malate-supplemented nonsynaptic brain mitochondria, and the rates of hydrogen peroxide and ATP production were measured. Intrastriatal administration of MPTP produced a pronounced decrease in striatal dopamine levels (p < 0.005) and a strong increase in 3,4 hydroxiphenylacetic acid/dopamine ratio (an indicator of dopamine catabolism; p < 0.005) in relation to controls, as evaluated by in situ microdialysis. MPTP addition to rat brain mitochondria increased hydrogen peroxide production by 90%, from 1.37+/-0.35 to 2.59+/-0.48 nanomoles of H2O2/minute . mg of protein (p < 0.01). The metabolite MPP+ produced a marked decrease on the rate of ATP production of brain mitochondria (p < 0.005). These findings support the mitochondria-oxidative stress-energy failure hypothesis of MPTP-induced brain neurotoxicity. PMID- 10731085 TI - Differences between lactate concentration of samples from ear lobe and the finger tip. AB - Blood lactate concentrations in capillary samples obtained from the ear lobe or from the finger tip are used indistinctly, since they are considered equivalents. The aim of the study reported in this paper was to verify whether that assumption is valid due to the practical implications which any possible differences between these two sampling sites would have in the planning and assessing of an athletic training program. Twenty six healthy male athletes competing in different sports at the national level (9 rowers, 7 cyclists and 10 runners) were studied during the performance of a graded exercise test up to the point of exhaustion, on specific ergometers. In each group, capillary blood samples were obtained simultaneously from both the ear lobe and the finger tip at three different times during the test: 1) in resting conditions; 2) when exercising at a submaximal work load and 3) seven minutes after the point of exhaustion. Significant differences were found between the blood lactate concentrations of samples obtained from the ear lobe and from the finger tip (p < 0.001). The method error of repeated measurements for lactate concentrations from paired samples obtained in resting conditions was 27%, when exercising at a submaximal work load, 16% and at maximal work load, 3%. Capillary blood samples collected from the finger tip consistently showed higher values in lactate concentration than those obtained, at the same time, from the ear lobe. PMID- 10731086 TI - Determination of plasma melatonin levels by enzyme-linked immunosorbent assay (EIA) in turbot (Scophtalmus maximus L.) and tench (Tinca tinca L.). AB - An enzymoimmunoassay (EIA) kit for plasma melatonin (MLT) measurements was employed in tench (Tinca tinca) and in turbot (Scophtalmus maximus). Tench and turbot plasma samples were purified with a C18 reversed phase extraction columns because this kit is designed for human serum measurements. The lowest detection limit of the technique was 11.48 pg/well with a sensitivity at 50% binding of 100 pg/well. Intra-assay and inter-assay CV (%) were always less than 5% (n=8), and 9% (n=6) in tench plasma samples, and less than 5% (n=8) and 13% (n=5) in turbot plasma samples, respectively. Correlation coefficients between EIA and RIA measurements in tench and turbot plasma samples were 0.93 and 0.89 (p<0.001) respectively. Diurnal and nocturnal plasma melatonin mean levels were 14.7+/-2.1 pg/ml and 87.4+/-11 pg/ml in tench (n=15), and 3.5+/-0.4 pg/ml and 28.1+/-2.1 pg/ml in turbot (n=15). These species showed a melatonin circadian rhythm as in other animals studied. The results suggest that the commercial kit used in this experiment could be a suitable and alternative method to RIA for plasma MLT determinations in tench and turbot although it is necessary to increase volumes (1ml) and concentrate daytime samples. PMID- 10731087 TI - Pfeiffer syndrome caused by haploinsufficient mutation of FGFR2. AB - Mutations of the fibroblast growth factor receptors (FGFRs) cause several dominantly inherited congenital skeletal disorders and syndromes. Recently, these mutations have been suggested to cause either ligand-independent activation of the receptor or a dominant negative inactivation. The analysis of two Japanese patients with Pfeiffer syndrome and postaxial polydactyly of the hand now shows that both carried the same 1119-2A-to-G transition of the FGFR2 gene and this nonsense mutation caused skipping of exon 9(B) and haploinsufficiency of FGFR2. PMID- 10731088 TI - GAPO syndrome: a new case of this rare syndrome and a review of the relative importance of different phenotypic features in diagnosis. AB - The case of GAPO syndrome reported here is the 24th recorded case, 23 cases having been published previously. The 29-year-old male under discussion presents all the typical features of the syndrome, having short stature, dysmorphic craniofacial features, total alopecia and pseudoanodontia. Orally, the erupted primary dentition was extremely worn and on radiographic examination, the second mandibular molars were found to be unerupted, together with the entire permanent dentition. Cephalometry revealed the absence of facial pneumatisation, a deficient cranial base with diminished upper face height and maxillary and mandibular hypoplasia with a prognathic skeletal pattern. Histological examination of an extracted primary incisor and its surrounding root bone revealed extensive ankylosis. This paper describes in detail the clinical findings and reviews, and discusses previously published cases in relation to the present one. As with prior cases, parental consanguinity was present in the pedigree. PMID- 10731089 TI - Occurrence and temporal variation in matrix metalloproteinases and their inhibitors during murine secondary palatal morphogenesis. AB - Extracellular matrix (ECM) molecules are known to play a pivotal role in morphogenesis of the secondary palate, and changes in their composition and distribution, not attributable to changes in synthesis, are known to occur during palatogenesis. The present study was undertaken to determine if the enzymes responsible for mediating their degradation, the matrix metalloproteinases (MMP), and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMP), are temporospatially regulated during murine palatal shelf morphogenesis. Palatal shelves were harvested at gestational days (gd) 12, 13 and 14. MMPs were identified by gelatin zymography, with and without inhibitors, and the identity of specific bands confirmed by Western blot analysis. TIMPs were identified by reverse zymography. MMP and TIMP messages were detected using RT-PCR with specific primers to MMPs 2, 3, 7, 9 and 13 and TIMPs 1 and 2. Zymography revealed bands of molecular weights corresponding to MMPs 2, 7, 9 and 13 at all ages examined; the intensity of these bands increased with developmental age. Western blot analysis established the presence of MMP-3 and its developmental variation in expression. RT-PCR demonstrated the presence of mRNA for all MMPs and TIMP at all sampling times and all but MMP-2 showed developmental variation. Whereas increases in mRNA were detected for MMPs 3, 9, and 13, MMP-7 mRNA decreased between gd 12 and 14. The results of this study demonstrate that MMPs 2, 3, 7, 9 and 13 and TIMPs 1 and 2 and their messages are present during the course of palatal shelf remodelling and that their expression is temporally regulated. PMID- 10731090 TI - Expression of CSF-1 receptor on TRAP-positive multinuclear cells around the erupting molars in rats. AB - Bone resorption overlying a developing tooth is a necessary event in the creation of an eruption pathway. The formation and function of osteoclasts, which play a major role in bone resorption, are controlled by several factors. Although CSF-1 and its mRNA are expressed in dental follicle cells required for eruption, little is known about the contribution of CSF-1 to osteoclast formation on the bony crypt around the tooth germ. The receptor protein of the CSF-1 encoded proto oncogene c-fms was identified on multinucleated cells adjacent to the dental follicle, in conjunction with TRAP staining as a marker enzyme for osteoclasts in rat. c-Fms was highly expressed in TRAP-positive multinuclear cells at 3 days postnatal and the number of c-Fms-expressing cells was reduced thereafter. Administration of IL-1alpha, which enhances formation and function of osteoclasts, caused an increase in the number of c-Fms and TRAP-positive cells in rat. On the contrary, injection of calcitonin, which depresses osteoclast formation, caused a decrease in the number. It is obvious that the receptor of CSF-1 is expressed on the surface of osteoclasts around the tooth germ, on the dental follicle. These findings suggested that CSF-1 directly enhances the influx of osteoclasts adjacent to the erupting tooth, resulting in the formation of an eruption pathway. PMID- 10731091 TI - Facial skeletal growth in growing "toothless" osteopetrotic (op/op) mice: radiographic findings. AB - The defective bone resorption in the osteopetrotic (op/op) mouse brings about failure of tooth eruption. Furthermore, the op/op mouse has been studied as a "toothless" mouse in recent morphological and physiological investigations of the relationship between mastication and masseter muscle development. The present study was conducted to examine in detail the nasal bone and the premaxillary bone in this mutant mouse and to assess the roles of incisor growth and the mechanical stress of mastication in nasal bone and premaxillary bone growth. The forms of the nasal bone and the premaxillary bone were observed using roentgenography in both toothless op/op and normal (control) mice. In the op/op mouse, the nasal bone and the premaxillary bone show remarkable deformity. In contrast, the normal mouse appears well developed. This suggests that growth of the incisor root is important to normal upper jaw growth in the mouse. Furthermore, it is proposed that the upper facial phenotype seen in the op/op mice results from not only decreased bone resorption, but also from absence of the mechanical stress provided by normal mastication. PMID- 10731092 TI - Morphometry of the normal human ear: a cross-sectional study from adolescence to mid-adulthood. AB - The objective of this study was to supply information about: (1) normal gender based dimensions of ears (linear distances and ratios, area); (2) left right symmetry; and (3) growth changes between adolescence and mid-adulthood. The three dimensional co-ordinates of several soft-tissue landmarks on the ears and face were obtained by an electromagnetic digitizer in 40 male and 33 female adolescents aged 12-15 years. 73 young female and 89 young male adults aged 19-30 years, and 41 male and 38 female adults aged 31-56 years. From the landmarks, paired car width and length were calculated and averaged for age and gender, as well as the relevant ratios, ear areas and angles relative to the facial midline, and indices of left right symmetry. Comparisons were performed by factorial analysis of variance. All ear dimensions were significantly larger in men than in women (P < 0.005). A significant effect of age was found (P < 0.005), with larger values in older individuals. On average, the three-dimensional position of ears was symmetric, with symmetry coefficients ranging between 94 and 96%. Data collected in the present investigation could serve as a database for the quantitative description of human ear morphology during normal adolescent and adult growth. PMID- 10731093 TI - Henry Drysdale Dakin (1880-1952): early studies on radical and 2-electron oxidation of amino acids, proteins and fatty acids. PMID- 10731094 TI - Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation. Joo H, Lin Z, Arnold FH. Nature 1999; 399: 670-673. PMID- 10731095 TI - Redox reactions related to indoleamine 2,3-dioxygenase and tryptophan metabolism along the kynurenine pathway. AB - The heme enzyme indoleamine 2,3-dioxygenase (IDO) oxidizes the pyrrole moiety of L-tryptophan (Trp) and other indoleamines and represents the initial and rate limiting enzyme of the kynurenine (Kyn) pathway. IDO is a unique enzyme in that it can utilize superoxide anion radical (O2*- ) as both a substrate and a co factor. The latter role is due to the ability of O2*- to reduce inactive ferric IDO to the active ferrous form. Nitrogen monoxide (*NO) and H2O2 inhibit the dioxygenase and various inter-relationships between the nitric oxide synthase- and IDO-initiated amino acid degradative pathways exist. Induction of IDO and metabolism of Trp along the Kyn pathway is implicated in a variety of physiological and pathophysiological processes, including anti-microbial and anti tumor defense, neuropathology, immunoregulation and antioxidant activity. Antioxidant activity may arise from O2*- scavenging by IDO and formation of the potent radical scavengers and Kyn pathway metabolites, 3-hydroxyanthranilic acid and 3-hydroxykynurenine. Under certain conditions, these aminophenols and other Kyn pathway metabolites may exhibit pro-oxidant activities. This article reviews findings indicating that redox reactions are involved in the regulation of IDO and Trp metabolism along the Kyn pathway and also participate in the biological activities exhibited by Kyn pathway metabolites. PMID- 10731096 TI - News and views on thioredoxin reductases. PMID- 10731097 TI - Tobacco smoke induces mitochondrial depolarization along with cell death: effects of antioxidants. AB - Smoking has been associated with a large number of diseases, in particular cancers. Among the many substances identified in tobacco smoke, reactive oxygen species (ROS) are major carcinogens. We have previously reported that exposure of mammalian cells to tobacco smoke induces the expression of stress proteins, as well as apoptosis (programmed cell death). Here we examined the effects of tobacco smoke on mitochondrial membrane potential (deltapsim), since mitochondria have been proposed to control the effector phase of apoptosis. We used normal human monocytes for these experiments, with the prospect for application of deltapsim as a biomarker of oxidative stress. Tobacco smoke induced mitochondrial depolarization at 3 h, and apoptosis (or necrosis for higher concentrations) after 16 h. Apoptosis was assessed by both a functional approach (annexin V binding) and morphological analysis (electron microscopy). N-acetyl-cysteine prevented tobacco smoke-induced deltapsim disruption and apoptosis, while the caspase inhibitor Z-VAD.Fmk did not affect deltapsim, though preventing apoptosis, and superoxide dismutase had no effect. Our data designate mitochondria as a target for ROS-mediated effects of tobacco smoke exposure. PMID- 10731098 TI - Assessing the effect of reactive oxygen species on Escherichia coli using a metabolome approach. AB - A two-dimensional thin-layer chromatographic analysis of [14C]-labelled metabolites in Escherichia coli was employed to follow metabolic shifts in response to superoxide stress. Steady-state challenge with paraquat at concentrations inducing SoxRS-controlled genes resulted in several alterations in metabolite pools, including a striking increase in valine concentration. Elevated valine levels, together with increased glutathione and alkylperoxidase, are proposed to constitute an intracellular protection mechanism against reactive oxygen species. As shown by this example of metabolome analysis, novel cellular responses to environmental challenge can be revealed by following the total complement of metabolites in a cell. PMID- 10731099 TI - Derangement of Kupffer cell functioning and hepatotoxicity in hyperthyroid rats subjected to acute iron overload. AB - Liver oxidative stress, Kupffer cell functioning, and cell injury were studied in control rats and in animals subjected to L-3,3',5-tri-iodothyronine (T3) and/or acute iron overload. Thyroid calorigenesis with increased rates of hepatic O2 uptake was not altered by iron treatment, whereas iron enhanced serum and liver iron levels independently of T3. Liver thiobarbituric acid reactants formation increased by 5.8-, 5.7-, or 11.0-fold by T3, iron, or their combined treatment, respectively. Iron enhanced the content of protein carbonyls independently of T3 administration, whereas glutathione levels decreased in T3- and iron-treated rats (54%) and in T3Fe-treated animals (71%). Colloidal carbon infusion into perfused livers elicited a 109% and 68% increase in O2 uptake in T3 and iron-treated rats over controls. This parameter was decreased (78%) by the joint T3Fe administration and abolished by gadolinium chloride (GdCl3) pretreatment in all experimental groups. Hyperthyroidism and iron overload did not modify the sinusoidal efflux of lactate dehydrogenase, whereas T3Fe-treated rats exhibited a 35-fold increase over control values, with a 54% reduction by GdCl3 pretreatment. Histological studies showed a slight increase in the number or size of Kupffer cells in hyperthyroid rats or in iron overloaded animals, respectively. Kupffer cell hypertrophy and hyperplasia with presence of inflammatory cells and increased hepatic myeloperoxidase activity were found in T3Fe-treated rats. It is concluded that hyperthyroidism increases the susceptibility of the liver to the toxic effects of iron, which seems to be related to the development of a severe oxidative stress status in the tissue, thus contributing to the concomitant liver injury and impairment of Kupffer cell phagocytosis and particle-induced respiratory burst activity. PMID- 10731100 TI - Substrate-dependent effect of phenolic antioxidants on Ca2+ accumulation by rat liver mitochondria. AB - The effect of different phenolic antioxidants on mitochondrial Ca2+ capacity (maximum amount of Ca2+ mitochondria can accumulate) was studied. Butylated hydroxytoluene substantially enhanced the Ca2+ capacity in mitochondria oxidizing succinate, butylated hydroxyanisole had a moderate effect while 2,5-di-(t-butyl)- 1,4 benzohydroquinone did not affect Ca2+ capacity at all. The analysis of Ca2+ accumulation in mitochondria oxidizing succinate in the presence of 2,5-di-(t butyl)-1,4 benzohydroquinone revealed inhibition of the rate of Ca2+ accumulation. This effect was absent when ATP hydrolysis or NAD+-dependent substrate oxidation supported Ca2+ transport. Direct measurements of oxygen consumption revealed the concentration-dependent inhibition of succinate oxidation by increasing concentrations of 2,5-di-(t-butyl)- 1,4 benzohydroquinone. When succinate was substituted by NAD+-dependent respiratory substrates, the Ca2+ capacity of mitochondria with 2,5-di-(t-butyl)-1,4 benzohydroquinone was even higher than in the presence of butylated hydroxytoluene. PMID- 10731101 TI - Local estrogen biosynthesis in males and females. AB - It is now apparent that in men and in postmenopausal women, estrogens have important physiological and pathophysiological roles. However, importantly, these actions are at a local level, namely paracrine, autocrine, and even 'intracrine' rather than endocrine in the classical sense. Thus for example local estrogen biosynthesis in the bones of men plays a hitherto unsuspected role in the maintenance of bone mineralization and in epiphyseal fusion; and in the testes, estrogen is essential for male germ cell development. On the other hand, in postmenopausal women, the mesenchymal cells of the breast are the major source of estrogen responsible for breast cancer development. This realization points to the importance of circulating C19 precursors in the maintenance of adequate estrogen biosynthesis in extragonadal sites and suggests the possibility of new therapies to block estrogen synthesis in a tissue-specific fashion. PMID- 10731102 TI - The regulation of aromatase activity in breast fibroblasts: the role of interleukin-6 and prostaglandin E2. AB - Prostaglandin E2 (PGE2) and cytokines, such as interleukin-6 (IL-6) or tumour necrosis factor a (TNFalpha) can regulate aromatase activity. In the present study we have compared their abilities to stimulate aromatase activity in fibroblasts derived from 'normal' breast adipose tissue proximal to a tumour or breast tumours. PGE2, TNFalpha and IL-6 plus its soluble receptor (IL-6sR) all increased aromatase activity in these cells. Basal aromatase activity and the degree of aromatase stimulation by these factors were greater in fibroblasts derived from 'normal' breast tissue than from breast tumours. The ability of IL 6+IL-6sR to increase aromatase activity was only marginally reduced by the PG synthesis inhibitor, indomethacin, indicating that IL-6+IL-6sR does not appear to act via induction of PG synthesis. The ability of PGE2 to stimulate aromatase activity in fibroblasts derived from 'normal' breast tissue was potentiated by IL 6sR suggesting that PGE2 may act via induction of IL-6. This was confirmed by measurement of IL-6 in conditioned medium collected from these cells. A significant increase in IL-6 concentrations was detected in conditioned medium collected from cells treated with PGE2. It is concluded that in some fibroblasts PGE2 may exert part of its regulatory effect on breast tissue aromatase activity via induction of IL-6. PMID- 10731103 TI - Breast tumor aromatase: functional role and transcriptional regulation. AB - Aromatase has been shown to be expressed at a higher level in human breast cancer tissue than in normal breast tissue, by means of enzyme activity measurement, immunocytochemistry, and RT-PCR analysis. Cell culture including MCF-7 breast cancer cells, animal experiments using aromatase-transfected breast cancer cells, and transgenic mouse studies have demonstrated that estrogen production in situ plays a more important role than circulating estrogens in breast tumor promotion. In addition, tumor aromatase is believed to be able to stimulate breast cancer growth through both autocrine and paracrine pathways, as demonstrated by a three dimensional cell culture study. RT-PCR and gene transcriptional studies have revealed that the aromatase promoter is switched from a glucocorticoid-stimulated promoter, I.4, in normal tissue to cAMP-stimulated promoters, I.3 and II, in cancerous tissue. Recently, we identified and characterized a cAMP-responsive element (CREaro) upstream from promoter I.3 by DNA deletion and mutational analyses. Our results from promoter functional analysis also demonstrated an interaction between the CREaro and the silencer element (S1) that was identified previously in our laboratory. In the presence of cAMP, the positive regulatory CREaro can overcome the action of the silencer on the function of promoter I.3. On the basis of results generated from our own and other laboratories, we propose that, in normal breast adipose stromal cells and fibroblasts, aromatase expression is driven by promoter I.4 (glucocorticoid dependent), and that the action of promoters I.3 and II is suppressed by the silencer negative regulatory element. However, in cancer cells and surrounding adipose stromal cells, the cAMP level increases, and aromatase promoters are switched to cAMP-dependent promoters - I.3 and II. Furthermore, we applied the yeast one-hybrid screening method to search for proteins interacting with the silencer element, S1. The major protein identified was ERRalpha-1; however, SF-1, which is present in the ovary, is not detected in breast cancer tissue. Using a reporter plasmid with the aromatase genomic fragment containing promoter I.3 and S1, in breast cancer SK-BR-3 cells, ERRalpha-1 was found to have a positive regulatory function. It is believed that the silencer element in the human aromatase gene may function differently in different tissues, as a result of distinct expression patterns of transcription factors. PMID- 10731104 TI - Aromatase within the breast. AB - In situ aromatization and enhanced uptake of estradiol from plasma are two potential mechanisms for maintenance of high concentrations of estradiol found in breast tumors of postmenopausal patients. To test the relative importance of these two mechanisms, a nude mouse model was established by inoculating aromatase (A+) and/or sham (A-) transfected MCF-7 cells into ovariectomized mice. Postmenopausal hormonal status was simulated by providing estradiol Silastic implants which clamped plasma estradiol levels at 5-20 pg/ml. We demonstrated that in situ aromatization rather than the uptake mechanism is the key determinant of tumor estradiol levels and tumor growth rate under conditions reflecting the postmenopausal state. The importance of intratumoral aromatase was also suggested by the findings that long-term estrogen deprivation increases sensitivity to estradiol and enhances aromatase activity in MCF-7 cells. The results of our in vivo and in vitro studies suggest that complete blockade of in situ aromatization in the breast would provide added benefit to postmenopausal breast cancer patients, especially those who relapse from antiestrogen therapy. PMID- 10731106 TI - Serum oestradiol and breast cancer risk. AB - Breast cancer risk is increased by early menarche and late menopause, suggesting that the long duration of exposure of the breasts to the high levels of ovarian steroids in premenopausal women increases risk. Recent prospective studies have shown that postmenopausal women who develop breast cancer have significantly greater prediagnostic serum concentrations of oestradiol than postmenopausal women who remain healthy. Estimation of long-term oestradiol concentrations in premenopausal women is difficult, and few data are available from prospective studies, but these are compatible with the hypothesis that relatively high oestradiol concentrations in premenopausal women are also associated with an increase in breast cancer risk. Women in populations with low breast cancer rates have low serum oestradiol concentrations both before and after the menopause. The serum concentration of oestradiol is probably a major determinant of breast cancer risk, but more data are needed to confirm this and to investigate the possible roles of other sex hormones. PMID- 10731105 TI - Aromatase and breast cancer susceptibility. AB - Based on experimental and epidemiological evidence it is hypothesized that estrogen increases breast cancer risk by increasing mitotic activity in breast epithelial cells. Aromatase is crucial to the biosynthesis of estrogens and may therefore play a role in breast cancer development. Supporting data for an etiological role of aromatase in breast tumor biology are several-fold. First, the association between weight and postmenopausal breast cancer risk may be mediated by aromatase. Secondly, a pilot study found a higher aromatase expression in normal breast adipose tissue from breast cancer cases as opposed to healthy women. Thirdly, experimental data in animals suggest that aromatase activity predisposes mammary tissue to preneoplastic and neoplastic changes. In a multiethnic cohort study conducted in Los Angeles and on Hawaii we investigated (i) whether the plasma estrone to androstenedione (E1/A) ratio in different ethnic groups was associated with ethnic differences in breast cancer incidence, and (ii) whether genetic variation in the CYP19 gene encoding the P450 aromatase protein was associated with breast cancer risk. The age- and weight-adjusted ethnic specific E1/A ratios x 100 among women without oophorectomy were 7.92 in African-Americans, 8.22 in Japanese, 10.73 in Latinas and 9.29 in non-Latina Whites (P=0.09). The high E1/A ratio in Latina women was not associated with a high breast cancer incidence; in fact Latina women had the lowest breast cancer incidence in the cohort observed so far. We found no consistent association of an intronic (TTTA)n repeat polymorphism with breast cancer risk in different ethnic groups. This polymorphism was not associated with differences in the plasma E1/A ratio in a way that would predict its functional relevance. We describe a newly identified TTC deletion in intron 5 of the CYP19 gene that is associated with the (TTTA)n repeat polymorphism. Neither this polymorphism, nor a polymorphism at codon 264 in exon VII of the CYP19 gene, was associated with breast cancer. We did not identify any genetic variation in exon VIII in 54 African-American subjects. We identified rare genetic variants of unknown functional relevance in the promoter 1.4 of the CYP19 gene in 3 out of 24 Latina women. Further investigation into the role of aromatase in breast cancer etiology is important, given that the potential use of aromatase inhibitors as breast cancer chemopreventives depends on these results. PMID- 10731107 TI - Drug and hormone interactions of aromatase inhibitors. AB - The clinical development of aromatase inhibitors has been largely confined to postmenopausal breast cancer patients and strongly guided by pharmacological data. Comparative oestrogen suppression has been helpful in circumstances in which at least one of the comparitors has caused substantially non-maximal aromatase inhibition. However, the triazole inhibitors, letrozole and anastrozole, and the steroidal inhibitor, exemestane, all cause >95% inhibition. Comparisons between these drugs therefore require more sensitive approaches such as the direct measurement of enzyme activity by isotopic means. None of these three agents has significant effects on other endocrine pathways at its clinically applied doses. Pharmacokinetic analyses of the combination of tamoxifen and letrozole have revealed that these drugs interact, resulting in letrozole concentrations approximately 35-40% lower than when letrozole is used alone. PMID- 10731108 TI - Biology of aromatase inhibitors: pharmacology/endocrinology within the breast. AB - Both mammary adipose tissue and breast cancers have the ability to aromatize androgens into oestrogens. Such potential may maintain the growth of hormone dependent tumours. It has therefore been important to determine the effects of new aromatase inhibitors such as formestane, exemestane, anastrozole and letrozole on oestrogen biosynthesis and concentrations of endogenous hormones within the breast. Studies based on in vitro incubations of breast cancer and cultures of mammary adipose tissue fibroblasts demonstrate that these drugs are highly effective inhibitors, with IC50 values ranging between 1 and 50 nM (although the relative efficacy varies between tissues and test systems). Despite this potential, in vitro incubations of breast tissues from patients treated with type II inhibitors such as aminoglutethimide and letrozole can display paradoxically high aromatase activity; this appears to be caused by the reversible nature of the inhibition, coupled with induction/stabilization of the aromatase enzyme. To assess in situ effects within the breast, postmenopausal women with large primary breast cancers have been treated neoadjuvantly with aromatase inhibitors using a protocol that included (i) breast biopsy before treatment, (ii) definitive surgery after 3 months of treatment and (iii) infusion of [3H]androstenedione and [14C]oestrone in the 18 h immediately before biopsy and surgery. With this study design, it has been shown that drugs such as letrozole profoundly inhibit in situ aromatase activity and reduce endogenous oestrogens within the breast. PMID- 10731109 TI - Effects of aromatase inhibitors on the pathobiology of the human breast, endometrial and ovarian carcinoma. AB - It is very important to examine the influence of inhibition of in situ estrogen production on the pathobiology of human sex steroid-dependent tumors in order to understand the clinical effects of aromatase inhibitors. We have examined the biological changes before and after aromatase inhibitor treatment in vitro (endometrial and ovarian cancer) and in vivo (breast cancer). First, we analyzed these changes using histoculture of 15 human endometrial cancers and 9 ovarian cancers. Five of the fifteen endometrial cancers and four of the nine ovarian cancers demonstrated decreased [3H]thymidine uptake or Ki67 labeling index after 14alpha-hydroxy-4-androstene-3,6,17-trione (NKS01) treatment. In ovarian cancer cases, the responsive cases tended to be associated with higher aromatase and estrogen receptor alpha (ER) expression compared with the other cases but this was not seen in the endometrial cancer cases. There were no changes in ER and aromatase expression before and after NKS01 treatment in either ovarian or endometrial cancer cases. We then studied the same primary human breast tumors before and after aminoglutethimide (AMG, n=3) and 4-hydroxyandrostenedione (4 OHA, n=3) treatment. Tumor aromatase activity increased in 3 cases and decreased or was unchanged in 3 cases but aromatase immunoreactivity in stroma and adipocytes was unaltered in 5 cases. There were no changes in the ER labeling index before or after treatment. Five of the six cases including the responsive cases tended to be associated with decreased cell proliferation or Ki67 expression and increased apoptosis when examined by the TUNEL method. These results indicate that aromatase inhibitors may exert their effects on human breast and other cancers through decreasing proliferation and increasing apoptosis, possibly without altering ER status. PMID- 10731110 TI - Aromatase inhibitors and their antitumor effects in model systems. AB - The potential of aromatase (estrogen synthetase) within the breast to provide a significant source of estrogen mediating tumor proliferation is suggested by studies reporting 4- to 6-fold higher estrogen levels in tumors than in plasma of postmenopausal patients with breast cancer. Recent studies in our laboratory have identified aromatase and its mRNA in tumor epithelial cells using immunocytochemistry and in situ hybridization. In addition, significant aromatase activity, which was stimulated 7-fold by dexamethasone, was measured in metastatic cells isolated from a breast cancer patient. Increase in proliferation, as measured by proliferating cell nuclear antigen immunostaining in tumor sections and by thymidine incorporation into DNA in response to testosterone, was observed in histocultures of breast cancer samples. This latter effect could be inhibited by 4-hydroxyandrostenedione. These results imply that intratumoral aromatase has functional significance and may be an important target for successful inhibitor treatment of breast cancer patients. To investigate treatment strategies with aromatase inhibitors and antiestrogens, we developed an intratumoral aromatase model to simulate the hormone responsive postmenopausal breast cancer patient. Tumors of estrogen receptor positive human breast carcinoma cells (MCF-7) transfected with the human aromatase gene are grown in ovariectomized nude mice. These cells synthesize sufficient estrogen to stimulate tumor formation. We have utilized this model to investigate the effects on tumor growth of the antiestrogens, tamoxifen and ICI 182780, and the aromatase inhibitors, letrozole and anastrozole (arimidex), alone and in combination. Both the aromatase inhibitors and the antiestrogens were effective in suppressing tumor growth. However, letrozole was significantly more effective than the antiestrogens. When the aromatase inhibitors were combined with the antiestrogen, tamoxifen, tumor growth was suppressed to about the same extent as with the aromatase inhibitors alone. Furthermore, the results do not suggest any benefit from combining tamoxifen with the pure antiestrogen, ICI 182780. Thus sequential use of these agents is likely to be more advantageous to the patient in terms of longer duration of effective treatment. PMID- 10731111 TI - Aromatase inhibitors and enzyme stability. AB - The effects of two steroidal (4-hydroxyandrostenedione and atamestane) and three non-steroidal (fadrozole, vorozole, and pentrozole) aromatase inhibitors on the levels of aromatase mRNA and protein were examined in vitro and in vivo. Immunocytochemistry revealed increased quantities of immunoreactive aromatase in human choriocarcinoma-derived JEG-3 cells in response to pretreatment with the non-steroidal inhibitors. To elucidate this effect in detail, aromatase protein in JEG-3 cells after treatment with various inhibitors was quantified using an enzyme-linked immunosorbent assay (ELISA). A time-dependent increase in aromatase protein in the cells was observed with all the aromatase inhibitors except 4 hydroxyandrostenedione, whereas aromatase mRNA levels in the cells remained unchanged during the inhibitor treatment. The three non-steroidal agents caused an approximately fourfold increase in aromatase protein in the cells 24 h after the treatment, as compared with untreated controls. The increase in aromatase protein in the cells was not blocked by treatment with cycloheximide, an inhibitor of protein synthesis. The inhibitors also appeared to block the rapid degradation observed in JEG-3 cells after induction by forskolin. In vivo, daily injection of the inhibitors into adult female mice caused increases in levels of both aromatase mRNA and protein in the ovary. The increase in aromatase mRNA in this in vivo study could be explained by an increase in gonadotropin concentrations in response to decreased plasma concentrations of estrogens. In conclusion, we suggest that aromatase inhibitors increase aromatase protein through stabilization and reduced protein turnover. PMID- 10731112 TI - Role of aromatase inhibitors in advanced breast cancer. AB - A number of potent and selective non-steroidal aromatase inhibitors are now available for treatment of advanced breast cancer in postmenopausal women, of which anastrozole and letrozole, in particular, represent a significant advantage over the earlier agents in terms of both efficacy and tolerability. These agents are rapidly becoming established as the second-line therapy of choice in postmenopausal women with advanced disease, progressing on tamoxifen, and data on their efficacy as first-line treatment compared with tamoxifen will be available in the near future. Exemestane, a new, steroidal aromatase inhibitor which potentially lacks cross-resistance with non-steroidal agents is still in clinical development. The full potential of the new-generation aromatase inhibitors in the treatment of breast cancer is currently being investigated in a large program of clinical trials evaluating their use as adjuvant treatment following surgery in postmenopausal patients with early disease. PMID- 10731113 TI - Lessons from the use of aromatase inhibitors in the neoadjuvant setting. AB - Postmenopausal patients with oestrogen receptor-positive locally advanced T4b, N0 1, M0 and large operable breast cancers T2>3 cm, T3, T4, N0-1 and M0 have been treated with 2.5 mg letrozole (12 patients), 10 mg letrozole (12 patients), 1 or 10 mg anastrozole (24 patients) and 20 mg tamoxifen (65 patients). There was no apparent difference in response rate between 2.5 and 10 mg letrozole. Only 17 patients with anastrozole have so far completed the 3-month treatment period. Median clinical, mammographic and ultrasound reductions in tumour volumes for patients treated with letrozole were 81% (95% confidence interval (CI) 66-88), 77% (95% CI 64-82) and 81% (95% CI 69-86) respectively and for anastrozole, values were 87% (95% CI 59-97), 73% (95% CI 58-82) and 64% (95% CI 52-76) respectively. This compares with a median reduction in tumour volume for tamoxifen-treated patients as assessed by ultrasound of 48% (95% CI 27-48). There were seven complete clinical responses (CR), sixteen patients who achieved 50% or greater reduction in tumour volume (PR) and one no change (NC) for letrozole and four CRs, twelve PRs and one progressive disease for anastrozole. Best radiological responses were one CR, twenty PRs and three NCs for letrozole and one CR, fifteen PRs and one NC for anastrozole. This study has shown that the new aromatase inhibitors, letrozole and anastrozole, are highly effective agents in the neoadjuvant setting and they should now be compared with tamoxifen as first line treatment in a randomised study. PMID- 10731115 TI - The potential of aromatase inhibitors in breast cancer prevention. AB - Substantial evidence supports the concept that estrogens cause breast cancer in animals and in women but the precise mechanism is unknown. The most commonly held theory is that estrogens stimulate proliferation of breast cells and thus statistically increase the chances for genetic mutations which could result in cancer. Another theory is that estrogen metabolism generates oxygen-free radicals and quinones which produce both stable and unstable DNA adducts. Both result in genetic mutations which accumulate and could ultimately cause cancer. A major criticism of the latter hypothesis is that breast tissue contains insufficient concentrations of estrogen for accumulation of genotoxic metabolites. Our hypothesis is that breast tissue estrogen levels, as a result of in situ synthesis, are much higher than previously thought. We and others have shown that estrogen can be made in the breast itself through conversion of androgens to estrogens, a process catalyzed by the enzyme aromatase. The levels of estrogen in the breast increase when aromatase is overexpressed. With sufficient amounts of aromatase in breast tissue, enough estradiol as substrate should be available to allow formation of substantial amounts of genotoxic metabolites. We postulate that aromatase overexpression may in this way cause breast cancer. As evidence supporting this concept, four animal models of aromatase overexpression and either breast cancer or premalignant lesions have been described. We have provided evidence that normal breast tissue can make estrogen and that certain stimulatory compounds can increase aromatase activity in the breast by nearly 10,000-fold. If our concepts are correct, it might be possible to prevent breast cancer by blocking the aromatase enzyme. Drugs are currently available to inhibit aromatase nearly completely without causing significant side-effects. Aromatase inhibitors might be more effective than antiestrogens in preventing breast cancer because of their dual role to block both initiation and promotion of breast cancer. To inhibit the initiation process, these inhibitors would reduce levels of the genotoxic metabolites of estradiol by lowering estradiol concentrations in tissue. At the same time, aromatase inhibitors would inhibit the process of tumor promotion by lowering tissue levels of estradiol and thus blocking cell proliferation. These concepts provide a strong rationale for studies of aromatase inhibitors to prevent breast cancer. PMID- 10731114 TI - Use of aromatase inhibitors in the adjuvant treatment of breast cancer. AB - The value of endocrine treatment of early breast cancer has been illustrated by the antioestrogen, tamoxifen, which has now been available for nearly 30 years. However, if the recognised side effects and pharmacological properties of tamoxifen are taken into consideration, it is possible that other endocrine treatments that are now available can provide equal or superior efficacy, along with improved tolerability. One such group of agents is the aromatase inhibitors specifically the new-generation triazole aromatase inhibitors, such as anastrozole and letrozole, which have both shown tolerability and efficacy advantages over standard treatments in postmenopausal women with advanced breast cancer. There are convincing reasons why the new generation of aromatase inhibitors have advantages over tamoxifen. For instance, from their agonist properties, the effects on the endometrium and tumour stimulation seen with tamoxifen would not be expected, nor would the visual disturbances that have been associated with the triphenylethylene compounds, including tamoxifen. A number of aromatase inhibitors, for instance, anastrozole, letrozole and exemestane, are currently being investigated for treatment of early breast cancer. The results of the trials of aromatase inhibitors and tamoxifen will, in the next few years, define whether or not the new-generation aromatase inhibitors have a role to play in the treatment of postmenopausal women with early breast cancer. PMID- 10731116 TI - Aromatase inhibitors: a dose-response effect? PMID- 10731117 TI - Cross-resistance to different aromatase inhibitors in breast cancer treatment. AB - Recent studies have documented the biochemical and clinical efficacy of novel aromatase inhibitors. These drugs belong to one of two classes: non-steroidal and steroidal drugs, known to be different with respect to enzyme binding site and their effect on the aromatase enzyme. Several studies have now confirmed a lack of complete cross-resistance to drugs of the two classes. While some of these observations may be explained by a more potent aromatase inhibition caused by some aormatase inhibitors compared to others, this is not always the case. Such observations therefore focus on the importance of alternative mechanism of action like a differential influence on the intratumour aromatase enzyme by the different drugs. Current and future studies should aim at exploring the mechanism of cross-resistance and evaluate optimal use of different aromatase inhibitors in sequence or probably, also in concert. PMID- 10731118 TI - Aromatase inhibitors and their use in the sequential setting. AB - Over the past decade several novel aromatase inhibitors have been introduced into clinical practice. The discovery of these drugs followed on from the observation that the main mechanism of action of aminogluthemide was via inhibition of the enzyme aromatase thereby reducing peripheral levels of oestradiol in postmenopausal patients. The second-generation drug, 4-hydroxyandrostenedione (formestane), was introduced in 1990 and although its use was limited by its need to be given parenterally it was found to be a well-tolerated form of endocrine therapy. Third-generation inhibitors include vorozole, letrozole, anastrozole and exemestane, the former three being non-steroidal inhibitors, the latter being a steroidal inhibitor. All are capable of inhibiting aromatase action by >95% compared with 80% in the case of 4-hydroxyandrostenedione. The sequential use of different generations of aromatase inhibitors in the same patients is discussed. Studies suggest that an optimal sequence of these compounds may well result in longer remission in patients with hormone receptor positive tumours. PMID- 10731119 TI - Combination hormonal therapy involving aromatase inhibitors in the management of women with breast cancer. AB - Numerous comparative clinical trials have been conducted evaluating combination hormonal therapy involving the aromatase inhibitor aminoglutethimide, but there is no evidence for any superiority of this approach over single-agent therapy alone. The advent of new aromatase inhibitors with greater potency, selectivity, and better tolerability has prompted a reconsideration of the combined therapy approach, with attention being focused on pharmacologic and endocrinologic clinical research. The value of combining newer aromatase inhibitors with other hormonal agents remains to be established. PMID- 10731120 TI - Aromatase inhibitors in relation to other forms of endocrine therapy for breast cancer. AB - Most endocrine therapies for breast cancer inhibit tumor growth by depriving the cell of estrogen or by blocking its receptor. However, some drugs, such as tamoxifen, can bind to the estrogen receptor (ER) and have both estrogenic and antiestrogenic effects, depending on the tissue, cell, or promoter context. These mixed properties may be explained by new information on ER function at the molecular level. Whether a synthetic drug acts as an estrogen or antiestrogen on a specific gene may be dictated by the particular ensemble of ER subtype, receptor interacting proteins, other transcription factors, or specific elements within the promoter of estrogen-regulated genes. Alterations in these other factors may also play a role in resistance to hormonal therapies. Aromatase inhibitors, like ovarian ablation, inhibit growth by lowering the estrogen concentration in blood or in the tumor tissue itself. Aromatase inhibitors are effective even in postmenopausal women with low estrogen concentrations - probably because of the ability of the tumor to become hypersensitive to estrogen after prolonged estrogen deprivation. Given that the ER itself is the prime target for endocrine manipulation, the ideal endocrine therapy may be one that reduces or eliminates ER from the tumor cell. Pure steroidal antiestrogens are then of great interest because, not only do they inhibit ER-induced transactivation of estrogen regulated genes, they also induce ER degradation. Additional clinical trials are necessary to identify the optimal endocrine therapy and optimal sequence of available therapies. PMID- 10731121 TI - Status of aromatase inhibitors in relation to other breast cancer treatment modalities. AB - Aromatase is one of the key enzymes possibly linked with the perpetuation or even initiation of breast cancer. Modulation of its activity by the new generation inhibitors has resulted in increased responses and improved therapeutic ratio compared with those of parent aromatase inhibitors. More recent trials have shown promising results with regard to improved therapeutic ratio compared with what is seen with presently accepted second-line hormonal approaches. Present data and laboratory research indicate that new aromatase inhibitors have the potential to play an important role as adjuvants, and possibly in the prevention of human breast cancer. It is probable that it may be as adjuvants that their real therapeutic strength in terms of a beneficial impact on survival may be realized. The absence of estrogen agonist activity of new aromatase inhibitors on lipid and bone metabolism calls for more clinical studies having late mortality in breast cancer survivors as the ultimate outcome objective; in this regard, interaction of new aromatase inhibitors with new selective estrogen receptor modulators looks promising. Achievement of these outcomes, and understanding of interactions with other therapies, await the termination of present trials and the start of new initiatives. PMID- 10731122 TI - Estrogen production in endometriosis and use of aromatase inhibitors to treat endometriosis. AB - Estrogen is the most important known factor that stimulates the growth of endometriosis. Estrogen delivery to endometriotic implants was classically viewed to be only via the circulating blood in an endocrine fashion. We recently uncovered an autocrine positive feedback mechanism, which favored the continuous production of estrogen and prostaglandin (PG)E2 in the endometriotic stromal cells. The enzyme, aromatase, is aberrantly expressed in endometriotic stromal cells and catalyzes the conversion of C19 steroids to estrogens, which then stimulate cyclooxygenase-2 to increase the levels of PGE2. PGE2, in turn, is a potent inducer of aromatase activity in endometriotic stromal cells. Aromatase is not expressed in the eutopic endometrium. Aromatase expression in endometriosis and its inhibition in eutopic endometrium are controlled by the competitive binding of a stimulatory transcription factor, steroidogenic factor-1, and an inhibitory factor, chicken ovalbumin upstream promoter-transcription factor to a regulatory element in the aromatase P450 gene promoter. In addition, we find that endometriotic tissue is deficient in 17beta-hydroxysteroid dehydrogenase type 2, which is normally expressed in eutopic endometrial glandular cells and inactivates estradiol-17beta to estrone. This deficiency is another aberration that favors higher levels of estradiol-17beta in endometriotic tissues in comparison with the eutopic endometrium. The clinical relevance of local aromatase expression in endometriosis was exemplified by the successful treatment of an unusually aggressive form of recurrent endometriosis in a postmenopausal woman using an aromatase inhibitor. PMID- 10731123 TI - Use of aromatase inhibitors in precocious puberty. AB - During puberty, estrogen causes breast maturation and growth of the uterine lining in girls, and accelerates linear growth and bone maturation in both boys and girls. Decreasing the biosynthesis of estrogen can attenuate these processes. In 12 girls with the McCune-Albright syndrome (MAS), in which precocious puberty is due to production of estrogen from ovarian cysts, testolactone (40 mg/kg per day) decreased the volume of ovarian cysts, the frequency of menses, and the rates of growth and bone maturation, for periods of 1-4 years. In a 6-month pilot study of 12 children (eight boys; four girls) with congenital adrenal hyperplasia, testolactone, in combination with an antiandrogen (flutamide), a mineralocorticoid (fludrocortisone acetate, Florinef), and a reduced glucocorticoid dose, improved the control of growth and bone maturation compared with conventional therapy. In a 6-year study of 10 boys with familial male precocious puberty, testolactone, in combination with an antiandrogen (spironolactone), decreased rates of growth and bone maturation, and increased predicted adult height. All patients who developed evidence for gonadotropin dependent puberty were also treated with a GnRH analog. Testolactone had no important adverse effects in any group of patients, although the need for a four times-daily dosing schedule made compliance difficult for many families. We conclude that suppressing of estrogen with testolactone was effective therapy, and that more potent and specific inhibitors of aromatase could further improve the treatment of these disorders. PMID- 10731124 TI - Aromatase overexpression and breast hyperplasia, an in vivo model--continued overexpression of aromatase is sufficient to maintain hyperplasia without circulating estrogens, and aromatase inhibitors abrogate these preneoplastic changes in mammary glands. AB - To test directly the role of breast-tissue estrogen in initiation of breast cancer, we have developed the aromatase-transgenic mouse model and demonstrated for the first time that increased mammary estrogens resulting from the overexpression of aromatase in mammary glands lead to the induction of various preneoplastic and neoplastic changes that are similar to early breast cancer. Continued overexpression of aromatase that leads to increased breast-tissue estrogen contributes to a number of epigenetic changes in mammary tissue such as alteration in the regulation of genes involved in apoptosis, activation of genes involved in cell cycle and cell proliferation, and activation of a number of growth factors. Our current studies show aromatase overexpression is sufficient to induce and maintain early preneoplastic and neoplastic changes in female mice without circulating ovarian estrogen. Preneoplastic and neoplastic changes induced in mammary glands as a result of aromatase overexpression can be completely abrogated with the administration of the aromatase inhibitor, letrozole. Consistent with complete reduction in hyperplasia, we have also seen downregulation of estrogen receptor and a decrease in cell proliferation markers, suggesting aromatase-induced hyperplasia can be treated with aromatase inhibitors. Our studies demonstrate that aromatase overexpression alone, without circulating estrogen, is responsible for the induction of breast hyperplasia and these changes can be abrogated using aromatase inhibitors. PMID- 10731125 TI - Aromatase and gynecomastia. AB - An imbalance between estrogen action relative to androgen action at the breast tissue level results in gynecomastia. Enhancement of aromatization of androgens to estrogens is important in the pathogenesis of gynecomastia associated with obesity, aging, puberty, liver disease, thyrotoxicosis, 17-oxosteroid reductase deficiency. Klinefelter's syndrome, and neoplasms of the testes, adrenals and liver. A primary aromatase excess syndrome with exuberant gynecomastia had been found both sporadically and in a familial setting. Although aromatase inhibition would appear to be an important class of drugs to treat gynecomastia, relatively little published data with these drugs exist and most concern the use of delta1 testolactone, which reduces the size of the breast glandular tissue, but does not completely ameliorate the problem. Studies with the newer generation of more potent aromatase inhibitors need to be carried out. PMID- 10731126 TI - Risks versus benefits in the clinical application of aromatase inhibitors. AB - Third-generation aromatase inhibitors are able to reduce circulating plasma estrogen concentrations in postmenopausal women to below detectable limits and significantly inhibit aromatase, the enzyme responsible for estrogen synthesis, in normal breast tissue and breast tumors. Their role in the treatment of advanced breast cancer is well established and their use in adjuvant therapy is currently being explored. On the basis of these trials, evaluation of these inhibitors in the prevention of breast cancer may be appropriate. Aromatase inhibitors have non-specific toxic side effects including (but not limited to): asthenia, headache, nausea, peripheral edema, fatigue, vomiting and dyspepsia. In addition, certain endocrinological side effects in postmenopausal women are notable, namely hot flushes and vaginal dryness. In advanced breast cancer, these side effects result in treatment withdrawal in few (<4%) women. Of concern, however, are the potential long-term endocrinological side effects in women receiving treatment as first-line adjuvant therapy or in sequence or combination with tamoxifen or other selective estrogen receptor modulators (SERMs). Current studies of adjuvant treatments for breast cancer in healthy women are carefully evaluating, in addition to general toxicities, the effects on bone, lipid metabolism, cardiovascular risk, quality of life and menopausal symptoms. Careful evaluation of all-cause morbidity and mortality is necessary to plan trials and justify long-term use of aromatase inhibitors in the treatment or prevention of breast cancer in healthy women. PMID- 10731127 TI - Recent developments in the understanding of the pathogenesis of psoriasis. AB - Recent studies have provided new insights into the molecular mechanisms involved in the pathogenesis of psoriasis. Patients may inherit a predisposition to psoriasis, although the disease is expressed only after being triggered by certain environmental or antigenic factors. There is considerable genetic heterogeneity in psoriasis and several genetic loci associated with the occurrence of the disease have been identified. Although the underlying abnormality in psoriasis has not been definitively identified, recent evidence suggests that activated T lymphocytes play an important role in the pathogenesis of the disease. The activation of T lymphocytes can be mediated through antigen presenting cells or through autoimmunity, and is influenced by cytokines. Recently developed animal models and in vitro studies of psoriasis have provided new evidence for the role of pathogenic lymphocytes in the initiation of the disease process. Further research will help to identify the susceptibility genes involved in psoriasis and provide a more complete understanding of the immunological basis of the disease. This should lead to the development of targeted therapies with improved efficacy and tolerability compared with some currently available treatments. PMID- 10731128 TI - Optimizing therapy: tazarotene in combination with phototherapy. AB - Phototherapy is often used in combination with other antipsoriatic treatments in an attempt to improve efficacy and reduce patients' cumulative exposure to radiation. Although this aim has been achieved with some combinations, the additional therapies often introduce a potential risk of other tolerability and safety problems. The efficacy of tazarotene reported in clinical trials to date suggest that this drug may help to improve the efficacy of phototherapy, and perhaps reduce the ultraviolet light exposure required without introducing additional, clinically significant problems. Preliminary results from the first 10 patients in a clinical trial investigating such combination therapy are reported here. They demonstrate that the addition of tazarotene to UVB phototherapy increases the percentage of patients achieving treatment success (> or = 50% global improvement in psoriasis) from 60% to 100% at Day 81. The UVB plus tazarotene combination achieved consistently greater reductions in the elevation and scaling of difficult-to-treat psoriatic plaques than UVB phototherapy alone or UVB phototherapy plus vehicle gel. The tazarotene combination therapy also achieved initial treatment success in less than half the time needed with phototherapy alone (median of 32 vs. 67 days). Combining UVB phototherapy with tazarotene treatment appears to offer a valuable therapeutic option that is more efficacious and faster than UVB phototherapy alone. PMID- 10731129 TI - Novel retinoids with receptor selectivity and functional selectivity. AB - Retinoids mediate a number of physiological pathways through their effects on cellular growth and differentiation. Upon binding to retinoic acid receptors (RARs) and retinoid X receptors (RXRs), retinoids regulate cellular processes by directly modulating the expression of responsive genes. The wide-ranging effects of retinoid action are attributable to two main factors-the ubiquitous distribution of several subtypes and isoforms of RARs and RXRs, and the ability of these receptors to regulate numerous genes upon ligand activation. The broad range of effects mediated by retinoids means not only that they have many potential therapeutic applications but also that non-selective retinoids are associated with a high incidence of adverse effects. The design of retinoids that are receptor-selective and function-selective is a strategy that is proving successful in developing novel retinoids that offer not only good efficacy but also good tolerability. Tazarotene, a receptor-selective retinoid indicated for the topical treatment of psoriasis, is at the forefront of this new generation of retinoids. In the near future, other receptor-selective retinoids may prove useful for the treatment of other dermatological diseases, cancer, and diabetes. PMID- 10731130 TI - Combination therapy with tazarotene plus a topical corticosteroid for the treatment of plaque psoriasis. AB - Although tazarotene monotherapy is generally efficacious and well tolerated, studies show that both the efficacy and the tolerability of tazarotene therapy can be further improved when it is used in combination with certain topical corticosteroids. The studies reported here evaluate the usefulness of two potential combination regimens. In one regimen, a corticosteroid is added to tazarotene treatment. In the other regimen, corticosteroid treatment alternates on a daily basis with tazarotene treatment. The results of the first study, which involved 300 patients, showed that additive combination therapy using tazarotene plus a mid- or high-potency topical corticosteroid significantly increased the percentage of plaques achieving treatment success at the end of the treatment period, compared with tazarotene plus placebo (91% and 95% vs. 80%, respectively; P<0.05 for both). Similarly, tazarotene plus a mid- or high-potency topical corticosteroid reduced the incidence of patient withdrawals compared with tazarotene plus placebo (5.5% and 9.6% vs. 13.3%). The results of the second study, which involved 398 patients, showed that a combination regimen that alternates between tazarotene and a high-potency topical corticosteroid treatment each day, significantly increased the treatment success rate compared with regimens using tazarotene alternating with a mid-potency corticosteroid or placebo (75% vs. 55% and 54%, respectively, at the end of the treatment period; P<0.05 for both). In addition, there was a trend towards a lower incidence of treatment-related adverse events as corticosteroid potency increased (from 42% with tazarotene plus placebo to 36%, 32%, and 31% with tazarotene plus the low-, mid-, and high-potency corticosteroid, respectively). Both treatment regimens are potentially useful and offer a rational approach to optimizing the efficacy and tolerability of tazarotene treatment for plaque psoriasis. PMID- 10731131 TI - The role of tazarotene in the treatment of psoriasis. AB - The recent availability of tazarotene, the first receptor-selective retinoid, provides a much-needed addition to the therapeutic armamentarium for mild-to moderate plaque psoriasis. Tazarotene gel offers a welcome combination of good efficacy and cosmetic acceptability, with minimal risk of systemic adverse effects. The selectivity of tazarotene for the beta and gamma subtypes of retinoic acid receptors suggests a targeted action on psoriatic keratinocytes, which may help to minimize the risk of adverse effects. The potential for adverse effects is further minimized by the limited transcutaneous absorption of tazarotene, its rapid metabolism into hydrophilic metabolites, and its rapid elimination from the body. These pharmacokinetic features ensure that plasma levels of tazarotene and its main metabolite, tazarotenic acid, are minimized- thus limiting systemic exposure. The hydrophilicity of the metabolites also limits systemic exposure by ensuring that accumulation does not occur in lipophilic tissues. PMID- 10731133 TI - A whole-genome assembly of Drosophila. AB - We report on the quality of a whole-genome assembly of Drosophila melanogaster and the nature of the computer algorithms that accomplished it. Three independent external data sources essentially agree with and support the assembly's sequence and ordering of contigs across the euchromatic portion of the genome. In addition, there are isolated contigs that we believe represent nonrepetitive pockets within the heterochromatin of the centromeres. Comparison with a previously sequenced 2.9- megabase region indicates that sequencing accuracy within nonrepetitive segments is greater than 99. 99% without manual curation. As such, this initial reconstruction of the Drosophila sequence should be of substantial value to the scientific community. PMID- 10731132 TI - The genome sequence of Drosophila melanogaster. AB - The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the approximately 120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes approximately 13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity. PMID- 10731135 TI - A brief history of Drosophila's contributions to genome research. AB - The sequence of the Drosophila melanogaster genome presented in this issue of Science is the latest milestone in nine decades of research on this organism. Genetic and physical mapping, whole-genome mutational screens, and functional alteration of the genome by gene transfer were pioneered in metazoans with the use of this small fruit fly. Here we look at some of the instances in which work on Drosophila has led to major conceptual or technical breakthroughs in our understanding of animal genomes. PMID- 10731136 TI - The Drosophila genome sequence: implications for biology and medicine. AB - The 120-megabase euchromatic portion of the Drosophila melanogaster genome has been sequenced. Because the genome is compact and many genetic tools are available, and because fly cell biology and development have much in common with mammals, this sequence may be the Rosetta stone for deciphering the human genome. PMID- 10731134 TI - Comparative genomics of the eukaryotes. AB - A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease. PMID- 10731138 TI - A Drosophila complementary DNA resource. AB - Collections of nonredundant, full-length complementary DNA (cDNA) clones for each of the model organisms and humans will be important resources for studies of gene structure and function. We describe a general strategy for producing such collections and its implementation, which so far has generated a set of cDNAs corresponding to over 40% of the genes in the fruit fly Drosophila melanogaster. PMID- 10731137 TI - From sequence to chromosome: the tip of the X chromosome of D. melanogaster. AB - One of the rewards of having a Drosophila melanogaster whole-genome sequence will be the potential to understand the molecular bases for structural features of chromosomes that have been a long-standing puzzle. Analysis of 2.6 megabases of sequence from the tip of the X chromosome of Drosophila identifies 273 genes. Cloned DNAs from the characteristic bulbous structure at the tip of the X chromosome in the region of the broad complex display an unusual pattern of in situ hybridization. Sequence analysis revealed that this region comprises 154 kilobases of DNA flanked by 1.2-kilobases of inverted repeats, each composed of a 350-base pair satellite related element. Thus, some aspects of chromosome structure appear to be revealed directly within the DNA sequence itself. PMID- 10731139 TI - Spontaneous ordering of oxide nanostructures AB - We report the spontaneous formation of uniformly distributed arrays of "tips" (tall conical hillocks) upon oxidation of palladium (Pd) thin films. The formation of the palladium oxide tips depended on the thickness and granularity of the Pd film and on annealing and oxidation conditions. As the Pd film thickness increased from 40 to 200 nanometers, the average height of the tips increased from 0.5 to 1.2 micrometers, their height distribution became broader, and their density decreased from 55 x 10(6) to 12 x 10(6) per square centimeter. Enhanced photoelectron emission from locations corresponding to the tips suggests their possible use in field emission applications. PMID- 10731140 TI - Generation of femtosecond pulses of synchrotron radiation AB - Femtosecond synchrotron pulses were generated directly from an electron storage ring. An ultrashort laser pulse was used to modulate the energy of electrons within a 100-femtosecond slice of the stored 30-picosecond electron bunch. The energy-modulated electrons were spatially separated from the long bunch and used to generate approximately 300-femtosecond synchrotron pulses at a bend-magnet beamline, with a spectral range from infrared to x-ray wavelengths. The same technique can be used to generate approximately 100-femtosecond x-ray pulses of substantially higher flux and brightness with an undulator. Such synchrotron based femtosecond x-ray sources offer the possibility of applying x-ray techniques on an ultrafast time scale to investigate structural dynamics in condensed matter. PMID- 10731141 TI - A class of microstructured particles through colloidal crystallization AB - Microstructured particles were synthesized by growing colloidal crystals in aqueous droplets suspended on fluorinated oil. The droplets template highly ordered and smooth particle assemblies, which diffract light and have remarkable structural stability. The method allows control of particle size and shape from spheres through ellipsoids to toroids by varying the droplet composition. Cocrystallization in colloidal mixtures yields anisotropic particles of organic and inorganic materials that can, for example, be oriented and turned over by magnetic fields. The results open the way to controllable formation of a wide variety of microstructures. PMID- 10731142 TI - Resonant oscillations between the solid earth and the atmosphere AB - Continuously excited free oscillations of the whole Earth have been found in the ground noise in a frequency band of long-period surface waves. Here we report evidence of an annual variation of this phenomenon, indicating that the excitation source is not within the solid Earth. There is evidence that these seismic free oscillations resonate with acoustic free oscillations of the atmosphere. The observed amplitudes suggest that the excitation source is at or just above Earth's surface. PMID- 10731143 TI - Simulation of early 20th century global warming AB - The observed global warming of the past century occurred primarily in two distinct 20-year periods, from 1925 to 1944 and from 1978 to the present. Although the latter warming is often attributed to a human-induced increase of greenhouse gases, causes of the earlier warming are less clear because this period precedes the time of strongest increases in human-induced greenhouse gas (radiative) forcing. Results from a set of six integrations of a coupled ocean atmosphere climate model suggest that the warming of the early 20th century could have resulted from a combination of human-induced radiative forcing and an unusually large realization of internal multidecadal variability of the coupled ocean-atmosphere system. This conclusion is dependent on the model's climate sensitivity, internal variability, and the specification of the time-varying human-induced radiative forcing. PMID- 10731144 TI - Rapid extinction of the moas (Aves: Dinornithiformes): model, test, and implications. AB - A Leslie matrix population model supported by carbon-14 dating of early occupation layers lacking moa remains suggests that human hunting and habitat destruction drove the 11 species of moa to extinction less than 100 years after Polynesian settlement of New Zealand. The rapid extinction contrasts with models that envisage several centuries of exploitation. PMID- 10731145 TI - The initial domestication of goats (Capra hircus) in the Zagros mountains 10,000 years ago. AB - Initial goat domestication is documented in the highlands of western Iran at 10,000 calibrated calendar years ago. Metrical analyses of patterns of sexual dimorphism in modern wild goat skeletons (Capra hircus aegagrus) allow sex specific age curves to be computed for archaeofaunal assemblages. A distinct shift to selective harvesting of subadult males marks initial human management and the transition from hunting to herding of the species. Direct accelerator mass spectrometry radiocarbon dates on skeletal elements provide a tight temporal context for the transition. PMID- 10731146 TI - Molecular linkage underlying microtubule orientation toward cortical sites in yeast. AB - Selective microtubule orientation toward spatially defined cortical sites is critical to polarized cellular processes as diverse as axon outgrowth and T cell cytotoxicity. In yeast, oriented cytoplasmic microtubules align the mitotic spindle between mother and bud. The cortical marker protein Kar9 localizes to the bud tip and is required for the orientation of microtubules toward this region. Here, we show that Kar9 directs microtubule orientation by acting through Bim1, a conserved microtubule-binding protein. Bim1 homolog EB1 was originally identified through its interaction with adenomatous polyposis coli (APC) tumor suppressor, raising the possibility that an APC-EB1 linkage orients microtubules in higher cells. PMID- 10731147 TI - Positioning of the mitotic spindle by a cortical-microtubule capture mechanism. AB - Correct positioning of the mitotic spindle is critical for cell division and development. Spindle positioning involves a search-and-capture mechanism whereby dynamic microtubules find and then interact with specific sites on the submembrane cortex. Genetic, biochemical, and imaging experiments suggest a mechanism for cortical-microtubule capture. Bim1p, located at microtubule distal ends, bound Kar9p, a protein associated with the daughter cell cortex. Bim1p is the yeast ortholog of human EB1, a binding partner for the adenomatous polyposis coli tumor suppressor. EB1 family proteins may have a general role in linking the microtubule cytoskeleton to cortical polarity determinants. PMID- 10731148 TI - Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction. AB - To elucidate mechanisms that control and execute activity-dependent synaptic plasticity, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPA Rs) with an electrophysiological tag were expressed in rat hippocampal neurons. Long-term potentiation (LTP) or increased activity of the calcium/calmodulin dependent protein kinase II (CaMKII) induced delivery of tagged AMPA-Rs into synapses. This effect was not diminished by mutating the CaMKII phosphorylation site on the GluR1 AMPA-R subunit, but was blocked by mutating a predicted PDZ domain interaction site. These results show that LTP and CaMKII activity drive AMPA-Rs to synapses by a mechanism that requires the association between GluR1 and a PDZ domain protein. PMID- 10731149 TI - Response of Schwann cells to action potentials in development. AB - Sensory axons become functional late in development when Schwann cells (SC) stop proliferating and differentiate into distinct phenotypes. We report that impulse activity in premyelinated axons can inhibit proliferation and differentiation of SCs. This neuron-glial signaling is mediated by adenosine triphosphate acting through P2 receptors on SCs and intracellular signaling pathways involving Ca2+, Ca2+/calmodulin kinase, mitogen-activated protein kinase, cyclic adenosine 3',5' monophosphate response element binding protein, and expression of c-fos and Krox 24. Adenosine triphosphate arrests maturation of SCs in an immature morphological stage and prevents expression of O4, myelin basic protein, and the formation of myelin. Through this mechanism, functional activity in the developing nervous system could delay terminal differentiation of SCs until exposure to appropriate axon-derived signals. PMID- 10731150 TI - A BAC-based physical map of the major autosomes of Drosophila melanogaster. AB - We constructed a bacterial artificial chromosome (BAC)-based physical map of chromosomes 2 and 3 of Drosophila melanogaster, which constitute 81% of the genome. Sequence tagged site (STS) content, restriction fingerprinting, and polytene chromosome in situ hybridization approaches were integrated to produce a map spanning the euchromatin. Three of five remaining gaps are in repeat-rich regions near the centromeres. A tiling path of clones spanning this map and STS maps of chromosomes X and 4 was sequenced to low coverage; the maps and tiling path sequence were used to support and verify the whole-genome sequence assembly, and tiling path BACs were used as templates in sequence finishing. PMID- 10731151 TI - Rapid progression to AIDS in HIV+ individuals with a structural variant of the chemokine receptor CX3CR1. AB - Human immunodeficiency virus (HIV) enters cells in vitro via CD4 and a coreceptor. Which of 15 known coreceptors are important in vivo is poorly defined but may be inferred from disease-modifying mutations, as for CCR5. Here two single nucleotide polymorphisms are described in Caucasians in CX3CR1, an HIV coreceptor and leukocyte chemotactic/adhesion receptor for the chemokine fractalkine. HIV-infected patients homozygous for CX3CR1-I249 M280, a variant haplotype affecting two amino acids (isoleucine-249 and methionine-280), progressed to AIDS more rapidly than those with other haplotypes. Functional CX3CR1 analysis showed that fractalkine binding is reduced among patients homozygous for this particular haplotype. Thus, CX3CR1-I249 M280 is a recessive genetic risk factor in HIV/AIDS. PMID- 10731152 TI - Replacing the mercury sphygmomanometer. Requires clinicians to demand better automated devices. PMID- 10731153 TI - Heart and heart-lung transplantation in Down's syndrome. The lack of supportive evidence means each case must be carefully assessed. PMID- 10731154 TI - Specialist registrar training. Some good news at last. PMID- 10731155 TI - Healthy People 2010: objectives for the United States. Impressive, but unwieldy. PMID- 10731156 TI - Total knee replacement: the joint of the decade. A successful operation, for which there's a large unmet need. PMID- 10731158 TI - Relatives to be told if organs are to be retained after postmortem PMID- 10731157 TI - Drug resistant tuberculosis can be controlled, says WHO. PMID- 10731159 TI - Privy Council sets aside GMC ruling. PMID- 10731161 TI - In brief PMID- 10731162 TI - New clinical guidelines for stroke published. PMID- 10731160 TI - Half of Bangladesh population at risk of arsenic poisoning. PMID- 10731163 TI - Genetics commission consults UK population PMID- 10731164 TI - Blair throws down challenge to GPs over NHS changes. PMID- 10731165 TI - EU advisers to declare their interests PMID- 10731166 TI - UK junior doctors to vote on pay offer PMID- 10731167 TI - Saliva test could diagnose cancers PMID- 10731168 TI - Japan to allow in foreign nurses to care for old people. PMID- 10731169 TI - Coordinated global action could end malnutrition in young children. PMID- 10731171 TI - Canada turns to france for staff to treat cancer patients PMID- 10731170 TI - Cloning of pigs bring xenotransplants closer. PMID- 10731172 TI - Dutch insurers flouting law on genetic disease, researchers say PMID- 10731173 TI - Immediate and long term effects of weight reduction in obese people with asthma: randomised controlled study. AB - OBJECTIVE: To investigate the influence of weight reduction on obese patients with asthma. DESIGN: Open study, two randomised parallel groups. SETTING: Private outpatients centre, Helsinki, Finland. PARTICIPANTS: Two groups of 19 obese patients with asthma (body mass index (kg/m(2)) 30 to 42) recruited through newspaper advertisements. INTERVENTION: Supervised weight reduction programme including 8 week very low energy diet. MAIN OUTCOME MEASURES: Body weight, morning peak expiratory flow (PEF), forced vital capacity (FVC), forced expiratory volume in one second (FEV(1)); and also asthma symptoms, number of acute episodes, courses of oral steroids, health status (quality of life). RESULTS: At the end of the weight reducing programme, the participants in the treatment group had lost a mean of 14.5% of their pretreatment weight, the controls 0.3%. The corresponding figures after one year were 11.3% and a weight gain of 2.2%. After the 8 week dieting period the difference in changes in percentage of predicted FEV(1) from baseline in the treatment and control groups was 7.2% (95% confidence interval 1.9% to 12.5%, P=0. 009). The corresponding difference in the changes in FVC was 8.6% (4. 8% to 12.5%, P<0.0001). After one year the differences in the changes in the two groups were still significant: 7.6% for FEV(1) (1. 5% to 13.8%, P=0.02) and 7.6% for FVC (3.5% to 11.8%, P=0.001). By the end of the weight reduction programme, reduction in dyspnoea was 13 mm (on a visual analogue scale 0 mm to 100 mm) in the treatment group and 1 mm in the control group (P=0.02). The reduction of rescue medication was 1.2 and 0.1 doses respectively (P=0.03). After one year the differences in the changes between the two groups were -12 for symptom scores (range -1 to -22, P=0.04) and 10 for total scores (-18 to -1, P=0.02). The median number of exacerbations in the treatment group was 1 (0-4) and in the controls 4 (0-7), P=0.001. CONCLUSION: Weight reduction in obese patients with asthma improves lung function, symptoms, morbidity, and health status. PMID- 10731174 TI - Trainee satisfaction before and after the Calman reforms of specialist training: questionnaire survey. AB - OBJECTIVES: To evaluate the impact of the Calman reforms of higher specialist training on trainee satisfaction. DESIGN: Questionnaire surveys using portable electronic survey units, two years apart. SETTING: Postgraduate, teaching, district general, and community NHS trusts in North Thames. North Thames deanery includes London north of the Thames, Essex, and Hertfordshire. PARTICIPANTS: Trainees in all grades and all specialties: 3078 took part in the first survey and 3517 in the second survey. MAIN OUTCOME MEASURES: Trainees' satisfaction with training in their current post, including educational objectives, training agreements, induction, consultant feedback, hands on experience acquired, use of log books, consultant supervision, and overall satisfaction with the post. RESULTS: In the second survey respondents were more likely to have discussed educational objectives with their consultant, used a log book, and had useful feedback from their consultant. They were more likely to give high ratings to induction, consultant supervision, and hands on experience acquired in the post. Each of these elements was associated with increased satisfaction with the post overall. Improvements were most noticeable at the level of specialist registrar, but changes in the same direction were also seen in more junior grades. CONCLUSIONS: After the reforms of specialist training, trainees in all grades reported greater satisfaction with their current posts. The changes required extra training time and effort from consultants. PMID- 10731175 TI - Recognition of television images as a developmental milestone in young children: observational study. AB - OBJECTIVES: To determine the age at which children with apparently normal development can recognise the television image of a cat, dog, or baby. DESIGN: Observational study. SETTING: District general hospital in north London. SUBJECTS: 797 children with apparently normal development aged between 8 and 23 months and 26 children with Down's syndrome aged 18 months. OUTCOME MEASURES: Whether or not the child recognised the television image of a cat, dog, or baby by naming, imitating, or pointing at the image. RESULTS: By 18 months of age 96% (95% confidence interval 94% to 98%) of normal children recognised the television image of a cat, dog, or baby compared with 5 of 26 (19%) children with Down's syndrome. CONCLUSION: Recognition of the television image of a cat, dog, or baby is a simple milestone, which may help in the developmental assessment of young children. PMID- 10731176 TI - Waiting times for cancer patients in England after general practitioners' referrals: retrospective national survey. PMID- 10731177 TI - Relation between infants' birth weight and mothers' mortality: prospective observational study. PMID- 10731178 TI - Cohort study of birthweight, mortality, and disability. PMID- 10731179 TI - Count sponges and instruments PMID- 10731182 TI - The internet may not be the biggest change in the past 150 years PMID- 10731180 TI - Association between teenage pregnancy rates and the age and sex of general practitioners: cross sectional survey in Trent 1994-7. AB - OBJECTIVE: To examine variations in teenage pregnancy rates in Trent region and to determine possible associations with local general practice characteristics such as the age and sex of the doctors. DESIGN: Cross sectional survey. SETTING: All 826 general practices in Trent region in existence between 1994 and 1997. SUBJECTS: All pregnancies of teenagers aged 13 to 19 between 1994 and 1997 that resulted in an admission to an NHS hospital. MAIN OUTCOME MEASURES: Pregnancy rates for teenagers aged 13 to 19 and general practice characteristics: presence of a female or young doctor (under 36 years old), number of whole time equivalent practice nurses, Townsend score, vocational training status, list size per whole time equivalent doctor, fund-holding status, and partnership size. RESULTS: On multivariate analysis, lower teenage pregnancy rates were associated with the presence of a female or young doctor and more nurse time. Practices in deprived areas had higher teenage pregnancy rates. CONCLUSION: General practices with female doctors, young doctors, or more nurse time had lower teenage pregnancy rates. The findings may have implications for the mix of health professionals within primary care. PMID- 10731183 TI - A surprising diagnosis PMID- 10731181 TI - Changing carriage rate of Neisseria meningitidis among university students during the first week of term: cross sectional study. AB - OBJECTIVE: To determine the rates of, and risk factors for, meningococcal carriage and acquisition among university students. DESIGN: Repeated cross sectional study. PARTICIPANTS: 2,507 students in their first year at university. MAIN OUTCOME MEASURES: Prevalence of carriage of meningococci and risk factors for carriage and acquisition of meningococci. RESULTS: Carriage rates for meningoccoci increased rapidly in the first week of term from 6.9% on day 1, to 11.2% on day 2, to 19.0% on day 3, and to 23.1% on day 4. The average carriage rate during the first week of term in October among students living in catered halls was 13.9%. By November this had risen to 31.0% and in December it had reached 34. 2%. Independent associations for acquisition of meningococci in the autumn term were frequency of visits to a hall bar (5-7 visits: odds ratio 2.7, 95% confidence interval 1.5 to 4.8), active smoking (1.6, 1.0 to 2.6), being male (1.6, 1.2 to 2.2), visits to night clubs (1. 3, 1.0 to 1.6), and intimate kissing (1.4, 1.0 to 1.8). Lower rates of acquisition were found in female only halls (0.5, 0.3 to 0.9). The most commonly acquired meningococcal strain was C2a P1.5 (P1.2), which has been implicated in clusters of invasive meningococcal disease at other UK universities. CONCLUSIONS: Carriage rates of meningococci among university students increase rapidly in the first week of term, with further increases during the term. The rapid rate of acquisition may explain the increased risk of invasive meningococcal disease and the timing of cases and outbreaks in university students. PMID- 10731184 TI - Recent advances: Dermatology. PMID- 10731185 TI - ABC of arterial and venous disease: Chronic lower limb ischaemia. PMID- 10731187 TI - Plant sterol and stanol margarines and health. PMID- 10731188 TI - Blair's billions: where will he find the money for the NHS? PMID- 10731186 TI - For and against: The male menopause--does it exist? PMID- 10731189 TI - Xenotransplantation. This new form of treatment might benefit millions. PMID- 10731190 TI - GPs can improve their hand washing habits. PMID- 10731191 TI - System to detect tuberculosis in new arrivals to UK must be improved. PMID- 10731192 TI - Antibiotic prophylaxis after percutaneous endoscopic gastrostomy insertion. All encompassing study is needed PMID- 10731193 TI - Honesty about new screening programmes is best policy. PMID- 10731194 TI - Higher rate of organ procurement can be achieved in UK. PMID- 10731195 TI - Eugenics debate. Eugenics principles are there. PMID- 10731196 TI - The changing doctor-patient relationship. Diagnoses are made from careful history and examination. PMID- 10731197 TI - Choice is a gift from the patient to the doctor, not the other way around. PMID- 10731198 TI - Continuity of supply of drugs is critical. PMID- 10731199 TI - Time to switch from whole cell to acellular pertussis vaccines? PMID- 10731201 TI - Obituaries PMID- 10731202 TI - Junior doctors oppose idea of new non-consultant grade PMID- 10731200 TI - The other Dr Finlay is commemorated in Cuban stamps. PMID- 10731204 TI - Antimony in medical history PMID- 10731203 TI - The Clinician's practical guide to attention-Deficit/Hyperactivity disorder PMID- 10731205 TI - Champion of women and the unborn: horatio robinson storer, MD PMID- 10731206 TI - NHS on the web PMID- 10731207 TI - Deaths on the operating table PMID- 10731208 TI - How to party PMID- 10731209 TI - The young as patients and doctors PMID- 10731212 TI - Recognition of television images may be a milestone in children's development PMID- 10731210 TI - Weight reduction alleviates asthma in obese asthmatic people PMID- 10731211 TI - Calman trainees are satisfied with specialist training PMID- 10731214 TI - Teenage pregnancy rates tend to be lower in general practices with young or female doctors PMID- 10731213 TI - Current referral times for cancer vary greatly PMID- 10731215 TI - Meningococci spread rapidly among new students in first week of university PMID- 10731216 TI - The mysterious orbitofrontal cortex. foreword PMID- 10731217 TI - The organization of networks within the orbital and medial prefrontal cortex of rats, monkeys and humans. AB - This paper reviews architectonic subdivisions and connections of the orbital and medial prefrontal cortex (OMPFC) in rats, monkeys and humans. Cortico-cortical connections provide the basis for recognition of 'medial' and 'orbital' networks within the OMPFC. These networks also have distinct connections with structures in other parts of the brain. The orbital network receives sensory inputs from several modalities, including olfaction, taste, visceral afferents, somatic sensation and vision, which appear to be especially related to food or eating. In contrast, the medial network provides the major cortical output to visceromotor structures in the hypothalamus and brainstem. The two networks have distinct connections with areas of the striatum and mediodorsal thalamus. In particular, projections to the nucleus accumbens and the adjacent ventromedial caudate and putamen arise predominantly from the medial network. Both networks also have extensive connections with limbic structures. Based on these and other observations, the OMPFC appears to function as a sensory-visceromotor link, especially for eating. This linkage appears to be critical for the guidance of reward-related behavior and for setting of mood. Imaging and histological observations on human brains indicate that clinical depressive disorders are associated with specific functional and cellular changes in the OMPFC, including activity and volume changes, and specific changes in the number of glial cells. PMID- 10731218 TI - The anatomical connections of the macaque monkey orbitofrontal cortex. A review. AB - The orbitofrontal cortex (OfC) is a heterogeneous prefrontal sector selectively connected with a wide constellation of other prefrontal, limbic, sensory and premotor areas. Among the limbic cortical connections, the ones with the hippocampus and parahippocampal cortex are particularly salient. Sensory cortices connected with the OfC include areas involved in olfactory, gustatory, somatosensory, auditory and visual processing. Subcortical structures with prominent OfC connections include the amygdala, numerous thalamic nuclei, the striatum, hypothalamus, periaqueductal gray matter, and biochemically specific cell groups in the basal forebrain and brainstem. Architectonic and connectional evidence supports parcellation of the OfC. The rostrally placed isocortical sector is mainly connected with isocortical areas, including sensory areas of the auditory, somatic and visual modalities, whereas the caudal non-isocortical sector is principally connected with non-isocortical areas, and, in the sensory domain, with olfactory and gustatory areas. The connections of the isocortical and non-isocortical orbital sectors with the amygdala, thalamus, striatum, hypothalamus and periaqueductal gray matter are also specific. The medial sector of the OfC is selectively connected with the hippocampus, posterior parahippocampal cortex, posterior cingulate and retrosplenial areas, and area prostriata, while the lateral orbitofrontal sector is the most heavily connected with sensory areas of the gustatory, somatic and visual modalities, with premotor regions, and with the amygdala. PMID- 10731219 TI - Orbitofrontal cortex pathology in Alzheimer's disease. AB - The orbitofrontal cortex has been examined in Alzheimer's disease (AD) from the viewpoint of neurofibrillary tangle (NFT) pathology, its laminar distribution and topography. NFT pathology in the orbitofrontal cortex is extensive in AD. In cases with extensive cortical pathology, NFTs extend from the pole of the frontal lobe to the orbitoinsular junction. In lesser affected cases, the anterior granular part of the orbital cortex is less invested by NFTs. Layers III and V contain the greatest density of NFTs and these are most dense in the dysgranular areas, posterior to the transverse orbital sulcus. Posterior and medial orbitofrontal areas, forming area 13 and the posterior tip of the paraolfactory gyrus, are the most severely damaged, as are the smaller agranular fields that surround the olfactory tract and cortex. The widespread orbitofrontal damage in AD affecting projection neurons suggests that this pathology may contribute heavily to the many non-memory-related behavior changes observed in this disorder. PMID- 10731220 TI - Inhibitory control and affective processing in the prefrontal cortex: neuropsychological studies in the common marmoset. AB - The orbitofrontal cortex has been ascribed a role in the inhibitory control, as well as in the emotional control, of behaviour. While damage to the orbitofrontal cortex in humans and non-human primates can cause inflexibility, impulsiveness and emotional disturbance, the relationship between these effects are unclear. Excitotoxic lesion studies in marmosets comparing the effects of cell loss within specific regions of the prefrontal cortex on performance of a range of behavioural tests reveal that mechanisms of response inhibition are not unique to the orbitofrontal cortex. Instead they are present in distinct cognitive domains for lowerorder as well as higher-order processing throughout the prefrontal cortex. Thus, the lateral prefrontal cortex is involved in the selection and control of action based upon higher-order rules while the orbitofrontal and medial prefrontal cortex may be involved in different but complementary forms of lower-order rule learning, their roles dissociable, as a result of their differential contribution to different types of associative learning. PMID- 10731221 TI - Delay activity of orbital and lateral prefrontal neurons of the monkey varying with different rewards. AB - We examined neuronal activity in the orbitofrontal cortex (OFC) in relation to reward expectancy and compared findings with those of the lateral prefrontal cortex (LPFC) in the monkey. Activity of OFC neurons was examined in a delayed reaction time task where every four trials constituted one block within which three kinds of rewards and no reward were delivered in a fixed order. More than half of OFC delay neurons were related to the expectancy of delivery or nodelivery of a reward as the response outcome, while some neurons showed nature of-reward-specific anticipatory activity changes. These delay-related activities reflected the preference of the animal for each kind of reward and were modulated by the motivational state of the animal. LPFC neurons are reported to show nature ofreward-specific anticipatory activity changes in a delayed response task when several different kinds of rewards are used. Such rewarddependent activity is observed in LPFC delay neurons both with and without spatially differential delay (working memory-related) activity. Although reward expectancy-related activity is commonly observed in both OFC and LPFC, it is suggested that the OFC is more concerned with motivational aspects, while the LPFC is related to both the cognitive and motivational aspects of the expectancy of response outcome. PMID- 10731222 TI - Reward processing in primate orbitofrontal cortex and basal ganglia. AB - This article reviews and interprets neuronal activities related to the expectation and delivery of reward in the primate orbitofrontal cortex, in comparison with slowly discharging neurons in the striatum (caudate, putamen and ventral striatum, including nucleus accumbens) and midbrain dopamine neurons. Orbitofrontal neurons showed three principal forms of reward-related activity during the performance of delayed response tasks, namely responses to reward predicting instructions, activations during the expectation period immediately preceding reward and responses following reward. These activations discriminated between different rewards, often on the basis of the animals' preferences. Neurons in the striatum were also activated in relation to the expectation and detection of reward but in addition showed activities related to the preparation, initiation and execution of movements which reflected the expected reward. Dopamine neurons responded to rewards and reward-predicting stimuli, and coded an error in the prediction of reward. Thus, the investigated cortical and basal ganglia structures showed multiple, heterogeneous, partly simultaneous activations which were related to specific aspects of rewards. These activations may represent the neuronal substrates of rewards during learning and established behavioral performance. The processing of reward expectations suggests an access to central representations of rewards which may be used for the neuronal control of goaldirected behavior. PMID- 10731223 TI - The orbitofrontal cortex and reward. AB - The primate orbitofrontal cortex contains the secondary taste cortex, in which the reward value of taste is represented. It also contains the secondary and tertiary olfactory cortical areas, in which information about the identity and also about the reward value of odors is represented. The orbitofrontal cortex also receives information about the sight of objects and faces from the temporal lobe cortical visual areas, and neurons in it learn and reverse the visual stimulus to which they respond when the association of the visual stimulus with a primary reinforcing stimulus (such as a taste reward) is reversed. However, the orbitofrontal cortex is involved in representing negative reinforcers (punishers) too, such as aversive taste, and in rapid stimulus-reinforcement association learning for both positive and negative primary reinforcers. In complementary neuroimaging studies in humans it is being found that areas of the orbitofrontal cortex (and connected subgenual cingulate cortex) are activated by pleasant touch, by painful touch, by rewarding and aversive taste, and by odor. Damage to the orbitofrontal cortex in humans can impair the learning and reversal of stimulus- reinforcement associations, and thus the correction of behavioral responses when these are no longer appropriate because previous reinforcement contingencies change. This evidence thus shows that the orbitofrontal cortex is involved in decoding and representing some primary reinforcers such as taste and touch; in learning and reversing associations of visual and other stimuli to these primary reinforcers; and in controlling and correcting reward-related and punishment-related behavior, and thus in emotion. PMID- 10731224 TI - Emotion, decision making and the orbitofrontal cortex. AB - The somatic marker hypothesis provides a systems-level neuroanatomical and cognitive framework for decision making and the influence on it by emotion. The key idea of this hypothesis is that decision making is a process that is influenced by marker signals that arise in bioregulatory processes, including those that express themselves in emotions and feelings. This influence can occur at multiple levels of operation, some of which occur consciously and some of which occur non-consciously. Here we review studies that confirm various predictions from the hypothesis. The orbitofrontal cortex represents one critical structure in a neural system subserving decision making. Decision making is not mediated by the orbitofrontal cortex alone, but arises from large-scale systems that include other cortical and subcortical components. Such structures include the amygdala, the somatosensory/insular cortices and the peripheral nervous system. Here we focus only on the role of the orbitofrontal cortex in decision making and emotional processing, and the relationship between emotion, decision making and other cognitive functions of the frontal lobe, namely working memory. PMID- 10731225 TI - Dissociable functions in the medial and lateral orbitofrontal cortex: evidence from human neuroimaging studies. AB - Recent imaging studies show that the orbitofrontal cortex (OFC) is activated during a wide variety of paradigms, including guessing tasks, simple delayed matching tasks and sentence completion. We suggest that, as with other regions of the prefrontal cortex, activity in the OFC is most likely to be observed when there is insufficient information available to determine the appropriate course of action. In these circumstances the OFC, rather than other prefrontal regions, is more likely to be activated when the problem of what to do next is best solved by taking into account the likely reward value of stimuli and responses, rather than their identity or location. We suggest that selection of stimuli on the basis of their familiarity and responses on the basis of a feeling of 'rightness' are also examples of selection on the basis of reward value. Within the OFC, the lateral regions are more likely to be involved when the action selected requires the suppression of previously rewarded responses. PMID- 10731226 TI - Addiction, a disease of compulsion and drive: involvement of the orbitofrontal cortex. AB - Understanding the changes in the brain which occur in the transition from normal to addictive behavior has major implications in public health. Here we postulate that while reward circuits (nucleus accumbens, amygdala), which have been central to theories of drug addiction, may be crucial to initiate drug self administration, the addictive state also involves disruption of circuits involved with compulsive behaviors and with drive. We postulate that intermittent dopaminergic activation of reward circuits secondary to drug self-administration leads to dysfunction of the orbitofrontal cortex via the striato-thalamo orbitofrontal circuit. This is supported by imaging studies showing that in drug abusers studied during protracted withdrawal, the orbitofrontal cortex is hypoactive in proportion to the levels of dopamine D2 receptors in the striatum. In contrast, when drug abusers are tested shortly after last cocaine use or during drug-induced craving, the orbitofrontal cortex is hypermetabolic in proportion to the intensity of the craving. Because the orbitofrontal cortex is involved with drive and with compulsive repetitive behaviors, its abnormal activation in the addicted subject could explain why compulsive drug self administration occurs even with tolerance to the pleasurable drug effects and in the presence of adverse reactions. This model implies that pleasure per se is not enough to maintain compulsive drug administration in the drugaddicted subject and that drugs that could interfere with the activation of the striato-thalamo orbitofrontal circuit could be beneficial in the treatment of drug addiction. PMID- 10731227 TI - Orbital and medial prefrontal cortex and psychostimulant abuse: studies in animal models. AB - One approach to pursuing questions about the neural substrates that support substance abuse-related behaviors involves the use of animal models. Carefully controlled animal experiments can be conducted without the confounds commonly found in studies of human addicts, such as polydrug abuse, variable drug history and premorbid psychiatric conditions. The present paper considers the orbitofrontal and related limbic prefrontal cortex in the context of such models of substance abuse. First, the importance of recognizing the heterogeneous structural and functional nature of orbitofrontal cortex in both rodents and primates is addressed, and the results of studies involving the prefrontal cortex in substance abuse-related behaviors are considered in light of this diversity. Second, data from metabolic mapping studies are described that indicate that the pattern of functional activity within medial and orbitofrontal cortex shifts as the duration of exposure to drugs such as cocaine is extended. These functional differences, in turn, may reflect progressive phases of the addictive process. In order to understand the neurobiological consequences of long-term drug use, it will be important to establish the differing roles played by distinct anatomical territories within orbital and medial prefrontal cortex during the course of chronic substance abuse. PMID- 10731228 TI - Orbitofrontal cortex and human drug abuse: functional imaging. AB - The orbitofrontal cortex (OFC) plays a central role in human behavior. Anatomically connected with association areas of all sensory modalities, limbic structures, prefrontal cortical regions that mediate executive function and subcortical nuclei, this brain region can serve to integrate the physical and emotional attributes of a stimulusobject and establish a motivational value based on estimation of potential reward. To the extent that addictive disorders reflect a dysregulation of the ability to evaluate potential reward against harm from drug self-administration, it would be anticipated that substance abuse disorder might reflect dysfunction of the OFC. With the application of brain imaging techniques to the study of human substance abuse, evidence has been obtained that activity in the OFC and its connections plays a role in several components of the maladaptive behavior of substance abuse, including expectancy, craving and impaired decision making. PMID- 10731229 TI - Prenatal screening in rural Bangladesh: from prediction to care. AB - The role of antenatal care is being increasingly questioned, particularly in resource poor environments. The low predictability of antenatal markers for adverse maternal outcomes has led some to reject antenatal care as an efficient strategy in the fight against maternal and perinatal mortality. Few studies, however, have assessed the predictability of adverse outcomes other than dystocia or perinatal death, and most studies have been hospital based. This population based cohort study was undertaken to assess whether prenatal screening can identify women at risk of severe labour or delivery complications in a rural area in Bangladesh. Antenatal risk markers, signs and symptoms were assessed for their association with severe maternal complications including dystocia, malpresentation, haemorrhage, hypertensive diseases, twin delivery and death. The results of the study suggest that antenatal screening by trained midwives fails to adequately distinguish women who will need special care during labour and delivery from those who will not need such care. The large majority of the women with dystocia or haemorrhage had no warning signs during pregnancy. A single blood pressure measurement and the assessment of fundal height, on the other hand, may detect a substantial number of women with hypertensive diseases and twin pregnancies. In addition, women who had an antenatal visit were four times more likely to deliver with a midwife than women who had no antenatal visit. Antenatal care may not be an efficient strategy to identify those most in need for obstetric service delivery, but if promoted in concurrence with effective emergency obstetric care, and delivered in skilled hands, it may become an effective instrument to facilitate better use of emergency obstetric care services. PMID- 10731230 TI - The quality of risk factor screening during antenatal consultations in Niger. AB - A decade after the first International Conference on Safe Motherhood, maternal mortality remains very high in most West African countries, even in capital cities. The detection of high risk pregnancies, known as the risk approach, during antenatal consultations has been the basis of most maternal and child health programmes over the last decade. The effectiveness of antenatal care as a tool to prevent or predict obstetric complications is being questioned more and more. In addition to the scarcity of reliable data about the predictivity of most risk factors, the quality of the screening must be questioned. The goal of this study was to assess the frequency of risk factors among a sample of pregnant women attending antenatal care in Niger and to assess the quality of the screening of those risk factors. Overall, 330 pregnant women were enrolled in the study. Each woman was examined twice: the first time by a midwife, the second time by one of the authors but without knowledge of the results of the first consultation. Fifty-five percent of pregnant women had at least one risk factor, 31% had more than one. Ninety-one percent of the risk factors were detected at interview. The following risk factors were not systematically searched for by midwives: height (48.5%), blood pressure (43.6%), glycosuria (40.6%), vaginal bleeding (38.2%), oedema (37.3%), parity (17%), age (16%), previous caesarean section (15.2%), previous stillbirth (15.2%) and previous miscarriages (14.8%). This study has shown that, in Niger, the quality of screening for risk factors during antenatal consultation is poor. In the urban settings where this study took place, lack of personnel, lack of equipment, lack of time and poor compliance by women cannot be made responsible for this situation. While screening of these risk factors continues as policy, the quality of screening must be dramatically improved. PMID- 10731231 TI - HIV counselling--a luxury or necessity? AB - WHO and UNAIDS have consistently promoted HIV counselling as a routine part of HIV testing in developing countries. Nevertheless, in many countries counselling is not considered a crucial accompaniment of testing services, and patients are tested without access to counselling during and after testing. Thus, information on the need for and results of counselling is needed to convince policy-makers and service managers to give greater priority to the development of counselling services. This qualitative study describes informational, social and emotional needs and problems of newly diagnosed seropositive patients attending public health services in Zimbabwe. Their basic factual information on HIV/AIDS was reasonable, but many patients equalled HIV to AIDS and conceptualized their infection as 'social and physical death'. This seriously impeded their capacity to use knowledge of their test results in a constructive way, and stimulated coping by denial and/or secrecy about their HIV status. These avoidant coping strategies discouraged clients from using condoms, seeking social support and taking measures to protect their vulnerable health. The complex and changing nature of clients' needs indicates that common short-cuts in counselling (e.g. giving brief information before and after the HIV test) are seriously flawed as a strategy to prepare clients for effective coping. Comprehensive pre- and post test counselling are an essential preparation for coping effectively during and immediately after testing. Availability of supportive counselling beyond this first phase is essential to assist clients with needs and problems which will appear over time. Development of counselling interventions should be guided by research into their effectiveness and by national policy guidelines. Replacing fear-inducing HIV campaigns with interactive, constructive information about HIV prevention and care will increase the preparedness of the community as a whole for effective living with HIV. PMID- 10731232 TI - Experiences of breastfeeding and vulnerability among a group of HIV-positive women in Durban, South Africa. AB - While international guidelines are currently being drawn up about HIV and infant feeding practices, and national and regional guidelines are under discussion in South Africa, there have been remarkably few studies that have sought to elicit HIV-positive mothers' experiences of breastfeeding and of paediatric infection. There is an urgent need to document this 'grass roots' knowledge in different sites, and for this data to be used to inform policy development, and for advocacy and counselling purposes. This qualitative investigation reports on the experiences and decisions taken around breastfeeding by a peer support group of 13 HIV-positive mothers meeting at King Edward VIII Hospital, Durban. In this study, the particular focus of information-giving and decision-making as to breast or formula feed is concerned with the impact on individual HIV-positive women and their babies. The most significant finding is that at no stage during their pregnancy were any of these mothers given information about the risks of HIV transmission through breastmilk. The study data were elicited in an in-depth group discussion, and individual women were invited to re-enact their stories in a follow-up discussion for clarification purposes. The women also discussed how they dealt with problems surrounding confidentiality in cases where few have been able to disclose their status to the extended family. There have been renewed calls for further investment in counsellors, with an enhanced role for community activists as peer educators. While there are severe resource constraints and low morale among many overworked nurses, one of the general problems in hospital settings remains the vertical health paradigm. This does not accommodate women's experiences, preferences, social networks and lay knowledge, and inhibits many women from becoming full participants in decisions affecting their own and their family's health. PMID- 10731234 TI - The influence of husbands on contraceptive use by Bangladeshi women. AB - This study uses the 1993-94 Bangladesh DHS to evaluate the effect of the woman's perception of her husband's approval of family planning on her current and future use of modern contraception, after controlling for selected socioeconomic and demographic factors. While most husbands support family planning, contraceptive use among those whose husbands do not approve of family planning is much lower. In some areas of Bangladesh, however, husband's disapproval of family planning is still a major deterrent factor for woman's fertility control. As husband's approval does appear to be a major determinant of contraceptive uptake in similar developing countries in the region, more effective male targeting may be necessary for maintaining the success of the family planning programme in future. PMID- 10731233 TI - Prevention of mother-to-child transmission of HIV in developing countries: recommendations for practice. The Ghent International Working Group on Mother-To Child Transmission of HIV. AB - OBJECTIVES: Different approaches to prevent mother-to-child transmission of HIV are being evaluated in developing countries. The first trials using a short regimen of zidovudine have been successful in Thailand, Cote d'Ivoire and Burkina Faso. International and local strategies are now being considered. The Ghent International Working Group on Mother-to-Child Transmission of HIV developed public health policy options to integrate these interventions into basic and maternal and child health (MCH) services. METHODS: The following tasks were undertaken: a critical review of randomized trials; an international pooled analysis of late postnatal transmission of HIV through breastfeeding; a review of the cost-effectiveness and cost-benefit of antiretroviral prophylaxis; a feasibility assessment of preventive strategies, including a postal survey on HIV voluntary counselling and testing (VCT) of pregnant women; the identification of requirements and research priorities for prenatal, obstetric and paediatric care. These projects provided the background for a three-day workshop in Ghent, Belgium, in November 1997. Conclusions were further refined, based on 1998 research findings. RESULTS: A summary of relevant evidence and ten public health recommendations are reported. VCT for pregnant women, a short regimen of zidovudine together with alternatives to breastfeeding currently represent the best option to reduce vertical transmission in most developing countries. The primary goal of the integrated package supporting these interventions is to alleviate overall maternal and infant morbidity and mortality. CONCLUSION: Prevention of mother-to-child transmission of HIV should now be considered for integration into basic health and MCH services of selected countries, with the involvement of governments and donor agencies. PMID- 10731235 TI - The World Bank and pharmaceuticals. AB - Within less than a decade the World Bank has become the largest single source of finance (loans) for health in low and middle income countries as well as a major player in the field of pharmaceuticals. Often 20-50% of the recurrent government health budget in developing countries is used to procure drugs. Drugs are among the most salient and cost-effective elements of health care and often a key factor for the success of a health sector reform. However, pharmaceuticals are frequently being used irrationally, mainly due to market imperfections in health care, such as information asymmetries, leading to serious health problems and a heavy financial burden on the health system. Lending priorities set by the World Bank could be used to promote public health sector reform, leading to the rational use of affordable and available drugs of good quality in developing countries. This report provides the first analysis of World Bank activity in the pharmaceutical sector worldwide. The analysis of 77 staff appraisal reports, describing the planning phase of World Bank country projects, shows that 16% of the total World Bank health, nutrition and population budget, or approximately US$1.3 billion, has been committed to loans or credits supporting pharmaceutical activities in the programme countries between 1989-95. Roughly US$1.05 billion has been committed to procurement of drugs and medical equipment. Only 5% of the total pharmaceutical sector lending is committed to software components such as drug policy work and rational use of drugs. No more than 45% of the projects were developed in collaboration with pharmaceutical expertise. The World Bank is recommended to improve its pharmaceutical sector involvement by promoting drug policy research and development including national and international dialogue on pharmaceutical issues to ensure rational use of both drugs and loans. In this, the World Bank has an advantage given its experience from working with both the private and the public sector, its in-house expertise in health economics, and lastly its ability to be listened to by governments through its power. PMID- 10731236 TI - User fees and drugs: what did the health reforms in Zambia achieve? AB - The article reports on exploratory research into the effects and prospects of health reforms in Zambia. The research, which was qualitative, was carried out in two rural and two urban health centres and their surrounding catchment populations. The authors focus on four principles of health reform: community involvement (including cost sharing), prevention, equity and quality of care. One of their main conclusions is that many people criticize the introduction of cost sharing because it does not improve the quality of care, by which they first of all mean the availability of drugs. The authors plead for a humane implementation of user fees in public health care that is directly linked to a more efficient provision of essential medicines. PMID- 10731237 TI - Cost recovery in Mauritania: initial lessons. AB - Cost recovery was introduced in Mauritania in 1993. Analysis of the Mauritanian experience provides a number of key points to the discussion surrounding the contribution of user fees to health care systems. Initial results appear to be largely positive regarding the improvement of the quality of health care and the overall level of utilization of basic health establishments. They suggest that users are globally willing to pay when the quality of health care improves, and that, contrary to a frequently voiced concern, EPI activities have increased. Several elements tend to show that cost recovery accompanied by a fair supply of essential drugs and by a better motivated staff has contributed to improve the efficiency of the health system. But a coherent price structure is needed to guide patients more efficiently to the different levels of the health pyramid. It is therefore vital that user fees are extended, as the government intends, to the second and third levels of the health system. The analysis conducted here also suggests that cost recovery has probably had no major negative effects as far as equity is concerned, although further investigation is necessary before a more precise judgement can be made. PMID- 10731238 TI - The impact of financing and quality changes on health care demand in Niger. AB - This paper assesses the demand effects of a cost recovery and quality improvement pilot study conducted in Niger in 1993. Direct user charges and indirect insurance payments were implemented in government health care facilities in different parts of the country, and were preceded or accompanied by quality changes in these facilities. Decision-making by patients is modelled as a three stage process of reporting an illness, seeking treatment and choice of provider; and multinomial nested logit techniques are used to estimate the parameters of the decision-tree. Overall, the results give a reasonably favourable impression of the policy changes. In neither case is there evidence of serious reductions in access or increases in cost. Particularly notable is that despite an increase in formal user charges, the observed decline in rates of visits is statistically insignificant, suggesting the success of measures to improve quality of health care in public facilities. The observed increase in the probability of formal visits in the district with indirect payments is also striking. Both contrast with the control region of Illela, where neither user charges were introduced nor were any efforts made to improve quality. The data suggest that higher utilization of formal care, probably due to improvements in quality, outweighed the decrease in utilization that may have come about due to introduction of cost recovery, so that the net effect of the policy changes was an increase in utilization. Quality considerations appear to be important in ensuring the long term success of cost sharing. PMID- 10731239 TI - Future prospects of voluntary health insurance in Thailand. AB - Voluntary health insurance schemes in Thailand are still under development and have yet to seriously address the questions of equity and efficiency, while private health insurance is limited to people who can afford the premium. One form of insurance, commonly known as the health insurance card scheme, was first introduced as the Health Card Program in 1983. This program is based on risk sharing of health expenditures, with no cost sharing, in a voluntary health insurance prepayment scheme. With the uncertain performance of the Thai economy, program sustainability and the efficient use of resources are major concerns. The Health Card Program needs enough enrollees to ensure a sufficient pool of risks. This study looks at health card purchase and utilization patterns, using data from Khon Kaen Province, and finds that employment, education levels and the presence of illness are significant factors influencing card purchase. The last factor is related to the problem of adverse selection of the program; families with symptoms of sickness are more likely to buy cards, resulting in greater use of health services. The results also show an improvement in accessibility to health care and a high level of satisfaction among card holders, both key objectives of the program. It is suggested that changes in the health card system could enable it to evolve into a community-based compulsory health insurance scheme for rural areas. PMID- 10731240 TI - Public and private hospitals in Bangladesh: service quality and predictors of hospital choice. AB - This study compares the quality of services provided by public and private hospitals in Bangladesh. The premise of the paper was that the quality of hospital services would be contingent on the incentive structure under which these institutions operate. Since private hospitals are not subsidized and depend on income from clients (i.e. market incentives), they would be more motivated than public hospitals to provide quality services to patients to meet their needs more effectively and efficiently. This premise was supported. Patient perceptions of service quality and key demographic characteristics were also used to predict choice of public or private hospitals. The model, based on discriminant analysis, demonstrated satisfactory predictive power. PMID- 10731241 TI - Historical analysis of the development of health care facilities in Kerala State, India. AB - Kerala's development experience has been distinguished by the primacy of the social sectors. Traditionally, education and health accounted for the greatest shares of the state government's expenditure. Health sector spending continued to grow even after 1980 when generally the fiscal deficit in the state budget was growing and government was looking for ways to control expenditure. But growth in the number of beds and institutions in the public sector had slowed down by the mid-1980s. From 1986-1996, growth in the private sector surpassed that in the public sector by a wide margin. Public sector spending reveals that in recent years, expansion has been limited to revenue expenditure rather than capital, and salaries at the cost of supplies. Many developments outside health, such as growing literacy, increasing household incomes and population ageing (leading to increased numbers of people with chronic afflictions), probably fueled the demand for health care already created by the increased access to health facilities. Since the government institutions could not grow in number and quality at a rate that would have satisfied this demand, health sector development in Kerala after the mid-1980s has been dominated by the private sector. Expansion in private facilities in health has been closely linked to developments in the government health sector. Public institutions play by far the dominant role in training personnel. They have also sensitized people to the need for timely health interventions and thus helped to create demand. At this point in time, the government must take the lead in quality maintenance and setting of standards. Current legislation, which has brought government health institutions under local government control, can perhaps facilitate this change by helping to improve standards in public institutions. PMID- 10731242 TI - A critical review of 'a critical review': the methodology of the 1993 World Development Report, 'Investing in Health'. AB - Since its publication in 1993, the World Bank's World Development Report, Investing in Health, has been subjected to much criticism, particularly over the way it proposes to measure the health losses summarized in the concept of the 'burden of disease', and to establish priorities for health interventions according to the reduction in mortality and disability they could produce and what they would cost. Some of these criticisms are justified, and are recognized by the WDR; others arise from misunderstanding or misapplication of the concepts. Sifting these criticisms to arrive at a better understanding requires looking at what kind of analysis is involved, how the subjective elements of the exercise were determined, and how they can be used to choose which interventions deserve priority when a country cannot meet all its citizens' health needs. PMID- 10731243 TI - Moderating discussions on the web: opportunities, challenges and lessons learned. PMID- 10731244 TI - Inherent Abnormalities of Fat Cells from Massively Obese Individuals. AB - In vitro investigations into adipose cell dynamics have revealed intrinsic characteristics of massively obese individuals' cells that could contribute to a relatively intractable expanded tat mass. Morbidly corpulent peoples' preadipocytes replicate to a greater degree than those from lean individuals. Coupled with exaggerated differentiation this enhanced growth would result in a greater number of tat cells which would increase adipose tissue mass. The relative resistance to de-differentiation that adipocytes from the massively obese demonstrate would contribute to stability of an increased number of adipocytes further exacerbating the problem. The increased message of an energy sensing protein, the obese gene product, suggests that the morbidly obese are insensitive to its action. Together these attributes provide a strong argument for a significant genetic role in the pathogenesis of obesity. PMID- 10731245 TI - A Predicted Future for Bariatric Surgery: Using the Surgical Model. AB - Bariatric surgery is the only currently available, effective, long-term method for controlling morbid obesity. 'Long-term' non-surgical, medical, voluntary weight loss data is rare; where it exists, it averages only 5-10% of patients' total body weight - of minimal long-term value for the morbidly obese - and has a consistently high failure rate, with many patients ultimately progressing to disability, End-Stage Obesity Syndrome or premature death. The author predicts that the time will come when most members of the lay and medical communities accept that bariatric surgery is the conservative approach to treatment for the morbidly obese. The mechanism will be by bariatric surgeons gathering and reporting data on their morbidly obese patients from the options presented. Data collection may take the form of controlled, randomized, descriptive, case or group studies of surgical and nonsurgical therapies upon patients' psycho socioeconomic-physical-behavioral-medical co-morbidities. Stressed is the concept that all bariatric surgeons have the capability and the implied duty to analyze their patients' data and disseminate the results, in part to achieve the predicted improvement in medical and lay acceptance of bariatric surgery as a conservative concept. PMID- 10731246 TI - Drug Distribution in Obesity and following Bariatric Surgery: A Literature Review. AB - BACKGROUND: The pharmacokinetic variables of drug clearance and volume of distribution are usually corrected for body weight or surface area. Only recently have the relationships which exist between body size, physiologic function and pharmacokinetic variables been evaluated in the obese population. These effects are not widely known, and data on this and the effects of bariatric surgical procedures are scantily documented in the surgical literature. METHODS: Literature review. RESULTS: Drugs with a low or moderate affinity for adipose tissue have a moderate increase in the volume of distribution (Vd), and this correlates with the increase in lean body mass (LBM). Highly lipophilic drugs, with some exceptions, show the expected increase in Vd and prolongation of elimination half-life, indicating a marked distribution into adipose tissue. Drug absorption, in general, is slowed by delayed gastric emptying and is normal when gastric emptying is normal or increased. Most drug absorption occurs in the small intestine where duration of drug/mucosal contact is the most important factor. CONCLUSIONS: Drugs whose distribution is restricted to LBM should utilize a loading dose based on ideal body weight (IBW). For those drugs which distribute freely into adipose tissue, the loading dose should be based on total body weight (TBW). Adjustment of the maintenance dose depends on clearance rates. In a few cases dosage adjustment depends on pharmacodynamic data, since drug clearance does not conform to these recommendations, for reasons which remain to be defined. Following bariatric surgery, in the absence of delayed gastric emptying or uncontrolled diarrhea, drug absorption rates are usually comparable to the non operated patient. PMID- 10731247 TI - MMPI Profiles of Female Candidates for Obesity Surgery: A Cluster Analytic Approach. AB - BACKGROUND: Early attempts to characterize personality in obese persons led to the conclusion that the severely obese are not homogeneous on personality measures. More recently, cluster analysis procedures have been applied to identify meaningful personality subtypes in populations of persons with severe obesity; some progress has been made in this effort, but researchers have indicated the need for replication in other settings. METHODS: In the present study, the Minnesota Multiphasic Personality Inventory (MMPI) was administered to 163 female candidates for intestinal bypass surgery. RESULTS: Eight clusters were identified. Of the MMPI profiles, 71% were in one of four normal limit clusters. The remainder were distributed across four clusters, all beyond normal limits. CONCLUSIONS: Present and previous results appear to converge on representative personality subtypes in severe obesity. Identification of such meaningful subgroups of candidates for obesity surgery may enhance the prediction of surgery outcomes, and yield a better understanding of personality in severely obese women. PMID- 10731248 TI - Measurement of Functional Pouch Volume following the Gastric Bypass Procedure. AB - BACKGROUND: The cottage cheese test was developed in an attempt to find a simple way to measure functional pouch volume and to better understand the fate of the tiny proximal pouch following the gastric bypass procedure. METHODS: Our patients were asked to eat cottage cheese in a structured fashion before their return visits from 3 months to 2 years postoperatively. RESULTS: We found there was a step-wise progression of increase in functional pouch volume with statistical significance between each time interval. Also, we compared the patients' excess weight loss at 1, 2, and 3 years postoperatively to their pouch size at 1 year postoperatively. Although there is a wide range (2.5-9.0 oz) of pouch sizes at 1 year, there is no significant difference in excess weight loss between the smaller and larger pouches. CONCLUSIONS: The pouches enlarge by the orderly process of hyperplasia. Within the 2.5-9 oz volume variation, the pouch volume alone is not a predictor of weight loss. Rather, how the patient uses the pouch/tool, in addition to the other behavior modifications, determines the degree of weight loss. This data strongly suggests that the surgeon's understanding of and teaching of the optimal use of the pouch/tool may be more important than previously thought. PMID- 10731249 TI - Staple Disruption and Marginal Ulceration in Gastric Bypass Procedures for Weight Reduction. AB - BACKGROUND: Staple disruption is a frequent and serious complication of the segmentation procedures of gastric bypass surgery for weight reduction. Staple line failure is frequently followed by weight gain and often marginal ulceration. METHODS: We reviewed the literature and analyzed our own series of 623 consecutively performed gastric bypass procedures for staple-line disruption and peptic ulceration. Gastric bypass procedures were broadly categorized by orientation of staple-line and location of the gastric pouch. RESULTS: Vertical pouches created along the lesser curvature appear to have the highest incidence of staple-line disruption and peptic ulceration. The incidence of staple disruption and marginal ulceration is significantly diminished but not eliminated when gastric segments are completely divided. In our series no cases of staple disruption have been encountered since the gastric pouch has been separated from the excluded stomach by a limb of jejunum. CONCLUSIONS: The high incidence of staple disruption in gastric bypass procedures with vertically oriented, lesser curvature pouches is likely the result of the greater musculature, lower compliance and increased motility of this region of the stomach. The increased incidence of peptic ulceration with lesser curvature gastric pouches may be explained by the high concentration of parietal cells in this area of the stomach. Staple disruption in gastric bypass procedures can be effectively reduced by transaction of gastric segments and possibly eliminated by separation of the pouch from the excluded stomach with a limb of jejunum. PMID- 10731250 TI - Is Routine Invasive Monitoring Indicated in Surgery for the Morbidly Obese? AB - BACKGROUND: Morbid obesity is generally considered to be a surgical and anesthetic risk. Some surgeons have advised the routine use of invasive monitoring for morbidly obese individuals undergoing surgery. The purpose of this study was to identify morbidly obese individuals undergoing primary gastric bypass procedures who required central or other forms of invasive monitoring for their management. METHODS: We reviewed a series of 521 morbidly obese individuals undergoing consecutively performed primary vertical banded gastroplasty-gastric bypasses, a form of gastric bypass (performed at two community hospitals), for patients who had central, arterial, or urinary catheters placed during their hospital course for monitoring purposes. The patient population was also analyzed for age, preoperative co-morbidities, body mass index, length of operation, and for whether technical complications were encountered intraoperatively. RESULTS: At one of the two hospitals, 10% of patients had arterial catheters placed intraoperatively. In each case, the catheters were removed in the recovery room. At the second hospital, no patient had invasive monitoring intraoperatively. In the entire study group, only five patients required the use of invasive monitoring postoperatively. In each of these patients, technical perioperative complications occurred. The five patients and two hospital groups did not differ significantly in age, sex, number of co-morbidities or preoperative BMI from the study group as a whole. CONCLUSION: Morbid obesity itself is not an indication for invasive monitoring. The majority of morbidly obese individuals can be safely managed through primary gastric bypass procedures without invasive monitoring. PMID- 10731251 TI - Laparoscopic Gastric Bypass, Roux-en-Y: Experience of 27 Cases, with 3-18 Months Follow-up. AB - Gastric Bypass, Roux en-Y, has been performed laparoscopically in 27 patients from October 1993 to June 1995. Weight loss ranged from a mean of 46% of excess body weight at 3 months (n = 27) to 101% at 18 months (n = 5). Pre-operative co morbidities were relieved or improved in nearly all instances: Gastroesophageal reflux was completely relieved in 21 of 22 patients. Diabetes mellitus was normalized in 13 of 13 patients. Morbidity is comparable to the open operation, while length of stay and disability are less. Cosmetic results are excellent. Laparoscopic Gastric Bypass, Roux en-Y is a feasible and valuable procedure. PMID- 10731252 TI - Biliopancreatic Diversion in Prader-Willi Syndrome Associated with Obesity. AB - BACKGROUND: Prader-Willi Syndrome (PRWS) is an uncommon neuroendocrine disorder of genetic origin, described in 1956 by Prader, Labhart and Willi. The main clinical manifestations in the adult are mental retardation, hyperphagia with gross obesity, hypogonadismcriptorhismus and short stature. The life expectancy of the affected individual ranges between 20 and 30 years rarely beyond - due to complications related to excessive obesity. Sustained dieting combined with behavior modification programs, as well as gastric restrictive surgery for obesity, proved to have a high failure rate in PRWS, due to the patients' inability to cooperate in changing their eating habits. METHODS: Biliopancreatic Diversion (BPD), which does not require the patient's cooperation in changing eating habits after surgery, was performed in two PRWS patients (13- and 22-years old), both with excessive obesity, severe respiratory distress, day sleepiness and limited mobility. RESULTS: Two years after surgery, the 13-year-old had lost 80% of her overweight, while the 22-year-old, after 1 year, had lost 34%. Recent laboratory tests showed normal data in both patients. Their respiratory distress had subsided completely, their mobility improved dramatically, and their self image and alertness enhanced. CONCLUSION: BPD resulted in an improved quality of life in these patients. PMID- 10731253 TI - Reversal of Hair Loss following Vertical Gastroplasty when Treated with Zinc Sulphate. AB - BACKGROUND: Nutritional complications following surgery for morbid obesity include both vitamin and mineral deficiency. Severe cases of zinc deficiency can lead to alopecia, diarrhea, emotional disorders, weight loss, intercurrent infection, bullous-pustular dermatitis and hypogonadism in males. Hair loss may occur after vertical gastroplasty (VG). METHODS: Diffuse hair loss occurred in 47 out of 130 patients who underwent VG. All patients had been routinely advised to take a multivitamin supplement, but 47 developed hair loss despite taking the supplement. These patients were then prescribed Zinc Sulphate 200 mg three times a day. There was no alteration in the vitamin supplementation. RESULTS: Arrest of hair loss and regrowth occurred in all patients. However, five patients reported recurrence of hair loss after stopping zinc. This loss was reversed within 6 months of recommencing zinc 600 mg daily. Ten control patients had no hair loss after gastrointestinal surgery. CONCLUSION: Significant hair loss occurred in about one-third of patients after VG, and was reversed by zinc supplementation. PMID- 10731254 TI - Linitis Plastica Presenting as Pouch Outlet Stenosis 13 Years after Vertical Banded Gastroplasty. AB - A 53-year-old woman, 13 years after vertical banded gastroplasty (VBG), presented with recently increased vomiting and high-grade pouch outlet stenosis, and was confirmed at laparotomy to have linitis plastics, for which she underwent total gastrectomy. Her pathology is unfavorable. Question is raised as to the incidence of gastric carcinoma subsequent to VBG and other gastric reductive and/or bypass procedures. Comparison is made of the increased incidence of gastric carcinoma over time, after gastric resective procedures for benign disease, versus the 'normal' incidence of gastric carcinoma, and the worse prognosis of gastric stump carcinoma. Noted is the recent observation, in a small series, of worrisome mucosal changes by endoscopic biopsies in patients after VBG, in the area of the pouch outlet. Long-term follow-up, with interval endoscopic biopsies and careful case reporting, will be important if we are to know the risk of eventual gastric carcinoma in these patients. PMID- 10731255 TI - Detection and analysis of intracoronary artery stent after PTCA using contrast enhanced three-dimensional electron beam tomography. AB - We evaluated several three-dimensional methods of contrast-enhanced electron beam tomography (EBT) for evaluation of intracoronary stent characteristics after percutaneous transluminal coronary angioplasty (PTCA). Twenty-six patients who had undergone PTCA and stenting (31 stents) were investigated by using contrast enhanced, electrocardiographically (ECG) triggered EBT scanning. EBT results were compared with conventional selective coronary arteriography (SCA). Overall, 27 intracoronary stents had been identified on EBT images (87.1%), while 4 stents could not be evaluated because of impaired image quality. EBT correctly identified the only case in which there was a residual stenosis at the distal portion of the stent. As compared with SCA results, EBT showed an over-estimation of intracoronary stent diameter (p < 0.001) and accurate measurement on the visualized length of the stents (p > 0.05). Fifty Hu, 100 Hu and 150 Hu were used as the observed window levels on three-dimensional (3-D) images; the accuracy for stent assessment increased with ascending window levels. EBT can reliably characterize coronary artery stents and may become a useful non-invasiveive method for patency evaluation of stented coronary segments after PTCA. PMID- 10731256 TI - Implantation of stents covered by autologous arterial grafts in human coronary arteries: a new technique. AB - A renewed interest in radial artery conduits for bypass surgery has emerged during the last few years. In order to combine a surgical with a percutaneous technique, stents were covered by radial arterial grafts. The purpose of this study was to evaluate the feasibility of, and the immediate and long-term results after, intracoronary implantation of stents covered by autologous arterial grafts. METHODS: A graft was removed from the radial artery. A conventional stent was then covered by the arterial graft. The autologous arterial graft was stabilized on the stent by sutures. Fifteen covered stents were implanted in 15 patients (56.1 +/- 17.3 years old) in all coronary vessels. RESULTS: The procedure of stent covering was feasible and short in duration. Procedural success was 100% with no in-hospital stent thrombosis, Q-wave myocardial infarction or death. In 14 patients (93.3%), including the patients with clinical restenosis, a repeat angiography was performed (minimum lumen diameter immediately after procedure: 3.01 +/- 0.22 mm, at follow-up: 2.56 +/- 0.90 mm). Target lesion revascularization was required in 2 patients. The event-free survival rate at 2 years was 87%. CONCLUSION: Intracoronary implantation of stents covered by an autologous arterial graft may be performed safely with excellent long-term results. A multicenter study is required to assess the efficacy. PMID- 10731257 TI - Influence of vessel size on early and late outcomes after primary angioplasty for acute myocardial infarction. AB - Small vessel size is associated with worse outcomes after elective angioplasty, but the effect of vessel size on outcomes after primary angioplasty for acute myocardial infarction has not been studied. We evaluated outcomes in 1,490 consecutive patients treated with primary angioplasty comparing patients with small (< 3.0 mm) versus large ( 3.0 mm) vessels. Outcomes were worse in patients with small vessels with lower procedural success rates (92% versus 96%; p = 0. 002), higher rates of reinfarction (5.5% vs. 3.4%; p = 0.07), more late reocclusion (12.5% vs. 4.1%; p = 0.002), less improvement in ejection fraction (1.8% vs. 4.2%; p = 0.04), lower follow-up ejection fraction (53.7% vs. 56.5%; p = 0.03), and higher 30-day and late mortality (12.5% vs. 6.4%; p = 0.0002). The higher mortality can be explained by a higher baseline risk profile combined with worse procedural results and higher rates of reocclusion and reinfarction. These data stress the importance of developing new strategies to improve procedural and late outcomes after primary angioplasty in patients with small vessels. PMID- 10731258 TI - Intervening on small infarct-related arteries: what does the future hold? PMID- 10731259 TI - CT-Based coronary angiography. PMID- 10731260 TI - A new technique to guarantee access to the sidebranch during bifurcational coronary stenting. AB - Two consecutive patients are treated using a new technique whereby a stent is implanted across a sidebranch but access to the sidebranch is never lost, thereby guaranteeing further treatment with balloon dilatation or stenting as required. PMID- 10731261 TI - The deadly double infarct syndrome treated by two-vessel primary angioplasty and stenting. AB - Percutaneous transluminal primary angioplasty (PTCA) has become an accepted therapy for treatment of acute myocardial infarction. Coronary stenting in the setting of acute myocardial infarction has been found to have several advantages, and has been successfully performed routinely at various centers. However, we are unaware of any report that describes successful angioplasty performed on two simultaneously occluded coronary arteries (the deadly double infarct syndrome). This is the first report of successful primary angioplasty with stenting performed on a patient with occlusion of two vessels simultaneously. We review the literature and suggest that this may prove to be a viable alternative in an emergency in two-vessel coronary artery bypass grafting. PMID- 10731262 TI - Percutaneous transluminal coronary angioplasty and stent insertion in a single left coronary artery. AB - Anomalous coronary arteries are rare and single coronary arteries are very rare congenital abnormalities. Significant stenoses within these arteries have been described, but interventional procedures on such anomalies are very infrequently performed. We describe a patient with angina pectoris and a single left coronary artery with multiple lesions treated with percutaneous transluminal coronary angioplasty (PTCA) and intra-coronary stent implantation. Our case shows that when the anatomy is not too abnormal, even multivessel PTCA and stent employment can be performed successfully in single coronary arteries. PMID- 10731263 TI - Innovations in stenting to treat diverse problems in interventional cardiology. PMID- 10731264 TI - Successful non-operative management of right coronary artery perforation during percutaneous coronary intervention in a patient receiving abciximab and aspirin. AB - We report a case of successful non-operative treatment of coronary artery perforation that occurred during percutaneous transluminal coronary angioplasty. The patient was receiving abciximab, aspirin and heparin. The perforation was recognized immediately and managed by partial reversal of heparin and inflation of a non-compliant balloon at the perforation site. The perforation sealed completely. The patient was discharged asymptomatic the following day. PMID- 10731265 TI - The ARTISTIC and ARREST trials. PMID- 10731266 TI - Access strategies for carotid artery intervention. PMID- 10731267 TI - Transseptal approach for stent implantation in right internal carotid artery stenosis. AB - Carotid artery stenting is emerging as an effective measure to prevent strokes in patients with significant carotid artery stenosis. We report a case of right internal carotid artery stenosis in which we used a transseptal approach for successful carotid artery stenting. This patient had concomitant stenosis at all three coronary arteries, including the left main coronary artery. A transseptal approach was adopted due to the sharply angled take-off of the right brachiocephalic artery from the tortuous aortic arch, where advancement of an extra-stiff wire into the right common carotid artery was not possible by a femoral artery approach. PMID- 10731268 TI - Coronary stenting without predilatation in a broad spectrum of clinical and angiographic situations. AB - BACKGROUND: Direct coronary stenting without balloon predilatation has the potential benefits of a reduced risk of extended dissections, reduced fluoroscopy exposure, reduced procedural time, and potential cost savings. With the availability of superior stents and delivery systems, we review our experience with direct stent deployments in a large selected patient cohort. METHODS: We prospectively studied 158 consecutive cases of attempted coronary stenting without balloon predilatation over a 1-year period. RESULTS: Indications for the procedure included unstable angina (71%), stable angina (24%) and acute myocardial infarction (5%). Target vessels were the anterior descending artery (56%), right coronary artery (30%), circumflex artery (8%), and vein grafts (6%). Pre-procedural TIMI flows were grade 3 (91.7%), grade 2 (5.8%), grade 1 (1.3%) and 0 (1.3%). Lesion types were graded A (27%), B (61%), and C (12%). Stent types included NIR Primo (80.4%), ACS Multi-Link RX Duet (10.1%), NIR (5.1%), and others (4.4%). Stents ranged from 3.0Eth 4.0 mm in diameter (median = 3 mm) and 8Eth 32 mm in length (median = 16 mm). Stents were successfully deployed in 155 cases (98.1%); 2 (1.3%) required predilatation and 1 (0.6%) deployed stent could not be expanded and was opened by rotablation. All except the rotablator case achieved final TIMI grade 3 flow, and 66% did not require further balloon dilatation. There were no deaths or cases of urgent bypass surgery. Case duration and radiation exposure in direct stenting (32.8 +/- 18.9 minutes and 6.7 +/- 6.2 minutes, respectively) compared favorably to those in a matched group of patients in conventional stenting (47 +/- 18 minutes and 11 +/- 7 minutes) and coronary angioplasty (40 +/- 12 minutes and 10 +/- 6 minutes) for single-lesion cases (p < 0.005). Creatine kinase elevation related to the procedure was observed in 5 patients. Subacute stent thrombosis occurred in 2 (1.3%) cases. CONCLUSION: Direct coronary stenting was successfully and safely accomplished in a variety of clinical situations in selected patients. PMID- 10731269 TI - Effects of intracoronary nicardipine, diltiazem and verapamil on coronary blood flow. AB - BACKGROUND: Intracoronary (IC) calcium channel blockers (CCB) such as diltiazem and verapamil are frequently utilized during percutaneous coronary interventions to maximize coronary blood flow. Their use, however, may be limited by systemic side effects such as hypotension and bradyarrhythmias. The vasoselective dihydropyridines, such as nicardipine, may be more effective at increasing coronary blood flow with fewer systemic side effects. This study compares the effects of nicardipine, diltiazem and verapamil on coronary blood flow, heart rate and blood pressure. METHODS: IC nicardipine (200 mcg), diltiazem (1 mg) and verapamil (200 mcg) were serially administered in a randomized, double-blinded fashion in minimally diseased (< 30% stenosis) left anterior descending or left circumflex arteries in nine patients. Epicardial coronary artery diameter (ECAD) was determined by quantitative coronary angiography and coronary blood flow velocity (CBFV) was measured by Doppler Flowire in each patient before and after each medication. RESULTS: Nicardipine significantly increased CBFV (p < 0.05) and had a longer duration of effect (p < 0.05), but had no difference in ECAD compared with diltiazem and verapamil. No differences were noted between CCB in changes in heart rate or mean arterial blood pressure. However, two patients had transient episodes of Type I second degree AV block after receiving diltiazem. CONCLUSION: When compared with diltiazem and verapamil, nicardipine appears to offer more potent and more prolonged vasodilatation with less risk of serious systemic side effects. Future studies are needed to assess the efficacy of IC nicardipine in patients with no-reflow. PMID- 10731270 TI - Vascular complications after percutaneous coronary interventions using abciximab (Reopro ): prospective evaluation with Doppler color flow imaging. AB - The aim of our study was to assess the incidence and consequences of vascular access site complications (VASC) associated with abciximab treatment in patients undergoing percutaneous transluminal coronary angioplasty (PTCA). PATIENTS AND METHODS: Between July 1996 and November 1997, 120 patients underwent PTCA with adjunctive abciximab (n = 60) or without it (n = 60). VASC were assessed within 24 hours of PTCA completion by physical examination and Doppler color flow imaging (DCFI) of the groin. RESULTS: A total of 24 VASC were observed mostly in abciximab patients (30% vs. 10%; p = 0.006) and only half of them were suspected on physical examination. Abciximab administration [odds ratio (OR) = 3.1; 95% confidence interval (CI) = 1.03Eth 9.0; p = 0.001], platelet fall > 40% (OR = 5.0; CI = 1. 03Eth 24.6; p = 0.045), and double-wall femoral artery puncture (OR = 5.3; CI = 1.9Eth 14.7; p = 0.001) carried a high probability of VASC after multivariate analysis. Vascular repair was needed in 10% of abciximab patients. CONCLUSIONS: Aggressive antiplatelet drugs adjunctive to the anticoagulation treatment may result in an increased rate of VASC after PTCA. The use of refined catheterization techniques is advised to avoid VASC associated with abciximab administration. DCFI is recommended in the patients with abnormal groin finding or significant platelet fall after PTCA. PMID- 10731271 TI - Bleeding risks and vascular complications following abciximab therapy for percutaneous coronary intervention: a new look at an old problem. PMID- 10731272 TI - Escape from true stent jail by use of the rotablator. AB - We describe the use of the Rotablator device to allow sidebranch access via the side-wall of a stent when it has proved impossible to pass even the lowest profile balloon catheter through the struts N a situation of "true stent jail". PMID- 10731273 TI - Removal of a ruptured, detached, and entrapped angioplasty balloon after coronary stenting. AB - Entrapment of balloon catheters after coronary angioplasty or stent implantation is a complication during these procedures. We describe the removal of a ruptured angioplasty balloon which had become disconnected from the proximal shaft of the balloon catheter and entrapped after coronary stenting by using a 4 mm Amplatz "Goose Neck" Microsnare retrieval system (Microvena, White Bear Lake, Minnesota). The retrieval was accomplished without any complications. Retrieval systems should be on hand in all interventional catheterization laboratories. PMID- 10731274 TI - Rupture of an angioplasty balloon after treatment of a recurrent in-stent restenosis-lesion of the left anterior descending coronary artery with directional coronary atherectomy. PMID- 10731275 TI - Uneventful use of tirofiban as an adjunct to coronary stenting in a patient with a history of abciximab-associated thrombocytopenia 10 months earlier. AB - A 52-year-old male undergoing a percutaneous coronary intervention after an inferior infarction treated with thrombolytic therapy was given abciximab with development of early severe reversible thrombocytopenia (platelet count nadir of 50,000 per mm3). The same patient underwent another percutaneous coronary intervention 10 months later and was given tirofiban without development of thrombocytopenia. This observation suggests that the development of thrombocytopenia with abciximab is not necessarily generalizable to other unique IIb/IIIa receptor antagonists. Tirofiban may be an appropriate consideration for patients who would benefit from a IIb/IIIa receptor antagonist and who are not candidates for abciximab therapy due to an episode of abciximab-associated thrombocytopenia. Further investigation of thrombocytopenia associated with IIb/IIIa receptor antagonists is warranted to determine appropriate approaches to the management of patients with a history of abciximab-associated thrombocytopenia. PMID- 10731276 TI - The effect of 32P beta-radiotherapy on both vessel remodeling and neointimal hyperplasia after coronary balloon angioplasty and stenting: a three-dimensional intravascular ultrasound investigation. AB - Intracoronary radiation is a promising therapy to decrease restenosis after percutaneous intervention. The aim of this pilot study was to determine the mechanism of intracoronary beta-radiation after balloon angioplasty and stenting in a double-blind placebo-controlled randomized fashion. Twenty-six patients were randomized to either placebo (n = 6) or 3 doses (28, 35 and 42 Gy) of beta radiation (n = 20) using the Guidant brachytherapy system (27 mm long 32P source wire). Of these, 21 patients underwent post-procedure and 6-month follow-up three dimensional intravascular ultrasound (IVUS) assessment. Volumetric quantification was performed by means of a semi-automated contour detection system after an ECG gated motorized pullback IVUS imaging and three-dimensional reconstruction. We compared the volumetric changes (Delta) of total vessel volume (TVV), plaque volume (PV) and lumen volume (LV) after 6 months between placebo (dummy wire) and irradiated patients. In addition, the volume of neointimal hyperplasia was quantified within the stented segments. There was an opposite behavior of TVV and LV change between placebo (DeltaTVV = -24 mm3 and DeltaLV = -42 mm3) and irradiated (DeltaTVV = +18 mm3 and (DeltaLV = +5 mm3) patients. The mean neointimal formation within the stented segment in the irradiated patients (n = 7) was 1.9 mm3 (1.5%). Our results suggest that beta-radiation affects vessel remodeling after percutaneous intervention and inhibit neointimal formation in stented patients. PMID- 10731277 TI - Elevation of defibrillation thresholds with propofol during implantable cardioverter-defibrillator testing. AB - A 26-year-old man with severe dilated cardiomyopathy and frequent ventricular tachycardia treated with propafenone plus mexiletine underwent implantable cardioverter-defibrillator testing one day after dual chamber defibrillator implantation. There were no significant changes in the R-wave and lead position on chest x-ray. Routine defibrillator threshold (DFT) testing after 320 mg intravenous propofol demonstrated high DFTs (greater than 31 Joules and requiring 360 Joules externally for termination). Change in polarity failed to improve DFTs. After about 20 minutes of washout, conscious sedation was performed with 20 mg of midazolam and repeat DFTs were equal to 21 Joules. This case demonstrates the potential for an acute dose dependent rise in DFT with propofol. PMID- 10731279 TI - Coronary vasomotor tone: determinants and effect on coronary angioplasty. AB - BACKGROUND: Coronary artery reference diameters increase during coronary angioplasty (PTCA). However, in clinical practice, balloon selection is often based on a preceding diagnostic coronary angiogram. It is common to find that the initially selected balloon is undersized due to resting vasomotor tone. This may contribute to a suboptimal angioplasty result. METHODS: Quantitative coronary angiography (QCA) was used to determine the magnitude of coronary artery vasodilatation over baseline angiography and its impact on balloon size choice. Pre-PTCA clinical and treatment variables were analyzed for their potential contribution to resting vasomotor tone. RESULTS: QCA of reference coronary diameters was performed in a group of 103 patients undergoing PTCA. Post PTCA proximal and distal reference diameters significantly increased over baseline. The average increase of the proximal segment was 0.368 mm (13.6%) p < 0. 001 and of the distal segment 0.567 mm (24.8%) p < 0.001. The initial nominal balloon diameter was smaller than the post PTCA proximal segment by an average of 0.34 mm (12.6%) p < 0.001. Of the clinical and treatment variables examined age < 65 years and pre-PTCA beta blocker use, significantly affected baseline vasomotor tone p < 0.05. CONCLUSIONS: Routine diagnostic angiography underestimates the true diameter of the coronary artery. Due to baseline vasomotor tone, coronary reference segments can be expected to increase approximately 13% in diameter during successful PTCA. Patients under 65 years of age and those using beta blockers may have a significantly increased baseline vasomotor tone. Underestimation of coronary artery diameter based on initial angiography necessitated a second, larger balloon in 16.5% of cases. PMID- 10731278 TI - Combination of abciximab with primary stenting in patients with acute myocardial infarction: a community hospital experience. AB - Abciximab administration during primary coronary angioplasty in patients with acute myocardial infarction (AMI) reduces death, reinfarction, and the need for urgent target vessel revascularization (TVR). Stenting in AMI reduces the rate of repeat in-hospital TVR. There is limited information on the effectiveness and one year clinical event rate of combined abciximab and primary stenting in patients with AMI at community hospitals. We evaluated the outcome of 40 consecutive patients treated with both abciximab and primary stenting at our institution. All patients underwent primary stenting of the infarct-related artery. All patients received abciximab, aspirin, ticlopidine, and heparin. TIMI grade 3 flow was established in all 40 patients. No patient required urgent TVR. There was no in hospital reinfarction or cardiac-related death. All patients were followed for at least one year, and no patient died after hospital discharge. Thallium exercise stress test revealed no evidence of ischemia at 12 months follow-up. We conclude that combined abciximab and primary stenting in this series of patients with AMI was associated with excellent results. PMID- 10731280 TI - Coronary heart disease in Filipino and Filipino-American patients: prevalence of risk factors and outcomes of treatment. AB - BACKGROUND: Very little has been published on Filipino (F) or Filipino-American (FA) health. Nothing has been written about coronary risk factors and their relationship to outcomes of percutaneous coronary intervention or cardiac surgical treatment in this group. The purpose of this study was to analyze prospectively collected data at a center treating coronary artery disease in a large series of Filipino patients. METHODS: From January 1, 1992 to December 1, 1996, 527 consecutive FA patients and 3,176 Caucasians (C) were identified from an ongoing cardiac database. In-hospital and late outcomes post discharge were evaluated and results between the FA and C groups were compared. RESULTS: The FA population had a higher incidence of hypertension (79% vs. 61%, p < 0.0001) and diabetes (34.7% vs. 24.1%, p < 0.001) compared to C patients. Hypercholesterolemia was similar in both groups. Obesity (FA 12.2% vs. C 18.3%, p < 0.0001) and current smoking (FA 15.8% vs. C 21.5%, p < 0.001) were more common in the C patients. Age at presentation did not differ between groups. Morbidity and mortality were higher in the FA patients following intervention in the catheterization lab (4.2% vs. 1.3%, p < 0.01). Logistic regression showed that FA ethnicity was an independent predictor of death after catheterization laboratory intervention (p < 0.01), along with emergency procedure, depressed ejection fraction, history of myocardial infarction (MI) and age greater than 65. For coronary bypass surgery, mortality and rate of MI was similar in both groups. Late follow-up post discharge (mean 17 months, range 12Eth 68) was obtained on 90% and 89% of eligible FA and C patients, respectively. Occurrence of late death and MI did not differ between the groups. However, need for any reintervention (catheterization laboratory or surgical) was significantly higher in the FA patients (21.2% vs. 14. 9%, p < 0.001). Cox proportional hazard regression modeling showed that FA ethnicity was an independent predictor of need for late reintervention (p < 0.01), along with type of initial treatment, history of diabetes, presence of triple vessel disease, initial presentation with acute MI and age greater than 65. CONCLUSION: Filipino-Americans have a higher prevalence of hypertension and diabetes, and a lower prevalence of smoking and obesity compared to Caucasians. FA ethnicity is an independent predictor of higher mortality after catheterization laboratory intervention and increased need for late reintervention. However, the rate of late MI and death in FA was similar to C patients. These results suggest that FA patients, especially those presenting with diabetes for CAD treatment, need to be followed closely after percutaneous intervention or cardiac surgery procedures. PMID- 10731281 TI - Transradial catheterization: a word of caution. AB - The radial artery has been used as an access for transcatheter procedures as well as a source for arterial conduit during coronary bypass surgery. It has been reported that 5Eth 13% of radial arteries may be damaged during transradial catheterization. The damage can be irreversible, and may therefore prohibit the radial arteryOs subsequent utilization as a coronary conduit. PMID- 10731282 TI - Intracoronary blood sampling using the multifunction probing catheter does not cause platelet activation. AB - We sought to test the hypothesis that intracoronary blood sampling in patients with angiographically demonstrated coronary artery disease could be performed using the multifunction probing catheter (Schneider, Bulach, Switzerland), without causing ex vivo platelet activation. PMID- 10731283 TI - Type A dissection of the ascending thoracic aorta during percutaneous coronary intervention. AB - Retrograde dissection of the aorta is extremely rare during percutaneous coronary intervention (PCI), but is a recognized and potentially life-threatening complication. We describe a case in which retrograde dissection of the aorta, necessitating urgent surgical repair, occurred during an attempt to open a chronically occluded right coronary artery. Initially localized, the dissection extended during an attempt to seal the right coronary ostium. Our experience suggests that if localized aortic retrograde dissection occurs, the management will depend on the stability of the distal coronary vessel. If stable, a conservative approach may be preferable to an attempt to seal the dissection. PMID- 10731284 TI - Reversible left anterior descending artery spasm, prolonged cardiac arrest and left main thrombosis during a PTCA attempt of the circumflex artery. AB - The authors report a case of percutaneous transluminal coronary angioplasty of the circumflex artery complicated by occlusion of the non-diseased left anterior descending artery by spasm. During advanced cardiac life support, required for the subsequent cardiac arrest, intra-coronary nitrates and calcium antagonists were administered. After 45 minutes, the spasm resolved, but N probably as a result of prolonged blood stasis N a thrombus appeared in the left main artery. While attempting to stent the left main, the thrombus was mechanically dislodged, leaving the epicardial coronary tree free, with a good flow. PMID- 10731285 TI - Femoral endarteritis as a complication of percutaneous coronary intervention. AB - Infectious complications following percutaneous coronary interventions are extremely unusual, with a reported frequency of less than 1%. This report describes a patient who developed septic endarteritis as a complication of percutaneous coronary intervention and reviews the literature of this complication. PMID- 10731286 TI - Catheter-induced spasm of the left main coronary artery. AB - Catheter-induced spasm of the left main coronary artery is rare during catheterization. We present several cases in which the occurrence of both ostial left main and right coronary spasm suggests that multiple sites of spasm may be a clue to this important diagnosis. PMID- 10731287 TI - The impact of stent design and delivery upon the long-term efficacy of radioisotope stents. AB - Both gamma and beta irradiation delivered via a radioactive catheter-based line source have been shown to have efficacy in reducing restenosis. However, these catheter-based treatments have some limitations, including the safety of handling sources ranging from 30 mCi to 500 mCi. Alternatively, one could use a stent as the platform for local radiation delivery as a means to prevent restenosis. Experimental studies have demonstrated that stents ion implanted with the b particle emitter 32P can reduce neointima formation. Clinical evaluation of the radioisotope stent began in the fall of 1996. Dose escalation studies have now been completed in approximately 250 patients with 32P, b-particle emitting stents ranging from 0.5 microCi to 24 microCi. Overall, these feasibility trials have demonstrated a clear, dose-dependent reduction of neointimal hyperplasia within the stent structure, but with an unanticipated finding of a relatively high incidence of restenosis at the stent margins. The purpose of this paper is to review the current status of radioactive stents, with an emphasis on the key elements of stent design and stent delivery that could impact the long-term efficacy of this device. PMID- 10731288 TI - Revealing data retrieved from an implantable loop recorder. AB - This patient is an 80-year-old man with a history of recurrent syncope of unknown etiology who underwent an extensive cardiac and neurologic evaluation which was essentially negative. The patient subsequently received an implantable loop recorder (Reveal , Medtronic, Inc., Minneapolis, Minnesota). Eight months after implantation, this device was activated by the patient following a syncopal episode. The episode revealed over 30 seconds of sinus arrest with only 3 ventricular escape complexes observed during that time. The patient activated this device and was subsequently referred for a dual chamber permanent pacemaker, which was implanted without complications. This case illustrates the advantages of a non-vascular, implantable loop recorder for helping to define the etiology of recurrent unexplained syncope. PMID- 10731289 TI - Combination enoxaparin and abciximab therapy during percutaneous coronary intervention: "NICE guys finish first". AB - Data from randomized clinical trials support the administration of both enoxaparin and platelet glycoprotein IIb/IIIa blockade to patients who present with non-ST segment evaluation acute coronary syndromes. Enoxaparin does not activate platelets, has a more predictable dose response that facilitates weight adjusted dosing and may have enhanced antithrombotic (increased anti-Xa activity) and safety (reduced anti-IIa activity) properties when compared with unfractionated heparin. Abciximab administration during percutaneous coronary intervention reduces the incidence of ischemic adverse outcomes and may improve survival in long-term follow-up. The preliminary experience with combining abciximab and intravenous enoxaparin during percutaneous coronary intervention in the NICE-4 Trial demonstrates a low incidence of minor/major bleeding (TIMI definition) and transfusion and infrequent major cardiac events to 30 days follow up. Future algorithms to facilitate the transition of patients from the clinical service who have received subcutaneous administration of enoxaparin to the cardiac catheterization laboratory prior to percutaneous coronary intervention are forthcoming and will provide seamless integration of "optimal" adjunctive pharmacology through the course of hospitalization for patients with non-ST elevation acute coronary syndromes. PMID- 10731290 TI - Low-molecular-weight heparin and platelet glycoprotein IIb/IIIa receptor blockade in the treatment of acute coronary syndromes: complementary or competing therapies? AB - Clinical trials have reported the beneficial effects of platelet glycoprotein (GP) IIb/IIIa receptor antagonists and low-molecular-weight heparins in patients presenting with unstable angina or non-ST elevation myocardial infarction. In an overview of all trials comparing GP IIb/IIIa antagonists with control treatment (i.e., heparin or placebo on a background of aspirin therapy), the incidence of death/myocardial infarction at 30 days was reduced by 11%, from 13% to 11.7% (p = 0.008).Overall, the trials of low-molecular-weight heparin have shown a non significant 10% reduction in the incidence of death/myocardial infarction at 30 days (4.8% versus 5.5% with unfractionated heparin; p = 0.2). There has been no evidence of heterogeneity (p = 0.1) in these trials. In contrast to the examination of all the low-molecular-weight heparins, when the results of the two trials of enoxaparin were combined, there was an 18% reduction in death/myocardial infarction at 43 days (7.1% versus 8.6%; p = 0.02). Given the differing mechanisms of action of GP IIb/IIIa antagonists and low-molecular weight heparins, and these encouraging trial results, the use of both agents was tested in a small pilot study of patients randomized to receive tirofiban plus either unfractionated heparin or enoxaparin. There was a trend for greater inhibition of platelet aggregation and shorter bleeding times in those who had received tirofiban plus enoxaparin. These data support the theoretical potential of this combination for the treatment of patients with acute coronary syndromes, but prospective randomized trials will need to be conducted to assess efficacy and safety. PMID- 10731291 TI - Angiogenesis: a "breakthrough" technology in cardiovascular medicine. AB - The identification of angiogenic growth factors has generated the opportunity for novel therapies in the treatment of a variety of diseases. Antibodies and/or naturally occurring angiogenesis inhibitors are being investigated clinically to antagonize key angiogenic factors. The strategy is designed to eliminate the vascular infrastructure and thereby minimize the extent of pathological consequences to the patient. A complementary strategy is likely to emerge for the treatment of cardiovascular diseases. Clinical trials of therapeutic angiogenesis have already been initiated in patients with myocardial ischemia and peripheral vascular disease. These include trials of recombinant protein therapy as well as gene transfer. While preliminary applications of gene therapy have established proof of the concept that angiogenic growth factors can augment collateral artery development in human subjects, many questions remain to be answered. PMID- 10731292 TI - Vascular brachytherapy: update on clinical trials. AB - The ultimate test for any emerging technology to become a standard of care depends on the outcome of the clinical trials. In the field of vascular brachytherapy, we are currently in the midst of multi-center, randomized clinical trials, and at the same time, data is being collected regarding long-term outcome of patients that enrolled early on in the pilot trials. These reports have recently reached three years of clinical and angiographic information on patients who were treated with intracoronary radiation for the prevention of restenosis. These data may give a clue to potential side effects and to the ultimate question: will dose radiation therapy be a standard of care for prevention of restenosis? PMID- 10731293 TI - Inflammation and embolization in ischemic heart disease. PMID- 10731294 TI - Glycoprotein IIb/IIIa antagonist and fibrinolytic agents: new therapeutic regimen for acute myocardial infarction. PMID- 10731295 TI - Combining treatment strategies in the 21st century: low-molecular-weight heparins in acute coronary syndromes. PMID- 10731296 TI - Facilitated percutaneous coronary intervention: validating the open artery hypothesis. PMID- 10731297 TI - Pharmacologic therapies in the treatment of peripheral vascular disease: clinical experience with reteplase, a third-generation thrombolytic. PMID- 10731298 TI - The disaster in Turkey--a personal account. PMID- 10731299 TI - Pulmonary alveolar proteinosis in Israel: ethnic clustering. AB - BACKGROUND: Pulmonary alveolar proteinosis is a rare disease in which a surfactant-like phospholipid-rich protein accumulates in the lungs. The disease is amenable to effective therapy by total lung lavage. OBJECTIVES: To investigate the prevalence, ethnic distribution and course of PAP in Israel. METHODS: A countrywide survey was conducted during which pulmonologists were questioned about patients with PAP. The patients were examined and their charts, radiological images, pathological slides and physiological data were reviewed. RESULTS: The survey yielded 15 patients (8 females) during the period 1976-98 (14 in the last decade), giving a prevalence of 3.7 x 10(6) and an incidence of 0.36 x 10(6)/year. Mean age of the patients was 33 +/- 13 years (range 0.5-46 years). Seven patients were North African (two were siblings), four were from Iraq and two were Arabs; there was only one Ashkenazi Jew (a child). Symptoms at the onset were dyspnea and chest pain. Spontaneous remission occurred in at least 3 patients, and 10 patients required 1-4 bronchoalveolar lavage treatments. The subjective and physiological response was favorable, but there was less consistent radiological improvement. CONCLUSION: The prevalence of PAP in Israel is approximately 3.7 x 10(6). Most cases occurred in Jews who had immigrated from North Africa or Iraq, and two were siblings. The prevalence among the Arab population appears to be similar. This clustering suggests the existence of a genetic predisposition. The course of the disease appears to be similar to that reported elsewhere. PMID- 10731300 TI - Drug testing in elite athletes--the Israeli perspective. AB - BACKGROUND: The use of performance-enhancing drugs by athletes, in particular anabolic steroids, is probably one of the major problems in sports today. During the early 1990s the Israeli Sports Federation and Olympic Committee established the Israeli Sports Anti-Doping Committee. OBJECTIVES: To present a follow-up on tests for use of performance-enhancing drugs among elite Israeli athletes from 1993 until the present. METHODS: Since 1993, 273 drug tests (urine samples) were performed in elite Israeli athletes. These tests were done during major competitions, and at random during the regular training season without prior notice to the athletes. The urine samples were sent for analysis to an official drug laboratory of the Olympic Committee in Cologne, Germany. RESULTS: Since 1993, seven (2.7%) male Israeli elite athletes (5 weight lifters, a javelin thrower, and a sprinter) tested positive for performance-enhancing drugs--all of them for anabolic steroids, and two for diuretics as well. DISCUSSION: These findings suggest that the phenomenon of performance-enhancing drug use by elite athletes has also entered Israeli sports, and probably represent the tip of the iceberg among Israeli sportsmen. Therefore, more drug tests should be performed, especially at random without prior notice and during the regular season. Athletes in the most popular sports such as soccer and basketball should also be tested. The concern over the use of these agents is both medical and ethical. PMID- 10731301 TI - Balneotherapy at the Dead Sea area for knee osteoarthritis. AB - BACKGROUND: Balneotherapy at the Dead Sea area has been applied in various inflammatory rheumatic diseases such as rheumatoid arthritis and psoriatic arthritis. The efficacy of balneotherapy at the Dead Sea area for the treatment of degenerative rheumatic diseases has not yet been formally evaluated. OBJECTIVE: To evaluate the efficacy of balneotherapy at the Dead Sea area in patients suffering from osteoarthritis of the knees. METHODS: Forty patients were randomly allocated into four groups of 10 patients. Group I was treated by bathing in a sulphur pool, group 2 by bathing in the Dead Sea, group 3 by a combination of sulphur pool and bathing in the Dead Sea, and group 4 served as the control group receiving no balneotherapy. The duration of balneotherapy was 2 weeks. RESULTS: Significant improvement as measured by the Lequesne index of severity of osteoarthritis was observed in all three treatment groups, but not in the control group. This improvement lasted up to 3 months of follow-up in patients in all three treatment groups. CONCLUSION: Balneotherapy at the Dead Sea area has a beneficial effect on patients with osteoarthritis of the knees, an effect that lastas at least 3 months. PMID- 10731302 TI - Financial impact of the introduction of erythropoietin in the treatment of anemia of premature infants in Israel. AB - BACKGROUND AND OBJECTIVE: Very low birthweight infants (< 1,500 g birthweight) often develop significant anemia that requires multiple blood transfusions, which carry a significant risk. Erythropoietin therapy is known to reduce the need for blood transfusions in preterm VLBW infants. Analysis of cost had been reported in prospective studies with conflicting results. No studies comparing the cost effectiveness of EPO have been reported during routine use in preterm VLBW infants. METHODS: We compared the cost of treating anemia of prematurity in two consecutive 12-month periods: before and after the introduction of EPO therapy in our unit. The cost of blood bank charges as well as disposable items and the cost of EPO were compared. RESULTS: A significantly smaller number of infants required blood transfusions in the EPO group (2 of 25 vs. 9/21 before EPO was introduced). The cost of therapy for anemia of prematurity was significantly smaller in the EPO group (128 +/- 168 US$ per infant vs. 151 +/- 189 US$ per infant before the introduction of EPO). CONCLUSION: We conclude that EPO is an efficient and cost effective alternative to blood transfusions in VLBW infants. PMID- 10731303 TI - Nimesulide-induced hepatitis and acute liver failure. AB - BACKGROUND: Nimesulide is a relatively new non-steroidal anti-inflammatory drug that is gaining popularity in many countries because it is a selective cyclooxygenase 2 inhibitor. Occasionally, treatment is associated with mild elevation of liver enzymes, which return to normal upon discontinuation of the drug. Several cases of nimesulide-induced symptomatic hepatitis were also recently reported, but these patients all recovered. OBJECTIVES: To report the characteristics of liver injury induced by nimesulide. PATIENTS AND METHODS: We report retrospectively six patients, five of them females with a median age of 59 years, whose aminotransferase levels rose after they took nimesulide for joint pains. In all patients nimesulide was discontinued, laboratory tests for viral and autoimmune causes of hepatitis were performed, and sufficient follow-up was available. RESULTS: One patient remained asymptomatic. Four patients presented with symptoms, including fatigue, nausea and vomiting, which had developed several weeks after they began taking nimesulide (median 10 weeks, range 2-13). Hepatocellular injury was observed with median peak serum alanine aminotransferase 15 times the upper limit of normal (range 4-35), reversing to normal 2-4 months after discontinuation of the drug. The remaining patient developed symptoms, but continued taking the drug for another 2 weeks. She subsequently developed acute hepatic failure with encephalopathy and hepatorenal syndrome and died 6 weeks after hospitalization. In none of the cases did serological tests for hepatitis A, B and C, Epstein-Barr virus and cytomegalovirus, as well as autoimmune hepatitis reveal findings. CONCLUSIONS: Nimesulide may cause liver damage. The clinical presentation may vary from abnormal liver enzyme levels with no symptoms, to fatal hepatic failure. Therefore, monitoring liver enzymes after initiating therapy with nimesulide seems prudent. PMID- 10731304 TI - Nedocromil and exercise-induced asthma: acute and chronic effects. AB - BACKGROUND: Nedocromil sodium confers both acute and chronic protective effects in patients with bronchial asthma, the interactions of which are unknown. OBJECTIVE: To examine to what extent and for how long nedocromil sodium prevents exercise-induced asthma when given immediately before exertion compared to chronic administration. PATIENTS AND METHODS: Eighteen asthmatic patients were given 4 mg NS at 30 min or 3.5 hours before exertion. We compared the resultant effect with that of the same protocol measured after 2 and 4 weeks of continuous treatment with the drug. RESULTS: Nedocromil sodium decreased exercise-induced asthma similarly at both points when given acutely. Chronic treatment of up to 4 weeks did not improve this protective effect at either interval following the inhalation. CONCLUSION: Nedocromil sodium most likely reaches its maximal effect on exercise-induced asthma upon the first administration, although treatment for longer than 4 weeks might be required to prove a chronic effect of the drug. PMID- 10731305 TI - Phenytoin-induced severe cutaneous drug reactions: suspected interactions with corticosteroids and H2-blockers. AB - BACKGROUND: Cutaneous drug reactions are attributed usually to one culprit drug, however, some CDRs may be associated with drug interactions. OBJECTIVES: To present a case series of four patients with phenytoin-induced severe CDRs, including toxic epidermal necrolysis (2 patients), exanthematous eruption (1 patient) and hypersensitivity syndrome (1 patient). In all patients the reactions appeared following the combined intake of phenytoin, corticosteroids and H2 blockers. CONCLUSIONS: Our case series may imply the role of drug interactions between phenytoin, corticosteroids and H2-blockers in the induction of severe CDRs. PMID- 10731306 TI - The need for a post-marketing surveillance program in Israel. PMID- 10731307 TI - Hughes syndrome--the syndrome behind the name (otherwise known as antiphospholipid syndrome). PMID- 10731308 TI - Interventional cardiology--promises and challenges. AB - The current explosion in coronary stent use for the treatment of coronary artery disease has helped to better define who really benefits from coronary stenting. It is clear that plain old balloon angioplasty can be at least as efficacious as stenting in certain patient groups. Patients with reference vessel diameter of < 2.5 mm, a small post-stent minimal lumen diameter, lesion length > 30 mm, diabetes mellitus and end-stage renal failure--all associated with a high incidence of diffuse in-stent restenosis--should not be stented routinely. The judicious use of stents in percutaneous revascularization will improve patient outcomes and decrease cost. The progress that has been achieved in coronary stent designs has led to many new questions, such as which patients benefit the most from stenting and whether adjunctive therapies or stent coatings will effectively prevent restenosis. Despite the advances in stent/balloon technology, antiplatelet agents, and interventional operator skill, the problems of in-stent restenosis remain. Novel therapeutic strategies for the management of severe coronary artery disease are evolving rapidly. Radiation therapy shows promise on this front. This therapy may reduce rates of restenosis by inhibiting smooth muscle proliferation and migration. However, important issues regarding the type of radiation and the matter of edge-restenosis are yet to be resolved, and the results of ongoing prospective randomized trials should provide valuable information on the efficacy of this modality. Patients with severe, non revascularizable coronary artery disease and debilitating symptoms represent another frustrating clinical problem. PMR performed in the cardiac catheterization laboratory may improve symptoms of angina in patients with end stage coronary artery disease. The early results are propitious. Among patients who have undergone this procedure 80-90% have improved from class IV (the most severe chest pain) to class I or II, enabling them to live relatively normal lives. Most investigators believe that PMR may be used only for those who are unsuitable candidates for percutaneous or surgical revascularization. Finally, gene therapy, by stimulating angiogenesis and improving coronary collateral development, may prove to be the most optimal treatment. Each of these strategies is currently under active investigation to confirm the results of the pilot studies, identify patient subgroups likely to respond to therapy, and determine long-term safety. Together with its challenges, the future of interventional cardiology is indeed bright. PMID- 10731310 TI - Post-marketing surveillance and adverse drug reactions. PMID- 10731309 TI - Inflammatory and immune aspects of atherosclerosis. PMID- 10731311 TI - Tick bite-induced facial cellulitis and posterior auricular lymphadenopathy. PMID- 10731312 TI - Bronchoalveolar carcinoma in a patient with recurrent familial Mediterranean attacks, fibrothorax, and treatment with colchicine. PMID- 10731313 TI - Eosinophilic pleural effusion due to Mycoplasma pneumoniae infection. PMID- 10731314 TI - Perforated appendicitis in shigellosis. PMID- 10731315 TI - Radiotherapy for plasmocytoma of the larynx. PMID- 10731316 TI - The pilot and the physician. PMID- 10731317 TI - The basket catheter. PMID- 10731319 TI - Can the Israeli health system support "sustainable medicine"? PMID- 10731318 TI - Consensus conference on the treatment and rehabilitation of hip fractures in the elderly. PMID- 10731320 TI - Dyspnea and left atrial myxoma. PMID- 10731321 TI - Samuel the physician: a talmudic authority. II. Diagnostics and experimentation. PMID- 10731322 TI - Acute respiratory distress syndrome in children: a 10 year experience. AB - BACKGROUND: Acute respiratory distress syndrome is a well-recognized condition resulting in high permeability pulmonary edema associated with a high morbidity. OBJECTIVES: To examine a 10 year experience of predisposing factors, describe the clinical course, and assess predictors of mortality in children with this syndrome. METHODS: The medical records of all admissions to the pediatric intensive care unit over a 10 year period were evaluated to identify children with ARDS. Patients were considered to have ARDS if they met all of the following criteria: acute onset of diffuse bilateral pulmonary infiltrates of non-cardiac origin and severe hypoxemia defined by < 200 partial pressure of oxygen during > or = 6 cm H2O positive end-expiratory pressure for a minimum of 24 hours. The medical records were reviewed for demographic, clinical, and physiologic information including PaO2/forced expiratory O2, alveolar-arterial O2 difference, and ventilation index. RESULTS: We identified 39 children with the adult respiratory distress syndrome. Mean age was 7.4 years (range 50 days to 16 years) and the male:female ratio was 24:15. Predisposing insults included sepsis, pneumonias, malignancy, major trauma, shock, aspiration, near drowning, burns, and envenomation. The mortality rate was 61.5%. Predictors of death included the PaO2/FIO2, ventilation index and A-aDO2 on the second day after diagnosis. Nonsurvivors had significantly lower PaO2/FIO2 (116 +/- 12 vs. 175 +/- 8.3, P < 0.001), and higher A-aDO2 (368 +/- 28.9 vs. 228.0 +/- 15.5, P < 0.001) and ventilation index (43.3 +/- 2.9 vs. 53.1 +/- 18.0, P < 0.001) than survivors. CONCLUSIONS: Local mortality outcome for ARDS is comparable to those in tertiary referral institutions in the United States and Western Europe. The PaO2/FIO2, A aDO2 and ventilation index are valuable for predicting outcome in ARDS by the second day of conventional therapy. The development of a local risk profile may allow early application of innovative therapies in this population. PMID- 10731323 TI - A case-control study of malignant melanoma in Israeli kibbutzim. AB - BACKGROUND: Incidence rates for malignant melanoma in Israel are rising steadily, and the kibbutz population is at increased risk for this malignancy. OBJECTIVES: To assess the risk factors for malignant melanoma among kibbutz members compared to matched healthy controls. METHODS: We conducted a case-control study of 168 malignant melanoma patients and 325 healthy controls, matched by age and gender. Data were collected on three categories of risk: demographic, personal (e.g., skin, eye and hair color), and environmental/behavioral (e.g., sun exposure, use of sunscreens). RESULTS: There were no differences between the groups regarding sociodemographic data. Significantly more patients than controls had fair, vulnerable skin (P < 0.001), light eyes (P < 0.05), and fair hair (P < 0.001). There was no difference in family history of malignant melanoma or other cancers. Patients with malignant melanoma had significantly more additional skin lesions (e.g., keratoses) (P < 0.001). More patients than controls recalled having been exposed to the sun for long periods when they were 6-13 years of age. A conditional logistic regression analysis showed that fair hair, fair vulnerable skin, and additional skin lesions were independently associated with malignant melanoma (P < 0.01). CONCLUSIONS: The main target population for interventions to reduce the incidence of malignant melanoma among kibbutz members should be individuals with these risk factors. A history of increased exposure to the sun from age 6 to 13 should also be taken into account as an independent risk factor. PMID- 10731324 TI - Parental knowledge of the treatment of fever in children. AB - BACKGROUND: Although the onset of fever in children often prompts parents to seek immediate treatment, the general level of parental knowledge on pediatric fever and administration of antipyretic medications is unknown. Parents without a basic understanding of treatment principles may give their children incorrect doses of medication. Overdosing may cause drug toxicity, while underdosing may lead to unnecessary, repeated clinic and/or emergency room visits. OBJECTIVES: To assess parental decision-making with regard to treating fever in children, and its effectiveness, and to suggest methods for improving the level of treatment. METHODS: In this cross-sectional self-reported survey, questionnaires were completed by 650 parents who sought medical assistance for a child under the age of 10 years. Parents represented various socioeconomic levels, educational backgrounds and religious affiliations. RESULTS: Ninety-six percent of parents treated fevers that reached 38.5 degrees C, and 77.6% treated fevers of only 38 degrees C. Acetaminophen was the treatment of choice for 96% and dipyrone for 4%. Parental sources of information for managing and administering antipyretic drugs were medical personnel (40.7%), mother's or grandmother's experience (30%), and the enclosed leaflet or instructions on the bottle (29.3%). Forty-three percent of the parents administered the recommended dosage (10-20 mg/kg), whereas 24.3% used less and 32.7% used more; 11% exceeded a daily dosage of 120 mg/kg. CONCLUSIONS: A total of 57% of parents treated children with incorrect doses of antipyretic drugs. In 11% of the children treated, the daily dose was at a level that could cause severe toxicity. Parental knowledge of the treatment of fever must be improved. PMID- 10731325 TI - Microscopic alterations in the physis of long bones in response to hind limb immobilization in the rat. AB - BACKGROUND: The growth plate increases its activity in response to exercise. Likewise, decreased physical activity exerts a negative effect on bone growth and development, leading to rarefaction of the subepiphyseal bone. Limb immobilization inhibits the growth plate's activity, indirectly shown by a recorded arrest in longitudinal growth of the long bones. However, there is no direct evidence concerning the growth plate itself. OBJECTIVE: To determine whether the growth plate exhibits measurable microstructural changes in response to decreased levels of physical activity. METHODS: Histomorphometric analysis was used to qualitatively and quantitatively assess the changes in the epiphyseal plate in response to single hind limb immobilization in the rat. In 16 of 25 Sprague-Dawley male rats the left hind limb was immobilized for 3 weeks; the remaining 9 rats served as controls. The left proximal tibia of each animal was examined by computerized image analysis. RESULTS: There was a decrease in epiphyseal height, cell column density and subepiphyseal trabecular area--all indices of growth plate activity. Metaphyseal cortical thickness was also depressed, thereby confirming the efficacy of the immobilization method applied. CONCLUSIONS: Limb immobilization in the rat induces inhibitory histological changes in the epiphyseal growth plate, which are in contrast to the excitatory microscopic changes seen with exercise. These changes can be assessed quantitatively. Their potential for reversibility remains to be determined by future experiments. PMID- 10731326 TI - Quantitative 15 steps exercise oximetry as a marker of disease severity in patients with chronic obstructive pulmonary disease. AB - BACKGROUND: Hypoxemia is a common complication of chronic obstructive pulmonary disease and a major factor in patients' prognosis and quality of life. The response to exercise has been evaluated by various means but no standardization has been accepted. OBJECTIVES: To suggest a simple outpatient technique for evaluating the response of arterial oxygen saturation to exercise for use as a marker of disease severity. PATIENTS AND METHODS: Ninety-six patients with various degrees of COPD were divided into three groups: mild (forced expiratory volume in 1 sec > 65%), moderate (FEV1 between 50 and 65%), and severe (FEV1 < 50%). Using continuous oximeter recording we measured oxygen saturation during 15 steps of climbing, and quantified oxygen desaturation by measuring the "desaturation area," defined as the area under the curve of oxygen saturation from the beginning of exercise through the lowest desaturation point and until after recovery to the baseline level of oxygen percent saturation. Desaturation was correlated to spirometry, lung gas volumes, blood gas analysis, and 6 min walking distance. RESULTS: A good correlation was found between severity of COPD and baseline SaO2, lowest SaO2, recovery time, and desaturation area. A negative correlation was found between desaturation area and FEV1 (r = -0.65), FEV1/forced vital capacity (r = -0.58), residual volume to total lung capacity (r = 0.52), and diffusing lung capacity for carbon monoxide (r = -0.52). In stepwise multiple regression analysis only FEV1 correlated significantly to desaturation area. A good correlation was noted between 6 min walking distance and desaturation area with the 15 steps technique (r = 0.56). CONCLUSIONS: In patients with severe COPD, arterial hypoxemia during exercise can be assessed by simple 15 steps oximetry. This method can serve both as a marker for disease severity and to determine the need for oxygen supplementation. PMID- 10731327 TI - Factors contributing to physicians' success in the advanced trauma life support program in Israel. AB - BACKGROUND: Since its introduction in Israel, more than 4,000 physicians from various specialties and diverse medical backgrounds have participated in the Advanced Trauma Life Support course. OBJECTIVES: To analyze the factors that influence the success of physicians in the ATLS written tests. METHODS: A retrospective study was conducted of 4,475 physicians participating in the Israeli ATLS training program between 1990 and 1996. Several variables in the records of these physicians were related to their success or failure in the final written examination of the course. RESULTS: Age, the region of medical schooling, and the medical specialty were found to significantly influence the successful completion of the ATLS course. CONCLUSIONS: Physicians younger than 45 years of age or with a surgical specialty are more likely to graduate the ATLS course. The success rate could be improved if the program's text and questionnaires were translated into Hebrew. PMID- 10731328 TI - Is living on a kibbutz a risk factor for malignant melanoma? PMID- 10731329 TI - Physicians' success in the advanced trauma life support program in Israel. PMID- 10731330 TI - MMR vaccination, Crohn's disease and autism: a real or imagined "stomach ache/headache?". PMID- 10731331 TI - The changing face of autoimmune disease: from JAMA to IMAJ. PMID- 10731332 TI - Autism, viral infection and measles-mumps-rubella vaccination. PMID- 10731333 TI - Advances in the diagnosis and treatment of malignant brain tumors. AB - Malignant brain tumors are tough to treat. The plethora of novel technological advances and the multitude of experimental therapies are indicative of this reality. However, it becomes clear that no magical panacea will provide the cure, and only step-wise progression of research and improved technological advances to enhance the efficacy of surgical and adjuvant therapies will eventually improve the outcome of brain tumor patients. PMID- 10731334 TI - Pericardial effusion accompanying isolated hereditary hypoparathyroidism. PMID- 10731335 TI - Pleural empyema with Salmonella mendoza following splenic abscesses in a patient with myelodysplastic syndrome. PMID- 10731336 TI - Spontaneous rupture of mesenteric hematoma with hemorrhagic shock as a complication of oral anticoagulant treatment. PMID- 10731337 TI - Thrombolytic therapy in a heart transplant recipient with acute myocardial infarction. PMID- 10731338 TI - Renal excretion of oral gastrografin seen on an abdominal film in a patient with incomplete intestinal obstruction due to multiple bezoars. PMID- 10731339 TI - Response of elderly women with thyrotoxicosis to treatment for tardive dyskinesia. PMID- 10731340 TI - Negatively charged static electricity stimulation as a possible mechanism for enhancing the involution of hypertrophic and keloid scars. PMID- 10731341 TI - Short stature. PMID- 10731342 TI - Melanonychia--a perplexing differential diagnosis. PMID- 10731343 TI - Rationing: how and who? AB - Rationing health services, in the sense of denying care deemed of positive benefit by at least some health system actors, is a problem that politicians would like to avoid. Health policy analysis has offered a number of approaches, such as global budgeting, technology assessment, managed competition, legal recourse and public participation as palliatives for this difficult problem. Each of these approaches on its own falls short, but no country has yet designed a process for explicit rationing. Israel, in the context of its recent health reform, has gone as far as any country in this direction. However, significant political leadership will be required to frame the public discussion of these difficult issues. PMID- 10731345 TI - Samuel the physician: a talmudic authority. 3. Pathology and therapeutics. PMID- 10731344 TI - "Women and smoking towards the millennium". PMID- 10731346 TI - Successful use of the medicinal leech (Hirudo medicinalis) for the treatment of venous insufficiency in a replanted digit. PMID- 10731347 TI - Nimesulide-induced hepatitis and acute liver failure. PMID- 10731348 TI - Chlamydia preumoniae in ischemic heart disease. AB - BACKGROUND: Previous work has suggested an association between Chlamydia pneumoniae infection and coronary artery disease. The infection was demonstrated by titers of antibodies--enzyme-linked immunosorbent assay or immunofluorescence, and polymerase chain reaction--and by the findings of C. pneumoniae in the atherosclerotic plaque. OBJECTIVES: To evaluate the association between chronic infection with C. pneumoniae, as measured by a high titer of IgG antibody, and CAD. Our study was designed to explore the relationship between seropositivity to C. pneumoniae and serious coronary events, and to assess whether or not there may be an additional association between established cardiovascular factors and infection with this organism. METHODS: The serum of 130 patients with proven CAD was tested for the presence of IgG antibodies to C. pneumoniae using an ELISA test. A titer < or = 1:64 using the microinfluorescence method, the recognized "gold standard," correlates with a positive result when using the ELISA method. The mean age was 57 (40-65 years). The patients, 82% male and 18% female, had either myocardial infarction (n = 109) or unstable angina (n = 21) 6 months before the investigation (range 3-24 months). The serum for the control group was obtained from 98 blood donors from the same area matched for age 52 (40-58 years) and sex. The donors had no known cardiac history. RESULTS: In the CAD group 75% of patients were positive for C. pneumoniae compared to 33% in the control group (P = 0.001). No increased correlation could be demonstrated between traditional risk factors and C. pneumoniae infection, except in those patients with diabetes mellitus. We found a lower prevalence of IgG antibody to C. pneumoniae in the diabetes subgroup than in other subgroups (P < 0.006), but a higher prevalence than in the control group. CONCLUSIONS: We demonstrated a more than twofold increase in seropositivity to C. pneumoniae among patients suffering serious coronary events, and this trend was independent of gender, age or ethnic group. These findings suggest that chronic C. pneumoniae infection may be a significant risk factor for the development of CAD, but this correlation should be investigated further. PMID- 10731349 TI - Increased plasma oxidizability and decreased erythrocyte and plasma antioxidative capacity in patients with NIDDM. AB - BACKGROUND: Atherosclerosis and microvascular complications in patients with non insulin-dependent diabetes have been linked to increased oxidative stress. The glutathione redox cycle is a major determinant of the antioxidative capacity of plasma and its constituents. METHODS: We attempted to investigate plasma oxidation and plasma and erythrocyte glutathione and glutathione enzymes in 20 patients with NIDDM, compared with euglycemic matched controls. Plasma oxidation was analyzed both basally (without) and as induced by 2,2'-azobis,2-amidopropane hydrochloride measured by the generation of thiobarbituric acid reactive substances and lipid peroxides. RESULTS: There was a significant increase in oxidation both basally (without) and as induced by AAPH. Plasma glutathione was lowered by 50% (P < 0.01) and erythrocyte glutathione peroxidase, glutathione s transferase and glutathione reductase activities were lower by 30%, 27% and 46%, respectively (P < 0.01) in the patients with NIDDM. CONCLUSIONS: Confronted by increased oxidation, patients with NIDDM show an abnormal plasma and erythrocyte antioxidative capacity, which may result in an accelerated rate of complications. PMID- 10731350 TI - Results of gastrointestinal evaluation in 90 hospitalized iron deficiency anemia patients. AB - BACKGROUND: Chronic occult blood loss from the gastrointestinal tract is widely accepted as a major cause of iron deficiency anemia. OBJECTIVES: To evaluate the diagnostic yield of gastroscopy, colonoscopy and fecal occult blood testing of hospitalized IDA patients, plus follow-up. METHODS: IDA was defined as hemoglobin < 12.5 g/dl (men) and 11 g/dl (women), and serum iron < 50 g/dl. The study group comprised 90 patients (42% male) with a mean age of 65 +/- 15 years and mean Hb 8.1 g/dl. RESULTS: Gastroscopy and colonoscopy revealed a bleeding source in 28.8% and 14.4% respectively. Gastrointestinal symptoms were found in 23% of patients with diseases of the upper gastrointestinal tract and in 15.3% of the lower. The sensitivity of fecal occult blood tests in detecting lesions in the lower and upper GI tracts was 100% and 30.7% respectively. Forty-four patients (48.9%) were discharged from the hospital with IDA of unknown origin. Over the following year, 20 of the 44 patients required further hospitalization, and of these, 13 were found to have anemia. Of the remaining 24 patients who were not hospitalized again, 15 had anemia. Four patients (9%) had significant gastrointestinal lesions and two died during the follow-up. CONCLUSIONS: Fecal occult blood is a sensitive examination for lower but not for upper GI tract lesions. PMID- 10731351 TI - Correlation of work structure and job satisfaction among Israeli family physicians. AB - BACKGROUND: Physicians need a professional environment that is conducive to efficient and satisfying work. Little has been published about the effect of work structure on the satisfaction that family physicians derive from their work. OBJECTIVES: To assess the structure and the positive and negative job components of family physicians in Israel, as well as the effect of these components on their satisfaction with their work. METHODS: A questionnaire was sent to a random selection of members of the Israel Society of Family Physicians (n = 225). RESULTS: Altogether 183 questionnaires were returned. Specialist family doctors, practice medical directors and salaried doctors were involved in more activities than non-specialist doctors, trainees and self-employed doctors. Overall satisfaction was highest for specialists and lowest for non-specialists. Work overload, insufficient resources and abundant paperwork were most frequently cited as negative work components. The opportunity to utilize medical knowledge, challenging work and work variety scored highest as positive components. CONCLUSIONS: The more professionally active physicians were also the more satisfied. Clinical work and teaching provided the most satisfaction, while administrative work and lack of time were the main causes of dissatisfaction. PMID- 10731352 TI - Hallway medicine: prevalence, characteristics and attitudes of hospital physicians. AB - BACKGROUND: Hallway medicine is an integral part of physicians' medical culture, but little is known about it. OBJECTIVE: To characterize the practice of hallway medicine among hospital physicians, both as providers and consumers. METHODS: We conducted a survey of 112 randomly chosen hospital physicians at the Soroka Medical Center in Beer Sheva, Israel between November 1997 and May 1998. A self administered 39-item questionnaire was used that included sociodemographic data, the extent to which hallway medicine is practiced, and satisfaction from and attitudes to it. RESULTS: Of the 112 selected physicians, 111 responded (99.1%). Of these, 91 (82%) had been asked by their colleagues to provide hallway medicine. Most of them (91%) agreed because of "willingness to help," because "it's unpleasant to refuse," or "it's the acceptable thing to do." Most of the requests (72%) were unscheduled and time consuming (41% up to 10 minutes and 21% more than 20 minutes). Records were kept in only 36% of the cases and follow-up in 62%. Physicians who provided hallway medicine were also consumers of it (P < 0.001), based on personal acquaintance, time saved and easy accessibility. In general, the attitude to hallway medicine was negative (54%) or ambiguous (37%). Most requests for hallway medicine were made to Israeli-trained physicians, surgeons or gynecologists, and senior physicians. CONCLUSIONS: Hallway medicine is practiced frequently among hospital physicians. A formal organization of health care service within medical centers might provide physicians with better medical care and reduce potential ethical, medical, legal, psychosocial and economic problems. PMID- 10731353 TI - Takayasu's arteritis identified by computerized tomography: revealing the submerged portion of the iceberg? AB - BACKGROUND: Takayasu's arteritis is a rare, probably underdiagnosed disorder in Israel. OBJECTIVE: To evaluate the contribution of computerized tomography to the diagnosis of Takayasu's arteritis. METHODS: A retrospective analysis of the diagnostic process was recently conducted in three consecutive patients diagnosed over the last 3 years. RESULTS: Three females of Arab origin with Takayasu's arteritis were recently identified by CT. In two of the three patients the imaging procedure was performed for different working hypotheses, and the radiological findings (wall thickening, perivascular edema, and segmental intraluminal obliteration of the aorta and its major branches) were unexpected. In these two patients, repeated physical examination following the imaging procedure disclosed initially missed findings that could have led to an earlier consideration of Takayasu's arteritis (bruits above the epigastrium, subclavian and carotid arteries, and absent brachial pulses). Retrospective analysis of the patients' symptoms following CT revealed the true nature of the patients misinterpreted complaints (e.g., typical abdominal angina replaced a faulty obtained history compatible with renal colic or dyspepsia). In the third patient CT was performed for the evaluation of an epigastric bruit associated with constitutional complaints. The diagnosis of aortitis, based upon the presence of diffuse aortic wall thickening and edema of the surrounding fat, without intraluminal narrowing, could have been missed by angiography, the traditional "gold standard" diagnostic procedure. All three patients complained of ill defined epigastric abdominal pain and had epigastric tenderness during examination. CONCLUSIONS: CT has the potential for detecting Takayasu's disease and may be superior to angiography, particularly at the early non-obliterative stage. Since the diagnosis of Takayasu's disease is rarely considered, the expanding use of CT and MRI technologies may reveal missed cases that are evaluated for other plausible diagnoses. The true incidence of Takayasu's arteritis in Israel may be much higher than reported, particularly in the Arab population. Our findings suggest that epigastric tenderness, originating from active inflammatory reaction in the abdominal aortic wall, should be considered as a diagnostic criterion of Takayasu's aortitis. PMID- 10731354 TI - Prenatal lead exposure in Israel: an international comparison. AB - BACKGROUND: Prenatal lead exposure (umbilical cord blood lead concentration > 10 (micrograms/dl) may impair cognitive development. Childhood lead poisoning is infrequent in Israel, and there are no data on lead exposure in immigrants to Israel from the former Soviet Union. OBJECTIVES: To evaluate prenatal blood lead concentrations in Israeli newborns whose mothers were born in Israel and in those whose mothers recently immigrated from Russia, and to compare data of prenatal lead exposure in Israel with those reported from other countries. METHODS: We compared the UCBLC of 35 newborns of new immigrants from Russia with a group of 35 newborns whose mothers were born in Israel. Venous BLC was also measured in 50 mothers. Data are compared with similar reports on prenatal lead exposure internationally. RESULTS: The UCBLC in all 70 newborns (mean +/- SD) was 3.53 +/- 1.6 micrograms/dl, and mothers' BLC (mean +/- SD) was 3.90 +/- 1.39 micrograms/dl. UCBLC and BLC in the 50 mother-newborn pairs correlated (r = 0.36, P < 0.01). All newborns except one had UCBLC < 8.0 micrograms/dl. There was no significant difference between UCBLC in the two groups. CONCLUSIONS: Prenatal lead exposure among the study subjects in both groups was low. In this sample the newborns of mothers born in Israel and those whose mothers recently immigrated from Russia were not found to be at risk for lead poisoning. Prenatal lead exposure in this sample was low compared to that reported from various parts of the world. PMID- 10731355 TI - Duplications of the gastrointestinal tract. AB - BACKGROUND: Gastrointestinal duplications are rare, benign congenital lesions that may occur at any location along the alimentary tract and generally require surgical intervention. Presenting symptoms may be quite varied even among patients with the same anomaly. OBJECTIVE: To review the clinical presentation of gastrointestinal duplications and present our experience with such lesions over the past decade. METHODS: The records of all patients treated for gastrointestinal duplications at a tertiary hospital during 1987 through 1996 were collected, and relevant published literature reviewed. RESULTS: In the nine patients with gastrointestinal duplications, six were in the small bowel and one each in the cecum, colon and esophagus. Presenting clinical features were varied and often subtle. Perinatal ultrasonography, radioscintography and computerized tomography were useful in some cases, while in others the correct diagnosis was established only at surgery. CONCLUSIONS: Alimentary tract duplications are uncommon, and may present as solid or cystic tumors, intussusception, perforation or gastrointestinal bleeding. A high index of suspicion is required when dealing with such cases. Appropriate investigations, including imaging techniques, should be directed toward adequate and planned surgery. PMID- 10731356 TI - A look at family medicine in Israel: which physicians are more satisfied and why? PMID- 10731357 TI - Takayasu's arteritis is no iceberg. PMID- 10731358 TI - Fluid resuscitation in low intensity conflict: when theory meets reality. PMID- 10731359 TI - Controlling extracellular matrix degradation: is the promised land in sight? Or why I take a matrix metalloproteinase inhibitor every morning. PMID- 10731360 TI - Skeletal manifestations in Gaucher disease: presentation and treatment. PMID- 10731361 TI - Atherogenic effects of chronic infections: the role of heat shock protein 60 in autoimmunity. PMID- 10731362 TI - An obscure cause of odynophagia (or, "check the wrapper"). PMID- 10731363 TI - A walk in the woods with Tunga penetrans. PMID- 10731364 TI - Pathologic fractures, anemia, hypercalcemia and hypocalciuria: an association between celiac disease and hyperparathyroidism. PMID- 10731365 TI - Cerebellopontine angle lipoma and its evaluation. PMID- 10731366 TI - Chronic mesenteric ischemia secondary to sarcoid involving mesenteric lymph nodes. PMID- 10731367 TI - Schmidt's syndrome: autoimmune polyglandular disease of the adrenal and thyroid glands. PMID- 10731368 TI - Ocular injuries caused by elastic cords. PMID- 10731369 TI - Hickman catheter rupture and embolization--a life-threatening complication. PMID- 10731370 TI - S-nitrosoderivative of a recombinant fragment of von Willebrand factor (S-nitroso Ar545c): a unique antithrombotic agent. PMID- 10731371 TI - Military fluid resuscitation: to infuse or transfer? PMID- 10731372 TI - European Society of Vascular Surgery, 3-5 September 1999, Denmark. PMID- 10731373 TI - The medicinal leech in Jewish writings. AB - Leeches were widely used for healing throughout antiquity and the Middle Ages. The rabbinic responsa literature acknowledges the use of leeching for the prevention and treatment of certain illnesses, and classic Jewish sources including the Bible, Talmud, and Codes of Jewish Law describe leeches and mention their medicinal use. Although the swallowing of a leech is considered dangerous and may lead to abdominal swelling, the Talmud describes an oral concoction containing leeches in wine for patients with enlarged spleens. It is also documented that engorged leeches placed in salt quickly discharge their blood and can be used again. Modern plastic and microsurgery have rediscovered the medicinal leech, Hirudo medicinalis, to reduce venous congestion. Clearly, the value of this annelid worm lies in its secretion of an anticoagulant known as hirudin, which has several advantages over heparin. One cannot help but marvel at the wisdom and ingenuity of our forefathers. PMID- 10731374 TI - [Catecholamines and beta-blockers in heart failure: some physiopathological and pharmacological reflections]. AB - Although in the past the use of beta-blockers was considered inadvisable in patients with chronic heart failure, recent evidence strongly recommends the use of these drugs for chronic heart failure due to their positive effects on mortality and hospitalization. This paper analyzes some of the possible mechanisms responsible for the positive action of beta-blockers in heart failure. In fact, the effects of reducing sympathetic activity by beta-blockers on the myocardium, skeletal muscle, inflammatory system, metabolism of free fatty acids and glucose, insulin resistance, serum electrolytes, renal function and oxygen free radical generation are illustrated and discussed. PMID- 10731375 TI - [Electrocardiography and myocardial viability]. AB - After an acute myocardial infarction, the cardiologist cannot discard data regarding myocardial viability. The most frequently used diagnostic methods for this are echocardiographic or scintigraphic techniques; unfortunately, these techniques are operator sensitive or expensive. However, also by simple electrocardiography is it possible to obtain important information about myocardial viability, in an objective and economic way. There are three electrocardiographic modifications potentially related to myocardial viability: 1) spontaneous modifications after an acute myocardial infarction: early or late T wave normalization, and Q wave regression; 2) exercise-induced modifications: ST segment elevation, T wave normalization, ventricular arrhythmias; 3) dobutamine-induced modifications: ST segment elevation and T wave normalization. In this editorial, the authors report the literature data on the various electrocardiographic signs and analyze their accuracy and utility in myocardial viability assessment. PMID- 10731376 TI - [Is a more efficient operative strategy feasible for the emergency management of the patient with acute chest pain?]. AB - Patients with acute chest pain are a common problem and a difficult challenge for clinicians. In the United States more than 5 million patients are examined in the emergency department on a yearly basis, at a cost of 6 billion dollars. In the CHEPER registry the prevalence of patients with chest pain in the Emergency Department was 5.3%. Similarly, in 1997 at our institution the prevalence was 4.8%. Only 50% of the patients are subsequently found to have cardiac ischemia as the cause of their symptoms and 50-60% of them showed a non-diagnostic electrocardiogram (ECG). Twenty-five-50% of chest pain patients are not appropriately admitted to the hospital and despite this conservative approach, acute myocardial infarction is misdiagnosed up to 8% of patients with acute chest pain who are released from the emergency department without further evaluation, accounting for approximately 20% of emergency department malpractice in the United States. Important diagnostic information is covered by the patient's medical history, physical examination, and ECG, but often this approach is inadequate for a definitive diagnosis. Creatine kinase (CK) and CK isoenzyme- cardiac muscle subunit (CK-MB)--are traditionally obtained in the emergency department in patients admitted for suspected acute coronary syndrome. Mass measurements of CK-MB have improved sensitivity and specificity, and to date this is the gold standard test for diagnosis of acute myocardial infarction. CK-MB, however, is not a perfect marker because it is not totally cardiac specific and does not identify patients with unstable angina and minimal myocardial damage. There are no controlled clinical impact trials showing that these tests are effective in deciding whether to discharge or to appropriately admit the patient with suspected acute coronary syndrome. Relevant investigative interest has recently been focused on new markers for myocardial injury, including myoglobin, cardiac troponins T and I. Myoglobin, a sensitive but not specific marker for cardiac damage, increases earlier than CK-MB and cardiac troponins. It should be used early after symptom onset and in conjunction with a more specific marker of myocardial damage. Cardiac troponins T and I are highly specific markers for cardiac damage, rise parallel to CK-MB and remain elevated longer, up to 5 to 9 days. They are useful for detection of less severe degrees of myocardial injury, which may occur in several patients with unstable angina who are at higher risk of cardiac events. Recent studies suggest that cardiac troponins have good diagnostic performance and prognostic value in the heterogeneous population of patients seen in the Emergency Department with acute chest pain. Despite these promising data, several analytical and interpretative problems in the routine use of cardiac troponins must be solved. Incremental value of echocardiography in acute chest pain patients is still uncertain. Echocardiography can be recommended as an adjunctive test if readily available during acute chest pain or prolonged pain, especially in patients without previous myocardial infarction. Rest myocardial radionuclide imaging has been studied in the emergency department setting and although the overall diagnostic performance and prognostic value of sestamibi has been found to be promising, it is not suitable, in our country, for extensive clinical use. ECG exercise stress test in the emergency department population has been shown to be safe and it has a good negative predictive value for cardiac events. It should be recommended that any institution identify specific and shared protocol and strategies for management of patients with chest pain. These should include basal clinical evaluation, serial ECG and the use of specific and sensitive myocardial markers. Adjunctive tests, such as echocardiography, nuclear studies and stress tests should be employed when indicated taking into account local facilities. PMID- 10731377 TI - [The clinical use of the GPIIb/IIIa inhibitors eptifibatide and tirofiban in the treatment of acute coronary syndromes of the "non-ST elevation" type]. AB - Acute coronary syndromes not associated with ST-segment elevation, i.e. unstable angina and non-Q wave myocardial infarction, represent a heterogeneous group of clinical disorders sharing similar pathogenic mechanisms, clinical presentation and medical management. Current guidelines recommended an early anti-thrombotic and anti-ischemic treatment in these patients, as well as their prompt risk evaluation based on easily available clinical and instrumental data, to identify those subjects at greater risk in whom a more aggressive management is warranted. Despite the association of aspirin, heparin and anti-ischemic drugs, the 30-day rate of death or myocardial infarction remains high (9-15%) in patients with markers of greater risk (i.e. Braunwald class III, ST-segment depression, abnormal creatine kinase or troponin values). Moreover, in patients with acute coronary syndromes undergoing percutaneous coronary interventions (PCI), complex coronary lesions increase the peri-procedural risk of thrombotic complications. Regardless of the agonist responsible for platelet activation and aggregation, platelet glycoprotein (GP) IIb/IIIa receptor activation is the key factor in thrombosis formation. Several clinical trials in the past few years have documented the beneficial value of GP IIb/IIIa inhibitors in patients treated with aspirin and heparin, with a significant reduction in the cumulative end point of death and/or myocardial infarction at 48-96 hours (odds ratio--OR 0.81, 95% confidence interval--CI 0.71-0.92, p < 0.01). Such therapeutical benefit is still present at 30 days (OR 0.88, 95% CI 0.81-0.97, p < 0.001) and 6 months (OR 0.88, 95% CI 0.79-0.97, p < 0.001). In patients treated with abciximab, eptifibatide or tirofiban, undergoing early PCI, a remarkable relative reduction in the risk of death and non-fatal acute myocardial infarction was shown before PCI (-34%, p < 0.001). The pre-PCI administration of GP IIb/IIIa inhibitors is associated with a significant reduction in peri-procedural complications (-41% relative reduction of death or acute myocardial infarction in the 48 hours after PCI, p < 0.001). In this subset of patients the benefit correlates with abnormal pre-PCI values of troponin, a reliable surrogate marker of active thrombosis. The greatest clinical benefit from GP IIb/IIIa inhibitors is expected in patients presenting high-risk features (early post-infarction angina; older age with a history of left ventricular dysfunction or diabetes; heart failure symptoms, ST segment depression, abnormal troponin, creatine kinase, and C-reactive protein values at admission) as well as in patients with recurrent ischemic attacks and those undergoing early PCI. Although the combination of GP IIb/IIIa inhibition and standard doses of unfractionated heparin is associated with an increased risk of major bleeding, such risk can be remarkably reduced adopting simple technical suggestions. PMID- 10731378 TI - [Therapeutic strategies in the treatment of endothelial dysfunction: facts and outlook]. AB - Endothelial cells play a key role in cardiovascular homeostasis by producing several vasoactive agents, which modulate basal vascular tone and structure. Traditional risk factors of atherosclerosis contribute to endothelial dysfunction through different mechanisms such as oxidative stress, modulation of constitutive nitric oxide synthase, activation of angiotensin-converting enzyme and presumably endothelin-1. The purpose of this review was to evaluate the results of experimental and human studies on several treatment options that could improve endothelial function. However, further studies are needed to evaluate whether these different classes of drugs may improve both vascular injury and cardiovascular morbidity and mortality. PMID- 10731379 TI - [Aortic dissection. A diagnostic hypothesis that is frequently discussed]. AB - Aortic dissection is a dramatic event which too often carries an ominous prognosis. The characteristic clinical presentation has been well described in medical texts and cannot be misdiagnosed. However, in some not infrequent cases, symptoms and signs may be so misleading and subtle that a subsequent catastrophic evolution then seems unexpected. The diagnosis may be easily confirmed or excluded by modern diagnostic tools such as transesophageal echocardiography, magnetic resonance imaging or spiral computed tomography, which all offer such accurate anatomic images of the aortic wall that nowadays it is possible to diagnose even those minimal lesions that can precede dissection, such as intramural hemorrhages or penetrating ulcers. However, these techniques are complex, costly and require experienced operators for optimum sensitivity and accuracy. Their use in patients with suspected acute aortic syndromes is of proven necessity. However, how often is all this feasible in a crowded Emergency Department where hundreds of patients with aspecific and overlapping symptoms and signs all require immediate attention? Furthermore, how often is a subtle intriguing initial presentation then followed by fatality, which might also come about some days later? Can failing to make an early diagnosis be cause for prosecution for having given a faulty diagnosis or might it be accepted as a risk related to the imprecise, probabilistic nature of the medical approach to the diagnosis? How can an Emergency Department doctor produce a reliable document of his way of proceeding in order to offer verifiable legal proof of his methodological integrity and thus be able to avoid misinterpretation of guilt? It is all too easy to judge overlooked clinical recognition when the clear and "simple" pathological diagnosis is available, if one does not consider the complexity of the disease and its possible manifestations in the single patient. In order to answer these questions it is necessary to collect the experience of doctors and others involved in this field. It is the aim of this paper and the clinical case presented to stimulate discussion and initiate the task in hand. PMID- 10731380 TI - [The tilt test enhanced with oral nitroglycerin in patients with unexplained syncope: the Italian Protocol. The Working Group on Syncope of the Associazione Italiana di Aritmologia e Cardiostimolazione--the Arrhythmia Area of the Associazione Nazionale Medici Cardiologi Ospedalieri]. AB - The head-up tilt testing potentiated with oral nitroglycerin, devised by an Italian group, is a simple, safe and powerful, but still not a standardized, diagnostic tool. In fact, owing to its rapid diffusion, the original protocol received, often arbitrarily, subsequent modifications. Thus a task force of experienced cardiologists defined the best methodology for the test on strictly evidence-based criteria. Particular attention was reserved to some controversial topics, such as the tilting angle, the duration of the passive and pharmacological stages of the test, the type and dosage of the drug utilized. We hope that the enclosed protocol, named by us as "The Italian Protocol", will be accepted as the standard methodology of the head-up tilt testing potentiated with oral nitrates. PMID- 10731381 TI - [Troponin I in acute ischemic cardiopathy]. AB - BACKGROUND: The present study was aimed at investigating cardiac troponin I values in the first week of acute myocardial infarction and in non-infarct acute coronary ischemic syndromes. METHODS: Eighty-two patients, 42 with acute myocardial infarction, 10 with stable angina and 30 with primary unstable angina, were enrolled in the study. Blood was collected within 6 hours of symptom onset and, in the group with acute myocardial infarction, after 24 and 48 hours, and on day 7. RESULTS: Serum troponin I increased within the first 6 hours of myocardial infarction, reached the peak after 24 hours, at 48 hours it decreased, and remained above the normal range until day 7. However, troponin I values 1) were constantly higher in patients who died, in those > 60 years old and in those with antero-lateral necrosis than in survivors, in those < 60 years old and in those with infero-posterior necrosis, respectively; 2) returned to normal range on day 7 in survivors and in patients with infero-posterior acute myocardial infarction; 3) were significantly higher in fibrinolysed patients than in those who did not undergo thrombolysis; 4) were higher in patients classified as Killip class > 2. Serum troponin I values were in the normal range in non-infarct acute coronary ischemic syndromes, but were higher in unstable than in stable angina. CONCLUSIONS: The lesser increase and the early return to the normal range of cardiac troponin I levels in the subgroups of patients with myocardial infarction having a better clinical course could be regarded as a favorable prognostic sign. Since the persistent higher values of cardiac troponin I in fibrinolysed subjects are associated with the angiographic finding of patent coronary arteries, it can be suggested that the large and persistent post-thrombolysis release of cardiac troponin I from the myocardium represents a biochemical marker of a successful reperfusion. The persistent high cardiac troponin I values in patients with advanced Killip class suggest that the neuropeptide is an index of ongoing myocyte injury and hemodynamic impairment as well. The higher values of cardiac troponin I in unstable angina are probably due to focal areas of myocardial necrosis undetectable by conventional enzymatic serum markers. PMID- 10731382 TI - [The effect of cardiovascular rehabilitation on the variability of the RR cycle after a first uncomplicated acute myocardial infarct]. AB - BACKGROUND: The aim of this study was to verify the changes in the autonomic balance by means of heart rate variability assessment in patients with myocardial infarction referred for cardiac rehabilitation. METHODS: We studied 122 patients (79 males, 43 females, mean age 56 +/- 5 years), with a first uncomplicated myocardial infarction (anterior 48, thrombolysis 72), Killip class 1, preserved left ventricular function (ejection fraction 49 +/- 6%). All patients were free of inducible residual ischemia. Four weeks after myocardial infarction, patients were randomized into two groups; Group 1 (n = 58) referred for an 8 week cardiac rehabilitation program (scheduled: 24 sessions); Group 2 (n = 64): normal daily physical activity. During a 24-hour Holter ECG monitoring the following parameters were calculated in pharmacological wash-out at randomization (T0) and at the end of cardiac rehabilitation/control period (T1): mean value of RR intervals (RR), its standard deviation (SDNN), pNN50, rMSSD in the time domain; low frequency (LF) and high frequency (HF) value and the LF/HF ratio in the frequency domain. T1-T0 changes in percent values (delta %) were considered and compared between the two groups. RESULTS: Thirty-one patients were excluded from the study either for insufficient adhesion to the cardiac rehabilitation program (< 13 sessions, 22 patients) or recurrent ischemia (3 Group 1 patients and 3 Group 2 patients) and non-assessable 24-hour Holter ECG monitoring (3 patients). Thirty-one Group 1 patients and 60 Group 2 patients completed the study with a first and a second 24-hour Holter ECG monitoring performed at 30 +/- 3 days and 60 +/- 4 days respectively. At the same time an ergospirometric test was performed to evaluate cardiopulmonary function by means of exercise time, maximum oxygen consumption, anaerobic threshold, exercise time at the anaerobic threshold, and maximum oxygen consumption at the anaerobic threshold. Twenty eight Group 1 patients and 44 Group 2 patients completed the study with a first and a second ergospirometric test. Baseline heart rate variability parameters were comparable in the two groups. During the observation period only in Group 1 patients heart rate variability parameters changed significantly: RR (Group 1 = +18.3 +/- 21.3; Group 2 = +4.2 +/- 5.2, p = 0.000), pNN50 (Group 1 = 45.0 +/- 38.9; Group 2 = +24.2 +/- 34.7, p = 0.011), HF (Group 1 = +81.6 +/- 124; Group 2 = -28.7 +/- 75.4, p = 0.014) and LF/HF ratio (Group 1 = -26.0 +/- 16.1; Group 2 = -4.9 +/- 6.1, p = 0.062). There were no significant differences in SDNN, rMSSD and LF. A linear correlation between delta LF/HF ratio and baseline LF/HF ratio values was found in Group 1 (r = 0.489, p = 0.006), whereas no correlation was found between this parameter and age, ejection fraction, creatine phosphokinase, and infarct localization. Group 1 patients had a significant improvement in exercise tolerance compared to Group 2 patients. CONCLUSIONS: A cardiac rehabilitation program positively modifies the sympatho-vagal balance in patients with uncomplicated myocardial infarction, increasing the parasympathetic tone and exercise tolerance. PMID- 10731383 TI - [The long-term effects of dual-chamber stimulation in 8 patients with hypertrophic obstructive cardiomyopathy and symptoms refractory to medical therapy]. AB - BACKGROUND: The issue of DDD pacing as a therapeutic option for patients with obstructive hypertrophic cardiomyopathy is still under debate. Moreover, some authors stress the concept of the placebo effect of electrical therapy in this particular setting. METHODS: We retrospectively evaluated 8 symptomatic patients with obstructive hypertrophic cardiomyopathy despite medical therapy, who underwent DDD pacemaker implantation as an adjunctive therapeutic strategy. All patients were evaluated with a two-dimensional/Doppler echocardiogram at baseline, shortly after the beginning of DDD pacing and at follow-up. In 3 patients dobutamine stimulation was necessary to elicit the intraventricular gradient. RESULTS: At follow-up (21 +/- 19 months, range 1-54 months) the peak gradient declined from 86 +/- 27 to 34 +/- 27 mmHg (55.2%). In 4 patients the peak gradient sharply declined after pacemaker implantation with active pacing and remained stable throughout the follow-up. In 2 patients we noted a continuous reduction in the peak gradient during the follow-up, while in 2 patients it returned to baseline values after 1 year and 1 month, respectively, despite an early reduction with DDD pacing. All patients experienced symptomatic amelioration throughout the follow-up. Two patients developed angina at the end of our observation together with an increase in the peak gradient. CONCLUSIONS: We believe that DDD pacing may be considered as a practical therapeutic option for patients with obstructive hypertrophic cardiomyopathy who would otherwise be regarded as candidates for surgery. PMID- 10731384 TI - [Is calcium antagonist administration necessary after aortocoronary bypass with the radial artery?]. AB - Calcium channel blocker therapy after total arterial revascularization is very frequent. The purpose of this study was to verify the real efficacy of calcium channel blocker therapy after coronary artery bypass graft with radial artery conduit. We have divided our patients (n = 50) into two groups: the first was discharged with calcium channel blocker therapy for 6 months; the second was discharged without calcium channel blocker therapy. Angiography and clinical evaluation showed no difference between the two groups regarding wall reactivity of radial artery conduit. However, we think that the efficacy of coronary artery bypass graft with radial artery conduit and calcium channel blocker therapy will require long-term follow-up. PMID- 10731385 TI - [Congenital quadricuspid aortic valve]. AB - A case of a 17-year-old asymptomatic man is reported. The patient had no other cardiac congenital abnormalities. Transthoracic echocardiography revealed a rare quadricuspid aortic valve malformation without aortic regurgitation. PMID- 10731386 TI - [The single coronary artery. 2 clinical cases]. AB - Single coronary artery is a rare congenital abnormality, characterized by a common origin of both right and left coronary arteries from a single aortic ostium. Recognizing the anatomic relationship between the single coronary artery and the great vessels is of prognostic importance: the variant in which the first part of the coronary artery passes between the aorta and the pulmonary trunk has the worst prognosis, and may be associated with sudden death during exercise. We describe 2 cases of single coronary artery: the first is of the more unusual left type, in which a Swan-Ganz catheter in the pulmonary artery helped to recognize the interarterial course subtype. The second case is a right type, belonging to the "septal" subtype, in which the left main trunk has a partial intramyocardial course: this variant has a good prognosis and can be distinguished from the "interarterial" subtype by the presence of a septal branch originating from the left main trunk. PMID- 10731387 TI - [Simultaneous coronary angioplasty and percutaneous mitral valvuloplasty: a case report]. AB - A patient with symptomatic pliable mitral stenosis and a significant lesion of the left coronary artery underwent combined interventional procedures during a single session. From the femoral approach a percutaneous transluminal coronary angioplasty was performed, with unsatisfactory results and necessitating stent implantation. Thereafter, mitral valve stenosis was relieved by percutaneous balloon valvotomy. PMID- 10731388 TI - [A postinfacrt pseudoaneurysm of the left ventricle with an unusually large entry opening]. PMID- 10731389 TI - [Unusual echocardiographic images of aortic dissection]. PMID- 10731390 TI - [A splenic infarct due to cardiac thromboembolism in the course of nonrheumatic atrial fibrillation]. PMID- 10731391 TI - [Echocardiography and arterial hypertension: the evidence and hopes]. PMID- 10731392 TI - Prolongation of life with angiotensin converting enzyme inhibitor therapy. PMID- 10731393 TI - Chlamydia pneumoniae and lipoprotein(a): the right combination for atherosclerosis? PMID- 10731394 TI - The international angiographic compression study. PMID- 10731395 TI - Videodensitometric vs edge-detection quantitative angiography. Insights from intravascular ultrasound imaging. PMID- 10731396 TI - Stents, antithrombotic agents and vascular complications. Does site of arterial access make a difference? PMID- 10731397 TI - Determining prognosis early after a myocardial infarction. PMID- 10731398 TI - Assessment of cardiovascular risk: time to apply genetic risk factors? PMID- 10731399 TI - Systematic review of the management of atrial fibrillation in patients with heart failure. AB - AIMS: To systematically review the management of atrial fibrillation (AF) in patients with heart failure. METHODS: Studies investigating the management of AF in patients with heart failure published between 1967 to 1998 were identified using MEDLINE, the Cochrane register and Embase databases. Reference lists from relevant papers and reviews were hand searched for further papers. RESULTS: Eight studies pertaining to acute and twenty-four pertaining to chronic AF were identified. For patients with acute AF ventricular rate control, anticoagulation and treatment of heart failure should be pursued simultaneously before cardioversion is attempted. Digoxin is relatively ineffective at controlling ventricular response and for cardioversion. Intravenous diltiazem is rapidly effective in controlling ventricular rate and limited evidence suggests it is safe. Amiodarone controls ventricular rate rapidly and increases the rate of cardioversion. There are insufficient data to conclude that immediate anti coagulation, trans-oesophageal echocardiography to exclude atrial thrombi followed by immediate cardioversion is an appropriate strategy. Patients with chronic AF should be anti-coagulated unless contra-indications exist. It is not clear whether the preferred strategy should be cardioversion and maintenance of sinus rhythm with amiodarone or ventricular rate control of AF combined with anticoagulation to improve outcome including symptoms, morbidity and survival. Electrical cardioversion has a high initial success rate but there is also a high risk of early relapse. Amiodarone currently appears the most effective and safest therapy for maintaining sinus rhythm post-cardioversion. Digoxin is fairly ineffective at controlling ventricular rate during exercise. Addition of a beta blocker reduces ventricular rate and improves symptoms. Whether digoxin is required in addition to beta-blockade for the control of AF in this setting is currently under investigation. If pharmacological therapy is ineffective or not tolerated then atrio-ventricular node ablation and permanent pacemaker implantation should be considered. CONCLUSION: There is a paucity of controlled clinical trial data for the management of AF among patients with heart failure. The interaction between AF and heart failure means that neither can be treated optimally without treating both. Presently treatment should be on a case by case basis. PMID- 10731400 TI - Searching for a better assessment of the individual coronary risk profile. The role of angiotensin-converting enzyme, angiotensin II type 1 receptor and angiotensinogen gene polymorphisms. AB - BACKGROUND: Polymorphisms within renin angiotensin system genes have been investigated as risk factors for coronary artery disease in different populations with contradicting results. The aim of this study was to investigate the genotype distribution and the allele frequencies of ACE, AT1R and AGT gene polymorphisms as coronary artery disease factors and their synergistic effects on coronary risk in an Italian population. METHODS AND RESULTDS: In this study ACE, AT1R and AGT gene polymorphisms were investigated in 205 consecutive coronary artery disease patients and in 209 controls. These polymorphisms were analysed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The ACE D and AGT 235T allele, but not AT1R C allele, frequency was statistically significant in patients. An association between coronary artery disease and ACE DD, AT1R CC and AGT TT genotype, was found by univariate analysis (OR 2.06 P=0.0007, OR 2.49 P=0.009, OR 1.87 P=0. 019, respectively). At multivariate analysis ACE DD and AT1R CC genotype (OR 1.81 P=0.011, OR 2.61 P=0.011, respectively) remained associated with coronary heart disease. Subjects carrying the ACE DD genotype and AT1R C allele showed a stronger association with myocardial infarction (OR=4.02, P<0.0001). CONCLUSION: Our report indicates the increased risk of coronary artery disease in the presence of ACE DD and AT1R CC genotypes independent of other risk factors, in Italian patients. The present study stresses the relevance of screening for genetic risk factors. PMID- 10731401 TI - The proatherogenic properties of lipoprotein(a) may be enhanced through the formation of circulating immune complexes containing Chlamydia pneumoniae specific IgG antibodies. AB - AIMS: To investigate a possible relationship between the atherogenic properties of lipoprotein(a) (Lp(a)) and Chlamydia pneumoniae infections. METHODS AND RESULTS: The study population was nested within the Vasterbotten Intervention Program or the WHO MONICA project. In this incident case-control study, 78 patients who had suffered acute myocardial infarction and 156 matched controls were included. The contents of circulating immune complexes were analysed for C. pneumoniae IgG antibodies and Lp(a). A significantly larger proportion of cases than controls had >/=13 mg. l(-1)Lp(a) and a C. pneumoniae specific IgG antibody titre >/=1/2 in circulating immune complexes (odds ratio=3.8). CONCLUSION: The proatherogenic effects of Lp(a) may be enhanced and/or partly mediated through the formation of circulating immune complexes containing C. pneumoniae -specific IgG antibodies. The connection between chronic C. pneumoniae infections and atherosclerosis may, at least in part, be explained by an interaction with Lp(a) through the formation of circulating immune complexes. PMID- 10731402 TI - New Q waves on the presenting electrocardiogram independently predict increased cardiac mortality following a first ST-elevation myocardial infarction. AB - AIMS: The prognostic significance of pathological Q waves appearing in the acute phase of myocardial infarction has not been determined. We investigated whether new Q waves on the presenting electrocardiogram of patients with acute ST-segment elevation were independently associated with a worse outcome after a first myocardial infarction. METHODS AND RESULTS: The presence or absence of new Q waves on the presenting electrocardiogram was assessed in 481 patients who presented within 4 h of symptom onset and were randomized to receive either captopril or placebo within 2 h of streptokinase therapy for myocardial infarction. Ventriculography was performed at 22+/-6 days and mortality status was obtained at a median follow-up of 5.6 years. New Q waves were associated with a lower ejection fraction (51+/-13% vs 61+/-12%, P<0.0001), a larger end-systolic volume index (37 ml vs 28 ml, P<0.001), and increased cardiac mortality at 30 days (7% vs 2%, P=0.01) and at follow-up (17% vs 7%, P=0.002). On multivariate analysis, age (P<0.01), new Q waves at presentation (P<0.01) and a history of angina (P=0.046) were independent predictors of cardiac mortality, whereas randomization to captopril and the time from symptom onset to streptokinase administration were not. CONCLUSION: New Q waves at presentation are independently associated with a worse outcome after a first myocardial infarction. The presence of new Q waves on the presenting electrocardiogram allows very early identification of patients at risk of increased cardiac mortality. PMID- 10731403 TI - Videodensitometric quantitative angiography after coronary balloon angioplasty, compared to edge-detection quantitative angiography and intracoronary ultrasound imaging. AB - AIMS: To assess the value of videodensitometric quantification of the coronary lumen after angioplasty by comparison to two other techniques of coronary artery lumen quantification. METHODS AND RESULTS: Videodensitometric quantitative angiography, edge detection quantitative angiography and 30 MHz intracoronary ultrasound imaging were performed after successful balloon angioplasty in 161 patients. Lumen cross-sectional areas were mean (SD) 2.82 (1.15) mm(2)for edge detection quantitative angiography, 3.67 (1.5) mm(2)for videodensitometric quantitative angiography and 5.32 (1.75) mm(2)for intracoronary ultrasound imaging (P<0.001). The correlation between intracoronary ultrasound imaging and videodensitometric quantitative angiography (r=0.44) was almost similar to that of intracoronary ultrasound imaging and edge detection quantitative angiography (r=0. 47). The correlation between the three techniques was not significantly influenced by the presence of ruptures and dissections on intracoronary ultrasound imaging. The absence of calcifications improved the correlation between videodensitometry and intracoronary ultrasound imaging. CONCLUSIONS: The luminal dimensions as measured by videodensitometric quantitative angiography matched intracoronary ultrasound imaging derived dimensions more closely than edge detection quantitative angiography. Videodensitometric quantitative angiography represents an on-line alternative to intracoronary ultrasound imaging for quantitative analysis regardless of the degree of vessel damage. PMID- 10731404 TI - Vascular complications and clinical outcome after coronary angioplasty with platelet IIb/IIIa receptor blockade. Comparison of transradial vs transfemoral arterial access. AB - AIMS: Vascular complications associated with femoral artery access for interventional cardiological procedures may increase morbidity especially in patients receiving anticoagulants, aspirin, ticlopidine and platelet glycoprotein IIb/IIIa receptor inhibitors. The use of radial arterial access has the potential to reduce the incidence of access site bleeding complications. The purpose of this study was to compare outcomes after the radial and femoral approaches in patients treated with the platelet IIb/IIIa inhibitor, abciximab. METHODS AND RESULTS: One hundred and fifty consecutive patients treated by abciximab underwent angioplasty by the radial or femoral approach in 83 and 67 cases, respectively. Outcome variables were major cardiac events and major access site bleeding at 1-month follow-up. Freedom from major cardiac events at 1-month follow-up occurred in 78 (93.9%) and 63 (94.0%) patients in the radial and femoral groups, respectively (P=0.99). There were no major access site bleeding complications in the radial group, as opposed to five (7.4%) in the femoral group, P=0.04. Postprocedure length of stay, days (3.7+/-6.0 radial vs 3.7+/-2.6 femoral, P=0.96) as well as total hospital length of stay (5.0+/-4.3 radial vs 4.9+/-3.0 femoral, P=0.72) were similar in both groups. CONCLUSION: Coronary angioplasty in patients treated by abciximab using the transradial approach is efficacious with fewer major access site complications than with the transfemoral approach. PMID- 10731405 TI - American College of Cardiology/ European Society of Cardiology international study of angiographic data compression phase I. The effects of lossy data compression on recognition of diagnostic features in digital coronary angiography. AB - OBJECTIVES: This study intended to determine the effect of varying degrees of lossy Joint Photographic Experts Group (JPEG) compression on detection of coronary angiographic features. Background Compression of digital coronary angiograms facilitates playback of images and decreases cost. There are little data on the effect of compression on the accuracy of coronary angiography. METHODS: At six centers, 71 angiographers each reviewed a set of 100 angiographic sequences. The 100 sequences were divided into four, 25-sequence subsets. Each subset of 25 was displayed either as original images or at one of three compression ratios (CRs) (6:1, 10:1 or 16:1). The effect of lossy compression on the sensitivity and specificity for detection of diagnostic features was determined. The effect of compression on subjective measures of image quality graded by the angiographers was also examined. RESULTS: Lossy compression at a ratio of 16:1 decreased the sensitivity for the detection of diagnostic features (76% vs. 80%P=0.004). The largest effect was in the detection of calcification (52% vs. 63% at 16:1 compression vs. original images, P<0.001). Subjective indicators of image quality indicated a reduction in confidence in interpretation at CRs of 10:1 and 16:1. CONCLUSIONS: With increased ratios of lossy compression, a degradation of digital coronary angiograms occurs that results in decreased diagnostic accuracy. The sensitivity for detection of common diagnostic features was decreased, and subjective assessment of image quality was impaired. Caution is warranted in the interpretation of coronary angiograms that have been subjected to lossy JPEG compression beyond a ratio of 6:1. PMID- 10731406 TI - American College of Cardiology/ European Society of Cardiology international study of angiographic data compression phase II. The effects of varying JPEG data compression levels on the quantitative assessment of the degree of stenosis in digital coronary angiography. AB - OBJECTIVES: This report describes whether lossy Joint Photographic Experts Group (JPEG) image compression/decompression has an effect on the quantitative assessment of vessel sizes by state-of-the-art quantitative coronary arteriography (QCA). BACKGROUND: The Digital Imaging and Communications in Medicine (DICOM) digital exchange standard for angiocardiography prescribes that images must be stored loss free, thereby limiting JPEG compression to a maximum ratio of 2:1. For practical purposes it would be desirable to increase the compression ratio (CR), which would lead to lossy image compression. METHODS: A series of 48 obstructed coronary segments were compressed/decompressed at CR 1:1 (uncompressed), 6:1, 10:1 and 16:1 and analyzed blindly and in random order using the QCA-CMS analytical software. Similar catheter and vessel start- and end points were used within each image quartet, respectively. All measurements were repeated after several weeks using newly selected start- and end-points. Three different sub-analyses were carried out: the intra-observer, fixed inter compression and variable inter-compression analyses, with increasing potential error sources, respectively. RESULTS: The intra-observer analysis showed significant systematic and random errors in the calibration factor at JPEG CR 10:1. The fixed inter-compression analysis demonstrated systematic errors in the calibration factor and recalculated vessel parameter results at CR 16:1 and for the random errors at CR 10:1 and 16:1. The variable inter-compression analysis presented systematic and random errors in the calibration factor and recalculated parameter results at CR 10:1 and 16:1. Any negative effect at CR 6:1 was found only for the calibration factor of the variable inter-compression analysis, which did not show up in the final vessel measurements. CONCLUSIONS: Compression ratios of 10:1 and 16:1 affected the QCA results negatively and therefore should not be used in clinical research studies. PMID- 10731407 TI - American College of Cardiology/ European Society of Cardiology international study of angiographic data compression phase III. Measurement Of image quality differences at varying levels of data compression. AB - OBJECTIVES: We sought to investigate up to which level of Joint Photographic Experts Group (JPEG) data compression the perceived image quality and the detection of diagnostic features remain equivalent to the quality and detectability found in uncompressed coronary angiograms. BACKGROUND: Digital coronary angiograms represent an enormous amount of data and therefore require costly computerized communication and archiving systems. Earlier studies on the viability of medical image compression were not fully conclusive. METHODS: Twenty one raters evaluated sets of 91 cine runs. Uncompressed and compressed versions of the images were presented side by side on one monitor, and image quality differences were assessed on a scale featuring six scores. In addition, the raters had to detect pre-defined clinical features. Compression ratios (CR) were 6:1, 10:1 and 16:1. Statistical evaluation was based on descriptive statistics and on the equivalence t -test. Results At the lowest CR (CR 6:1), there was already a small (15%) increase in assigning the aesthetic quality score indicating "quality difference is barely discernible-the images are equivalent.' At CR 10:1 and CR 16:1, close to 10% and 55%, respectively, of the compressed images were rated to be "clearly degraded, but still adequate for clinical use' or worse. Concerning diagnostic features, at CR 10:1 and CR 16:1 the error rate was 9.6% and 13.1%, respectively, compared with 9% for the baseline error rate in uncompressed images. CONCLUSIONS: Compression at CR 6:1 provides equivalence with the original cine runs. If CR 16:1 were used, one would have to tolerate a significant increase in the diagnostic error rate over the baseline error rate. At CR 10:1, intermediate results were obtained. PMID- 10731408 TI - ESC news and appointments PMID- 10731409 TI - Identification of the RecA protein-loading domain of RecBCD enzyme. AB - Genetic recombination in Escherichia coli is stimulated by the recombination hotspot Chi (chi), a regulatory element that modifies the activities of the RecBCD enzyme and leads to loading of the DNA strand exchange protein, RecA, onto the chi-containing DNA strand. The RecBC enzyme, which lacks the RecD subunit, loads RecA protein constitutively, in a manner that is independent of chi. Using a truncated RecBC enzyme lacking the 30 kDa C-terminal domain of the RecB subunit, we show that this domain is necessary for RecA protein-loading. We propose that this domain harbors a site that interacts with RecA protein, recruiting it to single-stranded DNA during unwinding. This ability of a translocating enzyme to deliver material (RecA protein) to a specific target site (the chi sequence) parallels that of other cellular motor proteins. PMID- 10731410 TI - NMR structure of activated CheY. AB - The CheY protein is the response regulator in bacterial chemotaxis. Phosphorylation of a conserved aspartyl residue induces structural changes that convert the protein from an inactive to an active state. The short half-life of the aspartyl-phosphate has precluded detailed structural analysis of the active protein. Persistent activation of Escherichia coli CheY was achieved by complexation with beryllofluoride (BeF(3)(-)) and the structure determined by NMR spectroscopy to a backbone r.m.s.d. of 0.58(+/-0.08) A. Formation of a hydrogen bond between the Thr87 OH group and an active site acceptor, presumably Asp57.BeF(3)(-), stabilizes a coupled rearrangement of highly conserved residues, Thr87 and Tyr106, along with displacement of beta4 and H4, to yield the active state. The coupled rearrangement may be a more general mechanism for activation of receiver domains. PMID- 10731411 TI - Low temperature regulated DEAD-box RNA helicase from the Antarctic archaeon, Methanococcoides burtonii. AB - DEAD-box RNA helicases, by unwinding duplex RNA in bacteria and eukaryotes, are involved in essential cellular processes, including translation initiation and ribosome biogenesis, and have recently been implicated in enabling bacteria to survive cold-shock and grow at low temperature. Despite these critical physiological roles, they have not been characterized in archaea. Due to their presumed importance in removing cold-stabilised secondary structures in mRNA, we have characterised a putative DEAD-box RNA helicase gene (deaD) from the Antarctic methanogen, Methanococcoides burtonii. The encoded protein, DeaD is predicted to contain a core element involved in ATP hydrolysis and RNA-binding, and an unusual C-terminal domain that contains seven perfect, trideca-peptide, direct repeats that may be involved in RNA binding. Alignment and phylogenetic analyses were performed on the core regions of the M. burtonii and other DEAD-box RNA helicases. These revealed a loose but consistent clustering of archaeal and bacterial sequences and enabled the generation of a prokaryotic-specific consensus sequence. The consensus highlights the importance of residues other than the eight motifs that are often associated with DEAD-box RNA helicases, as well as de-emphasising the importance of the "A" residue within the "DEAD" motif. Cells growing at 4 degrees C contained abundant levels of deaD mRNA, however no mRNA was detected in cells growing at 23 degrees C (the optimal temperature for growth). The transcription initiation site was mapped downstream from an archaeal box-A element (TATA box), which preceded a long (113 nucleotides) 5'-untranslated region (5'-UTR). Within the 5'-UTR was an 11 bp sequence that closely matches (nine out of 11) cold-box elements that are present in the 5'-UTRs of cold-shock induced genes from bacteria. To determine if the archaeal 5'-UTR performs an analagous function to the bacterial 5'-UTRs, the archaeal deaD 5'-UTR was transcribed in E. coli under the control of the cspA promoter and transcriptional terminator. It has previously been reported that overexpression of the cspA 5' UTR leads to an extended cold-shock response due to the 5'-UTR titrating cellular levels of a cold-shock repressor protein(s). In our hands, the cold-shock protein profiles resulting from overexpression of Escherichia coli cspA and M. burtonii deaD 5'-UTRs were similar, however they did not differ from those for the overexpression of a control plasmid lacking a 5'-UTR. In association with other recent data from E. coli, our results indicate that the role of the 5'-UTR in gene regulation is presently unclear. Irrespective of the mechanisms, it is striking that highly similar 5'-UTRs with cold-box elements are present in cold induced genes from E. coli, Anabaena and M. burtonii. This is the first study examining low temperature regulation in archaea and provides initial evidence that gene expression from a cold adapted archaeon involves a bacterial-like transcriptional regulatory mechanism. In addition, it provides the foundation for further studies into the function and regulation of DEAD-box RNA helicases in archaea, and in particular, their roles in low temperature adaptation. PMID- 10731412 TI - The nuclear poly(A) binding protein, PABP2, forms an oligomeric particle covering the length of the poly(A) tail. AB - The mammalian nuclear poly(A) binding protein, PABP2, controls the length of the newly synthesized poly(A) tail on messenger RNAs. To gain a better understanding of the mechanism of length control, we have investigated the structure of the PABP2.poly(A) complex. Electron microscopy and scanning force microscopy studies reveal that PABP2, when bound to poly(A), forms both linear filaments and discrete-sized, compact, oligomeric particles. The maximum diameter of the filament is 7 nm; the maximum diameter of the particle is 21(+/-2) nm. Maximum particle size is realized when the PABP2. poly(A) complex is formed with poly(A) molecules 200-300 nt long, which corresponds to the average length of the newly synthesized poly(A) tail in vitro and in vivo. The equilibrium between filaments and particles is highly sensitive to ionic strength; filaments are favored at low ionic strength, while particles predominate at moderate to high ionic strength. Nitrocellulose filter binding and gel mobility shift assays indicate that the PABP2.poly(A) particle formed on A(300) is not significantly more stable than complexes formed with smaller species of poly(A). These results are discussed in the context of the proposed functions for PABP2. PMID- 10731413 TI - Visualization of two binding sites for the Escherichia coli UmuD'(2)C complex (DNA pol V) on RecA-ssDNA filaments. AB - The heterotrimeric UmuD'(2)C complex of Escherichia coli has recently been shown to possess intrinsic DNA polymerase activity (DNA pol V) that facilitates error prone translesion DNA synthesis (SOS mutagenesis). When overexpressed in vivo, UmuD'(2)C also inhibits homologous recombination. In both activities, UmuD'(2)C interacts with RecA nucleoprotein filaments. To examine the biochemical and structural basis of these reactions, we have analyzed the ability of the UmuD'(2)C complex to bind to RecA-ssDNA filaments in vitro. As estimated by a gel retardation assay, binding saturates at a stoichiometry of approximately one complex per two RecA monomers. Visualized by cryo-electron microscopy under these conditions, UmuD'(2)C is seen to bind uniformly along the filaments, such that the complexes are completely submerged in the deep helical groove. This mode of binding would impede access to DNA in a RecA filament, thus explaining the ability of UmuD'(2)C to inhibit homologous recombination. At sub-saturating binding, the distribution of UmuD'(2)C complexes along RecA-ssDNA filaments was characterized by immuno-gold labelling with anti-UmuC antibodies. These data revealed preferential binding at filament ends (most likely, at one end). End specific binding is consistent with genetic models whereby such binding positions the UmuD'(2)C complex (pol V) appropriately for its role in SOS mutagenesis. PMID- 10731414 TI - Highly specific localization of promoter regions in large genomic sequences by PromoterInspector: a novel context analysis approach. AB - We present a new algorithm called PromoterInspector to locate eukaryotic polymase II promoter regions in large genomic sequences with a high degree of specificity. PromoterInspector focuses on the genetic context of promoters, rather than their exact location. Application of PromoterInspector can serve as a crucial pre processing step for other methods to locate exactly, or to analyze promoters. PromoterInspector does not depend on heuristics, because it is purely based on libraries of IUPAC words extracted from training sequences by an unsupervised learning approach. We compared PromoterInspector to in silico promoter prediction tools using the sequences from the review by J.W. Fickett. PromoterInspector compared favourably on Fickett's evaluation scheme. A true positive to false positive ratio of 2.3 was obtained, surpassing the best ratio of 0.6, reported for TSSG. The application of our method to several large genomic sequences of over 1.3 million base-pairs in total resulted in even more specific predictions. The coverage of annotated promoters was comparable to other in silico promoter prediction methods, while the true positive predictions increased by up to 100% of total matches. PromoterInspector scans 100 kb in less than one minute on a workstation, and thus is especially applicable for large genome analysis. The method is available at http://genomatix.gsf. de/cgi bin/promoterinspector/promoterinspector.pl. PMID- 10731415 TI - Influence of peroxisome proliferator-activated receptor alpha on ubiquinone biosynthesis. AB - The control of ubiquinone biosynthesis by peroxisome proliferators was investigated using peroxisome proliferator activated receptor alpha (PPARalpha) null mice. Administration of 2-(diethylhexyl)phthalate to control mice resulted in elevated ubiquinone levels in the liver, while dolichol, dolichyl-P and cholesterol concentrations remained unchanged. In PPARalpha-null mice, the level of these lipids were similar to control levels and administration of the peroxisome proliferator did not increase the levels of ubiquinone. The increase in ubiquinone levels was the result of increased synthesis. Induction was most pronounced in liver, kidney and heart, which have relatively high levels of PPARalpha. When the tissue concentration of hydrogen peroxide was elevated by inhibition of catalase activity with aminotriazole, the amount of ubiquinone was not increased, suggesting that the induction of ubiquinone synthesis occured through a direct mechanism. The activities of branch-point enzymes FPP-synthase, squalene synthase, cis-prenyltransferase, trans-prenyltransferase and NPHB transferase were substantially increased in control but not in PPARalpha-null mice after treatment with peroxisome proliferators. These data suggest that the induction of ubiquinone biosynthesis after administration of peroxisome proliferators is dependent on the PPARalpha through regulation of some of the mevalonate pathway enzymes. PMID- 10731416 TI - Visualization of the maturation transition in bacteriophage P22 by electron cryomicroscopy. AB - Large-scale conformational transitions are involved in the life-cycle of many types of virus. The dsDNA phages, herpesviruses, and adenoviruses must undergo a maturation transition in the course of DNA packaging to convert a scaffolding containing precursor capsid to the DNA-containing mature virion. This conformational transition converts the procapsid, which is smaller, rounder, and displays a distinctive skewing of the hexameric capsomeres, to the mature virion, which is larger and more angular, with regular hexons. We have used electron cryomicroscopy and image reconstruction to obtain 15 A structures of both bacteriophage P22 procapsids and mature phage. The maturation transition from the procapsid to the phage results in several changes in both the conformations of the individual coat protein subunits and the interactions between neighboring subunits. The most extensive conformational transformation among these is the outward movement of the trimer clusters present at all strict and local 3-fold axes on the procapsid inner surface. As the trimer tips are the sites of scaffolding binding, this helps to explain the role of scaffolding protein in regulating assembly and maturation. We also observe DNA within the capsid packed in a manner consistent with the spool model. These structures allow us to suggest how the binding interactions of scaffolding and DNA with the coat shell may act to control the packaging of the DNA into the expanding procapsids. PMID- 10731417 TI - A dimeric DNA interface stabilized by stacked A.(G.G.G.G).A hexads and coordinated monovalent cations. AB - We report on the identification of an A.(G.G.G.G).A hexad pairing alignment which involves recognition of the exposed minor groove of opposing guanines within a G.G.G.G tetrad through sheared G.A mismatch formation. This unexpected hexad pairing alignment was identified for the d(G-G-A-G-G-A-G) sequence in 150 mM Na(+) (or K(+)) cation solution where four symmetry-related strands align into a novel dimeric motif. Each symmetric half of the dimeric "hexad" motif is composed of two strands and contains a stacked array of an A.(G.G.G.G).A hexad, a G.G.G.G tetrad, and an A.A mismatch. Each strand in the hexad motif contains two successive turns, that together define an S-shaped double chain reversal fold, which connects the two G-G steps aligned parallel to each other along adjacent edges of the quadruplex. Our studies also establish a novel structural transition for the d(G-G-A-G-G-A-N) sequence, N=T and G, from an "arrowhead" motif stabilized through cross-strand stacking and mismatch formation in 10 mM Na(+) solution (reported previously), to a dimeric hexad motif stabilized by extensive inter-subunit stacking of symmetry-related A.(G.G.G.G).A hexads in 150 mM Na(+) solution. Potential monovalent cation binding sites within the arrowhead and hexad motifs have been probed by a combination of Brownian dynamics and unconstrained molecular dynamics calculations. We could not identify stable monovalent cation-binding sites in the low salt arrowhead motif. By contrast, five electronegative pockets were identified in the moderate salt dimeric hexad motif. Three of these are involved in cation binding sites sandwiched between G.G.G. G tetrad planes and two others, are involved in water-mediated cation binding sites spanning the unoccupied grooves associated with the adjacent stacked A.(G.G.G.G).A hexads. Our demonstration of A.(G. G.G.G).A hexad formation opens opportunities for the design of adenine-rich G-quadruplex-interacting oligomers that could potentially target base edges of stacked G.G.G.G tetrads. Such an approach could complement current efforts to design groove-binding and intercalating ligands that target G-quadruplexes in attempts designed to block the activity of the enzyme telomerase. PMID- 10731418 TI - The crystal structure of thymidylate synthase from Pneumocystis carinii reveals a fungal insert important for drug design. AB - Thymidylate synthase from Pneumocystis carinii (PcTS) is an especially important drug target, since P. carinii is a fungus that causes opportunistic pneumonia infections in immune-compromised patients and is among the leading causes of death of AIDS patients. Thymidylate synthase (TS) is the sole enzyme responsible for the de novo production of deoxythymidine monophosphate and hence is crucial for DNA replication in every organism. Inhibitors selective for P. carinii TS over human TS would be greatly beneficial in combating this disease. The crystal structure of TS from P. carinii bound to its substrate, dUMP, and a cofactor mimic, CB3717, was determined to 2.6 A resolution. A comparison with other species of TS shows that the volume of the closed PcTS active-site is 20 % larger than that of five other TS closed active-sites. A two-residue proline insert that is strictly conserved among all fungal species of TS, and a novel C-terminal closing interaction involving a P. carinii-specific tyrosine residue are primarily responsible for this increase in volume. The structure suggests several options for designing an inhibitor specific to PcTS and avoiding interactions with human TS. Taking advantage of the residue substitutions of P. carinii TS over human TS enables the design of a selective inhibitor. Additionally, the larger volume of the active-site of PcTS is an important advantage for designing de novo inhibitors that will exclude the human TS active-site through steric hindrance. PMID- 10731419 TI - The crystal structure of dihydrofolate reductase from Thermotoga maritima: molecular features of thermostability. AB - Two high-resolution structures have been obtained for dihydrofolate reductase from the hyperthermophilic bacterium Thermotoga maritima in its unliganded state, and in its ternary complex with the cofactor NADPH and the inhibitor, methotrexate. While the overall fold of the hyperthermophilic enzyme is closely similar to monomeric mesophilic dihydrofolate reductase molecules, its quaternary structure is exceptional, in that T. maritima dihydrofolate reductase forms a highly stable homodimer. Here, the molecular reasons for the high intrinsic stability of the enzyme are elaborated and put in context with the available data on the physical parameters governing the folding reaction. The molecule is extremely rigid, even with respect to structural changes during substrate binding and turnover. Subunit cooperativity can be excluded from structural and biochemical data. Major contributions to the high intrinsic stability of the enzyme result from the formation of the dimer. Within the monomer, only subtle stabilizing interactions are detectable, without clear evidence for any of the typical increments of thermal stabilization commonly reported for hyperthermophilic proteins. The docking of the subunits is optimized with respect to high packing density in the dimer interface, additional salt-bridges and beta sheets. The enzyme does not show significant structural changes upon binding its coenzyme, NADPH, and the inhibitor, methotrexate. The active-site loop, which is known to play an important role in catalysis in mesophilic dihydrofolate reductase molecules, is rearranged, participating in the association of the subunits; it no longer participates in catalysis. PMID- 10731420 TI - Crystal structures of Urtica dioica agglutinin and its complex with tri-N acetylchitotriose. AB - Urtica dioica agglutinin is a small plant lectin that binds chitin. We purified the isolectin VI (UDA-VI) and crystal structures of the isolectin and its complex with tri-N-acetylchitotriose (NAG3) were determined by X-ray analysis. The UDA-VI consists of two domains analogous to hevein and the backbone folding of each domain is maintained by four disulfide bridges. The sequence similarity of the two domains is not high (42 %) but their backbone structures are well superimposed except some loop regions. The chitin binding sites are located on the molecular surface at both ends of the dumbbell-shape molecule. The crystal of the NAG3 complex contains two independent molecules forming a protein-sugar 2:2 complex. One NAG3 molecule is sandwiched between two independent UDA-VI molecules and the other sugar molecule is also sandwiched by one UDA-VI molecule and symmetry-related another one. The sugar binding site of N-terminal domain consists of three subsites accommodating NAG3 while two NAG residues are bound to the C-terminal domain. In each sugar-binding site, three aromatic amino acid residues and one serine residue participate to the NAG3 binding. The sugar rings bound to two subsites are stacked to the side-chain groups of tryptophan or histidine and a tyrosine residue is in face-to-face contact with an acetylamino group, to which the hydroxyl group of a serine residue is hydrogen-bonded. The third subsite of the N-terminal domain binds a NAG moiety with hydrogen bonds. The results suggest that the triad of aromatic amino acid residues is intrinsic in sugar binding of hevein-like domains. PMID- 10731421 TI - Plasminogen activator inhibitor 1. Structure of the native serpin, comparison to its other conformers and implications for serpin inactivation. AB - The crystal structure of a constitutively active multiple site mutant of plasminogen activator inhibitor 1 (PAI-1) was determined and refined at a resolution of 2.7 A. The present structure comprises a dimer of two crystallographically independent PAI-1 molecules that pack by association of the residues P6 to P3 of the reactive centre loop of one molecule (A) with the edge of the main beta-sheet A of the other molecule (B).Thus, the reactive centre loop is ordered for molecule A by crystal packing forces, while for molecule B it is unconstrained by crystal packing contacts and is disordered. The overall structure of active PAI-1 is similar to the structures of other active inhibitory serpins exhibiting as the major secondary structural feature a five-stranded beta sheet A and an intact proteinase-binding loop protruding from the one end of the elongated molecule. No preinsertion of the reactive centre loop is observed in this structure.A comparison of the present structure with the previously determined crystal structures of PAI-1 in its alternative conformations reveals that, upon cleavage of an intact form of PAI-1 or formation of latent PAI-1, the well-characterised rearrangements of the serpin secondary structural elements are accompanied by dramatic and partly unexpected conformational changes of helical and loop structures proximal to beta-sheet A. The present structure explains the stabilising effects of the mutated residues, reveals the structural cause for the observed spectroscopic differences between active and latent PAI-1, and provides new insights into possible mechanisms of stabilisation by its natural binding partner, vitronectin. PMID- 10731422 TI - The BPTI decamer observed in acidic pH crystal forms pre-exists as a stable species in solution. AB - Bovine pancreatic trypsin inhibitor (BPTI) crystallizes under acidic pH conditions in the presence of thiocyanate, chloride and sulfate ions, yielding three different polymorphs in P2(1), P6(4)22 and P6(3)22 space groups, respectively. In all three crystal forms, the same decamer is found in the packing (ten BPTI molecules organized through two perpendicular 2-fold and 5-fold axes as a well-defined and compact object) in contrast to the monomeric crystal forms observed at basic pH conditions. The crystallization of BPTI under acidic conditions (pH 4.5) was investigated by small angle X-ray scattering with both under- and supersaturated BPTI solutions. Data showed the oligomerization of BPTI molecules under all investigated conditions. Accordingly, various mixtures of discrete oligomers (n=1 to 10) were considered. Calculated scattering curves were obtained using models based on the crystallographic structures, and the experimental patterns were analyzed as a linear combination of the model curves using a non-linear curve fitting procedure. The results, confirmed by gel filtration experiments, unambiguously demonstrate the co-existence of two different BPTI particles in solution: a monomer and a decamer, with no evidence of any other intermediates. Moreover, using both approaches, the fraction of decamers was found to increase with increasing salt concentration, even beyond the solubility curve. We therefore propose that at acidic pH, BPTI crystallizes following a two step process: decamers are first built in under- and supersaturated solutions, upon which crystal growth proceeds by decamer stacking. Indeed, those BPTI crystals should best be described as "BPTI decamer" crystals. PMID- 10731423 TI - The ultrahigh resolution crystal structure of ribonuclease A containing an isoaspartyl residue: hydration and sterochemical analysis. AB - Crystals of the deamidated form of bovine pancreatic ribonuclease which contains an isoaspartyl residue in position 67 diffract to 0. 87 A at 100 K. We have refined the crystallographic model using anisotropic displacement parameters for all atoms to a conventional crystallographic residual R=0.101 for all observed reflections in the resolution range 61.0-0.87 A. The ratio observations/parameters is 7.2 for the final model. This structure represents one of the highest resolution protein structures to date and interestingly, it is the only example containing more than one molecule in the asymmetric unit with a resolution better than 1.0 A. The non-crystallographic symmetry has been used as a validation check of the geometrical parameters and it has allowed an estimate for an upper limit of errors associated with this high resolution model. In the present structure it was possible to obtain a more accurate picture of the active site whose electron density was not clearly interpretable in the previous 1.9 A resolution structure. In particular, the P1 site is alternatively occupied either by a sulphate anion or by a water molecule network. Most of hydrogen atoms were visible in the electron density maps, including those involved in C(alpha) H(alpha).O interactions. Analysis of protein-solvent interactions has revealed the occurrence of an extensive cluster of water molecules, predominantly arranged in pentagonal fused rings and surrounding hydrophobic moiety of side-chains. Finally, in spite of the limited sample of residues, we have detected a clear dependence of backbone N-C(alpha)-C angle on residue conformation. This correlation can be fruitfully used as a valuable tool in protein structure validation. PMID- 10731424 TI - Measurements of cysteine reactivity during protein unfolding suggest the presence of competing pathways. AB - Evidence that proteins may unfold utilizing complex competing pathways comes from a new pulse-labeling protocol in which the change in reactivity of a single cysteine residue in a protein during unfolding is measured, making use of its easily monitored reaction with the Ellman reagent, dithionitrobenzoic acid. The kinetics of unfolding of two single cysteine-containing mutant forms of the small protein barstar, C82A, which contains only Cys40, and C40A, which contains only Cys82, have been studied. The data suggest that unfolding occurs via two parallel pathways, each forming competing intermediates. In one of these early intermediates, Cys40 and Cys82 are already as reactive as they are in the fully unfolded protein, while in the other intermediate, the Cys thiol groups are unreactive. One more long-lived intermediate also needs to be included on the pathway defined by the early intermediate with unreactive Cys thiol groups to account for the difference in the rates of fluorescence change and of change in Cys40 reactivity. The demonstration of multiple intermediates and pathways for unfolding indicates that protein unfolding reactions can be as complex as protein folding reactions. PMID- 10731425 TI - A new potent calmodulin antagonist with arylalkylamine structure: crystallographic, spectroscopic and functional studies. AB - An arylalkylamine-type calmodulin antagonist, N-(3, 3-diphenylpropyl)-N'-[1-R-(3, 4-bis-butoxyphenyl)ethyl]-propylene-diamine (AAA) is presented and its complexes with calmodulin are characterized in solution and in the crystal. Near-UV circular dichroism spectra show that AAA binds to calmodulin with 2:1 stoichiometry in a Ca(2+)-dependent manner. The crystal structure with 2:1 stoichiometry is determined to 2.64 A resolution. The binding of AAA causes domain closure of calmodulin similar to that obtained with trifluoperazine. Solution and crystal data indicate that each of the two AAA molecules anchors in the hydrophobic pockets of calmodulin, overlapping with two trifluoperazine sites, i.e. at a hydrophobic pocket and an interdomain site. The two AAA molecules also interact with each other by hydrophobic forces. A competition enzymatic assay has revealed that AAA inhibits calmodulin-activated phosphodiesterase activity at two orders of magnitude lower concentration than trifluoperazine. The apparent dissociation constant of AAA to calmodulin is 18 nM, which is commensurable with that of target peptides. On the basis of the crystal structure, we propose that the high-affinity binding is mainly due to a favorable entropy term, as the AAA molecule makes multiple contacts in its complex with calmodulin. PMID- 10731426 TI - Domain swapping in the sporulation response regulator Spo0A. AB - Adaptive responses of micro-organisms, such as chemotaxis and sporulation, are governed by two-component systems consisting of sensor kinases, that interpret environmental signals, and response regulators which activate the appropriate physiological responses. Signal transduction via response regulator proteins is mediated through transient phosphorylation of aspartic acid residues. In Spo0A, the key regulator of development (sporulation) in Bacillus, phosphorylation of the N-terminal receiver domain (N-Spo0A) at aspartate-55 switches on the transcription activation functions residing in the C-terminal effector domain. Here we report the crystal structure of N-Spo0A from Bacillus stearothermophilus at 1.6 A spacing, revealing a dimer formed by an alpha-helix swap. Comparison of this structure with the recently described structure of phosphorylated N-Spo0A shows that dimer formation results from a cis-trans isomerization of the Lys106- Pro107 peptide bond. The quaternary reorganization is associated with alterations in the active site stereochemistry which may have implications for signalling. Remarkably, this 3-D domain swapped N-Spo0A dimer has an identical topology to a hypothetical CheY-like dimer, recently proposed as an intermediate in the evolution of the family of periplasmic substrate binding proteins. PMID- 10731427 TI - Solution structure of hanatoxin1, a gating modifier of voltage-dependent K(+) channels: common surface features of gating modifier toxins. AB - The three-dimensional structure of hanatoxin1 (HaTx1) was determined by using NMR spectroscopy. HaTx1 is a 35 amino acid residue peptide toxin that inhibits the drk1 voltage-gated K(+) channel not by blocking the pore, but by altering the energetics of gating. Both the amino acid sequence of HaTx1 and its unique mechanism of action distinguish this toxin from the previously described K(+) channel inhibitors. Unlike most other K(+) channel-blocking toxins, HaTx1 adopts an "inhibitor cystine knot" motif and is composed of two beta-strands, strand I for residues 19-21 and strand II for residues 28-30, connected by four chain reversals. A comparison of the surface features of HaTx1 with those of other gating modifier toxins of voltage-gated Ca(2+) and Na(+) channels suggests that the combination of a hydrophobic patch and surrounding charged residues is principally responsible for the binding of gating modifier toxins to voltage gated ion channels. PMID- 10731428 TI - Two-state expansion and collapse of a polypeptide. AB - The initial phase of folding for many proteins is presumed to be the collapse of the polypeptide chain from expanded to compact, but still denatured, conformations. Theory and simulations suggest that this collapse may be a two state transition, characterized by barrier-crossing kinetics, while the collapse of homopolymers and random heteropolymers is continuous and multi-phasic. A new rapid-mixing flow technique has been used to resolve the late stages of polypeptide collapse, at time scales >/=45 microseconds. We have used a laser temperature-jump with fluorescence spectroscopy to resolve the complete time course of the collapse of denatured cytochrome c with nanosecond time resolution. We find the process to be exponential in time and thermally activated, with an apparent activation energy approximately 9 k(B)T (after correction for solvent viscosity). These results indicate that polypeptide collapse is kinetically a two state transition. Because of the observed free energy barrier, the time scale of polypeptide collapse is dramatically slower than is predicted by Langevin models for homopolymer collapse. PMID- 10731429 TI - Fast folding of Escherichia coli cyclophilin A: a hypothesis of a unique hydrophobic core with a phenylalanine cluster. AB - Escherichia coli cyclophilin A, a 164 residue globular protein, shows fast and slow phases of refolding kinetics from the urea-induced unfolded state at pH 7.0. Given that the slow phases are independent of the denaturant concentration and may be rate-limited by cis/trans isomerizations of prolyl peptide bonds, the fast phase represents the true folding reaction. The extrapolation of the fast-phase rate constant to 0 M urea indicates that the folding reaction of cyclophilin A is extraordinarily fast and has about 700 s(-1) of the rate constant. Interrupted refolding experiments showed that the protein molecules formed in the fast phase had already been fully folded to the native state. This finding overthrows the accepted view that the fast folding is observed only in small proteins of fewer than 100 amino acid residues. Examination of the X-ray structure of cyclophilin A has shown that this protein has only one unique hydrophobic core (phenylalanine cluster) formed by evolutionarily conserved phenylalanine residues, and suggests that this architecture of the molecule may be responsible for the fast folding behavior. PMID- 10731430 TI - Infrared dichroism of twisted beta-sheet barrels. The structure of E. coli outer membrane proteins. AB - The infrared dichroic ratios of the amide bands from oriented beta-barrels yield an experimental value for the mean orientation, beta, of the beta-strands, relative to the barrel axis. For a barrel of n strands, this then gives the shear number, S, that characterizes the stagger of the beta-sheet. Combining values of beta and n specifies the barrel geometry by using the optimized model of Murzin, Lesk & Chothia for regular barrels. Application to published infrared data on the Escherichia coli outer membrane protein, OmpA yields S=9-10 (n=8), a barrel radius of 0.81(+/-0.01) nm, and an internal free volume of 0.031 nm(3) per residue, where the average twist of the beta-sheets is theta approximately 28 degrees, and their coiling angle is epsilon approximately 1 degrees. Hydrophobic matching of the 2.6 nm transmembrane stretch partly determines the shear number of the OmpA beta-barrel. PMID- 10731431 TI - Cyclophilin-promoted folding of mouse dihydrofolate reductase does not include the slow conversion of the late-folding intermediate to the active enzyme. AB - Cyclophilins accelerate slow protein folding reactions in vitro by catalyzing the cis/trans isomerization of peptidyl-prolyl bonds. Cyclophilins were reported to be involved in a variety of cellular functions, including the promotion of protein folding by use of the substrate mouse dihydrofolate reductase (DHFR). The interaction of cyclophilin with DHFR has only been studied under limited conditions so far, not taking into account that native DHFR exists in equilibrium with a non-native late-folding intermediate. Here we report a systematic analysis of catalysis of DHFR folding by cyclophilins. The specific ligand methotrexate traps DHFR in its native state, permitting a specific analysis of the action of cyclophilin on both denatured DHFR with non-native prolyl bonds and denatured DHFR with all-native prolyl bonds. Cyclophilins from yeast and Neurospora crassa as well as the related prolyl isomerase b from Escherichia coli promote the folding of different forms of DHFR to the enzymatically active form, demonstrating the generality of cyclophilin-catalyzed folding of DHFR. The slow equilibrium between the late-folding intermediate and native DHFR suggests that prolyl isomerization may be required for this final phase of conversion to native DHFR. However, by reversible trapping of the intermediate, we analyze the slow interconversion between native and late-folding conformations in the backward and forward reactions and show a complete independence of cyclophilin. We conclude that cyclophilin catalyzes folding of DHFR, but surprisingly not in the last slow folding step. PMID- 10731433 TI - Radovan H. Zak 1931-1999 PMID- 10731432 TI - Insights into ligand-induced conformational change in Cel5A from Bacillus agaradhaerens revealed by a catalytically active crystal form. AB - Glycoside hydrolases are ubiquitous enzymes involved in a diverse array of biological processes, from the breakdown of biomass, through to viral invasion and cellular signalling. Endoglucanase Cel5A from Bacillus agaradhaerens, classified into glycoside hydrolase family 5, has been studied in a catalytically inactive crystal form at low pH conditions, in which native and complex structures revealed the importance of ring distortion during catalysis. Here, we present the structure of Cel5A in a new crystal form obtained at higher pH values in which the enzyme is active "in-crystal". Native, cellotriosyl-enzyme intermediate and beta-d-cellobiose structures were solved at 1.95, 1.75 and 2.1 A resolution, respectively. These structures reveal two classes of conformational change: those caused by crystal-packing and pH, with others induced upon substrate binding. At pH 7 a histidine residue, His206, implicated in substrate binding and catalysis, but previously far removed from the substrate-binding cleft, moves over 10 A into the active site cleft in order to interact with the substrate in the +2 subsite. Occupation of the -1 subsite by substrate induces a loop closure to optimise protein-ligand interactions. Cel5A, along with the unrelated family 45 and family 6 cellulases, provides further evidence of substantial conformational change in response to ligand binding for this class of hydrolytic enzyme. PMID- 10731434 TI - Myocardial oxygen consumption modulates adenosine formation by canine right ventricle in absence of hypoxia. AB - Myocardial adenosine formation varies with myocardial oxygen consumption (MVO(2)), but whether concurrent hypoxia is required for adenosine formation is uncertain. Changes in right coronary (RC) perfusion pressure (RCP) produce directionally similar alterations in right ventricular (RV) MVO(2)and in RC venous P O(2)(P(v)O(2)), an index of myocardial P O(2). RCP was varied in 10 anesthetized, open chest dogs to determine if, under these conditions, RV formation of adenosine would increase with MVO(2)in absence of myocardial hypoxia. Dialysis probes were implanted in the mid myocardium of RV free wall for collecting dialysate samples for HPLC analyses to estimate interstitial adenosine and other purines. Coronary venous blood was sampled from a superficial vein draining the RC artery (RCA) perfusion territory. At 115+/-3 mmHg baseline RCP, RC blood flow (RCBF)=0.51+/-0.04 ml/min/g, MVO(2)=4.6+/-0.5 ml/min/100 g, P(v)O(2)=34+/-1.5 mmHg, and dialysate adenosine=0. 27+/-0.03microM. When RCP was lowered to 61+/-1 mmHg by adjusting an occluder on the proximal RCA, RCBF decreased to 0.36+/-0.03 ml/min/g, MVO(2)fell to 3.7+/-0.4 ml/min/100 g, lactate uptake remained positive, P(v)O(2)fell to 30+/-1.7 mmHg, and dialysate adenosine decreased to 0.20+/-0.03microM. Reactive hyperemia of 1.25+/-0.13 ml/min/g was observed when the RCA constriction was released, although dialysate adenosine had fallen. When RCP was elevated to 164+/-2 mmHg by inflating a balloon catheter in the descending aorta, RCBF increased to 0.70+/-0.06 ml/min/g, MVO(2)increased to 5.8+/-1. 0 ml/min/100 g, P(v)O(2)rose to 39+/-2.3 mmHg, and dialysate adenosine increased to 0.33+/-0.04microM. These data indicate that (1) RV oxygen demand varies with RCP; (2) if RV ischemia is absent, myocardial adenosine formation is modulated by MVO(2), with no requirement for hypoxia; (3) pressure-flow autoregulation is relatively ineffective in the RC circulation, where adenosine does not mediate and may even blunt autoregulation. PMID- 10731435 TI - Changes in ultrastructural calcium distribution in goat atria during atrial fibrillation. AB - It has been suggested that Ca(2+)content of atrial cardiomyocytes is increased at the onset of atrial fibrillation (AF). Whether this phenomenon is transient is currently unknown. Therefore, in this study the time-related changes in Ca(2+)location in atrial myocytes from goats with chronic AF have been investigated. The distribution of calcium was assessed with the electron microscope using the cytochemical phosphate-pyroantimonate and oxalate pyroantimonate methods in atrial biopsies from goats in sinus rhythm and goats with 1-16 weeks of burst-pacing-induced AF. In atrial myocytes from control goats in sinus rhythm, a normal Ca(2+)distribution was observed, with regular deposits along the sarcolemma (an average of 3.4 deposits per microm at a regular distance). The number of sarcolemma-bound Ca(2+)deposits substantially increased after 1 and 2 weeks of atrial fibrillation. After this period the amount of Ca(2+)precipitate decreased at 4 and 8 weeks, and became below control level at 16 weeks. A similar time-related redistribution of Ca(2+)occurred in mitochondria. Whereas mitochondria from control goats displayed very few Ca(2+)deposits (average 4.0 deposits per micro m(2)), their number markedly increased after 1 and 2 weeks of atrial fibrillation, which indicates cellular Ca(2+)overload. From 4 weeks, Ca(2+)deposits reached control levels and were below control level after 16 weeks of atrial fibrillation (2.5 deposits per microm(2)). Our findings are consistent with the previously observed Ca(2+)overload early after the onset of atrial fibrillation. The present study shows that this overload persists for at least 2 weeks, after which the cardiomyocytes apparently adapt to a new Ca(2+)homeostasis, thereby avoiding Ca(2+)overload. This protection against Ca(2+)overload co-occurs with dedifferentiation like cellular remodeling. PMID- 10731436 TI - Decreased left ventricular ejection fraction in transgenic mice expressing mutant cardiac troponin T-Q(92), responsible for human hypertrophic cardiomyopathy. AB - The causality of mutant sarcomeric proteins in hypertrophic cardiomyopathy (HCM) is well established. The current emphasis is to elucidate the pathogenesis of HCM in transgenic animal models. We determined the left ventricular ejection fraction (LVEF) in transgenic mice expressing mutant cardiac troponin T (cTnT)-Q(92), known to cause HCM in humans. Transgenes were constructed by placing wild-type (R(92)) or mutant (Q(92)) full-length human cTnT cDNAs 3' into a 5.5-kb murine [alpha -myosin heavy chain (MyHC)] promoter injected into fertilized zygotes. Three wild-type and six mutant lines were produced. Transgene mRNA and proteins, detected using transgene-specific probes were expressed at high levels in all wild-type and three mutant lines. The total cTnT mRNA pool was increased by up to five-fold in transgenic mice, but the total cTnT protein remained unchanged. The mean values of LVEF, determined by(178)Ta radionuclide angiography, were 57.8+/ 6% (n=4) in non-transgenic littermate (NLM), 53.3+/-10 (n=6) in wild-type and 39. 4+/-6 (n=5) in mutant transgenic mice (P=0.009). The heart/body weight ratios and the number of cells stained with terminal deoxynucleotidyl transferase (TdT) mediated nick end-labeling were similar among the groups. Three mutant mice had myocyte disarray and excess interstitial collagen and two had normal myocardial structure despite having reduced LVEF. Thus, in vivo expression of the mutant cTnT-Q(92)protein, responsible for human HCM, impaired global cardiac systolic function in transgenic mice, which also occurred in the absence of myocyte disarray and increased interstitial collagen. PMID- 10731437 TI - Nitric oxide inhibition improved myocardial metabolism independent of tissue perfusion during ischemia but not during reperfusion. AB - Nitric oxide (NO) is one of the important regulators of cardiac metabolism and function as well as of tissue perfusion. Myocardial NO formation is increased during ischemia and reperfusion. We investigated the roles of endogenous NO in myocardial metabolism during ischemia and reperfusion independent of tissue perfusion changes. In an open-chest pig model, a bolus infusion of 20 mg/kg of N(G)-nitro l -arginine methyl ester (l -NAME), a NO synthase inhibitor, did not alter the regional myocardial perfusion compared with a control saline injection, as measured by colored microsphares. Using(31)P-nuclear magnetic resonance spectroscopy, we showed that the tissue levels of pH and adenosine triphosphate (ATP) but not those of creatine phosphate were significantly preserved in the l NAME group compared with the placebo group during the subsequent 15-min regional ischemia. Thus, l -NAME reduced myocardial ATP utilization during ischemia, and the mechanism underlying these effects is independent of tissue perfusion changes. However, l -NAME did not accelerate the recovery of ATP levels following reperfusion, suggesting distinct roles of endogenous NO during reperfusion. PMID- 10731438 TI - Telomerase activity in rat cardiac myocytes is age and gender dependent. AB - Telomerase replaces telomeric repeat DNA lost during the cell cycle, restoring telomere length. This enzyme is present only during cell replication and its activity reflects the extent of proliferation. Whether cardiac myocytes are terminally differentiated cells is still a highly controversial issue, and the possibility of myocyte division is frequently rejected. On this basis, telomerase was measured in pure preparations of myocytes, isolated from rats throughout their lifespan. Fetal and neonatal rat myocytes were used as positive control cells. Contrary to expectation, the authors report that telomerase activity was detectable in pure preparations of young adult, fully mature adult, and senescent ventricular myocytes, defeating the dogma that this cell population is permanent and irreplaceable. Aging decreased 31% telomerase activity in male myocytes. An opposite effect occurred in female myocytes in which this enzyme increased 72%. This differential adaptation between the two genders in the rat model may be relevant to observations in humans; myocyte loss occurs in men as a function of age, whereas myocyte number is preserved in women. The greater growth potential of female myocytes may be critical for the longer lifespan and decreased incidence of heart failure in women. PMID- 10731439 TI - Differential regulation of vascular smooth muscle nuclear factor kappa-B by G alpha q-coupled and cytokine receptors. AB - NF kappaB has been implicated as a downstream effector of G alphaq-coupled receptor signaling, but whether these and cytokine receptors activate NF kappaB similarly remains unclear. Stimulation of rat vascular smooth muscle cell G alphaq-coupled P2Y nucleotide receptors with UTP induces luciferase transcription from a sensitive and specific NF kappaB dependent promoter. However, these responses are only;15% of that to the reference cytokine IL-1 beta. IL-1 beta is a powerful stimulator of I kappaB alpha degradation, RelA nuclear import, and isoform specific NF kappaB enhancer binding in vitro, responses that are not detectable after P2Y receptor stimulation. Expression of two trans -dominant NF kappaB polypeptides suppresses induction of the NF kappaB reporter and also IL-1 beta stimulated monocyte chemoattractant-1 mRNA, which is not induced by UTP. In contrast, UTP induces higher expression of the endogenous COX-2 and IL-6 mRNAs than does IL-1 beta, implying that G alphaq-coupled receptor evokes additional NF kappaB-independent transcription factors in regulating these two genes. P2Y receptors are as effective as the reference growth factor PDGF-BB at inducing CREB, AP-1, SRE and NFAT transcription, which are largely unaffected by IL-1 beta treatment. NF kappaB is less efficiently activated then several other transcriptional effectors of G alphaq-coupled receptor signaling in vascular smooth muscle cells, and is instead preferentially activated by inflammatory cytokines. PMID- 10731440 TI - Differential acetylcholine release mechanisms in the ischemic and non-ischemic myocardium. AB - To understand better the pathophysiological roles of the vagal efferent system in ischemic heart diseases, we examined endogenous acetylcholine (ACh) release in the myocardium in vivo. Acute myocardial ischemia was induced in anesthetized cats by a 60-min occlusion of the left anterior descending coronary artery (LAD). We implanted dialysis probes in the left ventricular free wall and measured the dialysate ACh concentration using liquid chromatography. In the ischemic region, the ACh level increased from 0.68+/-0.12 to 12.3+/-3.3 n M (mean+/-S.E., P<0.01) by LAD occlusion. Bilateral vagotomy did not inhibit ischemia-induced ACh release (20.3+/-6.4 n M). In vagotomized animals, inhibition of the N-type Ca(2+)channel by intravenous administration of omega-conotoxin GVIA (10microg/kg) also failed to suppress ACh release (15.9+/-2.0 n M). However, the inhibition of intracellular Ca(2+)mobilization by local administration of 3,4,5 trimethoxybenzoic acid 8-(dietyl amino)-octyl ester (1 m M) suppressed ACh release (4.4+/-0.8 n M, P<0.05 compared with no pharmacological intervention). In the non-ischemic region, the ACh level increased from 1.9+/-0.4 to 6. 0+/-1.0 n M (P<0.05) by LAD occlusion, which was completely abolished by vagotomy. We concluded that ACh release in the ischemic region was mainly attributed to a local release mechanism, whereas that in the non-ischemic region depended on the presence of intact vagal activity. The local release mechanism would depend on intracellular Ca(2+)mobilization but not on N-type Ca(2+)channel opening. PMID- 10731441 TI - Is a functional sarcoplasmic reticulum necessary for preconditioning? AB - Several studies have shown that the protective effect of ischemic preconditioning (PC) is associated with decreased calcium release from the sarcoplasmic reticulum (SR). However, no study has yet demonstrated whether these changes are essential in the mechanism of PC. In order to investigate whether a functional SR was necessary for PC, we manipulated SR calcium handling using (i) 0.1microM ryanodine (RY), a concentration known to lock the SR calcium release channel in the open state and (ii) 50microM cyclopiazonic acid (CPA), a specific inhibitor of the SR calcium ATPase. Initial experiments confirmed that both RY and CPA eliminated the ability of the SR to accumulate calcium. Isolated rat hearts (n=6 7/group) were perfused normoxically for 30 min prior to either a further 40 min of perfusion [control (C)] or 4x[5 min ischemia (I) + 5 min reperfusion (R)] (PC). All hearts were then subjected to a further 40 min I + 40 min R. The C and PC protocols were then repeated in the presence of RY or CPA, introduced after 10 min of perfusion.(31)P-NMR was used to measure ATP, PCr, P(i)and intracellular pH. RY and CPA decreased developed pressure (DP) by 75% and 59%, respectively. Percentage recovery of LVDP was significantly higher in PC (72+/-8%), PC+RY (72+/ 7%) and PC+CPA (49+/-7%) groups compared with their respective controls (43+/-7%, 47+/-7% and 10+/-4%) (P<0.05). Thus, PC remains protective in the presence of a SR unable to accumulate calcium, suggesting that the changes in SR calcium release are not essential in the mechanism of preconditioning. PMID- 10731442 TI - Tacrolimus limits polymorphonuclear leucocyte accumulation and protects against myocardial ischaemia- reperfusion injury. AB - Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the pathogenesis of either human and experimental myocardial ischaemia. Tacrolimus, formerly known as FK506, has been previously shown to display cardioprotective effects on experimental ischaemia/reperfusion-induced myocardial damage. This study investigated whether cardioprotection induced by tacrolimus in myocardial ischaemia-reperfusion (MI/R) injury might be due to inhibition of the nuclear factor kappa B (NF- kappaB) that in turn causes reduced cardiac ICAM-1 expression and blunted polymorphonuclear leukocyte accumulation. Anaesthetized rats were subjected to total occlusion (45 min) of the left main coronary artery followed by 5 h reperfusion (MI/R). Sham myocardial ischaemia-reperfusion rats (Sham MI/R) were used as controls. Myocardial necrosis, myocardial myeloperoxidase activity, serum creatine kinase (CK) activity, cardiac mRNA for ICAM-1 reverse transcriptase polymerase chain reaction, the inhibitory protein of NF- kappaB I kappaB alpha (Western blot analysis) in the myocardium-at-risk, and left ventricle d P/d t(max)were evaluated. Myocardial ischaemia plus reperfusion in untreated rats produced marked myocardial necrosis, increased serum CK activity and myeloperoxidase activity (MPO, a marker of leukocyte accumulation) both in the area at risk and in the necrotic area, and reduced the left ventricle dP/d t(max). Furthermore, inhibitory protein I kappaB alpha levels decreased, and cardiac mRNA for ICAM-1 increased, after 0.5 and 5 h of reperfusion, respectively. Administration of tacrolimus (25, 50 and 100microg/kg as an i.v. infusion 5 min after reperfusion) lowered myocardial necrosis and myeloperoxidase activity in the area at risk and in necrotic area, decreased serum CK activity, increased left ventricle dP/d t(max), reduced the loss the of inhibitory protein I kappaB alpha and blunted the message for ICAM-1. The present data suggest that tacrolimus blocks the early activation of the transcription factor NF- kappaB, suppresses ICAM-1 gene activation, reduces leukocyte accumulation and protects against myocardial ischaemia-reperfusion injury. PMID- 10731443 TI - Effect of calcium on reactive oxygen species in isolated rat cardiomyocytes during hypoxia and reoxygenation. AB - It has been suggested that calcium (Ca(2+)) overload and oxidative stress damage the myocardium during ischemia and reperfusion. We investigated the possible effect of varying extracellular Ca(2+)and total cell Ca(2+)on reactive oxygen species (ROS) levels in resting adult rat cardiomyocytes. Cardiomyocytes were isolated by trypsin/collagenase digestion and exposed to 1 h of hypoxia (H) (95% N(2)/5% CO(2), no glucose) and 2 h of reoxygenation (R) (95% air/5% CO(2), glucose 5.5 m M) in suspension. Cell Ca(2+)was measured by uptake of(45)Ca(2+). ROS was measured by flow cytometry of ethidium's red fluorescence formed by oxidation of dihydroethidium mostly by superoxide anion. Cell viability decreased during H and R, expressed as uptake of trypan blue, loss of rod shape morphology and release of lactate dehydrogenase. Rapidly exchangeable cell Ca(2+)was closely correlated with extracellular Ca(2+)concentration. Cell Ca(2+)was unchanged during H but increased three to four times after R. This increase was attenuated by adding 3,4-dichlorobenzamil, 10 microm at R, and amplified by adding ouabain 1 m M (from start), respectively. Levels of ROS in hypoxic cells were unchanged or slightly reduced at the end of H and increased significantly by 20% compared to control after R. Levels of ROS were significantly decreased by lowering total extracellular Ca(2+)from 1 m M to 0.1 m M or by decreasing free extracellular Ca(2+)with EGTA 0.9 m M at the onset of R. Keeping extracellular Ca(2+)constant, ROS levels were neither affected by attenuating the increase in cell Ca(2+)by DCB nor by amplifying the increase in cell Ca(2+)by ouabain. In conclusion, ROS (superoxide anion) levels increase rapidly after reoxygenation, are correlated with extracellular-free Ca(2+)and are reduced by lowering extracellular-free Ca(2+). Levels of ROS are apparently not consistently correlated with total cell Ca(2+). PMID- 10731444 TI - The expression of SR calcium transport ATPase and the Na(+)/Ca(2+)Exchanger are antithetically regulated during mouse cardiac development and in Hypo/hyperthyroidism. AB - The mouse has been used extensively for generating transgenic animal models to study cardiovascular disease. Recently, a number of transgenic mouse models have been created to investigate the importance of sarcoplasmic reticulum (SR) Ca(2+)transport proteins in cardiac pathophysiology. However, the expression and regulation of cardiac SR Ca(2+)ATPase and other Ca(2+)transport proteins have not been studied in detail in the mouse. In this study, we used multiplex RNase mapping analysis to determine SERCA2, phospholamban (PLB), and Na(+)/Ca(2+) exchanger (NCX-1) gene expression throughout mouse heart development and in hypo/hyperthyroid animals. Our results demonstrate that the expression of SERCA2 and PLB mRNA increase eight-fold from fetal to adult stages, indicating that SR function increases with heart development. In contrast, the expression of the Na(+)/Ca(2+)-exchanger gene is two-fold higher in fetal heart compared to adult. Our study also makes the important observation that in hypothyroidic hearts the NCX-1 mRNA and protein levels were upregulated, whereas the SERCA2 mRNA/protein levels were downregulated. In hyperthyroidic hearts, however, an opposite response was identified. These findings are important and point out that the expression of NCX-1 is regulated antithetically to that of SERCA2 during heart development and in response to alterations in thyroid hormone levels. PMID- 10731445 TI - Mechano-electric feedback in right atrium after left ventricular infarction in rats. AB - Left ventricular myocardial infarction (MI) can lead to alterations in hemodynamic load conditions, thereby inducing right atrial hypertrophy and dilatation associated with phenotypic modulation of cardiomyocytes, electrical abnormalities, rhythm disturbances, and atrial fibrillation. However, there is limited information on the electrophysiological basis for these events. We investigated whether atrial stretch in the setting of chronic MI modulates the electrophysiological properties of cardiomyocytes via "mechano-electric feedback", providing a mechanism for atrial arrhythmia after ventricular infarction. Five weeks after left ventricular MI (n=37), action potentials (AP) were measured in right atrial tissue preparations using a current clamp scheme, and compared to sham-operated rats (SO, n=10). Contractile activity was recorded at a preload of 1 mN, and sustained stretch was applied via a micrometer. In SO, stretch of 1.75 mN shortened repolarization at 50% and prolonged it at 90%. In MI, mechanically-induced electrical alterations were observed at a significantly lower level of stretch than in SO (0.19 mN). Sustained stretch in MI prolonged AP at 90% repolarization giving rise to stretch-activated depolarizations (SAD) near 90% repolarization (SAD90). When reaching threshold for premature APs, electrical phenomena similar to atrial fibrillations were seen in some preparations. Moreover, we observed APs with prolonged duration at 25%, 50%, and 90% repolarization where stretch induced SAD near 50%. Gadolinium used at a concentration to inhibit stretch-activated channels (40microM) suppressed mechanically-induced electrical events. In conclusion, increased susceptibility after MI to mechanical stretch may predispose atrial cardiomyocytes to arrhythmia. These mechano-electrical alterations are sensitive to gadolinium suggesting involvement of stretch-activated ion channels. PMID- 10731446 TI - Further evidence for the cardiac troponin C mediated calcium sensitization by levosimendan: structure-response and binding analysis with analogs of levosimendan. AB - Levosimendan, an inodilatory drug discovered using troponin C as a target protein, has a cardiac effect deriving from the calcium sensitization of contractile proteins. The aim of this study was to give further evidence that levosimendan binds to cardiac troponin C and that the binding involves amino acid residues on helixepsilon of the N-terminal domain of this calcium-binding protein. Nine organic molecules, obtained by chemical modification of levosimendan, were tested both for their calcium-dependent binding to troponin C and troponin complex affinity HPLC columns, and for their ability to increase the calcium sensitivity of myofilaments in cardiac skinned fibers. A good correlation between the calcium sensitization and the calcium-dependent binding to troponin complex (r=0.90) and to cardiac troponin C (r=0.91) for the analogs of levosimendan was shown. In addition, the effect of levosimendan on the calcium induced conformational changes in native and point-mutated cTnC was studied. Cys84-->Ser, Asp87-->Lys and Asp88-->Ala point-mutated cTnC were shown to maintain a high affinity to calcium, but their Ca(2+)titration curves were not influenced by levosimendan as for the native protein. Finally, it was demonstrated that the NMR chemical shifts of the terminal methyl groups of Met47, Met81, and Met85 on calcium-saturated cTnC were changed after addition of levosimendan in water solution at pH 7.4. This effect was not seen when adding an analog of levosimendan, which did not bind to the troponin C affinity HPLC column and did not increase the calcium-induced tension in cardiac skinned fibers. PMID- 10731447 TI - Consequences of inspired oxygen fraction manipulation on myocardial oxygen pressure, adenosine and lactate concentrations: a combined myocardial microdialysis and sensitive oxygen electrode study in pigs. AB - Adenosine is a potent vasodilator whose concentration has been shown to increase in cardiac tissue in response to hypoxia. However, the time-dependent relationship between the levels of myocardial interstitial adenosine and tissue oxygenation has not yet been completely established. Therefore, the purpose of this study was to investigate the complex relationship between tissue myocardial oxygen tension (PtiO(2)) and interstitial myocardial adenosine and lactate concentrations by developing a new technique which combines a cardiac microdialysis probe and a Clark-type P O(2)electrode. The combined and the single microdialysis probes were implanted in the left ventricular myocardium of anesthetized pigs. The consequences of the combined use of microdialysis and P O(2)probes on myocardial PtiO(2)and microdialysis performances against glucose were evaluated. A moderate but significant reduction in the relative recovery against glucose of the combined probe was observed when compared to that of the single microdialysis probe (42+/-2 v 32+/-1%, mean+/-S.E. M.n=5 P<0.05), at 2microl/min microdialysis probe perfusion flow. Similarly, myocardial oxygen enrichment, measured by the P O(2)electrode, was negligible when microdialysis probe perfusion flow was 2microl/min. Systemic hypoxia (FiO(2)=0.08) resulted in a significant decrease in PtiO(2)from 30+/-4 to 11+/-2 mmHg, limited increase in coronary blood flow (CBF), and a significant increase in myocardial adenosine and lactate concentrations from 0.34+/-0.05 to 0.98+/-0.06micromol/l and from 0.45+/ 0.05 to 0.97+/-0.06 mmol/l respectively (P<0.05). Increasing the FiO(2)to 0.3 restored the PtiO(2)and hemodynamic parameters to baseline values with no changes in interstitial adenosine and lactate concentrations. Nevertheless, myocardial interstitial adenosine remained significantly higher than baseline values. In conclusion, this study demonstrates the ability of a combined probe to measure simultaneously regional myocardial PtiO(2)and metabolite concentration during hypoxia. The hypoxia-induced increase in myocardial adenosine persists after correction of hypoxia. The physiological significance of this observation requires further studies. PMID- 10731448 TI - Location as a determinant of myocardial infarction in rabbits. AB - Determinants of infarct size in the rabbit heart include risk zone size, regional myocardial blood flow (RMBF), temperature and duration of ischemia. However, other factors might contribute, such as the location of the risk zone (apex to base), independent of known factors. Occlusion of a large marginal branch of the circumflex artery in the rabbit produces a risk region that typically comprises the entire apex of the left ventricle with decreasing area involvement from apex to base. In a retrospective study of 65 rabbit hearts (subjected to 30 min of coronary artery occlusion) which had been sliced into six to eight cross sectional slices, average area at risk (AR) comprised 86+/-3% of the apical level, 68+/-2% of the middle level and 39+/-2% near the site of occlusion at the base of the heart. If necrosis were dependent on AR alone, then infarct size (area of necrosis/area at risk, AN/AR) would not vary by site. However, AN/AR in the apex was 54+/-3% while AN/AR near the base was 27+/-2%, P<0.0001. To test if this salvage of tissue at risk near the base was due to differences in regional myocardial blood flow, we measured RMBF during occlusion in additional rabbits (n=4). Average RMBF in the risk zone was 0.025 ml/min/g in the apex and 0.010 in the base, P=N.S. Nor was the salvage due to differences in temperature. During occlusion, temperature in the risk zone (n=5) was 38.1 degrees C+/-0.3 in the apex and 38.4+/-0.2 in the base (P=N.S.). When we examined this phenomenon in hearts that received a non-pharmacological intervention that decreases overall infarct size (ischemic preconditioning) and in hearts that received a pharmacological intervention that decreases overall infarct size, a similar pattern of decreasing infarct size as a percentage of the area at risk from apex to base was observed. In conclusion, infarct size as a percentage of the AR depends on whether the AR is at the apex or base of the heart. A larger part of the AR undergoes necrosis toward the apex of the heart. This phenomenon is independent of collateral flow or temperature, and suggests that other as yet unknown factors contribute to infarct size. PMID- 10731449 TI - Palmitate-mediated alterations in the fatty acid metabolism of rat neonatal cardiac myocytes. AB - During ischemia and reperfusion, increased palmitate oxidation is associated with diminished function of the myocardium. Palmitate, but not oleate, has been implicated in the induction of apoptosis in isolated neonatal rat ventricular myocytes. We report that extended incubation (20 h) of cultured neonatal rat cardiomyocytes, in the presence of palmitate, causes a decrease in the ability of these cells to oxidize fatty acids, an increase in cellular malonyl-CoA and a decrease in the activity of 5' AMP-activated protein kinase (AMPK) compared to myocytes incubated in the presence of oleate. While palmitate decreases the oxidative metabolism of fatty acids, it increases the formation of intracellular triglyceride and ceramide. Increased ceramide formation is associated with an increase in apoptosis in many cell systems and we also observe an increase in caspase-3 like activity and DNA-laddering in these cells. At the onset of cardiac failure, a switch in myocardial substrate utilization from fatty acids to glucose occurs. Our data suggest that decreased palmitate oxidation in cardiac myocytes in culture may signal the initiation of programmed cell death and ceramide elevation previously documented in ischemic, reperfused hearts. PMID- 10731450 TI - Deletion in the cardiac troponin I gene in a family from northern Sweden with hypertrophic cardiomyopathy. AB - The cardiac troponin I gene has been described to be associated with hypertrophic cardiomyopathy. Until now, mutations in this gene have been found only in the Japanese population. We now present the first non-Japanese family, from northern Sweden, with a mutation in the cardiac troponin I gene. Clinical diagnose was based on echocardiography, with a maximum left ventricular wall thickness of >13 mm, or major electrocardiographic abnormalities, excluding subjects with other known causes of cardiac hypertrophy. Mutation screening was performed with a single-strand conformation polymorphism analysis and identification of mutation by direct DNA sequencing. We have identified a 33-bp deletion in exon 8 encompassing the stop codon. Nine individuals in three generations were tested, and four were carriers of this deletion. The mother was genetically affected and died of heart failure aged 90. Echocardiography at 71 years of age revealed no hypertrophy, but the electrocardiogram showed signs of left ventricular hypertrophy. Her two sons, also genetically affected, had left ventricular hypertrophy, with maximum wall thickness of 15 and 16 mm, respectively. One daughter and four grandchildren were clinically unaffected, but one of them, a 27 year-old woman with maximum wall thickness of 8 mm and normal electrocardiogram, was found to be genetically affected. In conclusion, we describe a non-Japanese family in which hypertrophic cardiomyopathy is due to a genetic defect in the cardiac troponin I gene. This mutation is a deletion of 33 bp in the last exon, whereas the previously described mutations in this gene are single nucleotide changes and a single codon deletion. The deletion of the C-terminal part of the cardiac troponin I protein, seems in this particular family to be associated with a mild phenotypic expression of familial hypertrophic cardiomyopathy. PMID- 10731451 TI - Endogenous and exogenous coronary vasodilatation are attenuated in cardiac hypertrophy: a morphological defect? AB - Reactive hyperaemia (RH) following brief ischaemia is reduced in hypertrophied hearts, and this may contribute to reduced coronary flow reserve. We studied vasodilatation during RH and in response to exogenous stimuli in control and hypertrophied hearts and explored the mechanisms underlying RH. Vascular reactivity was assessed in isolated hypertrophied hearts (55+/-3 days after aortic banding or sham operation) by constructing dose-response curves to acetylcholine (ACh), sodium nitroprusside (SNP) and adenosine. Reactive hyperaemic vasodilatation was assessed after global ischaemia (5-120 s) in the presence/absence of L -NAME, 8-phenyltheophylline (8-PT) and glibenclamide. Purine release and NO overflow in the coronary perfusate were analysed. Aortic constriction increased heart/body weight ratio (47%), myocyte size (19%) and arteriolar wall thickness (51%), all P<0.01. Coronary reserve was reduced in hypertrophy (105+/-8%v 182+/-12%, P<0.01). Dose response curves for ACh, SNP and adenosine were reduced in hypertrophy (69%, 86% and 68%, all P<0.01) v shams; however ED(50)values were unchanged. The peak flow and duration of RH were also attenuated (50%, P<0.001) in hypertrophy. While purine washout during RH was related to the duration of preceding ischaemia, nitrate washout was not. RH experiments in the presence of L -NAME, 8-PT and glibenclamide indicated that RH is mediated by combined actions of K(ATP)channels>adenosine>NO in both groups. RH is mediated by similar mechanisms in control and hypertrophied hearts. All vasodilatation was similarly attenuated in hypertrophy, independent of endothelial activation. We hypothesize that increased arteriolar wall thickness may limit vasodilator responses to all stimuli in hypertrophy. PMID- 10731453 TI - Coronary heart disease. A message from preventive medicine and your physician. PMID- 10731452 TI - A randomized controlled trial of smoking cessation counseling after myocardial infarction. AB - BACKGROUND: Smoking cessation after myocardial infarction (MI) has been associated with a 50% reduction in mortality but in-hospital smoking cessation interventions are rarely part of routine clinical practice. METHODS: One hundred cigarette smokers consecutively admitted during 1996 with MI were assigned to minimal care or to a hospital-based smoking cessation program. Intervention consisted of bedside cessation counseling followed by seven telephone calls over the 6 months following discharge. Primary outcomes were abstinence rates measured at 6 months and 1 year post-discharge. RESULTS: At follow-up, 43 and 34% of participants in minimal care and 67 and 55% of participants in intervention were abstinent at 6 and 12 months. respectively (P<0.05). Abstinence rates were calculated assuming that participants lost to attrition were smokers at follow up. Intervention and self-efficacy were independent predictors of smoking status at follow-up. Low self-efficacy combined with no intervention resulted in a 93% relapse rate by 1 year (P<0.01). CONCLUSIONS: A hospital-based smoking cessation program consisting of inpatient counseling and telephone follow-up substantially increases smoking abstinence 1 year after discharge in patients post-MI. Patients with low self-efficacy are almost certain to relapse without intervention. Such smoking cessation programs should be part of the management of patients with MI. PMID- 10731454 TI - Cost-benefit of a nursing telephone intervention to reduce preterm and low birthweight births in an African American clinic population. AB - BACKGROUND: A cost-benefit analysis was performed to estimate the cost-savings obtained from a nursing telephone intervention delivered to pregnant women identified as being at risk for preterm or low-birthweight births. METHODS: After being screened for eligibility, a total of 1,554 women receiving prenatal care in a clinic located in Winston-Salem, North Carolina were randomized to intervention and control groups. Women in the intervention group received telephone calls from a registered nurse one or two times each week from the 24th through the 37th week of gestation. RESULTS: No clinical benefits were realized by Caucasian participants. The intervention reduced preterm and low-birthweight births, and resulted in cost savings, for African-American mothers ages 19 and over. No significant differences were seen in the rates of low-birthweight or preterm births and no cost savings were realized from intervention with women ages 18 and younger. CONCLUSIONS: A prenatal nursing support intervention in a clinic population of pregnant African American women was cost-beneficial for these adults (< or =19 years of age). PMID- 10731455 TI - Training effects of accumulated daily stair-climbing exercise in previously sedentary young women. AB - BACKGROUND: The health and fitness benefits associated with short, intermittent bouts of exercise accumulated throughout the day have been seldom investigated. Stair climbing provides an ideal model for this purpose. METHODS: Twenty-two healthy female volunteers (18-22 years) were randomly assigned to control (N = 10) or stair-climbing (N = 12) groups. Stair climbers then underwent a 7-week stair-climbing program, progressing from one ascent per day in week 1 to six ascents per day in weeks 6 and 7, using a public access staircase (199 steps). Controls were instructed to maintain their normal lifestyle. Standardized stair climbing tests were administered to both groups immediately before and after the program. Each paced ascent lasted 135 s, during which oxygen uptake (VO(2)) and heart rate (HR) were monitored continuously. Blood lactate concentration was also measured immediately following each test ascent. Fasting blood samples from before and after the program were analyzed for serum lipids. Data were analyzed using a two-way ANOVA with repeated measures. RESULTS: Relative to the insignificant changes in the control group, the stair-climbing group displayed a rise in HDL cholesterol concentration (P<0.05) and a reduced total:HDL ratio (P<0.01) over the course of the program. VO(2) and HR during the stair-climbing test were also reduced, as was blood lactate (all P<0.01). CONCLUSION: A short term stair-climbing program can confer considerable cardiovascular health benefits on previously sedentary young women, lending credence to the potential public health benefits of this form of exercise. PMID- 10731456 TI - Differences in tobacco assessment and intervention practices: a regional snapshot. AB - BACKGROUND: This research describes tobacco attitudes and practices of health care providers in the Upper Midwest. A baseline measure of preventive practices by providers was needed to plan effective tobacco intervention education programs. METHODS: Health care providers in a 16-county region received a mailed survey regarding tobacco assessment practices, intervention practices, attitudes, skills/knowledge, barriers, and desire for tobacco education. The survey was sent to all chiropractors, dentists, nurse practitioners/physician assistants, physicians (primary care and specialist), and public health nurses in the region. A total of 51.9% (n = 614) of all providers returned usable surveys. RESULTS: Significant differences were found between provider groups on all measured concepts. Primary care physicians, nurse practitioners/physician assistants, and public health nurses were more likely than specialist physicians, dentists, and chiropractors to assess, intervene, be supportive of tobacco cessation, have skills/knowledge about cessation, perceive fewer barriers, and want further education. CONCLUSIONS: In this region, provider groups differed in tobacco use assessment and treatment. All provider groups desired education regarding tobacco intervention. Region-wide tobacco cessation educational initiatives need to take into account differences between provider groups. PMID- 10731457 TI - Prevalence, treatment, control, and awareness of high blood pressure and the risk of stroke in Northwest England. AB - OBJECTIVES: The aim of this study was to examine the prevalence, treatment, control, and awareness of hypertension in patients with first-ever stroke and in controls sampled from the same primary care physician's population register. METHODS: A population-based case-control study was conducted in East Lancashire, England, using cases identified from the stroke register in 1994-1995. Information on blood pressure (BP) and other predefined factors was extracted from the practice medical records. Postal questionnaires were used for information on patients' awareness of hypertension. RESULTS: A total of 267 stroke cases and 534 controls were included. Sixty-one percent of cases and 43% of controls had BP >= 160/95 mm Hg on >= 2 occasions within 3 months or received antihypertensives. High proportions of cases (82%) and controls (85%) were on treatment. There was a continuous relationship between the risk of stroke and levels of BP control. Of 73 cases and 135 controls who were hypertensive and responded to the postal questionnaire, 56 and 83%, respectively, were aware of hypertension (P<0.01). CONCLUSIONS: The prevalence of hypertension was high among stroke patients. In those treated, <30% of patients had their BP adequately controlled to <140/90 mm Hg. Patient awareness of previous hypertension or high BP was very poor and attention needs to be paid to patient education. PMID- 10731458 TI - Eating habits, health status, and concern about health: a study among 1641 employees in the German metal industry. AB - BACKGROUND: Nutrition has been found to be associated with sociodemographic characteristics and concern about health. There is limited knowledge, however, of associations between blue-collar worker's diet, morbidity, and health care utilization. METHODS: We conducted a survey on eating habits, physical symptoms, health care utilization, health status, and concern about health in two German metal companies. A self-administered questionnaire was mailed to employees of whom 1641 participated in the study (response rate 54. 7%). RESULTS: Most employees were characterized by a combination of healthy and unhealthy eating elements. Using linear regression analyses adjusted for age, gender, and occupational status, healthy eating was negatively associated with stomach aches and headaches, but not with cardiovascular disease. Restricted activity days and days in hospital were associated with healthy eating, but self-assessed health status and physician consultations were not. Using stepwise multiple regression analysis, age, gender, and concern about health were strongly and morbidity was weakly related to diet. Occupational status, marital status, and number of children were not associated with nutrition. CONCLUSIONS: Health promotion programs should motivate younger and male employees to participate in and aim toward increasing concern about health. PMID- 10731459 TI - The economic effects of screening for obstructive airway disease: an economic analysis of the DIMCA program. AB - BACKGROUND: A large, population-based intervention (the DIMCA study) has shown substantial underdiagnosis of chronic obstructive pulmonary disease and asthma. Detection of undiagnosed patients by means of screening and subsequent monitoring was relatively inexpensive per detected patient, compared with other mass screening programs. The objectives of this study were to assess whether early detection according to the DIMCA protocol leads to increased utilization of health care resources and cost, other than the cost of the scheduled visits. METHODS: In a prospective randomized consent trial, the utilization of health care resources and cost were ascertained in two groups: a screened group (n = 416) and a control group (n = 462). In a subsample of 100 screened subjects, consultation frequency before screening was compared with the frequency after screening. Subjects were a random sample from the general population of between 25 and 70 years of age. RESULTS: During an average follow-up of 3.6 years, there were no significant differences in health care resource utilization and cost between the screened subjects and the controls. Resource utilization before screening was not significantly different from resource utilization after screening. Statistically significant differences were found within the screened group: subjects with a positive screening result (i.e., with signs or symptoms of obstructive airway disease) consulted their general practitioners 3.7 times more frequently (P = 0.001) for respiratory reasons than subjects with a negative screening result. The total health care cost due to respiratory disease in screen positive subjects was 6.4 times higher (P = 0.008). CONCLUSION: There were no indications that screening for obstructive airway disease led to increased cost, above that of average care. PMID- 10731460 TI - Increasing the fruit and vegetable consumption of fourth-graders: results from the high 5 project. AB - BACKGROUND: This study evaluated the effects of a school-based dietary intervention program to increase fruit and vegetable consumption among fourth graders. METHODS: Twenty-eight elementary schools were randomized to an immediate intervention condition or to a delayed intervention control condition. Measures of diet and psychosocial variables were collected at base line and 1 and 2 years post-baseline. The intervention included classroom, parent, and cafeteria components. RESULTS: Mean daily consumption of fruit and vegetables was higher for the intervention children compared with controls at Follow-up 1 (X(t) = 3.96, X(c) = 2.28) and at Follow-up 2 (X(t) = 3.20, X(c) = 2.21). Macro- and micronutrient changes favoring the intervention children were also observed at both Follow-up 1 and Follow-up 2. Mean daily consumption of fruit and vegetables was higher for intervention parents compared with controls at Follow-up 1 (X(t) = 4.23,X(c) = 3.94) but not at Follow-up 2. CONCLUSIONS: Strong effects were found for the High 5 intervention on fruit and vegetable consumption, on macro- and micro-nutrients, and on psychosocial variables. Future work is needed to enhance the intervention effects on parents' consumption and to test the effectiveness of the intervention when delivered by classroom teachers. PMID- 10731461 TI - Effect of cigarette promotions on smoking uptake among adolescents. AB - BACKGROUND: The purpose of this study was to evaluate the association between receptivity to cigarette promotions and smoking uptake in a cohort of adolescents. METHODS AND MEASURES: This was a prospective cohort study of 480 4th to 11th-grade students conducted in three rural Vermont K-12 schools. Cigarette use was determined by self-report at baseline, 12 months (survey 2), and 21 months (survey 3). Proportional odds models were used to evaluate smoking uptake as a function of baseline measures of cigarette use, receptivity to cigarette promotions, and confounding factors, including grade, parental education, peer smoking, and family smoking. Adolescents were receptive to cigarette promotions if they owned or were willing to use a personal item bearing a cigarette brand logo [cigarette promotional item (CPI)]. Smoking status was measured using a 6 point ordinal index that combined experience and attitudes: 0 = never smoker/not susceptible to smoking, 1 = never smoker/susceptible to smoking, 2 = puffer (1 cigarette or less in lifetime), 3 = non-current experimenter (2-99 cigarettes in lifetime/none in past 30 days), 4 = current experimenter, and 5 = smoker (> or =100 cigarettes in lifetime). RESULTS: The 480 students were equally distributed across grade at baseline. Environmental exposure to smoking was high, and 30% were receptive to cigarette promotions at baseline. Higher levels on the smoking index at baseline were associated with higher grade in school, peer smoking, and receptivity to cigarette promotions. One hundred eighty-five students (38.5%) had moved to a higher category on the smoking index by survey 3, of whom 30 had become smokers. Receptivity to cigarette promotions at baseline was significantly associated with higher smoking uptake, with 48.7% of receptive students moving up one or more categories on the smoking index [adjusted proportional odds 1.9 (95% CI 1.3, 2.9)]. Acquisition of receptivity to cigarette promotions was also associated with increased smoking uptake, with those becoming receptive in surveys 2 or 3 having significantly higher odds of progression [3.6 (1.8, 7.0) and 2.9 (1.5, 5.5), respectively] compared with those who did not change. Conversely, those who were receptive at one point but became non-receptive in surveys 2 or 3 had lower odds of progression [0.4 (0.2, 0.9) and 0.5 (0.3, 1.1), respectively]. CONCLUSIONS: This study supports a close linkage between tobacco promotional activities and uptake of smoking among adolescents beyond baseline descriptions of receptivity to cigarette promotions. Over time, the likelihood of smoking uptake is increased when an adolescent acquires a CPI or becomes willing to use one and is decreased when an adolescent who owns a CPI loses it or becomes unwilling to use it. This provides strong evidence that elimination of cigarette promotional campaigns could reduce adolescent smoking. PMID- 10731462 TI - Erectile dysfunction and coronary risk factors: prospective results from the Massachusetts male aging study. AB - BACKGROUND: Erectile dysfunction (ED), a wide spread and troublesome condition among middle-aged men, is partly vascular in origin. In the Massachusetts Male Aging Study, a random-sample cohort study, we investigated the relationship between baseline risk factors for coronary heart disease and subsequent ED, on the premise that subclinical arterial insufficiency might be manifested as ED. METHODS: Men ages 40-70, selected from state census lists, were interviewed in 1987-1989 and reinterviewed in 1995-1997. Data were collected and blood was drawn in participants' homes. ED was assessed from responses to a privately self administered questionnaire. Analysis was restricted to 513 men with no ED at baseline and no diabetes, heart disease, or related medications at either time. RESULTS: Cigarette smoking at baseline almost doubled the likelihood of moderate or complete ED at followup (24% vs. 14%, adjusted for age and covariates, P = 0.01). Cigar smoking and passive exposure to cigarette smoke also significantly predicted incident ED, as did overweight (body-mass index > or =28 kg/m(2)) and a composite coronary risk score. Weaker prospective associations were seen for hypertension and dietary intake of cholesterol and unsaturated fat. CONCLUSIONS: Erectile dysfunction and coronary heart disease share some behaviorally modifiable determinants in men who, like our sample, are free of manifest ED or predisposing illness. Open questions include whether modification of coronary risk factors can prevent ED and whether ED may serve as a sentinel event for coronary disease. PMID- 10731463 TI - Reducing noise pollution in the hospital setting by establishing a department of sound: a survey of recent research on the effects of noise and music in health care. AB - A proposal for a solution to reduce stress and anxiety in the hospital setting by combining the problems of excess noise in a hospital setting with the efficacy of music therapy is supported through an analysis of research in the field of noise, hospital noise pollution, and music medicine. Included in this overview are articles describing the effects of noise on health, the problems of noise pollution in the health care setting, and the benefits of replacing noise with music to reduce heart rate, blood pressure, breathing rate, emotional anxiety, and pain. By combining these areas of research, the authors propose the establishment of a department assigned to (1) control the amount of noise in a hospital and (2) provide a center of music therapy for all individuals in the hospital setting, including in-patients, out-patients, doctors, and staff. Due to the large specificity of these areas, this unifying source, or "Department of Sound," is suggested to aid in thoroughly addressing and combining these two concepts most effectively. PMID- 10731465 TI - Reply. PMID- 10731464 TI - Evaluation of smoking prevalence: will there be an end to self-administered questionnaires? PMID- 10731466 TI - Linkage analysis in the presence of errors IV: joint pseudomarker analysis of linkage and/or linkage disequilibrium on a mixture of pedigrees and singletons when the mode of inheritance cannot be accurately specified. AB - There is a lot of confusion in the literature about the "differences" between "model-based" and "model-free" methods and about which approach is better suited for detection of the genes predisposing to complex multifactorial phenotypes. By starting from first principles, we demonstrate that the differences between the two approaches have more to do with study design than statistical analysis. When simple data structures are repeatedly ascertained, no assumptions about the genotype-phenotype relationship need to be made for the analysis to be powerful, since simple data structures admit only a small number of df. When more complicated and/or heterogeneous data structures are ascertained, however, the number of df in the underlying probability model is too large to have a powerful, truly "model-free" test. So-called "model-free" methods typically simplify the underlying probability model by implicitly assuming that, in some sense, all meioses connecting two affected individuals are informative for linkage with identical probability and that the affected individuals in a pedigree share as many disease-predisposing alleles as possible. By contrast, "model-based" methods add structure to the underlying parameter space by making assumptions about the genotype-phenotype relationship, making it possible to probabilistically assign disease-locus genotypes to all individuals in the data set on the basis of the observed phenotypes. In this study, we demonstrate the equivalence of these two approaches in a variety of situations and exploit this equivalence to develop more powerful and efficient likelihood-based analogues of "model-free" tests of linkage and/or linkage disequilibrium. Through the use of a "pseudomarker" locus to structure the space of observations, sib-pairs, triads, and singletons can be analyzed jointly, which will lead to tests that are more well-behaved, efficient, and powerful than traditional "model-free" tests such as the affected sib-pair, transmission/disequilibrium, haplotype relative risk, and case-control tests. Also described is an extension of this approach to large pedigrees, which, in practice, is equivalent to affected relative-pair analysis. The proposed methods are equally applicable to two-point and multipoint analysis (using complex-valued recombination fractions). PMID- 10731468 TI - Comments on the role of fish-oil fatty acids in the prevention of cancer cachexia. PMID- 10731469 TI - The hyperinsulinaemic-euglycaemic glucose clamp: reproducibility and metabolic effects of prolonged insulin infusion in healthy subjects. AB - To examine the reproducibility of the hyperinsulinaemic-euglycaemic clamp technique at mid-physiological hyperinsulinaemia, seven healthy subjects ?age 50 (25, 59) years [median (range)], body mass index 23.1 (20.8, 25.5) kg.m(-2)? were investigated with three 2 h hyperinsulinaemic (60 micromol.l(-1))-euglycaemic (4.5 mmol.l(-1)) clamps performed 48 h and 14 days apart respectively. The third clamp was prolonged to 8 h in order to examine effects on glucose disposal during prolonged clamps. The glucose infusion rates (GIRs) during the three 2 h clamps were 7.41 (4.28, 10.96), 7.26 (5.38, 11. 02) and 6.63 (4.42, 10.3) mg.kg(-1).min( 1), with a median intra-individual coefficient of variation of 5.8 (2.6, 22) %. During the 8 h clamp a highly variable gradual increase in GIR was observed, reaching a plateau between 4 and 5 h at 32 (5, 101) % above the GIR between 1 and 2 h (P<0.05). This increase was correlated inversely with the GIR between 1 and 2 h (r=-0.82; P<0.05), and directly with age (r=0.86; P<0.05). Carbohydrate oxidation measured by indirect calorimetry was stable during the repeated 2 h clamps and the 8 h clamp. Endogenous glucose production measured by infusion of [6, 6-(2)H(2)]glucose was suppressed during the 8 h clamp. The 2 h hyperinsulinaemic-euglycaemic clamp is reproducible at a mid-physiological range of hyperinsulinaemia. If prolonged, it results in a delayed increase in non oxidative glucose disposal, which is most pronounced in subjects with low insulin sensitivity. The findings underline the importance of selecting age-matched controls in studies of insulin resistance. PMID- 10731467 TI - Linkage analysis in the presence of errors III: marker loci and their map as nuisance parameters. AB - In linkage and linkage disequilibrium (LD) analysis of complex multifactorial phenotypes, various types of errors can greatly reduce the chance of successful gene localization. The power of such studies-even in the absence of errors-is quite low, and, accordingly, their robustness to errors can be poor, especially in multipoint analysis. For this reason, it is important to deal with the ramifications of errors up front, as part of the analytical strategy. In this study, errors in the characterization of marker-locus parameters-including allele frequencies, haplotype frequencies (i.e., LD between marker loci), recombination fractions, and locus order-are dealt with through the use of profile likelihoods maximized over such nuisance parameters. It is shown that the common practice of assuming fixed, erroneous values for such parameters can reduce the power and/or increase the probability of obtaining false positive results in a study. The effects of errors in assumed parameter values are generally more severe when a larger number of less informative marker loci, like the highly-touted single nucleotide polymorphisms (SNPs), are analyzed jointly than when fewer but more informative marker loci, such as microsatellites, are used. Rather than fixing inaccurate values for these parameters a priori, we propose to treat them as nuisance parameters through the use of profile likelihoods. It is demonstrated that the power of linkage and/or LD analysis can be increased through application of this technique in situations where parameter values cannot be specified with a high degree of certainty. PMID- 10731470 TI - Magnesium regulates hypoxia-stimulated apoptosis in the human placenta. AB - Apoptosis (programmed cell death) in the human placenta is likely to play a major role in determining the structure and function of that organ. Fetal growth restriction (FGR) has been shown to be associated with increased levels of placental apoptosis. Altered regulation of apoptosis may play an important pathophysiological role in FGR. As reduced placental perfusion and reduced oxygenation are features of FGR, one aim of this study was to determine the effects of hypoxia on apoptotic activity, as assessed by DNA laddering, of placental tissue in vitro. In addition, levels of placental apoptosis may be affected by pharmacological agents routinely used in obstetric patient management. Thus an additional aim of this study was to determine the effects of several relevant pharmacological agents on the levels of DNA laddering during in vitro incubation of human placentae under hypoxic conditions. Incubation of normal placental explant tissue at 37 degrees C for 1-2 h under hypoxic conditions significantly increased placental DNA laddering compared with that in non-incubated tissue, whereas levels of DNA laddering during incubation for up to 2 h under normoxic conditions were not significantly higher than those in non incubated tissue. The DNA laddering activity of placental explants after 2 h of incubation under hypoxic conditions was significantly increased with increased concentrations of magnesium, but remained unchanged by the inclusion of pethidine, aspirin, nifedipine, dexamethasone, heparin or indomethacin in the incubation mixture. These results suggest that hypoxia may stimulate apoptotic activity in cultured human placental tissues, and that hypoxia-stimulated placental apoptosis may be further increased by increasing the extracellular magnesium concentration. PMID- 10731471 TI - Regulation of beta-adrenoceptor-mediated relaxation of the rat aorta is modulated by endogenous ovarian hormones. AB - The aim of this study was to assess the influence of the endogenous status of ovarian hormones on the relaxation induced by the beta-adrenoceptor agonists isoprenaline (isoproterenol) and dobutamine in thoracic aorta segments, precontracted with noradrenaline, from age-matched (13-week-old) virgin (oestrus) and ovariectomized (OVX) prepubertal female Wistar rats. Isoprenaline-induced relaxation was decreased in intact aortic segments from OVX rats compared with that in segments from oestrus rats. Relaxation was significantly reduced by endothelium removal, 1 micromol/l propranolol or 100 micromol/l N(G)-nitro-L arginine methyl ester (L-NAME). The beta(1)-adrenoceptor agonist dobutamine induced less relaxation in intact arteries from oestrus rats than did isoprenaline, and dobutamine-induced relaxation was markedly less in intact segments from OVX compared with oestrus rats. This dobutamine-induced relaxation was abolished by endothelium removal, and reduced by 1 micromol/l propranolol, 100 micromol/l L-NAME or 1 micromol/l yohimbine. Cholera toxin (an activator of the stimulatory G-protein G(s)) caused relaxation in intact arteries from oestrus rats; this relaxation was decreased by both deprivation of ovarian hormones and endothelium removal. Forskolin (a direct activator of the catalytic subunit of adenylate cyclase) and sodium nitroprusside (a nitric oxide donor and cGMP dependent vasodilator agonist) induced similar endothelium-independent relaxation in arteries from both oestrus and OVX rats. These results suggest that the relaxation elicited by endothelial beta-adrenoceptor activation in the rat thoracic aorta is impaired by deprivation of female ovarian hormones; this impairment is caused, at least in part, by decreases in both the endothelial release of NO and G(s) function. PMID- 10731472 TI - Metabolic response to feeding in weight-losing pancreatic cancer patients and its modulation by a fish-oil-enriched nutritional supplement. AB - Weight-losing patients with advanced cancer often fail to gain weight with conventional nutritional support. This suboptimal response might be explained, in part, by an increased metabolic response to feeding. It has been suggested that eicosapentaenoic acid (EPA) can modify beneficially the metabolic response to cancer. The aim of the present study was to examine the metabolic response to feeding in cancer and the effects of an EPA-enriched oral food supplement on this response. A total of 16 weight-losing, non-diabetic patients with unresectable pancreatic adenocarcinoma and six healthy, weight-stable controls were studied by indirect calorimetry in the fasting and fed states. Body composition was estimated by bioimpedence analysis. Cancer patients were then given a fish-oil enriched nutritional supplement providing 2 g of EPA and 2550 kJ daily, and underwent repeat metabolic study after 3 weeks of such supplementation. At baseline, resting energy expenditure whether expressed per kg body weight, lean body mass or body cell mass was significantly greater in the cancer patients compared with controls. Fat oxidation was significantly higher in the fasting state in cancer patients [median 1.26 g.kg(-1).min(-1) (interquartile range 0.95 1.38)] than in controls [0.76 g.kg(-1). min(-1) (0.62-0.92); P<0.05]. Over the 4 h feeding period, changes in insulin and glucose concentrations in cancer patients suggested relative glucose intolerance. In response to oral meal feeding, the percentage change in the area under the curve of energy expenditure was significantly lower in the cancer patients [median 7.9% (interquartile range 3.4-9.0)] than in controls [12.6% (9.9-15.1); P<0.01]. After 3 weeks of the EPA enriched supplement, the body weight of the cancer patients had increased and the energy expenditure in response to feeding had risen significantly [9.6% (6. 3 12.4)], such that it was no different from baseline healthy control values. Similarly, fasting fat oxidation fell to 1.02 g. kg(-1).min(-1) (0.8-1.18), again no longer significantly different from baseline healthy control values. While weight-losing patients with advanced pancreatic cancer have an increased resting energy expenditure and increased fat oxidation, the energy cost of feeding is, in fact, reduced. Provision of a fish-oil-enriched nutritional supplement results in some normalization of the metabolic response in both the fasted and fed states, in association with an improvement in nutritional status. PMID- 10731473 TI - Ethanol effects on cardiomyocyte contractility. AB - Little is known about the direct cardiac effects of socially common sub intoxication levels of ethanol. Previous studies evaluating the responses of normal cardiomyocytes to short-term ethanol exposure have utilized ethanol concentrations equivalent to extreme intoxication or lethal levels in vivo. The purpose of the present study was to investigate the contractile responses of isolated rat ventricular cardiomyocytes during exposure to relatively low concentrations of ethanol in the range 0.05-0.5% (v/v) (8.6-86 mM) under physiological conditions (3 Hz stimulation; 36 degrees C; BSA vehicle). High speed imaging techniques were used to study the kinetics of myocyte contraction, and shortening parameters were calculated for mechanistic evaluation. The concentration-response relationship was not linear and exhibited two plateau phases, suggesting at least two mechanisms of action of ethanol on cardiomyocyte contraction. At 0.05% (8.6 mM), ethanol treatment produced a 14.4% decrease in maximum myocyte shortening. The maximum rates of cell shortening and lengthening were similarly impaired, but there was no effect on contraction cycle timing at this low concentration. At 0.30% (51 mM), ethanol reduced maximum shortening by 40.2%, prolonged excitation-contraction coupling latency and abbreviated the contraction cycle time by 38%. The inotropic modulatory effect of ethanol was exaggerated in the absence of protein in the superfusion buffer. This is the first report which identifies ethanol at 0.05% (v/v) as a modulator of cardiac contractility. Kinetic analyses indicate that the mechanism of action involves disturbance of sarcoplasmic reticulum function, and this may contribute to arrhythmogenic vulnerability - especially in an in vivo context of heightened compensatory sympathetic drive. PMID- 10731474 TI - Na+/H+ exchanger activity and phosphorylation in temperature-sensitive immortalized proximal tubule cell lines derived from the spontaneously hypertensive rat. AB - Freshly isolated proximal tubules from the spontaneously hypertensive rat (SHR) demonstrate elevated Na(+)/H(+) exchanger (NHE) activity, but the underlying mechanism is unclear. Because of the difficulties in preparing sufficient numbers of proximal tubule cells for detailed biochemical studies, we have generated cell lines from SHR and Wistar-Kyoto rat (WKY) proximal tubule cells. Cell lines were obtained by transforming the cells with an origin-defective mutant of simian virus 40 encoding a heat-labile T antigen (tsA58 transformant). Such cells proliferate at the permissive temperature of 33 degrees C, but growth is abolished at the restrictive temperature of 39 degrees C. The predominant NHE isoform expressed was isoform 1, as determined by sensitivity to HOE-694 (3 methylsulphonyl-4-piperidinobenzoyl guanidine) and Western blotting using specific polyclonal antisera to NHE-1. NHE-3 protein was also present. Northern blots of poly(A) mRNA extracts of the cell lines revealed a low abundance of transcripts for NHE-2, -3 and -4, with no systematic difference between the lines. Although the intracellular pH was similar in the SHR and WKY lines, HOE 694-sensitive H(+) efflux due to NHE-1 was substantially elevated in SHR lines compared with WKY lines (95.0+/-2.8 and 39. 9+/-5.7 mmol.min(-1).l(-1) respectively; P<0.001; n=6). H(+) efflux due to non-Na(+)-dependent mechanisms were similar in lines from the two strains. Western blotting revealed that NHE-1 density was also very similar in SHR and WKY lines, and subcellular fractionation of homogenates indicated that NHE-1 was localized predominantly to plasma membranes. Thus the turnover number of NHE-1 was increased. Immunoprecipitation of (32)P-labelled phosphoproteins from these lines demonstrated an approximately 2-fold higher degree of phosphorylation of NHE-1 in SHR compared with WKY lines. These cell lines form a useful model for defining the biochemical mechanisms leading to the NHE-1 phenotype in the SHR kidney, in addition to investigations of other SHR phenotypic markers. PMID- 10731475 TI - Sequential changes in plasma cytokine and endotoxin levels in cirrhotic patients with bacterial infection. AB - To delineate the clinical roles of plasma cytokine or endotoxin levels in the natural course of infection in patients with decompensated cirrhosis, 66 cirrhotic patients were studied within a 1.5-year period. Plasma levels of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, IL-8 and endotoxin were determined on days 1, 4 and 7 after admission when hospital infection was suspected and 4 months later. A total of 24 patients (36.4%) were proven to be infected during hospitalization (group A), while 42 others were not infected (group B). Fever occurred in a very high proportion (22/24) of group A patients. Baseline levels of TNF-alpha (37.7+/-15.2 compared with 8.7+/-1.2 pg/ml; P<0.01) and IL-6 (180.5+/-20.5 compared with 24.6+/-7.5 pg/ml; P<0.0001) were higher in group A patients, while IL-1beta, IL-8 and endotoxin levels were not significantly different between the two groups. For patients with hospital infection, IL-6 levels determined during the episode were significantly higher than baseline levels. Using IL-6 >80 pg/ml as a baseline cut-off level to diagnose bacterial infection, the sensitivity, specificity and accuracy were 87.5, 100 and 95.5% respectively. The one-year cumulative probability of mortality (61.1% compared with 23.7%; P<0.001) and of bacterial re-infection (72.2% compared with 18.4%; P<0.0001) was higher in group A than in group B. Plasma TNF-alpha and IL-6 levels determined at 4 months were not different between the two groups. In conclusion, fever or elevated plasma IL-6 levels in patients with decompensated cirrhosis calls for early antibiotic treatment to prevent life-threatening bacterial infection. Bacterial infection is likely to recur in those patients with increased IL-6 levels, while severe episodes of infection occur in patients with increased TNF-alpha levels. PMID- 10731476 TI - alpha2-Macroglobulin, the main serum antiprotease, binds beta2-microglobulin, the light chain of the class I major histocompatibility complex, which is involved in human disease. AB - beta(2)-Microglobulin, a 12 kDa protein forming part of the class I HLA (histocompatibility locus antigen) major histocompatibility complex, has been used as a prognosis factor for multiple myeloma and as a marker of renal function, and has been shown to be involved in the pathogenesis of dialysis related amyloidosis. alpha(2)-Macroglobulin has the ability to bind a wide range of physiologically important molecules, thereby influencing their metabolic impact. In this study we show by Western blotting analysis that beta(2) microglobulin binds to alpha(2)-macroglobulin in vitro. This binding was confirmed by BIAcore analysis, and was shown by ELISA to be concentration dependent. The sequences of the binding peptides in the mature beta(2) microglobulin molecule were identified by Spot multiple peptide synthesis and alpha(2)-macroglobulin binding studies. In conclusion, beta(2)-microglobulin interacts specifically with the universal antiprotease a(2)-macroglobulin. The identification of this interaction brings into question some of the axioms on the metabolism of beta(2)-microglobulin, and may help to explain the clinical findings observed in b(2)-microglobulin-related diseases. PMID- 10731477 TI - Tumour necrosis factor alpha G-308-->A polymorphism and risk for coronary artery disease. PMID- 10731478 TI - Indexed glomerular filtration rate as a function of age and body size. AB - The conventional way in which to scale or index a measurement of glomerular filtration rate (GFR) is to express it in relation to body surface area (BSA). However, BSA may not be appropriate for infants and children because, as individuals increase in size, their relative BSA decreases. Several other whole body variables have been suggested as alternatives, including extracellular fluid volume (vECF). The purpose of the present study was to compare BSA and vECF as variables against which to index GFR, and in particular to look at this comparison in children versus adults. A total of 130 patients (age range 1-80 years; 40 patients <12 years) undergoing clinically indicated routine measurement of GFR using the bolus-injection single-compartment technique were included in the study. GFR was measured as the plasma clearance of [(51)Cr]EDTA as assessed from three peripheral venous blood samples taken between 2 and 4 h after injection of [(51)Cr]EDTA. Volume of distribution (V(d)) was obtained by extrapolation of the clearance curve to zero time. GFR was scaled to a BSA of 1.73 m(2). GFR and GFR/1.73 m(2) were corrected to account for the assumption of a single compartment. The rate constant of the exponential between 2 and 4 h was also corrected to give GFR/litre ECF. GFR and GFR/1.73 m(2) were both divided by GFR/litre ECF, to give vECF and vECF/1.73 m(2) respectively. Weight per unit BSA increases as a linear function of BSA. vECF is always less than V(d), on average by about 30%. vECF increased as an exponential function of BSA and as a linear function of body weight. vECF/70 kg body weight was higher in children (16. 2+/-3 litres) than adults (13.4+/-2.3 litres), but vECF/1.73 m(2) was lower in children (9.7+/-1.7 litres) compared with adults (12. 4+/-2 litres). vECV/1.73 m(2) increased as a function of both age and BSA, but vECF/kg decreased. GFR/12.5 litres vECF was higher than GFR/1.73 m(2) in children, but these values were similar in adults, with the ratio of these two forms of indexed GFR falling significantly with both age and BSA. Although this was not a normal population, but one with a wide range of renal function, GFR/vECF showed a strong inverse association with age, whereas for GFR/BSA the association was weak. In conclusion, these data provide further evidence that vECF is more valid physiologically for indexing GFR than is BSA, especially in children. Nevertheless, a GFR measurement in a child should ideally be expressed as a percentage of normal for that child's age. However, such normal values are not yet available. PMID- 10731479 TI - Chronic venous insufficiency in post-thrombotic patients. PMID- 10731480 TI - Venous reflux has a limited effect on calf muscle pump dysfunction in post thrombotic patients. AB - The purpose of the present study was to evaluate the relationship between calf muscle pump dysfunction (CMD) and the presence and location of valvular incompetence. Deep vein obstruction might influence CMD, and so venous outflow resistance (VOR) was measured. VOR and calf muscle pump function were measured in 81 patients, 7-13 years after venographically confirmed lower-extremity deep venous thrombosis. The supine venous pump function test (SVPT) measures CMD, and the VOR measures the presence of venous outflow obstructions, both with the use of strain-gauge plethysmography. Valvular incompetence was measured using duplex scanning in 16 vein segments of one leg. Venous reflux was measured in proximal veins using the Valsalva manoeuvre, and in the distal veins by distal manual compression with sudden release. Abnormal proximal venous reflux was defined as a reflux time of more than 1 s, and abnormal distal venous reflux as a reflux time of more than 0.5 s. No statistically significant relationship was found between the SVPT and either the location or the number of vein segments with reflux. Of the 81 patients, only nine still had an abnormally high VOR, and this VOR showed no relationship with the SVPT. In conclusion, venous reflux has a limited effect on CMD, as measured by the SVPT. The presence of a venous outflow obstruction did not significantly influence the SVPT. Duplex scanning and the SVPT are independent complementary tests for evaluating chronic venous insufficiency. PMID- 10731481 TI - Vitamin C modifies the cardiovascular and microvascular responses to cigarette smoke inhalation in man. AB - Both neutrophil margination and increases in the non-invasively assessed parameter, isovolumetric venous congestion cuff pressure (Pv(i)), are symptomatic of some inflammatory diseases. Neutrophil margination occurs primarily, though not exclusively, at the post-capillary endothelial surface. The local haemodynamic changes resulting from margination may be responsible for the observed increases in Pv(i). Smoke inhalation has been shown in animal studies to cause an increase in post-capillary neutrophil margination by mechanisms that can be blocked by oral vitamin C administration. We looked for indices of a relationship between margination and Pv(i) in man, using cigarette smoke inhalation as a pathophysiological challenge. We also examined the effect of prophylactic vitamin C on the response. Smoke inhalation was associated with highly significant increases in both Pv(i) and heart rate. After vitamin C pre treatment, no increase in Pv(i) was observed in response to the smoke inhalation; however, whilst heart rate still increased significantly, the duration of this response was attenuated. The results suggest that vitamin C affords protection against some of the cardiovascular and microvascular changes associated with cigarette smoke inhalation in man. They also support the notion that non-invasive assessment of changes in Pv(i) may provide a measurable index of systemic changes in inflammatory conditions. PMID- 10731482 TI - Tumour necrosis factor-alpha gene expression and production in human umbilical arterial endothelial cells. AB - Endothelial cells act as an interface between the blood and tissues, and are known to be involved in inflammatory processes. These cells are responsive to and produce different cytokines. Tumour necrosis factor-alpha (TNF-alpha) not only is one of the most important inflammatory peptides, but also can be induced by lipopolysaccharide (LPS). The focus of the present study was on TNF-alpha gene expression and production in human umbilical arterial endothelial cells (HUAEC), including the kinetics of this process. Interleukin-1alpha (IL-1alpha), LPS and TNF-alpha, which are all known to be elevated in septic shock, were used as stimulators at concentrations commonly found in patients with sepsis. Through the use of reverse transcriptase/PCR, immunohistochemical reactions and ELISA techniques, we showed that, in HUAEC, all three stimuli were able to induce gene expression and production of TNF-alpha. Furthermore, this induction by IL-1alpha, LPS and TNF-alpha occurred in a time- and concentration-dependent manner in these cells. TNF-alpha expression and production was induced by all three agents at concentrations commonly found in patients with sepsis. TNF-alpha mRNA was observed within 30 min regardless of the stimulus used, but the levels peaked at different times. Since it is well established that TNF-alpha is able to induce the synthesis of IL-1alpha in endothelial cells and, as shown in the present study, TNF-alpha and IL-1alpha are themselves able to induce the synthesis of TNF alpha in endothelial cells, an autocrine potentiation of cytokine release in sepsis can be proposed. This situation could lead to a locally acting 'vicious cycle' which, when considered in addition to the known ability of TNF-alpha to induce apoptosis, could mean that various organs will be damaged, a condition associated with sepsis. Thus these results provide further evidence for the important role played by the endothelium in inflammation. PMID- 10731483 TI - Effects of inter-alpha-inhibitor and several of its derivatives on calcium oxalate crystallization in vitro. AB - The bikunin peptide chain of the protease inhibitor inter-alpha-inhibitor (IalphaI) has been reported to be an inhibitor of calcium oxalate (CaOx) crystallization, and hence has been proposed as having a role in CaOx kidney stone formation. However, further experimental evidence is required to assess if fragments of IalphaI other than bikunin may play a role in the regulation of crystallization events in stone formation. The aim of the present study was to assess the effects of IalphaI and several of its derivatives on CaOx crystallization in a seeded inorganic system and to compare these effects with those of a known inhibitor of crystallization, prothrombin. IalphaI was purified from a preparation of human plasma and fragmented by alkaline hydrolysis, and two of its peptide chains, bikunin and heavy chain 1 (H1), were purified further by HPLC. Their purity was confirmed by SDS/PAGE. Using Coulter counter and [(14)C]oxalate analysis and scanning electron microscopy, IalphaI, its H1 chain and bikunin from urine and from plasma were shown to be relatively weak inhibitors of CaOx crystallization in vitro at expected physiological concentrations. It was concluded that members of the IalphaI family may not be as important in kidney stone formation as has been generally proposed, although further studies are required before a possible role for IalphaI and its fragments in stone formation can be unambiguously discounted. PMID- 10731484 TI - Localization of diuretic effects along the loop of Henle: an in vivo microperfusion study in rats. AB - In order to clarify the effects on sodium reabsorption in the loop of Henle of methazolamide (a carbonic anhydrase inhibitor), chlorothiazide and the loop diuretics frusemide and bumetanide, superficial loops were perfused in vivo in anaesthetized rats and the individual diuretics were included in the perfusate. Differentiation between effects in the pars recta and in the thick ascending limb of Henle (TALH) was achieved by comparing responses to the diuretics when using a standard perfusate, designed to mimic native late proximal tubular fluid, and a low-sodium perfusate, designed to block net sodium reabsorption in the pars recta. With the standard perfusate, methazolamide caused decreases in sodium reabsorption (J(Na)) and water reabsorption (J(V)); with the low-sodium perfusate, a modest effect on J(Na) persisted, suggesting that carbonic anhydrase inhibition reduces sodium reabsorption in both the pars recta and the TALH. The effects of chlorothiazide were very similar to those of methazolamide with both the standard and low-sodium perfusates, suggesting that chlorothiazide also inhibits sodium reabsorption in the pars recta and TALH, perhaps through inhibition of carbonic anhydrase. With the standard perfusate, both frusemide and bumetanide produced the expected large decreases in J(Na), but J(V) was also lowered. With the low-sodium perfusate, the inhibitory effects of the loop diuretics, particularly those of frusemide, were substantially reduced, while net potassium secretion was found. These observations indicate that a significant component of the effect of frusemide (and possibly of bumetanide) on overall sodium reabsorption is located in the pars recta, and that loop diuretics induce potassium secretion in the TALH. PMID- 10731485 TI - Comparative physiological study of arbutamine with exercise in humans. AB - Pharmacological stress testing may be used in the diagnosis of coronary artery disease when there are contra-indications to the use of conventional exercise protocols. The responses to such testing using arbutamine and to conventional treadmill exercise were compared in eight patients. Respiratory gas analysis and cardiovascular observations were performed during both tests. For an equivalent increment in heart rate, both protocols increased systolic blood pressure and serum lactate. Minute ventilation and oxygen consumption also rose during both protocols, but much more so with exercise. The end-tidal partial pressure of CO(2) [35.1 (S.D. 3. 1) to 30.8 (6.6) mmHg] and the dead space/tidal volume ratio (V(D)/V(T)) [0.37 (0.09) to 0.33 (0.08)] fell significantly during arbutamine infusion, but the respiratory exchange ratio did not change during either protocol. Oxygen pulse, a marker of stroke volume, did not change significantly after arbutamine, but rose markedly after exercise [arbutamine, 3.9 (1.1) to 3.37 (0.7) ml. min(-1).beat(-1); exercise, 4.7 (1.4) to 16.1 (4.6) ml.min(-1). beat( 1) (P<0.0001 compared with baseline); difference between peak responses: P<0.0001]. We conclude that arbutamine simulates some of the physiological responses to exercise, although a number of these responses are less marked than during conventional exercise, in particular cardiac output (oxygen pulse). An increase in ventilation is produced, possibly due to direct stimulation of arterial chemoreceptors. These data suggest that the main action of arbutamine is to increase central drive rather than to establish peripheral demand. PMID- 10731486 TI - Short-term analysis of the relationship between blood pressure and urinary sodium excretion in normotensive subjects. AB - The aim of this study was to investigate whether, in the short term, physiological blood pressure changes are coupled with changes in urinary sodium excretion in normotensive subjects, maintained at fixed sodium intake and under controlled postural and behavioural conditions. Twelve normotensive subjects were recruited. For each subject, seven urine samples were collected at fixed time intervals during an overall 26 h period: late afternoon (16.00-20.00 hours), evening (20.00-24.00 hours), night (24.00-06.00 hours), quiet wakefulness (06.00 09.00 hours), morning (09.00-12.00 hours), post-prandial (12.00-15.00 hours) and afternoon (15.00-18.00 hours). Blood pressure was monitored by an ambulatory blood pressure device during the whole 26 h period. Each urine sample was used to measure urinary sodium excretion and glomerular filtration rate (creatinine clearance). Blood pressure, heart rate, urinary sodium excretion and glomerular filtration rate recorded in the daytime were higher than those measured during the night-time. A significant positive correlation between mean blood pressure and urinary sodium excretion was found during the night, over the whole 26 h period, and during two subperiods of the daytime: quiet wakefulness and the post prandial period. The coefficient of the pressure-natriuresis curve was significantly decreased by postural changes. We conclude that, in normotensive subjects, blood pressure and urinary sodium excretion are coupled in the short term. The assumption of an upright posture can mask this relationship, presumably by activating neurohumoral factors. PMID- 10731487 TI - Increased left atrial thrombin generation in mitral stenosis is not reflected in arterial prothrombin fragment 1+2 levels. AB - A proportion of patients with mitral stenosis have increased left atrial thrombin generation, with elevated left atrial but normal peripheral venous levels of prothrombin fragment 1+2 (F1+2). Whether this pattern of left atrial and venous F1+2 levels is related to limited spillover of F1+2 from the left atrium into the systemic circulation, or to washout of increased left atrial F1+2 production into the arterial circulation with subsequent systemic clearance, is unclear. We examined the relationship between arterial and venous F1+2 levels in mitral stenosis patients without left atrial thrombus. The study group comprised 36 patients with either a normal (n=29) or prolonged (n=7) international normalized ratio (INR; a measure of clotting time) who were undergoing percutaneous balloon mitral valvuloplasty. Baseline arterial and venous blood samples were collected at the beginning of the valvuloplasty procedure, and left atrial and venous samples were collected after trans-septal puncture. The left atrial F1+2 level exceeded the corresponding venous level in patients with a normal INR (P<0.03); however, baseline arterial and venous F1+2 levels were similar. Arterial and venous F1+2 levels were also similar in the subgroup of patients with evidence of a regional increase in left atrial thrombin generation, and were not different from arterial and venous F1+2 levels in patients without such an increase. Baseline arterial and venous F1+2 levels were both lower in the presence of a prolonged INR. Thus the pattern of increased left atrial but normal venous F1+2 levels in mitral stenosis is due to limited spillover from the left atrium into the systemic circulation. PMID- 10731488 TI - Is homocysteine a biomarker for identifying women at risk of complications and adverse pregnancy outcomes? PMID- 10731489 TI - Microvascular disease and dementia in the elderly: are they related to hyperhomocysteinemia? PMID- 10731490 TI - Immunologic effects of yogurt. AB - Many investigators have studied the therapeutic and preventive effects of yogurt and lactic acid bacteria, which are commonly used in yogurt production, on diseases such as cancer, infection, gastrointestinal disorders, and asthma. Because the immune system is an important contributor to all of these diseases, an immunostimulatory effect of yogurt has been proposed and investigated by using mainly animal models and, occasionally, human subjects. Although the results of these studies, in general, support the notion that yogurt has immunostimulatory effects, problems with study design, lack of appropriate controls, inappropriate route of administration, sole use of in vitro indicators of the immune response, and short duration of most of the studies limit the interpretation of the results and the conclusions drawn from them. Nevertheless, these studies in toto provide a strong rationale for the hypothesis that increased yogurt consumption, particularly in immunocompromised populations such as the elderly, may enhance the immune response, which would in turn increase resistance to immune-related diseases. This hypothesis, however, needs to be substantiated by well-designed randomized, double-blind, placebo-controlled human studies of an adequate duration in which several in vivo and in vitro indexes of peripheral and gut associated immune response are tested. PMID- 10731491 TI - Introducing cancer nutrition to medical students: effectiveness of computer-based instruction. AB - BACKGROUND: Computer-based instruction has been introduced at the University of North Carolina at Chapel Hill to augment its nutrition course for first-year medical students. Seven program modules have been completed; 2 more are planned. Each module explains the biochemistry and physiology of nutrition through interactive lessons, exercises, and a video case study. OBJECTIVE: The goal of this study was to evaluate the instructional efficacy and acceptability of the nutrition and cancer module when used by first-year medical students. DESIGN: The module was used by 163 first-year medical students at the university's medical school as an obligatory component of the nutrition course. Before and after using the module, students were asked to answer multiple-choice questions concerning their knowledge and attitudes; each question had 5 possible answers. RESULTS: On average, students spent approximately 3 h studying the lessons. The percentage of correct responses to 20 knowledge questions increased from 22% before the module was used to 86% immediately after its use. When a randomly selected subsample of 25% of the students took the same test 3 mo later, they answered 62% of the questions correctly. The increase in the percentage of students who felt prepared to provide advice regarding nutrition's role in cancer prevention (from 5.7% to 66.9%) suggested a successful subjective learning experience. Neither the students' initial level of interest in cancer nutrition nor their acceptance of computer-based instruction was related to learning outcome. CONCLUSIONS: The tested module is a useful and effective aid for teaching nutritional principles of cancer prevention. The evaluation strategy helped identify areas for instructional improvement. PMID- 10731492 TI - The vitamin A spectrum: from deficiency to toxicity. AB - Dark adaptation has been used as a tool for identifying patients with subclinical vitamin A deficiency. With this functional test it was shown that tissue vitamin A deficiency occurs over a wide range of serum vitamin A concentrations. However, serum vitamin A concentrations >1.4 micromol/L predict normal dark adaptation 95% of the time. Other causes of abnormal dark adaptation include zinc and protein deficiencies. Stable isotopes of vitamin A and isotope-dilution techniques were used recently to evaluate body stores of vitamin A and the efficacy of vitamin A intervention programs in field settings and are being used to determine the vitamin A equivalences of dietary carotenoids. Vitamin A toxicity was described in patients taking large doses of vitamin A and in patients with type I hyperlipidemias and alcoholic liver disease. Conversely, tissue retinoic acid deficiency was described in alcoholic rats as a result of hepatic vitamin A mobilization, impaired oxidation of retinaldehyde, and increased destruction of retinoic acid by P450 enzymes. Abnormal oxidation products of carotenoids can cause toxicity in animal models and may have caused the increased incidence of lung cancer seen in 2 epidemiologic studies of the effects of high-dose beta carotene supplementation. Major issues that remain to be studied include the efficiency of conversion of carotenoids in whole foods to vitamin A by using a variety of foods in various field settings and whether intraluminal factors (eg, parasitism) and vitamin A status affect this conversion. In addition, the biological activity of carotenoid metabolites should be better understood, particularly their effects on retinoid signaling. PMID- 10731493 TI - Thigh adipose tissue distribution is associated with insulin resistance in obesity and in type 2 diabetes mellitus. AB - BACKGROUND: Adipose tissue (AT) content of the thigh is generally not considered to be associated with insulin resistance (IR), but it is unclear whether the distribution of AT in the thigh is a determinant of IR. OBJECTIVE: We investigated whether subcompartments of AT within the thigh are determinants of IR. DESIGN: Midthigh AT, muscle composition, and insulin sensitivity were compared in 11 obese patients with type 2 diabetes mellitus (DM); 40 obese, glucose-tolerant (GT) and 15 lean, GT volunteers; and 38 obese subjects who completed a weight-loss program. Midthigh AT area measured with computed tomography was partitioned into 3 components: subcutaneous AT (SCAT), AT beneath the fascia (SFAT), and AT infiltrating muscle groups (IMAT). Muscle attenuation characteristics were determined. RESULTS: Obese DM and obese GT subjects had lower insulin sensitivity than lean GT subjects. SCAT was greater in obesity, yet did not correlate with insulin sensitivity. SFAT was approximately 8% of total thigh AT and correlated with insulin sensitivity. IMAT was highest in obese DM, and although it accounted for only approximately 3% of thigh AT, it was a strong correlate of insulin sensitivity. Mean attenuation was highest in lean subjects and was associated with higher insulin sensitivity. Weight loss reduced the amount of thigh AT, the proportion of thigh IMAT, and the amount of low-density thigh muscle. CONCLUSIONS: SFAT and IMAT are markers of IR in obesity and DM although they are much smaller than SCAT, which does not predict IR. Muscle composition reflecting increased fat content is also associated with IR. PMID- 10731494 TI - Effects of familial predisposition to obesity on energy expenditure in multiethnic prepubertal girls. AB - BACKGROUND: The prevalence of childhood obesity is increasing and the causes of this are unknown. OBJECTIVE: The objective of this study was to determine whether energy expenditure (EE), measured by 24-h calorimetry and doubly labeled water, differed in normal-weight-for-height, multiethnic prepubertal girls with or without a familial predisposition to obesity. DESIGN: Normal-weight, prepubertal white (n = 52), African American (n = 30), and Hispanic (n = 19) girls with a mean (+/-SD) age of 8.5 +/- 0.4 y were studied according to parental leanness and overweight or obesity. The girls were grouped according to whether they had 2 lean parents (n = 30), 2 obese parents (n = 27), or 1 lean and 1 obese parent (n = 44). Basal metabolic rate (BMR), sleeping metabolic rate (SMR), 24-h EE, respiratory quotient, heart rate, and activity were measured by 24-h room calorimetry; free-living total EE (TEE), activity-related EE (AEE), and physical activity level were measured by doubly labeled water. EE was standardized by fat free mass (FFM). RESULTS: There were no significant differences among familial groups in weight, height, fat mass, FFM, or percentage body fat. African American girls had a higher FFM than did white or Hispanic girls (P < 0.05). BMR, SMR, 24 h EE, respiratory quotient, heart rate, and activity levels were not significantly different among familial groups. Additionally, there were no significant familial group differences in TEE, AEE, or physical activity level. However, BMR, SMR, and TEE were lower in African American girls than in white girls (P < 0.05). CONCLUSION: There was no significant difference in EE between normal-weight, multiethnic prepubertal girls predisposed to obesity and those not predisposed to obesity. PMID- 10731495 TI - Dietary composition and physiologic adaptations to energy restriction. AB - BACKGROUND: The concept of a body weight set point, determined predominantly by genetic mechanisms, has been proposed to explain the poor long-term results of conventional energy-restricted diets in the treatment of obesity. OBJECTIVE: The objective of this study was to examine whether dietary composition affects hormonal and metabolic adaptations to energy restriction. DESIGN: A randomized, crossover design was used to compare the effects of a high-glycemic-index (high GI) and a low-glycemic-index (low-GI) energy-restricted diet. The macronutrient composition of the high-GI diet was (as percent of energy) 67% carbohydrate, 15% protein, and 18% fat and that of the low-GI diet was 43% carbohydrate, 27% protein, and 30% fat; the diets had similar total energy, energy density, and fiber contents. The subjects, 10 moderately overweight young men, were studied for 9 d on 2 separate occasions. On days -1 to 0, they consumed self-selected foods ad libitum. On days 1-6, they received an energy-restricted high- or low-GI diet. On days 7-8, the high- or low-GI diets were consumed ad libitum. RESULTS: Serum leptin decreased to a lesser extent from day 0 to day 6 with the high-GI diet than with the low-GI diet. Resting energy expenditure declined by 10.5% during the high-GI diet but by only 4.6% during the low-GI diet (7.38 +/- 0.39 and 7.78 +/- 0.36 MJ/d, respectively, on days 5-6; P = 0.04). Nitrogen balance tended to be more negative, and energy intake from snacks on days 7-8 was greater, with the high-GI than the low-GI diet. CONCLUSION: Diets with identical energy contents can have different effects on leptin concentrations, energy expenditure, voluntary food intake, and nitrogen balance, suggesting that the physiologic adaptations to energy restriction can be modified by dietary composition. PMID- 10731496 TI - Soy sterol esters and beta-sitostanol ester as inhibitors of cholesterol absorption in human small bowel. AB - BACKGROUND: Plant sterols are natural dietary components with serum cholesterol lowering properties. The lowering of serum cholesterol by plant sterols is believed to be the result of an inhibition of cholesterol absorption in the small bowel, although increased bile acid excretion has also been suggested. The difference in effect of saturated and unsaturated plant sterols on cholesterol absorption needs to be elucidated further. OBJECTIVE: The primary aim of this study was to measure small-bowel cholesterol absorption and sterol excretion in addition to hepatic cholesterol synthesis after intake of soy sterol esters and beta-sitostanol ester corresponding to 1.5 g plant sterols/d. DESIGN: Seven ileostomy subjects were studied during a control period and 2 intervention periods when either soy sterol esters or beta-sitostanol ester was added to a basal diet. Ileostomy bags were collected every other hour and frozen immediately for analysis of nutrients and sterols. RESULTS: Cholesterol absorption was 56% (43-65%) in the control period and decreased to 38% (32-46%) in the soy sterol ester period (P = 0.00) and to 39% (30-48%) in the beta-sitostanol ester period (P = 0.00). CONCLUSION: Esterified soy sterols and beta-sitostanol inhibited cholesterol absorption equally, despite the different structures of the plant sterols. PMID- 10731497 TI - Postprandial chylomicrons and VLDLs in severe hypertriacylglycerolemia are lowered more effectively than are chylomicron remnants after treatment with n-3 fatty acids. AB - BACKGROUND: n-3 Fatty acids lower plasma triacylglycerols not only in the fasting state but also in the postprandial state. However, it is not known whether chylomicrons, chylomicron remnants, and VLDLs are all affected equally or whether some lipoprotein species are lowered preferentially. OBJECTIVE: Lipoproteins, including large and small chylomicron remnants, were determined specifically with the aid of a newly developed method involving a combination of size-exclusion chromatography and fluorometric determination of retinyl palmitate, which served as a marker for exogenous fat. DESIGN: Twelve hypertriacylglycerolemic men were treated for 6 wk with 4 capsules containing 85% fish-oil concentrate/d; each capsule contained 850 mg n-3 fatty acid ethyl esters (49.1% eicosapentaenoic acid by wt and 32.2% docosahexaenoic acid by wt). Oral-fat-tolerance tests were performed before and after the treatment. Blood samples were drawn in the fasting state and until 8 h postprandially. RESULTS: Treatment with n-3 fatty acids reduced the fasting VLDL-triacylglycerol concentration by 44% (P < 0.05) and postprandial chylomicrons and VLDLs at 4, 6, and 8 h (P < 0.05) by 49-64% and 36 43%, respectively. Chylomicron remnants were reduced only in the late postprandial phase: large chylomicron remnants by 19% at 6 h and by 43% at 8 h (P < 0.05) and small chylomicron remnants by 31% at 8 h (P < 0.05). CONCLUSION: n-3 Fatty acids effectively lower chylomicrons and VLDLs, but their effect on chylomicron remnants was observed only in the late postprandial phase. PMID- 10731498 TI - Carbohydrates, dietary fiber, and incident type 2 diabetes in older women. AB - BACKGROUND: Dietary carbohydrates may influence the development of type 2 (non insulin-dependent) diabetes, for example, through effects on blood glucose and insulin concentrations. OBJECTIVE: We examined the relations of baseline intake of carbohydrates, dietary fiber, dietary magnesium, and carbohydrate-rich foods and the glycemic index with incidence of diabetes. DESIGN: This was a prospective cohort study of 35988 older Iowa women initially free of diabetes. During 6 y of follow-up, 1141 incident cases of diabetes were reported. RESULTS: Total grain, whole-grain, total dietary fiber, cereal fiber, and dietary magnesium intakes showed strong inverse associations with incidence of diabetes after adjustment for potential nondietary confounding variables. Multivariate-adjusted relative risks of diabetes were 1.0, 0.99, 0.98, 0.92, and 0.79 (P for trend: 0.0089) across quintiles of whole-grain intake; 1.0, 1.09, 1.00, 0.94, and 0.78 (P for trend: 0.005) across quintiles of total dietary fiber intake; and 1.0, 0.81, 0.82, 0.81, and 0.67 (P for trend: 0.0003) across quintiles of dietary magnesium intake. Intakes of total carbohydrates, refined grains, fruit and vegetables, and soluble fiber and the glycemic index were unrelated to diabetes risk. CONCLUSION: These data support a protective role for grains (particularly whole grains), cereal fiber, and dietary magnesium in the development of diabetes in older women. PMID- 10731499 TI - Lactose does not enhance calcium bioavailability in lactose-tolerant, healthy adults. AB - BACKGROUND: There is evidence from animal studies that lactose has a beneficial effect on intestinal calcium absorption. However, data concerning the effect of lactose on calcium absorption in lactose-tolerant adults are inconclusive. OBJECTIVE: Our objective was to investigate the effect of lactose on calcium bioavailability in humans by the use of a stable-strontium test under controlled metabolic conditions. DESIGN: Eleven healthy, lactose-tolerant subjects (8 women, 3 men) randomly received a bolus of 2.27 mmol strontium alone (load A), the bolus with 35 g lactose (load B), or the bolus with 17.5 g glucose and 17.5 g galactose (load C). Blood samples were drawn at 0, 15, 30, 60, 90, 180, 240, and 300 min. Urine specimens were collected during the time intervals -2 to 0, 0-2, 2-4, 4-6, and 6-24 h. RESULTS: Pharmacokinetic parameters of strontium bioavailability were comparable for all 3 loads. In detail, fractional absorption at 240 min for loads A, B, and C was 12.1 +/- 0.7%, 13.0 +/- 1.1%, and 12.2 +/- 0.7%, respectively. Areas under the curve for 0-240 min were 70.8 +/- 6.3, 69.6 +/- 3.5, and 65.8 +/- 5.1 micromol*h/L for loads A, B, and C, respectively (NS). Moreover, fractional strontium excretion values of 5.1 +/- 0.8% (load A), 5.8 +/- 0.4% (load B), and 5.2 +/- 0.8% (load C) were not significantly different. CONCLUSIONS: Lactose does not have a beneficial effect on calcium bioavailability in lactose-tolerant adults. PMID- 10731500 TI - Estimation of nonheme-iron bioavailability from meal composition. AB - BACKGROUND: Considerable data are available on the individual effects of dietary factors on nonheme-iron absorption, but their combined effect when they are present in the same meal is not known. OBJECTIVE: Our objective was to predict the bioavailability of iron from complex meals that are consumed commonly in the United States on the basis of the contents of factors that are known to promote or inhibit food iron absorption. DESIGN: Radioisotopic measurements of nonheme iron absorption from 25 meals were made in 86 volunteer subjects by using extrinsic radioiron labeling. The meal contents of nonheme iron, calcium, ascorbic acid, polyphenols, and phytic acid were determined by biochemical analysis; energy and protein contents were estimated from food-composition tables. Animal tissue content was based on weight or was obtained from the manufacturer. RESULTS: After adjusting iron absorption for differences in iron status, the significant biochemical predictors of iron absorption as determined by multiple regression analysis were the contents of animal tissue (P = 0.0001), phytic acid (P = 0.0001), and ascorbic acid (P = 0. 0441). Collectively, these 3 variables accounted for 16.4% of the variation in absorption. On the basis of the multiple regression analysis, we developed the following equation to estimate iron absorption: Ln absorption, % (adjusted to serum ferritin concentration of 30 microg/L) = 1.9786 + (0.0123 x animal tissue in g) - (0.0034 x phytic acid in mg) + (0.0065 x ascorbic acid in mg). CONCLUSION: For the 25 meals evaluated, only the contents of animal tissue, phytic acid, and ascorbic acid were useful for estimating nonheme-iron absorption. PMID- 10731501 TI - Oligomeric but not monomeric silica prevents aluminum absorption in humans. AB - BACKGROUND: Soluble silica, a ubiquitous component of the diet, may be the natural ligand for dietary aluminum and may prevent its accumulation and toxicity in animals. However, previous studies on the inhibition of aluminum absorption and toxicity by soluble silica have produced conflicting results. We recently identified a soluble silica polymer, oligomeric silica, that has a much higher affinity for aluminum than does monomeric silica and that may be involved in the sequestration of aluminum. OBJECTIVE: By using (26)Al as a tracer, we investigated the effects of oligomeric and monomeric silica on the bioavailability of aluminum (study 1) and compared the availability of silicon from oligomeric and monomeric silica in the human gastrointestinal tract (study 2). DESIGN: In study 1, three healthy volunteers each ingested aluminum alone (control), aluminum with oligomeric silica (17 mg), and aluminum with monomeric silica (17 mg). In study 2, five healthy volunteers ingested both the oligomeric and monomeric forms of silica (34 mg). Serum and urine samples were analyzed for aluminum and silicon. RESULTS: Oligomeric silica reduced the availability of aluminum by 67% (P = 0.01) compared with the control, whereas monomeric silica had no effect (P = 0.40). Monomeric silica was readily taken up from the gastrointestinal tract and then excreted in urine (53%), whereas oligomeric silica was not detectably absorbed or excreted. CONCLUSIONS: The oligomeric, high aluminum-affinity form of soluble silica reduces aluminum availability from the human gastrointestinal tract. Its potential role in the amelioration of aluminum toxicity in other biological systems requires attention. PMID- 10731502 TI - Effect of beta-carotene supplementation and lactation on carotenoid metabolism and mitogenic T lymphocyte proliferation. AB - BACKGROUND: Information is lacking regarding the effects of beta-carotene supplementation, early lactation, or both on circulating carotenoid concentrations and T lymphocyte proliferation. OBJECTIVES: This study investigated the effects of short-term beta-carotene supplementation (30 mg/d for 28 d) during early lactation (days 4-32 postpartum) on circulating carotenoid concentrations and on the T lymphocyte proliferative response to phytohemagglutinin. DESIGN: Subjects aged 19-39 y were paired [lactating (4 d postpartum) and nonlactating (never pregnant, healthy women)] and randomly assigned to receive either beta-carotene or a placebo. During the study, subjects provided eight 24-h food records for analysis with the NUTRITIONIST IV and US Department of Agriculture carotenoid databases. Nonfasting blood samples were collected at baseline and at 28 d. Plasma analysis included quantification of alpha-carotene, beta-carotene, lutein, lycopene, retinol, and alpha-tocopherol, complete differential blood cell counts, and lymphocyte proliferative activity. RESULTS: beta-Carotene supplementation increased beta-carotene (P < 0.001) and alpha-carotene (P < 0.05) concentrations but did not affect lycopene concentrations significantly. Supplemented women showed significant decreases in plasma lutein (P < 0.03), as did lactating subjects (P < 0.02). Neither lactation nor beta-carotene supplementation affected the T lymphocyte proliferative response to phytohemagglutinin. CONCLUSIONS: Our results suggest that beta carotene supplementation as well as some events related to parturition, initiation of lactation, or both alter circulating concentrations of lutein. beta Carotene supplementation does not enhance T lymphocyte immune competence in healthy women. PMID- 10731503 TI - Changes in iron status during pregnancy in peruvian women receiving prenatal iron and folic acid supplements with or without zinc. AB - BACKGROUND: Iron deficiency anemia is the most prevalent nutrient deficiency during pregnancy, yet there are few data on the effect of prenatal iron supplementation in women in developing countries. OBJECTIVE: Our objective was to describe the effect of iron supplementation on hematologic changes during pregnancy, and the effect on those changes of adding zinc to the supplements. DESIGN: Pregnant women were enrolled in a randomized, double-masked study conducted at a hospital in a shantytown in Lima, Peru. Women were supplemented daily from 10-24 wk gestation to 4 wk postpartum with 60 mg Fe and 250 microg folic acid with or without 15 mg Zn. Hemoglobin and ferritin concentrations were measured in 645 and 613 women, respectively, at enrollment, at 28-30 and 37-38 wk gestation, and in the cord blood of 545 neonates. RESULTS: No differences in iron status were detected by supplement type, but hematologic changes were related to initial hemoglobin status. Women with anemia (hemoglobin <110 g/L) showed steady increases in hemoglobin concentration throughout pregnancy whereas women with relatively higher initial hemoglobin concentrations had declining values during mid pregnancy, then rising values by 37-38 wk gestation. Women with an initial hemoglobin concentration >95 g/L showed increases in serum ferritin by the end of the pregnancy. Despite supplementation, women with poorer hematologic status; who were younger, single, and multiparous; and who consumed fewer supplements were more likely to have anemia at the end of pregnancy. CONCLUSIONS: These hematologic changes are congruent with the effects of iron supplementation reported in placebo-controlled trials and the addition of zinc did not significantly affect them. PMID- 10731504 TI - Plasma total homocysteine, pregnancy complications, and adverse pregnancy outcomes: the Hordaland Homocysteine study. AB - BACKGROUND: Total homocysteine (tHcy) measured in serum or plasma is a marker of folate status and a risk factor for cardiovascular disease. OBJECTIVE: Our objective was to investigate associations between tHcy and complications and adverse outcomes of pregnancy. DESIGN: Plasma tHcy values measured in 1992-1993 in 5883 women aged 40-42 y were compared with outcomes and complications of 14492 pregnancies in the same women that were reported to the Medical Birth Registry of Norway from 1967 to 1996. RESULTS: When we compared the upper with the lower quartile of plasma tHcy, the adjusted risk for preeclampsia was 32% higher [odds ratio (OR): 1. 32; 95% CI: 0.98, 1.77; P for trend = 0.02], that for prematurity was 38% higher (OR: 1.38; 95% CI: 1.09, 1.75; P for trend = 0.005), and that for very low birth weight was 101% higher (OR: 2.01; 95% CI: 1.23, 3.27; P for trend = 0.003). These associations were stronger during the years closest to the tHcy determination (1980-1996), when there was also a significant relation between tHcy concentration and stillbirth (OR: 2.03; 95% CI: 0.98, 4.21; P for trend = 0.02). Neural tube defects and clubfoot had significant associations with plasma tHcy. Placental abruption had no relation with tHcy quartile, but the adjusted OR when tHcy concentrations >15 micromol/L were compared with lower values was 3.13 (95% CI: 1.63, 6. 03; P = 0.001). CONCLUSION: Elevated tHcy concentration is associated with common pregnancy complications and adverse pregnancy outcomes. PMID- 10731505 TI - Use of infrared thermographic calorimetry to determine energy expenditure in preterm infants. AB - BACKGROUND: Measurement of infant energy expenditure in the clinical setting is difficult and is rarely done. Both indirect and direct calorimetry require long measurement periods and frequent calibration. OBJECTIVE: The objective of this study was to validate in infants a newly developed method of determining energy expenditure, infrared thermographic calorimetry (ITC), against an established method, respiratory indirect calorimetry (IC). ITC measures mean infant body surface temperature. ITC was used in conjunction with heat loss theory to calculate radiant, convective, evaporative, and conductive heat losses and thereby determine total energy expenditure. DESIGN: Ten healthy preterm infants were studied by obtaining concurrent ITC and IC measurements over a 3.5-5.5-h study period. Continuous IC measurements were compared with ITC measurements taken every 10 min during study periods. IC values were summed over 10-min intervals covering the 5 min before and 5 min after each ITC measurement, to allow comparisons between the 2 methods. RESULTS: Comparison of paired ITC and IC mean measurements for all 10 infants over the entire study period showed no significant difference between the 2 methods. However, individual paired IC and ITC values were significantly different for 7 of 10 infants. The overall mean difference between the 2 methods was 1.3%. CONCLUSIONS: ITC is an accurate, noninvasive method for measurement of heat loss and energy expenditure in healthy preterm infants, and therefore it may be a useful clinical and research tool. PMID- 10731506 TI - Concentrations of riboflavin and related organic acids in children with protein energy malnutrition. AB - BACKGROUND: Riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) concentrations have been little studied in cases of malnutrition. OBJECTIVE: Our objective was to investigate the effects of malnutrition on riboflavin status and riboflavin's relation with thyroid hormones and concentrations of urinary organic acids. DESIGN: Malnourished children from the savannah in Benin (group S, n = 30) and the coast in Togo (group C, n = 30), as well as 24 control subjects from both regions, were studied. Blood riboflavin, FMN, and FAD were analyzed by HPLC; urinary organic acids were analyzed by gas chromatography-mass spectrometry. RESULTS: Children in group S were more severely malnourished than children in group C. Triiodothyronine concentrations were lower in group S than in group C or the control group (1.12 +/- 0.24 compared with 1.74 +/- 0.18 and 2.92 +/- 0.19 nmol/L, respectively; P < 0.0001). Plasma riboflavin concentrations in group S were higher than those in group C or the control group (66.90 +/- 12.75 compared with 28.09 +/- 9.12 and 20.08 +/- 3.03 nmol/L, respectively; P < 0.001). Plasma FAD concentrations in group S were lower than those in group C or the control group (31.57 +/- 10.19 compared with 59.02 +/- 5.60 and 65.35 +/- 5.23 nmol/L, respectively; P < 0.0001). Dicarboxylic aciduria was higher in group C than in group S or the control subjects. CONCLUSIONS: Children in group S had low triiodothyronine concentrations and low conversion of plasma riboflavin into its cofactors, leading to a plasma FAD deficiency. Plasma FAD was not correlated with urinary dicarboxylic acid concentrations. PMID- 10731507 TI - Mediterranean diet and age with respect to overall survival in institutionalized, nonsmoking elderly people. AB - BACKGROUND: In studies from Italy and Greece, a Mediterranean dietary pattern was shown to favorably affect life expectancy in the elderly population. This pattern is thought to reduce the risk of cancer in addition to being cardioprotective. OBJECTIVE: The objective of this study was to evaluate the interactive effects of the Mediterranean diet and age with respect to survival after controlling for several other variables that could be considered as confounders: age, sex, body mass index, albumin concentration, physical activity, self-assessment of health, and dieting in response to chronic conditions. DESIGN: This was a cohort study involving 161 nonsmoking elderly subjects (74 subjects aged <80 y and 87 subjects aged > or =80 y) living in Spain. The subjects were followed up for > or =9 y. Diet was assessed with a semiquantitative food-frequency questionnaire. RESULTS: A diet score based on 8 characteristics of the traditional diet in the Mediterranean region was associated with a significant reduction in overall mortality in elderly subjects aged <80 y but not in subjects aged > or =80 y. A unit increase in the diet score predicted a 31% reduction in mortality in subjects aged <80 y (95% CI: 7%, 57%). CONCLUSION: Efforts to promote adherence to the Mediterranean dietary pattern appear to be worthwhile in persons aged <80 y, in whom the diet predicts survival, but we do not have any available evidence that such a diet benefits subjects aged > or =80 y. PMID- 10731508 TI - Serum folate and the severity of atrophy of the neocortex in Alzheimer disease: findings from the Nun study. AB - BACKGROUND: Previous studies suggested that low concentrations of folate in the blood are related to poor cognitive function, dementia, and Alzheimer disease related neurodegeneration of the brain. OBJECTIVE: Our aim was to determine whether serum folate is inversely associated with the severity of atrophy of the neocortex. DESIGN: Nutrients, lipoproteins, and nutritional markers were measured in the blood of 30 participants in the Nun Study from one convent who later died when they were 78-101 y old (mean: 91 y). At autopsy, several neuropathologic indicators of Alzheimer disease were determined, including the degree of atrophy of 3 lobes of the neocortex (frontal, temporal, and parietal) and the number of neocortical Alzheimer disease lesions (ie, senile plaques and neurofibrillary tangles) as assessed by a neuropathologist. RESULTS: The correlation between serum folate and the severity of atrophy of the neocortex was -0.40 (P = 0.03). Among a subset of 15 participants with significant numbers of Alzheimer disease lesions in the neocortex, the correlation between folate and atrophy was -0.80 (P = 0.0006). Atrophy may be specific to low folate because none of the 18 other nutrients, lipoproteins, or nutritional markers measured in the blood had significant negative correlations with atrophy. CONCLUSIONS: Among elderly Catholic sisters who lived in one convent, ate from the same kitchen, and were highly comparable for a wide range of environmental and lifestyle factors, low serum folate was strongly associated with atrophy of the cerebral cortex. Definitive evidence for this relation and its temporal sequence awaits the findings of other studies. PMID- 10731509 TI - Mineral water as a source of dietary calcium: acute effects on parathyroid function and bone resorption in young men. AB - BACKGROUND: Calcium is a major component of mineralized tissues and is required for normal growth and maintenance of bone. Epidemiologic studies showed that a large percentage of the population fails to meet the currently recommended guidelines for optimal calcium intake. OBJECTIVE: The present study was designed to determine whether high-calcium mineral water is an efficient additional source of dietary calcium. DESIGN: Twelve healthy young men (mean +/- SD age: 21.1 +/- 1.2 y) ingested in a randomized order either 0.5 L of a mineral water containing 344 mg Ca/L or 0.5 L of a mineral water with a very low concentration of calcium (<10 mg/L) as a control. Blood samples were drawn before and 1, 2, 3, and 4 h after intake of the water. Urine was collected for 2 h before and every 2 h for 4 h after ingestion of the water. Serum concentrations of intact parathyroid hormone (iPTH) and serum concentrations and urinary excretion of a recently developed biochemical marker of bone resorption, type 1 collagen cross-linked C telopeptide (CTx), were measured. RESULTS: Serum iPTH was significantly (P < 0.002) lower after ingestion of high-calcium water than after ingestion of the control. There was a significant (P = 0.01) progressive decrease in urinary CTx after ingestion of the high-calcium water, whereas after ingestion of low-calcium water the changes were modest and not significant. The fall in serum CTx concentrations was 34.7% 3 h after ingestion of high-calcium water, compared with 17.6% with the control. The decreases in serum CTx concentrations were significantly (P < 0.05) lower 1, 2, 3, and 4 h after ingestion of high-calcium water than after ingestion of the control. CONCLUSION: The present study showed that one oral intake of water containing a very moderate dose of calcium (172 mg) acutely inhibited iPTH secretion and bone resorption. PMID- 10731510 TI - Tea drinking and bone mineral density in older women. AB - BACKGROUND: High caffeine intake is reportedly a risk factor for reduced bone mineral density (BMD) in women. Most studies, however, are from populations in which coffee drinking predominates and is the major caffeine source. Tea contains caffeine but also has other nutrients, such as flavonoids, that may influence bone mass in different ways. OBJECTIVE: We examined the relation between tea drinking and BMD in older women in Britain, where tea drinking is common. METHODS: We measured BMD at the lumbar spine, femoral neck, greater trochanter, and Ward's triangle in 1256 free-living women aged 65-76 y in Cambridge, United Kingdom. Tea drinking was assessed by self-completed questionnaire and women were categorized as tea drinkers or non-tea drinkers. RESULTS: There were 1134 tea drinkers (90.3%) and 122 non-tea drinkers (9.7%). Compared with non-tea drinkers, tea drinkers had significantly greater ( approximately 5%) mean BMD measurements, adjusted for age and body mass index, at the lumbar spine (0.033 g/cm(2); P = 0.03), greater trochanter (0.028 g/cm(2); P = 0.004), and Ward's triangle (0.025 g/cm(2); P = 0.02). Differences at the femoral neck (0.013 g/cm(2)) were not significant. These findings were independent of smoking status, use of hormone replacement therapy, coffee drinking, and whether milk was added to tea. CONCLUSIONS: Older women who drank tea had higher BMD measurements than did those who did not drink tea. Nutrients found in tea, such as flavonoids, may influence BMD. Tea drinking may protect against osteoporosis in older women. PMID- 10731511 TI - Arachidonic acid concentrations in patients with Crohn disease. PMID- 10731513 TI - Whole grains and coronary heart disease. PMID- 10731515 TI - Extracellular matrix components in retrocorneal fibrous membrane in comparison to corneal endothelium and Descemet's membrane. AB - PURPOSE: To investigate the extracellular matrix macromolecules found in Descemet's membrane and in retrocorneal fibrous membrane (RCFM), and to examine whether the corneal endothelium has the capacity to produce both basement and non basement membrane phenotypes. METHODS: Rabbit corneas with and without RCFM were analyzed by immunofluorescence using antibodies to 8 different collagens (basement membrane collagens: types IV and VIII; fibrillar collagens: types I and III; interfibrillar collagens: type VI and two spliced variant forms of type XII and one anchoring fiber: type VII), proteoglycans (perlecan and decorin), (beta)ig-h3 and laminin-1. RESULTS: Normal corneal endothelium stains positively for all of the tested collagen types except type VII collagen. On the other hand, Descemet's membrane reacts positively only to the type IV collagen antibody. When non-collagenous components in normal cornea were examined, corneal endothelium stained positively for perlecan, decorin, (beta)ig-h3 and laminin, whereas Descemet's membrane staining for these proteins was negative. When collagenous components of RCFM were examined, RCFM stained positively for all of the tested collagen types except type IV collagen. When non-collagenous components of RCFM were examined, RCFM demonstrated a strong positive staining with decorin, (beta)ig-h3 and laminin, while perlecan staining was weak. CONCLUSIONS: These observations suggest that corneal endothelium is able to produce both basement membrane phenotypes and non-basement membrane, fibrillar phenotypes. This in vivo study confirms our in vitro model of endothelial mesenchymal transformation, in which corneal endothelial cells are transformed to fibroblasts that are responsible for fibrosis. PMID- 10731516 TI - Apoptosis induced by chelation of intracellular zinc is associated with depletion of cellular reduced glutathione level in rat hepatocytes. AB - Zn(2+) has multiple implications in cellular metabolism, including free radicals metabolism and cell death by apoptosis. In the present study, we examined the role of Zn(2+) in the regulation of apoptosis in cultured rat hepatocytes. The chelation of Zn(2+) by a membrane permeable metal ion chelator, N, N, N', N' tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), induced apoptosis. Addition of ZnSO(4) prevented TPEN-induced apoptosis. Unlike the effect of TPEN, a membrane impermeable metal ion chelator, diethylenetriamine pentaacetic acid (DTPA), did not induce apoptosis, indicating that chelation of intracellular Zn(2+) was required to trigger apoptosis. Caspase-3-like proteolytic activity, a general biochemical mediator of apoptosis in a variety of cells and tissues, was also activated with the treatment of TPEN but not DTPA. TPEN treatment, but not DTPA, also resulted in the depletion of intracellular reduced glutathione (GSH) but addition of Zn(2+) recovered the GSH level. N-acetyl-L-cysteine (NAC), a thiol antioxidant, prevented TPEN-induced apoptosis. These results taken together suggest that intracellular Zn(2+) interfere with the apoptosis process, possibly through the regulation of cellular redox potential involving GSH. PMID- 10731517 TI - Differential inhibition of human CYP3A4 and Candida albicans CYP51 with azole antifungal agents. AB - The inhibition by azole antifungals of human cytochrome CYP3A4, the major form of drug metabolising enzyme within the liver, was compared with their inhibitory activity against their target enzyme, Candida albicans sterol 14alpha-demethylase (CYP51), following heterologous expression in Saccharomyces cerevisiae. IC(50) values for ketoconazole and itraconazole CYP3A4 inhibition were 0.25 and 0. 2 microM. These values compared with much lower doses required for the complete inhibition of C. albicans CYP51, where IC(50) values of 0.008 and 0.0076 microM were observed for ketoconazole and itraconazole, respectively. Additionally, stereoselective inhibition of CYP3A4 and CYP51 was observed with enantiomers of the azole antifungal compounds diclobutrazol and SCH39304. In both instances, the RR(+) configuration at their asymmetric carbon centres was most active. Interestingly, the SS(-) enantiomeric form of SCH39304 was inactive and failed to bind CYP3A4, as demonstrable by Type II binding spectra. PMID- 10731518 TI - Anti-oxidant, pro-oxidant properties of tannic acid and its binding to DNA. AB - Tannic acid has numerous food and pharmacological applications. It is an additive in medicinal products, and is used as a flavouring agent and as an anti-oxidant in various foods and beverages. We have previously shown that tannic acid in the presence of Cu(II) causes DNA degradation through generation of reactive oxygen species. On the other hand, it exhibits antimutagenic and anticarcinogenic activities, and induces apoptosis in animal cells. It is known that most plant derived polyphenolic anti-oxidants also act as pro-oxidants under certain conditions. In this paper, we compare the anti-oxidant and pro-oxidant properties of tannic acid and its structural component gallic acid. It is shown that tannic acid is the most efficient generator of the hydroxyl radical in the presence of Cu(II), as compared with gallic acid and its analogues syringic acid and pyrogallol. The anti-oxidant activity of tannic acid was studied by its effect on hydroxyl radical and singlet oxygen mediated cleavage of plasmid DNA. Again, tannic acid provided the maximum protection against cleavage, while gallic acid and its structural analogues were found to be non-inhibitory or partially inhibitory. The results suggest that the structural features of tannic acid that are important for its anti-oxidant action are also those that contribute to the generation of hydroxyl radicals in the presence of Cu(II). Restriction analysis of treated phage DNA and thermal melting profiles of calf thymus DNA indicated that tannic acid strongly binds to DNA. Indirect evidence indicates that modification of DNA bases may also occur. PMID- 10731519 TI - Orally administrated cerium chloride induces the conformational changes of rat hemoglobin, the hydrolysis of 2,3-DPG and the oxidation of heme-Fe(II), leading to changes of oxygen affinity. AB - The structure and oxygen affinity of hemoglobin from erythrocytes of CeCl(3) fed Wistar rats in the dose range of 0.2-20.0 mg/kg body weight/day were investigated by means of various spectroscopic methods. The changes in oxygen saturation curves of hemoglobin are dependent upon both feeding dose and feeding time. After 40 days feeding with 20 mg CeCl(3)/kg body weight/day, the curve changed to a double sigmoid shape and the oxygen affinity in low oxygen pressure increases. It regained the sigmoid form after 80 days feeding, but the degree of oxygen saturation in higher oxygen pressure became higher than that in the control. These results indicate that CeCl(3) can increase the oxygen affinity of hemoglobin of rat erythrocytes. This effect is further demonstrated by the analysis of Mossbauer spectra of erythrocytes. Increase of hemoglobin content in erythrocytes was found in rats fed with CeCl(3). It might be the offset response to the poor oxygen-releasing capability of the hemoglobin. CD and FT-IR deconvoluted spectra indicate that secondary structures of hemoglobin have remarkable changes, characterized by a gradual decrease of alpha-helix content, in a dose- and feeding time-dependent fashion. Meanwhile, the 31P NMR spectra demonstrate that the level of 2,3-diphosphoglyceric acid (2,3-DPG) in erythrocytes, an allosteric regulator of oxygen release from hemoglobin, decreases due to its hydrolysis. In addition, the Mossbauer and ESR spectra show clearly that a fraction of the heme-iron changes from Fe (II) to Fe (III) in CeCl(3) fed rats. The results indicate that the oral administration of CeCl(3) leads to a microenvironment changes of heme in intracellular hemoglobin. Oxygen affinity changes might be attributed to a series of events triggered by the binding of Ce (III) to hemoglobin and 2,3-DPG, including conformational changes of hemoglobin and 2,3-DPG hydrolysis, respectively and also the partial transformation from heme-Fe (II) to heme-Fe (III). PMID- 10731520 TI - Gender-specific metabolism of benz[a]anthracene in hepatic microsomes from Long Evans and Hooded Lister rats. AB - The cytochrome P450 isoforms responsible for the regio-selective metabolism of benz[a]anthracene (BA) are poorly defined but as with other polycyclic aromatic hydrocarbons (PAHs) may include members of the CYP2C sub-family. Since the expression of some of these is regulated in a gender-specific manner and may be altered by age, rat strain or by phenobarbital treatment, the effects of these variables on metabolism of BA to diols was investigated. These studies used hepatic, microsomal membranes from immature and adult Long-Evans rats and adult Hooded Lister rats. BA-diols were resolved by normal phase HPLC into three discrete peaks identified as benz[a]anthracene-5,6-diol (BA-5,6-diol), benz[a]anthracene-10, 11-diol (BA-10,11-diol) and a mixture of benz[a]anthracene 3,4- and -8,9-diols (BA-3,4-diol and BA-8,9-diol and termed Peak(3/8)). Significant gender-related differences were found in the rates of diol formation in adults of both the Long-Evans and Hooded Lister rat strains. Formation of BA 10,11-diol and to a lesser extent the components of Peak(3/8) were greater in the male compared to female animals by factors of at least 14 and two, respectively. An age-dependent effect is also observed in the Long-Evans rat since these differences are still apparent in prepubertal animals but to a lesser extent (gender ratio male:female BA-10,11-diol 9X; Peak(3/8) 1.4X). In contrast BA-5,6 diol was formed at similar rates by membranes from female and male rats whether mature (Long-Evans and Hooded Lister) or immature (Long-Evans). Phenobarbital treatment of the adult Long-Evans rats resulted in a moderate increase in the formation of each diol other than at the 10,11-position and the induction was not gender specific. The rate of formation of BA-10, 11-diol was decreased in phenobarbital-treated male rats suggesting modulation of a male specific isoform. Measurement of microsomal epoxide hydrolase revealed no gender or age differences and suggests that this enzyme is not rate limiting in BA-diol formation and thus is not responsible for the differences in BA-diol formation observed. The results suggest that CYP2C11 along with a male-specific isoenzyme not regulated by age are important in the formation of BA-10,11-diol and a component(s) of Peak(3/8) in males. CYPs 2B2 and/or 2C6 appear to be involved in formation of BA-5,6-diol in male and female. Identification of the CYPs involved in the regio-selective metabolism of BA may lead to an explanation of the lower carcinogenic potency of this PAH compared to dimethylbenz[a]anthracene and this study provides novel clues concerning the identities of the CYPs, which are important. PMID- 10731521 TI - Protective mechanism of metallothionein against copper-1, 10-phenanthroline induced DNA cleavage. AB - Metallothionein (MT) has been shown to protect DNA against cleavage induced by a variety of mutagenic agents. The mechanism has been attributed to its ability to either chelate transitional metals that participate in the Fenton reaction, or scavenge free radicals by means of the abundant cystenyl residues of the proteins. In the present study, the protective action of MT against DNA cleavage by the copper-1,10-phenanthroline [(OP)(2)Cu(+)] complex was studied in situ. At 0.1 microM, MT inhibited the (OP)(2)Cu(+) induced DNA cleavage by about 50% (IC(50) approximately 0.1 microM). At 2.5 microM, the cleavage activity was completely inhibited. Similar to MT, cysteine can protect against DNA cleavage by (OP)(2)Cu(+) (IC(50) of approximately 3 mM), however, its action was 1500-fold less efficient than MT. The combined action of MT and cysteine was additive. Reduced glutathione (1 and 10 mM) did not protect the (OP)(2)Cu(+) induced DNA cleavage. Sodium azide could inhibit the cleavage only at high concentrations (IC(40) approximately 25 mM). Spectrophotometric analysis showed that MT can inhibit the formation of the DNA[(OP)(2)Cu(+)] complex possibly by chelating Cu. It can also cause a dissociation of the complex after it was formed. In the later case, the mechanism through which MT protects against the DNA cleavage might occur when MT fitted in closely with the complex, competing with the hydroxyl groups of the nucleotides base for Cu, which, in turn, terminate the Fenton-like free radical reaction. PMID- 10731522 TI - Cytochrome P450 2E1 is the primary enzyme responsible for low-dose carbon tetrachloride metabolism in human liver microsomes. AB - We examined which human CYP450 forms contribute to carbon tetrachloride (CCl(4)) bioactivation using hepatic microsomes, heterologously expressed enzymes, inhibitory antibodies and selective chemical inhibitors. CCl(4) metabolism was determined by measuring chloroform formation under anaerobic conditions. Pooled human microsomes metabolized CCl(4) with a K(m) of 57 microM and a V(max) of 2.3 nmol CHCl(3)/min/mg protein. Expressed CYP2E1 metabolized CCl(4) with a K(m) of 1.9 microM and a V(max) of 8.9 nmol CHCl(3)/min/nmol CYP2E1. At 17 microM CCl(4), a monoclonal CYP2E1 antibody inhibited 64, 74 and 83% of the total CCl(4) metabolism in three separate human microsomal samples, indicating that at low CCl(4) concentrations, CYP2E1 was the primary enzyme responsible for CCl(4) metabolism. At 530 microM CCl(4), anti-CYP2E1 inhibited 36, 51 and 75% of the total CCl(4) metabolism, suggesting that other CYP450s may have a significant role in CCl(4) metabolism at this concentration. Tests with expressed CYP2B6 and inhibitory CYP2B6 antibodies suggested that this form did not contribute significantly to CCl(4) metabolism. Effects of the CYP450 inhibitors alpha naphthoflavone (CYP1A), sulfaphenazole (CYP2C9) and clotrimazole (CYP3A) were examined in the liver microsome sample that was inhibited only 36% by anti-CYP2E1 at 530 microM CCl(4). Clotrimazole inhibited CCl(4) metabolism by 23% but the other chemical inhibitors were without significant effect. Overall, these data suggest that CYP2E1 is the major human enzyme responsible for CCl(4) bioactivation at lower, environmentally relevant levels. At higher CCl(4) levels, CYP3A and possibly other CYP450 forms may contribute to CCl(4) metabolism. PMID- 10731524 TI - Reproductive medicine and research: contract laboratories vs. the university based researcher. PMID- 10731523 TI - Evaluating strategies for improving ovarian response of the poor responder undergoing assisted reproductive techniques. AB - OBJECTIVE: To assess the efficacy of various controlled ovarian hyperstimulation (COH) regimens in the prior poor-responder patient preparing for assisted reproductive techniques. DESIGN: English-language literature review. PATIENT(S): Candidates for assisted reproductive techniques who had been defined as having a prior suboptimal response to standard COH regimens. INTERVENTION(S): A variety of regimes are reviewed, including increased gonadotropin doses, change of gonadotropins, adjunctive growth hormone (GH), luteal phase (long) GnRH agonist (GnRH-a) initiation, early follicular phase (flare) GnRH-a initiation, low-dose luteal phase (ultrashort) GnRH-a initiation, progestin pretreatment, and microdose flare GnRH-a initiation. MAIN OUTCOME MEASURE(S): Maximal serum E(2) levels, follicular development, dose, and duration of gonadotropin therapy, cycle cancellation rates, oocytes retrieved, embryos transferred, and clinical and ongoing pregnancy rates. RESULT(S): A lack of uniformity in definition of the poor responder and of prospective randomized trials make data interpretation somewhat difficult. Of the varied strategies proposed, those that seem to be more uniformly beneficial are microdose GnRH-a flare and late luteal phase initiation of a short course of low-dose GnRH-a discontinued before COH. CONCLUSION(S): No single regimen will benefit all poor responders. General acceptance of uniform definitions and performance of large-scale prospective randomized trials are critical. Development of a reliable precycle screen will allow effective differentiation among normal responders, poor responders, and those who will not conceive with their own oocytes. PMID- 10731525 TI - Effects of human follicular fluid on the capacitation and motility of human spermatozoa. AB - OBJECTIVE: To investigate the capacitation and motility kinetics of spermatozoa treated with human follicular fluid (FF). DESIGN: Controlled, experimental laboratory study. SETTING: University-based gynecology unit. PATIENT(S): Human FF was collected from women undergoing assisted reproductive treatment. Semen samples were obtained from men visiting subfertility clinics. INTERVENTION(S): Spermatozoa were incubated with human FF under various experimental conditions. Spermatozoa incubated with Earle's balanced salt solution were used as the control. MAIN OUTCOME MEASURE(S): Chlortetracycline staining patterns and sperm motility parameters. RESULT(S): The rate of capacitation in the human FF-treated spermatozoa was significantly higher than that in the control spermatozoa after 1 hour and 3 hours of treatment. The percentage of acrosome-reacted spermatozoa also was significantly higher after human FF treatment than after control treatment. These effects of human FF were dose-dependent. Human FF-treated spermatozoa maintained their velocities at the zero-hour level for 5 hours, whereas the velocities of the control spermatozoa decreased significantly after 1 hour. Human FF treatment significantly increased the beat cross-frequency above the rate at zero hour for 5 hours. The hyperactivation of the human FF-treated spermatozoa remained stable for 3 hours, whereas that of the control spermatozoa decreased significantly after 1 hour of incubation. Significantly more human FF treated spermatozoa underwent hyperactivation than did control spermatozoa after 1 hour and 3 hours of treatment. The effects of human FF on beat cross-frequency and hyperactivation were dose-dependent. CONCLUSION(S): Human FF promotes capacitation and the acrosome reaction within a short period. It also stimulates or maintains various sperm motility parameters. PMID- 10731526 TI - The rate at which human sperm are immobilized and killed by mild acidity. AB - OBJECTIVE: To determine the rate at which mild acidity immobilizes and kills human sperm and to evaluate an acidic microbicide, BufferGel, for sperm immobilization. DESIGN: Controlled in vitro study. SETTING: An academic research university and hospital andrology lab. PATIENT(S): Eight volunteer male sperm donors. INTERVENTION(S): Semen samples were treated with hydrochloric acid (HCl) or BufferGel. MAIN OUTCOME MEASURE(S): Sperm motility was measured by using a computerized automated semen analyzer and video microscopy. Sperm membrane permeability and intracellular pH were measured by using fluorescent techniques. RESULT(S): In semen acidified with HCl to pH 4.0, sperm were rapidly immobilized (within 1 min) and were irreversibly immobilized (killed) within 10 minutes. The speed of immobilization and of killing were both linearly proportional to hydrogen ion activity over a pH range of 7.5-4.0. Across the same range, the intracellular pH of human sperm equilibrated to within 0.5 pH units of extracellular pH within 1-2 minutes. BufferGel immobilized sperm significantly faster than HCl from pH 4.0-6.0. CONCLUSION(S): Exposure to mild acidity rapidly acidifies the intracellular pH of human sperm and is rapidly spermicidal. BufferGel accelerates acid immobilization of sperm. PMID- 10731527 TI - Cryopreservation of human spermatozoa within human or mouse empty zona pellucidae. AB - OBJECTIVE: To compare the empty zona pellucidae (ZP) of different species for use in the cryopreservation of spermatozoa. DESIGN: Prospective study. SETTING: An IVF unit of a medical center. PATIENT(S): Patients with azoospermia, oligoasthenozoospermia, or normal spermatozoa. INTERVENTION(S): Human and mouse ZP were prepared by evacuating the cytoplasm of oocytes or embryos. The evacuated ZP were injected with spermatozoa from patients with severe oligoasthenozoospermia and from healthy, fertile men. After the freezing and thawing procedure, the spermatozoa were aspirated outside the ZP. MAIN OUTCOME MEASURE(S): The number of spermatozoa per ZP, the number of motile sperm before freezing and after thawing, the number of sperm lost per ZP after freezing, and the sperm recovery rate were compared according to the different origins of the ZP and the sperm. RESULT(S): The number of spermatozoa, number of motile sperm before freezing and after thawing, number of sperm lost per ZP, and sperm recovery rate were comparable in all groups. The total mean number of motile sperm before freezing and after thawing, the mean number of nonmotile sperm after thawing, the mean number of sperm lost after thawing, and the sperm recovery rate were 14.5%, 11. 8%, 1.0%, 1.5%, and 82%, respectively. CONCLUSION(S): Zona pellucidae are an ideal vehicle for the cryopreservation of sperm collected by testicular sperm extraction or microsurgical epididymal sperm aspiration or from patients with severe oligoasthenozoospermia. There were no differences when human and mouse ZP were used for sperm storage. PMID- 10731528 TI - Ultrastructural and immunohistochemical study of basal lamina of the testis in adolescent varicocele. AB - OBJECTIVE: To evaluate a possible involvement of the extracellular matrix (ECM) in the testes of adolescents with varicocele. DESIGN: Cross-sectional study. SETTING: University-based medical center. PATIENT(S): Twenty-four adolescents aged between 13 and 18 years underwent surgical treatment for repair of left idiopathic varicocele. INTERVENTION: A testis biopsy was performed at time of surgery. MAIN OUTCOME MEASURE(S): Transmission electron microscopy study of basal lamina and immunofluorescence studies of collagen type IV and laminin, two major components of basal lamina. RESULT(S): Transmission electron microscopy observations showed an uneven profile of the basal lamina with a variable thickness. Immunofluorescence studies showed an irregular immunofluorescent line that appeared interrupted in some observations. Collagen type IV showed some areas of strong immunostaining with other areas with reduced immunoreactivity. CONCLUSION(S): Our ultrastructural and immunohistochemical observations highlight focal damage at the level of peritubular basal lamina, but this damage is not as severe as that described in adult varicocele. Initial involvement of basal lamina could represent one of the mechanisms responsible for varicocele-induced histologic alterations of the testes. PMID- 10731529 TI - Telomerase activity and expression of telomerase subunits in the testicular tissue of infertile patients. AB - OBJECTIVE: Determination of telomerase activity and the expression of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) in the testicular tissue of patients with infertility arising from various causes. DESIGN: Prospective observational study. SETTING: A university hospital. PATIENT(S): Thirty-three patients with azoospermia arising from various causes. There were 12 testicular biopsy specimens from patients with Sertoli cell-only syndrome, 9 from patients with maturation arrest, and 12 from patients with obstructive azoospermia and normal histologic findings. INTERVENTION(S): Thirty three testicular biopsies. MAIN OUTCOME MEASURE(S): Correlation of histologic findings at testicular biopsy with telomerase activity, hTERT, and hTR. RESULT(S): All 12 biopsy specimens from patients with obstructive azoospermia were positive for telomerase activity, hTR, and hTERT. Biopsy specimens from the 9 patients with maturation arrest were positive for telomerase activity in 8 cases, hTR in 9 cases, and hTERT in 5 cases. None of the patients with Sertoli cell-only syndrome showed either telomerase activity or hTERT, but all of them showed hTR. CONCLUSION(S): Telomerase activity and evidence of hTERT in testicular tissue are highly sensitive and highly specific markers of gametogenesis, which could gain in importance as part of the fertility workup before microinjection procedures. PMID- 10731530 TI - Hypothalamic-pituitary-adrenal axis sensitivity to opioids in women with polycystic ovary syndrome. AB - OBJECTIVE: To evaluate the influence of the opioid system on the hypothalamic pituitary-adrenal axis in women with polycystic ovary syndrome (PCOS). DESIGN: Controlled clinical study. SETTING: Academic research environment. PATIENT(S): Eight lean and 12 obese women with PCOS, and seven lean and 5 obese control subjects. INTERVENTION(S): Each patient received an i.v. bolus of naloxone at a dose of 125 microgram per kilogram of body weight; 48 hours later, each patient received 16 mg of loperamide p.o. MAIN OUTCOME MEASURE(S): Samples were collected for 2 hours for the naloxone test and for 3 hours for the loperamide test. Levels of adrenocorticotropic hormone (ACTH) and cortisol were measured in all plasma samples. RESULT(S): The obese women with PCOS had a greater ACTH and cortisol response to opiate blockade than either the lean women with PCOS or the control subjects, but there was no difference between the lean or obese control subjects and the lean women with PCOS. There was no difference in the responsiveness of the hypothalamic-pituitary-adrenal axis to loperamide between the PCOS and control groups. CONCLUSION(S): The data indicate that the sensitivity of the hypothalamic-pituitary-adrenal axis to opioids cannot be altered in women with PCOS. However, abnormalities of the hypothalamic-pituitary-adrenal axis in women with PCOS could be central in origin, as suggested by the effects of naloxone administration, and probably are related to the anthropometric characteristics of these hyperandrogenic patients. PMID- 10731532 TI - Serum testosterone levels decrease in middle age in women with the polycystic ovary syndrome. AB - OBJECTIVE: To determine whether testosterone levels change as women with the polycystic ovary syndrome (PCOS) grow older. DESIGN: A follow-up cross-sectional study of a cohort of women with PCOS identified up to 20-25 years ago. SETTING: Women with PCOS were recruited primarily from practice records between 1970 and 1990. Voter registration tapes and household directories were used to identify age-, race-, and neighborhood-matched controls. PARTICIPANT(S): Eighty-four women with PCOS, 20-57 years of age, and 37 age-matched controls participating in a study of the risk for cardiovascular disease in women with PCOS. INTERVENTION(S): Clinical data were collected by questionnaire and fasting blood samples were obtained randomly throughout the menstrual cycle. MAIN OUTCOME MEASURE(S): Total and non-SHBG-bound testosterone levels. RESULT(S): Total and non-SHBG-bound testosterone levels were similar in women with PCOS who were 20-42 years of age but were reduced by approximately 50% among women 42-47 years of age and remained stable in women older than 47 years of age. Testosterone levels were increased in younger and older women with PCOS compared with controls but were similar to controls in women 42-47 years of age. CONCLUSION(S): Hyperandrogenism partly resolves before menopause in women with PCOS. This change may explain the tendency of women with PCOS to cycle regularly as they grow older. Testosterone levels remain elevated in older women with PCOS, however, and may contribute to their increased risk for cardiovascular disease, endometrial cancer, and other diseases. PMID- 10731531 TI - Comparison of Diane 35 and Diane 35 plus finasteride in the treatment of hirsutism. AB - OBJECTIVE: To compare the clinical efficacy of an original combined therapy with cyproterone acetate, 2 mg, and ethinylestradiol, 35 microgram (Diane 35), plus finasteride (5 mg) for 2 weeks per month with that of Diane 35 alone in hirsute women. DESIGN: Prospective randomized, single-blinded study. SETTING: Outpatients in an academic research environment. PATIENT(S): Fifty women with idiopathic hirsutism (IH) or the polycystic ovary syndrome (PCOS). INTERVENTION(S): Group 1 (n = 25) received Diane 35 alone and group 2 (n = 25) received Diane 35 plus finasteride. The latter drug was administered using a new therapeutic scheme: 14 consecutive days for each therapeutic cycle. MAIN OUTCOME MEASURE(S): Hormonal evaluation was done before beginning treatment and after 3 and 6 months of therapy. Hirsutism was graded at 3-month intervals. RESULT(S): The combination of Diane 35 plus finasteride for 14 days significantly decreased the hirsutism score after 3 months of therapy, while Diane 35 alone induced this effect after 6 months. CONCLUSION(S): Finasteride in combination with Diane 35 for 14 days is effective, well accepted, and safe in hirsute patients, as the amount of antiandrogenic drugs administered is much lower than that in conventional treatment. PMID- 10731533 TI - Androgen excess contributes to altered growth hormone/insulin-like growth factor 1 axis in nonobese women with polycystic ovary syndrome. AB - OBJECTIVE: To investigate the relationship between ovarian androgen excess and impaired growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis in nonobese women with polycystic ovary syndrome (PCOS). DESIGN: A prospective, controlled clinical study. SETTING: Reproductive Endocrine Unit, Department of Obstetrics and Gynecology, Jinling Hospital, Nanjing University School of Clinical Medicine. PATIENT(S): Six patients with PCOS with both clomiphene resistance and gonadotropin hyperreponsiveness and six controls with regular cycles, matched for age and body mass index (BMI). INTERVENTION(S): Bilateral ovarian wedge resection (OWR) was performed to induce ovulation surgically for these refractory women with PCOS. A GH stimulation test with oral L-dopa was arranged for controls and for patients with PCOS before and again 6 months later after OWR. MAIN OUTCOME MEASURE(S): Plasma GH, IGF-1, FSH, LH, testosterone, androstenedione, estradiol, progesterone, prolactin, insulin, and glucose. RESULT(S): Basal levels and areas under the response curve of GH and GH-IGF-1 ratio to L-dopa were significantly lower in patients with PCOS before surgery than those of controls. The OWR in patients with PCOS obviously reduced their androstenedione and testosterone levels and insulin-glucose ratios, and increased the GH and GH-IGF-1 responses to L-dopa. CONCLUSION(S): Impaired somatotrophic axis caused by a defect in central dopaminergic activity may be responsible for severe anovulation in these women with PCOS, which could be reversed by removing excessive androgens with OWR. PMID- 10731534 TI - Microsatellite polymorphism (tttta)(n) at -528 base pairs of gene CYP11alpha influences hyperandrogenemia in patients with polycystic ovary syndrome. AB - OBJECTIVE: To investigate the functional significance of CYP11alpha microsatellite polymorphism (tttta)(n) (-528 base pairs) in patients with polycystic ovary syndrome. DESIGN: Follow-up study. SETTING: Academic research center. PATIENT(S): Eighty patients and 90 controls. INTERVENTION(S): Body mass indices and waist-to-hip ratios were determined. Blood samples were obtained for DNA analysis and hormone measurements. MAIN OUTCOME MEASURE(S): CYP11alpha marker (tttta)(n) genotyping and serum total testosterone levels. RESULT(S): All the women were assigned to one of two genotype groups: 216+ (for women who had at least one copy of high frequency allele 216 with four repeat units) or 216- (for women who did not have allele 216). Fifty-nine patients (73.75%) had genotype 216+; their mean (+/-SD) total testosterone level was 78.0 +/- 19.8 ng/dL. Twenty one patients (26.25%) had genotype 216-; their mean (+/-SD) total testosterone level was 100.0 +/- 23.3 ng/dL. The difference in total testosterone levels was statistically significant. Seventy-eight controls (86.67%) had genotype 216+ and 12 controls (13.33%) had genotype 216-; the total testosterone levels of these two groups were similar (38.6 +/- 15.5 vs. 40.3 +/- 12.1 ng/dL). The difference in genotype distribution between the women with polycystic ovary syndrome and the controls (26.25% vs. 13.33% with genotype 216-) was statistically significant. CONCLUSION(S): CYP11alpha (tttta)(n) allelic variants were associated with both polycystic ovary syndrome and total testosterone levels in women with polycystic ovary syndrome, suggesting the existence of an epistasis phenomenon. PMID- 10731535 TI - Does anovulation induced by oral contraceptives favor pregnancy during the following two menstrual cycles? AB - OBJECTIVE: To compare IVF and pregnancy outcomes before and after anovulation induced by oral contraceptives. DESIGN: Observational clinical study. SETTING: Infertility clinic. PATIENT(S): Forty women with two intact ovaries (32 of 40 couples with male factor infertility and 8 with unknown causes of infertility) underwent 190 IVF treatment cycles (55 natural cycles and 135 clomiphene citrate stimulated cycles). INTERVENTION(S): If the women failed to conceive after 2-4 IVF treatment cycles, oral contraceptives were used to induce anovulation for 1 month before IVF was performed in two consecutive cycles. MAIN OUTCOME MEASURE(S): Rates of oocyte retrieval, fertilization, cleavage, preembryo formation, pregnancy, and implantation were compared before and after a period of anovulation. RESULT(S): The pregnancy rate per cycle of the first and second cycle combined (23%) and that of the second cycle alone (30%) after a period of anovulation were significantly higher than that observed before a period of anovulation (9%). CONCLUSION(S): Anovulation induced by oral contraceptives, showing bilateral ovarian quiescence, enhances pregnancy rates in the following two menstrual cycles of IVF treatment. PMID- 10731536 TI - Cost-effectiveness of in vitro fertilization and embryo transfer. AB - OBJECTIVE: Economic analyses in reproductive medicine often fail to take into account the chances of treatment-independent conception. We compared the cost effectiveness of several realistic strategies involving IVF using no treatment as the reference strategy. DESIGN: A decision tree was constructed for a subfertile couple in which the clinician had to decide whether to offer treatment with IVF. No treatment at all was used as the reference strategy. SETTING: An analytic decision-making framework. PATIENT(S): Hypothetical subfertile couples. INTERVENTION(S): Two potential treatment approaches: three IVF cycles performed as soon as possible, or no treatment performed initially and then three or four IVF cycles performed if a pregnancy resulting in a live birth does not occur naturally after 212 years. MAIN OUTCOME MEASURE(S): The cost of establishing one pregnancy that results in a live birth. RESULT(S): The cost-effectiveness of IVF depended not only on the costs and expected success rates of IVF itself but also on the couple-specific chances of treatment-independent conception. Consequently, the cost-effectiveness of IVF is strongly dependent on the age of the female partner. This finding corresponds with everyday clinical experience. CONCLUSION(S): Economic analyses in reproductive medicine should take into account the option of providing no treatment. PMID- 10731537 TI - Intrauterine insemination before transfer of frozen-thawed embryos may improve the pregnancy rate for couples with unexplained infertility: preliminary results of a randomized prospective study. AB - OBJECTIVE: To evaluate whether a combination of IUI and frozen-thawed embryo transfer (FT-ET) with ovulation induction would improve the PR in couples with unexplained infertility. DESIGN: Prospective, randomized study. SETTING: In Vitro Fertilization Unit, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel. PATIENT(S): Sixty-two patients with unexplained infertility were assigned into two groups. The study group was composed of 32 women (38 cycles) who received ovulation induction followed by IUI and FT-ET. The control group was composed of 30 women (33 cycles) who received ovulation induction followed by FT-ET. INTERVENTION(S): Clomiphene citrate (CC) and hCG, IUI, and FT-ET. MAIN OUTCOME MEASURE(S): Pregnancy rate (PR) per cycle, PR per ET. RESULT(S): In the study group, the PR per cycle and per ET were 36.8% (14 of 38) and 40.6% (13 of 32), respectively. In the control group, the PR per cycle and per ET were 12.1% (4 of 33) and 14.3% (4 of 28), respectively. Statistically significant differences were found between the two groups in the PR per cycle (P=.02) and PR per ET (P=.03). No statistically significant difference was found between the groups for the stage in which the embryos were cryopreserved, the survival cleavage rates after thawing, grading of thawed embryos, and number of embryos transferred. CONCLUSION(S): In couples with unexplained infertility, the PR may be improved by combining IUI and FT-ET with ovulation induction. Performing IUI before thawing may prevent treatment cancellation in cycles with no surviving embryos. PMID- 10731538 TI - Estradiol supplementation during the luteal phase may improve the pregnancy rate in patients undergoing in vitro fertilization-embryo transfer cycles. AB - OBJECTIVE: To evaluate the effect of adding E(2) to progestin supplementation during the luteal phase on pregnancy and implantation rates in patients undergoing IVF cycles. DESIGN: Prospective, randomized study. SETTING: An IVF unit in a university hospital. PATIENT(S): Patients who were undergoing IVF with controlled ovarian hyperstimulation using a GnRH analog and who had E(2)2,500 pg/dL at the time of hCG administration. INTERVENTION(S): Serum concentrations of E(2) and progesterone were measured in all patients on days 7, 10, and 12 after ET. MAIN OUTCOME MEASURE(S): The E(2) and progesterone profiles of the luteal phase and the pregnancy and implantation rates were documented. Data were analyzed for the entire study population and further stratified according to the GnRH analog protocol used (short or long). RESULT(S): Significantly higher E(2) levels were found during the luteal phase in the group that received E(2) supplementation. This effect was more pronounced in the patients who were treated with the long GnRH analog protocol. Significantly higher pregnancy and implantation rates were recorded in the patients who received E(2) supplementation and were treated with the long GnRH analog protocol. CONCLUSION(S): For patients who are treated with the long GnRH analog protocol for controlled ovarian hyperstimulation and for whom luteal support with hCG is contraindicated, the addition of E(2) to the progestin support regimen may have a beneficial effect on pregnancy and implantation rates. PMID- 10731539 TI - Embryo cryopreservation at the pronuclear stage and efficient embryo use optimizes the chance for a liveborn infant from a single oocyte retrieval. AB - OBJECTIVE: To estimate the potential for a liveborn in our program achieved through either fresh or frozen embryos derived from a single oocyte retrieval. DESIGN: Retrospective analysis. SETTING: A tertiary referral reproductive medicine unit. PATIENT(S): All consecutive patients undergoing oocyte retrieval from January 1, 1996, to June 30, 1997. INTERVENTION(S): All couples undergoing IVF-ET at our center are counseled about a specific embryo transfer number after oocyte retrieval based on demographic and historical factors. Only this specified number of embryos is retained in culture. All normally fertilized (2PN) oocytes exceeding this number are immediately cryopreserved at the pronuclear stage. For couples who do not conceive after fresh embryo transfers, frozen embryo transfers are subsequently performed by usually thawing only the number of embryos intended for transfer, thereby conserving remaining embryos for further potential frozen embryo cycles. MAIN OUTCOME MEASURE(S): Liveborn delivery per oocyte retrieval.39.0 years were 61.2%, 59.7%, and 18.5%, respectively. CONCLUSION(S): For women <39 years of age, the efficient use of embryo cryopreservation at the pronuclear stage and economical embryo utilization policies results in cumulative chances for a liveborn exceeding 60%. PMID- 10731540 TI - Spontaneous conception after a successful attempt at in vitro fertilization/intracytoplasmic sperm injection. AB - OBJECTIVE: To determine the incidence of spontaneous pregnancy in women who were not actively undergoing therapy after a successful attempt at IVF/intracytoplasmic sperm injection and to characterize its pattern of occurrence. DESIGN: Retrospective postal questionnaire. SETTING: An assisted reproduction unit at a university-based teaching hospital. PATIENT(S): Five hundred thirteen replies were received from 530 questionnaires mailed. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Spontaneous pregnancy. Women who did and did not conceive spontaneously after successful IVF treatment were compared in terms of their age, duration of infertility, previous reproductive history, and indication for treatment at the time of assisted reproduction. RESULT(S): The rate of spontaneous conception among the survey respondents was 20.7%. Younger women (16-18 mm in diameter by transvaginal ultrasonography. A modified FASIAR procedure was performed 22 to 28 hours after hCG injection. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate. RESULT(S): No clinical pregnancies were observed with the modified FASIAR technique. CONCLUSION(S): The FASIAR technique is still an attractive and economical technique. Our modification of the FASIAR technique, however, resulted in a suboptimal cycle fecundity. PMID- 10731554 TI - Serum mullerian-inhibiting substance levels during normal menstrual cycles. AB - OBJECTIVE: To measure serum levels of mullerian-inhibiting substance (MIS) during the normal menstrual cycle. DESIGN: Serum was collected from women during ovulation and the mid-luteal and early follicular phases of the menstrual cycles. It was stored frozen at -80 degrees C until assayed. SETTING: University of Louisville Departments of Obstetrics and Gynecology and Surgery. PATIENT(S): Twenty healthy women 22-35 years of age with normal menstrual cycles. INTERVENTION(S): Blood samples were collected on menstrual cycle day two or three and on the day of LH surge plus one and plus seven or eight. MAIN OUTCOME MEASURE(S): Serum MIS levels were measured by using an enzyme-linked immunosorbent assay. RESULT(S): Serum MIS levels ranged from a low of 1.4 +/- 0.9 ng/mL (mean [+/-SD]) in the early follicular phase, peaked mid-cycle at 1.7 +/- 1.1 ng/mL, and decreased to 1.4 +/- 0.9 ng/mL in the mid-luteal phase of the normal menstrual cycle. CONCLUSION(S): Fluctuations in serum MIS levels during the menstrual cycle suggest that MIS may have a regulatory role in folliculogenesis. PMID- 10731555 TI - Adenomyosis in a patient with the Rokitansky-Kuster-Hauser syndrome. PMID- 10731556 TI - Effects of human chorionic gonadotropin administration on testicular testosterone secretion during prolonged exercise. PMID- 10731557 TI - Parasite genomes PMID- 10731558 TI - The African trypanosome genome. AB - The haploid nuclear genome of the African trypanosome, Trypanosoma brucei, is about 35 Mb and varies in size among different trypanosome isolates by as much as 25%. The nuclear DNA of this diploid organism is distributed among three size classes of chromosomes: the megabase chromosomes of which there are at least 11 pairs ranging from 1 Mb to more than 6 Mb (numbered I-XI from smallest to largest); several intermediate chromosomes of 200-900 kb and uncertain ploidy; and about 100 linear minichromosomes of 50-150 kb. Size differences of as much as four-fold can occur, both between the two homologues of a megabase chromosome pair in a specific trypanosome isolate and among chromosome pairs in different isolates. The genomic DNA sequences determined to date indicated that about 50% of the genome is coding sequence. The chromosomal telomeres possess TTAGGG repeats and many, if not all, of the telomeres of the megabase and intermediate chromosomes are linked to expression sites for genes encoding variant surface glycoproteins (VSGs). The minichromosomes serve as repositories for VSG genes since some but not all of their telomeres are linked to unexpressed VSG genes. A gene discovery program, based on sequencing the ends of cloned genomic DNA fragments, has generated more than 20 Mb of discontinuous single-pass genomic sequence data during the past year, and the complete sequences of chromosomes I and II (about 1 Mb each) in T. brucei GUTat 10.1 are currently being determined. It is anticipated that the entire genomic sequence of this organism will be known in a few years. Analysis of a test microarray of 400 cDNAs and small random genomic DNA fragments probed with RNAs from two developmental stages of T. brucei demonstrates that the microarray technology can be used to identify batteries of genes differentially expressed during the various life cycle stages of this parasite. PMID- 10731559 TI - Genes and genomes of Necator americanus and related hookworms. AB - The human hookworms (Necator americanus and Ancylostoma duodenale) infect over one billion people. The phylogenetic relationships of the human hookworms suggest independent acquisition of the human host. The hookworms probably have a haploid chromosome number n = 6, and an XX-XO sex determination mechanism is likely to be used. Genetic and molecular research on hookworms is in its infancy, but several important genes and gene products have already been identified. Of note are cathepsin genes, a family of secreted proteins known as Ancylostoma activation associated proteins and a family of anticoagulants. The inception of an expressed sequence tag program on the human hookworm, N. americanus, promises to yield many new genes with novel functions in the biology of these important parasites. PMID- 10731560 TI - The conserved genome organisation of non-falciparum malaria species: the need to know more. AB - The current knowledge on genomes of non-falciparum malaria species and the potential of model malaria parasites for functional analyses are reviewed and compared with those of the most pathogenic human parasite, Plasmodium falciparum. There are remarkable similarities in overall genome composition among the different species at the level of chromosome organisation and chromosome number, conserved order of individual genes, and even conserved functions of specific gene domains and regulatory control elements. With the initiative taken to sequence the genome of P. falciparum, a wealth of information is already becoming available to the scientific community. In order to exploit the biological information content of a complete genome sequence, simple storage of the bulk of sequence data will be inadequate. The requirement for functional analyses to determine the biological role of the open reading frames is commonly accepted and knowledge of the genomes of the animal model malaria species will facilitate these analyses. Detailed comparative genome information and sequencing of additional Plasmodium genomes will provide a deeper insight into the evolutionary history of the species, the biology of the parasite, and its interactions with the mammalian host and mosquito vector. Therefore, an extended and integrated approach will enhance our knowledge of malaria and will ultimately lead to a more rational approach that identifies and evaluates new targets for anti-malarial drug and vaccine development. PMID- 10731561 TI - Mitochondrial genome diversity in parasites. AB - Mitochondrial genomes have been sequenced from a wide variety of organisms, including an increasing number of parasites. They maintain some characteristics in common across the spectrum of life-a common core of genes related to mitochondrial respiration being most prominent-but have also developed a great diversity of gene content, organisation, and expression machineries. The characteristics of mitochondrial genomes vary widely among the different groups of protozoan parasites, from the minute genomes of the apicomplexans to amoebae with 20 times as many genes. Kinetoplastid protozoa have a similar number of genes to metazoans, but the details of gene organisation and expression in kinetoplastids require extraordinary mechanisms. Mitochondrial genes in nematodes and trematodes appear quite sedate in comparison, but a closer look shows a strong tendency to unusual tRNA structure and alternative initiation codons among these groups. Mitochondrial genes are increasingly coming into play as aids to phylogenetic and epidemiologic analyses, and mitochondrial functions are being recognised as potential drug targets. In addition, examination of mitochondrial genomes is producing further insights into the diversity of the wide-ranging group of organisms comprising the general category of parasites. PMID- 10731562 TI - Chromatin diminution in the parasitic nematodes ascaris suum and parascaris univalens. AB - Chromatin diminution in Parascaris univalens and Ascaris suum undoubtedly represents an interesting case of developmentally programmed DNA rearrangement in higher eukaryotes. It is a complex mechanism involving chromosomal breakage, new telomere addition and DNA degradation, and occurs in all presomatic cells. The process is rather specific with respect to its developmental timing and the chromosomal regions that are eliminated. The functional significance of chromatin diminution still remains an enigma. The fact, however, that single-copy, protein coding genes are contained in the eliminated DNA demonstrates that in P. univalens and A. suum, there is a qualitative difference between germ-line and somatic genomes, and suggests that chromatin diminution may be used as a "throw away" approach to gene regulation. We present a hypothesis as to how, during evolution, a partial genome duplication might have been linked to the process of chromatin diminution, in order to provide a selective advantage to parasitic DNA eliminating nematodes. PMID- 10731563 TI - The genome of Entamoeba histolytica. AB - Estimation of genome size of Entamoeba histolytica by different methods has failed to give comparable values due to the inherent complexities of the organism, such as the uncertain level of ploidy, presence of multinucleated cells and a poorly demarcated cell division cycle. The genome of E. histolytica has a low G+C content (22.4%), and is composed of both linear chromosomes and a number of circular plasmid-like molecules. The rRNA genes are located exclusively on some of the circular DNAs. Karyotype analysis by pulsed field gel electrophoresis suggests the presence of 14 conserved linkage groups and an extensive size variation between homologous chromosomes from different isolates. Several repeat families have been identified, some of which have been shown to be present in all the electrophoretically separated chromosomes. The typical nucleosomal structure has not been demonstrated, though most of the histone genes have been identified. Most Entamoeba genes lack introns, have short 3' and 5' untranslated regions, and are tightly packed. Promoter analysis revealed the presence of three conserved motifs and several upstream regulatory elements. Unlike typical eukaryotes, the transcription of protein coding genes is alpha-amanitin resistant. Expressed Sequence Tag analysis has identified a group of highly abundant polyadenylated RNAs which are unlikely to be translated. The Expressed Sequence Tag approach has also helped identify several important genes which encode proteins that may be involved in different biochemical pathways, signal transduction mechanisms and organellar functions. PMID- 10731564 TI - The filarial genome project: analysis of the nuclear, mitochondrial and endosymbiont genomes of Brugia malayi. AB - The Filarial Genome Project (FGP) was initiated in 1994 under the auspices of the World Health Organisation. Brugia malayi was chosen as the model organism due to the availability of all life cycle stages for the construction of cDNA libraries. To date, over 20000 cDNA clones have been partially sequenced and submitted to the EST database (dbEST). These ESTs define approximately 7000 new Brugia genes. Analysis of the EST dataset provides useful information on the expression pattern of the most abundantly expressed Brugia genes. Some highly expressed genes have been identified that are expressed in all stages of the parasite's life cycle, while other highly expressed genes appear to be stage-specific. To elucidate the structure of the Brugia genome and to provide a basis for comparison to the Caenorhabditis elegans genome, the FGP is also constructing a physical map of the Brugia chromosomes and is sequencing genomic BAC clones. In addition to the nuclear genome, B. malayi possesses two other genomes: the mitochondrial genome and the genome of a bacterial endosymbiont. Eighty percent of the mitochondrial genome of B. malayi has been sequenced and is being compared to mitochondrial sequences of other nematodes. The bacterial endosymbiont genome found in B. malayi is closely related to the Wolbachia group of rickettsia-like bacteria that infects many insect species. A set of overlapping BAC clones is being assembled to cover the entire bacterial genome. Currently, half of the bacterial genome has been assembled into four contigs. A consortium has been established to sequence the entire genome of the Brugia endosymbiont. The sequence and mapping data provided by the FGP is being utilised by the nematode research community to develop a better understanding of the biology of filarial parasites and to identify new vaccine candidates and drug targets to aid the elimination of human filariasis. PMID- 10731565 TI - Genomes of Paragonimus westermani and related species: current state of knowledge. AB - There have been few investigations of genomes of Paragonimus westermani and related species. Most studies have focussed on questions such as the identities of species and relationships among them, origins and relationships of strains with different ploidy states, and the characterisation of genes producing immunologically significant proteins. In the context of these questions, work on the karyotypes, nuclear and mitochondrial genomes is reviewed. PMID- 10731566 TI - Impact of a plastid-bearing endocytobiont on apicomplexan genomes. AB - Both the chromosomal and extrachromosomal components of the apicomplexan genome have been supplemented by genes from a plastid-bearing endocytobiont: probably an algal cell. The sequence of the apicomplexan plastid's vestigial genome indicates that a large number (>100) of genes of endocytobiotic origin must have transferred laterally to the host cell nucleus where they control maintenance of the plastid organelle and supply its functional components by means of post translational protein trafficking. Should the nuclear genes prove to be less divergent phylogenetically than those left on the plastid genome, they might give better clues than we have at present to the origin of the plastid-bearing endocytobiont. Most of these nuclear genes still await discovery, but the on going genome sequencing project will reveal the function of the organelle, as well as many "housekeeping" processes of interest on a wider front. The plastid's own protein synthetic machinery, being cyanobacterial in origin, offers conventional targets for antibiotic intervention, and this is discussed here using a structural model of elongation factor Tu. Uncovering the vital function(s) of the plastid organelle will provide new drug targets. PMID- 10731567 TI - Chromosomal differentiation of the Schistosoma japonicum complex. AB - The C-banding pattern, location of telomere sequence and chiasma frequency of four species of the Schistosoma japonicum complex were compared with those of two African species, Schistosoma mansoni and Schistosoma haematobium. In the six species, C-banding patterns of seven autosomes and the two sex chromosomes (Z and W) showed relatively species-specific and geographical (Asian and African) differences. Particularly, a plausible pathway of alteration of chromosome 2 revealed a direction from the A-chromosome to the M- chromosome in terms of rearrangements of pericentric inversion and elimination of constitutive heterochromatin (AM inversion). This chromosome change suggested hypothetically that the S. japonicum complex is the original type, and the African species represents the derived type. Moreover, the mosaic construct of the Asian and African types in Schistosoma sinensium chromosomes prompted us to propose that the species might have been formed by hybrid speciation of the genomes of Asian and African species. Localisation of telomeric repeats enabled Asian and African schistosomes to be distinguished clearly by simple terminal location and by terminal and interstitial locations, respectively. Change of chiasma frequency in the S. japonicum complex might be caused by the reduction of interstitial chiasmate (Xi) in the larger chromosomes, 1 and Z (or W), and the change seems to have progressed to Japan from South East Asia. These data enabled us to predict a tentative evolutionary pathway of schistosomes at the cytogenetic level. PMID- 10731568 TI - The Schistosoma gene discovery program: state of the art. AB - Schistosoma are dioecious digenetic trematodes carrying a large (270 Mb) genome. Gaining knowledge about the genome of these parasites is of importance for the understanding of their biology, mechanisms of drug resistance and antigenic variation that determine escape from the host's immune system. This review will provide an update on the Schistosoma Gene Discovery Program, which is part of the Schistosoma Genome Project created in 1992. One of the main objectives of this program is the discovery and characterisation of new genes of Schistosoma mansoni and Schistosoma japonicum in an attempt to search for new targets for drugs and vaccine development. The success of the Schistosoma Gene Discovery Program is demonstrated by the number of catalogued genes, that now reaches 15 to 20% of the full gene complement of its genome. PMID- 10731569 TI - Theileria parva genomics reveals an atypical apicomplexan genome. AB - The discipline of genomics is setting new paradigms in research approaches to resolving problems in human and animal health. We propose to determine the genome sequence of Theileria parva, a pathogen of cattle, using the random shotgun approach pioneered at The Institute for Genomic Research (TIGR). A number of features of the T. parva genome make it particularly suitable for this approach. The G+C content of genomic DNA is about 31%, non-coding repetitive DNA constitutes less than 1% of total DNA and a framework for the 10-12 Mbp genome is available in the form of a physical map for all four chromosomes. Minisatellite sequences are the only dispersed repetitive sequences identified so far, but they are limited in distribution to 13 of 33 SfiI fragments. Telomere and sub telomeric non-coding sequences occupy less than 10 kbp at each chromosomal end and there are only two units encoding cytoplasmic rRNAs. Three sets of distinct multicopy sequences encoding ORFs have been identified but it is not known if these are associated with expression of parasite antigenic diversity. Protein coding genes exhibit a bias in codon usage and introns when present are unusually short. Like other apicomplexan organisms, T. parva contains two extrachromosomal DNAs, a mitochondrial DNA and a plastid DNA molecule. By annotating the genome sequence, in combination with the use of microarray technology and comparative genomics, we expect to gain significant insights into unique aspects of the biology of T. parva. We believe that the data will underpin future research to aid in the identification of targets of protective CD8+ cell mediated immune responses, and parasite molecules involved in inducing reversible host leukocyte transformation and tumour-like behaviour of transformed parasitised cells. PMID- 10731570 TI - The Giardia lamblia genome. AB - Giardia lamblia is a protozoan parasite of humans and other mammals that is thought to be one of the most primitive extant eukaryotic organisms. Although distinctly eukaryotic, it is notable for its lack of mitochondria, nucleoli, and perixosomes. It has been suggested that Giardia spp. are pre-mitochondriate organisms, but the identification of genes in G. lamblia thought to be of mitochondrial origin has generated controversy regarding that designation. Giardi lamblia trophozoites have two nuclei that are identical in all ways that have been studied. They are polyploid with at least four, and perhaps eight or more, copies of each of five chromosomes per organism and have an estimated genome complexity of 1.2x10(7)bp of DNA, and GC content of 46%. There is evidence for recombination at the telomeres of some of the chromosomes, and multiple size variants of single chromosomes have been identified within cloned isolates. However, the internal regions of the chromosomes demonstrate no evidence of recombination. For example, there is no evidence for control of vsp gene expression by DNA recombination, and no evidence for rapid mutation in the vsp genes. Single pass sequences of approximately 9% of the G. lamblia genome have already been obtained. An ongoing genome project plans to obtain approximately 95% of the genome by a random approach, as well as a complete physical map using a bacterial artificial chromosome library. The results will facilitate a better understanding of the biology of Giardia spp. as well as their phylogenetic relationship to other primitive organisms. PMID- 10731571 TI - The genome of Eimeria spp., with special reference to Eimeria tenella--a coccidium from the chicken. AB - Eimeria spp. contain at least four genomes. The nuclear genome is best studied in the avian species Eimeria tenella and comprises about 60 Mbp DNA contained within ca. 14 chromosomes; other avian and lupine species appear to possess a nuclear genome of similar size. In addition, sequence data and hybridisation studies have provided direct evidence for extrachromosomal mitochondrial and plastid DNA genomes, and double-stranded RNA segments have also been described. The unique phenotype of "precocious" development that characterises some selected lines of Eimeria spp. not only provides the basis for the first generation of live attenuated vaccines, but offers a significant entree into studies on the regulation of an apicomplexan life-cycle. With a view to identifying loci implicated in the trait of precocious development, a genetic linkage map of the genome of E. tenella is being constructed in this laboratory from analyses of the inheritance of over 400 polymorphic DNA markers in the progeny of a cross between complementary drug-resistant and precocious parents. Other projects that impinge directly or indirectly on the genome and/or genetics of Eimeria spp. are currently in progress in several laboratories, and include the derivation of expressed sequence tag data and the development of ancillary technologies such as transfection techniques. No large-scale genomic DNA sequencing projects have been reported. PMID- 10731572 TI - Toxocara canis: genes expressed by the arrested infective larval stage of a parasitic nematode. AB - Toxocara canis is a widely distributed nematode parasite which reaches maturity in dogs. However, eggs voided by canid animals are infective to a very wide range of paratenic hosts including humans. In noncanid hosts, infective larvae emerge from the eggs and invade the soft tissues, often entering the brain and musculature. Such larvae may remain for many months or years in these tissues without further growth or differentiation, and yet appear to evade inflammatory reactions or other modes of immune attack. To understand the ability of T. canis larvae to survive in the immunocompetent host, we have undertaken a molecular analysis of the major genes expressed at this stage. By a combination of protein sequencing, gene identification, and expressed sequence tag (EST) analysis we have characterised a range of potentially important gene products from this parasite. Some of these are homologues of prominent mammalian proteins such as C type lectins (represented by the secreted products TES-32 and TES-70), and mucins (TES-120), and additional products show strong similarities to known cysteine proteases, phosphatidylethanolamine-binding proteins and other ligands. A number of these proteins include a conspicuous 36-amino acid motif containing six cysteines. This domain (termed NC6 or SXC) appears to be an evolutionarily mobile module, which in T. canis is combined with a spectrum of diverse functional domains in different genes. In addition, we have identified a set of novel gene sequences that show no resemblance to any genes encoded by the free-living nematode C. elegans. Four of these are designated abundant novel transcripts, and collectively these account for nearly 20% of the cDNA isolated from the arrested infective stage. Such parasite-specific genes expressed at a high level by a stage that shows remarkable endurance may represent critical products necessary for the success of the parasitic mode of life. PMID- 10731573 TI - Genomic and genetic research on bursate nematodes: significance, implications and prospects. AB - Molecular genetic research on parasitic nematodes (order Strongylida) is of major significance for many fundamental and applied areas of medical and veterinary parasitology. The advent of gene technology has led to some progress for this group of nematodes, particularly in studying parasite systematics, drug resistance and population genetics, and in the development of diagnostic assays and the characterisation of potential vaccine and drug targets. This paper gives an account of the molecular biology and genetics of strongylid nematodes, mainly of veterinary socio-economic importance, indicates the implications of such research and gives a perspective on genome research for this important parasite group, in light of recent technological advances and knowledge of the genomes of other metazoan organisms. PMID- 10731574 TI - Molecular studies of Biomphalaria glabrata, an intermediate host of Schistosoma mansoni. AB - The freshwater gastropod Biomphalaria glabrata is one of the most important invertebrate hosts of the helminth parasite Schistosoma mansoni. Investigators are using different strategies to determine the molecular basis of this snail parasite relationship. Of particular interest are the identification of parasite resistance genes in the snail, and the application of molecular probes to better understand the epidemiology of schistosomiasis. This review will focus on recent advances that have been made on genome analysis of B. glabrata. Much of this work has centred on the use of random amplification of polymorphic DNA-PCR-based technology, with restriction fragment length polymorphism analysis and the generation of expressed sequence tags from the snail. A brief discussion of how parasite products may complicate this analysis is also given, along with an indication of the scope of the problems that lie ahead. PMID- 10731575 TI - The genomes of Onchocerca volvulus. AB - Onchocerca volvulus, the filarial parasite that causes onchocerciasis or river blindness, contains three distinct genomes. These include the nuclear genome, the mitochondrial genome and the genome of an intracellular endosymbiont of the genus Wolbachia. The nuclear genome is roughly 1.5x10(8) bp in size, and is arranged on four chromosome pairs. Analysis of expressed sequence tags from different life cycle stages has resulted in the identification of transcripts from roughly 4000 O. volvulus genes. Several of these transcripts are highly abundant, including those encoding collagen and cuticular proteins. Analysis of several gene sequences from O. volvulus suggests that the nuclear genes of O. volvulus are relatively compact and are interrupted relatively frequently by small introns. The intron-exon boundaries of these genes generally follow the GU-AG rule characteristic of the splice donor and acceptors of other vertebrate organisms. The nuclear genome also contains at least one repeated sequence family of a 150 bp repeat which is arranged in tandem arrays and appears subject to concerted evolution. The mitochondrial genome of O. volvulus is remarkably compact, only 13747 bp in size. Consistent with the small size of the genome, four gene pairs overlap, eight contain no intergenic regions and the remaining gene pairs are separated by small intergenic domains ranging from 1 to 46 bp. The protein-coding genes of the O. volvulus mitochondrial genome exhibit a striking codon bias, with 15/20 amino acids having a single codon preference greater than 70%. Intraspecific variation in both the nuclear and mitochondrial genomes appears to be quite limited, consistent with the hypothesis that O. volvulus has suffered a genetic bottleneck in the recent past. PMID- 10731576 TI - Cryptosporidium parvum: the many secrets of a small genome. AB - The coccidium Cryptosporidium parvum is an obligate intracellular parasite of the phylum Apicomplexa. It infects the gastrointestinal tract of humans and livestock, and represents the third major cause of diarrhoeal disease worldwide. Scarcely considered for decades due to its apparently non-pathogenic nature, C. parvum has been studied very actively over the last 15 years, after its medical relevance as a dangerous opportunistic parasite and widespread water contaminant was fully recognised. Despite the lack of an efficient in vitro culture system and appropriate animal models, significant advances have been made in this relatively short period of time towards understanding C. parvum biology, immunology, genetics and epidemiology. Until recently, very little was known about the genome of C. parvum, with even basic issues, such as the number and size of chromosomes, being the object of a certain controversy. With the advent of pulsed field gradient electrophoresis and the introduction of molecular biology techniques, the overall structure and fine organisation of the genome of C. parvum have started to be disclosed. Organised into eight chromosomes distributed in a very narrow range of molecular masses, the genome of C. parvum is one of the smallest so far described among unicellular eukaryotic organisms. Although fewer than 30 C. parvum genes have been cloned so far, information about the overall structure of the parasite genome has increased exponentially over the last 2 years. From the first karyotypic analyses to the recent development of physical maps for individual chromosomes, this review will try to describe the state-of-the-art of our knowledge on the nuclear genome of C. parvum and will discuss the available experimental evidence concerning the presence of extra chromosomal elements. PMID- 10731577 TI - Aging differentially modifies sensitivity of nerve blood flow to vasocontractile agents (endothelin-1, noradrenaline and angiotensin II) in sciatic nerve. AB - This study examined the influence of the vaso-constricting agents (noradrenaline, endothelin-1 and angiotensin II) in Sprague-Dawley rats aged 2, 6 and 24 months by evaluating epineurial arteriolar vasoreactivity in response to superfused teat agents. Nerve blood flow (NBF) was measured using microelectrode H2 polarography. In 24-month-old rats, NBF was decreased and vascular resistance (VR) was increased compared with 2- and 6-month-old rats. All of the constricting agents reduced NBF in the 2-, 6- and 24-month groups, however, the effects of the constricting agents reduced significantly with age. These results suggest that during aging, there is a decline of vasoconstrictive responses to noradrenaline, endothelin-1 and angiotensin II in peripheral nerve and that these changes may be due to altered function of receptors. PMID- 10731578 TI - Postnatal mortality from pneumonia. AB - Research has been conducted on the relationship between postnatal mortality from pneumonia and age, using data from the USA, Japan, former Czechoslovakia, Italy, Portugal and the UK during the period of 1979-1993. The logarithm of mortality caused by pneumonia fell linearly with the logarithm of age, during the interval of 1-10 years. This linear log-log dependence corresponds to the two-parametrical Weibull distribution, if the slope is greater than -1. However, the logarithm of mortality from pneumonia declined with the slope equal to -1. The mortality from pneumonia is inversely proportional to the age. The risk of death at age of 2 is one half of the risk at the age of 1, at the age of 3 it is one third of the risk of death at the age of 1ellipsis, etc. up to the age of 10. We assumed that no subpopulation susceptible to pneumonia exists and the risk of death from pneumonia applies in whole population. Consequently, there was applied another distribution function described at this paper. PMID- 10731579 TI - Age-related structural modulation of T lymphocyte-associated CD45 isoforms. AB - A monoclonal antibody, specific to all conventional CD45 isoforms, was employed in two-dimensional (2D) sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting to investigate possible age-related differential expression of these isoforms among immature and mature thymocytes as well as CD4+ and CD8+ T cell subpopulations in the periphery of newly-born, young and aged BALB/c mice. In young mice, and to a lesser degree in newly-born mice, intra thymic maturation seemed to be paralleled by the capacity of thymocytes to synthesize distinct CD45 isoforms constituted by extensively heterogeneous acidic charge entities. Thymocyte maturation in aged mice, on the other hand, was characterized by minimal heterogeneity, as the observed pattern was essentially similar to the immature population in 2D blots. As inferred from comparisons of 2D blots of sialylated and desialylated forms of the CD45 complex, age-related differences in isoforms expressed by the CD4+ and the CD8+ T cell subpopulations in the periphery resided mainly in the degree of sialylation of the constituent isoforms. Given the potential of the differential sialylation state of CD45 in altering the recognition properties of lymphocytes, regulation of CD45 sialylation with age may add another level of complexity to the lymphocyte surface phenotype, which in turn may be implicated in cell-cell interaction mechanisms during lymphocyte maturation and senescence. PMID- 10731580 TI - Endothelin-1 in monolayer cultures of articular chondrocytes from young and old rats: regulation by growth factors and cytokines. AB - The endothelin-1 (ET-1) concentrations were measured by RIA in the media of confluent monolayer cultures of rat articular chondrocyte (RAC) exposed to fetal calf serum (FCS) and several growth factors and cytokines. The cells were obtained from 1- and 18-month-old rats. First passage cells were starved in Dulbecco's modified Eagle's medium (DMEM) containing 0.2% FCS serum for 24 h and then incubated for 48 h in the same fresh medium with each of the following factors: fetal calf serum (FCS), transforming growth factor-beta (TGF-beta), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin like growth factor-1 (IGF-1), interleukin-1 beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), lipopolysaccharide (LPS), and NO donor, sodium nitroprusside (SNP). The following was found: the cells from 18-month-old animals accumulated about twice as much ET-1 per microg DNA under basal (low serum) and stimulated conditions as cells from young rats. All, but PDGF and SNP produced concentration-dependent rise in ET-1 levels, the most effective being 10% FCS, IL 1beta, TNF-alpha, EGF, IGF-1 and LPS. TGF-beta caused the smallest stimulation and PDGF was ineffective or slightly inhibitory at high concentrations. SNP caused concentration-dependent decrease of ET-1 concentrations. ET-1-specific mRNA was identified by RT-PCR in cells incubated with the above factors and its concentration paralleled that of the peptide. This suggests that ET-1 found in the culture media of RAC stems, at least in part, from the synthesis. Increased immunoreactive peptide concentration and mRNA expression with the age of the donor rat and its regulation by several growth factors and cytokines suggest the involvement of ET-1 in chondrocytes' physiology and possibly pathology. PMID- 10731581 TI - Age-dependent increase of heme oxygenase-1 gene expression in the liver mediated by NFkappaB. AB - Heme, the iron-porphyrin coordination complex, released from the degradation of hemoproteins, is a strong prooxidant. It is enzymatically degraded by heme oxygenase to free iron, carbon monoxide and biliverdin. Biliverdin and its reduced metabolite bilirubin are two potent physiological antioxidants. Here we show a progressive increase of steady-state levels of the mRNA encoding the inducible isoform of this enzyme (heme oxygenase-1) in the rat liver during aging. We had previously reported that aging is associated with increased activation of the nuclear factor kappaB (NFkappaB). We now provide evidence to establish that overexpression of NFkappaB in transfected liver-derived HepG2 cells can cause a marked induction of the endogenous heme oxygenase-1 (HO-1) mRNA and activation of the cotransfected HO-1 gene promoter. Taken together, these results support the conclusion that enhanced oxidative stress during aging is accompanied by compensatory induction of the antioxidant enzyme HO-1 through activation of the NFkappaB pathway. PMID- 10731582 TI - Cytogenetics of the chronic myeloid leukemia-derived cell line K562: karyotype clarification by multicolor fluorescence in situ hybridization, comparative genomic hybridization, and locus-specific fluorescence in situ hybridization. AB - The transformation of chronic myeloid leukemia (CML) from a chronic phase to an acute phase is frequently accompanied by additional chromosome changes. Extensive chromosome G-banded studies have revealed the secondary changes are nonrandom and frequently include trisomy 8, isochromosome 17q, trisomy 19, or an extra copy of the Philadelphia chromosome. In addition to these secondary chromosome changes, complex structural rearrangements often occur to form marker structures that remain unidentified by conventional G-banded analysis. The CML-derived cell line, K562, has been widely used in research since it was originally established in 1975. The K562 karyotype however, has remained incomplete, and marker structures have never been fully described. Recent advances in fluorescence in situ hybridization (FISH) technology have introduced the possibility of chromosome classification based on 24-color chromosome painting (M-FISH). In this study, we report a clarified karyotype for K562 obtained by a combination of the following molecular cytogenetic techniques: comparative genomic hybridization (CGH), FISH mapping using locus-specific probes, and M-FISH. Multicolor FISH has identified the marker structures in this cell line. The characteristic marker chromosome in K562 has been confirmed by this study to be a der(18)t(1;18). Multicolor FISH confirmed the identity of marker structures partially identified by G-banding as der(6)t(6;6),der(17)t(9;17),der(21)t(1;21),der(5)t(5;6). In addition M-FISH has revealed a deleted 20q and a complex small metacentric marker comprised of material from chromosomes 1, 6, and 20. A cryptic rearrangement was revealed between chromosomes 12 and 21 that produced a structure that looks like a normal chromosome 12 homologue by G-banding analysis. Finally, M-FISH detected regions from chromosome 13 intercalated into two acrocentric markers. PMID- 10731583 TI - Cytogenetic abnormalities during clinical, immunophenotypic, and molecular remission in pediatric acute lymphoblastic leukemia. AB - The significance of random cytogenetic abnormalities detected in pediatric acute lymphoblastic leukemia (ALL) during remission is unknown. We studied 10 of 72 consecutive ALL patients who developed cytogenetic abnormalities during clinical remission to determine their effect on remission status. The cytogenetic abnormalities occurred at a median of 14.5 months (range 5-72) from the initial diagnosis. Five abnormalities were designated as clonal (monosomy 21 in three metaphases and 64 approximately 69,XXY in three metaphases from one patient at different times, and del(20)(q12q13) in three metaphases, add(13)(q34) in two metaphases, and ?del(19)(p11) in two metaphases from three different patients). Seven abnormalities were previously described: del(5)(q12), del(5)(q33), -7, del(11)(q23), +12, and +13 each in one metaphase, and del(20)(q12q13) in three metaphases). The remaining cytogenetic abnormalities were nonclonal and random. Flow cytometry and analysis of IgH and TcR gene rearrangements showed that all evaluable patients were in immunophenotypic and molecular remission, respectively. Eight patients remain in clinical and molecular remission a median of 9 months (range 7-18) after detecting the cytogenetic abnormality, and the leukemia in two patients has relapsed. During remission, cytogenetic abnormalities may not be a harbinger of leukemia relapse in pediatric ALL; therefore, a wait-and-see approach is prudent. PMID- 10731584 TI - Silent Philadelphia chromosome: a distinct developmental stage in a philadelphia chromosome-positive chronic myeloproliferation? AB - In this paper, a patient is described who presented with peripheral blood and bone marrow features uncharacteristic of chronic granulocytic leukemia, which proved to be Philadelphia (Ph) chromosome-positive by metaphase and interphase cytogenetic analyses but lacked the p210 type of BCR/ABL fusion gene mRNA product by two different sensitive RT-PCR assays. In the course of the 32-month follow-up with a termination into a myeloblastic crisis, molecular investigations were performed four times. They indicated a constantly high rate of Ph positive cells and lack of BCR/ABL mRNA expression, except in the second investigation, when the patient showed reverse transcription polymerase chain reaction positivity with b3/a2 type of chimera, fusion gene mRNA expression, and a striking change in the bone marrow histology. Our findings might indicate that the dormant Ph chromosome state may exist not only at the primitive progenitor, but also at the entire peripheral blood cell compartment level. PMID- 10731585 TI - Acute monocytic leukemia with a novel 10;11 rearrangement resolved by fluorescence in situ hybridization. AB - Fluorescence in situ hybridization analysis in an adult with acute monocytic leukemia revealed the complex nature of a rearrangement between chromosomes 10 and 11, which resulted in disruption of the MLL gene. Using a combination of chromosome 10 and 11 paints, a 10 centromere-specific sequence, and a probe for the MLL locus at 11q23, the rearrangement was deduced to have involved a reciprocal translocation between chromosomes 10 and 11, followed by an inversion within the short arm of the derivative 10. To our knowledge, this novel rearrangement has not been described previously. PMID- 10731586 TI - Complex cytogenetic abnormalities in T-lymphoblastic lymphoma: resolution by spectral karyotyping. AB - We describe a case of T-lymphoblastic lymphoma (T-LBL) in a 13-year-old boy in which conventional cytogenetic analysis of lymph node tissue showed complex karyotypic aberrations including add(1p), add(2q), and multiple chromosomal deletions involving 5q, 7p, 7q, 13q, and 16q. Analysis by spectral karyotyping (SKY) refined add(1p) to a paracentric inversion of 1p, and add(2q) to an unbalanced translocation between chromosomes 2 and 4. The chromosomal deletions were simple deletions except del(5q), which was confirmed by SKY to be a cryptic unbalanced translocation between chromosomes 5 and 18. The present report illustrates that SKY technology is useful in identifying subtle translocations and resolving complex karyotypic aberrations in neoplastic disorders. PMID- 10731587 TI - Microdissection and FISH investigations in acute myeloid leukemia: a step forward to full identification of complex karyotypic changes. AB - Complex chromosomal rearrangements in malignant hemopathies frequently remain unclarified because of paucity of material for further fluorescence in situ hybridization analyses and/or lack of suitable probes. Chromosome microdissection (MD) can be an adequate approach to elucidate chromosome aberrations unrecognizable by conventional karyotyping. We applied MD in two patients with acute myeloid leukemia (AML) and unidentified chromosome changes at karyotype. Microdissection of a ring chromosome in an AML-M5 case revealed 21q polysomy. In an AML-M4 case, MD of an add(15p) disclosed a t(8;15) with over-representation of both 8q22 and 8q24 bands. YAC probes were helpful in showing duplication of the ETO gene at 8q22, and amplification of C-MYC, at 8q24. PMID- 10731588 TI - Jumping translocations of 11q in acute myeloid leukemia and 1q in follicular lymphoma. AB - Jumping translocation is a rare cytogenetic aberration in leukemia and lymphoma, and its etiologic mechanisms are not clearly known. We report two cases with jumping translocations. One had follicular lymphoma and jumping translocations of 1q onto the telomeric regions of 5p, 9p, and 15q in three cell lines, co-existing with the specific translocation t(14;18)(q32;q21). The second case had acute myeloid leukemia (AML) and jumping translocations of 11q as the sole aberration, onto multiple derivative chromosomes in each of the abnormal cells. A total of 17 telomeric regions were seen as the recipients of 11q in this case, and 9q was always involved as one of the recipients in all abnormal cells. Fluorescence in situ hybridization (FISH) confirmed the identification of 11q material in the derivative chromosomes. While 1q has been the most common donor of acquired jumping translocations, this is the first report on jumping translocations of 11q. Different from all previously reported jumping translocations which involve only one recipient in each cell line and lead to a mosaic trisomy, multiple recipients in most of the abnormal cells in this case had led to a tetrasomy, or a pentasomy of 11q. The pattern of chromosome involvement as the recipients of 11q appears to show a continuing evolutionary process of jumping, stabilization, and spreading of the donor material into other chromosomes. Somatic recombinations between the interstitial telomeric or subtelomeric sequences of a derivative chromosome and the telomeric sequences of normal chromosomes are believed to be the underlying mechanism of jumping translocations and their clonal evolution. PMID- 10731589 TI - Cytogenetic heterogeneity and clonal evolution in synchronous bilateral breast carcinomas and their lymph node metastases from a male patient without any detectable BRCA2 germline mutation. AB - Two synchronous bilateral breast carcinomas and their matched lymph node metastases from a 70-year-old man were cytogenetically analyzed. All four tumors were near-diploid, and except for the primary tumor from the right breast, had a 45,X,-Y clone in common. The loss of the Y chromosome was, however, common to all four tumors, whereas metaphase cells from peripheral blood lymphocytes showed a normal 46, XY chromosome complement. The primary tumor from the right breast was monoclonal, with loss of the Y chromosome and gain of 1q, whereas its metastasis had two related clones: the 45,X,-Y clone, and the other a more complex version of the clone in the primary tumor, with inv(3), -14, and del(16)(q13) as additional changes. The primary tumor from the left breast was polyclonal with three unrelated clones: 45,X,-Y/45,XY,-18/47,XY,+20, two of which were present in its metastasis. DNA flow cytometric studies showed diploidy for both primary tumors. No mutation in the BRCA2 gene was found on analysis of DNA from peripheral blood lymphocytes. The present findings show that del(16)(q13) is a recurrent finding among male breast carcinomas and that some of the primary cytogenetic abnormalities, as well as the pattern of chromosomal changes during the progression of sporadic breast carcinoma in the male, are similar to those in the female. In addition, the loss of the Y chromosome in the tumors but not in peripheral blood lymphocytes, suggests a possible role for this abnormality in the pathogenesis of male breast carcinoma. PMID- 10731590 TI - FISH analysis of an AML-M5a with segmental rearrangements involving 11q23-MLL region. AB - A 66-year-old man was diagnosed 10 years ago with thrombocytosis and treated with hydroxyurea. Recently, his condition deteriorated and he was found to have 68% blasts in the blood and AML-M5a. Conventional cytogenetic analysis and fluorescence in situ hybridization studies using 11-painting and MLL probes showed that chromosome 11 had duplications or triplications; the markers were also derived from the chromosome 11-MLL region. Therefore, we have demonstrated segmental rearrangement of chromosome 11 involving the MLL region resulting in multiple copies of the MLL gene. PMID- 10731592 TI - Losses of heterozygosity in oral and oropharyngeal epithelial carcinomas. AB - We analyzed 25 oral and oropharyngeal epithelial carcinomas for loss of heterozygosity (LOH) and microsatellite instability by using 55 oligonucleotide repeat markers located in 45 chromosomal regions. The aim was to identify which chromosomal regions and tumor-suppressor genes (TSGs) are preferentially lost in these tumors and to relate LOH at specific loci to clinicopathologic data. The analysis was performed on tumor tissue and on a corresponding normal tissue (blood lymphocytes) with the use of the polymerase chain reaction technique followed by microsatellite allele separation with denaturing gel electrophoresis. Thirty-two of 45 chromosomal regions demonstrated a significant (>/=20%) incidence of LOH. An allelic loss of >/=50% was found in 9p21 (77.8%), 8p22-23 (70%), 3p12 (61.5%), 1p36.1 and 12q22 (60%), 3q28 (57.1%), 5q23.3 (54.5%), 3p25 26, 3p24, and 7q35 (50%). We did not find any microsatellite instability. Our results suggest that in addition to a group of TSGs, pleiotropic for several tumor types, other suppressor genes are specifically involved in oral and oropharyngeal carcinogenesis. PMID- 10731591 TI - Spontaneous chromosomal instability in breast cancer families. AB - Spontaneous chromosomal instability has been correlated with cancer predisposition. In the present study, the phenomenon has been evaluated using two cytogenetic markers, namely, frequency of spontaneous sister chromatid exchanges (SCE) and spontaneous chromosomal aberrations (CA) in peripheral blood lymphocytes of hereditary breast cancer (HBC) patients (n = 11) and healthy blood relatives (HBR, n = 36). A statistically significant difference was observed for both the endpoints between HBC patients and controls (P < 0.001), HBC patients and HBR (P < 0.001), as well as HBR and controls (P < 0.001). Thus, 63.64% of the HBC patients and 25% of HBR showed a mean CA/cell value higher than the highest mean CA/cell value of the controls (0.11 CA/cell). Similarly, 81.81% of the HBC patients and 61.11% of HBR showed a mean SCE/cell value higher than the highest mean SCE/cell value of the controls (9.60 SCE/cell). Chromosomal aberrations were more frequently observed in the B and E group of chromosomes in HBC patients and HBR. These findings primarily indicate the high level of chromosomal instability in breast cancer families, and might be one of the predisposing factors for high risk of cancer in HBR. PMID- 10731593 TI - A case of chronic lymphocytic leukemia with a constitutional pericentric inversion of chromosome 1. AB - Chronic lymphocytic leukemia (CLL) has been reported to be associated with various chromosomal aberrations, the most common being trisomy 12 and structural rearrangements involving 13q, 11q, and 17p. We present a case of CLL with a constitutional pericentric inversion of chromosome 1. PMID- 10731594 TI - Trisomy of chromosome 10 in two cases of ovarian carcinoma. AB - Simple numerical chromosome aberrations have been observed in tumorigenesis and may point to indicative initiating or early events in tumorigenesis. We have identified two cases of ovarian carcinomas with trisomy of chromosome 10 using conventional GTG-banding and fluorescence in situ hybridization. This is, to our knowledge, the first report of trisomy 10 as a simple karyotypic abnormality observed in ovarian carcinoma. These results suggest that further studies investigating whether chromosome 10 genes are associated with the pathogenesis of some ovarian tumors are warranted. PMID- 10731595 TI - Trisomy 5 as a sole cytogenetic abnormality in pediatric acute lymphoblastic leukemia. AB - We describe a case of pediatric acute lymphoblastic leukemia (ALL) with trisomy 5 as a sole cytogenetic abnormality. A comparison is made with the two cases of adult acute lymphoblastic leukemia with trisomy 5 in the literature. This rare cytogenetic abnormality may portend an especially poor prognosis in patients with ALL. PMID- 10731596 TI - Suppression of X-ray-induced chromatid breaks in human tumor cells by introduction of normal chromosome 4. AB - Human tumor cells in culture frequently show an infinite lifespan and are characterized by an abnormally high frequency of chromatid breaks after x irradiation during G(2) phase of the cell cycle. This abnormally high frequency of persistent chromatid breaks results from deficient repair of the radiation induced DNA damage. In three of four tumor cell lines, addition of a single human chromosome 4 from normal cells by microcell fusion resulted in efficient repair as evidenced by suppression of radiation-induced chromatid breaks to the level in normal cells. With respect to senescence-related gene (s), two of these four tumor cell lines belonged to complementation group A and one each to groups C and D. Chromosome 4 restored DNA repair efficiency in only one of the two tumor cell lines of complementation group A. These results suggest that chromosome 4 carries a DNA repair gene or gene(s) that complement the repair deficiency in three of these four tumor lines. Furthermore, the gene(s) involved in cellular senescence on chromosome 4 appear to differ from the gene(s) for repair of radiation-induced DNA damage during G(2). PMID- 10731597 TI - A novel t(11;12)(q23-24;q24) in a case of minimally-differentiated acute myeloid leukemia (AML-M0). AB - Acute myeloid leukemia with minimal signs of myeloid differentiation (AML-M0) is a recent addition to the FAB group classification. Chromosome data is scarce, but existing reports describe a high incidence of complex karyotypes and myelodysplastic syndrome-like chromosome alterations, while single chromosome translocations have rarely been reported. We describe the case of a 60-year-old woman diagnosed with AML-M0 with a novel translocation t(11;12)(q23-24;q24) as the sole karyotypic marker. Fluorescence in situ hybridization analysis to assess MLL gene splitting did not show rearrangement of this oncogene. PMID- 10731598 TI - A malignant triton tumor with an unbalanced translocation (1;13)(q10;q10) and an isochromosome (8)(q10) as the sole karyotypic abnormalities. AB - The karyotype of a malignant nerve sheath tumor with rhabdomyosarcomatous differentiation (malignant triton tumor) of a 58-year-old woman is reported. The tumor revealed an isochromosome for the long arm of chromosome 8 and an unbalanced translocation (1;13)(q10;q10) leading to a gain of the long arm of chromosome 1 as the sole karyotypic abnormalities. PMID- 10731599 TI - Enterococcal-type glycopeptide resistance genes in non-enterococcal organisms. AB - Although the emergence of vancomycin-resistant enterococci can be attributed, in part, to the increasing use of vancomycin in clinical practice, and glycopeptide use in animal husbandry, the origins of the enterococcal vancomycin resistance genes are not clear. The vancomycin resistance-associated genes in Enterococcus gallinarum, Enterococcus casseliflavus/flavescens, Lactobacillus spp., Leuconostoc spp., Pediococcus spp., and Erysipelothrix rhusiopathiae, are not the source of the high-level vancomycin resistance-associated genes in enterococci. There are, however, environmental organisms which have been found to have gene clusters homologous to the enterococcal vanA, vanB and vanC gene clusters; these include the biopesticide Paenibacillus popilliae, and, to a lesser extent, the glycopeptide-producing organisms Amycolatopsis orientalis and Streptomyces toyocaensis. Still, the exact sources of the enterococcal vancomycin resistance genes remain a mystery. PMID- 10731600 TI - Exploration of deep intraterrestrial microbial life: current perspectives. AB - Intraterrestrial life has been found at depths of several thousand metres in deep sub-sea floor sediments and in the basement crust beneath the sediments. It has also been found at up to 2800-m depth in continental sedimentary rocks, 5300-m depth in igneous rock aquifers and in fluid inclusions in ancient salt deposits from salt mines. The biomass of these intraterrestrial organisms may be equal to the total weight of all marine and terrestrial plants. The intraterrestrial microbes generally seem to be active at very low but significant rates and several investigations indicate chemolithoautotrophs to form a chemosynthetic base. Hydrogen, methane and carbon dioxide gases are continuously generated in the interior of our planet and probably constitute sustainable sources of carbon and energy for deep intraterrestrial biosphere ecosystems. Several prospective research areas are foreseen to focus on the importance of microbial communities for metabolic processes such as anaerobic utilisation of hydrocarbons and anaerobic methane oxidation. PMID- 10731601 TI - Identification of a 36-kDa fibronectin-binding protein expressed by a virulent variant of Leptospira interrogans serovar icterohaemorrhagiae. AB - We investigated the ability of a virulent strain of Leptospira interrogans serovar icterohaemorrhagiae, its isogenic avirulent variant and a saprophytic strain to bind fibronectin using alkaline phosphatase-labelled fibronectin. A single 36-kDa fibronectin-binding protein was expressed only by the virulent strain and was located in the outer sheath according to proteinase K treatment results. The interaction of this protein with fibronectin was specific and the region of fibronectin bound to this potential adhesin overlapped the gelatin binding domain. The inability of a RGDS synthetic peptide to inhibit the binding of fibronectin indicated that the cell-binding domain was not involved in this interaction. Considering the wide distribution of fibronectin within a host and the diversity of mammals involved in the epidemiology of leptospirosis, its implication in the cell attachment process of virulent leptospires is coherent with the multiplicity of target cells. PMID- 10731602 TI - Cloning and overexpression of a tyrosinase gene mel from Pseudomonas maltophila. AB - The tyrosinase gene (mel), which is responsible for melanin formation, was isolated by shotgun cloning of SalI fragments of Pseudomonas maltophila DNA. A 0.7-kb SalI fragment in the recombinant plasmid pWSY8 imparted the ability to synthesize melanin to an Escherichia coli host HB101. The nucleotide sequence of this DNA fragment revealed an open reading frame of 504 bp, encoding a protein of 169 amino acids. The fragment containing the mel gene was then cloned into an expression plasmid pPAS1 under the control of a promoter isolated from the host, P. maltophilia AT18. This strain increased the melanin production by 70.6% compared with the strain HB101/pWSY8, in which the cloned mel gene was under the control of the lac promoter from the vector pUC18. PMID- 10731603 TI - Antibodies (IgG) to lipopolysaccharide of Vibrio cholerae O1 mediate protection through inhibition of intestinal adherence and colonisation in a mouse model. AB - An antiserum raised against purified lipopolysaccharide (LPS) of a Vibrio cholerae O1 strain (Co366) induced passive protection against challenge with the parent as well as other O1 organisms but not against O139 or non-O1/non-O139 organisms. A considerable level of protection against O1 strains was also observed with the IgG fraction of the antiserum which inhibited intestinal adherence and colonisation. The monovalent Fab(IgG) fragment, on the other hand, showed only a low level of protection. Interestingly, purified LPS failed to inhibit intestinal colonisation by the parent strain (Co366), thereby suggesting that the cell surface LPS moieties of vibrios may not be directly involved in the colonisation process. It may be concluded that the anti-LPS antibodies induce passive protection through microagglutination and/or immobilisation of vibrios which do not allow the organisms to adhere to and colonise the intestine. PMID- 10731604 TI - Processing of the Man(10)GlcNAc (M(10)) oligosaccharide by alpha-mannosidases from Candida albicans. AB - The hydrolysis of Man(10)GlcNAc (M(10)) by purified alpha-mannosidases and its further processing by a mixed membrane preparation from Candida albicans were studied. Incubation of the oligosaccharide with purified alpha-mannosidases I (E I) or II (E-II) from C. albicans released 1 and 2 mol of mannose per mol of M(10), respectively. This treatment converted M(10) into an acceptor substrate of further mannose residues from GDP-Man as catalyzed by membrane-bound mannosyltransferases. Elongation of E-I- or E-II-trimmed M(10) yielded a low molecular mass product (14-17 mannose residues added), and in the case of E-II, a minor amount of an additional product of a higher molecular mass. Our results indicate that purified alpha-mannosidases participate in N-glycan processing in C. albicans. PMID- 10731605 TI - Genetic diversity of the Campylobacter genes coding immunodominant proteins. AB - Three Campylobacter jejuni 72Dz/92 genes (cjaA (ompH1), cjaC (hisJ) and cjaD (omp18)) encoding immunodominant proteins are considered to be potential chicken vaccine candidates. The presence and conservation of cjaA, cjaC and cjaD genes among different Campylobacter clinical isolates were determined. The genes were detected in thirty Campylobacter strains using hybridization as well as Western blot analysis. However, PCR products of the predicted size were amplified only from ten out of thirty examined strains regardless of the employed primer pair. The nucleotide sequence of the C. jejuni 72Dz/92 genes was compared with the nucleotide sequences of their homologs cloned from other Campylobacter strains as well as with the whole genome sequence of C. jejuni NCTC 11168. The examined sequences revealed 0 to 16% divergence. Strain-dependent levels of divergence were observed. The polymorphism detected in cjaC was mainly within the 5' region of the gene, while the nucleotide substitutions in cjaA and cjaD are distributed uniformly along the whole genes. Most of the observed nucleotide substitutions occurred at the third base of the codons. This observation is consistent with the results of Western blot experiments. PMID- 10731606 TI - Novel growth characteristics and high rates of nitrate reduction of an Escherichia coli strain, LCB2048, that expresses only a periplasmic nitrate reductase. AB - Escherichia coli strain LCB2048 is a double mutant defective in the synthesis of the two membrane-associated nitrate reductases A and Z. This strain can grow anaerobically on a non-fermentable carbon source, glycerol, in the presence of nitrate even in media supplemented with high concentrations of tungstate. This growth was totally dependent upon a highly active, periplasmic nitrate reductase (Nap). Due to the presence of a previously unreported narL mutation, synthesis of the periplasmic nitrate reductase by this strain was induced during anaerobic growth by nitrate. We have also demonstrated that methyl viologen is an ineffective electron donor to Nap: its use leads to an underestimation of the contribution of Nap activity to the rate of nitrate reduction in vivo. PMID- 10731607 TI - Phospholipid and sterol analysis of plasma membranes of azole-resistant Candida albicans strains. AB - The phospholipid and sterol composition of the plasma membranes of five fluconazole-resistant clinical Candida albicans isolates was compared to that of three fluconazole-sensitive ones. The three azole-sensitive strains tested and four of the five resistant strains did not exhibit any major difference in their phospholipid and sterol composition. The remaining strain (R5) showed a decreased amount of ergosterol and a lower phosphatidylcholine:phosphatidylethanolamine ratio in the plasma membrane. These changes in the plasma membrane lipid and sterol composition may be responsible for an altered uptake of drugs and thus for a reduced intracellular accumulation of fluconazole thereby providing a mechanism for azole resistance. PMID- 10731608 TI - Protective effect of antioxidants against para-nonylphenol-induced inhibition of cell growth in Saccharomyces cerevisiae. AB - The cell growth-modulating activity of an endocrine disruptor, p-nonylphenol (NP), was estimated using the yeast Saccharomyces cerevisiae as a simple model of eukaryotic cells. NP caused a dose-dependent suppressive effect on cell growth of S. cerevisiae at 10, 25 and 50 microM. The NP-induced cell growth inhibition was restored when concomitantly lipophilic antioxidants such as alpha-tocopherol and beta-carotene were supplied, but not the hydrophilic antioxidants ascorbic acid or (-)epigallocatechin gallate (EGCG). The cellular oxygen consumption of S. cerevisiae was also inhibited in a dose-dependent fashion by the extracellular addition of NP, and pretreatment with alpha-tocopherol and beta-carotene suppressed NP-induced inhibition of cellular oxygen consumption, but ascorbic acid and EGCG were not effective. Furthermore, NP caused a marked generation of radical oxygen species (ROS) in S. cerevisiae, which was suppressed by treatment with alpha-tocopherol and beta-carotene, but not with ascorbic acid and EGCG. However, NP did not show a significant inhibitory effect on cell growth and survival of mitochondria-deficient petite mutant cells and they showed a relatively weak ROS-generating activity compared with parent yeast cells. These results suggest that NP-induced inhibition of cell growth and oxygen consumption in S. cerevisiae might be possibly associated with ROS generation in yeast mitochondria. The significance of this finding is discussed from the viewpoint of NP-induced oxidative stress against eukaryotic cells. PMID- 10731609 TI - Mutation of cytochrome bd quinol oxidase results in reduced stationary phase survival, iron deprivation, metal toxicity and oxidative stress in Azotobacter vinelandii. AB - Azotobacter vinelandii cydAB mutants lacking cytochrome bd lost viability in stationary phase, irrespective of temperature, but microaerobiosis or iron addition to stationary phase cultures prevented viability loss. Growth on solid medium was inhibited by a diffusible factor from neighbouring cells, and by iron chelators, In(III) or Ga(III); microaerobic growth overcame inhibition by the extracellular factor. Siderophore production and total Fe(III)-chelating activity were not markedly affected in Cyd(-) mutants, and remained responsive to iron repression. Cyd(-) mutants were hypersensitive to Cu(II), Zn(II), and compounds exerting oxidative stress. Failure to synthesise haemoproteins does not explain the complex phenotype since mutants retained significant catalase activity. We hypothesise that Cyd(-) mutants are defective in maintaining the near-anoxic cytoplasm required for reductive iron metabolism and nitrogenase activity. PMID- 10731610 TI - Hydroxyurea inhibits intracellular Toxoplasma gondii multiplication. AB - Tachyzoites of Toxoplasma gondii multiply within the parasitophorous vacuole (PV) until the lysis of the host cell. This study was undertaken to evaluate the effect of hydroxyurea (a specific drug that arrests cell division at G1/S phase) on the multiplication of T. gondii tachyzoites in infected Vero cells. Infected host cells were treated with hydroxyurea for periods varying from 5 to 48 h, and the survival and morphology of the parasite were determined. Hydroxyurea arrested intracellular T. gondii multiplication in all periods tested. After 48 h of incubation with hydroxyurea, intracellular parasites were not easily observed in Vero cells. Ultrastructural observations showed that infected host cells treated with hydroxyurea for 24 h or more presented disrupted intracellular parasites within the PV. However, the host cells exhibited a normal morphology. Our observations suggest that hydroxyurea was able to interfere with the cycle of the intracellular parasite, leading to the complete destruction of the T. gondii without affecting the host cells. PMID- 10731611 TI - Ribotyping and rapid identification of Staphylococcus xylosus by 16-23S spacer amplification. AB - Ninety-five strains of Staphylococcus xylosus isolated from goat milk, French sausage or mice were analyzed together with 35 Staphylococcus type strains by 16 23S spacer amplification and ribotyping. The results obtained by PCR amplification of the 16-23S spacer region permitted the distinction of each type strain and additionally generated a DNA banding pattern characteristic for 93 of the 95 Staphylococcus xylosus strains. Ribotyping proved to be an efficient epidemiological tool for Staphylococcus xylosus species as it clustered the 95 strains into 23 distinct types. PMID- 10731612 TI - Virulence-related DNA sequences and adherence patterns in strains of enteropathogenic Escherichia coli. AB - The presence of the genes for Escherichia coli adherence factor (EAF), attaching and effacing lesion (eae) and bundle-forming pili (bfp) in 72 strains identified as enteropathogenic E. coli (EPEC) by slide agglutination was evaluated using hybridization and PCR. The adherence property of these strains was assayed using 3h HeLa cells adherence assay. The results obtained indicated that virulence associated genes were present in 65% of the strains but only ten (13.9%) isolates were positive for all the three markers (typical EPEC), 37 (51.4%) isolates carried either one or two of these determinants (atypical EPEC) and the remaining 25 (34.7%) were negative for all these genes. In vitro adherence assay showed that 44 (61.1%) strains adhered to HeLa cells with a defined pattern, 13 (18.1%) isolates adhered loosely with no definite pattern and the remaining 15 (20.8%) were non-adherent. Analysis of the results showed a statistically significant association between the presence of the virulence-related genes with adherence of the strains with a defined pattern (P Lys(32 )is useful for biophysical and biochemical studies, and, particularly, for X-ray crystallography. PMID- 10731669 TI - Mice lacking a CDK inhibitor, p57Kip2, exhibit skeletal abnormalities and growth retardation. AB - p57Kip2, one of the cyclin-dependent kinase (CDK) inhibitors, has been suggested to be a tumor suppressor candidate. To elucidate its biological roles in mouse development and tumorigenesis, we created p57Kip2-deficient mice. The p57Kip2 deficient mice exhibited a cleft palate and defective bone formation resulting in severe dyspnea. Most of the p57Kip2-deficient mice died within 24 h after birth, while about 10% of them survived beyond the weaning period. All of the surviving mice showed severe growth retardation. The males showed immaturity of the testes, prostate and seminal vesicles, and the females showed vaginal atresia, immaturity of the uterus, and an increased number of atretic follicles. Although Yan et al. and Zhang et al. have already reported p57Kip2-deficient mice, they could not investigate the phenotypes of the surviving p57Kip2-deficient mice. Also, most of the symptoms of Beckwith-Wiedemann syndrome could not be reproduced in the mutant mice. Embryonic fibroblasts prepared from p57Kip2-deficient mice showed no differences in the proliferation rate and saturation density, suggesting that G1 arrest is largely independent of p57Kip2 function. Our results suggest that p57Kip2 plays a critical role in development, but do not support the hypothesis that the p57Kip2 gene is a tumor-suppressor gene or is responsible for Beckwith Wiedemann syndrome. PMID- 10731670 TI - Cloning and sequencing of the gene for a Tetrahymena fimbrin-like protein. AB - Tetrahymena F-actin-binding protein, which induces bundling of Tetrahymena F actin, was localized to a division furrow during cytokinesis. We report here the cloning and characterization of the gene and cDNA of a Tetrahymena F-actin binding protein. The cDNA encodes a protein comprising 579 deduced amino acids with a calculated molecular mass of 65.9 kDa. The predicted amino acid sequence shares 37.7, 41.8, and 39% identity with the sequences of yeast fimbrin, Arabidopsis thaliana fimbrin, and Dictyostelium discoideum plastin, respectively. The Tetrahymena F-actin-binding protein also shares two actin-binding domains previously identified in the fimbrin/plastin family, but lacks the EF-hand Ca2+ binding motif, suggesting that this protein is a novel-fimbrin-like protein in Tetrahymena. Moreover, we cloned a genomic DNA encoding the Tetrahymena fimbrin like protein and performed Southern and Northern hybridizations. The results indicate that the genomic DNA possesses 9 introns and that both the gene and transcript of Tetrahymena fimbrin-like protein are single. Thus, we suggest that Tetrahymena fimbrin-like protein localizes to the division furrow and probably cross-links actin filaments in a Ca(2+)-insensitive manner during cytokinesis. PMID- 10731671 TI - Purification and characterization of a novel protamine kinase in HL60 cells. AB - A protamine kinase from HL60 cells was purified to near homogeneity by DEAE Sephacel, protamine-agarose, Hydroxylapatite, and S-200 chromatography. It was purified by 75.8-fold through four chromatographic steps, and 0.67% of total activity was recovered. The purified enzyme had an apparent molecular mass of 120 kDa and was activated by Mg(2+) or Mn(2+), but inhibited by Ca(2+). Neither phospholipid nor phorbol ester significantly affected the enzyme activity. Staurosporine was the most potent inhibitor of the enzyme among the protein kinase inhibitors tested, K(252a), H(7), heparin, and staurosporine. The purified protamine kinase exhibited a maximum velocity of 5,000 pmol/min/mg and K(m) of 1.3 mM for protamine sulfate as a substrate. Myelin basic protein and protamine sulfate served as the best substrates for the protamine kinase among those tested. The activity of the protamine kinase remained unchanged upon treatment with PMA, retinoic acid, dimethyl sulfoxide, or 1,25 dihydroxy vitamin D(3) for 15 min, while treatment with a differentiating agent, 1,25 dihydroxy vitamin D(3), for one week increased its activity. These results suggest that protamine kinase in HL60 cells is involved in the late stage of the macrophage-monocytic differentiation pathway and may play a role in maintenance of the differentiation after HL60 cells are committed. PMID- 10731672 TI - Identification of human GATA-2 gene distal IS exon and its expression in hematopoietic stem cell fractions. AB - Transcription factor GATA-2 is essential for the proper function of hematopoietic stem cells and progenitors. Two first exons/promoters have been found in the mouse GATA-2 gene, and a distal IS promoter shows activity specific to hematopoietic progenitors and neural tissues. To ascertain whether the two promoter system is also utilized in the human GATA-2 gene, we isolated and analyzed a P1 phage clone containing this gene. The nucleotide sequence of the human GATA-2 gene 5' flanking region was determined over 10 kbp, and a human IS exon was identified in the locus through sequence comparison analysis with that of the mouse GATA-2 IS exon. RNA blotting and reverse-transcribed PCR analyses identified a transcript that starts from the IS exon in human leukemia-derived cell lines. The IS-originated transcript was also identified in CD34-positive bone marrow and cord blood mononuclear cells, which are recognized as clinically important hematopoietic stem cell-enriched fractions. Phylogenic comparison of the human and mouse GATA-2 gene sequences revealed several regions in the locus that exhibit high sequence similarity. These results demonstrate that the GATA-2 gene regulatory machinery is conserved among vertebrates. The fact that the human IS promoter is active in the hematopoietic stem cell/progenitor fraction may be an important clue for the design of a vector system that can specifically express various genes in hematopoietic stem cells and progenitors. PMID- 10731673 TI - Nitric oxide underlies the differentiation of PC12 cells induced by depolarization with high KCl. AB - Nitric oxide (NO) acts as a cytostatic agent to induce neuronal differentiation of PC12 cells after nerve growth factor (NGF) treatment. We newly subcloned PC12K cells that extended neurites after depolarization with high KCl. Here we present evidence that the neuronal differentiation of PC12K cells caused by depolarization with high KCl is mediated by endogenous NO. The outgrowth of neurites was significantly inhibited by 2 mM N-nitro-L-arginine methyl ester (L NMAE), and 10 mM L-NAME was necessary for complete inhibition. The inhibition of NGF-dependent neurite outgrowth by L-NAME was abolished by depolarization of cells with KCl. The expression of neuronal- and endothelial-NO-synthase in PC12K cells was confirmed by immuno-cytochemical and immuno-blotting analyses with the respective monoclonal antibodies. However, the expression of inducible-NO synthase was not observed in PC12K cells cultured with high KCl under the depolarization conditions with 45 mM KCl. We observed the increase of NO in the differentiated PC12K cells using diaminofluorescein, a novel fluorescent indicator for NO. PMID- 10731674 TI - Rapid turnover of tryptophan hydroxylase is driven by proteasomes in RBL2H3 cells, a serotonin producing mast cell line. AB - Previously we demonstrated that tryptophan hydroxylase (TPH) undergoes very fast turnover driven by ATP-dependent proteolysis in serotonin producing mast cells [Hasegawa et al. (1995) FEBS Lett. 368, 151-154]. We searched for the major proteases involved in the rapid degradation of TPH in RBL2H3 cells. Among various protease inhibitors tested, proteasome inhibitors MG115, MG101, MG132, and lactacystin effectively inhibited the intracellular degradation of TPH. Administration of the proteasome inhibitors to cultured cells caused more than a 5-fold accumulation of TPH. Administration of the inhibitors together with cycloheximide stabilized the amount of TPH with no appreciable increase or decrease. Although MG-series proteasome inhibitors could inhibit calpain, the involvement of calpain was excluded since the cysteine protease inhibitor E-64-d, which acts on calpain, had no effect. Extracts of RBL2H3 cells were shown to contain a protease that digests TPH in an ATP-dependent manner and is sensitive to proteasome inhibitors. The ubiquitination of TPH could be visualized by Western blot analysis using both anti-TPH and anti-ubiquitin antibodies. Based on these results, we conclude that 26S proteasomes are mainly involved in the degradation of TPH. In the reported amino acid sequences of TPH from various sources including human, rabbit, rat, and mouse, a PEST sequence that is widely shared among short-lived proteins has been recognized. It was noted that the PEST sequence lies within the most conserved portion of the enzyme, the pteridine binding site. PMID- 10731675 TI - A chimeric lectin formed from Bauhinia purpurea lectin and Lens culinaris lectin recognizes a unique carbohydrate structure. AB - Lectins are carbohydrate-binding proteins widely used in biochemical, immunochemical, and histochemical studies. Bauhinia purpurea lectin (BPA) is a leguminous lectin with an affinity for galactose and lactose. Nine amino acids, DTWPNTEWS, corresponding to the amino acid sequence from aspartic acid-135 to serine-143 in the primary structure of BPA were replaced with the corresponding amino acid residues from the mannose-binding Lens culinaris lectin (LCA), and the chimeric lectin obtained was expressed in Escherichia coli cells. The carbohydrate-binding specificity of the recombinant chimeric lectin was investigated in detail by comparing the elution profiles of various glycopeptides and oligosaccharides with defined carbohydate structures from immobilized lectin columns. Glycopeptides carrying three constitutive carbohydrate sequences of Galbeta1-3GalNAc-Ser/Thr and a complex-type biantennary glycopeptide, which show a high affinity for BPA or LCA, were shown to have no affinity for the chimeric lectin. In contrast, hybrid-type and high mannose-type glycopeptides with a Manalpha1-6(Manalpha1-3)Manalpha1-6Man sequence were found to have a moderate affinity for the chimeric lectin. This result demonstrates that a novel type of lectin with a unique carbohydrate-binding specificity can be constructed from BPA by substituting several amino acid residues in its metal-binding region with other amino acid residues. Additional lectin(s) with distinctly different carbohydrate-binding specificities will provide a powerful tool for many studies. PMID- 10731676 TI - Cyborg lectins: novel leguminous lectins with unique specificities. AB - Bauhinia purpurea lectin (BPA) is one of the beta-galactose-binding leguminous lectins. Leguminous lectins contain a long metal-binding loop, part of which determines their carbohydrate-binding specificities. Random mutations were introduced into a portion of the cDNA coding BPA that corresponds to the carbohydrate-binding loop of the lectin. An library of the mutant lectin expressed on the surface of lambda foo phages was screened by the panning method. Several phage clones with an affinity for mannose or N-acetylglucosamine were isolated. These results indicate the possibility of making artificial lectins (so called "cyborg lectins") with distinct and desired carbohydrate-binding specificities. PMID- 10731677 TI - Cloning of phosphatase I gene from a psychrophile, Shewanella sp., and some properties of the recombinant enzyme. AB - Psychrophilic phosphatase I from Shewanella sp. is a cold enzyme that was found as a novel protein-tyrosine-phosphatase (PTPase, EC 3. 1.3.48) with a histidine as its catalytic residue [Tsuruta and Aizono (1999) J. Biochem. 125, 690-695]. Here, we determined the nucleotide sequence of a DNA fragment (2,004 bp) containing the phosphatase I gene by cloning with polymerase chain reaction (PCR) and inverted PCR techniques. The deduced amino acid sequence, of the enzyme contained a conserved region of protein-serine/threonine-phosphatase (PPase). The 38.5 kDa-recombinant protein expressed in Escherichia coli was purified to homogeneity by glutathione-Sepharose 4B column chromatography, treatment with endoproteinase and Mono-Q column chromatography. The recombinant enzyme had a specific activity of 49.4 units and, like native psychrophilic phosphatase I, exhibited high catalytic activity at low temperature and PTPase activity. PMID- 10731678 TI - Expression of functional M2 muscarinic acetylcholine receptor in Escherichia coli. AB - The M2 muscarinic acetylcholine receptor mutant (M2 mutant), with a lack of glycosylation sites, a deletion in the central part of the third inner loop, and the addition of a six histidine tag at the C-terminus, was fused to maltose binding protein (MBP) at its N-terminus and expressed in Escherichia coli. The expression level was 0.2 nmol receptor per 100 ml culture, as assessed as [3H]L quinuclidinyl benzilate ([3H]QNB) binding activity, when the BL 21 strain was cultured at 37 degrees C to a late growth phase and the expression was induced by isopropyl beta-thiogalactoside at 20 degrees C. No [3H]QNB binding activity was detected when it was not fused to MBP or when expression was induced at 37 degrees C instead of 20 degrees C. The MBP-M2 mutant expressed in E. coli showed the same ligand binding activity as the M2 mutant expressed in the Sporodoptera frugiperda (Sf9)/baculovirus system, as assessed as displacement of [(3)H]QNB with carbamylcholine and atropine. The MBP-M2 mutant was solubilized, purified with Co2+-immobilized Chelating Sepharose gel and SP-Sepharose, and then reconstituted into lipid vesicles with G protein Go or Gi1 in the presence or absence of cholesterol. The reconstituted vesicles showed GTP-sensitive high affinity binding for carbamylcholine and carbamylcholine-stimulated [35S]GTP gamma S binding activity in the presence of GDP. The proportion of high affinity sites for carbamylcholine and the extent of carbamylcholine-stimulated [(35)S]GTP gamma S binding were the same as those observed for the M2 mutant expressed in Sf9 cells and were not affected by the presence or absence of cholesterol. These results indicate that the MBP-M2 mutant expressed in E. coli has the same ability to interact with and activate G proteins as the M2 mutant expressed in Sf9, and that cholesterol is not essential for the function of the M2 muscarinic receptor. PMID- 10731679 TI - Identification of key residues in rabbit liver microsomal cytochrome P450 2B4: importance in interactions with NADPH-cytochrome P450 reductase. AB - A cytochrome P450 2B4 (CYP2B4) model was used to select key residues supposed to serve in interactions with NADPH-cytochrome P450 reductase (P450R). Eight amino acid residues located on the surface of the hemoprotein were chosen for mutagenesis experiments with CYP2B4(Delta2-27) lacking the NH(2)-terminal signal anchor sequence. The mutated proteins were expressed in Escherichia coli, purified, and characterized by EPR- and CD-spectral analysis. Replacement of histidine 226 with alanine caused a 3.8-fold fall in the affinity for P450R with undisturbed reductive capacity of the system. Similarly, the K225A, R232A, and R253A variants exhibited P450R-directed activity that was depressed to about half that of the control enzyme, suggesting that the deletion of positive charges on the surface of CYP2B4(Delta2-27) resulted in impaired electrostatic contacts with complementary amino acids on the P450R protein. While the Y235A mutant did not show appreciably perturbed reduction activity, the conservative substitution with alanine of the phenylalanine residues at positions 223 and 227 gave a 2.1- to 6. 1-fold increase in the K(m) values with unchanged V(max); this was attributed to the disruption of hydrophobic forces rather than to global structural rearrangement(s) of the engineered pigments. Measurement of the stoichiometry of aerobic NADPH consumption and H(2)O(2) formation revealed the oxyferrous forms of the F223A, H226A, and F227A mutants to autoxidize more readily owing to less efficient coupling of the systems. Noteworthy, the F244A enzyme did not exhibit significant reduction activity, suggesting a pivotal role of Phe-244 in the functional coupling of P450R. The residue was predicted to constitute part of an obligatory electron transfer conduit through pi-stacking with Phe-296 located close to the heme unit. All of the residues examined reside in the putative G helix of CYP2B4, so that this domain obviously defines part of the binding site for P450R. PMID- 10731680 TI - Essential roles of carbohydrate signals in development, immune response and tissue functions, as revealed by gene targeting. AB - Knockout mice lacking glycosyltransferases or sulfotransferases provide unequivocal evidence that the carbohydrate moieties of glycoproteins, glycolipids, and proteoglycans play essential roles in various biological phenomena such as development, the immune response, and tissue functions. Examples of abnormalities of null mutants include arrest of embryogenesis due to deletion of N-acetylglucosaminyltransferase I or glucosylceramide synthase, failure of kidney formation in heparan sulfate 2-O-sulfotransferase deficiency, suppressed antibody production in alpha-2, 6-sialyltransferase deficiency, male sterility in GM2/GD2 synthase deficiency, and abnormalities in the function and stability of myelin in galactosylceramide deficiency. PMID- 10731681 TI - Concentration of mRNA for the natriuretic peptide receptor-C in hypertrophic chondrocytes of the fetal mouse tibia. AB - The roles of natriuretic peptides in cardiovascular homeostasis have been well characterized. A recent study revealed that mice lacking natriuretic peptide receptor-C (NPR-C) exhibit skeletal-overgrowth. We therefore, performed in situ hybridization with riboprobes to determine the localization of mRNAs for receptors for natriuretic peptides in the growth plate of the fetal mouse tibia The amount of mRNA for NPR-A was below the detectable level in the growth plate. The mRNA for NPR-B was detected predominantly in proliferating chondrocytes. By contrast, high levels of mRNA for NPR-C were found in hypertrophic chondrocytes. In other regions of the growth plate, the levels of mRNA for NPR-C were very low. The patterns of expression of mRNAs for NPR-B and NPR-C, namely, subtype switching during differentiation from proliferating chondrocytes to hypertrophic chondrocytes, suggest that these receptors might be involved in the growth and differentiation of the growth plate during fetal development in the mouse. PMID- 10731682 TI - Crystallization and preliminary X-ray diffraction analysis of the extracellular domain of the cell surface antigen CD38 complexed with ganglioside. AB - The cell surface antigen CD38 is a multifunctional ectoenzyme that acts as an NAD(+) glycohydrolase, an ADP-ribosyl cyclase, and also a cyclic ADP-ribose hydrolase. The extracellular catalytic domain of CD38 was expressed as a fusion protein with maltose-binding protein, and was crystallized in the complex with a ganglioside, G(T1b), one of the possible physiological inhibitors of this ectoenzyme. Two different crystal forms were obtained using the hanging-drop vapor diffusion method with PEG 10,000 as the precipitant. One form diffracted up to 2.4 A resolution with synchrotron radiation at 100 K, but suffered serious X ray damage. It belongs to the space group P2(1)2(1)2(1) with unit-cell parameters of a = 47.9, b = 94.9, c = 125.2 A. The other form is a thin plate, but the data sets were successfully collected up to 2.4 A resolution by use of synchrotron radiation at 100 K. The crystals belong to the space group P2(1) with unit-cell parameters of a = 57.4, b = 51.2, c = 101.1 A, and beta = 97.9 degrees, and contain one molecule per asymmetric unit with a VM value of 2.05 A(3)/Da. PMID- 10731683 TI - Macrophage protein kinase C: its role in modulating membrane microviscosity and superoxide in leishmanial infection. AB - Pretreatment of macrophages with, an agonist of PKC, showed diverse effects on degradation and survival of two virulent strains of Leishmania donovani promastigotes. Treatment of macrophages with PMA for 45 min at 37 degrees C generated significant amounts of superoxide anions and reduced the parasite burden of macrophages by up to 48 and 43% when AG83 and GE-1 strains were used for infection. Staurosporine, an inhibitor of PKC, inhibited PMA-dependent killing of the parasites, while tyrphostin AG 126, an inhibitor of protein tyrosine kinase, showed very little effect. Depletion of PKC by prolonged incubation with PMA drastically reduced the superoxide anion generation and increased the uptake and multiplication of the parasites. Finally, to understand the mechanism of higher uptake of the parasites by PKC-depleted macrophages, membrane microviscosity was measured by fluorescence depolarization. Membrane microviscosity was found to be approximately 40% lower in PKC-depleted macrophages than in normal macrophages, indicating the role of membrane fluidity in the infection process. Together, these data suggest PKC activation, superoxide generation, and membrane fluidity are essential factors in the efficient regulation of leishmanial infection. PMID- 10731684 TI - Influenza A virus-binding activity of glycoglycerolipids of aquatic bacteria. AB - As the aqueous sphere has been proposed to be an important source medium for the virus infection of land animals, the glycolipids of some aquatic organisms were examined for human influenza A virus-binding activity. Active compounds were not found among the eight echinoderm gangliosides, but two active non-sialylated glycoglycerolipids were isolated from an aquatic bacterium, Corynebacterium aquaticum. The structural formula of one of them, H632A, was elucidated to be 1 14-methyl-hexadecanoyl-3-alpha-D-galactopyranosyl-(1-->3)-6-(12-met hyl tetradecanoyl)-1-alpha-D-mannopyranosyl]-sn-glycerol. The latter together with reported one elsewhere, S365A, 1-14-methyl-hexadecanoyl-3-[alpha-D-mannopyranosyl (1-->3)-6-(12-meth yl-tetradecanoyl)-1-alpha-D-mannopyranosyl]-sn-glycerol, apparently bound to three human influenza viruses, A/PR/8/34 (H1N1), A/Aichi/2/68 (H3N2), and A/Memphis/1/71 (H3N2), exhibiting 7-12% (H632A) and 10-22% (S365A) of the activities of the control substances (Neu5Acalpha2-3-paragloboside and Neu5Acalpha2-6- paragloboside). Additionally, these glycolipids were assumed to have virus-neutralizing activities for the following two reasons: (i) The hemagglutination and hemolysis activities of the viruses were inhibited by the glycolipid. (ii) The leakage of a cytosolic enzyme (lactate dehydrogenase) from Madin-Darby canine kidney cells on virus infection was prevented by the glycolipids to nearly the same extent as by fetuin. This is the first evidence of the binding- and neutralizing-abilities of native glycoglycerolipids as to influenza viruses. PMID- 10731685 TI - Analysis of conformational changes at the unique loop adjacent to the ATP binding site of smooth muscle myosin using a fluorescent probe. AB - Recent crystallographic studies have shown that smooth muscle myosin has three highly conserved unique loops, loop B (320-327), loop M (687-699), and loop N (125-134), similar to other myosins, skeletal muscle and dictyostelium myosins. We previously demonstrated that the effect of actin is mediated by a conformational change in one of the loops, loop M comprising amino acids 677 to 689 of skeletal muscle myosin [Maruta and Homma (1998) J. Biochem. 124, 528-533]. In the present study, in order to clarify the role of these smooth muscle myosin loops in energy transduction, we specifically labeled the loops with a fluorescent photoreactive ADP analogue, 3'-O-(N-methylanthraniloyl)-8-azido-ADP (Mant-8-N(3)-ADP), and then measured the fluorescent polarization. When Mant-8 N(3)-ADP was trapped by aluminium fluoride or vanadate into the ATPase site, Mant 8-N(3)-ADP was covalently incorporated into loop N (125-134). In contrast, Mant-8 N(3)-ADP trapped by beryllium fluoride was covalently incorporated into both loop M (687-699) and loop N (125-134) at an almost equimolar ratio. Actin binding to smooth muscle myosin S1 (SMO-S1) labeled at only loop N (125-134) increased the polarization due to the viscosity of actin. In contrast, S1 labeled at both loops N and M showed a much smaller increase in polarization. Our results indicate that the probe at loop M (687-699) of smooth muscle myosin moved to a less hindered region, suggesting that actin binding induces conformational changes at loop M (687-699) similar to those of the corresponding loop (677-689) in skeletal muscle myosin, as previously demonstrated in our laboratory. PMID- 10731686 TI - Norepinephrine stimulates interleukin-6 mRNA expression in primary cultured rat hepatocytes. AB - Non-invasive immobilization stress causes an increase in the plasma interleukin (IL)-6 level accompanied by increased IL-6 mRNA expression and IL-6 immunoactivity in the liver [Biochem. Biophys. Res. Commun. (1997) 238, 707-711]. In the present study, using rat primary cultured hepatocytes and non-parenchymal liver cells, the effect of norepinephrine (NE) on IL-6 mRNA expression was determined. IL-6 mRNA expression in hepatocytes, but not in non-parenchymal liver cells, increased when the cells were treated with NE. The stimulatory effect of NE was inhibited by the combined use of alpha- and beta-adrenergic antagonists. IL-6 mRNA expression in hepatocytes also increased on incubation with the culture medium of non-parenchymal liver cells treated with NE. The effect of the medium was blocked by an IL-1 receptor antagonist. Moreover, exogenous IL-1beta stimulated IL-6 mRNA expression in hepatocytes. IL-1beta was present in the medium of non-parenchymal liver cells and increased with NE-treatment. These results suggest that NE released from sympathetic nerve terminals during stress can directly increase IL-6 mRNA expression in hepatocytes and indirectly through IL-1beta production from non-parenchymal liver cells. PMID- 10731687 TI - Accumulation and degradation in the endoplasmic reticulum of a truncated ER-60 devoid of C-terminal amino acid residues. AB - The accumulation and degradation in the endoplasmic reticulum (ER) of a truncated ER-60 protease, from which the C-terminal 89 amino acid residues have been deleted (K 417 ochre), was examined. K 417 ochre overexpressed in COS-1 cells is not secreted into the medium, but accumulates as insoluble aggregates in non ionic detergent without degradation in unusual clump membrane structures. K 417 ochre, stably expressed, forms soluble aggregates in non-ionic detergent and is distributed in the reticular structures of ER. Under these conditions, K 417 ochre is not secreted into the medium but is degraded with a half-life time of more than 8 h. Since K 417 ochre/C all S, in which all the Cys residues of K 417 ochre are replaced by Ser, also forms aggregates, an inter-disulfide bond appears unnecessary for aggregation. In both types of aggregates, Ig heavy chain binding protein, calnexin, glucose regulated protein 94, calreticulin, ERp72, and protein disulfide isomerase are scarcely found. Since degradation of the stably expressed K 417 ochre was not inhibited by lactacystin, leupeptin, NH(4)Cl, or cytocharasin B, but was inhibited by N-acetyl-leucyl-leucyl-norleucinal, the self-aggregated abnormal protein in the lumen of ER is assumed to be degraded by an unknown protease system other than proteasome, lysosome or autophagy. PMID- 10731688 TI - Some properties and the possible role of intrinsic ATPase of rat liver 80S ribosomes in peptide bond elongation. AB - The properties and role in peptide elongation of ATPase intrinsic to rat liver ribosomes were investigated. (i) Rat liver 80S ribosomes showed high ATPase and GTPase activities, whereas the GTPase activity of EF-1alpha and EF-2 was very low. mRNA, aminoacyl-tRNA, and elongation factors alone enhanced ribosomal ATPase activity and in combination stimulated it additively or synergistically. The results suggest that these translational components induce positive conformational changes of 80S ribosomes by binding to different regions of ribosomes. Translation inhibitors, tetracyclin and fusidic acid, inhibited ribosomal ATPase with or without elongational components. (ii) Two ATPase inhibitors, AMP-P(NH)P and vanadate, did not inhibit GTPase activities of EF 1alpha and EF-2 assayed as uncoupled GTPase, but they did inhibit poly(U) dependent polyphe synthesis of 80S ribosomes. (iii) Effects of AMP-P(NH)P and ATP on poly(U)-dependent polyphe synthesis at various concentrations of GTP were examined. ATP enhanced the activity of polyphe synthesis even at high concentrations of GTP, suggesting a specific role of ATP. At low concentrations of GTP, the extent of inhibition by AMP-P(NH)P was very low, probably owing to the prevention of the reduction of the GTP concentration. (iv) Vanadate inhibited the translocation reaction by high KCl-washed polysomes. These findings together indicate that ribosomal ATPase participates in peptide translation by inducing positive conformational changes of mammalian ribosomes, in addition to its role of chasing tRNA from the E site. PMID- 10731689 TI - Role of Atf1 and Pap1 in the induction of the catalase gene of fission yeast schizosaccharomyces pombe. AB - We examined the induction of the catalase gene (ctt1(+)) of fission yeast Schizosaccharomyces pombe in response to several stresses by using mutants of transcription factors (Atf1 and Pap1) and a series of deletion mutants of the ctt1(+) promoter region. A transcription factor, Atf1, and its binding site are necessary for the induction of ctt1(+) by osmotic stress, UV irradiation, and heat shock. Induction by menadione treatment, which produces superoxide anion, required element A, the region from -111 to -90 (numbered with the transcription start site as +1). The factor responsible for the induction of the gene by oxidative stress via element A was identified as the transcription factor Pap1. We also found that Atf1 is activated by menadione treatment in pap1 mutant cells, although it is not activated by menadione treatment in pap1(+) cells. The activity of catalase is not increased in pap1 cells by several stresses, despite mRNA induction, suggesting that Pap1 plays some role in the expression of catalase activity. PMID- 10731690 TI - Enzymic preparation of protein G-peroxidase conjugates catalysed by transglutaminase. AB - Transglutaminases (TGases, EC 2.3.2.13) have proved to be valuable enzymes for site-directed protein coupling via N(epsilon)-(gamma-L-glutamyl)lysine bonds. Their use in conjugate synthesis would overcome many problems caused by chemical reagents. In this approach, we show for the first time that two proteins with different functionalities, namely soybean peroxidase and protein G, can be cross linked by bacterial TGase with retention of their activities. Soybean peroxidase and protein G were chosen for the enzymic preparation of a bifunctional conjugate among a series of other TGase substrates detected by enzymic incorporation of small fluorescent or biotinylated molecules. The highest yields of conjugate were obtained with a 15-fold excess of peroxidase in phosphate buffer, pH 7.0. Size exclusion chromatography enabled both purification of the conjugates and recovery of the starting materials. Analysis of bifunctionality revealed the coupling of protein G with an average of three peroxidase molecules. PMID- 10731691 TI - Midkine enhances early stages of collagen gel contraction. AB - Midkine (MK) is a heparin-binding growth/differentiation factor implicated in the control of development and repair of various tissues. To investigate the roles of MK in embryogenesis and in regulation of wound healing, we utilized a system of collagen gel contraction by dermal fibroblasts, which provides an in vitro model for studying fibroblast-collagen interactions important in various physiological and pathological phenomena. MK enhanced gel contraction 8-24 h after plating, and its effect was inhibited by anti-MK antibody. The effect was reduced after 48 h, while TGF-beta continued to be active in the later stage. Morphologically, MK treated fibroblasts tended to be elongated more frequently than control fibroblasts. The effect of MK in the early stage of gel contraction suggests a role of MK as a modulator of cell-matrix interactions. PMID- 10731692 TI - Colocalization of GP125/CD98 with tropomyosin isoforms at the cell-cell adhesion boundary. AB - Two monoclonal antibodies designated as 1F6 and 4B10 were obtained on screening for reactivities to CD98-associated molecules by sandwich-type enzyme-linked immunosorbent assaying using hybridoma culture supernatants as the solid phase, cell lysates as an antigen source, and a mixture of biotinylated antibodies to CD98HC as a detector. Flow cytometric analysis with microspheres in combination with 1F6, 4B10, and anti-CD98HC also indicated the association of antibody defined antigen(s) with CD98. 1F6 and 4B10, stained fibrillate components in fixed and permeated cells but were not reactive with unfixed live cells, suggesting that epitopes reside in the cytoskeleton-associated structure in the intracellular region. Two-color immunostaining followed by confocal microscopy revealed the colocalization of the antigen with CD98 at the cell-cell adhesion boundary of HeLa cells. 1F6 detected proteins with relative molecular masses of 33,000 to 43,000 on immunoblotting analysis involving cell lysates of human and rat cell lines. Analysis with a purified tropomyosin specimen from rabbit skeletal muscle demonstrated that 1F6 and 4B10 recognize tropomyosin. Two dimensional gel electrophoresis followed by immunoblotting analysis revealed that 1F6 recognizes various tropomyosin isoforms. These results indicated that CD98 physically associates directly or indirectly with tropomyosin, and that this association is closely related to the cell-cell interaction. PMID- 10731694 TI - Midkine rescues Wilms' tumor cells from cisplatin-induced apoptosis: regulation of Bcl-2 expression by Midkine. AB - Midkine (MK) is a heparin-binding growth factor involved in diverse biological phenomena, e.g. neuronal survival, carcinogenesis, and tissue repair. MK expression is detected mainly in the kidney in adult mice. In this study, we show that, at a dose that can induce recoverable renal damage and induce apoptosis, cisplatin (CDDP) transiently suppressed MK expression in mouse kidney. In vitro, CDDP suppressed MK expression and induced apoptosis in cultured G401 cells, a Wilms' tumor cell line. Exogenous MK protein partially rescued G401 cells from CDDP-induced apoptosis. MK enhanced the expression of Bcl-2, but not that of Bcl x(L), in G401 cells in a dose-dependent manner, and it prevented the Bcl-2 reduction due to CDDP. Moreover, Bcl-2 expression in mouse kidney was also transiently suppressed by CDDP treatment, the expression profile being similar to that of MK. These results imply that MK exerts cytoprotective activity toward a damaging insult, presumably at least in part through enhancement of the expression of Bcl-2. PMID- 10731693 TI - Functional consequences of the deletion mutation deltaGlu160 in human cardiac troponin T. AB - To explore the functional consequences of a deletion mutation of troponin T (DeltaGlu160) found in familial hypertrophic cardiomyopathy, the mutant human cardiac troponin T, and wild-type troponins T, I, and C were expressed in Escherichia coli and directly incorporated into isolated porcine cardiac myofibrils using our previously reported troponin exchange technique. The mutant troponin T showed a slightly reduced potency in replacing the endogenous troponin complex in myofibrils and did not affect the inhibitory action of troponin I but potentiated the neutralizing action of troponin C, suggesting that the deletion of a single amino acid, Glu-160, in the strong tropomyosin-binding region affects the tropomyosin binding affinity of the entire troponin T molecule and alters the interaction between troponin I and troponin C within ternary troponin complex in the thin filament. This mutation also increased the Ca(2+) sensitivity of the myofibrillar ATPase activity, as in the case of other mutations in troponin T with clinical phenotypes of poor prognosis similar to that of Glu160. These results provide strong evidence that the increased Ca(2+) sensitivity of cardiac myofilament is a typical functional consequence of the troponin T mutation associated with a malignant form of hypertrophic cardiomyopathy. PMID- 10731695 TI - A spectrophotometric method for the determination of glycolate in urine and plasma with glycolate oxidase. AB - An enzymatic assay was developed for the spectrophotometric determination of glycolate in urine and plasma. Glycolate was first converted to glyoxylate with glycolate oxidase, and the glyoxylate formed was condensed with phenylhydrazine. The glyoxylate phenylhydrazone formed was then oxidized with K(3)Fe(CN)(6) in the presence of excess phenylhydrazine, and A(515) of the resulting 1, 5 diphenylformazan was measured. Since glycolate oxidase also acts on glyoxylate and L-lactate, the incubation of samples with glycolate oxidase was carried out in 120-170 mM Tris-HCl (pH 8.3) to obtain glyoxylate as its adduct with Tris. The pyruvate formed from lactate was removed by subsequent brief incubation with alanine aminotransferase in the presence of L-glutamate, and alpha-ketoglutarate formed was converted back to L-glutamate by glutamate dehydrogenase and an NADPH generating system. Thus the specificity of the assay relies principally on the substrate specificity of glycolate oxidase, and high sensitivity is provided by the high absorbance of 1,5-diphenylformazan at 515-520 nm. Plasma was deproteinized with perchloric acid, and then neutralized with KOH. Plasma and urine samples were then incubated with approximately 5 mM phenylhydrazine, and then treated with stearate-deactivated activated charcoal to remove endogenous keto and aldehyde acids as their phenylhydrazones. The normal plasma glycolate and urinary glycolate/creatinine ratio for adults determined by this method are approximately 8 microM and approximately 0.036, respectively. PMID- 10731696 TI - Difference in mechanism between glyceraldehyde- and glucose-induced insulin secretion from isolated rat pancreatic islets. AB - The effects of D-glyceraldehyde and glucose on islet function were compared in order to investigate the difference between them in the mechanism by which they induce insulin secretion. The stimulation of insulin secretion from isolated rat islets by 10 mM glyceraldehyde was not completely inhibited by either 150 microM diazoxide (an opener of ATP-sensitive K(+) channels) or 5 microM nitrendipine (an L-type Ca(2+)-channel blocker), whereas the stimulation of insulin secretion by 20 mM glucose was completely inhibited by either drug. The insulin secretion induced by glyceraldehyde was less augmented by 100 microM carbachol (a cholinergic agonist) than that induced by glucose. The stimulation of myo inositol phosphate production by 100 microM carbachol was more marked in islets incubated with the hexose than with the triose. The content of glyceraldehyde 3 phosphate, a glycolytic intermediate, in islets incubated with glyceraldehyde was far higher than that in islets incubated with glucose, whereas the ATP content in islets incubated with the triose was significantly lower than that in islets incubated with the hexose. These results suggest that glyceraldehyde not only mimics the effect of glucose on insulin secretion but also has the ability to cause the secretion of insulin without the influx of Ca(2+ )through voltage dependent Ca(2+) channels. The reason for the lower potency of the triose than the hexose in stimulating insulin secretion is also discussed. PMID- 10731698 TI - Presence of oxidized protein hydrolase in human cell lines, rat tissues, and human/rat plasma. AB - Oxidized protein hydrolase (OPH), an 80 kDa serine protease whose activity is inhibited by diisopropyl fluorophosphate (DFP), has been isolated from human erythrocytes [Fujino, T. et al. (1998) J. Biochem. 124, 1077-1085]. The presence of OPH in various biological samples was examined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting using an anti-OPH antibody raised against OPH purified from human erythrocytes, and by [(3)H]DFP-labeling and successive SDS PAGE/fluorography. Solubilized samples of human cell lines including K-562 cells, THP-1 cells and Jurkat cells, and rat tissues including brain, heart, liver, kidney, and testis, inhibited the anti-OPH antibody binding to OPH in ELISA. Immunoblotting of lysates of K-562 cells, THP-1 cells and Jurkat cells showed four immunoreactive protein bands including an 80 kDa protein. Immunoprecipitation of the [(3)H]DFP-labeled K-562 cell lysate and successive SDS PAGE/fluorography showed the presence of only the 80 kDa DFP-reactive protein with OPH antigenic activity. The level of the 80 kDa immunoreactive protein in K 562 cells rose as the cells differentiated toward erythrocytes. Immunoblotting of human and rat plasma showed two immunoreactive protein bands, including the 80 kDa protein, and SDS-PAGE/fluorography of [(3)H]DFP-labeled rat and human plasma showed the presence of only the 80 kDa DFP-reactive protein. The results indicate that OPH is present in a wide variety of biological samples. PMID- 10731697 TI - Caspases cleave the amino-terminal calpain inhibitory unit of calpastatin during apoptosis in human Jurkat T cells. AB - We have previously reported the activation of procalpain mu (precursor for low calcium-requiring calpain) in apoptotic cells using a cleavage-site-directed antibody specific to active calpain [Kikuchi, H. and Imajoh-Ohmi, S. (1995) Cell Death Differ. 2, 195-199]. In this study, calpastatin, the endogenous inhibitor protein for calpain, was cleaved to a 90-kDa polypeptide during apoptosis in human Jurkat T cells. The limited proteolysis of calpastatin preceded the autolytic activation of procalpain. Inhibitors for caspases rescued the cells from apoptosis and simultaneously inhibited the cleavage of calpastatin. The full length recombinant calpastatin was also cleaved by caspase-3 or caspase-7 at Asp 233 into the same size fragment. Cys-241 was also targeted by these caspases in vitro but not in apoptotic cells. Caspase-digested calpastatin lost its amino terminal inhibitory unit, and inhibited three moles of calpain per mole. Our findings suggest that caspases trigger the decontrol of calpain activity suppression by degrading calpastatin. PMID- 10731699 TI - Mechanistic study of beta-xylosidase from Trichoderma koningii G-39. AB - The catalytic mechanism of the beta-xylosidase purified from the culture filtrate of Trichoderma koningii G-39 was investigated. By NMR spectroscopy, the stereochemistry of the enzyme catalyzing the hydrolysis of 2,4-dinitrophenyl and p-nitrophenyl-beta-D-xylosides was found unequivocally to involve retention of the anomeric configuration. Based on the k(cat) values of a series of arylxylosides with leaving group pK(a)s in the range of 4-10, an extended Bronsted plot was constructed with a slope (beta(lg)) near zero. Enzymatic hydrolysis of aryl-beta-D-xylosides in acetate buffer (pH 4.0) containing 3 or 5% methanol showed a constant product ratio (methylxyloside/xylose), indicating the presence of a common intermediate, probably the xylosyl-enzyme intermediate. In the presence of DTT, the k(cat) values of p-cyanophenyl-beta-D-xylopyranoside and p-nitrophenyl-beta-D-xylopyranoside increased greatly. A two-step mechanism involving the formation and breakdown of the xylosyl-enzyme intermediate was therefore proposed. The rate-limiting step is the breakdown of the intermediate. The secondary deuterium kinetic isotope effect (k(H)/k(D)) measured for 2,4 dinitrophenyl-beta-D-xyloside was 1.02+/-0.01, suggesting that the transition state for breakdown of the xylosyl-enzyme intermediate is S(N)2-like. PMID- 10731700 TI - Purification of a 72-kDa protein-tyrosine kinase from rat liver and its identification as Syk: involvement of Syk in signaling events of hepatocytes. AB - Syk protein-tyrosine kinase (PTK) has been implicated in a variety of hematopoietic cell responses including immunoreceptor signaling. However, so far, there has been no evidence of the expression of Syk or Syk-related PTK in non hematopoietic tissues. In this study, we have purified from blood cell-depleted rat liver a 72-kDa cytoplasmic PTK which shows cross-reactivity with anti-Syk antibody. Partial amino acid sequence analysis revealed that this 72-kDa PTK is identical to Syk. Immunohistochemical and RT-PCR analyses demonstrated that Syk is expressed in human hepatocytes and two rat liver-derived cell lines, JTC-27 and RLC-16. Furthermore, Syk is significantly tyrosine-phosphorylated in response to angiotensin II in JTC-27 cells, and angiotensin II-induced MAP kinase activation is blocked by the treatment of cells with a Syk-selective inhibitor, piceatannol. These results suggest that Syk plays an important role in signaling events of hepatocytes, such as signaling steps leading to MAP kinase activation by G-protein-coupled receptors. This is the first report of the expression of Syk in non-hematopoietic tissue. PMID- 10731701 TI - Roles of functional loops and the C-terminal segment of a single-stranded DNA binding protein elucidated by X-Ray structure analysis. AB - The single-stranded DNA (ssDNA) binding protein from Escherichia coli (EcoSSB) plays a central role in DNA replication, recombination and repair. The tertiary structure of EcoSSB was determined at 2.2 A resolution. This is rather higher resolution than previously reported. Crystals were grown from the homogeneous intact protein but the EcoSSB tetramer in the crystals contains truncated subunits lacking a part of the C-terminal. The structure determined includes biologically important flexible loops and C-terminal regions, and revealed the existence of concavities. These concavities include the residues important for ssDNA binding. An ssDNA can be fitted on the concavities and further stabilized through interactions with the loops forming flexible lids. It seems likely to play a central role in the binding of ssDNA. PMID- 10731702 TI - Cocrystallization of a mutant aspartate aminotransferase with a C5-dicarboxylic substrate analog: structural comparison with the enzyme-C4-dicarboxylic analog complex. AB - A mutant Escherichia coil aspartate aminotransferase with 17 amino acid substitutions (ATB17), previously created by directed evolution, shows increased activity for beta-branched amino acids and decreased activity for the native substrates, aspartate and glutamate. A new mutant (ATBSN) was generated by changing two of the 17 mutated residues back to the original ones. ATBSN recovered the activities for aspartate and glutamate to the level of the wild type enzyme while maintaining the enhanced activity of ATB17 for the other amino acid substrates. The absorption spectrum of the bound coenzyme, pyridoxal 5' phosphate, also returned to the original state. ATBSN shows significantly increased affinity for substrate analogs including succinate and glutarate, analogs of aspartate and glutamate, respectively. Hence, we could cocrystallize ATBSN with succinate or glutarate, and the structures show how the enzyme can bind two kinds of dicarboxylic substrates with different chain lengths. The present results may also provide an insight into the long-standing controversies regarding the mode of binding of glutamate to the wild-type enzyme. PMID- 10731703 TI - Functional analysis of conserved aspartate and histidine residues located around the type 2 copper site of copper-containing nitrite reductase. AB - A heterologous expression system of the blue copper-containing nitrite reductase from Alcaligenes xylosoxidans GIFU1051 (AxgNIR) was constructed, and the purified recombinant enzyme was characterized. All the characteristic spectroscopic properties and enzyme activity of native AxgNIR were retained in the copper reconstituted recombinant protein expressed in Escherichia coli, indicating the correct coordination of two types of Cu (type 1 and 2) in the recombinant enzyme. Moreover, two conserved noncoordinate residues, Asp98 and His255, located near the type 2 Cu site were replaced to elucidate the catalytic residue(s) of NIR. The Asp98 residue hydrogen-bonded to the water molecule ligating the type 2 Cu was changed to Ala, Asn, or Glu, and the His255 residue hydrogen-bonded to Asp98 through the water molecule was replaced with Ala, Lys, or Arg. The catalytic rate constants of all mutants were decreased to 0.4-2% of those of the recombinant enzyme, and the apparent K(m) values for nitrite were greatly increased in the Asp98 mutants. All the steady-state kinetic data of the mutants clearly demonstrate that both Asp98 and His255 are involved not only in the catalytic reaction but also in the substrate anchoring. PMID- 10731704 TI - Refolding of firefly luciferase immobilized on agarose beads. AB - The renaturation yield of the denatured firefly luciferase decreased strongly with increasing protein concentration in a renaturation buffer, because of aggregation. In this study, firefly luciferase was immobilized on agarose beads at a high concentration. Although the protein concentration was extremely high (about 100-fold) compared to that of soluble luciferase, the renaturation yield was comparable with that for the soluble one. Thus, immobilization was shown to be effective for avoiding aggregation of firefly luciferase. It was also shown that the optimum buffer conditions for renaturation of the immobilized luciferase were the same as those for the renaturation in solution. Also, it was indicated that electrostatic interactions between a protein and the matrix have a negative effect on renaturation of the immobilized luciferase since the renaturation yield decreased at acidic pH only for the immobilized luciferase. These novel observations are described in detail in this paper. PMID- 10731705 TI - Effect of Arg145Gly mutation in human cardiac troponin I on the ATPase activity of cardiac myofibrils. AB - In order to determine the functional consequences of the Arg145Gly mutation in troponin I found in familial hypertrophic cardiomyopathy, human cardiac troponin I and its mutant were expressed in Escherichia coli and purified, and then their effects on the ATPase activity of porcine cardiac myofibrillar preparations from which both troponins C and I had been depleted were examined. Both the wild-type and mutant troponin Is suppressed the ATPase activity of the troponin C.I depleted myofibrils, but the maximum inhibition caused by mutant troponin I was weaker than that by wild-type troponin I. In the Ca(2)(+)-activation profile of the myofibrillar ATPase activity after reconstitution with both troponins I and C, the Ca(2)(+)-sensitivity with mutant troponin I was higher than that with wild type troponin I, whereas the maximum level of the ATPase activity with mutant troponin I was lower than that with wild-type troponin I. These findings strongly suggest that the Arg145Gly mutation in human cardiac troponin I modulates the Ca(2)(+)-regulation of contraction by impairing the interaction of troponin I with both actin-tropomyosin and troponin C. PMID- 10731706 TI - Evidence that the Aspergillus nidulans class I and class II chitin synthase genes, chsC and chsA, share critical roles in hyphal wall integrity and conidiophore development. AB - Although many chitin synthase genes have been identified in a broad range of fungal species, there have been only a few reports about their role in fungal morphogenesis. In most cases, single gene disruption or replacement did not reveal their function, possibly because of functional redundancy among them. We obtained null mutants of Aspergillus nidulans chsA and chsC genes encoding non essential class II and class I chitin synthases, respectively. The DeltachsA DeltachsC mutant exhibited growth defects on media supplemented with sodium dodecyl sulfate (SDS), high concentration of salts, chitin-binding dyes, or chitin synthase competitive inhibitors, suggesting loss of integrity of hyphal wall. Moreover, remarkable abnormalities of the double mutant were observed microscopically during its asexual development. The conidiophore population was drastically reduced. Interestingly, secondary conidiophores were occasionally produced from vesicles of the primary ones. The morphology of these conidiophores was similar to those of the A. nidulans developmental mutants, medusa (medA), abacus (abaA), and some kinds of bristle (brlA). In situ staining patterns suggested that chsA was mainly expressed in the metulae, phialides, and conidia, whereas chsC was expressed in hyphae as well as conidiophores. These results suggest that ChsA and ChsC share critical functions in hyphal wall integrity and differentiation. PMID- 10731707 TI - Synthesis of a new Cre recombinase gene based on optimal codon usage for mammalian systems. AB - The origin of the Cre recombinase gene is bacteriophage P1, and thus the codon usages are different from in mammals. In order to adapt this codon usage for mammals, we synthesized a "mammalian Cre recombinase gene" and examined its expression in Chinese hamster ovarian tumor (CHO) cells. Significant increases in protein production as well as mRNA levels were observed. When the recombination efficiency was compared using CHO cell transfectants having a cDNA containing loxP sites, the "mammalian Cre recombinase gene" recombined the loxP sites much more efficiently than the wild-type Cre recombinase gene. PMID- 10731708 TI - Identification of a response element for vitamin D3 and retinoic acid in the promoter region of the human fructose-1,6-bisphosphatase gene. AB - Fructose-1,6-bisphosphatase (FBPase) is a key gluconeogenic enzyme. The data herein show that both the enzyme activity and mRNA level of the human FBPase gene are enhanced by 9-cis retinoic acid (9cRA) and all-trans retinoic acid (atRA) as well as by 1,25-dihydroxyvitamin D3 (VD3) in human promyelocytic HL60 cells and normal monocytes in peripheral blood, which were used as an alternative source to liver for the DNA diagnosis of FBPase deficiency. To understand the molecular mechanism of this enhancing action, the 2.4 kb 5'-regulatory region of the human FBPase gene was isolated and sequenced. Using luciferase reporter gene assays, a 0.5 kb FBPase basal promoter fragment was found to confer induction by VD3, 9cRA, and atRA that was mediated by the vitamin D3 receptor (VDR), retinoid X receptor (RXR), and retinoic acid receptor (RAR). Within this region, a direct repeat sequence, 5'-TAACCTttcTGAACT-3' (-340 to -326), which functions as a common response element for VD3, 9cRA, and atRA, was identified. The results of electrophoretic mobility shift assays indicated that VDR-RXR and RAR-RXR heterodimers bind this response element. Collectively, these observations indicate that VD3 and RA are important modulators of the expression of the human FBPase gene in monocytic cells. PMID- 10731709 TI - Crystal structure of alkalophilic asparagine 233-replaced cyclodextrin glucanotransferase complexed with an inhibitor, acarbose, at 2.0 A resolution. AB - The product specificity of cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. #1011 is improved to near-uniformity by mutation of histidine-233 to asparagine. Asparagine 233-replaced CGTase (H233N-CGTase) no longer produces alpha-cyclodextrin, while the wild-type CGTase from the same bacterium produces a mixture of predominantly alpha-, beta-, and gamma cyclodextrins, catalyzing the conversion of starch into cyclic or linear alpha 1,4-linked glucopyranosyl chains. In order to better understand the protein engineering of H233N-CGTase, the crystal structure of the mutant enzyme complexed with a maltotetraose analog, acarbose, was determined at 2.0 A resolution with a final crystallographic R value of 0.163 for all data. Taking a close look at the active site cleft in which the acarbose molecule is bound, the most probable reason for the improved specificity of H233N-CGTase is the removal of interactions needed to form a compact ring like a-cyclodextrin. PMID- 10731710 TI - Nucleotide sequence of gene PBII encoding salivary proline-rich protein P-B. AB - The nucleotide sequence of gene PBII encoding salivary proline-rich protein P-B was determined. PBII is 7.1 kb long and contains 3 exons. PBII exhibits considerable nucleotide sequence homology not only in exons but also in introns with PBI (accession number D89501), the gene whose nucleotide sequence was determined previously [Isemura and Saitoh (1997) J. Biochem. 121, 1025-1030]. PBI and II constitute a gene family distinct from that to which the majority of salivary proline-rich protein ones belong. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession number AB031740. PMID- 10731711 TI - Comparative analysis of the genomic structures and promoter activities of mouse Siaalpha2,3Galbeta1,3GalNAc GalNAcalpha2, 6-sialyltransferase genes (ST6GalNAc III and IV): characterization of their Sp1 binding sites. AB - The genomic organization of the genes encoding the mouse N-acetylgalactosamine alpha2,6-sialyltransferase specific for Siaalpha2,3Galbeta1,3GalNAc (ST6GalNAc III and IV) has been determined. The ST6GalNAc III gene spans over 120 kilobases of genomic DNA with 5 exons; on the other hand, the ST6GalNAc IV gene spans over 12 kilobases of genomic DNA with 6 exons. But the exon-intron boundaries of these genes are very similar. The 5'-flanking regions of these genes do not contain a TATA- or CAAT-box but have three putative Sp1 binding sites for each promoter. Transient transfection experiments demonstrated functional promoter activity in an ST6GalNAc III-expressing cell line, P19, for the ST6GalNAc III promoter, and in an ST6GalNAc IV-expressing cell line, NIH3T3, for the ST6GalNAc IV promoter. Mobility shift assaying and mutational analysis of the promoter region indicated that two of the three Sp1 binding sites are involved in the transcriptional regulation of the ST6GalNAc III gene in P19 cells, while all three Sp1 binding sites are involved in the transcriptional regulation of the ST6GalNAc IV gene in NIH3T3 cells. PMID- 10731712 TI - Promotion of the uptake of PS liposomes and apoptotic cells by a product of growth arrest-specific gene, gas6. AB - Gas6, a ligand of receptor tyrosine kinases Axl, Sky, and Mer, potentiates cell proliferation and prevents cell death. It also contains g-carboxylglutamic acid residues that mediate the interaction of some blood coagulation factors with negatively charged phospholipids. In our previous study, we demonstrated that Gas6 specifically binds to phosphatidylserine (PS) and links Axl-expressing cells to the PS-coated surface. In this study, to further understand the biological role of the interaction of Gas6 with PS, we examined the effect of Gas6 on the uptake of PS liposomes by macrophages. In vitro phagocytosis studies showed that Gas6 enhanced the uptake of PS liposomes approximately threefold and that the interaction of Gas6 with the surface of macrophages was essential for this enhancement. Analyses of the mechanism of the uptake of PS liposome suggested that Gas6 interacts with PS liposome via its N-terminal Gla domain and with macrophages via its C-terminal domain. Like that of PS liposomes, the uptake of apoptotic cells by macrophages was also enhanced, approximately twofold, in the presence of Gas6. These findings suggest that Gas6 may help phagocytic cells recognize cells with PS exposed on their surfaces, which is considered to be one of the mechanisms for clearing away dying cells. Thus, Gas6 may play a critical role in homeostasis by facilitating the clearance of PS-expressing cells. PMID- 10731713 TI - Purification and characterization of a monoacylglycerol lipase from the moderately thermophilic Bacillus sp. H-257. AB - A thermostable monoacylglycerol lipase [MGLP, EC 3.1.1.23] was purified for the first time from a cell-free extract of the moderately thermophilic Bacillus sp. H 257. The enzyme was purified 3,028-fold to homogeneity by chromatography using Octyl-Sepharose CL-4B, Q-Sepharose FF, and Superose 12 columns. The molecular mass of the MGLP was estimated to be 25 kDa by gel filtration and 24 kDa by SDS PAGE, suggesting a monomeric protein. The isoelectric point was determined to be 4.66 by isoelectric focusing. The MGLP retained its full activity upon incubation at 60 degrees C for 10 min (pH 7. 3), and was stable at pH 7-10. The optimal temperature for activity at pH 7.5 was 75 degrees C, and the maximum activity was observed from pH 6-8. This enzyme hydrolyzes monoacylglycerols, with the highest activity occurring with 1-monolauroylglycerol. Di- and triacylglycerols, on the other hand, are essentially inert as substrates for the enzyme. The K(m) values for the hydrolysis of 1-monolauroylglycerol, 1-monooleoylglycerol, and 2 monooleoylglycerol were determined to be 140, 83 and 59 mM, respectively. The enzyme was not inhibited by cholate, but was slightly inhibited by Triton X-100 and deoxycholate. The amino acid sequence of the N-terminal region of the enzyme (16 residues) was also determined. PMID- 10731714 TI - N-Glycans protect proteins from protease digestion through their binding affinities for aromatic amino acid residues. AB - It was previously revealed [Yamaguchi, H., Nishiyama, T., and Uchida, M. (1999) J. Biochem. 126, 261-265] that N-glycans of both the high-mannose and complex types have binding affinity for aromatic amino acid residues. This study shows that free N-glycans protect proteins from protease digestion through their binding affinities for the aromatic amino acid residues exposed on protein molecules. Protease digestion of bovine pancreatic RNase A and bovine a lactalbumin was depressed in solutions (1 mM or so) of free N-glycans of both the high-mannose and complex types. The increasing order of the protective effects of the N-glycans paralleled that of their affinities for aromatic amino acid residues; and the presence of aromatic amino acids practically abolished the protective effects of the N-glycans. The N-glycans also depressed the protease digestion of metallothionein, an aromatic amino acid-free protein, in agreement with the observation that the N-glycans also interact with the solvent-exposed aromatic amino acid residues of the proteases. Thus it seems probable that the N glycans protect proteins from protease digestion by steric hindrance attributable to their binding affinity for the solvent-exposed aromatic amino acid residues of both substrate proteins and proteases. PMID- 10731715 TI - Convenient and efficient in vitro folding of disulfide-containing globular protein from crude bacterial inclusion bodies. AB - We investigated how the folding yield of disulfide-containing globular proteins having positive net charges from crude bacterial inclusion bodies was affected by additives in the folding buffer. In screening folding conditions for human ribonucleases and its derivative, we found that addition of salt (about 0.4 M) to a folding buffer increased the folding yield. This suggested that electrostatic interaction between polyanionic impurities such as nucleic acids and cationic unfolded protein led to the formation of aggregates under the low-salt conditions. Since inclusion bodies were found to contain nucleic acids regardless of the electrostatic nature of the expressed protein, the electrostatic interaction between phosphate moieties of nucleic acids and basic amino acid residues of a denatured protein may be large enough to cause aggregation, and therefore the addition of salt in a folding buffer may generally be useful for promotion of protein folding from crude inclusion bodies. We further systematically investigated additives such as glycerol, guanidium chloride, and urea that are known to act as chemical chaperons, and found that these additives, together with salt, synergistically improved folding yield. This study, suggesting that the addition of salt into the folding buffer is one of the crucial points to be considered, may pave the way for a systematic investigation of the folding conditions of disulfide-containing foreign proteins from crude bacterial inclusion bodies. PMID- 10731716 TI - Irreversible extrusion of the first loop facing the matrix of the bovine heart mitochondrial ADP/ATP carrier by labeling the Cys(56) residue with the SH-reagent methyl methanethiosulfonate. AB - The effect of the SH-reagent methyl methanethiosulfonate (MMTS) on the ADP/ATP carrier of bovine heart mitochondria was studied under various conditions. MMTS labeled predominately Cys(56) in the first loop facing the matrix (loop M1), and the labeling inhibited ADP transport via the carrier. The transport inhibition was found to be due to fixation of the carrier in the m-state conformation. MMTS labeling was suggested not to affect ADP binding to its major binding site. These features were the same as those of another commonly used SH-reagent, N ethylmaleimide (NEM). Although the van der Waals volume of the non-hydrogen bondable methylthio group of MMTS is much smaller than that of the ethylsuccinimide group of NEM, modification of Cys(56) inhibited the interconversion between the m- and c-state conformation. The mechanism by which MMTS inhibited the transport activity is discussed in terms of stabilization of conformation of the loop M1. PMID- 10731717 TI - Suppression of prostaglandin E(2)-mediated c-fos mRNA induction by interleukin-4 in murine macrophages. AB - When murine peritoneal macrophages were stimulated for 30 min with arachidonic acid, the growth-associated immediate early gene c-fos was induced in a concentration-dependent manner as assessed by Northern blot analysis. The arachidonic acid-induced c-fos mRNA expression was inhibited by a cyclooxygenase inhibitor, indomethacin, but not by a lipoxygenase inhibitor, nordihydroguaiaretic acid. Macrophages produced prostaglandin (PG) E(2) from arachidonic acid as determined by an enzyme immunoassay. Northern blot analysis revealed the expression of PGE receptor EP2 and EP4 subtypes, but not EP1 and EP3 in murine macrophages. PGE(2) brought about a marked elevation of cAMP, and c-fos mRNA expression was increased by PGE(2) and dibutyryl cAMP in these cells. These results suggest that arachidonic acid is transformed to PGE(2), which then binds to EP2 and EP4 receptors to increase intracellular cAMP and c-fos mRNA expression. Furthermore, the induction of c-fos by arachidonic acid, PGE(2), and cAMP was suppressed by pretreatment with interleukin (IL)-4. We also showed that the tyrosine phosphorylation of a Janus kinase, JAK3, is enhanced by IL-4 treatment, suggesting that the PGE(2)-mediated c-fos mRNA induction is inhibited by IL-4 through the tyrosine phosphorylation of JAK3. PMID- 10731718 TI - Bundle formation of smooth muscle desmin intermediate filaments by calponin and its binding site on the desmin molecule. AB - Smooth muscle basic calponin, a major actin-, tropomyosin-, and calmodulin binding protein, has been examined for its ability to interact with desmin intermediate filaments from smooth muscle cells using sedimentation analysis, turbidity changes, chemical cross-linking, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF/MS), and electron microscopic observations. Calponin interacted with desmin intermediate filaments in a concentration-dependent manner in vitro. The binding of calponin to desmin produced dense aggregates at 30 degrees C. The dense aggregates were observed by electron microscopy to be composed of large anisotropic bundles of desmin filaments, indicating that calponin forms bundles of desmin filaments. The addition of calmodulin or S100 to the mixture of calponin and desmin caused the removal of calponin from the desmin filaments and inhibited bundle formation in the presence of Ca(2+), but not in the presence of EGTA. Calponin-related proteins including G-actin, tropomyosin, and SM22, had little effect on the binding of calponin to desmin filaments, whereas tubulin weakly inhibited the binding. Desmin had little influence on the calponin-actin and calponin-tubulin interactions using the zero-length cross-linker, EDC. Domain mapping with chymotryptic digestion showed that the binding site of calponin resides within the central a-helical rod domain of the desmin molecule. The chemical cross linked products of calponin and synthetic peptides (TQ27, TNEKVELQELNDRFANYIEKVRFLEQQ; EE24, EEELRELRRQVDALTGQRARVEVE) derived from the rod domain were detected by MALDI TOF/MS. Furthermore, the calponin-desmin interaction was significantly inhibited by the addition of EE24, but only slightly by TQ27. These results suggest that calponin may act as a cross-linking protein between desmin filaments as well as among intermediate filaments, microfilaments and microtubules in smooth muscle cells. PMID- 10731719 TI - Macrophomate synthase: characterization, sequence, and expression in Escherichia coli of the novel enzyme catalyzing unusual multistep transformation of 2-pyrones to benzoates. AB - Macrophoma commelinae isolated from spots on leaves of Commelina communis has the ability to transform 5-acetyl-4-methoxy-6-methyl-2-pyrone (1) to 4-acetyl-3 methoxy-5-methylbenzoic acid (macrophomic acid, 2). This biotransformation includes the condensation of the 2-pyrone ring with a C3-unit precursor to form a substituted benzoic acid. We optimized conditions for induction of enzyme activity in M. commelinae, identified oxalacetate as a C3-unit precursor with cell extract, and purified the novel enzyme, macrophomate synthase. Oxalacetate inhibited the enzyme activity at a concentration higher than 5 mM, and magnesium chloride stimulated the enzyme activity. Kinetic analyses gave K(m) of 1.7 mM for 1 at 5 mM oxalacetate, K(m) of 1.2 mM for oxalacetate at 5 mM 1, and k(cat) of 0.46 s(-1) per subunit. Pyruvate was a weak substrate, with K(m) of 35.2 mM and k(cat) of 0.027 s(-1) at 5 mM 1. We cloned and sequenced a cDNA encoding the macrophomate synthase. The cDNA of 1,225 bp contained an open reading frame that encoded a polypeptide of 339 amino acid residues and 36,244 Da, the sequence of which showed no significant similarity with known proteins in a homology search with BLAST programs. Transformed E. coli cells carrying the cDNA encoding the mature protein of macrophomate synthase overproduced macrophomate synthase under the control of the T7 phage promoter induced by IPTG. The purified enzyme showed the same values of K(m) and optimum pH as the native macrophomate synthase. PMID- 10731720 TI - Expression and imprinting status of human PEG8/IGF2AS, a paternally expressed antisense transcript from the IGF2 locus, in Wilms' tumors. AB - A large imprinted gene cluster in human chromosome 11p15.5 has been implicated in Beckwith-Wiedemann syndrome and Wilms' tumor. We have identified a paternally expressed imprinted gene, PEG8/IGF2AS, in this locus. It is transcribed in the opposite direction to the IGF2 transcripts and some genomic regions are shared with the IGF2 gene, as in the case of the mouse imprinted Igf2as gene reported previously by T. Moore et al. As to the relationship between these genomic regions, the human and mouse genes are very similar but there is no homology in their middle parts. Interestingly, PEG8/IGF2AS and IGF2 were found to be overexpressed in Wilms' tumor samples, at levels over ten and a hundred times higher than that in normal kidney tissues neighboring the tumors, respectively. These findings indicate that PEG8/IGF2AS is a good marker of Wilms' tumor and also suggest the possibility of PEG8/IGF2AS being one of the candidate Wilms' tumor genes. PMID- 10731721 TI - Development of a new inhibitor of glucosylceramide synthase. AB - Analogs of the potent inhibitor of glucosylceramide (GlcCer) synthase, D-threo-1 phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), based on substitutions in the palmitoyl group were made by means of a stereo-selective synthetic method in order to elucidate the role of the hydrophobic portion in both the inhibitory action toward the enzyme and the biological effects. While P4 strongly inhibited GlcCer synthase with an IC(50) of 0.5 microM in vitro, it also inhibited cell growth by 50% at the concentration of 7 microM. The shorter N-acyl chain analogs including decanoyl, octanoyl, and hexanoyl groups showed similar IC(50) values for GlcCer synthase (around 2 microM) but the hexanoyl analog exhibited only a slight inhibitory effect on cell growth, showing the dissociation between GlcCer depletion and cell growth. Several compounds which exhibit similar hydrophobicity to the hexanoyl analog of P4 were subsequently designed. We found that D-threo-1 phenyl-2-benzyloxycarbonylamino-3-pyrrolidino-1-pr opanol (PBPP) was a most potent inhibitor, showing an IC50 of 0.3 microM. In cultured cells, PBPP was able to deplete glycosphingolipids without affecting cell growth or the ceramide level. PMID- 10731722 TI - Peroxiredoxin IV is a secretable protein with heparin-binding properties under reduced conditions. AB - Peroxiredoxins (PRxs) play a role in protecting protein free thiol groups against oxidative damage and thioredoxin-dependent peroxidase activity. This report describes the characteristics of the fourth member of the mammalian PRxs, PRx IV. Rat PRx IV produced in Sf21 cells by a baculovirus expression system has two bands with different electrophoretic mobilities, 31 and 27 kDa [Matsumoto et al. (1999) FEBS Lett. 443, 246-250]. The 27-kDa PRx IV lacks the NH(2)-terminal 36 amino acids which correspond to a predicted leader peptide, which is required for secretion from cells. Thus, the 31-kDa form is probably a precursor form, and the 27-kDa form, a secretable form which is enzymatically active. Pulse-chase experiments of PRx IV-transfected COS-1 cells showed that PRx IV is processed within 10 min and released from cells. The secretable form contains both reduced and oxidized forms. The reduced form binds to both a heparin affinity column and human umbilical vein endothelial cells, while the oxidized form does not. The equilibrium dissociation constants, K(D), for heparin and heparan sulfate as judged by surface plasmon resonance experiments were 19 and 870 nM, respectively. The secretable form corresponds to the major bands found in most tissues, as evidenced by immunoblot analysis. Within cells, secretable form was largely localized on the endoplasmic reticulum, as judged by colocalization with calreticulin. Moreover, PRx IV has glutathione-dependent peroxidase activity in addition to thioredoxin-dependent activity. These data indicate that PRx IV is a secretable protein and may exert its protective function against oxidative damage by scavenging reactive oxygen species in the extracellular space. PMID- 10731723 TI - Nucleocytoplasmic shuttling of the aryl hydrocarbon receptor. AB - The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that acts in concert with the AhR nuclear translocator (ARNT), and alters gene expression in response to environmental contaminants such as 2,3,7, 8 tetrachlorodibenzo-p-dioxin (TCDD). We have previously shown that AhR contains both a nuclear localization signal (NLS), AhR(13-39), and a nuclear export signal (NES), AhR(55-75), in its NH(2)-terminal region. In this study, we obtained direct evidence for the nucleocytoplasmic shuttling of AhR and show the biological significance of the shuttling in terms of the transcriptional activation of its target gene, CYP1A1. When AhR(13-75) fused with glutathione S transferase (GST)-green fluorescent protein (GFP) was microinjected into the nucleus of a polykaryotic of BHK21 cell, the GST-AhR(13-75)-GFP migrated from one nucleus to the other. This event, nucleocytoplasmic shuttling, was completely inhibited in the presence of leptomycin B (LMB). The interaction between chromosome region maintenance 1 (CRM1) and endogenous AhR was shown by immunoprecipitation with antibodies to AhR followed by immunoblot analysis with antibodies to CRM1. The inhibition of the nuclear export of AhR by LMB repressed the transcriptional activation of the CYP1A1 gene. The findings suggest that nuclear-cytoplasmic shuttling of AhR is essential for the inducible expression of the CYP1A1 protein. PMID- 10731724 TI - Inhibition of the production of rat cytokine-induced neutrophil chemoattractant (CINC)-1, a member of the interleukin-8 family, by adenovirus-mediated overexpression of IkappaBalpha. AB - Cytokine-induced neutrophil chemoattractant (CINC)-1, a counterpart of the human growth-regulated gene product (GRO) of the interleukin-8 family, has been suggested to play critical roles as a mediator of inflammatory reactions with neutrophil infiltration in rats. NF-kappaB has been speculated to be involved in the production of CINC-1, since the NF-kappaB-binding domain is important for the CINC-1 promoter activity in several of our reporter assays. In the present study, we examined the effects of an overexpression of IkappaBalpha, a specific natural inhibitor of NF-kappaB, on CINC-1 production. For this purpose, we constructed two recombinant adenoviruses, AxCAIkappaBalpha and AxCAmutantIkappaBalpha, which express respectively wild IkBa and a mutated nondegradable IkappaBalpha in which serine residues 32 and 36 are replaced by alanine residues. Transfecting wild and mutant IkBa by these adenovirus vectors inhibited NF-kappaB activation and CINC-1 production, which were both caused by IL-1beta stimulation in the normal rat kidney epithelial cell line NRK-52E. We also showed that the nondegradable mutant IkappaBalpha was approximately 30 times more potent than the wild type in inhibiting CINC-1 production. These findings demonstrate that CINC-1 production with NF-kappaB activation is primarily regulated by non-phosphorylated IkBa in the cytoplasm. PMID- 10731725 TI - Cloning of a rat glia maturation factor-gamma (rGMFG) cDNA and expression of its mRNA and protein in rat organs. AB - We isolated a rat glia maturation factor-gamma(rGMFG) cDNA and examined the tissue distribution of GMFG in rat by Northern and Western blot analyses. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein of 142 amino acid residues. The deduced amino acid sequence of the putative product is highly homologous (78.9%) to rat glia maturation factor-beta (rGMFB). Northern blot analysis indicated that a 0.9-kb mRNA is predominantly expressed in rat thymus, testis, and spleen. GMFG showed a different tissue distribution from GMFB, being present predominantly in proliferative and differentiative organs. PMID- 10731726 TI - Primary hypothyroidism presenting as a pituitary mass. AB - Pituitary enlargement secondary to primary hypothyroidism is a known but uncommon occurrence, which can be difficult to distinguish on computerized tomography (CT) and magnetic resonance imaging (MRI) from primary pituitary tumors. We describe a 33 year old female who was referred to a neurosurgeon for removal of a pituitary mass. The markedly elevated thyrotrophin stimulating hormone (TSH), absence of clinical features of hyperthyroidism, and low thyroid hormone values led to a diagnosis of pituitary enlargement secondary to primary hypothyroidism. The pituitary gland returned to normal size with thyroxine replacement therapy. Surgery was not indicated. PMID- 10731727 TI - Sonographic measurements of fetal parts to predict pulmonary maturity among twins and singletons. AB - To determine if sonographic examination of fetus can be readily utilized to predict a mature lecithin/sphingomyelin (L/S) ratio among twins and singletons. Twins (n = 36) undergoing amniocentesis for assessment of pulmonary maturity were matched with singleton (1:2) for maternal demographics, gestational age (GA), and indications for procedure. At the time of amniocentesis, twins and singletons with mature L/S ratios differed significantly in mean GA (33.2 +/- 2.7 vs 34.5 +/ 4.6 wks, p = 0.01), biparietal diameter (BPD), abdominal circumference (AC), femur length (FL) and estimate of birth weight (EFW). Based on ten receiver operating characteristics curves constructed, the following diagnostic thresholds predicted a mature L/S ratio with a true positive rate of 100% among twins and singletons, respectively: 1) BPD $84 and $92 mm; 2) head circumference $315 and $320 mm; 3) AC $295 and $350 mm; or 4) FL $64 and $72 mm; or 5) EFW $2400 and $3200 g. Using any one of these five criteria correctly identified pulmonary maturity among 59% of twins and 28% of singletons (p = 0.001). Sonographic measurement of fetal parts or EFW may be a noninvasive method to predict a mature L/S ration among twins as well as singletons. PMID- 10731728 TI - There's another doctor in the house--introducing Rep. Chester W. Masterson, M.D. PMID- 10731729 TI - Limitation--striking a fair balance. PMID- 10731730 TI - The work of the Criminal Cases Review Commission. PMID- 10731731 TI - Should the foetus have rights in law? PMID- 10731732 TI - Primary care groups. PMID- 10731733 TI - Mediation in the legal profession. PMID- 10731734 TI - Prospective multicenter studies on sevoflurane. Presentation. PMID- 10731735 TI - A prospective, randomized clinical comparison of sevoflurane and halothane in children. AB - BACKGROUND: The goal of the present multicenter investigation was to compare in a prospective and randomized study the induction, the maintenance and the recovery characteristics of halothane and sevoflurane when used in paediatric patients. METHODS: With the approval of the Ethical Committee and the parental written informed consent, 64 children aged 3-12 years, receiving general anaesthesia for urological, abdominal, and orthopaedic surgery, were studied. After oral flunitrazepam (0.05 mg kg-1), general anaesthesia was randomly induced by either sevoflurane (start: 1%, maximum: 7%, n = 32) or halothane (start: 0.5%, maximum: 4.5%, n = 32) and a 60% N2O in oxygen mixture until the loss of eyelash reflex (induction time). Then the trachea was intubated (if necessary, a muscle relaxant was administrated), and the concentrations of the anaesthetic vapours were adjusted in order to maintain cardiovascular stability until the end of surgery. The following times were recorded: time of extubation, time for having purposeful movements, time of eyes opening and readiness for discharge from the recovery area, as well as the occurrence of untoward events during either induction of, maintenance of, or recovery from anaesthesia. Before surgery and 24 hr after the procedure, blood was collected in order to measure serum creatinine and BUN. RESULTS: No differences in induction time, extubation time, side effects and postoperative renal function were observed between the two groups. Four patients in each group received muscle relaxants to perform intubation (p = NS). When compared to halothane group, children receiving sevoflurane had shorter times of showing purposeful movements (median: 9 min versus 15.5 min, p < 0.005), emergence from anaesthesia (median: 12 min versus 18 min, p < 0.05) and achieving readiness to be discharged (median: 18 min. versus 30 min, p < 0.005). Sevoflurane group also showed a more stable heart rate during the induction period than halothane one (p = 0.05). DISCUSSION: Sevoflurane is as effective as halothane in providing smooth and rapid induction of anaesthesia, while recovery is considerably faster and haemodynamic tolerance is better if compared to halothane; this suggests that sevoflurane could be an useful substitute for halothane in pediatric patients. PMID- 10731736 TI - Maintenance and recovery characteristics of sevoflurane anaesthesia in adult patients. A multicenter, randomized comparison with isoflurane. AB - BACKGROUND: The goal of the present multicenter, prospective, randomized clinical investigation was to compare the clinical efficacy and safety of sevoflurane and isoflurane during maintenance of and recovery from general anaesthesia in adult patients. METHODS: With the approval of the Ethical Committee and the patient informed consent, 143 ASA physical status I-II patients, aged 18-65 years, were randomized in order to receive either isoflurane (n = 71) or sevoflurane (n = 72) as the main general anaesthetic. After an oral diazepam (0.1-0.2 mg kg-1) and atropine (0.007-0.01 mg kg-1) premedication and a standardized intravenous induction, general anaesthesia was maintained by adjusting the end-tidal concentrations of the inhalational agent for the maintainance of cardiovascular stability. At the end of surgery the anaesthetic vapours were discontinued, and the neuromuscular block was reversed; the following times were recorded: time of eyes opening, time of command response and suitability for discharge from the recovery area. The occurrence of any untoward event was also recorded. Preoperatively and 24 hr after surgery, blood was collected in order to assess renal an hepatic functions. RESULTS: No differences in demography, duration of surgery, exposure to the inhalational agent and haemodynamic effects were observed between the two groups. Patients receiving sevoflurane showed shorter times for the achievement of extubation (median: 9 min versus 13 min, p = 0.002), eyes opening (median: 10 min versus 13 min, p = 0.002), command response (11 min versus 15 min, p = 0.002) and suitability for discharge from recovery room (median: 19 min versus 22 min, p < 0.05) than those receiving isofluorane. No intra- and intergroup differences were observed in pre- and post-operative laboratory testing of renal and hepatic function. DISCUSSION: We conclude that sevoflurane, when compared to isoflurane, provides a similarly safe maintenance but allows for a more rapid emergence from general anaesthesia. PMID- 10731737 TI - Maintenance of and recovery from anaesthesia in elderly patients. A clinical comparison between sevoflurane and isoflurane. AB - BACKGROUND: The goal of this multicenter, prospective, randomized clinical investigation was to compare the clinical efficacy and safety of sevoflurane and isoflurane during the maintenance of and the recovery from general anaesthesia in elderly patients. METHODS: With the approval of the Ethical Committee and the patient informed consent, 104 ASA physical status II-III patients, aged more than 65 years, were randomized in order to receive either isofluorane (n = 54) or sevoflurane (n = 50) as the main general anaesthetic. After an oral diazepam (0.1 0.2 mg kg-1) and intramuscular atropine (0.007-0.01 mg kg-1) premedication, anaesthesia was induced intravenously and then maintained by adjusting the end tidal concentrations of the inhalation agent for the maintainance of cardiovascular stability. At the moment of the last skin suture the inhalational agents were discontinued and the neuromuscular block was reversed. The following times were recorded: time of extubation, time of eyes opening, time of command response and readiness for discharge. The occurrence of untoward event throughout the study was also recorded. Before surgery and 24 hr after the procedure, blood was collected in order to assess renal function. RESULTS: No differences in demography, duration of surgery, exposure to the volatile anaesthetic, and renal function laboratory values were observed between the two groups. The time of extubation (median: 8 min versus 11 min, p < 0.01), emergence (median: 8.5 min versus 12.5 min, p < 0.01), command response (median: 10 min versus 15.5 min, p < 0.01), and suitability for discharge from the recovery area (median: 21 min versus 27.5 min, p < 0.01) were shorter in the sevoflurane group than in the isoflurane one. The success rate (absence of any event) during induction and maintenance periods was better in sevoflurane than isoflurane group (p < 0.02 and p < 0.001, respectively). Hypotension (systolic arterial blood pressure decrease > 30% of baseline values) was observed in 16 patients receiving isoflurane (29%) and only in 5 patients receiving sevoflurane (10%) (p < 0.02). DISCUSSION: When used in elderly patients undergoing operations of intermediate duration, sevoflurane provides a more rapid emergence from anaesthesia with a faster fulfillment of discharging criteria, and a more stable cardiovascular homeostasis than isoflurane. Renal function also appears to be equally well preserved with both anaesthetics. PMID- 10731738 TI - Prospective multicenter studies on sevoflurane. Overview. PMID- 10731739 TI - Nitric oxide and diabetes mellitus in humans. PMID- 10731740 TI - Thyroid nodules and Doppler ultrasonography. A new element for an old puzzle? AB - BACKGROUND: Whether Doppler ultrasonography (DU) may be of help in characterizing thyroid nodes is still under debate: both good and unsatisfactory results have been claimed, either in evaluating the presence of malignancy or in identifying hyperfunctional structures. METHODS: In this paper, the scintigraphic and Doppler patterns of 80 thyroid nodes have been compared, in the hypothesis that there may be some correlation between functional status and vascularity of the node; a small group of cyto-histological information were considered too. RESULTS: Perilesional flux seems to be, in general, somewhat more frequent than intranodal or mixed patterns: this is, however, true for all the groups of nodular functional conditions studied (peripheric flux was found in 70.2% of hyperfunctioning, 63.6% of isofunctioning and 59.3% of hypofunctioning nodes; intranodular flow was present in 18.9, 9 and 21.8% of hyper-, iso and hypofunctioning nodes respectively; mixed flow was found in 10.8% hyper, 27.2% iso and 18.7% hypofunctioning nodes). CONCLUSIONS: A prevalence of peripheric flow pattern is almost evenly distributed in all class of differently functioning nodes. Our data support the conclusion that no reliable correlations exist between scintigraphic and DU patterns: DU may not, therefore, be considered a valid substitute for nuclear imaging. PMID- 10731741 TI - Assessment of quality of life in recently post-menopausal women on dopaminergic therapy for pathological hyperprolactinaemia. AB - BACKGROUND: Psychological distress has been reported in pre-menopausal hyperprolactinaemic women. The aim of this study was to assess quality of life in a group of recently post-menopausal women with a long-term history of hyperprolactinaemia. METHODS: Thirty-one recently post-menopausal hyperprolactinaemic women (age range 46-59 years) and 37 control women matched for age and menopausal status. Hyperprolactinaemia had been diagnosed 2-22 years before the study. All hyperprolactinaemic women were on dopaminergic therapy. The self-rating Kellner Symptom Questionnaire (KSQ) and the Hamilton Depression Scale (HDS) were used to evaluate psychiatric profile. Evaluation of climacteric symptoms was performed with the ad-hoc self-rating 21-item Menopausal Rating Scale (MRS). Serum PRL, E2, LH, FSH, and free-thyroid hormones were evaluated. RESULTS: Hyperprolactinaemic women showed normal PRL on dopaminergic therapy. No difference was noted in PRL, LH, FSH, free-T4, and E2 levels between groups. Free T3 was significantly (p = 0.001) lower in hyperprolactinaemic than in control women. There was no difference in overall scores on the MRS between the groups. Only the item "rapid and strengthened heart-beat" was significantly (p = 0.04) lower in hyperprolactinaemic than in control women. Control women showed a significant correlation between the score for this item and free-thyroid hormone levels. Overall KSQ scores and subsection analysis of items did not show significant differences between groups. On HDS evaluation, depressive symptoms were similar in hyperprolactinaemic and control women. CONCLUSIONS: Quality of life seems unchanged in recently post-menopausal women with a long-term history of hyperprolactinaemia currently on dopaminergic therapy. The present study does not therefore support the differences in psychological profile reported in literature between untreated hyperprolactinaemic and control women unselected for age. PMID- 10731743 TI - [Sudden death by sleep apnea syndrome associated with myxedema. A case report and a review of the literature]. AB - The case is presented of a 48-year old, slightly overweight, brachymorphic male affected by undiagnosed myxedema, admitted for nocturnal dyspnea present for several years but worsened in the last few weeks. At the age of 19, a paranoid schizophrenia diagnosis was indicated leading to repeated admissions to psychiatric hospitals and continued pharmacological therapy. His sensorium was lucid albeit with a slight psycho-motor slowing down; pharyngeal edema and macroglossia were also apparent, blood O2 saturation was 97%. After the first emergency exams, a hypothyroid condition associated with multinodular goiter and tracheal dislocation was found. Administration of triiodothyronine p.o. and hydrocortisone i.v. was thus initiated. In the doubt of sleep apnea syndrome (SAS) occurrence, pulse oximetry was performed, but after 7 hours, the patient suddenly deceased. Data showed waves of deep O2 desaturation secondary to periods of prolonged apnea. A literature review shows that such a case has never been reported. A posteriori analysis of the patient's clinical management indicates that the obstructive form of SAS, associated with myxedema is a condition which needs to be promptly diagnosed; due to the possible seriousness of its functional evolution, the need for intensive or sub-intensive therapy, with continuous nasal airway positive pressure or with oro-tracheal intubation and assisted ventilation, should be carefully taken into consideration; continuous cardiac monitoring should also be carried out, given the risk for acute coronary complications and ventricular arrhythmias in the early phases of substitutive therapy with thyroid hormone. PMID- 10731742 TI - [Physiological significance and clinical utility of analytical methods for cardiac natriuretic peptides]. AB - It was well-established that the heart has an endocrine function because it is able to synthesize and secrete a family of related peptide hormones (known as cardiac peptide hormones) with potent diuretic, natriuretic and with complex interactions with the hormonal and nervous systems. Cardiac natriuretic peptides (ANP, BNP, and biologically active peptides of the N-terminal proANP1-98) are differently regulated in their production/secretion patterns and clearance rates; consequently, the assay for these peptides may provide complementary (or even different) pathophysiological and/or clinical information. The assay for cardiac natriuretic peptides has been utilized in clinical conditions associated with expanded fluid volume. In particular, this assay can be useful in discriminating between normal subjects and patients in different stages of heart failure and can also be considered as a prognostic indicator of long-term survival in patients with heart failure and/or after acute myocardial infarction. Non-competitive immunometric assays (such as two-site IRMAs), even if more expensive, seem to be preferable to RIAs for routinary assay of cardiac peptide hormones because they generally have a better degree of sensitivity, accuracy, and precision. The technical characteristics and the potential clinical usefulness of some of the methods for measuring these peptides are reviewed. PMID- 10731744 TI - Coverup. Philip Morris tries to polish its image. PMID- 10731745 TI - Rapid fire. PMID- 10731746 TI - It takes two. PMID- 10731747 TI - Seduced by smoke. PMID- 10731748 TI - Fighting smoke with fire. PMID- 10731749 TI - Logging on to 'liquid gold'. PMID- 10731750 TI - Where there's smoke, there's disease. The dangers of environmental tobacco smoke. PMID- 10731751 TI - Medicare leaves Minnesota seniors out in the cold "is it good medicine?". PMID- 10731752 TI - Health care taxes in Minnesota. An analysis. Minnesota Taxpayers Association. PMID- 10731753 TI - Trojan horse, Hobson's choice, pyrrhic victory and other thoughts. PMID- 10731754 TI - Lyme disease controversy. PMID- 10731755 TI - Waldeyer's ring lymphoma presenting as massive oropharyngeal hemorrhage. AB - A case report of a patient presenting with massive oropharyngeal hemorrhage originating from lymphoma of the tonsil and it's management is discussed. Locally advanced or recurrent squamous cell carcinoma may manifest with massive oropharyngeal hemorrhage, however, it is unusual for Waldeyer's ring lymphoma to present in this fashion. Management of oropharyngeal malignancies presenting in this manner includes airway control, control of hemorrhage, and biopsy of the tumor. Hemorrhage control is provided by surgical exploration or intraarterial embolization. Extranodal lymphoma of the head and neck is not uncommon and is thoroughly discussed in the medical literature. Hemorrhage into the upper aerodigestive tract is occasionally observed in the patient with head and neck cancer. It usually occurs, however, in patients with squamous cell carcinoma that have been previously treated or in patients with locally advanced cancers. A comprehensive review of the literature has revealed no other reports of head and neck lymphoma presenting with massive oropharyngeal hemorrhage. PMID- 10731756 TI - [Value of positron emission tomography with 18-fluorodeoxyglucose (FDG-PET) in diagnosis of recurrent bronchial carcinoma]. AB - The aim of the present investigation was to evaluate the diagnostic accuracy of positron emission tomography with 18-fluoro-2-deoxyglucose (FDG-PET) in the detection of recurrent lung cancer. PET was performed using an ECAT ART scanner (Siemens CTI) after i.v. injection of 220 +/- 50 MBq 18FDG. PET data were analysed by visual interpretation of coronal, sagittal and transversal slices. PET scans were interpreted independently by two experienced nuclear medicine physicians without prior knowledge of the results of other imaging studies or clinical data. 40 patients (= 41 cases) who had undergone primarily curative tumour treatment, were evaluated. In 29 of 35 cases with recurrent tumour, diagnosis was verified by pathologic means. FDG-PET correctly identified tumour recurrence in 34/35 cases. In 5/6 cases without prevent tumour recurrence PET gave true negative results. The overall accuracy of FDG-PET was 39/41 = 95% (95% confidence interval 83-99%). FDG-PET shows high diagnostic accuracy in detecting recurrent lung cancer in patients with prior curative tumour treatment, but cannot substitute the need for pathological diagnosis. PMID- 10731757 TI - [Adverse (cardio-)vascular effects of vinorelbine in non-small-cell bronchial carcinoma]. AB - Vinorelbin is an important tumouricidal substance. The (cardio-)vascular side effects are not well known. We report on four patients in highly palliative situations who were treated with vinorelbine for non-small cell lung cancer. Case one presented with myocardial infarction eleven days after onset of therapy. The second and third cases had to be admitted immediately after the beginning of vinorelbine treatment because of hypertension and angina pectoris. The fourth case suffered from angina abdominalis. A critical review of the literature showed 17 cardiac ischaemias with seven myocardial infarctions, three of them with lethal outcome. PMID- 10731758 TI - [Tumor simulating primary pulmonary cryptococcosis in an immunocompetent patient. A contribution to differential diagnosis]. AB - Case report of a primary cryptococcosis of the lung in a 78-year old non immunocompromised female. The patient presented with a mass in the right upper lobe, highly suspicious of lung cancer. Cryptococcus finally was detected on repeated biopsies from ulcerated and necrotic bronchial mucosa. A clinical work up showed no evidence of dissemination and no signs of immunoinsufficiency. Mass reduction in the lung was achieved under therapy with fluconazol. PMID- 10731759 TI - [Recommendations of the German Respiratory League on sports and physical training for patients with obstructive respiratory tract diseases]. PMID- 10731760 TI - [Fear of cortisone: in pulmonary rehabilitation it is less than expected. Results of a patient survey]. AB - BACKGROUND: The treatment of diseases of the upper and lower respiratory tracts can't be imagined without corticoids, applied systemically of locally. In specialist literature one can find indications of a certain reservation towards this kind of medication on the part of many patients. Sometimes, this reservation is so strong that it can be referred to as a regular fear of cortison. Bad compliance and with it, frequently, a deterioration of the symptoms, are inevitable consequences. The aim of the study was to find out how distinct the fear of cortison is with patients in pneumological rehabilitation and wether there are any relations with other spheres of attitude. METHOD: Patients with COPD and asthma with inhalative and/or systemic corticoid medication received an extensive questionnaire during their rehabilitation as in-patients for four weeks. 102 questionnaires were evaluated. RESULTS: The statements of the patients indicated high compliance regarding corticoid medication. As to the fear of such preparation, only 5/102 (4.9%) of those questioned produced results which suggest strong fear of cortison. Fear of cortison is, among other things, slightly related to general psychic-stress and a general negative attitude towards medicine. There is a negative correlation between compliance at the taking of medicine and fear of cortison. CONCLUSIONS: Contrary to statements in specialist literature of wide-spread reservations and fear towards cortison, we have found out that these play only a minor part, at least with the mostly elderly clientele of pneumological rehabilitation. Different explanations are discussed. PMID- 10731761 TI - [Patient behavioral management in COPD: concepts and efficacy]. PMID- 10731762 TI - [Non-allergic obstructive respiratory tract diseases in agriculture]. PMID- 10731763 TI - [Results of therapy in pulmonary tuberculosis: outcome monitoring in northern Lower Saxony]. AB - BACKGROUND: Despite it importance standardized treatment outcome-monitoring in tuberculosis patients is not officially done in Germany. METHODS: In this retrospective study we investigated tuberculosis outcome in 494 patients with pulmonary tuberculosis using the international recommended definitions. RESULTS: The median follow-up period was 62 month (36-180 month). A successful treatment could be observed in 378 (76.1%) of all patients. Treatment success was mostly documented as cure (n = 375). No documented treatment success was seen in 119 patients (23.9%). The most important reason for unsuccessful treatment was lost for follow up (transfer out) in 60/119 patients (51.2%), followed by death (24/119; 19.8%), an interrupted treatment (22/199; 18.3%) and treatment failure (13/119; 10.7%). No documented treatment success was significantly more common in retreatment cases compared to new cases (p = 0.0003) and in patients with at least a single drug resistance (p = 0.04). Beside these parameters treatment outcome was significantly superior in patients receiving a standard antituberculosis therapy including at least isoniazid and rifampin compared to patients treated with other regimens during both the initial phase (p = 0.0039) and the continuous phase (p = 0.0021) of therapy. CONCLUSION: In this retrospective study the use of the international definitions for outcome monitoring showed a substantial proportion of patients with unsuccessful therapy. For the evaluation of the success of the tuberculosis programme in Germany a prospective documentation of treatment outcome data in all patients using the international definitions seems essential. PMID- 10731764 TI - ["Biological weapons"--the return of epidemics?]. PMID- 10731765 TI - Guidelines for using human event-related potentials to study cognition: recording standards and publication criteria. AB - Event-related potentials (ERPs) recorded from the human scalp can provide important information about how the human brain normally processes information and about how this processing may go awry in neurological or psychiatric disorders. Scientists using or studying ERPs must strive to overcome the many technical problems that can occur in the recording and analysis of these potentials. The methods and the results of these ERP studies must be published in a way that allows other scientists to understand exactly what was done so that they can, if necessary, replicate the experiments. The data must then be analyzed and presented in a way that allows different studies to be compared readily. This paper presents guidelines for recording ERPs and criteria for publishing the results. PMID- 10731766 TI - An evaluation of methods for single-trial estimation of P3 latency. AB - This study investigated the validity of procedures for estimating the P3 complex in single trials. In "pseudo-real" simulations of the N1-P2 complex of the occipital visual-evoked potential, Mocks, Kohler, Gasser, and Pham (1988) had reported that their maximum-likelihood method (Pham, Mocks, Kohler, & Gasser, 1987) performed better than Woody's (1967) method. Using pseudo-real simulations of auditory oddball data, we wanted to know whether this finding also held true for the P3 complex. The performance of three methods was studied: peak picking, Woody's method, and Pham et al.'s method (as well as an extension of this latter method). Performance of all methods critically depended on the signal-to-noise ratio. There was some advantage for the more sophisticated methods, particularly when signal-to-noise ratios were realistic. "Good" trials may be selected by all methods, to improve the signal-to-noise ratio, but this selection entails the risk of bias. Further research should investigate whether these conclusions also hold true when the P3 complex consists of more than one component. PMID- 10731767 TI - Removing electroencephalographic artifacts by blind source separation. AB - Eye movements, eye blinks, cardiac signals, muscle noise, and line noise present serious problems for electroencephalographic (EEG) interpretation and analysis when rejecting contaminated EEG segments results in an unacceptable data loss. Many methods have been proposed to remove artifacts from EEG recordings, especially those arising from eye movements and blinks. Often regression in the time or frequency domain is performed on parallel EEG and electrooculographic (EOG) recordings to derive parameters characterizing the appearance and spread of EOG artifacts in the EEG channels. Because EEG and ocular activity mix bidirectionally, regressing out eye artifacts inevitably involves subtracting relevant EEG signals from each record as well. Regression methods become even more problematic when a good regressing channel is not available for each artifact source, as in the case of muscle artifacts. Use of principal component analysis (PCA) has been proposed to remove eye artifacts from multichannel EEG. However, PCA cannot completely separate eye artifacts from brain signals, especially when they have comparable amplitudes. Here, we propose a new and generally applicable method for removing a wide variety of artifacts from EEG records based on blind source separation by independent component analysis (ICA). Our results on EEG data collected from normal and autistic subjects show that ICA can effectively detect, separate, and remove contamination from a wide variety of artifactual sources in EEG records with results comparing favorably with those obtained using regression and PCA methods. ICA can also be used to analyze blink related brain activity. PMID- 10731768 TI - Age-related decline in inhibitory control contributes to the increased Stroop effect observed in older adults. AB - Past research has demonstrated an age-related increase in the Stroop effect. Some theorists have suggested that this increase results from a decline in the ability to inhibit word information on incongruent trials, whereas others have suggested that the decline reflects general slowing. These two hypotheses were evaluated using event-related brain potentials (ERPs) measured while younger and older adults performed the Stroop task. As expected, the Stroop effect was greater for older than younger adults. The ERP data revealed a selective age-related attenuation of two modulations reflecting the inhibition of word information on incongruent trials. Latency of the P3 wave did not increase to a greater extend for older than younger adults from the congruent to incongruent trials as expected based on the general slowing hypothesis. Taken together, these findings support the inhibitory deficit hypothesis by demonstrating an age-related decline in a conceptual level inhibitory process that supports the suppression of word information in the Stroop task. PMID- 10731769 TI - The visual N1 component as an index of a discrimination process. AB - Many previous studies have demonstrated that the visual N1 component is larger for attended-location stimuli than for unattended-location stimuli. This difference is observed typically only for tasks involving a discrimination of the attended-location stimuli, suggesting that the N1 wave reflects a discrimination process that is applied to the attended location. The present study tested this hypothesis by examining the N1 component elicited by attended stimuli under conditions that either required or did not require the subject to perform a discrimination. Specifically, the N1 elicited by foveal stimuli during choice reaction time (RT) tasks was compared with the N1 elicited by identical stimuli during simple-RT tasks. In three experiments, a larger posterior N1 was observed in choice-RT tasks than in simple-RT tasks, even when several potential confounds were eliminated (e.g., arousal and motor preparation). This N1 discrimination effect was observed even when no motor response was required and was present for both color- and form-based discriminations. Moreover, this discrimination effect was equally large for easy and difficult discriminations, arguing against a simple resource-based explanation of the present results. Instead, the results of this study are consistent with the hypothesis that the visual N1 component reflects the operation of a discrimination process within the focus of attention. PMID- 10731770 TI - Affective reactions to acoustic stimuli. AB - Emotional reactions to naturally occurring sounds (e.g., screams, erotica, bombs, etc.) were investigated in two studies. In Experiment 1, subjects rated the pleasure and arousal elicited when listening to each of 60 sounds, followed by an incidental free recall task. The shape of the two-dimensional affective space defined by the mean ratings for each sound was similar to that previously obtained for pictures, and, like memory for pictures, free recall was highest for emotionally arousing stimuli. In Experiment 2, autonomic and facial electromyographic (EMG) activity were recorded while a new group of subjects listened to the same set of sounds; the startle reflex was measured using visual probes. Listening to unpleasant sounds resulted in larger startle reflexes, more corrugator EMG activity, and larger heart rate deceleration compared with listening to pleasant sounds. Electrodermal reactions were larger for emotionally arousing than for neutral materials. Taken together, the data suggest that acoustic cues activate the appetitive and defensive motivational circuits underlying emotional expression in ways similar to pictures. PMID- 10731771 TI - Error processing and the rostral anterior cingulate: an event-related fMRI study. AB - The anterior cingulate is believed to play a crucial role in the regulation of thought and action. Recent evidence suggests that the anterior cingulate may play a role in the detection of inappropriate responses. We used event-related functional magnetic resonance imaging techniques to examine the neural responses to appropriate (correct rejects and correct hits) and inappropriate (errors of commission) behavioral responses during a go/no-go task. Analyses of the inappropriate responses revealed extensive activation in the rostral anterior cingulate cortex and in the left lateral frontal cortex. These areas were not activated for correctly classified trials (correct rejects and correct hits). These data suggest that the rostral anterior cingulate and left lateral frontal cortex are integral components of the brain's error checking system. PMID- 10731772 TI - Discrete and continuous prepulses have differential effects on startle prepulse inhibition and skin conductance orienting. AB - The effectiveness of different types of auditory prepulses in eliciting skin conductance orienting and in producing prepulse inhibition (PPI) of the acoustic startle eyeblink was studied in two experiments. A discrete white noise prepulse produced greater PPI than either a continuous white noise, a discrete tone, or a continuous tone. The discrete white noise advantage was not due to similarity in bandwidth to the startle pulse or to a refractory effect of the prepulse. Moreover, a dissociation between PPI and skin conductance orienting was seen in both experiments. PPI using auditory prepulses appears to be dependent primarily on the acoustic characteristics of the transient portion of the prepulse, whereas skin conductance orienting is more dependent on the sustained portions of the stimulus. PMID- 10731773 TI - Visuospatial attention and motor reaction in children: an electrophysiological study of the "Posner" paradigm. AB - To assess the processing stages involved in attention shifting and response selection tasks in children, we recorded event-related potentials (ERPs) and performance measures during a variant of the Posner paradigm. Subjects responded to visual targets, either preceded by a spatial cue (valid = same side; invalid = opposite side) or presented uncued. Valid targets evoked high-amplitude P1 responses, single-peaked P3s, and the shortest reaction times (RTs). Invalidity cued stimuli evoked delayed RTs, resulting in part from incorrectly oriented attention (decreased P1) leading to delayed target processing (belated N2-P3). Invalid targets also evoked a positive slow wave attributed to prolonged response selection due to cue/target incompatibility. Uncued stimuli elicited the longest RTs, unexplained by deficits in target detection or response selection, which likely resulted from a deficit in motor preparation due to the lack of warning signal. This method may be applied in clinical settings to disentangle selective processing deficits in target detection, response selection, or motor preparation stages. PMID- 10731774 TI - The relationship of respiratory sinus arrhythmia to the co-activation of autonomic and facial responses during the Rorschach test. AB - We studied the relationship of respiratory sinus arrhythmia (RSA) to the co activation of autonomic and facial expressive responses in 37 adult men during Rorschach testing. Interbeat interval (IBI), electrodermal activity, and facial electromyography were recorded continuously during the experiment. Prevailing mood and variability in the experience of valence and arousal were also measured. Bivariate intraindividual time series analysis was used to quantify spontaneously coupled phasic physiological responses. The results suggested that (a) spontaneous autonomic and expressive responses tend to parallel in time; (b) baseline, task-level, and task-induced change of RSA were positively related to coupled autonomic-expressive responses, a change in mood toward a more energetic state, and a tendency to have more variability in the emotional experience during the task; and (c) especially cardiac-expressive response coupling was related to mood change. It is concluded that the vagal system is related to the control of facial muscle activity and may mediate both tonic and phasic emotion-related changes in IBI. The role of phasic intraindividual response patterns in emotional and social behavior is highlighted. PMID- 10731775 TI - Early morning driver sleepiness: effectiveness of 200 mg caffeine. AB - Sleep-related vehicle accidents are prevalent early morning, especially in younger drivers. In two independent studies following a night of either restricted or nil sleep, young experienced drivers drove for 2 hr (0600-0800 h) continuously in an immobile car on an interactive, computer-generated, dull, and monotonous roadway. This exercise followed ingestion (at 0530 h) of 200 mg caffeine (= 2-3 cups coffee) versus placebo, counterbalanced, double blind. Driving incidents (lane drifting), subjective sleepiness, and 4-11 Hz electroencephalogram (EEG) activity were logged. In Study 1 (sleeping 0000-0500 h), caffeine significantly reduced incidents and subjective sleepiness throughout the 2-hr drive, and EEG power for the second 30-min period. In Study 2 (no sleep), sleepiness affected all measures profoundly, and driving was terminated after 1 hr. Nevertheless, caffeine reduced incidents significantly for the first 30 min and subjective sleepiness for the hour. This caffeine dose, feasibly taken via coffee, effectively reduces early morning driver sleepiness for about 30 min following nil sleep, and for around 2 hr after sleep restriction. PMID- 10731776 TI - Affective picture processing: the late positive potential is modulated by motivational relevance. AB - Recent studies have shown that the late positive component of the event-related potential (ERP) is enhanced for emotional pictures, presented in an oddball paradigm, evaluated as distant from an established affective context. In other research, with context-free, random presentation, affectively intense pictures (pleasant and unpleasant) prompted similar enhanced ERP late positivity (compared with the neutral picture response). In an effort to reconcile interpretations of the late positive potential (LPP), ERPs to randomly ordered pictures were assessed, but using the faster presentation rate, brief exposure (1.5 s), and distinct sequences of six pictures, as in studies using an oddball based on evaluative distance. Again, results showed larger LPPs to pleasant and unpleasant pictures, compared with neutral pictures. Furthermore, affective pictures of high arousal elicited larger LPPs than less affectively intense pictures. The data support the view that late positivity to affective pictures is modulated both by their intrinsic motivational significance and the evaluative context of picture presentation. PMID- 10731777 TI - Basic auditory dysfunction in dyslexia as demonstrated by brain activity measurements. AB - Although the generality of dyslexia and its devastating effects on the individual's life are widely acknowledged, its precursors and associated neural mechanisms are poorly understood. One of the two major competing views maintains that dyslexia is based primarily on a deficit in linguistic processing, whereas the other view suggests a more general processing deficit, one involving the perception of temporal information. Here we present evidence in favor of the latter view by showing that the neural discrimination of temporal information within complex tone patterns fails in dyslexic adults. This failure can be traced to early cortical mechanisms that process auditory information independently of attention. PMID- 10731778 TI - GE develops new nuclear medicine technology PMID- 10731779 TI - 131I therapy of toxic and non-toxic goiters. AB - For more than 50 years, 131I has been recognized as an effective tool for controlling thyroid hyperplasia and hyperactivity. The fixed dose administration is the simplest method with doses of 111-370 MBq (3 to 10 mCi) 131I being administered. More sophisticated methods aiming to deliver a well-defined amount of 131I per gram of thyroid tissue are handicapped by problems related to the evaluation of the goiter size and the prediction of the sensitivity of thyroid cells to radiation. The use of 131I in nontoxic multinodular goiter is to be reserved for specific situations. PMID- 10731780 TI - The role of nuclear medicine in the management of Graves' disease. AB - 131I therapy is safe and effective for most Graves' disease patients. It is being used more frequently in previously restricted populations such as children, adolescents, and women of childbearing age. It affords prompt, consistent relief of hyperthyroidism, but permanent hypothyroidism ensues in almost all patients. This complication is readily diagnosed with modern in vitro assays and inexpensive, life-long replacement therapy renders the patient asymptomatic and able to resume a normal lifestyle. Recent changes in NRC requirements have further liberalized the use of 131I for Graves' disease in the USA, permitting more patients to be treated with effective outpatient therapy. The controversial role of 131I in exacerbation of GO has been further clarified and preventive measures are available. PMID- 10731781 TI - Graves' ophthalmopathy and 131I therapy. AB - Graves' ophthalmopathy is an autoimmune process initiated and maintained by antigen(s) shared by the thyroid and the orbit. A matter of argument concerns the choice of the method of treatment for Graves' hyperthyroidism when clinically evident ophthalmopathy is present. Restoration of euthyroidism appears to be beneficial for ophthalmopathy. On the other hand the continuing disease activity associated with the recurrence of hyperthyroidism appears to adversely affect the course of ophthalmopathy. For these reasons it is our opinion that in patients with Graves' hyperthyroidism and ophthalmopathy the permanent control of thyroid hyperfunction by ablation of thyroid tissue should be obtained by radioiodine therapy or thyroidectomy. The rationale for an ablative strategy is the following: i) permanent control of hyperthyroidism avoids exacerbations of eye disease associated with recurrence of hyperthyroidism; ii) hypothyroidism, which follows thyroid tissue ablation, should be regarded as a therapeutic end point rather than as an undesirable result; iii) ablation of thyroid tissue may result in the removal of both the thyroid-orbit cross-reacting antigen(s) and the major source of thyroid-autoreactive lymphocytes. The relationship between radioiodine therapy and the course of GO is a matter of controversy, and some authors have suggested that radioiodine administration may be associated with a worsening of preexisting ophthalmopathy. This was not observed when radioiodine treatment was associated with a 3-month oral course of prednisone. The development or progression of GO after radioiodine therapy might be due to the release of thyroid antigens following radiation injury and to subsequent exacerbations of autoimmune reactions directed towards antigens shared by the thyroid and the orbit. The view that radioiodine therapy may be associated with a progression of ophthalmopathy is not shared by some authors who claim that the apparent link between progression of ophthalmopathy and radioiodine therapy might simply be coincidental, reflecting the natural history of the disease. The radioiodine associated exacerbation of eye disease might be used as an argument against the use of radioiodine therapy in patients with ophthalmopathy. We do not share this view, since the outward effects of radioiodine on eye disease can easily be prevented by concomitant administration of glucocorticoids. Glucocorticoid treatment should be limited, in our opinion, to patients with clinically evident eye disease and to those without ophthalmopathy but with other known risk factors, such as smoking. PMID- 10731782 TI - Quantitative radioiodine therapy in the treatment of differentiated thyroid cancer. AB - Radioiodine therapy is used in the treatment of patients with differentiated thyroid cancer both to ablate residual thyroid tissue after initial surgery and to treat residual, recurrent, or metastatic cancer. In most institutions, therapy remains based on empirically determined, fixed amounts of radioiodine that do not account for individual differences in the mass of tissue to be treated and in radioiodine kinetics. Over the last 25 years, we have developed and refined techniques based on pre-therapy, diagnostic quantitative radiation dosimetry and imaging with 131I that permit individualized treatment which balances the success of the treatment and the risk of serious acute adverse effects on the bone marrow and lungs. In this manuscript we discuss patient selection and preparation for radioiodine therapy and outline in detail methods for performing quantitative dosimetry studies. Guidelines for the application of these results to the treatment of individual patients are presented. PMID- 10731783 TI - 131I therapy of thyroid cancer patients. AB - Thyroid cancer is a rare malignancy with wide interethnic and geographic variations. In Germany thyroid carcinoma is the 13th most frequent malignancy (2.7 new cases yearly per 100,000 inhabitants). The overall temporal incidence is increasing slightly in recent years. The most common types of cancer are papillary (60-80%) and follicular cancers (10-20%). The relevant prognostic indicators are tumor stage and distant metastases. The mean survival rates in papillary thyroid cancer usually exceed 90%, whereas in follicular thyroid cancer they amount to approximately 80%. The standard treatment procedure in differentiated papillary and follicular thyroid cancer consists of total thyroidectomy followed by adjuvant ablative therapy with radioiodine. Only in papillary thyroid cancer stage pT1N0M0 lobectomy alone is considered to be appropriate. In patients with locally invasive differentiated thyroid cancers stage pT4 adjuvant percutaneous radiation therapy is a treatment option. Radioiodine therapy has to be performed under the stimulative influence of TSH. Usually TSH suppressive medication with Levothyroxine has to be withdrawn approximately 4 weeks prior to radioiodine therapy. In the future, exogenous stimulation by recombinant TSH may be used instead of thyroid hormone withdrawal. It has been proven by different studies that ablative radioiodine therapy reduces the frequency of recurrences and tumor spread in patients with thyroid cancer significantly. In patients with distant metastases, up to 50% of complete responses may be achieved with radioiodine treatment. PMID- 10731784 TI - Nuclear medicine therapy of neuroblastoma. AB - Specific targeting of radionuclides to neuroblastoma, a neural crest tumour occurring predominantly in young children and associated with a relatively poor prognosis, may be achieved via the metabolic route (MIBG), receptor binding (peptides) or immunological approach (antibodies). The clinical role of 131I-MIBG therapy and radioimmunotherapy in neuroblastoma is discussed. In recurrent or progressive metastatic disease after conventional treatment modalities have failed, 131I-MIBG therapy, with an overall objective response rate of 35%, is probably the best palliative treatment, as the invasiveness and toxicity of this therapy compare favourably with that of chemotherapy, immunotherapy and external beam radiotherapy. In patients presenting with inoperable stage III and IV neuroblastoma, 131I-MIBG therapy at diagnosis is at least as effective as combination chemotherapy but is associated with much less toxicity. In patients with recurrent disease 131I-MIBG therapy in combination with hyperbaric oxygen therapy proved feasible and encouraging effects on survival have been observed. Attempts to intensify the treatment in relapsed patients by combination of 131I MIBG therapy with high dose chemotherapy and/or total body irradiation have met with considerable toxicity. Developments in MIBG therapy aiming at improving the therapeutic index are mentioned. Early results of radioimmunotherapy using 131I UJ13A or 131I-3F8 monoclonal antibodies have shown moderate objective response and considerable side effects in patients with stage IV neuroblastoma, who had relapsed or failed conventional therapy. New developments in radioimmunotherapy of neuroblastoma include the use of chimaeric antibodies, the enhancement of tumour uptake by modulation of antigen expression or by increasing the tumour perfusion/vascularity/permeability, the use of other labels and multistep targeting techniques, e.g. using bispecific monoclonal antibodies. PMID- 10731785 TI - Nuclear medicine therapy of pheochromocytoma and paraganglioma. AB - Pheochromocytomas and paragangliomas are rare catecholamine-producing tumors which arise from chromaffin tissue. When a pheochromocytoma/paraganglioma is suspected, biochemical confirmation is based on 24-hour urinary excretion rates of catecholamines and their metabolites (metanephrines, VMA, etc.). Following biochemical confirmation non invasive imaging techniques such as CT and/or MR of the abdomen and 123I-MIBG scintigraphy are performed to localize the tumor. 111In octreotide may also be applied, mainly to localize head and neck chemodectomas. Malignant paragangliomas of either adrenal or extra-adrenal origin show a variable natural history: from a locally invasive indolent tumor to a highly aggressive malignancy. Surgery with complete resection or debulking of the primary tumor is the standard treatment. External radiotherapy and chemotherapy are usually scarcely effective. An alternative treatment is 131I-MIBG therapy which is performed with high specific activity 131I-MIBG. Usually a standardized dose ranging from 3.7 to 9.1 GBq of 131I-MIBG is administered by slow i.v. infusion. In advanced stage cases 131I-MIBG therapy aims at symptom palliation and tumor function reduction as well as at tumor arrest or tumor regression. In these cases MIBG therapy allows prolonged survival and good quality of life. In less advanced cases the purpose of MIBG therapy is to complement surgery and to achieve the total eradication of the tumor. Non functioning malignant paraganglioma can some time also concentrate MIBG and can be treated with high doses of the tracer. 131I-MIBG therapy is a safe treatment and is usually well tolerated by the patient (with rather low myelotoxicity). PMID- 10731786 TI - Therapy of neuroendocrine tumors with radiolabeled somatostatin-analogues. AB - Peptide receptor scintigraphy with the radioactive somatostatin-analogue [111In DTPA0]octreotide (DTPA = diethylenetriaminepentaacetic acid) is a sensitive and specific technique to show in vivo the presence and abundance of somatostatin receptors on various tumors. With this technique primary tumors and metastases of neuroendocrine cancers as well as of many other cancer types can be localised. A new application is the use of peptide receptor radionuclide therapy, administrating high doses of 111In- or 90Y-labeled octreotide-analogues. PRECLINICAL: We investigated the radiotherapeutic effect of 90Y- and 111In labeled [DOTA0,Tyr3]octreotide (DOTA = tetraazacyclododecanetetraacetic acid) or [111In-DTPA0]octreotide in Lewis rats bearing the somatostatin receptor-positive rat pancreatic tumor CA20948 in A) the flank or B) in the liver. PATIENTS: Thirty end-stage patients with mostly neuroendocrine progressing tumors were treated with [111In-DTPA0]octreotide, up to a maximal cumulative patient dose of about 74 GBq, in a phase 1 trial. PRECLINICAL RESULTS: A) Flank model: at least two 111MBq injections of [111In-DOTA0,Tyr3]octreotide were needed to reach tumor response, in 40% of the animals complete tumor remission was found after a follow-up period of 10 months. One or two injections of [90Y-DOTA0,Tyr3] octreotide yielded transient stable disease. B) Liver model: we found that peptide receptor radionuclide therapy is only effective if somatostatin receptors are present on the tumors, and is therefore receptor-mediated. High radioactive doses of 370 MBq [111In-DTPA0]octreotide or 93 MBq [90Y-DOTA0,Tyr3]octreotide can inhibit the growth of somatostatin receptor-positive metastases. CLINICAL RESULTS: There were no major clinical side effects after up to 2 years treatment, except that a transient decline in platelet counts and lymphocyte subsets can occur. Promising beneficial effects on clinical symptoms, hormone production and tumor proliferation were found. Of the 21 patients with progressive disease at baseline and who received a cumulative dose of more than 20 GBq [111In-DTPA0]octreotide, 8 patients showed stabilisation of disease and 6 other patients a reduction in size of tumors. There is a tendency towards better results in patients whose tumors have a higher accumulation of the radioligand. CONCLUSION: Radionuclide therapy with octreotide-derivatives is feasible, both with 111In and 90Y as radionuclides. PMID- 10731787 TI - Intracavitary therapy of craniopharyngiomas. AB - Craniopharyngiomas are benign cystic para-hypophyseal tumors often associated with hypopituitarism and visual-field abnormalities. Their therapy by surgery and external beam radiotherapy is imperfect. The intracavitary instillation of beta emitting colloid radiopharmaceuticals into the cysts permits the delivery of far higher radiation doses to the cyst lining than is possible by external beam radiotherapy. This technique permits destruction of the lining epithelium with resultant elimination of cyst fluid formation and cyst shrinkage in up to 80% of cases. PMID- 10731788 TI - Multiple endocrine neoplasia type 2A. Study of a family. AB - INTRODUCTION: Pheochromocytomas (Pheo) can occur sporadically, isolated or in association with other neuroendocrine lesions. In multiple endocrine neoplasia type 2A (MEN-2A), Pheo is associated to medullary thyroid carcinoma (MTC) or its precursor, C-cell hyperplasia (CCH) and parathyroid hyperplasia. Genetic screening provides early diagnosis and preventive treatment. In order to validate DNA analysis as a reliable method of early identification of gene carriers, we compared the results of genetic screening with clinical, biochemical, imaging and pathological findings in the members of an affected family. POPULATION AND METHODS: The diagnosis of a bilateral necrotic Pheo in a female patient led to the study of a family with four generations, aged 3 to 78 years (mean = 30.3 yrs). The study included a clinical examination; basal and pentagastrin stimulated calcitonin values; urinary catecholamines and their metabolites; serum calcium and a genetic study (direct sequence of PCR products from genomic DNA isolated from leucocytes using specific primers in exon 11 of the RET protooncogene of chromosome 10). The radiologic study, gammagraphic study (131I MIBG) and magnetic resonance study were performed in members with clinical suspicion of Pheo. RESULTS: Seven out of nine patients had a mutation on codon 634 of exon 11 of RET (TGC-CGC), leading to cysteine arginine substitution in the codified protein; all gene carriers had biochemical markers of MTC/CCH and four of Pheo. The Pheo patients underwent adrenalectomy (bilateral in three) and all the gene carriers underwent prophylactic thyroidectomy. The pathologic findings were: MTC in four (metastasized in one); CCH in three and parathyroid hyperplasia in one. CONCLUSIONS: Phenotypic penetration of RET mutation was 100% for MTC/CCH, but only 57% of the gene carriers had Pheo. Genetic screening allowed early prophylactic treatment in four out of seven patients; pathologic findings revealed several evolutionary stages of the disease. Patients not yet showing Pheo are under close clinical and laboratory surveillance. PMID- 10731789 TI - Multiple endocrine neoplasia: the paradigm of the arrival of molecular biology in clinical practice. PMID- 10731790 TI - Cardiac output quantification by Doppler echocardiography in intensive care- limitations and validation. AB - Cardiac output (CO) quantification is primordial to the evaluation of patients with heart failure who are on tailored therapy and under invasive hemodynamic monitoring. Doppler echocardiography can be used to access CO noninvasively, but the concordance between its results and those obtained by invasive methods in paired measurements is still controversial. To our knowledge, no previous studies have assessed the clinical relevance of Doppler echocardiography for CO serial evaluation in patients submitted to tailored therapy. AIM: To evaluate the usefulness of echo-Doppler in the assessment of CO and quantification of changes in CO, compared to thermodilution, in patients with advanced heart failure under hemodynamic monitoring to guide tailored therapy. METHODS: In 20 patients (14 male, 62 +/- 14 years old, all in sinus rhythm), with dilated cardiomyopathy and NYHA IV, admitted to the intensive care unit (ICU), CO was simultaneously determined by Doppler echocardiography (dpCO) and thermodilution (tdCO) in three serial evaluations (overall 60). The dpCO was calculated by multiplying the aortic orifice area by the velocity-time integral of aortic continuous wave Doppler flow and by the heart rate. A difference between tdCO and dpCO of more than 20% was considered a major error. RESULTS: In the overall evaluations, dpCO systematically overestimates tdCO (p = 0.026). The correlation between tdCO and dpCO was 0.81, the mean difference between measurements was 0.40 +/- 0.61 l/min (mean -2SD = -1.62 mean +2SD = 0.81) and 19 (32%) major errors occurred. No significant difference was found between CO percentual variation values assessed by both methods, with a stronger correlation (r = 0.92-p = 0.014) compared to that found for absolute values. On using the dpCO/tdCO ratio in the first evaluation to correct subsequent dpCO, the correlation was fairly good (r = 0.96 p = 0.0002 versus corrected dpCO). The mean difference between paired measurements was significantly lower (0.12 +/- 0.28 l/min-mean 2SD = -0.44 mean +2SD = 0.67), and there were no errors. CONCLUSIONS: CO estimated by Doppler echocardiography has a good correlation with thermodilution although with a weak concordance between paired results in patients with dilated cardiomyopathy and advanced heart failure admitted to the ICU for tailored therapy. Our results with dpCO percentual change in repeated evaluations and with corrected dpCO value after a single simultaneous invasive determination suggest that Doppler echocardiography is a valid method for clinical purposes, allowing us to propose a reduction in the time period of invasive hemodynamic monitoring. PMID- 10731791 TI - [The angioplasty of chronic coronary occlusions with the excimer laser for debulking followed by stent implantation]. AB - Coronary angioplasty (PCI) of chronic total occlusions (CTO), even when successful, are associated to less favourable long term results. The recent use of coronary stents has improved the long term outcome of those interventions. PURPOSE: To evaluate the short term results and long term occurrence of major adverse coronary events (MACE): death, MI, urgent revascularization and the need for a new target vessel revascularization (TVR) in patients with CTO who had previously been submitted to a PCI with excimer laser for plaque debulking followed by a provisional stent. POPULATION: From our database, we selected 19 patients with a mean age of 51 +/- 13 years (18 male) submitted to PCI between 1994 and 1998. Of those patients, 10 had had of a previous MI. Hypertension, smoking habits and hypercholesterolemia were present in 9 patients (42%). Two patients had diabetes. The main reason for PCI was stable angina in 16 patients (84%) and unstable in 3 patients (16%). Single-vessel disease was present in 18 patients (94%) and multiple-vessel in 1 patient (6%); left ventricular ejection fraction was preserved in 18 patients (94%). Single vessel PCI was performed in 16 patients (84%) and double vessel in 3 patients. Plaque debulking with excimer laser was performed in all patients, followed by 23 stents (Multilink--8; Gianturco Roubin--5; Palmaz Schatz--4; others--6). The mean clinical follow-up was 19 months. RESULTS: There were no major short-term clinical events (death, MI or urgent revascularization). During follow-up, TVR was only required in 5 patients (26%), all of them in the first 7 months after PCI. CONCLUSIONS: In the highly selected population, PCI for chronic total occlusion, with excimer laser plaque debulking followed by provisional stents, was a safe procedure with a very acceptable rate of new target vessel revascularization in the follow-up period. PMID- 10731792 TI - [Angioplasty in aortic coarctation and recoarctation in children and adolescents]. AB - STUDY OBJECTIVE: To determine the efficacy of percutaneous balloon angioplasty in children and adolescents with Coarctation and Recoarctation of the Aorta. DESIGN AND SETTING: We dilated native coarctation and recoarctation after a complete hemodynamic and angiographic assessment of the situation in a young population referred to a university hospital. PATIENTS AND INTERVENTION: In 22 patients with coarctation or recoarctation of the aorta, the systolic pressure gradient and the internal diameter of the stenotic area were measured before and after dilation, with a balloon catheter with an insufflation diameter no greater than the aortic diameter measured at the level of the diagram muscle. No infants were included nor patients with a rare aortic anatomy. RESULTS: A significant gradient reduction from 36.59 +/- 12.03 mm Hg to 10.45 +/- 7.52 mm Hg, as well an increase from 4.54 +/- 2.04 mm to 8.05 +/- 2.82 mm in aortic internal diameter, were achieved with no complications. CONCLUSIONS: Similar results were obtained both in native coarctation and recoarctation without significant complications. Age selection, aortic anatomy and balloon size were the major determinants of success. PMID- 10731793 TI - [The effects of endothelin-1 on myocardial function]. AB - INTRODUCTION: Endothelin-1 (ET-1) has potent vasoconstrictor, growth promoting and positive inotropic properties, with increased plasma levels in heart failure (HF). With regard to the cardiac effects of ET-1, most studies have not been able to differentiate its effects on the intrinsic properties of the myocardium from the secondary effects resulting from load changes and coronary vasoconstriction. This study investigated the myocardial effects of ET-1. METHODS: The study was performed on isolated rabbit papillary muscles (n = 9), before and after the addition of ET-1 (10 nM) to the superfusing solution (Krebs-Ringer; 1.25 nM Ca2+; 35 degrees C). One isotonic, one isometric and two afterloaded-isotonic twitches were recorded and analyzed. Only significant results (mean +/- SE, p < 0.05) are given, expressed as delta % baseline. RESULTS: ET-1 induced an increase of AT (147 +/- 33%), dT/dtmax (154 +/- 39%) and dT/dtmin (145 +/- 38%), while the duration of the twitch did not vary significantly. In addition, after ET-1, RT at the end of the isometric twitch decreased by 19 +/- 3%, when compared with the control and to its value at the beginning of the twitch. CONCLUSIONS: This study showed that, in addition to the well-known positive inotropic effect, ET-1 improves diastolic function by accelerating relaxation rate (dT/dtmin) and decreasing RT. These results may have important implications in the pathophysiology of HF. PMID- 10731794 TI - [Heart failure: symptomatic treatment versus prognostic modification]. AB - While the prognosis of patients with heart failure is related to the progression of left ventricular dysfunction and to the activation of several regulatory systems--neurohormones, cytokines and others--the symptoms relate to hemodynamic and skeletal muscle disturbances. This is why the relief of symptoms by pharmacological intervention is not always accompanied by an improvement in prognosis. Hemodynamic improvement is no longer the main issue of therapy. The antagonism of the neurohormonal and other regulatory systems, as a means to influence prognosis, stands as the main challenge for the future. Until now only ACE inhibitors, beta-blockers, spironolactone, the combination of nitrates and hydralazine, and heart transplantation have achieved this goal. PMID- 10731795 TI - [Secondary prevention after an acute myocardial infarct. Adrenergic beta blockers, angiotensin-converting enzyme inhibitors and statins for everyone?]. PMID- 10731796 TI - [The secondary prevention of coronary disease: ACE inhibitors, adrenergic beta blockers and statins for everyone? The arguments con]. PMID- 10731797 TI - Extra-adrenal pheochromocytoma (paraganglioma). AB - The authors describe the case of a 19-year-old female patient with an abdominal paraaortic extra-adrenal pheochromocytoma (paraganglioma), presenting arterial hypertension. The predominant catecholamine produced by the tumor was norepinephrine (4110 pg/ml; normal < 450 pg/ml). 131I metaiodobenzylguanidine (131I-MIBG), computed tomography and magnetic resonance imaging allowed location and characterization of the tumor. Histologically the tumor (weight = 34.2 g; 5.8 x 4 x 3 cm) was a typical pheochromocytoma. PMID- 10731798 TI - Holter-documented sudden death due to spontaneous rapid ventricular tachycardia followed by ventricular fibrillation. PMID- 10731799 TI - Progression of mitral regurgitation. A prospective Doppler echocardiographic study. PMID- 10731800 TI - [Sleep apnea and risk of traffic accidents]. PMID- 10731801 TI - [Animal cloning]. PMID- 10731802 TI - [General properties of human herpesviruses]. AB - The human herpesviruses share common structural and biological properties. After primary infection, they remain in a latent state during the life-time. Whereas infections are most often benign in immunocompetent individuals, severe infectious or tumoral complications may occur particularly in patients with cellular immunodeficiencies. Anti-viral drugs target viral replication, but have no effect on latency, which remains poorly understood. PMID- 10731803 TI - [Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2)]. AB - Herpes simplex virus (HSV) infections are prevalent worldwide. HSV are characterized by their ability to establish and maintain latent infections that can be reactivated. Various clinical presentations of HSV infections are described. Mostly asymptomatic, these infections may become life-threatening when occurring in neonates or when infecting the central nervous system. Accurate diagnosis of HSV infections is important and PCR is the most sensitive technique for detecting HSV. Type-specific serologies could be particularly useful for seroprevalence rates. Aciclovir is an efficient drug for the treatment of herpes simplex virus infections and resistance to this drug has been reported mainly in immunocompromised patients during the course of aciclovir treatment. There is a variety of potential vaccines for prophylaxis of HSV infection, but no vaccine is now available. PMID- 10731804 TI - [Varicella-zoster virus]. AB - Varicella-zoster virus, an ubiquitous human pathogen, causes vesicular rash during varicella, the primary infection of the host and zoster corresponding to reactivation. The symptoms could be various, nervous systems and lung being involved. Usually mild, varicella could be severe in immunocompromised patients, during pregnancy for the mother and the foetus, for the newborn and also for adults. Post herpetic neuralgia in old patient is the main complication of zoster. Various methods for virological diagnosis (culture, cytology, serology, PCR) with different sensibilities and specificities depending mainly of sample type are available. Various antiviral drugs are available, acyclovir being the reference one. PMID- 10731805 TI - [Epstein-Barr virus (EBV)]. AB - Epstein-Barr virus (EBV) is a B-lymphotropic human herpesvirus that infects the vast majority of the world's population; it establishes lifelong latency in the infected host. The virus enters through the oropharyngeal mucus and replicates in epithelial cells. The infiltrating B lymphocytes become infected and normal resting B cells activated by the virus result in immortalised B cell populations. This growth program utilises all of the latent genes and proteins. Following primary infection, the virus is found in saliva for weeks and chronic shedding participates in transmitting the infection from person to person through intimate kissing contact. EBV causes infectious mononucleosis, leads to EBV-induced malignant B-cell lymphoma in immunosuppressed patients, and is implicated in the pathogenesis of several cancers in the immunocompetent. Laboratory confirmation of infectious mononucleosis employing serological testing is generally required. Chronic infection and reactivation however need additional investigations such as nucleic acids testing. Quantitative polymerase chain reaction is now used as the major marker in B-cell proliferations either for assessing risk factors or for evaluating the efficacy of anti-EBV treatments. PMID- 10731806 TI - [Cytomegalovirus (CMV)]. AB - Human cytomegalovirus (CMV) is an ubiquitous agent. CMV infection is a common, usually subclinical world wide infection with a tendency for virulence in congenitally-infected infants and immunosuppressed patients including allograft recipients and patients with AIDS. Anti-CMV drugs are used for the treatment of severe infections in immunosuppressed patients. Developments of vaccine to protect pregnant women from primary infection are in progress. PMID- 10731807 TI - [Human herpesviruses 6 and 7 (HHV-6 and HHV-7)]. AB - Human herpesviruses 6 (HHV-6) and 7 (HHV-7) are world wide T lymphotropic viruses recently discovered. Their transmission is essentially by salivary route. Primary infections which occurred early in infancy, respectively during the first year of life for HHV-6 and in the second or third year for HHV-7, are followed by latency for life. HHV-7 is not actually associated with a disease. HHV-6 primary infection is often asymptomatic, if not it can induce exanthem subitum. HHV-6 reactivation can be symptomatic in immunodeficient subjects. The role of HHV-6 in the arising of lymphoproliferative or auto-immune diseases, discussed for a long time, is still to elucidate. HHV-6 infection is diagnosed by serodiagnosis in case of primary infection, but in the great number of cases, it would be realized by polymerases chain reaction. PMID- 10731808 TI - [Human herpesvirus 8 (HHV-8)]. AB - Human herpesvirus 8 (HHV-8) is associated with all epidemiological forms of Kaposi's sarcoma (KS), multicentric Castleman's disease and primary effusion lymphomas. HHV-8 is a gamma herpesvirus and his genome consists of an estimated 140 kb long unique coding region. Sero-epidemiologic studies support the findings that infection by HHV-8 in the occidental countries is not ubiquitous. The main routes of transmission for HHV-8 is sexual contact at least in homo-sexual men, however distribution of the prevalence of HHV-8 in Africa suggests that this virus could be transmitted by other ways such as mother to child or child to child. Analysis of the genome of HHV-8 identified an imprecedent numbers of genes implicated in cellular growth which have potentially a major role in tumorogenesis. Morphologic studies based on electron-microscopy, immunohistochemistry and in situ hybridization suggest that most of the cells in HHV-8-related tumours are latently infected. Efforts must concentrate on finding the best systems for growing the virus in cell cultures and in the strategy of treatment and (or) prevention of HHV-8-related tumours especially in organ transplant patients. PMID- 10731810 TI - [Care networks in the year 2000]. PMID- 10731809 TI - [Anti-herpes chemotherapy]. AB - With the increasing number of immunocompromised patients over the last two decades, disease pattern caused by Herpesviridae has changed. More virulent, more severe, herpesvirus diseases are more frequently treated and consequently the drug-resistant herpesvirus mutants have arisen in the clinic. All these events justify to explore future directions in drug development and herpesviral research as antisens strategy or immunotherapy. PMID- 10731811 TI - [Obsessional neurosis. Diagnosis, treatment]. PMID- 10731812 TI - [Skin topical corticosteroids. Principles and rules of their utilization]. PMID- 10731813 TI - [Primary adrenocortical insufficiency in adults. Etiology, diagnosis, treatment]. PMID- 10731814 TI - [Diplopia. Diagnostic orientation]. PMID- 10731815 TI - [Cerebral toxoplasmosis in an HIV-infected patient. Diagnosis, development, treatment and prevention]. PMID- 10731816 TI - [Measurement of public health status. Principle indicators of health, rate of incidence, prevalence, outbreak, morbidity and mortality. Risk factors, principle characteristics of morbidity and mortality in France]. PMID- 10731818 TI - [Urgent message]. PMID- 10731817 TI - [Shoulder injury. Diagnostic orientation and management of emergency situations]. PMID- 10731819 TI - [Dermatology, cosmetology and aesthetic medicine]. PMID- 10731821 TI - [In Process Citation] PMID- 10731820 TI - [Painters and color vision disorders]. PMID- 10731822 TI - [Management of first transient chest pain]. AB - Unstable angina is the first diagnosis to consider in a patient with transient chest pain; if managed in the setting of a coronary care unit, this condition leads to myocardial infarction in less than 5% of patients. Conversely half of acute myocardial infarctions have been heralded by unrecognised unstable anginal; thus a good proportion of the 120,000 infarctions occurring yearly in France could be avoided. Pre-hospital diagnosis is based on chest pain characterisation since physical examination and electrocardiogram are usually normal when the patient is pain free. The major concern being to avoid underdiagnosis, sensitivity is in this setting more important than specificity the patient should be hospitalised whenever a reasonable doubt exists. PMID- 10731823 TI - [Acute myocardial infarction: prehospital phase and first hospital days]. AB - During acute myocardial infarction, pre-hospital stage and the first hours after admission are crucial for initiating reperfusion therapy, which is determinant for short- and mid-term survival. Within 12 hours after onset of chest pain, either thrombolysis or primary angioplasty contribute to reduce mortality rate, the earlier being the better. Therapeutic advances, regarding either thrombolysis or angioplasty, are directed towards an earlier, more efficient and stable (without reocclusion) patency of the infarct-related artery. Most promising results so far have been obtained with platelet glycoprotein (GP) IIb/IIIa receptor blockers, in combination with thrombolysis or angioplasty. PMID- 10731825 TI - [Cardiac arrest outside the hospital]. AB - Out-of-hospital cardiac arrest remains a clinical problem with a survival rate of less than 5%. Prompt initiation of cardiopulmonary resuscitation and rapid delivery of advanced cardiac-life procedures are required. Combined in-hospital management by experienced cardiologists and intensive care specialists is recommended. Acute coronary-artery occlusion is frequent and poorly predicted by clinical and electrocardiographic findings. Accurate diagnosis by immediate coronary angiography can be followed if necessary by coronary angioplasty. Survivors undergo extensive work-up to determine the indications of an implantable defibrillator or coronary revascularization. PMID- 10731824 TI - [Management of severe acute pulmonary edema]. AB - Acute cardiogenic pulmonary oedema is a medical emergency. It generally result from an acute left ventricular insufficiency, itself resulting from systolic or diastolic dysfunction (alteration in relaxation or distensibility). Clinical presentation may be atypical, especially in the elderly. Treatment is based on oxygen, diuretics and nitrates. In severe cases, mechanical ventilation may be required. PMID- 10731826 TI - [Major pulmonary embolism]. AB - The diagnosis of major pulmonary embolism should be considered in case of acute respiratory distress, particularly when there is high thromboembolic risk. Although clinical symptoms are not specific, some are suggestive: syncope or dizziness with cyanosis and polypnoea, and especially arterial hypotension and cardiogenic shock. Diagnostic workup should be rapid and straight forward. Transthoracic echography is particularly useful to detect right heart thrombi and right ventricular overload. More information could be provided by helical computed tomography or perfusion lung scan or less commonly now by pulmonary angiography, depending on the patient's clinical condition and the available equipment. The mortality rate can reach 20 to 30%, and up to 65% after resuscitated cardiac arrest. Rapid desobstruction is justified through surgical embolectomy or intravenous thrombolysis favouring short duration protocols (alteplase over 2 h), in spite of the bleeding risk. PMID- 10731827 TI - [Cardiac tamponade]. AB - Cardiac tamponade is an emergency situation. Diagnosis is to be suspected when an elevation of systemic venous pressure, a decline in systemic arterial pressure and a clinical context of neoplasia or recent acute pericarditis are associated. Transthoracic echocardiogram is the gold-standard of diagnosis, and allows the accurate diagnosis of a large pericardial effusion: precise localisation and haemodynamic evaluation are needed before therapeutic decision. Pericardiocentesis is the only appropriate treatment. Surgical procedure, or less traumatic echo-guided pericardiocentesis, provide rapid haemodynamic relief of symptoms. Prognosis is determined by aetiology. PMID- 10731828 TI - [Cardiac syncopes]. AB - Cardiac causes explain 5 to 30% of the syncopes and their incidence increases with age. Prognosis is poor in case of misdiagnosis. Mechanical causes are related principally to obstruction of left ventricular outflow, and responsible for exertional syncope. Diagnosis is easy through clinical examination and echocardiography. Arrhythmic causes are due either to brady-cardia (diagnosed by surface electrocardiogram, 24-hour holter monitoring and electrophysiologic study in patients with bundle branch block) or to supraventricular or ventricular tachycardia. Arrhythmic syncope is easily prevented by antiarrhythmic drugs or non medical treatments such as radiofrequency ablation of tachycardia, pace-maker or defibrillator implantation. PMID- 10731829 TI - [Year 2000: The year of patient education?]. PMID- 10731830 TI - [Contact eczema. Etiology, physiopathology, diagnosis, treatment]. PMID- 10731831 TI - [Peripheral neuropathies. Etiology, diagnosis]. PMID- 10731832 TI - [Cancer of the hypopharynx and the larynx. Epidemiology, diagnosis, complications and long-term treatment]. PMID- 10731833 TI - [Osteoporosis. Epidemiology, etiology, diagnosis, prevention]. PMID- 10731834 TI - [Aseptic meningitis. Epidemiology, etiology, diagnosis, development, treatment]. PMID- 10731835 TI - [Polyarteritis nodosa (PAN). Diagnosis, development]. PMID- 10731836 TI - [Paranoid personality. Diagnosis]. PMID- 10731837 TI - The effect of choice on intention-behavior consistency. AB - Three studies were conducted with the aim of investigating whether choice of behavioral alternative increases intention-behavior consistency. In Study 1 undergraduates were asked to write and mail back an essay on a topic they either chose or were assigned. Demonstrating an effect of choice, significantly more subjects who chose an essay topic mailed back the essays. These subjects also rated their intention to write the essay as stronger, thus suggesting that the difference between choice and no choice is that the intention is self-generated. Supporting this interpretation, no differences in mail-back rates were observed in Studies 2 and 3 between a difficult and easy choice condition. Furthermore, an alternative hypothesis that choice induces implementation intentions did not receive support since independent effects were in Study 2 found of deciding when and where to write the essay and in Study 3 of making an outline of the essay. PMID- 10731838 TI - What's working in working memory: a role for the Central Executive. AB - Two experiments are reported which seek to explore the nature of the Central Executive of Baddeley's (Baddeley and Hitch, 1974) model of working memory. Experiment 1 uses a dual task methodology to compare performance on two storage tasks, making use of the two slave systems, the Phonological Loop (PL) and the Visuo-Spatial Scratch Pad (VSSP). Results suggest that storage can be thought of as based on the use of slave system independent resources. Experiment 2 uses a similar approach to compare performance on 2 processing tasks, again one PL based, the other VSSP based. These results suggest that processing is based on a central, shared set of resources. The two experiments support both a multiple resources model of working memory and more specifically identify a role for the Central Executive. PMID- 10731839 TI - Aggression and sociometric status among peers: do gender and type of aggression matter? AB - The connections between the use of different types of aggression (direct physical, direct verbal, and indirect) and sociometric status among same-sex and opposite-sex peers were studied. The subjects were 209 ninth-grade adolescents. Although an adolescent's aggression in general was related to being rejected by peers, a different picture emerged when the shared variance between types of aggression was controlled: The partial correlations showed that when the level of direct (physical and verbal) aggression was kept constant, increases in indirect aggression did not explain variance in peer rejection scores. On the contrary, the use of indirect aggression contributed (especially among boys) to social acceptance by peers. The direct (physical and verbal) forms of aggression were unrelated to adolescents' social acceptance scores. No clear differences were detected between girls' and boys' acceptance or rejection of their aggressive peers, despite the finding that boys seemed to tolerate indirect aggression better than girls did. PMID- 10731840 TI - A Swedish translation of the 20-item Toronto Alexithymia Scale: cross-validation of the factor structure. AB - The purpose of this study was to develop a new Swedish translation of the twenty item Toronto Alexithymia Scale (TAS-20) and to examine if the theoretical structure that underlies the factor structure of the English version of the TAS 20 could be recovered in this Swedish translation of the instrument. A sample of 157 undergraduate students of psychology was tested. Using confirmatory factor analysis, the previously established three-factor TAS-20 model was found to be replicable in this sample. In addition, the Swedish translation of the TAS-20 showed adequate internal reliability. The present study also illustrates the importance of using back translation methodology when transposing psychometric instruments from one language to another. PMID- 10731841 TI - Does P3 reflect attentional or memory performances, or cognition more generally? AB - The study evaluated the off-line relationship of attention, memory and other cognitive performances with the auditory event-related potentials P3 (P300) and N2. The sample comprised 200 middle-aged construction workers. Verbal, visuomotor and memory tests were administered. Attentional domains were examined using CogniSpeed software. Slowed reaction times in the test of sustained attention (vigilance) were associated with delayed P3 latency (p < 0.001) and decreased P3 amplitude (p = 0.005), as well as with delayed N2 latency (p < 0.001). Visuomotor slowing in Digit Symbol was also related to delayed P3 latency (p = 0.030) and decreased P3 amplitude (p = 0.014). In contrast, mild cognitive impairment, short and long-term memory, and concentrating or sharing attention with high working memory demands were not related to P3. The results suggest that P3 is linked to attentional performance with low working memory demands rather than to effortful working memory updating, retrieval from memory stores, or mild cognitive impairment. PMID- 10731842 TI - Icelandic norms for the Snodgrass and Vanderwart (1980) pictures: name and image agreement, familiarity, and age of acquisition. AB - This paper presents Icelandic norms for the widely used pictorial stimuli of Snodgrass and Vanderwart (1980). Norms are presented for name agreement, familiarity, imageability, rated and objective age-of-acquisition (AoA) of vocabulary, and word frequency. The ratings were collected from 103 adult participants while the objective AoA values were collected from 279 children, 2.5 11 years of age. The present norms are in many respects similar to those already collected for other language groups indicating that the stimuli will be useful for further psychological studies in Iceland. The rated AoA values show a high correlation with objective AoA (r = 0.718) thus confirming previous studies conducted with English speaking participants that rated AoA is a relatively valid measure of objective AoA. However, word frequency and familiarity are more closely correlated with rated AoA than with objective AoA indicating that these factors play some role in the ratings. Objective AoA norms are therefore to be preferred in studies of cognitive processes. PMID- 10731843 TI - Time-series analysis of the relationship between dizziness and stress. AB - Ten individuals with dizziness participated in a longitudinal study on the relationship between stress and dizziness. The participants rated dizziness and stress on visual analogue scales twice daily for a period of 28 days (in all 56 data points). The ratings of stress included physical stress, mental stress, emotional stress, and the presence of stressful events. The data was analysed by means of time-series analysis (ARIMA), and the temporal associations investigated by lagged correlations. Results showed concurrent associations between dizziness and mental and emotional stress. However, individual differences were observed indicating complex and diverse patterns of association between different forms of stress and dizziness. PMID- 10731844 TI - Defense mechanisms and morality: a link between isolation and moralization. AB - The relationship between morality and perceptual defense mechanisms was studied. Three new scales were constructed to measure different aspects of morality: moralism (the tendency to evaluate everything in terms of right and wrong), conscience (strength of feelings of right and wrong) and reparation (inclination to repair the damage one has caused). Perceptual defense mechanisms were measured with Kragh's Defense Mechanism Test (DMT). Three hypotheses about relationships between morality and defense mechanisms, derived from psychoanalytical literature, were tested on 54 male University students. Results show positive correlations between the defense mechanism isolation of affect and moralism, and between identification with the aggressor and reparation. Total amount of perceptual defense correlated positively with moralism. It is argued that the psychological study of morality should take unconscious processes into consideration. PMID- 10731845 TI - Cognitive distance of stairways: a multi-stairway investigation. AB - The present study had two major purposes. First it sought to determine to what extent in an earlier study of distance estimation in stairways (Hanyu & Itsukushima, 1995) would generalize to other types of stairway. Second, it sought to examine which hypothesis, information storage or effort, better explain the earlier results, in which people overestimated distance and traversed time estimates. We obtained four distance and time measures: distance estimate, traversal time estimate, mental walking time and actual traversal time. To measure information, we had participants rate each stairway for complexity (simple-complex) and effort (effortless-effortful) before and after the distance and time measurement tasks. The results revealed that the earlier findings (Hanyu & Itsukushima, 1995) did not fully generalize. The results also did not support either the information storage or the effort hypothesis. PMID- 10731846 TI - The role of movement and object in action memory: a comparative study between blind, blindfolded and sighted subjects. AB - Two experiments systematically compared four SPT conditions involving real/imaginary movement and real/imaginary object with one VT condition involving no enactment and no object. To test the effect of visual information on SPT memory, sighted subjects were compared with blindfolded subjects (in Experiment 1) and blind subjects (in Experiment 2). All subjects learned all SPTs and VTs. Free recall data showed no difference between the SPT conditions and between the groups of subjects; only blind subjects were found to be limited in the use of visualization strategy. All SPTs were recalled better than VTs, indicating that the enactment effect is not determined by either movement or object alone, rather both have an effective role and are equally involved for obtaining the enactment effect. The results provide no support for the motor encoding and multimodality views of SPTs, but are in line with the episodic integration view which assumes that neither movement nor object are of special importance, rather both have contribution in the enactment effect. PMID- 10731847 TI - Elder-caregiver care negotiations as dances of dependency. AB - Well-developed metaphors function as heuristic devices for the communication of scientific ideas. When applied to common, typical situations, metaphorical conceptualization enables a richer and deeper understanding. In this article, the routinized and taken-for-granted situation of elder-caregiver care negotiations is examined through the use of a dance metaphor, the dance of dependency. Metaphorical models of theory are intended to be elaborated and developed in ways that good poetic metaphors are not. Therefore, we systematically describe the general aspects of the dance (rules, facilitators, performances, aesthetics) and relate the specific elements of a dance to the dance of dependency. The picture that emerges is one of elder-caregiver care negotiations as performances, with elders as agents who actively and creatively engage caregivers in their dances of dependency. Participants in these dances sustain a complex organization of behavior to meet the situational demands and maintain themselves as fully integrated participants, based on standards set forth by the jointly negotiated working consensus of the dyad. The resulting description extends what is currently understood about everyday care negotiations, allowing an appreciation of the processes and dynamics by which care negotiations and elder-caregiver relational cultures in multiple contexts unfold, change, and are challenged. PMID- 10731848 TI - The DSM and child psychiatric nursing: a cautionary reflection. AB - The DSM is the key reference guide for clinicians and it continues to be the way in which practitioners apply discrete categories to behaviors that are sufficiently disruptive to social institutions to have come to the attention of tertiary care systems. As a way to classify and diagnose mental disorders, the DSM has been a source of controversy in the psychiatric community since its development. This article reviews some of the limitations of the DSM approach to psychiatric nosology with respect to practice with children. Alternative conceptualizations and approaches to childhood psychopathology are proposed and implications for child psychiatric nurses are discussed. PMID- 10731849 TI - Outcomes of perceived quality nursing care reported by oncology patients. AB - At a time when health care quality is being defined by providers, administrators, and third-party payers, it is more important than ever to ensure that patients' voices are heard. Patients can tell us not only what makes nursing care excellent; they can tell us how this care makes a difference. This article reports on the outcomes ascribed to quality nursing care by a group of purposively selected oncology patients who participated in a grounded theory study. One outcome was a sense of well-being comprising trust, optimism, and authenticity. A second outcome, increased fortitude, occurred when the patients felt that they had the readiness, strength, and stamina to undergo cancer treatment and bear its effects. The outcomes are described and discussed in relation to existing scholarly work. PMID- 10731850 TI - The effect on bonding behavior of giving a mother her premature baby's picture. AB - To investigate the effect on bonding behavior of giving mothers their premature baby's picture, 40 mother-infant dyads were randomly assigned to experimental (E) and control (C) groups. The E mothers were given a picture of their baby. The C mothers did not receive pictures. Both groups had visitation rights 24 hours a day. Two bonding observation measures were obtained--pretest and posttest. Results showed that E mothers exhibited significantly more bonding behaviors on posttest measured by (a) a bonding observation checklist, and (b) a physical examination observation checklist, than the C mothers. PMID- 10731851 TI - Impact of a research study a decade later: the use of pictures in a neonatal intensive care unit as a mode of nursing intervention to enhance maternal-infant bonding. AB - Approximately 12 years ago a preliminary study (Huckabay, 1987) was published on the use of giving mothers the picture of their premature babies as a mode of nursing intervention to enhance maternal-infant bonding. There is a mandate (Polit & Hungler, 1999) which stipulates that nurses need to be informed consumers of nursing research, be aware of the range of effective interventions that have been published, and use them in improving their nursing care. As reports of successful nursing interventions are published, a question is raised as to the impact of such studies on the actual care of patients or on furthering the knowledge base. To answer this question, an extensive computer search of the literature was done and neonatal intensive care unit (NICU) specialists of six major hospitals in southern California were interviewed to determine if they provide mothers the pictures of their premature babies as part of their nursing intervention to facilitate maternal-infant bonding. Findings based upon the NICU specialist interviews and the computer search of the literature are presented in the following order: (1) Current practices regarding use of pictures in six major hospitals, (2) Update of the incidence of premature births and the challenges they present, (3) Update of the importance of bonding theory and controversial issues surrounding its implementation, (4) A cross-cultural bonding study resulting from the use of the tool developed by Huckabay (1987) study, (5) Studies that have extended the use of pictures as a mode of nursing intervention in other situations, (6) Conclusion. PMID- 10731852 TI - [What is your diagnosis? Cutaneous larva migrans (creeping eruption)]. PMID- 10731853 TI - [Measuring blood pressure at the upper arm and wrist: are there differences?]. AB - Reliability and accuracy of a wrist and an upper arm sphygmomanometer were compared. Only in one of 27 persons were measurements at the wrist impossible. The 26 remaining volunteers showed good agreement between arm and wrist measurement (118/71 and 118/69 mmHg respectively). The variance was expectedly larger for systolic than diastolic values (10 vs 8 mmHg). As expected the correlation between the two methods was high (0.83 for systolic, 0.75 for diastolic values). Reproducibility of measurements was also equivalent for both methods. Devices for blood pressure measurements at the wrist are thus suitable. In order to identify individuals in which measurements at the wrist are impossible for technical reasons comparative measurements on the upper arm should precede the recommendation for a wrist device. PMID- 10731854 TI - ["I am dizzy"]. PMID- 10731855 TI - [SIADH with epileptic seizures and coma in fluoxetine therapy]. AB - Hyponatremia and the syndrome of inappropriate antidiuretic hormone secretion (SIADH) have been associated with several psychotropic drugs e.g., carbamazepine, neuroleptics, tricyclic antidepressants and more recently selective serotonin reuptake inhibiting (SSRI) antidepressants. SSRIs have gained widespread use in elderly depressed patients because of their favourable adverse effect profile. However, SSRIs have recently been associated increasingly with SIADH. We describe a 82-year old patient who was hospitalised after witnessed convulsions ten days after initiation of fluoxetine therapy for depression. She had hyponatremia and increased urine osmolarity suggesting SIADH. Using this case we discuss the clinical symptoms, aetiology, differential diagnosis and therapy of drug-induced SIADH. PMID- 10731856 TI - [Mastocytosis: an important differential diagnosis in anaphylactoid reaction to Hymenoptera sting. A case report and overview of clinical aspects, diagnosis and current therapy of mastocytosis]. AB - A 77-year old farmer presented with a history of three severe anaphylactoid reactions after hymenoptera stings for the last three years. No sensitisation to hymenoptera venoms could be shown on allergological work-up. The serum level of tryptase however was clearly elevated (37.1 micrograms/l; normal value: < 13.5 micrograms/l). The diagnosis of systemic mastocytosis could be confirmed by bone marrow biopsy. Patients with mastocytosis are at increased risk of anaphylactic/anaphylactoid reactions. They should be educated how to avoid trigger factors and they should always carry an emergency kit (H1 blocker, corticosteroid, adrenalin) and a "mastocytosis pass" with them. Venom immunotherapy is indicated in patients with proven hymenoptera allergy. Selected patients at very high risk of anaphylactic reactions may need a continuous prophylactic medication with H1 and H2 blocker and eventually a cyclooxygenase inhibitor. PMID- 10731857 TI - [25-year-old patient with headache and galactorrhea. Housewife born in 1974]. PMID- 10731858 TI - Pharmacotherapy for smoking cessation. PMID- 10731859 TI - Rapid resolution of antipsychotic-induced tardive dyskinesia with olanzapine. AB - Rapid improvement of tardive dyskinesia was identified following initiation of olanzapine in an elderly male patient formally treated with chlorpromazine. PMID- 10731860 TI - Child abuse reporting. PMID- 10731861 TI - Better childhood in the next millennium. PMID- 10731863 TI - Serum levels of antioxidant vitamins (alpha tocopherol, beta carotene, and ascorbic acid) in children with bronchial asthma. AB - We determined serum levels of alpha tocopherol, beta carotene, and ascorbic acid and lipid peroxidation products (thiobarbituric acid reactive substances--TBARS) in 14 children during an asthma attack and remission. Twelve healthy children served as controls. All antioxidant vitamins were significantly lower in asthmatics at remission compared to controls. Comparison of attack and remission periods in asthmatic patients failed to reveal any difference except in beta carotene (p = 0.03). The levels of all three vitamins correlated very significantly with each other (r = 0.89-0.95). TBARS levels were significantly higher at asthma attack compared to remission (p = 0.001). No correlation was observed between the antioxidant vitamins and lipid peroxidation products. This study shows that antioxidant vitamins are decreased in sera of asthmatic patients even during the asymptomatic periods of the disease, and that this decrease is not totally dependent on the increased oxidative stress as reflected by lipid peroxidation products. The role of antioxidant vitamins in prevention and/or treatment of asthma remains to be determined. PMID- 10731862 TI - Serum levels of eosinophilic cationic protein (ECP), myeloperoxidase (MPO), lipid peroxidation products, interleukin (IL)-5 and interferon (IFN)-gamma in children with bronchial asthma at acute asthma attack and remission. AB - We determined serum levels of myeloperoxidase (MPO), eosinophilic cationic protein (ECP), reactive oxygen species measured as thiobarbituric acid reactive substances (TBARS), interleukin (IL)-5, and interferon (IFN)-gamma in 14 asthmatic children during an asthma attack and remission. Twelve healthy children served as controls. In atopic asthmatics, asthma attack resulted insignificant elevations of ECP, MPO, and TBARS compared to remission. TBARS levels were also higher at remission compared to controls. However, there was a great deal of overlap in the values of asthmatics and controls. IL-5 and IFN-gamma were detectable at low levels and only in a few patients. These results provide further evidence for participation of eosinophils, neutrophils and reactive oxygen species in the pathogenesis of acute asthma, and suggest that their products may be used in monitoring asthma attack. Serum IL-5 and IFN-gamma levels are not appropriate for use in the follow-up of asthmatic children. PMID- 10731864 TI - Iron status in breast-fed full-term infants. AB - The aim of this study was to evaluate the iron status of full-term babies breast fed exclusively for four months and the importance of iron supplementation. One hundred sixteen term infants followed up since the newborn period by a well baby clinic were included in the study. Iron deficient and/or anemic infants were excluded from the study at four months. Some of the infants (51) were later given appropriate complementary food besides breast-feeding (Group A) and some (42) were given ferrous sulfate (1 mg/kg/d) (Group B). Blood count and serum iron and ferritin measurements were done at four and six months of age. At the 4th month, iron deficiency was found in 23 (19.8%) infants, 11 of which had iron deficiency anemia. At the 6th month, 23 (45%) infants in Group A were iron deficient and 11 (21.6%) of them had iron deficiency anemia. In Group B, three (7.1%) infants were iron deficient and one (2.4%) of them also had iron deficiency anemia (p < 0.0001). Significant iron deficiency and iron deficiency anemia have been found in four-month-old exclusively breast-fed full-term infants. It is observed that complementary food alone is insufficient; there is need for iron supplementation. PMID- 10731865 TI - Depression in children with hemophilic arthropathy and poliomyelitis: a preliminary report. AB - The aim of this study was to evaluate children with chronic disorders like hemophilia and poliomyelitis from the psychological perspective, to determine the frequency of depression, to identify the risk factors and to investigate the relation between disability and depression. Thirty-five patients with disability due to poliomyelitis and 12 patients with hemophilic arthropathy were included in the study. Thirty-six healthy children from the district schools served as controls. The Children's Depression Inventory (CDI) was used to assess the extent of depression. For the hemophilia group, joint scores proposed by the World Federation of Hemophilia were used to assess the degree of joint involvement. The poliomyelitis group was evaluated according to the level of ambulation and the need for orthoses. The CDI score was 10.57 +/- 5.87 in the poliomyelitis group, 11.00 +/- 5.64 in the hemophilic arthropathy group and 8.39 +/- 3.78 in the control group, but the difference was not statistically significant. Four of 35 patients with poliomyelitis (11.4%) and two of 12 hemophilic arthropathy patients (16%) exhibited depression. None of the children in the control group had depression. Since depression interferes with both medical compliance and rehabilitation potential, early diagnosis and treatment is important. Therefore, evaluation of the psychological status of chronically ill children must be a part of the rehabilitation program. PMID- 10731866 TI - Propranolol for primary and secondary prophylaxis of variceal bleeding in children with cirrhosis. AB - Variceal bleeding due to portal hypertension is a frequent and severe complication of cirrhosis in children as in adults. The prophylactic approach is important for these high mortality bleedings, both for the first and for recurrent attacks. Variceal bleeding/rebleeding rates were evaluated in sixty patients with cirrhosis who received 1-2 mg/kg/day propranolol p.o. for 1-14 years. According to Child-Pugh classification, 33 patients were Class A, 22 Class B, and five Class C. Patients were divided into two groups according to whether they had variceal bleeding before starting propranolol treatment (secondary prevention; 15 patients) or not (primary prevention; 45 patients). Seven (15.6%) of 45 patients experienced bleeding on propranolol therapy in the primary prevention group, while eight (53.3%) of 15 patients bled in the secondary prevention group (p < 0.01). Propranolol was found effective in primary and secondary prevention in Class A patients, while it was effective only for primary prevention in Class B and C patients. Propranolol administration is useful for preventing first and recurrent variceal bleeding in Class A cirrhotic patients. In Class B and C cirrhotic patients, it is effective only for preventing the first bleeding episode. PMID- 10731867 TI - Interferon-alpha treatment for chronic hepatitis C in children. AB - Interferon-alpha therapy has been proven efficient in chronic hepatitis C infection. Although it has been used as a standard therapy in adults, there are limited data on benefits of interferon treatment in children. We conducted a study of recombinant interferon-alpha therapy in 10 children with chronic hepatitis C. They had high aminotransferase values and positive antibodies to hepatitis C virus and HCV-RNA for at leas six months. Interferon-alpha was given at a dosage of 5 million units/m2 body surface three times a week for six months. At the end of therapy, five (50%) of the patients had complete response and two partial response. Three patients were nonresponders. Eight of the patients could be followed up for six months after stopping therapy, at which point one of the four complete responders and a partial responder relapsed. One of the three nonresponders had complete response at 12 months. Eventually, four (50%) of eight patients were complete responders. All of the nonresponders were the patients with previous malignant diseases. These findings suggest that interferon-alpha has beneficial effects in children with chronic hepatitis C, and a six month therapy seems to be reasonable. Patients with underlying malignant disease are not good candidates for interferon treatment. PMID- 10731868 TI - Bacterial nosocomial infections in mechanically ventilated children. AB - Of 480 patients admitted to the Pediatric Intensive Care Unit of the Institute of Child Health Children's Hospital in Istanbul, 97 required mechanical ventilation (MV). Sixty of these children were included in a retrospective analysis aiming to determine the frequency of and factors contributing to the development of nosocomial infections (NI). NI rate was 45 percent, ventilator-associated pneumonia (VAP) accounted for the greater part (66.7%) of the NI, followed by urinary tract infections (16.7%), septicemia (13.3%), and meningitis (3.3). Pseudomonas aeruginosa was the most frequent cause of VAP. The duration of the MV and invasive interventions were important risk factors for the development of VAP. PMID- 10731869 TI - Balloon dilatation angioplasty of stenosed systemic-pulmonary artery shunts. AB - Seven children and an adult patient with cyanotic congenital heart defects underwent balloon dilatation angioplasty (BDA) of a stenosed systemic-pulmonary artery shunt to improve arterial oxygen saturation. We attempted to perform BDA using the transvenous route in all patients in whom the aorta connected with the right ventricle, such as in tetralogy of Fallot or double outlet right ventricle, in an effort to avoid femoral artery injury. We could use the transvenous route (antegrade) in three children with tetralogy of Fallot and in one child with tetralogy of Fallot and pulmonary atresia (one of them was 6.6 kg). Following BDA, there was an increase in arterial oxygen saturation from a mean of 65.9 +/- 12.8% to a mean of 78.1 +/- 8.3% (p < 0.05). On follow-up three to 37 months (mean 16.5 +/- 11.2 months) after BDA, the condition of all patients had improved. Pulmonary hypertension developed in one patient during the follow-up period. It is concluded that BDA of stenosed systemic-pulmonary artery shunts is reasonable, effective and safe. Use of the transvenous route, if possible, to perform balloon dilatation angioplasty facilitates the safe advancement of the larger balloons in low-weight children. PMID- 10731870 TI - Regressed retinopathy of prematurity in children aged 5-8 years in Sivas, Turkey. AB - In this present study regressed retinopathy of prematurity has been investigated in children born prematurely (< 2300 g birth weight and < 34 weeks gestational age) in Sivas, Turkey during January 1989-January 1992. At the age of 5-8 years, 55 children born prematurely were examined; eye fundus information could be obtained by indirect ophthalmoscopy in all of them. The frequency of regressed retinopathy of prematurity was 35.45 percent for the whole group. Severe forms with optic atrophy, dragged optic disk, vitreoretinal scarring, retinal traction and temporal avascular retina were seen in 13.63 percent of cases. Moderate forms with pigmentary changes, vitreoretinal interphase changes and lattice degeneration were seen in 21.81 percent of cases. While the severe and moderate regressed premature retinopathy findings in premature children with gestational ages of 30-34 weeks were observed to be 12.0 and 14.0 percent, respectively, those in the 25-29 week-gestational-aged premature children were determined to be 5.0 and 28.33 percent, respectively. Although the incidence of both severe and moderate regressed premature retinopathy was higher in the 25-29 week gestational aged group when compared to that of the 30-34-week-gestational-aged group, the difference was not found to be statistically significant (p > 0.05). In conclusion, premature retinopathy should not only be followed up in the early stage with active changes but also later in infancy and childhood because of regressed premature retinopathy findings that may require treatment. PMID- 10731871 TI - Neurological soft signs and EEG findings in children and adolescents with Gilles de la Tourette syndrome. AB - Gilles de la Tourette syndrome (GTS) is a childhood-onset neuropsychiatric disorder, characterized by multiple motor and vocal tics. The presence of EEG abnormalities and neurological soft signs are reported in patients with GTS. In this study, conducted on 40 children and adolescents, non-specific EEG abnormalities and neurological soft signs were detected in 12 and 57.5 percent of cases, respectively. These findings are analyzed in comparison with other neuropsychological test results. A statistically significant association between EEG abnormalities, neurological soft signs and low-performance IQ results was detected. PMID- 10731872 TI - Solitary rectal ulcer syndrome in children. AB - The solitary rectal ulcer syndrome (SRUS) is an unusual disorder in childhood. Although well recognized in adult literature, the pediatric experience with this condition is limited, so SRUS often goes unrecognized or misdiagnosed. There are very few pediatric case reports in the English literature. This report describes four patients who presented with rectal bleeding, constipation, mucous discharge, and lower abdominal pain, with a diagnosis of SRUS. The diagnosis was made by rectoscopy, defecogram, anorectal manometry and histopathological evaluation. In two patients, defecogram showed a rectocele with both, the sphincter failed to relax to voluntary squeeze pressure on anorectal manometric examination. The histopathological finding in all patients was fibrous obliteration of the lamina propria with disorientation of muscle fibers. All of the patients responded well to conservative therapy, which included defecation training, laxatives, sulfasalazine, and application of rectal sucralfate enema, and remained asymptomatic on the follow-up. Although rare in the pediatric population, SRUS should be relatively easy to recognize in the child with rectal bleeding, after elimination of other causes. If suspected, the diagnosis of SRUS may be made at endoscopy and confirmed by rectal biopsy. PMID- 10731873 TI - Cyclic neutropenia complicated by renal AA amyloidosis. AB - Cyclic neutropenia is a rare disease characterized by regular cyclic fluctuations in the numbers of neutrophils. Patients with the disease suffer from recurrent infections at regular intervals of nearly three weeks. Recently, recombinant human granulocyte colony-stimulating factor (rhG-CSF) was reported to be an effective treatment for this disease. here we describe 17-year-old cyclic neutropenic female patient with a very rare association of renal amyloidosis of AA type who was under intermittent rhG-CSF treatment for the previous one and a half years. We conclude that although the disorder is usually benign, reactive amyloidosis may rarely develop in cases who remain untreated for a long period of time. However familial Mediterranean fever (FMF) type II should also be born in mind, particularly in predisposed populations. PMID- 10731874 TI - Severe lymphopenia in tuberculosis. A mere coincidence or a significant association? AB - A variety of infectious agents can cause secondary immunodeficient states. We herein present a one-year-old patient, admitted to the hospital with severe lymphopenia, who was subsequently diagnosed as tuberculosis. After the antituberculosis (anti TB) therapy was started, the clinical condition and the immunologic findings of the patient improved. We have thus concluded that the transient lymphopenia of the patient was due to Mycobacterium tuberculosis. We suggest that immunodeficiency should be investigated more often in children with tuberculosis and that further studies will shed light on the pathogenesis of this aspect of the disease. PMID- 10731875 TI - Allergic bronchopulmonary aspergillosis in two patients with cystic fibrosis. AB - Allergic bronchopulmonary aspergillosis (ABPA) is hypersensitivity to Aspergillus fumigatus which manifests as episodic wheezing, usually refractory to bronchodilator therapy, with fixed and transient pulmonary infiltrates, central bronchiectasis, blood eosinophilia, elevated serum IgE level, immediate skin reactivity to an A. fumigatus antigen and precipitating antibodies to A. fumigatus. It is an unusual complication of asthma and cystic fibrosis (CF). We present two cystic fibrosis patients with ABPA treated successfully with prednisone and, in Case 1 also with itraconazole. The physician should be alert to the possibility of ABPA whenever CF patients present with the new infiltrates, high serum total IgE and other positive parameters of A. fumigatus sensitization. Treatment with systemic steroids should be started in order to prevent irreversible lung damage. PMID- 10731876 TI - Malignant fibrous histiocytoma in a child. A case report and review of the literature. AB - Malignant fibrous histiocytoma (MFH) is the most common soft tissue sarcoma of late adult file. It is extremely rare in children, though, and its existence in the pediatric population remains controversial. Although the most common site of tumor in children is the extremities, which is similar to findings of adults' series, different sites have been reported in children. Because of the rarity of this tumor in childhood, the approach to treatment of MFH is based primarily on the experience with adults. We present the clinical and pathologic features of an eight-year-old boy with MFH located on his left retroperitoneum and also review the literature. PMID- 10731877 TI - Partial splenic embolization in beta-thalassemia major. A case report. AB - Partial splenic arterial embolization was performed in a thalassemic child for hypersplenism manifested by splenomegaly, leukopenia, thrombocytopenia, and anemia requiring frequent erythrocyte transfusion. During a follow-up period of 11 months, his hematological parameters improved significantly and the spleen size decreased. Partial splenic embolization could be an alternative therapy to surgical splenectomy for thalassemic children with hypersplenism. PMID- 10731879 TI - Inguinoscrotal hematocele of the newborn. AB - Neonatal inguinoscrotal hematocele is a very rare disease of the first few days of life. The cause of this pathology is thought to be related with the umbilical plastic clamp, with an incorrect clamping technique or with the infant's lying over the clamp. Surgical treatment is not necessary as long as testicular torsion is excluded. In this report, three cases of inguinoscrotal hematocele diagnosed at surgical exploration in our clinic are reported and the literature reviewed. PMID- 10731878 TI - Postoperative graft thrombosis in Fontan procedure. AB - Fontan repair could be used as the definitive palliation in many forms of complex cyanotic congenital heart disease. But thrombosis can occur after a modified Fontan operation (right atrium-right ventricular connection with a conduit). Appropriate management of this complication includes thrombolytic therapy, thrombectomy and revision (if surgically remediable causes of the thrombosis are identified) or redo operation. Two repair operations were performed for the treatment of thrombosis of the right side of the heart in patients in whom we had previously performed Bjork modification (right atrium-right ventricular connection with a conduit). The thromboses occurred 6 and 9 years after the operation, respectively. In both cases, the redo Fontan operation was successfully performed using a dacron tube graft. Patients were anticoagulated after the operation. Risk of thrombosis of the right side of the heart after the Fontan repair may be minimized with the use of prophylactic anticoagulation in high-risk patients soon after the operation. PMID- 10731880 TI - A rare coexistence of two gastric outlet obstructive lesions: infantile hypertrophic pyloric stenosis and organoaxial gastric volvulus. AB - Infantile pyloric stenosis is one of the most common conditions requiring surgery during the first few weeks of life. The association of infantile pyloric stenosis with gastric volvulus in an extremely uncommon occurrence. A 10-month-old male infant operated for infantile pyloric stenosis at two months of age is presented. His current problem was recurrent pulmonary infections and he was diagnosed to have organoaxial gastric volvulus and gastroesophageal reflux. The common features of presentation, radiological findings, surgical procedures and possible mechanisms of gastric volvulus associated with infantile pyloric stenosis are discussed. PMID- 10731881 TI - [Ambulatory management of thrombophlebitis]. PMID- 10731882 TI - [Therapy of venous thromboembolism with low-molecular-weight heparins]. AB - Low-molecular-weight heparins, nowadays already widely used for the prevention of thromboembolism, have now also become available for the treatment of deep-vein thrombosis. This article should serve to explain the rationale for this development and to demonstrate the clinically relevant advantages of the use of low-molecular-weight heparins. After briefly describing the characteristic properties of heparins the most relevant studies comparing the use of low molecular-weight heparin versus unfractionated heparin for the treatment of thromboembolism are discussed. In conclusion, clinical trials suggest that low molecular-weight heparins given subcutaneously can replace the hitherto standard intravenous application of unfractionated heparin in the initial treatment of deep-vein thrombosis, granting equal or even better efficacy and potentially lower rates of adverse side effects. Furthermore, the simplicity of this therapeutic regime allows for treatment of patients at home, thus offering patients mobility and also reducing the cost of treatment. PMID- 10731883 TI - [The concept of ambulatory therapy of proximal deep venous thrombosis]. AB - In recent studies outpatient treatment of proximal deep venous thrombosis with low-weight-molecular heparin is shown as safe and effective as the treatment within the hospital. The incidence of recurrent thromboembolism, major bleeding and mortality is not higher in an ambulatory setting than in the hospital. The comfort of the patient is higher, the costs can be reduced. The present review discusses the different conditions for ambulatory treatment of deep venous thrombosis. The question will be addressed which patients will probably be considered for outpatient treatment of deep vein thrombosis. PMID- 10731884 TI - Vascular surgery between the millenniums. AB - Modern diagnostic modalities as well as ongoing improvement of vascular prosthetic material and surgical techniques have stimulated progress in vascular surgery. New discoveries concerning the mechanism of endothelial function, atherosclerosis, developments in gene therapy and endovascular techniques will expand the future therapeutic spectrum of vascular surgery. Endoluminal implantation of stent grafts for the treatment of aortic aneurysm may be a reasonable alternative to conventional surgery, especially in high-risk patients. Long-term results of this procedure, however, are not yet available. Stenting of internal carotid artery stenosis may be considered as an experimental method of treatment. Its feasibility, efficacy, safety and long-term results must be analyzed before the application of the method may be restricted or recommended. Endoluminal irradiation (brachytherapy) reduces intimal hyperplasia/restenosis and can improve the long-term results of percutaneous transluminal angioplasty. Anti-atherosclerotic and anti-aggregatory therapy (with statins, estrogens, antibiotics, nitric oxide precursor/donors, glycoprotein IIb/IIIa receptor inhibitors) will play an important role in the prevention of ischemic diseases and improve the results of surgical/interventional treatment by reducing intimal hyperplasia and restenosis. Gene therapy opens new vistas in vascular medicine. Angiogenetic factors can be used for the treatment of patients with distal occlusion of the peripheral arteries. Gene transfer may be useful in the conservative treatment of progressive aortic aneurysms. A more unified vision toward vascular medicine might be the key for research and development in the future. PMID- 10731885 TI - [Angiology after-care after infrainguinal arterial reconstruction]. AB - This review article describes the natural course of infrainguinal bypasses, duplex ultrasound flow characteristics of stenosed vein bypasses, and the clinical relevance of duplex surveillance of infrainguinal vein bypasses. Among multiple factors bypass patency rates and limb salvage rates are influenced by the surgical technics of revascularization, graft material, localisation of the distal anastomosis, distal run-off, time interval between detection of bypass stenosis and revision. Preliminary duplex ultrasound criteria of a bypass dysfunction are a distal flow velocity < 45 cm/s, a bypass flow < 25 ml/min, a reduction of the inner diameter < 3 mm. The intrastenotic mean systolic peak velocity and the peak velocity ratio (PVR) are suitable for the grading of bypass stenoses. A vein bypass stenosis > 70% shows an intrastenotic mean peak velocity of 250 cm/s and a PVR > 3.3, a severe stenosis shows a mean peak velocity of 360 cm/s and a PVR > 7.2. PMID- 10731886 TI - Effect of balloon angioplasty and stenting following clips vs suture arterial stenosis: an experimental study. AB - BACKGROUND: Comparative evaluation of balloon angioplasty following intravascular stenting after experimental stenoses caused by arterial reconstruction with vascular clips and conventional sutures. MATERIALS AND METHODS: A total of 24 arteriotomies were carried out at the carotid and common iliac arteries of pigs following a 10 mm longitudinal arteriotomy and provocation of stenosis. Twelve of the arteries were reconstructed with vascular clips and 12 with conventional suture. Ultrasonography revealed stenosis fluctuating from 60-95% (PSV: 1.8-3.5 m/sec EDV: 1.3-1.47 m/sec PSV ratio > 3.5). After 8 weeks, following digital subtraction angiography, which revealed > 50% stenosis in all of the cases, balloon angioplasty followed by placement of intravascular stent was carried out. RESULTS: All the angioplasties remained angiographically and macroscopically patent two months after without thrombus formation. Rupture during dilatation occurred in one of the sutured cases. Histologically no degenerative changes, necrosis or remarkable intimal thickness were observed in either method. Focal inflammatory reaction was seen in 2 sutured and in 1 clipped cases while intimal ulceration was observed in 2 sutured cases. All cases with clips presented an intact endothelial surface. CONCLUSION: Early experimental results suggested that arterial stenosis provoked by clipped reconstruction could be managed successfully by balloon angioplasty followed by placement of intravascular stent. PMID- 10731887 TI - The histomorphologic changes at the saphenofemoral junction in varicosis of the greater saphenous vein. AB - BACKGROUND: Insufficiency of the greater saphenous vein starts at its junction with the femoral vein, so that the histomorphologic changes at the saphenofemoral junction are of special interest. METHOD: In this study we examined which histologic changes occur in the vein walls 5 cm distal to the junction of the greater saphenous vein into the femoral vein and whether these changes correlate to the clinical severity of venous disease. For this the morphologic changes of 28 venous specimens were described qualitatively and the two layers of the media were measured at their thickest and thinnest points. RESULTS: The three-layered structure of the vessel wall with intima, media and adventitia was destroyed in 21% of the cases, the intima and media were fused into one another due to loss of the membrana elastica interna. In 93% the intima was thickened at circumscribed areas. Sprouting of capillaries into the intima was found only if varicosis lasted for longer than 15 years. Aneurysms were found in 29% of the specimen. In part of severe aneurysms the media was completely missing. The thickness of the circular muscle layer of the media varied more than that of the longitudinal muscle layer (difference between minimal and maximal thickness 400 +/- 130 microM compared to 190 +/- 90 microM). With increasing clinical severity of the chronic venous insufficiency the difference between minimal and maximal thickness of the circular muscle layer increased (330 +/- 90 microM at stage I according to Widmer, 490 +/- 130 at stage III). CONCLUSION: The coexistence of muscular hypertrophy and atrophy in varicose veins is regarded by us as a reaction to altered perfusion and pressure in the veins. Considering the frequently severe damage to the veins, indication for a vein sparing therapy has to be viewed with care. PMID- 10731888 TI - Rapid intermittent compression increases skin circulation in chronically ischemic legs with infra-popliteal arterial obstruction. AB - BACKGROUND: Intermittent pneumatic compression (IPC) has been shown, by duplex, to increase popliteal artery flow in normal legs and in legs with superficial femoral artery occlusion. The objective of this study was to see if IPC improves distal circulation in legs with severe infra-popliteal disease. PATIENTS AND METHODS: Sixteen chronically ischemic legs with arteriographically demonstrated crural or pedal disease were studied during compression with an ArtAssist compression-device. This device delivers rapid compression of the foot and calf. Cutaneous laser-Doppler flux was measured continuously at the dorsal aspect of the distal forefoot. The findings were compared to those in thirteen normal controls of similar age. RESULTS: In ischemic legs, the spontaneous changes in skin-flux are minimal: mean resting flux in sitting position was 0.87 +/- 0.46 AU (Arbitrary Units). Upon activation of the compression device the maximum flux increased to 4.55 +/- 1.35 AU. The difference was statistically significant (p < 0.001). This response was similar to that in normal controls. CONCLUSION: Arterial flow augmentation upon compression is associated with increased skin flux. This response remains present in severe disease of the crural outflow arteries. Further investigation to define the role of intermittent compression for management of chronic arterial disease is warranted. PMID- 10731889 TI - Effects of vacuum compression therapy on skin microcirculation in patients suffering from lower limb ischaemia. AB - BACKGROUND: We investigated the short-term effect of vacuum compression (VC) treatment on skin microcirculatory perfusion in the foot of patients with lower limb ischaemia and healthy controls. PATIENTS AND METHODS: Ten patients with intermittent claudication or rest pain and 5 healthy controls underwent vacuum compression treatment for half an hour. The leg was positioned in an air-tight plexiglass cylinder in which hypobaric (-115 mm Hg) and hyperbaric (75 mm Hg) pressure could be generated alternately, in order to improve peripheral circulation. The effect on skin microcirculation was investigated using nailfold capillary microscopy (measuring nutritive perfusion), laser Doppler fluxmetry (LDF) (total skin perfusion) and transcutaneous oxygen tension measurements (TcpO2). RESULTS: A few patients experienced ischaemic symptoms during VC, probably because the leg was pinched off through inflation of the cuff. In both patients and controls capillary microscopic parameters did not change significantly. In some cases, microcirculatory perfusion decreased because the leg had cooled during the treatment. Application of a heating matrass annihilated this effect. Only in the patient group a few LDF- and TcpO2 parameters improved slightly, but significantly. CONCLUSION: Vacuum-compression therapy only slightly improves total skin perfusion and oxygenation, but not the nutritive microcirculation, being an essential factor in the occurrence of ischaemic symptoms. We therefore conclude that this instrument in its present form is not an aid in the treatment of lower limb ischaemia. PMID- 10731890 TI - The pole-pressure test: an easy alternative in patients with ischemic legs and incompressible arteries. AB - BACKGROUND: Systolic ankle artery pressure measured classically by cuff-manometry and Doppler ultrasound in patients with severely ischemic legs may be falsely elevated because of impaired compressibility of the arteries. We evaluated a hydrostatic blood pressure or so-called pole pressure test by correlating the ankle artery pressure as well as tcpO2 with the pole pressure. PATIENTS AND METHODS: We enrolled 29 patients (18 male, 11 female, 10 diabetics) with critical leg ischaemia. The pole pressure was obtained by elevating the leg, and measuring the height at which the Doppler signal disappears by means of a calibrated pole (recalculating cm H2O for mmHg). We used an 8 MHz cw-Doppler device. Only pole pressure values up to 70 cm H2O were included. In addition tcpO2 at 45 degrees C was recorded at the forefoot of the same leg. RESULTS: When we included the values of the ten diabetic patients the pole pressure of 57.9 +/- 16.2 mmHg (mean +/- SD) was significantly lower by 40.7 mmHg than the ankle pressure of 98. +/- 38.4 mmHg, p < 0.0001 (paired t-test). Without the diabetics the difference was 33.7 mmHg. The correlation coefficient between pole pressure and ankle pressure was 0.546 including the patients with diabetes, and 0.609 excluding the diabetics. The correlation of tcpO2 with the pole pressure, however, was 0.624 regardless of the inclusion of diabetics, while that between tcpO2 and ankle pressure was only 0.250. CONCLUSIONS: The pole pressure test is an easy, inexpensive and fast method providing a meaningful, quantitative measure independent of the compressibility of the arteries. PMID- 10731891 TI - Bacterial population of chronic crural ulcers: is there a difference between the diabetic, the venous, and the arterial ulcer? AB - BACKGROUND: At the Surgical Department of Surgery of the University Hospital Wurzburg microbiological examinations were performed of the ulcer grounds from patients with diabetic-neuropathic, diabetic-ischemic, venous, and arterial leg ulcers. The aim of the examination was to evaluate possible differences in the healing process of these ulcers based on the knowledge of their bacterial populations. PATIENTS AND METHODS: In a period of four months, 63 patients were consecutively examined by taking a bacteriological swab of their ulcer area. The healing process of their wounds was followed and related to the impact of bacterial colonisation and clinical signs of infection. RESULTS: 95% of the venous and arterial leg ulcers had a positive smear, whereas only 70% of diabetic ulcers were positive for bacterial growth. Bacterial population of the three ulcer entities, however did not differ significantly. 100% of the clinically infected venous and arterial ulcers but only 80% of the diabetic wounds revealed a positive smear. On the other hand, only 22% of the venous ulcers with a positive smear developed a clinical infection in contrast to 70% of the arterial and diabetic. Venous ulcers showed only in a few patients prolonged healing, even in cases of marked bacterial contamination. Despite of clinical signs of infection however, diabetic wounds sometimes did not reveal a positive wound smear (20%). All infected venous, but only 20% of the infected ischemic ulcers healed satisfactorily. Arterial wounds with no bacterial growth healed significantly better than contaminated wounds. This difference was not significant in the other entities. Radical removal of the infection by minor amputation increased the healing rate in diabetic ulcers over 80%, whereas ischemic wounds did not profit from this therapy. CONCLUSIONS: A positive bacterial wound smear is not inevitably correlated with a protracted leg ulcer healing. Nevertheless a fulminant infection often developed in diabetic ulcers despite the initial inability to demonstrate bacterial growth. In order to start antibiotic treatment as early as possible, a wound smear should be obtained routinely from patients with diabetic ulcers. In chronic venous ulcers, a routine swab does not appear to be indicated as it bears no clinical consequences. The same applies to patients with surgically fully treated peripheral arterial occlusive disease. As ischemia presents the limiting factor, antibiotic therapy in case of infection will not prevent imminent amputation. PMID- 10731892 TI - Hemangiosarcoma of the left hand in a patient with the rare combination of Maffucci's and Stewart Treves syndrome. AB - We describe a patient with the previously unseen combination of Maffucci's and Stewart Treves syndrome who presented with an angiosarcoma of the hand. Maffucci's syndrome is characterized by the presence of multiple enchondroma and soft tissue hemangioma. The syndrome is a rare nonhereditary condition with a usual onset in childhood. Malignant transformations are a common feature of this syndrome. In 1948, Stewart and Treves first described six cases of lymphangiosarcoma after radical mastectomy. This syndrome is an unusual form of angiosarcoma occuring as a complication of lymphedema. Chronic lymphedema and lymphangiectasia preceding lymphangiosarcoma may not only be induced by radical mastectomy with axillary lymph node dissection and postoperative radiation therapy. Posttraumatic, congenital or spontaneous chronic lymphedema may also be associated with lymphangiosarcoma. A time interval of many years seems to be required before malignant transformation develops. Generally the syndrome has a very poor prognosis. Both syndromes described above are of a rare frequency. We report this case because of prior unknown coincidence of both syndromes. PMID- 10731893 TI - Juxtaacetabular ganglion as a differential diagnosis in pulsating groin masses. AB - Ganglion cysts of the hip joint are uncommon synovial-lined fluid-filled juxtaarticular groin lesions. Whereas in the past the correct diagnosis was often made only at surgery there are now valuable imaging methods used for the diagnostic work-up. In experienced hands ultrasonography (US) combined with colour duplex Doppler ultrasonography (CDDS) as a real-time imaging technique easily performed at the patient's bedside is a valid alternative to more expensive or invasive investigations. We report on a patient who presented with a ganglion cyst and in whom first supported by conventional US an aneurysm of the femoral artery was suspected. The diagnosis of a juxtaarticular ganglion was subsequently correctly made at our institution by CDDS and magnetic resonance imaging, respectively, and the cyst was exstirpated successfully. The differential diagnosis of a pulsating groin mass as well as the most useful and specific imaging methods in the diagnostic work-up in this clinical setting are discussed. PMID- 10731894 TI - Double aortic arch: diagnosis missed for 29 years. AB - Double aortic arch is a rare vascular anomaly which causes tracheal and esophageal compression usually in the first months of life. Typical symptoms in the early childhood should lead to prompt diagnosis and surgical treatment of this malformation. In adults this anomaly is extremely rare. A case of a severely 29-year-old symptomatic woman is presented. Despite characteristic symptoms, the diagnosis was missed during childhood. The importance of different diagnostic procedures and operative therapy is discussed. Preoperative angiography can be replaced by the less invasive magnetic imaging and computed tomography. Surgical operation should also be performed in oligosymptomatic patients to prevent late complications. PMID- 10731896 TI - Vasospasmus: a rare manifestation of homocystinuria. AB - We report about a patient suffering from multiple spastic stenoses of varying degree of both iliac arteries and from occlusion of the left tibiofibular trunc. Laboratory investigations revealed increased levels of homocysteine and the diagnosis of homocystinuria was confirmed by fibroblast cell culture. The spasms responded well to vasodilative therapy with nitroglycerine, molsidomine (cGMP mediated) and prostaglandine E1 but not with nifedipine (Ca influx blocker). Our review of literature demonstrated that this arterial spastic abnormality is a very rare complication in patients suffering from homocystinuria. PMID- 10731895 TI - [Transposition of the subclavian artery with endovascular stent graft implantation for therapy of acute type B aortic dissection]. AB - We report about a patient with a thoracic aneurysm caused by an acute type-B dissection. Due to the concomitant affections the risk of surgery was distinctly increased, the reconstruction was performed endo vascular by stentgraft implantation (Talent, World Medical Systems, Sunrise FL) after transposition of the left subclavian artery to create a sufficient neck for the proximal stent placement. Endoluminal treatment seems to be a promising, less invasive alternative method in the treatment of acute aortic dissections. PMID- 10731897 TI - Aneurysms of the distal branches of the external carotid artery. AB - Aneurysms of the distal branches of the external carotid artery are rare and usually traumatic in origin. We present two cases which were treated in our clinic. The first case is about a traumatic aneurysm of the left superficial temporal artery (STA) in a young boy 8 years old. The young patient developed a pulsatile mass above his left eyebrow ten days after a bite by a boy of the same age. The second case is referred in a 36-year old woman with a pulsatile mass behind the right ear, which was an aneurysm of the posterior auricular artery. The treatment was ligation and resection under local anesthesia in the first case and under general anesthesia in the latter. Surgeons' familiarity with this entity is important for diagnosis and treatment. PMID- 10731898 TI - ["Digital subtraction angiography based on carbon dioxide, technique and clinical application"]. PMID- 10731899 TI - In search of the perfect water treatment method. PMID- 10731900 TI - The effects of gender and menstrual phase on carbohydrate utilization during acute cold exposure. AB - OBJECTIVE: The purpose of this study was to evaluate the effects of gender and menstrual cycle on the percent of carbohydrate (CHO) utilized during cold water immersion (20 degrees C). Previous research has suggested that males and females utilize CHO differently during submaximal exercise. This study examined whether this differential response is replicated during a submaximal elevation in metabolism, as demonstrated during thermogenesis (i.e., shivering during acute cold exposure). METHODS: Male and female subjects between the ages of 18 and 30 years were recruited for this study. Female subjects underwent the experimental trial once during the follicular phase and once during the luteal phase of their menstrual cycle. Subjects were immersed to the first thoracic vertebra until esophageal temperature reached 36.5 degrees C or for a maximum preocclusion period of 40 minutes. Peripheral temperature homeostasis via cuff occlusion (right arm and left leg) took place for 10 minutes, after which the pressure cuffs were released (postocclusion) and the subjects remained in the water for an additional 10 minutes. The following variables were measured: respiratory exchange ratio, percent of CHO utilization, and oxygen consumption (Vo2). RESULTS: Analysis of variance demonstrated no significant difference between genders or phases of the menstrual cycle in respiratory exchange ratio, percent CHO utilization, or Vo2 during cold water immersion. A significant difference was observed between men and women for absolute Vo2. CONCLUSIONS: These data suggest that although men and women differ with respect to absolute aerobic metabolism, this distinction does not cause a differential response with respect to substrate utilization during acute cold exposure. PMID- 10731901 TI - Microbiological water purification without the use of chemical disinfection. AB - OBJECTIVE: Point-of-use (POU) water treatment systems are self-contained units that can be used by recreational enthusiasts who normally obtain drinking water from untreated sources (i.e., rivers, lakes, etc). Microbiological water purifier units are capable of removing all waterborne pathogens. The purpose of this study was to evaluate a new technology (Structured Matrix) capable of micro biologically purifying the water without the use of chemical disinfectants or an external power requirement. METHODS: Each of 3 identical portable water filtration units were evaluated for their ability to remove Klebsiella terrigena, poliovirus type 1, rotavirus SA-11, and Cryptosporidium parvum oocysts. Units were operated according to the manufacturer's instructions to process 378 L of water. Each unit was challenged with test organisms after 0, 94, 190, 227, 284, 340, and 378 L had passed through it. For the 227-L and 284-L challenges, a "worst-case" water quality (4 degrees C, pH 9, and turbidity 30 NTU) was used that contained 1500 mg/L dissolved solids and 10 mg/L humid acid. At 340-L and 378-L challenges, worst-case water quality was adjusted to pH 5.0. Units were tested after stagnation for 48 hours following passage of 190, 340, and 378 L of water. RESULTS: The geometric average removal exceeded 99.9999% for bacteria, 99.99% for viruses, and 99.9% for Cryptosporidium parvum oocysts. CONCLUSION: These units comply with the criteria guidelines for microbial removal under the United States Environmental Protection Agency's "Guide Standard and Protocol for Testing Microbiological Water Purifiers." PMID- 10731902 TI - Subterranean medicine: an inquiry into underground medical treatment protocols in cave rescue situations in national parks in the United States. AB - Caving and spelunking have become increasingly popular over the years, with hundreds of thousands of amateur spelunkers across the country visiting caves. National parks in the United States offer hundreds of caves for all levels of spelunkers and, in fact, many national parks boast caves as either their main or major attraction. In an effort to increase visitor safety and establish subterranean medical treatment protocols, we began an investigation into cave rescue, medical protocols, previously published recommendations concerning cave safety, and visitor and rescue statistics in the national parks. Our inquiry provided little guidance from either the literature or the present US National Parks database for treating underground injuries. However, 2 predominant trends did appear. First, despite the nearly 2 million visitors to the caves in the 14 parks surveyed, there were only about 200 total calls for medical care. The vast number of those calls were for minor injuries. Second, no strict evidence-based treatment protocols for underground injuries exist, probably because they are not feasible. A caving incident database for the national parks would facilitate suggestions for preventative measures for the minor injuries and would help catalog the creative solutions for the rare serious subterranean medical incident. PMID- 10731904 TI - High-altitude global amnesia. AB - A variety of transient focal neurological signs presenting at high altitude have been described without associated acute mountain sickness or other concurrent illness. We report a case series of transient global amnesia at high altitude. The term high-altitude global amnesia (HAGA) is introduced to indicate this condition, and the pathophysiology is discussed. We hypothesize that because of the highly variable ventilatory response to hypoxia and to individual cerebral vasomotor reactivity, individuals with a marked hyperventilatory response could experience significant hypocapnic cerebral vasoconstriction that in turn could cause local hypoxia or ischemia to particular regions of the brain and resulting transient focal neurological impairment. PMID- 10731903 TI - Experimental use of a transportable hyperbaric chamber durable for 15 psi at 3700 meters above sea level. AB - A transportable hyperbaric chamber durable for 15 psi of pressure was used to treat a patient suffering from moderate acute mountain sickness at 3700 m above sea level. The symptoms were ameliorated a few minutes after pressurization in the chamber. After a 20-minute stay in the chamber, the patient was completely free of symptoms. Since the chamber can be inflated by using compressed air from a cylinder, no strenuous work was required of the operators. This transportable chamber seems to be useful for the treatment of high-altitude disorders at around 3000 m above sea level. PMID- 10731905 TI - Images. Patellofemoral pain syndrome (chondromalacia patellae) and mild acute mountain sickness. PMID- 10731906 TI - Rabies update. AB - Despite its relative rarity in industrialized countries, rabies continues to cause significant mortality worldwide with annual deaths estimated at over a hundred thousand. Recent epizootics in wild animals in the United States have renewed fears of rabies in this country. Additionally, nucleotide analysis to determine the source species for human rabies infections has led to new recommendations regarding exposure to bats. Fortunately, excellent human and animal vaccines are available and the development of new cell culture vaccines should help ease the current vaccine shortages. Travelers to developing countries should consider preexposure vaccination to avoid difficulties obtaining rabies immune globulin, which is in short supply worldwide, for postexposure prophylaxis should an exposure occur. PMID- 10731907 TI - Initiation of contemporary rabies postexposure therapy--a commentary. PMID- 10731908 TI - Amchhi Inji-ne, the English doctor: part 2. PMID- 10731909 TI - Staging simulated accidents. AB - Realistic simulated accidents are essential for wilderness emergency care training. Staging them requires good scene setting, acting, evaluation, and makeup. Training your students in moulage will not only help you stage simulated accidents for the class, it will also give the students valuable insights into emergency care by playing the roles of victims. Equipment can be cheap and simple, or more elaborate. With good scripts and good coaching of the victims, you can use moulage to help your students put all their skills together, bringing them as close as possible to the experience of a real wilderness emergency. PMID- 10731910 TI - The black bears of Yosemite National Park: bear-induced injuries--the role of improper food storage. PMID- 10731911 TI - Seizures and delirium in a trekker: the consequences of excessive water drinking? PMID- 10731912 TI - New aspects of surgical therapy of recurrent Crohn's disease. AB - Crohn's disease can neither be cured by surgery nor by medical therapy. Surgical therapy of recurrent Crohn's disease requires special precautions. The recurrence rate is 60% after 15 years. There are no certain data of the risk factors influencing the recurrence rate. The only clear facts are that wide resection out of the resection margins and smoking negatively influence recurrence. Hence, the major principles of therapy is a minimally-resected surgery. This mainly concerns strictures and stenosis. Strictures should be treated by stricturoplasty and stenosis by limited resection with Crohn-free resection margins. Just in case of interenteric and enterocutanous with a concomitant short bowel syndrome, in blind ending fistulas with an abscess or in enterovesical fistulas, we recommend immediate operation. The therapy of recurrent anorectal Crohn's disease underlies the same rules as primary therapy. If necessary, proctectomy remains the last option. Also, emergency surgery in recurrent Crohn's disease follows the same rules as in elective surgery. PMID- 10731913 TI - Primary malignant rhabdoid tumor of the brain: CT and MR findings. AB - PURPOSE: To describe the CT and MR findings of primary malignant rhabdoid tumor (MRT) of the brain, which is a rare but very aggressive neoplasm in childhood. MATERIALS AND METHODS: Retrospectively, we evaluated the CT and MR findings of 5 patients of primary MRT of the brain with a review of clinical records. RESULTS: The primary MRTs of the brain were large (n = 4) with a tendency to be associated with necrosis, hemorrhage (n = 2) and calcification (n = 2). Solid components of the tumor showed increased attenuation on precontrast CT scan and iso- or slightly hyper-signal intensity on T2-weighted images probably due to hypercellularity. Solid components of the tumor were also well enhanced on contrast-enhanced CT scan (n = 5) and MRI (n = 2). In 1 case with intratumoral bleeding, MR findings were variable on T1-weighted and T2-weighted images. Intracranial and intraspinal metastasis were found in 2 cases on preoperative MR studies. Follow-up CT and MR studies showed recurrence of the tumor and/or leptomeningeal metastasis in 3 cases. CONCLUSIONS: Although CT and MR findings of primary MRT of the brain are nonspecific, a tendency toward large size, calcification and intratumoral bleeding may be attributed to CT and MR findings. The solid components of tumors could present hyperdense on precontrast CT scan and iso- or slightly hyper-signal intensity on T2-weighted MR image. Preoperative and follow-up MR studies are important to detect metastatic foci. PMID- 10731914 TI - Uterine artery Doppler velocimetry in the prediction of adverse obstetric outcomes in unexplained MSAFP elevations. AB - Unexplained maternal serum-fetoprotein (MSAFP) elevation has been known to be associated with adverse obstetric outcomes, however it is not sufficiently useful as a screening test. This study was undertaken to determine whether uterine artery Doppler velocimetry could define a subset of patients with an elevated MSAFP level in whom complications of pregnancy might develop. The subjects included 179 women between 26 and 28 weeks' gestation with MSAFP > or = 2.5 multiples of the median, in whom either the presence of an early diastolic notch or a resistance index 0.6 was considered as an abnormal Doppler velocimetry finding. Those subjects who displayed abnormal Doppler velocimetry findings showed an increased incidence of preeclampsia, preterm birth, IUGR, and IUFD compared to those subjects with only elevated MSAFP (p < 0.05). No differences were observed in the incidence of LBW. Positive predictive values of adverse obstetric outcomes were significantly higher in the group having both elevated MSAFP and abnormal Doppler velocimetry compared to the group with only elevated MSAFP (p < 0.05). Uterine artery Doppler velocimetry in the second trimester can improve the value of unexplained MSAFP elevation in the prediction of adverse obstetric outcomes. PMID- 10731915 TI - Dietary iodine intake and urinary iodine excretion in patients with thyroid diseases. AB - This study was conducted to examine the usual iodine intake in patients with thyroid diseases and to compare iodine status with normal subjects. The dietary iodine intake was assessed using a semi-quantitative food frequency questionnaire, and urinary iodine excretion was measured in 184 patients diagnosed with thyroid diseases and 207 normal subjects. The average usual iodine intake of patients with thyroid diseases was 673.8 +/- 794.9 ug/day and that of normal subjects was 468.9 +/- 481.9 ug/day. Among the patients with thyroid diseases, higher values were found in the patients with thyroid cancer (1460.6 +/ 1044.8 ug/day) and lower values were found in patients with simple goiter (443.5 +/- 470.4 ug/day). The urinary iodine excretions of patients and normal subjects were 4.33 +/- 5.70 mg/L and 2.11 +/- 0.69 mg/L, respectively. The iodine intake and urinary iodine excretion of patients with thyroid diseases were significantly higher than those of normal subjects (p < 0.05). The dietary iodine intake and urinary excretion of patients with thyroid cancer were significantly higher than other patients with thyroid diseases and normal subjects because of the use of seaweed or seaweed-containing dietary supplements (p < 0.01). This study suggests that the habitual ingestion of seaweed-containing dietary supplements in addition to dietary iodine intake will have adverse effects due to its excessive iodine intake. PMID- 10731916 TI - Penile vibratory threshold changes with various doses of SS-cream in patients with primary premature ejaculation. AB - SS-cream made with extracts from natural products is a topical agent for treating premature ejaculation (PE). In order to elucidate the penile vibratory threshold changes and clinical effects of various doses of SS-cream, 53 patients with primary PE were investigated in a double-blind randomized placebo-controlled study. The mean age was 37.3 +/- 6.4 years and mean ejaculatory latency was 1.37 +/- 0.52 minutes. Neither the patients nor their sexual partners were satisfied with their sexual lives. Vibratory threshold at the glans penis, penile shaft, scrotum and index finger were measured using a biothesiometer twice during the screening period and three times one hour after the application of respective creams (SS-cream 0.05, 0.10. 0.15, 0.20 gm and placebo 0.10 gm) on the glans penis according to the order of the allocation table in a randomized fashion. The efficacy of SS-cream was defined as when the vibration threshold increased by more than 4 microns compared to the value tested during the screening period. The vibratory thresholds at the glans penis increased significantly in a dose dependent manner after the application of various doses (0.05, 0.10, 0.15, 0.20 gm) of SS-cream (p < 0.001), and the efficacy of SS-cream on the penile vibration threshold increased according to the increased dosage (penile shaft: 48.4, 51.6, 54.8, 64.5%, glans penis: 58.1, 67.7, 77.4, 83.9%, respectively). With these results, we concluded that SS-cream increased the penile sensory threshold dose dependently, and therefore it is clinically effective for treating the heightened penile sensory response in patients with PE. PMID- 10731917 TI - The changing epidemiology of hepatitis A in children and the consideration of active immunization in Korea. AB - Currently, Korea is a low endemicity country for HAV, especially in children. However, recent reports of hepatitis A outbreaks show that there has been a shift of disease incidence to adolescents and young adults, with 2 cases of acute liver failure in one reported outbreak. We need to study the immune status for HAV in order to provide information for the establishment of preventive measures and possible consequences of HAV in Korea. A total of 334 infants, children and adolescents less than 20 years of age living in rural areas of Kyonggi Province, Korea were evaluated for anti-HAV immune status in 1996. Five hundred and eighty four primary school children living in the same area were separately evaluated for the natural seroconversion rate between 1993 and follow-up samples taken in 1996. Anti-HAV IgG antibody was measured by enzyme immunoassay (HAVAB EIA kit, Abbott Laboratories, Chicago, Illinois, USA). In comparison with previous reports of seroprevalence rates, our data confirmed a dramatic drop in seroprevalence rates among children and adolescents under 20 years of age living in rural areas, from over 63.8% two decades ago to 4.6% in 1996. Natural acquisition of HAV antibody in primary school children rarely occurs, registering only 0.5% during three years. Several outbreaks in young adults during 1996-1998 suggested that immunity against HAV in this population is so low that massive outbreaks are unavoidable. Teenagers and young adults, especially soldiers, who are likely to be exposed to contaminated food or water, would also have a greater risk of hepatitis A. Immunizing children with HAV vaccine as a routine schedule should also be considered in Korea in the future, particularly if the disease burden could be estimated and the cost-effectiveness of the vaccine could be proved. PMID- 10731918 TI - Distribution of anti-HBs levels in Korean adults. AB - Exact titration of anti-HBs with mIU/mL unit is necessary in evaluating the success of HBV vaccination or in making a decision to increase the dose of HBV vaccination. Data of distribution of anti-HBs titers can contribute to cutting of public health costs by reducing unnecessary HBV booster doses. Moreover, anti-HBc is also an important marker for differentiation of vaccination-induced anti-HBs from infection-acquired anti-HBs. However, not much study about these subjects has been done in Korea. So we evaluated anti-HBs associated with anti-HBc and vaccination history. HBsAg and anti-HBs tests were done in 1,465 cases. The positive rates of HBsAg and anti-HBs were 4.5% and 74.6%, respectively. Anti-HBs positive rate was higher in the vaccinated group than that in the non-vaccinated group. The rates of anti-HBs positive cases with lower titers (10-< 100 mIU/mL) were 31.9%, while cases with higher titers (> or = 100 mIU/mL) were 68.1%. This suggested about 70% of anti-HBs-positive Korean adults (about 53% of the general adult population) have long-lasting immunity against HBV infection and may not require booster doses of HBV vaccination for a long time. Anti-HBs titers in the vaccine-induced anti-HBs group were higher than those in the infection-acquired anti-HBs group. No statistical differences were noted between male and female or among age groups. 25.7% of the HBsAg (-)/anti-HBs (-) group showed anti-HBc positive and HBV-DNA was detected in 11.1% among HBsAg (-)/anti-HBs (-)/anti HBcAb (+) cases. Further study about post vaccination anti-HBs titer decay in Korean should be performed to help cut vaccination costs. PMID- 10731919 TI - Estrogen affects vascular tone differently according to vasoactive substances in ovariectomized Sprague-Dawley rat. AB - The favorable effects of estrogen on cardiovascular diseases can be explained by several mechanisms such as changes in serum lipid profiles and thrombogenecity. Estrogen also affects the vascular tone, but there has been no report in which the effect of estrogen was tested comprehensively for several vasoactive substances, especially after long-term administration. Two weeks after bilateral ovariectomy in 8-week old female Sprague-Dawley rats, placebo or 17 beta estradiol (E2) pellets (0.5 mg; released over 3 weeks) were implanted subcutaneously. Two weeks after pellet implantation, organ chamber experiments were performed using aortae. Compared with control, E2-treated vessels showed impaired endothelium-dependent relaxation to acetylcholine. E2 enhanced the contraction to norepinephrine and U46619 and had no effect on endothelin-1 induced contraction. In contrast, the contraction to angiotensin (AT)-II was inhibited by E2. Northern blot analysis for AT1 receptor expression using cultured aortic smooth muscle cells showed no difference between control and E2 treated cells, suggesting that AT1 receptor downregulation is not the likely mechanism. These results suggest that E2 affects the vascular tone variably according to vasoactive substances. PMID- 10731920 TI - Bacteriologic characteristics and serotypings of Streptococcus pyogenes isolated from throats of school children. AB - To determine the carrier rate of beta-hemolytic streptococci (BHS), throat cultures were taken from healthy elementary school children in four separate areas of Korea from 1992 to 1995, including Inje, Nonsan, Seoul and Chinju. The strains of Streptococcus pyogenes had been serotyped with anti-T, -OF and -M sera. The isolation rates of BHS and S. pyogenes ranged from 14.1-32.4% and 10.9 18.5% respectively. More than half of the carriers showed heavy growth of BHS. M78 (48.6%) and M28 (22.2%) were most common in Inje, M12 (23.6%) and M5 (20.3%) in Nonsan, M12 (48.8%) and M5 (14.6%) in Seoul, and M12 (26.3%) and M22 (14.5%) in Chinju, respectively. About 15% of school children were positive for S. pyogenes in throat cultures, and the distribution of serotypes varied according to geographical regions. PMID- 10731921 TI - Effects of cyclosporin A on sex hormone and estrogen receptor in male rat with special reference to cyclosporin A-induced osteoporosis. AB - The mechanisms of high turnover bone loss induced by Cyclosporin A (CsA) are not clearly understood. Deficiencies in sex hormones result in high turnover osteoporosis, and not only androgen but also estrogen plays an important role in maintaining bone mass in men. To study whether or not there are any changes in the levels of sex hormones, aromatization, and the expression of estrogen receptors in CsA-induced osteoporosis, we treated 39 rats with vehicle, low-dose CsA (5 mg/kg) and high dose CsA (15 mg/kg) for 28 days, and measured sex hormone levels by radioimmunoassay. Aromatase activities in ROS cells and 3T3-L1 cells were determined by measuring the conversion rate of 3H-androstenedione into 3H estrone. ER and ER mRNA were measured by competitive RT-PCR in collected marrow cells and ROS cells. The levels of free testosterone in the serum in low-dose CsA treated rats were unchanged, but the levels were significantly decreased in those treated with high-dose CsA as previously reported. The levels of total estradiol in the serum were significantly increased in the low-dose CsA-treated group (5 mg/kg) and were comparable to levels of the control group in the high-dose CsA treated group (15 mg/kg). CsA increased the conversion of 3H-androstenedione to 3H-estrone in ROS cells, but not in 3T3-L1 cells. Meanwhile, CsA treatment did not change the rates of ER or ER mRNA expression in ROS cells or in collected bone marrow cells. In conclusion, CsA treatment decreased the level of free testosterone in the serum, but did not decrease the level of serum estradiol by enhancing aromatization. High-turnover osteoporosis induced by clinical dosage CsA treatment may not be caused by lowering the levels of circulating estrogen or by decreasing the expression of estrogen receptors. PMID- 10731922 TI - Expression of leptin receptor (Ob-R) in human atherosclerotic lesions: potential role in intimal neovascularization. AB - Neovascularization of the adventitial vasa vasorum with extension into the intima of atherosclerotic lesions is frequently observed, but its pathophysiological significance is still subject to debate. Recently, leptin, the product of the Ob gene, was identified. Leptin, via activation of the endothelial receptor (Ob-R), generates a growth signal involving a tyrosine kinase-dependent intracellular pathway and promotes angiogenic processes. We hypothesized that a high concentration of leptin within vasa vasorum and plaque itself, may influence inflammatory and vascular neovascularization coupling with functional upregulation of the vascular endothelial growth factor (VEGF). Microscopic computerized tomography was utilized for the spatial distribution of vasa vasorum and intimal neovascularization from atherosclerotic human coronary arteries. Atherosclerotic coronary arteries showed a dense plexus of microvessels in the adventitia and plaque itself. Microscopic analysis from human atherosclerotic aortas revealed an increase in the intimal thickness with neovascularization. The immunoreactivity for Ob-R, VEGF and matrix metalloproteinase (MMP) increased in atherosclerotic plaque, predominantly in the endothelial lining of the intimal neovessel and macrophages/foam cells. Our observation of a prominent colocalization between Ob-R, VEGF and MMP supports this hypothesis and these factors participate in the neovascularization of atherosclerotic lesions. The present study is the first report on vascular tissue and it opens a promising perspective concerning future investigations of leptin-dependent modulation of atherogenesis and vascular neovascularization under pathophysiolgical conditions. PMID- 10731923 TI - Retroviral-mediated IL-2 gene transfer into murine neuroblastoma. AB - We used retroviral-mediated gene transfer of the human interleukin (IL)-2 gene into murine neuroblastoma cells to investigate whether locally-secreted IL-2 is able to influence the generation of anti-tumor immune responses. Supernatant obtained from cultures of approximately 1 x 10(6) IL-2 gene-transduced, G-418 selected neuro-2a cells was assayed for human IL-2 production by ELISA kit. First, to estimate whether the local secretion of IL-2 from the genetically modified tumor cells would affect their tumorigenicity in vivo, IL-2-secreting neuro-2a cells were s.c. injected into A/J mice and tumor growth was measured weekly. And to estimate whether IL-2 transfected neuroblastoma cells protect mice from tumor development after wild-type tumor cell challenge, IL-2-secreting neuro 2a cells were s.c. injected into A/J mice. Seven days after IL-2 gene-transfected neuroblastoma cell injection, unmodified neuro-2a cells were s.c. injected into the contralateral site of A/J mice and tumor growth was measured weekly. Finally, to estimate IL-2 effect on pre-established large tumor burdens, IL-2-secreting neuro-2a cells were s.c. injected into A/J mice with established tumor and its growth was measured weekly. The IL-2 gene-transduced neuro-2a clones secreted 120.25-177.3 IU of IL-2 per ml per 10(6) cells during 24 hr. None of the mice injected with IL-2-secreting neuro-2a cells developed tumors within 6 weeks, while all of the mice injected with wild-type neuro-2a cells developed tumors. Immunization of mice with IL-2 gene-transfected, irradiated neuro-2a cells protected these animals against a subsequent challenge with wild-type tumor cells. Finally, the size of large neuroblastomas decreased after IL-2-secreting neuro-2a cell injection into mice. Local secretion of IL-2 gene-transduced tumor cells abrogates their tumorigenicity and induces protective immunity and may inhibit the growth of neuroblastoma. PMID- 10731924 TI - Coexpression of cyclooxygenase-2 and matrix metalloproteinases in human aortic atherosclerotic lesions. AB - Inflammation appears to have a major role in the development of atherosclerosis. Cyclooxygenase-2 (COX-2) is involved in the inflammatory response via the generation of prostanoids that, in turn, are involved in the production of matrix metalloproteinases (MMPs). This study aimed to investigate atherosclerosis in human aortas for in situ tissue distribution of COX-2, MMPs including MMP-9 and membrane type 1 MMP (MT1-MMP), and tissue inhibitor of metalloproteinase-2 (TIMP 2). Immunohistochemical studies were performed on atherosclerotic lesions of aortas from patients with aortic aneurysms (n = 4) and dissections (n = 3) by using antibodies to COX-2, MMP-9, MT1-MMP, and TIMP-2. Control tissues were obtained from traumatically dissected aortas (n = 2). All specimens from diseased aortas had atherosclerotic lesions ranging from fatty streak to atheromatous plaques. In control, there was no expression of COX-2, MMP-9, and MT1-MMP in all aortic layers. Immunoreactivity for COX-2 was predominantly noted in macrophages and smooth muscle cells (SMCs) of the intima including atherosclerotic plaque itself and the medial layer of the plaque base, as well as in SMCs and endothelial lining of the vasa vasorum in the adventitia. Immunoreactivity for MMP-9 and MT1-MMP was found in the same distribution as that of COX-2. Additionally, the expression of TIMP-2 increased in relation to MMP-9 expression. This study demonstrates that COX-2 is coexpressed with MMP-9 and MT1-MMP, not only by macrophages and SMCs in atherosclerotic lesions, but also in endothelial lining of the vasa vasorum of human aortas. Thus, vascular inflammatory reactions may influence extracellular matrix remodeling by coactivation of MMPs in the development of atherosclerosis and, in turn, the progression of disease. PMID- 10731925 TI - Carotid artery stenting in patients with symptomatic coronary artery disease. AB - Carotid artery stenting has been accepted as a potential alternative to carotid endarterectomy in patients with significant carotid artery stenosis. The objective of this study was to evaluate the feasibility, safety and long-term outcome of percutaneous stenting of carotid artery stenosis in patients with coexisting symptomatic coronary disease. Between May 1996 and May 1999, we performed carotid artery stenting at 48 lesions in 36 patients with carotid stenosis of 60% and symptomatic coronary artery stenosis. Twenty-one patients (58%) had neurologic symptoms. Carotid stenting was performed in 43 internal, 2 external and 3 common carotid lesions. We used Wallstent in 46 lesions, Palmaz stent in 2 lesions and Microstent II in 1 lesion. Staged or combined coronary intervention was performed in 18 patients (50%) and staged coronary artery bypass surgery was performed in 6 patients (17%). In the other 12 patients (33%), medical treatments were performed. Carotid stenting was successful in all lesions. Simultaneous bilateral carotid stenting was performed in 11 patients (31%). One major and 1 minor stroke developed during the procedure. There were no deaths during the procedures and within 30 days post-procedure. During the follow up of 14 +/- 7 (3 to 40) months, there were no deaths or neurological events. On follow-up (6 +/- 1 months) angiography and/or duplex sonography of 44 eligible lesions in 32 patients, there were 2 cases of asymptomatic restenosis (4.5%) which developed in Palmaz stents implanted at the external carotid artery and the common carotid artery, respectively. In conclusion, carotid artery stenting in patients with coexistent carotid and coronary artery disease is feasible, safe and shows favorable follow-up outcomes. PMID- 10731926 TI - Significance of rectosigmoid polyp as a predictor of proximal colonic polyp. AB - The association between rectosigmoid polyps and polyps in the more proximal colon is still a matter of debate, and the need for colonoscopy in patients with rectosigmoid polyps that are detected by flexible sigmoidoscopy is controversial. The aim of this study was to determine whether or not certain characteristics of rectosigmoid polyps are associated with the presence and characteristics of proximal colonic polyps. Seven hundred and twenty-eight patients who underwent total colonoscopy between October 1995 and June 1998 and who had colorectal polyps were retrospectively analyzed. Patients with inflammatory bowel diseases, familial adenomatous polyposis, or any advanced cancer were excluded. The odds ratio (OR) and 95% confidence interval (CI) of prevalence of proximal colonic polyps according to the patients age and sex, as well as the characteristics of rectosigmoid polyps, were calculated. Advanced adenoma was defined as an adenoma larger than 10 mm or an adenoma of any size with villous component, high-grade dysplasia or invasive carcinoma. Among 728 patients with colorectal polyps, 356 patients (48.9%) had polyps only in the rectosigmoid region, 193 patients (26.5%) had polyps only in the proximal colon, and 179 patients (24.6%) had polyps in both the rectosigmoid and proximal colon. In 535 patients with rectosigmoid polyps, the prevalence of proximal colonic polyps, neoplastic polyps and advanced adenomas were 33.4%, 27.3% and 2.9%, respectively. The prevalence of proximal colonic polyps in patients with rectosigmoid polyps was found to be significantly related to the male gender and elderly patients, in addition to the neoplastic histology of the rectosigmoid polyps. However, the prevalence of the proximal colonic polyps was not related to the size, number and shape of rectosigmoid polyps. In 179 patients with both rectosigmoid and proximal colonic polyps, the characteristics of proximal colonic polyps such as size, number and shape were similar to those of rectosigmoid polyps. We recommend total colonoscopic examination in all patients with rectosigmoid adenomas, regardless of the size, number, and shape, especially in elderly males. PMID- 10731927 TI - Endoscopic bone graft for delayed union and nonunion. AB - We performed endoscopic bone grafting for eight patients of delayed union and nonunion which developed after femoral and humeral shaft fractures. The mean interval from initial intervention to endoscopic bone grafting was 7.3 months. Six patients of delayed union and nonunion healed at 4.1 months on average. Two patients had unsatisfactory healing and eventually underwent non-endoscopic revisional surgery. There was no intraoperative on postoperative complication. Endoscopic bone grafting can be a less invasive alternative, obtaining rapid bone union in cases of compromised healing of the diaphyseal fracture. PMID- 10731928 TI - Spontaneous programmed cell death of peripheral blood mononuclear cells from HIV infected persons is decreased with interleukin-15. AB - Interleukin 15 (IL-15) is an important regulatory cytokine in cellular immunity. In vitro replacement of IL-15 has been shown to enhance immunity in Human immunodeficiency virus type 1 (HIV-1) infected lymphocytes. We evaluated the effect of IL-15 on the survival of peripheral blood mononuclear cells of HIV patients by examining in vitro lymphocyte apoptosis, and correlated the process with Bcl-2 and Fas gene regulation. Peripheral blood mononuclear cells (PBMC) from 21 HIV-infected adults and 24 HIV-seronegative healthy individuals were isolated and cultured to determine the effect of escalating doses of IL-15 (0, 1, 10, 100, 1000 ng/mL) on apoptosis. Lymphocyte proliferation assay with (3H) TdR was measured and Bcl-2 and Fas gene regulation was observed. The results were as follows: 1) IL-15 reduced culture induced lymphocyte apoptosis in HIV patients in a dose dependent manner, and reached a plateau level at a concentration of 100 ng/ml; 2) IL-15 significantly reduced the level of apoptosis after 3 days (14%) and 5 days (15%) of culture in HIV patients, while no difference was observed in HIV (-) donors; 3) The percentage of viable cells among the total number of lymphocytes was significantly enhanced by 25% in HIV patients with IL-15; 4) Bcl 2 expression was decreased in HIV patients (53.9 +/- 12.3%) compared to HIV (-) donors (93.0 +/- 3.7%), and IL-15 increased Bcl-2 expression by 21.2 +/- 5.2% in HIV patients; 5) Fas expression was increased in HIV patients (70.2 +/- 4.6%) compared to HIV (-) donors (32.4 +/- 4.3%), and IL-15 increased Fas expression by 8.4 +/- 1.2% in HIV (-) donors. Our findings indicate that IL-15 may influence immunologic abnormalities in HIV infection, particularly its ability to prevent apoptosis of lymphocytes by suppressing the down-modulation of Bcl-2. This may provide an experimental basis for IL-15 immunotherapy. PMID- 10731929 TI - Tissue plasminogen activator and plasminogen activator inhibitor-1 in human choledochal bile. AB - Fibrinolytic properties have been detected in animal and human gallbladder (GB) bile. Plasminogen activator inhibitor-1 (PAI-1) has been reported in greater concentration in GB stone bile and may be a nucleating factor in the pathogenesis of GB stone formation. It is unknown whether or not human choledochal bile has similar properties, which could have a role in choledocholithiasis. The aims of this study were to determine the presence of fibrinolytic properties of human choledochal bile and to compare those properties among normal, acalculous, and calculous-infected choledochal bile. Tissue plasminogen activator (t-PA) and PAI 1 of choledochal bile were measured by enzyme linked immunosorbent assay in patients with cholangitis due to acalculous bile duct obstructions (n = 9), choledocholithiasis with cholangitis (n = 20), and normal bile (n = 7). The t-PA concentration of choledochal bile was no different among the three groups (acalculous-infected bile, median 4.61 ng/ml, and calculous-infected bile, 4.61 ng/ml, versus normal bile, 7.33 ng/ml). PAI-1 was detected in choledochal bile in significantly greater concentrations in patients with acalculous cholangitis due to bile duct obstructions and choledocholithiasis with cholangitis (acalculous infected bile, median 0.36 ng/ml, and calculous-infected bile, 0.1 ng/ml, versus normal bile, 0.02 ng/ml, p < 0.05), but the bile concentration of PAI-1 was no different between the acalculous and calculous-infected choledochal bile. Human choledochal bile possesses t-PA and PAI-1. PAI-1 was present in greater concentrations in both acalculous and calculous-infected choledochal bile. Increased levels of PAI-1 may be an epiphenomenon of cholangitis rather than a factor in the pathogenesis of choledocholithiasis. PMID- 10731930 TI - Asymptomatic electrophysiologic carpal tunnel syndrome in diabetics: entrapment or polyneuropathy. AB - Electrophysiologic carpal tunnel syndrome (CTS) is common and is frequently asymptomatic in diabetics. In order to evaluate the clinical significance of asymptomatic electrophysiologic CTS, the nerve conduction studies (NCS) of 48 diabetics with asymptomatic electrophysiologic CTS were compared with those of 56 age and gender-matched controls, as well as 50 patients with symptomatic CTS without diabetes. Nerve conduction velocities of the ulnar, peroneal, and posterior tibial nerves were significantly slower in diabetics with asymptomatic electrophysiologic CTS than in normal controls. Compared to symptomatic non diabetic CTS, there was also significant slowing of the median and ulnar nerve conduction velocities in asymptomatic diabetic CTS. However, in diabetics with asymptomatic CTS, abnormalities of the distal segment of the median NCS were more prominent compared with those of all the other tested nerves. These findings suggested that asymptomatic electrophysiologic CTS in diabetics is a manifestation of increased vulnerability to the entrapment of the peripheral nerve. PMID- 10731931 TI - Decreased osteopontin expression in the rat kidney on a sodium deficient diet. AB - Osteopontin (OPN) is a secreted phosphoprotein that is constitutively expressed in the normal kidney and is induced by various experimental and pathologic conditions. Several possible functions of OPN have been suggested, however the mechanism and significance of OPN expression are still uncertain. Since high salt concentration or salt crystal have been known to enhance OPN expression in intact kidney or cultured renal cells, in the present study we examined whether or not a low salt condition had an effect on OPN expression in the kidney. Adult male Sprague-Dawley rats were fed either a normal sodium or a sodium deficient diet for 1 week. Kidneys were processed for in situ hybridization using a digoxigenin labeled riboprobe and for immunohistochemistry using antibodies to OPN, renin, and Na-K-ATPase. In rats fed a normal sodium diet, OPN mRNA and protein were expressed only in the descending thin limbs of Henle's loop (DTL) and in the papillary and pelvic surface epithelium (PSE). In rats fed a sodium deficient diet, there was a marked decrease in OPN immunoreactivity in the DTL, but no changes in PSE. In contrast, no changes were observed in OPN mRNA expression in the DTL by in situ hybridization, indicating that decreased OPN protein expression was a result of translational regulation. As expected, rats fed a sodium deficient diet were associated with increased immunoreactivity for Na-K ATPase and renin compatible with activation of the renin-angiotensin system. These results suggest that dietary sodium may be involved in the regulation of OPN expression in the DTL of the rat kidney. PMID- 10731932 TI - A case of Klinefelter syndrome with retroperitoneal teratoma. AB - Klinefelter syndrome (KS) is often associated with various neoplasms, especially germ cell tumors. Mediastinum is the most favored site of extragonadal germ cell tumors with KS, which is somewhat different from those without KS. The retroperitoneal germ cell tumor in KS is very rare. A five-month-old boy with an abdominal mass was found to have a retroperitoneal tumor. After surgical removal, he was diagnosed to have mature cystic teratoma. Cytogenetic study of his peripheral lymphocytes revealed that his karyotype was consistent with KS. This case suggests that patients with KS might be at risk of having germ cell tumors in sites other than mediastinum. It also suggests that all cases with these tumors should be screened for the presence of karyotypic abnormalities, and it might help to assess the exact correlation between germ cell tumors and KS, and to treat them accordingly. PMID- 10731933 TI - Primary carcinoma of the fallopian tube coexisting with benign cystic teratoma of the ovary. AB - Primary carcinoma of the fallopian tube is a rare malignancy of the female genital tract and infrequently diagnosed before an operation. The majority of patients have extensive disease at the time of diagnosis. We have experienced incidentally a case of a carcinoma of the fallopian tube coexisting with a benign cystic teratoma of the ovary in a 25-year-old woman. We report this case with a brief review of literatures. PMID- 10731934 TI - Radiologic findings of Mirizzi syndrome with emphasis on MRI. AB - We have reported a case of Mirizzi syndrome preoperatively diagnosed using MR cholangiopancreatography. MRCP and T2-weighted image using a single-shot fast spin-echo sequence accurately depicted all components of Mirizzi syndrome, including impacted stone in the neck of the gallbladder compressing the common hepatic duct and wall-thickening of the gallbladder without any evidence of malignancy. The combination of MRCP and T2-weighted image can be counted on to replace conventional modalities of diagnosing Mirizzi syndrome without any loss of diagnostic accuracy. PMID- 10731935 TI - Cutaneous hamartoma of the hand: MR imaging findings. AB - We report two cases of magnetic resonance imaging of the cutaneous hamartoma on the hand, which is a rare benign soft tissue tumor. PMID- 10731936 TI - A case of nephrogenic diabetes insipidus caused by obstructive uropathy due to prostate cancer. AB - Nephrogenic diabetes insipidus (DI) secondary to chronic urinary tract obstruction is a rare disease. The exact cause is unknown but it is likely that increased collecting duct pressures cause damage to the tubular epithelium, resulting in insensitivity to the action of arginine-vasopressin (AVP). A 77-year old man complaining of polyuria and polydipsia was treated with alpha glucosidase inhibitor under the impression of polyuria due to diabetes mellitus. But his symptoms did not improve. Water deprivation and AVP administration study revealed that the patient had nephrogenic DI. Urinary tract obstruction due to an enlarged prostate was suggested as a principal cause of nephrogenic DI. The patient underwent transurethral resection of the prostate and bilateral subcapsular orchiectomy. After surgery, the urine osmolarity was normalized and the patient became symptom-free. We report a case of nephrogenic DI due to obstructive uropathy which was cured by surgery eliminating obstruction. PMID- 10731937 TI - A case of congenital inverse Duane's retraction syndrome. AB - Inverse Duane's retraction syndrome is very uncommon. Congenital cases are even more unusual. A 6-year-old girl with convergent squint along with severe restriction on abduction is described. On attempted abduction, a narrowing of the palpebral fissure, upshoot and retraction of the eyeball were observed. Brain and orbit MRI demonstrated no intracranial or intraorbital mass, fracture, or entrapment of the medial rectus. Forced duction test was strongly positive. The primary lesion was found to be a tight medial rectus with shortening and soft tissue contracture. Surgical tenotomy of the medial rectus led to successful postoperative motility, but some limitation at full adduction and abduction persisted. This is a case reported with congenital medial rectus shortening, suggesting that this condition may be one of the etiologies of the rare inverse Duane's retraction syndrome. PMID- 10731938 TI - [Does residual mediastinal mass in children with completed treatment of Hodgkin's disease affect the incidence of relapses?]. AB - Four hundred and four children with Hodgkin's disease (stage I-IV) were treated in seven cooperating centers of Polish Paediatric Leukaemia/Lymphoma Study Group between 1988 and 1996. Mediastinal masses and/or hilar involvement were found in 261 (65%) patients. Remission was obtained in 256 (98%) of this group. In 31 (12%) children residual mediastinal/hilar masses were found after completing the treatment. For this reason in 13 cases the number of chemotherapy courses and/or the dose of radiation therapy were increased. In two cases thoracotomy or thoracoscopy were performed, and in one case gallium scan was performed. In none of these patients active disease was found. Relapses occurred in 4 (12.9%) from the group of 31 children with residual mediastinal/hilar involvement 8-15 months after cessation of the therapy. Twenty seven children have been in first remission for 5-113 months (median, 34). In 225 patients with a complete resolution of their mediastinal/hilar masses, relapses occurred in 13 (5.7%) cases. Patients with residual mediastinal mass should be carefully evaluated before making a decision to complete their treatment, including CT scan, MRI, and gallium scan. In doubtful cases histopathological verification should be done. PMID- 10731939 TI - [The analysis of failures in the treatment of children with non-B non-Hodgkin's lymphoma. Polish pediatric leukemia/lymphoma study group report]. AB - The aim of the study was to evaluate the results of treatment of 46 children with non B non-Hodgkin's Lymphoma registered in 7 centers of Polish Paediatric Leukaemia/Lymphoma Study Group from 1993 to 1998. The patients were treated according to BFM-90 protocol based on German regimen. The overall probability of event-free survival for the all children after 4 years of follow-up was 71%, for patients in stage III--65%, stage IV--70%. The achieved results were not as positive as in the BFM Study Group, what was related to: the great number of children with advanced stage of disease (54.3%), the late final diagnosis, the great number of recurrences (22.5%) and deaths caused by the toxicity of medication (6.5%) (infections, drug toxicity). PMID- 10731940 TI - [The efficacy of BFM-90 program in the treatment of acute lymphoblastic leukemia in children in the studies of Polish pediatric leukemia/lymphoma group]. AB - Between years 1993 and 1998, 113 children aged from 6 months to 18 years (41 girls and 72 boys) with first relapse of acute lymphoblastic leukaemia (ALL) were included in the study. All children were treated according to BFM-90 relapse protocol. Thirty-two cases were classified as very early relapses, 56 as early and 25 as late relapses. Sixty-one children had isolated bone marrow relapse, in 30 children extramedullary relapse occurred (in 21 children in central nervous system and in 16 children in testes). There were 23 combined relapses. Remission was achieved in 12 children with very early relapse (78.12%), 32 children with early relapse (85.71) and 19 children with late relapse (96%). Event-free survival in 30 months of follow-up was 29.2%, 59.0% and 73.2% for very early, early and late relapses, respectively. Sixteen children with relapsed ALL after chemotherapy according to BFM-90 relapse protocol underwent high-dose therapy with hematopoietic stem cell transplantation (in 3 cases autologous and in 13 cases allogeneic). In 6 children isolated bone marrow relapse occurred after transplantation, all of them died during subsequent chemotherapy. Ten children is alive and well from 2 to 43 months after transplantation. The results obtained with BFM-90 chemotherapy in children with first early relapses are not acceptable. Such patients require high-dose chemotherapy and transplantation of hematopoietic progenitor cells. PMID- 10731941 TI - [The analysis of failures in the treatment of children with chronic myelocytic leukemia in the studies of Polish pediatric leukemia/ lymphoma group]. AB - 94 children with chronic myelocytic leukaemia--CML treated in period 1975-1998 were included in the study. Twenty seven of 60 children were treated with hydroxyurea or busulfan with 6 MP. In 33 children aged 1, 5-17 years IFN (Interferon alfa) was applied at the dose of 3 millions units every second day subcutaneously. Our data showed that IFN alfa could be applied as an alternative treatment in children with CML, who have not a donor for allogenic BMT (bone marrow transplantation). PMID- 10731942 TI - [The analysis of failures in therapy of osteosarcoma in children treated according to SFOP-94 protocol in the studies of Polish pediatric solid tumors treatment group]. AB - Between December 1989 and April 1998 twenty eight children aged from 5 to 20 years (18 female and 10 male) suffering from osteosarcoma were treated according to the OS-SFOP-94 protocol. Twenty four patients presented with localized tumor of extremities and four with pulmonary metastases. The majority of primary tumors exceeded 150 ml of volume. The primary preoperative chemotherapy consisted of adriamycin (70 mg/m2 every four weeks) and high-dose methotrexate (12 g/m2 every week). In 20 patients limb-salvage surgery was applied, in three children- amputation and in one child tibia resection with genu arthrodesis was applied. Five of 28 patients died, one because of treatment related infection, 2 non responders with metastatic osteosarcoma due to progressive disease, and one because of local relapse with pulmonary metastasis non-responding to therapy, one because of treatment refusal. Twenty one from 25 children are alive from 5 to 51 months. Event frae survival of children with localized disease calculated according to Kaplan-Meier analysis was 64.17% in the 51st month. The main cause of failure in the treatment of osteosarcoma in children is primary and secondary progression of disease. The toleration and results of treatment for osteosarcoma in children according to the OS-SFOP-94 is satisfactory. PMID- 10731943 TI - [How to improve the results of treatment of myelodysplastic syndromes in the studies of Polish pediatric leukemia/lymphoma group]. AB - Fourty two children with myelodysplastic syndrome (MDS) treated in seven centres of The Polish Paediatric Leukaemia/Lymphoma Study Group in period 1975-1998 were included in the study. In 16 children RAEB-T, in 3 CMML, in 10 RA and in 13 RAEB were diagnosed. BMT is the best therapy for children with MDS. For children, who have not a donor for BMT, Roacutan therapy seems to be the most effective. PMID- 10731944 TI - [Results of the treatment for infant acute lymphoblastic leukemia treated in the program Dana-Faber Cancer Institute. Report of Polish Pediatric Study Group for Leukemia and Lymphoma]. AB - We present courses and treatment's results in 8 infants with acute lymphoblastic leukaemia (ALL) treated according Dana-Faber Cancer Institute protocol between 1994-1998 year. Complete remission (RC) was achieved in 8 children. Relapses were diagnosed between 6 to 20 month, only one achieved II RC. Four children are alive: in I RC-2, in II RC-1, in partial remission 1 and the treatment was ended in one. Treatment's results in infants with ALL are still unsatisfactory. PMID- 10731945 TI - [The efficacy of G-CSF and GM-CSF in the adjunctive treatment of infections complicating chemotherapy of acute leukemia in children]. AB - Growth factors (G-CSF Neupogen Roche i GM-CSF Leucomax Sandoz) have been applied in 133 therapeutic and prophylactic cycles in 88 children with acute leukaemias. GM-CSF and G-CSF were administered subcutaneously or intravenously at a dose of 2 to 8 micrograms/kg for 2 to 28 days. 45 prophylactic cycles had been administered in children with acute lymphoblastic leukaemia in high risk group and in relapses, which caused significant reduction in the number of infections, time of neutropenia and fever. Therapeutic cytokines cycles were applied when the absolute neutrophil count have fallen below 0.5 x 109/l. We observed significant reduction in duration of neutropenia in these cycles. Tolerance of GM-CSF and G CSF was good. Side effects were not observed. PMID- 10731946 TI - [The efficacy of G-CSF and GM-CSF in the adjunctive treatment of infections complicating chemotherapy of solid tumors in children. Report of Polish Pediatric Leukemia/Lymphoma Treatment Group]. AB - Total number of 306 cycles of GM-CSF-Leucomax Sandoz (5 mg/kg/day s.c.) and/or G CSF Filgrastim Hoffmann-La Roche (5-10 mg/kg/day s.c.) was applied in 146 children aged from 0.5-18 years during neutropenia associated with chemotherapy of solid tumours. Seventeen children with malignancies served as a historical control group. Our study have demonstrated after both G- and GM-CSF therapy shorter period of neutropenia, reduction of the number of febrile days and a decreased frequency of infectious complications and infection's duration. PMID- 10731947 TI - [The effectiveness of G-CSF and GM-CSF in the adjunctive treatment of infections complicating chemotherapy of non-Hodgkin's lymphoma in children. Report of Polish Pediatric Leukemia/Lymphoma Treatment Group]. AB - Total number of 252 cycles of GM-CSF-Leucomax Sandoz (5 mg/kg/day s.c.) and/or G CSF Filgrastim Hoffmann-La Roche (5-10 mg/kg/day s.c.) was applied in 124 children aged from 0.5-20 years during neutropenia associated with chemotherapy of non-Hodgkin's lymphoma (NHL). Twenty four children with NHL treated according to the same chemotherapy protocol but without G-CSF and GM-CSF served as a control group. Our study have demonstrated the good efficacy of both G-CSF and GM CSF therapy. They shortened the period of neutropenia, reduced the number of febrile days, infection's duration and decreased the frequency of infectious complications. PMID- 10731948 TI - [The role of local surgical and radiological control in the treatment of soft tissue sarcoma sensitive to chemotherapy in children. Report of Polish Pediatric Solid Tumors Treatment Group]. AB - In this paper the role local surgical and radiological control in the treatment of soft tissue sarcomas in children was analyzed. All children were treated according to CWS-91 and SIOP-IV protocols. Eighty three children with RMS A + E, EES/PNET, SS, UDS were included in the analysis. The primary surgery consisted of R0 (5%), R1 (18%) or R2 (16%) resection. In majority of cases (61%) primary surgical intervention was limited to diagnostic biopsy. Conventional or hyperfractionated radiotherapy was performed in 42.8%, 73.8% and 75% of children with disease stage II, III and IV, respectively. Delayed surgery was performed in 20 out of 53 (37.7%) children with stage III of the disease. In 5 patients without primary focus (urinary bladder in 3 and prostate in 2 cases) removed, progression of the disease occurred. In 5 children (stage IV) with progression of the disease no secondary surgery was performed. In 4 of them the primary tumor exceeded 10 cm in diameter. No delayed surgery was performed in 69% of relapsed children with stage III of the disease. Planned radiation therapy was not performed in 15.9% of cases. Primary local surgical control of primary tumor is of great importance for remission duration. In children who underwent delayed surgery the estimated EFS was of 0.7, in comparison with 0.5 EFS of those without secondary surgical treatment. PMID- 10731949 TI - [Clinical manifestation of toxic pancreas lesion in children with hematopoietic malignancies]. AB - From January 1994 until May 1997, 54 children with leukemia and non Hodgkin lymphoma were analyzed. The enzymatic function and ultrasound examination of pancreas were estimated. In 17 of 54 patients the clinical symptoms suggesting pancreatitis or toxic lesion of pancreas were observed. In 13 cases L asparaginase was administered. The main symptom of the pancreas disease was severe abdominal pain with vomiting. The typical ultrasound view of pancreatitis was observed in 4 cases, pancreas oedema was seen in 6 patients. The most serious course of pancreatitis was diagnosed in 3 children. Diabetes mellitus coexisted in two cases, in the third case osteoporosis was seen. Because of the toxic pancreas lesion in one patient the administration of L-asparaginase and cortical hormones was discontinued, in the remaining 2 children the therapeutic scheme was modified. In all 17 cases this side effect was completely reversible, as well as in 3 children with the most serious clinical course of pancreas lesions. PMID- 10731950 TI - [Magnetic resonance imaging: more sensitive method in the detection of leukemic central nervous system infiltration]. AB - PURPOSE: To define the ability of magnetic resonance imaging (MRI) in identifying leukemic infiltration of central nervous system (CNS) in children. METHODS: The MRI of CNS was carried out in 54 leukemic children (mean age 8.9 years). The results were compared with CSF cytology and neurological examinations. RESULTS: Leukemic involvement of CNS (1 intracerebral infiltration, 5 meningeal intracranial infiltration, and 5 cases of intraspinal infiltration) was found in 11 children (20.4%)--6 of them were before treatment. Blasts in CSF were found only in 3 examined children. CONCLUSIONS: The results of the study implicate the importance of routine MRI screening in leukemic children. We found especially important to perform the first examination before the onset of treatment because therapy may cause very quick regression of leukemic infiltration in CNS. PMID- 10731951 TI - [DNA index determined by flow cytometry in children with acute lymphoblastic leukemia as an additional diagnostic and prognostic element]. AB - The aim of the study was to establish the frequency of aneuploidy in bone marrow and peripheral blood cells in children with acute lymphoblastic leukemia (ALL) and to determine DNA index (DI), as an additional prognostic factor. The DNA content of cells was determined by flow cytometry using propidium iodide staining. The bone marrow and peripheral blood of 22 children were examined at the time of diagnosis. The diploid cell population (DI = 1) was found in 10 children (45%). Hiperdiploidy was found in 12(55%) children. DI > or = 1.16 was found in 7 children (32%) and 1 < DI < 1.6 in 5 children (23%). In one patient with DI > 1.16 two hiperdiploidal cell populations were found. In another one hipodiploidy with a hiperdiploidal cell populations coexisted. DI was compared with other prognostic factors--initial leukocyte count, immunophenotype, reaction to the treatment. PMID- 10731952 TI - [Carbonyl groups content on the basis of protein peroxidation analysis with total antioxidant status in blood of children with cancers]. AB - Toxic oxygen free radicals have been implicated as important pathologic mediators in carcinogenesis. Several reports have found antioxidants and enzymes related to the antioxidants function at increased or decreased levels in blood of patients with cancers. In previous publications we observed that antioxidant barrier can be different in healthy children and children with cancers. This time we were looking for a stable marker of damaging effect of free radicals reactions in association with antioxidant barrier in blood. Carbonyl group level in proteins has been introduced as a good marker of oxidative stress damage. For this study we selected 60 children at the age of 3 to 16, who had been diagnosed as suffering from cancers: malignant (m) bone tumors (t) (n = 25), m. brain t. (n = 20), lymphoma malignum (n = 5), m. liver t. (n = 5) and m. germ cell t. (n = 5). The control group consisted of 40 age-matched healthy children (22 boys and 18 girls). We investigated the concentration of carbonyl groups (CG) in serum spectrofotometrically, according to the method of Levine. Plasma total antioxidant status (TAS) was estimated colorometrically with radical cation ABTS (2,2'-Azino di-[3-ethylbenzthiazoline sulphonatel]), erythrocyte glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities were assessed according to the method of Paglia & Valentine and Minami & Yoshikawa respectively (Kits of Randox Laboratories Ltd.). In this study the content of CG in plasma proteins has shown a significant increase (1.60 +/- 0.77 vs 0.78 +/- 0.15 nmol/mg protein, p < 0.001) and erythrocyte GSH-Px activity has shown a significant decrease (16.3 +/- 7.9 vs 25.1 +/- 15.8 U/gHb, p < 0.02) in children with malignant tumors. No differences were observed in SOD activity and TAS between sick and healthy children. A possible interpretation of this data suggests an inadequate antioxidants' protection in children with cancers. The relationship between the oxidative damage and carcinogenesis requires further investigations. PMID- 10731953 TI - [Non-invasive evaluation of cardiovascular system in patients after 3 to 5 years since the completion of acute lymphoblastic leukemia treatment]. AB - In 34 patients, 25 males and 9 females, aged 12-24 years (mean 15.9) 3-5 years after the completion of acute lymphoblastic leukemia treatment the non-invasive evaluation of cardiovascular system was done with the use of electrocardiography, echocardiography and treadmill stress test. The cumulative anthracycline doses varied from 149 to 417 mg/m2, mean 250 mg/m2. The control group consisted of 28 healthy volunteers, 15 males and 13 females aged 9-25 years (mean 17.0). The history, physical examination and non-invasive tests' results did not revealed the cardiomyopathy signs, left ventricle contractility in the study and control groups did not differ significantly. Treadmill stress test showed lower maximal heart rate achieved and deeper ST segment depression in the study group. We conclude that the patients after leukemia treatment need further careful investigations. PMID- 10731954 TI - [BCL-2 antigen expression in blood cells in children suffering from leukemia]. AB - It was established that high level of BCL-2 antigen inhibited suicidal cell death and prevented apoptosis induced by antitumor drugs. Due to the fact that evaluation of BCL-2 expression in bone marrow and peripheral blood cells could be an important prognostic factor in patients with malignant hematopoietic diseases, we decided to study the number of BCL-2+ nuclear cells and assess this antigen expression in the cells. Using monoclonal FITC-conjugated antibody, antigen BCL-2 was found in peripheral blood cells in children with both lymphoblastic (ALL) and non-lymphoblastic leukemia (NLL). At the same time the percentage of PCNA+ blood nuclear cells was investigated. The cells were analysed with a flow cytometer. The study carried out in a group of 12 ill and 7 healthy children showed significantly increased percentage of lymphocytes and neutrophiles PCNA+ and BCL 2+ in ALL, in comparison with a healthy control group. Exclusively an increased percentage of BCL-2+ lymphocytes and neutrophiles was observed in NLL patients. Due to a relatively small group of investigated patients we can not make a certain conclusion but preliminary analysis suggests a correlation between a high number of BCL-2+ cells and a long time without remission. PMID- 10731955 TI - [Myelogenous system cells' activity in the bone marrow in children with acute leukemia remission]. AB - Myelogenic system cells' activity was evaluated in 12 bone marrow samples taken from the children in acute leukemia's remission period and 7 samples from the patients in whom malignant disease of the hematopoietic system was ruled out. Cell activity was evaluated with the nFMLP induced chemiluminiscence test (CL) in the luminol environment. In the bone marrow of the patients in the period of acute leukemia remission the cells' chemiluminiscence was, considerably higher than in the control material. It appears that in the acute leukemia's remission period a functional regeneration of the myeloid line cells occurs. PMID- 10731956 TI - [The assessment of hepatic enzymes' levels in children treated for leukemia and non-Hodgkin's lymphomas]. AB - The purpose of this study was to determine the frequent of serum aminotransferase elevation in children with leukemias and non-Hodgkin's lymphomas and define the cause of this pathology. In the serum of 43 children the bilirubin concentration, activities of aspartic aminotransferase (AspAT), alanine aminotransferase (AlAT) and gammaglutamylotranspeptidase (GGTP) were measured before treatment, during and after intensive chemotherapy. 43 patients 8 (65%) had bilirubin concentration above 1.2 mg/dl and/or aminotransferase activities above 100 U/l. The most possible causes of the liver damage in the patients were: hepatotoxicity of chemotherapy, virus or bacterial infections and leukemic or lymphomatous involvement of the liver. PMID- 10731957 TI - [Problems of phenotypic diagnosis of acute leukemia in children]. AB - The classification of acute leukemias based on the phenotype of leukemic cells was shown. The technical problems and difficulties in interpretation were discussed including atypical phenotypes, biphenotypic and mixed lineage forms of leukemias. PMID- 10731958 TI - [The evaluation of peripheral blood lymphoid cells in children in remission a year after the cessation of treatment for acute lymphoblastic leukemia and non Hodgkin's lymphoma type non-B]. AB - Immunophenotype of peripheral blood lymphocytes was studied in 10 children after the cessation of treatment acute lymphoblastic leukemia and in 3 after non Hodgkin's lymphoma non-B. They all were in complete remission. Peripheral blood lymphocytes cells were analysed by flow cytometric analysis. Estimate surface antigens were estimated: CD3, CD19, CD4, CD8, CD3-16+ 56+. The results were compared with data obtained in children not treated because of neoplastic diseases. Our results showed that the absolute number of lymphocytes T(CD3+), TS (CD8+) and NK cells were significantly increased in the study group. PMID- 10731959 TI - [Renal neoplasms in children with tuberous sclerosis]. AB - Two cases of 14 and 11 years old children with tuberous sclerosis were admitted to Department of Paediatric Surgery in Wroclaw and Department of Paediatric Hematology and Oncology in Lublin because of renal tumors. Radical nephrectomy was performed in both cases. Histopathological diagnosis revealed malignant rhabdoid tumor (MRT) in a 14 years old boy and benign angiomyolipoma in the latter case. The authors stress the rarity of MRT in older children and successful treatment due to nephrectomy and maintenance chemotherapy. PMID- 10731960 TI - [Results of treatment for angiosarcoma in children hospitalized in Department of Pediatric Hematology and Oncology (Wroclaw University School of Medicine) in 1989 1998]. AB - Angiosarcoma is a neoplasm included to sarcoma of soft tissue. In the period 1989 1998 9 children with angiosarcoma were treated in Department of Paediatric Hematology and Oncology. PMID- 10731961 TI - [Atypical sarcomas localized within a head and neck in children]. AB - Seven children treated surgically because of non-rhabdomyosarcomatous soft tissue sarcoma (NRSTS) localized extrameningeally on the head and neck were presented. Three of the patients were operated on haemangiopericytoma, two--fibrosarcoma, one child--neurofibrosarcoma and one--liposarcoma. The pre- and postoperative TNM classification was employed as a staging system. The surgical resectability--R was used to establish tumour margins. Four patients (two with fibrosarcoma and two with haemangiopericytoma) survived free of disease. The influence of the complete surgical resection on the outcome of the head and neck extrameningeal NRSTS was proved. PMID- 10731962 TI - [Malignant tumors of mandible in children]. AB - Primary bone tumors represent about 7% of paediatric malignancies. Osteosarcoma and Ewing's tumor are the most frequent ones, however they are rare in facial bones. Mandibular localization is slightly more frequent and of better prognosis than maxillary one. Until 1995 there were only about 70 cases reported in the medical literature, mainly in the oncological or dental periodics. Our material consists of two children with Ewing's tumor of the mandible and one patient with osteosarcoma. The diagnosis was based on histopathological or cytological studies. The combined treatment--chemotherapy and radiotherapy--was performed in two patients with Ewing's tumor. The recommended resection of the mandible including the tumor mass has not been performed. No facial asymmetry is seen after termination of the radiotherapy. The boy with osteosarcoma underwent primary mandibular partial resection; a two-year chemotherapy was introduced only when metastases in the regional lymph nodes occurred (BLM, CTX, ACT-D, ADM, CDDP). The mandible was reconstructed surgically in 5 years after termination of radiotherapy and the anatomical relationship in the masticatory organ was restored. All children are now in good condition under our long-term observation. We present these cases of mandibular tumors regarding their rare occurrence and positive results of the introduced treatment. PMID- 10731963 TI - [The treatment of central nervous system neoplasms]. AB - From August 1993 to March 1998 30 children (13 females and 17 males) at the age of 1-14 with central nervous system tumours were treated in Paediatric Hematology and Oncology Hospital in Wroclaw. In 11 patients we diagnosed medulloblastoma, in 4 ependymoma, 6 astrocytoma, 2 oligodendroglioma and in 7 other types of tumours. The histological diagnosis of 2 cases with tumours localized in the brain trunk was not available. All patients were directed to the chemotherapy after the surgery. Complete resection was achieved in 13 out of 30 patients. The chemotherapy was proceeded according to SIOP regimen. Children older than 3 years received additional radiotherapy. The effectiveness of treatment was controlled by constant neurological examinations with computed tomography and ultrasonography. The rate of failures was 10 patients with local recurrences and 1 patient with drug toxicity. 17 children remain still in observation--among them 5 are treated with chemotherapy and 2 present the progression of disease. PMID- 10731964 TI - [The surgical treatment of advanced stages of solid tumors in children]. AB - Hope for cure in children with advanced cancer came with introduction of chemo- and radiotherapy, however surgery is still important as a part of the multidirectional treatment. The aim of the review was to assess the impact of surgical treatment in children with advanced cancer. From 1991 to 1997, 30 patients aged from 6 months to 17 years were treated for soft tissue sarcomas (STS: stage III/8 pts, stage IV/2 pts), nephroblastoma (WT: stage IV/5 pts, stage V/3 pts), PNET/Ewing sarcoma (locally advanced/4 pts, metastatic/2 pts) and others (stage III/4 pts, stage IV/2 pts). All patients received pre- and postoperative chemotherapy, all but 6 were irradiated. Twenty one of 30 patients entered remission (CR) after radical surgery for local control: 12 relapsed locally, of whom 7 entered IICR after re-treatment and next surgery and 5 died. 3 of 9 patients who never had any local relapse, died of metastases. Nine of 30 patients never had any radical surgery, 8 died (including 2 toxic deaths) and 1 (stage V Wilms tumour) is in CR after chemotherapy and radiotherapy (12 Gy including both kidney with unresectable tumours). Advanced cancer does not imply the fatal outcome: 14/30 patients are in CR. Possibility of surgical resection of the disease focuses brings some hope for final cure even in initially disseminated disease: 13 such cases of 21 are in ICR or IICR (follow-up: 6 months 7 years) whereas only 1 of 9 those who have never had any radical surgery. PMID- 10731965 TI - [The factors influencing the effectiveness of treatment for brain tumors in children]. AB - In this paper we analysed the group of 110 children (60 boys and 50 girls) that between 1982-1992 has been operated and some of them treated multidisciplinary because of a brain tumor. The disease was mainly recognised in 3-7 year old children (49). The tumor was situated in the vermis cerebelli and in the IV ventricle (31), in the hemispherium cerebelli (20), in the region of the III ventrical (18), in the hemispherium cerebri (16), in the pons and medulla oblongata (11), and in the sellar region (11). In the histopathological report in most cases we recognised astrocytoma (32), medulloblastma (18), ependymoma (12) and craniopharyngiona (6). The tumor tissue was not taken for the histopathological examination because of a very dangerous approach in 34 patients. The malignancy state was low in 51 and high in 25 cases. The implantation of the shunt was carried on in 86 children. In 77 cases the resection of the tumor was done: total (9), subtotal (39), partial (24) and biopsy (5). The post operative radiotherapy was used in 71 and chemotherapy (Bloom's scheme) in 43 patients. According to treatment results (Karnofsky's scale) and analysis of survival curves (Log-Rank test) the most important influence in the final outcome have: malignancy, histological structure and localisation of a tumor, extent of resection and radiotherapy. PMID- 10731966 TI - [Ultrasonographic evaluation of thyroid in patients cured from Hodgkin disease]. AB - Long-term prognosis in patients cured from Hodgkin's disease (HD), diagnosed in childhood has dramatically improved. This is why, early detection and elimination of treatment side-effects has become essential. The study aimed at thyroid morphology evaluation in 46 patients remaining in complete remission more than five years after completion of treatment for HD. Thyroid morphology was assessed by palpation and ultrasonography. Control group consisted of 43 age- and sex matched healthy people. 17.4% of patients after HD treatment had abnormal thyroid morphology on physical examination (gland enlargement, increased consistency or presence of nodules). Thyroid volume in this group was significantly decreased comparing to control group (p < 0.005). 32.6% of patients had heterogenic echogenicity. Ultrasonography revealed pathological lesions in 21.7% of examined individuals. Single hypoechogenic lesions in 5 patients, multiple hypoechogenic in 4 patients and multiple hypo- and hyperechogenic in 1 existed. High frequency of pathological thyroid lesions in thyroid of patients cured from HD emphasizes the need for systematic ultrasonographic thyroid evaluation. PMID- 10731967 TI - [The evaluation of neoplasm markers in the diagnosis and treatment of germ cell tumors in children]. AB - In the Department of Paediatric Hematology and Oncology (University School of Medicine in Wroclaw) 25 patients, 1 to 19 years old, 8 boys and 17 girls, suffering from germ cell tumors were treated from June 1989 to August 1998. In the course of the treatment both surgery, chemotherapy and radiotherapy were used. The level of oncological markers: alpha-fetoprotein (AFP), lactic dehydrogenase (LDH), carcinoembryonal antigen (CEA), beta-choriongonadotropin (beta-HCG) and CA 125 was examined at the beginning, during the therapy, and at the end of the treatment. Increased levels of oncological markers were found: AFP in 17 children, LDH in 10 children, CEA in 3 children, beta-HCG in 3 children and CA 125 in 2 children. In all cases the level of AFP was decreasing during the therapy. Normal levels of AFP and LDH were observed in children with complete remission. The use of AFP in diagnostics and monitoring of germ cell tumors was proved. PMID- 10731968 TI - [Significance of immunological system examinations in the clinical course of Langerhans cell histiocytosis in children]. AB - The Langerhans cell histiocytosis (LCH) is an enigmatic disease, usually occurring in the young. Its etiology is unknown, its pathogenesis is not correctly understood and the clinical course is unpredictable. In the years from 1975 to 1998 in Department of Paediatric Haematology and Oncology (Wroclaw University School of Medicine) 32 children with diagnosis of the Langerhans Cell Histiocytosis (LCH) were treated. The study group included 19 boys and 13 girls, aged from 3 weeks to 14 years, median age 7.5 years. Investigations of immunological system were performed in the examined group. In all children lymphocytes' subsets were examined. We examined apoptosis with ELISA test in 3 patients, in 3--marked antigen CD44, in 12 marked sVCAM-1 by ELISA method. IL-1 in growing cell supernatants was determined in 17 children. In 15 children lower IL-1 production was observed before the beginning of therapy in comparison with control group. In all patients decreased concentrations of lymphocytes T were noted, but in 8 children lower CD4/CD8 rate was observed. In the study group higher expression of CD44 was noted. In 3 patients cell apoptosis was normal. In 12 patients lower expression of sVCAM-1 was observed. Abnormal expression of adhesion molecules may influence the pathogenesis of LCH. Disorders of immunological system were observed in the patients with LCH. PMID- 10731969 TI - [The role of leukemic cell apoptosis and adhesive molecule sICAM-1 in the clinical evolution of acute lymphoblastic leukemia in children]. AB - Total number of 160 children with ALL, 98 boys and 62 girls, aged from 0.5 to 18 years was included in the study. Apoptotic cells death acc. to annexin V and ICAM 1 levels in serum and cell culture supernatants according to conventional antibody sandwich ELISA Genzyme assay were studied. Blood samples were drawn from children at different stages of their disease: at the time of diagnosis, during intensive therapy (induction, consolidation), during maintenance therapy and after completing the treatment. Thirty seven healthy children served as the control group. ICAM-1 before therapy was higher than that obtained in control group of healthy children (5.619 v 2.53 g/l). ICAM-1 decrease (3.228) after starting of chemotherapy of ALL was noticed. It was found that in children with ALL during the whole period of therapy the ICAM-1 serum levels were significantly lower than that observed in the control group of healthy children (p < 0.005). After cessation of the therapy ICAM-1 grew up (6.27 g/l). Therapeutics that modulate the regulation of apoptosis and/or expression of sICAM and cytokines production provide a new opportunity for the treatment of childhood ALL. PMID- 10731970 TI - [Amifostine (ethyol) in the adjunctive treatment of children with acute lymphoblastic leukemia]. AB - Myelosuppression is one of the most common side effects during chemotherapy in children with leukemia and lymphoma. That is why the protection of patients against the acute and chronic toxicity of antineoplastic therapy has become a major concern of oncology centers. Amifostine (Ethyol--Schering-Plough) represents a new adjunct for the management of cancer patients receiving chemotherapy. It has the ability to protect selectively a range of tissues and bone marrow against acute and cumulative toxicity of chemotherapy. Because solid tumors tend to be hypovascular and more acid than normal tissue, amifostine may protect selectively normal tissue. Amifostine is believed to scavenge free radicals, repair radicals on essential molecules and from mixed disulfides to protect normal cells. In this trial we demonstrate how amifostine protects granulocytes, erythrocytes and platelets against toxicity of chemotherapy. PMID- 10731971 TI - [Ethyol (amifostine): application trial in pediatric oncology]. AB - Myelotoxicity is one of the major chemotherapeutic side effects. In some adult and paediatric studies it has been shown that amifostine protects bone marrow from toxic effects of alkylating agents and platinum compounds without reduction in overall cytotoxic action. AIM OF THE STUDY: To test an efficacy of amifostine as a myeloprotectant in multiagent chemotherapy containing alkylating agents or platinum analogues. Amifostine was used in 8 children from 3 to 15 yrs of age treated with chemotherapy (CHT) for cancers. It made total number of 28 courses. Amifostine was administered every other CHT course in a dose of 750 mg/m2. The degree of myelotoxicity measured at nadir was compared between with- and without amifostine CHT courses. Anaemia, leucopenia and thrombocytopenia of WHO grades I and II were qualified as mild toxicity while grades III and IV were recognized as severe. RESULTS: Severe anaemia, leucopenia and thrombocytopenia were found after 3/14 (21%), 8/14 (57%) and 6/14 (43%) courses with amifostine. Proportion of these side effects in identical CHT courses without amifostine in the same pts. was as followed: 1/14 (7%), 7/14 (50%) and 9/14 (64%). Differences among both groups were statistically significant (p = 0.025). Mild side effects (nausea, vomiting, transient hypotension) accompanied amifostine administration in 29% of courses (4/14). CONCLUSION: Preliminary results suggest that amifostine decreased the number of severe thrombocytopenias after CHT. The drug was well tolerated by children. PMID- 10731972 TI - [Cytoprotective effect of amifostine in children during induction therapy according to BFM-83: report on cases]. AB - Amifostine is the agent of proved cytoprotective activity against alkylating drugs and rubidomycine. Its protective effect against other cytotoxic drugs is doubtful. BFM-83 induction therapy for ANLL (ARA-C + RUB + VP-16) which is applied to children with acute non-lymphoblastic leukemia (ANLL) commonly contributes to severe adverse reactions. We administered amifostine to three children: 2 boys with ANLL (7 and 11 yrs) and 1 girl with MDS (3 yrs) during etoposide and rubidomycine induction therapy in order to decrease chemotherapy related adverse reactions. Doses of amifostine were 740 mg/m2, 910 mg/m2 and 910 mg/m2 respectively. Efficacy of the therapy was evaluated on the base of blast decline in the bone marrow, efficacy of the cytoprotection by myelo and nephrotoxicity symptoms analysis. Chemotherapy-related adverse effects in the children protected by amifostine were less severe and observed by the shorter periods as compared with the historical control group of 20 patients treated according to BFM-83 without cytoprotection. These cases show the potential beneficial effect of amifostine during BFM-83 induction therapy for ANLL. The further randomised clinical study of the proposed cytoprotection should be performed to establish its value. PMID- 10731973 TI - [Coagulation disorders during treatment with l-asparaginase preparations]. AB - Coagulation disturbances are noticed, during ALL treatment with L-asparaginase, carrying risk of clotting complications. We examined 38 children with ALL (20 boys and 18 girls) aged 2-16 y., treated in 1996-1997 y. according to BFM and New York programmes. They received L-asparaginase of 10,000 and 25,000 U/m2 per dose at the beginning of induction therapy. The therapy started with E.coli L asparaginase; in 16 cases the drug was changed to Erwinase. Decreasing of fibrinogen, antithrombin III concentration and prothrombin time was noticed. Infectious complications were established in 8 and clotting problems in 3 children. Substitution with antithrombin III was introduced in 15, with fibrinogen in 17 children because of low plasma concentration. In 21 patients treatment modifications according to decreasing of clotting factors concentration were done. Clotting problems strongly influence the treatment of children with ALL. Substitution therapy may improve the effectiveness of therapy. PMID- 10731974 TI - [New possibilities of treatment with PEG-L-asparaginase in patients with acute lymphoblastic leukemia sensitized to l-asparaginase E.coli and erwinase]. AB - L-asparaginase is widely used in the treatment of acute lymphoblastic leukemia in children and adults. Use of L-aspa E. Coli as well as Erwinase is not possible in all cases because of the side effects, mainly allergic reactions and disfunction of pancreas. Recently, the new form of the enzyme PEG-L-asparaginase was introduced. Binding L-asparaginase E. coli to polyethylene glycol a decreased its toxicity, extended its plasma half-live, not significantly affecting the efficacy. The aim of the study was to examine the results of PEG-L-asparaginase administration in five children with acute lymphoblastic leukemia, and the symptoms of intolerance to L-aspa E. Coli or Erwinase. There were three children with newly diagnosed ALL and two children with first relapse of ALL, treated according to New York Protocol and BFM-90 Protocol for ALL relapses respectively. PEG-L-asparaginase (Oncaspar) was administered in the dose of 2500 IU/m2. According to the protocol four children received 11 courses of treatment with the full dose of the drug. The number of doses for individual patient varied from one to six. The short-lived nettlerash was observed in one patient during two subsequent infusions of the drug. Hydrocortisone and antihistamine drugs were administered. Treatment with PEG-asparaginase was discontinued in one child, who developed dyspnea, nausea, vomiting and face rash during the third dose of the drug. Oncaspar is the valuable drug, which enabled continuation of treatment according to protocol in four out of five children with bad tolerance to routinely used L-asparaginase preparations. PMID- 10731975 TI - [The use of hematopoietic growth factors G-CSF/GM-CSF in the treatment of neutropenia in children with acute lymphoblastic leukemia and non-Hodgkin's lymphomas]. AB - Maximal intensification of antineoplastic therapy is currently a predominant trend in the treatment regimens for acute leukemias and lymphomas. However, by such approach myelosuppression and counteracting its sequelae become paramount problems. Hematopoietic growth factors G-CSF/GM-CSF play a great role in this aspect of the therapy. Effects of 35 courses of G-CSF/GM-CSF were evaluated in 19 children with ALL and NHL and compared with 21 episodes of neutropenia in 15 historical controls. In the treatment group time of neutropenia was approx. 3 times shorter as compared with a control group. Fever accompanying neutropenia occurred less frequently and lasted shorter in the treatment group. Also, symptoms of infection subsided faster. Subjective life quality was better in children receiving growth factors. PMID- 10731976 TI - [The assessment of the treatment of viral hepatitis C with interferon alpha in children with leukemias and lymphomas]. AB - Interferon alfa in dose 3-6 MU IM was administered during six months 3 times per week in 30 children with chronic hepatitis C with leukaemias and lymphomas. Chronic hepatitis was diagnosed basing on anti-HCV, RNA HCV by PCR, high activity of ALT and histopathologic examination of the liver. The patients were followed up for 12 months after IFN therapy. Sustained normalization of ALT levels, together with serum HCV RNA elimination was considered as a complete response and achieved in 33% children. Partial response (ALT normalization without serum HCV RNA elimination) was recorded in 57% patients. Anti HCV antibody was detected in all children during the time of observation. PMID- 10731977 TI - [Central venous lines in children with cancers]. AB - Authors analyzed clinical aspects of central venous lines in children with cancer. In 25 patients central venous catheters with subcutaneous ports and in 34 patients lines with external ending were inserted. Catheters were left in place respectively 62-836 days and 4-365 days. During that time 10 catheters were removed due to occlusion, leakage, local infection or sepsis. The causes of these complications were analyzed in discussion. PMID- 10731978 TI - [Long-term venous access "PORT": own experience]. AB - In the study we analyzed the methods and results of implantation of 100 ports. Children age ranged between 0.5 and 16 years. All children received chemotherapy during neoplasm treatment. In conclusion, ultrasound detector, radiological control and subclavian venous access are safe and less traumatic than external jugular vein. PMID- 10731979 TI - [Early and late complications of the use of life-cath type catheters in children with malignancies requiring urgent surgical intervention]. AB - Permanent access to the centrally positioned veins is necessary for the treatment of children with malignancy. The Broviac-Hickman's catheters and venous port system fulfill this require. Since January 1992 to February 1998, 276 intravenous life-cath devices were installed. In 7 patients life-threatening complications after catheter implantation occurred, which demanded urgent surgical intervention. The complications appeared in both early and late periods after catheter insertion. Pneumothorax (three children), bleeding to the mediastinum (one patient), fragmentation, disconnection and migration of a separate part of catheter into the right heart (two patients) were observed. The way of treatment and its results were described. PMID- 10731980 TI - [Alternative methods of central venous access insertion in pediatric oncology]. AB - Introduction of the central venous access (CVA) in children treated for malignancies is usually planned procedure. Some patients however require the CVA in emergency. Also in emergency, insertion of CVA should be feasible and safe. The standard technique of CVA insertion is through surgically approached internal jugular vein. This technique requires correct blood coagulation and general anaesthesia in younger patients. Alternative ways of the CVA insertion (via cephalic vein, cubital vein or external jugular vein) seem safe also in case of thrombocytopenia (< 40,000/mm3) and coagulopathy. Aim of the report is to compare efficacy (central tip of the catheter in the superior vena cava on X-ray) and safety (complication rate) of both techniques. PATIENTS: 166 children treated for malignancies aged from 0 to 16 years. The standard technique was used in 109 and in 107 was successful. The alternative technique was used in 57 and in 50 was successful. There were no acute complications, the late ones were observed in 7 of 107 in the standard technique group and in 9 of 50 in the alternative technique group. The alternative ways of the CVA introduction are effective and safe, however the longer X-ray monitoring is necessary. PMID- 10731981 TI - [Right atrial thrombus complicating chemotherapy by central venous catheterization in a child with Hodgkin's disease]. AB - The atrial catheter (subcutaneous port) was used in a 5 year old boy with Hodgkin's disease for the administration of antineoplastic agents and other drugs. Catheter was flushed daily or every 2 weeks with heparin solution (5 U/ml)). The complications including infection and occlusion were not observed in this patient during observation time. Routine echocardiography performed after eight months of usage of the catheter showed right atrial thrombus. Because of the failure of 14 days fibrinolytic therapy (Actylise) surgical thrombectomy was performed. Bacteriological culture of the surgical specimen was positive for Pseudomonas aeruginosa. The post-operative period passed without complications. Follow-up echocardiography findings were normal. The probable reason of thrombus formation could be damage of the right atrium by stream of drugs or balloting ending of the catheter. PMID- 10731982 TI - [Recanalization of the ductus arteriosus in a child with non-Hodgkin's lymphoma]. AB - Dissertation describes the case of 7.5-year-old boy with recanalization of the ductus arteriosus. In this patient treated because of the non-Hodgkin lymphoma during the chemotherapy the respiratory failure occurred and mechanical ventilation was required. After the respiratorotherapy in the controlled echocardiography examination the patency of previously closed ductus arteriosus, was described. There was no description of the similar case in available references. PMID- 10731983 TI - [The closure of persistent ductus arteriosus using coils in a child with Wilm's tumor]. AB - In Pediatric Institute of Medical University of Gdansk in all children with neoplasmatic diseases the echo examination is performed, due to exclude coexisting abnormalities of cardiovascular system. During this kind of procedure in 4.5-year-old girl with Wilms tumour the persistent ductus arteriosus was described. Because of the chemotherapy design and its potential cardiotoxicity the closure of PDA using coils was performed immediately. The successful closure of PDA gave the chance to apply the whole therapeutic protocol. PMID- 10731984 TI - [Acute leukemia in children with Down syndrome: analysis of cases]. AB - An analysis of clinical and laboratory parameters and the results of treatment of 14 children with Down Syndrome and acute leukaemia was performed. The children were treated between 1986-1997. Their age ranged from 1 day to 13 years (average 5.5). There were 9 girls and 5 boys. Four of them had congenital heart disease. ALL was observed in 10, AML in 3 and TAM (Transient Abnormal Myelopoesis) in 1. Half of the children with ALL was classified as L1 according to FAB with the majority of common phenotypes and M6 in ANLL group. Remission was achieved in all ALL patients, six of them are still free of symptoms, the remaining four died of brain haemorrhage as a consequence of myelosuppression. Only 1 of 3 children with ANLL achieved remission. The child died of cardiac arrest after induction phase of BFM 95 programme (ADE). The 2 remaining children with ANLL also died of circulation failure before initiation of chemotherapy. The children had complicated cyanotic heart disease. The neonate with TAM is in clinical and hematological remission. In conclusion all children with ALL achieved hematological remission but tolerance of treatment was a problem. The majority of patients had diminished bone marrow reserve. Mortality was frequently related to circulatory failure in children with associated heart defects. It seems necessary to discuss the modification of accepted programmes for leukemia for the treatment of children with Down Syndrome. PMID- 10731985 TI - [Acute lymphoblastic leukemia in children with Fanconi anemia]. AB - Fanconi anaemia (FA) is a rare autosomal recessive disorder. Manifestation of the disease is pleomorphic and may include many congenital malformations and marrow aplasia. Congenital disorders include: skeletal abnormalities, hypo- or hyperpigmentation of the skin, renal or heart anomalies and many others. FA is an invariably fatal disease owing to progressive marrow aplasia or the development of acute leukaemia or squamous cell carcinoma. We present two children with Fanconi anaemia who developed acute lymphoblastic leukaemia in the 4 and 12 year of life. PMID- 10731986 TI - [Late effects of chemo- and radiotherapy in two children treated for acute lymphoblastic leukemia]. AB - The endocrinological disorders are known sequelae of cancer therapy. The aim of our study was to evaluate the effects of chemo- and radiotherapy on growth and auxiological parameters in children treated for acute lymphoblastic leukaemia (ALL) since 1984 to 1996. We studied growth, pubertal development and endocrine functions in 32 patients who were treated for ALL. Two of them had endocrine abnormalities. Both took a different course of disease and treatment because of local relapse in uterus (the girl) and in testis (the boy). All patients received cranial preventive radiotherapy but two of them also received: abdominal irradiation (the girl) and direct testicular irradiation (the boy) with total doses 24 Gy. The girl had clinical and biochemical symptoms of primary thyroid failure, growth hormone deficiency and hypergonadotropic hypogonadism recognized after 8 years following the completion of therapy. The boy had biochemical symptoms of hypogonadism with low serum testosterone concentration responses to stimulation with human gonadotropin after 4 years of remission. CONCLUSION: We suggest that clinical and biochemical assessment of endocrinological functions including growth, pubertal development, thyroid function should be performed at regular intervals following chemotherapy and radiotherapy in patients with acute lymphoblastic leukaemia and another carcinomas. PMID- 10731988 TI - [Sepsis caused by Pseudomonas aeruginosa as the complication of induction treatment of acute non-lymphoblastic leukemia in a 10-year old girl]. AB - We present a girl with acute non-lymphoblastic leukaemia (ANLL) who developed sepsis during the treatment of the disease. She was treated with BFM-83 protocol for ANLL. Blood cultured revealed the presence of Pseudomonas aeruginosa. This type of opportunistic infections are common, especially in patients with neutropenia. PMID- 10731987 TI - [The osteoarthralgic symptoms preceding acute lymphoblastic leukemia]. AB - Osteoarthralgia is a common symptom at the beginning of acute lymphoblastic leukaemia (ALL). It occurs in 40% of cases. Still arthritis that precedes haematological malignancies is very rare. However in every case of arthritis of unknown origin or drug resistance arthritis the bone marrow biopsy is required. We present 3 cases of osteoarthralgia that precedes ALL by 2.5-7 months. PMID- 10731989 TI - [Tumor lysis syndrome in the course of neoplastic disease of hematopoietic system in children]. AB - Tumor lysis syndrome is characterized by hyperuricemia, hyperkalemia and hyperphosphatemia followed by hypocalcemia. Sometimes they are accompanied by hyperglycemia. It can lead to acute renal insufficiency, neurological and cardiological disorders. In the Clinic of Paediatric Haematology and Chemotherapy in Zabrze, in the period from Jan.01, 1990 to Dec.31, 1997, 305 children were treated due to neoplastic diseases of haematopoietic system: 169 with ALL, 34- ANLL, 48--NHL and 54--HD. Tumor lysis syndrome was found in 11 children, what was 3.6% of all patients. They included 3 children with ALL/1 from group SRG and 2 with HRG/, 2 with ANLL, 6 with NHL/2 with NB-NHL and 4 with B-NHL/. Tumor lysis syndrome did not occur in any patient with HD. Two symptoms of tumor lysis syndrome were found in 13 children (4.26%) including 3 with ALL (1 SRG and 2 HRG), 2 with ANLL and 8 NHL/3-NB-NHL and 5-B-NHL/. Diabetes occurred in two children with ALL. Acute renal insufficiency occurred in 6 children with ALL, 2 with ANLL and 8 with NHL. Two patients with B-NHL required dialysis. One renal insufficiency treated with hemodialyses was the first symptom of lymphoma. To prevent the consequences of tumor lysis syndrome hydration with alkalization, constant monitoring of the above mentioned biochemical parameters are necessary. Acute renal insufficiency, not responding to conservative treatment, should be immediately treated with hemodialyses and chemotherapy should be introduced under its 'shield'. PMID- 10731990 TI - [Acute renal insufficiency as the first symptom of malignant lymphoma in children]. AB - Acute renal insufficiency in children with malignant lymphoma may be the first symptom of illness. Intensive care with hemodialysis is the therapy of choice. It allows to eliminate the increased volume of liquid, toxins, continuing diagnostic and beginning chemotherapy. We present two children with NHL, who showed acute renal insufficiency. Intensive care with hemodialysis was required to treat renal failure before establishing diagnosis and during initial chemotherapy. PMID- 10731991 TI - [Coexistence of Langerhans cell histiocytosis and Niemann-Pick disease in children: a case report]. AB - Langerhans cell histiocytosis (LCH) and Niemann-Pick disease are very rare diseases. Both LCH and Niemann-Pick disease are distinguished by complex etiology, pathogenesis and broad spectrum of clinical manifestations. In our study we presented case report of 2.5 year old girl in who the coexistence of both diseases was noted. General symptoms that led to diagnosis were: hepatosplenomegaly, cholestatic jaundice, cutaneous lesions and mental development retardation. PMID- 10731992 TI - [Astrocytoma in a 6-year-old girl after the treatment for acute lymphoblastic leukemia]. AB - In recent years we observe the increasing number of event-free survivals of patients with acute lymphoblastic leukaemia. It is achieved with multidrug chemotherapy regiment. The important part of protocols is prophylaxis e.g. with radiation therapy of CNS. It is followed by drug toxicity and side effects of treatment (e.g. secondary CNS tumours). In our hospital we observed the case of 6 year-old girl with CNS tumour-astrocytoma. According to the regimen for ALL the radiation therapy of CNS was performed. Three months after neurosurgical operation the local recurrence was observed. The patient died of the progression of disease. PMID- 10731993 TI - [Functional reconstruction of the masticatory organ after combined therapy in a 9 year-old boy with osteosarcoma of the mandible]. AB - Resection of the considerable part of the arch of the mandible disturbs breathing, swallowing, speaking and alters the facial symmetry. One-staged reconstruction of the mandible is contraindicated in patients with malignant tumor and serious prognosis. The course of the combined treatment in 9-year-old boy with osteosarcoma of the mandible is presented (May 1987--resection of the anterior part of the body of the mandible and suprahyoid lymphadenectomy); the most severe postoperative functional disorders were treated immediately (tracheostomy, nasogastric tube for 3 weeks). The reconstruction of the mandible and restoration of the anatomical relationship in the masticatory organ were performed after 5 years. Because of the metastatic disease in the nuchal and cervical lymph nodes boy underwent chemotherapy (Jan 5th 1988-Feb 21st 1990) of the primary site of the tumor 7 months after surgery. The following cytostatic drugs were administered; BLM, CTX, ACT-D, ADM, CDDP. The functional rehabilitation, small correctional surgery and improvement in perception in the oral cavity facilitated the restoration of important functions of the masticatory organ (proved by the following studies: gustometric, manometric, logopedic, stereognostic, rentgenotelevision of the swallowing process). In addition, the self-perception and the boy's social status improved significantly after favourable change in patient's appearance. PMID- 10731994 TI - [Cyst of lower mediastinum in a 14-year-old boy with tumor of mediastinum in the course of Hodgkin's disease]. AB - Bilateral enlarged lymph nodes in supraclavian regions, bilateral large tumour of mediastinum and enlarged lymph nodes of hiluses of the lungs were detected in a boy with stage III of Hodgkin's disease. After chemotherapy (MVPP/B-DOPA) and radiotherapy partial remission was achieved. However abnormal mass in the lower mediastinum on the right side of the heart was still present. Results of USG and CT studies showed solid character of this mass. MRI revealed a mediastinal cyst at the place of this mass. The boy has been in the first remission for eight months. PMID- 10731995 TI - [The case of neurofibromatosis type ! in a 5-year-old boy]. AB - In the work we present the case of a 5-year-old boy who was admitted to the clinic because of the tumour in mediastinum. According to the results of the investigation the diagnosis of Recklinghausen disease was made. The tumour was situated in the region of pharynx, neck and mediastinum. The diagnosis was based on the characteristic features in the physical examination (cafe-au-lait spots, freckles in armpits and grains) and accessory investigations (x-ray, CT, NMR and histopathology). In the boy's family this disease was not noticed, although in his mother we observed cafe-au-lait spots on the skin of abdomen. Despite the fact that the diagnosed disease is known as a mild one (from the histopathological point of view) in the case of our patient it turned out to be much more clinically malignant because of the localization of the tumour. This situation forced us to the application of much more aggressive treatment (chemotherapy, operation). PMID- 10731996 TI - [Triton's tumor in children: a report of two cases]. AB - Triton's tumor is a rare neoplasm, consisting of both neurogenic and rhabdomyoblastic components. Triton's tumor does not respond well to chemo- or radiotherapy, resulting in its poor therapy effects. This paper reports on two cases of this malignancy diagnosed in our department. The first one was treated with CWS chemotherapy followed by radical tumor resection, which resulted in 4.5 year complete remission. The other patient was treated with chemotherapy according to CWS protocol, achieving partial regression of the tumor. We suggest that this response may be connected with the sensitivity of the RMS embryonal component of the tumor to chemotherapy. This partial remission may create a possibility of radical tumor resection. PMID- 10731997 TI - [Atypical picture of osteosarcoma situated in fibula of a 14-year-old boy]. AB - This paper presents the case of osteogenic sarcoma, situated in fibula of 14-year old boy. The X-ray picture of this tumor was typical of unicameral cyst, but histopathological examination showed traits of osteosarcoma. Only magnetic resonance suggested the presence of malignant neoplasm. PMID- 10731998 TI - [The problem of blood transfusion ++ in Jehova's Witnesses' children with oncological disease]. AB - We present 2 cases of Jehovah's Witnesses' children suffering from oncological diseases (non-Hodgkin Lymphoma and Acute Lymphoblastic Leukaemia). During their treatment we used erythropoietin and no blood products were transfused. PMID- 10732000 TI - Medical malaise. Is practicing medicine a noble profession, or is it becoming a skilled trade? PMID- 10731999 TI - [Family environment and psychological development of children with hematological malignancies]. AB - In the years 1994-1995, 20 children treated due to hyperplastic diseases of hemopoietic system in Pediatry and Hematology Department of Silesian University School of Medicine in Katowice, were included into the study. The children were examined at and after infant age. Methodology was based on the application of three scales of psychomotor development to elaborate the index of children development. The results show correct psychological development of children and the usefulness of the applied methods. PMID- 10732001 TI - Geriatrics photo quiz. Nephrotic syndrome. Proteinuria characterizes this condition, and treatment targets the underlying pathology. PMID- 10732002 TI - Enlarging growth on the lip. Prolonged sun exposure and smoking may promote development of the nodule. PMID- 10732003 TI - Rheumatoid arthritis. New disease-modifying and anti-inflammatory drugs. AB - Treatment options for rheumatoid arthritis (RA) are expanding as research has provided a more complete understanding of the pathophysiology of the disease. Three disease-modifying agents approved in the last 18 months for early intervention in RA are etanercept, leflunomide, and infliximab. For the relief of the signs and symptoms of RA, the new selective cyclooxygenase-2 (COX-2) inhibitors are joining the available nonsteroidal anti-inflammatory drugs. One COX-2 inhibitor is approved for use in RA, and another is under investigation for that indication. As a class, the COX-2 inhibitors offer efficacy similar to traditional NSAIDs but with less GI and platelet toxicity. PMID- 10732004 TI - Ischemic stroke. Clinical strategies based on mechanisms and risk factors. AB - Ischemic stroke is a common disorder associated with significant morbidity and mortality. Results of several pivotal clinical trials completed within the last decade have helped refine stroke prevention and treatment strategies. Endarterectomy for symptomatic carotid artery stenosis, anticoagulation in atrial fibrillation, and IV t-PA treatment of hyperacute ischemic stroke may reduce the burden of stroke. Ongoing studies are addressing newly recognized risk factors, such as aortic arch and intracranial atherosclerosis, as well as neuroprotective agents and locally delivered thrombolytics. Successful patient management requires a targeted clinical approach based on vascular localization and risk factor assessment. PMID- 10732005 TI - Pressure ulcers. Analysis of guidelines for treatment and management. AB - Although prevention is the most important step in pressure ulcer care, clinicians should be familiar with the best methods for treating and managing these lesions, because proper care and subsequent vigilance can prevent worsening of the wound. In 1994, the U.S. Department of Health and Human Services published clinical practice guidelines to help standardize and improve ulcer care. Six key areas of ulcer management were addressed. The section on ulcer care--which includes discussions of debridement, wound cleansing, dressings, and adjunctive therapies- is examined here. PMID- 10732006 TI - Update 2000. Guidelines for prescribing psychoactive drugs. AB - As patients age, a pattern develops in the primary care setting of underdiagnosis and undertreatment of psychiatric disorders, including dementing illnesses and depression. Choosing an appropriate psychoactive drug and dosage for an older patient is a complex task, due to the wide array of medications available and the need for careful titration. To address these problems, this article provides prescribing recommendations for antidepressants, antipsychotic medications, benzodiazepines (antianxiety/hypnotics), and other psychoactive medications. PMID- 10732007 TI - Use of plasma concentrations of 13,14-dihydro,15-keto-PGF2 alpha (PGFM) in the diagnosis of sub-clinical endometritis and its relationship to fertility in the postpartum dairy cow. AB - The objective of this study was to determine the value of using plasma concentrations of PGFM to diagnose subclinical endometritis in the dairy cow, and its relationship to subsequent fertility. A total of 274 cows between 24 to 29 d post partum was divided into 4 groups on the basis of clinical features of the uterus and ovary. Cows in Group 1 (n = 74) had a normal, involuting uterus and a CL on the ovary; cows in Group 2 (n = 51) had a normal, involuting uterus but no CL on the ovary; cows in Group 3 (n = 83) did not have a normal, involuting uterus but had a CL on the ovary; and cows in Group 4 (n = 66) did not have a normal, involuting uterus or a CL on the ovary. A blood sample was obtained from each cow on the day they were placed on the study, and plasma concentrations of PGFM and P4 were determined using RIA. Cows were artificially inseminated (AI) at the first observed estrus after Day 60 post partum, and pregnancy was determined by palpation of the uterus per rectum between 45 and 50 d postAI. Reproductive responses evaluated were conception rate to first service, days open, and percentage of cows pregnant by 90, 120, 150 and 180 d post partum. Data were analyzed using GLM procedures of SAS and a 2 x 2 factorial with contrast procedures. Polynomial regression analysis was used to determine the shape of the PGFM, P4 and fertility curves. There was no difference among mean PGFM concentrations of cows in each group. The rate of decline of plasma PGFM concentrations was lower in cows with an abnormal uterus and a CL on the ovary compared with those without a CL. A lower percentage of cows with abnormal uteri was pregnant by 90 d post partum compared with cows with normal uteri. From the results of this study, it was concluded that plasma PGFM concentrations between Days 24 to 29 post partum were not effective in identifying cows with subclinical endometritis. PMID- 10732008 TI - Measurement of vaginal temperature by radiotelemetry for the prediction of estrus in beef cows. AB - Peaks in vaginal temperature were assessed as predictors of estrus in 22 suckled beef cows during 2 breeding seasons. A flexible plastic anchor was used to retain a temperature radiotransmitter within the cow vagina and vaginal temperature was monitored at 4-min intervals. Blood was collected twice weekly for analysis of serum progesterone to confirm the occurrence of estrus and ovarian status was checked weekly using transrectal ultrasonography. Visual observations of estrous behavior were made for 20 min/h between 0400 and 0800 h and casually from 0800 to 1600 h (4 to 6 times). Values for vaginal temperature were available for 47 estrous periods. The prediction of estrus based on vaginal temperature was excellent when an estral peak in vaginal temperature was defined as an increase of at least 0.4 degree C for 3 or more consecutive hours using the corresponding hourly means of a 2 or 3-d baseline. Combining the results of 2 separate years and using a 3-d baseline, a peak in vaginal temperature was found for 42 of 47 confirmed estrus periods (detection sensitivity of 89.4% and prediction power positive of 85.7%). Corresponding detection sensitivity and prediction power positive for visual observations of standing estrus were 53.2 and 96.2%, respectively. The mean maximal increase in vaginal temperature at estrus was 0.9 +/- 0.3 degree C and the mean duration of the estrual peak in vaginal temperature was 6.5 +/- 2.7 h. In addition, vaginal temperature was found to be significantly depressed for 3 d prior to estrus and significantly elevated at mid-cycle. PMID- 10732009 TI - Effects of cumulus cell density during in vitro maturation of the developmental competence of bovine oocytes. AB - To determine the role of cumulus cells in oocyte maturation, we carried out an investigation on the effects of addition of cumulus cells to the maturation medium on the developmental competence of corona-enclosed oocytes and oocytes denuded from their somatic cells. The addition of cumulus cell (1.6 x 10(6) cells/mL) improved the development of bovine corona-enclosed oocytes, however, addition of a similar number of cumulus cells as cumulus-oocyte-complexes (COCs, cumulus cell density: 4.2 x 10(6) cells/mL) had no effect on the development of oocytes denuded from their somatic cells. To determine if corona-enclosed oocytes can obtain developmental competence without the addition of extra cumulus cells, the effects of cell density during in vitro maturation on the developmental competence were studied. A density of 1.6 to 3.2 x 10(6) cumulus cells/mL was the most effective for in vitro maturation of oocytes with intact gap junctions. The effects of the medium conditioned by COCs on the developmental competence of oocytes was also examined. It was demonstrated that COC-conditioned medium improved the development of bovine oocytes to the blastocyst stage. These data suggest that the developmental competence of bovine oocytes surrounded with corona cells is supported in a cell density-dependent manner in the maturation medium. In addition, the data indicate that cumulus cells benefit bovine oocyte development either by secreting soluble factors which induce developmental competence or by removing an embryo development-suppressive component from the medium. PMID- 10732010 TI - Ovarian activity and plasma concentrations of progesterone and estradiol during pregnancy in jennies. AB - The objectives of this study were to determine ovarian activity (with ultrasound) and plasma concentrations of progesterone and estradiol during pregnancy in jennies. There was considerable ovarian activity during the second month of pregnancy. Secondary corpora lutea (total of 2 to 7 per jenny) were formed (mainly by luteinization) starting on Day 41.8 +/- 1.0 (range Days 38 to 46; ovulation = Day 0). The echogenicity of the primary and secondary corpora lutea gradually decreased during pregnancy. Plasma progesterone concentrations increased between Days 0 and 10 (0.9 and 19.9 ng/mL, respectively), gradually decreased to Day 30 (12.1 ng/mL), increased between Days 30 and 40 (plateau, at approximately 17 ng/mL), gradually declined from Days 110 to 160 (nadir of approximately 6 ng/mL), and remained nearly constant until increasing again just before parturition. Plasma estradiol concentrations increased gradually from Day 65, peaked (1.2 ng/mL) on Day 165 (> or = 1 ng/mL on Days 150 to 210), and decreased thereafter, with very low concentrations during the last 20 d before parturition. Ovarian function and hormone profiles were generally similar to those previously reported during pregnancy in mares. PMID- 10732011 TI - Ultrasonographic evaluation of the conceptus from days 10 to 60 of pregnancy in jennies. AB - Daily ultrasound examinations were conducted from Days 10 to 60 (ovulation = Day 0) of pregnancy to monitor the conceptus in jennies (n = 12). The embryonic vesicle was first detected on Day 11.5 +/- 0.9 (mean +/- SD; range 10 to 13 d) and was mobile until movement ceased (fixation) on Day 15.5 +/- 1.4 (range, 13 to 18 d). The vesicle was spherical from Days 10 to 18 (mean growth rate, 3.2 mm/d), non spherical (irregular) with a reduced growth rate (0.5 mm/d) from Days 19 to 29, and then grew at a moderate rate (1.6 mm/d) up to Day 46. On average, detection of the embryo proper (consistently located on the ventral aspect of the yolk sac) and embryonic heartbeat were Days 20.7 +/- 1.2 and 23.5 +/- 1.3, respectively. Formation of the allantoic sac was first detected on Day 24.4 +/- 1.7 and was complete on Day 36.8 +/- 1.6. Descent of the fetus (and formation of the umbilical cord) began on Day 37.9 +/- 1.7 and was complete on Day 44.1 +/- 2.1. Crown-rump length averaged 3.7, 15.4, 22.7, 37.5 and 59.6 mm on Days 20, 30, 40, 50 and 60, respectively. In general, morphologic features and dates of occurrence were similar to those reported previously in the mare. PMID- 10732012 TI - Transrectal color Doppler sonography of the A. uterina in cyclic mares. AB - Color Doppler ultrasound was used transrectally in 6 mares to locate both the left and right Aa. uterinae and to obtain flow velocity waveforms at defined times (Days 0, 5, 10, 15 and 20) during 4 estrous cycles. Blood flow reflected by the resistance index (RI) was determined for both arteries on 120 occasions. As there was no significant difference and a high correlation in the RI values between the left and right arteries (paired Student's t-test, correlation coefficient r > 0.94; P < 0.05), the average RI value was used for subsequent analyses. There were correlations between RI values, mares and day of estrous cycle (P < 0.0001). The mean RI was higher (P < 0.05) in the 2 multiparous mares (age, 12 to 13 yr) than in the 4 younger maiden mares (age, 6 to 10 yr). During the estrous cycle mean RI values on Day 0 (day of ovulation) and Day 10 were higher (P < 0.05) than on Days 5, 15 and 20, whereas between estrous cycles within mares no differences (P > 0.05) could be measured. The results suggest that transrectal Color Doppler sonography is a noninvasive method for examining differences in impedance to uterine blood flow between different mares and cycle periods. PMID- 10732013 TI - Effect of hyaluronic acid on development of in vitro produced bovine embryos. AB - The present study was carried out to evaluate the effect of hyaluronic acid (HA) added to the culture medium on bovine embryo development to the blastocyst stage as well as embryo quality and viability after freezing and thawing. In vitro matured and fertilized (IVM/IVF) bovine oocytes from slaughterhouse ovaries were cultured for 8 d in SOFm supplemented with 4 mg/mL fatty acid-free BSA, either in the absence or presence of 1 or 0.5 mg/mL HA. There was a significant increase in blastocyst yield in the presence of 1 mg/mL HA (P < 0.01), whereas 0.5 mg/mL HA was ineffective. Cleavage rate and mean number of days to blastocyst formation were unaffected by HA at any concentration. At 1 mg/mL, HA did not affect either post-freeze survival of Grade 1 and 2 blastocysts or the number of nuclei per blastocyst. Supplementation with HA at 1 mg/mL also significantly enhanced embryo development up to the blastocyst stage (P < 0.05) in a chemically-defined culture medium without a protein source. It is concluded that supplementation of both semi-defined and defined culture media with 1 mg/mL HA improves the development of IVM/IVF bovine embryos to the blastocyst stage, without affecting embryo quality and post-freeze survival. These results open the possibility of including HA in culture media in order to increase the efficiency of in vitro blastocyst production from in vitro-matured bovine oocytes. PMID- 10732014 TI - Effect of sperm capacitation and fertilization media on IVF and early embryo development of prepubertal goat oocytes. AB - Experiments were carried out to develop an improved IVF system for prepubertal goat oocytes matured in vitro. Cumulus oocyte complexes (COC) were obtained by slicing ovaries from slaughtered prepubertal goats. Oocytes were matured in TCM199 supplemented with 20% estrous goat serum (EGS) + 10 micrograms/mL FSH + 10 micrograms/mL LH + 1 microgram/mL estradiol 17 beta for 27 h at 38.5 degrees C in 5% CO2 in air. In Experiments 1 and 2, freshly ejaculated spermatozoa were capacitated in 1 of 3 media: TALP/H, modified Defined Medium (mDM) and mH-M199 with 50 micrograms/mL heparin for 45 min. Matured oocytes were fertilized in TALP, mDM or mH-M199 in Experiment 1 and in TALP in Experiment 2. In Experiment 3, three media were used for sperm capacitation and fertilization: Treatment A (control group): spermatozoa were capacitated in mDM with 50 micrograms/mL heparin for 45 min and fertilized in TALP medium with 1 microgram/mL hypotaurine; Treatment B: spermatozoa were capacitated in mDM with 50 micrograms/mL heparin + 388 micrograms/mL caffeine for 30 min and fertilized in TALP medium without hypotaurine; Treatment C: spermatozoa were capacitated in mDM with 50 micrograms/mL heparin for 45 min and fertilized in TALP medium with PHE (20 microM penicillamine, 10 microM hypotaurine and 2 microM epinephrine). At 24 h post insemination, the ova were transferred to a granulosa cell monolayer, and early embryo development was evaluated until Day 8. In experiment 2, the results show, that mDM plus heparin for sperm capacitation and TALP medium with hypotaurine for oocyte fertilization provided the highest proportion of penetrated oocytes, both total number (79.6%) and normal fertilization (55.1%), whereas the use of caffeine (44.6 and 31.2%, total and normal fertilization rate, respectively) and PHE (31.8 and 20.6%, total and normal fertilization rate, respectively) as motility enhancers did not improve the results obtained in the control group (48.7% and 37.2%, total and normal fertilization rate, respectively). These were no differences for the results of morulae and blastocysts. PMID- 10732015 TI - A retrospective analysis of factors contributing to calf mortality and dystocia in beef cattle. AB - Records of 2191 calvings from the Clemson University Beef Physiology Unit between 1981 and 1993 were analyzed to determine factors affecting malpresentation, mortality and dystocia. Only 20 (0.91%) parturitions involved malpresentation: posterior presentation (n = 14), leg deviations (n = 3), head deviations (n = 2) and breech birth (n = 1). Dystocia affected calf mortality within 24 h of birth (P < 0.05), with mortality increasing as the severity of dystocia increased. There was an overall 4.5% death loss within 24 h of birth, with 4 and 7% mortality rates for calves from multiparous and primiparous dams, respectively (P < 0.05). Mortality was higher for bull vs heifer calves (P < 0.05). Ninety-four percent of calvings were unassisted, while 6% were assisted births. Dystocia was greater (P < 0.01) in primiparous (17%) than in multiparous dams (4%). In births involving dystocia, 28.1% required mild traction, 69.3% required heavy traction and 2.6% required Cesarean section. Birth weights associated with normal births and mild traction (36 and 36 kg) were lighter than those associated with heavy traction and Cesarean section (40 and 42 kg, respectively; P < 0.05). In conclusion, malpresentations were too few to be of significance, and dystocia influenced mortality within 24 h of birth. Calf birth weight and parity of dam explained most of the observed variations in dystocia. PMID- 10732016 TI - Production of sheep embryos in vitro and development of progeny following single and twin embryo transfers. AB - An in vitro culture system for producing ovine embryos is described, in which heat inactivated sheep serum was used as a protein source for maturation, fertilization and 7-d culture phases. Ovaries obtained from a commercial abattoir were used as the source of mature ewe (285) and lamb oocytes (356), which were subsequently cultured in this system to yield similar mean cleavage rates of 91 and 92%, respectively, but significantly different (P < 0.025) proportions for blastocysts/cleaved oocytes (46 and 18%). At Days 7 or 8 of culture, embryos from each source were transferred, either singly (ewe-derived) or in pairs (ewe- and lamb-derived), to hormonally synchronized recipient ewes, resulting in the birth of lambs ranging in weight from 2.5 to 8.8 kg for singletons and 2.6 to 8.0 kg for twins. Mean gestation length of 153.4 +/- 0.5 d (range 151 to 160) was slightly longer than the expected norm of about 150 d. The pregnancy rate was significantly higher after the transfer of embryo pairs (64%) than single (39%) embryos, while survival of lambs to weaning was greater for singleton (80%) than for twin lambs (43%). Some factor(s) in the culture media, such as growth factors in the sera, may have a mitogenic effect on embryonic cells, or it may alter the distribution of cells to the trophectoderm and inner cell mass, resulting in enhanced body growth rates. PMID- 10732017 TI - Effect of spermatozoal concentration and number on fertility of frozen equine semen. AB - Information on the number of motile spermatozoa needed to maximize pregnancy rates for frozen-thawed stallion semen is limited. Furthermore, concentration of spermatozoa per 0.5-mL straw has been shown to affect post-thaw motility (7). The objectives of this study were 1) to compare the effect of increasing the concentration of spermatozoa in 0.5-mL straws from 400 to 1,600 x 10(6) spermatozoa/mL on pregnancy rate of mares, and 2) to determine whether increasing the insemination dose from approximately 320 to 800 million progressively motile spermatozoa after thawing would increase pregnancy rates. Several ejaculates from each of 5 stallions were frozen in a skim milk-egg yolk based freezing medium at 2 spermatozoal concentrations in 0.5-mL polyvinyl-chloride straws. Half of each ejaculate was frozen at 400 x 10(6) cells/mL and half at 1,600 x 10(6) cells/mL. Insemination doses were based on post-thaw spermatozoal motility and contained approximately 320 x 10(6) (320 to 400) motile spermatozoa or approximately 800 x 10(6) (800 to 900) motile spermatozoa. Sixty-three mares were assigned to 1 of 4 spermatozoal treatments (1--low spermatozoal number, low concentration; 2--low spermatozoal number, high concentration; 3--high spermatozoal number, low concentration; 4--high spermatozoal number, high concentration) and were inseminated daily. Post-thaw spermatozoal motility was similar for cells frozen at both spermatozoal concentrations (P > 0.1). One-cycle pregnancy rates were 15, 40, 28 and 33%, respectively, for Treatments 1, 2, 3 and 4. Packaging spermatozoa at the high concentration tended to increase pregnancy rates vs packaging at the low concentration (37 vs 22%; P = 0.095). Furthermore, when the lower spermatozoal number was used, there tended (P < 0.1) to be a higher pregnancy rate if spermatozoa were packaged at the higher concentration. There was no increase in pregnancy rates when higher numbers of motile spermatozoa were inseminated (27 vs 31%; P > 0.1). Based on these results, a single 0.5-mL straw dose containing 800 x 10(6) spermatozoa should be used and each insemination dose should contain approximately 320 x 10(6) motile spermatozoa. Fertility trials utilizing other freezing extenders are necessary before recommending a single 0.5 mL insemination dose for all freezing extenders. PMID- 10732018 TI - Folliculogenesis, onset of puberty and fecundity of mink (Mustela vision Schreb) selectively bred for docility or aggressiveness. AB - It has been suggested that selective breeding of animals for docile behavior is correlated with early onset of puberty and improved fertility. We wished to test the hypothesis that mink bred for docility would show earlier onset of puberty and greater fecundity than mink bred for aggressiveness. We used farm-raised, 7 mo-old mink females that had been selectively bred for 7 to 10 generations on the basis of behavior towards humans. Onset of puberty was estimated once (between 15 and 20 December) by vaginal smears and was said to start wtih preponderance of cornified epithelial cells in the cytological specimen. Fecundity was measured by litter size and rate of folliculogenesis, with and without hCG stimulation, by histomorphometric examination of ovaries and uteri. A total of 43/100 (43%) docile females achieved proestrus and estrus as compared to 16/136 (12%) of the aggressive ones. Overall pregnancy rate, survival to 5 d after whelping and litter size did not differ between the docile and aggressive females. Docile females showed significantly higher numbers (P < 0.05 and < 0.001) of growing, maturing and atretic follicles than the aggressive ones, however the latter showed a highly significant (P < 0.001) folliculogenic response to hCG. The response of the ovary of aggressive females to hCG is particularly dramatic because in most aggressive females the ovaries contained none or only few follicles of any kind. The inhibition of folliculogenesis in aggressive mink is similar to that reported in Silverblue and Sapphire mink homozygous for the Stuart factor. The paradoxical response to hCG may be an indication that selective breeding for aggression may be correlated with the disturbance at the early stages of folliculogenesis which creates a deficiency of follicles that are sensitive to LH-type stimulation. PMID- 10732019 TI - Determination of early pregnancy and embryonic growth in goats by transrectal ultrasound scanning. AB - Ultrasonography has been shown to be a useful tool for pregnancy diagnosis and the study of embryonic growth in mammals. The objectives of this study were 1) to evaluate the use of real-time B-mode ultrasonography for early pregnancy diagnosis in goats, 2) to define criteria for accurate diagnosis of pregnancy, and 3) to monitor the embryonic growth ultrasonically until Day 40 after mating. Estrus was synchronized in 16 cyclic Anglo-Nubian goats with a single injection of cloprostenol (125 micrograms, i.m.). Estrous females were randomly assigned into 2 groups: 1) goats mated by a vasectomized male (n = 5; MV group), and 2) goats mated by an intact male of proven fertility (n = 11; MF group). Transrectal ultrasonographic examinations with a 5 MHz linear array transducer were performed from Days 13 to 40 post mating. The evaluated parameters included the appearance of nonechogenic areas in the uterus, presence of embryo(s), crown-rump length of embryo and embryonic heart rate (beats/min). On Day 18, the mean (+/- SEM) diameter of nonechogenic areas was 1.5 +/- 0.3 mm in the MV group and 4.0 +/- 0.5 mm in the MF group (P < 0.01). In 36% of the pregnant does these areas were less than 3 mm. The mean (+/- SEM) day of the first detection by means of heartbeats of at least 1 embryo was 20.7 +/- 0.5 d (range, Days 19 to 23). From Days 19 to 38 of pregnancy, crown-rump length was best represented by a linear regression (Y = -2.23 + 0.13X; r2 = 0.94; P < 0.05). Crown-rump length on the day of the first detection of an embryo was 5.3 +/- 0.3 mm, reaching 34.2 +/- 0.6 mm on Day 40. Mean (+/- SEM) heartbeat rate was 168.3 +/- 2.8 beats/min on Day 21, decreasing to 158.3 +/- 2.0 beats/min on Day 40. Detection of the caprine embryo by ultrasonography and confirmation of its viability by heartbeats was shown to be a reliable method for early pregnancy diagnosis in Anglo-Nubian goats. Ultrasonic measurement of crown-rump length was useful in predicting the age of the embryo. PMID- 10732020 TI - Role of media, protein and energy supplements on maintenance of morphology and DNA-synthesis of small preantral domestic cat follicles during short-term culture. AB - Small preantral follicles (40 to 90 microns in diameter) from domestic cats were cultured for 10 d using different media (M199 and Dulbecco's MEM) and protein (FCS and BSA) supplements. Culture efficacy was determined by Hoechst 33258 staining and estimation of Brom-desoxyuridine (BrdU)-incorporation into oocytes and granulosa cells. Culture in M199 + FCS and in DMEM + FCS resulted in 21.6% and 38.1%, respectively, of morphologically intact preantral follicles. Adding BSA increased the rate of normal follicles to 51.7% in M199 and to 58.6% in DMEM. Oocytes were found in 40% of the follicles, when DMEM and/or BSA supplementation was used, while M199 with FCS induced acute loss of oocytes in 85% of the follicles. About 10% of the oocytes contained degenerating chromatin. Measurement of BrdU-incorporation during culture allows for quick and effective assessment of follicle viability in vitro. Comparison of M199 and Dulbecco's MEM, both with FCS or BSA and DMEM with or without pyruvate/lactate, indicated that Dulbecco's MEM + BSA without pyruvate and lactate is the best medium for culture of cat follicles. However, further research of suitable medium supplements is needed. PMID- 10732021 TI - Comparison of sex ratio and cell number of IVM-IVF bovine blastocysts co-cultured with bovine oviduct epithelial cells or with Vero cells. AB - The influence of 2 co-culture systems (BOEC and Vero cells) on the development rates, quality grades and sex ratios of IVM-IVF bovine embryos were studied. Zygotes obtained after IVF were co-cultured in each co-culture system for 7 and 8 d (Day 0 = day of insemination) in B2 medium. No effect of the co-culture system was observed on development rates measured on Days 7 and 8. However, Vero cell co culture had a positive influence on embryo quality. Irrespective of their sex, embryos produced on Vero cells showed higher cells number than those co-cultured on BOEC (103.4 +/- 3.8 and 97 +/- 8.12 for BOEC vs 113.7 +/- 3.5 and 114 +/- 5.9 for Vero cells at Days 7 and 8, respectively; P < 0.05). The percentage of male embryos was increased in the two co-culture systems (60.7% males for BOEC; P < 0.05 vs 63% males for Vero cells; P < 0.01) on Day 7. In both co-culture systems the increase in the percentage of males was more obvious for embryos reaching the most advanced stage (expanded blastocysts). The results show that Vero cells improved the quality grade of bovine embryos produced in vitro, and thus are recommended for use as a safe co-culture system that does not contain pathogens. PMID- 10732022 TI - Comparison of preimplantation developmental competence after mouse oocyte growth and development in vitro and in vivo. AB - Culture systems for oocytes are essential for the experimental analysis of the basic mechanisms of oocyte development and, moreover, they will eventually find wide application in agriculture, the clinic, and wildlife preservation. Here, progress in mouse oocyte growth and development in vitro using oocyte-granulosa cell complexes from preantral follicles is reviewed. Oocyte-granulosa cell complexes were isolated from preantral (secondary) follicles of 12 day old mice, grown in vitro for 10 days, then matured and fertilized in vitro. The developmental competence of these oocytes was compared with oocytes grown in vivo and isolated from 22 day old mice, then matured and fertilized in vitro. In vitro grown oocytes did not achieve the same size as their in vivo-grown counterparts. However, when oocytes were grown in medium containing fetal bovine serum, their preimplantation developmental competence was equivalent to that of in vivo-grown oocytes. Surprisingly, more blastocysts per animal were produced when oocytes were grown in vitro than in vivo. There was no correlation between oocyte size and either preimplantation developmental competence or number of cells per blastocyst. Oocytes grown in serum-free medium did not achieve the same developmental competence as oocytes grown in medium supplemented with serum. Lastly, the health status as an adult of the only animal born after complete oocyte development in vitro is described and discussed. PMID- 10732023 TI - Monitoring meiosis in gametogenesis. AB - Meiotic recombination is essential to hold homologous chromosomes together so that they can separate accurately in the formation of gametes, thus preventing fetal loss due to aneuploidy. How do germ cells know when they have finished genetic recombination and that it is time to enter the meiotic division phase, and what are the elements that signal the onset of the division phase? During spermatogenesis there is no arrest at the end of meiotic prophase (as there is in oogenesis) and signals for progress into the meiotic division phase may be closely related to events of chromosome pairing and recombination. Methods for culture of male germ cells have been used to show that spermatocytes become competent for some aspects of the division phase by the early pachytene stage, long before they would normally enter division. Evidence suggests that establishment of homologous chromosome pairing is one aspect of acquiring competence. Activation of the cell cycle regulator MPF also appears to be important, and there is a requirement for activity of topoisomerase II in order for spermatocytes to exit prophase and enter the meiotic division phase. Understanding how these molecular entities tie into monitoring the completion of recombination and meiotic progress will be instructive about important gametic safeguards preventing aberrant chromosome segregation and resultant aneuploidy. PMID- 10732024 TI - Use of in vitro systems to study male germ cell development in neonatal rats. AB - The aim of this review is to summarize ways in which in vitro approaches have allowed us to investigate several aspects of gametogenesis in the male. In our laboratory, we have established both organ culture and cell co-culture methodologies and applied them to questions focused on cellular and molecular events important for development of primitive spermatogonia, or gonocytes, in testes of neonatal rats. We have described their postnatal reinitiation of mitosis and their migration to the basal lamina in anticipation of basal compartment formation and, through use of these in vitro systems, we have identified several mechanisms regulating these processes. These include matrix influence on mitosis and migration, adhesive mechanisms active between gonocytes and Sertoli cells, and involvement of the Kit receptor on germ cells and its ligand from Sertoli cells in supporting gonocyte migration, as described below. PMID- 10732025 TI - Activation of bovine and baboon primordial follicles in vitro. AB - Mammalian ovaries contain a large pool of non-growing, primordial follicles. The ability to initiate growth of this pool of resting follicles in vitro and to maintain follicular growth to a stage when the oocyte could be matured and fertilized would increase the reproductive potential of valuable domestic animals, endangered species and infertile women. This paper summarizes our progress to date in activating primordial follicles of cattle and baboons. Pieces of ovarian cortex, rich in primordial follicles, were obtained from fetal bovine and baboon ovaries during late gestation. Pieces were maintained in organ culture in serum-free medium containing ITS+ (insulin-transferrin-selenium-linoleic acid BSA) for up to 20 days and at various times during culture some pieces were fixed for histological morphometry. As early as 2 days of culture, the number of primordial follicles had decreased by 88% or 55%, whereas the number of primary follicles had increased 2.5- or 5-fold, compared to tissue freshly isolated from bovine or baboon ovaries, respectively (P < 0.01). In baboon cortical pieces a small number of secondary follicles developed during a 20-day culture period. The development of primary and secondary follicles was accompanied by an increase in diameter of both the granulosa cell layer and the oocyte. The addition of FSH (1, 10, or 100 ng/ml) had no effect on the development of follicles in bovine cortical pieces after 7 or 14 days of culture, relative to control cultures without FSH. These results show that a high percentage of primordial follicles from cattle and baboons can be activated to grow in serum-free medium in the absence of gonadotropins. Conditions that will support further growth in vitro of follicles from these species remain to be elucidated. The culture system we have developed could be used to develop such conditions and to explore factors that regulate the movement of primordial follicles into the pool of growing follicles. PMID- 10732026 TI - In vitro models for oocyte development. AB - The mammalian ovary has a large store of primordial follicles, which are a potential source of oocytes for in vitro production of embryos. Several culture systems have been developed to support the growth and development of oocytes from rodent primordial and preantral follicles and progress is slowly being made in modifying these techniques to support the in vitro growth of porcine and bovine follicles. Oocytes from porcine preantral follicles can acquire competence to resume meiosis and proceed to Metaphase II after in vitro growth (IVG) but fertilisation has yet to be demonstrated. This paper presents the current status of technology for the in vitro growth and development of immature mammalian oocytes. Culture systems used successfully to grow immature rodent oocytes are compared and adaptations of these methods to support porcine and bovine oocyte growth discussed. PMID- 10732027 TI - Interplay between CDC2 kinase and MAP kinase pathway during maturation of mammalian oocytes. AB - Two principal kinases, p34cdc2 kinase and MAP kinase play a pivotal role in maturation of mammalian oocytes. In the porcine and bovine oocytes both kinases are activated around the time of germinal vesicle breakdown (GVBD). Butyrolactone I (BL I), a specific inhibitor of cdk kinases, prevents effectively and reversibly resumption of meiosis in the porcine and bovine oocytes. Neither p34cdc2 kinase nor MAP kinase are activated in oocytes inhibited in the GV stage. The bovine oocytes maintained for 48 h in the medium supplemented with BL I, progress subsequently to metaphase II in 91%, their cumuli expand optimally and after in vitro fertilization they possess two pronuclei. When the cdc2 kinase is blocked in the porcine oocytes by BL I, MAP kinase, activated by okadaic acid treatment, is able to substitute cdc2 kinase and induce GVBD. The histone H1 kinase activity sharply decreases in the metaphase II oocytes treated by BL I and one or two female pronuclei are formed. These data indicate that BL I is a useful tool either for the two step in vitro culture of mammalian oocytes or for their activation in nuclear transfer experiments. PMID- 10732028 TI - Control of oocyte maturation in cows--biological factors. AB - Since bovine in vitro fertilization became possible in the early 80s, a lot of effort has been done to clarify the mechanisms of what seems more and more one of the crucial steps in this procedure, being oocyte maturation. Undoubtedly, many biological factors act together to prepare the immature oocyte for a successful development to a competent embryo after fertilization. Defects in oocyte maturation can possibly be caused by an inadequate nuclear or cytoplasmic maturation or even by a failure of both. There is a general agreement upon the fact that the origin of the oocyte can play an important role. Oocytes derived from very small follicles show a lower rate of maturation and lower blastocyst development with currently used maturation protocols. Parthenogenetic activation of small size follicle derived oocytes suggests that their poor development was not caused by fertilization problems but more likely by intrinsic oocyte factors. Similar developmental rates achieved through nuclear transfer and parthenogenetic activation suggests that the nucleus of the incompetent oocyte may not be the sole reason for a poor development. Another important factor appears to be the donor animal age. The younger the donor animal, the more impaired is its oocyte's developmental competence in most of the embryo IVP systems. Treatment with exogeneous gonadotropins can be beneficial in young donors on the oocyte cleavage rates but does not always increase the final blastocyst outcome. This review briefly documents some of the biological factors and their possible effects on the developmental capacities of the bovine oocyte in vitro. PMID- 10732029 TI - Controlling meiotic resumption in bovine oocytes: a review. PMID- 10732030 TI - Prolactin in follicular fluid and intracellular store calcium in follicular cells are related to morphological signs of ovarian follicle atresia in cows: work in progress. AB - It is known that prolactin (PRL) is the third pituitary hormone serving gonadotropic function in mammals. However, its role in the regulation of ovarian folliculogenesis and, in particular, its relationship to follicular atresia as well as the mechanism of its influence on follicular cells are poorly understood. We investigated PRL levels in follicular fluids (FFs) and intracellular store calcium ([Ca2+]is) in cell walls of bovine ovarian follicles with diameters of 10 to 20 mm and their relationship to follicular atresia. Ovarian follicles were categorized on the basis of macroscopic criteria and of microscopic examination of granulosa cell (GC) smears. Prolactin concentrations in FFs were measured by RIA and levels of [Ca2+]is in follicular cells were determined by using the fluorophore chlortetracycline. Compared to atretic follicles, morphologically normal follicles were characterized by higher concentrations of PRL in FFs (P < 0.001) and lower contents of [Ca2+]is in follicular cells (P < 0.01). Furthermore, follicles containing no more than 20% of pycnotic GCs had higher levels of PRL in their fluids than those containing over 40% of pycnotic GCs (P < 0.05). Finally, the direct effect of PRL on [Ca2+]is content in follicular cells was studied in vitro. Compared to control, PRL decreased (P < 0.001) the levels of [Ca2+]is in the cells after 24 h culture of follicular walls from morphologically normal follicles in TCM 199 supplemented by 10% fetal calf serum. Our findings suggest that the decline of PRL concentrations in FFs and the rise of [Ca2+]is contents in follicular cells are related to atresia of large bovine follicles and that there appears to be a relationship between the two biochemical parameters. PMID- 10732031 TI - In vitro survival and proliferation of porcine primordial germ cells. AB - Primordial germ cells (PGC) collected from the genital ridge of Day 25 porcine embryos were cultured on STO feeder cells in medium with or without supplemented growth factors. The effects on porcine PGC proliferation of leukemia inhibitory factor (LIF), LIF + stem cell factor (SCF) or LIF + SCF + basic fibroblast growth factor (bFGF), growth factors shown to be essential for in vitro survival and proliferation of murine PGC, were tested. After histochemical staining, both freshly collected and cultured PGC expressed alkaline phosphatase activity. With or without supplemented growth factors, porcine PGC survived and proliferated in culture for at least 5 d. None of the growth factors tested markedly enhanced in vitro growth of porcine PGC. These results suggest that growth factors provided by either the STO feeder layer or the cultured PGC themselves are sufficient to support in vitro survival and proliferation of porcine PGC. With the support of STO cells, addition of growth factors shown to be essential for the in vitro growth of murine PGC is not required for survival and proliferation of cultured porcine PGC. PMID- 10732032 TI - Cytogenetics of three breeds of river buffalo (Bubalus bubalis L.), with evidence of a fragile site on the X chromosome. AB - The cytogenetic study of 182 river buffalo (Bubalus bubalis L., 2n = 50) of Murrah, Mediterranean and Jaffarabadi breeds, from the State of Sao Paulo, was carried out to characterize their chromosomes and to detect possible chromosomal abnormalities. The karyotypes were indistinguishable with conventional staining as well as with C and replication R banding techniques. In about 44% of the sample (8 males and 72 females), an X marker chromosome due to a fragile site was shown. The frequency of metaphases expressing the fragility site on the X was highly variable, from 2.86 to 41.03%. In females, the fragile site, rarely appeared on both X chromosomes. Most of the metaphases showed only 1 marker chromosome. In R-banded metaphases using 5-bromodeoxyuridine (BrdU) treatment, it corresponded in general to the late replicating X chromosome. No correlation between the X fragile site and altered phenotype was found. Structural and numerical chromosome rearrangements were ruled out in the present sample of buffalo. PMID- 10732033 TI - Effect of undernutrition on the distribution of progesterone in the uterus of ewes during the luteal phase of the estrous cycle. AB - The effect of undernutrition on ovarian and uterine venous progesterone concentrations and endometrial progesterone content on Days 5 and 10 of the estrous cycle were studied. Forty ewes were synchronized using progestagen pessaries. At pessary withdrawal, the ewes were fed diets to provide either 1.5 or 0.5 times the daily maintenance requirement (Group H, n = 20 and Group L, n = 20, respectively). Ewes fed the low nutrition diet (Group L) had higher mean peripheral progesterone concentrations than those fed the high plane diet (Group H; P < 0.05) but lower endometrial progesterone content on Day 5 (P < 0.05). Neither ovarian nor uterine venous levels were affected by nutrition on either Day 5 or 10. Progesterone concentrations in blood samples collected ipsilateral to ovaries bearing a corpus luteum (CL) were higher than in the contralateral samples (P < 0.001). It is concluded that undernutrition can produce a reduction of endometrial content of progesterone the first week after mating. Since no differences in ovarian venous concentrations were observed, it remains to be shown whether this variation is due to other variables, such as the population of endometrial progesterone receptors or other nonhormonal factors. PMID- 10732034 TI - Loss of plasma membrane proteins of bull spermatozoa through the freezing-thawing process. AB - The widespread application of A. I. and realization of its full potential depends largely on the use of frozen semen. However, fertility resulting from A. I. is poorer than that from fresh semen in most species. The objective of this study was to compare the protein composition of fresh and frozen-thawed bull sperm plasma membrane surface. The effect of Tween 20 on protein removal from fresh and frozen sperm plasma membrane surface was studied and compared. The effect of incubation with different detergent concentrations on sperm motility and viability was examined. Approximately 2 x 10(8) frozen-thawed bull spermatozoa washed through a discontinuous Percoll gradient were incubated for 15 min at 20 degrees C with 0.01, 0.03 and 0.05% Tween 20. Sperm motility was completely eliminated at all 3 assayed detergent concentrations, while the initial sperm viability of 52% was decreased to 26, 10 and 5%, respectively, at the 3 concentrations. The removal of sperm plasma membrane proteins also increased from 0.72 mg to 2 mg with 0.05% Tween 20. Similar results were found with fresh semen samples. Although the amount of extracted proteins was significantly lower than that obtained with frozen spermatozoa, fresh sperm motility was likewise eliminated by the detergent treatment, and sperm viability was decreased. A semen sample with an initial sperm viability of 59% had a value of only 8% after treatment with 0.05% Tween 20. Comparative SDS-PAGE analysis of the extracted fractions from fresh and frozen-thawed semen treated with Tween 20 showed that the higher amount of extracted proteins in the frozen semen samples corresponded to the egg yolk lipoproteins in the cryoprotectant medium. However, it is worth noting that 4 more bands were found in the sample obtained from fresh semen than from frozen semen. These results indicate that some cell membrane proteins are lost through the freezing-thawing process. PMID- 10732035 TI - The effects of dose and duration of administration of pFSH during the first follicular wave on the ovulation rate of beef heifers. AB - Four experiments were carried out to examine the effects of administration of pFSH (Vetrepharm) from Day 3 of the estrous cycle in conjunction with PG on Day 5 on follicular populations and ovulation rate in heifers. In Experiment 1, 47 heifers were allocated to 1 of 4 treatment groups (n = 11 to 12 per group): a) control, b) 1.5 mg pFSH, c) 2.0 mg pFSH or d) 2.5 mg pFSH until estrus. Heifers assigned to the 3 treatments had a higher ovulation rate than the controls (P < 0.05). In Experiment 2, 45 heifers were allocated to 1 of 5 treatment groups (n = 8 to 10 per group): a) control, b) 1.0 mg pFSH until PG, c) 1.0 mg pFSH until estrus, d) 1.5 mg pFSH until PG or e) 1.5 mg pFSH until estrus. From Day 5, heifers assigned to pFSH treatments had more large follicles than the controls (P < 0.05). There was no effect of treatment on the incidence of twin ovulations. In Experiment 3, 43 heifers were assigned to 1 of 3 groups (n = 11 to 16 per group): a) control, b) 1.0 mg pFSH until estrus or c) 1.5 mg pFSH until estrus. At slaughter, 14 d after administration of PG, the incidence of twin ovulations was 0/11, 7/16 and 8/16 for Groups a, b and c, respectively (P = 0.011). In Experiment 4, pFSH (1.5 mg) was administered to 3 groups during the development of the first dominant follicle: a) growth phase (n = 19); b) static phase (n = 17); and c) decline phase (n = 17). All pFSH-treated heifers had a higher ovulation rate than the controls (P < 0.05); heifers assigned to Group c had a higher ovulation rate than those in Groups a or b (P < 0.05). More heifers assigned to Group c (7/17) superovulated than in the other 2 groups (P < 0.05). In conclusion, administration of 1.0 or 1.5 mg pFSH twice daily beginning at Day 3 of the estrous cycle in association with the induction of luteolysis increased the ovulation rate significantly when pFSH treatment was continued to onset of estrus. The ovulation rate and the occurrence of multiple ovulations were significantly higher when pFSH was administered at the time that the first dominant follicle was in decline. PMID- 10732036 TI - Influence of linoleic/linolenic acid ratio in the diet of periparturient cattle on plasma concentrations of PGF2 alpha metabolite and placental expulsion rate. AB - Forty-eight cows Holstein Friesian x Dutch Friesian (HF x DF) were randomly assigned to 2 groups fed 1 of 2 diets (isocaloric and isonitrogenous but different in linoleic/linolenic acid ratio) from 4 wk before expected parturition until 7 d after calving. Effects of the diet on plasma linoleic/linolenic acid ratio, plasma PGFM levels and placental explusion rate were studied. Dietary treatment resulted in significant differences in linoleic/linolenic acid ratio in blood plasma (1.00 +/- .22 vs 4.41 +/- .53). The placental expulsion rate was not significantly different between the 2 treatment groups. Plasma PGFM levels, as analyzed for 28 cows from 30 d before parturition until 1.5 d after parturition, were similar for the diets. Cows with a longer placental expulsion rate had lower PGFM levels at parturition (for instance, placental expulsion rate shorter (n = 11) and longer (n = 17) than 6 h, 1248 vs 2965 pg/ml, residual standard deviation 1185 pg/ml, P < 0.01). The results show that the dietary linoleic/linolenic acid ratio can influence the plasma linoleic/linolenic acid ratio without affecting the placental expulsion rate or plasma PGFM levels around parturition. PMID- 10732037 TI - Caliper and ultrasonographic measurements of bovine testicles and a mathematical formula for determining testicular volume and weight in vivo. AB - This study quantified the relationship between calibrated caliper and ultrasonographic derived measurements of bovine testicles in vivo with actual testicular length, width, volume and weight. The prolate spheroid formula was tested to accurately predict testicular volume and a modification to predict weight. Ten bulls were employed to derive caliper and ultrasound testicle (n = 20) length and width measurements in vivo. Caliper length measurements were more reliable than ultrasound derived lengths, with correlations of r2 = 0.8023; P < 0.05 and r2 = 0.5111; P < 0.05, respectively. Width for both the calipers and ultrasound measurements when compared to actual width measurements were r2 = 0.7313; P < 0.05 and r2 = 0.8310; P < 0.05, respectively. The prolate spheroid formula is reliable in determining testicle (n = 116) volume (r2 = 0.8928; P < 0.05). Testicular volume and weight are highly correlated (r2 = 0.9776; P < 0.05); therefore, a modification of the prolate spheroid formula was used to predict weight (r2 = 0.9084; P < 0.05) against the actual weight. Caliper-derived length and width measurements used in the prediction of volume and weight had correlation coefficients against actual volume and weight of r2 = 0.5497; P < 0.05 and r2 = 0.6340; P < 0.05, respectively. Ultrasound in vivo measurements for prediction of testicular volume and testicular weight had a correlation of r2 = 0.3276; P < 0.05 and r2 = 0.6249; P < 0.05, respectively. A testicular (n = 116) length to width ratio of 1.8:1 (SEM = 0.01) was determined for both slaughterhouse and castrated animals. Caliper measurements are reliable, inexpensive and much simpler to obtain than ultrasound determinations for in vivo testicle length, width, volume and weight. The two-dimensional measurement of length and width would be a more accurate predictor of testicle volume and weight than the one-dimensional measurement of scrotal circumference (SC), especially in bulls with variation in testicular shape. PMID- 10732038 TI - Changes in ovarian follicles following acute infection with bovine viral diarrhea virus. AB - Bovine viral diarrhea virus (BVDV) has been associated with several reproductive problems in cattle, including poor fertility, early embryonic deaths, abortion and congenital anomalies. Little is known about the cause of poor fertility in cows acutely infected with BVDV. The purpose of this study was to identify changes in ovarian function following acute infection with noncytopathic BVDV. The ovaries of 5 BVDV sero-negative and virus-negative pubertal heifers were monitored daily for 4 consecutive estrous cycles. The position and diameter of all follicles (> 5 mm) and luteal structures were recorded. Daily plasma samples were collected to measure peripheral progesterone and estradiol levels. Each heifer was infected intranasally with noncytopathic BVDV following ovulation of the second estrous cycle. The maximum diameter and growth rate of dominant anovulatory and ovulatory follicles were significantly reduced following acute BVDV infection. Similarly, the number of subordinate follicles associated with both the anovulatory and ovulatory follicle was reduced following infection. There were no significant differences in other follicle or luteal dynamic parameters or in peripheral progesterone or estradiol levels. Ovarian follicular growth was different during the first 2 estrous cycles following acute infection with BVDV when compared with the 2 estrous cycles preceding infection. These differences may be important in explaining reduced fertility in herds with acute BVDV infection. PMID- 10732040 TI - Effect of alpha-tocopherol and ascorbic acid on bovine in vitro fertilization. AB - The aim of this work was to study the effect of alpha-tocopherol (vitamin E) and ascorbic acid on the in vitro fertilization process. Frozen bovine semen was prepared using extenders with and without addition of vitamin E. Samples were capacitated with heparin in the fertilization medium. In vitro matured oocytes were inseminated with spermatozoa frozen with and without vitamin E and, after thawing, fertilized in TALP medium (control) and in TALP medium with vitamin E (1 mg/ml), with ascorbic acid (5 mM) and with vitamin E plus ascorbic acid. Gametes were incubated in the respective fertilization medium for 48 h; those frozen without vitamin E yielded 75, 76, 69 and 49% of fertilized oocytes in the control, vitamin E, ascorbic acid and vitamin E plus ascorbic acid media, respectively. The last value was significantly different (P < 0.01). In bovine sperm frozen with vitamin E, fertilization rates were 74, 50, 47 and 34%, respectively for the 4 groups. Values observed for the different supplements were significantly different inter se (P < 0.01), except between the media with vitamin E and with ascorbic acid. These results indicate that preserved antioxidant capacity of vitamin E impairs the success of the in vitro fertilization process. PMID- 10732039 TI - Local distributions of oviductal estradiol, progesterone, prostaglandins, oxytocin and endothelin-1 in the cyclic cow. AB - The cyclic patterns of hormones which regulate the activity of the oviduct in the cow have not been adequately reported. We studied progesterone (P4), estradiol 17 beta (E2), prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), oxytocin (OT) and endothelin-1 (ET-1) concentrations in the cow oviduct. Reproductive tracts from cyclic Holstein cows in the follicular phase (n = 5), post ovulation phase (n = 5) and luteal phase (n = 5) were collected at a slaughterhouse. Oviducts were separated from the uterus, the lumen vas washed with physiological saline, and the enveloping connective tissues were removed. The fimbria was then separated at first and then the rest was divided into 2 parts of equal length (proximal and distal). After extraction, levels of different hormones in the tissues were measured using double antibody enzyme immunoassays (EIAs). There were no differences in any hormone concentration between the 3 parts of the oviduct at any stage of the estrous cycle. The highest concentration of oviductal P4 was observed during the luteal phase and in the oviduct ipsilateral to the functioning CL. Oviductal OT was unchanged throughout the cycle. The highest E2 concentration was observed during the follicular phase in the oviduct ipsilateral to the dominant follicle. The oviduct ipsilateral to the dominant follicle during the follicular phase and ipsilateral to the ovulation site post ovulation showed higher levels of PGE2, PGF2 alpha and ET-1 than those on the contralateral side or during the luteal phase. The highest PGE2 was observed in the oviduct ipsilateral to the ovulation site during the post ovulation phase. The results suggest that the ovarian products (P4, OT and E2) and the local oviductal products (PGE2, PGF2 alpha, and ET-1) may synergistically control oviductal contraction for optimal embryo transport during the periovulatory period, and provide further evidence for the local delivery of ovarian steroids to the adjacent reproductive tract. PMID- 10732041 TI - Biostimulatory effects of estrous cows and bulls on resumption of ovarian activity in postpartum anestrous Zebu (Bos indicus) cows in the humid tropics. AB - Suckled postpartum Zebu (Bos indicus) cows were exposed either to estrous females alone or together with bulls to determine if this regimen would stimulate the resumption of ovarian activity. The study was carried out on 60 Zebu cows. The animals were allocated at parturition to 1 of 3 treatment groups. Group FB cows (n = 14) were exposed to progestogen treated females (approximately 3 cows per progestogen treated cow) and a bull for 42 d. Group F cows (n = 31) were exposed to progestogen treated females and after 21 d a bull was introduced and remained with the group until the end of the experiment 21 d later. Estrus was induced with Syncro Mate B treatment and the cows were in estrus when introduced into Groups FB and F. Group N cows (n = 15) were introduced to a bull 21 d after the treatments had begun in Groups FB and F, and the bull remained with these cows until the end of the study. On average, resumption of ovarian activity occurred in 57% (8/14), 19% (6/31) and 7% (1/15) of the cows in Group FB, F and N, respectively, within 21 d after the start of treatments. The difference was significant between Group FB and Groups F and N (P < 0.05). During the second part of the experiment, in which the bull was introduced to Groups F and N, an additional 32% (10/31) of the cows in Group F and 33% (5/15) in Group N resumed ovarian activity. No cows in Group FB resumed ovarian activity during this period. There were significant differences in body condition between cows that resumed ovarian activity and those that remained in anestrus (P < 0.05). We conclude that biostimulation effectively enhanced resumption of ovarian activity in anestrous suckled Zebu cows. This technique offers a potentially useful and practical way to improve reproductive efficiency in anestrous Zebu cattle in the tropics. PMID- 10732043 TI - Predictive markers in the late gestation period for retained placenta in black pied dairy cows under field conditions in France. AB - A prospective ecopathogical survey was conducted in French commercial dairy herds located in Brittany. Previous production and reproduction data and blood parameters were used to identify predictive indicators of risk for retained placenta (RP) in Black-Pied cows. All the cows had delivered a single calf after a dry period of at least 30 d and had produced milk for at least 30 d. The cows with and without retained placenta were allocated to groups according to herd and interval between antepartum blood sampling and calving. Two groups of cows with (RP-positive group, n = 45) and without (RP-negative group, n = 184) retained placenta were compared. Univariate analysis indicated lower plasma glucose concentration, lower monocyte count and higher red blood cell count in the RP positive group. A multiple logistic regression was run, with herd and blood sampling to calving interval as the fixed effects. It showed that a high red cell count and a low monocyte count were predictive indicators for retained placenta risk, which was found to be lower at third calving. Relationships of circulating indicators with placental retention etiology are discussed in terms of polyunsaturated fatty acid imbalance, its consequences on monocyte and erythrocyte functions, uterine motility and circulatory disturbances. PMID- 10732042 TI - Close synchrony of ovulation in superstimulated heifers that have a downregulated anterior pituitary gland and are induced to ovulate with exogenous LH. AB - The synchrony of ovulation was examined in superstimulated heifers that had a downregulated pituitary gland and which were induced to ovulate by injection of exogenous LH. The pituitary was downregulated and desensitized to GnRH by treatment with the GnRH agonist deslorelin. Nulliparous heifers (3.5 yr old) at random stages of the estrous cycle were assigned to 1 of 3 groups, and on Day -7 received the following treatments: Group 1 (control, n = 8), 1 norgestomet ear implant; Group 2 (GnRH agonist, n = 8); Group 3 (GnRH agonist-LH protocol, n = 8), 2 deslorelin ear implants. Ovarian follicle growth in all heifers was superstimulated with twice-daily intramuscular injections of FSH (Folltropin-V): Day O, 40 mg (80 mg total dose); Day 1, 30 mg; Day 2; 20 mg; Day 3, 10 mg. On Day 2, all heifers were given a luteolytic dose of PGF (7 A.M.), Norgestomet implants were removed from heifers in Group 1 (6 P.M.). Heifers in Group 3 were given an injection of 25 mg, i.m. porcine LH (Lutropin) on Day 4 (4 P.M.). Ovarian follicle status was monitored at 8-h intervals from Day 3 (8 A.M.) to Day 6 (4 P.M.) using an Aloka Echo Camera and 7.5 MHz transducer. Heifers in Groups 2 and 3 exhibited estrus earlier (P < 0.05) than heifers in Group 1. Heifers in Group 2 did not have a preovulatory LH surge and they did not ovulate. Individual control heifers in Group 1 ovulated between 12 A.M. on Day 5 and 8 A.M. on Day 6. Heifers with deslorelin implants and injected with LH in Group 3 ovulated between 4 P.M. on Day 5 and 8 A.M. on Day 6. It was confirmed that superstimulated heifers with GnRH agonist implants can be induced to ovulate with LH. It was also demonstrated that ovulation is closely synchronized after injection of LH. Thus, a single, fixed-time insemination schedule could be used in a GnRH agonist-LH superovulation protocol, with significant practical and economic advantages for superovulation and embryo transfer programs. PMID- 10732044 TI - High environmental temperature and humidity decrease oocyte quality in Bos taurus but not in Bos indicus cows. AB - Two experiments were conducted to assess the effects of environmental temperature and humidity on the quality and developmental capabilities of bovine oocytes. In Experiment 1, Bos taurus (Holstein and crossbred Angus) cows were subjected to 5 weekly sessions of ultrasound-guided follicle aspiration from February 16 through March 23 (cool season) and 5 sessions from May 22 through June 20 (hot season). In Experiment 2, Bos taurus (Holstein) and Bos indicus (Brahman) cows were superstimulated (Super-Ov) during the months of August (hot season) or January (cool season), and each cow was subjected to a single oocyte aspiration session. In each experiment, oocytes were classified as normal or abnormal based on ooplasm morphology and cumulus cell layers. In Experiment 1, oocytes classified as normal were in vitro matured and fertilized (IVM/IVF), and the resulting embryos cultured for 8 d. All oocytes recovered from superstimulated cows in Experiment 2 were matured and fertilized in vitro and the subsequent embryos cultured for 8 d, regardless of their morphological appearance. In Experiment 1, Bos taurus cows produced a higher (P = 0.02) percentage of normal oocytes during the cool season (75.9 +/- 8.0) than during the hot season (41.0 +/- 9.5). The percentage of fertilized oocytes developing to the 2-cell (82.4), 8-cell (65.4) and morula (46.6) stages were also greater (P < or = 0.06) during the cool season than the hot season (45.0, 21.2, 6.0 for 2-cell, 8-cell and morula stages, respectively). In Experiment 2, Bos taurus cows (Holstein) had a lower (P = 0.01) percentage of normal oocytes in the hot season (24.5 vs 80.0) and a lower (P < or = 0.003) percentage of fertilized oocytes developing to the 8-cell, morula and blastocyst stages. No difference (P > or = 0.57) in the percentage of normal oocytes or in embryo development was detected between seasons in Bos indicus (Brahman) cows. In conclusion, high environmental temperature and humidity resulted in a marked decline in the quality of oocytes retrieved from Bos taurus cows and markedly decreased their in vitro developmental capabilities. In contrast, a high percentage of oocytes retrieved from Bos indicus cows exhibited normal morphology and yielded a high proportion of blastocysts, regardless of season. PMID- 10732045 TI - Estrous behavior and the estrus-to-ovulation interval in Nelore cattle (Bos indicus) with natural estrus or estrus induced with prostaglandin F2 alpha or norgestomet and estradiol valerate. AB - Estrous behavior and the estrus-to-ovulation interval are essential for estimating the best time to artificially inseminate cattle. Because these parameters are not well characterized in the Nelore breed (Bos indicus), the main purpose of the this study was to determine the estrus-to-ovulation interval in Nelore heifers and cows with natural estrus or with estrus induced by treatments with PGF2 alpha or norgestomet and estradiol valerate (NEV). The cows and heifers were observed continuously (24 h a day) to determine the onset of estrus and to study estrous behavior in the cows. Ten hours after the start of estrus the ovaries were scanned every 2 h by ultrasonography to monitor the dominant follicle until ovulation. Blood samples were collected periodically to determine progesterone levels by RIA. Administration of PGF2 alpha (2 injections, 11 days apart) did not induce estrus in most Nelore females in spite of the presence of functional CL, indicated by progesterone concentrations above 6.0 ng/ml in 25 of 28 animals. Treatment with NEV induced high sexual receptivity in cows (10/11), but only 66% ovulated. Cows with natural or induced estrus exhibited behavioral estrus of 10.9 +/- 1.4 h, and ovulation occurred 26.6 +/- 0.44 h (n = 26) after the onset of estrus. In most of the cows (53.8%) estrus began at night (between 1801 and 600 h), and 34.6% it started and finished during the night. It is concluded that in Nelore females ovulation occurs approximately 26 h after the onset of estrus. Additionally, estrous behavior is shorter than in European breeds, and there is a high incidence of estrus at night, which makes it difficult to detect and, consequently, impairs Al in Nelore cattle. The observation that a high percentage of Nelore females with an active CL did not respond to usual dosages of PGF2 alpha warrants further investigation. PMID- 10732046 TI - Epididymal maturation affects calcium regulation in equine spermatozoa exposed to heparin and glucose. AB - Spermatozoal function is affected by the ability to regulate intracellular calcium concentrations ([Ca2+]i), and may be influenced by epididymal maturation as well as environmental components. Regulation of [Ca2+]i in ejaculated and epididymal stallion spermatozoa was monitored over time in various media. Spermatozoa from each of 5 pony stallions (3 ejaculate samples and 1 caput and cauda sample) were labeled with the fluorescent calcium indicator probe Indo-1 in a calcium-free modified Tyrode's buffer. Fluorescent emissions were monitored by a dual wavelength spectrofluorometer over 5 h. Calcium (1 mM) was added at T = 15 min, and heparin (HEP; 10 micrograms/ml) or heparin plus glucose (hGLUC; 5 mM in 10 micrograms/ml heparin) was added at T = 30 min. Spermatozoal Ca2+ content and regulation differed among males (P = 0.0066). Relative initial [Ca2+]i differed significantly among all stages of maturity (0.84 +/- 0.104, 0.76 +/- 0.023, 1.20 +/- 0.036 LSM of relative Ca2+ units for caput, cauda and ejaculate spermatozoa respectively; P = 0.001). Rate of Ca2+ uptake was similar for ejaculate and cauda spermatozoa (0.021 +/- 0.005 and 0.026 +/- 0.002 relative Ca2+ units/sec) but slower for caput spermatozoa (0.012 +/- 0.001; P = 0.0006). There was no immediate effect of HEP or hGLUC in any stage (P > 0.05), and caput spermatozoa did not differ from cauda spermatozoa for any treatment or time period. A significant increase in [Ca2+]i was seen in ejaculate spermatozoa treated with HEP from 2 h on (P < 0.05). This study demonstrates that both the absolute Ca2+ concentration and the rate of Ca2+ internalization in equine spermatozoa is dependent on the stage of maturation. Ejaculate spermatozoa respond to heparin through increased [Ca2+]i, which may play a role in the fertilizing ability of ejaculate spermatozoa. PMID- 10732047 TI - Ultrasonically guided transvaginal oocyte recovery from calves treated with or without GnRH. AB - We investigated the effect of GnRH given after gonadotropin stimulation on follicle growth and oocyte quality in young calves in a transvaginal oocyte recovery program. A 60 mg MPA pessary was inserted into each of nineteen 5-mo-old Friesian calves for 7 d; on Day 5 they received 140 mg, s.c. FSH (Folltropin) and 200 IU, i.m. PMSG and on Day 8 ten of the calves received 40 micrograms, i.m. GnRH (Fertagyl). Follicles were measured and aspirated on Day 9 using an ultrasound unit with a 6 MHz transvaginal probe (Toshiba). Oocytes from individual calves were recovered, graded and cultured in maturation media for 2 h (+GnRH group) or 22 h (-GnRH group), then fertilized and cultured for 6 d in SOF containing 0.8% BSA and amino acids. Oocyte viability (Class A,B or C) and embryo morphology were recorded. This procedure was repeated on the 19 calves plus 5 others 1 m.o. later, after random allocation to their respective groups. Approximately 70% of the calves responded to gonadotropin stimulation (> 2 follicles over 5 mm in diameter). Calves receiving GnRH tended to have both a higher number of follicles > 2 mm in diameter (27.1 vs 18.7) and of aspirated follicles (22.0 vs 14.1); however, there was a large variability between individuals (0 to 83 follicles and 0 to 73 aspirated). The total number of oocytes collected (10.8 vs 10.9) was not affected by GnRH treatment, probably due to the poor recovery rates in the highly stimulated calves from the +GnRH group, but GnRH did improve the proportion of viable oocytes (6.5 vs 4.1) due to a lower number of Class E oocytes (1.4 vs 4.5; P < 0.05). In the GnRH group, 40% of the viable oocytes had matured at the time of collection versus 0% in the group not treated with GnRH. The necessity of different culture runs between times and treatments prevented any meaningful comparison between groups for embryo development. Following the transfer of 19 morula/blastocyst-stage embryos to recipients, 3 pregnancies were detected by ultrasound examination on Day 60, with 1 oocyte originating from the +GnRH group and 2 from the -GnRH group. PMID- 10732048 TI - Nuclear staining and culture requirements for in vitro maturation of domestic bitch oocytes. AB - Oocyte nuclear staining and culture requirements for in vitro maturation (IVM) in the bitch have yet to be fully investigated. In the first part of this study we investigated 7 methods for labeling nuclear material (573 oocytes). The most favorable method involved fixation plus aceto-orcein staining and light microscopy. The influence of serum supplementation of the culture medium for IVM was then investigated (1292 oocytes). Culture was performed in media supplemented with no serum or with 5, 10 and 20% fetal calf serum (FCS) and 0.3 or 4% bovine serum albumin (BSA). Identifiable nuclear material was either a germinal vesicle (GV) or GV breakdown (GVBD). After 48 h in medium plus 0, 5, 10 or 20% FCS and 0.3 or 4% BSA, the percentage of oocytes matured to GVBD was 13, 9, 15, 23, 36 and 40%, and the percentage matured to metaphase I/anaphase I/metaphase II was 4, 12, 24, 14, 36 and 13%, respectively. After 96 h, maturation to GVBD was 31, 14, 21, 11, 50 and 38%, and to metaphase I/anaphase I/metaphase II it was 6, 5, 3, 19, 15 and 9%, respectively. Within the limits of this study, BSA or high concentrations of FCS appear to be optimal for bitch oocyte maturation in vitro. PMID- 10732050 TI - Effects of melatonin implants in pony mares. 1. Acute effects. AB - The effects of melatonin implant treatment over a four week period on LH, estradiol (E2) and progesterone (P4) secretion during the breeding season were studied in ovary-intact and ovariectomized pony mares. Mares with melatonin implants had significantly higher daytime melatonin concentrations than mares with sharm implants (P = 0.0065). In ovariectomized mares, LH secretion did not differ between mares with melatonin and sham implants. In ovary-intact mares, melatonin implants altered the pattern of LH secretion (P = 0.0023) in such a way that an increase in LH secretion was observed during the periovulatory period. Estradiol and P4 secretion were unaffected by melatonin implants. These results suggest that constant administration of melatonin may enhance the secretion of LH during the periovulatory surge but does not adversely affect E2, P4 or basal LH secretion in mares during the breeding season. PMID- 10732051 TI - Effects of melatonin implants in pony mares. 2. Long-term effects. AB - The effects of melatonin implant treatment over a 4 wk period at the summer solstice on the transition into and out of the following anovulatory season were evaluated in ovary-intact and ovariectomized mares. Melatonin implants tended to delay the timing of the final ovulation of the breeding season (P = 0.0797) in the ovary-intact mares. Although the decline in LH secretion associated with the end of the breeding season was parallel between treatments and ovarian statuses, the rate of LH secretion, as expressed by its mathematical accumulation, was lower in ovariectomized, melatonin-treated mares than in ovariectomized, control mares suggesting that melatonin administration advanced the offset of the breeding season in ovariectomized mares (P = 0.0001). The first ovulation of the subsequent breeding season was significantly delayed in the melatonin-treated mares as compared with that of control mares (P = 0.0031). During reproductive recrudescence, the time of the onset of the increase in LH secretion was similar among all 4 groups but the patterns of LH secretion were different for each treatment and ovarian status combination (P = 0.0112). Mares with melatonin implants had a slower rate of increase in LH secretion than control mares (P = 0.0001), and ovariectomized mares had a faster rate of LH increase than intact mares (P = 0.0001). These results suggest that melatonin implants during the summer solstice can alter the annual reproductive rhythm in mares and support the concept that endocrine patterns of reproductive recrudescence are not entirely independent of the ovary. PMID- 10732049 TI - Effects of follicular fluid on fertilization and embryonic development of bovine oocytes in vitro. AB - This study was conducted to evaluate the effect of bovine follicular fluid (BFF) on fertilizability and developmental capacity of bovine oocytes matured in vitro. Oocytes were collected from slaughterhouse ovaries, and matured in TCM199 supplemented with 5% superovulated cow serum (SCS), 2 mM pyruvate and 1 IU/mL PMSG. BFF was aspirated from small follicles (1 to 5 mm in diameter). In Experiment 1, BFF was added to the Brackett and Oliphant (BO) fertilization medium at concentrations of 0, 1, 5, 10 and 20%. After insemination with frozen thawed and heparin-treated (10 micrograms/mL, 15 min) bull spermatozoa for 18 h, some of the oocytes were fixed and stained to evaluate the fertilization rate. The rest of the oocytes were co-cultured in serum-free embryo culture medium (ECM; TCM199 supplemented with 5% SCS, 2 mM pyruvate and 5 micrograms/mL insulin) with bovine oviductal epithelial cells (BOEC) at 38.5 degrees C under 5% CO2 in air, and the developmental capacity of embryos was examined at 2, 7 and 9 d. In Experiment 2, BFF was added to the serum-free ECM with BOEC at 0, 5, 10 and 20% concentrations, and embryos were cultured for 9 d. Fertilization rates and blastocyst rates in low (1 and 5%) BFF in fertilization medium were not significantly different from the control (without BFF). However, high concentrations of BFF (10 and 20%) in the fertilization medium suppressed both fertilization rates and development. Large vesicles with fast monolayer formation were observed at all concentrations of BFF added to ECM with BOEC. There were no significant differences in cleavage or development to blastocyst in different concentrations of BFF added to ECM. However, the rate of development to hatched blastocysts in 20% BFF was significantly lower than that of the control (P < 0.05). The results of the present study indicate that BFF addition to fertilization medium and ECM with BOEC does not improve fertilizability or developmental capacity and that high concentrations of BFF reduce the rate of both fertilization and development. PMID- 10732052 TI - Cleavage, development and competence of sheep embryos fertilized by intracytoplasmic sperm injection and in vitro fertilization. AB - More abnormal fertilization has been found in sheep oocytes after intracytoplasmic sperm injection (ICSI) than after in vitro fertilization (IVF). Although the birth of a normal lamb has been reported, the efficiency of blastocyst production is low. We therefore evaluated the cleavage, development and viability of sheep embryos obtained from ICSI, IVF and sham injection. In vitro matured oocytes either injected or inseminated with spermatozoa were assessed for cleavage 1 and 4 d after injection or insemination, and for development to blastocyst after 7 d of culture. A total of 699 oocytes was injected (ICSI); 198 (30.6%) were activated and 55 (8.5%) developed to the blastocyst stage. Of the 17 recipient ewes with 1, 2, 3 or 4 embryos, 15 (88.2%) were pregnant on Day 18; of these 17 recipients, 7 (41.1%) and 6 (35.2%) ewes remained pregnant on Days 45 and 110, respectively. Two normal lambs were born, one ewe died on Day 110 with 2 normal male fetuses, another ewe aborted on Day 90 and 4 pregnancies were maintained. A total of 517 oocytes was inseminated (IVF); 296 (62%) were activated and 90 (18.8%) reached the blastocyst stage. A total of 19 ewes received 1, 2, 3 or 4 embryos; of these, 13 (68.4%) were pregnant on Day 18, 8 (42.1%) ewes remained pregnant on each of Days 45 and 110. Three ewes delivered 5 lambs. Five pregnancies were maintained. A total of 156 oocytes was sham injected, 38 (24.3%) were activated and no blatocysts were obtained after culture. The results of this study showed that blastocysts obtained after ICSI are potentially viable and are not a result of parthenogenesis. PMID- 10732053 TI - Effect of low temperatures on in-vitro matured bovine oocytes. AB - This research concerned effects of cooling in vitro matured bovine oocytes on subsequent fertilization and development in vitro. Oocytes were maintained at 39 degrees C (control), 20 degrees C, 10 degrees C or 0 degree C for 5, 10, or 20 min, then fertilized and cultured in vitro for 7 d. The proportion of fertilized oocytes that cleaved and developed to the morula/blastocyst stage was compared between different treatments. Duration of exposure had no effect on the results. Fertilization rate was higher (P < 0.05) for oocytes maintained at 39 degrees C (73.2%) than for oocytes cooled at 20 degrees C (58.6%), 10 degrees C (47.3%), or 0 degree C (36.9%). Cleavage rates were 58.3, 45.3, 15.7 and 7.0% for 39 degrees C, 20 degrees C, 10 degrees C and 0 degree C, respectively (P < 0.05). The lowest development rate to the blastocyst stage was obtained with oocytes cooled to 10 degrees C (0.0%) or 0 degree C (0.9%), followed by 20 degrees C (7.1%) and 39 degrees C (16.5%; P < 0.05). In a second experiment, the zona pellucida was removed after cooling but prior to fertilization (zona-free) from a portion of the in vitro- matured bovine oocytes in each treatment. When sperm penetration rates of zona-free oocytes were compared (percentage of oocytes exhibiting > or = 2 pronuclei), there was no difference (P > 0.05) between oocytes cooled at 0 degree C (59.7%) or 10 degrees C (67.9%). However, penetration rates in these 2 groups were lower (P < 0.05) when compared to zona-free oocytes cooled at 20 degrees C (83.1%) or those maintained at 39 degrees C (83.1%). Zona-free oocytes had higher penetration rates (P < 0.05) when cooled at 0 degree C (59.7%) or 10 degrees C (67.9%) than zona-intact oocytes cooled at 0 degree C (37.3%) or 10 degrees C (47.2%). However, there was no difference in the penetration rate when zona-free and zona-intact oocytes were cooled at 20 degrees C or maintained at 39 degrees C. These data demonstrate that cooling in vitro-matured bovine oocytes decreases the percentage of oocytes that undergo fertilization and subsequently develop in vitro. Moreover, at least part of the decrease in fertilization following oocyte cooling is due to effects on the zona pellucida. PMID- 10732054 TI - Alteration of follicular dynamics and superovulatory responses by gonadotropin releasing hormone and follicular puncture in cattle: a field trial. AB - A field experiment was conducted to determine the influence of follicular alteration on superovulatory responses. Ultrasonography was performed once daily over 4 d prior to gonadotropin treatment (Day 0), on the day of estrus during superstimulation, and on the day of embryo collection to monitor follicular development. Animals were superstimulated between Days 8 and 12 of the estrous cycle. Follicular status was altered 2 d prior to initiation of superstimulation (Day 0) with GnRH (Cystorelin, 200 micrograms i.m.) administered with (GnRH puncture group, n = 31) or without (GnRH-no puncture group, n = 52) concomitant removal of the largest follicle by follicular aspiration. Responses were compared with those of an untreated control group superovulated 8 to 12 d after estrus (n = 102). The proportion of animals with a high number (> or = 2) of large follicles (> = 7 mm) on Day 0 was lower (P < 0.001) in the 2 GnRH-treated groups than in the control group, while the increase in the number of medium size follicles (4 to 6 mm) on Day 0 was greater (P < 0.02) in the GnRH-puncture group. During superstimulation, the proportion of superovulatory cycles with a high follicular (> or = 10 follicles) response was similar in the control and GnRH-no puncture groups. Within the GnRH-treated animals, follicular and ovulatory responses were greater in the GnRH-puncture than in the GnRH-no puncture group (P < 0.001 to P < 0.02). Despite these changes in follicular and ovulatory responses, however, the mean number of embryos produced did not differ (P < 0.1) among treatments (4.3 +/- 0.4, 3.7 +/- 0.7, and 5.4 +/- 0.8 in control, GnRH-no puncture, and GnRH-puncture groups, respectively). This was due primarily to an increase in the mean numbers of unfertilized ova (P < 0.005) and in degenerated embryos (P < 0.06) in the GnRH-puncture group. Results indicate that the beneficial effects of treatment with GnRH and follicular puncture 2 d prior to superstimulation on follicular and ovulatory responses were limited by an increase in the number of unfertilized ova and degenerated embryos. PMID- 10732055 TI - Ovarian superstimulation after follicular wave synchronization with GnRH at two different stages of the estrous cycle in cattle. AB - The objective of this study was to evaluate superovulatory programs based on synchronization of follicular waves with GnRH at 2 different stages of the estrous cycle. Sixteen Holstein cows were randomly assigned to 1 of 3 groups and administered GnRH (Cystorelin, 4 ml i.m.) between Days 4 and 7 (Groups 1 and 3) or between Days 15 and 18 (Group 2) of the estrous cycle (estrus = Day 0). Four days after GnRH treatment, > or = 7-mm follicles were punctured in Groups 1 (n = 6) and 2 (n = 6) or were left intact in Group 3 (n = 4). All cows were superstimulated 2 d later (i.e., from Days 6 to 10 after GnRH treatment) with a total of 400 mg NIH-FSH (Folltropin-V) given twice daily in decreasing doses. The GnRH treatment caused a rapid disappearance of large follicles (P < 0.005), rapid decrease in estradiol concentrations (P < 0.003), and increase in the number of recruitable follicles (4 to 6 mm; P < 0.04), indicative of the emergence of a new follicular wave within 3 to 4 d of treatment. Between 4 and 6 d after GnRH treatment, the mean number of 4- to 6-mm follicles decreased (4.7 +/- 1.8 to 1.5 +/- 3.3) in the nonpunctured group but increased (3.9 +/- 1.0 to 7.3 +/- 1.9) in the punctured group of cows (P < 0.05). In response to FSH treatment, the increase in the number of > or = 7-mm follicles was delayed by approximately 2 d in the nonpunctured group (P < 0.006). Moreover, the mean number of > or = 7-mm follicles at estrus was higher (16.9 +/- 1.7 vs 11.5 +/- 3.0; P < 0.1) in the punctured than the nonpunctured group. The increase in progesterone concentration after estrus was delayed in the nonpunctured group (P < 0.1) compared with the punctured follicles. Mean numbers of CL as well as freezable (Grade 1 and 2) and transferable (Grade 1, 2 and 3) embryos were similar (P > 0.1) in punctured and nonpunctured groups. Spontaneous estrus did not occur prior to cloprostenol induced luteolysis in any group, and stage of the estrous cycle during which GnRH was given did not affect (P > 0.1) hormonal and follicular responses in the punctured groups. In conclusion, GnRH given at different stages of the estrous cycle promotes the emergence of a follicular wave at a predictable time. Puncture of the newly formed dominant follicle increases the number of recruitable follicles (4 to 6 mm) 2 d later and, in response to superstimulation with FSH, causes a greater number and faster entry of recruitable follicles into larger classes (> or = 7 mm) and a faster postovulatory increase in progesterone concentrations. PMID- 10732056 TI - Early development and location of embryos in the reproductive tract of Nili Ravi buffalo (Bubalus bubalis): a retrospective analysis. AB - One year data on embryo recovery were analyzed to study the development and descent of preimplantation embryos in Nili Ravi water buffalo. Forty-five superovulatory attempts were performed on 23 buffalo. A total of 45 embryos were recovered either nonsurgically or after slaughtering the animals at various time intervals (85 to 176 h) post estrus. Embryos were located in the oviducts at 85 h after estrus. At 108 h post estrus, most of them (78%) were recovered from the uteri. The embryos had 8 to 16 cells at 85 h post estrus, grew to morulae at 108 h and to compact morulae at 125 h post estrus. Early blastocysts were observed at 141 h post estrus. Blastocysts were predominant (69%) at 156 to 176 h after estrus; no hatched blastocysts were recovered during this time interval. Based on our findings, embryo recovery at around 150 h post estrus (i.e., Day 6 of the cycle) is recommended for compact morulae or blastocysts in the water buffalo. PMID- 10732057 TI - Serum testosterone concentration, efficiency of estrus detection and libido expression in androgenized beef cows. AB - Twenty multiparous, cyclic, nonlactating beef cows were blocked by dominance rank and randomly and equally allotted to 1 of 4 treatment groups: an untreated control group, a synovex-treated group which received 8 Synovex-H implants with no additional hormones, a testosterone-treated group which received 500 mg, i.m. and 1500 mg, s.c. testosterone enanthate on Day 1 with additional 1000 mg, s.c. doses of testosterone enanthate every 14 d, and a synovex + testosterone-treated group which received 8 Synovex-H implants with 500 mg, i.m. and 1500 mg, s.c. testosterone enanthate on Day 1 only. Blood samples were collected via jugular venipuncture once a week beginning 3 wk prior to start of treatment. In addition, samples were collected just prior to treatment; once a day for 1 wk after initiation of treatment; and then twice a week until 225 d after treatment. Efficiency of estrus detection was assessed 22 d prior to start of treatment and every 14 d thereafter for 98 d, using estrus detection trials with synchronized females or modified libido tests. Scores for estrus detection trials included total mounts in 1 h and the percentage of estrous cows detected. Libido was scored on a scale of 0 through 6. All testosterone treatments raised plasma testosterone concentrations above control and pretreatment levels (testosterone and synovex + testosterone > synovex > control; all P < 0.05). Synovex-, testosterone- and synovex + testosterone-treated females performed more mounts in 1 h than the controls (18, 9, 6 and 1, respectively; all P < 0.05). All testosterone-treated cows mounted a higher number of estrous females than the controls (P < 0.05). Only synovex + testosterone- and testosterone-treated cows received libido scores above pretreatment and control values. However, libido of testosterone-treated cows decreased over time, while that of synovex + testosterone-treated females remained high until Day 98. Libido scores correlated positively with the number of mounts in 1 h and the percentage of estrous females detected (0.70 and 0.44, respectively), and the correlation coefficient for these two factors was 0.63. In conclusion, the synovex + testosterone treatment was most effective for producing estrus detector females and libido testing was useful for evaluating sexual activity in androgenized females. PMID- 10732058 TI - Length of postpartum anestrus in goats in subtropical Mexico: effect of season of parturition and duration of nursing. AB - The aim of this study was to determine whether season of birth and length of nursing affected the duration of postpartum anestrus in Creole female goats maintained on a constant plane of nutrition in subtropical Mexico. Three experiments were conducted in the Laguna region in the State of Coahuila, Mexico (26 degrees N). In the first experiment, 34 goats gave birth in January; in the second, 31 females gave birth in May; and in the third, 22 goats kidded in October. At parturition, females were allocated to 1 of 3 groups based on body weight and date of parturition: kids were weaned at 2, 30 or 90 d according to their group. After weaning, females were milked manually once a day until the end of the study. All animals were kept in a shed and were fed alfalfa ad libitum and given 200 g of concentrate daily. Starting 1 wk after parturition, estrous behavior was detected twice daily using an apron-bearing male, and blood samples were obtained twice weekly to determine ovarian activity from the plasma progesterone levels. A strong effect of month of parturition was found on the duration of postpartum anestrus (P < 0.0001), which was longer in females kidding in January (about 200 d) than in those kidding in May (about 100 d) or October (about 50 d). A tendency for an interaction between season of parturition and length of nursing was observed in the length of anovulation (P < 0.07): for parturition in October, anestrus was longer when kids were weaned after 90 d than after 2 or 30 d (P < 0.01). Season of parturition also affected dates of reinitiation of ovulatory and estrous activity (P < 0.001). Proportions of normal, short and long cycles and of associations between estrous and ovulations were not influenced by season of parturition or the age of weaning. These data demonstrate that in subtropical latitudes, season of parturition can dramatically influence the duration of postpartum anestrus independently of the availability of food. PMID- 10732059 TI - Computer-assisted sperm head morphometry analysis (ASMA) of cryopreserved ram spermatozoa. AB - Normal sperm morphology has been shown to be indicative of male fertility; however, subjective methods of assessing morphology are highly variable. Computer assisted sperm morphometry analysis (ASMA) has been developed for the objective analysis of sperm head dimensions. Developing applicable protocols for sperm head morphometry analysis increases the efficiency of these systems. The objective of the current study was to develop accurate methods for employing ASMA of ram sperm heads. Staining methods, optimal sperm sample numbers microscopic magnification and sampling variation within and between technicians were assessed. Frozen semen from 10 fertile rams was thawed and prepared on slides for morphometric analysis. Staining spermatozoa with hematoxylin and rose bengal stains yielded the best results. Ram sperm head morphometry was accurately evaluated on at least 100 spermatozoa at x 40 objective magnification. Using these techniques, a sample could be analyzed in approximately 3 min. No significant differences in sperm head measurements were detected between 2 technicians. The system properly recognized and digitized ram spermatozoa 95% of the time. The morphometric measurements of sperm heads for all rams were as follows: length = 8.08 microns, width = 4.80 microns, width:length ratio = 0.59, area = 29.13 micron 2 and perimeter = 23.93 microns. The mean within analysis coefficients of variation for all individual analyses and parameters ranged from 4.8% for length to 6.0% for area. The variation between replicate analysis was 2.4% or less for both technicians. When applying proper sample preparation and analysis procedures no differences in measurements or variation were observed between the 2 system operators. PMID- 10732060 TI - Association of bovine embryos produced by in vitro fertilization with a noncytopathic strain of bovine viral diarrhea virus type II. AB - In the first experiment, heifers were infected experimentally with bovine viral diarrhea virus type II (BVDV-type II, strain CD87; characterized by high morbidity and mortality). Subsequently, in vitro fertilized embryos were produced from oocytes collected on Day 4, 8, and 16 post infection. In a total of 29 heifers, the infectious virus was detected in 55% of the samples of the follicular fluid, in 10% of the oviductal cells, in 10% of the uterine flushes and in 41% of the in vitro fertilized embryos. The highest number of embryos associated with the virus was detected in the group of animals slaughtered on Day 8 post infection (58%). The amount of the virus (10(1.5-2.0) TCID50/mL) associated with the washed single embryos generated from oocytes of heifers 8 and 16 d post infection was sufficient for disease transmission by intravenous inoculation to the seronegative recipients (6/15). In the second experiment, uninfected oocytes were exposed in vitro to BVDV (10(5) TCID50/mL) in the maturation medium and then fertilized and cultured prior to viral assay. Virus was detected in 4 of 7 samples containing embryos but not in samples of embryos produced from the control group of uninfected oocytes. The presence of BVDV in the IVF system did not affect embryonic development in vitro. In conclusion, it appears that BVDV-type II has the ability to be transferred with oocytes through the IVF system, resulting in infectious embryos with normal morphological appearance which may have a potential for disease transmission. PMID- 10732061 TI - Effect of delayed supplementation of fetal calf serum to culture medium on bovine embryo development in vitro and following transfer. AB - Supplementation of synthetic oviduct fluid (SOF) medium plus amino acids and bovine serum albumin (BSA) with either fetal calf serum (FCS) or charcoal-treated FCS (CT-FCS) from Day 5 of development was investigated to determine if either in vitro or post-transfer development was altered. Development to the compact morula stage or beyond was similar for all 3 treatments. However, blastocyst development at Day 7 was accelerated when serum was added to the medium (21.6, 40.1 and 39.4% blastocysts from cleaved embryos for BSA, FCS and CT-FCS, respectively; P < 0.01), but cell number of the resulting embryos was unaffected. Furthermore, addition of CT-FCS decreased the between replicate variation in embryo development and produced more Grade 1 and 2 quality embryos (25.8%) than BSA supplementation (18.1%; P < 0.05). The transfer of Grade 1 and 2 embryos at Day 7 following culture resulted in similar pregnancy and embryo survival rates for the 3 treatments, with a tendency for lower embryo survival of embryos cultured in FCS (embryo survival at Day 50 = 37.7% vs 53.3% and 57.6% for FCS, BSA and CT FCS, respectively; P = 0.1). Significant fetal loss from Day 50 to term occurred within all 3 treatments. There were no birth weight differences for calves amongst the 3 culture treatments; however, one of the sires produced calves that were significantly heavier than expected, suggesting a possible sire-by-embryo interaction. These results demonstrate that addition of FCS may promote blastocyst development; however, there was also a tendency for lower embryo survival. Thus charcoal treatment of FCS is recommended, because it decreases variability in embryo development between runs and results in embryo survival rates to term similar to that BSA-supplemented media. PMID- 10732062 TI - In vitro maturation of follicular oocytes of the Giant Panda (Ailuropoda melanoleuca): a case report. AB - The Giant Panda is an endangered species that would benefit from biotechnological assistance in reproduction. However, because there are only a few of these animals left in the world, scientists hesitate to use them for research procedures. We were fortunate to obtain ovaries from a Giant Panda that died of hepatic cirrhosis during the nonbreeding season. Oocytes were harvested within 4 h of death by dissecting the ovarian cortex in physiological saline and collecting the cumulus-oocyte complexes from the fluid, and then were classified into large (> 125 microns) and small (100 to 124 microns) follicular oocytes and placed in TCM199 supplemented with FSH (10 micrograms/mL) and LH (20 micrograms/mL). After culture for 22 h at 37 degrees C in air with 5% CO2, response was evaluated by growth of oocytes and presence of the first polar body. Of the 26 large follicular oocytes that were harvested, 12 were considered suitable for IVM, and 14 were degenerated, had a broken zona pellucida or had lost some cytoplasm. Of the 12 cultured oocytes, all grew to a mean diameter of 141.1(SD = +/- 6.7, n = 12), and 4 released the first polar body. None of the small follicular oocytes showed growth or other signs of maturation. We conclude from our preliminary results that it is possible to obtain functional Giant Panda oocytes from ovaries obtained post mortem during the nonbreeding season. PMID- 10732063 TI - Evaluation of two treatments in superovulation of mares. AB - The efficiency of superovulating mares with an enriched fraction of equine follicle-stimulating hormone (feFSH) and an equine pituitary extract (EPE) with similar FSH content but differing in the LH amount was compared. Mares were randomly assigned to an feFSH (n = 5) or EPE (n = 5) treatment. The experimental period was of 2 successive estrous cycles, with the first cycle as the control. At Days 6 and 7 of the estrous cycle, the mares received 250 micrograms i.m. cloprostenol. The treatments consisted of daily injections of 25 mg feFSH or EPE beginning on Day 6 post ovulation. Mares were inseminated every other day until the last ovulation was detected. When the mares in the control and treatment cycles developed at least 1 or 2 > or = 35-mm follicle, respectively, the treatment was interrupted, and a single injection of EPE (25 mg, i.v.) was administered to induce ovulation(s). Nonsurgical embryo recovery was performed 6 or 7 d after ovulation in both control and treatment cycles. The number of ovulations per mare was not significantly different (P > 0.05) between feFSH and EPE groups, but both were higher (P < 0.05) than that of the control cycle. The number of recovered embryos per ovulation was similar (P > 0.05) for control, feFSH and EPE groups. The high amount of LH presented in EPE did not affect the superovulatory response of the mares. Superovulatory treatments increased the ovulation rate of mares but did not affect the embryo recovery rate per ovulation. PMID- 10732064 TI - Caprine blastocyst development after in vitro fertilization with spermatozoa frozen in different extenders. AB - The feasibility of using frozen-thawed semen in caprine IVF outside the breeding season was investigated. Electroejaculated spermatozoa from a Nubian buck were washed twice and then frozen in skim milk- or in egg yolk-based extenders. Goat oocytes were matured and inseminated by frozen-thawed spermatozoa selected by swim-up. In vitro fertilization was performed in a modified defined medium (mDM), altered experimentally, for 24 h. Embryos were cultured in 50 microL of c-SOF + NEA for 9 d. The percentages of oocytes exposed to heparin-capacitated spermatozoa, (previously cryopreserved in skim milk-based extender) that cleaved, reached morula, blastocyst and expanded blastocyst stages were 82.8, 57.1, 35.7 and 30.0%, respectively. Without heparin treatment the rates for cleavage, morula, blastocyst and expanded blastocyst stages were 44.3, 31.4, 18.6 and 8.6%, respectively. Therefore, heparin treatment was included in sperm capacitation. Use of spermatozoa with BSA in the IVF medium yielded no cleavage. Although extenders containing 8 to 20% egg yolk enabled good sperm motility after cryopreservation, in vitro fertilizing ability was compromised under our conditions. By contrast, semen commercially processed in season in an egg yolk based diluent remained effective for IVF. The highest proportion of blastocysts resulted from the use of spermatozoa diluted in a skim milk extender, heparin capacitation, and insemination in medium containing lamb serum. PMID- 10732065 TI - Quantification of somatic and spermatogenic cell proliferation in the testes of ruminants, using a proliferation marker and flow cytometry analysis. AB - We compared 2 methods for the quantification of proliferation in somatic and spermatogenic compartments of post mortem-collected testes in cattle and roe deer. Proliferation was evaluated by estimation of the tissue polypeptid specific antigen (TPS) using an ELISA. This proliferation-specific marker was detected in homogenized cells after selective enrichment of different cell types by density gradient centrifugation. The haploid, diploid and tetraploid cells were monitored by one-parameter flow cytometry and analyzed for mitotic cell cycle. Somatic and spermatogenic cells were discriminated by dual-parameter flow cytometry after DNA staining with propidium iodide and selective labelling of stromatic cells with a vimentin antibody. The TPS was related to the ploidy of cells and their somatic or spermatogenic type. High concentrations of TPS were found in both species. The TPS values varied with different contents of spermatogenic and somatic cells in the fractions of the density gradient. The TPS was positively correlated with spermatogenic cells in the G2/M phase of mitotic cycle (r = 0.474; P < 0.01) and negatively correlated with somatic cells (r = -0.676; P < 0.0001) in roe deer (n = 40). Discrimination of germinative and stromatic cells in the G2-M phase showed their varying proliferation during the annual cycle in roe deer. The quantification of tetraploid spermatogenic cells allowed the calculation of an exact meiotic transformation (ratio haploid:tetraploid cells). In conclusion, TPS indicates proliferation in the germinative compartment of the testes. However, this marker provides only relative values, without information on the number and type of proliferating cells. Dual-parameter flow cytometry using specific staining for vimentin proves to be a better method for studying changing mitotic and meiotic steps during the involution and recrudescence of testes in seasonally breeding ruminants, as it relates proliferative processes directly to both spermatogenic and somatic cells. PMID- 10732066 TI - Preliminary experiments on the cryopreservation of penaeid shrimp (Penaeus japonicus) embryos, nauplii and zoea. AB - To improve availability of penaeid seedstock during periods of high demand, experiments were conducted to determine the feasibility of stockpiling embryos by freezing them. Embryos were screened for developmental stage; cryoprotectants, chilling effects, and freezing regimens were likewise evaluated. Juvenile forms (embryos, nauplii and zoea) of Penaeus japonicus were exposed to various cryoprotectants, including dimethyl sulfoxide, glycerol, methanol, ethylene glycerol and polyethylene glycol 300 under ambient temperature (25 degrees C). Following this bioassay, maximum safe concentrations of each cryoprotectant were tested on the juveniles under chilling to 0 degree C and with 42 freezing regimens. Methanol was found to be relatively nontoxic. Early developmental stages were the most sensitive to chilling. Initial attempts to freeze P. japonicus juveniles were reported. The survival rate of nauplii and zoea treated with 10% methanol in natural sea water (35 ppt salinity) and frozen to -15 degrees C was 85%, and some nauplii and zoea survived freezing to -25 and -196 degrees C. However, no treatment yielded normal nauplii or zoea after freezing. PMID- 10732067 TI - Increased risk of abortion following Neospora caninum abortion outbreaks: a retrospective and prospective cohort study in four dairy herds. AB - Explosive abortion outbreaks in 4 Dutch dairy herds during 1992 to 1994 are reported. In 50 of 51 fetuses submitted during the first 3 wk of the outbreaks characteristic histological lesions compatible for infection with Neospora caninum were seen. Diagnosis of infection was confirmed by immunohistochemistry in 40 fetuses (78%). No evidence for other abortifacients was found. The abortion risk of the herds was investigated in a prospective and retrospective cohort study. The prospective study showed that cows aborting during the outbreaks and N. caninum seropositive nonaborting cows had a two- to three-fold increased risk of abortion compared with N. caninum seronegative cows. Retrospective examination showed that seropositive nonaborting cows had an increased risk of abortion before the outbreaks, which may indicate that these animals were infected with N. caninum prior to the outbreaks. It is concluded that serostatus can be used for selective culling of cows to decrease future risk of abortion in dairy herds. PMID- 10732068 TI - Abortion risk in progeny of cows after a Neospora caninum epidemic. AB - A study was done of the descendants of cows from 4 dairy herds in which there had been N. caninum abortion outbreaks. Precolostral antibodies to N. caninum were demonstrated in 34 of 50 (68%) F1 calves and in 14 of 17 (82%) F2 calves from cows that aborted during the outbreaks. In 214 F1 progeny, N. caninum seroprevalence was nearly 50%, and there was a significant association between serostatus of the offspring and serostatus of dams. These observations indicated that congenital infection was an important mode of transmission after abortion outbreaks in these herds. A total of 52 abortions was recorded in 293 pregnancies of F1 progeny cows (1 to 3 pregnancies per animal). It was found that seropositive F1 cows had a three-fold increased abortion risk compared with seronegative F1 cows. In 2 of 10 abortions in seronegative cows evidence for N. caninum infection was found, suggesting that a low level of postnatal infection may also have occurred. It is concluded that N. caninum-infected calves should not be used as replacement stock, to decrease the future risk of abortion in dairy herds. PMID- 10732069 TI - Superstimulation of ovarian follicular growth with FSH oocyte recovery, and embryo production from Zebu (Bos indicus) calves: effects of treatment with a GnRH agonist or antagonist. AB - The capacity of heifer calves of a late sexually maturing Zebu (Bos indicus) genotype to respond to superstimulation with FSH at a young age and in vitro oocyte development were examined. Some calves were treated with a GnRH agonist (deslorelin) or antagonist (cetrorelix) to determine whether altering plasma concentrations of LH would influence follicular responses to FSH and oocyte developmental competency. Brahman calves (3-mo-old; 140 +/- 3 kg) were randomly assigned to 3 groups: control (n = 10); deslorelin treatment from Day -8 to 3 (n = 10); and cetrorelix treatment from Day -3 to 2 (n = 10). All calves were stimulated with FSH from Day 0 to 2, and were ovariectomized on Day 3 to determine follicular responses to FSH and to recover oocytes for in vitro procedures. Before treatment with FSH, heifers receiving deslorelin had greater (P < 0.001) plasma LH (0.30 +/- 0.01 ng/ml) than control heifers (0.17 +/- 0.02 ng/ml), while plasma LH was reduced (P < 0.05) in heifers treated with cetrorelix (0.13 +/- 0.01 ng/ml). Control heifers had a surge release of LH during treatment with FSH, but this did not occur in heifers treated with deslorelin or cetrorelix. All heifers had large numbers of follicles > or = 2 mm (approximately 60 follicles) after superstimulation with FSH, and there were no differences (P > 0.10) between groups. Total numbers of oocytes recovered and cultured also did not differ (P > 0.05) for control heifers and heifers treated with deslorelin or cetrorelix. Fertilization and cleavage rates were similar for the 3 groups, and developmental rates to blastocysts were also similar. Zebu heifers respond well to superstimulation with FSH at a young age, and their oocytes are developmentally competent. PMID- 10732070 TI - Comparison of three methods of acute administration of progesterone on ovarian follicular development and the timing and synchrony of ovulation in Bos indicus heifers. AB - The aim of this study was to induce the formation of a persistent dominant ovarian follicle and to compare the effects of 3 methods of acute administration of P4 on ovarian follicular development and on the timing and synchrony of ovulation. Stage of the estrous cycle was initially synchronized in Bos indicus heifers with a norgestomet implants (3 mg) for 10 d and with an analogue of PGF2 alpha (15 mg) on the first and last day of norgestomet treatment. Eight days after removal of the implants, heifers were randomly assigned to 4 groups. All heifers received a norgestomet implant (Day 0), which was removed 17 d later (Day 17); PGF2 alpha was administered on Days 0 and 4. Heifers in the control group (n = 5) received no other treatment. On Day 10 heifers in Group P4C (n = 5) were treated with a CIDR for 24 h; heifers in Group P4O (n = 5) were administered 100 mg i.m. of P4 in oil, while heifers in Group P4S (n = 5) were administered 100 mg i.m. of P4 in saline/alcohol. Data were analyzed using bootstrap estimates of location (mean) and spread (standard deviation; SD). Compared with the control heifers, day of emergence of the ovulatory follicle was delayed, and age and duration of dominance of the ovulatory follicle were reduced in the P4C and P4O heifers (P < 0.05) but not in the P4S heifers (P > 0.05). In all groups treated with P4 both the mean and variability (SD) in the timing of ovulation did not differ with that of the control group (P > 0.05) but there was less variability in the day of emergence, age, duration of dominance and diameter of the ovulatory follicle than in the control group (P < 0.05). Delayed timing and reduced synchrony (SD) of ovulation and greater age of the ovulatory follicle (P < 0.05) occurred in P4S heifers than in P4C heifers. We conclude that administration of 100 mg of P4 in oil is as effective as treatment with a CIDR for synchronizing emergence and ovulation of a newly recruited dominant follicle. However, reduced synchrony of ovulation, greater age of the ovulatory follicle and delayed timing of ovulation occurred following administration 100 mg of P4 in saline/alcohol compared with the CIDR device. PMID- 10732071 TI - Quantitative echotexture analysis of bovine corpora lutea. AB - A study was designed to evaluate the attributes of ultrasound images of bovine ovarian CL throughout the estrous cycle. The ovaries of 8 heifers were examined daily by transrectal ultrasonography for 2 interovulatory intervals (ovulation = Day 0). Ultrasonographic examinations of the ovaries were videotaped daily, and recorded images of the CL were digitized for computer analysis of echotexture (mean pixel value and heterogeneity). Blood samples were taken daily and to determine plasma progesterone concentrations. Corpora lutea were of 2 morphological types, those with a central fluid-filled cavity (n = 6) and those without (n = 9). No differences were detected between CL with or without a fluid filled cavity; therefore, data were combined. Mean pixel values of ultrasound images of the CL changed (P = 0.0001) during the interovulatory interval; values decreased (P < 0.05) from Day 0 to Day 3 during early growth of the CL, reached a plateau when increases in luteal diameter ceased, and decreased (P < 0.05) to minimal levels at the onset of regression of the CL. The mean pixel value subsequently increased (P < 0.05) after Day 17 to values similar to those at the beginning of the interovulatory interval. A time-dependent effect was not observed for heterogeneity of images of the CL (P > 0.5). The results supported the hypothesis that quantitative changes in luteal echotexture are reflective of changes in the physiologic status of the CL. PMID- 10732072 TI - The fertility of autumn calving suckler beef cows is increased by the addition of prostaglandin to progesterone and eCG estrus synchronization treatment. AB - The objective of this study was to determine the efficacy of PGF2 alpha treatment on pregnancy and calving rates in autumn-calving suckler beef cows synchronized with progesterone and eCG. The population studied consisted of 124 Charolais and 130 Limousin cows in 13 and 12 beef herds, respectively. In each herd, pairs of cows were formed according to parity, body condition score and calving difficulty. Group 1 received a progesterone releasing intravaginal device (PRID) for 12 d with a capsule containing 10 mg estradiol benzoate at implant insertion and 500 IU eCG at PRID removal (Day 0). Group 2 received the same treatment plus 25 mg i.m. dinoprost at Day -2. Each cow was artificially inseminated 56 h after PRID removal (Day 3). Plasma progesterone concentrations were measured to determine cyclicity prior to treatment in samples take on Days -22 and -12, to confirm the occurrence of ovulation (Day 13) and to determine the early pregnancy rate (Day 26). Serum pregnancy-specific protein B (PSPB) concentrations were determined to assess pregnancy rate at Day 39. The effects of variation factors on pregnancy and calving rates after treatment were studied using logistic mixed models and a Cox model, respectively. There were no significant differences between groups or breeds for the rate of cyclicity before treatment nor for ovulation rate (means, 74.1 and 95.7%, respectively). Cyclicity was, however, influenced by individual factors such as body condition score (OR = 3.36, P = 0.001), parity (OR = 5.4, P = 0.001) and herd factors such as stocking rate (OR = 5.62, P = 0.001). The use of a prostaglandin injection increased pregnancy rate at Day 26 (71.7 vs 56.7%, P = 0.01) and at 39 d (67.7 vs 54.3%, P = 0.02) and the calving rate at induced estrus (64.5 vs 48.5%, P = 0.01). We observed 9 twin calvings (5.6%) which occurred in cyclic cows only before treatment. Cows in Group 2 had a 1.5 greater chance of calving before 300 d following the first AI than cows in Group 1 (P = 0.03). In conclusion, the addition of PGF2 alpha injection, 48 h before PRID removal, increased reproductive efficiency in autumn calving Charolais and Limousin suckler beef cows compared to a classical estrus synchronization treatment using a PRID + eCG. PMID- 10732073 TI - Ovarian follicular wave synchronization and induction of ovulation in postpartum beef cows. AB - An experiment was designed to evaluate a) the effect of a progesterone-estradiol combined treatment on ovarian follicular dynamics in postpartum beef cows, and b) ovulation and the subsequent luteal activity after short-term calf removal and GnRH agonist treatment. Multiparous Angus cows (25 to 40 d after calving) were assigned to the following treatments: untreated (Control, n = 9); short term calf removal (CR, n = 8); progesterone (CIDR, n = 9) and progesterone plus estradiol 17 beta (CIDR + E-17 beta, n = 9). Progesterone treatment (CIDR) lasted 8 d and the day of device insertion was considered as Day 0. Cows in the CIDR + E-17 beta group also received an i.m. injection of 5 mg of E-17 beta on Day 1. On Day 8, calves were removed for 48 h (CR, CIDR and CIDR + E-17 beta groups) and 6 h before the end of calf removal these cows also received an i.m. injection of 8 micrograms of Busereline (GnRH). Anestrus was confirmed in all cows by the absence of luteal tissue and progesterone concentrations below 1 ng ml-1 at the beginning of the experiment. Although mean (+/- SEM) interval from the beginning of the experiment (Day 0) to wave emergence did not differ (P > 0.05) among treatment groups (Control, 1.9 +/- 1.0, range -2 to 7 d; CR, 3.9 +/- 0.7, range 0 to 6 d; CIDR, 2.8 +/- 0.5, range 0 to 4 d and CIDR + E-17 beta, 4.1 +/- 0.2, range 3 to 5), the variability was less (P < 0.05) in the CIDR + E-17 beta group. The proportion of cows ovulating 24 to 48 h after GnRH administration tended (P = 0.08) to be higher in cows from CIDR + E-17 beta group (8/9) than in those of CR (5/8) or CIDR (6/9) groups, respectively and was associated with a higher proportion (P < 0.05) of CIDR + E-17 beta treated cows (9/9) that had a dominant follicle in the growing/early static phase at the time of GnRH treatment compared to the other GnRH treated groups (5/8, and 4/9 for CR and CIDR groups, respectively). Two CR cows ovulated 0-24 h after GnRH and only one Control cow ovulated the day before the time of GnRH administration. Cows pretreated with progesterone had longer (P < 0.05) luteal lifespan (CIDR, 14.5 +/- 0.7, CIDR + E 17 beta, 13.9 +/- 0.6 d) than those not treated with CIDR (Control, 5, CR, 4.0 +/ 0.4). We conclude that progesterone plus estradiol treatment results in tightly synchronized wave emergence and high GnRH-induced ovulation rate with normal luteal activity in postpartum beef cattle. PMID- 10732075 TI - Heritability of scrotal circumference adjusted and unadjusted for body weight in Nellore bulls, using univariate and bivariate animal models. AB - Mixed model methodology under univariate and bivariate animal models was used to estimate genetic parameters of scrotal circumference (SC) unadjusted (h2U) and adjusted (h2A) for body weight, from field data on 875 Nellore males sired by 80 bulls. The models included random effect of animal, the fixed effects of age of dam, year of birth, and month and year of measurement, and the covariate age of the animal. In addition, one of the models also included the covariate body weight. Using univariate models, SC estimates of h2U were 0.18, 0.65, 0.77 and 0.60, and of h2A they were 0.15, 0.60, 0.71 and 0.50, at 9, 12, 18 and 24 m.o. of age, respectively. Bivariate estimates of heritability ranged from 0.18 to 0.77 for h2U, and from 0.13 to 0.70 for h2A. The results suggest that it would be better to consider unadjusted SC rather than adjusted SC in selection programs. Bivariate body weight estimates of heritability ranged from 0.15 to 0.45. Genetic correlations between SC and body weight ranged from 0.58 to 0.71 when body weight was not included in the model, and from 0.33 to 0.64 when body weight was included. These results suggest that simultaneous selection for body weight and SC is possible in Nellore cattle. PMID- 10732074 TI - Plasma levels of GH and PRL and concentrations in the fluids of bovine ovarian cysts and follicles. AB - Prolactin and GH have been detected within the ovary, and it has become increasingly evident that they have a role as intrafollicular regulatory factors. The aim of the present work was to gain an insight into the elements influencing intraovarian GH and PRL in bovine species and to see whether cystic degeneration was accompanied by abnormal bovine GH (bGH) and PRL (bPRL) plasma patterns. We followed the relationships between plasma and ovarian fluid bGH and bPRL concentrations over an entire year in Friesian cows whose ovaries showed distinct types of structures. To assess the presence of bGH and bPRL within ovarian cells, we assayed selected ovarian structures by immunohistochemistry. The results demonstrated that: 1) plasma and ovarian fluid hormonal concentrations were independent, and their ratio was independent of the ovarian structure classes, subclasses and period of the year; 2) in the majority of the cows the concentration of bGH in ovarian fluid was no more than 80% of the level in plasma, whereas in about half the animals bPRL concentrations were higher in the ovary than in peripheral plasma; 3) mean bPRL concentrations in ovarian fluids were significantly higher in summer than in winter; 4) immunoreactive bGH and bPRL were present within granulosa and luteal cells. Thus, it is suggested that in the cow bGH and bPRL levels in the ovary might be regulated in some way independently of the pituitary. PMID- 10732076 TI - Inbreeding and reproduction in mice divergently selected for response to a dietary toxin. AB - This study was conducted to determine whether inbreeding coefficients of selected parents or of progeny differed between lines of mice selected for increased or decreased responsiveness to a nutritional toxicosis. A second objective was to determine whether the influence of inbreeding of parents and/or progeny on reproductive traits differed between those lines. Mice were selected divergently for 8 generations for the effect on post-weaning growth of endophyte-infected fescue seed in their diet. Forty pairs (or in Generation 7, 20 pairs) were selected and mated per generation in each line. Inbreeding increased 0.5 to 0.6% per generation in both lines, a rate close to that predicted from genetic theory. Inbreeding coefficients of selected parents were not higher in the susceptible than in the resistant line. A difference would have been expected if the inbreeding coefficient had been correlated with susceptibility to toxicosis. The magnitudes of inbreeding depression for reproductive traits did not differ significantly between lines. The average inbreeding coefficient of the potential litter tended to be higher in nonfertile than fertile matings (P = 0.10), but inbreeding coefficients of sires and dams did not differ between successful and unsuccessful matings. Inbred litters tended to be born earlier than noninbred litters (P = 0.10). Inbred dams produced smaller litters than noninbred dams (main effect P < 0.05) but only when the litter also was inbred (interaction P < 0.01). Sex ratio was not influenced by inbreeding of sire, dam or litter, but there was a higher proportion of male progeny in the susceptible than in the resistant line (P = 0.01). To avoid reduced reproductive fitness, laboratory animal populations should be managed to minimize inbreeding of progeny and dam. PMID- 10732077 TI - Influence of flushing on LH secretion, follicular growth and the response to estrus synchronization treatment in suckled beef cows. AB - The effects of energy supplementation (flushing) on LH and estradiol secretion, follicular growth and the response to estrus synchronization treatment (Norgestomet + PMSG initiated 41.9 +/- 3.4 d after calving) were investigated in 16 suckled beef cows fed either 70% (Group C, n = 8) of energy requirements from calving to 3 wk after AI or fed the same restricted diet until 11 d before synchronization and then were supplemented with 2 kg concentrate until 3 wk after AI (Group S, n = 8). Concentrations of LH and estradiol 17 beta were measured from 3 sampling periods: 25 and 39 d after calving and between 29 and 49 h after implant removal. Ovaries were examined by ultrasonography 11 d before treatment to implant withdrawal (IR). The effects of energy level, day (or hour) of observation and corresponding interactions were tested on repeated measurements by split-plot ANOVA. No positive effect of flushing was observed on characteristics of LH secretion on Day 39. However, the size of the largest follicle and the number of large follicles were higher in Group S than in Group C cows, respectively, 7 and 9 d after the beginning of flushing to 2 d after the start of treatment. After IR, the estradiol secretion tended to be higher in Group S than in Group C cows (9.8 +/- 0.4 pg/mL vs 7.2 +/- 0.2 pg/mL; P = 0.06), but no effect on LH secretion was observed. After implant removal 12 cows ovulated (Group S: 7/8 vs Group C: 5/8; P > 0.05), 7 were pregnant at 21 d after AI (Group S: 6/8 vs Group C: 1/8; P < 0.05) and 4 at 45 d after AI (Group S: 4/8 vs Group C 0/8; P > 0.05). To conclude, flushing had a positive effect on follicular growth, which does not seem to be mediated by LH. In cows fed a restricted diet, flushing enhanced follicular growth, increased the fertilization rate and/or reduced early embryonic death. PMID- 10732078 TI - Factors affecting pregnancy rates following laparoscopic insemination of 28,447 Merino ewes under commercial conditions: a survey. AB - The results of laparoscopic insemination of 28,447 Australian Merino ewes with semen from 468 rams were used to study factors influencing pregnancy. The overall pregnancy rate was 71.7% (20,423/28,447). Pregnancy rates varied with type of progestagen implant, type and dosage of PMSG, fresh or frozen semen, wool type and number of ewes inseminated per hour. The pregnancy rate (64.6%) obtained with Medroxy-progesterone acetate (MAP) sponges, was significantly (P < 0.01) lower than with Fluorogestone acetate 30 mg (FGA 30; 74.7%) sponges, Fluorogestone acetate 40 mg (FGA 40; 72.1%) sponges, and Controlled Internal Drug Release (CIDR G; 71.7%) implants. A PMSG dose of 200 IU resulted in significantly (P < 0.05) lower pregnancy rates (62.4%) compared with 250 IU (72.9%), 300 IU (79.1%) and > or = 375 IU (69.4%). The mean pregnancy rate for ewes administered Folligon PMSG was 71.9%, which was significantly higher (P < 0.001) than that of ewes treated with Pregnecol PMSG (65.8%). The use of Pregnecol PMSG and MAP sponges was associated, and thus their conditional effects could not be calculated. Ewes inseminated with fresh semen were significantly (P < 0.001) more likely to become pregnant (82.2%) than those inseminated with semen frozen in pellets (69.5%) or straws (71.6%). Ewes inseminated during the months of March, April or May (fall, 71.5%) were just as likely to become pregnant as those ewes inseminated in November, December, January or February (69.6%). Significantly (P < 0.05) fewer strong wool ewes become pregnant to laparoscopic AI, (67.6%) than fine (71.7%), fine medium (73%) or medium wool ewes. Significantly (P < 0.0001) more pregnancies (77.6%) were achieved when more than 55 ewes were inseminated per hour compared with fewer than 35 ewes per hour (63.4%). PMID- 10732079 TI - Equine retained placenta: technique for and tolerance to umbilical artery injections of collagenase. AB - Under laboratory conditions and in clinical experiments, bacterial collagenase has proven to be effective in hydrolyzing placenta and detaching cotyledon from caruncle in the bovine species. Laboratory studies in which placental samples were incubated with collagenase have also demonstrated that collagenase is 3.7 times more effective in hydrolyzing equine placenta than bovine placenta. This led to the hypothesis that collagenase may be a potential treatment for mares with retained placenta. However, that collagenase may hydrolyze the uterine wall and perforate the uterus was a concern. It was the purpose of this study thus to determine any adverse effects of collagenase on the equine uterus and to develop a method for intraplacental injection of collagenase. Three normally expelled intact placentas from Arabian mares, 10 cyclic mixed-breed mares, and 4 mares of various breeds with retained placenta were used. Fluoroscein dye and latex were used to study the placental vasculature and to determine a suitable dose of collagenase; placentas were hydrolyzed by collagenase solution in vitro. Bacterial collagenase solution (40,000 units, 200 ml) was infused into the uterine lumen of each cyclic mare. Uterine biopsies were obtained from the mares before collagenase infusion and again at 16 h and 26 d after infusion. In the mares with retained placenta, each placenta was infused via its umbilical cord vessels with 200,000 units of bacterial collagenase in 1 L of saline. Results showed that none of the uteri from cyclic mares were damaged by collagenase treatment. During a 4-wk period of monitoring (including endoscopy) mares with retained placenta did not show any abnormalities. Retained placentas were expelled in less than 6 h after collagenase treatment. It was concluded that intraplacental injections of collagenase are a safe and potentially effective treatment for retained placenta in mares. PMID- 10732080 TI - The effects of perphenazine and bromocriptine on follicular dynamics and endocrine profiles in anestrous pony mares. AB - Nineteen anestrous pony mares were used in a project designed to determine the effects of altered prolactin concentrations on follicular dynamics and endocrine profiles during spring transition. The dopamine antagonist, perphenazine, was administered daily to mares (0.375 mg/kg body weight) in Group A (n = 6), while Group B mares (n = 7) received 0.08 mg/kg metabolic weight (kg75) dopamine agonist, 2-bromo-ergocriptine, intramuscularly twice daily. Mares in Group C (n = 6) received 0.08 mg/kg75, i.m., saline twice daily. Treatment began January 20, 1994, and continued until ovulation occurred. Mares were teased 3 times weakly with an intact stallion. The ovaries of the ponies were palpated and imaged weekly using an ultrasonic B-mode unit with a 5 Mhz intrarectal transducer until they either exhibited estrual behavior and had at least a 20-mm follicle, or had at least a 25-mm follicle with no signs of estrus. At this time, ovaries were palpated and imaged 4 times weekly. Blood samples were obtained immediately prior to ultrasonic imaging for measurement of prolactin, FSH and estradiol-17 beta. Perphenazine treatment advanced the spring transitional period and subsequent ovulation by approximately 30 d. Group A exhibited the onset of estrual behavior earlier (P < 0.01) than control mares. In addition, Group A mares developed large follicles (> 30 mm) earlier (P < 0.01) than Group B mares, with least square means for Groups A and B of 47.0 +/- 8.8 vs 88.1 +/- 8.2 d, respectively. Control mares developed 30-mm follicles intermediate to Groups A and B at 67.3 +/- 8.8 d. Bromocriptine decreased (P < 0.05) plasma prolactin levels throughout the study, while perphenazine had no significant overall effect. However, perphenazine treatment did increase (P < 0.05) mean plasma prolactin concentrations from Day 31 to 60 of treatment. There were no differences in mean plasma FSH or estradiol 17 beta between treatment groups. We concluded that daily perphenazine treatment hastened the growth of follicles and subsequent ovulation while bromocriptine treatment appeared to delay the growth of preovulatory size follicles without affecting the time of ovulation. PMID- 10732081 TI - Effect of mare's age and recovery methods on the recovery rate of equine follicular oocytes for IVM procedures. AB - Mares (n = 39) were classified according to age as young (less than 1.5 yr, n = 17) or old (more than 1.5 yr, n = 22) and sacrificed. Ovaries were measured and weighed, and the number of follicles and CL were counted. Follicle size and distribution were recorded (external: > 20 mm, < 20 mm; internal: > 5 mm, < 5 mm). External follicles were aspirated while internal follicles were sliced. The number and Type of oocytes recovered using each method were recorded. Oocyte recovery rates (oocytes/ovary) resulted in a mean of 0.92 oocytes by aspiration and 1.36 oocytes by additional slicing. The mean numbers of available follicles (8.11 and 5.02) oocytes recovered (4.94 and 3.02) and oocytes selected per ovary (3.29 and 2.32) were not significantly different in the young or old mares, respectively. PMID- 10732082 TI - Effect of sperm number and frequency of insemination on fertility of mares inseminated with cooled semen. AB - In this study, we tested the hypothesis that insemination of mares with twice the recommended dose of cooled semen (2 x 10(9) spermatozoa) would result in higher pregnancy rates than insemination with a single dose (1 x 10(9) spermatozoa) or with 1 x 10(9) spermatozoa on each of 2 consecutive days. A total of 83 cycles from 61 mares was used. Mares were randomly assigned to 1 of 3 treatment groups when a 40-mm follicle was detected by palpation and ultrasonography. Mares in Group 1 were inseminated with 1 x 10(9) progressively motile spermatozoa that had been cooled in a passive cooling unit to 5 degrees C and stored for 24 h. A second aliquot of semen from the same collection was stored for an additional 24 h and inseminated at 48 h after collection. Mares in Group 2 were inseminated once with 1 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. Group 3 mares were inseminated once with 2 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. All mares were given 2500 IU i.v. hCG at the first insemination. Pregnancy was determined by ultrasonography 12, 14 and 16 d after ovulation. On Day 16, mares were administered i.m. 10 mg of PGF2 alpha and, upon returning to estrus, were randomly reassigned to a group for repeated treatment. Semen was collected from one of 3 stallions every 3 d; mares with a 40-mm ovarian follicle were inseminated with semen from the stallion collected on the preceding day. Semen was allocated into doses containing 1 x 10(9) progressively motile spermatozoa, diluted with dried skim milk-glucose extender to a concentration of 25 x 10(6) motile spermatozoa/ml (total volume 40 ml), placed in a passive cooling unit and cooled to 5 degrees C for 24 or 48 h. Response was measured by number of mares showing pregnancy. Data were analyzed by Chi square. Mares inseminated twice with 1 x 10(9) progressively motile spermatozoa on each of two consecutive days had a higher pregnancy rate (16/25, 64%; P < 0.05) than mares inseminated once with 1 x 10(9) progressively motile spermatozoa (9/29, 31%) or those inseminated once with 2 x 10(9) progressively motile spermatozoa (12/29, 41%). Pregnancy rates did not differ significantly (P > 0.10) among stallions (69, 34 and 32%). Interval from last insemination to ovulation was 0.9, 2.0 and 2.0 d for mares in Groups 1, 2 and 3, respectively. Based on these results, the optimal insemination regimen is a dose of 1 x 10(9) progressively motile spermatozoa given on two consecutive days. However, a shorter interval (< or = 24 h rather than > 0.9 d) between insemination and ovulation may affect pregnancy rates, and needs to be investigated. PMID- 10732083 TI - Body condition influences maintenance of a persistent first wave dominant follicle in dairy cattle. AB - The objective of this study was to determine whether plasma concentrations of progesterone (P4) from a controlled internal drug releasing (CIDR) device (approximately 2 ng/ml) were adequate to sustain a persistent first wave dominant follicle (FWDF) in low body condition (LBC, body condition score [BCS] 1 = lean, 5 = fat [2.3 +/- 0.72, n = 4]) compared with high body condition (HBC, BCS = 4.4 +/- 0.12, n = 4) nonlactating dairy cows. On Day 7 of the estrous cycle (Day 0 = estrus), cows were treated with PGF2 alpha (25 mg i.m. Lutalyse, P.M., and Day 8 A.M.) and a used CIDR device containing P4 (1.2 g) was inserted into the vagina until ovulation or Day 16. Plasma was collected for P4 and estradiol (E2) analyses from Day 5 to Day 18 (or ovulation), and ovarian follicles were monitored daily by ultrasonography. Mean concentrations of plasma P4 were greater in HBC than LBC cows between Days 5 and 7 (4.6 > 3.4 +/- 0.37 ng/ml; P < 0.04). All LBC cows maintained the first wave dominant follicle and ovulated after removal of the CIDR device (18.3 +/- 0.3 d, n = 3; Cow 4 lost the CIDR device on Day 11 and ovulated on Day 15), whereas in the HBC cows ovulation occurred during the period of CIDR exposure (11.3 +/- 0.3 d; n = 3; a fourth cow developed a luteinized first wave dominant follicle that did not ovulate during the experimental protocol on Day 19). Mean day of estrus was 17 +/- 0.4 for LBC (n = 3) and 10 +/- 0.4 for HBC (n = 3) cows. Sustained concentrations of plasma E2 (12.9 +/- 2.8 pg/ml; Days 8 to 17) in LBC cows reflected presence of an active persistent first wave dominant follicle. The differential effect of BCS on concentrations of plasma P4 (y = ng/ml) was reflected by the difference (P < 0.01) in regressions: yLBC = 19.9 - 3.49x + 0.166x2 vs yHBC = 37.3 - 7.04x + 0.340x2 (x = day of cycle, Days 7 to 12). Although P4 concentration was greater for HBC cows prior to Day 8, a greater clearance of plasma P4 released from the CIDR device in the absence of a CL altered follicular dynamics, leading to premature ovulation in the HBC cows. A greater basal concentration of P4 was sustained in LBC cows that permitted maintenance of a persistent first wave dominant follicle. PMID- 10732084 TI - Evaluation of timed insemination during summer heat stress in lactating dairy cattle. AB - We wished to compare the effect of summer heat stress on pregnancy rate in cows that were inseminated at a set interval associated with a synchronized ovulation vs those inseminated upon routine estrus detection. The study was carried out on a commercial dairy farm in Florida from May to September 1995. Lactating dairy cows were given PGF2 alpha (25 mg i.m.) at 30 + 3 d postpartum and randomly assigned to be inseminated at a set time (Timed group) or when estrus was detected (Control group). Cows in the Timed group were synchronized by sequential administration of Buserelin (8 micrograms i.m.) on Day 0 at 1600 h, PGF2 alpha (25 mg i.m.) on Day 7 at 1600 h and Buserelin (8 micrograms i.m.) on Day 9 at 1600 h. They were inseminated on Day 10 between 0800 and 0900 h (Day 9 + 16 h). Cows in the Control group were given PGF2 alpha at 57 + 3 d postpartum and inseminated when detected in estrus. Estrus detection or insemination rate for control insemination cows was 18.1 +/- 2.5% versus 100% for time inseminated cows (P < 0.01). Mean interval from PGF2 alpha to insemination was shorter for time inseminated cows (3 +/- 2.1 d < 35.5 +/- 1.9 d; P < 0.01). Pregnancy rate was greater for time inseminated cows (13.9 +/- 2.6 > 4.8 +/- 2.5%; P < 0.01) as was overall pregnancy rate by 120 d postpartum (27.0 +/- 3.6 > 16.5 +/- 3.5%; P < 0.05). Number of days open for cows conceiving by 120 d postpartum was less for time inseminated cows (77.6 +/- 3.8 < 90.0 +/- 4.2 d; P < 0.05), as was interval to first service (58.7 +/- 2.1 < 91.0 +/- 1.9 d; P < 0.01). Services per conception were greater for time inseminated cows (1.63 +/- 0.10 > 1.27 +/- 0.11; P < 0.05). The timed insemination program did improve group reproductive performance. However, the timed insemination program will not protect the embryo from temperature-induced embryonic mortality, but management limitations induced by heat stress on estrus detection are eliminated. An economical evaluation of the timed insemination program indicates an increase in net revenue per cow with implementation of timed insemination for first service during the summer months. PMID- 10732085 TI - Effects of caffeine and/or heparin in a chemically defined medium with or without glucose on in vitro penetration of bovine oocytes and their subsequent development. AB - Bovine cumulus-enclosed oocytes were matured in culture for 22 to 24 h, freed from cumulus cells and inseminated with frozen-thawed spermatozoa in a chemically defined medium containing 1 mg polyvinylalcohol/ml with or without 5 mM caffeine and/or 10 micrograms heparin/ml and 13.9 mM glucose. Penetration of oocytes was observed only in the medium containing caffeine and/or heparin. Regardless of the presence of glucose, similar proportions of oocytes were penetrated in the medium containing heparin with (73 and 83%) or without (36 and 41%) caffeine. However, when the medium was supplemented with caffeine only, a higher penetration rate was observed in the presence (41%) than in the absence (27%) of glucose. When oocytes inseminated in medium containing caffeine and heparin with or without glucose were cultured in a chemically defined, protein-free medium, 72 and 90% and 9 and 21% of inseminated oocytes developed to the > or = 2-cell and blastocyst stages 48 and 192 h post insemination, respectively. These results, obtained using chemically defined conditions, indicate that glucose is required for stimulating fertilization in vitro of bovine oocytes and that synergistic action of caffeine and heparin appears independently of the reversing activity of caffeine on the inhibition of heparin-induced sperm capacitation by glucose. PMID- 10732086 TI - Cytogenetic analysis of bovine parthenotes after spontaneous activation in vitro. AB - We conducted a cytogenetic study of bovine parthenotes derived from oocytes matured and cultured in vitro. In vitro maturation was carried out by culturing follicular oocytes for 24 h in TCM199 supplemented with estrous cow serum (ECS) and hormones at 39 degrees C in 5% CO2. Matured oocytes were incubated for 20 h in sperm TALP without the addition of spermatozoa, after which they were cultured in maturation droplets for 48 to 72 h. Spontaneous activation occurred in 9.5% of the matured oocytes. Cytogenetic analysis of 24 parthenotes revealed that 62.5% exhibited a normal, diploid chromosome complement. The remaining 37.5% had various ploidy anomalies: haploidy (25%), triploidy (4.2%) and tetraploidy (8.3%). Parthenotes exhibited different developmental stages. The number of blastomeres ranged from 2 to 8 within a parthenote. Only 1 parthenote was comprised 9 to 16 cells. The results showed that spontaneous parthenogenetic activation which occurs in an IVM/IVF system may interfere with embryo production efficiency. PMID- 10732087 TI - Effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes. AB - The effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes was investigated. Oocytes with compact cumulus cells were cultured for 0, 6, 12 and 24 h in TCM199 supplemented with 5% fetal bovine serum (FBS) in 3% CO2 in air. The oocytes were first exposed to 20% ethylene glycol solution and were subjected to vitrification in a solution containing 40% ethylene glycol, 18% Ficoll-70 and 0.3 M sucrose. After warming in 20 degrees C water, oocytes which had been vitrified at less than 24-h of IVM were again cultured to complete the 24-h of IVM period. Oocytes were then incubated with frozen-thawed spermatozoa in Brackett and Oliphant (BO) medium containing 60 micrograms/ml heparin and 0.25% BSA for 20 h. In vitro fertilization rates of oocytes vitrified-warmed at 0, 6, 12 and 24-h IVM were 75.2, 68.0, 82.0 and 72.4%, respectively, comparable to the rates for unvitrified control oocytes (80.6%). A higher incidence of polyspermic fertilization was observed in oocytes vitrified at 24-h IVM (44.9 vs 22.6% in the control group, P < 0.05). Vitrification of oocytes at 12-h IVM seemed to be better than that of other IVM groups, since the normal fertilization rate of all treated oocytes was the highest (36.0%) among the vitrification groups. Developmental competence of the oocytes following vitrification and in vitro fertilization (12-h IVM group) was examined by cell-free culture of presumptive zygotes up to 9 d in modified synthetic oviduct fluid (mSOF) in 5% CO2, 5% O2 and 90% N2. The cleavage rate of zygotes from vitrified oocytes 48 h after insemination was 29.8%, which was lower than that of the control group (57.0%, P < 0.05). Development to blastocysts from the vitrified oocytes (4.8%) was much lower than that of the control group (27.0%, P < 0.05). These results indicate that cryopreservation of bovine oocytes by vitrification may be affected by their maturation stage in vitro, and that developmental competence to blastocysts of cleaved oocytes following vitrification may be impaired compared with unvitrified control oocytes. PMID- 10732088 TI - Release of proinflammatory cytokines related to luteolysis and the periparturient acute phase response in prostaglandin-induced parturition in cows. AB - An acute phase response was previously found in cows at parturition, which might be associated with uterine cytokine release. Five late pregnant cows were implanted with vascular catheters in both the maternal aorta and uterine vein. Blood samples were taken to study temporal relationships between changing plasma levels of proinflammatory cytokines and the periparturient acute phase response following prostaglandin (PG)-induced luteolysis at Day 275 of gestation. The plasma levels of three proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and interleukin-6 (IL-6), as well as progesterone (P4), PGFM and serum amyloid A (SAA) were measured every 4 h between PG induction and expulsion of the calf. In the arterial plasma, progesterone levels dropped to baseline levels within 10 h following PG treatment, indicative of complete luteolysis. Contrary to expectations, the uterine vein samples showed lower proinflammatory cytokine levels compared with the maternal aorta values. A classical acute phase response, as assessed by SAA, was observed during the expulsive stage, but not during luteolysis. PMID- 10732089 TI - Effects of passive immunization using antibody against an alpha-inhibin peptide on follicle-stimulating hormone concentrations and litter size in sows. AB - The objective of the experiment was to determine whether passive immunization against inhibin at weaning would increase FSH secretion and thereby influence postweaning reproductive performance in sows. Commercial Yorkshire sows (n = 173) were assigned within parity to 5 alpha-inhibin fragment antibody (alpha-IF-Ab) dosage groups: 0 (control), 3.25, 6.5, 13 and 26 RP-2 kU/kg alpha-IF-Ab. Antibody had been semipurified from ovine antisera raised against alpha-IF, a peptide that mimicked the N-terminal region of inhibin's alpha-subunit. A RP-2 U refers to a laboratory reference preparation. Sows were administered a single intramuscular injection of control solution or alpha-IF-Ab just before 21-d-old piglets were weaned. Blood samples were taken immediately before immunization and 24 h later. Sows were bred upon expression of estrus. Serum alpha-IF-Ab titers in sows 24 h following passive immunization increased (P < 0.001) with dosage. In control sows serum FSH concentrations decreased 24% by 24 h postweaning (P < 0.001). The decrease was diminished or prevented by alpha-IF-Ab treatment in a dose responsive manner (P < 0.001). Most (167/173) sows were bred within 10 d postweaning, and wean-to-service intervals tended (P < 0.1) to be shorter in the 13 and 26 RP-2 kU/kg alpha-IF-Ab dosage groups. Farrowing rate was 72% (124/173) and was similar among sows in the alpha-IF-Ab dosage groups. Litter size, expressed as total or live piglets born per sow or per sow farrowed, was unchanged by alpha-IF-Ab treatment. Results demonstrate that 1) inhibin plays a key role in regulating FSH secretion at weaning, and 2) blocking the acute postweaning drop in FSH secretion has little if any effect on subsequent reproductive performance. PMID- 10732090 TI - Effect of exogenous gonadotropins on the weaning-to-estrus interval in sows. AB - The efficacy of PG 600 (400 IU PMSG and 200 IU hCG) for accelerating the onset of estrus was determined for sows weaned during the summer. Yorkshire sows (average parity = 4.6), nursing 8.6 +/- 0.2 pigs (mean +/- SEM) were weaned after 27.7 +/- 0.4 d of lactation and were treated intramuscularly with either PG 600 (n = 35) or with 0.9% saline (n = 35). Sows were checked for estrus once daily in the presence of a mature boar. Treatment with PG 600 increased (P < 0.05) the percentage of sows in estrus within 7 d after weaning (97.1 vs 82.9%). Relative to controls, sows given PG 600 expressed estrus sooner (3.8 +/- 0.1 d vs 4.5 +/- 0.1 d; P < 0.01). Sows exhibiting estrus within 7 d after treatments were artificially inseminated 0 and 24 h after first exhibiting estrus. The percentage of inseminated sows that farrowed tended to be higher (P < 0.07) for control than for PG 600-treated sows (96.6 vs 82.3%). The number of pigs born live was similar (P > 0.1) for sows treated with PG 600 and with saline, and was 12.7 +/- 0.6 and 11.7 +/- 0.7, respectively. Pigs farrowed by saline-treated sows, however, tended to be heavier (P < 0.09) than pigs farrowed by sows treated with PG 600 (1.49 +/- 0.06 kg vs 1.34 +/- 0.06 kg). In summary, PG 600 accelerated the onset of estrus in sows weaned during the summer. Sows mated during the induced estrus, however, tended to have a lower farrowing rate and farrowed lighter pigs than control sows inseminated during a natural estrus occurring within 7 d after weaning. PMID- 10732092 TI - Effect of adrenalin and propranolol on progesterone and oxytocin secretion in vivo during the caprine estrous cycle. AB - The effects of jugular infusions of adrenalin and the beta-adrenergic receptor antagonist propranolol on plasma concentrations of progesterone and oxytocin were examined at 2 different stages of the caprine estrous cycle. Adrenalin (25 micrograms.kg-1h-1) significantly (P < 0.05) increased oxytocin secretion on Day 3 and Day 10 of the cycle (estrus = Day 0); progesterone concentrations were significantly (P < 0.05) elevated on Day 10 alone. Propranolol had no effect on progesterone secretion yet significantly (P < 0.05) reduced oxytocin concentrations on Day 3. These results suggest that there may be neuroendocrine involvement in the regulation of luteal oxytocin secretion in the goat. PMID- 10732091 TI - Treatment of swine summer infertility syndrome by means of oxytocin under field conditions. AB - Endogenous oxytocin is released by the sow at the time of mating in response to stimulation by the boar, which may explain, at least partially, the importance of the relationship between the boar's courting activity and the subsequent reproductive performance of the sow. The aim of this study was to determine the effects on reproductive performance of supplementing AI doses with exogenous oxytocin during the low fertility season. At an intensive piggery in northwest Spain 3 experimental groups were randomly formed and observed throughout the year. Group 1 sows were inseminated with semen supplemented with 4 IU oxytocin. Group 2 sows received 4 IU oxytocin injected through the vulvar lips mucosa at the time of insemination. Group 3 sows were inseminated without oxytocin and served as the controls. During the low fertility season the results for each group were as follows: farrowing rate 77.02, 56.25 and 54.39%, and litter size 10.77 +/- 0.28, 10.45 +/- 0.31 and 8.53 +/- 0.34 respectively. It is concluded that the addition of oxytocin to seminal doses just before AI is an easily applicable, effective method for increasing fertility and litter size during the summer months. PMID- 10732093 TI - Early preantral mouse follicle in vitro maturation: oocyte growth, meiotic maturation and granulosa-cell proliferation. AB - Mechanically isolated early preantral mouse follicles were cultured singly for 16 d and fully grown oocytes were obtained from these follicles. We then compared in vitro and in vivo follicle growth by trypsinising the follicles and counting their cell numbers in a Neubauer-counting chamber and recording the diameter and meiotic status of oocytes under an inverted microscope. As long as the granulosa cells were within the basal membrane, proliferation was slow. From Day 6, when granulosa cells had broken through the basal membrane, the proliferation rate progressed up to Day 10 and decreased thereafter to approximately 12,000 cells per culture droplet. Incorporation of BrdU revealed that proliferating cells were evenly distributed throughout the follicle until antrum formation. As granulosa cell differentiation progressed, proliferation of mural-granulosa cells ceased, while cells around the oocytes continued dividing. Oocyte diameter increased discontinuously in relation to follicle remodelling. During the first growth phase, diameters increased from 56.5 (+/- 4.4 microns) to 67 (+/- 4.1 microns) until the onset of antral-like cavity formation. The last growth phase started after Day 10, and by Day 14 oocyte diameters were not significantly different from those of 26-d-old in vivo control oocytes. The potential to resume meiosis after mechanical removal of granulosa cells was first reached on Day 8; thereafter, removal of the corona showed that all oocytes cultured with FSH remained arrested at the GV stage up to Day 16. After Day 8, approximately 70% of all oocytes underwent GVBD as a result of granulosa-cell removal, but only 23% of these reached MII after 24 h. The in vivo controls reached a comparable GVBD rate (66%) when the granulosa was removed, but most of the oocytes (82%) underwent first polar body extrusion 24 h later. These results suggest that although oocyte diameters after IVM are not different from those of the controls, culture conditions are not yet adequate to support complete meiotic maturation. PMID- 10732094 TI - Early pregnancy detection and the hormonal characterization of embryonic-fetal mortality in fallow deer (Dama dama). AB - The objectives of this investigation were to 1) determine serum concentrations of progesterone (P4), estrone sulfate (E1S) and pregnancy-specific protein B (PSPB) from estrus synchronization through mid-gestation in the fallow doe (Dama dama) and 2) characterize the hormonal profiles of does whose embryos or fetuses died in utero. Ten fallow does were synchronized for 14 d with an intravaginal P4 releasing device (CIDR) and were naturally mated after CIDR removal. Blood samples were collected at CIDR insertion, CIDR removal and at intervals through Day 203 post-CIDR removal for analysis of P4, E1S and PSPB by radioimmunoassay (RIA). Ultrasonography was performed on Days 49 and 69 post-CIDR removal. Serum P4 at the time of CIDR insertion was 4.8 +/- 0.6 ng/ml, and at CIDR withdrawal it was 6.2 +/- 0.3 ng/ml. Concentrations of E1S and PSPB were nondetectable at CIDR insertion. Serum E1S was highest at Day 93, and PSPB was first detectable in pregnant does at Days 27 to 30 post-CIDR withdrawal. Ultrasonography on Day 49 revealed that 6 does were pregnant, 2 were not pregnant and 2 others were diagnosed originally as early pregnant. At Day 69, ultrasonography revealed that 6 does (60%) were pregnant and 4 (40%) were not. A comparison of the ultrasonographic and hormonal data indicated that the 2 does diagnosed as early pregnant on Day 49 had conceived but had lost the pregnancy. A third doe which was pregnant on Day 69 lost the fetus later in gestation. Hormonal profiles of does whose embryo or fetus had died were characterized by erratic P4 and E1S profiles, with PSPB becoming undetectable in the 3 does by Days 49, 65 and 80 post-CIDR removal. These data 1) demonstrate the timing for the collection of serum samples for determining early pregnancy in fallow does using 3 hormonal methods and 2) characterize the hormonal profiles of 3 fallow does with embryonic fetal loss. PMID- 10732095 TI - Quantification of bull sperm characteristics measured by computer-assisted sperm analysis (CASA) and the relationship to fertility. AB - Two experiments were conducted to evaluate semen quality of bulls housed under controlled conditions at a large AI facility and relate results to fertility. In Experiment 1 semen was collected from six 6-yr-old bulls twice daily at 3- to 4-d intervals for 3 d. In Experiment 2 eleven 6- to 11-yr-old bulls were used. Extensive breeding information was available and semen was collected as in Experiment 1 but replicated 4 times. Standard semen analysis and computer assisted sperm analysis (CASA) with the Hamilton Thorne IVOS, model 10 unit, were performed on 36 first and second ejaculates in Experiment 1 and on 44 first ejaculates in Experiment 2. Sixteen fields (2 chambers with 8 fields per chamber) were examined per sample. In Experiment 1 the correlation between estimated sperm concentration by spectrophotometry and CASA was 0.91 (P < 0.01). Among bulls the range in the percentage of motile spermatozoa was 52 to 82 for CASA versus 62 to 69 for subjective measurements made by highly experienced technicians. Thus, CASA, with high repeatability, provided a more discriminating estimate of the percentage of motile sperm cells than did the subjective procedure. Bull effect was much greater than any other variable in the experiments. Chamber differences were small and so the results for the 2 chambers with 8 fields each were combined. One to five CASA values were correlated with bull fertility, defined as 59-day nonreturn rates corrected for cow and herd effects. The percentage of motile spermatozoa accounted for a small fraction of the total variation in fertility (r2 = 0.34). However higher r2 values (0.68 to 0.98) were obtained for 2 to 5 variables used in the multiple regression equations. The results are promising, and further testing will determine more precisely which of these CASA variables are most useful in estimating bull fertility potential. PMID- 10732096 TI - A pioneer in the artificial insemination industry: Thomas C. Webster 1896-1964. A tribute. PMID- 10732097 TI - Abnormal offspring following in vitro production of bovine preimplantation embryos: a field study. AB - Data on 944 calves from 2228 in vitro-produced (IVP) bovine preimplantation embryos were compared with data on 2787 AI calves born in the same herds in 1995. Bovine preimplantation embryos were produced in vitro following ovum pick up (OPU) from donor cows and pregnant heifers in an open nucleus breeding program. After 7 d of in vitro culture on a BRL cell monolayer in the presence of 10% FCS, frozen-thawed expanded blastocysts and fresh morulae to expanded blastocysts were transferred into recipient heifers and cows at 119 contracted farms throughout the Netherlands. The pregnancy rate, as confirmed by palpation per rectum between 90 and 150 d after transfer was 43.5% for both fresh and frozen embryos. Data on IVP and AI calves were registered by the farmers. The percentage of calves with a congenital malformation and the percentage of male calves were related to the total number of calves born. Gestation length, birth weight (measured by a balance), perinatal mortality and ease of calving were analyzed in a subdataset (699 IVP and 2543 AI calves, respectively) by a comparative analysis of variance (ANOVA). The ANOVA model included herd, month of calving, sire nested within AI or IVP, parity and breed of the inseminated cow/embryo recipient, sex of calf, type of calf (AI or IVP) and two-way interactions between type of calf and sex, parity and breed. The percentage of calves with congenital malformations was 3.2% and 0.7% for IVP and AI calves, respectively. An increased incidence of hydro allantois and abnormal spinal cords and limbs was observed in IVP calves. The percentage of male calves was significantly different between IVP and AI, 55.5% and 48.9%, respectively (Chi-square, 1 degree of freedom, P < 0.05). On the average, IVP calves showed a significant increase of birth weight by 10% (4-5 kg), a 3-d longer gestation period, 2.4% more perinatal mortality and a more difficult calving process compared to AI calves (P < 0.05). From these results it is concluded that calves produced by IVP deviate significantly from calves produced by AI. PMID- 10732098 TI - Current methods for stallion semen cryopreservation: a survey. AB - Various factors affect the success of AI with frozen-thawed semen in horses. Stallion variability is thought to be one of the major factors, but semen processing and evaluation techniques, thawing protocols, packaging systems and timing of insemination are far from standardized among laboratories. Our objective was to survey current methods for stallion semen cryopreservation used commercially around the world. From the answers to the questions in the survey, we attempted to provide an overview of procedures that are standard as well as those that are used by only few laboratories and to review critically the efficacy of these procedures. Twenty-five questionnaires were sent to individuals or laboratories in 14 countries that were i.v. involved in freezing stallion semen for commercial purposes. Questionnaires were returned from 10/14 countries with 21/25 (84%) of the addresses responding. From the responses, it became evident that most of prefreezing, freezing and thawing and post-thawing processing procedures were far from standardized. The great variety of procedures makes it difficult to accept any of them as reliable. In order to increase the credibility of AI technology in the horse, laboratories need to standardize processing methods as well as the record-keeping systems. In addition, it is evident that no group of research mares is large enough to provide meaningful fertility data. It is therefore imperative to have multicentered collaborative studies to record and disseminate information about methods and the corresponding fertility rate. to gain valuable information and be able to compare different protocols. PMID- 10732099 TI - Sperm transport and survival in the mare. AB - Following the deposition of semen in the mares uterus, spermatozoa must be transported to the site of fertilization, be maintained in the female tract until ovulation occurs, and be prepared to fertilize the released ovum. Sperm motility, myometrial contractions, and a spontaneous post-mating uterine inflammation are important factors for the transport and survival of spermatozoa in the mares reproductive tract. Fertilizable sperm are present in the oviduct within 4 hours after insemination. At this time, the uterus is the site of a hostile inflammatory environment. Our data suggest that spermatozoa trigger an influx of polymorphonuclear neutrophils (PMNs) into the uterine lumen via activation of complement. Furthermore, seminal plasma appears to have a modulatory effect on the post-mating inflammation through its suppressive effect on PMN chemotaxis and migration. Spermatozoa that safely have reached the oviduct can be stored in a functional state for several days, but prolonged sperm storage in the female tract is not required for capacitation and fertilization in the horse. The caudal isthmus has been proposed as a sperm reservoir in the mare. The pattern of sperm transport and survival of spermatozoa in the mares reproductive tract are different between fertile and subfertile stallions, between fertile and some infertile mares, and between fresh and frozen-thawed semen. Possible explanations for these differences include a selective phagocytosis of damaged or dead spermatozoa, impaired myometrial activity in subfertile mares, bio-physiological changes of spermatozoa during cryopreservation, and the removal of seminal plasma during cryopreservation of equine semen. PMID- 10732100 TI - Isolation and characterization of canine advanced preantral and early antral follicles. AB - This study was designed to develop preantral follicle isolation and classification protocols for the domestic dog as a model for endangered canids. Ovary donors were grouped by age, size, breed purity, ovary weight and ovary status. Ovaries were randomly assigned to 1 of 3 digestion protocols: A) digestion and follicle isolation on the day of spaying; B) storage at 4 degrees C for 18 to 24 h prior to digestion and follicle isolation; C) digestion on the day of spaying, then incubation at 4 degrees C for 18 h prior to follicle isolation. Minced tissue was placed in a collagenase/DNase solution at 37 degrees C for 1 h. Follicles were classified by oocyte size and opaqueness and by size and appearance of the granulosa cell layers. Preantral follicles contained small, pale oocytes. Preantral follicles containing grown oocytes with dense cytoplasmic lipid were designated as advanced preantral. Only advanced preantral and early antral follicles were examined and classified further. Group 1 follicles had incomplete or absent granulosa layers, Group 2 follicles had several intact granulosa layers, while Group 3 were vesicular (early antral) follicles. Misshapen or pale grown oocytes were classified as degenerated. The percentage of intact germinal vesicles (GV) was recorded for each Group. Digestion Protocol B produced the lowest percentage of degenerated follicles (P < 0.01). Prepubertal donors had fewer (P < 0.01) follicles in each Group and more (P < 0.001) degenerated follicles than older bitches. Larger ovaries yielded the highest total number of follicles (P < 0.05). Ovary status did not affect follicle yield. Oocytes from Group 1 follicles had fewer intact GVs than those from Group 2 or Group 3 (P < 0.0001). These findings provide an opportunity for quantitative studies of the factors regulating folliculogenesis in the domestic dog as a model for endangered canids. PMID- 10732101 TI - In vitro maturation of domestic dog oocytes cultured in advanced preantral and early antral follicles. AB - Initial studies in our laboratory demonstrated that a large proportion of domestic dog advanced preantral (APAN) and early antral (EAN) follicles contained grown oocytes that had acquired the dense cytoplasmic lipid characteristic of preovulatory oocytes. The objective of this study was to assess nuclear maturation of those oocytes after in vitro culture. Both APAN and EAN follicles (152 to 886 microns in diameter) were isolated from ovaries by treatment with collagenase and DNase. The follicles were cultured in Dulbecco's Modified Eagle's medium/nutrient mixture F-12 Ham culture medium supplemented with 20% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 1% (v/v) antibiotic-antimycotic, 1 microgram FSH/ml, 10 IU hCG/ml and 1 microgram estradiol/ml. Within each group (APAN or EAN), control follicles were not cultured (0 h), and 2 to 12 follicles per well were incubated under a humidified atmosphere of 5% CO2 in air at 37 degrees C for 24, 48 or 72 h. After 24 h of culture, significantly more (5.3%, 20/374; P < 0.05) oocytes from APAN follicles reached the metaphase I to metaphase II stages (MI to MII) than the percentage of control follicles observed at 0 h (0.9%, 3/318). Continued culture resulted in a further increase (P < 0.05) in the percentage of oocytes reaching MI to MII by 48 h (11.5%, 47/407), which remained unchanged at 72 h (9.9%, 40/404). The percentage of oocytes from EAN follicles reaching MI to MII did not significantly increase after 24 h of culture. However, there was an increase (P < 0.05) by 48 h of culture (8.7%, 11/126), which remained unchanged at 72 h (7.5%, 8/106). These results show that dog oocytes cultured within advanced preantral and early antral follicles in vitro are competent to resume meiosis to the metaphase stage. PMID- 10732102 TI - Melatonin treatment of embryo donor and recipient ewes during anestrus affects their endocrine status, but not ovulation rate, embryo survival or pregnancy. AB - Thirty-two Border Leicester x Scottish Blackface ewes that lambed in March were individually penned with their lambs from April 16th and given daily an oral dose of 3 mg melatonin at 1500 h (Group M). A further 32 acted as controls (Group C). Within each group half were used as embryo donors (Group D) following superovulation and half received embryos (Group R) following an induced estrus. Prior to weaning on 21 May ewes received ad libitum a complete diet providing 9 megajoules (MJ) of metabolizable energy and 125 g/kg crude protein. Thereafter each received 1.6 kg of the diet daily. In early June each ewe received an intravaginal device (300 mg progesterone) inserted for 12 d. Donors were superovulated with 4 i.m. injections of porcine FSH 12 h apart, commencing 24 h before progesterone withdrawal. Ovulation in recipients was induced with 800 IU PMSG injected i.m. at progesterone removal. Donor ewes were inseminated 52 h after progesterone withdrawal. Embryos were collected 4 d later and transferred to recipients. Melatonin suppressed plasma prolactin (P < 0.001) and advanced estrus (P < 0.05) and timing of the LH peak (P < 0.05). These events also occurred earlier in donors than in recipients (P < 0.01). Mean (+/- SEM) ovulation rates for melatonin-treated and control donors were 5.5 +/- 0.71 and 4.7 +/- 0.66, respectively (NS). Corresponding recipient values were 3.3 +/- 0.40 and 3.4 +/- 0.39 (NS). Mean (+/- SEM) embryo yields were 2.9 +/- 0.64 and 2.6 +/- 0.73 for melatonin-treated (n = 15) and control (n = 16) donors, respectively, and for the 12 ewes per treatment that supplied embryos, corresponding numbers classified as viable were 2.7 +/- 0.47 and 2.3 +/- 0.61 (NS). Following transfer, 57% of embryos developed to lambs when both donor and recipient received melatonin, 86% when only the donor received melatonin, 91% when only the recipient received melatonin, and 67% when neither received melatonin (NS). Thus, embryo survival following transfer was not improved by treating recipients with melatonin. Gestation length and lamb birthweights were unaffected by melatonin. Unlike nonpregnant control ewes, melatonin-treated recipients that failed to remain pregnant sustained estrous cyclicity following embryo transfer. PMID- 10732103 TI - The effect of oocyte size and bitch age upon oocyte nuclear maturation in vitro. AB - In vitro maturation in the bitch has yet to be fully investigated, and perfection of the technique is essential for future gamete salvage programs in endangered canine species. For optimal success with these techniques, knowledge of the individual animal and of oocyte effects upon maturational competence would be useful. Two factors were therefore studied using an aceto-orcein staining technique, which has been shown to be effective for monitoring nuclear maturation of canine oocytes following oocyte culture in medium supplemented with bovine serum albumin (BSA). Oocytes of different sizes were cultured in vitro and their nuclear maturation monitored. It was shown that the selection of oocytes which had acquired meiotic competence through adequate intrafollicular growth was important for in vitro maturation. In vitro maturation of oocytes from bitches aged 1 to 6 yr, and from those 7 yr and older was then compared, and it was found that oocytes from young bitches had a greater potential to mature than those collected from the older animals. PMID- 10732104 TI - The synchrony of prostaglandin-induced estrus in cows was reduced by pretreatment with hCG. AB - The induction of optimal synchrony of estrus in cows requires synchronization of luteolysis and of the waves of follicular growth (follicular waves). The aim of this study was to determine whether hormonal treatments aimed at synchronizing follicular waves improved the synchrony of prostaglandin (PG)-induced estrus. In Experiment 1, cows were treated on Day 5 of the estrous cycle with saline in Group 1 (n = 25; 16 ml, i.v., 12 h apart), with hCG in Group 2 (n = 27; 3000 IU, i.v.), or with hCG and bovine follicular fluid (bFF) in Group 3 (n = 21; 16 ml, i.v., 12 h apart). On Day 12, all cows were treated with prostaglandin (PG; 500 micrograms cloprostenol, i.m.). In Experiment 2, cows were treated on Day 5 of the estrous cycle with saline (3 ml, i.m.) in Group 1 (n = 22) or with hCG (3000 IU, i.v.) in Group 2 (n = 20) and Group 3 (n = 22). On Day 12, the cows were treated with PG (500 micrograms in Groups 1 and 2; 1000 micrograms in Group 3). Blood samples for progesterone (P4) determination were collected on Day 12 (Experiment 1) or on Days 12 and 14 (Experiment 2). Cows were fitted with heat mount detectors and observed twice a day for signs of estrus. Four cows in Experiment 1 (1 cow each from Groups 1 and 2; 2 cows from Group 3) had plasma P4 concentrations below 1 ng/ml on Day 12 and were excluded from the analyses. In Experiment 1, cows treated with hCG or hCG + bFF had a more variable (P = 0.0007, P = 0.0005) day of occurrence of and a longer interval to estrus (5.9 +/- 0.7 d, P = 0.003 and 6.2 +/- 0.8 d, P = 0.005) than saline-treated cows (3.4 +/- 0.4 d). The plasma P4 concentrations on Day 12 were higher (P < 0.0001) in hCG- and in hCG + bFF-treated cows than in saline-treated cows (9.4 +/- 0.75 and 8.5 +/- 0.75 vs 4.1 +/- 0.27 ng/ml), but there was no correlation (P > 0.05) between plasma P4 concentrations and the interval to estrus. In Experiment 2, cows treated with hCG/500PG and hCG/1000PG had a more variable (P = 0.0007, P = 0.002) day of occurrence of and a longer interval to estrus (4.2 +/- 0.4 d, P = 0.04; 4.1 +/- 0.4 d, P = 0.03) than saline/500PG-treated cows (3.2 +/- 0.1 d). The concentrations of plasma P4 on Days 12 and 14 of both hCG/500PG- and hCG/1000PG treated cows were higher (P < 0.05) than in saline/500PG-treated cows (7.3 +/- 0.64, 0.7 +/- 0.08 and 7.7 +/- 0.49, 0.7 +/- 0.06 vs 5.3 +/- 0.37, 0.5 +/- 0.03 ng/ml). The concentrations of plasma P4 on Days 12 or 14 and the interval to estrus were not correlated (P > 0.05) in any treatment group. The concentrations of plasma P4 on Days 12 and 14 of hCG/500PG- or hCG/1000PG-treated cows were correlated (r = 0.65, P < 0.05; r = 0.50, P < 0.05). This study indicated that treatment of cows with hCG on Day 5 of the estrous cycle reduced the synchrony of PG-induced estrus and that this reduction was not due to the failure of luteal regression. PMID- 10732105 TI - Induction of fertile estrus in bitches using a sustained-release formulation of a GnRH agonist (leuprolide acetate). AB - A single subcutaneous injection of a sustained-release formulation of a potent GnRH agonist, leuprolide acetate (LA; [D-Leu6, Pro9NEt]-GnRH), was evaluated as a method of inducing fertile estrus in 12 mature anestrous and 6 prepubertal beagle bitches. The bitches were treated with microencapsulated LA (100 micrograms/kg, s.c.) at 120 or 150 d post partum, or at 1 yr of age, followed by a GnRH-analogue (fertirelin; [Pro9NEt]-GnRH, 3 micrograms/kg, i.m.) on the first day of induced estrus. Signs of estrus were seen within 10.3 +/- 0.9 d after LA administration in all bitches. The interestrous interval in 120- and 150-d post-partum bitches was shortened (P < 0.05) to 191 +/- 3 and 222 +/- 3 d, respectively, compared with 264 +/- 11 d in control bitches. All LA treated dogs demonstrated behavioral estrus and mated. Three of 6 (50%) at 120 d post partum, 6 of 6 (100%) at 150 d post partum and 5 of 6 (83%) of prepubertal (1-yr old) bitches then became pregnant and produced a mean litter size of 4.1 +/- 0.8 pups. A normal circulating estrogen and progesterone response pattern was observed in mature anestrous bitches. A prepubertal bitch that failed to become pregnant had a similar estrogen response pattern but an insufficient progesterone profile. The results suggest that microencapsulated LA can be useful in inducing fertile estrus in the domestic dogs. PMID- 10732106 TI - Effects of long-term treatment with bovine somatotropin on follicular dynamics and subsequent oocyte and blastocyst yield in an OPU-IVF program. AB - Fourteen Holstein-Friesian heifers between 15 and 22 months of age with normal reproductive tracts were used in an experimental set up to investigate the effect of a long term rBST-treatment on the follicular population prior to transvaginal ovum pick-up (OPU). The estrous cycle of the animals was synchronized by means of a double injection of 2 ml cloprostenol 11 days apart. The heifers were divided in 2 equal groups (n = 7) in which the animals had the same average body weight, one group receiving a weekly subcutaneous injection of 640 mg recombinantly derived bovine somatotropin (rBST) on Mondays (rBST-treatment group) and a control group, being injected with 10 ml of saline. Heifers in both groups were submitted to OPU twice a week on Mondays and Thursdays using a 5 Mhz transducer and a disposable, 55 mm long, 20-g short bevelled needle at a vacuum pressure corresponding to approximately 13 ml water/min. The experimental period lasted for 10 weeks (April to June), each animal receiving a total of 10 injections and being submitted to OPU for 20 times. Oocytes were subsequently matured and cultured in a separate drop per cow following conventional IVF procedures. A blood sample was taken on heparin immediately after each OPU session, for determination of blood progesterone concentrations to assess the influence of treatment and OPU procedure on the cow's estrous cycle. Although results show a significant increase in the total number of follicles and medium sized follicles in the rBST-treated group, no statistically significant different number of retrieved oocytes between the rBST-treated and nontreated group could be detected. The average number of retrieved oocytes per session per cow was comparable, being 6.4 for the treated and 6.0 for the control group. Additionally, the average number of blastocysts per cow per session did not differ significantly between groups, being 1.41 in the rBST-treated group and 1.53 in the control group. The number of cultured oocytes which developed to the blastocyst stage was 22% in the rBST-treated group, which was not significantly different from 25% in the control group. Repeated OPU appeared to induce a certain degree of acyclicity in both treated and nontreated animals. PMID- 10732107 TI - Effect of eCG on the pregnancy rate of ewes transcervically inseminated with frozen-thawed semen outside the breeding season. AB - An experiment was conducted to determine whether factors affecting pregnancy rate out-of-season are associated more with transcervical artificial insemination (T AI) procedures or with the reproductive state of the ewe. Twenty Finncross ewes were treated with progesterone sponges, and at sponge removal (0 h) 10 ewes were treated with eCG. Blood samples were collected for LH and progesterone analyses, and follicular development was monitored using ultrasonography. Ewes were inseminated from 48 to 52 h with 200 million motile frozen-thawed spermatozoa. The incidence of estrus, LH surges and ovulation was greater (P < 0.01) and intervals to these responses were shorter (P < 0.01) in the eCG-treated ewes. The number of follicles > 5 mm was higher (P < 0.05) in eCG-treated than control ewes. Progesterone concentrations increased and remained elevated through Day 19 in 7 eCG-treated and in 1 control ewe, and these ewes were pregnant based upon ultrasonographic examination. The results demonstrate that the T-AI technique using frozen-thawed semen produces a relatively high (70%) pregnancy rate out-of season. The pregnancy rate was found to reflect primarily the reproductive condition of the ewe. PMID- 10732108 TI - Involvement of cumulus cells stimulated by FSH in chromatin condensation and the activation of maturation-promoting factor in bovine oocytes. AB - The effects of FSH-stimulated cumulus cells on the regulatory mechanisms of chromatin condensation and maturation-promoting factor (MPF) activation around the time of germinal vesicle breakdown (GVBD) in bovine oocytes were examined. Chromatin condensation occurred in oocytes arrested at the germinal vesicle (GV) stage by protein synthesis inhibitor, cycloheximide, but this condensation was blocked by FSH-stimulated cumulus cells. However, treatment with cyclic AMP (cAMP)-dependent protein kinase inhibitor, H-8, dramatically increased the proportion of oocytes possessing GVs with condensed bivalents. Under the condition of inhibited protein synthesis, the phosphorylation form of p34cdc2 kinase was not changed due to chromatin condensation, although the activity of histone H1 kinase was significantly increased compared with that of oocytes possessing GVs with filamentous bivalents. The cycloheximide-dependent GVBD block was overcome by okadaic acid (OA) in 48 and 13% of the oocytes in the absence and presence of FSH, respectively. An initial 6-h culture period critical for protein synthesis was necessary for OA to counteract the inhibitory effect exerted by cycloheximide on the induction of GVBD and activation of histone H1 kinase in the absence of FSH, whereas this first culture period was prolonged for 2 h in the presence of FSH. Furthermore, even in FSH-stimulated oocytes, H-8 facilitated an OA-counteracted overcome of the cycloheximide-dependent GVBD block after 2 h of initial culture for protein synthesis. From these results, it is concluded that cAMP-dependent protein kinase activity regulated by cumulus cells following FSH stimulation requests plays a role in the complex mechanism of chromatin condensation and MPF activation leading to meiotic resumption in bovine oocytes. PMID- 10732109 TI - Effect of steroid treatment of endometrial cells on blastocyst development during co-culture. AB - The objective of this study was to determine if treatment of endometrial cells with progesterone or progesterone plus estradiol would improve the development of bovine embryos to the blastocyst stage during co-culture. After IVF, bovine embryos were cultured with oviduct epithelial cells for 3 d. In Experiment 1 the embryos were cultured with a) oviduct epithelial cells; b) endometrial epithelial cells (EEC); c) EEC with 10 ng/ml progesterone (EEC + P); or d) EEC with 10 ng/ml progesterone and 10 pg/ml estradiol (EEC + PE) for 6 d. In Experiment 2 the embryos were cultured with a) oviduct epithelial cells; b) endometrial stromal cells (ESC); c) ESC with 10 ng/ml progesterone (ESC + P); or d) ESC with 10 ng/ml progesterone and 10 pg/ml estradiol (ESC + PE) for 6 d. Results from Experiment 1 showed that endometrial epithelial cells supported development to the blastocyst stage as effectively as the oviduct cells; however, the size of the blastocysts was smaller for the endometrial cells. There was no effect of steroid hormone treatment on development to the blastocyst stage or on the size of the blastocysts. Results from Experiment 2 showed that stromal cells supported development to the blastocyst stage as effectively as oviduct cells. The hatching rate was lower when the embryos were co-cultured with stromal cells than oviduct epithelial cells; but there was no effect of steroid treatment. These data show that untreated endometrial epithelial cells are as effective as oviduct cells in maintaining embryo development to the blastocyst stage. However, embryo development was not improved by steroid treatment of the cells. PMID- 10732110 TI - FSH bioactivity in commercial preparations of gonadotropins. AB - During the past 2 decades, commercial preparations of FSH have been extensively used to superovulate cattle. The problems that have been encountered in superovulation of cattle include high variability in the ovulation rate and subsequent yield of viable embryos. The lack of predictability in superovulatory trials has been attributed to difficulties in standardizing the potency of commercial FSH preparations. Traditionally, FSH potency has been tested in bioassays that utilize specific responses in whole animals or primary cell cultures. Whole animal bioassays lack sensitivity, while primary cell culture bioassays, which use fresh cells, have inherent variability within each preparation. An FSH bioassay that employed a stable chimeric cell line expressing the human FSH-R was used to provide an accurate measurement of FSH bioactivity. The hormonal potency of 2 commercial preparations of FSH used to superovulate cattle was determined using FSH immuno- and bioassays. Commercial FSH preparations differed in potency. One commercial product, prepared in 4 different years, showed no difference in the immunoactive levels of FSH. In the same product stored under identical conditions, FSH bioactivity varied from year to year. There was variability in FSH bioactivity both between and within commercial products. The lack of correlation between bioactivity and immunoactivity of commercial FSH preparations may explain, in part, the variability observed in superovulation of cattle. PMID- 10732111 TI - Cryopreservation of ovine embryos: slow freezing and vitrification. AB - Different methods for the cryopreservation of ovine embryos were evaluated in vitro (survival upon culture in vitro) and in vivo (pregnancy and lambing rates after transfer in field conditions). In the first 2 experiments, slow freezing conditions were evaluated. When glycerol and ethylene glycol were compared, no differences in the overall pregnancy rate were found (40.2 vs 51.3%), but better results were obtained with ethylene glycol than with glycerol in morulae (29.7 vs 59.4%, P < 0.05). In the second experiment, 2 methods of removing ethylene glycol were compared: a 1-step procedure using 0.5-M sucrose and a 3-step process for decreasing ethylene glycol concentration. There were no differences in the overall pregnancy rate (48.0 vs 48.0%) between the 2 methods. The last series of experiments were designed to compare 2 vitrification solutions: propylene glycol- glycerol (PG) and ethylene glycol--Ficoll 70--sucrose (EFS). There were no differences between the 2 vitrification solutions, based on the overall pregnancy rate (28.1 vs 40.0%). The vitrification technique and specially with EFS solution has resulted in good pregnancy rates. The EFS solution was particularly efficacious with morulae (55.5% pregnancy). These results demonstrate that vitrification with EFS can be used successfully for the cryopreservation of ovine embryos. PMID- 10732113 TI - Effects of unilateral ovariectomy on follicular development and ovulation in cattle. AB - In a study of 4 cyclic dry cows (Trial I) and 6 cyclic puberal heifers (Trial II), unilateral ovariectomy increased the number of ovulatory follicles, did not alter the hormone profile, cycle length or the number of follicular waves. Ovarian follicular development in all 4 cows was monitored daily using transrectal ultrasonography until the day of ovulation, during which period daily blood samples were also taken from the tail vein for determination of plasma FSH, LH and P4 concentrations. Unilateral ovariectomy was performed on the day after ovulation and ovarian activity was again monitored daily (ultrasonography and blood sampling for FSH, LH and P4) for 2 consecutive cycles (8 cycles in all). Estrus in all 6 heifers was synchronized using 2 injections of PGF2 alpha given 12 d apart. Similarly, ovarian activity in the 6 puberal heifers was monitored daily using ultrasonography and blood sampling for 1 complete control cycle. Following estrus and ovulation the left ovary was removed in all the animals, and thereafter 1 complete cycle was followed. Mean cycle length, FSH, LH and P4 concentrations before and after unilateral ovariectomy were compared using paired sample t-test. The results show that unilateral ovariectomy neither altered the cycle length nor the number of follicular waves in the cows, but it increased the number of ovulatory follicles (2 follicles developed and ovulated in 6 of the 8 cycles). The mean diameter of the largest follicle was 16.1 +/- 0.9 mm and the second largest 12.5 +/- 0.9 mm. No significant (P > 0.05) differences were observed in FSH (0.72 +/- 0.09 vs 0.71 +/- 0.07), LH (0.42 +/- 0.1 vs 0.37 +/- 0.07) and P4 (2.8 +/- 0.6 vs 2.6 +/- 0.4) levels before and after unilateral ovariectomy. Of the 6 heifers, 5 had 2 waves and 1 heifer had 3 waves of follicular growth during the control cycle, and this pattern did not change after the procedure. Mean cycle length (20.7 +/- 0.9 vs 21 +/- 0.9) did not differ before and after unilateral ovariectomy, and 4 of the 6 heifers ovulated twin follicles following ovariectomy. The mean diameter of the largest follicle was 14.5 +/- 0.7 mm and second largest measured 12.1 +/- 0.8 mm. No significant (P > 0.05) differences were observed in FSH (0.16 +/- 0.09 vs 0.21 +/- 0.07), LH (0.11 +/- 0.1 vs 0.15 +/- 0.07) and P4 levels (3.6 +/- 0.26 vs 3.8 +/- 0.29) before and after unilateral ovariectomy. Based on these results, we conclude that unilateral ovariectomy is an ideal method for obtaining twin ovulations in cows and heifers. PMID- 10732112 TI - Direct transfer of bovine embryos frozen-thawed in the presence of propylene glycol or ethylene glycol under on-farm conditions in an integrated embryo transfer program. AB - An integrated bovine embryo transfer program was conducted in collaboration with 11 Japanese prefectural livestock experiment stations. The program was conducted to evaluate the practicability of the direct transfer method for bovine embryos frozen-thawed in the presence of propylene glycol (PG) or ethylene glycol (EG) under on-farm conditions. Embryos at the compacted morula to expanded blastocyst stages were collected from superovulated donors on Day 7 or 8 after estrus and equilibrated in 1.6 M PG or 1.8 M EG in Dulbecco's phosphate-buffered saline (DPBS) supplemented with 20% heat-inactivated calf serum. Embryos were then loaded individually into a 0.25-ml straw and placed directly into a cooling chamber of a programmable freezer precooled to -7 degrees C. After 2 min, the straw was seeded, maintained at -7 degrees C for 8 min more, and then cooled to 30 degrees C either at 0.3 degree C/min or 0.5 degree C/min before being plunged into liquid nitrogen. Embryos at the same stages were also frozen in the presence of 1.4 M glycerol (GLY) by a conventional method, which served as a control. The frozen embryos were thawed by allowing the straws to stand in air for 5 to 10 sec and then immersing them in a 30 degrees C water bath. Embryos frozen-thawed in the presence of PG or EG were nonsurgically transferred into the uterine horn without diluting the cryoprotectant. Embryos frozen-thawed in the presence of GLY were nonsurgically transferred after removing GLY either by the stepwise method (GLY-I) or by in situ dilution with 0.3 M sucrose solution (GLY-II). A total of 1,273 (PG: 400, EG: 418, GLY-I: 177, GLY-II; 278) frozen-thawed embryos was transferred into recipients, yielding 545 pregnancies (overall: 42.8%, PG: 36.0%, EG; 44.7%, GLY-I; 48.6%, GLY-II; 46.0%). The pregnancy rate with PG was significantly lower than that with EG or GLY-II (P < 0.05). The pregnancy rate was affected by the type of cryoprotectant, the region where the embryo transfer program was carried out, the developmental stage of the embryos, the parity of the recipients, and corpus luteum (CL) quality of the recipients. There were no differences in rates of abortion and stillbirth among the 3 cryoprotectants. The present study demonstrates that EG can be effectively used as a cryoprotectant for freezing and direct transfer of bovine embryos, and that the direct transfer method is applicable under on-farm conditions. PMID- 10732114 TI - Effect of insemination dose on pregnancy rate in mares. AB - Different insemination doses have been used for artificial insemination(AI) in horses. Since the insemination dose can affect the pregnancy rate, it is important to ensure that an adequate dose be used regardless of the type of inseminationprotocol used. The aim of this study was to find out if it is possible to decrease the insemination dose from 500 x 10(6) progressively motile spermatozoa to 300 x 10(6) progressively motile spermatozoa and still maintain an acceptable pregnancy rate when using extended fresh semen. Thirteen stallions of known fertility and a well-defined group of 64 mares were used in the study. The mares were randomly assigned to 1 of 2 insemination groups. Examination for pregnancy was performed by ultrasonography per rectum approximately 16 d after the last insemination. When using an insemination dose of 300 x 10(6) progressively motile spermatozoa the pregnancy rate per cycle was 75%. With an insemination dose of 500 x 10(6) progressively motile spermatozoa the pregnancy rate per cycle was 64%. There was no significant difference in the pregnancy rate between the 2 insemination doses (P = 0.341). We conclude that when using fresh extended semen it is unlikely that an insemination dose of 300 x 10(6) progressively motile spermatozoa would yield a lower pregnancy rate than a dose of 500 x 10(6) progressively motile spermatozoa if stallions with good quality semen are selected. PMID- 10732115 TI - Ian Gordon, B.Sc., M.A., Ph.D. Recipient of the 1998 Embryo Transfer Pioneer Award. PMID- 10732116 TI - Practical measures to improve in vitro blastocyst production in the bovine. AB - Media, chemicals, sera and protocols used for in vitro production of bovine embryos are different from one laboratory to another. This paper describes some of the critical steps required to produce embryos in vitro, suggests quality control measures to lower variations in blastocyst yield and describes different tests which may be used when implementing new procedures in a routine production system. PMID- 10732117 TI - Production of embryos by assisted reproduction in the horse. AB - In vitro embryo production is not yet successful in the horse, largely due to low rates of fertilization in vitro. However, methods to produce embryos from isolated oocytes have been developed. Oocytes may be recovered from living mares by aspiration of the dominant preovulatory follicle by trans-abdominal puncture, and from both preovulatory and immature follicles by trans-vaginal ultrasound guided puncture. Transfer of in vivo-matured oocytes to the oviducts of bred recipient mares has resulted in good pregnancy rates (75-85%). Little work has been done on transfer of horse oocytes matured in vitro. Recovery rates of immature oocytes from mares in vivo are lower than those for cattle. In addition, work on oocytes recovered from horse ovaries post-mortem has shown that horse oocytes from smaller (< 20 mm diameter) viable follicles may not yet be meiotically competent. Methods for in vitro fertilization and for obtaining adequate numbers of competent immature oocytes from the mare must be developed before in vitro embryo production can become a useful clinical and research procedure in the horse. PMID- 10732118 TI - Practical considerations for the in vitro production of pig embryos. AB - The routine maturation, fertilization, and development of pig embryos in vitro has only recently been achieved. Many of the conditions for in vitro production of embryos have been undefined and thus difficult to replicate. The major problems of in vitro production of pig embryos have included maturation of oocytes, both nuclear and cytoplasmic, and development from one-cell to blastocyst. While these barriers have been at least partially overcome there is still a significant problem with polyspermy. Nevertheless, numerous offspring have been produced from in vitro maturation, in vitro fertilization, followed by a brief culture prior to embryo transfer. Here is provided a review of the literature encompassing the current status of the in vitro production of pig embryos. PMID- 10732119 TI - Technical and physiological aspects associated with the lower fertilization following intra cytoplasmic sperm injection (ICSI) in human. AB - The fertilization rates with ICSI range from 30% to 70% and suggest that, despite injecting sperm into mature oocytes, significant fertilization failure still occurs in humans. The objective of this study was to determine technical and physiological factors which may contribute to lower fertilization following ICSI. Eggs that failed to show two pronuclei (PN) 48 hours after ICSI were studied at two different time intervals: at ICSI program inception (group A) and after 8 months (group B). The eggs were analyzed by staining with DNA fluorochromes, Hoescht 33258 and DAPI. The extent of sperm head as well as maternal chromatin decondensation in unfertilized ICSI eggs was determined by high resolution fluorescence microscopy. The average fertilization rate (FR) from all ICSI cycles in these two groups was 45%. The FR in Groups A and B were 35% and 59%, respectively (P < 0.05). In Group A, 65% of the unfertilized eggs were characterized by condensed sperm chromatin with 11% showing partial decondensation. In Group B, only 28% of the unfertilized eggs demonstrated condensed sperm chromatin while 45% were partially decondensed. Sperm chromatin was not detected in 24% of all unfertilized eggs studied. The maternal chromatin remained at metaphase II in 84% of all unfertilized eggs analyzed. These observations suggest that the technical problem of deposition of the sperm inside the egg is not the major cause for failure of fertilization rates in ICSI cycles. The increased percentage of eggs undergoing sperm head decondensation may be related to subtle changes in technique as experience is gained over time. The failure of sperm head decondensation in some of the ICSI eggs may be associated with cytoplasmic immaturity but not nuclear maturity. PMID- 10732120 TI - Activation of development in mammals: is there a role for a sperm cytosolic factor? AB - In mammalian oocytes, fertilization-associated calcium [Ca2+]i oscillations are responsible for the activation of development. The mechanism(s) by which the sperm triggers the initial [Ca2+]i rise and supports long-lasting oscillations is not resolved. It has been proposed that the sperm may interact with receptors in the oocyte's plasma membrane and engage intracellular signaling pathways that result in Ca2+ release. A different line of investigation suggests that upon sperm-oocyte fusion, a sperm cytosolic factor is released into the oocyte which interacts with unknown cytosolic targets, and generates [Ca2+]i oscillations. We will discuss the most recent evidence for both lines of thought and demonstrate that injections of sperm crude extracts (SF) into mammalian oocytes trigger [Ca2+]i oscillations that support in vitro parthenogenetic development to the blastocyst stage. PMID- 10732121 TI - Biochemical and cellular insights into the temporal window of normal fertilization. AB - Normal development depends on both the timing of fertilization and gamete quality, especially in assisted reproductive procedures. Recent studies of the proteins involved in the polyspermy block and cell cycle progression provide a cellular and biochemical basis for the short fertilizable lifespan of the mammalian egg in several species. Specifically, the status of cortical granules, zona proteins, cell cycle kinases, and intracellular calcium stores form a powerful panel of assays to monitor egg activation competence in eggs undergoing maturation and spontaneous activation events in mature eggs. An understanding of how these indicators are influenced by in vitro conditions and exogenous follicular stimulation should provide useful information for optimizing assisted reproductive procedures. PMID- 10732122 TI - Remodeling the sperm nucleus into a male pronucleus at fertilization. AB - After fertilization, the dormant sperm nucleus undergoes morphological and biochemical transformations leading to the development of a functional nucleus, the male pronucleus. We have investigated the formation of the male pronucleus in a cell-free system consisting of permeabilized sea urchin sperm nuclei incubated in fertilized sea urchin egg extract containing membrane vesicles. The first sperm nuclear alteration in vitro is the disassembly of the sperm nuclear lamina as a result of lamin phosphorylation mediated by egg protein kinase C. The conical sperm nucleus decondenses into a spherical pronucleus in an ATP-dependent manner. The new nuclear envelope (NE) forms by ATP-dependent binding of vesicles to chromatin and GTP-dependent fusion of vesicles to each other. Three cytoplasmic membrane vesicle fractions with distinct biochemical, chromatin binding and fusion properties, are required for pronuclear envelope assembly. Binding of each fraction to chromatin requires two detergent-resistant lipophilic structures at each pole of the sperm nucleus, which are incorporated into the NE by membrane fusion. Targeting of the bulk of NE vesicles to chromatin is mediated by a lamin B receptor (LBR)-like integral membrane protein. The last step of male pronuclear formation involves nuclear swelling. Nuclear swelling is associated with import of soluble lamin B into the nucleus and growth of the nuclear envelope by fusion of additional vesicles. In the nucleus, lamin B associates with LBR, which apparently tethers the NE to the lamina. Thus male pronuclear envelope assembly in vitro involves a highly ordered series of events. These events are similar to those characterizing the remodeling of somatic and embryonic nuclei transplanted into oocytes. The relationship between sperm nuclear remodeling at fertilization and nuclear remodeling after nuclear transplantation is discussed. PMID- 10732123 TI - Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture. AB - Along with the transition from maternal to embryonic genome control the mammalian preimplantation embryo undergoes significant changes in its physiology during development. Concomitant with these changes are altering patterns of nutrient uptake and differences in the subsequent fate of such nutrients. The most significant nutrients to the developing mammalian preimplantation embryo are carbohydrates and amino acids, which serve not only to provide energy but also to maintain embryo function by preventing cellular stress induced by suboptimal culture conditions in vitro. It is subsequently proposed that optimal development of the mammalian embryo in culture requires the use of two or more media, each designed to cater for the changing requirements of the embryo. Importantly, culture conditions that maintain the early embryo are not ideal for the embryo post-compaction, and conditions that support excellent development and differentiation of the blastocyst can actually be inhibitory to the zygote. A marker of in vitro-induced cellular stress to the embryo is the relative activity of the metabolic pathways used to generate energy for development. Quantification of embryo energy metabolism may therefore serve as a valuable marker of embryo development and viability. PMID- 10732124 TI - Responses of oocytes and embryos to the culture environment. AB - Embryo development is strongly influenced by events occurring during oocyte maturation. Although many immature oocytes are capable of completing meiosis in vitro, only a small percentage of the original pool of immature oocytes is competent to continue development to the blastocyst stage and subsequently result in a pregnancy. This indicates that maturation of oocytes in vitro may not be occurring in an entirely normal manner. Cytoplasmic changes occurring during maturation, collectively termed cytoplasmic maturation, are essential for embryonic development. The cytoplasm of the oocyte may play a crucial role in assembling the correct metabolic machinery for production of sufficient energy for cellular functions during maturation, cleavage and blastocyst formation. A better understanding of the structural, functional and metabolic characteristics of the oocyte during maturation, and the consequence of changes in these parameters on developmental competence is needed. Understanding the role of cytoplasmic changes during oocyte maturation will help increase the efficiency of in vitro embryo production. Better embryo production strategies will facilitate basic research into the control of early development, improve implementation in endangered species, provide a source of high quality oocytes for nuclear transfer and transgenic technologies and benefit the commercial embryo transfer industry. PMID- 10732125 TI - Temporal patterns of embryonic gene expression and their dependence on oogenetic factors. AB - Successful development of a fertilized egg beyond early cleavage divisions requires the de novo initiation and subsequent regulation of embryonic transcription. The egg provides the specialized environment within which the newly formed zygotic nucleus initiates its developmental program and as a result plays an obligatory role in its regulation. Although the precise timing of the onset of embryonic transcription in mammals varies during early cleavage divisions, several common elements exist. In the present essay we review the current literature on the timing and control of embryonic gene expression in mammals, and discuss recent findings from our laboratory on gene expression patterns in bovine embryos and their relation to other species, and zygotic gene activation (ZGA). Lastly, we discuss the putative role of maternally inherited factors in conferring developmental competence to the blastocyst stage, and a method to identify such factors present in oocytes as mRNA. PMID- 10732126 TI - Cloning: new breakthroughs leading to commercial opportunities. AB - Research on cloning animals, again, came to the forefront of public attention in 1997. Most scientists involved in biomedical and agricultural research have emphasized the benefits, of which there are many, of cloning to the public. Basic studies on nuclear transfer have and will continue to contribute to our understanding of how genomic activation and cell cycle synchrony affect nuclear reprogramming and cloning efficiencies, specifically. Also, more basic information on actual mechanisms and specific factors in the oocyte causing nuclear reprogramming is forthcoming. As new molecular approaches in functional genomics are combined with nuclear transfer experiments, new genes involved in nuclear reprogramming will be found. The commercial potentials of products stemming from discoveries in cloning are vast. Cloning will be a more efficient, faster and more useful way of making transgenic fetuses for cell therapies, adult animals for protein production and organs for xenotransplantation. Clearly there are new opportunities in animal cloning technology that will produce many benefits to society. PMID- 10732127 TI - Commercialization of proteins produced in the mammary gland. AB - In the mid 1980's, few pioneering companies undertook the risk of developing methodologies for the production of complex human therapeutic proteins in the milk of transgenic animals. As we approach the end of the 1990's, the prospect of achieving this aim is becoming a reality as the first of these human therapeutic products, antithrombin III and alpha-1-antitrypsin are making their way through human clinical trials. It is projected that licensure by the Regulatory agencies and market launch for these transgenically produced therapeutics will occur around the year 2000. Although much has already been achieved, additional transgenic challenges await the basic embryo researcher and practitioner. The biopharming community recognizes the need for additional innovative methodologies (such as cloning, sperm sexing and retroviral mediated gene transfer etc.) to overcome the natural biological barriers and increase the efficiency of transgenic dairy animal production and rapid herd expansion. PMID- 10732128 TI - Embryonic stem cells as a model for studying regulation of cellular differentiation. AB - Mouse embryonic stem (ES) cells can be differentiated in vitro into near homogeneous populations of both neurons and skeletal muscle as well as other cell types. We previously showed that treatment of pluripotent ES cells with retinoic acid (RA) induced differentiation into highly enriched populations of gamma aminobutyric acid (GABA) expressing neurons. The reasons for generation of only GABA neurons as opposed to other neuronal cell types were not known. We have extended our previous work and now show that with RA induction of ES cells we not only obtain GABA neurons, but also dopaminergic neurons. Critical for the production of dopaminergic neurons after RA induction was the post-induction plating conditions used. No dopaminergic neurons were detected if cells were plated in serum-free media optimized for neuronal survival. However, significant numbers of dopamine neurons could be detected when cells were plated in media containing fetal calf serum. These observations support the conclusion that RA acts as a general neural inducing agent and that conditions post-induction either selectively support survival of a particular class of neuronal cells or that the conditions post-induction actually further instruct cells to differentiate into different types of neurons. PMID- 10732129 TI - Quantitative echotexture analysis of bovine ovarian follicles. AB - Computer-assisted image analysis was used to evaluate ultrasound images of bovine ovarian follicles. The ovaries of 8 sexually mature heifers were examined daily by transrectal ultrasonography for 2 estrous cycles. Ultrasonographic examinations of the ovaries were then videotaped, and the dominant and subordinate follicles of successive waves were individually identified and monitored. Recorded images of the dominant anovulatory follicle of the first wave (n = 15) and the ovulatory follicle of the last wave (n = 15) of the estrous cycle were subsequently digitized for computer analysis of echotexture (mean pixel value and pixel heterogeneity). Regions of the image spanning the breadth of the follicle wall were selected, and image analysis revealed that mean pixel value of the dominant anovulatory follicle changed over time (P = 0.0005). Mean pixel value decreased (P = 0.0005) dramatically during the early static phase (Days 6 to 8, Day 0 = day of ovulation), increased (P = 0.0005) at the onset of the regressing phase (Day 12), and reached maximal levels (P = 0.0005) on Day 14. Similarly, image echotexture of the ovulatory follicle revealed a time-dependent effect (P = 0.0001) due to a rapid decrease in mean pixel values between 7 and 4 d before ovulation, followed by an increase until the day before ovulation. The echotexture of images of the follicular antrum were also evaluated and with regard to the dominant anovulatory follicle, a time-dependent effect was not detected for mean pixel value (P = 0.62) but was observed for pixel heterogeneity (P = 0.02). In addition, there was a positive correlation between mean pixel value and heterogeneity (r = 0.61, P = 0.0001). Heterogeneity initially decreased (P = 0.02) and remained low until the emergence of the second follicular wave (mean Day 9). Values subsequently increased and became variable during the late static and regressing phases (> Day 9). Mean pixel value of the antrum of the dominant ovulatory follicle increased (P = 0.0001) as the day of ovulation approached. Heterogeneity did not change (P = 0.14), nor was there any correlation between mean pixel value and heterogeneity for the antrum of the ovulatory follicle (r = 0.06, P = 0.49). We concluded that changes in echotexture (mean pixel value and heterogeneity) of bovine ovarian follicles assessed by computer analysis of ultrasound images were temporally related to functional status (i.e., anovulatory versus ovulatory; growing, static or regressing). The results were strongly supportive of the concept that ultrasonographically detected image attributes are a reflection of physiologic status. PMID- 10732130 TI - Possible role for phosphatidylinositol 3-kinase in regulating meiotic maturation of bovine oocytes in vitro. AB - In this study 2 phosphatidylinositol 3-kinase (PI 3-kinase)-specific inhibitors, wortmannin and 2-[4-Morpholinyl]-8-phenyl-4H-1-benzopyran-4-one (LY294002), were used to investigate whether PI 3-kinase is involved in the signal transduction that leads to bovine oocyte maturation. Bovine follicular oocytes were cultured in vitro for 24 h in a basic medium consisting of tissue culture medium-199 supplemented with LH, FSH, fetal cow serum, Na-pyruvate and gentamicin. The oocytes were then examined for the stage of meiotic progression and degree of cumulus expansion. In Experiment 1, in cumulus-oocyte complexes (COCs), wortmannin, at any level tested (10(-8) M, 10(-7) M or 10(-6) M), had no effect on resumption of meiosis as judged by germinal vesicle breakdown and progression to prometaphase I or metaphase I. However, wortmannin significantly (P < 0.01) decreased the proportion of oocytes developing to metaphase II in a dose dependent manner. In Experiment 2, when denuded oocytes were cultured with wortmannin at 0, 10(-7) M and 10(-6) M concentrations, the same pattern of response for COCs was observed, with no effect on meiotic resumption and a significant (P < 0.01) decrease in the proportion of oocytes reaching metaphase II. In Experiment 3, half of the recovered COCs were denuded and both denuded and intact COCs were cultured in the presence of 0, 2.5 x 10(-5) M, 5.0 x 10(-5) M and 7.5 x 10(-5) M LY 294002 before being examined for meiotic progression. Whereas LY294002, at any examined level, had no effect on the percentage of oocytes developing to metaphase I, it significantly (P < 0.01) decreased the proportion of metaphase II oocytes when used at 5.0 x 10(-5) or 7.5 x 10(-5) M for both intact COCs and denuded oocytes. In Experiment 4, no significant difference in the degree of cumulus expansion was scored after the COCs were cultured in the presence of wortmannin or LY294002 or in the absence of either treatment. These results provide indirect evidence for a role of PI 3-kinase in the bovine oocyte itself in regulating meiotic progression beyond metaphase I. PMID- 10732131 TI - Superovulatory response of Murciana goats to treatments based on PMSG/Anti-PMSG or combined FSH/PMSG administration. AB - Superovulation in goats is frequently restricted by the cost of gonadotropin or the handling requirements. In this situation PMSG has the advantage of a lower cost and single dose protocol, but the variability of response obtained restricts its use. Thus, 2 alternative treatments with the advantages of PMSG were tested. In Experiment 1, we compared the ovulatory response of does treated with PMSG in combination or not with anti-PMSG antibodies at the onset of estrus, during season and out of season. In Experiment 2, we explored the effect of a partial substitution of FSH by PMSG at the end of treatment, comparing this treatment with a standard FSH protocol. Our results showed a significant (P < 0.01) seasonal effect on the incidence of corpora lutea (CL) regression in both experiments. The mean of viable embryos collected from does treated with anti PMSG antibodies (mean = 5.75) was significantly higher than in the control PMSG treated group (mean = 2.74) during spring (P < 0.05). Response during the fall was significantly lower regardless of treatment, and administration of antibody did not provide any significant improvement in superovulatory response (2.14 vs 1.77). In Experiment 2, the partial substitution of 3 doses of FSH by a single administration of PMSG did not reduce the number of CL or viable embryos, and no seasonal differences were observed, confirming that FSH provides a less variable response. From our results, it can be concluded that the use of PMSG antibodies for super-ovulating goats is an efficacious treatment which increases the number of viable embryos collected. However, partial replacement of FSH with PMSG at the end of treatment also did not compromise the number of embryos collected. Both approaches can be considered a valid alternative to treatments based on FSH. PMID- 10732132 TI - Effects of antioxidants on the development of bovine IVM/IVF embryos in various concentrations of glucose. AB - This study was undertaken to determine the effects of glucose, antioxidants and different oxygen tensions on the development of bovine embryos cultured in modified synthetic oviduct fluid (m-SOF) medium. In vitro matured (IVM) and fertilized (IVF) oocytes were incubated for 48 h. Embryos reaching at least the 4 cell stage were selected for further culture under various conditions for 6 d. Supplementing the m-SOF media with 4.5 mM glucose resulted in a significantly lower (P < 0.01) embryo developmental rate (21%; Day 8) than was obtained with 1.5 mM glucose (58%; Day 8) or no glucose (53%; Day 8). Antioxidants such as SOD, catalase and mannitol had no positive effect on embryo development in m-SOF medium supplemented with 1.5 mM glucose. However, in m-SOF medium supplemented with 4.5 mM glucose, SOD and mannitol significantly (P < 0.05) improved embryo development: SOD increased the developmental rate from 19 to 35% (Day 8), while mannitol increased it from 13 to 30% (Day 8). Low oxygen concentration improved embryo development significantly (P < 0.05) in m-SOF medium supplemented with 4.5 mM glucose (low O2: 31% vs high O2: 14%; Day 8) but not 0 mM glucose (low O2: 58% vs high O2: 55%; Day 8). Our data suggest that low concentration of glucose during culture of bovine embryos is beneficial, and that generation of free oxygen radicals is partly caused by a high concentration of glucose in the medium. PMID- 10732133 TI - Follicular activity and ovulation regulated by exogenous progestagen and PMSG in anestrous ewes. AB - Follicular dynamics and ovulation were compared in 3 groups of anestrous ewes: those treated with medroxyprogesterone acetate (MAP) sponges for 12 d, then with 750 IU PMSG at the time of sponge removal (P4 + PMSG, n = 6), or PMSG alone (n = 6) and untreated controls (n = 6). Waves of follicular activity were observed in all the animals. In the P4 + PMSG treatment group, MAP priming permitted more ovulatory follicles (P < 0.001) to be recruited without changing follicle growth rate; MAP priming also delayed the time of ovulation (P < 0.001) and the time of the LH surge (P < 0.01), which allowed for an increase in the size of ovulatory follicles (P < 0.05). Ovulation also resulted in normal luteal function after P4 + PMSG (P < 0.01) but not after PMSG alone, since premature luteal regression occurred in 80% of the cases and was related to the presence of follicles > 4 mm when P4 levels were < 1 ng/mL on the day following ovulation. The results showed that MAP priming increased the ovulation rate by increasing the number of follicles that responded to PMSG. PMID- 10732134 TI - Regulation of follicular activity and ovulation in ewes by exogenous progestagen. AB - The success of estrus synchronization programs using progestagen sponges, particularly for fixed-time AI, varies considerably. In view of the recent evidence in cattle that exogenous progestins alter follicular dynamics, it may be that the stage of the estrous cycle at which the synchronization protocol is begun affects the synchrony of ovulation. The goal of this study was to evaluate the effect of medroxyprogesterone acetate (MAP) intravaginal sponges on follicular dynamics, luteal function and interval to ovulation when inserted at 3 stages of the estrous cycle. Sponges were inserted for 12 d beginning on either Day 0, 6 or 12 (n = 5) following ovulation. Ovarian activity was monitored using real-time ultrasound imaging during the treatment and the post-treatment estrous cycles. Information from the post-treatment cycle was used as a baseline to compare with the treatment cycle. Most ewes (79%) in the post-treatment cycle exhibited 3 follicular waves in an estrous cycle of 16 d, with the second wave follicles having smaller diameter (P < 0.001). Treatment with MAP increased the number of follicular waves from 3 to 4 or 5 when sponges were inserted on Days 6 and 12, respectively. Size of the largest follicle was smaller (P > 0.01) in waves in the early and middle of the 12-d MAP treatment period when compared with the last 4 days. This effect was most pronounced when endogenous progesterone concentrations were elevated concurrently with the presence of the sponge. Persistence of the ovulatory follicle was increased (P < 0.001) when sponges were inserted on Day 12, the only treatment where these follicles were under the influence of MAP in the absence of functional corpora lutea. Follicles were regressing at sponge removal in the Day 6 treatment, which resulted in a delay in emergence of ovulatory follicles, the LH surge and ovulation (P < 0.08) in relation to Day 0 and Day 12. Treatment with MAP sponges does not adequately synchronize estrus and ovulation among cyclic ewes due to the different follicular patterns that result depending on the stage of cycle at the time of sponge insertion. PMID- 10732135 TI - Effect of protein synthesis inhibition before or during in vitro maturation on subsequent development of bovine oocytes. AB - The overall objective of this study was to assess the effect of maintaining meiotic arrest in bovine oocytes in vitro on developmental competence. In Experiment 1 the effect of inhibition of meiotic resumption using cycloheximide (CX), on subsequent was examined. Immature cumulus oocyte complexes (COCs, n = 804) were cultured in the absence (24 h) or presence of CX for 6, 12, 18 or 24 h. The control was inseminated 24 h later, while CX-treated oocytes were cultured for a further 24 h before insemination. In Experiment 2 the effect of exposing the oocyte (n = 1239) during meiotic arrest to putative stimulatory substances (pFSH and FCS) was examined. In Experiment 3, to study the importance of protein synthesis during maturation, synthesis was blocked for a 6-h period at various times (6, 12, 18 h) after start of culture (n = 1117). In Experiment 1, there was no difference in cleavage rate between treatments. However, the percentage of 5 to 8 cell embryos at 72 h post insemination was significantly lower after CX treatment (64 vs 42 to 51%; P < 0.05). This was reflected in a lower rate of blastocysts at Day 6 (9 to 15 vs 31%, P < 0.002). While the blastocyst rate at Day 8 was lower in CX-treated oocytes, the effect was only significant when CX was present for longer than 12 h. A marked decrease in development was noted following inhibition for 18 h or more compared with the control (17 to 19 vs 40%; P < 0.0002). In Experiment 2, addition of either FSH or FCS to oocytes in the presence of CX had no effect on any of the parameters studied, even though there was a positive effect in control oocytes. In Experiment 3, treatment with CX after the oocytes had matured for varying periods resulted in decreased blastocyst rates at Days 6 and 8 of culture. The most significant drop in development occurred when oocytes were cultured for 12 h before exposure to CX (15 vs 40%; P < 0.0001). In conclusion, CX-blocked oocytes retained their developmental competence, although final blastocyst yields were reduced. PMID- 10732136 TI - Premature elevation of progesterone shortens duration of ovulation in PMSG/hCG treated prepuberal gilts. AB - A surge of LH during the follicular phase triggers multiple pathways, including progesterone and prostaglandin synthesis before culminating in ovulation. Progesterone has been shown to be involved in the ovulatory process in many species. In prepuberal gilts treated with PMSG/hCG the follicular progesterone level has been shown to increase sharply before ovulation. This study was conducted to investigate whether premature elevation of progesterone can accelerate the ovulatory process in Large White PMSG/hCG-treated prepuberal gilts. Fifty-four Large White gilts were treated with 1000 IU, i.m. PMSG to stimulate follicular growth, followed 72 h later by 500 IU, i.m. hCG to induce ovulation. Gilts in the treatment group (n = 27) were given progesterone intermuscularly at 24 and 36 h after hCG. Ovaries were exteriorized to observe ovulation points during laparotomy under general anesthesia at 38 to 50 h after hCG. Ovulation in both groups commenced by 40.05 h after hCG and was completed by 47.71 h in the control group and by 42.87 h after hCG in the treated group. Progesterone shortened (P < 0.01) ovulation time by 4.84 h and the time required (P < 0.01) for the median proportion of follicles to ovulate (40.7 vs 43.5 h after hCG). Progesterone also increased (P < 0.01) the plasma progesterone concentration without altering follicular progesterone concentration. PMID- 10732137 TI - Effect of different prostaglandins on intrauterine pressure and uterine motility during diestrus in experimental cows. AB - Prostaglandins are widely used in herd management due to their luteolytic properties. They have also a direct effect on the myometrium. We hypothesized, that dissimilar prostaglandin preparations would differ as to their contractile effect. Intrauterine pressure was recorded during the diestrus of lactating dairy cows using a transcervically placed intraluminal pressure microtransducer. After recording physiologic uterine motility for 30 min, prostaglandins (dinoprost, DL cloprostenol, D-cloprostenol) or a placebo was administered intramuscularly, followed by a 2-h recording period. Significant differences were found for the area under the curve (P < or = 0.05) and mean amplitude (P < or = 0.05), whereas the number of spikes per 15 min and the baseline pressure during the last 3 min of every recording period did not differ significantly among treatments. Peak values for area under the curve and mean amplitude were found between 15 and 30 min after administration of DL-cloprostenol, while dinoprost yielded the steadiest plateau from this period until the end of the recording session. These results contrast with those of earlier studies comparing prostaglandins after intravenous administration. PMID- 10732138 TI - An abattoir survey of ovine reproductive tracts in Ireland. AB - Reproductive tracts were collected from 870 ewes slaughtered at an abattoir in County Dublin over a 9-m.o. period from September 1993 to June 1994. Ovarian activity was evident in 59.3% of the tracts examined. The mean ovulation rate was 1.26 +/- 0.045 (mean +/- SEM). The spatial relationship between corpora lutea (CL) and dominant follicles was established. Ten percent of the ewes examined were pregnant. Embryonic transuterine migration was observed in 12% of the single ovulating ewes and in all ewes pregnant with twins which had double ovulations on one ovary. Based on the observation of tracts with contemporaneous double ovulations and a single fetus, the prevalence of embryonic loss was 17.2%. Overall, 9.4% of the tracts showed evidence of pathological change, and 5.5% of the tracts had abnormalities likely to impair fertility. PMID- 10732139 TI - Hormonal and immunological changes in blood and mammary secretion in the sow at parturition. AB - The purpose of the present study was to record possible variations of estradiol 17 beta (E2) and cortisol concentrations, and parameters related to granulocyte phagocytosis in mammary secretions from healthy sows at parturition. The study was comprised 8 primiparous sows (Landrace x Yorkshire). Blood and mammary secretion samples were collected twice daily from 3 d before (only blood) until 3 d after farrowing. Estradiol-17 beta and cortisol concentrations were determined in plasma and in cell-depleted skimmed mammary secretions. Phagocytic capacity of polymorphonuclear cells (PMN) was assessed in whole blood and in cell suspensions derived from mammary secretions. Opsonic activity was assessed in serum and in cell-depleted skimmed mammary secretions. The 2 assays were based on chemiluminescence. Estradiol-17 beta concentration in plasma decreased (P < 0.001) directly after parturition. In skimmed secretions, the highest E2 concentration was recorded in the first sample after parturition and decreased (P < 0.01) thereafter. The highest cortisol concentration in plasma was recorded in the evening before parturition (P < 0.01). In skimmed secretions, there was no significant variation in cortisol concentration. The concentrations of both steroid hormones were lower in mammary secretions than in plasma. The phagocytic capacity of PMN in blood and mammary secretion, expressed as peak chemiluminescence per PMN, showed no significant change. This was also true for the opsonic activity in serum. In skimmed secretions the opsonic activity increased (P < 0.01) after parturition. These data emphasize the differences between plasma and mammary secretion concentrations of steroid hormones as well as between systemic and mammary gland immune competence. Regarding the phagocytosis process in mammary secretions, the part directly related to the PMN function seemed not to be altered at parturition compared with later on in lactation, whereas the part related to opsonic activity seemed to be impaired at parturition. The latter may play a role in the development of coliform mastitis at this time. PMID- 10732140 TI - The influence of insulin administration after weaning the first litter on ovulation rate and embryo survival in sows. AB - Primiparous crossbred sows (n = 43), lactating for an average of 21.1 +/- 0.1 d and weaning 8.7 +/- 0.1 pigs, were used to evaluate the influence of insulin on ovulation rate and embryo survival. The sows were maintained on 2.3 kg/head/d of a 14% protein gestation diet during pregnancy, fed ad libitum during lactation, given 2.7 kg/head/d from weaning until re-breeding and fed 2.3 kg/head/d after mating. Beginning the day after weaning (Day 0) sows were treated with 0.4 IU/kg body weight (BW) insulin (n = 21) or were administered an equivalent volume of saline (n = 22) for 4 d. Beginning on Day 3 and continuing until Day 14 after weaning, the sows were checked for estrus twice daily and were artificially inseminated using pooled semen from 2 fertile boars. At slaughter (days 30 to 40 of gestation), ovaries and uteri were collected, and the ovulation rate, embryo number and viability, and uterine weight and length were evaluated and recorded. Use of insulin decreased the average interval from weaning to estrus compared with saline by increasing percentage in estrus by Day 14 after weaning (5.0 +/- 0.57 vs 6.9 +/- 0.56 d, respectively; P < 0.03). Ovulation rate, number of embryos, embryo survival, and average uterine length and weight were not influenced by insulin treatment. Overall, insulin affected reproductive efficiency in primiparous sows by increasing the percentage of sows in estrus. PMID- 10732141 TI - Comparison of assessment of fowl sperm viability by eosin-nigrosin and dual fluorescence (SYBR-14/PI). AB - The kinetics of fowl sperm viability/mortality following short-term and long-term in vitro storage were studied using 2 different staining methods: eosin/nigrosin (observed under light microscopy) and SYBR-14/PI (dual fluorescence). Based on data obtained at 0, 30 min and at 2, 4 and 24 h (T0, T30, T2, T4, and T24) after in vitro storage (4 degrees C, agitated) of fresh or frozen-thawed semen, the dual association SYBR-14/PI was more effective than eosin/nigrosin (P < 0.05) staining for the detection of sperm viability/mortality at early stages (30 min) in nonfrozen ejaculates stored above 0 degree C. In cryopreserved preparations, the 2 techniques were comparable for assessing viable spermatozoa immediately after thawing, but higher percentages (P < 0.05) of nonviable spermatozoa were detected by the SYBR-14/PI procedure for up to 4 h of in vitro storage post thawing (4 degrees C, agitated). Finally, comparable results were observed between the 2 techniques 24 h after beginning in vitro storage post thawing. It is concluded that the dual association SYBR-14/PI procedure is more effective (or, at least, more rapid) than eosin/nigrosin staining for the assessment of sperm viability/mortality in both fresh and cryopreserved samples of fowl semen. However, in the latter case, the thawing stage needs to be followed by a period of in vitro storage lasting at least 4 h to allow for easier discrimination between viable and nonviable populations of spermatozoa. PMID- 10732142 TI - Cryopreservation of in vitro-produced bovine embryos: effects of protein type and concentration during freezing or of liposomes during culture on post-thaw survival. AB - Two experiments were conducted to examine the effect of membrane stabilization through the modification of in vitro culture medium or freezing medium on post thaw survival of in vitro-produced bovine embryos. In Experiment 1, Day 7 (Day 0 = day of IVF) late morulae and blastocysts that developed following culture in SOF/aa/BSA (IVC medium) were frozen slowly to -35 degrees C in the presence of 1.5 M ethylene glycol prepared in ovum culture medium (OCM) or in OCM supplemented with 10, 25 or 50% fetal calf serum (FCS) or 5, 10 or 25 mg/mL BSA. Post-thaw survival was assessed by re-expansion and/or hatching following 48 h of culture in IVC medium + 10% FCS. Overall, survival was significantly (P < 0.01) affected by embryo stage, with more hatched blastocysts surviving (71%) than blastocysts (59%) or late morulae (51%). Addition of FCS significantly (P < 0.01) reduced survival compared with control embryos or those frozen in BSA supplemented medium (50.48 vs 68.01 vs 63.53%, respectively). There was also a significant interaction between embryo stage and protein type (P < 0.05). The survival of late morulae/early blastocysts following freezing was improved in the presence of additional BSA but not FCS. In Experiment 2, the IVC medium was supplemented with liposomes containing lecithin, sphingomyelin and cholesterol. Sphingomyelin and cholesterol at ratios of 1:1, 1:4 and 4:1 were added to 50, 100 or 150 micrograms/mL lecithin to yield a final lipid concentration of 200 micrograms/mL. A further group contained 200 micrograms/mL lecithin only. Blastocysts were frozen in 1.5 M ethylene glycol in OCM, then thawed and assessed as in Experiment 1. The presence of liposomes during IVC did not affect the proportion of cleaved embryos that developed to blastocysts or survival following freezing. However, the survival of blastocysts that developed in the presence of 200 micrograms/mL lecithin only was significantly lower than in any other treatment (6%; P < 0.03). These studies demonstrate that the protein composition of the freezing medium can significantly affect survival after thawing and that the survival of late morulae can be improved with additional BSA. The presence of lecithin only in the liposome preparation did not affect embryo development, but significantly reduced survival after freezing, suggesting it can affect post-thaw embryo survival, perhaps by altering embryonic membrane composition. PMID- 10732143 TI - Pulsatility of systemic FSH and LH concentrations during follicular-wave development in cattle. AB - Changes in systemic FSH and LH pulsatility in temporal association with follicular-wave emergence and follicle deviation were studied in cattle. Wave emergence was defined as occurring when the future dominant follicle first reached 4 mm, as established retrospectively. Follicle deviation was defined as the beginning of a change in growth rates between the 2 largest follicles and occurred 60.4 +/- 4.2 h after wave emergence. Follicles were tracked by transrectal ultrasound scanning every 8 h, and blood samples for pulse characterization were collected every 20 min from before emergence until after deviation. Pulses were characterized by the Pulsar program applied to each 8-h increment, centered on the hour of follicle scanning in each heifer (n = 6). Pulsatility of FSH was not detected for any of the 8-h increments. The mean FSH concentrations for the 24 samples per 8 h increased (P < 0.05) between 8 h before and 8 h after wave emergence, followed by a decrease 40 to 16 h before deviation. The low mean values continued for 24 h after deviation. Pulses of LH were detected for all 8-h increments. The LH mean of all concentrations per 8 h and pulse frequency increased (P < 0.05) between the hour of wave emergence and 32 h after emergence and then pulse frequency plateaued at a mean interpeak interval of 1.3 h. Increased LH means for all concentrations per 8 h and basal concentration were reached 32 h before deviation. The results indicated that elevated concentrations of LH and reduced concentrations of FSH were present 32 to 16 h before to at least 24 h after the beginning of follicle deviation. However, an abrupt, short-term change in FSH concentrations or in LH pulsatility in close temporal association with follicle deviation that could act as an acute stimulus to initiate deviation was not found. PMID- 10732144 TI - Effect of oxytocin, prostaglandin F2 alpha, and clenbuterol on uterine dynamics in mares. AB - The effects of oxytocin, prostaglandin F2 alpha (PGF2 alpha), and clenbuterol on uterine contractility and tone during anestrus and diestrus, and during mobility and postfixation of the embryonic vesicle were studied in 51 pony mares. Contractility was assessed by scoring real-time ultrasound images, and tone was assessed by transrectal digital compression. Scoring was done by an operator who had no knowledge of treatment assignments. In anovulatory mares primed with progesterone for 16 d, oxytocin did not significantly alter contractility but did stimulate an increase in tone, whereas clenbuterol depressed both contractility and tone. The PGF2 alpha given on Days 12, 15, and 18 did not significantly alter uterine contractility in pregnant mares, but it increased contractility on all days in nonpregnant mares. Clenbuterol decreased both tone and contractility when given to pregnant mares on the day of embryonic-vesicle fixation, while it decreased tone but not contractility when given on Day 19. Clenbuterol treatment was associated with dislodgment of the fixed embryo in only 1 of 5 mares. However, on Day 19, clenbuterol treatment was associated with a change in shape of the conceptus when viewed in a cross section of the uterine horn. The conceptus shape became more circular rather than irregular or triangular, as indicated by a significant decrease in the variation in the distances between adjacent walls measured in 4 different directions. Results indicated that: 1) oxytocin increased uterine tone but did not alter contractility in progesterone primed anestrous mares; 2) on Days 12, 15 and 18, PGF2 alpha increased uterine contractility in nonpregnant mares but not in pregnant mares; 3) clenbuterol decreased both tone and contractility at all reproductive states except for a lack of a decrease in contractility on Day 19 of pregnancy; and 4) reduction in uterine tone from clenbuterol treatment on Day 19 was associated with a change in the two-dimensional shape of the in situ conceptus from irregular to a more circular form. PMID- 10732145 TI - Characterization of lower temperature storage limitations of fresh-extended porcine semen. AB - Irreversible damage caused by cold shock has been assumed to occur when boar semen is exposed to temperatures below 15 degrees C. Identification of the lower critical temperature at which extended boar semen undergoes cold shock, however, has yet to be defined. The aims of this study were to 1) identify the cold-shock critical temperature and time on extended boar semen as assessed by sperm motility and morphology, and 2) determine the effects on fertility of using extended porcine semen exposed to this critical temperature and time. For Objective 1, ejaculates from 18 boars were collected, analyzed and extended in Androhep to 50 x 10(6) sperm/mL. Doses (4 x 10(9) sperm) from each ejaculate were exposed to 5 storage temperatures (8, 10, 12, 14 and 17 degrees C). Sperm motility and morphology (including acrosomes) were assessed following collection and at 12-h intervals for 48-h. Decreases in sperm motility occurred within the first 12-h at all temperatures. Sample motility dropped below 70% within 12-h in the 8 degrees C group and by 48-h in the 10 degrees C group. Sample motility was > 75% in the 12, 14 and 17 degrees C (control) groups throughout the trial. The percentage of morphologically abnormal sperm cells, including acrosomes, did not change within or between treatment groups over the 48-h storage period. In Objective 2, boar ejaculates (n = 9) were handled as in the first objective and were equally divided into treated (12 degrees C for < or = 60-h) and control (17 degrees C for < or = 60-h) groups. Using a timed, double insemination technique, 135 sows were bred by AI using either 12 degrees C (n = 74) or 17 degrees C (n = 61) extended, stored semen. No differences were observed in the farrowing rate (93 vs 95%), total offspring born (11.58 vs 11.61) or number live born (10.68 vs 10.63) between 12 and 17 degrees C groups, respectively. The results demonstrate that acceptable fertility can be obtained with Androhep extended boar semen exposed to temperatures as low as 12 degrees C for up to 60-h, and that cold shock appears to occur in vitro when extended boar semen is exposed to storage temperatures below 12 degrees C. PMID- 10732146 TI - Effect of passive immunization against inhibin on FSH secretion, folliculogenesis and ovulation rate during the follicular phase of the estrous cycle in mares. AB - Physiological roles of inhibin in mares were investigated by means of passive immunization using an antiserum to inhibin that had been raised in a castrated goat. Eight mares were given an intravenous injection of either 100 mL (n = 4) or 200 mL (n = 4) of inhibin antiserum 4 d after a single intramuscular injection of PGF2 alpha on Day 8 after ovulation, 4 control mares were treated with 100 mL castrated goat serum in the same manner. Jugular vein blood samples were collected after treatment with the serum until 192 h post treatment. Follicular growth and ovulations were monitored by ultrasound examination at 24-h intervals. The ability of the inhibin antiserum to neutralize the bioactivity of equine inhibin was examined in vitro using a rat pituitary cell culture system. Suppression of secretion of FSH from cultured rat pituitary cells by equine follicular fluid was reversed by the addition of increasing doses of the inhibin antiserum, thereby indicating its bioactivity. Plasma levels of FSH and estradiol 17 beta were higher in mares treated with the inhibin antiserum. The ovulation rate was significantly higher in mares treated with antiserum (100 mL = 3.75 +/- 0.63; 200 mL = 4.50 +/- 0.65) than in control mares (1.25 +/- 0.25). These results demonstrate that inhibin is important in regulating FSH secretion and folliculogenesis in mares. They also show that neutralization of the bioactivity of inhibin may become a new method for the control of folliculogenesis and ovulation rate in mares. PMID- 10732147 TI - Bacteriology of preserved stallion semen and antibiotics in semen extenders. AB - Three experiments were conducted to evaluate the effects of different antibiotics in a milk-glucose semen extender on motility of equine sperm and elimination of bacteria following storage of extended semen in vitro. In Experiment 1, 7 antibiotics were compared: amikacin, gentamicin, streptomycin, potassium penicillin, sodium penicillin, ticarcillin, and polymixin B. In Experiment 2, 3 antibiotic treatments were compared: potassium penicillin G, amikacin, or a combination of potassium penicillin G and amikacin. In Experiment 3, 3 antibiotic treatments were compared: potassium penicillin G-amikacin, ceptiofur, and a combination of ticarcillin and clavulanic acid (Timentin). Control treatments (antibiotic-free extender) were included in each experiment. Six motility variables were evaluated: percentage of motile sperm; percentage of progressively motile sperm; percentage of rapidly-motile sperm; mean curvilinear velocity; mean average path velocity; and mean straight-line velocity. In Experiment 1, mean percentages of motile, progressively motile and rapidly motile sperm were lower (P < 0.05) in semen exposed to polymixin B then in other treatments. Mean average path velocity of sperm in extender containing polymixin B was lower (P < 0.05) than that of all other treatments, with exception of control or ticarcillin. Mean straight-line velocity of sperm in extender containing polymixin B was lower (P < 0.05) than that of all other treatments, with exception of control, streptomycin or ticarcillin. Semen samples containing gentamicin, amikacin, streptomycin, or potassium penicillin were more effective (P < 0.05) at eliminating bacterial growth than those samples containing polymixin B. Semen samples containing gentamicin were also more effective (P < 0.05) at eliminating bacterial growth than those samples containing ticarcillin or sodium penicillin. In Experiment 2, mean percentage of rapidly-motile sperm, and mean curvilinear, average-path, and straight-line velocities were greater (P < 0.05) for potassium penicillin amikacin than values for all other treatments. In 2 of 3 stallions, an effect of treatment on percentage of motile sperm was detected (P < 0.05). For one stallion, mean motility of potassium penicillin-amikacin was greater (P < 0.05) than that of all other treatment groups. For another stallion, mean motility of the control was lower (P < 0.05) than that of the other treatments. Following storage, potassium penicillin (16/18 [89%]) or potassium penicillin-amikacin (17/19 [94%]) were more effective (P < 0.05) at controlling aerobic and anaerobic bacterial isolates in semen specimens than was amikacin (10/18 [56%]). In Experiment 3, a difference among treatment groups for motility variables was not detected (P < 0.05). No bacterial growth was recovered in antibiotic-treated semen, with exception of Micrococcus sp. (2 colonies) which were isolated from one semen specimen treated with ceptiofur. PMID- 10732148 TI - Effects of repeated ultrasound-guided transvaginal follicular aspiration on bovine oocyte recovery and subsequent follicular development. AB - We wished to compare cumulus oocyte complex (COC) recovery and follicle development after single and repeated ultrasound-guided transvaginal follicle aspiration (aspiration). Aspirations were performed in Holstein-Friesian heifers every once weekly (every 7 d; n = 12) or twice weekly (every 3 or 4 d; n = 6) starting on Days 3 or 4 of the estrous cycle (estrus = Day 0) and continuing for 4 wk. During each session, all visible follicles > 2 mm were aspirated using an 7.5 MHz transducer to guide an 18 ga x 60 cm single lumen needle and applying 50 mm Hg vacuum which generated 25 mL/min. The COC's harvested from each follicle were counted and classified into 4 categories. Post-aspiration follicle wave emergence was traced by daily ultrasound examinations. A total of 1410 follicles were aspirated during 96 sessions, yielding 632 (45%)oocytes. There was no difference in average COC/follicle recovered between the single vs the repeated aspiration treatment. However, ovaries of heifers subjected to two aspirations per week yielded more follicles (17.2 +/- 5.7 vs 12.4 +/- 6.1; P < 0.01) and COC's (7.7 +/- 4.5 vs 5.4 +/- 3.7; P < 0.01) per session than those subjected to a single aspiration. Ovaries of heifers subjected to twice weekly aspirations at 4-d intervals resulted in a higher recovery rate (51.1 vs 38.6%), yielded more COC's (9.3 +/- 4.7 vs 6.2 +/- 3.8) and a higher number of viable COC's recovered per session (7.6 +/- 3.8 vs 5.2 +/- 3.3) than those aspirated every 3 d, all P < 0.01. Aspiration-induced follicle waves were indicated by an increased number of follicle > or = 4 mm seen within 2 d of the procedure. We conclude that follicle aspiration appears to induce and synchronize follicle waves, and when it is done twice a week it is associated with higher number of harvestable follicles and more oocytes recovered than when done once a week. These results can be attributed to the aspiration of a newly recruited pull of follicles 3 or 4 d after the first aspiration and before the establishment of follicular dominance and regression of subordinate follicles. PMID- 10732149 TI - Puberty and serum concentrations of ovarian steroids during prepuberal period in Friesian heifers artificially infected with Fasciola hepatica. AB - An experiment was conducted to study the effect of fascioliasis on puberty in 13 Friesian heifers divided into a control group (n = 7) and an infected group (n = 6). At 4 m.o. of age each infected heifer received 600 metacercariae of F. hepatica. Blood samples were taken twice a week, at which time permanent heat mount detectors were checked, from 8 m.o. of age until 2 estruses were detected in all heifers. Serum concentrations of progesterone (P4) and estradiol-17 beta (E2) were assayed by ELISA and RIA, respectively. There was a significant delay in the onset of puberty in infected heifers compared with controls. All animals reached puberty at similar body weights but at different ages. First estrus was delayed by 39 d in infected compared with control animals. Serum concentrations of E2 were significantly higher in the infected group than the control group, while P4 concentrations were lower. Fascioliasis alters the serum concentration of E2, which in turn results in an abnormally low concentration of P4, probably due to underdeveloped or absent corpora lutea. PMID- 10732150 TI - Serum progesterone and 17 beta-estradiol concentrations during pregnancy of Bactrian camel (Camelus bactrianus). AB - Serum progesterone and 17 beta-estradiol during pregnancy in the Bactrian camel were measured by radioimmunoassay. Serum progesterone concentrations increased by 15 d after artificial insemination (AI) and remained elevated throughout most of gestation, the mean concentrations (3.06 +/- 0.49 to 8.51 +/- 4.80 ng/mL) were similar to those reported for many species during the same stage of pregnancy. Serum 17 beta-estradiol increased significantly from 11 m.o. of pregnancy with peak mean concentrations of 617.47 +/- 32.56 pg/mL at the 11.5 m.o. PMID- 10732151 TI - The influence of fatness on the likelihood of early-winter pregnancy in muskoxen (Ovibos moschatus). AB - Among wild ruminants, muskoxen have an exceptional ability to fatten, but their pregnancy rates are variable and often low. To test whether the likelihood of pregnancy in muskoxen is associated with exceptionally good body condition, we used logistic regression analysis with data from 32 pregnant and 18 nonpregnant muskoxen > or = 1.5 yr of age shot in November (1989 to 1992) on Victoria Island in Arctic Canada. We assayed their serum for insulin-like growth factor-1 (IGF 1). All fatness and mass measures were positively related to the likelihood of pregnancy (P < 0.001), with the strongest associations for estimated total fat mass (80% of outcomes predicted correctly) and kidney fat mass (77%), and weaker models for body mass. Pregnancy was less likely to occur in lactating females than in nonlactating ones (P = 0.03). Although IGF-1 concentrations were higher (P = 0.001) in nonlactating females than in lactating ones (28.7 +/- 1.7 vs. 22.5 ng/ml), no association with pregnancy was detected (P = 0.57). Fatness associated with a 50% probability of pregnancy in muskoxen (22% of ingesta-free body mass or 32 kg fat in females > 3.5 yr old) is much higher than in caribou and somewhat higher than in cattle, and this may partly account for the low calving rates often observed in this species. PMID- 10732152 TI - Cryopreservation and ensuing in vitro fertilization ability of boar spermatozoa from epididymides stored at 4 degrees C. AB - The influence of prolonged storage of boar epididymides on post-thaw sperm motility, and in vitro fertilization was evaluated. Twenty pairs of epididymides were obtained from Large White boars, and spermatozoa from one of each of the pairs were immediately collected and frozen (control group). The remaining epididymides were cooled to 4 degrees C and stored for 1, 2 or 3 d, after which spermatozoa were collected and frozen (experimental groups Day 1, 2 and 3, respectively). Sperm motility was maintained throughout the dilution procedure and then dropped (P < 0.01) after freezing and thawing. During storage the motility of nonfrozen spermatozoa decreased significantly (P < 0.01), reaching a value equal to that of frozen-thawed spermatozoa on Day 3. In vitro fertilization experiments revealed significantly (P < 0.05) lower penetration rates using Day 1, 2 and 3 stored spermatozoa (12, 13 and 2%, respectively) than that of the control group (40%). Oocyte penetration ability seemed to be reflected by acrosome integrity. However, the motility of spermatozoa with the ability to penetrate oocytes in Day 1 and Day 2 groups did not differ from that of the controls. The motility of spermatozoa lacking penetration ability, on the other hand, gradually decreased as the storage period was prolonged. This suggests that the sperm motility and penetration ability are affected by different mechanisms during the cold storage of epididymides. Finally, control and experimental groups exhibited high incidences of monospermic penetration (64 to 90%) and of male pronuclear formation (67 to 71%). These data suggest that cryopreservation of spermatozoa from boar epididymides stored at 4 degrees C for 1 to 2 d can be used for conserving male germ cells when epididymal spermatozoa can not be collected immediately and cryopreserved. PMID- 10732153 TI - Verification of flow cytometorically-sorted X- and Y-bearing porcine spermatozoa and reanalysis of spermatozoa for DNA content using the fluorescence in situ hybridization (FISH) technique. AB - Flow cytometric sperm sorting based on X and Y sperm DNA difference has been established as the only effective method for sexing the spermatozoa of mammals. The standard method for verifying the purity of sorted X and Y spermatozoa has been to reanalyze sorted sperm aliquots. We verified the purity of flow-sorted porcine X and Y spermatozoa and accuracy of DNA reanalysis by fluorescence in situ hybridization (FISH) using chromosome Y and 1 DNA probe. Eight ejaculates from 4 boars were sorted according to the Beltsville Sperm Sexing method. Porcine chromosome Y- and chromosome 1-specific DNA probes were used on sorted sperm populations in combination with FISH. Aliquots of the sorted sperm samples were reanalyzed for DNA content by flow cytometry. The purity of the sorted X-bearing spermatozoa was 87.4% for FISH and 87.0% for flow cytometric reanalysis; purity for the sorted Y-bearing spermatozoa was 85.9% for FISH and 84.8% for flow cytometric reanalysis. A total of 4,424 X sperm cells and 4,256 Y sperm cells was examined by FISH across the 8 ejaculates. For flow cytometry, 5,000 sorted X spermatozoa and 5,000 Y spermatozoa were reanalyzed for DNA content for each ejaculate. These results confirm the high purity of flow sorted porcine X and Y sperm cells and the validity of reanalysis of DNA in determining the proportions of X- and Y-sorted spermatozoa from viewing thousands of individual sperm chromosomes directly using FISH. PMID- 10732154 TI - Rate of transport and development of preimplantation embryo in the superovulated buffalo (Bubalus bubalis). AB - This study was designed to ascertain the rate of transport and development of preimplantation embryo in the superovulated buffalo in order to determine the optimum time for their nonsurgical collection. Eighteen Murrah-type buffalo were superovulated with 600 mg NIH-FSH-P1. Luteolysis was induced by administration of PGF2 alpha at 72 (PG + 72) and 84 h (PG + 84) after initiating gonadotrophin treatment and fixed-time AI was done beginning at 36 h post PG + 72 administration and at 12-h intervals thereafter, upto 72 h. Six control buffalo received treatment similar to experimental group except that in place of FSH they received normal saline. For embryo collection, experimental animals were humanely killed at 6-h intervals corresponding to 156 (n = 2), 162 (n = 2), 168 (n = 2), 174 (n = 3), 180 (n = 3), 186 (n = 3) and 192 h(n = 3) after PG + 72 treatment, whereas the control animals were humanely killed at 156 (n = 2), 174 (n = 2) and 192 h (n = 2). Superovulated buffalo had higher number of ovulations than untreated controls (8.78 +/- 5.00 vs 0.67 +/- 0.51) and total ova/embryos recovered was 4.11 +/- 2.46 and 0.67 +/- 0.51, respectively. The high estradiol 17 beta (E2) levels with its prolonged rise may, by leading to reverse peristalsis in the oviduct with a consequent loss of some embryos in the peritoneal cavity, be one of the reasons for our inability to recover nearly 84/158 ova/embryos in the superovulated buffalo. In superovulated animals, nearly all the ova/embryos reached the uterus between 168 and 174 h post PG + 72 treatment or about 134 h (circa 5.5 d) after the onset of superovulatory estrus, suggesting that the ideal time for non-surgical embryo collection in the buffalo is between Days 7 to 8 after PG + 72 treatment or Days 5.5 to 6.0 of the superovulated cycle (estrus = Day 0). Embryo development of superovulated buffalo showed considerable variation as various stages of embryos (8 cell to expanded blastocyst) were recovered from the same donor buffalo, and the rate of development appeared to be 24 to 36 h faster than in cattle. PMID- 10732155 TI - Effect of kininase II inhibitors on bradykinin-stimulated bovine sperm motility. AB - It has been found that the enzyme kininase II present in the seminal plasma inactivates the kinins produced by the kallikreins, thus blocking their beneficial effects on the motility of post-ejaculated mammalian spermatozoa. In this study we examined the influence of 2 kininase II inhibitors (captopril and enalapril maleate) on bradykinin-induced bovine sperm motility. Ejaculates were collected from Holstein-Friesian bulls, and semen samples exhibiting reduced sperm motility (30 to 60%; mean 53 +/- 3.8%) were used. Each semen sample was divided into 6 portions: 1) control; 2) treated with bradykinin (M-8); 3) treated with captopril (M-2); 4) treated with enalapril maleate (M-3); 5) treated with bradykinin + captopril; and 6) treated with bradykinin + enalapril maleate. Total sperm motility was recorded over 4 h at 1-h intervals. It was found that in the second hour after treatment both the combinations of bradykinin + captopril and bradykinin + enalapril maleate considerably enhanced sperm motility compared with that of the controls (P < 0.01 and P < 0.05, respectively). Total sperm motility rates of semen samples treated with these combinations were also higher than in the samples treated either with inhibitors alone or with bradykinin alone. We concluded that the stimulatory effect of bradykinin on bovine sperm motility was considerably potentiated and prolonged by the addition of the kininase II inhibitors. PMID- 10732156 TI - Effects of superovulation, culture and microinjection on development of rabbit embryos in vitro. AB - Factors influencing the developmental potential of cultured rabbit zygotes and their ability to incorporate and integrate the WAP-hPC (human protein C) gene were investigated. Rabbit zygotes (n = 1053) were recovered from both superovulated and nontreated New Zealand White females. The hormonal treatment of rabbit donors resulted in a doubling of the number of recovered ova per donor when compared with the nontreated group (18 vs 9 ova). However, the quality of recovered zygotes (presence of both pronuclei) was significantly better in the nontreated group (99 vs 88%, Experiment 1). The effect of various culture media on the development of rabbit zygotes in vitro was evaluated after incubation under CO2-free conditions (Experiment 2). In serum-free, growth factor supplemented medium (BSEITS, DME/F12, 1.5% BSA, EGF, insulin, transferrin and sodium selenite) the percentage of morula/blastocyst stage embryos was significantly higher (88%) than in DME/FCS, (DME/F12, 10% fetal calf serum, 59%) or the control group (DME/F12, 1.5% BSA, 25%). In Experiment 3, zygotes were microinjected with the WAP-hPC gene and were examined after 72 h of culture. Zygote cleavage and the percentage of morula/blastocyst stage intact embryos were higher (79 and 58%, respectively) than in microinjected embryos (31.0 and 21.5%, respectively). Summarized data of the PCR assay of microinjected zygotes demonstrated positive signals for the integration of the WAP-hPC gene in 6.6% (34 of 515) of all the microinjected zygotes. PMID- 10732157 TI - Effects of temperature gradients on in vitro maturation of bovine oocytes. AB - The effect of temperature gradients on in vitro maturation of bovine oocytes was examined in this study. Six treatment groups were made by combining 3 different maturation periods (0 to 10 h, 10 to 18 h and 18 to 24 h) with 2 different culture temperatures (37.0 degrees C and 38.5 degrees C). The frequency of oocytes matured to the metaphase II stage was apparently gradually increased as the culture temperature was increased from 37.0 degrees C to 38.5 degrees C at 0, 10 and 18 h after the onset of culture (75.2 vs 80.5, 82.3 and 84.3%, respectively), but this difference was not significant. Neither was the minor decrease in the proportion of oocytes reaching metaphase II when the temperature was decreased from 38.5 degrees C to 37.0 degrees C at 10 and at 18 h after the onset of maturation (84.3 vs 82.4 and 78.0%, respectively). However, more oocytes cleaved (79.2%; P = 0.0653) and developed to morulae (43.6%; P = 0.0019) and blastocysts (27.4%; P = 0.1568) when they were in vitro matured at 38.5 degrees C between 0 and 10 h, and then at 37.0 degrees C from 10 to 24 h. Although only the morula group was statistically different, cleavage- (79.2 vs 69.8, 72.5, 74.2, 76.3, 74.3%, respectively) and blastocyst formation (27.4 vs 23.2, 24.6, 25.2, 19.6, 21.9%, respectively) from this group was the highest among the 6 treatments. PMID- 10732159 TI - The Italian Association for the Study of Glaucoma 14th annual meeting. Rapallo, Italy, 11-13 March 1999. Abstracts. PMID- 10732158 TI - Prevention of incubation behavior expression in turkey hens by active immunization against prolactin. AB - The consequences of active immunization against prolactin on expression of incubation, reproductive performance and hormonal profiles were evaluated in turkey hens. Hens were injected weekly for 4 wk starting 8 wk before being submitted to a stimulatory photoperiod and 3 times thereafter at intervals of 4 to 5 wk. The hens were injected i.d. with 0.5 mL of a mixture diluted half in Freund's adjuvant. The mixture was prediluted in .9% saline and contained 100 micrograms of a fusion protein (GST-tPRL), GST, oPRL or vehicle. The results indicate that active immunizations with GST-tPRL or oPRL both induce production of specific prolactin antibodies. The onset of egg production was unaffected but higher egg production was observed for the GST-tPRL immunized hens. No GST-tPRL immunized hens expressed incubation behavior, whereas 20 to 30% of hens in the other experimental groups did so. Apparent hyperprolactinemia was detected by RIA for the GST-tPRL immunized groups starting before photostimulation and lasting until Week 10 of egg production but not in other groups. No significant differences were observed in either plasma LH or estradiol concentrations of immunized and nonimmunized turkey hens. In conclusion, both GST-tPRL or oPRL induced the production of antibodies against prolactin in turkey hens. However, only active immunization using GST-tPRL induced higher antibody titers as well as full prevention of incubation behavior expression. Such a pharmacological approach is of great practical interest, although its uses need to be carefully evaluated under commercial conditions. PMID- 10732160 TI - 3rd International Conference on Therapies for Viral Hepatitis. Maui, Hawaii, USA. 12-16 December 1999. Abstracts. PMID- 10732161 TI - 1st International Conference on the Discovery and Clinical Development of Antiretroviral Therapies. St. Thomas, West Indies, US Virgin Islands. 13-17 December 1998. Abstracts. PMID- 10732162 TI - [X European meeting of the French Society of Cardiology. Paris, France, 19-22 January 2000. Abstracts]. PMID- 10732163 TI - Biochemical Society 670th Meeting, University College Cork. PMID- 10732164 TI - British Association of Dermatologists annual meeting. Edinburgh, United Kingdom, 29 June-3 July 1999. Abstracts. PMID- 10732165 TI - 18th Annual Conference of the United Leukodystrophy Foundation. Probing White Matter Disorders '98. Abstracts. PMID- 10732166 TI - Cumulative author and subject index for volumes 162 to 172. PMID- 10732168 TI - Microbial Genomes Conference IV: sequencing, functional characterization and comparative genomics. Chantilly, Virginia, USA. February 12-15, 2000. Abstracts. PMID- 10732167 TI - Society for Gynecologic Investigation 47th annual meeting. Chicago, Illinois, USA. March 22-25, 2000. Abstracts. PMID- 10732169 TI - Notes for guidance on the clinical administration of radiopharmaceuticals and use of sealed radioactive sources. Administration of Radioactive Substances Advisory Committee. PMID- 10732170 TI - 68th Annual meeting of the Swiss Society of Internal Medicine, annual meeting of the Swiss Societies of Hematology, Geriatrics, Clinical Pharmacology. Zurich, Switzerland, 9-11 March 2000. Abstracts. PMID- 10732171 TI - 5th World Congress on Trauma, Shock, Inflammation and Sepsis--Pathophysiology, Immune Consequences and Therapy. Munich, Germany, February 29-March 4, 2000. Abstracts. PMID- 10732172 TI - A comparison of the Allen Cognitive Level Test and the Wisconsin Card Sorting Test in adults with schizophrenia. AB - OBJECTIVE: This study examines the relationship among executive function, visuospatial problem solving, and measures of occupational functioning in 33 adult men with schizophrenia or schizoaffective disorder. METHOD: Three measures were administered in a one-time assessment session. The Wisconsin Card Sorting Test (WCST) was used to measure executive functioning, abstract reasoning, and problem-solving abilities. The Allen Cognitive Level (ACL) Test was used to measure learning, problem solving, and visuospatial abilities. The Routine Task Inventory (RTI) measured the level of performance in activities of daily living. RESULTS: Using multiple regression controlling for age, the WCST significantly predicted performance on the ACL the RTI. Perseverative errors on the WCST were negatively correlated with the ACL (r = -.47) and RTI scores (r = -.59). The ACL and RTI were also significantly correlated (r = .67). CONCLUSION: Both the WCST and the ACL are sensitive to similar domains of functioning and are predictive of task performance. The results support the use of the ACL as a quick measure of a person's cognitive and functional abilities. PMID- 10732173 TI - Clinical interpretation of "a comparison of the Allen Cognitive Level Test and the Wisconsin Card Sorting Test in adults with schizophrenia". PMID- 10732174 TI - Exploration of the perspectives of persons with schizophrenia regarding quality of life. AB - OBJECTIVE: This study is the first phase of a two-phase study aimed at exploring the perspectives of consumers with schizophrenia regarding quality of life and developing a quality-of-life assessment that addresses factors experienced as important by consumers. METHOD: Focus groups were used to explore the perspectives of 35 persons with schizophrenia regarding the meaning of quality of life and factors important to quality of life. RESULTS: Seven major factors that had an impact on quality of life resulted from inductive analysis, including activity, social interaction, time, disclosure, "being normal," finances, and management of illness. These factors relate to three overall themes: managing time, connecting and belonging, and making choices and maintaining control. CONCLUSIONS: Although the findings confirm the importance of factors included in existing quality-of-life assessments, they also highlight the need to look at new dimensions of commonly included factors and to include other factors. The findings support beliefs regarding occupation that are central to occupational therapy and the use of occupation as means and ends of therapy. PMID- 10732175 TI - Critical pathway development: an integrative literature review. AB - OBJECTIVE: The purpose of this integrative literature review was to summarize and analyze the methods used to develop critical pathways. METHOD: Relevant articles published in occupational therapy, physical therapy, nursing, and medical journals between 1992 and 1997 were reviewed to extract various methods and the steps or criteria used for each method. RESULTS: Nine approaches to critical pathway development and the steps or criteria involved in each method are presented in tabular format. The most detailed approach was used as a gold standard, and the other approaches were compared to it. CONCLUSION: This review should assist occupational therapy practitioners working with various diagnostic populations to understand the methods used, and steps involved, in the development of critical pathways. It should also serve as a resource for practitioners who have the opportunity to participate in critical pathway development. PMID- 10732176 TI - Occupational therapy benchmarks within orthopedic (hip) critical pathways. AB - OBJECTIVE: This study examined patient performance benchmarks for occupational therapy within orthopedic (hip) critical pathways. METHOD: Eight orthopedic (hip) critical pathways gathered from occupational therapy practitioners working in hospital and rehabilitation settings were examined to determine commonalities and differences of occupational therapy benchmarks, disciplines involved, and identified allowable variances. A comparison and contrast matrix was developed to provide a visual means of reviewing the data. RESULTS: Nursing, physical therapy, and occupational therapy were disciplines consistently involved in the pathways. Activities of daily living related to self-care were the most consistently used occupational therapy benchmark within the sample pathways, and functional transfers were the second most-used benchmark. The remaining occupational therapy benchmarks varied, and little commonality was found in their use. Frequency of use also varied among the eight pathways. Five of eight pathways specifically coded variances, with the remaining three providing space for explanation of the variance. CONCLUSION: Although these eight orthopedic (hip) critical pathways included occupational therapy benchmarks, further development and definition of the role of occupational therapy within subsequent orthopedic (hip) critical pathways is needed. PMID- 10732177 TI - Use of early tactile stimulation in rehabilitation of digital nerve injuries. AB - OBJECTIVE: Digital nerves are the most frequently injured peripheral nerve. To improve the recovery of functional sensibility of digital nerve injuries, a prospective randomized controlled study was conducted to see the effect of using early tactile stimulation in rehabilitation of digital nerve injuries. METHOD: Two specific tactile stimulators were made and prescribed for patients with digital nerve-injury. Twenty-four participants with 32 digital nerve injuries received the prescribed tactile stimulators (experimental group), and another 25 participants with 33 digital nerve injuries received only routine conventional therapy (control group). RESULTS: A significant difference (p < .05) was seen in the experimental group, although there were some variations between the different classes of associated injuries, with least benefit observed in the combined nerve, tendon, and bone injury class. CONCLUSION: Use of early tactile stimulation as described in this study can be considered an effective way to improve both quality and quantity of recovery of functional sensibility in digital nerve injuries without combined nerve, tendon, and bone injuries. PMID- 10732178 TI - Effects of body mechanics training on performance of repetitive lifting. AB - OBJECTIVE: To measure the efficacy of body mechanics instruction (BMI) in patients with low back pain. METHOD: The effect of BMI was measured in four participants with back injuries using a standardized lifting protocol. Static strength, weight lifted, number of lifts completed, and motion analysis data to describe the body mechanics were measured before and after work hardening to evaluate treatment effects. The participants' performances were compared with 52 controls from an earlier study. RESULTS: Starting postures, characterized by degrees of hip and knee flexion, varied by participant but favored a squat lift in three participants when compared with the controls. Dynamic motion synchrony of the hip and knee joints was similar to controls in three of the four participants. Posttest data revealed significant changes in static strength, dynamic endurance, and lifting speed. CONCLUSION: Intensive instruction in body mechanics provided during the work-hardening treatment produced major changes in lifting styles, in terms of both starting postures and dynamic aspects of repetitive lifting. The computerized measurement procedures used in this study permitted more careful and detailed analyses of body mechanics, particularly dynamic aspects, than is possible with observational methods. This study demonstrated some of the inherent intricacies in isodynamic lifting and suggests additional areas of performance that may be important to address in BMI. PMID- 10732179 TI - From hand twister to mind twister: computer-aided treatment in traumatic wrist fracture. AB - OBJECTIVE: The use of computers as a treatment modality in the occupational therapy hand clinic is, as yet, not common practice. A computer interface for wrist movements was developed, and a study to justify the application of such a device is presented. METHOD: Forty-seven patients in a day hand clinic who had traumatic fracture of one hand with limitation of wrist mobility participated in the study. Participants were divided into two treatment groups: computer-aided treatment (high technology) and traditional brush machine treatment (low technology). A device was developed based on the brush machine in which the brush machine's mechanism was converted into a medial-lateral joystick. Right-to-left movements were digitally transformed for the use of a computer game. Participants were treated for 5 weeks, and outcome measures included range of motion (ROM), grip strength, edema, and level of interest. RESULTS: Results showed significant improvement in ROM, grip strength, and edema across 5 weeks for all participants. Although no significant differences were found between the two groups in ROM, grip strength, and edema, the computer-aided group showed significantly more interest in treatment than did the brush machine group. Finally, the interaction between treatment group and the attitude toward computers was not significant. CONCLUSIONS: These results indicate the potential for more interesting motor treatment and rehabilitation of the wrist through the use of computer games. The efficacy of using computers in occupational therapy clinics needs further investigation. PMID- 10732180 TI - The experience of being an occupational therapist with a disability. AB - OBJECTIVE: This study addressed what it is like to practice as an occupational therapist with a disability. METHOD: Open-ended interviews using a phenomenological approach were conducted with 10 participants until data saturation was achieved. Each transcripted interview was coded for categories, and the common themes across transcripts were identified. RESULTS: These major themes were identified: "I am sensitive to their needs"; "The issue is how to cope with life"; and "Recognize your own strengths and limits." Each theme had several subcategories. CONCLUSIONS: Occupational therapists with disabilities approach their practice from a unique perspective and may be able to motivate and challenge clients in a different manner than therapists without disabilities. Therapists with disabilities perceive themselves as uniquely skilled persons who have developed successful strategies to cope with the experience of disability. PMID- 10732181 TI - Measuring knowledge of school-based occupational therapy. AB - OBJECTIVE: The primary purpose of this study was to develop a measure of Knowledge of Occupational Therapy in School settings (KNOTS) for occupational therapy students. A secondary purpose was to develop a scale of Self-Efficacy in School Settings (SESS) for occupational therapy students. METHOD: One hundred forty-five university students enrolled in either occupational therapy classes or education classes completed a questionnaire consisting (in part) of the 60-item KNOTS scale and the SESS scale. Analyses of internal reliability, group comparisons, and correlations were computed on the scores. RESULTS: The reliabilities of the KNOTS (.85) and SESS (.94) were reasonably high. Occupational therapy students performed significantly better than did education students, and senior occupational therapy students scored significantly higher than did juniors on the KNOTS. SESS scores were positively but not significantly correlated with KNOTS scores. CONCLUSION: If further investigation provides additional evidence for the reliability and validity of the KNOTS and SESS, both measures have a number of potential practical applications. PMID- 10732182 TI - Daily life for eight urban gay men with HIV/AIDS. AB - OBJECTIVE: The purpose of this qualitative research study was to gain an understanding of the daily life experiences of eight gay men with HIV/AIDS living alone in New York City. METHOD: The participants ranged in age from 25 to 50 years. Data were primarily collected using in-depth personal interviews in 1996 followed by telephone interviews 1 year later. Constant-comparison and thematic analyses were used to identify themes and subthemes. RESULTS: Two broad themes with related subthemes were generated from the data. "A Reasonably Stable Base" represented the emotional, physical, and environmental foundation that preexisted or was created as a consequence of living with HIV/AIDS. This theme played a particularly important role in the participants' lives, especially during periods of emotional and physical instability. "Finding and Maintaining Balance" was a second theme that illustrated strategies used for managing and readjusting daily routines, goals, and priorities, as well as how the participants experienced this readjustment process. CONCLUSIONS: All of the participants developed their own daily living strategies that were comparable to intervention methods provided in occupational therapy such as energy conservation and work simplification. The findings suggest that occupational therapy practitioners could potentially assist urban gay men with HIV/AIDS with finding and maintaining stability and balance in their daily lives. PMID- 10732183 TI - Consumer empowerment through occupational therapy: the Americans With Disabilities Act Title III. AB - OBJECTIVE: The purpose of this study was to determine whether occupational therapists (a) value a role educating consumers about the Americans With Disabilities Act of 1990 (ADA; Public Law 101-336); (b) are knowledgeable regarding Title III of the ADA; and (c) implement provisions and empower consumers who use wheelchairs to access public accommodations. METHOD: A random sample of 510 occupational therapists was surveyed, with 229 responding. Of those surveys returned, 152 respondents who serve clients who use wheelchairs met inclusion criteria. RESULTS: Although 90% of the participants agreed that occupational therapists should have ADA knowledge and should educate consumers, the mean score of ADA accessibility knowledge on a 10-point quiz was 1.85. The mean score of reported actions to implement ADA provisions with clients was 11.78 of a possible 40 points. There was a significant positive correlation between implementation and attitude (r = .3609, p = .01) and between implementation and knowledge (r = .3376, p = .01); however, the correlation between attitude and knowledge (r = .1673, p = .05) was not significant. CONCLUSION: Therapists' lack of knowledge and their self-reported inaction with regard to ADA Title III may affect the accessibility of the environment, independence, and empowerment of clients who are wheelchair mobile and, therefore, may impede progress toward fully inclusive communities. PMID- 10732184 TI - Authorship criteria: opening a dialogue. PMID- 10732185 TI - What is the best evidence to use in practice? PMID- 10732186 TI - The ICIDH-2: a new language in support of enablement. American occupational therapy foundation research advisory council. PMID- 10732187 TI - [Mechanisms of extreme states in human]. AB - Pathogenesis of extreme states during hypoxic hypoxia, exogenic and combined hyperthermia, aerial and immersion hypothermia is mainly associated with deterioration of reactivity and decline of functional reserves of the neuroendocrinal system, energetic dysbalance against more pronounced signs of anaerobic catabolism and recruitment of plastic resources of the body, activation of peroxide oxidation of lipids (POL), depletion of AOS potential with labilization of cellular membranes, and progressing changes in structural and functional relations within the antigenous/structural homeostasis. The determinacy of extreme state is influenced by initial status of the body functioning. The bodily extreme state has been found to depend on severity of asthenisation, peculiarities of the autonomous regulation, metabolic and immune status. Partial or full reversibility of decompensation disorders distinguishes extreme state from the critical (predeterminal) one. The relative adequacy of compensation and the principal possibility of a beneficial result are the necessary conditions for extreme state verification. Otherwise, in the lack of time for effective compensation typical syndromes of critical states may develop already during acute adaptation. There can develop partial insufficiency of a dominating functional system which will be compensated at the sacrifice of other organs and tissues. Sometimes extreme state may result in development of marginal or pathological states with human living activities preserved. PMID- 10732188 TI - [Radiation risk of malignant tumors in cosmonauts over life time as a result of participation in interplanetary and orbital missions]. AB - The paper considers model concepts of cell blast-transformation and oncogenesis in humans consequent to ionizing irradiation with varying dose rates and lengths of exposure. Presented are data of epidemiologic studies of oncologic risks for different organs and body tissues at different ages in a year since exposure calculated per a unit of absorbed dose (1 cGy). Probability of tumor development in males of different age due to chronic irradiation of various lengths was determined per a dose unit. Based on these data and with regard for possible dose loads on interplanetary and orbital crews, oncologic risk for cosmonauts was calculated in terms of life time. The dependence of radiation-induced oncologic risks on type and duration of space mission, shielding thickness, and solar cycle was analyzed. The authors compare values of the total radiation risk and oncologic risk for cosmonauts launched at various ages. PMID- 10732189 TI - [Radiation environment of orbital complex "Mir" during minimum of the 22-nd solar cycle (1994-1996)]. AB - Analysis of radiation dynamics on board the orbital complex MIR in the period of 1994 through to 1996 overlapping minimum of the 22nd solar cycle (SC) was performed. Radiation parameters in the working compartments during eight MIR missions (MIR 15-22) were evaluated. According to the solar dynamics during minimal activity with the lowest meanings of Wolf numbers (< 20), the daily average dose rate on MIR reached its peak over the whole 22nd SC and by a factor of more than three exceeded meanings typical for solar maximum. A statistical method was used to compute correlation coefficients and set up an equation of linear regression of the absorbed dose with cosmophysical parameters of the SC minimum. Meanings of the correlation coefficients for SC minimum were substantially lower as compared with SC maximum, that is there was no distinct correlation between dose rate and the parameters under study. Radiation doses to cosmonauts violated the admissible radiation limits for ground exposures but not radiation limits established for manned space missions. Reduction of dose loading on cosmonauts during SC minimum can be achieved by planning shorter missions or utilization of a program of radiation protection for traverse of SAA ERB. PMID- 10732190 TI - [Dynamics of interrelationship in a small group during long-term hypokinesia]. AB - Effects of low activity of test-subjects during antiorthostatic hypokinesia on the intragroup structure as well as the semantic composition and the directionality of estimation criteria selected by test-subjects to describe the system of attitudes to oneself and others, and their impact on individual status in the social structure of the group have been studied. It was shown that maintaining of a definite level of individual activity of members is important for the group structure and successful adaptation of test-subjects to the unfavourable factors of hypokinesia. The degree of the ego image integrity and dominance of criteria that infer empathy and positive feelings within the system of individual appraisements determine the social status of a group member. The psychologic signs of a "apet" (psychologically close) and a "outsider" (psychologically strange) members of the group have been revealed. PMID- 10732191 TI - [Thyroid gland development in Japanese quail embryos incubating in microgravity]. AB - The investigation of the progress of thyroid prenatal development in the Japanese quail has been first performed in the MIR microgravity environment. Development of thyroid glands in control and flight embryos was shown to pass same stages (trabecular and follicular). However, the flight embryos demonstrated retarded a development of the gland as compared to the control one. Slowing-down of thyroid organogenesis in the flight embryos is evidently related to retardation in formation of their blood system and a consequent decrease in blood inflow to the thyroid microcirculatory bed. PMID- 10732193 TI - [Peculiarities of growth and morphogenesis of syphoneal alga vaucheria sessilis in microgravity]. AB - The space experiment was aimed at studying the dependence on the gravity of intracellular processes in an autotrophic organism deprived of pronounced gravitropisms and gravitaxis. The experiment demonstrated an immediate gravitational effect on intercellular processes involving the syphoneal alga Vausheira sessilis cytoskeleton. In the absence of the force of gravity algal growth and morphogenesis are inhibited more significantly than under the condition of the Earth's gravity. PMID- 10732192 TI - [Changes in productivity of plant cells as result of cultivation under the space flight conditions]. AB - Effects of space flight on growth and biosynthetic features of plant cells were studied in two strains of ginseng (Panax ginseng) differing in growth and particularly biosynthetic activities, a strain of Lithospermum Erythrorhizon and a strain of Macrotomia Euchroma which produce biologically active naphroquin derived pigments. --and also differ in growth and biosynthetic properties. Following exposure aboard MIR and a Space shuttle, cells of the callosal cultures were subjected to callosal or suspension passaging. Biomass yield and biologically active substances--ginseng saponins ginsenoids and shikonin were determined in the cells cultures. There was no evidence for the biomass yield to be significantly altered by space flight; however, the content of biologically active substances was materially changed with the strain. PMID- 10732194 TI - [Choice of plant light status for space greenhouse: results of ground-based experience]. AB - To decide on the light status of plants in space greenhouse, a theoretical study was undertaken to correlate specific productivity of space greenhouse with illumination characteristics including vertical PAR flux density (I), photoperiod (tau), and crop leaf index (L). It was demonstrated that in pace with I the daily productivity per a volume unit tended to monotonously approach maximum at I = Ip, whereas the greenhouse energy efficiency ME peaked at I = IE, IK < IE < IP, where IK is a compensation point of the light curve of crop photosynthesis. Proposed are compromise criteria to optimize illumination as a maximum of linear combination of MV and ME and coefficients which account for the cost of a space station volume unit and a unit of board power supply, and as maximum of product Q = MV.ME. Experimental results serve as the basis for a technique for determination of the best, by the Q criterion, light status parameters for three types of space greenhouses: research growth chamber for synchronous cultivation of leaf mustard, wheat growth chamber with fixed crop density, and green conveyer for cultivation of Brassica pekinensis (Lour Rupor). For the last mentioned Q effective I and tau values differed with the conveyer step. The technique allows design of ground-based experiments aimed at determination of the most effective light status of space-grown crops. PMID- 10732195 TI - [Change in erythrocyte sedimentation during compression and decompression]. AB - It is determined that during compression and decompression of blood and blood suspension the main section plane of naturally sedimented erythrocytes assume virtually vertical position and, therefore, sedimentation rate of erythrocytes rises. Also, following compression blood density in vitro is irreversibly increased. The idea of existence of erythrocyte-bonded gas microcavities is discussed. PMID- 10732197 TI - [Spinal diseases and professional longevity of flying personnel]. AB - Analyzed were histories of medically discharged (n = 727), physically qualified (n = 807), pilots and navigators, and flying personnel (n = 372) who had been repeatedly tested in one and the same hospital at the age of 34-36 yrs and 38-41 yrs. Spondylosis deformans is diagnosed more frequently in medically discharged than qualified flyers but, according to the covariation analysis, this is explained by elder age of the discharged persons. Physically fit persons with SD diagnosed at the age of 30-40 are prone to be grounded earlier than healthy persons. Compared with persons having other diagnoses, flying career (FC) will have longer if SD was stated at the age of 30, same if SD was stated at the age of 35, and shorter if SD was diagnosed at the age of 40. These differences remain statistically significant after covariation correction for specialty, age of entering military school and number of chronic diseases. PMID- 10732196 TI - [Ultrasonic investigations in diagnostics of prostate diseases in flying personnel]. AB - Ultrasonic semiotics of the most common prostate problems was studied. Benefits from transabdominal and transrectal scanning to evaluation of the gland and surrounding tissues were highlighted. Prostate distresses were consistent with some unfavorable factors of air flight. The significance of incorporation of USI in the diagnostic algorithms of medical certification of flying personnel afflicted with prostate problems was stated. PMID- 10732198 TI - [Chronobiological characteristics of renal function under conditions of T4- and T3-hyperthyreosis]. AB - Hyperthyreosis in rats after 10 days of injecting T4 at the dose of 100 mg/kg of the body mass modified the chronostructure of renal functions by decreasing rhythms of the urinal pH mesor, and elevating rhythms of creatinine and protein excretion mesors, and the mesor and the amplitude of sodiuresis rhythms. Temporal readjustment of renal functions reflects peculiarities of the body adaptation to T4-induced hyperthyreosis. Injections of T3 (20 mg/kg of the body mass) during 10 days do not lead to any significant changes in the biorhythmic properties of the renal functions. PMID- 10732199 TI - [Classification of myocardial dystrophies in aviation medical certification]. AB - Myocardial dystrophy is one of the most common noncoronary lesions of the myocardium in pilots. However, the abundance of proposed classifications of myocardial dystrophies poses certain problems to medical experts. The authors attempt to consider the existing classifications from the standpoint of aviation medical certification and advocate the most acceptable, to their thinking, systematics by N.P. Paleev and L.I.Levitina (1991). Examples of establishing diagnoses of myocardial dystrophy are included. The best suited program for the aviation medical certification board to work out the final statement regarding the occupational fitness of pilots with myocardial dystrophy is proposed. PMID- 10732200 TI - [Sand-desert tenebrionid beetle Trigonoscelis gigas reitter: a promising biological model for space chronobiology]. AB - The beetle proposed for studying has a unique feature: unusually precise and reliable circadian clock that evolved as an advanced adaptation to extreme arid environment. Consequently this clock became functionally similar to that of vertebrates, i.e. it has a narrow range of entrainment, stable free-running period, strong endogenous component of rhythm. This beetle is also using due to its high viability, good tolerance to housing and handling, small size and safety in use. Space flight experiments with beetles on BION, PHOTON satellites and on MIR orbital station proved that parameters of circadian rhythms are dependent upon gravity. Future studies will focus on electrophysiology and comparative ecology of these beetles. This biological species together with developed methods represent a new promising technology of research in gravitational chronobiology. PMID- 10732201 TI - [Individual peculiarities of adaptation to long-term space flights: 24-hour heart rhythm monitoring]. AB - Presented are results of studying 24-hr variability of the cardiac rhythm which characterizes individual difference in reactions of two crew members to the same set of stresses during a 115-day MIR mission. Spacelab (USA) cardiorecorders were used. Data of monitoring revealed significantly different baseline health statuses of the cosmonauts. These functional differences were also observed in the mission. In one of the cosmonauts, the cardiac regulation changed over to a more economic functioning with the autonomous balance shifted towards enhanced sympathetic activity. After 2-3 months on mission he had almost recovered pre launch level of regulation. In the other, the regulatory system was appreciably strained at the beginning of the mission as compared with preflight baseline. Later on, on flight months 2-3, this strain kept growing till a drastic depletion of the functional reserve. On return to Earth, this was manifested by a strong stress reaction with a sharp decline in power of high-frequency and grow in power of very low frequency components of the heart rhythm. The data suggest that adaptation to space flight and reactions in the readaptation period are dependent on initial health status of crew members, and functional reserve. PMID- 10732202 TI - Heart rate variability during the 10-day Bulgarian-Russian space flight onboard the MIR station. PMID- 10732203 TI - [Coefficients of vertical vestibuloocular reflexes reactivity and vertical component of horizontal vestibular reflexes in the norm]. PMID- 10732204 TI - [Effect of the external electrostatic field on the conformation of a macromolecule containing charged groups]. AB - A method for calculating the free energy of a macromolecule containing charged groups in electrostatic field in aqueous solution was proposed. The non electrostatic component of free energy was calculated with consideration of van der Waals interactions between uncharged parts of the macromolecule. The electrostatic component of free energy was calculated with regard for the interactions of charged groups of the macromolecule with each other and with water molecules. It was found that, depending on the strength of external electric field, the free energy of the system passes through a minimum, whereas the internal energy passes through a maximum. By minimizing the free energy, relative changes in the mean radius 'r' and the distance between the termini of the macromolecule 'h' were calculated. It was found that, at some values of field strength, both 'r' and 'h' decrease. An increase in strength led to an increase in 'r' and 'h'. The regularities observed depend on the charge of the macromolecule and the spatial redistribution of macromolecules and counterions. PMID- 10732205 TI - [Phosphorylase kinase: mathematic modeling]. AB - A mathematical model of the dynamic behavior of phosphorylase kinase was devised. Based on the results obtained, the function of this protein is discussed. It is suggested that phosphorylase kinase doses in a cAMP-dependent manner additional portions of glucoso-l-phosphate, which the muscle cell receives in response to contraction. PMID- 10732206 TI - [Study of self-association of molecules of deoxyhexanucleotides 5'-d(CpGpTpApCpG) and 5'-d(CpGpCpGpCpG) in water solutions by NMR]. AB - The self-association of deoxyribohexanucleoside pentaphosphates 5'-d(CpGpTpApCpG) and 5'-d(CpGpCpGpCpG) in aqueous salt solutions was studied by 1 D- and 2 D homonuclear PMR and heteronuclear 1H-31P-spectroscopies. Signals from nonexchangeable protons of hexamers in NMR spectra were assigned using the available 2M-TOCSY, 2M-NOESY, and 1H-31P-(HMBS) spectra. The dependences of proton chemical shifts of deoxyhexanucleotides on concentration and temperature were measured. In terms of the two-states model (monomer-duplex), constants and thermodynamic parameters of self-association of hexamer molecules in solution were obtained based on these dependences. The values obtained correlate well with theoretical values calculated using the model of the "nearest neighbor" for the formation of duplexes of sequences d(CGTACG) and d(CGCGCG). PMID- 10732207 TI - [Mechanism of B-A transition in the DNA molecule with elastic interaction between sugars and nitrous bases]. AB - A two-component model with elastic interactions between subsystems was proposed, which describes conformational transitions in the DNA molecule. The model is applicable for the description of B-->A transitions in the DNA molecule. It is shown that conformational excitations propagate along the macromolecule via stable topological solutions having a definite velocity. Two types of solutions can be distinguished: exciting and reducing. The exciting solution realizes the transition of sugar from the ground state to a metastable state; in this process, the distance between complementary bases increases. The reducing solution is formed behind the exciting solution and realizes a reverse transition. PMID- 10732208 TI - [Hydration and structure of hemiprotonated polycytidylic acid in films]. AB - Structural transitions of poly(rC)-Ka+ in humid films with different water content were studied by infrared spectroscopy and piezogravimetry. From analysis of the hydration isotherms and the dependence of spectral parameters (frequencies and intensities of the main bands) on n the hydration sites of the polynucleotide were determined (C2O, O4', N4H2, N1, PO2-, C2'OH). It was found that the transition of the polynucleotide from the unordered state to a double-stranded complex poly(rC+).poly(rC) occurs in the interval of n from 2 to 8. The value n = 8 corresponds to the total hydration of poly(rC). A model of hydration of poly(rC+).poly(rC) based on the experimental results and known X-ray parameters of this double helix complex is proposed. The most important feature of the model is the presence of single water bridges between PO2(-)-groups in the first hydration shell of each chain and triple water bridges between O4', N4H2 and C2'OH- atomic groups of opposite chains. The experimental results obtained and the proposed structure of hydration environment of poly(rC+).poly(rC) suggest that the stabilization of this complex is stabilized by the intra- and inter chain water bridges and hydrogen bonds between pairs of cytosine bases. PMID- 10732209 TI - [Conformation and molecular and ionic transformations of polycytidylic acid immobilized in multilayer Langmuir and polyelectrolyte films]. AB - Multilayer films of complexes of polycytidylic acid with dioctadecyldimetylammonium were obtained by the Langmuir-Blodgett method (LB films), and complexes of poly(C) with polycations (poly-L-lysisne, polyethyleneimine, polyallylamine) were obtained by the method of alternate adsorption (polyionic assembly) from solutions of oppositely charged polyelectrolytes on the solid carrier (SA films). It was shown that poly(C) exists in SA films in a single-stranded state irrespective of whether in the starting solution it occurred in the single-stranded nonprotonated or double stranded protonated conformation. Conversely, in the LB film poly(C) preferred to be in a double protonated conformation. UV-spectra of water-insoluble LB and SA films at different pH values of surrounding water medium were investigated. Proton titration curves of poly(C) immobilized in LB films were obtained. The analysis of the shape of titration curves showed that the molecular-ionic transformation of poly(C) in LB films is accompanied by both the conformational transition of the polynucleotide and the molecular rearrangement in the whole film. Poly(C) was found to transform from the double- to single-stranded state and vice versa in the "deprotonation-protonation" cycle of LB film due to cooperative release/binding of hydrogen ions by cytosine bases. In contrast, poly(C) "protonation-deprotonation" in SA films occurred without conformational transitions of the polynucleotide. As opposed to poly(C) in solution a rather big hysteresis of forward and back titration curves was found for both types of multilayer films, indicating molecular rearrangements in films. The reason for the structural transformations of poly(C) upon fabrication of LB or SA films and the mechanism of molecular ionic transformations of poly(C) in films are discussed in terms of a simple model of ion exchange. An assumption about the nature of structural transformations of LB and SA films during their protonation deprotonation is put forward. PMID- 10732210 TI - [Mechanism of photosynthetic water oxidation in the dimeric oxygen-evolving complex of chloroplast photosystem II]. AB - Based on the analysis of the molecular organization and properties of an isolated oxygen-evolving complex of photosystem II of plant chloroplasts, a mechanism of water oxidation and oxygen release during photosynthesis was proposed. It is suggested that the photolysis of water occurs in a dimeric oxygen-evolving complex consisting of two core complexes. In the region of contact of these complexes, a hydrophobic "boiler" is formed where the conditions for screening and stabilization of Z-linanded manganese cations accumulating positive charges for the oxidation of water molecules are created. A prerequisite to the photolysis of water is the formation of a binuclear [Mn(3+)-OH ... HO-Mn3+] hydroxyl-manganese associate, which appears in the dimeric oxygen-evolving complex after the first two light flashes as a result of photohydrolysis of photochemically oxidized Z-liganded manganese cations. The process is accompanied by the release of the first water protons to the medium. The photosynthetic oxidation of water hydroxyls occurs at the next stage and is considered as synchronous detachment of four electrons from two bound OH-groups of the associate upon photooxidation of Mn3+ cations to Mn4+ cations after two subsequent light flashes. This process is accompanied by the disproportionation of electron density and the formation of a bond between oxygen atoms of hydroxyls followed by the evolution of molecular oxygen and protons, and regeneration of two starting Mn2+ cations and the primary state of the system. PMID- 10732211 TI - [Study of model protein-lipid systems by the energy transfer method]. AB - Complexes of ribonuclease, lysozyme, cytochrome c and hemoglobin with model phospholipid membranes composed of phosphatidylcholine and diphosphatidylglycerol (4:3, mol:mol) were investigated by the method of non-radiative fluorescence energy transfer. Evidence for the penetration of proteins in to the lipid bilayer interior was obtained. The size of the protein fragment incorporated into the polar membrane region was estimated. PMID- 10732212 TI - [Kinetics of resealing of lipid pores]. AB - The resealing of lipid pores in BLM at the temperature of phase transition was studied. It is show that the size of a pore correlates with the time of its resealing. The correlated coefficient of pore diffusion is close to that of lipid molecules in the gel state. PMID- 10732213 TI - [Allowing for polymer polydispersity--an essential condition for determination of aqueous pore diameters in cell walls and membranes using polymers]. AB - A method of allowing for polydispersion of polyethylene glycol (PEG) preparations was developed for the use of these preparations for the osmometrical evaluation of pore diameters with aqueous pores of Chara corallina cell walls as an example. The mass share of polyethylene glycol preparation fractions gamma p penetrating through the pores was determined using cellular "shadows", fragments of internodal cell walls tied up at the ends and filled with a 25% solution of nonpenetrating PEG 6000. When immersed into water, such "shadow" acquired a turgor (hydrostatic) pressure close to the cellular pressure and persistent over long time. The determination of gamma p for polyethylene glycols with different average molecular weights Mw was performed from the degree of pressure restoration after water was replaced by a 5-10% polymer solution. The kinetics of pressure changes was recorded using a mechanotronic dynamometer, which measures, in the quasi-isometric mode, the force necessary for partial compression of the "shadow" in its small fragment. By utilizing the dependence of the overall share of fractions with molecular weights Mi < Mk on Mk (data of [1]), we found that gamma p, for these polyethylene glycols corresponds to the threshold value of Mk = 800-1100 D (hydrodynamic radius of molecules rh = 0.85-1.05 nm). Thus, the effective diameter of the pores in the cell wall of Chara does not exceed 2.1 nm. It was shown that the smoothness of the sigmoid shape of the dependence of ionic channel conductivity on the Mw value of the polymer in the media is largely due to the polydispersion of polymer preparations, particularly, to the reduction in the share of fractions penetrating the channels as Mw is increased. The method normally used to estimate pore diameters in ionic channels which ignores the dispersion of polymer preparations, results in overestimated values. PMID- 10732214 TI - [Effect of neuraminidase and proteolytic enzymes on electrophoretic mobility of erythrocytes and their aggregation induced by La(3+)]. AB - The effect of neuraminidase, trypsin and pronase on the electrophoretic mobility of human erythrocytes and their aggregation induced by La3+ was studied in vitro. The aggregation of intact red cells differs from that of cells treated with neuraminidase and proteolytic enzymes. The above differences are observed in the range of La3+ suspension concentration from 20 to 330 microM. A possible mechanism of aggregation is discussed. PMID- 10732215 TI - [Self-organization of mitochondrial associates and effects of negative air ions]. AB - A convenient model for studying the mechanisms of biological self-organization is described by morphometric investigation of formation of mitochondrion associations in medium containing physiological concentration of potassium ions without nonpolar substances. Association formation was considerably better at 15 18 degrees C during isolation and storage than at 0 degree C. The existence of filamented mitochondria in homogenate was also shown by staining of succinate dehydrogenase. Formation of associations increased in medium pretreated with negative air ions carrying superoxide and is probably due to hydrogen peroxide. The effect of substances influencing the surface charge on association formation was studied. PMID- 10732216 TI - [Energetic aspects of propagation of action potential]. AB - On the basis of the electrical analogue of an action potential suggested previously, the distribution of electrical field outside and inside the membrane was obtained. The energy released upon the propagation of a single action potential along the nerve fibre was calculated. A comparison of the results with the available data showed the correctness of the calculations performed. PMID- 10732217 TI - [Structural modification of environment by microorganisms: formation of Liesegang rings around Dictyostelium discoideum population]. AB - The results of the experimental study of the environment modification--the emergence of Liesegang rings around a Dictyostelium discoideum population are presented. The formation of Liesegang rings induced by D. discoideum cells is observed on addition of glucose into the semi-solid nutrient medium (agar concentration 0.5-1.5%). We show that the emergence of Liesegang rings is attended with a redistribution of folic acid in the nutrient substrate. A pH decrease in the course of D. discoideum cultivation is shown to be a factor inducing the redistribution of folic acid. The mechanisms of structural modification of the D. discoideum environment are discussed. PMID- 10732218 TI - [How does environment modify the spatial structure of Dictyostelium discoideum population]. AB - The transformation of the spatial structure of a Dictyostelium discoideum population in response to environmental changes induced by this population was investigated. A comparative analysis of the spatial and temporal characteristics of the D. discoideum colony is given for two cases: (a) when the colony is cultivated on a bacterial lawn, i.e. under conditions close to natural, and (b) in the absence of the bacterial lawn when the colony grows on the nutrient substrate enriched with folic acid. It is shown that the environmental changes induced by cell metabolism modify the spatial structure of the D. discoideum population first, the rate of population propagation falls drastically, which correlates with a decrease in the substrate pH; second, the spatial redistribution of the D. discoideum cell density correlates with the redistribution of folic acid in the substrate. The mechanism of the environment impact on the D. discoideum colony transformation is discussed. PMID- 10732219 TI - [Effect of ischemic preconditioning on free radical centers of the isolated rat heart during ischemia and early reperfusion]. AB - The effect of ischemic preconditioning on the free-radical state of isolated rat myocardium fixed by rapid freezing at the 25th min of normothermic total ischemia and the 3rd min of reperfusion was studied by the EPR method. It was shown that EPR spectra registered at -40 degrees C consist of two free-radical signals: of the semireduced forms of ubiquinone and flavine coynzymes. It was found that during ischemia and at the beginning of reperfusion, the preconditioning results in a narrowing of the spectra (as compared with control) due to an increase in the narrow ubisemiquinone EPR signal portion, and a decrease in the total concentration of free-radical centers: by 16% in the case of ischemia, and 23% in the case of reperfusion. It was concluded that in both cases the changes were due to a decrease in the concentration of myocardial flavosemiquinones as a result of ischemic preconditioning. We registered the microvawe power saturation curves for these two stages, which corresponded to control and ischemic preconditioning. In the case of ischemia these dependences had similar shapes; however, in the case of reperfusion they differ from each other due to changes in the relative intensities of the EPR signals from ubisemiquinone and flavosemiquinones in the integral myocardial free-radical spectra. PMID- 10732221 TI - [Dependence of synergism of the combined effect of ultrasound and hyperthermia upon intensity of ultrasound]. AB - The inactivation of wild-type yeast Saccharomyces cerevisiae was studied after simultaneous treatment with ultrasound and hyperthermia. A temperature range was established within which ultrasound and hyperthermia exert a synergistic action. The effect was shown to depend on ultrasound intensity and the temperature at which the treatment takes place. The temperature range enhancing the ultrasound effect shifted forward higher temperature with increasing ultrasound intensity. For every intensity value, an optimal temperature exists at which the synergetic effect is maximum. The biophysical interpretation of the results obtained is based on the assumption that synergism is due to an additional lethal damage, which arises from the interaction of some sub-lesions induced by both agents. These sublesions are considered non-lethal if the agents are applied separately. PMID- 10732220 TI - [Spontaneous synchronous discharges in hippocampal slices. Simulation and experiment]. AB - Chaotic oscillations of extracellular potential of field-type nerve tissues are simulated by a 2D coupled map lattice. These tissues, say, the fields of the hippocampus, are represented by neural mass sheets consisting of current sources. The relationship between the source-sink ensembles and the extracellular field potential at each discrete instant of time t = 1, 2, ... is described by a single site map creating chaos. The 2D coupled map lattice is viewed as a network of diffusively coupled the maps creating spatiotemporal chaos. The conversion of chaotic oscillations into synchronous ones, which are typical for epileptiform discharges, is studied. The results obtained are in good agreement with those derived from hippocampal slices treated with picrotoxin. PMID- 10732222 TI - [Changes in serum alkaline phosphatase activity during in vitro exposure to amplitude-modulated electromagnetic field of ultrahigh frequency (2375 MHz) in guinea pigs]. AB - The activity of alkaline phosphatase by the action of pulse-modulated microwave radiation was studied. The carrier frequency of radiation was 2375 MHz, the range of modulation pulse rate was 10-390 Hz with the on-off time ratio 2, and the specific absorption rate was 8 and 0.8 microW/cm2. Time of exposure was 1 and 3 min under conditions of continuous temperature control. It was shown that the activity of alkaline phosphatase depends on both modulation frequency and intensity of superhigh-frequency electromagnetic radiation. At a frequency of 70 Hz, the activity of alkaline phosphatases increased 1.8-2.0 times. PMID- 10732223 TI - [Response of sensorimotor cortex neurons to weak disturbances of the magnetic field in Wistar rats. Cytochemical study]. AB - The influence of weak disturbances (up to 300 microT) of natural magnetic field on the protein metabolism in neurons of sensomotor cortex (layers III and V) in Wistar rats upon learning in a complex maze was studied. It was found that sensomotor neurons were very sensitive to weak disturbances of magnetic field. The protein content increased, while the nucleus-cytoplasm ratio and osmotic state of neurons remained unchanged. The specificity of neuron's reaction manifested itself in a sharp increase of nucleus and cytoplasm dimensions. In associative neurons (layer III), both the nucleus and cytoplasm were involved in the response; in efferent neurons (layer V), only nuclear parameters changed. The variance coefficients of all parameters of protein metabolism in sensomotor neurons, independently of their functional properties, were much higher than in control, which resulted in a wide diversity of cytochemical response. PMID- 10732224 TI - [Electromagnetic information in the phenomenon of life]. AB - The author's original experiments and ideas have been summarized, which concern the role of natural background radiation in maintenance of electromagnetic information essential for existence of the living organism as an integral whole. PMID- 10732225 TI - [Pathological integration in the nervous system]. PMID- 10732226 TI - [Arterial blood flow during deep breathing]. PMID- 10732227 TI - [Increased blood flow in the stomach as a possible mechanism of the anti-ulcer effect of Pro-Gly-Pro]. PMID- 10732228 TI - [Hypothesis of inhibition of the small intestine contraction by the vagus nerve]. PMID- 10732229 TI - [Restoration of interhemispheric symmetry of the bioelectrical brain activity in patients with neurasthenic syndrome by bioacoustic correction]. PMID- 10732230 TI - [Effect of trimetazidine on the brain metabolism during ischemia complicated by hypoxia]. PMID- 10732231 TI - [Effect of 25-hydroxycholesterol and progesterone on esterification of cholesterol, viscosity, and protein-lipid interactions in macrophage membranes]. PMID- 10732232 TI - [Correction of postischemic reperfusion injury complications by cytoflavin]. PMID- 10732233 TI - [Effect of parlodel on the electrical activity of the rat brain during experimental depressive syndrome]. PMID- 10732234 TI - [Changes in serotonin-modulated SMP-69 protein level in thrombocytes of patients with schizophrenia]. PMID- 10732235 TI - [Isolation of peripheral polypeptides from soybean lipid extracts and study of their effects on the phospholipid structure]. PMID- 10732236 TI - [Thyroxin-binding globulin as a marker of the body exposure to hazardous environmental factors]. PMID- 10732237 TI - [Surface-active properties of phosphatidylcholines--basis for surfactant replacement therapy]. PMID- 10732238 TI - [Effect of tetrahydrocortisol on protein biosynthesis in hepatocytes]. PMID- 10732239 TI - [Glutathione S-transferase activity in the liver of mice with various sensitivity to hepatocarcinogenic effect of o-aminoazotoluene]. PMID- 10732240 TI - [Intensification in vivo of free radical oxidation of low density lipoproteins in plasma from patients with myocardial ischemia treated by HMG-CoA-reductase pravastatin and suppression of lipid peroxidation by ubiquinone Q10]. PMID- 10732241 TI - [Interactions of low density lipoproteins with elastin in the intima and media from the intact and atherosclerotic human aorta]. PMID- 10732242 TI - [Effect of doxorubicin in vitro on energy-dependent calcium transport by rat brain mitochondria]. PMID- 10732243 TI - [Effects of progesterone in various dosage forms on lipid peroxidation and glutathione redox system in skin tissues in rats with experimental dermatitis]. PMID- 10732244 TI - [Effect of the gamma-aminobutyric acid derivative TZ-50-2 on the autonomic nervous system]. PMID- 10732245 TI - [Immunologic properties of preparations obtained from the skin]. PMID- 10732246 TI - [Calmodulin level in chondrosarcomas]. PMID- 10732247 TI - [Experimental ground for probiotic use in the complex treatment of destructive pancreatitis]. PMID- 10732248 TI - [Yersinioses in monkeys]. PMID- 10732249 TI - [Participation of components of the angiotensin II system in the regulation of DNA synthesis in the rat pyloric epithelium]. PMID- 10732250 TI - [Responses to the blue light in humans]. PMID- 10732251 TI - [Nitroxidergic neurons in respiratory organs]. PMID- 10732252 TI - [Immunohistochemical localization of major histocompatibility class 1 antigens in the human placenta]. PMID- 10732253 TI - [Pathomorphology of the subcutaneous implantation of samples of base polymer materials modified by plasma glow discharge]. PMID- 10732254 TI - [Estradiol-17beta receptors in the cytosol fraction in chondrosarcomas]. PMID- 10732255 TI - [Changes in cerebral vessels during experimental hyperlipidemia]. PMID- 10732256 TI - Family group decision making: protecting children and women. AB - With rising demands on child welfare, workers need to consider new options, including strategies that promote a collaborative effort of family, community, and government. Family group conferencing integrates efforts to advance child and adult safety and strengthens family unity while expanding its meaning. The conclusions in this article are based on family interviews and child protective services' file comparison from an outcome study of the Family Group Decision Making Project. PMID- 10732258 TI - The effectiveness of CASAs in achieving positive outcomes for children. AB - Using a quasiexperimental design, this study evaluated the effectiveness of CASAs in achieving positive outcomes for children, and examined the process variables believed to lead to permanency for children. Data were collected from court and CASA program files over a two-year period on 200 children, who were compared to children without CASA volunteers on outcome and process variables. Findings indicate that CASAs may have helped reduce the number of placements and court continuances children experienced, and that more services were provided to children with CASAs than to those without. Additional research is needed to further evaluate the impact of CASA services on children. PMID- 10732257 TI - Ready or not: assessing youths' preparedness for independent living. AB - This article discusses the utility of the Ansell-Casey Life Skills Assessment (ACLSA) in assessing life skills necessary for living successfully in the community upon emancipation from out-of-home care. ACLSA, completed by youths and their caregivers, identifies skills that have been mastered and those yet to be learned. Assessment information can be used for goal setting, strength identification, and relationship building, as well as to direct program planning and training in self-sufficiency services. PMID- 10732259 TI - Teaching social workers to use psychological assessment data. AB - Psychological assessments of children and parents are frequently used by social service caseworkers when making case planning decisions. Often, however, the two disciplines--psychology and social work--have difficulty collaborating in and coordinating their respective work. Caseworkers may lack formal training in how to understand and use psychological reports. Psychologists may lack formal training in forensic assessment and in understanding the constraints of the social service and child welfare systems. This article describes how caseworkers may become more sophisticated consumers of psychological assessments, and how collaborative relationships between evaluating psychologists and caseworkers can be fostered. PMID- 10732261 TI - Adrenal cancer. AB - Adrenal cancer is a rare neoplasm. Up to 1 in 1500 adrenal incidentalomas, however, may hide a carcinoma, which, if diagnosed late or left untreated, is associated with significant morbidity and mortality. Despite extensive investigation of the molecular mechanisms involved in adrenal carcinogenesis and significant improvements in diagnostic imaging, efforts to cure advanced adrenal cancer remain largely unsuccessful. This article reviews the recent advances in molecular understanding, clinical diagnosis, and treatment of adrenal cancer. PMID- 10732260 TI - Pathogenesis of adrenocortical incidentalomas and genetic syndromes associated with adrenocortical neoplasms. AB - The study of genetic syndromes associated with adrenocortical tumors (Beckwith Wiedemann, Li-Fraumeni, McCune-Albright, Carney, and multiple endocrine neoplasia type 1) has shed light on the molecular basis of tumorigenesis. Abnormalities at the 11p15 locus appear as crucial and frequent events found specifically in malignant, sporadic tumors, leading to overexpression of a growth-promoting factor and loss of expression of tumor suppressor genes. In benign tumors, the cAMP pathway can be exacerbated in an ACTH-independent manner when various membrane receptors of the seven transmembrane superfamily are "illegitimately" expressed. PMID- 10732262 TI - Radiology of the adrenal. AB - Adrenal incidentalomas are commonly noted on abdominal cross-sectional imaging studies. Most of these lesions are benign, non-functional adrenal adenomas. Certain adrenal lesions have such characteristic radiologic findings that their diagnosis can be made with virtual certainty. This article reviews the radiologic evaluation of adrenal tumors, with particular emphasis on incidentalomas. PMID- 10732263 TI - Subclinical Cushing's syndrome. AB - Classic Cushing's syndrome is a rare disease with an estimated incidence of 1 case per 100,000 persons. With routine use of imaging techniques such as ultrasound and CT, adrenal masses are being detected with increased frequency. A substantial percentage of these incidentalomas are hormonally active, with 5% to 20% of the tumors producing glucocorticoids. Autonomous glucocorticoid production without specific signs and symptoms of Cushing's syndrome is termed subclinical Cushing's syndrome. With an estimated prevalence of 79 cases per 100,000 persons, subclinical Cushing's syndrome is much more common than classic Cushing's syndrome. Depending on the amounts of glucocorticoids secreted by the tumor, the clinical spectrum ranges from slightly attenuated diurnal cortisol rhythm to complete atrophy of the contralateral adrenal gland with lasting adrenal insufficiency after unilateral adrenalectomy. Patients with subclinical Cushing's syndrome lack the classical stigmata of hypercortisolism but have a high prevalence of obesity, hypertension, and type 2 diabetes. All patients with incidentally detected adrenal masses scheduled for surgery must undergo testing for subclinical Cushing's syndrome to avoid postoperative adrenal crisis. The best screening test to uncover autonomous cortisol secretion is the short dexamethasone suppression test. Because the adrenal origin of a pathologic cortisol secretion is anticipated, the author prefers a higher dexamethasone dose (3 mg instead of 1 mg) to reduce false-positive results. A suppressed serum cortisol level of less than 3 micrograms/dL (80 nmol/L) after dexamethasone excludes significant cortisol secretion by the tumor. A serum cortisol level greater than 3 micrograms/dL requires further investigation, including confirmation by high-dose dexamethasone (8 mg) suppression testing, a CRH test, and analysis of diurnal rhythm. Determination of urinary free cortisol is less useful because increased values are a late finding usually associated with emerging clinical signs of Cushing's syndrome. Patients with suppressed plasma ACTH in response to CRH generally have adrenal insufficiency after surgery and require adequate perioperative and postoperative substitution therapy. Whether patients with subclinical Cushing's syndrome should undergo adrenalectomy is a matter of debate. The author performs surgery in young patients (< 50 years), in patients with suppressed plasma ACTH, and in patients with a recent history of weight gain, substantial obesity, arterial hypertension, diabetes mellitus, and osteopenia. In completely asymptomatic patients with normal plasma ACTH concentrations and in patients older than 75 years, the author recommends a nonsurgical approach. A large prospective randomized study is necessary to evaluate the benefits of surgery versus conservative treatment in patients with subclinical Cushing's syndrome. PMID- 10732264 TI - Surgery of the adrenal glands. AB - Because widespread use of imaging techniques has led to the frequent detection of incidentalomas, radiologists, endocrinologists, and endocrine surgeons must be knowledgeable about the appropriate evaluation of patients, and the selection of the appropriate surgical approach, including conventional open and laparoscopic adrenalectomy. This article reviews the authors' preferences based on experience with nearly 200 laparoscopic adrenalectomies. PMID- 10732265 TI - Diagnosis and treatment of adrenal incidentaloma. A cost-effectiveness analysis. AB - Methods of meta-analysis, decision analysis, and cost-effectiveness analysis were applied to the adrenal incidentaloma dilemma. It was shown that the life expectancy of patients with adrenal incidentalomas is decreased by a mean of about 1 year if left undiagnosed and untreated--more in cases of larger incidentalomas. Overall, selective analysis of adrenomedullary hormonal function (by urinary metanephrines) is the most cost-effective strategy. Other strategies, such as more extensive hormonal testing, imaging, and fine needle aspiration cytology may provide better cost-effectiveness in subgroups of patients, identified by signs, symptoms, and incidentaloma morphology. Full adrenal hormonal analysis is indicated in patients with larger (> or = 6 cm) incidentalomas and if the combination of hypertension and hypokalemia suggests Conn's disease. Small or medium-sized adrenal incidentalomas may be ignored if MR imaging or other tests suggest benign pheochromocytoma disorder, or patients are elderly, or both. PMID- 10732266 TI - The diagnostic dilemma of incidentalomas. Working through uncertainty. AB - The clinical evaluation of incidentally found nodules in the adrenal, thyroid, and pituitary glands is a challenge for physicians, regardless of their level of expertise. Choosing the most direct and cost-effective diagnostic approach and deciding when to treat or not to treat are common dilemmas in clinical practice. This article outlines one diagnostic approach using medical decision-making techniques such as heuristic thinking, critical appraisal of the literature, treatment threshold probability assessment, Bayes' theorem, and discriminant properties of diagnostic tests. These skills are usually discussed in postgraduate training curricula. Nevertheless, they often seem foreign to many clinicians. Evidence suggests that training in these techniques can improve clinical decision making. Use of the skills outlined herein provides a framework to work through the diagnostic uncertainty common in the evaluation of incidentalomas. This approach does not provide perfect answers, as noted in examplar 3 in which two experts argued about the actual pretest probability and treatment thresholds for pituitary incidentalomas. Even if there were no such disagreement, each patient presents unique issues, and there will always be some uncertainty. Nevertheless, this approach provides a starting point from which critical decisions can be made for individual patients. PMID- 10732267 TI - Management approaches to adrenal incidentalomas. A view from Ancona, Italy. AB - The definition of adrenal incidentaloma encompasses a heterogeneous spectrum of pathologic entities, including primary adrenocortical and medullary tumors, benign or malignant lesions, hormonally active or inactive lesions, metastases, and infections. This article provides an overview of the diagnostic clinical approach and management of the incidentally discovered adrenal masses. Approaches are based on data collected in more than 1000 cases of the Collaborative Study Group on Adrenal Incidentaloma of the Italian Society of Endocrinology and the authors' experience. PMID- 10732268 TI - Management approaches to adrenal incidentalomas. A view from Ann Arbor, Michigan. AB - Adrenal masses are discovered incidentally in 1 to 3% of abdominal CT scans performed for investigation of nonadrenal-related abdominal complaints. Although most incidentally discovered masses are clinically silent, 10% are hormone secreting and are associated with subtle symptoms of hormone excess. The major concern is the possibility that such a mass is malignant and requires surgical intervention. Benign adrenal cortical adenomas are 60 times more common than primary adrenal cortical carcinomas, which are rare, and many of the lesions that are malignant are metastatic from extra-adrenal neoplasms. Size is a significant factor in determining the probability that the lesion is benign or malignant, and there is concensus that most benign lesions are smaller than 3 cm, whereas most malignant lesions are larger than 6 cm. Uncertainty remains, however, as to the potential malignant character of masses measuring 3 to 6 cm; thus, size alone is insufficient for determining if an incidentally-found adrenal mass is benign or malignant, and information obtained from other diagnostic techniques, such as CT, MR imaging, and adrenal scintigraphy with 131I 6 beta-iodomethylnorcholesterol, should be considered. Fine needle biopsy of an adrenal mass is not recommended unless there is strong suspicion that the mass is metastatic from an extra adrenal neoplasm. PMID- 10732269 TI - Management approaches to adrenal incidentalomas (adrenalomas). A view from Athens, Greece. AB - We believe the management of adrenalomas should include the following: 1. A detailed history and physical examination to detect subtle evidence of hormonal hypersecretion or the possibility of metastatic carcinoma 2. Hormonal studies, such as The short dexamethasone suppression test (2 mg of dexamethasone) followed by a high-dose dexamethasone suppression test (8 mg), CRH assay, and analysis of the diurnal cortisol rhythm if serum cortisol is greater than 3 micrograms/dL 24 Hour (or spot) urinary catecholamine metabolites (metanephrine and normetanephrine) Plasma aldosterone level and renin activity in the hypertensive or normotensive patient with serum potassium less than 3.9 nmol/L 3. Additional imaging studies, such as MR imaging 131I-iodocholesterol (NP59) scanning to detect a subclinically functioning adenoma or carcinoma (MIBG scanning is rarely indicated) The role of FNA is limited. This modality may be helpful only in the patient with coexistent extradrenal carcinoma to confirm adrenal metastasis. Although genetic and molecular biology studies do not have wide clinical application, they should be encouraged and supported. Once all of these data are collected, the clinician should perform the following two steps: 1. All clearly nonfunctioning adrenalomas that are not suspicious for malignancy should be observed for several years, mainly with hormonal studies, until their secretory and benign nature is confirmed. 2. All patients with adrenalomas with evidence of subclinical function or potential for malignancy (using size, imaging, FNA, and molecular biology criteria) should undergo laparoscopic adrenalectomy for definitive diagnosis and therapy. The age, the overall medical condition, and the anxiety of the patient should be considered in the decision to operate on a patient with an adrenaloma. PMID- 10732270 TI - Management approaches to adrenal incidentalomas. A view from Rochester, Minnesota. AB - Two biproducts of the revolution in diagnostic imaging techniques are unintended discoveries, and uncertainty for the patient and the clinician. To address the uncertainty associated with adrenal incidentalomas, clinicians need to understand the definition, differential diagnosis, and options for assessment with respect to functional status and malignancy potential. This article presents an algorithmic approach that addresses these issues. PMID- 10732271 TI - Thyroid incidentalomas. Prevalence, diagnosis, significance, and management. AB - Thyroid incidentalomas are common, always impalpable, often less than 1.5 cm in size, and frequently benign. The authors recommend that low-risk patients with incidentalomas be followed up with clinical palpation in 6 to 12 months and not be subjected to routine testing with US-FNA. In the authors' strategy, fine needle aspiration is reserved for an impalpable nodule and is performed under ultrasonographic guidance in the high-risk group of patients in whom either the imaging features or the clinical history is worrisome for malignancy. It does not seem necessary, practical, or cost-effective to perform biopsy or to excise surgically all impalpable nodules. Because of the high prevalence of thyroid incidentalomas, most of which are benign, a nonsurgical approach is logical. PMID- 10732272 TI - Pituitary incidentalomas. AB - The optimal strategy for hormonal screening of a patient with any incidentally discovered pituitary mass is unknown. The authors' review of the endocrinologic literature supports the view that such patients are at slightly increased risk for morbidity and mortality. This risk implies a benefit of early diagnosis for at least for some of the disorders, suggesting the importance of case finding. Nevertheless, the data in Table 1 illustrate that clinically diagnosed hormone secreting pituitary tumors are far less common than incidentalomas. Clinically, one cannot accurately determine the approximately 0.5% of patients with incidentaloma who are at increased risk among the vast majority who are not. Given the limitations of diagnostic tests, effective hormonal screening requires a sufficiently high pretest probability to limit the number of false-positive results. This condition is met to varying degrees in the patient with a small incidentally discovered pituitary mass but no signs or symptoms of hormone excess. Even the more common lesions, such as prolactinoma, are relatively rare. [table: see text] Subjecting patients to unnecessary testing and treatment is associated with risk. In addition to its initial cost, testing may result in further expense and harm as false-positive results are pursued, producing the "cascade effect" described by Mold and Stein as a "chain of events (which) tends to proceed with increasing momentum, so that the further it progresses the more difficult it is to stop." The extensive evaluations performed for some patients with incidentally discovered masses may reflect the unwillingness of many physicians to accept uncertainty, even in the case of an extremely unlikely diagnosis. This unwillingness may be driven, in part, by fear of potential malpractice liability, the failure to appreciate the influence of prevalence data on the interpretation of diagnostic testing, or other factors. The major justification for further evaluation of these patients is not so much to avoid morbidity and mortality for the rare patient who truly is at increased risk but to reassure patients in whom further testing is negative and the physician. Physicians must take care not to create inappropriate anxiety in patients by overemphasizing the importance of an incidental finding unless it is associated with a realistic clinical risk. The authors' recommendations are based on currently available information to minimize the untoward effects of the cascade. As evidence accumulates, these recommendations may need to be revised. The benefit of the diagnosis of an adrenal or pituitary disorder must be considered in the context of the patient's overall condition. Additional studies are needed to analyze the clinical utility of hormonal screening for these common radiologic findings. Data from these studies can be used to identify critical gaps in knowledge and to adopt the epidemiologic methods of evaluation of evidence that have been applied to preventive measures. One must be careful to recognize lead time bias, in which survival can appear to be lengthened when screening simply advances the time of diagnosis, lengthening the period of time between diagnosis and death without any true prolongation of life; and length bias, which refers to the tendency of screening to detect a disproportionate number of cases of slowly progressive disease and to miss aggressive cases that, by virtue of rapid progression, are present in the population only briefly. Physicians must avoid the pitfalls of overestimation of disease prevalence and of the benefits of therapy resulting from advances in diagnostic imaging. Clinical judgment based on the best available evidence should be complemented and not replaced by laboratory data. PMID- 10732273 TI - Management of pituitary incidentalomas. A survey of British and American endocrinologists. AB - The results of a survey of endocrinologists concerning their approaches to the evaluation of a patient with an incidentally discovered pituitary mass are presented in this article. The practices of British and American endocrinologists are compared. The wide variation in diagnostic approaches to the practice of ordering tests in the United Kingdom and the United States highlights the need for research and debate regarding the most appropriate management of patients with such findings. PMID- 10732274 TI - [Adaptation, ecological stability and the evolution of diploid organisms]. AB - The possibility of the existence of an organism under different environmental conditions is determined by its ecological stability. This parameter can be expressed as the product of the average life span corresponding species and the probability of an organism's participation in reproduction. If ecological conditions are not substantially altered, regulatory selection provides an increase in fitness of an organism in a certain direction of adaptation. It is supposed that the process of regulatory selection is accompanied by the accumulation of mutations occurring in regulatory genes and mutations in regulatory regions of structural genes which correct the effect of the former mutations. An alteration in ecological stability occurs when the conditions of population existence are changed and is usually accompanied by a decrease in the fitness level earlier achieved. Thus, an increase in organisms' ecological stability is achieved by hybridization between populations of different origin and is accompanied by a decrease in fitness due to outbreeding depression. Under conditions of inbreeding, ecological stability is decreased due to the segregation, in the homozygous state, of recessive alleles of adaptive genes that have not yet reached the stage of evolutionary fixation. Diploidy is a factor allowing organisms to improve their ecological stability in every new generation. PMID- 10732275 TI - [Intraspecies polymorphism for the structural organization of ribosomal gene cluster in Blattella germanica]. AB - Intraspecific HindIII restriction fragment length polymorphism (RFLP) for the structural organization of the ribosomal gene cluster was described in Blattella germanica. In this species, homologous chromosomes were shown to contain ribosomal DNA (rDNA) repeats that differ in structure. The pattern of inheritance was determined for various structural variants of rDNA. PMID- 10732276 TI - [RNA interference in Escherichia coli cells: the expression of molecules that are complementary to the lon gene mRNA in parallel orientation]. AB - To study the effect of RNA interference (RNAi) on the activity of gene lon in Escherichia coli, genetic constructs were used that could express RNA molecules complementary to the 5' region of lon mRNA in the same direction. These RNAs were termed parallel RNAs (pRNAs). Two approaches were used to control expression. In one approach, lon gene activity was estimated genetically, based on the effect of the Lon protease on bioluminescence determined by the Vibrio fischeri lux regulon. The other approach was direct testing of ATP-dependent proteolysis in vitro. It was found that pRNA considerably suppressed lon expression. The antiparallel RNA (apRNA) was a less effective suppressor of this gene. The specific RNAi was found to decay gradually by the 40th generation. The data obtained indicate that Eubacterium cells have mechanisms for specific regulation of gene activity that are sensitive to the formation of both parallel and antiparallel RNA duplexes involving mRNA of the given gene. PMID- 10732277 TI - [Constructing of system of genetic analysis in Pseudomonas mendocina]. AB - A Tn10-containing variant of the pRK2013 plasmid, pRK2013-7, was used in the genetic analysis of Pseudomonas mendocina as chromosome-mobilizing inheritable factor that is able to integrate into the bacterial chromosome and transfer genetic markers with a frequency ranging from 3.2 x 10(-7) to 3.5 x 10(-3). The results of interrupted matings allowed localization of 10 genetic markers. This system of genetic analysis is suitable for P. mendocina mapping. PMID- 10732278 TI - [Genetic structure of species community of macromycetes of the genus Leccinum Gray (Basidiomycetes, Boletaceae) of the right-bank Poles'ye]. AB - The biochemical gene marking of the macromycete community of the genus Leccinum revealed nine reproductively isolated genetically differentiated forms instead of 2-5 forms traditionally recognized in Poles'ye. From 20 to 85% of the loci studied were found to have genetic distinctions and the degree of genetic variation did not always correlate with the morphological features of the forms identified. The variation of the locus number was found to be an important differentiating factor characteristic for this genus. In the group of L. scabrum s. I., every second locus was duplicated, whereas multiple isozymes were absent in L. aurantiacum s. I. The extremely high level of allozyme variation in macromycetes was found. The equilibrium between the expected and observed genotypes in the fruit body samples was established and discussed. It was the evidence of the fact that a panmictic model can be applied to the mushrooms of this genus, and that each fruit body is an individual organism. PMID- 10732279 TI - [Identification and certification of common wheat cultivars using RAPD and SSRP methods of analysis]. AB - Based on RAPD and SSRP analysis of wheats of different ecological-geographical origin, the procedures for composing genetic formulas of cultivars and their certification have been elaborated. The application of RAPD and SSRP analysis to the identification of wheat cultivars is discussed. PMID- 10732280 TI - [Morphometric and cytogenetic characteristics of haploid tomato plants]. AB - Stable differences have been found between haploid and diploid Mikado tomato plants with respect to quantitative characteristics of their vegetative and generative organs, whereas these plants were phenotypically similar with respect to qualitative characteristics, such as the bush habitus and the shapes of leaves, flowers, and fruit. A simple indirect method based on counting the chloroplasts in guard cells has been suggested to diagnose haploids. Analysis of meiosis during microspore formation in haploids has revealed considerable anomalies leading to heterogeneity and a high sterility rate of pollen, which has been confirmed by differences in DNA content. The data obtained suggest that formation of fertile gametes is accounted for by the absence of reduction division. It has been hypothesized that meiosis in haploids induces the genotypic variation that is subject to selection. PMID- 10732281 TI - [Selective value and gene geography of the alleles of Barley beta- amylase locus Bmy1]. AB - Polymorphism for locus Bmy 1 was studied in 406 cultivars of spring barley, 189 of which were regionalized in the former Soviet Union between 1929 and 1990. Three alleles were detected: Bmy 1 Ar, Bmy 1 Br, and Bmy 1 Al. The frequencies of these alleles were 35.59, 60.46, and 3.45%, respectively. In contrast to Bmy 1 Ar, the allele Bmy 1 Br was shown to correlate with an increase in the amylolytic activity of malt. Barley plants differing in the Bmy 1 alleles were found to differ in seed productivity. Allele Bmy 1 Ar was shown to prevail in cultivars from the northern regions of the former Soviet Union, in which its frequency exceeds 50%. Toward the south, its frequency gradually decreases to 14-17%. Conversely, the frequency of allele Bmy 1 Br increases from 38.6% (north) to 85.7% (south). The pattern of allele distribution depends on the following factors: temperature pattern during the growing season, moisture supply, and climate continentality. PMID- 10732282 TI - [The transfer of genes for Brown Rust resistance from Aegilops umbellulata Eig. to wheat (Triticum aestivum L.) genome]. AB - The potential of a genome-substituted form Avrolata (AABBUU) as a genetic system in genomic and chromosome manipulations for gene transfer from the wild species Aegilops umbellulata Eig. (UU) to cultivated wheat was studied. It was shown that plants combining resistance to leaf brown rust with high productivity may be produced from this form by classical hybridization procedures. The resistance gene introduced to line R-12 is dominant and probably identical to the Lr9 gene. By N-banding, chromosome staining technique and gliadin electrophoresis, the structural changes in chromosomes 1A, 2A, 4B, 6B, 7B, 1D, and 2D of the resistant line R-12 were revealed. PMID- 10732283 TI - [Genetic studies of house mice from the hybrid area of the Primorskii Region]. AB - Cytogenetic and allozyme differentiation of house mice Mus musculus L. was studied in 21 samples of Primorskii krai (region) from 1984 to 1998. The use of Q H- and C-banding made it possible to reveal a high variation in the content and distribution of pericentromeric heterochromatin and high frequencies of marker autosomes 5, 7, 10, 12, 15, and 17-19 in the karyotypes of mice from some samples. The presence of marker alleles that are typical of the subspecies groups castaneus and musculus was shown for protein loci Idh, Sod-1, Aat-1, and Hbb. The data obtained indicate that the fauna of house mice of Primorye is characterized by subspecific mosaicism and hybrid origin. Apparently, subspecies M. m. castaneus, M. musculus ssp., M. M. homourus, and M. m. gansuensis contributed, to different degrees, to the formation of this fauna. PMID- 10732284 TI - [Haplotypes of two diallelic Y chromosome loci in the indigenous and migrant populations of Siberia]. AB - Two diallelic Y-chromosome markers, the Y Alu polymorphism (YAP) and the T-C transition (Tat), were analyzed in the indigenous (Tuvinian, Buryat, Northern Altaic, and Tatar) and migrant (Slavic) populations of Siberia. A high frequency of the allele C was revealed in several indigenous populations (25-55%) and in Russians (20.8%). The YAP+ allele occurred at a surprisingly high frequency (31.4%) and was completely linked with the C allele in Buryats. The YAP+ chromosome was also found in the Tuvinian population (1.5%). The two diallelic loci showed a marked linkage disequilibrium (D = 92.4%) in the total sample. The YAP-/T and YAP-/C haplotypes prevailed in both indigenous and migrant populations: their respective frequencies were 80.4 and 19.6% in the Slavic population and 71.8 and 19.9%, respectively, in the indigenous one. The YAP+/C (7.8%) and YAP+/T (0.5%) haplotypes were found only in the indigenous population. An appreciable heterogeneity in haplotype frequency distribution between regional subpopulations was revealed in Russians, Tuvinians, and Buryats. The origin and evolution of Y-chromosome lines in Northern Asia are considered. PMID- 10732285 TI - [Genetic demographic processes in Ukrainian population in 1990. The marriage structure of the Donetsk population]. AB - Analysis of records of the marriages that were contracted in the city of Donetsk revealed that, between 1960 and 1992, the marriage rate in the city decreased from 14.8 to 4.7 per 1000 people, the migration rate (m) decreased from 0.71 to 0.34, and the endogamy index increased from 0.123 to 0.458. Between 1960 and 1985, outbreeding in the Donetsk population increased, which was expressed in an increase in the frequency of interethnic marriages, migration range, and the average marital distance, as well as a decrease in the proportion of the indigenous ethnic group (Ukrainians). By 1992, outbreeding decreased: the population became more homogeneous ethnically (93% Slavic), the proportion of Ukrainians and the frequency of monoethnic marriages increased, and the average marital distance decreased. In 1960 and 1992, the coefficients of marriage contingency (K) with respect to ethnicity were 0.34 and 0.22, respectively. Regarding birthplaces, the population became almost panmictic (in 1960 and 1992, the K values were 0.15 and 0.10, respectively). Marriage structure with respect to occupation remained almost unchanged (the K values were 0.22 and 0.23, respectively). Throughout the entire period studied, the most pronounced population subdivision was that with respect to the education level (the K values were 0.36 and 0.39, respectively). PMID- 10732286 TI - [Specific dermatoglyphic traits of patients with occupational allergic bronchitis]. AB - Dermatoglyphic traits proved to be closely associated with chronic mechanic bronchitis (CMB). With the most informative traits identified, it is possible to estimate the individual predisposition to this occupational disorder and thereby to increase the efficiency of preliminary and follow-up medical examinations. Comparison with data on dermatoglyphic traits predisposed to various types of pneumoconiosis supported the hypothesis on a single mechanical lung disease or "general dust lung disease". PMID- 10732287 TI - [Diversity of the 16126C types of mitochondrial DNA in eastern slavs from Magadan]. AB - Nucleotide sequences of the 16126C types of hypervariable segment I from the major noncoding region of human mitochondrial DNA in eastern Slavs from Magadan (N = 19) were analyzed. The mitochondrial DNA sequences were subsumed within three Caucasoid-specific mitochondrial haplogroups, T, J, and JT*. Haplogroup T in eastern Slavs proved to be the most rich in different hypervariable segment I types (nucleotide diversity 1.03%). Haplogroup J was remarkably less heterogeneous (0.28%). Data on the frequency distribution of haplogroup T and J subgroups among eastern Slavs in comparison with some other populations of Eastern Europe are presented. PMID- 10732288 TI - Relation between malnutrition and development of diabetes mellitus. PMID- 10732289 TI - Presence and extent of extrapancreatic fluid collections are indicators of severe acute pancreatitis. AB - BACKGROUND: It has been suggested that early localization of both necrosis and extrapancreatic fluid collections by contrast-enhanced computed tomography (CT) can predict the outcome in severe acute pancreatitis. These two assumptions were evaluated. PATIENTS AND METHODS: This study comprises 228 patients with a first attack of acute pancreatitis admitted to our clinic from 1987 to 1995 and for whom the prognostic value of a contrast-enhanced CT obtained within 72 h of admission was prospectively evaluated. These CTs were retrospectively re evaluated for the localization of pancreatic necrosis and extrapancreatic fluid collections. The indication for dialysis and artificial ventilation, the development of pancreatic pseudocysts, the necessity for surgery (necrosectomy), and mortality were used as clinical parameters. RESULTS: There was a significant correlation between the presence of pancreatic necrosis and extrapancreatic fluid collections versus the clinical parameters. The localization of pancreatic necrosis was of no importance for the outcome of the disease, whereas the increasing amount of extrapancreatic fluid collections paralleled the severity of acute pancreatitis. CONCLUSION: Pancreatic necrosis and extrapancreatic fluid collections are indicators for severe acute pancreatitis. Whereas the localization of pancreatic necrosis is not important for the outcome of the disease, the extent of extrapancreatic fluid collections is significantly correlated with a severe course. PMID- 10732290 TI - Cytokine gene expression in peripheral blood mononuclear cells reflects a systemic immune response in alcoholic chronic pancreatitis. AB - BACKGROUND: Recent data provide evidence of a systemic inflammatory response in severe acute pancreatitis; in contrast, the exact immune mechanisms underlying chronic pancreatitis remain unclear. METHODS: To investigate the immune response in the clinical features of chronic pancreatitis, we investigated the gene expression of tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor receptor (TNFR)-p55 and -p75 and inducible nitric oxide synthase (iNOS) in peripheral blood mononuclear cells (PBMC) of 18 patients with late-stage alcoholic chronic pancreatitis of different disease activity (Balthazar criteria). RESULTS: Semiquantitative reverse transcriptase-polymerase chain reaction revealed a significantly enhanced gene expression of TNF-alpha (P < 0.05), TNFR-p55 (P < 0.05) and TNFR-p75 (P < 0.01) in unstimulated PBMC of patients with advanced chronic pancreatitis (11/18 with calcifications) compared to healthy controls (n = 8). No significant difference was found between patients with mild acute pancreatitis and patients with an inactive quiescent pancreatitis. Moreover, no expression of inducible nitric oxide synthase was detectable. CONCLUSIONS: The enhanced gene expression of TNFR-p75, TNFR-p55 and TNF-alpha in unstimulated PBMC demonstrates an enhanced leucocyte activation in patients with late-stage chronic pancreatitis and suggests a pathogenetic role of the cytotoxic TNF-alpha pathway in the clinical features of alcoholic chronic pancreatitis. The pathogenetic role of nitric oxide in chronic pancreatitis remains to be fully elucidated. PMID- 10732291 TI - The endothelin antagonist bosentan does not improve survival in severe experimental pancreatitis in rats. AB - BACKGROUND: Severity of pancreatitis seems to be aggravated by impairment of vascular perfusion of the gland. Early mortality occurs within the first few days from the acute consequences of pancreatic injury with subsequent inflammatory response. Because vasoactive substances, including endothelin, seem to contribute to early mortality in acute pancreatitis, we tested the hypothesis that the inhibition of endothelin action could alter the outcome after severe experimental pancreatitis. METHODS: In two groups of rats, pancreatitis was induced by intraductal infusion into the pancreatic duct of 1 microL/g body weight (b.w.) of either a 4% or a 5% sodium taurocholate solution. The mixed endothelin A and endothelin B receptor antagonist bosentan (20 mg/kg b.w.) or vehicle was injected intravenously in 12-h intervals for 3 d starting 1 h after induction of bile acid pancreatitis. This dose of bosentan is known to completely inhibit the effect of exogenous endothelin. The survival rate was monitored for 7 d. Thereafter, the surviving rats were sacrificed and the pancreas was prepared for histological and biochemical evaluation. RESULTS: Irrespective of the treatment protocol (bosentan versus saline), survival was not different in animals challenged with either 4% or 5% sodium taurocholate. The corresponding survival rates were 62% with bosentan and 77% without bosentan in the 4% sodium taurocholate group. In the 5% sodium taurocholate group, the survival rates were 20% with and 27% without bosentan. Morphological and biochemical alterations were identical in control as well as in endothelin-antagonist-treated rats. CONCLUSION: Therapy with the mixed endothelin A and endothelin B receptor antagonist bosentan does not influence the outcome after severe experimental pancreatitis. Therefore, blockade of endothelin A and B receptor subtypes may not be of major importance as a therapeutic principle in this model of experimental pancreatitis. PMID- 10732292 TI - Detection of extrapancreatic nerve plexus invasion of pancreatic adenocarcinoma. Cytokeratin 19 staining and K-ras mutation. AB - BACKGROUND: Neural invasion is known to be one of the aggressive characteristics of pancreatic adenocarcinoma. However, there have been no systematic studies on intraoperative examination of neural invasion of pancreatic carcinomas after wide dissection of the retroperitoneum, particularly at the surgical margin. METHODS: We performed intraoperative immunostaining on the frozen sections of several excised plexus specimens, using peroxidase-labeled anti-cytokeratin 19 antibody in 17 cases of resectable pancreatic carcinoma. Postoperatively, we also tried to detect occult micrometastasis by direct sequencing of the K-ras gene in the same samples. RESULTS: Intraoperative staining for cytokeratin 19 was positive in 4 of 17 (23.5%) cases. Patients with margin-positive neural invasion had significantly worse prognosis than patients who were margin negative (P < 0.05). One patient had micrometastasis in the nerve plexus, revealed by K-ras mutation, whereas neither cytokeratin 19 staining nor postoperative pathological investigation detected involvement of the analyzed portion. In the four patients margin positive for cytokeratin 19 staining, the diagnosis of neural invasion by cytokeratin 19 staining was in agreement with the K-ras gene analysis. CONCLUSION: Intraoperative staining for cytokeratin 19 is useful for detecting pancreatic cancer involvement of the neural plexus margin. The results can be also utilized as a prognostic indicator during the follow-up period. PMID- 10732293 TI - An immunohistochemical study of the expression of bcl-2 and p53 oncoproteins in pancreatic intraepithelial neoplasia and pancreatic cancer. AB - BACKGROUND: The bcl-2 and p53 gene deregulation is frequently involved in several types of malignancies. The purpose of this study was to evaluate the expression of bcl-2 and p53 genes in various types of pancreatic intraepithelial proliferation and in pancreatic cancer and to answer the question of whether they interact in the process of intraductal epithelial proliferation. METHODS: Immunohistochemical staining for p53 and bcl-2 was performed on paraffin embedded sections from 56 patients operated on for pancreatic carcinoma, chronic pancreatitis, and other conditions. RESULTS: Pancreatic cancer in 100% of cases showed p53 expression and in 27.7% bcl-2 expression. The p53 gene was expressed already in pancreatic intraductal neoplasia and its frequency was significantly rising with an increasing degree of hyperplasia. Normal epithelium of pancreatic ducts and ductules showed a high expression of bcl-2, which was decreasing in the process of intraductal proliferation. CONCLUSIONS: Pancreatic cancer is characterized by a high expression of p53 and a low expression of bcl-2. In pancreatic cancers and pancreatic intraepithelial neoplasia, there is an inverse relationship between the expression of bcl-2 and p53. Malignant behavior of pancreatic cancer may be associated with the phenotype bcl-2-/p53+. PMID- 10732294 TI - Reduced basal and stimulated leukocyte adherence in tumor endothelium of experimental pancreatic cancer. AB - BACKGROUND: The interaction between immunocompetent cells and tumor endothelium is essential for an effective immunological response. In the present study, we evaluated resting and CD11b/CD18-mediated leukocyte adhesion in tumor vessels of experimental pancreatic cancer and in healthy pancreatic venules in the rat. METHODS: Solid tumor fragments (1 mm3) were interposed intrapancreatically between inert transparent polymethylmetacrylate plates for intravital microscopy (n = 12) by which tumor microcirculation, leukocyte-tumor-endothelium interaction, and the effect of the chemoattractants N-formyl methioninleucylphenylalanine (fMLP) and platelet-activating factor (PAF) on leukocyte adherence was investigated. RESULTS: Leukocyte adhesion in pancreatic tumor vessels was significantly reduced compared to healthy pancreatic venules. Both fMLP and PAF dramatically increased leukocyte adherence in normal pancreatic venules. No change in leukocyte adhesion was present in tumor vessels after exposure to these chemotactic substances. CONCLUSION: Resting and stimulated integrin-dependent leukocyte adhesion is strongly reduced in malignant vessels of experimental pancreatic cancer, which may be an important mechanism to escape immune control. PMID- 10732295 TI - Effects of oxytetracycline on the rat exocrine pancreas. AB - OBJECTIVES: Given the appearance of pancreatitis attributed to tetracycline, as described in the literature, we have investigated its effect on the enzymatic content of pancreas and duodenal fluid and on pancreatic ultrastructure. We have evaluated possible differences between sexes and the relation of our findings with those described in the initial phases of acute pancreatitis, in the context of the acinar hypothesis. METHODS: With 128 Wistar rats (63 male and 65 female), 3 groups were established: control (group I) experimental animals treated with oxytetracycline intramuscularly, 15 and 30 mg/kg/d (groups II and III, respectively). Before sacrifice, half of the rats in each group were stimulated with cholecystokinin. Blood, pancreatic tissue (for enzyme dosage and morphological study), and duodenal fluid were extracted following anesthesia. RESULTS: The stimulated males of group III presented lower amylase levels in pancreatic tissue and duodenal fluid (P < 0.003). Just the opposite occurred in female rats. A similar tendency was observed with other enzymes (lipase and trypsin). Zymogen granule counts, appearance of immature granules, and dilation of ergastoplasm were more frequent in the stimulated animals. CONCLUSIONS: Oxytetracycline seems to induce morphofunctional changes in rat pancreas, which differ according to sex. In the female, enzyme accumulation that could predispose intracellular activation seems to exist, as well as the ultrastructural findings described in initial phases of acute experimental pancreatitis. This agrees with the greater frequency of pancreatitis in women undergoing tetracycline treatment described in the literature. In contrast, for males the findings were more compatible with decrease of protein synthesis. This would make them less susceptible to crinophagy phenomena and, thus, to acute pancreatitis in the context of the acinar or lysosome hypothesis. PMID- 10732296 TI - A case of acute pancreatitis complicating Salmonella enteritis. AB - We report a case of acute pancreatitis complicating Salmonella enteritis. A 43-yr old woman who was admitted to our department because of Salmonella enteritis developed clinical acute pancreatitis with laboratory and radiographic signs on the fourth hospital day. She was free from symptoms on the eighth hospital day, but her elevated serum amylase and lipase levels persisted for more than 2 m.o. In this case, clinical acute pancreatitis was a complication of bacterial enteritis caused by Salmonella enteritidis, and it was characterized by onset a few days after the onset of enteritis and by sustained elevation of serum pancreatic enzyme levels. PMID- 10732297 TI - Kaposi's sarcoma of the pancreas mimicking pancreatic cancer in an HIV-infected patient. Clinical diagnosis by detection of HHV 8 in bile and complete remission following antiviral and cytostatic therapy with paclitaxel. AB - BACKGROUND: Diagnosis of pancreatic cancer is usually made by endoscopic retrograde cholangiopancreatography (ERCP) and corresponding findings in computed tomography (CT) or magnetic resonance imaging. Kaposi's sarcoma, a frequent tumor in individuals with a late-stage HIV infection, can be located in the gastrointestinal tract and cause identical symptoms to carcinoma of the same site. A close correlation of this tumor to human herpes virus 8 (HHV 8) has been known for several years and there are reports of successful antiproliferative therapy. METHODS: Aspirated pancreatic juice and bile was investigated for the presence of HHV 8 by polymerase chain reaction. The clinical course of the patient under antiviral therapy and treatment with paclitaxel was studied. RESULTS: A 47-yr-old HIV-infected man with a history of Kaposi's sarcoma of skin and lungs caused by obstructive jaundice in the years before was admitted. ERCP showed a typical double-duct sign and CT revealed a tumorous infiltration of the pancreatic head, highly suspicious for pancreatic adenocarcinoma. A mutation of the ki-ras gene could be ruled out and molecular analysis of bile identified HHV 8 by PCR. Intensive antiviral therapy, including foscarnet and treatment with paclitaxel led to a complete remission within 8 m.o. CONCLUSION: Kaposi's sarcoma of the pancreas possibly mimics pancreatic cancer in HIV-infected subjects. Diagnosis may be made by identification of HHV 8 in pancreatic juice or bile, and successful clinical outcome is possible by intensive antiviral and cytostatic treatment with paclitaxel. PMID- 10732298 TI - A pancreatic anaplastic carcinoma of spindle-cell form. AB - A 56-yr-old man with complaint of abdominal pain and body weight loss was admitted to our hospital. Contrast-enhanced computed tomography on admission revealed a pancreatic tumor. Contrast-enhanced computed tomography 1 m.o. after admission revealed rapid growth of this tumor. We performed exploratory laparotomy with only a needle biopsy of the unresectable tumor because of extensive spread, including liver metastases. Further histological and immunohistochemical examination revealed that both the pancreatic tumor and the hepatic metastases featured malignant spindle-shaped cells. Despite radiotherapy, the patient died 40 d after laparotomy. PMID- 10732299 TI - Birth of St. Mary (St. Anne's parturition) in the light of messages from medical education: three examples from Croatian sacral heritage. AB - Christian tradition treats birth in several characteristic motives. The most frequent is the Birth of Jesus Christ, while much rarer is the apocryphal motive of the birth of St. Mary. By analyzing three paintings from Croatian 16-17th century sacral-art heritage, depicting the parturition of St. Anne (St. Mary's Mother), the authors of this paper are trying to define the role of ethnomedical notions in the formation of modern medical and general culture. PMID- 10732300 TI - Are color and pulsed Doppler sonography safe in early pregnancy? AB - Diagnostic ultrasound has been used for many years with a remarkable history of safety during the standard clinical practice. Introduction of color and pulsed Doppler modes resulted with higher levels of transmitted and absorbed ultrasonic energy. This fact raised the question for the safety of its use in early pregnancy. This article presents the pros and contras regarding the safety and summarized actual guidelines and safety limits suggested and prescribed by several instances that supervise the use of ultrasound in medicine (WFUMB, ECMUS, ECURS, AIUM/NEMA). In addition, different clinical and experimental applications of Doppler ultrasound in early pregnancy are discussed regarding the safety limits. Generally, there are no strictly defined limits for the use of Doppler ultrasound in the early pregnancy. However, there is an unequivocal demand for carefulness that is best expressed by the ALARA principle. The prudent use of Doppler takes into account benefits against the possible theoretical risks, rather than prohibiting clinically useful technology or applications. PMID- 10732301 TI - European Community multi-Center Trial "Fetal ECG Analysis During Labor": ST plus CTG analysis. AB - This report form part of the European Community Multi-Center Trial "Fetal ECG Analysis during Labor". Aim of this prospective trial was to identify changes in the fetal ECG waveform with cases of verified fetal hypoxia. In this paper we also report on the use of a newly developed automatic system for identification of ST waveform changes (ST Log). All ECG were recorded with the STAN recorder (Neoventa Medical AB, Gothenburg, Sweden). The ECG information was not displayed during labor in order not to influence the clinical management. This report includes data from 320 cases and include six cases of fetal intrapartum hypoxia. Twenty seven cases showed changes in ST waveform. All five cases with the most marked ST change (a rise in T/QRS of > 0.10 units and lasting more then 10 minutes) had signs of ongoing intrapartum hypoxia. Six out of six cases with evidence of intrapartum asphyxia, showed ST changes. On the basis of our multi center trial it appears that the combined analysis of CTG and ST waveform changes provides an accurate way to identify adverse events during labor. The work is continuing with a new STAN recorder developed by Neoventa Medical in Goteborg and currently being tested in a Swedish randomized, controlled multi-center trial. PMID- 10732302 TI - Prenatal and perinatal risk factors for autism. AB - AIM: To identify pre- and perinatal risk factors for autism. METHOD: Case control study. We matched names of patients from North Dakota who met DSM criteria for autism, a pervasive developmental disorder, and autistic disorder with their birth certificates. Five matched controls were selected for each case. RESULTS: Univariate analysis of the 78 cases and 390 controls identified seven risk factors. Logistic modeling to control for confounding produced a five variable model. The model parameters were chi 2 = 36.6 and p < 0.001. The five variables in the model were decreased birth weight, low maternal education, later start of prenatal care, and having a previous termination of pregnancy. Increasing father's age was associated with increased risk of autism. CONCLUSION: This methodology may provide an inexpensive method for clinics and public health providers to identify risk factors and to identify maternal characteristics of patients with mental illness and developmental disorders. PMID- 10732303 TI - Rupture of membranes before 26 weeks of gestation: outcome of 148 consecutive cases. AB - AIMS: To assess the outcome of preterm prelabor rupture of the membranes (PPROM) before 26 weeks of gestation and to develop a prediction model for survival. METHODS: 148 consecutive cases of PPROM before 26 weeks of gestation, collected between 1988 and 1996, were retrospectively analyzed. A multivariate analysis (generalized estimating equations) of 21 process and 5 short and long term outcome variables was performed. RESULTS: 40 out of 148 children (27%) died before or during birth, 57 (38.5%) of the children survived more than 28 days, from which still 5 died after one month. Amongst the 52 survivors (35.1%), the Bayley Mental Development Index and Psychomotor Development Index at a corrected age of 7 months was normal in respectively 85.7% and 75.5% of the cases. At 6-7 years of age, 24 out of 33 children (73%) performed adequately at school. Sex, gestational age at PPROM, birth weight, the administration of steroids and interactions of steroid administration with sex and with gestational age at the time of PPROM largely determined the chances of survival. CONCLUSIONS: Overall fetal survival after PPROM before 26 weeks of gestation was 35.1%. Survival can be predicted with an accuracy of 75%, a sensitivity of 85% and a specificity of 60%. More than 70% of the survivors behaved and performed adequately at school age, but 27% require special long-term attention and care. PMID- 10732304 TI - Coagulation and fibrinolysis in viable mid-trimester pregnancies of normal, intrauterine growth retardation, chromosomal anomalies and hydrops fetalis and their eventual obstetric outcome. AB - A total of 71 pregnant women diagnosed by ultrasound to have viable fetus in late mid- trimester pregnancies of normal, IUGR, hydrops fetalis and chromosomal anomalies were studied for their coagulation, fibrinolytic and inhibitor levels with association on eventual obstetrics outcome. A hypercoagulable state was observed in all the pregnancies studied. However, higher hypercoagulation evidenced by significantly raised prothrombin formation and clot elasticity together with higher levels of D-dimer, uPA antigen and PAI-1 than observed in normal pregnancy suggests a hyperfibrinolytic/inhibitor state in hydrops fetalis pregnancy associated with bad obstetric outcome. In IUGR pregnancy associated with good outcome further enhanced clot elasticity was seen whilst no significant differences were observed in pregnancy with chromosomal anomalies when compared to uncomplicated normal pregnancy. Our study suggests that in hydrops fetalis pregnancy, further enhanced prothrombin formation and hyperfibrinolysis/inhibitor at late mid-trimester is associated with a poor obstetric outcome. PMID- 10732305 TI - Determinants of energy expenditure in ventilated preterm infants. AB - The purpose of this study was to determine oxygen consumption (VO2), carbon dioxide production (VCO2), and energy expenditure (EE) in a group of preterm ventilated infants during the first 3 weeks of life, and to determine the major factors that influence EE. Thirty-eight indirect calorimetry studies were performed in 18 ventilated infants with mean gestational age of 27.9 +/- 0.6 (SEM) weeks. The relationship of demographic factors, nutrient intake, and severity of illness assessments of EE were determined by regression analysis. Repeated measure analysis was performed for the effect of multiple studies in the same patient. Although VO2, VCO2, and EE all tended to increase over the first 3 weeks of life, there was a wide range of values. EE was best predicted by nonprotein calorie intake and postnatal age, while there was no correlation with birthweight, weight at the time of study, gestational age, protein intake, or severity of illness. Multiple regression analyses demonstrated a strong interaction between PNA and EI. In this population EE is best predicted by PNA and EI. The interactive effect between PNA and EI on EE is probably explained by the clinical practice of daily increments in substrate intake in these patients. PMID- 10732306 TI - Perinatal outcome and management of single fetal death in twin pregnancy: a case series and review. AB - OBJECTIVE: To determine neonatal outcome of single fetal death in twin pregnancy. METHODS: Retrospective study was performed on 30 cases of single fetal death in twin pregnancy during the period from August 1971 to December 1997 at Kitasato university hospital. RESULTS: The incidence of single fetal death in twin pregnancy was 6.2% among total number of 481 twins. Prognosis of surviving infants was satisfactory except in one case, with malformation in 10 cases of dichorionic twins (total 242 sets). In contrast, neonatal death occurred in 6 cases of 20 monochorionic twins (total 239 sets), 4 of which were among 7 cases of twin-to-twin transfusion syndrome (TTTS). Cerebral palsy occurred in 2 infants of monochorionic twins. One case was diagnosed as periventricular leukomalacia (PVL) within one week after birth, and this was the case of delivery 1 day after one twin fetal death. CONCLUSION: Single fetal death itself occurs more often in monochorionic twins. There seems to be a benign outcome for the surviving dichorionic twins. On the other hand, there may be a grave outcome, though infrequent, for monochorionic twins when concomitant TTTS has existed. It is very important to identify the chorionicity by ultrasound examination in early pregnancy. PMID- 10732307 TI - Comparison between creatine kinase brain isoenzyme (CKBB) activity and Sarnat score for prediction of adverse outcome following perinatal asphyxia. AB - AIM: To assess whether plasma creatine kinase brain isoenzyme (CKBB) levels or Sarnat scores are more accurate for prediction of poor neurological outcome in babies with suspected birth asphyxia. METHODS: In a retrospective study of 97 babies CKBB levels were compared to the presence of severe hypoxic ischaemic encephalopathy (HIE) as a predictive test for these outcomes: developmental delay, cerebral palsy, visual problems, deafness or death from perinatal asphyxia. The tests were compared using positive predictive values (PPV) and likelihood ratios (LR) with confidence intervals (CI). RESULTS: 3 babies had died from perinatal asphyxia and 14 survivors were found to have neurological or developmental problems. CKBB was elevated in babies with severe HIE (p = 0.0004). A receiver operator characteristic (ROC) curve showed the optimal discriminating value for CKBB to be 21 IU/L but the CKBB was a poor predictive test. For prediction of adverse outcome: CKBB > 21 sensitivity 76%, specificity 40%, PPV 21% and LR 1.3 (95% CI 0.8-1.7). Severe HIE sensitivity 53%, specificity 95%, PPV 69% and LR 10.6 (95% CI 3.8-29.2). CONCLUSION: CKBB is elevated following birth asphyxia but is a poor predictor of adverse neurological outcome. PMID- 10732308 TI - Neonatal outcome in small for gestational age infants: do they really better? AB - BACKGROUND: There still is a controversy as to the neonatal outcome of small for gestational age (SGA) infants compared to a appropriate for gestational age (AGA) preterm infants. As a part of a randomized multicenter trial on timing of bovine surfactant therapy, we aimed at investigating short-term outcome variables in SGA infants compared with AGA-infants. METHODS: SGA-infants were classified weighing below the 10th percentile at birth and were compared to AGA-infants in terms of prenatal and neonatal characteristics and neonatal outcome. RESULTS: A total of 317 infants were enrolled, 59 SGA- and 258 AGA-infants. Both groups did not differ in gestational age, however, SGA-infants had a lower birth weight. Preterm premature rupture of fetal membranes was observed more frequently in AGA-, preeclampsia in SGA-infants. The rate of intubation, severity of RDS, rate of surfactant administration, pulmonary airleaks and days on the ventilator did not differ between both groups. However prolonged nasal CPAP, supplemental oxygen therapy and chronic lung disease at 28 days and 36 weeks was diagnosed more often in SGA-infants. Furthermore mortality was significantly higher in SGA-infants as well as total NICU and total hospital days. CONCLUSION: As SGA-infants have an increased mortality rate and an increased risk for developing chronic lung disease, further studies should focus on prevention of intrauterine growth restriction and its complications. PMID- 10732309 TI - Chronic lung disease and survival in 4 tertiary neonatal units. AB - AIM: To compare mortality and respiratory morbidity in preterm infants born at 4 United Kingdom centers during 1994 and 1995. METHOD: Collection of CRIB scores, respiratory parameters and mortality rates from unit databases. RESULTS: Mortality in center A was 27% (actual number of deaths 36/135), in center B was 30% (39/130), in center C was 28% (51/182), in center D was 39% (60/156). The rate of chronic lung disease (36 week definition) in center A was 16%, in center B was 12%, in center C was 13%, in center D was 15%. The predicted number of deaths by CRIB scores in center A was 54 (95% confidence intervals 45-63), in center B was 33 (25-41), in center C was 53 (43-63), in center D was 46 (37-56). CONCLUSION: Center A had a lower than predicted mortality. Center D had a higher than predicted mortality. There is an urgent need for a national neonatal database to allow comparison between center and to identify reasons for variation in outcomes. PMID- 10732310 TI - Follow-up studies of newborn-babies with congenital ventriculomegaly. AB - The authors present the results of their follow-up studies of data from the last four years on patients with congenital ventriculomegaly. OBJECTIVE: To study the psychomotor and mental developmental outcome of neonates with congenital ventriculomegaly diagnosed prenatally. In addition, patients were also classified into subgroups with subgrouping based on the etiology and complications of congenital ventriculomegaly and on the absence of ventriculoperitoneal shunt placement, and the findings compared between the different subgroups. METHODS: Level 3 fetal ultrasonography was used for the prenatal diagnosis of congenital ventriculomegaly in 30 infants. Using neurological examination and the modified Brunet-Lezine infant test performed postnatal follow-up of motor and sensory development and intelligence, respectively. RESULTS: The results show thirteen symptomless, well-developing patients, ten moderately handicapped patients and seven severely handicapped patients. Newborns with isolated, moderate ventriculomegaly have the best outcome. PMID- 10732311 TI - Relationship of ion channel activity to control of myometrial calcium. AB - This article reviews the contribution of ion channels to membrane potential, the ion channels expressed in myometrium, and the effect of ion channel activity on the control of myometrial intracellular free calcium. Plasma membranes constitute barriers to permeability that establish concentration gradients of ions inside versus outside the cell. Na+, CA2+, and Cl- are normally in higher concentration outside than inside cells, whereas K+ is higher inside. In myometrium, Ca2+ entry into cells mediates myometrial membrane potential changes and serves as the internal signal for contraction. K+ efflux is thought to promote repolarization after an action potential and to participate in setting the resting membrane potential. Ions cross the cell membrane through channels that have different regulated properties and selectivities. Ion movement has been measured by a number of techniques, including radiolabeled ion flux, use of intracellular indicators, and patch-clamp methodology. A number of myometrial Ca2+ channels have been described, including voltage-regulated L-type channels and Ca2+ entry in response to intracellular Ca2+ store depletion. Fast Na+ channels may contribute to cation entry late in pregnancy. K+ channels in myometrium include Ca(2+)-activated channels, a delayed rectifier, and an inward rectifier. A Ca(2+) activated Cl- channel is also present in myometrium. In addition to being regulated by Ca2+, the activity of a number of these channels can be regulated by uterine contractants and relaxants. Regulation of ion channel activity can affect intracellular free Ca2+ concentrations in the myometrium. Therefore, control of ion channel activity represents one of several approaches for controlling myometrial contractile activity. PMID- 10732312 TI - Can murine uterine natural killer cells give insights into the pathogenesis of preeclampsia? AB - These studies aimed to advance understanding of the functions of pregnancy associated uterine lymphocytes of the natural killer (NK) cell lineage. The approach was morphometric analysis of implantation sites from timed pregnancies in genetically modified mice deficient in NK cells or in signaling associated with their major product, the cytokine interferon-gamma. In four different strains of pregnant, NK cell--deficient mice, the major decidual arterioles failed to undergo modifications to their smooth-muscle coats and displayed endothelial cell damage. Decidua lacked normal cell density. This pathology was observed by the end of the first trimester, before placental differentiation. By midgestation in these strains, placentas were smaller than in control strains. In normal mice, many uterine NK cells are perivascular in location and appear to be activated because they are the major sources of interferon-gamma and of the interferon-gamma--regulated enzyme inducible nitric oxide synthase. During pregnancy in mice genetically ablated for interferon-gamma, the interferon-gamma receptor chain-alpha or the transcription factor interferon regulatory factor-1, uterine NK cells differentiate but appear to be abnormal both morphologically and functionally. In these three strains, failure of pregnancy-induced vascular modifications and overt necrosis of decidua occur. Thus, in mice, lymphocytes of the NK cell lineage make specialized contributions to pregnancy-associated modification of the uterine vasculature and to maintenance of decidua. These contributions are achieved through interferon-gamma--mediated gene regulation and appear to enhance subsequent placental growth. Human CD56 bright decidual lymphocytes may have analogous functions. If so, changes in numbers or levels of activity of human uterine NK cells or mutations in genes regulated by uterine interferon-gamma could contribute to initiation of preeclampsia. PMID- 10732313 TI - Preeclampsia prevention and management. AB - Preeclampsia is a multisystem disorder specific to pregnancy with a high maternal and perinatal morbidity and mortality. The cause of this disorder is unknown. Preeclampsia likely represents the clinical end point of multiple contributory factors, and it is unlikely that any single cause will be found. The blueprint for the development of preeclampsia is laid down early in pregnancy, and delivery of the fetus and placenta remains the only effective treatment. Efforts to prevent preeclampsia in women at high risk have been largely unsuccessful. Until the pathogenesis of preeclampsia is well defined, it is unlikely that effective preventive strategies will be developed. PMID- 10732314 TI - Prostanoid stimulation of cytokine production in an amnion-derived cell line: evidence of a feed-forward mechanism with implications for term and preterm labor. AB - OBJECTIVE: To test the hypothesis that amnion cytokine production might be regulated by prostanoids. METHODS: Amnion-derived WISH cells were treated with a range of prostanoids and their effects on production of interleukin (IL)-6 and IL 8 were determined by enzyme-linked immunosorbent assay and Northern analysis. The effects of thromboxane inhibitors on cytokine production by term primary amnion explants also were examined. RESULTS: Prostaglandin (PG)A2, PGD2, PGF2 alpha, PGE2, PGJ2, and the PGI2 analogue carbaprostacyclin (1-1000 nmol/L) exhibited no significant effects on cytokine production. However, the thromboxane A2 (TXA2) agonist U46619 and carbocyclic (c)TXA2 both stimulated WISH cytokine production with similar potencies under basal or cytokine-stimulated conditions. Significant stimulation of IL-6 production was observed at concentrations > or = 8 nmol/L (P < .05 by analysis of variance), whereas IL-8 production was stimulated significantly but to a lesser extent. The effects of U46619 and cTXA2 were rapid; maximal stimulation of cytokine production occurred within 4 to 8 hours of treatment. U46619 augmented IL-1 beta-stimulated IL-6 and IL-8 mRNA expression within 2 hours of treatment. In amnion explants inhibitors of TX synthesis and action abrogated the stimulatory effects of IL-1 beta on cytokine production. CONCLUSION: These results are consistent with the presence of a feed-forward loop in amnion involving TXA2 and cytokines, which could play a significant role in the progression of the inflammatory response involved in the mechanism of infection-driven preterm labor. PMID- 10732315 TI - Hyperthermia prevents metabolic and cerebral flow responses to hypoxia in the fetal sheep. AB - OBJECTIVE: Fetal brain temperature has been found to decrease during hypoxia, strongly suggesting a reduction in cerebral O2 consumption and increases in cerebral blood flow. These responses may protect the brain in part against hypoxic injury. This study was undertaken to examine whether these compensatory mechanisms are lost during fetal hyperthermia. METHODS: Intermittent fetal hypoxemia was induced by administering low-O2 gas mixtures to nine near-term ewes. Fetal brain and body core temperature responses were measured with and without fetal hyperthermia induced by circulating warm water through a plastic coil looped about the fetus in utero. RESULTS: In normothermic fetuses, fetal brain temperature relative to core decreased during a 30-minute period of hypoxia and then returned to normal during recovery. This response may be explained by a combination of cerebral hypometabolism and increased cerebral blood flow. However, in hyperthermic fetuses (intrauterine warming for 1 hour, raising body core and brain temperatures 0.66 +/- 0.06 and 0.61 +/- 0.10 C, respectively) a subsequent period of hypoxia no longer induced a reduction in brain temperature relative to body core. CONCLUSION: When temperature of the fetal sheep is elevated, as may occur with maternal fever, prolonged exercise, and elevated environmental temperatures, the fetal brain is less well protected against hypoxic injury. PMID- 10732316 TI - Periovulatory and interleukin-1 beta-dependent up-regulation of intraovarian vascular endothelial growth factor (VEGF) in the rat: potential role for VEGF in the promotion of periovulatory angiogenesis and vascular permeability. AB - Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen and permeability factor the role of which in ovarian angiogenesis has been the subject of increasing interest. It was the objective of this communication to explore the possibility that interleukin (IL)-1 may regulate the in vitro expression of rat ovarian VEGF mRNA, as well as to study the in vivo expression of rat ovarian VEGF transcripts during follicular maturation, ovulation, and corpora lutea formation. Taken together, our findings 1) reaffirm the rat ovary as a site of VEGF expression; 2) document an in vivo increase in VEGF transcripts before ovulation; 3) disclose a marked dependence of VEGF on IL-1 beta; 4) reveal the IL-1 beta effect to be receptor mediated and dose and time dependent and to be shared by at least two growth factors--epidermal growth factor and basic fibroblastic growth factor; and 5) demonstrate a lack of VEGF effect on ovarian progesterone biosynthesis as assessed in cultured isolated granulosa cells. It is tempting to speculate that the up-regulatory effect of IL-1 beta on VEGF transcripts may be relevant to the marked angiogenesis and increased vascular permeability displayed by the hyperemic ovarian Graafian follicle during the terminal stages of follicular development. In this context, VEGF may be joined by other IL-1-dependent angiogenesis promoters such as IL-6 or transforming growth factor beta 1. Thus, IL-1-mediated VEGF induction may constitute one of several end points through which IL-1 may coordinate and perhaps amplify the ovulatory cascade. PMID- 10732317 TI - Expression of 17 beta-hydroxysteroid dehydrogenase type 5 in human ovary: a pilot study. AB - OBJECTIVE: Conversion of androstenedione to testosterone, the most potent androgen secreted by the ovary, is carried out by androgenic 17 beta hydroxysteroid dehydrogenase (17 beta-HSD) activity. The molecular basis for this is unclear. We tested the hypothesis that type 5 17 beta-HSD (17 beta-HSD5) is responsible for testosterone formation from androstenedione in the human ovary. METHODS: We used primers specific for each type of 17 beta-HSD to identify quantitatively and directly sequence the polymerase chain reaction products of a human ovary library. RESULTS: 17 beta-HSD1, 17 beta-HSD4, and 17 beta-HSD5 were detected in the library lysate, but not 17 beta-HSD2 or 17 beta-HSD3. 17 beta HSD5 was the predominant androgenic form of 17 beta-HSD expressed in human ovary. CONCLUSION: These data suggest that 17 beta-HSD5 may play a major role in testosterone biosynthesis by the human ovary. Further investigation of the regulation of 17 beta-HSD5 gene expression is warranted with regard to ovarian testosterone secretion in normal and abnormal states of ovarian function, such as polycystic ovary syndrome. PMID- 10732318 TI - Effect of parity on pituitary prolactin response to metoclopramide and domperidone: implications for the enhancement of lactation. AB - OBJECTIVE: The gastrointestinal motility agents metoclopramide and domperidone are known to increase pituitary prolactin (PRL) secretion and breast milk production. This study compared the effect of single doses of two strengths of metoclopramide and a single dose of domperidone on PRL secretion. METHODS: Ten nonpregnant women had baseline evaluation of serum PRL concentrations. The PRL concentrations were then determined after random oral administration of metoclopramide 10 mg, metoclopramide 5 mg, and domperidone 10 mg. Blood samples were drawn in the first 7 days of the menstrual cycle, at 13 time points over a 6 hour period (0, 15, 30, 45, 60, 75, 90, 120, 150, 180, 240, 300, and 360 minutes), with the zero time point beginning at 0800 hours. Variables such as weight, height, age, gravidity, parity, and oral contraceptive use were recorded. RESULTS: Baseline PRL concentrations showed the natural circadian rhythm. Metoclopramide and domperidone both caused a significant increase in PRL. However, PRL secretion was most influenced by parity. Nulliparous women had the quickest and highest PRL secretion with metoclopramide 10 mg, compared with the PRL response with metoclopramide 5 mg and domperidone 10 mg. Conversely, multiparous women had PRL secretion patterns that were equivalent between the medications. CONCLUSIONS: The PRL response to the medications was most influenced by parity. Therefore, we suggest that the medication therapy of choice for enhancing lactation may not be the same in all women, but may instead be determined by parity. PMID- 10732319 TI - CD44-9v and CD44-10v are potential molecular markers for squamous cell carcinoma of the vulva. AB - OBJECTIVE: CD44 is a cell surface glycoprotein widely distributed in the extracellular matrix. CD44 isoforms arising from alternative mRNA splicing are implicated in tumor metastases. The aim of this study is to investigate the expression of CD44s and two splice variants, CD44-9v and CD44-10v, in squamous cell carcinoma (SCC) of the vulva as well as its correlation with lymph node (LN) metastases and disease-free survival. METHODS: Thirty-five SCC vulvar tumors were evaluated for CD44s, CD44-9v, and CD44-10v expression by immunocytochemistry. One nonmetastatic LN was studied also. In cases with LN metastases, the metastatic LN as well as a nonmetastatic LN from the same patient were evaluated. RESULTS: CD44s and CD44-9v were expressed in all epithelia--normal, dysplastic, and SCC. However, intensity and distribution of expression of 9v isoforms changed within the tissue containing invasive cancer. CD44-9v expression was downregulated in the most differentiated cells within the carcinoma, mainly in patients who had disease recurrence or eventually died of disease (P = .031). All metastatic tumor to LNs was immunoreactive also for CD44-9v. CD44-10v expression was present in 78% of tumors and 56% of normal epithelium. Interestingly, CD44-10v membrane expression, but not cytoplasmic expression, correlated with disease recurrence (P = .035). CONCLUSION: Our findings warrant larger multi-institutional studies to determine the potential of CD44-9v and CD44-10v as molecular markers of disease recurrence in vulvar carcinoma. We propose to test the use of anti-CD44-9v monoclonal antibody for radioimmunoimaging of occult LN metastases. PMID- 10732320 TI - Estrogen and progesterone receptors: an overview from the year 2000. AB - Our current understanding of the steroid/thyroid superfamily of ligand-dependent nuclear transcription factors now involves specific domains for steroid ligand binding, receptor homo- and heterodimer formation, the association of transcription mediators or adaptors that activate, repress, or integrate competing influences, interaction with the hormone response element, transcription modulation based on the components of the transcriptional complex, and intracellular trafficking. Various ligands, including selective hormone receptor modulators, interact with transcriptional mediators in both ligand- and tissue-specific manners, thus multiplying the variety and complexity of interactions substantially. Transcriptional mediators, selective hormone receptor modulators, and varying isoforms of nuclear receptors and their tissue-specific interactions will be a major focus of research in the future. This symposium assembles many of the investigators presently elucidating our understanding of nuclear receptors as we move into the next millennium. PMID- 10732321 TI - Novel aspects of estrogen action. AB - The discovery of a second estrogen receptor, ER beta, in 1996 introduced a new paradigm in our understanding of estrogen action. ER alpha predominates in the uterus and mammary gland, whereas ER beta has significant roles in the central nervous, cardiovascular, and immune systems, urogenital tract, bone, kidney, and lung. ER beta appears to be the only from expressed in the embryonal central nervous system. Photoestrogens may be good candidates for physiologic ligands of ER beta. Coactivator proteins play critical roles in regulating estrogen. Female ER beta-deleted mice (beta ERKO) develop follicular arrest and anovulation. PMID- 10732322 TI - Selective estrogen receptor modulators (SERMs): A first step in the development of perfect hormone replacement therapy regimen. AB - Until 1986, our understanding of estrogen receptor (ER) action was based on information derived from in vitro biochemical analyses and in vivo correlations. With the cloning of the human ER cDNA, the reconstitution of ER responsive transcription units in heterologous cells has permitted the genetic dissection of the ER signal transduction pathway. The recent discovery of ER beta and a multitude of adaptor proteins (coactivators and corepressors) has expanded the potential explanation for tissue-selective activities. The current concept of ER action includes a rheostat-like action of the receptor due to conformational changes in the ligand receptor complex that depend on the nature of the bound ligand. This conformational change also determines subsequent adaptor protein interactions. Recognition of the tissue-specific activities of tamoxifen, the first selective ER modulator (SERM), led to the development of new SERMs (raloxifene and toremifene) with greater tissue selectivities. A knowledge of the key adaptor proteins expressed within each ER target cell will allow mechanism based screening of selective ER modulators. These future "designer estrogens" of the next millennium will be used for specific applications in the central nervous, cardiovascular, bone and reproductive systems. PMID- 10732323 TI - Estrogen receptor knock-out mice: molecular and endocrine phenotypes. AB - Using gene-targeting techniques, transgenic, knock-out (KO) mice, homozygous for the disrupted estrogen receptor alpha (alpha ERKO) and beta gene (beta ERKO) have been produced. Estrogen receptor (ER) beta expression does not appear to be dependent on ER alpha expression. Progesterone receptor (PR) mRNA is detected in alpha ERKO mice and appears to have both estrogen-independent and -dependent gene regulation. The phenotypic differences in alpha ERKO and beta ERKO mice are described. Additional studies will further define the specific roles of ER alpha and ER beta in various tissues. PMID- 10732324 TI - Genetic mutations resulting in loss of aromatase activity in humans and mice. AB - Aromatase enzyme is the product of the CYP19 gene. Human aromatase deficiency is a rare disorder and is usually caused by single base-pair changes resulting in amino acid substitution or premature stop codons. In most cases, the affected mother presents with virilization in the third trimester of pregnancy. Affected female newborns have pseudohemaphrodism with clitoromegaly and hypospadias. The cause of these presentations in pregnancy is the inability to convert fetal dehydroepiandrosterone to estrogen in the placenta and subsequent conversion to androgens in the periphery. Affected male newborns present with tall stature secondary to failed epiphyseal fusion. They also have delayed bone age, osteopenia, and undermineralization, which can be corrected with the addition of estrogen, highlighting estrogen's critical role in men as well as women. The aromatase knock-out male mouse (ArKO) has shortened femur length and bone undermineralization. Female ArKO mice at 10-12 weeks have multiple ovarian follicles arrested in the antral phase and stromal hyperplasia. By 1 year the ovaries become grossly dysmorphic with numerous cystic follicles and fibrous stroma. Male ArKO mice testes demonstrate arrest of spermatogenesis at the level of round spermatids and Leydig cell hyperplasia. ArKO mice also exhibit evidence of insulin resistance and visceral adiposity. PMID- 10732326 TI - Reproductive functions of the progesterone receptor. AB - Progesterone plays a central coordinate role in regulating reproductive events associated with the establishment and maintenance of pregnancy including ovulation, uterine and mammary gland development and tumorigenesis, and neurobehavioral expression associated with sexual responsiveness. The effects of progesterone are mediated by two receptor proteins (PR), termed A and B, that arise from a single gene and act as ligand-activated transcription factors to regulate the expression of reproductive target genes. Null mutation of both proteins in mice leads to pleiotropic reproductive abnormalities. This review summarizes the structure and functional properties of the PR isoforms and how functional differences between these proteins are likely to impact on the overall physiologic role of the receptor in reproductive systems. PMID- 10732325 TI - Novel mechanisms of progesterone antagonists and progesterone receptor. AB - The progesterone receptor (PR), as a member of the nuclear receptor superfamily of ligand-dependent transcription factors, activates gene transcription through binding to specific palindromic progesterone response elements (PRE) in the promoter region of progestin-responsive genes. The progesterone antagonists ZK98299 (Onapristone) and RU 486 (Mifepristone) inhibit the transcriptional activity of PR by complex mechanisms at concentrations much lower than the progestins. Altered conformation is central to antagonist inhibition of the transcriptional activity of PR. Antagonists also promote inappropriate association of PR with corepressors. We speculate that the different PR conformations induced by agonist and antagonists results in an asymmetric agonist/antagonist heterodimer that binds inefficiently to palindromic PREs. PR, under the same cellular conditions but with different promotor contexts, can repress (beta-casein) or enhance (3 beta-HSD) signal transducer and activator of transcription (Stat5)-mediated gene activation. The beta-casein promoter appears to contain a composite DNA-binding element for PR and Stat5 and that occupancy by PR in response to progestins or antagonists suppresses Stat5 transactivation function. PMID- 10732327 TI - Mechanisms of action and cross-talk between estrogen receptor and progesterone receptor pathways. AB - The intriguing biology of estrogens and progestins in their diverse target cells is determined by the structure of the hormonal ligand, the receptor subtype or isoform involved, the nature of the hormone-responsive gene promoter, and the character and balance of coactivators and corepressors that modulate the cellular response to the receptor-ligand complex. Estrogens regulate the growth, differentiation, and functioning of diverse target tissues, both within and outside of the reproductive system. Most of the actions of estrogens appear to be exerted through the estrogen receptor (ER) of target cells, an intracellular receptor that is a member of a large superfamily of proteins, which function as ligand-activated transcription factors, regulating the synthesis of specific RNAs and proteins. To understand how the ER discriminates between estrogen ligands, which activate the ER, and antiestrogen ligands, which fail to effectively activate the ER, we have generated and analyzed human ERs with mutations or other alterations in portions of the receptor. These studies provide evidence for the promoter-specific and cell-specific actions of the estrogen-occupied and antiestrogen-occupied ER, highlight a regional dissociation of the hormone binding and transcription activation functions in domain E of the receptor, and indicate that some of the contact sites of estrogens and antiestrogens in the ER are likely different. In addition, multiple interactions among different cellular signaling pathways are involved in the regulation of gene expression and cell proliferation by the ER. In several cell types, protein kinase activators and some growth factors enhance the transcriptional activity of the ER. Cyclic AMP also alters the agonist/antagonist balance of some antiestrogens. Estrogens, and antiestrogens to a lesser extent, as well as protein kinase activators and growth factors, increase phosphorylation of the ER and possibly other proteins involved in the ER-specific response pathway, suggesting that changes in cellular phosphorylation state will be important in determining the biologic activity of the ER and the effectiveness of antiestrogens as estrogen antagonists. The ER also has important interrelationships with the progesterone receptor (PR) system in modulation of biologic responses. Liganded PR-A and PR-B can each suppress estradiol-stimulated ER activity, with the magnitude of repression dependent on the PR isoform, progestin ligand, promoter, and cell type. These findings underscore the mounting evidence for the importance of interactions between members of the steroid hormone receptor family. PMID- 10732328 TI - Selective estrogen receptor modulators (SERMs) in clinical practice. AB - The objective of this literature review is to familiarize the reader with the clinical data on selective estrogen receptor modulators (SERMs) and antiestrogens currently in use in the US, excluding data on breast effects. Four compounds in the SERM and antiestrogen families are presently in clinical use in the US: clomiphene (CC), tamoxifen (TAM), toremifene (TOR), and raloxifene (RLX). The clinical database on these compounds is among the largest available. Each compound demonstrates a specific profile for its target tissue effects, and this may differ between premenopausal and postmenopausal women. CC is the most widely used agent for ovulation induction. TAM is indicated in the management of breast cancer and for prevention in women at high risk. TAM may have additional effects on the cardiovascular and skeletal systems. TOR also is used for its effects on breast tissue and may have positive cardiovascular effects. RLX is approved in the management of osteoporosis with data supporting favorable effects on the cardiovascular system and breast tissue. TAM and TOR appear to have stimulatory effects on the uterus and endometrium, whereas RLX is neutral. Few adverse events have been attributed to these agents, with hot flashes being the most common one. There appears to be an increased risk of thromboembolic events with continuous use of TAM, TOR, and RLX. SERMs and antiestrogens continue to be studied extensively. Their evolving profiles support key roles for these agents in modern day medicine, particularly in the management of postmenopausal women's health. PMID- 10732329 TI - Antiestrogens: clinical applications of pharmacology. AB - Selective estrogen receptor modulators (SERMs) appear to reduce the incidence of breast cancer in high-risk women. Five years of tamoxifen administration after the diagnosis of breast cancer results in a 50% reduction in the incidence of contralateral breast cancer. This reduction is maintained for 5 years after therapy is discontinued. The Study of Tamoxifen And Raloxifene (STAR), presently ongoing, will address the questions of breast cancer prevention, risk of endometrial cancer, the incidence of bone fractures, and coronary artery disease in women treated with these SERMs. PMID- 10732330 TI - Clinical uses of antiprogestogens. AB - Progesterone is essential to the establishment and maintenance of pregnancy. Compounds that inhibit the synthesis of progesterone, or antagonize progesterone action, have multiple effects including the induction of abortion, labor induction, contraception, and cervical ripening. They also may be used in the treatment of endometriosis, fibromyomata, meningiomas, Cushing syndrome, and glaucoma. Antiprogesterone compounds will play important roles in manipulating reproductive function for fertility control and in the treatment of hormone dependent disease. This article reviews the potential of antiprogesterone compounds. PMID- 10732331 TI - New progestins and potential actions. AB - Newer, nonsteroidal, orally active, tissue-selective progestins are being developed through a molecular approach to compound selection with human progesterone receptor (hPR) serving as the molecular target. The co-transfection and binding assays are used to test receptor selectivity and cross reactivity with a panel of receptors. Transcriptional products are used to further profile new progestin compounds. Desirable new progestins will suppress estrogen-induced endometrial stimulation, show no or minimal proliferative activity, maintain pregnancy, inhibit ovulation, contain no androgenic, mineralocorticoid or glucocorticoid activity, and possess minimal adverse physiologic effects. Newer progestins that possess many of these desirable properties are in development. PMID- 10732332 TI - [Computer prediction of DNa binding sites involved in interaction with different nuclear matrix elements]. PMID- 10732333 TI - [Distribution of 4.5S1 RNA among rodents and its conserved nucleotide sequence]. PMID- 10732334 TI - [Synthesis of chimeric IgE in vivo after ballistic transfection of immunoglobulin genes in various mouse tissues]. PMID- 10732335 TI - [Polymorphism of D6S2414 marker located in HLA (6p21.31) locus and genetic predisposition to Grave's disease in Moscow population]. PMID- 10732336 TI - [Plasmid instability in methylotrophic yeast Hansenula polymorpha: the capture of chromosomal DNA fragments by integrative plasmids]. PMID- 10732337 TI - [Plasmid instability in methylotrophic yeast Hansenula polymorpha: the capture of chromosomal DNA fragments by replicative plasmids]. PMID- 10732338 TI - [Proteins interacting with fragments of pre-mRNA and mRNA of the human ribosomal protein S26]. PMID- 10732339 TI - [Modified ribonuclease gene provides efficient positive selection in molecular cloning]. PMID- 10732340 TI - [Affinity separation of HeLa chromatin proteins and their selective modification in the regions of GT repeats]. PMID- 10732341 TI - [Interaction of RNA polymerase of bacteriophage T7 with affinity modifier analogs of nucleoside triphosphates]. PMID- 10732342 TI - [Secondary structure of 5'-terminal region of mRNA of human multidrug resistance gene]. PMID- 10732343 TI - [Quantitative RT-PCR analysis of short-lived RNAs and their decay products]. PMID- 10732344 TI - [The second DNA-methyltransferase of BstF5I restriction-modification system is homologous to the C-terminal domains of FokI and StsI methylases]. PMID- 10732345 TI - [Interaction of thiopyrylium salts with DNA]. PMID- 10732346 TI - [Interaction of cobalt phthalocyanine complex with DNA duplexes]. PMID- 10732347 TI - [The hinge region of human IgG3 is rod-like under acidic pH conditions]. PMID- 10732348 TI - [The study of domain structure of chaperone Caf1M from Yersinia pestis]. PMID- 10732349 TI - [Conformation of delta-endotoxin from Bacillus thuringiensis var.tenebrionis induced by methanol]. PMID- 10732350 TI - [Effect of delta-endotoxin from Bacillus thuringiensis var.tenebrionis on phospholipid vesicules]. PMID- 10732351 TI - [Tightly bound matrix DNA probably plays an important role in organization of chromosome centromeres]. PMID- 10732352 TI - [Identification of genes activated by mutant forms of p53]. PMID- 10732354 TI - [Structural-functional organization of Cowpox virus GRI-90 genome.I. Isolation of clones of DNA fragments of complete Cowpox virus genome]. PMID- 10732353 TI - [Unfolding of viscumin A-subunit during the intracellular transport of toxin]. PMID- 10732355 TI - [Cloning of full-length cDNA of the shallots virus X genome and infectivity of its transcripts in sugar beet protoplasts]. PMID- 10732356 TI - [Biological properties and genome structure of the Kazakh isolate K1 of Tobacco Mosaic virus]. PMID- 10732357 TI - [Methods of sperm microinjection as an instrument of assisted reproduction and a study of the biology of fertilization]. AB - Recent success in assisted fertilization mainly depended on the development of sperm microinjection methods: intracytoplasmic sperm injection and subzonal insemination. Some basic mechanisms that underlie fertilization were revealed by using intracytoplasmic sperm injection. In respect to this, problems of fertility, oocyte activation, formation of pronuclei and practical aspects of intracytoplasmic sperm injection are discussed. PMID- 10732358 TI - [The effects of exogenous actin-binding casein kinase injected into the oocytes and ova of the clawed toad]. AB - The effects of microinjections of exogenous casein kinase 2 on the structural organization of the maturing oocytes and eggs were studied in Xenopus laevis. Kinase inhibited the progesterone-stimulated oocyte maturation and induced dislocation of pigment granules. The morphological effect was shown to be dose dependent. The results obtained are discussed in the light of the possible influence of casein kinase 2 on the organization of the cortical actin cytoskeleton through phosphorylation of actin-binding proteins. PMID- 10732359 TI - [An imbalance in the natural cation ratio in the water as a factor affecting the synthesis of lipids and fatty acids in fish eggs]. AB - The effects on the lipid status of developing embryos of a disturbed natural ratio of cations in water as a result of the pollution of water bodies by waste with a high potassium content (130-140 mg/I) were studied in the laboratory. The results obtained confirm the indication of reduced lipid synthesis and altered formation of phospholipids in embryos developing in a medium with a disturbed natural ratio of cations. In addition, the lysophospholipid fraction increased in these embryos, which indicates activation of phospholipid hydrolysis. It was also found that changes in the salt regime lead to a decreased content of cholesterol, the main membrane thickener. It was proposed that the changes discovered lead to disturbed stability and permeability of the membranes of fish eggs, with the subsequent death of embryos. PMID- 10732360 TI - [The simulation of the cooperative effect of development in a culture of early mouse embryos after irradiation with electromagnetic waves in the millimeter range]. AB - We have found that two-cell mouse embryos cultured in vitro can be stimulated by electromagnetic irradiation in the millimeter range. After 30 min of exposure, they acquire the ability to develop in culture on their own and can reach the stage of blastocyst in a relatively large volume of Whitten cultural medium (150 microliters) without serum or growth factors. It is proposed that millimeter range electromagnetic waves activate metabolic processes and specifically the synthesis of factors controlling early embryonic development in culture. PMID- 10732361 TI - [The action of catecholamine-synthesis inhibitors and of spiperone on sea urchin and mouse embryos]. AB - We studied the effects of three inhibitors of catecholamine synthesis on the development of sea urchins Sphaerechinus granularis and Paracentrotus lividus. These drugs affected the early embryogenesis, which was expressed in inhibition of the cleavage divisions, appearance of abnormal embryos, and developmental arrest. The addition of arachidonic acid amide and dopamine to the incubation medium weakened the effects of the inhibitors. Spiperone induced developmental defects in preimplantation mouse embryos and sea urchin embryos. Arachidonic acid amide with dopamine exerted a protective effect against spiperone when introduced to sea urchin embryos at the blastula or late gastrula stages, rather than after fertilization. In murine embryos, this amide induced developmental defects and arrest itself and its effect was reversible. Possible mechanisms underlying the effects of these drugs are discussed. PMID- 10732362 TI - [The enzymatic activity of the antioxidant system in the ontogeny of mutant norflurazone-tolerant Arabidopsis thaliana]. AB - The activities of superoxide dismutase and guaiacol-dependent peroxidase were studied in the ontogenesis of recessive homozygous mutants of Arabidopsis thaliana Heynh. le-2 and nfz24, which are characterized by two- to threefold increases in tolerance to the herbicide norflurazone. The mutants le-2 and nfz24 differed from the initial race Dijon in some phenotypic features, duration of ontogenetic stages, and dynamics of the superoxide dismutase and peroxidase activities in ontogenesis. A single treatment of plants with norflurazone induced an accelerated increase in the level of both enzymes in the mutants as compared to the wild type plants. Under the conditions of multiple treatment with norflurazone, the mutants le-2 and nfz24 displayed a higher tolerance to the bleaching effect of the herbicide and were characterized by a higher level of superoxide dismutase. The data obtained suggest that the superoxide dismutase and peroxidase activities are controlled by both ontogenetic factors and stress signals. Mutations in the lines le-2 and nfz24 increase sensitivity to a stress signal or increase efficiency of an adaptive response due to long-term maintenance of a high level of the antioxidant enzymes under the conditions of stress. PMID- 10732363 TI - [Changes in the K562 cell sensitivity to nonspecific lysis by human and rat leukocytes under the influence of sodium butyrate, dimethyl sulfoxide and phorbol 12-myristate-13-acetate]. AB - We studied the ability of inducers and inhibitors of erythroid differentiation of K562 leukemia cells, such as sodium butyrate, dimethyl sulfoxide, and phorbol-12 myristate-13-acetate, respectively, to modulate sensitivity of these cells to non specific lysis (non-restricted with respect to antigens of the major histocompatibility complex) mediated by natural human or rat killer cells. Unfractionated leukocytes from human peripheral blood or rat splenocytes were used as sources of natural killers. The induction of erythroid differentiation by sodium butyrate was accompanied by a significant increase in cell sensitivity to lysis with human peripheral blood lymphocytes; incubation of K562 cells in the mixture of sodium butyrate and dimethyl sulfoxide did not change cell sensitivity to lysis by both types of effector cells. The inhibition of sodium butyrate induced erythroid differentiation with high doses of phorbol-12-myristate-13 acetate (100 nM; incubation was in the presence of both these agents simultaneously) resulted in an increased cell sensitivity to lysis with rat splenocytes. Incubation of K562 cells in a mixture of sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate (100 nM) produced greater lysis by human leukocytes, as compared with incubation in the mixture of sodium butyrate and dimethyl sulfoxide. PMID- 10732364 TI - [The ontogenetic correlations of noradrenaline level and adrenergic receptor density in the rat brain]. AB - We studied the level of noradrenaline and the density of alpha 2- and beta adrenoreceptors in the brain stem and cerebral cortex of 12-day- and 21-day-old rat fetuses, as well as of rats at the ages of 1, 2, 5, 7, 9, 16, 21, 35, and 70 days. We found a positive correlation between the level of noradrenaline in the brain stem and the density of beta-receptors in the cerebral cortex, and between the amount of alpha 2- and beta receptors in the cerebral cortex, as well as between the values of each of these indices of the neurochemical system and body weight. Significant negative correlations (r = -0.72 and r = -0.88, respectively) were found between the amount of alpha 2-adreno-receptors in the brain stem and the content of noradrenaline in this brain region, as well as in the cerebral cortex. Explanations of these positive and negative correlations between the level of noradrenaline and the amount of adrenergic receptors in the rat brain during ontogenesis are discussed. PMID- 10732365 TI - [The characteristics of embryogenesis and oogenesis in Adalia bipunctata L. and Harmonia axyridis Pall. beetles in all-female families]. AB - We describe abnormal embryonic development leading to death of 50% of embryos in all-female lines of Adalia and Harmonia infected with an androcide Spiroplasma. The arrest of embryonic development takes place at different stages throughout embryogenesis. Oogenesis of the infected beetles proceeds without any significant morphological changes. PMID- 10732366 TI - [A connective tissue tumor in the mussel Mytilus trossulus from a polluted region of Nakhodka Bay, the Sea of Japan]. AB - A tumor was found for the first time in a mussel Mytilus trossulus from a heavily polluted area of Nakhodka Bay, Sea of Japan. Tumor cells were found in the connective tissue of different organs and also in gill vessels and hemal sinuses of the visceral mass. They were both attached and diffuse. The tumor was at an advanced stage, replacing the normal connective tissue cells, and formed nodes. The tumor cells were polymorphic, with a high nucleocytoplasmic ratio, and had a prominent nucleolus. The size of their nuclei was three to five times that of the nuclei of agranular hemocytes. The mitotic activity of the tumor cells was more than an order of magnitude higher than in the normal cells: the mean mitotic index was 1.4 +/- 0.5%, ranging from 0.97 to 2.3% in different organs. The mitotic indices in the connective tissue cells of three normal mussels were 0, 0, and 0.12%. A significant proportion (up to 78%) of the mitotic cells were at metaphase. The frequency of abnormal mitoses was 17%. Metaphases with displaced (often multiple) chromosomes constituted 71% of abnormal mitoses; anaphases, 8%; and tri- and tetrapolar mitoses, 11%. The tumor described is similar to diffuse sarcomatoid diseases of mussels from other geographical regions. PMID- 10732367 TI - Application of experimental poverty measures to the aged. AB - The U.S. Census Bureau recently released new, experimental measures of poverty based on a National Academy of Sciences (NAS) panel's recommendations. This article examines the effects of the experimental measures on poverty rates among persons aged 65 or older in order to help inform policy debate. Policymakers and analysts use poverty rates to measure the successes and failures of existing programs and to create and defend new policy initiatives. The Census Bureau computes the official rates of poverty using poverty thresholds and definitions of countable income that have changed little since the official poverty measure was adopted in 1965. Amid growing concerns about the adequacy of the official poverty measure, a NAS panel undertook a study of the concepts, methodology, and data needed to measure poverty. The panel concluded in its 1995 report that the current measure no longer provides an accurate picture of relative rates of poverty for different groups in the population or of changes in poverty over time. The panel recommended changes in establishing the poverty thresholds, defining family resources, and obtaining the required data. The Census Bureau report shows how estimated levels of poverty would differ from the official level as specific recommendations of the NAS panel are implemented individually and how estimated trends would differ when many recommendations are implemented simultaneously. It computes nonstandardized and standardized poverty rates. (The latter constrains the overall poverty rate under the experimental measures to match the official rate.) This article reports poverty rates that have not been standardized and provides considerably more detail than the Census report about the effects of the experimental measures on poverty among the aged. It examines the effects of changing the poverty thresholds and the items included or excluded from the definition of available resources. It also explores the effects of the experimental measures on persons aged 65 or older by age group, gender, race and ethnicity, and marital status. Results indicate that: Poverty rates in 1997 for persons aged 65 or older under the experimental NAS poverty measure are 17.3 percent, compared with 10.5 percent under the official poverty measure. This 65 percent increase is largely driven by the NAS-based measure's subtraction of medical out-of-pocket (MOOP) expenses from resources. Under the NAS-based measures, poverty rates increase for all major groups of older persons, and increase the most for groups for whom the incidence of official poverty is the lowest. The experimental NAS poverty measure shows narrower differences between genders, racial and ethnic groups, and among persons of different marital statuses than the official poverty measure. For example, white Hispanic women aged 65 or older have poverty rates that are 450 percent higher than those for white non-Hispanic men under the official poverty measure and 181 percent higher under the NAS measure. The NAS-based measure's subtraction of MOOP expenses from resources has a disproportionate effect on poverty rates among non-Hispanic whites and men as compared with other groups. However, changes in relative poverty between groups appear to be most influenced by the NAS midpoint equivalence scale. Because this scale decreases poverty rates for persons who live alone or with unrelated individuals and increases them for persons who live with others, poverty rates differ meaningfully under the NAS and official measures among demographic groups. This article highlights issues concerning the elements of the experimental NAS poverty measure that are particularly important to the measurement of poverty among the aged population. Results suggest that the research community's future efforts to refine, enhance, and build upon the NAS panel's recommendations will yield important insights about poverty among the older population. PMID- 10732368 TI - Using data for couples to project the distributional effects of changes in Social Security policy. AB - Under Social Security program rules, the aged receive Social Security benefits either as retired workers, spouses, divorced spouses, or widow(er)s. Retired worker benefits are paid to workers who have 40 quarters of coverage over their lives. Auxiliary benefits are paid to spouses, divorced spouses, and widow(er)s of retired workers. Spouse benefits are computed using the earnings history of the current spouse for individuals who are married when they apply for benefits. Divorced spouse and widow(er) benefits are computed using the earnings history of the ex-spouse or deceased spouse with the highest PIA. A large number of retired women are entitled to auxiliary benefits. Some women receive only auxiliary benefits, while the majority of women have their retired-worker benefit supplemented by auxiliary benefits. Because the level of Social Security benefits can reflect the relative lifetime earnings of both spouses, as a couple, using individual data to estimate Social Security benefits will tend to underestimate actual benefits, particularly for women. However, detailed data for couples are often difficult to obtain. There is currently no known single data source that includes both marital and earnings history information. As a result, many researchers resort to estimating Social Security benefits using individual data or aggregate data, such as the average earnings of men and women. The Social Security Administration's Office of Research, Evaluation, and Statistics, with substantial assistance from the Brookings Institution, the Urban Institute, and the RAND Corporation, is developing a model that overcomes this problem by using the marital and earnings histories of both marital partners to estimate Social Security benefits. The Modeling Income in the Near Term (MINT) model projects retirement income (Social Security benefits, pension income, asset income, and earnings of working beneficiaries) from 1997 through 2031 for current and future Social Security beneficiaries using a unique data source--the Survey of Income and Program Participation (SIPP)--matched to Social Security Administration records. Using MINT data, this article establishes the importance of using data for couples rather than individuals by examining the impact of changing Social Security benefits to reflect 40 years of lifetime earnings rather than the 35 years required under current law. We compare the effect of this policy change on married women by estimating their benefits with data for couples and with individual data. Results indicate that: Using individual data overestimates the projected reduction in retirement benefits brought about by the policy change and makes the effects on women look more severe than they actually are. Because older birth cohorts are more likely than younger cohorts to receive auxiliary benefits based on their husbands' average lifetime earnings, the bias created by using individual data is projected to be much larger for older cohorts than for younger cohorts. This article emphasizes the importance of using data for couples to estimate Social Security benefits, particularly for women. Although our focus is on married women, using data for couples is just as important for calculating the retirement benefits of divorced and widowed individuals. For individuals who are divorced or widowed at retirement, their Social Security benefits are based on their own earnings history, as well as the earnings histories of each of their previous spouses. PMID- 10732369 TI - Characteristics of individuals with integrated pensions. AB - Employer pensions that integrate benefits with Social Security have been the focus of relatively little research. Since changes in Social Security benefit levels and other program characteristics can affect the benefit levels and other features of integrated pension plans, it is important to know who is covered by these plans. This article examines the characteristics of workers covered by integrated pension plans, compared to those with nonintegrated plans and those with no pension coverage. Integrated pension plans are those that explicitly adjust their benefit structure to help compensate for the employer's contributions to the Social Security program. There are two basic integration methods used by defined benefit (DB) plans. The offset method causes a reduction in employer pension benefits by up to half of the Social Security retirement benefit; the excess rate method is characterized by an accrual rate that is lower for earnings below the Social Security taxable maximum than above it. Defined contribution (DC) pension plans can be integrated along the lines of the excess rate method. To date, research on integrated pensions has focused on plan characteristics, as reported to the Bureau of Labor Statistics (BLS) through its Employee Benefits Survey (EBS). This research has examined the prevalence of integration among full-time, private sector workers by industry, firm size, and broad occupational categories. However, because the EBS provides virtually no data on worker characteristics, analyses of the effects of pension integration on retirement benefits have used hypothetical workers, varying according to assumed levels of earnings and job tenure. This kind of analysis is not particularly helpful in examining the potential effects of changes in the Social Security program on workers' pension benefits. However, data on pension integration at the individual level are available, most recently from the Health and Retirement Study (HRS), a nationally representative survey of individuals aged 51-61 in 1992. This dataset provides the basis for the analysis presented here. The following are some of the major findings from this analysis. The incidence of pension integration in the HRS sample is 32 percent of all workers with a pension (14 percent of all workers). The HRS can also identify integrated DC plans, a statistic that is not available from BLS data. The rate of integration for workers with only DC plans is 8 percent. After controlling for other variables, several socio-demographic characteristics are significantly related to the incidence of integration. The probability of having an integrated pension is 4.6 percentage points less for men compared to women. Non-Hispanic blacks are 6.4 percentage points less likely than non-Hispanic whites to have integrated pensions. Union members are 14 percentage points less likely to have integrated pensions, while workers with less than a graduate level education are at least 15 percentage points more likely to have a pension that is integrated. Some earnings and pension characteristics are also significantly correlated with pension integration. Earnings are positively related, with the probability of having an integrated pension increasing by 2 percentage points for an increase of $1,000 in annual pay. An even larger effect comes from earning at or above the Social Security taxable maximum. Workers at or above this income level are 10 percentage points more likely to have an integrated plan, but for those with more than one plan the probability of pension integration goes up by 13 percentage points. PMID- 10732370 TI - Improving return-to-work strategies in the United States disability programs, with analysis of program practices in Germany and Sweden. AB - The General Accounting Office (GAO) has made recommendations for improving the disability programs by citing practices that have been successful in Germany, Sweden, and the private sector. This issue is important in the United States because the number of disability beneficiaries is growing rapidly, program costs are increasing proportionately, and few disability recipients are leaving the disability rolls to resume work activity. GAO points out that the estimated lifetime savings for removing an additional 1 percent of the disabled beneficiaries from the rolls of the Disability Insurance (DI) and the Supplemental Security Income (SSI) programs each year will ultimately reach $3.0 billion. GAO cites three specific practices as showing the most promise for returning the disabled to work. They are (1) intervening as soon as possible after a disabling event to promote and facilitate return to work, (2) identifying and providing necessary return-to-work assistance and managing cases to achieve return-to-work goals, and (3) structuring cash and health benefits to encourage people with disabilities to return to work. This article examines these suggestions to improve the rate of rehabilitation of disabled workers using research by experts on return-to-work practices in Germany, Sweden, and the United States. Experts caution that any consideration of borrowing practices from other countries needs to take into account the unique economic, social, and political elements in each country. Although other countries appear to be very successful in their rehabilitation programs, practices that are successful in one country may not necessarily work well in another. Countries have different definitions of disability and payment structures. The existence of temporary and partial awards in Germany and Sweden may ensure a number of easily rehabilitated individuals, while the U.S. vocational rehabilitation (VR) agencies have been mandated to focus on only the most severely disabled individuals. Public expenditures for vocational rehabilitation, work for the disabled, and disability benefits are much higher as a percentage of gross domestic product in Germany and Sweden than they are in the United States. Compared with the United States, Germany spent twice as much for VR, and Sweden spent 2.6 times more. Impediments to GAO's suggestions include divergent goals of the Social Security program and VR agencies, lack of availability of VR services, the timing of VR referral (which is significantly later than the onset of the disability), and little incentive for return to work built into the payment structure. The Work Incentives Improvement Act of 1999 is currently being considered by a Congressional conference committee. The bill would establish a Ticket to Work and Self-Sufficiency program and would require or authorize the Social Security Administration to demonstrate and evaluate different ways of encouraging return to work. In designing these demonstrations, early intervention after a potentially disabling illness or injury is an approach that merits serious attention. PMID- 10732371 TI - Who is "62 enough"? Identifying respondents eligible for Social Security early retirement benefits in the Health and Retirement Study. AB - Workers are not instantly eligible for Social Security retirement benefits on their 62nd birthdays, nor can they receive benefits in the month they turn 62. This note discusses how well researchers can do using data from the Health and Retirement Study (HRS) to identify respondents old enough to receive and report early Social Security retirement benefits. It shows that only some workers aged 62 at the time of an HRS interview will be "62 enough" to have received a Social Security benefit and reported it in the survey. In general, workers become eligible for a retirement benefit the month after they turn 62, and they may receive their first payment the month after that. Until recently, payments were received very early in the month, but in mid-1997 and later, the Social Security Administration (SSA) staggered benefit payments over the course of a month. Therefore, many beneficiaries will not be able to report the receipt of their first benefit payment until the third month after their birthday in more recent HRS interviews. This note describes the best approach for approximating the pool of HRS respondents who are old enough to have reported the receipt of their first retirement benefit. It then applies the procedure to an analysis by Burkhauser, Couch, and Phillips, who used the 1994 HRS data to distinguish between those who took early retirement benefits upon turning 62 and those who postponed the receipt of benefits. Because these authors did not provide for respondents who were not "62 enough" to receive a benefit at the time of the interview, they understated the proportion of respondents who took retirement benefits at age 62. PMID- 10732372 TI - [Surgery for Barrett esophagus]. PMID- 10732373 TI - [A case of leiomyosarcoma of Meckel's diverticulum]. AB - The authors report a case of mesenchymal tumor (leiomyosarcoma) arising in a Meckel's diverticulum, with an history of digestive bleeding. Different kinds of Meckel's diverticulum tumors, their symptomatology and diagnostic difficulties are then discussed. PMID- 10732374 TI - [Ileal endometriosis. A clinical case and a review of the literature]. AB - Ileal localization of intestinal endometriosis is not extremely rare, but there are always problems related to the preoperative diagnosis. Symptoms of intestinal endometriosis offer often problems to the surgeon in the differential diagnosis with many more pregnant illnesses like appendicitis, diverticulitis, inflammatory bowel diseases and abdominal neoplasms. The authors report a case of endometriosis of the ileum clinically mimicking Crohn's disease. Crohn's disease of the ileum is the most frequently reported illness that is considered in the differential diagnosis. The review of the literature shows that a correct preoperative diagnosis of endometriosis of the ileum is very seldom done. All common diagnostic tools are not able to resolve diagnostic doubts. Also during explorative surgery the diagnosis of endometriosis is not easy. The authors conclude that endometriosis of the ileum has to be more often considered as possible cause of common intestinal symptoms mimicking inflammatory bowel disease and that this illness induces to reevaluate the importance for a correct diagnosis of an accurate clinical history. PMID- 10732375 TI - [Intestinal occlusion due to a giant lipoma of the cecum]. AB - Lipomas of the colon are localised in 90% of cases at submucous level, they are usually solitary and may be sessile or pedunculated. They are almost always asymptomatic; only when they are of a certain size they become manifest, causing alterations of the alveus, rectorrhagia, abdominal pain or intestinal transit alteration. They are often discovered by chance during endoscopic or radiological examination. PMID- 10732376 TI - [Accidents in laparoscopic surgery: Mirizzi's syndrome]. AB - It's still much discussed if Mirizzi syndrome must be operated in laparoscopy. Many authors stress the importance to make laparoscopy but others refuse absolutely such possibility and they only work in the classic manner. Nobody has still described the injury of right hepatic duct for Mirizzi syndrome Csendes type I during laparoscopic cholecystectomy. The authors report such a case that has been converted in the classic manner due to the importance of the injury for unknown Mirizzi syndrome. PMID- 10732377 TI - [The inferior nonrecurrent laryngeal nerve: a report of 7 cases observed since 1987]. AB - The non-recurrent inferior laryngeal nerve is a rare anomaly (0.34% on th right side, extremely rare on the left side) that increases the risk of damage to the nerve during thyroid and parathyroid surgery. Seven cases of non-recurrent laryngeal nerve are reported, emphasizing the diagnostic accuracy of angio-MR for the anatomical identification of the vascular anomaly that invariably occurs with the nerve malformation. PMID- 10732378 TI - [The chance finding of gallbladder carcinoma during cholecystectomy for long-term symptomatic lithiasis. A clinical case]. AB - The case of a patient who underwent conventional cholecystectomy for gallbladder adenocarcinoma is reported. Gallbladder carcinoma is rare, but since laparoscopic cholecystectomy has quickly emerged as a popular alternative to open cholecystectomy an increasing number of gallbladder carcinomas are discovered. Most of the time it is detected as an incidental tumor, the patient being with no or few symptoms and often the lesion too far advanced for any type of curative surgery. The most important questions related to the therapy are focused and a partial review of the literature is made. PMID- 10732379 TI - [Agenesis of the gallbladder]. AB - The authors report a case of agenesis of gallbladder associated with lithiasis of common bile duct. A review of the literature confirms the rarity of this congenital malformation. In 40-60% of cases it is associated with other gastrointestinal, skeletal, cardiovascular and genitourinary malformations. In 20 50% of cases it can be associated with common bile duct stones. Patients are asymptomatic in the majority of cases. A preoperative diagnosis is difficult despite of high accuracy of the modern diagnostic imaging techniques. PMID- 10732380 TI - [A new case of hepatic adenomatosis. A critical review of the literature]. AB - Liver adenomatosis is a rare disease--only 22 cases are previously described- characterized by multiple adenomas (more than ten) in a normal parenchyma. After a literature review the authors cannot exclude a link between the estroprogestatives use and the development of adenomatosis. Most important risks are bleeding and development of new adenomas, after the operation. On the basis of an extensive literature review, diagnosis features, treatment and follow-up are discussed. The aim of this report is to describe a case of multiple adenomas in a 38 years old housewife treated for seven years with oral contraceptives, admitted to hospital for spontaneous subcapsular haemorrhage due to rupture of adenomas, macroscopically considered suggestive of angiomas during the operation. Right lobectomy was performed and microscopic examination diagnosed multiple adenomas (liver adenomatosis). A repeated CT scan eight months later showed more than three new nodules in the liver. A new right lobectomy and tumorectomy of the nodules in III liver segment (remained after the first operation for the erroneous diagnosis) was performed and over a period of 20 months the patient remains in excellent condition. PMID- 10732381 TI - [Lung metastases from follicular thyroid cancer: a report of a clinical case and a review of the literature]. AB - The authors report a case of follicular thyroid cancer in "hot nodule" with micronodular synchronous lung metastases. The treatment consisted in TT associated with radioiodine therapy at the dose of 170 mCi with remission of cancer after six months of follow-up. Follicular variant is a prognostic unfavorable factor such as synchronous or metachronous pulmonary metastases. In those patients prognostic unfavorable variants are also age over 40 years, a poor radiofixation capacity, and a lesion of diameter up 1 cm. A strictly instrumental follow-up is mandatory to obtain diagnosis in a subclinical stage. In such cases the radiometabolic therapy agree to a remission of cancer with high survival after many years. TT yet represents a "minimal" surgical treatment for an accurate radioisotopic diagnosis. PMID- 10732382 TI - [Differential diagnostic problems between nodular fasciitis and soft-tissue sarcomas. A review of the literature and a case report]. AB - After a review of literature and the report of a case with intrathoracic location, the authors consider present criteria for a correct diagnosis of low grade soft tissue sarcoma, when the possibility of nodular fasciitis is under evaluation. Nodular fasciitis is a benign pathology up to day not well characterised from the clinical and pathological point of view. The authors conclude that differential diagnosis between low grade soft tissue sarcoma and nodular fasciitis is frequently possible only on the basis of clinical course evaluation; otherwise the management may be inadequate. PMID- 10732383 TI - [Double-breasted alloplasty in the treatment of a midline laparocele]. AB - The authors describe a new original surgical technique in repairing midline incisional hernias with polypropylene mesh, considering technical and functional advantages. Twenty-six cases are presented as well as early and late postoperative complications after 12 months follow-up. PMID- 10732384 TI - [Facial rejuvenation with chemical peeling using phenol (Exoderm)]. AB - The authors report their personal experience in the treatment of the resurfacing by means of phenol chemical peeling using a new solution described by Dr. Yoram Fintsi and called Exoderm. They report dressing technique and possible contraindications as well. PMID- 10732385 TI - [Surgical indications in the phlebopathic patient. The role of air plethysmography]. AB - A previous history of deep venous thrombosis (DVT) must be carefully evaluated in every patient affected by chronic venous insufficiency (CVI); the assessment of deep venous system patency is the goal of preoperative diagnosis. Air plethysmography examination allows an outpatient noninvasive testing of calf pump function and deep venous outflow. The authors report the results of a study on 37 patients (40 limbs) with a previous history of DVT, suffering from CVI; in 18 limbs air plethysmography showed a marked increase of calf volume on test exercise, suggesting residual venous obstruction. The authors believe that air plethysmography examination is mandatory before operation in patients with a previous history of DVT. PMID- 10732387 TI - In defense of publishing research findings in JAOA. PMID- 10732386 TI - [Angioneurosis due to vibrating tools with special reference to plethysmographic and Doppler vascular diagnosis]. AB - Angioneurosis, together with bones and joints disorders, represents the most frequent clinical syndrome due to vibrating instruments. The authors report a general presentation of the disease, particularly focused on indications, usefulness and methods of plethysmography and Doppler C.W., as noninvasive diagnostic procedures. PMID- 10732388 TI - Paradox answered. PMID- 10732389 TI - Physician recalls unforgettable evening in her early career. PMID- 10732390 TI - Importance of empiricism in medicine. PMID- 10732391 TI - Beyond MEDLINE. PMID- 10732392 TI - Benign prostatic hyperplasia treated with saw palmetto: a literature search and an experimental case study. AB - European physicians treat benign prostatic hyperplasia (BPH) with saw palmetto extract (SPE), while American physicians generally disregard SPE because "research is lacking." The authors investigated this discrepancy with a literature search and a clinical trial. The literature search began with MEDLINE, then expanded to "alternative" databases, including AGRICOLA, EMBASE, IBIS, and Cochrane, plus a manual search of unindexed herbal journals. The clinical trial was an experimental case study in which a 67-year-old man with symptomatic BPH was randomly administered SPE (160 mg standardized extract twice daily) or placebo. Outcome measures included the American Urological Association Symptom Index (AUASI), serum prostate-specific antigen, and prostate volume. Our expanded literature search revealed 58 clinical trials, whereas MEDLINE yielded only 19 clinical trials, or 33% of the total. Our clinical trial measured a baseline AUASI score of 20, which improved to 7 after unblinded administration of SPE. Subsequent double-blinded placebo produced a score of 14, and final single blinded allotment of SPE produced a score of 11. Prostate-specific antigen was 10.3 ng/mL at baseline and 10.7 ng/mL at trial's conclusion. Baseline prostatic volume was 92 mL, and end volume was 75 mL. In conclusion, MEDLINE proved inadequate as a stand-alone search engine for locating information about an herbal medicine. Our experimental case study, similar to N = 1 research methodology, proved suitable for clinical evaluation of an herbal medicine in a rural private practice. SPE improved the patient's BPH. Unstandardized look-alike herbs may act as nontherapeutic placebos and may undermine consumer confidence in herbal medicine. PMID- 10732393 TI - Reiter's syndrome and reactive arthritis. AB - Reactive arthritis is an infection-induced systemic illness characterized by an inflammatory synovitis from which no viable microorganisms can be cultured. The paradigm of reactive arthritis is an infectious trigger that occurs in a genetically susceptible individual in whom the immune system continues to be stimulated or "react" to an ongoing or cleared infection, resulting in intermittent exacerbations of a variety of immune-mediated signs and symptoms. Although the terms Reiter's syndrome and reactive arthritis are sometimes used interchangeably, Reiter's syndrome is actually the complete clinical triad of urethritis, conjunctivitis, and arthritis. Some patients will never fulfill all three components of Reiter's triad, however, and although the term incomplete Reiter's syndrome is often used in such cases, the authors believe that the term reactive arthritis should be used, as it is less confusing and is inclusive of any extra-articular manifestations. PMID- 10732394 TI - The Comprehensive Osteopathic Medical Licensing Examination, COMLEX-USA: a new paradigm in testing and evaluation. AB - Medical licensure in the United States demands a dynamic and current means to evaluate the competency of physicians seeking to practice medicine. A systematic measuring tool is required--one that is based on actual patient encounters and how physicians should apply their knowledge and skills to the clinical setting according to their level of training and professional development. Osteopathic physicians have a distinctive approach to healthcare, applying the biopsychosocial model with emphasis on the neuromusculoskeletal system. A component of this distinctive approach is a high level of knowledge and skill in the application of osteopathic manipulative treatment. Developed by the National Board of Osteopathic Medical Examiners, COMPLEX-USA is the new sequential three level examination process for osteopathic medical licensure in the United States. The examination process is interdisciplinary and highly clinical, with even basic science components tested within a clinical context. Examination content is based on wide expert consensus and data consistent with osteopathic medical education, training, and practice. Its design is a novel multidimensional structure that emphasizes clinical problem-solving skills and osteopathic principles and practice within the context of life cycle, gender, ethnicity, and points of service. Design schemata and blueprints are included along with descriptions of strategic research and development. COMPLEX-USA represents the most appropriate pathway for initial licensure for a distinctive and unique professional: the osteopathic physician in the United States. PMID- 10732395 TI - Passing of the "Old Doctor". 1918. PMID- 10732396 TI - Gastroesophageal reflux disease update for the primary care physician. AB - Gastroesophageal reflux disease (GERD) is one of the most common ailments that can decrease quality of life to below that of patients with congestive heart failure. Patients can present with typical, atypical, or serious symptoms, either alone or in combination. History, esophagogastroduodenoscopy, and 24-hour pH monitoring can help with the diagnosis; but all are not needed for each patient. This clinical review will help primary care physicians to make a rapid diagnosis, guide subsequent treatment, and indicate when to order additional testing or referral, or both. PMID- 10732397 TI - Current management of Helicobacter pylori infection. AB - Helicobacter pylori infection continues to be one of the hottest topics in gastroenterology, challenging the primary care physician daily with the clinical application of its new developments and frequency of involvement in differential diagnosis. This article offers the primary care physician a practical approach to the dyspeptic office patient that emphasizes the differential diagnosis of H pylori. Included is a strategy for rapid patient evaluation through key questions in the history, simple maneuvers in the physical examination, and a logical approach to testing and therapy based on the latest literature. The recommendations for all these office-based steps are designed to be efficient, cost-effective, and clinically relevant. PMID- 10732398 TI - NSAIDs and the gastrointestinal tract: an update on recent progress. AB - This article outlines the mechanisms involved in specific cyclooxygenase-1 (COX 1) and cyclooxygenase-2 (COX-2) inhibition as they relate to the safety of nonsteroidal anti-inflammatory drug (NSAID) use. New advancements in COX-2 technology appear to improve the safety profile of NSAID therapy. Gastroenterologic issues such as ulcer disease and liver disease as related to these novel new medications are reviewed. PMID- 10732399 TI - Inflammatory bowel disease: strategies for assessment, treatment, and prevention of relapse. AB - Inflammatory bowel disease is a chronic, lifelong disorder characterized by alternating periods of flare-ups and clinical remission. The majority of patients experience relapses several times a year. Prevention and treatment of these relapses are a major challenge to the practicing physician. Therefore, this article provides strategies that the primary care physician can use in the diagnosis and treatment of inflammatory bowel disease and prevention of relapses. PMID- 10732400 TI - [Neogenesis of beta cells and islet formation]. PMID- 10732401 TI - [Non-insulin dependent diabetes and apoptosis of beta-cells]. PMID- 10732402 TI - [Growth and regeneration potential abnormalities of pancreatic beta-cells in GK rats, NIDDM mellitus model]. PMID- 10732403 TI - [Death and survival mechanisms in beta-cells]. PMID- 10732404 TI - [Apollinaire Bouchardat Award 1999. Development of endocrine pancreas cells]. PMID- 10732405 TI - [Biochemical bases of aging: does glucose play a role?]. PMID- 10732406 TI - [Is aging associated with a diminution of insulin sensitivity? Roles of IGF1 and dehydroepiandrosterone]. PMID- 10732407 TI - [Diabetes in the elderly individual: point of view of the diabetologist]. PMID- 10732408 TI - [Diabetes in the elderly individual: point of view of the geriatrician]. PMID- 10732409 TI - [Diabetes in the elderly patient: point of view of the nephrologist]. PMID- 10732411 TI - [Role of glucokinase (GK) in the maintenance of glucose homeostasis. Specific disruption of the gene by the Cre-loxP technique]. PMID- 10732410 TI - [Insulin and its receptor: lessons learned from the disruption of their gene in mice]. PMID- 10732412 TI - [Insulin signalization and molecular mechanisms of insulin resistance]. PMID- 10732413 TI - [Management of retrograde ejaculation]. PMID- 10732414 TI - [My approach to increased liver enzymes in the diabetic patient]. PMID- 10732415 TI - [Which visual aid for the visually impaired]. PMID- 10732416 TI - [My approach to the management of a type I diabetic treated with short-acting insulin analogue]. PMID- 10732417 TI - [Isolated hypertriglyceridemia in a diabetic patient]. PMID- 10732418 TI - [Insulin-like effects of vanadium and other metallic ions. Potential use in the treatment of diabetes]. PMID- 10732419 TI - [Thiazolidinediones]. PMID- 10732420 TI - [C-peptide, new discoveries and therapeutic implications]. PMID- 10732422 TI - 6th Keio International Symposium for Life Sciences and Medicine. Ischemic Blood Flow in the Brain. Japan, November 24-27, 1999. Abstracts. PMID- 10732421 TI - [Metformin in the UKPDS study. Metabolic and vascular results]. PMID- 10732423 TI - Safe motherhood by AD 3000? PMID- 10732424 TI - Sentinel node and breast cancer: a new paradigm? PMID- 10732425 TI - Use of the logistic organ dysfunction system to study mortality in an Indian intensive care unit. AB - BACKGROUND: Mortality in Indian intensive care units has not been well studied. Scoring systems are used to predict mortality of patients admitted to such units. Some scoring systems predict hospital mortality while others predict mortality in intensive care units. We used the logistic organ dysfunction system to study the hospital and intensive care unit mortalities in our intensive care unit. METHODS: We prospectively studied 527 consecutively admitted patients in 1997 to the medical intensive care unit in St John's Medical College Hospital, Bangalore. The outcomes studied were death in hospital and death in the intensive care unit. Using standardized mortality ratios, we compared our observed hospital and intensive care unit mortalities with the hospital mortality predicted by the logistic organ dysfunction system. RESULTS: The standardized mortality ratios for hospital deaths was 1.3 with a confidence interval of 1.17-1.49 and for intensive care unit deaths it was 1.0 with a confidence interval of 0.89-1.18. The hospital mortality rates in our setting are significantly higher (p < 0.05) than the predicted hospital mortality rates of the published western model for intensive care unit patients. The intensive care unit mortality rates are not significantly different from the predicted hospital mortality rates of the published western model for intensive care unit patients. CONCLUSION: Our intensive care unit mortality rate is comparable to the western hospital mortality rate. However, after transfer of patients out of the unit, the hospital mortality is higher. PMID- 10732426 TI - Screening for chronic impairments using medical interns in rural Haryana, India. AB - BACKGROUND: With the increase in life expectancy, prevalence of impairments and disabilities are expected to increase in India. However, there have been very few studies to estimate the magnitude of the problem in rural India. This is essential, if appropriate rehabilitation services are to be planned in the country. METHODS: The study was done in the rural field practice area of the All India Institute of Medical Sciences at Ballabgarh, Haryana. The survey was conducted by successive batches of interns posted at Ballabgarh as a part of their compulsory rotating internship programme. The diagnostic criteria were based on history and simple clinical examination done at the domiciliary level. RESULTS: A total population of 25,509 in twelve villages were screened. The total impairment rate was 5.4% with no significant men/women difference. The prevalence of physical impairment was 4.7 per 1000 population. The prevalence of corneal opacity in children below 15 years of age was 4.7 per 1000. Prevalence of cataract was almost 35% in the population over 60 years of age and 15% in the population between 45 to 60 years. Auditory impairment was 19.6 per 1000 as ascertained by history. Three-fourths of this was conductive deafness and was found mainly in people above 60 years of age. CONCLUSION: Utilizing the rural field practice areas of medical colleges for collection of data on issues of national health importance would not only strengthen the health system in the country but also improve medical education. There is a need for a comprehensive preventive, promotive, curative and rehabilitative approach to disabilities in India. PMID- 10732427 TI - Erythrocyte indices for discriminating thalassaemic and non-thalassaemic microcytosis in Indians. AB - BACKGROUND: Microcytosis is a common red cell change seen in anaemias of varied aetiology. These include iron deficiency, thalassaemia, chronic disease and sideroblastic anaemias. The microcytosis of heterozygous beta-thalassaemia needs to be distinguished from non-thalassaemic microcytosis for its role in thalassaemia control. Red cell indices derived from automated red cell analysers have been used to discriminate between microcytic patients with a high probability of thalassaemia minor from those with a low probability. There is a controversy on the choice of red cell indices to be used and the cut-off values for this distinction, because the prevalence of iron deficiency as a cause of non thalassaemic microcytosis is variable. Since no Indian study using receiver operator characteristic (ROC) curves was available to determine the above, we conducted this study. METHODS: Red cell indices (mean corpuscular volume, total red blood cell count, red cell distribution width, linear discriminant function), serum iron, total iron binding capacity and haemoglobin A2 were estimated in 640 adults with microcytosis (mean corpuscular volume 80 fl). The ROC curves were plotted in all. RESULTS: Total red blood cell count was observed to be the most efficient single test followed by linear discriminant function and Bessman index. Mean corpuscular volume had the least efficacy. The cut-off values obtained for the Indian population were mean corpuscular volume < or = 76 fl, total red blood cell count > or = 4.9 x 10(12)/L and red cell distribution width > or = 18% and a positive linear discriminant function. These were different from those observed in the West, possibly because of the high prevalence of iron deficiency in India. CONCLUSION: In countries with a high prevalence of iron deficiency, cut-off values for red cell indices should be recalculated using ROC curves. PMID- 10732428 TI - Immunogenicity and reactogenicity of an inactivated hepatitis A vaccine in Indian adults. AB - BACKGROUND: In India, a possible decrease in the prevalence of anti-HAV (hepatitis A virus) antibodies in adults has increased their susceptibility to HAV infection. We evaluated the immunogenicity and reactogenicity of an inactivated hepatitis A vaccine administered in a 3-injection protocol. METHODS: Thirty-five healthy adult volunteers, seronegative for anti-HAV IgG, were administered 720 ELISA units/ml of the inactivated hepatitis A vaccine intramuscularly at days 0, 30 and 180. Anti-HAV IgG was determined at days 30, 60, 90 and 180 to assess the efficacy of the vaccine and adverse reactions were noted to evaluate its reactogenicity. RESULTS: The mean (SD) age of the volunteers was 33.1 (12.3) years and the man:woman ratio was 19:16. An overall seroprotection of 37.2% (13/35) was obtained at day 30, 57.1% (20/35) at day 60 and 85.7% (30/35) at day 90. By day 180, all the vaccinees (35/35; 100%) achieved protective seroconversion. The vaccine in general was well tolerated and no serious side-effects were observed. Only 8.6% (3/35) of subjects developed minor self-resolving adverse reactions such as local pain, erythema and/or low-grade fever. CONCLUSIONS: The inactivated hepatitis A vaccine in a three-injection protocol (0, 30 and 120 days) is safe, well tolerated and highly immunogenic in adult Indian subjects. PMID- 10732429 TI - Biomaterials: an overview. PMID- 10732430 TI - The impact of new technologies on vaccines. AB - Vast changes are taking place in vaccinology consequent to the introduction of new technologies. Amongst the vaccines included in the Expanded Programme of Immunization (EPI), the pertussis vaccine has been replaced by acellular purified fractions devoid of side-effects. Non-pathogenic but immunogenic mutants of tetanus and diptheria toxins are likely to replace the toxoids. An effective vaccine against hepatitis B prepared by recombinant technology is in large-scale use. Conjugated vaccines against Haemophilus influenzae b, S. pneumococcus and meningococcus are now available, as also vaccines against mumps, rubella and measles. Combination vaccines have been devised to limit the number of injections. Vaccine delivery systems have been developed to deliver multiple doses of the vaccine at a single contact point. A genetically-engineered oral vaccine for typhoid imparts better and longer duration of immunity. Oral vaccines for cholera and other enteric infections are under clinical trials. The nose as a route for immunization is showing promise for mucosal immunity and for anti inflammatory experimental vaccines against multiple sclerosis and insulin dependent diabetes mellitus. The range of vaccines has expanded to include pathogens resident in the body such as Helicobacter pylori (duodenal ulcer), S. mutans (dental caries), and human papilloma virus (carcinoma of the cervix). An important progress is the recognition that DNA alone can constitute the vaccines, inducing both humoral and cell-mediated immune responses. A large number of DNA vaccines have been made and shown interesting results in experimental animals. Live recombinant vaccines against rabies and rinderpest have proven to be highly effective for controlling these infections in the field, and those for AIDS are under clinical trial. Potent adjuvants have added to the efficacy of the vaccines. New technologies have emerged to 'humanize' mouse monoclonals by genetic engineering and express these efficiently in plants. These recombinant antibodies are opening out an era of highly specific and safe therapeutic interventions. Human recombinant antibodies would be invaluable for treating patients with terminal tetanus and rabies. Antibodies are already in use for treatment of cancer, rheumatoid arthritis and allergies. An advantage of preformed antibodies directed at a defined target and given in adequate amounts is the certainty of efficacy in every recipient, in contrast to vaccines, where the quality and quantum of immune response varies from individual to individual. PMID- 10732431 TI - Laparoscopic appendicectomy. AB - The clinical diagnosis of appendicular pathology has an unacceptably high false positive rate, particularly in premenopausal women. Diagnostic laparoscopy is a significant aid towards accurate diagnosis. Laparoscopic appendicectomy can be safely and economically performed as a logical sequel to diagnostic laparoscopy. We describe our method of safe and cost-effective laparoscopic appendicectomy. PMID- 10732432 TI - Laboratory investigations in rheumatology: a practical approach. PMID- 10732433 TI - Of care and care-givers. PMID- 10732434 TI - The western world pandemic of obesity--how is Africa faring? PMID- 10732435 TI - Caesarean section deliveries on the rise in Kerala. PMID- 10732436 TI - Ocular toxicity--a rare side-effect of cyclophosphamide. PMID- 10732437 TI - Indicators of iodine deficiency disorders. PMID- 10732438 TI - Polio eradication in India: are we on the correct path? PMID- 10732439 TI - Mortality due to adverse drug reactions in a large general hospital. PMID- 10732440 TI - Report of Brain Tumor Registry of Japan (1969-1993). PMID- 10732441 TI - Ring opening photoreactions of cytosine and uracil with ethylamine. AB - The photochemical reactions of cytosine (Cyt) and uracil (Ura) with ethylamine, an analog of the side chain of the amino acid lysine, have been studied. After irradiation of Cyt in aqueous ethylamine at lambda = 254 nm, N-(N' ethylcarbamoyl)-3-aminoacrylamidine (Ia) and N-(N'-ethylcarbamoyl)-3 ethylaminoacrylamidine (Ib) were isolated as products, while irradiation of Ura gave N-(N'-ethylcarbamoyl)-3-aminoacrylamide (IIa) and N-(N'-ethylcarbamoyl)-3 ethylaminoacrylamide (IIb) as products. Studies in which Ia and IIa were incubated with ethylamine at various pH values indicate that Ib and IIb are secondary products produced via thermal reactions of Ia and IIa with ethylamine. Heating of Ia and Ib leads to ring closure with the resultant formation of 1 ethylcytosine; small amounts of 1-ethyluracil are also produced. Heating of IIa and IIb produces 1-ethyluracil as the sole product. Spectroscopic properties were determined for each of these opened ring products, as well as for N-(N' ethylcarbamoyl)-3-amino-2-methylacrylamidine (III) and N-(N'-ethylcarbamoyl)-3 amino-2-methylacrylamide (IV). Quantum yield measurements showed that Ia was formed with a phi of 1.6 x 10(-4) at pH 9.8, while phi for formation of IIa was 7.2 x 10(-4) at pH 11.5. A profile of the relative quantum yield for formation of Ia, determined as a function of pH, showed that the maximum quantum yield occurs at around pH 9.5; the analogous profile for IIa shows a maximum quantum yield at pH 11.3 and above. Acetone sensitization does not produce Ia in the Cyt ethylamine system, which indicates that the known triplet state of Cyt is not involved in reactions leading to this opened ring product. PMID- 10732442 TI - DNA damage induced by 4,6,8,9-tetramethyl-2H-furo[2,3-h]quinolin-2-one, a new furocoumarin analog: photochemical mechanisms. AB - Some photochemical and photobiological properties of 4,6,8,9-tetramethyl-2H furo[2,3-h]quinolin-2-one (HFQ) were studied in comparison with its isomer 1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2-one (FQ) and 8-methoxypsoralen (8 MOP). The HFQ photobinds to DNA forming furan-side monoadducts (MAHFQ) that have molecular structure very similar to those of FQ (MAFQ). Unlike MA8-MOP and MAFQ, MAHFQ no longer photoreact. The HFQ, like FQ, produces moderate amounts of singlet oxygen but no superoxide anions. The HFQ and FQ induce numbers of DNA protein cross-links (DPC), much more plentiful than those of 8-MOP (about two and seven times, respectively) but no interstrand cross-links. The mechanism of DPC formation was studied in vivo in mammalian cells by alkaline elution and in vitro using a new test mixing histones and DNA from calf thymus. The latter is a very useful technique for the double irradiation protocol. The DNA (or histones) are separately exposed to a first UVA dose in the presence of the sensitizer; then, after its unbound molecules have been removed, histones (or DNA) are added to assemble the chromatin-like complex that is irradiated again. According to in vitro and in vivo methods, DPC appear to be formed by FQ and 8-MOP by a biphotonic process that starts with monoadduct induction in DNA, followed by their conversion into DPC. In the resulting lesions, the sensitizer molecule forms a covalent bridge between the two macromolecules (DPC at length greater than zero). Instead, HFQ induces DPC by a monophotonic process; thus, HFQ is probably not a physical part of the bridge between DNA and proteins, which may be linked together directly, like DPC at zero length induced by UVC. PMID- 10732443 TI - DNA damage induced by 4,6,8,9-tetramethyl-2H-furo[2,3-h]quinolin-2-one, a new furocoumarin analog: biological consequences. AB - 4,6,8,9-Tetramethyl-2H-furo[2,3-h]quinolin-2-one (HFQ) and its isomer FQ (1,4,6,8 tetramethyl-2H-furo[2,3-h]quinolin-2-one) showed very strong antiproliferative activity in mammalian cells, about two times greater than 8-methoxypsoralen (8 MOP). Both compounds induced DNA-protein cross-links (DPC) but not interstrand cross-links. The FQ generated DPC in a biphotonic process, yielding a new kind of diadduct, whereas HFQ induced DPC by a monophotonic one, probably without its physical participation in the covalent bridge. These lesions gave different toxic responses. Sensitization of FQ led to extensive DNA fragmentation and to a number of chromosomal aberrations. Conversely, HFQ seemed to be completely inactive and 8-MOP gave intermediate results. A strict relationship between DPC formation and induction of chromosomal aberrations was observed. The HFQ did not induce light skin erythemas, whereas FQ was more phototoxic than 8-MOP, thus suggesting that FQ lesions, DPC in particular, may be implicated in skin phototoxicity. Ehrlich ascites cells, a transplantable mouse tumor, inactivated by furoquinolinone sensitization and injected into healthy mice, protected them from a successive challenge by viable tumor cells. This response appeared to be based on an immune mechanism. Comparable amounts of base substitution revertants were scored when testing furoquinolinones and 8-MOP in bacteria but no DPC were detected. This suggests that classic mutagenesis tests on bacteria are insufficient to give adequate information on furocoumarin genotoxicity. Given its features, HFQ can be regarded as an interesting new agent for psoralen plus UVA photochemotherapy and photopheresis. PMID- 10732444 TI - Polyethylene glycol matrix reduces the rates of photochemical and thermal release of nitric oxide from S-nitroso-N-acetylcysteine AB - S-nitrosothiols have many biological activities and may act as nitric oxide (NO) carriers and donors, prolonging NO half-life in vivo. In spite of their great potential as therapeutic agents, most S-nitrosothiols are too unstable to isolate. We have shown that the S-nitroso adduct of N-acetylcysteine (SNAC) can be synthesized directly in aqueous and polyethylene glycol (PEG) 400 matrix by using a reactive gaseous (NO/O2) mixture. Spectral monitoring of the S-N bond cleavage showed that SNAC, synthesized by this method, is relatively stable in nonbuffered aqueous solution at 25 degrees C in the dark and that its stability is greatly increased in PEG matrix, resulting in a 28-fold decrease in its initial rate of thermal decomposition. Irradiation with UV light (lambda = 333 nm) accelerated the rate of decomposition of SNAC to NO in both matrices, indicating that SNAC may find use for the photogeneration of NO. The quantum yield for SNAC decomposition decreased from 0.65 +/- 0.15 in aqueous solution to 0.047 +/- 0.005 in PEG 400 matrix. This increased stability in PEG matrix was assigned to a cage effect promoted by the PEG microenvironment that increases the rate of geminated radical pair recombination in the homolytic S-N bond cleavage process. This effect allowed for the storage of SNAC in PEG at -20 degrees C in the dark for more than 10 weeks with negligible decomposition. Such stabilization may represent a viable option for the synthesis, storage and handling of S nitrosothiol solutions for biomedical applications. PMID- 10732446 TI - On the photochromism of spiro AB - The recently synthesized spiro[cyclohexadiene-dihydroacridines] consisting of perpendicularly arranged aroylcyclohexadiene and N-methyl-dihydroacridine moieties were found to have photochromic properties. The reversible photoisomerization from the spiro compound toward a colored merocyanine caused by C-C bond cleavage in the cyclohexadiene was studied by stationary and time resolved measurements of their optical spectra. The course of the absorption under UV and visible irradiation, respectively, and HPLC analysis of the photoproducts result in the determination of excitation energy-dependent quantum yields for the merocyanine formation and, in reverse, the ring closure, as well as degradation. Whereas the thermal back reaction completely recovers the spiro compound (k approximately 6.8 x 10(-4) s-1, T = 22 degrees C), degradation of the merocyanine under irradiation at 480 nm has a probability of about 6%. Picosecond resolved measurements of the fluorescence and the transient absorption show that photoisomerization occurs via the first excited singlet state within 100 ps depending on the activation barrier. PMID- 10732445 TI - Spectroscopic properties of fluoroquinolone antibiotics and nanosecond solvation dynamics in aerosol-OT reverse micelles. AB - Among fluoroquinolone antibiotics, ofloxacin (OFL) and norfloxacin (NOR) have piperazinyl groups but flumequine (FLU) does not have this substitutent. The emission spectra of OFL and NOR are strong, broad structureless bands with large Stokes' shifts in water but the emission intensities are very weak in organic solvents. Thus we find that these compounds exist as different chemical species in various solvents. A continuous red shift in the emission bands for OFL and NOR is observed as the water concentration within the aerosol-OT (AOT; sodium 1,4 bis[2-ethylhexyl]sulfosuccinate) micelle increases or temperature of this solution rises. From the fluorescence anisotropy measurements of OFL and NOR, we assume the intramolecular charge transfer after excitation from the nitrogen of the piperazinyl group to the keto oxygen. Theoretical calculations further support this observation. Multifrequency phase and modulation experiments and time-resolved emission spectra clearly show the occurrence of intramolecular charge transfer and the subsequent nanosecond water reorganization around OFL or NOR in the AOT micelle. Upon increasing the water concentration within the AOT micelle, the relaxation rate increases because of the large amount of free water. The emission spectra of FLU do not exhibit any significant response to the physical properties of their environment. PMID- 10732447 TI - Interaction of the excited singlet state of neutral red with aromatic amines AB - Quenching of neutral red (NR; neutral form of the dye) fluorescence by a number of aromatic amines has been investigated in acetonitrile solutions. The bimolecular quenching constants (kq) obtained from steady-state and time-resolved measurements for a particular donor-acceptor pair are seen to be the same within experimental error. Correlation of the changes in the kq values with the oxidation potentials of the donors (amines) indicates that electron transfer (ET) is the mechanism operative in the present systems. Direct evidence for ET has been obtained from picosecond transient absorption studies on a suitable amine-NR pair. Experimentally determined kq values are seen to correlate well with the free energy changes (delta G0) for the ET reactions, within the frame-work of the Marcus outer sphere ET theroy. From the correlation between the experimentally determined and theoretically calculated kq values, it appears that solvent reorganization plays a major role in governing ET dynamics in the systems investigated. PMID- 10732448 TI - Hydroxyterephthalate as a fluorescent probe for hydroxyl radicals: application to hair melanin. AB - The known photoreactions of melanin include production of melanin free radicals and oxygen consumption. While qualitative descriptions of the intermediates and products of these processes have been published, no quantitative procedures have been reported that allow the convenient measurement of the products of oxygen reduction. We have used complementary fluorescence and electron spin resonance techniques to study free radical production from extracted hair melanin irradiated at wavelengths above 320 nm. As a comparison, sepia was also studied. Irradiation of aerated suspensions of melanin in the presence of terephthalic acid dianion (TA)++ gives rise to the characteristic fluorescence spectrum of the 2-hydroxyterephthalate ion (HTA, lambda e lambda = 315 nm, lambda em = 425 nm). Production of HTA has been studied as a function of time (at constant light flux) and in the presence of other substrates including hydroxyl radical sources and scavengers. The use of TA as a fluorescent probe can be conveniently adapted to other systems. PMID- 10732449 TI - Evaluation of the protective effect of sunscreens on in vitro reconstructed human skin exposed to UVB or UVA irradiation. AB - We have previously shown that skin reconstructed in vitro is a useful model to study the effects of UVB and UVA exposure. Wavelength-specific biological damage has been identified such as the formation of sunburn cells (SBC) and pyrimidine dimers after UVB irradiation and alterations of dermal fibroblasts after UVA exposure. These specific effects were selected to evaluate the protection afforded by two sunscreens after topical application on the skin surface. Simplified formulations having different absorption spectra but similar sun protection factors were used. One contained a classical UVB absorber, 2 ethylhexyl-p-methoxycinnamate. The other contained a broad-spectrum absorber called Mexoryl SX, characterized by its strong absorbing potency in the UVA range. Both filters were used at 5% in a simple water/oil vehicle. The evaluation of photoprotection on in vitro reconstructed skin revealed good efficiency for both preparations in preventing UVB-induced damage, as shown by SBC counting and pyrimidine dimer immunostaining. By contrast, only the Mexoryl SX-containing preparation was able to efficiently prevent UVA-specific damage such as dermal fibroblast disappearance. Our data further support the fact that skin reconstructed in vitro is a reliable system to evaluate the photoprotection provided by different sunscreens against specific UVB and UVA biological damage. PMID- 10732450 TI - P53-dependent UVB responsiveness of human keratinocytes can be altered by cultivation on cell cycle-arrested dermal fibroblasts. AB - In cultured human keratinocytes, the tumor suppressor p53 acts as a control element in the protective response to UVB radiation and is affected by a variety of factors linked to cellular adhesion and differentiation. Because keratinocytes within the epidermis are not a homogeneous population but differ in their proliferative capacity and differentiation status, we compared the UVB responsiveness of primary keratinocyte populations isolated from various skin biopsies using p53 expression as a marker for their sensitivity to UVB. Besides keratinocytes exhibiting a UVB dose- and time-dependent upregulation of p53, keratinocyte populations were detected with high p53 expression levels even without irradiation. Such keratinocytes did not regulate p53 expression in response to UVB. Furthermore their p53-mediated UVB response was influenced by cocultivation with human dermal fibroblasts (HDF) but not with cell cycle arrested human normal keratinocytes or HaCaT keratinocytes. When these cells were cultivated together with arrested HDF, they did not only reveal increased p53 expression levels after UVB treatment but also a more pronounced transcriptional activation of the p53 downstream target gene p21. These findings indicate that the UVB response of keratinocytes, specifically the activation of the tumor suppressor p53, is heterogeneous and can be affected by growth conditions. PMID- 10732451 TI - Characterization of the autofluorescence of polymorphonuclear leukocytes, mononuclear leukocytes and cervical epithelial cancer cells for improved spectroscopic discrimination of inflammation from dysplasia. AB - Fluorescence spectroscopy has the potential to improve the in vivo detection of intraepithelial neoplasias; however, the presence of inflammation can sometimes result in misclassifications. Inflammation is a common and important pathologic condition of epithelial tissues that can exist alone or in combination with neoplasia. It has not only been associated with the presence of cancer but also with the initiation of cancer by damage induced due to the oxidative activity of inflammatory cells. Microscopic examination of cervical biopsies has shown increased numbers of polymorphonuclear and mononuclear leukocytes in inflamed tissues mostly confined to the stroma. The purpose of this study was to characterize the fluorescence properties of human polymorpho- and mononuclear leukocytes and compare their fluorescence to that of cervical cancer cells. Human neutrophils were purified from peripheral blood and their fluorescence characterized over an excitation range of 250-550 nm. There are four notable excitation emission maxima: the tryptophan peak at 290 nm excitation, 330 nm emission; the NAD(P)H peak at 350 nm excitation, 450 nm emission, the FAD peak at 450 nm excitation, 530 nm emission and an unidentified peak at 500 nm excitation, 530 nm emission. Treatment of these peripheral blood neutrophils with 40 nM phorbol myristate acetate or with the chemotactic peptide formyl-Met-Leu Phe (1 M) demonstrated a significant increase in NAD(P)H fluorescence. Isolated mononuclear cells have similar emission peaks for tryptophan and NAD(P)H and a small broad peak at 450 nm excitation, 530 nm emission suggestive of FAD. Comparison of the fluorescence from leukocytes to epithelial cancer cell fluorescence has demonstrated the presence of these fluorophores in different quantities per cell. The most notable difference is the high level of tryptophan in cervical epithelial cancer cells, thus offering the potential for discrimination of inflammation. PMID- 10732452 TI - Time-dependent biodistribution of tetra(m-hydroxyphenyl)chlorin and benzoporphyrin derivative monoacid ring A in the hamster model: comparative fluorescence microscopy study. AB - The pharmacokinetics of the photosensitizer used play a key role in the understanding of the mechanism of photodynamic therapy-induced damage. Fluorescence microscopy was used to compare time-dependent biodistribution of tetra(m-hydroxyphenyl)chlorin (mTHPC) and benzoporphyrin derivative monoacid ring A (BPD-MA) in different hamster tissues, including an early, chemically induced, squamous cell carcinoma. Following injection of 0.5 mg/kg body weight of mTHPC and 2.0 mg/kg BPD-MA, groups of three animals were sacrificed at different time points and a series of fluorescence micrographs from different excised organs were analyzed. The highest fluorescence intensities of mTHPC were observed at 96 h for squamous epithelia and skin and at 48 h for smooth muscle. There is no real peak of BPD-MA fluorescence between 30 min and 3 h in the basal epithelial layers, fibroconnective tissue, muscles or blood vessels. At 4 h after injection, the fluorescence level of BPD-MA decreased and at 24 h it had returned to background level in all observed tissues. The significantly faster clearance of BPD-MA is the principal advantage as compared to mTHPC. However, similar localization patterns in different tissues with essentially vascular affinity represent a possible disadvantage for treating early malignancies with BPD-MA as compared to mTHPC, which is mainly localized in various epithelia. For both photosensitizers no significant selectivity between early squamous cell carcinoma and healthy mucosae is seen. Pharmacokinetic studies of different photosensitizers in an appropriate animal model are essential for selecting new generation photosensitizers with the most favorable localization for photodynamic therapy of early malignancies in hollow organs. PMID- 10732453 TI - Plasma membrane properties involved in the photodynamic efficacy of merocyanine 540 and tetrasulfonated aluminum phthalocyanine. AB - Merocyanine 540 (MC540)-mediated photodynamic damage to erythrocytes was strongly reduced when illumination was performed at pH 8.5 as compared to pH 7.4. This could be explained by high pH-mediated hyperpolarization of the erythrocyte membrane, resulting in decreased MC540 binding at pH 8.5. In accordance, the MC540-mediated photooxidation of open ghosts was not inhibited at pH 8.5. Photoinactivation of vesicular stomatitis virus (VSV) was not inhibited at pH 8.5. This suggests that illumination at increased pH could be an approach to protect red blood cells selectively against MC540-mediated virucidal phototreatment. With tetrasulfonated aluminum phthalocyanine (AIPcS4) as photosensitizer, damage to erythrocytes, open ghosts and VSV was decreased when illuminated at pH 8.5. A decreased singlet oxygen yield at high pH could be excluded. The AIPcS4-mediated photooxidation of fixed erythrocytes was strongly dependent on the cation concentration in the buffer, indicating that the surface potential may affect the efficacy of this photosensitizer. This study showed that altering the environment of the target could increase both the efficacy and the specificity of a photodynamic treatment. PMID- 10732454 TI - Stabilization of the optical tracer agent indocyanine green using noncovalent interactions. AB - Indocyanine green is a medically useful dye that absorbs and fluoresces in the near infrared and has been sporadically employed clinically as an optical tracer agent for liver function evaluation and cardiac output measurements. The poor stability of this dye in aqueous solution, especially at the high concentrations needed for bolus injection, has been a hindrance in clinical application. However, by using carefully chosen macromolecular additives, the stability of these aqueous dye solutions may be enhanced significantly. Such noncovalent binding between dye and carrier molecules was found to preserve substantially the dye in aqueous solutions for several weeks with no apparent changes in the measured in vivo biological properties. PMID- 10732455 TI - Monitoring of impending myocardial damage after pleuropneumonectomy and intraoperative photodynamic therapy for malignant pleural mesothelioma using biochemical markers. AB - In five patients who were treated for malignant pleural mesothelioma (MPM) with pleuropneumonectomy and intraoperative photodynamic therapy (IPDT), impending myocardial damage was monitored using ECG, the classical biochemical markers (creatine kinase [CK], total activity; CKMB, mass; and myoglobin), and the new cardiac markers troponin I (cTnI) and troponin T (cTnT). In the peroperative and postoperative period all classical markers were elevated, in contrast to cTnI and cTnT, because of the concomitant skeletal muscle damage. Sequential electrocardiogram monitoring showed no signs of myocardial damage. From this study in patients with MPM treated with pleuropneumonectomy and IPDT it can be concluded that measurement of cTnI and cTnT for the detection of myocardial damage is more suitable than measurement of the classical markers. PMID- 10732456 TI - [Main trends in the formation of population health in the Russian Federation, 1998]. PMID- 10732457 TI - [Morbidity of young children depending on conditions of rearing]. AB - Disease incidence among infants is analyzed from the consultation rate and data of medical check-ups of children at infant homes and day nurseries and of children brought up at home. The highest morbidity according to consultation rate was observed in infant homes: 4015.8 per 1000. However, the incidence of acute diseases notably decreased after a long stay of the infant in the collective body. Time course of disease incidence in other groups was different. The detected regularities should be taken into consideration when planning prophylactic measures. PMID- 10732458 TI - [Cesarean section as a means of prevention of child disability]. AB - Analysis of the results of operations performed at the Ural Institute of Maternal and Neonatal Health Protection in 1988-1998 showed that women with high-risk pregnancy are purposefully hospitalized at the Perinatal Center, and therefore high percentage of abdominal deliveries is justified, because this operation is aimed at prevention of disabling diseases of the central nervous system in newborns and infants. PMID- 10732459 TI - [Risk factors in sudden infant death syndrome]. AB - Preterm babies are at risk of sudden death. Determination of the risk factors and prediction of the probability of sudden death syndrome in infants will help the physicians in purposeful observation of the risk-group children, improve the efficacy of prophylactic check-ups, and decrease neonatal mortality. A prognostic table for rapid diagnosis of the risk of sudden death syndrome is proposed for practical public health. PMID- 10732460 TI - [Current problems in public health reform in Russia]. AB - Transformation of state treatment and prophylaxis institutions into private institutions is analyzed on the basis of collective opinions of the heads of institutions which were expressed during sociological interviews. The rating of the major negative consequences of commercial transformation of public health and of preferences of respondents in the sphere of paid services is determined. Relative significance of factors affecting the price (tariff) of medical services is evaluated. PMID- 10732461 TI - [Experience in introduction of general practice]. AB - The author analyzes experience gained in preparation and introduction of general practitioner's practice in the Krasnodar region and tries to find the optimal approach to transfer to this system through a staged model. PMID- 10732462 TI - [Methodical approaches to studies of the efficiency and quality of the State Sanitary and Epidemiological Surveillance]. AB - Current methodology and organization of evaluations of the efficiency of specialists and subdivisions of sanitary epidemiological institutions of the first level of managing are presented. The authors propose a method for quantitative evaluation of the efficiency for comparison of compatible and equivalent values. Definitions essential for evaluation of the efficiency the State Sanitary and Epidemiological Surveillance are formulated. A demonstration model of computer processing of the data for estimations of efficiency of the State Sanitary and Epidemiological Surveillance has been developed. PMID- 10732463 TI - [Use of health care quality indicators in the study of the therapeutic-diagnostic process]. AB - Definition of the notions "medical care quality" (MCQ) and "medical error" (ME) and their range depending on the negative effect of ME on the status of essential signs of MCQ, obligatory registration and possibility of universal description of all detected ME and their negative consequences by means of automated expert methods helped the authors develop quantitative parameters of the state of 3 components of MCQ. Based on these data, integral value of MCQ state and the structure of facultative MCQ were estimated. PMID- 10732464 TI - [Expert assessment of the therapeutic-diagnostic process in the evaluation of health care quality]. AB - Introduction of automated system of screening expert evaluation of the quality of treatment and diagnostic process provides universal approaches to internal and external regulation of medical care and helps develop (at the level of a public health monitoring organ) an automated system for regulation of the quality of medical care at a territorial level. This, in turn, will help define the optimal criteria (standards) of medical care quality for medical institutions, control the adherence to these standards, detect the causes of neglect thereof, and adopt optimal managing decisions aimed at improvement of public health activities in general. PMID- 10732465 TI - [Problem of health evaluation]. AB - The author suggests the establishment and introduction of an arbitrary mnemonic "unit of health" as a formal characteristic of health for describing and evaluating the ratio of any morbid status or structures offering medical services and highly formal elements of the marketing public health system. PMID- 10732466 TI - [Ways of restructuring ambulatory stomatological care network]. AB - The most important problems in modern Russian dentistry are defined. An obligatory condition for their successful solution is clear-cut orientation at the priority use of economic and medicolegal approaches to reformation of the service. PMID- 10732467 TI - [Specialized care rendered to adolescents at pediatric ambulatory polyclinic institutions]. AB - Organization of the activities of institutions engaged in medical care of children (pediatric outpatient health centers) is discussed in connection with transfer of adolescents aged 15-17 years under the care of pediatric services. Based on the results of experimental research and practical designs, the authors discuss the obligatory preparatory measures introducing this process, pressing problems of therapeutic, prophylactic, and medicosocial care rendered to adolescents aged 10-17 years (according to WHO classification), and specific features in the activities of specialists new for these health centers, such as psychologist/medical psychologist, adolescent therapist, pediatric gynecologist, social worker, etc. PMID- 10732468 TI - [Problems in transition to health insurance system in Russia]. PMID- 10732469 TI - [Experience in the management of municipal public health system]. PMID- 10732470 TI - [Professional liability insurance in public health]. PMID- 10732471 TI - [Teaching of pathological anatomy at the Moscow University in the 1st half of the 19th century (the 150th anniversary of the Pathological Anatomy Department of the I. M. Sechenov Moscow Medical Academy). 2. First stage in introduction of pathological anatomy in the curriculum of the Medical Department (1811-1825)]. PMID- 10732472 TI - [State measures to control plague in Moscow, 1771]. PMID- 10732473 TI - [Contribution of L. F. Ragozin to the organization of medical-sanitary work in pre-revolutionary Russia]. PMID- 10732474 TI - [On guard for the health of military missile specialists]. PMID- 10732475 TI - [The organization and work experience of the Central Certification Commission]. PMID- 10732476 TI - [What is to be the technical training of the military physician?]. PMID- 10732477 TI - [The prevention of influenza and other acute respiratory infections among the troops]. PMID- 10732478 TI - [Agents for combined pharmacotherapy in severe trauma to the brain and ENT organs]. AB - Early complex treatment of a critical brain traumas should be aimed not only at men's life and personality saving, but also at their performance' rehabilitation. Complicity and to some degree individual nature of a critical trauma pathogenesis depend on the associative active methods of surgery interference and conservative pharmacotherapy. The article deals with well-practiced means and methods of symptomatic pharmacotherapy at the early and later stages of the brain traumas with vestibular and hearing system injures and with theoretical and clinical base for modern "quick-action adaptogens" usage in addition to psychoneurocorrectors. PMID- 10732479 TI - [Anesthesiological accompaniment for the wounded during transport]. AB - The authors discuss some traits of anesthesia maintenance during transportation of wounded. The study of 292 cases evacuated from Afghanistan and Chechnya, divided by two groups with different types of the anesthetics. The first group members were given by 1 ml of 2% promedol solution, while the second was sedative controlled (associative usage of mydazolam and phentanil). When the first group suffered from pain, the second group patients were practically free from the pain stress until they reached their hospitals. PMID- 10732480 TI - [Photorefractive keratectomy as the operation of choice for correcting ametropia in servicemen]. PMID- 10732481 TI - [Current treatment problems in burn strictures of the esophagus]. AB - Examination study of 156 cases with esophagus burns. The patients were sent to a specialize hospital with 2-3 mouths' delay despite their continuous dysphagia complains 4 patients suffered from critical complications, 15 patients had gastrostomas and 98 patients lost their body weight to a marked degree. Applied methods of treatment were esophagectomy, bougienage esophagus and surgery. 55.5% patients had good treatment results. 40.7%--satisfactory and 3.7%--bad results. PMID- 10732482 TI - [The dynamics of the mineral metabolic indices in patients with spinal osteochondrosis under the influence of peloid therapy]. PMID- 10732483 TI - [A comprehensive approach to the therapy of mycoses in middle-aged and elderly persons]. PMID- 10732484 TI - [The diagnostic criteria for different forms of viral hepatitis C in young persons]. PMID- 10732485 TI - [The mechanisms of central regulation disorder under combat exposure conditions]. AB - Results of research in central regulation system's disorders in the local wars' participants suffering from environmental psychoemotional and traumatic stress. It has been established, that there was an upsetting of interaction between neurodynamic processes in the sub-dominant hemisphere and a reduction of the functional reserve of the brain. Neurophysiology disorders discovered come from the pathological system that had been formed up in the central nervous system preserve their persistence for several years after the war. PMID- 10732486 TI - [The plant cover of Russia in the area of the Braun-Blanquet syntaxonomy: the development of an approach and the results]. AB - Theoretical basis of ecologo-floristic classification according to Braun-Blanquet approach is discussed with the special attention to the contribution of Russian scientists. Principal results concern presentation of vegetation continuum in discrete classification units: polymodal conception of plant communities, principle of plural syntaxonomic decisions, pragmatic approach to classification, method of homological rows of plant communities, etc. Current ecologo-floristic classification of vegetation in Russia consists of 77 classes. This system is a good basement for estimation of rarity of plant communities and biodiversity protection. The first variant of "Red Book" of plant communities of Russia and other eastern European countries is discussed. PMID- 10732487 TI - [Transabiotic factors in an aqueous environment (a review)]. AB - Excretion of metabolites is a characteristic feature of any alive organism. A big group of these products--second metabolites--because of their variability, quantity and physico-biological activity have a special importance in aquatic environment. Exometabolites of some organisms become an important part of environment for the others. The authors discussed the origin and evolution of exometabolites from simple waste products to biologically active substances. Quantitative and qualitative aspects of metabolic excretion by organisms in water conditions are analysed. The data on composition, origin and biological function of some second metabolites of different groups of aquatic organisms are presented. The authors propose a classification of second metabolites according to their functional significance. The role of metabolites and decay products in the development of chemical information streams in hydrobiocoenosis is analysed. Metabolites (soluble organic substances) form a field of chemical information for biotic community. The most important functions of this field are communication and conditioning. The authors emphasize the importance of investigations of chemical bioinformation field in aquatic ecosystems. PMID- 10732488 TI - [The distribution of the macrobenthos of the White Sea littoral in different spatial scales]. AB - Spatial distribution of macrobenthos of middle intertidal zone was studied in scale from centimetres to 30 kilometres along the coastline. The community structure and distribution of the 5 most abundant species (Hydrobia ulvae, Mya arenaria, Macoma baltica. Peloscolex benedeni, Arenicola marina) were considered. Spatial heterogeneity of macrobenthos, estimated as mean dissimilarity between samples, kept constant in scale of centimetres--meters, but increased significantly when enlarged area is considered. Patterns of many species changed with scale from random mosaic to more or less pronounced patchiness, whereas the density of H. ulvae and the structure of the whole community demonstrated fractal (self-similar) patch pattern in wide range of scale from dozens of meters to several kilometres. Spatial correlations between species (the composition of assemblages) and between species and environmental factors were also scale dependent. Some possible effects of scale on the observed spatial distribution of benthos are discussed, and multiscaled analysis of biotic heterogeneity is concluded to be very fruitful. PMID- 10732489 TI - [Light-dependent changes in the enzyme activity of the ascorbate-glutathione cycle and ascorbate oxidase in the leaves of pea]. AB - Light-determined activation of ferments of ascorbate-glutation cycle, ascorbate oxidase in chloroplasts and cytosol is demonstrated as well as ascorbate peroxidase, monodehydroascorbate-reductase, glutation-reductase and ascorbate oxydase in mitochondria. On the other hands activity of mitochondrial dehydroascorbate-reductase increased on reduction of light most likely due to function of electron transport from glutation to dehydroascorbate in mitochondria. Glutation metabolism is proved to be endogenic catalytic process where the amount reconstructed glutation changes slowly with a delay and gradually follow light changes. Light dependable changes of glutation content in chloroplasts ensure resistance of ferment system again hydrogen peroxide and superoxide radicals that generate intensively at light. PMID- 10732490 TI - [Continuous-discrete models of the dynamics of an isolated population and of 2 competing species]. AB - The paper presents the analysis of various mathematical models for dynamics of isolated population and for competition between two species. It is assumed that mortality is continuous and birth of individuals of new generations takes place in certain fixed moments. Influence of winter upon the population dynamics and conditions of classic discrete model "deduction" of population dynamics (in particular, Moran-Ricker and Hassel's models) are investigated. Dynamic regimes of models under various assumptions about the birth and death rates upon the population states are also examined. Analysis of models of isolated population dynamics with nonoverlapping generations showed the density changes regularly if the birth rate is constant. Moreover, there exists a unique global stable level and population size stabilizes asymptotically at this equilibrium, i.e. cycle and chaotic regimes in various discrete models depend on correlation between individual productivity and population state in previous time. When the correlation is exponential upon mean population size the discrete Hassel model is realized. Modification of basis model, based on the assumption that during winter survival/death changes are constant, showed that population size at global level is stable. Generally, the dependence of population rate upon "winter parameters" has nonlinear character. Nonparametric models of competition between two species does not vary if the individual productivity is constant. In a phase space there are several stable stationary states and population stabilizes at one or other level asymptotically. So, in discrete models of competition between two species oscillation can be explained by dependence of population growth rate on the population size at previous times. PMID- 10732491 TI - [The correlation of the size of the pinna and the auditory bulla in specialized desert rodents]. AB - Relation between size of pinna and auditory bulla in specialized desert rodents (families Allactagidae, Dipodidae, Gerbillidae, Heteromyidae, and genus Selevinia) is statistically significant and described by hyperbola. The empirical distribution of respective sizes can be interpreted as a total of two main trends. One is enlargement of the pinna, the bulla remains small (most of Allactagidae), another is enlargement of the bulla, the pinna remains small (Gerbillidae, Heteromyidae, Selevinia, most Dipodidae). A few species display moderate enlargement of both pinna and bulla, while in Euchoreutes they both are hypertrophied. Biomechanical preconditions are supposed to be following. Enlargement of the pinna is responsible for increasing of both sensitivity and locatory abilities of the hearing organ, while enlargement of the bulla is responsible for the first function only. So, the two main trends are results of selection of development of the hearing organ at the level of either pinna or bulla. It is shown by dispersion and correlation analyses that size of the parts of the hearing organ under investigation are correlated mostly with mobility and escaping mode. It is concluded therefore that the rodents characterized by active and speedy escaping from predation display the first of the above trends, while the rodents characterized by passive escaping from predation display the second trend. PMID- 10732492 TI - Cranial kinesis in the amphibia: a review. AB - All extant orders of amphibians are characterized by kinetic skulls. Main type of intracranial movability in amphibians is pleurokinetism, that is supplemented in different amphibian groups by various types of rhyncho- and prokinetism. The most primitive pattern of cranial kinesis is revealed in the stegocrotaphic gymnophions. More paedomorphic species retain general cranial flexibility that is characteristic of larval skull. That is unfavourable for evolution of well regulated (adult) cranial kinesis and related feeding adaptations. Kinetism is also reduced in the species with heavily ossified skulls. Adaptive role and evolution of cranial kinesis in amphibians are discussed. PMID- 10732493 TI - [2 parallel approaches to the analysis of plant cover]. AB - Two parallel approaches to the analysis of vegetation based on plant geography are being developed in modern science. In European countries, there is ecologo morphological classification (EMC), in Russia--comparative floristics (CF). The development of CF in many theoretical aspects is similar to EMC. One of the reason of parallel development is late penetration of EMC ideas into Russia. The objective reason of this phenomenon is extremely low endemism of floras that were traditionally studied by Russian geobotanic schools, viz. steps and forests. The high level of endemism in central European floras (especially in Alps) allows to transform the principles of floristic division to vegetation classification. During last 20 years the Russian deviate of EMC demonstrates deviation with theoretical basement of EMC--conception of typical species, using non differentiate block of diagnostic species. That does not provide any new content either to syntaxa and coenofloras that exist in European classifications or to the method of partial floras in CF. PMID- 10732494 TI - Experimental treatments. Unapproved but not always unavailable. Last option for the desperately ill. PMID- 10732495 TI - The healing power of placebos. PMID- 10732496 TI - Are bioengineered foods safe?. Interview by Larry Thompson. PMID- 10732497 TI - It's convenient and private, but beware of 'rogue sites'. PMID- 10732498 TI - Joint program pools food research resources. PMID- 10732499 TI - Unapproved body fat tester fetches weighty fine. PMID- 10732500 TI - A new sight-saving option. PMID- 10732501 TI - Vaginal health after menopause. PMID- 10732503 TI - Help when it's hard to swallow. PMID- 10732502 TI - What your genes can and can't tell you. PMID- 10732504 TI - Minimizing OTC drug risks. PMID- 10732505 TI - Can radiofrequency catheter ablation cure an arrhythmia? PMID- 10732506 TI - I have burning and stinging sensations in my feet almost constantly. What can be done about it? PMID- 10732507 TI - Are generic and brand name drugs different in terms of quality? PMID- 10732508 TI - Rural health: a human right. PMID- 10732510 TI - 'Swallowing the medicine': determining the present and desired modes for delivery of continuing medical education to rural doctors. AB - A National Needs Assessment of the continuing professional educational needs of Australian rural medical practitioners was conducted on a model previously successfully used in Queensland. The objective was to describe and quantify the continuing education needs of rural medical practitioners in order that service providers might more expeditiously set educational curricula in continuing medical education (CME) provision. A major component of the study was to determine which modes of CME delivery rural doctors currently use and which they would prefer to use. Rural doctors were perceived to be slow to access new technologies and training may be required in order for the doctors to use them. PMID- 10732509 TI - Community participation in the recruitment and retention of rural doctors: methodological and logistical considerations. AB - Two Queensland rural communities with histories of poor general practitioner (GP) recruitment and retention participated in a process aimed at developing broadly based community action plans to recruit and retain GPs. Despite their very different physical and social characteristics, the two communities developed many similar objectives and strategies, which had the possibility of being implemented more widely. The community participation process can be both time- and cost effective if consideration is given to a variety of methodological and logistical issues. The process is a means by which communities, Divisions of General Practice, government, academic institutions and others can work together to recruit and retain medical practitioners. PMID- 10732511 TI - Social construction of Anangu disability. AB - The usual and common-sense definition of disability is based on a medical model that sees disability as a limitation or lack of competence on the part of the individual. This definition fails to acknowledge that in some cultures disability as a concept does not exist. This paper, based on research undertaken in 1994/1995, examines how the social construction of disability among the Anangu of the cross border region of Western Australia, South Australia and the Northern Territory, takes into consideration the important factors of history, culture and language. The theoretical underpinning of the research was symbolic interactionism, which led to collaborative ethnography becoming the methodology employed to collect and analyse the data. The findings of the research indicate that the social construction of disability among the Anangu occurs in three historical phases: the impairment, oppression and empowerment phases. The paper's purpose is to offer service providers to Aboriginal people with disabilities, information that will help them understand some of the attitudes, customs, mores and beliefs of their clientele. PMID- 10732512 TI - Establishing a new university department of rural health: the first 2 years of the South Australian Centre for Rural and Remote Health. AB - This study aimed to describe the establishment of a new University Department of Rural Health (UDRH) in South Australia and to report early achievements. In May 1997, the UDRH was established, key staff were recruited, infrastructure was developed and in April 1998 a Joint University Committee on Rural and Remote Health was formed. By mid-1999, 14 full-time equivalent staff were employed in Whyalla and Adelaide. Early achievements include: review of undergraduate rural placements; increased rural clinical placements by 1000 student-weeks; partnership with the Dental School resulting in training opportunities and falling public waiting lists; multidisciplinary teaching practices in four rural sites; priority public health projects established; competitive research grants won; and a capital grant to strengthen Aboriginal health services infrastructure secured. These early achievements demonstrate UDRH potential to have a real impact on health worker education, service delivery, and public health status in rural and remote areas. This strong foundation must now be built on. PMID- 10732513 TI - A review of 'traditional' aboriginal health beliefs. AB - Western health professionals often experience difficulties in service delivery to Aboriginal people because of the disparity between Aboriginal and Western health belief systems. This article reviews the literature which considers 'traditional' Aboriginal health beliefs and medical systems. The traditional Aboriginal model of illness causation emphasises social and spiritual dysfunction as a cause of illness. Supernatural intervention is regarded as the main cause of serious illness. There are gender divisions in Aboriginal society that impact on the delivery of Western healthcare. Management strategies such as preventative care, bush medicine, and the role of traditional healers are discussed. These belief systems are considered with particular reference to their interactions and implications with regard to the Western medical system. This information provides a framework to allow improved understanding by health professionals of the health related decisions made by Aboriginal people. PMID- 10732514 TI - Diabetic retinal photographic screening: a model for introducing audit and improving general practitioner care of diabetic patients in a rural setting. AB - This paper describes the addition of diabetic retinal screening using retinal photography to an existing immunisation audit by a General Practitioner (GP) Network in a semi-rural area 60-min drive from central Wellington, New Zealand. The employment of a nurse-facilitator who visited practices to assist the setting up of diabetic registers and the subsequent auditing of patterns of referral for retinal photography was seen as a first step in a process that would lead to audit of the care of diabetic patients by GPs in the Network. This should lead to a measurable improvement in health-care delivery to diabetic patients in this area and is a model that could be adapted by any group of rural or semirural GPs within a defined geographical area. PMID- 10732515 TI - Rural Health Support, Education and Training Program (RHSET): where to now? AB - The Rural Health Support Education and Training (RHSET) Program is a Commonwealth Government grants program established in 1990 to enhance the access to rural communities to effective health services. The emphasis has been on professional workforce issues. Up until December 1997, 431 applications for funding had been approved and close to $37 million allocated. This article considers the grants awarded in that period according to their main topic of interest within three broad groupings: policy and tertiary service provision; health discipline specific groups; and special interest groups such as Aboriginal and Torres Strait Islanders and community organisations. Each subgroup is introduced and its contents outlined. It also suggests that despite heightened government interest in rural and remote health, a niche can still be found for RHSET. It further argues that the time has come for a major evaluation of project activity to ensure non-duplication and to develop performance indicators for evaluating projects addressing rural and remote area workforce issues. PMID- 10732517 TI - The development of the advanced role of rural nurses in New Zealand. AB - This paper offers an informative insight into the development of the advanced role of rural nursing practice in New Zealand. Provided is a futuristic vision of nurses' contribution for the provision of primary rural health care. The concept of advanced nursing practice will be discussed within the context of the interdisciplinary healthcare team. It is argued that as nurses take on advanced practice, it is essential they receive appropriate clinical and theoretical skills to ensure they are in a position to provide competent and clinically safe, effective health care in an ethical, efficient manner. A description of a survey, undertaken by the author, studying rural nurses' skills provides the recommendation for the development of an appropriate postgraduate course at Masters level, designed specifically for primary rural nurses for the advanced role. The provision of this advanced education together with preparation and support, can pave the way for the highly skilled and knowledgeable nurses of the future working in collaboration with the interdisciplinary rural healthcare team and rural community. PMID- 10732516 TI - Asthma management in rural Australia. AB - This article explores the question of whether rural location influences the diagnosis, management and outcome for patients with asthma. Three cases of asthma respiratory arrest are presented to highlight some of the difficulties of asthma management in rural communities, followed by a review of the current literature relevant to rural asthma. PMID- 10732518 TI - Pain assessment and management. AB - Little research is currently available related to pain management by neuroscience nurses. However, due to concerns about the potential for altering neurological status, some neurosurgery patients may not receive optimal pain management. This paper describes findings from a pain related survey which was distributed during the Canadian Association of Neuroscience Nurses June 1998 national conference. The survey was intended to assess Canadian neuroscience nurses pain management knowledge and to explore pain management techniques after intracranial surgery. While 60% of respondents answered four pain assessment and management case study related questions correctly, some respondents rated pain differently when it was expressed by a smiling or grimacing patient. The most common methods for pain control after intracranial surgery included intermittent codeine and/or morphine, often by intramuscular injection. Findings from this study suggest that some neuroscience nurses require further education about pain management and that many patients do not receive optimal pain management after intracranial surgery. PMID- 10732519 TI - Cerebral aneurysms: a review and what's new. PMID- 10732520 TI - Management of epilepsy throughout the reproductive cycle--an overview of treatment issues. PMID- 10732521 TI - Management of migraine throughout the reproductive cycle--an overview of treatment issues. PMID- 10732522 TI - Personality & behaviour changes following spinal cord injury: self perceptions- partner's perceptions. AB - There has been little specific investigation of personality and behaviour changes following spinal cord injury (SCI) and only limited consideration of the possible impact of concurrent traumatic brain injury (TBI). By mail-out questionnaire, we evaluated personality and behaviour changes in a married group (n = 9) with traumatic SCI, who knew their partners prior to injury, and who had not been identified as having concurrent TBI on referral to the Canadian Paraplegic Association. Both the person with SCI (and the partner) completed the revised Adjective Checklist and by their combined report, there were significant personality and behaviour changes. Unexpectedly, five individuals described post traumatic amnesia (PTA) > or = 3 days. Subsequently, participants' reports were further divided into two groups--"longer PTA" and "shorter PTA". The "longer PTA" group self-reported less change and more positive change than did their partners. The "longer PTA" partners described changes that are consistent with the profile of TBI. The "shorter PTA" group described themselves more negatively than did their partners. Given the size of the groups (n = 5, n = 4), these findings are presented to illustrate trends and to stimulate further research. PMID- 10732523 TI - Are you ready (to be a neuroscience nurse)? AB - On the pediatric neurosciences unit of British Columbia's Children's Hospital we are in the throes of a nursing staff crisis. In the last year alone we hired ten new graduates to work as casuals on our unit. With a two and a half day hospital orientation, five preceptor shifts, and a Competency Based Education Plan in hand, we send them off to the trenches. We know these nurses have little nursing experience and even less Neuroscience nursing experience. Yet, we expect them to care for patients and families whose problems they may not understand. For a preceptor, and senior colleague, this is a disturbing situation. We recognize that in their orientation shifts they have not even begun to experience the challenges of a Neuroscience unit. Have they cared for a child who has had a postoperative laminectomy? Do they recognize subtle seizures? Have they sat with a family who have just learned that their child has a brain tumor? No. We expect them to care for patients and families with minimal support. This is the reality of nursing today. Many of us pride ourselves on being committed pediatric Neuroscience nurses. Our physicians rely upon our assessment skills and they trust our intuition! We believe we have earned that trust. How can we convey our enthusiasm and excitement to our perceptees so they are motivated to stay and become experienced pediatric Neuroscience nurses? In this presentation we outline our paper to remedy this situation. PMID- 10732524 TI - Assessing hereditary breast cancer risk. PMID- 10732525 TI - Veiled yet vulnerable. Breast cancer screening and the Muslim way of life. AB - OBJECTIVES: While multiple reports have been published in the literature that describe the effect of religion and spirituality on healthcare practices, few have been reported that attempt to describe the effect of the fundamental beliefs and customs of the followers of Islam. A qualitative study aimed toward gaining an understanding of the degree to which Islamic beliefs and customs influence followers' breast cancer screening practices was, therefore, undertaken. MATERIALS AND METHODS: The study was designed with semistructured focus groups as the primary means of gathering data. A purposefully selected sample of nine Muslim women were recruited to participate in the study. Throughout the focus group, attempts were made to engage the women in a manner that stimulated in depth discussion of the beliefs and customs influencing breast cancer screening. RESULTS: Data revealed that the religious beliefs and customs of the Muslim women participating in the focus group significantly influenced their participation in breast cancer screening. Despite their knowledge of the benefits of regular breast cancer screening, the women indicated that they choose not to participate in available breast cancer screening programs, given that they were not structured in a manner that was consistent with the beliefs and customs of Islam. CONCLUSIONS: This study illustrates the manner and degree to which Islamic beliefs and customs can influence the breast cancer screening behavior of Muslim women and the importance of carefully designing breast health programs tailored to the needs of this population of women. PMID- 10732526 TI - Assessing cultural sensitivity in printed cancer materials. AB - OBJECTIVES: Printed cancer education materials (PCEMs) (pamphlets and fact sheets) are used to educate individuals about cancer prevention and awareness. Culture is an important variable affecting the retention and use of information in printed materials to African Americans that has not been fully addressed in the literature. MATERIALS AND METHODS: The major goal of the Cancer Prevention Materials for African Americans project, funded by the Texas Cancer Council, was to assess cultural sensitivity of currently disseminated PCEMs targeting African Americans. The project consisted of conducting focus groups, forming an advisory committee, and developing and using the Printed Cancer Education Materials for African Americans Cultural Sensitivity Assessment Tool. RESULTS: Cultural sensitivity of PCEMs was assessed in terms of format, visual message, and written message. The majority of the PCEMs (56.2%) were culturally insensitive, with the visual message being the weakest component of all the materials. CONCLUSION: Future PCEMs targeting African Americans should include culturally sensitive visual messages to be more effective in delivering the cancer prevention message. The use of the assessment tool can assist cancer control specialists in developing culturally sensitive materials. PMID- 10732527 TI - Self-advocacy training for cancer survivors. The Cancer Survival Toolbox. AB - OBJECTIVES: With the advent of managed healthcare, self-advocacy has been identified as an essential skill for cancer survivors. This article describes a self-advocacy training program, the Cancer Survival Toolbox, developed through a unique collaborative effort by the National Coalition for Cancer Survivorship, the Association of Oncology Social Work, and the Oncology Nursing Society. MATERIALS AND METHODS: Self-advocacy training is provided in audiotape format, as well as through the Internet and in interactive groups. The need for this training was established through surveys completed by 569 cancer survivors and 833 oncology professionals. Essential skills were identified through a literature review, and the content of the training was pilot tested with bicoastal groups of cancer survivors and with feedback from representatives of 15 national cancer organizations. RESULTS: While the majority of the 569 respondents to the survivor survey were highly educated and between the ages of 31 and 60 years, fewer than half reported that when they first received a diagnosis of cancer they were able to communicate their needs effectively, had the skills necessary to make decisions, or were able to negotiate with healthcare providers, insurers, and employers. Results of the survey of professional oncology nurses and social workers also supported the need for self-advocacy training. Fewer than one third of the 833 respondents to the professional survey reported that their patients who had received new diagnoses of cancer had essential self-advocacy skills. CONCLUSIONS: This self-advocacy training program is currently available on audiotape in English and Spanish. It is available in print in Chinese on the Internet. Data from the pilot groups indicate the program effectively addresses the self-advocacy skills of communication, information seeking, problem solving, decision making, and negotiating. Data are currently being collected to assess the efficacy of the audiotape format and the impact of the training on survivors and professionals. Future plans include training oncology professionals to teach these skills and to work collaboratively with survivors in advocacy efforts. Additional programs designed to address the needs of the uninsured as well as older adult and pediatric survivor populations are in development. PMID- 10732528 TI - Multidisciplinary care of hepatocellular carcinoma. AB - OBJECTIVES: Multidisciplinary care of cancer patients in varied settings is well described in the literature, but there is little specifically describing the multidisciplinary care of the patient with hepatocellular carcinoma (HCC). The purpose of this article is to describe HCC and the multidisciplinary approach at the Philadelphia Veterans Affairs Medical Center (PVAMC). MATERIALS AND METHODS: HCC is one of the most common solid tumors in the world, but it is rare in North America. It is associated with environmental carcinogens identified in animal studies, hepatitis B and C, cirrhosis of any etiology, and various metabolic diseases. No reliable therapy has been established for HCC. Surgical resection is the best treatment, but it is possible only in the patient with adequate hepatic reserve and limited-stage cancer. From January 1995 to May 1998, 22 patients at PVAMC received a diagnosis of primary HCC. One patient was a candidate for surgery, two patients received radiation therapy, and one patient underwent chemoembolization. Eighteen patients presented with an advanced-stage disease and comorbidities. RESULTS: Therapy goals in these 18 patients were limited to supportive care and enhancement of quality of life. A multidisciplinary team provided care to this challenging patient population. The multidisciplinary team treating HCC at PVAMC consisted of physicians, nurses, pharmacists, social workers, and a chaplain. Most care occurred in the outpatient setting. Supportive therapy included the controlling of ascites and abdominal discomfort, hepatic encephalopathy, and pruritus. Opioids relieved abdominal pain. Psychiatric support and counseling helped patients and families cope with the poor prognosis. CONCLUSIONS: A multidisciplinary team approach helped provide care for this challenging population. Through anecdotal reports, patients and family expressed satisfaction with their care. Research is needed to systematically test interventions designed to enhance quality of life in patients with HCC. PMID- 10732529 TI - Body image and quality of life after head and neck cancer surgery. AB - OBJECTIVES: The purpose of this paper is to describe the process of body image reintegration as it relates to quality of life in the surgical head and neck cancer patient. MATERIALS AND METHODS: To illustrate the course of body image reintegration after head and neck cancer surgery, the results of two major studies are presented. In the first study, a cohort sample of 75 adults about to undergo disfigurative surgery for head and neck cancer was examined to determine levels of anxiety before and after the procedure. The State Trait Anxiety Inventory was administered before and after surgery on postoperative days 4 through 6. In the large study, from which the cohort sample was derived, 117 adults about to undergo disfigurative surgery were investigated to determine the relationship between coping with surgical disfigurement/dysfunction and length of hospital stay. The Ways of Coping Checklist was administered before surgery. Disfigurement/dysfunction, as measured by the Disfigurement/Dysfunction Scale, and postoperative coping behaviors, as measured by the Coping Behaviors Score, were accounted for in both samples. Self-care and resocialization behaviors were observed and measured on postoperative days 4 through 6. RESULTS: In the cohort study, anticipation of disfigurative facial surgery was associated with extremely high levels of anxiety. In the large sample, coping effectiveness was diminished. The degree of surgical deficit ranged from mild to severe disfigurement and dysfunction. Preoperative coping effectiveness predicted postoperative coping behavior. It was concluded that body image reintegration was characterized by self-care, resocialization, and reduced anxiety. Further, that these components, when taken together, constitute assimilation of the surgical defect into self. CONCLUSIONS: Body image reintegration is critical to subsequent quality of life after head and neck cancer surgery. When disfigurement/dysfunction is associated with treatment, quality of life may be profoundly and adversely affected. Findings from the studies presented indicate a significant correlation between postoperative self-care and reduction in level of anxiety. The implications of this findings are important to care during the early period after surgery and to long-term follow-up after hospital discharge. PMID- 10732531 TI - A new transmucosal fentanyl for breakthrough cancer pain. PMID- 10732530 TI - Planning a patient-centered resource room. PMID- 10732532 TI - Ensuring quality care. The issue for the millennium. PMID- 10732533 TI - When a cancer patient commits suicide: psychosocial issues faced by patients, families, and caregivers. PMID- 10732534 TI - The sexual rehabilitation of persons with cancer. AB - OBJECTIVES: This article presents cognitive and behavioral sexual rehabilitation interventions to assist persons with cancer in understanding and adjusting to the physical changes caused by cancer treatment. MATERIALS AND METHODS: Interventions described here include educating patients about the phases of sexual functioning and the impact of treatment, giving patients permission to explore their ability to respond to sexual stimulation by using self-pleasuring exercises, teaching sensate focus exercises that structure noncoital foreplay; and suggesting changes in coital positions. RESULTS: Sexual functioning is an important aspect of quality of life for people with cancer that continues to be difficult for oncology professionals to address. Familiarity with these interventions can prepare the properly trained oncology social workers, nurses, and other mental health professionals to assist cancer patients with their concerns about sexual functioning. CONCLUSIONS: All oncology professionals should be aware of the importance of recognizing and addressing issues of sexual functioning in patients with cancer to validate the patient's experience and make the proper referral to a trained sex therapist. PMID- 10732535 TI - Daily fatigue patterns and effect of exercise in women with breast cancer. AB - OBJECTIVES: Cancer treatment-related fatigue is a common and disruptive side effect of chemotherapy. Exercise is an intervention proposed to reduce fatigue in cancer patients. The purpose of this study was to describe the patterns of daily fatigue in women with breast cancer who did and did not exercise while receiving the first three cycles of adjuvant chemotherapy. MATERIALS AND METHODS: Women received instruction to follow an 8-week home-based exercise program and to maintain daily exercise and fatigue diaries. Functional ability (12-minute walk) was measured pretest and post-test. RESULTS: Several distinct patterns of fatigue emerged. The most common pattern of fatigue after chemotherapy demonstrated a sharp rise in fatigue. However, several women demonstrated a chaotic pattern with erratic and wide swings in their fatigue throughout the entire study period. Women who adopted exercise experienced fewer days of high fatigue levels and more days of low levels of fatigue for both average and worst levels of fatigue. Women who did not exercise experienced more bad days (high fatigue) and fewer good days (low fatigue). CONCLUSIONS: Exercise appears to reduce the levels of average and worst fatigue and may help women recognize their pattern of fatigue. Exercise may reduce the intensity of fatigue by reorganizing women's interpretation of fatigue. Routine clinical assessment and education about fatigue by health professionals can help patients to understand their pattern of fatigue and may help them to manage the symptom. PMID- 10732536 TI - Cognition and the cancer experience. Clinical implications. AB - OBJECTIVES: The authors demonstrate the application of cognitive therapy in oncology care by presenting a brief review of theory and relevant case studies. MATERIALS AND METHODS: In light of the life and death nature of the fears evoked by cancer, it is important for the oncology clinician to recognize the role that cognition plays in patient responses to the diagnosis and treatment of cancer. After presentation of a theory base that creatively links cognition and the cancer experience, key cognitive concepts are defined and discussed. Case material illustrates the application of these concepts and how oncology clinicians can use select interventions adapted from the brief mental health treatment modality of cognitive therapy to promote adjustment to cancer. RESULTS: Patient and family views about cancer have emotional and behavioral consequences, influence ability to cope with diagnosis and treatment, and serve to focus clinical intervention. Cognitive interventions can help patients and families think about cancer in objective, adaptive ways. Focusing on perceptions and questions of meaning, clinicians can be effective using cognitive lines of questioning that expand patient stories and elicit beliefs about cause, control, and responsibility for their cancer. CONCLUSIONS: Cognitive interventions are brief and solution-focused interventions that acknowledge and build on generalist interviewing skills common to each discipline. As such, they are particularly useful in physical health settings where work is fast paced and clinicians are faced with the challenge of dealing in a collaborative manner with patient and family coping responses as they apply to the medical problem and care plan. PMID- 10732537 TI - Colorectal cancer screening beliefs. Focus groups with first-degree relatives. AB - OBJECTIVES: The purpose of this paper is to describe the perceived benefits and barriers to colorectal cancer screening reported by first-degree relatives of colorectal cancer patients. MATERIALS AND METHODS: In this study, the authors used focus groups to identify perceived benefits and barriers to colorectal cancer screening among parents and children of colorectal cancer patients. Four focus groups were conducted with relatives of colorectal cancer patients seen at two university medical centers in the Midwest. The groups ranged in size from five to eight members each and were stratified by gender. RESULTS: Four benefits of colorectal cancer screening were identified by participants: finding colorectal cancer early, decreasing the chances of dying from colorectal cancer, freedom from worry about colorectal cancer, and reassurance that one was cancer free. Four main barriers were identified that applied to all four types of colorectal cancer screening or to colorectal cancer screening in general. These included inadequate public awareness of colorectal cancer, inconsistent recommendations from healthcare providers, concerns about the efficacy of screening tests, and embarrassment. Barriers unique to each screening test also were identified. CONCLUSIONS: Understanding individual beliefs about the benefits and barriers to colorectal cancer screening will allow clinicians and researchers to develop effective interventions to increase screening. Results from the focus groups have been used to develop an instrument to measure benefits and barriers to colorectal cancer screening, which now needs to be tested with more culturally and socioeconomically diverse groups. PMID- 10732538 TI - A qualitative study of patient perspectives on colorectal cancer. AB - OBJECTIVES: The purpose of this study was to use qualitative methods to contribute to a complete patient perspective on the psychosocial impact of colorectal cancer. MATERIALS AND METHODS: A qualitative descriptive study was conducted in 20 patients attending a gastrointestinal follow-up clinic at the Toronto-Sunnybrook Regional Cancer Centre. The data documented included patient satisfaction and perceptions regarding to quality of care, information received, involvement in decision making, and long-term management of the illness. RESULTS: Overall, patients were satisfied with their treatment, including the quality and timeliness of the information they received, the quality of their healthcare, and the level of involvement in decision making. However, some patients were dissatisfied with information concerning long-term management of their illness. Patient care, including information and social support, was provided by cancer specialists, family physicians, family, and friends. Patients looked to cancer specialists as their primary source of information, but relied on family physicians to fill in gaps in understanding, to provide support, to manage overall care, and to act as a sounding board for ideas and treatment options. Social support was also provided by family and friends. All patients had a relatively positive outlook on their illness experience, although those with colostomies had some added difficulty. Despite the focus on positive change, many patients acknowledged difficulty coping with the side effects of treatment. CONCLUSIONS: These data indicate that patient information needs to be provided in the most common terms and the most straightforward language. Information also may need to be repeated and should include attention to long-term management of the illness. Health professionals should assume that patients may have difficulty in illness management and should encourage a discussion of patients' concerns. PMID- 10732539 TI - Complementary/alternative medicine and cancer research. A national initiative. PMID- 10732540 TI - Drug assistance programs for the cancer patient. PMID- 10732542 TI - The pace is hotting up. PMID- 10732541 TI - Raltitrexed for advanced colorectal cancer. The story so far. PMID- 10732543 TI - 2020 vision. PMID- 10732544 TI - Preventing and managing the problem of obesity. PMID- 10732545 TI - Role of emollients in dry skin conditions. PMID- 10732546 TI - Sweetening the burden of a diabetes diagnosis. PMID- 10732547 TI - Know how. Going home after stoma surgery. AB - Returning home after stoma surgery can be fraught with difficulties. Identifying potential problems and dealing with them promptly is an important part of the stoma care nurse's role. This article is the third in a series of three guides on stoma care. PMID- 10732548 TI - A recipe for tackling malnutrition. PMID- 10732549 TI - Female urinary catheterisation. PMID- 10732550 TI - Hepatitis and typhoid vaccine: killing two birds with one stone. PMID- 10732551 TI - A look at where we are up to with nurse prescribing. PMID- 10732552 TI - How to protect the skin of incontinent patients. PMID- 10732553 TI - Fungating wounds: their diagnosis and management. PMID- 10732554 TI - Give practice nurses their due. PMID- 10732555 TI - National asset. PMID- 10732556 TI - Nurses will face challenge of meningitis campaign. PMID- 10732557 TI - Overcoming nutritional problems in the under-fives. PMID- 10732558 TI - Achieving home-based care for deep vein thrombosis. PMID- 10732559 TI - Preventing domestic accidents in the elderly. PMID- 10732560 TI - Scalp psoriasis: topical therapies. PMID- 10732561 TI - Diabetes patients could control condition better. PMID- 10732562 TI - Secret agent holds the key to drug development. PMID- 10732563 TI - Know how. Coming to terms with stoma surgery. AB - Patients undergoing stoma surgery need a lot of information and support beforehand. The role of the stoma care nurse in helping patients adjust to the situation is paramount. This article is the first in a series of three guides on stoma care. PMID- 10732565 TI - Role of good nutrition in effective stoma care. PMID- 10732564 TI - Bridging the hospital and community gap. PMID- 10732566 TI - Helping a wound along the road to recovery. PMID- 10732567 TI - Budget busters. PMID- 10732568 TI - The real horrors. PMID- 10732569 TI - Dietary and lifestyle changes for menopausal women. PMID- 10732570 TI - Alleviating the distress of urinary incontinence. PMID- 10732571 TI - Knowing how. Recovering after stoma surgery. AB - The postoperative period after stoma surgery involves helping patients to come to terms with their new condition. Effective management of any problems that may arise is crucial to the rehabilitative process. PMID- 10732572 TI - How to assess leg ulceration. PMID- 10732573 TI - The role of the diabetes facilitator in providing care. PMID- 10732574 TI - Unison's campaign to prevent needlestick injuries. PMID- 10732575 TI - Needlestick injuries: a guide on prevention. PMID- 10732576 TI - Making the right dressing choice: what the survey said. PMID- 10732577 TI - The journey from laboratory to the bathroom cabinet. PMID- 10732578 TI - The challenge of managing heavily exuding wounds. PMID- 10732579 TI - Taking the right steps towards healthier legs. PMID- 10732580 TI - Failure to thrive: early intervention to address dietary issues is vital. PMID- 10732581 TI - Providing relief from the itching of atopic eczema. PMID- 10732582 TI - The role of sleep clinics in helping children get a good night's rest. PMID- 10732583 TI - Nursing's feminine perspective. PMID- 10732584 TI - 'How has your feminine perspective influenced your work'? PMID- 10732585 TI - Gender's role in healthcare leadership. Interview by Marjorie Jamieson. PMID- 10732586 TI - Nursing and feminism: an uneasy alliance. Interview by Lisa Legge. PMID- 10732587 TI - Sailing a career to calmer waters. PMID- 10732588 TI - Interdisciplinary rounds. PMID- 10732590 TI - [Comparative evaluation of four methods of an endotracheal tube holder. Preliminary results]. AB - OBJECTIVE: To compare four different types of endotracheal tube holders. PATIENTS: Patients admitted to Intensive Care Unit in Thertiary Hospital with 800 patients admitted/year. The minimum sample was 188 to obtain statistically significant differences. During the first 6 months, 68 consecutive patients non selected were randomly assigned to one of four types of tube holders. PARAMETERS: Each 8 hours were evaluated the presence of lesions in skin or mucose membrane, colocation facility and patients commodity. Also security and cost analysis parameters were included. STATISTIC ANALYSIS: SPSS statistical package were used, Kaplan-Meier curve, Kruskal-Wallis test; and making statistical contrasts between the different types of tube holders. We considered significant a p < 0.05. RESULTS AND CONCLUSIONS: The obtained results till this moment showed significant differences in time free of lesions, between Haid holder and the rest of tube holders. Likewise, in the subjective parameters for the nurse and patient evaluations the Haid holder obtained a superior mind in facility of colocation and commodity of patients, consecutive gauze bandage. PMID- 10732591 TI - [Study of the work climate in four critical care units in a hospital]. AB - A study was made of the work climate, which was understood to be the overall group of factors that influence nursing professionals in critical-care units. A descriptive study was made by carrying out an anonymous survey among the staff of four adult critical-care units: intensive care unit, coronary unit, reanimation unit, and cardiac postoperative care unit. The survey included seven factors: occupational satisfaction, occupational stress, occupational pressures, work relations, professional skills, professional training, and sociodemographic variables. Of 132 questionnaires completed, 80% of the persons who answered accepted their tasks, but only 51% indicated that they planned to continue in the critical-care unit. Sixty percent of the persons surveyed indicated that their work was stressful and that they felt physically fatigued as the workday progressed. Similarly, 60% reported that their work sometimes had a negative impact on family life and 35% indicated that it affected their health. Relations among members of the health-care team were reported as good by 90%. Seventy-five percent were capable of working independently and resolving situations at their own initiative. Ninety-five percent of the persons who completed the survey considered the possibilities for professional advancement to be scant and 88% were interested in the development of specialization. It was interesting that one third of the persons in the sample wanted to leave the hospital. Unit ergonomics had a negative effect on work activities. Staff felt a lack of information in spite of good relations with superiors and other team members. They felt the need for more social and professional recognition, as well as better economic remuneration, but they expected specialization to palliate these needs to some degree. PMID- 10732589 TI - [Endotracheal aspiration: respirator vs. manual resuscitation as method for hyperoxygenation and hyperinflation]. AB - Endotracheal aspiration protocols (EAT) include hyperoxygenation and hyperinflation to minimize the negative effects of the technique. No conclusive studies have determined the most effective hyperoxygenation and hyperinflation method. This study had two aims: to compare the effects on patient oxygenation and hemodynamics during endotracheal aspiration of secretions using, respectively, a respirator or manual resuscitator as the hyperoxygenation and hyperinflation method. Tidal volume (TV) and FIO2 with the manual resuscitator were quantified. The study was based on 172 aspiration sessions carried out under artificial ventilation in the immediate postoperative period of 26 patients who had undergone cardiac surgery without lung damage. Hyperinflation and hyperoxygenation before, during and after aspiration were carried out with and artificial ventilator in group I and with a manual resuscitator in group II. In all aspiration interventions, an analysis was made of hemodynamic parameters (MAP, MPAP, HR, CO and arrhythmias), ventilation and oxygenation parameters (HR, FIO2, SpO2, and SvO2), and the influence of the method on the appearance of atelectasis. Both methods produced small increases in all hemodynamic parameters, and significant differences in HR (p < 0.001) and MPAP (p < 0.002), although no clinical repercussions were observed. No severe arrhythmias were observed. No statistically significant differences between the two methods were found in the evolution of SpO2 and SvO2, which remained above baseline levels throughout both procedures. Analysis of the effectiveness of the manual resuscitator (the second aim) under the conditions established yielded a mean FIO2 of 0.86 and a mean tidal volume of 153% in relation to baseline tidal volume. Both methods of hyperoxygenation and hyperinflation prevent hypoxia and maintain hemodynamic stability in patients without producing lung damage. The effectiveness of the manual resuscitator for administering high oxygen concentration and large volumes was confirmed. PMID- 10732592 TI - The use of available social support networks by older blacks. AB - Research has indicated that strong social networks positively influence the health of older Black adults. However, scales used in studies to measure social support do not reveal whether these networks are actually activated. The purpose of this descriptive study was to describe the reasons older Blacks with chronic illnesses did not utilize available support networks in their health care management. Following a community-based survey of 383 older Blacks, where each participant had a social support score completed from the Lubben Social Network Scale, a smaller group of 116 Blacks was selected to participate in a qualitative interview. It was found that 59 out of the 116 older Blacks had high social support scores on the Lubben Scale, but only 40 of these individuals reported the use of support networks for the maintenance of their health regimen. These findings illustrated that some older Blacks with strong support networks chose to function independently of their social support networks in the maintenance of their health. PMID- 10732593 TI - Barriers to prostate cancer screening in African American men. AB - Prostate cancer is one of the most significant health problems facing men today, especially African American men. Decreased participation in prostate cancer screening by African American men is a serious problem, as decreased survival rates occur when the diagnosis of prostate cancer is delayed. This descriptive correlational study focuses on identifying the relationship between perceived barriers and participation in a free prostate cancer screening program. A purposive sample of African American men (n = 1,395) was drawn from multiple community sites in the southeast United States. All significant variables (age, income group, marital status, and educational intervention were used as covariants for the multiple logistic regression. With the addition of the covariants, the barrier suggesting "would be embarrassed" remained significant (p = 0.03). Two other barriers, "no way to get there" and "refuse to go" approached significance (p = 0.08 and p = 0.09, respectively). Nurses can use knowledge about barriers identified in this study to develop interventions aimed at increasing participation in prostate cancer screening among African American men. PMID- 10732594 TI - Student nurses' perceptions of elderly care. AB - The purpose of this study was to investigate the perceptions of student nurses towards the care of the elderly. Using King's Theory of Goal Attainment, this experimental study used a convenience sample of approximately 100 associate degree nursing students (ADN) from a nursing program in a southern state. Approval from the dean of the nursing department and IRB was obtained. Kogan's (1961) Attitude Toward Old People's Scale was used to measure students' attitudes before and after their clinical experiences. Student nurses were given the questionnaires during scheduled class time of each program before any lecture content and after content has been given to students. PMID- 10732595 TI - The role of family: perceptions of children with sickle cell. AB - The purpose of this study was to determine the perceptions of the quality of family relationships between children with sickle cell disease and their non diseased siblings. In this study, 28 subjects were interviewed in their homes. The quality of family relations was assessed utilizing the Family Environment Scale (Moos & Moos, 1981). There was no significant difference in cohesion, expressiveness, conflict, or organization. Control was higher for boys than girls. Implications for family nursing practice are presented. PMID- 10732596 TI - Cultural diversity and transcultural nursing as they impact health care. AB - This article examines and critiques what can be broadly defined as "culturally sensitive nursing practice." The authors argue that both past and present research on culturally sensitive nursing practice, regardless of which branch of such practice it subscribes to (transcultural, cross-cultural, multicultural, intercultural, international, and so forth), is not sufficiently based in a sociopolitical context. As a result, there is not significant positive improvement in health care outcomes for patients from traditionally under represented social membership groups (i.e., groups based on race, gender, socioeconomic class, etc.). Realizing sincere and affective culturally sensitive nursing practice is contingent upon the development and implementation of a comprehensive, sociopolitically conceptualized multicultural nursing education curriculum and pedagogy. PMID- 10732597 TI - The meaning of AIDS: African American nursing students' perceptions of providing care to AIDS patients. AB - A qualitative study was conducted to explore the perceptions of African American nursing students as they provided care to acquired immunodeficiency syndrome (AIDS) patients. This research addressed the following question: What are the lived experiences of African American nursing students when they provide care to AIDS patients? The participants in this study were a convenience sample of 12 associate degree nursing students enrolled full-time at a historically Black college and university located in Virginia. Data were collected through audio taped interviews. Five dominant themes emerged: 1) fear of exposure and transmission; 2) hopelessness about the situation; 3) self-identification with the individual; 4) empathy toward persons with AIDS (PWAs); 5) and spirituality and belief in an afterlife. Female nursing students were able to identify with their AIDS patients while male nursing students were not. Many of the nursing students felt ostracized by their peers, and a lack of knowledge hindered the nursing students' deliverance of compassionate nursing care. PMID- 10732598 TI - Evaluating preceptorship experiences in a distance nursing program. AB - Clinically based education for registered nursing students is a major component of a nursing curriculum. Nursing educational programs are becoming increasingly reliant on clinicians in hospitals and health care agencies to be preceptors for their students. The concept of preceptorship in baccalaureate nursing education continues to be endorsed as a viable alternative clinical teaching strategy (Myrick & Barrett, 1994). Preceptorship programs are necessary interventions to facilitate integrating and enacting the professional nurse role (Allanach & Jennings, 1990). The educational clinical preceptorship provides the opportunity for collaboration and the development of collegial relationships between members of academia and nursing service (Patton & Dowd, 1994). The highly competitive nature of today's health care institutions and the rapid changes in health and nursing care increase the need for nurse leaders. Students must acquire leadership and management skills and be competent as the role of the nurse changes to a participant in the organizational leadership and management process. The purpose of this study was to evaluate students' experiences regarding preceptor effectiveness in upper division leadership and management courses in a Bachelor of Science in Nursing (BSN) distance nursing program. Specifically, this article describes experiences from participating students enrolled in a performance/clinical rotation in the Spring 1996 semester throughout the state of California. For a successful clinical experience, it is important to evaluate the major components of the preceptorship experience. PMID- 10732599 TI - Take perfection off your "to-do" list. PMID- 10732600 TI - Keep the quest for perfection in perspective. PMID- 10732601 TI - Commit to your own personal quest. PMID- 10732602 TI - Expand your commitment to excellence. PMID- 10732603 TI - Human perfection is an oxymoron. PMID- 10732604 TI - Entrepreneurship redefines happiness and perfection. PMID- 10732605 TI - The imperfect path to happiness. PMID- 10732606 TI - A low pay award will not attract much-needed experienced nurses. PMID- 10732607 TI - Sex, lives and education. PMID- 10732608 TI - Eat, drink and be merry. PMID- 10732609 TI - Consuming passion. PMID- 10732610 TI - Divided they fall. PMID- 10732611 TI - What is the pre-booked admission scheme? PMID- 10732612 TI - Pilgrims' progress. PMID- 10732613 TI - Complements of the staff. PMID- 10732614 TI - Students rush to join nursing degree courses. PMID- 10732615 TI - Check the quality not the length. PMID- 10732616 TI - Inside job. Interview by Eileen Fursland. PMID- 10732617 TI - I wish.... PMID- 10732618 TI - Open-door policy. PMID- 10732619 TI - Nurses must be made to keep basic skills updated. PMID- 10732620 TI - Seen and not heard. PMID- 10732621 TI - All change. PMID- 10732622 TI - A tale of two tribes. PMID- 10732623 TI - A nursing student on a hospital placement. PMID- 10732624 TI - Continuing care of burn-injured patients. PMID- 10732625 TI - Tuberculosis: a menace reborn. PMID- 10732626 TI - Keeping teams together. PMID- 10732627 TI - Diabetes care: getting your patients on a sure footing. PMID- 10732628 TI - A bewildering time. PMID- 10732630 TI - The future is here. PMID- 10732629 TI - Spreading the word on insulin therapy. PMID- 10732631 TI - International wound care ambassadorship. PMID- 10732632 TI - Sacral and ischial pressure ulcers: evaluation, treatment, and differentiation. AB - Although the cost of caring for pressure ulcers is enormous, few accepted standards of care exist in the fragmented specialty of wound care. This review attempts to establish a standardized approach to the evaluation and treatment of sacral and ischial wounds, and to describe some important differences. Topics reviewed include important elements of the history, physical, and laboratory studies of patients with wounds. Treatment issues are also addressed including wound care, pressure relief, and nutrition. The authors hope that the principles discussed will stimulate caregivers to examine their practices, and will serve as a starting point for formation of more exacting accepted care. While each patient and wound may require subtle nuances of care in certain areas, a more uniform set of standards will be the first step in creating delivery systems that are geared for large-scale prospective randomized studies that are so desperately needed in most areas of wound care. PMID- 10732633 TI - A clinical evaluation of 3M no sting barrier film. AB - The maintenance of healthy skin depends on factors such as moisture, the nutritional status of the patient, and mechanical forces. A barrier film can be a valuable adjunct in the prevention and treatment of injury to skin. One hospital evaluated barrier film, a new alcohol-free film-forming liquid skin protectant, in its geriatric and spinal cord rehabilitation units. A total of 33 patients were enrolled in the study. The purpose of the evaluation was to determine if barrier film reduced redness, assisted in the adhesion of dressing and condom catheters, prevented or reduced maceration, prevented or reduced skin stripping, and/or had any adverse effects on patients. Redness was reduced in 96% of patients who were at risk. Maceration was prevented in 94% of subjects, and skin stripping was prevented in 100% of patients. Dressing adhesion improved significantly in 90% of subjects. For all subjects, regardless of method of application, barrier film was easy to apply. No patient experienced any adverse effects during the study. On the basis of this clinical trial, we found barrier film to be an effective liquid skin sealant and protectant. PMID- 10732634 TI - Examination of care-planning needs for elderly newly admitted to an acute care setting. AB - This study examined characteristics of elderly people newly admitted to an acute care setting who should be included in routine care planning. The mean serum albumin for the 137 participants was 3.0 g/dL, and 75% (n = 102) needed assistance with activity/transfer and going up stairs. Of the participants, 32.4% (n = 44) experienced some degree of either fecal or urinary incontinence. Use of the nutritional, functional, and cognitive profile developed from this study will enable nurses and medical personnel to better plan care to prevent functional decline and improve nutritional status during hospitalization of elderly patients. PMID- 10732635 TI - Understanding the Balanced Budget Act of 1997. AB - The Balanced Budget Act of 1997 contained sweeping changes to Medicare that directly affect healthcare professionals in all clinical settings. If clinicians are to be successful in today's healthcare marketplace, they must have a working knowledge of these changes and the impact they have on their employers and patients. Economic viability of our country's healthcare providers depends on their ability to comply with federal, state, and local regulations while providing improved patient outcomes using fewer medical resources and healthcare dollars. Clinical practice, and healthcare professionals who provide it, must quickly adapt to these changes. PMID- 10732636 TI - Nonhealing wounds: recalcitrant, chronic, or not understood? PMID- 10732637 TI - Considerations for the global assessment and treatment of patients with recalcitrant wounds. AB - Many factors, both intrinsic and extrinsic, may contribute to wound recalcitrance. For example, arterial circulation may be impaired by atherosclerosis, vasospastic disorders, microemboli, thromboangiitis obliterans, vasculitis, sickle cell anemia, and antiphospholipid syndrome, all of which may impair healing. Inflammatory disorders that may lead to recalcitrance include pyoderma gangrenosum and necrobiosis lipoidica. Chronic venous insufficiency, infection, diabetes mellitus, systemic malignancy, malnutrition, and exposure to pressure and shear prolong the healing process. Wounds secondary to primary skin carcinoma will not heal. Calciphylaxis, a life-threatening metabolic disorder, leads to multiple ulcerations that are especially difficult to heal. Knowledge of common factors that lead to wound recalcitrance is essential to the wound care clinician, as accurate diagnosis results in appropriate treatment. To arrive at the diagnosis, the wound care clinician must perform a thorough history and physical examination and order relevant investigative studies. Treatment is based on correction of the identified underlying condition. By utilizing a systematic approach in the management of each patient with a chronic wound, the wound care clinician increases the probability of achieving wound closure. PMID- 10732638 TI - Managing the oncology wound: reconstructive issues in breast cancer. AB - Oncology wounds are related to both the patient's cancer and the treatment of the cancer. Patients who have oncology wounds suffer from significantly impaired wound healing. Treating these patients and their wounds successfully requires overcoming their healing impairment. Reconstructive surgeons are increasingly playing a role in oncologic wound management, particularly among breast cancer patients. Breast reconstruction is perhaps the best contemporary model of conquering healing impairment in cancer to successfully restore the patient. PMID- 10732639 TI - Wound healing at the local level: the stunned wound. AB - Many factors can negatively impact the wound-healing process leading to recalcitrant wounds. Biochemical and cellular commonalties can be identified across a number of wound etiologies. Healing data for various wound etiologies are presented, but these outcomes fail to show significant differences. Tissue perfusion, debridement, and wound fluid management can all impact healing. This review article discusses these topics and introduces a theoretical concept, the "stunned wound." PMID- 10732640 TI - Moisture and healing: beyond the jargon. AB - Moist wound healing is one of the most frequently used, but least understood terms in wound care. Although no reliable operational definitions exist of too little or too much wound surface moisture, a low dressing water vapor transmission rate (WVTR) has proven to be a reliable measure of a dressing's capacity to retain moisture and provide an environment that supports healing. Dressing WVTR is a powerful linear predictor of both full- and partial-thickness healing of standardized acute wounds in vivo (alpha < 0.0001). Moreover, differences in the ability to produce an optimal moist wound environment within a dressing category, such as hydrocolloid dressings, are strongly correlated with dressing WVTR, suggesting that dressings in the same product categories may be associated with significantly different environments for healing. Even though the correlation between dressing WVTR and healing rates is more difficult to ascertain, clinical healing percentages during similar time-frames and healing times of similar wounds also follow patterns predicted by dressing moisture retention. These results suggest that, when other variables are held constant, use of more moisture-retentive dressings generally achieves environments supportive of earlier healing outcomes when compared to less moisture-retentive dressings. Maceration, an unwelcome occurrence with moisture-retentive dressing use on highly exuding wounds, is not consistently associated with increased adverse events. Evidence further suggests that greater dressing moisture retention is associated with fewer clinical infections, greater patient comfort, and reduced scarring. PMID- 10732641 TI - Contact sensitivity and recalcitrant wounds. AB - Allergic contact dermatitis is a common and under-recognized phenomenon in patients with recalcitrant wounds. There are many possible causes including components of numerous agents used to treat these wounds, such as topical antibiotics, adhesives in dressings, emollients, emulsifiers, and self administered medicaments. Considering allergic contact dermatitis when a wound is recalcitrant is important in the management of wounds. Etiologic agents, controversies, pathogenesis, diagnosis, and management of contact sensitivity are discussed. PMID- 10732642 TI - Immunology, microbiology, and the recalcitrant wound. AB - Wounding of normal skin initiates an acute inflammatory response that ordinarily contributes to the healing process. The underlying process is orchestrated by the specific and nonspecific immune response. Inflammatory cells provide growth factors and stimulate the deposition of matrix proteins and phagocytose debris. However, the maturation and resolution of a wound may be complicated by micro organisms. The effects of micro-organisms on oxygen consumption and pH, or toxin production, may interrupt the natural course of wound healing. Thus, a wound may not progress from the acute phase and heal, but may become a nonhealing chronic or recalcitrant wound as long as the antigens from micro-organisms or underlying pathology remain. Depending on the underlying disease pathology and the micro organism's virulence, microbial growth in acute or chronic wounds may lead to invasive wound infection. Wound infection is a complex interaction involving the host as well as the numbers and types of micro-organisms present. The literature suggests that micro-organisms alter the course of acute wound healing, and a body of evidence exists that suggests that large numbers of organisms in chronic wounds delay the healing process. However, other evidence suggests that, despite bacteria, most chronic wounds progress toward healing, depending upon the wound care strategy employed. Current therapy seeks to alter the relationships between microbial colonization and host defenses by providing an environment that tips the balance in favor of healing. The role of bacteria in acute and chronic wounds may span the spectrum from initiation of inflammation and the healing process, to colonization, invasive infection, systemic sepsis, organ system failure, and death. Understanding the interaction of the wound, wound micro-organisms, and the immune response is central to understanding how best to develop therapeutic approaches. PMID- 10732644 TI - [New federal nursing legislation] PMID- 10732643 TI - Wound fluids: a reflection of the state of healing. AB - Analyzing acute and chronic wound fluids provides an important and intriguing insight into the wound milieu. This review outlines some of the salient features of wound repair and the wound fluid environment. Most studies support the premise that the contents of the wound fluid reflect the status of the wound and can be indicative of whether a wound is on the course of a normal or impaired response to injury. For example, chronic wound fluids often differ from acute wound fluids in their proliferative effects on cells active in healing as well as their proteolytic effects. The authors discuss various cytokines, growth factors, proteinases, and protease inhibitors within wound fluids as well as their effect on wound repair. This review also presents confounding factors affecting interpretation of wound fluid studies, suggesting that further studies need to elucidate mechanisms whereby wound fluids either enhance or inhibit wound repair. So far, wound fluid analysis has yielded tantalizing glimpses of the teeming wound environment. What wound fluid contents tell us about the wound or its clinical care is not yet certain. PMID- 10732645 TI - [To be ill and a foreigner in health care institutions]. AB - The majority of the people from foreign countries who search for help in the national health system are living in poverty and come from the rural areas near the mediterranean sea. They are here either as refugees for political reasons or because they are looking for work. Thus they have come to meet accultural processes of different sizes. In a subjective way they react to a failed acculturation by showing off a ruined self-confidence and a false motivation. Their situation towards the national health system is defined by various factors: 1. Original culture influence, 2. Ruined self-confidence and they are therefore prone to health defects, 3. Special form of symptom (pathoplastic), 4. Changed department to institutional things. In comparison with that the cultural situation of the institute's staff shows characteristic symptoms of life in a modern capitalistic society--which appears normal to them. If we compared their situation with the one of the migrants--always keeping the theory of cultural relativism in mind--we have to consider this relation as highly problematic. This lecture wants to critically illustrate the global comparison between the two basic positions of the migrants and the staff working for health institutions and draw conclusions. PMID- 10732646 TI - [From the foreign to the familiar: the discourse on intercultural care and its impact on nursing science]. AB - This article concerns the discourse on intercultural nursing which can stimulate the further development of nursing theories and practices. In introduction, the article outlines the core of the discourse and some formal aspects. Subsequently, the overlapping discourses concerning the situation of migrant people in the social sciences and in society are discussed and the conclusion is prompted that the discourse needs to become part of general nursing theories to avoid a "culturalization" of ist subject. PMID- 10732647 TI - [The understanding of health, illness and care by Turkish immigrant women. An empirical study]. AB - The opportunities for and parameters of individual care are determined by an understanding of the patient's sociocultural and biographical context. As a presupposition for patient-centred nursing care nurses, therefore, need a soundly based knowledge of the manifold cultural ways of coping with everyday demands, especially in crises as brought on by illness. In a qualitative study on the understanding of illness, health and care 10 Turkish women, all of whom are experienced in lay care within their own families, were interviewed. The interviews were analysed with reference to the "Development Research Sequence" of James Spradley. This method filters out cultural themes from the subjective views of the informants that centre round the topics of health, illness and care within the family, views that determine both the everyday knowledge and the activities of the women. The study is based on the two assumptions that firstly patient specific variables, sociocultural factors, the family and the social network have to be taken into consideration if professional nursing care is to succeed and secondly that the process of immigration has effects on the understanding of illness, health and care as well as on the lay care practices which are specific to each generation and become significant for the care of family members in immigrant families. Examples will be used in writing up the results of the enquiry. PMID- 10732648 TI - [Immigrants in geriatric institutional care]. AB - To an increasing extent, elder immigrants make use of institutional caring support. Immigrants in need of care--a new challenge or chance? Taking nursing homes as an example, it is shown that nursing even within our own cultural context is characterized by many phenomena of strangeness and unfamiliarity. Confronted with strangeness, nurses react with feelings of anxiety and fascination. Old people react predominantly with retreat in fantasies and confusion. Will the situation change fundamentally when elder immigrants are increasingly admitted into nursing homes? Which conditions should be created, so that we can accept strangeness as a ubiquitous phenomenon in nursing homes? And is there not a chance to introduce fundamental changes to our present concepts of nursing in general if we are willing to confront ourselves with issues of multicultural nursing? PMID- 10732649 TI - [The importance of cultural coding in nursing practice, nursing research and nursing science--components of a critical concept of culture]. AB - Based on the question, on which conditions the understanding of the strange is possible at all, this article points out--illustrated by research material on nursing practice, nursing education and nursing science--collective interpretative patterns that are based on binary codings like "nature-culture", "body-mind", "female-male", "strange-familiar", foreign-native", "you-we", "sick healthy", "black people-white people", etc. These patterns and the value judgements expressed by them are social constructions within specific in certain historical and cultural contexts. Yet people neither experience collective interpretative patterns nor recognize them as such, but deal with them as if they were natural facts. This (unrecognised) process of naturalization of social cultural phenomena provokes contradictions and conflicts in all fields of practical life--in nursing practice and nursing education too, which will be shown by examples. To describe and analyse these complex circumstances, an approach to a critical concept of culture is developed in this article. The approach includes social-cultural events, processes and structures as well as their implicated symbolization. However, this heuristic construct itself must not be naturalized and "essentialized". PMID- 10732650 TI - ["A small piece of normal life". A qualitative study on health care for chronically ill children in home care]. AB - This qualitative study deals with the subjective experiences of families with chronically-ill children and with experiences of the External Care Services (EPD) in Berlin, who exclusively conduct home-care for children. Learning to cope with chronic illness is a process that often demands professional help that extends beyond the time spent in hospital. Essential for the analysis performed here are the systemic positions put forward by v. Uexkull, Wesiack (psychosomatic medicine) and Friedemann (nursing science) who stress the connection between health and relationship. The parents' positive experiences reflect the patient oriented understanding of health-care developed by the takes in the families in many respects. Without the care offered by the EPD some children would have been torn out of their family environment due to hospitalizations. This would have resulted in the damaging of the family health. PMID- 10732652 TI - [Notes on the exchange about the study "Massage of the foot reflex zones" by A. Kesselring et al. in "Pflege" (12) 2/1999]. PMID- 10732651 TI - [Effect of various dressings on the point of entry of an intravenous drip]. AB - In modern health care systems nosocomial infection is a major cause of patient suffering and leads to high financial costs. Overall nursing care that is part of nursing staff's daily routine is not brought under scrutiny often enough. The goal of this research project is to look at a small part only of optimal intravenous drip care. The focus of the paper is the influence that the use of two different kinds of dressings and two different types of cleansing methods have on the general appearance, as well as the microbiological results at the point of entry of the i.v. drip into the central veins. In order to examine this, test patients from different types of intensive care units were used for an experiment under highly controlled conditions. The results show that a variation in nursing care of the i.v. drip leads to a variation in germ development and in the general appearance at the point of entry. PMID- 10732653 TI - Blue genes and the monoamine hypothesis of depression. PMID- 10732654 TI - Mood stabilizers during breastfeeding: a review. AB - BACKGROUND: The postpartum period is an exceptionally high-risk time for recurrence of depression, mania, or psychosis for women with bipolar disorder. Puerperal prophylaxis with mood stabilizers decreases this risk. To allow patients and clinicians to make informed decisions about mood-stabilizer use during breastfeeding, there is a need for a critical review and analysis of the data. DATA SOURCES: A search of MEDLINE (1966-1998) and the Lithium Database, Madison Institute of Medicine, was conducted to obtain articles about lithium, valproate, carbamazepine, gabapentin, or lamotrigine use during lactation. Search terms used were pregnancy, teratogenesis, breastfeeding, lactation, breast milk levels and lithium, anticonvulsants, mood stabilizers. No other search restrictions were used. Unpublished data on gabapentin and lamotrigine were provided by the manufacturers. RESULTS: The search revealed 11 cases of lithium use during breastfeeding, 8 of which reported infant serum levels. Two cases reported symptoms consistent with lithium toxicity in the infants. Thirty-nine cases of valproate use during breastfeeding were found, 8 of which reported infant serum levels. There was 1 report of thrombocytopenia and anemia in an infant. Fifty cases of carbamazepine use during breastfeeding were found, 10 of which reported infant serum levels. Two infants experienced hepatic dysfunction. One unpublished study of gabapentin in breast milk was found. Three reports of lamotrigine use during breastfeeding were found. DISCUSSION: Available information remains limited to uncontrolled studies and case reports. Carbamazepine and valproate, but not lithium, have generally been considered compatible with breastfeeding. The overall paucity of data, data confounded by polypharmacy and infant age differences, and adverse reactions reported with all established mood stabilizers dictate a reassessment of these recommendations. We propose that a woman's historical response to medication and the clinical circumstances be the primary considerations when choosing a mood stabilizer during breastfeeding, rather than strict adherence to categorical assignments. PMID- 10732655 TI - Prior benzodiazepine use and buspirone response in the treatment of generalized anxiety disorder. AB - BACKGROUND: An earlier preliminary report suggested that prior treatment with benzodiazepines might predict a reduced response to buspirone in patients diagnosed with generalized anxiety disorder (GAD). To confirm or refute this hypothesis, the present data analysis was conducted. METHOD: One large data set (N = 735) of GAD patients (DSM-III) treated with buspirone, a benzodiazepine, and a placebo was analyzed by dividing all patients into 3 prior benzodiazepine (BZ) treatment groups: no prior BZ treatment, recent (< 1 month) BZ treatment, and remote (> or = 1 month) BZ treatment. Using an intent-to-treat last-observation carried-forward (LOCF) data set, acute 4-week treatment response was assessed in terms of clinical improvement, attrition, and adverse events as a function of these 3 prior benzodiazepine treatment groups. RESULTS: Patient attrition was significantly higher (p < .05) in the recent BZ treatment group than in the remote and no prior BZ treatment groups with lack of efficacy given as the primary reason by patients receiving buspirone but not benzodiazepine or placebo. In the buspirone group, adverse events occurred more frequently in the recent BZ treatment group than in the remote BZ treatment and no prior BZ treatment groups. Finally, clinical improvement with buspirone was similar to benzodiazepine improvement in the no prior BZ treatment and remote BZ treatment groups, but less than benzodiazepine improvement in the recent BZ treatment group, leading to the smallest buspirone/placebo differences in improvement in the recent BZ treatment group. CONCLUSION: These data suggest that the initiation of buspirone therapy in GAD patients who have only recently terminated benzodiazepine treatment should be undertaken cautiously and combined with appropriate patient education. PMID- 10732656 TI - Randomized, double-blind comparison of venlafaxine and sertraline in outpatients with major depressive disorder. Venlafaxine 631 Study Group. AB - BACKGROUND: This 8-week, double-blind, randomized trial compared the efficacy and tolerability of venlafaxine and sertraline in patients with major depression. METHOD: Outpatients (N = 147) with DSM-IV major depressive disorder and a baseline 21-item Hamilton Rating Scale for Depression (HAM-D) score of at least 18 were randomly assigned to venlafaxine, 37.5 mg b.i.d., or sertraline, 50 mg once daily. From day 15, the doses could be increased to venlafaxine, 75 mg b.i.d., or sertraline, 50 mg b.i.d. Efficacy was assessed with the 21-item HAM-D, the Montgomery-Asberg Depression Rating Scale (MADRS), and the Clinical Global Impressions scale (CGI) using a modified intent-to-treat analysis. RESULTS: No significant differences were noted between treatments for mean HAM-D, MADRS, or CGI scores. At week 8, the HAM-D response rate was 83% with venlafaxine (N = 75) and 68% with sertraline (N = 72) (p = .05). A HAM-D score less than 10 was recorded in 68% of venlafaxine-treated and 45% of sertraline-treated patients at week 8 (p = .008). Among patients who increased their dose, the remission rate (HAM-D score < 10) was 67% with venlafaxine and 36% with sertraline at week 8 (p < .05). The overall discontinuation rate was 21% with venlafaxine and 17% with sertraline. The most common adverse events with venlafaxine were nausea, headache, and sweating and with sertraline were nausea, headache, and diarrhea. CONCLUSION: Among patients who increased their dose, approximately twice as many experienced a remission with venlafaxine, which is a more clinically relevant endpoint than response and represents the proportion of patients who have recovered or are well. PMID- 10732657 TI - Psychosocial functioning in women with premenstrual dysphoric disorder before and after treatment with sertraline or placebo. AB - BACKGROUND: The objective of this study was to evaluate the pretreatment psychosocial functioning of women with premenstrual dysphoric disorder (PMDD) and the effect of sertraline treatment on psychosocial functioning in these patients. METHOD: Two hundred forty-three women recruited from 12 university-affiliated sites and meeting DSM-IV criteria for PMDD completed 1 cycle of single-blind placebo and were randomly assigned to flexible dose sertraline or placebo for 3 cycles. Psychosocial functioning was assessed by the Daily Record of Severity of Problems (DRSP), the Social Adjustment Scale (SAS), and the Quality of Life Enjoyment and Satisfaction Questionnaire (Q-LES-Q). RESULTS: SAS scores during the follicular phase were similar to SAS scores of community norms, whereas the pretreatment SAS and Q-LES-Q scores during the luteal phase were similar to scores of women with depressive disorders. Sertraline was significantly more effective than placebo in improving psychosocial functioning as measured by the SAS, the Q-LES-Q, and the 3 DRSP items of impaired productivity, interference with social activities, and interference with relationships with others. Improvement in psychosocial functioning assessed by SAS and Q-LES-Q correlated with improvement in symptomatology assessed by the Clinical Global Impressions Improvement (CGI-I) scale and the Hamilton Rating Scale for Depression (HAM-D). Remitters (CGI-I score of 1) were more likely to function better at baseline and showed larger improvements in functioning and quality of life with treatment compared with nonremitters. CONCLUSION: Sertraline was superior to placebo in improving psychosocial functioning in women with PMDD as reflected by SAS, Q-LES Q, and DRSP measures. Functional improvement correlated with improvement in premenstrual symptomatology and was apparent by the second cycle of treatment. Comparison of pretreatment SAS scores in women with PMDD with the scores of other populations of women documents the degree of luteal phase functional impairment in women with PMDD and a relative absence of follicular phase impairment. PMID- 10732658 TI - Treatment of the interictal psychoses. AB - BACKGROUND: The interictal "schizophrenia-like" psychoses of epilepsy conventionally are treated with antipsychotic medication with uncertain results. In patients with these psychoses, a preceding and concomitant dysphoric disorder usually can be documented. Effectiveness of the pharmacologic treatment by the combination of drugs that is effective for severe interictal dysphoric disorders is demonstrated in a series of patients with interictal psychosis. METHOD: Patients were treated with the combination of a tricyclic antidepressant and a selective serotonin reuptake inhibitor, enhanced if necessary by a small amount of the atypical neuroleptic risperidone. The series consisted of 8 consecutive patients with interictal psychosis seen over a 20-month period. Two additional patients seen over the past 10 years who required a different therapeutic intervention were also included. RESULTS: Five of the 8 consecutive patients achieved full remission of their psychosis; 3 patients could not be reached for the full treatment effort. One patient with a malignant psychosis had been treated successfully (prior to the series reported) by surgical removal of a left frontal epileptogenic zone; a second patient (treated after the series) recovered only upon elimination of the antiepileptic drug that had suppressed clinical seizures but had resulted in an alternating psychosis. CONCLUSION: Interictal psychoses can be viewed as severe interictal dysphoric disorders with psychotic features. The same combination of psychotropic medication that is effective for severe interictal dysphoric disorders serves as the primary therapy for interictal psychoses. The interictal psychiatric disorders presumably result from seizure-suppressing mechanisms that are the targets of the proconvulsant drugs. Upon suppression of seizures, some patients with interictal psychosis may require modification of the antiepileptic medication responsible for excessive inhibition. Complete surgical removal of the epileptogenic zone can eliminate a chronic interictal psychosis upon postoperative fading of inhibitory mechanisms. PMID- 10732659 TI - Use of sodium valproate in violent and aggressive behaviors: a critical review. AB - BACKGROUND: Valproate was initially introduced as an antiepileptic agent in 1967, but has been used over the years to treat a variety of psychiatric disorders. Its use in the treatment of patients exhibiting aggressive and violent behaviors has been reported in the literature as far back as 1988. However, these reports are uncontrolled, which is in marked contrast to the actual wide and established use of valproate for the treatment of aggressive behaviors. The aim of this report is to critically review the available data on valproate's use in nonbipolar patients with aggressive and violent behaviors. DATA SOURCES: The MEDLINE and PsycLIT databases were searched for all reports published from 1987-1998 containing the keywords valproate, the names of all commercial preparations, aggression, and violence. STUDY FINDINGS: Seventeen reports with a total of 164 patients were located. Ten of these were case reports with a total of 31 patients. Three were retrospective chart reviews with 83 patients, and 3 were open-label prospective studies with a total of 34 patients. No double-blind, placebo-controlled study could be found. An overall response rate of 77.1% was calculated when response was defined as a 50% reduction of target behavior. Most frequent diagnoses recorded were dementia, organic brain syndromes, and mental retardation. The antiaggressive response usually occurred in conjunction with other psychotropic medication. The dose and plasma valproate level required for response appeared to be the same as in the treatment of seizure disorders. DISCUSSION: While valproate's general antiaggressive effect is promising, in the absence of controlled data, conclusions are limited at this time. Specific recommendations for study design are given to obtain interpretable data for this indication. PMID- 10732660 TI - The alpha1-adrenergic antagonist prazosin ameliorates combat trauma nightmares in veterans with posttraumatic stress disorder: a report of 4 cases. AB - BACKGROUND: Central nervous system (CNS) adrenergic hyperresponsiveness may be involved in the pathophysiology of posttraumatic stress disorder (PTSD). Two Vietnam combat veterans with PTSD prescribed the centrally active alpha1 adrenergic antagonist prazosin for symptoms of benign prostatic hypertrophy unexpectedly reported elimination of combat trauma nightmares. This observation prompted an open-label feasibility trial of prazosin for combat trauma nightmares in chronic combat-induced PTSD. METHOD: Four consecutively identified combat veterans with chronic DSM-IV PTSD and severe intractable combat trauma nightmares participated in an 8-week open trial of escalating-dose prazosin. Nightmare severity response was rated using the nightmare item of the Clinician Administered PTSD Scale and the Clinical Global Impressions-Change scale. RESULTS: The 2 patients who achieved a daily prazosin dose of at least 5 mg were markedly improved, with complete elimination of trauma nightmares and resumption of normal dreaming. The 2 subjects limited to 2 mg of prazosin to avoid excessive blood pressure reduction were moderately improved with at least 50% reduction in nightmare severity. CONCLUSION: These clinical observations, together with neurobiological evidence for alpha1-adrenergic regulation of CNS neurobiological systems relevant to PTSD, provide rationale for placebo-controlled trials of prazosin for PTSD combat trauma nightmares. PMID- 10732661 TI - Twelve-month outcome in bipolar patients with and without personality disorders. AB - BACKGROUND: We studied the 12-month course of illness after hospitalization for patients with a DSM-III-R diagnosis of bipolar disorder, manic or mixed episode, to identify the impact of a co-occurring personality disorder on measures of outcome. METHOD: Fifty-nine patients with bipolar disorder hospitalized for the treatment of a manic or mixed episode were recruited. Diagnostic, symptomatic, and functional evaluations were obtained at the index hospitalization. Personality disorders were assessed using the Structured Clinical Interview for DSM-III-R, personality disorders version (SCID-II). Patients were then reevaluated at 2, 6, and 12 months after discharge to assess syndromic, symptomatic, and functional recovery. Factors associated with outcome were identified using multivariate analyses. RESULTS: Survival analyses showed that in the 12-month follow-up period, subjects with bipolar disorder and co-occurring personality disorder were significantly less likely to achieve recovery. Logistic regression analyses indicated that both a diagnosis of personality disorder and noncompliance with treatment were significantly associated with lack of syndromic recovery. CONCLUSION: Co-occurring personality disorders in patients with bipolar disorder are associated with poor outcome after hospitalization for mania. PMID- 10732662 TI - Clozapine in the treatment of neuroleptic-induced blepharospasm: a report of 4 cases. AB - BACKGROUND: Blepharospasm, the forcible closure of eyelids, is an infrequent consequence of neuroleptic treatment that, when severe, can interfere with the ability to walk, drive, or work. Like tardive dyskinesia, blepharospasm can be disfiguring and aesthetically distressing, contributing to the increased stigmatization of patients. CASE REPORTS: We report 4 patients with DSM-IV schizoaffective disorder, paranoid schizophrenia, or chronic undifferentiated schizophrenia who developed neuroleptic-induced blepharospasm. In all patients, blepharospasm remitted without the reemergence of psychosis within 3 to 5 months of treatment with clozapine, 100-200 mg/day. CONCLUSION: The results suggest that clozapine may successfully treat neuroleptic-induced blepharospasm without the reemergence of psychosis in patients with schizophrenia, schizoaffective disorder, or schizophreniform disorder. PMID- 10732663 TI - Lack of desipramine toxicity with citalopram. PMID- 10732664 TI - Subtyping micromanic episodes. PMID- 10732665 TI - Clarifying the safety of the MAOI diet and pizza. PMID- 10732666 TI - A case report of serotonin syndrome associated with combined nefazodone and fluoxetine. PMID- 10732667 TI - Sensory phenomena in obsessive-compulsive disorder and Tourette's disorder. AB - BACKGROUND: Recent studies have suggested that obsessive-compulsive disorder (OCD) is a heterogeneous disorder with some forms related to tics and Tourette's disorder. The present study was undertaken to investigate the sensory phenomena in patients with OCD and/or Tourette's disorder to determine if these phenotypic features represent valid clinical indices for differentiating tic-related OCD from non-tic-related OCD. METHOD: We evaluated 20 adult outpatients with OCD, 20 with OCD plus Tourette's disorder, and 21 with Tourette's disorder, using a semistructured interview designed to assess several definitions of sensory phenomena reported in the literature. DSM-III-R criteria were used for the OCD and Tourette's disorder diagnoses. RESULTS: Sensory phenomena including bodily sensations, mental urges, and a sense of inner tension were significantly more frequent in the 2 Tourette's disorder groups when compared with the OCD alone group. Feelings of incompleteness and a need for things to be "just right" were reported more frequently in the OCD plus Tourette's disorder group compared with the other 2 groups. CONCLUSION: Sensory phenomena may be an important phenotypic measure for grouping patients along the OCD-Tourette's disorder spectrum. Sensory phenomena include bodily and mental sensations. Bodily sensations include focal or generalized body sensations (usually tactile, muscular-skeletal/visceral, or both) occurring either before or during the patient's performance of the repetitive behaviors. These sensations are more frequently found in patients with OCD plus Tourette's disorder than in patients with OCD alone. Mental sensations include urge only, energy release (mental energy that builds up and needs to be discharged), incompleteness, and just-right perceptions. They are all more frequently found in patients with OCD plus Tourette's disorder than in patients with OCD alone. PMID- 10732668 TI - The 51,409-bp R-plasmid pTP10 from the multiresistant clinical isolate Corynebacterium striatum M82B is composed of DNA segments initially identified in soil bacteria and in plant, animal, and human pathogens. AB - The 51,409-bp DNA sequence of the multiresistance plasmid pTP10 from the gram positive opportunistic human pathogen Corynebacterium striatum M82B has been determined. Fully automated genome interpretation led to the identification of 47 ORFs. Analysis of the genetic organization of pTP10 suggests that the plasmid is composed of eight DNA segments, the boundaries of which are represented by transposons and insertion sequences. The DNA segments of pTP10 are highly similar to (1) a plasmid-encoded erythromycin resistance region from the human pathogen Corynebacterium diphtheriae; (2) a chromosomal DNA region from Mycobacterium tuberculosis; (3) a plasmid-encoded chloramphenicol resistance region from the soil bacterium Corynebacterium glutamicum; (4) transposable elements from phytopathogenic gram-negative Pseudomonas, Xanthomonas and Erwinia species; and (5) a plasmid-encoded aminoglycoside resistance region from the gram-negative fish pathogen Pasteurella piscicida. The complete DNA sequence of pTP10 provides genetic information regarding the mechanisms of resistance to 16 antimicrobial agents that belong to six structural classes. In addition, the mosaic structure of pTP10 represents the evolutionary consolidation into a single plasmid molecule of antimicrobial resistances from microorganisms found in different habitats by means of mobile elements, resulting in the generation of a multiresistant bacterium that can infect humans. PMID- 10732669 TI - HD-Zip proteins of families I and II from rice: interactions and functional properties. AB - Proteins of the closely related homeodomain-leucine zipper (HD-Zip) families I and II in plants are putative transcription factors that interact with similar pseudopalindromic DNA recognition sites. We have previously described the Oshox1 gene from rice, which encodes an HD-Zip II protein. To identify further rice HD Zip proteins, one-hybrid screens were performed in yeast strains containing a HIS3 reporter gene with upstream HD-Zip recognition sites. This resulted in the isolation of six new cDNAs encoding HD-Zip proteins belonging to family I (Oshox4, -5, -6) or family II (Oshox2, -3, -7). In transient assays, using rice suspension-cultured cells transformed by particle bombardment, we showed previously that Oshox1 can transcriptionally repress the activity of reporter gene constructs with upstream HD-Zip binding sites. Here, we confirm the repression properties of Oshox1 by showing that the repression function can be conferred on a heterologous DNA-binding domain. This portable functional domain (residues 1-155) is located proximal to the HD-Zip domain. In yeast, the same region of the Oshox1 protein was found to confer transcriptional activation instead of repression, pointing to the possibility that cell type-specific factors may determine the functional properties of the Oshox1 protein in rice. Like Oshox1, another HD-Zip family II protein (Oshox3) was also found to function as a transcriptional repressor in rice cells. In contrast, two HD-Zip I family proteins (Oshox4 and -5) appeared to act as activators in both rice and yeast cells. Results of two-hybrid assays and electrophoretic mobility shift assays strongly suggest that all HD-Zip proteins of families I and II can form homodimers and also heterodimers with all HD-Zip proteins of the same family. Heterodimerization across the HD-Zip families I and II apparently does not to occur. PMID- 10732670 TI - Intrachromosomal homologous recombination in Arabidopsis induced by a maize transposon. AB - In plants, the frequency of spontaneous intrachromosomal homologous recombination is low. Here, we show that a maize transposable element greatly stimulates intrachromosomal homologous recombination between direct repeat sequences in Arabidopsis. Plants were transformed with a construct (GU-Ds-US) containing a Ds (Dissociation) transposable element inserted between two partially deleted GUS reporter gene segments. Homologous recombination between the overlapping GUS fragments generates clonal sectors visible upon staining for GUS activity. Plants containing the GU-Ds-US construct and a source of Ac (Activator) transposase showed an over 1000-fold increase in the incidence of recombination relative to plants containing the same construct but lacking transposase. Transposon-induced recombination was observed in vegetative and floral organs, and several germinally transmitted events were recovered. Transposon-induced recombination appears to be a general phenomenon in plants, and thus may have contributed to genome evolution by inducing deletions between repeated sequences. PMID- 10732671 TI - Rapid systemic accumulation of transcripts encoding a tobacco WRKY transcription factor upon wounding. AB - An immediate-early, transiently activated wound-responsive gene was identified in tobacco by fluorescent differential display screening. The full-length cDNA encodes a polypeptide of 356 amino acids with a relative molecular mass of 39,082 Da. The deduced amino acid sequence shows two characteristic features; a leucine zipper motif found in the more N-terminal region and a WRKY domain containing a zinc-finger motif located in the central region. The gene was designated as wizz (wound-induced leucine zipper zinc finger). Northern analysis showed that upon wounding wizz transcripts were locally and systemically accumulated within 10 min, reached a maximum level by 30 min, and decreased thereafter to the basal level. Analyses of a WIZZ-GFP fusion protein clearly indicated that WIZZ is a nuclear factor. WIZZ specifically binds to sequences containing two TTGAC core motifs that are separated by a spacer of appropriate length. The binding activity was dependent on bivalent cations, most probably zinc. In transient reporter assays, however, WIZZ did not show transactivation activity in tobacco suspension cells, suggesting that it functions together with other components. The results indicate that WIZZ is a new transcription factor which participates in early stages of the wound response. PMID- 10732672 TI - Histone hyperacetylation facilitates chromatin remodelling in a Drosophila embryo cell-free system. AB - We found that Drosophila embryo extract contains a protein activity (or activities) that can destabilize nucleosomes, resulting in increased sensitivity to DNase I, release of nucleosomal supercoiling, high levels of conformational flexibility of DNA and more diffuse micrococcal nuclease digestion patterns. Incorporation of histone H1 did not significantly affect this nucleosome remodelling. Remodelling occurs more efficiently in hyperacetylated chromatin. It was shown previously that hyperacetylated chromatin, reconstituted in a Drosophila embryo cell-free system, exhibits increased DNase I sensitivity and a high degree of conformational flexibility of DNA. The present data suggest that the more diffuse structure of acetylated chromatin is a result of chromatin remodelling by protein activities in the Drosophila embryo extract. PMID- 10732673 TI - Activation of basal transcription by a mutation in SIN4, a yeast global repressor, occurs through a mechanism different from activator-mediated transcriptional enhancement. AB - The Saccharomyces cerevisiae protein Sin4 has been suggested to affect the transcription of various genes by locally altering chromatin structure. Previous studies have defined two classes of promoters: those which are activated by loss of SIN4 function (termed sin4-responsive promoters) and those which are not activated by sin4 mutations (termed sin4 non-responsive promoters). We analyzed the mechanism of this differential response of the two classes of promoters to a sin4 mutation. The sin4 non-responsive promoters were activated when upstream elements in the promoter region were eliminated. The upstream elements of sin4 non-responsive promoters were, in turn, found to repress the activity of the sin4 responsive promoters in an orientation-independent manner. The sin4-mediated activation was repressed by the Rme1- but not by the Tup1-Ssn6-mediated repression system. Activation of sin4-responsive promoters by Pho4 and the sin4 mutation was additive, and enhancement of transcription driven by sin4-responsive promoters was found to be due to an increase in the basal rate of transcription. The upstream regions in the sin4 non-responsive promoters contained elements that were able to inhibit activation of basal transcription. Based on these observations, we suggest that activation of basal transcription by a mutation in a gene for a global repressor, SIN4, occurs through a mechanism that differs from that responsible for activator-mediated transcriptional enhancement, and we therefore propose that basal transcription and activator-mediated transcription are repressed by different mechanisms. PMID- 10732674 TI - Mutations in CDC14 result in high sensitivity to cyclin gene dosage in Saccharomyces cerevisiae. AB - We screened for mutations that resulted in lethality when the G1 cyclin Cln2p was overexpressed throughout the cell cycle in Saccharomyces cerevisiae. Mutations in five complementation groups were found to give this phenotype, and three of the mutated genes were identified as MEC1, NUP170, and CDC14. Mutations in CDC14 may have been recovered in the screen because Cdc14p may reduce the cyclin B (Clb) associated Cdc28 kinase activity in late mitosis, and Cln2p may normally activate Clb-Cdc28 kinase activity by related mechanisms. In agreement with the idea that cdc14 mutations elevate Clb-Cdc28 kinase activity, deletion of the gene for the Clb-Cdc28 inhibitor Sic1 caused synthetic lethality with cdc14-1, as did the deletion of HCT1, which is required for proteolysis of Clb2p. Surprisingly, deletion of the gene for the major B-type cyclin, CLB2, also caused synthetic lethality with the cdc14-1 mutation. The clb2 cdc14 strains arrested with replicated but unseparated DNA and unseparated spindle pole bodies; this phenotype is distinct from the late mitotic arrest of the sic1::TRP1 cdc14-1 and the cdc14-1 hct1::LEU2 double mutants and of the cdc14 CLN2 overexpressor. We found genetic interactions between CDC14 and the replication initiator gene CDC6, extending previous observations of interactions between the late mitotic function of Cdc14p and control of DNA replication. We also describe genetic interactions between CDC28 and CDC14. PMID- 10732675 TI - RAD52-dependent and -independent homologous recombination initiated by Flp recombinase at a single FRT site flanked by direct repeats. AB - We have devised a system for isolating yeast DNA sequences that are able to act as initiators of recombination leading to deletions in mitotically growing yeast cells. This system has allowed us to identify the FRT site of the 2-micron site specific recombinase Flp as such a sequence. We show that Flp is able to initiate recombination leading to deletions at a single FRT site in cir(o) strains. These results indicate that Flp is able to cleave a single FRT site, supporting the observation that the mechanism of cleavage by Flp is trans-horizontal. Interestingly, Flp can induce homologous recombination in both a RAD52-dependent and RAD52-independent manner. Our work provides a new system for the study of homologous recombination leading to deletions, in which the initiation step can be efficiently controlled. We discuss the possibility that Flp-induced, RAD52 independent events occur by single-strand annealing. PMID- 10732676 TI - Involvement of the inverted repeat of the yeast 2-micron plasmid in Flp site specific and RAD52-dependent homologous recombination. AB - Site-specific recombination within the Saccharomyces cerevisiae 2-micron DNA plasmid is catalyzed by the Flp recombinase at specific Flp Recognition Target (FRT) sites, which lie near the center of two precise 599-bp Inverted Repeats (IRs). However, the role of IR DNA sequences other than the FRT itself for the function of the Flp reaction in vivo is not known. In the present work we report that recombination efficiency differs depending on whether the FRT or the entire IR serves as the substrate for Flp. We also provide evidence for the involvement of the IR in RAD52-dependent homologous recombination. In contrast, the catalysis of site-specific recombination between two FRTs does not require the function of RAD52. The efficiency of Flp site-specific recombination between two IRs cloned in the same orientation is about one hundred times higher than that obtained when only the two FRTs are present. Moreover, we demonstrate that a single IR can activate RAD52-dependent homologous recombination between two flanking DNA regions, providing new insights into the role of the IR as a substrate for recombination and a new experimental tool with which to study the molecular mechanism of homologous recombination. PMID- 10732677 TI - Acquisition of a potential marker for insect transformation: isolation of a novel alcohol dehydrogenase gene from Bactrocera oleae by functional complementation in yeast. AB - The alcohol dehydrogenase genes make up one of the best studied gene families in Drosophila, both in terms of expression and evolution. Moreover, alcohol dehydrogenase genes constitute potential versatile markers in insect transformation experiments. However, due to their rapid evolution, these genes cannot be cloned from other insect genera by DNA hybridization or PCR-based strategies. We have therefore explored an alternative strategy: cloning by functional complementation of appropriate yeast mutants. Here we report that two alcohol dehydrogenase genes from the medfly Ceratitis capitata can functionally replace the yeast enzymes, even though the medfly and yeast genes have evolved independently, acquiring their enzymatic function convergently. Using this method, we have cloned an alcohol dehydrogenase gene from the olive pest Bactrocera oleae. We conclude that functional complementation in yeast can be used to clone alcohol dehydrogenase genes that are unrelated in sequence to those of yeast, thus providing a powerful tool for isolation of dominant insect transformation marker genes. PMID- 10732678 TI - Functional analysis of the minor subunits of S fimbrial adhesion (SfaI) in pathogenic Escherichia coli. AB - S fimbrial adhesins I and II (SfaI and II), produced by extraintestinal Escherichia coli pathogens that cause urinary tract infections (UTI) and newborn meningitis (NBM), respectively, mediate bacterial adherence to sialic acid containing glycoprotein receptors present on host epithelial cells and extracellular matrix. The S fimbrial adhesin complexes consist of four proteins: SfaI-A, the major subunit protein and the minor subunit proteins SfaI-G, SfaI-S and SfaI-H. Sialic acid-specific binding is mediated by the minor subunit protein SfaI-S. In order to determine whether the minor subunit proteins SfaI-G, -S and H play a role in the modulation of adherence and the degree of fimbriation, a trans-complementation system was developed. A non-adhesive E. coli K-12 derivative, harbouring the sfaI-A gene but lacking sfaI-G, -S and -H, was transformed with sfaI-G, -S or -H. Only SfaI-S was able to increase the degree of fimbriation and to confer adhesion properties on the recombinant E. coli K-12 strains. Amino acid residues in SfaI-S that are involved in modulation of fimbriation as well as in receptor recognition were localized by random and site directed mutagenesis. PMID- 10732679 TI - Alternative silencing effects involve distinct types of non-spreading cytosine methylation at a three-gene, single-copy transgenic locus in rice. AB - We investigated transgene silencing in a line of rice plants that carries a single-copy 6.6-kb transgenic locus comprising three heterologous transgenes: bar, hpt and gusA. We identified at least three distinct types of silencing effects associated with different methylation patterns, including a novel form of transcriptional silencing involving methylation of cytosine residues only at non conventional acceptor sites in the coding region. Silencing arose de novo in individual R1, R2 and R3 plants despite the stability of the transgenic locus, although the basic structure of the locus, transgene dosage and position effects remained constant within the line. We found that different silencing effects could occur concurrently in adjacent heterologous transgenes in the same plant, with no evidence for spreading of silenced states or methylation patterns from one transgene to another. PMID- 10732680 TI - Thermostability, oligomerization and DNA-binding properties of the regulatory protein ArgR from the hyperthermophilic bacterium Thermotoga neapolitana. AB - The hexameric regulatory protein ArgR formed by arginine-mediated dimerization of identical trimers governs the expression of genes required for arginine metabolism and some other genes in mesophilic and moderately thermophilic bacteria. We have cloned the argR gene from two hyperthermophilic bacteria of the genus Thermotoga. The two-domain ArgR proteins encoded by T. neapolitana and T. maritima share a low degree of sequence similarity with other bacterial arginine repressors. The ArgR protein from T. neapolitana binds to an operator located just upstream of its coding sequence and, therefore, the argR gene may be autoregulated. The protein has extremely high intrinsic thermostability and tolerance to urea. Moreover, its binding to target DNA increases the melting temperature by approximately 15 degrees C. The formation of oligomeric ArgR-DNA complexes is a function of protein concentration, with hexameric complexes being favoured at higher concentrations. In the presence of arginine the hyperthermophilic ArgR protein binds to its own operator, argRo, only by forming hexamer ArgR-DNA complexes, whereas both trimer-DNA and hexamer-DNA complexes are detected in the absence of arginine. However, the affinity of T. neapolitana ArgR for DNA has been found to be higher for a mixture of trimers and non-bound hexamers than for arginine-bound hexamers. Our data indicate that genes for arginine biosynthesis are clustered in a putative operon, which could also be regulated by the ArgR protein, in the hyperthermophilic host. PMID- 10732681 TI - SecG is an auxiliary component of the protein export apparatus of Escherichia coli. AB - SecY, SecE and SecG form the membrane-embedded core complex of the Escherichia coli protein export apparatus. These three proteins co-purify and can be co immunoprecipitated, demonstrating that they are closely associated. While SecE and SecY are generally accepted as essential components of translocase, the role of SecG is more ambiguous. It is commonly believed that deletion of secG causes a cold-sensitive phenotype and a severe defect in export, even though some reports have indicated otherwise. However, we demonstrate that deletion of secG does not produce a cold-sensitive phenotype or a strong export defect in most genetic backgrounds. The more common result is that deletion of secG causes only a mild export defect and does not result in conditional lethality. We propose that the role of SecG is not fundamental to the export process, but is merely auxiliary- as suggested previously by biochemical data--and is physiologically important only when cells are otherwise compromised. PMID- 10732682 TI - Genetic characterization of the 44D-45B region of the Drosophila melanogaster genome based on an F2 lethal screen. AB - We have performed an F2 genetic screen to identify lethal mutations that map to the 44D-45B region of the Drosophila melanogaster genome. By screening 8500 mutagenized chromosomes for lethality over Df(2R)Np3, a deficiency which encompasses nearly 1% of the D. melanogaster euchromatic genome, we recovered 125 lines with lethal mutations that represent 38 complementation groups. The lethal mutations have been mapped to deficiencies that span the 44D-45B region, producing an approximate map position for each complementation group. Lethal mutations were analyzed to determine the phase of development at which lethality occurred. In addition, we have linked some of the complementation groups to P element-induced lethals that map to 44D-45B, thus possibly providing new alleles of a previously tagged gene. Some of the complementation groups represent potentially novel alleles of previously identified genes that map to the region. Several genes have been mapped by molecular means to the 44D-45B region, but do not have any reported mutant alleles. This screen may have uncovered mutant alleles of these genes. The results of complementation tests with previously identified genes in 44D-45B suggests that over half of the complementation groups identified in this screen may be novel. PMID- 10732683 TI - The mus308 locus of Drosophila melanogaster is implicated in the bypass of ENU induced O-alkylpyrimidine adducts. AB - The mus308 locus of D. melanogaster was originally characterized by virtue of a mutant phenotype that resulted in specific hypersensitivity to cross-linking agents. However, the gene product has also been implicated in the repair of lesions other than cross-links. The gene was recently sequenced, and it encodes a protein with motifs characteristic of both DNA polymerases and helicases. We present mutability studies, using the recessive lethal (RL) test, which show that N-ethyl-N-nitrosourea (ENU) induces hypermutability in mus308-deficient conditions, although only in early broods. Further studies elucidated the role of MUS308 in repair processes by characterizing the spectrum of molecular mutations induced by in vivo ENU in postmeiotic germ cells, in mus308 conditions. These revealed that, in comparison to repair-proficient conditions, there is an increase in the frequency of GC --> AT and AT --> GC transitions, and AT --> TA transversions. Moreover, frameshift mutations, which have not previously been reported to form part of the ENU spectrum, were also found. These results indicate that MUS308 is needed to process ENU-induced lesions, and support the hypothesis that the mus308 gene plays a role in post-replication bypass of O alkylpyrimidines, probably mediated by recombination, which serves to increase the time available for error-free repair of these persistent and highly mutagenic lesions. PMID- 10732684 TI - Minicircular plastid DNA in the dinoflagellate Amphidinium operculatum. AB - Plastid DNA was purified from the dinoflagellate Amphidinium operculatum. The genes atpB, petD, psaA, psbA and psbB have been shown to reside on single-gene minicircles of a uniform size of 2.3-2.4 kb. The psaA and psbB genes lack conventional initiation codons in the expected positions, and may use GTA for translation initiation. There are marked biases in codon preference. The predicted PsbA protein lacks the C-terminal extension which is present in all other photosynthetic organisms except Euglena gracilis, and there are other anomalies elsewhere in the predicted amino acid sequences. The non-coding regions of the minicircles contain a "core" region which includes a number of stretches that are highly conserved across all minicircles and modular regions that are conserved within subsets of the minicircles. PMID- 10732685 TI - The lipopolysaccharide of recipient cells is a specific receptor for PilV proteins, selected by shufflon DNA rearrangement, in liquid matings with donors bearing the R64 plasmid. AB - Shufflon DNA rearrangement selects one of seven PilV proteins with different C terminal segments, which then becomes a minor component of the thin pili of Escherichia coli strains bearing the plasmid R64. The PilV proteins determine the recipient specificity in liquid matings. A recipient Escherichia coli K-12 strain was specifically recognized by the PilVA', -C, and -C' proteins, while E. coli B was recognized only by the PilVA' protein. To identify specific PilV receptors in the recipient bacterial cells, R64 liquid matings were performed using various E. coli K-12 waa (rfa) mutants and E. coli B transformants as recipient cells. E. coli K-12 waa mutants lack receptors for specific PilV proteins. E. coli B cells carrying waaJ or waaJKL genes of E. coli K-12 were recognized by donors expressing the PilVC' protein or the PilVC and -C' proteins, respectively, in addition to the PilVA' protein. Addition of E. coli K-12 or B lipopolysaccharide (LPS) specifically inhibited liquid matings. We conclude that the PilV proteins of the thin pili of R64-bearing donors recognize LPS molecules located on the surface of various recipient bacterial cells in liquid matings. PMID- 10732686 TI - Mapping of centromeric regions on the molecular linkage map of rice (Oryza sativa L.) using centromere-associated sequences. AB - Genetic mapping of centromeres has been a challenge for plant geneticists. The objective of this study was to develop a new strategy for determining the locations of centromeric regions on genetic maps by mapping centromere-associated sequences, to make it possible to define the centromeric region of each chromosome as a single Mendelian locus on the molecular linkage map. Two DNA probes containing sequences specifically associated with the centromeres of grass species were used for genetic mapping. The centromere-associated sequences for all 12 rice chromosomes were mapped on the molecular map with either or both of the probes, and flanking molecular markers on one or both sides were localized 0 to 8 cM away. The map locations of the centromere-associated markers corresponded very well with the positions of centromeric regions determined previously using trisomic analyses for 11 of the 12 chromosomes. The precise mapping of the centromeric regions using these probes makes the molecular map a more complete and informative tool for genomic studies, which will facilitate studies of the structure and function of the rice centromeres. The simplicity of this technique, together with the fact that these probes are also associated with the centromeric regions in other grass species, may provide a general approach to the mapping of centromeric regions in the genomes of other cereal crops. PMID- 10732688 TI - Computerized classification of corpus cavernosum electromyogram signals by the use of discriminant analysis and artificial neural networks to support diagnosis of erectile dysfunction. AB - Corpus cavernosum electromyogram (CC-EMG) provides diagnostic information on cavernous autonomic innervation and a measure of the degree to which the cavernous smooth muscle cells are intact. The complicated CC-EMG is evaluated and used in the diagnosis of patients suffering from erectile dysfunction. The evaluation procedure has been simplified by applying digital signal processing techniques. Since mathematically-based interpretations require quantitative data, spectral analysis was performed. The derived biosignals were analyzed by fast Fourier transform (FFT). Besides various other spectral parameters, specific frequency bands were determined in the power spectrum using factor analysis. The parameters were used for the computerized classification of normal and pathological CC-EMG data and the classification was performed using two independent methods: discriminant analysis (DA) and artificial neural networks (ANN). A medical expert analyzed a total of 200 CC-EMG recordings from patients with and without erectile dysfunction and separated these into normal (136) and pathological (64) cases. Although each independent method had already resulted in a relatively high number of correct classifications, the classification success rate could be slightly improved by using a combination of both classification methods. A total of 72.79% and 77.94% were successfully classified using DA and ANN, respectively. The combination of both methods increased the classification success to 80.15%. The results of this study enabled impartial evaluation of the CC-EMG signals for clinical diagnostic purposes of erectile dysfunction. This method provided an objective and easy way to analyze the CC-EMG. Furthermore, this results in patient diagnosis becoming an easier task for less experienced doctors, since little knowledge of the raw signal is needed. PMID- 10732687 TI - Interstitial fluid pressure is increased in renal cell carcinoma xenografts. AB - The purpose of this study was to test the hypothesis that renal cell carcinoma (RCC) exhibits an increased intratumoral interstitial fluid pressure (IT-IFP). Therefore, resected tumors from human primary (n = 23) or metastatic RCC (n = 3) were xenografted in SCID mice. The IFP of single tumor nodules (n = 65) and normal mouse tissue (n = 195) was measured by means of the "wick-in-needle" technique. Data demonstrate that the mean IT-IFP at neoplasia was 35 times greater than in normal tissue, and decreased precipitously at the tumor boundary. IT-IFP values tended to increase with the grade of malignancy of the tumor cells and tumor size. The mean IT-IFP of xenografts derived from primary RCC was twice as high as that from metastatic RCC tissue. These findings indicate a biophysical barrier to drug delivery in RCC; this may, in concert with cellular-based drug resistance mechanisms, be an additional explanation for resistance of the tumor to certain blood-borne anticancer therapies. PMID- 10732689 TI - Impaired endothelium-dependent and neurogenic relaxation of corpus cavernosum from diabetic rats: improvement with L-arginine. AB - This study describes the relaxant response to acetylcholine, electrical field stimulation and sodium nitroprusside after contraction by phenylephrine (10(-5) M) in corpus cavernosum from control and diabetic rats. The response to acetylcholine (10(-9)-10(-3) M) and electrical field stimulation (0.5-64 Hz) is decreased and can be restored by the addition of nitric oxide synthetase substrate, L-arginine(10(-5) M). The response to sodium nitroprusside is not changed in diabetic rats compared to control rats. NADPH-diaphorase staining was enhanced in a diabetic preparation compared to control preparations. The findings suggest a role for the depletion of L-arginine in diabetes mellitus. The enhanced NADPH-diaphorase staining may be due to a deficiency of NOS substrate L-arginine in the endothelium and nerves of diabetic tissues. PMID- 10732690 TI - Seminal arginase activity in infertility. AB - Arginase (Arg) activity in seminal plasma and sperm cells from infertile men and healthy fertile donors was measured. There were no statistically meaningful differences in seminal plasma Arg activity between the two groups whereas sperm cells from oligospermic infertile men had a higher Arg activity compared with the controls. Some important correlations were established between sperm count and Arg activity (negative values) and sperm motility and Arg activity (positive values) in both sperm cells and plasma samples from infertile men. Results suggest that the arginine-nitric oxide pathway within sperm cells from oligospermic infertile men is disturbed by enhanced Arg activity. We think that this may play a part in sperm dysfunction and male infertility. PMID- 10732691 TI - Myofibroblasts in adolescent varicocele: an ultrastructural and immunohistochemical study. AB - Myofibroblasts of the testes play an important role in the morphofunctional integrity of the seminiferous tubule. Previous studies in adults with varicocele have demonstrated an involvement of this cell population that tends to transform into fibroblasts. The aim of the present study was to try and verify the morphological features of myofibroblasts in the adolescent with left idiophatic varicocele. Twenty-two testicular biopsies were obtained from adolescents (aged 13-18 years, mean 15.8) and operated for left idiophatic varicocele. Biopsies were processed for electron microscopy (TEM) and immunofluorescence studies. The latter determined the level of myofibroblasts by using alpha-smooth muscle isoactin, a specific marker of myofibroblasts and, hence, excluding fibroblasts. TEM observations revealed a normal ultrastructure of myofibroblasts that was similar to that for the controls but an increased presence of extracellular matrix. The immunofluorescence study always demonstrated strong cell positivity to anti alpha-smooth muscle isoactin as also seen in the controls. This study demonstrates that adolescents with varicocele have well preserved myofibroblasts and do not show any evidence of transformation into fibroblasts, this has already been demonstrated in adult varicocele. These observations could represent an important factor for the understanding of the reversal of growth failure of the testes observed after early treatment. PMID- 10732692 TI - Morphometric analysis of symptomatic benign prostatic hyperplasia with and without bladder outlet obstruction. AB - To investigate the histological features of benign prostatic hyperplasia (BPH) with and without bladder outlet obstruction (BOO), 32 patients with symptomatic BPH, 22 with BOO and ten without BOO, were studied. The area density of smooth muscle, fibrous tissue, glandular epithelium, and glandular lumen in BPH adenoma resected by transurethral surgery was estimated using morphometric analysis. No differences were found in the area density of stromal and glandular components between the two groups. In the glandular component, neither the epithelium nor lumen differed between the two groups. However, the area density of smooth muscle in the obstructed group showed a decrease compared to that in the unobstructed group (P < 0.01). We conclude that a decreased proportion of smooth muscle in BPH adenoma may play an important role in BOO. PMID- 10732693 TI - Effect of aging on bladder function and the response to outlet obstruction in female rats. AB - Bladder dysfunction in the aging population is a significant problem. However the concomitant presence of other diseases in many patients can make it difficult to distinguish between changes in bladder function and other influences. The present study was designed to study, in aging rats, bladder function and the effect of partial bladder outlet obstruction (BOO) on bladder function. Cystometrics were performed in awake, female Fischer 344 rats of four age groups (6, 12, 18 and 24 months) following subcutaneous implantation of a mediport catheter. Cystometric evaluations were carried out in control rats or those subject to three weeks of BOO. Bladder compliance significantly decreased with aging, which reflected an increase in threshold pressure without changes in bladder capacity. Partial BOO caused development of severe bladder instability. Following BOO, bladder capacity and compliance were significantly increased in all age groups. Threshold pressure was lower in obstructed animals, except for 6-month rats. Younger animals were able to generate a higher contraction pressure to compensate for the BOO, whereas older animals did not. Using an awake model of cystometric measurement, we have demonstrated that aging, by itself can affect bladder function. Furthermore, aged animals respond differently to BOO than younger animals. These results demonstrate that both aging and disease can contribute to bladder dysfunction, and suggest that treatment of bladder dysfunction may require a combination of therapies targeted to multiple etiologies. PMID- 10732695 TI - Experimental defect in rabbit urethra repaired with acellular aortic matrix. AB - Urethral reconstruction following failed hypospadias repair or post-traumatic chronic stricture requires adequate amounts of tissue. Many surgical techniques utilizing different types of biological tissues have been attempted: (a) vascularized skin flaps from the prepuce, scrotum or penile shaft; (b) full thickness free skin grafts; (c) vesical or buccal mucosa grafts; (d) ureter; artery; vein and appendix tissue. More recently, biodegradable polymers have also been used as delivery vehicles of urothelial cells in animals. It has been demonstrated that the implant of an acellular tissue matrix in the bladder can guide the regeneration of urothelium, blood vessels, smooth muscle and nerves. The aim of this study was to create an experimental model of urethral defect, and then repair it by implanting homologous acellular aortic grafts as urethral substitutes. An acellular matrix was obtained by detergent enzymatic treatment of rabbit thoracic aorta. The growth of urethral epithelium was verified in vitro, and homologous acellular vessels were then implanted in rabbits, bridging a previous surgical urethral defect. The outcome of reconstructive surgery was evaluated histologically at 10 days, 3 weeks, 3 and 12 months. As the time after surgery increased, the neourothelium became less thick, signs of inflammatory response disappeared, and the orientation of collagen fibrils and smooth muscle fascicles resembled that of a normal urethra. The implants displayed abundant vascularization, and the luminal surface started to become irregular. Acellular blood vessels may represent a promising approach to urethral defect therapy for different reasons: (a) unlimited availability, (b) readily obtainable in different lengths and gauges, (c) the potential for being organized as tissue bank, and (d) that just one simple surgical procedure is needed. Nevertheless, before this technique can be applied in humans, it must be tested in more species and animals. PMID- 10732694 TI - Effects of ovarian hormones on beta-adrenergic receptor-mediated relaxation in the female rabbit bladder. AB - The effects of ovarian hormones on beta-adrenergic receptor-mediated responses in female rabbit detrusor smooth muscles were investigated. Ovariectomized mature female New Zealand white rabbits were untreated or treated with estrogen and/or progesterone for 2 weeks. The contractile responses to carbachol and KCl in the detrusor strips were not significantly different in all groups. As compared with dobutamine and GS-332, isoproterenol and procaterol significantly relaxed the detrusor strips derived from all groups on KCl-induced tonic contractions. Combined with estrogen treatment, isoproterenol, procaterol and GS-332 caused a significant increase in this muscle relaxation. Furthermore, estrogen treatment caused a significant increase in relaxation as a result of forskolin and the cyclic AMP (cAMP) production that was induced by isoproterenol, procaterol and GS 332. However, estrogen treatment did not affect the relaxant response to dibutyryl cyclic AMP. Progesterone treatment did not affect beta-adrenergic receptor-mediated responses. These results suggest that estrogen treatment causes the increased relaxant responses mediated by beta2- and beta3-adrenergic receptor subtypes, which may be related to the increased cAMP content in female rabbit detrusor smooth muscles. PMID- 10732696 TI - Clinical, histopathological and flow-cytometric properties of incidental renal cell carcinomas. AB - Medical records of 63 patients operated on for renal cell carcinoma (RCC) between 1986 and 1996 in the Karlovac General Hospital were studied retrospectively. In 23 (36.5%) patients, the tumor was incidentally detected. The median patient age was 62 in the incidental group and 64 years in the symptomatic group (P > 0.05). Ultrasonography was the leading technique for incidental detection of RCC. The median tumor diameter was 6 cm in the incidental group and 9 cm in the symptomatic group (P < 0.001). Incidental carcinomas had a lower stage (P = 0.022) and a lower nuclear grade (P < 0.001) than the symptomatic ones. The incidental cases were associated with a more favorable ploidy status (P = 0.027) and a lower proliferative activity (P = 0.005). The 5-year survival rate was significantly higher in incidental (81.4%) than in symptomatic cases (44.3%) (P = 0.020). Univariate analysis showed that tumor stage, ploidy status, and proliferative activity were good prognostic parameters, while patient age, tumor size, and nuclear grade were not. Tumor stage was the only independent prognostic parameter in multivariate analysis. In conclusion, the incidentally detected RCC show more favorable clinical, histopathological, and flow-cytometric characteristics and their prognosis is significantly better than in symptomatic cases. PMID- 10732697 TI - Chromosomal numerical aberrations of exfoliated cells in the urine detected by fluorescence in situ hybridization: clinical implication for the detection of bladder cancer. AB - Numerical aberrations of chromosomes 7 and 9 of exfoliated cells from the urinary tract were examined using fluorescence in situ hybridization (FISH). To minimize contamination with inflammatory, squamous and normal transitional cells all samples were stained using Giemsa's solution and clusters of transitional cells were selected for FISH analysis. Samples collected from 21 patients who had cystoscopic monitoring for bladder cancer were used in this study. Cystoscopy was positive in seven patients and five biopsy-proven transitional cell carcinomas were identified. Among the patients with cancer recurrence, numerical chromosomal aberrations were detected in four cases (sensitivity: 80%) and positive cytology in two cases. This preliminary pilot study shows FISH can be a useful tool for the detection of recurrence during a follow up of bladder cancer. PMID- 10732698 TI - Differential binding activities of lectins and neoglycoproteins in human testicular tumors. AB - Testicular germ-cell tumors, a morphologically and clinically diverse group of malignancies provide an ideal model for investigating the biology of glycoconjugates because the biosynthesis of oligosaccharide chains of glycoproteins monitored by plant/invertebrate lectins often changes during tumorigenesis, tumor progression, and metastasis. To investigate such changes in germ-cell tumors, we analyzed 67 surgical specimens from 31 seminomas, 32 embryonic carcinomas, and four choriocarcinomas using glyco- and immunohistochemistry that involved five plant/invertebrate lectins, 16 neoglycoproteins, and galectin-1 antibody. The results showed that some of these markers, such as melibiose-, lactose-, and beta-N-acetylgalactosamine-BSA-biotin were clearly differentially expressed amongst these tumors and between primary and metastatic embryonic carcinomas. The differences in staining for positivity, intensity, and heterogeneity indicate that the differential display of glycoconjugates in tumor cells may be important in tumor growth, metastasis, or prognosis because subtypes of these tumors behave quite differently from one another. Furthermore, we also found identical staining for positivity between most neoglycoproteins and their corresponding lectins, though the staining intensity of neoglycoproteins was weaker. This suggests that neoglycoproteins may be useful markers to replace their plant lectins. PMID- 10732699 TI - Abortive alloantigen presentation by donor dendritic cells leads to donor specific tolerance: a study with a preoperative CTLA4lg inoculation. AB - Donor dendritic cells (DCs) within allografts initiate the induction of an allospecific T cell response, while an abortive alloantigen presentation by DCs may induce allospecific unresponsiveness. We thus investigated the tolerogenic effect of donor DCs that were made incompetent in alloantigen presentation by treatment of CTLA4Ig. When we treated rats with donor DCs (2 x 10(6)/rat i.v.) on the preoperative day, nine rejected allografts in an accelerated manner (5.0 +/- 2.2 vs. 8.2 +/- 1.6 days in the control group). Preoperative inoculation of DCs pulsed with CTLA4Ig, a procedure which suppresses an allogeneic mixed lymphocyte reaction (MLR), also provoked an accelerated rejection (5.6 +/- 1.7 days). When DCs and CTLA4Ig (500 microg/rat i.p. on days -9, -7 and -5) were concomitantly inoculated, allograft survival was significantly prolonged (>38.7 +/- 40.0 days); a preoperative CTLA4Ig inoculation alone failed to do so (7.5 +/- 1.2 days). Long term graft survivors tolerated skin grafts from the donor but not from those from a third party. These results indicate that abortive alloantigen presentation by donor DCs, upon which an accessory signal pathway is suppressed by CTLA4Ig, leads to prolonged graft survival and donor-specific tolerance. PMID- 10732700 TI - Electron-microscopic immunocytochemical study of the intracellular transport of the viral glycoproteins in ts1, a mutant of Moloney murine leukemia virus. AB - The transport and localization of env-proteins of ts1 virus (a paralytogenic temperature-sensitive mutant of Moloney murine leukemia virus) in infected cells of the TB cell line have been studied at the ultrastructural level. It was found that the envelope precursor polyprotein gPr80-env of ts1 was inefficiently transported out of the endoplasmic reticulum at the restrictive temperature. It was speculated that inefficient transport correlates with inefficient processing of gPr80env into gp70 and Prp15E and leads to paralytic disease. PMID- 10732701 TI - Phase I and phase II xenobiotic biotransformation in different inbred strains of rats: study in immobilized perfused hepatocytes. AB - The present study was designed to compare phase I and phase II biotransformation reactions in immobilized perfused hepatocytes as a cellular system obtained from inbred rat strains which represent models for some cardiovascular diseases, namely, spontaneously hypertensive rats (SHR), rats sensitive and resistant to isoprenaline-induced myocardial lesions (IS and IR, respectively) as compared to Wistar rats (W). The biotransformation kinetics for hexobarbital (HX), 7 ethoxycoumarin (7-EC), 1-chloro-2,4-dinitrobenzene (CDNB) and 4-nitrophenol (4 NP) were followed up in the hepatocyte perfusate. W and SHR rat hepatocytes have metabolized HX at a higher rate than those of the IR and IS strains. Hepatocytes from the W strain exhibited a higher rate of 7-EC deethylation activity compared to hepatocytes obtained from the IR or IS strains. Hepatocytes obtained from SHR and IR rats showed the highest glutathione-S-transferase (GST) activity towards CDNB compared to the IS or W strain. 4-NP disappearance was higher in the perfusion medium of hepatocytes obtained from the W and IS strains compared to the IR strain. These significant differences in drug biotransformation between various studied strains, which may be genetically determined, can be well demonstrated by using an efficient drug metabolizing model of the immobilized perfused hepatocytes. The importance of these differences should be considered during the study of the experimental therapy of the relevant disease as obtained from the specific experimental strain, where it may be expected that the pharmacokinetic profile of a drug in vivo and consequently its pharmacodynamic or toxic effects will be strain dependent. PMID- 10732702 TI - Different activity of adenylyl cyclase in prefrontal cortex in three rat strains. The effect of amphetamine. AB - Since the literature data do not provide enough information on the effects of amphetamine on the prefrontal cortex and since many controversial findings were reported in various rat strains we decided to compare adenylyl cyclase activity in the prefrontal cortex in various rat strains and test the effects of chronic amphetamine treatment (for 14 days) on the activity of this enzyme. Basal adenylyl cyclase activity was lower in Wistar rats than in Sprague-Dawley and Lewis rat strains. Amphetamine treatment produced in Wistar rats a substantial decrease in basal adenylyl cyclase activity. In Sprague-Dawley rats, we observed the highest enzyme activity which was slightly reduced after amphetamine treatment. In Lewis rats which had basal activity close to the activity of Wistar rats, amphetamine produced an increase in enzyme activity. The total adenylyl cyclase activity, estimated in the presence of forskolin, was the lowest in Wistar rats. The highest stimulation was observed in Lewis rats. Amphetamine treatment caused a very significant inhibition of total adenylyl activity in Wistar rats and a smaller inhibition in Sprague-Dawley rats. However, in Lewis rats amphetamine treatment increased the dose-response curve of forskolin stimulation. These results show that Lewis rats, compared to the other two strains, develop not only quantitatively but also qualitatively different responses. PMID- 10732703 TI - Predictive value of HLA class II PCR typing for the outcome of mixed lymphocyte reaction. AB - The use of HLA-DRB1 and -DQB1 polymerase chain reaction-sequence-specific primer (PCR-SSP) typing at different levels of resolution for MLR prediction was assessed in 54 HLA-A and -B matched donor/recipient unrelated pairs and 89 HLA-A and -B identical siblings. Graft-versus-host (GvH) direction one-way MLR was evaluated unless stated otherwise. The typing of DRB1 alleles satisfactory for MLR prediction in HLA identical siblings (P = 0.0015) does not appear to be sufficient in matched unrelated pairs (P = 0.2407). Using more discriminatory PCR SSP typing, the disparity in DRB1 allelic subtypes was predominantly found in the category of DRB1 allele compatible, MLR positive unrelated pairs. Besides, DRB1 allelic subtype mismatches were revealed in five of the forty-one DRB1 allele compatible, MLR negative unrelated pairs. More discriminatory typing made the correlation between DRB1 compatibility and MLR negativity extremely significant (P = 0.0001). As for these five exceptional cases, the reciprocal host-versus graft (HvG) direction MLR was considered, too. This allowed HLA-D disparity to be disclosed in two of them. An uninterpretable result reflecting defective MLR reactivity occurred in one case. Negative reciprocal MLR in the last two DRB1 allelic subtype incompatible pairs is hardly to explain without postulation of MLR silent DRB1 allelic subtype mismatches. An analysis in unrelated pairs showed a role of some DQB1 gene products in the proliferative response too. GvH direction positive MLR was found in two HLA identical siblings among the 89 tested. The DPB1 incompatibility detected in one of them could be a potential cause of proliferative response but MLR reactivity in the other, DPB1 identical, pair cannot be interpreted easily. PMID- 10732704 TI - Cytogenetic changes in Sprague-Dawley rat lymphomas in the course of in vivo passages. AB - Changes in the karyotype in three spontaneous Sprague-Dawley rat lymphomas (SD7/95, SD8/96, SD9/96) have been studied in the course of in vivo passages. In individual lymphomas karyological findings of primary disease from lymph nodes were compared with changes found in the 1st and 10th passages of the lymphoma and bone marrow samples. Chromosome studies were performed on direct preparations using the G-banding technique. Chromosome counts of all specimens studied were near diploidy, the majority of metaphase cells being pseudodiploid. In later passages of two lymphomas, the tendency in selection to hyperdiploid karyotype, particularly in bone marrow was observed. The examination revealed an increased percentage of breaks in lymph node cells of primary disease and the existence of nonrandom change, derivative chromosome 11, which occurred in structural variability in all three lymphomas studied. The aberration involving chromosome 11 was evaluated as the addition of unknown material at chromosome band 11q11 or as a duplication or triplication of segment 11q12-q23. If this structural aberration was not found, the excessive derivative chromosome 11 or translocation t(11;13) was proved to be present. Further, rearrangements of chromosomes 13 and 7 were nonrandom chromosome abnormalities revealed in later passages of the lymphomas. The results are in accordance with our previous observations in 14 cases of SD lymphomas that showed nonrandom occurrence of rearrangements concerning chromosome 11 and also relatively frequent translocation involving chromosome 13. PMID- 10732705 TI - Linkage mapping of the Fos cellular oncogene (Fos) to rat chromosome 6 and its possible role in the regulation of compensatory renal growth. AB - Using the analysis of HXB/BXH recombinant inbred (RI) strains, we have recently detected a suggestive association between the D6Mit9 marker and a compensatory growth of the remnant kidney after unilateral nephrectomy (P = 6 x 10(-)4). Based on conserved synteny between mice, rats, and humans, the putative quantitative trait locus (QTL) might map to a region with several positional candidate genes: for instance, the Fos oncogene was implicated as a possible candidate gene for compensatory renal growth (CRG). In the current study, we mapped the Fos gene in RI strains 27 cM apart from the position where the putative CRG regulatory QTL was detected. Despite relatively wide confidence intervals for mapping QTLs with modest effects in a small population of RI strains, this finding probably excludes the Fos as a positional candidate for CRG. PMID- 10732706 TI - Angiogenesis of extra- and intraembryonic blood vessels is associated with expression of nestin in endothelial cells. AB - For a long time, intermediate filament protein nestin was recognized as a specific constituent of the cytoskeleton of developing neural cells and skeletal muscle. Recent reports by other investigators demonstrated that nestin may also be expressed in other cell types. Here we give evidence that nestin is expressed by endothelial cells of developing blood vessels in the rat. Using anti-Rat-401 monoclonal antibody we identified high levels of nestin in the endothelium lining all blood vessels of E14-15 rat foetuses. Immunoreactivity for nestin was detected in both extraembryonic (chorion, placenta, umbilical cord) and intraembryonic blood vessels. In the body of the foetus, we observed nestin positive endothelial cells in vessels located in the areas of the mesenchyme as well as in vessels supplying the developing organs (central nervous system, liver, lung, spleen, heart, digestive tube). The strongest positive signal was given by the endothelium of newly vascularized tissues. In contrast to the developing tissues, expression of nestin by vascular endothelial cells was greatly reduced in adult tissues. PMID- 10732707 TI - The pyrAb gene coding for the large subunit of carbamoylphosphate synthetase from Bacillus stearothermophilus: molecular cloning and functional characterization. AB - The Bacillus stearothermophilus pyrAb gene, encoding the large subunit of carbamoylphosphate synthetase, was isolated and characterized. The DNA sequence is a 3195-nucleotide long reading frame coding for a polypeptide of 1064 amino acids and deduced Mr approximately 116,160 Da. The pyrAb gene is part of the B. stearothermophilus pyrimidine biosynthesis operon pyr. The 5' end of thepyrAb gene overlaps with the 3' end of the pyrAa gene coding for the small subunit of carbamoylphosphate synthetase, while the 3' end of the pyrAb gene is overlapped with the 5' end of the pyrD gene, which by analogy with the B. subtilis genomic organization, codes for dihydroorotate dehydrogenase. The deduced amino acid sequence of the large subunit of B. stearothermophilus carbamoylphosphate synthetase was compared with the known sequences of the large subunit of carbamoylphosphate synthetase enzymes from other organisms via the NCBI database. Extremely high (98%) identity in amino acid sequence with the large subunit of carbamoylphosphate synthetase from Bacillus caldolyticus was detected. PMID- 10732708 TI - The effect of irradiation on FRTL-5 cell morphology. AB - The morphological changes and proliferative activity of FRTL-5 cells irradiated in vitro with one dose of 0, 8, 12, 16, 24 or 32 Gy were studied morphometrically 7, 14 and 21 days after irradiation. The number of cells irradiated with 24 Gy and 32 Gy decreased during the 3 weeks of the experiment. Morphometrical analysis confirmed that the cells repaired their injuries up to 16 Gy of irradiation, while doses of 24 and 32 Gy damaged them irreversibly. Thyroglobulin was detected in cells after all doses of irradiation. PMID- 10732710 TI - Blasticidin resistance cassette in symmetrical polylinkers for insertional inactivation of genes in Dictyostelium. AB - In Dictyostelium discoideum inactivation of developmentally regulated genes via homologous recombination has become an important tool in studying systematically the entire developmental program of this model organism. The Dictyostelium genome is very A/T-rich,which presents obstacles to the preparation of knockout constructs. The coding regions offer few suitable restriction sites and the low complexity intergenic regions do not guarantee specificity of recombination. We present here the preparation of plasmids pBsR479, pBsR503, and pBsR519, in which a blasticidin resistance-cassette is positioned in the center of various symmetrical polylinkers. This design simplifies the cloning process and gives more flexibility in positioning the selectable marker within the coding regions. PMID- 10732709 TI - Interaction of bull, stallion and boar seminal plasma proteins and sperms with acidic polysaccharides. AB - The interaction of seminal plasma proteins, sperms and detergent-released sperm proteins of three species with different types of acidic polysaccharides was studied. Heparin-binding activity of boar, bull and stallion seminal plasma proteins, sperms and sperm proteins was compared with their ability to interact with polysaccharides differing in the presence of the sulfate groups or in their saccharide moiety (chondroitin sulfate, dextran sulfate, fucoidan, hyaluronic acid). Bull seminal plasma proteins were characterized by higher affinity to heparin, fucoidan and dextran sulfate, while significant differences between different types of polysaccharides were detected in the case of boar proteins. Sperm protein interactions with acidic polysaccharides in bull and stallion were analogous to the binding of seminal plasma proteins. Bull and stallion seminal plasma proteins inhibited the interaction of corresponding sperm proteins with acidic polysaccharides. PMID- 10732711 TI - Endotoxin response in spontaneously hypertensive rats: a role of the TNFalpha gene region. AB - We have shown previously that administration of endotoxin induces a smaller decrease of body temperature in spontaneously hypertensive rats (SHR) than in normotensive Brown Norway (BN) rats. Several studies have suggested that tumor necrosis factor alpha (TNFalpha) is one of the mediators of the body-temperature response to endotoxin. To test whether the TNFalpha gene could be involved in determination of the observed difference in the body-temperature response to endotoxin, we studied SHR (n = 6) and a congenic strain, SHR.1N (n = 5), which differs from SHR by a segment of chromosome 20 originating from BN and containing the TNFalpha gene. Body temperature was recorded continuously by means of radiotelemetry. We showed that, in both strains, an intraperitoneal injection of endotoxin (500 microg/kg of body weight) induces a rapid hyperthermic phase (20 40 minutes post-injection), which is followed, first, by a hypothermic phase (100 120 minutes post-injection) and, then, by a late hyperthermic phase (seven hours). Although both strains demonstrated a similar trend in the response, a significant difference was observed between the two response curves (P = 0.0001). Further analysis at each time point revealed that the two strains differed significantly at a peak of the hypothermic phase (P = 0.035) and the late hyperthermic phase (P = 0.035). In conclusion, these data indicate that the differential chromosomal segment of SHR.1N contains a gene(s) causally related to the body-temperature response to endotoxin. In the light of previously published data, the TNFalpha gene appears to be the most likely candidate gene within the segment. PMID- 10732712 TI - IL-2 gene-modified tumour vaccines: monitoring of IL-2 levels in serum and peritoneal cavity of vaccinated mice. AB - IL-2 kinetics was assessed in mice vaccinated with irradiated syngeneic tumour vaccines carrying an inserted IL-2 gene and producing constitutively IL-2. For comparison, the kinetics of i.v. administered recombinant IL-2 was also examined. During regular time intervals after the vaccination or administration of recombinant IL-2, samples of serum and peritoneal fluid were collected and examined, using CTLL bioassay or its MTT modification. After i.p. administration of irradiated IL-2-producing plasmacytoma (X63-m-IL-2) vaccine, the levels of IL 2 were substantially higher in the peritoneal fluid than in the serum. Both in the peritoneal fluid and in the serum, the IL-2 level was increasing up to 60 min after administration and then it gradually decreased. The last time point when IL 2 was still detectable both in the peritoneal fluid and in the serum was 30 h. Almost identical results were obtained when the IL-2 levels were detected by the conventional CTLL assay, in which DNA synthesis was monitored by 3H-thymidine labeling, and by the isotope-free MTT modification of the CTLL assay, in which the DNA synthesis was monitored by staining. The MTT modification has the advantage of an isotope-free method. Comparison of two different IL-2-producing vaccines, a murine plasmacytoma X63-m-IL-2, with high IL-2 production, and murine sarcoma MC12-IL-2, with low IL-2 production, revealed that whereas after i.p. administration of the high producers, the peak of IL-2 was reached both in the peritoneal fluid and in the serum after 1 h, the administration of low producers gave the peak level of IL-2 later, 5 h after i.p. administration. Comparison of IL-2 levels obtained after i.p. administration of live and irradiated X63-m-IL-2 vaccine revealed that the irradiated vaccine produced both in vitro and in vivo higher amounts of IL-2. As compared to i.p. administration, the kinetics after i.v. administration of the X63-m-IL-2 vaccine was different. The maximum level of recombinant IL-2 was reached 10 min after administration and IL-2 was undetectable after 5 h. When the injections of recombinant IL-2 were repeated, the elimination of IL-2 from the circulation was substantially faster. PMID- 10732713 TI - Changes in immunochemical localization of cytoskeletal proteins in human and boar spermatozoa before and after acrosome reaction. AB - Several cytoskeletal proteins (alpha-tubulin, beta-tubulin, actin, spectrin, tropomyosin, vimentin and cytokeratin) were studied in human and boar spermatozoa. Their localization was observed by means of specific antibodies using indirect immunofluorescence technique. Immunocytochemical results were confirmed by the Western blot technique. Cytoskeletal proteins were examined in ejaculated spermatozoa before and after acrosome reaction induced by the ionophore A23187. The immunofluorescence assay revealed that localization of the studied cytoskeletal proteins in human and boar spermatozoa differ remarkably. In human spermatozoa, the localization of actin and spectrin changed after acrosome reaction; on the other hand, in boar spermatozoa, the changes of localization concerned alpha-tubulin, beta-tubulin, actin and spectrin. The type of changes differs in the studied species and follows probably the course of acrosome reaction. The results suggest that cytoskeleton participates in the process of acrosome reaction of mammalian spermatozoa. PMID- 10732714 TI - Enhanced IL-10 and decreased IL-2 production after orthotopic corneal transplantation in mice. AB - Corneas from mice incompatible at both major histocompatibility complex (MHC) and non-MHC antigens were grafted orthotopically to unmodified and high-risk recipients. Production of interleukin-2 (IL-2) and IL-10 by cells from spleens and draining lymph nodes from corneal graft recipients was determined in vitro. Over 90% of corneal allografts suffered alloantigen-induced inflammatory reaction within the second or third week after surgery. However, only 56 % of grafts in unmodified and 75 % of grafts in high-risk recipients were irreversibly rejected. Cells obtained from draining lymph nodes from the vicinity of the eye of the corneal graft recipients produced significantly elevated amounts of IL-10 and decreased amounts of IL-2. This shift to the Th2 type cytokine response was observed after stimulation of the cells with graft donor MHC antigens, but not after stimulation with donor non-MHC or third-party alloantigens. No changes in cytokine production were detected in spleen. The enhancement of IL-10 production in the vicinity of the eye was a consequence of corneal grafting and did not correlate with the fate of the graft. The results thus show that orthotopic corneal transplantation induces a local shift to the Th2 type cytokine response, which might be considered another factor that contributes to the unique characteristics of the immunity in the eye and to the tolerance of an unusually high percentage of corneal allografts. PMID- 10732715 TI - Frequencies of HLA-DRB1, -DQB1 and -DPB1 alleles in Czech population. AB - The frequencies of phenotypes, alleles and allelic subtypes of DRB1 and DQB1 HLA loci in 420 unrelated individuals from the Czech population were determined. The frequencies of DPB1 alleles of the HLA locus were determined in 92 individuals. The assays were performed using the polymerase chain reaction (PCR) method or the restriction fragment length polymorphism (RFLP) analysis. The most frequent DRB1 allele was *07, the most frequent DQB1 allele was *03 and the most frequent DPB1 allele detected was *04. These assays define the extent of polymorphism of the HLA system and are useful for determining the selection strategy of HLA-identical donor-recipient pair suitable for bone marrow transplantation. PMID- 10732716 TI - The frequency of HLA-DRB1 and -DQB1 alleles in cervical cancer cases in the Czech Republic. AB - The distribution of HLA class II DRB1 and DQB1 alleles in cervical carcinoma patients was compared with the frequency of these alleles found in healthy population living in the Czech Republic. The RFLP analysis and PCR-SSP were used for DNA typing. Although the differences in the frequency of DRB1*03, DQB1*02 and DQB1*0303 alleles between the cases and the controls were rather large, corrected P values did not reach significance. PMID- 10732717 TI - Monoclonal antibody VI-10 specific for vimentin. PMID- 10732718 TI - Interleukin 2 gene therapy of surgical minimal residual tumour disease: characterization of cytolytic effector cells from tumour progressors and regressors. AB - Experiments were designed to characterize cytolytic effector cells from mice with SMRTD treated with IL-2 gene therapy. Mice were inoculated with syngeneic murine MK16 carcinoma cells. When the tumours reached 8-12 mm in diameter, they were excised and the operated mice were randomized into two groups. The first group without any further treatment was designated as operated-only; the second group, vaccinated 3 days after the operation with IL-2-producing tumour vaccine, is referred to as operated-vaccinated. Tumour recurrence rate in the operated-only mice was 90 percent; in the operated-vaccinated group the recurrence rate was 38.5 percent (progressors). The remaining 61.5 percent of mice were permanently protected (regressors). On day 53, the tumour progressors, regressors and healthy controls were sacrificed, and their spleen cells were used for 51Cr microcytotoxicity assay. Splenocytes from any group of mice were not cytolytic when allowed to react with MK16, YAC-1 (NK sensitive) and C1498 (NK resistant) targets. However, when grown for 3 days in IL-2-containing medium, the splenocytes from all groups of mice could develop cytolytic activity. The cytolytic activity of splenocytes from tumour progressors and regressors was substantially lower then that of splenocytes from healthy controls. In addition, significantly lower cytolytic activity was observed with IL-2-activated splenocytes from tumour progressors as compared to that of tumour regressors. Depletion of NK1.1+ cells or CD4+ plus CD8+ cells prevented the induction of significant IL-2-stimulated cytotoxicity directed against MK16 and C1498 targets in spleen cell cultures from tumour progressors, regressors, and healthy control mice, indicating that both, NK1.1+ and CD4+ plus CD8+, cells participate in the antitumour effect of IL-2 gene therapy. This was further supported by the finding that after depletion of CD4+ plus CD8+ cells, a residual cytolytic activity directed exclusively against NK-sensitive YAC-1 cells was observed. PMID- 10732719 TI - A threshold level of gag expression is required for particle formation in rat cells transformed by avian retroviruses. AB - To test the hypothesis that the block of polyprotein precursor processing and particle formation in RSV-transformed mammalian cells is due to a low level of pr76gag expression, rat tumor cell lines with different amounts of precursor molecules were used. The wild-type forms of pr76gag have been expressed at a high level by use of SV40-based vector and thirty-two stable transfected cell clones were isolated. The gag protein expression was detected in the cell lysate by immunoblotting. Untransfected cells released no proteins that could be detected by immunoprecipitation with anti-RSV serum. Membrane-enclosed gag precursor polyprotein molecules and infectious virus particles from different stably transfected clones have been found in the medium. Both immature and mature virions of type C morphology were directly detected by transmission electron microscopy. Surprisingly, virus-like particles of morphology similar to mature type C retroviruses were found enclosed within intracellular membranes in a stably transfected nonproducing clone. PMID- 10732720 TI - The asymmetric distribution of interphasic silver-stained nucleolus organizer regions in human and rat proerythroblasts. AB - The distribution of SSPs representing AgNORs was studied in human as well as rat proerythroblasts to provide information on the distribution of these nucleolar components in highly immature and proliferating non-neoplastic cells. The distribution of SSPs was asymmetric and most of the cells contained one nucleolus which possessed a larger number of these nucleolar components than the remaining nucleoli. Such nucleolus might be functionally dominant, since the number of nucleolar SSPs is apparently related to the nucleolar biosynthetic activity. On the other hand, when a proerythroblast possessed only one nucleolus, the number of SSPs in such a cell was very similar to the sum of SSPs in a polynucleolar cell. The asymmetric distribution of SSPs characteristic for most proerythroblasts disappeared in the terminal stages of the erythroblastic development. Cells in such stages, as described previously, were characterized by the presence of a limited number of single SSPs. PMID- 10732721 TI - Hematocrit and hemoglobin values are negatively correlated with insulin resistance in spontaneous hypertension. AB - It has been recently reported that increased hematocrit and hemoglobin values often accompany insulin resistance and compensatory hyperinsulinemia in humans. In the current study, we analyzed the relationship between hematocrit/hemoglobin on the one hand and insulin resistance, dyslipidemia, and hypertension on the other hand in HXB/BXH recombinant inbred (RI) strains derived from the spontaneously hypertensive rat (SHR) and the Brown Norway (BN) rat. The SHR progenitor strain had a significantly increased hematocrit values and it was also hypertensive and insulin-resistant when compared with the BN progenitor. The distribution of hematocrit and hemoglobin values among RI strains was continuous, suggesting a polygenic mode of inheritance. Analysis of RI strains revealed that hemoglobin was negatively correlated with insulin and insulin/glucose ratio, and that hematocrit was negatively correlated with insulin-stimulated glucose uptake in isolated adipocytes. There was no relationship between hematological parameters and blood pressure or lipid phenotypes in RI strains. The findings of the current study suggest that hematocrit and hemoglobin values might be added to the clustering variables related to the insulin resistance syndrome in the SHR strain. PMID- 10732722 TI - Sexing of chicken feather follicle, blastodermal and blood cells. AB - The production of chicken chimeras using donor and acceptor cells which can be of opposite sex has necessitated the utilization of methods developed to distinguish the sex of chickens. We demonstrate one of these methods, based on the polymerase chain reaction which amplifies the EcoRI repeat unit of the fowl W chromosome, and how this technique may be used to sex various cell types in chickens as well as small numbers of blastodermal cells. Our results demonstrate the ability to sex chickens using EcoRI primers, specific for the W chromosome, from as little as 2 ng of female genomic DNA isolated from blood and feathers--the latter being the result of DNA extraction from only one feather. Also evident in this study is the detection of the W chromosome by PCR from approximately 50 blastodermal cells originating from the developing blastodisc at stage X. PMID- 10732723 TI - CD10 expression in cultured human osteoblast-like cells. AB - Morphological features, bone nodule formation and alkaline phosphatase activity are currently used to identify osteoblasts. CD10 (cALLa antigen) is a glycoprotein with endopeptidase activity and it is present on the surface of many cell types. We have studied the expression of CD10 in osteoblast-like cells by immunocytochemistry and flow cytometry in order to identify other markers of the osteoblast lineage. We isolated osteoblast-like cells from specimens obtained in the course of oral surgery. Expression of the cALLa antigen (CD10) may also be an indicator of the osteoblast phenotype. PMID- 10732724 TI - Granulocyte-macrophage colony-stimulating factor gene-modified vaccines for immunotherapy of cancer. PMID- 10732725 TI - Therapeutic effect of heat shock on T-cell lymphoma in inbred Sprague-Dawley rat. AB - Anticancer effect of heat shock, either alone or in combination with the drug PMEDAP, and cold water immersion stress were studied in an in vivo model of s.c. transplanted rat T-cell lymphomas in an inbred Sprague-Dawley rat line (SD/cub). Significant anticancer effect was induced by repeated sessions of heat shock; decrease of s.c. lymphoma weight and prolongation of survival time of treated rats was found to be dependent on the number of HS sessions. Much stronger therapeutic effect was observed after repeated heat shock in combination with PMEDAP administration. Light and electron microscopy studies were performed to characterize the alterations within the lymphomas. Morphologically, cellular alterations corresponding with apoptosis were observed in lymphoma cells after repeated heat shock. Indirect immunoperoxidase technique was used to detect HSP 72/73 protein(s), p53 and Bcl2 proteins in lymphomas heated directly or indirectly. The induction of HSP 72/73 protein(s) was found in the lymphoma tissues from autopsied animals exposed to heat shock; the intensity of its expression was dependent on the experimental design. The expression of p53 and BcL2 proteins was not changed in lymphoma cells of HS treated animals as compared to that of untreated lymphoma bearing controls; the Bcl2 protein was present in both treated and untreated lymphomas, and the p53 protein remained undetectable in all samples. Contrary to the heat shock, the cold stress did not suppress growth of lymphomas and, furthermore, accelerated the infiltration of parenchymatous organs with lymphoma cells. PMID- 10732726 TI - Essential fatty acids and related molecular and cellular mechanisms in multiple sclerosis: new looks at old concepts. AB - Essential polyunsaturated fatty acids, their metabolism and their effects represent a ubiquitous system important for cellular membrane stability, for immunological and inflammatory regulations, for inter- and intracellular signalling. There are complex links to other regulatory and effector mechanisms - to the reactive oxygen species, nitric oxide and peroxynitrite anion, to eicosanoids and related compounds. The relation of polyunsaturated acids to multiple sclerosis was extensively discussed some time ago. Recently, a number of data placing these concepts in a new light have emerged, which are reviewed in the present article. The recent knowledge indicates that this topic deserves further attention in both the basic and the clinical research of multiple sclerosis. PMID- 10732727 TI - EGF receptor-like determinants on human spermatozoa and their possible cytoskeletal association. AB - The study was focused on the localization of human sperm epidermal growth factor receptor and its resistance to Triton X-100 extraction, indicating possible cytoskeletal association. Human spermatozoa were subjected to immunofluorescence and pre-embedding immunoelectron microscopy using anti-EGF receptor monoclonal antibody as a probe. In detergent-untreated cells, the entire head was stained with intensity declining towards the acrosomal tip. Triton-extracted spermatozoa were stained predominantly in the equatorial segment region. Electron microscopy performed after extraction revealed that the label was also present in the equatorial segment and localized to electron-dense material overlying the acrosomal remnants. This Triton-resistant staining could imply cytoskeletal association of the EGF receptor in the equatorial segment, with possible importance in fertilization. PMID- 10732728 TI - Detection of alpha(beta)-N-acetyl-D-galactosamine-binding sites in kidney- relation to Henoch-Schonlein-associated IgA nephropathy. AB - Henoch-Schonlein purpura is the most common vasculitis of childhood, accompanied by the deposition of IgA1 immunoglobulins into the glomerular mesangium. The actual molecular mechanism of IgA deposition is not clear, but the altered glycosylation of O-linked oligosaccharides of the hinge region of IgA1 is generally considered as the crucial etiopathogenic factor. The oligosaccharides of this glycoprotein from healthy persons are principally of mucin-type Galbeta1,3GalNAcalpha-O-glycan core structure, frequently sialylated. The patient's IgA hinge region saccharide is an incomplete GalNAcalpha-O-glycan only. This study investigates the presence of binding sites for alpha-GalNAc and beta GalNAc in frozen sections of kidney with and without nephropathy prompted by the possibility for a lectin mechanism of IgA deposition to mesangium. Neoglycoproteins prepared as conjugates with derivatized alpha- or beta-GalNAc moieties as histochemically crucial ligands and biotinylated bovine serum albumin as a carrier were employed for this purpose. The result of the experiments demonstrated expression of specific and accessible binding sites for both alpha- and beta-GalNAc in tubules but not in glomeruli of kidney samples both with and without nephropathy. These findings imply no involvement of a lectin mechanism of IgA1 binding to mesangium, unless a temporary alteration of accessibility of binding sites for probes in glomeruli occurs or the linkage region beyond the monosaccharide is pivotal for a receptor whose binding site may accommodate a peptide epitope in addition to the O-linked alpha-GalNAc residue. PMID- 10732729 TI - Diversity among wild type and vaccination strains of Trichophyton verrucosum investigated using random amplified polymorphic DNA analysis. AB - We initially tested 20 primers for their ability to amplify genomic DNA of Trichophyton verrucosum using RAPD. Six of these were selected for further study aimed at discrimination of wild type and vaccination strains of T. verrucosum. The results indicate that RAPD successfully distinguished all strains included in the study. In addition, results of corrected cluster analysis were consistent with the fact that the avirulent vaccination strains (T. verrucosum TV-M9 and T. verrucosum TV-M-130) were prepared by ultraviolet (UV) light induced mutagenesis of the standard wild type strain T. verrucosum Stradznice. No marker for a/virulence was detected. These outcomes suggest new possibilities for epidemiological analyses, for discrimination among different vaccination strains and studies of fungal population in vaccinated/infected hosts. PMID- 10732730 TI - Simultaneous detection of endogenous lectins and their binding capacity at the single-cell level--a technical note. AB - Endogenous lectins are proteins/glycoproteins which selectively recognize distinct saccharide ligands and are different from immunoglobulins and carbohydrate-utilizing enzymes. Expression of these molecules can be detected immunohistochemically using nonblocking monoclonal antibodies (A1D6: anti galectin-3, MR-15-2-2: anti-175 kD mannose receptor). Alternatively, biotinylated (neo)glycoconjugates which are recognized by a studied lectin can be employed as convenient probes to demonstrate specific binding of sugar epitopes by carbohydrate recognition domains (CRD) of endogenous lectins. In this study, we describe a new procedure for immunocytochemical visualization of the expression of endogenous lectins and glycochemical visualization of the reactivity of carbohydrate recognition domain(s), performed simultaneously at the single-cell level. PMID- 10732731 TI - Monoclonal antibody NF-09 specific for neurofilament protein NF-M. PMID- 10732732 TI - Two point mutations in the U3 region of the long terminal repeat convert a subgroup A transformation-defective rous sarcoma virus to a cytopathic virus. AB - To elucidate the mechanism of cytopathicity of the transformation-defective avian retrovirus tdPH2010, we examined the function of two point mutations we had previously found in the long terminal repeat U3 region of this virus. Our previous studies showed that the U3 region was responsible for the cytopathic effects. These mutations were a G-to-T mutation at position -126 from the transcription start site and a G-to-A mutation at -23. Site-directed mutagenesis was performed on a noncytopathic, wild-type virus BSU to alter the nucleotides at these two positions, one at a time, to those of tdPH2010. Cell growth assay using the altered viruses revealed that host cell growth was retarded only when both of these mutations were present. The two additional mutations previously found in the direct repeat 1-polypurine tract (DR1-PPT) region of tdPH2010 were present also in the noncytopathic strain tdPH2013. Site-directed mutagenesis confirmed that these two mutations had indeed no role in the cytopathic effect of tdPH2010. None of these mutations influenced virus production from the infected cells. We conclude that the cytopathic effects by tdPH2010 are ascribed to the two point mutations in the U3 region. PMID- 10732733 TI - Attempts to induce melanosome degradation in vivo. AB - Contradiction between repeatedly reported electron microscopic and histochemical observation of melanosome disintegration in the presence of lysosomal enzymes in vivo and failing attempts to induce such degradation by biochemical means in vitro with the aim to explain chemical mechanism(s) of this process belongs to chronically challenging problems in biochemistry of melanin structures. Attempts have been made to bring about melanosome disintegration in vivo by inoculating melanosomes isolated from dog hair into the tissue of amelanotic Bomirski melanoma and into peritoneal cavities of DBA/2 and C57BL/6J mice, and by repeated injection of melanosomes isolated from Bomirski pigmented melanoma into Syrian hamster foot pads. Both histological and electron microscopic observations demonstrated that melanosomes were phagocytized by macrophages and sporadically by fibroblasts. In peritoneal cavities the injected foreign melanosomes remained mostly extracellularly, were surrounded by foreign body multinuclear cells and formed granulomas. There were no convincing signs of degradation of the hair melanosomes, which behaved like inert foreign bodies. Only some phagocytized Bomirski hamster melanoma melanosomes tended to lose their integrity. Our data suggest that melanosomes devoid of their limiting membranes are not necessarily prone to extensive disintegration in vivo. The earlier reported association of lysosomal enzymes with disintegrating melanosomes does not constitute an evidence for their participation in melanosome degradation. Considering the structure of melanins, redox mechanisms (analogous with the metabolism of polycyclic hydrocarbons (DePierre and Ernster, 1978)) seem to be more probably involved in pigment degradation than hydrolytic reactions. PMID- 10732734 TI - Oviduct secretion contributes to the establishment of species specific barrier preventing penetration of oocytes with foreign spermatozoa. AB - Ovulated oocytes can be fertilized in vivo almost exclusively with spermatozoa of its own species only, while the species specificity of zona pellucida of in vitro matured oocytes is less restrictive. Our present experiments were undertaken to determine whether estrous oviductal fluid modifies the interaction between gametes of unrelated species. After incubation of in vitro matured sheep oocytes with bull spermatozoa, the penetration rate was 75.0%, whereas when the oocytes were matured in medium supplemented with 15% sheep oviductal fluid collected using the permanent indwelling oviductal cannulae, the penetration rate decreased to 4.8 % (4/84). In reverse combination, 70.4 % (38/54) cattle oocytes matured in vitro were penetrated with ram spermatozoa. The addition of oviductal fluid caused a drop in penetration by ram sperm to 38% (19/50). In parallel experiments, no penetration was recorded when in vivo matured sheep oocytes were incubated with bull spermatozoa; high fertilization rates (79.4% - 27/34) were recorded when such eggs were incubated with ram spermatozoa, irrespective to the presence or to the absence of oviductal fluid in the medium. The results suggest that the properties of zonae pellucidae of ovulated and in vitro matured oocytes are not identical and may be modified by contact with estrous oviductal fluid. PMID- 10732735 TI - Zona pellucida autoantibodies in women undergoing ART. AB - The presence of ZP autoantibodies in serum and Ffl samples of 109 women attending the ART Center in Kiev was investigated using IIF and ELISA. Positive serum and Ffl samples examined by both methods were found in 20 (18.34%) and in 19 (17.43%) patients respectively; 31 (28.44%) serum samples and 33 (30.27 %) Ffl samples analyzed by IIF were positive; of the samples analyzed by ELISA 21 (19.26%) and 20 (18.34%), respectively, were positive. No relationship was found between ZP autoantibody incidence and the type and cause of infertility. A significant prevalence of ZP autoantibodies detected by ELISA in Ffl was found in patients with fertilization failure (39.13%) and with low fertilization rate (42.85%) when compared to patients with middle fertilization rate (5.71%) and high fertilization rate (8.1%). The clinical significance of ZP autoantibodies in Ffls for in vitro fertilization outcome was suggested. PMID- 10732736 TI - Morphometrical and stereological analysis of myocardial mast cells in myocarditis and dilated cardiomyopathy. AB - Mast cells play a certain role in inflammation and immunological reactions. Cardiac mast cells, shown by sodium sulfate-alcian blue staining, were evaluated in endomyocardial biopsy specimens in patients with unexplained congestive heart failure. The results of histopathological analysis were consistent with active myocarditis according to the Dallas criteria in 10 patients (15%), borderline myocarditis in 9 (13.8%), and dilated cardiomyopathy in 25 patients (38.5%); these results were compared with a control group of 10 traffic accident victims. The highest numerical areal density of mast cells was found in active myocarditis (3.92 counts/mm2, SD = 1.84), followed by borderline myocarditis (2.76 counts/mm2, SD = 1.66), dilated cardiomyopathy (1.56 counts/mm2, SD = 0.45) and control group (0.77 counts/mm2, SD = 0.19). Degranulation involved 27% (SD = 3.6) of mast cells in active myocarditis, 18% (SD = 4.5) of mast cells in borderline myocarditis, 10.8% (SD = 3.12) of mast cells in dilated cardiomyopathy and 4% (SD = 2.0) of mast cells from autopsy tissue. The differences among the four groups were statistically significant (P <0.001). The increased number of mast cells and the higher degree of their degranulation in myocarditis compared to dilated cardiomyopathy and to control group indicate that they were activated. The mast cells could be involved in modulation of fibrous response, since they tended to be associated with areas of fibrosis. Likewise, numerical areal density and degree of degranulation of mast cells could also be used as additional diagnostic criteria for acute myocarditis, since a higher numerical areal density and degree of degranulation were present in myocarditis vs. dilated cardiomyopathy and control group. PMID- 10732737 TI - Binding of anti-CD4 monoclonal antibody to D1 domain (CR1, CR2 epitopes) of CD4 molecule. PMID- 10732738 TI - The promise of anti-angiogenic cancer therapy. PMID- 10732739 TI - H19 and IGF-2 allele-specific expression in hepatoblastoma. AB - Patterns of allele-specific expression of H19 and insulin-like growth factor-2 (IGF-2) were examined in tissue obtained from 30 children diagnosed with hepatoblastoma. All informative tumours demonstrated monoallelic expression of H19. In contrast, variable patterns of allele-specific expression of IGF-2 were seen in tumours from children of different ages. PMID- 10732740 TI - No association between P53 codon 72 polymorphism and risk of squamous cell carcinoma of the head and neck. AB - An initial report suggested that patients homozygous for the arginine allele at codon 72 of P53 were at increased risk for human papillomavirus (HPV)-related cervical cancer, but other groups have not confirmed this finding. Since approximately 18-36% of head and neck cancers are HPV-related, we examined the genotypic frequencies at that locus in 163 cases with squamous cell carcinoma of the head and neck (SCCHN) and 163 ethnically matched controls. We found no significant excess of arginine homozygotes in cases compared to controls (P= 0.50). No significant differences in allele frequencies were observed when the data were stratified by tobacco exposure or by cancer site. These findings suggest a limited role, if any, for this P53 polymorphism in SCCHN. PMID- 10732741 TI - Requirement for expert histopathological assessment of ovarian cancer and borderline tumors. AB - The distinction between borderline ovarian tumours (BOT) and ovarian carcinoma is made by histopathological assessment. Of 64 patients managed according to institutional BOT protocols, 27 (42%) had been referred with a diagnosis of ovarian carcinoma that was subsequently changed to BOT following histopathological review. The 70% 6-year event-free survival of the patients with a revised diagnosis was not significantly different from those who were referred with a diagnosis of BOT. This change in diagnosis is important as it avoids the need for chemotherapy for most patients and results in patients receiving appropriate information concerning prognosis. Interestingly, 24 patients (38.1%) reported a family history of epithelial cancer, a finding that has not been reported previously. Campaign PMID- 10732742 TI - Analysis of radon-associated squamous cell carcinomas of the lung for a p53 gene hotspot mutation. AB - Squamous cell lung carcinomas (SCC) from former employees of the Wismut uranium mining company (Saxony, Germany) were obtained from the Stollberg Archive in order to screen for p53 tumour suppressor gene codon 249 arg-->met hotspot mutations, a putative molecular bio-dosimeter of alpha-particle (radon) exposure (Taylor et al (1994) Lancet 343: 86-87; McDonald et al (1995) Cancer Epidemiol Biomarkers Prevent 4: 791-793). Of the 29 archived samples of SCC meeting quality criteria for DNA analysis by polymerase chain reaction (PCR) and Haelll restriction enzyme digestion, two tumours were found that harboured this mutation. DNA sequencing confirmed the presence of a G to T base substitution within the Haelll site spanning codons 249 and 250 of the p53 gene that results in replacement of arginine (wild-type) by methionine at residue 249. When these data are combined with those from our previous study of tumours from the Stollberg Archive in which 50 lung tumours were examined, (including nine SCCs), we conclude that the G-->T (arg-->met) codon 249 mutation prevalence in the Wismut miner cohort is not sharply elevated in lung cancers in general (two mutations/79 tumours), or specifically in SCCs of the lung (two mutations/38 SCC) when compared to data from lung cancer patients with no reported occupational exposure to radon gas. PMID- 10732743 TI - A prolonged methoxymorpholino doxorubicin (PNU-152243 or MMRDX) infusion schedule in patients with solid tumours: a phase 1 and pharmacokinetic study. AB - The aim of this phase I study was to assess feasibility, pharmacokinetics and toxicity of methoxymorpholino doxorubicin (MMRDX or PNU-152243) administered as a 3 h intravenous infusion once every 4 weeks. Fourteen patients with intrinsically anthracycline-resistant tumours received 37 cycles of MMRDX. The first cohort of patients was treated with 1 mg m(-2) of MMRDX. The next cohorts received 1.25 mg m(-2) and 1.5 mg m(-2) respectively. Common toxicity criteria (CTC) grade III/IV nausea and vomiting were observed in 1/18 cycles at 1.25 mg m(-2) and in 2/11 cycles at 1.5 mg m(-2). Transient elevation in transaminases up to CTC grade III was observed in 2/16 cycles at 1.25 mg m(-2) and 4/11 cycles at 1.5 mg m(-2). No cardiotoxicity was observed. At 1.25 mg m(-2) CTC grade IV neutropenia occurred in 1/17 cycles. At 1.5 mg m(-2) CTC grade III neutropenia was seen in 2/7 and grade IV in 3/7 evaluable cycles. Thrombocytopenia grade III was observed in 2/9 and grade IV in 1/9 evaluable cycles. One patient treated at 1.5 mg m(-2) died with neutropenic fever. Therefore, dose-limiting toxicity was reached and 1.25 mg m(-2) was considered the maximum tolerated dose for MMRDX as 3 h infusion. No tumour responses were observed. Pharmacokinetic parameters showed a rapid clearance of MMRDX from the circulation by an extensive tissue distribution. Renal excretion of the drug and its metabolite was negligible. In conclusion, prolongation of MMRDX infusion to 3 h does not improve the toxicity profile as compared with bolus administration. PMID- 10732744 TI - Immunochemotherapy with interleukin-2, interferon-alpha and 5-fluorouracil for progressive metastatic renal cell carcinoma: a multicenter phase II study. Dutch Immunotherapy Working Party. AB - In patients with metastatic renal cell carcinoma response rates of 7-26% have been achieved with immunotherapy. A high response rate of 48% in 35 patients has been reported for treatment with the combination of interferon-alpha (IFN-alpha), interleukin-2 (IL-2) and 5-fluorouracil (5-FU) (Atzpodien et al (1993a) Eur J Cancer29A: S6-8). We conducted a multicentre phase II study to confirm these results. Metastatic renal cell carcinoma patients were treated as outpatients with an 8-week treatment cycle. Recombinant human IL-2 20 MU m(-2) was administered subcutaneously (s.c.) three times a week (t.i.w) in weeks 1 and 4 and 5 MU m(-2) t.i.w. in weeks 2 and 3. Recombinant human IFN-alpha 2a 6 MU m(-2) was administered s.c. once in weeks 1 and 4 and t.i.w. in weeks 2 and 3, and 9 MU m(-2) t.i.w. in weeks 5-8. 5-FU (750 mg m(-2)) was given as a bolus injection intravenous once a week in weeks 5-8. The treatment cycle was repeated once in case of response or minor response. Fifty-two patients entered the study. All had undergone a nephrectomy and had progressive metastatic disease. The median WHO performance status was 1, the median number of metastatic sites was 2 (range 1-5) and the median time between the diagnosis of the primary tumour and the start of treatment was 12.9 months (range 1-153). Among the 51 patients, including four patients with early progressive disease, who were evaluable for response, the response rate was 11.8% (95% confidence interval (CI) 2.9-20.7%), with no complete responses. Median duration of response was 8.3 (range 3.8-22.4+) months. Median survival was 16.5 (range 1.8-30.5+) months. Grade 3/4 toxicity (WHO) occurred in 29/52 (55.8%) of the patients in cycle 1 and in 6/16 (37.5%) of the patients in cycle 2. It consisted mainly of anorexia, fatigue, nausea, fever and leucocytopenia. We cannot confirm the high response rate in patients with metastatic renal cell carcinoma treated with the combination of IFN-alpha, IL-2 and 5-FU, as described by Atzpodien et al. PMID- 10732745 TI - Subclinical cardiotoxicity following adjuvant dose-escalated FEC, high-dose chemotherapy, or CMF in breast cancer. AB - We compared adjuvant chemotherapy-related myocardial damage by antimyosin scintigraphy in patients who received either nine cycles of FEC (fluorouracil, epirubicin and cyclophosphamide) where the doses of epirubicin and cyclophosphamide were escalated according to the leucocyte nadir (group I, n = 14), three cycles of FEC followed by high-dose chemotherapy with alkylating agents (CTCb) given with the support of peripheral blood stem cell transplantation (group II, n = 14), or six cycles of standard intravenous CMF (cyclophosphamide, methotrexate and fluorouracil; group III, n = 8). The cardiac uptake of In-111-antimyosin-Fab (R11D10) antibody was measured and the heart-to lung ratio (HLR) calculated 8-36 months after the last dose of chemotherapy. Cardiac antimyosin antibody uptake was considerably higher among patients treated with nine cycles of dose-escalated FEC than among those who were treated with three cycles of FEC and high-dose CTCb (HLR, median 1.98; range 1.36-2.24 vs median 1.51; range 1.20-1.82; P < 0.001), or those treated with CMF (median 1.44; range 1.15-1.68; P < 0.001). The difference between groups II and III was not significant (P > 0.1). A linear association was found between the cumulative dose of epirubicin and the cardiac antimyosin uptake (P < 0.001). We conclude that subclinical cardiac damage caused by three cycles of conventional-dose FEC followed by one cycle of high-dose CTCb chemotherapy is small as compared with the damage caused by dose-escalated FEC. PMID- 10732746 TI - Cysteine proteinase cathepsin H in tumours and sera of lung cancer patients: relation to prognosis and cigarette smoking. AB - In order to evaluate the role of cysteine peptidase cathepsin H (Cath H) in human lung cancer its protein levels were determined in 148 pairs of lung tumour tissue and adjacent non-tumourous lung parenchyma using the enzyme-linked immunosorbent assay technique. Additionally, Cath H levels were determined in sera of 171 patients with malignant tumours, 34 patients with benign lung diseases and 47 healthy controls. The median level of Cath H in tumour tissue was 0.64 times that in the corresponding lung parenchyma. Relating tumour levels with histological type we found higher Cath H levels in small-cell and adenocarcinomas and lower levels in squamous cell carcinoma, large-cell carcinoma and secondary tumours. A significant difference in Cath H level between lung tumour tissue and non tumourous lung parenchyma was associated with the group of cigarette smokers (156 vs 263 ng mg(-1) protein, P < 0.001). For this group of patients Cath H tumour levels correlated with the survival rate, while for the entire patient population this was not the case. Smokers with high tumour levels of Cath H experienced poor survival. Cath H was significantly higher in sera of patients with malignant and benign lung diseases than in control sera (P < 0.001). The increase was significant for all histological types, being the highest in small-cell and squamous cell carcinomas. Our study reveals that in lung tumours there is different behaviour of Cath H compared with other cysteine peptidases, e.g. cathepsin B and cathepsin L. Variations between tissue and serum levels of Cath H indicate either reduced expression or enhanced secretion of this enzyme in lung tumours. PMID- 10732747 TI - Fatigue, sexual function and mood following treatment for haematological malignancy: the impact of mild Leydig cell dysfunction. AB - Fatigue, sexual dysfunction, anxiety and depression are all more common in patients who have previously been treated with cytotoxic chemotherapy and radiotherapy (XRT) for haematological malignancies. Following therapy, a significant proportion of men have biochemical evidence of Leydig cell dysfunction, defined by a raised luteinizing hormone level in the presence of a low/normal testosterone level. We postulated that mild testosterone deficiency may account for some of the long-term side-effects of treatment, and we have therefore assessed fatigue, mood and sexual function by questionnaire in 36 patients with Leydig cell dysfunction (group 1), and also in a group of 30 patients (group 2) with normal hormone levels who underwent the same treatment for cancer. There was no significant difference in anxiety and depression scores between the two groups although anxiety scores were higher than those previously reported for normal men. Eighty-seven per cent of group 2 were sexually active compared with only 69% of group 1 (P= 0.1), and patients in group 1 engaged less in sexual activity than those in group 2 (mean of 1.8 times per week compared with 3.2 times per week; P = 0.02) Fatigue scores were significantly higher in both groups compared with normal men, but there were no significant differences in any of the fatigue subscales between the two groups. We conclude that mild Leydig cell insufficiency following treatment with cytotoxic chemotherapy +/- XRT is not associated with higher levels of fatigue and anxiety but may result in reduced sexual function. These results do not provide a convincing argument that androgen replacement therapy is mandatory to improve quality of life in the majority of these patients, although it may be beneficial in a minority. To establish criteria for selection of patients for a trial of androgen therapy a randomized placebo-controlled study will be necessary. PMID- 10732748 TI - Complete shutdown of microvascular perfusion upon hepatic cryothermia is critically dependent on local tissue temperature. AB - Since microvascular dysfunction with complete circulatory arrest and, thus, prolongation of tissue ischaemia is considered a potential mechanism for cell necrosis following hepatic cryosurgery, we determined the temperature necessary for induction of complete nutritive perfusion failure in cryothermia-treated rat livers. After localization of the cryoprobe with seven thermocouples and application of a single or double freeze-thaw cycle, in vivo fluorescence microscopy of the cryoinjured left lobe was performed over a 2-h period using a computer-controlled stepping motor, which guaranteed analysis of the identical liver tissue segments with exact allocation of the thermocouples and thus determination of tissue temperature. Cryothermia resulted in a central non perfused part of injury, surrounded by a heterogeneously perfused peripheral zone. The non-perfused area after single and double freezing continuously increased over the first 90-min period due to a successive shutdown of perfusion within the peripheral border zone. Analysis of the thermocouples' temperature at the end of freezing revealed the 0 degrees C-front at 11.7 mm (single freeze-thaw cycle) and 12.1 mm (double freeze-thaw cycle) distant from the centre of the cryoprobe, which exactly corresponds with the initial (30 min) expansion of the area with nutritive perfusion failure. The increased non-perfused tissue area at 2 h conformed a critical border temperature between 8.29 +/- 1.63 degrees C and 9.07 +/- 0.24 degrees C. From these findings, we conclude that freezing of liver tissue to temperatures of at least < 0 degrees C causes complete/irreversible perfusion failure, which consequently will result in cell death and tissue necrosis, and may thus be supposed as a prerequisite for the safe and successful application of cryosurgery in hepatic tumour ablation. PMID- 10732749 TI - Development and validation of the Cancer Dyspnoea Scale: a multidimensional, brief, self-rating scale. AB - Dyspnoea is one of the most frequent and refractory symptoms in cancer patients. Lack of an appropriate assessment tool for dyspnoea seems to disturb establishment of management strategy. The purpose of this study was to develop and validate a brief self-rating scale to assess the multidimensional nature of dyspnoea in cancer patients. We developed a 12-item scale, the Cancer Dyspnoea Scale (CDS), composed of three factors (sense of effort/sense of anxiety/sense of discomfort), by using factor analysis. One hundred and sixty-six patients with advanced or recurrent lung cancer participated in the validation phase. The CDS showed good feasibility (average time required to complete it was 140 s). Construct validity, confirmed by repeating factor analysis, was good. Convergent validity, confirmed by a relation to Visual Analogue Scale of dyspnoea and modified Borg's scale, was also good (average: r= 0.57 and 0.52, respectively, and both P < 0.001). The CDS had good internal consistency (average Cronbach's alpha = 0.86) and stability (average test-retest reliability r = 0.66, P < 0.005). The present study demonstrated that the CDS is a brief, valid and feasible scale for assessing the multidimensional nature of dyspnoea in cancer patients. PMID- 10732750 TI - Activity of high-dose epirubicin combined with gemcitabine in advanced non-small cell lung cancer: a multicenter phase I and II study. AB - The aim of the study was to evaluate efficacy and tolerance of epirubicin and gemcitabine as first-line chemotherapy in patients with advanced non-small-cell lung cancer. A phase I study was performed with the combination of escalating doses of epirubicin intravenously on day 1 and a fixed dose of gemcitabine on days 1 and 8 of a 21 -day cycle. Eighteen patients were included in the phase I part of the study before the maximum tolerated dose was found. Dose-limiting toxicity was febrile neutropenia. The phase II part of the study was continued with epirubicin 100 mg m(-2) on day 1 and gemcitabine 1125 mg m(-2) on days 1 and 8 of a 21-day cycle. Forty-three chemotherapy-naive patients were included. The median age of the patients was 60 years (range 26-75). Most patients (74%) were in stage IV. Granulocytopenia CTC grade 4 occurred in 32.5% and thrombocytopenia grade 4 in 11.6% of cycles. Febrile neutropenia occurred in six patients. Non haematological toxicity was mainly mucositis CTC grade 2 and 3 in 35% of patients. The tumour response rate was 49% (95% confidence interval (CI) 35-63%). The median survival time for the patients was 42 weeks (95% CI 13-69). PMID- 10732751 TI - Continuous low dose Thalidomide: a phase II study in advanced melanoma, renal cell, ovarian and breast cancer. AB - To grow and metastasize, solid tumours must develop their own blood supply by neo angiogenesis. Thalidomide inhibits the processing of mRNA encoding peptide molecules including tumour necrosis factor-alpha (TNF-alpha) and the angiogenic factor vascular endothelial growth factor (VEGF). This study investigated the use of continuous low dose Thalidomide in patients with a variety of advanced malignancies. Sixty-six patients (37 women and 29 men; median age, 48 years; range 33-62 years) with advanced measurable cancer (19 ovarian, 18 renal, 17 melanoma, 12 breast cancer) received Thalidomide 100 mg orally every night until disease progression or unacceptable toxicity was encountered. Three of 18 patients with renal cancer showed partial responses and a further three patients experienced stabilization of their disease for up to 6 months. Although no objective responses were seen in the other tumour types, there were significant improvements in patients' sleeping (P < 0.05) and maintained appetite (P < 0.05). Serum and urine concentrations of basic fibroblast growth factor (bFGF), TNF alpha and VEGF were measured during treatment and higher levels were associated with progressive disease. Thalidomide was well tolerated: Two patients developed WHO Grade 2 peripheral neuropathy and eight patients developed WHO grade 2 lethargy. No patients developed WHO grade 3 or 4 toxicity. Further studies evaluating the use of Thalidomide at higher doses as a single agent for advanced renal cancer and in combination with biochemotherapy regimens are warranted. PMID- 10732752 TI - Individuals with presumably hereditary uveal melanoma do not harbour germline mutations in the coding regions of either the P16INK4A, P14ARF or cdk4 genes. AB - In familial cutaneous malignant melanoma (CMM), disruption of the retinoblastoma (pRB) pathway frequently occurs through inactivating mutations in the p16 (p16INK4A/CDKN2A/MTS1) gene or activating mutations in the G1-specific cyclin dependent kinase 4 gene (CDK4). Uveal malignant melanoma (UMM) also occurs in a familial setting, or sometimes in association with familial or sporadic CMM. Molecular studies of sporadic UMM have revealed somatic deletions covering the INK4A-ARF locus (encoding P16INK4A and P14ARF) in a large proportion of tumours. We hypothesized that germline mutations in the p16INK4A, p14ARF or CDK4 genes might contribute to some cases of familial UMM, or to some cases of UMM associated with another melanoma. Out of 155 patients treated at the Institut Curie for UMM between 1994 and 1997, and interviewed about their personal and familial history of melanoma, we identified seven patients with a relative affected with UMM (n = 6) or CMM (n = 1), and two patients who have had, in addition to UMM, a personal history of second melanoma, UMM (n = 1), or CMM (n = 1). We screened by polymerase chain reaction single-strand conformation polymorphism the entire coding sequence of the INK4A-ARF locus (exon 1alpha from p16INK4A, exon 1beta from p14ARF, and exons 2 and 3, common to both genes), as well as the exons 2, 5 and 8 of the CDK4 gene, coding for the functional domains involved in p16 and/or cyclin D1 binding. A previously reported polymorphism in exon 3 of the INK4A-ARF locus was found in one patient affected with bilateral UMM, but no germline mutations were detected, either in the p16INK4A, p14ARF or CDK4 genes. Our data support the involvement of other genes in predisposition to uveal melanoma. PMID- 10732753 TI - Mutation analysis of P73 and TP53 in Merkel cell carcinoma. AB - The p73 gene has been mapped to 1p36.33, a region which is frequently deleted in a wide variety of neoplasms including tumours of neuroectodermal origin. The p73 protein shows structural and functional homology to p53. For these reasons, p73 was considered as a positional and functional candidate tumour suppressor gene. Thus far, mutation analysis has provided no evidence for involvement of p73 in oligodendrogliomas, lung carcinoma, oesophageal carcinoma, prostatic carcinoma and hepatocellular carcinoma. In neuroblastoma, two mutations have been observed in a series of 140 tumours. In view of the occurrence of 1p deletions in Merkel cell carcinoma (MCC) and the location of p73 we decided to search for mutations in the p73 gene in five MCC cell lines and ten MCC tumours to test potential tumour suppressor function for this gene in MCC. In view of the possible complementary functions of p73 and TP53 we also examined the status of the TP53 gene. Sequence analysis of the entire coding region of the p73 gene revealed previously reported polymorphisms in four MCCs. In one MCC tumour, a mis-sense mutation located in the NH2-terminal transactivation region of the p73 gene was found. These results show that p73, analogous to neuroblastoma, is infrequently mutated in MCC. This is also the first report in which the role of TP53 in MCC has been investigated by sequencing the entire coding region of TP53. TP53 mis sense mutations and one non-sense mutation were detected in three of 15 examined MCCs, suggesting that TP53 mutations may play a role in the pathogenesis or progression of a subset of MCCs. Moreover, typical UVB induced C to T mutations were found in one MCC cell line thus providing further evidence for sun-exposure in the aetiology of this rare skin cancer. PMID- 10732754 TI - Desmoids in familial adenomatous polyposis are monoclonal proliferations. AB - Desmoids are poorly-understood, locally aggressive, non-metastasizing fibromatoses that occur with disproportionate frequency in patients with familial adenomatous polyposis (FAP). Their nature is controversial with arguments for and against a neoplastic origin. Neoplastic proliferations are by definition monoclonal, whereas reactive processes originate from a polyclonal background. We examined clonality of 25 samples of desmoid tissue from 11 female FAP patients by assessing patterns of X-chromosome inactivation to calculate a clonality ratio. Polymerase chain reaction (PCR) amplification of a polymorphic CAG short tandem repeat (STR) sequence adjacent to a methylation-sensitive restriction enzyme site within the human androgen receptor (HUMARA) gene using fluorescent-labelled primers enabled analysis of PCR products by Applied Biosystems Genescan II software. Twenty-one samples from nine patients were informative for the assay. Samples from all informative cases comprised a median of 66% (range 0-75%) clonal cells but from the six patients with a clonality ratio < or =0.5 comprised a median of 71% (65-75%) clonal cells. FAP-associated desmoid tumours are true neoplasms. This may have implications in the development of improved treatment protocols for patients with these aggressive tumours. PMID- 10732755 TI - Expression of telomerase-associated protein 1 and telomerase reverse transcriptase in hepatocellular carcinoma. AB - To know whether two protein components of human telomerase (human telomerase associated protein 1 (hTEP1) and human telomerase reverse transcriptase (hTERT) are useful markers for telomerase activation in human liver diseases, we examined mRNA levels of these and telomerase activity in human liver samples. Twenty-three human hepatocellular carcinomas (HCCs) and corresponding adjacent livers were analysed for hTEP1 and hTERT expression by semiquantitative reverse transcription polymerase chain reaction, and for telomerase activity by a telomeric repeat amplification protocol assay. Thirteen liver samples (ten HCCs and three dysplastic nodules) that were biopsied with 21-gauge needles were analysed for hTERT expression. hTEP1 was expressed in all samples examined. No correlation between hTEP1 expression and telomerase activity was observed. hTERT expression significantly correlated with telomerase activity (P< 0.001). The positivity of hTERT for HCC and corresponding non-cancerous liver was 100% and 30.4% respectively (P < 0.001). Seventy-four per cent (17/23) of HCCs showed strong hTERT expression, but none of the non-cancerous liver tissues did. hTERT expression of the 21-gauge needle biopsied specimens showed no significant difference from that of the surgical samples. The present study revealed that hTERT is strongly expressed in most HCCs, and that hTERT but not hTEP1 is a key component regulating telomerase activity in human liver. PMID- 10732756 TI - Abnormalities of the FHIT gene in human oral carcinogenesis. AB - The abnormalities of the fragile histidine triad (FHIT) gene in tissue samples of oral squamous cell carcinomas (SCCs) along with several leukoplakias and an erythroplakia were examined to determine whether the FHIT gene is actually a frequent target in vivo for alteration during oral carcinogenesis. Abnormal transcripts of the FHIT gene were found in eight of 15 oral SCCs. Although these abnormal transcripts varied widely, deletion patterns incorporating a deletion of exon 5 were the most common. Loss of heterozygosity (LOH) analysis demonstrated that the abnormal FHIT transcripts found in cancer cells were attributable to abnormalities of the FHIT gene. Abnormal FHIT transcripts were also observed in two of seven premalignant lesions. Interestingly, in the case of one patient with a premalignant lesion showing an abnormal FHIT transcript, subsequent oral SCC developed during a 3-year follow-up period. On the other hand, in the two patients from whom both leukoplakia and SCC samples were taken simultaneously, abnormal FHIT transcripts were found only in the SCCs. Although the functional role of FHIT remains to be clarified, these results suggest that the FHIT alteration is actually involved in carcinogenesis of the oral epithelium. PMID- 10732757 TI - Relationship of vascular maturation in breast cancer blood vessels to vascular density and metastasis, assessed by expression of a novel basement membrane component, LH39. AB - Angiogenesis, the formation of new vessels, has been demonstrated to be an indicator of prognosis in breast cancer patients. The extent of differentiation of the tumour vessels may affect access of peripheral white cells and egress or invasion of tumour cells. This has not been assessed in relation to tumour microvessel density or other variables and may be a marker of vascular remodelling. LH39 is a monoclonal antibody recognizing an epitope located at the lamina lucida of mature small veins and capillaries but not in newly formed vessels. To study vascular differentiation in breast tumours, we examined the vascular maturation index (VMI) in 12 normal and 50 breast carcinomas and this was correlated with different clinicopathological variables including angiogenesis. Mature vessels were defined by staining with antibodies to both LH39 and to CD31, using double immunohistochemistry, whereas immature vessels stained only for CD31. VMI was defined as the % fraction of mature vessels (LH39 positive) / total number of vessels (CD31-positive). The VMI was significantly higher in normal (54-68.5%; median 66.5%) than in tumours (0-47%; median 8.8%) (P = 0.0005). There was a significant inverse correlation between the tumour VMI and nodal status (Fisher's exact test, P = 0.01) and between high VMI and low thymidine phosphorylase (TP) expression (Mann-Whitney U-test, P= 0.01). No significant association between VMI and tumour size, oestrogen receptor, epidermal growth factor receptor, grade, angiogenesis, patient age, or E-selectin was seen. There was a significant reduction in relapse-free survival (P = 0.01) with high angiogenesis. These findings show that the VMI gives new information on the mechanism of tumour angiogenesis independently from microvessel quantitation, there is a wide variation in the differentiation of tumour vasculature but the degree of capillary differentiation is not associated with quantitative angiogenesis. The VMI identifies a subset of patients who have a high chance of regional node involvement. PMID- 10732758 TI - Association of CYP1A1 and microsomal epoxide hydrolase polymorphisms with lung squamous cell carcinoma. AB - Lung cancer is the leading cause of death among cancers in Taiwan. Although the etiology of lung cancer has yet to be defined, genetic variability in activities of metabolic enzymes has been correlated with lung cancer. In the present study, the possibility of association of CYP1A1 and microsomal epoxide hydrolase (HYL1) genetic polymorphisms with lung cancer was examined among 132 lung cancer patients and 259 controls in Taiwan. No significant association was observed for either CYP1A1 or HYL1 polymorphism alone and the overall incidence of lung cancer after adjusting for age, gender and smoking status. When cases were stratified according to histological type, there was significant association between CYP1A1*2A homozygote and squamous cell carcinoma (SCC) (odds ratio (OR) 2.86; 95% confidence interval (CI) 1.33-6.12). Similarly, the proportion of HYL1 genotypes corresponding to high or normal enzyme activities was higher in SCC than in controls (OR 1.96; 95% CI 1.04-3.70). A combination of susceptible CYP1A1 and HYL1 genotypes was found to be highly associated with lung cancer, especially with SCC (OR 6.76; 95% CI 2.29-19.10). Our results suggest that the combination of CYP1A1 and HYL1 polymorphisms is an important risk factor for lung SCC. PMID- 10732759 TI - Markers of bone turnover for the management of patients with bone metastases from prostate cancer. AB - Although increased bone formation is a prominent feature of patients with osteosclerotic metastases from prostate cancer, there is also some evidence for increased bone resorption. The aim of this study was to compare the clinical utility of new bone resorption markers to that of bone formation in patients with bone metastases from prostate cancer before and after bisphosphonate treatment. Thirty-nine patients with prostate cancer and bone metastasis, nine patients with prostate cancer without bone metastases, nine patients with benign prostatic hyperplasia and 355 healthy age-matched men were included. Urinary non-isomerized (alpha CTX) and beta isomerized (beta CTX) type I collagen C-telopeptides (CTX) and a new assay for serum CTX were used to assess bone resorption. Bone formation was determined by serum osteocalcin, serum total (T-ALP) and bone (BAP) alkaline phosphatase and serum type I collagen C-terminal propeptide (PICP). Fourteen patients with bone metastases were also evaluated 15 days after a single injection of the bisphosphonate pamidronate (120 mg). Levels of all bone formation and bone resorption markers were significantly (P < 0.006-0.0001) higher in patients with prostate cancer and bone metastasis than in patients with benign prostatic hyperplasia, patients with prostate cancer without bone metastases and healthy controls. In patients with bone metastases the median was increased by 67% for serum osteocalcin, 128% for T-ALP, 138% for BAP, 79% for PICP, 220% for urinary alpha CTX, 149% for urinary beta CTX and 214% for serum CTX. After bisphosphonate treatment all three resorption markers significantly decreased by an average of 65% (P = 0.001), 71% (P = 0.0010) and 61% (P = 0.0015) for urinary alpha CTX, urinary beta CTX and serum CTX, respectively, whereas no significant change was observed for any bone formation markers. Patients with prostate cancer and bone metastases exhibit a marked increase in bone resorption, which decreases within a few days of treatment with pamidronate. These findings suggest that these new resorption markers may be useful for the management of these patients. PMID- 10732760 TI - Role and new perspectives of transforming growth factor-alpha (TGF-alpha) in adenocarcinoma of the gastro-oesophageal junction. AB - The incidence of gastro-oesophageal junction (GEJ) adenocarcinoma is increasing in Western countries and prognosis is poor since metastasis is most often present at diagnosis. We examined samples from 87 resected type II GEJ adenocarcinomas, 30 of these with endoscopic diagnostic biopsy material, to evaluate transforming growth factor alpha (TGF-a) expression and p53 overexpression by immunohistochemistry and in situ hybridization (for TGF-alpha), in relation to biological and clinical behaviour. TGF-alpha messenger RNA (mRNA) and protein were detectable in neoplastic cells in 56% and 64% cases respectively. TGF-alpha mRNA was detected in intra- and peritumoral lymphocytes and those of metastatic lymph nodes. TGF-alpha protein expression was significantly associated with tumour progression (P= 0.025) and lymph node metastasis (P < 0.05). The strong TGF-alpha expression found in neoplastic cells inside blood and lymphatic vessels and in metastatic localizations suggests that TGF-a-positive GEJ adenocarcinomas could have a more aggressive biological phenotype. The expression of TGF-alpha mRNA and protein in both inflammatory and neoplastic cells indicates that TGF alpha is directly synthesized by both cell compartments. Finally, since TGF-alpha expression was associated with lymph node metastasis, its detection in preoperative perendoscopic biopsies might identify patients with more aggressive tumours who may need additional therapy, including neo-adjuvant treatment. PMID- 10732761 TI - Extensive molecular screening for hereditary non-polyposis colorectal cancer. AB - The genetic abnormalities underlying hereditary non-polyposis colorectal cancer (HNPCC) are germline mutations in one of five DNA mismatch repair genes or in the TGFbetaRII gene. The aim of our study was to evaluate the significance of simple tests performed on tumours to select appropriate candidates for germline mutational analysis. We studied three groups of patients, HNPCC kindreds fulfilling the International Collaborative Group (ICG) criteria (n = 10), families in which at least one of the criteria was not satisfied (n = 7) and sporadic colorectal cancer (CRC) diagnosed before the age of 50 (n = 17). We searched for microsatellite instability (MSI), presence of hMSH2 and hMLH1 germline mutations, expression of hMSH2, hMLH1 and p53 proteins in tumoural tissue samples by immunostaining. Fifteen out of 17 (88%) of HNPCC and incomplete HNPCC cases were MSI and eight pathogenic germline mutations in hMSH2 or hMLH1 were detected in these two groups (53%). All the 17 early-onset sporadic cases were MSS and no germline mutations were detected among the seven investigated cases. Thirteen out of 15 (81%) familial cases were MSI and p53 protein-negative, whereas 13/14 (93%) sporadic cases were MSS and strongly p53 protein-positive. This extensive molecular investigation shows that simple tests such as MS study combined with hMSH2 and hMLH1 protein immunostaining performed on tumoural tissues may provide valuable information to distinguish between familial, and probably hereditary, and sporadic CRC cases. PMID- 10732762 TI - Activity of growth factors in the IL-6 group in the differentiation of human lung adenocarcinoma. AB - The role of the interleukin-6 (IL-6) group of cytokines in differentiation of two lung adenocarcinoma cell lines has been examined using induction of alkaline phosphatase and expression of surfactant protein A. Oncostatin M was the most active and potent for alkaline phosphatase in A549 cells, with IL-6 having similar activity but less potency. Neither cytokine induced alkaline phosphatase in NCI-H441 cells, although induction was obtained with lung fibroblast conditioned medium. Surfactant protein A was induced in NCI-H441 cells by conditioned medium and dexamethasone and, to a much lesser extent, by oncostatin M or IL-6. Induction of alkaline phosphatase and surfactant protein A were both dexamethasone-dependent, though some induction of surfactant protein A was obtained with interferon-alpha in the absence of dexamethasone. The activity present in lung fibroblast-conditioned medium suggests paracrine control, but this appears not to be due to oncostatin M or IL-6 as disabling antibodies to either cytokine were not inhibitory, and, although alkaline phosphatase was induced in A549 by both cytokines, it was only induced by conditioned medium in NCI-H441 cells. Furthermore, surfactant protein A was induced in H441 by conditioned medium to a much greater extent than by oncostatin M or IL-6. These data demonstrate that cytokines of the IL-6 group have potential as differentiation inducers in lung adenocarcinoma cells and that there is an equivalent paracrine factor(s) in lung fibroblast conditioned medium. As the production of this factor by fibroblasts is not enhanced by glucocorticoid, although the response of the target cell is, it would appear to be distinct from the fibrocyte pneumocyte factor previously described by Post et al 1984. PMID- 10732763 TI - Hepatocyte growth factor modulates motility and invasiveness of ovarian carcinomas via Ras-mediated pathway. AB - Hepatocyte growth factor (HGF) is a multifunctional growth factor which has pleiotrophic biological effects on epithelial cells such as proliferation, motogenesis, invasiveness and morphogenesis. Peritoneal dissemination is critical for the progression of ovarian cancer, and our study revealed that HGF induces migration and invasion of ovarian cancer cells. We also demonstrated that HGF stimulates autophosphorylation of its receptor, followed by activation of the Ras MAP (mitogen-activated peptide) kinase cascade. Moreover, infection of ovarian cancer cells with Ras dominant-negative adenovirus reduced the HGF-induced motogenic and invasive activities. Additionally, both MEK and P13-kinase pathways downstream of Ras were involved in HGF-stimulated ovarian cancer cell invasiveness. PMID- 10732764 TI - The growth pattern and microvasculature of pancreatic tumours induced with cultured carcinoma cells. AB - Pancreatic cancer is one of the most frustrating problems in gastroenterological surgery, since there is little we can do to improve the survival of patients with current treatment strategies. If one is to elucidate factors related to carcinogenesis, tumour biology, diagnostics and new treatment modalities of this malignant disease, then it is essential to develop a suitable animal model. In the present study we investigated rat pancreatic tumour growth after intrapancreatic injection of cultured pancreatic carcinoma cells (DSL-6A/C1), originally derived from an azaserine-induced tumour, as well as the features of tumour microcirculation using the microangiography technique. After intrapancreatic inoculation, tumours were detected in 64% of animals. A 1 cm3 tumour volume was reached within 20 weeks after inoculation. The tumours were ductal adenocarcinomas. Larger tumours showed invasive growth and spreading into the surrounding tissues, mainly into spleen and peritoneum. Microangiography revealed that the pancreatic tumours had an irregular and scanty vessel network and there were avascular areas in the center of the tumour. The area between normal pancreas and the induced tumour had dense vascularization. Intrapancreatic tumour induction with cultured pancreatic carcinoma cells produced a solid and uniformly growing tumour in Lewis rats and it thus provides a possible model for pancreatic cancer studies. PMID- 10732765 TI - Manumycin inhibits ras signal transduction pathway and induces apoptosis in COLO320-DM human colon tumour cells. AB - The aim of the present study was to assess the cytotoxicity of manumycin, a specific inhibitor of farnesyl:protein transferase, as well as its effects on protein isoprenylation and kinase-dependent signal transduction in COLO320-DM human colon adenocarcinoma which harbours a wild-type K-ras gene. Immunoblot analysis of isolated cell membranes and total cellular lysates of COLO320-DM cells demonstrated that manumycin dose-dependently reduced p21 ras farnesylation with a 50% inhibitory concentration (IC50) of 2.51 +/- 0.11 microM and 2.68 +/- 0.20 microM, respectively, while the geranylgeranylation of p21 rhoA and p21rap1 was not affected. Manumycin dose-dependently inhibited (IC50 = 2.40 +/- 0.67 microM) the phosphorylation of the mitogen-activated protein kinase/extracellular regulated kinase 2 (p42MAPK/ERK2), the main cytoplasmic effector of p21ras, as well as COLO320-DM cell growth (IC50 = 3.58 +/- 0.27 microM) without affecting the biosynthesis of cholesterol. Mevalonic acid (MVA, 100 microM), a substrate of the isoprenoid synthesis, was unable to protect COLO320-DM cells from manumycin cytotoxicity. Finally, manumycin 1-25 microM for 24-72 h induced oligonucleosomal fragmentation in a dose- and time-dependent manner and MVA did not protect COLO320-DM cells from undergoing DNA cleavage. The present findings indicate that the inhibition of p21ras processing and signal transduction by manumycin is associated with marked inhibition of cell proliferation and apoptosis in colon cancer cells and the effect on cell growth does not require the presence of a mutated ras gene for maximal expression of chemotherapeutic activity. PMID- 10732766 TI - Sensitivity to CPT-11 of xenografted human colorectal cancers as a function of microsatellite instability and p53 status. AB - Biological parameters influencing the response of human colorectal cancers (CRCs) to CPT-11, a topoisomerase 1 (top1) inhibitor, were investigated using a panel of nine CRCs xenografted into nude mice. CRC xenografts differed in their p53 status (wt or muf) and in their microsatellite instability phenotype (MSI+ when altered). Five CRC xenografts were established from clinical samples. All five had a functional p53, two were MSI+ and three were MSI-. Tumour-bearing nude mice were treated intraperitonealy (i.p.) with CPT-11. At 10 mg kg(-1) of CPT-11, four injections at 4-day intervals, four of the five xenografts responded to CPT-11 (growth delay of up to 10 days); the non-responder tumour was MSI-. At 40 mg kg( 1) of CPT-11, six injections at 4-day intervals, the five CRCs displayed variable but marked responses with complete regressions. In order to assess the role of p53 status in CPT-11 response, four CRC lines were used. HT29 cell line was MSI /Ala273-mutp53, its subclone HT29A3 being transfected by wtp53. LoVo cell line was MSI+/wtp53, its subclone X17LoVo dominantly expressed Ala273-mutp53 after transfection. LoVo tumours (MSI+/mutp53) were more sensitive than X17LoVo (MSI+/mutp53. HT 29 tumours (MSI-Imutp53), were refractory to CPT-11 while HT29A3 tumours (MSI-/wtp53) were sensitive, showing that wtp53 improves the drug response in these MSI- tumours. Levels of mRNA expression of top1, fasR, TP53 and mdr1 were semi-quantified by reverse transcription polymerase chain reaction. None of these parameters correlated with CPT-11 response. Taken together, these observations indicate that MSI and p53 alterations could be associated with different CPT-11 sensitivities; MSI phenotype moderately influences the CPT-11 sensitivity, MSI+ being more sensitive than MSI(-)CRC freshly obtained from patients, mutp53 status being associated with a poor response to CPT-11. PMID- 10732767 TI - Dipyridamole potentiates the in vitro activity of MTA (LY231514) by inhibition of thymidine transport. AB - The novel pyrrolopyrimidine-based antifolate LY231514 (MTA), inhibits multiple folate-requiring enzymes including thymidylate synthase, glycinamide ribonucleotide formyltransferase and dihydrofolate reductase. Both thymidine and hypoxanthine are required to reverse MTA growth inhibition in leukaemia and colon cancer cells. Prevention of MTA growth inhibition by thymidine and/or hypoxanthine was investigated in two human lung (A549, COR L23) and two breast (MCF7, T47D) tumour cell lines, and the effect of the nucleoside/base transport inhibitor dipyridamole (DP) on thymidine and hypoxanthine rescue defined. MTA IC50 values (continuous exposure three population doublings) were: A549-640 nM, COR L23-28 nM, MCF7-52 nM and T47D-46 nM. Thymidine (1 microM) completely prevented growth inhibition at the MTA IC50 in all cell lines. At 10 x IC50, growth inhibition was only partially reversed by thymidine (< or = 10 microM); both thymidine and hypoxanthine (30 microM) being required for complete reversal, reflecting the multi-targeted nature of MTA. Growth inhibition by MTA was not affected by hypoxanthine alone. A non-toxic concentration (1 microM) of DP prevented thymidine/hypoxanthine rescue of MTA indicating that DP may potentiate MTA activity by preventing nucleoside and/or base salvage. Thymidine transport was inhibited by > or = 89% by 1 microM DP in all cell lines, whereas hypoxanthine transport was inhibited only in A549 and MCF7 cells. Therefore, prevention of end-product reversal of MTA-induced growth inhibition by DP can be explained by inhibition of thymidine transport alone. PMID- 10732768 TI - In vivo inhibition of cysteine proteinases delays the onset of growth of human pancreatic cancer explants. AB - An animal model was used to study the effects of oral treatment with a small molecular selective inhibitor of cysteine proteinases, Z-Phe-Arg fluoromethylketone (Z-Phe-Arg-FMK) on primary tumour development. Poorly differentiated rapidly growing and moderately differentiated slowly growing human pancreatic tumours were implanted in the neck of nude mice that were orally treated or not with the inhibitor. Growth rates of the tumours were determined during 38 days after implantation. The poorly differentiated tumours were not affected by treatment with the inhibitor. Development of the moderately differentiated tumours was inhibited significantly by Z-Phe-Arg-FMK treatment. Moreover, the amount of stroma was increased and the volume of cancer cells was reduced in the moderately differentiated tumours that had grown in the treated animals. Reduction in size of the tumours was not achieved by reduction in growth rate but in a delay of the onset of growth. It is concluded that cysteine proteinases play a transient role at the start of tumour development only when cancer cells are surrounded by stroma as was the case in the moderately differentiated but not in the poorly differentiated pancreatic tumours. However, this role of cysteine proteinases can easily be taken over by other proteinases. PMID- 10732769 TI - Alteration of tumour response to radiation by interleukin-2 gene transfer. AB - We have previously shown that BALB/c-derived EMT6 mammary tumours transfected with interleukin (IL)-2 have decreased hypoxia compared to parental tumours, due to increased vascularization. Since hypoxia is a critical factor in the response of tumours to radiation treatment, we compared the radiation response of IL-2 transfected tumours to that of parental EMT6 tumours. Because the IL-2 tumours have an altered host cell composition, which could affect the interpretation of radiation sensitivity as measured by clonogenic cells, we employed flow cytometric analysis to determine the proportion of tumour cells vs host cells in each tumour type. Using this approach, we were able to correct the plating efficiency based on the number of actual tumour cells derived from tumours, making the comparison of the two types of tumours possible. We also excluded the possibility that cytotoxic T-cells present in EMT6/IL-2 tumours could influence the outcome of the clonogenic cell survival assay, by demonstrating that the plating efficiency of cells derived from EMT6/IL-2 tumours remained unchanged after depletion of Thy-1+ cells. The in vivo radiation response results demonstrated that IL-2-transfected tumours were more sensitive to radiation than parental EMT6 tumours. The hypoxic fraction of the EMT6/IL-2 tumours growing in vivo was markedly decreased relative to parental EMT6 tumours thus the increased sensitivity results from the increased vascularity we have previously observed in these tumours. These results indicate the potential therapeutic benefit of combining radiation and immunotherapy in the treatment of tumours. PMID- 10732770 TI - Increased expression of HGF and c-met in rat small intestine during recovery from methotrexate-induced mucositis. AB - Chemotherapy or radiotherapy often cause mucosal damage in the gut (gut mucositis) in cancer patients. As a step to investigate mechanisms underlying subsequent intestinal repair, we have examined the expression profiles of hepatocyte growth factor (HGF) and its receptor c-met, two molecules previously implicated in tissue repair, in comparison to the histopathological and proliferative changes in a rat model of methotrexate-induced small intestinal mucositis. Histological analysis of the intestinal specimens revealed crypt loss and villus atrophy with damage maximal on day 5 after methotrexate injection, and normalization of mucosal structure commencing on day 6. Crypt cell proliferation was decreased dramatically on day 3, normalized on day 4 and up-regulated on days 5 and 6. HGF and c-met protein/mRNA expression was up-regulated between days 4 and 7, with the mRNA co-localizing to the crypt and lower villus epithelium. Therefore, following methotrexate injection, a decrease in crypt cell proliferation preceded histological damage, and conversely, crypt cell hyperproliferation preceded mucosal regeneration. Up-regulation of HGF and c-met coincided with crypt hyperproliferation and mucosal recovery, suggesting a role for HGF in intestinal repair following acute injury. The crypt epithelial localization of HGF and c-met implies an autocrine or paracrine mechanism of HGF action. PMID- 10732771 TI - Selective initial in vivo homing pattern of 5T2 multiple myeloma cells in the C57BL/KalwRij mouse. AB - One of the main characteristics of multiple myeloma cells is their predominant localization in the bone marrow. It is, however, unclear whether this is due to a selective initial entry, or whether this entry is more random and other processes like survival and/or growth stimulation, only present in the medullar microenvironment, are unique. To investigate this, in vivo homing kinetics of murine 5T2MM cells shortly after injection were assessed in bone marrow, liver, spleen, lungs, heart, intestines, kidney and testis by tracing of radiolabelled cells, by immunostaining of isolated cells and by polymerase chain reaction analysis. We demonstrated the presence of 5T2MM cells in bone marrow, spleen and liver with all other organs being negative. Adhesion assays of 5T2MM cells to different types of endothelial cells demonstrated a selective adhesion of 5T2MM cells to bone marrow and liver and not to lung endothelial cells. We here demonstrate that the specific in vivo localization of the 5T2MM cells is a result of the combination of a selective entry/adhesion of the 5T2MM cells in the bone marrow, spleen and liver, and a selective survival and growth of these tumour cells in the bone marrow and spleen but not in the liver. PMID- 10732772 TI - Increased type-IV collagenase (MMP-2 and MMP-9) activity following preoperative radiotherapy in rectal cancer. AB - The aim of this study was to investigate the effect of preoperative high-dose radiotherapy (25 Gy in 5 fractions over 5 days) on the type-IV collagenase protein profile, in patients with resectable rectal cancer, by gelatin zymography. Biopsy samples of tumour and distant normal mucosa from 12 patients with resectable rectal cancer were obtained pre- and post-radiotherapy. Expression of type-IV collagenases (both pro- and active forms) was studied using gelatin zymography. Enzyme levels were normalized for total protein content of each sample. Rectal cancer specimens expressed both pro (72 kDa) and active (62 kDa) forms of MMP-2 but only the pro form of MMP-9 (92 kDa). Normal mucosa showed expression of the pro forms of MMP-2 and MMP-9 while no active form of either enzyme was detected in any of the samples. A significant three- to fourfold increase (P < 0.01) of active matrix metalloproteinases (MMP)-2 (62 kDa) was seen in malignant rectal mucosa after radiotherapy. The effect of radiotherapy also led to a twofold increase (P = 0.047) of pro MMP-2 (72 kDa) and a two- to threefold increase (P = 0.03) of the precursor form of MMP-9 (92 kDa). In contrast, in normal mucosa expression of the precursor form of MMP-9 (92 kDa) did not change after radiation, and no significant effect on the levels of pro MMP-2 (72 kDa) was observed. Preoperative high-dose radiotherapy leads to an increase in activity of type-IV collagenases in patients with resectable rectal cancer. Type-IV collagenase inhibition may be a useful therapeutic adjunct to radiotherapy in rectal cancer. PMID- 10732773 TI - Relationships between hydrophobicity, reactivity, accumulation and peripheral nerve toxicity of a series of platinum drugs. AB - Previous work has shown platinum drugs to differ in their effects on the peripheral nervous system. To test whether their differential toxicity was due to differences in their partitioning into the peripheral nervous system, we correlated the hydrophobicity, reactivity, tissue accumulation and neurotoxicity of a series of eight platinum analogues. Neurotoxicity was detected by measuring sensory nerve conduction velocity (SNCV) in Wistar rats treated twice per week at the maximum tolerated dose. Tissue platinum concentrations were measured by inductively coupled plasma mass spectrometry. Hydrophobicity (log P) was measured using an octanol-aqueous shake-flask method. The half-life of platinum drug binding to plasma proteins in vitro was determined. The cumulative dose causing altered SNCV ranged from 15 to > 2050 micromol kg(-1). Ranking of the compounds by their neurotoxic potency in rats (oxaliplatin > R,R-(DACH)PtC4 > ormaplatin > S,S-(DACH)PtCl4 > S,S-(DACH)Pt oxalato > cisplatin > carboplatin > JM216) correlated with the frequency of neurotoxicity in patients (r> 0.99; P < 0.05). Ranking the compounds by their peripheral nerve accumulation was cisplatin > carboplatin > oxaliplatin > R,R-(DACH)PtCl4 = S,S-(DACH)PtCl4 and did not correlate with neurotoxicity. Log P ranged from - 2.53 to -0.16 but did not correlate with neurotoxicity. Log P correlated inversely with platinum accumulation in dorsal root ganglia (r2 = 0.99; P = 0.04), sural nerve (r2 = 0.85; P = 0.025), sciatic nerve (r2 = 0.98; P= 0.0012), spinal cord (r2 = 0.97, P= 0.018) and brain (r2 = 0.98, P= 0.001). Reactivity correlated with neurotoxicity potency in rats (r2 = 0.89, P = 0.0005) and with the frequency of neurotoxicity in patients (r2 = 0.99, P = 0.0002). The hydrophilicity of platinum drugs correlates with platinum sequestration in the peripheral nervous system but not with neurotoxicity. Differences in the reactivity of platinum complexes accounts for some of the variation in their neurotoxicity. PMID- 10732774 TI - TNF-alpha augments intratumoural concentrations of doxorubicin in TNF-alpha-based isolated limb perfusion in rat sarcoma models and enhances anti-tumour effects. AB - We have shown previously that isolated limb perfusion (ILP) in sarcoma-bearing rats results in high response rates when melphalan is used in combination with tumour necrosis factor alpha (TNF-alpha). This is in line with observations in patients. Here we show that ILP with doxorubicin in combination with TNF-alpha has comparable effects in two different rat sarcoma tumour models. The addition of TNF-alpha exhibits a synergistic anti-tumour effect, resulting in regression of the tumour in 54% and 100% of the cases for the BN175-fibrosarcoma and the ROS 1 osteosarcoma respectively. The combination is shown to be mandatory for optimal tumour response. The effect of high dose TNF-alpha on the activity of cytotoxic agents in ILP is still unclear. We investigated possible modes by which TNF-alpha could modulate the activity of doxorubicin. In both tumour models increased accumulation of doxorubicin in tumour tissue was found: 3.1-fold in the BN175 and 1.8-fold in the ROS-1 sarcoma after ILP with doxorubicin combined with TNF-alpha in comparison with an ILP with doxorubicin alone. This increase in local drug concentration may explain the synergistic anti-tumour responses after ILP with the combination. In vitro TNF-alpha fails to augment drug uptake in tumour cells or to increase cytotoxicity of the drug. These findings make it unlikely that TNF alpha directly modulates the activity of doxorubicin in vivo. As TNF-alpha by itself has no or only minimal effect on tumour growth, an increase in local concentrations of chemotherapeutic drugs might well be the main mechanism for the synergistic anti-tumour effects. PMID- 10732776 TI - In vitro modelling of epithelial and stromal interactions in non-malignant and malignant prostates. AB - To study the effects of stromal epithelial cell interactions on prostate cancer metastasis, we have used primary human prostatic stromal cells derived from malignant and non-malignant tissues and established epithelial cell lines from normal (PNT1a and PNT2-C2) and tumour (PC-3, DU145 and LNCaP) origins. The effects of stromal cells on epithelial cell growth were studied in direct and indirect (using culture inserts) co-culture and by exposure to stromal cell conditioned medium (assessed by MTT assay). The influence of stromal cells on epithelial cell invasion was measured using matrigel invasion chambers and on epithelial cell motility using time lapse microscopy. Results indicated that epithelial cell line growth was similarly unaffected or inhibited by stromal cells derived from malignant (n = 8) or non-malignant tissue (n = 8). In contrast, PNT2-C2 and PC-3 cells were found to be the least and the most invasive and motile epithelia respectively. Stromal cultures enhanced the invasion of both epithelial cells, but no differences were observed between the use of malignant and non-malignant tissues. All stromal cultures modestly stimulated PNT2-C2 motility but displayed a greater stimulation of PC-3 cell motility, while stromal cells derived from malignant tissue stimulated PNT2-C2 and PC-3 cell motility more than stromal cultures from non-malignant tissues. PMID- 10732777 TI - Neurofibromatosis 2, radiosurgery and malignant nervous system tumours. PMID- 10732775 TI - Inhibition of apoptosis in human tumour cells by the tumour-associated serpin, SCC antigen-1. AB - The squamous cell carcinoma antigen (SCC Ag) is a tumour-associated protein and a member of the serine protease inhibitor (serpin) family. The SCC Ag has been used as a serologic tumour marker for SCC progression, and its elevated serum levels are a risk factor for disease relapse. However, the biologic significance of this intracytoplasmic protein in cancer cells remains unknown. In this report, we demonstrated that apoptosis induced by 7-ethyl-10-hydroxycamptothecin, tumour necrosis factor-alpha (TNF-alpha) or interleukin (IL)-2-activated natural killer (NK) cells was significantly inhibited in tumour cells transduced with the SCC Ag 1 cDNA, as compared to control cells in vitro. Also, inhibition of the SCC Ag-1 expression in tumour cells by transfection of antisense SCC Ag-1 cDNA was accompanied by significantly increased sensitivity of these cells to apoptosis induced by etoposide or TNF-alpha. The mechanism of protection of tumour cells from apoptosis involved inhibition of caspase-3 activity and/or upstream proteases. In vivo, tumour cells overexpressing the SCC Ag-1 formed significantly larger tumours in nude mice than the SCC Ag-1-negative controls. Thus, overexpression of the SCC Ag-1, a member of the serpin family, in human cancer cells contributed to their survival by mediating protection from drug-, cytokine- or effector cell-induced apoptosis. PMID- 10732778 TI - Assessing apoptosis: a critical survey. AB - Much progress has been made in the understanding of the process of apoptosis and there is a burgeoning literature pointing to it being a key phenomenon in tissue homeostasis. Moreover it is clear that derangements of the apoptotic cascade and its regulation can occur in a variety of disease states including in cancer. Many methods exist for the demonstration of apoptosis but some are not quantifiable. Others, such as enumeration of apoptotic bodies or end-labelling techniques, lend themselves to quantification. However, poor methods of quantification are sometimes employed. In this review, the methodological problems that can affect the assessment of apoptosis are discussed and suggestions made for more rigorous approaches. PMID- 10732779 TI - Pathological studies of apoptosis in the normal breast. AB - Apoptosis in the normal breast has been demonstrated to be related to the menstrual cycle. The factors that influence apoptosis in the breast are reviewed, together with related variations in the degree of apoptosis to disease pathogenesis. Evidence for a relationship between mitosis and apoptosis is provided, and the implications of pregnancy and prolonged postlactational involution are discussed. Future progress will depend on active collaboration between molecular biologists and pathologists to dissect the complex signalling pathways that appear to control the apoptotic and mitotic cascades and their inter-relationships. PMID- 10732780 TI - Apoptosis in breast cancer: relationship with other pathological parameters. AB - Apoptosis is a frequent phenomenon in breast cancer and it can be detected by light microscopy in conventional histopathological sections or by special staining techniques. The number of apoptotic cells as a percentage of cells present, or the number of apoptotic cells per square millimetre of neoplastic tissue, is usually described as the apoptotic index (AI). In breast cancer, the AI is not related to tumour size, axillary lymph node metastasis or distant metastasis at diagnosis. It is greater in invasive ductal carcinomas than in other histological types. High AI is also related to high histological grade, high nuclear grade, comedo-type necrosis, lack of tubule formation, and dense infiltration of the tumour by lymphocytes. Sex steroid receptor-negative tumours have greater AIs than the sex steroid receptor-positive ones. Aneuploid breast cancers with high S-phase fractions (SPFs) also have high AI values compared with diploid tumours with low SPFs. p53-Positive breast cancers have high AIs, whereas tumours that are Bcl-2 positive have low AIs. The AI shows a strong positive correlation to all direct or indirect indicators of cell proliferation, such as mitotic index and Ki67 immunolabelling. Univariate survival analyses show that a high AI is linked with unfavourable disease outcome in axillary lymph node negative and -positive breast cancer, but multivariate analyses indicate that AI is not an independent prognostic factor. In conclusion, a high AI is related to malignant cellular features and indicators of invasiveness and cell proliferation in breast cancer. PMID- 10732781 TI - Cytometric analyses to distinguish death processes. AB - The morphological changes typical of apoptosis, as well as the loss of integrity of the plasma membrane and the breakdown of nuclear DNA provide numerous features that permit recognition of apoptotic cell death by various methods. Flow cytometry (FCM) and laser scanning cytometry (LSC) allow for accurate and rapid measurement of apoptosis in both cultures and clinical samples (e.g. solid tumors, bone marrow aspirates, peripheral blood etc.). Furthermore, both FCM and LSC enable one to correlate the apoptosis with the position of the dying cell in the cell cycle. Discussion includes the cytometric identification and quantitation of apoptotic or necrotic cells, based on the analysis of a particular biochemical or molecular feature that is characteristic for either necrosis or apoptosis. PMID- 10732782 TI - Survival signals within the tumour microenvironment suppress drug-induced apoptosis: lessons learned from B lymphomas. AB - The suppression of apoptosis is one mechanism by which tumours become drug resitant. Extracellular signals from the germinal centre (GC) of secondary lymphoid tissue can rescue B cells from physiological- and chemotherapy-induced apoptosis. Such survival signals include CD40 receptor ligation, interleukin-4 (IL-4) receptor stimulation and the interaction of the integrin ligand VCAM-1 with its receptor. The GC environment was modelled in vitro by providing B lymphoma cells with these survival signals. JLP119 B lymphoma cells underwent apoptosis after exposure to the topisomerase II inhibitor etoposide and this was dramatically reduced when the cells were cultured in the GC system. CD40 receptor ligation resulted in increased levels of Bcl-XL. Etoposide diminished the binding between Bax and Bcl-XL but this was restored by IL-4 and VCAM-1 triggered signals. These data demonstrate combined effects of three microenvironmental signals on the Bcl-2 family and illustrate the potential importance of such signalling pathways in drug resistance of tumour cells. PMID- 10732783 TI - Clinical studies of apoptosis and proliferation in breast cancer. AB - The interaction between cell death and cell proliferation determines the growth dynamics of all tissues. Studies are described here which relate the changes in proliferation and apoptosis that occur in human breast cancer during medical therapeutic manoeuvres. Xenograft studies strongly support the involvement of increased apoptosis as well as decreased proliferation after oestrogen withdrawal, and limited studies in clinical samples confirm the involvement of both processes. Cytotoxic chemotherapy induces increases in apoptosis within 24 h of starting treatment. However, after 3 months therapy the residual cell population shows apoptotic and proliferation indices much below pretreatment levels. Further molecular studies of this "dormant" population are important to characterise the mechanism of their resistance to drug therapy. The early changes in proliferation and apoptosis may provide useful intermediate response indices. PMID- 10732784 TI - Prognostic significance of apoptosis regulators in breast cancer. AB - Dysregulation of normal programmed cell death mechanisms plays an important role in the pathogenesis and progression of breast cancer, as well as in responses of tumors to therapeutic intervention. Overexpression of anti-apoptotic members of the Bcl-2 family such as Bcl-2 and Bcl-X(L) has been implicated in cancer chemoresistance, whereas high levels of pro-apoptotic proteins such as Bax promote apoptosis and sensitize tumor cells to various anticancer therapies. Though the mechanisms by which Bcl-2 family proteins regulate apoptosis are diverse, ultimately they govern decision steps that determine whether certain caspase family cell death proteases remain quiescent or become active. To date, approximately 17 cellular homologs of Bcl-2 and at least 15 caspases have been identified in mammals. Other types of proteins may also modulate apoptotic responses through effects on apoptosis-regulatory proteins, such as BAG-1-a heat shock protein 70 kDa (Hsp70/Hsc70)-binding protein that can modulate stress responses and alter the functions of a variety of proteins involved in cell death and division. In this report, we summarize our attempts thus far to explore the expression of several Bcl-2 family proteins, caspase-3, and BAG-1 in primary breast cancer specimens and breast cancer cell lines. Moreover, we describe some of our preliminary observations concerning the prognostic significance of these apoptosis regulatory proteins in breast cancer patients, contrasting results derived from women with localized disease (with or without node involvement) and metastatic cancer. PMID- 10732785 TI - Induction of cell death by radiotherapy. AB - Ionising radiation remains one of the most effective tools in the therapy of cancer. It combines the properties of an extremely efficient DNA-damaging agent with a high degree of spatial specificity. Nonetheless, there remain considerable differences in the outcome for treatment of tumours of differing histological type treated by radiotherapy. Tumours arising from lymphoid or germ cells are significantly more radiocurable than most solid tumours of epithelial origin. The molecular mechanisms underlying such differences in cellular radiosensitivity are the subject of current research. When normal mammalian cells are subjected to stress signals--e.g. radiation, chemotherapeutic drugs, oxygen deficiency--a range of gene products involved in the sensing and signalling of such stresses are activated. The response of eukaryotic cells to ionising radiation includes activation of DNA repair pathways and cell cycle checkpoints, with subsequent full 'biological' recovery or cell death. Radiation induces two different modes of cell death termed mitotic or clonogenic cell death, and apoptosis. Until recent years, there was surprisingly little mechanistic understanding of the events following induction of physical damage by radiation and biological outcome for the cell. There have been recent major advances in our understanding of the signal transduction pathways involved in determining the fate of cells after irradiation. PMID- 10732786 TI - Activation of p53 by oncogenes. AB - p53 is activated by a variety of cellular stresses, including DNA damage, hypoxia, and mitogenic oncogenes, but the extent to which each signal engages p53 as a tumour suppressor remains unknown. In non-immortal cells, the adenovirus E1A oncogene activates p53 to promote apoptosis, whereas oncogenic ras activates p53 to promote cellular senescence. Inactivation of p53 prevents E1A-induced apoptosis or Ras-induced senescence, allowing proliferation to continue unabated. In each instance, the ability of the oncogene to activate p53 involves the same functions as are required for their transforming potential, implying that p53 activation acts as a fail-safe mechanism to counter hyperproliferative signals. Furthermore, p19(ARF) is strictly required for oncogene signalling to p53. The fact that ARF--itself a tumour suppressor--acts as an intermediary in this response argues that the tumour suppressor activity of p53 can arise from its ability to eliminate oncogene-expressing cells. PMID- 10732787 TI - Regulation of mammary epithelial cell phenotypes by the helix-loop-helix protein, Id-1. AB - Mammary epithelial cells undergo cycles of proliferation, invasion, differentiation and apoptotic cell-death throughout adult life. The molecular mechanisms that regulate these complex and co-ordinated developmental programs are poorly understood. We have identified Id-1 protein, a negative regulator of basic helix-loop-helix transcription factors, as a critical regulator of these normal mammary epithelial cell phenotypes. We also found that Id-1 is an important regulator of the aggressive and invasive phenotype, as well as mediator of the effects of sex steroid hormones, in human breast cancer cells. PMID- 10732788 TI - Clinical studies of p53 in treatment and benefit of breast cancer patients. AB - This article describes p53 as a prognostic and predictive factor, together with some information on how best to determine the p53 status. By December 1998, 13,000 articles on p53 were identified on Pub Med, the National Library of Medicine. Within one week a further 62 articles were recorded making it difficult to give the complete p53 story. This review article will focus on discussing p53 in relation to its predictive potential. So far, no firm conclusions can be made based on the articles studied. This may be, in part, because many studies have used less than optimal techniques for determination of the p53 status, together with the fact that the studies lacked power to detect a potential difference in outcome from specific therapy in relation to p53 status. PMID- 10732789 TI - Clinical studies of Bcl-2 and treatment benefit in breast cancer patients. AB - Interest in translational studies aimed at investigating the role of biologic markers in predicting clinical outcome of breast cancer patients and, in particular, response to specific treatments, has progressively increased. Among biologic variables presently under investigation, apoptosis markers, in particular Bcl-2 and Bax expression, are receiving much attention for their relationship with the cellular response to genotoxic damage in experimental tumors. Retrospective, independent studies were carried out by several research groups on about 5000 patients with breast cancer at different stages and with an adequate follow-up. The outcome of separate analyses as a function of treatment generally demonstrated that Bcl-2 overexpression, which correlates with biologic features of a differentiated phenotype (slow proliferation, high steroid receptor levels, absence of p53 and c-erB-2 expression), is associated with a favorable outcome. Such a finding is mainly evident following surgery as well as endocrine treatment. Conversely, no or weak Bcl-2 expression, alone or in association with bax overexpression, appears indicative of a radiation response, and preliminary emerging evidence supports the involvement of such an association of apoptosis related markers even as predictors of long-term response to neoadjuvant cytotoxic treatment. Although the findings of an involvement of Bcl-2 and Bax as determinants of treatment response should be confirmed within the context of randomized clinical trials, they indicate a combined consideration of proteins that negatively and positively regulate apoptosis in translational studies on the effect of chemical and physical agents at a cellular level. PMID- 10732790 TI - Clinical aspects of cell death in breast cancer: the polyamine pathway as a new target for treatment. AB - Because intracellular polyamines have a critical role in cell proliferation and death pathways, the polyamine metabolic pathway represents a potential target for intervention in cancers. A number of polyamine analogues have been identified that downregulate polyamine synthesis and enhance polyamine catabolism, thereby depleting intracellular polyamines. Treatment of human breast cancer cell lines in culture with these analogues has been shown to decrease cell proliferation and induce programmed cell death. Phase I studies with one analogue are now complete, setting the stage for phase II trials to determine efficacy, in addition to preclinical studies to examine combinations of polyamine analogues and conventional cytotoxics. PMID- 10732791 TI - Use of aromatase inhibitors in breast carcinoma. AB - Aromatase, a cytochrome P-450 enzyme that catalyzes the conversion of androgens to estrogens, is the major mechanism of estrogen synthesis in the post-menopausal woman. We review some of the recent scientific advances which shed light on the biologic significance, physiology, expression and regulation of aromatase in breast tissue. Inhibition of aromatase, the terminal step in estrogen biosynthesis, provides a way of treating hormone-dependent breast cancer in older patients. Aminoglutethimide was the first widely used aromatase inhibitor but had several clinical drawbacks. Newer agents are considerably more selective, more potent, less toxic and easier to use in the clinical setting. This article reviews the clinical data supporting the use of the potent, oral competitive aromatase inhibitors anastrozole, letrozole and vorozole and the irreversible inhibitors 4-OH androstenedione and exemestane. The more potent compounds inhibit both peripheral and intra-tumoral aromatase. We discuss the evidence supporting the notion that aromatase inhibitors lack cross-resistance with antiestrogens and suggest that the newer, more potent compounds may have a particular application in breast cancer treatment in a setting of adaptive hypersensitivity to estrogens. Currently available aromatase inhibitors are safe and effective in the management of hormone-dependent breast cancer in post-menopausal women failing antiestrogen therapy and should now be used before progestational agents. There is abundant evidence to support testing these compounds as first-line hormonal therapy for metastatic breast cancer as well as part of adjuvant regimens in older patients and quite possibly in chemoprevention trials of breast cancer. PMID- 10732792 TI - The role of cytokines in both the normal and malignant ovary. AB - Normal ovarian tissue is rich in cytokines. Cytokines and chemokines are important in the physiology of ovarian function and of ovulation. Cytokines and chemokines may recruit cytokine-producing lymphocytes to the site of a developing follicle, and cytokines appear to play an important role in pre and post follicle development. Most of the same cytokines that are found in normal ovarian tissue are also found in association with malignancy in contrast to their functions in normal tissues. It is reasonable to assume that the functions of cytokines associated with malignancy may serve to promote the unregulated growth if tumor cells and metastasis. It is also likely that cytokines produced by tumors will modulate immune responses that favor tumor progression. In the following review, we have highlighted those functions of cytokines that have been identified as having the most significant impact on tumor growth and development. By examining activities of these cytokines in normal and in malignant ovarian tissues, it is hoped that future possible avenues for investigation may be opened up and that the results of these investigations will lead to strategies that can modulate the production or the activity of the cytokines leading to the growth of tumors or their metastases. Such strategies now fall under the general discipline of bioimmunotherapy. This is an expanding discipline as more is learned about growth regulation in cancer, and with the availability and rapid development of new molecules for therapeutic approaches. PMID- 10732793 TI - Prognostic variables of papillary and follicular thyroid carcinoma patients with lymph node metastases and without distant metastases. AB - From 1977 through 1995, 1,013 thyroid carcinoma patients received treatment and were followed up at Chang Gung Medical Center in Taiwan. To evaluate the prognostic variables of papillary and follicular thyroid carcinomas with limited lymph node metastases, a retrospective review of these patients was performed. Of these patients, 910 had papillary or follicular thyroid carcinoma, and 119 patients were categorized as clinical stage 2 with limited neck lymph node metastases only at the time of diagnosis. The patients were categorized into two groups as no recurrence and local recurrence or distant metastasis at the end of 1997. After the operations, radioactive iodide (131I) treatments were performed in 114 patients and external radiotherapy for neck region or distant metastases in 18 patients. The median follow-up period of these patients was 5.4 years. Clinical variables were coded in our computer for statistical analysis. After the treatments, 93 patients remained disease-free; 10 were in stage 2; 5 in stage 3; and 11 aggravated to stage 4. Of the clinical variables, age, post-operative first 1311 uptake scans, and 1-month post-operative thyroglobulin levels revealed statistically significant differences between the group which improved and the group which did not. During the follow-up period, five patients died; three patients died of thyroid cancer and two died of intercurrent diseases. Patients with papillary thyroid carcinoma revealed a higher percentage of lymph node metastases. Although limited lymph node metastases did not influence survival rate, patients with poor prognostic factors need more aggressive treatment to avoid progression of the cancer. PMID- 10732794 TI - Ectopic macroprolactinoma mimicking a chordoma: a case report. PMID- 10732795 TI - Noninvasive localization procedures in ectopic hyperfunctioning parathyroid tumors. AB - In primary hyperparathyroidism (pHPT), parathyroidectomy is the treatment of choice, but anatomic variations of ectopic glands may cause surgical failure. Reliable preoperative noninvasive localization procedures would have a positive impact on the operative time and increase recovery rate. We retrospectively evaluated 186 patients with pHPT who were studied before successful parathyroidectomy by double tracer scintigraphy (99mTc-pertechnetate+201TI chloride or 99mTc-pertechnetate +99mTc-sestamibi, 160 patients), ultrasonography (148 patients) and computerized tomography (CT) scan (92 patients). During bilateral neck exploration, 159 (85.5%) single adenomas, 6 (3.2%) parathyroid carcinomas, and 3 (1.6%) double adenomas were found. Moreover, 18 (9.7%) patients had diffuse chief cells parathyroid hyperplasia. Removed parathyroid glands were in ectopic sites in 41 (22.0%) cases, mainly localized in the upper mediastinum or behind the esophagus. The overall sensitivity was 83.5 and 85.2% for 99mTc pertechnetate+201TI chloride and 99mTc-pertechnetate+99mTc-sestamibi scintigraphy respectively, 80.4% for CT scan and 81.1% for ultrasonography. In patients with ectopic glands, sensitivity was 81.2, 79.5, 73.3 and 81.6% respectively. In 36 out of 41 patients with ectopic glands in whom the removed parathyroids were correctly localized, mean operative time was 95 min, and in 5 patients without preoperative localization it was 260 min. In conclusion, in pHPT, preoperative localization of an enlarged parathyroid is helpful, especially in ectopic adenomas and in anatomic variations in location, and it has been proved to reduce operative time and morbidity rate. PMID- 10732796 TI - Angelman syndrome: how many genes to remain silent? AB - The clinical features of Angelman syndrome (AS) include microcephaly, severe mental retardation, "puppet-like" ataxic gait with jerky arm movements, hyperactivity, bouts of inappropriate laughter, EEG abnormalities, and seizures. The frequency of occurrence of AS is in the range of 1/10,000 to 1/20,000 births. The AS locus maps to the imprinted chromosome 15q11-q13 region and the disease is caused by the absence of a normal maternal contribution to this region. The genetic complexity of AS is revealed by the existence of at least four molecular classes. A candidate AS gene, ubiquitin protein ligase 3A (UBE3A/E6-AP), has been identified, and mutations of this gene have been detected in several cases of AS. Moreover, UBE3A is expressed predominantly from the maternal allele in brain, strongly supporting its causative role in AS. However, there is evidence to suggest that, in addition to UBE3A, another gene(s) may be involved either directly in AS and/or indirectly by regulating UBE3A expression. PMID- 10732797 TI - Isolation and characterization of trinucleotide repeat containing partial transcripts in human spinal cord. AB - We report the isolation of 41 partial transcripts containing trinucleotide repeats (TNRs) (CAG/CCG or CAA) from human spinal cord using a polymerase chain reaction-based method. The sequence analysis and database search at the nucleic acid and protein level revealed several classes of TNR-containing partial transcripts (TNRPTs). The TNRPTs included 16 known genes and 10 contained or partially overlapped with 13 expressed sequence tags (ESTs), some of which are known to contain TNRs and others which have previously not been shown to contain these repeats (e.g., clone 54 with homology for the homeobox protein HOX-A5). A further 15 partial transcripts showed no homologies in the databases and therefore may be unique. The validity of this approach is supported by the detection of nervous system-specific genes (e.g., glial fibrillary acid protein) and genes known to show trinucleotide expansions in disease (e.g., AAD10 associated with spino cerebellar ataxia type 2). This method provides a simple approach for the isolation of TNRPTs, from which full-length transcripts can be obtained and the discovery of TNR-containing genes may be facilitated. TNRPTs can also be used to study quantitative gene expression at the transcriptional level, to construct TNR-enriched cDNA libraries, and to make larger contigs from ESTs. PMID- 10732798 TI - The mutation properties of spinal and bulbar muscular atrophy disease alleles. AB - We studied the gene for the trinucleotide repeat disorder X-linked spinal and bulbar muscular atrophy (SBMA) to quantify the spectrum of mutations and gain insight into genetic anticipation. This analysis was performed using single sperm typing from an affected individual. This method allows the quantification of large numbers of meioses and therefore provides accurate information about genetic instability of the CAG repeat expansions which cause SBMA. Among 198 X chromosome-containing sperm cells, 20% had a CAG repeat number equal to the donor's somatic DNA of 49 CAG repeats, 56% were expansions, and 24% contractions. Most of the expansions (84%) and contractions (94%) were between 1 and 3 CAG repeats. These results are consistent with those obtained from one previously studied SBMA patient and reveal greater CAG repeat instability in sperm than in somatic tissue. Our results indicate that in SBMA, in contrast to sperm typing analysis of Huntington's disease, there is relative stability of the CAG repeat number during paternal transmissions and that the spectrum of mutations is narrow. These results are in agreement with the limited available clinical data and suggest that anticipation may not be a significant feature of this disease. PMID- 10732799 TI - The correlation of clinical phenotype in Friedreich ataxia with the site of point mutations in the FRDA gene. AB - Most cases of Friedreich ataxia (FRDA) are due to expansions of a GAA trinucleotide repeat sequence in the FRDA gene coding for frataxin, a protein of poorly understood function which may regulate mitochondrial iron transport. However, between 1% and 5% of mutations are single base changes in the sequence of the FRDA gene, causing missense, nonsense, or splicing mutations. We describe three new mutations, IVS4nt2 (T to G), R165C, and L182F, which occur in patients in association with GAA expansions. These cases, and a further five reported cases of point mutations causing FRDA, demonstrate that splicing, nonsense, or initiation codon mutations (which cause a complete absence of functional frataxin) are associated with a severe phenotype. Missense mutations, even in highly evolutionally conserved amino acids, may cause a mild or severe phenotype. PMID- 10732800 TI - The human small conductance calcium-regulated potassium channel gene (hSKCa3) contains two CAG repeats in exon 1, is on chromosome 1q21.3, and shows a possible association with schizophrenia. AB - Mutations in various ion channel genes are responsible for neuromuscular and other neurological disorders. We have previously identified the human small conductance calcium-activated potassium channel gene (hSKCa3) which has two tandemly arranged CAG repeats in its 5' region. Here we have isolated the first genomic clones containing the gene and have shown that both repeats are in exon 1. Homology to the previously localized sequence tagged site G16005 indicated that the gene may be on chromosome 22q, however using polymerase chain reaction amplification of somatic cell hybrid DNA and fluorescence in situ hybridization of two P1 artificial chromosome clones, we physically localized the gene to chromosome 1q21.3. We previously found an association between the highly polymorphic second (more 3') CAG repeat and schizophrenia in 98 patients and 117 controls. We have now genotyped an additional 19 patients with schizophrenia and have performed statistical analyses on the entire group of patients and controls to investigate the possible effect of age of onset, family history, and gender of the patients on the observed association. None of these factors were found to influence the results. Both CAG repeats have been typed in 86 bipolar I disorder patients, and no significant difference in allele distribution was observed between our bipolar disorder patients and controls. PMID- 10732801 TI - Mutation and polymorphism analysis in the tuberous sclerosis 2 (TSC2) gene. AB - Tuberous sclerosis complex (TSC) is an autosomal dominant multi-system disorder with two known disease loci on chromosomes 9q34 (TSC1) and 16p13.3 (TSC2). TSC has a prevalence of approximately 1 in 5,000-6,000, exhibits incomplete penetrance, and occurs in all racial groups. Our laboratory has undertaken the complete mutation analysis of the TSC2 gene in 42 TSC families using single strand conformation polymorphism analysis and reverse transcription-polymerase chain reaction. Of the total of 42 families, 16 show evidence of linkage to the chromosome 16 TSC2 locus and 26 are either sporadic or too small to establish chromosome linkage. The TSC2 gene spans at least 45 kilobases of genomic DNA, has 41 known exons, and codes for a 5,474-base pair transcript. After complete gene analysis, 16 TSC2 mutations have been identified, including DNA insertions, deletions, splice site mutations, and amino acid substitutions. The majority of putative TSC2 mutations were found in sporadic rather than TSC2-linked families. We have also detected 15 polymorphisms which occur in the TSC2 gene. PMID- 10732802 TI - Identification of a novel missense mutation of the SMN(T) gene in two siblings with spinal muscular atrophy. AB - Spinal muscular atrophy (SMA) is a motor neuron disease caused by mutations in the telomeric copy of the survival motor neuron (SMN(T)) gene. Over 90% of SMA patients harbor a deletion of SMN(T), but relatively few base-pair mutations have been reported. We report here a novel G279C mutation with a G to T transversion on exon 7 (nucleotide position 868) of SMN(T). Another missense mutation has been reported recently on position 869. The fact that two mutations on the same codon both result in SMA suggest a functional significance of this amino acid within the SMN protein. PMID- 10732803 TI - Paternal age is a risk factor for Alzheimer disease in the absence of a major gene. AB - We compared the parental age at birth of patients with Alzheimer disease (AD) with that of cognitively healthy control subjects. Within 206 carefully diagnosed AD patients, two groups were distinguished according to the likelihood of carrying a major gene for AD (MGAD). This likelihood was calculated by applying a Bayesian approach which incorporates data on aggregation of the disease, age at onset, and "censoring" ages within the family. All AD patients were ranked by MGAD probability. According to the sample's quartiles, two subgroups were defined representing the 52 individuals with the lowest and the 52 with the highest MGAD probability. Age at onset of dementia, education, and apolipoprotein E epsilon4 allele frequencies were not statistically different between the two groups. Fathers of patients with a low MGAD probability were significantly older (35.7+/ 8.1 years) than fathers of both other groups (high MGAD probability 31.3+/-6.9 years, P=0.004; controls 32.6+/-6.8 years, P=0.04, n=50). The differences for mothers were less pronounced and not statistically significant. These findings suggest that increased paternal age is a risk factor for AD in the absence of a major gene, whereas increased maternal age and AD are associated only weakly and independently of genetic disposition. PMID- 10732804 TI - The Y-chromosomal genes SRY and ZFY are transcribed in adult human brain. AB - Sexual differentiation of the brain is thought to be regulated by hormonal signals from the developing male gonad. However, more-recent experimental and clinical data throw some doubt on the general validity of the "classical" steroid hypothesis and suggest that additional intervening factors or mechanisms need to be considered. In particular, it is now envisaged that neurons are capable of acquiring sex-specific properties independently of their hormonal environment. Here we show that two Y-chromosomal genes involved in sex determination of the gonad, SRY and ZFY, are transcribed in hypothalamus, and frontal and temporal cortex of the adult male human brain. These genes are candidates for male specific transcriptional regulators that could confer upon human brain cells the potential for hormone-independent realization and maintenance of genetic sex. PMID- 10732805 TI - Genomic structure of human anion exchanger 3 and its potential role in hereditary neurological disease. AB - Alterations in ion channel permeability or selectivity have been shown to cause neurological defects in humans. Anion exchanger isoform 3 (AE3) is prominently expressed in the brain and performs an electroneutral exchange of chloride and bicarbonate ions. In order to study the potential role of AE3 in human neurological disease, we characterized AE3 genomic structure and performed mutational analysis on patients with an episodic movement disorder that maps to the same genetic locus. AE3 genomic organization, including the nucleotide sequence of the 5'-untranslated region and intron/ exon boundaries, is highly conserved between humans and homologs from mouse and rat. Mutational analysis revealed no disease-causing defect in patients with familial paroxysmal dyskinesia, although several benign polymorphisms were identified. AE3 variation may prove useful for further genetic studies, such as finer resolution mapping. Characterization of genomic structure will facilitate mutational analysis of AE3 in studies of neurological diseases mapped to the same locus. PMID- 10732807 TI - Friedreich's ataxia presenting as adult-onset spastic paraparesis. AB - We have studied a man with an atypical form of Friedreich's ataxia (FRDA), who presented at age 26 years with a 2-year history of unsteadiness and clumsiness. The predominant feature of his initial neurological examination was a spastic paraparesis, along with a mild distal weakness and hyperreflexia of the upper limbs. He also displayed limb ataxia. Frataxin GAA repeat sizes were 1,040/690. This unusual FRDA presentation is not dissimilar to that of Acadian spastic ataxia. PMID- 10732806 TI - A novel mutation in the predicted TM2 domain of the presenilin 2 gene in a Spanish patient with late-onset Alzheimer's disease. AB - Mutations in the presenilin-2 (PS-2) gene are less frequent than mutations in the PS-1 gene. All mutations described in the PS-1 gene were found in early-onset Alzheimer's disease (AD) patients. At present, there are two missense mutations described for the PS-2 gene in some AD pedigrees. We have therefore analyzed transmembrane 2 (TM2) and TM5 domains of the PS-2 gene in AD patients and in a group of age-matched healthy controls. In a patient who was clinically diagnosed as having late-onset AD, we found a novel missense mutation consisting of a G->A substitution on exon 5 of the PS-2 gene, which results in a Val to Ile substitution at codon 148 within the predicted TM2 domain of the PS-2 protein. This is the third mutation described in the PS-2 gene and the first presenilin mutation detected in a Spanish AD patient. Both, the N141I mutation and the V148I mutation described here are located within the predicted TM2 domain and both were found in late-onset AD kindreds, whereas the mutation within the predicted TM5 domain was found in an early-onset AD pedigree. Carriers of mutations within TM2 of PS-1 have a mean age at onset of 40 years, while the other mutations in PS-1 occur in families with a mean age at onset of 47 years. In summary, we report here the first mutation in a presenilin gene in a Spanish AD case, which is the third mutation detected for the PS-2 gene. PMID- 10732808 TI - Muscle apoptosis in humans occurs in normal and denervated muscle, but not in myotonic dystrophy, dystrophinopathies or inflammatory disease. AB - Recent data suggest that death of muscle cells during development and in selected pathological conditions occurs via apoptosis. We investigated the occurrence of apoptosis in normal and pathological human skeletal muscle, using in situ end labeling (ISEL) to detect DNA fragmentation, and immunohistochemistry for the expression of tissue transglutaminase and interleukin-1beta-converting enzyme (ICE)-like proteases. In normal subjects, apoptotic myonuclei were occasionally observed as evidence of normal tissue turnover. Myonuclear apoptosis due to a deficit of trophic support from nerve cells also occurred in spinal muscular atrophies. No apoptosis of muscle cells was found in dystrophinopathies, myotonic dystrophy and inflammatory myopathies, suggesting that death of myofibers in those conditions is not due to activation of a gene-directed program of death. In dystrophinopathies and inflammatory myopathies, apoptosis was found in interstitial mononuclear cells, as a likely mechanism of clearance of the inflammatory infiltrates. PMID- 10732809 TI - Confirmation of a second locus for CMT2 and evidence for additional genetic heterogeneity. AB - The Charcot-Marie-Tooth (CMT) neuropathies are a group of disorders exhibiting neurophysical, pathological and genetic heterogeneity. CMT2 is a diagnostic subtype of this group of disorders characterized by variable expression and age of-onset and normal or slightly diminished nerve conduction velocities. Previously, linkage and heterogeneity had been reported in CMT2 with linked families localizing to chromosome 1p (CMT2A). Recently a second CMT2 locus has been described on chromosome 7 in a single large CMT2 family (CMT2D). We have performed pedigree linkage analysis on 15 CMT2 families (N = 371 individuals, 106 affected family members) and have confirmed linkage to chromosome 7. Furthermore, using both admixture and multipoint linkage analysis we show conclusive evidence for additional heterogeneity within this clinical subtype with evidence of families that exclude linkage to both the CMT2D and CMT2A regions. In addition, unlike the previous report we found no obvious consistent clinical differences between the linked family types. PMID- 10732810 TI - Locus heterogeneity, anticipation and reduction of the chromosome 2p minimal candidate region in autosomal dominant familial spastic paraplegia. AB - We examined 11 Caucasian pedigrees with autosomal dominant 'uncomplicated' familial spastic paraplegia (SPG) for linkage to the previously identified loci on chromosomes 2p, 14q and 15q. Chromosome 15q was excluded for all families. Five families showed evidence for linkage to chromosome 2p, one to chromosome 14q, and five families remained indeterminate. Homogeneity analysis of combined chromosome 2p and 14q data gave no evidence for a fourth as yet unidentified SPG locus. Recombination events reduced the chromosome 2p minimum candidate region (MCR) to a 3 cM interval between D2S352 and D2S367 and supported the previously reported 7 cM MCR for chromosome 14q. Age of onset (AO) was highly variable, indicating that subtypes of SPG are more appropriately defined on a genetic basis than by AO. Comparison of AO in parent-child pairs was suggestive of anticipation, with a median difference of 9.0 years (p<0.0001). PMID- 10732811 TI - Characterization of the rat spinocerebellar ataxia type 3 gene. AB - Machado-Joseph disease (MJD) belongs to a group of clinically and genetically heterogeneous neurodegenerative disorders characterized by progressive cerebellar ataxia. The disease-causing mutation has recently been identified as an unstable and expanded (CAG)n trinucleotide repeat in a novel gene of unknown function. In Caucasians, repeat expansions in the MJD1 gene have also been found in patients with the clinically distinct autosomal dominant spinocerebellar ataxia type 3 (SCA3). In order to gain insight into the biology of the MJD1/SCA3 gene we cloned the rat homologue and studied its expression. The rat and human ataxin-3 genes are highly homologous with an overall sequence identity of approximately 88%. However, the C-terminal end of the putative protein differs strongly from the published human sequence. The (CAG)n block in the rat cDNA consists of just three interrupted units suggesting that a long polyglutamine stretch is not essential for the normal function of the ataxin-3 protein in rodents. The expression pattern of the SCA3 gene in various rat and human tissues was investigated by Northern blot analyses. The mature transcript is approximately 6 kb in length. In rat testis, a smaller transcript of 1.3 kb was identified. Transcription of rsca3 was detected in most rat tissues including brain. Analyzing the expression level of the SCA3 gene in several human brain sections revealed no significant higher mRNA level in regions predominantly affected in MJD. Thus additional molecules and/or regulatory events are necessary to explain the exclusive degeneration of certain brain areas. PMID- 10732812 TI - A missense mutation in the SOD1 gene in patients with amyotrophic lateral sclerosis from the Kii Peninsula and its vicinity, Japan. AB - Unusually high incidences of amyotrophic lateral sclerosis (ALS) have been observed in the natives of the Kii Peninsula of Japan as well as the indigenous Chamorro people of Guam. Given the relatively high incidence of familial onset of the disease in the Kii Peninsula, we performed mutational analyses of the SOD1 gene of 23 patients (three familial cases and 20 sporadic cases) with ALS from the Kii Peninsula and its vicinity. In two of the 23 patients, we identified the same missense mutation (substitution of Thr for Ile 113) in exon 4 as a heterozygous state. The Ile 113Thr mutation in the SOD1 gene has been identified in some familial as well as sporadic cases with ALS, as a mutation with a low penetrance. This mutation has been reported to be associated with the formation of neurofibrillary tangles in an English family, which is a characteristic feature of ALS in the Kii Peninsula. These results suggest that the Ile113Thr mutation is a characteristic and relatively prevalent mutation in this area. PMID- 10732813 TI - Mutations in the X-linked form of Charcot-Marie-Tooth disease in the French population. AB - The present study reports eight additional mutations in the connexin32 gene associated with the X-linked form of Charcot-Marie-Tooth disease. One of these mutations was found twice in two apparently unrelated families. This form of the disease is demyelinating and dominant. However, patient selection for mutational screening should not be limited to these criteria since presentation can either be familial or sporadic, and some patients may be incorrectly classified as suffering from an 'axonal' form. These new mutations complete our previously published work on 12 other mutations and enable meaningful observation in a representative sample of the French population. Mutations are found in all regions of the gene. The most frequently observed mutations were those affecting arginines and mainly involved CpG sequences. Compared with other sources, some of the mutations were present at a higher frequency in the French population. PMID- 10732814 TI - Two previously unrecognized splicing mutations of GCH1 in Dopa-responsive dystonia: exon skipping and one base insertion. AB - We describe two previously unrecognized splice site mutations of GCH1 in Dopa responsive dystonia (DRD). Both mutations affect consensus splice acceptor (AG) sites. The first mutation is an A-->G transition at position -2 of intron 1 of GCH1. This mutation results in skipping of exon 2. Fusion of exons 1 and 3 causes a frame shift that generates a premature stop codon. The second mutation is an A- >G transition at position -2 of intron 2. The mutation generates a new splice acceptor site AG one base pair upstream of the wild-type splice site. This, together with a pyrimidine stretch upstream of the new splice site, renders this site functional and generates a transcript with the insertion of one base, i.e. the G of the wild-type splice site. This in turn causes a frame shift including the introduction of a premature stop codon. The two different mutations generate truncated GTP cyclohydrolase polypeptides. PMID- 10732815 TI - Homozygosity mapping of giant axonal neuropathy gene to chromosome 16q24.1. AB - Giant axonal neuropathy (GAN) is a rare autosomal recessive disorder described as a symmetrical distal neuropathy, with peripheral axons dilated by accumulation of 10 nm neurofilaments (NF) and a severe course of the disease. The observation of kinky or curly hairs is not a constant finding. The GAN1 locus was localized by homozygosity mapping to chromosome 16 q24.1 in a 3 (4) cM interval flanked by the markers D16S3073 and D16S505 (D16S511) in three non-related Tunisian families, showing a genetic homogeneity in these families. Two point lod-score calculation between the linked haplotype and the disease locus was 14.2 at theta(max) = 0. The patients share a slow course of the disease. The differences in the course of the disease between Tunisian and non-Tunisian patients suggest a possible genetic heterogeneity, which is why the present linkage has been referred to as GAN1. The biochemical defect in GAN1 should help to understand the mechanisms involved in NF accumulations as in other neurological diseases (ALS, SMA). PMID- 10732817 TI - Molecular diagnosis of non-deletion SMA patients using quantitative PCR of SMN exon 7. AB - The telomeric survival motor neuron (SMN(T)) gene is a valuable molecular diagnostic tool for childhood-onset spinal muscular atrophy (SMA) as homozygous deletions of SMN(T) exon 7 (delta7SMN(T)) are present in approximately 94% of patients. In this report, we provide the first comprehensive study of 32 unrelated non-deletion SMA patients. Quantitative polymerase chain reaction (PCR) studies established that 90% had two intact copies of SMN(T) exon 7 suggesting that these patients do not have 5q SMA. Once 5q SMA is confirmed, the SMN(T) gene can be screened for subtle mutations. Using single strand conformation analysis, we identified two missense mutations (P245L and Y272C) in exon 6 of the SMN(T) gene of two SMA patients shown to have a single copy of SMN(T) exon 7. Y272 is most likely critical for SMN(T) function as it is a target for recurring mutations and is associated with type I SMA. These results emphasize the need for dosage analysis in the differential diagnosis of 5q SMA in nondeletion patients, consistent with extensive clinical heterogeneity and some genetic heterogeneity in this disease. Homozygosity or heterozygosity for a delta7SMN(T) allele confirms the diagnosis of 5q SMA with greater precision than clinical examination alone. PMID- 10732816 TI - Emerin, deficiency of which causes Emery-Dreifuss muscular dystrophy, is localized at the inner nuclear membrane. AB - X-linked recessive Emery-Dreifuss muscular dystrophy (EDMD) is an inherited muscle disorder characterized by the clinical triad of progressive wasting of humero-peroneal muscles, early contractures of the elbows, Achilles tendons and postcervical muscles, and cardiac conduction block with a high risk of sudden death. The gene for EDMD on Xq28 encodes a novel protein named emerin that localizes at the nuclear membrane of skeletal, cardiac and smooth muscles and some other non-muscle tissues. To investigate a possible physiological role for emerin, we examined the ultrastructural localization of the protein in human skeletal muscle and HeLa cells, using ultrathin cryosections. We found that the immune-labeled colloidal gold particles were localized on the nucleoplasmic surface of the inner nuclear membrane, but not on the nuclear pore. Emerin stayed on the cytoplasmic surface of the nuclear lamina, even after detergent treatment that solubilizes membrane lipids and washes out membrane proteins. These results suggest that emerin anchors at the inner nuclear membrane through the hydrophobic stretch, and protrudes from the hydrophilic region to the nucleoplasm where it interacts with the nuclear lamina. We speculate that emerin contributes to maintain the nuclear structure and stability, as well as nuclear functions, particularly in muscle tissues that have severe stress with rigorous contraction relaxation movements and calcium flux. PMID- 10732818 TI - The thermolabile variant of methylenetetrahydrofolate reductase (MTHFR) is not a major risk factor for neural tube defect in American Caucasians. The NTD Collaborative Group. AB - Mutations in the gene for methylenetetrahydrofolate reductase (MTHFR) have been implicated as a risk factor in the formation of neural tube defects. We investigated this gene in a series of 65 sporadic American Caucasian patients with lumbosacral NTD and their unaffected parents, using both case-control design and assessment of linkage disequilibrium. We found no evidence to support mutations in MTHFR as a significant risk factor for NTD in this population. PMID- 10732819 TI - Further exclusion of FSHD1B from the telomeric region of 10q. AB - The localization of the gene for the majority of cases of facioscapulohumeral muscular dystrophy is established as 4q35-qter (FSHD1A), although locus heterogeneity has been demonstrated with a minority of families unlinked to 4q. In FSHD1A, the disease is associated with a deletion of 3.3 kb repeats from a tandem repeat located near the as-yet-unidentified gene. This repeat cross hybridizes with a telomeric region on 10q, making this cross-hybridizing region a feasible candidate gene for FSHD1B. We have tested the most telomeric marker on 10q (sAVA4) and excluded approximately 17 cM on either side of this marker as harboring the FSHD1B gene. PMID- 10732820 TI - Imaging the parathyroid glands using 99mTc-tetrofosmin: current status. PMID- 10732821 TI - Riedel's lobe of the liver and its clinical implication. PMID- 10732822 TI - Reactive arthritis or Reiter's syndrome and B51-associated seronegative spondyloarthropathy. PMID- 10732823 TI - Chemokine/receptor dynamics in the regulation of hematopoiesis. AB - Recently, a close association between hematopoiesis and chemokines and their receptors has been demonstrated. Much attention has been focused on the regulation of hematopoiesis by chemokines, because of the possibility of direct consequence for clinical practice in cell therapies such as hematopoietic stem cell transplantation. This report reviews the physiological roles of chemokines and their receptors in the context of hematopoietic cell regulation, with particular focus on the involvement of stromal cell derived factor (SDF)-1/pre-B cell growth stimulating factor (PBSF) and its receptor CXCR4. The proliferation, differentiation, migration, and engraftment of hematopoietic stem cells are discussed, and thereby raise a number of unresolved problems and points-to consider. PMID- 10732824 TI - Technetium-99m tetrofosmin parathyroid imaging in patients with primary hyperparathyroidism. AB - OBJECTIVE: To confirm the clinical significance of 99mTc-tetrofosmin imaging for the localization of hyperfunctioning parathyroid glands in patients with primary hyperparathyroidism. METHODS: All patients were imaged with 99mTc-tetrofosmin at 10 minutes and 2 hours after radiotracer injection, and with ultrasonography (US), computed tomography (CT), and magnetic resonance imaging (MRI). The parathyroid/thyroid uptake ratio of 99mTc-tetrofosmin (P/T uptake ratio) was calculated. PATIENTS: Twenty patients with primary hyperparathyroidism were referred to our clinic, underwent surgical neck exploration or mediastinotomy and were diagnosed as having parathyroid adenoma. These patients were investigated for the preoperative localization by 99mTc-tetrofosmin scintigraphy. RESULTS: 99mTc-tetrofosmin imaging demonstrated focal uptake in 19 out of 20 patients with parathyroid adenoma. Two of the lesions were ectopic. US identified 17 parathyroid glands. CT and MRI initially detected 17 parathyroid glands. However, two additional parathyroid glands were localized on repeated CT and MRI in tandem with the results of the 99mTc-tetrofosmin imaging. Thus, the sensitivity and specificity of tetrofosmin imaging were 95% (19/20) and 95% (19/20); US, 85% (17/20) and 94% (16/17); initial CT, 85% (17/20) and 94% (16/17); and initial MRI, 88% (17/20) and 94% (16/17), respectively. The P/T uptake ratio at 2 hours after tetrofosmin injection was correlated with the serum concentration of intact PTH (rs=0.47, p<0.05) and the resected tumor weight (rs=0.53, p<0.05). CONCLUSION: 99mTc-tetrofosmin scintigraphy is useful for localization of parathyroid adenoma. Tetrofosmin uptake depends on the tumor weight and serum intact PTH levels. PMID- 10732825 TI - Effects of dietary supplementation with n-3 fatty acids compared with n-6 fatty acids on bronchial asthma. AB - OBJECTIVE: The effects of perilla seed oil (n-3 fatty acids) on bronchial asthma were compared with the effects of corn oil (n-6 fatty acids) in relation to the pulmonary function and the generation of leukotriene B4 (LTB4) and C4 (LTC4) by leucocytes. METHODS AND SUBJECTS: 14 asthmatic subjects were divided randomly into two groups: one group (7 subjects) consumed perilla seed oil-rich supplementation and the other group (7 subjects) consumed corn oil-rich supplementation for 4 weeks. Generation of LTs by leucocytes and respiratory function were compared between the two groups. RESULTS: The generation of LTB4 and LTC4 by leucocytes tended to increase in subjects (N=7) with corn oil-rich supplementation, and decrease in subjects (N=7) with perilla seed oil-rich supplementation. Significant differences between the two groups were observed in the generation of LTB4 at 2 weeks (p<0.05) and LTC4 at 2 weeks (p<0.05) after dietary supplementation. Significant increases in the value of PEF (p<0.05), FVC (p<0.01), FEV(1.0) (p<0.05) and V(25) (p<0.05) were found in subjects who received perilla seed oil supplementation for 4 weeks. And significant differences in the value of FVC (p<0.05) and FEV(1.0) (p<0.05) were observed between the two groups after 4 weeks of dietary supplementation. CONCLUSION: These results suggest that perilla seed oil-rich supplementation is useful for the treatment of asthma in terms of suppression of LTB4 and LTC4 generation by leucocytes, and improvement of pulmonary function. PMID- 10732826 TI - Hepatitis C virus infection in patients with B-cell non-Hodgkin's lymphoma. AB - OBJECTIVE: As a causative role of hepatitis C virus (HCV) in B-cell lymphoproliferative disorders (LPD) has been suggested by several reports, we investigated the prevalence of HCV infection among patients with LPD at our hospital with the aim of clarifying the clinical features and the outcome for HCV antibody-positive patients with non-Hodgkin's lymphoma (NHL). METHODS: Retrospective chart review. PATIENTS: A total of 123 patients with B-cell LPD (4 with chronic lymphocytic leukemia, 17 with multiple myeloma, and 100 with B-cell NHL), 38 patients with non-B-cell LPD (5 with adult T-cell lymphoma, 8 with Hodgkin's disease, and 25 with non-B-cell NHL) and 516 patients with miscellaneous diseases other than liver diseases or LPD (control) were studied. RESULTS: HCV infection was detected in 17 of 100 patients with B-cell NHL versus none of 25 patients with non-B-cell NHL (p=0.023) and in 34 patients (6.6%) in the control group with miscellaneous diseases (p=0.0011). In HCV-positive B-cell NHL, primary liver involvement was detected in 3 of 17 patients compared to none of 83 HCV-negative patients (p=0.0019). Intermediate-grade lymphoma (Working Formulation) was the most frequent histology. Eleven of 15 HCV-positive patients achieved complete remission after chemotherapy, and 6 of 7 deaths were caused by liver-related diseases. CONCLUSION: The prevalence of HCV infection was higher in patients with B-cell NHL than in those with non-B-cell NHL and the control group. Primary liver involvement and liver-related causes of death were frequent in HCV positive patients with B-cell NHL. PMID- 10732827 TI - Clinical and pathophysiological features of amaurosis fugax in Japanese stroke patients. AB - OBJECTIVE: It has been emphasized that amaurosis fugax (AmF) is caused by thromboembolism due to atheromatous lesions of the extracranial carotid artery (EC-CA) in Caucasian populations. However, there have been few studies of AmF in Japan. We analyzed the clinical and pathophysiologic features of AmF in 43 Japanese AmF patients. SUBJECTS AND METHODS: Forty-three patients presented with AmF from a group of 2,056 Japanese patients with acute ischemic stroke. We investigated angiographic and transcranial Doppler findings, precipitating factors, medical treatment and prognosis, to elucidate the pathogenetic mechanism of AmF. RESULTS: Angiographic findings revealed an intracranial lesion in 22 patients (51%), extracranial lesion in 16 (37%), and no abnormality in 5 (12%). Blood flow in the ophthalmic artery (OA) examined by the transcranial Doppler ultrasonography (TCD) showed normal antegrade flow in 24 patients and reversed flow in 7. Precipitating factors for AmF were seen in 7 out of 43 patients. Regarding the pathogenesis of AmF, the micro-thromboembolism originated from the internal carotid artery (ICA) in 25 patients, the thromboembolism was via the external carotid artery (ECA) in 7, the hemodynamic retinal vascular insufficiency in 6 patients showed various atheromatous changes in the intracranial carotid artery (IC-CA) or EC-CA, and the cause was unknown in 5. CONCLUSION: In this series of patients, AmF was mainly caused by thromboembolism from IC-CA atheromatous lesions. Micro-thromboemboli from the ECA or hemodynamic retinal vascular insufficiency, although less frequent, should also be considered as possible etiologies for AmF. PMID- 10732828 TI - The sera from GM1 ganglioside antibody positive patients with Guillain-Barre syndrome or chronic inflammatory demyelinating polyneuropathy blocks Na+ currents in rat single myelinated nerve fibers. AB - OBJECTIVE: To determine the possible role of anti-GM1 ganglioside antisera from patients with Gullain-Barr*e syndrome (GBS) or chronic inflammatory demyelinating polyneuropathy (CIDP) in the development of nerve dysfunction. METHODS: The effect of the anti-GM1 antibody positive antisera obtained from 4 GBS patients and 1 CIDP patient on membrane potential and ionic currents in rat single myelinated nerve fibers was investigated using the voltage clamp technique and compared with that of the anti-GM1 negative antisera obtained from 3 healthy controls and 2 GBS patients. RESULTS: In the presence of active complement, anti GM1 positive antisera from 5 patients including 4 GBS patients and 1 CIDP patient significantly suppressed Na+ current more than anti-GM1 negative antisera. CONCLUSION: This study supports the notion that anti-GM1 antibody is one of the causative factors of conduction abnormality in GBS patients. PMID- 10732829 TI - Abnormal IL-1 receptor antagonist production in patients with polymyositis and dermatomyositis. AB - OBJECTIVE: To examine the relationship between serum levels of interleukin-1 receptor antagonist (IL-1Ra) and its gene expression in peripheral blood mononuclear cells (PBMC) from patients with polymyositis and dermatomyositis (PM/DM). METHODS: IL-1Ra levels in sera from patients and supernatants of unstimulated monocyte cultures were measured by enzyme-linked immunosorbent assay. Expression of IL-1Ra mRNA was analyzed by Northern blotting, and an 86 base pair variable repeat polymorphism in intron 2 of the IL-1Ra gene was determined by polymerase chain reaction. RESULTS: Serum IL-1Ra was significantly elevated in 27 patients with active-stage PM/DM when compared with levels in 16 patients with inactive-stage PM/DM and 19 normal controls. Serum concentrations of IL-1Ra were correlated with PM/DM disease activity. IL-1Ra mRNA was detected in freshly isolated PBMC from patients with active-stage PM/DM, but not in controls. Moreover, IL-1Ra concentrations were increased significantly in unstimulated monocytes from patients with active-stage PM/DM compared with monocytes from normal controls. However, there were no significant differences in IL-1Ra allele frequencies between patients and normal controls. CONCLUSION: Elevation of both IL-1Ra mRNA and protein in sera of patients with active-stage PM/DM suggest that higher levels of serum IL-1Ra may reflect increased IL-1Ra production in myositis, and that IL-1Ra may regulate IL-1-mediated muscle fiber damage in PM/DM. PMID- 10732830 TI - Riedel's lobe of the liver evaluated by multiple imaging modalities. AB - An 81-year-old Japanese woman visited our hospital because of abdominal discomfort. Physical examination revealed that she had an abdominal mass. A combination of ultrasonography, computed tomography, magnetic resonance imaging (MRI), and hepatic asialoglycoprotein scintigraphy was utilized to make a diagnosis. We found that she had a downward elongated hepatic lobe or Riedel's lobe which did not appear to be common in our district. The prevalence of Riedel's lobe in the Asian population has not been studied. Furthermore, this is the first report that describes the MRI and hepatic asialoglycoprotein scintigraphy features of Riedel's lobe of the liver. PMID- 10732831 TI - Successful resection of intracardiac invasive thymoma with right ventricular inflow tract occlusion. AB - A 72-year-old man presenting with the superior vena cava syndrome and intracardiac mass was admitted to our hospital. The mass was resected and confirmed to be invasive thymoma. Three years later, he was re-admitted with recurrence into the intracardiac space without any changes in the anterior mediastinum mass. The mass occupied the right atrial cavity and protruded into the right ventricle, causing right ventricular inflow tract obstruction. He underwent re-operation and irradiation. His postoperative course was uneventful, and he has remained alive. Invasive thymoma with intracardiac extension is extremely rare. PMID- 10732832 TI - Antibody-mediated insulin resistance treated by cessation of insulin administration. AB - A 45-year-old Japanese man was referred to our hospital because of hyperglycemia despite the administration of as much as 120 U/day of human insulin. He had no history of injecting animal insulin. Free insulin was below 5 microU/ml, but a high titer of total insulin (about 3,000 microU/ml) was observed, suggesting the presence of antibodies against human insulin. Scatchard analysis showed an increased insulin binding capacity in the plasma characterized by a higher affinity for insulin. He was successfully treated by cessation of insulin administration. A Scatchard analysis series showed that a reduction in the insulin binding capacity of antibodies paralleled the improvement in glycemic control. PMID- 10732833 TI - Type 1 diabetes mellitus caused by treatment with interferon-beta. AB - A 57-year-old man was referred to our outpatient clinic after interferon-beta (IFN-beta) treatment for 7 weeks. While IFN-beta therapy was continued in our outpatient clinic, his blood glucose level increased gradually, and he was admitted to our hospital for hyperglycemia. The patient was prescribed a 1,600 kcal diet and intensive insulin therapy was performed. GAD antibody became positive 15 months after the start of IFN therapy, and disappeared 27 months after the start of IFN therapy. Insulin secretion was depleted and the patient had HLA-DR4, B54, and DRB1*0405. This appears to be a case of type 1 diabetes mellitus induced by administration of IFN-beta alone. PMID- 10732834 TI - Tubulointerstitial nephritis associated with Legionnaires' disease. AB - A 47-year-old man was admitted to our hospital for community-acquired pneumonia complicated with acute renal failure. Legionella pneumophila serogroup type 1 was grown in BCYE (buffered charcoal yeast extract) agar for sputum culture. Although his respiratory illness responded to intravenous erythromycin therapy, renal failure worsened and necessitated hemodialysis. Renal biopsy showed profound tubulointerstitial nephritis. After initiation of steroid therapy his renal function improved and he was discharged thereafter. These findings suggest that in Legionnaires' disease with acute renal failure, tubulointerstitial nephritis should also be considered and steroid therapy may be an effective modality for the renal complication. PMID- 10732835 TI - Diabetes mellitus associated with rapidly progressive glomerulonephritis with perinuclear antineutrophil cytoplasm antibodies. AB - A 55-year-old woman who had been treated for diabetes mellitus for twenty-five years developed interstitial pneumonia and rapidly progressive glomerulonephritis (RPGN). The findings of light microscopy revealed fibrocellular crescent formation in all glomeruli and infiltration of lymphoid cells in interstitium. There were no deposits in the intracapillary area and mesangial area on both immunofluorescence and electron microscopy. Her interstitial pneumonia improved with pulse therapy of methylprednisolone and her hematuria disappeared with mix treatment of cyclophosphamide and double filtration plasmapheresis (DFPP). Her serum creatinine level improved from 2.2 mg/dl to 1.5 mg/dl. Interstitial pneumonia and hematuria did not recur at twelve months after the first hospitalization. This report presents a rare case with RPGN associated with diabetes mellitus who recovered with combination therapy of cyclophosphamide, steroid and DFPP. PMID- 10732836 TI - Nuclear hypersegmentation precedes the increase in blood eosinophils in acute eosinophilic pneumonia. AB - In this case of acute eosinophilic pneumonia (AEP), eosinophils with hypersegmented nuclei emerged in the blood before the increase of eosinophil count. An 18-year-old woman complaining of fever, cough and dyspnea was admitted because of diffuse ground-glass opacities in her chest roentgenogram. On admission, her blood cell count revealed a marked increase of neutrophils. Although the number of eosinophils was normal, some of them contained three- or four-lobed nuclei. She was diagnosed to have AEP through bronchoalveolar lavage and transbronchial lung biopsy. The combination with acute clinical course, pulmonary infiltration and the presence of hypersegmented eosinophils in blood may imply the diagnosis of AEP. PMID- 10732837 TI - Mucoid impaction caused by monokaryotic mycelium of Schizophyllum commune in association with bronchiectasis. AB - A 51-year-old female was admitted to our hospital because of fever, cough, and hemoptysis. A chest radiograph showed a partial collapse of the left upper division and infected bullae in the left upper lobe. Bronchoscopic examination showed thick mucous plugs in the left upper bronchus. The isolates of the plugs proved to be Schizophyllum commune. Neither accumulation of eosinophils nor Charcot-Leyden crystals were present in the plugs. Mild ectatic changes of the left upper bronchus had been observed 17 years previously. We describe the first case of mucoid impaction, which was independent of the immunological reactions, caused by S. commune in association with bronchiectasis. PMID- 10732838 TI - Air leak syndrome as one of the manifestations of bronchiolitis obliterans organizing pneumonia. AB - A 46-year-old man developed respiratory distress with air leak syndrome (ALS), including pneumothorax, pneumomediastinum, and subcutaneous emphysema. Open lung biopsy was performed and revealed the histopathologic evidence of bronchiolitis obliterans organizing pneumonia (BOOP), which responded well to steroid treatment. As far as we know, this appears to be the first case of BOOP presenting with ALS as one of its major complications. PMID- 10732839 TI - A diffuse alveolar hemorrhage in a human T-lymphotropic virus type I carrier with acute cerebellar ataxia and interstitial pneumonitis: an autopsy case report. AB - A 76-year-old HTLV-I-positive male with acute cerebellar ataxia was suffering from dyspnea on exertion. Chest CT suggested interstitial pneumonitis. Methylprednisolone pulse therapy improved his symptoms and chest CT findings. Twelve months after discharge, when the prednisolone dose was tapered to 5 mg every other day, his lung lesion recurred. The lesion responded initially to steroid therapy. However, hypoxemia intractable to steroid pulse therapy developed and the patient died of respiratory failure. The autopsy revealed diffuse alveolar hemorrhage with no finding of vasculitis. This is the first case report of diffuse alveolar hemorrhage in an HTLV-I carrier. PMID- 10732840 TI - IgA-lambda/IgG-kappa biclonal myeloma in which two clones proliferated in individual sites. AB - A 72-year-old man was admitted to our hospital because of lumbago and numbness of legs. Tumor invasion at the fourth lumbar vertebra was revealed. Immunohistochemistry using specific antibodies against each heavy and light chain of immunoglobulin revealed that the myeloma cells in bone marrow were all IgA lambda type whereas they were all positive for IgG-kappa type in a tumor of the fourth lumbar vertebra. These data indicate that the patient had IgG-kappa/IgA lambda biclonal myeloma. Different phenotypes of M-proteins and distinct proliferating sites for two clones suggest that they may have resulted from two independent transforming events. PMID- 10732841 TI - Necrotizing myopathy in a patient with chronic hepatitis C virus infection: a case report and a review of the literature. AB - We describe a 61-year-old man presenting with necrotizing myopathy associated with chronic active hepatitis due to hepatitis C virus (HCV) infection. Thirteen patients with HCV-associated myopathy have been reported previously. In most of these cases, varying degrees of inflammatory changes were observed in the muscle tissue. In 2 patients, myopathy developed after initiation of interferon therapy for chronic HCV hepatitis. Our case was unusual due to long-standing elevation of creatine kinase values which improved following interferon therapy and the non inflammatory features of the muscle tissue where the HCV RNA minus strand, a marker for replicative intermediates of the virus, was undetectable. The association of myopathy with HCV infection might represent a unique clinical entity, although the underlying pathological mechanisms remain unknown. PMID- 10732842 TI - Reiter's syndrome associated with HLA-B51. AB - A 22-year-old Japanese man developed polyarthritis with fever and urethritis. He was diagnosed as Reiter's syndrome since he was found to have uveitis and persistent aseptic pyuria. Although, he was negative for HLA-B27 or any other HLA B27 cross-reactive MHC class I antigens, he was positive for HLA-B51. The laboratory examination showed significant elevation of serum IgG and IgA anti Chlamydia antibodies. He was successfully treated with a combination of doxycycline, naproxen, salazosulfapyridine and methotrexate with a decrease in IgG and IgA anti-Chlamydia antibodies. Previous studies provided evidence that HLA-B51 itself might be involved in the development of Behcet's disease, which shares common features with Reiter's syndrome, such as uveitis, skin lesions, and polyarthritis. It is therefore suggested that combination of Chlamydia infection and HLA-B51 might play a role in the pathogenesis of Reiter's syndrome in our patient. PMID- 10732843 TI - Psoriatic arthritis complicating lung cancer. AB - Psoriatic arthritis is an inflammatory arthritis associated with psoriasis. While an elevated incidence of lung cancer has been observed in patients with RA or psoriasis, there has been no report of psoriatic arthritis associated with lung cancer. We here report the first case of psoriatic arthritis which developed lung cancer. In this case, it was suspected that a combination of cigarette smoking, pulmonary fibrosis, and low-dose methotrexate therapy might have promoted the development of lung cancer. PMID- 10732844 TI - Aspergillus fumigatus isolated from blood samples of a patient with pulmonary aspergilloma after embolization. AB - Aspergillus DNA was detected by PCR in the serum sample of a 78-year-old man and galactomannan antigen of Aspergillus by sandwich ELISA was found. However, the infiltrative hyphae were not detected by the histopathologic examination of the lung. He developed hemoptysis, which required embolization of bronchial arteries. Aspergillus fumigatus was isolated from blood samples after embolization by the lysis centrifugation method. To our knowledge, this is probably the first case in which Aspergillus spp. has been isolated from the systemic circulatory blood in a patient with pulmonary aspergilloma after embolization. PMID- 10732845 TI - Cost analysis between stent and conventional balloon angioplasty. AB - The present study evaluated the cost of coronary stenting compared with conventional balloon angioplasty in Japan. Procedural cost was estimated as the sum of the procedural fee and the cost of devices such as angioplasty balloon and stent. The data such as the number of balloon catheters and stents used, and the rate of crossovers that was shown by the Stent Restenosis Study (STRESS) were applied to calculate the costs of stenting and conventional balloon angioplasty. For the estimation of hospital room and nursing costs, the length of the in hospital stay was estimated at 7 days. The costs of procedures such as laboratory and radiological tests were determined based on routine coronary intervention at Chiba University Hospital. The rates of target lesion revascularization in the STRESS trial (conventional balloon angioplasty: 21%, stenting: 15%) were used to calculate the cost during follow up. The in-hospital costs of conventional balloon angioplasty and stenting were estimated to be Yens 982,300 and Yens 1,416,893, respectively. The overall costs, including follow-up cost, of conventional balloon angioplasty and stenting were estimated to be Yens 1,188,583 and Yens 1,564,238, respectively. The in-hospital cost of stenting is higher compared with conventional balloon angioplasty because of greater balloon use and direct stent cost. Lower target lesion revascularization reduces the cost difference between conventional balloon angioplasty and stenting, but the higher initial cost of stenting is not fully offset. PMID- 10732846 TI - Improvement in corrected QT dispersion by physical training and percutaneous transluminal coronary angioplasty in patients with recent myocardial infarction. AB - The aim of the present study was to assess whether physical training and percutaneous transluminal coronary angioplasty (PTCA) improve the corrected QT (QTc) dispersion in patients with recent myocardial infarction (MI). Twenty-four patients with recent MI were allocated to one of 3 groups: training (n = 8), PTCA (n = 7) or controls (n = 9). Physical training as well as PTCA decreased QTc dispersion, whereas QTc dispersion increased in the control group. Changes in QTc dispersion after physical training or PTCA were inversely correlated with exercise-induced ST depression at the baseline test. These observations suggest that physical training, as well as PTCA, could improve QTc dispersion and electrical instability in patients with recent MI, possibly due to improvement of myocardial ischemia. PMID- 10732847 TI - Elevated levels of soluble intercellular adhesion molecule-1 in the coronary circulation of patients with coronary organic stenosis and spasm. AB - The cell surface expression of intercellular adhesion molecule-1 (ICAM-1) is upregulated following activation during inflammatory responses, mediating both cell migration and activation. The involvement of inflammation in unstable angina is suggested by the presence of activated circulating leukocytes. To examine whether plasma soluble ICAM-1 (sICAM-1) levels increase in the coronary circulation of patients with coronary organic stenosis and coronary spasm, plasma sICAM-1 levels were measured in the coronary sinus (CS) and the aortic root (Ao) simultaneously in 10 patients with 90% or more coronary narrowing and coronary spasm (coronary spastic angina (CSA) with organic stenosis), in 11 patients with coronary spasm and no significant coronary narrowing (CSA without organic stenosis), in 16 patients with stable exertional angina, and in 13 control subjects. The plasma sICAM-1 levels (ng/ml) in the CS increased in CSA with organic stenosis (230+/-26) as compared with CSA without organic stenosis (158+/ 14), stable exertional angina (130+/-9) and control subjects (121+/-10) (p<0.01). The levels in the Ao also increased in CSA with organic stenosis (208+/-24) as compared with CSA without organic stenosis (149+/-13), stable exertional angina (130+/-11) and control subjects (121+/-10) (p<0.01). Furthermore, the plasma sICAM-1 levels were higher in the CS than in the Ao only in CSA with organic stenosis. These results suggest that activation of leukocytes occurs through the induction of ICAM-1 in the coronary circulation in the patients with CSA with organic stenosis. PMID- 10732848 TI - Effect of long-term cholesterol-lowering treatment with HMG-CoA reductase inhibitor (simvastatin) on myocardial perfusion evaluated by thallium-201 single photon emission computed tomography. AB - Fifteen patients with either angina pectoris or old myocardial infarction, who had positive 201Tl single photon emission computed tomography (SPECT) imaging and coronary sclerosis of more than 50%, were treated with an HMG-CoA reductase inhibitor (simvastatin) for more than 1 year. They were compared with an untreated control group (n = 25). Total cholesterol decreased 22% and high density lipoprotein (HDL) increased 9% with simvastatin; both changes were significantly different from those in controls. Long-term simvastatin induced improvement of myocardial perfusion on 201Tl SPECT images both during exercise and at rest, which was also significantly different from controls. In addition, the improvement of myocardial perfusion on 201Tl SPECT images was clearly related to the improvements in cholesterol values, especially nonHDL cholesterol. Thus, the greater the decrease in nonHDL cholesterol, the greater the improvement in myocardial perfusion at rest or during exercise with long-term treatment using an HMG-CoA reductase inhibitor. These findings indicate that the improvements in cholesterol values caused by HMG-CoA reductase inhibitor therapy are related to improvements of myocardial perfusion seen on 201Tl SPECT images. PMID- 10732849 TI - Myocardial viability detected by dobutamine echocardiography in patients with chronic coronary artery disease, and long-term outcome after coronary angioplasty. AB - Viable but dysfunctional myocardium detected by dobutamine echocardiography (DE) predicts early improvement in regional left ventricular (LV) function after percutaneous transluminal coronary angioplasty (PTCA). Whether DE can predict the long-term (>2 years) outcome after PTCA is still unclear. Thus, 50 patients (age 60.4+/-9.5 years) with chronic coronary artery disease and regional LV dysfunction who underwent DE 1 week before PTCA to assess myocardial viability were followed for 4.0+/-0.8 years. Regional LV function and LV ejection fraction (LVEF) were evaluated by 2-dimensional echocardiography in patients who remained event-free (cardiac death or myocardial infarction or unstable angina pectoris) after PTCA. At late follow-up (>2 years after PTCA), 29 patients showed regional LV function improvement, 15 showed no improvement, 3 showed worsening and 3 patients had cardiac events (1 nonfatal myocardial infarction and 2 unstable angina pectoris). LVEF improved (0.53+/-0.09 to 0.60+/-0.09, p<0.001) in patients with improved regional LV function, but deteriorated (0.38+/-0.03 to 0.30+/-0.03) in the 3 patients with worsened regional LV function. Of the 29 patients with improvement, 27 (93%) had viable myocardium, whereas only 3 (20%) of the 15 with no improvement had viable myocardium and all 6 of those with poor outcomes (3 with cardiac events and 3 with worsening) had viable myocardium (chi2 = 28.9, p<0.001). Patients with viable myocardium and a poor outcome had a lower mean LVEF before PTCA, and at 1 week and 3 months after PTCA (p = 0.004, <0.001, and =0.001, respectively), and a higher restenosis rate (p = 0.007) than patients with viable myocardium and without a poor outcome. It is concluded that viable myocardium detected by DE may predict long-term improvement in regional and global LV function after PTCA. However, patients with viable myocardium and persistent low LVEF are at risk for cardiac events or worsening of LV function. PMID- 10732850 TI - Effects of 'cool-down' during exercise recovery on cardiopulmonary systems in patients with coronary artery disease. AB - The effects of 'cool-down' during exercise recovery on cardiovascular and respiratory systems have not been fully clarified. The recovery of respiratory gasses was compared in cardiac patients after maximal exercise during which subjects either performed a cool-down or rested. Twenty-one patients (61+/-10 years) with coronary artery disease performed 2 symptom-limited incremental exercise tests on a cycle ergometer: one with a cool-down and the other without during recovery from the maximal exercise test. Expired gasses were analyzed on a breath-by-breath basis throughout the test and for 6min of recovery. Without a cool-down, the ventilatory equivalent for O2 (VE/O2) increased dramatically during recovery compared with the resting values or those of peak exercise: 44.5+/-7.7 at rest, 44.0+/-10.6 at peak exercise and 63.3+/-14.5 after 2min of recovery. End-tidal PO2 (P(ET)O2) also increased significantly during recovery. However, the overshoot phenomenon of these variables was attenuated when cool down exercise was performed during recovery. The high ratio of VE/VO2 reflects ventilation perfusion (VA/Q) unevenness and P(ET)O2 is an index of arterial PO2. Thus, it is suggested that cool-down exercise during recovery after maximal exercise testing provides beneficial effects on the respiratory system by decreasing the VA/Q unevenness and relative hyperventilation that are observed when cool-down exercise is not performed. PMID- 10732851 TI - Changes in autonomic nervous activity prior to spontaneous coronary spasm in patients with variant angina. AB - Although the autonomic nervous system has been implicated in the genesis of coronary spasm, the precise mechanism by which it serves as the trigger of coronary spasm remains unclear. The aim of this study was to investigate changes in autonomic nervous activity associated with ischemic episodes in patients with variant angina (VA). Heart rate variability (HRV) on Holter monitoring was analyzed during 17 ischemic episodes in 11 patients with VA. The parameters of HRV were measured during a 2-min period at various time intervals prior to the onset of ST-segment elevation. The low frequency (LF) and high frequency (HF) components of the HRV, LF/HF ratio, mean RR interval, and the coefficient of the RR interval variation (CV) were calculated for each time interval. Both the HF and the CV increased significantly in the 2 min prior to the onset of ST-segment elevation, suggesting heightened vagal activity. The LF/HF ratio, a measure of cardiac sympatho-vagal balance, did not change. The LF, a measure of sympathetic activity with vagal modulation, also did not change. The RR interval decreased significantly in the 2 min prior to the onset of ST-segment elevation. These results suggest that enhancement of both the vagal and the sympathetic nervous activity plays an important role in the initiation of coronary spasm. PMID- 10732852 TI - Tissue harmonic imaging: experimental analysis of the mechanism of image improvement. AB - Tissue harmonic scanning visually improves echocardiographic image quality. The aim of the present study was to objectively assess the improvement in harmonic image quality under controlled laboratory conditions. A tissue-mimicking phantom that contained 8-mm-diameter cystic lesions at depths ranging from 2 to 12 cm was used. Harmonic scans (1.7 MHz transmit, 3.4 MHz receive) of the phantom were obtained and lesion detectability was compared to that in scans acquired with 2 fundamental frequencies (2.0 and 3.3 MHz). A 2 cm-thick ethanol layer was also used to simulate the nonlinear effect of human fat. Cyst detectability was quantified by measurement of the contrast-to-speckle ratio (CSR). The results indicated no significant difference in the CSR between harmonic and fundamental images obtained without the ethanol layer. With images obtained with the ethanol layer, a relative increase of the CSR during harmonic imaging was observed with respect to fundamental imaging (p<0.05). In conclusion, a fat layer, here simulated by ethanol, plays a significant role in determining the resulting image quality. Without this layer, the contribution of the second harmonic mode was not significant. Thus, in a slim patient, the harmonic mode may not be as beneficial to image improvement as in an obese patient. PMID- 10732853 TI - Coronary artery bypass grafting in patients with hypopituitarism. AB - Three patients with angina pectoris and hypopituitarism underwent coronary artery bypass grafting. The patients received perioperative replacement steroid and thyroid hormone therapy, and there were no complications. Careful perioperative hormonal management is necessary for patients with hypopituitarism. PMID- 10732854 TI - Arrhythmogenic right ventricular cardiomyopathy with an initial manifestation of severe left ventricular impairment and normal contraction of the right ventricle. AB - A case of arrhythmogenic right ventricular cardiomyopathy (ARVC) with an initial manifestation of severe impairment of the left ventricle (LV) and normal contraction of the right ventricle (RV) is presented. A 43-year-old man was admitted to hospital because of congestive heart failure following a common cold. The LV function was diffusely and severely hypokinetic. Coronary arteriogram revealed normal vessels. An endomyocardial biopsy specimen obtained from the RV septum revealed mild infiltration of lymphocytes with focal myocytes necrosis and so healing myocarditis was suspected. The specimen did not include any fatty replacement of myocytes. Since then, the patient suffered from recurrent congestive heart failure as well as nonsustained ventricular tachycardia and required frequent hospitalization. Progressive impairment, dilation, and thinning of both ventricles were observed on serial echocardiographic examinations. Although the RV gradually enlarged and became impaired, severe dilatation and impairment of the LV has always been predominant in the patient's clinical course. After medical follow-up for 10 years, he died suddenly of ventricular fibrillation and pump failure. The autopsy revealed extensive fibrofatty replacement of myocytes in both the ventricles, extending from the outer layer to the inner layer of myocardium in the RV and to the middle layer in the LV. These features were compatible with arrhythmogenic right ventricular cardiomyopathy or perimyocarditis, although only the rightsided bundle of the interventricular septum was completely replaced by fatty tissue, which can not be explained as a sequel of perimyocarditis. Moreover, apoptosis was present in the myocyte nuclei of the myocardial layers bordering the area of fatty replacement. Therefore, myocarditis may have triggered or accelerated the process of apoptosis leading to ARVC. PMID- 10732855 TI - Acute myocardial infarction in a patient with anomalous left coronary artery origin and primary antiphospholipid syndrome. AB - Anomalous left main coronary artery (LMCA) originating from the right coronary sinus and running between the aorta and pulmonary trunk is a rare congenital condition. Although this disease is known to be associated with myocardial infarction and sudden death, the precise mechanism is uncertain. A 14-year-old male with this anomaly developed myocardial infarction during exercise complicated by primary antiphospholipid syndrome. He was admitted to hospital with persistent chest pain and sudden cardiac collapse that occurred while he was running. Cardiac catheterization demonstrated a narrowed segment in the LMCA and impaired blood flow, prompting a diagnosis of extensive anterior myocardial infarction. Emergency bypass surgery was performed using a single saphenous vein graft to the left anterior descending artery. Postoperative angiography showed the presence of an anomalous LMCA arising from the right sinus of Valsalva and running between the great vessels. The aortic samples were pathologically normal. He was discovered to also have primary antiphospholipid syndrome and was discharged without symptoms after warfarin therapy. Complicated primary antiphospholipid syndrome may trigger myocardial infarction in asymptomatic patients with this type of coronary anomaly. PMID- 10732856 TI - Delayed onset of pulmonary hypertension associated with an appetite suppressant, mazindol: a case report. AB - The use of the appetite suppressant agents aminorex and fenfluramine derivatives has been reported as a risk factor for the development of pulmonary hypertension. A 29-year-old female developed pulmonary hypertension suspected to be due to an amphetamine-like appetite suppressant agent, mazindol ((+/-)-5-(p-chlorophenyl) 2,5-dihydro-3H-imidazo [2,1-a] isoindol-5-ol). She was admitted to Sapporo Medical University Hospital with dyspnea due to severe pulmonary hypertension. Twelve months prior to admission, she had taken mazindol continuously for a period of 10 weeks. As yet, her pulmonary hypertension has not completely improved. This is the first reported case of mazindol-associated pulmonary hypertension, which developed after a long latent interval, and it suggests that mazindol is also a risk factor for the development of pulmonary hypertension, making long-term follow-up necessary for patients taking this anorectic agent. PMID- 10732858 TI - Apical hypertrophic cardiomyopathy associated with life-threatening paroxysmal atrial flutter with a slow ventricular response: a case report. AB - A 58-year-old male patient had apical hypertrophic cardiomyopathy (HCM) associated with a life-threatening tachycardia due to atrial flutter. Following palpitation and dyspnea for 2-3 h, he became unconscious because of circulatory catastrophe, but was fully resuscitated. An electrocardiogram recorded just before the loss of consciousness revealed atrial flutter at a rate of 260 beats/min with a 2:1 ventricular response. He was diagnosed as having apical HCM based on the echocardiographic and left ventriculographic findings. Atrial stimulation at a rate of 150 pacings/min for 1 min caused a marked drop in systemic systolic blood pressure from 170 to 120 mmHg. The patient was treated with 150 mg of cibenzoline per day to prevent supraventricular tachyarrhythmias and to improve left ventricular diastolic function. At the time of the recent follow-up at 2 and a half years, he felt quite well. PMID- 10732857 TI - Primary cardiac sarcoma: two case reports. AB - Two case reports of primary cardiac sarcoma, which is uncommon, are presented. The first case, a 38-year-old male, complained of chest tightness. Chest roentgenograms showed enlargement of the cardiac shadow and left pleural effusion. Transthoracic echocardiography and chest magnetic resonance imaging showed a tumor in the right atrium, and pericardial effusion. The tumor involved the right atrial wall and interatrial septum, and was partially resected. Pathohistological examination revealed angiosarcoma. He died 1 month later. The second case, a 19-year-old male complained of dyspnea and orthopnea. Chest roentgenograms showed pulmonary congestion. Transthoracic echocardiography showed a large mobile mass in the left atrium. An emergency operation was performed and the tumor was totally resected. Pathohistological examination demonstrated leiomyosarcoma. The postoperative course was uneventful, but the tumor rapidly recurred. Second and third operations were performed at intervals of 2 months. After the third operation, he was treated with radiotherapy. Local recurrence was not found but multiple distant metastases were found 2 months after completion of radiation therapy. PMID- 10732859 TI - Heart disease in Friedreich's ataxia: observation of a case for half a century. AB - A case of Friedreich's ataxia was followed for 47 years, beginning in 1930; this patient had an abnormal electrocardiogram (flat or inverted T waves in leads II and III with prolonged QT interval) from the very beginning of the onset of neurological symptoms. Cardiac and neurological disturbances progressed slowly but steadily, and the patient died suddenly at the age of 67. The autopsy revealed typical findings of Friedreich's ataxia and hypertrophic cardiomyopathy with thickened left ventricular wall and myocardial fiber disarray. To the authors' knowledge, this is the longest continuous follow-up study of Friedreich's ataxia, and it will provide invaluable information on the natural history and development of the cardiac and neurological disorders in this condition. PMID- 10732860 TI - Cells producing recombinant retrovirus with thymidine kinase gene from Herpes simplex virus suitable for human cancer gene therapy. AB - Therapeutic cells producing amphotropic retrovirus, which are able to transduce in vivo thymidine kinase gene of Herpes simplex virus were prepared. Single-cell clone cells with high virus productivity (PA-3 17JH5c113) were obtained by cell cloning. The cells were found free of replication competent retrovirus, they were non-tumorigenic in xenogeneic host and highly sensitive to ganciclovir treatment in vitro and in vivo. The therapeutic efficacy of PA-317JH5c113 cells was tested in rat brain tumor model. Increase in survival in the group of treated versus untreated rats was observed. Therefore, these cells are suitable for application in human clinical trial. PMID- 10732861 TI - Reversal of carboplatin resistance in human laryngeal carcinoma cells. AB - The effectiveness of carboplatin in the treatment of patients with tumors is limited by drug resistance. Because of that, there is a great interest to find a way to revert the resistance and improve the success of cancer treatment. The aim of the present study was to examine five potential modulators of carboplatin resistance with different mode of action: buthionine sulfoximine, ethacrinic acid, amphotericine B, cyclosporine A and aphidicoline. The effect of these compounds on the sensitivity of human laryngeal parental (HEp2) and carboplatin resistant (HEp7T) cells to carboplatin was examined by MTT spectrophotometric assay. The results have shown that buthionin sulfoximine and, to a lesser extent, ethacrinic acid reduced the resistance of HEp7T cells to carboplatin. Aphidicolin increased the sensitivity of both HEp2 and HEp7T cells to carboplatin, but this effect was more expressed in parental HEp2 cells. Our data suggest that human laryngeal carcinoma cells treated with clinically relevant doses of carboplatin became resistant to this drug due to multifactorial molecular mechanisms. Accordingly, the resistance to carboplatin could be reduced by different modulators. PMID- 10732862 TI - Lack of correlation between repair of DNA interstrand cross-links and differential sensitivity of G0 and proliferating CD4+ lymphocytes towards cisplatin. AB - G0 cells in a tumor are insensitive to the chemotherapeutical agents. The nature of this resistance is not completely understood. One of the factors modulating sensitivity of cells may be DNA repair of drug induced DNA damage. In this study we have compared gene-specific formation and repair of cisplatin-induced interstrand cross-links (ICL) in human G0 and proliferating CD4+ lymphocytes. Cisplatin killing of G0 CD4+ lymphocytes is inefficient, and these cells resemble those in a tumor. After exposure to cisplatin under similar conditions, the frequency of ICL introduced is twice as high in the proliferating compared to the resting lymphocytes. Repair of ICL was measured in the housekeeping gene, dihydrofolate reductase (DHFR), in the proliferation inducible c-myc gene, and in the inactive delta-globin gene. We observed similar relative rates and extent of ICL repair in all three genes studied, in G0 or proliferating CD4+ lymphocytes. The mechanisms responsible for the resistance of G0 CD4+ lymphocytes towards cisplatin are discussed. PMID- 10732863 TI - Flow cytometric analysis of some activation/proliferation markers on human thymocytes and their correlation with cell proliferation. AB - We immunophenotyped cells from ten human thymuses with emphasis on expression of the CD38 and CD71 antigens. These antigens play role in activation cells and increased expression of them was observed in some leukemia. Simultaneously, certain attention has also been devoted to some further activation markers, e.g. CD25, CD26 and HLA-DR. The classification of leukemia is based on comparison of normal and pathological cells. The study of expression of CD38, CD71 and other markers on thymocytes simultaneously with DNA analysis can be useful for answer if expression of CD38 and CD71 on pathologic cells is a sign of their proliferative ability, a part of immature phenotype in some leukemia, or it is a case of aberrant immunophenotype. In our study, 94% thymocytes were CD38+ and only 16% were CD71+. From our immunophenotypic results including MESF (molecules of equivalent soluble fluorochrome) values and analysis of the cell cycle, the conclusion could be drawn that antigen CD71 can participate in regulation of thymocyte development and presence of both -CD38 and CD71 on pathologic cells will be in all probability the case of aberrant phenotype. We observed a clear correlation of the percentage and MESF values of CD71-positive cells with the cell proliferation only after in vitro thymocytes stimulation with PHA and IL-2. In summary, a strong parallelism was observed regarding the positive relationship between the proliferative rate (assessed by the number of S-phase cells) of stimulated thymocytes and the quantitative (% and MESF) values of some markers - CD71, CD25, CD26 and HLA-DR and negative one with CD38 marker values. PMID- 10732864 TI - Reduction of genotoxic effects of MNNG by butylated hydroxyanisole. AB - Butylated hydroxyanisole (BHA) is a food preservative with markedly contradictory effects. On one side many studies showed its antimutagenic and anticarcinogenic effects but on the other side dietary levels of BHA were reported to cause gastrointestinal hyperplasia in rodents. We studied the influence of BHA on cytotoxicity, mutagenicity, and DNA-damaging activity of N-methyl-N'-nitro-N nitrosoguanidine (MNNG) in Chinese hamster V79 cells cultured in vitro. Our results showed that BHA significantly reduced the frequency of 6-thioguanine resistant (6-TGr) mutations and micronuclei induced in V79 cells by MNNG. These antimutagenic effects of BHA were, however, accompanied by a very marked increase of MNNG toxicity and also slightly increased level of MNNG-induced DNA damage. For evaluation of toxicity we used three methods: (i) trypane blue exclusion; (ii) plating efficiency; and (iii) intensity of cellular macromolecule synthesis. The level of DNA damage was measured by the comet assay. On the basis of obtained results we suggest that BHA, which induces phase II detoxifying enzymes, probably doesn't reduce the level of DNA damage induced in time of MNNG-treatment but it reduces the level of DNA damage created during a long-term period needed for expression of 6-TGr mutations and micronuclei. PMID- 10732865 TI - 7-ethoxyresorufin O-deethylase (EROD) activity is not capable of reflecting the overall malignant potential of breast cancer tissue. AB - 7-Ethoxylesorufin O-deethylase (EROD) (mainly catalyzed by cytochrome P450 (CYP) 1A1 and used as a marker for CYP 1A1) activity was measured in the breast tumor and surrounding tumor free (normal) tissues of 37 female breast cancer patients with infiltrating ductal carcinoma. About 11% of the tumor and normal breast tissue samples lacked the enzyme activity. Large interindividual variations in the activities of EROD were found in both tumor and normal tissues ranging from 0 to 283 and 0 to 801 fmol/mg/min, respectively. However, no significant difference was noted between the mean EROD activities of tumor and normal breast tissues. This tendency did not change with the stage and grade of the malignancy and menopausal status. No significant correlation was observed between the EROD activity and stage or grade of malignancy (p > 0.05). Thus, it appears that EROD activity is not capable of reflecting the overall malignant potential of breast cancer tissue. PMID- 10732866 TI - Flow cytometry of p53 protein expression in some hematological malignancies. AB - p53 is a tumor suppressor gene encoding a nuclear phosphoprotein that plays an important role in the control of normal cell proliferation. We have tried to establish the value of the p53 protein expression in peripheral blood (PB) and/or bone marrow (BM) cells of patients with some hematological malignancies. A recently developed fixation/permeabilization method was modified for flow cytometric assessment of p53 protein expression using two anti-p53 monoclonal antibodies. p53 quantitation expressed as molecules of equivalent soluble fluorochrome per cell (MESF) providing valuable data contributing to a more precise definition of leukemic cells, was also applied. Our findings showed higher percentage of p53 expression in cells of AML patients at the time of diagnosis opposite to the controls. These data, in association with immunophenotype of cells, accompanied diagnosis of relapse or definition of remission after allogeneic BM transplantation. We observed also elevated levels of p53 protein at initial diagnosis of early B-ALL. According to our results quantitation of p53 protein allows better characterization of selected population of BM cells and should be used for the monitoring of blast persistence during and after therapy and might also be one of the methods to indicate early relapse. Percentage of p53 protein positivity varied in our group of B-CLL patients tested in connection with progression of disease. We documented also one case of Burkitt's lymphoma with high percentage of p53 positivity. Measurement of p53 protein expression by flow cytometry may be of clinical importance by indicating levels of positivity. Our results suggest, that p53 alteration is frequently involved at initial diagnosis of AML, in some T-cell disorders and on the contrary more frequently during early B-ALL relapse, in advanced stages of B-CLL and in Burkitt's lymphoma. p53 protein quantitation is of value to ascertain malignancy and provides additional parameter suitable for the evaluation of residual disease and for the monitoring of therapy. PMID- 10732867 TI - Incidence of micronuclei in cytokinesis-blocked lymphocytes of medical personnel occupationally exposed to ultrasound. AB - In order to investigate possible DNA damaging effects of ultrasound, the micronucleus assay on cytokinesis blocked human lymphocytes was performed. Preparations were stained by conventional Giemsa staining technique combined with additional staining techniques using fluorescent dye DAPI and silver nitrate. Blood samples were taken from medical personnel employed on ultrasonic scanning in medical diagnosis and unexposed control subjects from general population. Lymphocytes were cultivated in vitro at 37 degrees C. Cytochalasin-B in final concentration of 6 microg/ml was added 44 h after mitogen stimulation and cultures were harvested 28 h thereafter. Staining with both additional techniques can be used to distinguish micronuclei originating from breakage or mitotic loss of certain human chromosomes bearing DAPI-positively stained or silver-positively stained regions. The results obtained indicate statistically significant increases in total number of micronuclei and changes in their distribution in exposed subjects compared to control. Based on different intensity of DAPI staining signal-positive and signal-negative "type" of micronuclei are distinguished, while silver staining has revealed Ag-NOR+ and Ag-NOR- micronuclei. In exposed subjects a prevalence in number of Ag-NOR+ micronuclei over Ag-NOR-micronuclei compared to control was observed, indicating greater susceptibility of chromosomes from D and G groups to damage caused by continuous occupationally exposure to ultrasound. In spite of their limitations, our results indicate that combination of conventional Giemsa staining of micronuclei with fluorescent dye DAPI and silver nitrate staining techniques can be valuable complement to the standard micronucleus assay. PMID- 10732868 TI - p53 status in breast carcinomas revealed by FASAY correlates well with p53 protein accumulation determined by immunohistochemistry. AB - The prognostic and predictive value of p53 mutation in breast cancer is still conflicting. The choice of the p53 status detection method may account for some discrepancies. In this pilot study we compared two differently-based methods for detection of p53 alteration in 32 breast carcinoma samples: the immunohistochemical method using Bp53, DO1 and DO11 monoclonal antibodies for analysis of the p53 protein accumulation in cell nuclei and the functional method FASAY. FASAY - functional analysis of the separated alleles in yeast - tests the capability of the human p53 to transactivate a reporter with a p53 binding site RGC driving the ADE2 gene in yeast. In our group the percentage of breast cancers with accumulated p53 protein was 50%, as well as percentage of mutant p53 scored by FASAY was 50%. Although the agreement of both methods, when comparing the results of individual patients was high (94%), our results show that immunohistochemistry does not reflect the p53 status quite exactly. PMID- 10732869 TI - Heterogeneity of keratin intermediate filaments expression in human glioma cell lines. AB - Keratin intermediate filaments (Ifs) are specific for epithelial cell differentiation. This study demonstrates the presence of keratin in two recently established human glioblastoma cell lines 8-MG-BA and 42-MG-BA. Immunofluorescence staining was performed on cells within passage 230 to 235 using monoclonal pan-cytokeratin antibodies. The cells were analyzed during several DIV at different cell density. Keratin-positive stained cells reached 5 to 7% in 8-MG-BA and less than 0.1% in 42-MG-BA cell line. The presence of keratin-positive cells was independent on cell density and days in vitro. Keratin positive cells appeared unevenly distributed in both cell lines. They were observed as single or areas of keratin-positive cells. The morphological features of keratin-positive and keratin-negative cells were similar. The results are discussed with respect to previous studies on glial fibrillary acidic protein (GFAP) and vimentin to show the heterogeneity of IFs expression in glioma cell lines. PMID- 10732870 TI - Comparison of two non-anthracycline-containing regimens for elderly patients with diffuse large-cell non Hodgkin's lymphoma--possible pitfalls in results reporting and interpretation. AB - Age over 65 years is a risk factor per se for doxorubicin administration, and coexisting diseases pose additional problems. There is still controversy whether chemotherapy regimens for elderly patients with aggressive NHL should be full dose doxorubicin containing or whether development of non-anthracycline containing regimens is warranted. In this prospective study, 47 patients aged over 65 years with diffuse large cell NHL clinical Stage I/IE bulky-IV and no other initial exclusion criteria were randomized to receive either BCNU 120 mg/m2 d. l, VP 16 60 mg/in2 d.2-4, procarbazine 85 mg/m2 d. 2-8 (arm A, 27 patients) or mitoxantrone 6 mg/m2 d. l. with VP16 and procarbazine in the same dosage and schedule (Arm B, 20 patients). Partial responders received additional irradiation treatment if feasible. Arms were well balanced according to age, sex, clinical stage and performance status. Ten patients from arm A and 13 from arm B had PS 2 or 3; 14 patients from arm A and 8 from arm B had clinically significant antecedent and/or concomitant disease (SACD: cardiac, vascular, cerebrovascular, neurological, renal or other). On the intent-to-treat basis, the results were the following. ARM A: median number of cycles 3 (range 1--6); early death 3 patients; 16/27 responses (59%), 7 complete (30%). ARM B: median number of cycles 3 (range 1-6); early death 4 patients; 12/20 responses (60%), 3 complete (15%). There was no difference either in response rate or survival between the two arms, and pooled results from the two arms displayed a plateau on the survival curve from the 20-th month onwards on the probability level of 0.40. Clinical stage of NHL, bulky disease, age and sex did not influence survival. Initial performance status did influence survival at the significance level of p = 0.045. Although presence of SACD did not influence initial performance status, it had a strong negative impact on survival p = 0.0004). The results point to the existence of two prognostic categories of elderly patients with large cell NHL, one with a poor survival, the other achieving a significant response rate and relapse free survival. Comorbidity (SACD) apparently accounts for the poor survival in a subpopulation of elderly patients. Clinical trials with elderly patients with NHL with PS 0 or 1 and no serious coexisting disease as inclusion criteria, analyzed on an evaluable patients basis, target only to a prognostically better subpopulation among these patients. PMID- 10732871 TI - Should researchers accept funding from the tobacco industry? PMID- 10732872 TI - Holding the present and future accountable to the past: history and the maturation of clinical ethics as a field of the humanities. AB - Clinical ethics, like bioethics more generally, until recently has tended to focus on the present and future, with little attention to the history of moral thought about health care that preceded bioethics. As a consequence, clinical ethics and bioethics lack maturity as fields of the humanities. The papers in this year's clinical ethics issue of the Journal put contemporary clinical ethics in critical dialogue with the past, making the former accountable to the latter. The six papers in this issue of the Journal are briefly described, with an emphasis on how they contribute to the maturation of clinical ethics as a field of the humanities. PMID- 10732873 TI - Patient consent and negotiation in the Brooklyn gynecological practice of Alexander J.C. Skene: 1863-1900. AB - The prevailing view in bioethics is that the relationship between doctors and their patients was largely a silent one before the landmark court decisions of the twentieth century. Some have proposed that this was not always the case. This paper provides historical evidence of consent and negotiation in one nineteenth century gynecological practice. The Clinical Records and writings of Dr. Alexander J.C. Skene, who practiced in Brooklyn, New York from 1863 to 1900, have been examined for evidence of discussion, consent and even negotiation with patients. Although this evidence comes from only one practice, it is especially significant because it was largely a gynecological practice with women who were varied in socioeconomic status and ethnic origin. The importance of documenting physician-patient relationships which included patients in decision-making before Schloendorff (1914) established the legal doctrine of informed consent cannot be underestimated. PMID- 10732874 TI - Behind closed doors: accountability and responsibility in patient care. AB - In this paper, I examine the notion of accountability and its historical evolution in health care. Using medical mistakes and adverse patient outcomes as my focus, I examine the interests served by particular models of accountability and argue for a model of collective fiduciary responsibility in U.S. health care today. PMID- 10732875 TI - A strategy of clinical tolerance for the prevention of HIV and AIDS in China. AB - HIV infection and AIDS create many dilemmas in Chinese AIDS/HIV prevention policy. A strategy of clinical tolerance is proposed to address these dilemmas. The immediate purpose of the strategy of clinical tolerance is to win the cooperation of members of stigmatized groups at high risk for contracting HIV infection and AIDS, which occurs as a result of acts done in private and thus beyond the reach of regulation. The strategy of clinical tolerance differs from both tolerance as liberal tolerance and tolerance as a moral ideal of tolerance. A strategy of clinical tolerance does not ask the government, health worker, health official or the public to change either laws or the disapproval of prostitution, homosexuality and drug use. A strategy of clinical tolerance asks, instead, that we weigh what we may regard as the wrong involved in prostitution, homosexuality, and drug use against the greater evil of an HIV/AIDS epidemic. A strategy of clinical tolerance offers the most effective and practical way to confront a growing and significant public health problem in China. PMID- 10732876 TI - Bluffing, puffing and spinning in managed-care organizations. AB - I argue that because bluffing, puffing, and spinning are features of corporate life, they are likely to characterize the doctor-patient relationship in managed care medicine. I show that managed-care organizations (MCOs) and the physicians who contract with them make liberal use of puffing and spinning. In this way, they create a context in which it is likely that patients will also use deceptive mechanisms. Unfortunately, patients risk their health when they deceive their doctors. Using the warranty theory of truth I argue that although bluffing may be ethical in business because all participants agree to it and business has not warranted truth-telling, it is not ethical in a medical context because physicians and MCOs have warranted truth-telling and the quality of medical care depends on it. PMID- 10732877 TI - What would John Dewey do? The promises and perils of pragmatic bioethics. AB - Recent work done at the intersection of classical American pragmatism and bioethics promises much: a clarified self-understanding for bioethics, a modus vivendi for progress, and liberation from misguided and misguiding theories and principles. The revival of pragmatism outside bioethics in the past twenty years, however, has been of a distinctly anti-realist orientation. Richard Rorty, for example, has urged that there is no objective truth or good for philosophy to be concerned with. I ask whether the work in Pragmatic Bioethics follows this perilous Rortyan trend. It will move towards anti-realism if its account of the good abandons any notion of truth or objectivity, and if, in its discussion of specific problems, it divides these problems into public and the private, urging consensus as the goal of the one, and an unconstrained notion of happiness as the goal of the other. In a final section, I suggest that bioethics done in the spirit of Royce's Philosophy of Loyalty might have much to offer to those dissatisfied with anti-realism. PMID- 10732878 TI - Genetic ignorance, moral obligations and social duties. AB - In a contribution to The Journal of Medicine and Philosophy, Professor Rosamond Rhodes argues that individuals sometimes have an obligation to know about their genetic disorders, because this is required by their status as autonomous persons. Her analysis, which is based on Kant's concept of autonomy and Aristotle's notion of friendship, is extended here to consequentialist concerns. These are of paramount importance if, as we believe and Professor Rhodes herself implies, the Kantian and Aristotelian doctrines can be helpful only in the sphere of private morality, not in the public realm. Better tools for assessing the right to genetic ignorance as an issue of public policy can, we contend, be found in Mill's ideas concerning liberty and the prevention of harm. Our own conclusion, based on the Millian way of thinking, is that individuals probably do have the right to remain in ignorance in the cases Professor Rhodes presents as examples of a duty to know. PMID- 10732879 TI - 31st Bethesda Conference. Emergency Cardiac Care. Task force 1: cardiac arrest. PMID- 10732880 TI - 31st Bethesda Conference. Emergency Cardiac Care. Task force 2: Acute coronary syndromes: Section 2A--Prehospital issues. PMID- 10732881 TI - 31st Bethesda Conference. Emergency Cardiac Care. Task force 2: Acute coronary syndromes: Section 2B--Chest discomfort evaluation in the hospital. PMID- 10732882 TI - 31st Bethesda Conference. Emergency Cardiac Care. Task force 3: Special aspects of research conduct in the emergency setting: Waiver of informed consent. PMID- 10732883 TI - The Angiotensin-converting Enzyme Inhibition Post Revascularization Study (APRES). AB - OBJECTIVE: This study was performed to assess the effect of treatment with ramipril on the incidence of cardiac events after invasive revascularization in patients with asymptomatic moderate left ventricular dysfunction. BACKGROUND: In patients with angina pectoris and left ventricular dysfunction, both invasive revascularization and treatment with angiotensin-converting enzyme inhibitors reduce cardiac mortality and morbidity. Whether there is a benefit from combining the two treatment strategies has never been evaluated prospectively. METHODS: After invasive revascularization, 159 patients with preoperative chronic stable angina pectoris, left ventricular ejection fraction between 0.30 and 0.50 and no clinical heart failure were randomly assigned to receive double-blind treatment with either ramipril or placebo and subsequently followed for a median of 33 months. RESULTS: Ramipril reduced the incidence of the triple-composite end point of cardiac death, acute myocardial infarction or clinical heart failure (risk reduction 58%; 95% confidence interval 7% to 80%, p = 0.031). The incidence of the quadruple-composite end point of cardiac death, acute myocardial infarction, clinical heart failure or recurrent angina pectoris was not altered with ramipril. These findings were consistent across subgroups with respect to left ventricular ejection fraction below or above 0.40, and whether coronary artery bypass grafting or percutaneous transluminal coronary angioplasty was performed. CONCLUSIONS: In patients with angina pectoris and asymptomatic moderate left ventricular dysfunction, long-term treatment with ramipril after invasive revascularization significantly reduced the incidence of the composite end point of cardiac death, acute myocardial infarction or clinical heart failure, indicating that the beneficial effects of angiotensin-converting enzyme inhibitor treatment may be extended to include treatment of this patient group. PMID- 10732884 TI - Does coronary angiography before emergency aortic surgery affect in-hospital mortality? AB - OBJECTIVES: To study the relationship between coronary angiography and in hospital mortality in patients undergoing emergency surgery of the aorta without a history of coronary revascularization or coronary angiography before the onset of symptoms. BACKGROUND: In the setting of acute ascending aortic dissection warranting emergency aortic repair, coronary angiography has been considered to be desirable, if not essential. The benefits of defining coronary anatomy have to be weighed against the risks of additional delay before surgical intervention. METHODS: Retrospective analysis of patient charts and the Cardiovascular Information Registry (CVIR) at the Cleveland Clinic Foundation. RESULTS: We studied 122 patients who underwent emergency surgery of the aorta between January 1982 and December 1997. Overall, in-hospital mortality was 18.0%, and there was no significant difference between those who had coronary angiography on the day of surgery compared with those who had not (No: 16%, n = 81 vs. Yes: 22%, n = 41, p = 0.46). Multivariate analysis revealed that a history of myocardial infarction (MI) was the only predictor of in-hospital mortality (relative risk: 4.98 95% confidence interval: 1.48-16.75, p = 0.009); however, coronary angiography had no impact on in-hospital mortality in patients with a history of MI. Furthermore, coronary angiography did not significantly affect the incidence of coronary artery bypass grafting (CABG) during aortic surgery (17% vs. 25%, Yes vs. No). Operative reports revealed that 74% of all CABG procedures were performed because of coronary dissection, and not coronary artery disease. CONCLUSIONS: These data indicate that determination of coronary anatomy may not impact on survival in patients undergoing emergency surgery of the aorta and support the concept that once diagnosed, patients should proceed as quickly as possible to surgery. PMID- 10732886 TI - Optimal management of non-ST segment elevation myocardial infarction remains unclear. PMID- 10732885 TI - Early angiography versus conservative treatment in patients with non-ST elevation acute myocardial infarction: MITI Investigators. Myocardial Infarction Triage and Intervention. AB - OBJECTIVES: To compare short- and long-term outcome after early invasive or conservative strategies in the treatment of non-ST segment elevation acute myocardial infarction (AMI). BACKGROUND: It is uncertain whether or not there is benefit from emergent invasive diagnosis and treatment of AMI in patients without ST segment elevation on the admission electrocardiogram (ECG). METHODS: In a cohort of 1,635 consecutive patients with AMI who presented to hospitals without ST segment elevation on their admission ECG, we compared treatments, hospital course and outcome in 308 patients who presented to hospitals whose initial strategy favored early angiography and appropriate intervention when indicated versus 1,327 similar patients who presented to hospitals that favor a more conservative initial approach. RESULTS: At baseline, patients admitted to hospitals favoring an early invasive strategy were younger, more predominately Caucasian and had less comorbidity. Early coronary angiography occurred in 58.8% versus 8% (p < 0.001), and early angioplasty was performed in 44.8% versus 6.1% (p < 0.001) in the two different cohorts. Patients treated in hospitals favoring the early invasive strategy had a lower 30-day (5.5% vs. 9.5%, p = 0.026) and four-year mortality (20% vs. 37%, p < 0.001). Multivariate analysis showed a trend towards lower hospital mortality (OR = 0.56, 95% CI: 0.29 to 1.09) and a significant lower long-term mortality (hazard ratio = 0.61, 95% CI: 0.47 to 0.80) in patients admitted to hospitals favoring an early invasive strategy. CONCLUSIONS: These data suggested that an early invasive strategy in patients with AMI and nondiagnostic ECG changes is associated with lower long-term mortality. PMID- 10732887 TI - Quality of life after coronary angioplasty or continued medical treatment for angina: three-year follow-up in the RITA-2 trial. Randomized Intervention Treatment of Angina. AB - OBJECTIVES: We sought to evaluate the impact of percutaneous transluminal coronary angioplasty (PTCA) and medical treatment on self-perceived quality of life among patients with angina. BACKGROUND: The second Randomized Intervention Treatment of Angina trial (RITA-2) implemented initial policies of PTCA or continued medical treatment in patients with angina, allowing assessment of long term health consequences. METHODS: A total of 1,018 patients were randomly assigned (504 to PTCA and 514 to medical treatment). The short form 36 (SF-36) self-administered quality-of-life questionnaire was completed at randomization and three months, one year and three years later. To date, 98% of patients reached one year and 67% reached three years. RESULTS: The PTCA group had significantly greater improvements in physical functioning, vitality and general health at both three months and one year, but not at three years. These quality of-life scores were strongly related to breathlessness, angina grade and treadmill exercise time both at baseline and at one year. The treatment differences in quality of life are explained by the PTCA group's improvements in breathlessness, angina and exercise time. The attenuation of treatment difference at three years is partly attributed to 27% of medically treated patients receiving nonrandomized interventions in the interim. For both groups, there were also improvements in ratings of physical role functioning, emotional role functioning, social functioning, pain and mental health, but for these the superiority of PTCA over medical treatment was less pronounced. After one year, 33% and 22% of the PTCA and medical groups, respectively, rated their health much better. CONCLUSIONS: Coronary angioplasty substantially improves patient perceived quality of life, especially physical functioning and vitality, as compared with continued medical treatment. These differences are attributed to alleviation of cardiac symptoms (specifically, breathlessness and angina), but must be balanced against the small procedure-related risks of PTCA. PMID- 10732888 TI - Effect of glycoprotein IIb/IIIa receptor blockade with abciximab on clinical and angiographic restenosis rate after the placement of coronary stents following acute myocardial infarction. AB - OBJECTIVES: In the Intracoronary Stenting and Antithrombotic Regimen-2 trial (ISAR-2), we sought to investigate the effect of abciximab on angiographic and clinical restenosis after stenting following acute myocardial infarction (AMI). We also intended to assess the impact of abciximab on clinical outcome in this setting. BACKGROUND: It is unclear whether abciximab reduces neointima formation after stenting. Such an effect may be particularly prominent in thrombus containing lesions. METHODS: Patients undergoing stenting within 48 h after onset of AMI were randomly assigned to receive either standard-dose heparin or abciximab plus reduced-dose heparin. Of 401 patients randomized, 366 without 30 day adverse events were eligible for six-month angiographic follow-up. Scheduled angiography was performed in 80% of these patients. RESULTS: By 30 days, the composite clinical end point of death, reinfarction, and target lesion revascularization (TLR) was reached in 5.0% of the abciximab group and in 10.5% of the control group (p = 0.038). At one year, absolute reduction in the composite clinical end point by abciximab was still 5.7% but had lost its statistical significance. Our primary end point, late lumen loss, was 1.26+/-0.85 mm with abciximab and 1.21+/-0.74 mm with standard heparin (p = 0.61), and binary angiographic restenosis rates were 31.1% and 30.6%, respectively (p = 0.92). CONCLUSIONS: In patients undergoing stenting following AMI, abciximab exerted beneficial effects by substantially reducing the 30-day rate of major adverse cardiac events. During one-year follow-up, there was no additional benefit from a reduction in TLR nor did abciximab reduce angiographic restenosis. PMID- 10732889 TI - Abciximab reduces mortality in diabetics following percutaneous coronary intervention. AB - OBJECTIVES: We sought to determine whether abciximab therapy at the time of percutaneous coronary intervention (PCI) would favorably affect one-year mortality in patients with diabetes. BACKGROUND: Diabetics are known to have increased late mortality following PCI. METHODS: Data from three placebo controlled trials of PCI, EPIC, EPILOG, and EPISTENT, were pooled. The one-year mortality rate for patients with a clinical diagnosis of diabetes mellitus was compared with the rate for nondiabetic patients treated with either abciximab or placebo. RESULTS: In the 1,462 diabetic patients, abciximab decreased the mortality from 4.5% to 2.5%, p = 0.031, and in the 5,072 nondiabetic patients, from 2.6% to 1.9%, p = 0.099. In patients with the clinical syndrome of insulin resistance--defined as diabetes, hypertension, and obesity--mortality was reduced by abciximab treatment from 5.1% to 2.3%, p = 0.044. The beneficial reduction in mortality with abciximab use in diabetics classified as insulin-requiring was from 8.1% to 4.2%, p = 0.073. Mortality in diabetics who underwent multivessel intervention was reduced from 7.7% to 0.9% with use of abciximab, p = 0.018. In a Cox proportional hazards survival model, the risk ratio for mortality with abciximab use compared with placebo was 0.642 (95% confidence interval 0.458 0.900, p = 0.010). CONCLUSIONS: Abciximab decreases the mortality of diabetic patients to the level of placebo-treated nondiabetic patients. This beneficial effect is noteworthy in those diabetic patients who are also hypertensive and obese and in diabetics undergoing multivessel intervention. Besides its potential role in reducing repeat intervention for stented diabetic patients, abciximab therapy should be strongly considered in diabetic patients undergoing PCI to improve their survival. PMID- 10732890 TI - Immediate and long-term outcome of intracoronary stent implantation for true bifurcation lesions. AB - OBJECTIVES: The aim of this study was to evaluate the immediate and long-term outcome of intracoronary stent implantation for the treatment of coronary artery bifurcation lesions. BACKGROUND: Balloon angioplasty of true coronary bifurcation lesions is associated with a lower success and higher complication rate than most other lesion types. METHODS: We treated 131 patients with bifurcation lesions with > or =1 stent. Patients were divided into two groups; Group (Gp) 1 included 77 patients treated with a stent in one branch and percutaneous transluminal coronary angioplasty (PTCA) (with or without atherectomy) in the side branch, and Gp 2 included 54 patients who underwent stent deployment in both branches. The Gp 2 patients were subsequently divided into two subgroups depending on the technique of stent deployment. The Gp 2a included 19 patients who underwent Y stenting, and Gp 2b included 33 patients who underwent T-stenting. RESULTS: There were no significant differences between the groups in terms of age, gender, frequency of prior myocardial infarction (MI) or coronary artery bypass grafting (CABG), or vessels treated. Procedural success rates were excellent (89.5 to 97.4%). After one-year follow-up, no significant differences were seen in the frequency of major adverse events (death, MI, or repeat revascularization) between Gp 1 and Gp 2. Adverse cardiac events were higher with Y-stenting compared with T-stenting (86.3% vs. 30.4%, p = 0.004). CONCLUSIONS: Stenting of bifurcation lesions can be achieved with a high success rate. However, stenting of both branches offers no advantage over stenting one branch and performing balloon angioplasty of the other branch. PMID- 10732891 TI - Immediate and late outcomes after direct stent implantation without balloon predilation. AB - OBJECTIVES: The aim of our study was to compare the in-hospital and long-term clinical outcomes of direct coronary stenting with balloon predilation followed by stent placement. BACKGROUND: With improvement in stent designs, the practice of direct stenting without balloon predilation has become more widespread. METHODS: We analyzed the Mayo Clinic Coronary Intervention data base between January 1, 1995 and March 5, 1999 and identified 777 patients who were treated with direct stenting (DS) and 3,176 patients treated with balloon angioplasty plus stenting (BA+S). RESULTS: The procedural success rates between the DS and BA+S groups were not significantly different (96.3% vs. 96.4%). The ability to deliver the stent in a subgroup of patients who had DS was 95%, with 5% requiring crossover to predilation. Multivariate analysis showed no significant differences with respect to in-hospital death (odds ratio [OR] 0.9, 95% confidence interval [CI] 0.5 to 1.8), in-hospital myocardial infarction (OR 0.9, 95% CI 0.6 to 1.2) or revascularization (OR 0.7, 95% CI 0.4 to 1.5) in the DS compared with the BA+S group. Long-term outcomes were not significantly different between the DS and BA+S groups. The procedural duration was significantly shorter in the DS group, and there was a decreased utilization of contrast agent, balloons and wires. CONCLUSIONS: The in-hospital and long-term clinical outcomes in patients undergoing a coronary intervention are equivalent when comparing stenting without balloon predilation with balloon angioplasty followed by stenting. Direct stenting is associated with decreased utilization of contrast agent and equipment and shorter procedure times. A randomized study should be performed to better determine the impact of this technique on short- and long-term procedural outcomes. PMID- 10732892 TI - Percutaneous revascularization of the internal mammary artery graft: short- and long-term outcomes. AB - OBJECTIVES: We evaluated the short- and long-term clinical outcomes after percutaneous revascularization of the internal mammary artery (IMA) graft. BACKGROUND: Previous reports in a relatively small number of patients have indicated the safety of balloon angioplasty for the treatment of stenoses in the IMA graft. However, the use of alternative interventional techniques and their long-term results have not yet been evaluated. METHODS: We analyzed the in hospital and one-year clinical outcomes of 174 consecutive patients who underwent percutaneous revascularization of 202 lesions located in the IMA graft, by either balloon angioplasty or stenting. RESULTS: Anastomotic lesions were evident in 128 cases (63%), and they were more commonly treated with balloon angioplasty (116/128, 91%), whereas lesions located at the ostium (n = 16, 8%) were more frequently treated with stents (11/16, 69%). Procedural success was 97% with excellent in-hospital outcome: 0.6% mortality rate, no Q-wave myocardial infarction (MI) and 0.6% rate of urgent bypass surgery. Cumulative one-year rates were: mortality 4.4%, MI 2.9% and target lesion revascularization (TLR) 7.4%. CONCLUSIONS: Revascularization of the IMA graft can be performed safely, with high procedural success and a low rate of in-hospital complications. Long-term follow-up showed very low TLR rate. PMID- 10732893 TI - Pressure-derived fractional collateral blood flow: a primary determinant of left ventricular recovery after reperfused acute myocardial infarction. AB - OBJECTIVES: We evaluated the relation between pressure-derived fractional collateral flow (PDCF) and left ventricular (LV) recovery after reperfused acute myocardial infarction (AMI). BACKGROUND: The functional significance of collateral flow remains uncertain in AMI. METHODS: The PDCF was measured in 70 patients with first AMI (pain onset <12 h) treated with primary angioplasty (PA), being determined by simultaneous measurement of mean aorta pressure (Pa), distal coronary pressure during the balloon occlusion (Poc), and central venous pressure (CVP): (Poc - CVP)/(Pa - CVP)*100. Sufficient collateral (group I) was defined as PDCF index >24% and insufficient collateral (group II) as PDCF index <24%. Echocardiography was performed before, and on day 3, day 7, and day 30 after PA. Wall-motion recovery index (RI) was obtained by dividing the number of improved wall-motion segments (>grade 1) at follow-up by the number of abnormal wall motion segments within the infarct zone at baseline. RESULTS: Baseline characteristics were similar between both groups. Peak levels of creatine kinase were lower in group I than in group II (2,600+/-1,900 U/liter vs. 4,100+/-3,000, p < 0.05). At one month, infarct zone wall-motion score index (1.65+/-0.54 vs. 2.31+/-0.46, p < 0.01) and LV volume indexes were smaller in group I than in group II, whereas, LV ejection fraction was higher in group I than in group II (52.8+/-8.3 vs. 45.9+/-9.0, p < 0.01). The PDCF index was the strongest predictor of RI at one month (r = 0.61, p < 0.01). Time to reperfusion was not related to RI at one month. However, it was significantly related to RI in group II (r = 0.34, p < 0.05). CONCLUSIONS: The LV recovery after reperfused AMI is primarily determined by PDCF and is less dependent on time to reperfusion in patients with sufficient collaterals. PMID- 10732894 TI - Serum neopterin and complex stenosis morphology in patients with unstable angina. AB - OBJECTIVES: We sought to assess the relation between serum neopterin concentration and complex coronary artery stenosis in patients with unstable angina. BACKGROUND: Monocyte activation is associated with acute atheromatous plaque disruption and acute coronary syndromes. Angiographically demonstrated complex coronary stenosis is often an expression of plaque disruption. Increased serum concentration of neopterin, a pterydine derivative secreted by macrophages after stimulation by interferon-gamma, has been observed in patients with acute coronary syndromes as compared with control subjects and patients with stable angina pectoris. METHODS: We studied 50 patients with unstable angina (32 men) who underwent coronary angiography after hospital admission. All coronary stenoses with > or =30% diameter reduction were assessed and classified as "complex" (irregular or scalloped borders, ulceration or filling defects suggesting thrombi) or "smooth" (absence of complex features). Serum neopterin levels were assessed within 24 h of hospital admission using a commercially available immunoassay (enzyme-linked immunosorbent assay kit, IBL, Hamburg, Germany). RESULTS: Thirty-nine patients were classified in Braunwald class IIIb, four in class IIb and seven in class Ib. The number of complex lesions per patient was 2.6+/-1.8 (mean +/- SD). The mean neopterin concentration was 7.76+/ 3.62 nmol/liter. A significant correlation was observed between neopterin serum concentration and the presence of complex coronary stenoses (r = 0.35, p = 0.015). Multiple regression analysis showed that serum neopterin (p < 0.0001) was independently associated with the number of complex lesions. Other variables associated with complex lesions were the number of vessels with > or =75% stenosis (p < 0.0001), plasma creatinine (p = 0.003), triglycerides (p = 0.014) and a history of unstable angina (p = 0.032). CONCLUSIONS: Serum neopterin concentration is associated with the presence of angiographically demonstrated complex lesions in patients with unstable angina and may represent a marker of coronary disease activity. PMID- 10732895 TI - Plaque inflammation in restenotic coronary lesions of patients with stable or unstable angina. AB - OBJECTIVES: To evaluate immunohistochemically various parameters of inflammation in coronary atherectomy specimens obtained from restenotic culprit lesions of patients presenting with either stable or unstable angina (UA). BACKGROUND: There is no information regarding the relationship between atherosclerotic plaque inflammation and the severity of the coronary syndromes in patients with restenotic coronary lesions. METHODS: A total of 37 patients with either stable angina or UA underwent directional coronary atherectomy for restenotic coronary lesions. Cryostat sections of atherectomy specimen were immunohistochemically stained with monoclonal antibodies CD68 (macrophages [MACs]), CD3 (T-lymphocytes) and alpha-actin (smooth muscle cells [SMCs]). Smooth muscle cell contents and MAC contents were planimetrically quantified as the percentage immunopositive tissue area of the total tissue area. T-lymphocytes were counted at 100-X magnification throughout the entire section and expressed as number of cells per mm2. RESULTS: Restenotic coronary lesions of patients with UA or stable angina showed no significant difference in SMC areas (31.9%+/-16.3% vs. 38.5%+/-18.8%, respectively; p = NS). However, restenotic coronary lesions of patients presenting with unstable angina contained significantly more MACs (24.4%+/-15.1% vs. 10.5%+/-5.8%, p = 0.001) and T-lymphocytes (18.8 cells/mm2+/-15.1 cells/mm2 vs. 8.6 cells/mm2+/-9.8 cells/mm2; p = 0.034) than patients with stable angina. CONCLUSIONS: These results suggested that inflammation appears to affect plaque instability in restenotic coronary lesions resulting in unstable coronary syndromes. PMID- 10732896 TI - Expression of vascular endothelial growth factor in patients with acute myocardial infarction. AB - OBJECTIVE: The purpose of this study was to investigate the clinical significance of vascular endothelial growth factor (VEGF) in acute myocardial infarction (AMI). We also examined the involvement of peripheral blood mononuclear cells (PBMCs), which are a possible source of VEGF in AMI. BACKGROUND: VEGF is a potent endothelial cell-specific mitogen and could affect the outcome of AMI. METHODS: Thirty patients with AMI were used for this study. Serum and PBMCs were isolated from peripheral blood on days 1, 7, 14 and 21 after the onset of AMI. PBMCs were cultured at a density of 5 x 10(6) cells/ml for 24 h. VEGF levels in serum and the culture media were measured by enzyme-linked immunosorbent assay using a specific anti-human VEGF antibody. RESULTS: Serum VEGF levels elevated gradually after the onset of AMI and reached a peak on day 14. VEGF levels in the culture medium of PBMCs after incubation for 24 h (PBMC-VEGF) were maximally elevated 7 days after the onset. Maximum serum VEGF levels showed significant positive correlations with maximum creatine phosphokinase (CPK) levels (r = +0.70, p < 0.001), but maximum PBMC-VEGF levels did not correlate with maximum CPK levels. Patients showing improvement in left ventricular systolic function during the course of AMI showed significantly higher PBMC-VEGF levels than patients without improvement. CONCLUSIONS: The extent of myocardial damage contributes to the elevation of serum VEGF levels in AMI. VEGF produced by PBMCs may play an important role in the improvement of left ventricular function by promoting angiogenesis and reendothelialization after AMI. PMID- 10732897 TI - Utilization of coronary angiography and revascularization after acute myocardial infarction in men and women risk stratified by the American College of Cardiology/American Heart Association guidelines. AB - OBJECTIVES: We sought to determine whether men and women are equally likely to receive coronary angiography and revascularization after acute myocardial infarction (AMI) when they are risk stratified according to American College of Cardiology/American Heart Association (ACC/AHA) practice guidelines for post-MI care. BACKGROUND: Several previous studies have suggested that women may undergo angiography and revascularization procedures less frequently than men. METHODS: In 439 consecutive patients admitted to a public hospital with AMI, rates of coronary angiography and revascularization were compared in men and women categorized, according to ACC/AHA practice guidelines, as having strong (class I or IIa) or weaker (class IIb) indications for angiography. RESULTS: Women were older and more likely to be diabetic or hypertensive, but men and women were equally likely to meet class I/IIa criteria for post-MI angiography (both 51%). Angiography rates were nearly identical in men and women overall (63% vs. 64%), as well as in patients in class I/IIa (80% vs. 82%) and class IIb (46% vs. 46%) (all p > 0.80, with >80% power to detect important differences); the only multivariate predictors of post-MI angiography were age and ACC/AHA class. Significant coronary artery disease was equally prevalent in men and women undergoing angiography, and men and women were equally likely to undergo revascularization, whether they were in class I/IIa (both 55%, p = 0.90) or class IIb (59% vs. 58%, p = 0.88). No significant differences in mortality were noted between men and women. CONCLUSIONS: Despite being older and having more risk factors than men, women were equally likely to undergo coronary angiography and revascularization procedures after AMI, and they had in-hospital clinical outcomes that were at least as favorable. PMID- 10732898 TI - Stress Doppler echocardiography for identification of susceptibility to high altitude pulmonary edema. AB - OBJECTIVE: This prospective single-blinded study was performed to quantitate noninvasive pulmonary artery systolic pressure (PASP) responses to prolonged acute hypoxia and normoxic exercise. BACKGROUND: Hypoxia-induced excessive rise in pulmonary artery pressure is a key factor in high-altitude pulmonary edema (HAPE). We hypothesized that subjects susceptible to HAPE (HAPE-S) have increased pulmonary artery pressure response not only to hypoxia but also to exercise. METHODS: PASP was estimated at 45, 90 and 240 min of hypoxia (FiO2 = 12%) and during supine bicycle exercise in normoxia using Doppler-echocardiography in nine HAPE-S and in 11 control subjects. RESULTS: In the control group, mean PASP increased from 26+/-2 to 37+/-4 mm Hg (deltaPASP 10.3+/-2 mm Hg) after 90 min of hypoxia and from 27+/-4 to 36+/-3 mm Hg (deltaPASP 8+/-2 mm Hg) during exercise. In contrast, all HAPE-S subjects revealed significantly greater increases (p = 0.002 vs. controls) in mean PASP both during hypoxia (from 28+/-4 to 57+/-10 mm Hg, deltaPASP 28.7+/-6 mm Hg) and during exercise (from 28+/-4 to 55+/-11 mm Hg, deltaPASP 27+/-8 mm Hg) than did control subjects. Stress echocardiography allowed discrimination between groups without overlap using a cut off PASP value of 45 mm Hg at work rates less than 150 W. CONCLUSIONS: These data indicate that HAPE-S subjects may have abnormal pulmonary vascular responses not only to hypoxia but also to supine bicycle exercise under normoxic conditions. Thus, Doppler echocardiography during supine bicycle exercise or after 90 min of hypoxia may be useful noninvasive screening methods to identify subjects susceptible to HAPE. PMID- 10732899 TI - Balloon angioplasty of native coarctation: clinical outcomes and predictors of success. AB - OBJECTIVES: We sought to investigate the clinical impact of balloon angioplasty for native coarctation of the aorta (CoA) and determine predictors of outcome. BACKGROUND: Balloon dilation of native CoA remains controversial and more information on its long-term impact is required. METHODS: Hemodynamic, angiographic and follow-up data on 69 children who underwent balloon angioplasty of native CoA between 1988 and 1996 were reviewed. Stretch, recoil and gain of CoA circumference and area were calculated and related to outcomes. RESULTS: Initial systolic gradients (mean +/- SD, 31+/-12 mm Hg) fell by -74+/-27% (p < 0.001), with an increase in mean CoA diameters of 128+/-128% in the left anterior oblique and 124+/-87% in the lateral views (p < 0.001). Two deaths occurred, one at the time of the procedure and one 23 months later, both as a result of an associated cardiomyopathy. Seven patients had residual gradients of >20 mm Hg. One patient developed an aneurysm, stable in follow-up, and four patients had mild dilation at the site of the angioplasty. Freedom from reintervention was 90% at one year and 87% at five years with follow-up ranging to 8.5 years. Factors significantly associated with decreased time to reintervention included: a higher gradient before dilation, a smaller percentage change in gradient after dilation, a small transverse arch and a greater stretch and gain, but not recoil. CONCLUSION: Balloon dilation is a safe and efficient treatment of native CoA in children. Greater stretch and gain are factors significantly associated with reintervention, possibly related to altered elastic properties and vessel scarring. PMID- 10732900 TI - Repaired coarctation: a "cost-effective" approach to identify complications in adults. AB - OBJECTIVES: The study was done to determine the most "cost-effective" approach to follow adults after repair of coarctation of the aorta. BACKGROUND: Recoarctation and/or aneurysm formation following surgical repair or angioplasty for coarctation of the aorta carry a significant morbidity and mortality. Various screening tests to detect such complications are used, but little is known of their sensitivities and specificities; as a consequence, the most "cost effective" approach to follow such patients is undefined. METHODS: Retrospective analysis was done on the sensitivity and specificity of symptomatology, physical examination, electrocardiogram, chest radiograph, exercise testing and transthoracic echocardiography to detect recoarctation and/or aneurysm formation in 84 adult patients following surgical repair or angioplasty of coarctation of the aorta, using magnetic resonance imaging (MRI) as the gold standard test. RESULTS: Echocardiography had the highest sensitivity in detecting recoarctation (87%) and chest radiograph the highest sensitivity in detecting aneurysm formation (67%). Combined clinical visit and echocardiography had a high sensitivity for diagnosing recoarctation and/or aneurysm formation (97%), but performing a clinical visit and an MRI on every patient without any prior screening test emerged as the most "cost-effective" strategy. CONCLUSIONS: The most "cost-effective" approach to diagnose complications at the site of repair in patients after surgical repair or balloon angioplasty of coarctation of the aorta appears to be the combination of clinical assessment and MRI scan on every patient. If MRI resources are scant, performing a clinical assessment plus a transthoracic echocardiography and an MRI on patients with positive results is an acceptable alternative. PMID- 10732901 TI - "Repaired" aortic coarctation in adults: not a "simple" congenital heart defect. PMID- 10732902 TI - Early and intermediate-term complications of self-expanding stents limit its potential application in children with congenital heart disease. AB - OBJECTIVES: We report on the early and intermediate-term follow-up results of self-expanding Wallstent (Schneider, Switzerland) implanted in children with congenital heart disease. BACKGROUND: The inherent shortcomings of balloon expandable stents prompted the trial of an alternative stent. METHODS: Twenty patients underwent 22 implantations of 25 self-expanding Wallstents between December 1993 and June 1997 in two institutions. The mean age and weight were 10.8+/-4.5 years and 30.5+/-14.2 kg, respectively. The patients were divided into two groups: 1) Group I comprised 17 patients with pulmonary arterial stenoses, 2) Group II comprised four patients with venous stenoses (one belonged to both groups). Sixteen patients underwent recatheterization at a median of 5.8 months (range 0.5 to 31, mean 8.1 months) after stenting. Hemodynamic and angiographic changes after the interventional procedures and complications were documented. RESULTS: All the stents were successfully deployed in the intended position. In Group I, the narrowest diameter of the stented vessel increased from 4.1+/-1.5 to 8+/-2 mm (95% increase, p < 0.0001) while the systolic pressure gradient across decreased from 24.6+/-15.8 to 12.1+/-11.4 mm Hg (51% decrease, p = 0.001). In Group II, the dimensional changes of the narrowest segment increased from 4.3+/ 0.5 to 7.5+/-0.4 mm (75% increase, p = 0.003), and the pressure gradient reduced from 5.0+/-2.9 to 0.9+/-1.0 mm Hg (82% decrease, p = 0.04) across the stented venous channel. Distal migration of two optimally positioned stents occurred within 24 h of implantation. At recatheterization, significant neointimal ingrowth (>30% of the expanded diameter) was noted in 7 (28%) of the 25 implanted stents. This responded poorly to balloon dilation. Predisposing factors for the neointimal ingrowth included stents of smaller diameter (<9 mm) and longer period after implantation. CONCLUSIONS: Self-expanding Wallstent could be deployed easily and safely to relieve vascular stenoses in children. The complications of distal migration, significant neointimal ingrowth and its unyielding design to overdilation limit its application to this patient group. PMID- 10732903 TI - Further experience with transcatheter closure of the patent ductus arteriosus using the Amplatzer duct occluder. AB - OBJECTIVE: The aim of this study was to report further experience with transcatheter closure of the patent ductus arteriosus (PDA) using the Amplatzer duct occluder (ADO). BACKGROUND: The design of previously used devices is not ideal for this purpose, and their use has been associated with several drawbacks, especially in large PDAs. METHODS: Forty-three patients, aged 0.3 to 33 years (mean 6.4+/-6.7 years), with a moderate to large, type A to E PDA, underwent attempted transcatheter closure using the ADO. The device is a plug-shaped repositionable occluder made of 0.004-in. nitinol wire mesh. It is delivered through a 5F to 6F long sheath. The mean PDA diameter (at the pulmonary end) was 3.9+/-1.2 mm (range 2.2 to 8 mm). All patients had color flow echocardiographic follow-up (6 to 24 months) at 24 h, 1 and 3 months after closure, and at 6-month intervals thereafter. RESULTS: The mean ADO diameter was 6.1+/-1.4 mm (range 4 to 10 mm). Complete angiographic closure was seen in 40 of 43 patients (93%; 95% confidence interval [CI] 85.4% to 100%). The remaining three patients had a trivial angiographic shunt through the ADO. At 24 h, color flow mapping revealed no shunt in all patients. A 9F long sheath was required for repositioning of a misplaced 8-mm device into the pulmonary artery. The mean fluoroscopy time was 7.9+/-1.6 min (range 4.6 to 12 min). There were no complications. No obstruction of the descending aorta or the pulmonary artery branches was noted on Doppler follow-up studies. Neither thromboembolization nor hemolysis or device failure was encountered. CONCLUSIONS: Transcatheter closure using the ADO is an effective and safe therapy for the majority of patients with patency of the arterial duct. Further studies are required to establish long-term results in a larger patient population. PMID- 10732904 TI - Early postoperative changes in regional systolic and diastolic left ventricular function after transmyocardial laser revascularization: a comparison of holmium:YAG and CO2 lasers. AB - OBJECTIVES: The purpose of this study was to determine the short-term effects of transmyocardial laser revascularization (TMR) on regional left ventricular systolic and diastolic function, myocardial blood flow (MBF) and myocardial water content (MWC). BACKGROUND: Clinical studies of TMR have noted a significant incidence of cardiac complications in the early postoperative period. However, the early post-treatment effects of laser therapy on the myocardium and their potential contribution to postoperative cardiac morbidity are unknown. METHODS: Swine underwent holmium:yttrium-aluminum-garnet (holmium:YAG) (n = 12) or carbon dioxide (CO2) (n = 12) laser TMR. Regional systolic function for the lased and nonlased regions was quantitated using preload recruitable work area (PRWA) and regional diastolic function with the ventricular stiffness constant alpha. RESULTS: Preload recruitable work area was significantly decreased in the lased regions both 1 (59.8+/-13.0% of baseline, p = 0.02) and 6 h (64.2+/-9.4% of baseline, p = 0.02) after holmium:YAG TMR. This decreased PRWA was associated with a significant reduction in MBF to the lased regions (13.2% reduction at 1 h, p = 0.02; 18.4% decrease at 6 h post-TMR, p = 0.01). These changes were not seen after CO2 laser TMR. A significant increase in MWC (1.4+/-0.3% increase with holmium:YAG, p = 0.004; 1+/-0.2% increase with CO2, p = 0.002) and alpha (217.4+/ 44.2% of baseline 6 h post-holmium:YAG TMR, p = 0.05; 206+/-36.7% of baseline 6 h post-CO2 TMR, p = 0.03) was seen after TMR with both lasers. CONCLUSIONS: In the early postoperative setting, impaired regional systolic function in association with regional ischemia is seen after TMR with a holmium:YAG laser. Both holmium:YAG and CO2 lasers are associated with increased MWC and impaired diastolic relaxation in the lased regions. These changes may explain the significant incidence of early postoperative cardiac morbidity. The impact of these findings on anginal relief and long-term outcome are not known. PMID- 10732905 TI - Electromagnetic guidance for catheter-based transendocardial injection: a platform for intramyocardial angiogenesis therapy. Results in normal and ischemic porcine models. AB - OBJECTIVES: To test the feasibility of myocardial angiogenic gene expression using a novel catheter-based transendocardial injection system. BACKGROUND: Angiogenesis has been induced by direct injection of growth factors into ischemic myocardium during open-heart surgery. Catheter-based transendocardial injection of angiogenic factors may provide equivalent benefit without need of surgery. METHODS: A new guidance system for intramyocardial therapy utilizes magnetic fields and catheter-tip sensors to locate a position in space and reconstruct three-dimensional left ventricular (LV) electromechanical maps without using fluoroscopy. A retractable 27G needle was coupled with the guidance system for LV transendocardial injection. In 12 pigs, the catheter was used to inject 0.1 ml of methylene-blue (MB) dye and 8 pigs had myocardial injections of adenoviral vector (1 x 10(10) particles per site) containing the LacZ transgene. Ten pigs underwent catheter-based transendocardial injection and six pigs were injected using transepicardial approach with the gene encoding adenovirus vascular endothelial growth factor-121 (Ad.VEGF121; 1 x 10(10) viral particles x 6 sites) and sacrificed at 24 h. Injection sites were identified with ultraviolet light by coinjection of fluorescent beads. RESULTS: Overall, 138 of 152 attempted injection MB tracks (91%) were found after sacrifice. Tissue staining was 7.1+/ 2.1 mm in depth and 2.3+/-1.8 mm in width. No animal had pericardial effusion or tamponade. In Ad.LacZ injected animals, gross pathology showed positive staining in injected zones, and histology confirmed positive myocyte staining. Adenovirus vascular endothelial growth factor-121 injected sites showed high levels of VEGF121 production that was of similar magnitude whether injected using the transendocardial (880.4+/-412.2 pg VEGF121/mg protein) or transepicardial (838.3+/-270 pg VEGF121/mg protein) delivery approach (p = 0.62). CONCLUSIONS: Using this magnetic guidance catheter-based navigational system, transgenes can effectively be transfected into designated myocardial sites. Thus, if it is determined that direct intramyocardial injection of angiogenic factors enhances collateral function in patients, this less invasive catheter-based system offers a similar gene delivery efficiency and, thus, may have clear advantages compared with the surgically-based transepicardial injection approach. PMID- 10732906 TI - Local adenovirus-mediated transfer of C-type natriuretic peptide suppresses vascular remodeling in porcine coronary arteries in vivo. AB - OBJECTIVE: This study was designed to examine whether or not adenovirus-mediated gene transfer of C-type natriuretic peptide (CNP) can prevent coronary restenotic changes after balloon injury in pigs in vivo. BACKGROUND: Gene therapy to prevent restenosis after percutaneous transluminal coronary angioplasty (PTCA) might be useful but requires a method applicable for in vivo gene delivery into the coronary artery as well as the efficient vector encoding a potent antiproliferative substance. We tested whether the adenovirus-mediated gene transfer of CNP by use of an infiltrator angioplasty balloon catheter (IABC) might prevent the coronary restenotic changes after balloon injury. METHODS: Balloon angioplasty was performed in the left anterior descending and the left circumflex coronary artery in pigs. Immediately after the balloon injury, adenovirus solution encoding either CNP (AdCACNP) or beta-galactosidase (AdCALacZ) gene was injected with IABC into the balloon-injured coronary segments. Expression of CNP was assessed by immunohistochemical staining and cyclic guanosine 3',5'-monophosphate (cGMP) measurement. Coronary restenotic changes were evaluated by both angiographic and histological examinations. RESULTS: CNP was highly expressed in the media and the adventitia of the coronary artery at the AdCACNP-transfected but not at the AdCALacZ-transfected segment. In the AdCALacZ-transfected segment, vascular cGMP levels tended to be reduced as compared with the untreated segment, whereas in the AdCACNP-transfected segment, vascular cGMP levels were restored. Angiographic coronary stenosis was significantly less at the AdCACNP-transfected than at the AdCALacZ-transfected segment. Histological examination revealed that this was achieved primarily by the marked inhibition of the geometric remodeling of the coronary artery by the CNP gene transfer. CONCLUSIONS: Adenovirus-mediated CNP gene transfer with the IABC system may be a useful gene therapy to prevent restenosis after PTCA in vivo. PMID- 10732907 TI - President's page: the U.S. Healthcare System 2010: problems, principles, and potential solutions. PMID- 10732908 TI - The ACC at 50: a giant grew in Bethesda. The impact of the ACC on cardiovascular medicine. American College of Cardiology. PMID- 10732909 TI - Demographics and cardiology, 1950-2050. PMID- 10732910 TI - Technological advances and the next 50 years of cardiology. PMID- 10732911 TI - Technological advances and the next 50 years of cardiology: ethics and privacy. PMID- 10732912 TI - Technological advances and the next 50 years of cardiology: glossary. PMID- 10732913 TI - The future of cardiology: utilization and costs of care. PMID- 10732914 TI - The role of the American College of Cardiology in promoting and maintaining the delivery of quality cardiovascular care in the future. PMID- 10732915 TI - Stress and genital herpes recurrences in women. PMID- 10732916 TI - Please don't pass the paella: eating disorders upset Spain. PMID- 10732917 TI - Device manufacturers consider constraints. PMID- 10732918 TI - Adequacy of clinical trials questioned at IOM meeting. Institute of Medicine. PMID- 10732919 TI - From the Centers for Disease Control and Prevention. Importation of wild poliovirus into Qinghai Province--China, 1999. PMID- 10732920 TI - From the Centers for Disease Control and Prevention. Prevalence of selected risk factors for chronic disease and injury among American Indians and Alaska Natives- United States, 1995-1998. PMID- 10732921 TI - From the Centers for Disease Control and Prevention. Elevated blood lead levels among internationally adopted children--United States, 1998. PMID- 10732922 TI - Scoring the quality of clinical trials. PMID- 10732923 TI - Scoring the quality of clinical trials. PMID- 10732924 TI - Scoring the quality of clinical trials. PMID- 10732925 TI - Sources of bias in the economic analysis of new drugs. Health Outcomes Committee Pharmaceutical Research and Manufacturers of America, Washington, DC. PMID- 10732926 TI - Sources of bias in the economic analysis of new drugs. PMID- 10732927 TI - Sources of bias in the economic analysis of new drugs. PMID- 10732928 TI - Sources of bias in the economic analysis of new drugs. PMID- 10732929 TI - Bisphosphonate therapy and vascular calcification. PMID- 10732930 TI - Vitamin D deficiency in women with hip fracture. PMID- 10732931 TI - Tobacco dependence curricula in medical schools. PMID- 10732932 TI - Causes and severity of ischemic stroke in patients with internal carotid artery stenosis. AB - CONTEXT: Therapeutic trials generally have not distinguished outcomes of stroke according to cause. OBJECTIVE: To determine whether stroke and subsequent disability was of large-artery, lacunar, or cardioembolic origin in patients with different degrees of symptomatic and asymptomatic carotid stenosis. DESIGN: Observational study of prospective data collected from the North American Symptomatic Carotid Endarterectomy Trial between 1987 and 1997. SETTING AND PATIENTS: A total of 2885 patients from 106 sites in the United States and abroad (median age, 67 years; 70% male) who had symptomatic internal carotid artery stenosis. MAIN OUTCOME MEASURE: Risk of stroke from each of the 3 causes at 5 years by territory and degree of stenosis. RESULTS: During an average follow-up of 5 years, 749 patients had 1039 strokes, including 112 of cardioembolic, 211 of lacunar, 698 of large-artery, 17 of primary intracerebral hemorrhage, and 1 of subarachnoid hemorrhage origin. The 5-year risk of first stroke after entry into the trial in any territory was 2.6% of cardioembolic cause, 6.9% of lacunar cause, and 19.7% of large-artery cause. The proportion of cardioembolic strokes in the territory of the symptomatic artery was 12.0% and 6.9% in 60% to 69% and 70% to 99% arterial stenosis, respectively; large-artery strokes predominated (78.4%) at 70% to 99% arterial stenosis. With 70% to 99% arterial stenosis, the proportion of strokes of cardioembolic and lacunar origin was 43.5% and 21.6% in asymptomatic and symptomatic arteries, respectively. A total of 67.6% of cardioembolic, 16.7% of lacunar, and 33.0% of large-artery strokes in the territory of the asymptomatic artery were disabling or fatal. CONCLUSIONS: Our data suggest that approximately 20% and 45% of strokes in the territory of symptomatic and asymptomatic carotid arteries with 70% to 99% stenosis, respectively, are unrelated to carotid stenosis. The cause of subsequent strokes in similar types of patients should be considered when making treatment decisions involving carotid endarterectomy for patients with asymptomatic carotid stenosis, since lacunar and cardioembolic strokes cannot be prevented by endarterectomy. PMID- 10732933 TI - Systematic implementation of an advance directive program in nursing homes: a randomized controlled trial. AB - CONTEXT: Although advance directives are commonly used in the community, little is known about the effects of their systematic implementation. OBJECTIVES: To examine the effect of systematically implementing an advance directive in nursing homes on patient and family satisfaction with involvement in decision making and on health care costs. DESIGN: Randomized controlled trial conducted June 1, 1994, to August 31, 1998. SETTING AND PARTICIPANTS: A total of 1292 residents in 6 Ontario nursing homes with more than 100 residents each. INTERVENTION: The Let Me Decide advance directive program included educating staff in local hospitals and nursing homes, residents, and families about advance directives and offering competent residents or next-of-kin of mentally incompetent residents an advance directive that provided a range of health care choices for life-threatening illness, cardiac arrest, and nutrition. The 6 nursing homes were pair-matched on key characteristics, and 1 home per pair was randomized to take part in the program. Control nursing homes continued with prior policies concerning advance directives. MAIN OUTCOME MEASURES: Residents' and families' satisfaction with health care and health care services utilization over 18 months, compared between intervention and control nursing homes. RESULTS: Of 527 participating residents in intervention nursing homes, 49% of competent residents and 78% of families of incompetent residents completed advance directives. Satisfaction was not significantly different in intervention and control nursing homes. The mean difference (scale, 1-7) between intervention and control homes was -0.16 (95 % confidence interval [CI], -0.41 to 0.10) for competent residents and 0.07 (95% CI, -0.08 to 0.23) for families of incompetent residents. Intervention nursing homes reported fewer hospitalizations per resident (mean, 0.27 vs 0.48; P = .001) and less resource use (average total cost per patient, Can $3490 vs Can $5239; P = .01) than control nursing homes. Proportion of deaths in intervention (24%) and control (28%) nursing homes were similar (P = .20). CONCLUSION: Our data suggest that systematic implementation of a program to increase use of advance directives reduces health care services utilization without affecting satisfaction or mortality. PMID- 10732934 TI - Rifampin and pyrazinamide vs isoniazid for prevention of tuberculosis in HIV infected persons: an international randomized trial. Terry Beirn Community Programs for Clinical Research on AIDS, the Adult AIDS Clinical Trials Group, the Pan American Health Organization, and the Centers for Disease Control and Prevention Study Group. AB - CONTEXT: Because of problems with adherence, toxicity, and increasing resistance associated with 6- to 12-month isoniazid regimens, an alternative short-course tuberculosis preventive regimen is needed. OBJECTIVE: To compare a 2-month regimen of daily rifampin and pyrazinamide with a 12-month regimen of daily isoniazid in preventing tuberculosis in persons with human immunodeficiency virus (HIV) infection. DESIGN: Randomized, open-label controlled trial conducted from September 1991 to May 1996, with follow-up through October 1997. SETTING: Outpatient clinics in the United States, Mexico, Haiti, and Brazil. PARTICIPANTS: A total of 1583 HIV-positive persons aged 13 years or older with a positive tuberculin skin test result. INTERVENTIONS: Patients were randomized to isoniazid, 300 mg/d, with pyridoxine hydrochloride for 12 months (n = 792) or rifampin, 600 mg/d, and pyrazinamide, 20 mg/kg per day, for 2 months (n = 791). MAIN OUTCOME MEASURES: The primary end point was culture-confirmed tuberculosis; secondary end points were proven or probable tuberculosis, adverse events, and death, compared by treatment group. RESULTS: Of patients assigned to rifampin and pyrazinamide, 80% completed the regimen compared with 69% assigned to isoniazid (P<.001). After a mean follow-up of 37 months, 19 patients (2.4%) assigned to rifampin and pyrazinamide and 26 (3.3%) assigned to isoniazid developed confirmed tuberculosis at rates of 0.8 and 1.1 per 100 person-years, respectively (risk ratio, 0.72 [95% confidence interval, 0.40-1.31]; P = .28). In multivariate analysis, there were no significant differences in rates for confirmed or probable tuberculosis (P = .83), HIV progression and/or death (P = .09), or overall adverse events (P = .27), although drug discontinuation was slightly higher in the rifampin and pyrazinamide group (P = .01). Neither regimen appeared to lead to the development of drug-resistant tuberculosis. CONCLUSIONS: Our data suggest that for preventing tuberculosis in HIV-infected patients, a daily 2 month regimen of rifampin and pyrazinamide is similar in safety and efficacy to a daily 12-month regimen of isoniazid. This shorter regimen offers practical advantages to both patients and tuberculosis control programs. PMID- 10732935 TI - Sedation in the intensive care unit: a systematic review. AB - CONTEXT: Sedation has become an integral part of critical care practice in minimizing patient discomfort; however, sedatives have adverse effects and the potential to prolong mechanical ventilation, which may increase health care costs. OBJECTIVE: To determine which form of sedation is associated with optimal sedation, the shortest time to extubation, and length of intensive care unit (ICU) stay. DATA SOURCES: A key word search of MEDLINE, EMBASE, and the Cochrane Collaboration databases and hand searches of 6 anesthesiology journals from 1980 to June 1998. Experts and industry representatives were contacted, personal files were searched, and reference lists of relevant primary and review articles were reviewed. STUDY SELECTION: Studies included were randomized controlled trials enrolling adult patients receiving mechanical ventilation and requiring short term or long-term sedation. At least 2 sedative agents had to be compared and the quality of sedation, time to extubation, or length of ICU stay analyzed. DATA EXTRACTION: Data on population, intervention, outcome, and methodological quality were extracted in duplicate by 2 of 3 investigators using 8 validity criteria. DATA SYNTHESIS: Of 49 identified randomized controlled trials, 32 met our selection criteria; 20 studied short-term sedation and 14, long-term sedation. Of these, 20 compared propofol with midazolam. Most trials were not double-blind and did not report or standardize important cointerventions. Propofol provides at least as effective sedation as midazolam and results in a faster time to extubation, with an increased risk of hypotension and higher cost. Insufficient data exist to determine effect on length of stay in the ICU. Isoflurane demonstrated some advantages over midazolam, and ketamine had a more favorable hemodynamic profile than fentanyl in patients with head injuries. CONCLUSION: Considering the widespread use of sedation for critically ill patients, more large, high-quality, randomized controlled trials of the effectiveness of different agents for short-term and long-term sedation are warranted. PMID- 10732936 TI - Projected cost-effectiveness of pneumococcal conjugate vaccination of healthy infants and young children. AB - CONTEXT: Pneumococcal conjugate vaccine for infants has recently been found effective against meningitis, bacteremia, pneumonia, and otitis media. OBJECTIVE: To evaluate the projected health and economic impact of pneumococcal conjugate vaccination of healthy US infants and young children. DESIGN: Cost-effectiveness analysis based on data from the Northern California Kaiser Permanente randomized trial and other published and unpublished sources. SETTING AND PATIENTS: A hypothetical US birth cohort of 3.8 million infants. INTERVENTIONS: Hypothetical comparisons of routine vaccination of healthy infants, requiring 4 doses of pneumococcal conjugate vaccine (at 2, 4, 6, and 12-15 months), and catch-up vaccination of children aged 2 to 4.9 years requiring 1 dose, with children receiving no intervention. MAIN OUTCOME MEASURES: Cost per life-year saved and cost per episode of meningitis, bacteremia, pneumonia, and otitis media prevented. RESULTS: Vaccination of healthy infants would prevent more than 12000 cases of meningitis and bacteremia, 53000 cases of pneumonia, 1 million episodes of otitis media, and 116 deaths due to pneumococcal infection. Before accounting for vaccine costs, the vaccination program would save $342 million in medical and $415 million in work-loss and other costs from averted pneumococcal disease. Vaccination of healthy infants would result in net savings for society if the vaccine cost less than $46 per dose, and net savings for the health care payer if the vaccine cost less than $18 per dose. At the manufacturer's list price of $58 per dose, infant vaccination would cost society $80000 per life-year saved or $160 per otitis media episode prevented (other estimated costs would be $3200 per pneumonia case prevented, $15000 for bacteremia, and $280000 for meningitis). The cost-effectiveness of an additional program to administer 1 dose of vaccine to children aged 2 to 4.9 years would vary depending on the children's ages, relative risks of pneumococcal disease, and vaccine cost. CONCLUSIONS: Pneumococcal conjugate vaccination of healthy US infants has the potential to be cost-effective. To achieve cost savings, its cost would need to be lower than the manufacturer's list price. In addition to tangible costs, the vaccine should be appraised based on the less tangible value of preventing mortality and morbidity from pneumococcal disease. PMID- 10732937 TI - Glucosamine and chondroitin for treatment of osteoarthritis: a systematic quality assessment and meta-analysis. AB - CONTEXT: Glucosamine and chondroitin preparations are widely touted in the lay press as remedies for osteoarthritis (OA), but uncertainty about their efficacy exists among the medical community. OBJECTIVE: To evaluate benefit of glucosamine and chondroitin preparations for OA symptoms using meta-analysis combined with systematic quality assessment of clinical trials of these preparations in knee and/or hip OA. DATA SOURCES: We searched for human clinical trials in MEDLINE (1966 to June 1999) and the Cochrane Controlled Trials Register using the terms osteoarthritis, osteoarthrosis, degenerative arthritis, glucosamine, chondroitin, and glycosaminoglycans. We also manually searched review articles, manuscripts, and supplements from rheumatology and OA journals and sought unpublished data by contacting content experts, study authors, and manufacturers of glucosamine or chondroitin. STUDY SELECTION: Studies were included if they were published or unpublished double-blind, randomized, placebo-controlled trials of 4 or more weeks' duration that tested glucosamine or chondroitin for knee or hip OA and reported extractable data on the effect of treatment on symptoms. Fifteen of 37 studies were included in the analysis. DATA EXTRACTION: Reviewers performed data extraction and scored each trial using a quality assessment instrument. We computed an effect size from the intergroup difference in mean outcome values at trial end, divided by the SD of the outcome value in the placebo group (0.2, small effect; 0.5, moderate; 0.8, large), and applied a correction factor to reduce bias. We tested for trial heterogeneity and publication bias and stratified for trial quality and size. We pooled effect sizes using a random effects model. DATA SYNTHESIS: Quality scores ranged from 12.3% to 55.4% of the maximum, with a mean (SD) of 35.5% (12%). Only 1 study described adequate allocation concealment and 2 reported an intent-to-treat analysis. Most were supported or performed by a manufacturer. Funnel plots showed significant asymmetry (P< or =.01) compatible with publication bias. Tests for heterogeneity were nonsignificant after removing 1 outlier trial. The aggregated effect sizes were 0.44 (95% confidence interval [CI], 0.24-0.64) for glucosamine and 0.78 (95% CI, 0.60-0.95) for chondroitin, but they were diminished when only high-quality or large trials were considered. The effect sizes were relatively consistent for pain and functional outcomes. CONCLUSIONS: Trials of glucosamine and chondroitin preparations for OA symptoms demonstrate moderate to large effects, but quality issues and likely publication bias suggest that these effects are exaggerated. Nevertheless, some degree of efficacy appears probable for these preparations. PMID- 10732938 TI - Time to reevaluate risk stratification guidelines for medically supervised exercise training in patients with coronary artery disease. PMID- 10732939 TI - Multiple potential risks for stroke. PMID- 10732940 TI - Advance directives for nursing home residents: achieving compassionate, competent, cost-effective care. PMID- 10732941 TI - Glucosamine and chondroitin for treating symptoms of osteoarthritis: evidence is widely touted but incomplete. PMID- 10732942 TI - JAMA Patient Page: advance directives. PMID- 10732943 TI - Analysis of underivatized amphetamines and related phenethylamines with high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. AB - Amphetamine, methamphetamine, illicit designer phenethylamines (MDA, MDEA, MDMA, MBDB, and BDMPEA), and other phenethylamines (benzyl-1-phenylethylamine, cathinone, ephedrine, fenfluramine, norfenfluramine, phentermine, 1 phenylethylamine, phenylpropanolamine, and propylhexedrine) were extracted from serum using a solid-phase extraction procedure. The extracts were examined with high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS). The drugs were separated on ODS column in acetonitrile/50 mM ammonium formate buffer (pH 3.0) (25:75) as a mobile phase. Full-scan mass spectra of drugs examined by means of APCI with collision-induced dissociation showed protonated molecular ions and fragments typical for particular drugs. LC-APCI-MS allowed an unequivocal differentiation of all drugs involved. The quantitation was performed using selected ion monitoring of protonated molecular ions and fragments of drugs involved and their deuterated analogues. The limits of detection ranged from 1 to 5 microg/L serum, and the recoveries ranged from 58 to 96%. A linear response was observed for all drugs in the range from 5 to 500 microg/L. The method was applied for routine determination of amphetamine, MDMA, MDA, and MDEA in one run. Solid-phase extraction used assured simultaneous isolation of various groups of basic drugs of forensic interest (opiates, cocaines, phenethylamines, and benzodiazepines) from biofluids. PMID- 10732944 TI - Fatal poisoning with a new phenylethylamine: 4-methylthioamphetamine (4-MTA). AB - There has been much publicity in the United Kingdom regarding a new phenylethylamine-based compound called 4-methylthioamphetamine (4-MTA), also known as para-methylthioamphetamine (p-MTA), MTA or "Flatliner". Chemically, 4 MTA is an amphetamine derivative and is a non-neurotoxic potent serotonin releasing agent and reversible inhibitor of rat monoamine oxidase-A. Analysis of postmortem blood and urine specimens in a case implicating 3,4 methylenedioxymethamphetamine revealed the presence of 4-MTA at a concentration of 4.6 mg/L in femoral blood and 87.2 mg/l in the urine. The concentration of 4 MTA in perimortem blood was measured at 4.2 mg/L. This is the first reported case of death involving 4-MTA in the United Kingdom and the first case known to involve 4-MTA only. PMID- 10732945 TI - Fatal aluminum phosphide poisoning. AB - A 39-year-old man committed suicide by ingestion of aluminum phosphide, a potent mole pesticide, which was available at the victim's workplace. The judicial authority ordered an autopsy, which ruled out any other cause of death. The victim was discovered 10 days after the ingestion of the pesticide. When aluminum phosphide comes into contact with humidity, it releases large quantities of hydrogen phosphine (PH3), a very toxic gas. Macroscopic examination during the autopsy revealed a very important asphyxia syndrome with major visceral congestion. Blood, urine, liver, kidney, adrenal, and heart samples were analyzed. Phosphine gas was absent in the blood and urine but present in the brain (94 mL/g), the liver (24 mL/g), and the kidneys (41 mL/g). High levels of phosphorus were found in the blood (76.3 mg/L) and liver (8.22 mg/g). Aluminum concentrations were very high in the blood (1.54 mg/L), brain (36 microg/g), and liver (75 microg/g) compared to the usual published values. Microscopic examination revealed congestion of all the organs studied and obvious asphyxia lesions in the pulmonary parenchyma. All these results confirmed a diagnosis of poisoning by aluminum phosphide. This report points out that this type of poisoning is rare and that hydrogen phosphine is very toxic. The phosphorus and aluminum concentrations observed and their distribution in the different viscera are discussed in relation to data in the literature. PMID- 10732946 TI - Solid-phase microextraction in the determination of methadone in human saliva by gas chromatography-mass spectrometry. AB - Solid-phase microextraction (SPME) with a 100-microm polydimethylsiloxane film fiber was applied to the determination of methadone and 2-ethylidine-3,3 diphenylpyrrolidine (EDDP) by GC-MS in human saliva and compared with liquid liquid extraction. A shorter extraction time of 30 min with the fiber was obtained, speeding up the total analysis time. Linearity was found for SPME from 0.05 to 2.0 microg/mL (r = 0.9976 for methadone; r = 0.9988 for EDDP) with precision between 0.7 and 4.3% for saliva spiked with 0.2 and 1.5 microg/mL of methadone and EDDP. The limit of detection using SPME was 0.04 microg/mL for methadone and 0.008 microg/mL for EDDP. Analytical recoveries of SPME and liquid liquid extraction ranged from 98.8 to 103.6%. The use of deuterated internal standard by both methods have yielded comparable results. Thus, the SPME method is highly accurate, precise, and useful for determination of methadone and EDDP in saliva. PMID- 10732947 TI - SPEC disc solid-phase extraction for rapid broad-spectrum drug screening in urine. AB - Broad-spectrum drug screening requires that all relevant substances be isolated, detected, and identified, regardless of their structure and/or polarity. To this end, systematic solid-phase extraction (SPE) approaches for drug isolation from biological fluids are required. Because speed and cost effectiveness are key issues in analytical toxicology, we have evaluated a disc-format extraction device for this purpose and compared the latter with an existing packed-bed column-format method. The discs were SPEC.PLUS.C18AR/MP3 cartridges with 10-mL solvent reservoirs, providing hydrophobic and cation exchange interactions. Blank human urine was spiked at 2 microg/mL with a selection of acidic, neutral, and basic drugs representing a variety of relevant drug classes. Urine specimens (2 mL) were diluted with 2 mL 0.1 M phosphate buffer (pH 5.0) and then applied to the preconditioned disc. Washing was done with 1 mL water. Acidic and neutral drugs were eluted with 1 mL ethyl acetate/acetone (1:1), and basic drugs were eluted with 1 mL ammoniated ethyl acetate. The eluates were collected separately, evaporated down to about 0.1 mL, and analyzed by gas chromatography-flame ionization detection to check cleanliness, recoveries, and reproducibilities. The discs showed good extraction properties for all drugs and were easy to handle. Recoveries were 75-100% with coefficients of variation of around 5%. The resulting eluates showed only a few matrix interferences. As compared to our standard SPE method with packed-bed columns, the disc procedure allowed reductions in elution volumes and total processing time of approximately 60-65%. PMID- 10732948 TI - Evaluation of testosterone/epitestosterone ratio influential factors as determined in doping analysis. AB - The ratio of the concentration of testosterone glucuronide to the concentration of epitestosterone glucuronide (T/E ratio) as determined in urine is the most frequently used method to prove testosterone abuse by athletes. A T/E ratio higher than 6 has been considered as proof of abuse in the past; however, cases of naturally occurring higher T/E ratios have been described. Since the introduction of the T/E ratio in doping analysis, the parameters that may or may not influence the T/E ratio, possibly leading to false-positive results, have been debated. To achieve more insight on the influencing circumstances, an overview is given to obtain an objective view on the merits of the urinary T/E ratio. Relevant analytical aspects of the T/E ratio, potential parameters of endogenous and exogenous origins, as well as some alternative methods to determine testosterone abuse, such as the urinary testosterone/luteinizing hormone ratio, gas chromatography-combustion-isotope-ratio mass spectrometry, hair analysis, and high-performance liquid chromatography-mass spectrometry, are discussed. PMID- 10732949 TI - Quantitative determination of paraquat in a fatal intoxication by HPLC-DAD following chemical reduction with sodium borohydride. AB - A fatality due to a massive ingestion of paraquat is presented. Screening by enzyme-multiplied immunoassay of postmortem blood and urine disclosed the presence of tricyclic antidepressants (in urine only), benzodiazepines, cotinine, and caffeine. Further analysis of blood, urine, and stomach contents with thin layer chromatography, high-performance liquid chromatography (HPLC), and gas chromatography confirmed the results found in the preliminary routine screening. It also revealed the presence of paraquat in blood, urine, and stomach contents, of diethyl parathion in urine and stomach contents, and of mevinphos in blood and stomach contents. Quantitation of paraquat was performed using HPLC with diode array detection. Sample preparation involved a protein-precipitation step using trichloroacetic acid (necessary only for blood and tissue homogenate), followed by a chemical reduction with sodium borohydride of the fully ionized paraquat to a diene, which is amenable to solvent extraction. Quantitative results were obtained for all postmortem matrices available: blood, 5.05 mg/L; urine, 6.00 mg/l; stomach contents, 17.2 g/L; liver, 4.86 mg/kg; and kidney, 80.6 mg/kg. The paraquat distribution in this case is compared with analytical findings reported in the literature. As would be expected, concentrations found in fatal paraquat intoxications display large differences. The data presented illustrate the outspoken lethal nature of the herbicide paraquat and the ongoing appearance of this compound in deadly accidental and suicidal poisonings. PMID- 10732950 TI - GCD quantitation of opiates as propionyl derivatives in blood. AB - We describe a method using a gas chromatograph with electron ionization detection (GCD) for the simultaneous determination of morphine, codeine, 6 monoacetylmorphine, ethylmorphine, and dihydrocodeine in blood. The method employs propionic anhydride in the presence of triethylamine to propionylate free hydroxyl groups of the opiates in blood. The quantitation is achieved by using GCD with selected ion monitoring of the two most characteristic ions for each analyte. The quantitation limit was 0.01 mg/L and the linearity was 0.01-10 mg/L for dihydrocodeine, ethylmorphine, and 6-monoacetylmorphine. For the other investigated opiates, the quantitation limit was 0.025 mg/L and linearity was 0.025-10 mg/L. The intraday relative standard deviation (RSD) varied from 7.2 to 10% at the 0.5 mg/L level, and the day-to-day RSDs varied from 7.5 to 11% at the 0.85 mg/L level. PMID- 10732951 TI - The determination of delta9-tetrahydrocannabinol and 11-nor-9-carboxy-delta9 tetrahydrocannabinol in whole blood using solvent extraction combined with polar solid-phase extraction. AB - A method for the determination of delta9-tetrahydrocannabinol (THC) and 11-nor-9 carboxy-delta9-tetrahydrocannabinol (THCCOOH) in whole blood using conventional solvent extraction followed by a cleanup using polar solid-phase cartridges is described. The method uses 0.5 mL of blood, and detection is by benchtop gas chromatography-mass selective detection using selected ion monitoring. The limit of detection is better than 1 ng/mL, and extraction efficiencies are greater than 80% for THC and 70% for THCCOOH. The method is robust and highly reproducible. PMID- 10732952 TI - Urinary excretion of codeine and morphine following the administration of codeine containing cold syrup. AB - The concentrations of codeine ([C]) and morphine ([M]) excreted in urine obtained from nine subjects at various time intervals after the ingestion of 10-40 mg of both simple and compound codeine doses three times per day for three days were determined by gas chromatography-mass spectrometry analysis. Four distinct [C]/[M] distribution patterns (phases) were observed: a, [C] > [M] from ingestion of first dose to 78-108 h after ingestion of the first dose (18-48 h after the ingestion of the last dose); b, [C] approximately [M] 78-90 h after ingestion of the first dose (18-30 h after ingestion of the last dose); c, [C] < [M] 78-102 h after ingestion of first dose (18-42 h after ingestion of the last dose); and d, [C] <0.05 microg/mL and [M] > 0.05 microg/mL 84-96 h after ingestion of the first dose (24-36 h after ingestion of the last dose with [M] ranging from 0.10 to 0.30 microg/mL) toward the terminal phase. No single individual exhibited all four [C]/[M] distribution characteristics during the entire excretion period and intervals monitored in this study. These data are of reference value for differentiating codeine and morphine/heroin ingestion. PMID- 10732953 TI - Interpretation and enantiomer analysis of methamphetamine abusers' urine and illegally brewed methamphetamine crystals. AB - This study deals with the high-performance liquid chromatographic identification of methamphetamine (MAMP) and amphetamine (AMP) enantiomers (d- and l-forms) in five illicit MAMP crystals and in urine specimens from 30 Japanese MAMP abusers. The analysis revealed that two of the types of crystals have a different optical purity ratio (l/d) and the other three have a single crystal of either the d- or l-enantiomer. The l/d ratios of two types of crystals were 0.04 and 49.4, and no racemic form (l/d = 1.00) was found. The urinary analysis showed that nonmetabolized MAMP and its demethylated metabolite, AMP, were present in urine specimens of all addicts. The stereoisomeric profiles in urine can be classified roughly into five groups according to the detected amount and the l/d ratio of MAMP and AMP enantiomers collected at one time point. In the first group, only d MAMP and d-AMP were detected (in 16 cases). In the second group, only l-MAMP and l-AMP were detected (in one case). In the third group, the amount of the l enantiomer detected, for both MAMP and AMP, was less than that of the d enantiomer, and the l/d ratio was between 0.004 and 0.54 for MAMP and between 0.01 and 0.07 for AMP (in five cases). In the fourth group, the l-enantiomer of MAMP and AMP was found to be more abundant than the d-enantiomer, and the l/d ratio was between 2.63 and 30.11 for MAMP and between 1.23 and 31.30 for AMP (in four cases). In the fifth group, the amount of l-MAMP detected was greater than that of d-MAMP, and less l-AMP than d-AMP was detected. The l/d ratios were between 1.13 and 8.82 for MAMP and between 0.17 and 0.82 for AMP (in four cases). These results might be suitable for identification and the forensic toxicological investigation of AMP analogues. PMID- 10732954 TI - Determination of beta2-agonists in bovine urine: comparison of two extraction/clean-up procedures for high-resolution gas chromatography-mass spectrometry analysis. AB - Two extraction/clean-up analytical procedures were investigated and compared regarding their recovery and matrix-purification efficiency for screening beta2 agonist residues in fortified bovine urine by high-resolution gas chromatography mass spectrometry (GC-MS). The first procedure, based on an analytical method originally developed for detecting anabolic steroids, consists of the employment of the nonionic resin, Amberlite XAD-2, a styrene-divinylbenzene copolymer for solid-phase extraction (SPE), followed by liquid-liquid extraction with diethyl ether. The second focuses on the use of a mixed SPE cartridge (reversed-phase and ion-exchange sorbent, Bond Elut Certify). In both cases, the trimethylsilylated derivatives were analyzed by GC-MS with an ion-trap detector. Clenbuterol, salbutamol, and terbutaline were used to spike urine samples during the comparison experimental phase. Afterwards, tulobuterol, mabuterol, mapenterol, cimbuterol, and brombuterol were included in the evaluation of the second procedure (the Bond Elut Certify procedure). At this stage, the detection was accomplished by GC-MS (quadrupole mass analyzer) with selective ion monitoring acquisition. The isotopic dilution method with the hexadeuterated analogues of clenbuterol and salbutamol was applied to prepare calibration curves and calculate recovery percentages. With XAD-2 resin, terbutaline and salbutamol (resorcinol and phenol-type beta2-agonists, respectively) could not be detected at 20 ng/mL or at 40 ng/mL. In spite of clenbuterol having been detected at 20 ng/mL, the results obtained were not reproducible. The use of the reversed-phase and ion-exchange sorbent Bond Elut Certify allowed multiresidue detection and showed several advantages for the screening of clenbuterol such as higher recoveries, cleaner final extracts, reduced sample preparation time, less labor intensive, and easier solvent consumption and disposal. Recoveries over 88% (concentrations ranging from 0.5 to 10 ppb) and limits of detection equal to 0.5 ppb were met for all the beta2-agonists studied with the last method. PMID- 10732955 TI - Redistribution of methamphetamine in the early postmortem period. PMID- 10732956 TI - The frequency of breast cancer in industrialized societies continues to escalate world-wide. PMID- 10732957 TI - The evaluation of high risk and pre-invasive breast lesions and the decision process for follow up and surgical intervention. AB - Atypical epithelial hyperplasia, lobular carcinoma in situ (lobular neoplasia), radial scar, and ductal carcinoma in situ are considered high-risk lesions that predispose toward the future development of non-invasive or invasive breast cancer. Generally, those women with atypical epithelial hyperplasia, radial scar, or lobular carcinoma in situ can be managed conservatively by close surveillance. The minority of women may consider prophylactic mastectomy. Ductal carcinoma in situ can usually be managed by lumpectomy with or without radiation, with some patients requiring mastectomy due to extensive disease. PMID- 10732958 TI - The management of ductal and lobular breast cancer. PMID- 10732959 TI - Assessing margin status. AB - As little time ago as 1991 the NIH Consensus conference could not agree on the need for negative margins. Today, negative margin status has become a prerequisite for BCT recognizing that positive margins impact negatively on local recurrence rates. The science of margin evaluation is fast becoming recognized to play a key role in providing patients with the opportunity for breast conservation therapy as well as the best possible cosmetic result. Preoperative factors that predict a greater likelihood of failure to obtain margins such as larger tumor size and positive lymph nodes are fixed and can only be dealt with by taking larger biopsies. RODEO-MRI can preoperatively predict probability of success or failure and can actual better define tumor dimensions and extent and help plan excisions. Use of intraoperative US may be a future tool used to facilitate the excision of non-palpable and possibly palpable tumors. Intraoperative pathological assessment should not be performed by frozen section but consideration given to cytological assessment so as to allow feedback to the surgeon intraoperatively as to which margin needs more attention. Finally, using all the above methods of obtaining negative margins, the surgeon may have the ability to impact the outcome of breast cancer surgery and recurrence. PMID- 10732960 TI - State-of-the-art approaches to sentinel node biopsy for breast cancer: study design, patient selection, technique, and quality control at Memorial Sloan Kettering Cancer Center. AB - Sentinel lymph node (SLN) biopsy has the potential to become a standard operation for most patients with operable breast cancer, but raises a number of issues for the surgeon and institution wishing to start a SLN program. We began to perform SLN biopsy for breast cancer at Memorial Sloan-Kettering Cancer Center in September of 1996. Based on a pilot study of 60 patients, detailed analyses of our first 500 and 1000 cases, and a cumulative experience of 1500 procedures, we report the lessons learned in starting a SLN program, emphasizing study design, case selection, technique and quality control. All patients had clinical T1-2N0 breast cancers and isotope plus blue dye mapping. The combination of blue dye and isotope localization was superior to either method alone, unfiltered technetium sulfur colloid was superior to filtered, and optimum isotope localization was achieved with a low-volume, low-dose intradermal injection. In our first 1000 cases, 14% of SLN were found by isotope alone, and 8% by dye alone. 10% of positive SLN were found by isotope alone, and 11% by dye alone. Failed and false negative SLN procedures were less frequent as experience increased. PMID- 10732961 TI - Adjuvant systemic management of early stage carcinoma of the breast. AB - In the last three decades, there has been a gradual, though significant change in the treatment of early stage breast cancer. For almost a century, physicians advocated an "anatomical view" of the dissemination of this disease, which justified a more radical and mutilating treatment strategy. Finally in the mid 1970s, results from large randomized trials began to show that either mastectomy or lumpectomy with radiation therapy were appropriate treatment for women with early stage disease. These results suggested that breast cancer can actually be a systemic disease ("biological view") even in early stages. This hypothesis was confirmed when large randomized clinical trials demonstrated the effectiveness of adjuvant systemic therapy in controlling micrometastatic disease in women with node-positive and node negative disease. As we approach the end of this century, most patients with early stage disease will be offered some form of adjuvant systemic therapy, before or after local treatment with surgery, with or without local radiation therapy. There has been a lot of interest on the proper sequence of the therapeutic modalities, in particular with the recent publication of larger randomized trials of primary systemic therapy. This specific topic is discussed elsewhere in this issue by Singletary. PMID- 10732962 TI - The evolving role of specific estrogen receptor modulators (SERMs) AB - Estrogens are the most effective therapy for women with postmenopausal problems. However, relatively few women use estrogen and then often for a limited time because of the fear of its carcinogenic effects on the uterus and breast; in addition, estrogen is not advised for women who have had breast cancer. Selective estrogen receptor modulators (SERMs) are agents with antagonist action on the uterus and breast and agonist action on the bones, cardiovascular system, and brain. Unlike estrogens, however, existing SERMs do not help alleviate the vasomotor and urogenital problems associated with menopause. A comprehensive review of the literature published from January 1995 to June 1999 was conducted. Reports were identified using Medline and Cancer Lit. The effect of menopausal problems on the health of women and the socioeconomic effects of menopause are discussed. All currently available and investigational SERMs are reviewed and discussed, including their mechanism of action, metabolism, dose scheduling, antitumor activity, and potential role in maintaining the health of menopausal women and in preventing breast cancer. PMID- 10732963 TI - Bioaccumulation and localization of exogenous cadmium in a teleost by electron microscopy (TEM) and its specific quantitation by electron probe X-ray microanalysis (EPMA). AB - A cadmium bioconcentration study was carried out in a fresh water teleost, Colisa fasciatus, to study the bioaccumulation kinetics and fate of exogenous cadmium (Cd) in biological tissues. Study shows that on exposure of the fish to a sublethal concentration of cadmium in test water, Cd uptake results in its bioconcentration in gills, liver and muscle tissues. To explore whether the accumulated Cd reaches the membranes or inside the cells, transmission electron microscopy (TEM) of the thin sections of tissues was done after histochemical localization of Cd in cells by modified SST method. TEM studies of sections of gills, liver and muscle tissues showed the deposits of exogenous Cd (visualized as dense clouds) in biological cells. This suggests the presence of free or loosely bound Cd on the membranes and inside the cells, which in the presence of Na2S is converted into insoluble metal sulfides. Electron probe X-ray microanalysis (EPMA) studies confirmed the presence of Cd on the membrane surface as well as inside the cells of bioindicator organs suggesting involvement of membrane transport of exogenous Cd inside the cells and its deposition as loosely bound insoluble metal complexes. PMID- 10732964 TI - Substituted 3-amino and/or 3-aminomethyl-3,4-dihydro-2H-1-benzopyrans: synthesis and biological activity. AB - A series of new 3-amino, 3-aminomethyl-5-alkoxy-3,4-dihydro-2H-1-benzopyran and 5'-alkoxy-3',4'-dihydrospiro-[piperazine-2.3'(2'H)-benzopyran] derivatives was prepared and evaluated for affinity at 5-HT1A, 5-HT2A and D2 receptors. Two of the compounds (1f and 2b) can be considered as potent and selective 5-HT2A ligands. One compound (1g) demonstrated high affinity for 5-HT1A and D2 receptor binding sites and one compound (1d) proved to be a mixed 5-HT1A/5-HT2A ligand. PMID- 10732965 TI - Acetylcholinesterase inhibitors for potential use in Alzheimer's disease: molecular modeling, synthesis and kinetic evaluation of 11H-indeno-[1,2-b] quinolin-10-ylamine derivatives. AB - Continuing our work on tetracyclic tacrine analogues, we synthesized a series of acetylcholinesterase (AChE) inhibitors of 11H-indeno-[1,2-b]-quinolin-10-ylaminic structure. Selected substituents were placed in synthetically accessible positions of the tetracyclic nucleus, in order to explore the structure-activity relationships (SAR) and the mode of action of this class of anticholinesterases. A molecular modeling investigation of the binding interaction of the lead compound (1a) with the AChE active site was performed, from which it resulted that, despite the rather wide and rigid structure of 1a, there may still be the possibility to introduce some small substituent in some positions of the tetracycle. However, from the examination of the experimental IC50 values, it derived that the indenoquinoline nucleus probably represents the maximum allowable molecular size for rigid compounds binding to AChE. In fact, only a fluorine atom in position 2 maintains the AChE inhibitory potency of the parent compound, and, actually, increases the AChE-selectivity with respect to the butyrylcholinesterase inhibition. By studying the kinetics of AChE inhibition for two representative compounds of the series, it resulted that the lead compound (1a) shows an inhibition of mixed type, binding to both the active and the peripheral sites, while the more sterically hindered analogue 2n seems to interact only at the external binding site of the enzyme. This finding seems particularly important in the context of Alzheimer's disease research in the light of recent observations showing that peripheral AChE inhibitors might decrease the aggregating effects of the enzyme on the beta-amyloid peptide (betaA). PMID- 10732966 TI - A novel biotinylated suicide inhibitor for directed molecular evolution of lipolytic enzymes. AB - A bifunctional activity label (8) for directed molecular evolution of lipolytic enzymes has been designed and synthesized. The structure is composed of a 4 nitrophenyl activated phosphonate, that is, a suicide substrate of lipases/esterases, connected to a biotin moiety through a spacer containing a disulfide bridge. The phosphonate (3) was prepared by Michaelis-Arbuzov reaction of trimethylsilyl-protected 11-bromoundecanol (2) with triethyl phosphite. The deprotected omega-hydroxyalkylphosphonate (4) was transformed into an active N hydroxysuccinimide carbonate (5) followed by 4-nitrophenyl activation of the phosphonate using standard procedures. The biotinylated phosphonate inhibitor (8) was then synthesised by coupling the phosphonate inhibitor (6) to the epsilon amino-caproic acid and cystamine containing biotinyl spacer (7). The function of all relevant groups of the final activity label (8) (biotin-label, cleavable disulfide bridge, phosphonate-inhibitor) have been successfully tested with the commercial lipase Lipolase (Novo Nordisk). Hence, a tool for directed molecular evolution of lipolytic enzymes has been developed. PMID- 10732967 TI - Synthesis, characterization and cytotoxicity of new platinum(IV) axial carboxylate complexes: crystal structure of potential antitumor agent [PtIV(trans 1R,2R-diaminocyclohexane)trans(acetate)2Cl2]. AB - A series of new platinum(IV) complexes of the type [PtIV(DACH)trans(L)2Cl2] (where DACH = trans-1R,2R-diaminocyclohexane, and L = acetate, propionate, butyrate, valerate, hexanoate, or heptanoate) bearing the carboxylate groups in the axial positions have been synthesized and characterized by elemental analysis, IR, and 195Pt NMR spectroscopy. The crystal structure of the analogue [PtIV(DACH)trans(acetate)2Cl2] was determined by single crystal X-ray diffraction method. There were two crystallographically independent molecules, both of which lie on crystallographic two-fold axes. The bond lengths and bond angles of both the molecules were the same within the experimental error. The compound crystallizes in the monoclinic space group C2, with a = 11.180(2) A, b = 14.736(3) A, c = 10.644(2) A, beta = 112.38(3) degrees, Z = 4 and R = 0.0336, based upon a total of 1648 collected reflections. In this complex, the platinum had a slightly distorted octahedron geometry owing to the presence of a geometrically strained five-member ring. The two adjacent corners of the platinum plane were occupied by the two amino nitrogens of DACH, whereas the other two equatorial positions were occupied by two chloride ions. The remaining two axial positions were occupied by the oxygens of acetate ligands. The DACH ring was in a chair configuration. An intricate network of intermolecular hydrogen bonds held the crystal lattice together. These analogues were evaluated in vitro and demonstrated cytotoxic activity against the human ovarian 2008 tumor cell line (IC50 = 0.001-0.06 microM). Structure-activity study revealed that activity was highest for the analogue where L = butyrate. PMID- 10732968 TI - Synthesis of a netropsin conjugate of a water-soluble epi-quinocarcin analogue: the importance of stereochemistry at nitrogen. AB - The efficient synthesis of a water-soluble C11a-epi-analogue (6b) of quinocarcin is described. This substance, and a netropsin amide conjugate (8) lack the capacity to inflict oxidative damage on DNA due to the stereoelectronic geometry of their oxazolidine nitrogen atoms. The capacity of these substances to alkylate DNA through the generation of an iminium species has been examined. Both compounds were found to be unreactive as DNA alkylating agents. The results of this study are discussed in the context of previous proposals on the mode of action of this family of antitumor alkaloids. PMID- 10732969 TI - Synthesis of new 1,2,3-benzotriazin-4-one-arylpiperazine derivatives as 5-HT1A serotonin receptor ligands. AB - A series of novel 1,2,3-benzotriazin-4-one derivatives was prepared and evaluated as ligands for 5-HT receptors. Radioligand binding assays proved that the majority of the novel compounds behaved as good to excellent ligands at the 5 HT1A receptor, some of which were selective with respect 5-HT2A and 5-HT2C receptors. Six analogues (1a, 2a, 2b, 2c, 2e and 2i) were selected and further evaluated for their binding affinities on D1, D2 dopaminergic and alpha1-, alpha2 adrenergic receptors. A o-OCH3 derivative (2e) bound at 5-HT1A sites with subnanomolar affinity (IC50 = 0.059 nM) and shows high selectivity over all considered receptors and may offer a new lead for the development of therapeutically efficacious agents. PMID- 10732970 TI - Peptide T-araC conjugates: solid-phase synthesis and biological activity of N4 (acylpeptidyl)-araC. AB - Due to the capability of peptidyl derivatives of araC to behave as prodrugs of this antimetabolite, and because of the well known biological properties of peptide T and its analogues (in particular that of targeting CD4+ cells), new peptide T-araC conjugates were prepared and tested in vitro for antiproliferative activity. The aim was that of specifically delivering the antitumor drug to CD4+ cells. N4-(Acylpeptidyl)-derivatives of araC were synthesized by a new general approach involving solid-phase synthesis, which allows mild conditions, avoids the usually required protection of the glycoside portion of nucleosides and affords high yields of the final products. After the demonstration that peptide T araC conjugates were able to activate chemotaxis by binding CD4 receptor on monocyte membranes, the antiproliferative activity was evaluated against a panel of leukemia lymphoma and carcinoma cell lines derived from human tumors, three CD4+ cell lines included. Title compounds resulted effective as antiproliferative agents at concentrations 4- to 10-fold higher than those of araC alone, did not preferentially inhibit CD4+ cells and proved stable not only in cell culture medium containing 20% FCS, but also in human plasma. All this suggests their potential utility in vivo. PMID- 10732971 TI - Reversible modulation of human factor Xa activity with phosphonate esters: media effects. AB - Enantiomers of 4-nitrophenyl 4-X-phenacyl methylphosphonate esters (X = H, PMN; CH3 and CH3O) inactivate human factor Xa with rate constants 8-86 M(-1)s(-1) at pH 6.75 in 0.025 M Hepes buffer, 0.15 M NaCl and 2 mM CaCl2 at 7.0+/-0.1 degrees C. The stereoselectivity of the inactivation of factor Xa is 2-10 and favors the levorotatory enantiomers. The pH-dependence of inactivation of factor Xa by (-) PMN is sigmoidal and consistent with the participation of a catalytic residue with a pKa of 6.2+/-0.1. Factor Xa reactivates from its phosphonyl adducts through a self-catalyzed intramolecular reaction, which is much influenced by the presence of phospholipids. The rate of reactivation in the absence of phospholipids is not pH dependent at pH <9, but it increases very much at pH >9. In the presence of phospholipids, the pH dependence of the rate constant for reactivation is sigmoidal in the pH 6.5-10.3 range and levels off at pH >9 indicating that the enzyme catalyzes its reactivation. The kinetic pKa for the recovery of factor Xa from its adducts with the PMNs is in the range of 6.7-8.1 and is consistent with the participation of the catalytic His57 in the reactivation process. PMID- 10732972 TI - Novel allocolchicinoids with an eight membered B-ring: design, synthesis and inhibition of tubulin assembly. AB - Several B-ring variations of O-methyl androbiphenyline (8), newly accessible from (-)-(M,7S)-colchicine via photooxygenation and subsequent endoperoxide transformation, were synthesized and evaluated for their inhibitory effects on tubulin assembly in vitro. The amino-allocolchicinoid (9), a key compound in this study, was transformed to the highly potent ketone 10 and by oxidation with H2O2/Na2WO4 to a mixture of syn/anti-oximes, like 11 and 12. These could easily be transformed to hitherto unknown allocolchicinoids 13 and 14 with an eight membered B-ring lactam obtained via a Beckmann rearrangement. Surprisingly both do not notably affect tubulin assembly, despite obvious structural similarities with active analogues of the thiocolchicine- and azasteganacin-series. PMID- 10732973 TI - Benzophenone boronic acid photoaffinity labeling of subtilisin CMMs to probe altered specificity. AB - A transition state analogue inhibitor, boronic acid benzophenone (BBP) photoprobe, was used to study the differences in the topology of the S1 pocket of chemically modified mutant enzymes (CMMs). The BBP proved to be an effective competitive inhibitor and a revealing active site directed photoprobe of the CMMs of the serine protease subtilisin Bacillus lentus (SBL) which were chemically modified with the hydrophobic, negatively charged and positively charged moieties at the S1 pocket S166C residue. As expected, in all cases BBP bound best to WT SBL. BBP binding to S166C-SCH2C6H5 and S166C-CH2-c-C6H11, with their large hydrophobic side chains, was reduced by 86-fold and 9-fold, respectively, compared to WT. Relative to WT, BBP binding to the charged CMMs, S166C-S CH2CH2SO3- or S166C-S-CH2CH2NH3+, was reduced 170-fold and 4-fold respectively. Photolysis of the WT-SBL-BBP enzyme inhibitor (EI) complex, inactivated the enzyme and effected the formation of a covalent crosslink between WT and BBP. The crosslink was identified at Gly127 by peptide mapping analysis and Edman sequencing. Gly127 is located in the S1 hydrophobic pocket of SBL and its modification thus established binding of the benzophenone moiety in S1. Photolysis of the EI complex of S166C-SCH2C6H5, S166C-S-CH2CH2SO3-, or S166C-S CH2CH2NH3+ and BBP under the same conditions did not inactivate these enzymes, nor effect the formation of a crosslink. These results corroborated the kinetic evidence that the active site topology of these CMMs is dramatically altered from that of WT. In contrast, while photolysis of the S166C-CH2-c-C6H11-BBP EI complex only inactivated 50% of the enzyme after 12 h, it still effected the formation of a covalent crosslink between the CMM and BBP, again at Gly127. However, this photolytic reaction was less efficient than with WT, demonstrating that the S1 pocket of S166C-CH2-c-C6H11 is significantly restricted compared to WT, but not as completely as for the other CMMs. PMID- 10732974 TI - Efficient syntheses, human and yeast farnesyl-protein transferase inhibitory activities of chaetomellic acids and analogues. AB - Chaetomellic acids are a class of alkyl dicarboxylic acids that were isolated from Chaetomella acutiseta. They are potent and highly specific farnesyl pyrophosphate (FPP) mimic inhibitors of Ras farnesyl-protein transferase. We have previously described the first biogenetic type aldol condensation-based total synthesis of chaetomellic acid A. Modification of the later steps of that synthesis resulted in the efficient syntheses of chaetomellic acids A and B in three steps with 75-80% overall yield. In this report, details of the original total syntheses of chaetomellic acids A, B and C, the new syntheses of acids A and B and structure-activity relationship of these compounds against various prenyl transferases including human and yeast FPTase and bovine and yeast GGPTase I are described. Chaetomellic acids are differentially active against human and yeast FPTase. Chaetomellic acid A inhibited human and yeast FPTase activity with IC50 values of 55 nM and 225 microM, respectively. In contrast, chaetomellic acid C showed only a 10-fold differential in inhibitory activities against human versus yeast enzymes. In keeping with molecular modeling-based predictions, the compounds with shorter alkyl side chains (C-8) were completely inactive against FPTase. PMID- 10732975 TI - 3-Aryl-2-carbomethoxybicyclo[3.2.1]oct-2-enes inhibit WIN 35,428 binding potently and selectively at the dopamine transporter. AB - The search for medications for cocaine abuse has focused upon the design of potential cocaine antagonists or cocaine substitutes which interact at the dopamine transporter of mammalian systems. This manuscript describes the synthesis and biological evaluation of 8-substituted 2-carbomethoxy-3 arylbicyclo[3.2.1]oct-2-enes. These compounds prove potent and selective inhibitors of the dopamine transporter. Their selectivity results primarily from a reduced inhibitory potency toward the serotonin transporter. This work supports the notion that the orientation of the 3-aryl ring in the bicyclo[3.2.1]octane system affects the interaction of these molecules with the serotonin transporter far more markedly than it affects the interaction with the dopamine transporter. PMID- 10732976 TI - Syntheses of R and S isomers of AF-DX 384, a selective antagonist of muscarinic M2 receptors. AB - Enantiomers of 5,11-dihydro-11-[2-[2-[(N,N-dipropylaminomethyl)piperidin-1- yl]ethylamino]-carbonyl]-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one (AF-DX 384) 1, have been synthesized from (S)-(+) and (R)-(-)-2-[N,N dipropylaminomethyl]piperidine 4. The enantiomeric excess of 1 has been determined by capillary electrophoresis by using the alpha-highly sulphated cyclodextrin (alpha-HSCD) as chiral selector within the running electrolyte. (S) (+)-(4) was prepared from (S)-(-)-pipecolic acid in a 4-step procedure (overall yield: 30%, ee: 99%) and (R)-(-)-AF-DX 384 from (R)-(+)-pipecolic acid. The (R)-( ) isomer exhibited in vitro a 23-fold higher affinity than its enantiomer (S)-(+) towards muscarinic receptors of subtype 2. PMID- 10732977 TI - Structure-activity relationships of acyloxyamidine cytomegalovirus DNA polymerase inhibitors. AB - This paper describes the structure activity relationships of a new class of cytomegalovirus DNA polymerase inhibitors having two aryl groups joined by an acyloxyamidine linker. Examination of a series of analogues in which the terminal groups are varied revealed a very narrow SAR around the 2,4-dichlorophenyl group of the lead compound, but a variety of replacements for the benzothiazole ring are compatible with activity. The most notable of these is the isoxazole ring of compound 78, which provides a 30-fold enhancement in potency compared to the lead compound. We also describe the design, synthesis and evaluation of 10 analogues in which the acyloxyamidine linker is modified or replaced by an isosteric group. Structure-activity relationship studies identified the linker -NH2 group as a critical pharmacophoric element. Ab initio molecular orbital calculations combined with qualitative estimates of steric interaction energies suggest that the lowest energy conformations of the acyloxyamidine linker are characterized by an extended planar CAr-C=N-O-C arrangement and either a syn-periplanar or anti periplanar N-O-C-C(Ar') arrangement. Only the anti-periplanar conformation was observed in the crystal structures of three acyloxyamidines. The most active of the linker-modified compounds designed on the basis of these studies is the amidine carbamate 20, which is approximately one-third as potent in the cytomegalovirus DNA polymerase inhibition assay as the comparator acyloxyamidine 53. The activity of 20 suggests that acyloxyamidines may bind to the cytomegalovirus DNA polymerase via an anti-periplanar conformation similar to that observed in the crystal structure of acyloxyamidine 36. PMID- 10732978 TI - Generation and evaluation of putative neuroregenerative drugs. Part 1: virtual point mutations to the polyketide rapamycin. AB - This paper explores the use of computational methods to direct engineered biosynthesis based on the desired properties of the target compounds. The immunosuppressive properties of rapamycin are a result of the formation of the complex FKBP12-rapamycin-FRAP. Neuroregenerative properties are exhibited by the complex or complexes of rapamycin with FKBP proteins. Our objective has been to design biosynthetically available analogues of rapamycin that bind tightly to FKBP12 but not to FRAP. This has been carried out by successive single ketide deletions from the effector domain of rapamycin. The approach described here has yielded modified rapamycin analogues (RP2 and RP3) as targets for biosynthesis by modified polyketide synthases. RP2 and RP3 have an identical binding affinity (linear interaction energy calculation) to FKBP12 as rapamycin but little or no affinity for binding to FRAP. PMID- 10732979 TI - Generation and evaluation of putative neuroregenerative drugs. Part 2: screening virtual libraries of novel polyketides which possess the binding domain of rapamycin. AB - The use of computational methods to direct engineered biosynthesis toward candidates based on the desired properties of the target compounds has been explored. The objective for this study has been the modification of rapamycin in order to eliminate its immunosuppressive activity and retain its neuroregenerative abilities. We have designed analogues of rapamycin which have truncated effector domains but retain the ability to bind to FKBP proteins, which is a prerequisite for the neuroregenerative abilities of the drugs. The procedures described here consist of the screening of large virtual libraries of molecules which retain the binding domain of rapamycin but in which different substitute ketide units replace the effector domain. These methods have provided analogues of rapamycin that cannot retain the immunosuppressive abilities of rapamycin, have a binding affinity to FKBP12 identical to that of rapamycin (by linear interaction energy calculations), and are suitable for synthesis by modified polyketide synthases. PMID- 10732980 TI - Protease inhibitors. Part 8: synthesis of potent Clostridium histolyticum collagenase inhibitors incorporating sulfonylated L-alanine hydroxamate moieties. AB - A series of hydroxamates was prepared by reaction of alkyl/arylsulfonyl halides with N-2-chlorobenzyl-L-alanine, followed by conversion of the COOH moiety to the CONHOH group, with hydroxylamine in the presence of carbodiimides. Other structurally related compounds were obtained by reaction of N-2-chlorobenzyl-L alanine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by the similar conversion of the COOH into the CONHOH moiety. The new compounds were assayed as inhibitors of the Clostridium histolyticum collagenase, ChC (EC 3.4.24.3), a bacterial zinc metallo-peptidase which degrades triple helical collagen as well as a large number of synthetic peptides. The prepared hydroxamate derivatives proved to be 100-500 times more active collagenase inhibitors than the corresponding carboxylates. Substitution patterns leading to best ChC inhibitors (both for carboxylates as well as for the hydroxamates) were those involving perfluoroalkylsulfonyl- and substituted arylsulfonyl moieties, such as pentafluorophenylsulfonyl; 3- and 4-protected aminophenylsulfonyl-; 3- and 4-carboxyphenylsulfonyl-; 3-trifluoromethyl phenylsulfonyl; as well as 1- and 2-naphthyl-, quinoline-8-yl- or substituted arylsulfonylamidocarboxyl moieties among others. Similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P2' and P3' sites, in order to achieve tight binding to the enzyme. This study also proves that the 2 chlorobenzyl moiety, investigated here for the first time, is an efficient P2' anchoring moiety for obtaining potent ChC inhibitors. PMID- 10732981 TI - Origin of rate-acceleration in ester hydrolysis with metalloprotease mimics. AB - Mimics of carboxypeptidase A, a prototypical metalloprotease, were synthesized by linking macrocyclicpolyamines to the primary side of beta-cyclodextrin followed by complexing with Zn(II). These enzyme mimics exhibit saturation kinetics in hydrolysis of p-nitrophenyl acetate (PNPA) and enhance the rate of hydrolysis reaction by almost 300-fold. The effective molarities (EM) of the mimics range from 0.2 to 1.9 M. Origin of the rate acceleration was examined: the reactivity of Zn(II) complexes of [12]aneN3 [12]aneN4, and [14]aneN4 for hydrolyzing PNPA increases with increase in basicity of the zinc bound hydroxides [Zn(II)-OH], yielding a linear Bronsted plot. Free hydroxide fits well on this plot. A similar plot was obtained with the enzyme mimics. The Bronsted relationships indicate that the Zn(II)-OH in the catalytic systems hydrolyzes the ester by direct nucleophilic attack on the ester carbonyl of cyclodextrin-bound but not Zn(II) coordinated PNPA. PMID- 10732982 TI - Docking study of enantiomeric fonofos oxon bound to the active site of Torpedo californica acetylcholinesterase. AB - Molecular interaction between enantiomeric fonofos oxon (O-ethyl S-phenyl ethylphosphonothiolate) and acetylcholinesterase (AChE) of Torpedo californica was evaluated by using the Cerius2 program. It was suggested that the difference in the inhibitory activity of the two enantiomers of fonofos oxon on AChE is due to the steric hindrance in binding to the AChE active site. PMID- 10732983 TI - Synthesis and evaluation of C-9 modified N-acetylneuraminic acid derivatives as substrates for N-acetylneuraminic acid aldolase. AB - Several C-9 modified N-acetylneuraminic acid derivatives have been synthesised and evaluated as substrates of N-acetylneuraminic acid aldolase. Simple C-9 acyl or ether modified derivatives of N-acetylneuraminic acid were found to be accepted as substrates by the enzyme, albeit being transformed more slowly than Neu5Ac itself. 1H NMR spectroscopy was used to evaluate the extent of the enzyme catalysed transformation of these compounds. Interestingly, the chain-extended Neu5Ac derivative 16 is not a substrate for N-acetylneuraminate lyase and behaves as an inhibitor of the enzyme. PMID- 10732984 TI - 5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4-pentynoic acid and its derivatives inhibit ionotropic gamma-aminobutyric acid receptors by binding to the 4'-ethynyl 4-n-propylbicycloorthobenzoate site. AB - Acyclic noncompetitive antagonists of ionotropic gamma-aminobutyric acid (GABA) receptors, bearing an ester or ether linkage, were designed, synthesized, and assayed for their inhibition of the specific binding of [3H]4'-ethynyl-4-n propylbicycloorthobenzoate (EBOB), a radiolabeled noncompetitive antagonist, to rat brain and housefly head membranes. 5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4 pentynoic acid (DBCPP), a butyl benzoate analogue, was found to competitively inhibit the binding of [3H]EBOB in rat brain membranes, with an IC50 of 88 nM. The potency conferred by the p-substituent decreased in the order C(triple bond)C(CH2)2COOH > C(triple bond)C(CH2)2COOCH3 > C(triple bond) CH > Br. Pentyl phenyl ethers were equally potent compared with butyl benzoates, while phenyl pentanoates and benzyl butyl ethers were less pont. These compounds were generally less active in housefly head membranes than in rat brain membranes. The introduction of an isopropyl group into the 1-position of the 3,3-dimethylbutyl group of a butyl benzoate and two benzyl butyl ethers caused an increase in potency in housefly GABA receptors, whereas this modification at the corresponding position of other compounds led to an unchanged or decreased potency. In the case of rat receptors, this modification resulted in a decrease in potency except for a phenyl pentanoate. To confirm that DBCPP interferes with GABA receptor function, we performed whole-cell patch clamp experiments with rat dorsal root ganglion neurons in the primary culture. Repeated co-applications of GABA and DBCPP suppressed GABA-induced whole-cell currents with an IC50 of 0.54 microM and a Hill coefficient of 0.7. These findings indicate that DBCPP and its derivatives inhibit ionotropic GABA receptors by binding to the EBOB site and that there might be structural difference in the noncompetitive antagonist binding site between rat and housefly GABA receptors. PMID- 10732985 TI - Specific inactivation of Escherichia coli tRNA(Phe) by antisense DNA-treatment under Mg2+-deficient conditions. AB - The preparation of an Escherichia coli tRNA mixture lacking several specific species may be useful for applications ranging from cell-free protein preparation to protein engineering. We have already demonstrated that tRNA(Asp) can be inactivated, or 'knocked out', with practical specificity by an antisense strategy. In the present study, we synthesized five tRNA(Phe)-targeted antisense oligonucleotides and tested if this tRNA can also be inactivated specifically. The salt conditions used previously for the tRNA(Asp) inactivation were not applicable to tRNA(Phe). Instead, Mg2+-deficient conditions were found to be useful for the inactivation of tRNAPhe by the antisense oligonucleotides. These conditions were also applicable to the inactivation of tRNA(Asp). The susceptibility to the antisense DNAs can change drastically, depending on the concentration of Mg2+. PMID- 10732986 TI - A simple and rapid method for cloning insect vitellogenin cDNAs. AB - We describe a simple and rapid method for cloning insect vitellogenin (Vg) cDNAs. The method relies on the facts that insect Vg amino acid sequences can be aligned confidently along their entire lengths and that a short, highly conserved GL/ICG motif and up to nine cysteine residues that follow at conserved locations are present near the C-termini. An adaptor-ligated double-strand cDNA library is constructed from poly(A)+ RNA prepared from vitellogenic female fat body tissues using a commercial kit, and subjected to PCR with each of the degenerate nucleotide sequences for the GL/ICG motif and the adaptor sequence as primers. The PCR products (0.7-0.9 kb, representing the 3' portion) are cloned, the nucleotide sequences are determined, and the deduced amino acid sequences are aligned with the known insect Vg sequences starting from the GL/ICG motif. Gene specific primers corresponding to the sequences near the 5'-termini of the initial clones and the adaptor sequence are employed to obtain the remaining 5' portion of the Vg cDNAs. The method was successfully applied to the bean bug Plautia stali (Heteroptera), revealing three Vg genes. PMID- 10732987 TI - Isolation and characterization of Dorin M, a lectin from plasma of the soft tick Ornithodoros moubata. AB - A lectin with high hemagglutinating activity, which we have named Dorin M, was identified in the plasma of the soft tick Ornithodoros moubata. The activity of the plasma lectin could be efficiently inhibited by sialic acid, N-acetyl-D hexosamines and sialoglycoproteins. Dorin M was purified to homogeneity using two different isolation systems: affinity chromatography on a column of bovine submaxillary mucin conjugated to Sepharose 4B with specific elution by N-acetyl-D glucosamine and chromatography on Blue-Sepharose followed by anion exchange FPLC on a MonoQ column. The purified lectin is a glycoprotein which, in the native state, forms aggregates with molecular mass of about 640 kDa. Non-reducing SDS PAGE revealed that the lectin consists of two noncovalently bound subunits migrating closely around 37 kDa. Dorin M is a glycoprotein, probably modified by N-type glycosylation. After chemical deglycosylation, only one band of about 32 kDa was detected. Dorin M is the first lectin purified from ticks. PMID- 10732988 TI - Alpha-amylases of the coffee berry borer (Hypothenemus hampei) and their inhibition by two plant amylase inhibitors. AB - The adult coffee berry borer (Hypothenemus hampei Ferrari [Coleoptera: Scolytidae]), a major insect pest of coffee, has two major digestive alpha amylases that can be separated by isoelectric focusing. The alpha-amylase activity has a broad pH optimum between 4.0 and 7.0. Using pH indicators, the pH of the midgut was determined to be between 4.5 and 5.2. At pH 5.0, the coffee berry borer alpha-amylase activity is inhibited substantially (80%) by relatively low levels of the amylase inhibitor (alphaAI-1) from the common bean, Phaseolus vulgaris L., and much less so by the amylase inhibitor from Amaranthus. We used an in-gel zymogram assay to demonstrate that seed extracts can be screened to find suitable inhibitors of amylases. The prospect of using the genes that encode these inhibitors to make coffee resistant to the coffee berry borer via genetic engineering is discussed. PMID- 10732989 TI - cDNAs of aminopeptidase-like protein genes from Plodia interpunctella strains with different susceptibilities to Bacillus thuringiensis toxins. AB - Aminopeptidase N has been reported to be a Bacillus thuringiensis (Bt) Cry1A toxin-binding protein in several lepidopteran insects. cDNAs of aminopeptidase like proteins from both Bt-susceptible RC688s and Bt-resistant HD198r strains of the Indianmeal moth, Plodia interpunctella, were cloned and sequenced. They contain 3345 and 3358 nucleotides, respectively, and each has a 3048 bp open reading frame that encodes 1016 amino acids. Putative protein sequences include 10 potential glycosylation sites and a zinc metal binding site motif of HEXXH, which is typical of the active site of zinc-dependent metallopeptidases. Sequence analysis indicated that the deduced protein sequences are most similar to an aminopeptidase from Heliothis virescens with 62% sequence identity and highly similar to three other lepidopteran aminopeptidases from Plutella xylostella, Manduca sexta, Bombyx mori with sequence identities of 51-52%. Four nucleotide differences were observed in the open reading frames that translated into two amino acid differences in the putative protein sequences. Polymerase chain reaction (PCR) confirmed an aminopeptidase gene coding difference between RC688s and HD198r strains of P. interpunctella in the PCR amplification of a specific allele (PASA) using preferential primers designed from a single base substitution. The gene mutation for Asp185-->Glu185 was also confirmed in two additional Bt-resistant P. interpunctella strains. This mutation is located within a region homologous to the conserved Cry1Aa toxin binding regions from Bombyx mori and Plutella xylostella. The aminopeptidase-like mRNA expression levels in the Bt-resistant strain were slightly higher than those in the Bt susceptible strain. The sequences reported in this paper have been deposited in the GenBank database (accession numbers AF034483 for susceptible strain RC688s and AF034484 for resistant strain HD198r). PMID- 10732990 TI - Biosynthesis of L-alanine, a major amino acid of fibroin in Samia cynthia ricini. AB - The derivation of alanine in fibroin was investigated using NMR and selective isotopic labelling. 2H2O infused orally into 5th instar larvae was incorporated into the proton of the methyl group of alanine in fibroin. Proton exchange among alanine, glycine and serine was also found. Incorporation of 13C from [2 (13)C]acetate into alanine C2 and C3 and glycine C2 in fibroin, and also C4 of free glutamine plus glutamate was observed in vivo. Hemolymph contained a peak for C4 of glutamate plus glutamine, and an alanine C3 peak appeared transiently. Thus, it is suggested that the C-skeleton of alanine formed was derived from L malate via the TCA-cycle, and that this alanine is utilized in part for fibroin synthesis. Spectra of the hemolymph extract of larvae infused orally with [15N2]urea showed no 15N-compounds, whereas those of larvae injected subcutaneously showed only one peak of urea, whose intensity decreased with time, as shown in the in vivo spectra of a living larva infused with [15N2]urea. The solution NMR spectrum of fibroin showed no 15N-labelled compounds. Temporal changes in the peak intensities of six compounds in the spectra of a living larva infused with [15N]ammonium demonstrated a process in which 15N was incorporated into fibroin containing 15N-alanine through the amide group of glutamine and the amino group of glutamate. Thus, alanine biosynthesis from the TCA-cycle originates mainly from water, L-malate and ammonium. The fact that no 15N-urea was detected in the hemolymph extract of larvae infused with [15N]ammonium suggests that 15N-urea found in the above in vivo spectra may be that accumulated in the hindgut. Thus, excess ammonium in the body causes the production of urea by the urea-cycle. In Samia larvae, urea was not reutilized but excreted. The metabolic relationships between the assimilation of ammonium and the function of the urea-cycle are discussed. PMID- 10732991 TI - Chemosensory proteins of Locusta migratoria (Orthoptera: Acrididae). AB - We describe a family of proteins abundantly expressed in the chemoreception sensory organs, the antennae and legs, of the desert locust, Locusta migratoria (Orthoptera, Acrididae). Using polymerase chain reaction-based approaches and homology screenings, "OS-D"-like proteins were identified in L. migratoria. The different sub-types (LmigOS-Ds) are very similar to each other and share about 50 70% identity with OS-Ds from Drosophila melanogaster and Periplaneta americana. A similar degree of identity was also observed with moth OS-Ds. Northern blot analysis revealed a strong expression of the LmigOS-Ds in the antennae and legs, suggesting their involvement in chemosensory processes. Despite the lack of direct evidence for their role in chemosensation, LmigOS-Ds and their homologs seem to constitute a large protein family, characterized by a striking abundance and diversity among insect chemosensory organs. PMID- 10732992 TI - Substrate specificity of the brush border K+-leucine symport of Bombyx mori larval midgut. AB - L-leucine uptake into membrane vesicles from Bombyx mori larval midgut was tested for inhibition by 55 compounds, which included sugars, N-methylated, alpha-, beta , gamma-, delta-, epsilon-amino acids, primary amines, alpha-amino alcohols, monocarboxylic organic acids and alpha-ketoacids. Based on cis-inhibition experiments performed at the high pH (10.8) characteristic of the midgut luminal content in vivo, we find that the carrier binding site interacts with molecules which possess a well-defined set of structural features. Amino acids are preferentially accepted as anions and the ideal inhibitor must have an hydrophobic region and a polar head constituted by a chiral carbon atom bearing two hydrophilic groups, a deprotonated amino-group and a dissociated carboxylic group. Binding is reduced if one of the two hydrophilic groups is removed. Lowering the pH to less alkaline value (8.8) only affects the affinity of delta- and epsilon-amino acids, which are excluded from binding because of their positively charged side-chain. Modifications of the potassium electrochemical gradient increased the affinity constant values of the molecules, but have little effect on the rank of specificity. Physiological implications of the data reported are discussed. PMID- 10732993 TI - Genomic copy number of intracellular bacterial symbionts of aphids varies in response to developmental stage and morph of their host. AB - Buchnera, endosymbiotic bacteria of aphids possess many genomic copies per cell. In this study, we estimated genomic copy number per Buchnera cell from host insects at various developmental stages and of two different morphs, apterae and alatae, by fluorimetry and real-time quantitative PCR. The results indicated that the genomic copy number of Buchnera increased during postembryonic development of insects to adulthood, and that it decreased during the host's ageing. In Buchnera from alatae, the genomic copy number per cell was about twice as many as in those from apterae. DAPI-staining showed that the distribution of the genomic DNA in the Buchnera cells from old insects tended to aggregate, suggesting that intracellular structure of the genomic DNA of Buchnera varies in response to the physiological conditions of their host. PMID- 10732994 TI - Toxicity of cinnamomin--a new type II ribosome-inactivating protein to bollworm and mosquito. AB - The toxicity of cinnamomin, a new type II ribosome-inactivating protein purified from the seeds of camphor tree (Cinnamomum camphora), to bollworm (Helicoverpa armigera) and mosquito (Culex pipines pallens) during larval stage was tested. The LC50 of cinnamomin to bollworm larvae fed on diet containing cinnamomin was 1839 ppm and the LC50 to larvae of mosquito was 168 ppm. The gut extract of bollworm larvae could apparently hydrolyze cinnamomin. The inhibition of protein synthesis by cinnamomin was tested in in vitro translation system of bollworm larvae, and its LC50 was determined to be approx. 14 nM. Bollworm larvae ribosome treated with cinnamomin produced a specific RNA fragment (R-fragment) characterized on urea-denatured polyacrylamide gel. Evidence was provided that hidden breaks exist in the largest ribosomal RNA of bollworm larvae. PMID- 10732995 TI - Potassium signalling in the brain: its role in behaviour. AB - This paper examines evidence that glial cells respond to changes in extracellular potassium ([K+]e) in ways that contribute to modulation of neuronal activity and thereby behaviour. Glial cells spatially (and probably directionally) redistribute potassium from regions of increasing concentration to those with a lesser concentration. This redistribution is largely responsible for slow potential shifts associated with behavioural responses of animals. These slow shifts are related in amplitude to the level of 'arousal' of an animal, and its motivational state. In addition, glia, especially astrocytes, respond to changes in [K+]e, the presence of transmitters like nor-adrenaline and glutamate and at least some hormones with changes in their metabolism and/or the morphological characteristics of the cell. The ionic, metabolic and morphological responses of glia to changes in extracellular potassium after neuronal activity have been associated with at least some forms of learning, including habituation, one trial passive avoidance learning and changes associated with enriched environments. The implication of these effects of potassium signalling in the brain is that there is considerable involvement of glia in a number of processes crucial to neuronal activity. Glia may also form another route for information distribution in the brain that is at least bi-directional, though less specific than its neuronal counterparts. It is evident that the Neuroscience of the future will have to incorporate much more study of neuron-glial interactions than hitherto. PMID- 10732996 TI - Role of astrocytes in the clearance of excess extracellular potassium. AB - The development of concepts describing potassium clearance mechanisms in the mammalian central nervous system in the last 35 years is reviewed. The pattern of excess potassium in the extracellular space is discussed as are the implications of these potassium levels for neuronal excitability. There is a systematic description of the available evidence for astrocytic involvement in situ. The three possible astrocytic potassium clearance mechanisms are introduced: spatial buffer mechanism; carrier-operated potassium chloride uptake as well as channel operated potassium chloride uptake. The three mechanisms are compared and their compatibility is discussed. Evidence is now available showing that at least two of these if not all three mechanisms co-exist and complement each other. Finally, it is concluded that these potassium movements are not used as a signal system, only as a homeostatic feedback mechanisms. Such a genuine signal system involving glial elements exists--but it is based on calcium waves. PMID- 10732997 TI - Colocalization of neurotransmitter receptors on astrocytes in explant cultures of rat CNS. AB - In recent years evidence has accumulated that astrocytes express functional receptors for a variety of neurotransmitters/neuromodulators. By means of electrophysiological and combined autoradiographic and immunohistochemical methods we have demonstrated the colocalization of cholinergic, adrenergic and peptidergic receptors on astrocytes in explant cultures from various regions of rat central nervous system. A great number of biochemical and electrophysiological studies from other laboratories have shown that most of the neurotransmitters exert their effects on second messenger systems and on Ca2+ activated K+-channels. Furthermore, certain neurotransmitters are involved in the regulation of energy metabolism by stimulating enzymatic breakdown of glycogen in astrocytes. It was suggested that there is a cross-talk between the various neurotransmitter receptors on the glial membrane and that these receptors act in a synergistic or antagonistic way. The coexistence of cholinergic and peptidergic receptors on astrocytes is of great interest since both neurotransmitter systems are involved in cognitive functions and are impaired in patients with Alzheimer's dementia. The question is therefore raised whether not only neurones but also astrocytes might be involved in neurodegenerative disorders such as Alzheimer's disease. PMID- 10732998 TI - Quantitative immunochemistry on neuronal loss, reactive gliosis and BBB damage in cortex/striatum and hippocampus/amygdala after systemic kainic acid administration. AB - Cell specific markers were quantified in the hippocampus, the amygdala/pyriform cortex, the frontal cerebral cortex and the striatum of the rat brain after systemic administration of kainic acid. Neuron specific enolase (NSE) reflects loss of neurons, glial fibrillary acidic protein (GFAP) reflects reactive gliosis, and brain levels of serum proteins measures blood-brain-barrier permeability. While the concentration of NSE remained unaffected in the frontal cerebral cortex and the striatum, their GFAP content increased during the first three days. In the hippocampus and amygdala, NSE levels decreased significantly. GFAP levels in the hippocampus were unaffected after one day and decreased in the amygdala/pyriform cortex. After that, GFAP increased strikingly until day 9 or, in the case of amygdala/pyriform cortex, even longer. This biphasic time course for GFAP was accompanied by a decrease of S-100 during days 1-9 followed by a significant increase at day 27 above the initial level. The regional differences in GFAP and S-100 could result from the degree of neuronal degeneration, the astrocytic receptor set-up and/or effects on the blood-brain barrier. PMID- 10732999 TI - Quantitative autoradiography of Na+-dependent [3H]L-aspartate binding to L glutamate transporters in rat brain: structure-activity studies using L-trans pyrrolidine-2,4-dicarboxylate (L-t-PDC) and 2-(carboxycyclopropyl)-glycine (CCG). AB - Sodium-dependent binding of [3H]L-aspartate was studied in thaw-mounted horizontal sections of fresh-frozen (i.e. not fixed) rat brain. After the incubation with [3H]L-aspartate, the sections were exposed against a 3H-sensitive film and the resulting autoradiograms were evaluated by quantitative densitometry. Effects of several inhibitors were examined and their potency expressed as IC50 and nH. Together with previously published data, the present study supports the view that [3H]L-aspartate binding to fresh-frozen sections of rat brain represents interaction of the radioligand with the substrate-binding sites on glutamate transporters. The most potent inhibitors were (2S,3S,4R)-2 (carboxycyclopropyl)-glycine (L-CCG III) and (2S,4R)-4-methylglutamate. In contrast, L-anti,endo-3,4-methanopyrrolidine dicarboxylate (L-a,e-MPDC) was about an order of magnitude less potent. Only subtle regional variations in the characteristics of inhibitors of [3H]L-aspartate binding were detected. It is not certain whether these differences reflect regional variations in the distribution of individual glutamate transporters or regional peculiarities in their pharmacological characteristics. In particular, (2S,4R)-4-methylglutamate, shown previously to differentiate between GLT-1 (principal glutamate transporter in the forebrain) and GLAST (expressed mainly in the cerebellum), did not strongly differentiate between the binding of [3H]L-aspartate in forebrain and cerebellum. Computer-assisted molecular modelling using selected glutamate analogues with restricted conformation (L-trans-pyrrolidine-2,4-dicarboxylate and four isomers of 2-(carboxycyclopropyl)-glycine: L- and D-CCG I, L-CCG III and L-CCG IV) identified at least one area of unfavourable steric interaction. We conclude that the quantitative autoradiographic studies using [3H]L-aspartate or other transporter-specific ligands, will be a useful tool to study the pharmacology of substrate binding sites on glutamate transporters in the mammalian brain in situ. PMID- 10733000 TI - Acidosis and astrocyte amino acid metabolism. AB - The relationship between acidosis and the metabolism of glutamine and glutamate was studied in cultured astrocytes. Acidification of the incubation medium was associated with an increased formation of aspartate from glutamate and glutamine. The rise of the intracellular content of aspartate was accompanied by a significant decline in the extracellular concentration of both lactate and citrate. Studies with either [2-(15)N]glutamine or [15N]glutamate indicated that there occurred in acidosis an increased transamination of glutamate to aspartate. Studies with L-[2,3,3,4,4-(2)H5]glutamine indicated that in acidosis glutamate carbon was more rapidly converted to aspartate via the tricarboxylic acid cycle. Acidosis appears to result in increased availability of oxaloacetate to the aspartate aminotransferase reaction and, consequently, increased transamination of glutamate. The expansion of the available pool of oxaloacetate probably reflects a combination of: (a) Restricted flux through glycolysis and less production from pyruvate of acetyl-CoA, which condenses with oxaloacetate in the citrate synthetase reaction; and (b) Increased oxidation of glutamate and glutamine through a portion of the tricarboxylic acid cycle and enhanced production of oxaloacetate from glutamate and glutamine carbon. The data point to the interplay of the metabolism of glucose and that of glutamate in these cells. PMID- 10733001 TI - Modulation of glutamine uptake and phosphate-activated glutaminase activity in rat brain mitochondria by amino acids and their synthetic analogues. AB - Uptake of L-[14C]Gln and phosphate-activated glutaminase (PAG) activity were measured in nonsynaptic mitochondria isolated from rat cerebral hemispheres, in the presence of protein and nonprotein amino acids and their synthetic structural analogues and derivatives. The uptake was inhibited by > 50% in the presence of a 10-fold excess of His, homocysteine (Hcy), Trp, Leu, Tyr, Ile, Thr, Ala, Phe, Met, Ser, by > 20% in the presence of a 10-fold excess of Val, Arg, Glu, and was not affected by a 10-fold excess of Orn, alpha-ketoglutarate, Tau and Pro. Uptake of L-[14C] Leu differed from Gln uptake by its resistance to Arg, Glu, and a relatively high sensitivity to the reference inhibitor of the plasma membrane transport of large neutral amino acids (L-system)--BCH (2 aminobicyclo[2.2.1]heptane-2-carboxylic acid), and a number of natural L-system substrates. A newly synthesized alanine analogue, 2'-cyano-(biphenyl) alanine, referred to as MRC01, was the only compound tested that inhibited Gln uptake more strongly than Leu uptake. The strongest Gln uptake inhibitors: MRC01, His, Hcy and Leu, inhibited PAG activity by > 50% when added at the inhibitor/Gln concentration ratio of 1:2. PAG activity was not affected by Tau, Lys or Pro, compounds which did affect Gln uptake. The results suggest that a number of natural amino acids function as common endogenous modulators of cerebral mitochondrial Gln uptake and its degradation. MRC01, because of its inhibitory potency towards both mitochondrial Gln uptake and PAG activity, may become a convenient tool in studying the role of Gln transport in its mitochondrial metabolism in intact CNS cell and tissues. PMID- 10733002 TI - Compartmentation of TCA cycle metabolism in cultured neocortical neurons revealed by 13C MR spectroscopy. AB - Cultured neocortical neurons were incubated in medium containing [U-13C]glucose (0.5 mM) and in some cases unlabeled glutamine (0.5 mM). Subsequently the cells were "superfused" for investigation of the effect of depolarization by 55 mM K+. Cell extracts were analyzed by 13C magnetic resonance spectroscopy and gas chromatography/mass spectrometry to determine incorporation of 13C in glutamate, GABA, aspartate and fumarate. The importance of the tricarboxylic acid (TCA) cycle for conversion of the carbon skeleton of glutamine to GABA was evident from the effect of glutamine on the labeling pattern of GABA and glutamate. Moreover, analysis of the labeling patterns of glutamate in particular indicated a depolarization induced increased oxidative metabolism. This effect was only observed in glutamate and not in neurotransmitter GABA. Based on this a hypothesis of mitochondrial compartmentation may be proposed in which mitochondria associated with neurotransmitter synthesis are distinct from those aimed at energy production and influenced by depolarization. The hypothesis of mitochondrial compartmentation was further supported by the finding that the total percent labeling of fumarate and aspartate differed significantly from each other. This can only be explained by the existence of multiple TCA cycles with different turnover rates. PMID- 10733003 TI - Acutely isolated astrocytes as models to probe astrocyte functions. AB - Neuroscientists have become increasingly aware and accepting of the concept that astrocytes likely have many important functions in the CNS. One limitation in establishing these functions is the usual problem of what constitutes suitable experimental approaches. A major experimental step for functional studies of astrocytes has been the widespread use of primary astrocyte cultures, an approach that Leif Hertz pioneered. However, it is now becoming clear that, building on this work, an experimental paradigm shift is now needed. Namely, to increasingly study preparations corresponding to in situ conditions, such as slices. An alternative experimental system where the cells have some of the technical advantages of primary astrocyte cultures is freshly isolated astrocytes. Recent experiments from our laboratory have shown metabotropic glutamate receptor expression by such cells. Examples are given of how functional receptor studies and channel activity measured by patch clamp electrophysiology can be combined with single cell RT-PCR to define further the receptor or channel type are described to illustrate the uses of such preparations. PMID- 10733004 TI - Expression of interleukin-1 alpha, tumor necrosis factor alpha and interleukin-6 genes in astrocytes under ischemic injury. AB - Astrocytes form an integral part of the blood brain barrier and are the first cell type in the central nervous system to encounter insult if there is an ischemic attack. The immunologic reaction of astrocytes to an ischemic insult would be affective to the subsequent responses of other nerve cells. We previously showed that ischemia caused an increase in the levels of interleukin 1alpha (IL-1alpha), tumor necrosis factor alpha (TNF alpha), and interleukin 6 (IL-6) in the culture medium of mouse cerebral cortical astrocyte. We did not have evidence on the source of these cytokines. This study aimed to investigate the expressions of these cytokine mRNAs in the astrocytes under ischemia. Results demonstrated that ischemia could induce necrosis and apoptosis in astrocytes. By using the RT-PCR method, we demonstrated for the first time that the mRNA levels of IL-1alpha, TNF alpha and IL-6 in normal astrocyte was very low, but their expressions could be induced quickly under ischemia. These cytokines might be interactive as indicated by the difference in time course of their expressions, with IL-1alpha being the earliest and IL-6 being the latest. The result provided some understanding of the induction and progression of these immunologic responses in astrocytes under ischemia. It also supported our previous findings that astrocytes contributed to the cytokines released under ischemia. PMID- 10733005 TI - Reduced [3H]IP3 binding but unchanged IP3 receptor levels in the rat hippocampus CA1 region following transient global ischemia and tolerance induction. AB - Changes in inositol (1,4,5)-trisphosphate (IP3) binding properties and the protein level of the IP3 receptor have been reported in different pathological conditions in the brain, e.g. cerebral ischemia, Alzheimer's disease, and Huntingtons disease. We used the 4-vessel occlusion model in rat brain to investigate the effect of transient ischemia insults on the IP3 receptor mRNA level, the IP3 receptor protein level and [3H]IP3 binding. Recirculation periods were limited (1-72 h) to avoid the development of delayed neuronal death. We found that the IP3 receptor mRNA levels were decreased after damage-inducing ischemia (9 min) in the hippocampus CA1 and CA3 regions. The mRNA levels were unaltered after tolerance-inducing ischemia (3 min). However, [3H]IP3 binding was significantly reduced after both damage- and tolerance-inducing ischemia in the hippocampus CA1 region. Furthermore, all investigated brain areas showed a decreased [3H]IP3 binding when tolerance-inducing ischemia was followed by a second ischemic insult (3 + 8.5 min ischemia). The IP3 receptor protein levels remained constant in all investigated brain areas. These results indicate that a reduced [3H]IP3 binding capability in the particularly vulnerable areas occurs as an early consequence of cerebral ischemia, before IP3 receptor protein levels are reduced in these areas. Structural or conformational changes altering IP3 binding may be of necessity on the pathway leading to down-regulation of IP3 receptor protein levels, as observed by others. PMID- 10733006 TI - Effect of ammonia on GABA uptake and release in cultured astrocytes. AB - While the pathogenesis of hepatic encephalopathy (HE) is unclear, there is evidence of enhanced GABAergic neurotransmission in this condition. Ammonia is believed to play a major pathogenetic role in HE. To determine whether ammonia might contribute to abnormalities in GABAergic neurotransmission, its effects on GABA uptake and release were studied in cultured astrocytes, cells that appear to be targets of ammonia neurotoxicity. Acutely, ammonium chloride (5 mM) inhibited GABA uptake by 30%, and by 50-60% after 4-day treatment. GABA uptake inhibition was associated with a predominant decrease in Vmax; the Km was also decreased. Ammonia also enhanced GABA release after 4-day treatment, although such release was initially inhibited. These effects of ammonia (inhibition of GABA uptake and enhanced GABA release) may elevate extracellular levels of GABA and contribute to a dysfunction of GABAergic neurotransmission in HE and other hyperammonemic states. PMID- 10733007 TI - Glial cells and volume transmission in the CNS. AB - Although synaptic transmission is an important means of communication between neurons, neurons themselves and neurons and glia also communicate by extrasynaptic "volume" transmission, which is mediated by diffusion in the extracellular space (ECS). The ECS of the central nervous system (CNS) is the microenvironment of neurons and glial cells. The composition and size of ECS change dynamically during neuronal activity as well as during pathological states. Following their release, a number of neuroactive substances, including ions, mediators, metabolites and neurotransmitters, diffuse via the ECS to targets distant from their release sites. Glial cells affect the composition and volume of the ECS and therefore also extracellular diffusion, particularly during development, aging and pathological states such as ischemia, injury, X irradiation, gliosis, demyelination and often in grafted tissue. Recent studies also indicate that diffusion in the ECS is affected by ECS volume inhomogeneities, which are the result of a more compacted space in certain regions, e.g. in the vicinity of oligodendrocytes. Besides glial cells, the extracellular matrix also changes ECS geometry and forms diffusion barriers, which may also result in diffusion anisotropy. Glial cells therefore play an important role in extrasynaptic transmission, for example in functions such as vigilance, sleep, depression, chronic pain, LTP, LTD, memory formation and other plastic changes in the CNS. In turn, ECS diffusion parameters affect neuron-glia communication, ionic homeostasis and movement and/or accumulation of neuroactive substances in the brain. PMID- 10733008 TI - The astrocytic ("peripheral-type") benzodiazepine receptor: role in the pathogenesis of portal-systemic encephalopathy. AB - An increasing body of evidence supports the notion that activation of astrocytic (peripheral-type) benzodiazepine receptors contributes to the pathogenesis of the central nervous system symptoms which are characteristic of portal-systemic encephalopathy (PSE). Binding site densities for the PTBR ligand [3H-PK11195] are increased in autopsied brain tissue from PSE patients as well as in the brains of animals with experimental chronic liver failure. In the case of the animal studies, increased PTBR sites resulted from increased PTBR gene expression. Exposure of cultured astrocytes to ammonia or manganese (two neurotoxic agents which under normal circumstances are removed by the hepatobiliary system and which are found to accumulate in brain in PSE) results in increased densities of [3H-PK11195] binding sites. Activation of PTBR is known to result in increased cholesterol uptake and increased synthesis in brain of neurosteroids some of which have potent positive allosteric modulator properties on the GABA-A receptor system. Accumulation of such substances in the brain in chronic liver failure could explain the neural inhibition characteristics of PSE. PMID- 10733009 TI - Cytokine chemokine expression in contused rat spinal cord. AB - Spinal cord injury within the first few hours, is complicated by inflammatory mechanisms, including the influx of monocyte/macrophages as well as the activation of resident spinal microglia and astrocytes. Numerous studies have suggested that the initial infiltration of the hematogenous cells may be due to the secretion of cytokines and chemokines in the injured CNS. In order to elucidate which chemotactic factors may be expressed following traumatic spinal cord contusion, the presence of mRNA for a number of cytokines, chemokines and growth factors was examined in contused rat spinal cord by reverse transcriptase polymerase chain reaction and immunohistochemistry. Spinal injury was accompanied by an increase in glial fibrillary acidic protein mRNA suggesting astrocyte activation and astrogliosis. TNFalpha message levels were upregulated as early as 1 h post injury and returned to baseline levels by 3 days post injury (DPI). By immunocytochemistry, staining for TNFalpha increased at 1 and 3 dpi and was predominantly diffuse in the necrotic tissue. The chemokines IP-10, MCP-1, and MIP-1alpha were also detected in the injured spinal cord. mRNA levels of IP-10 peaked around 6 h post injury and were upregulated up to 7 dpi. MCP-1 mRNA was detected at 1 h post injury and its levels returned to baseline by 14 dpi. An increase in MCP-1 staining was observed from 1 to 7 dpi. The staining was also diffuse in the necrotic tissue and also localized to cells near the site of injury. The presence of aFGF and bFGF was also detected in the injured spinal cord. mRNA for aFGF was detected at 0 time, increased at 6 h post injury, peaked at 3 days, and remained elevated up to 21 days. bFGF mRNA was initially detected at 1 h post injury, increased between 6 h and 3 days, declined thereafter and returned to baseline levels by 21 days. PMID- 10733010 TI - 5-Hydroxytryptamine2B receptors stimulate Ca2+ increases in cultured astrocytes from three different brain regions. AB - The expression of 5-hydroxytryptamine-2B (5-HT2B) receptor mRNA has recently been shown in cultured astrocytes. Here the expression of functional 5-HT2B receptors has been studied in cultured astrocytes from rat cerebral cortex, hippocampus, and brain stem. Fluo-3- and fura-2-based microspectrofluorometry was used for measuring changes in intracellular free calcium concentrations ([Ca2+]i). The 5 HT2B agonist alpha-methyl 5-HT (40 nM) produced rapid transient increases in [Ca2+]i in astrocytes from all three brain regions studied, and these responses were blocked by the selective 5-HT2B antagonist rauwolscine (1 microM). The specificity of the responses to alpha-methyl 5-HT was further demonstrated by the failure of 4-(4-fluorobenzoyl)-1-(4-phenylbutyl)-piperidine oxalate (1 microM), a specific 5-HT2A/5-HT2C antagonist, to block these responses. The 5-HT2B-induced increases in [Ca2+]i persisted in Ca2+-free buffer, indicating that the increase in [Ca2+]i results from mobilization of intracellular Ca2+ stores. The expression of 5-HT2B receptors on astroglial cells was further verified immunohistochemically and by Western blot analysis. These results provide evidence of the existence of 5-HT2B receptors on astrocytes in primary culture. PMID- 10733011 TI - The effects of isofagomine, a potent glycogen phosphorylase inhibitor, on glycogen metabolism in cultured mouse cortical astrocytes. AB - A novel inhibitor of liver glycogen phosphorylase, isofagomine, was investigated as a possible inhibitor of the enzyme in the brain and in cultured astrocytes. Additionally, the effect of the drug on norepinephrine (NE) induced glycogen degradation in astrocytes was studied. Astrocytes were cultured from mouse cerebral cortex and homogenates were prepared from the cells as well as from mouse brain. Isofagomine dose-dependently inhibited glycogen phosphorylase when measured in the direction of glycogen degradation in both preparations with IC50 values (mean +/- SEM) of 1.0 +/- 0.1 microM and 3.3 +/- 0.5 microM in brain and astrocyte homogenates, respectively. Moreover, isofagomine at a concentration of 400 microM completely prevented NE induced depletion of glycogen stores and the concomitant lactate production in intact astrocytes. It is suggested that this novel glycogen phosphorylase inhibitor may be a valuable tool to investigate the functional importance of glycogen in astrocytes and in the brain. PMID- 10733012 TI - Cytokine and intracellular signaling regulation of tissue factor expression in astrocytes. AB - There is evidence that inflammatory cytokines such as IL-1beta, TNFalpha, and IL 6 are involved in the pathogenesis of cerebrovascular disorders including stroke. One action of cytokines that contributes to diseases in peripheral tissues is upregulation of the procoagulant receptor tissue factor (TF). In the CNS, astrocytes are the primary cells that express TF; although little is known about how TF is regulated in these cells. Experiments were performed to evaluate the effect of cytokine treatment on TF activity in primary cultures of murine cortical astrocytes and in the human astrocytoma cell line (CCF). IL-1beta treatment induced a 2.5-fold increase in TF activity in the primary astrocytes and a 3-fold induction in the astrocytoma cells. TNFalpha treatment induced a 2.5 fold increase in TF activity in both the primary astrocytes and astrocytoma cells. IL-6 upregulated TF activity 2-fold in primary astrocytes, however, it had no effect on TF activity in the astrocytoma cells. The signaling pathways regulating TF expression in these cells were examined by using staurosporine, a broad spectrum inhibitor of serine-threonine protein kinases, and by examining the effects of intermediates in the sphingomyelin signaling pathway. Staurosporine inhibited IL-1beta-induced TF activity in the primary astrocytes but did not effect IL-1beta- or TNFalpha-induced TF activity in the astrocytoma cells. TF activity in the astrocytoma cells was upregulated 1.5-fold over constitutive levels by a ceramide analogue or the enzyme sphingomyelinase, however the ceramide analogue had no effect on TF activity in the primary astrocytes. These results suggest inflammatory cytokines can upregulate TF activity in astrocytes and the astrocytoma CCF cell line although the two cell types appear to utilize different signaling pathways to mediate TF expression. Further studies will be important to more completely define the signaling regulation of TF in astrocytes since alterations in brain TF levels may play a key role in CNS pathophysiology. PMID- 10733013 TI - Mitochondrial malic enzyme activity is much higher in mitochondria from cortical synaptic terminals compared with mitochondria from primary cultures of cortical neurons or cerebellar granule cells. AB - Most of the malic enzyme activity in the brain is found in the mitochondria. This isozyme may have a key role in the pyruvate recycling pathway which utilizes dicarboxylic acids and substrates such as glutamine to provide pyruvate to maintain TCA cycle activity when glucose and lactate are low. In the present study we determined the activity and kinetics of malic enzyme in two subfractions of mitochondria isolated from cortical synaptic terminals, as well as the activity and kinetics in mitochondria isolated from primary cultures of cortical neurons and cerebellar granule cells. The synaptic mitochondrial fractions had very high mitochondrial malic enzyme (mME) activity with a Km and a Vmax of 0.37 mM and 32.6 nmol/min/mg protein and 0.29 mM and 22.4 nmol/min mg protein, for the SM2 and SM1 fractions, respectively. The Km and Vmax for malic enzyme activity in mitochondria isolated from cortical neurons was 0.10 mM and 1.4 nmol/min/mg protein and from cerebellar granule cells was 0.16 mM and 5.2 nmol/min/mg protein. These data show that mME activity is highly enriched in cortical synaptic mitochondria compared to mitochondria from cultured cortical neurons. The activity of mME in cerebellar granule cells is of the same magnitude as astrocyte mitochondria. The extremely high activity of mME in synaptic mitochondria is consistent with a role for mME in the pyruvate recycling pathway, and a function in maintaining the intramitochondrial reduced glutathione in synaptic terminals. PMID- 10733014 TI - The glutathione content of retinal Muller (glial) cells: effect of pathological conditions. AB - Maintenance of isolated retinal Muller (glial) cells in glutamate-free solutions over 7 h causes a significant loss of their initial glutathione content; this loss is largely prevented by the blockade of glutamine synthesis using methionine sulfoximine (5 mM). Anoxia does not reduce the glutathione content of Muller cells when glucose (11 mM), glutamate and cystine (0.1 mM each) are present. In contrast, simulation of total ischemia (i.e., anoxia plus removal of glucose) decreases the glutathione levels dramatically, even in the presence of glutamate and cystine. Less severe effects are caused by high extracellular K+ (40 mM). Reactive oxygen species are generated in the retina under various conditions, such as anoxia, ischemia, and reperfusion. One of the crucial substances protecting the retina against reactive oxygen species is glutathione, a tripeptide constituted of glutamate, cysteine and glycine. It was recently shown that glutathione can be synthesized in retinal Muller glial cells and that glutamate is the rate-limiting substance. In this study, glutathione levels were determined in acutely isolated guinea-pig Muller cells using the glutathione sensitive fluorescent dye monochlorobimane. The purpose was to find out how the glial glutathione content is affected by anoxia/ischemia and accompanying pathophysiological events such as depolarization of the cell membrane. Our results further strengthen the view that glutamate is rate-limiting for the glutathione synthesis in glial cells. During glutamate deficiency, as caused by e.g., impaired glutamate uptake, this amino acid is preferentially delivered to the glutamate-glutamine pathway, at the expense of glutathione. This mechanism may contribute to the finding that total ischemia (but not anoxia) causes a depletion of glial glutathione. In situ depletion may be accelerated by the ischemia-induced increase of extracellular K+, decreasing the driving force for glutamate uptake. The ischemia-induced lack of glutathione is particularly fatal considering the increased production of reactive oxygen species under this condition. Therefore the therapeutic application of exogenous free radical scavengers is greatly recommended. PMID- 10733015 TI - Neuronal expression of glutamine synthetase in Alzheimer's disease indicates a profound impairment of metabolic interactions with astrocytes. AB - A considerable body of evidence indicates that the activity of glutamine synthetase is decreased in the cerebral cortices of brains affected by Alzheimer's disease. It is difficult to discern the reason for this decrease because it is not known whether the cellular distribution of glutamine synthetase is altered in Alzheimer's disease. Therefore the present study has used immunocytochemistry to compare the cellular distributions of glutamine synthetase in the inferior temporal cortices of six Alzheimer's diseased brains and six age matched, non-demented brains. Double-label immunocytochemistry has been used to examine whether the distribution of cellular glutamine synthetase is influenced by the distribution of senile plaques. It was found that glutamine synthetase expression in astrocytes is diminished in Alzheimer's disease, particularly in the vicinity of senile plaques. The most striking finding of the present study was that glutamine synthetase was expressed in a subpopulation of pyramidal neurons in all six Alzheimer's diseased brains, whereas glutamine synthetase was not observed in any neurons from control brains. The changed expression of glutamine synthetase may be triggered by toxic agents in senile plaques, a reduced noradrenergic supply to the cerebral cortex, and increased brain ammonia levels. That such dramatic changes occur in the distribution of this critical, and normally stable enzyme, suggests that the glutamate-glutamine cycle is profoundly impaired in Alzheimer's disease. This is significant because impairments of the glutamate-glutamine cycle are known to cause alterations of mood and behaviour, disturbance of sleeping patterns, amnesia, confusion and reduced awareness. Since these behavioural changes are also seen in Alzheimer's disease, it is speculated that they might be attributable to the reduced expression of glutamine synthetase or to impairments of the glutamate-glutamine cycle. PMID- 10733016 TI - Mammalian oviduct and protection against free oxygen radicals: expression of genes encoding antioxidant enzymes in human and mouse. AB - Genetic expression of five antioxidant enzymes involved in mechanisms protecting embryos against reactive oxygen species (ROS) was studied in human and mouse oviducts. The presence of transcripts encoding for gamma-glutamylcysteine synthetase (GCS), glutathione peroxidase (GPX), Cu-Zn-superoxide dismutase (Cu-Zn SOD), Mn-superoxide dismutase (Mn-SOD) and catalase was analysed by use of the reverse transcription-polymerase chain reaction (RT-PCR). Different expression profiles of transcripts encoding for these enzymes were observed between human and mouse oviducts. In the mouse, all transcripts encoding for the enzymes tested were present in oviduct. In human, only transcripts encoding for GPX, Cu-Zn-SOD and catalase were also detected in oviduct. However, GCS and Mn-SOD transcripts were never observed in human oviduct. Cu-Zn-SOD transcripts are relatively highly expressed whatever species. These results suggest that different gene expression patterns of these antioxidant enzymes between human and mouse may reflect the variations in the ability of embryos to develop in vivo and in vitro. However, hormone related-expression of the missing transcripts in human cannot be ruled out. PMID- 10733017 TI - Leuprorelin depot 3.75 mg versus lynestrenol in the preoperative treatment of symptomatic uterine myomas: a multicentre randomised trial. AB - OBJECTIVES: To compare the effect of the gonadotrophin-releasing hormone agonist leuprorelin and progestin lynestrenol, given prior to surgical treatment of symptomatic uterine myomas, on the pre-operative symptoms, tolerance, and operative blood loss. STUDY DESIGN: Fifty-six women were randomly selected to receive, during 16 weeks, either monthly subcutaneous injections of leuprorelin 3.75 mg sustained release (n=33) or lynestrenol 5 mg two tabs per day (5th to the 25th menstrual cycle) (n=23). RESULTS: Intent-to-treat analysis of the main efficacy criterion, namely ultrasonographic reduction of myoma(s) diameter, showed a significant difference in favour of leuprorelin (P=0.02) with a mean decrease of 26.5+/-4.5% (n=29) as opposed to 7.3+/-5% in the lynestrenol group (n=17). Clinical improvement was satisfactory in both groups. Hematocrit decrease between the preoperative value and the value measured 48 h postoperatively was significantly lower in the leuprorelin group than in the lynestrenol one (P=0.02) (for hemoglobin: P=0.07). CONCLUSION: Leuprorelin was more effective than lynestrenol because of its more intense antigonadotropic activity. The tolerance was good, reflecting each drug mechanism of action. PMID- 10733018 TI - Factors that may predispose to rupture of tubal ectopic pregnancy. AB - OBJECTIVE: The purpose of our study was to evaluate factors that may predispose to rupture of tubal ectopic pregnancy. The study included 99 cases of ectopic pregnancies that were treated during the 5-year period 1992-1996. RESULTS: It was found that: (1) tubal rupture is encountered more often in women with no history of ectopic pregnancy and in those who have full-term pregnancy, suggesting that ectopic pregnancy is less suspected in these women; (2) tubal rupture is encountered less often in lower age ectopic pregnancy; and (3) serum beta-hCG level does not predict tubal rupture. CONCLUSIONS: Rupture of the tube is more often observed in women with a history of ectopic pregnancy and in women with full-term pregnancy. Such observations suggest that ectopic pregnancy is less suspect in these cases. PMID- 10733019 TI - Maternal serum levels of macrophage colony-stimulating factor are associated with adverse pregnancy outcome. AB - OBJECTIVE: The aim of this study was the measurement of maternal serum levels of M-CSF throughout pregnancy, in a low risk obstetrical population, to examine the relationship of M-CSF and pregnancy outcome. STUDY DESIGN: Maternal serum was obtained at various stages of pregnancy and post partum, M-CSF levels were measured by ELISA, pertinent clinical data tabulated, and pregnancy outcome was determined. RESULTS: In 564 pregnancies studied, 22% of 260 nulliparous pregnancies and 10% of 304 multiparous pregnancies were hypertensive. Preeclampsia occurred in 1.5% of nulliparous and in 1% of the multiparous women. In apparently normal pregnancies with good outcome, M-CSF levels rose throughout pregnancy. No cases of preeclampsia occurred if maternal serum M-CSF levels increased more than 100% throughout pregnancy. CONCLUSIONS: This study suggests that absolute levels and relative changes in maternal serum M-CSF levels during pregnancy are associated with adverse pregnancy outcomes. PMID- 10733020 TI - Fetal heart rate in relation to its variation in normal and growth retarded fetuses. AB - OBJECTIVES: (1) to assess the relationship of basal fetal heart rate (FHR) with both long term (LTV) and short term (STV) FHR variation in low-risk pregnancies, longitudinally from 24 weeks gestation onwards and (2) to investigate the relationship of FHR with LTV and STV in intrauterine growth retarded (IUGR) fetuses. STUDY DESIGN: Computerised FHR recordings were made in twenty-nine uncomplicated pregnancies (n=224) and in twenty-seven IUGR fetuses who were selected retrospectively from three databases (n=135). Nomograms of FHR variation with FHR and GA were constructed using multilevel analysis. RESULTS AND CONCLUSIONS: There was a strong negative relationship of FHR with both LTV and STV in the control group (R2=53% and 52%, respectively). In the IUGR fetuses, FHR was generally higher than in normal fetuses whereas LTV and STV were lower. The relationship of FHR with LTV and STV in the IUGR group was less strong (for both: R2=18%). Correction of FHR variation for basal FHR in the IUGR fetuses only resulted in a slight reduction in the number of recordings with a variation below the normal range. As it does not improve the recognition of fetuses being considered at the highest risk, such a correction of FHR variation for basal FHR is therefore not necessary. Intrafetal consistency, known to be present in healthy fetuses, was also present in the IUGR fetuses with a low FHR variation. PMID- 10733021 TI - The biochemical functions of the renal tubules and glomeruli in the course of intrahepatic cholestasis in pregnancy. AB - Biochemical functions of kidney glomeruli and tubules were estimated in pregnancy complicated by cholestasis. The investigated group consisted of 72 women with pregnancy complicated by cholestasis and 30 healthy pregnant patients as a control group. Biochemical assays were performed for the deamination of amino acids, carbonic acid dissociation and creatinine metabolism. Statistical analysis was carried out using the t-test and P<0.05 was considered to be significant. In diurnal urine samples collected from pregnant patients with cholestasis, decreased concentrations of NH4+ (42.0+/-8.9 versus 50.3+/-7.6 mmol/24 h), H+ (19.0+/-7.0 versus 25.0+/-5.0 mmol/24 h), creatinine (1.15+/-0.2 versus 1.43+/ 0.3 mmol/24 h) as well as lower levels of creatinine clearance (89.0+/-23.0 versus 135.0+/-30.0 ml/min) and normal levels of potassium and sodium were observed. Serum creatinine and uric acid concentrations were elevated (86.6+/ 7.07 versus 66.3+/-4.42 micromol/l and 32.1+/-8.3 versus 19.0+/-3.57 micromol/l). Diurnal urine volume was lower in patients with cholestasis than in the control group (995+/-313 versus 1264+/-426 ml/24 h). Disturbances of biochemical functions of kidney glomeruli and tubules, regarding creatinine metabolism and deamination of amino acids, and dissociation of carbonic acid, were seen in patients with cholestasis during pregnancy. PMID- 10733022 TI - Postpartum, also a risk period for domestic violence. AB - OBJECTIVE: In the first European study of its kind the prevalence of physical and sexual violence postpartum was estimated in a random sample of 207 Swedish born women attending antenatal clinics. STUDY DESIGN: The Severity of Violence Against Women scale (SVAW) was used to measure the frequency of threats and severity of physical and sexual abuse by means of a postal questionnaire covering a period of 8 weeks postpartum. In the sample of women the same research tool had been employed during the preceding pregnancy in the form of a personal interview. RESULTS: The non-response rate was 75/207 (36%) with a small difference in the rates of drop-out between women who had been abused or not abused during their pregnancy. Of the 132 women answering the questionnaire, 32 reported threats, physical or sexual abuse postpartum. Of those 32 women, 22 (69%) stated that they had not been subject to abuse previously. Women who were abused postpartum were older and were married to a higher extent than those who had been abused prior to and during pregnancy. CONCLUSIONS: Abuse does not appear to be restricted to a specific socio-demographic group of women or to a specific period in a woman's reproductive life. Therefore, questions to women regarding both threats and physical violence should be part of all clinical practices. PMID- 10733023 TI - Amniotic fluid nitric oxide metabolite levels and nitric oxide synthase localization in feto-placental tissues are modified in association with human labor. AB - Nitric oxide (NO) has a relaxant effect on uterine smooth muscle and may be implicated in maintaining uterine quiescence during pregnancy. In order to investigate the role of nitric oxide in human parturition, we have measured NO metabolite levels in maternal and fetal compartments in association with labor, both at term and preterm. We have also examined the localization and distribution of NO synthase (NOS) isoforms in placentas and fetal membranes after term and preterm delivery by means of immunohistochemistry. Although no differences were present in maternal and fetal blood and in maternal urine among groups, we found that NO metabolite concentrations were higher in amniotic fluid collected from women in labor than in non-laboring patients, both at term (15.4+/-1.6 vs. 6.8+/ 0.6 microM/mg creatinine) and preterm (16.7+/-2.0 vs. 7.0+/-0.8 microM/mg creatinine). Ir-bNOS staining appeared to be decreased in fetal membranes collected after spontaneous labor at term and preterm. In contrast, a stronger staining for iNOS was detected in trophoblast cells of fetal membranes from women in labor than in those from non-laboring women. We suggest that NOS isoenzymes in fetal placental tissues are differently regulated and might play different roles during pregnancy. PMID- 10733024 TI - Stage-related superoxide anion production of granulocytes of gynecologic cancer patients. AB - OBJECTIVE: To measure superoxide anion production of polymorphonuclear leukocytes (PMNLs) in 58 gynecologic cancer patients and compare to that of healthy controls. METHODS: PMNLs were separated from peripheral blood samples by Ficoll and subsequent Percoll gradient sedimentation. Baseline and phorbol-dibutyrate (100 nmol/l) stimulated superoxide anion production was measured spectrophotometrically as superoxide dismutase inhibitable reduction of ferricytochrome c (50 micromol/l) absorbance. Differences between the mean superoxide anion production of different patient groups and the control group were assessed by Student's t-test. RESULTS: The mean superoxide anion production of PMNLs of healthy controls was 1.855 nM/min/3 x 10(5) cells (SD=0.211 nM/min/3 x 10(5) cells). Superoxide anion production of gynecologic cancer patients and healthy controls varied in a wide range. PMNLs of patients had lower baseline and stimulated activity than those of healthy volunteers. The frequency of a mean superoxide production at least 2 x SD below the control showed a parallel increase with advancing stage. CONCLUSION: Granulocytes of gynecologic cancer patients have reduced capacity and inducibility of superoxide anion production already at an early stage of disease. PMID- 10733026 TI - Liver invasion by recurrent granulosa cell tumour of the ovary: imaging findings. AB - Granulosa cell tumour of the ovary is a rare neoplasm of low malignant potential, late recurrences, local spread and high survival rates. We report the MR imaging appearance of invasion of the liver parenchyma by recurrent granulosa cell tumour of the ovary 15 years after initial diagnosis. PMID- 10733025 TI - Surgical decompression and radiation therapy in epidural metastasis from cervical cancer. AB - Spinal cord compression by epidural metastasis is considered an exceptional complication in patients with cervical carcinoma. We report three patients treated for a cervical carcinoma who developed epidural metastasis with spinal cord compression at 9, 25 and 48 months after primary treatment of the uterine malignancy. All patients had poorly-differentiated adenocarcinomas with lymphovascular space invasion, and two had lymph node metastasis. All patients underwent emergency decompressive laminectomy followed by radiotherapy and a partial recovery of the neurological function was achieved. In two patients the spinal cord was the only site of recurrent disease, whereas the other had lung and brain metastasis at the time of epidural involvement diagnosis. All three patients, however, died of disseminated disease. Surgical decompression followed by radiation therapy may result in a complete preservation of the neurologic functions in patients with spinal cord compression secondary to metastatic carcinoma of the uterine cervix. Considering the propensity for disseminated disease, long term survival might be achieved only with the use of effective chemotherapy. PMID- 10733027 TI - The PR interval-fetal heart rate relationship during repetitive umbilical cord occlusions in immature fetal sheep. AB - OBJECTIVE: To evaluate the relationship of the PR interval and fetal heart rate during repetitive umbilical cord occlusions in immature sheep fetuses. STUDY DESIGN: In seven chronically cannulated immature sheep fetuses [gestational age 90.6 days (mean)], we analyzed continuous fetal electrocardiogram recordings during repetitive cord occlusions for 2 out of every 5 min until fetal mean arterial pressure dropped to 50% of baseline value. PR interval-fetal heart rate correlation coefficients (Pearson) was measured on consecutive blocks of 2.5 min. R-values of the baseline and the repetitive occlusion period were compared by Fisher's exact test. RESULTS: Repetitive cord occlusions resulted in acidosis and hypotension. Two fetuses died at the end of the repetitive occlusion period. Four out of seven fetuses showed a significant change from a negative relationship between the PR interval and fetal heart rate during baseline to a predominantly positive relationship during the repetitive occlusion period. CONCLUSION: In immature fetal sheep, a change from a negative relationship between the PR interval and fetal heart rate to a predominantly positive relationship between the PR interval and fetal heart rate was observed in four out of seven fetuses following the initiation of repetitive umbilical cord occlusions. PMID- 10733028 TI - Acardiac fetus in a triplet pregnancy: ultrasound pitfalls. A case report. AB - This communication aims at illustrating ultrasound diagnostic difficulties in early pregnancy with acardiac fetus. Our case concerns a spontaneously conceived triplet pregnancy. It was diagnosed as a twin pregnancy at 11 weeks of amenorrhea. One and a half months later the patient was referred to our center for spontaneous premature rupture of membranes with the diagnosis of a fetal demise in a triplet pregnancy. The definite diagnosis of acardia was assessed sonographically by the presence of a reverse blood flow through the umbilical cord, reflex movements, limbs anomalies and discordance between femoral and crown rump length. Two days after admission, the patient developed chorioamnionitis and the three fetuses were expelled. PMID- 10733029 TI - The Zavanelli manoeuvre in shoulder dystocia: case report and review of published cases. AB - A case of severe shoulder dystocia is reported in which, after other methods had failed, cephalic replacement succeeded but was very difficult. The infant suffered severe damage. A review of methods to relieve shoulder dystocia is given, with special attention to published cases of the Zavanelli manoeuvre. It could only be used when all other methods fail. PMID- 10733030 TI - Aneurysm of the umbilical vein: case report and review of literature. AB - We report a case of aneurysm of the umbilical vein, causing fetal death at 41 weeks gestation. We conclude that these aneurysms are a complication of congenital thinning of the vessel wall and want to emphasize that in stillbirths the cause of death may only be revealed by careful placental examination, including the umbilical cord. PMID- 10733031 TI - Disseminated carcinomatosis after laparoscopic surgery for presumably benign ruptured ovarian teratoma. AB - The authors reported the intraperitoneal carcinomatosis after laparoscopic surgery for presumably benign ruptured ovarian teratoma in a 28-year-old woman. A 28-year-old female patient exhibited intraperitoneal carcinomatosis after a laparoscopic surgery for ruptured mature teratoma of the ovary with occult malignant transformation. The complication was found two months after initial laparoscopic surgery. Laparoscopic surgery was smooth including oophorectomy, and removing all spilled specimens within the abdominal cavity. At the end of the laparoscopic surgery, cleaning the abdominal cavity and irritating the port site were also performed. Cytology of the abdominal cavity and all removed specimens did not show evidence of malignancy. She followed up regularly and uneventfully except for persistently abdominal fullness and erythematous change of umbilical portal site. Evidence demonstrated intestinal obstruction associated with ascites after a detailed evaluation. Although the patient received supportive treatment the symptom exacerbated. Therefore, the patient was treated with exploratory laparotomy. Pathology proved with intraperitoneal carcinomatosis caused by squamous cell carcinoma. All tumor evaluations including tumor markers, a thorough physical examination, imaging studies and evaluations of the nuclear medicine were negative except of intraperitoneal carcinoma, origin to be determined. The patient is dead 14 months' post-treatment by exploratory laparotomy. Although it was not clear that the laparoscopic approach or the disease itself worsened indeed the prognosis because the disease was already disseminated before the laparoscopy, we still emphasized the possible limitation of laparoscopic surgery if diagnosis at original surgery is impaired, of if excision is incomplete and the delay between the laparoscopic procedure and the carcinomatosis. PMID- 10733032 TI - Complex congenital heart disease and pre-eclampsia. A case report. PMID- 10733033 TI - Successful preoperative diagnosis of massive ovarian edema aided by comparative imaging study using magnetic resonance and ultrasound. AB - Massive ovarian edema (MOE) is a rare disease. Therefore, preoperative diagnostic method of massive ovarian edema (MOE) has not been established. We have succeeded in making a preoperative diagnosis of MOE aided by ultrasonogram and magnetic resonance imaging (MRI) and the patient's ovaries were preserved. Characteristics and proposed diagnostic imaging criteria for MOE are discussed. PMID- 10733034 TI - Maternal-fetal conflict. AB - Advances in prenatal care have brought about a greater understanding as to the special status of the fetus to the point that it is considered a patient in its own regard. Pregnant women generally follow the medical recommendations of their physicians that are intended for the benefit of their baby. Any situation where maternal well-being or wishes contradict fetal benefit constitutes a maternal fetal conflict. Such situations include a broad range of possible interventions, non-interventions, and coercive influences. In such cases, the attending physician is expected to attain an attitude that involves either the respect of the woman's autonomy and right to privacy, which precludes any approach other than to accept her decision, or to modify this absolute for the beneficence of the fetus. Current ethical viewpoints range from absolute respect for maternal autonomy with no persuasion allowed, to gentle persuasion and to others which permit intervention and overriding of the woman's autonomy. Court-ordered decisions enforcing the pregnant woman to undergo a procedure in order to improve fetal outcome have been criticized as an invasion of a woman's privacy, limitation of her autonomy, and taking away of her right to informed consent. PMID- 10733035 TI - Anthropometry in relation to prostate cancer risk in the Netherlands Cohort Study. AB - In the Netherlands Cohort Study, the authors investigated whether anthropometry is associated with prostate cancer risk. At baseline in 1986, 58,279 men aged 55 69 years completed a self-administered questionnaire on diet, anthropometry, and other risk factors for cancer. After 6.3 years of follow-up, 681 cases were available with complete data on height and weight at baseline, and for 523 cases, there were data for weight at age 20 years. In both age-adjusted and multivariate case-cohort analyses (adjusted for age, family history of prostate cancer, and socioeconomic status), height, body mass index (BMI; kg/m2), and lean body mass (kg) at baseline were not associated with prostate cancer risk. The rate ratios of prostate cancer for men with a BMI at age 20 of less than 19, 19-20.9, 21 22.9, 23-24.9, and 25 or greater were 1.00 (reference), 1.06, 1.09, 1.39, and 1.33, respectively (p for trend = 0.02). For gain in BMI from age 20 years to age of the cohort at baseline, an inverse trend in risk was found (p for trend = 0.01), which did not persist after additional adjustment for BMI at age 20 (p for trend = 0.07). In subgroup analyses, no clear associations between anthropometry and advanced prostate cancer were found. Our findings suggest that body composition in young adulthood may already exert an effect on later risk of prostate cancer. PMID- 10733036 TI - Invited commentary: do anthropometric measures predict risk of prostate cancer? PMID- 10733037 TI - Respiratory cancer in a cohort of copper smelter workers: results from more than 50 years of follow-up. AB - Several studies have linked inhalation of airborne arsenic with increased risk of respiratory cancer, but few have analyzed the shape of the exposure-response curve. In addition, since inhaled airborne arsenic affects systemic levels of inhaled arsenic, there is concern that inhaled arsenic may be associated with cancers of the skin, bladder, kidney, and liver, which have been linked to ingested arsenic. The authors followed 8,014 white male workers who were employed for 12 months or more prior to 1957 at a Montana copper smelter from January 1, 1938 through December 31, 1989. A total of 4,930 (62%) were deceased, including 446 from respiratory cancer. Significantly increased standardized mortality ratios (SMRs) were found for all causes (SMR = 1.14), all cancers (SMR = 1.13), respiratory cancer (SMR = 1.55), diseases of the nervous system and sense organs (SMR = 1.31), nonmalignant respiratory diseases (SMR = 1.56), emphysema (SMR = 1.73), ill-defined conditions (SMR = 2.26), and external causes (SMR = 1.35). Internal analyses revealed a significant, linear increase in the excess relative risk of respiratory cancer with increasing exposure to inhaled airborne arsenic. The estimate of the excess relative risk per mg/m3-year was 0.21/(mg/m3-year) (95% confidence interval: 0.10, 0.46). No other cause of death was related to inhaled arsenic exposure. PMID- 10733038 TI - Carpal tunnel syndrome: a nested case-control study of risk factors in women. AB - Risk factors for the development of carpal tunnel syndrome in women were studied by means of a nested case-control analysis of a prospective cohort study of the health effects of oral contraception in British women. A total of 1,264 women who had a diagnosis of carpal tunnel syndrome reported by their general practitioner between 1968 and 1993 were compared with 1,264 age-matched control women who did not have this diagnosis. The syndrome was associated in older women with some hormonal factors, notably past use of oral contraception (adjusted odds ratio in women aged 40 years and over = 1.38, 95 percent confidence interval: 1.08, 1.76) and more generally with obesity (adjusted odds ratio = 1.68, 95 percent confidence interval: 1.29, 2.18). However, the strongest link was with a previous history of another musculoskeletal complaint for which consultation had been sought (adjusted odds ratio = 1.98, 95 percent confidence interval: 1.61, 2.42). Previous findings of a higher risk in women with diabetes and myxoedema were confirmed, but these contribute only a small proportion of all cases in women. There was no link with psychologic problems or nonmusculoskeletal pain complaints. The previously described increased incidence of carpal tunnel syndrome in women may be partly due to hormonal factors, but is also related to an underlying propensity to musculoskeletal problems and their higher overall frequency in women. PMID- 10733039 TI - Risk factors for abdominal aortic aneurysm: results of a case-control study. AB - Abdominal aortic aneurysms (AAAs) have historically been considered to be a manifestation of atherosclerosis. However, there are epidemiologic and biochemical differences between occlusive atherosclerotic disease and aneurysmal disease of the aorta. A case-control study was performed to investigate risk factors for AAA at the two tertiary care hospitals in Winnipeg, Manitoba, Canada, between June 1992 and December 1995 to investigate risk factors for AAA. Newly diagnosed cases of AAA (n = 98) were compared with non-AAA controls (n = 102), who underwent ultrasound for indications similar to those of the cases. Compared with that for never smokers, the adjusted odds ratio (OR) was 2.75 (95% confidence interval (CI): 0.85, 8.91) for 1-19 pack-years, 7.31 (95% CI: 2.44, 21.9) for 20-34 pack-years, 7.35 (95% CI: 2.40, 22.5) for 35-49 pack-years, and 9.55 (95% CI: 2.81, 32.5) for 50 or more pack-years. Other factors significantly associated with AAA were male gender (OR = 2.68, 95% CI: 1.26, 5.73), diastolic blood pressure (OR per 10 mmHg = 1.88, 95% CI: 1.31, 2.69), and family history of AAA (OR = 4.77, 95% CI: 1.26, 18.1). There was an inverse association between diabetes mellitus and AAA (OR = 0.32, 95% CI: 0.12, 0.88). Neither clinical hypercholesterolemia nor serum levels of total cholesterol, low density lipoprotein cholesterol, and high density lipoprotein cholesterol was associated with AAA. The results of this study suggest that the risk factors for AAA differ from those for atherosclerosis and that atherosclerosis per se is not an adequate explanation as the cause of AAAs. PMID- 10733040 TI - Longitudinal study of risk factors for coronary heart disease across the menopausal transition. AB - The patterns of change in blood lipids, diastolic blood pressure, body mass index, smoking and drinking behaviors, and exercise were examined in an ongoing longitudinal study from 1991 to 1995 of 150 middle-aged Melbourne, Australia, women as they passed through menopause. Changes in risk factors over time were examined with reference to time of the final menstrual period (FMP). Random effects models were fitted with adjustments for repeated measures and other covariates, including age. There were overall net increases between 3 years before and the 3 years after menopause of 0.25 mmol/liter for low density lipoprotein cholesterol, 0.05 mmol/liter for high density lipoprotein cholesterol (HDL cholesterol), 0.34 mmol/liter for triglycerides, 0.12 kg/m2 for body mass index, and 0.48 mmHg for diastolic pressure. The proportion of drinkers decreased by 13%, that of smokers increased by 17%, and that of women who exercised at least once a week increased by 6%. The only change dependent on the FMP was a significant decrease in HDL cholesterol (counterbalanced by a similar rise in HDL cholesterol in the year before the FMP), and the rate of decrease was maximal around 9 months after menses ceased, with an instantaneous estimate of slope of 0.55 mmol/liter per year. PMID- 10733041 TI - Perinatal factors associated with blood pressure during childhood. AB - This study aims to determine whether variables reflecting an adverse intrauterine environment are associated with childhood blood pressure. The authors conducted a secondary analysis of data from a prospective cohort of children born to healthy, nulliparous women enrolled in a randomized controlled trial. A total of 518 children were traced in 1995-1996 from 614 eligible children born in a clinic in Rosario, Argentina. The outcome was systolic blood pressure at 5-9 years. Hemoglobin during pregnancy was positively associated with children's pressure. Other maternal characteristics during pregnancy (blood pressure, smoking, weight gain, weight at 20 weeks' gestation, and glycemia) and size at birth (birth weight, ponderal index, head circumference/length ratio, and small for gestational age) were not associated with children's pressure. Among children in the upper quartile of body mass index, there was a weak inverse correlation between birth weight and systolic pressure, and systolic pressure was 14.8 mmHg (95 percent confidence interval: 3.3, 26.4) higher in low birth weight children than in others. The main predictors of childhood pressure were childhood body mass index and maternal pressure outside pregnancy. In this healthy population, the authors found weak support for an association between variables reflecting an adverse fetal environment and childhood blood pressure. Low birth weight was a risk factor for high blood pressure only in overweight children. PMID- 10733042 TI - Association of very low birth weight with exposures to environmental sulfur dioxide and total suspended particulates. AB - This paper presents results of a population-based case-control study of the association between maternal exposures to environmental sulfur dioxide and total suspended particulates (TSP) and risk for having a very low birth weight (VLBW) baby, i.e., one weighing less than 1,500 g at birth. The study, which took place between April 1, 1986 and March 30, 1988, comprised 143 mothers of VLBW babies and 202 mothers of babies weighing 2,500 g or more living in Georgia Health Care District 9. Environmental exposure estimates (microg/m3) were obtained through environmental transport modeling that allowed us to assign environmental sulfur dioxide and TSP exposure estimates at the birth home of each study subject. Exposures less than or equal to 9.94 microg/m3, the median of TSP and sulfur dioxide exposures for the controls, were considered as referent exposures. Exposures to atmospheric TSP and sulfur dioxide above the 95th percentile (56.75 microg/m3) yielded an adjusted odds ratio of 2.88 (95% confidence interval (CI): 1.16, 7.13), that from above the 75th to the 95th percentile (25.18-56.75 microg/m3) yielded an adjusted odds ratio of 1.27 (95% CI: 0.68, 2.37), and that from above the median (9.94 microg/m3) to the 75th percentile, an adjusted odds ratio of 0.99 (95% CI: 0.51, 1.72). The trend demonstrated in these adjusted estimates suggests an association between VLBW and maternal exposures to high levels of air pollution. PMID- 10733043 TI - Sex, birth weight, and the risk of stillbirth in Scotland, 1980-1996. AB - The aim of this study was to determine whether the risk of stillbirth associated with male fetal sex was modified by fetal growth. The study group consisted of all singleton first births weighing greater than 500 g delivered between 28 and 43 weeks gestation in Scotland in 1980-1996 (n = 469,152). Overall, male fetuses were at an increased risk of stillbirth (relative risk = 1.19, 95 percent confidence interval: 1.10, 1.29). There was a significant negative interaction between male sex and increasing birth weight quintile in term, but not preterm, births. The interaction was virtually identical when calculated independently for births in the periods 1980-1987 and 1988-1996. There were linear decreases in the proportion of stillbirths and the proportion of birth weights in the lowest quintile over the period 1980-1996. Adjustment for year of birth did not affect the relation between male sex and stillbirth. However, adjustment for birth weight resulted in a loss of the association between year of birth and risk of stillbirth. The authors concluded that 1) the association between male sex and stillbirth diminishes with increasing birth weight quintile, and 2) there was a fall in the proportion of stillbirths in Scotland between 1980 and 1996, which may have been due to a fall in the proportion of small babies over the same period. PMID- 10733045 TI - Dietary sodium intake and cataract: the Blue Mountains Eye Study. AB - A population-based cross-sectional study (n = 2,873) was conducted near Sydney, Australia, from January 1992 to January 1994 to assess the relation between dietary sodium intake and risk of cataract. Photographs of subjects' lenses were graded for cortical, nuclear, and posterior subcapsular cataracts. Dietary sodium intake was assessed with a food frequency questionnaire. The study found that higher sodium intakes were associated with greater risk of posterior subcapsular cataract (p for trend = 0.006). The adjusted relative risk was 2.0 (95% confidence interval: 1.2, 3.4) for subjects in the highest versus the lowest quintile of sodium intake. These findings suggest that a high-salt diet may increase the risk of posterior subcapsular cataract. PMID- 10733044 TI - Age at menopause and childbearing patterns in relation to mortality. AB - Several studies have reported increased mortality risk with early natural menopause. More recently, mortality risk was reported to be reduced among women who gave birth at age > or =40 years. The association between reproductive history and mortality was explored among 826 women in a prospective study involving 18,959 person-years of follow-up (from age 50 to 1990-1991) and 108 deaths. After adjustment for age and other covariates, the risk ratio among parous women was 1.53 (95% confidence interval: 0.58, 4.07) for natural menopause at age < or =45 years compared with > or =51 years. In contrast to a previous report, however, the highest estimated mortality risk was seen among women who gave birth in their forties (adjusted risk ratio = 2.14, 95% confidence interval: 1.05, 4.38) compared with having a last birth at ages 30-34 years. PMID- 10733046 TI - Concordance among measures of pregnancy outcome based on fetal size and duration of gestation. AB - Epidemiologic and clinical studies of pregnancy outcome often consider a variety of related, overlapping outcome measures. The overlap among these measures was analyzed using data from the Mount Sinai Hospital Perinatal Data Base, New York City, New York. A total of 52,621 births from 1986 through 1996 were included, with information on gender, ethnicity, birth weight, and gestational age assigned based on last menstrual period or early ultrasound. The authors considered very low birth weight (VLBW) (<1,500 g), low birth weight (LBW) (<2,500 g), degrees of preterm delivery (less than 32, 34, and 37 weeks' gestation), and small for gestational age (less than the 10th percentile of weight for gestational age) births. Infants at the extremes of gestational age (<32 or 34 weeks' gestation) were almost always LBW (97.6 and 91.7%, respectively), and those who were VLBW were almost always preterm (99.2%). However, only 69.2% of LBW infants were preterm, and 50.2% of preterm infants were LBW (kappa = 0.54). Only for VLBW and less than 32 weeks' gestation were both measures of overlap at least 70% (kappa = 0.98). The lack of concordance among measures suggests that multiple outcome measures be considered and that results from analyses using disparate measures not be compared directly. PMID- 10733047 TI - Re: "Dietary flavonoid intake and risk of cardiovascular disease in postmenopausal women". PMID- 10733048 TI - Prevalent vertebral deformities predict mortality and hospitalization in older women with low bone mass. Fracture Intervention Trial Research Group. AB - OBJECTIVES: To determine the relationship between prevalent vertebral deformities and the risk of mortality and hospitalization in older women with low bone mass. DESIGN: A prospective cohort study. SETTING: Eleven clinical centers in the United States. PARTICIPANTS: A total of 6459 community-dwelling women with low bone mass aged 55 to 81 participated in the Fracture Intervention Trial (FIT), a multicenter clinical trial of alendronate that enrolled women into one of two study arms based solely on the presence or absence of existing radiographic vertebral deformities. There were 2027 women with at least one vertebral deformity enrolled in the vertebral fracture arm of FIT and followed prospectively for an average of 2.9 years, whereas 4432 women with no vertebral deformity were enrolled in the clinical fracture arm of FIT and followed prospectively for an average of 4.2 years. MEASUREMENTS: Determination of prevalent vertebral deformities on baseline lateral thoracic and lumbar spine radiographs was made at the coordinating center using a combination of radiographic morphometry by digitization and semiquantitative radiologic interpretation. Deaths were confirmed by obtaining copies of original death certificates of all participants who died. Episodes of hospitalization were captured through adverse event reporting; hospitalizations resulting solely from adverse events containing the words "fracture" or "trauma" were excluded from the analyses. RESULTS: During the follow-up period, 122 women died, and 1676 women were hospitalized on at least one occasion for reasons not related solely to fracture. Compared with women without prevalent vertebral deformities, those women with prevalent deformities had higher risks of mortality (age- and treatment assignment-adjusted relative risk 1.60, 95% confidence interval (CI), 1.10-2.32) and hospitalization (age- and treatment assignment-adjusted relative risk 1.18, 95% CI, 1.06-1.31). In addition, further adjustment for other factors, including smoking status, physical activity, hypertension, coronary heart disease, obstructive lung disease, any fracture since the age of 50, health status, total hip BMD, and body mass index did not alter the association between prevalent vertebral deformities and risk of mortality substantially (multivariate relative risk 1.49, 95% CI, 1.05-2.21). Adjustment for all these factors and diabetes also did not change the relationship between prevalent vertebral deformities and hospitalization (multivariate relative risk 1.14, 95% CI, 1.02 1.27). Rates of mortality and hospitalization increased with increasing number of prevalent vertebral deformities (tests for trend P < .01). CONCLUSIONS: Prevalent vertebral deformities in older women with low bone mass are associated with increased risks of mortality and hospitalization. Only a portion of this increased risk was explained by other known predictors of these outcomes. PMID- 10733049 TI - Relationship between low blood pressure and depressive symptomatology in older people. AB - OBJECTIVES: To determine if low blood pressure is associated with a definable constellation of somatic and psychological symptoms in older persons. DESIGN: A population-based study. SETTING: In-home interviews in five southwestern states. PARTICIPANTS: A total of 2723 Mexican Americans aged 65 or older not living in institutions. MEASURES: Blood pressure, Center for Epidemiologic Studies Depression Scale (CES-D), global self-rating of health, and self-esteem. RESULTS: Bivariate analyses indicate a significant relationship between low blood pressure and increased depressive symptomatology; for example, systolic hypotensive subjects scored a CES-D mean of 12.07 +/- .67 compared to 8.99 +/- .95 for normotensives (P < .01). Regression analyses supported these findings when controlling for confounders such as gender, age, and use of antihypertensive medications. Subjects with low blood pressure also scored lower on self-esteem and global self-reported health and reported more days waking up feeling tired. CONCLUSIONS: These data support the existence of a relationship between low blood pressure and higher levels of depressive symptomatology as well as a constellation of somatic and psychosocial symptoms. PMID- 10733050 TI - Coronary artery calcification in older adults with minimal clinical or subclinical cardiovascular disease. AB - BACKGROUND: Coronary artery calcification (CAC) reflects the extent of coronary artery atherosclerosis. The extent of coronary artery calcification is not well described in older adults. OBJECTIVE: To determine the extent of CAC in older adults participating in a large population study of cardiovascular disease (CVD), especially those characterized as having minimal clinical or subclinical cardiovascular disease. DESIGN: An observational epidemiologic study. POPULATION: Participants in the Cardiovascular Health Study Cohort, mean age 78 years, who had electron beam computed tomography (EBT) scan of the heart (n = 133); included were 106 persons with no prior evidence of clinical or subclinical CVD. MEASUREMENTS: Total CAC score was measured using cardiac EBT. Cardiovascular disease and risk factors, as well as carotid ultrasound, electrocardiogram, echocardiogram, and ankle-arm index, had been measured previously to define subclinical disease. Previous cerebral magnetic resonance imaging was also evaluated. RESULTS: Overall, the CAC scores were higher in those with clinical cardiovascular disease or evidence of subclinical cardiovascular disease than in those with no evidence of disease. For the 106 participants without evidence of clinical or subclinical disease, the median score was 176, compared with 367 in those with subclinical disease and 923 in those with clinical CVD. Seventeen persons had scores of zero. There was little difference in risk factors across quartiles of CAC in the subgroup of 106 with prior characterization of minimal CVD despite the broad range of CAC scores. There was a higher proportion of those with white matter grade > or = 2 by magnetic resonance imaging among those with higher CAC scores (P = .025). Infarct-like lesions prevalence ranged from 12.5% in the lowest group to 47.1% in the highest CAC group (P = .019). CONCLUSIONS: Older adults with evidence of clinical or subclinical CVD have higher total CAC scores. Though the extent of coronary artery calcification was lower in those with minimal evidence of CVD, the range was broad and not explained by CVD risk factors. PMID- 10733051 TI - Glycemic control of older adults with type 2 diabetes: findings from the Third National Health and Nutrition Examination Survey, 1988-1994. AB - BACKGROUND: Although nearly half of all people who have diabetes are aged 65 or older, glycemic control of older adults with diabetes has not been well described. METHODS: We conducted a cross-sectional study of 1,482 participants with self-reported type 2 diabetes in the Third National Health and Nutrition Examination Survey, 1988-1994 (NHANES III), a nationally representative sample of the US noninstitutionalized civilian population. Variables included in this analysis included age, sociodemographic factors, drug treatment, and level of glycemic control. RESULTS: The mean % (+/-SE) HbA1c was 7.78 +/- 0.21, 7.64 +/- 0.18, 7.71 +/- 0.14, and 7.27 +/- 0.14 in persons aged 20 to 54, 55 to 64, 65 to 74 and > or = 75 years, respectively. The mean mg/dL (+/-SE) fasting plasma glucose (FPG) was 175.9 +/- 7.6, 164.5 +/- 6.1, 183.3 +/- 5.3, and 158.5 +/- 5.5 in the four age groups and older, respectively. When controlling for race, gender, education, and duration of diabetes, age was not significantly associated with levels of HbA1c [P (trend) =0.17] or FPG [P (trend) =0.19]. Among NHANES III participants aged 65 or older, ADA guidelines for glycemic control (HbA1c < 7%) were achieved by 71%, 44%, and 27% of persons using no drug therapy, oral hypoglycemic agents, and insulin, respectively. CONCLUSIONS: Although many older adults with type 2 diabetes do not achieve targets for glucose control, there is no evidence to suggest that community-dwelling older adults with diabetes are treated less vigorously than younger persons with diabetes. PMID- 10733052 TI - The impact of metrifonate therapy on caregivers of patients with Alzheimer's disease: results from the MALT clinical trial. Metrifonate in Alzheimer's Disease Trial. AB - OBJECTIVE: To assess the impact on burden reported by caregivers of patients with mild to moderate Alzheimer's disease (AD) who were treated with metrifonate during a randomized double blind clinical trial. DESIGN: Randomized clinical trial, with a 2-week screening period and a 26-week double blind, placebo controlled, treatment phase. Caregivers were assessed at baseline, at 12 weeks, and at end of trial. SETTING: Caregivers were interviewed at clinics as part of the assessment of the patients. PARTICIPANTS: Six hundred and three caregivers of AD patients who were enrolled in the MALT trial; 591 (98%) provided data suitable for analysis at baseline, and 546 (91%) provided data allowing for inclusion in the analysis of change scores. MEASUREMENTS: The Caregiver Burden Assessment consisted of the Screen for Caregiver Burden, including both subjective (SCB subj) and objective (SCB-obj) scores; the cognitive subscale of Poulshock and Deimling (PD); an abridged version of the Relatives Stress Scale (aRSS); assessments of time spent in providing care, including the Caregiver Activity Time Scale (CATS); and demographic and background variables on both the patient and caregiver. RESULTS: Treatment of mild to moderate AD patients with metrifonate for a duration of 26 weeks significantly reduced the psychological burden of care to the caregivers, as measured by the SCB-subj, the PD, and the aRSS. There were no statistically significant differences on the measures assessing the time spent in caregiving, except for the caregiver's subjective impression of the change in time spent providing care during the trial. When comparing individual dose groups, most of the measures of burden showed the largest benefits in burden for the 60/80 mg group, followed by the 40/50 mg group, and then the placebo group. However, there was no statistically significant dose effect. CONCLUSIONS: This study provides the first evidence from a randomized clinical trial of any acetylcholinesterase inhibitor used in the treatment of AD demonstrating a positive impact on the patient's caregiver as well as benefits to the patient. These results were shown consistently across several measurement scales and were observed after six months of treatment. These findings reinforce the clinical significance of research that has shown that metrifonate has beneficial impacts on the cognitive, behavioral, and functional abilities of AD patients. Because caregiver burden is a leading factor in the decision for institutional care placement, the ability to favorably impact that burden through pharmacological treatment of the patient is important. PMID- 10733053 TI - Can the control of bodily orientation be significantly improved in a group of older adults with a history of falls? AB - OBJECTIVE: To determine the short-term effectiveness of a biofeedback-based, computerized intervention that applied the principles of the ecological theory of perception and control of bodily orientation to the rehabilitation of older adults with a history of falling. PARTICIPANTS: Forty-five older adults (X = 78.5 years) with a history of falls were assigned to either a control (n = 21) or intervention group (n = 24). DESIGN: A 2 x 2 (Group x Time) mixed factorial design with repeated measures on the second factor. SETTING: A motor control research laboratory in a university setting. MEASUREMENT: Impairments in multiple dimensions of balance (i.e., dynamic postural control and sensory organization) were measured before and after an 8-week balance intervention using the 100% Limits of Stability Test and Sensory Organization Test. More functional limitations in balance and/or mobility were measured using two clinical tests: Berg Balance Scale and Timed Up and Go test. MAIN RESULTS: Repeated measures multivariate analyses and follow-up testing indicated significant improvements in the dynamic balance and sensory integration capabilities in the case of the intervention group only (P < .03). Significant improvements were also observed in the two clinical measures of balance and mobility evaluated before and after the intervention for the intervention group only. CONCLUSIONS: Theory-driven rehabilitation programs that focus on manipulating individual, task, and environmental constraints concurrently can significantly improve the control of bodily orientation of older adults with a previous history of falls in both static and dynamic action environments. Moreover, the significant improvements in functional performance observed for the intervention group suggest that interventions emphasizing task-specific practice are not the only means by which an older adult's ability to perform daily activities requiring postural control can be improved. PMID- 10733054 TI - Mortality and institutionalization following hip fracture. AB - OBJECTIVES: To identify determinants of mortality and institutionalization after hip fracture and to identify those older hip fracture patients at high risk of death or institutionalization after hip fracture. DESIGN: Population-based prospective inception cohort study of hip fracture patients; patients were assessed in the hospital and at 3 months following the hip fracture. SETTING: Edmonton area hip fracture patients admitted to one of two Edmonton, Alberta, Canada, acute care centers between July 10, 1996, and August 31, 1997. PARTICIPANTS: Patients were residents of the Edmonton area and over the age of 64. Those who had previously fractured the same hip within the past 5 years or had some pathological condition underlying the hip fracture were excluded. Of 610 eligible patients, 558 contributed some baseline information and were included in the mortality analysis; the institutionalization analysis was restricted to the 338 patients who lived in the community before fracture, survived the 3-month period postfracture, and had completed a 3-month follow-up interview. MEASUREMENTS: The baseline interview was done in the hospital to assess mental status, prefracture physical function, prefracture health perception, and prefracture social support. The 3-month follow-up interview was done by phone to assess physical function, health perception, and social support 3 months postfracture. Demographic and comorbidity information was collected from medical records. RESULTS: Low mental status in hospital was found to increase the chances of mortality and institutionalization, and male gender was found to increase mortality risk fourfold. Each additional 10 years of age increased the risk of institutionalization approximately 2.5 times. Patients with lower postfracture physical function had at least five times the risk of institutionalization compared to patients with high postfracture physical function. CONCLUSIONS: Cognitive impairment, older age, and gender were associated with increased risk of poor outcome following hip fracture. The socioeconomic variables--social support and health perception--did not contribute significant additional information in explaining mortality or institutionalization risk. While demographic factors cannot be modified, physical function 3 months postfracture may be amenable to intervention and may reduce the risk of institutionalization. Intervening to increase postfracture physical function may be particularly beneficial to older patients, or to those who are cognitively impaired. PMID- 10733055 TI - Extent and determinants of hospitalization in a cohort of older disabled people. AB - OBJECTIVES: The purpose of this prospective cohort study was to determine the extent and determinants of hospitalization in a population sample of older disabled people. DESIGN: A longitudinal cohort study. SETTING: Noninstitutionalized disabled people in the region of Augsburg, South Germany. PARTICIPANTS: The study population included 2427 persons, aged 60 years and older, who, between 1991 and 1993, applied for benefits from the statutory health insurance system provided to the most severely handicapped noninstitutionalized persons. MEASUREMENTS: Baseline variables were derived from a standardized medical examination. Information about hospitalization and mortality until June 30, 1996, was extracted from records of the pertinent health insurance plan. Rate ratios (RRs) of hospitalization were derived from a multivariable Poisson regression model corrected for the correlation of multiple hospitalizations for each person. RESULTS: The overall absolute hospitalization rate was 6.7 hospitalizations per 10 person-years at risk, with a mean length of stay per hospitalization of 19.2 days. Multivariable analysis showed an inverse association of age and hospitalization. Persons cared for primarily by their children had a lower rate of hospitalization (RR = 0.83) than persons relying mainly on professional home care. Medical causes of disability and dependency with respect to activities of daily living were unrelated to the hospitalization rate. A total of 1415 persons (58.3%) died during follow-up. The rate of hospitalization was twice as high among these subjects compared with others. The hospitalization rate increased rapidly during the last year of life, with a peak increase in the last 3 months. CONCLUSIONS: This study expands the current inadequate database on hospitalization in Europe. Further expansion is necessary for the efficient allocation of medical resources to older people, a group steadily increasing in numbers. PMID- 10733057 TI - Patterns of care in the early stages of Alzheimer's disease: impediments to timely diagnosis. AB - OBJECTIVE: Description of factors associated with delay in diagnosis of Alzheimer's disease (AD). DESIGN: A self-administered mail questionnaire. SETTING: Households including someone with AD identified through a nationwide marketing database. PARTICIPANTS: A total of 1480 caregivers of patients diagnosed with AD. MEASUREMENTS: There were two measures of delay examined through caregiver reports: (1) duration in years from first AD signs until determination of a definite problem, and (2) duration from problem recognition to first physician consultation. Also, caregivers were categorized by time since patient's diagnosis and relationship to patient. Within-group analyses examined the impact of these characteristics on delay measures. RESULTS: Mean lag in years from observation of first symptoms to problem recognition for those diagnosed in the past 12 months, the past 13 to 48 months, and the past 49 months or more was, respectively, 1.20, 1.56, and 2.25 (P < .001). The timing of diagnosis also influenced lag from problem recognition to first physician consultation so that subgroups with recent, less recent, and distant diagnosis reported delays in years of .82, .84, and 1.31 (P < .001). Caregiver relationship was not significantly related to these lags. Correct diagnosis of AD was reported by caregivers in only 38% of cases at initial physician consultation. CONCLUSIONS: These results suggest that both caregivers and physicians lack ready understanding of the difference between memory processes in aging and AD. Ongoing public and professional education is needed to convey the basics of the diagnosis of AD. In addition, routine screening for dementia should be considered to surmount attitudinal and logistical barriers. PMID- 10733056 TI - Subjective memory complaint in relation to cognitive performance and depression: a longitudinal study of a rural Chinese population. AB - OBJECTIVES: To examine the associations of subjective memory complaint (SMC) in old age with (a) objective test performance, (b) past and subsequent cognitive decline, and (c) depression. DESIGN: A group of community residents were examined twice during a 3-year period. SETTING: Two townships on a rural Chinese islet. PARTICIPANTS: A total of 543 men and women aged 65 years and older. MEASUREMENTS: During each examination, neurologists interviewed and examined all participants for dementia and asked the question, "Do you have trouble with your memory?" In addition, research assistants administered (a) the Cognitive Abilities Screening Instrument (CASI) to assess cognitive abilities, including long-term memory (LTM) and short-term memory (STM), and (b) the Geriatric Depression Scale-Short Version (GDS-S) to assess symptoms of depression. RESULTS: At each examination, almost half of the subjects acknowledged having trouble with their memory (the SMC+ group). At both examinations, the SMC+ group scored significantly lower on the CASI and significantly higher on the GDS-S than the SMC- group. However, the presence of SMC was not associated with faster cognitive decline over the past or subsequent 3 years. There were no consistent associations between SMC and the demographic variables of age, gender, and education at the two examinations. Logistic regression analysis showed that SMC was associated with poorer memory test scores after controlling for gender, age, education, and depression. CONCLUSIONS: SMC was associated with poorer objective memory performance even after controlling the effect of depression and demographic data, but SMC did not predict faster cognitive decline or dementia over 3 years. PMID- 10733058 TI - A randomized trial of weighted vest use in ambulatory older adults: strength, performance, and quality of life outcomes. AB - BACKGROUND: Lower extremity weakness is a major risk factor for falls and hip fractures. Aging muscle is capable of responding to strengthening techniques. Strategies for providing accessible, inexpensive, safe, and effective strengthening programs for older adults are needed. OBJECTIVE: To evaluate whether use of a weighted vest improved strength, physical performance, markers of bone turnover, or health-related quality of life. DESIGN: A 27-week randomized, controlled, unmasked clinical trial. The primary outcome was peak isokinetic knee extensor strength at follow-up, adjusted for baseline strength. SETTING: Home-based program. PARTICIPANTS: A total of 62 women and men, mean age 74 years. INTERVENTIONS: Subjects were randomized to: no vest (n = 21), 3% body weight (BW) vest (n = 19), or 5% BW vest (n = 22). The vest is a nylon garment with pockets that are loaded with adjustable weights. The vest was prescribed for 2 hours daily, 4 days per week. No specific physical activities were mandated. MEASUREMENTS: All measures were made at baseline and 27 weeks. These included: knee strength and endurance by isokinetic dynamometer; timed physical performance tests; serum osteocalcin and urinary N-telopeptides; and health-related quality of life scales. RESULTS: Follow-up values of muscular strength and endurance, physical performance, bone turnover markers, and health-related quality of life did not differ by treatment assignment. The final study visit was attended by 19 (90%), 15 (80%), and 20 (91%) of the control, 3%, and 5% groups, respectively. Three permanent discontinuations of vest use occurred. CONCLUSIONS: Weighted vest use did not result in improvement in multiple domains of strength and function and did not affect bone turnover markers. We conclude that the training stimulus afforded by the vest (at the dosage tested) was below the required amount to produce strength gains or bone stimulation. PMID- 10733059 TI - Risk factors for symptomatic peripheral arterial disease in older persons in an academic hospital-based geriatrics practice. AB - OBJECTIVE: To investigate risk factors for symptomatic peripheral arterial disease (PAD) in older persons. DESIGN: A retrospective analysis of charts from all older persons seen from January 1, 1998, through June 15, 1999, at an academic, hospital-based geriatrics practice. SETTING: An academic, hospital based geriatrics practice staffed by fellows in a geriatrics training program and full-time faculty geriatricians. PATIENTS: A total of 467 men, mean age 80 +/- 8 years, and 1444 women, mean age 81 +/- 8 years, were included in the study. MEASUREMENTS AND MAIN RESULTS: Symptomatic PAD was present in 93 of 467 men (20%) and in 191 of 1444 women (13%) (P = .001). Significant risk factors for symptomatic PAD by univariate analysis were: age (P = .021 in women); cigarette smoking, hypertension, diabetes mellitus, serum total cholesterol, serum high density lipoprotein (HDL) cholesterol (inverse association), and serum low density lipoprotein (LDL) cholesterol (P < .001 in men and women); obesity (P = .013 in men and .002 in women); and serum triglycerides (P = .027 in women). Significant independent risk factors for symptomatic PAD by stepwise logistic regression analysis were: age (odds ratio = 1.052 in men and 1.025 in women); cigarette smoking (odds ratio = 2.552 in men and 4.634 in women); hypertension (odds ratio = 2.196 in men and 2.777 in women); diabetes mellitus (odds ratio = 6.054 in men and 3.594 in women); serum HDL cholesterol (odds ratio = .948 in men and .965 in women); and serum LDL cholesterol (odds ratio = 1.019 in men and women). CONCLUSIONS: Significant independent risk factors for symptomatic PAD in older men and women were age, cigarette smoking, hypertension, diabetes mellitus, serum HDL cholesterol (inverse association), and serum LDL cholesterol. PMID- 10733060 TI - Hodgkin's disease survival by stage and age. AB - OBJECTIVE: Prior reports on Hodgkin's disease have suggested a biologic behavior difference between young and old patients. A study of 35,033 patients could confirm that older patients do not do as well as young patients regardless of age. METHODS: The National Cancer Data Base provided data from U.S. tumor registries on 35,033 patients newly diagnosed with Hodgkin's disease from 1985 through 1994. For analysis the patients were divided into two time periods, 1985 1989 and 1990-1994. The earlier period provided survival data to assess the impact of age and stage. RESULTS: The overall disease-specific, 5-year survival rate for the 1985-1989 period was 84.9%. For stages I and II, it reached almost 90%. For both observed survival based on all deaths and disease-specific survival, the duration of survival decreased with increasing age. This decrease with age occurred for all stages of the disease. CONCLUSIONS: The data reflect the actual status of management of Hodgkin's disease in the United States rather than the best attainable results. The decreasing survival with increasing age and in all stages further supports the concept of a difference in biologic behavior of Hodgkin's disease associated with age. PMID- 10733061 TI - Exercise and older patients: guidelines for the clinician. AB - Sedentary persons who improve their physical fitness are less likely to die of all causes and of cardiovascular disease than are those who remain sedentary. There now exists a wealth of data demonstrating that physical activity and exercise may ameliorate disease and delay decline in function in the geriatric population. We review evidence that exercise can improve body composition, diminish falls, increase strength, reduce depression, reduce arthritis pain, reduce risks for diabetes and coronary artery disease, and improve longevity. However, many healthcare professionals do not feel adequately prepared to design and prescribe exercise programs for their patients. This review provides a basic overview of the benefits of exercise in the geriatric population and guidelines indicating how to prescribe and facilitate adherence to an exercise program. Healthcare providers are strongly encouraged to promote a less sedentary life style for their older patients, which may augment quality of life in these older individuals. PMID- 10733062 TI - Annotated bibliography of palliative care and end of life care. PMID- 10733063 TI - Colon cancer screening (USPSTF recommendation). U.S. Preventive Services Task Force. PMID- 10733064 TI - Low blood pressure and depression: comorbidity and competing outcomes. PMID- 10733065 TI - Excess mortality following vertebral fracture. PMID- 10733066 TI - Diabetes mellitus in older adults: underdiagnosis and undertreatment. PMID- 10733067 TI - Subclinical cardiovascular disease and atherosclerosis are not inevitable consequences of aging. PMID- 10733068 TI - A matter of age. PMID- 10733069 TI - Home hospital care. PMID- 10733070 TI - ERT and cognitive decline. PMID- 10733071 TI - A loading dose of tamoxifen followed by a standard dose in frail older women with breast cancer is feasible and effective. PMID- 10733072 TI - Recurrent paraparesis in an octogenarian. PMID- 10733073 TI - Pancytopenia and valproic acid: a dose-related association. PMID- 10733074 TI - Erythrocyte plasma membranes obtained from centenarians show different functional properties. PMID- 10733075 TI - Your privacy or your health--will medical privacy legislation stop quality health care? PMID- 10733076 TI - Privacy and health information: the need for a fine-grained account. PMID- 10733077 TI - Privacy and confidentiality concerns--are we up to the challenge? PMID- 10733078 TI - Evaluation of the red blood cell and plasma transfusion guidelines. AB - OBJECTIVES: To determine physicians' preferred content and format for the Guidelines for Red Blood Cell and Plasma Transfusion for Adults and Children before development in order to guide the development process. To obtain physicians' recommendations about the Guidelines' content, format, dissemination and future directions after development to guide future work on the Guidelines. DESIGN: Pre-post Guidelines qualitative design using 20 focus groups (nine pre and 11 post). STUDY PARTICIPANTS: One hundred and seven physicians (5-11 per group) who used at least 1 unit of blood over the past 6 months; with a minimum of 1 year in practice and not retired. A total of 24 physicians attended both pre and post focus groups. In general, specialties of participants included: general practice, surgery pediatrics, obstetrics/gynecology and hematology/oncology. Years of practice ranged from 1 to 35 years (mean = 7 years). MAIN OUTCOME MEASURES: Pre Guidelines focus groups made recommendations on the content and format of the Guidelines; post Guidelines focus groups made recommendations on the same areas along with dissemination and future directions. RESULTS: Three main components of the Guidelines were evaluated: content, format and dissemination. The content, which followed as much as possible that recommended by pre Guidelines focus groups, was deemed appropriate by post Guidelines focus groups, with minor gaps noted (e.g. information on alternatives to blood products). The format addressed many of the concerns raised by pre Guidelines focus groups; however, the post Guidelines focus groups were concerned about the lack of visual aids and categorization of information. The dissemination strategy was successful at diffusing the Guidelines, with some concerns expressed about reaching specific physician target groups. CONCLUSION: The pre-post focus group method was useful in conducting an evaluation of the Guidelines and this method which examined content, format and dissemination could contribute to evaluations of other guidelines. PMID- 10733079 TI - Patient questionnaires in primary health care. Validation of items used in asthma care. AB - OBJECTIVE: To evaluate each item in a patient questionnaire for the purpose of investigating whether the validity of each item is acceptable. DESIGN: The questionnaire was completed by the patients at an ordinary follow-up visit for their asthma, and within 1 week a nurse interviewed them by telephone with the aim of analysing the validity of each item through the use of predetermined criteria. SETTINGS: Patients with asthma in primary health care settings in Sweden. STUDY PARTICIPANTS: Fifty-one patients were consecutively included from three different primary health care units. RESULTS: Nine of 13 items had an acceptable validity. The four items that were not found to have acceptable validity were developed further. CONCLUSION: Evaluating each item in a questionnaire by means of interviews with the specific patient population is a useful method of assuring that the intention of the patient questionnaire has been met. PMID- 10733080 TI - From diagnosis to drug taking: staff compliance with guidelines and patient compliance to prescriptions in Burkina Faso. AB - OBJECTIVE: We studied compliance with guidelines and prescriptions for six steps of the health care process to identify the step with the greatest need for improvement. DESIGN: In a cross-sectional study we used hidden observation in health centres and counting of remaining drugs in home visits. We assessed provider compliance with guidelines for medical history, physical examination, drug choice, and explanation of drug dosing, and patient compliance for drug buying and drug taking. SETTING: The study took place in six rural health centres in Burkina Faso. MAIN OUTCOME MEASURES: We measured unconditional (UPC), conditional (CPC) and accumulated proportions of compliant procedures (APC). UPC determined the proportion of compliant procedures independent from earlier steps. CPC was defined as the proportion of compliant procedures among those which were compliant in all previous steps. APC was the proportion of procedures compliant in all steps including the step concerned. RESULTS: Twenty-three per cent UPC medical history, 27% UPC (CPC = 39%) clinical examination, 59% (83%) drug choice, 22% (40%) explanation of dosing, 71% (75%) drug buying, and 63% (67%) drug taking compliance. Two per cent of the patients had compliant procedures for all steps of the process (APC). CONCLUSION: The majority of patients did not get treatment compliant with guidelines. Diagnosis had the largest need for improvement. UPC, CPC and APC were useful to identify steps with the greatest need for improvement and to assess quantitatively aspects of quality of care. PMID- 10733081 TI - Predictors of satisfaction with surgical treatment. AB - OBJECTIVE: To investigate prospectively which medical, psychosocial or treatment related factors predicted treatment satisfaction and to evaluate the adequacy of a preceding retrospective study which had examined the same factors. Furthermore, to examine the predictors and the stability of the major determinants of patient treatment satisfaction. DESIGN: Assessments made before admission, at discharge and 2 and 4 months after discharge were used to predict both the level and the rate of change in satisfaction with different aspects of treatment. SETTING: Three surgical departments at a University Hospital. STUDY PARTICIPANTS: Four hundred and eighty-two patients electively admitted for several surgical conditions. RESULTS: The central treatment-related measures were the same in the retrospective and prospective studies: global satisfaction with treatment (GS), perceived quality of contact with the nursing (QCN) and medical staff (QCM) and provision of adequate treatment information (INF). More of the variance in GS was explained in the prospective study (48.7% versus 36.3%). GS was most influenced by treatment-related factors with QCN as the strongest predictor in both studies. Only a small portion of the variance in QCN and QCM could be accounted for by the characteristics of the patients. INF was predicted by characteristics of the patients, their illness and life situation and by treatment-related factors. QCN was the strongest predictor of INF. The relationships with the nursing and medical personnel appear to be the major determinants of both patient treatment satisfaction and patients' reception of adequate information about their condition and its treatment. PMID- 10733083 TI - Effect of discharge letter-linked diagnosis registration on data quality. AB - OBJECTIVE: Diagnostic data are essential for the assessment of medical practice: they are needed for retrieval of clinical cases and describing co-morbidity and complications. In most Western countries, diagnosis registration in hospital information systems is based mainly on completing forms after patient discharge. As this registration plays no role in patient care, data quality is usually unsatisfactory. To improve data quality, we redesigned the process of diagnosis registration at a paediatric department, and now paediatricians provide diagnoses with codes in a separate registration heading of the discharge letter. We compared the quality of this discharge letter-linked diagnosis registration with the quality of the previous form based registration. DESIGN: Retrospective study with blinded before and after measurement. Re-abstracted diagnosis descriptions of the text of discharge letters were taken as gold standard. SETTING: A paediatric department in an academic medical centre. STUDY PARTICIPANTS: From each registration period, 60 admissions were selected randomly. Mean age of the patients was 4.5 (SD +/- 5.5) and 5.2 (SD +/- 5.2) years for the old and new situation respectively. Mean length of stay was 8.8 (SD +/- 11.0) and 7.2 (SD +/- 12.4) days. INTERVENTION: Discharge letter-linked diagnosis registration. MAIN OUTCOME MEASURES: Completeness and accuracy, both at three-digit level of ICD-9 CM. RESULTS: Completeness of form-based diagnosis registration was 51% (95% CI, 44-58%) and of discharge letter-linked diagnosis registration 54% (95% CI, 47 60%). Accuracy was 65% (95% CI, 58-72%) and 67% (95% CI, 60-74%) respectively. CONCLUSIONS: The discharge letter-linked diagnosis registration does not provide a better basis for assessment of medical practice than the form based diagnosis registration. PMID- 10733082 TI - An association between length of stay and co-morbidity in chronic airflow limitation. AB - OBJECTIVE: To examine factors which impact on the length of stay and readmission for patients with chronic airflow limitation at a South Australian hospital from December 1996 until March 1998. DESIGN: Discharges from Flinders Medical Centre for patients aged > or = 18 years, where chronic airflow limitation was an active problem, and including a subset with a primary diagnosis of chronic airflow limitation, were identified, retrospectively, by the center's Clinical Coding Service from the hospital's in-patient separation database. SETTING: Flinders Medical Centre, Adelaide, South Australia. OUTCOME MEASURES: Length of stay; number of co-morbidities; readmission within 28 days. RESULTS: Five-hundred and twenty discharges (male:female, 258:262) with a primary diagnosis of chronic airflow limitation (ANDRG-3 177, chronic obstructive airways disease) were identified. Readmission within 28 days was related to the number of co morbidities and to age. A relationship between length of stay and the number of co-morbidities was identified. A mean length of stay of 6.39 days was found for patients with less than five co-morbidities, 5.36 at their first admission to Flinders Medical Centre and 3.25 at their first admission to Flinders Medical Centre with no co-morbidities. These mean lengths of stay fall below overseas data previously published and are consistent with Kong's estimate of an ideal mean length of stay of 3.2 days when a clinical management guideline is used in low-risk chronic airflow limitation patients. CONCLUSIONS: Length of stay and readmission to hospital within 28 days of patients with a primary diagnosis of chronic airflow limitation is at least partly related to the number of co morbidities and to age. The study has highlighted the difficulty of relying on changes to aggregate data as outcome measures for these patients. PMID- 10733084 TI - Setting national standards for practice equipment. Presence of equipment in Estonian practices before and after introduction of guidelines with feedback. AB - BACKGROUND: During the last few years quality assurance has received increasing attention in Estonian health care as well as in family medicine, which is a new speciality in Estonia (since 1993). The modest equipment that district doctors (former primary care doctors) had at their disposal, appeared inadequate considering family doctors' work tasks. AIM: To determine the type of equipment available in primary care practices before setting a standard, and 1 year after the minimal standard of practice equipment was introduced. To follow how well family doctors adhere to this standard. METHOD: A questionnaire was sent to a random sample of district doctors (n=157) in 1992 and to all family doctors practising as independent contractors in 1998 (n=376). Quality of equipping was assessed against the standard set by consensus of the representatives of Family Doctors' Society, based on the country's needs and family doctor's job description. RESULTS: The level of primary care doctors' office equipment in 1992 was quite low. However, by the spring of 1998, substantial improvement of the equipment in family doctors' practices was estimated. The number of doctors possessing instruments for otorhinolaryngological, ophthalmological and gynaecological work as well as for taking care of children had increased two- to three-fold. About one-half of the family doctors reported that they had all the instruments listed in the standard. CONCLUSIONS: Setting a national standard helps to improve practice equipment--an important structural aspect of quality of care. Although improvement of equipment alone cannot guarantee quality of care, it may provide an important first step towards promoting it. PMID- 10733085 TI - Reducing negative appendectomy: evaluation of ultrasonography and computer tomography in acute appendicitis. AB - OBJECTIVE: To study the sensitivity and the specificity for ultrasonography and computed tomography in patients with suspected appendicitis, and their value to the clinician. MAIN OUTCOME MEASURES: The negative appendectomy rate and the sensitivity and the specificity for ultrasonography and computed tomography in patients with suspected appendicitis. RESULT: The diagnostic accuracy was 88% (men 95%, women 80%). Two hundred and thirty-nine patients were examined by ultrasonography preoperatively. The sensitivity for ultrasonography was 0.82 and the specificity was 0.97. Forty-nine patients were examined by computed tomography preoperatively. The sensitivity for computer tomography was 0.88 and the specificity was 0.95. CONCLUSIONS: We conclude that ultrasound and computed tomography investigations on patients with suspected appendicitis are of great value. Computed tomography seems to have a higher sensitivity than ultrasound and a high specificity. In fertile women, where unnecessary surgery is best avoided, we believe that computed tomography investigation or ultrasound examination are better alternatives to surgical intervention. PMID- 10733086 TI - Identifying drug safety issues: from research to practice. AB - PURPOSE: Adverse drug events (ADEs), or injuries due to drugs, are common and often preventable. However, identifying ADEs, potential ADEs, and medication errors can be a major challenge. In this review, we describe methodologies that have been used to identify these events and give strategies for identification in non-study settings. RESULTS: Methods such as voluntary reporting, chart review, and computerized monitoring for events have been most commonly used in studies of ADEs in inpatients. However, voluntary reporting, the method most hospitals currently use, has a very low yield of events. Chart review is much more sensitive but the costs are prohibitive. Computerized monitoring for ADEs (using rules or triggers) is a high yield and relatively inexpensive strategy that should be adopted by organizations. A limitation of this strategy, however, is that it identifies few medication errors and potential ADEs, which are also important. These can be captured through pharmacy logs, chart review, and direct observation. Once events have been identified, they can be classified by type of event, severity, and preventability. In non-study settings, the most practical method for identifying ADEs is computerized monitoring, and for identifying prescribing errors it is pharmacy logs of interventions. Once problems are found, a structure (either individual or committee) must be in place to classify them, identify opportunities for improvement, and carry out the necessary changes. CONCLUSIONS: Health care organizations have the technology to significantly improve their detection of ADEs, medication errors, and potential ADEs. Identification and subsequent classification of events is crucial for quality efforts to improve patient safety. PMID- 10733088 TI - Regulatory roles for FcgammaRIII (CD16) and FcgammaRII (CD32) in the development of T- and B-lineage lymphoid cells. AB - IgG Fc receptors (FcgammaR) occur on all hematopoietic lineages and participate in a diversity of functions that reflect the combined effects of the molecular heterogeneity of FcgammaR and the inherent specialization of the FcgammaR+ cells. An extensive literature describes the functions of FcgammaR on mature myeloid and lymphoid cells in humans and mice but little has been published about FcgammaR on lineage progenitor cells. Several studies suggest that FcgammaR can influence leukocyte development and that FcgammaRII (CD32) and FcgammaRIII (CD16) can regulate murine T- and B-lineage development at stages before the expression of clonal antigen receptors. The nominal ligand of FcgammaR is IgG and the physiologically relevant ligand is the IgG-antigen complex, but it is also known that alternative, non-Ig ligands exist for Fc receptors. A role for FcgammaR in the regulation of leukocyte development has potential relevance for clinical situations in which the levels of nominal and/or alternative ligands of FcgammaR are elevated, or the production of soluble forms of FcgammaR is increased. PMID- 10733087 TI - Insights from mouse models of colitis. AB - Emerging studies using mouse models of experimental colitis are defining the nature of the immunological disturbances that initiate inflammation and destruction of the intestine. A better understanding of disease-promoting and suppressing CD4+ T cells is providing insight into the mechanisms controlling immune responses within the intestinal compartment. Moreover, a role for distinct T cell populations, including intraepithelial gammadelta T cells, in maintaining the physical integrity of the intestine was suggested by recent studies. Cytokine gene-knockout mice and anti-cytokine treatments remain important tools to define the pro- and anti-inflammatory functions of cytokines. These advances are fostering the design and evaluation of new therapeutic approaches that may eventually be applied to treat human inflammatory bowel disease. PMID- 10733089 TI - Ecto-enzymes: physiology meets pathology. AB - Ecto-enzymes are catalytic membrane proteins with their active sites outside the cell. They include cholinesterase, which inactivates acetylcholine, and angiotensin-converting enzyme, which converts angiotensin I to biologically active angiotensin II, and numerous other peptidases, transpeptidases, nucleotidases, phosphodiesterases, and phosphatases. Many CD antigens of leukocytes are ecto-enzymes; some CD antigens for which no function is currently known are probably ecto-enzymes. Expression is highly regulated and correlated with differentiation and activation. Some are highly restricted in distribution; others are ubiquitous. Many are shared between leukocytes and non-hematogenous cells. Biological functions appear to depend on the type and location of the cell in which expression occurs, and include recycling of nutrients, local control of response to cytokines and hormones, bone formation, cell mobility, invasion, and metastasis. Many novel regulatory functions of ecto-enzymes remain to be discovered, and may reveal new mechanisms of local extracellular control of cellular function. PMID- 10733090 TI - Activation of macrophages by food antigens: enhancing effect of gluten on nitric oxide and cytokine production. AB - Macrophages play an important role in effector mechanisms of various chronic inflammatory diseases. We studied the effect of gluten, the agent inducing celiac disease, and other food antigens on the activation of macrophages. Nitric oxide (NO) and cytokine production were followed as markers of activation, using cultured murine peritoneal macrophages. None of the food antigens tested caused direct inducible nitric oxide synthase (iNOS) activation in macrophages. Unlike other food antigens gluten, gliadin, and their proteolytic fragments significantly enhanced NO production when applied together with interferon-gamma (IFN-gamma), the most efficient being fragments originating from 25- to 45-min peptic digestion. The activation pathway was mediated via direct stimulation of tumor necrosis factor alpha (TNF-alpha) secretion. The NO-enhancing effect was confirmed at the level of iNOS mRNA transcription. In case of sustained local inflammatory reaction connected with increase of IFN-gamma, gluten and its proteolytic fragments may thus elevate NO production. Increased NO level could consequently participate in the development of mucosal lesions in the gut of celiac patients. PMID- 10733091 TI - Gender difference in cell-mediated immunity after thermal injury is mediated, in part, by elevated levels of interleukin-6. AB - The gender difference in normal immune function has been well documented, however, there is only limited information regarding whether such a difference occurs after injury. To investigate this, we examined cell-mediated immune responses in male and female mice given a 15% total body surface area dorsal scald or sham injury. Both delayed-type hypersensitivity (DTH) and splenocyte proliferative responses were significantly suppressed in males at 1 day and in females at 7 and 10 days post burn (P < 0.01). The decreased splenocyte proliferation was found to be macrophage-dependent and suppression of both immune parameters corresponded with elevated interleukin-6 (IL-6) levels. Furthermore, post-burn treatment with an anti-IL-6 antibody partially restored the DTH response in males at 1 day and females at 10 days post injury and completely restored splenocyte proliferation. These data demonstrate a possible mechanism for the gender difference in cell-mediated immune responses after thermal injury. PMID- 10733092 TI - Regulation of ICAM-1/CD54 expression on human endothelial cells by hydrogen peroxide involves inducible NO synthase. AB - Expression of the inducible isoform of nitric oxide synthase (iNOS) is stimulated by cytokines in human epithelial cells. This work indicates that incubation of human umbilical cord endothelial cells with combinations of interleukin-1beta, tumor necrosis factor alpha, and interferon-gamma stimulated the synthesis of iNOS mRNA, as detected by reverse transcriptase-polymerase chain reaction. It is important to note that 50, 100, and 200 microM hydrogen peroxide was able to stimulate iNOS directly. Furthermore, 100 microM H2O2 enhanced synthesis of the oxidation products, nitrite (NO2-) and nitrate (NO3-) at 12 and 36 h. iNOS protein, detected by Western blot analysis, as well as L-citrulline levels, were also increased. When endothelial cell monolayers were incubated for 1 h with 100 microM H2O2 and subsequently with cytokines, iNOS mRNA was further augmented. Under the same conditions, we regularly observed an inhibition (25%) of intercellular adhesion molecule-1 (ICAM-1/CD54) expression. The latter was reversed when the NOS inhibitor N(G)-monomethyl-L-arginine was added, as shown by flow cytometry. These data suggest a specific effect of endogenous hydroperoxides on the biosynthesis and processing of the human endothelial iNOS isoform. We propose that H2O2 induces a temporary NO-dependent modulation of adhesion molecule expression to limit the tissue destruction that accompanies the vascular recruitment of leukocytes. PMID- 10733093 TI - A beneficial aspect of a CB1 cannabinoid receptor antagonist: SR141716A is a potent inhibitor of macrophage infection by the intracellular pathogen Brucella suis. AB - The psychoactive component of marijuana, delta9-tetrahydrocannabinol (THC) suppresses different functions of immunocytes, including the antimicrobicidal activity of macrophages. The triggering of cannabinoid receptors of CB1 and CB2 subtypes present on leukocytes may account for these effects. We investigated the influence of specific CB1 or CB2 receptor antagonists (SR141716A and SR144528, respectively) and nonselective CB1/CB2 cannabinoid receptor agonists (CP55,940 or WIN 55212-2) on macrophage infection by Brucella suis, an intracellular gram negative bacteria. None of the compounds tested affected bacterial phagocytosis. By contrast, the intracellular multiplication of Brucella was dose-dependently inhibited in cells treated with 10-500 nM SR141716A and 1 microM SR141716A induced cells exerted a potent microbicidal effect against the bacteria. SR144528, CP55,940, or WIN 55212-2 did not affect (or slightly potentiated) the growth of phagocytized bacteria. However, CP55,940 or WIN 55212-2 reversed the SR141716A-mediated effect, which strongly suggested an involvement of macrophage CB1 receptors in the phenomenon. SR141716A was able to pre-activate macrophages and to trigger an activation signal that inhibited Brucella development. The participation of endogenous cannabinoid ligand(s) in Brucella infection was discussed. Finally, our data show that SR141716A up-regulates the antimicrobial properties of macrophages in vitro and might be a pharmaceutical compound useful for counteracting the development of intramacrophagic gram-negative bacteria. PMID- 10733094 TI - Nitric oxide regulates nucleoside transport in activated B lymphocytes. AB - Activation of human B lymphocytes by lipopolysaccharide (LPS) or phorbol 12 myristate 13-acetate (PMA) results in the differential regulation of nucleoside uptake [Soler, C., Felipe, A., Mata, J. F., Casado, F. J., Celada, A., Pastor Anglada, M. (1998) J. Biol. Chem. 273, 26939-26945]. Because nitric oxide (NO) is involved in the modulation of the apoptotic response of B cells, the effects of NO on the regulatory responses of these transport systems to phorbol esters has been studied in Raji cells by a combination of approaches that involve arginine depletion, inhibition of nitric oxide synthase, and non-enzymatic production of NO using a donor. Human B lymphocytes express three transport systems involved in nucleoside uptake: N1 and N5, which are concentrative and Na+-dependent, and the nitrobenzylthioinosine-sensitive equilibrative system es. Raji cells do not express significant amounts of iNOS mRNA or protein; thus, NO production is presumably constitutive. The data are consistent with a role of NO in maintaining the basal transport activities of the three systems: N1, N5, and es. However, the up-regulatory effect of PMA on N1 and N5 does not require NO, whereas the inhibition of es transport activity does. In summary, NO differentially modulates nucleoside transport systems in activated human B lymphocytes and thus, NO may also be involved in the regulation of nucleoside (i.e., adenosine) disposal by activated B cells. PMID- 10733095 TI - Human mast cell migration in response to members of the transforming growth factor-beta family. AB - Mast cells are known to accumulate at sites of inflammation, however, the chemotaxins involved remain largely undefined. Transforming growth factor-beta (TGF-beta) isoforms regulate numerous cellular functions, including cell growth and differentiation, formation of extracellular matrix, and the immune response. In this study we have compared the potency of different members of the TGF-beta family as human mast cell chemotaxins, and analyzed the expression of TGF-beta binding proteins on human mast cells. We were able to demonstrate that the maximal chemotactic response was attained at approximately 40 fM for the three TGF-beta isoforms, with TGF-beta3 being more effective than TGF-beta1 and TGF beta2 at this concentration. This effect was observed in both the HMC-1 human mast cell line and in cultured primary mast cells. In addition, TGF-beta1, TGF beta2, and less efficiently, TGF-beta3 inhibited the proliferation of HMC-1 cells. The migratory response is probably mediated through interaction with the TGF-beta serine/threonine type I and II receptors that were found to be expressed on the cells. No expression of TGF-beta type III receptor, endoglin, or the endothelial TGF-beta type I receptor ALK-1 could be detected. These results provide evidence that TGF-beta isoforms are highly potent chemotaxins for human mast cells and can play an important role in the recruitment of mast cells in inflammatory reactions. PMID- 10733096 TI - Effects of heme proteins on nitric oxide levels and cell viability in isolated PMNs: a mechanism of toxicity. AB - Isolated human PMNs served as a model to determine oxyhemoglobin (oxyHb) binding and the effects of oxymyoglobin (oxyMb) or oxyHb on production of both nitric oxide (NO*) and superoxide (O2*-) and the resulting cytotoxicity. Physiologically relevant concentrations of NO* and H2O2 oxidized, to a similar extent, 2,7 dichlorodihydrofluorescein (DCFH) loaded into polymorphonuclear neutrophils (PMNs). Activation of PMNs with phorbol 12-myristate 13-acetate (PMA) markedly increased the internalization of extracellular oxyHb (10-250 microg/mL). OxyMb (10-300 microg/mL) or oxyHb (30-300 microg/mL) enhanced DCFH oxidation by a concentration-dependent mechanism in unstimulated, lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-alpha)-, and PMA-stimulated PMNs. This increased DCFH oxidation was eliminated by NO* synthase inhibitors, glutathione and ascorbate, and was reduced by albumin. Nitrite accumulation in PMN filtrates mirrored NO*-induced DCF fluorescence. OxyMb-induced increases in NO* levels paralleled alterations in DNA and cell membrane damage and ATP levels in PMNs and co-cultured lymphocytes, and were attenuated by NO* synthase inhibitors. OxyMb eliminated extracellular O2*- at protein concentrations 100- to 1000-fold above those of superoxide dismutase. These results suggest that heme proteins bind and internalize into PMNs and increase NO*-induced damage in neighboring cells by inhibiting O2*(-)-scavenging of NO*. We propose a mechanism whereby heme protein induced NO* levels may contribute to immunosuppression and increased infection rates associated with transfusions and cellular damage during inflammation. PMID- 10733097 TI - Increased expression of the tetraspanins CD53 and CD63 on apoptotic human neutrophils. AB - The recently discovered tetraspanin superfamily comprises a group of cell-surface proteins that are suggested to be involved in cell activation and signal transduction as well as in cell adhesion, motility, and metastasis. In this study, we have assessed the expression of two tetraspanins, CD53 and CD63, and two principal leukocyte adhesion molecules, CD11b and CD62L, on human apoptotic neutrophils. After aging of human neutrophils for 20 and 40 h in vitro, apoptosis was analyzed by light microscopy and flow cytometry. The binding of monoclonal antibodies directed against CD11b, CD62L, CD53, and CD63 on apoptotic and nonapoptotic cells was determined by dual-color flow cytometry. Aging of neutrophils in vitro resulted in a significant (P < 0.05) down-regulation of expression of the selectin CD62L, and a significantly increased expression of the two tetraspanins CD53 and CD63. The selective analysis of apoptotic versus nonapoptotic cells proved that both the increased expression of the tetraspanins and the loss of CD62L were restricted to the apoptotic subpopulation. An identical pattern of surface molecule expression was detected at 12 h after induction of apoptosis by an agonistic anti-Fas IgM monoclonal antibody. Further studies are required to clarify whether tetraspanins participate in the recognition of apoptotic circulating or extravasated neutrophils by macrophages. PMID- 10733098 TI - Changes in susceptibility to Fas-mediated apoptosis during differentiation of HL 60 cells. AB - The Fas-mediated pathway has been implicated as an important cellular pathway mediating apoptosis in diverse cell types. We conducted studies to examine the susceptibility to Fas-mediated apoptosis of HL-60 cells treated with differentiation-inducing factors such as dimethyl sulfoxide (DMSO), retinoic acid (RA), and 1alpha, 25 dihydroxyvitamin D3 (VD3). Although the expression of Fas antigen (Ag) and its mRNA showed a marked increase in HL-60 cells with cell differentiation, that of Bcl-2 protein and its mRNA revealed the reverse. The expression of caspase proteins such as caspases-3 and -8 was also enhanced during cell differentiation. DNA fragmentation, annexin V binding, and caspase activities increased in differentiated HL-60 cells with the addition of anti-Fas Ag antibody. These findings were more clearly demonstrated in DMSO- or RA-induced neutrophil-like cells than in VD3-induced monocyte-like cells. Therefore, susceptibility to Fas-mediated apoptosis showed an increase with differentiation of HL-60 cells, especially in the neutrophil lineage. These results suggest that the difference of susceptibility to Fas-mediated apoptosis among cell populations depends on the expression of Fas Ag, Bcl-2, and caspases. Cell maturation and susceptibility to Fas-mediated apoptosis may be linked in hematopoietic cells. PMID- 10733099 TI - Serum amyloid A is an activator of PMN antimicrobial functions: induction of degranulation, phagocytosis, and enhancement of anti-Candida activity. AB - Serum amyloid A (SAA) is a 12-kDa protein secreted in large amounts by liver cells during microbial infections or inflammatory diseases. We have recently reported that SAA induces chemotaxis of polymorphonuclear cells (PMN), monocytes, and T lymphocytes and stimulates their adhesion to endothelial monolayers. In this study, we investigated whether SAA regulates PMN antimicrobial activities. We found that recombinant SAA (rSAA), at concentrations comparable to serum levels attained during an acute phase response, is a potent activator of PMN. Stimulation of PMN by rSAA results in a rapid and transient increase of cytosolic calcium concentration and up-regulation of cell-surface expression of antigens involved in adhesion and microbial recognition such as CD11c and CD16. In addition, stimulation of PMN with rSAA increases secretion of lactoferrin, an antimicrobial protein that is contained in specific granules of PMN and enhances PMN phagocytic activity against heat-killed Candida albicans. Finally, activation of PMN with rSAA enhances their anti-Candida activity within 30 min of stimulation. These results suggest that SAA is involved in up-regulating PMN antimicrobial activities and that high circulating concentrations of SAA as seen in the acute phase response may constitute a potential host defense mechanism against fungal infections. PMID- 10733100 TI - Differential regulation of TSG-14 expression in murine fibroblasts and peritoneal macrophages. AB - Tumor necrosis factor (TNF)-stimulated gene 14 (TSG-14, also termed PTX3) encodes a secreted glycoprotein whose carboxy-terminal half shares sequence similarity with the pentraxin family of acute phase proteins (C-reactive protein and serum amyloid P component). We compared TSG-14 mRNA expression in cultures of murine BALB/c 3T3 fibroblasts and thioglycollate-elicited peritoneal macrophages. TNF and interleukin-1 (IL-1) potently induced TSG-14 expression in 3T3 fibroblasts but not in peritoneal macrophages. Lipopolysaccharide (LPS) elicited TSG-14 expression in both cell types, but induction in 3T3 cells and macrophages showed several distinct characteristics. Whereas in 3T3 fibroblasts TSG-14 mRNA was rapidly up-regulated by LPS, expression in macrophages was substantially delayed. Furthermore, cycloheximide greatly reduced LPS-induced TSG-14 mRNA up-regulation in macrophages but not in 3T3 cells. Finally, interferon-gamma (IFN-gamma; but not IFN-alpha/beta) inhibited LPS-induced TSG-14 expression in macrophages and not in 3T3 fibroblasts. The antioxidant pyrrolidine dithiocarbamate inhibited LPS induced nuclear factor-kappaB (NF-kappaB) activation and TSG-14 expression in macrophages. In contrast, IFN-gamma did not inhibit NF-kappaB function as measured by IkappaB-alpha and IkappaB-beta degradation, IkappaB-alpha resynthesis, or electrophoretic mobility shift analysis. Inhibition of LPS induced TSG-14 mRNA expression by IFN-gamma in macrophages was also observed in the presence of cycloheximide and in cells from STAT1 null mice, suggesting that IFN-gamma inhibits TSG-14 expression through an unconventional mechanism. PMID- 10733101 TI - Phorbol myristate acetate induces neutrophil NADPH-oxidase activity by two separate signal transduction pathways: dependent or independent of phosphatidylinositol 3-kinase. AB - The neutrophil NADPH-oxidase can be activated by protein kinase C (PKC) agonists such as phorbol myristate acetate (PMA), resulting in superoxide anion release. This superoxide release is independent of phosphatidylinositol 3-kinase (PI 3 kinase) because the inhibitor wortmannin does not affect the response. In this study, PMA is shown to also induce a wortmannin-sensitive NADPH-oxidase activation, however, not resulting in release of superoxide but in intracellular production of the radical. This indicates that two pools of NADPH-oxidase, one localized in the plasma membrane and the other in the granule membranes, are separately regulated and the signal transduction pathways leading to activation of these pools differ regarding involvement of PI 3-kinase. Activation of both pools was dependent on ERK/MAPK kinase (MEK) activity and protein phosphatase 1 and/or 2A. As the two oxidase responses were differently affected by the inhibitor Go-6850, different PKC isozymes are suggested to take part in the two signal transduction pathways. PMID- 10733102 TI - Phosphatidylinositol 3-kinase and mTOR mediate lipopolysaccharide-stimulated nitric oxide production in macrophages via interferon-beta. AB - Bacterial lipopolysaccharide (LPS) elicits responses by macrophages that help the body repel infections. Recent evidence indicates that phosphatidylinositol 3 kinase (PI 3-kinase) may mediate some of these responses. Here, we show that exposing macrophages to LPS rapidly increased membrane-associated PI 3-kinase activity and also elevated p70 S6 kinase activity. Inhibitors of PI 3-kinase or the mammalian target of rapamycin (mTOR) fully blocked p70 S6 kinase activation, implying that this kinase is controlled by PI 3-kinase and mTOR. These inhibitors also substantially reduced LPS-induced nitric oxide (NO) production. This inhibition was, in part, attributable to impaired LPS-stimulated secretion of interferon-beta, an autocrine co-factor for NO production. However, the addition of exogenous interferon-beta did not fully restore NO production, indicating that the NO response was being inhibited by another mechanism as well. Together, these data suggest that PI 3-kinase, mTOR, and possibly p70 S6 kinase mediate LPS induced NO production by regulating the secretion of interferon-beta and by a second undefined mechanism. PMID- 10733103 TI - Effects of selective protein kinase C inhibitors on the proteolytic down regulation of L-selectin from chemoattractant-activated neutrophils. AB - The signaling factors that direct the rapid shedding of L-selectin from neutrophils upon chemoattractant stimulation are poorly understood. Protein kinase C (PKC) has been implicated, yet previous studies have relied on the use of phorbol esters and nonselective kinase inhibitors. We treated neutrophils with various selective kinase inhibitors to evaluate their effects on N-formyl methionyl-leucyl-phenylalanine (fMLP)-induced L-selectin shedding. We found that three selective inhibitors of PKC, structurally related to staurosporine, largely blocked both fMLP- and phorbol 12-myristate 13-acetate (PMA)-induced L-selectin shedding; however, these inhibitors did not affect fMLP-induced up-regulation of Mac-1 (CD11b/CD18) expression, which has been shown not to involve PKC. Other selective serine, threonine, and tyrosine kinase inhibitors were found not to block fMLP-induced L-selectin shedding. These findings provide more definitive evidence for the role of PKC in chemoattractant-induced L-selectin proteolysis. It is interesting that certain highly selective PKC inhibitors, not structurally related to staurosporine, were found to directly induce L-selectin shedding from neutrophils. PMID- 10733104 TI - Isolation of MYADM, a novel hematopoietic-associated marker gene expressed in multipotent progenitor cells and up-regulated during myeloid differentiation. AB - A large number of hematopoietic cytokines and their receptors as well as transcription factors have been shown to be involved in maturation of blood cells. However, many of the genes important for the differentiation of multipotent stem cells to specific cellular lineages are still unknown. To identify novel genes involved in lineage selection of myeloid cells, we have applied differential display analysis during commitment toward granulocytes and macrophages of an IL-3-dependent multipotent progenitor cell line, FDCP-mix. One regulated cDNA represented a novel gene with restricted expression pattern within the hematopoietic system and was strongly up-regulated when FDCP-mix cells differentiated in GM-CSF, G-CSF, and M-CSF. The expression appears to be differentiation stage-specific in myeloid cells and is absent in B and T lymphocytes. Thus we found expression in normal mouse bone marrow enriched for stem cells and multipotent progenitors (c-kit+Sca-1+Lin- cells). When these cells were induced to differentiate toward myeloid cells, MYADM was up-regulated. In contrast, during conditions known to favor the development of B cell progenitors, the gene was down-regulated. The gene, termed MYADM for myeloid-associated differentiation marker gene, shows 100% identity to expressed sequence tags from early mouse embryonic development as well as from the mouse lung and from activated mouse macrophages. The predicted 32-kDa MYADM protein contains multiple hydrophobic putative transmembrane segments and has several potential consensus sites for phosphorylation. In view of its expression pattern, MYADM could serve as a new marker gene for hematopoietic differentiation. Although the function is unknown, antisense oligonucleotides were able to inhibit colony formation of c kit+ Lin- bone marrow cells, suggesting an important role for MYADM in myeloid differentiation. PMID- 10733105 TI - Transcriptional regulation of HIV-1 LTR during antigen-dependent activation of primary T cells by dendritic cells. AB - Numerous factors are known to bind human immunodeficiency virus (HIV) long terminal repeat (LTR) and activate viral transcription, but little is known as to how they function in naturally activated T cells and to what extent their binding is relevant to HIV replication in vivo. To characterize the HIV LTR-binding factors responsible for antigen-dependent activation of HIV, we examined replication of LTR mutant viruses in CD4+ T cells activated by different stimuli. NF-kappaB or Sp1 mutant virus replicated well in CD4+ T cells activated by phorbol ester and calcium ionophore. When they were activated by antigen-pulsed dendritic cells, the replication of the Sp1-deleted virus was severely impaired in CD45RA+, but not in CD45RO+ T cell subsets that dominantly produce interleukin 2 (IL-2). Stimulation via CD3/CD28 induced a high level of IL-2 production in both T cell subsets, but Sp1-deleted virus poorly replicated in CD45RA+ subset. The level of NF-kappaB and Sp1-binding factors did not differ between these subsets. Our results suggest that additional cofactors distinct from IL-2 inducing signaling molecules are important for LTR activation during antigen dependent T cell activation. PMID- 10733106 TI - Receptor reserve analysis of the human CCR3 receptor in eosinophils and CCR3 transfected cells. AB - A novel pharmacological study of CCR3 receptor reserve in a CCR3-transfected cell (CREM3) and human eosinophils was done; functional responses measured were increases in intracellular calcium and chemotaxis. Eotaxin, eotaxin-2, monocyte chemoattractant protein-4 (MCP-4), RANTES, and MCP-3 induced similar maximal eosinophil chemotaxis, whereas MCP-3 and RANTES induced submaximal calcium responses in eosinophils compared to eotaxin, MCP-4, and eotaxin-2. This suggested a receptor reserve in the chemotaxis response. Receptor reserve was quantitated for eotaxin. Occupancy of all CCR3 receptors was required for a maximal calcium response in both CREM3 and eosinophils (reserve = 1.0 or 0.17, respectively); the stimulus-calcium response relationship was linear, indicating no receptor reserve. In contrast, in eosinophils a large receptor reserve (6.5) was found for chemotaxis, where occupancy of 15% receptors drove half-maximal responses. These studies indicate that CCR3 interacts with G-proteins that are poorly coupled to the calcium response, whereas coupling efficiency and/or amplification to the chemotaxis apparatus in human eosinophils is significantly greater. PMID- 10733107 TI - Practice parameters for the treatment of sigmoid diverticulitis. The Standards Task Force. The American Society of Colon and Rectal Surgeons. PMID- 10733108 TI - Practice parameters for the treatment of sigmoid diverticulitis--supporting documentation. The Standards Task Force. The American Society of Colon and Rectal Surgeons. AB - It should be recognized that these guidelines should not be deemed inclusive of all proper methods of care or exclusive of methods of care reasonably directed to obtaining the same results. The ultimate judgment regarding the propriety of any specific procedure must be made by the physician in light of all of the circumstances presented by the individual patient. PMID- 10733109 TI - Double-blind crossover study of sacral nerve stimulation for fecal incontinence. AB - PURPOSE: Patients with fecal incontinence not amenable to simple repair may have to undergo major reconstructive surgery or resort to a stoma. Sacral nerve stimulation is an alternative approach that may diminish incontinence by altering sphincter and rectal motor function. This study is the first double-blind trial examining the effectiveness of this therapy. METHODS: Two patients with passive fecal incontinence who had been implanted for nine months with a permanent sacral nerve stimulator and electrode were studied using fecal incontinence diaries, anorectal physiological tests, and quality-of-life assessments (SF-36 health survey). The trial period consisted of two two-week periods, with the stimulator turned on for two weeks and off for two weeks. The main investigator and the patients were blinded to the status of the stimulator. RESULTS: There was a dramatic difference between the number and severity of episodes of incontinence when the stimulator was turned on vs. turned off (Patient 1, 20 vs. 2 episodes; Patient 2, 4 vs. 0 episodes; off vs. on). There was an increase in squeeze pressure (Patient 1, 70 vs. 100 cm H2O; Patient 2, 60 vs. 90 cm H2O; off vs. on), with moderate increases in resting pressure and rectal threshold and urge volumes. Quality-of-life measurements showed a marked improvement prestimulation vs. nine months after permanent stimulation. CONCLUSIONS: There is a marked, unequivocal improvement in symptoms of fecal incontinence with sacral nerve stimulation shown in this double-blind crossover trial. Sacral nerve stimulation improves the quality of life in selected patients with fecal incontinence. PMID- 10733110 TI - Role of virtual computed tomographic colonography in patients with colorectal cancers and obstructing colorectal lesions. AB - PURPOSE: The aim of this study was to assess the ability of computed tomographic colonography to diagnose colorectal masses, stage colorectal cancers, image the proximal colon in obstructing colorectal lesions, and evaluate the anastomoses in patients with previous colorectal surgery. METHODS: We prospectively performed computed tomographic colonography examinations in 34 patients (20 males; mean age, 64.2; range, 19-91 years): 20 patients had colorectal masses (defined at endoscopy as intraluminal masses 2 cm or larger), 7 patients had benign obstructing colorectal strictures, and 7 patients had a prior colorectal resection. Final tumor staging was available in all 16 patients who had colorectal cancers and 15 patients were referred after incomplete colonoscopy. The ability of computed tomographic colonography to stage colorectal cancers, identify synchronous lesions in patients with colorectal masses, and image the proximal colon in patients with obstructing colorectal lesions was assessed. RESULTS: Computed tomographic colonography identified all colorectal masses, but overcalled two masses in patients who were either poorly distended or poorly prepared. Computed tomographic colonography correctly staged 13 of 16 colorectal cancers (81 percent) and detected 16 of 17 (93 percent) synchronous polyps. Computed tomographic colonography over-staged two Dukes Stage A cancers and understaged one Dukes Stage C cancer. A total of 97 percent (87/90) of all colonic segments were adequately visualized at computed tomographic colonography in patients with obstructing colorectal lesions compared with 60 percent (26/42) of segments at barium enema (P < 0.01). Colonic anastomoses were visualized in all nine patients, but in one patient, computed tomographic colonography could not distinguish between local tumor recurrence and surgical changes. CONCLUSION: Computed tomographic colonography can accurately identify all colorectal masses but may overcall stool as masses in poorly distended or poorly prepared colons. Computed tomographic colonography has an overall staging accuracy of 81 percent for colorectal cancer and is superior to barium enema in visualizing colonic segments proximal to obstructing colorectal lesions. PMID- 10733111 TI - Extensive surgery after high-dose preoperative chemoradiotherapy for locally advanced recurrent rectal cancer. AB - PURPOSE: This was a pilot study of high-dose preoperative concurrent radiation and chemotherapy before extensive surgery in patients with locally advanced recurrent rectal cancer. Here we report on curative resectability, acute toxicities during chemoradiotherapy, surgical complications, local control, and three-year survival rates achieved with this aggressive multimodal regimen. METHODS: Between 1994 and 1997, 35 previously nonirradiated patients with pelvic recurrence of rectal cancer were entered in the study. All patients presented with tumor contiguous or adherent to adjacent pelvic organs and were not deemed amenable to primary curative surgery. A total radiation dose of 50.4 Gy with a small-volume boost of 5.4 to 9 Gy was delivered in conventional fractionation (single dose, 1.8 Gy). 5-Fluorouracil was scheduled as a continuous infusion of 1,000 mg/m2/day on Days 1 to 5 and 29 to 33. Six weeks after completion of chemoradiotherapy, patients were reassessed for resectability, and radical surgery was attempted whenever feasible. RESULTS: After preoperative chemoradiotherapy 28 of 35 patients (80 percent) underwent resection with curative intent. In 16 of 35 patients (57 percent) extended resection of adjacent organs was performed. Resections with negative margins were achieved in 17 patients (61 percent); 9 patients had microscopic, and 2 patients had gross residual disease. There was no postoperative mortality. Fourteen patients (44 percent) experienced postoperative complications. Toxicity from chemoradiotherapy occurred mainly as diarrhea (National Cancer Institute Common Toxicity Criteria Grade 3; 23 percent), dermatitis (Grade 3; 11 percent), and leucopenia (Grade 3; 11 percent). One patient died of tumortoxic multiple organ failure during chemoradiotherapy. With a median follow-up of 27 months, local re-recurrence after curative resection was observed in only three patients (18 percent); six patients developed distant metastases. Three-year actuarial survival rate was significantly improved after complete resection (82 percent) as compared with noncurative surgery (38 percent; P = 0.03). CONCLUSION: A combination of high dose preoperative chemoradiotherapy followed by extended surgery can achieve clear resection margins in more than 60 percent of patients with recurrent rectal tumor not amenable to primary surgery. An encouraging trend evolved for this multimodal treatment to improve long-term local control and survival rate. PMID- 10733113 TI - Safety and advantages of laparoscopic vs. open colectomy in the elderly: matched control study. AB - PURPOSE: The aim of this study was to determine rates of complications and extent of benefits for laparoscopic-assisted colectomy compared with open colectomy in patients older than age 75. METHODS: Forty-two patients undergoing laparoscopic assisted colectomy (1992-1998) were matched to 42 open colectomy patients for gender, age, year of surgery, operating surgeon, and procedure. Health status (American Society of Anesthesiology score), previous abdominal surgery, conversion rate, surgical outcome, and need for assistance at admission and dismissal (independence vs. home with assistance vs. nursing facilities) were reviewed. RESULTS: Mean ages were 81.2 and 80.5 years for laparoscopic-assisted colectomy and open colectomy, respectively (P = not significant). Twenty-one laparoscopic-assisted colectomy and 23 open colectomy patients were females. American Society of Anesthesiology scores were comparable, as were rates of previous abdominal surgery (57 percent for laparoscopic-assisted colectomy vs. 62 percent for open colectomy; P = not significant). Mean operative times were longer for laparoscopic-assisted colectomy (190 minutes for laparoscopic-assisted colectomy vs. 142 minutes for open colectomy; P < 0.001); operating room times progressively decreased from 221 minutes in 1992 to 1995 to 147 in 1998 for laparoscopic right hemicolectomy (P < 0.001). The conversion rate for laparoscopic-assisted colectomy was 14.3 percent. There were no deaths in either group, and laparoscopic-assisted colectomy was associated with fewer morbidities (14.3 percent for laparoscopic-assisted colectomy vs. 33.3 percent for open colectomy; P = 0.04), narcotic usage (2.7 vs. 4.8 days; P < 0.001), time to return to bowel movements (3.9 vs. 5.9 days; P < 0.001), and length of hospital stay (6.5 vs. 10.2 days; P < 0.001). Independent status at admission in 37 laparoscopic-assisted colectomy and 38 open colectomy patients was maintained at discharge by 35 laparoscopic-assisted colectomy vs. 29 open colectomy patients (P = 0.025). CONCLUSIONS: Laparoscopic-assisted colectomy is safe and beneficial, including preservation of postoperative independence, to the elderly when compared with open colectomy. PMID- 10733112 TI - Laparoscopic ultrasound for the detection of hepatic metastases during laparoscopic colorectal cancer surgery. AB - PURPOSE: The search for liver metastases before surgery forms an accepted part of colorectal cancer surgical practice. Intraoperative ultrasound and manual palpation of liver together form the criterion standard as far as screening for metastases is concerned. However, extracorporeal imaging, such as ultrasound and magnetic resonance imaging, are also widely used. The purpose of this study was to demonstrate the efficacy of laparoscopic ultrasound scan in detection of liver metastases during laparoscopic colorectal cancer surgery by comparison with conventional imaging modalities. METHODS: A prospective, controlled study was undertaken. A total of 76 consecutive patients undergoing laparoscopic colorectal resections for malignancy were recruited. Patients underwent preoperative liver ultrasound scan and intraoperative blinded laparoscopic ultrasound scan examination performed by a single surgeon. Contrast-enhanced magnetic resonance imaging was performed within 30 days of surgery. RESULTS: Conventional ultrasound scan was negative in all cases. Metastases were identified during simple laparoscopic inspection of the liver in one case. Two cases shown by laparoscopic ultrasound scan to have definite metastases were confirmed by magnetic resonance imaging. In seven further instances laparoscopic ultrasound scan identified suspicious liver masses. In three cases these were confirmed to be metastases at magnetic resonance imaging; one was confirmed as a cyst, and the remaining three suspicious lesions were confirmed at serial magnetic resonance imaging scans to be benign and of no significance. CONCLUSION: Laparoscopic ultrasound scan with a flexible-tipped probe permits satisfactory hepatic examination. It is superior to conventional ultrasound scan and seems to be as effective as magnetic resonance imaging, although the latter modality is still required to delineate identified lesions. PMID- 10733114 TI - Prokinetic effect of erythromycin after colorectal surgery: randomized, placebo controlled, double-blind study. AB - PURPOSE: Nausea and vomiting three to seven days after an elective operation on the colon and rectum remain a persistent clinical problem. Erythromycin, a safe, inexpensive drug that stimulates intestinal motilin receptors, has previously been shown to accelerate gastric emptying significantly after upper gastrointestinal surgery. We aimed to evaluate the effect of postoperative intravenous erythromycin on postoperative ileus in patients undergoing elective surgery for primary colorectal cancer. METHODS: Between May 1998 and April 1999, 150 patients undergoing primary resection of colon or rectal cancer were enrolled in this prospective, randomized, placebo-controlled trial. One hundred thirty four patients completed the study. Patients were excluded if they had extensive metastatic disease, were taking medications known to interact with erythromycin, or if they required an ileostomy. Patients received either 200 mg of intravenous erythromycin or placebo every six hours. Clinical endpoints were recorded and continuous end-points are presented as mean +/- standard deviation. RESULTS: There were no significant complications related to erythromycin. The erythromycin (n = 65) and placebo (n = 69) groups were comparable regarding demographic and operative factors. The erythromycin group had a slightly shorter length of time to passage of flatus (4.1 +/- 1.3 vs. 4.4 +/- 1.1 days; P = 0.03). There was no significant difference between erythromycin and placebo in time to first solid food (5.6 +/- 1.9 vs. 5.4 +/- 1.8 days), time to first bowel movement (5.2 +/- 1.9 vs. 5.4 +/- 1.3 days), or time to discharge from hospital (7.5 +/- 2.0 vs. 7.6 +/- 2.8 days). There was no difference in the rate of clinically significant nausea (26 vs. 26 percent; P = 0.99), vomiting (17 vs. 16 percent; P = 0.88), or nasogastric tube placement (9 vs. 7 percent; P = 0.68). CONCLUSIONS: Erythromycin does not seem to alter clinically important outcomes related to postoperative ileus in patients undergoing resection for colorectal cancer. PMID- 10733115 TI - Local staging of rectal cancer with transrectal ultrasound and endorectal magnetic resonance imaging: comparison with histologic findings. AB - PURPOSE: The aim of the present study was to compare the accuracy of endorectal coil magnetic resonance imaging with transrectal ultrasound in staging rectal carcinoma. METHODS: Twenty-six consecutive patients with rectal carcinoma, histologically proven by endoscopic biopsy, were staged with both endorectal coil magnetic resonance imaging and transrectal ultrasound and then underwent radical surgery. The preoperative staging was compared with histologic findings of the operative specimen according to TNM classification. RESULTS: Endorectal coil magnetic resonance imaging showed better results but was not statistically significantly different from transrectal ultrasound in evaluating T (accuracy, 84.6 vs. 76.9 percent): four overstaged and no understaged cases for the former and five overstaged cases and one understaged case for the latter. Both procedures showed similar results in evaluating N: 81 percent sensitivity and 66 percent specificity for endorectal coil magnetic resonance imaging and 72 percent sensitivity and 80 percent specificity for transrectal ultrasound. CONCLUSIONS: An accurate locoregional staging of rectal cancer is essential for the planning of optimal therapy for rectal cancer. Endorectal coil magnetic resonance imaging and transrectal ultrasound showed similar results; the former is more expensive, whereas the latter is operator dependent. At present the use of endorectal coil magnetic resonance imaging seems to be justified only in selected low rectal cancers where transrectal ultrasound yielded doubtful results. However, a more extensive study is necessary to compare the advantages of these diagnostic techniques. PMID- 10733116 TI - Angiogenesis, proliferation, and apoptosis in anal high-grade squamous intraepithelial lesions. AB - PURPOSE: Management of anal high-grade squamous intraepithelial lesions is controversial. Anal and cervical high-grade squamous intraepithelial lesions are similar in that they occur in transitional squamous epithelium, are associated with human papilloma virus infection, and have increased incidence in the immunocompromised population. Ablation of cervical high-grade squamous intraepithelial lesions is preferred, but similar ablation or excision of anal high-grade squamous intraepithelial lesions may compromise bowel control; thus, there is a need to define the malignant potential of anal high-grade squamous intraepithelial lesions. METHODS: We analyzed 50 paraffin sections of normal anoderm, anal low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and anal squamous-cell carcinoma. Microvessels were detected immunohistochemically with von Willebrand factor and counted manually along the epithelial-stromal junction. Proliferation and apoptosis were determined in the epithelial cells with MIB-1 antibody immunostaining and the terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling, respectively. RESULTS: Microvascular density was significantly greater in anal high-grade squamous intraepithelial lesions (mean, 0.50 vessels/cm) vs. normal anoderm (mean, 0.21 vessels/cm; P = 0.0017, Mann-Whitney U test). The proliferative percentages were greater in low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and squamous-cell carcinoma (mean, 20.4, 21.8, and 23.6 percent) vs. normal anoderm (mean, 14.4 percent), although not significantly (P = 0.06, Kruskal-Wallis statistic). Although the mean proliferative proportions were similar in low-grade squamous intraepithelial lesions and high-grade squamous intraepithelial lesions, the apoptotic proportion was lower for high-grade squamous intraepithelial lesions than low-grade squamous intraepithelial lesions (10.13 vs. 19.96 percent, respectively; P = NS, Mann Whitney U test). CONCLUSIONS: Angiogenesis, increased proliferation, and decreased apoptosis occur in anal high-grade squamous intraepithelial lesions as they do in the cervix before the development of malignancy. These biologic markers support the importance of anal high-grade squamous intraepithelial lesions as a potential premalignant lesion warranting surgical intervention. PMID- 10733117 TI - Hereditary nonpolyposis colorectal cancer (Lynch syndrome II) in Uruguay. AB - PURPOSE: We updated an Uruguayan family with hereditary nonpolyposis colorectal cancer first described in 1977, incorporating knowledge of how the hMLH1 germline mutation has been established and shown to segregate in accord with the expected autosomal dominant mode of genetic transmission. METHODS: DNA-based molecular genetic testing was performed in conjunction with genetic counseling. Individuals were provided with their genetic test results, so that at-risk family members would be able to benefit from targeted management programs. RESULTS: We counseled 19 members of this kindred, 13 of whom were positive for the hMLH1 germline mutation. Specific recommendations for surveillance and management were provided. We were able to describe follow-up, including anecdotal cancer survival and pathology findings extending from the initial 1977 report of this family to the present. A remarkable sibship within this kindred was comprised of eight siblings, six of whom underwent resections for colorectal carcinoma between 1963 and 1971. Colon carcinomas before 1977 in this sibship were treated with classic hemicolectomies. Of those who had hemicolectomies for their first primary colorectal cancers, two had a second colon cancer primary, and two had a third colon cancer primary. CONCLUSIONS: Attention given to this extended family with hereditary nonpolyposis colorectal cancer has had a positive impact on the physician community in Uruguay, leading to the identification of additional families with hereditary nonpolyposis colorectal cancer. PMID- 10733118 TI - Desmoid disease in patients with familial adenomatous polyposis. AB - PURPOSE: The aim of this retrospective study was to review the clinical features, and surgical and medical management of patients with familial adenomatous polyposis-associated desmoid tumors. METHODS: From 1980 to 1997, 97 of 780 patients with familial adenomatous polyposis developed desmoid disease. Clinical and demographic data; operative notes; and histologic, radiologic, and follow-up reports were retrieved from patients' medical records. Risk factors for desmoid disease, such as prior surgery, age at desmoid tumor diagnosis, pregnancy, and family history were sought. The outcome after noncytotoxic and cytotoxic therapy was evaluated with respect to improvement of symptoms. RESULTS: There were 38 males with a mean age of 32.1 years and 59 females with a mean age of 29.1 years. A family history of desmoid tumors was found in 41 patients (42 percent), and a history of pregnancy was documented in 33 females (56 percent). The most common clinical presentation was small-bowel obstruction (58 percent). One-half of the desmoids were located in the mesentery, and 32 percent were located in the mesentery and the abdominal wall. Desmoids developed after colectomy in 77 cases (80 percent), after a mean time of 4.6 years. Partial resection of desmoid tumor was performed in 46 patients (47 percent), resection of extra-abdominal desmoid tumors was performed in 17 cases (17 percent), and biopsy only was performed in 34 patients (35 percent). Postoperative morbidity was 23 percent after desmoid tumor resection. Eight patients (8 percent) died of their intra-abdominal desmoid. Mean follow-up time was 5.3 years. Sulindac, tamoxifen, or toremifene therapy was able to alleviate symptoms in only 4 of 31 patients. Symptomatic improvement was noted after chemotherapy in six of ten patients with extremely complex desmoids. CONCLUSION: Desmoid disease was found in 12.4 percent of our patients with familial adenomatous polyposis. In view of the high rate of morbidity, indication for surgery should be limited mainly to acute or chronic small-bowel obstruction, because resection triggers a high recurrence rate. Noncytotoxic therapy was not effective for progressive desmoid tumors, whereas chemotherapy was effective in aggressive cases of intra-abdominal desmoid tumors. PMID- 10733119 TI - Effects of antiadhesive agents on the healing of intestinal anastomosis. AB - PURPOSE: The effects of antiadhesive agents on the healing of intestinal anastomosis were investigated. METHODS: Eighty rats were divided into eight groups. Colotomy and anastomosis were performed to all rats. Saline solution (control), carboxymethylcellulose, aprotinin, verapamil, tenoxicam, cyclosporine, and dextran 70 were administered intraperitoneally. Vitamin E was administered intramuscularly. The rats were killed 15 days later. Anastomotic healing was assessed by bursting pressure and the hydroxyproline content of the anastomotic tissues. The results were evaluated by Mann-Whitney U test. RESULTS: The mean (+/ standard deviation) bursting pressures of carboxymethylcellulose, cyclosporine, and aprotinin groups (108 +/- 6.73, 122.5 +/-14.39, and 127 +/- 20.23, respectively) were significantly lower than those of the control group (234 +/- 6.19). The mean level of hydroxyproline in the anastomotic tissues was significantly lower in the carboxymethylcellulose and cyclosporine groups (8.92 +/- 0.6 and 8.32 +/- 0.63) than that in the control group (16.33 +/- 0.68). CONCLUSION: These findings indicate that carboxymethylcellulose and cyclosporine had adverse effects on intestinal anastomosis in rats. PMID- 10733120 TI - Botulinum toxin in the treatment of outlet obstruction constipation caused by puborectalis syndrome. AB - PURPOSE: Puborectalis syndrome has been difficult to treat. We investigated the efficacy of botulinum toxin in treating patients with puborectalis syndrome who had previously failed to respond to electromyographic biofeedback sessions and who refused to use anal dilators. METHODS: Of a group of 50 patients with chronic outlet obstruction constipation, four patients with puborectalis syndrome were included in the study. The patients were studied using anorectal manometry, defecography, and electromyography and then treated with 30 units of Type A botulinum toxin, injected into two sites on either side of the puborectalis muscle, under ultrasonographic guidance. RESULTS: One patient was lost to follow up. After treatment in other patients, the frequency of natural bowel movements increased from zero to six per week and laxatives were needed by only one patient. Anorectal manometry demonstrated decreased tone during straining from (mean +/- standard deviation) 96.2 +/- 12 mmHg to 42.5 +/- 13 mmHg at four weeks (P = 0.003) and 63.2 +/- 22 mmHg at eight weeks (P = 0.009). Defecography performed eight weeks after treatment showed improvement in the anorectal angle, which increased from 94 +/- 11 degrees to 114 +/- 13 degrees (P = 0.01), and evacuation of barium paste. Electromyography demonstrated mild paradoxical contraction. However, 16 weeks after treatment one of these three patients suffered symptomatic recurrence. This patient was re-treated with 50 units of toxin; eight months later he required a further 60 units. Seven months after the last injection he reported normal daily bowel movements without the use of laxatives. CONCLUSIONS: Botulinum toxin injection should be considered as a simple therapeutic approach in patients with puborectalis syndrome. The use of higher dosage and a more precise method of toxin injections under transrectal ultrasonography account for the long-term higher success rate. However, because the effects of the toxin wear off within three months of administration, repeated injections could be necessary to maintain the clinical improvement. PMID- 10733121 TI - Local somatothermal stimulation inhibits motility of the internal anal sphincter through nitrergic neural release of nitric oxide. AB - PURPOSE: A somatoanal reflex had been demonstrated in our previous work. Because nitric oxide plays an important role in mediating relaxation of the internal anal sphincter, our purpose was to examine whether and how local somatothermal stimulation inhibits the function of the internal anal sphincter by stimulating nitric oxide release via nitrergic neurons and to elucidate the possible mechanism. METHODS: The activity of the internal anal sphincter in anesthetized rabbits was measured by use of continuously perfused, open-tip manometric methods. Local somatothermal stimulation was achieved by applying an electroheating rod 1 cm away from the skin area at the right popliteal region. The responses were further manipulated by pre-treating the rabbits with agonists or antagonists linked to nitric oxide synthesis. RESULTS: The motility of the internal anal sphincter before and during local somatothermal stimulation was significantly different (tonic pressure (mean +/-standard error of the mean), 5.4 +/- 0.3 vs. 4.9 +/- 0.3 mmHg, P = 0.0195; phasic pressure, 3.9 +/- 0.6 vs. 2.9 +/ 0.4 mmHg, P = 0.0002; frequency distribution of the phasic contractions (peak-to peak interval), 28.9 +/- 3.7 vs. 65.3 +/- 10.4 seconds, P = 0.0001). The response began at approximately one minute after local somatothermal stimulation when the skin temperature was 41 +/- 0.3 degrees C. No anal response was observed when local somatothermal stimulation was applied at the control area. The local somatothermal stimulation-induced internal anal sphincter relaxation was not inhibited by pretreatment with atropine, propranolol, or phentolamine (tonic pressure, 5.8 +/- 1 vs. 5.2 +/- 0.8 mmHg, P = 0.038; phasic pressure, 4.2 +/- 0.9 vs. 3.1 +/- 0.6 mmHg, P = 0.020; peak-to-peak interval, 27.2 +/- 4.3 vs. 52.9 +/- 14.5 seconds, P = 0.043) but was completely blocked by pretreatment with a nitric oxide synthesis inhibitor. The effect of the nitric oxide synthesis inhibitor could be reversed by pretreatment with L-arginine (tonic pressure, 6 +/- 0.7 vs. 5.6 +/- 0.7 mmHg, P = 0.047; phasic pressure, 4.7 +/- 0.7 vs. 3.9 +/- 0.5 mmHg, P = 0.048; peak-to-peak interval, 23.8 +/- 3 vs. 33 +/- 3.7 seconds, P = 0.048), but not by D-arginine. CONCLUSION: Local somatothermal stimulation inhibits internal anal sphincter motility through the activation of nonadrenergic noncholinergic neural release of nitric oxide. This procedure may represent a simplified approach for the treatment of anorectal diseases with hypofunction of the L-arginine/nitric oxide pathway. [Key words: Local somatothermal stimulation; Nitric oxide; Internal anal sphincter; Motility; Moxibustion] Jiang J-K, Chiu J H, Lin J-K. Local somatothermal stimulation inhibits motility of the internal anal sphincter through nitrergic neural release of nitric oxide. PMID- 10733122 TI - Membrane-type 1 matrix metalloproteinase mRNA expression in colorectal cancer. AB - PURPOSE: Membrane-type matrix metalloproteinases are recently described proteolytic enzymes belonging to the matrix metalloproteinase family. Initial studies have indicated that membrane-type matrix metalloproteinases are involved in tumor invasion and metastasis. Membrane-type 1 matrix metalloproteinase is the first membrane-type matrix metalloproteinase to be described. The aim of this study was to investigate the expression of membrane-type 1 matrix metalloproteinase mRNA in colorectal cancer. METHODS: Samples were collected from surgical specimens of patients with colorectal adenocarcinoma and were immediately frozen in liquid nitrogen and stored at -80 degrees C until processed. Both normal and cancer tissue was taken from each patient. TNM stage, tumor differentiation, mucin production, and vascular invasion were assessed. Northern blotting was used to quantify membrane-type 1 matrix metalloproteinase mRNA levels in the samples using a membrane-type 1 matrix metalloproteinase cDNA clone. X-ray film images were digitized and densitometry was used to quantify bands. All samples were normalized against 18S rRNA levels. Results are expressed as the ratio of cancer to normal tissue levels. Statistical analysis was performed using analysis of variance, with P < 0.05 accepted as the level of significance. RESULTS: A total of 32 samples were prospectively analyzed. The correlation between TNM stage and increased expression of membrane-type 1 matrix metalloproteinase mRNA in cancer tissue over normal tissue is expressed in the mean ratio of cancer to normal tissue expression for Stages I through IV, respectively: 1.4 +/- 0.2 (12 patients); 4.1 +/- 2.6 (8 patients); 3.4 +/- 3 (7 patients); and 4.5 +/- 5 (5 patients). Stage I is significantly different from Stages II and IV (P < 0.05). These preliminary results show an overall increasing trend in membrane-type 1 matrix metalloproteinase expression with increasing tumor stage. However, there was no correlation between membrane-type 1 matrix metalloproteinase expression and mucin production, degree of tumor differentiation, or vascular invasion. CONCLUSION: Preliminary results indicate that membrane-type 1 matrix metalloproteinase levels correlate with increasing tumor stage. PMID- 10733123 TI - Immunohistochemical localization of cathepsin D in colorectal tumors. AB - PURPOSE: Although it has been suggested that cathepsin D, a lysosomal protease, is involved in tumor invasion and metastasis in human colorectal cancers, conflicting studies have also been reported recently. In addition, this issue has been only rarely studied in human colorectal tumors by use of immunohistochemical methods. The aim of the study presented here was to clarify not only the correlation between cathepsin D expression and tumor invasion or metastasis but also the correlation between the intracellular immunostaining pattern of cathepsin D and tumor invasion and metastasis in human colorectal tumors. METHODS: Thirty-four primary colorectal adenocarcinomas and 24 adenomas were immunostained by use of an anticathepsin D antibody. Both the incidence and the immunostaining patterns of cathepsin D were investigated in all tissue samples. RESULTS: Three different immunostaining patterns, i.e., supranuclear, basal, and diffuse, were observed in samples containing cathepsin D. Although the incidence of cathepsin D-positive carcinomas was not correlated with tumor progression, invasion, or metastasis, the immunostaining pattern was significantly correlated with lymphatic invasion. CONCLUSIONS: The results of this study suggest that abnormal cathepsin D immunostaining patterns (basal or diffuse) can be used to predict a potential for lymphatic invasion in colorectal carcinoma. PMID- 10733124 TI - Selective augmentations of intratumoral 5-fluorouracil concentration by local immunotherapy with OK-432 and fibrinogen. AB - PURPOSE: Pyrimidine nucleoside phosphorylase is an enzyme that converts 5'-deoxy 5-fluorouridine into its active metabolite, 5-fluorouracil. In colorectal cancer tissue pyrimidine nucleoside phosphorylase has been proven to be produced by macrophages in the cancer stroma despite presence of the cancer cells. We reported that local immunotherapy with OK-432 and fibrinogen induced aggregation of macrophages in the cancer stroma and enforced their pyrimidine nucleoside phosphorylase expression. Thus it was hypothesized that if colon cancer were treated with 5'-deoxy-5-fluorouridine, the 5-fluorouracil concentration in cancer tissues would be enhanced by local immunotherapy. The present study was conducted to investigate whether local immunotherapy for colon cancer could increase the intratumoral 5-fluorouracil concentration in patients given chemotherapy with 5' deoxy-5-fluorouridine. METHODS: Twenty patients with resectable colorectal cancer were examined in this study. They were given 5'-deoxy-5-fluorouridine (600 mg/day) orally for seven days preoperatively. Nine randomly selected patients underwent intratumoral injection of OK-432 mixed with fibrinogen, which was performed on the third preoperative day (OK-432 and fibrinogen plus 5'-deoxy-5 fluorouridine group); eleven patients were given oral 5'-deoxy-5-fluorouridine only (5'deoxy-5-fluorouridine group). The 5-fluorouracil concentration in tumor tissue and normal colon mucosa tissue was measured, and the influence of the local immunotherapy was assessed. RESULTS: The 5-fluorouracil concentration in the cancer tissue was increased by the local immunotherapy, whereas that in the normal colon mucosa was not influenced. Thus, the influence of local immunotherapy was selective to the cancer tissue where the mixture of OK-432 and fibrinogen was injected. CONCLUSION: In patients with colorectal cancer, selective high 5-fluorouracil concentration in the cancer tissue could be achieved by a combination of 5'-deoxy-5-fluorouridine and local immunotherapy with a mixture of OK-432 and fibrinogen. PMID- 10733125 TI - Aneuploidy and columnar cuff surveillance after stapled ileal pouch-anal anastomosis in ulcerative colitis. AB - PURPOSE: A stapled pouch-anal anastomosis without mucosectomy is widely used in restorative proctocolectomy. Uncertainty exists about the longer-term outcome of retaining a columnar cuff of epithelium in the anal canal and about the need for surveillance of the columnar cuff. The aim of this article was to assess the ability to obtain biopsies of the columnar cuff, to assess the risk of dysplasia, and to search for the presence of aneuploidy as an early of marker of dysplasia in nondysplastic epithelium. METHOD: A total of 457 biopsy specimens were taken during 203 examinations of 113 patients. All biopsy specimens were stained with hematoxylin and eosin and examined by microscopy. One hundred thirty-two of these biopsy specimens from 67 patients were frozen and analyzed by flow cytometry for aneuploidy. RESULTS: Mean follow-up after pouch formation was 2.5 years, and the time after diagnosis of ulcerative colitis was 10.1 years. Successful columnar cuff biopsies were done on 93 percent of patients. There was no dysplasia. Two biopsy specimens from one patient had aneuploidy. CONCLUSION: To date, neoplastic change in the columnar cuff is rare. A selective policy of surveillance biopsies is recommended that includes patients greater than ten years after the diagnosis of ulcerative colitis and patients with dysplasia or cancer in their proctocolectomy specimen, but long-term follow-up data are needed. PMID- 10733126 TI - Sigmoid volvulus in Department of Veterans Affairs Medical Centers. AB - PURPOSE: Sigmoid volvulus is the third leading cause of large-bowel obstruction. The optimal management strategy remains controversial. This study was undertaken to evaluate the care of patients with sigmoid volvulus recently treated at Department of Veterans Affairs hospitals. METHODS: All patients with the International Classification of Diseases, Ninth Revision, Clinical Modification, Third Edition code for colonic volvulus during the period 1991 to 1995 were identified in the computerized national Department of Veterans Affairs database. Data on patient demographics, clinical course, and outcomes were analyzed. RESULTS: Two hundred twenty-eight patients had volvulus of the sigmoid colon and sufficient clinical data for evaluation. The mean age was 70; all were males. Endoscopic decompression was attempted in 189 of 228 (83 percent) patients and was successful in 154 of 189 (81 percent). Management included celiotomy in 178 of 228 (78 percent) patients. There were no intraoperative deaths. Twenty-five of 178 (14 percent) patients died within 30 days of surgery. The mortality rate was 24 percent for emergency operations (19/79), and 6 percent for elective procedures (6/99). Mortality was correlated with emergent surgery (P < 0.01) and necrotic colon (P < 0.05). Among those 50 patients managed by decompression alone, six (12 percent) died during the index admission. Ten of the remaining 44 (23 percent) patients eventually developed recurrent volvulus requiring further treatment, and 2 of 10 (20 percent) patients died. CONCLUSIONS: In this cohort sigmoid volvulus often presents as a surgical emergency. Initial endoscopic decompression resolves the acute obstruction in the majority of cases. Surgical intervention carries a substantial risk of mortality, particularly in the setting of emergent surgery or in the presence of necrotic colon. PMID- 10733127 TI - Femoral neuropathy: an infrequently reported postoperative complication. Report of four cases. AB - Postoperative femoral neuropathy is an uncommon complication of abdominal surgery. We present four cases occurring after colectomy at our institution and discuss the diagnosis and treatment. PMID- 10733128 TI - Dieulafoy's lesion of the anal canal: a new clinical entity. Report of two cases. AB - Dieulafoy's lesion is an unusual source of massive lower gastrointestinal hemorrhage. It is characterized by severe bleeding from a minute submucosal arteriole that bleeds through a punctate erosion in an otherwise normal mucosa. Although Dieulafoy's lesions were initially described only in the stomach and upper small intestine, they are being identified with increasing frequency in the colon and rectum. To our knowledge, however, Dieulafoy's lesion of the anal canal has not been described previously. We present two patients with Dieulafoy's lesion of the anal canal who presented with sudden onset of massive hemorrhage. The clinicopathologic features of this unusual clinical entity are discussed and suggestions are made for diagnosis and management. PMID- 10733129 TI - Keratoacanthoma in the differential diagnosis of anal carcinoma: difficult diagnosis, easy therapy. Report of three cases. AB - Three male patients who had an anal keratoacanthoma are described. Anal keratoacanthoma is a rapidly growing skin tumor that invades the dermis but remains locally and can mimic clinically and histologically a squamous-cell carcinoma. PMID- 10733130 TI - Nifedipine for treatment of anal fissures. PMID- 10733131 TI - Long-term survival after resection of metastatic melanoma from the colon. PMID- 10733132 TI - Vesicular blood flow after ligation of the internal iliac arteries in low anterior resection or abdominoperineal resection. PMID- 10733133 TI - Effect of adaptive plasticity of linear vestibulo-ocular reflex upon angular vestibulo-ocular reflex. AB - OBJECTIVE: The vestibulo-ocular reflex (VOR) produces compensatory eye movements during head movements. The VOR consists of the angular VOR and the linear VOR. The VOR is under adaptive control that corrects VOR performance when visual vestibular mismatch arises during head movements. Most experiments concerning plasticity of the VOR have used the angular VOR. So far, there has been no investigation on the effect of adaptive plasticity of the linear VOR on the angular VOR. In this study, using a linear sled, we tested the hypothesis that the angular VOR is altered by changes in the linear VOR. METHODS: To modify the linear VOR, we used an adaptation paradigm. Eight subjects were translated sinusoidally at 0.125 Hz and 0.16 g peak acceleration along their inter-aural axis for 40 min, while viewing earth-fixed vertical stripes on the wall while wearing the binocular magnifying lenses. Six of the eight subjects showed an increase in linear VOR sensitivity after adaptation. Each of the six subjects who showed an increase in linear VOR sensitivity after adaptation also showed an increased angular VOR gain at 0.125 Hz. CONCLUSION: These results suggest that the linear VOR and the angular VOR share common neural pathways in such a way that a change in the synaptic efficacy of one pathway is accompanied by a commensurate change in the other. PMID- 10733134 TI - The human vertical optokinetic afternystagmus and the effect of gravity during pitch tilt. AB - BACKGROUND: Findings concerning human vertical optokinetic afternystagmus (VOKAN) are often not consistent and in some cases even contradictory. METHOD: VOKAN was studied in nine normal subjects using corneo-retinal potential technique and an infrared video camera detection apparatus (ISCAN). VOKAN responses were measured at three angles of pitch head (and body) tilt: 0 degrees (upright), 90 degrees (supine) and 135 degrees (45 degrees below horizontal). The optokinetic stimulus was stripe movement with a velocity of 60 degrees/s. RESULTS: Upward slow phase velocity (SP-Up) VOKAN was evoked in 6/9 subjects (67%) at 0 degrees tilt and at 90 degrees tilt, and in 4/9 (44%) at 135 degrees tilt. Downward slow phase velocity (SP-Down) VOKAN was elicited in 3/9 subjects (33%) at 0 degrees tilt; in 2/9 subjects (22%) at 90 degrees tilt and in 0/9 subjects (0%) at 135 degrees tilt. CONCLUSION: The pitch tilt, as a gravity effect, influences significantly on the appearance and the duration of VOKAN. PMID- 10733135 TI - Loudness discomfort levels in patients with conductive and mixed hearing loss. AB - OBJECTIVE: The purpose of this study is to investigate the effects of conductive component on the loudness discomfort level (LDL) judgments. The relation between LDL value and the magnitude of air-bone gap in such subjects was also assessed. METHODS: LDLs were obtained from 100 ears of 50 hearing-impaired subjects. Twenty five subjects (50 ears) had either conductive or mixed hearing loss for both ears. Another 25 subjects (50 ears) with bilateral sensorineural hearing loss served as control. LDL measurements were performed using the method reported by Hawkins et al. in 1987. LDL data were plotted and analyzed as a function of hearing loss for three stimulus frequencies (0.5, 1.0 and 2 kHz). Also, LDL values were plotted as a function of air-bone gap in the conductive group. RESULTS: LDLs were significantly higher in subjects with conductive or mixed hearing loss for all three tested frequencies. There is a significant positive correlation between LDL value and the magnitude of air-bone gap for all three test frequencies. Considerable intersubject variability was found in LDLs obtained from subjects with conductive component which prevented the accurate prediction of LDLs from threshold data for such patients. CONCLUSION: The large intersubject variation in LDL data strongly suggests the need for individualized LDL measurements in patients with conductive or mixed hearing loss. Higher LDLs indicates that higher maximal power output can be prescribed for such patients without worrying about loudness intolerance. PMID- 10733136 TI - The effects of changing middle ear pressure and gas partial pressure on mucosal blood flow and vascular permeability in the chinchilla. AB - OBJECTIVE: To determine if middle ear (ME) gas composition and/or total pressure regulates local mucosal blood flow (MBF) and vascular permeability. The hypotheses tested are: (1) relatively high local CO2 tensions and/or low O2 tensions increase the ME MBF and vascular permeability; and (2) sub-atmospheric total ME pressure provokes similar effects. METHODS: The responses of ME MBF and vascular permeability parameters were measured during 60 min exposures of chinchilla MEs to one of two test gas mixtures (16.3% O2, 5.1% CO2, balance N2, or 5.3% O2, 15.6% CO2, balance N2) applied at different levels of underpressure (ref. ambient). In the first set of experiments (n = 19), mucosal perfusion parameters were recorded using a Laser Doppler Flowmeter for 60 min before and 60 min after exposure to the experimental conditions. In the second set of experiments (n = 19 chinchillas, 38 ears), the MEs were exposed to the gas mixtures and then maintained for 60 min at ambient pressure or at negative pressures of -200, -400, -600 mmH2O. Fifty minutes into the experiment, the animals were injected intravenously with 60 mg/kg of horseradish peroxidase (HRP). The animals were killed and existing effusion was aspirated, its volume recorded and then analyzed for total protein. From surface preparations of the ME mucosa, vascular leakage sites were measured as percent total surface area using an image analysis program with the threshold window set to discriminate HRP stain. RESULTS: Throughout the 120 min in the first set of experiments, the measured MBF parameters decreased in all exposure groups. Comparisons among groups for the absolute magnitude of the change from baseline showed that high local CO2 partial pressures decreased MBF and ME underpressures increased MBF, but the effects did not achieve statistical significance. The results of the second set of experiments demonstrated no effect of gas composition on any of the measured parameters of vascular permeability. All measures of permeability were linearly related to the magnitude of the underpressure. CONCLUSION: These data support a role for total ME pressure, but not CO2 partial pressure in regulating ME MBF and vascular permeability. PMID- 10733138 TI - Long-term observation on hearing change in patients with chronic otitis media. AB - OBJECTIVE: Very few reports are available in the literature to clarify the natural long course of hearing change in an individual patient with chronic otitis media (COM). We reviewed hearing change in patients suffering from perforated COM without an operation. METHODS: Eighty-seven ears of 70 patients had COM with tympanic perforation and were observed without an operation. The mean follow-up period was 10.7 years (5-22 years) and the mean age at the first examination was 51.3 years. To minimize the effect of aging, 23 patients with normal ear drums on the other side were further analyzed. RESULTS: All 87 ears tended to show deterioration of hearing gradually under long observation. In 23 patients, hearing deterioration was 0.13 dB/year in the control side and 0.61 dB/year in the COM side (P < 0.02). Although elevation of bone conduction hearing level tended to be larger at high frequencies than at low and middle frequencies, there was no significant difference between COM and normal ears. CONCLUSIONS: Air conduction hearing levels deteriorated with the passage of time and surgery is recommended at the early stage of COM to prevent progress of hearing loss. PMID- 10733137 TI - The prevalence of allergy in young children with an acquired cholesteatoma. AB - STUDY OBJECTIVE: To determine whether allergic sensitisation occurs more frequently in young children with an acquired cholesteatoma in comparison with young children without ear diseases. DESIGN: In this retrospective case-control study the allergic sensitisation of 43 children (age 0-10 years) who were operated for an acquired cholesteatoma was compared with the allergic sensitisation of children without ear pathology. SETTING: Otology department of the ORL clinic of the Academic Medical Centre of University of Amsterdam in the Netherlands. MEASUREMENTS: From all subjects a complete history examination was performed and stored in a database. To assess for the presence of allergic sensitisation in patients who underwent radical ear surgery because of an acquired cholesteatoma, and the control group a multiantgen radioallergosorbent test (RAST) was performed. The GraphPad Prism v 2.0. statistical software was used to perform the analyses. RESULTS: Eighteen subjects (41.8%) demonstrated sensitivity to allergens and this is statistically significant (P < 0.0047) higher than the control group and expected for the general paediatric population. 80% of the patients with a positive multiantgen radioallergosorbent test were boys. In all positive cases an allergy for the house dust mite was determined. CONCLUSION: Allergy is statistically frequently present in paediatric patients with an acquired cholesteatoma. The house dust mite seems to be the most important allergen when allergy is involved with an acquired cholesteatoma. Cholesteatoma with an positive allergy test occurs much more often in boys than girls. This could mean that young boys with a series of inflammatory middle ear diseases and allergic sensitisation are probably more at risk to develop an acquired cholesteatoma in childhood. PMID- 10733139 TI - Morphometric study of the medial aspect of the human maxillary sinus with special reference to the nasal fontanelle. AB - OBJECTIVE: The purpose of this study was to clarify configurations of the nasal fontanelle (NF) from the morphometrical point of view, especially variations of its four margins (anterior, posterior, superior, and inferior), for clinical application. METHODS: We used 136 sides of hemi-sectioned heads that were obtained from 119 donated Japanese cadavers (66 men and 53 women with an average age of 77.6+/-12.0). After mucosal examination, the specimens were boiled with a small amount of powdered soap and treated with protease. The residual mucous membrane was then gently removed. These specimens were originally made for our previous study describing variations of the uncinate process (Isobe M, Murakami G, Kataura A. Variations of the uncinate process of the lateral nasal wall with clinical implications, Clin. Anat. 1998;11:295-303). Different series of measurements were conducted based on surgical approaches and angles of observation. RESULTS: The superior margin of the NF is difficult to identify because the ethmoidal infundibulum, which leads the anterior end of the margin upward, often interrupts the superior margin. Because the inferior and posterior margins are modified by thin paper-like bony structures and because the anterior margin is disturbed by variations of the lacrimal bone and/or the inferior turbinate, they are also difficult to identify. Knowing these variables, we evaluated the NF morphometrically. The NF was located 12.6+/-4.3 mm posterior to the anterior nasal spine and 6.6+/-2.2 mm anterior to the sphenopalatine foramen. The size of the NF was 17.9+/-3.2 mm (anteroposterior axis) x 11.5+/-3.0 mm (inferosuperior axis). The lowest orbital floor was located 10 mm below the superior margin of the anterior NF (-10 mm), and the distance was therefore measured as -3.7+/-2.4 mm on average. Viewed from the maxillary sinus, the location of the NF varied along the anteroposterior axis, whereas it was located consistently at the most superior portion of the medial aspect of the sinus wall. CONCLUSION: During endoscopic sinus surgery for tumor resection in the maxillary sinus, a large and primary window should be prepared in the posterior NF, including partial removal of the uncinate process. Preparation of an additional window in the inferior meatus is preferable to enlargement of the primary window. Approaching the anterior NF should be avoided, if possible, due to its complicated configuration as well as its proximity to the orbital floor. PMID- 10733140 TI - Immunocytochemical distribution of the c-H-ras protein in nasal polyps: localization to the terminal bars. AB - OBJECTIVE: This study was performed to investigate the role of proto-oncogene products, c-H-ras and c-erbB-2, in the pathophysiology of nasal polyps. METHODS: An immunocytochemical investigation of the expression of proto-oncogene products of c-H-ras and c-erbB-2 was performed on 16 nasal polyps and 15 nasal mucosa specimens. RESULTS: The c-H-ras protein was found to be localized to the terminal bars of apical cells of the epithelium in 11 of 16 nasal polyps and in seven of 15 nasal mucosa. Localized staining at the terminal bars for the c-H-ras protein was significantly more frequently observed in glandular-cystic type than in edematous type nasal polyps. Such localized staining was significantly more frequently seen in polyp specimens with invaginated epithelia than in those with no invaginated epithelium. Our findings confirmed that the c-erbB-2 protein was mainly localized to the plasma membrane in all samples. CONCLUSION: These results suggest that the c-H-ras protein localized to the terminal bars plays an important role in the pathophysiology of glandular-cystic type nasal polyps and that it may be related to invagination of nasal polyp epithelium. PMID- 10733141 TI - Carbon dioxide laser vaporization for turbinate: optimal conditions and indications. AB - Carbon dioxide (CO2) laser vaporization for turbinate has rapidly gained acceptance in Japan for the treatment of allergic rhinitis. OBJECTIVE: The aim of this study was to examine the effects of laser output, patient age, and the presence of a deviated nasal septum on treatment outcome in patients with intractable allergic rhinitis. METHODS: The inferior turbinates were irradiated at an output of 3 or 5 W for 0.1 s. RESULTS: Of 67 patients, 43 (64.2%) were judged to have symptoms which improved markedly or moderately after a single treatment. Treatment was more effective with a laser output of 5 W than with an output of 3 W. However, treatment was judged less effective in patients aged 15 years or less than in older patients. The presence of a slightly deviated nasal septum had no effect on treatment outcome. CONCLUSION: Although assessing outcome on the basis of symptoms is difficult, we believe that these findings will suggest the optimal conditions and indications for laser surgery for allergic rhinitis. PMID- 10733142 TI - Changes in unilateral nasal airflow in patients with seasonal allergic rhinitis measured in and out of season. AB - OBJECTIVE: Nasal congestion is a common complaint of patients suffering from seasonal allergic rhinitis yet there are very few investigations which have studied the objective changes in nasal airflow in and out of season. The aim of this study was to investigate the changes in nasal airflow in and out of season. METHODS: In the present study unilateral nasal airflow was measured in and out of the pollen season in 13 patients with seasonal allergic rhinitis. Unilateral nasal airflow was measured using posterior rhinomanometry at an inspiratory reference pressure of 75 Pa. RESULTS: There was no significant difference between median total nasal airflow in season (325 cm3/s) and out of season (324 cm3/s) (P = 0.15, n = 13) yet there was a significant difference between maximum unilateral airflow in season (278 cm3/s) and out of season (234 cm3/s) (P = 0.007, n = 26). CONCLUSION: The results of the present study indicate that although patients with seasonal allergic rhinitis may experience a sensation of nasal congestion at the start of the pollen season there is little or no change in total nasal airflow and unilateral nasal airflow may be increased. PMID- 10733143 TI - Nuclear expression of the p16CDKN2 gene product during senescence of human pharyngeal epithelial cells. AB - OBJECTIVE: It has been proposed that a gene mapping to human chromosome 9p21 plays a critical role in cellular senescence. CDKN2 is a tumor suppressor gene on chromosome 9p21. It encodes a nuclear protein, p16, which inhibits the D-type cyclin/cyclin-dependent kinase complexes that phosphorylate the retinoblastoma protein, thus blocking cell cycle progression through G1. The purpose of this study was to determine whether nuclear p16 expression is altered during the senescence of human pharyngeal epithelial cells (HPECs) m vitro. METHODS: An immunocytochemical study was performed to examine a panel of cultured HPECs with a finite lifespan for the nuclear p16 expression. All HPEC cultures were also subjected to a histochemical assay that identifies senescent cells by their positive beta-galactosidase activity. RESULTS: Nuclear p16 was undetectable when HPECs were initially cultured in serum-free low-calcium medium. However, nuclear p16 was clearly detected when the cultured HPECs exhibited beta-galactosidase activity in the same medium. CONCLUSION: These results suggest that immunocytochemically detectable amounts of nuclear p16 are associated with senescence of HPECs in vitro. PMID- 10733144 TI - Radiotherapy outcome and prognostic factors in early glottic carcinoma. AB - OBJECTIVE: To evaluate radiotherapy outcome and prognostic factors in early glottic carcinoma. METHODS: The medical records of 76 patients with glottic T1N0 or T2N0 squamous cell carcinoma diagnosed at Tampere University Hospital from 1970 to 1991 and treated with primary radiotherapy were retrospectively reviewed. Except for three patients treated during the last years of the study period, radiotherapy was delivered by split-course technique with a pause of 1-3 weeks in the middle of the treatment. Primary locoregional control and disease-specific survival were analysed by the Kaplan-Meier method, and the log rank test was applied to compare the survival functions. Prognostic factors were evaluated by uni- and multivariate Cox regression analysis. RESULTS: The 10-year locoregional control rate after radiotherapy was 83 and 48% for patients with T1 and T2 tumours, respectively (P = 0.0005). The 10-year disease-specific survival was 91% for T1 and 69% for T2 disease (P = 0.0018). The larynx could be preserved in 87% of T1 and 44% of T2 cases. Tumour extent expressed by the number of vocal cord thirds involved was the only factor with significant prognostic value in the multivariate analysis of primary locoregional control (hazard ratio (HR) 3.2, 95%CI 1.8-5.8, P = 0.0001). Involvement of the posterior vocal cord third (HR 8.4, 95%CI 1.0-69.5, P = 0.04899) and T-category (HR 3.0, 95% CI 0.9-10.2, P = 0.0790) were connected with poorer prognosis in the multivariate analysis of disease-specific survival. In the multivariate analysis of T1 cases only, higher number of vocal cord thirds involved (HR 5.9, 95%CI 2.2 16.2, P = 0.0005) and longer treatment duration (HR 1.1, 95%CI 1.0-1.3, P = 0.0188) indicated worse locoregional control. Treatment duration (HR 1.2, 95%CI 1.0-1.3, P = 0.0384) together with posterior cord involvement (HR 9.9, 95% CI 1.1-92.7, P= 0.0437) signified poorer survival. CONCLUSION: Our findings indicate that the extent of the tumour is the most important predictor of radiotherapy outcome in early glottic carcinoma. This suggests that a classification based on the actual size of the tumour could be a better prognostic indicator than the conventional T grouping. Although treatment duration was significant only in separate analysis of T1 cases, the split-course regimen resulting in long treatment times may be considered a major contributor to our relatively low local control rate also in T2 disease. PMID- 10733145 TI - Type IV collagenase and immunostaining of type IV collagen in human thyroid tumors. AB - OBJECTIVE: Required stages in the processes of malignant tumor invasion and metastasis are known to include the destruction of cell stroma and vascular basement membrane. It has been suggested that type IV collagenase can degrade type IV collagen, a main component of basement membrane. In our study, type IV collagenase activity was compared with the grade of destruction of type IV collagen by thyroid tumors. METHODS: Type IV collagenase activity was measured in human thyroid tissue obtained surgically from four patients with Graves' disease, four with follicular adenoma, six with papillary carcinoma, and four with follicular carcinoma. Six normal thyroid tissue specimens were also studied. The grade of destruction in the diseased regions of thyroid tumors and surrounding intact tissues were determined immunochemically by anti-type IV collagen antibody staining. RESULTS: Tumors with high type IV collagenase activity exhibited extracapsular invasion, lymph node metastasis and very weak discontinuity of immunostaining for type IV collagen from the basement membrane in diseased regions. In addition, surrounding intact tissues exhibited weak immunostaining for type IV collagen. Tumors with low type IV collagenase activity exhibited neither extracapsular invasion nor lymph node metastasis, but did exhibit weak immunostaining for type IV collagen from the basement membranes in diseased regions. However, the surrounding intact tissues had preserved type IV collagen in follicular basement membranes. CONCLUSION: These findings suggest that staining for type IV collagen can be considered a biochemical marker for prediction of the aggressiveness of invasion and metastasis. PMID- 10733146 TI - Clinical study of elderly patients with Meniere's and related diseases. AB - In order to study the characteristics of elderly patients (70 or over at onset) with endolymphatic hydrops disorders, we selected six patients with Meniere's disease (MD), five patients with delayed endolymphatic hydrops (DEH), and two patients with syphilis of the labyrinth (SL) from 7112 patients who had visited the Neurotological Clinic of the Toyama Medical and Pharmaceutical University Hospital during 1979-1998. Clinical features were characterized as follows: (1) Since 1990, the proportion of elderly patients increased as lifespans lengthened. (2) A narrow fluctuation in hearing, low positive rate on glycerol test, high positive rate of dominant-SP in EcochG, and the moderate abnormal rate of the oculomotor system tests were found. (3) The prognosis was relatively good. From these neurotological observations, it could be concluded that elderly patients with endolymphatic hydrops disorders were characterized by the multipathological finding of the endolymphatic hydrops and the complications of CNS disorders. PMID- 10733147 TI - Facial neuroma in the internal auditory canal. AB - Authors presented two cases of facial neuromas in the internal auditory canal, one without facial palsy and the other with facial palsy. In both cases neuromas were occult and undiagnosed. Although in the first case neuroma was greater than the other, facial palsy was not developed. The mechanism of the facial palsy due to neuromas could not be clearly clarified. PMID- 10733148 TI - Invasive verrucous carcinoma: a temporal bone histopathology report. AB - Only nine cases of primary verrucous carcinoma of the temporal bone have been reported in the English literature. We describe histopathologic findings in a 78 year-old man dying of intracranial complications of primary verrucous carcinoma of the external auditory canal. Following autopsy the temporal bone was prepared for light microscopic examination. The temporal bone was serially sectioned horizontally after fixation, decalcification, and embedding, and each 10th section was stained with hematoxylin and eosin and examined by light microscopy. The carcinoma originated from the external auditory canal, infiltrating the mastoid cavity, the middle ear, tissue adjacent to the internal carotid artery, and the posterior cranial fossa, where it invaded the right cerebellum and produced an abscess. The labyrinth and internal auditory canal were not infiltrated. Metastasis to lymph nodes or distant sites was not identified. In the present case, the verrucous carcinoma originating from the external auditory canal extended into the posterior cranial fossa, while it did not invade the membranous labyrinth. PMID- 10733149 TI - A case of giant keratoacanthoma of the auricle. AB - Although keratoacanthomas are not rare in the head and neck area, patients with this type of tumor rarely consult an otolaryngologists for treatment. Keratoacanthoma should be considered in the differential diagnosis of squamous cell carcinoma. This tumor grows rapidly, usually attaining a size of about 10-20 mm in approximately 6 weeks. This is followed by slow involution over a period of 2-6 months. A keratoacanthoma larger than 20-30 mm is called as 'giant keratoacanthoma' and it is scarce. We encountered a case of giant keratoacanthoma (50 mm in diameter) on the right auricle of 84-year-old Japanese woman with a 3 year history of gradual tumor growth. Several clinical and histopathological factors made the diagnosis difficult. The tumor was completely removed by surgery and diagnosed as a keratoacanthoma by histopathological examination. PMID- 10733150 TI - Influence of extracellular Na+ removal on cytosolic Ca2+ concentration in smooth muscle cells of rabbit cerebral artery. AB - There are some controversies over the contribution of Na+/Ca2+ exchanger (NCX) to the regulation of cytosolic Ca2+ concentration ([Ca2+]c) in smooth muscle. To prove the functional role of Na+/Ca2+ exchanger, we examined whether the removal of extracelluar Na+ could affect [Ca2+]c of rabbit cerebral arterial smooth muscle. The fluorescence ratio of fura-2 (R(340/380)) was measured in the single myocyte of rabbit middle cerebral artery and Na+ was substituted with the same concentration of NMDG+ or Li+. In 21 out of 230 cells tested, Na+ removal increased R(340,380) (deltaR(340/380)) by 115 +/- 16.5% of the deltaR(340/380) induced by 10 mM caffeine in the same cell. The Na+ removal-induced deltaR(340/380) was blocked by a selective inhibitor of cardiac type NCX exchanger (KB-R7943, (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea, 10 microM). In those cells where the Na+ removal by itself did not increase R(340/380), the caffeine-induced deltaR(340/380) was increased by Na+-removal (130 +/- 9.8% of control response, n=30). Under the whole-cell patch clamp condition, short application of caffeine induced transient increase of outward current (I(K,Ca)-transient) which reflect the change of subsarcolemmal [Ca2+]. The application of KB-R7943 increased the amplitude of I(K,Ca)-transient (n=4). These results suggest the functional existence of NCX in rabbit cerebral artery smooth muscle. PMID- 10733151 TI - Inhibition of vasoconstriction and Ca2+ currents mediated by neuropeptide Y Y2 receptors. AB - The effects of neuropeptide Y (NPY) and agonists selective for NPY Y1 and Y2 receptors were studied on contraction and Ca2+ currents in arterial smooth muscle. In isolated arterioles from the guinea pig small intestine, small brief constrictions were evoked by depolarising the arteriolar smooth muscle using high K+ solution applied from a micropipette. The constrictions were reduced in amplitude by the Y2-selective agonists PYY(13-36) and N-acetyl[Leu28, Leu31]NPY (24-36) in concentrations from 20-100 nM. NPY or the Y1 selective agonist [Leu31 Pro34]NPY in concentrations from 50 pM to 100 nM increased the amplitude of the constrictions, with a maximum effect at 10 nM. Smooth muscle cells were isolated from rat small mesenteric arteries, and voltage-activated Ca2+ currents measured by whole cell patch clamping. The peak amplitude of the Ca2+ currents was decreased by N-acetyl[Leu28, Leu31]NPY-(24-36), and by NPY (100 nM). [Leu31, Pro34]NPY either had no effect or slightly increased the Ca2+ currents. We conclude that Y2 receptors on vascular smooth muscle can reduce Ca2+ currents induced by depolarisation, and thus oppose constriction caused by smooth muscle depolarisation. PMID- 10733152 TI - Supersensitivity by AF64A, a novel inhibitor of neuronal choline-uptake, of the rat iris sphincter smooth muscle. AB - Effects of ethylcholine mustard aziridinium ion (AF64A) on contractile responses to agonists or transmural nerve stimulation (TNS) were examined in rat iris sphincter muscle. The responses to TNS of isolated sphincter muscle were abolished within 1 hr after the addition of 0.1 mM AF64A to the bathing solution, while responses to acetylcholine (ACh) and 5-hydroxytryptamine (5-HT) were not changed significantly. In sphincter muscles which were isolated 3 days after the micro-injection of 100 nmol AF64A into the anterior chamber of eyes in vivo, the response to TNS were decreased to about 30% of the control. The injection of AF64A at higher concentrations often resulted in serious chronic damage of the eye. When 100 nmol AF64A was injected twice with an interval of 3 days, the response to TNS was decreased to about 20% of the control. Maximum responses and sensitivities to ACh and 5-HT were markedly enhanced in sphincters from eyes which had been treated with AF64A twice. The sensitivity of depolarized sphincters to external Ca2+ was also increased significantly. Seven weeks after the second injection, responses to TNS recovered to more than 50% of the control and the effects of AF64A mostly disappeared. In conclusion, the acute inhibition of parasympathetic transmission without postsynaptic changes can be achieved within 1 hr after bath-application of 0.1 mM AF64A. The reduction of parasympathetic nerve activity by treatment with AF64A in vivo induces nonspecific supersensitivity in iris sphincter, whereas effects of AF64A were mostly reversible under the present experimental conditions. PMID- 10733154 TI - Alpha1-adrenoceptors in the guinea pig thoracic aorta. AB - In the present study, we tried to determine which alpha1-adrenoceptor subtypes are involved in the guinea pig thoracic aorta by using in vitro functional analysis. In first, we tried to estimate the pA2 values of some key alpha1 adrenoceptor antagonists (prazosin, 5-methylurapidil, WB4101, BMY7378 and tamsulosin) against responses to norepinephrine in the thoracic aorta of guinea pigs. The concentration-response curves of norepinephrine were rightward shifted by the presence of prazosin, 5 methylurapidil, WB4101, BMY7378 and tamsulosin. The pA2 values for these antagonists against norepinephrine were 7.83, 7.78, 8.20, 5.73 and 9.57, respectively. In second, we tried to compare the estimated pA2 values obtained in the present study with reported pKi and pA2 values for cloned and native alpha1-adrenoceptor subtypes. In rabbit mesenteric artery, trigone, urethra, prostate and human lower urinary tract which were proposed to contain the putative alpha1L-adenoceptor, we obtained the good correlation for the pA2 values reported in these tissues with pA2 values estimated in guinea pig thoracic aorta. Moreover, regression lines were close to the line of identity. These results suggest that the alpha1-adenoceptors mediating contraction of guinea pig thoracic aorta are similar pharmacologically to the putative alpha1L adenoceptor subtype in rabbit mesenteric artery, trigone, urethra, prostate and human lower urinary tract. As a final point, guinea pig thoracic aorta may be able to use as a tool to develop the new alpha1-adrenoceptor antagonist which is therapeutically advantageous in the treatment of urinary tract obstruction (e. g., in benign prostatic hyperplasia). PMID- 10733153 TI - Effects of AF64A on the mRNA levels of muscarinic receptor subtypes in the rat iris sphincter. AB - The reduction of parasympathetic nerve activity by the treatment with ethylcholine mustard aziridinium ion (AF64A) in vivo induced both specific and non-specific supersensitivities in the rat iris sphincter (Tanaka et al., 1999). Changes in the expression of muscarinic receptor subtypes, which could be a cause of specific supersensitivity induced by the treatment with AF64A, were examined using competitive PCR techniques. Muscarinic receptor population is composed of m2, m3, and m4 subtypes in the rat iris (Furuta et al., 1998). Interestingly, m4 mRNA was much more abundantly expressed than m2 and m3 in the rat iris sphincter. The treatment with AF64A significantly increased the mRNA levels of m2 and m3 subtypes to 370 and 330% of the control but not that of m4 (approximately 90% of the control). In addition, the total protein contents were increased to approximately 125% of the control. The up-regulation of the mRNA levels of m2 and m3 subtypes by the treatment with AF64A was significant when they were compensated for the increase in total protein contents. The down regulation of m4 mRNA expression was not significant even after being corrected for the protein content. These results suggest that the up-regulation of the mRNA levels of m2 and m3 subtypes may be, at least in part, responsible for the supersensitivity to muscarinic agonists after the treatment with AF64A in vivo. PMID- 10733155 TI - Development of a new catheter-tip pressure transducer. AB - Two major disadvantages of the conventional catheter-tip transducer have been incapability to calibrate baseline pressure and to adjust sensitivity after insertion. A vast majority of conventional catheter-tip transducers are classified as gauge type. Due to their structure they are inherently incapable of recalibrating after insertion. We have overcome this problem by developing a new differential type catheter-tip transducer equipped with a lumen that connects a small chamber at the backside of the transducer to another external port. This lumen is capable of pressure passage. The output of this type depends on the difference between the two imposed pressures. This passage makes a baseline standard possible, when the end of the lumen is exposed to atmospheric pressure. When pneumatic pressure is imposed to the end of the lumen using a syringe, for example, the transducer output shifts up and down in accordance to that pressure, enabling baseline pressure recalibration and verifying the degree of sensitivity after insertion. By obtaining the following data, we confirmed the stability and availability of this transducer: Baseline drift less than 0.04 mmHg/8 hour, frequency characteristics flat up to 60 Hz, and common mode rejection ratio more than 46 dB. PMID- 10733156 TI - Palpability of breast tumors--correlation with ultrasonic findings. AB - With the recent development of high-resolution real-time ultrasonic devices and their application to clinical examination and breast screening, small non palpable breast lesions have frequently been detected and controversy has arisen concerning their diagnosis and treatment. In order to evaluate the relationship between ultrasonic B-mode images and palpability of breast tumors, 71 breast tumors including 21 cancers were analyzed. Five basic measurements on tumor size and location were performed followed by calculation of secondary parameters of palpability. Larger size (D and W), less distance from the skin surface (S-T), less embeddedness within or more protrusion from the mammary gland (G-T) and greater relative size of tumor with respect to whole breast thickness (D/(S-P)) were found to be significantly related to greater palpability of breast tumors. Among the qualitative diagnostic criteria for breast tumors, an irregular shape, rough border, thick boundary echoes, heterogeneous internal echoes and indirect signs were statistically more frequent in palpable than non-palpable tumors, and in malignant than in benign tumors. In cases of cancer, palpability was related significantly only to size and to heterogeneous internal echoes. For early detection of breast cancer, identification of breast cancer among small non palpable breast lesions less than 1 cm in size in ultrasonic B-mode images is considered to be essential. PMID- 10733157 TI - A case report: tumorectomy for brain metastasis of hepatocellular carcinoma. AB - Described here is a rare case of an operation for brain metastasis of hepatocellular carcinoma (HCC) after hepatectomy. We admitted a 45-year-old male patient complaining chiefly of a visual field disturbance and headaches. He had undergone a hepatectomy due to HCC one year prior to hospitalization. He was diagnosed with a brain metastasis with intratumoral hemorrhage. He underwent a tumorectomy and hemorrhage excision. After the surgery, his neurological symptoms improved, and he was temporarily rehabilitated. The decision to operate in this case could be questionable because the patient already had a pulmonary metastasis. Nevertheless, we concluded that local therapy for such a brain metastatic lesion would be effective in alleviating his discomforts and improving his quality of life as long as the primary lesion was under control. PMID- 10733158 TI - Availability of Japanese subsidies for international telemedicine projects. AB - In this article, the authors report the methods for obtaining subsidies for overseas telemedicine projects from Japanese sources based on their own personal experiences. The Japanese Government is already subsidizing such specialized NGOs (Non-Governmental Organizations) as the Telemedicine Society of Japan and Basic Human Needs and, Japanese trading companies are also hiring telemedicine experts. Prospective methods for obtaining subsidies are outlined as under the following headings: Assistance without compensation, Technology transfer, Grass-roots grant assistance, the Telecommunications Advancement Organization, Postal Savings for International Voluntary Aid, Venture business development funds provided by the Ministry of International Trade and Industry, Mission demonstration satellites by the National Space Development Agency of Japan, the Sasakawa Pacific Island Nations Fund, and International Communications Foundation. Key points of the applications are noted under (1) Degree of contribution to local residents, (2) Significance of project continuation and (3) Novelty and economic impact. PMID- 10733159 TI - Effect of exogenous tetragastrin on gastric myoelectrical activity in humans. AB - It is known that cutaneous electrogastrography (EGG) undergoes a change after food ingestion, showing increases in frequency and amplitude compared with preprandial values, but the factors regulating such changes remain to be elucidated. Paying special attention to gastrin, one of gastrointestinal peptides released after food ingestion, we administered tetragastrin in an exogenous manner and evaluated its effects on EGG in the present study. In healthy subjects, the intramuscular injection of tetragastrin significantly increased EGG frequency dose-dependently, but caused no significant change in amplitude. These results suggest that the increase in endogenous gastrin release is one of the mechanisms which underlies the increase in EGG frequency after food ingestion. PMID- 10733160 TI - The effects of lesions in the nucleus basalis of Meynert and physostigmine on rat frontal cortex acetylcholine level. AB - To clarify the relationship between acetylcholine (ACh) level in the frontal cortex and the area of lesion in the nucleus basalis of Meynert (nbM), we used ibotenic acid to bilaterally lesion the substantia innominata (SI), including the nbM, in rats and measured extracellular ACh by microdialysis. Baseline level of ACh (ACh-baseline) averaged 0.14 +/- 0.07 ng/40 microl/20 min (n=13) in the steady state. We also measured the size of the lesioned areas in the SI (nbM lesion) by using a computed image analysis system. Both ACh level and nbM-lesion varied widely among animals. The bilateral nbM-lesion averaged 28 +/- 30% (n=13). The correlation coefficient between ACh-baseline and bilateral nbM-lesion was 0.871 (p<0.01). After intraperitoneal injection of physostigmine, maximum ACh levels increased to 0.18 +/- 0.06 ng/40 microl/20 min, and deltaACh (ACh-max/ACh baseline) averaged 132 +/- 19% (n=8). There was no apparent correlation between deltaACh and bilateral nbM-lesion. These results show that the size of lesioned area in the bilateral SI was strongly correlated with the baseline ACh level in the frontal cortex, and indicate that the nbM is the major functional source of ACh in the frontal cortex. Our results also suggests that physostigmine possibly affects not only spared nbM-originated cholinergic fibers but also non-nbM originated cholinergic fibers in the rat with the bilateral nbM lesion. PMID- 10733161 TI - Survey for Trypanosoma cruzi infection in a municipality in northeast Brazil. AB - An epidemiological survey of Trypanosoma cruzi infection was carried out in Bodoco, located in the western part of the State of Pernambuco, Brazil. Two hundred and forty-one individuals were parasitologically and immunologically screened. Although hemoculture did not reveal the presence of parasites in the blood, the sera of 5 individuals were scored as positive by the indirect fluorescence antibody test and the enzyme-linked immunosorbent assay. Seropositivity in individuals above and below the age of 40 was 14.8 and 0.5%, respectively. These results indicate that recent infections with T. cruzi are rare in this area. However, since a T. cruzi-infected triatomid (Triatoma brasiliensis) was captured in a school classroom, this area must be considered endemic. When triatomid feces containing parasites were inoculated into a jird (mongolian gerbil), parasitemia appeared 10 days later. Immunohistochemical staining, using monoclonal antibody specific for T. cruzi, labeled organisms in jird tissues. These observations demonstrate that the jird is a suitable host for experimental T. cruzi infections and that monoclonal antibody is effective for detection of the parasite in host tissues. PMID- 10733162 TI - Saving the cost and energy by an interactive multimedia system with ISDN 128 Kbps for telemedicine. AB - The telemedicine systems offer many potential advantages for health care delivery. In 1997, we implemented Phoenix, the interactive multimedia system of the Nippon Telegraph and Telephone Corporation (NTT) for clinical application involving teleconsultations over a wide area for delivery of special care in emergency medicine at Tokai University linking with eight hospitals via the Integrated Service Digital Network (ISDN) 128 Kbps. This study was designed to determine the potential saving of the cost and energy through the interactive multimedia network. By using the interactive multimedia system with some modifications, we achieved a satisfactory real-time contact regarding clinical matters. We believe that this network has allowed appropriate transfer of information between medical centers. This system has also significantly reduced the estimated cost for clinical meetings. PMID- 10733163 TI - HIV-DNA vaccination following transfer of a large number of activated T cells enhances immunoresponses against HIV-1. PMID- 10733164 TI - HIV gp120 plus specific peptides are recognized in a similar manner to specific HLA plus peptide by HLA-restricted antigen-specific T-cell lines. AB - HIV induces disease only following chronic activation of the immune system. Other retroviruses such as the mouse mammary tumour virus (MMTV) activate a large percentage of T cells by encoding a superantigen (SAg). To date there is no evidence that HIV encodes a SAg. An alternative way to induce pan-activation of the immune system is by allogeneic stimulation, which occurs following transplantation. Here we extend previous work which demonstrated that HIVpg120 could bind peptides in a similar manner to HLA, by demonstrating that human antigen presenting cells (APCs) expressing gp120 (but not DR1) can present a DR1 restricted peptide to induce proliferation of a DR1-restricted peptide-specific T cell line in a similar manner to the same peptide presented by a DR1 expressing APC. Our data provide strong support for the hypothesis that the HLA-like regions of gp120 encode functional properties shared with HLA, and could explain the extraordinary clinical and immunological similarities between AIDS and chronic graft versus host disease. PMID- 10733166 TI - Characterization of genetically engineered mengoviruses in mice. AB - We have shown that genetically engineered mengoviruses containing artificially shortened 5' noncoding poly(C) tracts (e.g., C0 or C13UC10) are dramatically attenuated in adult Swiss/ICR mice when compared to wild-type virus or to a genetically engineered virus containing a wild-type length poly(C) tract (C44UC10). To explore further the relationship between poly(C) tracts and virulence, we have conducted more extensive characterizations of several engineered viruses in the murine model. Both short and long poly(C) tract viruses were highly virulent in newborn mice, underscoring the importance of age in poly(C)-mediated attenuation. Virus vMC24, with a tract sequence of C13UC10, was as attenuated in 4-week-old BALB/c, C.C3-H2k/LiMcdJ, and DBA/2 mice as in Swiss/ICR mice. But it was more pathogenic for C57BL/6 mice, and highly virulent for C3H/Hej and C3H/Hen mice, demonstrating the importance of murine genotype. As expected from its virulence in all mouse strains, vMwt, with a poly(C) of C44UC10, induced higher levels of viremia than vMC24. The vMwt also induced higher levels of circulating interferon and had reduced pathogenicity in chemically immunosuppressed Swiss/ICR mice. Similar immunosuppression did not increase the virulence of vMC24. Collectively, the data suggest that endogenous immune components and the immune competence of the host play significant roles in determining the susceptibility of mice to mengovirus infection. PMID- 10733165 TI - HIV-1 infects and alters immune function of a monocyte subset expressing low CD14 surface phenotype. AB - Monocytes represent a leukocyte subset that express high levels of CD14 on their surface (CD14-high). These cells play a critical role in the pathogenesis of HIV 1 infection. In the present study, we have identified a monocyte subset expressing an extremely low level of CD14 (CD14-low), and examined their susceptibility to HIV-1 infection. Phenotypic analysis by flow cytometry of these cells revealed a low level of CD4, but the absence of CD3, CD14, CD19, and CD83 surface markers. Both CD14-low and CD14-high cell populations expressed CD13 and CD33 markers on their surface, suggesting these cells to be of myeloid origin. Morphologically, CD14-low cells were indistinguishable from CD14-high cells. CD14 low cells were susceptible to infection with a monocytotropic strain of HIV-1 (HIVADA). However, like CD14-high monocytes, CD14-low cells could not be productively infected with a T cell tropic strain of HIV-1 (H9/HTLV(IIIB)). Similar to CD14-high monocytes, CD14-low cells were capable of inducing antigen stimulated CD4+ T-cell proliferation. HIV-1 infection substantially reduced their ability to induce antigen-stimulated T-cell proliferation. These data indicate that CD14-low cells belong to the monocyte lineage and may play an important role in the immunopathogenesis of HIV-1 infection. PMID- 10733167 TI - Alloreactive cytotoxic CD4+ responses elicited by cytomegalovirus-infected endothelial cells: role of MHC class I antigens. AB - Cytomegalovirus (CMV) has been associated with chronic graft rejection in solid organ transplant patients. To elucidate the mechanism by which CMV leads to graft rejection, we hypothesized that CMV infection of endothelial cells could stimulate alloreactive cytotoxic T lymphocytes (CTL). This hypothesis was explored using the following experimental model: peripheral blood mononuclear cells (MNC) obtained from normal hosts were grown on monolayers of umbilical vein endothelial cells (UVEC) infected with CMV (CMV-UVEC) or not (control) and tested for CTL activity against uninfected UVEC. We showed that CMV-UVEC-stimulated MNC have significant CTL activity against uninfected UVEC. The CTL activity elicited by CMV-UVEC stimulation was significantly higher compared with that stimulated by uninfected UVEC or by ganciclovir-treated CMV-UVEC, indicating the critical role of productive CMV infection. The CTL activity was specific for the UVEC used as stimulators and did not affect MHC-unrelated UVEC. However, lymphoblastoid lines (LBL) major histocompatibility complex (MHC)-identical with the stimulator UVEC were also killed by the CMV-UVEC-stimulated MNC. CTL killed identical UVEC and LBL in a competitive fashion. Blocking experiments with monoclonal antibodies (mAbs) identified CD4 cells as the main effector of CTL activity and MHC class I as the antigenic target of CTL. Although natural killer (NK) cells did not significantly contribute to the CTL activity of CMV-UVEC-stimulated MNC, their presence in the MNC cultures during the stimulation process was critical for the development of CTL. This model offers a framework for understanding the role of CMV infection in graft rejection and for devising preventative strategies. PMID- 10733168 TI - Vaccination of seropositive subjects with CHIRON CMV gB subunit vaccine combined with MF59 adjuvant for production of CMV immune globulin. AB - The safety and immunogenicity of four different regimens of CHIRON cytomegalovirus (CMV) gB subunit vaccine combined with MF59 adjuvant and administered to seropositive plasma donors were evaluated to ascertain whether vaccination of seropositive subjects would significantly increase antibody titer to gB glycoprotein. This was done to select the best vaccination regimen for generating high-titered plasma for manufacture of CMV immune globulin. No serious adverse events were attributed to this vaccine, and the vaccine was well tolerated. Only the first dose of vaccine in each regimen stimulated a four-fold or greater antibody response to gB glycoprotein and each regimen induced similar antibody titers. However, initial vaccination followed by a 1 week rest from plasmapheresis and two booster vaccinations at 8 and 24 weeks, each followed with another 1 week rest from plasmapheresis, maintained the highest geometric mean gB ELISA titer of the four regimens over the 34-week post-vaccination period. CMVIG manufactured from a pool of high titered plasma units from two of four subject groups had gB ELISA and neutralizing antibody titers nine and six times higher, respectively, compared to Cytogam, indicating that vaccination of seropositive subjects with CHIRON gB vaccine combined with MF59 adjuvant prior to harvesting plasma can enhance functional antibody in a CMVIG product. PMID- 10733169 TI - The immunogenicity of subunit vaccines for respiratory syncytial virus after co formulation with aluminum hydroxide adjuvant and recombinant interleukin-12. AB - The effects of recombinant interleukin-12 (rIL-12) on immune responses generated by subunit vaccines for respiratory syncytial virus (RSV) were evaluated in BALB/c mice. Parenteral co-administration of rIL-12 with F/AlOH or F/PBS resulted in accelerated clearance of infectious virus from the lungs 4 days after challenge. The immune responses elicited by 0.03 microg of F protein plus 10 ng of rIL-12 adsorbed to AlOH were more efficacious than those induced by 3 microg of F protein co-formulated with 1,000 ng of rIL-12 in PBS alone. Adsorption to AIOH prolonged the presence of rIL-12 in the sera. The resultant systemic humoral immune responses after vaccination with F/AlOH or G/AlOH were dependent on the dose of rIL-12 and characterized by heightened serum immunoglobulin G2a (IgG2a) antibody titers. Co-administration of rIL-12 with F/AlOH was also associated with diminished protein-specific IgE titers, elevated neutralizing antibody titers, and interferon-gamma and (IFN-gamma) in the sera, and enhanced antigen-dependent killer cell activity in the lungs after challenge. For maximum benefit, the data suggested that rIL-12 must be co-administered with F/AlOH. Collectively, the results indicated that rIL-12 directed immune responses toward a type 1 phenotype. However, examination of pulmonary inflammatory cells after challenge suggested that the type 1 phenotype was not absolute. Co-formulation with rIL-12 did not diminish pulmonary eosinophilia upon challenge of naive mice primed with F/AlOH, G/AlOH, or FI-RSV, and CD4+ T cells were expanded relative to the CD8+ T cell compartment. These results are important for the future design of subunit vaccines against RSV. PMID- 10733170 TI - Induction of cytotoxic T lymphocytes of heterogeneous specificities by immunization with a single peptide derived from influenza A virus. AB - We examined whether immunization with a single peptide induces cytotoxic T lymphocytes (CTLs) of heterogeneous specificities in vivo. Immunization of BALB/c mice with the peptide H2:529-537, which corresponded to amino acid residues 529 537 on the HA2 subunit transmembrane region of influenza A/Jap virus (H2N2) and possessed an H-2Kd-binding motif, induced CD8+CD4- CTLs. These CTLs lysed influenza A/Jap virus-infected target cells as well as those pulsed with the H2:529-537 peptide. H2:529-537 peptide-induced CTLs also lysed to lower but significant levels the target cells pulsed with the H1:533-541 peptide, which corresponded to amino acid residues 533-541 on the HA2 subunit transmembrane region of influenza A/PR/8 virus (H1N1) and were compatible to H2:529-537. Immunization with the H1:533-541 peptide, which also possessed an H-2Kd-binding motif, induced CTLs in vivo. H1:533-541-induced CTLs lysed influenza A/PR/8 virus infected target cells and those pulsed with the peptide H1:533-541. Subtype cross reactive CTLs to the H2:529-537 peptide were not induced by immunization with the H1:533-541 peptide. Two peptides, H2:3S and H2:7S, which had one amino acid substitution, serine at the third and seventh positions, respectively, induced CTLs that lysed target cells pulsed with the respective peptides to the highest levels. These results indicate that immunization with a single peptide induces CTLs of heterogeneous specificities in vivo. PMID- 10733171 TI - Neutralizing monoclonal antibody to the E1 glycoprotein epitope of rubella virus mediates virus arrest in VERO cells. AB - The best-known mechanism of action of antibody-mediated virus neutralization is to impede the entrance of viruses to host cells, as determined by neutralization assays. Antibodies may also inhibit the exit of rubella virus (RV) from infected host cells; in this case, the interaction of the antibodies with their domains must occur on the plasma membrane, because antibodies cannot enter the cells. In the present study, we were able to block temporally the exit of virions from RV infected cells by the binding of monoclonal antibody (mAb) H3 to their surface. The objective was accomplished in three steps: first, we determined the duration of the viral replication cycle; then we established the kinetics of the presence of the domains defined by our mAbs in the cytoplasm of RV-infected VERO cells; and, finally, we assessed the release of viral particles to the supernatant of infected VERO cells in the presence or absence of mAbs or positive and negative mice sera. RV-specific mice sera and mAb H3, which binds to the amino acid sequence 208-239 of the RV-E1 glycoprotein, were able to delay for 24 hours the release of virions from infected cultures, suggesting that the reaction of mAb H3 with its epitope may arrest any change necessary for the assembly and/or release of virions. In conclusion, the neutralizing domain recognized by mAb induces antibodies that can block the viral replication by several mechanisms of action, such as the obstruction of virus entry into cells and the delay of viral release. All of these mechanisms are intimately involved in the critical virus-host cell interactions that allow self-limitation of the infection. PMID- 10733172 TI - Characterization of protection against coronavirus infection by noninternal image antiidiotypic antibody. AB - Previously, we have reported protective vaccination of mice against a coronavirus infection using rabbit polyclonal noninternal image Ab2gamma anti-idiotypic (anti Id) antibody specific for a virus-neutralizing and protective monoclonal antibody (mAb) 7-10A against the viral surface S glycoprotein. To characterize further the mechanisms involved in the induction of protective immunity by this noninternal image anti-Id, plasma and splenocytes from Ab2gamma-immunized BALB/c mice were passively transferred to naive BALB/c mice, followed by viral challenge. A reproducible significant delay in mortality observed in mice to which plasma was passively transferred, together with the presence of specific in vitro neutralizing antiviral Ab3 identified the humoral immune response as the major element responsible for protection. The activation of specific and cross-reactive T lymphocytes by both virus and anti-Id in immunized mice and the absence of adoptive transfer of protection by splenocytes suggested the participation of T helper activity in the induction of protective virus-neutralizing Ab3. To obtain more defined monoclonal reagents for a better understanding of anti-Id-induced protection, mAb2 were generated against the same mAb1 7-10A and characterized. We report the successful generation of mAb2 of the gamma type. However, unlike the polyclonal Ab2gamma, they were not capable of inducing a protective immune response. PMID- 10733173 TI - Homing studies on distribution of ectromelia (mousepox) virus-specific T cells adoptively transferred into syngeneic H-2d mice: paradigm of lymphocyte migration. AB - Mousepox (infectious ectromelia) may be used as a model for studies on the cellular immune response and pathogenesis of generalized viral infections. Ectromelia virus (EV) initially replicates in the footpad (f.p.) skin at the site of infection, next in draining lymph nodes, and then in the spleen and liver where the virus may induce extensive necrotic process with inflammatory reaction. We show in this study that after recipient BALB/c mice (H-2d) f.p. infection with EV prior to the adoptive transfer of syngeneic donor EV-specific cytotoxic T lymphocytes interferon-gamma-positive (IFN-gamma-+), interleukin-2-positive (IL 2+), and IL-4+ of both phenotypes, CD8+ approximately 70%, and CD4+ approximately 30%) preferentially migrated to the inguinal and auxiliary lymph nodes, spleen, liver, and skin at the site of infection (f.p.). Many particles of EV with the morphology characteristic for orthopoxviruses and virus-specific immunofluorescence within the cells of inguinal and auxiliary lymph nodes, liver, spleen, and skin have been observed using high-resolution transmission electron microscopy and fluorescence antibody technique, respectively. Results presented in this article support the concept that immune T cells adoptively transferred into infected recipient mice are able not only to specific migration in the host and homing in the sites of virus replication, but also to develop immunoprotection in the transferred animals. PMID- 10733176 TI - 21st century audiology. PMID- 10733174 TI - Lung surfactant protein A provides a route of entry for respiratory syncytial virus into host cells. AB - Lung surfactant protein A (SP-A) has a central role in host defense mediated by the interaction of surface carbohydrates of inhaled pathogens with the lectin domains of SP-A. Respiratory syncytial virus (RSV), the most important viral pathogen of neonates and infants, encodes a highly glycosylated attachment protein, G. Binding studies were performed with G-protein from RSV (human, A2 strain) and human SP-A. The effect of SP-A on the interaction between RSV and host cells was determined by two methods: an infectivity study with monolayers of Hep-2C cells and by interleukin-8 (IL-8) release from buffy coat (BC) cells. SP-A binds to RSV G-protein in a concentration-dependent manner that is inhibitable by both ethylenediamine tetraacetic acid (EDTA) and mannan, indicating that binding is through the carbohydrate recognition domain of the SP-A and a carbohydrate moiety of the G-protein. The level of RSV infection of Hep-2C cells increases with increasing concentrations of SP-A. The amount of IL-8 released by BC cells in the presence of RSV is increased with SP-A concentrations of 2.9 microg/mL or greater. Our results show that SP-A enhances the attachment of RSV and subsequent entry into host cells. The effect of SP-A on viral uptake by epithelial cells and macrophage may determine both innate and adaptive immune responses to RSV. PMID- 10733175 TI - Maternal IgG avidity and IgM detected by blot as diagnostic tools to identify pregnant women at risk of transmitting cytomegalovirus. AB - In this study, we determined the avidity index (AI) of anticytomegalovirus (CMV) immunoglobulin G (IgG) and the anti-CMV immunoglobulin M (IgM) profile in 124 pregnant women, 87 of whom were considered at risk of transmitting CMV infection to their offspring and 37 of whom were at no risk. IgG avidity and blot for IgM were performed on two serum samples from each woman, at 6-18 weeks' gestation and at 20-23 weeks' gestation. Pregnancy outcomes were monitored. The results obtained showed that the determination of anti-CMV IgG avidity at 6-18 weeks' gestation can identify all women who would have an infected fetus/newborn (100% sensitivity), whereas IgM detected by blot had poorer results (69% sensitivity). Interestingly, at 20-23 weeks' gestation, the sensitivity of IgM detection by blot was higher than that obtained by avidity (75 % and 63%, respectively) and the combination of IgG avidity and IgM by blot yielded the best results (81% sensitivity). PMID- 10733177 TI - In pursuit of ceruminolytic agents: a study of earwax composition. AB - OBJECTIVE: To assess the protein and simple sugar content of earwax in pursuit of better ceruminolytic agents. STUDY DESIGN: Collected earwax specimens were tested in several media for dissolution before being analyzed for amino acid and carbohydrate content. PATIENTS: The samples were obtained from eight random patients requiring ear plug removal. RESULTS: The amino acid composition differs considerably from hair and stratum corneum of glabrous skin. Sugar analysis revealed high levels of galactosamine and galactose. CONCLUSIONS: This analysis of proteins and carbohydrates further characterizes earwax. Future ceruminolytic agents must dissolve lipid, keratin, and a monolayer of lipid covalently bound to the epidermal cells, which encourages cellular aggregation. PMID- 10733178 TI - Biologic activity of mitochondrial metabolites on aging and age-related hearing loss. AB - HYPOTHESIS: Compounds that upregulate mitochondrial function in an aging model will improve hearing and reduce some of the effects of aging. BACKGROUND: Reactive oxygen metabolites (ROM) are known products of oxidative metabolism and are continuously generated in vivo. More than 100 human clinical conditions have been associated with ROM, including atherosclerosis, arthritis, autoimmune diseases, cancers, heart disease, cerebrovascular accidents, and aging. The ROM are extremely reactive and cause extensive DNA, cellular, and tissue damage. Specific deletions within the mitochondrial DNA (mtDNA) occur with increasing frequency in age and presbyacusis. These deletions are the result of chronic exposure to ROM. When enough mtDNA damage accrues, the cell becomes bioenergetically deficient. This mechanism is the basis of the mitochondrial clock theory of aging, also known as the membrane hypothesis of aging. Nutritional compounds have been identified that enhance mitochondrial function and reverse several age-related processes. It is the purpose of this article to describe the effects of two mitochondrial metabolites, alpha-lipoic acid and acetyl L-carnitine, on the preservation of age-related hearing loss. METHODS: Twenty-one Fischer rats, aged 24 months, were divided into three groups: acetyl-l carnitine, alpha-lipoic acid, and control. The subjects were orally supplemented with either a placebo or one of the two nutritional compounds for 6 weeks. Auditory brainstem response testing was used to obtain baseline and posttreatment hearing thresholds. Cochlear, brain, and skeletal muscle tissues were obtained to assess for mtDNA mutations. RESULTS: The control group demonstrated an expected age-associated threshold deterioration of 3 to 7 dB in the 6-week study. The treated subjects experienced a delay in progression of hearing loss. Acetyl-l carnitine improved auditory thresholds during the same time period (p<0.05). The mtDNA deletions associated with aging and presbyacusis were reduced in the treated groups in comparison with controls. CONCLUSIONS: These results indicate that in the proposed decline in mitochondrial function with age, senescence may be delayed by treatment with mitochondrial metabolites. Acetyl-l-carnitine and alpha-lipoic acid reduce age-associated deterioration in auditory sensitivity and improve cochlear function. This effect appears to be related to the mitochondrial metabolite ability to protect and repair age-induced cochlear mtDNA damage, thereby upregulating mitochondrial function and improving energy-producing capabilities. PMID- 10733179 TI - Hearing problems in Mexican American elderly. AB - OBJECTIVE: To investigate hearing problems in a sample of elderly Mexican Americans. STUDY DESIGN: A longitudinal field study of a cohort of 3,050 subjects with in-person baseline and a 2-year follow-up. Population-based, cross sectional, weighted data were analyzed. SETTINGS AND SUBJECTS: Hispanic Established Populations for Epidemiologic Studies of the Elderly (H-EPESE) consisting of Mexican Americans aged 65 and older provided basic health data using area probability sampling in five southwestern states during 1993 and 1994. MAIN OUTCOME MEASURES: Information was collected regarding demographics, medical conditions, smoking, and alcohol consumption. Hearing problems were identified through a series of self-perceived hearing problem questions, hearing aid use, and inability to hear a normal voice. RESULTS: A hearing problem was identified in 24.5% of this cohort (weighted, 748/3,049). Statistical analysis using a multiple logistic regression model was performed to identify factors jointly associated with hearing problems. Age group (odds ratio [OR] = 2.7, p<0.0001), male sex (OR = 1.9, p< 0.0001), hypertension (OR = 1.4, p<0.001), arthritis (OR = 1.5, p<0.0005), significant depressive symptomatology (OR = 1.4, p<0.002), and ever having consumed alcohol (OR = 1.4, p<0.005) were jointly statistically significantly associated with hearing problems. Number of cigarettes smoked daily (e.g., 0, 1-10, 11-20, etc.) was nearly significantly associated with a hearing problem in the multivariate model (OR = 1.1 for each increased in category, p<0.07). CONCLUSIONS: Hearing problems are common in this population. Control of hypertension, an amelioration of arthritis, and decreasing the consumption of alcohol and cigarettes may lower the likelihood of development of a hearing problem. Initial depressive symptomatology may have occurred subsequent to the hearing loss. A longitudinal study would allow determination of the direction of causation. PMID- 10733180 TI - Causes of unilateral sensorineural hearing loss screened by high-resolution fast spin echo magnetic resonance imaging: review of 1,070 consecutive cases. AB - OBJECTIVE: Evaluation of the ability of screening high-resolution, nonenhanced, fast spin echo (FSE) T2-weighted magnetic resonance imaging (MRI) of the internal auditory canal (IAC) and cerebellopontine angle (CPA) to detect nonacoustic schwannoma causes of unilateral sensorineural hearing loss (SNHL). FSE-MRI is equally sensitive in detecting acoustic (vestibular) schwannoma as gadolinium enhanced MRI, but sensitivity to other causes of hearing loss is unknown. STUDY DESIGN: Retrospective review of screening FSE-MRI studies. SETTING: Academic otology/neurotology and neuroradiology practices. PATIENTS: There were 1,070 patients with unilateral SNHL who underwent radiologic screening for retrocochlear pathology. RESULTS: Normal findings were found in 944 cases. Typical (acoustic) vestibular schwannoma were found in 56 patients. Seventy additional lesions were identified: 27 CPA lesions, 29 inner ear lesions, and 12 intraaxial lesions including 9 infarctions, 1 multiple sclerosis case, 1 mesial temporal lobe sclerosis, and 1 colloid cyst. CONCLUSIONS: High-resolution T2 FSE MRI of the IAC and CPA is a highly sensitive screening tool for unilateral SNHL, which can detect a variety of lesions in addition to vestibular schwannomas. To our knowledge in 2 years of follow-up in these patients screened for IAC/CPA lesions, no other lesions causing SNHL have been found. High-resolution FSE screening technique, used in conjunction with appropriate clinical prescreening and referral, can provide an equally sensitive method of evaluating unilateral SNHL compared to gadolinium-enhanced T1 MRI while reducing costs and providing distinct advantages in evaluating nonacoustic schwannoma causes of SNHL. PMID- 10733181 TI - The phenotype of DFNA13/COL11A2: nonsyndromic autosomal dominant mid-frequency and high-frequency sensorineural hearing impairment. AB - OBJECTIVE: To study nonsyndromic progressive sensorineural hearing impairment in patients with a COL11A2 mutation (DFNA13) in a Dutch kindred. STUDY DESIGN: Survey. SETTING: Department of otorhinolaryngology of a university hospital. PATIENTS: Twenty-one living members of a Dutch family (150 relatives in 5 generations; 49 were studied) with autosomal dominant nonsyndromic sensorineural hearing impairment showed linkage to the DFNA13 locus. Mutation analysis revealed a missense mutation in the COL11A2 gene. METHODS: History was taken, hearing threshold levels were measured in all affected persons, and penetrance was evaluated. Longitudinal analysis (using linear regression analysis of threshold on-age data) was possible in only 1 case. Cross-sectional analysis comprised the comparison of threshold levels between different generations of the family. Also, hearing thresholds were corrected for median presbyacusis to find out whether the hearing impairment characteristic of this trait is stationary or progressive. Vestibular functions were evaluated with electronystagmography, by use of rotatory and caloric tests. RESULTS: Most of the affected persons dated their first hearing impairment symptoms to the second to third decade of life. A possibly reduced penetrance was found. Anamnestically, 4 affected persons had hearing impairment symptoms from early childhood onward. Longitudinal analysis in the proband (IV26) revealed the exceptional combination of congenital offset and substantial early progression. Most of the threshold levels differed significantly between the frequencies within each age group and, at the high frequencies (2-8 kHz), between the two age groups covered by generations IV and III (ages 30-38 years and 58-74 years, respectively); the latter could be attributed to presbyacusis. Various caloric abnormalities, including areflexia, were found in about half (8/17) of the tested subjects with sensorineural hearing impairment. CONCLUSIONS: Correction for age, hearing impairment-presumably present and stationary from an early age onward-showed the worst or second-worst threshold usually at 1, 2, and 6 kHz (3540 dB) or 8 kHz (50 dB), whereas the best or second-best threshold was found at 0.25 to 0.5 and 4 kHz (25 dB). Presbyacusis, presumably from the fourth decade of life onward, caused a change of the typically shaped audiogram described as midfrequency hearing impairment with additional high-frequency impairment. PMID- 10733182 TI - Profound hearing loss associated with hydrocodone/acetaminophen abuse. AB - OBJECTIVES: To describe profound hearing loss associated with hydrocodone overuse and the successful rehabilitation of these patients with cochlear implantation. STUDY DESIGN: Retrospective review. SETTING: A tertiary otologic referral center. PATIENTS: Twelve patients with rapidly progressive hearing loss and a concurrent history of hydrocodone overuse. INTERVENTIONS: Comprehensive medical histories, physical findings, audiometric tests, and, in those patients undergoing cochlear implantation, postimplantation performance data were reviewed. MAIN OUTCOME MEASURES: Clinical characteristics of hydrocodone-related hearing loss and open set word and sentence performance in those patients undergoing cochlear implantation. RESULTS: Hydrocodone overuse was associated with rapidly progressive sensorineural hearing loss in 12 patients. In four patients the initial presentation was unilateral, and two of the patients experienced vestibular symptoms. None of the 12 patients experienced improved thresholds after high-dose prednisone. Seven of the eight patients undergoing cochlear implantation have demonstrated early success with their devices. CONCLUSIONS: Hydrocodone is frequently prescribed in combination with acetaminophen for the relief of pain and has a side effects profile similar to other medications in its class. Although not described previously, overuse or abuse can be associated with a rapidly progressive sensorineural hearing loss. These patients can be successfully rehabilitated with cochlear implantation. PMID- 10733183 TI - Assessment of vestibular ototoxicity of ear drops by recording of vestibular evoked potentials to acceleration impulses. AB - INTRODUCTION: The cochlear ototoxicity of several ear drops is well documented in the literature, but very few studies exist on the vestibular ototoxicity of these topical drugs. GOAL OF STUDY: To develop an animal model for the assessment of the vestibular ototoxicity of ear drops. MATERIALS AND METHODS: Two animal groups, consisting of five fat sand rats (FSRs) each, underwent unilateral labyrinthectomy. Normal saline was topically applied into the middle ear cavity of rats in the first group for 7 days (control group). Rats in the second group were treated in the same way by topical gentamicin solution. Cochlear function was assessed by the recording of auditory evoked potential (ABPs) thresholds, and vestibular function was assessed by the recording of vestibular evoked potentials (VsEPs) to angular accelerations. RESULTS: In the control group, except for the amplitude of the first wave, there was no significant difference in the VsEPs recorded before and after topical application. In the gentamicin group, VsEPs could not be recorded after 7 days, and ABPs were recorded in one case only, with a threshold of 100 dB sound pressure level (SPL). CONCLUSION: VsEPs seem to be a reliable measure for evaluating the vestibular ototoxicity of topical ear drops. PMID- 10733184 TI - Human studies of a piezoelectric transducer and a microphone for a totally implantable electronic hearing device. AB - OBJECTIVE: For the surgical treatment of patients with moderate and severe sensorineural hearing loss, the authors have developed a totally implantable hearing device, the totally integrated cochlea amplifier (TICA). To evaluate the effectiveness of transducer and microphone of this device, three separate human studies were conducted. STUDY DESIGN: The first study using transducer prototypes involved self experiments in investigators with normal hearing. The second study used the transducer prototypes in patients with hearing loss, and the third study involved the temporary implantation of the final transducer prototype and microphone in patients undergoing otologic surgery. PATIENTS: In routine middle ear surgery, transducer prototypes were coupled to the ossicular chain of 28 patients. In addition to the transducer, in 5 patients the microphone was placed beneath the skin of the auditory canal, allowing the skin to cover the microphone membrane completely. RESULTS: The piezoelectric transducer reached an equivalent sound pressure level of 145 dB SPL < or =10 kHz. The dynamics for music reached 32 dB, which was identical with the results of the preoperative investigations using high-fidelity headsets (33 dB). The low nonlinear distortions of <0.1% and the frequency range of 10 kHz are reflected in the positive evaluation of the sound quality by 84% of the patients involved. When phonetically balanced speech material and music were presented under free field conditions at a sound level of 65 dB SPL, understanding of the phonetically balanced speech material was 100%. Most patients judged the presentations of music as clear and undistorted with all broadband components. CONCLUSIONS: Data in humans on the performance of the two main components of the TICA implant, the transducer and the microphone, are reported. PMID- 10733185 TI - Comparison of electrode position in the human cochlea using various perimodiolar electrode arrays. AB - OBJECTIVE: This study was conducted to evaluate the insertion properties and intracochlear trajectories of three perimodiolar electrode array designs and to compare these designs with the standard Cochlear/Melbourne array. BACKGROUND: Advantages to be expected of a perimodiolar electrode array include both a reduction in stimulus thresholds and an increase in dynamic range, resulting in a more localized stimulation pattern of the spiral ganglion cells, reduced power consumption, and, therefore, longer speech processor battery life. METHODS: The test arrays were implanted into human temporal bones. Image analysis was performed on a radiograph taken after the insertion. The cochleas were then histologically processed with the electrode array in situ, and the resulting sections were subsequently assessed for position of the electrode array as well as insertion-related intracochlear damage. RESULTS: All perimodiolar electrode arrays were inserted deeper and showed trajectories that were generally closer to the modiolus compared with the standard electrode array. However, although the precurved array designs did not show significant insertion trauma, the method of insertion needed improvement. After insertion of the straight electrode array with positioner, signs of severe insertion trauma in the majority of implanted cochleas were found. CONCLUSIONS: Although it was possible to position the electrode arrays close to the modiolus, none of the three perimodiolar designs investigated fulfilled satisfactorily all three criteria of being easy, safe, and atraumatic to implant. PMID- 10733186 TI - A silastic positioner for a modiolus-hugging position of intracochlear electrodes: electrophysiologic effects. AB - HYPOTHESIS: It was postulated that an electrode array that achieved a close modiolar proximity would result in reduced threshold levels and amplitude slopes, as measured with electrically evoked auditory brainstem responses (EABRs). BACKGROUND: Quality and quantity of auditory information transmitted by a cochlear implant to patients with sensorineural hearing loss depend on spatial and temporal resolution achieved by the electrical intracochlear stimulation. METHODS: To improve spatial resolution, a new electrode system was developed by Advanced Bionics Corp., with the intention of obtaining greater modiolar proximity. The implant version specified for animal experiments consists of a straight electrode array of seven embedded platinum discs and a so-called Silastic-positioner. The Silastic positioner is shaped to follow the dimensions of the scala tympani with a concave (triangular) inner side, which fits the form of the electrode array. The aim of the study was to evaluate the influence of a modiolus-hugging electrode position in contrast to a conventional electrode position on EABR in short-term animal experiments. Short-term electrophysiologic studies were performed on six adult cats. After local intracochlear application of neomycin solution (50 mg/mL), electrodes were inserted into the scala tympani. Electrically evoked auditory brainstem response threshold levels and EABR amplitude slopes were systematically investigated with and without the positioner. RESULTS: Electrically evoked auditory brainstem response measurements revealed a distinct apicobasal threshold shift, with increasing thresholds toward the basal end of the electrode. After insertion of the positioner, this shift diminished or was inverted and EABR thresholds and amplitude slopes were reduced significantly. CONCLUSIONS: Threshold and amplitude slope data emphasize the functional benefit of the positioner system, especially for the stimulation of electrodes in the more basal channels. PMID- 10733187 TI - Electrode to modiolus proximity: a fluoroscopic and histologic analysis. AB - HYPOTHESIS: This study was conducted to evaluate the electrode to modiolus proximity of two commonly used electrodes (Nucleus and Clarion) and a new prototype electrode. BACKGROUND: Theoretical advantages of modiolus-hugging cochlear implant electrodes include reduced stimulus energy requirements, more efficient power management and longer battery life, more discreet ganglion cell population stimulation, and reduced facial nerve stimulation. METHODS: Fluoroscopic analysis and histologic surface preparation analysis of electrode position and trauma. RESULTS: The Nucleus straight electrode occupied an outer wall position in the scala tympani. The Clarion electrode occupied a more intermediate position. The actively coiling prototype electrode was modiolus hugging but caused more trauma than the other two electrodes tested. CONCLUSIONS: The optimal modiolus-hugging atraumatic intracochlear electrode does not currently exist. PMID- 10733188 TI - The Clarion electrode positioner: temporal bone studies. AB - OBJECTIVE: To study the relationship of the Clarion electrode to the modiolus when using an intracochlear positioner. BACKGROUND: There are theoretical advantages to positioning a cochlear implant electrode in close proximity to the modiolus. This may allow more focused, discrete fields of electrical current, reducing both requirements to achieve threshold and the channel interactions associated with the simultaneous and nonsimultaneous stimulation of closely spaced electrodes. METHODS: Ten fresh temporal bones were used to assess the position of the electrode in the scala tympani with the positioner in place. The bones were X-rayed after implantation. The relationship of the electrode to the modiolus was studied by calculating a ratio between the curve assumed by the electrode in relationship to the outer wall of the cochlea. The depth of insertion was evaluated in degrees or number of turns around the modiolus. RESULTS: The electrode was brought closer to the modiolus and a greater depth of insertion was achieved in all cases with the positioner. CONCLUSION: The intracochlear positioner is capable of bringing the electrode consistently closer to the neural elements within the modiolus. PMID- 10733189 TI - Benign paroxysmal positional vertigo and canalith repositioning: clinical correlations. AB - OBJECTIVE: To correlate patient response and recurrence rate after canalith repositioning to clinical presentation and cause of BPPV. STUDY DESIGN: This was a retrospective chart review. SETTING: This study was performed at a tertiary referral center. PATIENTS: The study included patients with a diagnosis of benign paroxysmal positional vertigo based on history and the presence of a positive response to the Dix-Hallpike maneuver (affected ear down) who were treated at the University of Arkansas for Medical Sciences between January 1993 and February 1997 using canalith repositioning. There were 52 patients (13 men, 39 women) with an average age of 63 years. INTERVENTION: Canalith repositioning was performed without the use of vibration, with the maneuver repeated up to three times as necessary at the initial session until vertigo and nystagmus were abolished. MAIN OUTCOME MEASURE: Initial response, defined as complete response (elimination of nystagmus and symptoms), improved response (elimination of nystagmus but with some residual symptoms), or no response (continued nystagmus and symptoms), as well as recurrence rate. RESULTS: Initial complete response was seen in 66% of patients, and 33% showed improved response. This difference was believed to be caused by the pathophysiology. Recurrence rates appeared to be correlated to cause, with higher rates reflecting ongoing inner ear injury, such as with Meniere disease. CONCLUSIONS: Canalith repositioning was effective in 99% of patients based on elimination of nystagmus. Partial responders probably experienced resolution naturally over time because of a difference in pathophysiology. Higher recurrence rates can be expected when the cause is thought to involve an ongoing process. PMID- 10733190 TI - Neurotologic manifestations and treatment of multiple spontaneous tegmental defects. AB - OBJECTIVE: To describe the causes, histopathologic features, manifestations, and treatment of symptomatic multiple spontaneous tegmental defects. STUDY DESIGN: Retrospective review of three clinical cases and one temporal bone histopathology report. CLINICAL FEATURES: Varied, including spontaneous cerebrospinal fluid otorhinorrhea, conductive hearing loss, chronic headaches, pneumocephalus, extradural abscess, and meningitis. A notable common feature was multiple (8-15) tegmental defects, 1 to 6 millimeters in diameter. Three of the four cases also included associated dural defects and small meningoencephaloceles or arachnoid granulations. Imaging studies generally underestimated the number of defects. INTERVENTION AND OUTCOMES: Successful middle cranial fossa repair with temporalis fascia was accomplished in the three clinical cases. Extension of exposure anteriorly and medially was necessary. Closure of the defects with a bone graft or equivalent synthetic material was not always possible, given the anatomic and pathologic features. Our data suggest that there are both congenital and acquired causes of the tegmental dehiscences. CONCLUSIONS: Multiple tegmen defects constitute a special entity. Successful repair requires a middle fossa craniotomy with extended exposure. PMID- 10733191 TI - Otogenic cerebrospinal fluid rhinorrhea: a new technique for closure of cerebrospinal fluid leak. AB - OBJECTIVE: To describe a new technique for control of cerebrospinal fluid (CSF) rhinorrhea and to review alternative approaches. STUDY DESIGN: Five cases and literature review. SETTING: Tertiary referral center. PATIENTS: Five case study patients. INTERVENTION: Surgical. MAIN OUTCOME MEASURE: Control of CSF rhinorrhea. RESULTS: CSF rhinorrhea can be controlled through eustachian tube ligation in the nasopharynx, distal to all known pathways through which CSF leaks may occur from the temporal bone into the eustachian tube. CONCLUSION: Transoral eustachian tube ligation appears to be a safe and effective alternative to other techniques for controlling CSF rhinorrhea and should be included in the otologist's repertoire. PMID- 10733192 TI - Evaluation of three intraoperative auditory monitoring techniques in acoustic neuroma surgery. AB - OBJECTIVE: To assess the efficacy of three intraoperative auditory monitoring techniques: auditory brainstem response (ABR), electrocochleography (ECoG), and direct eight nerve monitoring (DENM). STUDY DESIGN: A retrospective review of the intraoperative recordings of ABR, ECoG, and DENM was performed. SETTING: A private, neurotologic practice. PATIENTS: Sixty-six patients with usable preoperative hearing underwent intraoperative auditory monitoring with ABR, ECoG, or DENM. INTERVENTIONS: Intraoperative auditory monitoring. MAIN OUTCOME MEASURES: Postoperative pure-tone hearing threshold and word recognition scores. RESULTS: Of the 66 patients, 16 (24%) had postoperative serviceable hearing. Five (18%) of the 28 with ABR monitoring, 3 (17%) of the 18 with ECoG monitoring, and 8 (40%) of the 20 with DENM monitoring had serviceable hearing after surgery. CONCLUSIONS: This study suggests that DENM may be more effective than ABR or ECoG, although the differences in hearing preservation rates are not statistically significant. PMID- 10733193 TI - Acoustic neuroma surgery: use of cochlear nerve action potential monitoring for hearing preservation. AB - OBJECTIVES: To compare the hearing preservation results obtained with use of two intraoperative eighth nerve monitoring methods, cochlear nerve action potential (CNAP) and auditory brainstem response (ABR), during complete acoustic neuroma (AN) resection. STUDY DESIGN: Retrospective. SETTING: Tertiary referral center. PATIENTS: Thirty-three consecutive patients who underwent hearing preservation AN surgery. INTERVENTIONS: Intraoperative monitoring by CNAP and/or ABR during AN resection. MAIN OUTCOME MEASURE: Postoperative hearing. Hearing preservation was considered achieved for pure-tone average < or =50 dB and speech discrimination > or =50%. RESULTS: Thirty-two patients met inclusion criteria for the study. Monitoring was successfully performed in 23 of 25 patients (92%) who underwent attempted CNAP monitoring and 13 of 27 (48%) who underwent attempted ABR monitoring. When tumor size was < or =20 mm in greatest dimension, hearing preservation was achieved in 12 of 18 patients (67%) monitored with CNAP, versus 2 of 8 patients (25%) not monitored with CNAP (p = 0.05). Monitoring by ABR did not improve hearing preservation rates compared with those not monitored with ABR (40% vs. 63%). At the completion of surgery, the presence or absence of CNAP predicted the presence or absence of hearing preservation in 18 of 23 cases (p = 0.01), while ABR successfully predicted hearing results in 10 of 13 cases (p = 0.05). CONCLUSIONS: When CNAP and ABR monitoring techniques during AN surgery were compared, CNAP was more frequently obtainable. Monitoring by CNAP was significantly associated with a higher chance of hearing preservation. Monitoring by ABR did not have a positive influence on hearing preservation results. Both ABR and CNAP were useful for predicting postoperative hearing. PMID- 10733194 TI - Neurotologic follow-up after radiation of posterior fossa tumors. AB - OBJECTIVE: Stereotactic radiation treatment, also known as gamma knife surgery or radiosurgery, has come into acceptance as a treatment alternative to surgical removal for posterior fossa tumors. The purpose of this article is to describe the role of the neurotologist in the optimal management of neurotologic complications after stereotactic radiation, as illustrated by five patients. STUDY DESIGN: Retrospective chart review. PATIENTS: Five patients who underwent stereotactic radiation of posterior fossa tumors. MAIN OUTCOME MEASURES: Presence or absence of neurotologic complications (tumor growth, hearing loss, imbalance/ataxia, vertigo, and facial paralysis) or neurosurgical complaints (facial numbness, motor weakness, headache, hydrocephalus, and subarachnoid cysts). RESULTS: Postradiation neurotologic complaints included vertigo, imbalance/ataxia, and progressive hearing loss in four of the five patients. Continued tumor growth occurred in two patients; two patients had no growth; in one patient the tumor became smaller. The complications of facial nerve paralysis, facial numbness, motor weakness, headache, hydrocephalus, cerebellar edema, and posterior fossa arachnoid cyst formation occurred less frequently. CONCLUSIONS: Stereotactic radiation of posterior fossa tumors can produce significant neurotologic problems. It is imperative that neurotologists remain involved in the follow-up care of patients with posterior fossa tumors to offer optimal treatment alternatives for the neurotologic disorders. PMID- 10733195 TI - Prevention of cerebrospinal fluid rhinorrhea in neurotologic surgery. AB - OBJECTIVE: To determine the efficacy and safety of quick-setting hydroxyapatite cement in eliminating cerebrospinal fluid (CSF) rhinorrhea following neurotologic surgery. STUDY DESIGN: A prospective study of 40 consecutive patients undergoing neurotologic surgery in whom the dura was opened. SETTING: All patients were treated as hospital inpatients at a tertiary referral center. PATIENTS: 25 men and 15 women between the ages of 20 and 72 years (mean age 51 years) underwent neurotologic surgery at the parent institution. INTERVENTION: Various neurotologic procedures were performed for the resection of 25 acoustic tumors, 5 meningiomas, 3 glomus tumors, 2 vestibular nerve sections, 2 chordomas, 1 epidermoid tumor, and 1 meningoencephelocele, and for 2 patients referred to our institution with known CSF leaks following acoustic tumor surgery. A new form of quick-setting hydroxyapatite cement, which that hardens within 3 to 5 minutes was used to seal the air cell tracts of the temporal bone in all cases. MAIN OUTCOME MEASURE: The presence of CSF rhinorrhea postoperatively. RESULTS: CSF rhinorrhea occurred in 2 patients following acoustic tumor surgery, the first through an occult air cell tract at the margin of the drilled internal auditory canal, and the second via an oval window fistula 1 month after a translabyrinthine approach. CONCLUSIONS: This form of hydroxyapatite cement appears safe, reliable, effective, and economical for the prevention of CSF rhinorrhea following neurotologic surgery. CSF rhinorrhea cannot be eliminated unless our ability to identify all potential air cell tract communications improves. PMID- 10733196 TI - Conservative facial nerve management in jugular foramen schwannomas. AB - OBJECTIVE: Although transposition of the facial nerve is crucial in infiltrative vascular lesions involving the jugular foramen, the objective was to show that a conservative approach to management of the facial nerve is sufficient with jugular foramen neuromas because of their noninfiltrative, less vascular nature and medial location in the jugular foramen. STUDY DESIGN: Retrospective case review. SETTING: Tertiary, private, multiphysician, otologic practice. PATIENTS: Sixteen patients with jugular foramen schwannoma (18 procedures) treated between January 1975 and October 1995. The 8 male and 8 female patients ranged in age from 13 to 66 years (mean age 47.7 years). INTERVENTION: One-stage, total jugular foramen neuroma removal without transposition of the facial nerve, using a variety of surgical approaches. MAIN OUTCOME MEASURES: Facial nerve transposition (yes or no), House-Brackmann facial nerve grade, lower cranial nerve status, complications. RESULTS: One-stage total tumor removal was accomplished in all the cases. In 13 (72%) of the neuromas, removal was accomplished without facial nerve transposition. Transposition was performed in 2 revision cases in which scar tissue from a previous operation prevented complete control of the carotid artery and safe removal, 2 cases with large tumor extension anteriorly to the petrous apex, and 1 case with extensive involvement of the middle ear. A House-Brackmann facial nerve Grade I or II was obtained in 16 of the 18 procedures, with 1 Grade III and 1 case that remained Grade V, as it was preoperatively. CONCLUSIONS: One stage, total tumor removal can be achieved with excellent control of the important vascular structures and without transposition of the facial nerve in a majority of jugular foramen schwannomas. PMID- 10733198 TI - Incudomalleolar joint separation. PMID- 10733197 TI - Oculoplastic surgical techniques for protection of the eye in facial nerve paralysis. AB - OBJECTIVE: To describe an algorithm for medical and surgical management of corneal exposure secondary to seventh cranial nerve paresis. STUDY DESIGN: A retrospective review of 54 patients requiring surgical intervention for seventh cranial nerve paresis was performed. SETTING: Patients underwent outpatient procedures at a tertiary care facility. RESULTS: 31 men and 23 women, with a mean age of 55.7 years, were included in this study. They included 45 patients with involvement of the facial nerve at the time of surgery, 3 with inflammatory processes, 2 with central palsies, 1 with a traumatic paralysis, and 3 with idiopathic palsies. All 54 patients were treated both medically and surgically. Fifty-two patients underwent gold weight placement, and 39 patients underwent surgical repositioning of their lower eyelids; 24 of those patients had supplemental ear cartilage grafts inserted as well. Five patients had brow ptosis repairs, and 13 had tarsorrhaphies (9 temporary and 4 permanent). Twelve patients had confluent epithelial defects of >50% of their corneal surface, and 33 patients had smaller disruptions of their corneal epithelium. Three patients had minor complications secondary to surgical intervention. Thirty-seven patients had isolated seventh nerve palsies, and 17 patients also had fifth nerve pareses affecting their ocular surfaces. The average preoperative lagophthalmos was 6.3 mm, and the average postoperative lagophthalmos after all ocular procedures was 1.6 mm. The mean follow-up time was 19.7 months. CONCLUSIONS: Treatment options for seventh cranial nerve deficits are reviewed. A decision-making process for the treatment steps is proposed and analyzed. PMID- 10733200 TI - Endoscopy of the middle ear. PMID- 10733199 TI - Bone dust in the middle ear. PMID- 10733201 TI - All in a name: schwannomin versus merlin. PMID- 10733202 TI - Gadolinium-enhanced MRI for idiopathic sudden sensorineural hearing loss. PMID- 10733203 TI - Detection of simian immunodeficiency virus (SIV)-specific T-cell-mediated cytotoxicity in the peripheral blood from infected cynomolgus monkeys. AB - We have previously demonstrated that peptide immunization restimulates the memory CD4 T-cell response, but fails to induce cytotoxic T lymphocyte (CTL) in cynomolgus macaques. To examine the nature of protective immunity to simian immunodeficiency virus (SIV) in this study, freshly isolated peripheral blood mononuclear cells (PBMC) from four infected juvenile cynomolgus macaques and from three uninfected control macaques were assessed for CTL activity monthly for 9 consecutive months, beginning 1 month after detection of infection. Target cells consisted of major histocompatibility (MHC) haploidentical parental PBMC which were stimulated with mitogen and then pulsed with heat-killed SIVcyn. CTL activity was demonstrated in PBMCs from all four infected animals. The effector cells are T cells which mediate cytotoxicity against SIVcyn-pulsed target cells in an MHC-restricted manner. Furthermore, the cytotoxicity is virus specific and predominantly, if not exclusively, mediated by CD8+ T cells; it is also MHC class I restricted. Incubation of target cells with pepstatin A during antigen pulsing prior to the cytotoxic assay inhibited target cell generation, suggesting that viral antigens are processed via an endocytic pathway. PMID- 10733204 TI - Morphologic and immunophenotypic characteristics of malignant lymphomas in SIV infected rhesus macaques. AB - Eight cases of extranodal non-Hodgkin's lymphoma in simian immunodeficiency virus (SIV)-infected rhesus macaques, aged 4-9 years, were phenotypically and immunologically characterized, using the updated Kiel classification, in order to determine both the differences and the similarities between these types of lymphoma in immunodeficient rhesus macaques (Macaca mulatta) and man. The high grade malignant tumors were of B-cell origin, with a predilection for extranodal growth in viscera and periorbital tissues. Immunophenotypical characterization showed that the monkey lymphomas were similar in many aspects to human immunodeficiency virus-associated lymphomas. The number of Ki67 positive cells varied from case to case and ranged from 50 to 90%. A serological screening for the simian equivalent of the Epstein-Barr virus (sEBV) by immunofluorescence assay revealed a prevalence of 92% of the sEBV antibodies in our cohort. The presence of Ebstein-Barr virus nuclear antigen (EBNA-2) could be demonstrated by immunohistochemistry in four out of eight cases. In situ hybridization revealed the presence of small EBV-encoded RNAs (EBER-1, EBER-2) in six of the eight cases. Further studies should define the precise role of herpesvirus infection for lymphomagenesis in SIV-induced immunodeficiency. PMID- 10733205 TI - Measurement of serum bone-specific alkaline phosphatase activity in cynomolgus macaques. AB - Serum levels of bone-specific alkaline phosphatase activity (B-ALP) in cynomolgus monkeys were evaluated as an index of elevated bone turnover following ovariectomy. The enzyme immunoassay 96-well microtiter plate B-ALP assay, developed by Metra Biosystems (Mountain View, CA) for human use, was employed and compared with a standard automated assay measuring total serum levels of alkaline phosphatase activity (T-ALP). The B-ALP assay was first validated for use in these monkeys. Ovariectomy led to increased bone turnover as indicated by approximately 2-fold higher activity in both assays and this elevation was inhibited by daily estradiol administration. Although both assays provided generally similar results, several monkeys were observed to have greatly elevated values of T-ALP but not B-ALP. This discrepancy is believed to result from high levels of the liver isoform of alkaline phosphatase in monkeys with hepatic dysfunction, which are not detected by the B-ALP assay. PMID- 10733206 TI - The effect of dietary protein on the mineral status of vervet monkeys with special reference to the impact of milk solids on calcium excretion. AB - This study assessed the impact of Westernised and traditional African diets on mineral metabolism in general and calcium status in particular in vervet monkeys. Twelve adult male vervet monkeys (Cercopithecus aethiops), with an average weight of 5+/-0.58 kg each, were divided into two groups of six individuals each and fed traditional diets containing largely maize + legumes (17.4%) or Westernised diets containing milk solids (17.2%) as the source of high crude protein for 8 weeks. Blood was taken at 2-week intervals, the animals were weighed, while urine and stool samples were collected over 24 hours. The monkeys on the milk solids diet had diarrhoea for 6 weeks post-dietary intervention, and produced significantly greater quantities (P<0.02) of stool. These animals also produced significantly more urine (P<0.02). There was no difference in the degree of calciuresis of the two groups, but the monkeys on maize + legume proteins absorbed significantly more calcium during weeks two and six (P<0.04). Furthermore, both groups of monkeys showed a significant decline in plasma calcium levels over the experimental period (P<0.001). The diets had no effect on phosphate levels in the plasma or urine. However, both groups of animals absorbed less phosphate (P<0.09). There was an increasing loss of urinary magnesium (P = 0.03) in both groups, with the milk solids group showing lower plasma levels of this element (P = 0.09). However, the milk solids group lost less magnesium through the stool (P<0.03). In addition, the animals on milk solids showed significant natriuresis (P<0.05), while plasma sodium levels in both groups declined over time (P<0.03). Both diets induced a state of urinary potassium loss (P = 0.0003) and decrease in plasma potassium (P<0.0002). Urinary pH and plasma urea were unaffected by the diets, but the monkeys on maize + legumes excreted significantly less (P<0.001) urinary urea. This study indicates that the milk solids diet compromised mineral homeostasis by interfering with gut and renal functioning, while the traditional African diet did not induce these effects. PMID- 10733207 TI - Clinical outcome of a protocol to produce immunosuppression in rhesus monkeys (Macaca mulatta): application to infectious disease and gene therapy studies. AB - Induced immunosuppression is required for a number of studies using rhesus monkeys (Macaca mulatta). This report describes the clinical outcome and safety of a dose-finding experiment that determined doses of cyclophosphamide and prednisone that could be used to induce a state of immunosuppression in rhesus monkeys. After determining the optimum dose of immunosuppressive agents, the protocol was then used on animals participating in infectious disease and gene therapy studies. Splenectomy was performed in some animals to increase the severity of immunosuppression. The onset, duration, and severity of lymphopenia and leukopenia were consistent in all animals. In most animals, physical examination findings and clinical serum chemistry profiles demonstrated only transient abnormalities. With proper clinical monitoring, combination treatment with cyclophosphamide and prednisone is an effective and safe method for inducing immunosuppression in rhesus monkeys. PMID- 10733208 TI - Strategies to improve the sensitivity in capillary electrophoresis for the analysis of drugs in biological fluids. AB - Capillary electrophoresis (CE) is a useful method to quantify drugs in biological fluids. However, especially for blood or plasma samples, the sensitivity is not sufficient to quantify drugs and their metabolites as they often need to be quantified in the lower microg/L range. To overcome this limitation and to increase the sensitivity, two strategies are applied: first, to increase the amount of analyte added to the capillary and, second, to increase the sensitivity on the detector site. To improve the sensitivity on the detector site, alternative detection techniques to UV detection, e.g., laser-induced fluorescence detection (LIF) or mass spectroscopy (MS), can be applied. However, LIF detection can only be used for fluorescent analytes and the current equipment for CE-MS coupling provides only small improvements in sensitivity compared to UV detection. The detection window for UV detection can be enhanced using capillaries with an extended light path (bubble cell) or Z-shaped capillaries. Sensitivity improvements up to a factor of 10 have been reported. Increasing the amount of analyte in the capillary can be done either by chromatographic or by electrokinetic methods. Chromatographic methods such as on-capillary membrane preconcentration have been used for several analytes. However, no validated application has been reported to date. In contrast, several validated examples can be found in which electrokinetic techniques like sample stacking have been applied to achieve limits of quantification in the lower microg/L range. In conclusion, to date, electrokinetic techniques such as field-amplified sample injection offer the most promising results in achieving a sufficient sensitivity to quantify drugs in biological fluids. PMID- 10733209 TI - Determination of taurine in plasma by capillary zone electrophoresis following derivatisation with fluorescamine. AB - A novel capillary zone electrophoresis method is described for the determination of taurine in plasma. The method is rapidly executed and is highly selective for taurine as separation is based on the difference in ionisation of this amino acid from that of other amino acids. Following addition of homotaurine as internal standard, plasma proteins were precipitated with acetonitrile and the supernatant was derivatised with fluorescamine in the presence of a borate buffer. Capillary electrophoresis (CE) separations were carried out in reverse polarity mode at 27.5 kV on a Beckman P/ACE MDQ CE instrument, equipped with a diode array detector (DAD) set at 266 nm. The sample tray was cooled to 5 degrees C and separations were carried out at 20 degrees C. The fused-silica capillary was 50.2 cm in length (40.2 cm to detector) with an internal diameter of 75 microm. A capillary conditioning solution was applied daily in order to suppress the residual electroosmotic flow (EOF). The method, which was validated using feline plasma as the blank matrix, was shown to be linear and reproducible over the concentration range 2.5-100 microg/mL. The coefficients of variation (CVs) of replicate analyses were less than 4.5% at 1 microg/mL taurine in feline plasma and less than 3% for 2.5 microg/mL in human plasma. Recovery was estimated at 99.2% with a CV of 4.85%. It has been demonstrated that quantitation in aqueous solution yields similar results to those obtained by interpolation on a plasma calibration curve provided that subtraction for the taurine peak in unspiked plasma is carried out and that a suitable internal standard is employed. PMID- 10733210 TI - Measurement of bilirubin partition coefficients in bile salt micelle/aqueous buffer solutions by micellar electrokinetic chromatography. AB - The partition coefficients for the distribution of bilirubin between aqueous phosphateborate buffer and cholic, taurocholic, taurodeoxycholic, and taurochenodeoxycholic micelles have been measured by micellar electrokinetic chromatography at pH 8.5. Determination of the partition coefficients required that the critical micelle concentration and partial specific volumes be determined for each bile salt. Critical micelle concentrations were slightly higher for the trihydroxy bile salts. Partial specific volumes of the bile salt micelles differed very little from each other, and for each bile salt they were constant over the concentration range studied, which was typically from slightly above the critical micelle concentration to 35 mM. Capacity factors were corrected for the effects of applied voltage by extrapolation of the capacity factor to zero applied volts. The free solution mobility of bilirubin, determined in the absence of bile salt, was also corrected for the effects of applied voltage. Plots of extrapolated capacity factor versus phase ratio yield the partition coefficient as the slope of a linear fit to the data. Partition coefficients for bilirubin were significantly higher for dihydroxy bile salts than for trihydroxy bile salts. PMID- 10733211 TI - Micellar electrokinetic chromatography separations of dynorphin peptide analogs. AB - Prodynorphin is a precursor that has multiple cleavage sites to release various dynorphin opioid peptides. The dynorphin analogs used in this study have 18 amino acid residues. A series of dynorphin-like peptides, differing by a single residue (alanine substitution) were assembled by Fmoc solid-phase procedures and purified by preparative high performance liquid chromatography (HPLC). Separation of the Ala-scan dynorphin analogs was investigated by micellar electrokinetic chromatography (MEKC) employing anionic, cationic and zwitterionic surfactants. The role of electrostatic and hydrophobic forces in analyte-surfactant interactions is discussed with respect to the observed elution patterns. Separation of all dynorphin analogs by MEKC using a zwitterionic surfactant shows this technique to be powerful for separating closely related peptide species. It also demonstrates the potential for using MEKC for the prescreening of peptide libraries to determine their biological activity toward specific receptors. Results from the separation of dynorphin analogs by free solution and ion-pairing capillary electrophoresis are also presented. PMID- 10733212 TI - Separation of D-lysergic acid diethylamide derivatives using micellar electrokinetic capillary chromatography. AB - By adjusting column temperature and applied electric field, a fast separation in micellar electrokinetic capillary chromatography was developed for the separation of D-lysergic acid diethylamide derivatives. A baseline separation of nine derivatives was accomplished with a run time of less than 12 min by utilizing elevated column temperature (60 degrees C) and an applied electric field of 387 V/cm. The number of plates generated per unit time for the separations completed at elevated temperatures was significantly higher when compared to separations at the same applied electric field but at lower temperatures (20 degrees C). PMID- 10733213 TI - Therapeutic drug monitoring of albendazole: determination of albendazole, albendazole sulfoxide, and albendazole sulfone in human plasma using nonaqueous capillary electrophoresis. AB - A nonaqueous capillary electrophoretic method (NACE) for the fast determination of plasma levels of albendazole (ABZ), albendazole sulfoxide (ABZSO), and albendazole sulfone (ABZSO2) is described. The assay is based upon liquid/liquid extraction of these compounds using dichloromethane at pH 10.2 (recovery between 63 and 98%), followed by a NACE separation performed within 8 min employing a 0.036 M borate buffer (apparent pH 9.9) in a mixture of methanol and N methylformamide (1:3) and on-column absorbance detection at 280 nm. Using 0.5 mL of plasma and extract reconstitution in 200 microL N-methylformamide, drug levels between 1.0-10 microM were found to provide linear calibration graphs. Intraday and interday imprecisions evaluated from peak area ratios (n = 5) were <10% and <12%, respectively. Corresponding imprecisions of detection times (n = 5) were <1% and <6%, respectively. The limit of detection (LOD) for ABZ, ABZSO and ABZSO2 was 8 x 10(-7) M. The reliability of the method developed was verified via analysis of 45 plasma samples obtained from patients treated with ABZ. Good agreement was obtained between the levels of ABZSO and those determined by routine HPLC. ABZ was found to be undetectable in all patient samples, whereas the levels of ABZSO2 were below or close to LOD. PMID- 10733214 TI - Separation of related opiate compounds using capillary electrochromatography. AB - Capillary electrophoretic separations have been investigated for six controlled narcotic analgesic compounds having related structures. Owing to the similar charge-to-mass ratios of these compounds, capillary zone electrophoresis failed to provide a satisfactory separation, whereas a baseline-resolved separation was achieved in 10 min using micellar electrokinetic chromatography. Column efficiencies of 40,000-150,000 plates/m were obtained with a 50 cm long, 50 microm inner diameter (ID) capillary using 50 mM sodium dodecyl sulfate (SDS) in a 50 mM borate solution containing 12% isopropanol. In contrast, separation of this mixture by capillary electrochromatography proved to be significantly superior. The capillary was 15 cm long, with an ID of 75 microm, and was packed with 1.5 microm nonporous octadecyl silica (ODS) particles. The mobile phase consisted of 80% 10 mM tris(hydroxymethyl)aminomethane (Tris) and 20% acetonitrile, and contained 5 mM SDS. A complete separation was obtained in 2.5 min with an efficiency of 250,000-500,000 plates/m. PMID- 10733215 TI - Separation of hemoglobin variants with similar charge by capillary isoelectric focusing: value of isoelectric point for identification of common and uncommon hemoglobin variants. AB - Clinical assays for the primary evaluation of congenital hemoglobin (Hb) disorders must detect and identify a variety of Hb variants. We analyzed hemolysates containing Hb variants with similar charge to evaluate the diagnostic sensitivity and specificity of automated capillary isoelectric focusing (CIEF). Peak separation was observed for each variant in samples containing Hb S, D, and G. The calculated isoelectric points (pI) of these variants were significantly different such that each could be identified in a single run with pI as the sole criterion of identification. The pI of Hb C was significantly different from that of Hb E, C-Harlem, and O-Arab. Hb E, C-Harlem, and O-Arab had similar pI and were not readily differentiated. Hb Koln, M-Saskatoon, Aida, and S/Aida hybrid were readily separated from common Hb variants and detected by CIEF. We conclude that CIEF exhibits both diagnostic sensitivity and specificity, and that pI is an objective and specific criterion of Hb variant identification. PMID- 10733216 TI - Hemoglobin A2 quantification by capillary zone electrophoresis. AB - Hemoglobin A2 (HbA2) comprises about 2.2% of the total hemoglobin in the erythrocytes. The separation and quantitation of this minor hemoglobin by capillary electrophoresis (CE) using an arginine Tris buffer is described. Some of the variables affecting the accuracy and precision of HbA2 quantification are investigated. Furthermore, the quantification of this hemoglobin by CE is compared to that of a microcolumn chromatography method. The CE method is better suited than the microcolumn method for measuring HbA2 in the sickle cell trait. PMID- 10733217 TI - Changes of alpha1-acid glycoprotein microheterogeneity in acute inflammation stages analyzed by isoelectric focusing using serum obtained postoperatively. AB - The relationship between variations of alpha1-acid glycoprotein (orosomucoid, AGP) microheterogeneity detected from isoelectric focusing (IEF) patterns and clinical stage of acute inflammation based on serum C-reactive protein (CRP) levels and interleukin-6 (IL-6) levels was investigated. Serum samples were obtained from healthy subjects, and from patients with esophageal or stomach carcinoma before and after operation. Samples without neuraminidase treatment were used for AGP microheterogeneity analysis, and samples with neuraminidase treatment for AGP heterogeneity analysis. In AGP microheterogeneity, nine bands were detected in the range of pI 3.18-3.57 in sera obtained from healthy subjects. In patients, AGP microheterogeneity changed the first day after operation; the percentage of bands surrounding pI 3.5 increased, and the highest value appeared in sera taken the first or second day after operation and then decreased quickly. These bands showed reactivity for concanavalin A (Con A). The increase in Con A-reactive AGP occurred later than the increase in IL-6, and occurred earlier than the increase in CRP. On the seventh day after operation, the percentage of bands around pI 3.2 increased. These bands showed the reactivity for Datura stramonium agglutinin. On the other hand, in samples with neuraminidase treatment, little change of AGP heterogeneity was observed in most samples, which did not reflect the stage of inflammation. These findings suggested that AGP microheterogeneity detection was a useful marker for the clinical stage of inflammation. PMID- 10733218 TI - Gel electrophoretic distinction between Congo Red nonreactive beta-amyloid (1-42) and beta-amyloid (1-40). AB - Congo-Red nonreactive beta-amyloid (1-42) exhibits in gel electrophoresis (pH 8.82, 0.01 M ionic strength, 2 degree C) a surface charge density larger than that of the corresponding peptide of length (1-40), and a size indistinguishable from that of (1-40). PMID- 10733219 TI - Preparative-scale isoelectric focusing separation of enantiomers using a multicompartment electrolyzer with isoelectric membranes. AB - The IsoPrime multicompartment electrolyzer, equipped with a series of isoelectric membranes with closely spaced pI values, was used for the first time for the preparative-scale separation of the enantiomers of dansyl phenylalanine with hydroxypropyl beta-cyclodextrin as resolving agent. The final separation conditions could be established easily in three successive experiments by rationally narrowing the pH steps between the neighboring isoelectric membranes. The final separation yielded products with an enantiomeric excess greater than 99.9%, at production rates of about 0.1 mg/h. The greatest experimental difficulty was caused by the relatively high salt content of the hydroxypropyl beta-cyclodextrin used, which resulted in high conductivity and limited the maximum field strength one could use. PMID- 10733220 TI - Single-cell analysis using capillary electrophoresis: influence of surface support properties on cell injection into the capillary. AB - Capillary electrophoresis (CE) is an important tool of chemical cytometry. Whole cell analysis using CE starts with cell injection into the capillary by either siphoning or electroosmosis. However, strong adherence of the cell to the support surface can prevent efficient cell injection and lead to irreproducible analysis. Here we evaluated several surfaces as potential cell supports for HT29 cells (human colon adenocarcinoma). These cells strongly adhered to the surface of untreated glass or polystyrene. Hydrophobic coating with dimethyldichlorosilane (DMS) or Sigmacote did not significantly reduce cell adhesion. In contrast, cell adhesion was reduced significantly when the surface was modified with hydrophilic polymers (hydrogels) such as poly(2-hydrohyethyl methacrylate) (PHEMA) and polyvinyl alcohol (PVA). In addition to their pronounced antiadhesive properties, PHEMA and PVA coatings were the most biocompatible (had highest survival of cells in contact with surface). Hydrogel-coated polystyrene plates were tested as a commercial alternative to hydrogel-coated glass slides. The cell adhesive properties of such plates were similar to those of PHEMA and PVA. However, the biocompatibility of the plates was lower than that of the other surfaces tested. Moreover, in contrast to PHEMA- and PVA-coated glass slides, the plates were sensitive to UV light and therefore should not be used when fluorescent image microscopy with UV excitation precedes CE. The analyses of the data obtained showed that PHEMA- and PVA-coated glass slides were the most suitable cell supports for cell injection into the capillary. PMID- 10733221 TI - Quantitative determination of glutathione in single human erythrocytes by capillary zone electrophoresis with electrochemical detection. AB - Glutathione (GSH) in single human erythrocytes is determined by capillary zone electrophoresis with end-column amperometric detection at a gold/mercury amalgam microelectrode. A capillary of 10 microm inner diameter is suitable for determination of GSH in an individual erythrocyte with a good signal-to-noise ratio. The limit of detection is 1 x 10(-7) mol/L or 26 amol and the linear dynamic range is 2 x 10(-7) to 2 X 10(-5) mol/L for the capillary. In this method, the calibration line is obtained with a capillary adsorbed before a certain amount of hemoglobin can be used for the quantification of GSH in the external standardization. The whole cell injection and the lack of necessity of a derivatization reaction lead to more accurate and precise results, which are closer to the macroscopic values of glutathione in human red blood cell (i.e., hemolysate) than those determined by indirect laser-induced fluorescence detection. PMID- 10733222 TI - Screening for the beta-39 mutation in thalassemia by capillary electrophoresis in free solution in strongly acidic, isoelectric buffers. AB - A novel method is reported for screening for point mutations in genomic DNA: free zone capillary electrophoresis in very acidic buffers. This method exploits the charge difference among the four different bases (C, T, A, G) in a pH window between 2.5 and 3.5, where the four titration curves fan out. The method is applied to the detection of the beta-39 missense mutation in the beta-globin gene in thalassemias. A 60-mer fragment straddling the mutation site has been amplified. In an isoelectric buffer (iminodiacetic acid) of pH 3.3, partial resolution between the wild type and mutated strands is obtained. In a pH 3.0 phosphate buffer, baseline resolution is achieved between the two strands in a heterozygous individual. Due to the short size of the amplified fragment, this method can only be applied to routine screening for known mutations because resolution was lost in a fragment 100 bases long. PMID- 10733223 TI - Single-strand conformation polymorphism analysis by capillary zone electrophoresis in neutral pH buffer. AB - Sensitivity of single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products was reported to be lower in capillary zone electrophoresis (CZE) compared to conventional slab gel electrophoresis. We examined the effects of buffer ion type, pH, and temperature in an attempt to improve the mutation detectability in the SSCP-CZE mode. It was noted that, by utilizing short-chain polyacrylamide as sieving media while simultaneously lowering the temperature, there was no improvement of conformer detectability. On the contrary, there was a large increment in conformers' resolution by running samples in a lower-pH buffer system. The effects of different buffering ions and pH values were investigated. By using a new buffer system, consisting of 35 mM 2 (N-morpholino)propanesulfonic acid (MES), 30 mM tris(hydroxymethyl)aminomethane (Tris), 1 mM ethylene diaminetetraacetic acid (EDTA), pH 6.8, and keeping constant all the other conditions, such as temperature, sieving, applied voltage, capillary length, and inner diameter (ID), there was a remarkable improvement in resolution and the sensitivity became comparable to that of slab gel systems. PMID- 10733224 TI - Quantitative analysis of leptin mRNA using competitive reverse transcription polymerase chain reaction and capillary electrophoresis with laser-induced fluorescence detection. AB - Leptin, the protein hormone product of the obese (ob) gene, functions in the regulation of appetite, energy expenditure, and reproduction in animals and humans. Since changes in the level of circulating leptin can have marked physiological consequences, it is important to be able to accurately quantify leptin gene expression. Toward this goal, we have constructed a chicken leptin RNA competitor and successfully employed it as an internal standard in the development of a quantitative-competitive reverse transcription polymerase chain reaction (QC-RT-PCR) assay for leptin mRNA. Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) was utilized for the separation and analysis of chicken leptin target (261 bp) and competitor (234 bp) dsDNA products from QC-RT-PCR assay samples. Leptin amplicons were separated using a DB-1 coated capillary (27 cm x 100 microm ID) at a field strength of 300 V/cm in a replaceable sieving matrix consisting of 0.5% hydroxypropylmethyl cellulose (HPMC) in 1 x TBE (89 mM Tris-base, 89 mM boric acid, 2 mM EDTA, pH 8.3) buffer with 0.5 microg/mL EnhanCE fluorescent intercalating dye. Samples were diluted 1:100 with deionized water and introduced into the capillary by electrokinetic injection. QC-RT-PCR/CE-LIF was used to quantify leptin mRNA in liver and adipose tissue from 8-week-old male and female broiler chickens. This study is the first report of quantitative analysis of leptin gene expression using QC-RT-PCR/CE-LIF. PMID- 10733226 TI - Alternative base-calling algorithm for DNA sequencing based on four-label multicolor detection. AB - A simple base-calling scheme based on four-label multicolor detection is suggested for DNA sequencing. The entire spectra of the dye labels were used for identification. Specifically, the maxima of the emission spectra rather than the intensity ratios at selected wavelengths are used to provide excellent discrimination. Capillary gel electrophoresis was used for the separation of DNA fragments. Data acquisition and analysis compatible with fast and high-throughput imaging detection was accomplished. The accuracy of base calling of PGEM/U DNA from the raw data obtained with 5 nm and 7 nm spectroscopic resolution were 98.4% for 386 bases and 98.4% for 385 bases. Base calling of M13mp18 DNA showed 98.3% accuracy for 420 bases. PMID- 10733225 TI - On-line identification of depurinating DNA adducts in human urine by capillary electrophoresis--fluorescence line narrowing spectroscopy. AB - The benzo[a]pyrene (BP)-derived 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) depurinating one-electron oxidation adduct was identified in the urine extracts of coal-smoke-exposed humans for the first time. Urine samples were prepared by solid-phase extraction and reversed-phase high-performance liquid chromatography. Subsequently, the BP-6-N7Gua adduct was identified on-line with capillary electrophoresis-- fluorescence line narrowing spectroscopy (CE-FLNS) at 4.2 K. The daily excretion of BP-6-N7Gua in human urine of individuals exposed to coal smoke was approximately 226 pmol per micromol of creatinine. Due to the high level of excretion we propose that BP-6-N7Gua adducts found in urine could serve as effective biomarkers for risk assessment of BP exposure. The results demonstrate that CE-FLNS allows for on-line separation and DNA adducts identification in complex fluid extracts. PMID- 10733229 TI - Genetic modification of the human mitotic clock by telomerase: a matter of life and death PMID- 10733227 TI - Rapid genotyping of factor V Leiden mutation using single-tube bidirectional allele-specific amplification and automated ultrathin-layer agarose gel electrophoresis. AB - We report a novel, high-throughput genotyping method by single nucleotide polymorphism (SNP) analysis using bidirectional allele-specific amplification with polymerase chain reaction (PCR) in a single-step/single-tube format. Blood coagulation factor V G1691A (also referred to as Leiden) mutation was chosen as a model system for SNP detection, as this is one of the most common inherited risk factors of thrombosis, effecting 2-5% of the human population. The rationale of our method is the production of allele-specific PCR fragments, different in size, which was achieved by bidirectional amplification, starting from the position of the mutation. Thus, both homozygosity and heterozygosity were readily identified from a single reaction by simply determining the sizes of the resulting PCR products. The advantage of our assay, compared to other single-tube systems, is that this method did not require the use of pre-PCR labeled (fluorophore) primers or probes. Preferential production of the allele-specific products was achieved by a hot-start, time release PCR system. Specificity was increased by introducing a mismatch in the 3'-antepenultimate position of the allele-specific primers. This method made possible the large-scale screening for the factor V Leiden mutation using single-tube PCR followed by automated ultrathin-layer agarose gel electrophoresis, with real-time detection of the "in migratio" ethidium-bromide labeled fragments. PMID- 10733228 TI - Caspases and neurodegeneration: on the cutting edge of new therapeutic approaches. AB - Unregulated apoptosis underlies many pathological conditions, including neurodegenerative diseases. In this review, we focus on the role of cysteine aspartate-specific proteases (caspase) activity in Huntington disease (HD) and Alzheimer disease (AD) as two representative neurodegenerative disorders that normally manifest in mid- to late-life. Caspases appear to be involved in the molecular pathology of HD by directly cleaving huntingtin and generating toxic protein fragments containing the polyglutamine tract, and by being recruited and activated by polyglutamine-containing aggregates composed mainly of truncated huntingtin fragments. Several proteins involved in AD, including beta-amyloid precursor protein (APP) and presenilins (PSs), are also cleaved by caspases. For APP, caspase cleavage may contribute to toxicity by generating toxic fragments or by shifting APP processing toward an amyloidogenic pathway. For PSs, caspase cleavage disables antiapoptotic functions attributed to PS C-terminal fragments. These observations suggest that caspases actively contribute to the molecular pathogenesis of these diseases and support the development of caspase inhibitors as potential therapeutic approaches for chronic neurodegenerative disorders. PMID- 10733230 TI - The success of gene therapy in correcting the failings of advancing age PMID- 10733231 TI - The molecular regulation of myogenesis. AB - Over the past years, several studies have unraveled important mechanisms by which the four myogenic regulatory factors (MRFs: MyoD, Myf-5, myogenin, and MRF4) control the specification and the differentiation of the muscle lineage. Early experiments led to the hypothesis that these factors were redundant and could functionally replace one another. However, recent experiments using in vivo and in vitro models have demonstrated that in fact different aspects of the myogenic program are controlled by different factors in vivo, suggesting that these factors play distinct roles during myogenesis. The activity of the MRFs during proliferation and differentiation of muscle precursor cells has clearly been demonstrated to be dependent on specific cell-cycle control mechanisms as well as distinct interactions with other regulatory molecules, such as the ubiquitously expressed E proteins and several other transcription factors. Furthermore, the observation that the MRFs can recruit chromatin remodeling proteins has shed some light on the mechanisms by which the MRFs activate gene expression. Recently, a functional role for MyoD during satellite cell activation and muscle repair has been identified in vivo, which cannot be substituted for by the other MRFs. This has put forward the hypothesis that these factors also play specific biological roles following muscle injury and repair. PMID- 10733232 TI - Genetic landmarks through philately--Crick, Watson and Wilkins: the scientists behind DNA structure. PMID- 10733233 TI - Support for linkage of familial combined hyperlipidemia to chromosome 1q21-q23 in Chinese and German families. AB - We examined familial combined hyperlipidemia (FCHL) families from nonisolated regions in Germany and China to see if we could corroborate support for a chromosome 1q FCHL locus in more general populations. We recruited 24 German families with 137 members, 92 of whom met the criteria of affected in terms of the low density lipoprotein (LDL) and triglyceride levels in excess of the 90th percentile for age and gender. In China, we recruited 12 families with a total of 81 members. All affected persons had total cholesterol concentrations >240 mg/dl and triglyceride concentrations >250 mg/dl. We examined the markers APOA2, D1S1677, D1S104, D1S194, D1S426, and D1S196. Two-point linkage analysis allowing for heterogeneity gave a maximum linkage of disorder score (HLOD) of 2.60 right over D1S194, estimating the proportion of linked families at 36%. This marker is adjacent to D1S104. The evidence for linkage was roughly the same both in the German (HLOD 1.40) and Chinese families (HLOD 1.52). Marker D1S194 is close to the retinoid X receptor (RXR) gene locus, which was found to be linked to triglyceride levels in an earlier twin study from our laboratory. We interpret our observations as encouraging support for the recent findings indicating the presence of a gene for FCHL on chromosome 1q. Furthermore, since DIS194 is adjacent to the gene for the RXR, we suggest that RXR is an attractive candidate for involvement in FCHL. PMID- 10733234 TI - Impact of age and body size on inter-individual variation in measures of lipid metabolism: influence of gender and apolipoprotein E genotype. AB - This study was undertaken in 1695 adult subjects (870 women and 825 men) in order to further document the complexity of the influence of the apolipoprotein (apo) E genotypes on the mean levels and intragenotypic variability of seven measures of lipid metabolism. In addition, the statistical relationships between variability in these traits and variation in age, body mass index (BMI) and waist-to-hip ratio (WHR) were assessed. The contribution of variation in age and body size to inter-individual variation was found to be dependent on context, defined by gender and apo E genotype. Our findings are consistent with the reality that it is neither genes nor environments, but their interactions that are responsible for the variation in risk of cardiovascular disease. PMID- 10733235 TI - Assessment of education and counselling offered by a familial colorectal cancer clinic. AB - We have evaluated whether or not client expectations, in terms of education and information needs, have been met by a multi-disciplinary familial colorectal cancer clinic. The study used a pre- and post-clinic questionnaire design and 126 (84 women, 42 men) clients of the clinic participated. The most common reason for coming to the clinic is to 'find out whether there is a gene for colorectal cancer in the family', followed by 'to reduce risk for bowel cancer' and 'concern for children's risk'. Clients would have preferred to receive more information before attendance at the clinic to help with preparation. Information given during the clinic increased knowledge of bowel cancer genetics and had a positive effect on the accuracy of some clients' perceptions of their risk of developing cancer. In multivariate analysis, higher risk perceptions, higher education level and greater baseline knowledge predicted post-clinic knowledge of bowel cancer genetics and an increase in knowledge. Client reports of the presence of a genetic counsellor or medical geneticist in the session also predicted post clinic knowledge and an increase in knowledge. Most participants felt they received enough information during the clinic session on various aspects of familial colorectal cancer, but the desire for more written information on prevention, including lifestyle actions, was expressed by many. PMID- 10733236 TI - Partial CFTR genotyping and characterisation of cystic fibrosis patients with myocardial fibrosis and necrosis. AB - Myocardial necrosis and fibrosis is a rare complication of cystic fibrosis (CF) causing sudden and unexpected death in infancy due to cardiac arrest. Characteristic morphological lesions are recognisable postmortem. The 18 CF patients with this complication had varied clinical features including mild pulmonary involvement, early onset severe pancreatic insufficiency, and profound electrocardiogram (ECG) changes. In this group of patients, 5 were deltaF508 homozygotes, 1 was deltaF508/ N1303K and 1 was a deltaF508/M compound heterozygote. A pair of affected siblings (deltaF508 homozygotes) were fully concordant for myocardial involvement and for the general course of the disease. The co-existence of a genetic predisposition to myocardial lesions resulting most probably from severe cystic fibrosis transmembrane (CFTR) genotypes (such as deltaF508/deltaF508, deltaF508/N1303K) and deficiency of certain trophic factors necessary for metabolism of the myocardium, are postulated to cause myocardial complications in CF leading to circulatory failure and early death. PMID- 10733237 TI - An SRY-negative XX male with Huriez syndrome. AB - This report studies a 42-year-old 46,XX patient affected by palmoplantar keratoderma. clinically classified as Huriez syndrome. The patient showed a male phenotype with apparently normal male features including testicular development. Cytogenetic and chromosomal painting analysis excluded the presence of translocation of the Y chromosome. PCR analysis of genomic DNA failed to detect the presence of the testis-determining gene, SRY. The presence of other Y chromosome genes, known to be involved in testicular maturation and spermatogenesis, has also been analyzed. The data suggest that the sex reversal in this 46,XX male patient is due to a defect on a yet unidentified autosomal or X-linked sex-determining gene. The relationship between the sex reversion and the presence of sclerotylosis is discussed. PMID- 10733238 TI - Identification of three novel mutations in the insulin receptor gene in type A insulin resistant patients. AB - Type A insulin resistance syndrome is characterized by the association of ovarian hyperandrogenism, acanthosis nigricans, and severe insulin resistance. We have identified three novel mutant alleles of the insulin receptor gene in 3 patients with type A syndrome, a severe form of insulin resistance. Two of the patients were sisters (A1, A2), 1 of them was a compound heterozygote for a mutation at the 3'-splice acceptor site of intron 21 (AG-->AA), and a missense mutation Val140Leu in exon 2. Her sister was a simple heterozygote for the 3'-splice acceptor mutation. The third patient (A3) was heterozygous for the missense mutation Ala1028Val in exon 17, in the consensus sequence for ATP binding. PMID- 10733239 TI - An unaffected individual from a breast/ovarian cancer family with germline mutations in both BRCA1 and BRCA2. AB - Currently many centers offer testing for three specific mutations, 185delAG, 5382insC, and 6174delT, in the BRCA1 and BRCA2 genes to Ashkenazi Jewish individuals at high risk for breast and ovarian cancer. We recently tested members of a family with multiple cases of breast and ovarian cancer (Family R014). The proband in this family tested positive for the 185delAG mutation. The unaffected sister of the proband tested positive for both the 185delAG and the 6174delT mutations. Further testing and review of the family history suggest that both mutations may have come from a maternal grandfather and passed down for two generations. Counseling of the unaffected double heterozygote individual in this family is complicated by lack of information on the risk of breast, ovarian, and other cancers in such individuals. A better understanding of these risks will depend on the identification and study of more individuals carrying mutations in both the BRCA1 and BRCA2 genes. Our study emphasizes the importance of testing Ashkenazi Jewish individuals from high-risk breast and ovarian cancer families for all three common BRCA1 and BRCA2 mutations identified in this ethnic group. PMID- 10733240 TI - Correlations between individual clinical manifestations and CTG repeat amplification in myotonic dystrophy. AB - Myotonic dystrophy (DM) is a multisystemic disease caused by the expansion of a CTG repeat, located in the 3'-untranslated region of the DMPK gene. The number of CTG repeats broadly correlates with the overall severity of the disease. However, correlations between CTG repeat number and presence/absence or severity of individual clinical manifestations in the same patients are yet scarce. In this study the number of CTG repeats detected in blood cells of 24 DM subjects was correlated with the severity of single clinical manifestations. The presence/absence of muscular atrophy, respiratory insufficiency, cardiac abnormalities, diabetes, cataract, sleep disorders, sterility or hypogonadism is not related to the number of CTG repeats. Muscular atrophy and respiratory insufficiency are present with the highest frequency, occurring in 96 and 92% of the cases, respectively. A significant correlation was found with age of onset (r = -0.57, p<0.01), muscular disability (r = 0.46, p<0.05), intellective quotient (r = -0.58, p<0.01) and short-term memory (r= -0.59, p<0.01). Therefore, the CTG repeat number has a predictive value only in the case of some clinical manifestations, this suggesting that pathogenetic mechanisms of DM may differ depending on the tissue. PMID- 10733241 TI - The Haspeslagh syndrome (MIM 177980) is caused by an unbalanced reciprocal 6q/9p translocation. PMID- 10733242 TI - A further Angelman syndrome patient with UPD15 due to paternal meiosis II nondisjunction. PMID- 10733243 TI - The International Federation of Human Genetics Societies. PMID- 10733244 TI - 'Novel' immunodeficiency syndrome may be a previously described entity. PMID- 10733245 TI - Microbiological safety evaluations and recommendations on sprouted seeds. National Advisory Committee on Microbiological Criteria for Foods. AB - In 1997, the National Advisory Committee on Microbiological Criteria for Foods (NACMCF/the Committee) was asked to review the current literature on sprout associated outbreaks: identify the organisms and production practices of greatest public health concern: prioritize research needs: and provide recommendations on intervention and prevention strategies. In response to this charge, the Fresh Produce Work Group (FPWG) documented the relevant epidemiology and microbial ecology of sprout-associated outbreaks and reviewed current industry practices and initiatives related to the growing of seed and the production of sprouts. Sprouts have been identified as a special problem because of the potential for pathogen growth during the sprouting process. If pathogens are present on or in the seed, sprouting conditions may favor their proliferation. There is no inherent step in the production of raw sprouts to reduce or eliminate pathogens. Contaminated seed is the likely source for most reported sprout-associated outbreaks. Research has been initiated on methods to reduce or eliminate pathogenic bacteria on seeds and sprouts and some treatments show promise. However, to date, no single treatment has been shown to completely eliminate pathogens under experimental conditions used. Finally, the Committee found that, at the time of the charge, there was a lack of fundamental food safety knowledge along the continuum from seed production through sprout consumption. More recently, many have become aware of the potential for this food to be a vehicle for foodborne illness and the need for appropriate controls: however, such awareness is not universal. Although seed appears to be the most likely source of contamination in sprout associated outbreaks, practices and conditions at the sprouting facility may also impact on the safety of the finished product. In recent sprout-associated outbreak investigations, facilities associated with outbreaks did not consistently apply seed disinfection treatments prior to sprouting. Conversely, facilities that used seed from the same lot as an implicated facility, but had not been associated with reported illnesses, appear to have been consistently using seed disinfection treatments, such as 20,000 ppm calcium hypochlorite, to disinfect seed prior to sprouting. The Committee has developed a number of specific recommendations, including: 1. The knowledge of all interested parties pertaining to the microbiological safety of sprouted seeds must be enhanced; government and industry should develop education programs for seed and sprout producers on basic principles for microbiological food safety, good agricultural practices, good manufacturing practices, and hazard analysis and critical control point (HACCP) systems. 2. Good agricultural practices should be systematically implemented to reduce the potential for microbial contamination of seeds for sprout production. 3. Seed cleaning, storage, and handling practices that minimize the potential for microbial contamination should be developed and implemented. 4. Seeds should be treated with one or more treatments that have been shown to reduce pathogenic bacteria that may be present. Intervention strategies that deliver less than a given reduction (at this time, 5-log) in levels of Salmonella spp. and enterohemorrhagic Escherichia coli O157 should be coupled with a microbiological testing program. 5. Establish good manufacturing practices and food safety systems, including regulatory oversight, microbial testing, adoption of HACCP, and improved traceback, that systematically look for means to prevent seeds from serving as the vehicle for foodborne disease, and 6. Conduct research related to the microbiological safety of sprouted seeds, particularly in the areas of pathogen reduction or elimination, sources of contamination and its prevention, and preventing or retarding pathogen growth during sprouting. PMID- 10733247 TI - Efficiency of four secondary enrichment protocols in differentiation and isolation of Listeria spp. and Listeria monocytogenes from smoked fish processing chains. AB - Four secondary enrichment protocols (conventional methods: UVM II, Fraser 24 h and Fraser 48 h: Impedimetric method: Listeria electrical detection medium) were studied for their ability to isolate Listeria spp. and Listeria monocytogenes from fish and environmental samples collected along the processing chain of cold smoked fish. From all methods, Listeria spp. and L. monocytogenes were respectively present in 56 and 34 of 315 samples analysed. Fraser broth incubated for 48 h gave the fewest false negative Listeria spp. results [4/56; (7.1%)], but concurrently only 15/34 (44.1%) samples were correctly identified as containing L. monocytogenes, Listeria electrical detection (LED) medium detected only 36/56 (64.3%) Listeria spp. positive samples. Despite this lower isolation rate, LED identified 20/34 (58.8%) L. monocytogenes positive samples correctly and gave fewer false positive results. The overall conclusion was that more than one isolation method is needed to accurately estimate L. monocytogenes contamination rates. PMID- 10733246 TI - Arginine, citrulline and ornithine metabolism by lactic acid bacteria from wine. AB - The catabolism of arginine, an amino acid found in grape juice and wine, citrulline and ornithine was investigated in four lactic acid bacteria. Only Lactobacillus hilgardii X1B catabolized arginine and excreted citrulline into the medium. The recovery of arginine as ornithine was lower than the expected theoretical value. The arginase-urease pathway was not detected indicating that the amino acid degradation was carried out only by the arginine dihydrolase pathway. Oenococcus oeni m, a strain not able to utilize arginine, degraded citrulline that was completely recovered as ornithine, ammonia and CO2. Lactobacillus hilgardii X1B catabolized citrulline but it was only 44% recovered as ornithine. The citrulline utilization by Oenococcus oeni m may be important for two reasons: it can gain extra energy for growth from citrulline metabolism, and the amino-acid diminution could avoid the possibility of ethyl carbamate formation from the citrulline naturally present in wine. PMID- 10733248 TI - Use of the RAPID ID 32 A system for rapid identification of Clostridium species important in food hygiene. AB - The identification of Clostridium species using conventional biochemical reactions or commercially available miniaturized ready-to-use test kits often yields uncertain results. One of these test kits, the RAPID ID 32 A identification system for anaerobes (bioMerieux, Marcy-l'Etoile, France), is based on the evaluation of the action of preformed bacterial enzymes and allows classification to the species level within 4 h. This study intended to assess the suitability and reliability of this system for the rapid identification of Clostridium species relevant to food hygiene. For this purpose, 122 test strains of 18 different Clostridium species were examined via RAPID ID 32 A. Of these, 110 (90.2%) were correctly identified to the species level within 4 h. In addition, six strains were successfully classified after examination with supplementary biochemical reactions suggested by the manufacturer, which required an overall examination time of 72 h. The identity of five Clostridium strains could not be determined and in one case the test kit yielded a wrong species classification. Altogether, a correct identification was possible for 116 (95.1%) of the isolates, the total error rate was 4.9%. Using the RAPID ID 32 A system a great number of Clostridium species which are relevant to food hygiene and are often difficult to identify can be correctly and reliably classified within 4 h. Nevertheless, the test system has to be regarded as not yet completely satisfactory, e.g., because C. tyrobutyricum isolates were only insufficiently identified. Further improvements of the system are desirable in order to make it universally applicable in food microbiology. PMID- 10733249 TI - Incidence of highly genetically diversified Vibrio parahaemolyticus in seafood imported from Asian countries. AB - Vibrio parahaemolyticus is an important pathogen of humans and aquacultured animals, especially in Asian countries. In this study, we examined 686 samples of seafood imported from Hong Kong, Indonesia, Thailand and Vietnam for V. parahaemolyticus. V. parahaemolyticus was recovered from 315 (45.9%) samples. The incidence of V. parahaemolyticus in products from Hong Kong and Thailand was markedly higher than the incidence in products from Indonesia and Vietnam. The incidence rates in shrimp, crab, snail, lobster, sand crab, fish and crawfish were 75.8, 73.3, 44.3, 44.1, 32.5, 29.3 and 21.1%, respectively. None of the isolates possessed the hemolysin genes (tdh, trh). The chromosomal DNA of 121 randomly selected imported isolates and three local environmental strains was digested with SfiI and analyzed by pulsed-field gel electrophoresis (PFGE). These isolates were then grouped into 96 different but mostly unique PFGE patterns. After hierarchical cluster analysis, these patterns could be arbitrarily grouped into twenty-two PFGE types (type A to V). A wide range of PFGE types were identified in isolates from different origin. Moreover, the PFGE types were not specifically associated with the origin or kind of seafood. These results reveal the high genetic diversity in V. parahaemolyticus isolated from seafood. PMID- 10733250 TI - Effect of added proteinases and level of starter culture on the formation of biogenic amines in raw milk Manchego cheese. AB - The influence of two proteinases (Bacillus subtilis neutral proteinase and Micrococcus sp. cysteine proteinase) and two starter culture levels (0.1% and 1%) on biogenic amine formation has been studied in raw ewes' milk Manchego cheese. Amino acid decarboxylating micro-organisms were determined on tyrosine enriched selective media. Biogenic amines were analysed by capillary electrophoresis in citrate buffer at pH 3.6. Addition of proteinases and level of starter culture did not influence the population of micro-organisms with amino acid decarboxylating activity, which represented on average 1% of the bacterial population in 30-day-old cheeses. Tyramine and histamine were detected in all batches of cheese from day 30. Concentrations of tyramine and histamine were higher in cheeses made from milk with neutral proteinase (up to 356 and 284 mg kg(-1), respectively, after 90 days) than in cheeses made from milk with cysteine proteinase (up to 269 and 189 mg kg(-1), respectively) or with no proteinase added (up to 305 and 226 mg kg(-1), respectively). Formation of tyramine and histamine was also favoured in cheeses made with 1% starter culture with respect to cheeses made with only 0.1% starter culture, probably due to the higher pH values of the former cheeses. After 90 days of ripening, concentrations of 10-20 mg kg(-1) phenylethylamine were observed in 9 of the 12 batches, and levels < 10 mg kg(-1) tryptamine were only detected in 3 batches, with no significant relationship between the concentration of these amines and proteinase addition or level of starter culture. PMID- 10733252 TI - Bibliography of food microbiology. PMID- 10733251 TI - Effect of acidification and oil on the thermal resistance of Bacillus stearothermophilus spores heated in food substrate. AB - The effect of the addition of vinegar and/or oil to a food homogenate (tomato sauce, tuna and vegetables) on the thermal resistance of Bacillus stearothermophilus spores was studied. The results indicated that the food substrate without the addition of vinegar and oil and a pH value of 5.28 reduced the thermal resistance of B. stearothermophilus spores compared with that obtained in double-distilled water, (D121 = 1.41 and 3.08 min respectively). The addition of vinegar reduced the pH of the substrate (4.81) and consequently the D values were reduced (D121 = 1.28 min). The addition of soya oil and vinegar to substrate until a pH of 4.81, further reduced the thermal resistance of the spores, giving a D121 value of 0.93 min. PMID- 10733253 TI - Atomic force microscopy imaging of living cells: progress, problems and prospects. AB - Recent development of atomic force microscopy (AFM) applications in imaging living cells is reviewed, focusing on technical progress and application advancements made in the following major areas: (i) high-resolution imaging of cellular structures, (ii) real-time monitoring of cellular dynamic processes, and (iii) detecting micromechanical properties of the cell. Technical and experimental difficulties frequently encountered in AFM applications in above areas are presented and possible strategies for overcoming these obstacles are discussed. Significant advances in the AFM study of living cells can be achieved from further development of AFM technology, sample preparation, and innovative applications. PMID- 10733255 TI - A method for three-dimensional coculture of cancer cells combined to any other type of cells maintained organotypically. AB - A three-dimensional cell coculture method is presented where cancer cells can be maintained alone or combined with other cell types in longterm culture in order to reconstitute some of the interactions between the different cell elements in tumors in vivo. The cells are accumulated by centrifugation to form 'nodules' which are cultivated on a semisolid agar medium at medium/air interface. The nodules are not mere cell aggregates, they are able to develop morphological and functional differentiation as well as tissue-like membrane junctions. Studies on short-term and long-term effects of anticancer treatments are possible and their long-term regrowth can be obtained. Especially, in nodules containing cell mixtures, the localization of the different cell types can be determined and their specific differentiation. An example showing stroma-like formations and collagen production in breast cancer cell and breast fibroblast containing nodules is presented. PMID- 10733254 TI - An improved method for the purification of stellate cells from rat liver with dichloromethylene diphosphate (CL2MDP). AB - Hepatic perisinusoidal cell population consists of hepatic stellate cells, Kupffer cells, endothelial cells, and Pit cells. These cells are isolated by enzymic digestion and purified by density gradient centrifugation. With isolation of stellate cells, conventional method is unable to eliminate the contamination of Kupffer cells because the densities of these two cells are similar. We report here an improved method for isolation of highly purified hepatic stellate cells, using dichloromethylene diphosphate (CL2MDP), which has selective cytotoxicity of Kupffer cells. Three days after the single intravenous administration of liposome encapsulated CL2MDP, the Kupffer cells disappeared almost completely from the liver. Following Percoll density gradient centrifugation, the purity of the hepatic stellate cells exceeded 98% without any contamination of the Kupffer cells. Kupffer cells are reported to affect the physiological functions of stellate cells. The availability of highly purified stellate cells will facilitate the investigation of their functions in primary culture. PMID- 10733256 TI - A comparative study of the isolation of type II epithelial cells from rat, hamster, pig and human lung tissue. AB - Data is reported on the reproducibility and purity of alveolar type II cell isolations from 4 species. Human and pig type II cells were isolated using a tissue slice method to remove blood and contaminating cells, whilst rat and hamster cells were isolated using the method of protease instillation. All cells were purified on Percoll gradients and by differential attachment. Cell type purity was assessed by phase contrast microscopy, electron microscopy (EM), percentage of cells alkaline phosphatase (AP) positive and percentage of cells staining strongly for NADPH dependent nitro blue tetrazolium reductase (NBT). These enzymes are considered as markers for type II and Clara cells respectively. The purity of all cell preparations was enhanced following 24 h culture on a biomatrix and whilst plating efficiency was similar for all species, the human tissue consistently yielded the highest purity of type II cells. All cells with lamellar bodies did not contain AP, and activity was variable between species. Further studies are needed to determine if NBT is equally nonspecific as a cell marker enzyme. In summary, sufficient type II cells of high purity can be isolated thus permitting interspecies comparative studies to investigate the effects of selective and non-specific pulmonary toxins, but more specific marker enzymes are required to identify Type II and Clara cells. PMID- 10733257 TI - Preparation of primary cell cultures from lamprey. AB - The lamprey is an important model for studies of evolution and comparative biology. The ability to culture cells from lamprey tissues makes it possible to employ an in vitro approach to address basic questions in these areas. Methods are described for the initiation of cell cultures derived from tissues of adult and larval sea lamprey (Petromyzon marinus). Primary cultures initiated from gill, muscle, gut, brain, ovary, heart and kidney were viable for up to eight months and several of the cultures were propagated for multiple passages. Most cultures were initiated from tissue explants in basal nutrient medium supplemented with fetal bovine and trout sera on a culture surface treated with fibronectin and collagen. Variations of these culture conditions to meet the specific growth requirements of certain cell types are discussed. PMID- 10733258 TI - Characterization of a macrophage-based system for studying the activation of latent TGF-beta. AB - TGF-beta has been implicated in scarring and tissue fibrosis. Most cells secrete TGF-beta as a high molecular weight, latent complex that must be processed to a lower molecular weight, biologically active form. A number of molecules are involved in this activation step including the mannose 6-phosphate/insulin-like growth factor-II receptor, tissue transglutaminase, thrombospondin, plasmin, and others. Here we describe a rapid macrophage-based system for TGF-beta1 activation, which could be used for screening potential anti-fibrotic agents. The system employs transformed mouse peritoneal macrophages treated with lipopolysaccharide as a cell line capable of activating latent TGF-beta. The activation mechanism in our system involves mannose 6-phosphate/insulin-like growth factor-II receptor and transglutaminase. The activation of latent TGF-beta in this system can be prevented by the addition of mannose-6-phosphate but not mannose-1-phosphate. In addition, transglutaminase inhibitors, antibodies to thrombospondin, insulin-like growth factor-II in the presence of its binding protein IGFBP-2, but not IGFBP-1, suppressed the activation of TGF-beta. Anti inflammatory molecules, such as hydrocortisone, when added to LPS-treated macrophages, inhibited TGF-beta activation by a mechanism, that may involve downregulation of transglutaminase expression. In summary, this new, rapid and reproducible system allows testing molecules for their ability to inhibit TGF beta activation, thus providing a screening method for potential anti-scarring molecules. PMID- 10733259 TI - Rapid and efficient retrovirus-mediated gene transfer into B cell lines. AB - Murine B cell lines such as WEHI-231, BAL17 and M12.4.1 are frequently used as model systems to study signal transduction, cell cycle regulation, and apoptosis. Dissection of these processes often involves expressing exogenous genes in these cells. Electroporation is an inefficient method to express genes in B cell lines and requires several weeks to isolate and analyze clones, followed by an additional one to two weeks to grow sufficient cells for biochemical experiments (e.g. immunoprecipitations). In this report, we describe an optimized procedure for retroviral-mediated gene transfer into murine B cell lines that allows one to obtain a pure population of cells expressing an exogenous gene within 4 days. Two days post-infection, between 10% (BAL17 and M12.4.1 cells) and 70% (WEHI-231 cells) of the cells express the exogenous gene. Culturing the cells for an additional 48 hours with puromycin kills all the non-infected cells and yields a pure population of cells that express the exogenous gene. Sufficient cells for biochemical experiments can be obtained by expanding the cell culture for an additional 5 to 7 days. This rapid and efficient retroviral-mediated gene transfer procedure can greatly expedite the study of signal transduction and other processes in B cells. PMID- 10733260 TI - Beta-blockers for congestive heart failure: what is the current consensus? AB - Despite the availability of angiotensin converting enzyme (ACE) inhibitors for patients with congestive heart failure (CHF), mortality and morbidity remains unacceptably high. CHF is thought to progress as a result of activation of endogenous neurohormonal systems which are activated by the initial myocardial injury. The 2 neurohormonal systems which seem to be important in CHF are the sympathetic nervous system (SNS), and the renin-angiotensin-aldosterone system (RAAS). While stimulation of the SNS has important circulatory support functions in the short term, long term activation appears to have deleterious effects on cardiac function and outcomes. The purpose of this article is to review the literature on the use of beta-blockers in patients with CHF. The published randomised clinical trials of beta-blockers in patients with CHF have shown very promising effects on mortality and morbidity. Several systematic overviews of these trials also suggest beneficial effects on mortality, hospitalisation for CHFE need for transplant, and ejection fraction. The effect of beta-blockers on exercise tolerance. New York Heart Association Function Class (NYHA-FC) and quality of life remain equivocal. The recent presentation of the results from several large-scale trials which were terminated early because of significant survival benefit, has removed any concern over the robustness of the mortality data. Available evidence suggests that a wide variety of patients with CHF, including the elderly, should be considered for beta-blocker therapy. Caution is warranted in the initiation and titration of therapy, as symptoms of CHF may transiently worsen. Whether all beta-blockers are equally efficacious remains unknown. PMID- 10733261 TI - Management of chronic lymphocytic leukaemia. AB - Chronic lymphocytic leukaemia (CLL) is the most common form of adult leukaemia in Western countries. The diagnosis requires mature-appearing lymphocytes in the peripheral blood to >5 x 10(9)/L. The immunophenotype typically includes B cell antigens CD19, CD20 and CD23, low expression of surface immunoglobulin and CD5+, with other T cell antigens absent. Bone marrow biopsy, although not required for diagnosis, must show at least 30% lymphocytes. Cytogenetic abnormalities are frequent in patients with CLL, and may be associated with poor prognosis. Clinically, most patients are asymptomatic at presentation, with incidental lymphadenopathy and/or hepatosplenomegaly in the routine physical examination. Infections by opportunistic pathogens are the major cause of death. Aggressive transformation occurs in 10% of patients with CLL, most commonly prolymphocytic leukaemia (PLL) and Richter's syndrome. PLL de novo must be differentiated from PLL of an aggressive transformation. The incidences of autoimmune diseases and solid or haemopoietic secondary malignancies are increased in patients with CLL. Clinical stage is the strongest prognostic factor in CLL. There is no indication for early intervention. The current recommendation to start treatment includes disease-related symptoms, massive and/or progressive hepatosplenomegaly or lymphadenopathy, increasing bone marrow failure, autoimmune disease, and recurrent infections. Alkylating agents (e.g. chlorambucil) and nucleoside analogues (e.g. fludarabine) are the most active agents for CLL. Fludarabine induces higher response rates, but no improvement in overall survival has been observed. Fludarabine is the drug of choice for the majority of patients with CLL. Chlorambucil may be helpful for elderly patients with poor performance, and for patients who do not tolerate fludarabine. No drug combination is better than single agents. For patients refractory to initial treatment, referral to a clinical trial is the best choice. Other salvage therapy includes retreatment with the same initial agent (chlorambucil or fludarabine) if initial response was observed, or fludarabine for patients refractory to chlorambucil. Promising new approaches include cycle-active agents, nelarabine, biological therapy such as anti-CD52 monoclonal antibody, bone marrow transplantation, including the use of submyeloablative preparative regimens ('minitransplant') to induce graft-versus leukaemia effect, and gene therapy. Prophylactic antibacterials and intravenous immunoglobulin should not be used routinely during supportive care. Epoetin may be helpful for patients who have anaemia without obvious cause. Assessment of response to therapy in CLL has been updated by the National Cancer Institute Working Group, and these guidelines are used worldwide for clinical trials. PMID- 10733262 TI - Insulin-Like growth factor I: implications in aging. AB - According to the somatomedin model, growth hormone (GH)-dependent hepatic synthesis is responsible for maintaining circulating insulin-like growth factor (IGF)-I levels. On the other hand, the local autocrine/paracrine IGF-I expression in peripheral tissue is generally GH-independent and reflects the effects of various and tissue-specific trophic hormones. Circulating IGF-I levels undergo important age-related variations increasing at puberty and decreasing, thereafter, to low levels in the elderly. Low IGF-I levels in the elderly mainly reflect impaired somatotroph secretion but the decline in gonadal sex steroid levels, some protein and micronutrients malnutrition as well as age-dependent variations in IGF-binding proteins may also play a role in the age-related decrease in IGF-I activity. This, in turn, partially accounts for age-related changes in bones, muscles, cardiovascular system, central nervous system and the immune system. However, it is currently unclear whether treatment with exogenous IGF-I can retard or reverse age-related changes in body structure and function. PMID- 10733264 TI - Comparative tolerability of the newer generation antiparkinsonian agents. AB - In recent years, the treatment of Parkinson's disease has undergone an immense amount of research, resulting in the development of multiple new medications. This has largely been fuelled by dissatisfaction over the development of motor complications secondary to long term levodopa therapy. Different treatment approaches are applied depending on the stage of Parkinson's disease. In early and mild Parkinson's disease, selegiline offers a limited symptomatic effect. Its neuroprotective effect, although at present theoretical, has questionable clinical relevance. Increased mortality associated with selegiline has been reported, although a meta-analysis of 5 different trials did not support this finding. The newer, non-ergoline dopamine agonists, pramipexole and ropinirole, have undergone extensive studies to evaluate their efficacy as monotherapy in early Parkinson's disease. These newer agonists are ideal initial symptomatic medications, primarily because they delay the onset of levodopa-induced motor fluctuations. Efficacy of the newer dopamine agonists in advanced disease seems to be comparable to that of the older agents, bromocriptine and pergolide. Adverse effects can be reduced by starting the medication at a very low dose and then slowly titrating upward. Catechol-O-methyl transferase (COMT) inhibitors are indicated for the treatment of motor fluctuations in advanced disease, particularly the 'wearing-off' phenomenon. Tolcapone, a peripheral and central COMT inhibitor, appears to be quite effective, producing a 47% reduction in 'off' time. Unfortunately, 3 deaths have been observed, which are presumably secondary to tolcapone therapy. The drug has been withdrawn in many countries, and liver enzyme testing is mandatory in the US. Entacapone, a purely peripheral COMT inhibitor with a lower potency than tolcapone, has also proved to be effective and has not been associated with liver damage, obviating the need for testing. PMID- 10733263 TI - Thrombolytic therapy for stroke: a review with particular reference to elderly patients. AB - Clinical trials in the 1990s of intravenous thrombolysis for ischaemic stroke have involved over 3000 patients. Alteplase given within 3 hours of onset significantly reduces the combined end-point of death and disability. Although alteplase appears safe when given up to 6 hours after onset, individual trials have failed to confirm efficacy beyond 3 hours. Meta-analysis indicates that intravenous alteplase given up to 6 hours after stroke onset significantly reduces death or dependence 3 months after stroke. Two trials of intra-arterial pro-urokinase confirm benefits of treatment up to 6 hours in highly selected patients with angiographically confirmed proximal middle cerebral occlusion. Streptokinase increased the risk of early death significantly in 3 trials, with no overall reduction in eventual death and disability. Patients over 80 years have been excluded from most trials of alteplase, and experience in this age group is minimal. Increased incidence and poorer functional outcome in the elderly mean that thrombolysis may have greater absolute benefit in this group than in the young, but there is also a higher prevalence of absolute or relative potential contraindications to treatment (ranging from increased use of anticoagulant drugs to higher prevalence of atrial fibrillation). Further trials are necessary to address age restrictions and other important issues in the use of alteplase. Thrombolysis is likely to remain feasible for a minority of stroke patients of all ages, and there is a need for other acute treatment options. PMID- 10733266 TI - Home phototherapy: report on a workshop of the British Photodermatology Group, December 1996. AB - Phototherapy is a popular and effective treatment for many patients with skin diseases. However, repeated journeys to hospital for phototherapy can be inconvenient and expensive. If it were available, many patients might prefer home based phototherapy as long as it was safe and effective. Indeed, many psoriasis patients already self-treat with ultraviolet A sunbeds at home. This report represents a consensus view from a British Photodermatology Group workshop held in December 1996, the purpose of which was to examine the potential role of home based phototherapy in dermatological practice. We conclude that home-based therapy represents a suboptimal treatment with greater attendant risks than phototherapy in a hospital environment. The level of medical supervision of the home treatment is crucial to its safety and effectiveness. Until further studies are forthcoming, home phototherapy should be largely restricted to those with overwhelming difficulties in attending hospital. PMID- 10733265 TI - Bone and joint infections in the elderly: practical treatment guidelines. AB - Two types of haematogenous osteomyelitis that are seen in the elderly are vertebral and long bone osteomyelitis. Osteomyelitis secondary to contiguous foci of infection can occur in older adults without vascular insufficiency (secondary to pressure ulcers) or with vascular insufficiency due to diabetes mellitus or peripheral vascular disease from atherosclerosis. Most cases of osteomyelitis can be reasonably treated with adequate drainage, thorough debridement, obliteration of dead space, wound protection, and antimicrobial therapy. Patients are initially given a broad spectrum antimicrobial that is changed to specific antimicrobial therapy based on meticulous bone cultures taken at debridement surgery or from deep bone biopsies. Surgical management is often required in the treatment of osteomyelitis and includes adequate drainage, extensive debridement of all necrotic tissue, obliteration of dead spaces, stabilisation, adequate soft tissue coverage, and restoration of an effective blood supply. Bone repair and bone mineral density may be significantly retarded and may be corrected by eliminating risk factors, supplementing the diet with calcium, bisphosphonates, and/or vitamin D, and treating with testosterone and/or estrogen when deficient. Sodium fluoride treatment and anabolic steroids may be used as alternatives. Septic arthritis is a medical emergency, and prompt recognition and rapid and aggressive treatment are critical to ensuring a good prognosis. The treatment of septic arthritis includes appropriate antimicrobial therapy and joint drainage. Adverse effects of prescribed antibacterials occur more often in the elderly patient than in young adults. The physician can help to minimise the incidence of adverse effects and improve outcomes by being aware of the principles of clinical pharmacology, the characteristics of specific drugs, and the special physical, psychological and social needs of older patients. PMID- 10733267 TI - Contact allergens, but not irritants, alter receptor-mediated endocytosis by human epidermal Langerhans cells. AB - Allergic contact dermatitis is a T-cell-mediated inflammation, induced by contact with sensitizers and occurring through the release of epidermal cytokines and the activation of epidermal Langerhans cells (LCs). The aim of this study was to analyse early events of LC activation induced either by contact allergens or by irritants devoid of any contact allergenic properties. in order to obtain an in vitro method to discriminate between these two groups of molecules. Various contact sensitizers and irritants were studied for their effects on the endocytosis of major histocompatibility complex class II (MHC-II) molecules by freshly-isolated human epidermal LCs. As observed by flow cytometry, a spontaneous decrease in the surface expression of MHC-II (HLA-DR) molecules, linked to spontaneous internalization of the MHC-II molecules by LCs, was obtained by moving freshly-isolated LCs from 4 degrees C to 37 degrees C. Pre incubation of LCs with either sensitizers or irritants increased the spontaneous internalization of HLA-DR molecules with a similar magnitude, but no clear discrimination between sensitizer and irritant effects was obtained by flow cytometry analysis. In contrast, confocal microscopy enabled discrimination between the effects of sensitizers and irritants: sensitizer-treated samples showed internalized HLA-DR molecules aggregated in large vesicles with very bright fluorescence; irritant-treated samples were not different from untreated controls and showed compact HLA-DR molecules in small vesicles with diffuse fluorescence, and mostly localized in the submembranous zone. Electron microscopy demonstrated that sensitizer-treated LCs internalized HLA-DR molecules preferentially in lysosomes collected near the nucleus, whereas the irritant treated and non-treated LCs internalized these molecules in the prelysosomes only near the cell membrane. We conclude that contact allergens and irritants induce distinct patterns of HLA-I)R endocytosis, which may be useful for the development of in vitro screening tests. PMID- 10733268 TI - Apoptosis in primary cutaneous amyloidosis. AB - Amyloid deposits in primary cutaneous amyloidosis (PCA) may be initially derived from cytokeratin. possibly after keratinocyte death. However, the mechanism of keratinocyte death remains obscure. To investigate the potential role of apoptosis in the pathogenesis of PCA, a retrospective study was conducted on the skin tissues from 20 Chinese patients with PCA. We used a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) method for detecting the apoptotic cells. Immunohistochemical staining was performed to examine the expression of the B-cell leukemia/lymphoma 2 gene (bcl-2) and Fas. Apoptotic cells were shown in 11 of 20 cases (55%) by TUNEL. Histological sections showed that dyskeratotic cells and vacuolar alteration of the basal cells were more commonly observed in the TUNEL-positive group. In all cases of PCA, epidermal expression of bcl-2 was minimal, while expression of Fas was observed on keratinocytes in the basal to granular layers: however, these findings were not different from those in normal skin. Our results suggest that the keratinocyte destruction in PCA may occur as an initial result of apoptosis, which in turn leads to the amyloid formation. PMID- 10733269 TI - Wool follicle matrix cells: culture conditions and keratin expression in vitro. AB - Wool follicle matrix cell cultures were initiated as explants from Tukidale (carpet wool) sheep primary follicle bulbs after removal of the outer root sheath. Successful explantation required coculture on collagen with intact dermal papillae. Cells had a typical epidermal morphology (pavements of flattened. polyhedral cells). Extracellular matrix from dermal papillae, conditioned media, separation of dermal papilla from bulb matrices by tissue culture inserts and feeder layers were unable to support matrix cell explantation. Cultures could be maintained for up to 14 passages during which time the cells became larger with an increased cytoplasmic/nuclear ratio and irregular outline. Proliferation of matrix cells was greater on laminin than with either collagen type I or type IV. Proliferation was considerably reduced under serum-free conditions. This was most apparent at low calcium (0.09 mmol/L). By Northern hybridization matrix cells were found to express keratin K18 at all stages of culture. Keratin K 1.15 expression was evident by the tenth passage. The wool-specific keratin K2.10 was not detected. The data demonstrate that successful wool matrix cell culture is achievable. Keratin gene expression occurs in these cells and varies with the stage of culture. PMID- 10733271 TI - Fluorescence localization of K+ channels in the membrane of squid giant axons. PMID- 10733270 TI - High levels of 8-hydroxy-2'-deoxyguanosine appear in normal human epidermis after a single dose of ultraviolet radiation. AB - Major photoproducts induced by carcinogenic ultraviolet (UV) radiation are the cylobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4 PPs). 8-Hydroxy-2 -deoxyguanosine (8-OHdG) is also a DNA base-modified product generated by reactive oxygen species in conditions of ultraviolet stress, Although UVB-induced CPDs and 6-4 PPs have been investigated in animal and human skin, little is known about the role of 8-OHdG in UVB-induced human skin damage or carcinogenesis. Normal human skin from three volunteers was exposed to UV radiation, and the time course of induction and removal of 8-OHdG was examined by immunohistochemical analysis with catalysed signal amplification on formalin fixed paraffin sections. Formation of CPDs and 6-4 PPs was also examined by immunostaining on the same skin specimens. Control epidermis with no exposure to UV radiation showed little nuclear staining of 8-OHdG, but an increased level of 8-OHdG was clearly observed in epidermis biopsied after irradiation. Induced 8 OHdG can rapidly be removed from nucleus during the first 24-48 h, as the staining intensity diminished gradually, almost reaching the control level by 72 96 h after irradiation. Staining for CPDs or 6-4 PPs revealed induction of these photoproducts in human skin, although 6-4 PP-positive cells disappeared more rapidly than those that stained for CPDs or 8-OHdG. Together with protective effect of antioxidants, our results indicate that not only CPDs and 6-4 PPs but also 8-OHdG may play a significant part in UV carcinogenesis. PMID- 10733272 TI - Physiological characterization of supramedullary/dorsal neurons of the cunner, Tautogolabrus adspersus. PMID- 10733274 TI - Web alert. Development. PMID- 10733273 TI - Paper alert. Neurobiology. PMID- 10733275 TI - Development: neural development at the millennium. PMID- 10733276 TI - Of neurons and gene chips. PMID- 10733277 TI - C-terminal propeptides and vacuolar sorting by BP-80-type proteins: not all C terminal propeptides are equal. PMID- 10733278 TI - Proceedings of the European Seminars in Respiratory Medicine--Update 1999. St Moritz, Switzerland, July 2-6, 1999. Part 1. PMID- 10733279 TI - [The homeostasis of the microbial communities in soils polluted by heavy metals]. AB - Homeostatic potentialities of microbial communities in soils contaminated with heavy metals have been studied. To estimate the effect of contaminants on the soil microflora it was proposed to use the indices characterizing homeostasis of microbial community: microorganism survivability in contaminated soil and the period of restoration of their number, sensitivity to certain contaminants. In the model experiments on dark-grey podzolized soil it was shown that contamination with heavy metals (Cu2+, Cd2+, Sr2+, Pb2+, Hg2+) in the doses of 2 and 4 maximum permissible concentrations provokes a short-term inhibition of microorganisms development after which their number restores. Introduction of the mixture of metals evokes more intensive inhibition and extends the period of microorganism number restoration as compared with the effect of certain metals in the same doses. PMID- 10733280 TI - [The role of the carbohydrate composition of the glycocalyx in some species of lactobacilli in the manifestation of their adhesive properties]. AB - Availability of certain monosaccharides in the composition of glycocalyx of lactic acid bacteria (Lactobacillus plantarum--strains 337D and 11/16; Streptococcus thermophilus--strains S1 (nonmucous race) and S5 (mucous race), Enterococcus faecium (K-50) has been investigated with the help of plant lectins with certain carbohydrate specificity labelled by colloid gold. All the microorganisms under investigation were characterized by the presence of N-acetyl D-galactosamine and N-acetyl-D-glucosamine in rather insignificant amounts. Glycocalyx of lactic acid bacteria was also characterized by availability of essential amount of L-fructose and low amount of sialic acid (except for S. thermophilus S5 (mucous race). Presence of alpha-N-acetyl-D-galactosamine, alpha D, beta-D-galactose, alpha-D-glucose, alpha-D-mannose in the composition of the lactic acid bacteria glycocalyx composition evidences for the additional role of these monosaccharides in the process of the microorganism adhesion on the human and animal intestine mucosa. It has been confirmed that availability of certain monosaccharides in the composition of surface glycopolymers of lactic acid bacteria was connected with adhesive properties of cells and their existence conditions. PMID- 10733281 TI - Increased aldehyde lipid peroxidation products in premature infants with chronic lung disease. PMID- 10733282 TI - Incubator oxygen concentrations during free flow oxygen treatment. PMID- 10733283 TI - Clinical and radiological diagnosis of spinal cord birth injury. PMID- 10733284 TI - Maternal-fetal conflict, Moses Maimonides, and the Christian Church. PMID- 10733285 TI - Proceedings of the International Workshop on the Health Effects of Ultraviolet Radiation. Tokyo, Japan, February 17-19, 1999. PMID- 10733286 TI - GABA GABA A: modulating sensitivity to drugs and alcohol. PMID- 10733288 TI - A cellular basis for the adipost. PMID- 10733287 TI - Orexins, sleep and arousal. PMID- 10733290 TI - Report from the 21st Congress of the European Society of Cardiology, Barcelona, Spain, 28 August-1 September 1999. PMID- 10733289 TI - Bornagain? Neurotrophic viruses in neuropsychiatry. PMID- 10733291 TI - Neural features of the reach and grasp. AB - The concept of canonical representations within the motor system has been both supported and refuted using a variety of behavioral studies. Here, based upon neurophysiological data, I discuss the relationship amongst those neuronal substrates of action and the behavioral components of a movement. A novel view of reaching and grasping has been proposed which predicts that movements with similar kinematic and dynamic properties have a similar representation within the nervous system (Smeets & Brenner, 1999). However this is broadly inconsistent with a variety of neurophysiological findings that emphasize the independence amongst representations of action. PMID- 10733292 TI - How strongly is muscle activity associated with joint moments? AB - In this commentary we question whether the relationship between muscle activity and joint moments is the same for natural motor tasks as for controlled experimental situations. An important consideration in this regard is the identification of the correct electromechanical delay (EMD) for comparing EMG and joint moment data. Data from recent cycling studies are used to illustrate the importance of EMD, and how changing task constraints can alter the relation between muscle activity and joint moment balance for bi-articular antagonist pairs. PMID- 10733293 TI - Guess what! Subungual squamous cell carcinoma mimicking chronic paronychia. PMID- 10733294 TI - Guess what! Urticaria factitia (itching urticarial dermographism) released by suction disks of an electrocardiograph. PMID- 10733295 TI - Vital statistics: memorial volume of selections from the reports and writings. 1885. PMID- 10733296 TI - Dehydroepiandrosterone (DHEA) and testosterone concentrations in human hair after chronic DHEA supplementation. PMID- 10733297 TI - Olanzapine-induced psychotic mania in bipolar schizo-affective disorder. PMID- 10733298 TI - [CSE (cholesterol-synthesis enzyme) inhibitors in the treatment of hypercholesterolemia in primary biliary cirrhosis]. PMID- 10733299 TI - [The skeletal manifestations of hemosiderosis]. PMID- 10733300 TI - [The law on the severely disabled]. PMID- 10733301 TI - [Familial hypercholesterolemia]. PMID- 10733302 TI - [Do adrenal cortical hormones influence the pathogenesis of stress ulcer?]. PMID- 10733304 TI - 7th International Meeting on Neural Transplantation and Repair (INT-7). Odense, Denmark, August 26-30, 1999. Abstracts. PMID- 10733303 TI - 12th International Symposium on Endoscopic Ultrasonography. Monte Carlo, Monaco, 11-13 February 2000. Abstracts. PMID- 10733305 TI - Images in reproductive medicine. Endometrial cavity after microwave endometrial ablation. PMID- 10733306 TI - Laparoscopy-assisted Ruge procedure for the creation of a neovagina in a patient with Mayer-Rokitansky-Kuster-Hauser syndrome. AB - OBJECTIVE: To describe the successful use of a laparoscopy-assisted Ruge procedure for the reconstruction of a vagina in a patient with Mayer-Rokitansky Kuster-Hauser syndrome. DESIGN: Case report. SETTING: A university hospital. PATIENT(S): A 19-year-old Japanese woman with Mayer-Rokitansky-Kuster-Hauser syndrome. INTERVENTION(S): Creation of a neovagina by a laparoscopy-assisted Ruge technique. MAIN OUTCOME MEASURE(S): Clinical examinations were performed during the follow-up period. The depth and diameter of the neovagina were measured by vaginography. Patient satisfaction also was determined. RESULT(S): The neovagina was 12 cm in length and 4 cm in diameter. The mucosa of the neovagina was pinkish and had a moist surface. No intraoperative or postoperative complications were observed. CONCLUSION(S): The use of an isolated segment of the sigmoid colon for vaginal construction has the advantages of providing a sufficient length of neovagina and not requiring immediate postoperative self-dilatation. We believe that our procedure has various advantages in addition to those of the original Ruge method, including its minimally invasive nature and excellent cosmetic results. Further, a laparoscopy-assisted operation allows for the diagnosis of uterine defects and the creation of a neovagina at the same time. PMID- 10733307 TI - Management of spontaneous abortion-medical or surgical. PMID- 10733308 TI - Thrombophilia and recurrent fetal loss-related? PMID- 10733309 TI - Micronized vs. intramuscular progesterone for IVF--more work to be done? PMID- 10733310 TI - Inactivating mutations in the LH receptor-as rare as "a hen with teeth"or as frequent as polycystic ovary syndrome? PMID- 10733311 TI - Inactivating mutations in the LH receptor-as rare a "a hen with teeth" or as frequent as polycystic ovary syndrome? PMID- 10733313 TI - Surveys are important research tools. PMID- 10733312 TI - Testing for Chlamydia antibodies in recurrent spontaneous abortion. PMID- 10733314 TI - Research abstracts: publish or perish. PMID- 10733315 TI - Role of technology in health care is debated. PMID- 10733316 TI - Colorectal neoplasia in acromegaly: the reported increased prevalence is overestimated. PMID- 10733317 TI - Diversion colitis: a trigger for ulcerative colitis in the instream colon. PMID- 10733318 TI - Gastric antral vascular ectasia and its relation with portal hypertension. PMID- 10733319 TI - Methyl bromide 1-year dietary study in dogs. AB - Average human exposure resulting from consumption of methyl I bromide (MB) fumigated food has been estimated to be 0.00125 mg/kg/day. A 1-yr feeding study in beagle dogs was conducted as a safety study in which the high-dose diet was intended to yield a methyl bromide dose of at least 100 times the calculated human dietary exposure. Diets were fumigated with MB and fed to the dogs daily, except for weekends and holidays. MB consumption each feeding day was calculated as a time weighted average (TWA) that accounted for the rate of degassing from the fumigated diet and the rate of feed consumption during the feeding period. TWA compound consumption in the loss-, mid- and high-dose groups, respectively, averaged 0.06 ? 0.02, 0.13 ? 0.03 and 0.28 ? 0.08 mg/kg/day in males and 0.07 ? 0.03, 0.12 ? 0.03 and 0.27 ? 0.09 mg/kg/day in females. Clinical observations, body weight and feed consumption, ophthalmology, clinical pathology, urinalysis, organ weights and macroscopic and microscopic pathology were comparable in control and MB-treated dogs. Under the conditions of this study. the no-observed effect level (NOEL) for MB was at least 0.28 mg/ kg/day, or approximately 200 times the expected average human dietary exposure. PMID- 10733320 TI - The virtual double-headedmicroscope: telepathology for all? PMID- 10733321 TI - CD34 expression in calcifying fibrous pseudotumours. PMID- 10733322 TI - Chromophobe cell renal carcinomas with sarcomatoid areas. PMID- 10733323 TI - Intraoperative assessment of sentinel nodes in breast cancer. PMID- 10733324 TI - Myoid cells in the fibrosarcomatous variant of dermatofibrosarcoma protuberans. PMID- 10733325 TI - Filiform and signet-ring cells in large B-cell lymphoma: ultrastructural interpretation. PMID- 10733326 TI - Predominant Paneth cell differentiation in a colonic adenocarcinoma. PMID- 10733327 TI - Extraskeletal primitive neuroectodermal tumour with massive osteo-cartilaginous metaplasia. PMID- 10733328 TI - Majocchi's granuloma trichophyticum in an immunocompromised patient. PMID- 10733330 TI - What's in a name? PMID- 10733329 TI - Botulinum toxin therapy for hyperhidrosis. PMID- 10733332 TI - [Health plans: dream, mirage or nemesis]. PMID- 10733331 TI - [The reed is ready but the basket has to be made: evaluating the SPT-strategy in Spain]. PMID- 10733333 TI - [Health plans]. PMID- 10733334 TI - [Study of the comparability of the 9th and 10th revision of the International Classification of Diseases (ICD) for causes of death in Catalonia]. PMID- 10733335 TI - [Entangled in chaos: digital management of knowledge in public health]. PMID- 10733336 TI - [Assumption of maximal indetermination: an absolute error or a relative error in calculation of sample size?]. PMID- 10733337 TI - Simple preparation method of PCR fragments for automated DNA sequencing. AB - In an effort to find a simple and inexpensive purification method of polymerase chain reaction (PCR) reaction before cycle sequencing reaction, we compared a commercial system with a precipitation protocol performed in our laboratory. We found that, particularly with small PCR products, our method works with greater success than the method compared. Our precipitation method may be used on a larger PCR fragment before cycle sequencing reaction as well. Furthermore, it has the advantage of being simple as the well-known dilution method; in contrast to the dilution method, the precipitation method removes excess primers as well as possible primer dimers. PMID- 10733338 TI - Noggin expression in a mesodermal pluripotent cell line C1 and its regulation by BMP. AB - Osteoblasts and chondrocytes are derived from mesodermal stem cells and their differentiation is under the control of coordinated interaction among signaling molecules. Noggin is one of the signaling molecules which bind to and inactivate BMPs to induce neural tissues and dorsal mesoderm in Xenopus. However, its expression and regulation in mammalian cells has not been known. In this study, we investigated expression of noggin in murine pluripotent mesodermal cell line, C1. Noggin expression was very low in these C1 cells before they were induced to differentiate. When C1 cells were induced to differentiate into chondrocytes in aggregate cultures in the presence of dexamethasone(dex), noggin expression was significantly increased. In a sharp contrast, when the C1 cells were induced to differentiate into osteoblastic cells by the treatment with beta glycerophosphate (betaGP) and ascorbic acid (AA), noggin mRNA expression remained to be barely detectable. Noggin expression was also observed in the developing cartilage of vertebrae in 15.5 dpc mouse embryos. The noggin mRNA level in C1 cells in monolayer cultures was enhanced significantly by the treatment with BMP4/7 in a dose-dependent manner with a maximal effect at 100 ng/ml. The BMP4/7 effect on noggin expression was time dependent starting within 12 h and peaked at 24 h. These results indicate that noggin is expressed in the pluripotent mesodermal cell line C1 and that its expression is regulated by BMP. PMID- 10733339 TI - Promotion of chondrocyte proliferation by versican mediated by G1 domain and EGF like motifs. AB - We have previously demonstrated that versican stimulated NIH3T3 fibroblast proliferation. Since versican is expressed in cartilage, we investigated whether versican plays a role in chondrocyte proliferation. We developed a technique to stably express a recombinant versican mini-gene in chicken chondrocytes, and its effect on chondrocyte proliferation was analyzed by the increase in cell number. The effect of cell adhesion on cell proliferation was tested. Finally, the versican mini-gene was truncated to assess the role of EGF-like motifs in cell proliferation. Expression of the recombinant versican mini-gene stimulated chondrocyte proliferation. Antisense oligonucleotides complementary to versican inhibited chondrocyte proliferation. The G1 domain of versican stimulated chondrocyte proliferation by destabilizing chondrocyte adhesion. Furthermore, deletion of the two EGF-like motifs from the G3 domain also reduced the function of versican in stimulating cell proliferation. Versican enhances chondrocyte proliferation through a mechanism involving its G1 and G3 domains. This finding may have implications for our understanding of the pathogenesis of various joint diseases. PMID- 10733340 TI - Effects of serum on calcium mobilization in the submandibular cell line A253. AB - The effects of serum on inositol 1,4,5-trisphosphate (IP3) formation and Ca2+ mobilization in the human submandibular cell line A253 were studied. Exposure of A253 cells to fetal bovine serum (FBS) elicited a 3.3-fold increase in IP3 formation and a concentration-dependent transient increase in cytosolic free Ca2+ concentration ([Ca2+]i), which was similar in Ca2+-containing and Ca2+-free media. Newborn bovine serum (NBS), but not bovine serum albumin (BSA), induced a similar response. The Ca2+ release triggered by FBS was significantly (88%) reduced by the phospholipase C inhibitor U73122, indicating that Ca2+ release induced by FBS is through the PLC pathway. Pretreatment with the tyrosine kinase inhibitor genistein abolished the FBS- and NBS-induced Ca2+ release, suggesting that tyrosine kinase plays an important role in mediating the Ca2+ release. Pre exposure to ATP or thapsigargin (TG) significantly reduced the FBS-induced [Ca2+]i increase, indicating that Ca2+ release caused by FBS is from the TG- or ATP-sensitive Ca2+ store. While FBS exposure elicited a large Ca2+ release, it reduced Ca2+ influx. Furthermore, FBS significantly inhibited the Ca2+ influx activated by the depletion of intracellular stores by ATP or TG. These results suggest that (1) serum elicits Ca2+ release from ATP- and TG-sensitive stores, which is mediated by IP3; (2) the serum-induced Ca2+ release may be modulated by a tyrosine kinase-associated process; and (3) serum strongly inhibits Ca2+ influxes including the store depletion-activated Ca2+ influx. PMID- 10733341 TI - G protein G alpha q/11 and G alpha i1,2 are activated by pancreastatin receptors in rat liver: studies with GTP-gamma 35S and azido-GTP-alpha-32P. AB - In the liver, pancreastatin exerts a glycogenolytic effect through interaction with specific receptors, followed by activation of phospholipase C and guanylate cyclase. Pancreastatin receptor seems to be coupled to two different G protein systems: a pertussis toxin-insensitive G protein that mediates activation of phospholipase C, and a pertussis toxin sensitive G protein that mediates the cyclic GMP production. The aim of this study was to identify the specific G protein subtypes coupling pancreastatin receptors in rat liver membranes. GTP binding was determined by using gamma-35S-GTP; specific anti-G protein alpha subtype sera were used to block the effect of pancreastatin receptor activation. Activation of G proteins was demonstrated by the incorporation of the photoreactive GTP analogue 8-azido-alpha-32P-GTP into liver membranes and into specific immunoprecipitates of different Galpha subunits from soluble rat liver membranes. Pancreastatin stimulation of rat liver membranes increases the binding of gamma-35S-GTP in a time- and dose-dependent manner. Activation of the soluble receptors still led to the pancreastatin dose-dependent stimulation of gamma-35S GTP binding. Besides, WGA semipurified receptors also stimulates GTP binding. The binding was inhibited by treatment with anti-Galphaq/11 (85%) and anti-Galphai1,2 (15%) sera, whereas anti-Galphao,i3 serum failed to affect the binding. Finally, pancreastatin stimulates GTP photolabeling of particulate membranes. Moreover, it specifically increased the incorporation of 8-azido-alpha-32P-GTP into Galphaq/11 and Galpha, but not into Galphao,i3 from soluble rat liver membranes. In conclusion, pancreastatin stimulation of rat liver membranes led to the activation of Galphaq/11 and Galphai1,2 proteins. These results suggest that Galphaq/11 and Galphai1,2 may play a functional role in the signaling of pancreastatin receptor by mediating the production of IP3 and cGMP respectively. PMID- 10733342 TI - Nitric oxide regulation of cGMP production in osteoclasts. AB - Bone resorption by osteoclasts is modified by agents that affect cyclic guanosine monophosphate (cGMP), but their relative physiological roles, and what components of the process are present in osteoclasts or require accessory cells such as osteoblasts, are unclear. We studied cGMP regulation in avian osteoclasts, and in particular the roles of nitric oxide and natriuretic peptides, to clarify the mechanisms involved. C-type natriuretic peptide drives a membrane guanylate cyclase, and increased cGMP production in mixed bone cells. However, C-type natriuretic peptide did not increase cGMP in purified osteoclasts. By contrast, osteoclasts did produce cGMP in response to nitric oxide (NO) generators, sodium nitroprusside or 1-hydroxy-2-oxo-3,3-bis(3-aminoethyl)-1-triazene. These findings indicate that C-type natriuretic peptide and NO modulate cGMP in different types of bone cells. The activity of the osteoclast centers on HCI secretion that dissolves bone mineral, and both NO generators and hydrolysis-resistant cGMP analogues reduced bone degradation, while cGMP antagonists increased activity. NO synthase agonists did not affect activity, arguing against autocrine NO production. Osteoclasts express NO-activated guanylate cyclase and cGMP-dependent protein kinase (G-kinase). G-kinase reduced membrane HCI transport activity in a concentration-dependent manner, and phosphorylated a 60-kD osteoclast membrane protein, which immunoprecipitation showed is not an H+-ATPase subunit. We conclude that cGMP is a negative regulator of osteoclast activity. cGMP is produced in response to NO made by other cells, but not in response to C-type natriuretic peptide. G-kinase modulates osteoclast membrane HCI transport via intermediate protein(s) and may mediate cGMP effects in osteoclasts. PMID- 10733343 TI - Regulation of plasma membrane blebbing by the cytoskeleton. AB - When neuroblastoma cells are exposed to lysophosphatidic acid (LPA), they undergo a vigorous, but transient blebbing phase. The effect is sensitive to inhibition by staurosporine, KT 5926 (an inhibitor of myosin light chain kinase), and cytochalasin B, suggesting that LPA activates the phosphorylation of myosin light chain and increases the contractile activity of the actomyosin network. Cell contractions increase the intracellular pressure driving bleb formation. Calyculin, an inhibitor of protein phosphatase2A, also causes blebbing which continues as long as the drug is present, presumably by keeping myosin light chain in the phosphorylated state. Blebbing of neuroblastoma cells is regulated by the status of all three cytoskeletal systems: disassembly of microtubules by nocodazole and of intermediate filaments by acrylamide increased the number of blebbing cells. Cytochalasin B, on the other hand, prevents bleb retraction and, after prolonged incubation, bleb formation. These results are discussed in terms of a model viewing the cytoskeleton as an integrated network transmitting force throughout the cell. Bleb retraction was studied by transfecting neuroblastoma cells with a vector containing the gene for gamma-cytoplasmic actin fused to the green fluorescent protein EGFP (EGFP-actin). EGFP-actin was not detected on the membranes of extending blebs, but started accumulating along the cytoplasmic surface of blebs as soon as the extension phase came to an end and retraction set in. These results confirm earlier suggestions that actin polymerization is required for bleb retraction and for the first time directly relate the two events. PMID- 10733344 TI - Comparative effects of estrogen and antiestrogens on differentiation of osteoblasts in mouse bone marrow culture. AB - Estrogens as well as some antiestrogens have been shown to prevent bone loss in postmenopausal women. These compounds seem to inhibit bone resorption, but their anabolic effects have been less explored. In this study, bone marrow cultures were used to compare the effect of 17beta-estradiol (E2), and two triphenylethylene derivatives, tamoxifen (TAM), and FC1271a, and a benzothiophene derivative raloxifene (RAL) on differentiation of osteoblasts. All enhanced osteoblastic differentiation of 21-day cultures as indicated by increased mineralization and bone nodule formation. All, except RAL, stimulated cell proliferation during the first 6 days of the culture. However, in the presence of RAL the content of total protein was increased in 13-day cultures. SDS-PAGE and autoradiography of [14C]-proline labeled proteins revealed elevated level of the newly synthesized collagen type I. The pure antiestrogen ICI 182,780 abolished the increase of the specific activity of alkaline phosphatase by E2, TAM, and FC1271a but not the effect of RAL on protein synthesis. Our results show that E2 as well as TAM, FC1271a, and RAL stimulate bone formation in vitro but the mechanism of the anabolic action of RAL in bone clearly differs from that of E2, TAM, and FC1271a. PMID- 10733345 TI - Characteristics of EGFR family-mediated HRG signals in human ovarian cancer. AB - The ability of the epidermal growth factor receptor (EGFR) family members, EGFR, HER2, HER3, and HER4, to form homo- and heterodimers after interaction with different ligands expands the signal diversity of these proteins. We investigated their mechanism of activation by exogenous EGF and heregulin (HRG) in human ovarian carcinoma cell lines which express different amounts and combinations of the four receptors. Consistently the predominant interaction after EGF treatment was between EGFR and HER2, whereas activation of HER3 and HER4 depended on the relative abundance of the four receptors in the cells. Remarkably HER3 activation by HRG could occurs independent of HER2, and in one cell line almost no HER4 activation by HRG was detected despite high levels expression. Both EGF and HRG induced activation of mitogen-activated protein kinase (MAPK), but the time course of MAPK activation differed depending on the hetero-dimers induced. EGF and HRG mediated cell growth through the EGFR/HER2 heterodimer and HER4, respectively, but not through HER3 when it was the only HRG receptor expressed and phosphorylated in the cells. These findings reveal a distinct pattern of HRG induced EGFR family interaction in ovarian cancer that is distinct from that described in human breast cancer. Moreover EGF and HRG can exert distinct biological functions depending on the receptor complexes induced in a given ovarian cancer cell line. PMID- 10733346 TI - Malignant astrocytoma cell attachment and migration to various matrix proteins is differentially sensitive to phosphoinositide 3-OH kinase inhibitors. AB - Phosphoinositide 3-OH kinase (PI3-K) has been shown to play an important role in the signaling pathway necessary for cytoskeletal reorganization in non-astrocytic cells. We investigated the role of PI3-K in U-251 MG human malignant astrocytoma cell adhesion and migration. Attachment of U-251 MG cells to vitronectin, fibronectin, laminin, and collagen was inhibited in a concentration-dependent manner by two specific inhibitors of PI3-K (Wortmannin and LY294002). Attachment to vitronectin, fibronectin, and laminin was more sensitive to inhibition of PI3 K (45% inhibition at 10 nM Wortmannin) than attachment to collagen (25% inhibition at 100 nM Wortmannin). Similarly, migration toward these substrates showed differential sensitivity to inhibition. Attachment of the cells to these matrix proteins resulted in an increase in PI3-K activity, as compared to that of cells in suspension, with attachment to vitronectin resulting in the greatest increase in PI3-K activity. p125 focal adhesion kinase (p125FAK) was found to co immunoprecipitate with PI3-K from the NP40-soluble cell fraction of a 1% NP40 detergent lysate of cells in the early stages of adhesion to vitronectin and fibronectin, but not during adhesion to collagen. The expression of p125FAK protein and level of phosphorylation were similar on adherence to all three substrates. These data indicate that the sensitivity of U-251MG cell attachment and migration to PI3-K inhibitors is substrate-dependent, and that complex formation of PI3-K and p125FAK correlates with this sensitivity to PI3-K inhibitors. Our data suggest a role for PI3-K and p125FAK complex formation in PI3-K activation. PMID- 10733347 TI - Mitochondrial DNA deletions are associated with ischemia and aging in Balb/c mouse brain. AB - Deletions in the mitochondrial DNA (mtDNA) of Balb/c mouse cerebrums, resembling deletions found in elderly humans or in patients with certain disorders, were detected by PCR. Analysis was carried out on mice of various ages and on mice in which the bilateral common carotid arteries had been incompletely ligated to reconstruct cerebral ischemia. A 3,867 bp mtDNA deletion was present only in old or ischemic mouse groups. Among the non-ischemic groups, it was found in 0 of 12 weaning, 0 of 12 young, and four of eight old mice. Among the ischemic groups, it was found in 12 of 17 young and 11 of 11 old mice. Moreover, the percentage of total mtDNA containing deletions was 22% for the old non-ischemic group, 37% for the young ischemic group, and 69% for the old ischemic group. In addition, PCR analysis detected two other deletions of 3,726 bp and 4,236 bp in 4 of the 11 old ischemic cerebrums. The results indicate that mtDNA deletions are associated with aging, that ischemia increases the incidence of mtDNA deletions, and that mtDNA deletions resulting from ischemia are more likely to occur in old mice than in young mice. PMID- 10733349 TI - Electronic denture marking--an aid for identification. PMID- 10733348 TI - Regulation of thyroid hormones on the production of testosterone in rats. AB - The effects of a thyroidectomy and thyroxine (T4) replacement on the spontaneous and human chorionic gonadotropin (hCG)-stimulated secretion of testosterone and the production of adenosine 3',5'-cyclic monophosphate (cAMP) in rat testes were studied. Thyroidectomy decreased the basal levels of plasma luteinizing hormone (LH) and testosterone, which delayed the maximal response of testosterone to gonadotropin-releasing hormone (GnRH) and hCG in male rats. T4 replacement in thyroparathyroidectomized (Tx) rats restored the concentrations of plasma LH and testosterone to euthyroid levels. Thyroidectomy decreased the basal release of hypothalamic GnRH, pituitary LH, and testicular testosterone as well as the LH response to GnRH and testosterone response to hCG in vitro. T4 replacement in Tx rats restored the in vitro release of GnRH, GnRH-stimulated LH release as well as hCG-stimulated testosterone release. Administration of T4 in vitro restored the release of testosterone by rat testicular interstitial cells (TICs). The increase of testosterone release in response to forskolin and androstenedione was less in TICs from Tx rats than in that from sham Tx rats. Administration of nifedipine in vitro resulted in a decrease of testosterone release by TICs from sham Tx but not from Tx rats. The basal level of cAMP in TICs was decreased by thyroidectomy. The increased accumulation of cAMP in TICs following administration of forskolin was eliminated in Tx rats. T4 replacement in Tx restored the testosterone response to forskolin. But the testosterone response to androstenedione and the cAMP response to forskolin in TICs was not restored by T4 in Tx rats. These results suggest that the inhibitory effect of a thyroidectomy on the production of testosterone in rat TICs is in part due to: 1) the decreased basal secretion of pituitary LH and its response to GnRH; 2) the decreased response of TICs to gonadotropin; and 3) the diminished production of cAMP, influx of calcium, and activity of 17beta HSD. T4 may enhance testosterone production by acting directly at the testicular interstitial cells of Tx rats. PMID- 10733350 TI - Taking the lead on a national nutrition policy. PMID- 10733351 TI - Documenting your future: how to write a business plan. PMID- 10733352 TI - [Retinal degeneration]. PMID- 10733353 TI - [Oculomotor paralyses in the course of diabetes--concerning 12 cases]. AB - PURPOSE: Our purpose is to study the clinical features of the oculomotor paralyses in the course of the diabetes, to assess the risk factors and to show the importance of the diagnosis and the cerebral imaging. MATERIAL AND METHODS: We report a series of 12 diabetic patients with OMP collected at the department of ophthalmology in UHC Ibn Rochd in Casablanca between 1995 and 1998. RESULT: Our study was about 9 men and 3 women, the mean age was 58 years 6 months. The diabetes was type I in 2 cases and type II in 10 cases. All the patients complained of diplopia with headache. The paralysis of VI has been noticed in 8 cases, it was bilateral in 5 cases. Unilateral Ill palsy occurred in 4 cases. Brain computed tomography was normal in 10 cases and pathologic in 2 cases. Treatment consisted in alternating occlusion in all patients. Concomitance was achieved, after 4 to 6 months of treatment. CONCLUSION: The metabolic abnormalities due to hyperglycemia and the ischemic phenomena due to the diabetic macroangiopathy explain these palsies. PMID- 10733354 TI - [The new JFO is one year old]. PMID- 10733355 TI - [Accidental laser burn]. AB - A student suffered an accidental parafoveal laser burn during a physics experiment. We discuss here the clinical features and management and follow-up. This case presents a good example of photic injuries which can occur in many different settings. PMID- 10733356 TI - [Complications of cataract surgery]. PMID- 10733357 TI - [Cataract surgery after refractive surgery. Part I: practical surgical consequences]. AB - Prior refractive surgery has little incidence on the phako-exeresis technique. Most of the precautions taken are based more on theoretical considerations rather than extensive clinical experience. Excepting cases of high myopy corrected with IOL where early opacification may have occurred, mo st of the population undergoing refractive surgery has not yet reached the age of cataracts. In case of prior corneal surgery (radial keratotomy, PKR, Lasik or intracorneal ring segments) the incision tunnel must remain behind or away from the anatomically modified sectors. In case of prior refractory surgery, explantation must always precede the procedure. A large diameter (6mm or more) lens is always chosen in order to ava avoid optical consequences of modifications induced by the aspheric cornea. PMID- 10733358 TI - [Cataract surgery after refractive surgery: Part II: the problem of implant calculation]. AB - Refractive surgery is a growing technique in a selected population of young patients who no longer want to wear corrective lenses. As this population becomes older and reaches an age where cataracts may develop, we decided to examine the problems encountered in calculating the IOL. Certain techniques may perturb keratometry oe measurement of axis length, two elements essential for calculating the implant. In certain cases where no solution is currently available for making these measurements, we propose archiving keratometry and axial length measurements for candidates for refractive surgery. PMID- 10733359 TI - [Secondary intraocular lens implantation: methods and complications]. AB - PURPOSE: We report a review of the literature on complications of secondary lens implantation without capsular support. METHODS: We assessed results and complications after trans-sulcus sclera l fixation (SSIOL), open-loop anterior chamber intraocular lens (ACIOL) and iris-claw lens (ICIOL). RESULTS: Series published in the literature showed variable results. The rate of increased visual acuity ranged from 71% to 92% for SSIOL, 77% to 92% for ACIOL, 83% to 100% for ICIOL. Complications included: decentration and tilt of th IOL in 0-15.3% for SSIOL, 0-8.6% for ACIOL, 1.8-4.8 % for ICIOL; retinal detachment in 1.1-6% for SSIOL, 0-3.3% for ACIOL; cystoid macular edema in 5.8-23% for ACIOL, 4.8%-5.2% for ICIOL; bullous keratopathy in 0-26.3% for SSIOL, 0-14.2% for ACIOL, 4.8% for ICIOL. Vitreous hemorrhage and suture erosion were specific complications report ed for SSIOL in 1.1-25% and 15-20% of cases respectively. CONCLUSION: Secondary IOL implication is a good alternative for correction of aphakia eyes without a posterior capsule. This analysis shows no one procedures offers more safety than the others. The decisive facto r for choosing among the different types of IOL appears to be surgical experience. PMID- 10733360 TI - [Infectious complications of cataract surgery: revisiting acute endophthalmitis]. AB - We reviewed management procedures for endophthalmitis on the basis of epidemiological, clinical, microbiological and experimental data, mainly obtained from two prospective multi-center studies in France (Groupement d'Etudes Epidemiologiques et Prophylactiques, GEEP) and the American Endophthalmitis Vitrectomy Study (EVS). We analyzed medical history of the patient, recent history involving the eye, and examination and laboratory findings to search for a better treatment protocol for each patient and to assess evidence-based prophylaxis for cataract surgery. PMID- 10733361 TI - [Retinal complications of cataract surgery]. AB - Main retinal complications of cataract extraction are retinal detachment and cystoid macular edema. The incidence of retinal detachment after ex traction is 1 %, and may increase after Nd: YAG laser capsulotomy to 4 %. Cystoid macular edema in aphakic or pseudophakic patients is unusually transient, with a decrease of visual acuity in about 3 % of cases; a long lasting cystoid macular edema with prolonged visual deficiency is described in only 1 % of cases. The incidence of photic macular injuries is estimated at between 7 and 28 % of cases; most photoretinal injuries are asymptomatic or minimally symptomatic because of their extrafoveolar location. Finally, the visual prognosis after cataract surgery is poor in diabetic patients, because of the frequency of cystoid macular edema and progression of diabetic retinopathy. PMID- 10733362 TI - [In memory of Pierre Amalric]. PMID- 10733363 TI - [Glaucoma and pterygium]. PMID- 10733364 TI - [French Society of Perinatal Medicine 29th annual meeting. Monaco, 1999. Abstracts]. PMID- 10733365 TI - Reports of the Systolic and Pulse Pressure (SYPP) Working Group. 1st Meeting of the SYPP Working Group. Rome, Italy, April 1999. PMID- 10733366 TI - Proceedings of the Workshop on the Prevention of Leprosy. Pohnpei, Federated States of Micronesia. 25-27 May 1999. PMID- 10733367 TI - Nifedipine and haemolytic anaemia. PMID- 10733368 TI - Proceedings of the 11th International Conference on Periodontal Research. Goteborg, Sweden, 17-19 June 1999. PMID- 10733369 TI - Reply to Meyer on the convergent validity of the MMPI and Rorschach. PMID- 10733370 TI - [On history writing, comment II: Immunosuppressive agents are more important than tissue typing]. PMID- 10733371 TI - C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. AB - BACKGROUND: Since inflammation is believed to have a role in the pathogenesis of cardiovascular events, measurement of markers of inflammation has been proposed as a method to improve the prediction of the risk of these events. METHODS: We conducted a prospective, nested case-control study among 28,263 apparently healthy postmenopausal women over a mean follow-up period of three years to assess the risk of cardiovascular events associated with base-line levels of markers of inflammation. The markers included high-sensitivity C-reactive protein (hs-CRP), serum amyloid A, interleukin-6, and soluble intercellular adhesion molecule type 1 (sICAM-1). We also studied homocysteine and a variety of lipid and lipoprotein measurements. Cardiovascular events were defined as death from coronary heart disease, nonfatal myocardial infarction or stroke, or the need for coronary-revascularization procedures. RESULTS: Of the 12 markers measured, hs CRP was the strongest univariate predictor of the risk of cardiovascular events; the relative risk of events for women in the highest as compared with the lowest quartile for this marker was 4.4 (95 percent confidence interval, 2.2 to 8.9). Other markers significantly associated with the risk of cardiovascular events were serum amyloid A (relative risk for the highest as compared with the lowest quartile, 3.0), sICAM-1 (2.6), interleukin-6 (2.2), homocysteine (2.0), total cholesterol (2.4), LDL cholesterol (2.4), apolipoprotein B-100 (3.4), HDL cholesterol (0.3), and the ratio of total cholesterol to HDL cholesterol (3.4). Prediction models that incorporated markers of inflammation in addition to lipids were significantly better at predicting risk than models based on lipid levels alone (P<0.001). The levels of hs-CRP and serum amyloid A were significant predictors of risk even in the subgroup of women with LDL cholesterol levels below 130 mg per deciliter (3.4 mmol per liter), the target for primary prevention established by the National Cholesterol Education Program. In multivariate analyses, the only plasma markers that independently predicted risk were hs-CRP (relative risk for the highest as compared with the lowest quartile, 1.5; 95 percent confidence interval, 1.1 to 2.1) and the ratio of total cholesterol to HDL cholesterol (relative risk, 1.4; 95 percent confidence interval, 1.1 to 1.9). CONCLUSIONS: The addition of the measurement of C-reactive protein to screening based on lipid levels may provide an improved method of identifying persons at risk for cardiovascular events. PMID- 10733372 TI - Primary angioplasty versus thrombolysis for acute myocardial infarction. PMID- 10733373 TI - Effect of mode of delivery on neonatal intracranial injury. PMID- 10733374 TI - Effect of mode of delivery on neonatal intracranial injury. PMID- 10733375 TI - Mortality among patients on dialysis, patients on dialysis awaiting transplantation, and transplant recipients. PMID- 10733376 TI - Miltefosine for visceral leishmaniasis. PMID- 10733377 TI - Miltefosine for visceral leishmaniasis. PMID- 10733378 TI - Infections associated with diabetes mellitus. PMID- 10733379 TI - Infections associated with diabetes mellitus. PMID- 10733380 TI - Gestational diabetes. PMID- 10733381 TI - Blindness in a strict vegan. PMID- 10733382 TI - Funding crisis for Indian biotech centre. PMID- 10733383 TI - Republican candidates clash on fetal tissue in research. PMID- 10733384 TI - Biomedical centre memorial to victims of Nazi research. PMID- 10733385 TI - Gene therapy institute denies that errors led to trial death. PMID- 10733386 TI - Calls for international biotech panel. PMID- 10733388 TI - For discussion purposes only. Indicators of excellence. PMID- 10733387 TI - What's behind your smile? AB - Dentistry in the 1990s has seen an evolution in new cosmetic materials and techniques. In order to appreciate and use these methods and materials properly, it is important to have an understanding of cosmetic dentistry and its origins. This article gives a brief history of esthetic dentistry and guidelines for cosmetic dentists of the future. PMID- 10733389 TI - Circumcision--the debate goes on. PMID- 10733390 TI - Circumcision--the debate goes on. PMID- 10733391 TI - Circumcision--the debates goes on. PMID- 10733392 TI - Circumcision--the debate goes on. PMID- 10733393 TI - Circumcision--the debate goes on. PMID- 10733394 TI - Circumcision--the debate goes on. PMID- 10733395 TI - Circumcision--the debate goes on. PMID- 10733396 TI - Circumcision--the debate goes on. PMID- 10733397 TI - Circumcision--the debate goes on. PMID- 10733398 TI - Circumcision--the debate goes on. PMID- 10733399 TI - Circumcision--the debate goes on. PMID- 10733400 TI - Bull horn lesions in childhood. PMID- 10733401 TI - Offer infants water. PMID- 10733402 TI - Values. PMID- 10733403 TI - [Pelagia noctiluca jellyfish poisoning on the Mediterranean coast]. PMID- 10733404 TI - [From evaluation to accreditation]. PMID- 10733405 TI - [Ambulatory operations: as hospital service or personal discounts possible]. PMID- 10733406 TI - [Visual hallucinations after brain operation: prompt CT assessment]. PMID- 10733407 TI - [The conscience of Dr. Efimyc. From: "Ward No. 6" by Anton Chekhov]. PMID- 10733408 TI - Alteration of plasma hypoxanthine concentration during ischaemia in the forelimb of the pig. AB - Ischaemia in the forelimb of pigs was induced by clamping the main artery of an 'isolated leg' preparation. In plasma from the brachial vein the hypoxanthine concentration increased linearly between 10 and 25 min of ischaemia. The average increase in plasma hypoxanthine concentration in ten experiments was 25-0 micromol/1 after 30 min of ischaemia. It is concluded that the plasma hypoxanthine level might be a useful indicator of tissue hypoxia caused by impairment of peripheral blood flow. PMID- 10733409 TI - A method of determining lipoprotein-lipase activity in human adipose tissue. AB - A method of determining lipoprotein-lipase activity (LPLA) in human adipose tissue specimens (weighting 5-50 mg) is described. The specimens were incubated at 37 degrees C in a reaction medium based on a glycine buffer (pH 8.3, ionic strength 0.08), in which the enzyme was stablized and the velocity of release of fatty acids was constant during the incubation. The enzyme activity was increased, as is characteristic of lipoprotein-lipase (LPL), three to four-fold by the addition of serum. The inhibitions by NaCl, protamine sulphate and apolipoprotein C-III were as for LPL, when analysed in both a serum-activated and non-activated reaction medium. The apparent LPLA was about six times greater when analysed in a reaction medium based on a glycine buffer in the presence of heparin (1 g/l) than when analysed in a reaction medium based on a Tris buffer. An analysis of the influence of a high (1 g/l) and a low (0.05 g/l) concentration of heparin on the properties of the enzyme activity was carried out, using LPL of bovine skim milk as a reference enzyme. A phospholipid/soybean-oil emulsion was used as substrate, with [3H]triolein as a trace substance. The emulsion was stable for 5 months. The adipose tissue specimens were stored in liquid nitrogen. The analytical error was 15%, which was reduced to 11% (=within-day variation) when intra-individual comparisons were made. PMID- 10733410 TI - Hepatitis C virus, the E2 envelope protein, and alpha-interferon resistance. PMID- 10733411 TI - Chinese stone tools reveal high-tech Homo erectus. PMID- 10733412 TI - AIDS research. Novel protein delivers HIV to target cells. PMID- 10733413 TI - Stem cells. Protest leads Europeans to confess patent error. PMID- 10733415 TI - Research facilities. Glittering future for Yale Medical School. PMID- 10733414 TI - Global health. Group urges action on Third World drugs. PMID- 10733416 TI - Microbial genomics. Culling genes early yields rich harvest. PMID- 10733417 TI - Conflict of interest. NEJM admits breaking its own tough rules. PMID- 10733418 TI - Magnetic resonance imaging. Detecting enzyme activity in live animals. PMID- 10733419 TI - Evolution. In search of vertebrate origins: beyond brain and bone. PMID- 10733420 TI - Findings from undersea to outer space. Going deep for an unearthly microbe. PMID- 10733421 TI - Findings from undersea to outer space. Power from pond scum. PMID- 10733422 TI - Findings from undersea to outer space. The brains behind the face. PMID- 10733423 TI - Managing the Rhesus supply. PMID- 10733425 TI - A question of permanence. PMID- 10733424 TI - Statistics of ancestral roots. PMID- 10733426 TI - Clinical research. PMID- 10733427 TI - Essays on science and society: the spirit of discovery. PMID- 10733428 TI - Molecular biology. A sting in the tail of electron tracks. PMID- 10733429 TI - Signal transduction. The calcium entry pas de deux. PMID- 10733430 TI - Wnt signaling in oncogenesis and embryogenesis--a look outside the nucleus. PMID- 10733431 TI - Neuronal nicotinic acetylcholine receptors: from structure to therapeutics. PMID- 10733433 TI - Obituary: Vale - Professor Peter Nansen. PMID- 10733432 TI - Endothelins are potent vasoconstrictors, and much more besides. PMID- 10733434 TI - [Russia's first Department of Roentgenology (the 80th anniversary of the Department of Roentgenology with a course on Pediatric Roentgenology, St. Petersburg Medical Academy for Postgraduate Education)]. PMID- 10733435 TI - [N.E. Shtern, founder of Saratov roentgenology]. PMID- 10733436 TI - [16th Annual meeting of the German Association for the Study of the Liver. Koln, 28-29 January 2000. Abstracts]. PMID- 10733437 TI - European perspectives in the control of infectious diseases, Malta, 16 April 1999. PMID- 10733438 TI - Typhoid fever vaccines. PMID- 10733439 TI - Potential of DNA vaccines delivered by influenza virosomes. PMID- 10733440 TI - Hyperkalemia. PMID- 10733441 TI - Continuous intravenous epoprostenol for pulmonary hypertension due to the scleroderma spectrum of disease. A randomized, controlled trial. AB - BACKGROUND: Pulmonary hypertension is a progressive and often fatal complication of the scleroderma spectrum of disease for which no treatment has been proven effective in a randomized trial. OBJECTIVE: To determine the effect of epoprostenol on pulmonary hypertension secondary to the scleroderma spectrum of disease. DESIGN: Randomized, open-label, controlled trial. SETTING: 17 pulmonary hypertension referral centers. PATIENTS: 111 patients with moderate to severe pulmonary hypertension. INTERVENTION: Epoprostenol plus conventional therapy or conventional therapy alone. MEASUREMENTS: The primary outcome measure was exercise capacity. Other measures were cardiopulmonary hemodynamics, signs and symptoms of pulmonary hypertension and scleroderma, and survival. RESULTS: Exercise capacity improved with epoprostenol (median distance walked in 6 minutes, 316 m at 12 weeks compared with 270 m at baseline) but decreased with conventional therapy (192 m at 12 weeks compared with 240 m at baseline). The difference between treatment groups in the median distance walked at week 12 was 108 m (95% CI, 55.2 m to 180.0 m) (P < 0.001). Hemodynamics improved at 12 weeks with epoprostenol. The changes in mean pulmonary artery pressure for the epoprostenol and conventional therapy groups were -5.0 and 0.9 mm Hg, respectively (difference, -6.0 mm Hg [CI, -9.0 to -3.0 mm Hg), and the mean changes in pulmonary vascular resistance were -4.6 and 0.9 mm Hg/L per minute, respectively (difference, -5.5 mm Hg/L per minute [CI, -7.3 to -3.7 mm Hg/L per minute). Twenty-one patients treated with epoprostenol and no patients receiving conventional therapy showed improved New York Heart Association functional class. Borg Dyspnea Scores and Dyspnea-Fatigue Ratings improved in the epoprostenol group. Trends toward greater improvement in severity of the Raynaud phenomenon and fewer new digital ulcers were seen in the epoprostenol group. Four patients in the epoprostenol group and five in the conventional therapy group died (P value not significant). Side effects of epoprostenol therapy included jaw pain, nausea, and anorexia. Adverse events related to the epoprostenol delivery system included sepsis, cellulitis, hemorrhage, and pneumothorax (4% incidence for each condition). CONCLUSIONS: Continuous epoprostenol therapy improves exercise capacity and cardiopulmonary hemodynamics in patients with pulmonary hypertension due to the scleroderma spectrum of disease. PMID- 10733442 TI - Inhaled iloprost to treat severe pulmonary hypertension. An uncontrolled trial. German PPH Study Group. AB - BACKGROUND: Inhaled aerosolized iloprost, a stable prostacyclin analogue, has been considered a selective pulmonary vasodilator in the management of pulmonary hypertension. OBJECTIVE: To assess the efficacy of inhaled iloprost in the treatment of life-threatening pulmonary hypertension. DESIGN: Open, uncontrolled, multicenter study. SETTING: Intensive care units and pulmonary hypertension clinics at six university hospitals in Germany. PATIENTS: 19 patients who had progressive right-heart failure despite receiving maximum conventional therapy (12 with primary pulmonary hypertension, 3 with pulmonary hypertension related to collagen vascular disease without lung fibrosis, and 4 with secondary pulmonary hypertension). INTERVENTION: Inhaled iloprost, 6 to 12 times daily (50 to 200 microg/d). MEASUREMENTS: Right-heart catheterization and distance walked in 6 minutes at baseline and after 3 months of therapy. RESULTS: During the first 3 months of therapy, New York Heart Association functional class improved in 8 patients and was unchanged in 7 patients. Four patients died, 3 of right-heart failure and 1 of sepsis. The acute hemodynamic response to inhaled iloprost was predominant pulmonary vasodilatation with little systemic effect at baseline and at 3 months (data available for 12 patients). Hemodynamic variables were improved at 3 months, and the distance walked in 6 minutes improved by 148 m (95% CI, 4.5 to 282 m; P = 0.048). Of the 15 patients who continued to use inhaled iloprost, 8 stopped: Four had lung transplantation, 1 switched to intravenous prostacyclin therapy, and 3 died. Seven patients are still receiving inhaled iloprost (mean +/ SD) duration of therapy, 536 +/- 309 days; mean dosage, 164 +/- 38 microg/d). CONCLUSIONS: Inhaled iloprost may offer a new therapeutic option for improvement of hemodynamics and physical function in patients with life-threatening pulmonary hypertension and progressive right-heart failure that is refractory to conventional therapy. PMID- 10733443 TI - Calcific constrictive pericarditis: is it still with us? AB - BACKGROUND: The presence of pericardial calcification on a plain radiograph strongly suggests constrictive pericarditis in patients with heart failure. However, calcific constrictive pericarditis is considered rare in the United States since tuberculosis incidence has decreased, and doubt has therefore been raised about the importance of this radiologic finding in modern cardiovascular practice. OBJECTIVE: To determine the clinical and prognostic significance of pericardial calcification on radiography in patients with constrictive pericarditis. DESIGN: Retrospective cohort study. SETTING: Tertiary referral center. PATIENTS: A consecutive series of 135 patients (mean age +/- SD, 56 +/- 16 years) who from 1985 through 1995 had constrictive pericarditis confirmed surgically (n = 133) or by autopsy (n = 2). Patients were divided into two groups: those with pericardial calcification on chest radiography (group I) and those without (group II). MEASUREMENTS: Clinical and diagnostic findings were compared in both groups, and outcome was compared in 132 patients who had pericardiectomy. RESULTS: Pericardial calcification was seen in 36 patients (27%). The cause of constrictive pericardial disease was indeterminate in 67% of patients in group I and in 21% of patients in group II (P < 0.001). Patients in group I had had symptoms for a longer period and were more likely to have pericardial knock, larger atrial size, and atrial arrhythmia. Significantly more perioperative deaths were seen in group I, but incidence of late survival and incidence of noncalcific disease were similar in both groups. CONCLUSIONS: Pericardial calcification is a common finding in patients with constrictive pericarditis. It is often associated with idiopathic disease and other markers of disease chronicity and is an independent predictor of increased perioperative mortality rates. PMID- 10733444 TI - Understanding economic and other burdens of terminal illness: the experience of patients and their caregivers. AB - BACKGROUND: Terminal illness imposes substantial burdens--economic and otherwise- on patients and caregivers. The cause of these burdens is not understood. OBJECTIVE: To determine the mechanism for economic and noneconomic burdens of terminal illness and to identify potential ameliorating interventions. DESIGN: In person interviews of terminally ill patients and their caregivers. SETTING: Six randomly selected U.S. sites: Worcester, Massachusetts; St. Louis, Missouri; Tucson, Arizona; Birmingham, Alabama; Brooklyn, New York; and Mesa County, Colorado. PARTICIPANTS: 988 terminally ill patients and 893 caregivers. MEASUREMENTS: Needs for transportation, nursing care, homemaking, and personal care; subjective perception of economic burden; expenditure of more than 10% of household income on health care costs; caregiver depression and sense of interference with his or her life; and patient consideration of euthanasia or physician-assisted suicide. RESULTS: Of all patients, 34.7% had substantial care needs. Patients who had substantial care needs were more likely to report that they had a subjective sense of economic burden (44.9% compared with 35.3%; difference, 9.6 percentage points [95% CI, 3.1 to 16.1]; P = 0.005); that 10% of their household income was spent on health care (28.0% compared with 17.0%; difference, 11.0 percentage points [CI, 4.8 to 17.1]; P < or = 0.001); and that they or their families had to take out a loan or mortgage, spend their savings, or obtain an additional job (16.3% compared with 10.2%; difference, 6.1 percentage points [CI, 1.4 to 10.6]; P = 0.004). Patients with substantial care needs were more likely to consider euthanasia or physician-assisted suicide (P = 0.001). Caregivers of these patients were more likely to have depressive symptoms (31.4% compared with 24.8%; difference, 6.6 percentage points [CI, 0.4 to 12.8]; P = 0.01) and to report that caring for the patients interfered with their lives (35.6% compared with 24.3%; difference, 11.3 percentage points [CI, 5.0 to 17.7]; P = 0.001). Caregivers of patients whose physicians listened to patients' and caregivers' needs had fewer burdens. CONCLUSIONS: Substantial care needs are an important cause of the economic and other burdens imposed by terminal illness. Through empathy, physicians may be able to ameliorate some of these burdens. PMID- 10733445 TI - Update in hepatology. PMID- 10733446 TI - Assisted suicide: finding common ground. PMID- 10733447 TI - Assisted suicide compared with refusal of treatment: a valid distinction? University of Pennsylvania Center for Bioethics Assisted Suicide Consensus Panel. AB - The continuing debate over the deeply controversial issue of physician-assisted suicide has been complicated by confusion about how this practice resembles or differs from refusal of life-sustaining treatment. Perspectives on ethics and policy hinge on the contested issue of whether a valid distinction can be made between assisted suicide and withdrawal of treatment. This paper uses three illustrative cases to examine leading arguments for and against the recognition of a fundamental distinction between these practices. The first case involves assisted suicide by ingestion of prescribed barbiturates, the second involves withdrawal of artificial nutrition and hydration, and the third involves a decision to stop eating and drinking. On theoretical and practical grounds, this paper defends the position that there is a valid distinction between assisted suicide and refusal of treatment. PMID- 10733448 TI - The role of guidelines in the practice of physician-assisted suicide. University of Pennsylvania Center for Bioethics Assisted Suicide Consensus Panel. AB - Oregon has legalized and implemented physician-assisted suicide, while observers argue about the moral import of attempting to formulate guidelines; the utility any set of guidelines can have for physician practice, health care providers, patients, or families; and whether guidelines can really protect against harm or abuse. What were once theoretical questions have taken on new urgency. The debate over the value and power of guidelines includes the following questions: What has been the experience of efforts to implement physician-assisted suicide using consensus guidelines? What goals are guidelines intended to serve? Who should formulate guidelines? What features should be reflected in any proposed guidelines to make them practical and to permit achievement of their goals? Are there any fundamental obstacles to the creation or implementation of guidelines? Is dying a process that is amenable to direction under guidelines, be they issued by physicians, departments of health, blue ribbon panels, or other regulatory bodies? This paper explores these questions as physician-assisted suicide becomes legal. PMID- 10733449 TI - Should assisted suicide be only physician assisted? University of Pennsylvania Center for Bioethics Assisted Suicide Consensus Panel. AB - Discussions in the media, courts, legislatures, and professional societies generally assume assistance with suicide to be a physician's task; in these venues it is commonly referred to as "physician-assisted suicide." This paper defines both the necessity and the limits of the physician's role in assisted suicide by asking the question: Should assisted suicide be only physician assisted? Although physician involvement is necessary, we argue that it is not sufficient to ensure that patients requesting assisted suicide receive the best care. Assisted suicide requires physician involvement, but physicians' limited competence in performing the full range of tasks, the competencies of other professions, and the possibility that other professions could expand their authority in this area suggest that physician-assisted suicide is a far too narrow construct of the task. The willingness of other professionals--including nurses, social workers, and clergy--to participate and even take the lead in assisting suicides is critical to meet society's interest that assisted suicide should be humane, effective, and confined to appropriate cases. As long as legislation and guidelines focus exclusively on the physician's role, our laws and regulations will fall short of meeting societal expectations. PMID- 10733450 TI - Palliative treatments of last resort: choosing the least harmful alternative. University of Pennsylvania Center for Bioethics Assisted Suicide Consensus Panel. AB - Comprehensive palliative care, as exemplified by many state-of-the-art hospice programs, is the standard of care for the dying. Although palliative care is very effective, physicians, nurses, patients, families, and loved ones regularly face clinically, ethically, legally, and morally challenging decisions throughout the dying process. This is especially true when terminally ill patients are ready to die in the face of complex, difficult-to-treat suffering and request assistance from their health care providers. Although physician-assisted suicide has received the most attention as a potential last-resort response, this practice remains illegal in the United States except in Oregon, and even there it is relatively infrequent. More commonly, decisions are made about accelerating opioid therapy for pain, foregoing life-sustaining therapy, voluntarily stopping eating and drinking, and administering terminal sedation in response to unacceptable suffering. The moral distinctions between these practices are critical to some but relatively inconsequential to others. This paper illustrates, through summaries of real clinical cases, how each of these practices might be used in response to patients in particular clinical circumstances, keeping in focus the patient's values as well as those of families, other loved ones, and health care providers. The challenge is to find the least harmful solution to the patient's problem without abandoning patients and their loved ones to unacceptable suffering or to acting in a more deleterious way on their own. PMID- 10733451 TI - Responding to legal requests for physician-assisted suicide. University of Pennsylvania Center for Bioethics Assisted Suicide Consensus Panel. AB - In 1998, 15 terminally ill Oregon residents ended their lives with overdoses of medications supplied legally by their physicians. Many more people consider this possibility. This paper examines the ways in which the physician's response to requests for assisted suicide may change in an era of legalization, articulates some of the resulting conceptual challenges, and provides practical advice to physicians facing such requests. In areas where it is legal, assisted dying becomes one of the many options that can be freely considered for terminally ill patients with extreme suffering. Some patients even view assisted death as a right that can be expected on demand. We consider the ethical implications of disclosing assisted dying to patients as an option of last resort and suggest that physicians working in environments where assisted dying is legal are obliged to do so. However, we conclude that physicians should not encourage patients to hasten death even when practicing in jurisdictions that allow assisted dying. Furthermore, without abandoning the model, we suggest that strict informed consent does not fully address patients' needs at this time. Physicians must also focus on patients' broader biopsychosocial concerns and help them identify solutions through empathic listening and emotional support. We provide a framework and vocabulary for physicians to use when responding to requests for assisted suicide. Physicians should clarify the request, explore and address the patient's concerns, achieve a shared understanding of the goals of treatment, search for less harmful alternatives, express to the patient what they are willing to do, discuss the relevant legal issues, and share their decision making with colleagues. PMID- 10733452 TI - Epoprostenol (prostacyclin) and pulmonary hypertension. PMID- 10733454 TI - Toward evidence-based statistics. PMID- 10733455 TI - Toward evidence-based statistics. PMID- 10733453 TI - Death with dignity: a case study. PMID- 10733456 TI - Toward evidence-based statistics. PMID- 10733457 TI - Cost-effectiveness of low-molecular-weight heparins for deep venous thrombosis. PMID- 10733458 TI - Antiretroviral therapy for pregnant HIV-infected women. PMID- 10733459 TI - A new role of upper gastrointestinal endoscopy. PMID- 10733460 TI - Methylphenidate for cerebral palsy with choreoathetosis. PMID- 10733461 TI - FK506-induced autoimmune diabetes. PMID- 10733462 TI - Research on humans faces scrutiny; new policies adopted. PMID- 10733463 TI - May the drug transporter P glycoprotein affect the antiviral activity of human immunodeficiency virus type 1 proteinase inhibitors? PMID- 10733464 TI - DNA pooling in mutation detection with reference to sequence analysis. AB - We discuss pooling methods of mutation detection for identifying rare mutations. We provide mathematical formulae for obtaining the optimal pool size as a function of the mutation frequency in the study population and the specificity of the test. The optimal pool size depends strongly on the specificity of the test. With a test that has 99% specificity, pooling can reduce the number of tests that need to be performed by 80%, whereas, with a test with 95% specificity, pooling reduces the number of samples that must be tested by only 50%. We used the software PHRED to call mutations after sequencing of pooled samples with known STK11 mutations. We found that, when the area under the curve for the less prominent peak was used to call mutations, we were able to pool pairs of samples and correctly identify mutations. Pooling of three samples did not lead to an adequately specific test for the basic automated allele-calling procedures that we used. We discuss methods by which the specificity may be improved to permit pooling of three or more samples when testing for mutations by sequencing. PMID- 10733465 TI - Evidence for variable selective pressures at MC1R. AB - It is widely assumed that genes that influence variation in skin and hair pigmentation are under selection. To date, the melanocortin 1 receptor (MC1R) is the only gene identified that explains substantial phenotypic variance in human pigmentation. Here we investigate MC1R polymorphism in several populations, for evidence of selection. We conclude that MC1R is under strong functional constraint in Africa, where any diversion from eumelanin production (black pigmentation) appears to be evolutionarily deleterious. Although many of the MC1R amino acid variants observed in non-African populations do affect MC1R function and contribute to high levels of MC1R diversity in Europeans, we found no evidence, in either the magnitude or the patterns of diversity, for its enhancement by selection; rather, our analyses show that levels of MC1R polymorphism simply reflect neutral expectations under relaxation of strong functional constraint outside Africa. PMID- 10733466 TI - The Ile198Thr and Ala379Val variants of plasmatic PAF-acetylhydrolase impair catalytical activities and are associated with atopy and asthma. AB - The platelet-activating factor (PAF) represents a phospholipid with complex biological functions, including involvement in inflammatory processes. The degrading enzyme PAF acetylhydrolase (PAFAH) represents a candidate for asthma and other atopic diseases. Two loss-of-function mutations of PAFAH are associated with severe asthma in Japanese individuals. Our aim was to look for further PAFAH variants in white populations, their possible association with atopic and asthmatic phenotypes, and their functional importance. We picked up three common variants in the PAFAH gene: Arg92His (exon 4), Ile198Thr (exon 7), and Ala379Val (exon 11). The known loss-of-function mutations were not seen. The variant allele Thr198 was found to be highly associated with total IgE concentrations in an atopic population (P=.009) and with "atopic asthma" in an asthmatic population (P=.008). The variant allele Val379 was found to be highly associated with "specific sensitization" in the atopic population (P=.002) and with "asthma" in the asthmatic population (P=.003). By use of recombinant PAFAH enzymes, the variant Val379 showed increased (14 microM) and Thr198 markedly increased (42 microM) KM values compared to the wild type (7 microM); furthermore, Vmax of Val379 was highly increased (132%). Thr198 and Val379 influence plasmatic PAFAH toward lower substrate affinities and therefore are very likely to prolong the activities of PAF. At the same time, they are associated with an increased risk to develop asthma and atopy. Thus, two PAFAH variants seem to play a key role in atopic and asthmatic processes in Caucasian populations. PMID- 10733467 TI - Women with a reduced ovarian complement may have an increased risk for a child with Down syndrome. AB - Advanced maternal age is the only well-established risk factor for trisomy 21 Down syndrome (DS), but the basis of the maternal-age effect is not known. In a population-based, case-control study of DS, women who reported surgical removal of all or part of an ovary or congenital absence of one ovary were significantly more likely to have delivered a child with DS than were women who did not report a reduced ovarian complement (odds ratio 9.61; 95% confidence interval 1.18 446.3). Because others have observed that women who have had an ovary removed exhibit elevated levels of FSH and similar hallmarks of advanced maternal age, our finding suggests that the physiological status of the ovary is key to the maternal-age effect. In addition, it suggests that women with a reduced ovarian complement should be offered prenatal diagnosis. PMID- 10733468 TI - http://www.ilar.org A source of information for rheumatologists, allied health professionals, medical students and the general public PMID- 10733470 TI - A self limiting tumour. PMID- 10733469 TI - Cytokines and systemic lupus erythematosus. PMID- 10733471 TI - Sequential changes of KL-6 in sera of patients with interstitial pneumonia associated with polymyositis/dermatomyositis. AB - OBJECTIVE: KL-6 is a mucin-like high molecular weight glycoprotein, which is strongly expressed on type II alveolar pneumocytes and bronchiolar epithelial cells. It has been demonstrated that the KL-6 antigen is a useful marker for estimating the activity of interstitial pneumonia. In this study, it is hypothesised that serum KL-6 is a useful marker to evaluate the activity of interstitial pneumonia associated with polymyositis/dermatomyositis (PM/DM). METHODS: KL-6 was measured in sera in 16 patients diagnosed with PM/DM. Five had non-specific interstitial pneumonia (NSIP), three had diffuse alveolar damage (DAD), and eight had no pulmonary involvement, and 10 were normal non-smokers as a control group. The correlation was also evaluated between the KL-6 level and each clinical course in patients with pulmonary involvement associated with PM/DM. Immunohistochemical analysis using monoclonal anti-KL-6 antibody was also performed. RESULTS: KL-6 concentrations in sera of patients with interstitial pneumonia associated with PM/DM were significantly high compared with those of PM/DM without interstitial pneumonia, and normal non-smokers. KL-6 concentrations in sera in patients with DAD significantly increased compared with those of other groups. KL-6 values in sera changed according to the progression or improvement of interstitial pneumonia. Immunohistochemical study using pulmonary tissues obtained from patients with DAD demonstrated that the hyaline membrane, proliferating type II pneumocytes, bronchial epithelial cells and some endothelial cells in pulmonary veins were stained by antihuman KL-6 antibody. CONCLUSION: These data demonstrate that measurement of serum KL-6 was a useful marker to evaluate the activity of acute interstitial pneumonia associated with PM/DM. PMID- 10733473 TI - Vertebral fractures and bone mineral density in idiopathic, secondary and corticosteroid associated osteoporosis in men. AB - OBJECTIVE: To investigate bone mineral density (BMD) in men with symptomatic osteoporosis and compare BMD in patients with idiopathic, secondary and corticosteroid associated osteoporosis. METHODS: Age, number of vertebral fractures at presentation and BMD were investigated in men presenting to a bone metabolism clinic with idiopathic (n=105; group 1), secondary (n=67; group 2) and corticosteroid osteoporosis (n=48; group 3). BMD was measured in 176 healthy men (controls). Osteoporosis was diagnosed if there was >/=20% vertebral deformity. RESULTS: Age at peak BMD in controls was 20-29 years at spine (LS-BMD) and femoral neck (FN-BMD). LS-BMD did not change with age but FN-BMD decreased in controls and groups 1 and 2. Mean (SD) age was similar in groups 1 (62.8 (11.5) years, 2 (60.2 (11.0)) years and 3 (62.7 (10.4) years with 45%, 51% and 40% of patients respectively presenting before 60 years. Back pain, present for up to 12 months, was the commonest cause of referral. Vertebral fractures at presentation averaged mean (SD) 2.51 (1.9) in group 1, 2.76 (2.2) in group 2 and 2.48 (1.8) in group 3. LS-BMD Z scores and T scores were more negative in group 1 patients with /= 1 fracture. PMID- 10733472 TI - Differential Th1/Th2 cytokine patterns in chronic arthritis: interferon gamma is highly expressed in synovium of rheumatoid arthritis compared with seronegative spondyloarthropathies. AB - OBJECTIVE: To investigate possible differences in Th1 and Th2 cytokine mRNA expression in the synovial tissue (ST) of patients with rheumatoid arthritis (RA) and seronegative spondyloarthropathies (SpA) with diagnostic and/or pathogenic interest. METHODS: Eleven RA patients and 14 SpA patients (10 with undifferentiated spondyloarthropathy (USpA), two with ankylosing spondylitis (AS) and two with psoriatic arthritis (PsA)) were included. Th1 (interferon gamma, interleukin 2) and Th2 (interleukin 4, interleukin 5 and interleukin 10) cytokine mRNA levels from arthritic knee ST were quantified by using an optimised polymerase chain reaction method with a computerised analysis system. Protein levels of proinflammatory cytokines (interleukin 1, tumour necrosis factor alpha and interleukin 6) in synovial fluid were quantified with a specific ELISA test. RESULTS: Th1 cytokines were detected in all of RA ST samples in contrast with 58% (interferon gamma) and 71% (interleukin 2) of SpA samples. Th2 cytokines were expressed in 90% of RA ST samples, but the findings in SpA were interleukin 10 in 90%, interleukin 4 in 60% and interleukin 5 in 40% of ST samples. However, when the mRNA levels of each cytokine were quantified and corrected for T cell mRNA levels, only interferon gamma levels were significantly higher in RA than in SpA (p<0.003). Thus, the Th1/Th2 cytokine ratio in RA was fivefold that of SpA. Synovial fluid interleukin 1beta concentrations were higher in RA than in SpA (p<0. 05); there were also higher synovial fluid levels of tumour necrosis factor alpha in RA than in SpA, but without statistical significance. CONCLUSION: This study has detected both Th1 and Th2 cytokine gene expression in ST from RA and SpA patients. Synovium interferon gamma mRNA levels and SF interleukin 1beta protein levels were significantly higher in RA than in SpA, so reflecting the known proinflammatory activity of interferon gamma through macrophage activation. Thus, the Th1 (interferon gamma)/Th2 (interleukin 4) ratio is significantly higher in RA than in SpA ST. These data confirm previous studies on ST Th1/Th2 balance in RA and extend previous work in comparing ST RA with subgroups of SpA distinct of ReA. PMID- 10733474 TI - Psychological, behavioural, and social adjustment in children and adolescents with juvenile chronic arthritis. AB - OBJECTIVE: To assess the psychological, behavioural and social adjustment of children (7-11 years) and adolescents (12-16 years) with juvenile chronic arthritis (JCA). Higher rates of maladjustment were expected to be found in these patients. METHODS: Self report questionnaires were used within the context of personal interviews. Family functioning and social support were studied as well. Forty seven patients with JCA, 52 healthy peers and their respective parents participated in the study. RESULTS: Self esteem, perceived competence and body image in patients with JCA were as positive as they were in healthy participants. There were no differences between ill and healthy youngsters with respect to the incidence of psychopathology. Patients with JCA, in general, perceived themselves as socially competent, but they seemed to have somewhat less opportunity or energy to participate in social activities. Children with JCA showed a high level of aspiration to cope with social expectations. This aspiration seemed to be even stronger in case the disease caused more strains, for example, in periods of inflammation and in the systemic onset type. The high level of social adjustment in children with JCA seemed to be supported by highly cohesive family structures. Generally, adolescents with JCA experienced much social support. CONCLUSIONS: In contrast with our expectation, children and adolescents with JCA seemeed to cope quite well with the psychological and social consequences of their long term condition. For future studies, it is hypothesised that the high levels of adaptation might imply an enduring psychological strain, which is reflected in an altered function of the autonomic nervous system. PMID- 10733475 TI - Monocytes from systemic lupus erythematous patients are severely altered in phenotype and lineage flexibility. AB - OBJECTIVE: Cells of the myeloid lineage comprise a very heterogeneous population with many phenotypes and functional activities including macrophages and dendritic cells. To investigate the status, differentiative potential and lineage commitment of monocytic cells in systemic lupus erythematosus (SLE) patients, this study isolated and cultured peripheral blood monocytes from patients and healthy donors. METHODS: Monocytes were isolated by gradient centrifugation and adherence to plastic dishes. The cells were then cultured for three days, partially supplemented with GM-CSF and interleukin 4 (IL4) to obtain dendritic cells. The differentiation status was monitored by the expression of surface markers using flow cytometry and cytokine secretion. RESULTS: Monocytes from SLE patients expressed significantly lower numbers of the monocytic marker CD14 and HLA-DR while secreting significantly more tumour necrosis factor alpha (TNFalpha) than monocytes from healthy donors. The addition of GM-CSF and IL4 resulted in an inhibition of TNFalpha secretion, but was not sufficient to generate monocytederived dendritic cells. CONCLUSION: Monocytes from SLE patients are severely altered in phenotype and function and have a limited differentiation flexibility towards the accessory type of monocytic cells. PMID- 10733476 TI - Assessing the prevalence of hand osteoarthritis in epidemiological studies. The reliability of a radiological hand scale. AB - OBJECTIVE: The hands are often involved in the osteoarthritic disease process. A radiological grading scale is presented, derived from a published atlas, to assess the prevalence of hand osteoarthritis (OA) involvement in clinical and epidemiological studies and its reproducibility is studied. METHODS: This hand scale is based on the radiological feature "joint space narrowing", which represents the macromorphological process of cartilage loss. Osteophytes and sclerosis are less important unless seen in conjuction with joint space narrowing. Nine individual joints per hand (four proximal interphalangeal joints (PIP), four distal interphalangeal joints (DIP), first carpometacarpal joint (CMC 1)) are scored dichotomously for the presence of OA. To save time and to increase reliability a severity grading of radiological features is not performed. To determine inter-rater and intra-rater reliability of the individual joints and the presence of OA in two separate joint groups (>/= 2 PIP or DIP and at least one CMC-1, used to define "generalised OA" in the ongoing Ulm Osteoarthritis Study) 50 pairs of anteroposterior hand radiographs were read by two investigators twice within one month. The kappa coefficient was calculated to quantify the strength of associations. RESULTS: On average five minutes were needed to score one hand radiograph. Both raters were able to reproduce their own readings in all individual joints and for the presence of OA in two separate joint groups after one month. Reliability was highest for the PIP joints (kappa: 0.56-1. 00) it was slightly lower for the DIP joints (0.38-0.87), for the CMC-1 joints (0.58-0.69) and for OA in two separate joint groups (0. 54). The values for inter-rater agreement were good as well, kappa coefficients ranged from 0.52 to 0.92. CONCLUSION: This grading scale was shown to be reliable within and between readers for all the individual joints as well as for the presence of OA in two separate joint groups. Scoring a limited number of joints dichotomously makes this scale efficient and therefore useful for clinical and epidemiological trials, when dealing with large patient samples. PMID- 10733477 TI - Association of the TNFa13 microsatellite with systemic sclerosis in Japanese patients. AB - OBJECTIVES: To elucidate the contribution of microsatellite polymorphisms of TNFa and TNFb alleles to the pathogenesis of systemic sclerosis (SSc) by comparing the allele distribution among populations with different HLA susceptibility genes in SSc. METHODS: TNFa and TNFb microsatellite polymorphisms were determined by PCR in 54 Japanese and 50 German SSc patients and in normal controls. HLA-DR genotyping was carried out by PCR-SSCP. RESULTS: The frequency of TNFa13 was significantly increased in Japanese SSc (p=0. 011, OR=8.53, 95% confidence intervals (95%CI)=2.46, 32.51, and p<1. 0 x 10E-5, OR=10.35, 95%CI=4.88, 22.09) and SSc with antitopoisomerase I antibody (a-Scl-70) (p=0.021, OR=33.25, 95%CI=3. 39, 800.76, and p<1.0 x 10E-5, OR=24.42, 95%CI=8.40, 72.83), compared with the German patient group and German controls, respectively. This increase was not only attributable to a higher prevalence of TNFa13 in Japanese compared with Germans (p=0.005, OR=3.55, 95%CI=1.60, 7.85) but was also caused by an increase in SSc, especially in the a-Scl-70 positive patients (p=0.028, OR=6.88, 95%CI=1.16, 22.60) compared with Japanese controls. TNFa13 was positively in linkage disequilibrium with HLA-DRB1*1502 (LD=0.053, t=2.69). Association analysis indicated that both TNFa13 and DRB1*1502 might have comparable probabilities of being susceptibility factors for SSc with a-Scl-70 in Japanese. Prevalences of TNFa6 and 13 were significantly increased and prevalences of TNFa2, and 7 were significantly decreased in Japanese controls as compared with German controls. CONCLUSION: TNFa13 is a genetic marker for SSc with a-Scl-70 in Japanese patients. Various differences in the prevalences of TNFa alleles between Japanese and German controls were established. PMID- 10733478 TI - Circulating concentrations of soluble L-selectin (CD62L) in patients with primary Sjogren's syndrome. AB - OBJECTIVE: Serum concentrations of soluble (s) L-selectin (CD62L) were measured in patients with primary Sjogren's syndrome (SS) to relate these concentrations to clinical and immunological features of SS. METHODS: The study included 40 consecutive patients (38 women and two men) with a mean age of 61 years (range 24 78) who fulfilled four or more of the preliminary diagnostic criteria for SS proposed by the European Community Study Group in 1993, and 33 healthy blood donors from the hospital blood bank. A sandwich enzyme linked immunosorbent assay (ELISA) was used to detect the soluble form of human sL-selectin (CD62L). RESULTS: The mean (SEM) values of sL-selectin (CD62L) were 861 (66) microg/ml for patients with SS and 986 (180) microg/ml for healthy blood donors, but there was no significant difference. In patients with primary SS, serum sL-selectin (CD62L) concentrations were significantly higher in patients with Raynaud's phenomenon (1275 (112) microg/ml versus 789 (69) microg/ml, p=0.007), autoimmune thyroiditis (1162 (113) microg/ml versus 787 (69) microg/ml, p=0.02) and rheumatoid factor (993 (95) microg/ml versus 684 (70) microg/ml, p=0.01) when compared with patients without these features. CONCLUSION: The presence of Raynaud's phenomenon, autoimmune thyroiditis and rheumatoid factor is associated with higher concentrations of circulating sL-selectin (CD62L) in the sera of patients with primary SS. PMID- 10733479 TI - Prevalence of Chlamydia trachomatis in urine of male patients with ankylosing spondylitis is not increased. AB - OBJECTIVE: To compare the prevalence of Chlamydia trachomatis infections in ankylosing spondylitis (AS) patients with controls, using DNA amplification assays in urine specimens. METHODS: The prevalence of C trachomatis infections was assessed in 32 male AS patients and 120 age and sex matched controls. Urine specimens were tested by ligase chain reaction and polymerase chain reaction. In addition, blood samples of AS patients were tested on serum antibodies to C trachomatis (IgA and IgG) by a specific peptide based solid phase enzyme immunoassay. A questionnaire was used to assess the differences in sexual behaviour and ethnic origin between the two groups. AS patients were also asked about disease characteristics. RESULTS: No significant differences were found between cases and controls in the prevalence of C trachomatis infections. No associations were found between C trachomatis antibodies and disease characteristics, except for acute anterior uveitis (AAU). Four of eight (50%) AS men positive for IgG had a history of AAU in comparison with three of 24 (12.5%) IgG negative men (OR = 7.0; 95% confidence intervals: 1.1, 44.1). CONCLUSION: The prevalence of Chlamydia trachomatis infections, as detected by commercially available DNA amplification assays in urine specimens, in AS patients is not higher compared with male controls of the same age. However, there seems to be an association between specific antibodies to C trachomatis and AAU. PMID- 10733480 TI - Activation of the transcription factor NF-kappaB in the rat air pouch model of inflammation. AB - OBJECTIVE: To investigate the activation of NF-kappaB in the carrageenan rat air pouch model of inflammation in a time course experiment, and the effect of dexamethasone on NF-kappaB activation. METHODS: Air pouch tissue treated with carrageenan (inflamed tissue) was obtained from rats killed at days 1, 2, 3, 6, 14, 21, 28 and 35 after carrageenan challenge. Tissue was also taken from non carrageenan treated pouches (non-inflamed tissue) at day 3, and from inflamed tissues treated with dexamethasone. Tissue sections were wax embedded and stained with an "activity specific" monoclonal antibody raised against the nuclear localisation signal (NLS) of the p65 sub-unit of NF-kappaB. RESULTS: Cells containing activated NF-kappaB were detected in the intimal and sub-intimal regions of the air pouches as early as day 1. There was a significant increase in cells staining for activated NF-kappaB as the inflammation progressed. Initially cells staining were more prominent in the intimal versus sub-intimal region (p<0.001 for day 1) and at later time points the pattern was reversed (p<0.001 for day 6). There was a significant reduction in the number of cells staining for activated NF-kappaB in tissue taken from dexamethasone treated rats, compared with inflamed pouches alone (p<0.001). At no time point was positive staining for activated NF-kappaB observed in blood vessels. CONCLUSIONS: Activated NF-kappaB is present in the inflamed air pouch and the activation is associated with the inflammatory response to carrageenan. Treatment with dexamethasone resulted in reduced numbers of cells staining for activated NF-kappaB. PMID- 10733481 TI - Bone mineral density and biochemical parameters of bone metabolism in female patients with systemic lupus erythematosus. AB - OBJECTIVE: To evaluate bone mineral density and biochemical parameters of bone metabolism in ambulatory premenopausal female patients with systemic lupus erythematosus (SLE). METHODS: 30 women who fulfilled the ARA criteria for the classification of SLE were studied. Lumbar and femoral bone mineral density was determined by dual energy x ray absorptiometry. Various laboratory parameters including serum calcium, serum phosphorus, alkaline phosphatase, bone specific isoform of alkaline phophatase, propeptide of type 1 procollagen, deoxypyridinoline excretion, telopeptide of type 1 collagen, serum creatinine, osteocalcin, parathyroid hormone, 25-OH vitamin D, testosterone, progesterone, estradiol, follicle stimulating hormone and luteinotropic hormone were measured. RESULTS: According to the WHO criteria 39% of all patients with SLE studied had normal bone mineral density, 46% had osteopenia and 15% had osteoporosis at the lumbar spine; at the femoral neck 38.5% had normal bone mineral density, 38.5% had osteopenia and 23% suffered from osteoporosis. Significantly lower osteocalcin levels were found in SLE patients. All other bone resorption and formation markers measured were not statistically different, but higher serum albumin corrected calcium and lower phosphorus values were found in the SLE group. Of all sex hormones tested lower testosterone and higher follicle stimulating hormone concentrations were seen in patients with SLE. CONCLUSION: A high incidence was found of osteopenia and osteoporosis in premenopausal patients with SLE. Bone diminution in SLE seems to be attributable, at least in part, to decreased bone formation in SLE patients. PMID- 10733482 TI - Response to methotrexate in early rheumatoid arthritis is associated with a decrease of T cell derived tumour necrosis factor alpha, increase of interleukin 10, and predicted by the initial concentration of interleukin 4. AB - OBJECTIVE: This study was performed to assess whether there is any change in the T cell cytokine pattern in early rheumatoid arthritis (RA) patients treated with methotrexate (MTX) and whether the lymphocytic cytokine pattern correlates with disease activity. METHODS: Eight patients with RA (disease duration < six months) were studied serially before, after three, and after six to nine months of treatment with MTX for the cytokines tumour necrosis factor alpha (TNFalpha), interferon gamma (IFNgamma), interleukin 4 (IL4) and interleukin 10 (IL10) by intracellular staining of T cells derived from peripheral blood. Response to treatment was assessed by the modified disease activitiy score. RESULTS: The clincial response was accompanied by a significant decrease of TNFalpha positive CD4(+) T cells from a median of 8.53% (interquartile range 5.83-10.91%) before treatment to 6.17% (2.15-6.81%) after six to nine months of treatment (p=0.021). Inversely, IL10 positive T cells increased from a median of 0.65% (interquartile range 0.6-0.93%) to a median of 1. 3% (1.22%-1.58%) after six to nine months of treatment (p=0.009). No significant change in the percentage of INFgamma positive T cells and a small decrease of IL4 positive T cells during treatment were observed. The percentage of IL4 positive CD4(+) T cells before treatment correlated with disease activity after six to nine months (r= -0.7066; p=0.05). CONCLUSIONS: During treatment of RA with MTX the percentage of TNFalpha producing T cells decreases whereas that of IL10 producing T cells increases. This may affect macrophage activation and, therefore, may represent a regulatory mechanism relevant to disease remission. Furthermore, the percentage of IL4 positive CD4(+) T cells at disease onset may be a useful prognostic marker. PMID- 10733483 TI - Urokinase-mediated fibrinolysis in the synovial fluid of rheumatoid arthritis patients may be affected by the inactivation of single chain urokinase type plasminogen activator by thrombin. AB - BACKGROUND: Excessive fibrin deposition within the inflamed joints of rheumatoid arthritis (RA) patients suggests that local fibrinolysis is inefficient, which seems to be in contrast with the observed increased levels of urokinase type plasminogen activator (u-PA). Thrombin-mediated inactivation of single chain u-PA (scu-PA) into an inactive form called thrombin-cleaved two chain u-PA (tcu-PA/T) may provide a possible explanation for this contradiction. AIM: To assess the occurrence of tcu-PA/T in the synovial fluid of patients with RA and with osteoarthritis (OA), and in the synovial fluid of controls to find support for thrombin-mediated inactivation of scu-PA in RA. METHODS: Levels of scu-PA and tcu PA/T were measured in the synovial fluid of 20 RA patients, nine OA patients and 14 controls using sensitive bioimmunoassays. Total urokinase antigen was quantified by a urokinase ELISA. RESULTS: tcu-PA/T was found in the synovial fluid of all RA and OA patients. Only in seven of 14 control samples, levels of tcu-PA/T could be measured above the detection limit of the assay (0.2 ng/ml). The concentrations of tcu-PA/T, scu-PA and u-PA:Ag were significantly higher in the synovial fluid of the RA and OA patients as compared with the controls, while the RA patients had significantly higher levels of tcu-PA/T and u-PA:Ag than the OA patients. In RA, tcu-PA/T seemed to account for more than 40% of total urokinase antigen, while the contribution of tcu-PA/T to total urokinase antigen was only minor in OA and the controls (9.0% and 6.6%, respectively). CONCLUSION: A significant part of the high total urokinase antigen in the synovial fluid of RA patients can be attributed to tcu-PA/T, implying that a large amount of scu-PA is not available for fibrinolysis because of its inactivation by thrombin. Thus, thrombin may promote the inflammation process in RA by inhibiting the fibrinolytic system and preventing the removal of fibrin. PMID- 10733484 TI - A missense mutation in the heavy subunit of gamma-glutamylcysteine synthetase gene causes hemolytic anemia. AB - gamma-Glutamylcysteine synthetase (GCS) catalyzes the initial and rate-limiting step in the biosynthesis of glutathione. gamma-GCS consists of a heavy and a light subunit encoded by separate genes. Hereditary deficiency of GCS has been reported in 6 patients with hemolytic anemia and low erythrocyte levels of glutathione and gamma-glutamylcysteine. In addition, 2 patients also had generalized aminoaciduria and developed neurologic symptoms. We have examined a Dutch kindred with 1 suspected case of GCS deficiency. The proband was a 68-year old woman with a history of transient jaundice and compensated hemolytic anemia. One of her grandchildren was also GCS deficient; he was 11 years old and had a history of neonatal jaundice. The enzyme defect was confirmed and GCS activity was found to be less than 2% of normal in the erythrocytes of both patients. The complementary DNA (cDNA) for the heavy subunit of GCS was sequenced in these patients and in several members of the family. The proband and her GCS- deficient grandson were identified as homozygous for a 473C-->T substitution, changing codon 158 from CCC for proline into CTC for leucine. Several family members with half-normal GCS activity in their erythrocytes were heterozygous for the mutation. PMID- 10733485 TI - Selective elimination of leukemic CD34(+) progenitor cells by cytotoxic T lymphocytes specific for WT1. AB - Hematologic malignancies such as acute and chronic myeloid leukemia are characterized by the malignant transformation of immature CD34(+) progenitor cells. Transformation is associated with elevated expression of the Wilm's tumor gene encoded transcription factor (WT1). Here we demonstrate that WT1 can serve as a target for cytotoxic T lymphocytes (CTL) with exquisite specificity for leukemic progenitor cells. HLA-A0201- restricted CTL specific for WT1 kill leukemia cell lines and inhibit colony formation by transformed CD34(+) progenitor cells isolated from patients with chronic myeloid leukemia (CML), whereas colony formation by normal CD34(+) progenitor cells is unaffected. Thus, the tissue-specific transcription factor WT1 is an ideal target for CTL-mediated purging of leukemic progenitor cells in vitro and for antigen-specific therapy of leukemia and other WT1-expressing malignancies in vivo. PMID- 10733486 TI - Molecular cloning of a novel type 1 cytokine receptor similar to the common gamma chain. AB - In a complementary DNA (cDNA) screening of murine Th2-skewed lymphocytes with our recently developed signal sequence trap method termed SST-REX, a novel type 1 cytokine receptor, Delta1 (delta1), was identified. Although delta1 is ubiquitously expressed in multiple tissues, the expression level is higher in Th2 skewed lymphocytes than in Th1-skewed ones. The delta1 cDNA encodes a 359-amino acid type 1 membrane protein. The extracellular domain of 206 amino acids showed 24% identity with the murine common gamma receptor that is shared among the receptors for interleukin(IL)-2, IL-4, IL-7, IL-9, and IL-15. The membrane proximal region of delta1 includes a box1 motif, which is important for association with Janus kinases (JAKs), and showed a significant homology with that of the mouse erythropoietin receptor (EPOR). A box2 motif was also found in close proximity to the box1 region. Dimerization of the cytoplasmic region of delta1 alone did not transduce proliferative signals in IL-3-dependent cell lines. However, the membrane-proximal region of delta1 could substitute for that of human EPOR in transmitting proliferative signals and activating JAK2. These results suggest that delta1 is a subunit of cytokine receptor that may be involved in multiple receptor systems and play a regulatory role in the immune system and hematopoiesis. PMID- 10733487 TI - Transformation of mycosis fungoides: clinicopathological and prognostic features of 45 cases. French Study Group of Cutaneious Lymphomas. AB - The course of mycosis fungoides (MF) is indolent except when transformation to a large T-cell lymphoma occurs. The diagnosis of transformed MF (T-MF) relies on the presence of more than 25% of large cells on biopsy of an MF lesion. We analyzed 45 patients with T-MF recorded by the French Study Group on Cutaneous Lymphomas to better determine clinicopathological features of MF transformation and to analyze their impact on prognosis. Median time from diagnosis of MF to transformation was 6.5 years. Extracutaneous progression was present in 20 patients. Mean survival from transformation to death was 22 months. In univariate analysis, only an extracutaneous progression was associated with a worse prognosis (5-year actuarial survival: 7.8% versus 32%). Neither sex, age, clinical and skin disease stage at transformation, transformation speed, nor percentage of large cells or CD30 expression (14 of 45) had a prognostic value. When performing multivariate analysis, age (at least 60 years), and extracutaneous spreading were found to be associated with a poor prognosis. There was no difference between survival curves of patients with T-MF and with pleomorphic large T-cell CD30- lymphomas. The main diagnostic pitfall was "histiocytic-rich" MF, requiring CD68 staining for the diagnosis of T-MF. Out of 45 patients, 6 presented an histologic transformation before clinical progression, suggesting that an early histopathological diagnosis may be performed by histological follow-up. The prognostic value of such early histopathological diagnosis must be confirmed by prospective studies. PMID- 10733488 TI - Unrelated donor marrow transplantation for chronic myelogenous leukemia: 9 years' experience of the national marrow donor program. AB - Over a period of 8.5 years (February 1988 to October 1996), 1423 patients with chronic myelogenous leukemia (CML) underwent unrelated donor (URD) bone marrow transplants (BMTs) facilitated by the National Marrow Donor Program (NMDP) at 85 transplant centers. One hundred thirty-seven evaluable (9.9%) patients failed to engraft, and an additional 83 (6.6%) evaluable patients experienced late graft failure. Grade III/IV acute graft-versus-host disease (GVHD) developed in 33% of patients (95% confidence interval [CI], 30%-36%). The incidence of extensive chronic GVHD was 60% (95% CI, 56%-63%) at 2 years. Only 5.7% of patients (95% CI, 3.6%-7.8%) transplanted in chronic phase developed hematologic relapse at 3 years. Several factors were independently associated with improved disease-free survival (DFS), including transplant in chronic phase, transplant within 1 year of diagnosis, younger recipient age, a cytomegalovirus seronegative recipient, and development of no or mild acute GVHD. The combined effect of these factors on outcome is manifest in a subset (n = 157) of young (less than 35 years), chronic phase patients transplanted within 1 year of diagnosis using HLA-matched donors who had 63% (95% CI, 53%-73%) DFS at 3 years. URD BMT therapy for CML is both feasible and effective with more frequent and more rapid identification of suitable donors. Early URD transplant during chronic phase yields good results and should be considered in CML patients otherwise eligible for transplant but without a suitable related donor. PMID- 10733489 TI - Splenectomy in myelofibrosis with myeloid metaplasia: a single-institution experience with 223 patients. AB - In a 20-year period, 223 patients (median age, 64.8 years) with myelofibrosis with myeloid metaplasia (MMM) had therapeutic splenectomy at our institution. Primary indications for surgery were transfusion-dependent anemia (45.3%), symptomatic splenomegaly (39. 0%), portal hypertension (10.8%), and severe thrombocytopenia (4.9%). Operative mortality and morbidity rates were 9% and 31%, respectively. The 203 survivors of surgery had a median postsplenectomy survival time (PSS) of 27 months (range, 0-155). Among preoperative variables, thrombocytopenia (platelet count less than 100 x 10(9/)L) and nonhypercellular bone marrow were identified as independent risk factors for decreased PSS. Durable remissions in constitutional symptoms, transfusion-dependent anemia, portal hypertension, and severe thrombocytopenia were achieved in 67%, 23%, 50%, and 0% of the patients, respectively. Histologic or cytogenetic features of bone marrow obtained before splenectomy did not predict a response in cytopenias. After splenectomy, substantial enlargement of the liver and marked thrombocytosis occurred in 16.1% and 22.0% of the patients, respectively. The thrombocytosis was associated with an increased risk of perioperative thrombosis and decreased PSS. The rate of blast transformation (BT) was 16.3%, and the risk of BT was higher in the presence of increased spleen mass and preoperative thrombocytopenia. However, the PSS of patients with BT was not significantly different from that of patients without BT. We conclude that presplenectomy thrombocytopenia in MMM may be a surrogate for advanced disease and is associated with an increased risk of BT and inferior PSS. However, the development of BT after splenectomy may not affect overall survival and does not undermine the palliative role of the procedure for the other indications. PMID- 10733491 TI - Increased transplant-related morbidity and mortality in CMV-seropositive patients despite highly effective prevention of CMV disease after allogeneic T-cell depleted stem cell transplantation. AB - We evaluated the efficacy, toxicity, and outcome of preemptive ganciclovir (GCV) therapy in 80 cytomegalovirus (CMV)-seropositive patients allografted between 1991 and 1996 and compared their outcome to 35 seronegative patients allografted during the same period. Both cohorts were comparable with respect to diagnosis and distribution of high- versus standard-risk patients. All patients received a stem cell graft from an HLA-identical sibling donor, and grafts were partially depleted of T cells in 109 patients. Patients were monitored for CMV antigenemia by leukocyte expression of the CMV-pp65 antigen. Fifty-two periods of CMV reactivation occurring in 30 patients were treated preemptively with GCV. A favorable response was observed in 48 of 50 periods, and only 2 patients developed CMV disease: 1 with esophagitis and 1 with pneumonia. Ten of 30 treated patients developed GCV-related neutropenia (less than 0.5 x 10(9)/L), which was associated with a high bilirubin at the start of GCV therapy. Overall survival at 5 years was 64% in the CMV-seronegative cohort and 40% in the CMV-seropositive cohort (P =.01). Increased treatment-related mortality accounted for inferior survival. CMV seropositivity proved an independent risk factor for developing acute graft-versus-host disease, and acute graft-versus-host disease predicted for higher treatment-related mortality and worse overall survival in a time dependent analysis. We conclude that, although CMV disease can effectively be prevented by preemptive GCV therapy, CMV seropositivity remains a strong adverse risk factor for survival following partial T-cell-depleted allogeneic stem cell transplantation. PMID- 10733490 TI - Engraftment, clinical, and molecular follow-up of patients with multiple myeloma who were reinfused with highly purified CD34+ cells to support single or tandem high-dose chemotherapy. AB - Eighty-two patients with advanced multiple myeloma (MM) were enrolled in 2 sequential clinical studies of 1 or 2 courses of myeloablative therapy with stem cell support. Conditioning regimens consisted of high-dose melphalan (MEL) with or without total body irradiation (TX1 = 35) and MEL as the first preparative regimen, followed within 6 months by busulfan and melphalan (TX2 = 47). On the basis of adequate stem cell harvest, 31 patients (TX1 = 13; TX2 = 18) were transplanted with highly purified CD34+ cells. Positively selected stem cells did not adversely affect hematopoietic reconstitution compared with unmanipulated peripheral blood stem cell. Overall, the complete remission (CR) rate of evaluative patients was 13.8% and 41% for single and double autotransplant, respectively (P =.04). Moreover, 3 patients undergoing TX2 achieved molecular remission and 2 remain PCR-negative after 36 and 24 months from autograft. The median event-free survival (EFS) durations for TX1 and TX2 were 17 and 35 months, respectively (P =.03). Actuarial 3-year overall survival for patients treated with 1 or 2 transplants are 76% and 92%, respectively (P = NS). On multivariate analysis, superior EFS was associated with low beta2 microglobulin (beta2-M) level at diagnosis and TX2, whereas overall survival was correlated with beta2-M. Positive selection of CD34+ cells did not influence the achievement of clinical or molecular CR, as well as remission duration or survival of MM patients. Thus, whereas multiple cycles of high-dose therapy may be beneficial for patients with myeloma, the clinical impact of tumor cell purging remains highly questionable. PMID- 10733492 TI - Comparison of chemotherapy to radiotherapy as consolidation of complete or good partial response after six cycles of chemotherapy for patients with advanced Hodgkin's disease: results of the groupe d'etudes des lymphomes de l'Adulte H89 trial. AB - The treatment of advanced Hodgkin's disease (HD) with chemotherapy (CTx) alone or combined modality treatments has been controversial. In 1989, we designed a randomized study to compare 2 cycles of CTx to (sub)total nodal irradiation (RTx) as consolidation treatments for patients with stage IIIB/IV HD in complete remission (CR) or good partial response after 6 cycles of CTx. A total of 559 patients were randomized to receive 6 cycles of MOPP/ABV (mechlorethamine, vincristine, procarbazine, prednisone/Adriamycin [doxorubicin], bleomycin, vinblastine) hybrid (n = 266) or ABVPP (n = 267). After induction treatment, 418 patients could be evaluated for the consolidation phase. With a median follow-up of 48 months, the 5-year disease-free survival estimates were 80% for 8 cycles of MOPP/ABV, 82% for 6 cycles of MOPP/ABV plus RTx, 68% for 8 cycles of ABVPP, and 75% for 6 cycles of ABVPP plus RTx (P =.01). The 5-year disease-free survival estimates did not differ between CTx and RTx, 74% and 79%, respectively (P =.07). After MOPP/ABV, the 5-year overall survival estimates did not differ between CTx and RTx, 85% and 88%, respectively (P =.2). After ABVPP, the 5-year survival estimates were 94% for CTx and 78% for RTx (P =.002). These results showed that RTx was not superior to CTx consolidation after doxorubicin-induced CR for patients with advanced HD. Because of the uncertainty of obtaining a prolonged second remission for patients relapsing after CTx and RTx and the possible long term effects of RTx, we prefer 8 cycles of CTx as standard treatment when a CR has been achieved after 6 cycles. PMID- 10733493 TI - Significance of cyclin D1 overexpression for the diagnosis of mantle cell lymphoma: a clinicopathologic comparison of cyclin D1-positive MCL and cyclin D1 negative MCL-like B-cell lymphoma. AB - Mantle cell lymphoma (MCL) is a distinct clinicopathologic entity of non Hodgkin's lymphoma, characterized by a monotonous proliferation of small to medium-sized lymphocytes with co-expression of CD5 and CD20, an aggressive and incurable clinical course, and frequent t(11;14)(q13;q32) translocation. We examined 151 cases of lymphoma with MCL morphology from a viewpoint of cyclin D1 overexpression, which is now easily detectable by immunohistochemistry. 128 cases (85%) showed positive nuclear staining for cyclin D1, while the remaining 23 (15%) were negative. Except for cyclin D1 immunohistochemistry, current diagnostic methods, including morphological and phenotypical examinations, could not make this distinction. Although both the cyclin D1-positive and -negative groups were characterized by male predominance, advanced stages of the disease, frequent extranodal involvement, and low CD23 reactivity, the cyclin D1-positive group showed a higher age distribution (P =.04), larger cell size (P =.02), higher mitotic index (P =.01), more frequent gastrointestinal involvement (P =.05), higher international prognostic index score (P =.05), and lower p27(KIP1) expression (P <.0001). Of particular interest is that cyclin D1-positive MCL showed significantly worse survival than cyclin D1-negative lymphoma (5-year survival: 30% versus 86%, P =.0002), which was confirmed by multivariate analysis to be independent of other risk factors. These data suggest that cyclin D1 positive and -negative groups may represent different entities and that the former closely fits the characteristics of classical, typical MCL. We therefore propose that cyclin D1-positivity should be included as one of the standard criteria for MCL, and that innovative therapies for this incurable disease should be explored on the basis of the new criteria. PMID- 10733494 TI - Mapping of a syndrome of X-linked thrombocytopenia with Thalassemia to band Xp11 12: further evidence of genetic heterogeneity of X-linked thrombocytopenia. AB - X-linked thrombocytopenia with thalassemia (XLTT; Online Mendelian Inheritance in Man [OMIM] accession number 314050) is a rare disorder characterized by thrombocytopenia, platelet dysfunction, splenomegaly, reticulocytosis, and unbalanced hemoglobin chain synthesis. In a 4-generation family, the gene responsible for XLTT was mapped to the X chromosome, short arm, bands 11-12 (band Xp11-12). The maximum lod score possible in this family, 2.39, was obtained for markers DXS8054 and DXS1003, at a recombination fraction of 0. Recombination events observed for XLTT and markers DXS8080 and DXS8023 or DXS991 define a critical region that is less than or equal to 7.65 KcM and contains the gene responsible for the Wiskott-Aldrich syndrome (WAS; OMIM accession number 301000) and its allelic variant X-linked thrombocytopenia (XLT; OMIM accession number 313900). Manifestations of WAS include thrombocytopenia, eczema, and immunodeficiency. In WAS/XLT the platelets are usually small, and bleeding is proportional to the degree of thrombocytopenia. In contrast, in XLTT the platelet morphology is normal, and the bleeding time is disproportionately prolonged. In this study no alteration in the WAS gene was detected by Northern blot or Western blot analysis, flow cytometry, or complimentary DNA dideoxynucleotide fingerprinting or sequencing. As has been reported for WAS and some cases of XLT, almost total inactivation of the XLTT gene-bearing X chromosome was observed in granulocytes and peripheral blood mononuclear cells from 1 asymptomatic obligate carrier. The XLTT carrier previously found to have an elevated alpha:beta hemoglobin chain ratio had a skewed, but not clonal, X-inactivation pattern favoring activity of the abnormal allele. Clinical differences and results of the mutation analyses make it very unlikely that XLTT is another allelic variant of WAS/XLT and strongly suggest that X-linked thrombocytopenia mapping to band Xp11 12 is a genetically heterogeneous disorder. PMID- 10733495 TI - Pharmacologic doses of granulocyte colony-stimulating factor affect cytokine production by lymphocytes in vitro and in vivo. AB - Peripheral blood stem cell (PBSC) transplantation is successful in improving engraftment without increasing acute graft-versus-host disease (GVHD), despite much larger numbers of T cells in unmanipulated PBSCs than in bone marrow grafts. In mouse models and retrospective human studies, granulocyte colony-stimulating factor (G-CSF) therapy has been associated with less acute GVHD. We studied the effect of G-CSF on interferon (IFN)-gamma and IL-4 expression in CD4(+) lymphocytes. CD4(+) cells co-cultivated with G-CSF and stimulated with PHA or CD3 monoclonal antibodies showed significant decreases in IFN-gamma and increases in IL-4 expression (n = 13; P <. 01). G-CSF appeared to have a direct effect on CD4(+) cells independent of monocytes present in the culture because purified CD4(+) cells exposed to G-CSF, washed, and cocultivated with untreated monocytes demonstrated similar changes in IFN-gamma and IL-4 expression, whereas untreated CD4(+) cells cocultured with G-CSF-stimulated monocytes behaved as controls. We then studied peripheral blood mononuclear cells (PBMCs) from G-CSF-mobilized PBSC donors. When their PBMCs were cultured with PHA or CD3 monoclonal antibody, the percent of IFN-gamma-expressing cells decreased by a mean of 55% and 42%, respectively, whereas the percent of IL-4-containing cells increased by a mean of 39% and 58%, respectively, following G-CSF stimulation. Increased apoptosis of IFN-gamma-producing CD4(+) cells was not responsible for the shift in TH1/TH2 subsets. G-CSF-R mRNA was present in both CD4(+) and CD8(+) cells. These results suggest that G-CSF decreases IFN-gamma and increases IL-4 production in vitro and in vivo and likely modulates a balance between TH1 and TH2 cells, an effect that may be important in PBSC transplantation. PMID- 10733496 TI - Vascular endothelial growth factor synergistically enhances bone morphogenetic protein-4-dependent lymphohematopoietic cell generation from embryonic stem cells in vitro. AB - The totipotent mouse embryonic stem (ES) cell is known to differentiate into cells expressing the beta-globin gene when stimulated with bone morphogenetic protein (BMP)-4. Here, we demonstrate that BMP-4 is essential for generating both erythro-myeloid colony-forming cells (CFCs) and lymphoid (B and NK) progenitor cells from ES cells and that vascular endothelial growth factor (VEGF) synergizes with BMP-4. The CD45(+) myelomonocytic progenitors and Ter119(+) erythroid cells began to be detected with 0.5 ng/mL BMP-4, and their levels plateaued at approximately 2 ng/mL. VEGF alone weakly elevated the CD34(+) cell population though no lymphohematopoietic progenitors were induced. However, when combined with BMP-4, 2 to 20 ng/mL VEGF synergistically augmented the BMP-4-dependent generation of erythro-myeloid CFCs and lymphoid progenitors from ES cells, which were enriched in CD34(+) CD31(lo) and CD34(+) CD45(-)cell populations, respectively, in a dose-dependent manner. Furthermore, during the 7 days of in vitro differentiation, BMP-4 was required within the first 4 days, whereas VEGF was functional after the action of BMP-4 (in the last 3 days). Thus, VEGF is a synergistic enhancer for the BMP-4-dependent differentiation processes, and it seems to be achieved by the ordered action of the 2 factors. PMID- 10733497 TI - Expansion of hematopoietic stem cells in the developing liver of a mouse embryo. AB - The activity of hematopoietic stem cells in the developing liver of a C57BL/6 mouse embryo was quantified by a competitive repopulation assay. Different doses of fetal liver cells at days 11 to 18 of gestation were transplanted into irradiated mice together with 2 x 10(5) adult bone marrow cells. A long-term repopulation in myeloid-, B-cell, and T-cell lineage by fetal liver cells was evaluated at 20 weeks after transplantation. At day 12 of gestation multilineage repopulating activity was first detected in the liver as 50 repopulating units (RU) per liver. The number of RU per liver increased 10-fold and 33-fold by day 14 and day 16 of gestation, and decreased thereafter, suggesting a single wave of stem cell development in the fetal liver. A limiting dilution analysis revealed that the frequency of competitive repopulating units (CRU) in fetal liver cells at day 12 of gestation was similar to that at day 16 of gestation. Because of an increase of total fetal liver cell number, the absolute number of CRU per liver from days 12 to 16 of gestation increased 38-fold. Hence, the mean activity of stem cells (MAS) that is given by RU per CRU remained constant from days 12 to 16 of gestation. From these data we conclude that hematopoietic stem cells expand in the fetal liver maintaining their level of repopulating potential. PMID- 10733498 TI - Human megakaryocytes and platelets contain the estrogen receptor beta and androgen receptor (AR): testosterone regulates AR expression. AB - Gender differences in vascular thromboses are well known, and there is evidence that platelets may be involved in these differences and that sex hormones affect platelet function. We characterized the expression of the estrogen receptor alpha (ER alpha), estrogen receptor beta (ER beta), progesterone receptor (PR), and androgen receptor (AR) in the megakaryocyte lineage. Megakaryocytes generated ex vivo from normal human CD34(+) stem cells contained RNA for ER beta and AR, which increased with cell differentiation. Platelets and human erythroleukemia (HEL) cells also contained ER beta and AR transcripts. No ER alpha or PR messenger RNA or protein was detected in the megakaryocyte lineage. Immunofluorescence microscopy showed that ER beta protein was present in glycoprotein (GP) IIb(+) megakaryocytes and the HEL megakaryocytic cell line in a predominantly cytoplasmic location. AR showed a cytoplasmic and nuclear distribution in GPIIb(+) and GPIIb(-) cells derived from CD34(+) cells and in HEL cells. Western immunoblotting confirmed the presence of ER beta and AR in platelets. Megakaryocyte and HEL AR expression was up-regulated by 1, 5, and 10 nmol/L testosterone, but down-regulated by 100 nmol/L testosterone. These findings indicate a regulated ability of megakaryocytes to respond to testosterone and suggest a potential mechanism through which sex hormones may mediate gender differences in platelet function and thrombotic diseases. PMID- 10733499 TI - The alpha(E)C domain of human fibrinogen-420 is a stable and early plasmin cleavage product. AB - Human fibrinogen-420, (alpha(E)betagamma)(2), was isolated from plasma and evaluated for its ability to form clots and for its susceptibility to proteolysis. Clotting parameters, including cross-linking of subunit chains, of this subclass and of the more abundant fibrinogen-340 (alphabetagamma)(2), were found to be similar, suggesting little impact of the unique alpha(E)C domains of fibrinogen-420 on coagulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmic digestion patterns revealed production from fibrinogen-420 of the conventional fibrinogen degradation products, X, Y, D, and E, to be comparable to that from fibrinogen-340 in all respects except the presence of at least 2 additional cleavage products that were shown by Western blot analysis to contain the alpha(E)C domain. One was a stable fragment (alpha(E)CX) comigrating with a 34-kd yeast recombinant alpha(E)C domain, and the other was an apparent precursor. Their release occurred early, before that of fragments D and E. Two bands of the same mobility and antibody reactivity were found in Western blots of plasma collected from patients with myocardial infarction shortly after the initiation of thrombolytic therapy. PMID- 10733500 TI - Eicosanoid regulation of angiogenesis: role of endothelial arachidonate 12 lipoxygenase. AB - Angiogenesis, the formation of new capillaries from preexisting blood vessels, is a multistep, highly orchestrated process involving vessel sprouting, endothelial cell migration, proliferation, tube differentiation, and survival. Eicosanoids, arachidonic acid (AA)-derived metabolites, have potent biologic activities on vascular endothelial cells. Endothelial cells can synthesize various eicosanoids, including the 12-lipoxygenase (LOX) product 12(S)-hydroxyeicosatetraenoic acid (HETE). Here we demonstrate that endogenous 12-LOX is involved in endothelial cell angiogenic responses. First, the 12-LOX inhibitor, N-benzyl-N-hydroxy-5 phenylpentanamide (BHPP), reduced endothelial cell proliferation stimulated either by basic fibroblast growth factor (bFGF) or by vascular endothelial growth factor (VEGF). Second, 12-LOX inhibitors blocked VEGF-induced endothelial cell migration, and this blockage could be partially reversed by the addition of 12(S) HETE. Third, pretreatment of an angiogenic endothelial cell line, RV-ECT, with BHPP significantly inhibited the formation of tubelike/cordlike structures within Matrigel. Fourth, overexpression of 12-LOX in the CD4 endothelial cell line significantly stimulated cell migration and tube differentiation. In agreement with the critical role of 12-LOX in endothelial cell angiogenic responses in vitro, the 12-LOX inhibitor BHPP significantly reduced bFGF-induced angiogenesis in vivo using a Matrigel implantation bioassay. These findings demonstrate that AA metabolism in endothelial cells, especially the 12-LOX pathway, plays a critical role in angiogenesis. PMID- 10733501 TI - Phenotypic and functional characteristics of hematopoietic cell lineages in CD69 deficient mice. AB - AIM/CD69 is the earliest leukocyte activation antigen and is expressed mainly by activated T, B, and natural killer (NK) cells. It is also constitutively expressed by platelets, by bone marrow myeloid precursors, and by small subsets of resident lymphocytes in the secondary lymphoid tissues. The engagement of CD69 by specific antibodies induces intracellular signals, including Ca(++) flux, cytokine synthesis, and cell proliferation. To investigate the physiological relevance of CD69, we generated mice deficient in CD69 (CD69-/-) by gene targeting in embryonic stem cells. CD69 (-/-) mice showed largely normal hematopoietic cell development and normal T-cell subpopulations in thymus and periphery. Furthermore, studies of negative- and positive-thymocyte selection using a T-cell receptor transgenic model demonstrated that these processes were not altered in CD69 (-/-) mice. In addition, natural killer and cytotoxic T lymphocyte cells from CD69-deficient mice displayed cytotoxic activity similar to that of wild-type mice. Interestingly, B-cell development was affected in the absence of CD69. The B220(hi)IgM(neg) bone marrow pre-B cell compartment was augmented in CD69 (-/-) mice. In addition, the absence of CD69 led to a slight increase in immunoglobulin (Ig) G2a and IgM responses to immunization with T dependent and T-independent antigens. Nevertheless, CD69-deficient lymphocytes had a normal proliferative response to different T-cell and B-cell stimuli. Together, these observations indicate that CD69 plays a role in B-cell development and suggest that the putative stimulatory activity of this molecule on bone marrow-derived cells may be replaced in vivo by other signal transducing receptors. PMID- 10733502 TI - A novel nuclear phosphoprotein, GANP, is up-regulated in centrocytes of the germinal center and associated with MCM3, a protein essential for DNA replication. AB - Antigen (Ag) immunization induces formation of the germinal center (GC), with large, rapidly proliferating centroblasts in the dark zone, and small, nondividing centrocytes in the light zone. We identified a novel nuclear protein, GANP, that is up-regulated in centrocytes. We found that GANP was up-regulated in GC B cells of Peyer's patches in normal mice and in spleens from Ag-immunized mice. GANP-positive cells appeared in the light zone of the GC, with coexpression of the peanut agglutinin (PNA) (PNA)-positive B220-positive phenotype. The expression of GANP was strikingly correlated with GC formation because Bcl6 deficient mice did not show the up-regulation of GANP. GANP-positive cells were mostly surrounded by follicular dendritic cells. Stimulation with anti-micro and anti-CD40 induced up-regulation of ganp messenger RNA as well as GANP protein in B220-positive B cells in vitro. GANP is a 210-kd protein localized in both the cytoplasm and nuclei, with a homologous region to Map80 that is associated with MCM3, a protein essential for DNA replication. Remarkably, GANP is associated with MCM3 in B cells and MCM3 is also up-regulated in the GC area. These results suggest that the up-regulation of GANP might participate in the development of Ag driven B cells in GCs through its interaction with MCM3. PMID- 10733503 TI - Interferon-gamma-induced membrane PAF-receptor expression confers tumor cell susceptibility to NK perforin-dependent lysis. AB - Perforin is known to display a membranolytic activity on tumor cells. Nevertheless, perforin release during natural killer (NK)-cell activation is not sufficient to induce membrane target-cell damage. On the basis of the ability of perforin to interact with phospholipids containing a choline phosphate headgroup, we identify the platelet-activating factor (PAF) and its membrane receptor as crucial components in tumor cell killing activity of human resting NK cells. We demonstrate for the first time that upon activation, naive NK cells release the choline phosphate-containing lysolipid PAF, which binds to perforin and acts as an agonist on perforin-induced membrane damage. PAF is known to incorporate cell membranes using a specific receptor. Here we show that interferon-gamma (IFN gamma) secreted from activated NK cells ends in PAF-receptor expression on perforin-sensitive K562 cells but not on perforin-resistant Daudi cells. In order to prove the capacity of PAF to interact simultaneously with its membrane PAF receptor and with perforin, we successfully co-purified the 3 components in the presence of bridging PAF molecules. The functional activity of this complex was further examined. The aim was to determine whether membrane PAF-receptor expression on tumor cells, driven to express this receptor, could render them sensitive to the perforin lytic pathway. The results confirmed that transfection of the PAF-receptor complementary DNA into major histocompatibility complex class I and Fas-receptor negative tumor cells restored susceptibility to naive NK cells and perforin attack. Failure of IFN-gamma to induce membrane PAF receptor constitutes the first described mechanism for tumor cells to resist the perforin lytic pathway. PMID- 10733504 TI - Transgenic expression of granulocyte-macrophage colony-stimulating factor induces the differentiation and activation of a novel dendritic cell population in the lung. AB - The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the differentiation of dendritic cells (DCs) during pulmonary viral infection was investigated by using a mouse model of GM-CSF transgene expression established with an adenoviral vector (AdGM-CSF). GM-CSF gene transfer resulted in increased levels of GM-CSF in the lung, which peaked at day 4 and remained increased up to day 19. A striking cellular response composed predominantly of macrophage-like cells was observed in the lung receiving AdGM-CSF but not control vector. By FACS analysis, the majority of these cells were identified at an early time point as macrophages and later as mature/activated myeloid DCs characterized by CD11b(bright), CD11c(bright), MHC class II(bright), and B7.1(bright). In contrast, GM-CSF had a weak effect on a small DC population that was found present in normal lung and was characterized by CD11c(bright) and CD11b(low). By immunohistochemistry staining for MHC II, the majority of activated antigen presenting cells were localized to the airway epithelium and peribronchial/perivascular areas in the lung. A concurrently enhanced Th1 immune response was observed under these conditions. The number of CD4 and CD8 T cells was markedly increased in the lung expressing GM-CSF, accompanied by increased release of interferon (IFN)gamma in the lung. Furthermore, lymphocytes isolated from either lung parenchyma or local draining lymph nodes of these mice but not the control mice released large amounts of IFNgamma on adenoviral antigen stimulation in vitro. These findings reveal that GM-CSF promotes the differentiation and activation of a myeloid DC population primarily by acting on macrophages during pulmonary immune responses. PMID- 10733505 TI - In vitro growth inhibition of a broad spectrum of tumor cell lines by activated human dendritic cells. AB - Dendritic cells (DCs) are critical subsets of leukocytes providing antigen presentation for initiation of humoral and cellular immune responses. Their role as effector cells in tumor resistance, however, is less known. We report here that human DCs generated by culturing plastic-adherent peripheral blood monocytes in the presence of granulocyte-monocyte colony-stimulating factor (GM-CSF) and interleukin-4 have potent growth-inhibition activity in vitro on a wide spectrum of human tumor lines of different tissue origin. Proinflammatory stimuli lipopolysaccharide (LPS) and interferon-gamma, but not tumor necrosis factor alpha and CD40 signaling, can further enhance DC-mediated inhibition of tumor growth. The growth inhibition requires contact between DCs and tumor cells while LPS treatment enhances the antitumor activity in DC culture supernatants. Our results suggest that in addition to their predominant role as regulatory cells, activated DCs are also potential effector cells in tumor immunity. PMID- 10733506 TI - Granule exocytosis, and not the fas/fas ligand system, is the main pathway of cytotoxicity mediated by alloantigen-specific CD4(+) as well as CD8(+) cytotoxic T lymphocytes in humans. AB - We investigated the cytotoxicity mechanisms of alloantigen-specific human CD4(+) and CD8(+) cytotoxic T lymphocytes (CTLs) using cells from family members with the Fas gene mutation. Alloantigen-specific CD4(+) and CD8(+) CTL bulk lines and clones were generated from 2 individuals by stimulation of their peripheral blood lymphocytes with allogeneic Fas(-/-) or Fas(+/-) cell lines that were established from B-lymphocytes of a patient with Fas deficiency and her mother, respectively. Both CD4(+) and CD8(+) CTL bulk lines and clones directed against allogeneic HLA antigens exerted cytotoxicity against Fas(-/-) and Fas(+/-) cells to almost the same degree. The cytotoxicity of CD4(+) and CD8(+) CTLs appeared to be Ca(2+) dependent and was completely inhibited by concanamycin A, an inhibitor of perforin-mediated cytotoxicity. Messenger RNAs for the major mediators of CTL cytotoxicity, Fas ligand, perforin, and granzyme B were all detected in these CD4(+) CTLs with the use of the reverse transcriptase polymerase chain reaction. The majority of CD4(+) CTL clones that showed Fas-independent cytotoxicity were T(H)0, as determined by their cytokine production profile. These data, obtained with the use of a novel experimental system, clearly show that the main pathway of cytotoxicity mediated by alloantigen-specific human CD4(+) as well as by CD8(+) CTLs is granule exocytosis, and not the Fas/Fas ligand system. PMID- 10733507 TI - Retroviral transfer of the hENT2 nucleoside transporter cDNA confers broad spectrum antifolate resistance in murine bone marrow cells. AB - Antifolate drugs such as methotrexate are commonly used in cancer chemotherapy. It may be possible to increase the antitumor activity of antifolates by the coadministration of drugs that inhibit nucleoside transport, thereby blocking the capacity of tumor cells to salvage nucleotide precursors. An important limitation of this approach is severe myelosuppression caused by many of these drug combinations. For this reason, we have developed a gene therapy strategy to protect bone marrow cells against combined treatment with antifolates and nitrobenzylmercaptopurine riboside (NBMPR), a potent inhibitor of the es nucleoside transporter. A retroviral vector (MeiIRG) was constructed that expressed the NBMPR-insensitive ei transporter, hypothesizing that transduced bone marrow cells would survive drug treatment because of the preservation of nucleoside salvage pathways. In vitro clonogenic assays confirmed that the MeiIRG vector did protect myeloid progenitors against the toxic effects of 3 different antifolates when each was combined with NBMPR. On testing this system in vivo, decreased myelosuppression was observed in mice transplanted with MeiIRG transduced bone marrow cells and subsequently treated with trimetrexate and NBMPR P. In these mice, significant increases were noted in absolute neutrophil count nadirs, reticulocyte indices, and the numbers of myeloid progenitors in the bone marrow. Furthermore, a survival advantage was associated with transfer of the MeiIRG vector, indicating that significant dose intensification was possible with this approach. In summary, the MeiIRG vector can decrease the toxicity associated with the combined use of antifolates and NBMPR-P and thereby may provide a strategy for simultaneously sensitizing tumor cells while protecting hematopoietic cells. PMID- 10733508 TI - Cell cycle regulatory gene abnormalities are important determinants of leukemogenesis and disease biology in adult acute lymphoblastic leukemia. AB - To test the hypothesis that cell cycle regulatory gene abnormalities are determinants of clinical outcome in adult acute lymphoblastic leukemia (ALL), we screened lymphoblasts from patients on a Southwest Oncology Group protocol for abnormalities of the genes, retinoblastoma (Rb), p53, p15(INK4B), and p16(INK4A). Aberrant expression occurred in 33 (85%) patients in the following frequencies: Rb, 51%; p16(INK4A), 41%; p53, 26%. Thirteen patients (33%) had abnormalities in 2 or more genes. Outcomes were compared in patients with 0 to 1 abnormality versus patients with multiple abnormalities. The 2 groups did not differ in a large number of clinical and laboratory characteristics. The CR rates for patients with 0 to 1 and multiple abnormalities were similar (69% and 54%, respectively). Patients with 0 to 1 abnormality had a median survival time of 25 months (n = 26; 95% CI, 13-46 months) versus 8 months (n = 13; 95% CI, 4-12 months) for those with multiple abnormalities (P <.01). Stem cells (CD34+lin-) were isolated from adult ALL bone marrows and tested for p16(INK4A) expression by immunocytochemistry. In 3 of 5 patients lymphoblasts and sorted stem cells lacked p16(INK4A) expression. In 2 other patients only 50% of sorted stem cells expressed p16(INK4A). By contrast, p16 expression was present in the CD34+ lin- compartment in 95% (median) of 9 patients whose lymphoblasts expressed p16(INK4A). Therefore, cell cycle regulatory gene abnormalities are frequently present in adult ALL lymphoblasts, and they may be important determinants of disease outcome. The presence of these abnormalities in the stem compartment suggests that they contribute to leukemogenesis. Eradication of the stem cell subset harboring these abnormalities may be important to achieve cure. PMID- 10733509 TI - Delineation of a minimal interval and identification of 9 candidates for a tumor suppressor gene in malignant myeloid disorders on 5q31. AB - Interstitial deletion or loss of chromosome 5 is frequent in malignant myeloid disorders, including myelodysplasia (MDS) and acute myeloid leukemia (AML), suggesting the presence of a tumor suppressor gene. Loss of heterozygosity (LOH) analysis was used to define a minimal deletion interval for this gene. Polymorphic markers on 5q31 were identified using a high-resolution physical and radiation hybrid breakpoint map and applied to a patient with AML with a subcytogenetic deletion of 5q. By comparing the DNA from leukemic cells to buccal mucosa cells, LOH was detected with markers D5S476 and D5S1372 with retention of flanking markers D5S500 to D5S594. The D5S500-D5S594 interval, which covers approximately 700 kb, thus represents a minimal localization for the tumor suppressor gene. Further refinement of the physical map enabled the specification of 9 transcription units within the encompassing radiation hybrid bins and 7 in flanking bins. The 9 candidates include genes CDC25, HSPA9, EGR1, CTNNA1, and 5 unknown ESTs. Reverse-transcription polymerase chain reaction confirms that all of them are expressed in normal human bone marrow CD34(+) cells and in AML cell lines and thus represent likely candidates for the MDS-AML tumor suppressor gene at 5q31. PMID- 10733510 TI - HMBA induces activation of a caspase-independent cell death pathway to overcome P glycoprotein-mediated multidrug resistance. AB - Multidrug resistance (MDR) is often characterized by the expression of P glycoprotein (P-gp), a 170-kd ATP-dependent drug efflux protein. As well as effluxing xenotoxins, functional P-gp can confer resistance to caspase-dependent apoptosis induced by a range of different stimuli, including Fas ligand, tumor necrosis factor, UV irradiation, and serum starvation. However, P-gp-positive cells remain sensitive to caspase-independent death induced by cytotoxic T-cell granule proteins, perforin, and granzyme B. It is, therefore, possible that agents that induce cell death in a caspase-independent manner might circumvent P gp-mediated MDR. We demonstrated here that hexamethylene bisacetamide (HMBA) induced equivalent caspase-independent cell death in both P-gp-positive and negative cell lines at concentrations of 10 mmol/L and above. The HMBA-induced death pathway was marked by release of cytochrome c from the mitochondria and reduction of Bcl-2 protein levels. In addition, we show that functional P-gp specifically inhibits the activation of particular caspases, such as caspases-8 and -3, whereas others, such as caspase-9, remain unaffected. These studies greatly enhance our understanding of the molecular cell death events that can be regulated by functional P-gp and highlight the potential clinical use of drugs that function via a caspase-independent pathway for the treatment of MDR tumors. PMID- 10733511 TI - Variant genotypes of FcgammaRIIIA influence the development of Kaposi's sarcoma in HIV-infected men. AB - Disturbances in inflammatory cytokine production and immune regulation coupled with human herpesvirus-8 (HHV-8) infection underlie the current understanding of the pathogenesis of Kaposi's sarcoma (KS), the most common HIV-associated malignancy. The low affinity Fc gamma receptors (FcgammaR) for IgG link humoral and cellular immunity by mediating interaction between antibodies and effector cells, such as phagocytes and natural killer cells. We examined the frequency of polymorphic forms of the low affinity FcgammaRs, FcgammaRIIA, FcgammaRIIIA, and FcgammaRIIIB in 2 cohorts of HIV-infected men with KS and found that the FcgammaRIIIA genotype exerts a significant influence on susceptibility to or protection from KS. The FF genotype was underrepresented in patients with KS, whereas the VF genotype was associated with development of KS. A similar association was observed between FcgammaRIIIA genotypes and HHV-8 seropositivity. These observations suggest a possible role for FcgammaRIIIA in the development of KS during HIV infection. PMID- 10733512 TI - Butyrate-induced erythroid differentiation of human K562 leukemia cells involves inhibition of ERK and activation of p38 MAP kinase pathways. AB - Butyrate induces cytodifferentiation in many tumor cells of different origin, suggesting that an as yet unidentified common mechanism inherent to malignant cells is the target of butyrate action. This study determined the role of different mitogen-activated protein (MAP) kinase signal transduction pathways in butyrate-induced erythroid differentiation of K562 human leukemia cells. Using a panel of anti-ERK, JNK, and p38 phosphospecific antibodies, the study showed that phosphorylation of ERK and JNK is decreased following treatment of cells with butyrate, whereas phosphorylation of p38 is increased. In contrast, a K562 subline defective in butyrate-mediated induction of erythroid differentiation did not reveal these changes in phosphorylation patterns. Inhibition of ERK activity by UO126 induces erythroid differentiation and acts synergistically with butyrate on hemoglobin synthesis and inhibition of cell proliferation, whereas inhibition of p38 activity by SB203580 completely abolished induction of hemoglobin expression by butyrate. Taken together, our data suggest a model in which butyrate induces erythroid differentiation of K562 cells by inhibition of ERK and activation of p38 signal transduction pathways. PMID- 10733513 TI - Bidirectional induction of the cognate receptor-ligand alpha4/VCAM-1 pair defines a novel mechanism of tumor intravasation. AB - Engagement of cell surface adhesion receptors with extracellular constituents and with cellular counter-receptors is crucial for the extravasation of blood-borne neoplastic cells and their seeding at distant sites; however, the early events of tumor dissemination-ie, the intravasation step(s)-have been largely neglected. A role for the alpha4beta7 integrin was hypothesized to explain the high leukemogenicity exhibited by one (NQ22) among several T-cell lymphomas studied. To clarify the mechanisms of early aggressivity, the behavior of highly and poorly leukemogenic cell lines were compared in vitro. Cocultivation of physically separated leukemic cells with resting endothelial cells resulted in the up-regulation of VCAM-1 expression. NQ22 cells expressed mRNA of different cytokines that up-regulate VCAM-1 and at higher levels than cells of a nonaggressive lymphoma, and they migrated more efficiently through an activated endothelial cell layer. With the use of neutralizing antibodies against interferon-gamma, granulocyte macrophage colony-stimulating factor, and tumor necrosis factor (TNF)-alpha, it was determined that TNF-alpha is one of the soluble factors released by NQ22 cells involved in the up-regulation of VCAM-1. The finding that vascular cells within the early local growth were strongly positive for VCAM-1 indicated that NQ22 cells could activate endothelial cells also in vivo. Finally, cocultivation of preleukemic alpha4(-)NQ22 cells with TNF alpha-activated endothelial cells induced the expression of alpha4 integrins on the former cells. Reciprocal up-regulation and engagement of alpha4/VCAM-1 pairs determined the sequential transmigration and intravasation steps, and similar mechanisms might affect the aggressivity of human T lymphoblastic lymphomas. PMID- 10733514 TI - Regulation of polymorphonuclear leukocyte phagocytosis by myosin light chain kinase after activation of mitogen-activated protein kinase. AB - Polymorphonuclear leukocyte (PMNL) phagocytosis mediated by FcgammaRII proceeds in concert with activation of the mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase ERK2. We hypothesized that myosin light chain kinase (MLCK) could be phosphorylated and activated by ERK, thereby linking the MAP kinase pathway to the activation of cytoskeletal components required for pseudopod formation. To explore this potential linkage, PMNLs were challenged with antibody-coated erythrocytes (EIgG). Peak MLCK activity, 3-fold increased over controls, occurred at 4 to 6 minutes, corresponding with the peak rate of target ingestion and ERK2 activity. The MLCK inhibitor ML-7 (10 micromol/L) inhibited both phagocytosis and MLCK activity to basal values, thereby providing further support for the linkage between the functional response and the requirement for MLCK activation. The MAPK kinase (MEK) inhibitor PD098059 inhibited phagocytosis, MLCK activity, and ERK2 activity by 80% to 90%. To directly link ERK activation to MLCK activation, ERK2 was immunoprecipitated from PMNLs after EIgG ingestion. The isolated ERK2 was incubated with PMNL cytosol as a source of unactivated MLCK and with MLCK substrate; under these conditions ERK2 activated MLCK, resulting in phosphorylation of the MLCK substrate or of the myosin light chain itself. Because MLCK activates myosin, we evaluated the effect of directly inhibiting myosin adenosine triphosphatase using 2,3-butanedione monoxime (BDM) and found that phagocytosis was inhibited by more than 90% but MLCK activity remained unaffected. These results are consistent with the interpretation that MEK activates ERK, ERK2 then activates MLCK, and MLCK activates myosin. MLCK activation is a critical step in the cytoskeletal changes resulting in pseudopod formation. PMID- 10733515 TI - Polarization and interaction of adhesion molecules P-selectin glycoprotein ligand 1 and intercellular adhesion molecule 3 with moesin and ezrin in myeloid cells. AB - In response to the chemoattractants interleukin 8, C5a, N-formyl-methionyl-leucyl phenylalanine, and interleukin 15, adhesion molecules P-selectin glycoprotein ligand 1 (PSGL-1), intercellular adhesion molecule 3 (ICAM-3), CD43, and CD44 are redistributed to a newly formed uropod in human neutrophils. The adhesion molecules PSGL-1 and ICAM-3 were found to colocalize with the cytoskeletal protein moesin in the uropod of stimulated neutrophils. Interaction of PSGL-1 with moesin was shown in HL-60 cell lysates by isolating a complex with glutathione S-transferase fusions of the cytoplasmic domain of PSGL-1. Bands of 78- and 81-kd were identified as moesin and ezrin by Western blot analysis. ICAM 3 and moesin also coeluted from neutrophil lysates with an anti-ICAM-3 immunoaffinity assay. Direct interaction of the cytoplasmic domains of ICAM-3 and PSGL-1 with the amino-terminal domain of recombinant moesin was demonstrated by protein-protein binding assays. These results suggest that the redistribution of PSGL-1 and its association with intracellular molecules, including the ezrin radixin-moesin actin-binding proteins, regulate functions mediated by PSGL-1 in leukocytes stimulated by chemoattractants. PMID- 10733516 TI - Lysophosphatidic acid opens a Ca(++) channel in human erythrocytes. AB - Lysophosphatidic acid (LPA) is a lipid-derived second messenger that mobilizes many cells of the circulatory and vascular systems to assist in thrombus development and wound healing. LPA, however, has not been tested on human erythrocytes, largely because erythrocytes are considered to be both biologically inert and inactive in intercellular communication. To test this presumption, we have examined the impact of LPA on signaling reactions within the human red blood cell (RBC). Using both (45)Ca(++) and a Ca(++)-sensitive fluorescent probe (Fluo 3), we demonstrated that LPA, but not phosphatidic acid or the closely related sphingosine-1-phosphate, stimulates the influx of micromolar quantities of extracellular Ca(++) into fresh RBCs. This Ca(++) influx was shown to be channel mediated rather than leak promoted because the influx was observed at LPA concentrations too low to perturb membrane integrity, it was inhibited by P-type but not L-type Ca(++) channel blockers, it was inhibited by broad-specificity protein kinase inhibitors, and it was not induced by inactive analogues of LPA. Further characterization reveals that only approximately 25% of the RBCs participate in LPA-induced Ca(++) entry and that within this active population, Ca(++) gating occurs in an all-or-nothing manner. Because the stimulation of Ca(++) uptake occurs at LPA concentrations (1-5 micromol/L) known to occur near a developing thrombus and because the internalized Ca(++) can potentially promote prothrombic properties in the stimulated RBCs, we conclude that RBCs are not insensitive to signals released from other cells. PMID- 10733517 TI - Immunization of allogeneic bone marrow transplant recipients with tumor cell vaccines enhances graft-versus-tumor activity without exacerbating graft-versus host disease. AB - Allogeneic bone marrow transplantation (BMT) induces 2 closely associated immune responses: graft-versus-tumor (GVT) activity and graft-versus-host disease (GVHD). We have previously shown that pretransplant immunization of allogeneic BMT donors with a recipient-derived tumor cell vaccine increases both GVT activity and lethal GVHD because of the priming of donor T cells against putative minor histocompatibility antigens (mHAgs) on the tumor vaccine cells. The work reported here tested the hypothesis that tumor cell vaccination after BMT would produce an increase in GVT activity without exacerbating GVHD. C3H.SW donor bone marrow and splenocytes were transplanted into major histocompatibility complex matched, mHAg-mismatched C57BL/6 recipients. One month after BMT, recipients were immunized against either a C57BL/6 myeloid leukemia (C1498) or fibrosarcoma (205). Immunized recipients had a significant increase in survival and protection against tumor growth in both tumor models, and significant tumor protection was seen even in recipients with preexisting micrometastatic cancer before immunization. Alloreactivity appeared to contribute to the in vitro anti-tumor cytolytic activity, but in vivo immunity was tumor specific, and no exacerbation of GVHD was observed. Although the immunodominant mHAg B6(dom1) was shown to be expressed by all B6 tumors tested and was largely responsible for the alloreactivity resulting from tumor immunization of donors, the in vitro alloreactivity of immune recipients was more restricted and was not mediated by recognition of B6(dom1). In conclusion, post-transplant tumor immunization of allogeneic BMT recipients against either a leukemia or a solid tumor can increase GVT activity and survival without exacerbating GVHD. PMID- 10733518 TI - Blockade of CD134 (OX40)-CD134L interaction ameliorates lethal acute graft-versus host disease in a murine model of allogeneic bone marrow transplantation. AB - Expression of CD134 (OX40) on activated CD4(+) T cells has been observed in acute graft-versus-host disease (GVHD) after human and rat allogeneic bone marrow transplantation (BMT). We investigated the role of interaction between CD134 and CD134 ligand (CD134L) in a murine model of acute GVHD by using a newly established monoclonal antibody (mAb) against murine CD134L. Acute GVHD was induced by transfer of bone marrow cells and spleen cells into lethally irradiated recipients in a parent (C57BL/6) to first filial generation (C57BL/6 crossed with DBA/2) BMT. Administration of anti-CD134L mAb significantly reduced the lethality of acute GVHD and other manifestations of the disease, such as loss of body weight, hunched posture, diarrhea, and patchy alopecia. The survival rate 80 days after BMT in mice treated with the mAb was about 70%, whereas all mice treated with control antibodies died within 43 days. Histologic examinations revealed that inflammatory changes in target organs such as the liver, gut, and skin were also ameliorated in mice treated with the mAb compared with control mice. An in vitro assay of T-cell proliferation showed a marked hyporesponsiveness to host alloantigen in samples from mice treated with anti CD134L mAb. In addition, low levels of interferon gamma and transiently elevated levels of interleukin 4 and IgE in serum samples were found in mice treated with anti-CD134L mAb. These results suggest that CD134-CD134L interactions have an important role in the pathogenesis of acute GVHD. PMID- 10733519 TI - Human natural killer T cells acquire a memory-activated phenotype before birth. AB - Natural killer T (NKT) cells have recently been shown to play an important role in the rejection of malignant tumors and in the regulation of autoimmune diseases. Potent antitumor effects of the marine sponge-derived NKT cell ligand KRN7000 were observed in mice. Therefore, the elucidation of the natural ligand of NKT cells, which is currently still unknown, might have important clinical consequences for the treatment of cancer and autoimmune diseases. Analysis of cord blood mononuclear cells from healthy term infants demonstrated that in sharp contrast with the vast majority of cord blood lymphocytes, human NKT cells have already acquired a memory-activated phenotype before birth. This observation indicates that NKT cells encounter a natural ligand during fetal life and that this ligand is unlikely to be of microbial origin. PMID- 10733520 TI - Coinfection of multiple strains of Epstein-Barr virus in immunocompetent normal individuals: reassessment of the viral carrier state. AB - This study reassesses the occurrence of Epstein-Barr virus (EBV) diversity and coinfection versus dominance of a single viral strain within immunocompetent normal carriers. Polymerase chain reaction analysis of several different polymorphic loci of the EBV genome was performed on collections of peripheral blood mononuclear cells and multiple lymphoid and epithelial tissues of the same individuals. Autopsy specimens from 15 individuals who died of causes unrelated to EBV infection served as normal viral carriers. Unexpectedly, coinfection of multiple distinct strains of EBV of the same type (usually type 1) and less frequently of both types 1 and 2 was found to be very high within individual viral carriers. These data indicate that coinfection with multiple EBV strains is much more prevalent in normal carriers than previously appreciated, which in turn has direct implications on EBV persistence, host-viral interaction and pathogenesis. PMID- 10733521 TI - Genetic control of hematopoietic stem cell frequency in mice is mostly cell autonomous. AB - Previously we reported that the size of the stem cell compartment (measured as LTC-IC) is 11-fold greater in DBA/2 than in C57BL/6 mice, and we identified genes that regulate the size of the stem cell pool. To determine whether stem cell intrinsic or extrinsic events account for these differences, we created chimeras by aggregating morulae from the strains C57BL/6 and DBA/2. In these chimeras stem cells of both genotypes are exposed to a common mixed environment. Thus, an equalization of stem cell frequencies is expected if stem cell extrinsic effects dominate. Conversely, the parental ratio of LTC-IC should be preserved if the regulation is stem cell autonomous. For each chimera, individual LTC-IC were genotyped on the clonal levels by analyzing their progeny. We found that most of the difference that regulates the size of the stem cell compartment was intrinsic. PMID- 10733522 TI - X-linked genetic factors regulate hematopoietic stem-cell kinetics in females. AB - X inactivation makes females mosaics for 2 cell populations, usually with an approximate 1:1 distribution. Skewing of this distribution in peripheral blood cells is more common among elderly women. The depletion of hematopoietic stem cells followed by random differentiation may explain the acquired skewing with age. However, an animal model suggests that selection processes based on X-linked genetic factors are involved. We studied peripheral blood cells from 71 monozygotic twin pairs aged 73 to 93 years and from 33 centenarians, and we found that with age, 1 of the cell populations becomes predominant for most women. We also observed a strong tendency for the same cell line to become predominant in 2 co-twins. This suggests that X-linked genetic factors influence human hematopoietic stem cell kinetics. The fact that females have 2 cell lines with different potentials could be one of the reasons women live longer than men. PMID- 10733523 TI - Controlling TGF-beta signaling. PMID- 10733524 TI - Involvement of the MAP kinase cascade in resetting of the mammalian circadian clock. AB - Although the suprachiasmatic nucleus (SCN) is the major pacemaker in mammals, the peripheral cells or immortalized cells also contain a circadian clock. The SCN and the periphery may use different entraining signals-light and some humoral factors, respectively. We show that induction of the circadian oscillation of gene expression is triggered by TPA treatment of NIH-3T3 fibroblasts, which is inhibited by a MEK inhibitor, and that prolonged activation of the MAPK cascade is sufficient to trigger circadian gene expression. Therefore, such prolonged activation of MAPK by entraining cues may be involved in the resetting of the circadian clock. PMID- 10733525 TI - Essential function of Wnt-4 in mammary gland development downstream of progesterone signaling. AB - Female reproductive hormones control mammary gland morphogenesis. In the absence of the progesterone receptor (PR) from the mammary epithelium, ductal side branching fails to occur. We can overcome this defect by ectopic expression of the protooncogene Wnt-1. Transplantation of mammary epithelia from Wnt-4(-)/(-) mice shows that Wnt-4 has an essential role in side-branching early in pregnancy. PR and Wnt-4 mRNAs colocalize to the luminal compartment of the ductal epithelium. Progesterone induces Wnt-4 in mammary epithelial cells and is required for increased Wnt-4 expression during pregnancy. Thus, Wnt signaling is essential in mediating progesterone function during mammary gland morphogenesis. PMID- 10733526 TI - The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1. AB - The ordered progression through the cell cycle depends on regulating the abundance of several proteins through ubiquitin-mediated proteolysis. Degradation is precisely timed and specific. One key component of the degradation system, the anaphase promoting complex (APC), is a ubiquitin protein ligase. It is activated both during mitosis and late in mitosis/G(1), by the WD repeat proteins Cdc20 and Cdh1, respectively. These activators target distinct sets of substrates. Cdc20 APC requires a well-defined destruction box (D box), whereas Cdh1-APC confers a different and as yet unidentified specificity. We have determined the sequence specificity for Cdh1-APC using two assays, ubiquitination in a completely defined and purified system and degradation promoted by Cdh1-APC in Xenopus extracts. Cdc20 is itself a Cdh1-APC substrate. Vertebrate Cdc20 lacks a D box and therefore is recognized by Cdh1-APC through a different sequence. By analysis of Cdc20 as a substrate, we have identified a new recognition signal. This signal, composed of K-E-N, serves as a general targeting signal for Cdh1-APC. Like the D box, it is transposable to other proteins. Using the KEN box as a template, we have identified cell cycle genes Nek2 and B99 as additional Cdh1-APC substrates. Mutation in the KEN box stabilizes all three proteins against ubiquitination and degradation. PMID- 10733527 TI - mus304 encodes a novel DNA damage checkpoint protein required during Drosophila development. AB - Checkpoints block cell cycle progression in eukaryotic cells exposed to DNA damaging agents. We show that several Drosophila homologs of checkpoint genes, mei-41, grapes, and 14-3-3epsilon, regulate a DNA damage checkpoint in the developing eye. We have used this assay to show that the mutagen-sensitive gene mus304 is also required for this checkpoint. mus304 encodes a novel coiled-coil domain protein, which is targeted to the cytoplasm. Similar to mei-41, mus304 is required for chromosome break repair and for genomic stability. mus304 animals also exhibit three developmental defects, abnormal bristle morphology, decreased meiotic recombination, and arrested embryonic development. We suggest that these phenotypes reflect distinct developmental consequences of a single underlying checkpoint defect. Similar mechanisms may account for the puzzling array of symptoms observed in humans with mutations in the ATM tumor suppressor gene. PMID- 10733528 TI - CLOCK, an essential pacemaker component, controls expression of the circadian transcription factor DBP. AB - DBP, the founding member of the PAR leucine zipper transcription factor family, is expressed according to a robust daily rhythm in the suprachiasmatic nucleus and several peripheral tissues. Previous studies with mice deleted for the Dbp gene have established that DBP participates in the regulation of several clock outputs, including locomotor activity, sleep distribution, and liver gene expression. Here we present evidence that circadian Dbp transcription requires the basic helix-loop-helix-PAS protein CLOCK, an essential component of the negative-feedback circuitry generating circadian oscillations in mammals and fruit flies. Genetic and biochemical experiments suggest that CLOCK regulates Dbp expression by binding to E-box motifs within putative enhancer regions located in the first and second introns. Similar E-box motifs have been found previously in the promoter sequence of the murine clock gene mPeriod1. Hence, the same molecular mechanisms generating circadian oscillations in the expression of clock genes may directly control the rhythmic transcription of clock output regulators such as Dbp. PMID- 10733529 TI - E2f3 is critical for normal cellular proliferation. AB - E2F is a family of transcription factors that regulate both cellular proliferation and differentiation. To establish the role of E2F3 in vivo, we generated an E2f3 mutant mouse strain. E2F3-deficient mice arise at one-quarter of the expected frequency, demonstrating that E2F3 is important for normal development. To determine the molecular consequences of E2F3 deficiency, we analyzed the properties of embryonic fibroblasts derived from E2f3 mutant mice. Mutation of E2f3 dramatically impairs the mitogen-induced, transcriptional activation of numerous E2F-responsive genes. We have been able to identify a number of genes, including B-myb, cyclin A, cdc2, cdc6, and DHFR, whose expression is dependent on the presence of E2F3 but not E2F1. We further show that a critical threshold level of one or more of the E2F3-regulated genes determines the timing of the G(1)/S transition, the rate of DNA synthesis, and thereby the rate of cellular proliferation. Finally, we show that E2F3 is not required for cellular immortalization but is rate limiting for the proliferation of the resulting tumor cell lines. We conclude that E2F3 is critical for the transcriptional activation of genes that control the rate of proliferation of both primary and tumor cells. PMID- 10733531 TI - The zinc ribbon domains of the general transcription factors TFIIB and Brf: conserved functional surfaces but different roles in transcription initiation. AB - The function of the conserved zinc-binding domains in the related Pol II- and Pol III-specific factors TFIIB and Brf was investigated. Three-dimensional structure modeling and mutagenesis studies indicated that for both factors, the functional surface of the zinc ribbon fold consists of a small conserved patch of residues located on one face of the domain comprised mainly of the second and third antiparallel beta strands. Previous studies have shown that the TFIIB zinc ribbon is essential for recruitment of Pol II into the preinitiation complex. In contrast, Pol III recruitment assays and in vitro transcription demonstrate that the disruption of the Brf zinc ribbon does not lead to a defect in Pol III recruitment but, rather, a defect in open complex formation. Therefore, the same conserved surface of the zinc ribbon domain has been adapted to serve distinct roles in the Pol II and Pol III transcription machinery. PMID- 10733530 TI - PERP, an apoptosis-associated target of p53, is a novel member of the PMP-22/gas3 family. AB - The p53 tumor suppressor activates either cell cycle arrest or apoptosis in response to cellular stress. Mouse embryo fibroblasts (MEFs) provide a powerful primary cell system to study both p53-dependent pathways. Specifically, in response to DNA damage, MEFs undergo p53-dependent G(1) arrest, whereas MEFs expressing the adenovirus E1A oncoprotein undergo p53-dependent apoptosis. As the p53-dependent apoptosis pathway is not well understood, we sought to identify apoptosis-specific p53 target genes using a subtractive cloning strategy. Here, we describe the characterization of a gene identified in this screen, PERP, which is expressed in a p53-dependent manner and at high levels in apoptotic cells compared with G(1)-arrested cells. PERP induction is linked to p53-dependent apoptosis, including in response to E2F-1-driven hyperproliferation. Furthermore, analysis of the PERP promoter suggests that PERP is directly activated by p53. PERP shows sequence similarity to the PMP-22/gas3 tetraspan membrane protein implicated in hereditary human neuropathies such as Charcot-Marie-Tooth. Like PMP 22/gas3, PERP is a plasma membrane protein, and importantly, its expression causes cell death in fibroblasts. Taken together, these data suggest that PERP is a novel effector of p53-dependent apoptosis. PMID- 10733532 TI - The carboxyl terminus of phage HK022 Nun includes a novel zinc-binding motif and a tryptophan required for transcription termination. AB - The amino-terminal arginine-rich motif of the phage HK022 Nun protein binds phage lambda nascent mRNA transcripts while the carboxy-terminal domain binds RNA polymerase and arrests transcription. The role of specific residues in the carboxy-terminal domain in transcription termination were investigated by mutagenesis, in vitro and in vivo functional assays, and NMR spectroscopy. Coordination of zinc to three histidine residues in the carboxy-terminus inhibited RNA binding by the amino-terminal domain; however, only two of these histidines were required for transcription arrest. These results suggest that additional zinc-coordinating residues are supplied by RNA polymerase in the context of the Nun-RNA polymerase complex. Substitution of the penultimate carboxy-terminal tryptophan residue with alanine or leucine blocks transcription arrest, whereas a tyrosine substitution is innocuous. Wild-type Nun fails to arrest transcription on single-stranded templates. These results suggest that Nun inhibition of transcription elongation is due in part to interactions between the carboxy-terminal tryptophan of Nun and double-stranded DNA, possibly by intercalation. A model for the termination activity of Nun is developed on the basis of these data. PMID- 10733533 TI - A novel pairing process promoted by Escherichia coli RecA protein: inverse DNA and RNA strand exchange. AB - Traditionally, recombination reactions promoted by RecA-like proteins initiate by forming a nucleoprotein filament on a single-stranded DNA (ssDNA), which then pairs with homologous double-stranded DNA (dsDNA). In this paper, we describe a novel pairing process that occurs in an unconventional manner: RecA protein polymerizes along dsDNA to form an active nucleoprotein filament that can pair and exchange strands with homologous ssDNA. Our results demonstrate that this "inverse" reaction is a unique, highly efficient DNA strand exchange reaction that is not due to redistribution of RecA protein from dsDNA to the homologous ssDNA partner. Finally, we demonstrate that the RecA protein-dsDNA filament can also pair and promote strand exchange with ssRNA. This inverse RNA strand exchange reaction is likely responsible for R-loop formation that is required for recombination-dependent DNA replication. PMID- 10733534 TI - Chronic viral hepatitis and the human genome. PMID- 10733535 TI - Host genetic factors influence disease progression in chronic hepatitis C. AB - Progressive hepatic fibrosis and cirrhosis develops in 20% to 30% of patients with chronic hepatitis C virus (HCV). We propose that host genetic factors influencing fibrogenesis may account for some of the variability in progression of this disease. In progressive fibrosis of other organs, particularly heart and kidney, production of the profibrogenic cytokine, transforming growth factor beta1 (TGF-beta1), may be enhanced by angiotensin II, the principal effector molecule of the renin-angiotensin system. The inheritance of polymorphisms in TGF beta1, interleukin 10 (IL-10), tumor necrosis factor alpha (TNF-alpha), and genes of the renin-angiotensin system was examined in 128 patients with chronic HCV. The influence of genotypes on the stage of hepatic fibrosis was tested after adjustment for potential confounders (age, gender, alcohol consumption, portal inflammation, and steatosis), which may have independent effects on histological severity. The stage of fibrosis was 0 in 30 (23.4%), 1 in 44 (34.4%), 2 in 27 (21.1%), and 3 or 4 in 27 (21.1%). A statistically significant relationship was seen between inheritance of high TGF-beta1- and angiotensinogen (AT)-producing genotypes and the development of progressive hepatic fibrosis. This association persisted after correcting for potential confounders. Patients who inherited neither of the profibrogenic genotypes had no or only minimal fibrosis. Knowledge of these polymorphisms may have prognostic significance in patients with chronic HCV and may direct more aggressive therapy towards those patients with an increased risk of disease progression. The documentation of a significant relationship between AT genotype and fibrosis raises the novel suggestion that angiotensin II may be another mediator of extracellular matrix production in the liver. PMID- 10733536 TI - The cost-effectiveness of hepatitis A vaccination in patients with chronic hepatitis C. AB - Infection with hepatitis A virus (HAV) occasionally leads to acute liver failure and has a higher fatality rate in patients with chronic hepatitis C virus (HCV). Vaccination of patients with HCV against HAV is effective and well tolerated. This study examines the cost-effectiveness of HAV vaccination in North American patients with chronic HCV. A decision analysis model was constructed to compare 3 HAV vaccination strategies in adult patients with chronic HCV over a period of 5 years: (1) vaccinate no patients (treat none); (2) vaccinate only susceptible (anti-HAV negative) patients (selective); or (3) vaccinate all patients without prior testing of immune status (universal). Probabilities and direct costs were estimated from hospital data and the literature. The cost per patient for the 3 vaccination strategies were: treat none, $2.00; selective, $56.00; and universal, $82.00. For every 1,000,000 patients with HCV vaccinated over a 5-year period, the selective strategy prevented 128 symptomatic cases of HAV, 3 liver transplantations, and 3 deaths owing directly to HAV compared with the treat none strategy. In addition, the selective strategy costs an additional $427,000 per patient with HAV prevented, and $23 million per HAV-related death averted, compared with the treat none strategy. The results were most sensitive to the incidence of HAV infection; vaccination increased costs if the annual rate of infection was less than 0.56% (baseline, 0.01%). Vaccination of North American patients with chronic HCV against HAV infection is not a cost-effective therapy. PMID- 10733537 TI - Prospective validation of the CLIP score: a new prognostic system for patients with cirrhosis and hepatocellular carcinoma. The Cancer of the Liver Italian Program (CLIP) Investigators. AB - Prognosis of patients with cirrhosis and hepatocellular carcinoma (HCC) depends on both residual liver function and tumor extension. The CLIP score includes Child-Pugh stage, tumor morphology and extension, serum alfa-fetoprotein (AFP) levels, and portal vein thrombosis. We externally validated the CLIP score and compared its discriminatory ability and predictive power with that of the Okuda staging system in 196 patients with cirrhosis and HCC prospectively enrolled in a randomized trial. No significant associations were found between the CLIP score and the age, sex, and pattern of viral infection. There was a strong correlation between the CLIP score and the Okuda stage. As of June 1999, 150 patients (76.5%) had died. Median survival time was 11 months, overall, and it was 36, 22, 9, 7, and 3 months for CLIP categories 0, 1, 2, 3, and 4 to 6, respectively. In multivariate analysis, the CLIP score had additional explanatory power above that of the Okuda stage. This was true for both patients treated with locoregional therapy or not. A quantitative estimation of 2-year survival predictive power showed that the CLIP score explained 37% of survival variability, compared with 21% explained by Okuda stage. In conclusion, the CLIP score, compared with the Okuda staging system, gives more accurate prognostic information, is statistically more efficient, and has a greater survival predictive power. It could be useful in treatment planning by improving baseline prognostic evaluation of patients with HCC, and could be used in prospective therapeutic trials as a stratification variable, reducing the variability of results owing to patient selection. PMID- 10733538 TI - Fractional allelic loss in non-end-stage cirrhosis: correlations with hepatocellular carcinoma development during follow-up. AB - Hepatocellular carcinoma (HCC) is usually preceded by cirrhosis whose genetic background is still poorly understood. The aim of this study was to evaluate, in non-end-stage cirrhosis, the fractional allelic loss (FAL) at loci mostly reported to be altered in HCC and the microsatellite instability (MSI). Twenty cases of cirrhosis were retrospectively selected. Eleven had developed an HCC during the follow-up (HCC-prone group), while 9 remained HCC-free (HCC-free group). Microdissected hepatocellular cirrhotic nodules from basal liver biopsies, were studied at 20 loci (on the chromosomal arms 1p and 1q, 3p, 4q, 6q, 7q, 8p, 13q, and 18q) and with the mononucleotide repeats BAT26 and TGFbIIR. Genetic changes were detected in both groups. Overall, the FAL index was statistically increased in the HCC-prone group (0.213) as compared to the HCC free group (0.094; P =.044). Allelic loss at chromosomal arms 1p, 4q, 13q, 18q, and concurrent losses at more than 3 loci were confined to the HCC-prone group. In both groups, MSI was never ascertained using BAT26 and TGFbIIR. In conclusion, an increased FAL index and the lack of MSI characterize the non-end-stage cirrhosis of patients undergoing HCC during follow-up. These data emphasize the role of early clonal changes in chronic liver disease, and their potential predictive significance for clinical use. PMID- 10733539 TI - Hepatocyte transplantation in rats with decompensated cirrhosis. AB - Hepatocyte transplantation improves the survival of laboratory animals with experimentally induced acute liver failure and the physiological abnormalities associated with liver-based metabolic deficiencies. The role of hepatocyte transplantation in treating decompensated liver cirrhosis, however, has not been studied in depth. To address this issue, cirrhosis was induced using phenobarbital and carbon tetrachloride (CCL(4)) and animals were studied only when evidence of liver failure did not improve when CCL(4) was held for 4 weeks. Animals received intrasplenic transplantation of syngeneic rat hepatocytes (G1); intraperitoneal transplantation of syngeneic rat hepatocytes (G2); intraperitoneal transplantation of a cellular homogenate of syngeneic rat hepatocytes (G3); intraperitoneal transplantation of syngeneic rat bone marrow cells (G4); or intrasplenic injection of Dulbecco's modified Eagle medium (DMEM) (G5). After transplantation, body weight and serum albumin levels deteriorated over time in all control (G2-G5) animals but did not deteriorate in animals receiving intrasplenic hepatocyte transplantation (G1) (P <.01). Prothrombin time (PT), total bilirubin, serum ammonia, and hepatic encephalopathy score were also significantly improved toward normal in animals receiving intrasplenic hepatocyte transplantation (P <. 01). More importantly, survival was prolonged after a single infusion of hepatocytes and a second infusion prolonged survival from 15 to 128 days (P <.01). Thus, hepatocyte transplantation can improve liver function and prolong the survival of rats with irreversible, decompensated cirrhosis and may be useful in the treatment of cirrhosis in humans. PMID- 10733540 TI - Effect of cisapride on intestinal bacterial overgrowth and bacterial translocation in cirrhosis. AB - Deranged intestinal motility, which occurs in cirrhosis, may facilitate the development of intestinal bacterial overgrowth (IBO), which can lead to bacterial translocation (BT). To assess the effect of cisapride on IBO and BT in cirrhosis, cirrhotic rats received cisapride or a placebo for 7 days, and measurements of jejunal bacterial content and BT studies were performed. In addition, jejunal fluid from 46 cirrhotic patients was obtained for quantitative bacterial culture. Those patients in whom gram-negative IBO was detected were randomized to receive or not to receive cisapride (20 mg twice per day) for 1 week. Cisapride significantly reduced IBO in cirrhotic rats. In addition, no BT was documented in treated animals, whereas it occurred in 40% in nontreated cirrhotic rats. Total IBO was documented in 23 of 46 cirrhotic patients, which was caused by gram negative organisms in 10 cases. Orocecal transit time (OCT) significantly decreased after cisapride therapy, and was associated with the abolishment of bacterial overgrowth caused by gram-negative organisms in 4 out of 5 treated patients, whereas it persisted in nontreated cases. Cisapride administration to cirrhotic rats resulted in a reduction of the IBO, which is associated with a marked decrease in BT. On the other hand, cisapride facilitates the abolition of IBO caused by gram-negative organisms in cirrhotic patients. PMID- 10733541 TI - A model to predict poor survival in patients undergoing transjugular intrahepatic portosystemic shunts. AB - Transjugular intrahepatic portosystemic shunts (TIPS) may worsen liver function and decrease survival in some patients. The Child-Pugh classification has several drawbacks when used to determine survival in such patients. The survival of 231 patients at 4 medical centers within the United States who underwent elective TIPS was studied to develop statistical models to (1) predict patient survival and (2) identify those patients whose liver-related mortality post-TIPS would be 3 months or less. Among these elective TIPS patients, 173 had the procedure for prevention of variceal rebleeding and 58 for treatment of refractory ascites. Death related to liver disease occurred in 110 patients, 70 within 3 months. Cox proportional-hazards regression identified serum concentrations of bilirubin and creatinine, international normalized ratio for prothrombin time (INR), and the cause of the underlying liver disease as predictors of survival in patients undergoing elective TIPS, either for prevention of variceal rebleeding or for treatment of refractory ascites. These variables can be used to calculate a risk score (R) for patients undergoing elective TIPS. Patients with R > 1.8 had a median survival of 3 months or less. This model was superior to both the Child Pugh classification, as well as the Child-Pugh score, in predicting survival. Using logistic regression and the same variables, we also developed a nomogram that indicates which patients survive less than 3 months. Finally, the model was validated among an independent set of 71 patients from the Netherlands. This Mayo TIPS model may predict early death following elective TIPS for either prevention of variceal rebleeding or for treatment of refractory ascites. PMID- 10733542 TI - Mild hypothermia delays the onset of coma and prevents brain edema and extracellular brain glutamate accumulation in rats with acute liver failure. AB - Mild hypothermia is effective in the prevention of brain edema associated with cerebral ischemia and traumatic brain injury. Brain edema is also a serious complication of acute liver failure (ALF). To assess the effectiveness of hypothermia in ALF, groups of rats were subjected to hepatic devascularization (portacaval anastomosis, followed 48 hours later by hepatic artery ligation), and body temperatures were maintained at either 35 degrees C (hypothermic) or 37 degrees C (normothermic). Mild hypothermia resulted in a significant delay in the onset of severe encephalopathy and in reduction of brain water content compared with normothermic ALF rats (control [n = 8] 80.22%; ALF-37 degrees C [n = 8] 81.74%; ALF-35 degrees C [n = 8] 80.48% [P <.01 compared with ALF-37 degrees C]). This protective effect was accompanied by a significant reduction of cerebrospinal fluid (CSF) (but not plasma) ammonia concentrations (CSF ammonia: control: 0.05 mg/dL; ALF-37 degrees C: 1.01 mg/dL; ALF-35 degrees C: 0.07 mg/dL, P <.01 compared with ALF-37 degrees C). In vivo cerebral microdialysis studies revealed that mild hypothermia resulted in a significant reduction of extracellular glutamate concentrations in the brains of rats with ALF (control: 1. 06 micromol/L; ALF-37 degrees C: 2.74 micromol/L; ALF-35 degrees C: 1.49 micromol/L [P <.01 compared with ALF-37 degrees C]). These findings suggest that: 1) mild hypothermia is an effective approach to the prevention of the central nervous system consequences of experimental ALF; and that 2) the beneficial effect of hypothermia is mediated via mechanisms involving reduced blood-brain transfer of ammonia and/or reduction of extracellular brain glutamate concentrations. Mild hypothermia may be an effective approach to delay the onset of brain edema in patients with ALF awaiting liver transplantation. PMID- 10733543 TI - Detection of circulating antibodies against malondialdehyde-acetaldehyde adducts in patients with alcohol-induced liver disease. AB - Acetaldehyde and malonildialdehyde can form hybrid protein adducts, named MAA adducts that have strong immunogenic properties. The formation of MAA adducts in the liver of chronic alcohol-fed rats is associated with the development of circulating antibodies that specifically recognized these adducts. The aim of this study was to examine whether MAA adducts might participate in the immune response associated with human alcohol-induced liver disease. Circulating antibodies against MAA adducts were evaluated in 50 patients with alcohol-induced hepatitis or cirrhosis, in 40 patients with non-alcohol-induced liver disease, in 15 heavy drinkers without liver damage and in 40 healthy controls by enzyme linked immunosorbent assays (ELISA). Immunoglobulin G (IgG) reacting with MAA modified proteins were significantly increased in the patients with alcohol induced cirrhosis or hepatitis. The individual levels of anti-MAA IgG in those patients were associated with the severity of liver damage. Anti-MAA antibodies were also positively correlated with the levels of IgG recognizing epitopes generated by acetaldehyde and malonildialdehyde. However, competitive inhibition experiments indicated that the anti-MAA antibodies were unrelated to those against acetaldehyde- or malonildialdehyde-derived antigens and mainly recognized a specific, cyclic MAA epitope. Some degree of immune reactivity towards MAA adducts was also observed in patients with non-alcohol-induced liver injury. However, competitive ELISA showed that the antigens recognized by these sera were not the cyclic MAA adducts. Altogether, these results showed the formation of MAA antigens during alcohol-induced liver disease and suggest their possible contribution to the development of immunologic reactions associated with alcohol related liver damage. PMID- 10733544 TI - Detection of adenovirus and initiation of apoptosis in hepatocellular carcinoma cells after Ad-p53 treatment. AB - Transcription of the p53 gene can regulate progression of apoptosis in a wide variety of tissues. Three categories of human hepatocyte culture have been used to show the initiation of apoptosis after treatment with p53-bearing adenovirus. Chang liver cells are derived from normal liver tissue and express native p53, whereas hepatocellular carcinoma (HCC)-derived cell lines were Hep3B (p53 deleted) and PLC/PRF/5 (p53-mutant). Cultures were infected with Ad-p53 (15 particles per cell; 36 hours), and after treatment, morphological changes in all cell categories were observed by electron microscopy. Infection was evident in the cytoplasm of all treated cell types: after entry across the plasma membrane viruses translocated and came to rest surrounding and adjacent to nuclei, cytoplasm proximal to nuclear membranes became dense with virus- and membrane derived debris, but intact viruses did not enter nuclei. Apoptosis, recognized morphologically by characteristic chromatin and cytoplasmic condensation, occurred more frequently in HCC-derived cells, and the ultimate fate of apoptotic bodies was phagocytosis and degradation by neighboring cells. PMID- 10733545 TI - Probability of hepatocellular carcinoma of small hepatocellular nodules undetectable by computed tomography during arterial portography. AB - Recent advances in imaging modalities enable the identification of small hepatocellular nodules. Among the imaging techniques currently used for detecting hepatocellular carcinomas (HCC), computed tomography (CT) during arterial portography (CTAP) is one of the most sensitive techniques available for detecting hemodynamic change. Even so, well-differentiated HCCs that display only limited hemodynamic change, a feature shared with nonmalignant hepatocellular nodules, are not always detectable by CTAP. To improve our ability to distinguish well-differentiated HCCs from nonmalignant hepatocellular nodules, we have attempted to clarify how the characteristics of the nodules are shown by each imaging technique. We studied the imaging and pathological characteristics of 31 nodules (in 22 patients) detected by ultrasonography (US), but not by CTAP. Histological diagnoses were as follows: HCC, 17 of 31 nodules (55%); high-grade dysplastic nodules, 1 of 31 (3%); and nonmalignant nodules, 13 of 31 (42%). Neither digital substraction angiography (DSA) nor CT arteriography (CTA) were able to detect any of the nodules. Detection rates for plain CT were: 5 of 17 (29%) HCC, 1 of 1 (100%) high-grade dysplastic nodules, and 1 of 13 (8%) nonmalignant nodules. Detection rates for T1/T2-weighted magnetic resonance imaging (MRI) were: 4 of 17 (24%) HCC, 1 of 1 (100%) high-grade dysplastic nodules, and 3 of 13 (23%) nonmalignant nodules. Dynamic CT and dynamic MRI provided no additional information. In conclusion, there is some probability that hepatocellular nodules detected by US, but not by CTAP, are HCC. Presently, it is difficult to distinguish between benign nodules and malignant ones with these imaging techniques, and our findings indicate that biopsy may be advisable for nodules detected under these conditions. PMID- 10733546 TI - Primary liver resection and salvage transplantation or primary liver transplantation in patients with single, small hepatocellular carcinoma and preserved liver function: an outcome-oriented decision analysis. AB - Two treatments are accepted for patients with solitary hepatocellular carcinoma SAP8 > SAP6 > SAP3 was observed in an in vitro model of cutaneous candidiasis based on reconstituted human epidermis. Transcripts of SAP1 and SAP2 were detected during initial invasion of the stratum corneum by C. albicans. Deeper, extensive penetration of the corneal layer was accompanied by additional SAP8 mRNA. SAP6 expression occurred concomitantly with germ tube formation and extensive hyphal growth in the strata granulosum, spinosum, and basale. Ultrastructural studies using specific polyclonal antibodies directed against the gene products of SAP1-3 and SAP4-6 revealed predominant expression of Sap1-3. The protective effect of the aspartic proteinase inhibitor pepstatin A during infection of the epidermis and an attenuated virulence phenotype of SAP-deficient mutants suggest that the observed SAP expression correlates with tissue damage in the skin. PMID- 10733679 TI - Differentiation-dependent alternative splicing and expression of the extracellular matrix protein 1 gene in human keratinocytes. AB - The human extracellular matrix protein 1 (Ecm1) gene is located at chromosome band 1q21 close to the epidermal differentiation complex and is transcribed in two discrete mRNAs: a full length Ecm1a and a shorter, alternatively spliced, Ecm1b transcript, the expression of which is restricted to tonsils and skin. The chromosomal localization and the Ecm1b expression in skin prompted us to investigate the role of Ecm1 in keratinocyte differentiation. In this study, we provide evidence for the existence of a relationship between keratinocyte differentiation and expression of the Ecm1b transcript. Cultures of subconfluent undifferentiated normal human keratinocytes express only Ecm1a. Upon reaching confluence, the cells start to differentiate, as measured by keratin K10 mRNA expression. Concomitantly Ecm1b mRNA expression is induced, although expression of Ecm1a mRNA remains unchanged. In addition, treatment of undifferentiated normal human keratinocyte cells with 12-O-tetradecanoyl-phorbol-13-acetate strongly induces the expression of Ecm1b mRNA. Expression of Ecm1b can also be induced by coculturing normal human keratinocytes with lethally irradiated feeder cells and by a diffusible factor secreted by stromal cells. In adult human skin, Ecm1a mRNA is expressed throughout the epidermis with the strongest expression in the basal and first suprabasal cell layers, whereas expression of Ecm1b mRNA is predominantly found in spinous and granular cell layers. Immunohistochemically, Ecm1a expression is almost completely restricted to the basal cell layer, whereas Ecm1b is detected in the suprabasal layers. These results are strongly suggestive of a role for Ecm1b in terminal keratinocyte differentiation, which is also supported by the localization of the Ecm1 gene at 1q21. Refinement of its genomic localization, however, placed Ecm1 centromeric of the epidermal differentiation complex. PMID- 10733680 TI - Bcl-2 antisense oligonucleotides (G3139) inhibit Merkel cell carcinoma growth in SCID mice. AB - Merkel cell carcinoma was first described in 1972 by Toker and is an aggressive neuroendocrine skin tumor with a high metastatic potential. Merkel cell carcinoma is thought to derive from the neuroendocrine (Merkel) cells of the skin, although in contrast to fetal and especially adult Merkel cells, Merkel cell carcinomas express high levels of the Bcl-2 oncoprotein. Bcl-2 is capable of blocking programmed cell death and has been shown to play an important role in normal cell turnover, tumor biology, and chemoresistance. High Bcl-2 expression leading to prolonged survival of cells may therefore be of importance in the biological and clinical characteristics of Merkel cell carcinoma. In a SCID mouse xenotransplantation model for human Merkel cell carcinoma, we investigated the influence of the bcl-2 antisense oligonucleotide G3139 (Genta) on tumor growth in comparison with control oligonucleotides or cisplatin. Bcl-2 antisense treatment, targeting the first six codons of the bcl-2 mRNA, resulted in either a dramatic reduction of tumor growth or complete remission, whereas reverse sequence and two base mismatch control oligonucleotides or cisplatin had no significant antitumor effects compared with saline-treated controls. Apoptosis was enhanced 2.4-fold in the bcl-2 antisense treated tumors compared with the saline-treated group, and no other treatment showed a comparable increase in apoptosis. Our findings suggest that bcl-2 antisense treatment may be a novel approach to improve treatment outcome of human Merkel cell carcinoma. PMID- 10733681 TI - arg-cys substitution at codon 1246 of the human myosin Va gene is not associated with Griscelli syndrome. AB - Myosin Va is an actin-associated motor protein involved in organelle transport such as melanosomes and neuron synaptic vesicles and has always been proposed as the candidate gene for the autosomal recessive Griscelli-Prunieras syndrome, one of the silvery hair syndromes, which is a lethal disease combining immunodeficiency and neurologic and pigmentary abnormalities. Thus far, two mutations in the myosin Va gene have been described to be associated with this syndrome. One of these mutations was a homozygous mis-sense mutation causing an arginine to cysteine alteration at codon 1246. Because we also found this particular substitution after mutation analysis of a Griscelli patient, we checked its relevance in a control group of 124 unrelated healthy individuals and found it to be present, even in homozygous state, in normal unaffected individuals. It is clear that this arg1246cys substitution is a polymorphism occurring in the human population and not occurring in association with Griscelli syndrome. Distinguishing a polymorphism from a bona fide mutation is of utmost importance and has major ethical implications with regard to prenatal genetic counseling in affected families. PMID- 10733682 TI - Sleep organization in the first year of life: developmental trends in the quiet sleep-paradoxical sleep cycle. AB - The night sleep of 48 healthy drug-free infants, aged 1-54 weeks, was recorded and analysed in order to show how cycles contribute to sleep episode organization and how the balance among different sleep states (i.e. quiet sleep, paradoxical sleep and ambiguous sleep) within cycles changes as a function of age. A greater proportion of time spent in cycles (TCT) on total sleep time (TST), as a result of the lengthening of sleep cycles, was found in older infants, whereas sleep out of cycles decreases with age. The internal structure of the sleep cycles also changes with age, because of the increase in the proportion of quiet sleep (QS), the appearance of slow wave sleep (SWS) from the 21st week onwards, and the decrease in ambiguous sleep. The proportion of paradoxical sleep (PS), however, remains stable throughout the first year of age. The improvement of sleep organization across the first year of life is paralleled by an internal restructuring of the cycle, involving mainly changes in QS. We may speculate that both changes, one involving the lengthening of cycle and the other involving the increase in QS component, contribute to the improvement of biological and psychological sleep functions during development. PMID- 10733683 TI - Sensitivity and specificity of the multiple sleep latency test (MSLT), the maintenance of wakefulness test and the epworth sleepiness scale: failure of the MSLT as a gold standard. AB - Excessive daytime sleepiness (EDS) is an important symptom that needs to be quantified, but there is confusion over the best way to do this. Three of the most commonly used tests: the multiple sleep latency test (MSLT), the maintenance of wakefulness test (MWT) and the Epworth sleepiness scale (ESS) give results that are significantly correlated in a statistical sense, but are not closely related. The purpose of this investigation was to help clarify this problem. Previously published data from several investigations were used to calculate the reference range of normal values for each test, defined by the mean+/-2 SD or by the 2.5 and 97.5 percentiles. The 'rule of thumb' that many people rely on to interpret MSLT results is shown here to be misleading. Previously published results from each test were also available for narcoleptic patients who were drug free at the time and who by definition had EDS. This enabled the sensitivity and specificity of the three tests to be compared for the first time, in their ability to distinguish the EDS of narcolepsy from the daytime sleepiness of normal subjects. The receiver operator characteristic curves clearly showed that the ESS is the most discriminating test, the MWT is next best and the MSLT the least discriminating test of daytime sleepiness. The MSLT can no longer be considered the gold standard for such tests. PMID- 10733684 TI - Cyclic alternating pattern and spectral analysis of heart rate variability during normal sleep. AB - The natural arousal rhythm of non-rapid eye movement (NREM) sleep is known as the cyclic alternating pattern (CAP), which consists of arousal-related phasic events (Phase A) that periodically interrupt the tonic theta/delta activities of NREM sleep (Phase B). The complementary condition, i.e. non-CAP (NCAP), consists of a rhythmic electroencephalogram background with few, randomly distributed arousal related phasic events. Recently, some relation between CAP and autonomic function has been preliminarily reported during sleep in young adults by means of spectral analysis of heart rate variability (HRV). The present study was aimed at analysing the effects of CAP on HRV in a group of normal children and adolescents. Six normal children and adolescents (age range 10.0-17.5 y) were included in this study. All-night polygraphic recordings were performed after adaptation to the sleep laboratory. Six 5-min epochs were selected from sleep Stage 2 and six from Stages 3 and 4 (slow-wave sleep), both in CAP and NCAP conditions. From such epochs, a series of parameters describing HRV was then calculated, in both time and frequency domains, on the electrocardiographic R-R intervals. Statistical comparison between CAP and NCAP epochs revealed a significant difference for most of the frequency domain parameters (increase of the low-frequency band, increase of the low-frequency/high-frequency ratio and decrease in the high-frequency band during CAP) both in Stage 2 and in slow-wave sleep. Our results demonstrate that the physiological fluctuations of arousal during sleep described as CAP are accompanied by subtle, but significant, changes in balance between the sympathetic and vagal components of the autonomic system. PMID- 10733685 TI - The role of the spindle in human information processing of high-intensity stimuli during sleep. AB - Sleep spindles are 12-14 Hz oscillations in EEG, which are thought to inhibit or 'gate' information processing. Event-related potentials may be employed to probe the extent of information processing during sleep. Previous research indicates that event-related potentials elicited by moderate intensity stimuli show increased positivity (or further removal of negativity) when stimuli are presented concurrent with spindles. However, the effectiveness of spindles to inhibit the processing of much louder stimuli remains unknown. The purpose of the present study was to investigate the extent of this gating, by using a range of stimuli including those that are loud and intrusive. Eight good sleepers were recorded during a single night. Auditory stimuli were delivered randomly at 0, 60, 80 or 100 dB SPL. Trials were sorted off-line by sleep stage, stimulus intensity and spindle characteristic (i.e. spindle absent, spindle present). During the sleep-onset period, the often-reported changes in event-related potentials were observed - N1 decreased and P2 increased in amplitude. In Stage 2 sleep, P2 was affected by the presence of spindles, particularly when stimulus intensity was loud. Its amplitude was greatest when spindles occurred following the onset of the stimulus. Scalp-recorded spindles might, therefore, be a consequence of the prior thalamic inhibition of information processing, especially when confronted by loud, intrusive external stimuli. PMID- 10733686 TI - Age differences in the spontaneous termination of sleep. AB - The stage from which the spontaneous ending of sleep occurred was investigated in 138 sleep episodes obtained from 14 younger (19-28 years) and 11 older (60-82 years) individuals. The possible influences of circadian phase and quality of the preceding sleep period, as well as the impact of aging on characteristics of sleep termination were examined. Under experimental conditions in which subjects were isolated from time cues, and behavioral options to sleep were limited, no age-associated differences in the duration of sleep periods, or in the number or duration of REM episodes were observed. Despite similar percentages of NREM (stages 2-4) and REM sleep across age groups, younger subjects awakened preferentially from REM while older subjects did not. Of the sleep episodes obtained from older subjects, those with sleep efficiencies higher than the median were more likely to terminate from REM than those with lower sleep efficiencies. For all subjects, the REM episodes from which sleep termination occurred were truncated relative to those that did not end the sleep period. In addition, nonterminating REM episodes that were interrupted by a stage shift were most often interrupted by brief arousals to stage 0. Such arousals within nonterminating REM episodes occurred, on average, after a similar duration as the terminating point of sleep-ending REM episodes. The results from this study demonstrate that there are age-related differences in the sleep stage from which spontaneous awakenings occur, and that these differences may be due in part to the quality of the sleep period preceding termination. Findings regarding the characteristics of both terminating and nonterminating REM episodes are consistent with the notion that the neural and biochemical context of REM sleep may facilitate a smooth transition to wakefulness. It is speculated that age associated changes in sleep continuity may render unnecessary the putative role of REM sleep in providing a 'gate' to wakefulness. PMID- 10733687 TI - Prevalence of insomnia in a survey of 12,778 adults in France. AB - This study was an epidemiological questionnaire survey of a representative sample of the French population that included 12 778 individuals and in which adapted DSM-IV criteria for the definition of insomnia were used. Our goals were not only to assess the prevalence of 'insomnia' using these criteria, but also to compare the results obtained with those of prior studies using different definitions of 'insomnia'. The aim of this study was also to identify where areas of agreement and disagreement existed, as we believe that it is important to emphasize these points because DSM-IV recommendations are supposedly reflected in clinical practice. Seventy-three per cent of the individuals surveyed complained of a nocturnal sleep problem, but only 29% reported at least one sleep problem three times per week for a month, and 19% (2428 subjects) had at least one sleep problem three times per week for a month and complained of daytime consequences (DSM-IV criteria). Only 9% had two or more nocturnal sleep problems with daytime consequences and were classified as 'severe insomniacs'. Our study indicates that if DSM-IV criteria are used, the diagnosis of 'insomnia' is lower than in other epidemiological studies. The DSM criteria have an advantage in that they emphasize the daytime consequences of nocturnal sleep disturbances, which seem to be responsible for the most important socio-economic costs of the problem. PMID- 10733689 TI - Familial incidence of insomnia. AB - This study evaluated the familial incidence of sleep disturbances among individuals with insomnia complaints. The sample consisted of 285 patients evaluated for insomnia at a sleep disorders clinic. All patients completed a sleep survey and underwent a semistructured clinical interview as part of their initial evaluation of insomnia. Information on the presence and nature of sleep disturbances among their family members (first- and second-degree relatives) was obtained from a sleep survey. The findings indicate that 35% of patients consulting for insomnia had a positive family history of sleep disturbances. Insomnia was the most common type of sleep disturbance identified (76%) and the mother was the most frequently afflicted family member. Reports of sleep disturbances among a family member were more prevalent when the onset of insomnia was before 40-years-old than when it was later in life. A positive family history was slightly higher when the insomnia complaint involved sleep-onset difficulties relative to sleep-maintenance or mixed insomnias. Although the present findings suggest that a positive family history of insomnia may be a potential risk factor for insomnia, it is unclear whether this reflects a genetic predisposition or a social learning phenomenon. PMID- 10733688 TI - Subjective measurement of insomnia and quality of life in depressed inpatients. AB - Insomnia and major depressive episodes (MDE) have each been associated with quality of life (QOL) deficits. In this study we examined insomnia as an independent predictor of QOL deficits during MDE, and used a secondary analysis of cross-sectional data. The study was based at the inpatient psychiatric ward and included 88 adults (mean age 53; 78% women). We assessed insomnia severity with the 21-item Hamilton Rating Scale for Depression (HRSD) and the Beck Depression Inventory (BDI). Measurements of QOL in the week prior to admission included activities of daily living (ADLs), instrumental ADLs (IADLs), daily living and role functioning, and relation to self and colleagues (the last two both subscales of the Basis 32). Linear regression models used the insomnia items as independent variables and the QOL measures as the dependent variables, after adjusting for age and nonsleep related depression severity. The results showed that 93% of patients endorsed insomnia on the observer-rated HRSD, and 97% endorsed sleep disturbance in the self-rated BDI. However, the insomnia items on the HRSD and BDI showed poor concurrent validity. Increasing severity of insomnia on the HDRS was associated with better QOL, while increasing severity of insomnia on the BDI was associated with worse QOL. We conclude that the BDI and HRSD do not produce equivalent measures of insomnia severity in depressed inpatients, and each insomnia measure has a unique relationship with QOL. PMID- 10733690 TI - Effect of low and high frequency thalamic stimulation on sleep in patients with Parkinson's disease and essential tremor. AB - Continuous high frequency stimulation of the ventral intermediate nucleus of the thalamus (Vim), delivered through surgically implanted quadripolar electrodes, alleviates tremor in Parkinson's disease (PD) and essential tremor (ET). The Vim is adjacent to the thalamic reticular nuclei, where sleep spindles originate according to animal models. In order to determine whether Vim stimulation affects sleep spindles, six patients (4 PD, 2 ET), aged 60-69 years, were recorded on a control night and a stimulation night (130 Hz, 2-3 V; right stimulation in five patients and bilateral stimulation in one patient). Stimulation did not modify sleep quality or architecture. Sleep spindles were present and symmetrical in five out of six patients under stimulation. However, in one patient with a sustained 'thalamotomy-like effect' that abolished tremor, spindles were asymmetrical even without stimulation. In each patient, spindle density was similar on both nights (mean+/- SEM: 2.25+/-0. 61 spindles per min of stage 2 sleep vs. 1.84+/-0.31). In an attempt to promote sleep two different patterns of stimulation were applied in the region of ventrooralis posterior and reticularis nuclei in five patients in the awake state. Continuous low frequency stimulation (5 Hz, 0.1 V), and repeated trains of 15 Hz for 1 s every 15 s mimicking the pattern of physiological spindles, each failed to induce sleep or cortical synchronization. We conclude that Vim stimulation, unlike thalamotomy, selectively reduces tremor without altering sleep or sleep spindles. Our results also suggest that low frequency stimulation applied in the region of the reticular nuclei does not induce sleep. PMID- 10733691 TI - FAST TRACK: daytime sleepiness in Parkinson's disease. AB - We describe multiple sleep latency test (MSLT) results in 27 adult patients with idiopathic Parkinson's disease (PD). Pathological sleepiness (i.e. mean sleep latency 0.05). PMID- 10733729 TI - Malaria control in an agro-industrial settlement of Rondonia (Western Amazon region, Brazil). AB - A malaria control pilot project was developed in the Urupa agro-industrial farm that is situated in the State of Rondonia (Western Amazon Region, Brazil). Around 180 inhabitants had been surveyed for the past five years. The control measures were based on (1) training a community agent to perform on the spot microscopical diagnosis of malaria and to treat the uncomplicated cases of malaria; (2) limiting the use of insecticides to a short period before the high transmission season. This resulted in a significant reduction in the time between the onset of clinical symptoms and specific chemotherapy which fell from 3.5 to 1.3 days. In relation to the previous three reference years the total number of malaria cases was reduced to 50% in the first year and to 25% in the second year. The introduction of these measures coincided with pronounced reduction in the frequency of Plasmodium falciparum infections but this was less marked for P. vivax infections. In the second year of the pilot experiment there was no P. falciparum transmission on the farm. During the last decade there was a general decrease in the endemicity of malaria in the State of Rondonia. The linear regression coefficient values indicate that the decline was more pronounced in Urupa than in the general municipality and that the falciparum malaria API in Urupa farm is significantly lower than in the general municipality of Candeias were the farm is situated. PMID- 10733731 TI - Population dynamics and feeding behavior of Triatoma brasiliensis and Triatoma pseudomaculata, main vectors of chagas disease in Northeastern Brazil. AB - Biological parameters of Triatoma brasiliensis and T. pseudomaculata that could influence the epidemiological importance of these insects as vectors of Trypanosoma cruzi were compared. The parameters studied were incubation period, interval between hatching or moulting and first feeding, number of blood meals, development time, mortality, net reproductive rate, instantaneous daily reproductive rate, time-lapse before starting feeding, duration of feeding, blood ingestion capacity, occurrence of defecation and blood ingestion velocity. Most aspects of feeding were similar for the two species, although T. pseudomaculata had a longer life cycle than T. brasiliensis producing one and two generations per year, respectively. The two species had similar instantaneous daily rates of population growth. PMID- 10733732 TI - Trypanosomes of non-human primates from the National Centre of Primates, Ananindeua, State of Para, brazil. AB - Trypanosome infections were sought in 46 non-human primates captured principally in Amazonian Brazil. Twenty-two (47.8%) were infected with four Trypanosoma species: T. cruzi, T. minasense, T. devei and T. rangeli. These preliminary results confirmed the high prevalence and diversity of natural infections with trypanosomes in primates from Brazilian Amazon and were the first formal record of simian infections with trypanosomes in the State of Acre. The presence of T. cruzi-like and T. rangeli-like parasites are recorded in four new hosts. PMID- 10733733 TI - Redescription of Tejidotaenia appendiculata (Baylis, 1947) (Cestoda: proteocephalidea), a parasite of Tupinambis teguixin (Sauria: teiidae) from South America. AB - The species Tejidotaenia appendiculata (Baylis, 1947), a parasite found in teju, Tupinambis teguixin is redescribed and a new diagnosis is provided. The species is characterized by the anterior position of the ovary and the peculiar shape of suckers. It is the first record of this species in Brazil. PMID- 10733734 TI - Mepraia spinolai in the Southeastern Pacific Ocean coast (Chile) - first insular record and feeding pattern on the Pan de Azucar island. AB - In a field collection performed at Pan de Azucar Island in Northern Chile, 95 specimens representing all instars of Mepraia spinolai were collected. The intestinal contents of 55 specimens were examined for Trypanosoma cruzi infection and were found to be negative. This is the first record of an insular habitat for M. spinolai, where the insects had fed mainly on seabirds (78%), some on marine mammals (5%), and some on reptiles (7%). PMID- 10733735 TI - Identification of planorbids from Venezuela by polymerase chain reaction amplification and restriction fragment length polymorphism of internal transcriber spacer of the RNA ribosomal gene. AB - Snails of the genus Biomphalaria from Venezuela were subjected to morphological assessment as well as polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. Morphological identification was carried out by comparison of characters of the shell and the male and female reproductive apparatus. The PCR-RFLP involved amplification of the internal spacer region ITS1 and ITS2 of the RNA ribosomal gene and subsequent digestion of this fragment by the restriction enzymes DdeI, MnlI, HaeIII and MspI. The planorbids were compared with snails of the same species and others reported from Venezuela and present in Brazil, Cuba and Mexico. All the enzymes showed a specific profile for each species, that of DdeI being the clearest. The snails were identified as B. glabrata, B. prona and B. kuhniana. PMID- 10733736 TI - New species records for the blackfly (Diptera-simuliidae) fauna of Argentina with description of adults, pupa and larva of Simulium oyapockense s. l. And S. seriatum. AB - Two blackfly species Simulium (Cerqueirellum) oyapockense Floch & Abonnenc and S. (Hemicnetha) seriatum Knab are recorded from Argentina, representing the most southern register for both species. S. oyapockense is a species epidemiologically very important, as a vector of onchocerciasis in the Amazonian focus. Both species are described and illustrated and their distribution are reported, in similarity to others like S. roraimense Nunes de Mello and S. ganalesense Vargas et al. in reference to S. oyapockense and S. mexicanum Bellardi similar to S. seriatum are discussed. PMID- 10733737 TI - Minicircle kDNA microheterogeneity in Endotrypanum indicate diversity within this genus. AB - A comparison of kDNA restriction-endonuclease fragment patterns from strains representing selected Endotrypanum zymodemes was done by schizodeme analysis. As the degree of heterogeneity within mini-circles varied among species or strains of Endotrypanum, the fingerprint obtained with each of the restriction enzymes was unique for each of these parasites. The data have revealed that this trypanosomatid genus is much more complex than it was originally thought to be. PMID- 10733738 TI - Canine experimental infection: intradermal inoculation of Leishmania infantum promastigotes. AB - Five mixed breed dogs were inoculated intradermally (ID) with cultured virulent stationary phase promastigotes of Leishmania infantum Nicole, 1908 stocks recently isolated. Parasite transformations in the skin of ID infected dogs were monitored from the moment of inoculation and for 48 h, by skin biopsies. Anti Leishmania antibody levels were measured by indirect immunofluorescence assay, counterimmunoelectrophoresis and direct agglutination test, and clinical conditions were examined. Thirty minutes after ID inoculation the first amastigotes were visualised and 3 to 4 h after inoculation the promastigotes were phagocytized by neutrophils and by a few macrophages. These cells parasitised by amastigotes progressively disappeared from the skin and 24 h after inoculation parasites were no longer observed. Local granulomes were not observed, however, serological conversion for antibodies anti-Leishmania was achieved in all dogs. Direct agglutination test was the only technique positive in all inoculated dogs. Amastigotes were found in the popliteal lymph node in one dog three months after inoculation. This work demonstrates that, with this inoculum, the promastigotes were transformed into amastigotes and were up taken by neutrophils and macrophages. The surviving parasites may have been disseminated in the canine organism, eliciting a humoral response in all cases. PMID- 10733739 TI - Is there a role for autoimmunity in immune protection against malaria? AB - Much remains to be known about the mechanisms involved in protective immunity against malaria and the way it is acquired. This is probably the reason why, in spite of so much progress, it has not yet been possible to develop an anti malaria vaccine able to induce parasite specific antibodies (Ab) and/or T-cells. It has been considered in the early 80s that the induction of efficient protection against the blood stage forms of Plasmodium falciparum would not be possible without simultaneously eliciting an autoimmune (AI) response against erythrocytes, even at the price of inducing an AI pathology. Despite the description of the reciprocal relationship, i.e. the protective effect of malaria on the development of AI diseases _ demonstrated since 1970 _ no effort has been made to verify the possible involvement of the AI response in protection against malaria. PMID- 10733740 TI - Cell populations in lesions of cutaneous leishmaniasis of Leishmania (L.) amazonensis- infected rhesus macaques, Macaca mulatta. AB - The cellular nature of the infiltrate in cutaneous lesion of rhesus monkeys experimentally infected with Leishmania (L.) amazonensis was characterized by immunohistochemistry. Skin biopsies from infected animals with active or healing lesions were compared to non-infected controls (three of each type) to quantitate inflammatory cell types. Inflammatory cells (composed of a mixture of T lymphocyte subpopulations, macrophages and a small number of natural killer cells and granulocytes) were more numerous in active lesions than in healing ones. T cells accounted for 44.7 +/- 13.1% of the infiltrate in active lesions (versus CD2+ = 40.3 +/- 5.7% in healing lesions) and T-cell ratios favor CD8+ cells in both lesion types. The percentage of cells expressing class II antigen (HLA-DR+) in active lesions (95 +/- 7.1%) was significantly higher (P < 0.005) from the healing lesions (42.7 +/- 12.7%). Moreover, the expression of the activation molecules CD25 (@ 16%), the receptor for interleukin-2, suggests that many T cells are primed and proliferating in active lesions. Distinct histopathological patterns were observed in lesions at biopsy, but healing lesions contained more organized epithelioid granulomas and activated macrophages, followed by fibrotic substitution. The progression and resolution of skin lesions appears to be very similar to that observed in humans, confirming the potential for this to be used as a viable model to study the immune response in human cutaneous leishmaniasis. PMID- 10733741 TI - Role of CD8+ T cells in endogenous interleukin-10 secretion associated with visceral leishmaniasis. AB - This study examined the role and source of endogenous interleukin-10 (IL) secretion in visceral leishmaniasis (VL). The amounts of endogenous and Leishmania specific IL-10 and interferon-gamma (IFN) secreted by peripheral blood mononuclear cells (PBMC) from VL patients were compared. The correlation coefficient between endogenous IL-10 secretion and Leishmania specific IFN-gamma was -0. 77, suggesting a major role for endogenous IL-10 secretion in VL. The effects of CD4+ and CD8+ T cell clones, isolated from a treated VL patient, on IL 10 secretion were assayed by mixing the clones with autologous, inactivated PBMC. The CD8+ clones mediated increased levels of IL-10 secretion in the presence of PBMC alone suggesting that CD8+ T cells may mediate endogenous IL-10 secretion. PMID- 10733742 TI - A simple method for human peripheral blood monocyte isolation. AB - We describe a simple method using percoll gradient for isolation of highly enriched human monocytes. High numbers of fully functional cells are obtained from whole blood or buffy coat cells. The use of simple laboratory equipment and a relatively cheap reagent makes the described method a convenient approach to obtaining human monocytes. PMID- 10733743 TI - Polymerase chain reaction amplification of genomic fragments of bovine herpesvirus-1. AB - Especial conditions were developed for the amplification of five DNA segments from US region of BHV-1 by polymerase chain reaction. In order to eliminate most nonspecific products it was found that addition of three cosolvents DMSO, glycerol and NP 40 was a simple method for increasing the specificity of amplification. PMID- 10733744 TI - Identification of environmental Serratia plymuthica strains with the new combo panels type 1S. AB - Automated systems are required when numerous samples need to be processed, offering both high through put and test of a multiple simultaneously. This study was performed to compare the MicroScan WalkAway automated identification system in conjunction with the new MicroScan Combo Neg Panels Type 1S with conventional biochemical methods for identifying ten environmental Serratia plymuthica strains. High correlation between both methods were observed for all the 21 tests evaluated, and the MicroScan system was found capable of correctly identifying all S. plymuthica strains tested. In all tests, the percentage of correlation was 100%, except in raffinose test (91%). PMID- 10733745 TI - An in vitro system from Plasmodium falciparum active in endogenous mRNA translation. AB - An in vitro translation system has been prepared from Plasmodium falciparum by saponin lysis of infected-erythrocytes to free parasites which were homogeneized with glass beads, centrifuged to obtain a S-30 fraction followed by Sephadex G-25 gel filtration. This treatment produced a system with very low contamination of host proteins (<1%). The system, optimized for Mg2+ and K+, translates endogenous mRNA and is active for 80 min which suggests that their protein factors and mRNA are quite stable. PMID- 10733746 TI - Frequency of CCR5 delta-32 mutation in human immunodeficiency virus (HIV) seropositive and HIV-exposed seronegative individuals and in general population of Medellin, Colombia. AB - Repeated exposure to human immunodeficiency virus (HIV) does not always result in seroconversion. Modifications in coreceptors for HIV entrance to target cells are one of the factors that block the infection. We studied the frequency of Delta-32 mutation in ccr5 gene in Medellin, Colombia. Two hundred and eighteen individuals distributed in three different groups were analyzed for Delta-32 mutation in ccr5 gene by polymerase chain reaction (PCR): 29 HIV seropositive (SP), 39 exposed seronegative (ESN) and 150 individuals as a general population sample (GPS). The frequency of the Delta-32 mutant allele was 3.8% for ESN, 2.7% for GPS and 1.7% for SP. Only one homozygous mutant genotype (Delta-32/Delta-32) was found among the ESN (2.6%). The heterozygous genotype (ccr5/Delta-32) was found in eight GPS (5.3%), in one SP (3.4%) and in one ESN (2.6%). The differences in the allelic and genotypic frequencies among the three groups were not statistically significant. A comparison between the expected and the observed genotypic frequencies showed that these frequencies were significantly different for the ESN group, which indirectly suggests a protective effect of the mutant genotype (Delta-32/Delta-32). Since this mutant genotype explained the resistance of infection in only one of our ESN persons, different mechanisms of protection must be playing a more important role in this population. PMID- 10733748 TI - Antimicrobial susceptibility of Campylobacter jejuni subsp. jejuni assessed by E test and double dilution agar method in Southern Chile. AB - The susceptibility patterns of 108 Campylobacter jejuni subsp. jejuni clinical strains, to six antimicrobial agents was determined by using the E-test and the double dilution agar methods. Using both methods, no strain was found to be resistant to ciprofloxacin, erythromycin and gentamicin, but two (1.8%) were resistant to tetracycline and all to aztreonam. Seven (6.5%) strains were resistant to ampicillin by the E-test and five (4.6%) by the double dilution agar method and by both methods. No great discrepancies were observed between both methods. PMID- 10733747 TI - Hepatitis B virus exposure in human immunodeficiency virus seropositive Cuban patients. AB - In order to estimate the prevalence of serological markers of exposure to Hepatitis B Virus (HBV), 295 subjects were selected at random from the National Registry of human immunodeficiency virus positive subjects. Evidence of exposure to HBV was defined as: testing Hepatitis B surface antigen (HBsAg) and anti Hepatitis B core antigen (anti-HBc) positive or anti-HBc positive only. Overall, 133 (45.5%) were positive for anti-HBc and 15 (5.1%) resulted positive to HBsAg. Significant statistical association was found between male sex and exposure to HBV (p<0.01). Homosexual or bisexual behavior was found to be strongly associated to HBV exposure (p<0.001). In conclusion, the prevalence of HBV serological markers is higher in Cuban HIV positive subjects compared to the Cuban general population. PMID- 10733749 TI - Aspects related to productivity for four generations of a Lutzomyia longipalpis laboratory colony. AB - A closed colony of Lutzomyia longipalpis was established with specimens collected in the Raposa - Serra do Sol indian reservoir, one of the main foci of visceral leishmaniasis in the State of Roraima, Brazil. Biological observations were made on four generations of a L. longipalpis colony with emphasis on productivity. Aspects studied were the number of laid and retained eggs, and the number of adults (male and female) per generation. During the four generations the percentage of engorged females that laid eggs varied from 64.2% (third generation F3) to 90.3% (second generation-F2). The mean number of eggs laid per female varied from 23.6 (F3) to 39. 9 (first generation-F1). The maximum number of eggs laid per female varied from 84 (F3) to 124 (F1). The mean number of retained eggs per female was 12.7 (parental generation-P and F1) to 22.1 (F2). The number of females exceeded the number of males in all generations. However, significant difference for male/female ratio was found only for F3. Fecundity rates were between 42.1 (F3) and 58.3 (F2). From a total of 439 blood-fed females, 355 females laid 12,257 eggs that yield 5,354 adults (2,525 males and 2,829 females) in four generations. F2 presented maximum productivity and fecundity rates. PMID- 10733750 TI - Age structure and abundance in populations of muscoid flies from a poultry facility in Southeast Brazil. AB - Muscina stabulans, M. domestica, Chrysomya putoria, C. megacephala and Stomoxys calcitrans were the most abundant muscoid flies captured in a poultry facility in southeastern Brazil. We examined the gonadotrophic profiles of the females caught at different sites and different times and found that Mu. stabulans and M. domestica, the predominant species, presented similar gonadotrophic profiles only when captured on the manure under the cages, but very different and sometimes opposite gonadotrophic profiles when sampled from wooden posts, vegetation or electric cords. We also determined sex ratios and relative abundance for these two species and found significant differences between them. More than 50% of the females of both species of Chrysomya captured on manure carried eggs or exhibited signs of recent oviposition. The vast majority of S. calcitrans presented ovaries with eggs or signs of recent oviposition. A small proportion of them had ovaries in the recent emerged condition. Our data on ovarian stages, sex ratio and relative abundance allowed us to associate different gonadotrophic profiles with each site and characterize each site as a resting, ovipositing or mating site. PMID- 10733751 TI - Ultrastructural study of the TG180 murine sarcoma cell invasion by Toxoplasma gondii: comparison between in vivo and in vitro cell cultures. AB - Infection of non-adherent TG180 murine sarcoma cells with Toxoplasma gondii was compared, at the ultrastructural level, in both in vivo and in vitro conditions. Suspensions of 3.0 x 10(6) TG180 cells infected in vitro with 1.0 x 10(6) parasites of the RH strain were harvested between the first and 6th day post infection and processed for transmission electron microscopy. In vivo infection was made by intraperitoneal inoculation in mice of 1.0 x 10(6) TG180 cells, that were co-inoculated with a parasite suspension at the same cell concentration. Cells were harvested 10, 20, 30 min and 24, 48 h post-inoculation and processed for transmission electron microscopy at the same conditions of the in vitro culture. It was observed TG180 murine sarcoma cells with intense and equivalent intracellular parasitism in both conditions. Host cells with parasitophorous vacuoles containing up to 16 parasites, as well as parasites undergoing mitoses or presenting a bradyzoite-like morphology, were frequently seen in both culture methods. PMID- 10733753 TI - Combination vaccines: development, clinical research and approval PMID- 10733752 TI - Nuclear phenotype changes after heat shock in Panstrongylus megistus (Burmeister). AB - The nuclear phenotypes of Malpighian tubule epithelial cells of male nymphs of the blood-sucking insect, Panstrongylus megistus, subjected to short- and long duration heat shocks at 40oC were analyzed immediately after the shock and 10 and 30 days later. Normal nuclei with a usual heterochromatic body as well as phenotypes indicative of survival (unravelled heterochromatin, giants) and death (apoptosis, necrosis) responses were observed in control and treated specimens. However, all nuclear phenotypes, except the normal ones, were more frequent in shocked specimens. Similarly altered phenotypes have also been reported in Triatoma infestans following heat shock, although at different frequencies. The frequency of the various nuclear phenotypes observed in this study suggests that the forms of cell survival observed were not sufficient or efficient enough to protect all of the Malpighian tubule cells from the deleterious effects of stress. In agreement with studies on P. megistus survival following heat shock, only long-duration shock produced strongly deleterious effects. PMID- 10733754 TI - Human factors and cardiac surgery: a multicenter study. AB - OBJECTIVE: To study the role of human factors on surgical outcomes, with a series of 243 arterial switch operations performed by 21 surgeons taken as a model. METHODS: The following data were collected: patient-specific and procedural variables, self-assessment questionnaires, and a written report from a human factors researcher who observed the operation. The relationship of patient specific variables to outcomes (death and death and/or near miss) was used to develop a multivariable baseline model to analyze the role of human factors after adjustment for these variables. RESULTS: The overall mortality was 6.6% with 24.3% of cases resulting in death and death and/or near misses. The self assessment questionnaires were found to be unhelpful. Major and minor human failures were extracted from the written report. Major negative events were potentially life-threatening failures, whereas minor events were failures that, in isolation, were not expected to have serious consequences. Major events were closely related to death (P <.001) and death and/or near misses (P <.001). Appropriate compensation, however, sharply reduced the risk of death (P =.003). The total number of minor events was also closely related to both death and death and/or near misses (P <.001). CONCLUSION: The study highlights the role of human factors in negative surgical outcomes. Even in the most eventful circumstances, however, appropriate human factors defense mechanisms can lead to a successful outcome. PMID- 10733755 TI - A clinical trial combining donor bone marrow infusion and heart transplantation: intermediate-term results. AB - BACKGROUND: Donor chimerism (the presence of donor cells of bone marrow origin) is present for years after transplantation in recipients of solid organs. In lung recipients, chimerism is associated with a lower incidence of chronic rejection. To augment donor chimerism with the aim to enhance graft acceptance and to reduce immunosuppression, we initiated a trial combining infusion of donor bone marrow with heart transplantation. Reported herein are the intermediate-term results of this ongoing trial. METHODS: Between September 1993 and August 1998, 28 patients received concurrent heart transplantation and infusion of donor bone marrow at 3.0 x 10(8) cells/kg (study group). Twenty-four contemporaneous heart recipients who did not receive bone marrow served as controls. All patients received an immunosuppressive regimen consisting of tacrolimus and steroids. RESULTS: Patient survival was similar between the study and control groups (86% and 87% at 3 years, respectively). However, the proportion of patients free from grade 3A rejection was higher in the study group (64% at 6 months) than in the control group (40%; P =.03). The prevalence of coronary artery disease was similar between the two groups (freedom from disease at 3 years was 78% in study patients and 69% in controls). Similar proportions of study (18%) and control (15%) patients exhibited in vitro evidence of donor-specific hyporesponsiveness. CONCLUSIONS: The infusion of donor bone marrow reduces the rate of acute rejection in heart recipients. Donor bone marrow may play an important role in strategies aiming to enhance the graft acceptance. PMID- 10733756 TI - Pulmonary hemodynamics contribute to indicate priority for lung transplantation in patients with cystic fibrosis. AB - OBJECTIVE: Lung transplantation is a viable option for patients with cystic fibrosis. The current strategy of selection, based on spirometry and deterioration of quality of life, results in a high mortality on the waiting list. We reviewed the case histories of patients with cystic fibrosis accepted for lung transplantation to ascertain whether pulmonary hemodynamics could contribute to predict life expectancy. METHODS: Forty-five patients with cystic fibrosis were accepted: 11 died on the waiting list (group I), 24 underwent transplantation (group II), and 10 are still waiting (group III). During evaluation we recorded spirometry, oxygen requirement, ratio of arterial oxygen tension to inspired oxygen fraction (PaO (2)/FIO (2)), arterial carbon dioxide tension (PaCO (2)), 6-minute walk test results, right ventricular ejection fraction, echocardiography, and pulmonary hemodynamics. We compared data from group I, II, and III patients. A comparison was also made within group II between the data collected at the time of evaluation and at the time of transplantation to quantify the deterioration during the waiting time. RESULTS: The waiting time, spirometry, 6-minute walk test results, and right ventricular ejection fraction did not differ among the three groups. A statistically significant difference was found for PaO (2)/FIO (2), PaCO (2), mean pulmonary artery pressure, cardiac index, pulmonary arterial wedge pressure, and intrapulmonary shunt between groups I and II. Groups I and III showed statistically significant differences for mean pulmonary artery pressure, PaO (2)/FIO (2), and systemic vascular resistance indexed. No differences were observed between groups II and III. The comparison within group II showed a significant deterioration of pulmonary hemodynamics during the waiting time. CONCLUSIONS: Pulmonary hemodynamics are worst in patients dying on the waiting list and deteriorate significantly during the waiting time. They may thus contribute to establish priority for lung transplantation in patients with cystic fibrosis. PMID- 10733757 TI - Transgenic swine lungs expressing human CD59 are protected from injury in a pig to-human model of xenotransplantation. AB - BACKGROUND: Pulmonary xenotransplantation is currently limited by hyperacute rejection mediated in part by xenoreactive natural antibody and complement. Transgenic swine organs that express the human complement regulatory protein CD59 have demonstrated improved survival in models of pig-to-primate xenotransplantation. OBJECTIVE: The purpose of this study was to evaluate transgenic swine lungs that express the human complement regulatory protein CD59 in a model of pig-to-human xenotransplantation. METHODS: Transgenic swine lungs (n = 5, experimental group) and outbred swine lungs (n = 6, control group) were perfused with fresh, whole human blood through a centrifugal pump on an ex vivo circuit. Functional data were collected throughout perfusion. Immunoglobulin and complement studies were performed on perfusate samples, and both histologic and immunofluorescent analyses were performed on tissue sections. RESULTS: Mean lung survival for the experimental group was increased when compared with controls, 240 +/- 0 minutes versus 35.3 +/- 14.5 minutes, respectively, with a P value of less than.01. A decreased rise in pulmonary vascular resistance at 15 minutes was observed in the experimental group (343 +/- 87 mm Hg. L(-1). min(-1), in contrast to the control group (1579 +/- 722 mm Hg. L(-1). min(-1); P <.01). Pulmonary compliance at 15 minutes was improved for the experimental group versus control group (9.31 +/- 1.41 mL. cm(-2) H(2)O and 4.11 +/- 2.84 mL. cm(-2) H(2)O, respectively; P <.01). SC5b-9 generation in the plasma perfusate was delayed for the experimental group versus the control group. Immunofluorescent examination of tissue sections demonstrated equivalent deposition of immunoglobulin G, immunoglobulin M, C1q, and C3 in both groups, with reduced deposition of C9 in the experimental group. CONCLUSIONS: Transgenic swine pulmonary xenografts that express the human complement regulatory protein CD59 demonstrated improved function and survival in an ex vivo model of pig-to-human xenotransplantation. PMID- 10733758 TI - Transforming growth factor beta in relation to cardiac allograft vasculopathy after heart transplantation. AB - BACKGROUND: Cardiac allograft vasculopathy is a frequent sequel to cardiac transplantation, but the role of cytokines on the subsequent development of vasculopathy is still largely unknown. METHODS: We retrospectively studied 172 heart transplant recipients to investigate the relationship between the development of vasculopathy and various factors including the presence of transforming growth factor (TGF-beta) in the graft. Endomyocardial biopsy specimens were stained with antibodies for TGF-beta and CD+68, and a TGF-beta staining score was derived. Vasculopathy was diagnosed by angiography and rejection was graded according to the International Society of Heart and Lung Transplantation classification. TGF-beta(1) genotype was determined by polymerase chain reaction analysis of DNA. RESULTS: After a mean follow-up period of 68 +/- 32 months, the prevalence of significant vasculopathy was 52%. The TGF-beta staining score was higher in patients with more severe vasculopathy (95% confidence interval = 8.9-12.1) than in those who showed minimal or mild vasculopathy score changes of more than 7 (95% confidence interval = 3.4-5.1), P =.0001. TGF-beta expression correlated with the degree of vasculopathy (r = 0.73, P <.0007) during the study period. Risks for vasculopathy were recipient homozygous TGF-beta genotype, recurrent rejection, recipient history of ischemic heart disease, donor male sex, old donor age (years), and donor history of subarachnoid hemorrhage. CONCLUSION: A strong association exists between the expression of TGF-beta in cardiac biopsy specimens and the development of vasculopathy. TGF-beta in the cardiac allograft is related to its genotype and to the number of rejection episodes. Strategies to down-regulate TGF-beta production might improve the outcome of cardiac allografts. PMID- 10733759 TI - Mechanisms of tolerance induction and prevention of cardiac allograft vasculopathy in miniature swine: the effect of augmentation of donor antigen load. AB - OBJECTIVE: Cotransplantation of a donor kidney along with a heart allograft can induce tolerance to both organs and prevent cardiac allograft vasculopathy in miniature swine. To determine whether the tolerogenic effect of donor kidney cotransplantation was due to an effect specific to the kidney graft or to an increase in donor antigen load, we compared heart-kidney recipients with recipients receiving two class I disparate hearts or with recipients receiving donor peripheral mononuclear cells at the time of isolated heart transplantation. METHODS: Recipients of major histocompatibility complex class I disparate allografts received 12 days of cyclosporine (INN: cyclosporin; 10-13 mg/kg administered intravenously on days 0-11). Group 1 animals received a heart alone (n = 5). Group 2 animals received heart and kidney allografts (n = 4). Group 3 animals received two major histocompatibility complex-matched heart allografts (n = 4). Two double-heart recipients were thymectomized 21 days before transplantation. Group 4 animals received a heart allograft and an infusion of high-dose donor peripheral blood leukocytes (2.5 x 10(9) cells/kg, n = 2). RESULTS: Vasculopathy developed in group 1 recipients and the allografts were rejected within 55 days. Group 2 recipients accepted their heart and kidney allografts indefinitely without vasculopathy. Euthymic recipients from group 3 accepted their hearts long-term (>190 and >197 days), but vascular lesions developed. In thymectomized recipients from group 3, the hearts were rejected in 63 and 96 days with severe vasculopathy. Group 4 recipients demonstrated transient macrochimerism but their hearts were rejected within 47 and 63 days. CONCLUSIONS: The beneficial effects of donor kidney cotransplantation on cardiac allograft survival and prevention of cardiac allograft vasculopathy are likely to involve both an increase in donor antigen load and an effect specific to the kidney allograft. PMID- 10733760 TI - Hypoplastic left heart syndrome: valuing the survival. AB - OBJECTIVE: To examine the survival, developmental status, quality of life, and direct medical costs of children with hypoplastic left heart syndrome who have undergone stage I, II, and III reconstructive surgery. METHODS: A total of 106 children underwent staged repair for classic hypoplastic left heart syndrome between February 1990 and March 1999 (stage I: 106; stage II: 49; stage III: 25; 4 converted to heart transplantation). Survival was analyzed by the Kaplan-Meier method. In a cross-sectional study, parents assessed quality of life by completing the Infant/Toddler Child Health Questionnaire or Child Health Questionnaire Parent Format-28; they assessed developmental progress by completing the Ages and Stages Questionnaire. The ratio-of-costs-to-charges method was used to derive hospital costs, and payments were used to capture physician time and wholesale pricing for outpatient medications. RESULTS: Institutional 1-year and 5-year actuarial survivals were 58% and 54%. Birth weight, the need for preoperative inotropic drugs, and surgical experience were predictors of survival. Norwood I patients achieved fewer developmental benchmarks than those who survived to subsequent stages. Child Health Questionnaire Parent Format-28 mean summary scores for physical and psychosocial health were 48.5 +/- 6.3 and 42.8 +/- 9.9. The median inpatient costs for stage I, II, and III repairs were $51,000, $33,892, and $52,183, respectively. Monthly outpatient and readmission costs were less than 10% of total costs. CONCLUSION: A prospective, large-scale study of the comprehensive outcomes of staged repair and transplantation is needed. This study will need to address the longer-term developmental and quality-of-life outcomes, as well as the long-term cost effectiveness of these procedures. PMID- 10733761 TI - Tissue-engineered valved conduits in the pulmonary circulation. AB - OBJECTIVE: Bioprosthetic and mechanical valves and valved conduits are unable to grow, repair, or remodel. In an attempt to overcome these shortcomings, we have evaluated the feasibility of creating 3-leaflet, valved, pulmonary conduits from autologous ovine vascular cells and biodegradable polymers with tissue engineering techniques. METHODS: Endothelial cells and vascular medial cells were harvested from ovine carotid arteries. Composite scaffolds of polyglycolic acid and polyhydroxyoctanoates were formed into a conduit, and 3 leaflets (polyhydroxyoctanoates) were sewn into the conduit. These constructs were seeded with autologous medial cells on 4 consecutive days and coated once with autologous endothelial cells. Thirty-one days (+/-3 days) after cell harvesting, 8 seeded and 1 unseeded control constructs were implanted to replace the pulmonary valve and main pulmonary artery on cardiopulmonary bypass. No postoperative anticoagulation was given. Valve function was assessed by means of echocardiography. The constructs were explanted after 1, 2, 4, 6, 8, 12, 16, and 24 weeks and evaluated macroscopically, histologically, and biochemically. RESULTS: Postoperative echocardiography of the seeded constructs demonstrated no thrombus formation with mild, nonprogressive, valvular regurgitation up to 24 weeks after implantation. Histologic examination showed organized and viable tissue without thrombus. Biochemical assays revealed increasing cellular and extracellular matrix contents. The unseeded construct developed thrombus formation on all 3 leaflets after 4 weeks. CONCLUSION: This experimental study showed that valved conduits constructed from autologous cells and biodegradable matrix can function in the pulmonary circulation. The progressive cellular and extracellular matrix formation indicates that the remodeling of the tissue engineered structure continues for at least 6 months. PMID- 10733762 TI - Transcaval repair of the sinus venosus syndrome. AB - BACKGROUND: Surgical correction of the sinus venosus syndrome has been associated with sinus node dysfunction and venous obstruction postoperatively. We present the long-term follow-up of a lateral transcaval approach, which closes the atrial communication and corrects the partial anomalous pulmonary venous connection to the superior vena cava with the use of a simple pericardial patch. METHODS: The records of 66 patients undergoing repair between April 1981 and April 1997 were examined. Mean age at repair was 10.2 years (range, 1.5-65 years; median, 5 years). Six patients had a left superior vena cava, 4 had an additional atrial septal defect, and 2 had coronary artery bypass grafts. Immediate and long-term follow-up included physical examination, electrocardiography, transthoracic echocardiography, and use of a 24-hour ambulatory Holter monitor. Sinus node function, incidence of significant arrhythmia, and evidence of mechanical venous obstruction were assessed. RESULTS: Follow-up data were available for 64 (97%) patients for a mean follow-up of 4.1 years (range, 1-9 years). There were no deaths. No evidence of residual atrial septal defect, superior vena cava, or venous obstruction were found by echocardiography. On electrocardiography all patients were in sinus rhythm, with no arrhythmia seen. Holter monitoring was performed at a mean of 7.3 years postoperatively. All patients had normal sinus node function, and no sustained atrial arrhythmia was seen. CONCLUSION: Transcaval repair is a simple technique that does not interfere with sinus node function. There is no evidence to suggest that this approach leads to venous obstruction. PMID- 10733764 TI - Commentary PMID- 10733763 TI - Intracardiac thrombus formation after the Fontan operation. AB - OBJECTIVES: Intracardiac thrombus formation is suspected to be a specific sequela after the Fontan operation and is difficult to determine by means of routine transthoracic echocardiography. The aim of our study was to evaluate the occurrence of intracardiac thrombi in the different types of Fontan modifications and to identify predisposing risk factors. METHODS: We evaluated 52 patients who had undergone a Fontan-type operation and were free of symptoms regarding thrombosis as determined by transesophageal echocardiography. RESULTS: In 17 (33%) patients thrombus formation could be found without clinical evidence of thromboembolic complications. Neither underlying morphologic disease nor age at operation, type of Fontan operation, sex, follow-up interval, arrhythmias, or laboratory or hemodynamic findings could be identified as predisposing risk factors. CONCLUSION: In patients having had a Fontan operation with inadequate or without anticoagulation medication, we would recommend routine transesophageal echocardiography to exclude eventual thrombi. Because of the high incidence of thrombi, we suggest oral anticoagulation therapy in all patients. PMID- 10733766 TI - Commentary PMID- 10733765 TI - Re-creation of sinuses is important for sparing the aortic valve: a finite element study. AB - OBJECTIVE: The treatment of choice for aortic valve insufficiency due to root dilatation has become root replacement with aortic valve sparing. However, root replacement with a synthetic graft may result in altered valve stresses. The purpose of this study was to compare the stress/strain patterns in the spared aortic valve in different root replacement procedures by means of finite element modeling. METHODS: Our finite element model of the normal human root and valve was modified to simulate and evaluate three surgical techniques: (1) "cylindrical" graft sutured below the valve at the anulus, (2) "tailored" graft sutured just above the valve, and (3) "pseudosinus" graft, tailored and sutured below the valve at the anulus. Simulated diastolic pressures were applied, and stresses and strains were calculated for the valve, root, and graft. Leaflet coaptation was also quantified. RESULTS: All three root replacement models demonstrated significantly altered leaflet stress patterns as compared with normal patterns. The cylindrical model showed the greatest increases in stress (16%-173%) and strain (10%-98%), followed by the tailored model (stress +10% 157%, strain +9%-36%). The pseudosinus model showed the smallest increase in stress (9%-28%) and strain (2%-31%), and leaflet coaptation was closest to normal. CONCLUSION: Valve-sparing techniques that allow the potential for sinus space formation (tailored, pseudosinus) result in simulated leaflet stresses that are closer to normal than the cylindrical technique. Normalized leaflet stresses in the clinical setting may result in improved longevity of the spared valve. PMID- 10733767 TI - Long-term results of simultaneous carotid endarterectomy and myocardial revascularization with cardiopulmonary bypass used for both procedures. AB - OBJECTIVE: Controversy continues about the treatment of patients with a concomitant occlusive disease of the coronary and carotid arteries. Our operative strategy in these patients is to do simultaneous carotid endarterectomy and myocardial revascularization in conjunction with cardiopulmonary bypass with mild hypothermia. We report our experience with this kind of one-stage procedure and its retrospective long-term results. METHODS: From February 1985 to September 1998, 340 patients underwent simultaneous carotid endarterectomy and myocardial revascularization. The average age of the patients was 65.3 years; 45.6% were neurologically symptomatic, and 44.4% had bilateral carotid stenosis. The indication for carotid endarterectomy was lumen diameter reduction of more than 75%, angiographic signs of thrombogenic endovascular morphology, or both. Carotid endarterectomy was performed in conjunction with cardiopulmonary bypass with mild hypothermia, hemodilution, systemic heparinization, and controlled hemodynamics under pulsatile perfusion for additional cerebral protection. RESULTS: There were 16 perioperative neurologic complications (4.7%), 11 permanent deficits (3.2%), and 9 cardiac complications (2.6%). Early mortality was 2.6% (SE 0.8%): 2 patients had a stroke and 2 had a myocardial infarction. The 5-year survival was 78.9% (SE 2.6%), and freedom from ipsilateral stroke and cardiac event were 93.2% (SE 1.5%) and 87.5% (SE 2.1%), respectively. The predictor for early death was age over 70 years, and predictors for late death were age over 70 years, previous myocardial infarction, previous stroke, and bilateral carotid stenosis of greater than 90%. CONCLUSION: On the basis of our long-term results, we believe that simultaneous carotid endarterectomy and myocardial revascularization in conjunction with cardiopulmonary bypass is a method safe enough to prefer its routine use with acceptable low operative risk and satisfactory long-term morbidity. PMID- 10733768 TI - Commentary PMID- 10733769 TI - Ring annuloplasty prevents delayed leaflet coaptation and mitral regurgitation during acute left ventricular ischemia. AB - OBJECTIVE: Incomplete mitral leaflet coaptation during acute left ventricular ischemia is associated with end-diastolic mitral annular dilatation and ischemic mitral regurgitation. Annular rings were implanted in sheep to investigate whether annular reduction alone is sufficient to prevent mitral regurgitation during acute posterolateral left ventricular ischemia. METHODS: Radiopaque markers were inserted around the mitral anulus, on papillary muscle tips, and on the central meridian of both mitral leaflets in three groups of sheep: control (n = 5), Physio ring (n = 5) (Baxter Cardiovascular Div, Santa Ana, Calif), and Duran ring (n = 6) (Medtronic Heart Valve Div, Minneapolis, Minn). After 8 +/- 1 days, animals were studied with biplane videofluoroscopy before and during left ventricular ischemia. Annular area was calculated from 3-dimensional marker coordinates and coaptation defined as minimal distance between leaflet edge markers. RESULTS: Before ischemia, leaflet coaptation occurred just after end diastole in all groups (control 17 +/- 41, Duran 33 +/- 30, Physio 33 +/- 24 ms, mean +/- SD, P >.2 by analysis of variance). During ischemia, regurgitation was detected in all control animals, and leaflet coaptation was delayed to 88 +/- 8 ms after end-diastole (P =.02 vs preischemia). This was associated with increased end-diastolic annular area (8.0 +/- 0.9 vs 6.7 +/- 0.6 cm(2), P =.004) and septal lateral annular diameter (2.9 +/- 0.1 vs 2.5 +/- 0.1 cm, P =.02). Mitral regurgitation did not develop in Duran or Physio sheep, time to coaptation was unchanged (Duran 25 +/- 25 ms, Physio 30 +/- 48 ms [both P >.2 vs preischemia]), and annular area remained fixed. CONCLUSION: Mitral annular area reduction and fixation with an annuloplasty ring eliminated delayed leaflet coaptation and prevented mitral regurgitation during acute left ventricular ischemia after ring implantation. PMID- 10733770 TI - Postinfarction ventricular septal rupture: early repair through the right atrial approach. AB - OBJECTIVE: Early repair of posterior ventricular septal rupture associated with myocardial infarction by means of transinfarct ventriculotomy is technically challenging and can be associated with significant mortality and morbidity. An alternative route of exposing the septum is through the right atrium. This technique, which avoids direct incision of the ventricle in select patients, reduces postrepair bleeding and impairment of ventricular contractile function. METHODS: The results of 12 patients operated on over a 20-year period were reviewed and analyzed. Late follow-up was obtained in all patients who survived the operation. There were 9 men and 3 women, with a mean age of 69.9 years. The mean time between acute myocardial infarction and surgery was 7.3 days (range, 2 16 days). Six patients were in New York Heart Association class IV, and 3 patients presented for surgery in cardiogenic shock. One patient had previously undergone a coronary artery bypass. The surgical technique included a standard sternotomy approach with a transatrial approach to the septal rupture. In all patients the septal rupture was repaired with a Dacron patch. RESULTS: There were 3 early deaths and 1 late death; one patient was reoperated on for a residual shunt. Postoperative complications included low cardiac output, acute renal tubular necrosis, and supraventricular arrhythmia. Eight patients are alive and undergoing echocardiographic investigation, and only 1 patient had a small residual shunt. CONCLUSION: Our experience shows that a posterior ventricular septal rupture can be safely repaired through a transatrial approach. Avoiding additional damage to the ventricle, it reduces the risks of the postoperative bleeding and enhances survival. PMID- 10733771 TI - Preservation of the right atrial appendage improves reduced plasma atrial natriuretic peptide levels after the maze procedure. AB - OBJECTIVES: The present study was conducted to determine whether preservation of the right atrial appendage lessens the decrease of plasma atrial natriuretic peptide levels after the maze procedure and whether the increase of plasma atrial natriuretic peptides improves the ability of the kidneys to excrete the fluid load after the operation. METHODS: We evaluated 42 patients who underwent the maze procedure. The right atrial appendage was preserved in 22 patients but not in 20. Blood samples were obtained before and after the operation for measurement of atrial natriuretic peptides. To evaluate the influence of atrial natriuretic peptides on the ability of the kidneys, we also measured body weight, fluid balance, and the doses of furosemide and dopamine administered after the operation. RESULTS: The restoration to sinus rhythm at 1 month after was comparable in the two groups. Plasma atrial natriuretic peptide levels significantly increased after the operation in patients in whom the right atrial appendage was preserved (1 day after: 23.4 +/- 17.8 vs 3 days after: 42.7 +/- 23.6 and 7 days after: 36.3 +/- 23.7 pg/mL, P <.05) but not in patients in whom the right atrial appendage was not preserved (1 day after: 20.0 +/- 19.6, 3 days after: 28.5 +/- 19.3, and 7 days after: 23.0 +/- 16.1 pg/mL). Furthermore, plasma atrial natriuretic peptide levels were significantly lower in patients in whom the right atrial appendage was not preserved than in patients in whom the right atrial appendage was preserved at 3 and 7 days after the operation. The fluid balance during the first 7 days of the postoperative period was comparable in the two groups, although the total dose of dopamine used in the same period was significantly smaller in patients in whom the right atrial appendage was preserved than in patients in whom the right atrial appendage was not preserved (155.3 +/- 119.0 vs 244.9 +/- 129.0 microg/kg, P <.05). CONCLUSIONS: The present study showed that preservation of the right atrial appendage lessens the decrease of plasma atrial natriuretic peptide levels after the maze procedure and that increased plasma atrial natriuretic peptides may improve the ability of the kidneys to excrete the fluid load after the operation. PMID- 10733772 TI - Paclitaxel-induced apoptosis in non-small cell lung cancer cell lines is associated with increased caspase-3 activity. AB - OBJECTIVE: Our objective was to determine whether paclitaxel-induced apoptosis in human lung cancer cells is Fas dependent. METHODS: Human lung cancer cell lines were evaluated for morphologic evidence of apoptosis, DNA fragmentation (TUNEL positivity), and caspase-3 activation after paclitaxel treatment. Human lung adenocarcinoma, squamous cell carcinoma, undifferentiated lung carcinoma, and bronchoalveolar carcinoma cell lines were each cultured in 10 micromol/L paclitaxel. RESULTS: After 24 hours of culture in paclitaxel, a 22% to 69% increase in the number of apoptotic cells was evident by means of methylene blue azure A-eosin staining with characteristic blebbing and nuclear condensation. TUNEL assay also confirmed an increase of 19.9% to 73.0% of cells with nuclear fragmentation. Caspase-3 activity, assayed by Z-DEVD cleavage, increased from 20% to 215% (P <.05). ZB4, an antagonistic anti-Fas antibody, did not block paclitaxel induction of caspase-3 activity (155.8 vs 165.8 U, not significant). Apoptotic morphologic changes were inhibited in cells cultured in the presence of paclitaxel and Ac-DEVD-CHO, a caspase-3 inhibitor. CONCLUSIONS: Paclitaxel induces apoptosis in lung cancer cell lines, as assessed by a consistent increase in caspase-3 activity, DNA laddering, and characteristic morphologic changes. Paclitaxel-induced apoptosis in human lung cancer cells is associated with caspase-3 activation but is not Fas dependent. PMID- 10733773 TI - Increased vascular endothelial growth factor and vascular endothelial growth factor-c and decreased nm23 expression associated with microdissemination in the lymph nodes in stage I non-small cell lung cancer. AB - OBJECTIVE: We examined a microdissemination of cancer cells in lymph nodes and assessed its clinical and biologic characteristics. METHODS: Both primary tumors and lymph nodes (2030 nodes) were obtained from 122 patients with primary stage I lung cancer who underwent curative operations with routine systematic nodal dissection of both the hilar and the mediastinal nodes. Immunohistochemical anticytokeratin staining was used to detect nodal microdissemination of cancer cells. Vascular endothelial growth factor, vascular endothelial growth factor type C, and nm23 expression at primary sites were also immunohistochemically studied. RESULTS: In total, 35 patients (29%) had cytokeratin-positive cells in lymph nodes. Increased expression of vascular endothelial growth factor (P =.0001) and vascular endothelial growth factor type C (P <. 0001) at primary sites were significantly associated with nodal microdissemination, and nm23 was inversely correlated with microdissemination (P =.008). The 3- and 5-year survivals for the patients with nodal microdissemination were 57% and 54%, respectively, which was a significantly worse prognosis as compared with those prognoses (83% and 76%) for the patients without nodal microdissemination (P =.006). The independent prognostic impact of nodal microdissemination was not clear; however, vascular endothelial growth factor retained independent significance. CONCLUSION: All of these findings lead us to conclude that the microspread of tumor cells in nodes detected by immunohistochemical anticytokeratin staining is definitely a metastasis with a high risk of systemic disease. PMID- 10733774 TI - Survival related to lymph node involvement in lung cancer after sleeve lobectomy compared with pneumonectomy. AB - OBJECTIVE: The purpose of this study was to compare the outcomes after sleeve lobectomy and pneumonectomy for patients with non-small cell lung cancer distributed according to their nodal involvement status. METHODS: Of 1172 patients in whom primary non-small cell lung carcinoma, including mediastinal lymph nodes, was completely excised, 151 patients underwent sleeve lobectomy and 60 underwent pneumonectomy. For bias reduction in comparison with a nonrandomized control group, we paired 60 patients undergoing sleeve lobectomy with 60 patients undergoing pneumonectomy by using the nearest available matching method. RESULTS: The 30-day postoperative mortality was 2% (1/60) in the pneumonectomy group and 0% in the sleeve lobectomy group. Postoperative complications occurred in 13% of patients in the sleeve lobectomy group and in 22% of those in the pneumonectomy group. Local recurrences occurred in 8% of patients in the sleeve lobectomy group and in 10% of those in the pneumonectomy group. The overall 5- and 10-year survivals for the sleeve lobectomy group were 48% and 36%, respectively, whereas those for the pneumonectomy group were 28% and 19%, respectively (P =.005). Multivariable analysis showed that the operative procedure, T factor, and N factor were significant independent prognostic factors and revealed that survival after sleeve lobectomy was significantly longer than that after pneumonectomy (P =.03). CONCLUSIONS: These data suggest that sleeve lobectomy should be performed instead of pneumonectomy in patients with non-small cell lung cancer regardless of their nodal status whenever complete resection can be achieved because this is a lung-saving procedure with lower postoperative risks and is as curative as pneumonectomy. PMID- 10733775 TI - The early response genes c-jun and HSP-70 are induced in regional cardiac stunning in conscious mammals. AB - OBJECTIVES: A reversible contractile dysfunction without necrosis after transient myocardial ischemia has been termed stunning. The molecular mechanisms underlying this phenomenon are only now beginning to be unraveled. It is conceivable that the expression of early-response genes may play a crucial role in stunning. METHODS: The expression of HSP-70, c-jun, and GRP-94 was investigated in a chronically instrumented dog model (n = 9). The left anterior descending coronary artery was occluded temporarily for 10 minutes after the animals had fully recovered from instrumentation. The wall thickening fraction was measured in the left anterior descending coronary artery and the nonischemic ramus circumflex of the left coronary artery-perfused region. When the wall thickening fraction of the left anterior descending coronary artery had recovered to 50% of preocclusion values, tissue samples were obtained from the areas perfused by the left anterior descending coronary artery and the nonischemic ramus circumflex of the left coronary artery. RESULTS: The messenger RNA of HSP-70 was increased to 214% +/- 26% in the area perfused by the left anterior descending artery compared with that perfused by the nonischemic ramus circumflex of the left coronary artery. There was no difference in the messenger RNA of GRP-94. The HSP-70 content was elevated to 130% +/- 14% in the left anterior descending artery compared with the area perfused by the ramus circumflex of the left coronary artery, and the c-jun protein content was 70% +/- 25% higher in the ischemic area compared with the control area. CONCLUSIONS: The induction of early-response genes observed here may indicate that they play an adaptive role in myocardial stunning, even in conscious mammals. PMID- 10733776 TI - Rabbit ventricular myocyte volume changes as a direct result of crystalloid cardioplegia in congestive heart failure induced by aortic regurgitation. AB - OBJECTIVES: We hypothesized that the cell volume of ventricular myocytes isolated from hearts in volume-overload congestive failure would respond differently to hypothermic cardioplegia than would sham-operated cohorts. METHODS: Adult rabbits underwent either valvotomy and aortic regurgitation-induced heart failure or sham surgery. Congestive failure was confirmed clinically and by means of echocardiography. Cell volumes of isolated myocytes were measured by digital video microscopy. After equilibration in 37 degrees C physiologic solution, cells were suprafused with 9 degrees C standard or low-Cl(-) St Thomas' Hospital solution followed by reperfusion in 37 degrees C physiologic solution. RESULTS: Exposure to cold St Thomas' Hospital solution for 20 minutes caused sham myocytes to swell by 8% (n = 9); cell volumes fully recovered on normothermic reperfusion. In contrast, congestive failure myocytes (n = 9) maintained their cell volume in cold St Thomas' Hospital solution and during reperfusion. Lowering the [K(+)][Cl( )] product of St Thomas' Hospital solution by partially replacing Cl(-) with an impermeant anion prevented cellular edema in the sham group (n = 8) but caused a 4% swelling in failure myocytes (n = 10) on reperfusion. Osmotically shrinking the failure cells (n = 9) converted their behavior to that of sham cells. CONCLUSIONS: In the absence of ischemia, congestive failure myocytes are less sensitive to cardioplegia-induced edema than sham cells. Low-Cl(-) cardioplegia, which prevents edema and protects the normal heart, induced swelling and may be detrimental in myopathic hearts. Differences in volume regulation in failure and sham myocytes may be due to activation of volume-sensitive channels that are turned off by osmotic shrinkage. PMID- 10733777 TI - Ten weeks of rapid ventricular pacing creates a long-term model of left ventricular dysfunction. AB - OBJECTIVE: Rapid ventricular pacing produces a reliable model of heart failure. Cessation after 4 weeks of rapid ventricular pacing results in rapid normalization of left ventricular function, but the left ventricle remains persistently dilated. We present novel data that show that prolonged rapid ventricular pacing (10 weeks) creates a model of chronic left ventricular dysfunction. METHODS: In 9 dogs undergoing 10 weeks of rapid ventricular pacing, left ventricular function and volumes were serially assessed by using 2 dimensional echocardiography and pressure-volume analysis for 12 weeks after cessation of pacing. RESULTS: Increased end-diastolic volume and decreased systolic and diastolic function were seen at the end of pacing. By 2 weeks of recovery from rapid ventricular pacing, end-diastolic volume and ejection fraction were partially recovered but did not improve further thereafter. Load independent and load-sensitive indices of function obtained by pressure-volume analysis at 8 and 12 weeks of recovery confirmed a persistence of both systolic and diastolic dysfunction. In addition, left ventricular mass increased with pacing and remained elevated at 8 and 12 weeks of recovery. Four of these dogs studied at 6 months of recovery showed similar left ventricular abnormalities. CONCLUSION: Ten weeks of rapid ventricular pacing creates a long-term model of left ventricular dysfunction. PMID- 10733778 TI - Adenosine triphosphate-dependent potassium channel modulation and cardioplegia induced protection of human atrial muscle in an in vitro model of myocardial stunning. AB - OBJECTIVES: Although adenosine triphosphate-dependent potassium channel openers have been shown to enhance cardioplegic protection in animal myocardium, there is a lack of data on human cardiac tissues. We aimed at determining, on human atrial muscle, whether adenosine triphosphate- dependent potassium channels are involved in protection caused by high-potassium cardioplegia and whether adenosine triphosphate-dependent potassium channel activation might improve cardioplegic protection in an in vitro model of myocardial stunning. METHODS: Human atrial trabeculae were obtained from adult patients undergoing cardiac operations. In an organ bath at 37 degrees C, the preparations were subjected to 60 minutes of hypoxia at a high stimulation rate either in Tyrode solution (control, n = 17) or in St Thomas' Hospital solution without additives (n = 6) or associated with 100 nmol/L bimakalim (n = 7) or 1 micromol/L glibenclamide (n = 7), followed by 60 minutes of reoxygenation and 15 minutes of positive inotropic stimulation with 1 micromol/L dobutamine. RESULTS: Atrial developed tension was reduced by hypoxia to 27% +/- 5% of baseline and incompletely recovered after reoxygenation to 38% +/- 7%, whereas dobutamine restored contractility to 74% +/- 7% of basal values. St Thomas' Hospital solution with or without bimakalim improved developed tension after reoxygenation and dobutamine (P <.0001 vs control), whereas glibenclamide inhibited these protective effects of cardioplegic arrest (P =.001 vs St Thomas' Hospital solution). After reoxygenation, the protective effect of bimakalim disappeared at a high pacing rate (400- and 300-ms cycle length) but recovered during dobutamine superfusion. CONCLUSIONS: Adenosine triphosphate-dependent potassium channels are likely involved in the cardioprotective effects of cardioplegia in human atrial trabeculae and adenosine triphosphate-dependent potassium channel activation with bimakalim used as an additive to cardioplegia enhanced protection. PMID- 10733779 TI - Right-sided aortic arch with bilateral ductus: a rare case of nonconfluent pulmonary arteries without associated cardiac anomalies. PMID- 10733780 TI - Pulmonary vascular compromise in a child with postpneumonectomy syndrome. PMID- 10733781 TI - Leiomyosarcoma of the bronchus: report of two cases of resection with long-term follow-up. PMID- 10733783 TI - A possible explanation for the failure to improve the internal jugular venous oxygen saturation with balloon pump-induced pulsatile perfusion PMID- 10733782 TI - A possible explanation for the failure to improve the internal jugular venous oxygen saturation with balloon pump-induced pulsatile perfusion. PMID- 10733784 TI - Vasodilatation after cardiac surgery. PMID- 10733785 TI - Posterior aortoventricular bleeding after aortic valve replacement with the stentless Cryolife-O'Brien valve. PMID- 10733786 TI - A new editor for the journal PMID- 10733787 TI - The Effect of Basic Fibroblast Growth Factor on the Reversion of Omental Adipocytes from Lean and Obese Patients. AB - BACKGROUND: this study was designed to characterize some of the biochemical and molecular genetic changes during reversion of human fat cells. METHODS: mature adipocytes were isolated from greater omental fat tissue of eight lean and 14 massively obese persons by established methodology. RESULTS: at day 7 of adherence to Leighton tubes, there was appreciable depletion of triacylglycerol, as well as assumption of an elongated contour. Relatedly, there was an increase in the expression of Beta-actin mRNA and a significant decrease in the specific activity of cytosolic glycerophosphate dehydrogenase. The decrement in the specific activity of glycerophosphate dehydrogenase, after 7 days in culture, was significant at p < 0.001. Basic fibroblast growth factor at 10 ngml(1) accelerated significantly (p < 0.03) the decrease in the specific activity of glycerophosphate dehydrogenase in adipose cells from lean subjects. In contrast, basic fibroblast growth factor had no significant influence on cells from massively obese persons. CONCLUSION: such resistance may contribute to the intractability of massive obesity. PMID- 10733788 TI - The Effects of Fat Distribution on Resting Energy Expenditure in Premenopausal Morbidly Obese Females. AB - BACKGROUND: upper body, or abdominal, distribution of body fat is associated with a number of metabolic and hormonal aberrations that could influence resting energy expenditure REE. The purpose of our study was to examine the effects of fat distribution on REE of 96 morbidly obese premenopausal females. METHODS: the study population consisted of three groups of study subjects, 32 with lower body fat distribution (LBD) and waist-to-hip circumference ratios WHR < 0.80, 20 with intermediate (INT) fat distribution and WHR between 0.80 and 0.85 and 34 females with upper body distribution of fat (UBD) and WHR > 0.85. Indices measured included: (1) REE; (2) maximal oxygen consumption during an exercise tolerance test (VO&inf2; max); (3) basal respiratory quotient (RO); (4) fasting blood glucose; and (5) serum cholesterol and triglycerides. RESULTS: we found that morbidly obese women who store fat abdominally (WHR > 0.80) have significantly (p < 0.01) higher REE (kcal per h per BSA) than those with lower body obesity. Levels of triglyceride and glucose of the UBD group were also higher than those of the LBD subjects, i.e. 35% and 23%, respectively. VO&inf2; max and RO were similar between the study groups, suggesting that the elevated REE of the patients with abdominal adiposity were likely not the result of their greater muscle mass or differences in substrate utilization. CONCLUSION: fat distribution affects REE in morbidly obese premenopausal females, and further research is needed to identify the various entities regulating REE in the morbidly obese. PMID- 10733789 TI - Patient-Controlled Analgesia Following Vertical Gastroplasty: a Comparison with Intramuscular Narcotics. AB - BACKGROUND: patient-controlled analgesia PCA is a rapidly spreading approach to the management of post-operative pain. The suitability of this method for the morbidly obese patient undergoing bariatric surgery has not yet been determined. METHODS: in the present study we randomly compared two groups of patients undergoing silastic ring vertical gastroplasty. One group received PCA (12 patients) and the other (11 patients) received intermittent doses of pethidine intramuscularly. RESULTS: the cumulative morphine use during the first post operative day was 52.71 +/- 1.83 mg by the PCA group and an equivalent of 24.55 +/- 3.42 mg morphine by the IM pethidine group (p = 0.0002). The analgesic and sedative effects by the PCA were found to be superior. There were no significant differences between the groups in the incidence of side-effects or complications, except a higher, unexplained incidence of wound infection in the PCA group. CONCLUSION: use of PCA in patients undergoing bariatric surgery has obvious advantages and appears to be a safe procedure. PMID- 10733790 TI - Vertical Banded Gastroplasty in the Severely Obese under Age Twenty-One. AB - BACKGROUND: The severely obese under 21 years of age are at high risk of missing normal development during a crucial period of life and should be considered for surgical treatment. Vertical banded gastroplasty allows patients to be treated effectively while continuing to have normal digestion and absorption without the risks of complex operations. METHODS: This was a retrospective outcome review of 47 severely obese who were under age 21 when surgically treated with VBG. RESULTS: There were no operative mortalities, leaks, or wound infections. Body mass index in 25 patients followed 5 years decreased from an average operative 48.1 to 36.2 kg m(2). Equally for 14 patients followed 10 years, BMI decreased from an average operative 49.6 to 39.2 kg m (2). Both patient groups had 74% follow-up. CONCLUSIONS: Sustained weight reduction improved general health and allowed participation in life activities that would otherwise not have been possible. Adherence to recommended operative technique and intraoperative measurement of pouch volume is necessary to avoid excessive enlargement of the pouch, with resulting weight gain, reflux, and need for revision. PMID- 10733791 TI - Patients Accept a Shorter Hospital Time for Vertical Banded Gastroplasty in a Short Stay Unit: a Randomized Study. AB - BACKGROUND: vertical banded gastroplasty can be performed with a short Post operative hospital stay without this giving rise to more complications. An increased number of patients can be expected after the NIH consensus statement on Bariatric Surgery. Before cost-effective short stay units are employed for such surgery, patients' attitudes to short-term care should be investigated. METHODS: we compared patient satisfaction in two groups of patients. They had been randomized to have a vertical banded gastroplasty in either a normal ward or a short stay unit, open Monday 7 AM to Friday 1 PM. RESULTS: there were no differences in patient satisfaction with either length of stay or quality of stay, despite the fact that short-stay unit patients stayed significantly shorter post-operatively (3.25 (0.62) days vs. 4.70 (0.95); p = 0.0004; mean (SD)). CONCLUSION: it appears that vertical banded gastroplasty can be performed with a short postoperative hospital stay without discomfort to the patient. PMID- 10733792 TI - Weight Loss and Food Intake 18 Months following Vertical Banded Gastroplasty or Gastric Bypass for Severe Obesity. AB - BACKGROUND: vertical banded gastroplasty (VBG) and gastric bypass Roux-en-Y (GBP) are adjunctive to lifelong commitment to energy restricted diet in the attempt by the severely obese to lose weight and maintain weight loss. METHODS: the outcome of 48 subjects (36 VBG and 12 GBP) is presented. RESULTS: 18 months nutritional counseling and follow-up indicated VBG and GBP to be equally effective in maintaining appreciable weight loss. Achievement of 'functional weight', such as minimum 50% loss of excess body weight for at least 12 months Post-operatively occurred in the majority of patients. Excess weight loss by GBP and VBG was 77% and 54% respectively during the first 6 months, with 7-15% additional loss during the next 12 months. BMI decreased from an average 43 to 27 kg m(2) after 12 months. During the first 3 months, energy intake was approximately 2930 kJ, increasing to;4605 kJ at 6 months, to; 5860 kJ at 12 months and then stabilizing. Intake of;50% of the Recommended Daily Allowance (RDA) for most vitamins and minerals was reached. Hemoglobin, iron, folic acid and thiamin values were in the normal range for the entire 18 months follow-up, while serum vitamin B12 levels decreased to deficiency levels during the same period. The pre-operative moderately elevated triglycerides, cholesterol, glucose and insulin levels returned to normal range, thereby alleviating the need for medication and reducing the risk of obesity-related morbidity. Most subjects were quickly satiated with small amounts of solid foods and did not report hunger feelings for the first 6 months post-operatively. The main significant changes in food preferences in the first 6 months were the decrease in starch-based products and the increase in semi-solid milk products and eggs. CONCLUSION: taken together these observations suggest that the subjects should be strongly advised to partake in structured counseling for an extended period of time. PMID- 10733793 TI - Simultaneous Cholecystectomy: to be or not to be. AB - BACKGROUND: the argument concerning the justification for doing cholecystectomy at the same time as a gastric limiting procedure for morbid obesity continues to be a lively one. This study is an attempt to resolve this issue by reviewing the data of a single surgeon over the 10 year period from January 1983 through April of 1993. METHODS: the first 3 years of the study (386 patients) involved primary gastroplasty with or without cholecystectomy. In 1986, the author began doing Roux-Y gastric bypass which has included 673 consecutive patients. At the same time, our criteria for simultaneous cholecystectomy changed from not only cholelithiasis and cholesterolosis of the gallbladder, but also to a strong family history of biliary tract disease which necessitated surgery and clinical evidence of chronic cholecystitis at surgery, i.e. thickening of the gallbladder wall and more than the usual amount of adhesions. FINDINGS: in the 1983-1986 group, 13% needed a second operation for symptomatic binary tract disease sometime between 1 and 9 years after surgery. In the latter group from 1986 to 1993, 9% have necessitated cholecystectomy, usually several years following the original Roux-Y gastric bypass. CONCLUSIONS: we conclude that simultaneous cholecystectomy be based on the above criteria rather than being done routinely. PMID- 10733794 TI - Gastric Bypass and Vertical Banded Gastroplasty- a Prospective Randomized Comparison and 5-Year Follow-up. AB - BACKGROUND: a prospective randomized study was undertaken to compare the outcome of vertical banded gastroplasty (VBG) and gastric bypass (GBP) in patients with clinically severe obesity. METHODS: eligibility criteria included Class IV obesity, < 50 years old and a history of at least one attempt of non-operative weight loss. Patients were managed conservatively for 3 months prior to surgery. Patients were followed post-operatively and monitored for early and late complications and their weight loss outcome for up to 5 years. RESULTS: 44 patients were recruited. Two patients withdrew within 4 weeks and were excluded. Twenty subjects had a GBP and 22 a VBG. There were no significant differences with respect to age, gender, maximum or pre-operative weight between the groups (p > 0.05). Patients who underwent GBP demonstrated significantly greater post operative weight loss (p < 0.05) which was apparent from 6 months onwards. There were no deaths, pulmonary emboli, post-operative leaks or wound dehiscence. There were no instances of staple-line disruption. Symptomatic ulcer disease, confirmed endoscopically, developed in 25% of GBP patients. Nine patients developed gallstones post-operatively of whom five were in the VBG and four in the GBP group. CONCLUSIONS: weight loss following GBP was maintained, while VBG patients slowly regained. PMID- 10733795 TI - Gastric Banding for Treatment of Morbid Obesity: Preliminary Results. AB - BACKGROUND: gastric banding (GB) has been used for treatment of morbid obesity. METHODS: a banding device, introduced by Broadbent and consisting of a self blocking nylon strip covered with a silicone tube, was used in 13 patients who have completed 1-year follow-up. This device was used for its mechanical properties, biocompatibility, ease of insertion and low cost. RESULTS: at 1 year, mean excess weight loss was 51.6%, with all but one patient losing more than 25% of excess weight. Associated illnesses resolved. There were two complications (15%): one patient required band removal for self-induced vomiting and one patient required repair of an incisional hernia. CONCLUSIONS: GB has had good results thus far. Reported differences depend on materials, stoma diameter, pouch size, and developing techniques. PMID- 10733796 TI - Laparoscopic Placement of Adjustable Silicone Gastric Band in the Treatment of Morbid Obesity: How to Do It. AB - BACKGROUND: laparoscopic placement of the adjustable silicone gastric band (ASGB) was begun in our institution in 1992. METHODS: this work started on the animal model first. In the animal laboratory, details of laparoscopic dissection around the pig's stomach were defined. A new prototype of the adjustable silicone band for laparoscopic use was devised. The first human laparoscopic ASGB procedure was performed in our institution on September 1, 1993; 37 patients have undergone this operation by May, 1994. There were 33 women and four men. The average pre operative weight was 114 kg (92160 kg). The mean BMI was 42 kg m(2) (37-50 kg m(2)). RESULTS: no major operative difficulty has been encountered. Immediate post-operative outcome was uneventful except for one patient. CONCLUSION: the technique of laparoscopic ASGB is described. Preliminary weight loss is comparable to open ASGB and vertical gastroplasty, provided that the surgeon has mastered laparoscopy and open bariatric surgery. PMID- 10733797 TI - Laparoscopic Placement of Adjustable Silicone Gastric Banding: Early Experience. AB - BACKGROUND: the authors describe a laparoscopic technique for the positioning of stoma adjustable silicone gastric banding (SASGB), which respects the main steps of the open procedure. METHODS: (1) patient position: supine with thighs abducted and 30 degrees reverse Trendelenburg; (2) Four 10 mm trocars (supra-umbilical, sub-xiphoid, right upper quadrant, left upper quadrant) and an 18 mm trocar (left subcostal); (3) exposure of the subcardial area; (4) measurement of the pouch; (5) dissection of the lesser and greater curvatures; (6) retrogastric tunnel; (7) introduction and placement of the band; (8) band closure and stoma calibration; (9) retention sutures. RESULTS: results obtained in a first (1992) series of five patients who underwent the laparoscopic application of the regular SASGB and results of a second series (1993-1994) of seven patients in whom the new LAP-ASGB was utilized are reported. CONCLUSION: this new approach can represent a major achievement in bariatric surgery, as it combines the minimal invasiveness of the laparoscopic approach with the reversibility of SASGB. PMID- 10733798 TI - New Approach in Surgical Treatment of Morbid Obesity: Laparoscopic Gastric Banding. AB - BACKGROUND: gastric banding has been performed for morbid obesity, with the last nine patients having a laparoscopic approach. MATERIALS: forty-five patients who had undergone primary operations for morbid obesity between 1986 and 1993 were selected for retrospective analysis. All patients had undergone gastric banding. Average pre-operative BMI was 50.9 (kg m(2)) and average pre-operative weight was 135.1 kg. RESULTS: the 3 year mean post-operative BMI reached 28.7 and the 3 year mean post-operative weight loss was 55.7 kg. Blood pressure significantly decreased from the mean 151/ 96 mmHg to l32/90 mmHg at 1-year follow-up. There were no significant changes noted in the levels of RBC, electrolytes and transaminase. There were post-operative wound-healing complications in 18.1% of the patients, wound discharge in 8.8% and incisional hernia in 8.8% of the patients. In 1993 we commenced laparoscopic gastric banding which enabled us to shorten the hospital stay and decrease post-operative complications. CONCLUSION: we are achieving the same good weight-loss results with the laparoscopic technique as after 'open' laparotomy gastric banding. PMID- 10733799 TI - Laparoscopic Vertical Banded Gastroplasty: The Milwaukee Experience. AB - BACKGROUND: laparoscopic techniques are being developed for bariatric surgery. METHODS: eleven morbidly obese patients underwent laparoscopic vertical banded gastroplasty in 1993-1994. RESULTS: average length of hospital stay was 3.9 days, mean operating time was 202 min, and the average hospital charges were $12 800. These numbers were compared to the most recent open gastric bypass patients, where average length of stay was 7.4 days, mean operating time was 105 min, and the average hospital charges were $9800 (adjusted value of $16 700). There were no post-operative complications in the laparoscopically-performed VBG patients. CONCLUSION: laparoscopic VBG is feasible and cost-saving. Weight loss and long term results await ongoing follow-up. PMID- 10733800 TI - Prevention and Reversal of Liver Damage following Bypass Diversion for Obesity. PMID- 10733801 TI - Is Adolescent Gastric-Restrictive Antiobesity Surgery Warranted? AB - BACKGROUND: There is a paucity of information about adolescent morbid obesity and bariatric surgery. METHODS: We interviewed 78% of an adolescent ( 50 kg m(2)), the success rate was 63% with 100% follow-up at 40 months. Major morbidity occurred in six of the 160 patients who underwent 195 operations (the trial period and subsequent year). There were no deaths and follow-up was 98%. CONCLUSIONS: The ideal gastric operation based on this study emphasizes the following requirements: a small pouch (< 15 ml) totally separated from the stomach, a pouch not dependent on staples, placed in the dependent position to prevent stasis, constructed without foreign material and with an anastomosis which permits ingestion of solid food. PMID- 10733804 TI - Invited Commentary. PMID- 10733805 TI - Treatment of Vitamin B12 Deficiency after Gastric Surgery for Severe Obesity. AB - BACKGROUND: Vitamin B12 deficiency after gastric surgery for obesity is due to a failure of separation of vitamin B12, from protein foodstuffs and to a failure of absorption of crystalline vitamin B12 in the presence of intrinsic factor. The purpose of this study was to determine which of four oral doses of crystalline vitamin B12 was most effective in treating vitamin B12 deficiency in 102 patients. METHODS AND RESULTS: At time of entry into the study, the patients had a serum vitamin B12, < 100 pmol L(1), were 29.9 +/- 21.7 months post-op, were 37 +/- 8 years old and had a body mass index of 30 +/- 6 kg m (2). Eight (8%) had had a vertical banded gastroplasty and 94 (92%) a gastric bypass. For the first 3 months all patients received 350 ug per day of crystalline vitamin B12, and all increased their serum vitamin B12 levels to over 100 pmol L(1). The patients were then assigned to receive for a further 3 month period one of four oral doses of crystalline vitamin B12-100 ug, 250 ug, 350 ug and 600 ug. Serum vitamin B12 levels were greater than 150 pmol L(1) after 6 months in 83.3% of patients who received 100 ug; 92.3% of patients who received 250, ug; 94.7% after 350 ug and 95.2% after 600 ug (p%0.525). CONCLUSION: At least 350 ug per day is the appropriate oral dose of crystalline vitamin B12 after gastric surgery for obesity to correct low serum vitamin B12 levels in 95% of patients. PMID- 10733806 TI - An Exploration of the Outcomes of Gastric Bypass Surgery for Morbid Obesity: Patient Characteristics and Indeces of Success. AB - BACKGROUND: The search for replicable predictors of the outcomes of surgical intervention for obesity has proven challenging, with patient selection being a recurrent theme in bariatric literature. METHODS: In this study, 20 gastric bypass patients were interviewed at an average of 2 years following surgery. Subjects provided extensive data relating to their characteristics and experiences. RESULTS: Statistical analyses were undertaken to establish likely predictors of success, both in terms of percentage of excess weight lost and in broader terms. It was found that self-esteem, some scales of the Adjective Checklist, and valuing of food and eating related to a successful outcome. CONCLUSION: Recommendations are made regarding an interview schedule for pre operative assessment, and a prospective study to evaluate its predictive power is proposed. PMID- 10733807 TI - Reoperation Due to Complications after Gastric Restriction Operation. AB - BACKGROUND: In the past 4 years we performed operations on 90 patients who suffered from morbid obesity. Five different operative techniques were available, vertical banded gastroplasty (VBG), silastic ring vertical gastroplasty (SRVG), gastric banding operation, adjustable silicone gastric banding (ASGB) and biliopancreatic diversion (BPD). METHODS: Two of these operations were mainly utilized. The ASGB was done routinely. The SRVG was used particularly for patients with hiatal hernia. Only one patient, who had a deformed pylorus, underwent BPD. RESULTS: Eleven patients had to be reoperated due to complications after the first operation of gastric restriction; They were divided into three groups depending on the type of complication: reoperation due to lack of compliance, due to technical failure, or due to other complications. In the last group we observed three patients with band perforation into the stomach without epigastric pain. This complication has, to our knowledge, only been described in very few cases. CONCLUSION: In some patients it remained difficult to reach adequate compliance, although we kept close contact with them after the operation. We do not yet know the reasons for the band perforation observed in three patients. PMID- 10733808 TI - Necrotizing Sialometaplasia after Silastic Ring Vertical Gastroplasty: Case Report and Review of Literature. AB - Necrotizing sialometaplasia (NS) is a self-limiting benign condition of unknown origin that affects mainly the minor salivary glands on the hard palate. In its clinical and histological appearance, it mimics malignancy. This report presents a patient with NS who suffered from frequent bouts of vomiting subsequent to silastic ring vertical gastroplasty which resulted in remarkably low pH of the oral cavity. The literature pertaining to NS appearing in conjunction with gastrointestinal disorders was reviewed, and the possible contribution of recurrent emesis to the pathogenesis of NS is discussed. PMID- 10733809 TI - Gastric Bypass after Cardiac Transplantation. AB - A 39-year-old morbidly obese male presented with severe and poorly controlled diabetes mellitus (DM) and hypertension (HBP) as well as moderately severe obesity hypoventilation syndrome (OHS) that threatened the survival of his cardiac graft which had been transplanted 7 years previously. A gastric bypass procedure with a 45-cm Roux-limb was performed. His OHS resolved, his DM became undetectable off medication, and his HBP medication was significantly reduced with good control. His maximum excess weight loss was 95%. Importantly, his Cyclosporine and lmuran anti-rejection therapy was maintained without any difficulty attributable to the gastric bypass procedure until his sudden death in late November 1994, 2.75 years following the gastric bypass procedure. PMID- 10733810 TI - Sixth Time Bariatric-Related Surgery for Intractable Nausea, Vomiting, Abdominal Pain, Diarrhea and Weight Loss Failure. AB - A 42-year-old patient with a sixth bariatric-related surgical procedure is presented. The steps taken for the management of her intractable nausea, vomiting, abdominal pain, unacceptable weight loss and diarrhea are described, together with the rationale therefor. Bariatric reoperative surgical procedure and outcome are discussed, together with some alternative considerations. PMID- 10733811 TI - Success in Surgical Intervention for Morbid Obesity: Is Weight Loss Enough? AB - Varieties of gastric surgery have increasingly been used in the management of morbid obesity. Generally, however, research and commentary in this area have related to surgical technique, with weight loss or morbidity being regarded as the most important dependent measures. In the context of the publication of several papers relating to the effects of surgery in the long-term, we believe that it is timely for surgeons to examine their criteria for success. In this paper, we argue that weight loss is inadequate as a primary criterion for success in this context, and that the value of the intervention should be measured against a multidimensional concept of success. PMID- 10733812 TI - Obesity Surgery in Children. AB - BACKGROUND: There have been few articles about bariatric surgery for morbidly obese children. Nevertheless, children who suffer clinically severe obesity also suffer poor social acceptance and an inability to participate in sports or other life activities. Since 1983 the author has performed vertical banded gastroplasty (VBG), Roux-en-Y gastric bypass (RYGBP), or biliopancreatic diversion (BPD) on 22 children, ages 8 - 18 years. METHODS: This was a retrospective review of 22 children, 11 with sleep apnea and 11 without sleep apnea. There were nine males and 13 females. The procedures were VBG-5; RYGBP-14; and BPD-4. RESULTS: There were no operative deaths, infections, or other serious immediate complications. Body mass index (BMI) in those with sleep apnea decreased from a mean of 67.8 preoperatively to 46.5 kg/m(2) at an average follow-up of 32 months. Likewise, for those without sleep apnea, BMI decreased from 56.4 preoperatively to 35.5 kg/m(2) at an average follow-up of 50 months. All patients with sleep apnea had this condition resolve with adequate weight loss. Furthermore, these patients have been able to stay awake in school and have made better grades. Postoperative complications included protein deficiency in three BPD patients, and Vitamin A and D deficiency, folic acid deficiency, gallstones, kidney stones, postoperative laryngeal edema, and incisional hernia in one patient each. There were two late deaths; one at 15 months and one at 3.5 years postoperatively. CONCLUSIONS: Clinically severely obese children can safely undergo bariatric operations usually offered to adults. Furthermore, most patients have sustained significant weight loss. Those patients with sleep apnea have had resolution of their sleep apnea. Complications can be minimized with adequate vitamin, mineral, and trace element supplementation. Long-term results are not yet known. PMID- 10733813 TI - The Effect of Follow-up on Reporting Success for Obesity Surgery. AB - BACKGROUND: Much is written about the importance of follow-up in determining the effect of surgical treatment for obesity upon weight loss. When patients are lost to follow-up, it has been suggested that these patients should be considered as failures. This study was undertaken to determine the effect of incorporating patients not followed in a definition of success for weight loss at one year. METHODS: Data from 34 surgical practices were used to study the effect of using two different denominators, patients followed (Df) or patients eligible (De), to define success. The numerator used in both methods was the number of patients with /= 40 ml (n = 47). RESULTS: Of the 198 patients operated with VBG, pouch volume measurement was successful in 158 patients. Mean pouch volume was 32 ml at 50.5 cm of water. Loss of body mass index at 6, 12, 24, 48, 60 months did not significantly differ in the three groups. The rate of late reoperative procedures was also similar in the three groups. The incidence of staple-line breakdown (SLB) and endoscopically verified esophagitis was higher in the >/= 40 ml group. CONCLUSION: We have been unable to demonstrate a difference in weight loss after VBG for differing pouch volumes. There is an increased rate of SLB and esophagitis in the group with largest pouch volume; however, length of follow-up was longest for this group. PMID- 10733831 TI - Gastric Mucosal Changes following Gastroplasty: A Comparative Study Between Vertical Banded Gastroplasty and Silastic Ring Vertical Gastroplasty. AB - BACKGROUND: Long-term changes of gastric mucosa following surgery for morbid obesity have never been studied, to the best of our knowledge. As 31 patients in our series presented with various gastrointestinal complaints following surgery, we used this opportunity to study mucosal changes. METHODS: Thirty-seven gastroscopies were performed on 31 patients, 20 patients following Vertical Banded Gastroplasty (VBG) and 11 patients following Silastic Ring Vertical Gastroplasty (SRVG) with various gastrointestinal complaints. Macroscopic appearance of the gastric mucosa was examined and biopsies taken from the proximal gastric pouch, the transitional zone and distal stomach. RESULTS: In most patients, macroscopic appearance of the proximal and distal pouches was normal. Pathological findings were mainly located in the transitional zone and were found mainly in the VBG group. CONCLUSIONS: This study indicates that damage to the gastric mucosa is related to the surgical technique, and mainly to the strip of mesh used in the VBG patients. Since hyperplasia and metaplasia were among the microscopic findings, a question is raised about the possibility of malignant transformation. We suggest that routine post-operative gastroscopies be considered, especially following VBG. PMID- 10733832 TI - Long-term Results on Quality of Life of Surgical Treatment of Obesity with Vertical Banded Gastroplasty. AB - BACKGROUND: Few papers assess quality of life after vertical banded gastroplasty (VBG). METHODS: 100 patients with severe obesity (preoperatively mean BMI 41.7 kg m(2)) answered an interview 60 (+/- 2.5) months after VBG. RESULTS: There was no fatal outcome. Nine patients had pulmonary embolus; ten patients required reoperation because of stomal stenosis. Of the 89 patients that still bore a gastroplasty at the moment of the interview, 65 had lost more than 40% of their excess weight (= "success'). Improvement in quality of life of these 89 patients was reflected by significant diminution of depression and back pains. Significant diminution of arterial hypertension and improvement of professional satisfaction, and of social, physical, and sexual activity was significantly related to weight loss. CONCLUSION: VBG resulted generally in a favorable long-term effect on quality of life. However, patients should be informed preoperatively about potential side-effects such as possible persistent vomiting after several years, esophagitis and gastritis, restriction in the choice of foods and prolongation of meals. PMID- 10733833 TI - Invited Commentary. PMID- 10733834 TI - Vertical Gastroplasty with Artificial Pseudopylorus: Analysis of 106 Cases. AB - BACKGROUND: Between January 1991 and December 1994, 106 consecutive morbidly obese patients (19 males and 87 females) underwent Vertical Gastroplasty with Artificial Pseudopylorus (VGAP). METHODS: The mean age of the patients was 34 years, range 20-58), the mean percent excess body weight 122% (range 80-234), and the mean Body Mass Index (BMI) 51 kg/m(2) (range 41-81). The technique of gastroplasty was relatively simple, avoiding the creation of the circular stapled window and the mesh banding of the outlet. The partition of the stomach was done by the use of three double-row staplers vertically- downwards from His angle parallel to the lesser curvature of the stomach. The pseudopylorus was constructed by the use of two bands of silk No. 0, which were completely covered by stomach seromuscular coat. RESULTS: Percent excess weight loss (+/- SD) on the 3rd, 6th, 18th, 24th, 36th and 48th month postoperatively was 32 +/- 10, 51 +/- 12, 71 +/- 12, 78 +/- 12, 82 +/- 9, 80 11 and 81 +/- 10 respectively. Postoperative mean BMI SD) at the same times were 40 +/- 7, 34 +/- 7, 29 +/- 6, 27 +/- 5, 28 +/- 3, 28 +/- 4 and 28 +/- 3 respectively. The early complication rate was 4.7% and the late, including incisional hernias, 6.6%. One patient (0.9%) died of massive pulmonary embolism. CONCLUSION: VGAP is a relatively simple and safe method which gives satisfactory results in body weight reduction, and avoids some complications of the other forms of vertical gastroplasty. PMID- 10733835 TI - Results of Bariatric Surgery for Morbid Obesity in Patients Older than 50 Years. AB - BACKGROUND: Surgery is increasingly used for weight loss in morbidly obese patients. The authors evaluated the safety and efficacy of bariatric surgery in patients older than 50 years. METHODS: Prospective data on 62 consecutive patients (Male = 13, Female = 49) undergoing bariatric procedures between 1985 1994 were reviewed. Mean follow-up was 30 +/- 2 months (3-48 months). All data are mean +/- SEM. RESULTS: Age was 57 +/- 1 year (range 50-71 years). Patients had a mean preoperative weight of 125 + 4 kg (275 +/- 9 lb) and 119 +/- 6% excess body weight. A total of 68 procedures were performed: vertical banded gastroplasty (VBG = 23), Roux-en-Y gastric bypass (RYGB = 43), and billopancreatic diversion (BPD = 2). Six patients were converted to RYGB (5) and BPD (1) after failed VBG. Hospital mortality was nil. Complications were wound infection (5), pulmonary (4) gastric leak (2) abscess (1) and others (4). Mean weight loss at 3 year; was 55 +/- 7 and 33 +/- 6% of percent excess body weight for RYGB and VBG, respectively. Postoperative use of medications for arthritis, diabetes mellitus and asthma was reduced by 23%, 62% and 100%, respectively. Satisfaction with the outcome of treatment and weight loss was reported by 81 % of patients. Six patients that were converted from jejunoileal bypass (metabolic complications) to VBG gained weight. CONCLUSIONS: Bariatric surgery is safe and well tolerated in morbidly obese patients older than 50 years. Weight loss parallels that of younger populations and is greater in patients treated with RYGB in this subgroup. Age should not be a contraindication to bariatric surgery provided the patient has obesity-related medical morbidity. Control of obesity related co-morbid conditions is improved by weight loss. PMID- 10733836 TI - Nutrient Intake following Vertical Banded Gastroplasty or Gastric Bypass. AB - BACKGROUND: This study explored eating habits, nutrient intake, and blood vitamin and mineral levels to determine whether severely obese subjects (BMI 40-50kg m(2)) post-vertical banded gastroplasty (VBG) or gastric bypass Roux-en-Y (GBR) are at risk of developing compounded under-nutrition. METHODS: A dietary follow up of 36 VBG and. 19 GBR was maintained for 18 months via 7-day food intake diaries and 24-h recalls. Food intake was analyzed for energy and nutrient composition and for its relative amount to recommended dietary allowances (RDA). RESULTS: Weight loss was greatest during the first 6 months, continued at a slower rate for the next 6 months, nearly ceasing thereafter. The results following GBR were not substantially different from those following VBG 18 months postoperatively. The median weight loss at 1 year postoperatively was 48, 46, 48 and 36 kg; expressed as residual excess body weight: 0.2, 16, 13 and 22% for GBR Men, Women, VBG Men, Women, respectively. According to the classification proposed by Reinhold, all subjects achieved excellent treatment outcomes 18 months postoperatively. Despite the relatively low reported energy intake (2050% below RDA), no correlation was found between rate of weight loss and energy intake at 6 months postoperatively. The intake of most vitamins and minerals was below 50% of RDA during the 18 months follow-up. The increase in energy intake did not improve the level of the nonenergy-contributing nutrients. Compliance to multivitamin and mineral supplement intake deteriorated with time. CONCLUSION: The low to within-normal range of blood vitamin and mineral levels 12 months postoperatively suggests the slow development of subclinical nutritional deficiency which could jeopardize the subjects' long-term health status. PMID- 10733837 TI - Biliopancreatic Diversion with a New Type of Gastrectomy: Some Previous Conclusions Revisited. AB - BACKGROUND: In 1990, we modified Scopinaro's billopancreatic diversion (BPD); instead of a distal gastrectomy and gastroileal anastomosis, a parietal gastrectomy was performed with nutrients diverted through a duodenal switch. Also, the length of the common channel (50 cm) was doubled to 100 cm, while the nutrient limb remained 250 cm. In 1991, we reported initial results after 16 months, weight loss was as expected following BPD, but patients reported fewer side-effects and the prevalence of excessive malabsorption was less. This cohort of patients had their duodenum stapled shut to construct the duodenal switch. This staple-line failed insidiously in some patients, allowing the duodenum to recanalize partially or completely. This resulted in an incomplete BPD. METHODS: Since 1992, the duodenal switch has been constructed with a complete transaction of the duodenum to prevent recanalization. We report here on the first 61 patients who underwent this definitive procedure. RESULTS: At 16 months, we observed a mean weight loss of 84% of initial excess weight, the number of daily stools at 2.9 +/- 1.6 and the prevalence of diarrhea at 10%. Twenty per cent of patients experienced mild anemia, hypocalcemia, or hypoalbuminemia, which required added supplements. CONCLUSIONS: BPD with parietal gastrectomy, duodenal switch and longer common channel improved weight loss and decreased gastrointestinal side-effects without an increased prevalence of excessive malabsorption. The parietal gastrectomy may contribute to weight loss by increasing satiety, and decreasing side-effects by regulating gastric emptying. PMID- 10733838 TI - 'Hybrid' Bariatric Surgery: Bilio-pancreatic Diversion and Duodenal Switch- Preliminary Experience. AB - BACKGROUND: Hybrid, combined or mixed bariatric surgery is the combination of a degree of 'malabsorption' (as achieved by the intestinal bypass) with a 'restriction' (as achieved by gastric bypass or gastroplasty), thereby simultaneously reducing the absorption of fats in the small bowel and decreasing the intake of food. METHODS: A modification of the bilio-pancreatic diversion (BPD) with a duodenal switch procedure, vertical lineal gastrectomy and preservation of the pylorus, has been used in 23 patients. The antropyloric pump and 4 cm of the duodenum are left intact to preserve physiologic gastric emptying and to prevent anastomotic ulcer. The use of staplers and continuous running sutures reduces surgical risks and operative time. RESULTS: One patient, converted from a vertical gastroplasty, had an intrathoracic esophageal perforation and died of multi-systemic organ failure, a mortality rate of 4.5%. One patient had a partial dehiscence of the laparotomy wound. Three patients developed subcutaneous seromas. Mean weight losses during the first 4 months were 13, 11, 6 and 5 kg, with a loss of 70% of excess weight in patients approaching 1 year. No patient needs treatment for diarrhea. No serious secondary side-effects have been detected. CONCLUSION: This operation appears to result in very satisfactory weight loss, improved quality of life, and a low incidence of complications. PMID- 10733839 TI - Modifications of Intestinal Permeability Test Induced by Biliopancreatic Diversion: Preliminary Results. AB - BACKGROUND: Biliopancreatic Diversion (BPD), excluding the jejunum and part of the ileum from the transit of the food, reduces the absorptive intestinal area available to 250 cm of the distal ileum. The Intestinal Permeability Test (IPT) with Lactulose/Mannitol is performed to assess the intestinal mucosa function. The aim of this study was to investigate the effect of intestinal anatomical modifications induced by BPD on IPT in a group of severely obese patients. METHODS: Group A: 18 severely obese subjects who underwent BPD; the IPT was performed before (T0) and 8.2 +/- 0.9 days after BPD (T1). Group B: nine subjects of Group A; IPT was repeated 96.1 +/- 12.7 days (T2) and 180.4 +/- 19.7 days (T3) after BPD, respectively. IPT was expressed as the %Lactulose/%Mannitol ratio in the urine collected during 5 h after oral administration (normal %L/%M < 0.04). RESULTS: Data from Group A (paired Student's t-test) exhibited significantly higher values in T, with respect to T0 for %L/%M (p < 0.05) and for %L (p < 0.05), and significantly lower values in T, with respect to T0 (p < 0.001) for %M. In the Group B analysis of Variance from T0-T1-T2-T3 resulted statistically significant (p < 0.05) for % L/ % M and % M. CONCLUSIONS: The reduction of intestinal absorption surface induced by BPD causes a significant increase of the Lactulose/Mannitol IPT values, showing an intestinal mucosa function impairment. The IPT values improve progressively at 3 and 6 months after this surgical procedure. PMID- 10733840 TI - Pathogenesis of extracranial cerebrovascular disease. AB - The extracranial vasculature is subject to a vast array of disease processes including atherosclerosis, dissection, inflammation, and mechanical compression. Our current understanding of the pathogenesis of these diseases is discussed. PMID- 10733841 TI - Prospective randomized studies for carotid endarterectomy. AB - The clinical trial has become the standard method used to evaluate surgical procedures. Regarding carotid endarterectomy, clinical trials have reformed the indications for surgery as a means of decreasing the risk of stroke. The methodology and results from significant trials for the symptomatic and asymptomatic patient with carotid stenosis are described. Critical evaluation of these trials is necessary for the discerning surgeon to form a rational approach to clinical practice in carotid disease. PMID- 10733842 TI - Indications for treatment of symptomatic atherosclerotic carotid artery disease. AB - Although the incidence of stroke and stroke mortality have declined during the past few decades, stroke still ranks third as a cause of death and a primary cause of long-term disability in most industrialized societies. Atherosclerotic disease at the carotid bifurcation accounts for approximately 20% to 30% of all ischemic strokes. In this article, the indications for treatment of symptomatic internal carotid artery stenosis are reviewed. PMID- 10733843 TI - Indications for treatment of asymptomatic carotid stenosis. AB - Atherosclerosis of the carotid bifurcation is an observable sign of systemic disease driven by key risk factors and resulting in an epidemic of stroke, myocardial infarction, and vascular death worldwide. Aggressive integrative preventive interventions of controlling hypertension, hyperlipidemia, diabetes mellitus, smoking, systemic inflammation/infarction, depression, and hyperhomocyst(e)imia are needed in the medical management of these high-risk patients. Surgical indications for asymptomatic surgery may be recalled through the acronym CAROTID, which emphasizes knowledge of risk benefit to a particular patient, adequate disclosure, and physician--patient equipoise. PMID- 10733844 TI - Anatomic considerations in the treatment of extracranial cerebrovascular disease. AB - This article outlines the anatomic relationships and variants of structures encountered during surgical approaches for the treatment of extracranial cerebrovascular disease. PMID- 10733845 TI - Carotid endarterectomy: general principles and surgical technique. AB - In this article, the authors describe their standard technique for carotid endarterectomy and discuss the various surgical options and different variations of the procedure. Although there are numerous ways to perform carotid endarterectomy, several basic principles of carotid reconstruction must be adhered to. The surgeon must have complete preoperative knowledge of the patient's vascular anatomy, must maintain complete vascular control at all times, must have sufficient working anatomic knowledge to prevent harm to adjacent structures, and must assure the patient a patent repair that is free of technical errors. The most important factor in assuring a technically acceptable carotid surgery is the availability of a skilled cerebrovascular surgeon who has demonstrable morbidity and mortality rates below 3% and a proper understanding of both vascular principles and cerebral physiology. PMID- 10733846 TI - Carotid endarterectomy using regional anesthesia. AB - The author has had considerable experience with the use of general anesthesia and regional anesthesia for patients undergoing carotid endarterectomy. His experience and a review of the literature indicate that the use of regional anesthesia significantly reduces the incidence of nonsurgical complications following operation. A particularly robust effect is placed on reducing cardiopulmonary complications. PMID- 10733847 TI - Surgical treatment of nonatherosclerotic lesions of the extracranial carotid artery. AB - Nonatherosclerotic disorders of the extracranial carotid artery, such as dissections, aneurysms, and carotid body tumors, are diverse in their causes, presentations, and modes of treatment. The surgical treatment of these lesions often will require a higher exposure of the extracranial carotid than is typical for a carotid endarterectomy. The prevention of perioperative ischemic injury assumes a major role in determining the surgical strategy for treating these lesions. This article offers a brief description of several of these disorders, a review of the general surgical approach to the high cervical artery, and a brief description of lesion-specific surgical techniques. PMID- 10733848 TI - Angioplasty and stenting for carotid atherosclerotic disease. AB - Based on current available data, carotid endarterectomy remains the procedure of choice for stroke risk reduction at the cervical carotid bifurcation, provided that certain inclusion and exclusion criteria are met. There exists, however, a group of patients who do not fulfill criteria related to the various studies for carotid occlusive disease, and these patients may be candidates for other procedures to achieve stroke risk reduction such as carotid angioplasty and stenting. This article provides a brief history of angioplasty percutaneous revascularization and discusses patient selection, methodology, and clinical results of carotid angioplasty and stenting. PMID- 10733849 TI - Endovascular treatment of noncarotid extracranial cerebrovascular disease. AB - The last two decades have witnessed a growing application of endovascular techniques for the treatment of atherosclerotic disease of the extracranial vertebral arteries, subclavian arteries, and brachiocephalic artery. Beginning with simple balloon angioplasty, these minimally invasive techniques have now progressed to the use of stent-supported angioplasty. Stent-supported angioplasty is currently providing a therapeutic alternative to traditional methods of open surgery for revascularization of these vessels and increasing the therapeutic options available for patients who have failed maximal medical therapy. Additionally, endovascular techniques are also being used successfully to treat a variety of nonatherosclerotic diseases affecting the noncarotid extacranial arteries, such as inflammatory, radiation, and anastomotic-graft strictures; acute intimal dissection; traumatic and spontaneous arteriovenous fistulas; and aneurysms or pseudoaneurysms. Continued innovation and refinement of endovascular devices and techniques will inevitably improve technical success rates, reduce procedure-related complications, and broaden the endovascular therapeutic spectrum for extracranial cerebrovascular disease. PMID- 10733850 TI - Carotid endarterectomy prevention strategies and complications management. AB - Perioperative complications of carotid endarterectomy (CEA) are uncommon but potentially devastating. The authors review strategies aimed at minimizing morbidity of surgical treatment of carotid occlusive disease. Multiple components of the perioperative course of patients who undergo CEA must be tightly controlled to maintain an acceptably low complication rate. These factors comprise appropriate patient selection, including careful assessment of techniques aimed at prevention and monitoring of intraoperative complications and postoperative care. PMID- 10733851 TI - Emergency management of ischemic stroke. AB - The accurate diagnosis of acute ischemic stroke is possible using clinical skills and diagnostic tools that are familiar to all neurosurgeons. Avoidance of immediate complications relies on the fundamentals of critical care. Effective treatment for ischemic stroke is available in the form of intravenous thrombolysis, but many stroke patients are denied this therapy because of the narrow window of opportunity for safe administration. Intra-arterial delivery may extend this benefit to a greater number of patients and may eventually prove more effective than intravenous treatment. Surgical treatment in a small number of ischemic stroke patients can be lifesaving and may afford reasonable functional recovery. This article discusses typical clinical presentations and differential diagnosis, diagnostic imaging for ischemic stroke, and possible treatments. PMID- 10733852 TI - Outcomes analysis of the treatment of carotid artery disease. AB - The author presents a brief review of the methodologies of outcomes analysis. Recent large database outcomes studies on patients undergoing carotid endarterectomy are also reviewed and compared with the data generated from two prospective randomized studies, and large database outcomes analysis are discussed. The efforts of the American Association of Neurological Surgeons and the Congress of Neurological Surgeons Outcomes Committee to perform online, multicenter outcomes studies are reviewed. PMID- 10733854 TI - GUEST EDITORS' INTRODUCTION. PMID- 10733853 TI - Diagnostic modalities for carotid artery disease. AB - Stroke secondary to atherosclerosis is one of the most common causes of death in the United States. A major cause of thromboembolic stroke is atherosclerotic narrowing and ulceration in the region of the carotid bifurcation. The indication for carotid surgery may or may not include the presence of symptoms, but all indications include the presence of an atherosclerotic plaque that may result in embolic or occlusive disease. Because the protective results of surgery depend on accurately defining the presence of these plaques and the degree of carotid artery stenosis, it is essential to understand the diagnostic methods used in determining carotid disease and grading carotid artery stenosis. An understanding of their reliability and associated risks is essential for the proper management of patients with carotid bifurcation disease. PMID- 10733855 TI - Accuracy, Scope, and Flexibility of Models. AB - Traditionally, models are compared on the basis of their accuracy, their scope, and their simplicity. Simplicity is often represented by parameter counts; the fewer the parameters, the simpler the model. Arguments are presented here suggesting that simplicity has little place in discussions of modeling; instead, the concept of flexibility should be substituted. When comparing two models one should be wary of the possibility of their differential flexibility. Several methods for assessing relative flexibility are possible, as represented in this special issue of the Journal of Mathematical Psychology. Here, the method of cross-validation is applied in the comparison of two models, a linear integration model (LIM) and the fuzzy-logical model of perception (FLMP), in the fitting of 44 data sets concerning the perception of layout seen among three panels with the presence or absence of four sources of information for depth. Prior to cross validation the two models performed about equally well; after cross-validation LIM was statistically superior to FLMP, but the overall pattern of fits remained nearly the same for both models. Copyright 2000 Academic Press. PMID- 10733856 TI - How to Assess a Model's Testability and Identifiability. AB - Formal definitions are given of the following intuitive concepts: (a) A model is quantitatively testable if its predictions are highly precise and narrow. (b) A model is identifiable if the values of its parameters can be ascertained from empirical observations. (c) A model is redundant if the values of some parameters can be deduced from others or if the values of some observables can be deduced from others. Various rules of thumb for nonredundant models are examined. The Counting Rule states that a model is quantitatively testable if and only if it has fewer parameters than observables. This rule can be safely applied only to identifiable models. If a model is unidentifiable, one must apply a generalization of the Counting Rule known as the Jacobian Rule. This rule states that a model is quantitatively testable if and only if the maximum rank (i.e., the number of linearly independent columns) of its Jacobian matrix (i.e., the matrix of partial derivatives of the function that maps parameter values to the predicted values of observables) is smaller than the number of observables. The Identifiability Rule states that a model is identifiable if and only if the maximum rank of its Jacobian matrix equals the number of parameters. The conclusions provided by these rules are only presumptive. To reach definitive conclusions, additional analyses must be performed. To illustrate the foregoing, the quantitative testability and identifiability of linear models and of discrete state models are analyzed. Copyright 2000 Academic Press. PMID- 10733857 TI - An Introduction to Model Selection. AB - This paper is an introduction to model selection intended for nonspecialists who have knowledge of the statistical concepts covered in a typical first (occasionally second) statistics course. The intention is to explain the ideas that generate frequentist methodology for model selection, for example the Akaike information criterion, bootstrap criteria, and cross-validation criteria. Bayesian methods, including the Bayesian information criterion, are also mentioned in the context of the framework outlined in the paper. The ideas are illustrated using an example in which observations are available for the entire population of interest. This enables us to examine and to measure effects that are usually invisible, because in practical applications only a sample from the population is observed. The problem of selection bias, a hazard of which one needs to be aware in the context of model selection, is also discussed. Copyright 2000 Academic Press. PMID- 10733858 TI - Akaike's Information Criterion and Recent Developments in Information Complexity. AB - In this paper we briefly study the basic idea of Akaike's (1973) information criterion (AIC). Then, we present some recent developments on a new entropic or information complexity (ICOMP) criterion of Bozdogan (1988a, 1988b, 1990, 1994d, 1996, 1998a, 1998b) for model selection. A rationale for ICOMP as a model selection criterion is that it combines a badness-of-fit term (such as minus twice the maximum log likelihood) with a measure of complexity of a model differently than AIC, or its variants, by taking into account the interdependencies of the parameter estimates as well as the dependencies of the model residuals. We operationalize the general form of ICOMP based on the quantification of the concept of overall model complexity in terms of the estimated inverse-Fisher information matrix. This approach results in an approximation to the sum of two Kullback-Leibler distances. Using the correlational form of the complexity, we further provide yet another form of ICOMP to take into account the interdependencies (i.e., correlations) among the parameter estimates of the model. Later, we illustrate the practical utility and the importance of this new model selection criterion by providing several real as well as Monte Carlo simulation examples and compare its performance against AIC, or its variants. Copyright 2000 Academic Press. PMID- 10733859 TI - Bayesian Model Selection and Model Averaging. AB - This paper reviews the Bayesian approach to model selection and model averaging. In this review, I emphasize objective Bayesian methods based on noninformative priors. I will also discuss implementation details, approximations, and relationships to other methods. Copyright 2000 Academic Press. PMID- 10733860 TI - Cross-Validation Methods. AB - This paper gives a review of cross-validation methods. The original applications in multiple linear regression are considered first. It is shown how predictive accuracy depends on sample size and the number of predictor variables. Both two sample and single-sample cross-validation indices are investigated. The application of cross-validation methods to the analysis of moment structures is then justified. An equivalence of a single-sample cross-validation index and the Akaike information criterion is pointed out. It is seen that the optimal number of parameters suggested by both single-sample and two-sample cross-validation indices will depend on sample size. Copyright 2000 Academic Press. PMID- 10733861 TI - Model Selection Based on Minimum Description Length. AB - We introduce the minimum description length (MDL) principle, a general principle for inductive inference based on the idea that regularities (laws) underlying data can always be used to compress data. We introduce the fundamental concept of MDL, called the stochastic complexity, and we show how it can be used for model selection. We briefly compare MDL-based model selection to other approaches and we informally explain why we may expect MDL to give good results in practical applications. Copyright 2000 Academic Press. PMID- 10733862 TI - Statistical Tests for Comparing Possibly Misspecified and Nonnested Models. AB - Model selection criteria (MSC) involves selecting the model with the best estimated goodness-of-fit to the data generating process. Following the method of Vuong (1989), a large sample Model Selection Test (MST), is introduced that can be used in conjunction with most existing MSC procedures to decide if the estimated goodness-of-fit for one model is significantly different from the estimated goodness-of-fit for another model. The MST extends the classical generalized likelihood ratio test, is valid in the presence of model misspecification, and is applicable to situations involving nonnested probability models. Simulation studies designed to illustrate the concept of the MST and its conservative decision rule (relative to the MSC method) are also presented. Copyright 2000 Academic Press. PMID- 10733863 TI - Model Comparisons and Model Selections Based on Generalization Criterion Methodology. AB - The purpose of this article is to formalize the generalization criterion method for model comparison. The method has the potential to provide powerful comparisons of complex and nonnested models that may also differ in terms of numbers of parameters. The generalization criterion differs from the better known cross-validation criterion in the following critical procedure. Although both employ a calibration stage to estimate parameters, cross-validation employs a replication sample from the same design for the validation stage, whereas generalization employs a new design for the critical stage. Two examples of the generalization criterion method are presented that demonstrate its usefulness for selecting a model based on sound scientific principles out of a set that also contains models lacking sound scientific principles that are either overly complex or oversimplified. The main advantage of the generalization criterion is its reliance on extrapolations to new conditions. After all, accurate a priori predictions to new conditions are the hallmark of a good scientific theory. Copyright 2000 Academic Press. PMID- 10733864 TI - The Importance of Complexity in Model Selection. AB - Model selection should be based not solely on goodness-of-fit, but must also consider model complexity. While the goal of mathematical modeling in cognitive psychology is to select one model from a set of competing models that best captures the underlying mental process, choosing the model that best fits a particular set of data will not achieve this goal. This is because a highly complex model can provide a good fit without necessarily bearing any interpretable relationship with the underlying process. It is shown that model selection based solely on the fit to observed data will result in the choice of an unnecessarily complex model that overfits the data, and thus generalizes poorly. The effect of over-fitting must be properly offset by model selection methods. An application example of selection methods using artificial data is also presented. Copyright 2000 Academic Press. PMID- 10733865 TI - Key Concepts in Model Selection: Performance and Generalizability. AB - What is model selection? What are the goals of model selection? What are the methods of model selection and how do they work? Which methods perform better than others and in what circumstances? These questions rest on a number of key concepts in a relatively underdeveloped field. The aim of this paper is to explain some background concepts, to highlight some of the results in this special issue, and to add my own. The standard methods of model selection include classical hypothesis testing, maximum likelihood, Bayes method, minimum description length, cross-validation, and Akaike's information criterion. They all provide an implementation of Occam's razor, in which parsimony or simplicity is balanced against goodness-of-fit. These methods primarily take account of the sampling errors in parameter estimation, although their relative success at this task depends on the circumstances. However, the aim of model selection should also include the ability of a model to generalize to predictions in a different domain. Errors of extrapolation, or generalization, are different from errors of parameter estimation. So, it seems that simplicity and parsimony may be an additional factor in managing these errors, in which case the standard methods of model selection are incomplete implementations of Occam's razor. Copyright 2000 Academic Press. PMID- 10733866 TI - TELEGRAPHIC REVIEWS. PMID- 10733867 TI - Do TNF-alpha-insensitive cancer cells escape alpha-tubulin nitrotyrosination? AB - TNF-alpha induces tumor-selective cytotoxicity in certain cancers, but many tumors are resistant to the effects of this inflammatory cytokine. This brief hypothesis outlines my views that nitric oxide-mediated alpha-tubulin posttranslational nitrotyrosination may be a major mechanism through which TNF alpha exerts its cytotoxic effects on cancer cells. Additionally, I propose that tumor cells that are resistant to the effects of TNF-alpha may be so because of suppressed levels of "tubulin tyrosine ligase," which is responsible for the posttranslational tyrosination of alpha-tubulin. PMID- 10733868 TI - Interactions of nitric oxide with lipid peroxidation products under aerobic conditions: inhibitory effects on the formation of malondialdehyde and related thiobarbituric acid-reactive substances. AB - Under aerobic conditions, exposure of peroxidized lipids to nitric oxide (NO) was found to result in a rapid decrease in the levels of thiobarbituric acid-reactive substances (TBARS). Addition of 10-100 microM NO to rat brain homogenates preincubated for 2 h at 37 degrees C caused up to a 20% decrease in the levels of TBARS compared to controls. A similar inhibitory effect was observed on TBARS produced by Fe(2+)-induced decomposition of 15-hydroperoxyeicosatetraenoic acid (15-HPETE), due apparently to NO-induced decomposition of the hydroperoxide (ferrous oxidation/xylenol orange assay). Prostaglandin G(2) (PGG(2), 35 microM), as a model bicyclic endoperoxide, and malondialdehyde (MDA, 20 microM), the main component of TBARS, proved also susceptible to degradation by NO or NO donors (diethylamine NONOate, DEA/NO) at concentrations of 100 microM or higher in 0.05 M phosphate buffer, pH 7.4, and at 37 degrees C, as indicated by the reduced response to the TBA assay. No significant effect on TBARS determination was caused by nitrite ions. These and other data indicate that NO can inhibit TBARS formation by decomposing primary lipid peroxidation products, chiefly 15-HPETE and related hydroperoxides, and, to a lesser extent, later stage TBARS precursors, including bicyclic endoperoxides and MDA, via nitrosation and other oxidative routes, without however affecting chromogenic reactions during the assay. PMID- 10733869 TI - Endogenous production of nitric oxide and effects of nitric oxide and superoxide on melanotrope functioning in the pituitary pars intermedia of Xenopus laevis. AB - Previous studies have focused on the immunohistochemical detection of a nitric oxide (NO)-cyclic 3',5'-monophosphate (cGMP) pathway in the brain and pituitary of the aquatic toad Xenopus laevis. We here investigate the endogenous production and possible involvement of NO signaling in the regulation of melanotrope cell activity in the pituitary pars intermedia of this amphibian. Using immunohistochemical staining of cultured cells with a polyclonal antiserum against inducible NO synthase (iNOS), immunoreactivity was observed both in melanotropes and in stellate-shaped cells. Part of these stellate-shaped cells is characterized as folliculo-stellate cells by their capacity of beta-Ala-Lys N(epsilon)-AMCA uptake. Using chemiluminescence detection we demonstrate the presence of NO and reaction products like nitrite (NO(-)(2)) or peroxynitrite (ONOO(-)) in the incubation medium of cultured melanotropes. Bacterial lipopolysaccharide (LPS) stimulates the generation of NO and reaction products, the effect of which was blocked by S-methyl-l-thiocitrulline hydrochloride, a potent general NOS inhibitor. With [(3)H]lysine incorporation and a superfusion technique, it is shown that peptide release from melanotropes is stimulated by administration of superoxide dismutase (SOD), which was added to the superfusion medium to prevent scavenging of NO by superoxide anions. Pretreating the cells with the general NOS inhibitor l-nitroarginine methyl ester for 48 h attenuated the SOD-induced stimulation, but did not affect the stimulation by sodium nitroprusside (SNP) or 3-morpholinylsydnoneimine chloride (SIN-1), whereas hemoglobin blocked the combined effect of SOD plus NO donors. The soluble guanylate cyclase inhibitor 1H-[1,2, 4]oxadiazolo[4,3a]-quinoxaline-1-one did not inhibit but even significantly potentiated the effect of NO donors on peptide release without affecting the SOD-induced stimulation of peptide release. In addition to the previously described neuronal NOS (nNOS) immunoreactivity in nerve fibers in the pars intermedia of Xenopus, the present data reveal iNOS and nNOS as potential sources of endogenous NO production in cultured cells of the pars intermedia. Our study shows that also in nonmammalian vertebrates endogenous NO production may be physiologically relevant under conditions where protection against oxidative damage is needed. The endocrine cells of the pars intermedia themselves, as well as the folliculo-stellate cells, under such conditions may dispose of a protective mechanism against oxidative stress. The sensitivity of the endogenous NO production to LPS suggests that NO may also play a role during systemic inflammation. PMID- 10733870 TI - Does diabetes mellitus affect the progress of tolerance to isosorbide dinitrate (ISDN) in corporal tissue? AB - For erection to take place, the penile arteries and sinusoids have to dilate, thereby increasing the blood flow into the penis. There is increasing evidence that release of l-arginine derived nitric oxide (NO) from nonadrenergic noncholinergic (NANC) nerves and from the sinusoidal endothelium is a major event in penile smooth muscle relaxation and promotes the endogenous formation of cyclic guanosine monophosphate (cGMP). Nitrovasodilators can be attributed to the activation of soluble guanylate cyclase, resulting in an increase in intracellular level of cyclic guanosine monophosphate, but prolonged exposure to high levels of nitroglycerine and other organic nitroesters induces tolerance against the cardiovascular effect. In this study, the aim was to determine the effect of diabetes on the corporal smooth muscle relaxant effect of ISDN and the effect of diabetes on the process of tolerance to the drug. For this purpose, alloxan-induced diabetic rabbits were used to form diabetes group. The responses of the corpus cavernous strips obtained from control and alloxan-induced diabetic rabbit were studied in organ chamber. In conclusion, prolonged in vitro exposure of corpus cavernosum strips obtained from control and diabetic groups to high concentrations of ISDN caused significant desensitization to the relaxant effect the drug. So, prolonged exposure of corporal tissue to the agents like nitroglycerine, used for treatment of impotence, may render ineffective the therapy in diabetic erectile impotence. However, intolerance to nitric oxide provides a rationale for the concept of using nitro oxide agents (like SNP) in the treatment of diabetic erectile dysfunction. PMID- 10733871 TI - Production and excretion of nitrate by human newborn infants: neonates are not little adults. AB - Endothelium-derived relaxing factor, identified as nitric oxide or its adducts, is metabolized to nitrate and excreted in the urine. Since blood pressures are lower in newborn infants compared to adults, we hypothesized that newborn infants would have increased excretion of nitrate on the day of birth. Neonatal urine was collected before 24 h of age when exogenous intake of nitrate was low. Two different analytical methods showed that nitrate accounted for >99% of nitrogen oxides in urine of healthy neonates and adults. The absolute micromolar concentration of nitrate in urine from infants was significantly below that of adults. When nitrate content was standardized for the reduced renal function in the newborn infant (creatinine content) and body mass (kilogram weight), the concentration of nitrate in neonatal urine was significantly higher than that of adults. Nitrate concentrations in the urine of prematurely born infants were twice that of nitrate measured in urine from term infants. These findings suggested that nitric oxide is produced in larger intravascular quantities in newborn infants versus adults. Thus, we postulated that nitric oxide released from a nitrosothiol would be metabolized to nitrate more readily by neonatal erythrocytes compared to red blood cells obtained from adults. Neonatal erythrocytes, suspended at concentrations of 8, 12, or 16 g per deciliter of hemoglobin, produced 1.7- to 2.1-fold more nitrate than equivalent hemoglobin concentrations of adult erythrocytes that were each incubated with S-nitroso-N acetylpenicillamine (100 microM) over a 2-h period. Taken together, the studies of urinary nitrate in newborn infants and the ability of neonatal erythrocytes to generate nitrate are consistent with a robust production of nitric oxide immediately after birth. PMID- 10733872 TI - Unidirectional and oscillatory shear stress differentially modulate NOS III gene expression. AB - Atherosclerotic plaques preferentially develop in regions exposed to a low mean shear stress and cyclic reversal of flow direction (oscillatory flow). This contrasts with plaque-free zones where endothelial cells are exposed to unidirectional flow. Previous works from our laboratory using a unique experimental flow system demonstrated the existence of a differential regulation of endothelial nitric oxide synthase (NOS III) gene expression by unidirectional and oscillatory flow patterns. We further studied the possible mechanisms responsible for selective unresponsiveness of NOS III gene regulation to oscillatory flow. The results obtained demonstrate that (i) induction of the activity of the 1600-base-pair NOS III gene promoter by unidirectional and oscillatory shear stress is modulated by similar mechanisms that involve NF kappaB activation, but do not involve Ras-dependent MAP kinase activation, and (ii) the lack of induction of NOS III gene regulation by oscillatory shear stress can be attributed to the activation of a yet unidentified negative cis-acting element present in the NOS III gene. PMID- 10733873 TI - Kinetics and mechanism of the decomposition of S-nitrosoglutathione by l-ascorbic acid and copper ions in aqueous solution to produce nitric oxide. AB - S-Nitrosothiols serve as a good source of nitric oxide ((*)NO) mainly due to the ease of cleavage of the S-N bond which consequently produces (*)NO. The reductive decomposition of S-nitrosoglutathione (GSNO) by l-ascorbic acid (vitamin C) yields (*)NO which was monitored both electrochemically (using NO-probe) and spectrophotometrically. The rate of reaction and (*)NO release was found to be pH dependent in a manner which drastically increases with pH demonstrating that the l-ascorbic acid dianion (A(2-)) is by far the most reactive species of l-ascorbic acid (H(2)A). The derived rate expression (measuring the disappearance of the absorption at ca. 336 nm due to GSNO) was established as rate = -d[GSNO](t)/dt = ((k(a)[H(+)](2) + k(b)[H(+)]K(1) + k(c)K(1)K(2))/([H(+)](2) + K(1)[H(+)] + K(1)K(2)))[GSNO](t)[H(2)A](t). k(a), k(b), and k(c) are second-order rate constants via the H(2)A, HA(-), and A(2-) pathways, respectively, while K(1) and K(2) represent the first and second equilibrium dissociation constants of l ascorbic acid. There is little or no reaction at low pH (below 5.5), where H(2)A is a predominant species, and as a result the rate constant (k(a)) via this route was found to be negligible. At 25 degrees C, k(b) = 5.23 +/- 1.47 x 10(-3) dm(3) mol(-1) s(-1) and k(c) = 1.22 +/- 0.04 x 10(3) dm(3) mol(-1) s(-1), activation parameters DeltaH(double dagger)(b) = 54.4 +/- 4.3 kJ mol(-1), DeltaS(double dagger)(b) = -106 +/- 16 J K(-1) mol(-1), DeltaH(double dagger)(c) = 80.5 +/- 7.5 kJ mol(-1), DeltaS(double dagger)(c) = 84 +/- 7 kJ mol(-1). The experimental rate and activation parameters suggest that this redox process follows an outer-sphere electron transfer mechanism. GSNO is relatively stable in the dark, aqueous medium and even in the presence of trace quantities of Cu(2+). Induced catalytic decomposition of GSNO only becomes significant above ca. 10 microM Cu(2+), but after this it shows linear dependency. To nullify any catalysis by Cu(2+) or any other transition metal ions, EDTA was added to all experimental reactions except those where catalysis by Cu(2+) was studied. PMID- 10733874 TI - Modification of the cadmium reduction assay for detection of nitrite production using fluorescence indicator 2,3-diaminonaphthalene. AB - The measurement of nitric oxide (NO) is important for characterizing the regulatory roles of NO in various biological systems. In this communication we report that cadmium (Cd) reduction of nitrate (NO(-)(3)) to nitrite (NO(-)(2)) can be quantitated by using the fluorescence indicator, 2,3-diaminonaphthalene (DAN) to detect the sum of NO(-)(3) and NO(-)(2) (NO(-)(x)) from endothelial cells. This assay is at least 10-fold more sensitive than when Cd reduction is coupled with the spectrophotometric Greiss reaction and can be used to quantitate the small amounts of NO(-)(x) generated from the constitutive form of endothelial nitric oxide synthase (eNOS). In addition various P(2) purinoceptor agonists and antagonists do not interfere the Cd reduction/DAN assay. Thus the Cd reduction/DAN assay can be used not only to characterize P(2) purinoceptor release of NO(-)(x) from cultured endothelial cells but also to quantitate NO( )(x) levels in serum. PMID- 10733875 TI - Electrode materials for nitric oxide detection. AB - Nitric oxide oxidation signals were compared for uniform test electrodes of platinum, iridium, palladium, rhodium, ruthenium, gold, graphite, and a nickel porphyrin on graphite in deaerated phosphate-buffered saline (pH 7.0) at 35 degrees C. All tested materials detected NO(*) amperometrically. Current densities (A/M/cm(2) +/- S.D.) were Ir (0.021 +/- 0.002), Rh (0.088 +/- 0.012), graphite (0.117 +/- 0.018), Pd (0.118 +/- 0.033), Au (0.149 +/- 0. 039), Pt (0.237 +/- 0.117), Ni (II)-tetra(3-methoxy-4-hydroxyphenyl) porphyrin on graphite (0.239 +/- 0.009), and Ru (0.680 +/- 0.058). NO(*) oxidation current on ruthenium was maximal at 675 mV (vs Ag/AgCl), nearly three times that on the next-best materials, platinum and Ni-porphyrin on graphite poised at 800 mV. The measured limit of detection for NO(*) on Ru was below 3 nM. Enhanced NO(*) oxidation current on ruthenium is apparently due to formation of nitrosyl- or chloronitrosyl-ruthenium complexes at the electrode surface. At fixed potentials above 675 mV, ruthenium exhibited an even larger NO(*) response, characterized by current flow opposite in polarity to an oxidation, which we hypothesize reflects suppression of the oxidative background current (presumably due to chloride oxidation or to the electrolysis of water) by a film consisting of nitrosyl- or chloronitrosyl-ruthenium complexes. The sensitive response of the ruthenium electrode to the direct oxidation of NO(*) may be useful in sensors for biomedical applications. PMID- 10733876 TI - Isolation and subunit composition of native sterol carrier protein 2/3-oxoacyl coenzyme A thiolase from normal rat liver peroxisomes. AB - In the present report we describe a method for the complete purification of native sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCP-2/thiolase) from normal rat liver peroxisomes. The isolation procedure is based on the alteration in chromatographic properties of the enzyme in the presence of low concentrations of CoA. The purified preparation of SCP-2/thiolase consisted of 58- and 46-kDa polypeptides. Peroxisomes prepared freshly from normal rat liver contained three SCP-2/thiolase isoforms, separable by conventional chromatography. Immunochemical, molecular sieving, and chemical cross-linking experiments indicated that these isoforms represent thiolytically active homo- and heterodimeric combinations of the 46- and 58-kDa subunits (2 x 58, 58-46, and 2 x 46-kDa proteins). PMID- 10733877 TI - Extremely thermostable elongation factor G from Aquifex aeolicus: cloning, expression, purification, and characterization in a heterologous translation system. AB - The fus gene of the translation factor G (EF-G) from the hyperthermophilic bacterium Aquifex aeolicus was cloned under control of a phage promoter and overexpressed in Escherichia coli with the T7 RNA polymerase system. A heat denaturation step at 95 degrees C was used to purify the protein from the cell extract. This approach simplified the chromatographic procedures and decreased the protein loss since most of Escherichia coli proteins were denatured and precipitated. Ten milligrams of the highly purified protein was isolated from 4 liters of induced culture. The overproduced EF-G was active in ribosome-dependent GTP hydrolysis and a poly(U)-directed polyphenylalanine translation system with E. coli 70S ribosomes. The method presented here might facilitate functional and structural studies of important components of the protein biosynthesis system. PMID- 10733878 TI - Overexpression, purification, and refolding of a Porphyromonas gingivalis cysteine protease from Escherichia coli. AB - This paper describes the overexpression of the Rgp-1 (arginine) protease domain from Porphyromonas gingivalis. This protease and the related Kgp (lysine) protease, both of which display trypsin-like specificity, have been implicated as major virulence factors and may play a significant role in the etiology of periodontal disease. Both Rgp-1 and Kgp are initially translated as polyproteins, each containing a protease domain and multiple adhesin domains. The Rgp-1 protease domain was expressed in E. coli, purified, refolded, and assayed for activity. These expression studies demonstrated that prior to the formation of inclusion bodies in the E. coli cytoplasm, the protease was proteolytically active and could hydrolyze a specific synthetic substrate. When the Rgp-1 protease domain was purified from inclusion bodies and refolded, it was found to be autolytically active and displayed specific catalytic activity. This is the first report on the expression and purification of active Rgp-1 from E. coli. Polyclonal antisera raised against recombinant protein recognized the native form of the protease in the P. gingivalis strain W50, indicating that the recombinant protein contained some of the antigenic determinants of the native protease. PMID- 10733879 TI - Large-scale purification of myeloperoxidase from HL60 promyelocytic cells: characterization and comparison to human neutrophil myeloperoxidase. AB - A large-scale purification procedure was developed for the isolation of myeloperoxidase from HL60 promyelocytic cells in culture. Initial studies showed the bulk of peroxidase-positive myeloperoxidase activity to be located in the cetyltrimethylammonium bromide solubilized particulate fraction of cell homogenates. The myeloperoxidase was then chromatographically purified using concanavalin A followed by gel filtration. SDS-PAGE analysis of the final preparation showed the presence of only two proteins with molecular masses of approximately 55 and 15 kDa, corresponding to the large and small subunits of myeloperoxidase. These data, along with Reinheit Zahl (RZ) values (A(430)/A(280)) of greater than or equal to 0.72, indicate that the myeloperoxidase prepared by this method is apparently homogeneous. Preparations routinely yielded 12-20 mg of pure myeloperoxidase per 10 ml of cell pellet. The HL60 myeloperoxidase was shown to be indistinguishable from purified human neutrophil myeloperoxidase by size exclusion chromatography, analytical ultracentrifugation, SDS-PAGE, Western blot, and NH(2)-terminal sequence analysis. The activities of the two myeloperoxidase samples, as measured using either the tetramethylbenzidine or the taurine chloramine assay, were indistinguishable. Finally, both enzymes responded identically to dapsone and aminobenzoic acid hydrazide, known inhibitors of myeloperoxidase. A protocol is presented here for the rapid, large-scale purification of myeloperoxidase from cultured HL60 cells, as well as evidence for the interchangeability of this myeloperoxidase and that purified from human neutrophils. PMID- 10733880 TI - Purification and characterization of recombinant forms of murine Tcl1 proteins. AB - The TCL1 gene, which is located on chromosome 14, plays a major role in human hematopoietic malignancies and encodes a 14-kDa protein whose function has not been determined. This gene is expressed in pre-B cells, in immature thymocytes, and, at low levels, in activated T cells but not in peripheral mature B cells and in normal cells. The Tcl1 protein is similar in its primary structure to a protein encoded by the mature T-cell proliferation gene (MTCP1). The MTCP1 gene is located on the X chromosome and has been shown to be involved in rare chromosomal translocations in T-cell proliferative diseases. The murine TCL1 gene resides on mouse chromosome 12 and is homologous to the human TCL1 and MTCP1 genes. Murine Tcl1 protein has 116 amino acid residues and shares 50% sequence identity with human Tcl1, while the human and mouse Mtcp1 are nearly identical, with conservative differences in only six residues. The TCL1 and MTCP1 genes appear to be members of a family of genes involved in lymphoid proliferation and T-cell malignancies. Our laboratory has undertaken the study of the Tcl1 and Mtcp1 proteins to determine the structure and the function of these related proteins. In the present report, we have produced, using a bacterial expression system, the purified murine Tcl1 protein and a mutant form of murine Tcl1 protein containing a cysteine to alanine mutation at amino acid position 85. The recombinant proteins were purified by chromatography on a Ni-NTA resin followed by reverse-phase FPLC using a buffer system at pH 7.9 and a polymer-based reverse phase column. The murine Tcl1 recombinant protein displays limited solubility and forms disulfide-linked dimers and oligomers, while the mutant murine Tcl1 C86A protein has increased solubility and does not form higher order oligomers. The purified recombinant murine proteins were characterized by N-terminal sequence analysis, mass spectrometry, and circular dichroism spectroscopy. Initial results indicate that the mutant murine Tcl1 C86A protein is suitable for both NMR and X ray crystallographic methods of structure determination. PMID- 10733881 TI - Expression, purification, and characterization of histidine-tagged mouse monoglyceride lipase from baculovirus-infected insect cells. AB - Monoglyceride lipase (MGL) has been produced with the baculovirus-insect cell system. The mouse MGL cDNA was subcloned into a baculovirus transfer vector in frame with a sequence encoding an N-terminal stretch of six histidine residues. Purification to apparent homogeneity was obtained by nickel-chelating chromatography. The final yield was 3 mg of pure enzymatically active MGL per liter of Sf9 cell suspension culture. Analysis by SDS-PAGE and mass spectrometry showed that the recombinant histidine-tagged enzyme had the expected molecular mass. With monoolein as substrate, the specific activity and the apparent K(m) were close to those of rat MGL of adipose tissue. PMID- 10733882 TI - Optimized production and purification of Bacillus anthracis lethal factor. AB - Bacillus anthracis lethal factor (LF) is a 90-kDa zinc metalloprotease that plays an important role in the virulence of the organism. LF has previously been purified from Escherichia coli and Bacillus anthracis. The yields and purities of these preparations were inadequate for crystal structure determination. In this study, the genes encoding wild-type LF and a mutated, inactive LF (LF-E687C) were placed in an E. coli-Bacillus shuttle vector so that LF was produced with the protective antigen (PA) signal peptide at its N-terminus. The resulting vectors, pSJ115 and pSJ121, express wild-type and mutated LF fusion proteins, respectively. Expression of the LF genes is under the control of the PA promoter and, during secretion, the PA signal peptide is cleaved to release the 90-kDa LF proteins. The wild-type and mutated LF proteins were purified from the culture medium using three chromatographic steps (Phenyl-Sepharose, Q-Sepharose, and hydroxyapatite). The purified proteins were greater than 95% pure and yields (20 30 mg/L) were higher than those obtained in other expression systems (1-5 mg/L). These proteins have been crystallized and are being used to solve the crystal structure of LF. Their potential use in anthrax vaccines is also discussed. PMID- 10733883 TI - High-throughput assay for inorganic pyrophosphatases using the cytosolic enzymes of Saccharomyces cerevisiae and human as an example. AB - This paper describes the development of a new, malachite green based, enzymatic assay for the identification of specific inhibitors of inorganic pyrophosphatase (iPPase) from Saccharomyces cerevisiae for antifungal drug discovery. The human iPPase was used as counterscreen. The coding regions of both enzymes were amplified, cloned into a vector providing a His-tag at the C-terminus, expressed in Escherichia coli, and purified by metal chelate affinity chromatography. Since the complete human sequence had not been published previously, the human iPPase was cloned on the basis of expressed sequence tag data. The human sequence was confirmed and showed about 55% amino acid identity with the yeast enzyme and 95% identity with an already published bovine enzyme. Both recombinant iPPases were characterized with regard to their biochemical properties, showing that the His tag did not influence the specific activity, pH optimum, inhibitor profile, or dimerization. The enzyme activity was determined by quantifying released phosphate by complex formation with malachite green. The resulting complex was quantified spectrophotometrically. The assay was adapted to a microtiter plate format. Thus, it is possible to screen a large compound pool for iPPase inhibitors in a short period of time. PMID- 10733884 TI - Bacterial expression and purification of the Arabidopsis NADPH-cytochrome P450 reductase ATR2. AB - An N-terminally modified form of the Arabidopsis NADPH-cytochrome P450 ATR2 (ATR2mod) was expressed from the tactac promoter in Escherichia coli to obtain high yields of the enzyme. The N-terminal modification eliminates the predicted chloroplast transit peptide of ATR2 allowing for more efficient expression. ATR2mod was purified from membrane extracts using a 2',5'-ADP-agarose affinity column. The specific activity of the purified ATR2mod for cytochrome c reduction was 9.4 micromol min(-1) mg(-1) and the K(m) for cytochrome c reduction was 15 +/ 2 microM. The purified NADPH-cytochrome P450 reductase was able to support function of CYP79B2. PMID- 10733885 TI - Expression of human cardiac-specific homeobox protein in Escherichia coli. AB - Human cardiac-specific homeobox protein cDNA (hCsx) was cloned into expression plasmid pET32a and fused with Escherichia coli thioredoxin (Trx). The Trx-Csx fusion protein was under the control of bacteriophage T7 promoter. When expressed in E. coli BL21(DE3), about half of the recombinant Trx-Csx products existed in the form of insoluble inclusion bodies. When coexpressed with human protein disulfide isomerase, more than 90% of Trx-Csx products accumulated in the soluble form in the cell lysate. The recombinant Csx fusion protein was purified by one step metal-chelating affinity chromatography. PMID- 10733886 TI - Expression in Escherichia coli and simple purification of human Fhit protein. AB - The fragile histidine triad (Fhit) protein is a homodimeric protein with diadenosine 5',5"'-P(1),P(3)-triphosphate (Ap(3)A) asymmetrical hydrolase activity. We have cloned the human cDNA Fhit in the pPROEX-1 vector and expressed with high yield in Escherichia coli with the sequence Met-Gly-His(6)-Asp-Tyr-Asp Ile-Pro-Thr-Thr followed by a rTEV protease cleavage site, denoted as "H6TV," fused to the N-terminus of Fhit. Expression of H6TV-Fhit in BL21(DE3) cells for 3 h at 37 degrees C produced 30 mg of H6TV-Fhit from 1 L of cell culture ( approximately 4 g of cells). The H6TV-Fhit protein was purified to homogeneity in a single step, with a yield of 80%, using nickel-nitrilotriacetate resin and imidazole buffer as eluting agent. Incubation of H6TV-Fhit with rTEV protease at 4 degrees C for 24 h resulted in complete cleavage of the H6TV peptide. There were no unspecific cleavage products. The purified Fhit protein could be stored for 3 weeks at 4 degrees C without loss of activity. The pure protein was stable at -20 degrees C for at least 18 months when stored in buffer containing 25% glycerol. Purified Fhit was highly active, with a K(m) value for Ap(3)A of 0.9 microM and a k(cat)(monomer) value of 7.2 +/- 1.6 s(-1) (n = 5). The catalytic properties of unconjugated Fhit protein and the H6TV-Fhit fusion protein were essentially identical. This indicates that the 24-amino-acid peptide containing the six histidines fused to the N-terminus of Fhit does not interfere in forming the active homodimers or in the binding of Ap(3)A. PMID- 10733887 TI - Fermentation, purification, and efficacy of a recombinant vaccine candidate against botulinum neurotoxin type F from Pichia pastoris. AB - A recombinant vaccine candidate was developed that protected mice against botulinum neurotoxin serotype F (BoNTF) intoxication. A synthetic gene encoding BoNTF fragment C (rBoNTF(H(c))) was designed, constructed, and inserted into a plasmid for expression in the yeast Pichia pastoris. A total cell protein content of 2.9 g was obtained per liter of fermentation broth. Recombinant rBoNTF(H(c)) was purified from the soluble yeast extract in two chromatographic steps. The process employed Mono S cation exchange chemistry followed by Alkyl-Superose hydrophobic interaction chromatography, producing material judged to be greater than 98% pure by SDS-PAGE. The recovery of purified product from cell extract was estimated to be greater than 42%, with a yield of 140 mg/kg of cell paste. rBoNTF(H(c)) was also purified from the insoluble fraction of the yeast cell lysate. Because the fragment C in the pellet was 35% of the total insoluble protein, only a Mono S cation exchange chromatography step was necessary to achieve a purity greater than 98%. Mice that received three injections of 0.2 microgram of purified soluble rBoNTF(H(c)) were completely protected when challenged with 1000 mouse ip LD(50) of BoNTF toxin. Similarly, three doses of 1 microgram of purified resolubilized rBoNTF(H(c)) completely protected mice from a challenge of 5000 mouse ip LD(50) of BoNTF toxin. Individual serum antibody ELISA titers of mice injected with soluble rBoNTF(H(c)) correlated with survival as all 34 mice with ELISA titers of 100 or greater survived toxin challenge. The work presented here demonstrates that purified rBoNTF(H(c)) is able to protect against a high challenge dose of neurotoxin. PMID- 10733888 TI - Highly purified recombinant varicella Zoster virus thymidine kinase is a homodimer. AB - Recombinant varicella zoster virus (VZV) thymidine kinase (TK) was isolated in a fast and gentle two-step procedure from Escherichia coli. The TK was expressed as a PreScission-cleavable fusion protein and purified by glutathione and ATP affinity chromatography, yielding homogeneous, highly pure VZV TK. The purified enzyme displays enzymatic activities with K(m) values of 0.3 +/- 0.06 microM for the natural substrate thymidine and 11.6 +/- 3.2 microM for ATP, indicating the biochemical equivalence with the viral VZV TK expressed in infected cells. Determinations of the native molecular weight by size exclusion chromatography and native polyacrylamide gel electrophoresis revealed that the pure enzyme is biologically active as a homodimer. PMID- 10733889 TI - tRNA-assisted overproduction of eukaryotic ribosomal proteins. AB - Structural studies of eukaryotic ribosomes are complicated by the tendency of their constituent proteins to be expressed at very low levels in Escherichia coli. We find that this is mainly due to their exceptionally high content of AGA/AGG arginine codons, which are poorly utilized by the bacterial translational machinery. In fact, we could overcome this limitation by the combined use of a T7 RNA polymerase expression vector and a plasmid carrying the E. coli gene argU, which encodes the minor tRNA(Arg) species that reads AGA/AGG codons. In this system, five cytoplasmic ribosomal proteins from three different eukaryotic lineages (Saccharomyces cerevisiae S8, L13, and L14; Arabidopsis thaliana L13; and Homo sapiens L7) could be overexpressed to up to 50% of total bacterial protein and were purified to homogeneity in tens of milligrams amounts. The purification procedure simply involved metal affinity chromatography followed, in some cases, by an additional heparin chromatography step. Recombinant polypeptides bound RNA with high affinity (K(d) between 50 and 300 nM). This novel overexpression/purification strategy will allow the production of high amounts of most eukaryotic ribosomal proteins in a form suitable for structural and functional studies. Coupled with recently completed and ongoing whole-genome sequencing projects, it will facilitate the molecular characterization of the eukaryotic ribosome. PMID- 10733890 TI - Overexpression and purification of the Escherichia coli inner membrane enzyme acyl-acyl carrier protein synthase in an active form. AB - Acyl-acyl carrier protein synthase (Aas) is widely used to synthesize thioester adducts of fatty acids between 8 and 18 carbons in length enzymatically to the phosphopantetheine group of acyl carrier protein. The enzyme is an 80.6-kDa inner membrane protein that functions in vivo as a 2-acylglycerophosphoethanolamine acyltransferase. The E. coli aas open reading frame was inserted into the expression plasmid pET28a so that, upon expression, a 21-amino-acid extension containing 6 consecutive histidine residues was added to the carboxyl terminus. The plasmid was designated pAasH. The activity of Aas in membranes was assessed from several cell lines. Membranes from the commonly used host line BL21(DE3) containing pAasH accumulated 30-fold and 38-fold more Aas activity than membranes from BL21(DE3) cells lacking the plasmid, when induced with isopropyl beta-d thiogalactopyranoside (IPTG) or lactose, respectively. When pAasH was expressed under IPTG induction in cell line C41(DE3), a previously described cell line selected to enhance the expression of membrane proteins, Aas levels accumulated to 135-fold higher levels than in the cell line lacking the plasmid. Functional Aas can be isolated from either BL21(DE3) or C41(DE3) cell lines by differential centrifugation, followed by detergent extraction with Triton X-100 and nickel nitrilotriacetic acid affinity chromatography. The overexpression of Aas in cell line C41(DE3) is noteworthy compared to cell line BL21(DE3) because it results in a 3- to 4-fold higher accumulation of active enzyme in the membrane fraction and a lower proportion of inactive protein in the inclusion body. PMID- 10733891 TI - Genetic immunization for the recovery and purification of recombinant proteins. AB - A system that uses genetic immunization for recombinant protein recovery and purification is described. The genetic sequence encoding a target protein is subcloned into both a eukaryotic and a prokaryotic vector. With the eukaryotic construct, a rabbit is genetically immunized and specific polyclonal antibodies to the encoded protein raised. The prokaryotic construct is used for bacterial transformation and expression of recombinant protein. Recovery and purification of target recombinant protein are obtained by passing the lysate of expressing bacteria through an immunoaffinity column prepared with the polyclonal antibodies raised in the genetically immunized animal. This method allows purification of recombinant protein without fusion tails and can be applied to purify any protein whose encoding genetic sequence is known. PMID- 10733892 TI - Expression and purification of imidazole glycerol phosphate synthase from Saccharomyces cerevisiae. AB - Imidazole glycerol phosphate (IGP) synthase is a glutamine amidotransferase that catalyzes the formation of IGP and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) from N(1)-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-car boxamide ribonucleotide (PRFAR). This enzyme represents a junction between histidine biosynthesis and de novo purine biosynthesis. The recent characterization of the HIS7 gene in the yeast Saccharomyces cerevisiae IGP synthase established that this protein is bifunctional, representing a fusion between the N-terminal HisH domain and a C-terminal HisF domain. Catalytically active yeast HIS7 was expressed in a bacterial system under the control of T7 polymerase promoter. The recombinant enzyme was purified to homogeneity and the native molecular weight and steady-state kinetic constants were determined. The yeast enzyme is distinguished from the Escherichia coli IGP synthase in its utilization of ammonia as a substrate. HIS7 displays a higher K(m) for glutamine and a lower turnover in the ammonia-dependent IGP synthase activity. As observed with the E. coli IGP synthase, HIS7 shows a low basal level glutaminase activity that can be enhanced 1000-fold in the presence of a nucleotide substrate or analog. The purification and characterization of the S. cerevisiae enzyme will enable a more detailed investigation of the biochemical mechanisms that mediate the ammonia-transfer process. The fused structural feature of the HIS7 protein and the development of a high-level production system for the active enzyme elevate the potential for determination of its three-dimensional structure through X-ray crystallography. PMID- 10733893 TI - Caspase 8: an efficient method for large-scale autoactivation of recombinant procaspase 8 by matrix adsorption and characterization of the active enzyme. AB - A gene coding for a truncated form of human procaspase 8 has been cloned and expressed in Escherichia coli. This construct contains M(206) through D(479) of human procaspase 8, preceded by an N-terminal polyhistidine tag. The recombinant protein, containing 286 amino acids, was expressed in high yield in the form of inclusion bodies (IB). The IB were solubilized in guanidinium chloride and dialyzed against 50% acetic acid. The solution was mixed with 9 volumes of H(2)O and then rapidly diluted from the acidic medium to one containing 1.0 M Tris, pH 8.0, and 5 mM DTT. SDS-PAGE analysis of the soluble, dilute protein solution (20 30 microgram of protein/ml) showed a single 33-kDa band corresponding to the nonprocessed, inactive procaspase 8. Concentration of the dilute protein to levels as high as 2 mg/ml resulted in only modest (1-10%) autocatalytic conversion to the 19- and 11-kDa polypeptide subunits which are characteristic of the activated enzyme. Further concentration of these protein solutions to a near dry state on the ultrafiltration membrane, followed by washing of the membrane with buffer, led to extracts containing high yields of enzyme showing a specific activity of 8.43 micromol/min/mg against the chromogenic substrate Ac-IETD-pNA. SDS-PAGE, protein sequencing, and mass spectrometric analysis of these extracts showed complete conversion of the 33-kDa procaspase 8 to the 19- and 11-kDa subunits of activated caspase 8. This method allows for preparation of 100-mg quantities of highly pure and active recombinant human caspase 8. Enzyme activity was shown to be associated with a heterotetrameric complex that is converted to an inactive dimer upon storage. PMID- 10733894 TI - Refolding and purification of Zymomonas mobilis levansucrase produced as inclusion bodies in fed-batch culture of recombinant Escherichia coli. AB - Zymomonas mobilis levansucrase was overproduced by the fed-batch culture of recombinant Escherichia coli harboring a novel expression system that is constitutively expressed by the promoter from the Rahnella aquatilis levansucrase gene. Most of the levansucrase was produced as inclusion bodies in the bacterial cytoplasm, accounting for approximately 20% of the total cellular protein. Refolding after complete denaturation by high concentrations of urea or guanidine hydrochloride was not successful, resulting in large amounts of insoluble aggregates. During the development of the refolding method, it was found that direct solubilization of the inclusion bodies with Triton X-100 reactivated the enzyme, with a considerable refolding efficiency. About 65% of inclusion body levansucrase was refolded into active levansucrase in the renaturation buffer containing 4% (v/v) Triton X-100. The in vitro refolded enzyme was purified to 95% purity by single-step DEAE-Sepharose ion exchange chromatography. Triton X 100 was removed by this ion exchange chromatography. PMID- 10733895 TI - Short, hydrophobic, alanine-based proteins based on the basic region/leucine zipper protein motif: overcoming inclusion body formation and protein aggregation during overexpression, purification, and renaturation. AB - GCN4 is a yeast transcriptional regulatory protein; its DNA-binding domain is a basic region/leucine zipper (bZIP) structure that comprises a dimer of alpha helices capable of high-affinity, sequence-specific recognition of the DNA major groove. We are exploiting what nature has evolved by manipulating the bZIP motif as a molecular recognition scaffold; thus we reduced the elegantly minimal bZIP to an even more simplified structure by substitution with alanine residues-hence, a generic, Ala-based, helical scaffold. These Ala-based mutants are unusual proteins for expression as they are short ( approximately 100 amino acids) and hydrophobic (Ala-mutated basic regions, leucine-zipper dimerization domains). Hydrophobicity posed a major problem throughout the expression, isolation, and purification stages; inclusion body formation and protein aggregation were significant hurdles throughout protein production. We describe measures that solved these problems, including use of high concentrations of denaturant in all steps of protein isolation and purification and use of temperature-dependent renaturing techniques to obtain folded, functional protein. Despite these difficulties, we ultimately retrieved 5-10 mg/L of broth of active, correctly folded protein after the complete purification procedure. Homogeneity of the proteins was established by chromatography, electrophoresis, and mass spectrometry. Furthermore, characterization by circular dichroism and DNase footprinting analysis demonstrates that these alanine-based mutants retain the structure and function of the native GCN4 DNA-binding domain. Remarkably, the most heavily mutated protein, containing 24 alanines of 27 total amino acids in the DNA-binding basic region, still binds the AP-1 site, the target of native GCN4. PMID- 10733896 TI - Estrogen receptors: how do they control reproductive and nonreproductive functions? AB - Three aspects of recent development in estrogen receptor research will be discussed in this review. First, since the discovery of the second estrogen receptor, ERbeta, a new era has begun in this field. The presence of another receptor for estrogen having different tissue distribution and molecular specificity has posed a question as to the authenticity of the hitherto believed interpretation of the diverse actions of estrogen in different organs of both sexes. Ongoing studies, however, seem on the way of clarifying these new complex puzzles caused by the appearance of the new actor. Recent data with knockout mice for these genes are analyzed and discussed. Second, the mechanism of estrogen receptor action as a ligand-dependent transcription factor has been much more clarified these several years since the discovery of coactivators of steroid receptors which transmit the effect of ER to the transcription initiation complex. This may also open a way to understand the remodeling of chromatin to an active form which has long been sought. Third, the downstream genes of ER are now being isolated and characterized, which is mandatory for the global understanding of the estrogen action during the development and function of an individual animal. This approach, which has been most difficult, will now become more popular in future as newer technologies for this develop. PMID- 10733897 TI - Prostaglandin F(2alpha) (PGF(2alpha)) induces cyclin D1 expression and DNA synthesis via early signaling mechanisms in Swiss mouse 3T3 cells. AB - Prostaglandin F(2alpha) (PGF(2alpha)), a mitogen for Swiss 3T3 cells, triggers cyclin D1 mRNA/protein expression prior to cellular entry into the S phase, but fails to raise cdk4 or cyclin D3 levels, while 1-oleoyl-2-diacylglycerol (OAG), a protein kinase C (PKC) and tyrosine kinase (TK) activator, induces only cyclin D1 expression with no mitogenic response. In contrast, in PKC-depleted or -inhibited cells, PGF(2alpha), but not OAG, increases cyclin D1 expression with no mitogenic response. Finally, OAG, in the presence of orthovanadate (Na(3)VO(4)) or TGF(beta1), induces DNA synthesis. Thus, it appears that PGF(2alpha) triggers cyclin D1 expression via two independent signaling events that complement with TGF(beta1)-triggered events to induce DNA synthesis. PMID- 10733898 TI - Expression of retinoid receptors during human monocyte differentiation in vitro. AB - 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)VD(3)) and retinoic acid (RA) modulate the activation of monocytes (MO) and their differentiation into macrophages (MAC). As these effects are mostly mediated by heterodimers or homodimers of the specific nuclear receptors for 1,25(OH)(2)VD(3) and RA, we investigated the expression of the retinoic acid receptors (RAR) alpha, beta, and gamma and the retinoid X-receptor (RXR) alpha in MO during differentiation into MAC or dendritic cells (DC). The mRNA of all investigated receptors except RARbeta was detected in short-term cultured MO. During differentiation of MO to MAC the mRNA expression of the RA receptors decreased. In contrast, along the differentiation pathway of MO to DC, only the mRNA expression of RARgamma declined, whereas RARalpha and RXRalpha were constantly expressed at a high level. Despite the strong expression of RARalpha and RXRalpha at mRNA level in MO derived DC, the protein expression of the receptors was low in these cells. However, MO and MO-derived MAC showed a strong expression of these receptors at protein level. This suggests that a posttranscriptional or posttranslational mechanism of receptor regulation is occurring in these cells, and in particular in the DC. The inverse regulation of RA receptor expression and protein levels between MAC and DC may control the responsiveness of these cells to 1,25(OH)(2)VD(3) and RA. PMID- 10733899 TI - Viral interferon regulatory factor 1 of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) binds to, and inhibits transactivation of, CREB-binding protein. AB - Kaposi's sarcoma-associated herpesvirus (KSHV) contains many cellular homologue genes. The K9 open reading frame (ORF) of KSHV encodes a virus-encoded interferon regulatory factor (vIRF) which functions as a repressor for cellular interferon mediated signal transduction, and as an oncogene to induce cell growth transformation. In addition, KSHV vIRF plays an important role in the regulation of gene expression. From genetic and biochemical analysis, we demonstrate that KSHV vIRF1 binds to a transcriptional coactivator CREB-binding protein (CBP) in vivo and in vitro. KSHV vIRF1 binds to the KIX domain and CH/3 region of CBP. The CH/3 region of CBP coincides with the binding region of adenovirus E1A. We also show that vIRF1 inhibits the transactivational activity of CBP in HeLa cells. These results demonstrate that vIRF1 can modulate gene expression by inhibiting the transactivation function of coactivator CBP. PMID- 10733900 TI - Differential interaction of CrkII adaptor protein with platelet-derived growth factor alpha- and beta-receptors is determined by its internal tyrosine phosphorylation. AB - CrkII is an intracellular adaptor protein involved in signal transduction by various growth factors. Activation of PDGF alpha-receptor resulted in its association with CrkII in vivo. In contrast, binding of CrkII to the PDGF beta receptor was negligible, despite its becoming prominently phosphorylated. Bacterially expressed GST-CrkII SH2 domain specifically bound to Tyr-762 and Tyr 771 in the activated PDGF alpha- and beta- receptors, respectively. GST fusion protein of full-length CrkII also bound to the activated PDGF beta-receptor. However, tyrosine phosphorylation of GST-CrkII diminished its binding to the beta receptor. CrkI, a truncated version of CrkII lacking the phosphorylatable tyrosine residue, could bind to both PDGF alpha- and beta-receptors in vivo. In conclusion, tyrosine phosphorylation of CrkII negatively affects its binding to the PDGF receptors. The differential binding of CrkII to the PDGF alpha- and beta receptors may be a rationale for functional diversity between the two receptors. PMID- 10733901 TI - Molecular cloning and characterization of Xenopus RGS5. AB - We identified six genes that encode putative RGS proteins (XRGSI-VI) in developing Xenopus embryos using PCR amplification with degenerate primers corresponding to the conserved region (RGS domain) of known RGS proteins. RT-PCR analysis revealed that mRNAs of these XRGSs are differentially expressed during embryogenesis. At stage 1, only XRGSII mRNA was detected. On the other hand, expression of XRGSVI mRNA increased apparently at stage 14 and expression of three of other XRGS (III, IV, V) elevated between stage 25 and 40. To further characterize XRGS proteins expressed in Xenopus embryos, we isolated a cDNA clone for XRGSIII. Based on determined nucleotide sequence, XRGSIII was considered as a Xenopus homologue of mammalian RGS5 (XRGS5). Genetic analysis using the pheromone response halo assay showed that expression of XRGS5 inhibits yeast response to alpha-factor, suggesting that XRGS5 negatively regulates the G-protein-mediated signaling pathway in developing Xenopus embryos. PMID- 10733902 TI - Identification of cDNAs encoding two subtypes of vitamin D receptor in flounder, Paralichthys olivaceus. AB - cDNAs encoding two subtypes of the vitamin D receptor (VDR) are cloned from a teleost (flounder, Paralichthys olivaceus). This is the first report of VDR subtypes in fish. Flounder VDR (fVDR) a and b share 86% identity at the amino acid level. With human (h), rat, mouse, quail, and Xenopus VDRs, fVDRa shares 72%, 71%, 71%, 69%, and 71% identity, and fVDRb shares 70%, 69%, 69%, 67%, and 68% identity, respectively. The peptide sequences of the DNA-binding domain (DBD) and hormone-binding domain (HBD) of both subtypes have particularly high homology to those of the tetrapods; e.g. 92% identity for DBP and 74% for HBD between fVDRa and hVDR. In an evolutionary tree constructed with peptide sequences of VDRs and related members of the nuclear receptor superfamily, fVDRa and b are more closely related to each other than to other molecules, and situated in the cluster of VDRs at a position which corresponds well with the evolutional position of fish in the vertebrates. Additional independent genome duplication which is thought to have occurred in ray-finned fish phylogeny may explain the existence of two subtypes of VDR in flounder. PMID- 10733903 TI - Localization of the type I restriction-modification enzyme EcoKI in the bacterial cell. AB - To localise the type I restriction-modification (R-M) enzyme EcoKI within the bacterial cell, the Hsd subunits present in subcellular fractions were analysed using immunoblotting techniques. The endonuclease (ENase) as well as the methylase (MTase) were found to be associated with the cytoplasmic membrane. HsdR and HsdM subunits produced individually were soluble, cytoplasmic polypeptides and only became membrane-associated when coproduced with the insoluble HsdS subunit. The release of enzyme from the membrane fraction following benzonase treatment indicated a role for DNA in this interaction. Trypsinization of spheroplasts revealed that the HsdR subunit in the assembled ENase was accessible to protease, while HsdM and HsdS, in both ENase and MTase complexes, were fully protected against digestion. We postulate that the R-M enzyme EcoKI is associated with the cytoplasmic membrane in a manner that allows access of HsdR to the periplasmic space, while the MTase components are localised on the inner side of the plasma membrane. PMID- 10733904 TI - Purification, identification, and characterization of an osmotic response element binding protein. AB - Kidney cells, especially the epithelial cells lining the collecting tubules in the inner medulla, are constantly exposed to concentrated urine. They are protected from the osmotic effect of high levels of sodium ion and urea by accumulating compatible osmolytes such as sorbitol, betaine, and myo-inositol. These osmolytes are involved in maintaining cell volume and electrolyte contents because they do not perturb the protein structure and function over a wide range of concentrations. Sorbitol is produced via the reduction of glucose by aldose reductase (AR), while betaine and myo-inositol are transported into the cells through specific transporters. Under hyperosmotic stress, transcriptions of genes encoding these proteins are highly induced. The induction of transcription was found to be mediated through the osmotic response elements (OREs) located in the 5' flanking sequences of these genes. We had earlier identified the OREs in human AR gene. In this study we purified and identified the osmotic response element binding protein (OREBP). OREBP is a transcription factor of approximately 200 kDa in size, characterized by a Rel-like DNA binding domain and a glutamine-rich transactivation domain. Dominant negative OREBP significantly diminished hyperosmotic AR gene induction. Immunohistochemical analysis showed that this transcription factor is rapidly translocated into the nucleus upon hyperosmotic stress. PMID- 10733905 TI - High density lipoprotein inhibits assembly of amyloid beta-peptides into fibrils. AB - The extracellular deposition of amyloid beta (Abeta) in senile plaques constitutes one of the defining hallmarks of Alzheimer's disease. Abeta peptides can aggregate spontaneously to highly insoluble amyloid fibrils, but several components are likely to influence the kinetics of fibrillogenesis in vivo. We report here that high density lipoprotein (HDL), the predominant lipoprotein in the human brain, reduces amyloid formation in vitro as determined by thioflavin T fluorescence and high speed sedimentation assays. The inhibition occurred in a dose dependent manner, and with concentrations of HDL above 1% resulting in more than 70% inhibition. We also examined the combined effect of apolipoprotein E (apoE) and HDL on Abeta fibrillogenesis. We found that HDL particles enriched with any of the three apoE isoforms inhibited Abeta fibrillogenesis as their native counterparts. Taken together, these findings suggest that HDL-like particles in the brain may prevent the formation of Abeta fibrils. PMID- 10733906 TI - Isolation of a novel PDZ-containing myosin from hematopoietic supportive bone marrow stromal cell lines. AB - Stromal cells in bone marrow provide an optimal microenvironment for hematopoiesis. The established stromal cell lines from bone marrow showed various cellular heterogeneities and differed in their hematopoietic supportive ability. By a differential display method, we cloned a gene whose expression levels were correlated with the hematopoietic supportive ability of stromal cells. Its deduced amino acid sequence shows a structure similar to myosins, except that it lacks an actin binding site. Interestingly, it contains a KE-rich sequence and a PDZ domain in the NH(2)-terminal, which are protein-protein interaction domains; therefore we termed this novel myosin Myosin containing PDZ domain (MysPDZ). Western blot analysis showed that its protein levels positively correlated with the supportive ability of stromal cells and immunostaining suggested that MysPDZ was present at cytoskeleton in a filamentous and/or network form. Thus MysPDZ may be involved in the maintenance of the stromal cell architectures required for cell to cell contact. PMID- 10733907 TI - Induction of apoptosis by all-trans-retinoic acid and C2-ceramide treatment in rat stromal-vascular cultures. AB - Apoptosis of preadipocytes and adipocytes contributes to the balance of adipose tissue mass by reducing adipocyte number. To address this phenomenon, we treated cultured rat S-V cells with all-trans-retinoic acid (RA) (10 microM) or C2 ceramide (50 microM) during adipogenesis. Gel electrophoresis of DNA from treated cells cultured in serum-free medium showed that 10 microM RA or 50 microM ceramide induced a distinct laddering pattern of DNA fragments. Cellular caspase 3 activity, another marker of apoptosis, was increased by RA (10 microM) (P < 0.05), but not by 50 microm C2-ceramide. RT-PCR results showed that RA (10 microM) decreased the expression of Bcl-2 mRNA. These results suggest that fat cell loss by apoptosis can be regulated, in part, by RA (10 microM) which increases caspase 3 activity and decreases Bcl-2 expression in rat S-V cells. C2 ceramide apparently works through a different cellular mechanism to induce apoptosis. PMID- 10733908 TI - Operon structure and functional analysis of the genes encoding thermophilic desulfurizing enzymes of Paenibacillus sp. A11-2. AB - Paenibacillus A11-2 can efficiently cleave two carbon&bond;sulfur bonds in dibenzothiophene (DBT) and alkyl DBTs, which are refractory by conventional petroleum hydrodesulfurization, to remove sulfur atom at high temperatures. An 8.7-kb DNA fragment containing the genes for the DBT desulfurizing enzymes of A11 2 was cloned in Escherichia coli and characterized. Heterologous expression analysis of the deletion mutants identified three open reading frames that were required for the desulfurization of DBT to 2-hydroxybiphenyl (2-HBP). The three genes were designated tdsA, tdsB, and tdsC (for thermophilic desulfurization). Both the nucleotide sequences and the deduced amino acid sequences show significant homology to dszABC genes of Rhodococcus sp. IGTS8, but there are several local differences between them. Subclone analysis revealed that the product of tdsC oxidizes DBT to DBT-5,5'-dioxide via DBT-5-oxide, the product of tdsA converts DBT-5,5'-dioxide to 2-(2-hydroxyphenyl) benzene sulfinate, and the product of tdsB converts 2-(2-hydroxyphenyl)benzene sulfinate to 2-HBP. Cell-free extracts of a recombinant E. coli harboring all the three desulfurization genes converted DBT to 2-HBP at both 37 and 50 degrees C. In vivo and in vitro exhibition of desulfurization activity of the recombinant genes derived from a Paenibacillus indicates that an E. coli oxidoreductase can be functionally coupled with the monooxygenases of a gram-positive thermophile. PMID- 10733909 TI - Rat receptor-activity-modifying proteins (RAMPs) for adrenomedullin/CGRP receptor: cloning and upregulation in obstructive nephropathy. AB - Adrenomedullin (AM) is a potent vasorelaxing peptide originally isolated pheochromocytoma. Recently, a family of receptor-activity-modifying proteins (RAMPs 1-3) were identified in humans. Associated with the calcitonin receptor like receptor (CRLR), RAMP2 or RAMP3 may function as the AM receptor. Here we cloned rat RAMP family, analyzed their distribution in rat tissues, and examined regulation of their expression in the kidney using an obstructive nephropathy model. Northern blot analyses revealed that the RAMP family genes are expressed in various tissues with different tissue specificity; RAMP1 is abundantly expressed in the brain, fat, thymus, and spleen, RAMP2 in the lung, spleen, fat, and aorta, while RAMP3 is most abundant in the kidney and lung. After ureteral obstruction, RAMP1, RAMP2, and CRLR gene expressions in the obstructed kidney were markedly upregulated, whereas RAMP3 expression was unchanged. Thus, RAMPs are regulated differently in obstructive nephropathy, suggesting their distinct roles in renal pathophysiology. PMID- 10733910 TI - DNA induces apoptosis in electroporated human promonocytic cell line U937. AB - Experimental gene transfer has permitted a wide variety of studies on gene regulation and function. However, possible effects of the introduced DNA on cellular metabolism are not well understood. Here we demonstrated that introduction of DNA into a promonocytic cell line, U937, by electroporation caused extensive cell death. The toxicity of DNA was concentration-dependent. Various DNAs including plasmid and genomic DNAs all caused cell death, indicating that the toxicity is nucleotide sequence-independent. DNA-induced cell death was associated with internucleosomal DNA fragmentation, a decrease in cell size, and a considerable proportion of cells outside cell cycle. From these results, we concluded that cells died by apoptosis. Our findings have experimental implication for an important issue concerning the interpretation of experiments using gene transfer. In addition, we propose that our observed phenomenon may be relevant to an important immune defense mechanism in monocytes/macrophages that facilitates a response to certain viral infections. PMID- 10733911 TI - Expression, purification, and crystallization of the Escherichia coli selenomethionyl beta-ketoacyl-acyl carrier protein synthase III. AB - Bacterial beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, also called FabH) catalyzes the condensation and transacylation of acetyl-CoA with malonyl-ACP. In order to understand the mode of enzyme/substrate interaction and design small molecule inhibitors, we have expressed, purified, and crystallized a selenomethionyl-derivative of E. coli KAS III. Several lines of evidence confirmed that purified selenomethionyl KAS III was homogenous, stably folded, and enzymatically active. Dynamic light scattering, size exclusion chromatography, and mass spectrometry results indicated that selenomethionyl KAS III is a noncovalent homodimer. Diffraction quality crystals of selenomethionyl KAS III/acetyl-CoA complex, which grew overnight to a size of 0.2 mm(3), belonged to the tetragonal space group P4(1)2(1)2. PMID- 10733912 TI - Different regulation of vascular endothelial growth factor expression by the ERK and p38 kinase pathways in v-ras, v-raf, and v-myc transformed cells. AB - Here we show that vascular endothelial growth factor (VEGF) mRNA expression is up regulated in oncogene transformed rat liver epithelial (RLE) cell lines and that the extracellular signal-regulated kinase (ERK) and p38 kinase differentially regulate the oncogene-mediated stimulation of VEGF. The highest level of VEGF mRNA expression was observed in the v-H-ras transformed RLE cell line, followed by the v-raf and v-myc transformed lines. The PD98059 MEK inhibitor was used to block the ERK pathway and SB203580 inhibitor to block the p38 pathway. The parent and the v-H-ras transformed RLE cell lines showed up-regulation of VEGF RNA expression through the ERK pathway and down-regulation of VEGF through the p38 pathway. VEGF was regulated in a comparable manner in a human breast carcinoma cell line. In the v-raf and v-myc transformed RLE lines, positive regulation of VEGF was transduced through the p38 pathway. These findings suggest that (1) oncogenic ras differs from raf and myc in the recruitment of the MAPK signaling pathways for VEGF regulation; (2) that VEGF is regulated in ras transformed and human cancer cell lines in a positive and negative manner by the ERK and p38 signaling pathways. PMID- 10733913 TI - The effects of RAD52 epistasis group genes on various types of spontaneous mitotic recombination in Saccharomyces cerevisiae. AB - The role of RAD52 epistasis group genes on spontaneous mitotic recombination was examined using three different types of spontaneous mitotic recombination in Saccharomyces cerevisiae. The spontaneous recombination between homologous sequences in a plasmid and a chromosome was essentially unaffected by null mutations in any of the RAD52 epistasis group genes. Recombination between genes in separate autonomously replicating plasmids was reduced 833-fold in a rad52 null mutant, but only 2- to at most 20-fold in rad50, 51, 54, 55, 57 null mutants. Recombination between tandemly repeated heteroalleles in an autonomously replicating plasmid was reduced almost 100-fold in a rad52 null mutant, but is either unaffected or slightly increased in rad50, 51, 54, 55, 57 null mutants. The finding that RAD50, 51, 54, 55, 57 are dispensable or marginally involved in these spontaneous recombinations suggests further that spontaneous mitotic recombination in S. cerevisiae might be processed by other than RAD52 epistasis group. PMID- 10733914 TI - A mouse fibroblast cell model to study human papillomavirus-associated tumorigenesis. AB - Cervical cancer represents the second most common cancer in women worldwide. About 90% of cervical cancer contain high-risk human papillomavirus (HPV) DNA, most often HPV type 16. Animal models and mostly laboratory mice are excellent for carrying out diverse immunological studies. We transfected a fibroblast cell line, 3T3-A31, with human papillomavirus type 16 genome to develop an in vivo/in vitro malignant transformant model. Isolated clones inoculated to immunocompetent mice displayed a tumorigenic phenotype. Small clusters of metastatic cells were found in the liver of animal 45 days after receiving the inoculum. Integrated viral DNA and expression of E7 viral oncogene from the high-risk HPV-16 were demonstrated both in transfectans and tumor-derived cells. The observed high grade neovascularization was correlated with the upregulation of vascular endothelial growth factor (VEGF) mRNA on HPV-16 transformed fibroblast cells. These observations emphasize the association between papillomavirus expression and progression to malignancy. PMID- 10733915 TI - Physicochemical modeling of the role of free radicals in photodynamic therapy. AB - A new kinetic approach is suggested and experimentally supported for quantification of the spin-trapping procedure. Accordingly, the concentration of the spin adduct formed in the interaction between the spin-trap DMPO and various free radicals (cyanopropylperoxy, cumylperoxy, phenylethylperoxy, and hydroperoxy radicals) generated by the initiated oxidation of the parent molecules is followed by kinetic ESR spectrometry. The initial sections of the corresponding kinetics are linear indicating that during this period the consumption of the adduct is negligible and thus the rate of accumulation (W(A)) approximates the rate of formation (W(f)): W(A) approximately W(f) = k(ST)[Rad(*)][DMPO], supported also by the fact that the rate of initiation of oxidation equals W(A) at high [DMPO]. In addition, the circulatory experimental apparatus enables calculation of the rate of molecular decomposition of the adduct by stopping circulation (W(f) becomes negligible) and following the decrease of the ESR signal. Corresponding rate constants are summarized. PMID- 10733916 TI - Cysteine 3 is not the site of in vitro palmitoylation on G(salpha). AB - Several studies have examined the role of palmitoylation of G protein alpha subunits by nonenzymatic in vitro acylation using palmitoyl-CoA. Here, we investigated nonenzymatic palmitoylation of purified G(salpha) in vitro. GDP bound G(salpha) was stoichiometrically autoacylated on cysteine residue(s) with micromolar concentrations of palmitoyl-CoA. The acylation led to a complete loss of steady-state GTPase activity and GTPgammaS binding to G(salpha). Mutation of Cys 3 to Ala in G(salpha) did not prevent either palmitoylation or its consequent functional alterations. However, stoichiometric palmitoylation of His(6) G(salpha) did not alter its GTPase activity or GTPgammaS binding. Isoelectric focusing of tryptic peptides from autoacylated wild type, His(6)-tagged, and C3A mutant of G(salpha) showed that Cys 160 is the site of in vitro palmitoylation. Therefore, we conclude that in vitro palmitoylation of G(salpha) occurs on Cys 160 and this modification decreases the ability of the protein to exchange GTP for GDP; N-terminus elongation of G(salpha) prevents this latter effect without altering palmitoylation. PMID- 10733917 TI - Potent and selective inhibition of squalene epoxidase by synthetic galloyl esters. AB - n-Alkyl esters (ethyl, octyl, dodecyl, and cetyl) of gallic acid were evaluated as enzyme inhibitors of recombinant rat squalene epoxidase (SE), a rate-limiting enzyme of cholesterol biogenesis. Dodecyl (6) (IC(50) = 0.061 microM) showed the most potent inhibition, which was far more potent than those of previously reported naturally occurring gallocatechins. Octyl gallate (5) (IC(50) = 0.83 microM) and cetyl gallate (7) (IC(50) = 0.59 microM) also showed good inhibition, while gallic acid (IC(50) = 73 microM) itself was not so active. In addition, chemically synthesized galloyl ester of cholesterol (9) (IC(50) = 3.9 microM), farnesol derivative (10) (IC(50) = 0.57 microM), and dodecyl galloyl amide (8) (IC(50) = 3.0 microM) were also potent inhibitors of SE. Inhibition kinetics revealed that dodecyl gallate inhibited SE in competitive (K(I) = 0.033 microM) and no-time-dependent manner. The potent inhibition of the flavin monooxygenase would be caused by specific binding to the enzyme, and by scavenging reactive oxygen species required for the epoxidation reaction. PMID- 10733918 TI - Homologous expression of recombinant cellobiose dehydrogenase in Phanerochaete chrysosporium. AB - Cellobiose dehydrogenase (CDH) is a novel extracellular hemoflavoenzyme from Phanerochaete chrysosporium and is produced only in cultures supplemented with cellulose. In this report, CDH from P. chrysosporium has been homologously expressed in cultures supplemented with glucose as the sole carbon source when no endogenous CDH is expressed. This was achieved by placing the cdh-1 gene under the control of the D-glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter (1.1 kb) fused upstream of the ATG start codon of cdh-1. The gpd promoter-chd-1 construct was inserted into the multiple cloning site of the expression vector pOGI18, which contained the Schizophyllum commune ade5 as a selectable marker. The P. chrysosporium ade1 auxotrophic strain OGC107-1 was transformed with the pAGC1 construct, and the prototrophic transformants were assayed for CDH activity. Approximately 50% of the Ade(+) transformants exhibited CDH activity in the extracellular medium of stationary cultures. At least one of the transformants produced high levels (500-600 U/liter) of recombinant CDH (rCDH). Purification by ammonium sulfate precipitation, Sephacryl S-200 chromatography, and FPLC using a Mono-Q 5/5 column yielded homogeneous rCDH. Physical, spectral, and kinetic characteristics of purified homologously expressed rCDH were similar to those of wild-type CDH. This expression system will enable site-directed mutagenesis studies to be carried out on CDH. PMID- 10733919 TI - The molecular structure of the fastest myosin from green algae, Chara. AB - Chara myosin in green algae, Chara corallina, is the fastest myosin of all those observed so far. To shed light on the molecular mechanism of this fast sliding, we determined the primary structure of Chara myosin heavy chain (hc). It has a motor domain, six IQ motifs for calmodulin binding, a coiled-coil structure to dimerize, and a globular tail. Chara myosin hc is very similar to some plant myosins and has been predicted to belong to the class XI. Short loop 1 and loop 2 may account for the characteristics of mechanochemical properties of Chara myosin. PMID- 10733920 TI - A salt bridge between an N-terminal coiled coil of gp41 and an antiviral agent targeted to the gp41 core is important for anti-HIV-1 activity. AB - HIV-1 envelope glycoprotein transmembrane subunit gp41 play a critical role in the fusion of viral and target cell membranes. The gp41 C-terminal heptad repeat region interacts with the N-terminal coiled-coil region to form a six-stranded core structure. Peptides derived from gp41 C-terminal heptad repeat region (C peptides) are potent HIV-1 entry inhibitors by binding to gp41 N-terminal coiled coil region. Most recently, we have identified two small organic compounds that inhibit HIV-1-mediated membrane fusion by blocking the formation of gp41 core. These two active compounds contain both hydrophobic and acidic groups while the inactive compounds only have hydrophobic groups. Analysis by computer modeling indicate that the acidic groups in the active compounds can form salt bridge with Lys 574 in the N-terminal coiled-coil region of gp41. Asp 632 in a C-peptide can also form a salt bridge with Lys 574. Replacement of Asp 632 with positively charged residues or hydrophobic residues resulted in significant decrease of HIV 1 inhibitory activity. These results suggest that a salt bridge between an N terminal coiled coil of the gp41 and an antiviral agent targeted to the gp41 core is important for anti-HIV-1 activity. PMID- 10733921 TI - Interferon-dependent activation of the serine kinase PI 3'-kinase requires engagement of the IRS pathway but not the Stat pathway. AB - Several signaling pathways are activated by interferon alpha (IFNalpha) in hematopoietic cells, including the Jak-Stat and the insulin receptor substrate (IRS) pathways. It has been previously shown that IFNalpha activates the phosphatidylinositol (PI) 3'-kinase via an interaction of the p85 subunit of PI 3'-kinase with IRS proteins. Other studies have proposed that Stat-3 also functions as an adapter for p85. We sought to identify the major pathway that regulates IFNalpha activation of the PI3'-kinase in hematopoietic cells. Our data demonstrate that IFNalpha induces the interaction of p85 with IRS-1 or IRS-2, but not Stat-3, in various hematopoietic cell lines in which IRS-1 and/or IRS-2 and Stat-3 are activated by IFNalpha. In addition, inhibition of PI 3'-kinase activity by preincubation of cells with the PI 3'-kinase inhibitor LY294002 does not affect IFN-dependent formation of SIF complexes that contain Stat-3. To determine whether phosphorylation of tyrosine residues in the IFN receptor is required for activation of the PI 3'-kinase, we performed studies using mouse L929 fibroblasts transfected with mutated human IFNAR1 and/or IFNAR2 subunits of the Type I IFN receptor, lacking tyrosine phosphorylation sites. The serine kinase activity of the PI-3K was activated by human IFNalpha in these cells, suggesting that phosphorylation of the Type I IFN receptor is not essential for PI3K activation. We then determined whether IFNalpha activates the Akt kinase, a known downstream target for PI 3'-kinase that mediates anti-apoptotic signals. Akt was activated by insulin or IGF-1, but not IFNalpha, in the IFNalpha sensitive U-266 myeloma cell line. Altogether, our data establish that the IRS pathway and not the Stat pathway, is the major pathway regulating engagement of PI 3'-kinase in hematopoietic cells. Furthermore, the selective activation of Akt by insulin/IGF-1 suggests the existence of distinct regulatory activities of PI3' kinase in growth factor versus interferon signaling. PMID- 10733922 TI - In vivo gene gun-mediated DNA delivery into rodent brain tissue. AB - Various types of gene transfer into live tissues have been tried. However, in vivo gene transfer into brain tissue or neuronal cells without virus vector has required a great effort. Particle-mediated gene transfer into live brain tissue was thought to be impossible because of its fragility and the mechanical problem of a previous type of gene gun. In addition, particle-mediated DNA transfer into monolayer-cultured cells without mechanical damage has been difficult. We successfully transferred DNA into rodent live brain tissue and also into monolayer-cultured cells without mechanical damage by using a new type of gene gun and also confirmed gene expression in the brain. This new method represents another variation of gene transfer into the brain. PMID- 10733923 TI - Nuclear localization signals of the BRCA2 protein. AB - BRCA2 is a tumor suppressor gene whose germline mutations increase the lifetime risk of breast cancer. BRCA2 encodes a large nuclear protein involved in DNA repair, but the location of its functional domain has been unclear. Here, we report nuclear localization signals (NLSs) of the BRCA2 protein. By expressing various portions of the BRCA2 protein tagged with enhanced green fluorescent protein in HeLa cells, we show that the C-terminal domain is necessary for nuclear localization. Two regions in the C-terminal domain were identified with functional NLSs by site-directed mutagenesis analyses. The NLSs locate between the germline mutation found in the most downstream position and the polymorphic stop codon, suggesting that defects in the proper nuclear transport of the BRCA2 protein are causative of carcinogenesis. Our data thus provide a possible explanation for the high frequency of frame-shift and nonsense mutations in BRCA2 of hereditary breast cancer patients. PMID- 10733924 TI - In situ localization of agouti signal protein in murine skin using immunohistochemistry with an ASP-specific antibody. AB - Switching between production of eumelanin or pheomelanin in follicular melanocytes is responsible for hair color in mammals; in mice, this switch is controlled by the agouti locus, which encodes agouti signal protein (ASP) through the action of melanocortin receptor 1. To study expression and processing patterns of ASP in the skin and its regulation of pigment production in hair follicles, we have generated a rabbit antibody (termed alphaPEP16) against a synthetic peptide that corresponds to the carboxyl terminus of ASP. The specificity of that antibody was measured by ELISA and was confirmed by Western blot analysis. Using immunohistochemistry, we characterized the expression of ASP in the skin of newborn mice at 3, 6, and 9 days postnatally. Expression in nonagouti (a/a) black mouse skin was negative at all times examined, as expected, and high expression of ASP was observed in 6 day newborn agouti (A/+) and in 6 and 9 day newborn lethal yellow (A(y)/a) mouse skin. In lethal yellow (pheomelanogenic) mice, ASP expression increased day by day as the hair color became more yellow. These expression patterns suggest that ASP is delivered quickly and efficiently to melanocytes and to hair matrix cells in the hair bulbs where it regulates melanin production. PMID- 10733925 TI - An N-terminal 33-amino-acid-deletion variant of hsp25 retains oligomerization and functional properties. AB - The mechanism(s) by which heat shock protein 25 (hsp25) protects cells from stress may involve one or more of the biochemical properties attributed to hsp25 and other small M(r) hsp. In this report, structural and functional properties of an N-terminal 33 amino acid deletion variant of hsp25 (termed hsp25.c) were considered by comparison with hsp25. 6-His tagged recombinant hsp25 and hsp25.c (termed (H6)hsp25.a and (H6)hsp25.c) were expressed and purified. Oligomeric proteins formed and possessed properties previously attributed to hsp25. The 33 amino acid deletion represented by hsp27.c did not affect the ability of the recombinant protein to act as an inhibitor of elastase, as a molecular chaperone in the refolding of denatured citrate synthase, or as an actin-binding protein. The overexpression of either hsp25 or hsp25.c, enhanced the stress resistance of stable transformed eukaryotic cells. This N-terminal variant protein may be used in further cellular and biochemical assessment of hsp25 oligomerization and function. PMID- 10733927 TI - EPR spin-trapping of a myeloperoxidase protein radical. AB - Incubation of myeloperoxidase (MPO) with H(2)O(2) in the presence of the spin trap DBNBS (3,5-dibromo-4-nitrosobenzenesulfonic acid) results in the EPR detectable formation of a partially immobilized protein radical. The radical was only formed in the presence of both MPO and H(2)O(2), indicating that catalytic turnover of the protein is required. The changes in the EPR spectrum of the adduct upon treatment with pronase confirm that the spin trap is bound to a protein residue. These results establish that MPO, like lactoperoxidase [Lardinois, O. M., Medzihradszky, K. F., and Ortiz de Montellano, P. R. (1999) J. Biol. Chem. 274, 35441-35448], reacts with H(2)O(2) to give a protein radical intermediate. The protein radical may have a catalytic role, may be related to covalent binding of the prosthetic heme group to the protein, or may reflect a process that leads to inactivation of the enzyme. PMID- 10733926 TI - Mapping of a protective helper T cell epitope of human influenza A virus hemagglutinin. AB - The synthetic peptide comprising the 317-341 region of human influenza A virus (H1N1 subtype) hemagglutinin elicits peptide-specific antibody and helper T cell responses and confers protection against lethal virus infection. Molecular mapping of the 317-329 region, which encompasses the epitope recognized by peptide-specific T cells, revealed that the minimal size required for T cell activation was the 317-326 segment. The most likely peptide alignment, which placed 320Leu to pocket 1 of the I-E(d) peptide binding groove, was predicted by molecular mechanics calculations performed with the parental and with the Ala substituted analogs. In line with the prediction data, the results of the peptide binding assay, where the relative binding efficiency to I-E(d) molecules expressed on the surface of antigen-presenting cells was monitored, identified the 320-326 core sequence interacting with the major histocompatibility class II peptide binding groove. Functional analysis of Ala-substituted variants by functional assays and by calculating the surface-accessible areas of the single peptidic amino acids in the I-E(d)-peptide complexes demonstrated that 324Pro is a primary contact residue for the T cell receptor. Our results show that this type of analysis offers a suitable tool for molecular mapping of helper T cell epitopes and thus provides valuable data for subunit vaccine design. PMID- 10733928 TI - Modification of adipocyte membrane adenylyl cyclase activity by NAD: evidence against NAD-induced endogenous ADP-ribosylation of Gsalpha protein. AB - Treatment of saponin-permeabilized adipocytes with NAD enhanced adenylyl cyclase activity stimulated by GTgammaS, [Al/F(4)](-), isoproterenol, and forskolin in membrane fractions and potentiated isoproterenol-induced cAMP accumulation in whole cells. In parallel, when permeabilized adipocytes were incubated with [(32)P]NAD, there was significant incorporation of [(32)P]ADP-ribose in a 44-kDa acceptor membrane protein. This reaction was inhibited by l-arginine and was enhanced by the addition of GTPgammaS. Surprisingly, this 44-kDa protein could not be identified as Gs protein: (1) It was not recognized by Gsalpha specific antibody; (2) it did not comigrate with the major cholera toxin substrates in either 10% SDS-PAGE or two-dimensional electrophoresis; (3) a pretreatment of adipocytes with NAD did not decrease cholera toxin-mediated ADP-ribosylation of Gsalpha proteins on membrane fractions. Our results indicate that NAD did not induce endogenous ADP-ribosylation of Gsalpha in permeabilized rat adipocytes but nonetheless modified the adenylyl cyclase response. PMID- 10733929 TI - Blockade of JAK2 by tyrphostin AG-490 inhibits antigen-induced eosinophil recruitment into the mouse airways. AB - We studied the effect of tyrphostin AG-490, a specific Janus kinase 2 (JAK2) inhibitor, on antigen-induced eosinophil recruitment into the airways of sensitized mice and on IL-5-induced chemokinesis and adhesiveness of eosinophils. The in vivo administration of AG-490 prevented antigen-induced eosinophil infiltration in the airways of sensitized mice in a dose-dependent manner. However, the administration of AG-490 did not affect antigen-induced IL-5 production in the airways nor in vitro antigen-induced IL-5 production and T cell proliferation of spleen cells. Furthermore, AG-490 inhibited IL-5-induced chemokinesis and beta1-integrin adhesiveness of eosinophils in vitro. Because antigen-induced eosinophil recruitment into the airways is mediated by IL-5, these results indicate that JAK2 activation is critical for antigen-induced, IL-5 dependent mobilization of eosinophils into the tissue. PMID- 10733930 TI - Sepsis-induced muscle proteolysis is prevented by a proteasome inhibitor in vivo. AB - Sepsis-induced muscle proteolysis mainly reflects ubiquitin-proteasome-dependent protein degradation. The effect of in vivo administration of a proteasome inhibitor on muscle protein breakdown during sepsis is not known. We treated rats with the proteasome inhibitor N-benzyloxycarbonyl-Ile-Glu-(O-t-butyl)-Ala leucinal (PSI) or corresponding volume of vehicle i.p. 2 h before sham-operation or induction of sepsis by cecal ligation and puncture. The sepsis-induced increase in total and myofibrillar muscle protein breakdown was inhibited in rats treated in vivo with PSI and a maximal effect was seen following 15 mg/kg of the proteasome inhibitor. Results from in vitro experiments in which incubated muscles were treated with 100 microM PSI suggest that the drug has a direct effect on muscle and that the effect is specific for the proteasome. The results are important because they suggest that it may be possible to prevent or improve the cachectic response in skeletal muscle during sepsis by treatment with a proteasome inhibitor. PMID- 10733931 TI - FYVE-DSP1, a dual-specificity protein phosphatase containing an FYVE domain. AB - Dual-specificity protein phosphatases (DSPs) dephosphorylate proteins at Ser/Thr and Tyr. FYVE domain is a double zinc finger motif which specifically binds phosphatidylinositol(3)-phosphate. Here, we report a novel dual specificity phosphatase that contains a FYVE domain at the C-terminus. We designate the protein FYVE-DSP1. Molecular cloning yielded three isoforms of the enzyme presumably derived from alternate RNA splicing. Sequence alignment revealed that the catalytic phosphatase domain of FYVE-DSP1 closely resembled that of myotubularin, while its FYVE domain has all the conserved amino acid residues found in other proteins of the same family. Recombinant FYVE-DSP1 is partitioned in both cytosolic and membrane fractions. It dephosphorylates proteins phosphorylated on Ser, Thr, and Tyr residues and low molecular weight phosphatase substrate para-nitrophenylphosphate. It shows typical characteristics of other DSPs and protein tyrosine phosphatases (PTPs). These include inhibition by sodium vanadate and pervanadate, pH dependency, and inactivation by mutation of the key cysteinyl residue at the phosphatase signature motif. Finally, PCR analyses demonstrated that FYVE-DSP1 is widely distributed in human tissues but different spliced forms expressed differently. PMID- 10733932 TI - Substance P regulates PTH secretion through the neurokinin-1 receptor. AB - The primary regulator of PTH secretion is serum ionized Ca(2+); however, neuropeptide-containing nerve fibers have been localized to the parathyroid gland. The purpose of this study was to determine whether or not substance P (SP) regulates PTH secretion. In dispersed porcine parathyroid cells, SP reversibly inhibited 0.5 mM CaCl(2)-induced PTH secretion (IC(50) = 0.29 nM) and had no effect at CaCl(2) concentrations of 1.5 mM and greater. At 0.5 mM CaCl(2), treatment with a NK-1 selective receptor agonist resulted in a concentration dependent decrease in PTH secretion (IC(50) = 0.21 nM). In contrast, NK-2 and NK 3 receptor agonists were approximately 100-fold less active than SP or the NK-1 receptor selective agonist. An enantiospecific reversal of the effects of SP on PTH secretion was observed with LY306740, a potent selective NK-1 receptor antagonist (K(i) = 0.125 nM). In porcine parathyroid cells, expression of mRNA for the NK-1 receptor was observed using RT-PCR. In summary, a novel neuroendocrine pathway is described whereby the neuropeptide, SP, regulates PTH secretion through NK-1 receptors. PMID- 10733933 TI - Role of hepatocyte nuclear factor 1alpha and 1beta in the transcriptional regulation of human dipeptidyl peptidase IV during differentiation of Caco-2 cells. AB - Caco-2 cells undergo differentiation to an enterocytic-like cell when maintained in a post-confluent state for 1-2 weeks. During this period Caco-2 cells begin to express high levels brush border membrane associated enzymes such as dipeptidyl peptidase IV. Using the dipeptidyl peptidase IV gene promoter in electrophoretic mobility shift assays, we have shown for the first time that levels of hepatocyte nuclear factor 1alpha increase three- to fourfold during Caco-2 cell differentiation. Transient cotransfection experiments with 3T3 cells using dipeptidyl peptidase IV promoter constructs and expression vectors containing hepatocyte nuclear factor 1alpha and beta show that the ratio of alpha and beta modulates reporter gene expression. These results suggest that the increase in levels of hepatocyte nuclear factor 1alpha that occur during intestinal cell differentiation, are important for expression of dipeptidyl peptidase IV and other intestinal proteins. PMID- 10733934 TI - The mevalonate/isoprenoid pathway inhibitor apomine (SR-45023A) is antiproliferative and induces apoptosis similar to farnesol. AB - Apomine (SR-45023A) is a new antineoplastic compound which is currently in clinical trials and representative of the family of cholesterol synthesis inhibitors 1,1-bisphosphonate esters. Apomine inhibits growth of a wide variety of tumor cell lines with IC(50) values ranging from 5 to 14 microM. The antiproliferative activity of apomine was studied in comparison with that of other inhibitors of the mevalonate/isoprenoid pathway of cholesterol synthesis, simvastatin, farnesol, and 25-hydroxycholesterol. All these compounds inhibit 3 hydroxy-3-methylglutaryl-coenzyme A reductase activity. Apomine (IC(50) = 14 microM), simvastatin (IC(50) = 3 microM), farnesol (IC(50) = 60 microM), and 25 hydroxycholesterol (IC(50) = 2 microM) inhibited HL60 cell growth. Growth inhibition due to simvastatin was reverted by mevalonate, whereas the antiproliferative activity of apomine, farnesol, and 25-hydroxycholesterol was not. Apomine triggered apoptosis in HL60 cells in less than 2 h. Apomine and farnesol induced caspase-3 activity at concentrations similar to their IC(50) values for cell proliferation, whereas a 10-fold excess of simvastatin was necessary to trigger apoptosis compared to its potency on proliferation. Caspase 3 activity was not induced by 25-hydroxycholesterol. The overall similar profile on mevalonate synthesis inhibition, cell growth inhibition, and apoptosis suggests that apomine acts as a synthetic mimetic of farnesol. PMID- 10733935 TI - Firefly luciferase generates two low-molecular-weight light-emitting species. AB - A bioluminescent D-luciferin-luciferase mixture is separated by gel filtration during the time course of the reaction. A simultaneous analysis with an UV visible diode array detector and an on-line luminometer gives nonsuperimposable chromatograms. Luminescence recordings display three peaks, one associated with the enzyme (light-emitting species 1: LES(1)), and two other species free from the luciferase: LES(2), with a luciferyl-adenylate-like spectrum and LES(3). Production of these two species is nucleotide (ATP or 2'-dATP)- and pH-dependent. The chromatographic data presented here could lead to reconsideration of the generally assumed emission mechanism, which involves one emitter only. It could also suggest that each free emitting species is related to a colour of emission corresponding to the two defined wavelengths previously described ( approximately 575 and approximately 620 nm). PMID- 10733936 TI - p-aminohippuric acid transport at renal apical membrane mediated by human inorganic phosphate transporter NPT1. AB - Organic anions are secreted into urine via organic anion transporters across the renal basolateral and apical membranes. However, no apical membrane transporter for organic anions such as p-aminohippuric acid (PAH) has yet been identified. In the present study, we showed that human NPT1, which is present in renal apical membrane, mediates the transport of PAH. The K(m) value for PAH uptake was 2.66 mM and the uptake was chloride ion sensitive. These results are compatible with those reported for the classical organic anion transport system at the renal apical membrane. PAH transport was inhibited by various anionic compounds. Human NPT1 also accepted uric acid, benzylpenicillin, faropenem, and estradiol-17beta glucuronide as substrates. Considering its chloride ion sensitivity, Npt1 is expected to function for secretion of PAH from renal proximal tubular cells. This is the first molecular demonstration of an organic anion transport function for PAH at the renal apical membrane. PMID- 10733938 TI - A novel protein MAJN binds to Jak3 and inhibits apoptosis induced by IL-2 deprival. AB - To find a possible signal interacting with the Jak3 N-terminal, we screened the human peripheral blood cDNA library through both a two-hybrid system and a tyrosine-phosphorylation-modified two-hybrid system using the N-terminal of Jak3 as bait. Results showed that one new homologue of myosin heavy chain, designated MAJN (molecule associated with Jak3 N-terminal), could bind to Jak3 in a tyrosine phosphorylation-independent manner. The interaction between Jak3 and MAJN was further confirmed by immunoprecipitation in BAF-B03 beta cells. To investigate the function of MAJN, we have constructed the BAF-B03 beta/MAJN cell line that stably expresses MAJN and found that overexpression of MAJN can partially inhibit the apoptosis induced by interleukin-2 deprival. Further studies are needed to elucidate how MAJN executes its function to antagonize BAF-B03beta cell death in the absence of IL-2. PMID- 10733937 TI - Sex steroid influence on cannabinoid CB(1) receptor mRNA and endocannabinoid levels in the anterior pituitary gland. AB - Recent studies have demonstrated the occurrence of endocannabinoid synthesis and of gene expression and immunoreactivity for the cannabinoid CB(1) receptor in the anterior pituitary gland. Since the activity of this gland is under the influence of circulating sex steroids, the present study was designed to elucidate whether expression of the CB(1) receptor gene in the anterior pituitary gland is also under the influence of these steroids. To this aim, we first examined the possible changes in the levels of CB(1) receptor-mRNA transcripts in the anterior pituitary gland of intact male rats and normal cycling female rats at the different stages of the ovarian cycle. We observed that males had higher levels of CB(1) receptor-mRNA transcripts than females. In addition, these transcripts fluctuated in females during the different phases of the ovarian cycle, with the highest values observed on the second day of diestrus and the lowest on estrus. In these animals, we also measured the content of endocannabinoids in the anterior pituitary gland and the hypothalamus. We observed that females had higher contents of anandamide than males in both cases. The content of anandamide in females also fluctuated during the ovarian cycle in both the anterior pituitary gland and the hypothalamus. The highest values in the anterior pituitary gland were found in the estrus and the lowest on the first day of diestrus and proestrus, whereas the inverse tendency was found in the hypothalamus. No changes were observed in the other major endocannabinoid, 2 arachidonoyl-glycerol, between males and females and during the ovarian cycle. To further explore the potential influence of circulating sex steroids on CB(1) receptor gene expression in the anterior pituitary gland, as a second objective, we examined the possible changes in the amount of transcripts for this receptor in gonadectomized and sex steroid-replaced gonadectomized rats of both sexes. We observed that orchidectomy (ORCHX) in males reduced CB(1) receptor-mRNA levels, whereas replacement with dihydrotestosterone also maintained low levels of this messenger. In females, estradiol-replaced ovariectomized (OVX) rats exhibited significantly lower CB(1) receptor-mRNA levels than OVX animals that had not been replaced with this estrogen. In this experiment, we also examined if the previously reported response of the CB(1) receptor gene in the anterior pituitary lobe to chronic administration of Delta(9)-tetrahydrocannabinol (Delta(9)-THC) is under sex steroid influence. We observed that chronic Delta(9)-THC treatment decreased CB(1) receptor-mRNA levels in intact and ORCHX males, but not in dihydrotestosterone-replaced ORCHX males. In females, Delta(9)-THC treatment produced no effect in both OVX- and estradiol-replaced OVX rats. In summary, these data collectively support that expression of the CB(1) receptor gene in the anterior pituitary gland is regulated by sex steroids in both males and females. Furthermore, gonadal steroids appear to affect the response of this gene to chronic cannabinoid administration. We have also observed that anandamide contents in the anterior pituitary gland and the hypothalamus might be controlled by circulating sex steroids. The functional implications of these data are discussed. PMID- 10733939 TI - CD39 modulates IL-1 release from activated endothelial cells. AB - The activation of endothelial cells (EC) and monocyte-macrophages (Mφ) by lipopolysaccharide (LPS) is considered an important element of the vascular injury observed in endotoxemia. Interleukin-1 (IL-1) beta release from Mφ in response to LPS, appears to be mediated by the autocrine/paracrine release of ATP via P2X7 receptor activation. In EC, similar nucleotide-mediated signaling pathways may be influenced by high levels of expression of CD39, the vascular nucleoside triphosphate diphosphohydrolase (NTPDase; ENTPD I). To determine whether CD39 modulates ATP-mediated release of IL-1 from EC, we stimulated human EC with LPS and measured levels of ATP secretion and IL-1 release. LPS triggered ATP secretion from EC that was soon followed by IL-1alpha release. Overexpression of CD39 following infection with recombinant CD39 adenoviral vectors (AdCD39) abrogated the initial phase of ATP secretion and inhibited IL-1alpha release; comparable results were obtained with soluble NTPDase. These data demonstrate that CD39/NTPDase modulates IL-1alpha release from LPS stimulated human EC. PMID- 10733940 TI - Engineering of a mini-trichosanthin that has lower antigenicity by deleting its C terminal amino acid residues. AB - Trichosanthin is a ribosome-inactivating protein that possesses antitumor and antiviral activities. Clinical trials of trichosanthin on AIDS patients, however, elicit anaphylactic reactions. To reduce the antigenicity of trichosanthin as a drug while preserving its biological activity, the C-terminal domain (residues 203 to 247), which contains a putative antigenic site, was systemically deleted. We have found that the minimum length of trichosanthin that can fold into an active conformation is residue 1 to 240. The mini-trichosanthin (C7) generated by deleting the last seven C-terminal amino acid residues has 2.7-fold decrease in antigenicity, 10-fold reduction in in vitro ribosome-inactivation activity, and in vivo cytotoxicity toward K562 cells, and 2-fold reduction in abortificient activity. Structural analyses of C7 indicate decrease in the helix content, increased exposure of Trp192, and lower thermodynamic stability. The deletion of the C-terminal residues (Leu241 to Ala247) probably perturbs local structure of the C-terminal antigenic epitope that results in the decrease in antigenicity and activities of C7. PMID- 10733941 TI - Molecular cloning and partial characterization of a plant VAP33 homologue with a major sperm protein domain. AB - In a search for proteins interacting with the resistance protein Cf9 from tomato, a new cDNA was cloned and characterized. Protein sequence database searches suggested that the 120 residue-N terminal domain of the encoded protein (named VAP27) is highly similar to the VAP33 protein family from animals, to uncharacterized plant proteins, and to a lower extent, to the major sperm protein (MSP) from nematodes. The second half of the protein is similar to VAMP and to the VAP33 N-terminus comprising a predicted coiled-coil region followed by a transmembrane segment. The sequence/structure comparison of VAP27 with the crystal structure of AsMSP1 from Ascaris suum, using molecular modeling with the threading method, suggested that the N-terminus of VAP27 does possess a MSP-like domain that might participate in the formation of a protein-protein network. The coiled-coil region of VAP27 was modeled based on the structure of the VAP- and VAMP-containing SNARE complex. The coiled-coil region might also be involved in protein-protein interactions similar to VAP-VAMP interactions. PMID- 10733942 TI - Characterization of the functionally related sites in the neural inducing gene noggin. AB - Previously we have shown that blocking bone morphogenetic protein (BMP) receptor signaling by a dominant negative BMP receptor causes neurogenesis in Xenopus animal caps (ACs), whereas the physiological neural inducer noggin acts as a homodimer physically binding to BMP-4 and disrupting its signaling at the ligand level. The present study attempted to elucidate the relationship between the structure and function of noggin. By replacing some cysteine residues with serine residues through a site-directed mutagenesis strategy, we generated three noggin mutants, C145S, C205S, and C(218, 220, 222)S (3CS). Although mRNAs encoded by these mutants were translated as efficiently as wild-type (WT) noggin mRNA, they behaved differently when expressed in vivo. Expression of WT noggin or C205S in Xenopus ACs converted the explants (prospective ectoderm) into neural tissue, indicated by the neural-like morphology and expression of the pan neural marker NCAM in the ACs. In contrast, ACs expressing C145S or 3CS sustained an epidermal fate like the control caps. Similar results were observed in the mesoderm where C205S (but not C145S and 3CS) displayed dorsalizing activity as well as WT noggin. Altogether, our results suggest that Cys145 alone or Cys(218, 220, 222) as a whole in noggin protein is required for the biological activities of noggin, probably participating in the dimerization of noggin with BMP-4 or itself. PMID- 10733943 TI - Activation of protease-activated receptor-2 (PAR-2) triggers mucin secretion in the rat sublingual gland. AB - Protease-activated receptor-2 (PAR-2) is distributed throughout the gastrointestinal systems. The present study investigated the role for PAR-2 in the rat salivary glands. PAR-2 mRNA was detected in the sublingual, submaxillary, and parotid glands by a reverse-transcriptase polymerase chain reaction. In the isolated sublingual gland that exhibited the strongest signal for PAR-2, Ser-Leu Ile-Gly-Arg-Leu-NH(2), a PAR-2-activating peptide, and trypsin, a PAR-2 activating enzyme, but not thrombin that can activate PARs 1, 3, and 4, triggered secretion of N-acetylneuraminic acid, an indicator of mucin, that was a unique major sialic acid detectable after hydrolysis of the sublingual mucin with 0.1 N HCl. The PAR-2-mediated secretion of mucin was attenuated by genistein, a tyrosine kinase inhibitor, but not by inhibitors of protein kinase C and phosphatidyl inositol 3'-kinase. Thus, PAR-2 is expressed by the three distinct salivary glands in the rat, and sublingual PAR-2 appears to play a role in triggering mucin secretion, at least in part, via activation of tyrosine kinase. PMID- 10733944 TI - Transcriptional repression of p21((Waf1/Cip1/Sdi1)) gene by c-jun through Sp1 site. AB - Previously, we found that c-jun represses the tumor suppressor p21((Waf1/Cip1/Sdi1)) (p21) gene expression. In this study, we further investigated the mechanism of the inhibitory effect of c-jun on p21. After analysis of a series of deletion and point mutants of p21 promoter, we found that Sp1-3 site (-77 and -83) relative to the transcription start site played an important role for c-jun-repressing-responsive element in the p21 promoter. Both Sp1 and Sp3 transcription factors were the key factors for this event. However, the data from electrophoretic mobility shift assay indicated that c-jun did not change the Sp1 DNA-binding affinity, suggesting that additional factors may be involved in the repression of p21 by c-jun. Furthermore, c-jun could inhibit butyrate-inducing p21 gene expression through Sp1, indicating at least one common pathway whereby p21 expression is affected by c-jun and butyrate in opposing actions. Moreover, the hyperphosphorylated retinoblastoma protein (Rb) increased in c-jun expressing cells, indicating that phosphorylated Rb may play a role in regulating Sp1 to repress p21 expression. This is the first demonstration of how housekeeping factors and oncogene product counteract the function of tumor suppressor genes to control cell cycle progression. PMID- 10733945 TI - Inhibition of the 26S proteasome induces expression of GLCLC, the catalytic subunit for gamma-glutamylcysteine synthetase. AB - The majority of short- and long-lived cellular proteins are degraded by the activities of the 26S proteasome, a large multi-catalytic protease. Its unique function places it as a central regulatory activity for many important physiological processes. Lactacystin is a very specific 26S proteasome inhibitor and represents an excellent tool for demonstrating that a pathway exhibits proteasome-dependent biochemical regulation. Exposure of HepG2 cells to lactacystin resulted in robust elevation of GLCLC mRNA levels, followed by an increase in GSH concentrations. GLCLC is the gene that encodes the catalytic subunit for gamma-glutamylcysteine synthetase, the rate-limiting enzyme for the synthesis of glutathione (GSH). Inhibition of non-proteasome, protease activities did not induce GLCLC. Gel mobility shift assays and expression of CAT activity from heterologous reporter vectors identified Nrf2 mediation of the GLCLC antioxidant response element, ARE4, as the mechanism by which lactacystin induced GLCLC. These studies have identified 26S proteasome activity as a central regulatory pathway for glutathione synthesis. PMID- 10733946 TI - Possible involvement of orexin in the stress reaction in rats. AB - We examined whether corticotropin-releasing factor (CRF) was involved in orexin induced grooming and face-washing behaviors, and whether orexin was involved in the stress reaction. Administration of alpha-helical CRF, CRF antagonist, alone had no behavioral effect, but it blocked the orexin-induced grooming and face washing behaviors in rats. The level of corticosterone increased in a dose dependent manner 15 min after icv injection of orexin, and it remained high for at least 60 min. In 2-month-old rats, 1 h of immobilization stress increased orexin mRNA levels, but not the melanin-concentrating hormone (MCH) mRNA, in the lateral hypothalamic area (LHA). In 6-month-old rats, 30 min of cold stress increased the expression of orexin mRNA in the LHA. Unlike in the 2-month-old rats, immobilization stress did not change orexin mRNA expression in 6-month-old rats. These results suggest that CRF is involved in orexin-induced behaviors, and that orexin may play an important role in some stress reactions. PMID- 10733947 TI - Induction of heme oxygenase-1 modulates cis-aconitase activity in lens epithelial cells. AB - Heme oxygenase-1 is the heme catabolic enzyme induced in human dermal fibroblasts by environmental stress. We report an increase of heme oxygenase-1 message in lens epithelial cells after exposure to UVA radiation, followed by a 10-fold increase of protein expression. The size of message was larger than previously demonstrated for fibroblasts. The relationship between heme oxygenase-1 activation and iron metabolism was investigated by measurement of activities of both cytosolic and mitochondrial cis-aconitase enzymes. A 2-fold increase in mitochondrial cis-aconitase activity in UVA-exposed cells coincided with the time of maximal heme oxygenase-1 expression. We propose that modulation of cis aconitase activity at the translational level by an increase of cellular iron is an important consequence of heme oxygenase-1 activation. This might be a novel aspect of the protective role of heme oxygenase-1 in modulating the response of cells challenged with oxidative stress. PMID- 10733948 TI - In vivo (31)P-NMR diffusion spectroscopy of ATP and phosphocreatine in rat skeletal muscle. AB - The aim of this study was to measure the diffusion of ATP and phosphocreatine (PCr) in intact rat skeletal muscle, using (31)P-NMR. The acquisition of the diffusion-sensitized spectra was optimized in terms of the signal-to-noise ratio for ATP by using a frequency-selective stimulated echo sequence in combination with adiabatic radio-frequency pulses and surface coil signal excitation and reception. Diffusion restriction was studied by measuring the apparent diffusion coefficients of ATP and PCr as a function of the diffusion time. Orientation effects were eliminated by determining the trace of the diffusion tensor. The data were fitted to a cylindrical restriction model to estimate the unbounded diffusion coefficient and the radial dimensions of the restricting compartment. The unbounded diffusion coefficients of ATP and PCr were approximately 90% of their in vitro values at 37 degrees C. The diameters of the cylindrical restriction compartment were approximately 16 and approximately 22 microm for ATP and PCr, respectively. The diameters of rat skeletal muscle fibers are known to range from 60 to 80 microm. The modelling therefore suggests that the in vivo restriction of ATP and PCr diffusion is not imposed by the sarcolemma but by other, intracellular structures with an overall cylindrical orientation. PMID- 10733949 TI - Theoretical studies of the response of a protein structure to cavity-creating mutations. AB - We have investigated the response of a protein structure to cavity-creating mutations by molecular dynamics (MD) simulations for the wild-type and the five mutants of phage T4 lysozyme. Essential dynamics (ED) analysis and the methods for calculating different components of local interaction energies are used to examine the structural and energetic characteristics associated with the mutations. In agreement with the x-ray results, it is found that the structural changes due to the replacements of a bulky side chain such as Leu or Phe with Ala within the hydrophobic core can be characterized as slight adjustments rather than substantial reorganization of the protein. The relative stability of different mutant structures can be related with the extent of structural readjustments in response to the mutation. The destabilization of the mutant Leu- >Ala proteins relative to the wild-type is closely related with the loss of van der Waals contacts due to the cavity-creating mutations. PMID- 10733950 TI - Molecular dynamics simulation of dipalmitoylphosphatidylcholine membrane with cholesterol sulfate. AB - Using the molecular dynamics simulation technique, we studied the changes occurring in a dipalmitoylphosphatidylcholine (DPPC):cholesterol (CH) membrane at 50 mol% sterol when cholesterol is replaced with cholesterol sulfate (CS). Our simulations were performed at constant pressure and temperature on a nanosecond time scale. We found that 1) the area per DPPC:CS heterodimer is greater than the area of the DPPC:CH heterodimer; 2) CS increases ordering of DPPC acyl chains, but to a lesser extent than CH; 3) the number of hydrogen bonds between DPPC and water is decreased in a CS-containing membrane, but CS forms more water hydrogen bonds than CH; and 4) the membrane dipole potential reverses its sign for a DPPC CS membrane compared to a DPPC-CH bilayer. We also studied the changes occurring in lipid headgroup conformations and determined the location of CS molecules in the membrane. Our results are in good agreement with the data available from experiments. PMID- 10733951 TI - The phase behavior of cationic lipid-DNA complexes. AB - We present a theoretical analysis of the phase behavior of solutions containing DNA, cationic lipids, and nonionic (helper) lipids. Our model allows for five possible structures, treated as incompressible macroscopic phases: two lipid-DNA composite (lipoplex) phases, namely, the lamellar (L(alpha)(C)) and hexagonal (H(II)(C)) complexes; two binary (cationic/neutral) lipid phases, that is, the bilayer (L(alpha)) and inverse-hexagonal (H(II)) structures, and uncomplexed DNA. The free energy of the four lipid-containing phases is expressed as a sum of composition-dependent electrostatic, elastic, and mixing terms. The electrostatic free energies of all phases are calculated based on Poisson-Boltzmann theory. The phase diagram of the system is evaluated by minimizing the total free energy of the three-component mixture with respect to all the compositional degrees of freedom. We show that the phase behavior, in particular the preferred lipid-DNA complex geometry, is governed by a subtle interplay between the electrostatic, elastic, and mixing terms, which depend, in turn, on the lipid composition and lipid/DNA ratio. Detailed calculations are presented for three prototypical systems, exhibiting markedly different phase behaviors. The simplest mixture corresponds to a rigid planar membrane as the lipid source, in which case, only lamellar complexes appear in solution. When the membranes are "soft" (i.e., low bending modulus) the system exhibits the formation of both lamellar and hexagonal complexes, sometimes coexisting with each other, and with pure lipid or DNA phases. The last system corresponds to a lipid mixture involving helper lipids with strong propensity toward the inverse-hexagonal phase. Here, again, the phase diagram is rather complex, revealing a multitude of phase transitions and coexistences. Lamellar and hexagonal complexes appear, sometimes together, in different regions of the phase diagram. PMID- 10733952 TI - Molecular mass and volume in radiation target theory. AB - Radiation target analysis is based on the action of ionizing radiation directly on macromolecules. Interactions of this radiation with the molecules leads to considerable structural damage and consequent loss of biological activity. The radiation sensitivity is dependent on the size of the macromolecules. There has been confusion and discrepancy as to whether the molecular mass or the molecular volume was the determinant factor in the sensitivity. Some proteins are known to change their hydrodynamic volume at low pH, and this characteristic can be utilized to compare the radiation sensitivities of these proteins in the two states. The results show that the radiation sensitivity of proteins depends on the mass of the molecule and is independent of the molecular volume/shape. PMID- 10733953 TI - Two-dimensional fluorescence intensity distribution analysis: theory and applications. AB - A method of sample analysis is presented which is based on fitting a joint distribution of photon count numbers. In experiments, fluorescence from a microscopic volume containing a fluctuating number of molecules is monitored by two detectors, using a confocal microscope. The two detectors may have different polarizational or spectral responses. Concentrations of fluorescent species together with two specific brightness values per species are determined. The two dimensional fluorescence intensity distribution analysis (2D-FIDA), if used with a polarization cube, is a tool that is able to distinguish fluorescent species with different specific polarization ratios. As an example of polarization studies by 2D-FIDA, binding of 5'-(6-carboxytetramethylrhodamine) (TAMRA)-labeled theophylline to an anti-theophylline antibody has been studied. Alternatively, if two-color equipment is used, 2D-FIDA can determine concentrations and specific brightness values of fluorescent species corresponding to individual labels alone and their complex. As an example of two-color 2D-FIDA, binding of TAMRA-labeled somatostatin-14 to the human type-2 high-affinity somatostatin receptors present in stained vesicles has been studied. The presented method is unusually accurate among fluorescence fluctuation methods. It is well suited for monitoring a variety of molecular interactions, including receptors and ligands or antibodies and antigens. PMID- 10733954 TI - Stability of a melittin pore in a lipid bilayer: a molecular dynamics study. AB - We have investigated the configuration and the stability of a single membrane pore bound by four melittin molecules and embedded in a fully hydrated bilayer lipid membrane. We used molecular dynamics simulations up to 5.8 ns. It is found that the initial tetrameric configuration decays with increasing time into a stable trimer and one monomer. This continuous transformation is accompanied by a lateral expansion of the aqueous pore exhibiting a final size comparable to experimental findings. The expansion-induced formation of an interface between the pore-lining acyl chains of the lipids and the pore water ("hydrophobic pore") is transformed into an energetically more favorable toroidal pore structure where some lipid heads are translocated from the rim to the central part of the interface ("hydrophilic pore"). The expansion of the pore is supported by the electrostatic repulsion among the alpha-helices. It is hypothesized that pore growth, and hence cell lysis, is induced by a melittin-mediated line tension of the pore. PMID- 10733955 TI - Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells. AB - This paper describes the combined use of atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRFM) to examine the transmission of force from the apical cell membrane to the basal cell membrane. A Bioscope AFM was mounted on an inverted microscope, the stage of which was configured for TIRFM imaging of fluorescently labeled human umbilical vein endothelial cells (HUVECs). Variable-angle TIRFM experiments were conducted to calibrate the coupling angle with the depth of penetration of the evanescent wave. A measure of cellular mechanical properties was obtained by collecting a set of force curves over the entire apical cell surface. A linear regression fit of the force indentation curves to an elastic model yields an elastic modulus of 7.22 +/- 0. 46 kPa over the nucleus, 2.97 +/- 0.79 kPa over the cell body in proximity to the nucleus, and 1.27 +/- 0.36 kPa on the cell body near the edge. Stress transmission was investigated by imaging the response of the basal surface to localized force application over the apical surface. The focal contacts changed in position and contact area when forces of 0.3-0.5 nN were applied. There was a significant increase in focal contact area when the force was removed (p < 0.01) from the nucleus as compared to the contact area before force application. There was no significant change in focal contact coverage area before and after force application over the edge. The results suggest that cells transfer localized stress from the apical to the basal surface globally, resulting in rearrangement of contacts on the basal surface. PMID- 10733956 TI - Mechanics of living cells measured by laser tracking microrheology. AB - To establish laser-tracking microrheology (LTM) as a new technique for quantifying cytoskeletal mechanics, we measure viscoelastic moduli with wide bandwidth (5 decades) within living cells. With the first subcellular measurements of viscoelastic phase angles, LTM provides estimates of solid versus liquid behavior at different frequencies. In LTM, the viscoelastic shear moduli are inferred from the Brownian motion of particles embedded in the cytoskeletal network. Custom laser optoelectronics provide sub-nanometer and near-microsecond resolution of particle trajectories. The kidney epithelial cell line, COS7, has numerous spherical lipid-storage granules that are ideal probes for noninvasive LTM. Although most granules are percolating through perinuclear spaces, a subset of perinuclear granules is embedded in dense viscoelastic cytoplasm. Over all time scales embedded particles exhibit subdiffusive behavior and are not merely tethered by molecular motors. At low frequencies, lamellar regions (820 +/- 520 dyne/cm(2)) are more rigid than viscoelastic perinuclear regions (330 +/- 250 dyne/cm(2), p < 0.0001), but spectra converge at high frequencies. Although the actin-disrupting agent, latrunculin A, softens and liquefies lamellae, physiological levels of F-actin, alone (11 +/- 1.2 dyne/cm(2)) are approximately 70-fold softer than lamellae. Therefore, F-actin is necessary for lamellae mechanics, but not sufficient. Furthermore, in time-lapse of apparently quiescent cells, individual lamellar granules can show approximately 4-fold changes in moduli that last >10 s. Over a broad range of frequencies (0.1-30, 000 rad/s), LTM provides a unique ability to noninvasively quantify dynamic, local changes in cell viscoelasticity. PMID- 10733957 TI - Biophysical characterization of changes in amounts and activity of Escherichia coli cell and compartment water and turgor pressure in response to osmotic stress. AB - To obtain turgor pressure, intracellular osmolalities, and cytoplasmic water activity of Escherichia coli as a function of osmolality of growth, we have quantified and analyzed amounts of cell, cytoplasmic, and periplasmic water as functions of osmolality of growth and osmolality of plasmolysis of nongrowing cells with NaCl. The effects are large; NaCl (plasmolysis) titrations of cells grown in minimal medium at 0.03 Osm reduce cytoplasmic and cell water to approximately 20% and approximately 50% of their original values, and increase periplasmic water by approximately 300%. Independent analysis of amounts of cytoplasmic and cell water demonstrate that turgor pressure decreases with increasing osmolality of growth, from approximately 3.1 atm at 0.03 Osm to approximately 1.5 at 0.1 Osm and to less than 0.5 atm above 0.5 Osm. Analysis of periplasmic membrane-derived oligosaccharide (MDO) concentrations as a function of osmolality, calculated from literature analytical data and measured periplasmic volumes, provides independent evidence that turgor pressure decreases with increasing osmolality, and verifies that cytoplasmic and periplasmic osmolalities are equal. We propose that MDO play a key role in periplasmic volume regulation at low-to-moderate osmolality. At high growth osmolalities, where only a small amount of cytoplasmic water is observed, the small turgor pressure of E. coli demonstrates that cytoplasmic water activity is only slightly less than extracellular water activity. From these findings, we deduce that the activity of cytoplasmic water exceeds its mole fraction at high osmolality, and, therefore, conclude that the activity coefficient of cytoplasmic water increases with increasing growth osmolality and exceeds unity at high osmolality, presumably as a consequence of macromolecular crowding. These novel findings are significant for thermodynamic analyses of effects of changes in growth osmolality on biopolymer processes in general and osmoregulatory processes in particular in the E. coli cytoplasm. PMID- 10733958 TI - Modeling a dehalogenase fold into the 8-A density map for Ca(2+)-ATPase defines a new domain structure. AB - Members of the large family of P-type pumps use active transport to maintain gradients of a wide variety of cations across cellular membranes. Recent structures of two P-type pumps at 8-A resolution have revealed the arrangement of transmembrane helices but were insufficient to reveal the architecture of the cytoplasmic domains. However, recent proposals of a structural homology with a superfamily of hydrolases offer a new basis for modeling these domains. In the current work, we have extended the sequence comparison for the superfamily and delineated domains in the 8-A density map of Ca(2+)-ATPase. The homology suggests a new domain structure for Ca(2+)-ATPase and, specifically, that the phosphorylation domain adopts a Rossman fold. Accordingly, the atomic structure of L-2 haloacid dehalogenase has been fitted into the relevant domain of Ca(2+) ATPase. The resulting model suggests the existence of two ATP sites at the interface between two domains. Based on this new model, we are able to reconcile numerous results of mutagenesis and chemical cross-linking within the catalytic domains. Furthermore, we have used the model to predict the configuration of Mg.ATP at its binding site. Based on this prediction, we propose a mechanism, involving a change in Mg(2+) liganding, for initiating the domain movements that couple sites of ion transport to ATP hydrolysis. PMID- 10733959 TI - Comparison of Ca(2+) sparks produced independently by two ryanodine receptor isoforms (type 1 or type 3). AB - The molecular determinants of a Ca(2+) spark, those events that determine the sudden opening and closing of a small number of ryanodine receptor (RyR) channels limiting Ca(2+) release to a few milliseconds, are unknown. As a first step we investigated which of two RyR isoforms present in mammalian embryonic skeletal muscle, RyR type 1(RyR-1) or RyR type 3 (RyR-3) has the ability to generate Ca(2+) sparks. Their separate contributions were investigated in intercostal muscle cells of RyR-1 null and RyR-3 null mouse embryos. A comparison of Ca(2+) spark parameters of RyR-1 null versus RyR-3 null cells measured at rest with fluo 3 showed that neither the peak fluorescence intensity (DeltaF/F(o) = 1.25 +/- 0.7 vs. 1.55 +/- 0.6), spatial width at half-max intensity (FWHM = 2.7 +/- 1.2 vs. 2.6 +/- 0.6 microm), nor the duration at half-max intensity (FTHM = 45 +/- 49 vs. 43 +/- 25 ms) was significantly different. Sensitivity to caffeine (0.1 mM) was remarkably different, with sparks in RyR-1 null myotubes becoming brighter and longer in duration, whereas those in RyR-3 null cells remained unchanged. Controls performed in double RyR-1/RyR-3 null cells obtained by mice breeding showed that sparks were not observed in the absence of both isoforms in >150 cells imaged. In conclusion, 1) RyR-1 and RyR-3 appear to be the only intracellular Ca(2+) channels that participate in Ca(2+) spark activity in embryonic skeletal muscle; 2) except in their responsiveness to caffeine, both isoforms have the ability to produce Ca(2+) sparks with nearly identical properties, so it is rather unlikely that a single RyR isoform, when others are also present, would be responsible for Ca(2+) sparks; and 3) because RyR-1 null cells are excitation-contraction (EC) uncoupled and RyR-3 null cells exhibit a normal phenotype, Ca(2+) sparks result from the inherent activity of small clusters of RyRs regardless of the participation of these RyRs in EC coupling. PMID- 10733960 TI - Chloride channels of glycine and GABA receptors with blockers: Monte Carlo minimization and structure-activity relationships. AB - GABA and glycine receptors (GlyRs) are pentameric ligand-gated ion channels that respond to the inhibitory neurotransmitters by opening a chloride-selective central pore lined with five M2 segments homologous to those of alpha(1) GlyR/ ARVG(2')LGIT(6')TVLTMTTQSSGSR. The activity of cyanotriphenylborate (CTB) and picrotoxinin (PTX), the best-studied blockers of the Cl(-) pores, depends essentially on the subunit composition of the receptors, in particular, on residues in positions 2' and 6' that form the pore-facing rings R(2') and R(6'). Thus, CTB blocks alpha(1) and alpha(1)/beta, but not alpha(2) GlyRs (Rundstrom, N., V. Schmieden, H. Betz, J. Bormann, and D. Langosch. 1994. Proc. Natl. Acad. Sci. U.S.A. 91:8950-8954). PTX blocks homomeric receptors (alpha(1) GlyR and rat rho(1) GABAR), but weakly antagonizes heteromeric receptors (alpha(1)/beta GlyR and rho(1)/rho(2) GABAR) (Pribilla, I., T. Takagi, D. Langosch, J. Bormann, and H. Betz. 1992. EMBO J. 11:4305-4311; Zhang D., Z. H. Pan, X. Zhang, A. D. Brideau, and S. A. Lipton. 1995. Proc. Natl. Acad. Sci. U.S.A. 92:11756-11760). Using as a template the kinked-helices model of the nicotinic acetylcholine receptor in the open state (Tikhonov, D. B., and B. S. Zhorov. 1998. Biophys. J. 74:242-255), we have built homology models of GlyRs and GABARs and calculated Monte Carlo-minimized energy profiles for the blockers pulled through the pore. The profiles have shallow minima at the wide extracellular half of the pore, a barrier at ring R(6'), and a deep minimum between rings R(6') and R(2') where the blockers interact with five M2s simultaneously. The star-like CTB swings necessarily on its way through ring R(6') and its activity inversely correlates with the barrier at R(6'): Thr(6')s and Ala(2')s in alpha(2) GlyR confine the swinging by increasing the barrier, while Gly(2')s in alpha(1) GlyR and Phe(6')s in beta GlyR shrink the barrier. PTX has an egg-like shape with an isopropenyl group at the elongated end and the rounded end trimmed by ether and carbonyl oxygens. In the optimal binding mode to alpha(1) GlyR and rho(1) GABAR, the rounded end of PTX accepts several H-bonds from Thr(6')s, while the elongated end enters ring R(2'). The lack of H-bond donors on the side chains of Phe(6')s (beta GlyR) and Met(6')s (rho(2) GABAR) deteriorates the binding. The hydrophilic elongated end of picrotin does not fit the hydrophobic ring of Pro(2')s/Ala(2')s in GABARs, but fit a more hydrophilic ring with Gly(2')s in GlyRs. This analysis provides explanations for structure-activity relationships of noncompetitive agonists and predicts a narrow pore of LGICs in agreement with experimental data on the permeation of organic cations. PMID- 10733961 TI - General anesthetic binding to gramicidin A: the structural requirements. AB - There is a distinct possibility that general anesthetics exert their action on the postsynaptic receptor channels. The structural requirements for anesthetic binding in transmembrane channels, however, are largely unknown. High-resolution (1)H nuclear magnetic resonance and direct photoaffinity labeling were used in this study to characterize the volatile anesthetic binding sites in gramicidin A (gA) incorporated into sodium dodecyl sulfate (SDS) micelles and into dimyristoylphosphatidylcholine (DMPC) bilayers, respectively. To confirm that the structural arrangement of the peptide side chains can affect anesthetic binding, gA in nonchannel forms in methanol was also analyzed. The addition of volatile anesthetic halothane to gA in SDS with a channel conformation caused a concentration-dependent change in resonant frequencies of the indole amide protons of W9, W11, W13, and W15, with the most profound changes in W9. These frequency changes were observed only for gA carefully prepared to ensure a channel conformation and were absent for gA in methanol. For gA in DMPC bilayers, direct [(14)C]halothane photolabeling and microsequencing demonstrated dominant labeling of W9, less labeling of W11 and W13, and no significant labeling of W15. In methanol, gA showed much less labeling of any residues. Inspection of the 3-D structure of gA suggests that the spatial arrangements of the tryptophan residues in the channel form of gA, combined with the amphiphilic regions of lipid, create a favorable anesthetic binding motif. PMID- 10733962 TI - Role of Mg(2+) in Ca(2+)-induced Ca(2+) release through ryanodine receptors of frog skeletal muscle: modulations by adenine nucleotides and caffeine. AB - Mg(2+) serves as a competitive antagonist against Ca(2+) in the high-affinity Ca(2+) activation site (A-site) and as an agonist of Ca(2+) in the low-affinity Ca(2+) inactivation site (I-site) of the ryanodine receptor (RyR), which mediates Ca(2+)-induced Ca(2+) release (CICR). This paper presents the quantitative determination of the affinities for Ca(2+) and Mg(2+) of A- and I-sites of RyR in frog skeletal muscles by measuring [(3)H]ryanodine binding to purified alpha- and beta-RyRs and CICR activity in skinned fibers. There was only a minor difference in affinity at most between alpha- and beta-RyRs. The A-site favored Ca(2+) 20- to 30-fold over Mg(2+), whereas the I-site was nonselective between the two cations. The RyR in situ showed fivefold higher affinities for Ca(2+) and Mg(2+) of both sites than the purified alpha- and beta-RyRs with unchanged cation selectivity. Adenine nucleotides, whose stimulating effect was found to be indistinguishable between free and complexed forms, did not alter the affinities for cations in either site, except for the increased maximum activity of RyR. Caffeine increased not only the affinity of the A-site for Ca(2+) alone, but also the maximum activity of RyR with otherwise minor changes. The results presented here suggest that the rate of CICR in frog skeletal muscles appears to be too low to explain the physiological Ca(2+) release, even though Mg(2+) inhibition disappears. PMID- 10733963 TI - Proton mobilities in water and in different stereoisomers of covalently linked gramicidin A channels. AB - Proton conductivities in bulk solution (lambda(H)) and single-channel proton conductances (g(H)) in two different stereoisomers of the dioxolane-linked gramicidin A channel (the SS and RR dimers) were measured in a wide range of bulk proton concentrations ([H], 0.1-8000 mM). Proton mobilities (micro(H)) in water as well as in the SS and RR dimers were calculated from the conductivity data. In the concentration range of 0.1-2000 mM, a straight line with a slope of 0.75 describes the log (g(H))-log ([H]) relationship in the SS dimer. At [H] > 2000 mM, saturation is followed by a decline in g(H). The g(H)-[H] relationship in the SS dimer is qualitatively similar to the [H] dependence of lambda(H). However, the slope of the straight line in the log(lambda(H))-log([H]) plot is 0.96, indicating that the rate-limiting step for proton conduction through the SS dimer is not the diffusion of protons in bulk solution. The significant difference between the slopes of those linear relationships accounts for the faster decline of micro(H) as a function of [H] in the SS dimer in relation to bulk solution. In the high range of [H], saturation and decline of g(H) in the SS dimer can be accounted for by the significant decrease of micro(H) in bulk solution. At any given [H], g(H) in the RR dimer is significantly smaller than in the SS. Moreover, the g(H)-[H] relationship in the RR stereoisomer is qualitatively different from that in the SS. Between 1 and 50 mM [H], g(H) can be fitted with an adsorption isotherm, suggesting the presence of a proton-binding site inside the pore (pK(a) approximately 2), which limits proton exit from the channel. At 100 mM < [H] < 3000 mM, g(H) increases linearly with [H]. The distinctive shape of the g(H)-[H] relationship in the RR dimer suggests that the channel can be occupied simultaneously by more than one proton. At higher [H], the saturation and decline of g(H) in the RR dimer reflect the properties of micro(H) in bulk solution. In the entire range of [H], protons seem to cross the SS and RR channels via a Grotthuss-like mechanism. The rate-limiting step for proton transfer in the SS dimer is probably the membrane-channel/bulk solution interface. It is also proposed that the smaller g(H) in the RR dimer is the consequence of a different organization and dynamics of the H-bonded network of water molecules inside the pore of the channel, resulting in a slower proton transfer and multiple pore occupancy by protons. PMID- 10733964 TI - Effects of cytoplasmic and luminal pH on Ca(2+) release channels from rabbit skeletal muscle. AB - Ryanodine receptor (RyR)-Ca(2+) release channels from rabbit skeletal muscle were incorporated into lipid bilayers. The effects of cytoplasmic and luminal pH were studied separately over the pH range 5-8, using half-unit intervals. RyR activity (at constant luminal pH of 7.5) was inhibited at acidic cytoplasmic pH, with a half-inhibitory pH (pH(I)) approximately 6.5, irrespective of bilayer potential and of whether the RyRs were activated by cytoplasmic Ca(2+) (50 microM), ATP (2 or 5 mM), or both. Inhibition occurred within approximately 1 s and could be fully reversed within approximately 1 s after brief inhibition or within approximately 30-60 s after longer exposure to acidic cytosolic pH. There was no evidence of any hysteresis in the cytoplasmic pH effect. Ryanodine-modified channels were less sensitive to pH inhibition, with pH(I) at approximately 5.5, but the inhibition was similarly reversible. Steady-state open and closed dwell times of RyRs during cytoplasmic pH inhibition suggest a mechanism where the binding of one proton inhibits the channel and the binding of two to three additional protons promotes further inhibited states. RyR activity was unaffected by luminal pH in the pH range 7.5 to 6.0. At lower luminal pH (5-5.5) most RyRs were completely inhibited, and raising the pH again produced partial to full recovery in only approximately 50% of cases, with the extent of recovery not detectably different between pH 7.5 and pH 9. The results indicate that isolated skeletal muscle RyRs are not inhibited as strongly by low cytoplasmic and luminal pH, as suggested by previous single-channel studies. PMID- 10733965 TI - Local movement in the S2 region of the voltage-gated potassium channel hKv2.1 studied using cysteine mutagenesis. AB - The positively charged S4 region of voltage-dependent potassium channels moves outward during depolarization, leading to channel opening, but possible movement of the negatively charged S2 region may be more complex. Here we have studied possible movement of the S2 region of the slowly activating human voltage dependent potassium channel hKv2.1. For this, cysteine mutants in the S2 region were expressed in Xenopus oocytes by injection of cRNA. Whole-cell currents were measured using the two-electrode voltage-clamp technique, and the effect of the membrane-impermeable cysteine-binding reagent parachloromercuribenzenesulfonate (PCMBS) was studied. For mutant S223C (located just outside the membrane in the S2 region), PCMBS inhibited currents and caused faster deactivation of tail currents. The time course of reactivity of PCMBS on tail current amplitudes was faster at more negative holding potentials. There was no effect of PCMBS on potassium channel currents for mutants D225C, N226C, A230C, and V232C. These data suggest that residue S223 is exposed to the extracellular phase at normal resting potentials, making it accessible to PCMBS, but upon depolarization there is a conformational change, making it less accessible, possibly by a local rather than global movement of S2 residues into the membrane. Voltage-dependent movements of nearby residues could also explain the results. PMID- 10733966 TI - Mutation in pore domain uncovers cation- and voltage-sensitive recovery from inactivation in KAT1 channel. AB - Effects of threonine substitution by glutamine at position 256 in the pore of the KAT1 channel have been investigated by voltage-clamp, using heterologous gene expression in Xenopus oocytes. The major discrepancy in T256Q from the wild-type channel (wt) was cation specific. While K(+) currents were reduced in a largely scalar fashion, the NH(4)(+) current exhibited slow, voltage-dependent inhibition during hyperpolarization. The same effects could be induced in wt, or intensified in T256Q, by addition of the impermeant cation methylammonium (MA(+)) to the bath. This stresses that both the mutation and MA(+) affect a mechanism already present in the wt. Assuming that current inhibition could be described as entry of the channel into an inactive state, we modeled in both wt and in T256Q the relaxation kinetics of the clamp currents by a C-O-I gating scheme, where C (closed) and I (inactivated) are nonconductive states, and O is an open state allowing K(+) and NH(4)(+) passage. The key reaction is the transition I-O. This cation-sensitive transition step ensures release of the channel from the inactive state and is approximately 30 times smaller in T256Q compared to wt. It can be inhibited by external MA(+) and is stimulated strongly by K(+) and weakly by NH(4)(+). This sensitivity of gating to external cations may prevent K(+) leakage from cation-starved cells. PMID- 10733967 TI - Apparent change in ion selectivity caused by changes in intracellular K(+) during whole-cell recording. AB - In whole-cell recordings from HEK293 cells stably transfected with the delayed rectifier K(+) channel Kv2.1, long depolarizations produce current-dependent changes in [K(+)](i) that mimic inactivation and changes in ion selectivity. With 10 mM K(o)(+) or K(i)(+), and 140-160 mM Na(i,o)(+), long depolarizations shifted the reversal potential (V(R)) toward E(Na). However, similar shifts in V(R) were observed when Na(i,o)(+) was replaced with N-methyl-D-glucamine (NMG(+))(i, o). In that condition, [K(+)](o) did not change significantly, but the results could be quantitatively explained by changes in [K(+)](i). For example, a mean outward K(+) current of 1 nA for 2 s could decrease [K(+)](i) from 10 mM to 3 mM in a 10 pF cell. Dialysis by the recording pipette reduced but did not fully prevent changes in [K(+)](i). With 10 mM K(i,o)(+), 150 mM Na(i)(+), and 140 mM NMG(o)(+), steps to +20 mV produced a positive shift in V(R), as expected from depletion of K(i)(+), but opposite to the shift expected from a decreased K(+)/Na(+) selectivity. Long steps to V(R) caused inactivation, but no change in V(R). We conclude that current-dependent changes in [K(+)](i) need to be carefully evaluated when studying large K(+) currents in small cells. PMID- 10733968 TI - Gating of amiloride-sensitive Na(+) channels: subunit-subunit interactions and inhibition by the cystic fibrosis transmembrane conductance regulator. AB - In search of the structural basis for gating of amiloride-sensitive Na(+) channels, kinetic properties of single homo and heterooligomeric ENaCs formed by the subunits with individual truncated cytoplasmic domains were studied in a cell free planar lipid bilayer reconstitution system. Our results identify the N terminus of the alpha-subunit as a major determinant of kinetic behavior of both homooligomeric and heterooligomeric ENaCs, although the carboxy-terminal domains of beta- and gamma-ENaC subunits play important role(s) in modulation of the kinetics of heterooligomeric channels. We also found that the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits amiloride-sensitive channels, at least in part, by modulating their gating. Comparison of these data suggests that the modulatory effects of the beta- and gamma-ENaC subunits, and of the CFTR, may involve the same, or closely related, mechanism(s); namely, "locking" the heterooligomeric channels in their closed state. These mechanisms, however, do not completely override the gating mechanism of the alpha-channel. PMID- 10733969 TI - pH modification of human T-type calcium channel gating. AB - External pH (pH(o)) modifies T-type calcium channel gating and permeation properties. The mechanisms of T-type channel modulation by pH remain unclear because native currents are small and are contaminated with L-type calcium currents. Heterologous expression of the human cloned T-type channel, alpha1H, enables us to determine the effect of changing pH on isolated T-type calcium currents. External acidification from pH(o) 8.2 to pH(o) 5.5 shifts the midpoint potential (V(1/2)) for steady-state inactivation by 11 mV, shifts the V(1/2) for maximal activation by 40 mV, and reduces the voltage dependence of channel activation. The alpha1H reversal potential (E(rev)) shifts from +49 mV at pH(o) 8.2 to +36 mV at pH(o) 5.5. The maximal macroscopic conductance (G(max)) of alpha1H increases at pH(o) 5.5 compared to pH(o) 8.2. The E(rev) and G(max) data taken together suggest that external protons decrease calcium/monovalent ion relative permeability. In response to a sustained depolarization alpha1H currents inactivate with a single exponential function. The macroscopic inactivation time constant is a steep function of voltage for potentials < -30 mV at pH(o) 8.2. At pH(o) 5.5 the voltage dependence of tau(inact) shifts more depolarized, and is also a more gradual function of voltage. The macroscopic deactivation time constant (tau(deact)) is a function of voltage at the potentials tested. At pH(o) 5.5 the voltage dependence of tau(deact) is simply transposed by approximately 40 mV, without a concomitant change in the voltage dependence. Similarly, the delay in recovery from inactivation at V(rec) of -80 mV in pH(o) 5.5 is similar to that with a V(rec) of -120 mV at pH(o) 8.2. We conclude that alpha1H is uniquely modified by pH(o) compared to other calcium channels. Protons do not block alpha1H current. Rather, a proton-induced change in activation gating accounts for most of the change in current magnitude with acidification. PMID- 10733970 TI - Critical determinants of Ca(2+)-dependent inactivation within an EF-hand motif of L-type Ca(2+) channels. AB - L-type (alpha(1C)) calcium channels inactivate rapidly in response to localized elevation of intracellular Ca(2+), providing negative Ca(2+) feedback in a diverse array of biological contexts. The dominant Ca(2+) sensor for such Ca(2+) dependent inactivation has recently been identified as calmodulin, which appears to be constitutively tethered to the channel complex. This Ca(2+) sensor induces channel inactivation by Ca(2+)-dependent CaM binding to an IQ-like motif situated on the carboxyl tail of alpha(1C). Apart from the IQ region, another crucial site for Ca(2+) inactivation appears to be a consensus Ca(2+)-binding, EF-hand motif, located approximately 100 amino acids upstream on the carboxyl terminus. However, the importance of this EF-hand motif for channel inactivation has become controversial since the original report from our lab implicating a critical role for this domain. Here, we demonstrate not only that the consensus EF hand is essential for Ca(2+) inactivation, but that a four-amino acid cluster (VVTL) within the F helix of the EF-hand motif is itself essential for Ca(2+) inactivation. Mutating these amino acids to their counterparts in non inactivating alpha(1E) calcium channels (MYEM) almost completely ablates Ca(2+) inactivation. In fact, only a single amino acid change of the second valine within this cluster to tyrosine (V1548Y) supports much of the functional knockout. However, mutations of presumed Ca(2+)-coordinating residues in the consensus EF hand reduce Ca(2+) inactivation by only approximately 2-fold, fitting poorly with the EF hand serving as a contributory inactivation Ca(2+) sensor, in which Ca(2+) binds according to a classic mechanism. We therefore suggest that while CaM serves as Ca(2+) sensor for inactivation, the EF-hand motif of alpha(1C) may support the transduction of Ca(2+)-CaM binding into channel inactivation. The proposed transduction role for the consensus EF hand is compatible with the detailed Ca(2+)-inactivation properties of wild-type and mutant V1548Y channels, as gauged by a novel inactivation model incorporating multivalent Ca(2+) binding of CaM. PMID- 10733971 TI - Sphingomyelin interfacial behavior: the impact of changing acyl chain composition. AB - Sphingomyelins (SMs) containing homogeneous acyl chains with 12, 14, 16, 18, 24, or 26 carbons were synthesized and characterized using an automated Langmuir-type film balance. Surface pressure was monitored as a function of lipid molecular area at constant temperatures between 10 degrees C and 30 degrees C. SM containing lauroyl (12:0) acyl chains displayed only liquid-expanded behavior. Increasing the length of the saturated acyl chain (e.g., 14:0, 16:0, or 18:0) resulted in liquid-expanded to condensed two-dimensional phase transitions at many temperatures in the 10-30 degrees C range. Similar behavior was observed for SMs with lignoceroyl (24:0) or (cerotoyl) 26:0 acyl chains, but isotherms showed only condensed behavior at 10 and 15 degrees C. Insights into the physico mechanical in-plane interactions occurring within the different SM phases and accompanying changes in SM phase state were provided by analyzing the interfacial area compressibility moduli. At similar surface pressures, SM fluid phases were less compressible than those of phosphatidylcholines with similar chain structures. The area per molecule and compressibility of SM condensed phases depended upon the length of the saturated acyl chain and upon spreading temperature. Spreading of SMs with very long saturated acyl chains at temperatures 30-35 degrees below T(m) resulted in condensed films with lower in plane compressibilities, but consistently larger cross-sectional molecular areas than the condensed phases achieved by spreading at temperatures only 10-20 degrees below T(m). This behavior is discussed in terms of the enhancement of SM lateral aggregation by temperature reduction, a common approach used during domain isolation from biomembranes. PMID- 10733972 TI - Effects of vitrified and nonvitrified sugars on phosphatidylcholine fluid-to-gel phase transitions. AB - DSC was used to study the ability of glass-forming sugars to affect the gel-to fluid phase transition temperature, T(m), of several phosphatidylcholines during dehydration. In the absence of sugars, T(m) increased as the lipid dried. Sugars diminished this increase, an effect we explain using the osmotic and volumetric properties of sugars. Sugars vitrifying around fluid phase lipids lowered T(m) below the transition temperature of the fully hydrated lipid, T(o). The extent to which T(m) was lowered below T(o) ranged from 12 degrees to 57 degrees, depending on the lipids' acyl chain composition. Sugars vitrifying around gel phase lipids raised T(m) during the first heating scan in the calorimeter, then lowered it below T(o) in subsequent scans of the sample. Ultrasound measurements of the mechanical properties of a typical sugar-glass indicate that it is sufficiently rigid to hinder the lipid gel-to-fluid transition. The effects of vitrification on T(m) are explained using the two-dimensional Clausius-Clapeyron equation to model the mechanical stress in the lipid bilayer imposed by the glassy matrix. Dextran and polyvinylpyrrolidone (PVP) also vitrified but did not depress T(m) during drying. Hydration data suggest that the large molecular volumes of these polymers caused their exclusion from the interbilayer space during drying. PMID- 10733973 TI - L-Type Ca(2+) channel charge movement and intracellular Ca(2+) in skeletal muscle fibers from aging mice. AB - In this work we tested the hypothesis that skeletal muscle fibers from aging mice exhibit a significant decline in myoplasmic Ca(2+) concentration resulting from a reduction in L-type Ca(2+) channel (dihydropyridine receptor, DHPR) charge movement. Skeletal muscle fibers from the flexor digitorum brevis (FDB) muscle were obtained from 5-7-, 14-18-, or 21-24-month-old FVB mice and voltage-clamped in the whole-cell configuration of the patch-clamp technique according to described procedures (Wang, Z.-M., M. L. Messi, and O. Delbono. 1999. Biophys. J. 77:2709-2716). Total charge movement or the DHPR charge movement was measured simultaneously with intracellular Ca(2+) concentration. The maximum charge movement (Q(max)) recorded (mean +/- SEM, in nC microF(-1)) was 53 +/- 3.2 (n = 47), 51 +/- 3.2 (n = 35) (non-significant, ns), and 33 +/- 1.9 (n = 32) (p < 0.01), for the three age groups, respectively. Q(max) corresponding to the DHPR was 43 +/- 3.3, 38 +/- 4.1 (ns), and 25 +/- 3.4 (p < 0.01) for the three age groups, respectively. The peak intracellular [Ca(2+)] recorded at 40 mV (in microM) was 15.7 +/- 0. 12, 16.7 +/- 0.18 (ns), and 8.2 +/- 0.07 (p < 0.01) for the three age groups, respectively. No significant changes in the voltage distribution or steepness of the Q-V or [Ca(2+)]-V relationship were found. These data support the concept that the reduction in the peak intracellular [Ca(2+)] results from a larger number of ryanodine receptors uncoupled to DHPRs in skeletal muscle fibers from aging mammals. PMID- 10733974 TI - The C-terminus of tubulin increases cytoplasmic dynein and kinesin processivity. AB - In motor movement on microtubules, the anionic C-terminal of tubulin has been implicated as a significant factor. Our digital analyses of movements of cytoplasmic dynein- and kinesin-coated beads on microtubules have revealed dramatic changes when the C-terminal region (2-4-kDa fragment) of tubulin was cleaved by limited subtilisin digestion of assembled microtubules. For both motors, bead binding to microtubules was decreased threefold, bead run length was decreased over fourfold, and there was a dramatic 20-fold decrease in diffusional movements of cytoplasmic dynein beads on microtubules (even with low motor concentrations where the level of bead motile activity was linear with motor concentration). The velocity of active bead movements on microtubules was unchanged for cytoplasmic dynein and slightly decreased for kinesin. There was also a decrease in the frequency of bead movements without a change in velocity when the ionic strength was raised. However, with high ionic strength there was not a decrease in run length or any selective inhibition of the diffusional movement. The C-terminal region of tubulin increased motor run length (processivity) by inhibiting "detachment" but without affecting velocity. Because the major motor binding sites of microtubules are not on the C-terminal tail of tubulin (), we suggest that the changes are the result of the compromise of a weakly attached state that is the lowest affinity step in both motors' ATPase cycles and is not rate limiting. PMID- 10733975 TI - Stretching of single collapsed DNA molecules. AB - The elastic response of single plasmid and lambda phage DNA molecules was probed using optical tweezers at concentrations of trivalent cations that provoked DNA condensation in bulk. For uncondensed plasmids, the persistence length, P, decreased with increasing spermidine concentration before reaching a limiting value 40 nm. When condensed plasmids were stretched, two types of behavior were observed: a stick-release pattern and a plateau at approximately 20 pN. These behaviors are attributed to unpacking from a condensed structure, such as coiled DNA. Similarly, condensing concentrations of hexaammine cobalt(III) (CoHex) and spermidine induced extensive changes in the low and high force elasticity of lambda DNA. The high force (5-15 pN) entropic elasticity showed worm-like chain (WLC) behavior, with P two- to fivefold lower than in low monovalent salt. At lower forces, a 14-pN plateau abruptly appeared. This corresponds to an intramolecular attraction of 0.083-0.33 kT/bp, consistent with osmotic stress measurements in bulk condensed DNA. The intramolecular attractive force with CoHex is larger than with spermidine, consistent with the greater efficiency with which CoHex condenses DNA in bulk. The transition from WLC behavior to condensation occurs at an extension about 85% of the contour length, permitting looping and nucleation of condensation. Approximately half as many base pairs are required to nucleate collapse in a stretched chain when CoHex is the condensing agent. PMID- 10733976 TI - Monte Carlo implementation of supercoiled double-stranded DNA. AB - Metropolis Monte Carlo simulation is used to investigate the elasticity of torsionally stressed double-stranded DNA, in which twist and supercoiling are incorporated as a natural result of base-stacking interaction and backbone bending constrained by hydrogen bonds formed between DNA complementary nucleotide bases. Three evident regimes are found in extension versus torsion and force versus extension plots: a low-force regime in which over- and underwound molecules behave similarly under stretching; an intermediate-force regime in which chirality appears for negatively and positively supercoiled DNA and extension of underwound molecule is insensitive to the supercoiling degree of the polymer; and a large-force regime in which plectonemic DNA is fully converted to extended DNA and supercoiled DNA behaves quite like a torsionless molecule. The striking coincidence between theoretic calculations and recent experimental measurement of torsionally stretched DNA (Strick et al., Science. 271:1835, 1996; Biophys. J. 74:2016, 1998) strongly suggests that the interplay between base stacking interaction and permanent hydrogen-bond constraint takes an important role in understanding the novel properties of elasticity of supercoiled DNA polymer. PMID- 10733977 TI - Binding mechanisms of TATA box-binding proteins: DNA kinking is stabilized by specific hydrogen bonds. AB - One of the common mechanisms of DNA bending by minor groove-binding proteins is the insertion of protein side chains between basepair steps, exemplified in TBP (TATA box-binding protein)/DNA complexes. At the central basepair step of the TATA box TBP produces a noticeable decrease in twist and an increase in roll, while engaging in hydrogen bonds with the bases and sugars. This suggests a mechanism for the stabilization of DNA kinks that was explored here with ab initio quantum mechanical calculations and molecular dynamics/potential of mean force calculations. The hydrogen bonds are found to contribute the energy necessary to drive the conformational transition at the central basepair step. The Asn, Thr, and Gly residues involved in hydrogen bonding to the DNA bases and sugar oxygens form a relatively rigid motif in TBP. The interaction of this motif with DNA is found to be responsible for inducing the untwisting and rolling of the central basepair step. Notably, direct readout is shown not to be capable of discriminating between AA and AT steps, as the strength of the hydrogen bonds between TBP and the DNA are the same for both sequences. Rather, the calculated free energy cost for an equivalent conformational transition is found to be sequence-dependent, and is calculated to be higher for AA steps than for AT steps. PMID- 10733978 TI - Mechanical stability of single DNA molecules. AB - Using a modified atomic force microscope (AFM), individual double-stranded (ds) DNA molecules attached to an AFM tip and a gold surface were overstretched, and the mechanical stability of the DNA double helix was investigated. In lambda phage DNA the previously reported B-S transition at 65 piconewtons (pN) is followed by a second conformational transition, during which the DNA double helix melts into two single strands. Unlike the B-S transition, the melting transition exhibits a pronounced force-loading-rate dependence and a marked hysteresis, characteristic of a nonequilibrium conformational transition. The kinetics of force-induced melting of the double helix, its reannealing kinetics, as well as the influence of ionic strength, temperature, and DNA sequence on the mechanical stability of the double helix were investigated. As expected, the DNA double helix is considerably destabilized under low salt buffer conditions ( alpha(I) conformational transition occurs. The photocycle rate increases with increasing temperature, but its efficiency is found to be drastically reduced as the transition takes place. A large shift is observed in the all-trans left arrow over right arrow 13-cis equilibrium due to the increased stability of the 13-cis isomer in alpha(I) form. This, together with the increase in the rate of dark adaptation as the temperature increases, leads to a large increase in the 13-cis isomer concentration in bR in the alpha(I) form. The fact that 13-cis retinal has a much-reduced absorption cross section and its inability to pump protons leads to an observed large reduction in the concentration of the observed photocycle intermediates, as well as the proton gradient at a given light intensity. These results suggest that nature might have selected the alpha(II) rather than the alpha(I) form as the helical conformation in bR to stabilize the all-trans retinal isomer that is a better light absorber and is capable of pumping protons. PMID- 10733982 TI - Ultracentrifuge and circular dichroism studies of folding equilibria in a retro GCN4-like leucine zipper. AB - Equilibrium ultracentrifuge and circular dichroism (CD) studies of a retropeptide of a GCN4-like leucine zipper in neutral saline buffer are reported as functions of temperature. Ultracentrifuge results indicate the presence of three oligomeric species: monomer, dimer, and tetramer, in quantifiable amounts, and the data provide values for the standard DeltaG, DeltaH, and DeltaS for interconversion. CD at 222 nm displays the strong concentration dependence characteristic of dissociative unfolding, but also shows a helicity far below that of the parent propeptide. Remarkably enough, the CD at 222 nm shows an extremum in the region between 0 and 20 degrees C. At higher T, the usual cooperative unfolding is observed. Comparable data are presented for a mutant retropeptide, in which a single asparagine residue is restored to the characteristic heptad position it occupies in the propeptide. The mutant shows marked differences from its unmutated relative in both thermodynamic properties and CD, although the oligomeric ensemble also comprises monomers, dimers, and tetramers. The mutant is closer in helicity to the parent propeptide but is less stable. These findings do not support either of the extant views on retropeptides. The behavior seen is consistent neither with the view that retropeptides should have the same structure as propeptides nor with the view that they should have the same structure but opposite chirality. The simultaneous availability of oligomeric population data and CD allows the latter to be dissected into individual contributions from monomers, dimers, and tetramers. This dissection yields explanations for the observed extrema in curves of CD (222 nm) versus T and reveals that the dimer population in both retropeptides undergoes "cold denaturation." PMID- 10733983 TI - Molecular diffusion into ferritin: pathways, temperature dependence, incubation time, and concentration effects. AB - The detailed kinetics of permeation and effusion of small nitroxide spin probe radicals with the protein shells of horse spleen ferritin (HoSF) and human H chain ferritin (HuHF) and a 3-fold channel variant D131H+E134H of HuHF were studied by electron paramagnetic resonance spectroscopy and gel permeation chromatography under a variety of experimental conditions. The results confirm that the permeation of molecular species of 7-9-A diameter into ferritin is a charge selective process and that the threefold channels are the likely pathways for entry into the protein. Studies with holoHoSF show that increased temperature increases the rates of penetration and effusion and also increases the concentration of positively charged spin probe accumulated within the protein in excess of that in the external solution. The interior of HoSF is much more accessible to small molecules at physiological temperature of approximately 40 degrees C than at room temperature. The large activation energy of 63-67 kJ/mol measured for the effusion/penetration and the small diffusion coefficient, D approximately 5 x 10(-22) m(2)/s at 20 degrees C, corresponding to a time of approximately 60 min for traversing the protein shell, is consistent with the kinetics of diffusion being largely controlled by the restrictive porosity of the protein itself. An inverse dependence of the first-order rate constant for effusion out of the protein channel on the incubation time used for radical penetration into the protein is attributed to increased binding of the radical within the funnel-shaped channel. PMID- 10733984 TI - Interactions and aggregation of apoferritin molecules in solution: effects of added electrolytes. AB - We have studied the structure of the protein species and the protein-protein interactions in solutions containing two apoferritin molecular forms, monomers and dimers, in the presence of Na(+) and Cd(2+) ions. We used chromatographic, and static and dynamic light scattering techniques, and atomic force microscopy (AFM). Size-exclusion chromatography was used to isolate these two protein fractions. The sizes and shapes of the monomers and dimers were determined by dynamic light scattering and AFM. Although the monomer is an apparent sphere with a diameter corresponding to previous x-ray crystallography determinations, the dimer shape corresponds to two, bound monomer spheres. Static light scattering was applied to characterize the interactions between solute molecules of monomers and dimers in terms of the second osmotic virial coefficients. The results for the monomers indicate that Na(+) ions cause strong intermolecular repulsion even at concentrations higher than 0.15 M, contrary to the predictions of the commonly applied Derjaguin-Landau-Verwey-Overbeek theory. We argue that the reason for such behavior is hydration force due to the formation of a water shell around the protein molecules with the help of the sodium ions. The addition of even small amounts of Cd(2+) changes the repulsive interactions to attractive but does not lead to oligomer formation, at least at the protein concentrations used. Thus, the two ions provide examples of strong specificity of their interactions with the protein molecules. In solutions of the apoferritin dimer, the molecules attract even in the presence of Na(+) only, indicating a change in the surface of the apoferritin molecule. In view of the strong repulsion between the monomers, this indicates that the dimers and higher oligomers form only after partial denaturation of some of the apoferritin monomers. These observations suggest that aggregation and self-assembly of protein molecules or molecular subunits may be driven by forces other than those responsible for crystallization and other phase transitions in the protein solution. PMID- 10733985 TI - Molecular basis for the polymerization of octopus lens S-crystallin. AB - S-Crystallin from octopus lens has a tertiary structure similar to sigma-class glutathione transferase (GST). However, after isolation from the lenses, S crystallin was found to aggregate more easily than sigma-GST. In vitro experiments showed that the lens S-crystallin can be polymerized and finally denatured at increasing concentration of urea or guanidinium chloride (GdmCl). In the intermediate concentrations of urea or GdmCl, the polymerized form of S crystallin is aggregated, as manifested by the increase in light scattering and precipitation of the protein. There is a delay time for the initiation of polymerization. Both the delay time and rate of polymerization depend on the protein concentration. The native protein showed a maximum fluorescence emission spectrum at 341 nm. The GdmCl-denatured protein exhibited two fluorescence maxima at 310 nm and 358 nm, respectively, whereas the urea-denatured protein showed a fluorescence peak at 358 nm with a small peak at 310 nm. The fluorescence intensity was quenched. Monomers, dimers, trimers, and polymers of the native protein were observed by negative-stain electron microscopic analysis. The aggregated form, however, showed irregular structure. The aggregate was solubilized in high concentrations of urea or GdmCl. The redissolved denatured protein showed an identical fluorescence spectrum to the protein solution that was directly denatured with high concentrations of urea or GdmCl. The denatured protein was readily refolded to its native state by diluting with buffer solution. The fluorescence spectrum of the renatured protein solution was similar to that of the native form. The phase diagrams for the S-crystallin in urea and GdmCl were constructed. Both salt concentration and pH value of the solution affect the polymerization rate, suggesting the participation of ionic interactions in the polymerization. Comparison of the molecular models of the S crystallin and sigma-GST suggests that an extra ion-pair between Asp-101 and Arg 14 in S-crystallin contributes to stabilizing the protomer. Furthermore, the molecular surface of S-crystallin has a protruding Lys-208 on one side and a complementary patch of aspartate residues (Asp-90, Asp-94, Asp-101, Asp-102, Asp 179, and Asp-180) on the other side. We propose a molecular model for the S crystallin polymer in vivo, which involves side-by-side associations of Lys-208 from one protomer and the aspartate patch from another protomer that allows the formation of a polymeric structure spontaneously into a liquid crystal structure in the lens. PMID- 10733987 TI - Proteins with similar architecture exhibit similar large-scale dynamic behavior. AB - We have investigated the similarities and differences in the computed dynamic fluctuations exhibited by six members of a protein fold family with a coarse grained Gaussian network model. Specifically, we consider the cofactor binding fragment of CysB; the lysine/arginine/ornithine-binding protein (LAO); the enzyme porphobilinogen deaminase (PBGD); the ribose-binding protein (RBP); the N terminal lobe of ovotransferrin in apo-form (apo-OVOT); and the leucine/isoleucine/valine-binding protein (LIVBP). All have domains that resemble a Rossmann fold, but there are also some significant differences. Results indicate that similar global dynamic behavior is preserved for the members of a fold family, and that differences usually occur in regions only where specific function is localized. The present work is a computational demonstration that the scaffold of a protein fold may be utilized for diverse purposes. LAO requires a bound ligand before it conforms to the large-scale fluctuation behavior of the three other members of the family, CysB, PBGD, and RBP, all of which contain a substrate (cofactor) at the active site cleft. The dynamics of the ligand-free enzymes LIVBP and apo-OVOT, on the other hand, concur with that of unliganded LAO. The present results suggest that it is possible to construct structure alignments based on dynamic fluctuation behavior. PMID- 10733988 TI - Proton electron nuclear double resonance from nitrosyl horse heart myoglobin: the role of His-E7 and Val-E11. AB - Electron nuclear double resonance (ENDOR) spectroscopy has been used to study protons in nitrosyl horse heart myoglobin (MbNO). (1)H ENDOR spectra were recorded for different settings of the magnetic field. Detailed analysis of the ENDOR powder spectra, using computer simulation, based on the "orientation selection" principle, leads to the identification of the available protons in the heme pocket. We observe hyperfine interactions of the N(HisF8)-Fe(2+)-N(NO) complex with five protons in axial and with eight protons in the rhombic symmetry along different orientations, including those of the principal axes of the g tensor. Protons from His-E7 and Val-E11 residues are identified in the two symmetries, rhombic and axial, exhibited by MbNO. Our results indicate that both residues are present inside the heme pocket and help to stabilize one particular conformation. PMID- 10733986 TI - Protein dynamics in an intermediate state of myoglobin: optical absorption, resonance Raman spectroscopy, and x-ray structure analysis. AB - A metastable state of myoglobin is produced by reduction of metmyoglobin at low temperatures. This is done either by irradiation with x-rays at 80 K or by electron transfer from photoexcited tris(2, 2'-bipyridine)-ruthenium(II) at 20 K. At temperatures above 150 K, the conformational transition toward the equilibrium deoxymyoglobin is observed. X-ray crystallography, Raman spectroscopy, and temperature-dependent optical absorption spectroscopy show that the metastable state has a six-ligated iron low-spin center. The x-ray structure at 115K proves the similarity of the metastable state with metmyoglobin. The Raman spectra yield the high-frequency vibronic modes and give additional information about the distortion of the heme. Analysis of the temperature dependence of the line shape of the Soret band reveals that a relaxation within the metastable state starts at approximately 120 K. Parameters representative of static properties of the intermediate state are close to those of CO-ligated myoglobin, while parameters representative of dynamics are close to deoxymyoglobin. Thus within the metastable state the relaxation to the equilibrium is initiated by changes in the dynamic properties of the active site. PMID- 10733990 TI - Global analysis of fluorescence lifetime imaging microscopy data. AB - Global analysis techniques are described for frequency domain fluorescence lifetime imaging microscopy (FLIM) data. These algorithms exploit the prior knowledge that only a limited number of fluorescent molecule species whose lifetimes do not vary spatially are present in the sample. Two approaches to implementing the lifetime invariance constraint are described. In the lifetime invariant fit method, each image in the lifetime image sequence is spatially averaged to obtain an improved signal-to-noise ratio. The lifetime estimations from these averaged data are used to recover the fractional contribution to the steady-state fluorescence on a pixel-by-pixel basis for each species. The second, superior, approach uses a global analysis technique that simultaneously fits the fractional contributions in all pixels and the spatially invariant lifetimes. In frequency domain FLIM the maximum number of lifetimes that can be fit with the global analysis method is twice the number of lifetimes that can be fit with conventional approaches. As a result, it is possible to discern two lifetimes with a single-frequency FLIM setup. The algorithms were tested on simulated data and then applied to separate the cellular distributions of coexpressed green fluorescent proteins in living cells. PMID- 10733989 TI - Effects of temperature on calcium-sensitive fluorescent probes. AB - The effect of temperature on the binding equilibria of calcium-sensing dyes has been extensively studied, but there are also important temperature-related changes in the photophysics of the dyes that have been largely ignored. We conducted a systematic study of thermal effects on five calcium-sensing dyes under calcium-saturated and calcium-free conditions. Quin-2, chlortetracycline, calcium green dextran, Indo-1, and Fura-2 all show temperature-dependent effects on fluorescence in all or part of the range tested (5-40 degrees C). Specifically, the intensity of the single-wavelength dyes increased at low temperature. The ratiometric dyes, because of variable effects at the two wavelengths, showed, in general, a reduction in the fluorescence ratio as temperature decreased. Changes in viscosity, pH, oxygen quenching, or fluorescence maxima could not fully explain the effects of temperature on fluorescence. The excited-state lifetimes of the dyes were determined, in both the presence and absence of calcium, using multifrequency phase-modulation fluorimetry. In most cases, low temperature led to prolonged fluorescence lifetimes. The increase in lifetimes at reduced temperature is probably largely responsible for the effects of temperature on the physical properties of the calcium-sensing dyes. Clearly, these temperature effects can influence reported calcium concentrations and must therefore be taken into consideration during any investigation involving variable temperatures. PMID- 10733991 TI - A maximum entropy analysis of protein orientations using fluorescence polarization data from multiple probes. AB - Techniques have recently become available to label protein subunits with fluorescent probes at predetermined orientation relative to the protein coordinates. The known local orientation enables quantitative interpretation of fluorescence polarization experiments in terms of orientation and motions of the protein within a larger macromolecular assembly. Combining data obtained from probes placed at several distinct orientations relative to the protein structure reveals functionally relevant information about the axial and azimuthal orientation of the labeled protein segment relative to its surroundings. Here we present an analytical method to determine the protein orientational distribution from such data. The method produces the broadest distribution compatible with the data by maximizing its informational entropy. The key advantages of this approach are that no a priori assumptions are required about the shape of the distribution and that a unique, exact fit to the data is obtained. The relative orientations of the probes used for the experiments have great influence on information content of the maximum entropy distribution. Therefore, the choice of probe orientations is crucial. In particular, the probes must access independent aspects of the protein orientation, and two-fold rotational symmetries must be avoided. For a set of probes, a "figure of merit" is proposed, based on the independence among the probe orientations. With simulated fluorescence polarization data, we tested the capacity of maximum entropy analysis to recover specific protein orientational distributions and found that it is capable of recovering orientational distributions with one and two peaks. The similarity between the maximum entropy distribution and the test distribution improves gradually as the number of independent probe orientations increases. As a practical example, ME distributions were determined with experimental data from muscle fibers labeled with bifunctional rhodamine at known orientations with respect to the myosin regulatory light chain (RLC). These distributions show a complex relationship between the axial orientation of the RLC relative to the fiber axis and the azimuthal orientation of the RLC about its own axis. Maximum entropy analysis reveals limitations in available experimental data and supports the design of further probe angles to resolve details of the orientational distribution. PMID- 10733992 TI - A theoretical investigation of single-molecule fluorescence detection on thin metallic layers. AB - In the present paper, the excitation and detection of single-molecule fluorescence over thin metallic films is studied theoretically within the framework of classical electrodynamics. The model takes into account the specific conditions of surface plasmon-assisted optical excitation, fluorescence quenching by the metal film, and detection geometry. Extensive numerical results are presented for gold, silver, and aluminum films, showing the detectable fluorescence intensities and their dependence on film thickness and the fluorescent molecule's position under optimal excitation conditions. PMID- 10733993 TI - Photobleaching in two-photon excitation microscopy. AB - The intensity-squared dependence of two-photon excitation in laser scanning microscopy restricts excitation to the focal plane and leads to decreased photobleaching in thick samples. However, the high photon flux used in these experiments can potentially lead to higher-order photon interactions within the focal volume. The excitation power dependence of the fluorescence intensity and the photobleaching rate of thin fluorescence samples ( approximately 1 microm) were examined under one- and two-photon excitation. As expected, log-log plots of excitation power versus the fluorescence intensity and photobleaching rate for one-photon excitation of fluorescein increased with a slope of approximately 1. A similar plot of the fluorescence intensity versus two-photon excitation power increased with a slope of approximately 2. However, the two-photon photobleaching rate increased with a slope > or =3, indicating the presence of higher-order photon interactions. Similar experiments on Indo-1, NADH, and aminocoumarin produced similar results and suggest that this higher-order photobleaching is common in two-photon excitation microscopy. As a consequence, the use of multi photon excitation microscopy to study thin samples may be limited by increased photobleaching. PMID- 10733994 TI - Protein-bound water molecule counting by resolution of (1)H spin-lattice relaxation mechanisms. AB - Water proton spin-lattice relaxation is studied in dilute solutions of bovine serum albumin as a function of magnetic field strength, oxygen concentration, and solvent deuteration. In contrast to previous studies conducted at high protein concentrations, the observed relaxation dispersion is accurately Lorentzian with an effective correlation time of 41 +/- 3 ns when measured at low proton and low protein concentrations to minimize protein aggregation. Elimination of oxygen flattens the relaxation dispersion profile above the rotational inflection frequency, nearly eliminating the high field tail previously attributed to a distribution of exchange times for either whole water molecules or individual protons at the protein-water interface. The small high-field dispersion that remains is attributed to motion of the bound water molecules on the protein or to internal protein motions on a time scale of order one ns. Measurements as a function of isotope composition permit separation of intramolecular and intermolecular relaxation contributions. The magnitude of the intramolecular proton-proton relaxation rate constant is interpreted in terms of 25 +/- 4 water molecules that are bound rigidly to the protein for a time long compared with the rotational correlation time of 42 ns. This number of bound water molecules neglects the possibility of local motions of the water in the binding site; inclusion of these effects may increase the number of bound water molecules by 50%. PMID- 10733995 TI - Imaging and tracking of single GFP molecules in solution. AB - Visualization and tracking of single fluorescent molecules is a recent development in optical microscopy holding great promise for the study of cell biological processes. However, all experimental strategies realized so far confined the observation to extremely thin interfacial layers. The detection and characterization of single molecules in three-dimensionally extended systems such as living cells has yet to be accomplished. We show, here, for the first time that single protein molecules can be visualized and tracked in three-dimensional (3D) samples at room temperature. Using a wide-field fluorescence microscope equipped with an Ar(+)-laser and a low-light-level CCD camera, single molecules of the green fluorescent protein (GFP) were detected in gels and viscous solutions at depths of up to approximately 10 microm from the interface. A time resolution of 5 ms was achieved by a high-speed framing mode. The two-dimensional localization accuracy was determined to be approximately 30 nm. The number of photons emitted by single GFP molecules before photodestruction was found to be < or = 4 * 10(5). Freely diffusing GFP molecules could be tracked over up to nine images acquired at a frame rate of approximately 80 Hz. From the trajectories, the diffusion coefficients of single GFP molecules were derived and found to agree well with expectation and microphotolysis measurements. Our results imply that the visualization and tracking of single molecules in living cells is possible. PMID- 10733996 TI - F-actin retains a memory of angular order. AB - Modifications can be made to F-actin that do not interfere with the binding of myosin but inhibit force generation, suggesting that actin's internal dynamics are important for muscle contraction. Observations from electron microscopy and x ray diffraction have shown that subunits in F-actin have a relatively fixed axial rise but a variable twist. One possible explanation for this is that the actin subunits randomly exist in different discrete states of "twist, " with a significant energy barrier separating these states. This would result in very slow torsional transitions. Paracrystals impose increased order on F-actin filaments by reducing the variability in twist. By looking at filaments that have recently been dissociated from paracrystals, we find that F-actin retains a "memory" of its previous environment that persists for many seconds. This would be consistent with slow torsional transitions between discrete states of twist. PMID- 10733997 TI - Enumerating neurology. PMID- 10733998 TI - The incidence and lifetime prevalence of neurological disorders in a prospective community-based study in the UK. AB - Over an 18-month period, all incident cases of neurological disorders were ascertained prospectively in an unselected urban population based in 13 general practices in the London area by a General Practice Linkage Scheme with the National Hospital for Neurology and Neurosurgery. In three of these practices, the lifetime prevalence of neurological disorders was also assessed. A population of 100 230 patients registered with participating general practices was followed prospectively for the onset of neurological disorders. Multiple methods of case finding were used to maintain accuracy. The age- and sex-adjusted incidence rates of neurological disorders were calculated. The lifetime prevalence of neurological disorders was surveyed in 27 658 of the patients. The age- and sex adjusted incidence rates were calculated for major neurological conditions. [These are expressed as rates per 100 000 persons per annum, with 95% confidence intervals (CI) in parentheses]. The commonest of these were first cerebrovascular events, 205 (CI: 183, 230); shingles, 140 (CI: 104, 184); diabetic polyneuropathy, 54 (CI: 33, 83); compressive neuropathies, 49 (CI: 39, 61); epilepsy, 46 (CI: 36, 60); Parkinson's disease, 19 (CI: 12, 27); peripheral neuropathies, 15 (CI: 9, 23); CNS infections, 12 (CI: 5, 13); post-herpetic neuralgia, 11 (CI: 6, 17); and major neurological injuries, 10 (CI: 4, 11). Lifetime prevalence rates are also reported (expressed as rate per 1000 persons with 95% CI). The most prevalent conditions were: completed stroke, 9 (CI: 8, 11); transient ischaemic attacks, 5 (CI: 4, 6); active epilepsy, 4 (CI: 4, 5); congenital neurological deficit, 3 (CI: 3, 4); Parkinson's disease, 2 (CI: 1, 3); multiple sclerosis, 2 (CI: 2, 3); diabetic polyneuropathy, 2 (CI: 1, 3); compressive mononeuropathies, 2 (CI: 2, 3); and sub-arachnoid haemorrhage, 1 (CI: 0.8, 2). Overall, the onset of 625 neurological disorders was observed per 100 000 population annually. Six percent of the population had at some time had a neurological disorder. This is the first study of the incidence and lifetime prevalence of neurological disorders in recent times; we found that these disorders give rise to significant morbidity in the community. PMID- 10733999 TI - Frequency analysis of EMG activity in patients with idiopathic torticollis. AB - The pathophysiology of idiopathic dystonic torticollis is unclear and there is no simple test that confirms the diagnosis and excludes a psychogenic or voluntary torticollis in individual patients. We recorded EMG activity in the sternocleidomastoid (SCM) and splenius capitis (SPL) muscles of eight patients with rotational torticollis and eight age-matched controls, and analysed the signals in the frequency and time domains. All control subjects but one showed a significant peak in the autospectrum of the SPL EMG at 10-12 Hz, which was absent in all patients with torticollis. Conversely, patients with torticollis had evidence of a 4-7 Hz drive to the SPL and SCM that was absent in coherence spectra from controls. The pooled cumulant density estimates revealed a peak in both groups, and within the patient group there was a second narrow subpeak with a width of 13 ms. The activity in the SCM and SPL was in phase in the patients but not in the controls. The lack of any phase difference and the suggestion of short-term synchronization between SCM and SPL are consistent with an abnormal corticoreticular and corticospinal drive in dystonic torticollis. Clinically, the pattern of SPL EMG autospectra and of SCM-SPL coherence may provide a sensitive and specific feature distinguishing dystonic from psychogenic torticollis. PMID- 10734000 TI - Cytokine-induced modulation of cellular adhesion to human cerebral endothelial cells is mediated by soluble vascular cell adhesion molecule-1. AB - Tumour necrosis factor-alpha ( TNF-alpha) has been proposed as one of the key mediators of inflammatory diseases of the CNS such as multiple sclerosis. It has been shown to induce the expression of adhesion molecules which is a prerequisite for the transmigration of immune cells through the blood-brain barrier. We therefore investigated the role of TNF-alpha in the expression and release of vascular cell adhesion molecule-1 (VCAM-1) in cultures of human cerebral endothelial cells (HCEC) in comparison with peripheral blood mononuclear cells (PBMC). A time- and dose-dependent expression of VCAM-1 and release of soluble VCAM-1 was detected in HCEC but not PBMC. TNF-alpha-induced release of soluble VCAM-1 was further increased by cotreatment with interferon-beta (IFN-beta), while IFN-beta alone did not affect VCAM-1 expression or the release of soluble VCAM-1. In addition, we observed that preincubation of PBMC with soluble VCAM-1 completely blocked their adhesion to HCEC. In conclusion, the proinflammatory effect of TNF-alpha on HCEC, which involves the induction of VCAM-1 expression and cellular adhesion, is followed by the consecutive effects of soluble VCAM-1 release in blocking adhesion and downregulating further cellular infiltration. Increasing soluble VCAM-1 release during active inflammation could be another mechanism by which IFN-beta treatment exerts protective effects in multiple sclerosis patients. PMID- 10734001 TI - Dexamethasone regulation of matrix metalloproteinase expression in CNS vascular endothelium. AB - Matrix metalloproteinases (MMPs) have been implicated in the early breakdown of the blood-brain barrier in neuroinflammatory disease. Although expression of these enzymes by resident glial cells and recruited immune cells has been described, altered expression of MMPs by the CNS vascular endothelial cells may also contribute to barrier disruption. In the present study, the in vitro expression of MMP-2 and -9 as well as tissue inhibitor of metalloproteinase (TIMP)-2 by rat CNS microvascular endothelial cells has been determined and compared with that by endothelial cell lines derived from rat aorta and high endothelial venules. Primary cultures of rat brain microvascular endothelial cells as well as the rat brain (GP8/3.9) and rat retinal endothelial (JG2/1) cell lines constitutively expressed MMP-2, -9 and TIMP-2. In vitro activation of CNS endothelium with the pro-inflammatory cytokines, tumour necrosis factor-alpha and interleukin-1beta, resulted in selective upregulation of MMP-9 activity, whereas no significant changes were seen in MMP-2 or TIMP-2 levels at 24 h. The addition of dexamethasone partially inhibited the cytokine-induced upregulation of MMP-9. Treatment of GP8/3.9 brain endothelial cells with active MMP-9 caused subtle but distinct alterations in the expression of the junctional protein, ZO-1. Quantitative differences found between CNS and non-CNS endothelial cells in the expression of both MMP-2 and -9, and in the expression of TIMP-2 demonstrate that CNS vascular endothelium is functionally distinct from non-CNS endothelium. These results suggest that cytokine-induced upregulation of MMP-9 expression by the CNS vascular endothelium may play a role in the pathogenesis of blood-brain and blood retinal barrier breakdown in vivo. PMID- 10734002 TI - Long-term prognosis of neuropathy associated with anti-MAG IgM M-proteins and its relationship to immune therapies. AB - Many data point to a pathogenetic role for IgM antibodies to the myelin associated glycoprotein (MAG) in the neuropathy associated with IgM monoclonal gammopathy, supporting the use of immune therapies in affected patients. Almost 50% of patients have been reported to improve with these therapies, but the effect of treatment on the long-term prognosis of the neuropathy remains unclear. We analysed the outcome of 25 of the 26 patients (mean age at entry 65 years, range 45-85 years) with neuropathy and high anti-MAG IgM, first examined by us between 1984 and 1994. By January 1999, after a mean follow-up of 8.5 years (range 2-13 years) and a mean duration of neuropathy symptoms of 11.8 years (range 3-18, >10 years in 16), 17 patients (68%) (aged 58-84 years, mean 73.4) were alive, while eight (32%) (aged 69-78 years, mean 73.1) had died 3-15 years (mean 10.6) after neuropathy onset; in none of them was death caused by the neuropathy, although in three it was possibly related to the therapy for the neuropathy. By the time of last follow-up or patients' death, 11 patients (44%) were disabled by severe hand tremor, gait ataxia or both. The disability rates at 5, 10 and 15 years from neuropathy onset were 16, 24 and 50%, respectively. Of the 19 patients treated during the follow-up for 0.5-11 years (mean 4 years) with various immune therapies, five reported a consistent and four a slight improvement in the neuropathy (total 47%) after one treatment or more, but in only one patient was improvement persistent throughout, to the end of follow-up. In 10 patients (53%), severe adverse events, possibly related to therapy, occurred during treatment and were considered responsible for the patient's death in three. The neurological impairment did not differ between treated and untreated patients at the end of a similar follow-up. Our findings indicate that (i) the majority of patients with neuropathy and anti-MAG IgM have a favourable prognosis even after several years, and (ii) current immune therapies, though temporarily effective in half of the patients, are associated with considerable side effects which limit their prolonged use and efficacy, suggesting that until more effective or safer therapies become available, they should probably be reserved for patients impaired in their daily life or in a progressive phase of the disease. PMID- 10734003 TI - The natural history of hereditary neuralgic amyotrophy in the Dutch population: two distinct types? AB - On investigation of 101 attacks in 24 patients with hereditary neuralgic amyotrophy (HNA) from nine different families, we found that HNA can run two distinct courses: a 'classic' relapsing-remitting and a chronic undulating type with exacerbations. Only one type occurred per family, suggesting genetic heterogeneity. This is supported by the finding that only in a family with 'classic type' HNA are data of linkage analysis compatible with linkage to the 17q24-q25 interval which harbours a locus for the disease. The average number of attacks per patient during a follow-up of 26 years was four in the classic form of HNA and five in the chronic undulating type. All patients suffered from residual symptoms on follow-up, with a median Rankin score of 2 in both groups, showing that long-term prognosis is less favourable than previously reported. PMID- 10734004 TI - A role for the substantia nigra pars reticulata in the gaze palsy of progressive supranuclear palsy. AB - We examined the topography and degree of cell loss within basal ganglia structures commonly involved in progressive supranuclear palsy in order to identify any relationship between degeneration in these nuclei and gaze palsy. Serial section analyses and unbiased quantitative techniques were applied to brain tissue from six cases with progressive supranuclear palsy (four with gaze palsy and two without) and six controls with no neurological or neuropathological abnormalities. The total number of nucleolated neurons within the substantia nigra pars compacta (SNc) and reticulata (SNr), the subthalamic nucleus, and the internal and external segments of the globus pallidus was determined for all subjects and the data expressed as percentages of control values to compare degeneration across these basal ganglia structures. The density of neurofibrillary tangles was also evaluated within these structures. Despite significant subcortical neurofibrillary tangle formation in all cases, there was considerable variability in the degree of neuronal cell loss in all basal ganglia regions, except the SNc which was consistently affected. There was no correlation between the ranked density of neurofibrillary tangles and the degree of neuronal cell loss in any basal ganglia region. Comparisons between cases with and without gaze palsy revealed a 40% greater decrease in the number of SNr neurons in cases with gaze palsy (75 +/- 8% loss) compared with those without (35 +/- 14% loss). This was the largest difference between these cases. As the SNr projects to the superior colliculus, degeneration of this basal ganglia structure may disrupt eye movements in progressive supranuclear palsy. PMID- 10734005 TI - Hallucinations in Parkinson's disease: prevalence, phenomenology and risk factors. AB - Hallucinations, mainly of a visual nature, are considered to affect about one quarter of patients with Parkinson's disease. They are commonly viewed as a side effect of antiparkinsonian treatment, but other factors may be involved. The aim of this study was to determine the phenomenology, prevalence and risk factors of hallucinations in Parkinson's disease. Two-hundred and sixteen consecutive patients fulfilling clinical criteria for Parkinson's disease were studied. Demographic and clinical variables were recorded, including motor and cognitive status, depressive symptoms and sleep-wake disturbances. Patients with and without hallucinations were compared using non-parametric tests, and logistic regression was applied to significant data. Hallucinations had been present during the previous 3 months in 39.8% of the patients, and fell into three categories: minor forms, consisting of a sensation of a presence (person), a sideways passage (commonly of an animal) or illusions were present in 25.5% of the patients (an isolated occurrence in 14.3%), formed visual hallucinations were present in 22.2% (isolated in 9.3%) and auditory hallucinations were present in 9.7% (isolated in 2.3%). Patients with minor hallucinations had a higher depression score than non-hallucinators but did not differ in other respects. Logistic regression analysis identified three factors independently predictive of formed visual hallucinations: severe cognitive disorders, daytime somnolence and a long duration of Parkinson's disease. These findings indicate that, when minor hallucinations are included, the total prevalence is much higher than previously reported. A simple side-effect of dopaminergic treatment is not sufficient to explain the occurrence of all visual hallucinations. The main risk factor in treated patients is cognitive impairment, although sleep-wake cycle disturbances, and possibly other factors related to the duration of the disease, act as cofactors. PMID- 10734006 TI - Relationship of lesion location to cognitive outcome following microelectrode guided pallidotomy for Parkinson's disease: support for the existence of cognitive circuits in the human pallidum. AB - Current models of basal ganglia anatomy posit the existence of multiple parallel, anatomically segregated circuits. Anatomical data from non-human primates suggest that the circuits subserving motor functions are segregated from those subserving cognitive functions. Here we present data that demonstrate that, in humans, motor and cognitive frontosubcortical circuits are segregated. We studied a group of patients with Parkinson's disease undergoing surgical lesioning of the globus pallidus internus for relief of their symptoms. Lesion location along an anteromedial-to-posterolateral axis was found to be related to postsurgical outcome on both cognitive and motor measures. Performance on several neuropsychological measures, including the generation of category exemplars and continuous mental addition, was linearly related to distance along this axis, with anteromedial lesions leading to postsurgical impairment, intermediate lesions having little effect and posterolateral lesions leading to an improvement on several measures. The same relationship was found between memory performance under conditions of proactive interference and lesion location within the globus pallidus internus. In contrast, bradykinesia, assessed as the speed of finger tapping, had a non-linear relationship to lesion location, intermediate lesions leading to greater postsurgical improvement than lesions in more extreme anteromedial or posterolateral locations. These data demonstrate that the cognitive effects of pallidotomy can be dissociated from the motor effects. These effects depend upon the placement of the lesions within the globus pallidus internus, supporting the segregation of functionally distinct circuits in the human pallidum. PMID- 10734007 TI - Parallel visuomotor processing in the split brain: cortico-subcortical interactions. AB - We tested nine patients with callosal pathology in a simple reaction time task with and without redundant targets in the same or opposite visual hemifield. Four patients showed large facilitation (redundancy gain) in the presence of a redundant target, exceeding probability summation models (neural summation). Five patients showed redundancy gain not exceeding probability models. Violation of probability models was not associated with a specific type of callosal lesion. Neural summation, which probably occurs at collicular level, may be modulated by cortical activity. To test this hypothesis, we used functional MRI. During detection of redundant simultaneous targets, activations in the extrastriate cortex were observed in a patient with callosal agenesis and redundancy gain violating probability models, but not in a patient with callosal agenesis and redundancy gain not exceeding probability models. We conclude that cortical activity in the extrastriate cortex may be a modulating factor in the magnitude of the redundancy gain during parallel visuomotor transforms. PMID- 10734008 TI - Visual confrontation naming and hippocampal function: A neural network study using quantitative (1)H magnetic resonance spectroscopy. AB - Prior research on the relationship between visual confrontation naming and hippocampal function has been inconclusive. The present study examined this relationship using quantitative (1)H magnetic resonance spectroscopy ((1)H-MRS) to operationalize the function of the left and right hippocampi. The 60-item Boston Naming Test (BNT) was used to measure naming. Our sample included 46 patients with medically intractable, focal mesial temporal lobe epilepsy who had been screened for all pathology other than mesial temporal sclerosis. Statistics included Pearson correlations and neural network analysis (multilayer perceptron and radial basis function). Baseline BNT performance correlated significantly with left (1)H-MRS hippocampal ratios. Thirty-six per cent of the variance in baseline BNT performance was explained by a neural network model using left and right (1)H-MRS ratios(creatine/N-acetylaspartate) as input. This was elevated to 49% when input from the right hippocampus was lesioned mathematically. In a second model, left (1)H-MRS hippocampal ratios were modelled using measures of semantic and episodic memory as input (including the BNT). Explained variance in left (1)H-MRS hippocampal ratios fell from 60.8 to 3.6% when input from BNT and another semantic memory measure was degraded mathematically. These results provide evidence that the speech-dominant hippocampus is a significant component of the overall neuroanatomical network of visual confrontation naming. Clinical and theoretical implications are explored. PMID- 10734009 TI - Cerebral activation during micturition in normal men. AB - Specific cerebral lesions have shown the crucial role of the brain in the control of micturition. The precise identification of the anatomical cerebral structures involved in micturition can contribute to a better understanding of the control of micturition and the development of therapeutic models. Various neuropathological and animal studies have referred to the medulla oblongata, pons, limbic system, superior frontal lobe and premotor cortical regions as areas implicated in micturition control. The aim of this study was to investigate whether the activity of these areas during micturition can be confirmed by PET in normal men. The distribution of the regional cerebral blood flow after bolus injection of (15)O water was used as an indirect measure of cerebral activation. PET scans were performed during the following three conditions: (i) at rest with the bladder empty; (ii) during simulated micturition after instillation of isotonic saline into the urinary bladder; and (iii) the withholding of urine (saline). Normal micturition using this model was achieved in eight out of 12 right-handed normal subjects. The changes in bladder contraction, bladder pressure and intra-abdominal pressure were monitored on-line during the whole scanning session by a cystometry device. The images were analysed using statistical parametric mapping at a significance threshold of P < 0.05 with correction for multiple independent comparisons. Micturition versus rest was associated with bilateral activation of areas close to the postcentral gyrus, inferior frontal gyrus, globus pallidus, cortex cerebelli, vermis and midbrain. On the left side, activation of the middle frontal gyrus, superior frontal gyrus, superior precentral gyrus, thalamus and the caudal part of the anterior cingulate gyrus was seen, while on the right side we found activation in the supramarginal gyrus, mesencephalon and insula. When the threshold value was lowered to P < 0.001 (Z > 3.09) without correction for multiple comparisons, we found additional activation in the medial pontine tegmentum, mesencephalon, right thalamus, right middle frontal gyrus and left insula. When urine- withholding was compared with rest, the left insula showed a tendency to activate. We conclude from this study, in which urinary bladder contraction was verified cystometrically, that the onset and maintenance of micturition in normal men is associated with a vast network of cortical and subcortical regions, confirming observations from clinical and animal studies. PMID- 10734010 TI - Essential role of the right superior parietal cortex in Japanese kana mirror reading: An fMRI study. AB - Functional magnetic resonance imaging (fMRI) was used to investigate the neural substrates responsible for Japanese kana mirror reading. Japanese kana words, arranged vertically from top to bottom, were used in the mirror reading task in 10 normal right-handed Japanese adults. Since both mirror-reversed and normally oriented kana items are read in the same (top to bottom) direction, it was possible to minimize the oculomotor effects which often occur in the process of mirror reading of alphabetical language. By using the SPM96 random effect analysis method, a significant increase in the blood oxygen level-dependent signal during mirror reading relative to normal reading was detected in multiple brain regions, including the bilateral superior occipital gyri, bilateral middle occipital gyri corresponding to Brodmann area (BA) 18/19, bilateral lingual gyri (BA 19), left inferior occipital gyrus (BA 18), left inferior temporal cortex (BA 37), bilateral fusiform gyri (BA 19), right superior parietal cortex (SPC) (BA 7), left inferior frontal gyrus (BA 44/45) and an inferior part of the left BA 6. In addition to these cortical regions, the right caudate nucleus and right cerebellum were also activated. The activation found in the right SPC and the left inferior temporal region is consistent with the hypothesis that mirror reading involves both the dorsal visuospatial and ventral object recognition pathways. In particular, a significant correlation was found between the fMRI signal change in the right SPC and the behavioural performance (error index) in the task. This may reflect increased demand on the right SPC for the spatial transformation which is required for the accurate recognition of mirror-reversed kana items. This relationship between the haemodynamic response in a specific brain area and the behavioural data provides new evidence for the essential role of the right SPC in Japanese kana mirror reading. PMID- 10734011 TI - Differential cognitive effects of colloid cysts in the third ventricle that spare or compromise the fornix. AB - A series of twelve cases, all of whom had received surgery for the removal of a colloid cyst in the third ventricle, was examined on a series of memory tests. The only consistent predictor of poor memory performance that could be detected from MRIs was the presence of bilateral interruption of the fornix, which occurred in three of the subjects. Although these three cases were poor on tests of learning and recall, there was evidence that recognition was less impaired. The subjects were also tested on a set of recognition and concurrent discriminations that closely matched tests given to non-human primates. Clear parallels were found between the apparent effects of fornix damage in these clinical cases and those observed following more selective surgery in non-human primates. These findings not only indicate that fornix damage is sufficient to induce anterograde amnesia but also support the validity of recent animal tests that are thought to capture aspects of episodic memory. PMID- 10734012 TI - Mediodorsal thalamic function in scene memory in rhesus monkeys. AB - Three monkeys were trained preoperatively in a scene memory task which is analogous, in some ways, to human episodic memory. The same animals were also trained in object-reward association memory. Following bilateral ablations of almost the entire magnocellular division of the mediodorsal thalamic nucleus, the animals were impaired both in scene memory and in object-reward association memory. These results, combined with recent results in object recognition memory from monkeys with mediodorsal thalamic lesions, show that the impairment produced by this lesion is more general, affecting a broader range of memory tasks, than the impairment which is produced in monkeys by lesions restricted to the hippocampus-fornix-mamillary system. It is also more severe than the effect of lesions limited to the medial part of the magnocellular division of the mediodorsal thalamic nucleus. These findings extend the evidence that the magnocellular division of the mediodorsal thalamic nucleus has an important and general role in memory, and they are consistent with the proposal that lesions of the magnocellular division of that nucleus have their effect by disrupting the function of prefrontal cortex. PMID- 10734014 TI - Inverse correlation between frontal lobe and cerebellum sizes in children with autism. AB - Certain cognitive and behavioural deficits suggest that the frontal lobe functions abnormally in patients with autism, but little anatomical research is available to either verify or refute this. In contrast, several neuropathological and neuroimaging studies have demonstrated anatomical abnormalities in the cerebellum in autistic patients. The current study shows that frontal lobe cortex volume is increased in a subset of patients with autism and that this increase correlates with the degree of cerebellar abnormality. This evidence of concurrent structural abnormalities in both the frontal lobe and the cerebellum has important implications for understanding the development and persistence of the autistic disorder. PMID- 10734013 TI - Saccadic eye movement disturbances in whiplash patients with persistent complaints. AB - In order to analyse the possible basis of subjective complaints following whiplash injury, horizontal eye movements were examined in subjects with persistent complaints ('symptomatic group') and subjects who had completely recovered ('recovered group'). The results for the symptomatic and recovered groups were compared with those for age-matched, healthy volunteers (control group). A battery of different saccade paradigms was employed: two were reflexive saccade tasks including a gap and an overlap task, and two were intentional saccade tasks consisting of an antisaccade and a memory-guided saccade task. In addition, the symptomatic and recovered groups also underwent psychiatric evaluation in a structured clinical interview, and all groups were assessed for emotional functioning using the Beck Depression Inventory (BDI). The recovered group did not differ significantly from the control group in saccade performance and emotional functioning. The symptomatic group showed dissociation of their performances of reflexive and intentional saccade tasks: performance in reflexive saccade tasks was normal, but in intentional saccade tasks the symptomatic group showed significantly impaired inhibition of unwanted reflexive saccades, impaired saccade triggering (i.e. increased latency) and a higher percentage error in amplitude in memory-guided saccades. Based on clinical interviews, no signs of major depression or dysthymia were found in any of the groups. Compared with the other two groups, the symptomatic group had significantly higher overall BDI scores, but these resulted from BDI dimensions that were non-specific to depression, viz. 'physiological manifestations' (e.g. fatigue, sleep disturbance) or 'performance difficulty' (e.g. work inhibition). In summary, in the symptomatic group the pattern of eye movement disturbances together with normal performance in reflexive saccade tasks and impaired performance in the intentional saccade tasks, especially impaired inhibitory function, suggests dysfunction of prefrontal and frontal cortical structures. PMID- 10734015 TI - Ten secrets for development of drugs for functional gastrointestinal diseases. PMID- 10734016 TI - Interleukin 10 treatment reduces fibrosis in patients with chronic hepatitis C: a pilot trial of interferon nonresponders. AB - BACKGROUND & AIMS: Interleukin (IL)-10 is a cytokine that down-regulates the proinflammatory response and has a modulatory effect on hepatic fibrogenesis. The aim of this study was to determine the effect of IL-10 on hepatic injury in patients with chronic hepatitis C. METHODS: Twenty-four patients with chronic hepatitis C who had not previously responded to interferon-based therapy were enrolled in a randomized, double-blinded 2-dose trial in which they received either 4 or 8 microgram/kg IL-10 subcutaneously daily for 90 days. Liver biopsies were performed before and at the end of therapy. RESULTS: IL-10 was well tolerated with 22 patients completing the study. Serum ALT levels normalized in 19 of 22 patients by the end of therapy and were sustained in 5 of 22. Hepatic inflammation decreased in 19 of 22 patients, with 11 having a decrease by >/=2. Fibrosis decreased in 14 of 22 patients (mean change, 3.6-2.6; P = 0.001). There was no change in serum HCV RNA levels. IL-10 therapy was associated with changes in serological markers, suggesting a reduction of immune response and fibrogenesis. CONCLUSIONS: IL-10 therapy is safe and well tolerated in patients with chronic hepatitis C. Although it has no apparent antiviral activity, IL-10 normalizes serum ALT levels, improves liver histology, and reduces liver fibrosis in a large proportion of patients receiving treatment. Therefore, IL-10 may have therapeutic potential in patients with chronic hepatitis C patients who do not respond to interferon-based therapy. PMID- 10734017 TI - Long-term omeprazole treatment in resistant gastroesophageal reflux disease: efficacy, safety, and influence on gastric mucosa. AB - BACKGROUND & AIMS: The efficacy and safety of long-term acid suppression remains a subject for debate. We report data from patients with refractory reflux esophagitis who were undergoing maintenance therapy with >/=20 mg omeprazole daily for a mean period of 6.5 years (range, 1.4-11.2 years). METHODS: Patients with severe reflux esophagitis resistant to long-term therapy with H(2)-receptor antagonists and who were not eligible for surgery were evaluated at least annually for endoscopic relapse and histological changes in the gastric corpus. RESULTS: In 230 patients (mean age, 63 years at entry; 36% were >/=70 years), there were 158 relapses of esophagitis during 1490 treatment years (1 per 9.4 years), with no significant difference in relapse rates between Helicobacter pylori-positive and -negative patients. All patients rehealed during continued therapy with omeprazole at the same or higher dose. The annual incidence of gastric corpus mucosal atrophy was 4.7% and 0.7% in H. pylori-positive and negative patients, respectively, which was mainly observed in elderly patients who had moderate/severe gastritis at entry. In patients with baseline moderate/severe gastritis, the incidences were similar: 7.9% and 8.4%, respectively. Corpus intestinal metaplasia was rare, and no dysplasia or neoplasms were observed. The adverse event profile was as might be expected from this elderly group of patients. CONCLUSIONS: Long-term omeprazole therapy (up to 11 years) is highly effective and safe for control of reflux esophagitis. PMID- 10734018 TI - Endoscopic mucosal resection of early cancer and high-grade dysplasia in Barrett's esophagus. AB - BACKGROUND & AIMS: In view of the mortality and morbidity rates of esophagectomy and the relatively large group of inoperable patients, local therapeutic techniques are required for high-grade dysplasia and early Barrett's cancer. METHODS: A prospective investigation of endoscopic mucosal resection was conducted in 64 patients (mean age, 65 +/- 10 years) who had early carcinoma (61 patients) or high-grade dysplasia (3 patients) in Barrett's esophagus. Thirty five patients met the criteria for low risk: macroscopic types I, IIa, IIb, and IIc; lesion diameter up to 20 mm; mucosal lesion; and histological grades G1 and G2 and/or high-grade dysplasia (group A). The remaining 29 patients were included in group B (high risk). RESULTS: A total of 120 resections were performed, with no technical problems encountered. The mean number of treatment sessions per patient was 1. 3 +/- 0.6 in group A and 2.8 +/- 2.0 in group B (P < 0.0005). Only one major complication occurred, a case of spurting bleeding, which was managed endoscopically. Complete local remission was achieved significantly earlier (P = 0.008) in group A than in group B. In May 1999, complete remission had been achieved in 97% of the patients in group A and in 59% of those in group B; however, 1 patient in group A and 9 in group B are still undergoing treatment or awaiting the first check-up. During a mean follow-up of 12 +/- 8 months, recurrent or metachronous carcinomas were found in 14%. CONCLUSIONS: Endoscopic mucosal resection of early carcinoma in Barrett's esophagus is associated with promisingly low morbidity and mortality rates. The procedure may offer a new minimally invasive therapeutic alternative to esophagectomy, especially in low risk situations. Comparisons with surgical results will need to be done when the long-term results of this procedure become available. PMID- 10734019 TI - Development and validation of a self-report symptom inventory to assess the severity of oral-pharyngeal dysphagia. AB - BACKGROUND & AIMS: The aim of this study was to develop and evaluate the validity and reliability of a self-report inventory to measure symptomatic severity of oral-pharyngeal dysphagia. METHODS: Test-retest reliability and face, content, and construct validity of a prototype visual analogue scale inventory were assessed in 45 patients who had stable, neuromyogenic dysphagia. RESULTS: Normalized scores varied over time by -0.5% +/- 17.6% (95% confidence interval, 9.2% to 8.2%). Factor analysis identified a single factor (dysphagia), to which 18 of 19 questions contributed significantly, that accounted for 56% of total variance (P < 0.0001). After deletion of 2 questions with poor face validity and patient compliance, this proportion increased to 59%; mean test-retest change was -2% (95% confidence interval, -11% to 7%); and total score correlated highly with an independent global assessment severity score (r = 0.7; P < 0.0001). A mean 70% reduction in score (P < 0.0001) was observed after surgery in patients with Zenker's diverticulum (discriminant validity). CONCLUSIONS: Applied to patients with neuromyogenic dysphagia, the 17-question inventory shows strong test-retest reliability over 2 weeks as well as face, content, and construct validity. Discriminant validity (responsiveness) has been demonstrated in a population with a correctable, structural cricopharyngeal disorder. Responsiveness of the instrument to treatment in neuromyogenic dysphagia remains to be quantified. PMID- 10734020 TI - Increased frequency of transient lower esophageal sphincter relaxation induced by gastric distention in reflux patients with hiatal hernia. AB - BACKGROUND & AIMS: This study aimed to determine if hiatal hernia influences vulnerability to reflux and transient lower esophageal sphincter relaxation (tLESR) during gastric distention in patients with gastroesophageal reflux disease (GERD). METHODS: Eight normal subjects and 15 patients with GERD were studied. A metal clip attached to the squamocolumnar junction (SCJ) was beneath the hiatus in all control subjects. Eight GERD patients with >/=1-cm SCJ-hiatus separation were considered hernia patients, and 7 with <1-cm separation were considered nonhernia patients. Manometry and esophageal pH were recorded for 30 minutes, after which the stomach was loaded with acid dextrose and the recording continued for 2 hours with intragastric air infusion of 15 mL/min. RESULTS: Baseline reflux was comparable among groups. Gastric distention increased the frequency of reflux by the tLESR mechanism in all groups. Controls and nonhernia patients had median increases of 4.0 and 4.5 in tLESR frequency, respectively, and hernia patients had a median increase of 9.5/h. tLESR frequency was highly correlated with the SCJ-hiatus separation (r = 0.76; P < 0.001). CONCLUSIONS: Gastric air infusion was a potent stimulus for tLESR and reflux. The resultant tLESR frequency was directly proportional to the separation between the SCJ and hiatus, suggesting that the perturbed anatomy associated with hiatal hernia predisposed to eliciting tLESRs in patients with GERD. PMID- 10734021 TI - Intravenous pantoprazole rapidly controls gastric acid hypersecretion in patients with Zollinger-Ellison syndrome. AB - BACKGROUND & AIMS: Parenteral control of gastric acid hypersecretion in conditions such as Zollinger-Ellison syndrome (ZES) or idiopathic gastric acid hypersecretion is necessary perioperatively or when oral medications cannot be taken for other reasons (e.g., during chemotherapy, acute upper gastrointestinal bleeding, or in intensive care unit settings). METHODS: We evaluated the efficacy and safety of 15-minute infusions of the proton pump inhibitor pantoprazole (80 120 mg every 8-12 hours) in controlling acid output for up to 7 days. Effective control was defined as acid output >10 milliequivalents per hour (mEq/h) (<5 mEq/h in patients with prior acid-reducing surgery) for 24 hours. RESULTS: The 21 patients enrolled had a mean age of 51.9 years (range, 29-75) and a mean disease duration of 8.1 years (range, <0.5-21); 13 were male, 7 had multiple endocrine neoplasia syndrome type I, 4 had undergone acid-reducing surgery, 2 had received chemotherapy, and 13 had undergone gastrinoma resections without cure. Basal acid output (mean +/- SD) was 40.2 +/- 27.9 mEq/h (range, 11.2-117.9). In all patients, acid output was controlled within the first hour (mean onset of effective control, 41 minutes) after an initial 80-mg intravenous pantoprazole dose. Pantoprazole, 80 mg every 12 hours, was effective in 17 of 21 patients (81%) for up to 7 days. Four patients required upward dose titration, 2 required 120 mg pantoprazole every 12 hours, and 2 required 80 mg every 8 hours. At study end, acid output remained controlled for 6 hours beyond the next expected dose in 71% of patients (n = 15); mean acid output increased to 4.0 mEq/h (range, 0-9.7). No serious or unexpected adverse events were observed. CONCLUSIONS: Intravenous pantoprazole, 160-240 mg/day administered in divided doses by 15-minute infusion, rapidly and effectively controlled acid output within 1 hour and maintained control for up to 7 days in all ZES patients. PMID- 10734022 TI - Pharmacogenomics and metabolite measurement for 6-mercaptopurine therapy in inflammatory bowel disease. AB - BACKGROUND & AIMS: The effects of 6-mercaptopurine (6-MP) are mediated via its intracellular conversion to 6-thioguanine (6-TG) and 6-methylmercaptopurine (6 MMP) nucleotide metabolites, the latter genetically controlled by thiopurine methyltransferase (TPMT). We sought to determine optimal therapeutic 6-MP metabolite levels and their correlation with medication-induced toxicity and TPMT genotype. METHODS: Therapeutic response was determined in 92 pediatric patients with inflammatory bowel disease coincidentally with hematologic, pancreatic, and hepatic laboratory parameters, and compared with erythrocyte metabolite levels and TPMT genotype. RESULTS: Clinical response was highly correlated with 6-TG levels (P < 0.0001) but not with any other variable, including 6-MMP levels, drug dose, gender, and concurrent medications. The frequency of therapeutic response increased at 6-TG levels > 235 pmol/8 x 10(8) erythrocytes (P < 0.001). Hepatotoxicity correlated with elevated 6-MMP levels (>5700 pmol/8 x 10(8) erythrocytes; P < 0.05). Although leukopenia was associated with higher 6-TG levels (P < 0.03), it was observed in only 8% of responders. Patients heterozygous for TPMT (8/92) had higher 6-TG levels (P < 0.0001), and all responded to therapy. CONCLUSIONS: 6-MP metabolite levels and TPMT genotyping may assist clinicians in optimizing therapeutic response to 6-MP and identifying individuals at increased risk for drug-induced toxicity. PMID- 10734023 TI - Impaired nitrergic innervation in rat colitis induced by dextran sulfate sodium. AB - BACKGROUND & AIMS: The pathophysiological role of neuronal nitric oxide synthase (nNOS) in colitis remains unknown. METHODS: We investigated colonic transit, nonadrenergic, noncholinergic (NANC) relaxation, nNOS activity, and nNOS synthesis in the myenteric plexus in dextran sulfate sodium (DSS)-induced colitis in rats. RESULTS: Oral administration of 5% DSS for 7 days induced predominant distal colitis and delayed colonic transit. In the proximal colon, carbachol-, sodium nitroprusside-, and electrical field stimulation (EFS)-induced responses were not different between control and DSS-treated rats. In the distal colon, EFS evoked cholinergic contraction, NANC relaxation, and orphanin FQ-induced contraction were significantly impaired in DSS-treated rats compared with those in control rats, but carbachol- and sodium nitroprusside-induced responses remained intact in DSS-treated rats. The number of nNOS-immunopositive cells, catalytic activity of NOS, and nNOS synthesis in the colonic wall were significantly reduced in the distal colon of DSS-treated rats. In contrast, the number of PGP 9.5-immunopositive cells and PGP 9.5 synthesis in the colonic wall remained intact in the distal colon of DSS-treated rats. CONCLUSIONS: These results suggest that impaired NANC relaxation in the distal colon is associated with reduced activity and synthesis of nNOS in the myenteric plexus in DSS induced colitis. PMID- 10734024 TI - The luminal short-chain fatty acid butyrate modulates NF-kappaB activity in a human colonic epithelial cell line. AB - BACKGROUND & AIMS: The transcription factor nuclear factor-kappaB (NF-kappaB) plays a central role in regulating immune and inflammatory responses. Because butyrate deficiency has been associated with inflammatory bowel disease, we examined the effect of butyrate on NF-kappaB activity in the human HT-29 colonic cell line. METHODS: The influence of butyrate (4 mmol/L) on NF-kappaB activity was determined using the gel mobility shift assay. The effect of butyrate on the expression of NF-kappaB subunits and inhibitory proteins was determined by immunoblotting. NF-kappaB-regulated gene expression was assayed by primer extension of intercellular adhesion molecule 1 and Mn superoxide dismutase messenger RNA, and by analysis of a transfected luciferase reporter. RESULTS: Exposure of HT-29 cells to butyrate eliminated their constitutive NF-kappaB, p50 dimer activity. This inhibition corresponded with a reduction in p50 nuclear localization, without a reduction in expression. Butyrate also selectively modulated activation of NF-kappaB, suppressing its activation by tumor necrosis factor alpha and phorbol ester more than 10-fold, without affecting the activity induced by interleukin (IL)-1beta. Butyrate did, however, enhance formation of the stronger p65-p50 transcriptional activator in IL-1beta-stimulated cells. The changes in NF-kappaB activation did not correlate with changes in IkappaBalpha levels. Gene expression reflected DNA binding. The influence of butyrate on NF kappaB may result in part from its ability to inhibit deacetylases because the specific deacetylase inhibitor trichostatin A has a similar effect. CONCLUSIONS: These findings suggest that the influences of butyrate on colonic inflammatory responses may result in part from its influence on NF-kappaB activation. This activity of butyrate apparently involves its ability to inhibit deacetylases. PMID- 10734025 TI - Molecular mechanism of interferon alfa-mediated growth inhibition in human neuroendocrine tumor cells. AB - BACKGROUND & AIMS: Although human neuroendocrine tumors respond to interferon (IFN)-alpha treatment in vivo, the underlying mechanisms of growth inhibition are poorly understood. To characterize the antiproliferative effects at a molecular level, we explored the growth-regulatory action of IFN-alpha in the human neuroendocrine tumor cell lines BON and QGP1. METHODS: IFN-alpha receptor expression and signal transduction were examined by reverse-transcription polymerase chain reaction, immunoblotting, subcellular fractionation, and transactivation assays. Growth regulation was evaluated by cell numbers, soft agar assays, and cell cycle analysis using flow cytometry. Expression and activity of cell cycle-regulatory molecules were determined by immunoblotting and histone H1-kinase assays. RESULTS: Both cell lines expressed IFN-alpha receptor mRNA transcripts. Ligand binding initiated phosphorylation of Jak kinases and Stat transcription factors, resulting in Stat activation, nuclear translocation, and transcription from an ISRE-reporter construct. Prolonged IFN-alpha treatment dose-dependently inhibited both anchorage-dependent and -independent growth. Cell cycle analysis of IFN-alpha-treated, unsynchronized cultures revealed an increased S-phase population, which was further substantiated in G(1) synchronized QGP1 cells. IFN-alpha-treated cells entered S phase in parallel to control cultures, but their progress into G(2)/M phase was delayed. Both cellular cyclin B levels and CDC 2 activity were substantially reduced. The extent and time course of this reduction corresponded to the observed S-phase accumulation. CONCLUSIONS: IFN-alpha directly inhibits growth of human neuroendocrine tumor cells by specifically delaying progression through S phase and into G(2)/M. These cell cycle changes are associated with inhibition of cyclin B expression, resulting in reduced CDC2 activity. PMID- 10734026 TI - Induction and maintenance of immune effector cells in the gastric tissue of mice orally immunized to Helicobacter pylori requires salivary glands. AB - BACKGROUND & AIMS: Helicobactor pylori mostly colonizes the gastric mucus that contains salivary antibodies. We studied the role of saliva in the induction and maintenance of gastric immunity conferred by oral vaccination against H. pylori. METHODS: C57BL/6 mice underwent a sialoadenectomy before and after intragastric immunization using whole-cell sonicates of H. pylori and cholera toxin as an adjuvant. At 1 and 6 months after oral inoculation, we assessed the density of the H. pylori colonizing the stomach, specific antibodies in gastric secretion and sera, and the constituents of cellular infiltrates in the tissue. RESULTS: A sialoadenectomy before, but not after, immunization abrogated protection by the vaccination at 1 month after inoculation. Protected mice had more neutrophils, plasma cells, and lymphocytes, but fewer eosinophils, in the gastric tissue than nonprotected mice. Protected mice had a greater increase of immunoglobulin (Ig) G1 specific to H. pylori than IgG2a in sera. At 6 months after inoculation, oral immunization was less effective in mice who had a sialoadenectomy than in control immunized mice. The antibody titers in both gastric secretion and in sera did not correlate with the density of bacteria colonizing the stomach. CONCLUSIONS: It is suggested that, in intragastric immunization against H. pylori, saliva is necessary for both the induction and maintenance of optimal immunity in the stomach. Effective immunity was associated with an increased number of neutrophils and lymphocytes in gastric tissue. PMID- 10734027 TI - Natural history of hepatitis C virus carriers with persistently normal aminotransferase levels. AB - BACKGROUND & AIMS: Some patients with serum hepatitis C virus (HCV) have persistently normal aminotransferase (ALT) levels and are affected by cirrhosis. This study prospectively evaluated progression of the disease in a group of anti HCV-positive patients with persistently normal ALT levels. METHODS: Thirty-seven subjects were studied. Each subject underwent liver biopsy at baseline and after 5 years of follow-up. At baseline, serum samples were tested for genotypes and HCV RNA load. ALT levels and serum HCV RNA were tested every other month and every 6 months, respectively. Patients with increased ALT were discharged from the study and treated with IFN. Five years after the end of IFN therapy, a liver biopsy was performed. RESULTS: Liver biopsy at baseline showed chronic hepatitis in 34 patients and normal histology in 3 patients, 2 of whom were negative for HCV RNA and 1 positive. HCV genotypes were distributed as follows: 2a, 56%; 1b, 41%; and 1a, 3%. At the end of 7-year follow-up, 73% of the patients still had normal ALT values. Liver histology after 5 years was comparable to that observed at entry to study. CONCLUSIONS: Most patients with persistently normal ALT serum levels have very mild chronic hepatitis. However, healthy anti-HCV-positive subjects exist. In patients with HCV-related chronic hepatitis associated with persistently normal ALT levels, the grade of disease activity does not increase over years and progression to cirrhosis is slow or absent. PMID- 10734028 TI - Selective alteration of agonist-mediated contraction in hepatic arteries isolated from patients with cirrhosis. AB - BACKGROUND & AIMS: Impaired pressor function in cirrhosis may be specific to certain agonists and vascular territories. This investigation determined whether responses to arginine vasopressin (AVP) and 5-hydroxytryptamine (5-HT) were impaired in hepatic arteries from cirrhotic patients. METHODS: Cumulative concentration-response curves were produced for AVP (10(-11) to 3 x 10(-6) mol/L), 5-HT (10(-9) to 3 x 10(-5) mol/L), and potassium chloride (2.5 -120 mmol/L) in hepatic arteries from liver donors (noncirrhotic) and recipients (cirrhotic). The receptor stimulated by AVP was identified using a V(1)-receptor antagonist (d[CH(2)](5)Tyr[Me]AVP) and a selective V(2)-receptor agonist (desmopressin [DDAVP]). RESULTS: Cirrhotic patients had a high heart rate (98 +/- 4 beats/min) and cardiac output (9.87 +/- 0.51 L/min) but low peripheral vascular resistance (711 +/- 35 dyn. s/cm(5)). None of the arteries had a functional endothelium. Maximal contraction (but not sensitivity) to AVP was smaller (P = 0.0002) in hepatic arteries from recipients (34.03% +/- 3.42% KCl) than donors (60.69% +/- 5.56% KCl). 5-HT-mediated contraction was enhanced in recipient hepatic arteries (88.81% +/- 5.43% KCl vs. 71.63% +/- 4. 46% KCl; P = 0.01), but sensitivities were similar (P = 0.20). KCl-mediated contractions were similar (P = 0.87) in both groups. Arteries did not respond to DDAVP, but d(CH(2))(5)Tyr(Me)AVP produced a concentration-dependent rightward shift in the response to AVP. CONCLUSIONS: These results demonstrate a selective impairment of V(1) receptor-mediated contraction in denuded hepatic arteries from cirrhotic patients, suggesting an abnormality within the vascular smooth muscle. PMID- 10734029 TI - Impaired biliary cholesterol secretion and decreased gallstone formation in apolipoprotein E-deficient mice fed a high-cholesterol diet. AB - BACKGROUND & AIMS: Because apolipoprotein E (apoE) is a key cholesterol transport molecule involved in the hepatic uptake of chylomicron cholesterol, it may play a critical role in controlling bile cholesterol elimination and cholesterol gallstone formation induced by dietary cholesterol. To test this hypothesis, we studied biliary lipid secretion and gallstone formation in apoE-deficient mice fed cholesterol-rich diets. METHODS: Bile lipid outputs and gallstone sequence events were analyzed in apoE-deficient mice fed a high-cholesterol diet or a lithogenic diet compared with control animals. RESULTS: A high-cholesterol diet increased biliary cholesterol secretion and gallbladder bile cholesterol concentration in wild-type mice; the increase in bile cholesterol secretion was significantly attenuated in apoE-deficient mice. ApoE knockout mice fed a high cholesterol lithogenic diet had a markedly lower frequency of gallbladder bile cholesterol crystal and gallstone formation than wild-type mice, which was most likely a result of the decreased cholesterol saturation index found in gallbladder bile of apoE-deficient mice. CONCLUSIONS: These results show that apoE expression is an important factor for regulating both biliary secretion of diet-derived cholesterol as well as diet-induced cholesterol gallstone formation in mice. PMID- 10734030 TI - Does alcohol directly stimulate pancreatic fibrogenesis? Studies with rat pancreatic stellate cells. AB - BACKGROUND & AIMS: Activated pancreatic stellate cells have recently been implicated in pancreatic fibrogenesis. This study examined the role of pancreatic stellate cells in alcoholic pancreatic fibrosis by determining whether these cells are activated by ethanol itself and, if so, whether such activation is caused by the metabolism of ethanol to acetaldehyde and/or the generation of oxidant stress within the cells. METHODS: Cultured rat pancreatic stellate cells were incubated with ethanol or acetaldehyde. Activation was assessed by cell proliferation, alpha-smooth muscle actin expression, and collagen synthesis. Alcohol dehydrogenase (ADH) activity in stellate cells and the influence of the ADH inhibitor 4-methylpyrazole (4MP) on the response of these cells to ethanol was assessed. Malondialdehyde levels were determined as an indicator of lipid peroxidation. The effect of the antioxidant vitamin E on the response of stellate cells to ethanol or acetaldehyde was also examined. RESULTS: Exposure to ethanol or acetaldehyde led to cell activation and intracellular lipid peroxidation. These changes were prevented by the antioxidant vitamin E. Stellate cells exhibited ethanol-inducible ADH activity. Inhibition of ADH by 4MP prevented ethanol-induced cell activation. CONCLUSIONS: Pancreatic stellate cells are activated on exposure to ethanol. This effect of ethanol is most likely mediated by its metabolism (via ADH) to acetaldehyde and the generation of oxidant stress within the cells. PMID- 10734031 TI - The efficacy and safety of long-term omeprazole treatment for gastroesophageal reflux disease. PMID- 10734033 TI - An advance in the treatment of portal hypertension? PMID- 10734032 TI - Maintaining gut homeostasis: the butyrate-NF-kappaB connection. PMID- 10734034 TI - Salmonella pathogenesis: the trojan horse or the New York shuttle? PMID- 10734035 TI - Laparoscopic colorectal surgery PMID- 10734037 TI - Chlorophyll binding to peptide maquettes containing a retention motif. AB - The motif Glu-X-X-His/Asn-X-Arg is conserved in the first and third membrane spanning domains of all light-harvesting chlorophyll a/b- and a/c-binding proteins in chloroplasts. Molecular modeling of synthetic peptides containing the sequence Glu-Ile-Val-His-Ser-Arg, a motif found in the apoprotein of the major light-harvesting complex in plants, generated a loop structure formed by intrapeptide, electrostatic attraction between Glu and Arg. His, Asn, and charge compensated Glu-Arg pairs are known ligands of the magnesium atom in chlorophyll. The prediction that this structure should bind two molecules of chlorophyll was confirmed experimentally with an assay based on fluorescence resonance energy transfer between peptides and chlorophyll a. Motifs with both potential ligands bound approximately two times the amount of chlorophyll as one in which His was replaced by Ala. These results support the conclusion that formation of this intermediate, within membranes of the envelope, is a crucial step in assembly of light-harvesting complexes and a mechanism that regulates import of the apoproteins into the chloroplast. PMID- 10734038 TI - Protein trans-splicing and cyclization by a naturally split intein from the dnaE gene of Synechocystis species PCC6803. AB - A naturally occurring split intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) has been shown to mediate efficient in vivo and in vitro trans-splicing in a foreign protein context. A cis-splicing Ssp DnaE intein construct displayed splicing activity similar to the trans-splicing form, which suggests that the N- and C-terminal intein fragments have a high affinity interaction. An in vitro trans-splicing system was developed that used a bacterially expressed N-terminal fragment of the Ssp DnaE intein and either a bacterially expressed or chemically synthesized intein C-terminal fragment. Unlike artificially split inteins, the Ssp DnaE intein fragments could be reconstituted in vitro under native conditions to mediate splicing as well as peptide bond cleavage. This property allowed the development of an on-column trans-splicing system that permitted the facile separation of reactants and products. Furthermore, the trans-splicing activity of the Ssp DnaE intein was successfully applied to the cyclization of proteins in vivo. Also, the isolation of the unspliced precursor on chitin resin allowed the cyclization reaction to proceed in vitro. The Ssp DnaE intein thus represents a potentially important protein for in vivo and in vitro protein manipulation. PMID- 10734039 TI - Activated protein C directly activates human endothelial gelatinase A. AB - Angiogenesis (formation of new blood vessels) occurs in a number of diseases such as cancer and arthritis. The matrix metalloproteinase (MMP), gelatinase A, is secreted by endothelial cells and plays a vital role during angiogenesis. It is secreted as a latent enzyme and requires extracellular activation. We investigated whether activated protein C (APC), a pivotal molecule involved in the body's natural anti-coagulant system, could activate latent gelatinase A secreted by human umbilical vein endothelial cells (HUVEC). APC induced the fully active form of gelatinase A in a dose (100-300 nM)- and time (4-24 h)-responsive manner. The inactive zymogen, protein C, did not activate gelatinase A when used at similar concentrations. APC did not up-regulate membrane type 1 MMP (MT1-MMP) mRNA in HUVEC. In addition, the MMP inhibitor, 1, 10-phenanthroline (10 nM), was unable to inhibit APC-induced activation. These results suggested that MT1-MMP was not involved in the activation process. APC activation of gelatinase A occurred in the absence of cells, indicating that it acts directly. APC may contribute to the physiological/pathological mechanism of gelatinase A activation, especially during angiogenesis. PMID- 10734040 TI - Notch responds differently to Delta and Wingless in cultured Drosophila cells. AB - Notch, a cell surface receptor, is required for producing different types of cells during development of Drosophila melanogaster. Notch activates expression of one set of genes in response to ligand Delta and another set of genes in response to ligand Wingless. The means by which Notch initiates these different intracellular activities was examined in this study. Cultured cells expressing Notch were treated with Delta or Wingless, and the effect on Notch was examined by Western blotting. Treatment of cells with Delta resulted in accumulation of approximately 120-kDa Notch intracellular domain molecules in the cytoplasmic fraction. This form of Notch did not accumulate in cells treated with Wingless, but the approximately 350-kDa full-length Notch molecules accumulated. These results indicate that N responds differently to binding by Delta and Wingless, and suggest that although the Delta signal is transduced by the Notch intracellular domain released from the plasma membrane, the Wingless signal is transduced by the Notch intracellular domain associated with the plasma membrane. PMID- 10734041 TI - Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway. AB - A productive angiogenic response must couple to the survival machinery of endothelial cells to preserve the integrity of newly formed vessels. Angiopoietin 1 (Ang-1) is an endothelium-specific ligand essential for embryonic vascular stabilization, branching morphogenesis, and post-natal angiogenesis, but its contribution to endothelial cell survival has not been completely elucidated. Here we show that Ang-1 acting via the Tie 2 receptor induces phosphorylation of the survival serine-threonine kinase, Akt (or protein kinase B). This is associated with up-regulation of the apoptosis inhibitor, survivin, in endothelial cells and protection of endothelium from death-inducing stimuli. Moreover, dominant negative survivin negates the ability of Ang-1 to protect cells from undergoing apoptosis. The activation of anti-apoptotic pathways mediated by Akt and survivin in endothelial cells may contribute to Ang-1 stabilization of vascular structures during angiogenesis, in vivo. PMID- 10734042 TI - The Akt proto-oncogene links Ras to Pak and cell survival signals. AB - The Ras oncogene regulates cellular proliferation, differentiation, transformation, and survival through multiple downstream signals. Ras signals through its effector phosphoinositide 3 (PI3) kinase to the Pak protein kinase (p65(pak)), but the steps from Ras to Pak remain to be elucidated. PI3 kinase can stimulate the small G protein, Rac, a direct activator of Pak, as well as the Akt proto-oncogene, a serine-threonine protein kinase. We found that activated Akt stimulated Pak, whereas a dominant negative Akt inhibited Ras activation of Pak in transfection assays. Akt stimulation of Pak was not inhibited by dominant negative mutants of either Rac or Cdc42 suggesting that Akt activated Pak through a GTPase-independent mechanism. We also developed a novel cell-free system to study Ras activation of Pak. In this system Ras activated Pak only in the presence of a crude cell extract but failed to activate Pak when Akt was immunodepleted from the extract. Akt protects cells from apoptosis through phosphorylation of downstream targets such as the Bcl-2 family member, Bad. We found that activated Pak decreased apoptosis and increased phosphorylation of Bad, whereas dominant negative Pak increased apoptosis and decreased phosphorylation of Bad. These studies define a new oncogene-mediated cell survival signal. PMID- 10734043 TI - Phosphoinositides are required for store-mediated calcium entry in human platelets. AB - We have recently observed that small GTP-binding proteins are important for mediation of store-mediated Ca(2+) entry in human platelets through the reorganization of the actin cytoskeleton. Because it has been shown in platelets and other cells that small GTP-binding proteins regulate the activity of phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase, whose products, phosphoinositides, play a key role in the reorganization of the actin cytoskeleton, we have investigated the role of these lipid kinases in store mediated Ca(2+) entry. Treatment of platelets with LY294002, an inhibitor of phosphatidylinositol 3- and phosphatidylinositol 4-kinases, resulted in a concentration-dependent inhibition of Ca(2+) entry stimulated by thapsigargin or the physiological agonist, thrombin. In addition, wortmannin, another inhibitor of these kinases, which is structurally unrelated to LY294002, significantly reduced store-mediated Ca(2+) entry. The inhibitory effect of LY294002 was not mediated either by blockage of Ca(2+) channels or by modification of membrane potential. LY294002 inhibited actin polymerization stimulated by thrombin or thapsigargin. These results indicate that both phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase are required for activation of store-mediated Ca(2+) entry in human platelets and that the mechanism could involve the reorganization of the actin cytoskeleton. PMID- 10734044 TI - I(ARC), a novel arachidonate-regulated, noncapacitative Ca(2+) entry channel. AB - Along with the inositol trisphosphate-induced release of stored Ca(2+), a receptor-enhanced entry of Ca(2+) is a critical component of intracellular Ca(2+) signals generated by agonists acting at receptors coupled to the activation of phospholipase C. Although the simple emptying of the intracellular Ca(2+) stores is known to be capable of activating Ca(2+) entry via the so-called "capacitative" mechanism, recent evidence suggests that Ca(2+) entry at physiological agonist concentrations, where oscillatory Ca(2+) signals are typically observed, does not conform to such a model. Instead, a noncapacitative Ca(2+) entry pathway regulated by arachidonic acid appears to be responsible for Ca(2+) entry under these conditions. Using whole-cell patch clamp techniques we demonstrate that low concentrations of arachidonic acid activate a Ca(2+) selective current that is superficially similar to the store-operated current I(CRAC), but which also demonstrates certain distinct features. We have named this novel current I(ARC) (for arachidonate-regulated calcium current). Importantly, I(ARC) can be readily activated in cells whose Ca(2+) stores have been maximally depleted. I(ARC) represents a novel Ca(2+) entry pathway that is entirely separate from those activated by store depletion and is specifically activated at physiological levels of stimulation. PMID- 10734045 TI - Dissociation of the high density lipoprotein and low density lipoprotein binding activities of murine scavenger receptor class B type I (mSR-BI) using retrovirus library-based activity dissection. AB - The murine class B, type I scavenger receptor (mSR-BI) is a receptor for both high density lipoprotein (HDL) and low density lipoprotein (LDL) and mediates selective, rather than endocytic, uptake of lipoprotein lipid. We have developed a "retrovirus library-based activity dissection" method to generate mSR-BI mutants in which some, but not all, of the activities of this multifunctional protein have been disrupted. This method employs three techniques: 1) efficient in vitro cDNA mutagenesis (here error-prone PCR was used), 2) efficient retroviral delivery and high expression of single mutant cDNAs into individual cells, and 3) isolation of infected cells expressing the desired mutant phenotype using high sensitivity positive/negative screening by two-color fluorescence activated cell sorting. A set of mutants, all having arginine substitutions at two common sites (positions 402 or 401 and position 418), were isolated and characterized. Mutation at either site alone did not generate as strong a mutant phenotype (loss of DiI uptake from DiI-HDL) as did the double mutations. "Activity-dissected" double mutants were as effective as wild-type mSR-BI in functioning as LDL receptors, mediating high affinity LDL binding and uptake of metabolically active cholesterol from LDL, but they lost most of their corresponding HDL receptor activity. Thus, these mutants provide support for the proposal that the interaction of SR-BI with HDL differs from that with LDL. Examination of the in vivo function of such mutants may provide insights into the differential roles of the LDL and HDL receptor activities of SR-BI in normal lipoprotein metabolism and in SR-BI's ability to protect against atherosclerosis. PMID- 10734046 TI - Cloning and characterization of a functional peroxisome proliferator activator receptor-gamma-responsive element in the promoter of the CAP gene. AB - c-Cbl-associating protein (CAP) is a multifunctional signaling protein that interacts with c-Cbl, facilitating the tyrosine phosphorylation of c-Cbl in response to insulin. In 3T3-L1 adipocytes and diabetic rodents, CAP gene expression is stimulated by activators of peroxisome proliferator activator receptor gamma (PPARgamma), such as thiazolidinediones (TZDs), resulting in increased insulin-stimulated c-Cbl phosphorylation. Sequence analysis of 2.5 kilobases of the 5'-flanking region of the CAP gene reveals a predicted peroxisome proliferator response element (PPRE) from -1085 to -1097. The isolated promoter was functional in 3T3 fibroblasts and adipocytes. Co-transfection of the CAP promoter with PPARgamma and retinoic acid X receptor alpha caused fold stimulation of promoter activity. The TZD rosiglitazone produced an additional 2 3-fold stimulation of the promoter. Deletion of the predicted PPRE from the CAP promoter abolished its ability to respond to rosiglitazone. Gel shift analysis of the putative PPARgamma site demonstrates direct binding of PPAR/retinoid X receptor heterodimers to the PPRE in the CAP gene. These data demonstrate that TZDs directly stimulate transcription of the CAP gene through activation of PPARgamma. PMID- 10734047 TI - Vesicular Ca(2+) participates in the catalysis of exocytosis. AB - Effects of vesicular monoamine transporter inhibitors on catecholamine release from bovine chromaffin cells have been examined at the level of individual exocytotic events. As expected for a depletion of vesicular stores, release evoked by depolarizing agents was decreased following 15-min incubations with reserpine and tetrabenazine, as evidenced by a decrease in exocytotic frequency and amount released per event. In contrast, two reserpine derivatives, methyl reserpate and reserpic acid, were much less effective. Surprisingly, the incubations also decreased the accompanying rise in intracellular Ca(2+) evoked by depolarizing agents. Subcellular studies revealed that reserpine and tetrabenazine at concentrations near their K(i) values not only could increase cytoplasmic catecholamines but also could displace Ca(2+) from vesicles. Furthermore, transient exposure to tetrabenazine and reserpine, but not methyl reserpate and reserpic acid, induced exocytotic release of catecholamines. Reserpine induced a rise in intracellular Ca(2+), as detected by whole-cell measurements with Fura-2. It could induce exocytosis, albeit at a lower frequency, in Ca(2+)-free solutions, supporting an internal Ca(2+) source. Depletion of endoplasmic reticulum and mitochondrial Ca(2+) pools did not eliminate the reserpine-activated release. These results indicate that vesicular Ca(2+) can play an important role in exocytosis and under some conditions may be involved in initiating this process. PMID- 10734049 TI - Dominant negative role of the glutamic acid residue conserved in the pyruvate kinase M(1) isozyme in the heterotropic allosteric effect involving fructose-1,6 bisphosphate. AB - Pyruvate kinase M(1), a nonallosteric isozyme, lacks heterotropic allosteric effect involving fructose-1,6-bisphosphate (FBP). To explore the molecular basis for this, a series of mutants were prepared and characterized, in which the possible candidate, Glu-432, was replaced in the rat M(1) isozyme and its allosteric mutant with the replacement of Ala-398 by Arg. Although these single mutants of Glu-432 remained nearly fully active, similar to the wild type, only the mutants with replacements by Lys and Ala were more efficiently activated by FBP when the enzymes were inhibited by L-phenylalanine. Kinetic analyses and ligand-induced fluorescence quenching studies using the allosteric double mutants indicated that the loss of a negative charge at residue 432 led to a dramatic decrease in the apparent activation constant and apparent K(d) for FBP. Furthermore, this enhancement was found to be associated with the modification of the FBP-binding site rather than the alteration of the subunit assembly. These findings suggest that Glu-432 hinders the heterotropic allosteric effect by preventing the binding of FBP through a repulsive electrostatic interaction and thereby contributes to its unique unregulated properties, independent of the shifted allosteric transition. PMID- 10734048 TI - Involvement of nuclear factor of activated T cells activation in UV response. Evidence from cell culture and transgenic mice. AB - Mammalian cells respond to UV radiation by signaling cascades leading to activation of transcription factors, such as activated protein 1, NFkappaB, and p53, a process known as the "UV response." Nuclear factor of activated T cells (NFAT) was first identified as an inducible nuclear factor in immune response and subsequently found to be expressed in other tissues and cells. To date, however, the regulation and function of NFAT in tissues and cells, other than the immune system, are not well understood. In this study, we demonstrate that UV radiation activates NFAT-dependent transcription through a calcium-dependent mechanism in mouse epidermal JB6 cell lines, as well as in the skin of NFAT-luciferase reporter transgenic mice. Exposure of JB6 cells to UV radiation leads to the transactivation of NFAT in a dose-dependent manner. A23187 had a synergistic effect with UV for NFAT induction, whereas pretreatment of cells with nifedipine, a calcium channel blocker, dramatically impaired the NFAT activity induced by either UV or UV plus A23187. Calcium-dependent activation of NFAT by UV was further confirmed by an in vivo study using NFAT-luciferase reporter transgenic mice. These results demonstrated that UV radiation is a strong activator for skin NFAT transactivation through calcium-dependent pathways, suggesting that NFAT activation may be a part of the UV response. PMID- 10734050 TI - Proline- and alanine-rich Ste20-related kinase associates with F-actin and translocates from the cytosol to cytoskeleton upon cellular stresses. AB - Proline- and alanine-rich Ste20-related kinase (PASK) is a Ste20-related protein kinase isolated from rat brain. Cell fractionation studies showed that PASK was present both in the cytosol and in Triton X-100-insoluble cytoskeletal fraction in rat tissues. In brain, PASK associated with protein complexes that contained actin and tubulin, confirming the association of PASK with the cytoskeleton in vivo. Glutathione S-transferase-PASK fusion protein cosedimented with F-actin, indicating that PASK binds to F-actin. In contrast to rat tissues, PASK was detected only in the Triton X-100-soluble cytosolic fraction in cultured PC12 and NIH 3T3 cells. Cytosolic PASK translocated to the cytoskeleton when these cells were stimulated with severe cellular stresses such as hypertonic sodium chloride, hydrogen peroxide, and heat shock at 45 degrees C. Our results suggest that PASK may be involved in the regulation of the cytoskeleton in response to cellular stresses such as hyperosmotic shock. PMID- 10734051 TI - The binding of oxidized low density lipoprotein to mouse CD36 is mediated in part by oxidized phospholipids that are associated with both the lipid and protein moieties of the lipoprotein. AB - There is growing evidence that CD36 has an important physiological function in the uptake of oxidized low density lipoprotein (OxLDL) by macrophages. However, the ligand specificity and the nature of the ligands on OxLDL that mediate the binding to CD36 remain ill defined. Results from recent studies suggested that some of the macrophage scavenger receptors involved in the uptake of OxLDL recognized both the lipid and the protein moieties of OxLDL, but there was no conclusive direct evidence for this. The present studies were undertaken to test whether a single, well characterized OxLDL receptor, CD36, could bind both the lipid and protein moieties of OxLDL. COS-7 cells transiently transfected with mouse CD36 cDNA bound intact OxLDL with high affinity. This binding was very effectively inhibited ( approximately 50%) both by the reconstituted apoB from OxLDL and by microemulsions prepared from OxLDL lipids. The specific binding of both moieties to CD36 was further confirmed by direct ligand binding analysis and by demonstrating reciprocal inhibition, i.e. apoB from OxLDL inhibited the binding of the OxLDL lipids and vice versa. Furthermore, a monoclonal mouse antibody that recognizes oxidation-specific epitopes in OxLDL inhibited the binding of intact OxLDL and also that of its purified protein and lipid moieties to CD36. This antibody recognizes the phospholipid 1-palmitoyl 2-(5'-oxovaleroyl) phosphatidylcholine. This model of an oxidized phospholipid was also an effective competitor for the CD36 binding of both the resolubilized apoB and the lipid microemulsions from OxLDL. Our results demonstrate that oxidized phospholipids in the lipid phase or covalently attached to apoB serve as ligands for recognition by CD36 and, at least in part, mediate the high affinity binding of OxLDL to macrophages. PMID- 10734052 TI - Deoxyhypusine synthase generates and uses bound NADH in a transient hydride transfer mechanism. AB - Deoxyhypusine is a modified lysine residue. It is formed posttranslationally in the precursor of eukaryotic initiation factor 5A (eIF5A) by deoxyhypusine synthase, employing spermidine as a butylamine donor. In the initial step of this reaction, deoxyhypusine synthase catalyzes the production of NADH through dehydrogenation of spermidine. Fluorescence measurements of this reaction revealed a -22-nm blue shift in the emission peak of NADH and a approximately 15 fold increase in peak intensity, characteristics of tightly bound NADH that were not seen by simply mixing NADH and enzyme. The fluorescent properties of the bound NADH can be ascribed to a hydrophobic environment and a rigidly held, open conformation of NADH, features in accord with the known crystal structure of the enzyme. Considerable fluorescence resonance energy transfer from tryptophan 327 in the active site to the dihydronicotinamide ring of NADH was seen. Upon addition of the eIF5A precursor, utilization of the enzyme-bound NADH for reduction of the eIF5A-imine intermediate to deoxyhypusine was reflected by a rapid decrease in the NADH fluorescence, indicating a transient hydride transfer mechanism as an integral part of the reaction. The number of NADH molecules bound approached four/enzyme tetramer; not all of the bound NADH was available for reduction of the eIF5A-imine intermediate. PMID- 10734053 TI - Syndecan-2 is involved in the mitogenic activity and signaling of granulocyte macrophage colony-stimulating factor in osteoblasts. AB - We previously showed that granulocyte-macrophage colony-stimulating factor (GM CSF) binds to heparan sulfate proteoglycans expressed at the surface of osteoblastic cells and that the mitogenic activity of this cytokine is dependent on the presence of fully sulfated proteoglycans. In this study, we determined if GM-CSF interacts with syndecans, a family of cell surface heparan sulfate proteoglycans. Human primary osteoblasts were found to express syndecan-2 and -4 but few syndecan-1 transcripts and proteins. Recombinant human GM-CSF coupled to biotin was found to bind to syndecan-2. Immunocytochemical transmission electron microscope analysis showed co-localization of syndecan-2 and GM-CSF at the cell membrane surface. Syndecan-2 also co-localized at the cell surface and co immunoprecipitated with the GM-CSF receptor alpha chain, suggesting a strong interaction between the cytokine, its receptor, and syndecan-2. Phosphorylation of tyrosine residues in syndecan-2 associated with the alpha chain of the GM-CSF receptor was increased after cell stimulation by GM-CSF. Antisense oligonucleotides that reduced specifically the expression of syndecan-2 inhibited the mitogenic activity of GM-CSF and the activation of extracellular signal regulated kinase-1 induced by the cytokine. Our results indicate functional interactions between syndecan-2 and GM-CSF in osteoblasts, and we propose that syndecan-2 plays a role as a co-receptor for this cytokine. PMID- 10734054 TI - Regulation by neurotransmitter receptors of serotonergic or catecholaminergic neuronal cell differentiation. AB - The murine F9-derived 1C11 clone exhibits a stable epithelial morphology, expresses nestin, an early neuroectodermal marker, and expresses genes involved in neuroectodermal cell fate. Upon appropriate induction, 100% of 1C11 precursor cells develop neurite extensions and acquire neuronal markers (N-CAM, synaptophysin, gammagamma-enolase, and neurofilament) as well as the general functions of either serotonergic (1C11*(/5HT)) (5HT, 5-hydroxytryptamine) or noradrenergic (1C11**(/NE)) (NE, norepinephrine) neurons. The two programs are shown to be mutually exclusive. 1C11 thus behaves as a neuroepithelial cell line with a dual bioaminergic fate. 1C11*(/5HT) cells implement a functional 5-HT transporter and thereby a complete serotonergic phenotype within 4 days, whereas 5-HT(1B/D), 5-HT(2B), and 5-HT(2A) receptors are sequentially induced. The accurate time schedule of catecholaminergic differentiation was defined. Catecholamine synthesis, storage, and catabolism are acquired within 4 days; the noradrenergic phenotype is complete at day 12 and includes a functional norepinephrine transporter and an alpha(1D)-adrenoreceptor (day 8). The time dependent onset of neurotransmitter-associated functions proper to either program is similar to in vivo observations. Along each pathway, the selective induction of serotonergic or adrenergic receptors is shown to be an essential part of the differentiation program, since they promote an autoregulation of the corresponding phenotype. PMID- 10734055 TI - Gonadotropin-releasing hormone receptor initiates multiple signaling pathways by exclusively coupling to G(q/11) proteins. AB - The agonist-bound gonadotropin-releasing hormone (GnRH) receptor engages several distinct signaling cascades, and it has recently been proposed that coupling of a single type of receptor to multiple G proteins (G(q), G(s), and G(i)) is responsible for this behavior. GnRH-dependent signaling was studied in gonadotropic alphaT3-1 cells endogenously expressing the murine receptor and in CHO-K1 (CHO#3) and COS-7 cells transfected with the human GnRH receptor cDNA. In all cell systems studied, GnRH-induced phospholipase C activation and Ca(2+) mobilization was pertussis toxin-insensitive, as was GnRH-mediated extracellular signal-regulated kinase activation. Whereas the G(i)-coupled m2 muscarinic receptor interacted with a chimeric G(s) protein (G(s)i5) containing the C terminal five amino acids of Galpha(i2), the human GnRH receptor was unable to activate the G protein chimera. GnRH challenge of alphaT3-1, CHO#3 and of GnRH receptor-expressing COS-7 cells did not result in agonist-dependent cAMP formation. GnRH challenge of CHO#3 cells expressing a cAMP-responsive element driven firefly luciferase did not result in increased reporter gene expression. However, coexpression of the human GnRH receptor and adenylyl cyclase I in COS-7 cells led to clearly discernible GnRH-dependent cAMP formation subsequent to GnRH elicited rises in [Ca(2+)](i). In alphaT3-1 and CHO#3 cell membranes, addition of [alpha-(32)P]GTP azidoanilide resulted in GnRH receptor-dependent labeling of Galpha(q/11) but not of Galpha(i), Galpha(s) or Galpha(12/13) proteins. Thus, the murine and human GnRH receptors exclusively couple to G proteins of the G(q/11) family. Multiple GnRH-dependent signaling pathways are therefore initiated downstream of the receptor/G protein interface and are not indicative of a multiple G protein coupling potential of the GnRH receptor. PMID- 10734056 TI - Regulation of the human chemokine receptor CCR1. Cross-regulation by CXCR1 and CXCR2. AB - To investigate the regulation of the CCR1 chemokine receptor, a rat basophilic leukemia (RBL-2H3) cell line was modified to stably express epitope-tagged receptor. These cells responded to RANTES (regulated upon activation normal T expressed and secreted), macrophage inflammatory protein-1alpha, and monocyte chemotactic protein-2 to mediate phospholipase C activation, intracellular Ca(2+) mobilization and exocytosis. Upon activation, CCR1 underwent phosphorylation and desensitization as measured by diminished GTPase stimulation and Ca(2+) mobilization. Alanine substitution of specific serine and threonine residues (S2 and S3) or truncation of the cytoplasmic tail (DeltaCCR1) of CCR1 abolished receptor phosphorylation and desensitization of G protein activation but did not abolish desensitization of Ca(2+) mobilization. S2, S3, and DeltaCCR1 were also resistant to internalization, mediated greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization, and were only partially desensitized by RANTES, relative to S1 and CCR1. To study CCR1 cross-regulation, RBL cells co expressing CCR1 and receptors for interleukin-8 (CXCR1, CXCR2, or a phosphorylation-deficient mutant of CXCR2, 331T) were produced. Interleukin-8 stimulation of CXCR1 or CXCR2 cross-phosphorylated CCR1 and cross-desensitized its ability to stimulate GTPase activity and Ca(2+) mobilization. Interestingly, CCR1 cross-phosphorylated and cross-desensitized CXCR2, but not CXCR1. Ca(2+) mobilization by S3 and DeltaCCR1 were also cross-desensitized by CXCR1 and CXCR2 despite lack of receptor phosphorylation. In contrast to wild type CCR1, S3 and DeltaCCR1, which produced sustained signals, cross-phosphorylated and cross desensitized responses to CXCR1 as well as CXCR2. Taken together, these results indicate that CCR1-mediated responses are regulated at several steps in the signaling pathway, by receptor phosphorylation at the level of receptor/G protein coupling and by an unknown mechanism at the level of phospholipase C activation. Moreover selective cross-regulation among chemokine receptors is, in part, a consequence of the strength of signaling (i.e. greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization) which is inversely correlated with the receptor's susceptibility to phosphorylation. Since many chemokines activate multiple chemokine receptors, selective cross-regulation among such receptors may play a role in their immunomodulation. PMID- 10734057 TI - Protein phosphatase 2A inhibitors, I(1)(PP2A) and I(2)(PP2A), associate with and modify the substrate specificity of protein phosphatase 1. AB - Recombinant I(1)(PP2A) and I(2)(PP2A) did not affect the activity of the catalytic subunit of protein phosphatase 1 (PP1(C)) with (32)P-labeled myelin basic protein, histone H1, and phosphorylase when assayed in the absence of divalent cations. However, in the presence of Mn(2+), I(1)(PP2A) and I(2)(PP2A) stimulated PP1(C) activity by 15-20-fold with myelin basic protein and histone H1 but not phosphorylase. Half-maximal stimulation occurred at 2 and 4 nM I(1)(PP2A) and I(2)(PP2A), respectively. Moreover, I(1)(PP2A) and I(2)(PP2A) reduced the Mn(2+) requirement by about 30-fold to 10 microM. In contrast, PP1(C) activity was unaffected by I(1)(PP2A) and I(2)(PP2A) in the presence of Co(3+) (0.1 mM), Mg(2+) (2 mM), Ca(2+) (0.5 mM), and Zn(2+) (0.1 mM). Following gel filtration chromatography on Sephacryl S-200 in the presence of Mn(2+), PP1(C) coeluted with I(1)(PP2A) and I(2)(PP2A) in the void volume. However, when I(1)(PP2A) and I(2)(PP2A) or Mn(2+) were omitted, PP1(C) emerged with a V(e)/V(0) of approximately 1.6. The results demonstrate that I(1)(PP2A) and I(2)(PP2A) associate with and modify the substrate specificity of PP1(C) in the presence of physiological concentrations of Mn(2+). A novel role is suggested for I(1)(PP2A) and I(2)(PP2A) in the reciprocal regulation of two major mammalian serine/threonine phosphatases, PP1 and PP2A. PMID- 10734058 TI - Evolutionary conservation of C-terminal domains of primary sigma(70)-type transcription factors between plants and bacteria. AB - Three different cDNAs coding for putative plant plastid sigma(70)-type transcription initiation factors have recently been cloned and sequenced from Arabidopsis thaliana. We have analyzed the evolutionary conservation of function(s) of the N-terminal and C-terminal halves of these three sigma factors by in vitro transcription studies using heterologous transcription systems and by complementation assays using Escherichia coli thermosensitive rpoD mutants. Our results indicate differences and similarities of the three plant factors and their prokaryotic ancestors. The functions of the N-terminal parts of the plant sigma factors are considerably different from the function of the N-terminal part of the principal sigma(70) factor of E. coli. On the other hand, the C-terminal parts have kept at least two characteristics when compared with their prokaryotic ancestors: 1) they can distinguish between different promoter structures, and 2) one of them is capable of fully complementing E. coli rpoD mutants, i.e. recognizing all essential E. coli promoters that are used by the E. coli principal sigma(70) factor. This shows for the first time in vivo a strong evolutionary conservation of cis- and trans-acting elements between the prokaryotic and the plant plastid transcriptional machinery. PMID- 10734059 TI - Tumor necrosis factor-alpha and interleukin-1beta inhibit apolipoprotein B secretion in CaCo-2 cells via the epidermal growth factor receptor signaling pathway. AB - In inflammatory conditions of the gut, cytokines are released into the mucosa and submucosa propagating and sustaining the inflammatory response. In CaCo-2 cells, we have shown that various inflammatory cytokines interfere with the secretion of lipids, an effect that is likely caused by the release of a ligand to the epidermal growth factor (EGF) receptor. In the present study, the role of the EGF receptor signaling pathway and the effects of the cytokines tumor necrosis factor alpha (TNF-alpha) and and interleukin 1beta (IL-1beta) on triacylglycerol-rich lipoprotein secretion were investigated. CaCo-2 cells were incubated with oleic acid to enhance triacylglycerol-rich lipoprotein secretion. TNF-alpha and IL 1beta significantly decreased the basolateral secretion of apolipoprotein B (apoB) mass, with IL-1beta being more potent. Tyrphostin, an inhibitor of the EGF receptor intrinsic tryosine kinase, prevented or markedly attenuated the decrease in apoB secretion by TNF-alpha or IL-1beta. Both cytokines increased the phosphorylation of the EGF receptor by 30 min. Moreover, phosphotyrosine immunoblots of the EGF receptor demonstrated an increase in tyrosine residues phosphorylated by 0.5 and 6.5 h. At both these time points, TNF-alpha and IL 1beta also decreased the binding of EGF to its cell surface receptor. At 6.5 h, activation of the EGF receptor was sustained. In contrast, the early activation of the receptor was only transient as receptor phosphorylation and binding of EGF to its receptor returned to basal levels by 2 h. Preventing ligand binding to the EGF receptor by a receptor-blocking antibody attenuated receptor activation observed after 6.5 h. This did not occur at 0.5 h, suggesting that early activation of the EGF receptor was non-ligand-mediated. Similarly, apoB secretion was inhibited by an early non-ligand-mediated process; whereas at the later time, inhibition of apoB secretion was ligand-mediated. Thus, the inflammatory cytokines TNF-alpha and IL-1beta interfere with the secretion of triacylglycerol rich lipoproteins by both early and delayed signaling events mediated by the EGF receptor signaling pathway. PMID- 10734060 TI - Direct extracellular contact between integrin alpha(3)beta(1) and TM4SF protein CD151. AB - Previously we established that the alpha(3)beta(1) integrin shows stable, specific, and stoichiometric association with the TM4SF (tetraspannin) protein CD151. Here we used a membrane impermeable cross-linking agent to show a direct association between extracellular domains of alpha(3)beta(1) and CD151. The alpha(3)beta(1)-CD151 association site was then mapped using chimeric alpha(6)/alpha(3) integrins and CD151/NAG2 TM4SF proteins. Complex formation required an extracellular alpha(3) site (amino acids (aa) 570-705) not previously known to be involved in specific integrin contacts with other proteins and a region (aa 186-217) within the large extracellular loop of CD151. Notably, the anti-CD151 monoclonal antibody TS151r binding epitope, previously implicated in alpha(3) integrin association, was mapped to the same region of CD151 (aa 186 217). Finally, we demonstrated that both NH(2)- and COOH-terminal domains of CD151 are located on the inside of the plasma membrane, thus confirming a long suspected model of TM4SF protein topology. PMID- 10734061 TI - Three new familial hemiplegic migraine mutants affect P/Q-type Ca(2+) channel kinetics. AB - Missense mutations in the pore-forming human alpha(1A) subunit of neuronal P/Q type Ca(2+) channels are associated with familial hemiplegic migraine. We studied the functional consequences on P/Q-type Ca(2+) channel function of three recently identified mutations, R583Q, D715E, and V1457L after introduction into rabbit alpha(1A) and expression in Xenopus laevis oocytes. The potential for half maximal channel activation of Ba(2+) inward currents was shifted by > 9 mV to more negative potentials in all three mutants. The potential for half-maximal channel inactivation was shifted by > 7 mV in the same direction in R583Q and D715E. Biexponential current inactivation during 3-s test pulses was significantly faster in D715E and slower in V1457L than in wild type. Mutations R583Q and V1457L delayed the time course of recovery from channel inactivation. The decrease of peak current through R583Q (30.2%) and D715E (30. 1%) but not V1457L (18.7%) was more pronounced during 1-Hz trains of 15 100-ms pulses than in wild type (18.2%). Our data demonstrate that the mutations R583Q, D715E, and V1457L, like the previously reported mutations T666M, V714A, and I1819L, affect P/Q-type Ca(2+) channel gating. We therefore propose that altered channel gating represents a common pathophysiological mechanism in familial hemiplegic migraine. PMID- 10734062 TI - Caspase-8-mediated intracellular acidification precedes mitochondrial dysfunction in somatostatin-induced apoptosis. AB - Activation of initiator and effector caspases, mitochondrial changes involving a reduction in its membrane potential and release of cytochrome c (cyt c) into the cytosol, are characteristic features of apoptosis. These changes are associated with cell acidification in some models of apoptosis. The hierarchical relationship between these events has, however, not been deciphered. We have shown that somatostatin (SST), acting via the Src homology 2 bearing tyrosine phosphatase SHP-1, exerts cytotoxic action in MCF-7 cells, and triggers cell acidification and apoptosis. We investigated the temporal sequence of apoptotic events linking caspase activation, acidification, and mitochondrial dysfunction in this system and report here that (i) SHP-1-mediated caspase-8 activation is required for SST-induced decrease in pH(i). (ii) Effector caspases are induced only when there is concomitant acidification. (iii) Decrease in pH(i) is necessary to induce reduction in mitochondrial membrane potential, cyt c release and caspase-9 activation and (iv) depletion of ATP ablates SST-induced cyt c release and caspase-9 activation, but not its ability to induce effector caspases and apoptosis. These data reveal that SHP-1-/caspase-8-mediated acidification occurs at a site other than the mitochondrion and that SST-induced apoptosis is not dependent on disruption of mitochondrial function and caspase-9 activation. PMID- 10734063 TI - Mechanism by which phosphonoformic acid resistance mutations restore 3'-azido-3' deoxythymidine (AZT) sensitivity to AZT-resistant HIV-1 reverse transcriptase. AB - The development of phosphonoformic acid (PFA) resistance against a background of 3'-azido-3'-deoxythymidine (AZT) resistance in human immunodeficiency virus type 1 (HIV-1) restores viral sensitivity to AZT. High level AZT resistance requires multiple mutations (D67N/K70R/T215F/K219Q). In order to characterize the mechanism of PFA resistance-mediated resensitization to AZT, the A114S mutation associated with PFA resistance was introduced into the reverse transcriptase (RT) of both wild type and drug-resistant virus. We previously showed that pyrophosphorolytic removal of chain-terminating AZT is the primary mechanism of the AZT resistance phenotype (Arion, D., Kaushik, N., McCormick, S., Borkow, G., and Parniak, M. A. (1998) Biochemistry 37, 15908-15917). Introduction of A114S into the AZT resistance background significantly diminishes both the enhanced pyrophosphorolytic activity and the DNA synthesis processivity associated with the AZT-resistant RT. The A114S mutation also alters the nucleotide-dependent phosphorolysis activity associated with AZT resistance. The presence of the A114S mutation therefore severely impairs the mutant enzyme's ability to excise chain terminating AZT. The decrease in phosphorolytic activity of RT conferred by the PFA resistance A114S mutation resensitizes AZT-resistant HIV-1 to AZT by allowing the latter to again function as a chain terminator of viral DNA synthesis. These data further underscore the importance of phosphorolytic removal of chain terminating AZT as the primary mechanism of HIV-1 AZT resistance. PMID- 10734064 TI - Structural defects underlying protein dysfunction in human glucose-6-phosphate dehydrogenase A(-) deficiency. AB - The enzyme variant glucose-6-phosphate dehydrogenase (G6PD) A(-), which gives rise to human glucose-6-phosphate dehydrogenase deficiency, is a protein of markedly reduced structural stability. This variant differs from the normal enzyme, G6PD B, in two amino acid substitutions. A further nondeficient variant, G6PD A, bears only one of these two mutations and is structurally stable. In this study, the synergistic structural defect in recombinant G6PD A(-) was reflected by reduced unfolding enthalpy due to loss of beta-sheet and alpha-helix interactions where both mutations are found. This was accompanied by changes in inner spatial distances between residues in the coenzyme domain and the partial disruption of tertiary structure with no significant loss of secondary structure. However, the secondary structure of G6PD A(-) was qualitatively affected by an increase in beta-sheets substituting beta-turns related to the lower unfolding enthalpy. The structural changes observed did not affect the active site of the mutant proteins, since its spatial position was unmodified. The final result is a loss of folding determinants leading to a protein with decreased intracellular stability. This is suggested as the cause of the enzyme deficiency in the red blood cell, which is unable to perform de novo protein synthesis. PMID- 10734065 TI - Ribosome-mediated folding of partially unfolded ricin A-chain. AB - After endocytic uptake by mammalian cells, the cytotoxic protein ricin is transported to the endoplasmic reticulum, whereupon the A-chain must cross the lumenal membrane to reach its ribosomal substrates. It is assumed that membrane traversal is preceded by unfolding of ricin A-chain, followed by refolding in the cytosol to generate the native, biologically active toxin. Here we describe biochemical and biophysical analyses of the unfolding of ricin A-chain and its refolding in vitro. We show that native ricin A-chain is surprisingly unstable at pH 7.0, unfolding non-cooperatively above 37 degrees C to generate a partially unfolded state. This species has conformational properties typical of a molten globule, and cannot be refolded to the native state by manipulation of the buffer conditions or by the addition of a stem-loop dodecaribonucleotide or deproteinized Escherichia coli ribosomal RNA, both of which are substrates for ricin A-chain. By contrast, in the presence of salt-washed ribosomes, partially unfolded ricin A-chain regains full catalytic activity. The data suggest that the conformational stability of ricin A-chain is ideally poised for translocation from the endoplasmic reticulum. Within the cytosol, ricin A-chain molecules may then refold in the presence of ribosomes, resulting in ribosome depurination and cell death. PMID- 10734066 TI - Sur1 knockout mice. A model for K(ATP) channel-independent regulation of insulin secretion. AB - Sur1 knockout mouse beta-cells lack K(ATP) channels and show spontaneous Ca(2+) action potentials equivalent to those seen in patients with persistent hyperinsulinemic hypoglycemia of infancy, but the mice are normoglycemic unless stressed. Sur1(-/-) islets lack first phase insulin secretion and exhibit an attenuated glucose-stimulated second phase secretion. Loss of the first phase leads to mild glucose intolerance, whereas reduced insulin output is consistent with observed neonatal hyperglycemia. Loss of K(ATP) channels impairs the rate of return to a basal secretory level after a fall in glucose concentration. This leads to increased hypoglycemia upon fasting and contributes to a very early, transient neonatal hypoglycemia. Whereas persistent hyperinsulinemic hypoglycemia of infancy underscores the importance of the K(ATP)-dependent ionic pathway in control of insulin release, the Sur1(-/-) animals provide a novel model for study of K(ATP)-independent pathways that regulate insulin secretion. PMID- 10734067 TI - Damage-mediated phosphorylation of human p53 threonine 18 through a cascade mediated by a casein 1-like kinase. Effect on Mdm2 binding. AB - The p53 tumor suppressor protein is stabilized in response to ionizing radiation and accumulates in the nucleus. Stabilization is thought to involve disruption of the interaction between the p53 protein and Mdm2, which targets p53 for degradation. Here we show that the direct association between a p53 N-terminal peptide and Mdm2 is disrupted by phosphorylation of the peptide on Thr(18) but not by phosphorylation at other N-terminal sites, including Ser(15) and Ser(37). Thr(18) was phosphorylated in vitro by casein kinase (CK1); this process required the prior phosphorylation of Ser(15). Thr(18) was phosphorylated in vivo in response to DNA damage, and such phosphorylation required Ser(15). Our results suggest that stabilization of p53 after ionizing radiation may result, in part, from an inhibition of Mdm2 binding through a phosphorylation-phosphorylation cascade involving DNA damage-activated phosphorylation of p53 Ser(15) followed by phosphorylation of Thr(18). PMID- 10734068 TI - Arrestin binding to the M(2) muscarinic acetylcholine receptor is precluded by an inhibitory element in the third intracellular loop of the receptor. AB - Desensitization of G protein-coupled receptors (GPCRs) involves the binding of members of the family of arrestins to the receptors. In the model system involving the visual GPCR rhodopsin, activation and phosphorylation of rhodopsin is thought to convert arrestin from a low to high affinity binding state. Phosphorylation of the M(2) muscarinic acetylcholine receptor (mAChR) has been shown to be required for binding of arrestins 2 and 3 in vitro and for arrestin enhanced internalization in intact cells (Pals-Rylaarsdam, R., and Hosey, M. M. (1997) J. Biol. Chem. 272, 14152-14158). For the M(2) mAChR, arrestin binding requires phosphorylation at multiple serine and threonine residues at amino acids 307-311 in the third intracellular (i3) loop. Here, we have investigated the molecular basis for the requirement of receptor phosphorylation for arrestin binding. Constructs of arrestin 2 that can bind to other GPCRs in a phosphorylation-independent manner were unable to interact with a mutant M(2) mAChR in which the Ser/Thr residues at 307-311 were mutated to alanines. However, although phosphorylation-deficient mutants of the M(2) mAChR that lacked 50-157 amino acids from the i3 loop were unable to undergo agonist-dependent internalization when expressed alone in tsA201 cells, co-expression of arrestin 2 or 3 restored agonist-dependent internalization. Furthermore, a deletion of only 15 amino acids (amino acids 304-319) was sufficient to allow for phosphorylation independent arrestin-receptor interaction. These results indicate that phosphorylation at residues 307-311 does not appear to be required to activate arrestin into a high affinity binding state. Instead, phosphorylation at residues 307-311 appears to facilitate the removal of an inhibitory constraint that precludes receptor-arrestin association in the absence of receptor phosphorylation. PMID- 10734069 TI - Basolateral sorting signals differ in their ability to redirect apical proteins to the basolateral cell surface. AB - Polarized sorting of membrane proteins in epithelial cells is mediated by cytoplasmic basolateral signals or by apical signals in the transmembrane or exoplasmic domains. Basolateral signals were generally found to be dominant over apical determinants. We have generated chimeric proteins with the cytoplasmic domain of either the asialoglycoprotein receptor H1 or the transferrin receptor, two basolateral proteins, fused to the transmembrane and exoplasmic segments of aminopeptidase N, an apical protein, and analyzed them in Madin-Darby canine kidney cells. Whereas both cytoplasmic sequences induced endocytosis of the chimeras, only that of the transferrin receptor mediated basolateral expression in steady state. The H1 fusion protein, although still largely sorted to the basolateral side in biosynthetic surface transport, was subsequently resorted to the apical cell surface. We tested whether the difference in sorting between trimeric wild-type H1 and the dimeric aminopeptidase chimera was caused by the number of sorting signals presented in the oligomers. Consistent with this hypothesis, the H1 signal was fully functional in a tetrameric fusion protein with the transmembrane and exoplasmic domains of influenza neuraminidase. The results suggest that basolateral signals per se need not be dominant over apical determinants for steady-state polarity and emphasize an important contribution of the valence of signals in polarized sorting. PMID- 10734070 TI - The nonbilayer/bilayer lipid balance in membranes. Regulatory enzyme in Acholeplasma laidlawii is stimulated by metabolic phosphates, activator phospholipids, and double-stranded DNA. AB - In membranes of Acholeplasma laidlawii a single glucosyltransferase step between the major, nonbilayer-prone monoglucosyl-diacylglycerol (MGlcDAG) and the bilayer forming diglucosyl-diacylglycerol (DGlcDAG) is important for maintenance of lipid phase equilibria and curvature packing stress. This DGlcDAG synthase is activated in a cooperative fashion by phosphatidylglycerol (PG), but in vivo PG amounts are not enough for efficient DGlcDAG synthesis. In vitro, phospholipids with an sn glycero-3-phosphate backbone, and no positive head group charge, functioned as activators. Different metabolic, soluble phosphates could supplement PG for activation, depending on type, amount, and valency. Especially efficient were the glycolytic intermediates fructose 1,6-bisphosphate and ATP, active at cellular concentrations on the DGlcDAG but not on the preceding MGlcDAG synthase. Potencies of different phosphatidylinositol (foreign lipid) derivatives differed with numbers and positions of their phosphate moieties. A selective stimulation of the DGlcDAG, but not the MGlcDAG synthase, by minor amounts of double-stranded DNA was additive to the best phospholipid activators. These results support two types of activator sites on the enzyme: (i) lipid-phosphate ones close to the membrane interphase, and (ii) soluble (or particulate)-phosphate ones further out from the surface. Thereby, the nonbilayer (MGlcDAG) to bilayer (DGlcDAG) lipid balance may be integrated with the metabolic status of the cell and potentially also to membrane and cell division. PMID- 10734071 TI - Caspase-8 activation and bid cleavage contribute to MCF7 cellular execution in a caspase-3-dependent manner during staurosporine-mediated apoptosis. AB - There are at least two distinct classes of caspases, initiators (e.g. caspases-8, -9, and -10) and effectors (e.g. caspase-3). Furthermore, it is believed that there are two distinct primary apoptotic signaling pathways, one of which is mediated by death receptors controlled by caspases-8/10, and the other by the release of cytochrome c and activation of a caspase-9/Apaf1/cytochrome c apoptosome. However, several recent reports have demonstrated that caspase-8, and its substrate Bid, are frequently activated in response to certain apoptotic stimuli in a death receptor-independent manner. These results suggest that significant cross-talk may exist between these two distinct signaling arms, allowing each to take advantage of elements unique to the other. Here we provide evidence that activation of caspase-8, and subsequent Bid cleavage, does indeed participate in cytochrome c-mediated apoptosis, at least in certain circumstances and cell types. Furthermore, the participation of activated caspase-3 is essential for activation of caspase-8 and Bid processing to occur. Although caspase-8 activation is not required for the execution of a cytochrome c-mediated death signal, we found that it greatly shortens the execution time. Thus, caspase 8 involvement in cytochrome c-mediated cell death may help to amplify weaker death signals and ensure that apoptosis occurs within a certain time frame. PMID- 10734073 TI - Identification of caspase 3-mediated cleavage and functional alteration of eukaryotic initiation factor 2alpha in apoptosis. AB - Induction of apoptosis in a variety of cell types leads to inhibition of protein synthesis. Recently, the cleavage of eukaryotic translation initiation factor 4G (eIF4G) by caspase 3 was described as a possible event contributing to translation inhibition. Here, we report the cleavage of another initiation factor in apoptotic cells, eIF2alpha, that could contribute to regulation of translation during apoptosis. This cleavage event could be completely inhibited by pretreatment of HeLa cells with Z-VAD-fmk. In vitro analysis using purified eIF2 and purified caspases showed cleavage of eIF2alpha by caspase 3, 6, 8, and 10 but not 9. Caspase 3 most efficiently cleaved eIF2alpha and this could be inhibited by addition of Ac-DEVD-CHO in vitro. Comparison of cleavage of phosphorylated versus nonphosphorylated eIF2alpha revealed a modest preference of the caspases for the nonphosphorylated form. When eIF2. 2B complex was used as substrate, only caspase 3 was capable of eIF2alpha cleavage, which was not affected by phosphorylation of the alpha subunit. The eIF2.GDP binary complex was cleaved much less efficiently by caspase 3. Sequence analysis of the cleavage fragment suggested that the cleavage site is located in the C-terminal portion of the protein. Analysis showed that after caspase cleavage, exchange of GDP bound to eIF2 was very rapid and no longer dependent upon eIF2B. Furthermore, in vitro translation experiments indicated that cleavage of eIF2alpha results in functional alteration of the eIF2 complex, which no longer stimulated upstream AUG selection on a mRNA containing a viral internal ribosome entry site and was no longer capable of stimulating overall translation. In conclusion, we describe here the cleavage of a translation initiation factor, eIF2alpha that could contribute to inhibition or alteration of protein synthesis during the late stages of apoptosis. PMID- 10734072 TI - From androgen receptor to the general transcription factor TFIIH. Identification of cdk activating kinase (CAK) as an androgen receptor NH(2)-terminal associated coactivator. AB - The androgen receptor (AR), like other steroid receptors, modulates the activity of the general transcription machinery on the core promoter to exert its function as a regulator. Co-immunoprecipitation of prostate cancer LNCaP cell extract using protein A-Sepharose coupled with anti-AR antibody indicates that the AR interacts with the general transcription factor TFIIH in a physiological condition. Co-transfection of cdk activating kinase (CAK), the kinase moiety of TFIIH, enhanced AR-mediated transcription in a ligand-dependent manner in human prostate cancer PC-3 and LNCaP cells, and in a ligand-independent manner in human prostate cancer DU145 cells. Detailed interaction studies further revealed that the AR NH(2)-terminal domain interacting with CAK was essential for the CAK induced AR transactivation. Together, our data suggest that the AR may interact with TFIIH for efficient communication with the general transcription factors/RNA polymerase II on the core promoter. PMID- 10734074 TI - PDZ-dependent activation of nitric-oxide synthases by the serotonin 2B receptor. AB - Taking advantage of three cellular systems, we established that 5-HT(2B) receptors are coupled with NO signaling pathways. In the 1C11 serotonergic cell line and Mastomys natalensis carcinoid cells, which naturally express the 5 HT(2B) receptor, as well as in transfected LMTK(-) fibroblasts, stimulation of the 5-HT(2B) receptor triggers intracellular cGMP production through dual activation of constitutive nitric-oxide synthase (cNOS) and inducible NOS (iNOS). The group I PDZ motif at the C terminus of the 5-HT(2B) receptor is required for recruitment of the cNOS and iNOS transduction pathways. Indeed, the 5-HT(2B) receptor-mediated NO coupling is abolished not only upon introduction of a competitor C-terminal 5-HT(2B) peptide in the three cell types but also in LMTK( ) fibroblasts expressing a receptor C-terminally truncated or harboring a point mutation within the PDZ domain. The occurrence of a direct functional coupling between the receptor and cNOS activity is supported by highly significant correlations between the binding constants of drugs on the receptor and their effects on cNOS activity. The 5-HT(2B)/iNOS coupling mechanisms appear more complex because neutralization of endogenous Galpha(13) by specific antibodies cancels the cellular iNOS response while not interfering with cNOS activities. These findings may shed light on physiological links between the 5-HT(2B) receptor and NO and constitute the first demonstration that PDZ interactions participate in downstream transductional pathways of a G protein-coupled receptor. PMID- 10734075 TI - Intracellular activation of rat hepatic lipase requires transport to the Golgi compartment and is associated with a decrease in sedimentation velocity. AB - Hepatic lipase (HL) is an N-glycoprotein that acquires triglyceridase activity somewhere during maturation and secretion. To determine where and how HL becomes activated, the effect of drugs that interfere with maturation and intracellular transport of HL protein was studied using freshly isolated rat hepatocytes. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), castanospermine, monensin, and colchicin all inhibited secretion of HL without affecting its specific enzyme activity. The specific enzyme activity of intracellular HL was decreased by 25 50% upon incubation with CCCP or castanospermine, and increased 2-fold with monensin and colchicin. Glucose trimming of HL protein was not affected by CCCP, as indicated by digestion of immunoprecipitates with jack bean alpha-mannosidase. Pulse labeling experiments with [(35)S]methionine indicated that conversion of the 53-kDa precursor to the 58-kDa form, nor the development of endoglycosidase H resistance, were essential for acquisition of enzyme activity. In sucrose gradients, HL protein from secretion media sedimented as a homogeneous band of about 5.8 S, whereas HL protein from the cell lysates migrated as a broad band extending from 5.8 S to more than 8 S. With both sources, HL activity was exclusively associated with the 5.8 S HL protein form. We conclude that glucose trimming of HL protein in the endoplasmic reticulum is not sufficient for activation; full activation occurs during or after transport from the endoplasmic reticulum to the Golgi and is associated with a decrease in sedimentation velocity. PMID- 10734076 TI - TASK-3, a new member of the tandem pore K(+) channel family. AB - We have isolated from the rat cerebellum cDNA library a complementary DNA encoding a new member of the tandem pore K(+) channel family. Its amino acid sequence shares 54% identity with that of TASK-1, but less than 30% with those of TASK-2 and other tandem pore K(+) channels (TWIK, TREK, TRAAK). Therefore, the new clone was named TASK-3. Reverse transcriptase-polymerase chain reaction analysis showed that TASK-3 mRNA is expressed in many rat tissues including brain, kidney, liver, lung, colon, stomach, spleen, testis, and skeletal muscle, and at very low levels in the heart and small intestine. When expressed in COS-7 cells, TASK-3 exhibited a time-independent, noninactivating K(+)-selective current. Single-channel conductance was 27 pS at -60 mV and 17 pS at 60 mV in symmetrical 140 mM KCl. TASK-3 current was highly sensitive to changes in extracellular pH (pH(o)), a hallmark of the TASK family of K(+) channels. Thus, a change in pH(o) from 7.2 to 6.4 and 6.0 decreased TASK-3 current by 74 and 96%, respectively. Mutation of histidine at position 98 to aspartate abolished pH(o) sensitivity. TASK-3 was blocked by barium (57%, 3 mM), quinidine (37%, 100 microM), and lidocaine (62%, 1 mM). Thus, TASK-3 is a new member of the acid sensing K(+) channel subfamily (TASK). PMID- 10734077 TI - The fungal CPCR1 protein, which binds specifically to beta-lactam biosynthesis genes, is related to human regulatory factor X transcription factors. AB - Here we report the isolation and characterization of a novel transcription factor from the cephalosporin C-producing fungus Acremonium chrysogenum. We have identified a protein binding site in the promoter of the beta-lactam biosynthesis gene pcbC, located 418 nucleotides upstream of the translational start. Using the yeast one-hybrid system, we succeeded in isolating a cDNA clone encoding a polypeptide, which binds specifically to the pcbC promoter. The polypeptid shows significant sequence homology to human transcription factors of the regulatory factor X (RFX) family and was designated CPCR1. A high degree of CPCR1 binding specificity was observed in in vivo and in vitro experiments using mutated versions of the DNA binding site. The A. chrysogenum RFX protein CPCR1 recognizes an imperfect palindrome, which resembles binding sites of human RFX transcription factors. One- and two-hybrid experiments with truncated versions of CPCR1 showed that the protein forms a DNA binding homodimer. Nondenaturing electrophoresis revealed that the CPCR1 protein exists in vitro solely in a multimeric, probably dimeric, state. Finally, we isolated a homologue of the cpcR1 gene from the penicillin-producing fungus Penicillium chrysogenum and determined about 60% identical amino acid residues in the DNA binding domain of both fungal RFX proteins, which show an overall amino acid sequence identity of 29%. PMID- 10734078 TI - Changes in the inactivation of rat Kv1.4 K(+) channels induced by varying the number of inactivation particles. AB - N-type inactivation of rat Kv1.4 channels with one, two, or four inactivation balls was investigated using homogeneous populations of channels expressed in Xenopus oocytes. Tandem dimeric and tetrameric constructs of Kv1.4 were made. Channels encoded by tandem cDNAs Kv1. 4-Kv1.4Delta1-145 and Kv1.4-[Kv1.4Delta1 145](3) have two or only one tethered inactivation ball, respectively, whereas Kv1.4 itself encodes channels having four inactivation balls. The time constants for inactivation of macroscopic currents were increased significantly as the number of inactivation balls was decreased, whereas the time constants for recovery from inactivation were not modified. The ratios of the rate constants of inactivation (k(inact)) of Kv1.4-Kv1.4Delta1-145 and Kv1.4-[Kv1.4Delta1-145](3) channels to that of the Kv1.4 channel were 0.65 and 0.4, respectively, whereas the ratios of the rate constant of recovery (k(rec)) of these channels to that of Kv1.4 were almost unity. The rate constants k(inact) for channels having two and four inactivation balls are smaller than those that would be expected if inactivation balls on each channel are independent, suggesting some interaction occurs between inactivation balls. Furthermore, noninactivating current became apparent as the number of inactivation balls on a channel was decreased. PMID- 10734079 TI - Acute stimulation with long chain acyl-CoA enhances exocytosis in insulin secreting cells (HIT T-15 and NMRI beta-cells). AB - Non-insulin-dependent diabetes mellitus is associated with, in addition to impaired insulin release, elevated levels of free fatty acids (FFA) in the blood. Insulin release is stimulated when beta-cells are acutely exposed to FFA, whereas chronic exposure may inhibit glucose-induced insulin secretion. In the present study we investigated the direct effects of long chain acyl-CoA (LC-CoA), the active intracellular form of FFA, on insulin exocytosis. Palmitoyl-CoA stimulated both insulin release from streptolysin-O-permeabilized HIT cells and fusion of secretory granules to the plasma membrane of mouse pancreatic beta-cells, as measured by cell capacitance. The LC-CoA effect was chain length-dependent, requiring chain lengths of at least 14 carbons. LC-CoA needed to be present to stimulate insulin release, and consequently there was no effect following its removal. The stimulatory effect was observed after inhibition of protein kinase activity and in the absence of ATP, even though both kinases and ATP, themselves, modulate exocytosis. The effect of LC-CoA was inhibited by cerulenin, which has been shown to block protein acylation. The data suggest that altered LC-CoA levels, resulting from FFA or glucose metabolism, may act directly on the exocytotic machinery to stimulate insulin release by a mechanism involving LC-CoA protein binding. PMID- 10734080 TI - Regulation of xanthine oxidase by nitric oxide and peroxynitrite. AB - Xanthine oxidase (XO) is a central mechanism of oxidative injury as occurs following ischemia. During the early period of reperfusion, both nitric oxide (NO(*)) and superoxide (O-*(2)) generation are increased leading to the formation of peroxynitrite (ONOO(-)); however, questions remain regarding the presence and nature of the interactions of NO(*) or ONOO(-) with XO and the role of this process in regulating oxidant generation. Therefore, we determined the dose dependent effects of NO(*) and ONOO(-) on the O-*(2) generation and enzyme activity of XO, respectively, by EPR spin trapping of O-*(2) using 5 (diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide and spectrophotometric assay. ONOO(-) markedly inhibited both O-*(2) generation and XO activity in dose dependent manner, while NO(*) from NO(*) gas in concentrations up to 200 microM had no effect. Furthermore, we observed that NO(*) donors such as NOR-1 also inhibited O-*(2) generation and XO activity; however, these effects were O-*(2) dependent and blocked by superoxide dismutase or ONOO(-) scavengers. Finally, we found that ONOO(-) totally abolished the Mo(V) EPR spectrum. These changes were irreversible, suggesting oxidative disruption of the critical molybdenum center of the catalytic site. Thus, ONOO(-) formed in biological systems can feedback and down-regulate XO activity and O-*(2) generation, which in turn may serve to limit further ONOO(-) formation. PMID- 10734081 TI - Zinc and cadmium can promote rapid nuclear translocation of metal response element-binding transcription factor-1. AB - Metal response element-binding transcription factor-1 (MTF-1) is a six-zinc finger protein that plays an essential role in activating metallothionein expression in response to the heavy metals zinc and cadmium. Low affinity interactions between zinc and specific zinc fingers in MTF-1 reversibly regulate its binding to the metal response elements in the mouse metallothionein-I promoter. This study examined the subcellular distribution and DNA binding activity of MTF-1 in cells treated with zinc or cadmium. Immunoblot analysis of cytosolic and nuclear extracts demonstrated that in untreated cells, about 83% of MTF-1 is found in the cytosolic extracts and is not activated to bind to DNA. In sharp contrast, within 30 min of zinc treatment (100 microM), MTF-1 is detected only in nuclear extracts and is activated to bind to DNA. The activation to bind to DNA and nuclear translocation of MTF-1 occurs in the absence of increased MTF 1 content in the cell. Furthermore, immunocytochemical localization and immunoblotting assays demonstrated that zinc induces the nuclear translocation of MTF-1-FLAG, expressed from the cytomegalovirus promoter in transiently transfected dko7 (MTF-1 double knockout) cells. Immunoblot analysis of cytosolic and nuclear extracts from cadmium-treated cells demonstrated that concentrations of cadmium (10 microM) that actively induce metallothionein gene expression cause only a small increase in the amount of nuclear MTF-1. In contrast, an overtly toxic concentration of cadmium (50 microM) rapidly induced the complete nuclear translocation and activation of DNA binding activity of MTF-1. These studies are consistent with the hypothesis that MTF-1 serves as a zinc sensor that responds to changes in cytosolic free zinc concentrations. In addition, these data suggest that cadmium activation of metallothionein gene expression may be accompanied by only small changes in nuclear MTF-1. PMID- 10734083 TI - Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139. AB - Histone H2AX is a ubiquitous member of the H2A histone family that differs from the other H2A histones by the presence of an evolutionarily conserved C-terminal motif, -KKATQASQEY. The serine residue in this motif becomes rapidly phosphorylated in cells and animals when DNA double-stranded breaks are introduced into their chromatin by various physical and chemical means. In the present communication we show that this phosphorylated form of H2AX, referred to as gamma-H2AX, appears during apoptosis concurrently with the initial appearance of high molecular weight DNA fragments. gamma-H2AX forms before the appearance of internucleosomal DNA fragments and the externalization of phosphatidylserine to the outer membrane leaflet. gamma-H2AX formation is inhibited by N benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and the inhibitor of caspase activated DNase, and it is induced when DNase I and restriction enzymes are introduced into cells, suggesting that any apoptotic endonuclease is sufficient to induce gamma-H2AX formation. These results indicate that gamma-H2AX formation is an early chromatin modification following initiation of DNA fragmentation during apoptosis. PMID- 10734082 TI - Protein phosphatase 2A and phosphatidylinositol 3-kinase regulate the activity of Sp1-responsive promoters. AB - The transcription factor Sp1 regulates the activity of a large number of eukaryotic gene promoters, including early SV40 and human immunodeficiency virus type 1 (HIV-1). Here, we report that expression of SV40 small tumor antigen (small t) in quiescent CV-1 cells transactivates two Sp1-responsive promoters, including a deletion mutant of HIV-1 LTR, through specific inhibition of endogenous AC and ABalphaC forms of protein phosphatase 2A (PP2A). Expression of a small t mutant, lacking the PP2A-binding domain, failed to transactivate Sp1. Overexpression of the B56alpha, B56beta, and B56gamma1 regulatory PP2A subunits strongly inhibited the ability of small t, but not the phosphatase inhibitor, okadaic acid, to enhance Sp1-driven gene expression. Using inhibitors and co expression of kinase-deficient mutants, we also show that functional phosphatidylinositol 3-kinase (PI 3-kinase) and atypical protein kinase C zeta are required for small t-induced Sp1-dependent promoter transcriptional activation. Moreover, two inhibitors of PI 3-kinase, wortmannin and LY294002, inhibit the initiation of SV40 DNA replication in quiescent CV-1 cells. Taken together, these results suggest that PP2A and PI 3-kinase contribute to the ability of small t to regulate Sp1 activity, stimulate early SV40 DNA replication, and enhance the transformation of resting cells during SV40 infection. PMID- 10734084 TI - Interaction of thrombospondin-1 and heparan sulfate from endothelial cells. Structural requirements of heparan sulfate. AB - Cell surface-associated heparan sulfate proteoglycans, predominantly perlecan, are involved in the process of binding and endocytosis of thrombospondin-1 (TSP 1) by vascular endothelial cells. To investigate the structural properties of heparan sulfate (HS) side chains that mediate this interaction, the proteoglycans were isolated from porcine endothelial cells and HS chains obtained thereof by beta-elimination. To characterize the structural composition of the HS chains and to identify the TSP-1-binding sequences, HS was disintegrated by specific chemical and enzymatic treatments. Cell layer-derived HS chains revealed the typical structural heterogeneity with domains of non-contiguously arranged highly sulfated disaccharides separated by extended sequences containing predominantly N acetylated sequences of low sulfation. Affinity chromatography on immobilized TSP 1 demonstrated that nearly all intact HS chains possessed binding affinity, whereas after heparinase III treatment only a small proportion of oligosaccharides were bound with similar affinity to the column. Size fractioning of the bound and unbound oligosaccharides revealed that only a specific portion of deca- to tetradecasaccharides possessed TSP-1-binding affinity. The binding fraction contained over 40% di- and trisulfated disaccharide units and was enriched in the content of the trisulfated 2-O-sulfated L-iduronic acid-N sulfated-6-O-sulfated glucosamine disaccharide unit. Comparison with the disaccharide composition of the intact HS chains and competition experiments with modified heparin species indicated the specific importance of N- and 6-O-sulfated glucosamine residues for binding. Further depolymerization of the binding oligosaccharides revealed that the glucosamine residues within the TSP-1-binding sequences are not continuously N-sulfated. The present findings implicate specific structural properties for the HS domain involved in TSP-1 binding and indicate that they are distinct from the binding sequence described for basic fibroblast growth factor, another HS ligand and a potential antagonist of TSP-1. PMID- 10734085 TI - Cloning and expression of the bioluminescent photoprotein pholasin from the bivalve mollusc Pholas dactylus. AB - Pholasin is the photoprotein responsible for luminescence in the bivalve Pholas dactylus and consists of a luciferin tightly bound to a glycosylated protein. It is a sensitive indicator of reactive oxygen species. A full-length clone encoding apopholasin was isolated from a P. dactylus light organ cDNA library. The unprocessed apoprotein contained 225 amino acids, starting with a signal peptide of 20 amino acids, 3 predicted N-linked glycosylation sites, 1 O-linked site, no histidines, and 7 cysteines. The recombinant apoprotein was expressed in cell extracts and insect cells. The size of the apoprotein expressed in cell extracts and the cytosol of insect cells was 26 kDa but that of the fully processed protein was 34 kDa, as was native pholasin. Both the processed and unprocessed recombinant apoproteins were recognized by a polyclonal antibody raised against native pholasin. Acid methanol extracts from Pholas added to recombinant apoprotein resulted in chemiluminescence triggered by sodium hypochlorite but not photoprotein formation. These results have important implications in understanding the molecular evolution of bioluminescence and will allow the development of recombinant pholasin as an intracellular indicator of reactive oxygen species. PMID- 10734086 TI - Heparan sulfate chains from glypican and syndecans bind the Hep II domain of fibronectin similarly despite minor structural differences. AB - Numerous functions of heparan sulfate proteoglycans are mediated through interactions between their heparan sulfate glycosaminoglycan chains and extracellular ligands. Ligand binding specificity for some molecules, including many growth factors, is determined by complex heparan sulfate fine structure, where highly sulfated, iduronate-rich domains alternate with N-acetylated domains. Syndecan-4, a cell surface heparan sulfate proteoglycan, has a distinct role in cell adhesion, suggesting its chains may differ from those of other cell surface proteoglycans. To determine whether the specific role of syndecan-4 correlates with a distinct heparan sulfate structure, we have analyzed heparan sulfate chains from the different surface proteoglycans of a single fibroblast strain and compared their ability to bind the Hep II domain of fibronectin, a ligand known to promote focal adhesion formation through syndecan-4. Despite distinct molecular masses of glypican and syndecan glycosaminoglycans and minor differences in disaccharide composition and sulfation pattern, the overall proportion and distribution of sulfated regions and the affinity for the Hep II domain were similar. Therefore, adhesion regulation requires core protein determinants of syndecan-4. PMID- 10734087 TI - Endogenous ADP-ribosylation of the G protein beta subunit prevents the inhibition of type 1 adenylyl cyclase. AB - Mono-ADP-ribosylation is a post-translational modification of cellular proteins that has been implicated in the regulation of signal transduction, muscle cell differentiation, protein trafficking, and secretion. In several cell systems we have observed that the major substrate of endogenous mono-ADP-ribosylation is a 36-kDa protein. This ADP-ribosylated protein was both recognized in Western blotting experiments and selectively immunoprecipitated by a G protein beta subunit-specific polyclonal antibody, indicating that this protein is the G protein beta subunit. The ADP-ribosylation of the beta subunit was due to a plasma membrane-associated enzyme, was sensitive to treatment with hydroxylamine, and was inhibited by meta-iodobenzylguanidine, indicating that the involved enzyme is an arginine-specific mono-ADP-ribosyltransferase. By mutational analysis, the target arginine was located in position 129. The ADP-ribosylated beta subunit was also deribosylated by a cytosolic hydrolase. This ADP ribosylation/deribosylation cycle might be an in vivo modulator of the interaction of betagamma with specific effectors. Indeed, we found that the ADP ribosylated betagamma subunit is unable to inhibit calmodulin-stimulated type 1 adenylyl cyclase in cell membranes and that the endogenous ADP-ribosylation of the beta subunit occurs in intact Chinese hamster ovary cells, where the NAD(+) pool was labeled with [(3)H]adenine. These results show that the ADP-ribosylation of the betagamma subunit could represent a novel cellular mechanism in the regulation of G protein-mediated signal transduction. PMID- 10734088 TI - Eosinophil-specific regulation of gp91(phox) gene expression by transcription factors GATA-1 and GATA-2. AB - The glycoprotein gp91(phox) is an essential component of the phagocyte NADPH oxidase and is expressed in eosinophils, neutrophils, monocytes, and B lymphocytes. We previously suggested an eosinophil-specific mechanism of gp91(phox) gene expression. To elucidate the mechanism, we performed functional assays on deletion mutants of the gp91(phox) promoter in various types of gp91(phox)-expressing cells. A 10-base pair (bp) region from bp -105 to -96 of the promoter activated transcription of the gene in eosinophilic cells, but not in neutrophilic, monocytic, or B-lymphocytic cells. A 2-bp mutation introduced into the GATA site spanning bp -101 to -96 (-98GATA site) of the fragment abolished its activity. Gel shift assays using a GATA competitor and specific antibodies demonstrated that both GATA-1 and GATA-2 specifically bound to the 98GATA site with similar affinities. Individual transfection of GATA-1 and GATA-2 into Jurkat cells, which have neither endogenous GATA-1 nor GATA-2, activated the -105/+12 construct in a -98GATA site-dependent manner. Combined transfection of GATA-1 and GATA-2 activated the promoter less than transfection of GATA-1 alone. These results suggest that GATA-1 is an activator and that GATA-2 is a relative competitive inhibitor of GATA-1 in the expression of the gp91(phox) gene in human eosinophils. PMID- 10734089 TI - Binding of Rab3A to synaptic vesicles. AB - Prenylated Rab GTPases cycle between membrane-bound and soluble forms. Membrane bound GDP-Rabs interact with GDP dissociation inhibitor (GDI), resulting in the dissociation of a Rab.GDI complex, which in turn serves as a precursor for the membrane re-association of Rabs. We have now characterized the binding of Rab3A to synaptic vesicles in vitro using either purified complexes or rat brain cytosol as source for GDI.Rab3A. Binding of Rab3A results in the immediate release of GDI from the membrane. Furthermore, binding does not require the presence of additional guanine nucleotides (GDP or GTP) or of cytosolic factors. Although nucleotide exchange follows binding, binding is initially reversible, suggesting that binding of GDP-Rab3A and nucleotide exchange are separate and independent events. Comparison with the binding of Rab1B revealed that both Rab proteins bind preferentially to their respective resident membranes although some promiscuity was observable. Binding is saturable and involves a protease sensitive binding site that is tightly associated with the vesicle membrane. PMID- 10734090 TI - Induction of the peroxisomal glycerolipid-synthesizing enzymes during differentiation of 3T3-L1 adipocytes. Role in triacylglycerol synthesis. AB - The glycerophosphate backbone for triglyceride synthesis is commonly believed to be created through the conversion of dihydroxyacetone phosphate (DHAP) by glycerophosphate dehydrogenase (GPD) to sn-glycerol 3-phosphate (GP), which is then converted by glycerophosphate acyltransferase (GPAT) to 1-acyl-GP. Consistent with this, GPD and GPAT are highly induced during differentiation of mouse 3T3-L1 preadipocytes. While the acyl dihydroxyacetone phosphate (acyl-DHAP) pathway for glycerolipid synthesis is commonly believed to be involved only in glycerol ether lipid synthesis, we report here that during conversion of 3T3-L1 preadipocytes to adipocytes, the specific activity of peroxisomal DHAP acyltransferase (DHAPAT) is increased by 9-fold in 6 days, while acyl-DHAP:NADPH reductase is induced by 5-fold. A parallel increase in the catalase (the peroxisomal marker enzyme) activity is also seen. In contrast, the specific activity of alkyl-DHAP synthase, the enzyme catalyzing the synthesis of the ether bond, is decreased by 60% during the same period. Unlike microsomal GPAT, the induced DHAPAT is found to have high activity at pH 5.5 and is resistant to inhibition by sulfhydryl agents, heat, and proteolysis. On subcellular fractionation, DHAPAT is found to be associated with microperoxisomes whereas GPAT activity is mainly present in microsomes. Northern blot analyses reveal that induction of DHAPAT can be largely explained through increases in DHAPAT mRNA. A comparison of microsomal and peroxisomal glycerolipid synthetic pathways, using D [3-(3)H, U-(14)C]glucose as the precursor of the lipid glycerol backbone shows that about 40-50% of triglyceride is synthesized via the acyl-DHAP pathway. These results indicate that the acyl-DHAP pathway is important not only for the synthesis of ether lipids, but also for the synthesis of triacylglycerol and other non-ether glycerolipids. PMID- 10734091 TI - Thioredoxin activation of phosphoribulokinase in a bi-enzyme complex from Chlamydomonas reinhardtii chloroplasts. AB - The activation of oxidized phosphoribulokinase either "free" or as part of a bi enzyme complex by reduced thioredoxins during the enzyme reaction was studied. In the presence of reduced thioredoxin, the product of the reaction catalyzed by phosphoribulokinase within the bi-enzyme complex does not appear in a linear fashion. It follows a mono-exponential pattern that suggests a slow dissociation process of the bi-enzyme complex in the assay cuvette. A plot of the steady state of product appearance against thioredoxin concentration gave a sigmoid curve. On the basis of our experimental results, we propose a minimum model of the activation of phosphoribulokinase by reduced thioredoxin. Reduced thioredoxin may act on the phosphoribulokinase, either within the complex or in the dissociated metastable form. However, the time required to activate the enzyme as part of the complex is shorter (about 20 s) than that required to activate the dissociated form (about 10 min). This might be of physiological relevance, and we discuss the role of the interactions between phosphoribulokinase and glyceraldehyde-3 phosphate dehydrogenase in the regulation of the Calvin cycle. PMID- 10734092 TI - Calcium influx through calcium leak channels is responsible for the elevated levels of calcium-dependent proteolysis in dystrophic myotubes. AB - To estimate calpain proteolysis, we measured the hydrolysis rate of a fluorogenic calpain substrate in individual resting normal and dystrophic mdx mouse myotubes in culture. Hydrolysis rates were high during myoblast and myotube alignment and fusion. After alignment and fusion ceased, hydrolysis rates declined. For normal myotubes, hydrolysis remained low after the development of contractile activity. In contrast, after the development of contractile activity, dystrophic mdx myotubes had abnormally high levels of hydrolysis that were dependent on external calcium and that could be abolished by calpeptin, an inhibitor of calpain. We eliminated the direct effects of contraction during measurements of hydrolysis by the addition of tetrodotoxin. Substrate hydrolysis by lysosomes or proteosomes was controlled for using NH(4)Cl and clasto-lactacystin beta-lactone, respectively. Increased activity of the calcium-activated protease in mature mdx myotubes was linked to the abnormal activity of calcium-specific leak channels because an antagonist of these channels reduced the higher levels of hydrolysis in dystrophic myotubes to nearly normal levels. The abnormal activity of these channels is linked to an increased frequency of transient sarcolemmal disruptions in the more fragile mdx myotubes (, ). Treatment of mdx myotubes with a pro-drug of methylprednisolone also reduced calpain substrate hydrolysis to nearly normal levels. However, this inhibition only required 2.5 h of pretreatment, which was not long enough to act by the known effects of prednisolone on calcium homeostasis. PMID- 10734093 TI - The co-repressor mSin3A is a functional component of the REST-CoREST repressor complex. AB - The repressor REST/NRSF restricts expression of a large set of genes to neurons by suppressing their expression in non-neural tissues. We find that REST repression involves two distinct repressor proteins. One of these, CoREST, interacts with the COOH-terminal repressor domain of REST (Andres, M. E., Burger, C., Peral-Rubio, M. J., Battaglioli, E., Anderson, M. E., Grimes, J., Dallmanm J., Ballas, N. , and Mandel, G. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9873 9878). Here we show that the co-repressor mSin3A also interacts with REST. The REST-mSin3A association involves the NH(2)-terminal repressor domain of REST and the paired amphipathic helix 2 domain of mSin3A. REST forms complexes with endogenous mSin3A in mammalian cells, and both mSin3A and CoREST interact with REST in intact mammalian cells. REST repression is blocked in yeast lacking Sin3 and rescued in its presence. In mammalian cells, repression by REST is reduced when binding to mSin3A is inhibited. In mouse embryos, the distribution of mSin3A and REST transcripts is largely coincident. The pattern of CoREST gene expression is more restricted, suggesting that mSin3A is required constitutively for REST repression, whereas CoREST is recruited for more specialized repressor functions. PMID- 10734095 TI - Structure and mechanism of 3-deoxy-D-manno-octulosonate 8-phosphate synthase. AB - 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate. KDO8P is the phosphorylated precursor of 3 deoxy-D-manno-octulosonate, an essential sugar of the lipopolysaccharide of Gram negative bacteria. The crystal structure of the Escherichia coli KDO8P synthase has been determined by multiple wavelength anomalous diffraction and the model has been refined to 2.4 A (R-factor, 19.9%; R-free, 23.9%). KDO8P synthase is a homotetramer in which each monomer has the fold of a (beta/alpha)(8) barrel. On the basis of the features of the active site, PEP and A5P are predicted to bind with their phosphate moieties 13 A apart such that KDO8P synthesis would proceed via a linear intermediate. A reaction similar to KDO8P synthesis, the condensation of phosphoenolpyruvate, and erythrose 4-phosphate to form 3-deoxy-D arabino-heptulosonate 7-phosphate (DAH7P), is catalyzed by DAH7P synthase. In the active site of DAH7P synthase the two substrates PEP and erythrose 4-phosphate appear to bind in a configuration similar to that proposed for PEP and A5P in the active site of KDO8P synthase. This observation suggests that KDO8P synthase and DAH7P synthase evolved from a common ancestor and that they adopt the same catalytic strategy. PMID- 10734094 TI - Dimerization of Escherichia coli DNA-gyrase B provides a structural mechanism for activating the ATPase catalytic center. AB - DNA-gyrase exhibits an unusual ATP-binding site that is formed as a result of gyrase B subunit dimerization, a structural transition that is also essential for DNA capture during the topoisomerization cycle. Previous structural studies on Escherichia coli DNA-gyrase B revealed that dimerization is the result of a polypeptidic exchange involving the N-terminal 14 amino acids. To provide experimental data that dimerization is critical for ATPase activity and enzyme turnover, we generated mutants with reduced dimerization by mutating the two most conserved residues of the GyrB N-terminal arm (Tyr-5 and Ile-10 residues). Our data demonstrate that the hydrophobic Ile-10 residue plays an important role in enzyme dimerization and the nucleotide-protein contact mediated by Tyr-5 side chain residue helps the dimerization process. Analysis of ATPase activities of mutant proteins provides evidence that dimerization enhances the ATP-hydrolysis turnover. The structure of the Y5S mutant of the N-terminal 43-kDa fragment of E. coli DNA GyrB subunit indicates that Tyr-5 residue provides a scaffold for the ATP-hydrolysis center. We describe a channel formed at the dimer interface that provides a structural mechanism to allow reactive water molecules to access the gamma-phosphate group of the bound ATP molecule. Together, these results demonstrate that dimerization strongly contributes to the folding and stability of the catalytic site for ATP hydrolysis. A role for the essential Mg(2+) ion for the orientation of the phosphate groups of the bound nucleotide inside the reactive pocket was also uncovered by superposition of the 5'-adenylyl beta-gamma imidodiphosphate (ADPNP) wild-type structure to the salt-free ADPNP structure. PMID- 10734096 TI - Three-dimensional structure of ryanodine receptor isoform three in two conformational states as visualized by cryo-electron microscopy. AB - Using cryo-electron microscopy and single particle image processing techniques, we present the first three-dimensional reconstructions of isoform 3 of the ryanodine receptor/calcium release channel (RyR3). Reconstructions were carried out on images obtained from a purified, detergent-solubilized receptor for two different buffer conditions, which were expected to favor open and closed functional states of the channel. As for the heart (RyR2) and skeletal muscle (RyR1) receptor isoforms, RyR3 is a homotetrameric complex comprising two main components, a multidomain cytoplasmic assembly and a smaller ( approximately 20% of the total mass) transmembrane region. Although the isoforms show structural similarities, consistent with the approximately 70% overall sequence identity of the isoforms, detailed comparisons of RyR3 with RyR1 showed one region of highly significant difference between them. This difference indicated additional mass present in RyR1, and it likely corresponds to a region of the RyR1 sequence (residues 1303-1406, known as diversity region 2) that is absent from RyR3. The reconstructions of RyR3 determined under "open" and "closed" conditions were similar to each other in overall architecture. A difference map computed between the two reconstructions reveals subtle changes in conformation at several widely dispersed locations in the receptor, the most prominent of which is a approximately 4 degrees rotation of the transmembrane region with respect to the cytoplasmic assembly. PMID- 10734097 TI - The PDZ domains of zonula occludens-1 induce an epithelial to mesenchymal transition of Madin-Darby canine kidney I cells. Evidence for a role of beta catenin/Tcf/Lef signaling. AB - The integrity of cell-cell contacts such as adherens junctions (AJ) and tight junctions (TJ) is essential for the function of epithelia. During carcinogenesis, the increased motility and invasiveness of tumor cells reflect the loss of characteristic epithelial features, including cell adhesion. While beta-catenin, a component of AJ, plays a well characterized dual role in cell adhesion and signal transduction leading to epithelial cell transformation, little is known about possible roles of tight junction components in signaling processes. Here we show that mutants of the TJ protein zonula occludens protein-1 (ZO-1), which encode the PDZ domains (ZO-1 PDZ) but no longer localize at the plasma membrane, induce a dramatic epithelial to mesenchymal transition (EMT) of Madin-Darby canine kidney I (MDCKI) cells. The observed EMT of these MDCK-PDZ cells is characterized by a repression of epithelial marker genes, a restricted differentiation potential and a significantly induced tumorigenicity. Intriguingly, the beta-catenin signaling pathway is activated in the cells expressing the ZO-1 PDZ protein. Ectopic expression of the adenomatous polyposis coli tumor suppressor gene, known to down-regulate activated beta-catenin signaling, reverts the transformed fibroblastoid phenotype of MDCK-PDZ cells. Thus, cytoplasmic localization of the ZO-1 PDZ domains induces an EMT in MDCKI cells, most likely by modulating beta-catenin signaling. PMID- 10734099 TI - HSP25, a small heat shock protein associated with dense bodies and M-lines of body wall muscle in Caenorhabditis elegans. AB - HSP25, a previously uncharacterized member of the alpha-crystallin family of small heat shock proteins in Caenorhabditis elegans, has been examined using biochemical and immunological techniques. HSP25 is the second largest of 16 identifiable small heat shock proteins in the nematode and is expressed at all developmental stages under normal growth conditions. Recombinant HSP25 produced in Escherichia coli exists predominantly as small oligomers (dimers to tetramers) and possesses chaperone activity against citrate synthase in vitro. In C. elegans, HSP25 is localized to dense bodies and M-lines in body wall muscle, to the lining of the pharynx, and to the junctions between cells of the spermathecal wall. Affinity chromatography of nematode extracts on a column of immobilized HSP25 resulted in specific binding of vinculin and alpha-actinin but not actin, as revealed by Western blotting. These results suggest a role for HSP25 in the organization or maintenance of the myofilament lattice and adherens junctions in C. elegans. PMID- 10734098 TI - NF-kappaB activation by camptothecin. A linkage between nuclear DNA damage and cytoplasmic signaling events. AB - Activation of the transcription factor NF-kappaB by extracellular signals involves its release from the inhibitor protein IkappaBalpha in the cytoplasm and subsequent nuclear translocation. NF-kappaB can also be activated by the anticancer agent camptothecin (CPT), which inhibits DNA topoisomerase (Topo) I activity and causes DNA double-strand breaks during DNA replication to induce S phase-dependent cytotoxicity. Here we show that CPT activates NF-kappaB by a mechanism that is dependent on initial nuclear DNA damage followed by cytoplasmic signaling events. NF-kappaB activation by CPT is dramatically diminished in cytoplasts and in CEM/C2 cells expressing a mutant Topo I protein that fails to bind CPT. This response is intensified in S phase cell populations and is prevented by the DNA polymerase inhibitor aphidicolin. In addition, CPT activation of NF-kappaB involves degradation of cytoplasmic IkappaBalpha by the ubiquitin-proteasome pathway in a manner that depends on the IkappaB kinase complex. Finally, inhibition of NF-kappaB activation augments CPT-induced apoptosis. These findings elucidate the progression of signaling events that initiates in the nucleus with CPT-Topo I interaction and continues in the cytoplasm resulting in degradation of IkappaBalpha and nuclear translocation of NF-kappaB to attenuate the apoptotic response. PMID- 10734101 TI - Platelet-derived growth factor receptor beta regulates migration and DNA synthesis in metanephric mesenchymal cells. AB - Platelet-derived growth factor (PDGF) B-chain and PDGF receptor beta (PDGFR beta) are essential for glomerulogenesis. Mice deficient in PDGF B-chain or PDGFR beta exhibit an abnormal glomerular phenotype characterized by total lack of mesangial cells. In this study, we localized PDGFR beta in the developing rat kidney and explored the biological effects of PDGF in metanephric mesenchymal cells in an attempt to determine the mechanism by which PDGF regulates mesangial cell development. Immunohistochemical and in situ hybridization studies of rat embryonic kidneys reveal that PDGFR beta localizes to undifferentiated metanephric mesenchyme and is later expressed in the cleft of the comma-shaped and S-shaped bodies and in more mature glomeruli in a mesangial distribution. We also isolated and characterized cells from rat metanephric mesenchyme. Metanephric mesenchymal cells express vimentin and alpha-smooth muscle actin but not cytokeratin. These cells also express functional PDGFR beta, as demonstrated by autophosphorylation of the receptor as well as activation of phosphatidylinositol 3 kinase in response to PDGF B-chain homodimer. PDGF B-chain also induces migration and proliferation of metanephric mesenchymal cells. Taken together with the fact that PDGF B-chain is expressed in the glomerular epithelium and mesangial area, as demonstrated in the human embryonic kidney, we suggest that PDGF B-chain acts in a paracrine fashion to stimulate the migration and proliferation of mesangial cell precursors from undifferentiated metanephric mesenchyme to the mesangial area. PDGF B-chain also likely stimulates proliferation of mesangial cell precursors in an autocrine fashion once these cells migrate to the glomerular tuft. PMID- 10734100 TI - Dissecting the energetics of the apoflavodoxin-FMN complex. AB - Many flavoproteins are non-covalent complexes between FMN and an apoprotein. To understand better the stability of flavoproteins, we have studied the energetics of the complex between FMN and the apoflavodoxin from Anabaena PCC 7119 by a combination of site-directed mutagenesis, titration calorimetry, equilibrium binding constant determinations, and x-ray crystallography. Comparison of the strength of the wild type and mutant apoflavodoxin-FMN complexes and that of the complexes between wild type apoflavodoxin and shortened FMN analogues (riboflavin and lumiflavin) allows the dissection of the binding energy into contributions associated with the different parts of the FMN molecule. The estimated contribution of the phosphate is greatest, at 7 kcal mol(-1); that of the isoalloxazine is of around 5-6 kcal mol(-1) (mainly due to interaction with Trp 57 and Tyr-94 in the apoprotein) and the ribityl contributes least: around 1 kcal mol(-1). The stabilization of the complex is both enthalpic and entropic although the enthalpy contribution is dominant. Both the phosphate and the isoalloxazine significantly contribute to the enthalpy of binding. The ionic strength does not have a large effect on the stability of the FMN complex because, although it weakens the phosphate interactions, it strengthens the isoalloxazine-protein hydrophobic interactions. Phosphate up to 100 mM does not affect the strength of the riboflavin complex, which suggests the isoalloxazine and phosphate binding sites may be independent in terms of binding energy. Interestingly, we find crystallographic evidence of flexibility in one of the loops (57-62) involved in isoalloxazine binding. PMID- 10734102 TI - Allosteric activation of acid alpha-glucosidase by the human papillomavirus E7 protein. AB - Changes in the cellular carbohydrate metabolism are a hallmark of malignant transformation and represent one of the earliest discernible events in tumorigenesis. In the early stages of certain epithelial cancers, a metabolic switch is regularly observed, in which slowly growing glycogenotic cells are converted to highly proliferating basophilic cells. This step is accompanied by a rapid depletion of the intracellular glycogen stores, which in liver carcinogenesis results from the activation of the enzyme acid alpha-glucosidase by an as yet unknown mechanism. We show here that acid alpha-glucosidase is a target for the E7 protein encoded by human papillomavirus type 16, a human tumor virus that plays a key role in the genesis of cervical carcinoma. We show that expression of E7 induces the catalytic activity of acid alpha-glucosidase in vivo and wild type E7, but not transformation-deficient mutants bind directly to acid alpha-glucosidase and increase the catalytic activity of the enzyme in vitro. The data suggest that the E7 protein encoded by human papillomavirus type 16 can act as an allosteric activator of acid alpha-glucosidase. PMID- 10734103 TI - Identification and characterization of a dimerization domain in CED-6, an adapter protein involved in engulfment of apoptotic cells. AB - Phagocytosis of apoptotic cells is a key step in the completion of programmed cell death that occurs throughout life in multicellular organisms. The molecular events involved in clearance of apoptotic cells are just beginning to be elucidated. Recently, CED-6, an adapter protein involved in engulfment has been cloned in Caenorhabditis elegans and in humans. CED-6 is composed of a phosphotyrosine-binding (PTB) domain and a proline-rich C-terminal domain with no apparent catalytic domain. Since PTB domains, originally identified in Shc, mediate intracellular signaling downstream of cell surface receptors, CED-6 has also been proposed to mediate intracellular signals leading to engulfment. In this report, we demonstrate that CED-6 dimerizes through a leucine zipper domain that is immediately adjacent to the PTB domain. Several lines of evidence based on co-immunoprecipitation studies, yeast two-hybrid assays, and gel filtration studies suggest that CED-6 exists as a dimer in vivo. Through mutational analyses, we show that the leucine zipper is necessary and sufficient for CED-6 dimerization and that this dimerization is conserved among C. elegans, rodent, and human CED-6 proteins. We propose that dimerization may have unique implications for ligand binding via CED-6 and its function during the phagocytosis of apoptotic cells. PMID- 10734104 TI - CCR11 is a functional receptor for the monocyte chemoattractant protein family of chemokines. AB - Chemokines mediate their diverse activities through G protein-coupled receptors. The human homolog of the bovine orphan receptor PPR1 shares significant similarity to chemokine receptors. Transfection of this receptor into murine L1.2 cells resulted in responsiveness to monocyte chemoattractant protein (MCP)-4, MCP 2, and MCP-1 in chemotaxis assays. Binding studies with radiolabeled MCP-4 demonstrated a single high affinity binding site with an IC(50) of 0.14 nM. As shown by competition binding, other members of the MCP family also recognized this receptor. MCP-2 was the next most potent ligand, with an IC(50) of 0.45 nM. Surprisingly, eotaxin (IC(50) = 6.7 nM) and MCP-3 (IC(50) = 4.1 nM) bind with greater affinity than MCP-1 (IC(50) = 10.7 nM) but only act as agonists in chemotaxis assays at 100-fold higher concentrations. Because of high affinity binding and functional chemotactic responses, we have termed this receptor CCR11. The gene for CCR11 was localized to human chromosome 3q22, which is distinct from most CC chemokine receptor genes at 3p21. Northern blot hybridization was used to identify CCR11 expression in heart, small intestine, and lung. Thus CCR11 shares functional similarity to CCR2 because it recognizes members of the MCP family, but CCR11 has a distinct expression pattern. PMID- 10734105 TI - Interaction of the somatostatin receptor subtype 1 with the human homolog of the Shk1 kinase-binding protein from yeast. AB - Interaction between the C terminus of a G-protein-coupled receptor and intracellular constituents may represent a crucial step in regulating signal transduction. To identify potential interacting candidates the C terminus of the somatostatin receptor subtype 1 was used as bait in a yeast two hybrid screen of a human brain cDNA library. We identified the human Skb1 sequence (Skb1Hs) as interacting protein, which is homologous to the yeast protein known Skb1 to down regulate mitosis in Schizosaccharomyces pombe via binding to the Shk1 protein kinase; the latter is a homolog to the mammalian p21(cdc42/Rac)-activated protein kinases. Interaction required almost the entire C terminus of the somatostatin receptor subtype 1 including the conserved NPXXY motif of transmembrane region seven; in the case of the Skb1Hs most of the N terminus and an S adenosylmethionine binding domain were mandatory, whereas the C terminus was not essential. Interaction was verified by coexpression experiments in human embryonic kidney cells. As revealed by immunocytochemical analysis Skb1Hs expressed alone aggregates in large cytosolic clusters. When coexpressed, receptor subtype 1 and Skb1Hs were colocalized at the cell surface; these cells showed a strong increase in somatostatin binding compared with cells expressing the receptor only. This may suggest that Skb1Hs acts like a chaperone by correctly targeting the receptor to the cell surface. PMID- 10734106 TI - A novel H(+)-coupled oligopeptide transporter (OPT3) from Caenorhabditis elegans with a predominant function as a H(+) channel and an exclusive expression in neurons. AB - We have cloned and functionally characterized a novel, neuron-specific, H(+) coupled oligopeptide transporter (OPT3) from Caenorhabditis elegans that functions predominantly as a H(+) channel. The opt3 gene is approximately 4.4 kilobases long and consists of 13 exons. The cDNA codes for a protein of 701 amino acids with 11 putative transmembrane domains. When expressed in mammalian cells and in Xenopus laevis oocytes, OPT3 cDNA induces H(+)-coupled transport of the dipeptide glycylsarcosine. Electrophysiological studies of the transport function of OPT3 in Xenopus oocytes show that this transporter, although capable of mediating H(+)-coupled peptide transport, functions predominantly as a H(+) channel. The H(+) channel activity of OPT3 is approximately 3-4-fold greater than the H(+)/peptide cotransport activity as determined by measurements of H(+) gradient-induced inward currents in the absence and presence of the dipeptide using the two-microelectrode voltage clamp technique. A downhill influx of H(+) was accompanied by a large intracellular acidification as evidenced from the changes in intracellular pH using an ion-selective microelectrode. The H(+) channel activity exhibits a K(0.5)(H) of 1.0 microM at a membrane potential of 50 mV. At the level of primary structure, OPT3 has moderate homology with OPT1 and OPT2, two other H(+)-coupled oligopeptide transporters previously cloned from C. elegans. Expression studies using the opt3::gfp fusion constructs in transgenic C. elegans demonstrate that opt3 gene is exclusively expressed in neurons. OPT3 may play an important physiological role as a pH balancer in the maintenance of H(+) homeostasis in C. elegans. PMID- 10734107 TI - The beta(2)-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor. AB - Many G protein-coupled receptors (GPCRs) activate MAP kinases by stimulating tyrosine kinase signaling cascades. In some systems, GPCRs stimulate tyrosine phosphorylation by inducing the "transactivation" of a receptor tyrosine kinase (RTK). The mechanisms underlying GPCR-induced RTK transactivation have not been clearly defined. Here we report that GPCR activation mimics growth factor mediated stimulation of the epidermal growth factor receptor (EGFR) with respect to many facets of RTK function. beta(2)-Adrenergic receptor (beta(2)AR) stimulation of COS-7 cells induces EGFR dimerization, tyrosine autophosphorylation, and EGFR internalization. Coincident with EGFR transactivation, isoproterenol exposure induces the formation of a multireceptor complex containing both the beta(2)AR and the "transactivated" EGFR. beta(2)AR mediated EGFR phosphorylation and subsequent beta(2)AR stimulation of extracellular signal-regulated kinase (ERK) 1/2 are sensitive to selective inhibitors of both EGFR and Src kinases, indicating that both kinases are required for EGFR transactivation. beta(2)AR-dependent signaling to ERK1/2, like direct EGF stimulation of ERK1/2 activity, is sensitive to inhibitors of clathrin mediated endocytosis, suggesting that signaling downstream of both the EGF activated and the GPCR-transactivated EGFRs requires a productive engagement of the complex with the cellular endocytic machinery. Thus, RTK transactivation is revealed to be a process involving both association of receptors of distinct classes and the interaction of the transactivated RTK with the cells endocytic machinery. PMID- 10734108 TI - The VT+ and VT- isoforms of the fibroblast growth factor receptor type 1 are differentially expressed in the presumptive mesoderm of Xenopus embryos and differ in their ability to mediate mesoderm formation. AB - Previously, we cloned a variant form of the type 1 fibroblast growth factor receptor (FGFR1), FGFR-VT-, from Xenopus embryos (Gillespie, L. L., Chen, G., and Paterno, G. D. (1995) J. Biol. Chem. 270, 22758-22763). This isoform differed from the reported FGFR1 sequence (FGFR-VT+) by a 2-amino acid deletion, Val(423) Thr(424), in the juxtamembrane region. This deletion arises from the use of an alternate 5' splice donor site, and the activity of the VT+ and VT- forms of the FGFR1 was regulated by phosphorylation at this site. We have now investigated the expression pattern and function of these two isoforms in mesoderm formation in Xenopus embryos. Cells within the marginal zone are induced to form mesoderm during blastula stages. RNase protection analysis of blastula stage embryos revealed that the VT+ isoform was expressed throughout the embryo but that the VT isoform was expressed almost exclusively in the marginal zone. The ratio of VT+:VT- transcripts in the marginal zone indicated that the VT+ form was predominant throughout blastula stages except for a brief interval, coinciding with the start of zygotic transcription, when a dramatic increase in VT- expression levels was detected. This increase could be mimicked in part by treatment of animal cap explants with FGF-2. Overexpression of the VT+ isoform in Xenopus embryos resulted in development of tadpoles with severe reductions in trunk and tail structures, while embryos overexpressing the VT- isoform developed normally. A standard mesoderm induction assay revealed that a 10-fold higher concentration of FGF-2 was required to reach 50% induction in VT+-overexpressing animal cap explants compared with those overexpressing the VT- isoform. Furthermore, little or no expression of the panmesodermal marker Brachyury (Xbra) was detected in VT+-overexpressing embryos, while VT--overexpressing embryos showed normal staining. This demonstrates that VT+ overexpression had a negative effect on mesoderm formation in vivo. These data are consistent with a model in which mesoderm formation in vivo is regulated, at least in part, by the relative expression levels of the VT+ and VT- isoforms. PMID- 10734109 TI - Definition of the interaction domain for cytochrome c on the cytochrome bc(1) complex. Steady-state and rapid kinetic analysis of electron transfer between cytochrome c and Rhodobacter sphaeroides cytochrome bc(1) surface mutants. AB - The interaction domain for cytochrome c on the cytochrome bc(1) complex was studied using a series of Rhodobacter sphaeroides cytochrome bc(1) mutants in which acidic residues on the surface of cytochrome c(1) were substituted with neutral or basic residues. Intracomplex electron transfer was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 72 (Ru-72 Cc). Flash photolysis of a 1:1 complex between Ru-72-Cc and cytochrome bc(1) at low ionic strength resulted in electron transfer from photoreduced heme c to cytochrome c(1) with a rate constant of k(et) = 6 x 10(4) s(-1). Compared with the wild-type enzyme, the mutants substituted at Glu-74, Glu-101, Asp-102, Glu 104, Asp-109, Glu-162, Glu-163, and Glu-168 have significantly lower k(et) values as well as significantly higher equilibrium dissociation constants and steady state K(m) values. Mutations at acidic residues 56, 79, 82, 83, 97, 98, 213, 214, 217, 220, and 223 have no significant effect on either rapid kinetics or steady state kinetics. These studies indicate that acidic residues on opposite sides of the heme crevice of cytochrome c(1) are involved in binding positively charged cytochrome c. These acidic residues on the intramembrane surface of cytochrome c(1) direct the diffusion and binding of cytochrome c from the intramembrane space. PMID- 10734110 TI - Requirement of ryanodine receptor subtypes 1 and 2 for Ca(2+)-induced Ca(2+) release in vascular myocytes. AB - While the roles of subtypes 1 and 2 of the ryanodine receptors (RYRs) have been studied in cellular systems expressing specifically one or the other of these subtypes (i.e. skeletal and cardiac muscle), the function of these receptors has not been evaluated in smooth muscles. We have previously reported RYR-mediated elementary (Ca(2+) sparks) and global Ca(2+) responses in rat portal vein myocytes. Here, we investigated the respective roles of all three RYR subtypes expressed in these cells as revealed by reverse transcriptase-polymerase chain reaction. Antisense oligonucleotides targeting each one of the three RYR subtypes were shown to specifically inhibit the expression of the corresponding mRNA and protein without affecting the other RYR subtypes. Confocal Ca(2+) measurements revealed that depolarization-induced Ca(2+) sparks and global Ca(2+) responses were blocked when either RYR1 or RYR2 expression was suppressed. Caffeine-induced Ca(2+) responses were partly inhibited by the same antisense oligonucleotides. Neither the corresponding scrambled oligonucleotides nor the antisense oligonucleotides targeting RYR3 affected depolarization- or caffeine-induced Ca(2+) responses. Our results show that, in vascular myocytes, the two RYR1 and RYR2 subtypes are required for Ca(2+) release during Ca(2+) sparks and global Ca(2+) responses, evoked by activation of voltage-gated Ca(2+) channels. PMID- 10734111 TI - Mammalian Notch1 is modified with two unusual forms of O-linked glycosylation found on epidermal growth factor-like modules. AB - Notch is a large cell-surface receptor known to be an essential player in a wide variety of developmental cascades. Here we show that Notch1 endogenously expressed in Chinese hamster ovary cells is modified with O-linked fucose and O linked glucose saccharides, two unusual forms of O-linked glycosylation found on epidermal growth factor-like (EGF) modules. Interestingly, both modifications occur as monosaccharide and oligosaccharide species. Through exoglycosidase digestions we determined that the O-linked fucose oligosaccharide is a tetrasaccharide with a structure identical to that found on human clotting factor IX: Sia-alpha2,3-Gal-beta1, 4-GlcNAc-beta1,3-Fuc-alpha1-O-Ser/Thr. The elongated form of O-linked glucose appears to be a trisaccharide. Notch1 is the first membrane-associated protein identified with either O-linked fucose or O-linked glucose modifications. It also represents the second protein discovered with an elongated form of O-linked fucose. The sites of glycosylation, which fall within the multiple EGF modules of Notch, are highly conserved across species and within Notch homologs. Since Notch is known to interact with its ligands through subsets of EGF modules, these results suggest that the O-linked carbohydrate modifications of these modules may influence receptor-ligand interactions. PMID- 10734112 TI - Activation of p38 and c-Jun N-terminal kinase pathways and induction of apoptosis by chelerythrine do not require inhibition of protein kinase C. AB - Chelerythrine, a natural benzophenanthridine alkaloid, has been reported to mediate a variety of biological activities, including inhibition of protein kinase C (PKC). Here we report that chelerythrine induced time- and dose dependent activation of JNK1 and p38 in HeLa cells, which was mediated the upstream kinases, MEKK1 and MKK4. However, treatment with two other potent and selective PKC inhibitors, GF-109203X and Go6983, or down-regulation of PKC activity by prolonged treatment with phorbol 12-myristate 13-acetate had no effect on JNK1 and p38 activities. Furthermore, under the conditions where JNK1 and p38 were activated, we did not observe any significant inhibitory effect of chelerythrine on the activities of PKC isozymes present in HeLa cells. Interestingly, pretreatment with the antioxidants, N-acetyl-L-cysteine, dithiothreitol, and glutathione, impaired chelerythrine-induced JNK1 and p38 activation. In addition, chelerythrine induced apoptosis that was blocked by the antioxidants and the dominant-negative mutants of MEKK1, MKK4, JNK1, and p38. Together, these results uncover a novel biochemical property of chelerythrine, i.e. activation of MEKK1- and MKK4-dependent JNK1 and p38 pathways through an oxidative stress mechanism, which mediate the induction of apoptosis, but are independent of PKC inhibition. PMID- 10734113 TI - Src family kinases negatively regulate platelet-derived growth factor alpha receptor-dependent signaling and disease progression. AB - We tested the hypothesis that Src family kinases (SFK) contribute to c-Cbl mediated degradation of the platelet-derived growth factor (PDGF) alpha receptor (alphaPDGFR). Using either a receptor mutant that does not engage SFKs (F72/74), or cells that that lack SFKs, we found that SFKs contributed to degradation of the alphaPDGFR. Overexpression of c-Cbl also reduced the receptor half-life, but only if the receptor was able to engage SFKs. In cultured cells, prolonging the half-life of the receptor correlated with enhanced signaling and more efficient S phase entry, whereas accelerating receptor degradation had the opposite effect. Consistent with these tissue culture findings, there was a statistically significant increase in the onset of a proliferative retinal disease when animals were injected with cells expressing the F72/74 receptor, as compared with cells expressing the WT receptor. Our findings suggest that SFKs cooperate with c-Cbl to negatively regulate the alphaPDGFR, and that the SFK/c-Cbl suppression of alphaPDGFR output is relevant to the onset and progression of a proliferative disease. PMID- 10734114 TI - Cross-talk between phosphatidylinositol 3-kinase and sphingomyelinase pathways as a mechanism for cell survival/death decisions. AB - Peptide hormones act to regulate apoptosis through activation of multiple pro- and anti-apoptotic signaling cascades of which lipid signaling events represent an important facet of the cellular rheostat that determines survival and death decisions. Activation of sphingomyelinase, which generates ceramide, is an intermediate in cellular stress responses and induction of apoptosis in many systems. Conversely, phosphatidylinositol 3-kinase (PI3K) is a critical signaling molecule involved in regulating cell survival and proliferation pathways. In the present study, we investigate cross-talk between the PI3K and sphingomyelinase pathways as a mechanism for regulation of cell survival/death decisions. We show that phorbol ester, insulin-like growth factor 1, and a constitutively active PI3K suppress both tumor necrosis factor-induced apoptosis and ceramide generation. Conversely, inhibition of the PI3K pathway with expression of a kinase-dead PI3K both prevented survival signaling and enhanced tumor necrosis factor-induced ceramide generation. The ability of exogenous sphingomyelinase to induce ceramide generation was partially suppressed by expression of constitutively active PI3K and enhanced by inhibition of PI3K suggesting that cross-talk between PI3K and ceramide generation within cells is regulated subsequent to activation of sphingomyelinase. PMID- 10734115 TI - The Bloom's syndrome gene product interacts with topoisomerase III. AB - Bloom's syndrome is a rare genetic disorder associated with loss of genomic integrity and a large increase in the incidence of many types of cancer at an early age. The Bloom's syndrome gene product, BLM, belongs to the RecQ family of DNA helicases, which also includes the human Werner's and Rothmund-Thomson syndrome gene products and the Sgs1 protein of Saccharomyces cerevisiae. This family shows strong evolutionary conservation of protein structure and function. Previous studies have shown that Sgs1p interacts both physically and genetically with topoisomerase III. Here, we have investigated whether this interaction has been conserved in human cells. We show that BLM and hTOPO IIIalpha, one of two human topoisomerase III homologues, co-localize in the nucleus of human cells and can be co-immunoprecipitated from human cell extracts. Moreover, the purified BLM and hTOPO IIIalpha proteins are able to bind specifically to each other in vitro, indicating that the interaction is direct. We have mapped two independent domains on BLM that are important for mediating the interaction with hTOPO IIIalpha. Furthermore, through characterizing a genetic interaction between BLM and TOP3 in S. cerevisiae, we have identified a functional role for the hTOPO IIIalpha interaction domains in BLM. PMID- 10734116 TI - Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase. AB - Adult human mesenchymal stem cells are primary, multipotent cells capable of differentiating to osteocytic, chondrocytic, and adipocytic lineages when stimulated under appropriate conditions. To characterize the molecular mechanisms that regulate osteogenic differentiation, we examined the contribution of mitogen activated protein kinase family members, ERK, JNK, and p38. Treatment of these stem cells with osteogenic supplements resulted in a sustained phase of ERK activation from day 7 to day 11 that coincided with differentiation, before decreasing to basal levels. Activation of JNK occurred much later (day 13 to day 17) in the osteogenic differentiation process. This JNK activation was associated with extracellular matrix synthesis and increased calcium deposition, the two hallmarks of bone formation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as did transfection with a dominant negative form of MAP kinase kinase (MEK-1). Significantly, the blockage of osteogenic differentiation resulted in the adipogenic differentiation of the stem cells and the expression of adipose-specific mRNAs peroxisome proliferator-activated receptor gamma2, aP2, and lipoprotein lipase. These observations provide a potential mechanism involving MAP kinase activation in osteogenic differentiation of adult stem cells and suggest that commitment of hMSCs into osteogenic or adipogenic lineages is governed by activation or inhibition of ERK, respectively. PMID- 10734117 TI - Phosphoinositide-dependent activation of the ADP-ribosylation factor GTPase activating protein ASAP1. Evidence for the pleckstrin homology domain functioning as an allosteric site. AB - The ADP-ribosylation factor (Arf) family of GTP-binding proteins are regulators of membrane traffic and the actin cytoskeleton. Both negative and positive regulators of Arf, the centaurin beta family of Arf GTPase-activating proteins (GAPs) and Arf guanine nucleotide exchange factors, contain pleckstrin homology (PH) domains and are activated by phosphoinositides. To understand how the activities are coordinated, we have examined the role of phosphoinositide binding for Arf GAP function using ASAP1/centaurin beta4 as a model. In contrast to Arf exchange factors, phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P(2)) specifically activated Arf GAP. D3 phosphorylated phosphoinositides were less effective. Activation involved PtdIns-4,5-P(2) binding to the PH domain; however, in contrast to the Arf exchange factors and contrary to predictions based on the current paradigm for PH domains as independently functioning recruitment signals, we found the following: (i) the PH domain was dispensable for targeting to PDGF induced ruffles; (ii) activation and recruitment could be uncoupled; (iii) the PH domain was necessary for activity even in the absence of phospholipids; and (iv) the Arf GAP domain influenced localization and lipid binding of the PH domain. Furthermore, PtdIns-4,5-P(2) binding to the PH domain caused a conformational change in the Arf GAP domain detected by limited proteolysis. Thus, these data demonstrate that PH domains can function as allosteric sites. In addition, differences from the published properties of the Arf exchange factors suggest a model in which feedforward and feedback loops involving lipid metabolites coordinate GTP binding and hydrolysis by Arf. PMID- 10734118 TI - Domain one of the high affinity IgE receptor, FcepsilonRI, regulates binding to IgE through its interface with domain two. AB - The high affinity receptor for IgE, FcepsilonRI, binds IgE through the second Ig like domain of the alpha subunit. The role of the first Ig-like domain is not well understood, but it is required for optimal binding of IgE to FcepsilonRI, either through a minor contact interaction or in a supporting structural capacity. The results reported here demonstrate that domain one of FcepsilonRI plays a major structural role supporting the presentation of the ligand-binding site, by interactions generated within the interdomain interface. Analysis of a series of chimeric receptors and point mutants indicated that specific residues within the A' strand of domain one are crucial to the maintenance of the interdomain interface, and IgE binding. Mutation of the Arg(15) and Phe(17) residues caused loss in ligand binding, and utilizing a homology model of FcepsilonRI-alpha based on the solved structure of FcgammaRIIa, it appears likely that this decrease is brought about by collapse of the interface and consequently the IgE-binding site. In addition discrepancies in results of previous studies using chimeric IgE receptors comprising FcepsilonRIalpha with either FcgammaRIIa or FcgammaRIIIA can be explained by the presence or absence of Arg(15) and its influence on the IgE-binding site. The data presented here suggest that the second domain of FcepsilonRI-alpha is the only domain involved in direct contact with the IgE ligand and that domain one has a structural function of great importance in maintaining the integrity of the interdomain interface and, through it, the ligand-binding site. PMID- 10734119 TI - Molecular cloning, genomic organization, and biochemical characterization of myristoyl-CoA:protein N-myristoyltransferase from Arabidopsis thaliana. AB - Myristoyl-CoA:protein N-myristoyltransferase (NMT, EC 2.3.1.97) catalyzes the co translational addition of myristic acid to the amino-terminal glycine residue of a number of important proteins of diverse functions. We have isolated a full length Arabidopsis thaliana cDNA encoding NMT (AtNMT1), the first described from a higher plant. This AtNMT1 cDNA clone has an open reading frame of 434 amino acids and a predicted molecular mass of 48,706 Da. The primary structure is 50% identical to the mammalian NMTs. Analyses of Southern blots, genomic clones, and database sequences suggested that the A. thaliana genome contains two copies of NMT gene, which are present on different chromosomes and have distinct genomic organizations. The recombinant AtNMT1 expressed in Escherichia coli exhibited a high catalytic efficiency for the peptides derived from putative plant myristoylated proteins AtCDPK6 and Fen kinase. The AtNMT was similar to the mammalian NMTs with respect to a relative specificity for myristoyl CoA among the acyl CoA donors and also inhibition by the bovine brain NMT inhibitor NIP(71). The AtNMT1 expression profile indicated ubiquity in roots, stem, leaves, flowers, and siliques (approximately 1.7 kb transcript and approximately 50 kDa immunoreactive polypeptide) but a greater level in the younger tissue, which are developmentally very active. NMT activity was also evident in all these tissues. Subcellular distribution studies indicated that, in leaf extracts, approximately 60% of AtNMT activity was associated with the ribosomal fractions, whereas approximately 30% of the activity was observed in the cytosolic fractions. The NMT is biologically important to plants, as noted from the stunted development when the AtNMT1 was down-regulated in transgenic Arabidopsis under the control of an enhanced CaMV 35S promoter. The results presented in this study provide the first direct molecular evidence for plant protein N-myristoylation and a mechanistic basis for understanding the role of this protein modification in plants. PMID- 10734120 TI - The accessibility of a novel reentrant loop of the glutamate transporter GLT-1 is restricted by its substrate. AB - The excitatory neurotransmitter glutamate is removed from the synaptic cleft by several related sodium- and potassium-coupled transporters. They thereby restrict the neurotoxicity of this transmitter. Based on the accessibility of single cysteines to the large sulfhydryl reagent 3-N-maleimidyl(propionyl)biocytin, we have proposed a topological model for the astroglial glutamate transporter GLT-1 (Grunewald, M., Bendahan, A. and Kanner, B. I. (1998) Neuron 21, 623-632). Because of several unexpected observations, we have investigated the topological disposition of 19 cysteine residues engineered into a loop proposed to be intracellular. We have probed the accessibility of these cysteines to small and large sulfhydryl reagents. The impermeant hydrophilic sulfhydryl reagent [(2 trimethylammonium)ethyl] methanethiosulfonate inhibits transport activity only at two of these positions, weakly at G365C and potently at A364C. Glutamate and its nontransportable analogue dihydrokainate markedly protect A364C transporters against this impermeant reagent. Using a biotinylated maleimide, we found that, among the 14 mutants tested with it, only A364C is accessible to it from the extracellular side. This, together with our previous observations, indicates that the loop-including amino acid residues 354, 359, 373, and 379-is largely intracellular, but a short region of it forms a reentrant pore-loop-like structure, the accessibility of which is dependent on the conformation of the transporter. PMID- 10734121 TI - Identification and characterization of a Na(+)-independent neutral amino acid transporter that associates with the 4F2 heavy chain and exhibits substrate selectivity for small neutral D- and L-amino acids. AB - A cDNA was isolated from the mouse brain that encodes a novel Na(+)-independent neutral amino acid transporter. The encoded protein, designated as Asc-1 (asc type amino acid transporter 1), was found to be structurally related to recently identified mammalian amino acid transporters for the transport systems L, y(+)L, x(C)(-), and b(0,+), which are linked, via a disulfide bond, to the type II membrane glycoproteins, 4F2 heavy chain (4F2hc), or rBAT (related to b(0,+) amino acid transporter). Asc-1 required 4F2hc for its functional expression. In Western blot analysis in the nonreducing condition, a 118-kDa band, which seems to correspond to the heterodimeric complex of Asc-1 and 4F2hc, was detected in the mouse brain. The band shifted to 33 kDa in the reducing condition, confirming that Asc-1 and 4F2hc are linked via a disulfide bond. Asc-1-mediated transport was not dependent on the presence of Na(+) or Cl(-). Although Asc-1 showed a high sequence homology (66% identity at the amino acid level) to the Na(+)-independent broad scope neutral amino acid transporter LAT2 (Segawa, H., Fukasawa, Y., Miyamoto, K., Takeda, E., Endou, H., and Kanai, Y. (1999) J. Biol. Chem. 274, 19745-19751), Asc-1 also exhibited distinctive substrate selectivity and transport properties. Asc-1 preferred small neutral amino acids such as Gly, L Ala, L-Ser, L-Thr, and L-Cys, and alpha-aminoisobutyric acid as substrates. Asc-1 also transported D-isomers of the small neutral amino acids, in particular D-Ser, a putative endogenous modulator of N-methyl-D-aspartate-type glutamate receptors, with high affinity. Asc-1 operated preferentially, although not exclusively, in an exchange mode. Asc-1 mRNA was detected in the brain, lung, small intestine, and placenta. The functional properties of Asc-1 seem to be consistent with those of a transporter subserving the Na(+)-independent small neutral amino acid transport system asc. PMID- 10734122 TI - Tumor necrosis factor-alpha inhibits expression of CTP:phosphocholine cytidylyltransferase. AB - We investigated the effects of tumor necrosis factor alpha (TNFalpha), a key cytokine involved in inflammatory lung disease, on phosphatidylcholine (PtdCho) biosynthesis in a murine alveolar type II epithelial cell line (MLE-12). TNFalpha significantly inhibited [(3)H]choline incorporation into PtdCho after 24 h of exposure. TNFalpha reduced the activity of CTP:phosphocholine cytidylyltransferase (CCT), the rate-regulatory enzyme within the CDP-choline pathway, by 40% compared with control, but it did not alter activities of choline kinase or cholinephosphotransferase. Immunoblotting revealed that TNFalpha inhibition of CCT activity was associated with a uniform decrease in the mass of CCTalpha in total cell lysates, cytosolic, microsomal, and nuclear subfractions of MLE cells. Northern blotting revealed no effects of the cytokine on steady state levels of CCTalpha mRNA, and CCTbeta mRNA was not detected. Incorporation of [(35)S]methionine into immunoprecipitable CCTalpha protein in pulse and pulse chase studies revealed that TNFalpha did not alter de novo synthesis of enzyme, but it substantially accelerated turnover of CCTalpha. Addition of N-acetyl-Leu Leu-Nle-CHO (ALLN), the calpain I inhibitor, or lactacystin, the 20 S proteasome inhibitor, blocked the inhibition of PtdCho biosynthesis mediated by TNFalpha. TNFalpha-induced degradation of CCTalpha protein was partially blocked by ALLN or lactacystin. CCT was ubiquitinated, and ubiquitination increased after TNFalpha exposure. m-Calpain degraded both purified CCT and CCT in cellular extracts. Thus, TNFalpha inhibits PtdCho synthesis by modulating CCT protein stability via the ubiquitin-proteasome and calpain-mediated proteolytic pathways. PMID- 10734123 TI - Oxidative phosphorylation, Ca(2+) transport, and fatty acid-induced uncoupling in malaria parasites mitochondria. AB - Respiration, oxidative phosphorylation, calcium uptake, and the mitochondrial membrane potential of trophozoites of the malaria parasite Plasmodium berghei were assayed in situ after permeabilization with digitonin. ADP promoted an oligomycin-sensitive transition from resting to phosphorylating respiration. Respiration was sensitive to antimycin A and cyanide. The capacity of trophozoites to sustain oxidative phosphorylation was additionally supported by the detection of an oligomycin-sensitive decrease in mitochondrial membrane potential induced by ADP. Phosphorylation of ADP could be obtained in permeabilized trophozoites in the presence of succinate, citrate, alpha ketoglutarate, glutamate, malate, dihydroorotate, alpha-glycerophosphate, and N,N,N',N'-tetramethyl-p-phenylenediamine. Ca(2+) uptake caused membrane depolarization compatible with the existence of an electrogenically mediated Ca(2+) transport system in these mitochondria. An uncoupling effect of fatty acids was partly reversed by bovine serum albumin, ATP, or GTP and not affected by atractyloside, ADP, glutamate, or malonate. Evidence for the presence of a mitochondrial uncoupling protein in P. berghei was also obtained by using antibodies raised against plant uncoupling mitochondrial protein. Together these results provide the first direct biochemical evidence of mitochondrial function in ATP synthesis and Ca(2+) transport in a malaria parasite and suggest the presence of an H(+) conductance in trophozoites similar to that produced by a mitochondrial uncoupling protein. PMID- 10734124 TI - Structural relationships and sialylation among meningococcal L1, L8, and L3,7 lipooligosaccharide serotypes. AB - Eighteen of 34 endemic meningococcal case strains were of the L8 lipooligosaccharide (LOS) type; four of these were both L3 and L7 (L3,7), and seven were L1. L1 structures arose by alternative terminal Gal substitutions of lactosyl diheptoside L8 structures, as determined by electrospray ionization and other mass spectrometric techniques, and enzymatic and chemical degradations (Structures L1 and L1a). [see text for structure] The more abundant molecule, designated L1, had a trihexose globosyl alpha chain; the less abundant one, designated L1a, had a beta-lactosyl alpha chain and a parallel alpha-lactosaminyl gamma chain. A P(k) globoside (Galalpha1-->4Galbeta1-->4 Glc-R) monoclonal antibody bound 9/10 L1 strains, but a P(1) globoside (Galalpha1-->4Galbeta1- >4GlcNAc-R) mAb bound none of them. alpha-Galactosidase caused loss of both L1 structures and creation of L8 structures; beta-galactosidase caused loss of the L8 determinant. The L1/P(k) glycose was partially sialylated. Some LOS also had unsubstituted basal beta-GlcNAc additions. These structural relationships explain co-expression of L8, L1, and L3,7 serotypes. PMID- 10734125 TI - Rho family proteins modulate rapid apoptosis induced by cytotoxic T lymphocytes and Fas. AB - Little is known about the role of Rho proteins in apoptosis produced by stimuli evolved specifically to produce apoptosis, such as granzymes from cytotoxic T lymphocytes (CTLs) and Fas. Here we demonstrate that all three Rho family members are involved in CTL- and Fas-induced killing. Dominant-negative mutants of each Rho family member and Clostridium difficile toxin B, an inhibitor of all family members, strongly inhibited the susceptibility of cells to CTL- and Fas-induced apoptosis. Fas-induced caspase-3 activation was inhibited by C. difficile toxin. Activated mutants of each GTPase increased susceptibility to apoptosis, and activation of Cdc42 increased within 5 min of Fas stimulation. In contrast, during the time required for CTL and Fas killing, no apoptosis was produced by dominant-negative or activated mutants or by C. difficile toxin alone. Inhibition of actin polymerization using latrunculin A reduced the ability of constitutively active GTPase mutants to stimulate apoptosis and blocked Fas-induced activation of caspase-3. Furthermore, the ability of Rac to enhance apoptosis was decreased by point mutations reported to block Rac induction of actin polymerization. Rho family proteins may regulate apoptosis through their effects on the actin cytoskeleton. PMID- 10734126 TI - The stability of the Cdc6 protein is regulated by cyclin-dependent kinase/cyclin B complexes in Saccharomyces cerevisiae. AB - The Saccharomyces cerevisiae Cdc6 protein is necessary for the formation of prereplicative complexes that are a prerequisite for firing origins during DNA replication in the S phase. In budding yeast, the presence of Cdc6 protein is normally restricted to the G(1) phase of the cell cycle, at least partly because of its proteolytic degradation in the late G(1)/early S phase. Here we show that a Cdc28-dependent mechanism targets p57(CDC6) for degradation in mitotic-arrested budding yeast cells. Consistent with this observation, Cdc6-7 and Cdc6-8 proteins, mutants lacking Cdc28 phosphorylation sites, are stabilized relative to wild-type Cdc6. Our data also suggest a correlation between the absence of Cdc28/Clb kinase activity and Cdc6 protein stabilization, because a drop in Cdc28/Clb-associated kinase activity allows mitotic-arrested cells to accumulate Cdc6 protein. Finally, we also show that cdc28 temperature-sensitive G(1) mutants accumulate Cdc6 protein because of a post-transcriptional mechanism. Our data suggest that budding yeast cells target Cdc6 for degradation through a Cdc28 dependent mechanism in each cell cycle. PMID- 10734127 TI - Translocation of the Rac1 guanine nucleotide exchange factor Tiam1 induced by platelet-derived growth factor and lysophosphatidic acid. AB - Several guanine nucleotide exchange factors for the Rho family of GTPases that induce activation by exchanging GDP for GTP have been identified. One of these is the tumor invasion gene product Tiam1, which acts on Rac1. In this study, we demonstrate that platelet-derived growth factor (PDGF) and lysophosphatidic acid induce the translocation of Tiam1 to the membrane fraction of NIH 3T3 fibroblasts in a time-dependent manner. Previously, we have shown that Tiam1 is phosphorylated by protein kinase C (PKC) and calcium/calmodulin kinase II (CaMK II) after stimulation with agonists. Here we show, by pretreatment of cells with kinase inhibitors, that CaMK II, but not PKC, is involved in the membrane translocation of Tiam1. Addition of the calcium ionophore ionomycin alone induced the translocation of Tiam1. However, the cell-permeable diacylglycerol oleoylacetylglycerol was without effect and did not enhance the effect of ionomycin. These data further indicated a role for CaMK II and not PKC. Inhibition of phosphoinositide 3-kinase by wortmannin had little effect on the translocation of Tiam1. The role of phosphorylation was further studied by comparing the phosphorylation pattern of Tiam1 in the membranes versus whole cell Tiam1. PDGF-induced phosphorylation of membrane-associated Tiam1 occurred more rapidly than that of the total Tiam1 pool, and CaMK II, but not PKC, played a significant role in this process. Furthermore, by using the p21-binding domain of PAK-3, we show that PDGF, but not lysophosphatidic acid, activates Rac1 in vivo and that this activation involves CaMK II and PKC, but not 3-phosphoinositides. Our results indicate that Tiam1 is translocated to and phosphorylated at membranes after agonist stimulation and that CaMK II, but not PKC, is involved in this process. Also, these kinases are involved in the activation of Rac in vivo. PMID- 10734128 TI - A novel activation function for NAB proteins in EGR-dependent transcription of the luteinizing hormone beta gene. AB - The EGR1/NGFI-A transcription factor directly activates the luteinizing hormone beta (LHbeta) subunit promoter, and female mice lacking EGR1 are infertile due to LHbeta deficiency. The NGFI-A-binding proteins NAB1 and NAB2 are corepressors of EGR1/NGFI-A and of the related proteins EGR2/Krox20 and EGR3. Here we report that at certain promoters, including LHbeta, NAB proteins display a novel ability to stimulate EGR-directed transcription. NAB coactivation requires the conserved NCD2 protein domain, previously implicated in NAB corepression, is strictly dependent upon EGR binding to the LHbeta proximal promoter and is independent of EGR activation domains. Furthermore, we report that NAB-activated promoters such as LHbeta contain EGR consensus sites that are fewer in number and lower in binding affinity than those found at NAB-repressed promoters such as basic fibroblast growth factor. Analysis of mutant and synthetic promoters confirms that both the strength and multiplicity of EGR-binding sites influence the transcriptional outcome of NAB recruitment. These results suggest a novel means by which EGR target genes could be differentially regulated in cells where EGR and NAB proteins are coexpressed. PMID- 10734129 TI - Physical proximity and functional association of glycoprotein 1balpha and protein disulfide isomerase on the platelet plasma membrane. AB - Platelet function is influenced by the platelet thiol-disulfide balance. Platelet activation resulted in 440% increase in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1balpha (GP1balpha). PDI contains two active site dithiols/disulfides. The active sites of 26% of the PDI on resting platelets was in the dithiol form, compared with 81% in the dithiol form on activated platelets. Similarly, GP1balpha presented one or more free thiols on the activated platelet surface but not on resting platelets. Anti-PDI antibodies increased the dissociation constant for binding of vWF to platelets by approximately 50% and PDI and GP1balpha were sufficiently close on the platelet surface to allow fluorescence resonance energy transfer between chromophores attached to PDI and GP1balpha. Incubation of resting platelets with anti-PDI antibodies followed by activation with thrombin enhanced labeling and binding of monoclonal antibodies to the N-terminal region of GP1balpha on the activated platelet surface. These observations indicated that platelet activation triggered reduction of the active site disulfides of PDI and a conformational change in GP1balpha that resulted in exposure of a free thiol(s). PMID- 10734130 TI - Expression of Fas ligand in activated T cells is regulated by c-Myc. AB - The transcription factor c-Myc is important for the control of cell cycle progression, neoplasia, and apoptotic cell death. c-Myc dimerizes with its partner Max to form an active transcription factor complex. Little is known, however, about the transcriptional targets of c-Myc and their roles in c-Myc induced cell death. Here we demonstrate that T cell activation-induced expression of Fas ligand (FasL, CD95-L, APO-1-L), which can induce apoptotic cell death in many different cell types, is regulated by c-Myc. Down-modulation of c-Myc protein via antisense oligonucleotides blocked activation-induced FasL mRNA and protein expression and functional FasL expression in activated T cells and T cell lines. Further, FasL promoter activity in T cells is driven by overexpression of c-Myc and inhibited by expression of dominant-negative mutants of c-Myc and Max. Our findings indicate that c-Myc controls apoptotic cell death in T cells through regulation of FasL expression. PMID- 10734131 TI - PU.1 and interferon consensus sequence-binding protein regulate the myeloid expression of the human Toll-like receptor 4 gene. AB - The protein product of the Toll-like receptor (TLR) 4 gene has been implicated in the signal transduction events induced by lipopolysaccharide (LPS). In mice, destructive mutations of Tlr4 impede the normal response to LPS and cause a high susceptibility to Gram-negative infection. Expression of TLR4 mRNA in humans is restricted to a small number of cell types, including LPS-responsive myeloid cells, B-cells, and endothelial cells. To investigate the molecular basis for TLR4 expression in cells of myeloid origin, we cloned the human TLR4 gene and analyzed its putative 5'-proximal promoter. In transient transfections a region of only 75 base pairs upstream of the major transcription initiation site was sufficient to induce maximal luciferase activity in THP-1 cells. The sequence of this region is similar in human and mouse TLR4 genes and lacks a TATA box, typical Sp1-sites or CCAAT box sequences. Instead, it contains consensus-binding sites for Ets family transcription factors, octamer-binding factors, and a composite interferon response factor/Ets motif. The activity of the promoter in macrophages was strictly dependent on the integrity of both half sites of the composite interferon response factor/Ets motif, which was constitutively bound by the myeloid and B-cell-specific transcription factor PU.1 and interferon consensus sequence-binding protein. These results indicate that the two tissue restricted transcription factors PU.1 and interferon consensus sequence-binding protein participate in the basal regulation of human TLR4 in myeloid cells. Cloning of the human TLR4 gene provides a basis for further investigation of the possible impact of genetic variations on the susceptibility to infection and sepsis. PMID- 10734132 TI - Fibrillar amyloid beta-protein mediates the pathologic accumulation of its secreted precursor in human cerebrovascular smooth muscle cells. AB - Cerebrovascular deposition of the amyloid beta-protein (Abeta) is a key pathologic lesion seen in patients with Alzheimer's disease and certain related disorders, including hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D). The deposition of Abeta has pronounced deleterious effects on smooth muscle cells within the cerebral vessel wall. We have previously shown that Abeta(1-40) possessing the E22Q HCHWA-D mutation extensively assembles into fibrils on the surface of cultured human cerebrovascular smooth muscle (HCSM) cells. This cell-surface Abeta fibril formation induces a series of pathologic responses in cultured HCSM cells, including a marked increase in the levels of cell-associated amyloid beta-protein precursor (AbetaPP) and cell death. In the present study, we investigated the relationship between HCSM cell-surface Abeta fibril formation and the striking increase in cell-associated AbetaPP. Time course studies showed that cell-surface HCHWA-D Abeta(1-40) fibril formation occurred rapidly, whereas both the increase in cell-associated AbetaPP and loss of cell viability were delayed responses. Domain analysis using site-specific antibodies indicated that the vast majority of the increase in cell-associated AbetaPP was secreted AbetaPP (sAbetaPP). Localization studies showed that the sAbetaPP was present on the HCSM cell surface. This result raised the possibility that sAbetaPP may bind back to HCSM cell-surface fibrils formed by HCHWA-D Abeta(1-40). Indeed, binding of biotinylated sAbetaPP to fibrillar HCHWA-D Abeta(1-40) was demonstrated by transmission electron microscopy. Furthermore, solid-phase binding assays showed that biotinylated sAbetaPP exhibited dose dependent, saturable binding to fibrillar (but not soluble) HCHWA-D Abeta(1-40) with k(d) approximately 28 nM. Exon deletion experiments further defined a fragment of sAbetaPP (AbetaPP(18-119)), encoded by AbetaPP exons 2 and 3, to contain the fibrillar Abeta-binding domain. In addition, AbetaPP(18-119) effectively blocked the cell-surface accumulation of sAbetaPP and subsequent cell death in HCSM cells treated with pathogenic Abeta. Together, these findings could explain the accumulation of AbetaPP in cerebrovascular Abeta deposits observed both in vitro and in vivo and may contribute to the pathologic responses evoked by pathogenic forms of Abeta in HCSM cells. PMID- 10734133 TI - Identification of tyrosine phosphatases that dephosphorylate the insulin receptor. A brute force approach based on "substrate-trapping" mutants. AB - Many pharmacologically important receptors, including all cytokine receptors, signal via tyrosine (auto)phosphorylation, followed by resetting to their original state through the action of protein tyrosine phosphatases (PTPs). Establishing the specificity of PTPs for receptor substrates is critical both for understanding how signaling is regulated and for the development of specific PTP inhibitors that act as ligand mimetics. We have set up a systematic approach for finding PTPs that are specific for a receptor and have validated this approach with the insulin receptor kinase. We have tested nearly all known human PTPs (45) in a membrane binding assay, using "substrate-trapping" PTP mutants. These results, combined with secondary dephosphorylation tests, confirm and extend earlier findings that PTP-1b and T-cell PTP are physiological enzymes for the insulin receptor kinase. We demonstrate that this approach can rapidly reduce the number of PTPs that have a particular receptor or other phosphoprotein as their substrate. PMID- 10734134 TI - RbAp48 belongs to the histone deacetylase complex that associates with the retinoblastoma protein. AB - The retinoblastoma susceptibility gene product, the Rb protein, is a key regulator of mammalian cell proliferation. One of the major targets of Rb is the S phase inducing E2F transcription factor. Once bound to E2F, Rb represses the expression of E2F-regulated genes. Transcriptional repression by Rb is believed to be crucial for the proper control of cell growth. Recently, we and others showed that Rb represses transcription through the recruitment of a histone deacetylase. Interestingly, we show here that the Rb-associated histone deacetylase complex could deacetylate polynucleosomal substrates, indicating that other proteins could be present within this complex. The Rb-associated protein RbAp48 belongs to many histone deacetylase complexes. We show here that the histone deacetylase HDAC1 is able to mediate the formation of a ternary complex containing Rb and RbAp48. Moreover, less deacetylase activity was found associated with Rb in cell extracts depleted for RbAp48 containing complexes, demonstrating that Rb, histone deacetylase, and RbAp48 are physically associated in live cells. Taken together, these data indicate that RbAp48 is a component of the histone deacetylase complex recruited by Rb. Finally, we found that E2F1 and RbAp48 are physically associated in the presence of Rb and HDAC1, suggesting that RbAp48 could be involved in transcriptional repression of E2F-responsive genes. PMID- 10734135 TI - Apoptosis signal-regulating kinase 1 (ASK1) induces neuronal differentiation and survival of PC12 cells. AB - Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen activated protein kinase kinase kinase that activates the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase signaling cascades. We report here that expression of constitutively active ASK1 (ASK1DeltaN) induces neurite outgrowth in the rat pheochromocytoma cell line PC12. We found that p38 and to a lesser extent JNK, but not ERK, were activated by the expression of ASK1DeltaN in PC12 cells. ASK1DeltaN-induced neurite outgrowth was strongly inhibited by treatment with the p38 inhibitor SB203580 but not with the MEK inhibitors, suggesting that activation of p38, rather than of ERK, is required for the neurite-inducing activity of ASK1 in PC12 cells. We also observed that ASK1DeltaN induced expression of several neuron-specific proteins and phosphorylation of neurofilament proteins, confirming that PC12 cells differentiated into mature neuronal cells by ASK1. Moreover, ASK1DeltaN-expressing PC12 cells survived in serum-starved condition. ASK1 thus appears to mediate signals leading to both differentiation and survival of PC12 cells. Together with previous reports indicating that ASK1 functions as a pro-apoptotic signaling intermediate, these results suggest that ASK1 has a broad range of biological activities depending on cell types and/or cellular context. PMID- 10734136 TI - Serum response factor-dependent regulation of the smooth muscle calponin gene. AB - Smooth muscle calponin is a multifunctional, thin filament-associated protein whose expression is restricted to smooth muscle cell lineages in developing and postnatal tissues. Although the physiology of smooth muscle calponin has been studied extensively, the cis-elements governing its restricted pattern of expression have yet to be identified. Here we report on smooth muscle-specific enhancer activity within the first intron of smooth muscle calponin. Sequence analysis revealed a proximal consensus intronic CArG box and two distal intronic CArG-like elements, each of which bound recombinant serum response factor (SRF) as well as immunoreactive SRF from smooth muscle nuclear extracts. Site-directed mutagenesis studies suggested that the consensus CArG box mediates much of the intronic enhancer activity; mutating all three CArG elements abolished the ability of SRF to confer enhancer activity on the smooth muscle calponin promoter. Cotransfecting a dominant-negative SRF construct attenuated smooth muscle-specific enhancer activity, and transducing smooth muscle cells with adenovirus harboring the dominant-negative SRF construct selectively reduced steady-state expression of endogenous smooth muscle calponin. These results demonstrate an important role for intronic CArG boxes and the SRF protein in the transcriptional control of smooth muscle calponin in vitro. PMID- 10734137 TI - SYNCRIP, a cytoplasmic counterpart of heterogeneous nuclear ribonucleoprotein R, interacts with ubiquitous synaptotagmin isoforms. AB - Synaptotagmins (Syts) are a large family of membrane proteins consisted of at least 12 isoforms. They are categorized in neuron-specific isoforms (I-V, X, and XI) and ubiquitous isoforms (VI-IX) based on their expression patterns. Syt-I, a neuron-specific and abundant isoform, has been well characterized and postulated to be the exocytotic Ca(2+) sensor. However, the functions of other isoforms remain obscure. Here, we report that ubiquitous isoforms of synaptotagmins, Syt VII, Syt-VIII, and Syt-IX, interacted with a cytoplasmic RNA-binding protein, SYNCRIP (Synaptotagmin-binding, cytoplasmic RNA-interacting protein), through their C2B domains. SYNCRIP was originally found in the Syt-II C2AB domain bound fraction from the mouse brain lysate. cDNA cloning of SYNCRIP cDNA revealed that the protein was highly homologous to heterogeneous nuclear ribonucleoprotein R (hnRNP R) recently identified. SYNCRIP protein was ubiquitously and constantly expressed in various tissues of mice parallel to hnRNP R. SYNCRIP indeed bound RNA with preference to poly(A) RNA; however, in contrast to the nuclear localization of hnRNP R, SYNCRIP was distributed predominantly in the cytoplasm as judged by both biochemical fractionation and immunohistochemical studies. In vitro binding experiments showed the potential interaction of SYNCRIP with C2B domains of Syts except for those of Syt-V, -VI, and -X. Furthermore, the interaction between SYNCRIP and Syt-VII, -VIII, or -IX was revealed by co immunoprecipitation experiments using COS cells transiently expressing each Syt isoform. These findings suggested that SYNCRIP was a target of ubiquitous type of Syts and implied the involvement of ubiquitous Syts in the regulation of dynamics of the cytoplasmic mRNA. PMID- 10734138 TI - Bovine papillomavirus E5 protein induces the formation of signal transduction complexes containing dimeric activated platelet-derived growth factor beta receptor and associated signaling proteins. AB - The bovine papillomavirus E5 protein binds to the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in constitutive activation of the receptor and cell growth transformation. By subjecting extracts from E5 transformed or PDGF-treated cells to velocity sedimentation in sucrose gradients, activated PDGF beta receptor complexes were separated from monomeric, inactive receptor. Rapidly sedimenting activated complexes contained oligomeric (apparently dimeric), tyrosine-phosphorylated PDGF beta receptor, the E5 protein, and associated cellular signaling proteins including the p85 subunit of phosphoinositol 3'-kinase, phospholipase Cgamma, and Ras-GTPase activating protein. These signaling proteins made the major contribution to the increased sedimentation rate of the activated receptor complexes. Pairwise analysis of components of these complexes indicated that multiple signaling proteins and the E5 protein were simultaneously present in the activated complexes. Our results also showed that the E5 protein and PDGF activated only a small fraction of the total PDGF beta receptor, that not all receptor molecules associated with the E5 protein were tyrosine-phosphorylated, and that signaling proteins could bind to hemiphosphorylated receptor dimers. On the basis of these results, we propose a model for the assembly of multiprotein, activated PDGF beta receptor complexes in response to the E5 protein. PMID- 10734139 TI - Inhibition of tumor necrosis factor-alpha transcription in macrophages exposed to febrile range temperature. A possible role for heat shock factor-1 as a negative transcriptional regulator. AB - We previously reported that expression of tumor necrosis factor-alpha (TNFalpha) was attenuated in macrophages exposed to febrile range temperatures. In this study, we analyzed the influence of temperature on TNFalpha transcription in the Raw 264.7 macrophage cell line during incubation at 37 and 39.5 degrees C. The initial activation of TNFalpha transcription in response to endotoxin (LPS) was comparable in the 37 and 39.5 degrees C cell cultures, peaking within 10 min of LPS stimulation. However, the duration of transcriptional activation was markedly reduced in the 39.5 degrees C cells (30-60 min) compared with the 37 degrees C cells (2-4 h). Deletion mapping of the TNFalpha gene revealed that the proximal 85-nucleotide promoter sequence and the 5'-untranslated region were sufficient for temperature sensitivity. This sequence contains six heat shock response element (HRE) half-sites but no complete HREs. Electrophoretic mobility shift and immunoblot assays demonstrated that nuclear transclocation of heat shock factor (HSF) and its activation to a DNA-binding form occurred in the 39.5 degrees C cells in the absence of heat shock protein-70 gene activation. The proximal TNFalpha promoter/5'-untranslated region sequence competed for HSF binding to a classic HRE. Overexpression of HSF-1 reduced activity of the TNFalpha promoter. These data suggest that partial activation of HSF-1 during exposure to febrile, sub-heat shock temperatures may block TNFalpha transcription by binding to its proximal promoter or 5'-untranslated region. PMID- 10734140 TI - Leukotriene D(4) triggers an association between gbetagamma subunits and phospholipase C-gamma1 in intestinal epithelial cells. AB - The proinflammatory mediator leukotriene D(4) (LTD(4)) binds to the seven transmembrane receptor CYSLT(1). Although this leukotriene plays an important biological role, its intracellular signaling pathways are only partly known. In previous experiments, we found that LTD(4) induced tyrosine phosphorylation and translocation of phospholipase (PLC)-gamma1 to a plasma membrane fraction in a human epithelial cell line (Int 407). In the present study, we further examined these signaling events and found that LTD(4) induced a rapid interaction between Gbetagamma subunits and PLC-gamma1; results obtained with GST fusion proteins of PLC-gamma1 suggest that this interaction is mediated via the pleckstrin homology domain of PLC-gamma1. Moreover, LTD(4) induced an increased association of c-Src with PLC-gamma1, and the selective Src family tyrosine kinase inhibitor PP1 blocked both LTD(4)-induced tyrosine phosphorylation of PLC-gamma1 and the association of PLC-gamma1 with Gbetagamma subunits. The relevance of these observations in intracellular calcium signaling was investigated by microinjecting cells with anti-Gbeta, anti-PLC-gamma1, or anti-c-Src antibodies and by pretreatment with PP1. LTD(4)-induced calcium mobilization was blocked by each of the indicated antibodies (but not isotype-matched control antibodies) and by PP1. Our data suggest that Gbetagamma subunits can, directly or indirectly, serve as membrane-bound partners for PLC-gamma1 and c-Src and that each of these proteins is essential for LTD(4)-induced downstream PLC-gamma1 signaling. PMID- 10734141 TI - The COOH-terminal transactivation domain plays a key role in regulating the in vitro and in vivo function of Pax3 homeodomain. AB - Efficient transcription activation by Pax3 requires binding to a complex DNA sequence element containing binding sites for both the paired domain and the Prd type homeodomain. Previously, we have shown that this requirement is lost in PAX3 FKHR, the product of a t(2;13) chromosomal translocation associated with alveolar rhabdomyosarcoma. In contrast to Pax3, the chimeric PAX3-FKHR, which acts as an oncogene, can efficiently activate a DNA sequence element containing only a homeodomain binding site (TAATAN(2-3)ATTA), despite the presence of an intact Pax3 paired domain. Here, we showed that this alteration in sequence-specific transcription activity was determined in part by the transactivation domain. First, we demonstrated that in intact Pax3, substitution of the Pax3 transactivation domain with an unrelated viral VP16 transactivation domain enabled Pax3 to transactivate homeodomain-specific DNA sequence, as well as to transform fibroblasts. Furthermore, we could abolish the homeodomain-dependent transcription and transforming activities of PAX3-FKHR by replacing its FKHR transactivation domain with Pax3 transactivation domain. Collectively, these results suggested that the transactivation domain influences the DNA binding specificity of Pax3. The translocation process increased the oncogenic potential of Pax3 by removing the inhibitory action of Pax3 transactivation domain on its homeodomain. PMID- 10734142 TI - The MutL ATPase is required for mismatch repair. AB - Members of the MutL family contain a novel nucleotide binding motif near their amino terminus, and the Escherichia coli protein has been found to be a weak ATPase (Ban, C., and Yang, W. (1998) Cell 95, 541-552). Genetic analysis has indicated that substitution of Lys for Glu-32 within this motif of bacterial MutL results in a strong dominant negative phenotype (Aronshtam, A., and Marinus, M. G. (1996) Nucleic Acids Res. 24, 2498-2504). By in vitro comparison of MutL-E32K with the wild type protein, we show the mutant protein to be defective in DNA activated ATP hydrolysis, as well as MutS- and MutL-dependent activation of the MutH d(GATC) endonuclease and the mismatch repair excision system. MutL-E32K also acts in dominant negative manner in the presence of wild type MutL in vitro, inhibiting the overall mismatch repair reaction, as well as MutH activation. As judged by protein affinity chromatography, MutL and MutL-E32K both support formation of ternary complexes that also contain MutS and MutH or MutS and DNA helicase II. These findings imply that the MutL nucleotide binding center is required for mismatch repair and suggest that the dominant negative behavior of the MutL-E32K mutation is due to the formation of dead-end complexes in which the MutL-E32K protein is unable to transduce a signal from MutS that otherwise results in mismatch-dependent activation of the MutH d(GATC) endonuclease or the unwinding activity of helicase II. PMID- 10734143 TI - The xeroderma pigmentosum group C protein complex XPC-HR23B plays an important role in the recruitment of transcription factor IIH to damaged DNA. AB - The xeroderma pigmentosum group C protein complex XPC-HR23B was first isolated as a factor that complemented nucleotide excision repair defects of XP-C cell extracts in vitro. Recent studies have revealed that this protein complex plays an important role in the early steps of global genome nucleotide excision repair, especially in damage recognition, open complex formation, and repair protein complex formation. However, the precise function of XPC-HR23B in global genome repair is still unclear. Here we demonstrate that XPC-HR23B interacts with general transcription factor IIH (TFIIH) both in vivo and in vitro. This interaction is thought to be mediated through the specific affinity of XPC for the TFIIH subunits XPB and/or p62, which are essential for both basal transcription and nucleotide excision repair. Interestingly, association of TFIIH with DNA was observed in both wild-type and XP-A cell extracts but not in XP-C cell extracts, and XPC-HR23B could restore the association of TFIIH with DNA in XP-C cell extracts. Moreover, we found that XPC-HR23B was necessary for efficient association of TFIIH with damaged DNA in cell-free extracts. We conclude that the XPC-HR23B protein complex plays a crucial role in the recruitment of TFIIH to damaged DNA in global genome repair. PMID- 10734144 TI - Anchoring of surface proteins to the cell wall of Staphylococcus aureus. Sortase catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH(2)-Gly(3) substrates. AB - Staphylococcus aureus sortase anchors surface proteins to the cell wall envelope by cleaving polypeptides at the LPXTG motif. Surface proteins are linked to the peptidoglycan by an amide bond between the C-terminal carboxyl and the amino group of the pentaglycine cross-bridge. We find that purified recombinant sortase hydrolyzed peptides bearing an LPXTG motif at the peptide bond between threonine and glycine. In the presence of NH(2)-Gly(3), sortase catalyzed exclusively a transpeptidation reaction, linking the carboxyl group of threonine to the amino group of NH(2)-Gly(3). In the presence of amino group donors the rate of sortase mediated cleavage at the LPXTG motif was increased. Hydrolysis and transpeptidation required the sulfhydryl of cysteine 184, suggesting that sortase catalyzed the transpeptidation reaction of surface protein anchoring via the formation of a thioester acyl-enzyme intermediate. PMID- 10734145 TI - Regulation of the NF-kappaB activation pathway by isolated domains of FIP3/IKKgamma, a component of the IkappaB-alpha kinase complex. AB - FIP3, isolated as a type 2 adenovirus E3-14.7-kDa interacting protein, is an essential component of the multimeric IkappaB-alpha kinase (IKK) complex and has been shown to interact with various components (Fas receptor-interacting protein, NF-kappaB-inducing kinase, IKKbeta) of the NF-kappaB activation pathway. FIP3 has also been shown to repress basal and tumor necrosis factor (TNF) alpha-induced NF kappaB activity as well as to induce cell death when overexpressed. The adenovirus E3-14.7-kDa protein (E3-14.7K) is an inhibitor of TNFalpha-induced cell death. In the current study, we generated deletion mutants to map the domains of FIP3, which are responsible for its various functions. The NF-kappaB inhibitory activity and the E3-14.7K binding domains were mapped at the carboxyl half of the FIP3 protein. We also found that the carboxyl-terminal half of FIP3 blocked TNFalpha-induced IkappaB-alpha phosphorylation and subsequent degradation, which suggests that the stabilization of the cytoplasmic inhibitor of NF-kappaB underlies the FIP3 inhibition of NF-kappaB activity. The amino terminal 119 amino acids were responsible for the FIP3-IKKbeta and FIP3-IKKalpha interaction, and the middle of the protein (amino acids 201-300) appeared to be both the FIP3 self-association domain as well as the FIP3-Fas receptor interacting protein interaction domain. Thus, FIP3 might serve as a scaffold protein to organize the various components of the IkappaB-alpha kinase complex. Whereas the full-length protein is required for efficient cell death, the amino terminal 200 amino acids are sufficient to cause rounding and detachment of the cells from the monolayer. PMID- 10734146 TI - In vivo gene modification elucidates subtype-specific functions of alpha(2) adrenergic receptors. AB - Mice with altered alpha(2)-adrenergic receptor genes have become important tools in elucidating the subtype-specific functions of the three alpha(2)-adrenergic receptor subtypes because of the lack of sufficiently subtype-selective pharmacological agents. Mice with a deletion (knockout) of the alpha(2A)-, alpha(2B)-, or alpha(2C)-gene as well as a point mutation of the alpha(2A)-gene (alpha(2A)-D79N) and a 3-fold overexpression of the alpha(2C)-gene have been generated. Studies with these mice indicate that most of the classical functions mediated by the alpha(2)-adrenergic receptor, such as hypotension, sedation, analgesia, hypothermia, and anesthetic-sparing effect, are mediated primarily by the alpha(2A)-subtype. The alpha(2B)-subtype is the principal mediator of the hypertensive response to alpha(2)-agonists, appears to play a role in salt induced hypertension, and may be important in developmental processes. The alpha(2C)-subtype appears to be involved in many central nervous system processes such as the startle reflex, stress response, and locomotion. Both the alpha(2A)- and alpha(2C)-subtypes are important in the presynaptic inhibition of norepinephrine release and appear to have distinct regulatory roles. The ability to study subtype-specific functions in different mouse strains by altering the same alpha(2)-adrenergic receptor in different ways strengthens the conclusions drawn from these studies. Although these genetic approaches have limitations, they have significantly increased our understanding of the functions of alpha(2) adrenergic receptor subtypes. PMID- 10734147 TI - Comparison of the effects of clozapine, risperidone, and olanzapine on ketamine induced alterations in regional brain metabolism. AB - The ability of subanesthetic doses of N-methyl-D-aspartate (NMDA) antagonists to induce positive, negative, and cognitive schizophrenia-like symptoms suggests that reduced NMDA receptor function may contribute to the pathophysiology of schizophrenia. An increasing body of evidence indicates that antipsychotic drugs, especially those with "atypical" properties, can antagonize the effects of NMDA antagonists in a variety of experimental paradigms. We demonstrated previously that clozapine, the prototype of atypical antipsychotics, but not haloperidol, the typical antipsychotic, blocked ketamine-induced alterations in brain metabolism. In this study, effects of clozapine were compared with two of the newer atypical antipsychotic drugs, risperidone and olanzapine, on ketamine induced alterations in regional [(14)C]2-deoxyglucose (2-DG) uptake. A subanesthetic dose of ketamine (25 mg/kg) induced robust increases in 2-DG uptake in limbic cortical regions, hippocampal formation, nucleus accumbens, and basolateral amygdala. Pretreatment of rats with risperidone (0.3 mg/kg) before ketamine administration did not alter the effects of ketamine. These data suggest that novel pharmacological properties may contribute to the effects of clozapine in this model, in addition to the well characterized actions at D(2) and 5HT(2A) receptors. In contrast to the results with risperidone, olanzapine blocked ketamine-induced increases in 2-DG uptake. However, a higher dose of olanzapine (10 mg/kg) was required to completely block the effects of ketamine than would be expected if D(2) and 5HT(2) receptor blocking properties of the drug were solely responsible for its action. The results suggest that the ketamine challenge 2-DG paradigm may be a useful model to identify antipsychotic drugs with atypical characteristics and to explore mechanisms of atypical antipsychotic action. PMID- 10734148 TI - Effect of beta-adrenoceptor blockers on sarcoplasmic reticular function and gene expression in the ischemic-reperfused heart. AB - Although beta-adrenoceptor (beta-AR) blockers are used for the treatment of ischemic heart disease, the mechanisms of their beneficial actions have not been fully elucidated. In view of the role of sarcoplasmic reticular (SR) abnormalities in cardiac dysfunction due to ischemia-reperfusion (I/R), we examined the effects of beta-AR blockers on the I/R-induced changes in SR Ca(2+) uptake and release, as well as the protein contents and gene expression of ryanodine receptor, SR Ca(2+)-pump, phospholamban, and calsequestrin. I/R in isolated rat hearts was induced by stopping the perfusion for 30 min and then reperfusing the ischemic hearts for 60 min. Hearts were treated with or without 10 microM atenolol, a beta(1)-specific blocker, or 10 microM propranolol, a nonspecific beta-blocker, 10 min before inducing ischemia as well as during the reperfusion period. I/R depressed cardiac performance, SR Ca(2+) uptake, and Ca(2+) release activities, protein contents, as well as Ca(2+)/calmodulin dependent protein kinase and cAMP-dependent protein kinase-mediated phosphorylations, significantly. The mRNA levels for SR Ca(2+) pump, ryanodine receptors, phospholamban, and calsequestrin were also reduced by I/R. All these changes due to I/R were partially prevented by beta-AR blocker treatment. The results indicate that the beneficial effects of beta-AR blockers on cardiac performance in the I/R hearts may be related to the prevention of changes in SR Ca(2+) uptake and release activities, protein contents, as well as Ca(2+)/calmodulin-dependent protein kinase and cAMP-dependent protein kinase phosphorylations of SR proteins. On the other hand, the protection of I/R-induced alterations in mRNA levels for SR proteins by beta-AR blockers suggests cardiac SR gene expression as a molecular site of their cardioprotective action. PMID- 10734149 TI - Induction of apoptosis by the O-hydroxyethyl-D(Ser)(8)-cyclosporine A derivative SDZ IMM 125 in rat hepatocytes. AB - The immunosuppressive cyclosporine A derivative, O-hydroxyethyl-D(Ser)(8) cyclosporine (SDZ IMM 125), was examined for its ability to induce apoptosis in rat hepatocytes cultured for 4 or 20 h. Four hours after SDZ IMM 125 treatment, chromatin condensation and fragmentation, and the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeled and Annexin V positive cells increased dose dependently without any observable lactate dehydrogenase leakage. The activity of the cysteine protease, caspase-3, was increased, but not that of caspase-1 and -6. The specific caspase-3 inhibitor, Ac Asp-Glu-Val-Asp-aldehyde, inhibited caspase-3 activation and attenuated SDZ IMM 125-induced apoptosis and lactate dehydrogenase leakage. After 20 h of SDZ IMM 125 incubation, the parameters of apoptosis were further increased. Decreased mitochondrial membrane potential (measured by rhodamine 123 uptake) and cytochrome c release went in parallel with ultrastructural mitochondrial changes, and might be regarded as early events that trigger the apoptotic cascade. Transmission electron microscopy showed cytoplasmic blebbing after 4 h of SDZ IMM 125 incubation. As observed by transmission electron microscopy, treatment with SDZ IMM 125 resulted in an increase in the number of necrotic cells after 20 h, but not after 4 h. Our findings suggest that in rat hepatocyte cultures, SDZ IMM 125 is a specific inducer of apoptosis after short-term incubation, and this overlaps with necrosis after longer treatment periods. It is very likely that the necrosis occurring later is the result of the early apoptotic events. PMID- 10734150 TI - Comparative pharmacodynamics of keliximab and clenoliximab in transgenic mice bearing human CD4. AB - Keliximab and clenoliximab are monkey/human chimeric CD4 monoclonal antibodies (mAbs) of the IgG1 and IgG4 isotypes, respectively. The pharmacokinetics (PK) and pharmacodynamics (PD) of these mAbs were evaluated in transgenic mice bearing human CD4 molecules on their T cells after a single i.v. administration at three dose levels (5-125 mg/kg). The PK of keliximab and clenoliximab were similar, dose-dependent, and adequately described by a two-compartment model with saturable elimination from both compartments. The enumeration of circulating CD4(+) T cells and density of CD4 on their surface were determined as the PD effects. An indirect response model was proposed to characterize the PD effects. With the increase in mAb dose, the maximum intensity (R(max)) of PD effects was increased, and the time to reach R(max) shifted to later times. At all three dose levels, keliximab caused a relatively rapid decline in the number of circulating CD4(+) T cells, which then recovered gradually. In contrast, clenoliximab at the lowest dose (5 mg/kg) did not produce a significant effect on CD4(+) T cell counts compared with the placebo group. At high doses, clenoliximab caused a significant decrease in the number of CD4(+) T cells. Keliximab appeared to be more potent and efficient in depleting CD4(+) T cells. Both mAbs produced similar down-modulation of CD4 at corresponding dose levels. The findings of this study are consistent with the results of a recent clinical trial that emphasize the importance of this transgenic mouse model for evaluating PK/PD to support clinical development of anti-human CD4 mAbs. PMID- 10734151 TI - A subtype of the gamma-aminobutyric acid(B) receptor regulates cholinergic twitch response in the guinea pig ileum. AB - The pharmacological profile of the gamma-aminobutyric acid (GABA)(B) receptor regulating cholinergic twitch contraction in the guinea pig ileum myenteric plexus-longitudinal muscle preparation was investigated. GABA and (-)-baclofen inhibited the contraction, exhibiting quite close potencies (pD(2) for GABA = 5.70; pD(2) for (-)-baclofen = 5.33). The compound CGP 47656 also reduced the cholinergic twitch concentration (pD(2) = 5.42), but its efficacy was significantly lower than that of (-)-baclofen or GABA. Added at varying concentrations, CGP 47656 modified the concentration-response curve of (-) baclofen as expected for a partial agonist. Phaclofen, CGP 36742, CGP 35348, and CGP 52432 behaved as competitive antagonists of (-)-baclofen, exhibiting the following pA(2) values: 3.90, 4.88, 5.02, and 7.82, respectively. The compound CGP 56999 behaved as a potent noncompetitive GABA(B) receptor antagonist. In comparing the pharmacological profile of the ileal receptor with those of the previously characterized pharmacological subtypes of the GABA(B) receptor present in the central nervous system, it can be seen that the GABA(B) receptor inhibiting cholinergic twitch contraction in guinea pig ileum myenteric plexus longitudinal muscle mostly resembles the receptor located on somatostatin human neocortex nerve terminals. PMID- 10734152 TI - Pyrrolo-1,5-benzoxazepines induce apoptosis in HL-60, Jurkat, and Hut-78 cells: a new class of apoptotic agents. AB - Some, but not all, of a series of novel pyrrolo-1,5-benzoxazepines (PBOXs) induce apoptosis as shown by cell shrinkage, chromatin condensation, and DNA fragmentation in three human cell lines, HL-60 promyelocytic, Jurkat T lymphoma, and Hut-78 s.c. lymphoma cells. This chemical selectivity, together with the lack of apoptotic activity against rat Leydig cells, argues against a general cell poisoning effect. PBOX-6, a potent member of the series, caused activation of a member of the caspase-3 family of proteases. In addition, the caspase-3-like inhibitor z-DEVD-fmk, but not the caspase-1-like inhibitor z-YVAD-fmk prevented PBOX-6-induced apoptosis, suggesting that caspase 3-like proteases are involved in the mechanism by which PBOX compounds induce apoptosis. The release of cytochrome c into the cytosol in HL-60 cells in response to PBOX-6 suggests that this cellular response may be important in the mechanism by which PBOX-6 induces apoptosis. However, reactive oxygen intermediates do not play a key role in PBOX 6-induced apoptosis because neither the free radical scavenger TEMPO nor the antioxidant N-acetylcysteine had any effect on PBOX-6-induced apoptosis. The apoptotic induction seems independent of the mitochondrial peripheral-type benzodiazepine receptor (PBR) that binds these pyrrolobenzoxazepines with high affinity, due to the lack of correlation between their affinities for the receptor and their apoptotic potencies, their high apoptotic activity in PBR deficient cells such as Jurkats, and their lack of apoptotic induction in PBR rich rat Leydig cells. These PBOXs also can overcome nuclear factor-kappaB mediated resistance to apoptosis. This suggests an important potential use of these compounds in drug-resistant cancers. PMID- 10734153 TI - Neuromuscular dysfunction in the jejunum and colon of human leukocyte antigen B27 transgenic rats. AB - HLA-B27 transgenic rats are a model of spontaneous gastrointestinal inflammation associated with expression of human leukocyte antigen (HLA) B27 and beta(2) microglobulin. Our goal was to investigate in vitro enteric nerve regulation and contractile activity in isolated longitudinal muscles from the jejunum and colon of HLA-B27 rats. Nontransgenic age-matched Fisher 344 rats were used as controls. Intestinal inflammation and tissue injury, quantified histologically and through tissue myeloperoxidase activity, were evident in both the jejunum and colon of HLA-B27 rats. Although resting tension and spontaneous activity of the jejunal and colonic muscles from HLA-B27 rats did not differ significantly from controls, responses to both enteric nerve stimulation or direct muscle activation were significantly inhibited. In muscles from HLA-B27 rats, electrical field stimulation (0.5 ms, 0.5-20 Hz) induced low-amplitude contractions (maximal reduction 60-65%) compared with respective controls. In the presence of atropine and guanethidine, nonadrenergic and noncholinergic contractile responses to higher frequencies of stimulation (8-20 Hz) were also of lower amplitude. These changes were accompanied by a shift in neurally mediated contractions from predominantly cholinergic in the jejunum and colon of Fisher 344 rats to predominantly nonadrenergic and noncholinergic in HLA-B27 rats. Furthermore, maximal contractions to carbachol or KCl depolarization were reduced (up to 2.7 fold) compared with respective controls. In the jejunum of HLA-B27 rats the EC(50) level for carbachol was decreased. The data indicate that gastrointestinal inflammation induced by expression of HLA-B27 is associated with hypocontractility and inhibition of enteric cholinergic control of the longitudinal muscle in both the small and large intestine. PMID- 10734154 TI - Potential role of the alpha4 and alpha6 nicotinic receptor subunits in regulating nicotine-induced seizures. AB - Several studies have shown that genetic factors influence sensitivity to nicotine induced seizures in the mouse. We used recombinant inbred (RI) strains derived from the Long-Sleep (LS) and Short-Sleep (SS) mouse lines to assess the possibility that polymorphisms associated with one or more of the nicotinic receptors cosegregate with differential sensitivity to nicotine-induced seizures. Restriction fragment length polymorphisms (RFLPs) associated with the alpha2, alpha3, alpha4, alpha5, and alpha6 nicotinic receptors were identified in the LS and SS mouse lines, but the RI strains were polymorphic for only the alpha4 and alpha6 RFLPs. The RI strains were tested for sensitivity to nicotine-induced seizures. Strain and gender effects on seizure sensitivity were obtained as assessed by ED(50) values and latency to seizure. Those RI strains with the LS like alpha4 RFLP were, on average, more sensitive to nicotine-induced seizures than were those RI strains with SS-like alpha4 RFLP. The alpha6 nicotine receptor may also play a role in modulating nicotine-induced seizures, but this effect is markedly influenced by gender. Females of the RI strains with the LS-like alpha6 RFLP were more sensitive to nicotine than were females of the strains with the SS like alpha6 RFLP. Similar trends were seen in the males, but these trends were not significant. Thus, these strain differences may be due to polymorphisms associated with both the alpha4 and alpha6 nicotinic receptors, but gender also plays an important role in regulating sensitivity to nicotine-induced seizure. PMID- 10734155 TI - Oxidative stress induced by tert-butyl hydroperoxide causes vasoconstriction in the aorta from hypertensive and aged rats: role of cyclooxygenase-2 isoform. AB - We analyzed the mechanisms involved in the effect of tert-butyl hydroperoxide (t BOOH) in isolated aortic rings with and without endothelium from normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) at 6, 18, and 24 months of age. t-BOOH (1 microM-10 mM) induced concentration-dependent contractions that were scarcely modified by aging and potentiated in SHR and by endothelium removal. The nitric oxide synthase and prostacyclin synthase inhibitors N(G)-nitro-L-arginine methyl ester (100 microM) and tranylcypromine (100 microM), respectively, increased both basal tone and the t-BOOH-induced contractions in intact segments from WKY, with these effects not observed in SHR. Indomethacin (10 microM), a nonspecific cyclooxygenase inhibitor, and SQ 29,548 (10 microM), a prostaglandin H(2)/thromboxane A(2) receptor blocker, abolished the t-BOOH-induced vasoconstriction, independent of age and hypertension. In both strains, these contractile responses were unaltered by the thromboxane synthase inhibitor imidazole (10 microM). The cyclooxygenase-2 inhibitor NS-398 (10 microM) abolished or markedly reduced the t-BOOH-induced contractions in segments with or without endothelium, respectively. In addition, expression of cyclooxygenase-2 protein was detected in aorta from WKY and SHR in either basal condition or after stimulation with t-BOOH. These results suggest that (1) t-BOOH induced vasoconstriction in the aorta from WKY and SHR is essentially mediated by cyclooxygenase-2 metabolites, different from thromboxane-A(2), probably prostaglandin-H(2), and/or isoprostanes; (2) aging scarcely modifies, whereas endothelium negatively modulates, these contractions in both strains; and (3) nitric oxide and prostacyclin exert a negative modulator role on the t-BOOH induced vasoconstriction in WKY, with this modulator role lost in SHR. PMID- 10734156 TI - Taurine and niacin block lung injury and fibrosis by down-regulating bleomycin induced activation of transcription nuclear factor-kappaB in mice. AB - The effects of taurine (T) and niacin (N) on bleomycin (BL)-induced increased production of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1alpha, IL-6, and transforming growth factor-beta (TGF-beta) levels in the bronchoalveolar lavage fluid (BALF), and increased collagen content and nuclear factor-kappaB (NF-kappaB) activation in the lungs were investigated in mice. The mice were intratracheally instilled with saline (SA) or BL (0.1 U/mouse/50 microliter) under ketamine and xylazine anesthesia. They had ad libitum access to diet containing 2.5% niacin (w/w) or the same control diet (CD) and water with and without taurine (1%) 3 days before intratracheal instillation and throughout the study. The mice were sacrificed at different times for collecting BALF and lungs, which were appropriately processed for various measurements. Treatment with taurine and niacin attenuated the BL-induced increases in proinflammatory cytokines such as IL-1alpha, TNF-alpha, IL-6, and TGF-beta in BALF and lung hydroxyproline content of the mice in BL + TN groups. Reverse transcription polymerase chain reaction analysis of total RNA from whole lung was performed to assess the induction of TNF-alpha and IL-1 mRNAs as markers of NF-kappaB activation. The NF-kappaB DNA-binding activity in whole-lung extract was evaluated by electrophoretic mobility shift assay. This revealed a progressive increase in NF-kappaB activation and IkBalpha depletion in lungs from mice in BL + CD groups from day 1 through day 21 compared with the corresponding SA + CD control groups. Treatment with taurine and niacin generally inhibited the BL induced increases in the nuclear localization of NF-kappaB and preserved IkappaBalpha protein in BL + TN groups. This may be one of the mechanisms for the antifibrotic effect of taurine and niacin. PMID- 10734157 TI - Dopamine decreases melatonin content in golden hamster retina. AB - Dopamine significantly decreased melatonin levels in Golden hamster retinas excised at noon and incubated under light. The effect of dopamine was reversed by spiperone and clozapine (selective antagonists for D(2) and for D(4)/D(2) dopaminergic receptors, respectively) but not by SCH 23390 (a selective D(1) dopamine receptor antagonist). Both clozapine and spiperone per se significantly increased melatonin levels, whereas SCH 23390 was ineffective. Quinpirole (an agonist for D(2)-subfamily dopaminergic receptor) decreased melatonin content in retinas excised at midday. Dopamine increased, whereas quinpirole decreased, cAMP accumulation in retinas excised at noon. Retinal dopaminergic turnover rate (assessed as the ratio of 3,4-dihydroxyphenylacetic acid to dopamine) was significantly higher at midday than at midnight. In retinas excised at midnight, melatonin content in vitro was unaffected by dopamine or quinpirole. At midnight, dopamine increased cAMP accumulation, whereas quinpirole was ineffective. When hamsters were kept under constant darkness for 48 h and sacrificed at subjective midday or midnight, dopamine increased cAMP accumulation at both times, whereas quinpirole decreased this parameter only at subjective midday. Dopaminergic turnover rate was significantly higher at subjective midday than at subjective midnight. These results show that dopamine regulates melatonin biosynthesis in the Golden hamster retina. PMID- 10734158 TI - Signaling mechanisms for muscarinic receptor-mediated coronary vasoconstriction in isolated rat hearts. AB - Signaling mechanisms for muscarinic receptor-mediated vasoconstriction in coronary resistance arteries were studied in potassium-arrested isolated rat hearts perfused at a constant flow rate. The cholinergic agonist bethanechol was given by bolus injection or constant infusion. Perfusion pressure was monitored as an indicator of coronary vascular resistance. Bolus injection of bethanechol evoked a phasic vasoconstriction in a dose-dependent manner, whereas infusion of bethanechol evoked a tonic vasoconstriction without producing tachyphylaxis. Bethanechol-induced phasic vasoconstriction was eliminated by perfusion with a Ca(2+)-free buffer. The L-type voltage-operated Ca(2+) channel blocker nifedipine decreased the maximal constrictor response to bethanechol by 59 +/- 2% (n = 4, P <.001), whereas the putative receptor-operated Ca(2+) channel blocker SK&F 96365 converted this vasoconstriction into vasodilation that was not mediated by nitric oxide. The protein kinase C inhibitor chelerythrine reduced the maximal phasic vasoconstrictor response to bethanechol by 78 +/- 2% (n = 6, P <.001) Bethanechol induced tonic vasoconstriction was rapidly converted to a sustained vasodilation during infusion of SK&F 96365 or nifedipine, whereas infusion of chelerythrine gradually attenuated the tonic response to bethanechol. Results from other experiments do not support a role for phospholipase A(2)-dependent mediators in generating coronary vasoconstrictor responses to bethanechol. It is concluded that voltage-independent receptor-operated Ca(2+) channels, voltage-operated Ca(2+) channels, and protein kinase C are major signaling components for muscarinic receptor-mediated contraction of rat coronary resistance arteries. PMID- 10734159 TI - Stimulation of the Na(+)-K(+) pump in cultured guinea pig airway smooth muscle cells by serotonin. AB - The effect of 5-hydroxytryptamine (5-HT) or serotonin on Na(+)-K(+) pump activity of airway smooth muscle was investigated by measuring (86)Rb(+) uptake in cultured guinea pig tracheal smooth muscle cells. (86)Rb(+) uptake consisted of three distinct components, one sensitive to ouabain, one to bumetanide, and one insensitive to either inhibitor. 5-HT induced a concentration-dependent increase in ouabain-sensitive (86)Rb(+) uptake (EC(50) = 21 nM) but had no effect on bumetanide-sensitive uptake, suggesting that it stimulates the Na(+)-K(+) pump but not the Na(+)-K(+)-Cl(-) cotransporter. Ouabain-sensitive uptake also was stimulated by the 5-HT(2A/2C) agonists 2,5-dimethoxy-4-iodoamphetamine and alpha methyl-5-HT, but not by the 5-HT(1) agonist 5-carboxamidotryptamine, the 5 HT(1A/1B/2C) agonist 1-(3-chlorophenyl)piperazine, or the 5-HT(3) agonist 1-(3 chlorophenyl)biguanide. 5-HT-stimulated (86)Rb(+) uptake was inhibited by the 5 HT(2A) antagonists ketanserin and spiperone, but not by the 5-HT(1A) antagonist NAN 190 or the 5-HT(3) antagonist Y25310. 5-HT-stimulated (86)Rb(+) uptake was inhibited by reducing extracellular Na(+) concentration and by the Na(+)-H(+) exchange inhibitors dimethylamiloride and 5-(N-methyl-N-isobutyl)-amiloride. These observations suggest that 5-HT stimulates the Na(+)-K(+) pump of airway smooth muscle via 5-HT(2A) receptors by a mechanism dependent on Na(+) influx, possibly through the Na(+)-H(+) exchanger. Because stimulation of the Na(+)-K(+) pump produces hyperpolarization, this may represent a negative-feedback mechanism that opposes contraction in response to 5-HT. PMID- 10734160 TI - Selected cysteine residues in transmembrane domains of mu-opioid receptor are critical for effects of sulfhydryl reagents. AB - The effects of sulfhydryl-specific methanethiosulfonate (MTS) derivatives on mu opioid receptor binding were examined in Chinese hamster ovary (CHO) cells that stably express mu-opioid receptors (HmuCHO). Three charged MTS derivatives inhibited the binding of [(3)H][D-Ala(2),N-MePhe(4),Gly-ol(5)]-enkephalin to mu opioid receptors with IC(50) values ranging from 0.12 to 13 mM. Further characterization of the mu-opioid receptor interactions with ethylammonium MTS (the most potent among tested MTS reagents) revealed that ethylammonium MTS inhibition of ligand binding to the receptor was irreversible, with both the maximal receptor binding (B(max)) and the binding affinity (K(d)) being changed. Preincubation of HmuCHO cells with [D-Ala(2),N-MePhe(4), Gly-ol(5)]-enkephalin or naloxone prevented the receptor inactivation normally caused by MTS derivatives, indicating that the reactions may occur within or near the ligand-binding pocket on the receptor. To identify the susceptible sulfhydryl groups, each of the cysteine residues in the mu-receptor transmembrane domains were substituted with serine by site-directed mutagenesis. All of the mutant receptors transiently expressed in COS cells had receptor binding properties similar to the wild-type receptors. However, four mutant receptors with a serine substitution in transmembrane domain III (C161S), IV (C192S), V (C237S), or VII (C332S) displayed significant resistance against MTS inhibition compared with the wild-type receptor. We conclude that these four cysteine residues react with MTS reagents and are responsible for the effect of the MTS reagents on mu-opioid receptor binding. PMID- 10734161 TI - Strain differences in basal and cocaine-evoked dopamine dynamics in mouse striatum. AB - In vivo microdialysis was used to characterize basal dopamine (DA) dynamics and cocaine-evoked DA levels in the striatum of 129/Sv-ter, C57BL/6J, DBA/2J, and Swiss-Webster mice. Basal dialysate levels of DA did not differ in the four strains tested. Similarly, the no net flux method of quantitative microdialysis revealed no difference in extracellular levels between strains. However, the in vivo extraction fraction of DA was significantly less in 129/Sv-ter (53%) mice compared with C57BL/6J (68%), DBA/2J (69%), and Swiss-Webster (67%) mice, indicating a lower rate of basal DA uptake in the 129/Sv-ter strain. Perfusion of K(+) (60 and 100 mM) through the microdialysis probe significantly increased dialysate DA levels and there was no difference between strains in the magnitude of this effect. The acute administration of cocaine (5-20 mg/kg i.p.) increased DA levels in the four strains tested. Cocaine-evoked DA levels (in nanomoles) were significantly greater in 129/Sv-ter compared with C57BL/6J, DBA/2J, or Swiss Webster mice after administration of either 5, 10, or 20 mg/kg cocaine. However, the percentage increase in DA did not differ across strains. These data demonstrate that there are strain-related differences in basal DA dynamics in the striatum of the mouse. Basal DA uptake was reduced in striatum of 129/Sv-ter mice compared with C57BL/6J, DBA/2J, or Swiss-Webster mice. In addition, the response of DA levels to cocaine may be enhanced in 129/Sv-ter compared with C57BL/6J, DBA/2J, or Swiss-Webster mice. PMID- 10734162 TI - Enhancement by epinephrine of benzylpenicillin transport in rat small intestine. AB - The perfusion of rat small intestinal lumen with epinephrine (0.1 mM) resulted in a significant increase in the amount of benzylpenicillin (BP) transported from the mucosal to the serosal side. In this study, the perfusion of the lumen with phenylephrine, clonidine, dobutamine, or salbutamol had no effect on BP transport. However, the combinations of phenylephrine and isoproterenol, clonidine and isoproterenol, and phenylephrine and salbutamol increased the BP transport to a similar extent as that observed with epinephrine alone. Tolazolin or propranolol inhibited the epinephrine-induced increase in BP transport. An increase in the intracellular concentration of cAMP in conjunction with specific activation of either alpha(1)- or alpha(2)-adrenoceptors induced an increase in BP transport similar to that observed in response to epinephrine alone. Staurosporine or N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide abolished the epinephrine-induced increase in BP transport. Peptides or either zwitterionic or anionic cephalosporins also blocked the effect of epinephrine on BP transport. The extent of BP uptake into brush border or basolateral membrane vesicles prepared from epinephrine-perfused intestinal loops was markedly greater than that into vesicles prepared from control loops. The perfusion of intestinal lumen with carbonyl cyanide p-trifluoromethoxy phenylhydrazone, amiloride, or ouabain inhibited epinephrine-induced BP transport. These results indicate that the interaction of epinephrine with both beta(2)-adrenoceptors and either alpha(1)- or alpha(2-)adrenoceptors markedly stimulates the BP transport, an effect likely mediated by the enhancement of the function in the brush border membrane of intestinal epithelial cells coupled with the generation of an H(+) gradient. PMID- 10734164 TI - Antinociceptive activity of [beta-methyl-2', 6'-dimethyltyrosine(1)]-substituted cyclic [D-Pen(2), D-Pen(5)]Enkephalin and [D-Ala(2),Asp(4)]Deltorphin analogs. AB - Research in our laboratories involves the development of selective opioid agonists and antagonists as: 1) pharmacological tools to elucidate the mechanisms of opioid antinociception, and 2) potential analgesics that possess therapeutic advantages over currently available drugs. We hypothesized that the selectivity of peptide agonists toward the opioid receptor types and subtypes is topographically dependent. The current results assess the antinociceptive activity and opioid receptor selectivity of a series of beta-methyl-2',6' dimethyltyrosine (TMT)-substituted cyclic [D-Pen(2),D-Pen(5)]enkephalin (DPDPE) and [D-Ala(2), Asp(4)]deltorphin (DELT I) analogs. Compounds were injected via the intracerebroventricular route into male ICR mice, and antinociception was assessed using the 55 degrees C warm water tail-flick test. Antinociceptive A(50) values ranged from 0.35 to 17 nmol for the DELT I analogs and from 7.05 to >100 nmol for the DPDPE analogs. To test for receptor selectivity, mice were treated with selective mu- and delta-opioid antagonists. In general, mu [beta funaltrexamine (beta-FNA)]- and delta(1) ([D-Ala(2),Leu(5), Cys(6)] enkephalin) antagonists blocked the antinociceptive actions of [TMT(1)]DPDPE analogs, whereas the antinociceptive actions of [TMT(1)]DELT I analogs were more sensitive to antagonism by the delta(2)-selective antagonist [Cys(4)]deltorphin and the mu antagonist beta-FNA. The antinociceptive actions of the [(2R, 3S)-TMT(1)]DELT I analog was suppressed by both [D-Ala(2),Leu(5), Cys(6)]enkephalin and beta-FNA. These results are in contrast to those found with the parent molecules DPDPE (primarily a delta(1) agonist) and DELT I (a mixed delta(1)/delta(2) agonist). These results demonstrate that topographical modification in position 1 of the DPDPE and DELT I peptides affects antinociceptive potency and opioid receptor selectivity. PMID- 10734163 TI - Effects of cannabinoid receptor agonist and antagonist ligands on production of inflammatory cytokines and anti-inflammatory interleukin-10 in endotoxemic mice. AB - Previous studies have shown that mice primed with Corynebacterium parvum produce higher levels of inflammatory cytokines than unprimed mice upon challenge with lipopolysaccharide (LPS). Herein, we describe experiments in which two cannabinoid (CB) agonists, WIN 55212-2 [(R)-(+)-[2, 3-dihydro-5-methyl-3-[(4 morpholinyl)methyl]pyrrolo[1,2,3-de]1, 4-benzoxazin-6-yl](1-naphthyl)methanone) and HU-210 [(-)-11-hydroxy-delta(8) tetrahydrocannabinol-dimethylheptyl], were examined for their effects on LPS-induced cytokines in C. parvum-primed and unprimed mice. These agonists have been reported to bind selectively to the CB2 and CB1 receptor subtypes, respectively. WIN 55212-2 (3.1-50 mg/kg i.p.) and HU 210 (0.05-0.4 mg/kg i.p.) decreased serum tumor necrosis factor-alpha and interleukin-12 (IL-12) and increased IL-10 when administered to mice before LPS. The drugs also protected C. parvum mice (but not unprimed mice) against the lethal effects of LPS. The protection afforded to C. parvum mice could not be attributed to the higher levels of IL-10 present in these mice after agonist treatment. The WIN 55212-2- and HU-210-mediated changes in the responsiveness of mice to LPS were antagonized by SR141716A [N-(piperdin-1-yl)-5-(4-chloropheny)-1 (2, 4-dichloropheny)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride], a selective CB1 receptor antagonist, but not by SR144528 [N-[(1S)-endo-1,3,3 trimethylbicyclo[2.2. 1]heptan-2-yl]5-(4-choro-3-methylphenyl)-1-(4 methylbenzyl)p yrazole-3 -carboxamide], a selective antagonist at the CB2 receptor. Therefore, both CB agonists modulated LPS responses through the CB1 receptor. Surprisingly, SR141716A itself modulated cytokine responses in a manner identical with that of WIN 55212-2 and HU-210 when administered alone to mice. The agonist-like effects of SR141716A, which were more striking in unprimed than in primed mice, suggested that the antagonist also could function as a partial agonist at the CB1 receptor. Our findings indicate a role for the CB1 receptor subtype in cytokine modulation by CB ligands. PMID- 10734165 TI - Effect of chronic ethanol and withdrawal on the mu-opioid receptor- and 5 Hydroxytryptamine(1A) receptor-stimulated binding of [(35)S]Guanosine-5'-O-(3 thio)triphosphate in the fawn-hooded rat brain: A quantitative autoradiography study. AB - Previous studies have shown that chronic ethanol influences the density of central mu-opioid receptors and serotonin(1A) (5-hydroxytryptamine(1A)) receptors. To determine whether the functional coupling of these two receptors to G proteins in the rat brain, particularly in mesocorticolimbic regions, is affected by ethanol, receptor-mediated [(35)S]guanosine-5'-O-(3-thio) triphosphate ([(35)S]GTPgammaS) binding stimulated by [D-Ala(2),N-MePhe(4),Gly ol(5)]-enkephalin (DAMGO) or L694,247 was used. By quantitative autoradiography, receptor-mediated [(35)S]GTPgammaS binding activated by the two agonists was mapped throughout brain sections at the level of the nucleus accumbens and hippocampus from groups of alcohol-preferring Fawn-Hooded (FH) rats after different ethanol consumption paradigms. Significant DAMGO (mu-opioid receptor agonist)-stimulated binding of [(35)S]GTPgammaS was obtained in the striatum, nucleus accumbens, and lateral septum, whereas L694,247 (5 hydroxytryptamine(1A/1B/1D) receptor agonist)-stimulated binding of [(35)S]GTPgammaS was observed in the lateral septum, amygdala, and cingulate cortex. Chronic ethanol self-administration significantly reduced DAMGO stimulated [(35)S]GTPgammaS binding in the nucleus accumbens (-19%), lateral septum (-15%), and striatum (-23%), which recovered toward control levels after ethanol withdrawal. However, chronic ethanol, as well as ethanol withdrawal, failed to produce any significant alteration in L694,247-stimulated [(35)S]GTPgammaS binding in all tested brain regions. The region-specific and receptor-specific alteration of agonist-stimulated [(35)S]GTPgammaS binding suggests that the change of functional coupling of mu-opioid receptors to G proteins induced by chronic ethanol drinking may have a pathophysiological role in the consequences of ethanol consumption. PMID- 10734166 TI - Effects of cigarette smoke on immune response: chronic exposure to cigarette smoke impairs antigen-mediated signaling in T cells and depletes IP3-sensitive Ca(2+) stores. AB - Chronic exposure of mice and rats to cigarette smoke affects T-cell responsiveness that may account for the decreased T-cell proliferative and T dependent antibody responses in humans and animals exposed to cigarette smoke. However, the mechanism by which cigarette smoke affects the T cell function is not clearly understood. Our laboratory has shown that chronic exposure of rats to nicotine inhibits the antibody-forming cell response, impairs the antigen mediated signaling in T cells, and induces T cell anergy. To determine the mechanism of cigarette smoke-induced immunosuppression and to compare it with chronic nicotine exposure, rats were exposed to diluted, mainstream cigarette smoke for up to 30 months or to nicotine (1 mg/kg b.wt./24 h) via miniosmotic pumps for 4 weeks, and evaluated for immunological function in vivo and in vitro. This article presents evidence suggesting that T cells from long-term cigarette smoke-exposed rats exhibit decreased antigen-mediated proliferation and constitutive activation of protein tyrosine kinase and phospholipase C-gamma1 activities. Moreover, spleen cells from smoke-exposed and nicotine-treated animals have depleted inositol-1, 4,5-trisphosphate-sensitive Ca(2+) stores and a decreased ability to raise intracellular Ca(2+) levels in response to T cell antigen receptor ligation. These results suggest that chronic smoking causes T cell anergy by impairing the antigen receptor-mediated signal transduction pathways and depleting the inositol-1,4, 5-trisphosphate-sensitive Ca(2+) stores. Moreover, nicotine may account for or contribute to the immunosuppressive properties of cigarette smoke. PMID- 10734167 TI - ATP inhibits glutamate synaptic release by acting at P2Y receptors in pyramidal neurons of hippocampal slices. AB - It has been proposed that extracellular ATP inhibits synaptic release of glutamate from hippocampal CA1 synapses after its catabolism to adenosine. We investigated the possibility that at least part of this effect is mediated by ATP itself acting on P2Y receptors. ATP and various analogs decreased the amplitude and duration of glutamate-mediated excitatory postsynaptic potentials in all tested neurons. This effect was reversible and concentration-dependent and had the following rank order of agonist potency: AMP = ATP = adenosine-5'-O-(3 thio)triphosphate > adenosine = ADP. alpha,beta-Methylene ATP, beta,gamma methylene ATP, 2-methylthioadenosine 5'-triphosphate, GTP, and UTP induced only a partial response. The depolarization induced by exogenous glutamate was not affected by ATP, indicating that this nucleotide acts presynaptically to inhibit glutamate-mediated excitatory postsynaptic potentials. Neither inhibition of ectonucleotidase activity with alpha,beta-methylene ADP, suramin, or pyridaxalphosphate-6-azophenyl-2',4'-disulfonic acid 4-sodium nor removal of extracellular adenosine (with adenosine deaminase) altered ATP effects. 8 Cyclopentyltheophylline competitively inhibited ATP effects, whereas P2 receptor antagonists (pyridaxalphosphate-6-azophenyl-2',4'-disulfonic acid 4-sodium, suramin, and reactive blue 2) were ineffective. ATP effects were by far more sensitive to pertussis toxin (PTX) than those of adenosine. After PTX, adenosine 5'-O-(3-thio)triphosphate induced only a partial response, and ATP concentration response curve was biphasic. The second phase of this curve was blocked by adenosine deaminase, implying that it is mediated by adenosine as a result of ATP catabolism. Under control conditions, however, catabolism of ATP is not required to explain its actions. In conclusion, ATP inhibits synaptic release of glutamate by direct activation of P2Y receptors that are PTX- and 8-cyclopentyltheophylline sensitive. PMID- 10734168 TI - Cocaine-induced seizure thresholds: quantitative trait loci detection and mapping in two populations derived from the C57BL/6 and DBA/2 mouse strains. AB - Seizures are a well known consequence of human cocaine abuse, and in rodent models, sensitivity to cocaine seizures has been shown to be strongly influenced by genotype. For example, several studies have reported significant differences between the C57BL/6 (B6) and DBA/2 (D2) inbred mouse strains in their sensitivity to cocaine-induced seizures. This prompted our use of the BXD recombinant inbred (RI) strain set and an F(2) population derived from the B6 and D2 progenitor strains for further genetic analyses and for gene mapping efforts in this study. Cocaine was infused into the lateral tail vein, and the doses needed to induce a running bouncing clonic seizure and a tonic hindlimb extensor seizure were recorded for each mouse. In the BXD RI set, a genome-wide search was carried out for QTLs (quantitative trait loci), which are sites on a chromosome containing genes that influence seizure susceptibility. An F(2) population (B6D2F2, n = 408) was subsequently used as a second, confirmation step. Based on both RI and F(2) results, three QTLs emerged as significant (P <.00005): one for clonic seizures on chromosome 9 (distal), and two for tonic seizures on chromosomes 14 (proximal to mid) and 15 (distal). Two additional QTLs emerged as suggestive (P <.0015), both associated with clonic seizures on chromosomes 9 (proximal) and 15 (distal). Both QTLs on chromosome 9 were sex-specific, with much larger effects on the phenotype seen in females than in males. PMID- 10734169 TI - Selectively bred lines of mice show response and drug specificity for genetic regulation of acute functional tolerance to ethanol and pentobarbital. AB - Genetic regulation of acute tolerance to ethanol may be associated with ethanol consumption and other ethanol-related behaviors in rodents. We have used lines of mice, selectively bred for high and low acute functional tolerance (HAFT and LAFT, respectively) to ethanol-induced loss of balance to test this hypothesis. Replicate HAFT and LAFT lines differ in AFT to ethanol-induced loss of balance by 4.4- and 5-fold, respectively. Frequency distributions and mean AFT scores for those lines, F(1), and backcrosses show a dominance for the HAFT phenotype. Time courses for acquisition and decay showed that AFT to ethanol-induced loss of balance developed rapidly, could be maintained up to 6 h with repeated doses, and decayed 6 h after peak tolerance and discontinuance of ethanol administration. The lines did not differ in initial sensitivity as measured by brain ethanol concentration at loss of balance, indicating that initial sensitivity and AFT to loss of balance were not coselected traits. Surprisingly, HAFT versus LAFT lines did not differ in development of AFT to loss of righting response, or hypothermia, indicating different mechanisms or neuronal systems mediate genetic influences on these measures. Voluntary ethanol consumption was low in both of the replicate lines, but HAFT lines consumed greater amounts of ethanol than LAFT lines. The HAFT and LAFT lines developed AFT to pentobarbital-induced loss of balance, however, there were no line differences in rates or extent of the AFT development. These results show that genetic regulation of AFT development is drug- as well as response-specific. PMID- 10734170 TI - Tertiapin potently and selectively blocks muscarinic K(+) channels in rabbit cardiac myocytes. AB - Tertiapin is a 21-residue peptide isolated from honey bee venoms. A recent study indicated that tertiapin is a potent blocker of certain types of inwardly rectifying K(+) (Kir) channels (). We examined the effect of tertiapin on ion channel currents in rabbit cardiac myocytes using the patch-clamp technique. In the whole-cell configuration, tertiapin fully inhibited acetylcholine (1 microM) induced muscarinic K(+) (K(ACh)) channel currents in atrial myocytes with the half-maximum inhibitory concentration of approximately 8 nM through approximately 1:1 stoichiometry. The potency of tertiapin in inhibiting K(ACh) channels was not significantly different at -40 and -100 mV. Tertiapin also inhibited the K(ACh) channel preactivated by intracellular guanosine 5'-O-(3-thiotriphosphate), a nonhydrolyzable GTP analog. A constitutively active Kir channel, the I(K1) channel, was at least 100 times less sensitive to tertiapin. Another Kir channel in cardiac myocytes, the ATP-sensitive K(+) channel, was virtually insensitive to tertiapin (1 microM). The voltage-dependent K(+) and the L-type Ca(2+) channels were not affected by tertiapin (1 microM). At the single-channel level, tertiapin inhibited the K(ACh) channel from the outside of the membrane by reducing the NP(o) (N is the number of functional channels, and the P(o) is the open probability of each channel) without affecting the single-channel conductance or fast kinetics. Therefore, tertiapin potently and selectively blocks the K(ACh) channel in cardiac myocytes in a receptor- and voltage-independent manner. Tertiapin is a novel pharmacological tool to identify the functional role of the K(ACh) channel in the parasympathetic regulation of the heart beat. PMID- 10734171 TI - Signal transduction cascade in staurosporine-induced prostaglandin E(2) production by rat peritoneal macrophages. AB - The possible participation of phosphatidylinositol (PI) 3-kinase, p44/42 mitogen activated protein (MAP) kinases and protein kinase C (PKC) in staurosporine induced prostaglandin E(2) (PGE(2)) production was investigated pharmacologically in rat peritoneal macrophages. When the cells were incubated in the presence of staurosporine (63 nM), phosphorylation of p44/42 MAP kinases and cytosolic phospholipase A(2) (cPLA(2)) was induced at 15 min and increased until 60 min, whereas PGE(2) production and expression of cyclooxygenase-2 (COX-2) protein began to increase at 2 h and increased thereafter. Both PD98059 and U0126, MAP kinase/extracellular signal-regulated kinase (ERK) kinase inhibitors, and LY294002, a PI 3-kinase inhibitor, inhibited staurosporine-induced phosphorylation of p44/42 MAP kinases and cPLA(2) and PGE(2) production. Moreover, U0126 inhibited staurosporine-induced arachidonic acid release at 1 h. Although PD98059 and U0126 at 30 microM partially inhibited staurosporine-induced COX-2 protein expression, they completely inhibited staurosporine-induced PGE(2) production. LY294002 at 100 microM did not inhibit staurosporine-induced expression of COX-2 protein. In contrast, Ro-31-8220, a PKC inhibitor, completely inhibited staurosporine-induced PGE(2) production and COX-2 protein expression at 8 h but did not inhibit staurosporine-induced phosphorylation of p44/42 MAP kinases and cPLA(2). These findings suggest that staurosporine induces PGE(2) production by two mechanisms. One is cPLA(2) phosphorylation through a signal transduction pathway from PI 3-kinase to p44/42 MAP kinases, by which arachidonic acid, a substrate for COX-1 and COX-2, is increased. The other is COX-2 protein expression, which is induced mainly by activation of PKC and partially by activation of p44/42 MAP kinases; thus, arachidonic acid is metabolized to PGE(2). PMID- 10734172 TI - Substance P in the dorsal motor nucleus of the vagus evokes gastric motor inhibition via neurokinin 1 receptor in rat. AB - Many gastrointestinal stimuli result in gastric fundic relaxation. This information is integrated at the interface of vagal afferents and efferents in the dorsal vagal complex. Substance P (SP) is present in this region, and the neurokinin(1) receptor (NK(1)R) is highly expressed in preganglionic neurons of the dorsal motor nucleus of the vagus (DMN). However, its functional effects on vagal motor output to the stomach have not been investigated. Therefore, we determined the gastric motor effects of stereotaxic microinjection of SP and selective tachykinin receptor agents into the DMN of anesthetized rats. Dose related decreases in intragastric pressure and antral motility were obtained on the microinjection of SP (135 and 405 pmol) into the DMN, without cardiovascular changes. Similar decreases in intragastric pressure were noted after the microinjection of [Sar(9),Met(O(2))(11)]SP (NK(1)R agonist; 135 pmol) but not senktide (NK(3)R agonist; 135 pmol) or vehicle. The gastric motor inhibition evoked by SP (135 pmol) was attenuated by prior microinjection of 2-methoxy-5 tetrazol-1-yl-benzyl-(2-phenyl-piperidin-3-yl)-a mine (GR203040; 1 nmol; NK(1)R antagonist). Vagotomy or hexamethonium (15 mg/kg i.v.) completely abolished the gastric relaxation evoked by SP (135 pmol) microinjected into the DMN. We conclude that SP acts on NK(1)R preganglionic cholinergic vagal neurons in the DMN, which control enteric nonadrenergic noncholinergic motor inhibition of the fundus. The potential relevance is that an antiemetic site of action of NK(1)R antagonists may be in the DMN to prevent excitation of neurons controlling fundic relaxation, which is an essential prodromal component of emesis. PMID- 10734173 TI - Diffusion of peroxynitrite into the human platelet inhibits cyclooxygenase via nitration of tyrosine residues. AB - Peroxynitrite (ONOO(-)), a reactive oxidant produced by the reaction between nitric oxide and superoxide, was found to diffuse into the platelet cytosol and inhibit arachidonic acid-induced platelet aggregations with IC(50) value of 5.8 +/- 1.2 microM. A fluorescence assay established that ONOO(-) diffused into the platelet cytosol in a manner that was inhibited (50-70%) by 4, 4' diisothiocyanatostilbene-2,2'-disulfonic acid, an inhibitor of HCO(3)(-)/Cl(-) anion exchanger. Treatment of platelets with (-)-epigallocatechin gallate (2 microM), a tea polyphenol and inhibitor of tyrosine nitration, abolished the inhibitory effect of ONOO(-) on arachidonate-induced aggregations by 88%. ONOO(-) (50-300 microM), added to platelets 1 min before arachidonic acid, inhibited (20 100%) formation of platelet cyclooxygenase (COX) products thromboxane A(2) and 12 hydroxyheptadecatrienoic acid. Interestingly, simultaneous addition of ONOO(-) and arachidonic acid stimulated eicosanoid production by 20 to 60%. The inhibition of thromboxane A(2) generation correlated with the 5- to 10-fold increase in the 3-nitrotyrosine levels of the platelet COX. Experiments with purified COX-1 and COX-2 also showed 9-fold increase of 3-nitrotyrosine levels, which correlated with decreased (93-98%) production of prostaglandin H(2) when ONOO(-) (50 microM) was added 1 min before arachidonic acid. However, the addition of ONOO(-) (50-100 microM) simultaneously with arachidonic acid increased prostaglandin H(2) formation by 30 to 60%. Thus, the inhibitory effect of ONOO(-) involved nitration of COX tyrosine residues, whereas the stimulatory effect was likely to be a result of ONOO(-) functioning as a peroxide activator of eicosanoid signaling. Increasing doses of ONOO(-) not only inhibited platelet COX but also induced formation of unique eicosanoids: iso-prostaglandin F(2alpha), epoxyhydroxyeicosatrienoic acid, and trans-arachidonic acids, suggesting that OH and NO(2) radicals were generated from ONOO(-) in platelets. Formation of ONOO(-) from NO and superoxide may function as a platelet hormone like COX regulatory mechanism in inflammatory processes in which large amounts of these molecules are produced. PMID- 10734175 TI - Internalization and recycling of delta-opioid receptor are dependent on a phosphorylation-dephosphorylation mechanism. AB - Internalization, recycling, and resensitization of the human delta-opioid receptor (hDOR) were studied in the neuroblastoma cell line SK-N-BE, endogenously expressing this receptor. Conventional and confocal fluorescence microscopy observations, corroborated by Scatchard analysis, indicated that after a 100 nM Eto treatment, 60 to 70% of hDOR were rapidly internalized (t(1/2) < 15 min). This agonist-triggered internalization was reversible for a treatment not exceeding 1 h and became irreversible for prolonged treatment (4 h), leading probably to the degradation and/or down-regulation of the receptor. The rapid internalization of hDOR was totally blocked in the presence of heparin, known as an inhibitor of G protein-coupled receptor kinases (Benovic et al., 1989), a result indicating that phosphorylation by these kinases is a critical step in desensitization (Hasbi et al, 1998) and internalization of hDOR (present study) in SK-N-BE cell line. Blockade of internalization by agents not interferring with phosphorylation, as hypertonic sucrose or concanavalin A, also blocked the resensitization (receptor functional recovering) process. Furthermore, blockade of dephosphorylation of the internalized hDOR by okadaic acid totally suppressed its recycling to the plasma membrane and its subsequent resensitization. These results indicate that regulatory events leading to desensitization, internalization, and recycling in a functional state of hDOR involve phosphorylation by a G protein-coupled receptor kinase, internalization via clathrin-coated vesicles, and dephosphorylation by acid phosphatases. PMID- 10734174 TI - Polymethoxylated flavones in orange juice are inhibitors of P-glycoprotein but not cytochrome P450 3A4. AB - The presence in orange juice of compounds that specifically inhibit the P glycoprotein (P-gp) drug efflux transporter, but not the cytochrome P450 (CYP) isozyme CYP3A4, was investigated. The uptake of [(3)H]vinblastine, a substrate of P-gp, by Caco-2 cells was measured. An ethyl acetate extract of orange juice did not affect the initial uptake rate of [(3)H]vinblastine but significantly increased the steady-state uptake, as did cyclosporin A (20 microM), an inhibitor of P-gp. No significant effect on the uptake of 3-O-[(3)H]methylglucose or [(14)C]phenylalanine by Caco-2 cells was found, compared with the control. When the extract was separated on a Cosmosil column, the eluate with 70% methanol showed the most potent ability to increase [(3)H]vinblastine uptake. Additional separation of the 70% methanol eluate on a silica gel column with hexane-acetone (3:1) gave 3,3',4',5,6,7,8-heptamethoxyflavone (HMF) and 4',5,6,7,8 pentamethoxyflavone (tangeretin). HMF, tangeretin, and 3',4',5,6,7,8 hexamethoxyflavone (nobiletin), another methoxyflavone contained in orange juice, all increased the steady-state uptake of [(3)H]vinblastine by Caco-2 cells in a concentration-dependent manner. The order of potency of these compounds at the concentration of 50 microM was tangeretin > HMF > nobiletin. None of these methoxyflavones inhibited 6beta-hydroxylation of testosterone catalyzed by CYP3A4. The ethyl acetate extract of orange juice and these methoxyflavones also increased steady-state [(3)H]vinblastine uptake by LLC-GA5-COL300 cells (a cell line transfected with human MDR1 cDNA). We conclude that these methoxyflavones enhanced vinblastine uptake by specifically inhibiting drug efflux via P-gp. They may have potential as agents for reversing multidrug resistance or for recovering the bioavailability of certain drugs. PMID- 10734176 TI - Plasma and lymph pharmacokinetics of recombinant human interleukin-2 and polyethylene glycol-modified interleukin-2 in pigs. AB - Modification of recombinant human interleukin-2 (IL-2) with polyethylene glycol (PEG-IL-2) decreases clearance and might favor absorption into the lymphatics, due to its increased molecular weight. In the present study, we compared the plasma and lymph concentrations of IL-2 and PEG-IL-2 in Yorkshire pigs. The IL-2 regimens were i.v. bolus (0.1-1.6 x 10(6) I.U., MIU/kg), 15-min i.v. infusion (0.1 MIU/kg), or s.c. bolus (0.1-3.0 MIU/kg). The PEG-IL-2 doses were 15-min i.v. infusion (0.01 MIU/kg) or s.c. bolus (0.01-0. 10 MIU/kg). Lymph and plasma data were analyzed using noncompartmental methods and NONMEM. Bioavailability of IL-2 was route- and dose-dependent. Bioavailability of i.v. bolus doses of >/=0.16 MIU/kg was complete but only 39% at 0.1 MIU/kg. For the infusion and s.c. doses, bioavailability was 28 and 42%, respectively. Noncompartmental and NONMEM estimates of clearance and volume of distribution at steady state agreed: 300 ml/h/kg and 570 ml/kg, respectively, for IL-2. The ratio of the area under the curve in lymph and plasma increased from 0.67 to 3.4 when comparing i.v. and s.c. routes, and the s.c. delivery advantage (ratio of dose-normalized ratio of the area under the curve in lymph after s.c. and i.v. administration) was 6.6 to 16. For PEG-IL-2, bioavailability was 100%, clearance was 5.9 ml/h/kg, and volume of distribution at steady state was 370 ml/kg. The ratio of the area under the curve in lymph and plasma increased from 0.33 (i.v.) to 1. 2 (s.c.), and the s.c. delivery advantage was 3.8. Subcutaneous dosing would be favored over i.v. dosing, and IL-2 would be favored over PEG-IL-2 to maximize lymph and minimize plasma exposure. Because IL-2 efficacy may be related to lymph concentrations, dosing regimens can now be designed to test this hypothesis. PMID- 10734177 TI - A novel angiotensin analog with subnanomolar affinity for angiotensin-converting enzyme. AB - This study demonstrates that a novel angiotensin I analog, angiotensinogen 3 11(Lys(11)), possesses a high affinity for angiotensin-converting enzyme (ACE), which is substantially greater than the endogenous substrates. This assessment is based on data derived from a variety of techniques. First, the binding characteristics of (125)I-angiotensinogen 3-11(Lys(11)) were examined. Equilibrium saturation isotherms utilizing guinea pig lung membranes revealed that (125)I-angiotensinogen 3-11(Lys(11)) bound a single high-affinity site in the presence of EDTA exhibiting a K(d) of 0.15 +/- 0.02 nM with a B(max) = 4295 +/- 535 fmol/mg of protein. Competition studies revealed the following rank order of binding affinity: (125)I-angiotensinogen 3-11(Lys(11)) >> bradykinin >> angiotensin I. Next, SDS-polyacrylamide gel electrophoresis analysis revealed that chemically cross-linked (125)I-angiotensinogen 3-11(Lys(11)) specifically bound a protein of M(r) 173,000 that had the same molecular weight as ACE. Utilizing in vitro autoradiography, the binding distributions of (125)I angiotensinogen 3-11(Lys(11)) and the ACE inhibitor, (125)I-351A, were also compared. These experiments demonstrated that the binding distributions of (125)I angiotensinogen 3-11(Lys(11)) and (125)I-351A are identical in the guinea pig lung and testes. Finally, the purification of ACE from guinea pig serum was monitored with (125)I-angiotensinogen 3-11(Lys(11)) and (125)I-351A binding. These results demonstrated that the binding site for (125)I-angiotensinogen 3 11(Lys(11)) and (125)I-351A copurified. These experiments indicate that the novel angiotensin I analog, (125)I-angiotensinogen 3-11(Lys(11)) binds to ACE and suggest that there are critical binding sites outside the catalytic domains of ACE that determine binding specificity and affinity. PMID- 10734178 TI - Ras/mitogen-activated protein kinase mediates norepinephrine-induced phospholipase D activation in rabbit aortic smooth muscle cells by a phosphorylation-dependent mechanism. AB - Phospholipase D (PLD) activity is regulated by phosphatidylinositol 4,5 biphosphate, protein kinase C (PKC), ADP-ribosylation factor, and Rho. The present study was designed to investigate the mechanism of norepinephrine (NE) mediated PLD activation in rabbit aortic vascular smooth muscle cells (VSMC). NE (10 microM) caused activation of PLD, as measured by the production of phosphatidylethanol in [(3)H]oleic acid-labeled cells. NE also increased PKC activity in VSMC. However, treatment of cells with bisindolylmaleimide, a PKC inhibitor, or long-term treatment with phorbol-12-myristate-13-acetate that depletes PKC did not decrease NE-induced activation of PLD. NE-stimulated PLD activity was attenuated by farnesyl transferase inhibitors (FPT III and SCH 56582), which reduce activation of both Ras and mitogen-activated protein (MAP) kinase. Moreover, transfection of VSMC with a dominant negative Ras resulted in inhibition of NE-stimulated MAP kinase and PLD activities. Treatment of cells with PD-98059, a MAP kinase kinase inhibitor, also reduced NE-stimulated PLD activity. These data suggest that NE-stimulated PLD activity is mediated via activation of Ras and MAP kinase in rabbit VSMC. To study the mechanism of activation of PLD by Ras/MAP kinase, NE-induced phosphorylation of PLD was examined. In VSMC, PLD of molecular mass 120 kDa was identified with polyclonal PLD antibody. Phosphorylation of PLD by NE, measured as (32)P incorporation into PLD, was inhibited by PD-98059. Moreover, PLD immunoprecipitated from VSMC lysates was phosphorylated in vitro by MAP kinase. Collectively, these results show a novel pathway for activation of PLD that appears to be mediated through Ras/MAP kinase pathway by a mechanism involving phosphorylation. PMID- 10734179 TI - Pharmacological characterization of (125)I-1229U91 binding to Y1 and Y4 neuropeptide Y/Peptide YY receptors. AB - 1229U91 (GW1229 or GR231118) [lle,Glu,Pro,Dpr,Tyr, Arg,Leu,Arg, Tyr-NH(2))2 cyclic (2,4'),(2'4)-diamide] has been reported by several research groups to be a potent antagonist at the Y1 neuropeptide Y (NPY) receptor subtype. However, 1229U91 also displaces (125)I-peptide YY (PYY) with high affinity from the Y4 subtype. Previously, we reported that 1229U91 had full agonist properties for the Y4 receptor. To characterize the pharmacological properties of 1229U91 directly, we had it radioiodinated with the chloromine-T method. (125)I-1229U91 bound to cell lines expressing the human Y1 and Y4 receptors with high affinity. The K(d) and B(max) for (125)I-1229U91 binding to Y1 were 14.9 pM and 1458 fmol/mg protein, respectively. The Y4 receptor bound (125)I-1229U91 with a K(d) of 12.5 pM and a B(max) of 1442 fmol/mg protein. When competing (125)I-1229U91 binding from Y1 and Y4 receptors, a similar rank order of potency was observed: 1229U91 > [Leu(31),Pro(34)]-NPY >/= [Leu(31),Pro(34)]-PYY > PYY >/= NPY > NPY(2-36) > PYY(3 36). Pancreatic polypeptide (PP) potently displaced (125)I-1229U91 from the Y4 receptor, but displayed little affinity for Y1. In autoradiographic studies with rat brain sections, (125)I-1229U91 bound with a distribution similar to that reported for the Y1 receptor when localized with (125)I-[Leu(31),Pro(34)]-PYY. Brain regions exhibiting binding sites for (125)I-PP were not detected with this radioligand. Those include the interpeduncular nucleus and the periventricular nucleus of the hypothalamus. Furthermore, (125)I-labeled rat PP was not displaced from these areas with 10 nM 1229U91. Thus, (125)I-1229U91 is a high affinity Y1 and Y4 radioligand and binds with a distribution in the rat brain consistent with the localization of the Y1 receptor. PMID- 10734180 TI - SB 239063, a potent p38 MAP kinase inhibitor, reduces inflammatory cytokine production, airways eosinophil infiltration, and persistence. AB - The anti-inflammatory/antiallergic activity of a novel second-generation p38 mitogen-activated protein kinase inhibitor, SB 239063[trans-1-(4 hydroxycyclohexyl) -4-(4-fluorophenyl)-5-(2-methoxypyridimidin-4-yl)imidazole], was investigated in vivo and in vitro. SB 239063 had an IC(50) of 44 nM for inhibition of recombinant purified human p38alpha. In lipopolysaccharide stimulated human peripheral blood monocytes, SB 239063 inhibited interleukin-1 and tumor necrosis factor-alpha production (IC(50) values = 0.12 and 0.35 microM, respectively). A role for p38 kinase in cytokine-associated inflammation in the mouse was shown by p38 activation in the lung and inhibition of lipopolysaccharide-induced tumor necrosis factor-alpha production by SB 239063 (ED(50) = 5.8 mg/kg p.o.). Antiallergic activity was demonstrated by essential abolition (approximately 93% inhibition) of inhaled ovalbumin (OA)-induced airway eosinophilia by SB 239063 (12 mg/kg p.o.), measured by bronchoalveolar lavage (BAL) in OA-sensitized mice. In addition, p38 kinase was found by Western analysis to be activated in guinea pig lung. Administration of SB 239063 (10 or 30 mg/kg p.o.) in conscious guinea pigs markedly reduced ( approximately 50% inhibition) OA-induced pulmonary eosinophil influx, measured by BAL 24 h after antigen. SB 239063 (10 mg/kg b.i.d. p.o.) administered after leukotriene D(4) inhalation, reduced by 60% the persistent airway eosinophilia seen at 4 days. Apoptosis of cultured eosinophils isolated from guinea pig BAL was increased by SB 239063 (1-10 microM) in the presence of interleukin-5. These results indicate that SB 239063 is a potent inhibitor of inflammatory cytokine production, inhibits eosinophil recruitment, in addition to enhancing apoptosis of these cells. Collectively, the results support the potential utility of p38 kinase inhibitors, such as SB 239063, for the treatment of asthma and other inflammatory disorders. PMID- 10734181 TI - Synthesis and characterization of a fluorescent substrate for the N arachidonoylethanolamine (anandamide) transmembrane carrier. AB - N-Arachidonoylethanolamine (AEA) is a proposed endogenous ligand of the central cannabinoid receptor (CB1). Previous studies indicate that AEA is translocated across membranes via a process that has the characteristics of carrier-mediated facilitated diffusion. To date, studies of this mechanism have relied on [(3)H]AEA as a substrate for the carrier. We have synthesized an analog of AEA, SKM 4-45-1, that is nonfluorescent in the extracellular environment. When SKM 4 45-1 is exposed to intracellular esterases, it is de-esterified and becomes fluorescent. We have carried out studies to demonstrate that SKM 4-45-1 accumulation in cells occurs via the AEA carrier. SKM 4-45-1 is accumulated by both cerebellar granule cells and C6 glioma cells. Uptake of SKM 4-45-1 into C6 glioma is inhibited by AEA (IC(50)=53.8 +/- 1.8 microM), arachidonoyl-3 aminopyridine amide (IC(50)=10.1 +/- 1.4 microM), and arachidonoyl-4 hydroxyanilineamide (IC(50)=6.1 +/- 1.3 microM), all of which also inhibit [(3)H]AEA accumulation. Conversely, [(3)H]AEA accumulation by cerebellar granule cells is inhibited by SKM 4-45-1 with an IC(50) of 7.8 +/- 1. 3 microM. SKM 4-45 1 is neither a substrate nor inhibitor of fatty acid amide hydrolase, an enzyme that catabolizes AEA. SKM 4-45-1 does not bind the CB1 cannabinoid receptor at concentrations <10 microM. In summary, the cellular accumulation of SKM 4-45-1 occurs via the same pathway as AEA uptake and provides an alternative substrate for the study of this important cellular process. PMID- 10734182 TI - Allopurinol prevents early alcohol-induced liver injury in rats. AB - Free radical formation caused by chronic ethanol administration could activate transcription factors such as nuclear factor-kappaB (NF-kappaB), which regulates production of inflammatory cytokines. Xanthine oxidase is one potential source of reactive oxygen species. Therefore, the purpose of this study is to determine whether allopurinol, a xanthine oxidase inhibitor and scavenger of free radicals, would affect free radical formation, NF-kappaB activation, and early alcohol induced liver injury in rats. Male Wistar rats were fed a high-fat diet with or without ethanol (10-16 g/kg/day) continuously for up to 4 weeks with the Tsukamoto-French enteral protocol. Either allopurinol or saline vehicle was administered daily. Allopurinol had no effect on body weight or the cyclic pattern of ethanol in urine. Mean urine ethanol concentrations were 271 +/- 38 and 252 +/- 33 mg/dl in ethanol- and ethanol + allopurinol-treated rats, respectively. In the control group, serum aspartate aminotransferase and alanine aminotransferase levels were approximately 40 I.U./l and 25 U/l, respectively. Administration of enteral ethanol for 4 weeks increased serum transaminases approximately 5-fold. Allopurinol blunted these increases significantly by approximately 50%. Ethanol treatment also caused severe fatty infiltration, mild inflammation, and necrosis. These pathological changes also were blunted significantly by allopurinol. Furthermore, enteral ethanol caused free radical adduct formation, values that were reduced by approximately 40% by allopurinol. NF-kappaB binding was minimal in the control group but was increased significantly nearly 2.5-fold by ethanol. This increase was blunted to similar values as control by allopurinol. These results indicate that allopurinol prevents early alcohol-induced liver injury, most likely by preventing oxidant dependent activation of NF-kappaB. PMID- 10734183 TI - R115866 inhibits all-trans-retinoic acid metabolism and exerts retinoidal effects in rodents. AB - All-trans-retinoic acid (RA) regulates epithelial differentiation and growth through activation of specific nuclear RA receptors (RARs). Because high-rate metabolism largely impairs the biological efficacy of RA, we have sought for compounds capable of inhibiting the metabolic breakdown of the retinoid. This study identifies R115866 as a novel inhibitor of the cytochrome P450 (CYP) mediated metabolism of RA. In vitro, nanomolar concentrations of R115866 inhibited the conversion of RA by CYP26, a RA-inducible RA metabolizing enzyme. In vivo, oral administration of R115866 (2.5 mg/kg) to rats induced marked and transient increases of endogenous RA levels in plasma, skin, fat, kidney, and testis. Consistent with its ability to enhance endogenous RA content in tissues, R115866 was found to exert retinoidal activities. Like RA, the title compound: 1) inhibited vaginal keratinization in estrogen-stimulated rats; 2) induced epidermal hyperplasia in mouse ear skin; 3) transformed mouse tail epidermis from a para- to an orthokeratotic skin type; and 4) up-regulated the CYP26 mRNA expression in rat liver. Furthermore, we found that the keratinization suppressive and CYP26-inducing activities of R115866 could be reversed by concomitant administration of the RAR antagonist, AGN193109. Our data characterize R115866 as a potent, orally active inhibitor of RA metabolism, capable of enhancing RA levels and displaying retinoidal actions. These activities are reversed by RAR antagonism, supporting the idea that the actions of R115866 result from increased availability of endogenous RA and improved RAR triggering. PMID- 10734184 TI - The morph server: a standardized system for analyzing and visualizing macromolecular motions in a database framework. AB - The number of solved structures of macromolecules that have the same fold and thus exhibit some degree of conformational variability is rapidly increasing. It is consequently advantageous to develop a standardized terminology for describing this variability and automated systems for processing protein structures in different conformations. We have developed such a system as a 'front-end' server to our database of macromolecular motions. Our system attempts to describe a protein motion as a rigid-body rotation of a small 'core' relative to a larger one, using a set of hinges. The motion is placed in a standardized coordinate system so that all statistics between any two motions are directly comparable. We find that while this model can accommodate most protein motions, it cannot accommodate all; the degree to which a motion can be accommodated provides an aid in classifying it. Furthermore, we perform an adiabatic mapping (a restrained interpolation) between every two conformations. This gives some indication of the extent of the energetic barriers that need to be surmounted in the motion, and as a by-product results in a 'morph movie'. We make these movies available over the Web to aid in visualization. Many instances of conformational variability occur between proteins with somewhat different sequences. We can accommodate these differences in a rough fashion, generating an 'evolutionary morph'. Users have already submitted hundreds of examples of protein motions to our server, producing a comprehensive set of statistics. So far the statistics show that the median submitted motion has a rotation of approximately 10 degrees and a maximum Calpha displacement of 17 A. Almost all involve at least one large torsion angle change of >140 degrees. The server is accessible at http://bioinfo.mbb.yale. edu/MolMovDB PMID- 10734185 TI - Characterization of the Saccharomyces cerevisiae cyclic nucleotide phosphodiesterase involved in the metabolism of ADP-ribose 1",2"-cyclic phosphate. AB - ADP-ribose 1",2"-cyclic phosphate (Appr>p) is produced in yeast and other eukaryotes as a consequence of tRNA splicing. This molecule is converted to ADP ribose 1"-phosphate (Appr-1"p) by the action of the cyclic nucleotide phosphodiesterase (CPDase). Comparison of the previously cloned CPDase from Arabidopsis with proteins having related cyclic phosphodiesterase or RNA ligase activities revealed two histidine-containing tetrapeptides conserved in these enzyme families. Using the consensus phosphodiesterase signature, we have identified the yeast Saccharomyces cerevisiae open reading frame YGR247w as encoding CPDase. The bacterially expressed yeast protein, named Cpd1p, is able to hydrolyze Appr>p to Appr-1"p. Moreover, as with the previously characterized Arabidopsis and wheat CPDases, Cpd1p hydrolyzes nucleosides 2',3'-cyclic phosphates (N>p) to nucleosides 2'-phosphates. Apparent K (m)values for Appr>p, A>p, U>p, C>p and G>p are 0.37, 4.97, 8.91, 12.18 and 14.29 mM, respectively. Site-directed mutagenesis of individual amino acids within the two conserved tetrapeptides showed that H(40)and H(150)residues are essential for CPDase activity. Deletion analysis has indicated that the CPD1 gene is not important for cellular viability. Likewise, overexpression of Cpd1p had no effect on yeast growth. These results do not implicate an important role for Appr>p or Appr-1"p in yeast cells grown under standard laboratory conditions. PMID- 10734186 TI - Degradation of ribosomal RNA precursors by the exosome. AB - The yeast exosome is a complex of 3'-->5' exonucleases involved in RNA processing and degradation. All 11 known components of the exosome are required during 3' end processing of the 5.8S rRNA. Here we report that depletion of each of the individual components inhibits the early pre-rRNA cleavages at sites A(0), A(1), A(2)and A(3), reducing the levels of the 32S, 20S, 27SA(2)and 27SA(3)pre-rRNAs. The levels of the 27SB pre-rRNAs were also reduced. Consequently, both the 18S and 25S rRNAs were depleted. Since none of these processing steps involves 3'- >5' exonuclease activities, the requirement for the exosome is probably indirect. Correct assembly of trans -acting factors with the pre-ribosomes may be monitored by a quality control system that inhibits pre-rRNA processing. The exosome itself degrades aberrant pre-rRNAs that arise from such inhibition. Exosome mutants stabilize truncated versions of the 23S, 21S and A(2)-C(2)RNAs, none of which are observed in wild-type cells. The putative helicase Dob1p, which functions as a cofactor for the exosome in pre-rRNA processing, also functions in these pre-rRNA degradation activities. PMID- 10734187 TI - An ATM homologue from Arabidopsis thaliana: complete genomic organisation and expression analysis. AB - ATM is a gene mutated in the human disease ataxia telangiectasia with reported homologues in yeast, Drosophila, Xenopus and mouse. Whenever mutants are available they all indicate a role of this gene family in the cellular response to DNA damage. Here, we present the identification and molecular characterisation of the first plant homologue of ATM. The genomic locus of AtATM ( Arabidopsis thaliana homologue of ATM ) spans over 30 kb and is transcribed into a 12 kb mRNA resulting from the splicing of 79 exons. It is a single copy gene and maps to the long arm of chromosome 3. Transcription of AtATM is ubiquitous and not induced by ionising radiation. The putative protein encoded by AtATM is 3856 amino acids long and contains a phosphatidyl inositol-3 kinase-like (Pi3k-l) domain and a rad3 domain, features shared by other members of the ATM family. The AtAtm protein is highly similar to Atm, with 67 and 45% similarity in the Pi3k-l and rad3 domains respectively. Interestingly, the N-terminal portion of the protein harbours a PWWP domain, which is also present in other proteins involved in DNA metabolism such as human mismatch repair enzyme Msh6 and the mammalian de novo methyl transferases, Dnmt3a/b. PMID- 10734188 TI - Test of intron predictions reveals novel splice sites, alternatively spliced mRNAs and new introns in meiotically regulated genes of yeast. AB - Correct identification of all introns is necessary to discern the protein-coding potential of a eukaryotic genome. The existence of most of the spliceosomal introns predicted in the genome of Saccharomyces cerevisiae remains unsupported by molecular evidence. We tested the intron predictions for 87 introns predicted to be present in non-ribosomal protein genes, more than a third of all known or suspected introns in the yeast genome. Evidence supporting 61 of these predictions was obtained, 20 predicted intron sequences were not spliced and six predictions identified an intron-containing region but failed to specify the correct splice sites, yielding a successful prediction rate of <80%. Alternative splicing has not been previously described for this organism, and we identified two genes (YKL186C/ MTR2 and YML034W) which encode alternatively spliced mRNAs; YKL186C/ MTR2 produces at least five different spliced mRNAs. One gene (YGR225W/ SPO70 ) has an intron whose removal is activated during meiosis under control of the MER1 gene. We found eight new introns, suggesting that numerous introns still remain to be discovered. The results show that correct prediction of introns remains a significant barrier to understanding the structure, function and coding capacity of eukaryotic genomes, even in a supposedly simple system like yeast. PMID- 10734190 TI - Role of RNA structure in non-homologous recombination between genomic molecules of brome mosaic virus. AB - Brome mosaic virus (BMV) is a tripartite genome, positive-sense RNA virus of plants. Previously it was demonstrated that local hybridization between BMV RNAs (RNA-RNA heteroduplex formation) efficiently promotes non-homologous RNA recombination. In addition, studies on the role of the BMV polymerase in RNA recombination suggested that the location of non-homologous crossovers depends mostly on RNA structure. As a result, a detailed analysis of a large number of non-homologous recombinants generated in the BMV-based system was undertaken. Recombination hot-spots as well as putative elements in RNA structure enhancing non-homologous crossovers and targeting them in a site-specific manner were identified. To verify these observations the recombinationally active sequence in BMV RNA3 derivative was modified. The results obtained with new RNA3 mutants suggest that the primary and secondary structure of the sequences involved in a heteroduplex formation rather than the length of heteroduplex plays the most important role in the recombination process. The presented data indicate that the sequences proximal to the heteroduplex may also affect template switching by BMV replicase. Moreover, it was shown that both short homologous sequences and a hairpin structure have to accompany a double-stranded region to target non homologous crossovers in a site-specific manner. PMID- 10734189 TI - Transcriptional repression by the insulator protein CTCF involves histone deacetylases. AB - The highly conserved zinc-finger protein, CTCF, is a candidate tumor suppressor protein that binds to highly divergent DNA sequences. CTCF has been connected to multiple functions in chromatin organization and gene regulation including chromatin insulator activity and transcriptional enhancement and silencing. Here we show that CTCF harbors several autonomous repression domains. One of these domains, the zinc-finger cluster, silences transcription in all cell types tested and binds directly to the co-repressor SIN3A. Two distinct regions of SIN3A, the PAH3 domain and the extreme C-terminal region, bind independently to this zinc finger cluster. Analysis of nuclear extract from HeLa cells revealed that CTCF is also capable of retaining functional histone deacetylase activity. Furthermore, the ability of regions of CTCF to retain deacetylase activity correlates with the ability to bind to SIN3A and to repress gene activity. We suggest that CTCF driven repression is mediated in part by the recruitment of histone deacetylase activity by SIN3A. PMID- 10734191 TI - The effect of mutations in the HIV-1 nucleocapsid protein on strand transfer in cell-free reverse transcription reactions. AB - Interactions between the nucleocapsid protein (NC) and reverse transcriptase of HIV-1 have been shown to promote the initiation of reverse transcription. We assayed the effect of NC on later events, using a strand transfer system with donor and acceptor HIV RNA templates and found that the presence of NC resulted in increased synthesis of full-length strand-transferred (FLST) DNA. This effect also occurred with mutated forms of NC that lacked both zinc fingers, or that contained a point mutation (histidine-->cysteine) at amino acid 23. In contrast, NC-derived proteins containing only the proximal or distal zinc fingers, or lacking the N- and C-termini, were all unable to catalyze the synthesis of FLST DNA. Band-shift assays using both the mutated and wild-type forms of these proteins revealed that all the NC proteins promoted strand association between ( ) strong-stop DNA [(-)ssDNA] and acceptor RNA. The zinc finger motifs were dispensable for full-length processive reverse transcription, and the N- and C termini were required; however, all NC domains were dispensable for association of (-)ssDNA and acceptor RNA. This suggests that annealing is a less stringent reaction than DNA polymerization. PMID- 10734192 TI - A further investigation and reappraisal of the thio effect in the cleavage reaction catalyzed by a hammerhead ribozyme. AB - We synthesized three types of 11mer substrate, namely the natural substrate S11O and the thio-substituted substrates S11 S pS and S11 R pS, in which the respective pro-S p and pro-R p oxygen atoms were replaced by sulfur, and subjected them to detailed kinetic analysis in the cleavage reaction catalyzed by a hammerhead ribozyme. In agreement with previous findings, in the presence of Mg(2+)or Ca(2+)ions the rate of ribozyme-catalyzed cleavage of S11 S pS was as high as that of S11O, whereas the corresponding rate for S11 R pS was nearly four orders of magnitude lower than that for either S11O or S11 S pS. However, the rate of the ribozyme-catalyzed reaction with each of the three substrates was enhanced by Cd(2+)ions. Such results have generally been taken as evidence that supports the direct interaction of the sulfur atom at the R p position of the cleavage site with the added Cd(2+)ion. However, our present analysis demonstrates that (i) the added Cd(2+)ion binds at the P9 site; (ii) the bound Cd(2+)ion at the P9 site replaces two Mg(2+)or two Ca(2+)ions, an observation that suggests a different mode of interaction with the added Cd(2+)ion; and, most importantly and in contrast to the conclusion reached by other investigators, (iii) the Cd(2+)ion does not interact with the sulfur atom at the R p position of the scissile phosphate either in the ground state or in the transition state. PMID- 10734194 TI - Design and optimization of effector-activated ribozyme ligases. AB - A selected ribozyme ligase, L1, has been engineered to respond to small organic effectors. Residues important for ribozyme catalysis were mapped to a compact core structure. Aptamers that bound adenosine and theophylline were appended to the core structure, and the resultant aptazymes proved to be responsive to their cognate effectors. Rational sequence substitutions in the joining region between the aptamer and the ribozyme yielded aptazymes whose activities were enhanced from 800-1600-fold in the presence of 1 mM ATP or theophylline, respectively. However, when an anti-flavin aptamer was appended to the core ribozyme structure flavin-responsivity was minimal. The joining region between the aptamer and the ribozyme core was randomized and a series of negative and positive selection steps yielded aptazymes that were activated by up to 260-fold in the presence of 100 microM FMN. The selected joining regions proved to be 'communication modules' that could be used to join other aptamers to the ribozyme core to form aptazymes. These results show that ribozyme ligases can be readily engineered to function as allosteric enzymes, and reveal that many of the techniques and principles previously demonstrated during the development of hammerhead aptazymes may be generalizable. PMID- 10734193 TI - NMR studies on functional structures of the AU-rich element-binding domains of Hu antigen C. AB - Hu antigen C (HuC) has three RNA-binding domains (RBDs). The N-terminal two, RBD1 and RBD2, are linked in tandem and bind to the AU-rich elements (AREs) in the 3' untranslated region of particular mRNAs. The solution structures of HuC RBD1 and RBD2 were determined by NMR methods. The HuC RBD1 and RBD2 structures are quite similar to those of Sxl RBD1 and RBD2, respectively. The individual RBDs of HuC, RBD1 and RBD2 in isolation can interact rather weakly with the minimal ARE motif, AUUUA, while the didomain fragment, RBD1-RBD2, of HuC binds more tightly to a longer ARE RNA, UAUUUAUUUU. Chemical shift perturbations by the longer RNA on HuC RBD1-RBD2 were mapped on and around the two beta-sheets and on the C-terminal region of RBD1. The HuC RBD1-RBD2 residues that exhibited significant chemical shift perturbations coincide with those conserved in Sxl RBD1-RBD2. These data indicate that the RNA-binding characteristics of the HuC and Sxl didomain fragments are similar, even though the target RNAs and the biological functions of the proteins are different. PMID- 10734195 TI - Hexahydrated magnesium ions bind in the deep major groove and at the outer mouth of A-form nucleic acid duplexes. AB - Magnesium ions play important roles in the structure and function of nucleic acids. Whereas the tertiary folding of RNA often requires magnesium ions binding to tight places where phosphates are clustered, the molecular basis of the interactions of magnesium ions with RNA helical regions is less well understood. We have refined the crystal structures of four decamer oligonucleotides, d(ACCGGCCGGT), r(GCG)d(TATACGC), r(GC)d(GTATACGC) and r(G)d(GCGTATACGC) with bound hexahydrated magnesium ions at high resolution. The structures reveal that A-form nucleic acid has characteristic [Mg(H(2)O)(6)](2+)binding modes. One mode has the ion binding in the deep major groove of a GpN step at the O6/N7 sites of guanine bases via hydrogen bonds. Our crystallographic observations are consistent with the recent NMR observations that in solution [Co(NH(3))(6)](3+), a model ion of [Mg(H(2)O)(6)](2+), binds in an identical manner. The other mode involves the binding of the ion to phosphates, bridging across the outer mouth of the narrow major groove. These [Mg(H(2)O)(6)](2+)ions are found at the most negative electrostatic potential regions of A-form duplexes. We propose that these two binding modes are important in the global charge neutralization, and therefore stability, of A-form duplexes. PMID- 10734196 TI - Differential regulation of gene activity and chromatin structure within the human serpin gene cluster at 14q32.1 in macrophage microcell hybrids. AB - The human gene encoding alpha1-antitrypsin (alpha1AT, gene symbol PI ) is highly expressed in the liver and in cultured hepatoma cells and, to a lesser extent, in macrophages, where transcription originates from a separate upstream promoter. alpha1AT maps to a region of human chromosome 14q32.1 that includes a related serine protease inhibitor (serpin) gene that encodes corticosteroid-binding globulin (CBG). We recently reported the chromatin organization of this approximately 130 kb region, as defined by DNase I hypersensitive sites (DHSs) and matrix-attachment regions, in expressing and non-expressing cells. Furthermore, we demonstrated that transfer of human chromosome 14 from non expressing fibroblasts to rat hepatoma cells resulted in activation of both alpha1AT and CBG transcription and gene activation was accompanied by long range chromatin reorganization of the entire region. In this study, we transferred human chromosome 14 from fibroblasts to mouse macrophages and documented activation of alpha1AT but not CBG gene expression. RT-PCR experiments indicated that transcription of the human alpha1AT gene in the microcell hybrids initiated at the macrophage promoter. Furthermore, DHS mapping experiments revealed a distinctive chromatin configuration of the locus that resembled the structure found in human macrophage-like cell lines, with many DHSs around alpha1AT but few in CBG. Thus, mouse macrophage cell lines will provide a useful cell type to study the effects of targeted modifications of the human alpha1AT-CBG locus on the regulation of cell-specific gene activity and chromatin structure. PMID- 10734197 TI - The RNA-binding domain of ribosomal protein L11 recognizes an rRNA tertiary structure stabilized by both thiostrepton and magnesium ion. AB - Antibiotics that inhibit ribosomal function may do so by one of several mechanisms, including the induction of incorrect RNA folding or prevention of protein and/or RNA conformational transitions. Thiostrepton, which binds to the 'GTPase center' of the large subunit, has been postulated to prevent conformational changes in either the L11 protein or rRNA to which it binds. Scintillation proximity assays designed to look at the binding of the L11 C terminal RNA-binding domain to a 23S ribosomal RNA (rRNA) fragment, as well as the ability of thiostrepton to induce that binding, were used to demonstrate the role of Mg(2+), L11 and thio-strepton in the formation and maintenance of the rRNA fragment tertiary structure. Experiments using these assays with both an Escherichia coli rRNA fragment and a thermostable variant of that RNA show that Mg(2+), L11 and thiostrepton all induce the RNA to fold to an essentially identical tertiary structure. PMID- 10734198 TI - Mapping of accessible sites for oligonucleotide hybridization on hepatitis delta virus ribozymes. AB - Semi-random libraries of DNA 6mers and RNase H digestion were applied to search for sites accessible to hybridization on the genomic and antigenomic HDV ribozymes and their 3' truncated derivatives. An approach was proposed to correlate the cleavage sites and most likely sequences of oligomers, members of the oligonucleotide libraries, which were engaged in the formation of RNA-DNA hybrids. The predicted positions of oligomers hybridizing to the genomic ribozyme were compared with the fold of polynucleotide chain in the ribozyme crystal structure. The data exemplified the crucial role of target RNA structural features in the binding of antisense oligonucleotides. It turned out that cleavages were induced if the bound oligomer could adapt an ordered helical conformation even when it required partial penetration of an adjacent double stranded region. The major features of RNA structure disfavoring hybridization and/or RNase H hydrolysis were sharp turns of the polynucleotide chain and breaks in stacking interactions of bases. Based on the predicted positions of oligomers hybridizing to the antigenomic ribozyme we chose and synthesized four antisense DNA 6mers which were shown to direct hydrolysis in the desired, earlier predicted regions of the molecule. PMID- 10734199 TI - Solution structure of the DNA decamer duplex containing a 3'-T x T basepair of the cis-syn cyclobutane pyrimidine dimer: implication for the mutagenic property of the cis-syn dimer. AB - The cis - syn dimer is the major DNA photoproduct produced by UV irradiation. In order to determine the origin of the mutagenic property of the cis - syn dimer, we used NMR restraints and molecular dynamics to determine the solution structure of a DNA decamer duplex containing a wobble pair between the 3'-T of the cis - syn dimer and the opposite T residue (CS/TA duplex). The solution structure of the CS/TA duplex revealed that the 3'-T x T base pair of the cis - syn dimer had base pair geometry that was significantly different from the canonical Watson Crick base pair and caused destabilization and conformational distortion of its 3'-region. However, a 3'-T x A base pair at the cis - syn dimer within this related DNA decamer maintains the normal Watson-Crick base pair geometry and causes little distortion in the conformation of its 3'-side. Our results show that in spite of its stable hydrogen bonding, the insertion of a T residue opposite the 3'-T of the cis - syn dimer is inhibited by structural distortion caused by the 3'-T x T base pair. This may explain why the frequency of the 3'-T- >A transversion, which is the major mutation produced by the cis - syn dimer, is only 4%. PMID- 10734201 TI - Discovering regulatory elements in non-coding sequences by analysis of spaced dyads. AB - The application of microarray and related technologies is currently generating a systematic catalog of the transcriptional response of any single gene to a multiplicity of experimental conditions. Clustering genes according to the similarity of their transcriptional response provides a direct hint to the regulons of the different transcription factors, many of which have still not been characterized. We have developed a new method for deciphering the mechanism underlying the common transcriptional response of a set of genes, i.e. discovering cis -acting regulatory elements from a set of unaligned upstream sequences. This method, called dyad analysis, is based on the observation that many regulatory sites consist of a pair of highly conserved trinucleotides, spaced by a non-conserved region of fixed width. The approach is to count the number of occurrences of each possible spaced pair of trinucleotides, and to assess its statistical significance. The method is highly efficient in the detection of sites bound by C(6)Zn(2)binuclear cluster proteins, as well as other transcription factors. In addition, we show that the dyad and single-word analyses are efficient for the detection of regulatory patterns in gene clusters from DNA chip experiments. In combination, these programs should provide a fast and efficient way to discover new regulatory sites for as yet unknown transcription factors. PMID- 10734200 TI - Identification of iron responsive genes by screening cDNA libraries from suppression subtractive hybridization with antisense probes from three iron conditions. AB - The goal of the present study is to identify genes that respond to iron availability. Suppression subtraction hybridization (SSH) was used to generate cDNA libraries from iron loaded and control human astrocytoma cells (SW1088). The cDNA libraries were screened with antisense cDNA probes obtained from mRNA isolated from astrocytoma cells exposed to three conditions: (i) normal media (control), (ii) deferox-amine treated (iron deficient) or (iii) iron loaded. The screening of the cDNA libraries with antisense probes from the three conditions enhanced the screening efficiency and decreased the number of false positives. Positive clones were identified and sequenced. The genes of interest were further analyzed by determining changes in hybridization signal on northern blots from astrocytoma cells exposed to iron or deferoxamine over different time intervals. Our analysis identified cDNAs corresponding to known iron responsive genes such as L-chain ferritin, but also revealed a number of mRNAs with novel sequences and mRNAs previously not known to be responsive to iron such as one of the ABC transporters and Thy-1 glycoprotein. Thus our results suggest that the expression of a number of genes may be influenced by changes in iron availability. PMID- 10734202 TI - Genomic organization of human GMEB-1 and rat GMEB-2: structural conservation of two multifunctional proteins. AB - The glucocorticoid modulatory element binding proteins 1 and 2 (GMEB-1 and GMEB 2) are of interest both for their multiple activities (e.g. modulation of transactivation by the glucocorticoid receptor and initiation of parvovirus replication) and their membership in the emerging family of KDWK proteins. The genomic sequence of these proteins was desired in order to begin studies on the control of GMEB expression and to pursue previous evidence for significant homologies between the GMEBs. We now report the genomic sequence of human GMEB-1 and rat GMEB-2. The structure of both genes, including portions of the introns, is highly conserved. However, GMEB-1 and GMEB-2 were found to reside on chromosomes 1 and 20, respectively, demonstrating that they are encoded by distinctly different genes. Several isoforms of the GMEBs have been reported or detected in this study, and the splicing patterns were determined. The tissue distribution of each GMEB is not the same and is highest in fetal and developing tissues, consistent with previous suggestions that both homo- and hetero oligomers may possess biological activity. The promoter region of both genes has been identified and both display high levels of transcription activity in transiently transfected cells when fused upstream of a promoterless reporter. These results indicate that the GMEBs are proteins that evolved from a single parent gene, have been highly conserved since the divergence of rats and humans and probably play important roles in development and differentiation. PMID- 10734203 TI - Determination of the recognition sequence of Mycobacterium smegmatis topoisomerase I on mycobacterial genomic sequences. AB - Mycobacterium smegmatis topoisomerase I has several distinctive features. The absence of the zinc finger motif found in other prokaryotic type I topoisomerases and the ability of the enzyme to recognise single-stranded and duplex DNA are unique characteristics of the enzyme. We have mapped the strong topoisomerase sites of the enzyme on genomic DNA sequences from Mycobacterium tuberculosis and M.smegmatis. The enzyme does not nick DNA in random fashion and DNA cleavage occurred at a few specific sites. Mapping of these sites revealed conservation of a pentanucleotide motif CG/TCT/T at the cleavage site (/ represents the cleavage site). The enzyme binds and cleaves consensus oligo-nucleotides having this sequence motif. The protein exhibits a very high preference for C or a G residue at the +2 position with respect to the cleavage site. Based on earlier and the present studies we propose that the enzyme functions in vivo mainly at these specific sites to carry out topological reactions. PMID- 10734204 TI - The repertoire of DNA-binding transcriptional regulators in Escherichia coli K 12. AB - Using a combination of several approaches we estimated and characterized a total of 314 regulatory DNA-binding proteins in Escherichia coli, which might represent its minimal set of transcription factors. The collection is comprised of 35% activators, 43% repressors and 22% dual regulators. Within many regulatory protein families, the members are homogeneous in their regulatory roles, physiology of regulated genes, regulatory function, length and genome position, showing that these families have evolved homogeneously in prokaryotes, particularly in E.coli. This work describes a full characterization of the repertoire of regulatory interactions in a whole living cell. This repertoire should contribute to the interpretation of global gene expression profiles in both prokaryotes and eukaryotes. PMID- 10734205 TI - A new label technology for the detection of specific polymerase chain reaction products in a closed tube. AB - A novel signal generation principle suitable for real time and end-point detection of specific PCR products in a closed tube is described. Linear DNA probes were labeled at their 5'-ends with a stable, fluorescent terbium chelate. The fluorescence intensity of this chelate is lower when it is coupled to single stranded DNA than when the chelate is free in solution. The synthesized probes were used in the real time monitoring of PCR using a prototype instrument that consisted of a fluorometer coupled to a thermal cycler. When the probe anneals to a complementary target amplicon, the 5'-->3' exonucleolytic activity of DNA polymerase detaches the label from the probe. This results in an enhanced terbium fluorescence signal. Since terbium has a long excited state lifetime, its fluorescence can be measured in a time-resolved manner, which results in a low background fluorescence and a 1000-fold signal amplification. The detection method is quantitative over an extremely wide linear range (at least 10 10(7)initial template molecules). The label strategy can easily be combined with existing label technologies, such as TaqMan 5'-exonuclease assays, in order to carry out multiplex assays that do not suffer from overlapping emission peaks of the fluorophores. PMID- 10734206 TI - Advanced method for oligonucleotide deprotection. AB - A new procedure for rapid deprotection of synthetic oligodeoxynucleotides has been developed. While all known deprotection methods require purification to remove the residual protective groups (e.g. benzamide) and insoluble silicates, the new procedure based on the use of an ammonia-free reagent mixture allows one to avoid the additional purification steps. The method can be applied to deprotect the oligodeoxynucleotides synthesized by using the standard protected nucleoside phosphoramidites dG(iBu), dC(Bz)and dA(Bz). PMID- 10734207 TI - Rational design of landmark probes for quantitative DNA fiber mapping (QDFM). AB - Rapid construction of high-resolution physical maps requires accurate information about overlap between DNA clones and the size of gaps between clones or clone contigs. We recently developed a procedure termed 'quantitative DNA fiber mapping' (QDFM) to help construct physical maps by measuring the overlap between clones or the physical distance between non-overlapping contigs. QDFM is based on hybridization of non-isotopically labeled probes onto DNA molecules that were bound to a solid support and stretched homogeneously to approximately 2.3 kb/microm. In this paper, we describe the design of probes that bind specifically to the cloning vector of DNA recombinants to facilitate physical mapping. Probes described here delineate the most frequently used cloning vectors such as BACs, P1s, PACs and YACs. As demonstrated in representative hybridizations, vector specific probes provide valuable information about molecule integrity, insert size and orientation as well as localization of hybridization domains relative to specifically-marked vector sequences. PMID- 10734208 TI - Mass spectrometry of single-stranded restriction fragments captured by an undigested complementary sequence. AB - In this report, we describe a simple and accurate method to analyze restriction fragments using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The two complementary strands of restriction fragments are separated through hybridization to a capture probe, which is a single-stranded undigested fragment. Using the biotin-streptavidin linkage, the hybrid is immobilized on streptavidin-coated magnetic beads. After conditioning the captured restriction fragments, they are eluted from the probe and their molecular weights are determined. The proposed method greatly improves the quality, and reduces the complexity of the mass spectrum by analyzing only one of the complementary strands of restriction fragments. PMID- 10734209 TI - MethyLight: a high-throughput assay to measure DNA methylation. AB - Cytosine-5 DNA methylation occurs in the context of CpG dinucleotides in vertebrates. Aberrant methylation of CpG islands in human tumors has been shown to cause transcriptional silencing of tumor-suppressor genes. Most methods used to analyze cytosine-5 methylation patterns require cumbersome manual techniques that employ gel electrophoresis, restriction enzyme digestion, radiolabeled dNTPs or hybridization probes. The development of high-throughput technology for the analysis of DNA methylation would significantly expand our ability to derive molecular information from clinical specimens. This study describes a high throughput quantitative methylation assay that utilizes fluorescence-based real time PCR (TaqMan) technology that requires no further manipulations after the PCR step. MethyLight is a highly sensitive assay, capable of detecting methylated alleles in the presence of a 10,000-fold excess of unmethylated alleles. The assay is also highly quantitative and can very accurately determine the relative prevalence of a particular pattern of DNA methylation. We show that MethyLight can distinguish between mono-allelic and bi-allelic methylation of the MLH1 mismatch repair gene in human colorectal tumor specimens. The development of this technique should considerably enhance our ability to rapidly and accurately generate epigenetic profiles of tumor samples. PMID- 10734210 TI - Oligonucleotide immobilization on micropatterned streptavidin surfaces. AB - We describe a simple procedure for photolithographic patterning of streptavidin on silicon substrates. Long wavelength UV (365 nm) light was used to direct the covalent attachment of photoactivatable biotin onto silylated silicon wafers. Fluorescently labeled streptavidin was found to bind only in areas exposed to the light. We used this procedure to selectively pattern streptavidin inside microwells etched in silicon, and we investigated the binding characteristics of biotinylated oligonucleotides of lengths, n = 16, 54 and 99 bases. The binding curves were found to fit the functional form of the Langmuir isotherm, with binding saturation proportional to n(-3/4). PMID- 10734211 TI - Fluorescence resonance energy transfer from pyrene to perylene labels for nucleic acid hybridization assays under homogeneous solution conditions. AB - We characterized the fluorescence resonance energy transfer (FRET) from pyrene (donor) to perylene (acceptor) for nucleic acid assays under homogeneous solution conditions. We used the hybridization between a target 32 mer and its complementary two sequential 16 mer deoxyribonucleotides whose neighboring terminals were each respectively labeled with a pyrene and a perylene residue. A transfer efficiency of approximately 100% was attained upon the hybridization when observing perylene fluorescence at 459 nm with 347-nm excitation of a pyrene absorption peak. The Forster distance between two dye residues was 22.3 A (the orientation factor of 2/3). We could change the distance between the residues by inserting various numbers of nucleotides into the center of the target, thus creating a gap between the dye residues on a hybrid. Assuming that the number of inserted nucleo-tides is proportional to the distance between the dye residues, the energy transfer efficiency versus number of inserted nucleotides strictly obeyed the Forster theory. The mean inter-nucleotide distance of the single stranded portion was estimated to be 2.1 A. Comparison between the fluorescent properties of a pyrene-perylene pair with those of a widely used fluorescein rhodamine pair showed that the pyrene-perylene FRET is suitable for hybridization assays. PMID- 10734212 TI - Multifunctional DNA conjugates for the in vitro selection of new catalysts. AB - DNA-substrate conjugates are required for the direct in vitro selection of novel DNA catalysts for reactions between two small reactants. Here we describe the introduction of all necessary features into ssDNA by a novel, multifunctional primer containing a flexible PEG spacer, an o-nitrobenzyl moiety allowing for selective photocleavage, and anthracene as a reactant, a fluorescence label and/or an immobilization tag. These components were checked individually and by a mock selection. PMID- 10734213 TI - One tube mutation detection using sensitive fluorescent dyeing of MutS protected DNA. AB - A novel, universal method for mutation detection utilising the ability of MutS protein to recognise DNA incomplementarities is proposed. The examined and reference DNA fragments are PCR amplified. The PCR products are purified, mixed, heated and cooled to form heteroduplexes. In the case of mutation the heteroduplex DNA containing mismatch is protected against exonuclease digestion by MutS, while the DNA without mismatches is degraded. The protection effect is visualised by the direct addition of a highly sensitive fluorescent dye (SYBR Gold) selectively binding DNA. The Thermus thermophilus recombined His-tagged MutS and 3'-5' exonuclease activity of T4 DNA polymerase were used in the assay. PMID- 10734214 TI - Amplifiable DNA from gram-negative and gram-positive bacteria by a low strength pulsed electric field method. AB - An efficient electric field-based procedure for cell disruption and DNA isolation is described. Isoosmotic suspensions of Gram-negative and Gram-positive bacteria were treated with pulsed electric fields of <60 V/cm. Pulses had an exponential decay waveform with a time constant of 3.4 micros. DNA yield was linearly dependent on time or pulse number, with several thousand pulses needed. Electrochemical side-effects and electrophoresis were minimal. The lysates contained non-fragmented DNA which was readily amplifiable by PCR. As the method was not limited to samples of high specific resistance, it should be applicable to physiological fluids and be useful for genomic and DNA diagnostic applications. PMID- 10734215 TI - Existence of a plant tyrosylprotein sulfotransferase: novel plant enzyme catalyzing tyrosine O-sulfation of preprophytosulfokine variants in vitro. AB - An in vitro assay system to detect tyrosylprotein sulfotransferase (TPST) activity of higher plant cells was established, using synthetic oligopeptides based on the deduced amino acid sequence of a phytosulfokine-alpha (PSK-alpha) precursor. TPST activity was found in microsomal membrane fractions of rice, asparagus and carrot cells and it was confirmed that acidic amino acid residues adjacent to the tyrosine residues of acceptor peptides were essential to the sulfation reaction. The asparagus TPST exhibited a broad pH optimum of 7.0-8.5, required manganese ions for maximal activity and appeared to be a membrane-bound protein localized in the Golgi apparatus. These enzymes should be defined as a new class of plant sulfotransferases that catalyze tyrosine O-sulfation of a PSK alpha precursor and other unknown proteins. PMID- 10734216 TI - Sterol metabolism and ERG2 gene regulation in the yeast Saccharomyces cerevisiae. AB - Certain exogenously-supplied sterols, like ergost-8-enol, are efficiently converted into ergosterol in yeast. We have taken advantage of this property to study the regulation of the Delta8-Delta7-sterol isomerase-encoding ERG2 gene in an ergosterol auxotrophic mutant devoid of squalene-synthase activity. Ergosterol starvation leads to an 8-16-fold increase in ERG2 gene expression. Such an increase was also observed in wild-type cells either grown anaerobically or treated with SR31747A a sterol isomerase inhibitor. Exogenously-supplied zymosterol is entirely transformed into ergosterol, which represses ERG2 transcription. By contrast, exogenously-supplied ergosterol has little or no effect on ERG2 transcription. PMID- 10734217 TI - Arabidopsis phytochromes C and E have different spectral characteristics from those of phytochromes A and B. AB - The red/far-red light absorbing phytochromes play a major role as sensor proteins in photomorphogenesis of plants. In Arabidopsis the phytochromes belong to a small gene family of five members, phytochrome A (phyA) to E (phyE). Knowledge of the dynamic properties of the phytochrome molecules is the basis of phytochrome signal transduction research. Beside photoconversion and destruction, dark reversion is a molecular property of some phytochromes. A possible role of dark reversion is the termination of signal transduction. Since Arabidopsis is a model plant for biological and genetic research, we focussed on spectroscopic characterization of Arabidopsis phytochromes, expressed in yeast. For the first time, we were able to determine the relative absorption maxima and minima for a phytochrome C (phyC) as 661/725 nm and for a phyE as 670/724 nm. The spectral characteristics of phyC and E are strictly different from those of phyA and B. Furthermore, we show that both phyC and phyE apoprotein chromophore adducts undergo a strong dark reversion. Difference spectra, monitored with phycocyanobilin and phytochromobilin as the apoprotein's chromophore, and in vivo dark reversion of the Arabidopsis phytochrome apoprotein phycocyanobilin adducts are discussed with respect to their physiological function. PMID- 10734218 TI - Vascular endothelial cells synthesize and secrete brain-derived neurotrophic factor. AB - Brain-derived neurotrophic factor (BDNF) is an abundant neurotrophin in brain and peripheral nerves, where it affects neural development, survival and repair after injury. BDNF has been detected in rat and human blood, but the source of circulating BDNF is not established. BDNF messenger and peptide were detected in cultured cells and in the culture medium of human umbilical vein endothelial cells. The expression of BDNF was up-regulated by elevation of intracellular cAMP and down-regulated by Ca(2+) ionophore, bovine brain extract and laminar fluid shear stress. These results suggest that vascular endothelial cells may contribute to circulating BDNF. PMID- 10734219 TI - The SixA phospho-histidine phosphatase modulates the ArcB phosphorelay signal transduction in Escherichia coli. AB - The Escherichia coli SixA protein is the first discovered prokaryotic phospho histidine phosphatase, which was implicated in a His-to-Asp phosphorelay. The sixA gene was originally identified as the one that interferes with, at its multi copy state, the cross-phosphorelay between the histidine-containing phosphotransmitter (HPt) domain of the ArcB anaerobic sensor and its non-cognate OmpR response regulator. Nevertheless, no evidence has been provided that the SixA phosphatase is indeed involved in a signaling circuitry of the authentic ArcB-to-ArcA phosphorelay in a physiologically meaningful manner. In this study, a SixA-deficient mutant was characterized with special reference to the ArcB signaling, which allows E. coli cells to respond to not only external oxygen, but also certain anaerobic respiratory conditions. Here evidence is provided for the first time that the SixA phosphatase is a crucial regulatory factor that is involved in the ArcB signaling, particularly, under certain anaerobic respiratory growth conditions. We propose a novel mechanism, involving an HPt domain and a phospho-histidine phosphatase, by which a given multi-step His-to-Asp signaling can be modulated. PMID- 10734220 TI - Identification of the regions involved in DNA binding by the mouse PEBP2alpha protein. AB - The polyomavirus enhancer binding protein 2alpha (PEBP2alpha) is a DNA binding transcriptional regulatory protein that binds conserved sites in the polyomavirus enhancer, mammalian type C retroviral enhancers and T-cell receptor gene enhancers. Binding of PEBP2alpha and homologous proteins to the consensus DNA sequence TGPyGGTPy is mediated through a protein domain known as the runt domain. Although recent NMR studies of DNA-bound forms of the runt domain have shown an immunoglobulin-like (Ig) fold, the identification of residues of the protein that are involved in DNA binding has been obscured by the low solubility of the runt domain. Constructs of the mouse PEBP2alphaA1 gene were generated with N- and C terminal extensions beyond the runt homology region. The construct containing residues Asp90 to Lys225 of the sequence (PEBP2alpha90-225) yielded soluble protein. The residues that participate in DNA binding were determined by comparing the NMR spectra of free and DNA-bound PEBP2alpha90-225. Analysis of the changes in the NMR spectra of the two forms of the protein by chemical shift deviation mapping allowed the unambiguous determination of the regions that are responsible for specific DNA recognition by PEBP2alpha. Five regions in PEBP2alpha90-225 that are localized at one end of the beta-barrel were found to interact with DNA, similar to the DNA binding interactions of other Ig fold proteins. PMID- 10734221 TI - A Schistosoma protein, Sh-TOR, is a novel inhibitor of complement which binds human C2. AB - Human complement regulatory (also called inhibitory) proteins control misdirected attack of complement against autologous cells. Trypanosome and schistosome parasites which survive in the host vascular system also possess regulators of human complement. We have shown Sh-TOR, a protein with three predicted transmembrane domains, located on the Schistosoma parasite surface, to be a novel complement regulatory receptor. The N-terminal extracellular domain, Sh-TOR-ed1, binds the complement protein C2 from human serum and specifically interacts with the C2a fragment. As a result Sh-TOR-ed1 pre-incubated with C2 inhibits classical pathway (CP)-mediated haemolysis of sheep erythrocytes in a dose-dependent manner. In CP-mediated complement activation, C2 normally binds to C4b to form the CP C3 convertase and Sh-TOR-ed1 has short regions of sequence identity with a segment of human C4b. We propose the more appropriate name for TOR of CRIT (complement C2 receptor inhibitory trispanning). PMID- 10734222 TI - A single amino acid change makes a rat neuronal sodium channel highly sensitive to pyrethroid insecticides. AB - Two amino acid substitutions in a housefly sodium channel, L1014F in domain IIS6 and M918T in the IIS4-S5 linker, have been identified in kdr and super-kdr pyrethroid-resistant phenotypes, respectively. Unlike their native insect counterparts, mammalian sodium channels are only weakly sensitive to pyrethroids. Do the sodium channels of mammal and pyrethroid-resistant housefly share similar structural characteristics that account for their low pyrethroid sensitivities? We report here that substitution of isoleucine for methionine at position 874 (equivalent to the super-kdr site 918 in the housefly) in the rat IIA alpha subunit causes a 100-fold increase in sensitivity. PMID- 10734223 TI - Structure-function relationships of the NMDA receptor antagonist peptide, conantokin-R. AB - Conantokin-R (con-R) is a gamma-carboxyglutamate-containing 27-residue neuroactive peptide present in the venom of Conus radiatus, and acts as a non competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor. This peptide features a single disulfide bond, a type of structural element found in most classes of conotoxins, but not in other conantokins. The NMDA receptor antagonist activity of chemically synthesized con-R was determined through an assay involving inhibition of the spermine-enhanced binding of the NMDA receptor channel blocker, [(3)H]MK-801, to rat brain membranes, and yielded an IC(50) of 93 nM. This value represents a 2-5 times better potency than con-G or con-T, the other two characterized conantokins. Circular dichroism (CD) analysis of the metal-free form of con-R is indicative of a low alpha-helical content. There is an increase in alpha-helicity upon the addition of divalent cations, such as Ca(2+), Mg(2+), or Zn(2+). Isothermal titration calorimetry experiments showed one detectable Mg(2+) binding site with a K(d) of 6.5 microM, and two binding sites for Zn(2+), with K(d) values of 150 nM and 170 microM. Residue-specific information of the conformational state of con-R was obtained by two-dimensional (1)H-NMR. Analyses of the alpha-proton chemical shifts, NOE patterns, and hydrogen exchange rates of the peptide indicated an alpha-helical conformation for residues 1-19. Synthetic con-R-derived peptide variants, containing deletions of 7 and 10 amino acid residues from the carboxy-terminus of the wild-type peptide, displayed unaltered cation binding and NMDA receptor antagonist properties. The alpha-helical secondary structures of the two truncation peptides were more stable than full-length con-R, as evidenced by CD measurements and reduced backbone hydrogen exchange rates. These results provide experimental evidence that the structural elements common to the three conantokins thus far identified are the primary determinants for receptor function and cation binding/secondary structure stability. PMID- 10734224 TI - Evidence for the existence of rhodanese (thiosulfate:cyanide sulfurtransferase) in plants: preliminary characterization of two rhodanese cDNAs from Arabidopsis thaliana. AB - The existence of rhodanese (thiosulfate:cyanide sulfurtransferase; EC 2.8.1.1) in plants has been highly controversial. We have isolated and characterized for the first time in plants two cDNAs encoding rhodanese isoforms in Arabidopsis thaliana, AtRDH1 and AtRDH2. Both cDNAs contained a full-length open reading frame, the expression of which increased the rhodanese activity of transgenic yeast. AtRDH1 protein was mitochondrial, while AtRDH2 was cytosolic. AtRDH1 and AtRDH2 genes originated from the duplication of a large genomic region in chromosome 1 which took place before the appearance of the Arabidopsis genus. Our results confirm the existence of rhodanese in plants. PMID- 10734225 TI - Polymyxin B binds to anandamide and inhibits its cytotoxic effect. AB - Anandamide (ANA), an endogenous cannabinoid, can be generated by activated macrophages during endotoxin shock and is thought to be a paracrine contributor to hypotension. We discovered that ANA in saline/ethanol solution and in serum was efficiently adsorbed in a polymyxin B (PMB)-immobilized beads column and eluted with ethanol. We confirmed the direct binding of PMB to ANA by using surface plasmon resonance. The adsorption of ANA by PMB may abolish the diverse effects of ANA such as hypotension, immunosuppression, and cytotoxicity, and may suggest a new therapeutic strategy for endotoxin shock. PMID- 10734226 TI - Genome microarray analysis of transcriptional activation in multidrug resistance yeast mutants. AB - The cDNA from activated mutants of the homologous transcription factors Pdr1p and Pdr3p was used to screen DNA microarrays of the Saccharomyces cerevisiae complete genome. Twenty-six overexpressed targets of the PDR1-3 and/or PDR3-7 mutants were identified. Twenty-one are new targets, the majority of which are of unknown function. In addition to well known ABC transporters, these targets appear to be involved in transport or in membrane lipids and cell wall biosyntheses. Several of the targets seem to contribute to the cell defence against a variety of stresses. Pdr1p and Pdr3p do not act similarly on all targets. Unexpectedly, the expression of 23 other genes appeared to be repressed in the PDR1-3 and/or PDR3-7 mutants. In contrast to the majority of the activated genes, none of the repressed genes contains pleiotropic drug resistance binding sites in their promoter. PMID- 10734227 TI - Distinct versus redundant properties among members of the INK4 family of cyclin dependent kinase inhibitors. AB - p16(INK4a), p15(INK4b), p18(INK4c) and p19(INK4d) comprise a family of cyclin dependent kinase inhibitors and tumor suppressors. We report that the INK4 proteins share the ability to arrest cells in G1, and interact with CDK4 or CDK6 with similar avidity. In contrast, only p18 and particularly p19 are phosphorylated in vivo, and each of the human INK4 proteins shows unique expression patterns dependent on cell and tissue type, and differentiation stage. Thus, the INK4 proteins harbor redundant as well as non-overlapping properties, suggesting distinct regulatory modes, and diverse roles for the individual INK4 family members in cell cycle control, cellular differentiation, and multistep oncogenesis. PMID- 10734228 TI - Biophysical characterization of interactions between the core binding factor alpha and beta subunits and DNA. AB - Core binding factors (CBFs) play key roles in several developmental pathways and in human disease. CBFs consist of a DNA binding CBFalpha subunit and a non-DNA binding CBFbeta subunit that increases the affinity of CBFalpha for DNA. We performed sedimentation equilibrium analyses to unequivocally establish the stoichiometry of the CBFalpha:beta:DNA complex. Dissociation constants for all four equilibria involving the CBFalpha Runt domain, CBFbeta, and DNA were defined. Conformational changes associated with interactions between CBFalpha, CBFbeta, and DNA were monitored by nuclear magnetic resonance and circular dichroism spectroscopy. The data suggest that CBFbeta 'locks in' a high affinity DNA binding conformation of the CBFalpha Runt domain. PMID- 10734229 TI - Folding of a synthetic parallel beta-roll protein. AB - Recently, the design of beta-sheet proteins and concomitant folding studies have attracted increasing attention. A unique natural all-beta domain occurs in a family of cytolytic bacterial toxins, the so-called RTX toxins. This domain consists of a variable number (about 6-45) of tandem repeats of a glycine-rich nine-residue motif with the consensus sequence GGXGXDX(L/I/F)X. The analysis of the three-dimensional structure of alkaline protease from Pseudomonas aeruginosa which possesses six of these repeats revealed that they fold into a novel 'parallel beta-roll' where calcium is bound within the turns connecting the beta strands. A 75-mer peptide of the sequence NH(2)-WLS-[GGSGNDNLS](8)-COOH was chemically synthesised. Circular dichroism spectroscopy showed that this polypeptide folds in the presence of Ca(2+) and polyethylene glycol into a beta structure which is presumably identical with the parallel beta-roll. This synthetic beta-roll behaves similarly to the isolated beta-roll domains from Escherichia coli haemolysin or Bordetella pertussis cyclolysin in terms of calcium binding and polymerisation behaviour. PMID- 10734230 TI - Fibromodulin and lumican bind to the same region on collagen type I fibrils. AB - Fibromodulin and lumican are closely related members of the extracellular matrix leucine-rich repeat glycoprotein/proteoglycan family. Similar to decorin, another member of this protein family, they bind to fibrillar collagens and function in the assembly of the collagen network in connective tissues. We have studied the binding of recombinant fibromodulin, lumican and decorin, expressed in mammalian cells, to collagen type I. Using a collagen fibril formation/sedimentation assay we show that fibromodulin inhibits the binding of lumican, and vice versa. Fibromodulin and lumican do not affect the binding of decorin to collagen, nor does decorin inhibit the binding of fibromodulin or lumican. Binding competition experiments and Scatchard plot analysis indicate that fibromodulin binds to collagen type I with higher affinity than lumican. PMID- 10734231 TI - Distinct function of the cytoplasmic tail in human D1-like receptor ligand binding and coupling. AB - To delineate the role of the cytoplasmic tail in the distinct binding and coupling properties of human dopamine D1-like receptors, chimeric receptors were generated in which the entire tail region of wild-type human D1A (or D1) and D1B (or D5) receptors was exchanged. The hD1A-D1BT, but not hD1B-D1AT, receptor expression was dramatically reduced compared with wild-type receptor expression. Swapping the cytoplasmic tail resulted in a full switch of dopamine binding affinity and constitutive activity, while dopamine potency decreased and agonist mediated maximal activation of adenylyl cyclase increased for both chimeras. Hence, the cytoplasmic tail plays a crucial role in D1-like receptor expression, agonist binding affinity and constitutive activation but regulates in a distinct fashion the formation of D1A and D1B receptor active states upon dopamine binding. PMID- 10734232 TI - A family of gamma-like calcium channel subunits. AB - The gamma subunit was initially identified as an auxiliary subunit of the skeletal muscle calcium channel complex. Evidence for the existence of further gamma subunits arose following the characterization of a genetic defect that induces epileptic seizures in stargazer mice. We present here the first account of a family of at least five putative gamma subunits that are predominantly expressed in brain. The gamma-2 and gamma-4 subunits shift the steady-state inactivation curve to more hyperpolarized potentials upon coexpression with the P/Q type alpha(1A) subunit. The coexpression of the gamma-5 subunit accelerates the time course of current activation and inactivation of the alpha(1G) T-type calcium channel. PMID- 10734233 TI - Mutually exclusive expression of beta(III)-tubulin and vimentin in adrenal cortex carcinoma SW13 cells. AB - During embryogenesis, the maturation of neuroblasts into neurones is accompanied by the down-regulation of vimentin and by the expression of neuronal microtubular proteins. Here, we show that human adrenal cortex SW13 cells express beta(III) tubulin, MAP2b and tau. Analysis of vimentin-positive and -negative subclones of SW13 cells revealed that, under defined cultured conditions, beta(III)-tubulin and MAP2b were present only in vimentin-deficient cells and that beta(III) tubulin repression occurred at the transcriptional level in vimentin-positive cells. These results suggest that vimentin repression and beta(III)-tubulin expression are co-ordinated by an upstream mechanism relevant to the control of cytoskeletal protein expression during neuronal development. PMID- 10734234 TI - Heme orientation affects holo-myoglobin folding and unfolding kinetics. AB - Native myoglobin (Mb) consists of two populations which differ in the orientation of the heme by 180 degrees rotation (as verified by nuclear magnetic resonance) but have identical absorption spectra and equilibrium-thermodynamic stability. Here, we report that these two fractions of native oxidized Mb (from horse) both unfold and refold (chemical denaturant, pH 7, 20 degrees C) in two parallel kinetic reactions with rate constants differing 10-fold. In accord, the oxidized heme remains coordinated to unfolded horse Mb in up to 4 M guanidine hydrochloride (pH 7, 20 degrees C). PMID- 10734235 TI - The survival motor neuron protein interacts with the transactivator FUSE binding protein from human fetal brain. AB - To identify interacting proteins of survival motor neuron (SMN) in neurons, a fetal human brain cDNA library was screened using the yeast two-hybrid system. One identified group of SMN interacting clones encoded the DNA transactivator FUSE binding protein (FBP). FBP overexpressed in HEK293 cells or endogenously expressed in fetal and adult mouse brain bound specifically in vitro to recombinant SMN protein. Furthermore, an anti-FBP antibody specifically co immunoprecipitated SMN when both proteins were overexpressed in HEK293 cells. These results demonstrate that FBP is a novel interacting partner of SMN and suggests a possible role for SMN in neuronal gene expression. PMID- 10734236 TI - Retinoblastoma protein dephosphorylation is an early event of cellular response to prooxidant conditions. AB - The modification of intracellular redox conditions with diethylmaleate (DEM), a glutathione-depleting agent, induces a p53-independent growth arrest mediated by the accumulation of p21(waf1) mRNA and protein. The same treatment also induces the retinoblastoma protein (pRb) dephosphorylation. This dephosphorylation (i) is very fast, being observed already 5 min after the exposure of the cells to DEM, (ii) is dependent on the prooxidant effects of DEM, being prevented by the treatment with N-acetylcysteine and (iii) is completely reversible, since the rephosphorylation of pRb is promptly obtained upon the removal of the glutathione depleting agent from the culture medium. The dephosphorylation of pRb is independent of the accumulation of p21(waf1) induced by DEM; in fact, p21(waf1) levels start to increase much later after DEM treatment and accordingly cyclin dependent kinase activities are not yet induced when pRb is already dephosphorylated following DEM treatment. Finally, pRb dephosphorylation is catalyzed by phosphatases activated by DEM treatment. PMID- 10734237 TI - Purification and characterization of dissimilatory nitrate reductase from a denitrifying halophilic archaeon, Haloarcula marismortui. AB - Dissimilatory nitrate reductase was purified from a denitrifying halophilic archaeon, Haloarcula marismortui, to an electrophoretically homogeneous state. The purified enzyme was inferred to be a homotetramer composed of a 63 kDa polypeptide. The electron paramagnetic resonance spectrum of the purified enzyme revealed typical rhombic signals which were ascribed to Mo(V) in the Mo molybdopterin complex. Like the bacterial membrane-bound (Nar-) enzyme, the purified enzyme supported the catalysis of chlorate. The enzyme was activated in extreme saline conditions and the values of k(cat) and K(m) toward nitrate were 145 s(-1) and 79 microM, respectively, in the presence of 2.0 M NaCl. PMID- 10734238 TI - The amyloid beta peptide abeta (25-35) induces apoptosis independent of p53. AB - Apoptosis of neuronal cells apparently plays a role in Alzheimer's disease (AD). The amyloid beta (Abeta) peptide derived from beta-amyloid precursor protein is found in AD brain in vivo and can induce apoptosis in vitro. While p53 accumulates in cells of AD brain, it is not known if p53 plays an active role in Abeta-induced apoptosis. We show here that inactivation of p53 in two experimental cell lines, either by expression of the papillomavirus E6 protein or by a shift to restrictive temperature, does not affect apoptosis induction by Abeta (25-35), indicating that Abeta induces apoptosis in a p53-independent manner. PMID- 10734239 TI - Human corneal thickness and its impact on intraocular pressure measures: a review and meta-analysis approach. AB - We determined the "normal" central corneal thickness (CCT) value in human corneas based on reported literature values for within-study average CCT values, and used this as a reference to assess the reported impact of physiological variables (especially age and diurnal effects), contact lens wear, pharmaceuticals, ocular disease, and ophthalmic surgery on CCT. With the expected CCT and its variance defined, it should be possible to determine the potential impact of differences in CCT in intraocular pressure (IOP) assessments, especially by applanation tonometry, using a meta-analysis approach. Some 600 sets of CCT data were identified from the worldwide literature over the period of 1968 through mid 1999, of which 134 included IOP measures as well. The within-study average CCT values and reported variance (SD) was noted along with the number of eyes and any special characteristics, including probable ethnic origin of the study subjects. Various sets of data were subjected to statistical analyses. From 300 data sets from eyes designated as normal, the group-averaged CCT was 0.534 mm. From 230 data sets where interindividual variance was reported, the group-averaged CCT was 0.536 mm (median 0.536 mm; average SD of 0. 031 mm, average coefficient of variation = 5.8%). Overall, studies using slit-lamp-based pachometry have reported marginally lower CCT values (average 0.530 mm, average SD 0.029 mm) compared to ultrasound-based studies (average 0.544, average SD 0.034 mm), which perhaps reflects the type of individual studied (non-surgical vs. pre-surgical patients) rather than the technique itself. A slight chronological increase in reported average CCT values (approximately 0.006 mm/decade) was evident, but a substantial chronological increase was evident for ultrasound pachometry studies (approximately 0.015 mm/decade). Within the meta-analysis-generated average and variance, age had no obvious impact on CCT measures for *whites, although an age related decline in CCT is evident for non-whites. Any diurnal effects are likely concealed within the expected variance in CCT. Contact lens wear and pharmaceuticals generally produced changes in CCT that were well within the expected variance in CCT. Of the ocular diseases, only those associated with collagen disorders (including keratoconus) or endothelial-based corneal dystrophies (e.g., Fuchs) were likely to result in decreases or increases, respectively, of CCT beyond the normal variance. Routine contact lens wear and diseases such as diabetes seem unlikely to produce changes in CCT of a magnitude that would justify pachometry as a monitoring method beyond routine slit-lamp evaluation. Increases in CCT beyond the expected variance were reported after a range of intraocular surgeries (cataract operations, penetrating keratoplasty), whereas photorefractive surgery produces a measurable decrease in CCT. A meta analysis of possible association between CCT and IOP measures of 133 data sets, regardless of the type of eyes assessed, revealed a statistically significant correlation; a 10% difference in CCT would result in a 3. 4 +/- 0.9 mm Hg difference in IOP (P or =0.5 x 109/l and the last platelet transfusion post transplant were 13 and 11 days, respectively, with 14 (22%) of patients having platelet recovery delayed beyond day 21. There was no significant difference between patients who had or had not received extensive chemo/radiotherapy or among the different mobilization regimens for time to neutrophil or platelet recovery or the number of platelet or red blood cell transfusions received post transplant. Threshold doses of the different cell types transplanted (per kg of patient weight) which predicted rapid platelet recovery were 2 x 106 CD34+ cells, 5 x 105 total CFC and 2.5 x 104CFC-Mk. Corresponding thresholds for progenitor activity measured in LTC could not be established. These results further support the view that standard mobilization regimens yield progenitor numbers that are, in most cases, nonlimiting for generating neutrophil and platelet recoveries within 2 to 3 weeks after myeloablative therapy. Assessment of the CD34+ cell and/or CFC content of leukapheresis collections may identify patients in whom platelet recovery is likely to be significantly delayed although CFC-Mk enumeration does not appear to offer any unique predictive advantage. PMID- 10734293 TI - Autologous bone marrow transplantation for childhood acute lymphoblastic leukaemia in second remission - long-term follow-up. AB - From 1984 to 1996, 31 consecutive children without sibling donors, aged 5-19 years (median 8) with acute lymphoblastic leukaemia (ALL) in second complete remission (CR), received unpurged autologous bone marrow transplantation (ABMT) after melphalan and single fraction total body irradiation (TBI). ABMT was performed using fresh unmanipulated marrow harvested after standard reinduction and consolidation therapy 2-11 months (median 5) after relapse. With a median survival of 2.9 years the probability of survival for all patients in continuing second CR was 45.1% (95% CI, 24%-62%) after 5 years. Regimen-related and non leukaemia mortality was 7% (95% CI, 2%-26%). The longest time to second relapse from ABMT was 3.1 years. Pituitary and gonadal dysfunction requiring hormonal replacement therapy occurred in the majority of long-term survivors. Twelve patients developed cataracts. ABMT with melphalan/single fraction TBI has proved an effective anti-leukaemia treatment with low regimen-related mortality but significant long-term morbidity. The current approach of allogeneic BMT from an unrelated donor when no sibling donor is available, following conditioning with cyclophosphamide/ fractionated TBI has resulted in a reduced relapse rate and improved short-term overall survival in the treatment of relapsed childhood ALL. However, long-term results are awaited. PMID- 10734294 TI - Allogeneic bone marrow transplantation for low-grade lymphoma and chronic lymphocytic leukemia. AB - Twenty-six patients with low-grade lymphoma (LGL) (n = 18) or chronic lymphocytic leukemia (CLL) (n = 8) received allogeneic BMTs between 1985 and 1998. Median age was 42 years, median interval from diagnosis to transplant 22 months and median number of prior treatments three. Twenty (77%) had stage IV disease; 22 (85%) had never achieved CR. Donor source was HLA matched sibling (n = 19, 73%), matched unrelated (n = 6, 23%) or syngeneic (n = 1). Conditioning therapy included total body irradiation in 23 patients and busulphan in three. Twenty-five received GVHD prophylaxis with cyclosporine A; + methotrexate (n = 19), + methylprednisolone (n = 2) or + T cell depletion of allograft +/- methotrexate (n = 4). Sixteen patients are alive, a median of 2.4 years post BMT. Death occurred due to transplant complications (n = 7) or underlying disease (n = 3). Eighteen (12 LGL, six CLL) of 22 evaluable patients (82%) achieved CR post BMT. Cumulative incidence of refractory/recurrent disease was 18% (95% confidence interval (CI) 7 42%). Overall and event-free survivals were 58% (95% CI 35-75%) and 54% (95% CI 32-72%), respectively. Allogeneic BMT for young patients with advanced LGL or CLL is feasible and can result in long-term disease-free survival. PMID- 10734295 TI - Matched unrelated bone marrow transplantation for combined immunodeficiency. AB - Bone marrow transplantation (BMT) from siblings is the treatment of choice for severe combined immunodeficiency (SCID). The objective of this study was to evaluate the efficiency of BMT from matched unrelated donors (MUD) in congenital immunodeficiencies when a sibling donor is unavailable. Sixteen consecutive patients with SCID (n = 9) and CID (n= 7), were referred for an unrelated donor search. Acceptable donors were found for all patients. Fifteen patients received busulfan and cyclophosphamide pretransplant conditioning. One patient had an early loss of graft and was reconditioned using cyclophosphamide and total body irradiation. The graft-versus-host disease (GVHD) prophylaxis used was methylprednisolone, cyclosporin A with or without methotrexate. Neutrophil engraftment was rapid and was achieved in all patients within a mean of 15.4 days. Only 13 episodes of fever were recorded shortly after BMT. GVHD of grade II or more was apparent in 2/9 (22%) of SCID patients and in 4/7 (57%) of CID patients. Overall survival was 75% with a mean follow-up of 47.4 months (range 18 101). Six out of nine SCID patients (67%) and 6/7 (86%) of CID patients are alive and well. Eleven patients had normal humoral immunity, and cell-mediated immunity as measured by flow cytometry and mitogenic responses, was intact in all patients. Intradermal candida skin test was positive in 9/10 patients tested. We conclude that BMT from MUD results in rapid engraftment and is therefore associated with a low rate of infection contributing to the improved survival rate. The protocol used is especially favorable for patients with combined immunodeficiency. PMID- 10734296 TI - Changes in T cell receptor repertoire associated with graft-versus-tumor effect and graft-versus-host disease in patients with relapsed multiple myeloma after donor lymphocyte infusion. AB - Recent reports of clinical responses following donor lymphocyte infusions (DLI) in patients with relapsed multiple myeloma (MM) after allogeneic BMT have demonstrated the ability of allogeneic cells to mediate a graft-versus-myeloma (GVM) effect, but the mechanisms involved have not been determined. To identify changes in the T cell compartment associated with DLI, we performed a molecular analysis of the T cell receptor (TCR) repertoire in four patients with relapsed MM who received infusions of CD4+ lymphocytes from HLA-identical sibling donors. Three of the four patients demonstrated a clinical anti-myeloma response following DLI but also developed graft-versus-host disease (GVHD). The TCR repertoire was examined after PCR amplification of 24 Vbeta gene subfamilies. This method determines the relative utilization of each Vbeta gene subfamily and also allows the identification of clonal and oligoclonal T cell populations through analysis of CDR3 regions for each TCR Vbeta gene subfamily. Serial blood samples were obtained over at least a 1 year period before and after DLI and results compared to 10 normal donors. Serial analysis of CDR3 size profiles demonstrated the appearance of clonal T cell populations after DLI in each of the three responding patients. The appearance of some clones was noted within the first 3 months after DLI and coincided with decreasing levels of monoclonal paraprotein indicating an ongoing GVM response. Other T cell clones appeared at later time points and coincided with the development of GVHD. These findings demonstrate that T cell clones with different patterns of onset can be identified in the peripheral blood of MM patients following DLI. Further functional characterization of these distinct clonal expansions will be required to determine whether these T cell clones are mediators of either anti-myeloma or anti-host activity. PMID- 10734298 TI - Resveratrol selectively inhibits leukemia cells: a prospective agent for ex vivo bone marrow purging. AB - Ex vivo purging of contaminating tumor cells may reduce the incidence of relapse in patients undergoing bone marrow transplantation. In this study we demonstrate that resveratrol, a phytoalexin with anti-oxidant and chemopreventive activity, exhibits anti-leukemic activity against mouse (32Dp210, L1210) and human (U937, HL-60) leukemic cell lines by inhibiting cell proliferation. Long-term exposure to resveratrol also inhibits the clonal growth of normal hematopoietic progenitor cells but at a higher IC50 of resveratrol than that for most of the leukemia cell lines tested. The inhibitory effect of resveratrol on hematopoietic progenitors is partially reversible, whereas the effect on leukemia cells is largely irreversible. The inhibition of leukemia cells by resveratrol involves nucleosomal DNA fragmentation (apoptosis). On the other hand, resveratrol does not induce or enhance spontaneously occurring apoptotic death in normal hematopoietic progenitor cells. In vivo experiments performed with untreated and resveratrol-treated bone marrow showed comparable hematopoietic reconstitution in lethally irradiated mice (10 Gy) as determined by survival, hematologic recovery, and the number of hematopoietic progenitor cells present in the marrow of reconstituted animals. Taken together, these results indicate the potential use of resveratrol for ex vivo pharmacological purging of leukemia cells from bone marrow autografts without significant loss in the hematopoietic activity of progenitor cells. PMID- 10734297 TI - Adsorption and pharmacokinetics of cyclosporin A in relation to mode of infusion in bone marrow transplant patients. AB - Two main factors that affect the pharmacokinetics of cyclosporin A (CsA) during 24-h durable intravenous (DIV) administration have been reported, namely physiological changes after bone marrow transplantation, and blood sampling through indwelling lines. In addition, it has been found that infusion sets made of polyvinyl chloride (PVC) markedly adsorb CsA. We conducted in vitro adsorption studies of CsA on infusion sets, and the administration routes that are used in the treatment of patients with bone marrow transplantation. We also examined the effects of administration route on CsA pharmacokinetics in clinical practice. The in vitro adsorption study using 30-mm segments of lumen from commercially available infusion sets showed that the degree of CsA adsorption per area of lumen made of PVC was significantly higher than that in those made of polyethylene (PE) or polybutadiene (PB), which showed no adsorption of CsA. Due to its adsorption, use of infusion sets made of PVC resulted in about a 40-50% loss of CsA dose, which affected the pharmacokinetic parameters during 24-h DIV, while those made of PE and PB did not. The use of non-PVC infusion sets should allow for accurate monitoring of CsA results, and provide cost benefit in the treatment of bone marrow transplantation. PMID- 10734299 TI - Thymus-independent expansion of T lymphocytes in children after allogeneic bone marrow transplantation. AB - The contribution of the thymus-dependent pathway and thymus-independent pathways for T cell regeneration after BMT in children is still unclear. We analyzed the kinetics of T cell regenerative pathways after allogeneic BMT. The number of CD4+CD45RA+ T cells, a thymus-dependent population, was very low until 3 months after BMT. The numbers of CD28- T cells and CD8+ T cells expressing CD8alpha/alpha homodimer (CD8alpha/alpha+ T cells), a thymus-independent population, increased shortly after BMT, beyond the levels of healthy children in some patients. The numbers of Vgamma9+Vdelta2+ and Valpha24+ T cells, which represent populations of extrathymic development, were less than 200/microl during the 6 months after BMT. There was a significant inverse correlation between the percentages of CD4+CD45RA+ and CD28-T cells at 1 month, and a positive correlation between the percentages of CD28- and CD8alpha/alpha+ T cells at 2 and 3 months after BMT. The mean age at BMT was higher in patients with a high level of CD8alpha/alpha+ T cells than in those without an increase in these cells, suggesting the influence of thymic function on the regenerative pathways. These results suggest that the thymus-independent pathway is the dominant source of T cells even in children shortly after allogeneic BMT. PMID- 10734300 TI - Early iron reduction programme for thalassaemia patients after bone marrow transplantation. AB - Thirty thalassaemia patients received iron reduction starting at around 3 months post transplant. Sixteen received desferrioxamine and nine had phlebotomy, five patients had desferrioxamine followed by phlebotomy. The desferrioxamine group had higher serum ferritin levels at the start of iron reduction as compared to the phlebotomy group (5292 vs 2453 microg/l, P EQ 0.001). After 444 and 407 days of iron reduction, serum ferritins at cessation of iron reduction in both groups was similar (665 vs 588 microg/l). The rate of decline of serum ferritin in both groups was similar. There was no graft rejection during the programme. Early institution of iron reduction in ex-thalassaemia is safe. PMID- 10734301 TI - Varicella-zoster infection after allogeneic bone marrow transplantation: incidence, risk factors and prevention with low-dose aciclovir and ganciclovir. AB - We examined the incidence of herpes varicella-zoster virus (VZV) infection in 151 patients undergoing allogeneic BMT between August 1990 and September 1997 and who survived at least 3 months. Median follow-up was 17 (range 3.3-80.7) months. Herpes simplex virus antibody positive (HSV+) patients received aciclovir 1200 mg p.o. daily or 750 mg i.v. daily, in divided doses from day 0 to engraftment. Ganciclovir (5 mg/kg i.v. three times per week) was given in CMV+ patients (or if the donor was CMV+) from engraftment to day 84. Ganciclovir was continued or recommenced if a dose of greater than 20 mg of prednisone was used for the treatment of GVHD otherwise aciclovir was recommenced. In HSV+ patients not receiving ganciclovir, aciclovir 600 mg p.o. daily in divided doses was given until at least 6 months after BMT. Thirty-two patients developed VZV infection from 4.1 to 28 months after transplant. The estimated cumulative incidence of VZV was 13% (95% confidence interval 6-19%) at 12 months, 32% (22-42%) at 24 months and 38% (27-50%) at 28 months, with no further cases beyond that time. No patient developed VZV whilst receiving aciclovir or ganciclovir (P < 0.0001). However, there was a rapid onset of VZV following cessation of antiviral therapy (33% (20 46%) at 1 year post cessation). The presence of GVHD and the prior duration of antiviral prophylaxis were significant and independent risk factors for the development of VZV. Age, underlying disease, conditioning therapy or donor type were not. We conclude that 3-6 months of low-dose aciclovir and ganciclovir are effective at delaying the onset of VZV after allogeneic BMT, but may not affect the overall incidence of infection. Prolonged prophylaxis may be warranted in patients at high risk of infection, for example those patients with GVHD. PMID- 10734302 TI - Reduced risk of persisting cytomegalovirus pp65 antigenemia and cytomegalovirus interstitial pneumonia following allogeneic PBSCT. AB - In order to evaluate the risk of cytomegalovirus (CMV) associated disease after allogeneic stem cell transplantation (SCT), 158 consecutive patients at risk for infection were analyzed. BMT was performed in 101 patients and peripheral blood stem cell transplantation (PBSCT) in 57 patients. CMV antigenemia was found in 57 cases (56%) after BMT and 27 cases (47%) after PBSCT, respectively. CMV antigenemia resistant to a 14-day course of GCV was found in 26 patients (26%) after BMT but in only four patients (7%) after PBSCT (P < 0.01). Eighteen patients (11%) developed CMV disease, 14 post BMT and four post PBSCT. Lethal CMV related interstitial pneumonia (CMV-IP) occurred in 13 cases of whom 12 patients were bone marrow recipients (P = 0.04). The subgroup of seronegative patients with a CMV seropositive donor had a significantly lower risk of developing CMV antigenemia, GCV-resistant CMV antigenemia (P < 0.01) and CMV-related disease (P = 0.01). In conclusion, the incidence of persistent CMV antigenemia and CMV-IP was significantly reduced when allogeneic transplantation was performed with peripheral blood stem cells instead of bone marrow. These findings suggest that our previous in vitro data on improved immune reconstitution after allogeneic PBSCT as compared to allogeneic BMT have clinical relevance. PMID- 10734303 TI - Acquiescence to adjunctive experimental therapies may relate to psychological distress: pilot data from a bone marrow transplant center. AB - Use of alternative therapy for breast cancer outside of the hospital setting has been identified as a marker of psychological distress. Whether acquiescence to experimental therapies within the medical setting might also be a sign of psychological distress is not well known. We therefore evaluated patients with breast cancer undergoing bone marrow transplantation (BMT), an experimental method for treatment, to determine if acquiescence to further adjunctive experimental therapy related to psychological distress. In order to do this, we studied psychological test results of 42 breast cancer patients undergoing BMT at the University of Florida between January and December 1997. These tests included the Medical Outcomes Short Form Health Survey, the Beck Depression Inventory, the State-Trait Anxiety Inventory and the Medical Coping Modes Questionnaire. Women who accepted adjunctive experimental therapy had significantly higher trait anxiety and poorer role functioning compared to women who did not (both P < 0.001). These findings suggest that psychological distress may be a factor in medical decision-making even within the medical setting and that prospective research in this area is warranted. PMID- 10734304 TI - One antigen mismatched related donor bone marrow transplant in a patient with acute lymphoblastic leukaemia and beta-thalassaemia major: potential cure of both marrow disorders. AB - We report a case of a 34-year-old man with T-ALL and beta-thalassaemia major who underwent a one antigen mismatched related donor bone marrow transplant. Five months post transplant chimeric studies revealed full donor haemopoiesis and the patient remains leukaemia and thalassaemia free at 12 months post transplant. Cumulative risk factors contributing to the increased transplant-related mortality in patients with two different marrow disorders are discussed. PMID- 10734305 TI - Plasma exchange as successful treatment of thrombotic thrombocytopenic purpura post autologous bone marrow transplant in a child. AB - We describe the case of a small child with stage IV neuroblastoma who developed thrombotic thromobocytopenic purpura (TTP) post autologous bone marrow transplant. Pneumococcal sepsis may have been the cause, a previously unreported association in transplant-associated TTP. Despite the child's size (10 kg) and the severity of the disease early intensive treatment with whole blood exchange and subsequently plasma exchange with cryosupernatant proved to be rapidly effective, in contrast to previous reports on its ineffectiveness in this setting. PMID- 10734306 TI - Potential allogeneic graft-versus-tumor effect in a patient with ovarian cancer. AB - A 33-year-old woman developed progressive ovarian cancer resistant to classical chemotherapy agents. We performed a bone marrow allograft after a myeloablative regimen. During hematological recovery, she developed acute graft-versus-host disease (GVHD). From this time her tumor diminished progressively. One year post transplant she has limited chronic liver GVHD and is still free of disease. The complete remission of advanced ovarian cancer was probably related to the GVHD which might therefore provide a new treatment option for this disease. PMID- 10734307 TI - Topical azathioprine in the combined treatment of chronic oral graft-versus-host disease. AB - This paper presents the first report of the use of topical azathioprine in the management of persistent symptomatic chronic oral graft-versus-host disease (GVHD). Topical azathioprine suspension was used as an oral rinse and was swallowed, maintaining the previously prescribed systemic dose of azathioprine, and resulted in improvement in a case of oral GVHD that was resistant to other approaches to management. Topical azathioprine may provide additional therapy in the management of immune-mediated oral mucosal disease. Clinical trials appear warranted based upon the results of topical azathioprine use as presented in this case report. PMID- 10734308 TI - HTLV-1 tax oncoprotein represses the p53-mediated trans-activation function through coactivator CBP sequestration. AB - The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein repressed the transcriptional activity of wild-type p53 through its N-terminal trans-activation domain. Although Tax did not directly bind to p53, this repression required the activation of CREB pathway by Tax. In contrast to a recent report by Pise-Masison et al. (1998a, b) we found that the phosphorylation of p53 on Ser 15 is not a major cause of the Tax-mediated inactivation of p53. However, Tax with a mutation in the coactivator CBP-binding site (K88A), which activates NF-kappaB but not the CREB pathway, could not repress the p53 trans-activation function. Moreover, Tax inhibited p53 binding to CBP in vitro and inhibited synergistic activation of transcription by CBP and p53. Thus, Tax is likely to compete with p53 in binding with CBP, thereby repressing its trans-activation function. PMID- 10734309 TI - Induction of erythroid differentiation by inhibition of Ras/ERK pathway in a friend murine leukemia cell line. AB - The role of Ras and MAP kinases (MAPKs) in the regulation of erythroid differentiation was studied using a cell line (SKT6) derived from Friend virus (Anemic strain)-induced murine erythroleukemia. This cell line undergoes differentiation in vitro in response to erythropoietin (EPO) or other chemical inducers such as dimethylsulfoxide (DMSO). When a constitutively active ras mutant (ras12V) was expressed in SKT6 cells, EPO-induced differentiation was inhibited. Conversely, a dominant negative ras mutant (ras17N) induced differentiation even in the absence of EPO, suggesting that the basal Ras activity is essential for the maintenance of the undifferentiated phenotype and proliferative potential in this cell line. Rapid inactivation of ERK was observed after expression of ras17N. Slow but significant inactivation of ERK was also observed during EPO-induced differentiation. Furthermore, overexpression of a constitutively active mutant of ERK-activating kinase (MAPKK) was found to suppress erythroid differentiation, while pharmacological inhibition of MAPKK induced differentiation. These findings suggest that down-regulation of Ras/ERK signaling pathway may be an essential event in EPO-induced erythroid differentiation in this system. PMID- 10734310 TI - Sustained recruitment of phospholipase C-gamma to Gab1 is required for HGF induced branching tubulogenesis. AB - A distinctive property of Hepatocyte Growth Factor (HGF) is its ability to induce differentiation of tubular structures from epithelial and endothelial cells (branching tubulogenesis). The HGF receptor directly activates PI3 kinase, Ras and STAT signalling pathways and phosphorylates the adaptator GRB2 Associated Binder-1 (Gab1). Gab1 is also phosphorylated in response to Epidermal Growth Factor (EGF) but is unable to induce tubule formation. Comparison of 32P-peptide maps of Gab1 from EGF- versus HGF-treated cells, demonstrates that the same sites are phosphorylated in vivo. However, while both EGF and HGF induce rapid tyrosine phosphorylation of Gab1 with a peak at 15 min, the phosphorylation persists for over 1 h, only in response to HGF. Nine tyrosines are phosphorylated by both receptors. Three of them (Y307, Y373, Y407) bind phospholipase C-gamma (PLC gamma). Interestingly, the overexpression of a Gab1 mutant unable to bind PLC gamma (Gab1 Y307/373/407F) did not alter HGF-stimulated cell scattering, only partially reduced the growth stimulation but completely abolished HGF-mediated tubulogenesis. It is concluded that sustained recruitment of PLCgamma to Gab1 plays an important role in branching tubulogenesis. PMID- 10734311 TI - Identification of genes differentially over-expressed in lung squamous cell carcinoma using combination of cDNA subtraction and microarray analysis. AB - In order to develop effective vaccine products against human cancer, we are interested in identifying genes over-expressed in tumor cells. Through a combination of cDNA library subtraction and microarray technology, we identified seventeen genes preferentially expressed in lung squamous cell carcinoma, including four novel genes. To date, expression profiles of these genes were confirmed by Northern and/or real-time analysis, and several genes were also found to be expressed in head and neck squamous tumors. Thus, these combined methods represent a high throughput approach for identifying tumor specific genes. Furthermore, the report of characterization on these genes will allow them to be exploited for their diagnostic, prognostic, and therapeutic potentials including immunotherapy and antibody based anticancer therapy. PMID- 10734312 TI - Constitutive activation of the Ras/MAP kinase pathway and enhanced TCR signaling by targeting the Shc adaptor to membrane rafts. AB - The Shc adaptor is responsible for coupling receptor tyrosine kinases and tyrosine kinase-associated receptors to the Ras/MAP kinase pathway. Shc is believed to be regulated by a change in subcellular localization from the cytosol to the plasma membrane, where it recruits Grb-2/Sos complexes and hence permits juxtaposition of the guanine nucleotide exchange factor Sos to Ras, resulting in GDP/GTP exchange and Ras activation. Shc has been recently shown to inducibly colocalize in detergent-resistant membrane rafts together with the activated TCR and associated signaling molecules. To understand whether Shc localization in membrane rafts is sufficient to regulate Shc function, we constructed a Shc chimera containing the Ras membrane localization motif at the C-terminus. We show that membrane targeted Shc was constitutively localized in the plasma membrane of T-cells, and was mostly compartmentalized in lipid rafts. Membrane targeted Shc was phosphorylated on tyrosine residues and bound Grb-2/Sos in the absence of TCR engagement. Furthermore, expression of membrane targeted Shc resulted in constitutive downstream signaling, including Erk2 activation and enhancement of TCR dependent activation of the TCR responsive transcription factor NF-AT. Hence localization of Shc in membrane rafts is sufficient for Shc to acquire a signaling competent state. Interestingly, a membrane targeted Shc mutant lacking both Grb-2 binding sites was not only incapable of signaling in the absence of TCR triggering, but transdominantly inhibited endogenous Shc, supporting a non redundant role for Shc in the activation of the Ras/MAP kinase pathway in T cells. PMID- 10734313 TI - The adenovirus E1A binding protein BS69 is a corepressor of transcription through recruitment of N-CoR. AB - BS69 was first identified as a protein that interacts directly with the transactivation domain (conserved region 3) of the 289R adenovirus type 5 E1A protein. We show here that BS69 is a potent repressor of transcription. BS69 mediates repression, at least in part, through interaction with the co-repressor N-CoR. BS69 interacts with N-CoR through a MYND domain in its carboxyl terminus. A recently cloned splice variant of BS69, designated BRAM1, is also capable of interacting with N-CoR and E1A, but unlike BS69, is not able to repress transcription, indicating that N-CoR interaction is necessary but not sufficient for BS69 repression. Expression of E1A inhibits repression mediated by BS69. Our data suggest that BS69 participates in transcriptional repressor complexes and that E1A can modulate these complexes through interaction with BS69. PMID- 10734314 TI - Somatic mutations of the MET oncogene are selected during metastatic spread of human HNSC carcinomas. AB - A metastatic cancer develops by accumulation of mutations in genes that control growth, survival and spreading. The latter genes have not yet been identified. In lymph node metastases of head and neck squamous cell carcinomas (HNSCC), we found mutations in the MET oncogene, which encodes the tyrosine kinase receptor for Scatter Factor, a cytokine that stimulates epithelial cell motility and invasiveness during embryogenesis and tissue remodeling. We identified two somatic mutations: the Y1230C, known as a MET germline mutation which predisposes to hereditary renal cell carcinoma, and the Y1235D that is novel and changes a critical tyrosine, known to regulate MET kinase activity. The mutated MET receptors are constitutively active and confer an invasive phenotype to transfected cells. Interestingly, cells carrying the MET mutations are selected during metastatic spread: transcripts of the mutant alleles are highly represented in metastases, but barely detectable in primary tumors. These data indicate that cells expressing mutant MET undergo clonal expansion during HNSCC progression and suggest that MET might be one of the long sought oncogenes controlling progression of primary cancers to metastasis. PMID- 10734315 TI - Evidence of epigenetic changes affecting the chromatin state of the retinoic acid receptor beta2 promoter in breast cancer cells. AB - Retinoic acid (RA)-resistance in breast cancer cells has been associated with irreversible loss of retinoic acid receptor beta, RARbeta, gene expression. Search of the causes affecting RARbeta gene activity has been oriented at identifying possible differences either at the level of one of the RARbeta promoters, RARbeta2, or at regulatory factors. We hypothesized that loss of RARbeta2 activity occurs as a result of multiple factors, including epigenetic modifications, which can pattern RARbeta2 chromatin state. Using methylation specific PCR, we found hypermethylation at RARbeta2 in a significant proportion of both breast cancer cell lines and primary breast tumors. Treatment of cells with a methylated RARbeta2 promoter, by means of the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR), led to demethylation within RARbeta2 and expression of RARbeta indicating that DNA methylation is at least one factor, contributing to RARbeta inactivity. However, identically methylated promoters can differentially respond to RA, suggesting that RARbeta2 activity may be associated to different repressive chromatin states. This supposition is supported by the finding that the more stable repressive RARbeta2 state in the RA resistant MDA-MB-231 cell line can be alleviated by the HDAC inhibitor, trichostatin A (TSA), with restoration of RA-induced RARbeta transcription. Thus, chromatin-remodeling drugs might provide a strategy to restore RARbeta activity, and help to overcome the hurdle of RA-resistance in breast cancer. PMID- 10734316 TI - Microsatellite instability and the PTEN1 gene mutation in a subset of early onset gliomas carrying germline mutation or promoter methylation of the hMLH1 gene. AB - High-frequent microsatellite instability (MSI-H) was detected in two of the 80 gliomas examined, whlie the other 78 gliomas showed microsatellite stable (MSS) phenotype. Both of the two MSI-H tumors were glioblastomas which developed in teenage patients. One of the patient was diagnosed as having Turcot's syndrome and had a germline mutation in the hMLH1 gene. Loss of expression due to promoter methylation was selectively observed in the wild type allele of the hMLH1 gene in the tumor of this patient. The other patient had neither a family history nor a past personal history of malignancy. Although no mutation in the mismatch repair genes was detected in the tumor of this patient, the level of expression of the hMLH1 gene was markedly decreased and the promoter sequence of the gene was highly methylated. In the tumor of this patient, the PTEN1 gene, one of the genes carrying microsatellite sequences in their coding regions, was altered by a slippage mutation within five adenine repeat sequences. These findings indicate that the genetic or epigenentic inactivation of the hMLH1 gene is involved in a subset of early-onset gliomas and the PTEN1 gene could be a downstream target for mutation as observed in glioblastoma without MSI. PMID- 10734317 TI - M6P/IGF2R is mutated in squamous cell carcinoma of the lung. AB - In addition to the intracellular sorting of lysosomal enzymes, the mannose 6 phosphate/insulin-like growth factor II receptor (M6P/IGF2R) plays a critical role in regulating the bioavailability of extracellular proteolytic enzymes and growth factors. It has also been shown to be mutated in a number of human cancers, and to suppress cancer cell growth. The purpose of this study was to determine if the M6P/IGF2R is mutated in lung cancer, a leading cause of cancer death worldwide. Archival pathology specimens were obtained on 22 patients with newly diagnosed, untreated squamous cell carcinoma of the lung. Two polymorphisms in the 3'-untranslated region of the M6P/IGF2R were used to screen lung tumors for loss of heterozygosity (LOH) by PCR amplification of DNA. Nineteen of 22 (86%) patients were informative (heterozygous), and 11/19 (58%) squamous cell carcinomas of the lung had LOH at the M6P/IGF2R locus. The remaining allele in 6/11 (55%) LOH patients contained mutations in either the mannose 6-phosphate or the IGF2 binding domain of the M6P/IGF2R. Thus, the M6P/IGF2R is mutated frequently in squamous cell carcinoma of the lung, providing further support for its function as a tumor suppressor. PMID- 10734318 TI - SPAF, a new AAA-protein specific to early spermatogenesis and malignant conversion. AB - A novel spermatogenesis associated factor (SPAF) was found to be aberrantly expressed at the malignant conversion stage in a clonal epidermal model of chemical carcinogenesis. Sequence analysis revealed two ATPase modules, classifying this gene as a new member of the AAA-protein family (ATPase associated with diverse activities). Immunohistochemical staining of mouse testis sections with SPAF antibody localized expression to spermatogonia and early spermatocytes in the basal compartment of the seminiferous tubules. Northern and Western analysis of SPAF expression in testes of mice at different developmental stages confirmed its expression at early stages of spermatogenesis. In view of a mitochondrial-localization-like signal, sequence similarities to membrane associated proteins, ATP binding properties, and intracellular expression patterns in testis, we speculate that SPAF protein may be involved in morphological and functional mitochondrial transformations during spermatogenesis. Ectopic expression of the SPAF gene in malignant epidermal cells may signify adoption of an early germ cell-like phenotype advantageous in malignant conversion. PMID- 10734319 TI - Down-regulation of bcl-2 is associated with p16INK4-mediated apoptosis in non small cell lung cancer cells. AB - We introduced a functional p16 cDNA into non-small cell lung cancer (NSCLC) cell lines expressing different combinations of normal and mutated p16, p53, and Rb genes via a recombinant adenovirus to determine the effect of exogenous p16 expression on cell growth. Analysis of p16-deficient cells infected with Adv/p16 identified growth arrest of the cells in the G0 - G1 phase early on. Apoptosis was identified to occur by the 5th day after infection which corresponded with increased p16 expression, reduced Rb expression, and increased Rb hypophosphorylation, but only occurred in cells expressing functional p53. Further analysis indicated that the expression of the anti-apoptotic protein bcl 2 was greatly reduced in the NSCLC cell lines H460 and A549 (both -p16, +p53, +Rb), again only by the 5th day after Adv/p16 infection, but no affect on Bax expression was observed. H1299 cells (-p16, -p53, +Rb) infected with Adv/p16 only exhibited apoptosis by an additional infection with Adv/p53 which also corresponded with a down-regulation of bcl-2. In addition, the infection of A549 cells with Adv/p16 followed by a subsequent infection with Adv/Rb lead to a significant decrease in apoptosis which correlated with an increase in bcl-2 expression. These studies suggest that p16 is capable of mediating apoptosis in NSCLC cell lines expressing wild-type p53, through a direct down-regulation of Rb and an indirect down-regulation of the anti-apoptotic protein bcl-2. PMID- 10734320 TI - IL-4 regulation of IL-6 production involves Rac/Cdc42- and p38 MAPK-dependent pathways in keratinocytes. AB - The stress-activated pathways leading to activation of p38 MAP kinase (p38 MAPK) and c-jun N-terminal kinases (JNK) have been shown to be activated by pro inflammatory cytokines, physical and chemical stresses as well as a variety of hematopoietic growth factors. One exception is interleukin (IL)-4, which does not activate this pathway in hematopoietic cell. We report here that in A431, a keratinocytic cell line, IL-4 activates Rac and Cdc42 and their downstream effector p21-activated kinase (PAK). Rac and Cdc42 appear to regulate a protein kinase cascade initiated at the level of PAK and leading to activation of p38 MAPK, since IL-4 stimulates tyrosine phosphorylation of p38 MAPK and increases its catalytic activity. As A431 cells are able to produce IL-6 in response to IL 4 stimulation, we assessed the involvement of p38 MAPK in IL-6 gene expression. A pyrimidazole compound, SB203580, a specific inhibitor of p38 MAPK, inhibits production and gene expression of IL-6. SB203580 reduced significantly the stability of IL-6 mRNA. Here we provide evidence that p38 MAPK is activated in response to IL-4 and is involved in IL-6 synthesis by stabilizing IL-6 mRNA. PMID- 10734321 TI - Demonstration in vivo that stromelysin-3 functions through its proteolytic activity. AB - Stromelysin-3 (ST3), a matrix metalloproteinase (MMP) expressed in aggressive carcinomas, has been shown to promote tumor development in different in vivo experimental models. However, the inability of its mature form to degrade extracellular matrix components casts doubt on whether ST3 functions in vivo as a protease. In this study, we evaluated whether the ST3 tumor-promoting effect could be ascribed to its proteolytic activity and whether this putative protease could be targeted with MMP inhibitors. Catalytically inactive mutant cDNA of human (h) ST3 or mouse (m) ST3 were generated and transfected into MCF7 cells. When injected into nude mice in the presence of matrigel, the mutant-bearing cells did not exhibit the enhanced tumorigenicity elicited by MCF7 cells transfected with wild-type ST3 cDNA. In a second approach, TIMP2 overproduction in MCF7 cells expressing hST3 was induced by retroviral infection. The co expression of ST3 and TIMP2 failed to enhance the tumorigenicity of MCF7 cells. Notably, matrigel depleted of low-molecular-weight proteins and growth factors failed to promote the tumorigenicity of ST3-expressing MCF7 cells. These findings provide the first in vivo evidence that ST3 is indeed a protease that can modulate cancer progression by remodeling extracellular matrix and probably by inducing it to release the necessary microenvironmental factors. Thus, ST3 represents an interesting target for specific MMP inhibition. PMID- 10734322 TI - Should a preterm breech go for vaginal delivery or caesarean section. AB - This study correlates the mode of breech delivery to the immediate neonatal outcome in preterm breeches. We had 9816 deliveries in the period between 1st January 1994 to 31st August 1996. The incidence of breech deliveries was 3.95% and the incidence of preterm breech deliveries was 1.9%. Totally 112 (69%) patients delivered vaginally and 50 (31%) underwent caesarean section. Between 30 36.6 weeks gestation the incidence of birth asphyxia was higher in the vaginal group. In this group the take home baby rate after vaginal delivery was 81% as compared to 86% in caesarean group. Head entrapment, cord prolapse, respiratory distress syndrome and intraventricular haemorrhage were the various complications seen with vaginal breech delivery. PMID- 10734323 TI - A comparison of continuous infusion of vecuronium and atracurium in midline and paramedian laparotomies. AB - This was a study to compare continuous intravenous infusion of atracurium with continuous intravenous infusion of vecuronium for intraoperative muscle relaxation in 62 ASA I / II patients. Scheduled for laparotomies and pelvic surgeries under general anaesthesia. They were randomly allocated in two groups to receive either vecuronium infusion of 50 microg/kg/hour following a bolus dose of 0.1 microg/kg, or atracurium infusion of 400 microg/kg/hour following a bolus dose of 0.5 microg/kg. The mean infusion dose of atracurium was 478 +/- 44.11 microg/kg/hour and that of vecuronium was 63.2 +/- 74 microg/kg/hour for adequate muscle relaxation. The depth of neuromuscular blockade was monitored by using peripheral nerve stimulator so that only one twitch of train of four was present, resistance to ventilation, surgical relaxation and haemodynamic changes. Vecuronium infusions produced more haemodynamic stability than atracurium infusions. Vecuronium produced lesser change in systolic blood pressure (mean change of 3. 46 +/- 3.33%) from baseline values as compared to atracurium (mean change of 5.81 +/- 3.73%) from baseline values ( p < 0.01) which was statistically significant. The difference in mean pulse rate change from baseline value in the atracurium group (4.78 +/- 2.745%) was less than that in the vecuronium group (5.99 +/- 2.67%), which was not statistically significant. Spontaneous recovery was faster with vecuronium (540.94 +/- 76.46 seconds) as compared to atracurium (596. 33 +/- 72.48 seconds). 84.4% of patients who received vecuronium fell within good to very good category of muscle relaxation as compared to 63.3% in atracurium group. There were no cost benefits when either agents were used in infusion form. PMID- 10734324 TI - Primary hypertrophic tuberculosis of the pyloroduodenal area: report of 2 cases. AB - Tuberculosis of the stomach and duodenum is rare in patients with pulmonary tuberculosis. Primary involvement is even rarer. Two cases of primary tuberculosis of the localised to the pyloro-duodenal area are presented. The most common symptoms are non-specific leading to a difficulty in establishing a pre operative diagnosis. A high degree of suspicion is therefore required for its diagnosis and to differentiate it from more frequent causes of gastric outlet obstruction such as chronic peptic ulcer disease and gastric carcinoma. The treatment of gastric tuberculosis is primarily medical with anti-tuberculous drug therapy. The role of surgery lies in the cases with obstruction following hypertrophic tuberculosis. The surgery done is usually a gastroenterostomy. With the relative rate of extra-pulmonary tuberculosis increasing, tuberculosis of the pyloro-duodenal area should be considered in the differential diagnosis of gastric outlet obstruction. PMID- 10734325 TI - Spontaneous oesophageal perforation due to mediastinal tuberculous lymphadenitis atypical presentation of tuberculosis. AB - Spontaneous non-traumatic oesophageal perforation secondary to bursting of a mediastinal tuberculous abscess into the oesophagus is rare. The diagnosis is delayed, as perforation remains localised due to mediastinal lymph nodes. Patient can be effectively managed by paraoesophageal drainage of the mediastinal abscess and oesophageal diversion. PMID- 10734326 TI - Pedunculated nasal glioma: MRI features and review of the literature. AB - Nasal gliomas are uncommon lesions, with approximately 100 cases reported in the literature. We present a case of intranasal glioma with a pedicle of glial tissue and defect in the cribriform plate of the ethmoid bone. Presence of such a pedicle is almost always present in the encephaloceles and is known to be distinctively uncommon in nasal gliomas. PMID- 10734327 TI - Imaging features of gossypiboma: report of two cases. AB - Recognition of postoperatively retained foreign body referred euphemistically as gossypiboma is essential but is very often considerably delayed. Legal implications as well as confusing configuration patterns cause considerable dilemma in the accurate diagnosis. We present computed tomographic features of gossypiboma in two patients who presented with symptoms of fever and pain in the immediate post-operative period. A prospective radiological diagnosis is essential for further management in these patients. PMID- 10734328 TI - Agenesis of corpus callosum - a rare case. AB - A case of corpus callosum agenesis associated with a chromosomal structural defect is described. PMID- 10734329 TI - Common structural elements in 'scorpion-toxin' type proteins. PMID- 10734330 TI - Perinatal outcome of twins in relation to chorionicity. AB - Twin gestation, a high-risk pregnancy is responsible for 10% of all perinatal mortalities. The high perinatal mortality of twins has been repeatedly stressed over the years. Studies have also revealed difference in the perinatal mortality rates in relation to chorionicity. Thus, a prospective study of 100 twin pregnancies was carried out at our institute to assess the morbidity and mortality of twins in relation to chorionicity and to analyse the factors responsible for the greater loss. The perinatal mortality rate of monochorionic twins was 17.64% and that of dichorionic twins was 8. 88%, which is statistically significant. Birth weight was found to be the most important factor correlating with mortality rates. The higher perinatal mortality of monochorionic twins was largely due to low birth weights (29.3% of monochorionic twins weighed less than 1500 gm as compared to 12.6% of dichorionic twins). Avoidable deaths were comparable in the two twin groups but dichorionic twins showed greater percentage of fresh stillbirths (40%) whereas in monochorionic twins, 50% were macerated stillbirths. Prematurity was another common factor responsible for greater perinatal mortality in monochorionic twins. Monochorionic twins showed increased incidence of discordant growth (34.8%) as compared to that of dichorionic twins (14.08%). The type of placentation did influence the perinatal outcome of twins making its antenatal diagnosis important. PMID- 10734331 TI - Does left ventricular function improve with L-carnitine after acute myocardial infarction? AB - A double blind randomized placebo controlled clinical trial was carried out to assess the efficacy and safety of L-carnitine in patients suffering from acute anterior wall myocardial infarction with respect to left ventricular function. Sixty patients (34 men, 26 women, mean age 56+11 yr.) with acute anterior wall myocardial infarction were randomized to placebo and L-carnitine. All the patients were given intravenous L-carnitine / placebo in the dose of 6gm/day for the first seven days followed by oral L-carnitine / placebo 3 gm/day in three divided doses for a period of three months. Echocardiography was performed for regional wall motion abnormality, left ventricular end systolic volume (ESV), end diastolic volume (EDV) and ejection fraction (EF) on admission, after seven days and after three months of the infarction. Forty-four patients completed the study. There were three deaths, two in the placebo and one in the L-carnitine group (p>0.05). Thirteen patients were lost to follow up. Echo parameters in both groups were comparable (p>0.05). The duration of chest pain prior to initiation of the I.V. L-carnitine was 7.5 + 5.2 hrs in the L-carnitine group and 7 + 4 hrs in the placebo group (p>0.05). There was no statistical difference in the EF, ESV and EDV on admission, at discharge and after three months in the L-carnitine and the placebo groups (p>0.05). No significant adverse effects were noted. L carnitine, though a safe drug, does not affect the left ventricular function in patients with myocardial infarction. PMID- 10734332 TI - Correlation between creatine kinase activity, lipid-peroxidation and water test in male infertility. AB - New approaches need to be pursued towards the assessment of sperm quality using biochemical markers. In order to help develop a good biochemical marker to assess sperm-membrane integrity, the enzyme creatine kinase (CK) was studied in semen of normal, oligospermic and azoospermic samples and correlated with sperm concentration, lipid-peroxidation (LP) and water test. Presence of isoforms of creatine kinase (CK-MB) was also seen. An inverse correlation was observed between CK activity and sperm concentration (p<0.001). Water test was seen to be inversely correlated with CK activity (p<0. 001). Lipid peroxidation showed positive correlation with CK activity (p<0.001). A significant correlation between loss of sperm function meditated by induction of peroxidative damage to sperm plasma membrane is indicated. Enzymes like CK can serve as good biochemical marker along with lipid peroxidation to confirm loss of sperm membrane integrity. The water test can be used as a preliminary screening test for sperm membrane integrity. PMID- 10734333 TI - Ruptured intracranial dermoids: magnetic resonance imaging. AB - Rupture of intracranial dermoids tumour is rare and carries with it the risk of significant morbidity as well as fatality. Three cases that presented with varying symptoms ranging from headache to chiasmatic compression and suspected to have rupture of dermoid tumour are described. The importance of MR imaging in their diagnosis is discussed. PMID- 10734334 TI - Multiple intracranial lipomas, hypogenetic corpus callosum and vestibular schwannoma: an unusual spectrum of MR findings in a patient. AB - We describe imaging findings of a patient with multiple intracranial lipomas, hypogenetic corpus callosum and a vestibular schwannoma. We did not find association of intracranial lipomas and vestibular schwannoma in English literature. PMID- 10734335 TI - Malignant hidradenoma: a rare sweat gland tumour. AB - Malignant hidradenoma is a rare sweat gland carcinoma, which can have an aggressive course with recurrence and/or metastasis. A case is reported, in an elderly male. The tumour had a histologic similarity to its benign counterpart, but exhibited additional features of infiltrative growth pattern and invasion of adjacent tissue. PMID- 10734337 TI - Diarrhoea and aging. PMID- 10734336 TI - Benzathine penicillin induced immune haemolytic anaemia. AB - Penicillin-induced immune haemolytic anaemia is very rare. A ten year-old-female with rheumatic mitral stenosis on benzathine penicillin prophylaxis presented with features of haemolytic anaemia and investigations supported the diagnosis of immune haemolytic anaemia. Patient responded to discontinuation of the drug and therapy with oral prednisolone. This is first such case reported from India. PMID- 10734338 TI - Martius' labial fat pad interposition and its modification in complex lower urinary fistulae. AB - OBJECTIVE: To assess the results of Martius' labial fat pad interposition and its modification using skin island in the repair of giant and recurrent vesicovaginal and urethrovaginal fistulae. PATIENTS AND METHODS: Fifteen patients of urethrovaginal and vesicovaginal fistulae underwent Martius' labial fat pad interposition and its skin island modification during 1996 to 1999. Ten of these were recurrent (66%) and five were giant fistulae (34%) i.e. more than five cms. RESULTS: Results were very gratifying with a successful repair in 14 patients (93%). Two patients had transient, low-grade stress incontinence, which did not need any additional procedure. In one patient, there was failure of repair, which was later successfully repaired using fat pad from opposite labia. CONCLUSION: Martius' fat pad interposition provides vascularity and surface for epithelialisation and also prevents overlapping of vesical/urethral and vaginal suture lines. Martius' repair has good results with low morbidity in the treatment of giant and recurrent urethrovaginal and vesicovaginal fistulae. PMID- 10734339 TI - A comparative study of clonidine versus a combination of diazepam and atropine for premedication in orthopaedic patients. AB - Sixty patients in the age group of 18-60 years of A.S.A. Grade I/II risk, scheduled for elective orthopaedic surgeries under general anaesthesia were studied for pre-medication with either oral clonidine or with combination of effects of diazepam & atropine. Patients in Group A (clonidine group) received tablet clonidine 100 mcg (1 tablet) if less than 50 kg in weight and 200 mcg if weighing more than 50 kg two hours before surgery. Patients in Group B (Diazepam atropine group) received one tablet of Diazepam (10 mg) orally two hours before surgery and injection atropine-sulphate 0.01 mg/kg half an hour preoperatively by intramuscular route. In our study, the sedative and anti-sialogogue effects of clonidine were comparable to those of diazepam-atropine combination, which are commonly used premedicants. The anti-anxiety effect of clonidine was found to be better than that of diazepam-atropine combination. Clonidine also proved to be a better agent for the attenuation of pressor response to laryngoscopy and intubation. Thus, oral clonidine is a better premedicant compared to atropine diazepam combination. Also, it is a more acceptable agent because of its oral route of administration. PMID- 10734340 TI - Induction Of ovulation. AB - Sixty-one patients with anovulation as a cause of infertility were selected for our study. Various ovulation-inducing drugs were used like clomiphene citrate, human menopausal gonadotropin (hMG), human chorionic gonadotropin (hCG), bromergocryptine and leptadene. The response of the different drugs was observed by serial sonography for ovulation. Indeed there was a good response to clomiphene citrate, but those patients who failed to respond to clomiphene citrate and were frustrated with the use of hMG and hCG due to the cost and the complications of the therapy were put on Aloe compound and leptadene - an ayurvedic drug which enhances fertility in different ways. PMID- 10734341 TI - MR imaging in children with ectopic pituitary gland and anterior hypopituitarism. AB - Posterior pituitary ectopia refers to an absent normal posterior pituitary bright spot within the sella with ectopic bright signal at another site (such as the median eminence) on a weighted magnetic resonance. We describe two children with idiopathic anterior hypopituitarism who showed an ectopic posterior pituitary and absent pituitary stalk on imaging. We emphasize the association of the absent pituitary stalk in ectopic pituitary gland and low growth hormone levels. PMID- 10734342 TI - MR imaging features in hypothalamic hamartoma: a report of three cases and review of literature. AB - Hypothalamic hamartomas are rare tumours of particular interest because of their unusual symptoms. Three cases of hypothalamic hamartomas are reported in children, who presented with precocious puberty and gelastic seizures. PMID- 10734343 TI - Duodenocolic fistula: case report and review of the literature. AB - Duodenocolic fistula is a rare complication of malignant and inflammatory bowel disease. It presents as diarrhoea and faeculent vomiting. The diagnosis is established with upper and lower gastrointestinal tract contrast studies. A case is reported and the optimal operative procedure is discussed. PMID- 10734344 TI - Crossed polydactyly. AB - A common and conspicuous congenital hand anomaly, polydactyly commonly involves only the hand or the foot. Polydactyly involving both hands and feet is rare. We herewith report two cases of Crossed Polydactyly (Type I) and review the literature. PMID- 10734345 TI - Leptospirosis - an under-diagnosed clinical condition. AB - Leptospirosis is a zoonotic disease caused by leptospiral spirochaete. Two cases in children are described presenting with hepatorenal dysfunction. PMID- 10734346 TI - Analgesia and sedation in paediatric intensive care unit. PMID- 10734347 TI - Prostaglandin E2 gel In ripening of cervix in induction of labour. AB - A study was done in 75 patients who underwent induction of labour with Prostaglandin E2 gel. All these patients had an unripe cervix. The commonest indications were post-datism, intrauterine growth retardation and pregnancy induced hypertension. All patients were primigravidas with singleton pregnancy and beyond 35 weeks of pregnancy. The mean Bishop score at the time of instillation was less than three. The improvement of another 2-3 points within six hours and by 7-8 points within 12 hours was found after instillation of the gel. 92% of the patients went into spontaneous labour and 8% required reinstillation. The incidence of failed induction was 1.33%. The mean duration of latent phase was 10.34 hours. Induction delivery time was 16.43 hours. 68.1% patients required augmentation of labour and 31.9% did not require augmentation of labour with oxytocin drip. The incidence of vaginal delivery was 81.33% and that of caesarean section was 17.33%. The commonest indication of caesarean section was foetal distress. PMID- 10734348 TI - Anthropometry, lipid profile and dietary pattern of patients with chronic ischaemic heart disease. AB - The anthropometry, lipid profile and dietary characteristics of 114 patients with chronic ischaemic heart disease (IHD) were evaluated. There were 91 (80%) men and the mean age was 56 +/- 9 years. The body mass index was near normal (24.4 +/- 3.4), but the waist: hip ratio was high (0.94 +/- 0.06) suggesting central obesity. This was well in accordance of the step II recommendations of the NCEP guidelines as regards their caloric intake and its break-up in terms of carbohydrate, protein and fat (including saturated, mono-unsaturated and poly unsaturated fatty acids) content. Their daily cholesterol intake (31 +/- 32 mg/day, range 4-180) was very low. The total cholesterol (212 +/- 37 mg%) was marginally elevated, HDL cholesterol (33 +/- 7.5 mg%) was low, LDL cholesterol (148 +/- 39 mg%) was high and the total: HDL ratio (6.8 +/- 2.0) was significantly abnormal. The serum triglyceride level (154 +/- 68 mg%) was on the higher side of normal. These observations give further credence to the recently evolving view that there are different and hitherto unrecognised risk factors of IHD in Indians, who seem to have the highest incidence of IHD amongst all ethnic groups of the world despite consuming a diet low in fat and cholesterol content. PMID- 10734349 TI - Hallervorden Spatz disease: MR imaging. AB - Three patients were diagnosed as suffering from Hallervorden Spatz disease, a rare disorder, on the basis of their clinical and MRI findings. PMID- 10734350 TI - Giant rheumatoid synovial cyst of the hip joint: diagnosed by MRI. AB - Synovial cysts are commonly found in the knee joint. Hip Joint is an infrequent site for formation of synovial cysts. The features of a large, synovial cyst on magnetic resonance imaging, occurring in the hip joint, are described. PMID- 10734351 TI - Superior sagittal sinus thrombosis: a rare complication of nephrotic syndrome. AB - A two and half year-old-male child, known case of steroid responsive nephrotic syndrome presented with fever and vomiting of acute onset. He was diagnosed to have superior sagittal sinus thrombosis on a contrast computerised tomographic scan of brain. Recovery was complete without anticoagulant therapy. Superior sagittal sinus thrombosis is an extremely rare complication of nephrotic syndrome. PMID- 10734352 TI - Mohr syndrome: a rare case and distinction from orofacial digital syndrome 1. AB - In view of the different modes of inheritance and the different prognoses of the two oro-facio-digital syndromes, type 1 and type 2, it is important to establish a correct diagnosis in these patients. A case of type II oro-facio-digital syndrome is being reported and the distinguishing clinicoradiological features with type I are compared. PMID- 10734353 TI - Xanthogranulomatous pyelonephritis presenting as emphysematous pyelonephritis: a rare association. AB - Xanthogranulomatous and emphysematous pyelonephritis are two rare variants of pyelonephritis. Their combined occurrence is a very rare condition, which has been documented in our case. PMID- 10734354 TI - Haemophilia: scope for rehabilitation in India. PMID- 10734355 TI - Changes in the cumulus-oocyte complex of subordinate follicles relative to follicular wave status in cattle. AB - We investigated factors that affect cumulus-oocyte complex (COC) morphology and oocyte developmental competence in subordinate follicles on different days after follicular wave emergence in beef heifers. In Experiment 1, heifers (n = 13) were assigned at random to COC aspiration during the growing/static (Days 1 to 3) or regressing (Day 5) phase of subordinate follicle development (follicular wave emergence = Day 0). Follicular wave emergence was induced by transvaginal ultrasound-guided follicular ablation, ovaries were collected at slaughter, all follicles > or = 2 mm except the dominant follicle were aspirated, and COC were microscopically evaluated for morphology. There was a greater percentage of COC with expanded cumulus layers on Day 5 (42.4%) than on Days 1 to 3 (2.2%). In Experiment 2, heifers (n = 64) at random stages of the estrous cycle had all follicles > or = 5 mm ablated and 4 d later, 2 doses of PGF were injected 12 h apart; heifers were monitored daily by ultrasonography for ovulation (Day 0 = follicular wave emergence). Heifers were assigned to the following time periods for oocyte collection from subordinate follicles: Days 0 and 1 (growing phase), Days 2, 3 and 4 (static phase), and Days 5 and 6 (regressing phase). Ovaries were individually collected at slaughter, and all follicles > or 2 mm except for the dominant follicle were aspirated. The COC were morphologically evaluated and then matured, fertilized and cultured in vitro. Expanded COC were more frequent during the regressing phase (53.4%) than the growing or static phase (14.4 and 17.8%, respectively; P < 0.05). While the proportions of COC with > or = 4 layers of cumulus cells and denuded oocytes were higher (P < 0.05) in the growing and static phases, the production of morulae was highest (P < 0.05) with COC collected from subordinate follicles during the regressing phase. In Experiment 3, heifers (n = 18) were assigned at random to oocyte collection from subordinate follicles 3 and 4 d (static phase) or 5 and 6 d (regressing phase) after follicular wave emergence. The heifers were monitored ultrasonically for ovulation (Day 0 = follicular wave emergence); COC were collected from all follicles (> or = 5 mm) except for the dominant follicle by transvaginal ultrasound-guided follicle aspiration 3 to 6 d later. Recovered oocytes were stained and examined microscopically to evaluate nuclear maturation. A higher proportion of oocytes collected on Days 5 and 6 showed evidence of nuclear maturation (50%) than on Days 3 and 4 (8.3%; P < 0.05). Results support the hypothesis that COC morphology and oocyte developmental competence change during the growing, static and regressing phases of subordinate follicle development. PMID- 10734356 TI - Effect of fetal age and method of recovery on isolation of preantral follicles from bovine ovaries. AB - The objective of this study was to compare enzymatic and mechanical methods, at distinct fetal ages, on isolation of different developmental stages of preantral follicles from bovine ovaries. Fetal ovaries were obtained from pregnant cattle at 150 to 270 d of gestation, and 135,521 preantral follicles at different stages of development were studied. The dissociation of ovaries with a mechanical procedure resulted in an average of 938.16 prenatral follicles. In contrast, 3,715.56 follicles were obtained when enzymatic digestion was used (P = 0.0001). Histological evaluation confirmed follicular stages and demonstrated that both mechanical and mechanical-enzymatic procedure did not affect the cellular integrity of the follicles. Granulosa cell-oocyte complexes surrounded by a basal membrane, were considered preantral follicles in this study. The ratio of different stages of isolated preantral follicles was significantly (P = 0.0001) correlated to fetal age. The earliest fetal age at which tertiary follicles were identified was at 210 d of gestation. The results confirm previous observation that follicular development and atresia are initiated during fetal development. These data provide information on methodologies to isolate intact bovine preantral follicles for investigating the control and regulation of follicular development and the growth of preantral follicles in vitro. PMID- 10734357 TI - Follicular growth, estrus and pregnancy after fixed-time insemination in beef cows treated with intravaginal progesterone inserts and estradiol benzoate. AB - An experiment was performed to compare the effects of 3 short-term treatments with progesterone and estradiol benzoate (EB) on follicular growth, synchrony of estrus and pregnancy rate after fixed-time insemination in lactating postpartum beef cows. In Treatment 1 (n = 46), each cow received a progesterone-containing intravaginal insert for 7 d with injection of EB (2 mg, i.m.) at the time of device insertion. In Treatment 2 (n = 46), the insert was used for only 5 d with injection of EB (2 mg, i.m.) at the time of insertion. Cows in Treatment 3 (n = 47) received an insert for 5 d with no EB at the time of insertion. Each cow in the 3 groups received PGF2 alpha (25 mg, i.m.) at the time of insert removal, followed by EB (1 mg, i.m.) 30 h later. The cows were then inseminated 28 to 30 h after treatment with EB (58 to 60 h after insert removal). Treatment with 2 mg EB terminated the growth of the largest ovarian follicle (> 5 mm in diameter) at device insertion in 16/16 and 14/15 cows in Treatments 1 and 2, respectively. Estrus was detected within an 8-h target period (48 to 56 h after insert removal) in 93, 87 and 81% of cows in Treatments 1, 2 and 3, respectively (P > 0.05). Pregnancy rates at 39 d post insemination were 60, 50 and 51% for Treatments 1, 2 and 3, respectively (P > 0.05). The pregnancy rates did not differ between cows that were anovulatory or those that had ovulated before the initiation of treatments (54%), or among cows that were 28 to 40, 41 to 60 or > 60 days post partum at insemination (43, 59 and 54%, respectively). Treatment with progesterone inserts for 5 or 7 d, PGF2 alpha at the time of insert removal and 1 mg EB 30 h later induced the high degree of synchrony of estrus and ovulation necessary for fixed-time insemination. PMID- 10734358 TI - Relationship between ultrasonic characteristics of the corpus luteum, plasma progesterone concentration and early pregnancy diagnosis in Friesian mares. AB - The purpose of the present study was to evaluate the change in cross-sectional area of the early corpus luteum (CL) and progesterone production in relation to subsequent pregnancy diagnosis. The cross-sectional area of the CL of 75 Friesian brood mares was measured by ultrasonography on Day 1 or 2 and Day 8 or 9 after ovulation. The change in cross-sectional area was expressed in a volume ratio. Plasma progesterone concentrations were measured on Days 8 to 9, and ultrasonography to determine pregnancy status was carried out on Day 17. The data obtained were analyzed by using a multiple logistic regression model. There were significant differences in the age, volume ratio and progesterone concentration between pregnant and nonpregnant mares. Pregnancy on Day 17 was related to the change in size of the CL up to Days 8 to 9 and progesterone concentration on Days 8 to 9. These differences between pregnant and nonpregnant mares might reflect the first luteal response to pregnancy. PMID- 10734359 TI - Increased LH pulse frequency and estrogen secretion associated with termination of anestrus followed by enhancement of uterine estrogen receptor gene expression in the beagle bitch. AB - The relationships among pulsatile LH secretion pattern, estrogen secretion, and expression of the uterine estrogen receptor gene were examined throughout the estrous cycle in beagle bitches. In Experiment 1, blood samples were collected from 30 bitches every 10 min for 8 h from a cephalic vein during different phases of the estrous cycle. An increase in the mean plasma levels of LH occurred from mid to late anestrus (P < 0.01). The LH pulse frequency increased (P < 0.01) from late anestrus to proestrus, and was strongly correlated (r = 0.96, P < 0.001) with the mean plasma level of estradiol-17 beta (E2). In Experiment 2, middle uterine samples, including the myometrium and endometrium, from 18 bitches were taken at 6 stages of the estrous cycle. The total number of estrogen receptors and nuclear estrogen receptor and its mRNA levels in the uterus also increased (P < 0.01) from late anestrus to proestrus. Mean plasma E2 level and the number of uterine estrogen receptor were positively correlated (r = 0.81, P < 0.05). In Experiment 3, nine bitches were ovariectomized in mid anestrus. Two weeks later they received a single injection of 10 or 50 micrograms/kg, i.m., estradiol benzoate. The number of uterine estrogen receptor and their mRNA levels for ovariectomized bitches were low, but increased (P < 0.05) after treatment with a low dose of estradiol benzoate. These results suggest that increases in LH pulse frequency and estrogen secretion are associated with termination of anestrus and that subsequent enhancement of uterine estrogen receptor expression may be up regulated by estradiol. PMID- 10734360 TI - Fertility in bitches artificially inseminated with extended, chilled semen. AB - Sixteen bitches were artificially inseminated with either fresh, 24 h-chilled or 48 h-chilled extended semen over 38 estrous cycles. A commercial system for extending, chilling and transporting semen commonly used in the equine industry was used Pregnancy rates and litter sizes of the bitches inseminated with extended, chilled semen (19/20, 95%; litter size = 7.1) were not significantly different from those observed in bitches inseminated with fresh semen (17/18, 94%; litter size = 7.2; P > or = 0.89). These results show that a commercial system for extending, chilling and transporting equine semen is an attractive and efficient method of shipping canine extended chilled semen. PMID- 10734361 TI - In vitro capacitation of dog spermatozoa as assessed by chlortetracycline staining. AB - We developed an assay for detecting capacitation and acrosome status in dog spermatozoa using chlortetracycline (CTC) as a fluorescent probe. Sperm cells were stained after incubation in modified canine capacitation medium (mCCM). Calcium ionophore A23187 permitted the induction of acrosomal exocytosis of capacitated sperm cells. Spermac staining and transmission electron microscopy were used as control tests to detect acrosome-reacted spermatozoa. Three different patterns of CTC distribution in the spermatozoa were found. These patterns were similar to those observed in other mammalian species. The CTC test was used to monitor the time course of the capacitation process in dogs. It was found that the kinetics of capacitation in canine sperm cells are similar to those observed in other mammals. PMID- 10734362 TI - Analysis of zona pellucida modifications due to cortical granule exocytosis in single porcine oocytes, using enhanced chemiluminescence. AB - The present study was carried out to determine whether modification of zona pellucida (ZP) of a single oocyte following the cortical granule (CG) exocytosis induced by electrical stimulation could be analyzed using enhanced chemiluminescent (ECL) detection of the biotinylated ZP in a porcine oocyte. When a biotinylated ZP derived from a single oocyte matured in vitro was subjected to SDS-PAGE, 3 major bands (ZP1, ZP2 and ZP3) were observed following ECL detection. In these oocytes, CGs staining with fluorescein isothiocyanate (FITC)-labeled peanut agglutinin (FITC-PNA) had formed a monolayer underlying the plasma membrane. Electrical stimulation to induce artificial activation caused a decline in the fluorescent intensity of the CGs with a concomitant decrease in the amounts of ZP1 and ZP2 bands. However, the mobility changes of ZP1 and ZP2 on SDS PAGE were not found under the inhibitory condition of the CG exocytosis in which oocytes were treated with ethylene glycol-bis(beta-aminoethyl ether) N, N, N',N' tetraacetic acid (EGTA) or 1, 2-bis(2-aminophenoxy)ethane-N, N, N', N' tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA/AM). In addition, when a time-dependent decrease in amounts of ZP1 and ZP2 bands on SDS-PAGE was observed in a single oocyte during activation, a maximum decrease in these bands was detected in oocytes incubated for at least 3.5 h after electrical stimulation. These results show that the method employed, ECL detection of the biotinylated ZP of a single oocyte, is a valuable tool for the analysis of ZP modification resulting from a decrease in amounts of ZP1 and ZP2 glycoproteins in combination with exocytosis of CGs, and that the prolonged period after activation is required for complete ZP modification in porcine oocytes. PMID- 10734363 TI - Effect of cooling rates on post-thaw sperm motility, membrane integrity, capacitation status and fertility of dairy bull semen used for artificial insemination in Sweden. AB - We studied the effects of 2 different cooling rates during equilibration of semen from room temperature to 4 degrees C, at 4.2 degrees C/min (control split sample) or at 0.1 degree C/min (treatment split sample) on in vitro sperm viability post thawing and fertility after AI. Forty batches of split-frozen semen from 14 dairy bulls (Swedish Red and White breed) aged 14 to 16 m.o. or 66 to 79 m.o. were evaluated post-thawing for sperm motility (visual and computer-assisted sperm analysis [CASA], membrane integrity (fluorescent microscopy and flow cytometry post-loading with the combined fluorophores Calcein AM/EthD-1 and SYBR-14/PI); acrosomal status (with Pisum sativum agglutinin [PSA] staining); and capacitation status (CTC-assay). Fertility values (56-d nonreturn rate) of the slow cooling batches (treatment) were 0.4% units higher than for faster cooled (control) batches, but the difference was not statistically significant. Fertility values for the older bulls were 1.6% units higher than for the group of younger sires. No statistically significant correlations were found between semen viability parameters assessed in vitro and 56-d nonreturn rate. Visually assessed sperm motility, membrane integrity, capacitation and acrosomal status post-thawing did not differ significantly between cooling procedures, however the percentage of motile spermatozoa and the kinetic characteristics of spermatozoa--average path velocity (VAP), straight path velocity (VSL) and curvilinear velocity (VCL)- assessed by CASA differed significantly between cooling procedures. The results indicate that most of the in vitro sperm viability parameters post-thawing and the fertility results for bulls after AI did not differ significantly between the 2 semen cooling procedures tested. PMID- 10734364 TI - Estimates of pregnancy outcomes based on selection of bovine embryos produced in vivo or in vitro. AB - The objective of this study was to estimate the degree of variation among experienced evaluators selecting in vivo- or in vitro-produced embryos for transfer and to determine how this affects both the proportion of recipients becoming pregnant after transfer, and the number of embryo transfers required per pregnancy. Data from 6 experienced evaluators who graded Day 7 embryos produced either in vivo (n = 15) or in vitro (n = 15) were used to estimate these effects. The evaluators viewed video recorded images of the embryos and classified each embryo for stage of development and quality grade (1 = excellent, 2 = good, 3 = fair, 4 = degenerated and nontransferable). The statistical model considered transfer of embryos of the following individual or combined grades: Grade 1 only, Grade 2 only, Grade 3 only, Grades 1 and 2, Grades 2 and 3, and Grades 1, 2 and 3. Probabilities of pregnancy after embryo transfer were based on pregnancy rates of recipients at the facility of 1 of the 6 evaluators where the percentages of heifers pregnant after the transfer of Grade 1, 2 and 3 embryos, by embryo source, were 76, 65 and 54% (in vivo), and 59, 45 and 30% (in vitro). Within most grades, the proportion of embryos selected for transfer differed (P < 0.05) among the 6 evaluators. Although no significant differences (P > 0.10) were found among evaluators in the proportion of recipients pregnant after transfer within any embryo grade, there was substantial variation among evaluators in the proportion of recipients becoming pregnant, especially for embryos produced in vitro. Estimated percentages of heifers becoming pregnant for embryos classified as Grade 1, 2 or 3 were 66 to 76, 62 to 69, and 54 to 60%, respectively, for in vivo produced embryos; and, 39 to 59, 15 to 45, and 24 to 32%, respectively, for in vitro-produced embryos. Approximately twice as many transfers were needed per pregnancy for embryos produced in vitro as for those produced in vivo regardless of the grade. PMID- 10734365 TI - Intracytoplasmic injection (ICSI) of in vivo or in vitro matured oocytes with fresh ejaculated or frozen-thawed epididymal spermatozoa and additional calcium ionophore activation in the pig. AB - In Experiment 1, we performed intracytoplasmic sperm injection (ICSI) of frozen thawed epididymal and fresh ejaculated in vitro-capacitated spermatozoa into in vivo and in vitro-matured porcine oocytes. Within each group, oocytes were sperm injected, sham-injected or served as handling controls. After subsequent in vitro culture for 48 h the number of unchanged, fragmented und cleaved oocytes was recorded. The best result (14% cleaved after ICSI vs 2 and 0% with the sham injection and handling controls; P < 0.01) was achieved with fresh in vitro capacitated spermatozoa injected into in vivo-matured oocytes. In vitro-matured oocytes displayed high fragmentation rates. In Experiment 2, in vitro matured oocytes were injected with freshly ejaculated in vitro-capacitated spermatozoa, followed by a 5 min-exposure to 0 (control), 50 or 100 microM calcium-ionophore. Comparable groups were sham injected or served as handling controls. It became apparent that Ca-ionophore treatment after injection of spermatozoa was ineffective at 100 microM, where at 50 microM a significant reduction in cleavage rate was observed (6 vs 26% with untreated controls, P < 0.01). Fluorescence staining with Hoechst 33342 revealed that in most cases of sperm-injected oocytes that remained unchanged after 48 h of in vitro-culture, sperm heads had not decondensed. Only few oocytes had continued to the pronucleus stage. In this context no favorable effect of Ca-ionophore was to be observed. PMID- 10734367 TI - Bull-specific effect on fertilization rate and viable embryo recovery in the superovulated buffalo (Bubalus bubalis). AB - This study was conducted to compare fertilization rate and viable embryo recovery rate in superovulated buffalo (n = 64) following insemination with semen from buffalo bulls (n = 5) having different fertility rates as determined by AI. Frozen-thawed semen from fertile bulls with similar post-thaw progressive motility and sperm morphology was used to inseminate buffalo at superovulatory estrus. Fertilization and viable embryo recovery rates differed among bulls, but this bull-specific effect was not related to the overall herd fertility rate as determined by AI in normal cyclic animals. These results indicate that individual bulls differ in their contribution to fertilization of superovulated donors and also to embryonic development, as determined by viable embryo recovery. Moreover, the results also suggest that buffalo bulls can be screened for optimal fertility and embryo recovery rates in superovulated donors. Further studies are warranted to ascertain the factors which contribute to such bull-specific effects. PMID- 10734366 TI - High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins. AB - We describe a bovine embryo culture system that supports repeatable high development in the presence of serum or BSA as well as under defined conditions in the absence of those components. In the first experiment, embryo development in SOF with amino acids (SOFaa), sodium citrate (SOFaac) and myo-inositol (SOFaaci) and with BSA or polyvinyl alcohol (PVA) was compared with that in a M199 granulosa cell co-culture (M199 co-culture). Subsequently, development and cell numbers of blastocysts cultured under defined conditions in SOFaaci with PVA (SOFaaci-PVA), or under undefined conditions in SOFaaci with 5% cow serum (SOFaaci-CS) or M199 co-culture were compared. The repeatability of culture results in SOFaaci-CS was checked by weekly replicates (n = 30) spread over 11 months. The viability of embryos developed in SOFaaci-PVA was estimated by transfer of morphologically good blastocysts (n = 10) to synchronized recipients. In the second experiment, the effect of omitting CS or BSA from IVM and IVM-IVF on subsequent embryo development in SOFaaci-PVA or in SOFaaci-CS was investigated. Blastocyst development in SOFaa-PVA, SOFaac-PVA, SOFaa-BSA and M199 was 16 +/- 3b, 23 +/- 2ab, 30 +/- 8a and 36 +/- 7a%, respectively (Pab < 0.05). Additional inclusion of myoinositol resulted in 42 +/- 1a% blastocysts in SOFaaci PVA vs 19 +/- 3b% in SOFaac-PVA, 47 +/- 7a% in SOFaac-BSA, and 36 +/- 7a% in M199 co-culture, respectively (Pab < 0.01). In 30 replicates, the average cleavage and blastocyst rates of oocytes in SOFaaci-CS were 87 +/- 4 and 49 +/- 5%, respectively. Five normal calves were produced after transfer of 10 blastocysts developed in defined culture medium (i.e., SOFaaci-PVA). Defined IVM or IVM-IVF (i.e., in absence of CS and BSA) reduced cleavage rates (83 +/- 3 and 55 +/- 3% vs 90 +/- 1% in presence of CS; P < 0.01). Subsequent embryo development in SOFaaci-CS was not affected in either of these defined conditions. However, cleavage and blastocyst rates under completely defined IVP conditions were 54 +/- 7 and 19 +/- 4%, respectively. It was concluded that under defined culture conditions, addition of citrate and myo-inositol improved blastocyst development to rates comparable to those obtained with serum, BSA or co-culture and that the quality of blastocysts was not affected by the absence of serum or BSA. However, serum was essential during IVM/IVF for normal fertilization and subsequent high blastocyst development. PMID- 10734368 TI - Repeated endoscopic ovum pick-up in sheep. AB - Endoscopy is an effective and minimally invasive technique which offers the possibility of repeated ovum pick-up (OPU). In this study 4 different treatment programs (Groups A, B, C and D) for repeated endoscopic OPU in sheep were investigated. The number of follicles and oocytes, quality of cumulus-oocyte complexes (COCs), and detectable effects on fertility of the donor ewes were compared. Each group consisted of 5 East Friesian Milksheep. In Group A, follicles were punctured twice a week, in Group B once a week, and in Group C once a week followed by administration of 1500 IU PMSG 48 h prior to OPU. In Group D follicles were punctured and the sheep stimulated with 1500 IU PMSG 48 h prior to OPU once every 2 weeks. The PMSG-stimulated sheep received anti-PMSG immediately after OPU. Over a period of 10 weeks 216 OPU-sessions were performed. A total of 1978 follicles was punctured, and 1098 oocytes were recovered, for a collection rate of 55.5%. In the Groups A, B, C and D an average of 6.8, 8.6, 12.2 and 14.9 follicles per animal and session was aspirated, and an average of 3.8, 4.9, 7.0 and 7.6 COCs per animal per session was recovered, respectively. No significant differences between groups were observed in the collection rates (51.1 to 57.1%) or in the quality of the COCs, and 65 to 70% of the COCs were suitable for in vitro production of ovine embryos. Seven sheep developed small adhesions between the ovary and infundibulum. After the study 15 ewes became pregnant following natural mating with the same fertile ram (5 from Group A, 1 from Group B, 4 from Group C and 5 from Group D). In conclusion, OPU once a week in PMSG/anti-PMSG treated ewes was found to be the most effective treatment program for oocyte collection. PMID- 10734369 TI - Early pregnancy diagnosis in goats by determination of pregnancy-associated glycoprotein concentrations in plasma samples. AB - Different RIA systems available for measuring the concentrations of pregnancy associated glycoproteins (PAGs) in dairy goats were compared in order to evaluate their accuracy in early pregnancy diagnosis. Plasma concentrations of PAGs were determined by 3 heterologous RIA systems with a bovine PAG standard and tracer in combination with antisera anti-ovine PAG (RIA 1), anti-caprine PAG55 + 62 (RIA 2), anti-caprine PAG55 + 59 (RIA 3), and by 2 homologous RIA systems that employed caprine PAG55 + 62 and caprine PAG55 + 59 and their specific antisera (RIAs 4 and 5, respectively). In all of the RIAs, the mean concentrations of PAGs were significantly higher (P < 0.01) in pregnant than in nonpregnant goats from Day 21 onwards after breeding. On Day 21, the accuracy rates of early pregnancy diagnoses were 56% (RIA 1), 96% (RIA 2), 99% (RIA 3), 95% (RIA 4) and 90% (RIA 5), whereas on Day 28 these rates were > 99% for RIAs 2, 3, 4 and 5. The RIAs for PAGs depend on proteins from the placenta being present in maternal plasma and require only a single sample of blood, to distinguish pregnant goats from those that fail to return to estrus for other reasons. The homologous and semi heterologous assays are highly accurate as early as Day 21 of pregnancy. PMID- 10734370 TI - Evidence for an annual reproductive rhythm independent of food availability in male Creole goats in subtropical northern Mexico. AB - The aim of this study was to determine if there is a seasonal pattern of sexual activity dependent on food availability in male Creole goats in subtropical Mexico. The study was conducted in the Laguna Region in the State of Coahuila, Mexico (26 degrees N). Male Creole goats (n = 8) were kept in a shed, fed alfalfa ad libitum and given 200 g of concentrate daily throughout the study. Live weight and testicular weight were determined every 2 wk. Sexual behavior and sperm production were determined monthly. Blood samples were obtained weekly to determine testosterone plasma concentrations. All variables were subjected to sinusoidal modeling procedures and showed important seasonal variations (P < 0.0001) with different phase angles for body weight, testicular weight and testosterone plasma concentrations. The nadir of live weight occurred in November and the peak in May. The lowest testicular weight (90 g) and testosterone plasma concentrations (0.1 ng/mL) were observed in January and February, respectively, while the peaks were observed in July and August (145 g and 10 ng/mL, respectively). Ejaculation latency also varied during the study, being low between May and November (96 sec) and reaching a peak in April (183 sec). Minimum number of spermatozoa per ejaculate occurred between February and April (1.4 x 10(9) cells/ejaculate) while the maximum number was observed between May and September (2.8 x 10(9) spermatozoa/ejaculate). Progressive sperm motility was low between January and April (3.04 on average) and high between May and November (about 3.55 on average). The percentage of live spermatozoa diminished between January and April (68% in April) and then increased to values around 80% between May and November. These results lead us to conclude that male Creole goats in Northern Mexico, fed constantly throughout the year, exhibit seasonality in their reproductive activity. Intense sexual activity occurred between May and December. PMID- 10734371 TI - Uterine luminal proteins and estrogens in gilts on a normal nutritional plane during the estrous cycle and on a normal or high nutritional plane during early pregnancy. AB - In gilts, a high plane of nutrition during early pregnancy often results in increased embryo mortality, possibly related to changes in embryo-uterine asynchrony at a critical stage of pregnancy (around Day 11). Therefore, in the present study, uterine luminal proteins and estrogens were studied between Days 5 and 16 after the onset of estrus in gilts on either a normal (2.5 kg/d, cyclic and pregnant gilts) or a high (4.0 kg/d, pregnant gilts only) feeding level. Conceptus recovery rate between Days 5 and 12 was not affected by the feeding level during early pregnancy, neither were systemic progesterone levels. Between Days 9 and 11, dramatic changes took place in the protein composition of the uterine luminal 10kD+ proteins, shifting from most (90%) of the acidic proteins at Day 5 and 7 to approximately 50% at Day 11/12, especially due to an increase in basic proteins with an iso-electrical point of more than 8. This shift occurred most rapidly for the pregnant gilts at the high feeding level and least rapidly in the cyclic gilts, resulting in significant differences in the relative amount of acidic proteins at Day 10 and 11 after the onset of estrus (P < 0.05). Similarly, levels of estrogens in the uterine flushings at Days 10, 11 and 12 were always highest for the pregnant gilts on the high feeding level and were always lowest in the cyclic gilts (P < 0.05); pregnant gilts on the normal feeding level showed intermediate estrogen levels. The fact that gilts on a high feeding level during early pregnancy show more rapid changes in the uterine luminal protein composition and embryonic estrogen production seems to suggest that the rate of these changes may be related to embryo survival. PMID- 10734372 TI - The fecundity of porcine semen stored for 2 to 6 days in Androhep and X-CELL extenders. AB - Extending the raw ejaculate prior to artificial insemination (AI) is beneficial, in part, due to the increased number of females that are bred from an ejaculate, along with prolonged shelf life of the semen. The objective of this study was to examine the affects of storage time on the fecundity of porcine semen diluted in 2 semen extenders, Androhep and X-CELL. A completely randomized design with a factorial arrangement of treatments was utilized in which 429 high quality, gel free ejaculates from 48 boars were used in a timed, double insemination of 1,431 first-service gilts. The gilts were divided into groups and inseminated with semen stored in Androhep or X-CELL for 2 to 3 d, 3 to 4 d, 4 to 5 d, or 5 to 6 d prior to use (day of collection = Day 0). Sperm age was identical, and both extenders were used concurrently each day of the trial. Farrowing rate and litter size data were recorded. Farrowing rates did not differ between extenders through Days 4 to 5 of storage. Gilts inseminated with Androhep diluted stored semen showed a decrease (P < 0.001) in farrowing rate compared with those inseminated with semen extended in X-CELL stored for 5 to 6 d. Mean litter sizes did not differ between extenders through Days 2 to 3 of storage. Compared with the X-CELL extended semen, gilts inseminated with Androhep extended semen produced smaller litters when semen was stored for 4 to 5 d (P < 0.05). Within the Androhep treatment, smaller mean litter sizes (P < 0.05) were evident when the semen was stored for 3 to 4 and 4 to 5 d. No differences were detected in litter size or farrowing rate for gilts bred with semen stored for 2 to 6 d in the X-CELL extender (P > 0.1). The results of this study indicate that extender type influences the fertility potential of fresh porcine semen stored for 2 to 6 d. For optimal fecundity in gilts, semen extended with Androhep extender should be used for AI within 3 d. The X-CELL extended semen can be used for up to 6 d without significant decrease in litter size or farrowing rate. These recommendations are dependent upon using high quality semen that is properly handled from collection through insemination. PMID- 10734373 TI - Effect of treatment with recombinant bovine somatotropin on responses to superovulatory treatment in swamp buffalo (Bubalus bubalis). AB - The objective of this study was to determine the effect of treatment with recombinant bovine somatotropin (rBST) on the response to superovulatory treatment in swamp buffalo. Estrous cycles of 16 buffalo cows were synchronized by intravaginal administration of progesterone and estradiol benzoate, and the cows were then randomly divided into 2 groups. The rBST-treated group received 250 mg of a sustained-release formula of rBST on Day 4 after progesterone implantation, whereas the control group did not receive rBST. Both groups were then given a superovulatory regimen of twice daily injections of FSH for 3.5 d (total dose of 260 mg, i.m.), between Days 9 and 11 after administration of progesterone. The cows were bred naturally 1 d after the last FSH injection, then 6 d after breeding they were slaughtered, and their reproductive tracts were removed. The numbers of corpora lutea (CL) and follicles were recorded, and embryos were flushed out of the uterine horns. There were no significant differences between the rBST-treated and control cows for the mean numbers (+/- SEM) of CL (6.0 +/- 2.2 vs 4.3 +/- 1.1), follicles (15.9 +/- 4.1 vs 19.8 +/- 2.9), or total embryos recovered per collection (4.5 +/- 1.6 vs 2.3 +/- 1.0). However, there were significant differences between rBST-treated and control cows for the numbers of transferable embryos per collection (3.0 +/- 1.0 vs 0.8 +/- 0.3; P < or = 0.05) and the overall proportion of transferable embryos (75 vs 33%; P < or = 0.01). The results of this study show that pretreatment of swamp buffalo with rBST significantly increases the production of transferable embryos in response to superovulation. PMID- 10734374 TI - Involvement of ovarian steroids in basal and oxytocin-stimulated prostaglandin (PG) F2 alpha secretion by the bovine endometrium in vitro. AB - It is assumed that exposure of endometrium to spontaneously secreted luteal hormones stimulates PGF2 alpha secretion and modifies oxytocin (OT) influence on the bovine uterus. At first, the time-dependent effect of endogenous luteal products on endometrial PGF2 alpha secretion was examined. Endometrial strips (100 mg) from slaughtered heifers (Days 11 to 17 of the cycle) were incubated alone or with luteal cells (1 x 10(5) cells/mL). The highest PGF2 alpha secretion by the endometrium under influence of hormones secreted from luteal cells was observed after 12 h of incubation compared with the control (P < 0.001). Then, endometrium (Days 11 to 17) was incubated with luteal cells and concomitantly with antagonists of P4 and OT. The P4 antagonist prevented the stimulatory effect of endogenous luteal hormones on PGF2 alpha secretion (P < 0.05), but the OT antagonist did not. Further, direct effects of exogenous P4, OT and estradiol (E2) on endometrial PGF2 alpha secretion (Days 11 to 17) were examined. Both OT and P4 increased PGF2 alpha secretion (P < 0.05); E2 alone had no effect on PGF2 alpha secretion, but it amplified the P4 effect (P < 0.05). Finally, we studied the effect of endogenous luteal products on OT-stimulated PGF2 alpha secretion from endometrium. When endometrium (Days 11 to 17) was incubated without luteal cells, OT stimulated PGF2 alpha secretion (P < 0.001), whereas incubation of endometrium with luteal cells abolished the stimulatory effect of OT on PGF2 alpha secretion (P < 0.001). These treatments did not affect PGF2 alpha secretion from the endometrium collected on Days 1 to 4. In conclusion, P4 stimulates PGF2 alpha secretion by the endometrium and E2 amplifies this effect. As long as the endometrium is under the influence of P4, ovarian OT does not affect PGF2 alpha secretion. PMID- 10734375 TI - Ovarian dynamics, serum estradiol and progesterone concentrations during the interovulatory interval in goats. AB - Ovarian changes determined by daily transrectal ultrasonic scanning, and its correlation with serum progesterone (P4) and estradiol (E2) concentrations were studied in seven cyclic Saanen goats. Estrous cycles were synchronized with 2 injections of a PGF2 alpha analogue 9 d apart. All follicles > or = 2 mm in diameter and CL were measured each day. One goat showed a longer interestrous interval, associated with development of a cystic-luteinized structure. The mean interovulatory interval for the other 6 goats was 20.8 +/- 0.4 d. The incidence of goats with 4, 3, and 2 follicular waves was 3, 1 and 2 respectively; follicular waves emerged on Days 0.5 +/- 0.6, 7.2 +/- 0.7, 10.7 +/- 0.5 and 13.7 +/- 0.8 for Wave 1, 2, 3 and the Ovulatory wave, respectively. The largest follicle of Wave 2 was smaller (4.9 +/- 0.1 mm) than the largest follicles of Wave 3 (6.2 +/- 0.1 mm; P < or = 0.01) and of the Ovulatory wave (7.0 +/- 0.5 mm; P < or = 0.01), and tended to be smaller than the largest follicle of Wave 1 (6.3 +/- 0.6 mm; P < or = 0.09). Interval between emergence of Wave 1 and Wave 2 was longer than interval between emergence of Wave 2 and Wave 3 (7.3 +/- 0.9 d vs 4.0 +/- 0.4 d; P < or = 0.01), and between Wave 3 and the Ovulatory wave (3.8 +/- 1.1 d; P < or = 0.05). Two days before ovulation, the diameter of the ovulatory follicle was larger (P < or = 0.01) than the first subordinate follicle. Serum E2 concentrations increased from the day of ovulation (2.7 +/- 0.3 pg/mL) to Day 2 (7.6 +/- 0.9 pg/mL; P < or = 0.01), associated with the early-mid growing phase of the largest follicle of Wave 1, and then decreased to basal levels on Day 5 (P < or = 0.01) and peaked again (16.5 +/- 2.4 pg/mL) 2 d before ovulation. The CL were detected ultrasonically on Day 3 post ovulation and attained a mean maximum diameter of 13.5 +/- 0.8 mm between Days 8 and 14. The following characteristics were observed: 1) ovarian follicular development in goats is wave-like; 2) increased P4 concentrations may be promoting follicular wave turnover; 3) it is suggested that the presence of follicular dominance and the production of E2 are different among waves. While in Wave 1 and in the Ovulatory wave, follicular dominance is present and production of E2 is consistent, no changes in serum E2 concentrations were found in other stages of the interovulatory interval. In the intervening waves, no indicators of follicular dominance could be firmly documented. PMID- 10734376 TI - Uterine contractility is necessary for the clearance of intrauterine fluid but not bacteria after bacterial infusion in the mare. AB - Bacteria were infused into the uteri of 5 estrous mares resistant to persistent mating-induced endometritis, first during a control cycle, and then during treatment with clenbuterol, a beta 2 agonist. Uterine cellular response was evaluated 48 h later by transrectal ultrasonography, followed by uterine lavage. During clenbuterol treatment all mares accumulated intrauterine fluid, whereas in the control cycle none of the mares retained fluid. There was no significant difference between the 2 cycles in the cloudiness of the lavage fluid, number of cells per milliliter, percentage of neutrophils and frequency of bacterial growth from the recovered fluid. We conclude that uterine contractility is important in the clearance of uterine fluid, but not necessarily for the elimination of bacteria, thus supporting the published evidence that impaired uterine contractility contributes to the pathogenesis of persistent mating-induced endometritis. PMID- 10734377 TI - Oxytocin gene expression and action in goat testis. AB - Gene expression, immunohistochemical localization, binding and effects of oxytocin (OT) on androgen production in the testis of adult goats were studied. Using polymerase chain reaction (PCR) analysis we were able to detect OT gene transcripts in the goat testis. Immunohistochemistry revealed that both OT and neurophysin epitopes were expressed together in the intratubular regions, especially in the Sertoli cells, suggesting the production of OT in these cells. However, enzyme immunoassay found no difference in OT concentration between testicular arterial and venous plasma. Saturable, specific [3H]-OT binding sites were present in membrane fractions of the goat testis. Scatchard analysis indicated an apparent affinity of 42 +/- 7 L/nmol and binding capacity of 24 +/- 4 fmol OT bound/mg DNA. In vitro treatment of goat testes for 6 h with 100 nM OT led to a 3.5-fold increase (P < 0.001) in 5 alpha-dihydrotestosterone (DHT) production in spite of the induced decrease in testosterone, suggesting that OT not only affects testosterone production but also modulates its conversion to DHT. These results indicate that the goat testis produces OT, which may be involved in the local control of androgen biosynthesis. PMID- 10734378 TI - Factors affecting reproductive performance in captive Mallard ducks. AB - Propagation of wild-strain Mallards (Anas platyrhynchos) in captivity is hindered by low egg fertility. Therefore, we studied the effects of captivity, age, mate choice and isolation on reproductive parameters of Mallards. Captive drakes had smaller immature testes than free-flying Mallard drakes. Captive yearling ducks weighed less than adult ducks at the beginning of the breeding season, but no differences were found between their initial clutch size, egg volume or number of clutches laid. Yearling pairs had lower egg fertility (7%) than adult pairs (80%). Egg fertility was higher (51 vs 21%) in self-chosen pairs than in randomly assigned pairs. Isolation of ducks, however did not influence egg production. PMID- 10734379 TI - The importance of porcine sperm parameters on fertility in vivo. AB - It would be desirable to use semen parameters to predict the in vivo fertilizing capacity of a particular ejaculate. In animal production, an ejaculate is divided into multiple doses for artificial insemination (AI); therefore, it would be economically beneficial to know the functional quality (i.e., fertility) of the semen before it is inseminated. To identify a predictive assay of the fertilizing capacity of a porcine ejaculate, we performed 4 rapid assays of sperm quality (motility, viability, physiological status as assessed by chlortetracycline fluorescence, and ATP content) on samples from 9 ejaculates, before and after a thermal stress test (42.5 degrees C, 45 min). These parameters were subsequently correlated with in vivo fertility resulting from AI with 2 sperm doses, 3 x 10(9) or 0.3 x 10(9) motile cells in 70 mL (optimal or suboptimal sperm number per insemination, respectively) from these same ejaculates. No parameter was correlated to the fertility rates obtained after inseminating with the optimal semen doses, either before or after the thermal stress test (P > 0.05). However, with respect to the animals inseminated with the suboptimal semen dose, sperm motility (the percentage of motile spermatozoa as assessed visually by microscopy) prior to thermal stress was well-correlated to fertility rates (r = 0.783, P = 0.01). The percentage of spermatozoa displaying the chlortetracycline Pattern AR (acrosome reaction) was also statistically related to fertility (r = 0.05, P = 0.04), but the biological importance of this relationship is questionable given the small variation among ejaculates (range: 0 to 2%). No other sperm parameter was significantly related to fertility rates in this group (P > 0.05). These data, therefore, indicate that sperm motility is a useful indicator of sperm fertilizing capacity in vivo. Moreover, to identify a predictor of semen fertility it is critical that the number of spermatozoa used during insemination is sufficiently low to detect differences in sperm fertilizing efficiency. PMID- 10734380 TI - Uterine clearance and resistance to persistent endometritis in the mare. AB - The objective of this article is to review the role of uterine defense mechanisms in natural resistance to chronic or persistent endometritis. A breakdown of uterine physical clearance mechanisms is currently believed to play a major role in susceptibility to persistent endometritis. Mares with increased susceptibility to persistent endometritis have impaired myometrial contractility in response to an acute inflammation, resulting in an accumulation of fluid and inflammatory products within the uterine lumen. The origin of this defect remains unknown. Recent studies have demonstrated that spermatozoa trigger PMN chemotaxis into the uterine lumen. This observation suggests that a transient endometritis is a normal physiological response to breeding. However, in mares with impaired uterine defense mechanisms, the condition may develop into a persistent endometritis and subsequent subfertility. In contrast to spermatozoa, seminal plasma has a suppressive effect on complement activation and PMN chemotaxis (65). The exact role of seminal components in breeding-induced inflammation needs further investigation. PMID- 10734381 TI - The effect of egg yolk, low density lipoproteins, methylxanthines and fertilization diluent on cryopreservation efficiency of northern pike (Esox lucius) spermatozoa. AB - The effect of egg yolk, low density lipoproteins (LDL) as well as methylxanthines (caffeine and theophylline) and fertilization diluent on cryopreservation efficiency of northern pike, Esox lucius, spermatozoa was tested. Milt was cryopreserved in pellets on dry ice then stored in liquid nitrogen. The extender consisted of 0.6 M sucrose + 15% DMSO supplemented with egg yolk or LDL fractions. The most effective results (77.3% hatched larvae vs 74.1% in the control group) were obtained from extender that contained only 0.6 M sucrose + 15% DMSO and was used for freezing, while the fertilization diluent was used for thawing. Addition of egg yolk or LDL to the extender did not improve the results. The presence of caffeine in the thawing solution significantly lowered fertilization rate of cryopreserved spermatozoa, whereas theophylline did not significantly affect the results. The addition of fertilization diluent to the eggs prior to insemination was superior to the other treatments. The proposed procedure constitutes a complete method for the efficient cryopreservation of northern pike semen. PMID- 10734382 TI - Recombinant bovine somatotropin: association with reproductive performance in dairy cows. AB - A clinical trial was performed to determine the effect of bST on reproductive performance in dairy cows on 4 Michigan dairy farms when bST was used according to labeled directions. Holstein cows (n = 555) at 4 Michigan dairy farms were randomly assigned to receive bovine somatotropin (bST) or to serve as untreated controls. Bovine somatotropin (500 mg, s.c.) was administered every 14 d beginning at 63 to 69 d of lactation and continuing until approximately 21 d prior to the end of lactation or until the producer removed the animal from the herd. A total of 229 (42.57%) animals consisting of 112 (48.91%) controls and 117 (51.09%) bST-treated cows were not diagnosed pregnant and were considered as having reproductive failure by 150 d of lactation and 92 (17.10%) of the animals consisting of 45 (48.91%) controls and 47 (51.09%) bST-treated cows had reproductive failure at the end of their lactation. No significant difference in the incidence of reproductive failure existed between the study treatment groups during either time frame. Cows with cystic ovarian disease had more instances of reproductive failure at 150 d of lactation than cows without this disease. Cows with dystocia, twin births, metritis, displaced abomasum and cystic ovarian disease had more instances of reproductive failure by the end of lactation than cows without these diseases. The bST-treated cows developed more nonspecific off feed conditions during the study lactation and had more twin births at the calving following bST administration compared to control cows. No differences were found between the study treatment groups in calculations of reproductive parameters. Logistic regression analysis found no significant effect of bST on reproductive failure; the odds ratios for the bST treatment effect on reproductive failure at 150 d of lactation and reproductive failure at the end of lactation were 0.95 (95% CI 0.67 to 1.36) and 0.95 (95% CI 0.59 to 1.54), respectively. Overall, bST was not found to have a significant impact on reproductive performance in the herds studied. PMID- 10734383 TI - Effect of linoleic acid-albumin in the culture medium on freezing sensitivity of in vitro-produced bovine morulae. AB - The objective of this study was to improve the survival of in vitro-produced bovine morulae after cry opreservation. In Experiment 1, presumptive zygotes at 20 h post-insemination (hpi) were cultured in a mixture of modified synthetic oviduct fluid (m-SOF)/0.3% BSA and m-SOF/0.3% linoleic acid-albumin from bovine serum (LAA) at 39.0 degrees C in 5% O2, 5% CO2 and 90% N2 (final LAA concentration: 0, 0.01, 0.03, 0.1 or 0.3%). Morulae harvested at 138 hpi were frozen and thawed in m-PBS/0.3% BSA containing 1.5 M ethylene glycol and were cultured for 96 h in m-SOF/10% FBS to assess further development. The post-thaw survival of morulae derived from culture in 0.1% LAA (60%, P < 0.01) and in 0.03% LAA (55%, P < 0.05) was higher than that in 0% LAA (32%). Lowering the LAA concentration below 0.1% resulted in similar rates of morula development as in m SOF/0.3% BSA. In Experiment 2, zygotes were cultured in m-SOF/0.1% LAA from 20 to 90 hpi and/or from 90 to 138 hpi. Post-thaw survival of morulae that had been exposed to LAA from 20 to 90 hpi (39%) or from 90 to 138 hpi (56%) was higher than that of morulae cultured without LAA from 20 to 138 hpi (12%, P < 0.02). These survival rates were lower than that of morulae cultured with LAA over a period of 20 to 138 hpi (76%, P < 0.001). The results indicate that cell-free culture of IVM/IVF bovine zygotes in m-SOF supplemented with LAA produces morula stage embryos relatively tolerant to the process of freezing and thawing. PMID- 10734384 TI - Effects of follicular status at treatment on follicular development and ovulation in response to FSH in Spanish merino ewes. AB - Cyclic Spanish Merino ewes were treated on Day 13 of the estrous cycle with 12 mg, i.m., FSH-P in saline (n = 9) or propylene glycol (n = 24), currently with 100 micrograms, i.m., Cloprostenol (Day 0). From Day-6 to Day 0, the ewes were observed daily by transrectal ultrasonography, after Day 0, ultrasonography was performed every 12 h for 72 h. Sizes and locations of > or = 2 mm follicles were recorded at each observation. The ovulation rate was determined by laparoscopy on Day 7 after estrus. The number of ovulations ranged from 0 to 6 in ewes treated with FSH-P in saline and from 0 to 16 in ewes receiving FSH-P in propylene glycol (P < 0.05). In the latter group, the response was bimodally distributed; about half of the females had 1 ovulation, whereas the remainder had > 4 with a mean of 7 ovulations. The ovulation rate was associated with 2 characteristics of the largest follicle present at treatment (Day 0). First, if the largest follicle on Day 0 had not changed in diameter from Day-1 to Day 0, then 7 of 9 ewes had > 3 ovulations; if the largest follicle had either increased or decreased, only 8 of 24 ewes had > 3 ovulations (P < 0.05). Second, there was a linear trend (P < 0.07) for ovulation rate to decrease as the persistence of the largest follicle at treatment increased; no ewe in which the largest follicle on Day 0 remained present for more than 36 h ovulated more than 6 follicles. As with the ovulation rate, the numbers of large follicles on Days 1.5, 2 and 2.5 varied with the interaction of change in diameter of the largest follicle on Day 0 from Day-1 to Day 0 and with vehicle. In summary, the superovulatory response was affected by the change in diameter from Day-1 to Day 0 of the largest follicle on Day 0 and the period required for that follicle to regress after treatment with FSH-P and cloprostenol. PMID- 10734385 TI - Cellular alteration after dilution of cryoprotective solutions used for the vitrification of in vitro-produced bovine embryos. AB - Embryo quality of in vitro-produced bovine blastocysts was assessed at several steps of a vitrification procedure in which glycerol and ethylene glycol were used as cryoprotectants (3-step equilibration with cryoprotectants followed by vitrification, dilution of the cryoprotectants in 0.85 M galactose then in embryo transfer freezing medium [ETF], and finally co-culture for periods). To visualize cell membrane alterations, double staining was performed using a cell permeant fluorochrome (bisbenzimide--BIS) and a nonpermeant one (propidium iodide--PI). In Experiment 1, the effect of the vitrification procedure on the hatching rate and total cell number was assessed 72 h after treatment. Hatching rate and the number of stained nuclei were decreased in comparison with untreated embryos when blastocysts were exposed to the whole procedure with or without vitrification (respectively 42 and 53% vs 76% for hatching and 128 +/- 17 and 141 +/- 17 vs 226 +/- 13 for stained nuclei). In Experiment 2, the effect of cryoprotectants and their dilution was evaluated on membrane permeability and total cell numbers at various steps of the vitrification procedure. Blastocysts exposed only to cryoprotectant solutions and stained immediately after dilution in galactose showed no modification. After dilution in ETF, the total number of stained nuclei decreased, and the number of blastomeres showing membrane permeabilization (PI stained) increased (respectively, 74 +/- 5 vs 110 +/- 5 and 32 +/- 2% vs 0.1 +/- 1.8%). In Experiment 3, we demonstrated that the total number of stained nuclei after ethanol fixation (membrane permeabilization) was higher when embryos treated up to dilution in ETF were stained with PI than when the same embryos were stained with BIS. This suggests that, for unknown reasons, some nuclei of the treated embryos were not stained with BIS. Membrane permeabilization and inability of BIS to stain some nuclei were the most obvious alterations probably induced by osmotic shock at dilution. This hypothesis is supported by the fact that the introduction of a further dilution step in 0.42 M galactose (Experiment 4) before dilution in ETF decreased the proportion of cells permeant to PI and increased the hatching rate after 72 h of co-culture. In conclusion, double staining with BIS and PI allowed for discrimination between different types of cellular injuries after the various steps of our vitrification protocol. It represents a useful tool for adjusting equilibration and dilution conditions during a cryopreservation procedure. PMID- 10734386 TI - Production of calves by transfer of nuclei from cultured somatic cells obtained from Japanese black bulls. AB - We investigated the possibility of producing calves from transferable bovine embryos obtained by nuclear transfer using somatic cell-derived cell lines. Muscle cells obtained from 2 Japanese Black bulls were dispersed in Hank's solution supplemented with collagenase Type-I. The separated muscle cells were cultured in Dulbecco's Modified Eagle's medium (D-MEM) supplemented with 10% fetal bovine serum (FBS) at 39 degrees C in an atmosphere of 5% CO2 in air. Cells were passaged at least 4 times, and for 5 d prior to nuclear transfer they (donor cells: karyoplasts) were cultured in D-MEM supplemented with 0.5% FBS (to induce quiescence) or 10% FBS. Recipient oocytes were produced by in vitro culture of bovine oocytes that were obtained at a slaughterhouse and then enucleated in modified phosphate buffered saline supplemented with cytochalasin B. Embryos were reconstructed by 3 protocols using karyoplasts cultured in the medium with 0.5% FBS. 1) Group A: recipient oocytes (cytoplasts; n = 157) were treated with Ca ionophore A 23187, ethanol and cycloheximide, and then a karyoplast was fused to an activated cytoplast. 2) Group B: karyoplasts were transferred to cytoplasts (n = 117), and the couplets were treated with electric stimulation and then Ca ionophore A 23187 and cycloheximide. 3) Group C: cytoplasts (n = 104) were cultured for a further 12 h before fusion, and then the couplets were treated with electric stimulation and cycloheximide. 4) Group D: in addition to the above 3 groups, karyoplasts cultured in the medium with 10% FBS were transferred to recipient cytoplasts (n = 137) and treated as in Protocol 2. Reconstructed embryos were cultured in modified CR1aa for 8 d, and the development of embryos was assessed. In total 73 blastocysts were obtained, and the frequency of development to the blastocyst stage in Group A (2.5%) was lower than that of Groups B, C and D (20.5, 18.3 and 19.0%, respectively; P < 0.01). Of these the sex of 21 blastocysts was determined by rapid Y-chromosome detection assay, and all were male, suggesting that nuclear replacement had been achieved successfully. When 26 blastocysts were transferred to 20 recipient cows, 8 of them became pregnant; 4 cows subsequently aborted about 60 d after embryo transfer while the remaining 4 cows calved. These results indicate that reconstructed embryos obtained by nuclear transfer of muscle cell-derived cell lines can develop to the blastocyst stage, and some are sufficiently competent to develop to term. Particularly important was the finding that special culture protocols for somatic cells prior to nuclear transfer were not necessary in our system. PMID- 10734387 TI - Effect of exogenous glutathione on the in vitro fertilization of bovine oocytes. AB - Increased amounts of reactive oxygen species (ROS) during in vitro culture may cause cytotoxic damage to gametes and embryos. The main purpose of this study was to investigate the effect of glutathione (GSH), a ROS scavenger, supplemented during IVF of bovine oocytes on embryo development using spermatozoa from different bulls. The following experiments were performed: 1) matured COCs were fertilized in the absence or presence of 1 mM GSH using semen from 4 bulls (Bulls A, B, C and D); 2) matured COCs were fertilized in the absence or presence of 1 mM GSH using semen from Bull C to examine sperm penetration, pronuclear formation and apposition; 3) COCs were fertilized with in the presence of either 0, 0.1, 1.0 or 10 mM GSH to examine the effect of GSH concentration using sperm from Bull C; 4) concentrations of GSH were measured both in the medium and in the oocytes during IVF. Glutathione at 1 mM in IVF medium affected the blastocyst formation, but not the cleavage rate. The effect on blastocyst formation was bull dependent: semen from Bull B and D had a negative, that from Bull C a positive and the one from Bull A no effect. The positive effect of Bull C semen increased the rate of blastocyst formation from 20.1 to 27.3% in control and GSH-treated samples, respectively. The increased rate was due to more zygotes reaching the 8-cell or greater stage by Day 4 after IVF. There was no change in the fertilization or cleavage rates. The GSH was still stable after 18 h incubation in IVF medium, and there was a dose-dependent increase in the GSH concentration in the oocytes. It is concluded that the effect of GSH during IVF on the proportion of blastocysts is dependent on both bull and GSH concentration. PMID- 10734388 TI - Establishment of pluripotent cell lines from porcine preimplantation embryos. AB - Embryonic stem (ES) cells are pluripotent cells isolated from in vitro culture of preimplantation embryos. Experiments were undertaken to identify preimplantation embryonic stages and culture conditions under which pluripotent, porcine embryo derived cell lines could be isolated. Cell lines were established from in vitro culture of intact, porcine early hatched blastocysts and isolated inner cell masses (ICM) from intermediate and late hatched blastocysts on feeder layers prepared from permanent mouse embryonic fibroblasts (STO). The cells of these porcine embryo-derived cell lines had a morphology similar to that of murine ES cells, but colony morphology was more epithelial-like. The cell lines retained a normal diploid karyotype, consistently expressed alkaline phosphatase activity, and survived cryopreservation. When subjected to in vitro differentiation, either spontaneous or induced, the embryo-derived cell lines differentiated extensively into a wide range of cell types representing the 3 embryonic germ layers. In vivo pluripotency of the cells was demonstrated by birth of a chimeric piglet, documented by pigmentation and DNA markers, and the ability to direct the development of nuclear-transfer embryos to the blastocyst stage. Such pluripotent embryo-derived cells provide a potential route for porcine genetic manipulation. PMID- 10734389 TI - Induction of pluripotency by injection of mouse trophectoderm cell nuclei into blastocysts following transplantation into enucleated oocytes. AB - Pluripotency of mouse trophectoderm (TE) cells was examined using a nuclear transfer technique. We transferred a TE cell to an enucleated oocyte and cultured the reconstituted oocyte to be blastocyst stage. Then a portion of the inner cell mass (ICM) isolated from the TE-origin blastocyst was injected into the cavity of a fertilized blastocyst to produce a chimeric embryo, which was transferred to a recipient female. Of 319 oocytes reconstituted with TE cells, 263 (82.4%) had a single nucleus (1PN), 3 (0.9%) had 2 nuclei (2PN) and 53 (16.6%) had a nucleus with a polar body (1PN1PB). Although the oocytes with 1PN and 2PN developed to blastocysts (81 of 263, 30.8% and 1 of 3, respectively), only those with 1PN were used to produce chimeric blastocysts. After the transfer of chimeric embryos to recipient females, 7 (28%) of 25 conceptuses analyzed at midgestation showed chimerism. Of those 5 (71%), 6 (86%) and 4 (57%) chimeric conceptuses showed distribution of donor nuclei in the fetus, membrane and placenta, and the distributions were 10 to 65, 10 to 50 and 10 to 15%, respectively. Of the 23 young obtained, 7 (30%; 2 males and 5 females) were coat color chimeras. The contributions of donor nuclei were detected in the brain, lung, heart, liver, kidney, testis, ovary and blood. Each coat-color chimeric mouse was mated with CD 1 male or female mice, but no germ line chimera was obtained. When ICM cells were used as the control nuclear donor, the contribution was equivalent to those of TE cells. In conclusion, pluripotency of mouse TE cells on a somatic line was induced, and chimeric young were obtained using a nuclear transplantation technique. PMID- 10734390 TI - Breeding soundness evaluation of extensively managed bulls in Costa Rica. AB - This paper describes the results of single breeding soundness evaluations (BSE) in 898 Bos indicus, Bos taurus and B. indicus x B. taurus bulls, 1 to 12 yr old, extensively reared in different climatic regions of Costa Rica and representing approximately 2% of the total breeding bull population. Thirty-three percent (n = 296) of the bulls were classified as unsound for breeding owing to clinical problems (9.1%, n = 82), low scrotal circumference (SC) being the most common finding, followed by unsatisfactory sperm morphology (23.9%, n = 214). The prevalence of bulls unsound for breeding was lowest in Bos indicus (29%, P < 0.01), intermediate in B. taurus (41%), and highest in B. indicus x B. taurus (48%). The percentages of abnormal sperm heads, acrosomes and midpieces tended to be higher in the ejaculates of bulls with softer testicular consistency (P < 0.001), a long scrotum (P < 0.01) or a low SC (P < 0.05), and such bulls were more often classified as being unsound for breeding (P < 0.05). Frequencies of sperm abnormalities were higher in bulls < 2 yr of age than in older males (P < 0.01) and were highest in B. indicus x B. taurus bulls (P < 0.001). The results confirm differences between species in their adaptability to a tropical environment and support earlier evidence of an association between SC, testicular consistency and scrotal length clinical parameters, and testicular function in bulls. PMID- 10734391 TI - Characteristics of Neospora caninum-associated abortion storms in diary herds in The Netherlands (1995 to 1997). AB - Abortion storms in 50 dairy herds in The Netherlands were reported in which there was a strong association with Neospora caninum-infection. The duration of the abortion storms ranged from 6 to 65 d (mean 41.5 d). The cumulative proportion of aborting cows ranged from 0.11 to 0.57 (mean 0.26) of the animals at risk. An apparent seasonal influence was noted as most abortion storms occurred during the summer and early autumn. The prevalence of antibodies to N. caninum in 50 herds which had had an abortion storm was compared with that of 100 control herds which had no history of an abortion storm. Seroprevalence was estimated by testing a 20% cross sectional herd sample using a tachyzoite lysate-based ELISA method. Seroprevalence in case herds (range 17 to 87%, mean 51.5%) was significantly higher than that in control herds (range 0 to 53%, mean 13.9%). For most herds the seroprevalence levels were equal across all age groups, which suggests that the infection had been perpetuated by vertical transmission. In these herds, the abortion storms appeared to be induced by factors causing recrudescence of a N. caninum-infection in chronically infected animals rather than being the result of a recent introduction. In 6 case herds the seroprevalence in the dairy cows was significantly higher than in the young stock, which may have been attributable to superimposed postnatal infection. PMID- 10734392 TI - Risk factors for Neospora caninum-associated abortion storms in dairy herds in The Netherlands (1995 to 1997). AB - A 2 to 1 matched case control study design was used to analyze herd level risk factors for Neospora caninum-associated abortion storms in 47 dairy herds. Data were obtained using a questionnaire regarding the state of affairs at the farms over the 2 years prior to the abortion storm. The questionnaire included 120 variables considered to be potential risk factors for either introduction of infection or recrudescence of chronic infection. The relationship between risk factors and case control pairs was analyzed by conditional logistic regression using a three-steps procedure. In addition, cross sectional serology was used to assess the possible role of concomitant infections. The main factors that were significant in the analysis and that were considered to have potential biological relevance were the presence of dogs, the presence of poultry, and the feeding of moldy maize-silage during summer. For both the presence of dogs and the presence of poultry on the farms, a linear relationship was found between the number of animals and the assessed risk for an abortion storm. These findings suggest a possible role of these species in the transmission of N. caninum. Further evidence for such a role of dogs was the significant association between the presence of dogs and the presence of seropositive cattle in the control herds. The feeding of moldy fodder is considered to be a factor which may induce recrudescence of a latent N. caninum-infection by mycotoxins causing immune suppression. We also found some evidence for a possible influence of management practices around calving and a high prevalence of retained afterbirths. No significant association was found for herd level prevalence of antibodies to bovine viral diarrhea virus, bovine herpesvirus 1, Leptospira hardjo or Salmonella dublin. PMID- 10734393 TI - Estrus synchronization and fertility in post-partum dairy cattle after administration of human chorionic gonadotrophin (HCG) and prostaglandin F2 alpha analog. AB - Human chorionic gonadotrophin (hCG) plus PGF2 alpha was compared with GnRH plus PGF2 alpha for estrus synchronization of dairy cows. There were 3 treatments: GnRH analog (Buserelin, 12.6 micrograms) plus PGF2 alpha analog (Cloprostenol, 150 micrograms) 6 d later (GnRH + PGF[Day 6]); hCG (2000 IU) plus PGF2 alpha 9 d later (hCG + PGF[Day 9]); and hCG plus PGF2 alpha 6 d later (hCG + PGF[Day 6]). Treatment occurred either Days 55 to 90 or Days 91 to 135 post partum. For responses during the first 10 d after PGF2 alpha administration, estrus synchronization (P = 0.24), efficacy (percentage of treated pregnant; P = 0.20) and conception (percentage of inseminated pregnant; P = 0.23) rates were not different among the 3 treatments. Cows treated between Days 55 and 90 had a higher rate (P < 0.05) of detected estrus during this period (69% for GnRH + PG [Day 6], 70% for hCG + PGF[Day 9] and 72% for hCG + PGF[Day 6]) compared with cows treated between Days 91 and 135 (52% for GnRH + PGF[Day 6], 50% for hCG + PGF[Day 9] and 57% for hCG + PGF[Day 6]). Efficacy of treatment was higher (P < 0.05) in animals treated between Days 55 and 90 (54% for GnRH + PGF[Day 6], 56% for hCG + PGF[Day 9] and 63% for hCG + PGF [Day 6]) compared to animals treated between Days 91 and 135 (36% for GnRH + PGF[Day 6], 35% for hCG + PGF[Day 9] and 47% for hCG + PGF[Day 6]). There were no significant differences in conception between Days 51 and 90 and Days 91 and 135. The interval between parturition first AI with conception was significantly (P < 0.001) shorter in GnRH + PGF (Day 6; 106 d), hCG + PGF (Day 9; 109 d) and hCG + PGF (Day 6; 103 d) treated cattle than in 106 untreated animals (136 d). Thus, GnRH plus PGF2 alpha or hCG plus PGF2 alpha treatments elicited similar effects in estrus synchronization, treatment efficacy, and conception rate in post-partum dairy cows. PMID- 10734394 TI - A preliminary study on the usefulness of huIL-8 in cervical relaxation of the ewe for artificial insemination and for embryo transfer. AB - The efficacy of using human interleukin 8 (huIL-8) as an agent for inducing cervical relaxation in estrous and diestrous sheep was assessed in a small pilot study. Multiparous, estrus-synchronized ewes were treated for either 2 or 5 consecutive days with vaginal suppositories with or without 5 micrograms cytokine. Cervical penetration with an insemination instrument was then assessed in vivo. After euthanasia, physical, histological and enzymological properties of the cervix were examined. Treatment of diestrous sheep with huIL-8 did not result in recruitment of neutrophils into the cervix. Treatment of estrous sheep with huIL-8 usually led to neutrophil recruitment to the cervix and to either full or partial penetration of the cervix. However, some animals receiving placebo treatment had neutrophil infiltration of both the vagina and cervix and, in one of these, partial penetration of the cervix was also achieved. Thus, treatment with IL-8 as the sole agent in the vaginal suppository was not sufficient to relax the cervix of the nonpregnant ewe in this study. PMID- 10734395 TI - Reactive oxygen species requirements for bovine sperm capacitation and acrosome reaction. AB - Sperm capacitation is necessary for the fertilization of oocytes. During capacitation intracellular and membrane changes occur, that culminate with an exocytotic event called the acrosome reaction. The aim of this work was to study the participation of the superoxide anion (O2-.) and of hydrogen peroxide (H2O2) in the capacitation process and acrosome reaction in spermatozoa from cryopreserved bovine semen. Samples were capacitated with heparin or treated with the xanthine-xanthine oxidase-catalase system (X-XO-C) for the production of O2-. The percentage of capacitated spermatozoa was determined using the chlortetracycline (CTC) technique, by means of epifluorescence microscopy. Addition of X-XO-C to the incubation medium significantly induced capacitation (P < 0.05), but there were no differences with samples incubated with heparin. When the medium contained heparin or the X-XO-C, addition of superoxide dismutase (SOD, 0.5 mg/mL) significantly inhibited capacitation (P < 0.05). In samples treated with heparin and with diverse concentrations of H2O2 (10, 25, 50 and 250 microM) in the incubation medium, the percentage of capacitated spermatozoa was significantly reduced (P < 0.05); however, acrosome reaction was produced at concentrations of 10 and 25 microM H2O2. At concentrations greater than 25 microM H2O2 a deleterious effect was observed on sperm motility. From these results it may be inferred that O2-. is required in the capacitation process and that H2O2 may participate as an inductor of the acrosome reaction in spermatozoa from cryopreserved bovine semen. PMID- 10734396 TI - The influence of enucleation on the ultrastructure of in vitro matured and enucleated cattle oocytes. AB - The enucleation of recipient oocytes in nuclear transfer experiments is generally carried out by aspirating one third of the ooplasm adjacent to the first polar body. It was supposed that this enucleation step affects the ultrastructure of the remaining cytoplast, resulting in a decline or destruction of its cellular compartments. Even if the transferred nucleus had the potential to support the development of a single-cell nucleus transfer embryo to the blastocyst stage, meiotic division could be stopped at any stage if the destruction of the ultrastructure of host cytoplasm resulted in a limited metabolism. The present study was conducted to investigate the influence of the enucleation procedure on the ultrastructure of the remaining ooplast. In vitro matured oocytes; in vitro matured and enucleated oocytes; and in vitro matured and enucleated oocytes that were subsequently cultivated in vitro for additional 4 h were prepared for transmission electron microscopy (TEM). An examination of ultra-thin sections showed that the arrangement of organelles in all matured oocytes was in accordance with that already described for normal oocyte development. Immediately after enucleation no major differences in the arrangement of cortical granules, mitochondria, smooth endoplasmic reticulum (SER), lipid droplets and vacuoles were found compared with nonmanipulated oocytes. After enucleation and 4 h of culture, 24- and 36-h matured oocytes differed from each other in the arrangement of large aggregates of SER surrounded by a wall of mitochondria and lipid droplets. These complexes were still found in the 24-h but not in 36-h matured, enucleated and cultivated oocytes. Clusters of SER, mitochondria and lipid droplets were described by different authors as having metabolic activity. The results of this study in connection with results from nuclear transfer experiments suggest that these aggregates and their metabolic activity can be transferred with cytoplasm from 24- but not 36-h matured oocytes. Only cytoplasm from the 24-h matured oocytes showed a development-supporting effect when fused to enucleated recipient cells before nuclear transfer. PMID- 10734397 TI - Hormonal control of estrous cyclicity and attempted superovulation in wood bison (Bison bison athabascae). AB - The wood bison (Bison bison athabascae) is a threatened Canadian species that has faced extinction twice in the last 100 yr. Development of assisted reproductive technologies could help ensure the long-term propagation and genetic management of this species. The objectives of this study were to refine estrus synchronization techniques and evaluate superovulatory responses after FSH or eCG administration. In Experiment 1, females were fitted with Syncro-mate B (SMB) implants for 9 d and received an injection of either estradiol valerate (E2V; n = 9) or cloprostenol (PGF; n = 9) at implant insertion (Day-9). In Experiment 2, estrus was synchronized with SMB implants and a PGF injection of Day-9, and superovulation was attempted on Day-2 with either 2500 IU eCG (n = 5) or 400 mg Folltropin-V (n = 5). In each experiment, biosin were examined daily for estrual behavior. Ultrasonography was used during the luteal phase to detect ovulation and assess ovarian status; feces were analyzed by ELISA for immunoreactive progestogens (P) to study ovarian endocrine responses. In Experiment 1, a closer synchrony of estrus was observed between Days 2 to 4 among the PGF-treated (77.8%) than the E2V-treated (66.7%) females. Corpora lutea (CL) were detected in 55% of E2V- and PGF-treated females. In Experiment 2, neither treatment successfully induced superovulation, with only a single female per treatment producing > or = 1 CL. In both experiments, progestogen profiles were similar for each treatment (P < 0.05). PMID- 10734398 TI - Changes in lipid content of fowl spermatozoa after liquid storage at 2 to 5 degrees C. AB - Quantitative and qualitative changes may occur in the lipids of spermatozoa during in vitro storage of gametes and may indicate possible degradations occurring within the cells under these conditions. The aim of the present study was to investigate such changes. The motility, viability, morphological integrity and lipid content were measured in fowl semen stored for 48 h at 2 to 5 degrees C and diluted 1:1 in Beltsville Poultry Semen Extender (BPSE) under aerobic agitation. The total lipid content of spermatozoa decreased (P < 0.05) from 820 to 620 micrograms/10(9) cells over 48 h. There was no significant evolution in the total lipid content of seminal plasma (1000 to 850 micrograms/10(9) cells). The proportion of phospholipids in spermatozoa decreased from 75 to 60% of the total lipids. Among the phospholipids, the proportions of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin decreased (P < 0.05) from 58, 13 and 10% at 0 h to 42, 10 and 9% at 48 h, respectively. In contrast, lysophosphatidylcholine, which was marginally represented at 0 h (2%), increased considerably (24%) at 48 h. During the same period, the proportion of motile spermatozoa decreased from 87.5 to 46% and the proportion of viable and morphologically normal cells decreased from 84 to 48%. These results indicate the occurrence of lipid lysis, peroxidation and/or endogenous metabolism able to modify the structure of the spermatozoal membranes and to alter their metabolism and fusion abilities. PMID- 10734400 TI - The ovine uterus as a host for in vitro-produced bovine embryos. AB - A series of experiments were conducted to determine whether bovine blastocysts would develop beyond the blastocyst stage in the ovine uterine environment. In Experiment 1, in vitro matured, fertilized and cultured (IVM/IVF/IVC) expanded bovine blastocysts were transferred into uteri of ewes on Day 7 or 9 of the estrous cycle and collected on Day 14 or 15 to determine if the bovine blastocysts would elongate and form an embryonic disk. Springtime trials with ewes that were synchronized with a medroxyprogesterone acetate (MAP) sponge resulted in a 78% blastocyst recovery rate, and 68% of the recovered spherical or elongated embryos had embryonic disks. In Experiment 2, transfer of 4-cell bovine embryos to the oviducts of ewes at Day 3 resulted in a lower recovery (47 vs 80%) than the transfer of blastocysts at Day 7 when embryos were recovered at Day 14. However, the percentage of embryos containing embryonic disks was higher for embryos transferred at the 4-cell stage (71%) than for embryos transferred as blastocysts (50%). In Experiment 3, IVF embryos from super-ovulated cows or Day 8 in vitro produced embryos transferred to cows were collected at Day 14 and were found to be similar in size to those produced by transfer to ewes in Experiment 2. In Experiment 4, the transfer of bovine blastocysts to ewes did not prolong the ovine estrous cycle. In Experiment 5, extension of the ovine estrous cycle by administration of a MAP releasing intravaginal device allowed bovine embryos to elongate extensively and to become filamentous. In Experiment 6, uterine flushings on Day 14 or Day 16 contained elevated levels of interferon-tau when bovine blastocyst were transferred on Day 7. Transfer of bovine embryos to the reproductive tract of a ewe allows some embryos to develop normally to advanced perimplantation stages and may be a useful tool for studying critical stages of embryo development and the developmental capacity of experimental embryos. PMID- 10734399 TI - Improved in vitro embryo development using in vivo matured oocytes from heifers superovulated with a controlled preovulatory LH surge. AB - In bovine in vitro embryo production, the IVM step is rather successful with 80% of the oocytes reaching the MII stage. However, the extent to which the process limits the yield of viable embryos is still largely unknown. Therefore, we compared embryonic developmental capacity during IVC of IVF oocytes which had been matured in vitro with those matured in vivo. In vitro maturation was carried out for 22 h using oocytes (n = 417) obtained from 2- to 8-mm follicles of ovaries collected from a slaughterhouse in M199 with 10% fetal calf serum (FCS), 0.01 IU/mL LH, and 0.01 IU/mL FSH. In vivo matured oocytes (n = 219) were aspirated from preovulatory follicles in eCG/PG/anti-eCG-superovulated heifers 22 h after a fixed time GnRH-induced LH surge; endogenous release of the LH surge was suppressed by a Norgestomet ear implant. This system allowed for the synchronization of the in vitro and in vivo maturation processes and thus for simultaneous IVF of both groups of oocytes. The in vitro developmental potential of in vivo matured oocytes was twice as high (P < 0.01) as that of in vitro matured oocytes, with blastocyst formation and hatching rates 11 d after IVC of 49.3 +/- 6.1 (SEM; n = 10 heifers) vs 26.4 +/- 1.0% (n = 2 replicates), and 39.1 +/- 5.1% vs 20.6 +/- 1.4%, respectively. It is concluded that IVM is a major factor limiting in the in vitro production of viable embryos, although factors such as the lack of normal preovulatory development of IVM oocytes contributed to the observed differences. PMID- 10734401 TI - Factors affecting pregnancy rate following nonsurgical embryo transfer in buffalo (Bubalus bubalis): a retrospective study. AB - The objectives of this study were to determine the pregnancy rate and factors affecting it following nonsurgical embryo transfer in buffalo. Donor buffalo were superovulated with FSH, and embryos collected nonsurgically were evaluated for stage of development and quality. They were transferred nonsurgically to 91 recipients on Days 5 to 7 of the natural (n = 52) or induced (n = 39) estrus (estrus = Day 0). The overall pregnancy rate of 24/91(26.4%) was higher than in earlier reports for buffalo but was much lower than in cattle. Pregnancy rates were not affected by season (autumn vs winter), side of transfer (right vs left uterine horn), or type of estrus (spontaneous vs induced). The pregnancy rate was high 11/27(40.7%) when donors and recipients were closely synchronized, while it was compromised when recipients were in estrus at +12 h (1/7, 14.3%) and at -12 h (5/27, 18.5%). Asynchrony beyond 12 h on either side resulted into conception failure. The pregnancy rate tended to increase with the increase in CL size of recipients, while stage of embryonic development had no effect. The transfer of an 8-cell embryo with a 16-cell embryo led to the birth of heterosexual twins, indicating that the uterine milieu of Day 5 to 6 recipients may be tolerated by the out-of-phase 8-cell embryo, at least in the presence of a more mature embryo. Embryo quality had the greatest effect on pregnancy rate as it was higher (P < 0.005) after the transfer of Grade I than Grade III embryos (6/10, 60.0% vs 3/36, 13.9%). Assessment of returns to estrus indicated that among nonpregnant recipients, 17/67 (25.4%) embryos never matured sufficiently to prevent luteolysis through maternal recognition of pregnancy (MRP), while 14/67 (20.8%) embryos probably died following MRP. These results indicate that efforts to increase pregnancy rate following embryo transfer in buffalo should include prevention of luteolysis during the first week of transfer and a reduction in the incidence of embryonic mortality. PMID- 10734402 TI - Comparison of fertility data from vaginal vs intrauterine insemination of frozen thawed dog semen: a retrospective study. AB - Fertility data from 327 artificial inseminations (AIs) using frozen-thawed dog semen are presented here. The AIs were performed in 274 bitches using semen from 185 males of 76 breeds. The data cover all AIs conducted during 1983 through 1995 at Cryogenetic Laboratories (CLONE) in the United States with AKC-registered and research bitches, and all AIs carried out at the Department of Obstetrics and Gynecology at the Swedish University of Agricultural Sciences in Uppsala, Sweden, using semen frozen by CLONE, in 0.5-mL straws. Semen was frozen using a standardized, three-step liquid nitrogen vapor freezing method. Whelping rates > 70% were obtained when post-thaw motility was 40% or higher. The inseminations were made either directly into the uterus using transcervical catheterization with the Norwegian catheter (NIU; 167 AIs) or a fiberoptic endoscope (EIU; 19 AIs), or in the cranial vagina (VAG; 141 AIs). Resulting whelping rates were 84.4% (NIU), 58.9% (VAG; P < 0.001), and 57.9% (EIU). Increasing the number of VAG AIs per cycle from 1 to 2 enhanced the whelping rate (P < 0.05). The mean interval from the first AI to whelping was 61.8 +/- 2.4 d, and was longer for VAG AIs (62.7 +/- 2.7 d) than for NIU AIs (61.2 +/- 2.1 d; P < 0.001). The mean interval from the last AI was 60.1 +/- 1.9 d, and did not differ between VAG AIs (60.2 +/- 2.2 d) and NIU AIs (60.0 +/- 1.6 d). Gestation length was not influenced by breed or litter size. A total of 1158 pups resulted from the 327 AIs. Litter size was 5.4 +/- 3.0 (NIU), 4.0 +/- 2.7 (VAG; P < 0.001), and 6.0 +/- 2.1 (EIU). Litter size was also influenced by breed (P = 0.006) and, for VAG AIs, by the number of inseminations performed per cycle (P = 0.009). This study is the largest that has been carried out on frozen-thawed dog semen AI. It shows that using a good method for cryopreservation, together with nonsurgical intrauterine AI employing the Norwegian catheter, can yield whelping rates and litter sizes similar to those reported from well-controlled natural matings. Furthermore, this is the first study to show that intrauterine deposition of frozen-thawed dog semen results in a significantly higher whelping rate and larger litter size than vaginal deposition. PMID- 10734403 TI - Evaluation of four mathematical functions to describe scrotal circumference maturation in Nellore bulls. AB - Four functions to characterize scrotal circumference (SC) growth in Nellore bulls were compared to identify which was the most suitable for biological interpretation. Nellore bulls (n = 532), born between September and December of 1992 to 1994 were used in the study. Measurements were made on fixed dates in January, April, July and October of each year. At the time of SC measurements, the ages of the bulls ranged from 200 to 1300 d. The functions used to describe the data were: Brody, SC = A (1 - B exp -kt); Logistic, SC = A/(1 + B exp -kt); Gompertz, SC = A exp(-B exp -kt) and Richards SC = A (1 + B exp -kt)m, where SC is the scrotal circumference at t days of age, A is the estimated SC at maturity, B is the integration constant established by the initial values of SC and t, k is the maturity constant, which equals the ratio between the maximum rate of growth and SC at maturity; m is the inflection point parameter for Richards function, which did not converge. The Brody, Gompertz and Logistic functions fitted the data in a similar fashion, with similar values for the statistics EMS and R2, and they reached convergence with similar computational costs. The Logistic function presented the best pattern of average prediction error, and therefore, it was selected for biological interpretation. For the Logistic function, estimated SC at maturity (A) was 37.95 cm at 72 mo of age. The maturity index (k) was .11 cm, and the inflection point (time of maximum growth) was reached at 13.09 mo of age at an average SC of 18.97 cm. PMID- 10734404 TI - Sexual dimorphism in IVM-IVF bovine embryos produced from X and Y chromosome bearing spermatozoa sorted by high speed flow cytometry. AB - The objective of this study was to examine preimplantation development and sperm aster characteristics of bovine male and female embryos produced by using spermatozoa sorted for the X or Y chromosome. In vitro matured oocytes were inseminated at 24 h of maturation with sorted X or Y chromosome-bearing spermatozoa, using either fresh or frozen-thawed semen. Samples were taken from each sperm group 12 h post insemination (hpi), fixed, and immunostained for the microtubule cytoskeleton. Confocal microscopy enabled visualization of sperm aster formation and microtubule characteristics of each zygote during early fertilization. Cultured embryos were checked for cleavage at 30, 35, 40 and 45 hpi, embryo development was examined daily until Day 8 of culture. Blastocyst cell numbers were determined at the end of the experiments. Reanalysis of the sorted sperm cells for DNA content showed purity rates of 90.1 and 92.1% for X and Y chromosome-bearing spermatozoa, respectively. Reduced fertilization and development rates were observed when sorted spermatozoa were used compared with fresh and frozen-thawed spermatozoa. Penetration rates at 12 hpi were 39.5, 44.7, 55.9 and 79.0%, while blastocyst formation rates at Day 8 were 26.7, 26.5, 31.7 and 40.7% for X and Y chromosome-bearing spermatozoa, using fresh and frozen thawed semen groups, respectively. Sperm aster size was larger in males than females, while the size of pronuclei and subjective grade of sperm aster quality showed no differences between sexes. In this study, a greater cleavage rate and sperm aster size in male embryos indicated a dimorphic pattern of development in male and female embryos during fertilization and first cleavage. PMID- 10734405 TI - Active immunization against follistatin and its effect on FSH, follicle development and superovulation in heifers. AB - Ovaries of heifers were examined daily by transrectal ultrasonography for one interovulatory interval before initiation of immunizations (control cycle, n = 14), and again after the fifth immunization with a sham-vaccine (Freund's adjuvant only; n = 7) or a recombinant porcine follistatin-vaccine (1 mg per vaccination; n = 7) to study the effect of follistatin on follicle dynamics. After the fifth immunization, 4 heifers had a follistatin antibody titer of > or = 1:3200, while the remaining 3 heifers had a titer of only 1:400. At wave emergence, the total number of follicles and the number of small follicles (3 to 5 mm) were higher (P < 0.05) in the follistatin group than in the control and sham groups. In addition, high-titer heifers had a greater (P < 0.05) number of follicles (total and small) per day than low-titer heifers. Plasma concentration of FSH remained unchanged after sham- or follistatin-immunization. Sham- and follistatin-vaccinated heifers were then given half the standard superovulatory dose of Folltropin (200 mg of FSH) 14 d after the sixth immunization. More ovulations were detected in follistatin- (10.9 +/- 2.4) than sham- (5.0 +/- 0.8) vaccinated heifers (P < 0.05). Moreover, heifers with a high titer had more ovulations (P < 0.02) than heifers with a low titer (15.0 +/- 2.5 vs 5.3 +/- 1.2). The number of ova-embryos classified as fertilized:unfertilized and transferable:discarded, and quality of the embryos were similar between sham and follistatin groups. By 80 d after the last immunization, when antibody titers were undetectable in the follistatin group, there was no difference in superovulatory response between sham (6.7 +/- 1.6) and follistatin (7.6 +/- 1.6) groups. In summary, follistatin immunization was associated with an increase in the number of small follicles at the time of wave emergence and a greater response to superovulatory treatment. The results suggest that effects of follistatin on follicular dynamics were not mediated through changes in pituitary secretion of FSH. PMID- 10734406 TI - Effects of season of birth on the prepubertal pattern of gonadotropin secretion and age at puberty in beef heifers. AB - There is an early transient rise in gonadotropin secretion in spring-born prepubertal heifers and there is an indication that this pattern is different in autumn-born heifers. The effect of season of birth on age and weight at puberty is equivocal. This study was designed to compare the temporal patterns of LH and FSH secretion between spring- and autumn-born heifers and to determine the effects of season of birth on age and weight at puberty. Blood samples from 2 groups of heifer calves born in spring (last week of March, n = 5) or autumn (last week of October, n = 5) were collected every other week from birth to puberty and every 15 min for 10 h at 6, 12, 18, 24 and 32 wk of age. Timing of puberty was determined by measuring progesterone in plasma samples collected every 2 to 3 d starting at 42 wk of age. Age and weight at onset of puberty did not differ between the 2 groups of heifers (P > 0.05); however, the autumn-born heifers tended to mature in a wider range of ages and weights. Based on the 10-h sampling periods, mean serum concentrations of LH and LH pulse frequency and amplitude were higher in spring-born heifers at 18 wk of age than in autumn-born heifers (P < 0.05). In spring-born heifers, LH pulse frequency increased over time to 32 wk of age, and LH pulse amplitude was higher at 12 and 18 wk than at 32 wk of age (P < 0.05). Autumn-born heifers had higher LH pulse frequency at 6 wk and showed a decrease in mean concentrations of LH at 12 and 18 wk of age (P < 0.05). The FSH pulse frequency of spring-born heifers was higher at 12 wk of age than in autumn-born heifers (P < 0.05), FSH pulse amplitude in autumn-born heifers decreased from 6 to 32 wk of age. It was concluded that although the mean age and weight at puberty did not differ between spring- and autumn-born heifers, the range in age and weight at puberty was wider in the autumn-born heifers. The patterns of LH secretion differed between spring- and autumn-born prepubertal heifers, with spring-born calves exhibiting an early rise in LH secretion, while mean serum concentrations of LH decreased during this period in autumn-born heifers. PMID- 10734407 TI - Development to live young from bovine small oocytes after growth, maturation and fertilization in vitro. AB - Bovine oocytes (90 to 99 microns in diameter) were isolated from early antral follicles (0.5 to 0.7 mm in diameter). Cumulus-oocyte complexes (COC) with pieces of parietal granulosa were embedded in collagen gels and cultured for 14 d. After in vitro growth culture, oocytes recovered from the collagen gels were further matured, fertilized and cultured in vitro, and then were transferred to recipient cows. After 14 d of growth culture, 37% of the oocytes (203/556) showed normal morphology in the collagen gels. The mean diameter of the oocytes was 110.1 +/- 6.0 microns, significantly larger (P < 0.01) than before growth culture (94.8 +/- 2.7 microns), and 77% were at the germinal vesicle stage while 23% had undergone germinal vesicle breakdown. After 24 h of maturation culture followed by insemination, 27% of in vitro-grown oocytes reached the second metaphase, and 42% of the oocytes were normally fertilized. After insemination, 18.2% of in vitro grown oocytes cleaved and 3.7% developed to the blastocyst stage. Three blastocysts obtained from in vitro-produced 90- to 99-micron oocytes were transferred to 3 recipients. One recipient subsequently became pregnant and delivered a live calf on Day 277. These results demonstrated for the first time that 90 to 99-micron oocytes from early antral follicles can complete growth and acquire full developmental competence in vitro so that live young can be produced after maturation, fertilization, subsequent culture in vitro, and transfer to recipient cows. PMID- 10734408 TI - Influence of noradrenaline on progesterone synthesis and post-translational processing of oxytocin synthesis in the bovine corpus luteum. AB - Noradrenaline (NA) influences secretory function of the bovine corpus luteum (CL), stimulating secretion of progesterone and ovarian oxytocin (OT). To study whether NA is able to stimulate progesterone synthesis and to affect post translational OT processing, different doses of NA alone or in combination with different doses of OT were added to bovine CL slices from 8 to 13 d of the estrous cycle. To determine which receptors NA affects, and if dopamine (DA) also affects CL function, we used NA or DA combined with a beta-antagonist (propranolol). The results indicated that NA stimulates both luteal progesterone and OT content; furthermore, it increased the activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and peptidyl glycine-alpha-amidating mono-oxygenase (PGA), terminal enzymes in synthesis of these 2 hormones. The stimulating effect of NA was inhibited by propranolol and by pre-treatment of CL slices with high OT doses. Post-translational processing of OT synthesis by PGA activation was also stimulated by DA, but this effect was inhibited by beta-receptor blocker. Thus DA acts in CL as a NA precursor. In conclusion, it can be assumed that the noradrenergic system affects CL secretory function on different levels of regulation. Furthermore, a high concentration of OT in CL prevents NA from activating PGA and thus decreases post-translational OT synthesis. PMID- 10734409 TI - Vitrification of mouse embryos in two cryoprotectant solutions. AB - The objective of this study was to compare the efficiency of 2 media on the vitrification of mouse compacted morulae, early blastocysts and expanded blastocysts after equilibration at room temperature of 4 degrees C. Embryos were equilibrated for 10 min in either 25% VS3 (Rall Equilibration Medium, REM) or 10% glycerol + 20% propylene glycol (Massip Equilibration Medium, MEM) in DPBS at 20 degrees C or 4 degrees C. For vitrification either 100% VS3 (Rall Vitrification Medium, RVM) or 25% glycerol + 25% propylene glycol (Massip Vitrification Medium, MVM) in DPBS was used. Embryos equilibrated at room temperature were loaded in 20 microL of vitrification media into 250 microL straws and then immediately (30 sec) plunged into liquid nitrogen (LN2). After equilibration at 4 degrees C the embryos were put into straws with 20 microL of precooled vitrification medium, and after 20 min at 4 degrees C they were plunged into LN2. Embryos from both groups were thawed in a 20 degrees C water bath for 20 sec, transferred to 1.0 M sucrose in DPBS for 5 min and then cultured for 24 to 48 h in Whitten's medium at 37 degrees C in 5% CO2 in air. In the groups of embryos prepared for vitrification at room temperature the survival rate of compact morulae vitrified in RVM was higher than those vitrified in MVM (65/70, 93% vs 49/74, 66%; P < 0.01). No difference was found in the survival rate of early blastocysts and expanded blastocysts vitrified in RVM or MVM (30/83, 36% vs 25/75, 33% and 4/66, 6% vs 4/76, 5%). No difference was found between the survival rate of compact morulae after equilibration with RVM or MVM at 4 degrees C (62/75, 83% vs 52/74, 70%). Both the early blastocysts and expanded blastocysts equilibrated at 4 degrees C MVM yielded a higher survival rate than RVM (28/74, 38% and 40/70, 57% vs 4/75, 5% and 4/77, 5%; P < 0.01). We conclude that, of the 3 developmental stages, compact morulae withstand the vitrification process best, and reduction of the temperature prior to plunging into LN2 is not required. A 10-fold increase in the survival rate of expanded blastocysts can be achieved using low temperature equilibration (4 degrees C) and MVM. PMID- 10734410 TI - An ultrasonographic study of luteal function in breeds of sheep with different ovulation rates. AB - Development and demise of luteal structures were monitored using daily transrectal ultrasonography in 2 breeds of sheep differing in ovulation rates (nonprolific Western white-faced cross-bred, n = 12 and prolific pure-bred Finn sheep, n = 7), during 1 estrous cycle in the mid-breeding season. Jugular blood samples were collected once a day for radioimmunoassay (RIA) of progesterone. The mean diameter of ovulatory follicles was higher in Western white-faced than in Finn ewes (6.4 +/- 0.2 and 5.3 +/- 0.2 mm, respectively; P < 0.001). The mean volume of luteal structures was higher (P < 0.05) in Western white-faced compared with Finn sheep from Days 5 to 15 of the cycle (Day 0 = day of ovulation). This accounted for the higher (P < 0.05) total luteal volumes recorded in Western white-faced ewes on Day 7 and from Days 11 to 15, despite the higher ovulation rate in Finn ewes (2.7 +/- 0.3 and 1.7 +/- 0.2, respectively; P < 0.05). Mean serum progesterone concentrations were higher (P < 0.05) in Western white-faced than in Finn ewes from Days 4 to 14. Daily total luteal volumes were positively correlated with daily serum progesterone concentrations throughout the cycle in Finn sheep (r > or = 0.40, P < 0.02), and during luteal growth and regression (r > 0.60, P < or = 0.00001) but not during mid-cycle in white-faced ewes (r = 0.16; P = 0.22). During the growth of the corpora lutea (CL), luteal tissue volume increased faster (P < 0.05) than serum progesterone concentrations in both breeds of sheep. During luteolysis, the decrease in luteal volumes parallelled that in serum progesterone concentrations in Finn (P = 0.11) but not in Western white faced ewes, where luteal volumes decreased more slowly (P = 0.02) in relation to progesterone secretion. Increased ovulation rate in prolific Finn ewes resulted in more but smaller CL, and lower serum progesterone levels compared with nonprolific Western white-faced ewes. We conclude that breed-specific mechanisms exist to control the formation of luteal tissue and progesterone secretion in cyclic ewes differing in prolificacy. The mechanisms may involve ovulation of Graafian follicles at different sizes and inhibitory paracrine effects of CL on co-existing CL. PMID- 10734411 TI - Ultrasound-guided follicle aspiration: effect of the frequency of a linear transvaginal probe on the collection of bovine oocytes. AB - The effect of the frequency of an ultrasonic linear transvaginal probe on the collection of bovine oocytes by transvaginal ultrasound-guided follicle aspiration was investigated. Probes with different frequencies (7.5 or 5.0 MHz) were applied to examine the clarity of follicles on the monitor using ovaries of slaughtered cows in Experiment 1. The follicles were visualized on the monitor and divided into small (3- to 5-mm diameter) and large (6- to 10-mm) groups. They were also divided into 2 groups according to the clarity of their outline (clear or obscure). The number of small follicles visualized with a clear outline was greater (P < 0.01) with the 7.5 MHz probe than with the 5.0 MHz probe (9.0 vs 3.2). Oocyte aspiration from live cows was performed using the 7.5 or 5.0 MHz probe in Experiments 2 and 3. The recovered oocytes were divided into 3 categories: cumulus-oocyte-complexes (COCs), denuded oocytes and all others. In Experiment 2, the number of oocytes collected per donor cow was assessed, and in Experiment 3 the number of oocytes per aspirated follicle was examined by aspirating a constant number of follicles per aspiration session. The numbers of oocytes and COCs per donor cow obtained with the 7.5 MHz probe (11.2 and 9.0, respectively) were greater (P < 0.01) than those obtained with the 5.0 MHz probe (4.3 and 3.5). This difference between probes was due to the greater clarity of the follicle images obtained with the 7.5 MHz probe. PMID- 10734412 TI - Uterine luminal proteins and estrogens in gilts on a normal nutritional plane during the estrous cycle and on a normal or high nutritional plane during early pregnancy. PMID- 10734413 TI - Effects of hormones and osmolarity in the culture medium on germinal vesicle breakdown of porcine oocytes. AB - The present study was conducted to examine effects of hormones and osmolarity on germinal vesicle breakdown (GVBD) and histone H1 kinase (H1K) activity in porcine oocytes cultured in vitro. The basic medium used for culture of oocytes was modified Tyrode's solution in which the osmolarity was adjusted to 134 to 495 mOsm by changing the concentration of sodium chloride (NaCl). When the hormones were present, osmolarity of medium that allows GVBD of oocytes was less than 400 mOsm. However, the range of osmolarity of medium that allows meiotic maturation of oocytes was 210 to 362 mOsm. On the other hand, without hormonal supplement, the incidence of GVBD in oocytes decreased as the osmolarity of the medium increased in the rage of 210 to 362 mOsm. By increasing the osmolarity of the medium from 210 to 362 mOsm by addition with sorbitol instead of NaCl, the incidence decreased from 89.1% to 13.3%. In oocytes cultured in medium of 210 mOsm without hormones, the percentage of oocytes that underwent GVBD and had increased H1K activity 20 h after culture was significantly higher (P < 0.05) than those of oocytes cultured in the same medium supplemented with hormones or medium of 362 mOsm. These results indicate that in vitro induction of GVBD in porcine oocytes is strongly affected by osmolarity of the medium in the absence of hormones. The results also suggest that, under low osmolarity (210 mOsm), GVBD is accelerated with rapid increase of H1K activity. PMID- 10734414 TI - Effect of cryopreservation of zona-binding capacity of canine spermatozoa in vitro. AB - The hemizona assay (HZA) was used as a functional test for zona pellucida binding capacity of fresh and frozen-thawed canine spermatozoa. We investigated 30 ejaculates from 3 dogs with sperm motility > 70% and sperm concentration > 5.10(8) cells per ejaculate with up to 20% abnormal and dead spermatozoa. Fifteen ejaculates were each divided into 2 portions: one portion was used for analysis of fresh semen, the other for cryopreserved semen. On the day of the experiments, in vitro-matured canine oocytes were bisected into 2 equal hemizonae. One half of the hemizonae were coincubated with fresh capacitated (control) spermatozoa, the other half of the hemizonae were coincubated with frozen-thawed (tested) spermatozoa at final concentration of 1 to 2 x 10(6) cells/mL in 200 microL droplets of BSA-supplemented Toyoda, Yokojama and Hoshi (TYH) medium at 37 degrees C, 5%, CO2 for 1 h. Sperm suspensions were examined kinesigraphically for post capacitation type of movement. The Student's t-test was used to compare differences between semen parameters. The data on HZA binding activity of fresh and frozen-thawed canine semen were analyzed by ANOVA and then by the Newman Keuls multiple range method. The results showed no differences in the initial semen quality parameters among the 3 dogs. After thawing, the semen from Dog 1 and Dog 2 demonstrated relatively uniform sperm parameters, while in Dog 3 sperm motility, and viability and the percentage of morphologically normal spermatozoa were significantly decreased. The binding activity of frozen-thawed spermatozoa from the 3 dogs was significantly reduced (29.40 +/- 9.02, 18.60 +/- 3.30, 8.20 +/- 4.49) compared with that (107.20 +/- 19.22, 109.80 +/- 20.75, 78.20 +/- 12.47; P < 0.01) of fresh spermatozoa. The results showed that semen samples with similar sperm parameters prior to cryopreservation displayed different sperm zona binding capacity after freezing. The HZI (value of sperm binding capacity of frozen-thawed vs fresh semen samples) was higher in Dog 1 (27.43) than in Dog 2 (16.90) or Dog 3 (10.40), and thus confirmed the variation of zona binding activity after thawing between dogs. The freezability of individual dog semen is discussed. In conclusion HZA may be a valuable tool for evaluating the post-thaw fertilizing ability of canine spermatozoa. PMID- 10734415 TI - A potential relationship between the 16;20 and 14;20 Robertsonian translocations and low in vitro embryo development. AB - A possible effect of 16;20 and 14;20 Robertsonian translocations on the development of bovine oocytes matured and fertilized in vitro was assessed on the basis of embryo yield and blastocyst formation. Oocytes fertilized with semen from 2 bulls (A and B), which were heterozygous for these translocations, showed a significantly lower cleavage rate (36.3 +/- 2.25%; 39.8 +/- 3.88%) and percentage of blastocysts (3.7 +/- 1.24%; 3.2 +/- 1.20%) than those fertilized with semen from a bull (C) with a normal karyotype (control, 58.1 +/- 2.14%; 20.1 +/- 1.92%; P < 0.01). There was also a difference in the rate of further blastocyst development between the tested bulls and the control. The rates of expanded blastocysts were 6.6 and 11.1% for Bulls A and B, respectively, and 38.7% for the control bull on Day 7; while on Day 8 these values were 41.7 and 55.5% vs 76.0%. These results demonstrated that in the bulls carrying the 16;20 and 14;20 translocations, in vitro preimplantation embryo development was reduced, probably due to genetically unbalanced spermatozoa. PMID- 10734416 TI - Effects of glutamine, proline, histidine and betaine on post-thaw motility of stallion spermatozoa. AB - The supplementation of the freezing diluent with 3 amino acids (glutamine, proline and histidine) and 1 amino acid-related compound (betaine) in preserving stallion spermatozoa diluted in INRA82 extender containing 2.5% (v/v) glycerol and 2% (v/v) egg yolk (control extender) during freezing and thawing was studied at 0, 40, 80, 120 and 160 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 1). Glutamine and proline were studied at 0, 10, 20, 30, 40, 50, 60, 70 and 80 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 2). In each experiment, spermatozoa were evaluated after thawing by computer automated sperm analyzer. The percentage of motile spermatozoa (faster than 30 microns/sec) was assessed. In addition, the velocity of the average path (VAP), the straight line velocity (VSL), the curvilinear velocity (VCL) and the amplitude of the lateral head displacement (ALH) were also measured. In Experiment 1, only glutamine (40 mM) significantly improved sperm motility (56.0% +/- 3.0 vs 49.7% +/- 1.6; P < 0.05) compared with the control extender, while velocities were unaffected at concentrations of 40 to 120 mM. However, at 160 mM, a significant decrease in motility and velocity was observed for all amino acids. In Experiment 2, motility in glutamine (range 41.1% +/- 3.8%; 42.4% +/- 3.6) and proline (43.0% +/- 3.7; 45.6% +/- 3.8) extenders compared with the control (34.7% +/- 1.6) was improved significantly (P < 0.05). Sperm velocity was improved at concentrations higher than 40 mM glutamine and 50 mM proline. PMID- 10734417 TI - Birth of piglets preselected for gender following in vitro fertilization of in vitro matured pig oocytes by X and Y chromosome bearing spermatozoa sorted by high speed flow cytometry. AB - The present study examined the ability to establish pregnancies after transfer of pig embryos derived from in vitro fertilization (IVF) of in vitro matured (IVM) oocytes by X and Y chromosome-bearing spermatozoa sorted by flow cytometry. Cumulus-oocyte complexes (COC) were cultured in BSA-free NCSU-23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/mL), epidermal growth factor (10 ng/mL), LH (0.5 microgram/mL) and FSH (0.5 microgram/mL) for 22 h, then the oocytes were cultured without hormonal supplements for an additional 22 h. Boar semen was collected and prepared by flow cytometry sorting of X and Y chromosome bearing spermatozoa. After IVM, cumulus-free oocytes were co-incubated with sorted X or Y spermatozoa (2 x 10(4)/mL) for 6 to 7 h in modified Tris buffered medium containing 2.5 mM caffeine and 0.4% BSA. After IVF, putative embryos were transferred to NCSU-23 medium containing 0.4% BSA for culture. A portion of the oocytes was fixed 12 h after IVF, the remainder were cultured up to 96 h. At 96 h after IVF, 8-cell to morula stage embryos (n = 30 to 35) from each gender were surgically transferred to the uterus of recipient gilts. Insemination of IVM pig oocytes with X- or Y-bearing sperm cells did not influence the rate of penetration (67 vs 80%), polyspermy (40 vs 53%), male pronuclear formation (95 vs 96%), or mean number of spermatozoa per oocyte (1.6 vs 1.6), respectively. Furthermore, no difference was observed between cleavage rates at 48 h after IVF (X, 49 vs Y, 45%). Transfer of embryos derived from X bearing spermatozoa to 18 recipients resulted in 5 pregnancies and delivery of 23 females and 1 male piglet. Similarly, transfer of embryos derived from Y-bearing sperm cells to 10 recipients resulted in 3 pregnancies, with 9 male piglets delivered. The results show that X- and Y-bearing spermatozoa sorted using USDA sperm sexing technology can be successfully used in an IVM-IVF system to obtain piglets of a predetermined sex. PMID- 10734418 TI - Effect of PGE2 on uterine contractility and tone in mares. AB - A technique for transvaginal, ultrasound-guided intrauterine injection was developed. After preliminary study using different approaches, the procedure was successful in 24 of 25 (96%) mares, based on detecting fluid in the uterine lumen during and after the injection. The technique was used to study the effect of PGE2, reportedly produced by the embryonic vesicle, on uterine contractility on Day 12 (Day 0 = ovulation). Uterine contractility was scored (1 = minimal, 4 = maximal) every 10 min for 1 h and every 30 min for the next hour by a continuous 1-min ultrasound examination of a longitudinal section of the uterine body without knowledge of group. In Experiment 1, the main effect of group (1-mL vehicle, n = 6; 0.25 microgram PGE2, n = 7) tended to be significant (P < 0.09), and the effect of time was significant (P < 0.008). The mean score was higher for the PGE2 group (2.0 +/- 0.1) than for the vehicle group (1.7 +/- 0.1). An increase in contractility occurred between 0 and 5 min in the vehicle group (P < 0.0004) and between 0 and 10 min in the PGE2 group (P < 0.04). In Experiment 2, there was a tendency (P < 0.08) for effect of group (control without injection, n = 6; 1-mL vehicle, n = 6; 0.025 microgram PGE2, n = 6). The PGE2 group (2.0 +/- 0.1) was different from the vehicle group (1.6 +/- 0.1) and the control group (1.6 +/- 0.1). An increase in contractility occurred between 0 and 20 min in the PGE2 group, and the changes were not significant in the other groups. However, scores were higher in the PGE2 group before treatment, and there were no significant effects when data were converted to percentage changes. The results for an effect of intrauterine treatment of PGE2 on uterine contractility are considered uncertain because of the transient increase in contractility from vehicle injections in Experiment 1 and the higher score in the PGE2 group before treatment, with no significant differences in percentages in Experiment 2. Indirectly, however, an effect of PGE2 was suggested by a shorter (P < 0.05) period of detectability of intrauterine fluid in the PGE2 groups (21 +/- 31 min) than in the vehicle groups (50 +/- 42 min). The shorter period was attributable to greater dispersion of the fluid as a result of increased contractility. In Experiment 3, PGE2 (10 mg, n = 5) and vehicle (4 mL, n = 5) were given intravenously. In addition to uterine contractility, uterine tone was scored (1 = minimal, 4 = maximal) by transrectal digital compression. The main effect of group was significant (P < 0.03) for uterine contractility score, which increased between 0 and 20 min after PGE2 injection. The time effect and interaction were highly significant (P < 0.0001) for uterine tone score, and tone increased in the PGE2 group between 0 and 20 min after injection. The results indicated that PGE2 should be considered as a potential stimulator of both uterine contractions and uterine tone during the time of embryo mobility in mares. PMID- 10734419 TI - Effect of cryoprotectants and their concentration on post-thaw survival and development of expanded mouse blastocysts frozen by a simple rapid-freezing procedure. AB - Experiments were conducted to develop a simple rapid-freezing protocol for expanded mouse blastocyst-stage embryos. The effect of type of cryoprotectant (ethylene glycol and propylene glycol) and its concentrations (4.5, 6.0 and 7.0 mol/L each with 0.5 mol/L sucrose) on morphological survival and development in vitro were studied. The survival and development of embryos frozen with best concentration of each cryoprotectant pre-exposed to either a low concentration (1.5 mol/L with 0.25 mol/L sucrose) of the respective cryoprotectant or ascending concentrations of sucrose were also compared. The in vivo development of embryos frozen with best protocol (pre-exposure to 1.5 mol followed by 7.0 mol ethylene glycol) was compared with nonfrozen embryos. The rate of re-expansion and hatching was influenced by the type and concentration of the cryoprotectant. A significantly higher re-expansion and hatching rate was achieved at 7.0 mol of both cryoprotectants compared with 4.5 and 6.0 mol of the respective cryoprotectants. When comparing 2 cryoprotectants, a higher (P < 0.05) rate of hatching was obtained with ethylene glycol at 7.0 mol compared with a similar concentration of propylene glycol. The highest re-expansion (91%) and hatching (86%) of expanded blastocysts was achieved with pre-exposure of embryos to a low concentration of ethylene glycol followed by freezing in the same cryoprotectant at 7.0 mol. The transfer of embryos frozen using this protocol resulted in the development of live fetuses. The proportion of live fetuses in the pregnant recipients with frozen-thawed embryos were not different from those transferred nonfrozen embryos (49 vs 57%). It may be concluded that simple rapid-freezing with dehydration in ascending sucrose concentrations or pre-equilibration in a low concentration of ethylene glycol or propylene glycol followed by exposure to the respective cryoprotectant at 7.0 mol resulted in high survival and development of expanded blastocysts. Ethylene glycol at 7.0 mol with pre equilibration is, however, most effective for cryopreservation of this stage in the mouse. PMID- 10734420 TI - Morphological classification of the ovaries in relation to the subsequent oocyte quality for IVF-produced bovine embryos. AB - Although some inferences have been made regarding the morphological aspects of the ovaries in relation to the subsequent oocyte developmental competence in an in vitro system, the influence of ovarian morphology, taken as a pair, has yet to be demonstrated. The present study addresses this limitation. Forty pairs of ovaries from 5 morphological classes were examined to determine whether their characteristics could influence oocyte yield and developmental competence in vitro. An ovary was designated as bearing a corpus luteum (CL) with a dominant follicle (DF) a cyst (CY) or none of these structures (NO). Thus, the paired classes considered in this study consisted of 1) CL-NO 2) CL-DF 3) CL + DF-NO 4) NO-DF and 5) NO-NO. Comparisons were made among the members of 3 subgroups CL, NO and DF. Within the CL-subgroup, the pairs of CL-NO ovaries resulted in higher (P < 0.01) number of oocytes, cleavage rates and blastocyst formation per ovary than in the other categories (CL + DF-NO and CL-DF), with the latter being superior (P < 0.01) to that of CL + DF-NO in terms of cleavage rates. In the NO-subgroup, NO CL pairs yielded higher (P < 0.01) rates of oocyte recovery and cleavage than the NO-DF pairs, and the latter was inferior (P < 0.05) to that of NO-NO ovaries for the 2 indices. Further, blastocyst rates from the NO-CL pairs was higher (P < 0.01) compared with those of NO-CL + DF, NO-DF, and NO-NO groups. And, in the DF subgroup, the DF-CL pairs gave a higher (P < 0.05) oocyte yield and cleavage rate (P < 0.01) than the pairs of DF-NO ovaries but not significantly different in blastocyst formation. The overall oocyte recovery, cleavage and blastocyst rates for the 5 classes were, in a decreasing order CL-NO; NO-NO; CL-DF; CL + DF-NO; and DF-NO. Our results suggest that the morphological classification of ovarian pairs could be a useful means for predicting the developmental competence of oocytes in vitro, and that the presence of a dominant follicle in either one or both ovaries of a pair has a negative effect on the IVF-produced bovine embryos. PMID- 10734421 TI - The suitability of echotexture characteristics of the follicular wall for identifying the optimal breeding day in mares. AB - Ultrasonically detected changes in the equine preovulatory follicle were characterized for the 3 d preceding ovulation early (n = 47) and late (n = 14) in the ovulatory season. Values for the following follicle end points increased progressively over the 3 d: diameter, incidence of nonspherical shape, echogenicity of the apparent granulosa layer, and prominence of an anechoic layer beneath the granulosa. The latter 2 echotexture end points were scored from 1 to 3 (minimal to maximal). Follicle diameter and the 2 echotexture characteristics were more prominent early than late in the ovulatory season. Early in the season, both echotexture characteristics were at the maximal score of 3 in 33/47 (70%) follicles on Day -1 (Day 0 = ovulation). None of the follicles ovulated before both characteristics reached a score of > or = 2. Use of follicle diameter alone to predict impending ovulation seemed ineffective because of a wide range in diameters on Day -1 (31 to 49 mm). The efficiency of a score of > or = 2 for both granulosa echogenicity and prominence of the anechoic layer as an echotexture indicator for the initiation of breeding early in the ovulatory season was compared to diameter indicators of > or = 30 mm, > or = 35 mm, or > or = 40 mm. Data were evaluated as though mares had been bred every other day beginning when an indicator was attained. If the echotexture and > or = 30 mm indicators had been used, none of 34 mares would have ovulated before breeding. However, the mean number of breedings per bred mare would have been greater (P < 0.05) for the > or = 30 mm indicator (2.1 +/- 0.1) than for the echotexture indicator (1.6 +/- 0.1 breedings). The number of breedings per mare would have been equivalent for the echotexture indicator and the diameter indicators of > or = 35 mm (1.5 +/- 0.1) and > or = 40 mm (1.4 +/- 0.2). However, 21 and 74% of the mares would have ovulated before breeding for the > or = 35 mm and > or = 40 mm indicators, respectively. Results suggested that the echotexture indicator would have been more efficient for initiation of breeding than any of the diameter indicators. PMID- 10734422 TI - Premature luteal regression in goats superovulated with PMSG: effect of hCG or GnRH administration during the early luteal phase. AB - Twenty-two goats were superovulated with PMSG; 84 h after the onset of estrus the goats were treated with saline solution (control group n = 7), hCG (hCG group, n = 7), or GnRH (GnRH group, n = 8). The ovaries of all the goats were laparoscopically examined 3 and 6 d after the onset of estrus. In each case the CL were counted and classified according to their appearance as normal-looking or as regressing. Blood samples for progesterone determination were collected every 12 h from Day 1 to Day 6. Premature luteal regression was considered to have occurred if progesterone concentrations declined to less than 1 ng/mL by Day 6. According to progesterone concentrations, 57.5, 0 and 37.5% of the goats underwent premature luteal regression in the control, hCG and GnRH groups, respectively. Progesterone concentrations were higher in the hCG group than in the other groups on Days 5 and 6 post estrus (P < 0.05). The control group was the only one in which there was a significant (P < 0.05) increase in the number of regressing CL between Day 3 (1.6 +/- 1.4) and Day 6 (7.3 +/- 1.4). It was also the only group in which there was a significant decrease in the number of normal looking CL between Day 3 (12.6 +/- 2.1) and Day 6 (2.6 +/- 2.1). On Day 6 the animals treated with hCG had significantly more normal-looking CL (12.0 +/- 2.3) than those in the control group (2.6 +/- 2.1). The number of large follicles present on the ovaries on Day 6 post estrus had negative correlations with progesterone concentrations (P = 0.05) and with the number of normal-looking CL (P < 0.05). It is concluded that the administration of hCG 84 h after the onset of estrus prevents premature luteal regression in goats superovulated with PMSG. PMID- 10734423 TI - Statistical models predicting embryo survival to term in cattle after embryo transfer. AB - Embryo survival to term in recipient cattle is highly variable. We examined calving data in the published literature to determine whether a model of binomial independence or a model which includes an embryo (e) and recipient term (r), adequately explain observed embryo survival rates following attempts to induce twin calving using transfer of two embryos. To achieve this we examined 32 published papers which provided us with 47 sets of data concerning 4560 recipients with either 0, 1 or 2 calves born. In each set of data, the observed embryo survival rate to term (p) (number of calves born/number of embryos) was calculated and the expected number of recipients with either 0, 1 or 2 calves born was determined, assuming a binomial distribution. Parameters for the second model were estimated using maximum-likelihood procedures. The model of embryo independence was rejected in 85% of the sets of data, suggesting that factors other than the embryo are important sources of variation in embryo survival or loss. The proposed e and r model of embryo survival adequately describes the published data in recipients receiving either single or twin embryos. In general, only 50-70% of embryos and recipients are sufficiently competent to result in a calving. Variation among laboratories producing either in vitro or in vivo derived embryos was due to variation in recipient and not embryo competence. It is argued that e rather than observed embryo survival rate, and r rather than observed pregnancy rate, should be used to compare differences among embryo treatments and groups of recipients, respectively. Acceptance of this proposition should permit faster progress in identifying the biology of superior embryos and recipients, which is a prerequisite to improving embryo survival rate in cattle. Collectively, the published data are not consistent with a model of embryo independence, and that a model of embryo survival to term which recognises recipient as well as embryo contributions to embryo survival may be more appropriate in cattle. PMID- 10734424 TI - Local increase of ovarian steroid hormone concentration in blood supplying the oviduct and uterus during early pregnancy of sows. AB - Countercurrent transfer in the ovarian vascular pedicle elevates the concentration of steroid hormones in blood supplying the oviduct and periovarian part of the uterus during the estrous cycle in the pig. This study was conducted to determine whether during early pregnancy the arterial blood supply to the oviduct and uterus carries greater concentration of steroid hormone than systemic blood. The concentration of ovarian steroid hormones (progesterone, estradiol-17 beta, estrone, androstenedione and testosterone) was measured in 40 gilts on Days 12, 18, 25 or 35 of pregnancy. Silastic catheters were inserted: a) into the jugular vein, b) into the branch of uterine artery close to the ovary (proximal to the ovary) and c) into the branch of the uterine artery close to the cervix (distal to the ovary). On the day following surgery simultaneous blood samples from cannulated vessels were collected every 20 min for 3 hours. The concentration of steroid hormones was determined by radioimmunoassay. The mean concentrations of studied hormones in branches of the uterine artery proximal and distal to the ovary were significantly greater than in the jugular vein (P < 0.001) by 18 to 69% and 7 to 31%, respectively. The concentrations of hormones in proximal and distal to the ovary branch of the uterine artery were also significantly different (P < 0.001). The increase in concentrations of the measured hormones did not differ considerably between investigated days of pregnancy. It is concluded that during maternal recognition of pregnancy, formation of the corpus luteum of pregnancy, implantation of the embryo and the placenta elongation the oviduct and uterus are supplied with locally elevated concentration of steroid hormones compared to systemic blood. PMID- 10734425 TI - Superovulation of rabbits with FSH alters in vivo development of vitrified morulae. AB - Morulae were flushed from the oviducts and uteri of New Zealand White (NZW) rabbits superovulated with either 6 (3 d) or 8 (4 d) injections of FSH and from non-superovulated controls. The percentages of embryos recovered from 4 d (100%, n = 8) donors was significantly higher (P < 0.01) than that of 3 d (76%, n = 16) and control (87%, n = 22) donors. Overall, fertilization rates were significantly lower for the 3 d embryos (P < 0.01). Most (86 to 90%) morulae were morphologically suitable for vitrification in an ethylene glycol-based solution. Following storage in liquid nitrogen, morulae were rapidly thawed and transferred to the uteri of pseudopregnant recipients. The total number of kits born for the 3 d, 4 d, and control groups was 40, 61 and 48, respectively. The percentage of live kits from morulae transferred was significantly lower for the 3 d (20%, n = 201) than either the 4 d (36%, n = 169; P < 0.01) or the control (31%, n = 157; P < 0.05) group. The mean number of kits born/recipient for the 3 d (2.4 +/- 2.9), 4 d (4.7 +/- 3.5), and control (3.0 +/- 2.2) protocols did not differ (P > 0.05). The estimated overall efficiency of producing kits based on normal morulae collected for control and 4 d groups, however, was nearly two-fold that for females given 6 FSH treatments. We conclude that the 4 d FSH superovulation regimen enhances the efficiency of rabbit reproductive biotechnology after embryo cryopreservation. These findings have important implications for rabbit colony management using embryo cryopreservation. PMID- 10734426 TI - Cryopreservation of in vitro-derived bovine blastocysts microinjected with foreign DNA at the pronuclear stage. AB - Days 6 and 7 bovine blastocysts derived from in vitro-fertilized and DNA-injected zygotes (day of IVF = Day 0) were cryopreserved either by conventional two-step freezing or by vitrification. Foreign DNA used for microinjection was the green fluorescent protein gene under the control of the immediate early promoter of human cytomegalovirus. All blastocysts were produced by an in vitro system and were harvested on Days 6 and 7. The proportion of DNA-injected zygotes developing into blastocysts on Days 6 and 7 (total 8%) was lower than that of nontreated zygotes (total 19%; P < 0.01). After cryopreservation in 1.5 M ethylene glycol, the survival rates of DNA-injected blastocysts assessed by re-expansion at 24 h of culture (Day 6: 59%, Day 7: 71%) were comparable with those of nontreated blastocysts (Day 6: 76%, Day 7: 71%). The post-thaw hatching rate within 72 h of culture of DNA-injected Day 7 blastocysts (38%) was not different from that of nontreated Day 7 blastocysts (40%), but the hatching rate of DNA-injected Day 6 blastocysts (23%) was lower than that of nontreated Day 6 blastocysts (47%; P < 0.05). After vitrification in 7.2 M ethylene glycol, 0.0026 M Ficoll-70 and 0.3 M sucrose, the survival and hatching rates of DNA-injected Day 7 blastocysts (61 and 28%, respectively) were similar to those of nontreated Day 6 (71 and 33%, respectively) and Day 7 (75 and 36%, respectively) blastocysts. However, the post warming survival rate of DNA-injected Day 6 blastocysts was only 30%, and none of the blastocysts hatched (P < 0.01). The mean cell number of DNA-injected Day 6 blastocysts (100.3 +/- 36.4 cells) was lower than that of nontreated Day 6 blastocysts (130.5 +/- 37.1 cells; P < 0.01), while those of DNA-injected and nontreated Day 7 blastocysts were not different (111.2 +/- 42.8 and 119.6 +/- 31.4 cells, respectively). These results indicate that Day 7 IVMFC bovine blastocysts derived from DNA-injected zygotes can be successfully cryopreserved by conventional two-step freezing or vitrification. PMID- 10734427 TI - Estrus synchronization with lower dose of PGF2 alpha and subsequent fertility in subestrous buffalo. AB - Two experiments were conducted to determine luteal regression, estrous response and fertility in buffalo receiving cloprostenol via 2 routes of administration. In Experiment 1, cyclic buffalo (n = 10) were assigned to 2 equal groups receiving either 500 micrograms i.m. cloprostenol (Estrumate, ICI) or 125 micrograms cloprostenol injected intravulvosubmucosal (ivsm) ipsilateral to the side of the corpus luteum (CL) on Day 11 of an induced estrous cycle. Serum progesterone (P4) concentrations were evaluated immediately before treatment and at 24, 48, 72, 96 and 120 h after PGF2 alpha administration. The decline in serum P4 concentrations was significantly different (P < 0.05) between groups up to 48 hrs after treatment. However, no significant difference (P > 0.05) was observed for the interval from treatment to the onset of estrus (94.9 +/- 10.7 vs 96.0 +/- 15.9 h) for 500 or 125 micrograms of cloprostenol groups, respectively. In Experiment 2, multiparous, lactating subestrous buffaloes (n = 137) were treated either with 125 micrograms ivsm cloprostenol or 500 micrograms i.m. cloprostenol (n = 28 vs 33, respectively) during peak breeding (September-February) or low breeding (March-August) season (n = 37 vs 39, respectively). Buffalo observed in estrus were inseminated twice with frozen-thawed semen at 12 and 22 h after the onset of estrus. Buffalo that failed to exhibit estrus were given a second equal dose of cloprostenol at an 11-d interval and underwent fixed-time insemination at 72 and 96 h. The interval to the onset of estrus was 85.0 +/- 4.4 vs 73.2 +/- 2.6 h during peak breeding and 96.1 +/- 2.6 vs 92.1 +/- 3.8 h during the low breeding season for buffalo treated with 125 and 500 micrograms cloprostenol, respectively. These intervals were different (P < 0.05) between seasons but not between treatments in the same season. Conception rates of 47.8 vs 53.1% during peak breeding and 23.5 vs 25.6% during low breeding season were also different (P < 0.05) between seasons but not between the treatments in the same season for buffalo treated with 125 and 500 micrograms cloprostenol, respectively. These results indicated that 125 micrograms ivsm and 500 micrograms i.m. cloprostenol were equally effective for synchronizing estrus in subestrous buffalo. No negative effect of a lower dose of cloprostenol was observed on estrus synchrony and subsequent fertility; however, season of treatment had a significant effect on conception rates. PMID- 10734428 TI - Factors influencing ovarian activity and sexual behavior of postpartum mares under farm conditions. AB - Management of the postpartum period is one of the most important factors of stud farm medicine. In horses, owing to the long gestation period, the time from parturition to repeat conception needs be short to maintain an optimal yearly foaling interval. For this reason the features of postpartum ovarian activity and sexual behavior were studied under farm conditions. During 2 consecutive breeding seasons, 107 mares on 5 commercial horse farms were monitored after parturition by regular teasing, transrectal ultrasonography and blood sampling for progesterone. Foalings took place from January 1 to June 15. Body condition scoring was carried out within 5 d and at 60 to 65 d after parturition. The first ovulation occurred within 20 d after foaling in 84.1% (90/107) of the mares. The mean intervals from foaling to the first and second ovulations were 17.8 +/- 1.6 d (+/- SEM) and 40.9 +/- 2.7 d (+/- SEM), respectively. The mean intervals from parturition to the first and second ovulation (P < 0.001), the interovulatory interval (P < 0.01), the second follicular phase (P < 0.001), and the time until the first overt estrus (P < 0.01) were significantly longer in mares foaling before the vernal equinox. In the beginning of the breeding season the intervals from parturition to the first ovulation (P < 0.01), to the second ovulation (P < 0.01), and to the first overt estrus (P < 0.001) were significantly longer for primiparous mares than for multiparous animals. There was a tendency for an increased interovulatory interval and for a longer second follicular phase in mares with decreased body condition after parturition (P = 0.069, P = 0.089, respectively). Suckling and breed had no effect on postpartum ovarian activity. We concluded that under field conditions the resumption of cyclic ovarian activity and sexual behavior in mares after foaling are strongly affected by the season of parturition and parity. In some cases, body condition change and other factors may also play a role in influencing postpartum reproductive function. PMID- 10734429 TI - Effects of isolation and culture of turkey primary follicular oocytes on morphology and germinal vesicle integrity. AB - A novel approach to the production of transgenic poultry is to use primary follicular oocytes (PFOs). However, fundamental information regarding the impact of isolation and culture procedures on PFO integrity is lacking. This study describes the isolation and culture of PFOs from mature turkeys and the effects of these procedures on PFO morphology and germinal vesicle (GV) integrity. To isolate PFOs, ovarian cortex was incubated in trypsin-EDTA alone or further incubated in collagenase plus hyaluronidase (CH). About 200 to 500 PFOs, ranging in size from less than 100 microns in diameter to 1,000 microns, were recovered from each ovary. The culture of PFOs less than 100 microns in diameter for 4 h resulted in blebbing of the oolemma followed by extrusion of ooplasm. Primary follicular oocytes 100 to 250 microns in diameter survived culture for 24 h whereas larger PFOs survived for up to 7 d. Those PFOs with intact granulosa cell investments survived longer than those fully or partially denuded of granulosa cells with CH. Co-culture of PFOs (100 to 250 microns in diameter) on a monolayer of granulosa cells derived from mature, yellow-yolk follicles augmented PFO survival rates. The rate of GV breakdown was not influenced by the isolation or culture of the PFO. These data provide the basis for developing procedures for the in vitro maturation and in vitro fertilization of isolated PFOs. PMID- 10734430 TI - Reproductive performance of lactating dairy cows treated with gonadotrophin releasing hormone (GnRH) and/or prostaglandin F2a (PGF2a) for synchronization of estrus and ovulation. AB - The objective of this study was to determine the reproductive performance of lactating dairy cows treated with GnRH and/or PGF2a for synchronization of estrus and ovulation. Between Days 43 and 57 post partum, a total of 374 dairy cows was divided into 4 groups. Cows in Group 1 (n = 62) were treated with 25 mg, i.m. PGF2a on Days 43 and 57; cows in Group 2 (n = 65) were not treated at this time; cows in Group 3 (n = 118) were treated with 100 ug, i.m. GnRH on Day 50, 25 mg, i.m. PGF2a on Day 57, 100 ug, i.m. GnRH on Day 59, and time-inseminated 16 h later; cows in Group 4 (n = 129) were treated with 25 mg, i.m. PGF2a once on Day 57. Cows in Groups 1 and 4 were inseminated at an induced estrus within 7 d after the last PGF2a treatment, and cows in Group 2 were inseminated at a noninduced estrus within a corresponding period of time. Conception rate, estrus detection rate and pregnancy rate were analyzed using logistic regression, and controlled for lactation number, body condition score and time of year. Days from calving to conception were analyzed using the GLM procedures of SAS, and the model included group, body condition score, lactation number, time of year, and their interactions. Cows in Group 3 had a significantly higher pregnancy rate than cows in Groups 1, 2 and 4. Orthogonal contrasts of mean days from calving to conception showed that cows in Group 3 had significantly (P < 0.01) less days from calving to conception than cows in Group 1 and Group 4. There was a significant effect of time of year on pregnancy rate and days from calving to conception, but there was no interaction between time of year and these reproductive characteristics. There was no effect of body condition score and lactation number on the reproductive characteristics evaluated. From the results of this study, it was concluded that better reproductive performance was observed in cows inseminated at a synchronized ovulation than in those inseminated at a synchronized estrous period. PMID- 10734431 TI - Effects of buserelin injection and deslorelin (GnRH-agonist) implants on plasma progesterone, LH, accessory CL formation, follicle and corpus luteum dynamics in Holstein cows. AB - The influence of Buserelin injection and Deslorelin (a GnRH analogue) implants administered on Day 5 of the estrous cycle on plasma concentrations of LH and progesterone (P4), accessory CL formation, and follicle and CL dynamics was examined in nonlactating Holstein cows. On Day 5 (Day 1 = ovulation) following a synchronized estrus, 24 cows were assigned randomly (n = 4 per group) to receive 2 mL saline, i.m. (control), 8 micrograms, i.m. Buserelin or a subcutaneous Deslorelin (DES) implant in concentrations of 75 micrograms, 150 micrograms, 700 micrograms or 2100 micrograms. Blood samples were collected (for LH assay) at 30 min intervals for 2 h before and 12 h after GnRH-treatment from cows assigned to Buserelin, DES-700 micrograms and DES-2100 micrograms treatments and thereafter at 4-h intervals for 48 h. Beginning 24 h after treatment, ovaries were examined by ultrasound at 2-h intervals until ovulation was confirmed. Thereafter, ultrasonography and blood sampling (for P4 assay) was performed daily until a spontaneous ovulation before Day 45. A greater release of LH occurred in response to Deslorelin implants than to Buserelin injection (P < 0.01). Basal levels of LH between 12 and 48 h were higher in DES-700 micrograms group than in DES-2100 micrograms and Buserelin (P < 0.05). The first wave dominant follicle ovulated in all cows following GnRH treatment. Days to CL regression did not differ between treatments, but return to estrus was delayed (44.2 vs 27.2 d; P < 0.01) in cows of DES-2100 micrograms group. All GnRH treatments elevated plasma P4 concentrations, and the highest P4 responses were observed in the DES-700 micrograms and DES-2100 micrograms groups. The second follicular wave emerged earlier in GnRH-treated than in control cows (9.9 vs 12.8 d; P < 0.01). However, emergence of the third dominant follicle was delayed in cows of DES-2100 micrograms treatment (37.0 d) compared with DES-700 micrograms (22.2 d), Buserelin (17.8 d) or control (19.0 d). In conclusion, Deslorelin implants of 700 micrograms increased plasma P4 and LH concentrations and slightly delayed the emergence of the third dominant follicle. On the contrary, Deslorelin implants of 2100 micrograms drastically altered the P4 profiles and follicle dynamics. PMID- 10734433 TI - Effect of column filtration upon the quality parameters of fresh dog semen. AB - We filtered dog semen through various resin columns to obtain a quick, simple system for improving semen quality. Fresh ejaculates were filtered through columns with either glasswool or a chemically-inert polypropylene network disc. The columns were filled with Sephadex G-15 (nonionic resin), Sephadex A-50 (anionic-exchange resin), Sephadex C-50 (cationic-exchange resin) or a combination of Sephadex A-50 and C-50. Filtration through glasswool improved semen quality, with a significant (P < 0.001) increase in the percentage of viability and decrease in the percentage of altered acrosomes (P < 0.001) and total abnormalities (P < 0.001). Total motility was not modified, but curvilinear velocity or linearity of the movement were improved using the glasswool bed. The effect of the glasswool was so intense that it masked the effects of the filtration resins. Substitution of glasswool by polypropylene discs resulted in an unmasking of the effects of the resins, although the polypropylene exerted slight effects on semen. Elution of the spermatozoa through Sephadex G-15 or Sephadex C-50 resulted in a decrease of altered acrosomes. However, filtration through Sephadex A-50 increased viability and decreased the percentage of altered acrosomes and total abnormalities. Combined filtration through Sephadex A-50 and C-50 yielded the combined results observed with the resins individually. Ultrastructural imaging of the interaction between spermatozoa and the beds and resins showed that the cells were loosely deposited upon the glasswool fibers and the Sephadex G-15 particles, whereas close interaction was observed between spermatozoa and Sephadex A-50 and C-50 particles. The whole of the sperm cell bound to C-50 particles, whereas spermatozoa were specifically bonded to A-50 particles in the apical region of the head and in segments of the tail, which were periodically distributed. The data suggest that filtration through glasswool or an anionic resin-exchange can significantly improve dog semen quality. PMID- 10734432 TI - Influence of deslorelin (GnRH-agonist) implant on plasma progesterone, first wave dominant follicle and pregnancy in dairy cattle. AB - The objectives of this study were to investigate the effect of a synthetic GnRH agonist (Deslorelin) implant on CL function and follicle dynamics when administered 48 h after PGF2 alpha, in a timed-insemination protocol, and to determine if the incorporation of a Deslorelin implant into a timed-insemination protocol to synchronize ovulation would be beneficial to the establishment of pregnancy. In Experiment 1, 15 non lactating cyclic Holstein cows received Buserelin (8 micrograms, i.m.) on Day-9, Lutalyse (25 mg, i.m.) on Day-2, and then on Day 0 received either a Deslorelin implant (700 micrograms, s.c.; n = 5), Buserelin (8 micrograms, i.m.; n = 5), or no treatment (control; n = 5). Blood samples were collected on Days-9, -2, 0 and thereafter daily until the next ovulation. Ovaries were scanned by ultrasound on Days-9, -2, 0, 1 (day of ovulation) and 3 times a week thereafter until a subsequent ovulation. From Days 0 to 15, the rate of increase of plasma progesterone (P4) was greater (P < 0.01) for Deslorelin than for control and Buserelin. Establishment of the first-wave dominant follicle (FWDF) as a Class 3 (> 9 mm) follicle was delayed (P < 0.01) with Deslorelin (14.2 +/- 1.3 d) compared with the control (4.6 +/- 1.3 d) and Buserelin (5.0 +/- 1.5 d) treatments. The FWDF resumed growth after Day 13 in all 5 Deslorelin-treated cows, and 2 cows ovulated spontaneously. In 1 Deslorelin treated cow, the FWDF regressed, and a second-wave dominant follicle ovulated, while 2 other Deslorelin cows failed to ovulate until after Day 36. The cumulative numbers of Class 2 and 3 follicles was lowest in the Deslorelin group (P < 0.01), while the cumulative number of Class 1 follicles was highest (Deslorelin > Buserelin > Control; P < 0.01). The number of days to CL-regression and days to subsequent estrus did not differ (P > 0.05) among treatments. In Experiment II, 16 lactating potentially subfertile (body condition score 2.25) cows received Cystorelin (100 micrograms, i.m.; Day-9), Lutalyse (25 mg, i.m.; Day-2), and either a Cystorelin injection (100 micrograms, i.m.; n = 8) or Deslorelin implant (700 micrograms, s.c.; n = 8) on Day 0 and inseminated 16 h later. Deslorelin-treated cows had a higher plasma P4 concentration between Days 0 and 16 (P < 0.05) than the 2 other groups, and 5 of the 8 cows in this group were pregnant (Day 45, palpation) compared with 1 of 8 cows in the Cystorelin group (P < 0.05). Incorporation of a Deslorelin implant into a timed-insemination protocol enhanced the pregnancy rate in cows of poor body condition. The results support the hypothesis that enhanced CL function and delayed establishment of the first-wave dominant follicle may enhance embryo survival. PMID- 10734434 TI - Plasma progesterone profiles and fertility status of anestrus Zebu cattle treated with norgestomet-estradiol-eCG regimen. AB - Zebu cattle are notorious for poor fertility characterized by late maturity and long intercalving intervals attributed to a variety of factors, including genetic, nutritional and climatic. The aim of the present investigation, therefore, was to induce fertile estrus in acyclic pubertal heifers and postpartum anestrous Zebu cows by hormonal intervention. Pubertal Hariana and Sahiwal anestrous heifers (n = 51) and postpartum cows (n = 55) were either assigned a placebo (controls, n = 6 for each breed and parity) or treated with 10 d norgestomet (3 mg) subcutaneous ear implants, with an initial injection of 3 mg, i.m. norgestomet + 5 mg estradiol valerate, followed by 500 IU eCG at implant withdrawal (NOR-treated groups). Jugular venous plasma samples were obtained from a total of 28 animals (controls: 4 heifers and 4 cows; NOR-treated: 12 heifers and 8 cows) on Days 0 (implant insertion), 3, 7, 9 and Day 10 (implant withdrawal), every 12 h on Days 11 and 12, and then once daily on Days 17, 24 and 31. All the samples were assayed for progesterone. Almost all (97%) heifers and 81% cows were induced to estrus, the majority (92% heifers and 79% cows) within 120 h of implant removal. Synchrony of the induced estrus was better in cows, but interval to estrus and estrus duration were significantly longer in heifers (P < 0.05). Post-treatment fertility, based on Day 28 nonreturn rate, first service, and overall conception rates, was better in heifers (78.9, 60.5 and 73.7%, respectively) than cows (77.1, 48.6 and 62.9%, respectively), but the differences were significant only for the overall pregnancy rate (71.8% for heifers and 51.2% for cows; P < 0.05). Low pre-treatment plasma progesterone values (< 0.5 ng/mL) were consistent with ovarian inactivity, confirming the true anestrus status of experimental animals. Controls failed to exhibit estrus and maintained low progesterone concentrations throughout the study. In treated animals, high progesterone values from Day 17 onwards suggested ovulatory estrus. These early luteal phase progesterone concentrations in nonpregnant (P = 0.06) and nonpregnant, nonreturn (P < 0.05) animals were low in comparison with those of pregnant animals. Good fertility resulting from breeding according to estrus, inspite of variable intervals to estrus and estrus duration, advocates its advantage over fixed-time insemination in norgestomet-treated anestrous Zebu cattle. PMID- 10734435 TI - The characterization of bovine embryos obtained from prepubertal calf oocytes and their viability after non surgical embryo transfer. AB - We have previously shown that the addition of epidermal growth factor (EGF) during in vitro maturation was capable of stimulating the cytoplasmic maturation of cow and calf oocytes. The aim of the present study was to compare calf and cow blastocysts produced in the presence of EGF in terms of total cell number and cell distribution between trophectoderm (TE) and inner cell mass (ICM), pattern of protein synthesis, and ability to establish pregnancy after embryo transfer to recipients. For all experiment, embryos at Day 7 were obtained from IVM/IVF/IVC oocytes. No significant differences were noted in total cell number (cow = 138 +/ 46 vs calf = 142 +/- 59; mean +/- SD) or ICM and TE cell number between calf (ICM = 35 +/- 19, TE = 107 +/- 52) and cow (ICM = 38 +/- 21, TE = 99 +/- 32) blastocysts, nor in the ICM/total cell number ratio (cow = 0.27 +/- 11, calf = 0.25 +/- 12). No differences were noted in the constitutive and the neosynthetic protein profiles between cow and calf embryos obtained in vitro. The results of embryo transfer, showed that there was higher pregnancy loss following transfer of calf compared with cow embryos. After Day 35, the rate of pregnancy decreases, with only 22% of calf embryos maintaining pregnancy until calving compared with 39% for cow embryos. In conclusion, it would seem that embryos originating from calf oocytes are less capable of establishing pregnancies than embryos obtained from adult oocytes, although this difference was not significant. This low viability cannot be explained by differences in cell number or by the protein profiles identified between these 2 groups of embryos. PMID- 10734436 TI - Viability assessment of dog spermatozoa using flow cytometry. AB - The percentages of living and dead spermatozoa in fresh dog semen samples were assessed by means of a dual staining technique using carboxifluorescein diacetate (CFDA) and propidium iodide (PI). Two ejaculates were obtained from dogs, each ejaculate was divided into 4 aliquots, and different proportions of freeze-killed cells were added to each aliquot. Data obtained by flow cytometry analysis of each sample were compared with those obtained by the microscopic evaluation under epifluorescence illumination and by phase-contrast microscopy evaluation of the samples stained with eosin-nigrosin. Regression analysis was used to compare the 3 methods for membrane integrity assessment of canine spermatozoa, and high correlation coefficients were found between the flow cytometry procedure and the 2 microscopy techniques. The results from this study validate the use of flow cytometry as a precise method for assessing the viability of dog spermatozoa. PMID- 10734437 TI - Factors affecting reproductive performance in Ontario dairy herds. AB - Data were collected on reproductive, health and production events in 45 dairy herds in southwestern and eastern Ontario, Canada, from July 1990 to July 1993. Mean and median days in milk at first estrus, at first service, and at conception were 85.1 and 83.9, 94.0 and 88.9, and 126.3 and 115.5 d, respectively. The mean estrus detection rate was 48.3%, with a minimum of 28.4% and a maximum of 64.2%. The mean overall conception rate was 46.7%, with a minimum of 29.8% and a maximum of 70.7%. The most commonly used used therapeutic agents were GnRH (11.5% lactational incidence risk) and PG (13.1% lactational incidence risk). Estrus management accounted for the largest portion of PG use in the herds studied (69.4%). Results of a two sample t-test indicated no significant difference in reproductive performance among herds housed in free-stall compared with those housed in tie-stall barns. Multivariate linear regression models of 3 reproductive performance measures indicated that cows with ovarian cysts and/or increased peak milk production had significantly longer calving-to-first estrus and calving-to-conception intervals as well as lower conception rates than average producing cows without cysts. Cows with metritis were found to have significantly shorter calving-to-conception intervals but no detectable difference in conception rate or calving-to-first estrus interval than their herdmates. As lactation number increased reproductive performance improved. Cows that were observed in estrus earlier conceived earlier. Animals receiving uterine infusions had significantly longer calving-to-conception intervals and lower conception rates than their herdmates. There was no detectable difference in reproductive performance for cows treated with GnRH or PG compared with the nontreated cows. An increased estrus detection rate was associated with shorter calving-to-conception intervals and lower conception rates. Based on the range of performance in the study herds, the factors with the greatest potential influence on calving-to-conception interval in the average herd were the estrus detection rate and conception rate. PMID- 10734438 TI - Activity of Chinchilla laniger spermatozoa collected by electroejaculation and cryopreserved. AB - Because reproductive studies and the application of assisted reproductive techniques are relevant issues for an endangered species such as Chinchila laniger, the availability of a source of viable spermatozoa becomes of utmost importance. In this paper, we evaluate several functional parameters (motility, viability, response to hypoosmotic swelling test and acrosomal integrity) of fresh or frozen-thawed spermatozoa. Electro-ejaculation trials (50-cyc/sec sinusoidal wave was applied for 5 of every 10 sec) were successful in all unanesthetized animals. After volume (108.3 +/- 12.0 microL, n = 15) and concentration (421.8 +/- 34.4 x 10(6) cells/mL, n = 15) measurements, the above mentioned parameters were determined. In frozen-thawed semen samples sperm motility, viability, hypoosmotic swelling test and acrosomal integrity were significantly lower than in fresh semen samples. The results clearly indicated that electro-ejaculation is a useful method for evaluating spermatozoa for genetic analysis or for used in Al in this species. In addition, the cryopreservation procedure in this study preserved adequate levels of functional sperm activity. PMID- 10734439 TI - Influence of cumulus cells and sperm concentration on cleavage rate and subsequent embryonic development of buffalo (Bubalus bubalis) oocytes matured and fertilized in vitro. AB - The objective of the present study was to investigate the effects of sperm concentration and presence or absence of cumulus cells on fertilization, cleavage rate and subsequent embryonic development upto the blastocyst stage in buffalo. Cumulus-oocyte-complexes (COCs) obtained from slaughterhouse ovaries were matured in vitro in TCM-199 + 10% FBS + 5 micrograms/mL FSH-P for 24 h. After maturation the COCs were either used as such (cumulus-intact) or freed from attached cumulus cells by repeated pipetting (cumulus-free). Frozen-thawed buffalo spermatozoa were treated with 10 micrograms/mL heparin and 2.5 mM caffeine for sperm capacitation. Oocytes were fertilized in vitro with 1 to 2, 4 to 5 or 9 to 10 million sperm/mL and the cleavage rate was recorded 42 to 44 h post insemination. The cleaved embryos were co-cultured with buffalo oviductal epithelial cells for 10 d post insemination, and the uncleaved oocytes were fixed and stained with aceto-orcein for determination of the penetration rate. The cleavage rate and the proportion of cleaved embryos that developed to morula and blastocyst stages were significantly higher (P < 0.05) whereas the proportion of degenerated oocytes and those that became arrested at the 2 to 16-cell stage were significantly lower (P < 0.05) with cumulus-intact than with cumulus-free oocytes at the 3 sperm concentrations. Increasing the sperm concentration increased the cleavage rate significantly (P < 0.05) from 1 to 2 million through 9 to 10 million sperm/mL but had no effect on the proportion of cleaved embryos that developed to morula and blastocyst stages. In conclusion, the results of the present study suggest that cumulus cells have a positive influence on fertilization, cleavage and subsequent embryonic development. Increase in sperm concentration increases cleavage rate without affecting subsequent embryonic development. PMID- 10734440 TI - The influence of short-term nutrient changes on follicle growth and embryo production following superovulation in beef heifers. AB - Acute decreases in nutrient intake can improve embryo quality in sheep, although reductions in ovulation rate can also occur. In cattle, short-term nutrient restriction prior to ovulation has been shown to increase subsequent pregnancy rates. Thus, the objective was to determine the effect of a severe reduction in food intake on follicle growth and embryo quantity and quality in heifers superovulated with FSH. Beef heifers (n = 61) were offered a diet of grass silage and concentrates (ratio of 5:1, on a fresh weight basis), which was adjusted to provide a predicted intake of 28.6 Mcal/kg ME/d (H) or 9.6 Mcal/kg ME/d (L). Heifers were synchronized with a progesterone-releasing device for 7 d. They were allocated to oocyte recovery (n = 16/treatment) after 3 (225 IU) or 8 (600 IU) injections of FSH given at 12-h intervals. Oocytes were matured, fertilized and cultured individually in vitro. The remaining heifers (n = 14/treatment) were superovulated using FSH (600 IU), and embryos were recovered 7 d after breeding. The embryos were morphologically graded and subsequently cultured for 24 h before differential staining to determine inner cell mass and trophectoderm cell numbers. Follicle numbers increased following 8 (16.6 +/- 2.0) compared with 3 (6.7 +/- 0.6) injections of FSH (P < 0.0001). Heifers on the L diet had more follicles than those on the H diet (13.5 +/- 2.4 vs 9.6 +/- 1.2; P < 0.06), which was predominantly due to an increase in the number of 7- to 10-mm follicles. However, this effect was only evident after 8 injections of FSH. There was no nutritional effect on cleavage rates in vitro (55.6 +/- 8.1 vs 53.8 +/- 9.0 for H vs L diets, respectively). However, cleavage rates were lower in oocytes collected after 8 than after 3 injections of FSH (31.3 vs 69.2%; P < 0.0001). There was no significant effect of nutrition on ovulation rate after FSH (14.4 +/ 1.9 vs 16.3 +/- 3.0 for H vs L diet, respectively). The number of embryos recovered was not different between heifers on H (10.4 +/- 1.3) and L (11.3 +/- 2.4) diets. Following culture for 24 h, a significantly higher proportion of embryos from heifers on the L diet developed to the blastocyst stage (72.9 vs 41.5%; P < 0.01). Total cell numbers on Day 8 were greater in embryos from heifers on the L diet (98.3 vs 75.4; P < 0.0001); yet the inner cell mass as a percentage of total cells was not different (21 vs 20%). These data indicate that low energy intake prior to and during superovulation resulted in more follicles and in improved embryo quality, as evident from the increased number of blastocysts formed and higher cell numbers. PMID- 10734441 TI - Efficacy of decreasing the dose of GnRH used in a protocol for synchronization of ovulation and timed AI in lactating dairy cows. AB - To determine the efficacy of reducing the dosage of GnRH used in a protocol for synchronization of ovulation and timed AI, primiparous and multiparous lactating Holstein cows (n = 237) were randomly assigned to 1 of 2 treatment groups. Ovulation was synchronized for cows in the first group using intramuscular injections of GnRH and PGF2 alpha as follows: Day 0, 100 micrograms GnRH; Day 7, 25 mg PGF2 alpha; Day 9, 100 micrograms GnRH. Ovulation was synchronized in the second group of cows using the same injection schedule and dosage of PGF2 alpha but only 50 micrograms GnRH per injection. All cows underwent a timed AI at 12 to 18 h after the second GnRH injection. The proportion of cows ovulating in response to the second GnRH injection (synchronization rate) and pregnancy status at 28 and 56 d post AI were determined using transrectal ultrasonography. The synchronization rate, double-ovulation rate, conception rate at 28 and 56 d post AI, and pregnancy loss from 28 to 56 d post AI did not differ statistically between treatment groups. For all cows, synchronization rate was 84.0%, and double-ovulation rate was 14.1%. Conception rates calculated using all cows receiving synchronization of ovulation were 41.1% at 28 d and 34.4% at 56 d post AI. Conception rates calculated for only synchronized cows were 47.6% at 28 d and 40.1% at 56 d post AI. For all cows, pregnancy loss from 28 to 56 d post AI was 13.5%, with an attrition rate of 0.5% per day. Estimated savings in hormone costs using 50 rather than 100 micrograms GnRH per injection for synchronizing ovulation were $6.40 per cow and $20.27 per pregnancy. Thus, decreasing the dosage of GnRH used for synchronization of ovulation and timed AI in lactating dairy cows reduces synchronization costs per cow and per pregnancy without compromising the efficacy of the synchronization protocol. PMID- 10734442 TI - Developmental kinetics of the first cell cycles of bovine in vitro produced embryos in relation to their in vitro viability and sex. AB - The development of bovine IVP-embryos was observed in a time-lapse culture system to determine cell cycle lengths of 1) embryos that developed into compact morulae (CM) or blastocysts (BL) within 174 h after insemination (viable), 2) embryos that arrested during earlier stages (nonviable) and 3) male and female embryos. In 4 replicates, inseminated oocytes were cultured on a microscope stage in 3 to 4 groups on a granulosa cell monolayer in supplemented TCM 199. Images were sequentially recorded and stored at 30-min intervals. All embryos that could be identified throughout the culture period were included (n = 392), and the times of cleavage events noted. After culture, 100 CM or BL were randomly selected for sexing by PCR. BL developed equally well in the time-lapse and control culture systems (36 vs 38%). The respective lengths of the first 4 cell cycles of viable embryos were 32.0 +/- 3.9, 8.8 +/- 1.6, 10.8 +/- 4.7 and 47.7 +/- 11.8 h. The subsequent intervals between the 9- to 16-cell, early morula, CM and BL stages lasted 16.2 to 18.2 h. Blastomeres of 2-, 4- and 8-cell embryos cleaved asynchronously with < 1, 2.6 +/- 2.5 and 9.2 +/- 4.5 h intervals, respectively, between the first and last blastomere to cleave. The interval from insemination to tight compaction and formation of a blastocoel was 128.4 +/- 10.7 and 145.8 +/ 12.5 h, respectively. The first 3 cell cycles were approximately 3 h shorter (P < 0.1) while the fourth cycle was 5 h shorter (P = 0.06) for the viable vs nonviable embryos. On this basis it was possible to define time windows in which the proportion of viable 2-, 3- to 4-, 5- to 8- and 9- to 16- cell embryos were at their highest. No differences were found between the cleavage intervals of male and female embryos. We conclude 1) that the time-lapse culture system allows for detailed observation of the developmental kinetics of several embryo groups at the same time, and 2) that these embryos can be manipulated at the end of culture, thus allowing a linkage between early cleavage events and other developmental parameters such as embryo sex or viability after transfer. PMID- 10734443 TI - Fertility comparison between breeding at 24 hours or at 24 and 48 hours after collection with cooled equine semen. AB - It has become a common practice in the equine breeding industry to send 2 insemination doses for breeding with transported cooled semen, one to be used for the initial insemination upon arrival, and the other to be held a second insemination the next day. One fertile stallion and 36 fertile mares were used to determine if breeding once with 1 dose of semen cooled for 24 h would improve fertility compared with breeding twice, 1 d apart, with half the dose of semen cooled for 24 h on the first day of breeding and half cooled for 48 h on the second day of breeding. Mares were given two intramuscular injections of 10 mg PGF2 alpha 14 d apart. Following the second injection, mares were teased with a stallion and their ovaries were scanned by transrectal ultrasonography daily. When a dominant follicle (> 35 mm diameter) was detected, 1500 units hCG were injected intravenously, and the mares were inseminated. Semen was collected in advance of anticipated breeding, mixed in nonfat dry milk solids-glucose extender to a concentration of 25 million sperm/mL, and placed in 2 commercial cooling containers for 24 or 48 h of storage prior to breeding. Mares were randomly assigned to 1 of 2 insemination treatment groups: 1) Group T1 (n = 18), in which mares were inseminated on the day of hCG injection with 500 million spermatozoa cooled for 24 h, or 2) Group T2 (n = 18), in which mares were inseminated on the day of hCG injection with 250 million spermatozoa cooled for 24 h, and again on the following day with 250 million spermatozoa cooled for 48 h. Pregnancy status was confirmed by transrectal ultrasonographic examination at 14 and 16 d after ovulation. Pregnancy rates were the same for both insemination treatment groups (12/18; 67%). There was no advantage to holding half of the insemination dose for rebreeding on the following day. PMID- 10734444 TI - Comparison of bacteriological qualities of various egg yolk sources and the in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or lecithin based diluents. AB - The addition of components of animal origin (egg yolk, milk) to most commercial diluents used to freeze bull semen represents a potential risk of contamination of the doses with bacteria or mycoplasma. A series of quantitative and qualitative analyses were performed to detect microbiological contamination observed in Biociphos plus (a new lecithin-glycerol based freezing salt buffer), in an egg yolk diluent (Triladyl) or in an egg yolk + milk-based (Laiciphos) diluent of bull semen. The 2 diluents containing animal products showed moderate (10 to 60 CFU/mL) contamination (17/17 samples) with bacteria or mycoplasma, or both, while no contamination was observed in the 6 examined batches of Biociphos plus. Biociphos plus was also compared with another commercial diluent (Laiciphos) for use in freezing bull semen intended for in vitro and/or in vivo fertilization. No difference (P > 0.05) could be detected between the 2 diluents for in vitro fertility rates (percentage of cleaved zygotes: 85.7% and 88.0%, respectively, for Laiciphos and Biociphos plus). Similarly, 2 series of comparisons conducted in dairy cows artificially inseminated with semen frozen in either Biociphos plus or Laiciphos showed no difference in fertilizing capacity (tested at 60 to 90 d; P > 0.05) irrespective of the age of the bulls (Trial 1, bulls aged 14 to 15 m.o.; Trial 2, bulls aged 2 to 5 yr, field trials). It is concluded that, in addition to maintaining the fertilizing capacity of bull semen at levels comparable to those observed with standard freezing diluent, Biociphos plus also prevents microbiological contamination by bacteria or mycoplasma, both of which are generally present in the various commercially available sources of egg yolk. PMID- 10734445 TI - Comparative effects of early weaning or once-daily suckling on occurrence of postpartum estrus in primiparous beef cows. AB - We concurrently evaluated early weaning and once-daily suckling as options for shortening postpartum interval to estrus in primiparous cows calving in good body condition. Over 2 consecutive years, a total of 77 primiparous cows were assigned to 1 of 3 treatments: control (ad libitum suckling); once-daily suckling (for 30 d prior to start of breeding); or early weaning (calves weaned 30 d prior to start of breeding). All cows had a body condition score (BCS) of > or = 5 at parturition and were maintained in good condition throughout the breeding season. Cows were observed for estrus twice daily and blood samples for subsequent measurement of progesterone were collected at 48-h intervals. For data analysis, cows were divided into early- (> or = 85 d post partum) and late-calving (< 85 d post partum) groups at the onset of the breeding season. All calves were weighed at birth, at the onset of treatment, at weekly intervals until the start of the breeding season, and within 45 d of 205 d of age. Early weaning or once-daily suckling shortened the postpartum interval to first estrus of late-calving primiparous cows by 17 (P < 0.01) and 12 d (P < 0.08), respectively, compared with control group cows. However, among early-calving cows, neither early weaning nor once-daily suckling influenced the postpartum interval. Postpartum intervals of once-daily suckled cows and early-weaned cows were similar (P > 0.10). PMID- 10734446 TI - Washing and trypsin treatment of in vitro derived bovine embryos exposed to bovine viral diarrhea virus. AB - Gametes, somatic cells and materials of animal origin in media are potential sources for introducing bovine viral diarrhea virus (BVDV) into systems for production of IVF bovine embryos. Further, the efficacy of washing and trypsin treatment for removal of BVDV from IVF embryos is questionable. Washing and trypsin treatments recommended by the International Embryo Transfer Society for in vivo-derived embryos were applied to in vitro-derived, virus-exposed, bovine embryos in this side-by-side comparison of treatments. Embryos for the study were produced in a virus-free system in which follicular oocytes were matured and fertilized in vitro and presumptive zygotes were co-cultured with bovine uterine tubal cells for 7 d. A total of 18 trials was performed, 9 using a noncytopathic BVDV and 9 using a cytopathic BVDV. In each trial, 4 equal groups of 10 or less, zona pellucida-intact embryos/ova were assembled, including 2 groups of morulae and blastocysts (M/B) and 2 groups of nonfertile or degenerated ova (NFD). Each group was prewashed and exposed to 10(4) to 10(6) TCID50/mL of either noncytopathic (SD-1) or cytopathic (NADL) BVDV for 2 h. Following in vitro viral exposure, one group of M/B and one group of NFD were washed. The other groups of M/B and NFD were trypsin-treated. Both treatments were consistent with IETS guidelines. After in vitro exposure to noncytopathic BVDV and washing, viral assays of 100% (9/9) and 78% (7/9) of the groups of M/B and NFD ova, respectively, were positive. After in vitro exposure to cytopathic BVDV and washing, viral assay of 33% (3/9) of the groups of both M/B and NFD ova were positive. After in vitro exposure to noncytopathic BVDV and trypsin treatment, viral assay of 44% (4/9) of groups of M/B and 67% (6/9) of groups of NFD ova were positive. Finally, after in vitro exposure to cytopathic BVDV and trypsin treatment, viral assay of 22% (2/9) of the groups of M/B and 44% (4/9) of the groups of NFD ova were positive. Contingency table analysis, in which data was stratified by embryo type and virus biotype, was used to compare results. While a difference existed between results of the 2 treatments of groups of M/B within the noncytopathic biotype (P = 0.01, Mantel Haenszel Chi-square), no difference was observed between comparison of treatment between all groups in both biotypes (P > 0.05). PMID- 10734447 TI - Peripuberal sexual development of Pantaneiro stallions. AB - Pantaneiro horses are a breed native to flood plains of Brazil, where they thrive with little human interference. The aims of this study were to characterize age associated changes in testicular size, serum testosterone and sexual behavior and to determine age at puberty as well as to evaluate daily sperm production in the Pantaneiro stallion. After weaning the males were kept in bachelor groups away from the females, in a separated area in the natural environment of flood plains. Infantile period ended at approximately 14.4 m.o. of age. Sexual interest and breeding capability first appeared between the ages of 15.6 to 27.5 m.o. The transition from prepuberal to postpuberal histological stage, occurred at approximately 27.8 m.o. of age. All animals were in the postpuberal period at 38.5 m.o. of age. Daily sperm production estimated with testicle homogenate, at 38.5 m.o. of age was low (1.4603 +/- 0.48 x 10(9) sperm) and serum testosterone levels were similar to adult levels by 27.8 m.o. In conclusion the 2 y old Pantaneiro stallions showed full sexual behavior and had adult testosterone levels, but at this age, the stage of development of the seminiferous epithelium was variable and the efficiency of sperm production was still low. PMID- 10734448 TI - Tubal transfer of bovine embryos: a simple endoscopic method reducing long-term exposure of in vitro produced embryos. AB - Although numerous trials had shown the need to define a procedure to get free access to the bovine oviduct, there was no adequate report of a technique which was accepted for the routine transfer of early tubal-stage embryos. We have now report an endoscopically mediated transvaginal method for transferring embryos into the oviduct. The in vitro produced embryos were loaded into a curved glass capillary tube which was connected to a perfusor tube plus 1-mL syringe. The capillary tube was directly inserted via the infundibulum into the ampulla. After first having checked the ovaries for the presence of a corpus luteum the embryos were deposited under visual guidance in about 20 to 50 microL medium. Twenty-four Simmental and Brown Swiss heifers received 26 embryos and 9 animals became pregnant, of which 7 recipients delivered 8 live calves. With practice, the time used for endoscopic transfer was reduced to less than 10 min. The results demonstrate that the described technique is suitable for practical application. Especially for the early transfer of IVP-derived embryos this technique might be advantageous. In conclusion, this method is also of great potential interest for the recovery of tubal-stage embryos and for the in vivo culture of embryos followed by conventional flushing at Day 7. PMID- 10734449 TI - Presence of beta-mercaptoethanol can increase the glutathione content of pig oocytes matured in vitro and the rate of blastocyst development after in vitro fertilization. AB - The present study examined the effect of beta-mercaptoethanol (BME) during in vitro maturation (IVM) of pig oocytes on in vitro fertilization (IVF) parameters, intracellular glutathione (GSH) concentration, subsequent embryo development and blastocyst cell numbers. Cumulus-oocyte complexes were cultured in North Carolina State University (NCSU)-23 medium containing porcine follicular fluid, cysteine, hormonal supplements and 0 to 50 microM BME for 20 to 22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20 to 22 h. After culture, cumulus-free oocytes were coincubated with frozen-thawed spermatozoa for 5 to 6 h. Putative embryos were transferred to NCSU-23 containing 0.4% BSA and cultured for 144 h (Experiment 1). In comparisons between the presence or absence of BME, no differences were observed in fertilization parameters. At 48 h, no mean differences were found in cleavage rates. However, at 144 h, compared with no addition (26%), the presence of 12.5 and 25 microM BME increased (P < 0.05) the proportion of blastocysts in a dose-dependent manner (34 and 41%). Further increase from 25 to 50 microM BME reduced (P < 0.05) the blastocyst development rate. Blastocysts derived from oocytes matured with 25 microM BME had the highest (P < 0.05) trophectoderm (TE) and total cell numbers. No difference was found in inner cell mass (ICM) cells among treatments. In Experiment 2, after IVM, oocytes were fixed to analyze the GSH concentration. Compared to no addition, a higher (P < 0.01) level of GSH was found in oocytes matured with 25 microM BME. Compared with 25 microM BME, GSH was low (P < 0.05) at 50 microM BME. The results show that at certain concentrations BME in IVM medium has beneficial effects on subsequent embryo development, and is correlated with intracellular GSH level in pig oocytes. PMID- 10734450 TI - In vitro evaluation and pregnancy rates after vitrification of in vitro produced bovine embryos. AB - The efficacy of different vitrification solutions to cryopreserve in vitro produced bovine blastocysts was evaluated based on in vitro development of embryos in culture and on in vivo development of embryos transferred into recipients. In the first experiment, 2 vitrification solutions were compared: propylene glycol + glycerol (Pg + Gly) and ethylene glycol + Ficoll + sucrose (EFS). Differences in the overall development and hatching rates in favor of EFS were found (56.4 vs 33.3% and 35.4 vs 13.3%; P < 0.05). In the second experiment, 3 vitrification solutions were compared: EFS, modified EFS (EFSm) and ethylene glycol + glycerol (Eg + Gly). The vitrification solutions EFSm and Eg + Gly yield higher hatching rates than did EFS (57.7 vs 59.6 vs 35.7%; P < 0.05). The last experiment was designed to compare in vivo 2 vitrification solutions: EFSm and Eg + Gly. There were no differences between them based on the results obtained after transfer (35.2 vs 43.7%). The vitrification solutions EFSm and Eg + Gly have resulted in good pregnancy rates. These results demonstrated that vitrification can be used successfully in the cryopreservation of in-vitro produced bovine embryos, and it might be considered for use in commercial programs. PMID- 10734451 TI - Noncytopathogenic bovine viral diarrhea virus (BVDV) reduces cleavage but increases blastocyst yield of in vitro produced embryos. AB - The growing application of in vitro embryo production systems that utilize slaughterhouse tissues of animals of unknown health status conveys the risk of disease transmission. One pathogen of concern in this regard is bovine viral diarrhea virus (BVDV), and the objective of this study was to investigate the effect of BVDV on in vitro embryonic development. A bovine in vitro embryo production system was experimentally infected with BVDV at 2 stages: prior to in vitro maturation by incubating cumulus-oocyte complexes (COC) with virus (strain Pe515; titer 10(6.2) tissue culture infective dose (TCID)50/mL) or vehicle for 2 h, and then during in vitro culture by the use of BVDV infected granulosa cells. Exposure to BVDV throughout in vitro production reduced cleavage rates (P = 0.01) but increased (P = 0.05) the number of embryos that reached the 8-cell stage when expressed as a percentage of cleaved oocytes. Blastocyst yield was increased by the presence of virus when expressed as a proportion of oocytes (P = 0.0034) or of those cleaved (P < 0.0001). The percentage of total blastocyst yield on Days 7, 8 and 9 for the control and virus treatments was 20, 51, 29 and 29, 41, and 29%, respectively, indicating that the rate of blastocyst development was nonsignificantly faster in the virus-treated group (P = 0.06). These results indicate that the presence of non-cytopathogenic BVDV in an in vitro production system may reduce cleavage rates but allow those cleaved to develop to blastocysts at a higher rate. PMID- 10734452 TI - Endometrial suppressor cells in beef cattle. AB - Endometrial cells were recovered post mortem from cyclic and pregnant crossbred beef cattle (n = 5 each) on Days 16 to 18 after estrus, and were evaluated for their ability to suppress lymphocyte responses and release suppressor factor into the culture medium. The suppressor factor was assessed for transforming growth factor-beta (TGF-beta) activity. In addition, Percoll was used to fractionate endometrial cells from Angus cows (n = 4) on Days 16 to 18 of pregnancy to determine the density of the suppressor cells. Endometrial cells from cyclic and pregnant cows suppressed lymphocyte proliferative responses and released suppressor factor into the culture medium. The suppressor factor exhibited TGF beta activity. Suppressor activities tended to be greatest for fractionated cells with densities of 1.01 and 1.095 g/mL. In conclusion, the bovine endometrium contains low- and high-density suppressor cells capable of releasing suppressor factor. The factor seems to be associated with TGF-beta. PMID- 10734453 TI - A procedure for Poitou jackass sperm cryopreservation. AB - We have tried to establish sperm banking for the endangered Poitou donkeys. No successful cryopreservation technique had been described for spermatozoa of this species; our preliminary work indicated that a particular medium and procedure may be effective for cryopreservation of Poitou jackass spermatozoa as evaluated by sperm motility, membrane integrity and pregnancy rate after AI with frozen thawed semen. We found that glutamine at 80 mM and 10% (v/v) quail egg yolk in a basal medium containing 4% (v/v) glycerol (T2-94 medium) improved the post-thaw total and progressive motility and velocity assessed with the automated analyzer ATS-M. The T2-94 medium also preserved the sperm nuclear, acrosom, and plasma membrane integrity as assessed with the acridine orange method, fluorescein conjugated Pisum sativum agglutinin (FITC-PSA) lectin procedure, and hypo-osmotic swelling test, respectively. Semen frozen-thawed in T2-94 medium as used to artificially inseminate. 13 Poitou jennies from the beginning of estrus to ovulation during 4 cycles at a rate of one AI per day. Heigh pregnancies and 3 foals were obtained, but only when the glycerol was removed from sperm before AI. We conclude that the cryopreservation of Poitou jackass semen for sperm banking may succeed by using the T2-94 medium and removing the glycerol post-thaw, but before AI. PMID- 10734454 TI - Sperm transport and survival in the mare: a review. AB - After the deposition of semen in the mare's uterus, spermatozoa must be transported to the site of fertilization, be maintained in the female tract until ovulation occurs, and be prepared to fertilize the released ovum. Sperm motility, myometrial contractions, and a spontaneous post-mating uterine inflammation are important factors for the transport and survival of spermatozoa in the mare's reproductive tract. Fertilizable sperm are present in the oviduct within 4 h after insemination. At this time, the uterus is the site of a hostile inflammatory environment. Our data suggest that spermatozoa trigger an influx of polymorphonuclear neutrophils (PMNs) into the uterine lumen via activation of complement. Furthermore, semen plasma appears to have a modulatory effect on the post-mating inflammation through its suppressive effect on PMN chemotaxis and migration. Spermatozoa that safely have reached the oviduct can be stored in a functional state for several days, but prolonged sperm storage in the female tract is not required for capacitation and fertilization in the horse. The caudal isthmus has been proposed as a sperm reservoir in the mare. The pattern of sperm transport and survival of spermatozoa in the mare's reproductive tract are different between fertile and subfertile stallions, between fertile and some infertile mares, and between fresh and frozen/thawed semen. Possible explanations for these differences include a selective phagocytosis of damaged or dead spermatozoa, impaired myometrial activity in subfertile mares, bio-physiological changes in spermatozoa during cryopreservation, and the removal of semen plasma during cryopreservation of equine semen. PMID- 10734455 TI - Concentrations of total protein, albumin and immunoglobulins in undiluted uterine fluid of gynecologically healthy mares. AB - Undiluted uterine fluid from 20 Warmblood/Standardbred mares (5 to 14 yr old) was recovered by absorption to an intrauterine tampon. The mares were considered gynecologically healthy based on a clinical examination including uterine swabs for cytology and bacteriology as well as endometrial biopsy examinations. The protein profiles (SDS-PAGE) and concentrations of total protein, albumin, and immunoglobulins (Ig) A and G in the uterine fluid were examined and compared with the same proteins in serum. Major peaks were identified on the obtained protein profiles, and there was a clear similarity between the serum profiles and uterine fluid profiles. Variability in protein concentrations among mares was considerably larger in uterine fluid than in serum. Concentrations of the various proteins in uterine fluid were 44 to 56% of those in serum, except for IgA, which had a similar concentration in both serum and uterine fluid. Concentration of the proteins corresponding to peak No. 3 (molecular weight 60 to 71 kDa) in uterine fluid was higher (P < 0.05) in younger mares than in older ones. Parity had no effect on the recorded protein concentrations. The present study of gynecologically healthy mares showed that there is a large individual variation in the protein composition of uterine fluid. The results suggest that age, but not parity, may affect this composition, and indicate further that there is considerable transudation to the uterine cavity. PMID- 10734456 TI - Effectiveness of two systems for transporting equine semen. AB - The storage and transport of cooled, liquid semen is an effective way of facilitating the use of desirable stallions for breeding mares located on distant farms. The Equitainer System is the most widely used transport container and it has been shown that it is possible to ship semen in this container and obtain good conception rates. However, the cost of Equitainers is high, and stud-farms that ship large quantities of semen have tended to rely on cheaper alternatives, even though little documentation exists concerning their reliability, especially under extreme temperature conditions. Two different containers for transporting equine semen (the Equitainer and a styrofoam box) were compared in their effectiveness at maintaining semen quality (i.e. sperm motility and plasma membrane integrity) during 24 h of storage. The transport containers were stored at 2 different environmental temperatures, i.e., room temperature (20 degrees C) and 37 degrees C. Thirty-seven ejaculates from 10 Standardbred stallions (3 to 6 samples per stallion) were examined. Sperm function and plasma membrane integrity were assessed using a Mika Motion Analyzer and a fluorescein stain (Calcein AM/Ethidium homodimer) in fresh diluted semen that had been stored for 24 h at room temperature (20 degrees C). Another 18 ejaculates from 5 stallions were examined using methods described above, but the transport boxes were kept at a high environmental temperature (37 degrees C). After storage at room temperature, there was no significant difference in total sperm motility and frequency of spermatozoa with an intact plasma membrane between the 2 types of transport boxes. A significant difference was seen in linear sperm motility, with the Equitainer being the better container. However, a significant difference was also seen in average path velocity, with the styrofoam box being the better container. After storage at 37 degrees C, the Equitaner maintained semen quality better. A significant difference was seen in total sperm motility, average path velocity, lateral head displacement and frequency of spermatozoa with an intact plasma membrane between the 2 types of transport boxes. Although, both transport containers were satisfactory when used under normal conditions. The Equitainer seemed superior under more extreme temperatures and during longer transport periods (> 24 to 30 h). PMID- 10734457 TI - Effect of presence of a dominant follicle on the superovulatory response in buffalo (Bubalus bubalis). AB - Ten buffalo were superovulated by administration of 8 doses of FSH in a descending schedule spread over 4 d (5.5/5.5, 4.5/4.5, 3.5/3.5 and 2.5/2.5 mL, i.m.; total dose of 64 AU in 32 mL) beginning on Day 10 of an unstimulated estrous cycle, and 30 and 20 mg Lutalyse was given alongwith the 5th and 6th injections of FSH, respectively, to induce luteolysis. The number of corpora lutea (CL) was determined on 6 d post estrus. The ovaries were examined daily by ultrasonography from Day -5 to Day 5 (Day 0 = day of start of superovulation). The animals were retrospectively classified into 2 groups depending upon the presence (n = 4) or absence of a dominant follicle (n = 6). The mean diameter of the largest follicle (F1) increased from 8.25 +/- 0.48 mm on Day -5 to 10.75 +/- 0.25 mm on Day 0 in the dominant group, whereas in the nondominant group the F1 follicle exhibited a progressive decrease from 9.00 +/- 0.45 mm to 7.00 +/- 0.65 mm during the same period, the difference in profiles between the 2 groups was significant (P = 0.042). The profile of the diameter of the second largest follicle (F2) and the difference in diameters between largest and second largest follicles (F1-F2) were not significantly different between the 2 groups. The profile of mean number of large (> or = 10 mm diameter), but not small (2 to 5 mm diameter) or medium (6 to 9 mm diameter) follicles differed significantly (P = 0.001) between the 2 groups from Day -5 to Day 5 (P = 0.030). The number of CL was not significantly different between nondominant (4.00 +/- 0.97) and dominant groups (3.25 +/- 1.31). The number of CL was positively correlated (P < 0.01) with the number of medium follicles and the total number of follicles on the day of initiation of superovulation, but not with follicles of any size category or total number of follicles on any previous day. The results of this study indicate that following the use of morphological criteria based on the size of the largest follicle alone, the superovulation response is not affected by the presence of a dominant follicle at the initiation of superovulation in buffalo. PMID- 10734458 TI - Serum immunoglobulin type G concentrations in calves produced by IVF and delivered by elective cesarean section. AB - Colostrum ingestion by neonatal calves is widely recognized to provide passive transfer of immunity. In this study immunoglobulin absorption from colostrum was evaluated in 54 IVF-produced calves. The IVF calves were delivered by Cesarean section on Days 275 to 277 of gestation, 24 h after the dams had been administered 30 mg dexamethasone. The calves suckled bottles or were force-fed 6 L of colostrum in the first 12 h of life. Colostrum was obtained from the first post-calving milking of recipient dams or from frozen storage reserves if dam secretion was not adequate. Immunoglobulin type G (IgG) content of both sources of colostrum was determined. Serum samples from the calves were collected at 0, 12 and 24 h of age and analyzed for IgG. Twenty dairy calves born vaginally served as the controls and were subjected to the same colostrum management protocol except that the colostrum was obtained only from frozen post-calving milk of dairy cows from the same farm. The control calves were also subjected to the same sampling protocol. The IVF group of calves ingested more IgG (P < 0.0001) and absorbed more IgG by 24 h of age (P < 0.0001) than their control group counterparts. Absorption of IgG was analyzed by comparing the g/kg body weight of IgG with serum IgG values at corresponding times after birth. Colostrum absorption efficiency was the same for both IVF and control groups of calves at 12 and 24 h of age. There was a maximum IgG dose above which additional increases in serum IgG were not realized. The slightly premature, Cesarean delivered IVF calves absorbed IgG from colostrum similarly to control calves delivered vaginally. PMID- 10734459 TI - Determination of acrosin activity of boar spermatozoa by the clinical method: optimization of the assay and changes during short-term storage of semen. AB - A clinical assay to evaluate total acrosin activity developed for human semen has been optimized for use in boar spermatozoa. The main modifications included a decrease of sperm number per assay from 1.0 to 10.0 x 10(6) to 12.5 to 75.0 x 10(3) spermatozoa, and the time of incubation from 180 to 60 min. Linearity of response for differing quantities of spermatozoa was maintained. Extensive washing of spermatozoa was necessary to eliminate seminal plasma, the source of acrosin inhibitors. Seminal plasma that was diluted 1000 times inhibited acrosin activity by about 50%. To abolish the inhibitory effect of seminal plasma it was necessary to use 25,000-fold dilution. Total acrosin activity of boar spermatozoa was about 100 times higher than that of human spermatozoa. Acrosin activity of boar spermatozoa in extended semen decreased during 7 d of storage. These results indicate that the clinical assay of acrosin activity can be used for boar spermatozoa to evaluate the quality of boar semen. PMID- 10734460 TI - Growth hormone priming as an adjunct treatment in superovulatory protocols in the ewe alters follicle development but has no effect on ovulation rate. AB - This study investigated the effect of FSH alone and rGH priming followed by FSH treatment on follicle populations, follicular fluid concentrations of components of the IGF system and steroids, and the ovulation rate in sheep. Estrus was synchronized with progestagen sponges. Ewes (n = 10/group) in Group 1 served as untreated controls, while those in Groups 2 to 5 received a standard superovulatory treatment of 1.1 mg i.m. oFSH twice daily for 4 d. In addition, ewes in Groups 3 and 5 were administered rGH (15 mg/d, i.m.) for the 7 d prior to FSH treatment. Groups 1, 2 and 3 were sacrificed just prior to the LH surge; Groups 4 and 5 were allowed to ovulate. Daily plasma samples were collected to monitor GH, IGF-1 and insulin levels. All follicles > or = 1.0 mm from Groups 1, 2 and 3 were counted, and follicular fluid from follicles > or = 2.5 mm was assayed for estradiol, testosterone, IGF-1 and IGFBPs. Compared with the control, treatment with rGH + FSH but not FSH alone increased (P < 0.001) plasma concentrations of GH, IGF-1 and insulin. The mean number of large-(> or = 4.5 mm) and medium-sized (2.5 to 4.0 mm) follicles was increased (P < 0.01), and the mean number of small (< or = 2.0 mm) follicles was decreased (P < 0.001) by FSH treatment. The mean number of medium-sized (2.5 to 4.0 mm) follicles was further increased (P < 0.05) by rGH priming. Estradiol concentration in medium but not in large estrogenic follicles was increased (P < 0.05) by rGH priming, whereas testosterone concentration in estrogenic follicles was not altered. Components of the IGF system in medium-sized estrogenic follicles were similar in all treatment groups; however, in large estrogenic follicles rGH increased IGF-1 concentrations (P < 0.05) and intensity of the 44-42 kDa IGFBP band (P < 0.01). Priming with rGH did not alter superovulatory responses. These results show that rGH priming, when used as an adjunct to FSH treatment in ewes, alters components of the IGF system in large estrogenic follicles and increases the number and physiological maturity of medium-sized follicles in the ovary; it does not however alter ovulation rate responses. PMID- 10734461 TI - Cellular composition and viability of demi- and quarter-embryos made from bisected bovine morulae and blastocysts produced in vitro. AB - The cellular composition and viability of intact, IVP embryos were compared with those of demi- and quarter-embryos produced by bisection of IVP morulae and blastocysts. Embryos were produced by established techniques from oocytes harvested from slaughterhouse ovaries. In Experiment 1, morulae at Day 6 or blastocysts at Day 7 were bisected on an inverted microscope using a microsurgical steel blade. Demi-embryos were then cultured without a zona pellucida until Day 8, when they were morphologically assessed for quality (viability). A higher proportion of demi-embryos made from blastocysts than from morulae were classified as viable (381/420, 91% vs 164/267, 61%; P < 0.001). In Experiment 2, only Day 7 blastocysts were bisected, and some of the resulting demi-embryos were bisected a second time 24 h later to produce quarter-embryos. The remaining demi-embryos, the quarter-embryos, and control intact embryos were cultured until Day 9, at which time they were assessed for quality and subjected to immunosurgery and differential staining to count inner cell mass (ICM) and trophectoderm cells. A higher proportion of demi-embryos than quarter-embryos was classified as viable (408/459, 89% vs 223/319, 70%, respectively; P < 0.001). Total cell numbers decreased with successive bisections, but the proportion of surviving cells found in the ICM was significantly (P < 0.05) higher in the best quality demi- and quarter-embryos (35 and 32%, respectively) than in the controls (22%). Transfer of all 12 quarter-embryos derived from 3 blastocysts, in pairs, into 6 recipient heifers resulted in 2 pregnancies, each with a single viable fetus at 90 d of gestation. The fetuses originated from 2 different blastocysts. The results suggest that bisection of intact IVP embryos into demi-embryos and bisection of those into quarter-embryos can increase the number of transferable embryos by as much as 178 and 235%, respectively. PMID- 10734462 TI - Use of prostaglandin E2 to ripen the cervix of the mare prior to induction of parturition. AB - Eleven light-breed pregnant mares (335 to 347 d gestaton) were used to evaluate the use of prostaglandin E2 as a cervical ripening agent prior to induction of parturition during the months of April and May. Six hours prior to induction, each mare's cervix was examined per vagina for softness and dilation. Each mare was then assigned to 1 of 2 treatment groups: Group PGE mares (n = 7) received 2.0 to 2.5 mg prostaglandin E2 deposited intracervically; Group SAL mares (n = 4) received 0.5 mL of sterile NaCl deposited intracervically. Six hours later, the mares were readied for parturition by wrapping the tail, scrubbing and rinsing the perineum and udder, and examining the cervix as previously described. Each mare was then administered 15 U, i.v. oxytocin at 15-min intervals until the chorioallantois ruptured. Intervals from initial oxytocin injection until rupture of the chorioallantois, from initial oxytocin injection until delivery of the foal, from delivery of the foal until the foal stood unassisted, and from delivery of the foal until the foal suckled were recorded. Mean cervical dilation immediately prior to induction of parturition tended to be greater in Group PGE mares (3.9 +/- 1.7 cm) than in Group SAL mares (1.9 +/- 1.9 cm; P = 0.10). Mean change in cervical dilation over the 6-h period prior to induction (3.4 +/- 1.9 cm vs 1.5 +/- 2.1 cm), mean number of injections of oxytocin required until the chorioallantois ruptured (1.9 +/- 0.7 vs 2.5 +/- 1.0), and mean intervals from initial injection of oxytocin to rupture of the chorioallantois (20 +/- 10 min vs 28 +/- 19 min) and delivery of the foal (28 +/- 7 min vs 34 +/- 22 min) were not different between Group PGE and SAL mares, respectively (P > 0.10). The proportion of foals that stood within 1 h of birth also did not differ between Group PGE foals (6/7; 86%) and Group SAL foals (3/4; 75%; Chi-square = 0.17; P > 0.10). The proportion of foals that nursed within 2 h of birth was higher in Group PGE foals (6/7; 86%) than in Group SAL foals (1/4; 25%; Chi-square = 4.02; P < 0.05). Premature separation requiring manual rupture of the chorioallantois at the vulvar labia occurred in 1 Group PGE mare (cervical dilation of 1.5 cm at time of induction) and 1 Group SAL mare (cervix closed and firm at time of induction). Foals born from the 2 mares with premature placental separation had the longest intervals from initial oxytocin injection to delivery, delivery to ability to stand unassisted, and delivery to suckling within their respective treatment groups. In summary, it appears that cervical ripening prior to induction of parturition favors shorter deliveries and foal vigor. Intracervical administration of prostaglandin E2 may prove useful for ripening the cervix of the mare prior to induction of parturition. Further studies are indicated to determine optimal dosage and method of administration of prostaglandin E2. PMID- 10734463 TI - Synchronization of estrus with PGF2 alpha administered 18 days after a progesterone treatment in lactating dairy cows. AB - In a previous study we showed that estrus synchronization with 2 treatments of PGF2 alpha 13 d apart reduced conception rate at the synchronized estrus and that this reduction occurred mainly in cows in the early luteal phase at the second PGF2 alpha treatment. The objective of the present study was to determine the efficacy of a synchronization regimen in which PGF2 alpha was administered during the mid- to late-luteal phase to cows that had previously been synchronized with progesterone. Spring-calving cows from 6 dairy herds were used in this study. On Day -32 (Day 1 = the start of the breeding season), cows that had calved 2 or more weeks ago were randomly assigned to a synchronization (S, n = 732) or control (C, n = 731) group. Cows in Group S were treated with an intravaginal progesterone device (CIDR) for 12 d from Day -32 to Day -20, while those in Group C were left untreated. Similar percentages of cows in Group S (80.6%) and C (82.9%) had cycled by Day -7. The CIDR treatment synchronized the onset of estrus, resulting in 92.9% of cows in estrus being detected within 7 d after CIDR removal. Cows in Group S that had cycled by Day -7 were treated with PGF2 alpha (25 mg, i.m., Lutalyse) on Day -2. Cows in both groups that were anestrous on Day -7 were treated with a combination of progesterone and estradiol benzoate (EB) to induce estrus and ovulation (CIDR and a 10 mg EB capsule on Day -7, CIDR removal on Day -2, and injection of 1 mg EB 48 h after CIDR removal). The PGF2 alpha treatment synchronized the onset of estrus in 87.5% of the cows. Group S and C cows had similar conception rates to first (61.0 vs 58.3%) and second (58.4 vs 60.9%) AI; similar pregnancy rates over the AI period (82.8 vs 79.2%) and over the whole breeding season (91.9 vs 90.6%); and required a similar number of services per pregnancy to AI (1.7 vs 1.8). The interval from the start of the breeding season to conception for cows conceiving to AI or to combined AI and natural mating was shorter (P < 0.001) by 5.7 and 6.2 d, respectively, for the Group S cows. It is concluded that the treatment regimen tested in the present study achieved satisfactory estrus synchronization, had no detrimental effect on fertility at the synchronized estrus, and shortened the interval from start of the breeding season to conception. PMID- 10734464 TI - Kinetics of nuclear maturation and protein profiles of oocytes from prepubertal and adult cattle during in vitro maturation. AB - The aim of this present study was to compare the kinetics of nuclear maturation between calf and cow oocytes in order to determine if there are differences between the 2 groups which could explain their disparate developmental capacity. The constitutive and neosynthetic protein patterns of cow and calf oocytes and of their corresponding cumulus cells were also compared during in vitro maturation. A total of 397 calf oocytes and 406 cow oocytes was matured in M199 + 10 ng/mL EGF. The first group of oocytes (n = 30) was immediately fixed and stained after removal from the follicle, and represent 0 h. The remaining oocytes were removed from the maturation medium at 4, 8, 12, 16, 20 and 24 h respectively. Half were denuded, fixed and stained for nuclear status; while the remainder were radiolabeled with methionine-(35S). Immediately after isolation, all the oocytes were at the GV stage. By 8 h, GVBD had occurred in most oocytes (calf: 97%; cow: 100%) and some had reached pro-metaphase I (calf: 49%; cow: 51%). By 12 h, most of the oocytes were at metaphase I (calf: 84%; cow: 94%). By 16 h, 54% of calf oocytes had reached telophase I or beyond compared with 71% of cow oocytes. This difference between the 2 groups became significant by 20 h, with 89% of cow oocytes (P < 0.05) at metaphase II and 71% of calf oocytes. By 24 h of culture, GVBD had occurred in all cases. Most oocytes completed meiosis I and were arrested at metaphase II with the first polar body extruded (calf: 72%; cow: 86%). No differences were noted in the constitutive and the neosynthetic protein profiles of cumulus cells in relation to the age of animal. Changes in neosynthetic protein patterns were observed both in cow and calf cumulus during IVM, and several proteins showed stage-specific synthesis. For the constitutive protein patterns of cow and calf oocytes, there were quantitative (38 and 40 kD) and qualitative (4, 10, 16, 17, 24, 25 and 26 kD) differences between the 2 groups. Only a few differences were observed in neosynthetic proteins between cow and calf oocytes, but there were changes in relation to nuclear status both in cow and calf oocytes. In conclusion, the difference in developmental capacity between cow and calf oocytes may be explained by a difference in the kinetics of nuclear maturation, which was significant at 20 h of culture (with 89% of cow oocytes at metaphase II and 71% of calf oocytes). At the biochemical level, our results indicate that nuclear progression during in vitro maturation of bovine oocytes is linked to changes in protein synthesis by the oocyte itself, while cumulus protein synthesis may either stimulate or modulate the process of oocyte maturation. PMID- 10734465 TI - Immunohistochemical localization of epidermal growth factor, transforming growth factor-alpha and growth factor-beta s in the caprine peri-implantation period. AB - Control over the action of steroid hormones in the uterus and conceptus during the initial period of gestation appears to be regulated locally by growth factors. This study involved immunohistochemical detection of epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) and transforming growth factor-beta s (TGF-beta s), to determine their role in the caprine peri implantation period. Epidermal growth factor was expressed in the luminal and glandular endometrial epithelium of goats on all days studied (Days 22 to 30 post coitum), but it was not detected in trophoblastic cells or in other embryonic structures. Between Days 22 and 30 post coitum, TGF-alpha was detected in the epithelial cells and superficial stroma of the uterus and in the trophoendodermic cells of the embryo. Transforming growth factor-beta s expression, observed in the endometrium, embryo and extraembryonic membranes on Day 22 post coitum, decreased by Day 24 post coitum and disappeared in the embryo by Day 30 post coitum, while remaining in the other structures. The presence of these growth factors during the peri-implantation period in the goat suggests their participation in proliferation and differentiation phenomena which occur during implantation and embryonic development. PMID- 10734466 TI - Thyrotropin stimulates progesterone secretion by luteal cells by activation of the cAMP/protein kinase A signaling system: a potential involvement of protein kinase C. AB - Although the corpus luteum (CL) is not known as a target tissue for thyrotropin (TSH), this hormone increases progesterone production by porcine luteal cells cultured in vitro. In this study we investigated the optimal conditions for TSH stimulated progesterone secretion as well as the involvement of protein kinase A (PKA) and protein kinase C (PKC) in the mechanism of TSH action on porcine luteal cells. To study the PKA and PKC signaling mechanisms, luteal cells collected from mature CL were incubated with the inhibitor of PKA and potent activators of both kinases: PKA-forskolin and PKC-phorbol ester 12-myriistate-13-acetate (PMA). The PKA inhibitor totally suppressed progesterone production in TSH alone, forskolin alone and in TSH plus forskolin-stimulated luteal cells. Forskolin increased basal (P < 0.05) and TSH-stimulated (P < 0.05) progesterone secretion and cAMP accumulation (P < 0.05). Forskolin and PMA added together to control (non-TSH treated) luteal cells had an additive effect on progesterone production. In TSH treated cells, the effect of PMA was statistically significant but did not show an additive effect with forskolin. Further PMA did not affect cAMP accumulation in control and TSH-treated luteal cells. Treatment of control and TSH-treated luteal cells with forskolin and PMA together showed the same increase in cAMP accumulation as with forskolin alone. This is the first demonstration that TSH acts on luteal cell steroidogenesis by activation of the cAMP/PKA second messenger system and also that the PKC signaling pathway may be involved in luteal TSH action on the corpus luteum. PMID- 10734467 TI - The relationship of increased susceptibility of sperm DNA to denaturation and fertility in the stallion. AB - The relationship between fertility and susceptibility of sperm DNA to denaturation was determined in a group of 84 actively breeding, clinically fertile stallions. Susceptibility of DNA to denaturation was determined using the sperm chromatin structure assay (SCSA). The SCSA measures, mean of alpha-t (mean alpha t), standard deviation of alpha-t (SD alpha t), and the COMP of alpha-t (cells outside the main population)] were significantly correlated with the percentage seasonal pregnancy rate (SPR; mean alpha t, r = -0.24, P < or = 0.05; % COMP alpha t, r = -0.27, P < or = 0.05); percentage pregnant per first cycle (FCP; SD alpha t, r = -0.30, P < or = 0.01; % COMP alpha t, r = -0.42, P < or = 0.0001); and the percentage pregnant per cycle (PC; mean alpha t, r = -0.31, P < or = 0.01; SD alpha t, r = -0.32, P < or = 0.01; % COMP alpha t, r = -0.41, P < or = 0.0001). This study describes detectable intrinsic variation in sperm chromatin structure among fertile stallions (SPR, mean = 83%; FCP, mean = 58%; PC, mean = 57%) in an active breeding population (number of mares bred/stallion/year, mean = 37), in the absence of overt reproductive abnormalities and apparent diseases such that an increase in the susceptibility of sperm DNA to denaturation is associated with reduced fertility, both in terms of efficiency of reproduction (FCP and PC) and seasonal pregnancy rate (SPR). Both COMP alpha t and mean alpha t were useful indicators of fertility, with COMP alpha t being the only SCSA value able to identify mean differences between fertility groupings for SPR and FCP, and overall it was the most reliable indicator of fertility in this group of stallions. The SCSA is able to evaluate a compartment of the spermatozoa which is different from that of traditional tests for sperm quality such as motility and morphology. PMID- 10734468 TI - Morphology of spermatozoa in the cauda epididymidis before and after electroejaculation and a comparison with ejaculated spermatozoa in the domestic cat. AB - When 2 ejaculates are collected by electroejaculation from the domestic cat within a period of 10 min the first ejaculate has a higher proportion of abnormal spermatozoa than the second. The reason for this difference is not known for the domestic cat, but in other species long-term epididymal storage results in a higher proportion of abnormal spermatozoa. The aims of this study were to determine the proportions of abnormal spermatozoa in the cauda epididymidis and to ascertain if electroejaculation affects this proportion. Therefore the proportions of spermatozoa in the cauda epididymidis with different morphological abnormalities were compared before and after ejaculation. In addition, the proportion of morphologically abnormal spermatozoa in the epididymis was compared with that in the ejaculate. Nine privately-owned domestic cats were anesthetized, and one testicle was surgically removed. An ejaculate was collected by electroejaculation, after which the remaining testicle was ectomized. There were no significant differences in the proportions of different sperm abnormalities between the cauda epididymidis removed before ejaculation and the one removed after ejaculation. A significantly (P = 0.009) higher proportion of spermatozoa with tail abnormalities was found in the ejaculates compared with the cauda epididymides (11.1 and 1.6%, respectively), while, as expected, there was a lower proportion of spermatozoa with distal droplets in the ejaculates than in the cauda epididymides (35.1 and 75.9%, respectively). This new information contributes to the understanding of the etiology of sperm defects in the domestic cat, and is of importance when evaluating a semen sample in this species. PMID- 10734470 TI - Gamete recovery and follicular transfer (graft) using transvaginal ultrasonography in cattle. AB - Current in vitro culture systems may not be adequate to support maturation, fertilization and embryo development of calf oocytes. Thus, we initiated a study to investigate an alternative method of assessing oocyte competence in vivo, initially using oocytes from adults. Experiment 1 was done to determine if follicle puncture would alter subsequent follicle development, ovulation and CL formation. In control (no follicle puncture, n = 3) and treated (follicle puncture, n = 3) heifers, ultrasound-guided transvaginal follicle aspiration was used to ablate all follicles > or = 5 mm at random stages of the estrous cycle to induce synchronous follicular wave emergence among heifers; PGF2 alpha was given 4 d later. Three days after PGF2 alpha, the preovulatory follicle in treated heifers was punctured with a 25-g needle between the exposed and nonexposed portions of the follicular wall, and 200 microL of PBS were infused into the antrum. There was no significant difference between control and treated heifers for mean diameter of the dominant follicle prior to ovulation, the interval to ovulation following PGF2 alpha, or first detection and diameter of the CL. Experiment 2 was designed to assess multiple embryo production following interfollicular transfer of oocytes (i.e., transfer of multiple oocytes from donor follicles to a single recipient preovulatory follicle). Follicular wave emergence was synchronized among control (no follicle puncture, n = 5), oocyte recipient (n = 7) and oocyte donor (n = 5) heifers as in Experiment 1. In control and oocyte recipient heifers, a norgestomet ear implant was placed at the time of ablation and removed 4 d later, at the second PGF2 alpha treatment. In oocyte donor heifers, FSH was given the day after ablation, and, 4 d later, oocytes were collected by transvaginal follicle aspiration, pooled and placed in holding medium. Five or 6 oocytes were loaded into the 25-g needle of the follicle infusion apparatus with < or = 200 microL of transfer medium. Puncture of the preovulatory follicle of recipient heifers was done as in Experiment 1. Immediately thereafter, LH was given to control and oocyte recipient heifers, but only the recipients were inseminated. Ovarian function was assessed by transrectal ultrasonography and control and oocyte recipient heifers were sent to the abattoir 2 or 3 d after ovulation, where excised oviducts were flushed. The interval between LH administration and ovulation (33 to 36 h) was highly synchronous within and among control and oocyte recipient heifers. Four of 5 (80%) ova were collected from controls and 16 of a potential 43 (37%) ova/embryos were recovered from oocyte recipients; 8 embryos from 3 heifers. Thus, the gamete recovery and follicular transfer procedure (GRAFT) did not alter ovulation or subsequent CL formation, and resulted in the recovery of multiple ova/embryos in which a total of 19 oocytes yielded as many as 8 early embryos, a 42% embryo production rate. PMID- 10734469 TI - Chilling sensitivity of carp (Cyprinus carpio) embryos at different developmental stages in the presence or absence of cryoprotectants: work in progress. AB - The unsolved problem of cryopreservation of the yolk-rich teleost embryos may be related, in part, to their sensitivity to chilling and cryoprotective agents. The aim of this study was to gain data on the sensitivity of carp embryos to low temperatures at different developmental stages and on the possible protective and toxic effects of cryoprotectants. A total of 86,400 morulae, half-epiboly and heartbeat-stage embryos was selected and then placed in water or in 1 M methanol, dimethyl sulfoxide (Me2SO), glycerol or 0.1 M sucrose solution at 0, 4 or 24 degrees C for 5 min or 1 h. Following these treatments, the embryos were held in a 24 degrees C water bath until the evaluation of hatching rates. In every developmental stage a significant decrease of hatching rates following exposure to 4 or 0 degree C was detected. Sensitivity to chilling changed significantly with development (heartbeat < morula < half-epiboly). Half-epiboly stage embryos were less sensitive to a short period of exposure to cryoprotectants than morula and heartbeat stages. A 1-h exposure to cryoprotectants revealed a stage dependent sensitivity. Toxicity increased in the order of methanol < Me2SO < glycerol in morula and half-epiboly stages, and methanol < glycerol < Me2SO in the heartbeat stage. The results show morulae are partially protected against chilling in Me2SO and sucrose, half-epiboly in Me2SO, sucrose and methanol, and heartbeat-stage in methanol and glycerol. The results further suggest that carp embryos are sensitive to chilling and that toxicity and protective effects against chilling of cryoprotectants are stage-dependent. The finding on the low chilling sensitivity of heartbeat-stage embryos and the protective effect of certain cryoprotectants may be useful in designing cryopreservation protocols. PMID- 10734471 TI - Computer assisted morphometric analysis of ram sperm heads: evaluation of different fixative techniques. AB - The recent development of automated systems for morphometric sperm head analysis has provided a series of objective parameters which have facilitated the standardization of morphological semen evaluation. This current work attempts to establish the optimum fixing conditions for the morphometric characterization of ram spermatozoa. Ejaculates were obtained from 5 Merino rams used for periodic collection of semen and were diluted at 1:50 with TEST medium. Air-dried smears were fixed either in ethanol-ether (1:1), 50% methanol, 2% glutaraldehyde or SUZA fixative, in which case the smear was pretreated with chloramine. The samples were then stained with commercial kit Hemacolor. Once the preparations had been mounted, they were analyzed with the Sperm Class Analyzer automatic sperm morphometry analysis system (ASMA). The minimum number of sperm cells analyzed per sample was 100. The parameters evaluated were the area, perimeter, length, width, shape factor and mass. The results showed significant differences in sperm head dimensions between the 4 fixation techniques, with the lowest values for all parameters corresponding to the SUZA fixative, followed by glutaraldehyde, methanol, and finally ethanol-ether. In addition, there were significant variations between animals. It can, therefore, be concluded that the working protocol must be defined when performing morphometric analysis of ram semen and that the results obtained under different conditions of fixation cannot be entirely extrapolated. Equally, the high variability among individuals suggests that, in a species like the ram with a low index of teratozoospermia, there is a need for a revision of the classic definition of normality, which should include morphometric data. PMID- 10734472 TI - Morphology of porcine cumulus-oocyte-complexes depends on the stage of preovulatory maturation. AB - The aim of this investigation was to determine the relationship between the morphology of the cumulus-oocyte-complexes (COCs) and the meiotic configuration of oocytes as an LH peak mimicked by hCG. Estrus was synchronized in a total of 29 crossbred Landrace gilts by feeding Regumate for 15 d and administering 1000 IU PMSG. The LH peak was simulated by treatment with 500 IU hCG at 80 h after PMSG. Endoscopic oocyte recovery was carried out 2 h before and 10, 22 and 34 h after hCG. Only macroscopically healthy follicles with a diameter of more than 5 mm were punctured. Altogether, 410 follicles from 57 ovaries were punctured and 251 COCs were aspirated. Oocyte recovery rate increased from 48.5% (P < 0.01) of the early, not yet preovulatory follicles (2 h before hCG) to 80.8% of late preovulatory follicles (34 h after hCG). Cumulus morphology in COCs recovered 2 h before and 10 h after hCG was heterogeneous, with most (72.9 to 57.4%; P < 0.01) showing a compact or slightly expanded cumulus. Starting at about 22 h after hCG, COC morphology changed dramatically (86.7% of COCs with expanded cumulus; P < 0.01), and 34 h after hCG, 98.3% of the COCs had only an expanded cumulus. The percentage of oocytes with a mature meiotic configuration increased (11.2; 7.1; 41.4 and 70.2%, respectively, n = 238 oocytes; P < 0.01) as the interval post hCG increased (-2, 10, 22, 34 h, respectively). Meiotic configuration was related to COC morphology: compact COCs--88.9% diplotene, expanded COCs--53.8% metaphase II (M-II), and denuded oocytes--69.2% degenerated chromatin. These results indicate that there is a relationship between oocyte recovery rate, COC morphology, and meiotic configuration and preovulatory follicle maturation after the application of hCG. PMID- 10734473 TI - Differences in uterine position of reproductively normal mares and those with delayed uterine clearance detected by scintigraphy. AB - The position of the uterus within the abdomen may affect a mare's ability to rapidly clear the uterine lumen of contamination. In this study, the position of the uterus was determined from left and right lateral flank scintigrams taken 1 and 2 h after intrauterine infusion of radiocolloid. Scintigraphy was performed during estrus in 44 mares, 24 were reproductively normal and 20 exhibited a delay in uterine clearance. Reproductively normal mares were nulliparous (n = 14) or pluriparous (n = 10), 3 to 21 yr of age, had no history of persistent uterine infections and cleared > 50% of a radiocolloid within 2 h of infusion into the uterus. Mares that exhibited a delay in uterine clearance were pluriparous (n = 18) or nulliparous (n = 2), 12 to 24 yr of age, had a history of endometritis and cleared < 30% of a radiocolloid within 2 h. The angle between the caudal-ventral aspect of the uterine image and cervix relative to horizontal as visualized on the scintigram was measured with a protractor. Results were analyzed by the General Linear Model System. The uterine-cervical angle relative to horizontal was more ventral in mares with delay in uterine clearance and was more horizontal in reproductively normal mares (mean +/- SEM-111.6 +/- 3.6 for delay in uterine clearance mares; 147.6 +/- 3.9 for reproductively normal mares; P < 0.0001). The mean angle for reproductively normal, pluriparous mares was steeper than that for nulliparous mares (141.1 +/- 2.9, 152.3 +/- 2.44 respectively; P = 0.004). There were no differences in angles between left and right lateral views within individuals. We conclude that a uterus that tilts ventrally in relation to the pelvic brim may contribute to the inability of delay in uterine clearance mares to rapidly clear their uterine lumen of contamination. Parity may contribute to the more ventral orientation of the uterus. PMID- 10734474 TI - Luteal function and estrus in peripubertal beef heifers treated with an intravaginal progesterone releasing device with or without a subsequent injection of estradiol. AB - The objectives of this experiment were to determine if treatment of beef heifers with progesterone (P4) using an intravaginal device alone or in combination with estradiol benzoate (EB) would induce estrus and cause development of corpora lutea (CL) with a typical life span. Peripubertal heifers (n = 311) were used when about 40% of the heifers had a functional CL. The heifers were assigned to receive one of the following treatments on Day 0: 1) a sham device for 7 d (C, n = 108); 2) an intravaginal device containing P4 for 7 d (P, n = 102); or 3) an intravaginal device containing P4 for 7 d plus an injection of 1 mg EB 24 to 30 h after device removal (PE, n = 101). Serum concentrations of P4 were determined on Days -7, 0, 8, 15 and 22. Weight and age of the heifers at the start of the trial averaged 292 +/- 45 kg and 365 +/- 38 d, respectively. A greater (P < 0.0001) proportion of the heifers from the PE than P group was in standing estrus (81 vs 37%) and formed normal CL (68 vs 44%) after device removal. Of the heifers exhibiting estrus, a greater (P < 0.05) proportion of PE (94%) than P (80%) heifers was active 1 to 3 d after implant removal. Short-term progesterone treatment increased the proportion of heifers in estrus and those forming normal CL, and adding EB to the progesterone treatment further enhanced these responses. PMID- 10734475 TI - Effect of injection of beta-carotene or vitamin E and selenium on fertility of lactating dairy cows. AB - Experiments tested whether supplemental antioxidants improved fertility. To test effects of beta-carotene, cows in a hot environment were injected with prostaglandin F2 alpha (PGF2 alpha) and were given 3 injections, i.m., of 800 mg beta-carotene or saline at Days -6 and -3 before the anticipated date of insemination and at insemination (n = 37-41 inseminated cows/group). There was no effect of beta-carotene on the proportion of cows detected in estrus following PGF2 alpha, timing of estrus after PGF2 alpha injection or pregnancy rate in inseminated cows. In a second trial, cows in a temperate climate received intramuscular injections of vitamin E (500 mg) and selenium (50 mg) at 30 d post partum (n = 97) or were untreated controls (n = 89). Treatment did not affect interval from calving to first insemination or the proportion of cows pregnant at first service, but it increased the pregnancy rate at second service (69.8 vs 52.1%; P = 0.07) and reduced services per conception (1.7 vs 2.0; P < 0.05) and interval from calving to conception (84.6 vs 98.1; P < 0.05). Thus, injection of vitamin E and selenium increased fertility in cattle that did not become pregnant at first service. PMID- 10734476 TI - Comparison of ultrasonography, bovine pregnancy-specific protein B, and bovine pregnancy-associated glycoprotein 1 tests for pregnancy detection in dairy cows. AB - At Days 26 to 58 after AI, 138 Holstein-Friesian dairy cows were repeatedly examined by ultrasonography, using a 7.5 MHz linear-array rectal transducer. The total calving rate was 37.6% (52/138), and late embryonic mortality occurred 8.6% of the cows (12/138). On the days of ultrasound scanning, blood samples were drawn from the jugular vein for measuring the concentration of bovine pregnancy specific protein B (bPSPB) and bovine pregnancy-associated glycoprotein 1 (bPAG 1). When compared with calving results, there were no significant differences in accurate diagnosis of pregnant cows were found between the 3 methods. However, when recognition of an embryo proper with a beating heart was used as the criterion for positive ultrasonographic diagnosis significantly fewer (P < 0.001) pregnant cows were correctly identified than by the other 2 tests. When compared with the noncalving cows, significantly fewer (P < 0.001) false positive diagnoses were made by the 2 ultrasonographic tests than by the PSPB and bPAG 1 tests, while significantly fewer (P < 0.001) false positive diagnoses were made by the bPSPB test than by the bPAG 1 test. The accuracy of detecting nonpregnant animals by both protein tests was limited by the relatively long half-life of these proteins after calving and by early embryonic mortality. PMID- 10734477 TI - Comparison of ultrasound-guided vs laparoscopic transvaginal ovum pick-up (OPU) in simmental heifers. AB - Transvaginal ovum pick-up (OPU) offers several advantages over standard embryo transfer procedures. For a systematic comparison of the ultrasound-guided (U-OPU) and the laparoscopic OPU (L-OPU) method, groups of Simmental heifers were subjected to 1 of the 2 OPU-methods for 8 wk (15 sessions) followed by a treatment-free interval of 11 wk and then another 8-wk period of OPU using the alternative method. Parameters that were evaluated included the number of follicles aspirated, number and morphology of recovered cumulus-oocyte complexes (COCs), and developmental capacity of oocytes after in vitro maturation (IVM) and fertilization (IVF). Blood samples were also taken from the donors twice a wk for progesterone measurement. To evaluate effects of long-term OPU on subsequent fertility of donors, all heifers (n = 14) were inseminated during the first natural estrus after the OPU experiment. The proportion of Class I oocytes was significantly (P < 0.001) higher after U-OPU than after L-OPU (38.7% vs 21.0%). Following IVM/IVF, this difference in oocyte quality was reflected by the cleavage rate (U-OPU: 58.1%; L-OPU: 52.1%; P < 0.05) and the rate of development to morulae and blastocysts (U-OPU: 27.1%; L-OPU: 13.9%; P < 0.001). Among other factors, the greater changes in vacuum pressure during L-OPU vs U-OPU might be responsible for the difference in oocyte quality. This problem may be overcome by a more flexible system for regulating the vacuum. Progesterone levels were higher during the L-OPU than the U-OPU periods. Seven donors (50%) were diagnosed pregnant by ultrasonography on Day 28 and by palpation per rectum on Day 42. PMID- 10734478 TI - Relationship between peripheral estrogen concentrations at insemination and subsequent fetal loss in cattle. AB - In a survey on pregnancy rate and embryonic losses in dairy cattle on 6 Israeli farms, cows (n = 78) were divided into 3 groups on the basis of ultrasonography at 21 d post insemination; pregnancy diagnosis at 40 to 50 d post insemination and blood progesterone (P4) levels at 21 d. The groups were either pregnant (P4 level > 1.0 ng/ mL); not pregnant (P4 < 0.5 ng/mL), or showed early embryo loss (P4 > 1.0 ng/mL and the presence of an embryonic vesicle on D 21 but later returned to estrus or were found not pregnant on D 40 to 50). On the day of insemination, peripheral estrogen was significantly higher (P < 0.05) in the early embryo loss group (15.3 +/- 1.1 pg/mL, n = 27) than in pregnant (9.4 +/- 0.6 pg/mL, n = 26) or not pregnant (9.6 +/- 0.7 pg/mL, n = 25) group. The cows on 3 farms which were fed 1 to 2 kg/d of vetch (Vicia sativa), an estrogenic legume, had higher estrogen concentrations on the day of insemination than cows (2 farms) fed other legumes (13.7 +/- 0.64, n = 58 vs 10.7 +/- 0.8 pg/mL, n = 42; P < 0.01). On one of the 3 farms, vetch was replaced with alfalfa after the first year. Following the cessation of vetch feeding the estrogen concentrations in the blood decreased from 32 +/- 5 pg/mL to 14 +/- 2 pg/mL (n = 9). These data suggest that high peripheral estrogen on the day of insemination is associated with early embryonic loss. These data also indicate that estrogen concentrations on the day of insemination can be influenced by diet. PMID- 10734479 TI - Exposure of in vitro-produced bovine embryos to foot-and-mouth disease virus. AB - The aim of this study was to investigate whether foot and mouth disease virus (FMDV) interacts with in vitro produced (IVP) bovine embryos. One milliliter of a suspension of FMDV (2 x 10(7) TCID50/mL) was added to several batches of these embryos 7 d after in vitro fertilization, by which time they had either developed to the morula/blastocyst stage (n = 256) or degenerated (n = 260). Six experiments were performed in which developed or degenerated batches of embryos were incubated with FMDV for periods of 1 h (3), 2 h (2) or 4h (1). After this, the embryos were washed 10 times according to the International Embryo Transfer Society (IETS), then pooled and ground up to form a suspension, and assayed on cell cultures for FMDV. The cell cultures were observed daily for cytopathic effects for 3 d post exposure. In addition to the cell culture method, the polymerase chain reaction (PCR) technique was used to assay for the presence of the virus in the washing fluids. Assays for FMDV were also conducted on the first and second wash and on the pooled sample constituting the eight, ninth and tenth wash. With the exception of the second wash from a batch of embryos exposed to FMDV for 2 h, all samples of the first and second wash produced FMDV cytopathic effects, but none occurred with the pooled samples of the 8th, 9th and 10th wash. FMDV was also isolated from all but 1 of the batches of embryos after 1 h of incubation, from 1 of 4 batches after 2 h of incubation and from all batches after 4 h incubation. By contrast, the presence of virus could not be demonstrated by PCR based on the technique used here. These results show that 7 d old IVP bovine embryos can retain FMDV after washing, unlike in vivo-derived embryos, which do not appear to carry risks of FMDV transmission when washed according to IETS recommendations. Stricter controls are, therefore, necessary when using IVP embryos from cattle in a non-FMD-free zone in domestic or international trade. PMID- 10734480 TI - Can spermatozoa with abnormal heads gain access to the ovum in artificially inseminated super- and single-ovulating cattle? AB - The collective efficiency of barriers in the female tract against spermatozoa with abnormal heads was studied. In Experiment 1, Day 6 ova/embryos were recovered nonsurgically from superovulated (n = 24) and single-ovulating (n = 44) cows following artificial insemination with semen of bulls selected for normal spermatozoal motility (> or = 50%) and high content (> 30%) of spermatozoa with misshapen heads, random nuclear vacuoles or the diadem defect. To assess characteristics of spermatozoa capable of traversing barriers in the female tract, accessory spermatozoa were classified morphologically (x 1250) and compared with those of the inseminate. Superovulated cows proved inadequate for assessment of accessory spermatozoa due to evidence of poor sperm retention in the zona pellucida; thus, only single-ovulating cows were used. Accessory spermatozoa (n = 479) from 31 ova/embryos recovered from 44 cows were more normal in head shape than those in the inseminate (76 vs 62%; P < 0.05). Spermatozoa with normal head shape, but with nuclear vacuoles appeared as accessory spermatozoa at the same frequency as they were found in the inseminate (20 vs 17%, respectively). Only sperm cells with subtly misshapen heads appeared as accessory spermatozoa. In Experiment 2, semen pooled from 4 bulls having large numbers of spermatozoa exhibiting a gradation from severely asymmetrically misshapen heads to subtly misshapen heads was evaluated. Again, the accessory sperm population (960 sperm cells recovered from 64 ova/embryos) was enriched with spermatozoa of normal head shape relative to the inseminate (53 vs 26%, respectively; P < 0.05). Sperm cells with only nuclear vacuoles and those with subtly misshapen heads were not different between the accessory and inseminate populations (11 vs 8%, and 20 vs 25%, respectively). We conclude that morphologically abnormal spermatozoa are excluded from the accessory sperm population based upon severity of head shape distortion. PMID- 10734481 TI - Normal calves from transfer of biopsied, sexed and vitrified IVP bovine embryos. AB - Data on biopsied, sexed and cryopreserved in vitro produced (IVP) bovine embryos, and their in vivo developmental competence are very limited. Two preliminary studies were conducted before the primary study. In Experiment 1, post-thaw in vitro developmental competence of biopsied and vitrified IVP embryos was evaluated using re-expansion as an endpoint. In Experiment 2, the pregnancy rates of biopsied fresh, frozen or vitrified embryos following single embryo transfer were compared. Since vitrified embryos resulted in a higher pregnancy rate than frozen-thawed embryos, in the primary study (Experiment 3), all IVP embryos were vitrified following biopsy and sexing (by DNA fingerprinting). In Experiment 3, we compared pregnancy initiation and calving results of heifers in the following treatments: 1) artificial insemination (AI); 2) AI plus contralateral transfer of a single embryo (AI + SET); 3) ipsilateral transfer of single embryo (SET); or 4) bilateral transfer of two embryos (DET). Birth weights, gestation lengths and dystocia scores were recorded. In Experiment 1, post-thaw re-expansion rate of biopsied and vitrified embryos was 85% (70/82). In Experiment 2, pregnancy rates (90 d) were 44% (7/16), 23% (3/13), and 50% (7/14) for vitrified, frozen and fresh embryos, respectively (P < 0.10). In Experiment 3, pregnancy rates of AI and SET were 65% (20/31) and 40% (16/40), respectively (P < 0.05). The pregnancy rate of AI + SET was 75% (27/36) with 11 carrying twins, and the pregnancy rate of DET was 72% (26/36) with 10 carrying twins. All AI fetuses were carried to term, but only half the SET fetuses were carried to term. Similar calving rates were observed in the AI + SET and DET groups, 76 and 70%, respectively, of those pregnant at Day 40. Mean birth weight, dystocia score and gestation length of AI calves were not different from those of SET calves. Mean birth weight and dystocia score of single-born calves were greater than those of twin born calves (P < 0.05). These data demonstrate that biopsied IVP bovine embryos can be successfully cryopreserved by vitrification and following post-thaw embryo transfer, acceptable rates of offspring with normal birth weights can be obtained without major calving difficulties. PMID- 10734482 TI - Transfer of fresh and cryopreserved IVP bovine embryos: normal calving, birth weight and gestation lengths. AB - In vitro and in vivo developmental competence of fresh and cryopreserved in vitro produced (IVP) bovine embryos was evaluated up to birth. Three experiments were done. The objective in the first experiment was to develop an optimal vitrification procedure for IVP bovine embryos by determining effects of exposure time (2, 5, 10, 20 min) and temperature (4, 22, 27 degrees C) in cryoprotective agents prior to vitrification on their post-thaw viability. The best combination was used in Experiments 2 and 3. In the second experiment, the importance of post thaw morphologic selection on pregnancy rates was determined by transferring either selected or unselected single embryos. In the third experiment, pregnancy initiation, maintenance and calving results of vitrified embryos were compared with fresh and conventionally frozen embryos. Fetal losses, birth weights, gestation lengths and frequency of dystocia in the third experiment were monitored. The interaction of exposure time and temperature on both post-thaw re expansion and hatching rates was significant (P < 0.01). Five minute exposure at 27 degrees C was optimal. In the second experiment, post-thaw selected vitrified embryos had higher pregnancy rates than unselected embryos (P < 0.05). In the third experiment, the pregnancy rate of vitrified embryos did not differ from that of fresh embryos (P > 0.05). However, pregnancy rate of conventionally frozen embryos was lower than that of fresh or vitrified embryos (P < 0.05). Of 92 calves born, 53 were male and 39 were female. Birth weights and dystocia scores of single-born calves did not differ between sexes (P > 0.05). Twin-born calves were lighter than single-born calves (P < 0.05). Overall, the data demonstrate that the transfer of vitrified IVP bovine embryos can result in healthy, apparently normal calves similar to those derived from transfer of fresh and conventionally frozen IVP bovine embryos. PMID- 10734483 TI - Effect of different glycerol treatments on frozen-thawed dog sperm longevity and acrosomal integrity. AB - Four different concentrations of glycerol in a Tris-fructose-citric acid extender for frozen dog semen and the effects of adding glycerol at 37 degrees C or 4 degrees C to the extender were studied by monitoring the post-thaw sperm longevity and acrosomal integrity during incubation at 39 degrees C. In the first part of this study, ejaculates from 13 dogs were pooled and divided into 4 aliquots, which were centrifuged and the sperm pellets rediluted with a Tris fructose-citric acid extender containing 2, 4, 6 and 8% (v/v) glycerol, respectively. Progressive motility by subjective estimation, live:dead spermatozoa ratio using eosin-nigrosin staining, and acrosomal integrity using phase contrast microscopy were evaluated before processing and at 0, 0.5, 1, 2 and 4 hours post-thawing incubating the semen samples in the dark at 39 degrees C. The experiment was performed using seven replicates and it was found that sperm motility and acrosomal integrity were superior following the use of 8% glycerol in the extender. In Experiment 2, 13 ejaculates from the same dogs used in the first experiment were pooled and divided into 3 aliquots, and an 8% glycerol diluent was added at 37 degrees C and 4 degrees C after 1 h of cooling or at 4 degrees C after 2 h of cooling, respectively. After freezing and thawing the same parameters as studied in the first experiment were assessed. The experiment was performed in 7 replicates, and no difference was found between treatments. PMID- 10734484 TI - Endocrine and thermal responses to GnRH treatment and prediction of sperm output and viability in holstein-Friesian breeding bulls. AB - A study was conducted to determine changes in serum LH and testosterone concentrations and in scrotal surface temperature (SST; measured with infrared thermography) following GnRH treatment and to predict the number of spermatozoa collected and the proportion that were viable. Holstein-Friesian breeding bulls (n = 22, average age, 24.3 m.o.; range, 15 to 41 m.o.) were examined twice 30 d apart. Concurrently, semen was collected twice weekly with an artificial vagina. Treatment with GnRH (100 micrograms, i.m.) increased (P < 0.0001) serum LH and testosterone concentrations and increased (P < 0.0001) SST (range 0.6 to 1.1 degrees C; P < 0.05) at the top and bottom of the scrotum. In regression models to predict the total number of spermatozoa, significant independent variables included ultrasonic echotexture of the testes (negative slope), scrotal width (positive slope) and SST at the bottom of the scrotum 45 min after GnRH treatment (positive slope). In regression models to predict the percentage of live spermatozoa, ultrasonic echotexture was a significant independent variable (negative slope). Measurement of testicular ultrasonic echotexture and SST after GnRH treatment augmented measurement of testicular size for predicting the number and percentage of live spermatozoa. PMID- 10734485 TI - Heterologous fertilization to characterize spermatozoa of the genus Bos. AB - Advances in assisted reproductive techniques, specifically, development of protocols for production of in vitro matured, fertilized and cultured domestic bovine embryos, offer opportunities to apply these techniques to nondomestic bovidae in species preservation. Domestic bovine oocytes were inseminated with nondomestic bovine spermatozoa. Effects of heparin concentration, sperm concentration and their interaction on total and normal in vitro fertilization rates and on subsequent embryo development were evaluated. In different replications, semen from 3 Bos bison, 2 Bos gaurus, 1 Bos grunniens, and 1 Bos javanicus bulls was used. Treatment of spermatozoa included 2 heparin levels (2 and 8 micrograms/mL) and 3 sperm concentrations (1, 3 and 5 x 10(6)/mL). The B. grunniens bull exhibited excessive polyspermy in all treatments; therefore, 1 replicate was completed using 2 levels of heparin (0 and 1 microgram/mL) and 2 sperm concentrations (1 and 2 x 10(6)/mL). After 18 to 22 h, cumulus cells were removed from presumptive zygotes, and a portion thereof was compressed between a slide and coverslip and fixed in acetic acid:ethanol solution. Light microscopy was used to visualize pronuclei and the second polar body as a determinant of fertilization. Remaining presumptive zygotes were placed into embryo culture medium, and blastocyst development was assessed on Days 7 and 8 (fertilization = Day 0). Percentages of total and normal fertilization and of blastocyst formation were analyzed by a logistic regression model, isolating effects due to bull, heparin and sperm concentration, and to their interaction. Work presented here suggests that, just as in Bos taurus, the nondomestic bulls in the Bos species seem to have individual heparin and sperm concentration requirements for successful IVF. We conclude that each bull, domestic or nondomestic, needs to be evaluated individually. Preliminary sperm characterization using domestic cattle oocytes would result in a greater potential for generating purebred embryos of the desired species should scarce female gametes become available. PMID- 10734486 TI - Preliminary results of hemizona assay (HZA) as a fertility test for canine spermatozoa. AB - The Hemizona assay (HZA) is considered to be an effective test for predicting the fertilizing potential of spermatozoa. It is a functional test that distinguishes the zona-binding capacity of spermatozoa from fertile and infertile males. The objective of this study was to validate the HZA for canine spermatozoa, as a test for diagnosing canine male fertility status. Various parameters that affect binding capacity were examined: the presence of an adequate number of capacitated and motile spermatozoa for an HZA, the influence of fertility status, sperm binding variability within fertile dogs over 60 d, variability in sperm-binding capacity of different oocytes, the lower limit number of spermatozoa binding to a zona from the fertile control, and evaluation of HZI to determine the fertilizing capacity of spermatozoa. Hemizonae were obtained from frozen oocytes of spayed bitches. The oocytes were manually cut into nearly equal halves. Spermatozoa were capacitated by swim-up and 1 h incubation at 37 degrees C in modified Ham's F10 medium. Spermatozoa and hemizonae were co-incubated in 100-microL drops at 37 degrees C for 1 h. Spermatozoa from 7 fertile and 3 infertile dogs were used for this study. The optimal sperm concentration for hemizona insemination was 1 x 10(6)/mL capacitated and motile spermatozoa. A significant difference (P < 0.001) was found the number of tightly zonabound spermatozoa between fertile and infertile dogs. Although there was a small difference in zona binding capacity between ejaculates of the same fertile dog (44 +/- 18.24), the main cause for the difference mentioned above was that of poor zona pellucida-binding capacity of spermatozoa from infertile dogs. We found a maximum of 14.28% bad oocytes when we compared sperm samples from 3 fertile and 3 infertile dogs in 56 HZA replicates. To avoid the effect of bad zona on sperm binding we calculated 37 (95% CI) bound spermatozoa from infertile dogs in 56 replicates. Thus, an HZA experiment in which a control dog had < 37 zona bound spermatozoa was repeated. Based on a minimum of 37 bound spermatozoa for fertile males (controls), a differential zona binding capacity and hemizona index (HZI) between fertile and infertile dogs and between 2 fertile dogs was determined. The binding differential between fertile and infertile dogs was 64.92 +/- 24.29, while between 2 fertile dogs it was 22.38 +/- 10.02 (P < 0.001). According to the HZI values, a value equal to or less than 41.11 indicated an infertile test dog, while an HZI value equal to or greater than 57.95 indicated a fertile test dog. Any value between these two could indicate either fertility or infertility. The evaluation of fertilizing potential of spermatozoa can be improved using the HZA protocols described above. PMID- 10734487 TI - Separation of motile spermatozoa from frozen-thawed buffalo semen: swim-up vs filtration procedures. AB - Three experiments were conducted to maximize the recovery rate of motile spermatozoa from frozen-thawed buffalo semen. In Experiment 1, the swim-up of motile spermatozoa was performed in the presence or absence of HEPES in TALP medium and CO2 in the environment. The recovery rate of motile spermatozoa in TALP medium (control), TALP + HEPES + CO2, TALP + HEPES and TALP + CO2 was 15, 18, 12 and 10%, respectively (P > 0.05), with sperm motility at 87, 89, 90 and 90%, respectively (P > 0.05). In Experiment 2, the pH of TALP medium was adjusted to 7.0, 7.5, 8.0, 8.5 and 9.0, and swim-up procedure was performed in the presence of HEPES and CO2. The recovery rate of motile spermatozoa at different pH was 14, 20, 24, 27 and 16%, respectively (P < 0.05). Motility of separated spermatozoa was 88, 91, 90, 89 and 90%, respectively (P > 0.05). In Experiment 3, the efficiency of ion-exchange filtration and Swim-up procedure in separating motile spermatozoa from frozen-thawed buffalo semen was compared. The recovery rate of motile spermatozoa was 95% in filtration procedure and 33% in swim-up procedure (P < 0.005). In all experiments, normal acrosomes did not vary due to treatments (P > 0.05). In conclusion, HEPES and CO2 had no significant effect on swim-up of buffalo spermatozoa. The pH 8.5 of TALP improved the recovery rate of motile spermatozoa in swim-up procedure. The ion-exchange filtration was found superior to swim-up procedure in harvesting maximum number of motile spermatozoa from frozen-thawed buffalo semen (95 vs 33%; P < 0.001). PMID- 10734488 TI - The isolation and in vitro culture of bovine preantral and early antral follicles of different size classes. AB - The ovary of cattle contains thousands of oocytes which are enclosed primarily in the preantral follicles. Methods of culturing preantral follicles are now being developed. The aim of this study was to investigate the effect of the size of bovine preantral and early antral follicles and culture media on their in vitro growth. Individual follicles isolated by microdissection of the ovarian slices were sorted into the following size classes: 75 to 124, 125 to 174, 175 to 224, 225 to 274, 275 to 324 and > or = 325 microns. The follicles were cultured individually in TCM 199 + fetal calf serum (FCS) + supplements (FSH, estradiol-17 beta, insulin, transferrin, sodium selenite, sodium pyruvate, 1-glutamine and hypoxanthine) or in Menezo B2 + FCS + supplements (Experiment 1) and in TCM 199 + steer serum (SS) with or without additional supplements (Experiment 2). The total number of isolated follicles of different size classes was similar in heifers and cows. No significant difference in the growth rate of follicles of different sizes was seen in the 2 media (TC 199 and B2). However, the culture of follicles in the TCM 199 that was supplemented only with SS and contained no other additives significantly reduced follicular survival and growth in comparison with follicles cultured in the supplemented medium. The survival time of follicles was related to their initial size at the beginning of culture. The longest period of growth was for follicles 275 to 324 microns in diameter (i.e., 10.7 +/- 5.7; 12.1 +/- 6.2 and 12.2 +/- 2.7 d, respectively, for culture in supplemented Menezo B2, TCM 199 + FCS and TCM 199 + SS). Survival and growth of some follicles was maintained for 23 d. PMID- 10734489 TI - Computer analysis of video and ultrasonographic images for evaluation of bull testes. AB - The objectives of this study were to determine relationships between scrotal size (SC; estimated from a video image) and testicular size, and between ultrasonographic echotexture of the testis and seminiferous tubule area in bulls. Video images of the scrotum of 49 Holstein-Friesian (H-F) bulls were recorded and digitized. Scrotal width and length were measured with custom software. After slaughter, scrotums (containing testes) were excised, SC and testicular height, width and volume were measured, and the testes were examined ultrasonographically. Correlations between SC and testicular width or volume (r = 0.86, P < 0.001 and r = 0.84, P < 0.001, respectively) were much higher than those between scrotal width and testicular width or volume (r = 0.23, P < 0.11 and r = 0.28, P < 0.06). Histological examination of the testes was performed in 31 of the bulls. Ultrasonographic echotexture of the testes (determined with custom software) was highly correlated (r = -0.5, P < 0.005) with seminiferous tubule area. Although SC was superior to video imaging for estimating testicular size, ultrasonographic imaging of the testes has considerable potential for the evaluation of testicular function in bulls. PMID- 10734490 TI - Delayed insemination is successful with a new extender for storing fresh equine semen at 15 degrees C under aerobic conditions. AB - Milk-based semen diluents are known to be practical and effective in protecting equine spermatozoa during storage before artificial insemination. Milk is a biological fluid with a complex composition and contains components which are beneficial or harmful to spermatozoa. The aim of this study was to test the fertility of stallion semen after long-term storage using different milk diluents (INRA 82 or Kenney's diluent) vs one diluent chemically defined (INRA 96), which is composed of efficient milk components and optimized for sperm survival and storage temperature. The milk fraction used was that which best maintained spermatozoal survival based on motility measured in previous studies. Four breeding trials were conducted to determine the influence of combination of new diluent and storage conditions on fertility of the stallion. We compared the standard protocol of storing semen in a skim milk diluent (INRA 82 or Kenney's diluent) at 4 degrees C under anaerobic conditions with the experimental protocol which consisted of storing in a chemically defined, milk-free diluent (INRA 96), at 15 degrees C, under aerobic conditions. After 4 breeding trials, in which the semen was stored for 24 h under the 2 protocols, we obtained 57% (n = 178) and 40% (n = 173) of fertility per cycle using the experimental and the standard protocol respectively (p < 0.001). Another breeding trial was conducted to determine the influence of storage time on the fertility of spermatozoa. We have compared the fertility of semen inseminated immediately (68% of fertility per cycle, n = 50) vs the fertility of semen stored under the experimental protocol for 72 h before insemination (48% of fertility per cycle, n = 52). The experimental protocol improved sperm fertility compared to the standard protocol and seems to be a particular alternative for stallions with cold shock sensitive spermatozoa. Storing semen for 72 h under the experimental protocol seems to be useful in the field. PMID- 10734492 TI - Effects of arterial infusions of adrenalin and acetylcholine on luteal secretion of progesterone and oxytocin in goats. AB - The effects of close intra-arterial infusion of acetylcholine and adrenalin on ovarian secretion of progesterone and oxytocin were examined on Day 10 of the estrous cycle in goats (estrus = Day 0). Acetylcholine (15 micrograms/min) was without effect, but adrenalin (10 micrograms/min) significantly (P < 0.001) raised both progesterone and oxytocin concentrations in ovarian vein plasma. These results show that luteal hormone secretion is enhanced in the goat by beta adrenergic stimulation and suggest that, as in the sheep and cow, there may be neuroendocrine involvement in the regulation of caprine luteal function. PMID- 10734491 TI - Pregnancy-specific elevations in fecal concentrations of estradiol, testosterone and progesterone in the domestic dog (Canis familiaris). AB - Estradiol (E2), testosterone (T) and progesterone (P4) concentrations were determined by enzyme-immunoassay in aqueous extracts of fecal samples obtained during anestrus, proestrus, estrus and metestrus of 11 nonpregnant and 11 pregnant bitches. Fecal hormone concentrations (ng/g) changed in relation to stage of cycle. Mean fecal steroid concentrations in 22 anestrous bitches and 3 ovariectomized bitches were low and similar for E2 (53 +/- 5 and 27 +/- 2), T (60 +/- 7 and 36 +/- 6), and P4 (62 +/- 6 and 86 +/- 15). Within 0 to 3 d of the ovulatory LH surge fecal E2 reached peak concentrations (301 +/- 38). The T peaks (281 +/- 41) were coincident or 1 to 3 d later. Fecal P4 was then elevated for approximately 2 m.o. Between Days 26 and 45 after ovulation, mean fecal P4 concentrations were higher (P < 0.05) in pregnant (401 +/- 60) than in nonpregnant bitches (164 +/- 23) and peak fecal P4 concentrations in individual animals were higher (P < 0.01) in pregnant (812 +/- 121) than in nonpregnant bitches (425 +/- 97). In the same period mean concentrations of E2 (117 +/- 13 vs 61 +/- 5) and T (102 +/- 10 vs 70 +/- 6) were also higher (P < or = 0.05) in pregnant than in nonpregnant bitches. Serum E2, T and P4 concentration were positively correlated (P = 0.1) with concentration in fecal samples obtained one day after serum collection. Although serial fecal ovarian steroid concentrations demonstrate the time course of ovulatory cycles, the diagnostic value of individual fecal samples appears limited. The ratios of peak to basal values were approximately 6, 5 and 7 for E2, T and P4, respectively, and were considerably lower than ratios of 12 to 50 previously reported for serum or plasma concentrations. The results demonstrate that there are pregnancy-specific increases in P4, E2 and T production reflected in fecal concentrations. While such increases are reflected in fecal samples, they are generally not evident in serum or plasma concentrations because of increased hemodilution, metabolism and clearance in pregnant bitches. The physiological stimulus for these increases, presumably ovarian in origin, or the potential role of prolactin is not known. PMID- 10734493 TI - Measurement of early pregnancy factor activity for monitoring the viability of the equine embryo. AB - The viability of embryos before flushing from donor mares (n = 5) and after transfer to recipient mares (n = 7) was monitored in mare serum by detecting early pregnancy factor (EPF) using the rosette inhibition test (RIT). The EPF activity was measured in donor mares before and after natural mating at natural estrus; after ovulation on Days 2, 5 and 8; and after embryo flushing (Day 8) on Days 8, 9, 10 and 13 after ovulation. The collected embryos were transferred immediately after flushing. The EPF activity in recipient mares were measured on the day of transfer and after embryo transfer on Days 1, 2, 3 and 5. Pregnancy was confirmed on Day 12 to 14 after embryo transfer. The mean EPF activity of donor mares was increased to the pregnant level (> an RI titer score of 10) on Day 2 after ovulation. Two days after flushing the embryos, the EPF activity of donor mares had decreased to the nonpregnant level. Among the 7 recipient mares, 3 mares were diagnosed pregnant on Day 12 after embryo transfer with ultrasound. The EPF activity of the pregnant recipient mares was increased above the minimum level observed in pregnant mares on Days 2 to 3 after transfer. However, among the nonpregnant recipient mares after embryo transfer, the EPF activity of 3 mares remained at the pregnant level only 2 to 3 d and then declined to the nonpregnant level. In one recipient mare, EPF activity did not reach the pregnant level throughout the sample collection. The results of this study indicated that equine EPF can be detected in serum of pregnant mares as early as Day 2 after ovulation. From our observation, we conclude that the measurement of EPF activity is useful for monitoring the in vivo viability of equine embryos and early detection of embryonic death. PMID- 10734494 TI - Fetal sex determination by ultrasonically locating the genital tubercle in ewes. AB - Accuracy of transrectal ultrasonographic determination of fetal sex by identifying and locating the genital tubercle was determined in 29 ewes with single fetus at 60 to 69 d post breeding. Examinations were conducted once under farm conditions utilizing a real-time diagnostic scanner equipped with a linear array 5 MHz transducer. Definite diagnoses were made after lambing. Total accuracy was 89%, and 100% (14/14) of the male fetuses and, 76% (10/13) of the female fetuses were correctly diagnosed. In 2 (7%) ewes, fetal sex was not determinable. PMID- 10734495 TI - Intracellular calcium responses in bovine oocytes induced by spermatozoa and by reagents. AB - The objectives of the present study were to clarify and compare the characteristics of the transient rises in intracellular calcium concentrations ([Ca2+]i) induced either by spermatozoa or by stimulation with artificial activators in bovine oocytes. These transient rises in [Ca2+]i in oocytes matured in vitro were recorded with Ca2+ imaging using the Ca2+ indicator fura-2. During fertilization, a series of transient rises in [Ca2+]i was observed. The first Ca2+ response peaked at a concentration of 521 +/- 39 nM (n = 20) and lasted for 4 min, while the subsequent Ca2+ responses were significantly smaller and shorter, with a peak of 368 +/- 13 nM (n = 23) and a duration of 2 min. Injection of inositol 1,4,5- triphosphate (InsP3) into unfertilized oocytes caused a transient rise in [Ca2+]i in a dose-dependent manner. The maximum response was induced by 20 nA x 1 sec injection of InsP3. Thimerosal, a sulfhydryl reagent, induced the repetitive transient rises in [Ca2+]i. The peak and the duration of the rises in [Ca2+]i induced by InsP3 or thimerosal were smaller and shorter, respectively, than those of the first rise induced by spermatozoa. Ethanol and Ca2+ ionophore IA23187, which are general parthenogenetic activators of unfertilized oocytes, each induced a single transient rise in [Ca2+]i. The duration of the rise in [Ca2+]i by ethanol or Ca2+ ionophore was significantly longer than that by spermatozoa at fertilization, although the peaks were smaller. These results clarified the characteristics of the rises in [Ca2+]i induced by spermatozoa and by several artificial reagents, and showed that the first rise in [Ca2+]i induced by spermatozoa had a higher peak [Ca2+]i and a longer duration compared with each the subsequent rises in [Ca2+]i and the rises in [Ca2+]i induced by artificial reagents. These indicate that a mode like as the first rise in [Ca2+]i induced by spermatozoa is an effective trigger for artificial activation of oocytes. PMID- 10734496 TI - Circadian and pulsatile variations in plasma levels of inhibin, FSH, LH and testosterone in adult Murrah buffalo bulls. AB - The present study investigated pulsatile and circadian variations in the circulatory levels of inhibin, gonadotrophins and testosterone. Six adult buffalo bulls (6 to 7 yr of age) were fitted with indwelling jugular vein catheters, and blood samples were collected at 2-h intervals for a period of 24 h and then at 15 min interval for 5 h. Plasma concentrations of inhibin, FSH, LH and testosterone were determined by specific radioimmunoassays. Plasma inhibin levels in Murrah buffalo bulls ranged between 0.201 to 0.429 ng/mL, with a mean of 0.278 +/- 0.023 ng/mL. No inhibin pulses could be detected during the 15-min sampling interval. Plasma FSH levels ranged between 0.95 to 3.61 ng/mL, the mean concentration of FSH over 24 h was 1.66 +/- 0.25 ng/mL. A single FSH pulse was detected in 2 of 6 bulls. The LH levels in peripheral circulation ranged between 0.92 to 9.91 ng/mL, with a mean concentration of 3.33 +/- 1.02 ng/mL. Pulsatility was detected in LH secretion with an average of 0.6 pulses/h. Plasma testosterone levels in 4 buffalo bulls ranged from 0.19 to 2.99 ng/mL, the mean level over 24 h were 1.34 +/- 0.52 ng/mL. Testosterone levels in peripheral circulation followed the LH secretory pattern, with an average of 0.32 pulses/h. The results indicate parallelism in inhibin, FSH and LH, and testosterone secretory pattern. Divergence in LH and FSH secretory patterns in adult buffalo bulls might be due to the presence of appreciable amounts of peripheral inhibin. PMID- 10734497 TI - Bacteria in semen used for IVF affect embryo viability but can be removed by stripping cumulus cells by vortexing. AB - Bacterial contamination of in vitro vs in vivo produced embryos presents a particular danger because of the alteration of the zona pellucida and the use of various biological products during culture. Our objective was to investigate the effects of semen contaminated with bacteria on IVF of bovine oocytes and to determine if removal of cumulus cells by vortexing as opposed to pipetting would reduce contamination and improve subsequent embryonic development. Semen from 5 bulls of the Native Korean breed (Bulls A, B, C, D, E) was used for IVF of matured oocytes. Preliminary studies had shown that the semen from Bulls A, B, D and E but not Bull C was contaminated with various species of common bacteria. After IVF, the cumulus cells surrounding the oocytes were removed either by pipetting or vortexing. Viability and cleavage rates of the resulting zygotes was assessed after 44 h in culture. When cumulus cells were removed by pipetting, only zygotes derived from oocytes that were fertilized with uncontaminated semen from Bull C developed to morula and blastocyst stages; zygotes derived from oocytes that were fertilized with contaminated semen from Bulls A, B, D and E started to degenerate, and the culture media became noticeably turbid. When cumulus cells were removed by vortexing, zygotes derived from oocytes fertilized with either contaminated or uncontaminated semen showed good rates of development (16 to 32%) to morula or blastocyst stages. From these results it can be concluded that the bacteria introduced with the semen contaminated the in vitro system and severely reduced the viability of the embryos. In contrast, complete removal of the cumulus cells with vortexing, as opposed to pipetting, reduced the contamination of the culture medium, allowing embryonic development to take place. PMID- 10734498 TI - The effect of reducing hindquarter elevation time after artificial insemination in bitches. AB - Hindquarter elevation time after artificial insemination in dogs was reduced from the common and arbitrarily used 10 min to only 1 min after insemination. Artificial insemination with fresh undiluted semen was conducted in 32 breedings using 15 hound bitches. The overall pregnancy rate was 91% (29/32), with an average litter size of 7.35 puppies per pregnancy. The pregnancy rate was not altered by reducing the 10-min (n = 14) hindquarter elevation time to 1 min (n = 18; P = 0.30). Similarly, the litter size was not different between groups (P = 0.40). PMID- 10734499 TI - Evaluation of fluids from cystic follicles for in vitro maturation and fertilization of bovine oocytes. AB - Follicular cysts are defined as cystic structures derived from unovulated follicles. The formation of the cysts appears to be related to failure of the oocyte to resume meiosis. The aim of this study was to evaluate in the bovine: 1) the ability of the fluid from cystic follicles to promote in vitro oocyte maturation and fertilization, 2) the predictive value of the morphology of oocytes derived from cystic follicles on the ability of the follicular fluid to promote in vitro maturation/fertilization as well as the oocytes to undergo maturation and fertilization. In Experiment 1, the ability of fluid from cystic (and normal) follicles from live and slaughtered cows (to promote) in vitro maturation and fertilization of bovine cumulus-oocyte-complexes (COC's) was assessed by cumulus expansion, sperm penetration, male pronucleus formation and polyspermy rates. Concentrations of progesterone (P4) and estradiol-17 beta (E2) were measured in the fluid from cystic follicles collected from live and slaughtered cows. In Experiment 2, we investigated the relationship of the morphology of COC's from cystic follicles, and the effect of the follicular fluids on oocyte maturation as well as P4 and E2 concentrations. In Experiment 1, although sperm penetration and male pronucleus formation were inhibited significantly by fluid from some cystic follicles collected from live and slaughtered cows, there were no significant differences in sperm penetration, male pronucleus formation and polyspermy rates between fluid from cystic follicles collected from live cows, from slaughtered cows and from control groups, regardless of the P4/E2 ratio. In Experiment 2, the morphology of cumulus oocyte complexes from cystic follicles varied and the pronucleus formation of oocytes after in vitro fertilization was abnormal. On the other hand, the male pronucleus formation rates were not significantly different between the cystic follicular fluids and control, regardless, of the P4/E2 ratio. The results of this study suggest that many of the bovine follicular fluids from cystic follicles possess the ability to induce cumulus expansion, nuclear maturation and male pronucleus formation following in vitro maturation and fertilization of bovine oocytes. The morphology of the cumulus-oocytes complexes from cystic follicles seems not to relate to the ability of the cystic follicular fluids to induce oocyte maturation, and oocytes from cystic follicles possess the ability to form male pronucleus even though most were abnormal after in vitro fertilization. PMID- 10734500 TI - Immunolocalization of P450C17 in the mare corpus luteum. AB - Although the mare corpus luteum (CL) is capable of aromatization, the expression of other enzymes involved in estradiol synthesis is not yet clear. This study examined the localization of P450C17 in the mare CL at different stages of its functional development. In ovaries from follicular phase mares P450C17 was localized in the theca cells of ovarian follicles. Following ovulation, no immunostaining for P450C17 was detected in the mature CLs of nonpregnant mares. In pregnant mares, no immunostaining for P450C17 was identified in the corpus luteum prior to secretion of eCG by the feto placental unit at Day 35 of pregnancy. The P450C17 was found to be expressed in CLs retrieved from Day 40 of pregnancy onwards. The changing expression of P450C17 raises the possibility that this may be a regulatory step for estrogen synthesis in the mare ovary. PMID- 10734501 TI - Urgent need to prevent abortion-related maternal deaths in Africa. PMID- 10734502 TI - Abortion: behaviour of adolescents in two districts in Kenya. AB - BACKGROUND: In Kenya the reported high rates of unwanted pregnancies (more than 90%), among adolescents have subsequently resulted in unsafely induced abortions with the associated high morbidity and mortality rates. OBJECTIVE: To evaluate the adolescents' behaviour regarding induced abortion. DESIGN: A cross-sectional, prospective study done from July 1995 to June 1996. SETTING: Schools and health facilities in Kiambu and Nairobi districts in Kenya. PARTICIPANTS: Interviews were conducted among adolescents aged 10-19 years in schools at the two districts and selected using a multi-stage random sampling procedure, as well as adolescent girls at two hospitals and two clinics in the immediate post-abortion period. MAIN OUTCOME MEASURES: The number of adolescents health programmes, aimed at reducing the dangers of unsafely induced abortion, which are designed and subsequently implemented. DATA COLLECTION: Demographic and health data, as well as data on behaviour regarding induced abortion were collected using a self administered questionnaire. RESULTS: The study sample comprised 1820 adolescents. These were 1048 school girls (SG), 580 school boys (SB) and 192 post-abortion (PA). Many adolescents were aware of abortion dangers, with the awareness being significantly lower among the SB whose girlfriends (GF) had aborted than those whose GF had not (p < 0.01). The practice of abortion was reported among 3.4% SG, 9.3% SBs' GF and 100% PA. Direct and indirect costs of abortion were heavy on the girls. Knowledge of the abortion dangers had no influence on the choice of the abortionist. Abortion encounter positively influenced approval by the adolescents, of abortion for pregnant school girls (p < 0.01). CONCLUSION: Despite the costs and awareness of abortion dangers by adolescents, they will take risks. PMID- 10734503 TI - Magnitude of abortion-related complications in Ethiopian health facilities: a national assessment. AB - OBJECTIVE: To describe the occurrence of abortion-related complications, available treatment and referral arrangements at various level of the Ethiopian health system, including those in remote areas. DESIGN: A cross sectional descriptive study. SETTING: The study was carried out in all but two of the regions in Ethiopia. SUBJECTS AND PARTICIPANTS: From the respective regions, health facilities were randomly selected. Health workers and traditional birth attendants were interviewed and operations registers were reviewed. RESULTS: In hospitals, the interviewees estimated that an average of 17 patients with post abortion complications were seen per month, in lower level health facilities providing post-abortion care the estimate was one patient per month. In lower level units not providing post-abortion care, 84% of the interviewees responded that they saw patients with complications due to abortion. The study shows that abortion complications occur in both urban and rural areas and are seen not only in hospitals, but also in health centres and at health stations, many of which are ill equipped to handle the patients. CONCLUSION: The results indicate a need to address services to improve treatment of abortion complications and other reproductive health services, as well as the provision of education to prevent unsafe abortions with both communities and lower level health facilities that serve them. PMID- 10734504 TI - Effect of misoprostol for cervical ripening prior to pregnancy interruption before twelve weeks of gestation. AB - BACKGROUND: Liberalization of the law in respect of legal abortions has led to a search for an appropriate technique for termination of pregnancy. The technique should be cheap, easy to perform and have minimal or no complications. AIM: To evaluate the effectiveness of performing manual vacuum aspiration (MVA) with and without the use of misoprostol to the procedure. DESIGN: Randomised control study. SETTING: Obstetrics and Gynaecology Department, University of Natal Medical School, South Africa. RESULTS: One hundred and thirty six women were recruited; 70 women were assigned to the misoprostol group. Of these, 11 (15%) did not show any change in cervical score. Their mean cervical dilatation was similar to the control group (3.3 versus 31; p > 0.06). In the group whose gestational age was less than eight weeks, the time taken to complete the procedure, quantity of products of conception and cervical dilatation, were different from that of the control group, and this reached statistical significance except quantity of products of conception in primigravidae. In pregnancies greater than eight weeks gestation, all parameters assessed, such as cervical dilatation, quantity of products of conception was significantly different from the control group, in both multi- and primigravidae. Pain score was similar for all gestations. CONCLUSION: Misoprostol is of specific value during MVA for voluntary interruption of pregnancy. PMID- 10734505 TI - Abortion: knowledge and perceptions of adolescents in two districts in Kenya. AB - BACKGROUND: Pregnancy among adolescents is unplanned in many instances. Although some pregnant adolescents carry the pregnancy to term, abortion, in many instances unsafely induced, is a commonly sought solution in Kenya. OBJECTIVE: To determine adolescents' perceptions of induced abortion. DESIGN: A cross-sectional descriptive study carried out between July 1995 and June 1996. SETTING: An urban and a rural district in Kenya. PARTICIPANTS: Adolescents aged 10-19 years in schools in Nairobi and Kiambu districts, and a group of immediate post-abortion adolescent girls in some health facilities in Nairobi. MAIN OUTCOME MEASURES: The number of health programmes formulated and put into use, which are adolescent friendly and providing information, education and communication on abortion issues. DATA COLLECTION: One thousand eight hundred and twenty adolescents were subjected to a self-administered questionnaire that collected demographic and health data as well as perceptions of induced abortion. Focus group discussions on perceptions of abortion were held with 12 groups of adolescents in schools and the information obtained recorded on paper and in a tape-recorder. RESULTS: One thousand nine hundred and fifty two adolescents, comprising of 1048 school girls (SG), 580 boys (SB), 192 post-abortion girls (PA) and 132 adolescents in the focus group discussions, formed the study sample. More than 90% were aware of induced abortion (IA). Knowledge of IA correlated positively with level of education (P < 0.01). Seventy one per cent of SG, 84% of PA and 40% of SB were aware of abortion-related complications, the most common being infections, death and infertility. Eighty three per cent of PA felt that complications were preventable by seeking care from a qualified doctor compared to one quarter each for the SB and SG. 56% PA, 69% SB and 72% SG felt that abortions were preventable. However, less than 40% proposed abstinence as a primary strategy. The most important source of information on abortion was the media followed by friends and teachers. CONCLUSION: Adolescents are aware of abortion and the related complications, but there is more variability in their knowledge and preventive measures. PMID- 10734506 TI - Serum free carnitine levels in children with kwashiorkor. AB - OBJECTIVE: To determine the serum free carnitine concentration in normally nourished children and in children with kwashiorkor and to relate the carnitine concentration to the ability to oxidise exogenous long chain fatty acids in the body. STUDY DESIGN: A cross-sectional comparative study of two age-matched groups. SUBJECTS: Forty seven children with kwashiorkor and 47 age-matched normally nourished children. MAIN OUTCOME MEASURES: Fasting blood samples were enzymatically analysed for free carnitine levels. 13C labelled hiolein was administered orally and the recovery of 13C from the breath air was monitored after administration of the feed. The cumulative per cent dose (CUMPD) recovery of 13C 16 hours after the ingestion of labelled hiolein was determined. RESULTS: Normal children had significantly higher free carnitine concentrations (mean = 60.7 mumol/l; 95% confidence interval of the mean = 42.7-77.8) than the kwashiorkor children (mean = 16.5 mumol/l; 95% confidence interval of the mean = 11.3-19.8)(p < 0.001). There was no correlation between serum free carnitine concentration and serum albumin in kwashiorkor subjects, but there was a significant correlation between serum free carnitine concentration and the degree of weight loss as indicated by the weight: weight for age and sex ratio. The greater the weight loss, the lower the serum carnitine concentration amongst the kwashiorkor children (r = 0.46; p < 0.01). There was a linear relationship between serum free carnitine and hiolein oxidation (r = 0.89; p < 0.001). CONCLUSION: There is carnitine deficiency in kwashiorkor, and that the impaired lipid oxidation in kwashiorkor is related to this deficiency. PMID- 10734507 TI - Symptomatic HIV-infection in infants according to serostatus of mothers during pregnancy. AB - OBJECTIVE: To ascertain the pattern of symptomatic HIV infections, HIV seropositivity and mother to child transmission (MTCT) rate during a two-year follow up period at 6, 12, 18, and 24 months according to mothers serostatus during pregnancy. DESIGN: A longitudinal cohort study with socio-economic, medical, and demographic history of all participating pregnant women from recruitment; and of the child at birth recorded separately on pre-constructed questionnaire. The medical condition of each infant was recorded at the periodic follow-ups. SETTING: The maternal and paediatric wards of the Harare Central Hospital, Harare, Zimbabwe. SUBJECTS: Three hundred and eighty six HIV seropositive and 372 HIV-seronegative Zimbabwean pregnant women attending maternal and paediatric wards of Harare Central Hospital. INTERVENTIONS: About 10 ml of umbilical (venous) blood was collected at birth, centrifuged after clotting and stored frozen until HIV tests were carried out. Also capillary blood for filter paper storage was taken at six and 16 months and venous blood at one and two years of age for HIV serology, using ELISA. HIV serostatus was confirmed using Western Blot (WB) technique. MAIN OUTCOME MEASURES: The HIV-serostatus of participating pregnant women and of infants at birth, patterns of symptomatic HIV infections and HIV seropositivity among infants and the number of deaths during a two year follow up at periodic intervals. RESULTS: A total of 108 infant deaths occurred within the follow up period. Significantly more deaths (82 versus 25, p < 0.0001; excluding one infant whose HIV serostatus was not determined), parotitis, palpable neck and groin lymph nodes occurred among infants born to HIV positive than those born to HIV negative mothers. Palpable axillary lymph nodes were significantly more common at 12, 18, and 24 months of follow up in infants born to HIV positive women. The maternal HIV serostatus during pregnancy and of infants at all periodic follow ups showed no association with persistent diarrhoea, fever and cough. However prolonged fever became significantly associated at 24 months. CONCLUSION: Mother to child transmission (MTCT) rate based on the HIV-serostatus of 272 infants; 17.4% in utero and 11.1% via breast milk respectively. Multivariate analysis showed that an infant presenting with faltering growth (Odds Ratio [OR] = 5.597), palpable neck (OR = 5.919) and axillary lymph nodes (OR = 4.197) was likely to be HIV positive at 24 months. PMID- 10734508 TI - Experience with high dose dexamethasone in the treatment of chronic symptomatic immune thrombocytopaenia. AB - OBJECTIVE: To evaluate the efficacy of high dose dexamethasone (HDD) as treatment for symptomatic chronic immune thrombocytopaenia (ITP). DESIGN: A non-randomised intervention study with final evaluation one year after treatment, comparing findings before and after intervention. SETTING: Tygerberg University Hospital, South Africa. PARTICIPANTS: A consecutive sample of six children with chronic (duration more than six months) ITP. The diagnosis of ITP was based on a platelet count of < 100 x 10(9)/1 together with appropriate clinical, laboratory and bone marrow findings. INTERVENTIONS: All children treated with dexamethasone 0.5 mg/kg/day intravenously for four days every 28 days for a total of six cycles. MAIN OUTCOME MEASURES: A rise in platelet count maintained for a least one year associated with the disappearance of symptoms due to thrombocytopaenia. RESULTS: Treatment was easy to administer and well tolerated with transient side effects in only two children. Three patients had a rise in platelet count of > 50 x 10(9)/1 during treatment and three had platelet counts of > 30 x 10(9)/1 after completion of therapy but only one at one month and one at six months after completion of the six courses respectively. None of the patients showed a sustained rise in platelet count during and after HDD treatment. CONCLUSION: HDD did not cause a significant sustained rise in the platelet count in children with chronic symptomatic ITP. If high dose prednisone and IVIG fail, a splenectomy should be considered in children over five years of age. PMID- 10734509 TI - Syndrome of cervical spondylosis in Blantyre, Malawi. AB - OBJECTIVE: To define the spectrum of clinical and radiological presentations of cervical spondylosis in Malawians. DESIGN: A prospective study. SETTING: The Queen Elizabeth Central Hospital, Blantyre which is the main referral hospital in Malawi and the teaching hospital of Malawi College of Medicine. SUBJECTS: Thirty seven patients (nineteen females and eighteen males) who presented with cervical spondylosis between February 1997 and September 1998. INTERVENTIONS: Treatment was conservative except one patient with cervical myelopathy who had laminectomy. RESULTS: Four of the patients were aged below 40 years; six were in the seventh decade, 13 in the fifth and fourteen in the sixth decades of life. All the 37 patients had discogenic disorder which manifested as brachial neuropathy. In addition, five (13.5%) had cervical myelopathy and two (5.4%) each had vertebro basilar artery insufficiency, pseudoangina and dysphagia. Anterior osteophytes were three times as common as posterior osteophytes. CONCLUSION: In view of the various manifestations of cervical spondylosis the term cervical syndrome can be aptly applied to this disease. PMID- 10734510 TI - Blunt abdominal trauma in children at Kenyatta National Hospital. AB - OBJECTIVE: To determine the pattern of intra-abdominal injuries arising from blunt abdominal trauma and evaluate the management of blunt abdominal trauma and its outcome. DESIGN: Retrospective study. SETTING: Paediatric Surgical Department, Kenyatta National Hospital (KNH), Nairobi. SUBJECTS: Fifty-five children who were admitted and treated for blunt abdominal trauma in KNH between January 1983 and December 1993. MAIN OUTCOME MEASURES: Morbidity as determined by operative management and complications, mortality and period of hospital stay. RESULTS: Incidence of trauma was high in males with male to female ratio of 2:1. Incidence of trauma was prevalent in seven year age group. Motor vehicle accidents accounted for the majority of injuries. Most patients arrived late in hospital. Sixty per cent underwent laparotomy. Three quarters of these had positive findings. The spleen was the organ most commonly injured. Hypovolaemic shock was the most frequent complication. One patient died during management. On average, patients stayed for seven days in the ward before discharge. CONCLUSION: Blunt abdominal trauma more commonly affected male children. Motor vehicle accidents were a major aetiologic factor in blunt abdominal trauma in children seen in Kenyatta National Hospital. Twenty-five per cent of the children were subjected to unnecessary operation. This was due to unavailability of sensitive diagnostic modalities. PMID- 10734511 TI - Circumcision in various Nigerian and Kenyan hospitals. AB - OBJECTIVE: To present and evaluate circumcision practices in Nigerian and Kenyan Africans. DESIGN: Continuous prospective study involving:- two hundred and forty nine consecutive circumcisions performed from 1981 to 1998 and; fifty patients with post circumcision complications referred for further management during the same period. SETTING: Lagos University Teaching Hospital, Duro-Soleye Hospital and First Foundation Medical Centre in Lagos, Nigeria; and Kenyatta National Hospital, Mater Misericordiae and Nairobi Hospital in Nairobi, Kenya. SUBJECTS: Males of all ages presenting for circumcision and males of all ages referred with post circumcision complications. RESULTS: One hundred and fifty-two (61.04%) of circumcisions performed were in adolescents and young adults for cultural initiation into 'manhood'. The remaining seventy-nine (31.73%) neonates were circumcised for religious, parental, cultural and medical reasons. Eighty per cent of the patients referred with circumcision complications were initially circumcised by unqualified traditional "surgeons". One patient (2%) died from septicaemia; two patients (4%) lost their penis from gangrene and five other patients (10%) remained with permanent disability from complete or partial amputation of the penis or glans. CONCLUSION: This study shows that circumcision is a commonly performed operation but can have serious, life shattering and prolonged complications sometimes resulting in death. It should therefore be performed only in medical institutions under sterile conditions and by suitably trained surgeons for specific medical indications. Ritual circumcision whether mass or single for cultural or religious reasons remains controversial without medical benefit and should be discouraged. PMID- 10734512 TI - Quinolones for short-term treatment of uncomplicated urinary tract infection. AB - OBJECTIVE: To investigate whether quinolones produce in vivo responses comparable to reported in vitro activity against the spectrum of organisms in uncomplicated urinary tract infection (UTI) in Nigeria. DESIGN: Equal numbers of patients with urine culture positive UTI were randomized to oral quinolone, perfloxacin 400 mg bd twice a day and ofloxacin 200 mg bd twice a day for five days. SETTING: Out patient clinics and wards at the University College Hospital, Ibadan. SUBJECTS: Sixty patients aged sixteen years and above with uncomplicated UTI. MAIN OUTCOME MEASURES: Number of isolates, number of patients with clinical and bacteriological cure one week after commencing therapy. Relative effectiveness and side effects of the drugs. RESULTS: Sixty- four bacterial isolates were obtained with the enterobacteriaceae comprising 86%. Sixty-two (97%) were sensitive to both drugs in vitro. Clinical cure occurred in 57 patients (95%), being 28 (93%) in those taking perfloxacin and 29 (97%) in those taking ofloxacin. Bacteriological cure occurred in 55 patients (92%), being 27 (90%) in those taking perfloxacin and 28 (93%) in those taking ofloxacin. Fifty-nine of the 64 isolates (92%) were eliminated in one week, 30 of 33 (91%) in those taking perfloxacin, and 29 of 31 (94%) in those taking ofloxacin; the difference was insignificant (p = 1.16). The enterobacteriaceae were generally susceptible to both drugs. Side effects were minor and infrequent. CONCLUSION: Quinolones were highly active against the common urinary pathogens in these Nigerian patients and can be reliably employed in treatment when culture results are unavailable. PMID- 10734513 TI - Malnutrition and xerophthalmia in rural communities of Ethiopia. AB - OBJECTIVE: To determine the level of malnutrition and xerophthalmia in pre-school children. DESIGNS: Non-randomised community based study. SETTINGS: Four different administrative regions: Harari, Tigray, Southern Nation Nationalities and people region (SNNPR) and Oromiya, with different eco-zones, were studied from May to June 1996. SUBJECTS: Fifteen thousand and eighty seven children, aged between six and 71 months, examined for clinical symptoms and signs of xerophthalmia. Anthropometry and blood samples were taken from every 20 children (n = 634) of same age, for serum retinol and nutritional determination. INTERVENTION MEASURES: Disease targeted approach of vitamin A supplementation was employed in the regions. RESULTS: The overall prevalence rates of night blindness and Bitot's spots exceeded WHO cut-off point for xerophthalmia as a public health problem, with higher prevalence rates in males (53%) than females (26%). The proportion of children with deficient serum retinol concentrations (SRC), and Bitot's spot were observed to be higher in Oromiya and Harari regions followed by Tigray, than SNNPR administrative regions. Most of the affected children were aged between 36 and 72 months. The greatest low SRC was also observed in the same age group of children in all regions. There was higher prevalence rate of stunting (60.1%) than wasting (12.2%) with an additional (8.8%) of children both stunted and wasted. The proportion of stunted children was high in Tigray followed by Oromiya, SNNPR and Harari regions. CONCLUSION: The high level of stunting and Bitot's spot, together with the low level of serum retinol concentrations found in these regions, indicates the need to strengthen this intervention strategy further with universal vitamin A capsule distribution, nutrition education and promotion of horticulture activities. PMID- 10734514 TI - Granulocytic sarcoma: report of three cases. AB - Granulocytic sarcoma (GS) is a rare extramedullary solid tumour composed of malignant immature cells of the granulocytic series. It may herald, accompany or signal acute myeloid leukaemia (AML) or chronic granulocytic leukaemia (CGL). GS may also occur in patients with myelodysplastic syndromes (MDS) where it is a sign of imminent disease progression. Three cases of GS are presented; the first one involving the pancreas and preceding AML, the second case affecting uterine cervix in stable phase CGL and the third case is GS of the breast accompanying AML. Any site of the body may be involved by the GS, and morbidity depends on the local organ/tissue affected in addition to the attending primary leukaemia or MDS. Treatment of GS involves surgery, radiotherapy and chemotherapy. The objective of this communication is to enhance awareness in personnel providing health care. Further, early diagnosis and treatment affects overall outcome. PMID- 10734515 TI - Severe malnutrition due to subtle neurologic deficits and epilepsy: report of three cases. AB - In southern and eastern Africa, where approximately eight per cent of households lack access to adequate food, children suffering from chronic infections such as tuberculosis, gastrointestinal parasites and human immunodeficiency virus, often present with severe protein energy malnutrition. Three cases are described of children presenting to Chikankata Salvation Army Hospital who required hospitalization and urgent feeding due to PEM. No underlying aetiology for their life-threatening PEM could initially be identified and they were all observed to gain weight while in the intensive feeding unit. After discharge, each re presented with recurrent failure-to-thrive and were found to have subtle neurologic deficits and underlying epilepsy. Epilepsy and developmental disabilities should be considered in patients with PEM for whom other aetiologies cannot be identified. PMID- 10734516 TI - Rheumatic heart disease. PMID- 10734517 TI - Prevalence of rheumatic heart disease among school children in Addis Ababa. AB - OBJECTIVE: To determine the prevalence of rheumatic heart disease (RHD), and the impact of potential risk factors on its occurrence among school children in Addis Ababa. DESIGN: Cross-sectional survey of school children for the presence or absence of RHD. SETTING: Randomly selected ten government and five private schools from the list of all government and private schools in Addis Ababa teaching grades 7 and 8. SUBJECTS: All school children attending grades 7 and 8 in the randomly selected schools. MAIN OUTCOME MEASURES: Clinical findings suggestive of RHD confirmed by echocardiographic and Doppler studies. RESULTS: Out of the 10,053 school children selected for the study 9,388 (93%) were examined. Sixty of the 9,388 school children examined were subsequently confirmed to have RHD giving an overall prevalence rate of 6.4 per 1000 children. The prevalence for government and private schools representing low and high socio economic groups were 7.1 and 1 per 1000 children respectively (Fisher's exact = 0.019). Crowding conditions at home, in schools and in the bedrooms were not associated with the risk of RHD (chi 2 = 4.968; p > or = 0.174). CONCLUSION: This study confirmed that the prevalence of RHD, at least in the low socio-economic group is among the highest in the world. This evidence highlights the need for an urgent control programme. PMID- 10734518 TI - Effects of passive smoking and breastfeeding on childhood bronchial asthma. AB - OBJECTIVE: To evaluate the effect of passive smoking and breastfeeding on the severity and age of onset of bronchial asthma. DESIGN: Cross-sectional study. SETTING: Paediatric observation ward and paediatric chest clinic, Kenyatta National Hospital. PATIENTS: Children aged between one and 120 months. RESULTS: More than fifty per cent of the children had their first wheeze at less than 12 months of age and 68% were categorised as having moderate to severe asthma. Passive smoking was positively significantly associated with early onset of wheezing (chi 2 = 6.22, p = 0.012, Odds ratio = 2.44, 95% CI 1.2,5.0), and also, at a non significant level, to severity of asthma (chi 2 = 2.8, p = 0.09, Odds ratio = 2.1, 95% CI 0.9,4.7). On the other hand, exclusive breastfeeding was significantly negatively associated with severity of bronchial asthma (chi 2 = 4.02, p = 0.045, Odds ratio = 0.4, 95% CI 0.14,0.98), but did not seen to have effect on age of onset of the disease (chi 2 = 0.0006, p = 0.94, Odds ratio = 0.9, 95% CI 0.4,2.2). CONCLUSION: Passive smoking is associated with early onset of asthma and possibly with development of severe asthma while exclusive breastfeeding is protective against development of severe asthma but does not seem to affect the age of onset of this disease. PMID- 10734519 TI - Megaloblastic marrow in macrocytic anaemias at Kenyatta National and M P Shah Hospitals, Nairobi. AB - OBJECTIVE: To determine the diagnostic value of megaloblastic marrow in patients with possible vitamin B12 and/or folate deficiency and to look for criteria that could be used in diagnosis of vitamin B12 deficiency. DESIGN: Prospective study. SETTING: Kenyatta National Hospital and M P Shah Hospital, Nairobi. SUBJECTS: Patients of all age groups and both sexes presenting with clinical and blood picture of macrocytic anaemia. MAIN OUTCOME MEASURES: Response to vitamin B12 injections. Vitamin B12 deficiency diagnosed by the following methods: Vitamin B12 serum level assays alone, bone marrow examination alone and both vitamin B12 assays and bone marrow examination. Response was measured clinically and by increase in haemoglobin level after three weeks of treatment. RESULTS: Three hundred and forty-three patients were evaluated; 156 (45.5%) were males, and 187 (54.5%) females. Age range was 13-80 years and a mean of 37.7 years. Initial investigations were as follows: 21.9% had vitamin B12 assays only, 59.2% bone marrow examination only and 19.9% both vitamin B12 assayed and bone marrow evaluated. Haemoglobin increase after a total of 6,000 mcg of vitamin B12 was between 2.6-4.6 g/dl in three weeks. Reticulocytes measured on day ten ranged between 12% and 17%. Uniform improvement in all patients was observed for white blood cells (WBC) and platelet counts. Other significant results and observations included clinical improvement in the signs and symptoms of anaemia after the 6,000 mcg injection of vitamin B12 given over three weeks. Serum folic acid level was determined in 21% of cases and in all these it was normal. CONCLUSION: The important practical points were response to vitamin B12 in all cases and the importance of re-assessing patients after a total of 6,000 mcg of vitamin B12. Folic acid deficiency was not detected. Patients in these two institutions with megaloblastic anaemia had vitamin B12 deficiency. It is proposed that a model consisting of oval macrocytosis on blood film and megablastic bone marrow be treated with vitamin B12 injections in settings where vitamin B12 serum level assays are not easily available. PMID- 10734521 TI - Antibody levels to Salmonella typhi and paratyphi in Nigerians. AB - OBJECTIVE: To determine the antibody titre levels to typhoid/paratyphoid fever organisms among apparently healthy volunteers. DESIGN: Cross sectional study. SETTING: General community and University Teaching Hospital. PARTICIPANTS: Volunteer sample of 323 apparently healthy individuals with body temperatures < or = 37.8 degrees C. MAIN OUTCOME MEASURES: Questionnaire administration to classify volunteers into three socio-economic status (SES). RESULTS: There were 35.29% of the apparently healthy population in Jos community with antibodies to typhoid/paratyphoid fever organisms. The presence of these antibodies were neither sex nor SES related. Normal antibody titres were up to 1:40 and 1:80 for O and H Salmonella antigens respectively. Contrary to the general belief, typhoid/paratyphoid fevers have not affected virtually everybody in Nigeria. The difference between those without previous history and those with previous history was significant (p < 0.05) with those in the former category having a higher percentage. CONCLUSION: For a single sero-diagnosis to have any diagnostic value in Jos community and its environs, only a four-fold rise to what has been found to be normal should be significant. This means that only titres of 1:160 and 1:320 and above for O and H antigens should be considered significant. PMID- 10734520 TI - Blood group distribution in an urban population of patient targeted blood donors. AB - BACKGROUND: The frequencies of ABO and Rhesus D blood groups vary from one population to another. OBJECTIVE: To determine the frequencies of the ABO and Rhesus D blood groups and to assess the preparedness of the hospital based patient targeted blood donor unit in the provision of blood and blood products and also to determine the blood groups of patients attending the antenatal clinic over the same period. DESIGN: A retrospective study using data from the hospital's blood transfusion unit. SETTING: The Aga Khan Hospital, Nairobi. SUBJECTS: All persons donating blood and attending the antenatal clinic between October 1997 and December 1997 were included in the study. RESULTS: Blood group O was found to be most frequent: 49% in indigenous African donors and ANC attendants, 45% in the general donor population and 34% among Asian donors. The frequency of blood group B was 33% in the Asian donors, 27% in all donors, 25% in African donors, and 24% in ANC attendants. Group AB was seven per cent in Asian donors, five per cent in both the general donor population and the ANC attendants and four per cent in the African donors. The frequency of blood group A was 26% in Asian donors, 23% in all donors and 22% in both the African donors and ANC attendants. Ninety four per cent of the indigenous African donors were Rhesus D positive, 97% of the ANC attendants were Rhesus D positive and 90% of the Asian donors were Rhesus D positive. CONCLUSION: The blood groups of blood and blood products available at the hospital based transfusion unit closely correlate with those of the general population. A positive bias towards the rarer blood groups AB and Rh D negative is noted. The Rhesus D antigen has a high prevalence in indigenous Africans. PMID- 10734523 TI - Diagnosis of tuberculosis in sputum negative patients in Dar es Salaam. AB - OBJECTIVE: To determine diagnostic criteria for tuberculosis among sputum smear acid fast bacilli negative patients with chronic cough, based on symptoms, signs and simple laboratory tests. DESIGN: A two-month prospective follow up study. SETTING: Muhimbili Medical Centre, Dar es Salaam, Tanzania. SUBJECTS: One hundred and seventy- eight consecutive patients admitted between 1st November, 1994 and 31st March, 1995 with chronic productive cough. MAIN OUTCOME MEASURES: Sputum smear acid fast bacilli (AAFB) negative with TB, discriminating ability of clinical and laboratory parameters. RESULTS: Forty three (24.2%) were sputum smear positive for acid fast bacilli (AAFB). In 90 (50.6%) patients, AAFB could be isolated in specimen other than sputum and in 45 (25.3%) no AAFB could be isolated. In a univariate analysis of all symptoms, signs and laboratory test results, cough of four or more weeks, haemoptysis, oral candidiasis, chest consolidation, pleural effusion, mid zone and upper zone chest x-ray opacities were significantly different between sputum AAFB negative TB and non TB patients. Discriminant analysis revealed six highly significant variables: Mantoux reaction, pleural effusion, Kaposi's lesion, cervical lymphadenopathy, matted lymph node, mid zone and upper zone CXR infiltrates. From these variables an equation was derived to calculate the probability that a sputum smear negative patient had tuberculosis. Then a scoring system was developed that classified correctly 84% of cases of sputum AAFB negative patients into tuberculosis or non tuberculosis. CONCLUSION: In sputum smear AAFB negative patient clinically suspected to have tuberculosis, Mantoux reaction, cervical lymphadenopathy, matted lymph nodes, absence of mid lower zone infiltration on CXR and presence of pleural effusion could be used for presumptive diagnosis, but they would not make a therapeutic trial unnecessary. PMID- 10734522 TI - Parasight-F dipstick antigen test in the diagnosis of falciparum malaria in Ethiopia. AB - OBJECTIVE: To determine the shelf life, rapidity and diagnostic performance of ParaSight-F. DESIGN: Prospective randomised study. SETTINGS: Malaria diagnostic and treatment posts and health centres in central-southern part of the Ethiopian Rift Valley. SUBJECTS: Three hundred and eighty two subjects randomly selected. MAIN OUTCOME MEASURES: Double blind evaluation of the sensitivity, specificity and predictive values of PFT versus CBF. RESULTS: The point prevalence was found to be 29.7% with species dominance of P. falciparum to P. vivax in the ratio of 4:1. The ParaSight-F test showed, considering P. falciparum only, a sensitivity of 92.5% and specificity of 93%. A remarkably high positive predictive value (PPV) of 82% as well as a negative predictive value (NPV) of 99% was obtained. Considering all species of Plasmodium, sensitivity was 78.6% and specificity 93%, with PPV and NPV of 82% and 91% respectively. Moreover, PFT was found to be rapid with a batch of ten-strips--a rack completed in 30 to 50 minutes, which was five to six times faster than CBF reading. In shelf-life studies, after one year of storage at room temperature, results were found similar to the original readings, indicating the stability and long durability of the test strips. CONCLUSION: In their durability and high diagnostic performances, both the microscopic slide readings and PFT were found comparable and interchangeable, and advantage in endemic areas where laboratory facilities are not available. The rapidity of PFT may be of greater value during malaria epidemics. But during non-epidemic seasons, the inability of PFT not to detect all forms of malaria remains a draw back. PMID- 10734524 TI - Clinical presentation of onchocerciasis among indigenous and migrant farmers in southwest Ethiopia. AB - OBJECTIVE: To show the magnitude of the problem and give a systematic account of the clinical manifestations of onchocerciasis in a rain forest ecology in southwest Ethiopia. DESIGN: A cross sectional clinico-epidemiological study. SETTING: Teppi Health Centre and Endris-Goji Clinic, Ethiopia. SUBJECTS: Four hundred and twenty one individuals aged ten years and above. RESULTS: Eighty four per cent of the examined people had a macrofilarial load ranging from one to 855 microfilariae per mg of skin snip. If the presence of any of skin microfilaria, nodule and/or leopard skin were taken as diagnostic criteria, the overall prevalence rate of onchocerciasis would be 88%. However, if we strictly followed stringent diagnostic criteria such as presence of unprompted itching, nodule(s), microfilaraedermia and skin dermatitis, the prevalence rate of onchocerciasis would be 23% (98/421). Hanging or pendular scrotum, which has been rarely reported in Africa but not until now in Ethiopia, was found in 19% (46/246) of the male patients. There was neither negative nor positive correlation between microfilarial load and severity of clinical manifestations of the disease. CONCLUSION: Onchocerciasis has been found to be one of the major health problems in this community and its diagnosis should not be based on selected signs and symptoms such as nodules and leopard skin alone that could undermine the magnitude of the problem. PMID- 10734526 TI - Re: An anti-rotation device in skin traction of the lower limb. PMID- 10734525 TI - Role of the spiritual churches in antenatal clinic default in Calabar, Nigeria. AB - OBJECTIVE: To establish the role played by spiritual churches in the observed increase rate of antenatal clinic default in Calabar, Nigeria. DESIGN: Cross sectional comparative study. SETTING: Calabar, an urban community, the capital of Cross River State in south-eastern part of Nigeria. SUBJECTS: Women who booked for antenatal care in the University of Calabar Teaching Hospital but delivered outside orthodox health facilities. RESULTS: A total of 44.3%, of our antenatal clinic defaulters delivered in spiritual churches. Fear of spiritual attack by wicked people and prophetic warning in church (48.3%) and high hospital bills (26.8%) were the major reasons for this act. Perineal tear, primary postpartum haemorrhage and prolonged labour were associated more with delivery in church than hospital delivery or home delivery or delivery by TBAs (p < 0.01). Birth asphyxia and birth trauma were also significantly higher in church than hospital and other unorthodox delivery centres (p < 0.01). All the maternal deaths occurred in church and perinatal mortality was higher in church than other delivery facilities. CONCLUSION: The spiritual churches play a major role in the increasing rate of antenatal clinic default in Calabar. The pregnancy outcome in these spiritual churches is worse than when delivered in hospital, by TBAs or at home. Adequate health education; reduction in delivery fees in hospitals and general improvement in the socio-economic status of the populace may help reduce these high maternal and perinatal morbidity and mortality in the spiritual churches. PMID- 10734527 TI - Naturalistic comparative study of outcome and cognitive effects of unmodified electro-convulsive therapy in schizophrenia, mania and severe depression in Nigeria. AB - BACKGROUND: Although unmodified electro-convulsive therapy (ECT) is widely used in Nigeria for schizophrenia, mania and severe depression failing to respond to adequate pharmacotherapy in the short-term, there are no prospective studies on its efficacy and side effects. OBJECTIVE: To compare the efficacy of electro convulsive therapy and standard pharmacotherapy in the treatment of schizophrenia, mania and severe depression. DESIGN: A prospective open label study. METHOD: Seventy subjects (mean age, 29.4) with mean duration of illness, 50.6 months, and diagnosis of schizophrenia (37.1%), mania (28.6%), severe depression (30%) and schizo-affective disorder (4.3%), were assessed pre-ECT and at seven other intervals in six months, using the Brief Psychiatric Rating Scale (BPRS), the Mini-Mental State Examination (MMSE) and other cognitive test batteries. They were compared with a matched group of patients who received only pharmacotherapy. RESULTS: Although the ECT group had suffered more treatment resistant disorders, there was full clinical recovery in two months, when all were discharged. The group had significantly shortened duration of hospitalisation, after commencement of ECT. Hence ECT facilitated recovery in this potentially drug treatment resistant psychotic subjects. Although frequency of complaints of subjective memory difficulty increased during ECT and normalised at follow up, objective tests showed steady cognitive improvement with clinical recovery. Complaints of muscle pain (31.4%), post-ECT confusion (15.7%) and post ECT headache (20%) in the first week of treatment, were not evident at follow up. CONCLUSION: Unmodified ECT combined with pharmacotherapy was safe and effective with non-enduring subjective memory difficulty for this potentially drug treatment resistant group of psychotic patients. PMID- 10734528 TI - Ethnomedical aspects of the commonly used toothbrush sticks in Ethiopia. AB - OBJECTIVE: To determine the botanical identity, cytotoxicity, and antibacterial property of the commonly used toothbrush sticks in Ethiopia. SETTING: Research laboratories of the departments of Biology and Chemistry, Addis Ababa University between December 1993 and May 1995. METHODS: The study was performed by purchasing the commonly used toothbrush sticks from street markets in various towns of Ethiopia. Voucher specimens were collected and their botanical identity was determined following floral keys. The toothbrush sticks were ground in a mill and soaked in absolute methanol for 24 hours and filtered. The filtrates were dried in a rotary evaporator and the crude extracts were stored at 4 degrees C. The crude methanol extracts were used to test their antibacterial activity by impregnating into filter paper discs and placing on test plates of Staphylococcus aureus and Bacillus cerues. Their lethality to brine shrimp (Artemia salina) was performed following standard procedures. RESULTS: Twenty different species of plant used as toothbrush were collected and their botanical identity determined. Crude methanol extracts of only Agave sisalana, Birbira and Hypericum revolutum test concentrations up to 500 micrograms/ml showed weak toxicity to brine shrimp. All the extracts showed antibacterial activity against Staphylococcus aureus and Bacillus cereus by agar diffusion method. CONCLUSION: Toothbrush sticks can be used by the vast majority of people who cannot afford buying the commercial toothbrush and toothpaste. The toothbrush sticks may be important for the oral and dental hygiene of the users and hence may be useful in decreasing dental caries. Further studies should focus on the effect of the toothbrush sticks against other common bacteria that are associated with dental diseases. PMID- 10734529 TI - Protruding urethral leiomyoma obscuring vaginal introitus: case report. AB - Leiomyoma is quite rare in the female urethra. We report an additional case to 29 reported in the literature. A multiparous woman presented with a complaint of a gradually enlarging mass causing dyspareunia for the last seven months. The mass was originating from the upper part of the distal urethra with a relatively thin stalk covered by squamous epithelia, and immunohistopathological examination confirmed it as leiomyoma. PMID- 10734530 TI - Acute complete puerperal inversion of the uterus following twin birth: case report. AB - A twenty-year old multiparous woman was admitted to our obstetric unit on February 13th 1998, with features of acute complete puerperal uterine inversion, two hours after twin birth at home. She was resuscitated and the inverted uterus repositioned using Johnson's method under general anaesthesia. The potential of twin birth as an important aetiological factor in acute/sub-acute puerperal inversion is discussed. Formal training of traditional birth attendants (TBAs) in developing countries where trained medical personnel are scarce, is emphasised. PMID- 10734531 TI - Re: Sexual life of medical students. PMID- 10734532 TI - Writing and publishing health sciences research: ethical considerations and experiences from EAMJ. PMID- 10734533 TI - Control of breast cancer using health education. AB - OBJECTIVE: To determine the efficacy of mass screening in the control of primary breast cancer among a selected group of women from Mpigi district, Uganda. DESIGN: Qualitative study by comparing the level of knowledge of risk factors, attitude and practice to breast cancer and the ability to carry out self breast examination before and after intervention study. SETTING: Nineteen static units in three counties of Mpigi district, Uganda. SUBJECTS: Three thousand and three women aged 15 years and above resident in the study area. INTERVENTION METHODS: Health education and self breast examination. MAIN OUTCOME MEASURES: Level of knowledge of risk factors, attitude and practice to breast cancer when one falls sick and ability to carry out self breast examination. RESULTS: Baseline study established that participants did not know the risk factors and could not associate any risk factor with breast cancer. Knowledge of the art of self breast examination was non-existent. After intervention, all the participants knew the risk factors and could therefore associate them with breast cancer. Health seeking behaviours greatly improved and more than 90% could describe self breast examination satisfactorily. CONCLUSION: Mass screening using health education can be carried out at grass root level by the health workers through various clinics such as maternal and child health clinics. These results can be extrapolated to other cancers where causes are not known and cure is not there. Mass screening should be included in primary health care programmes. PMID- 10734534 TI - Disease profile, complications and outcome in patients on maintenance haemodialysis at King Faisal University Hospital, Saudi Arabia. AB - OBJECTIVE: To determine the primary renal disease, acute complications and long term outcome of patients with end stage renal disease(ESRD) undergoing maintenance haemodialysis (MHD). DESIGN: A prospective descriptive study. SETTING: King Faisal Hospital of the University, Al-Khobar, Saudi Arabia. PATIENTS: Patients who had chronic renal failure with end stage renal disease (ESRD) and had been on regular dialysis for more than three months between September 1983 and September 1996 were included in the study. They were classified into three age groups, namely, group I comprising those aged below 29 years (48 patients); group II 30-59 years (147 patients), and group III, those aged 60 years and above (38 patients). Data were collected on disease characteristics, laboratory and radiological investigations, intra-dialytic complications and long-term outcome. The nature of renal disease was ascertained by review of medical data, clinical examination and laboratory investigations. RESULTS: Two hundred and thirty-three patients with ESRD on MHD were studied. The major causes of ESRD were chronic glomerulonephritis (42.9%) and diabetic nephropathy (27.9%). Hypotension, the most frequent acute complication was seen in 27% of dialytic treatments. Long-term complications resulted mainly from the vascular access and included thrombosis (60 cases), infection (50 cases), haemorrhage from access sites (20 cases) and aneurysmal dilatation (13 cases). These were more frequent in diabetics. Survival in groups I, II and III were 95%, 84% and 27% respectively at the end of the study. The 53 (22.7%) deaths that occurred in the whole study population were mostly due to cerebrovascular accidents (24.5%), cardiovascular events (15.1%), pulmonary oedema and sepsis, each contributed 13.2% of the deaths. CONCLUSION: In our patients with chronic renal failure on MHD, younger patients had better survival than the elderly. The main causes of death were cardiovascular and cerebrovascular diseases. Hypotension was the most frequent acute complication. Long-term complications were frequent, especially in diabetics. PMID- 10734535 TI - Palmar and digital dermatoglyphic patterns in Malawian subjects. AB - OBJECTIVE: To establish palmar and digital dermatoglyphic patterns. DESIGN: Cross sectional study of randomly selected black Malawian subjects. SETTING: Chichiri Secondary School and College of Medicine in Blantyre, Malawi. MAIN OUTCOME MEASURES: Total finger ridge count (TFRC), atd angle, a-b ridge counts, pattern intensity index (PII) and the variability of ridge patterns were determined. RESULTS: Arches were the most predominant digital pattern in both sexes, followed by radial loops in males and whorls in females. The sex differences between these digital patterns were not statistically significant and palmar patterns did not show sexual dimorphism. Females had significantly higher TFRC than males (p < 0.001) while males showed higher mean PII values than females. Females also had significantly higher atd angle than males while males had significantly higher a b ridge counts than females (p < 0.001). The TFRC, atd angle and a-b ridges count were significantly higher in Nigerians than Malawians (p < 0.001). The mean PII was also higher in Nigerians than in Malawians. CONCLUSION: For the first time the normal dermatoglyphic patterns of Malawians was established. PMID- 10734536 TI - Plantar dermatoglyphic features of the Urhobos of southern Nigeria. AB - OBJECTIVE: To establish the dermatoglyphic features of the Urhobos of southern Nigeria. DESIGN: Cross sectional study of randomly selected Urhobo subjects. SETTING: Department of Anatomy, College of Health Sciences, University of Port Harcourt, Port Harcourt, southern Nigeria. MAIN OUTCOME: Plantar and digital pattern types were determined and Dankmeijer's (DI) and pattern intensity (PII) indices were calculated. RESULTS: Males showed greater pattern types than females except in loops and whorls of the digits, where females showed greater numbers than males. The pattern intensity index was higher in females than in males. What appears characteristic to this group however, was the presence of few whorl distribution in the small toe in female subjects which was absent in male subjects. CONCLUSION: This study thus provide normal dermatoglyphic features for the Urhobos of Southern Nigeria and also highlighted possible distinguishing dermtoglyphic difference between the Ibos and Urhobos of Southern Nigeria. PMID- 10734537 TI - Causes and outcome of bladder injuries in Durban. AB - OBJECTIVE: To document our experience with the management of bladder injuries. DESIGN: A retrospective study. SETTING: King Edward VIII Hospital in Durban, South Africa. PATIENTS: One hundred and twenty patients with urinary bladder injuries. INTERVENTIONS: Patients with intraperitoneal bladder injuries underwent laparotomy whereas those with extraperitoneal bladder injuries were managed non operatively with a suprapubic catheter. RESULTS: The patients' median age was 28.5 years and the male to female ratio was 5:1. Sixty injuries were due to firearms, seven to stabs and fifty-three were due to blunt trauma. There were other associated injuries in sixty-six patients while fifty patients had isolated bladder injuries. Ninety-four intraperitoneal injuries were repaired while 26 extraperitoneal injuries were managed conservatively. The mortality rate was fourteen per cent (twenty-four per cent for patients with other associated injuries and two per cent for patients with isolated injuries). The mean hospital stay was 18.65 +/- 23.35 days (13.3 +/- 13.5 for firearms 30.56 +/- 33.39 for blunt injuries and 11.00 +/- 2.55 for stabs). CONCLUSION: The majority of bladder injuries were penetrating and the major cause was firearm wounds. Blunt injuries were associated with prolonged hospital stay. Isolated bladder injuries carried a low mortality rate even in patients with delayed diagnosis. Associated injuries were responsible for the high mortality. Suprapubic cystostomy for extraperitoneal bladder injuries led to no complications in our hands. PMID- 10734538 TI - Hydraulic urethral dilatation after optical internal urethrotomy: preliminary report. AB - OBJECTIVES: To determine the rate of early recurrence of urethral stricture in the first six months in patients who perform hydraulic urethral dilatation(HUD) after optical internal urethrotomy (OIU) and compare the early recurrence rate in patients who perform HUD after OIU with the recurrence rates in patients reported in the literature who undergo OIU without performing HUD. DESIGN: Prospective descriptive case series. SETTINGS: Urology clinic, Muhimbili Medical Centre, Dar es Salaam, Tanzania. SUBJECTS: Patients with symptoms of urethral stricture subsequently radiologically demonstrated to have urethral stricture that was deemed treatable by OIU. INTERVENTIONS: Evaluation included urethrography, renal biochemical profile and urethroscopy at the time of OIU. Patients with strictures no longer than 50 mm, no complete block or marked tortuousness, and no paraurethral sepsis were selected for OIU, urethral catheter for seven days followed by HUD) for one month. RESULTS: During a follow up period of five to seventeen months (mean 10.1 months), only two out of twenty three patients (8.7%) showed clinical evidence of stricture recurrence. The remaining patients reported normal voiding. CONCLUSION: This report suggests that HUD, a cost-free technique, is an effective method for preventing stricture recurrence after OIU. As follow up of these and subsequent patients continue, it is hoped that this not-so-well known technique will undergo evaluation at other centres in our region. PMID- 10734539 TI - Temporomandibular joint ankylosis in south western Nigeria. AB - OBJECTIVE: To determine the relationship between the aetiological factors, duration of ankylosis and the types of ankylosis and to evaluate the outcome of different treatment modalities used. DESIGN: Retrospective study of cases managed. SETTING: University teaching hospital at Ibadan, Nigeria. SUBJECTS: Thirty six cases treated between 1982 and 1997. RESULTS: Infection related aetiological factors were 66.6% while trauma was 27.8%. The relationship between aetiological factors and type of ankylosis classified by anatomic site, was statistically significant. Results suggest that infection-related aetiological factors are likely to give rise to extracapsular ankylosis, while trauma results in intracapsular ankylosis. Twenty five per cent reported for treatment within 12 months of onset of ankylosis while 72.2% reported after 24 months. The relationship between ankylosis of 24 months duration and above, and classification of ankylosis according to tissue type was also significant (p < 0.05) and suggests that ankylosis of such duration is likely to be bony or mixed fibrous and bony. Of the types of ankylosis, 77.8% had bony components while 22.2% were of the fibrous variety. As regards treatment, interpositional arthroplasty with the masseter muscle produced results that were more consistent and satisfactory than gap arthroplasty. CONCLUSION: The aetiological factors and duration of ankylosis influenced the type of ankylosis. PMID- 10734540 TI - In-patient management of leg ulcers. AB - OBJECTIVE: To review in-patient management of leg ulcers and to compare our experiences with those from other similar centres. DESIGN: A retrospective study. SETTING: University of Benin Teaching Hospital, Nigeria. PATIENTS: Forty five patients hospitalised with leg ulcers. RESULTS: There were 27 males and 18 females (MF 3:2). The age range was 8-90 years (mean 32.5 years). The commonest sites were around the ankle(55.6%) and the distal third of the leg (40%). Trauma, diabetes mellitus and sickle-cell disease accounted for the commonest aetiological factors(35.6%, 22.2% and 17.8% respectively). Coliforms and Staphylococcus aureus were the commonest organisms isolated on culture. All the patients had routine wound dressing and antibiotics but only 25 patients (56%) responded fully to this line of preliminary management. The other 20 patients had wound debridement in addition. Nineteen patients(42%) needed skin grafting while four patients (9%) had below-knee amputation. CONCLUSION: Our experience shows that leg ulcers whose nature portend an increase in morbidity and mortality are best managed as in-patients. The underlying causes should be addressed while local wound care is administered. Skin grafting and amputation should be employed when necessary. PMID- 10734541 TI - Obstructing duodenal ulcers in a tropical population. AB - OBJECTIVE: To characterise the pattern and pathology of obstructing duodenal ulcers in Zaria, Nigeria in the last seventeen years. DESIGN: A retrospective study. SETTING: Department of Surgery, Ahmadu Bello University Teaching Hospital, Zaria, Nigeria. PATIENTS: Fifty eight patients who had surgery for obstructing duodenal ulcer. RESULTS: The mean age was 36.6 years and male/female was ratio 3.6:1. The frequency of obstruction has been on an increase, reaching a peak in 1995 and has remained high. This period coincided with a fall in the frequency of perforation. Obstruction was due to cicatrisation in 48%, cicatrisation and adhesions in 28%, adhesions alone in 16% and inflammatory oedema in eight per cent. Three patients with adhesion had evidence of sealed or treated perforation. Seventy one per cent of the adhesions occurred during the period of rapid rise in the frequency of obstruction. Truncal vagotomy and drainage was performed after three to five days of nasogastric drainage of the stomach. Post-operative gastric atony occurred in one patient and mortality was 1.7%. There was one short term recurrence. CONCLUSION: Gastric outlet obstruction is now the commonest indication for duodenal ulcer surgery in this population. Obstruction need not be due to cicatrisation alone. Peripyloric adhesion, perhaps from previous perforation is often the cause of obstruction. Prolonged gastric drainage before and after surgery is not necessary. Truncal vagotomy and drainage is an effective treatment. PMID- 10734542 TI - Antimicrobial susceptibillty of Staphylococcus aureus strains at Muhimbili Medical Centre, Tanzania. AB - OBJECTIVE: To determine the antimicrobial susceptibility pattern of S. aureus isolates including the presence of methicillin resistant S. aureus strains. DESIGN: Cross-sectional study. SETTING: Department of Microbiology and Immunology, Muhimbili Medical Centre, Dar es Salaam, Tanzania between October 1997 and March 1998. PATIENTS: Two hundred and sixty patients consisting of 67 neonates, 114 children aged 18 years and below and 79 adults. MAIN OUTCOME MEASURES: Antimicrobial susceptibility to tetracycline, erythromycin, cefuroxime, methicillin and penicillin G and presence of mec A gene. RESULTS: Among the S. aureus strains, 97.3%, 68.1%, 37.3% and 6.5% were sensitive to cefuroxime, erythromycin, tetracycline and penicillin G respectively. Only one (0.4%) S. aureus isolate was resistant to methicillin using both the E test and presence of mec A gene. There was no significant difference between the sensitive S. aureus isolates from the neonates, children and adults. CONCLUSION: S. aureus strains are becoming more resistant to commonly used antimicrobial agents, the prevalence of methicillin resistant S. aureus strains in our study population is low compared with reported studies. PMID- 10734543 TI - Cytokine expression in patients with treated congenital hydrocephalus: a preliminary report of five patients. AB - BACKGROUND: Previous studies have described the elaboration of cytokines in experimental models of congenital hydrocephalus using rats or mice. However, there have been no reports of similar studies in humans. OBJECTIVE: To determine the cytokine expression pattern in the cerebrospinal fluid (CSF) of patients with treated congenital hydrocephalus. DESIGN: A prospective study. SETTING: Wentworth Hospital, Durban, South Africa. SUBJECTS: Five patients (three infants and two older patients) with congenital hydrocephalus treated by means of a ventriculoperitoneal shunt. INTERVENTIONS: Immunophenotyping of peripheral blood was performed on a flow cytometer. The isolation, in-vitro stimulation of peripheral blood and CSF mononuclear cells, and intracellular cytokine determination by flow cytometry were performed. MAIN OUTCOME MEASURES: Peripheral blood and CSF cytokine measurements. RESULTS: Although not statistically significant, all measured mean cytokine levels in the peripheral blood of the infant group were consistently higher than that of the adult group. CSF cytokine levels in both groups were similar and unremarkable. CONCLUSION: No clear pattern of CSF cytokine elaboration, either type-1 (T helper 1) (Th1) or Type-2 (T helper 2) (Th2), could be demonstrated in either of the groups. The significance of higher peripheral blood cytokine levels in the infants is unclear, but may be age related, and is not apparent in the CSF. PMID- 10734544 TI - Carcinoma of the thyroid in Ga-Rankuwa Hospital, Pretoria. AB - OBJECTIVE: To determine the pattern of thyroid carcinoma diagnosed at Ga-Rankuwa Hospital over a ten-year period. DESIGN: A retrospective study. SETTINGS: Ga Rankuwa Hospital; a teaching hospital for the Medical University of Southern Africa. SUBJECTS: Seventy one patients who were diagnosed as having carcinoma of the thyroid during the study period. RESULTS: The male to female ratio was 1:4.5. Papillary and follicular carcinoma constituted 42.3% and 40.9%, respectively. Anaplastic carcinoma constituted seven per cent and three cases of medullary carcinoma were seen. There were two cases of Hurthle cell carcinoma and two cases showed a mixed histology. CONCLUSION: All the histological subtypes of thyroid carcinoma do occur in our patient population. PMID- 10734545 TI - Fibrocalculous pancreatic diabetes in a child: case report. AB - A case of fibrocalculous pancreatic diabetes in a 12-year old boy is described. A medline literature search back to 1966 revealed that no cases had been reported from East Africa. We reviewed the literature and suggest that the condition is probably under-reported. PMID- 10734546 TI - Loss of vision from distant haemorrhage: report of four cases. AB - To describe the occurrence of visual loss from ischaemia of the optic nerve following distant haemorrhages. Four patients who sustained vision loss following distant haemorrhage, presenting to the neuro-ophthalmic clinic of the department of Ophthalmology, Addis Ababa University, from 1995 to 1997 were evaluated. The clinical presentation, management and prognosis are discussed. Post-haemorrhagic vision loss, other than being a rare occurrence, is under-reported due to the fact that these patients are very sick with massive blood loss and hence little attention is given to the vision loss by attending physicians and sometimes even the patients themselves. It is hoped that this paper will increase awareness about this condition among physicians attending to patients with severe bleeding and thus facilitate early diagnosis and referral. PMID- 10734547 TI - Suppurative granulomatous eosinophilic panniculitis: case report. AB - A case of 12 year-old Nigerian male is presented. He had an unusual variant of Weber-Christian disease and manifested massive subcutaneous indurations and nodules limited to the cheeks, lips, left pectoral, infraclavicular and supraclavicular areas. A wedge biopsy revealed suppurative granulomatous eosinophilic panniculitis. Despite exhaustive investigations, no obvious trigger of the panniculitis could be identified. Response to corticosteroids and to empirical trials with other drugs was poor, and the outcome was fatal. We believe this is the first report from Nigeria of this rare variant of Weber-Christian panniculitis in the paediatric age, and draw attention to the life-threatening nature of this disorder. PMID- 10734548 TI - Evaluation of microtitre spin agglutination assay in the diagnosis of bovine brucellosis. PMID- 10734549 TI - [Structural and functional characteristics of ATP-dependent Lon-proteinase from Escherichia coli]. AB - Enzymic and structural peculiarities of the ATP-dependent Lon protease from Escherichia coli and its mutant and modified forms were studied. Amino acid residues important for the function of proteolytic and ATPase sites and for the transmission of the interdomain signals of the activity coupling were found. It was shown that the protein substrates are hydrolyzed only by the full-size enzyme, whereas the isolated proteolytic domain displays a peptide-hydrolyzing activity. PMID- 10734550 TI - [Study of localization and molecular interactions of biologically active compounds in living cells and tissue slices based on confocal microspectroscopy and reconstruction of the spectral images]. AB - The confocal spectral imaging (CSI) technique is described, its basic principles are considered, and a brief review of its applications to the study of biologically active compounds (BAC) within living cells and in tissue slices is presented. This technique is based on measurements and analysis of fluorescence or resonance Raman spectra in each point of the specimen under microscope with a three-dimensional resolution of about cubic micrometer. This technique is applicable to the study of stained fluorescent and nonfluorescent compounds. Unlike the conventional approaches based on the optical microscopy, the CSI technique opens the opportunity for the identification of complexes and microenvironment of BAC in intact cells and thin tissue slices (slices or sections), as well as for the analysis of localization and distribution of compounds of interest and their complexes in cellular organelles and tissue structures. The use of CSI technique in combination with the conventional biochemical and cytological methods makes it possible to significantly expand the informativeness of investigation of modes of action of new BAC. PMID- 10734551 TI - [Mechanism of aspartyl proteinase action. VII. Noncovalent complexes of HIV-1 aspartyl proteinase with substrate and substrate-like inhibitors]. AB - A computer model of a noncovalent complex of HIV-1 aspartyl protease with substrate-like inhibitor JG-365 was a priori constructed by using the approaches of theoretical conformational analysis and molecular mechanics. The root mean square deviation of the calculated conformation of the inhibitor from the X-ray diffraction analysis data was 0.87 A. These results enabled the a priori calculation of the structure of noncovalent complex of HIV-1 protease with a hexapeptide fragment of its native specific substrate Ser-Gln-Asn-Tyr-Pro-Ile Val. The only possible orientation of the cleavable peptide bond in this and the nucleophilic water molecule relative to the catalytically active Asp residues of the enzyme (Asp25 and Asp125) was found that provides for the chemical transformation of the substrate to a tetrahedral intermediate. An action mechanism of enzymes of this class was proposed on the basis of the analysis of calculated distances. We showed that neither steric distortion of the cleavable bond nor the formation of unfavorable contacts in molecules of the enzymes and their substrates accompany the optimum orientation of substrate molecules at the active sites of HIV-1 aspartyl proteases and rhizopuspepsin. PMID- 10734552 TI - [Extracellular production of recombinant human epidermal growth factor (hEGF) in Escherichia coli cells]. AB - An expression plasmid pPTK-hEGF2 was constructed to provide for the extracellular production of recombinant human epidermal growth factor by the Escherichia coli cells. The plasmid contained two expression cassettes, one of which carried a tandem of the fused genes ompF-hegf under the control of the tac promoter, ensuring regulated secretion of hEGF into the E. coli periplasm, and another one contained the kil gene from the ColE1 plasmid under the control of lac promoter. The regulated low-level biosynthesis of Kil protein increased the permeability of E. coli outer membrane for periplasmic proteins. This enabled the recombinant proteins secreted into the cell periplasm to outflow into the cultural medium. As a result, the E. coli strains that harboured this plasmid construct produced effectively the recombinant hEGF into the cultural medium. The yields of hEGF produced by the nTG1(pPTK-hEGF2) and HB101(pPTK-hEGF2) strains reached 25 and 30 mg/l of cell culture after 14 and 18 h of cultivation, respectively. The hEGF preparation isolated possessed biological activity both in vivo and in vitro. PMID- 10734553 TI - [Lys35 spin-labeled cytotoxin II from Naja oxiana venom for study of its interaction with phospholipid membranes by EPR]. PMID- 10734554 TI - [Synthesis of novel fluorescent 9,10-bisphenylethynylanthracene-derived pseudonucleoside and its introduction into oligonucleotides]. PMID- 10734555 TI - [RNA polymerase II from Schizosaccharomyces pombe contains 12 various subunits: identification and characteristics of Rpb4 subunit]. PMID- 10734556 TI - Physical and biochemical characterization of a simple intermediate between fluidized and expanded bed contactors. AB - Physical and biochemical comparison has been made of the performance of a simple fluidized bed contactor and a commercial expanded bed contactor, characterized by identical dimensions, and operated at various settled bed heights with two anion exchange adsorbents. The contactors were tested with various feedstocks comprising bovine albumin in the absence and presence of 20 g dry cell weight biomass litre-1. Earlier classification of the simple contactor as a single stage, well mixed fluidized bed is reviewed. The relative merits of STREAMLINE DEAE and DEAE Spherodex LS as fluidizable, anion exchange adsorbents are discussed. PMID- 10734557 TI - On-line monitoring of the purification of GST-(His)6 from an unclarified Escherichia coli homogenate within an immobilised metal affinity expanded bed. AB - The use of a rapid chromatographic assay to monitor the level of a specific protein during its downstream processing by expanded bed adsorption is described. An expanded bed column (5 cm diameter) has been modified to allow the abstraction of liquid samples at various heights along the bed, in an automated, semi continuous manner throughout the separation. The withdrawn samples were filtered in-line and the level of the target protein assayed by a rapid on-line chromatographic method. Using this technique it was possible to monitor the development of adsorbate profiles during the loading, washing and elution phases of the application of an unclarified feedstock. The potential of the technique is demonstrated using the separation of histidine tagged glutathione s-transferase (GST-(His)6) from an unclarified Escherichia coli homogenate using an expanded bed of Ni2+ loaded STREAMLINE Chelating. The level of GST-(His)6 in the abstracted homogenate samples was measured using Zn2+ loaded NTA-silica as an affinity chromatographic sensor. The approach described demonstrates potential for the on-line monitoring and control of expanded bed separations and for providing a greater understanding of adsorption/desorption and hydrodynamic processes occurring within the bed. PMID- 10734558 TI - The effect of column verticality on separation efficiency in expanded bed adsorption. AB - The effect of column verticality on liquid dispersion and separation efficiency in expanded bed adsorption columns was investigated using 1 and 5 cm diameter columns. Column misalignment of only 0.15 degree resulted in the reduction of the Bodenstein number from 140 to 50 for the 1 cm dia. column and from 75 to 45 for the 5 cm dia. column. This degree of misalignment was not detectable by visual assessment of adsorbent particle movement within the column. Depending on the relative importance of transport limitations, kinetic limitations and dispersion to any specific separation, this increase in dispersion with column alignment can significantly affect separation efficiency. Pure protein breakthrough profiles resulting from the application of bovine serum albumin onto STREAMLINE Q XL demonstrated that, at 10% breakthrough, 7.8% more protein could be applied to a vertical 1 cm dia. column compared to the same column misaligned by 0.15 degree. When an unclarified yeast homogenate was applied to a 1 cm dia. vertical column packed with STREAMLINE DEAE, 10% breakthrough of glucose-6-phosphate dehydrogenase (G6PDH) corresponded to a load 55% greater compared to the same column aligned 0.185 degree off-vertical. The G6PDH breakthrough curves for vertical and 0.15 degree off-vertical runs performed using a 5 cm column were essentially indistinguishable. PMID- 10734559 TI - Evaluation of the effect of in-bed sampling on expanded bed adsorption. AB - An expanded bed adsorption (EBA) column (5 cm diameter) has been modified to allow the abstraction of liquid samples from various positions along the height of an expanded bed. As the adsorbent particles were fluidized, in-bed monitoring of key component concentrations during feedstock application, washing and elution was achieved by the withdrawal of liquid samples from the voids within the expanded bed through ports along the wall of the column. Component levels in the withdrawn streams can be assayed using on-line analytical chromatography or samples can be collected and assayed off-line. On-line monitoring can be used to control the duration of the loading stage and as a tool to provide information about the hydrodynamic and adsorption/desorption processes that occur during expanded bed adsorption. Studies of residence time distributions indicated that the modifications to the column do not significantly affect liquid dispersion. Using the adsorption of glucose-6-phosphate dehydrogenase from yeast homogenate on Streamline DEAE as a model system, comparison of breakthrough curves for runs when in-bed monitoring was and was not performed also suggested that separation efficiency is not appreciably affected by in-bed sampling. PMID- 10734560 TI - Simplified and more robust EBA processes by elution in expanded bed mode. AB - This paper illustrates the feasibility of eluting EBA columns in the expanded bed mode as an alternative to the generally used method of packed bed elution. It is shown that at linear flow rates of 1-3 cm/min the difference in total elution volume between expanded bed elution and packed bed elution is less than 20%. It is suggested that expanded bed elution offers a range of significant advantages, while the drawbacks will be insignificant in most applications. The key to the success of this method seems to be the use of EBA matrices with a relatively low degree of expansion (i.e. a high density) at the linear flow rates employed for elution of bound product. PMID- 10734561 TI - Cell/adsorbent interactions in expanded bed adsorption of proteins. AB - Expanded bed adsorption (EBA) is an integrated technology for the primary recovery of proteins from unclarified feedstock. A method is presented which allows a qualitative and quantitative understanding of the main mechanisms governing the interaction of biomass with fluidized resins. A pulse response technique was used to determine the adsorption of various cell types (yeast, Gram positive and Gram negative bacteria, mammalian cells and yeast homogenate) to a range of commercially available matrices for EBA. Cells and cell debris were found to interact with the ligands of agarose based resins mainly by electrostatic forces. From the adsorbents investigated the anion exchange matrix showed the most severe interactions, while cation exchange and affinity adsorbents appeared to be less affected. Within the range of biologic systems under study E. coli cells had the lowest tendency of binding to all matrices while hybridoma cells attached to all the adsorbents except the protein A affinity matrix. The method presented may be employed for screening of suitable biomass/adsorbent combinations, which yield a robust and reliable initial capture step by expanded bed adsorption from unclarified feedstock. PMID- 10734562 TI - Highly dense beaded sorbents suitable for fluidized bed applications. AB - New highly dense beaded sorbents suitable for fluidized bed applications of protein separations are presented. They are prepared using porous mineral oxides supporting functional hydrogels responsible for protein interaction. Beads of small diameter (70 microns) are selected to reduce mass transfer resistance. Zirconium oxide was the preferred mineral material due to its high density (5.9 g/ml) allowing high fluidizing liquid velocities (600 cm/h) into columns with a moderate bed expansion (lower than 3). Composite mineral--hydrogel sorbents are evaluated for their ability to rapidly adsorb proteins in fluidized bed and to separate with an appropriate resolution macromolecule mixtures in packed bed. Lysozyme dynamic capacities of 68 and 53 mg per ml of sedimented bed were obtained at fluidizing velocities of 450 and 900 cm/h. PMID- 10734563 TI - Characterization of very dense mineral oxide-gel composites for fluidized-bed adsorption of biomolecules. AB - Efficient design of fluidized-bed biomolecule adsorption from crude feed stock requires particles with elevated density, large adsorption capacity and broad chemical stability. Moreover, combinations of small particle diameters with high densities allow for high fluidization velocities while preserving a rapid mass transfer. This approach has been implemented by combining stable porous mineral oxide of high density (2.2, 4.7, 5.7, 9.4 g/ml) with functionalized hydrogels. The cross-linked hydrogel derivative fills the internal porosity of the beads and provides a high equilibrium binding capacity. Various porous mineral oxides (silica, titania, zirconia and hafnia) have been characterized in term of fluidization behavior, surface reactivity and chemical resistance to harsh CIP procedures. Porous zirconia particles were also modified into ion-exchangers by suitable surface modification and intraparticle polymerization of functionalized stable derivatives of acrylic monomers. Back-mixings in fluidized bed columns were analyzed by residence time distribution analysis of inert tracers. 328 and 218 mixing plates per meter were found for respectively, bed expansions of 1.7 and 2.9. The dynamic protein adsorption behaviors of zirconia-based polymeric anion-exchange sorbents were obtained in fluidized-bed, using BSA as model protein. A dynamic binding capacity of 62 mg/ml was observed at a fluidizing velocity of 320 cm/h. These investigations substantiate the favorable physical and chemical characteristics anticipated for dense composite beads for use as fluidized bed adsorbents. PMID- 10734564 TI - Expanded bed adsorption on supermacroporous cross-linked cellulose matrix. AB - Rigid spherical macroporous adsorbent beads (CELBEADS) prepared by cross-linking of cellulose were characterized and found eminently suitable for use as expanded bed affinity chromatography matrix. Chromatographic runs were performed on a 10 mm diameter column with three solutes tyrosine, papain and bovine serum albumin under non-retaining conditions on CELBEADS and Streamline DEAE, a commercial agarose based expanded bed matrix. Performance of the runs was measured in terms of height equivalent to theoretical plate, HETP. Variation in HETP with velocity on Streamline DEAE gave flat profiles in packed bed and increasing trend in expanded bed. On CELBEADS, the HETP curves in both packed and expanded bed modes followed profiles typical of macroporous adsorbents i.e. increasing and levelling with velocity. HETP values obtained for papain and bovine serum albumin on CELBEADS were lower than those obtained on Streamline DEAE at all velocities. Lactate dehydrogenase was purified from porcine muscle homogenate using Cibacron blue conjugated to CELBEADS using a protocol reported for supports with surface hydroxyl groups. Elution of the enzyme was investigated both in packed mode as well as in expanded mode at a flow rate of 1 ml min-1. The purification procedure took about 60 minutes and a purification fold of about 14 was achieved in both cases. The adsorbent could be cleaned in place with 5 M urea and used repeatedly without loss of performance. PMID- 10734565 TI - Characterisation of STREAMLINE phenyl. AB - STREAMLINE Phenyl is a new hydrophobic interaction chromatography support designed for use in expanded bed adsorption. The phenyl groups are linked to STREAMLINE matrix via highly stable ether linkages. Within this development project the chemical and chromatographic stability as well as the breakthrough capacity for human IgG has been studied. The chemical stability was monitored as the carbon leakage from the matrix to the storage solution, pH 1-14 at 20 and 40 degrees C. The carbon content in the supernatant was determined with Total Organic Carbon (TOC) technique. In the chromatographic stability study STREAMLINE Phenyl was stored in eight different storage solutions under ambient conditions for 12 weeks and then tested in a chromatographic function test. The results show that the adsorbent is chemically stable and that the chromatographic properties are retained under the tested conditions. The breakthrough capacity study demonstrates the importance of the bed height for obtaining maximal dynamic capacity. Further, there is a good correlation between breakthrough data generated from packed bed and expanded bed runs. PMID- 10734566 TI - Use of streamline chelating for capture and purification of poly-His-tagged recombinant proteins. AB - Expression of recombinant proteins with poly-histidine tags enables their convenient capture and purification using immobilized metal affinity chromatography (IMAC). The 6 x His-tagged protein binds to a chelating resin charged with metal ions such as Ni2+, Cu2+ or Zn2+, and can therefore be separated from proteins which have lower, or no, affinity for the resin. Two recombinant proteins, a malaria transmission-blocking vaccine candidate secreted extracellularly by S. cerevisiae and a modified diphtheria toxin produced intracellularly by E. coli, were expressed with 6 x His tags and could therefore be purified using IMAC. In an effort to further simplify the initial capture of these proteins, an expanded bed adsorption technique using a chelating resin (Streamline Chelating) was introduced. It was possible to capture the intracellular diphtheria protein from E. coli directly after cell lysis, without prior centrifugation or filtration. The extracellular malaria vaccine candidate was also directly captured from a high cell density yeast culture. Detailed information on the experimental work performed, and the capture processes developed, is provided. PMID- 10734567 TI - A simplified purification procedure of alpha-lactalbumin from milk using Ca(2+) dependent adsorption in hydrophobic expanded bed chromatography. AB - The technique of expanded bed adsorption is originally designed for a direct recovery of proteins from fermentor feedstocks. In this article we describe the use of expanded bed adsorption for the recovery of alpha-lactalbumins from defatted milk using the hydrophobic Streamline Phenyl gel. alpha-Lactalbumins are Ca(2+)-binding proteins. Upon Ca2+ removal, they undergo a significant conformation change rendering them more hydrophobic. Based on this unique property we develop a protocol for fast and efficient purification of alpha lactalbumin from milk. The use of this technique results in a reduction of the number of chromatographic purification steps. PMID- 10734568 TI - Expanded bed adsorption as a unique unit operation for the isolation of bacteriocins from fermentation media. AB - Expanded bed adsorption using a strong cation exchanger allowed the direct isolation of amylovorin L471, a bacteriocin from Lactobacillus amylovorus DCE 471, from the fermentation medium. The pH of the loading and elution buffer were optimised in a packed bed with cell-free culture supernatant. Bound bacteriocin was eluted with 1.0 M NaCl. The highest recovery (30%) was obtained at the lowest pH (3.6). At higher pH values the recovery was lower, namely 12%, 15% and 7% at pH 4.5, 6.5 and 8.0, respectively. In expanded bed mode, direct isolation of the bacteriocin from the fermentation medium at pH 3.6 (loading and elution) initially resulted in a recovery of 12%. After optimisation of the pH (loading and elution at pH 3.6 and 6.5, respectively), the recovery for amylovorin L471 increased up to 30% and higher. Recovery of enterocin A from Enterococcus faecium CTC 492 fermentation medium averaged 15% (loading and elution at pH 3.6 and 6.0, respectively). With pediocin, produced by Pediococcus acidilactici ATCC 8042, 26% recovery was obtained at a pH of 6.5 during loading and elution. Low recoveries can be ascribed to non-optimal operation conditions (pH of loading and elution buffer), inactivation of the bacteriocin on a cationic resin, and the formation of more insoluble and less active, strongly hydrophobic bacteriocin aggregates upon further purification. PMID- 10734569 TI - Pilot scale recovery of monoclonal antibodies by expanded bed ion exchange adsorption. AB - The aim of the investigations was to estimate the scale up properties of an efficient chromatographic first capture step for the recovery of murine IgG1 from undiluted and unclarified hybridoma cell culture broth using an ion exchange matrix in expanded bed mode. The tested new sulfopropyl-based ion exchange matrix (Streamline SP XL, Amersham Pharmacia Biotech) stands out due to its enhanced capacity compared to its precursor (Streamline SP). Defining the working pH in preliminary electrophoretic analyses (titration curve, SDS-PAGE) and small-scaled chromatographic binding studies showed, that the optimal value for the IgG purification was pH 4.6, where a co-chromatography of the medium supplement albumin (500 mg l-1, pI = 4.8) could not be avoided. Further scouting experiments dealt with the dynamic capacity of the matrix, which was evaluated by frontal adsorption analysis. In packed bed mode no break-through of the target protein was achieved even after 6.5 mg IgG per ml matrix were applied. These results could not be reproduced in expanded bed mode with cell-free supernatant, where the dynamic capacity was found to be only 1.5 mg IgG/ml SP XL. Processing cell containing broth resulted in an additional decrease of the value down to 0.5 mg ml-1, presumably caused by the remarkable biomass adsorption to the matrix. The search for the reasons led to the examination of the hydrodynamic conditions. Buffer experiments with a tracer substance (acetone) pointed out, that the flow in expanded bed was significantly more influenced by back-mixing effects and channel formations than in packed bed. These effects could be compensated with an enhanced viscosity of the liquid phase, which was achieved by the addition of glucose. As a result of the improved hydrodynamic conditions in the expanded bed, the dynamic capacity could be increased from 0.5 to more than 4.5 mg IgG/ml matrix for the processing of cell culture broth with 400 mM glucose. Finally, the scale up from a Streamline 25 to a Streamline 200 column was performed under conditions, which proved to be optimal: 100 L of unclarified hybridoma broth were concentrated with a binding rate of 95% in less than 3.5 hours. Loading the column no break-through of the target protein was achieved. However, the eluate still contained debris and cells, which points out the major disadvantage of the method: the biomass attachment to the matrix. PMID- 10734570 TI - Capture of human Fab fragments by expanded bed adsorption with a mixed mode adsorbent. AB - A novel group of mixed mode adsorbents has been developed for purification of monoclonal and polyclonal antibodies from a broad range of raw materials such as hybridoma cell culture, ascites fluid, animal sera, milk, whey and egg yolk. The aim of this study was to determine whether such mixed mode adsorbents were also useful for the recovery of recombinant proteins from microbial feedstocks. This paper describes the performance of one of these adsorbents for expanded bed capture of a human Fab fragment from recombinant E. Coli cell extracts. It is concluded that the mixed mode adsorbent binds the Fab fragment efficiently from crude extracts without any requirement for preconditioning the extract by for example de-salting or dilution. The capacity of the mixed mode adsorbent is approx. 12 mg Fab/ml matrix. The novel mixed mode adsorbent can be useful during production of highly purified Fab fragments as the first step in a purification scheme. In this respect the mixed mode adsorbent is advantageous over alternative commercially available ion-exchange materials which require pre-conditioning of cell extract for Fab' capture. Together with the concentration and clarification effect a significant enrichment of the Fab fragment is obtained in one single high yield operation. PMID- 10734571 TI - Purification of recombinant human serum albumin efficient purification using STREAMLINE. AB - Two inter-related challenges must be overcome to develop a recombinant human serum albumin process. One is purity; the other is cost. Regarding cost, our goal is to produce recombinant human serum albumin at least as economically as plasma derived human serum albumin. To control production costs, maximum quantities of albumin must be produced from minimum volumes of cell culture, followed by high efficiency, high-yield purification methods. By introducing STREAMLINE technology, we have improved productivity by roughly 50% in terms of processing time and 45% in terms of yield. Furthermore, the life time of the gel and column are very long. After more than 1000 process cycles, performance remained unchanged. PMID- 10734572 TI - Direct coupling of expanded bed adsorption with a downstream purification step. AB - In the course of developing a cost-effective, scaleable process for the purification of a recombinant protein from Chinese hamster ovary (CHO) suspension cell culture, we investigated direct capture of this molecule using expanded bed adsorption (EBA). EBA combines clarification, purification, and concentration of the product into a single step. The unclarified bioreactor material was directly applied to a STREAMLINE 25 column containing an affinity STREAMLINE adsorbent. This work focused on simplifying the EBA operations and minimizing the overall processing time by running the EBA column unidirectionally, eluting in the expanded bed mode, and coupling the EBA column directly with ion exchange or hydrophobic interaction chromatography. Unidirectional EBA was clearly a simpler unit operation and did not require the use of specialized equipment. The increase in the elution pool volume was insignificant, especially when the EBA column was eluted directly onto the downstream column. Scale-down was simple and could be automated. Coupling of unidirectional EBA with a downstream purification step reduced processing time, equipment requirements and cost. PMID- 10734573 TI - Production of plasmid DNA for human gene therapy using modified alkaline cell lysis and expanded bed anion exchange chromatography. AB - We describe a process for the commercial manufacture of therapeutic grade plasmid DNA. The industrially scaleable unit operations employed in this process are: (i) optimized alkaline lysis; (ii) bag filtration; (iii) expanded bed anion exchange chromatography; (iv) ultrafiltration, and (v) size exclusion chromatography. These steps are scaleable alternatives to current approaches to plasmid DNA isolation such as high speed centrifugation for feed-stock clarification and solvent precipitation for plasmid concentration, and an efficient alternative to conventional low through-put packed bed chromatography. The process produces plasmid DNA characterized by low level chromosomal DNA, RNA and endotoxin contamination without the use of flammable solvents or toxic reagents and is suitable for therapeutic administration. PMID- 10734574 TI - Development and scale up of a capture step (expanded bed chromatography) for a fusion protein expressed intracellularly in Escherichia coli. AB - A capture step was developed using the expanded bed adsorption technology to separate a protein of interest on a cation exchanger from a crude Escherichia coli homogenate. This method was developed in bench-top scale using a STREAMLINE 25 column (Amersham Pharmacia Biotech, Sweden) and STREAMLINE SP. The development was based on earlier experiments performed in a packed bed column (SP-Sepharose FF) to investigate the conditions for sample application, wash and elution. The packed bed method was transformed into an expanded bed method by slightly modifying the wash procedure and cleaning in place (CIP). This method was then scaled-up to pilot scale and used for production of the fusion protein according to cGMP. The yield over the step in pilot scale was 70-85% compared with only 30 50% in small scale. Pressure build-up, attachment of biomass to the adsorbent and collapses of the expanded bed were phenomena seen in small scale but not in pilot scale. The scale-up of the step significantly improved the performance of the step. PMID- 10734575 TI - On-line monitoring of glucose and/or lactate in a fermentation process using an expanded micro-bed flow injection analyser. AB - A novel flow injection biosensor system for monitoring fermentation processes has been developed using an expanded micro bed as the enzyme reactor. An expanded bed reactor is capable of handling a mobile phase containing suspended matter like cells and cell debris. Thus, while the analyte is free to interact with the adsorbent, the suspended particulate matter passes through unhindered. With the use of a scaled down expanded bed in the flow injection analysis (FIA) system, it was possible to analyse samples directly from a fermentor without the pretreatment otherwise required to extract the analyte or remove the suspended cells. This technique, therefore, provides a means to determine the true concentrations of the metabolites in a fermentor, with more ease than possible with other techniques. Glucose oxidase immobilised on STREAMLINE was used to measure glucose concentration in a suspension of dead yeast cells. There was no interference from the cell particles even at high cell densities such as 15 gm dry weight per litre. The assay time was about 6 min. Accuracy and reproducibility of the system was found to be good. In another scheme, lactate oxidase was covalently coupled to STREAMLINE for expanded bed operation. With the on-line expanded micro bed FIA it was possible to follow the fermentation with Lactobacillus casei. PMID- 10734576 TI - Binding assays in heterogeneous media using a flow injection system with an expanded micro-bed adsorption column. AB - Competitive binding assays have been performed in flow injection systems. To further increase the versatility of the system, and to enable it to deal with samples containing particulate matter, the adsorption step was designed as an expanded bed column. Immunochemical quantification of human serum albumin was chosen as a model system to use for the development of the technology. A competitive ELISA was set up using peroxidase labelled HSA as competing ligand. The introduction of the expanded bed immunosorption column made the system tolerant to samples containing suspended particulate matter. The analytical outcome is very similar to that from the packed bed system even though more time is required for each assay cycle. The capability of the system was tested by addition of increasing amounts of yeast cells. The results clearly indicate that the system is suitable e.g. for process monitoring of fermentations. PMID- 10734577 TI - Use of a micro-expanded bed containing immobilised lysozyme for cell disruption in flow injection analysis. AB - A method for cell disruption in Flow Injection Analysis (FIA) systems has been developed. The principle involves on-line cell disruption by means of immobilised lysozyme followed by an ultrasonic treatment. In order to avoid flow problems in the analytical system, the lysozyme was immobilised to Streamline that was used in an expanded bed in the flow system. Samples of suspensions of Micrococcus lysodeikticus were treated and the success of the treatment was evaluated in terms of released protein and as a decrease in the optical density at 450 nm. The new technology offers a powerful tool in flow injection analyses for quantification of intracellular compounds. The concept of integration, i.e. combining cell disruption with handling of cell debris and assay procedure in one continuous flow process facilitates its use and increases the probability of reaching reproducible and reliable results. PMID- 10734578 TI - Flow injection analysis of intracellular beta-galactosidase in Escherichia coli cultivations, using an on-line system including cell disruption, debris separation and immunochemical quantification. AB - A continuous integrated process for on-line quantification of intracellular components has been developed. By applying the concept of expanded micro-beds in a flow injection system it was possible to first perform on-line cell disintegration followed by an on-line binding assay for quantification of a reporter protein (beta-galactosidase) from the cell interior. The disintegration process involved the use of an expanded bed with immobilised lysozyme followed by ultrasonic treatment in a flow-through cell. The cell debris does not interfere in the binding assay as it is carried out in an expanded bed. The time for an assay cycle is at present approx. 35 min. This integrated system can be used for quantification of proteins down to at least 10(-7) mol/L. PMID- 10734579 TI - [Perspectives and limits in treatment of obesity]. AB - The widely propagated morbidity and mortality risks of obesity appear somewhat exaggerated, except for morbid obesity (BMI > 40 kg/m2) and for high risk obese subgroups concerning diabetes mellitus, hypertension, metabolic syndrome and obstructive sleep apnea syndrome. Non-medical reasons represent a major component of the social pressure that is presently experienced by obese persons in our society. Weight reduction represents the primary therapeutic approach in overweight patients with type 2 diabetes, hypertension, metabolic syndrome and obstructive sleep apnea, and it may be recommended in high-risk individuals for primary prevention of these diseases. Massive obesity is associated with excess mortality, especially in younger, physically inactive men with upper-body-segment obesity. It is widely assumed that weight reduction will lead to a reduction of excess mortality in these individuals; so far, however, there is no proof for this assumption. Non-medicamentous conservative therapeutic approaches to weight reduction have the advantage of safety, even though their long-term efficacy is generally disappointing. There are no randomized, controlled trials to prove a reduction of morbidity or mortality risks and of therapeutic safety for pharmacological, invasive or surgical methods to treat obesity. PMID- 10734580 TI - [20 years small intestinal bypass surgery. What is left?]. AB - The jejunoileostomy was a frequently used surgical procedure to treat morbid obese patients (obesity grade III) from 1970 to 1980. The observed weight loss was very good, 50-60 kg during the first 18 months after operation (n = 288). Even obesity-induced side effects were improved significantly. However, negative consequences and complications were seen all too often: chronic loss of electrolytes, protein and vitamins and diarrhea with perianal skin problems. Reoperations were often necessary due to renal stone formation, cholecystolithiasis or blind loop syndrome. Therefore this procedure was abandoned in favor of operations such as vertical banded gastroplasty or the adjustable gastric band. However, in special cases similar operations may be helpful if gastric restriction has failed. PMID- 10734581 TI - [Gastric bypass in morbid obesity]. PMID- 10734582 TI - [Laparoscopic gastric banding]. AB - The introduction of laparoscopic gastric banding appears to have revolutionized bariatric surgery. This review presents this new method in terms of indications, operative technique and preliminary results according to our own experience as well as reports in the literature. It seems that a long-term weight reduction of more than 50% excess weight can be achieved with a low morbidity rate. In the near future laparoscopic gastric banding can possibly become the procedure of choice for the treatment of morbid obesity. PMID- 10734583 TI - [Gene therapy possibilities in plastic surgery]. AB - Advances in gene technologies have meanwhile reached plastic surgery. Important contributions in this field (which are not all included in the paper) come not only from plastic surgeons, but also from neighboring specialities like dermatology, trauma surgery, orthopedics and vascular surgery. The uniting principle for all this work is improving wound healing and reconstructing tissue defects taking into consideration functional and aesthetic aspects. Gene-therapy is gaining further importance in the clinical field of plastic surgery. In this regard, every clinician has to be aware of the fact that progress in experimental and experimental-clinical work will be achieved only with the help of basic science. On the other hand, basic science needs the clinical input to get relevant patient-oriented studies started. Further intensive cooperation between clinicians and basic scientists is therefore mandatory. In plastic surgery, 2 years ago we founded a forum called ECSAPS (European Conference of Scientists and Plastic Surgeons), which takes place in European city every year. PMID- 10734584 TI - [Immune paralysis of T-lymphocytes and monocytes in postoperative abdominal sepsis. Correlation of immune function with survival]. AB - In vitro functions of stimulated peripheral T cells and monocytes were investigate in patients experiencing sepsis following major visceral surgery. Cell culture supernatants were analyzed by ELISA for IL-2, IFN-gamma, IL-4, IL 10, TNF-alpha, IL-1 beta, and IL-12p40. In addition, monocyte HLA class II expression was determined by flow cytometry. T cell secretion of IL-2, TNF-alpha, and in part IFN-gamma (but not IL-4) was significantly diminished in non survivors throughout the entire course of sepsis, compared to controls and sepsis survivors. Production of IL-1 beta and IL-12 p40 by monocytes was strongly reduced in both survivors and non-survivors at the onset of sepsis. Persistence of depressed monocyte cytokine secretion correlated with lethality. Thus, overall suppression of cytokine production by T cells and monocytes was already observed at the beginning of postoperative sepsis. HLA class II expression by monocytes exhibited a strong and sustained down-regulation with no significant differences between sepsis survivors and non-survivors. In summary, suppression of both T cell and monocyte functions develops early during postoperative sepsis. Recovery of immune functions and severity of immune defects are associated with outcome. PMID- 10734585 TI - [Classification and treatment of bile duct injuries after laparoscopic cholecystectomy]. AB - Iatrogenic bile duct lesions are serious complications during laparoscopic cholecystectomy and include biliary leakage and major bile duct injury. The incidence of biliary lesions following laparoscopic cholecystectomy is up to threefold higher than that of the open procedure. A total of 108 patients with bile duct lesions after laparoscopic cholecystectomy were treated at our institution. Endoscopic treatment was successful in 68 cases, 6 patients were treated by external drainage, and 34 patients required surgical therapy. Selection criteria for the type of treatment included the etiology, anatomical situation, and diagnostic interval of the biliary lesion. We suggest a classification of bile duct injury and a proposal for diagnosis and treatment of these complications. PMID- 10734586 TI - [Timely management of bile duct complications after laparoscopic cholecystectomy]. AB - INTRODUCTION: Bile duct complications after laparoscopic cholecystectomy occur twice to three times more frequently than after an open procedure. Four different types of lesions may be differentiated by the Siewert classification: postoperative bile fistulas (type I), late strictures (type II), tangential injuries of the bile duct (type III) and defect lesions (type IV). The diagnostic and therapeutic management is demonstrated in relation to our own experience and the literature. METHODS: Eleven patients (median age 43.8 +/- 17.2) with bile duct complications after laparoscopic cholecystectomy were operatively treated between November 1993 and December 1998. Nine patients (four type-II lesions, five type-IV lesions) were referred from another hospital; 2 defect lesions out of 410 laparoscopic cholecystectomies (0.5%) were documented in our own patient group. RESULTS: Four patients with late strictures were operatively treated with a hepaticocholedochostomy (n = 2) or hepaticojejunostomy (n = 2) after 14.3 +/- 8.4 months and were discharged from hospital after 10.6 +/- 3.8 days. In both cases with type-IV lesion and a short defect, an end-to-end anastomosis was successful (hospital stay 11.6 +/- 1.0 days). However, a retrocolic Roux-Y end-to side hepaticojejunostomy was performed in all cases with a larger defect (n = 5; hospital stay 14.8 +/- 2.0 days). The two defect lesions in our own group were detected by intraoperative cholangiography and immediately treated after conversion either with hepaticocholedochostomy or hepaticojejunostomy (hospital stay 11.2 +/- 0.6 days). CONCLUSIONS: The incidence of bile duct complications after laparoscopic cholecystectomy might be kept down by anatomic preparation, selective intraoperative cholangiography and early consideration of conversion to open procedure. The clinical course after biliary tract injury can be positively influenced only by a standard diagnostic and operative procedure and by an early transfer to a specialized center. PMID- 10734587 TI - [Benign bile duct stenoses. Treatment with flexible metal wire stent or silicone prostheses (Yamakava) in nonresectable stenoses or high surgical risk cases]. AB - INTRODUCTION: High benign biliary stenoses, which can occur as a complication of disease or surgery, may be difficult to correct by open surgery and may also carry a high risk. An alternative to surgery is the percutaneous implantation of a flexible metallic stent or silicon prosthesis at the affected site. METHODS: Twelve patients with benign biliary stenoses have been operated on in our department since March 1992, and the therapeutic success was reviewed in this study. RESULTS: Six received a Palmaz stent and six a silicon prosthesis, with a follow-up after completion of treatment of more than 7 years and more than 3 years, respectively. Fifty percent of the patients with a stent experienced no problems and 66% of the stents remained patent (including one requiring operative intervention to restore patency). No further surgery was necessary in any of the patients with a silicon prosthesis. CONCLUSION: When the advantages and disadvantages of the various procedures are considered, long-term splinting with a wide-lumened silicon prosthesis (Yamakava) appears to be a promising alternative to surgery for the treatment of benign biliary stenosis. PMID- 10734588 TI - [Does the new UICC classification allow better prognostic assessment for ductal pancreatic carcinoma?]. AB - INTRODUCTION: To compare treatment results an international established classification is necessary. In 1997 the TNM classification of 1992 was modified. METHODS: Between 1983 and 1997, 159 patients with a ductal carcinoma of the pancreas underwent resection. All data of the resected specimens were documented in standardized manner prospectively in a protocol that offered ready transfer of the collected data to a new classification. The TNM categories and stage groupings were transferred to the new UICC classification of 1997 and analyzed in comparison to the classification of 1992. RESULTS: The inclusion of a pT4 category equivalent to the other GI tumors made a new stage grouping necessary. Also division into pN1 a and pN1 b was established. According to the clinical experience only few tumors in early stages (pT1/2 and stage I) were observed in the new classification. There was a significant improval in the patient's distribution to the new stage grouping because of the homogeneous groups. In comparison to the 1992 classification the new stage II shows a relevant prognostic value and a significant difference to stage III. CONCLUSION: We conclude that the new UICC classification relates to prognosis better than the old classification. PMID- 10734590 TI - [Surgical therapy of colonic diverticulitis--how reliable is primary anastomosis?]. AB - INTRODUCTION: The aim of the study was to evaluate the modern principles of surgery in diverticulitis, e.g. early elective resection and primary anastomosis. METHODS: The data of 445 consecutive patients were retrospectively analysed after classifying all cases in four subgroups according to a modified Hinchey classification (stages 0-III). RESULTS: Within our study group the morbidity was 26.5% (n = 118) and the mortality was 1.6% (n = 7). In 96% (n = 425) of all cases and in 64% (21/33) of patients with perforated diverticulitis and peritonitis (stage III), a primary anastomosis was performed. Four patients of the study group showed insufficient anastomosis (0.9%). No leakage was observed from any of the anastomoses performed in stage III diverticulitis. Stage of inflammation and age of patient correlate with morbidity and mortality. Some 53% (94/177) of the patients in stage II and 67% (22/33) of the patients in stage III had never showed symptoms of diverticulitis before. CONCLUSION: Prophylactic surgery to avoid life-threatening situations, including abscess formation or perforation, is not possible in many cases. However, especially patients at risk (age, coexisting illness) should undergo early surgery. Primary anastomosis can be performed safely even at an advanced stage. PMID- 10734589 TI - [Treatment concept of adenomas of Vater's ampulla]. AB - INTRODUCTION: The most common benign ampullary tumors are adenomas (80%). They are considered as premalignant lesions with a transformation rate to carcinoma of up to 30%. METHODS: From 1 January 1997 to 28 February 1999 we treated 11 patients with adenoma of the ampulla of Vater. An ampullectomy was performed in 10 cases. One poor-risk patient could not be operated on. RESULTS: No operative mortality occurred. In two patients a pT1 adenocarcinoma was diagnosed postoperatively. One of the two patients with a high-risk carcinoma underwent a second operation, a Whipple pancreatoduodenectomy. Nine of 10 patients had no recurrence with a median follow-up of 12 months. CONCLUSION: One patient died of glioblastoma. We would therefore recommend ampullectomy as the first-line treatment for benign tumors of the ampulla of Vater. In low-risk pT1 carcinoma (G1/G2, L0) and R0 resection, local excision is acceptable. In high-risk pT1 carcinoma (G3 and/or L1) Whipple pancreatoduodenectomy is mandatory. PMID- 10734591 TI - [Acute aortic dissection. Diagnosis and therapy of ischemic complications]. AB - AIM OF THE STUDY: About 30% of the patients with acute aortic dissection suffer from organ or limb ischemia. We analyzed the influence of ischemic localization and method of operative treatment (aortic fenestration or extraanatomic bypass revascularization) on morbidity and mortality. PATIENTS AND METHODS: From 1 May 1987 to 31 December 1998 21 patients with 24 vascular complications such as renal or intestinal ischemia, lower extremity ischemia and paraplegia following acute aortic dissection were treated at our institution. Recruitment was retrospective in 16 and prospective in 5 patients. In 5 patients (24%) the complication was associated with Stanford A, in 16 (76%) with Stanford B dissection. Ten patients (48%) complained of malperfusion of only one region, whereas 11 patients (52%) suffered from ischemia of two or three different regions. Aortic fenestration and resection of the dissected membrane was performed in nine cases (37%). Fifteen patients (63%) were treated with extraanatomic bypass techniques. RESULTS: One third of the patients died, four of them due to aortic penetration or perforation and two due to visceral ischemia. During follow-up of 32 (1-110) months two patients developed aortic complications. One died of aortic perforation, while the other developed a thoracoabdominal aneurysm and had to be treated by a tube graft replacement. CONCLUSIONS: Outcome depended more on the spontaneous course of aortic dissection and on prompt diagnosis and therapy of the complications than on the different operative techniques. PMID- 10734592 TI - [Delirium after vascular surgery interventions. Intermediate-term results of a prospective study]. AB - INTRODUCTION: Postoperative delirium is a common psychic disturbance occurring acutely after various surgical procedures and typically presenting with a fluctuating course. These patients' recovery takes longer. In this study we analyze the incidence of postoperative delirium in patients undergoing vascular surgery and try to identify risk factors for its development. METHODS: Patients undergoing elective arterial operations were included. Their medical history, the specific vascular diagnosis and operation performed, the medication and laboratory data were monitored. Additionally the patients were preoperatively interviewed by a psychiatrist. Intraoperatively the drugs, infusions, possible transfusions, blood gases and pressures were monitored, as were the times of surgery and anesthesia. Postoperatively patients were seen daily by the psychiatrist and the surgeon for at least 7 days. Postoperative delirium was diagnosed according to DSM IV criteria, and mild, moderate and severe delirium were distinguished. RESULTS: Fifty-four patients entered the study. Twenty-one (38.9%) developed postoperative delirium (11 mild, 2 moderate, 8 severe). Patients with aortic operations developed delirium more frequently than those with non-aortic procedures(55.5 vs 22.2%, n = 27 each). Some preexisting diseases (hearing disturbance) increased the probability of postoperative delirium, while age was not identified as a risk factor. General psychopathological and depressive disturbances increased the likelihood of postoperative delirium. Patients who had a severe intraoperative course developed postoperative delirium more frequently. This was not seen in the absolute time of surgery or anesthesia nor in the intraoperative development of blood pressure or intraarterial gases, which did not differ between patients with and without postoperative delirium. More reliable parameters were an increased intraoperative need for crystalloid volume, intra- or postoperatively decreased hemoglobin values (Hb < 10 g/dl) and the development of acidosis that had to be treated. Patients with delirium had serious complications more often (8/21 = 38.1% vs 6/33 = 18.2%) and needed Intensive Care treatment longer (2.7 vs 2.1 days, only aortic surgery 3.2 vs 2.4 days). CONCLUSIONS: Postoperative delirium after vascular surgery is frequent. Patients undergoing aortic surgery, with specific concomitant medical disease, psychopathological disturbances and a severe intraoperative course, are at risk of developing postoperative delirium. PMID- 10734593 TI - [Large flank tumor after covered gallbladder perforation]. AB - We report on a 79-year-old woman patient with a tumor with a diameter of 18 cm in the right flank. The medical history of the patient was normal except for a single asymptomatic gallstone, which had been known since 12 years. Sonography and abdominal CT showed an enormous intra-abdominal mass filled with liquid with a permanent union with the gallbladder. We than performed an explorative laparotomy with a tumor resection and a cholecystectomy. The histological examination showed perforation of the gallbladder with a great pseudocyst filled with 1800 ml infected bile and with penetration through the abdominal wall. PMID- 10734594 TI - [Idiopathic segmental fatty tissue necrosis of the ligamentum teres hepatis. Variation on a theme...]. AB - INTRODUCTION: Idiopathic segmental infarction of the peritonealized intra abdominal fatty tissue is a rare cause of acute abdominal distress. Patients are operated for by suspected acute appendicitis or cholecystitis, and the true diagnosis is made intraoperatively. METHODS: A 32-year-old woman was admitted to our hospital with a 2-day history of pain in the right upper abdomen. Clinical presentation suggested acute cholecystitis, but laboratory evaluation and sonography revealed no pathological findings. Because of a distinctly palpable and very painful epigastric tumor 2 x 3 x 3 cm, a CT scan was performed, that showed a clearly circumscribed mass in the ligamentum teres hepatis with hyperattenuating, infiltrating streaks. Laparoscopy was performed, and a tumor was found, that was adherent to the stomach's antrum and could easily be resected. Twenty-four hours after surgery the patient only felt slight discomfort and could be dismissed on the second day. Pathology report revealed a hemorrhagic infarction of the fatty tissue, which can be histologically found in idiopathic segmental necrosis of the greater omenum or the appendices epiploicae. CONCLUSIONS: Laparoscopy is an excellent diagnostic tool and also has therapeutic possibilities. Resection of the necrotic tissue ensures faster recovery and pain control and should be performed to prevent complications such as bacterial superinfection with formation of an abscess or spontaneous bleeding. PMID- 10734595 TI - [Klippel-Trenaunay syndrome with involvement of the rectum. Surgical therapy after interventional-radiologic preparation]. AB - Abdominal organs such as the rectum and urinary bladder are rarely involved in Klippel-Trenaunay syndrome, but may occasionally be the source of severe blood loss. Since frequently no isolated source of bleeding is identified, severe blood loss can result in a critical condition. This article describes an unusual multimodal treatment concept for a patient with Klippel-Trenaunay syndrome associated with severe recurrent rectal bleeding. We present the case of a 39 year-old patient with Klippel-Trenaunay syndrome and a history of rectal bleeding since childhood requiring multiple blood transfusions over the years. He was referred to our department in a state of continuous rectal bleeding. Preoperative work-up revealed a complete alteration of the rectum and the distal parts of the sigmoid/colon by hemangiomas, with diffuse bleeding from the destroyed rectal mucosa. Preoperatively the superior rectal artery was embolized. After a 48-h interval, sphincter-preserving complete rectal excision including the sigmoid/colon was performed followed by a colon pouch anal anastomosis and protective loop ileostomy. PMID- 10734596 TI - [Modification of postoperative wound healing by showering]. AB - Usually postoperative wounds are kept dry until the stitches are removed. In a prospective randomized study early water contact was allowed in order to test postoperative wound healing in 817 patients operated on for varicose veins. Regardless of whether the wounds were kept dry or had water contact with or without shower foam from the second postoperative day, no infection was registered. PMID- 10734597 TI - [Therapy concept in femoral neck fractures. 1]. PMID- 10734598 TI - [Practice parameters, guidelines, standard]. PMID- 10734599 TI - [Recommendations for first aid for the patient with craniocerebral trauma in multiple trauma]. PMID- 10734600 TI - Molecular bases of color vision in vertebrates. PMID- 10734601 TI - Genome size as a mutation-selection-drift process. AB - A novel method for estimating neutral rates and patterns of DNA evolution in Drosophila takes advantage of the propensity of non-LTR retrotransposable elements to create nonfunctional, transpositionally inactive copies as a product of transposition. For many LINE elements, most copies present in a genome at any one time are nonfunctional "dead-on-arrival" (DOA) copies. Because these are off shoots of active, transpositionally competent "master" lineages, in a gene tree of a LINE element from multiple samples from related species, the DOA lineages are expected to map to the terminal branches and the active lineages to the internal branches, the primary exceptions being when the sample includes DOA copies that are allelic or orthologous. Analysis of nucleotide substitutions and other changes along the terminal branches therefore allows estimation of the fixation process in the DOA copies, which are unconstrained with respect to protein coding; and under selective neutrality, the fixation process estimates the underlying mutational pattern. We have studied the retroelement Helena in Drosophila. An unexpectedly high rate of DNA loss was observed, yielding a half life of unconstrained DNA sequences approximately 60-fold faster in Drosophila than in mammals. The high rate of DNA loss suggests a straightforward explanation of the seeming paradox that Drosophila has many fewer pseudogenes than found in mammalian species. Differential rates of deletion in different taxa might also contribute to the celebrated C-value paradox of why some closely related organisms can have very different DNA contents. New data presented here rule out the possibility that the transposition process itself is highly mutagenic, hence the observed linear relation between number of deletions and number of nucleotide substitutions is most easily explained by the hypothesis that both types of changes accumulate in unconstrained sequences over time. PMID- 10734602 TI - A note on the correlation between heterozygosity and recombination rate. AB - Mechanisms responsible for the correlation between heterozygosity and recombination rate have been thought to be background selection and selective sweep. In addition to these two, diversity enhancing selection and/or gene conversion is suggested to be contributing to the correlation, by surveying some examples of highly polymorphic loci that tend to locate in high-recombination regions. PMID- 10734603 TI - Model dependence of the phylogenetic inference: relationship among carnivores, Perissodactyls and cetartiodactyls as inferred from mitochondrial genome sequences. AB - Some previous analysis of mitochondrial proteins strongly support the Carnivora/Perissodactyla grouping excluding Cetartiodactyla (Artiodactyla + Cetacea) as an outgroup, but the support of the hypothesis remains equivocal from the analysis of several nuclear-encoded proteins. In order to evaluate the strength of the support by mitochondrial proteins, phylogenetic relationship among Carnivora, Perissodactyla, and Cetartiodactyla was estimated with the ML method by using the updated data set of the 12 mitochondrial proteins with several alternative models. The analyses demonstrate that the phylogenetic inference depends on the model used in the ML analysis; i.e., whether the site heterogeneity is taken into account and whether the rate parameters are estimated for each individual proteins or for the concatenated sequences. Although the analysis of concatenated sequences strongly supports the Carnivora/Perissodactyla grouping, the total evaluation of the separate analyses of individual proteins, which approximates the data better than the concatenated analysis, gives only ambiguous results, and therefore it is concluded that more data are needed to resolve this trichotomy. PMID- 10734604 TI - Further genetic studies on the Katsunuma population of Drosophila melanogaster. AB - Changes in the genetic structure of the Katsunuma natural population of Drosophila melanogaster have been examined during the past 35 years. The frequency of recessive lethal genes on the second chromosome once increased from 15% to 30% in the early 1970s, then decreased to about 24% in the late 1970s, and thereafter showed no significant changes. Sterility genes, the frequency of which is always less than the lethals, showed a similar tendency. The SD (segregation distorter) mutant gene disappeared but some others such as rbl (reduced bristle) and bw (brown) persisted in the population. The frequency of inversion-carrying chromosomes gradually decreased in the period, such that the standard chromosome frequency in the second and third chromosomes increased from about 40% to more than 80%. Coincident with these frequency changes is the invasion of a transposable element P into the Katsunuma population. The P element should have invaded into Katsunuma in the late 1960s. It spread over the population apparently inducing deleterious mutations, causing the decrease in the allelism rate, and hence increasing the effective population size. Soon, however, most flies became resistant to the P element-mediated transposition as they began to harbor defective P elements. During the course of spreading, the P element must also have induced deleterious mutations on the polymorphic inversions, breaking up the heterotic gene complexes along the chromosomes, which probably caused the reduction in the frequency of inversion chromosomes. Temporal invasion of D. simulans, a sibling species of D. melanogaster, into Katsunuma occurred several times after 1978, and the species seems to have been settled since 1990. This, however, did not have any effect on the genetic structure of D. melanogaster population. PMID- 10734605 TI - Complete nucleotide sequence of the prophage VT2-Sakai carrying the verotoxin 2 genes of the enterohemorrhagic Escherichia coli O157:H7 derived from the Sakai outbreak. AB - The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain RIMD 0509952, derived from an outbreak in Sakai city, Japan, in 1996, produces two kinds of verotoxins, VT1 and VT2, encoded by the stx1 and stx2 genes. In the EHEC strains, as well as in other VT-producing E. coli strains, the toxins are encoded by lysogenic bacteriophages. The EHEC O157:H7 strain RIMD 0509952 did not produce plaque-forming phage particles upon inducing treatments. We have determined the complete nucleotide sequence of a prophage, VT2-Sakai, carrying the stx2A and stx2B genes on the chromosome, and presumed the putative functions of the encoded proteins and the cis-acting DNA elements based on sequence homology data. To our surprise, the sequences in the regions of VT2-Sakai corresponding to the early gene regulators and replication proteins, and the DNA sequences recognized by the regulators share very limited homology to those of the VT2-encoding 933W phage carried by the EHEC O157:H7 strain EDL933 reported by Plunkett et al. (J. Bacteriol., p1767-1778, 181, 1999), although the sequences corresponding to the structural components are almost identical. These data suggest that these two phages were derived from a common ancestral phage and that either or both of them underwent multiple genetic rearrangements. An IS629 insertion was found downstream of the stx2B gene and upstream of the lysis gene S, and this might be responsible for the absence of plaque-forming activity in the lysate obtained after inducing treatments. PMID- 10734606 TI - Las21 participates in extracellular/cell surface phenomena in Saccharomyces cerevisiae. AB - Las21 (Yj1062W) is a member of the major facilitator super family, possessing multimembrane spanning domains. The LAS21 gene was identified as a responsible gene for a Saccharomyces cerevisiae mutan which shows sensitivity to a local anestheticum, tetracaine. The null las21 mutant (las21 delta) is viable but shows temperature sensitive growth. We found, in addition to this phenotype, that the las21 delta strain shows a number of defects; mating deficiency, calcofluor resistance, and formation of Zymolyase sensitive spores. Temperature sensitive growth of the las21 delta mutant was found to be suppressed by 0.1 M MgSO4. Two multicopy suppressors were obtained. They are ECM33 (YBR078W) and PIR2/HSP150 (YJR159W) both have some roles in an extracellular function. The common features of the suppressors, genetic and physiological, of the las21 delta mutation suggest that Las21 participates in a global activity of extracellular phenomena. The las 21 phenotypes are consistent with the idea that Las21/Gpi7 acts in metabolism of glycosylphosphatidylinositol. PMID- 10734607 TI - Sequence variation and evolution of the mitochondrial DNA control region in the musk shrew, Suncus murinus. AB - The complete mitochondrial DNA (mtDNA) control region was cloned and sequenced in the musk shrew, Suncus murinus, Insectivora. The general aspect was similar to that found in other mammals. We have found in two locations of this region the presence of arrays of tandem repeats like those in other shrew species. One array was located in the left domain containing the termination-associated sequences (TAS) and the length of a copy was 77 bp. The other repeats were situated upstream from the recognition site for the end of H-strand replication in the right domain and were 20 bp long. The left halves of the control region containing the former repeats were sequenced and compared in several laboratory lines and wild animals from different localities, variations in copy number of repeated sequences were found both among individuals and within an individual. A comparative study of repeated sequences provides useful indication for the origin and evolution of tandem repeated sequences. Strand slippage and mispairing during replication of mtDNA with concerted manner is currently regarded as a dominant theory to account molecular mechanism for tandemly repeated sequences, and the pattern of sequence and length variation in our study supports this theory. Our results, however, suggest that the evolution of the repeated sequences containing the TAS in the musk shrew might go through the process of two steps; at the first step one complete repeated and several incomplete repeated sequences had reproduced in common ancestor of the shrew, and the second stage step-up of complete repeated sequences occurred with concerted evolution after differentiation into continental and insular groups. PMID- 10734608 TI - Presence and phylogenetic analysis of HERV-K LTR on human X and Y chromosomes: evidence for recent proliferation. AB - The K group of human endogenous retroviruses (HERV-K) has been suggested to have a role in disease and has recently been shown to include long terminal repeat (LTR) elements that are human specific. Here we investigated the presence of HERV K LTRs on the human X and Y chromosomes with the use of PCR on a monochromosomal somatic cell hybrid DNA panel. We report twelve such sequences on the X chromosome and ten sequences on the Y chromosome. Phylogenetic analysis reveals that clones X2, 4, 5, 6, 7, 11, 15 from the X chromosome and clones Y4, 5, 7, 10 from the Y chromosome are closely related to the human-specific members of Medstrand and Mager's cluster 9. The sequence of clone Y7 from the Y chromosome is identical with human-specific HERV-K LTR element (AC002350) from chromosome 12q24. The findings suggest recent proliferation and transposition of HERV-K LTR elements on these chromosomes. Such events may have contributed to structural change and genetic variation in the human genome. We draw attention to evolutionarily recent changes in homologies between X and Y chromosomes as a method of further investigating such transpositions. PMID- 10734609 TI - Qualitative research--does it have a place in dental public health? PMID- 10734610 TI - An exploratory qualitative study examining the social and psychological processes involved in regular dental attendance. AB - OBJECTIVE: This study aimed to provide a description of the psychosocial process involved in regular dental attendance. METHODS: The study design was a qualitative cross-sectional study using unstructured and semistructured interviews and observations of regular dental visits. The study participants included 12 men and 18 women attending general dental practices and six men and four women attending an emergency dental service. The data were systematically recorded and subjected to line-by-line grounded theory coding around the main concerns of those attending the dentist. RESULTS: The main concern of those attending for a regular dental visit was checking their oral health. The six month recall was conceptualized as a checking cycle in six phases: recalling, responding, inducing (i), waiting, inducing (ii), and telling. The possible outcomes of the cycle were maintaining oral health, sustaining oral health, and a further checking cycle. Variations in checking cycles resulted from reordering and normalizing pressures within participants' lifestyles. CONCLUSIONS: The findings of this study suggest that people's patterns of dental attendance are similar to those of other chronic illnesses. An understanding of the dynamic psychosocial processes involved in frequent dental attendance may be achieved when further research into this phenomenon is conducted. PMID- 10734611 TI - Medication and dry mouth: findings from a cohort study of older people. AB - OBJECTIVES: The aim of this study was to examine the association between medication exposure and (1) unstimulated whole-salivary flow rate and (2) the severity of xerostomia among older people while adjusting for multiple medication use. METHODS: Data were obtained from participants remaining at the five-year follow-up phase of a cohort study of community-dwelling older South Australians. Medication exposure information was available at baseline and at five years, enabling examination of the effects on dry mouth of long-term exposure to medications. At the five-year follow-up, unstimulated salivary flow was estimated using the spit method, and xerostomia severity was estimated using the 11-item Xerostomia Inventory. Because of the potential difficulties posed by polypharmacy, a two-stage analytical approach was employed: (1) Classification and Regression Tree (CART) analysis was used as an exploratory device to elucidate the relationships among the dependent and independent variables, and (2) linear regression analysis was used as a complementary procedure. RESULTS: Unstimulated flow rate was lower among individuals who were female or taking antidepressants at both baseline and five years, and higher among smokers or people who were taking hypolipidemic drugs. Xerostomia severity was higher among females, or individuals taking: (1) an anginal at baseline and five years, (2) an anginal without a concomitant betablocker at five years, (3) thyroxine and a diuretic at five years, or (4) antidepressants or antiasthma drugs at both baseline and at five years. CONCLUSIONS: These results suggest that polypharmacy can be accounted for to a certain extent by using CART analysis in conjunction with more conventional approaches; and that the relationship between medications and dry mouth is a complex one, and differs according to which aspect of dry mouth is being examined. PMID- 10734612 TI - Dental services, costs, and factors associated with hospitalization for Medicaid eligible children, Louisiana 1996-97. AB - OBJECTIVE: This study compared types and costs of dental services rendered to children who had received care in a hospital operating room (H) with children who had not (NH). METHODS: The study population consisted of all children aged 1-5 years who received a dental service reimbursed by the Louisiana Medicaid EPSDT program from October 1996 through September 1997. Claim files were provided by the Louisiana Bureau of Health Services Financing. A treatment intensity index [TII = 3 x (# extractions) + 2 x (# pulpotomies + # crowns) + # simple restorations] was calculated for H children (n = 2, 142) and NH children (n = 38,423). Using logistic regression, a dichotomous hospitalization variable (H vs NH) was regressed against treatment intensity and selected personal and parish (county) characteristics for each of the five age groups. Total and average reimbursement per child were calculated for both groups of children, by age. RESULTS: The mean treatment intensity scores for H and NH children were 24.02 (SD = 11.82) and 2.16 (SD = 4.78), respectively. For all age groups, children with treatment intensity scores greater than 8 were at least 132 times more likely to be hospitalized than were children with scores less than or equal to 8. The mean cost for care provided to H children was $1,508 compared with $104 for NH. Total costs for dental care rendered to H children (5% of the study population) were $3,229,851 (45% of total dental costs for the study population). CONCLUSION: Reducing severe caries through early interventions could provide substantial cost savings. PMID- 10734613 TI - Medicaid costs associated with the hospitalization of young children for restorative dental treatment under general anesthesia. AB - OBJECTIVE: This paper examines the cost to the Iowa Medicaid program of hospitalizing young children for restorative dental care under general anesthesia, and describes the dental services received in this setting. METHODS: Medicaid dental claims for young children receiving restorative dental care under general anesthesia during fiscal year 1994 were matched with corresponding hospital and anesthesia claims. RESULTS: The total cost to the Medicaid program of treating a child in the hospital under general anesthesia was $2,009 per case. Less than 2 percent of Medicaid-enrolled children under 6 years of age who received any dental service accounted for 25 percent of all dollars spent on dental services for this age group, including hospital and anesthesia care. The most frequent type of procedure was stainless steel crowns (SSCs), with an average of almost six per case. CONCLUSIONS: Early identification, prevention, and intervention are critically important to prevent the costly treatment of children with ECC in hospital operating rooms. PMID- 10734614 TI - Professionally applied topical fluoride and restorative care in insured children. AB - OBJECTIVES: This study evaluates the association between use of professionally applied topical fluoride and use of interproximal restorations in primary and permanent teeth of children. METHODS: Insurance claims from 15,190 children, for treatment provided by 1,556 different dentists, were analyzed to look for associations between frequency of use of professionally applied topical fluoride and use of interproximal restorations. The average follow-up period for the children included in the analysis was 5.3 years, with the range from 3.0 to 7.9 years. RESULTS: Both tabular and regression results failed to demonstrate an association between frequency of use of professionally applied topical fluoride and use of interproximal restorations in either the primary or permanent dentition. The most powerful predictor of restorative care for these children was the overall propensity of the dentist to place restorations in children. CONCLUSIONS: In this group of insured children, we were unable to find an association between the frequency of use of professionally applied topical fluoride and restorative care. Further, despite numerous recommendations that professionally applied topical fluorides should be used only in moderate- and high-caries children, approximately two-thirds of these children received topical fluoride at every recall visit, nearly two times per year. PMID- 10734615 TI - Factors related to decisions to extract or retain at-risk teeth. AB - OBJECTIVES: Decisions to extract a tooth may be among the most critical in dentistry. The aim of this study was to prospectively investigate both clinical and nonclinical factors related to decisions to extract or retain teeth in private general dental practice. METHODS: A convenience sample of 196 Iowa dentists in private general dental practice reported on 549 cases where decisions were made to extract or retain teeth as they occurred in their practices during a one-month period in May 1997. Bivariate and multivariate analyses were used to identify factors that differentiated between cases where a tooth (or teeth) was extracted and cases where an at-risk tooth was retained. RESULTS: Of the 549 cases, 67 percent involved extraction, while the remainder involved alternatives to extraction. In comparing extraction cases to alternative treatment cases, we excluded 150 extraction cases because dentists reported that no alternative to extraction was available. Using Generalized Estimating Equations (GEE), we identified cost of treatment, presence of tooth mobility, poor prognosis of alternative treatment, and presence of gross caries as significant factors associated with extraction, while previous treatment of the tooth and concerns with patients' health were significantly associated with alternatives to extraction. CONCLUSIONS: For teeth at risk for extraction, cost, substantial periodontal disease, and several clinical factors were predictive of extraction. PMID- 10734617 TI - Oral cancer educational materials for the general public: 1998. AB - OBJECTIVES: Previous studies have shown that US adults are not well informed about oral cancers and only 15 percent ever have had an oral cancer examination. This study sought to determine the quantity and adequacy of educational materials designed to inform or educate US adults about risks for, and signs and symptoms of, oral cancer and the need for an oral cancer examination. METHODS: Letters requesting copies of oral cancer educational materials produced by the organization or agency--leaflets, fact sheets, pamphlets, videos, posters--were sent to 172 national and state organizations or agencies. To determine the adequacy of the items, a previously developed, tested, and used form based on current science was adapted for this study. In addition, the SMOG index was used to determine readability for printed items. RESULTS: Seventy-seven percent or 132 of the selected organizations responded to queries. A total of 59 items were received that focused on or included the topic of oral cancer. Twenty of these 59 items focused specifically on oral cancer; the balance, on other topics, but mentioned oral cancer. The readability ranged from sixth to 13th grade. CONCLUSIONS: This study demonstrates a dearth of educational materials about oral and pharyngeal cancers; most are written at too high a grade level for the general public. These findings may help to explain why the public is so uninformed about these neoplasms. PMID- 10734616 TI - The availability and distribution of dentists in rural ZIP codes and primary care health professional shortage areas (PC-HPSA) ZIP codes: comparison with primary care providers. AB - OBJECTIVE: This paper maps dentists, primary care physicians, physician assistants, nurse practitioners, and nurse midwives in rural areas and rural areas meeting criteria as underserved for primary health care. METHODS: Computer based mapping was used to localize health care provider groups by five-digit ZIP code. For each rural and each rural primary care health professional shortage area (PC-HPSA) ZIP code, the number of providers in each group was determined. The different combinations of providers were determined. RESULTS: All providers in rural areas were present at levels substantially lower than national averages, particularly in PC-HPSA areas. Dentists were approximately equal in number to primary care physicians in rural areas and the largest group in PC-HPSAs. Approximately 75 percent of rural residents lived in ZIP code areas with dentists available. Over 5.8 million rural residents and over 50 percent of rural PC-HPSA residents had no providers available in their ZIP code areas. CONCLUSIONS: Rural areas continue to have a short supply of primary care providers and dentists. Dentists were present in many areas where primary care provider presence was absent or very low. These data, including those relating to provider co-presence, can be used to develop strategies to overcome health care access problems due to provider shortages. PMID- 10734618 TI - A comparison of sealant placement techniques and 12-month retention rates. AB - OBJECTIVE: This study compared time required and 12-month retention for two methods of sealant placement: traditional acid-etch technique and air-abrasion technique without acid etch. METHODS: Sealants were placed on the permanent first molars of 84 children in grades 1-4 who were randomly assigned to treatment groups. All sealants were placed in a school setting by the same clinician. Chair time required for sealant placement was recorded, and retention rates were determined for each technique 12 months after placement. RESULTS: Mean chair time for placement of sealants on four first molars using the air-abrasion technique was significantly less than for the acid-etch technique (7:36 minutes vs 10:56 minutes). Fifty-eight children were available at 12-month follow-up; an examiner not involved in sealant placement and masked as to the technique used determined retention. Rates of complete retention for occlusal surfaces were not significantly different for the two techniques, although the rate for acid etch was higher than that for air abrasion (95% vs 87%). The complete retention rates for the acid-etch technique were significantly higher than air abrasion for buccal and distolingual surfaces. These rates were 65 percent and 58 percent, respectively, for acid etch and 6 percent and 28 percent, respectively, for air abrasion. CONCLUSION: Although more research is needed to improve air-abrasion applications, it does not appear that air abrasion without acid etching offers a significant advantage over traditional sealant placement methods and, in fact, appears to be inferior to the acid-etch technique for use in public health settings. PMID- 10734619 TI - Dean's fluorosis index: an assessment of examiner reliability. AB - OBJECTIVES: This report describes the interexaminer reliability achieved using Dean's Index in a study of dental fluorosis, and shows the effect on kappa values of assigning different weights to the various components of Dean's Index. METHODS: Three dentists conducted replicate fluorosis examinations on 202 children in Newburgh and Kingston, NY. Examiner reliability was assessed by computing percent agreement and weighted and unweighted kappa statistics. RESULTS: Agreement on the presence or absence of fluorosis using Dean's definition of fluorosis ranged from 92 to 97 percent and the respective kappa values ranged from 0.75 to 0.94. A comparison of subject-level severity scores for Dean's Index resulted in percent agreement ranging from 79.6 percent to 86.8 percent and kappa values ranging from 0.67 to 0.76. Weighting the kappa statistics improved agreement and reduced the differences. CONCLUSIONS: Examiners showed good to excellent agreement beyond chance in the use of the index. Subject level kappa scores were higher than tooth-level scores. PMID- 10734620 TI - [The use of cardiopulmonary exercise test in patients with mitochondrial myopathies]. AB - BACKGROUND: Exercise tests has been used in the diagnosis of metabolic myopathies. If there is an abnormal response pattern in mild mitochondrial myopathy (MM) and her role in the initial suspect diagnostic is unknown. SUBJECTS AND METHODS: Prospective study of 26 patients with mitochondrial myopathy (15 men, 11 women) and a control group of 14 sedentary volunteers (9 men, 5 woman) with similar antropometric characteristics. We have made pulmonary function tests and treadmill exercise with serial venous sampling of blood lactate, piruvate, ketone bodies, free fatty acids and creatinkinase. RESULTS: Patients with MM showed exercise limitation with lower maximal power (MM = 143 [47] vatts, C = 187 [40] vatts, p = 0.006), maximal oxygen uptake (MM = 27 [8] ml/min/kg, C = 40 [7] ml/min/kg, p = 0.001) and maximal oxygen pulse (MM = 11 [3] ml/beat, C = 14 [3] ml/beat, p = 0.006). For the same oxygen uptake the heart rate was higher and the anaerobic threshold was earlier in MM patients (MM = 48% [14], C = 62% [12], p = 0.01). We found a considerable slower maximal oxygen uptake in patients with lipid increase in muscle biopsy (n = 15). Acetoacetate curves, beta hydroxybutyrate, free fatty acids and creatinkinase were similar in both groups. The exercise test was pathologic in 17/26 of the patients. The sensitivity of the exercise test for maximal oxygen uptake was 0.65. CONCLUSIONS: The cardiopulmonary exercise test is a useful test in the suspect initial diagnostic of patients with mild mitochondrial myopathy because the majority of patients show a pathologic test. PMID- 10734621 TI - [Hematopoietic stem cell transplantation as salvage treatment in patients with aggressive non-Hodgkin's lymphoma]. AB - BACKGROUND: The indication of early hematopoietic stem cell transplantation (HSCT) in patients with aggressive non-Hodgkin's lymphoma (LNH) is controversial. PATIENTS AND METHODS: Retrospective analysis of 86 patients with aggressive NHL treated with MACOP/VACOP-B chemotherapy. HSCT was performed as salvage treatment to patients under 65 years of age with progressive disease or chemosensitive relapse. Progression free survival (PFS) and overall survival (OS) were determined by the Kaplan-Meier method. Rates of response and survival functions were compared between the International Prognostic Index (IPI) groups using the Chi-square and log-rank tests, respectively. RESULTS: Patients median age was 48 years; 22% had T cell NHL and 57% had intermediate-high and high risk (high risk) IPI. There were 6 toxic deaths (7%), and treatment failure was observed in 42 patients (48.8%). Thirty one of them were candidates for TPH due to age under 65 years, although 21 were finally transplanted (including 13 with high risk IPI). A significant association between PFS and IPI was observed, 61.9% for low risk (low and low-intermediate) versus 28.2% for high risk groups (p = 0.0007). With a median follow up of 4.8 years, OS was 64%; 80.5% for low risk versus 52.6% for high risk IPI groups (p = 0.01), and 83.7% versus 62% for the same groups in patients under 65 years of age (p = 0.02). The median follow up after failure to chemotherapy was 42.7 months. CONCLUSIONS: In this retrospective study, OS rate in high risk IPI patients with NHL using HSCT as salvage treatment is similar to that reported using HSCT during earlier phases of treatment. PMID- 10734622 TI - [Determinant factors of smoking cessation during pregnancy]. AB - BACKGROUND: To investigate sociodemographic factors and smoking habit characteristics associated with spontaneous smoking cessation during pregnancy. DESIGN: SUBJECTS AND METHODS: Cross-sectional study including 726 pregnant smoking women who attended the antenatal clinic of the Vall d'Hebron University Hospital in Barcelona between July and December 1997 and who agreed to participate were studied. Information was obtained by a structured questionnaire which included items on age, educational level, marital status, previous and current smoking habit, partner smoking habit and partner's attitude towards the smoking habit of the pregnant woman. RESULTS: At the first antenatal visit, 19.7% of women had given up smoking spontaneously. Light smokers (between 1 and 9 daily cigarettes; OR = 4.74) or moderate smokers (between 10 and 19 daily cigarettes; OR = 2.20) were more likely to stop smoking compared to heavy smokers (more than 20 daily cigarettes). If the partner did not smoke, smoking cessation was more likely among pregnant women (OR = 1.78). At the first antenatal visit, among those women who carried on smoking during pregnancy, there was a significative reduction in cigarette consumption of about half the usual daily number of cigarettes smoked. CONCLUSIONS: One out of five pregnant women give up smoking spontaneously during pregnancy from the moment they know they were pregnant until the first antenatal visit. Women who are less likely to stop smoking during pregnancy are those who smoke a larger number of cigarettes. Family environment is an important influential factor in the smoking habit of the pregnant woman. PMID- 10734623 TI - [Changes in epidemiological pattern of methicillin-resistant Staphylococcus aureus infections in a tertiary hospital. Experience of 5 years]. AB - OBJECTIVE: To describe the demographic pattern and tendency of the infections by MRSA between 1992 and 1997. DESIGN AND METHODS: Descriptive study of the infections by MRSA in a tertiary-care hospital. RESULTS: 267 MRSA infections, 131 infections were included within 58 buds and 136 cases isolated form. The more affected services were Internal Medicine, Urology, Neurology, Vascular surgery and intensive care unit. A tendency was observed to the increase in > 65 years cases and in medical services. CONCLUSIONS: The increase of elderly cases in medical services and > 65 years carriers in their nose could translate the existent situation in the community. PMID- 10734624 TI - [Mitochondrial diseases: still a difficult diagnosis]. PMID- 10734625 TI - [Critical care units: a difficult task of information]. PMID- 10734626 TI - [Apoptosis: to be or not to be, that is the question]. PMID- 10734627 TI - [Accidental post inoculation of acute hepatitis C in a health care professional with response to interferon treatment]. PMID- 10734628 TI - [Survival and quality of life in out-of-hospital cardiac arrest in a population without an emergency care system]. PMID- 10734629 TI - [Polyarteritis nodosa, cyclophosphamide and interstitial lung disease]. PMID- 10734630 TI - [Potential role of monocytes in the pathogenesis of thrombotic events in patients with deficiencies in clotting factors]. PMID- 10734631 TI - [A randomized, prospective and comparative study is a clinical trial]. PMID- 10734632 TI - [Metabolic engineering of microbial cells]. PMID- 10734633 TI - [A new branch of biotechnology--wound-healing substances based on polyaminosaccharides]. AB - This article concerns itself with the recently developed field of biotechnology dealing with the production of polyaminosaccharide-based wound-healing preparations. A method of obtaining the wound-healing preparation Mycoran with antiburn properties is presented. Based on the histologic findings provided by medical and biological tests of the preparation and the data available in the literature on the role of polysaccharides as signal molecules and glycoconjugates, the possible mechanism of stimulating fibroblast proliferation by fungal chitin and chitosan during the wound-healing process is discussed. PMID- 10734634 TI - [Physiologo-biochemical properties of a strain of Beijerinckia mobilis 1phi Phn+- a degrader of polycyclic aromatic hydrocarbons]. AB - Beijerinckia mobilis 1f capable of degrading polycyclic aromatic hydrocarbons (PAHs) was isolated from a soil contaminated with creosote. Strain 1f could utilize phenanthrene and naphthalene as the sole sources of carbon. The mean rate of phenanthrene degradation during culture growth was 7-8 micrograms/(ml h). After cultivation under nonselective conditions, strain 1f retained its ability to degrade phenanthrene. Cometabolism considerably widened the range of PAHs that could be transformed by strain 1f. The strain was able to grow in a mineral medium with creosote as the sole source of carbon. After 30 days of cultivation in this medium, the total concentration of PAHs decreased from 665.5 mg/l to 170 mg/l. PMID- 10734636 TI - [New classification of the T category for pancreatic cancer]. AB - The General Rules for the Study of Pancreatic Cancer (4th edition) is widely used in Japan, but several problems have been detected. One is the complexity of classification of the T category of peripancreatic invasion. This study was undertaken to simplify the classification of the T category. In 145 patients who underwent resection of pancreatic cancer, we analyzed the prognosis of those with invasion and without invasion based on seven factors of peripancreatic invasion. Furthermore, we selected three factors (S, invasion to the anterior pancreatic capsule; RP, invasion to the retroperitoneal tissue; and PL, invasion to the extrapancreatic nerve plexus) as prognostic factors by means of stepwise multivariate linear regression analysis. Subsequently we established a new classification of the T category: T1, without invasion in all three factors; T2, with invasion in only one of the three factors; and T3, with invasion in two or three factors. The prognoses of the same patients were analyzed retrospectively, and 1-, 3-, and 5-year survival rates were 100%, 88%, and 44% in T1, 76%, 30%, and 18% in T2, and 27%, 4%, 3% in T3 patients, respectively. In conclusion, we suggest that our new classification of the T category is simple and useful for the assessment of the prognosis in pancreatic cancer. PMID- 10734635 TI - [Histopathologic problems in the classification of pancreatic carcinoma]. AB - The major differences between the third and fourth edition of the classification of pancreatic carcinoma are that tumors of ductal origin are divided into intraductal tumors and invasive ductal carcinoma, and atypical hyperplasia is mentioned. A low papillary or flat carcinoma in the pancreatic duct which is regarded as an early feature of ductal adenocarcinoma is classified as an intraductal tumor. However, usage of this term is not suitable from the standpoint of the outline of pathology. Minimally invasive intraductal papillary mucinous carcinoma is classified as an intraductal tumor instead of invasive ductal carcinoma. It is often argued that minimally invasive intraductal papillary-mucinous carcinomas and invasive carcinomas derived from intraductal papillary adenocarcinoma should be distinguished. An intraductal papillary mucinous tumor represents cystic dilatation of the pancreatic duct due to mucin secretion and papillary projection and is sometimes misdiagnosed as a mucinous cystic tumor. However, the two tumors are different. This point should be revised in the next edition. The pathologic description of the classification of pancreatic carcinoma should reflect the exact pathophysiology of these tumors and be simple. PMID- 10734637 TI - [Is it able to simplify the classifications of staging and curability for pancreatic cancer?]. AB - This study was undertaken to simplify the classifications of the staging and curability of pancreatic cancer by the Japan Pancreas Society (JPS). A total of 461 patients who underwent surgical resection from 1968 to 1997 were analyzed in this study in accordance with our new classification of staging and curability. This staging mainly consists of three factors based on tumor extent:invasion to the retroperitoneum (rp); invasion to the extrapancreatic nerve plexus (pl); and lymph node metastasis (n). Each factor is simply judged as + (positive) or - (negative). Curability is also judged simply based on whether the presence of tumor is detected or not, and the degree of lymph node dissection is not included in curability. Our staging and curability assessment is strongly associated with survival rates after surgery. The 5-year survival rate of stage I patients is 50% and curability of A is > or = 50%. It is concluded that it is possible to simplify the classification of the staging and curability of pancreatic cancer by the JPS. PMID- 10734638 TI - [Evaluation of classification of pancreatic cancer by the Japan Pancreas Society and Union Internationale Contre le Cancer and proposal for a new international classification]. AB - Different classification systems of pancreatic cancer by the Japan Pancreas Society (JPS, 1996) and Union Internationale Contre le Cancer (UICC) (1997), both of which are based on the TNM system, have been hampering the exchange of data between Japan and Western countries. In the present study, both classifications were assessed using data from 67 patients with invasive ductal carcinoma of the pancreatic head who underwent resection at our clinic. We also propose a new TNM classification and stage grouping system that draws on the merits of both. The results showed that UICC stage grouping did not reflect outcome well, while the JPS system predicted outcome much better. The prognostic value of tha T category is sufficiently reliable in both classifications, but the major drawback of the JPS system is its complex structure and difficult handling. To meet the ideal standard for an international classification system, we propose a new classification: the T category is a minor modification of that of the UICC, and the N category is a modification of that of the JPS for much simpler grouping. Based on the data from our 67 resected patients cases, our new staging system reflects the outcome in the four stages well. Our new classification system may improve the staging classification and lead to the establishment of a more practical and universal staging system for ductal carcinoma of the pancreas. PMID- 10734639 TI - [The role of preoperative staging for pancreatic cancer]. AB - Despite the poor prognosis of pancreatic carcinoma patients, surgical resection remains the only potentially curative treatment. As it has been demonstrated that extended radical surgery does not lead to a better prognosis, accurate staging to select those patients who may benefit from resection is essential. According to the report of a national survey of pancreatic cancer in 1997, patients with S0 or S1, RP0 or RP1, and N0 or N1 disease have longer survival periods compared with patients with S2, RP2, and N2 disease. Therefore patients classified as Stage I, Stage II, or Stage III are recognized as candidates for surgical resection. Patients classified as Stage IVb because of positive P factor or positive H factor are selected for conservative treatment such as chemotherapy and irradiation. It remains to be clarified whether patients classified as Stage IVa should undergo surgical resection or not. Future prospective randomized studies of patients with Stage IVa disease will reveal whether surgical resection or chemoradiation is effective. Helical CT is useful to evaluate S and RP factors for definitive preoperative staging. CT-AP can reveal occult metastases to the liver. These preoperative evaluations should be evaluated along with histological findings. PMID- 10734640 TI - [Proposal of a new lymph node mapping system for pancreatic head cancer]. AB - The Japanese Pancreatic Society published the 4th edition of the general guidelines for the study of pancreatic cancer (JPS guidelines) in 1993. The current JPS guidelines differ from the UICC classification of lymph nodes. Thirty five lymph node stations are divided into three groups in the JPS guidelines. Group 1 (n1) corresponds closely with the regional lymph nodes in the UICC classification. Group 2 (n2) and group 3 (n3) are distant metastases in the UICC classification. To clarify practical lymph node mapping, 2,449 resected tubular adenocarcinomas of the pancreas head registered with the JPS were analyzed. Lymph node involvement was observed most frequently in posterior pancreatoduodenal (PPD) and anterior pancreatoduodenal (APD) and then in paraaortic (n2) nodes. Fifty-six patients who achieved 5-year survival were registered, of whom 19 had lymph node involvement. The sites of lymph node involvement were limited to the PPD and/or APD nodes in 14 cases. PPD and APD nodes differ from the other n1 in terms of high incidence of metastasis and fair prognosis. In conclusion, we propose a new practical lymph node mapping system in which PPD and APD nodes are the first station and the other n1 are the second station. PMID- 10734641 TI - [Difficulty in using RP, PL, and EW factors in the Japanese classification of pancreatic carcinoma]. AB - The classification of pancreatic carcinoma by the Japan Pancreas Society reflects the prognosis of each stage better than does the TMN classification. On the other hand, there are too many factors to examine in the Japanese system, some of which are difficult to use and have low accuracy in pre- and/or intraoperative diagnosis (RP and PL), and their analysis requires various specimen handling procedures (EW). We propose that: 1) a simple, easy decision flow chart be established for ew and 2) EW/ew be designated as (-) or (+) and for EW/ew (+) cases other factors (ly, v, pl, and direct tumor invasion) be added (for example, ew (+)-pl). PMID- 10734642 TI - [The general rules for the study of pancreatic cancer from the viewpoint of internal medicine]. AB - Useful nonsurgical treatments like chemoradiotherapy have been developed for the management of patients with unresectable pancreatic cancer. The General Rules for the Study of Pancreatic Cancer (Japan Pancreas Society) should therefore take diagnostic imaging techniques into account in order to deal with nonsurgical cases under the classification system as well as with surgical ones. The anatomic extent of disease based on preoperative dynamic CT images was evaluated in comparison with the results of pathology in a total of 35 patients with pancreatic cancer. The CT findings accurately represented the pathology for peripancreatic invasion, including vascular involvement, with a diagnostic accuracy ranging from 72% to 88%. However, it failed to detect almost half of lymphatic metastases. The staging of pancreatic cancer with dynamic CT was accurate in 66% of the patients, was underestimated in 25%, and was rarely overestimated. The detection of minute metastases is a crucial problem in the CT staging of tumors. The significant correlation of the RP factor (retroperitoneal tumor invasion) with lymphatic metastases as noted in this study offers a clue to solving this problem. PMID- 10734643 TI - [General rules for the study of pancreatic cancer by molecular biological aspect]. AB - Genetic changes in K-ras, p53, p16, DPC4, and telomerase activity are frequent in pancreatic adenocarcinoma. The incidence of these changes has been reported to be approximately 80% for K-ras, 50% for p53, p16, and DPC4, and 90% for telomerase activity. Genetic abnormalities of APC and microsatellite instability are relatively rare (less than 10%) in pancreatic carcinoma. Among these genetic abnormalities, K-ras and telomerase activity have been used as molecular markers for the diagnosis of pancreatic carcinoma. K-ras mutation could be considered as an early event in the progression to malignancy and thus it has no clear association with the prognosis of the carcinoma. In contrast, mutation of p53 could be a prognostic indicator. PMID- 10734644 TI - [Icteric-type hepatoma with liver cirrhosis successfully treated with hepatectomy after biliary drainage: case report]. AB - A 67-year-old man with liver cirrhosis and an icteric-type hepatoma involving the left main portal vein underwent left hepatic lobectomy after percutaneous transhepatic biliary drainage. Surgery was successful because of effective biliary drainage and meticulous assessment of liver function tests, including 99mTC-galactosyl human serum albumin scintiphotography. PMID- 10734645 TI - Preparticipation screening by assembly line. PMID- 10734646 TI - Sports medicine in pediatric practice: keeping pace with the changing times. PMID- 10734647 TI - The evolution of the female athlete: progress and problems. PMID- 10734648 TI - Preseason conditioning for the preadolescent athlete. PMID- 10734649 TI - Cardiac evaluation of the young athlete. PMID- 10734650 TI - Swimming: a case-based approach to exercise-induced asthma and rotator cuff tendonitis. PMID- 10734651 TI - Football: a case-based approach to mild traumatic brain injury. PMID- 10734652 TI - Soccer: a case-based approach to ankle and knee injuries. PMID- 10734653 TI - Resident's column: should sports medicine be part of the pediatric training curriculum? PMID- 10734654 TI - Adaptational process of parents of pediatric oncology patients. AB - This study, based on grounded theory, explores the adaptational process of parents of pediatric oncology patients. Thirty-two Taiwanese parents (26 mothers and 6 fathers) were interviewed. Data were collected through individual in-depth and focus group interviews, observations, medical chart review, nurses' note, and researchers' reflexive journals. The findings suggest that parents adapt to their children's cancer by a dynamic process; i.e., they modify their coping tasks and related strategies as clinical events (e.g., diagnosis, side effects, relapses, or death) occur. This adaptational process consisted of five components: confronting treatment, maintaining family integrity, establishing support, maintaining emotional well-being, and searching for spiritual meaning. Related factors such as coping tasks are described. PMID- 10734655 TI - Natural killer cell numbers and cytotoxic activity in pediatric Hodgkin disease. AB - In this study peripheral blood natural killer (NK) cell activity was evaluated in 17 pediatric cases with Hodgkin disease (HD) (9 untreated, 8 in remission) and 20 age-matched healthy children. Peripheral blood CD16 and CD56 molecule expressions were also examined. No difference related to NK cell numbers and cytotoxic activity was detected at either stage of the disease. In cases in which long-term remission has been achieved (> or = 5 years) NK cell activity was slightly but not significantly increased in parallel with remission duration. Finally, no relation between NK cell activity and the etiology, prognosis, and severity of the disease has been established in children with HD. PMID- 10734656 TI - Comparative genomic hybridization in pediatric acute lymphoblastic leukemia. AB - Comparative genomic hybridization (CGH) was used to clarify the chromosomal status of 15 patients diagnosed with childhood acute lymphoblastic leukemia (ALL). Bone marrow samples from 10 of the 15 patients were selected because no metaphases were obtained for cytogenetic analysis. Three patients with normal trypsin and giemsa banding (GTG) karyotypes were also studied by CGH to determine whether significant abnormalities might have been missed by banding analysis, and samples from an additional 2 patients with hyperdiploidy were also included. Seven of the 10 patients with failed GTG banding analysis were found to be chromosomally abnormal by CGH; 2 out of 3 patients with normal GTG band karyotypes were abnormal, indicating that the metaphases available for karyotyping were not malignant cells, and that CGH analysis of hyperdiploid samples provided more accurate resolution than karyotyping alone. The prognostic value of chromosomal aberrations detected by CGII and the efficiency of the technique suggest a central role for CGH in routine clinical cytogenetics. PMID- 10734657 TI - Should hemophiliac patients be circumcised? AB - Social and cultural integration of hemophilic boys into society is one of the most important cornerstones of modern hemophilia therapy. Circumcision, a traditional procedure, is an important ritual for Muslims and Jews and an important social problem for the hemophiliac patient and his family. The aim of this study was to evaluate the psychosocial dimension of circumcision and the opinions of parents and children. A total of 105 hemophiliac patients and parents were interviewed and surveyed. Of these, 94% of the parents of uncircumcised patients wanted circumcision for their children. Most parents saw circumcision as a mandatory procedure. Hemophilic boys (60%) and their parents (82%) have an inferiority complex because the boys are unable to be circumcised. Bleeding risk is the primary reason of anxiety (70%). The parents of all the circumcised patients were happy after circumcision. In conclusion, circumcision is an important social problem of hemophilic patients that needs to be solved. PMID- 10734659 TI - Prepubertal malignant melanoma: report of three cases. AB - Prepubertal malignant melanoma (MM) is an extremely rare tumor. In Slovenia, 13 MM cases were registered between 1968 and 1996 by the Cancer Registry of Slovenia. The diagnosis of MM was confirmed by histology in 3 children. In 3 other children the lesions initially diagnosed as MM were reclassified as Spitz nevus. In the remaining cases, the slides were not accessible for histological review, and the clinical course of disease corroborated the diagnosis of a benign nevus. In the present report, 3 of 13 cases with histologically confirmed prepubertal MM are described. The difficulties encountered in the diagnosis and management of this rare condition are discussed. PMID- 10734658 TI - Childhood acute promyelocytic leukemia: no benefit of all-trans-retinoic acid administered in a short-course schedule. AB - From January 1990 to August 1997, 29 consecutive patients were treated with newly diagnosed primary acute promyelocytic leukemia (APL) at the authors' Institution. Of these, 27 (16 boys and 11 girls) were evaluable. Median age at diagnosis was 6.3 (range: 1.9-15.7) years. This population was treated with two consecutive protocols: 13 patients were included in the AML-HPG-90 protocol and 14 in the AML HPG-95. The initial treatment was the same for both protocols: an induction 8-day phase with cytarabine, idarubicin, and etoposide was followed by a consolidation with cyclophosphamide, cytarabine, 6-mercaptopurine, vincristine, doxorubicin, and prednisone. Two courses of intensification with high-dose (HD) cytarabine and etoposide were given in the first study. Only one intensification course was administered in the second study, with HD cytarabine plus idarubicin or etoposide decided by randomization. Complete remission was achieved in 67% (18/27) of cases. Mortality on induction was quite high, 30% (8/27) mainly due to hemorrhages from disseminated intravascular coagulation (DIC). The event-free survival estimate for all patients was 0.47 (SE: 0.1). From April 1994, all-trans retinoic acid (ATRA) was administered just during the first days of the induction phase (median: 9, range: 2-27) to stop or prevent DIC. Eighteen patients received ATRA and 9 did not. Three patients developed signs of ATRA syndrome during the first days of administration but no one died due to this toxicity. The impact of a short course of ATRA on early control of DIC was studied by analyzing the number of platelet, cryoprecipitate, and fresh frozen plasma transfusions during the induction phase in both groups. No statistical differences in complete remission rate, early mortality, need of transfusion of blood components for DIC, and survival estimates could be established between patients who received ATRA and those who did not. ATRA used in a short-course schedule during induction of APL did not stop early mortality due to DIC. Moreover, survival results did not improve with this method of ATRA usage. Longer periods of ATRA administration during APL therapy are strongly recommended. PMID- 10734661 TI - Thrombosis during all-trans-retinoic acid therapy in a child with acute promyelocytic leukemia and factor VQ 506 mutation. AB - Acute promyelocytic leukemia (APL) is often associated with a severe hemostatic disorder, caused by the release of procoagulant and fibrinolytic substances from leukemic blasts. The coagulation profile may exhibit disseminated intravascular coagulation and fibrinolysis or proteolysis. Therefore, heparin and antifibrinolytic agents alone or in combination have been used to prevent severe bleedings. Remission induction with all-trans-retinoic acid (ATRA) is accompanied with rapid correction of hemostatic abnormalities. Thrombosis is a rare complication of APL and may be due to the alterations in hemostasis caused by the disease itself as well as ATRA and antifibrinolytics. Here, the occurrence of thrombosis during induction treatment with ATRA combined with aprotinin and chemotherapy is described in a patient who is homozygous for factor VQ 506 mutation. PMID- 10734660 TI - A variant of myelokathexis with hypogammaglobulinemia: lymphocytes as well as neutrophils may reverse in response to infections. AB - A 7-year-old boy with prolonged and marked leukopenia diagnosed at 6 months of age is described. The polymorphonuclear cells presented no hypersegmented nuclei or concentrated nuclear chromatin, although vacuolated myeloid cells appeared in bone marrow smears. Neutrophils reversed in response to administration of G-CSF. His leukocyte counts were 400-1000/microL during afebrile periods and increased to 2000-3000/microL in response to infections. The increased leukocyte was usually neutrophils, but lymphocytes also increased at EB-virus infection. The serum IgG decreased gradually and was 364 mg/dL at 7 years of age. Antibody responses were normal and recurrent otitis media has been the patient's only problem. Granulocytopenia with hypogammaglobulinemia of this patient mimics myelokathexis with hypogammaglobulinemia, and lymphocytes also increased at viral infections. PMID- 10734662 TI - Immune thrombocytopenia and hemolytic anemia as a presenting manifestation of Hodgkin disease. AB - A very unusual clinical presentation of Hodgkin disease with immune thrombocytopenia and autoimmune hemolytic anemia is reported. A 6.5-year-old boy presented with thrombocytopenia, Coombs' positive hemolytic anemia, and multiple small posterior cervical lymph nodes. After a course of high-dose methylprednisolone therapy with a diagnosis of Evans syndrome, complete response for thrombocytopenia and partial response for anemia was achieved. Six weeks later there was a sudden increase in the size of left posterior cervical lymph nodes and a biopsy was compatible with Hodgkin disease, mixed cellularity type. The child was successfully treated with chemotherapy and radiation therapy. He has been off therapy for 28 months and has no clinical or laboratory evidence of autoimmune cytopenia. A combination of immune thrombocytopenia and autoimmune hemolytic anemia may be associated with Hodgkin disease. The recognition of this clinical picture as a complication of Hodgkin disease has important implications. This complication appeares to be managed best by the definitive treatment of Hodgkin disease. PMID- 10734663 TI - Septic arthritis in hemophilia with central venous catheter: a case report. AB - A case is reported of septic arthritis in a child with human immunodeficiency virus-negative hemophilia A associated with a Staphylococcus aureus catheter associated septicemia. The infection occurred in relation to the use of a totally implantable central venous catheter. The organism was eventually eradicated with antibiotics injected via the catheter. With increasing use of such catheters in the hemophilic population, clinicians should be alerted to the possibility of septic arthritis for prompt diagnosis and treatment. PMID- 10734664 TI - Kasabach-Merritt syndrome in a Jehovah's Witness infant. PMID- 10734665 TI - Platelet release defect in a child with Ehlers-Danlos syndrome. PMID- 10734666 TI - Aldehydes (nonanal and hexanal) in rat and human bronchoalveolar lavage fluid after ozone exposure. AB - We hypothesized that exposure of healthy humans to ozone at concentrations found in ambient air causes both ozonation and peroxidation of lipids in lung epithelial lining fluid. Smokers (12) and nonsmokers (15) were exposed once to air and twice to 0.22 ppm ozone for four hours with exercise in an environmental chamber; each exposure was separated by at least three weeks. Bronchoalveolar lavage (BAL) was performed immediately after one ozone exposure and 18 hours after the other ozone exposure. Lavage fluid was analyzed for two aldehyde products of ozonation and lipid peroxidation, nonanal and hexanal, as well as for total protein, albumin, and immunoglobulin M (IgM) as markers of changes in epithelial permeability. Ozone exposure resulted in a significant early increase in nonanal (p < 0.0001), with no statistically significant relationship between increases in nonanal and lung function changes, airway inflammation, or changes in epithelial permeability. Increases in hexanal levels were not statistically significant (p = 0.16). Both nonanal and hexanal levels returned to baseline by 18 hours after exposure. These studies confirm that exposure to ozone with exercise at concentrations relevant to urban outdoor air results in ozonation of lipids in the airway epithelial lining fluid of humans. PMID- 10734667 TI - [Laparoscopy-assisted vaginal hysterectomy]. PMID- 10734668 TI - [The overall and step-by-step duration of cesarean section]. AB - The purpose of this study is to define the time for different steps of Caesarean section from the moment the women lays on the operating table to the last stitch on the skin. The study is prospective and include 82 elective and emergent CS. The traditional surgical technique is used without omitting any step. The mean stay of the women in the operating theater is 90 min. The preparation for the anesthesia/analgesia is 23 min (range 8-41). The proper time of the operation is 44.3 min. The laparotomy by Pannenstiel incision takes 3 min. The opening time of the uterus is 37 sec (10-190) and the closer on two layers is 17 min (10-35). The extraction of the foetus takes 53 sec (15-180). The exteriorization of the uterus doesn't affect the repair time. Leaving the visceral and parietal peritoneum unsutured can spare 5.5 min. The elective CS takes more time than the emergent one. The time from the beginning of the operation to the extraction of the foetus is longer in resection (7 min) than in first CS (5 min). PMID- 10734669 TI - [Single-layer or double-layer suturing of the uterine incision in cesarean section?]. AB - The aim of the study is to compare the early complications after '/' cesarean section due to the way of closure of a low transverse uterine incision--single layer or two layers. MATERIAL AND METHODS: We investigated retrospectively in the clinical data (history of birth) 450 women with cesarean section: 300 with single layer and 150--with two layers> The hysterotomy was closed by suture with cat-gut plain No. 2 or chromic No. 1 or No. 2. Results showed single layer repair of the uterus is connected with better postoperative prognosis: low rates of febrile morbidity and wound infection, shorter hospitalization time, better involution of the uterus, p < 0.05. We had no dehiscence and secondary bleeding in both ways of repair, so relaparotomy wasn't needed. CONCLUSION: Single-layer closure has advantages compared to two-layers closure that is why it is recommended for wider application. PMID- 10734670 TI - [Therapeutic regimens for treating bacterial vaginosis in pregnant women]. AB - The study comprises 128 pregnant women examined at different gestational weeks. The diagnosis of bacterial vaginosis was made using: a) the complex clinical criteria--vaginal discharge, vaginal pH, amine test and "clue cells" b) Nugent scoring system c) Spiegel criteria. Two therapeutic regimens were compared- intravaginal 2% clindamycin creme (Dalacin V) 5 g three consecutive days and intravaginal metronidazole (Flagyl) 500 mg once daily for 5 consecutive days. Control examination was carried out 5-7 days after completion of therapy using the same protocol. 28 women from the first group and 31 women from the second group had the control examination. Bacterial vaginosis was eradicated in 93% of women using intravaginal clindamycin and in 87% of women using intravaginal metronidazole. Both regimes were more effective compared to treatment with oral ampicillin for 7 days, where the cure rate was 62%. PMID- 10734671 TI - [Prognostic factors in endometrial carcinoma]. AB - In our study we tried to give more light and information about the most frequent prognostic factors in endometrial cancer. The evaluation of 500 patients and all possible prognostic factors gave us the opportunity to treat better and to choose better therapy for our patients. The results were similar like other western clinics. The clinical and therapeutical administration of the classic and histological factors, as well as the biological markers was not in such wide study cleared up. That is why we did this research in order to, satisfy these scientific needs. The results did not differ from other western clinics working in this field. PMID- 10734672 TI - [Translabial sonography of the lower uterine segment]. AB - The aim of this prospective study is to asses the possibilities of translabial (transperineal) sonography for observation the cervix uteri and to compare it with the transabdominal sonography. The material consists of 58 pregnant women in third trimester of pregnancy. We use abdominal transducer (3.5 Mhz) of Kranzbuhler ultrasound for abdominal and transperineal scanning. The ultrasound examination of the cervix includes: endocervical length, cervical weight, weight of the cervical canal and in case of tunneling its parameters. The satisfying image for ultrasound biometry of the cervix we achieved in 92% of the cases with transabdominal scanning and in 97% with translabial scanning. The mean values of the ultrasound examined parameters of the cervix are not significantly different with transabdominal and translabial measurement. The translabial ultrasound method is feasible and well accepted by the pregnant women. PMID- 10734673 TI - [Leukocytes in human seminal fluid]. AB - The peroxidase method is sufficient for quantification of granulocytes, but immunocytology is the standard for detection of white blood cells (WBC) in semen. Granulocytes are the most prevalent WBC in semen (50 to 60%) followed by macrophages (20 to 30%) and T-lymphocytes (2 to 5%). The frequency of leukocytospermia (> 106 WBC/ml) among male infertility patients is 29%. There are ample evidences for sperm damage by WBC: 1) Seminal WBC numbers were higher in infertile patients than among fertile men; 2) leukocytospermia was associated with decreased sperm numbers and impared sperm motility; 3) WBC damage sperm function and were an important prognostic factor for IVF-ET failure. Approximately 80% of leukocytospermic samples are microbiologically negative. In some cases Chlamydia trachomatis might have triggered a persistent inflammatory reaction. Genital tract inflammation facilitates the formation of sperm antibodies. PMID- 10734675 TI - [Hysterosalpingosonography by the vaginal route--a new method for assessing uterine tube patency]. AB - BUT: Introducing a new sonographic method for evaluation the patency of fallopian tubes. MATERIAL AND METHODS: Transvaginal hysterosalpingosonography (HSSG), using chlorocid as a contrast material was performed in 52 infertile women, 12 of with after ectopic pregnancy. RESULTS: Transvaginal hysterosalpingosonography showed as patent 24 fallopian tubes and 68 obturated. LSC and/or HSG pointed 30 patent and 62 obturated. Compared to LSC and HSG, transvaginal HSSG showed 100% sensitivity and 88% specificity. CONCLUSIONS: This investigation revealed the good diagnostic value of HSSG with chlorocid as an easy and not expensive method for the evaluation of the patency of fallopian tubes. PMID- 10734674 TI - [The effect of chlamydial infection on tubal patency]. AB - The study was carried out on 162 infertile women (110 with primary and 52 with secondary infertility), patients of the University Clinic of Gynecologic Endocrinology and Infertility, Sofia. The present study was designed to estimate the effect of chlamydial infection as a cause of tubal pathology in infertile women. Evidence of past chlamydial infection was determined by the presence of antichlamydial IgG antibodies by commercial ELISA test. 101 of the patients examined were positive for antichlamydial IgG antibodies (62.3%). 76 women were examined for tubal patience by means of hysterosalpingography and/or laparoscopy. 38 women had patient tubes and 38 women had tubal occlusion (20 had bilateral and 18 unilateral occlusion). 29 of the women with unilateral or bilateral tubal occlusion had a presence of IgG antichlamydial antibodies in their sera (76.3%) compared to 17 of the women with patient tubes (44.7%). The risk for tubal obstruction in women with positive serology for Chlamydia is 3.22 compared to 0.85 risk in the women with negative serology for chlamydia. The results of the present study indicate that women with evidence of past chlamydial infection are 4 times more likely to have obstructed fallopian tubes compared to women who had no such evidence. It is mandatory to examine the patient for chlamydial serology at the beginning of the diagnostic protocol in order to ensure adequate management prior to more invasive procedures such as hysterosalpingography or laparoscopy. PMID- 10734676 TI - [The preoperative preparation of the vagina with Betadine before abortion on demand]. AB - Recent evidence has associated bacterial vaginosis and trichomoniasis with several postoperative complications. We carried out a prospective study aiming to estimate the frequency of vaginitis in women wanting to make an artificial abortion and the possibility to influence this infections by local application of vaginal BETADINE suppositories in all forms of infectious vaginitis: Candida albicans, trichomonas vaginalis and bacterial vaginosis. PMID- 10734677 TI - [Laparoscopic fenestration of the ovaries in sterility and the Stein-Leventhal syndrome--is it a modern fad or correct procedure]. AB - The treatment of the sterility due to the Stein-Leventhal syndrome has been always object of many discussions. Sometimes, the approach towards this disease in the different departments is diametrically different. Some offer only conservative therapy other-only surgical interventions and some-combination between the two. The authors of this study present the results of operative laparoscopy in the treatment of sterility and polycystic ovary syndrome, in the department of gynaecology to the Higher Medical Institute, Pleven. Object of observation were 33 women in whom was performed laparoscopic fenestration of the ovaries and 20 women in whom was made resectio cuneiformis ovariorum. Comparative analysis of the results for one year period after the surgical interventions was made. The conclusion of the authors is that after failure of the conservative approach, the laparoscopic fenestration of the ovaries remains the only right therapeutic intervention. PMID- 10734678 TI - [The use of the preparation Anteovin in dysfunctional uterine hemorrhages and PCOS]. AB - The present prospective study is aimed at estimating the therapeutic effect of a biphasic oestrogen dominated contraceptive pill Anteovin in certain forms of dysfunctional uterine bleeding (DUB) and in polycystic ovary syndrome (PCOS) which is associated with dysfunctional bleeding. Two groups of women were studied -the first group (n = 34) consisted of women with DUB; the second group (n = 31) comprised PCOS. The nature and dynamics of uterine bleeding, contraceptive effectiveness and occurrence of side effects were followed up. In the second group the changes in the levels of testosterone (T), dehydroepiandrosterone sulfate (DHEA-S) and prolactin (Prl) were additionally investigated. RESULTS: A significant decrease in bleeding was observed in both groups. In the second group it is in parallel with a significant decrease of T even as early as on the third month and of DHEA-S on the sixth month of treatment. No significant changes in prolactin levels were found out during the treatment. CONCLUSION: The biphasic contraceptive pill Anteovin has a very good therapeutic effect in DUB and metrorrhagic forms of PCOS while at the same time the side effects are slightly expressed and transitory and an excellent contraception is achieved. PMID- 10734679 TI - [Condylomata acuminata. The correlation between affecting sexual partners and the risk of developing preneoplasia of the cervix uteri. The therapeutic potentials of the Nd-Yag laser]. AB - The present study summarized results from 206 patients (120 females and 80 males) with diagnosis condylomata acuminata. The diagnostic and surveillance methods used in the present work were: clinical, colposcopic, histologies and microbiological examination. 114 from all patients--46.5% females, and 62.2% males were sexual partners. In 25 women (43.8%) from that group HPV-lesions were found. In 8 patients with cervical lesions CIN I-III was diagnosed. Our results confirmed the previously announced literary data about an enhanced risk for CIN development in women with HPV infection, whose sexual partners had clinical data of Condylomata acuminata. Our therapeutic protocol with Nd-YAG laser treatment in a determined scheme and number of applications according to the stage of the disease and pathological diagnosis was very successful, as compared to data from the literature. PMID- 10734680 TI - [Primary carcinoma and precancerous lesions of the vagina--the diagnostic difficulties]. AB - A retrospective morphological research is made on material from vagina for a three years period of time. The results from 93 studied cases are divided in 3 groups: 1. Re-occurrences in vagina--52; 2. Vaginal cancer and VAIN--11; 3. Non malignant lesions--30. The diagnostic difficulties are discussed making parallel between primary vaginal neoplasia, the so-called lower genital tract neoplastic syndrome and postirradiation dysplasia. PMID- 10734681 TI - [Sentinel lymph node biopsy. The possibilities for axial staging in early breast cancer and the results in 187 patients]. AB - The authors studied the sentinel lymph node (SLN) identification rate and its accuracy in predicting axillary staging (N- or N+) in patients with early breast cancer. One hundred eighty seven mapping procedures were performed using a vital dye (Patent blue V, Drimaren Brilliant blue or Mitoxantrone) injected at the primary tumour site. The overall rate of identification of SLN was 81.3% (in 152 of 187 patients). The pathological status of the sentinel nodes was compared with that of the remaining axillary nodes. The accuracy of the sentinel biopsy was 89.5% (in 42 of 47 axillary positive patients). In 15 cases with lymph metastases, the SLN was the only site of disease. This experience indicates the sentinel lymphadenectomy should diminish staging morbidity and could improve surgical management of the axilla in women with early breast cancer. PMID- 10734682 TI - [Polycystic ovary syndrome. II. The current therapeutic approach]. PMID- 10734683 TI - [A rare case of generalized echinococcosis and pregnancy]. AB - The authors describe a rare case of combination of generalized echinococcosis and gravidity. It concerns about a 23-years-old patient in 10 lunar month in whom 10 days before the term, accidentally through ultrasound is found an Echinococcus cyst in the liver. The delivery performed with planned caesarean section without disturbance of the cyst, ended successfully. Two days after the surgery the patient complained of troubled respiration and oppression. The X-ray photograph showed 3 Echinococcus cysts more--one in the right end two in the left lungs. The problem was solved with three more operations after 2, 6 and 10 months, with which the Echinococcus cysts were eliminated. PMID- 10734684 TI - [Myopia and labor]. AB - The authors describe 7 cases of pregnant women with high myopia to 15 diopters, who have been delivered vaginally without worsening of the vision and especially of the myopia. That's why the authors practice and recommend a vaginal delivery to women with a myopia, including high-degree myopia. PMID- 10734685 TI - [A clinical study of the local contraceptive C-Film Lucchini]. AB - OBJECT: To evaluate the compliance and efficient of C-Film Lucchini. METHODS: 20 sexually active women were treated for the period of 3 months. There were used condoms in 1/3 of the women. It was made a microbiological study to all the patients on the vaginal flora colposcopy and it was controlled their kidney function. RESULTS: During the treatment with C-Film Lucchini 10% of the patients complained about a vaginal discharge 5% of the women have a problem with the application. CONCLUSION: C-Film Lucchini is a high quality and well-tolerated local spermicidal contraceptive with a few side effects. Despite the complaints of the local irritation the tolerance of the C-Film Lucchini is excellent. But the main problem with C-Film Lucchini is need of its insertion the 10-15 minutes before coitus and its application in front of the cervix. PMID- 10734686 TI - [The treatment of vaginal mycosis with Orungal]. AB - The authors report their results of the treatment of the vaginal mycosis with Orungal (Janssen-Cilag). Twenty two sexually active women are included in the study, all with clinical signs of fungal vaginitis. Microscopic examination of vaginal secretions and fungal cultures are performed for all of the patients. Twenty cultures show C.albicans as causing agent and 2-non-C.albicans. One-day treatment with Orungal 2 x 200 mg is prescribed. Control examination and mycologic cultures are performed on day 7-10 and 30 after treatment. In 20 patients (91%) there is not clinical signs and the cultures remains negatives. In 2 (9%) the signs of fungal vaginitis persists which evoke the giving of the same dose once again. Adverse reaction (nausea) has been notify by one woman. CONCLUSION: The treatment with Orungal of the vaginal mycosis is highly effective, with minimal adverse reactions and very good acceptance by women. PMID- 10734687 TI - [The use of the vasoconstrictor hemostatic Remestyp in surgical obstetrics]. AB - Terlipressin (Remestyp) or N-a-triglycyl-(8-lysme) vasopressin after parenteral application and slow enzymatic cleavage releases synthetic analog of vasopressin 8-lysine-vasopressin. It is potent myometrial stimulator in pregnant and non pregnant uterus and at the same time decreases myometrial and endometrial blood flow. Remestyp has synergistic effect with oxytocin and/or methergin. Our experience shows good effect of Remestyp in the complex treatment of cases with hypotonic uterus, placenta praevia or adherence during and after abdominal or vaginal birth. Injection of Remestyp around the fibroids during cesarean section significantly decrease the blood lost and make the myomectomy safer. We observe decrease of oozing at the incision of the skin and stopping initial subfascial hematoma. PMID- 10734688 TI - [A multicenter study of the antimicrobial effect of Macmiror and Macmiror Complex in the treatment of vaginal infections]. AB - The aim of the present multicentre study was to examine the therapeutic possibilities of the wide-spectrum medicament MACMIROR & MACMIROR COMPLEX for the treatment of the vaginal infections. MATERIALS AND METHODS: The study included 159 nonpregnant women among 15 and 54 years (middle age 35.6) with different by kind and intensity colpitis complaints. The following microbiological characteristic was established: in 26 cases Gardnerella vaginalis, in 46 Candida spp., and in the rest 87-mixed aerobic bacterial flora, with a combination of Gardnerella, yeast and Trichomonas. The treatment of the patients was done in combined scheme: peroral and vaginal administration, simultaneously with local treatment of the partner. The control examination was performed bistagely: on 7 10 day and on 30-40 day. RESULTS: The good clinical and microbiological influence of the treated patients was established, for the first control examination the effect was found in 88.1% and 86.8% and for the second--respectively in 81.1% and 82.4%. CONCLUSIONS: The received results give us a cause to approve, that the combination "Nifuratel and Nystatin" (Macmiror & Macmimor complex) has the good possibilities to influence the mixed forms of vaginal infection. PMID- 10734689 TI - Hiding information on "voice technology"? PMID- 10734690 TI - The relationship between teacher sign skills and student evaluations of teacher capability. AB - This study examines the extent to which deaf students' perceptions of their teachers' effectiveness and ease of communication in the classroom are related to the teachers' sign skills. Thirty-three faculty, teaching a variety of courses at the National Technical Institute for the Deaf (NTID), were rated on "teaching effectiveness" and "communication ease" by their students over a 2-year period. Faculty sign proficiency was evaluated independently using the Sign Communication Proficiency Interview (SCPI). Results indicate a moderate relation between students' perceptions of communication ease and teaching effectiveness, and a weak relation of these two variables to the teachers' assessed level of sign skill. The data also show that the students could clearly differentiate three levels of teacher sign skill in the classroom. Results are discussed in relation to the component parts of effective communication from the perspective of students in the classroom. PMID- 10734692 TI - Using technology to educate deaf and hard of hearing children in rural Alaskan general education settings. AB - Teachers in Alaskan rural schools were surveyed on their use of instructional technology. The surveyed teachers included the entire population of certified elementary and secondary school classroom teachers of deaf and hard of hearing students in these schools. Respondents generally were older, more experienced, elementary level female teachers who were relatively well educated. Almost half of the respondents held special education certification; about 25% had an endorsement in deaf education. Data analysis indicates that educators who use instructional technology, either in curriculum or broad agenda formats, may be older, hold an advanced degree and secondary education certification, benefit from in-service training on site, are connected to the Internet, and actively use the technology available at their schools. Most of the surveyed educators identified lack of training in the use and integration of software as a barrier to increased classroom use of technology. Based on the study findings, recommendations are made concerning the training needs of teachers in rural Alaska. PMID- 10734691 TI - The Central Asylum for the Instruction of the Deaf and Dumb, Canajoharie, New York, 1823-1835. AB - The Central Asylum for the Instruction of the Deaf and Dumb, Canajoharie, NY, was a public school for the Deaf that existed from 1823 to 1835. Little has been written about it. This study draws upon as much information as appears to be available on the history of this school. The history of the Central Asylum adds to the understanding of the beginnings of public education for the Deaf in the United States. PMID- 10734693 TI - Hiring criteria, duties, and professional growth opportunities for residential staff at schools for the deaf: a survey. AB - Seventy-three schools identifying themselves as having a residential program for deaf students were surveyed to determine demographic information about dormitory staff, qualifications required for employment in the residential program, opportunities for continuing education, and issues relating to the roles and responsibilities of dorm employees. The data showed that schools are committed to providing in-service education for the dorm workers but have minimal educational requirements for employment in their residential programs. The data were compared to the Conference of Educational Administrators of Schools and Programs for the Deaf (CEASD) Certification Requirements for Dormitory Counselors. Recommendations are made in regard to the updating of the CEASD certification requirements; the authors also recommend that schools make a stronger commitment to upholding those requirements. PMID- 10734694 TI - Preliminary results of the Annual Survey of Deaf and Hard of Hearing Children and Youth in Puerto Rico: the first wave. AB - Preliminary findings are provided from the data collected in Puerto Rico through the Annual Survey of Deaf and Hard of Hearing Children and Youth during the 1997 1998 school year. The study was conducted as a part of an initiative to increase participation in the Annual Survey among the deaf and hard of hearing school-age population in Puerto Rico. Demographic, instructional, etiological, audiological, and communication data on 336 deaf and hard of hearing school age children were collected and summarized. The findings suggest the existence of a heterogeneous deaf community rather than the traditionally conceived homogeneous community. The discussion emphasizes the description of those attributes that suggest heterogeneity and the urgent need to continue to collect the kind of data gathered in the survey. The authors urge that Puerto Rican educators and researchers be stimulated to address the educational and health-related needs of Puerto Rico's deaf and hard of hearing school-age population. PMID- 10734695 TI - Responses to interactive stress: infants who are deaf or hearing. AB - Nineteen infants who were deaf (D/H) and 19 infants who were hearing (H/H) were observed during face-to-face interactions with their hearing mothers. Infant behaviors were coded for repetitive physical activity and gaze aversion during two episodes of normal play which were interrupted by a "still-face" episode. Mothers' assessments of their infants as "difficult" or "easy" were derived from the Parenting Stress Index (Abidin, 1986). "Difficult" deaf infants displayed significantly more repetitive activity during the initial normal interaction and significantly more gaze aversion during the still-face episode, compared to "easy" deaf babies and both "easy" and "difficult" hearing babies. Implications of these findings are discussed in the context of parental perceptions of infant behaviors, and the importance of visual attention and nonverbal signals for the optimal development of infants who are deaf. PMID- 10734696 TI - [Variability of the femoral head and neck antetorsion angle in ultrasonographic measurements of healthy children and in selected diseases with hip disorders treated surgically]. AB - Changes in the angle of femoral head and neck antetorsion during childhood play an important role in the physiological development of the hip joint. In the course of some diseases the angle increases pathologically, necessitating surgical treatment. Recent technical advances have enabled the application of ultrasonography (USG) for the measurement of antetorsion angle. In this study the results of USG have been verified by comparison with results of direct measurement during varus derotational osteotomy of 163 femoral proximal ends in 104 children. Values for femoral head and neck antetorsion obtained by USG correlated closely with those obtained during surgery, justifying the use of ultrasonography for the further part of this study. An increase in antetorsion was observed in 56 joints (77%) in a group of 38 children with spastic cerebral palsy subjected to surgery. Mean angle of antetorsion was 37 degrees (SD +/- 11). The angle returned to its pre-operative values within 2-3 years from surgery. In the group of 25 children with Perthes disease, increased antetorsion was found in 11 (44%) joints subjected to surgery and in 8 (32%) normal joints. The angle changed during the observation period, confirming the opinion that the increase is a secondary event in this disease. The angle was much greater than normal for age in the group of 21 children with congenital hip dysplasia. Basing on the results of surgery it is concluded that corrective osteotomy of femoral proximal end in cases of increased antetorsion and valgity of femoral neck is not a sufficient procedure to prevent the angle from reverting to pre-operative values and should be supplemented by osteotomy of the pelvis. Furthermore, ultrasonography has emerged as the best method currently available for measurement of femoral head and neck antetorsion. The correlation coefficient for USG vs. direct (intraoperative) measurement was 0.9 in all groups, reaching 0.93 in the spastic cerebral palsy group, in which contractures and limited mobility are responsible for very low coefficients in the case of other methods. The use of USG for assessment of femoral antetorsion has revealed, particularly after longer observation periods, that the angle in the apparently normal contralateral extremity exceeded values normal for age. PMID- 10734697 TI - Approach to the patient with suspected vasculitis. AB - By taking a careful patient history, conducting a thorough physical examination, knowing the clinical features of vasculitis, and using selected laboratory tests, the physician can diagnosis vasculitis tentatively. Recognizing the pattern of organ involvement provides a clue to the type of vasculitis present. Serologic laboratory tests for ANCAs or hepatitis B or C may help confirm the presence of the underlying vasculitis, and a definitive diagnosis can be confirmed by a biopsy of involved tissue or by angiography. PMID- 10734698 TI - Surveillance of antibiotic resistance in Neisseria gonorrhoeae in the WHO Western Pacific Region, 1998. The WHO Western Pacific Gonococcal Antimicrobial Surveillance Programme. AB - Effective treatment of gonorrhoea in the World Health Organization's Western Pacific Region is hampered by the emergence and spread of antibiotic resistant strains of Neisseria gonorrhoeae. A programme of surveillance of gonococcal susceptibility to antibiotics (GASP) continued in the region in 1998. A high proportion of isolates in many participating countries was resistant to quinolones and penicillins, continuing trends observed by this programme since 1992. Resistance to the later generation cephalosporins and to spectinomycin was absent or infrequent. Options for effective treatment of gonorrhoea in the region have been severely compromised by antibiotic resistance. PMID- 10734699 TI - Surveillance data in CDI. PMID- 10734700 TI - Communicable diseases surveillance highlight. PMID- 10734701 TI - Leaders in medicine. Myra A. Peters, MD. PMID- 10734702 TI - Do physicians counsel smokers to quit? PMID- 10734703 TI - Consumer Assessment of Health Plans Survey (CAHPS) results for Oklahoma managed care Medicaid, 1997, 1998, and 1999. AB - Consumer satisfaction surveys have become an important source of information for purchasers and consumers of health care and health care organizations themselves. Individuals receiving health care provide valuable information regarding access, use of services, and satisfaction with care that can be used for multiple evaluative purposes. The Oklahoma Health Care Authority has adopted the Consumer Assessment of Health Plans Survey (CAHPS) to measure patient satisfaction for the SoonerCare managed care programs. The Oklahoma Foundation for Medical Quality administered the surveys. The adult and child CAHPS core questionnaires served as the basis for the general surveys in 1997 and 1998. The CAHPS for Children with Special Needs survey results and the Pediatric Adaptation of the CAHPS Behavioral Health Survey results were administered in 1999 for baseline measures. Results indicated an overall increase in consumer satisfaction levels across the two CAHPS core questionnaires. Baseline measures for special needs populations were also established. PMID- 10734704 TI - State of the state's health: still on the decline. PMID- 10734705 TI - Forecasting summertime surface-level ozone concentrations in the Lower Fraser Valley of British Columbia: an ensemble neural network approach. AB - Empirical models for predicting daily maximum hourly average ozone concentrations were developed for 10 monitoring stations in the Lower Fraser Valley (LFV) of British Columbia. According to data from 1991 to 1996, ensemble neural network models increased explained variance an average of 7% over multiple linear regression models using the same input variables. Without modification, all models performed poorly on days when the observed peak ozone concentration exceeded 82 parts per billion, the National Ambient Air Quality Objective. When numbers of extreme events in training data were increased using a histogram equalization process, models were able to forecast exceedances with improved accuracy. Modified generalized additive model (GAM) plots and associated measures of input variable importance and interaction were generated for a subset of the trained models and used to investigate relationships between input variables and ozone levels. The neural network models displayed a high degree of interaction among inputs, and it is likely the ability of these model types to account for interactions, rather than the nonlinearity of individual input variables, that explains their improved forecast skill. Inspection of GAM-style plots indicated that the relative importance of input variables in the ensemble neural network models varied with geographic location within the LFV. Four distinct groups of stations were identified, and rankings of inputs within the groups were generally consistent with physical intuition and results of prior studies. PMID- 10734706 TI - Development of a speciated, hourly, and gridded air pollutants emission modeling system--a case study on the precursors of photochemical smog in the Seoul metropolitan area, Korea. AB - A speciated, hourly, and gridded air pollutants emission modeling system (SHEMS) was developed and applied in predicting hourly nitrogen dioxide (NO2) and ozone (O3) levels in the Seoul Metropolitan Area (SMA). The primary goal of the SHEMS was to produce a systemized emission inventory for air pollutants including ozone precursors for modeling air quality in urban areas. The SHEMS is principally composed of three parts: (1) a pre-processor to process emission factors, activity levels, and spatial and temporal information using a geographical information system; (2) an emission model for each source type; and (3) a post processor to produce report and input data for air quality models through database modeling. The source categories in SHEMS are point, area, mobile, natural, and other sources such as fugitive emissions. The emission database produced by SHEMS contains 22 inventoried compounds: sulfur dioxide, NO2, carbon monoxide, and 19 speciated volatile organic compounds. To validate SHEMS, the emission data were tested with the Urban Airshed Model to predict NO2 and O3 concentrations in the SMA during selected episode days in 1994. The results turned out to be reliable in describing temporal variation and spatial distribution of those pollutants. PMID- 10734707 TI - Maximum ground-level concentrations with downwash--analysis. AB - Equations derived previously for critical downwind distance xc' wind speed uc' and plume rise zc' the values that produce maximum ground-level concentrations (MGLC) chi c under downwash conditions, have been solved. Tables of chi c' xc' uc' and zc' and graphs of the relationships among uc and zc, for a range of stack heights hs' and building heights hb' are presented. Results for two types of sources--a turbine and a reciprocating engine--are discussed. Some comparisons are made to the U.S. Environmental Protection Agency's (EPA) SCREEN3 model. PMID- 10734708 TI - Tomographic reconstruction of tracer gas concentration profiles in a room with the use of a single OP-FTIR and two iterative algorithms: ART and PWLS. AB - Computed tomographic (CT) reconstructions of air contaminant concentration fields were conducted in a room-sized chamber employing a single open-path Fourier transform infrared (OP-FTIR) instrument and a combination of 52 flat mirrors and 4 retroreflectors. A total of 56 beam path data were repeatedly collected for around 1 hr while maintaining a stable concentration gradient. The plane of the room was divided into 195 pixels (13 x 15) for reconstruction. The algebraic reconstruction technique (ART) failed to reconstruct the original concentration gradient patterns for most cases. These poor results were caused by the "highly underdetermined condition" in which the number of unknown values (156 pixels) exceeds that of known data (56 path integral concentrations) in the experimental setting. A new CT algorithm, called the penalized weighted least-squares (PWLS), was applied to remedy this condition. The peak locations were correctly positioned in the PWLS-CT reconstructions. A notable feature of the PWLS-CT reconstructions was a significant reduction of highly irregular noise peaks found in the ART-CT reconstructions. However, the peak heights were slightly reduced in the PWLS-CT reconstructions due to the nature of the PWLS algorithm. PWLS could converge on the original concentration gradient even when a fairly high error was embedded into some experimentally measured path integral concentrations. It was also found in the simulation tests that the PWLS algorithm was very robust with respect to random errors in the path integral concentrations. This beam geometry and the use of a single OP-FTIR scanning system, in combination with the PWLS algorithm, is a system applicable to both environmental and industrial settings. PMID- 10734709 TI - Penetration of evaporative emissions into a home from an M85-fueled vehicle parked in an attached garage. AB - The use of both oxygenated fuels in carbon monoxide (CO) nonattainment areas and reformulated gasoline in ozone nonattainment areas has been mandated by the 1990 Clean Air Act Amendments. Methanol has been proposed as an alternative fuel for CO nonattainment areas. Its use will potentially increase indoor methanol inhalation exposure resulting from the evaporation of methanol vapor from methanol-fueled vehicles parked in residential garages. Indoor air concentrations of methanol, benzene, and toluene were measured in a residential home with an attached garage. The effects of vehicle emission control devices (charcoal canister hose connection); home heating, ventilation, and air conditioning (HVAC) fans; ambient air, garage, and fuel tank temperatures; and wind speed were examined. The disconnection of the charcoal canister hose, which simulates a spent evaporative emission control device, resulted in elevated benzene, toluene, and methanol concentrations in the garage and attached home. Higher fuel tank temperatures resulted in higher benzene and toluene concentrations in the garage, but not methanol. The concentrations for all compounds in the garage and concentrations of benzene and toluene in the adjacent room were lower when the HVAC fan was on than when it was off, while the concentrations of all three compounds in the rest of the house were higher, although these differences were not statistically significant. Thus, the portion of the population that parks cars in garages attached to homes will experience increased methanol exposures if methanol is used as an automotive fuel. PMID- 10734710 TI - Development and evaluation of the PRIME plume rise and building downwash model. AB - A new Gaussian dispersion model, the Plume Rise Model Enhancements (PRIME), has been developed for plume rise and building downwash. PRIME considers the position of the stack relative to the building, streamline deflection near the building, and vertical wind speed shear and velocity deficit effects on plume rise. Within the wake created by a sharp-edged, rectangular building, PRIME explicitly calculates fields of turbulence intensity, wind speed, and streamline slope, which gradually decay to ambient values downwind of the building. The plume trajectory within these modified fields is estimated using a numerical plume rise model. A probability density function and an eddy diffusivity scheme are used for dispersion in the wake. A cavity module calculates the fraction of plume mass captured by and recirculated within the near wake. The captured plume is re emitted to the far wake as a volume source and added to the uncaptured primary plume contribution to obtain the far wake concentrations. The modeling procedures currently recommended by the U.S. Environmental Protection Agency (EPA), using SCREEN and the Industrial Source Complex model (ISC), do not include these features. PRIME also avoids the discontinuities resulting from the different downwash modules within the current models and the reported overpredictions during light-wind speed, stable conditions. PRIME is intended for use in regulatory models. It was evaluated using data from a power plant measurement program, a tracer field study for a combustion turbine, and several wind-tunnel studies. PRIME performed as well as or better than ISC/SCREEN for nearly all of the comparisons. PMID- 10734711 TI - An ex post cost-benefit analysis of the nitrogen dioxide air pollution control program in Tokyo. AB - The benefits and costs of past nitrogen dioxide (NO2) control policies were calculated for Tokyo, Japan, using environmental, economic, political, demographic, and medical data from 1973 to 1994. The benefits of NO2 control were estimated as medical expenses and lost work time due to hypothetical no-control air concentrations of NO2. Direct costs were calculated as annualized capital expenditures and 1 year's operating costs for regulated industries plus governmental agency expenses. The major findings were as follows: (1) Using Tokyo's average medical cost of pollution-related illness, the best net estimate of the avoided medical costs due to incidence of phlegm and sputum in adults was 730 billion yen ($6.08 billion; 1 U.S. dollar = 120 yen). (2) The best net estimate of the avoided medical costs due to incidence of lower respiratory illness in children was 93 billion yen ($775 million). (3) Using Tokyo's average duration of pollution-related illness and average wages, the best net estimate of the avoided costs of lost wages in workers was 760 billion yen ($6.33 billion). (4) The best net estimate of the avoided costs of lost wages in mothers caring for their sick children was 100 billion yen ($833 million). (5) Using Tokyo specific data, the best net costs were estimated as 280 billion yen ($2.33 billion). (6) Using human health and productivity benefits, and annualized capital cost and operating cost estimates, the best net benefits-to-costs ratio was 6:1 (upper limit 44:1; lower limit 0.3:1). Benefit calculations were sensitive to assumptions of mobile source emissions and certain health impacts that were not included. Cost calculations were highly dependent on assumptions of flue gas volume and fuel use. For comparative purposes, we identified other studies for air pollution-related illness. Assumptions that formed the basis for most of the inputs in the present study, such as duration of illness, medical treatment costs, per person illness in children, and lost wages for working mothers, were similar to those recommended in the literature. Lost wages in sick workers and per capita illness incidence in adults were higher than numbers reported elsewhere. Further advances in cost-benefit analysis (CBA) procedures to evaluate the economic effectiveness of NO2 controls in Tokyo are recommended to estimate impacts and values for additional human health benefits, ecosystem health and productivity effects, and nonliving system effects, as well as benefits of ancillary reductions in other pollutants. The present study suggests that Tokyo's past NO2 control policies in total were economically quite effective. PMID- 10734712 TI - Removal of BTEX vapor from waste gases by a trickle bed biofilter. AB - The system performance of a trickle bed biofilter for treating single and mixed benzene, toluene, ethylbenzene, and o-xylene (BTEX) vapors from waste gases was investigated under different gas flow rates and influent BTEX concentrations. When a single substrate was fed, removal efficiencies of greater than 90% could be achieved for the loads below 64 g benzene/m3/hr, 110 g toluene/m3/hr, 53 g ethylbenzene/m3/hr, and 55 g o-xylene/m3/hr. When a mixed substrate was fed, removal efficiencies of each compound could be above 90% at BTEX loads below 96 g/m3/hr. The trickle bed biofilter appears to be an effective treatment process for removing both single and mixed BTEX vapors with low to high loads. Under similar substrate loads, BTEX vapors were preferentially biodegraded in the order of toluene, benzene, o-xylene, and ethylbenzene. The volumetric removal rates (elimination capacities) of BTEX vapors for a single-substrate feed were higher than those for a mixed-substrate feed under similar substrate loads; these differences were enhanced at higher substrate loads and less significant for a preferred substrate. PMID- 10734713 TI - Size distribution and sources of aerosol in Launceston, Australia, during winter 1997. AB - As part of a pilot study into the chemical and physical properties of Australian fine particles, a suite of aerosol samples was collected at Ti Tree Bend in Launceston, Tasmania, during June and July 1997. This period represents midwinter in the Southern Hemisphere, a period when aerosol sources in Launceston are dominated by smoke from domestic wood burning. This paper describes the results from this measurement campaign, with the aim of assessing the effect of wood smoke on the chemical and physical characteristics of ambient aerosol. A micro orifice uniform deposit impactor (MOUDI) was used to measure the size distributions of the aerosol from 0.05 to 20 microns aerodynamic diameter. Continuous measurements of fine particle mass were made using a PM2.5 tapered element oscillating microbalance (TEOM) and light scattering coefficients at 530 nm were measured with nephelometers. Mass size distributions tended to be bimodal, with the diameter of the dominant mode tending to smaller sizes with increases in total mass. Non-sea salt potassium and polycyclic aromatic hydrocarbons (PAHs) were used as chemical tracers for wood smoke. Wood smoke was found to increase absolute particle mass (enough to regularly exceed air quality standards), and to concentrate mass in a single mode below 1 micron aerodynamic diameter. The acid-base equilibrium of the aerosol was altered by the wood smoke source, with free acidity hydrogen ion, non-sea salt sulfate, and ammonium concentrations being higher and the concentration of all species, including nitrate (to differing extents), focused in the fine particle size ranges. The wood smoke source also heavily influenced the aerosol scattering efficiency, adding to a strong diurnal cycle in both mass concentration and light scattering. PMID- 10734714 TI - The sensitivity of PM2.5 source-receptor relationships to atmospheric chemistry and transport in a three-dimensional air quality model. AB - Air quality model simulations constitute an effective approach to developing source-receptor relationships (so-called transfer coefficients in the risk analysis framework) because a significant fraction of particulate matter (particularly PM2.5) is secondary (i.e., formed in the atmosphere) and, therefore, depends on the atmospheric chemistry of the airshed. In this study, we have used a comprehensive three-dimensional air quality model for PM2.5 (SAQM AERO) to compare three approaches to generating episodic transfer coefficients for several source regions in the Los Angeles Basin. First, transfer coefficients were developed by conducting PM2.5 SAQM-AERO simulations with reduced emissions of one of four precursors (i.e., primary PM, sulfur dioxide (SO2), oxides of nitrogen (NOx), and volatile organic compounds) from each source region. Next, we calculated transfer coefficients using two other methods: (1) a simplified chemistry for PM2.5 formation, and (2) simplifying assumptions on transport using information limited to basin-wide emission reductions. Transfer coefficients obtained with the simplified chemistry were similar to those obtained with the comprehensive model for VOC emission changes but differed for NOx and SOz emission changes. The differences were due to the parameterization of the rates of secondary PM formation in the simplified chemistry. In 90% of the cases, transfer coefficients estimated using only basin-wide information were within a factor of two of those obtained with the explicit source-receptor simulations conducted with the comprehensive model. The best agreement was obtained for VOC emission changes; poor agreement was obtained for primary PM2.5. PMID- 10734715 TI - Superior pillared clay catalysts for selective catalytic reduction of nitrogen oxides for power plant emission control. AB - Fe(3+)-, Cr(3+)-, Cu(2+)-, Mn(2+)-, Co(2+)-, and Ni(2+)-exchanged Al2O3-pillared interlayer clay (PILC) or TiO2-PILC catalysts are investigated for the selective catalytic reduction (SCR) of nitric oxide by ammonia in the presence of excess oxygen. Fe(3+)-exchanged pillared clay is found to be the most active. The catalytic activity of Fe-TiO2-PILC could be further improved by the addition of a small amount of cerium ions or cerium oxide. H2O and SO2 increase both the activity and the product selectivity to N2. The maximum activity on the Ce-Fe TiO2-PILC is more than 3 times as active as that on a vanadium catalyst. Moreover, compared to the V2O5-WO3/TiO2 catalyst, the Fe-TiO2-PILC catalysts show higher N2/N2O product selectivities and substantially lower activities (by approximately 85%) for SO2 oxidation to SO3 under the same reaction conditions. A 100-hr run in the presence of H2O and SO2 for the CeO2/Fe-TiO2-PILC catalyst showed no decrease in activity. PMID- 10734716 TI - PM10 levels in the Lower Fraser Valley, British Columbia, Canada: an overview of spatiotemporal variations and meteorological controls. AB - Three years of hourly averaged PM10 (particulate matter less than 10 microns in diameter) tapered element oscillating microbalance (TEOM) data from 10 sites in the large coastal valley incorporating Greater Vancouver were used to investigate the spatiotemporal dimensions and air pollution meteorology of particulate pollution. During the period studied, the provincial "acceptable" objective daily concentration of 50 micrograms m-3 was exceeded at 7 of the 10 sites. The highest annual, seasonal, and maximum hourly concentrations were recorded at Abbotsford in the central valley. Mean seasonal PM10 concentrations were highest in the wintertime in the western Lower Fraser Valley (LFV) and in the summertime at the central and eastern valley locations. Within the network, interstation correlations of daily average concentrations exceed 0.8 at interstation distances less than 20 km and decrease thereafter. For daily maximum concentrations (hourly), interstation correlations decrease sharply with distance. Meteorological conditions responsible for elevated particulate concentrations in the LFV are associated with (1) short periods (1- to 3-hr duration) of reduced dispersion during summer nights at sites close to primary sources, (2) summer anticyclonic conditions when photochemical pollutant concentrations build up across the entire valley, and (3) occasional wintertime "gap wind" events in the eastern valley. PMID- 10734717 TI - Stabilization/solidification of salt from a waste incinerator. AB - A laboratory procedure was developed and verified for stabilizing salt produced by an industrial waste incinerator. This procedure is based on salt stabilization by means of an asphalt binder. Conductivity values and relevant anion contents in leachates of stabilized waste with an asphalt coating were near zero. The pH value of these leachates equaled the pH value of the water used, so that the stabilized waste salt represented inert material, posing no environmental hazard. An unusually significant reduction in the volume of processed salt occurred during stabilization. After compacting under 10.4 MPa pressure, the volume of test specimens was almost 55% smaller than the initial salt volume. In practice, this would mean more than a doubling of landfill waste capacity. Volume reduction was successfully explained by means of a mathematical model. PMID- 10734718 TI - Changing speed-VMT distributions: the effects on emissions inventories and conformity. AB - The emissions factor modeling component of the motor vehicle emissions inventory (MVEI) modeling suite is currently being revised by the California Air Resources Board (CARB). One of the proposed changes in modeling philosophy is a shift from using link-based travel activity data to trip-based travel data for preparing mobile emissions inventories. Also as part of the revisions, new speed correction factors (SCFs) will be developed by CARB for the revised model. The new SCFs will be derived from vehicle emissions on 15 new driving cycles, each constructed to represent a typical trip at a specific average speed. This paper discusses how the new SCFs will affect transportation conformity and emissions inventory development, and evaluates the differences in total emissions produced by trip based and link-based distributions of speed and vehicle miles of travel (VMT). We simulated both link-based and trip-based speed-VMT distributions using travel data from the Sacramento and San Diego travel demand models. On the basis of the simulation results, there is reason to expect that mobile emissions inventories constructed using the proposed trip-based philosophy will differ markedly from those constructed in the current manner. Noting that results may vary by region, increases are expected in the CO and HC inventory levels, with concomitant decreases in the NOx mobile emissions inventories. PMID- 10734719 TI - "The devil hath laughed at the physicians": witchcraft and medical practice in seventeenth-century New England. PMID- 10734720 TI - The origin of the dispute over the discovery of heparin. PMID- 10734721 TI - Benito Feijoo, medical disenchanter of Spain. PMID- 10734722 TI - Medicaid must change so that patients and doctors don't lose. PMID- 10734723 TI - Medicine 2000. What the future may bring to a practice near you. PMID- 10734724 TI - [Evolution of the concept of sustainable development from "our common future" to "a new WHO"]. PMID- 10734725 TI - [European perspectives of the reform of the nursing education with special reference to France and Germany]. PMID- 10734726 TI - [Occurrence of alveolar hydatid disease (multilocular echinococcosis) outside of Hokkaido and a proposal for its prevention]. AB - Alveolar hydatid disease (AHD), had been endemic only in Rebun Island prior to 1945, it is now prevalent throughout the mainland Hokkaido. AHD cases have been reported also in 21 prefectures outside of Hokkaido, e.g. Aomori, Tokyo, Miyagi, etc. Case reports of AHD (n = 135) were reviewed and 76 cases were identified in 21 prefectures outside of Hokkaido in 1926-96. These cases were classified in 4 groups: 1) cases supposedly infected where they lived (n = 19), 2) cases that were infected in Hokkaido (n = 23), 3) cases that were infected in foreign countries (n = 30), 4) cases infected in unidentified places (n = 4). Prefectural distribution and sex ratios were characteristic according to the groups. The first group was supposedly infected were they lived, but it was suggested that this group had some relationship with the epidemics of AHD in Hokkaido. Cases of the 1st and 2nd group were estimated to have been infected prior to early 1970, and it was suggested that these 2 groups will increase the number hereafter in the prefectures outside of Hokkaido because the number of AHD cases has been increasing in the mainland Hokkaido since late 1980. PMID- 10734727 TI - [Social interaction and mortality in a five year longitudinal study of the elderly]. AB - This study clarified the relationship between social interaction and mortality using a five year longitudinal study. The subjects were all 60 years or above who lived in a farming community near major urban centers in Japan (n = 1,069). A total of 153 subjects died within the five year period after the baseline survey. A questionnaire was utilized, the contents of which were about social interaction (using the "Index of Social Interaction" which consisted from 5 subscales: Independence, Social curiosity, Interaction, Feeling of Safety, and Participation in the society), health status, life style, and subjects' feeling about themselves. The results were as follows: 1) low score on "Index of Social Interaction" was significantly related to five-year mortality, 2) the mean score of "index of Social Interaction" of deceased was significantly lower then survived subjects aged 75 and over, 3) logistic regression analysis adjusted for age, sex, length of education, and health status revealed that odds of mortality were significantly high with lower score in the Index of Social Interaction. PMID- 10734728 TI - [Potential exposure to inorganic mercury in people living near a sewage sludge dumping site: urinary excretion of mercury, subjective symptoms and renal function]. AB - OBJECTIVE: The purpose of this study was to evaluate the presence of exposure to inorganic mercury and its health effects among people living near a sewage sludge dumping site in Nagasaki Prefecture, Japan. In this area, sewage sludge and industrial waste have been dumped since 1975, and total mercury levels exceeding the water quality standards (0.0006-0.0020 mg/l) have been detected in seeping water and river water since July 1997. METHODS: The population for the present study comprised 48 subjects (aged 11-91 years) living near a sewage sludge dumping site and 49 subjects (aged 10-82 years) living in a non-polluted area. In November and December 1998, subjective symptoms of inorganic mercury exposure, history of occupational exposure to inorganic mercury, frequency of fish intake, sources of drinking water and other health habits were inquired by a self administered questionnaire. Total mercury and total protein levels and N-acetyl beta-D-glucosaminidase (NAG) activity in morning urine specimens were also measured. RESULTS: Among males, the proportion of subjects who complained of tremor in the hands (P = 0.02) and increased irritability (P = 0.10) was higher in the polluted area than in the control area. In addition, the proportion of those who did not report being easily fatigued was lower in the polluted area than in the control area (P = 0.07). Among females there was no significant difference in the prevalence of self-reported symptoms related to the central nervous system disturbance between the two areas. After adjustment for gender and age using logistic regression analysis, the prevalence of increased irritability was significantly higher (P = 0.05) and the proportion of those who did not report being easily fatigued was significantly lower (P = 0.03) in the polluted area than in the control area. However, there was no significant difference in the geometric mean of urinary total mercury concentration (microgram/g creatinine) between the polluted area (0.66, 95% confidence interval [CI] 0.48 0.91 for men and 0.96, 95% CI 0.70-1.33 for women) and the control area (0.81, 95% CI 0.60-1.09 for men and 0.83, 95% CI 0.57-1.22 for women). There was no individual whose total mercury concentration in urine exceeded 30 micrograms/g creatinine, at which level of urinary total mercury toxic effects on the central nervous system have been reported in industrial workers. There was also no significant difference in the geometric means of urinary total protein level and NAG activity. CONCLUSION: There was no evidence of excessive exposure to inorganic mercury among residents in the polluted area. Thus, we concluded that the difference in the prevalence of subjective symptoms was not due to the direct effect of exposure to inorganic mercury. To prevent the contamination of water by taking measures against pollution sources, monitoring of the quality of drinking water, and finally to secure safe water supply by public waterworks are required. PMID- 10734729 TI - [Epidemiology of alveolar hydatid disease (AHD) and estimation of infected period of AHD in Rebun Island, Hokkaido]. AB - An epidemiological study was performed of endemic alveolar hydatid disease (AHD, multilocular echinococcosis), Rebun Island, Hokkaido and the period of AHD infection of patients was estimated. Death certificates of the residents of the island were analyzed, and 74 deaths (43 males and 31 females) by AHD were found out of the 3,126 deaths that occurred during the period from 1948 to 1990. The red fox population of the island was estimated on the basis of past researchs. The deaths due to AHD distributed around a major peak (n = 67) between 1948-1975 and there were 7 sporadic cases between 1976-90. The red fox population on the island had been estimated to be largest in 1935. The mean infection period from initial AHD infection to death was estimated to be 26 +/- 7 years (x +/- SD) on the basis of the period between the year in which the peak red fox population was observed (1935) and the major peak of patient death (1962). The mean symptomatic period was 5 +/- 5 years, and the mean latent period from infection to the onset of AHD was 21 +/- 7 years. Sex ratio (M/F = 28/13 = 2.15) was higher (P < 0.05) at the age groups below 10 and 26-45 years than the other age groups (15/18 = 0.83), and playing outdoors during childhood and working outdoors in the prime of life were assumed to be the causes of infection. PMID- 10734730 TI - [Tobacco-use prevention and cessation programs for high school students by a prefectural health center]. PMID- 10734731 TI - [Qualitative study of municipalities' maternal and child health promotion planning]. AB - From 1997, basic maternal and child health services have been provided through municipalities. The ministry of Health and Welfare has requested all municipalities to publish a MCH promotion plan. We conducted a qualitative analysis of important contents of the plans using an evaluation index of original dichotomous variables. Out of 3,256 municipalities, 2,873 developed the plans for MCH promotion. Most of the plans present descriptions of the significance of MCH promotion planning, goals of the plans, problems of current MCH statistics and services, and plans of providing services. The proportion was low for plans which have needs assessment of target population, indicators of evaluation of plans, description of the relationship between objectives and services, importance of objectives, action plans, specific chapter or evaluation of the plan, monitoring of the plan, and plans for informing public of the MCH plan. Therefore, the MCH promotion plans have weaknesses in evaluation and action. The MCH promotion plans of middle sized cities were more likely to have these important contents. We will continue to analyze the processes for producing of excellent MCH promotion plans to extract universal promoting factors for producing MCH promotion plans. PMID- 10734732 TI - [Programmed cell death (apoptosis) in excised subretinal neovascularization]. AB - BACKGROUND: The occurrence of apoptosis in choroidal neovascularizations (CNV) has only been described previously in a few cases. However, little is known about the extent and function of apoptosis. We analyzed the incidence of apoptosis in order to find new options in the therapy of CNV. Particular attention was given to the length of time that the process existed before surgery in patients with age-related macular degeneration (AMD). PATIENTS AND METHODS: In 34 patients (18 women, 16 men) ranging in age from 20 to 91 years (mean 70.3 years), CNV was detected by angiography. The majority of patients (n = 29) with CNV had AMD, which in most patients affected both eyes. In two patients CNV was due to post traumatic proliferative vitreoretinopathy, in two other patients CNV occurred after a penetrating bulbus injury, and in one patient pseudoxanthoma elasticum was found. Thirty-four CNV membranes were excised by pars plana vitrectomy via retinotomy, fixed in formalin and embedded in paraffin. Sections were stained with PAS and H&E and examined microscopically. The in situ cell-detection kit (TUNEL) was used for immunohistochemical detection of apoptotic cells from fragmented DNA. RESULTS: In 74% (n = 25) of all patients and in 76% (n = 22) of patients with AMD, disseminated or focal apoptotic cells were detectable in the connective tissue, retinal pigment epithelium and in the vascular endothelium. In AMD patients with apoptotic cells, the SNV existed 1-12 months (mean 5.5 months) before surgical intervention. In patients without apoptotic cells, the CNV were present 5-36 months (mean 17 months) before surgery. At the time of surgery, the loss of reading ability had on average been going on 3 months; in patients with apoptotic cells, the mean time from the onset of this symptom was 2.4 months (1 week to 11 months); and in patients without apoptotic cells it was 5 months (1-12 months). CONCLUSION: Programmed cell death as a regulating pathomechanism was observed in all tissue parts of CNV with variable extension. In patients with AMD, a correlation existed between the length of time that CNV existed before surgery and the incidence of apoptotic cells. Apoptosis was found more frequently in recent CNV than in long-standing lesions. The various activity found in CNV suggests that the success of treatment may depend on the moment of intervention. To characterize the role of apoptosis further inducing and inhibiting factors have to be analyzed. PMID- 10734733 TI - [Age-related maculopathy. Comparative studies of patients, their children and healthy controls]. AB - BACKGROUND: The aim of this study was to evaluate prospectively whether there are differences in spectrometrically measurable parameters of the fundus between patients with early and late age-related maculopathy (ARM), the children of the ARM patients (F1 generation) and normals. METHOD: Using the "Jenaer Imaging Spectrometer", retinal oxygen saturation, xanthophyll, and intrinsic fluorescence were measured; the spatial distribution of xanthophyll was determined using the Rodenstock SLO 101 model. RESULTS: Xanthophyll is reduced in late ARM as compared to the F1 generation and the control group (alpha < 0.01). The different fluorescence spectra, measured at shortwave and at longwave excitation, suggest the presence of more than one fluorophore. Furthermore, the components of the fluorophores seem to be different between patients with ARM and their F1 generation. The longwave autofluorescence is age-dependent only in late ARM (r2 = 0.81). For the first time, we found an alteration in oxygen saturation in retinal vessels in patients with ARM. CONCLUSIONS: Xanthophyll is reduced only in late ARM. Autofluorescence and oxygen saturation are different between ARM patients, the F1 generation and normals, however, we were not able to identify a genetically based predisposition concerning the parameters studied. PMID- 10734734 TI - [Long-term results of implantation of hydroxylapatite as artificial eye]. AB - BACKGROUND: Since its first implantation as an eyeball replacement in the United States in 1985, hydroxyapatite has been rarely used as an orbital implant in Germany. The aim of the study was to collect data on long-term tolerance, postoperative complications and the cosmetic end results. PATIENTS AND METHODS: In the spring of 1997, 52 patients underwent a follow-up examination at our hospital. The average post-operative follow-up period was 23 (12-41) months. RESULTS: The subjective tolerance was considered good by 3/4 of the patients. In the comparison of position and prominence of the artificial eye, 8 patients achieved complete equalization. As regards defective lid position, 8 patients had a ptosis, 6 an ectropium, and there was one case of entropium. Changes in conjunctiva stretched from muciferous secretion to follicular squamous hyperplasia. There was no case of implant extrusion in the series examined. With reference to the method of implantation, far superior motility was observed in primary implantations. Statistically significant motility differences could not be shown regarding muscle attachment because of the small number of the cases. CONCLUSIONS: In the series examined, hydroxyapatite proved to be a generally well tolerated orbital implant with few postoperative complications and good motility. Primary implantation with attachment of all six eye muscles gave the best results. PMID- 10734735 TI - [Amniotic membrane transplantation with or without limbal allografts in corneal surface reconstruction in limbal deficiency]. AB - PURPOSE: We examined whether amniotic membrane transplantation (AMT) is useful in preparing the perilimbal stroma to enhance the success of allograft limbal transplantation (ALT). METHODS: Forty-seven eyes of 42 consecutive patients with cytologically proven limbal deficiency (LD) were included in this prospective study. Based on the severity of LD, group A (mild) with 18 eyes received AMT alone, group B (moderate) with 13 eyes received AMT and ALT, and group C (severe) with 16 eyes received AMT, ALT and penetrating keratoplasty. All except for group A received continuous systemic cyclosporin A. RESULTS: Except for the two eyes with atopy, all amniotic membrane-covered surfaces showed rapid epithelialization in 2-4 weeks, reduced inflammation, vascularization and scarring, and became smooth. For the mean follow-up period of 23 months, 38 eyes (82.6%) showed visual improvement, consisting of > or = 6 lines (15 eyes), 4-5 lines (10 eyes), 1-3 lines (13 eyes). Visual improvement was noted in 16/18 eyes (88.9%) in group A, in 10/13 eyes (77%) in group B, and in 12/16 eyes (75%) in group C. In group C corneal graft rejection occurred in 12 of 16 eyes (75%). In group B and C, early reversible limbal allograft rejection was noted in 3 of 29 eyes (10.3%) and a recurrent limbal deficiency was observed in 8 of 29 eyes (27.6%). CONCLUSION: For partial LD with superficial involvement, AMT alone is sufficient and hence superior to ALT because of no need for using cyclosporin A. For total LD, additional ALT is needed and AMT helps reconstruct the perilimbal stroma with reduced inflammation and vascularization, which collectively may enhance ALT success. PMID- 10734736 TI - [Long-term results of combined glaucoma and cataract surgery. Intraocular pressure and visual acuity follow-up]. AB - BACKGROUND: The aim of this investigation was to ascertain the long-term results of intraocular pressure and visual acuity after combined glaucoma and cataract surgery. PATIENTS AND METHODS: Between 1994 and 1997, 65 eyes underwent Pham's sclerectomy and at the same time 65 eyes underwent Elliot's trephination with scleral flap. Both procedures were combined with phacoemulsification and implantation of a posterior chamber lens. The mean follow-up was 21.9 +/- 11.0 months. RESULTS: The IOP of eyes that underwent Pham's sclerectomy decreased from 21.4 +/- 3.3 mmHg to 14.9 +/- 2.7 mmHg and of eyes that underwent Elliot's trephination from 24.6 +/- 7.3 mmHg to 15.8 +/- 2.6 mmHg. Thirty percent of the eyes with Pham's sclerectomy and 25% with Elliot's trephination required an additional medication. In both procedures the overall visual acuity increased by three lines. CONCLUSIONS: These results of combined glaucoma and cataract surgery confirm their efficiency. From the economical aspect, combined glaucoma and cataract surgery has gained increasing significance. PMID- 10734737 TI - [Ocular cicatricial pemphigoid. Retrospective analysis of risk factors and complications]. AB - Cicatricial pemphigoid (CP) is a rare autoimmune disease characterized by subepidermal blistering and progressive cicatrization affecting the skin and mucosa. Ocular involvement occurs in approximately 70% of the patients. METHODS: The course of the disease, complications and putative risk factors in patients with ocular cicatricial pemphigoid (OCP) treated at the Departments of Ophthalmology and Dermatology were analyzed retrospectively from 1986 to 1998. RESULTS: Eighteen of 28 patients (64%) with CP demonstrated ocular involvement. The mean age of patients with OCP was 73 years; 61% were female. At the time of referral to our hospital, all patients had reached advanced stage III (83%) or IV (17%) of OCP. In 38% of patients vision was already reduced to < 20/200 at first presentation. Twenty-eight percent of patients additionally suffered from glaucoma. Two patients exhibited life-threatening extraocular manifestations of CP (larynx stricture, esophagus stricture). Conjunctival or mucosal biopsies were performed in 15 patients with OCP and showed typical immuno-deposits at the basement membrane zone in 12/15 patients. Therapy with dapsone (12 patients), oral steroids (11 patients), azathioprine (5 patients), cyclophosphamide (4 patients), colchicine (2 patients) and methotrexate (1 patient) was used concomitantly or consecutively. Complications of OCP including entropion, recurrent epithelial erosions, corneal ulcers, keratitis, and corneal perforations required multiple surgical interventions such as entropion surgery (8 patients), tarsorrhaphy (3 patients), mucous membrane grafting (1 patient), amniotic membrane transplantation (1 patient), tectonic keratoplasty (1 patient), keratoprosthesis (1 patient) and enucleation (1 patient). Despite control of the inflammatory process, further visual loss occurred in 53% of eyes. Reading visual acuity could only be maintained in 35% of eyes. DISCUSSION: Early diagnosis and therapy can prevent ocular complications of OCP. This study indicates that advanced stages of the disease often result in irreversible visual loss despite institution of immunosuppressive therapy. Whether or not the high association of OCP with glaucoma and/or anti-glaucomatous treatment in our patients represents part of the underlying disease process or plays a role in the pathogenesis of OCP must still be clarified. PMID- 10734738 TI - [Prognosis of postoperative endophthalmitis]. AB - BACKGROUND: The outcome of 20 patients is summarized in a retrospective study to identify clinical findings that influence the long-term prognosis of postoperative endophthalmitis. PATIENTS: Between 1991 and 1997 a total of 20 patients with postoperative endophthalmitis were admitted. Median age was 80 years (range: 9-95), 11 patients were male, 9 female. Sixteen pars-plana vitrectomies, 2 anterior vitrectomies and 2 rinsings of the anterior chamber without vitrectomy were performed. Furthermore, all patients received intraocular and systemic antibiotic treatment. For microbiological investigation, specimens from vitreous, anterior chamber and conjunctiva were sent in. Long-term outcome was controlled for an average of 14 months after treatment of the endophthalmitis (range: 4-36 months). RESULTS: At the end of treatment, 40% of patients had a visual acuity of 0.4 or better, 80% had 1/20 or better. Patients with a preoperative visual acuity of at least hand movement had a better postoperative visual outcome than patients with only light perception. Visual acuity was better in patients with chronic endophthalmitis than in patients with acute or subacute endophthalmitis. In patients with chronic or subacute endophthalmitis, improvement of visual acuity was found some months after the operation more often than in patients with acute endophthalmitis. However, in 40% of cases with an acute onset, no improvement or even worsening of the visual acuity was documented. Best postoperative results were found after infection with Staphylococcus epidermidis and Propionibacterium acnes. CONCLUSION: Important prognostic factors of postoperative endophthalmitis are visual acuity, the onset of the endophthalmitis (acute, subacute or chronic) and the microbiological findings. At the time of surgery and antibiotic treatment, visual acuity should be at least hand motion to expect an improvement in the visual outcome. PMID- 10734739 TI - [Consistent numerical calculation of optics of the pseudophakic eye]. PMID- 10734740 TI - [Functional results after surgical extraction or photocoagulation of choroid neovascularization (CNV) in age-related macular degeneration]. AB - BACKGROUND: SLO microperimetric examination after the extraction of choroidal neovascular membranes (CNV) in age-related macular degeneration (AMD) shows absolute scotoma in the area of pigment epithelial loss. Laser treatment also causes complete functional loss. The functional results of these two methods should be compared before the surgical procedure is expanded. METHODS AND PATIENTS: Five eyes of five patients with large subfoveal well-defined CNV were treated by photocoagulation following the MPS criteria. Functional results were compared with similar phenotypes from a group of 78 patients operated upon. Before and after the treatment visual acuity was tested following the ETDRS criteria. The need for magnification for reading was tested using the ZEISS charts. Fundus-controlled microperimetry was performed using the scanning laser ophthalmoscope (Rodenstock) to detect deep and relative scotomata. RESULTS: The recurrence rate (OP 2/5; ALK 1/5) was normal regarding the small number of patients. Visual results are slightly better in patients operated on (mean: pre 0.08; 6 weeks 0.09; 3 month 0.13; last 0.13) than in laser-treated patients (mean: pre 0.06; 6 weeks 0.07; 3 months 0.08; last 0.12). Deep scotoma can be reduced with surgical extraction of the CNV (mean factor: 6 weeks 0.6; last control 0.8) while laser treatment of the margins enlarges the scotoma (mean factor: 6 weeks 2.3; last control 2.2). CONCLUSIONS: Because of the minimal functional advantages of the surgical procedure we do not think it is the method of first choice. For both methods the treatment of well-defined CNV increases the possibility of low-vision rehabilitation. PMID- 10734741 TI - [Corneal tattooing. Present-day evaluation of a historic treatment method]. PMID- 10734742 TI - [Acute bilateral vision loss in pancreatitis]. PMID- 10734743 TI - [Vitreoretinal Update Meeting 1999. Conference of the American Academy of Ophthalmology, Orlando, Florida, 22-23/10/1999]. PMID- 10734744 TI - [Therapeutic strategies in recurrent erosion. Mechanical trauma, epithelial basal membrane dystrophy and idiopathic genesis]. PMID- 10734745 TI - [The education of specialists in cardiology in Spain]. PMID- 10734746 TI - [Coronary revascularization with arterial grafts only: a step forward or just speculation?]. PMID- 10734747 TI - [The long-term clinical results of the implanting of intracoronary stents in current practice]. AB - INTRODUCTION: Published Stress and Benestent studies, obtained in selected populations under technical conditions that do not reflect present practice with intercoronary stenting showed a clinical benefit of coronary stenting. OBJECTIVE: To assess clinical longterm outcome of coronary stenting in current practice and to compare coronary lesions with and without Stress/Benestent criteria. METHODS: 216 consecutive patients with successful placement of 279 stents in 256 lesions and no major in-hospital events. Mean clinical follow-up was 16.7 +/- 10.4 months. Standard technique included seven types, high pressure balloon inflation (15.6 +/- 2.2 atm) and post-stenting treatment of four weeks with aspirin and ticlopidine. RESULTS: Cumulative rates of target lesion revascularization were 9.7% at 6 months, 13.5% at 12 months and 15.1% at 18, 24 and 36 months. Cumulative rates of combined clinical end-point (death, myocardial infarction and target lesion revascularization) were 11.3%, at 6 months, 13.9% at 1 year, 19.3% at 2 years and 21.1% at 3 years. No Stress/Benestent lesions were 193; 75%, and had a higher incidence of target lesion revascularization (17.9 vs 7.5%, Log Rank = 0.015) and combined clinical end-point (22.4 vs 10%, Log Rank = 0.025), than Stress/Benestent lesions. CONCLUSION: Coronary stenting of no Stress/Benestent lesions have a less favourable clinical longterm outcome. However, clinical outcome in a nonselected population at the present time is similar to the old Stress/Benestent studies, probably because of technical improvements. PMID- 10734748 TI - [A comparison of the clinical and angiographic evolution of diabetic and nondiabetic patients treated by conventional angioplasty versus stent implantation in native coronary arteries]. AB - INTRODUCTION AND OBJECTIVES: Diabetic patients have a high restenosis risk after balloon coronary angioplasty. Stent implantation in these patients appears to be a potential beneficial therapeutic option. The aim of this study was to compare the clinical and angiographic outcome of diabetic patients vs non-diabetic patients, treated with conventional angioplasty vs stent implantation in lesions located in native coronary arteries. MATERIAL AND METHODS: A total of 302 patients (58 diabetics and 244 non-diabetics) underwent a coronary angioplasty of one vessel in native coronary arteries with initial success and after at least six months clinical and angiographic follow-up were included in the study. Of the total number of patients, 100 were treated with conventional balloon angioplasty and 202 with stent implantation. Major adverse clinical events and angiographic restenosis rate were evaluated at follow-up. RESULTS: Mean age of patients was 65 years and 74% were male. Angiographic restenosis rate was similar in diabetic vs non-diabetic patients with stent implantation (24% vs 23% respectively). Nevertheless, diabetic patients treated with balloon angioplasty compared to diabetic patients treated with stenting, evolved with a higher restenosis rate (64% vs 24%; p < 0.05), and at the end of follow-up diabetics had need a higher rate of target vessel revascularization (40% vs 24%; p < 0.05), a lower major event free survival (56% vs 70%; p < 0.05) and worse symptomatic status (72% vs 36%; p < 0.05). CONCLUSIONS: Diabetic patients treated with conventional one vessel coronary balloon angioplasty evolved with a high restenosis rate and a bad mid-term clinical outcome. Stent implantation was able reduce to the restenosis rate and improve the mid-term clinical outcome, in a comparable population of diabetic patients. PMID- 10734749 TI - [Coronary revascularization surgery with arterial conduits. The technic, results and 4-year follow-up in 1023 consecutive patients]. AB - OBJECTIVE: The main objective of the present study was to analyze the in-hospital and mid term results obtained in 1,023 consecutive patients undergoing coronary artery bypass surgery (CABG) in whom a combination of arterial grafts was used: radial arteries (RA) and one or both internal mammary arteries (IMA). METHODS: From May 1995 to May 1998, 1,023 consecutive patients underwent CABG alone, using arterial conduits (AC) (one or two IMA and RA) for myocardial revascularization. The left internal mammary artery (LIMA) was employed as an "in situ" graft, and the right internal mammary artery (RIMA) as a free graft or "in situ" both in combination with the RA. The latter was connected to the LIMA through a T or Y anastomosis, or emerged directly from the ascending portion of the aorta. RESULTS: An average of 3.2 bypasses per patient were performed. The LIMA was used in 100% of the patients. The RIMA was used in 21.7% and the RA in 100% of the cases. Operative mortality was 2.5% (26 patients) and 32 (3.1%) suffered perioperative acute myocardial infarction. The first 62 patients were angiographically re-studied before discharge, and a 98.4% patency of the AC used was found. Mean follow up time was 25.0 +/- 9.6 months (range, 1 to 48 months). CONCLUSIONS: a) myocardial revascularization procedures using a combination of mammary and RA grafts are safe; b) in-hospital and mid term morbidity and mortality are not higher than those observed with saphenous vein grafts; c) it is possible to achieve complete myocardial revascularization with only AC, even in patients with impaired left ventricular function, and d) AC can be used in elderly patients. PMID- 10734750 TI - [Patency study of internal mammary artery grafts: the usefulness of echo enhancers for identifying the flow signal by color Doppler echocardiography]. AB - OBJECTIVES: a) To study the capacity of the technique of high-frequency color Doppler to detect flow signal of left internal mammary artery grafts; b) to assess the usefulness of an echo-enhancer agent to facilitate the detection of the signal, and c) to evaluate the patency of the graft according to its pulsed Doppler velocity profile pattern. METHODS: 39 consecutive patients were studied. A Hewlett-Packard 5500 echocardiograph was used, with a high-frequency probe (S12) applied at the high left parasternal border. When a graft signal was not elicited after a predetermined 5-minute check period, an intravenous dose of 4 g of Levovist (Schering Espana) at 400 mg/ml was administrated. According to previous studies, a pulsed Doppler flow profile with a predominantly diastolic pattern was considered a normal graft patency, while a systolic one was deemed as abnormal. RESULTS: Graft flow was identified by color Doppler in 33/39 patients (85%). The additional use of an echo-enhancer in 6 patients with no detected signal increased this proportion to 38/39 (97%). Normal flow patterns were seen in 34/38 (89%). Among the four patients with abnormal pattern, 1 case of early myocardial infarction was observed, while angiographic studies showed distal occlusion of the graft in 1 or the presence of competitive flow in 2 patients. CONCLUSIONS: The high-frequency color Doppler technique allows the detection of a flow signal from internal mammary artery grafts in most patients. The administration of an echo-enhancer agent is useful in those with non detectable signals. An abnormal pulsed Doppler velocity pattern indicates graft malfunction. PMID- 10734751 TI - [The effect of the duration of the cardiac cycles on determining mitral valve area by means of pressure half-time]. AB - INTRODUCTION AND AIMS: To analyze the influence of variations in the length of cardiac cycle length of calculating mitral valve area by means of the pressure half time in patients with mitral valve stenosis and atrial fibrillation. METHODS: Fifty-nine patients with pure mitral valve stenosis and atrial fibrillation were subjected to transmitral flow measurements by continuous Doppler monitoring from the apical window. In each patient the pressure half time was quantified, corresponding to a minimum of 30 consecutive cycles. RESULTS: Considering all the measurements made in each patient, the correlation between pressure half time and cardiac cycle was significant in 20 cases (34%). The pressure half time variation coefficients were significantly greater when including the values corresponding to the shortest cycles. Thus, for cycle duration of > or = 800, 700, 600, 500 and 400 ms, the mean values were 0.096 +/- 0.041, 0.106 +/- 0.042 (NS), 0.128 +/- 0.032 (p < 0.05), 0.167 +/- 0.048 (p < 0.001) and 0.231 +/- 0.057 (p < 0.0001), respectively. Upon analyzing the relation between pressure half time and cardiac cycle with progressive exclusion of the longer cycles > or = 800, 700 and 600 ms the number of patients with significant correlation coefficients increased to 19/37 (51%), 12/23 (52%) and 4/6 (67%) on respectively excluding. CONCLUSIONS: Patients with mitral valve stenosis and atrial fibrillation show a variation in pressure half time that may complicate calculation of the mitral valve area. Variability is inherent to the measurement method, and is furthermore dependent upon cardiac cycle duration. This may be resolved by limiting determinations to cycles longer than 800 ms. PMID- 10734752 TI - [The use of anticoagulant treatment in patients with nonrheumatic atrial fibrillation]. AB - INTRODUCTION: The efficacy of anticoagulant treatment in the prevention of thromboembolic complications among patients with nonrheumatic atrial fibrillation is established. In our country, data on the use of this therapy in clinical practice are not available. OBJECTIVE: To examine anticoagulants use among patients with nonrheumatic atrial fibrillation and to analyze the influence of several thromboembolic risk factors in anticoagulant use. PATIENTS AND METHODS: We have studied, 302 patients retrospectively, with nonrheumatic atrial fibrillation. We determined the presence of heart failure, hypertension, previous thromboembolism, diabetes and left atrium dilation. We added age, sex, pattern of non-permanent arrhythmia and hospitalization and we conducted univariate and multivariate analyses to identify their influence the establishment of the anticoagulant treatment. RESULTS: 28.8% of patients were treated with oral anticoagulants, 83.7% were treated with oral anticoagulant or antiplatelet agents. Only three patients, out of 49, aged 80 years or older were treated with anticoagulants. Multivariate analysis showed that previous thromboembolism (odds ratio 4.03 [1.9-8.1]), permanent atrial fibrillation (odds ratio 2.6 [1.3-5.3]), left atrium dilation (odds ratio 2.3 [1.2-4.1]) and heart failure (odds ratio 1.9 [1.07-3.6]) were factors that predicted higher use of anticoagulant treatment. CONCLUSIONS: a) Anticoagulant treatment is underused among patients with nonrheumatic atrial fibrillation; b) previous thromboembolism, left atrium dilation and heart failure have conditioned higher probability of undergoing anticoagulant treatment, and c) patients aged 80 years and over and non permanent atrial fibrillation predicted less use of the therapy. PMID- 10734754 TI - [Guideline for the education of the specialist in cardiology in Spain. Comision Nacional de la Especialidad de Cardiologia]. AB - This article presents the program for training in cardiology. The document was elaborated by the National Committee of the Specialty of Cardiology, from the Ministry of Health and Ministry of Education, and describes the theoretical and practical aspects of training in cardiology prevailing at present in Spain. PMID- 10734753 TI - [The effect of triflusal on human platelet aggregation and secretion: the role of nitric oxide]. AB - INTRODUCTION AND AIMS: The thrombotic process is a multicellular phenomenon in which not only platelets are involved but also neutrophils are involved. Recent in vitro studies performed in our laboratory have demonstrated that triflusal reduced platelet aggregation by stimulating nitric oxide (NO) production by neutrophils. The aim of the present study was to evaluate whether the in vivo treatment with triflusal could also modify the ability of neutrophils to produce NO. Furthermore, the role of NO released by neutrophils on platelet aggregation and secretion was also tested. METHODS: The study was performed in 12 healthy volunteers of 32 +/- 6 years of age. The volunteers were treated with triflusal (600 mg/day) for 5 days and platelets and neutrophils were isolated before and after treatment. The ability of neutrophils to produce NO and the capacity of inhibiting platelet aggregation and secretion of transforming growth factor-beta (TGF-beta) were assessed. RESULTS: After the treatment with triflusal we obtained the following results: a) an increase in NO production by neutrophils; b) potentiation of the inhibition of platelet aggregation by neutrophils, an effect that was reverted by incubating neutrophils with an L-arginine antagonist, L NAME, and c) the presence of neutrophils reduced the release of TGF-beta by platelets measured as index of platelet secretion by a NO-independent mechanism. CONCLUSIONS: Triflusal (600 mg/day/5 days) stimulated NO production by neutrophils. After the treatment with triflusal, neutrophils inhibited both platelet aggregation and secretion. The antiaggregating effect of neutrophils was an NO-dependent mechanism while the inhibition of platelet secretion mediated by neutrophils after the treatment with triflusal was an NO-independent mechanism. PMID- 10734755 TI - [The practical clinical guidelines of the Sociedad Espanola de Cardiologia on interventional cardiology: coronary angioplasty and other technics]. AB - Interventional cardiology has had an extraordinary expansion in last years. This clinical guideline is a review of the scientific evidence of the techniques in relation to clinical and anatomic findings. The review includes: 1. Coronary arteriography. 2. Coronary balloon angioplasty. 3. Coronary stents. 4. Other techniques: directional atherectomy, rotational atherectomy, transluminal extraction atherectomy, cutting balloon, laser angioplasty and transmyocardial laser and endovascular radiotherapy. 5. Platelet glycoprotein IIb/IIIa inhibitors. 6. New diagnostic techniques: intravascular ultrasound, coronary angioscopy, Doppler and pressure wire. For the recommendations we have used the classification system: class I, IIa, IIb, III like in the guidelines of the American College of Cardiology and the American Heart Association. PMID- 10734756 TI - [The practical clinical guidelines of the Sociedad Espanola de Cardiologia on coronary surgery]. AB - Surgery in coronary disease, including myocardial revascularization and the surgery of mechanical complications of acute myocardial infarction, has shown to improve the symptoms, quality of life and/or prognosis in certain groups of patients. The expected benefit in each patient depend on many well-known factors among which the appropriateness of the indication for surgery is fundamental. The objective of these guidelines is to review current indications for cardiac surgery in patients with coronary heart disease through an evaluation of the degree of evidence of effectiveness in the light of current knowledge (systematic review of bibliography) and expert opinion gathered from various reports. Indications and the degree of recommendation for conventional coronary artery bypass grafting have been established for each of the most frequent anatomo clinical situations defined by clinical symptoms (stable angina, unstable angina and acute myocardial infarction) as well as by left ventricular function and extend of coronary disease. Furthermore, the subgroups with the greatest surgical risk and stratification models are described to aid the decision making process. Also we analyse the rational basis and indication for the new surgical techniques such as minimally invasive coronary surgery and total arterial revascularization. Finally, the indication and timing of surgery in patients with mechanical complications of acute myocardial infarction are considered. PMID- 10734757 TI - [Apoptosis in cardiovascular diseases]. AB - Apoptosis consists of a distinct form of cell death that displays characteristic alterations in cell morphology and cell fate which are different than death due to oncosis or necrosis. In terms of tissue kinetics, apoptosis may be considered a mechanism that counterbalances the effect of cell proliferation by mitotic division. In fact, deregulated apoptosis has been implicated in the development a wide variety of human diseases. Excessive apoptotic cell death may cause organ atrophy and organ failure. On the other hand, insufficient elimination of redundant cells may lead to organ and tissue structural remodeling. In recent years, apoptosis has become a highly fashionable and competitive area of research. Fortunately, it has not escaped the attention of the cardiovascular community. Sightings of apoptosis have been reported from every corner of cardiovascular medicine ranging from conduction system defects to congestive heart failure, and from atherosclerosis to aneurysms. There is no question that these sightings will eventually be converted into mechanistic etiopathogenic and physiopathological insights and will form the basis for designing new diagnostic modalities and novel therapies. PMID- 10734758 TI - [Sudden death (VI). The Brugada syndrome and right myocardiopathies as a cause of sudden death. The differences and similarities]. AB - In 1992 we described a new syndrome characterized by syncopal or sudden death episodes in patients with a structurally normal heart and a characteristic electrocardiogram 9 showing a pattern of right bundle branch block and ST segment elevation in right precordial leads V1 to V3. The disease is genetically determined with and autosomic dominant pattern of transmission. Until now three mutations and one polymorphism in the sodium cardiac channel gene have been identified in two families and one sporadic patient. As in many other genetically determined diseases, the disease is heterogeneous, caused by more than one gene. The syndrome has been identified in almost all countries in the world. Its incidence is difficult to evaluate, but it seems to be responsible for 4 to 10 sudden deaths per year per 10,000 inhabitants in areas like Laos or Thailand, and it represents the most frequent cause of death in young male adults in these countries. Up to 50% of all sudden deaths in patients with structurally normal heart are caused by the disease. The diagnosis can be easily made thanks to the characteristic electrocardiographic pattern. In some patients, the presence of concealed and intermittent forms might make the diagnosis more difficult. The electrocardiogram can be modulated by autonomic changes and administration of antiarrhythmic drugs. Beta-adrenergic stimulation normalizes the electrocardiogram, whereas ajmaline, flecainide or procainamide administration increase ST segment elevation. These drugs allow the unmasking of concealed or intermittent forms of the disease. Prognosis of patients with the syndrome is poor without an implantable defibrillator and antiarrhythmic drugs like amiodarone or betablockers do not protect against sudden death. The poor prognosis is similar in patients with a history of aborted sudden death or syncope and in asymptomatic patients in whom the abnormal electrocardiogram characteristic of the syndrome, was identified during a routine examination. PMID- 10734759 TI - [A giant rhabdomyoma of the left ventricle surgically treated]. PMID- 10734760 TI - [Aortic pseudocoarctation complicated by a giant pseudoaneurysm]. AB - Pseudocoarctation of the aorta is an infrequent anomaly caused by the elongation and the congenital kinking of the aorta. We present a case of pseudocoarctation of the aorta with saccular aneurysm complicated with a large pseudoaneurysm. Both, magnetic resonance and aortography showed an aneurysm of the aortic isthmus that extended/progressed as pseudoaneurysm towards the neck. The patient was asymptomatic but due to the high risk of rupture, she underwent surgical reparation. The revision of the literature and our experience with this case show that pseudocoarctation of the aorta is not always a benign pathology and that surgical reparation should be performed in all those cases that are complicated by aneurysm. PMID- 10734761 TI - [Sudden death and ventricular fibrillation of possible ischemic origin in a boy with hypertrophic myocardiopathy]. AB - It has been presented a case of an eleven-year-old patient admitted with a pattern of ventricular fibrillation and diagnosed as hypertrophic cardiomyopathy. Admission analysis and myocardia anatomy evolution suggested ischemic etiology. We checked the risk factors of sudden death, its relation with ischemic disease and the etiology of ischemia in the hypertrophic myocardiopathy. PMID- 10734762 TI - [Sudden death due to an anomalous origin of the left anterior descending coronary artery from the pulmonary trunk in a 9-year-old boy]. AB - A 9-year-old healthy boy is presented, who complained of dizziness and to being exhausted after climbing numerous stairs, collapsing immediately after. Advanced resuscitation efforts were unsuccessful. At autopsy an isolated congenital anomaly in the origin of the left anterior descending coronary artery from the pulmonary trunk was found, with the origin of the right and left circumflex coronary arteries from the aorta, accompanied by with cardiac hypertrophy and chronic ischemic changes in the myocardium irrigated by the anterior descending artery. This represents a very infrequent variant that allows a longer survival and less functional repercussion if compared with those cases in which the main left coronary is originated from the pulmonary trunk. PMID- 10734763 TI - [Right ventricular outflow obstruction due to a giant pseudoaneurysm of the anterior descending coronary artery in a patient with Behcet's disease]. AB - Giant pseudoaneurysms of coronary arteries in patients with Behcet's disease is a uncommon finding. It has been described exceptionally in the literature. We present a case of giant pseudoaneurysm of the left anterior descending coronary artery with obstruction of the right ventricular outflow in a patient with Behcet's disease. He improved after surgical resection and steroid treatment. PMID- 10734764 TI - [Listeria monocytogenes endocarditis of a prosthetic valve]. PMID- 10734765 TI - [The prophylactic administration of low-molecular-weight heparin before surgery]. PMID- 10734766 TI - [Eptifibatide in acute coronary syndromes. Comments on the practice guidelines in antithrombotic treatment]. PMID- 10734767 TI - Guest editor defends October symposium. PMID- 10734768 TI - Ethical capitalism is best. PMID- 10734769 TI - A moral obligation. PMID- 10734770 TI - Medical education is subsidized. PMID- 10734771 TI - Questioning electronic signatures. PMID- 10734772 TI - Is claims review necessary? PMID- 10734773 TI - MedBytes. PMID- 10734774 TI - Outlawing mistakes. PMID- 10734775 TI - Commentary: the role of quality improvement organizations in reducing medical errors. PMID- 10734776 TI - A new war. PMID- 10734777 TI - It could happen here. PMID- 10734778 TI - The courts of last resort. PMID- 10734779 TI - The insurance answer? PMID- 10734780 TI - Bright futures. PMID- 10734782 TI - Listeriosis, France. PMID- 10734781 TI - Perioperative assessment and management of patients with coronary artery disease. AB - Primary care physicians, surgeons, and anesthesiologists are all involved in assessing patients before surgery. The medical literature describes techniques to identify high-risk cardiac patients before elective surgery and strategies to reduce risk once these patients have been identified. Multifactorial indices can identify high-risk patients undergoing nonvascular surgery. Selective use of dipyridamole-thallium imaging based on clinical factors will identify high-risk patients before vascular surgery. Once high-risk patients have been identified, risk reduction can be attempted in various ways. We have no convincing evidence that prophylactic revascularization benefits high-risk patients. Perioperative beta-blockade reduces perioperative myocardial ischemia, mortality, and the incidence of cardiovascular complications for up to 2 years. Therefore, perioperative beta-blockers are recommended for high-risk patients undergoing elective noncardiac surgery. PMID- 10734783 TI - Global laboratory network for poliomyelitis eradication, 1997-1999. Development and expanding contributions. PMID- 10734784 TI - Maternal preferences for type of weaning multimixes used to feed young children. PMID- 10734785 TI - Life-threatening cutaneous reactions to thiacetazone-containing antituberculosis treatment in Kumasi, Ghana. AB - Antituberculosis treatment containing thiacetazone is associated with a high incidence of life-threatening cutaneous drug reactions in patients infected with the human immunodeficiency virus (HIV). In order to develop a local policy concerning the use of this drug, a study was undertaken to determine the incidence of such reactions in a total of 1063 Ghanaian adult patients treated for pulmonary tuberculosis (PTB) with thiacetazone-containing regimens. The incidence was retrospectively determined in 3 different treatment groups, comparing: (A) unselected use of thiacetazone; (B) exclusion of thiacetazone from all patients with positive HIV serology; (C) selective exclusion of thiacetazone from patients with clinical criteria suggesting HIV infection plus education of health workers and patients. Of the 408 patients in group A receiving thiacetazone, 9 (2.2%) developed life-threatening cutaneous reactions and 7 of these were HIV-positive. Overall, 6.8% of HIV-positive patients compared to 0.65% of HIV-negative patients developed severe reactions (P < 0.01; relative risk = 10.5). Six of the 9 patients with reactions died. All 379 patients in group B were screened for HIV antibodies and positive cases (23%) received a regimen in which thiacetazone was substituted by ethambutol. In contrast to Group A, only one HIV-negative patient (0.26%) developed a severe cutaneous reaction (P = 0.02). Among 276 patients in group C, thiacetazone was substituted with ethambutol only in those with clinical evidence of HIV infection (8%) and staff and patients were educated about early recognition of the side-effect. With this policy, these were no admissions with severe cutaneous reactions compared to 2.2% of those in group A (P = 0.01). In conclusion, a policy of selective use of thiacetazone in the treatment of PTB based on clinical criteria combined with patient and staff education was found to be a practical and cost-effective strategy combating severe cutaneous reactions to thiacetazone. PMID- 10734786 TI - A preliminary study of serum ferritin reference range in the adult Ghanaian. AB - Serum ferritin levels were determined in 85 Healthy Ghanaian adults (45 men and 39 women) with a Ciba Corning 125I-Ferritin procedure. Concentrations showed a non-guassian distribution. Values for males ranged from 5.6-273 ng/ml and from 0.74-135 ng/ml for females. The non-guassian nature of the distribution necessitated a logarithmic transformation of the data in order to calculate the serum ferritin reference range (Mean +/- 2SD) for the subjects. The reference range for the males (antilogarithm) was 8.5-306.5 ng/ml (mean = 51.0 ng/ml). The reference range for the Ghanaian women was 3-112 ng/ml (mean = 18.0 ng/ml). The reported reference range for the Caucasian male is 7-350 ng/ml (mean -51.0 ng/ml) and for the Caucasian female, 5-135 ng/ml (mean = 22.0 ng/ml). These Ghanaian reference ranges agree closely with the caucasian values. Data from this preliminary study suggest serum ferritin values of < 8.5 ng/ml and < 3 ng/ml could serve as cut-off points below which iron deficiency may be said to be present in the adult Ghanaian male and female respectively when the same method of analysis is used. A further study of ferritin levels in the Ghanaian is recommended. Pending further work, continued use of the conventional cut-off points of 20 ng/ml for males and 10 ng/ml for females to identify iron-deficient individuals is in order. These conventional cut-off points enhance chances for identifying iron-deficient individuals. PMID- 10734787 TI - Infectivity and transmission dynamics of simulium damnosum s. 1 around Owena dam (Ondo State). AB - A twelve month study on the infectivity and transmission dynamics of simulium damnosum s.1 around Owena dam was conducted as part of a longitudinal study of onchocerciasis in the community living in the vicinity of the dam. A total of 3,133 flies (49.6% nilliparous, 50.4% parous) were caught during the period of study and dissected. The annual biting rate per year was recorded as 33,663 flies while the parous biting rate was recorded as 16,780 for these flies. 0.4% of the flies were infected with Onchocerca volvulus larvae while 0.3% of these had the infective larvae. However 0.3% of the nulliparous flies were found to be infected with mermithid larvae and 0.5% with trematodes. The biting activity of the fly was observed to be bimodal with a small peak in the morning hours (10.00-11.00 hours) and a more pronounced peak in the evening (16.00-17.00 hours). An annual transmission potential of 109 larvae per man per year was recorded with 64% of the infection being recorded during the early dry season when the parous rate was very high. The implications of these findings to the study area are discussed. PMID- 10734788 TI - Causes and pattern of tooth extraction in children treated at the University College Hospital, Ibadan. AB - A study of 429 children age 0-15 years who attended the paedodontic clinic of the Department of preventive Dentistry for treatment from January 1995 to December 1995 with a view of assessing causes and pattern of tooth extraction was carried out. 51.52% of the children had extraction. Dental caries and its sequelae accounted for the highest indication for extraction (57.47%) while periodontal disease accounted for the least (0.45%). Deciduous teeth were mostly extracted with the deciduous molars being the teeth frequently extracted. The frequency of extractions shows that a challenging situation exists for those engaged in the organisation of dental health education and emphasis should be on prevention. PMID- 10734789 TI - Evidence for obstructive and restrictive lung pathology among tetra-ethyl lead handlers and petrol tanker fillers at a petrochemical industry in Nigeria. AB - Described here is a computer predicted and computer measured pulmonary function in a randomly selected population sample of 111 men. These consisted of 36 primary school teachers (control), 35 Tetra-ethyl Lead handlers (TEL) and 40 Tanker Loaders (TL) at a petrochemical industry in Warri, Delta State, Nigeria. Productive cough and headache were commoner among the TL (45%, 38%) and the TEL handlers (43%, 31%). TEL handlers were 5 times likely to develop insomnia and other sleep disturbances than the control group. TL were more than 3 1/2 times more likely to develop nervousness than the control group. Measured FEF25-75% was significantly lower than its predicted value among the TEL handlers (56%) and TL (55%) when compared with the control group (36%) (P < 0.05 respectively). Although there were distinct reductions in the measured values of FEF75-85% among the TEL handlers (41%) and the TL (40%) compared to the control group (28%), this difference did not reach a level of significance when the TEL or the TL were compared individually to the control group. Predicted and measured forced mid expiratory flow time (FMFT) was not significantly varied among the cases and the control. There was statistically significant disparities (P < 0.005) in the values of predicted and measured maximum voluntary ventilation (litres) of the TEL handlers (47%) and of the TL (46%) compared with that of the control group (29%). The data suggest obstruction of the big and small airways of those occupationally exposed at a petrochemical factory in Nigeria especially the tetra ethyl lead handlers and the tanker loaders. This data also suggests that pulmonary impairment may lead to an increased respiratory absorption of lead in its organic form. Efforts should be made to ascertain the degree of impairment and to treat the affected workers. PMID- 10734790 TI - Pulmonary tuberculosis in adults: factors associated with mortality at a Ghanaian teaching hospital. AB - Tuberculosis (TB) is a leading cause of global mortality. The aim of this study was to compare factors associated with poor outcome (death) and good outcome (cure) of adult patients receiving treatment for pulmonary TB (PTB) at the Komfo Anokye Teaching Hospital, Kumasi, Ghana. The case notes and chest radiographs of 80 such patients who died were retrospectively reviewed and compared to 80 patients who were cured during the same period. Patients who died were 3.4 times more likely to be HIV-positive (p < 0.001). Mortality was also associated with increased age (p < 0.001), residence in a rural area (p < 0.05) sputum smear negative disease (p < 0.01), and more prolonged symptom duration prior to initial diagnosis (p < 0.05). Furthermore, patients who died were 2.1 times more likely to have a history of previous TB treatment (p < 0.01), 2.0 times more likely to have previously defaulted from treatment ((p < 0.05), and 2.9 times more likely to have > or = 5 chest radiographic zones affected by disease (p < 0.001). In conclusion, although mortality among patients with PTB is strongly associated with HIV infection in this community, other factors identified reflect late diagnosis, poor treatment compliance and inadequate resources for diagnosis and treatment of TB in rural areas. Improved rates of diagnosis, enhancement of treatment compliance and decentralization of TB services to the district level may therefore help to reduce mortality from TB. PMID- 10734791 TI - Humoral immunity in Nigerians with Schistosoma haematobium infection. AB - Circulating soluble immune complexes (CIC), immunoglobulin classes (IgG, IgA, and IgM), acute phase proteins (alpha-l-fetoprotein and alpha-1-acid glycoprotein) and complement factors (C4 and C3c) levels were determined in 52 Nigerian patients with urinary schistosomiasis (USS) and 39 healthy subjects by polyethylene glycol precipitation and single radial immunodiffusion methods respectively. A considerably higher proportion of USS patients than the controls had elevated levels of soluble immune complexes. IgM, IgA and C3c levels were significantly higher in USS patients than the controls. However, serum concentration of C4 was significantly reduced in USS patients than the controls while that of IgG was not significantly reduced in these patients. The mean levels of alpha-l-fetoprotein and alpha-l-acid glycoprotein were comparable in the USS subjects and the controls. Correlation analysis showed association between S. haematobium egg numbers with serum concentration of IgM, C3c and CIC. These observations could be the results of increased rate of CIC formation, and inflammatory responses to schistosome antigens. The study concluded that IgM associated-CIC may be responsible for the immunopathology of chronic schistosomiasis. PMID- 10734792 TI - Severity and outcome of falls in children. AB - Three hundred and five patients with falls over a period of 4 years were reviewed with the aim to determine the pattern, severity and outcome of childhood injuries from falls in our environment. The results showed that falls accounted for 25% of all childhood injuries. There were more male than female in ratio 2:1. The age ranges from 5 months-15 years with mean age of 6.15 years + or - 3.63. About 40% of the patients were preschool children. Home was the commonest site of falls in 55.3% followed by school in 38% of the patients. Falls from height accounted for 25% and at the ground level in 75% of the patients. Falls from stairs and storey buildings accounted for 68.3% of falls from height, whereas falls while playing on a ground level occurred in 83% of those whose falls were at ground level. Musculoskeletal injuries with fractures of bones were the commonest injuries sustained followed by head injuries. Fracture of lower limb bones accounted for 48% of all the bone injuries commonest bone being femur. The upper limb bones fractured occurred in 45.6%. There were two cases of skull fracture. Head injuries occurred in 6.2% of the patients and severe in 10% of the head injured patients. Other systemic injuries occurred rarely. Most of the injuries were trivial with Injury Severity Score ranges from 2-26. The mean Injury Severity Score was 2 in 235 patients (77%), 10.5 in 64 (21%), 12.5 in 4 (1.4%) and 21.5 in 2 patients(0.6%). Seventy-eight percent of the cases were treated as outpatient. Hospital stay ranges from 1-45 days with mean of 3.8 days. The hospital stay correlated very well with the Injury Severity Score. Wound Infection occurred in only 1.4% of the patients with mortality in 2 patients (0.6%) with Injury Severity Score of 17 and 26 respectively. PMID- 10734793 TI - Uterine perforation from induced abortion at Korle Bu Teaching Hospital, Accra, Ghana: a five year review. AB - A total of 79 cases of perforated uterus from termination of pregnancy in Accra, an incidence of 3.6% of induced abortion were managed at Korle Bu Teaching Hospital during the period 1990-1994. The mean age and parity of patients who had perforated uterus were 21.6 +/- 2.3 years and 1.97 +/- 0.28 respectively. The specific case mortality associated with this calamity was 227.8 per 1000. The need to prevent unwanted pregnancies through improved contraceptive measures and sex education must be emphasised in developing countries. PMID- 10734794 TI - Comparison of the periodontal treatment needs of normal and handicapped children in Lagos. AB - The periodontal treatment needs of normal children and those who have mental and multiple handicaps are compared. There were 7.8% handicapped children who did not require any periodontal treatment compared with 1.5% of normal children. Similarly, a higher proportion of normal children (78.6%) compared with 59.4% of handicapped children needed professional oral prophylaxis and oral hygiene education. It was also found from the study that there was a greater need for professional oral prophylaxis in normal children and handicapped children living in institutions than in handicapped children staying with their parents at home. It was concluded that both normal and handicapped children require supervised oral hygiene measures till effective oral hygiene habit is established. PMID- 10734795 TI - Gene frequencies of ABO and Rh (D) blood group alleles in a healthy infant population in Ibadan, Nigeria. AB - The ABO and Rhesus blood group systems remain the most important blood group systems clinically. In order to provide gene frequency values for the ABO and Rh (D) alleles in a healthy infant population in south west Nigeria, 4748 healthy infants were typed for ABO and Rh (D) blood groups over a five year period (1988 1992). Overall, 2575 (54.2%) were blood group O, 1023 (21.6%) were blood group A, 1017 (21.4%) were blood group B and 133 (2.8%) were blood group AB. The distribution of the ABO blood groups did not differ significantly from those expected under the Hardy Weinberg equilibrium (Goodness-of-fit X2 = 6.09, df = 3, p = 0.1075). The proportions of the infants belonging to the various ABO blood groups did not vary significantly over the period of the study (X2 = 14.53, df = 12, p = 0.268). Overall gene frequencies for the O, A and B genes were 0.7398, 0.1305 and 0.1298 respectively. For the Rh (D) gene, 4520 (95.2%) were Rh positive while 228 (4.8%) were Rh-negative. However, the proportions of Rh (D) negative infants varied significantly over the period of the study, with a particular year (1991) having nearly twice the usual frequency of Rh-negative individuals (X2 = 31.17, df =, p < 0.001). The frequency of the Rh (D) gene was 0.7809. These figures are reported in the hope that they may find some use as reference for studies of ABO blood groups in health and disease, especially since they were obtained in an infant population in which it is expected that selection pressures should not have started to act to any significant extent. PMID- 10734796 TI - Trends of sinusitis in Ibadan, Nigeria. AB - Sinusitis occurs frequently, complications are infrequent since the introduction of antibiotics and mortality has also decreased significantly for same reason. A retrospective study of 90 patients with sinusitis seen over a 10 year period between 1988 and 1997 in the E. N. T. Department, University College Hospital, Ibadan was done to highlight the general direction and tendency of sinusitis in our environment. There were 50 (56%) males and 40 (44%) females. The average age was 33 years and 70% were under the age 40 years, showed that sinusitis affects mostly children and young adults. 38% in social class V while 10% social Class I. Chronic sinusitis was 93%, acute sinusitis 7% (both types unisinusitis 56%, pansinusitis 29%, multisinusitis 16%). All the cases of unisinusitis were maxillary sinusitis. 85 (94%) had surgical treatment (antrostomy 64%, Fronto ethmoi-dectomy 15%, Caldwell-Luc's 13%, antral lavage 13%). 80 (94%) patients are alive and well after 2 years of follow up, 5 (6%) lost to follow up. Complications arising from sinusitis was seen affecting the orbit in 41%--the largest, followed by sinus wall 32%, no ear complications seen. Thirteen (14%) of the sinusitis cases associated with allergy only, 24 (27%) with polyps only, 6 (1%) with both allergy and polyps. It stresses the interwoveness of these entities (allergy, Polyps, sinusitis)--one predisposes to the other. Thus with these trends of sinusitis being highlighted, it is hoped that this would enhance our knowledge of the pathology, diagnosis and treatment of sinusitis. PMID- 10734797 TI - Socio-economic implication of multi-drug resistant malaria in the community; how prepared is Nigeria for this emerging problem? AB - The control of faciparum malaria is becoming increasingly challenging in many endemic areas of the world, including Nigeria, due to the development of resistance to chloroquine and other anti-malarial drugs. Current rising health care costs demands that any preventive medicine and/or disease control program be judged according to its economic viability. As has been previously noted, any successful control of malaria will depend on socio-economic factors that influence its prevalence and management in the community. Cost-benefit or cost effectiveness analysis of proposed and current intervention strategic are thus justified. This paper presents a review of studies involving socio-economic evaluation of the morbidity and mortality consequences of malaria. Studies involving health facility utilization profile for malaria in Nigeria and elsewhere are also reviewed. The review finds no studies evaluating or determining an appropriate economic model/framework for malaria control in Nigeria and concludes that as enormous and challenging the problem of multi-drug resistant malaria is, it can still be contained if control and management strategies are adopted based on sound and practical socio-economic judgement. PMID- 10734798 TI - Cardiomyopathy in children. AB - The cardiomyopathies are a group of disorders of the myocardium associated with a decrease in cardiac function. This article describes the three functional types: (1) dilated or congestive; (2) obstructive, as in hypertrophic cardiomyopathy, and (3) restrictive as in infiltrative disorders such as the storage diseases. PMID- 10734799 TI - Radioactive seed embolization to the lung following prostate brachytherapy. AB - Over the past decade, prostate brachytherapy has been increasingly utilized as definitive management for early stage carcinoma of the prostate gland. One risk of the procedure is pulmonary seed embolization. In this article we report the incidence and timing of seed migration. Pulmonary embolization of radioactive seeds occurs in approximately 20% of patients undergoing prostate brachytherapy. To date, no acute or delayed detrimental effects have been reported which are attributable to the pulmonary embolization of the seeds; nevertheless, it is imperative that patients and health care providers be cognizant of this possible event. PMID- 10734800 TI - Adolescent all-terrain vehicle deaths in West Virginia, 1990-1998. AB - West Virginia had the third highest number of ATV-related deaths and the highest death rate among all states from 1990-98. Adolescents were identified as a particularly high-risk ATV user group. Fatality data from the U.S. Consumer Product Safety Commission indicated that one-quarter (25 of 101) of the state's ATV-related deaths occurred among children 16 and younger. Only one victim was wearing a helmet at the time of their fatal crash and nearly 70% of the victims were males. The average years of potential life lost was 64.2. WV's adolescent death rate of 0.67 per 100,000 was 5 times higher than the national rate. Young ATV drivers exacerbate the inherent dangers associated with ATVs through poor judgment and risk-taking. Health care providers should utilize "teachable moments" to emphasize ATV safety to their young patients and their parents, but more importantly, adults must become role models by embracing and teaching practical preventive measures. PMID- 10734801 TI - Pulmonary epitheloid hemangioendothelioma: a peculiar rare tumor of vascular origin. AB - An extremely rare case of pulmonary epitheloid hemangioendothelioma (PEH), previously known as intravascular bronchoalveolar tumor (IVBAT), in a 38-year-old female is presented. This patient had a history of rheumatoid arthritis and bilateral multiple small pulmonary nodules which progressed over the years. The histopathological diagnosis of PEH was confirmed by immunohistochemical stains. Prognosis of this tumor is very unpredictable. There is no effective treatment for pulmonary epitheloid hemangioendothelioma. PMID- 10734802 TI - Emergency contraceptives over the counter. Allowing easy access is important. PMID- 10734803 TI - Making condoms available in schools. The evidence is not conclusive. PMID- 10734804 TI - Adolescent injury prevention and clinicians: time for instant messaging. Physicians can help to reduce pointless and early deaths. PMID- 10734805 TI - The science and art of treating acne in adolescence. PMID- 10734806 TI - Evidence-based case review. Contraception for adolescents. PMID- 10734807 TI - Identifying and treating adolescent depression. PMID- 10734808 TI - A primary care approach to adolescent health care. PMID- 10734809 TI - Myths and variations in normal pubertal development. PMID- 10734810 TI - Should short children who are not deficient in growth hormone be treated? PMID- 10734811 TI - Medical complications occurring in adolescents with anorexia nervosa. PMID- 10734812 TI - Outpatient management of children with diabetes. Diabetes: don't immediately admit before evaluating the entire situation. PMID- 10734813 TI - Helping teens who live in violent communities. PMID- 10734814 TI - Dysuria in adolescents. PMID- 10734815 TI - When genital pain occurs in adolescent boys, what conditions should we be thinking about? PMID- 10734816 TI - How should doctors talk to teen patients? PMID- 10734817 TI - Myths and medicine. PMID- 10734818 TI - Medical myth: Analgesia should not be given to patients with an acute abdomen because it obscures the diagnosis. PMID- 10734819 TI - Adolescent sexuality and the media: a review of current knowledge and implications. PMID- 10734820 TI - Fibromyalgia. PMID- 10734821 TI - Unmasking mental disorders. PMID- 10734822 TI - Decision-making dialogue. PMID- 10734823 TI - Study supports medication for ADHD. PMID- 10734824 TI - A boost for St. John's Wort? PMID- 10734825 TI - Secretin for autism? Not so fast. PMID- 10734826 TI - Vitamin E shows no benefit in heart disease. PMID- 10734827 TI - Extra oxygen prevents surgical infections. PMID- 10734828 TI - A "direct hit" against liver tumors. PMID- 10734829 TI - Some asthma patients need medication adjustment. PMID- 10734830 TI - Light therapy for precancerous skin lesions. PMID- 10734832 TI - Why do I seem to get sick soon after a stressful event, such as my wife's hospitalization? PMID- 10734831 TI - Are foods sweetened with fruit juice any healthier than those sweetened with regular sugar? PMID- 10734833 TI - Are antidepressants addictive? PMID- 10734834 TI - Neck pain. When it's serious, when it's not. PMID- 10734835 TI - Health tips. How to find comfortable shoes. PMID- 10734836 TI - Grapefruit juice influences certain cholesterol drugs. PMID- 10734837 TI - New drugs to treat irritable bowel syndrome being tested. PMID- 10734838 TI - Assisted living. All the right moves. PMID- 10734839 TI - Psoriasis. Tailoring treatment may help you find relief. PMID- 10734840 TI - Bunions. Don't crowd your toes. PMID- 10734841 TI - What causes denture sores, and what can I do about them? PMID- 10734842 TI - I'm afraid to fly. How can I get over my fear? PMID- 10734843 TI - Lesbians in alcohol recovery surviving childhood sexual abuse and parental substance misuse. AB - Narratives of lives disrupted by abuse are essential data sources for understanding women's survival and healing in contexts of childhood sexual abuse. In this qualitative, feminist study of lesbians recovering from alcohol problems who have histories of childhood sexual abuse, a multiethnic sample of 20 women narrated their life stories in a series of three in-depth interviews. The purpose of this paper is to focus on parental substance misuse as it affected these women when they were growing up. Conditions and consequences of surviving childhood sexual abuse and parental substance misuse are analysed using narrative strategies and described using excerpts from the women's narratives. Loss was the overarching core theme that integrated participants' storied descriptions of parental substance misuse. They incurred severe losses in the absence of basic necessities for safe and healthy passages through childhood. In their abusive homes, they were not allowed innocence, and protected from violence, nor nurtured, guided, and loved. As many said, they lost their very childhoods. PMID- 10734844 TI - Evaluating the effect of assessment education with community psychiatric nurses using a file audit tool. AB - Within public mental health services in Australia, community mental health nurses are often the first point of contact for people presenting with mental health problems. Community mental health nurses are responsible for determining appropriate triage and assessing need for treatment. Community mental health staff identified a need to improve skills in assessment and documentation of psychiatric symptoms. This collaborative study sought to develop an educational package, based on Present State Examination-9, for initial assessment, and a reliable file audit tool to evaluate the effectiveness of the education. Twenty initial mental health assessments completed by twenty different community mental health nurses were audited approximately one month prior to, and two months after, attendance at the education programme. The audit indicated that the documentation of assessments improved in all aspects, although the most significant differences were achieved in recording history, mental state and treatment plan. These items were the main items emphasised in the education programme. It is suggested that the Present State Examination-9 provides a useful base for teaching clinicians how to incorporate the documentation of comprehensive and precise information into assessment formats and treatment planning and that the file audit tool, when linked with an assessment format provides a useful way of evaluating self and peer assessment documentation. PMID- 10734845 TI - User views of a regional secure unit--findings from a patient satisfaction survey. AB - This paper includes key findings of a patient satisfaction survey, conducted within an English Medium Secure Unit, offering care and treatment for 45 male and female mentally disordered offenders. It aims to identify patients' perceptions of the quality of their care and other aspects of their lives within the secure unit. Following permission from the local Ethics Committee, two interviewers collected data within a two week period, using a semi-structured interview schedule. All but three of the respondents agreed to be interviewed. Talking to nurses was perceived as highly satisfying. However, provision for specific cultural and women's needs, facilities for visitors, opportunities to practise religious beliefs and access to care plans were highlighted as unsatisfactory. The study concludes with recommendations for improvement, some of which have already been considered and implemented. PMID- 10734846 TI - The importance of theory in student nurses' psychiatric practicum. AB - This study aims to evaluate the importance of theory in enhancing student nurses' experiences in a rural psychiatric practicum and also identify positive, negative and potentially helpful factors which could be used to improve student nurses' psychiatric practicum. A pilot study of sixty-five undergraduate nursing students were given a questionnaire after their psychiatric practicum. Positive, negative and potentially helpful factors were derived from themes of their responses. Results indicated that theory played an important role in enhancing students' learning in their clinical practicum. Positive factors included interaction with clients; improvement of knowledge and understanding of mental health, thus dispelling myths; staff's professionalism, enthusiasm and acceptance of students; direct involvement with clients; diversity of learning opportunities and shift work. Negative factors related to nursing staff performance. Potentially helpful factors were those promoting goodness of fit between theory and practicum and characteristics of the clinical setting. PMID- 10734847 TI - Religion and mental health. PMID- 10734848 TI - The Behavioural Status Index (BSI) some preliminary reliability studies. AB - This paper reports results of reliability studies for an assessment instrument that has undergone extensive empirical investigation: the Behavioural Status Index. Results indicate acceptable levels of test re-test reliability, item analysis and internal consistency. Inter-rater reliability is reported as equivocal Future empirical studies are discussed. PMID- 10734849 TI - American Indian working women: correlates of subclinical depression among American Indian women. AB - The purpose of this study was to examine the extent to which role conflict, life satisfaction, self-esteem, instrumentality, expressiveness, age and education predicts ambulatory depression among a community based sample of urban American Indian working women. The convenience sample consisted of a total of 148 Midwestern American Indian working women, ranging in age from 18 to 65 years. Participants completed five instruments assessing gender-related traits of instrumentality and expressiveness, self-esteem, depression, life satisfaction, role conflict, and socio-demographic information. A forced entry stepwise multiple regression was conducted which included all the designated predictor variables. The significant negative Beta in Step 1 indicated that women scoring on the Masculinity (instrumentality) sub-scale were less likely to have a high depression score. The prediction of depression by the Masculinity sub-scale was no longer significant after the life satisfaction and self-esteem variables were added to the equation in Step 2. There was a significant inverse relationship for life satisfaction and self-esteem in predicting the criterion variable, depression. Thirty-two percent of the variance in the prediction of the dependent variable (depression scores) was accounted for by the six variables. In counselling urban American Indian women, supporting instrumentality, enhancing self-esteem and life satisfaction, can positively impact on ambulatory dysphoria. PMID- 10734850 TI - The process of transforming self in women veterans with post-traumatic stress disorder resulting from sexual abuse. AB - In recent years increasing attention has been given to the prevalence and impact of military sexual abuse. Symptoms of post-traumatic stress disorder (PTSD), a diagnostic entity originally restricted to those who had experienced wartime trauma, have been observed in women veterans who reported that they were sexually abused during military service. However, little research has been conducted in this area. The purpose of this study was to determine the recovery process in women veterans suffering from PTSD resulting from sexual abuse. Using grounded theory methods, the researchers interviewed 16 women veterans who were in active recovery. Constant comparison data analysis revealed the emerging basic social process of "transforming self." The findings indicate that participants transformed self by progressing through five stages: reacting to triggers; seeking validation; sorting through confusion; becoming intentional; and affirming self. The authors compare these findings to other research on recovery from sexual abuse. PMID- 10734851 TI - Southeast Asian refugee children: violence experience and depression. AB - The United Nations High Commission for Refugees (UNHCR) reports that there were approximately 8.2 million refugees world wide in 1980. Estimates today place the number of refugees to be at least 44 million. Although most refugees remain within the boarders of their homelands, others who are granted asylum immigrate to host countries. The largest number of refugees to enter the United States during the past 20 years are from Southeast Asia, following the end of the Viet Nam War in 1975. The majority of immigrating Southeast Asian refugees were children and adolescents. Empirical study of refugee children, who are known to have experienced catastrophic violence during war, escape from homelands and in camps of asylum, is relatively scarce. Some studies that have addressed this issue document association between violence experience, depression and post traumatic stress disorder. Even so, the findings are not always clear. The purpose of this paper is to document the frequency of pre-migration and post migration violence experiences reported by Southeast Asian refugee children in the U.S. and their relationship to depression. This study also addresses the emotional impact of violence experiences as described by the children. Previous studies have often neglected this important variable in accounting for variations in children' 5 mental health status. Nurses, internationally, will want to consider the multiple facets of violence experience when assessing children who may be at high risk for psycho-social adaptation problems following resettlement. PMID- 10734852 TI - [Pediatric nursing 2000--what it can do, what it does and future plans]. PMID- 10734853 TI - ["Quality advances in nursing"]. PMID- 10734854 TI - [Tasks and function of the nursing staff in child- and adolescent psychiatry]. PMID- 10734855 TI - [Possible variations of supine positioning of premature infants]. PMID- 10734856 TI - [The value of the first and second measles-mumps-rubella immunization according to the STIKO (Permanent Immunization Committee) recommendations. Interview by Marianne E Tippmann]. PMID- 10734857 TI - [The operation room statute--results of interdisciplinary cooperation in the operating room]. PMID- 10734858 TI - [Glaxo Wellcome, Germany, spends 60.000 DM for African HIV projects in Uganda and Malawi]. PMID- 10734859 TI - [Tourette syndrome and other tic disorders]. PMID- 10734860 TI - [Endoscopic methods in peripheral nerve surgery]. PMID- 10734862 TI - [New therapeutic concepts in the management of venous leg ulcer: a compression system enhances healing]. PMID- 10734861 TI - [Award winning drug]. PMID- 10734863 TI - [Violence against Alzheimer patients--causes, effects and solutions]. PMID- 10734864 TI - [A God who has a heart] [In Process Citation] PMID- 10734865 TI - Big brother is watching us. PMID- 10734866 TI - A good place to start. PMID- 10734867 TI - Home improvement. PMID- 10734868 TI - Let them eat cake. Interview by Charlotte Alderman. PMID- 10734869 TI - Clean-up operation. PMID- 10734870 TI - Prime movers. PMID- 10734872 TI - [In Process Citation] PMID- 10734871 TI - One giant step for a man. PMID- 10734873 TI - Ordinary people. PMID- 10734874 TI - Blistering attack. PMID- 10734875 TI - Guidelines on using gloves. PMID- 10734876 TI - The role of hope for relatives of critically ill patients: a review of the literature. PMID- 10734877 TI - Putting the debate on nursing language in context. PMID- 10734878 TI - Public involvement: working for better health. PMID- 10734879 TI - The nurse's role in antibiotic therapy. PMID- 10734880 TI - Rich rewards in childcare. PMID- 10734881 TI - Penny wise. PMID- 10734882 TI - People seeking a career in nursing need choices in the paths they take. PMID- 10734883 TI - Hop on, hop off routes. PMID- 10734884 TI - What is the UKCC's education commission? PMID- 10734885 TI - Too theoretical by half. PMID- 10734886 TI - Board here to fast track. PMID- 10734888 TI - Prepared for the worst. PMID- 10734887 TI - A lengthy list of what you need to know. PMID- 10734889 TI - Resistance fighters. PMID- 10734890 TI - Want to solve the recruitment and retention crisis? PMID- 10734891 TI - The government's carrot-and-stick approach. PMID- 10734892 TI - Reach for the sky not the corset. PMID- 10734893 TI - Sifting evidence. PMID- 10734894 TI - Return to sender. PMID- 10734895 TI - Guiding lights. PMID- 10734896 TI - Students of hardship. PMID- 10734897 TI - Eye contact. PMID- 10734898 TI - Taxing questions. PMID- 10734899 TI - Back to basics. PMID- 10734900 TI - The stuff of life. PMID- 10734901 TI - The future of information technology is on the web and the future of nursing is its students. PMID- 10734902 TI - Discharging patients with burn injuries. PMID- 10734903 TI - A cover-up. PMID- 10734904 TI - Malaria: a scourge of humankind. PMID- 10734905 TI - A person-focused system for people with learning disabilities. PMID- 10734906 TI - Primary care. A different direction. PMID- 10734907 TI - Primary care. The tough get going. PMID- 10734908 TI - Primary care. None the wiser. PMID- 10734909 TI - Primary care. We can work it out. PMID- 10734910 TI - Primary care. Long division. PMID- 10734911 TI - Primary care. Partners for the future. PMID- 10734912 TI - Tribalism is out. PMID- 10734913 TI - Administering a catheter maintenance solution--1. PMID- 10734914 TI - I have a dream... PMID- 10734915 TI - Post-term pregnancy. PMID- 10734916 TI - Primary Care Trusts. New opportunities for midwives in the new NHS? PMID- 10734917 TI - Highlights of the 6th Annual Report. Part 3: Big babies. PMID- 10734918 TI - Pierced nipples and breastfeeding. Achieving compromise. PMID- 10734919 TI - Meeting the needs of lesbian clients. PMID- 10734920 TI - Optimal fetal positioning. The experience of a shiatsu practitioner. PMID- 10734921 TI - An interpretation of midwives' views about the nature of midwifery. PMID- 10734922 TI - The more things change.... AB - The main messages from the survey were that maternity care varied widely from one district to another and often varied within communities and within hospitals. Much depended on both the attitudes and preferences of the individual providing care and whether a woman was offered care as part of an innovative pilots project. In a number of areas, Changing Childbirth pilots were closed down rather than rolled out across the district, despite good results. Evidence of more woman centred care suggests that attitudes towards women had changed more than the actual services. However, those women who were interested in a home birth were still experiencing major obstacles. Postnatal care and support for breastfeeding were identified as those aspects of care most in need of improvement. PMID- 10734923 TI - Making a difference in midwifery. A community perspective. PMID- 10734924 TI - Knowing the sex of the baby before birth. PMID- 10734925 TI - Puerperal fever. Disaster and triumph. PMID- 10734926 TI - Watch your language! PMID- 10734928 TI - Pleased to meet you. Petra ten Hoope-Bender. PMID- 10734927 TI - The great divide. PMID- 10734929 TI - Which course? The ALSO instructors' course. PMID- 10734931 TI - [In Process Citation] PMID- 10734930 TI - Born before arrival. PMID- 10734932 TI - [Nursing doctors and doctoral students: reasons for getting a master's degree in other areas]. AB - There are in Brazil only 37 nursing doctors and doctoral students who obtained the Master degree in other area. A set of questions--concerning the reasons of the choice, the enrollment in the Program and the suggestions related to the nurses in similar situation and to the Nursing Schools--was mailed to this group. The answers showed that the option, in spite of being determined by several circumstances, sets up a valuable experience and is part of the history of the nursing graduate programs in Brazil. PMID- 10734933 TI - [Preventive measures against AIDS and other sexually transmitted diseases known by students in the health field]. AB - This study was carried out in to identify the preventive measures against Aids and other Sexually Transmitted Diseases (STD) that undergraduate health students predominantly know and to identify if there is a significant difference among the measures mentioned, considering the sex of subjects studied. A questionnaire was answered by health undergraduate students randomly selected. The results show that most of the items mentioned as preventive for Aids and other STD are important measures for their prevention. However, when data are analysed considering the frequency, that is the number of students that mentioned correct measures, we can observe that it is less than it was expected, what is very worrying. Another point observed in the results is that, there was no statistical difference, according to the sex of subjects studied. Data analysis evidence that knowledge of most undergraduate students is not enough, reinforcing the need to implement continuous specific educational actions. PMID- 10734934 TI - [Diseases and the sick--understanding medical practices in capitalism]. AB - This article discusses the diseases and the sick as well as how the process from which to take care of them became object of medical practices. Based on Donnagelo and Goncalves contributions, the author discusses the process that explains medical practices appropriation by the capitalism. PMID- 10734935 TI - [Ratio scaling evaluation of nurses' professional prestige through a cross modality method]. AB - Professional prestige was determined using the psychophysical methods of magnitude estimation and cross-modality matching with line lengths and dynamometric forces as responses. The main results revealed that: (a) the rankings order resulting from all methods employed provide degrees of prestige which are in close agreement with the different professions (w = 0.94), (b) the non-metric continuum of professional prestige exhibits prothetic characteristics, (c) the variability of estimations is a linear function of the estimation of magnitudes, thus confirming that Ekman's law is also valid for non-metric continua, (d) the exponents obtained using matchings of number, line lengths and force of hangrip were not significantly different from the exponents predicted by the transitivity property of scales and, finally (e) the data furnish a scale of professional prestige which, at the level of ratio measurement, is valid, stable and consistent. PMID- 10734936 TI - [Chemical substances, work and neurologic changes: possible relationships between those variables]. AB - Authors searched 239 reports of patients admitted in a hospital who carried neurological pathologies with unknown etiologies in order to find the possible relationship between the disease and the occupational use of chemical substances. They found the occupation in 32 reports. 37.50% of the patients worked with agriculture; 34.37% were maids/worked at home; 12.50% were masons; one was a shoemaker, one a saddler; one made furniture and the others worked with machines. Considering their activities, they possibly used chemical substances. Few information relating this utilization and the presented disease showed the reduced importance given to these variables. PMID- 10734937 TI - [Analysis of the situation of occupational health in Chile]. AB - The authors make a critical analysis of the Occupational Health in Chile based on the conclusions obtained from a multidisciplinary workshop, attended by professionals of this area. The method used was strategic planning, exposing the weaknesses, strengths and expectations regarding the health reality of the region. A profile of the type of professionals that should participate in the programs was also discussed, elaborating a work plan that should reflect the future development in this area, as well as pointing out the role of the University in the work plan and analysing the future of Occupational Health. PMID- 10734938 TI - [Images in Venus' mirror: woman, nursing and modern times]. AB - This paper discusses the process of nursing professional identity construction in modernity. We utilize the ideas of category gender (Lobo, 1991) and modern person (Duarte, 1986). The modernity as a historical process establishes the emergence of work as a central category in the public X private relationship. Understanding the profession as form of construction and public presentation of the modern person, we discuss the nursing professional identity process in relation to its female basis. Working on nursing ethnographic bibliography, we identify a picture that gives to the female basis of the profession the greater cause of dilemmas such as: technical competence, vocation and identity. PMID- 10734939 TI - [Implementation on the operating level of the nursing process]. AB - The development of the nursing care attention process guided through the PDCA. (plan, do, check, act), provides a continuous improvement of our actual context. The meaning of PDCA is translated as VIDA for: view, implementation, discernment, at just. For its application it takes in view the change theory of Kurt Lewin. This paper provides the results of the Implementation stage using different approaches as technical care, education, administration and research. These strategies show that people did not express their feelings, beliefs and their own expectations and autonomy in nursing care. They even do not develop their citizenship, right of living and being able to grow. PMID- 10734941 TI - [Leprosy and sexuality: living with a difference]. AB - The present article is based on a research that analysed the social effects of leprosy in men and women, identifying the effects in their family life. 10 men and 10 women with leprosy registered in a specific health service were part of this study. The methodology was qualitative, through semi-structured interviews. Data analysis pointed out the fact of the disease perceived as a reason for family disorders and, also, impairing these subjects sexual relationships. PMID- 10734940 TI - [Perception of children by undergraduate nursing students]. AB - The purpose of the present study was to determine the perception of children by nursing undergraduate students in a Brazilian Public University. The students were asked to write about their perception of children on the first and last day of their academic activities in pediatric nursing. Using a qualitative approach, the authors analyzed the reports and identified the following thematic nuclei: "a small, fragile and innocent being," "citizen human being", "human being in the developmental phase", "a being who has fun", "the environment and child training", and "interaction of children with adults". Results show that at first the students perceived children as fragile, immature beings unprepared to face life and needing adult protection. At the end of the course, they started to see children as developing beings and as citizens with the same rights as adults. PMID- 10734942 TI - [The computer in nursing education--analysis of the attitudes of students of university schools]. AB - This study aims at identifying and analyzing the attitudes by students from different institutions regarding the use of the computer in Nursing teaching. Methodology consisted in the elaboration, application and analysis of Likert scales distributed among students of the eighth semester in higher education institutions at the cities of Ribeirao Preto and Bauru. Results indicated the computer as one of the main resources for nursing teaching and the simulation as an excellent bridge between theory and practice as well as the educational softwares useful and interesting. Authors verified the high level of interest and positive attitudes regarding the employment of computer resources in nursing teaching. PMID- 10734944 TI - [World health]. PMID- 10734943 TI - [The health care system and the state of nursing in Argentina]. PMID- 10734945 TI - [Innovation in higher nursing education and its contribution to the teaching design]. AB - The role of nursing is studied, as it is related to constant innovation and improvement of teaching quality, social requirements and new professional roles. As an alternative, the contribution given by the teaching model is considered. Such model offers educational bases on leisure activities about adequate learning environments, user population requirements and the actual educational system. Favourable results of the proposed innovation will need continuous evaluation shared by the faculty as a means to guarantee the consolidation of this exchange program. PMID- 10734946 TI - [Analysis of teaching practices of university professors in the health field]. AB - The study aims to describe teaching practices--in planning, execution and evaluation of the teaching-learning process--used by faculty of some courses in the health field in Brazil: Odontology, Medicine, Nursing, Pharmacy, Psychology, Nutrition, Social Work and Veterinary Medicine. The sample was made up by 29 faculties enrolled in a didactic subject course at graduate programs. They answered a questionnaire with semi-open questions. The answers obtained point to a "banking" education practice, according to Paulo Freire. This kind of education is characterized by: 1) emphasis on the transmission of information; 2) many hours of education; 3) little integration between courses and 4) lecture classes. PMID- 10734947 TI - [Teaching at the secondary level and professional practice in the context of Brazilian nursing]. AB - The formation of nursing professionals has been a concern through nursing history in our country. Within the context of development of nursing history, authors analysed the evolution of the legislation about the formation of nursing professionals at the secondary level as well as the legislation about their practice. PMID- 10734948 TI - [Care of hospitalized children: reflexions about the participation of parents]. AB - The care of a child admitted in the hospital is going through meaningful changes and, in a more actual approach, the main strategy seems to stimulate parents or relatives to stay with their children in the hospital. The purpose of this study is to analyse parents' participation in the process of staying in the hospital with their children. We have chosen the qualitative method and used as a theoretic--methodological reference the Model of Technological Work Organization. Based on the process of work, we have pointed out that bringing mothers to the ward is not so simple as it interferes in the reorganization of the work at the theoretical and practical levels. Concepts such as co-operation and partnership are built, however, sharing knowledge, power and room is not a simple action. It implies in changes of values and postures by parents and professionals. PMID- 10734949 TI - [Knowledge and opinions of a group of adolescents about the prevention of AIDS]. AB - This descriptive study was developed in a State School, located in the city of Natal-RN, with the purpose to identify the knowledge and opinion of a group of adolescents about AIDS prevention. 30 questionnaires were applied and among the main obtained results, the authors found out that the adolescents are not well informed regarding this disease. Among the mentioned prevention methods they found the use of condom, but not in a systematic way by 50% of the population studied. PMID- 10734950 TI - [Application of the nursing process based on Orem's theory: a case study with a pregnant adolescent]. AB - This is a descriptive study with a qualitative approach that aimed at applying the nursing process based in Orem Self-Care Theory, through a case study with a pregnant adolescent in order to identify the nursing diagnosis in the above mentioned clients, based on NANDA'S nursing diagnoses. Results obtained identified three nursing diagnoses: prejudiced adaptation, sleep disturb and familiar change process. The application of the nursing process based in Orem and the importance of the diagnosis identified for clients nursing care were evidenced. PMID- 10734951 TI - [Evaluation of the severity of clinical illness: a psychophysical approach]. AB - The severity of clinical illness was evaluated through the psychophysics method of magnitude estimation. The purpose of this experiment was: 1) to compare the scales derived from right judgement (magnitude estimation) used in three different Brazilian samples; 2) to verify the stability and agreement of severity estimations of clinical situations judged in Brazil, USA and England. In order to develop this study, professionals who work in the areas of psychology, nursing and medicine were invited. The lower coefficient of Pearson's correlation among the groups were of 0.88 the and the greater of 0.94. The exponents found between the Brazilian and American samples were of 1.22 and between the Brazilian and English samples of 1.15. PMID- 10734952 TI - [Voice rehabilitation of laryngectomized patients: cultural characteristics in the process]. AB - The objective of this study was to identify the characteristics of the process of vocal rehabilitation among laryngectomees focusing on culture. The study included four laryngectomees who attend the GARPO-Laryngectomees (Support Group For The Rehabilitation of Ostomized Patients--Laryngectomees) and communicate with esophageal or pharyngeal voice. The theme that emerged from the data was "to be born again", in agreement with the cultural value of the voice in our society. The study revealed as well the difficulties of the process, the importance of family participation and the stigma of the altered body image. PMID- 10734953 TI - [Educational activities on the prevention of AIDS in a municipal basic health unit: participation of a nurse]. AB - This is an exploratory descriptive study with a qualitative approach that had as its purpose to characterize the educational activities of the nurses that work in the prevention and control program for sexually transmittable diseases and AIDS in the municipality of Natal, RN. The study ws conducted in the basic health care units with 10 (ten) nurses that implemented educational activities in AIDS prevention. A structured interview was used and content analysis techniques were applied to the data. It was identified that the majority of the nurses understood health education as the transmission of information and that the AIDS prevention program had insufficient actions, lacked structure in the service settings and institutional support by the Municipal Secretariat of Health. The program activities were characterized as traditional limited, sporadic, and lacking human and material resources. PMID- 10734954 TI - [Pulse oximetry for the evaluation of oxygen transport in patients in critical condition]. AB - In this clinical investigation 35 patients under mechanical ventilation were studied. It was possible to establish the precision of two pulse oximeters of different brands. The performance of these equipments was evaluated by comparing data with the hemo-oximeter and a statistical analysis employed the student t test. Results showed that bias between oximeters reading and hemo-oximeter was similar for both instruments. Eventually critical patients may present conditions that limit the use of this technique. The study showed that the discrepancy in the results observed may be attributed to the presence of the methemoglobin and possible to anaemia associated to hypoxia. PMID- 10734955 TI - [Available electronic journals that can contribute to the dissemination of health education]. PMID- 10734956 TI - [Practice of undergraduate nursing students for the prevention and treatment of pressure ulcers]. PMID- 10734957 TI - [Characteristics of stress in undergraduate nursing students]. PMID- 10734958 TI - [Let us initiate research]. PMID- 10734959 TI - [Research in the nursing field]. PMID- 10734960 TI - [Paramedical research: viewpoint of hospital management]. PMID- 10734961 TI - [Results of seven years of nursing research at the Public Assistance Hospital of Paris]. PMID- 10734962 TI - [Research and the nursing project of the establishment]. PMID- 10734963 TI - [The Huriet law and the rules for biomedical research]. PMID- 10734965 TI - [Is nursing research obvious?]. PMID- 10734964 TI - [The hospital nurse and biomedical research]. PMID- 10734966 TI - [Nursing research: the point of view of the Association for Nursing Research]. PMID- 10734968 TI - [Practitioner-researcher, a new dimension of knowledge]. PMID- 10734967 TI - [Documentation in nursing care]. PMID- 10734969 TI - [M.a.n.a.g.e.m.e.n.t]. PMID- 10734970 TI - [Standards of competence for nursing and medical technology staff]. PMID- 10734971 TI - [Reflexion: the nurse in the project culture]. PMID- 10734972 TI - [Pedagogy: representation(s) and education in nursing care]. PMID- 10734973 TI - [The three educational programs: nurses' aides, nurses, nursing staff]. PMID- 10734974 TI - [Nursing education in Italy]. PMID- 10734975 TI - [Revision of the regulation from 15 March, 1993]. PMID- 10734976 TI - [The beginnings of Nursing Convergence upsets the landscape of French nursing unions. Interview by Anne Boiteux]. PMID- 10734977 TI - [The National Network of Hospital Documentalists. A network to discover]. PMID- 10734978 TI - [A new regulation for an "unique" competence]. PMID- 10734979 TI - [Writing, reading, caring]. PMID- 10734980 TI - [The challenge of writing]. PMID- 10734981 TI - [Writing and imagination]. PMID- 10734982 TI - [A writing studio]. PMID- 10734983 TI - [Writing as a mediator between life and the history of life in education]. PMID- 10734984 TI - [Writing, fragile sweet lure...]. PMID- 10734985 TI - [Teacher-tutor, what is the meaning of that function?]. PMID- 10734986 TI - [Accompanying the nurses' aides during their process of professionalization]. PMID- 10734987 TI - [ Multidisciplinary teaching in traumatology-orthopedics]. PMID- 10734988 TI - [Nurses' evaluation in the complex psychiatric milieu]. PMID- 10734989 TI - [Working conditions and operational thinking]. PMID- 10734990 TI - [Comparison of three bibliographic data bases]. PMID- 10734991 TI - [Hospital medical records technicians, working conditions]. PMID- 10734992 TI - [Writing a professional article]. PMID- 10734993 TI - [Regulatory facts]. PMID- 10734994 TI - [The legal responsibilities of nurses during their work]. PMID- 10734995 TI - [The Committee for Cooperation of Nursing and Staff Education is celebrating its 50th anniversary]. PMID- 10734997 TI - [Honor sports]. PMID- 10734996 TI - [Experiencing ones death is the ultimate moment of personal freedom. Interview by Bernadette Fabregas]. PMID- 10734998 TI - [The quality of life in hospitals as judged by the French]. PMID- 10734999 TI - [Sexuality and advanced age]. PMID- 10735000 TI - [Sexuality and emotions of the aged person]. PMID- 10735001 TI - [Survey of sexuality and emotionality in persons under long-term care]. PMID- 10735002 TI - [Sex behavior problems in the aged]. PMID- 10735003 TI - [The right to love until the end of life]. PMID- 10735004 TI - [Drug poisoning in the aged]. PMID- 10735005 TI - [The family of sugars of variable action]. PMID- 10735006 TI - [The aging of the skin]. PMID- 10735007 TI - [Dementia, relations, dignity, a special case based on the life of Marie-Laure]. PMID- 10735008 TI - [The chart of rights and liberties of the dependent aged]. PMID- 10735009 TI - [When home care for the aged person is no longer possible]. PMID- 10735010 TI - ["Animation" in the geriatric environment?]. PMID- 10735011 TI - An end-game for cancer. PMID- 10735012 TI - Cancer statistics, 2000: a benchmark for the new century. PMID- 10735013 TI - Cancer statistics, 2000. AB - The Surveillance Research Program of the American Cancer Society's Department of Epidemiology and Surveillance Research reports its annual compilation of estimated cancer incidence, mortality, and survival data for the United States in the year 2000. After 70 years of increases, the recorded number of total cancer deaths among men in the US declined for the first time from 1996 to 1997. This decrease in overall male mortality is the result of recent down-turns in lung and bronchus cancer deaths, prostate cancer deaths, and colon and rectum cancer deaths. Despite decreasing numbers of deaths from female breast cancer and colon and rectum cancer, mortality associated with lung and bronchus cancer among women continues to increase. Lung cancer is expected to account for 25% of all female cancer deaths in 2000. This report also includes a summary of global cancer mortality rates using data from the World Health Organization. PMID- 10735014 TI - American Cancer Society guidelines for the early detection of cancer. AB - This issue of CA inaugurates a yearly report on American Cancer Society guidelines for early detection of cancer in asymptomatic individuals. The current recommendations, which reflect almost 20 years of updates, cover screening recommendations for breast, colorectal, prostate, and cervical cancers, as well as for other cancers, depending on patient age, history, environmental and/or occupational exposures, etc. A key concept for both the general public and health providers is the distinction between public health recommendations regarding screening and decisions about early detection tests that might be undertaken on an individual basis. Although it is likely that current screening protocols will be supplanted by newer technologies, such as genetic and molecular markers of risk and disease, greater utilization of the technologies at hand will improve efforts toward establishing an organized and systematic approach to early cancer detection. PMID- 10735015 TI - Differences in breast cancer prognosis among African-American and Caucasian women. AB - This review explores factors potentially contributing to the disparity in survival after breast cancer between African-American and Caucasian women in the United States. A number of factors have been implicated as the cause of poorer survival for black women, including clinical and pathologic features of the disease that are indicative of poor prognosis, economic resource inequities, and differences in treatment access and efficacy. The latter is explored in detail using data from the National Surgical Adjuvant Breast and Bowel Project (NSABP), a nationwide multicenter clinical trials group for breast and colorectal cancers. Key studies into the disparity in breast cancer survival are reviewed according to proposed principal determinants of poorer outcome for black women. Results among black and white women participating in several randomized NSABP clinical trials are also presented. Primary endpoints in those studies were clinical and pathologic disease characteristics at study entry, time to disease progression or new cancers, and total survival time after breast cancer diagnosis and treatment. In most studies reported in the literature, the primary explanatory factor alone, such as stage of disease at diagnosis, did not fully account for differences in outcome between groups; when additional factors were taken into account, however, prognoses became more similar. Results from the NSABP clinical trials similarly indicated that when stage of disease and treatment were comparable, outcomes for blacks did not differ markedly from those of whites. In summary, black women, diagnosed at comparable disease stage as white women and treated appropriately, tend to experience similar breast cancer prognoses and survival. However, important clinical and pathologic disease characteristics may continue to place certain women at increased risk of poorer outcome, and warrant continued study. The opportunity for increased clinical trial participation by black women is encouraged. PMID- 10735016 TI - Mental health as a National Health Priority Area: focus on depression. PMID- 10735017 TI - Complementary and alternative medicine: an educational, attitudinal and research challenge. PMID- 10735018 TI - Measles in an era of measles control. PMID- 10735019 TI - Complementary therapies: have they become accepted in general practice? AB - OBJECTIVES: To describe Victorian general practitioners' attitudes towards and use of a range of complementary therapies. DESIGN: A self-administered postal survey sent to a random sample of 800 general practitioners (GPs) in Victoria in July 1997. PARTICIPANTS: 488 GPs (response rate, 64%). MAIN OUTCOME MEASURES: GPs' knowledge; opinions about harmfulness and effectiveness; appropriateness for GPs to practise; perceived patient demand; need for undergraduate education; referral rates to complementary practitioners; and training in and practice of each therapy. RESULTS: Acupuncture, hypnosis and meditation are well accepted by the surveyed GPs, as over 80% have referred patients patients to practitioners of these therapies and nearly half have considered using them. General practitioners have trained in various therapies--meditation (34%), acupuncture (23%), vitamin and mineral therapy (23%), hypnosis (20%), herbal medicine (12%), chiropractic (8%), naturopathy (6%), homoeopathy (5%), spiritual healing (5%), osteopathy (4%), aroma-therapy (4%), and reflexology (2%). A quarter to a third were interested in training in chiropractic, herbal medicine, naturopathy and vitamin and mineral therapy. General practitioners appear to underestimate their patients' use of complementary therapies. CONCLUSIONS: There is evidence in Australia of widespread acceptance of acupuncture, meditation, hypnosis and chiropractic by GPs and lesser acceptance of the other therapies. These findings generate an urgent need for evidence of these therapies' effectiveness. PMID- 10735020 TI - Australian oncologists' self-reported knowledge and attitudes about non traditional therapies used by cancer patients. AB - OBJECTIVE: To assess Australian radiation and medical oncologists' self-reported knowledge about and attitudes towards a range of non-traditional therapies used by people with cancer. DESIGN: Postal survey during May and June 1997 of all 265 radiation and medical oncologists practising in Australia. PARTICIPANTS: 161 oncologists returned surveys (61% response rate). MAIN OUTCOME MEASURES: Oncologists' own level of knowledge, and, for each known therapy, their perceptions of its likely harm or benefit in patients being treated curatively and palliatively, and of the prevalence of use among their patients. RESULTS: Oncologists reported knowing most about acupuncture, antioxidant therapy and meditation and least about cellular therapy, magnetotherapy and psychic surgery. The therapies most likely to be considered helpful were meditation, acupuncture and hypnotherapy. Those most likely to be considered harmful were coffee enemas, psychic surgery, Iscador therapy and diet therapies. Perceptions of patients' use of most therapies varied widely, with herbal therapies, antioxidant therapy and meditation considered the most commonly used. CONCLUSIONS: These results indicate self-identified gaps in oncologists' knowledge about non-traditional therapies their patients may use; they suggest a need to consider including education about these therapies in oncologists' training. PMID- 10735021 TI - Enhanced measles surveillance during an interepidemic period in Victoria. AB - OBJECTIVE: To describe results of the first two years of enhanced measles surveillance in Victoria. DESIGN: Case series identified through enhanced measles surveillance. PARTICIPANTS AND SETTING: All measles cases notified to the Disease Control Section, Department of Human Services, Victoria, in 1997 and 1998. MAIN OUTCOME MEASURES: Proportion of notified cases laboratory confirmed as measles, rubella, or human parvovirus infection; identification of clusters (two or more linked cases of measles); and utility of the National Health and Medical Research Council clinical case definition for suspected measles. RESULTS: Rates of laboratory testing of notified cases improved after introduction of a paediatric phlebotomy service in July 1997, from 21 of 90 notified patients (23%) in the preceding six months, to 258 of 317 notified patients (81%) between July 1997 and December 1998. Of the 317, only 19 (6%) were laboratory confirmed with measles, while a further 26 (8%) were laboratory confirmed with human parvovirus infection (18) or rubella (8). Three clusters of measles, involving 11 cases, were identified during 1998. Use of the NHMRC case definition did not greatly improve the positive predictive value for diagnosis of measles above that of notification alone (14% versus 8%). CONCLUSIONS: Circulation of measles virus in Victoria in 1997 and 1998 appeared minimal. In this interepidemic period most notified cases of measles were not measles; to identify true cases, surveillance during an interepidemic period must include laboratory testing of notified cases. PMID- 10735022 TI - The epidemic of asthma: too much allergen or not enough infection? AB - Asthma has generally been thought to result from exposure to allergens in infancy leading to atopy, and eventually to airway hyperresponsiveness. There is now evidence that implicates absence of childhood infections as a factor in development of asthma. Childhood infections seem to be important in normal maturation of the immune system, with asthma a manifestation of a persistent "immature" immune system. PMID- 10735023 TI - Cough in children. AB - Cough is a common presenting problem in children. A persistent moist or productive cough is always abnormal. The history, examination, chest x-ray and spirometry usually provide clues to specific causes of cough. Non-specific cough, defined as a non-productive or dry cough in the absence of identifiable respiratory disease, is poorly understood. We briefly discuss major controversies in non-specific cough and its management in children. PMID- 10735024 TI - Psychological impact of genetic testing for adult-onset disorders. An update for clinicians. AB - Testing for gene mutations that confer susceptibility to adult-onset disorders has potential benefits, but these must be balanced against the psychological harms, if any. We review published findings on the psychological effects of such testing, focusing on Huntington's disease, which has the most available data, and the hereditary cancer syndromes. Most of the evidence suggests that non-carriers and carriers differ significantly in terms of short-term, but not long-term, psychological adjustment to test results. The psychological impact of genetic testing depends more on pretest psychological distress than the test result itself. PMID- 10735025 TI - Evidence-based health policy-making, hospital funding and health insurance. AB - An important goal of health services research is to improve the efficiency and effectiveness of health services through a quantitative and evidence-based approach. There are many limitations to the use of evidence in health policy making, such as differences in what counts as evidence between the various disciplines involved, and a heavy reliance on theory in social science disciplines. Community and interest group values, ideological positions and political assessments inevitably intrude into government health policy-making. The importance of these factors is accentuated by the current absence of evidence on the impact of policy options for improving the health status of the community, and ensuring that efficiency and equity objectives for health services are also met. Analysis of recent hospital funding and private health insurance initiatives shows the limited role of evidence in the making of these decisions. Decision making about health policy might be improved in the future by initiatives such as greater exposure of health professionals to educational inputs with a policy focus; increased contribution of doctors to health services research via special postgraduate programs; and establishing a national, multidisciplinary centre for health policy research and evaluation. PMID- 10735026 TI - Chappel v Hart: the High Court considers causation of damage from a surgeon's negligent failure to warn. AB - In Chappel v Hart, the High Court upheld the decision of the Supreme Court of New South Wales that a surgeon was liable for failing to warn a patient of a previously unreported complication of a procedure. The patient, if properly warned, would have had the operation at another time, probably with a more experienced surgeon. The decision has important implications for doctors when advising patients of the risks of surgery. PMID- 10735027 TI - Disasters, the media and doctors. AB - Disasters and mass casualty incidents are traumatic events that are followed by a predictable media response. Managing the media response appropriately can avoid problems in a hospital's security, command, control and communications structures and mitigate the stress of hospital staff attempting to deal with the incident. PMID- 10735028 TI - Hyperbaric oxygen for carbon monoxide poisoning. PMID- 10735029 TI - Computed tomography screening for coronary disease. PMID- 10735030 TI - Hepatitis A in injecting drug users: a national problem. PMID- 10735031 TI - Outbreak of hepatitis A among illicit drug users in southeast Sydney. PMID- 10735032 TI - An epidemic of reactive arthritis in Vanuatu: a descriptive study. PMID- 10735033 TI - A complication of sclerotherapy. PMID- 10735034 TI - Effect of estradiol and progesterone on oviductal LH-receptors and LH-dependent relaxation of the porcine oviduct. AB - We have previously shown that the porcine oviduct possesses immunoreactive and functional LH receptors and that LH causes relaxation of the oviduct, especially during the periovulatory stage of estrous cycle. The current studies were undertaken to investigate the effects of estradiol and progesterone on LH receptor protein and LH-stimulated motility of the oviduct in steroid-primed ovariectomized gilts. Twenty-one cross-bred gilts were ovariectomized at 6 m.o. of age. Four weeks later gilts received daily intramuscular injection of either 2 mL corn oil (control n = 4), estradiol benzoate (EB) 1.5 mg (n = 6), progesterone 50 mg (n = 5), or 1.5 mg EB plus 50 mg progesterone (n = 6) for 4 consecutive days. The gilts were slaughtered on Day 5 after the first injection of steroids or vehicle. Rings of isthmus and ampulla were collected from each oviduct and placed in a tissue chamber perfused with Kreb's solution for 60 min. The mechanical activity was recorded for 30 min after LH treatment. Immunoreactivity of LHR in the Fallopian tube sections were detected in the epithelium of the tubal mucosa, smooth muscle cells and the blood vessel endothelium. Western blotting showed that porcine oviducts contain 75, 48 and 45 kDa immunoreactive LH receptor proteins, like the corpus luteum (CL). The lowest receptor expression was found in controls and in gilts treated with estradiol or progesterone. Combined treatment with estradiol and progesterone resulted in a significant increase of LH receptor protein concentrations when compared with control animals. In vitro LH treatment affected oviduct contractility of combined estradiol and progesterone treated gilts but not the oviduct of the remaining groups. It also caused a decrease in amplitude, frequency and areas under the curve (AUC) of ampulla (P < 0.05) and the amplitude and AUC of isthmus (P < 0.001) in combined estradiol and progesterone-primed gilts. These results indicate that estradiol and progesterone together, but not separately, increase LH receptor protein in the porcine oviduct and that combined estradiol and progesterone priming is necessary for LH-induced relaxation of the porcine oviduct. PMID- 10735035 TI - Ultrasonographic study of ovarian function during early pregnancy and after parturition in the ewe. AB - Transrectal ultrasonography of ovaries was performed in 11 ewes from Days 10 to 26 and on Day 30 of pregnancy to record the number, size, and position of ovarian follicles > or = 3 mm in diameter and corpora lutea (CL). Transrectal and/or transabdominal ultrasonography of the uterus was performed on Days 10 to 26, 30, 35, 40, 45 and 50 of gestation to ascertain the number and position of the conceptuses. In a second experiment, ultrasonography was conducted in 15 ewes on Days 10, 25, 30, 45 and 50 of pregnancy and from Days 13 to 29 after parturition. Ovarian data were classified into ovaries without CL (Group 1), ovaries with the CL in 1 ovary (Group 2), and ovaries with the CL in both ovaries during pregnancy (Group 3). In early pregnant ewes, the total number of follicles and the diameter of all follicles > or = 3 mm were smaller (P < 0.05) in the CL-bearing ovaries (both Group 2, n = 7 and Group 3, n = 8) than in the non-CL-bearing ovaries (Group 1, n = 7), while the largest follicle diameter was significantly smaller in Group 3 than in Group 1 or 2 ovaries. The number of 3-mm follicles in Group 2 ovaries was lower (P < 0.05) than in Group 1 or 3 ovaries, but the mean number of follicles > or = 5 mm in diameter was significantly lower in Group 3 than in Group 1. The total luteal volume per ovary was higher (P < 0.001) in Group 2 than in Group 3 ovaries of early pregnant ewes. The total follicle diameter and the number of follicles growing from 3 to > or = 5 mm in diameter was lower (P < 0.05) for Group 2 ovaries of ewes that carried twins (n = 3) compared with Group 2 ovaries from ewes with singletons (n = 4). There were no differences in follicular dynamics between Group 3 ovaries and the ovaries of Group 2 in ewes that carried twins. No follicles > 3 mm were seen in the ovaries of postpartum ewes that contained CL during gestation, until Days 21 and 25 postpartum for Groups 2 (n = 10) and 3 (n = 8), respectively, and follicles reaching > or = 5 mm in diameter were detected in only 2 ovaries (Group 2), on Days 27 and 28 postpartum, respectively. We conclude that during early pregnancy in ewes there is a suppression of antral follicle growth which appears to be exerted primarily by the developing conceptus but remains confined to CL-bearing ovaries. Residual local inhibition of follicular development extends into the postpartum period. PMID- 10735037 TI - Waves of follicle development during the estrous cycle in sheep. AB - The pattern of ovarian follicle development in maiden cyclic lambs was characterized using the definition of a follicle wave as the changes in the number of follicles among the days of the estrous cycle, as originally defined in cattle by Rajakoski in 1960. We also examined the steroid content relationships among follicles on Days 5 (Wave 1) and 14 (Waves 2 and 3) of the estrous cycle. In Experiment 1, the ovaries of 20 cyclic lambs (40 to 45 kg) were examined daily using transrectal ultrasonography for 1 or 2 estrous cycles (n = 31 cycles). The number of small (2 and 3 mm in diameter), medium (4 and 5 mm) and large (> or = 6 mm) follicles were aligned with the beginning and end of the average length estrous cycle and then compared among days. Identified follicles were defined as those that grew to > or = 4 mm and remained at > or = 3 mm for > or = 3 d. The number of identified follicles emerging (retrospectively identified at 2 or 3 mm) per ewe per day was also aligned with the average length estrous cycle. In Experiment 2, ewe lambs were ovariectomized on Day 5 (n = 6) or 14 (n = 5) of the estrous cycle, then follicle diameters and follicular fluid concentrations of estradiol and progesterone were compared among follicles. Data were analyzed by repeated measures ANOVA and compared among days using Fisher's LSD. In Experiment 1, either 2 (n = 10 cycles), 3 (n = 20 cycles) or 4 (n = 1 cycle) periods of emergence of identified follicles occurred during individual cycles, with estrous cycle lengths of 15.6 +/- 1.6, 16.1 +/- 1.1 and 17 d respectively. In animals with 2 or 3 periods of emergence of identified follicles, the total number of small, medium and large follicles differed (P < 0.05) among days of the estrous cycle showing a wave-like pattern. In Experiment 2, a single follicle collected on each of Days 5 and 14 of the cycle (6.2 +/- 0.2 and 3.9 +/- 0.2 mm in diameter) had a higher (P < 0.05) concentration of follicular fluid estradiol (36.2 +/- 4.4 and 50.9 +/- 21.6 ng/mL) than other follicles collected on the same day (next largest follicle: 4.3 +/- 0.3 and 3.5 +/- 0.4 mm; 4.3 +/- 0.9 and 18.2 +/- 6.7 ng/mL estradiol). The results showed that 1) there was a synchronous emergence of follicles associated with fluctuations in the number and size of follicles during the estrous cycle; 2) within a wave there was a hierarchy among follicles for diameter and steroid content; 3) ovarian follicle growth in ewe lambs occurred in 2 or 3 organized waves during the estrous cycle. PMID- 10735036 TI - Prolonging the MGA-prostaglandin F2 alpha interval from 17 to 19 days in an estrus synchronization system for heifers. AB - Our objective was to determine whether extending the interval from 17 to 19 d between removal of melengestrol acetate (MGA) feed and administration of PGF2 alpha would alter conception rates, pregnancy rates and the degree of synchrony in replacement beef heifers. A commercial heifer operation in north-central Kansas purchased 591 Angus x Hereford heifers from 12 sources. Prior to the spring breeding season, 14% of the heifers were culled. The remaining heifers were assigned randomly to 2 MGA-PGF2 alpha synchronization systems. All heifers were fed MGA (0.5 mg/head/d) for 14 d, and PGF2 alpha was administered either 17 or 19 d after the completion of MGA feeding. Heifers were inseminated artificially for 30 d followed by 30 d of natural mating. Based on each source, first-service conception rates ranged from 66 to 90%, whereas overall pregnancy rates ranged from 91 to 100%. Heifers given PGF2 alpha on Day 17 after MGA had first-service conception rates of 75.9% compared with 81.4% for heifers receiving PGF2 alpha on Day 19. In response to the PGF2 alpha injection, 99% of the Day 19 heifers that were detected in estrus were inseminated artificially by 72 h after the PGF2 alpha injection, whereas 74% of the heifers in the Day 17 treatment were inseminated by that time. Average interval to artificial insemination (AI) after PGF2 alpha was greater (P < 0.01) for the Day 17 heifers (73.1 +/- 1.1 h) than for the Day 19 heifers (56.2 +/- 1.1 h). No differences in conception rates or overall pregnancy rates occurred; however, heifers receiving PGF2 alpha on Day 19 after MGA had shorter intervals to estrus, and a greater proportion was inseminated within 72 h after PGF2 alpha, thus possibly facilitating successful timed insemination of the remaining heifers not yet inseminated by that time. PMID- 10735038 TI - Monosaccharides are not detected in whole or isthmic bovine oviductal fluid collected throughout the estrous cycle, as analyzed by HPLC. AB - In the bovine oviduct, monosaccharides may play a role in the preparation of gametes for fertilization. Sperm are sequestered in the isthmic region of the oviduct where capacitation, requisite biochemical changes in sperm membranes, may take place. Retention of spermatozoa in the oviductal isthmus is dependent on a carbohydrate recognition system between oviductal epithelium and sperm membrane lectins. The monosaccharide, fucose, has been found to be important to this recognition system. However, both gametes and epithelium are also bathed in oviductal fluid (ODF), and fucose or other monosaccharides may be constituents of ODF and so may be important to sperm binding to oviductal epithelium and subsequent preparation for fertilization. In this study, ODF from dairy cows was analyzed by HPLC for the presence of 5 monosaccharides (fucose, galactose, glucosamine, mannose and xylose). Both whole ODF, collected by cannulation of the entire oviduct of 1 cow over a complete estrous cycle, and regional staged ODF, collected and pooled from 13 cows from the isthmic region only at estrus, were analyzed. We report negligible concentrations of all 5 monosaccharides in both types of ODF analyzed. Because the detection limit of our assay was 10(8) times lower than fucose concentrations found to be physiologically important in earlier in vitro studies, we conclude that bovine ODF does not contain physiologically active levels of free fucose or other, similar monosaccharides at any time of the estrous cycle. PMID- 10735039 TI - Effect of mating system, flushing procedure, progesterone dose and donor ewe age on the yield and quality of embryos within a MOET program in sheep. AB - Multiple ovulation and embryo transfer (MOET) has the potential to increase the rates of genetic improvement in sheep. However, better realization of this potential requires a higher yield of transferable embryos. Thus we investigated some factors that may contribute to high embryo yield and quality under field conditions, as part of an ongoing MOET program. Comparison of the effects of 2 breeding systems (natural service plus laparoscopic intrauterine AI vs natural service only) on embryo yield and quality indicated that while AI did not affect embryo recovery, it significantly (P < 0.05) improved fertilization rate and embryo quality, and increased (P < 0.05) embryo survival rate after transfer to recipients. Two flushing procedures (the original semi-laparoscopic and a modified version) were compared for effects on embryo recovery. The modifications made to the original collection method increased (P < 0.001) embryo recovery from 69.0 +/- 2.4 to 83.2 +/- 0.6%. The effects of the progestagen priming dosage during superovulatory treatment and ewe age on MOET outcome were also investigated. Donor ewes primed with 30-mg progestagen sponges came into estrus 1.9 h earlier (P < 0.05) than those primed with 45-mg sponges, but there was no difference in ovulation rate or embryo recovery, or in embryo survival after transfer between the 2 regimens. However, Chi-square analysis indicated a significant benefit in favor of the higher progesterone dose on both fertilization (P < 0.01) and embryo quality (P < 0.001). Age of donor ewe did not significantly affect the timing of estrus, fertilization rate or embryo survival after transfer. While adult ewes had higher (P < 0.05) ovulation rates and embryo yields, shearling ewes produced a much higher proportion of Grade 1 embryos (P < 0.05). PMID- 10735040 TI - Estrogen and progesterone receptors in the ovine cervix during the postpartum period. AB - Cervical estrogen (E) and progesterone (P) receptors were characterized and quantified during the postpartum period in Corriedale ewes lambing in the late breeding season. Cervices and uteri were collected after ovariohysterectomy at 1 d (n = 2), 5 d (n = 4), 17 d (n = 2) or 30 d (n = 2) post partum. The estrogen and progesterone receptors were measured using binding assays with tritiated hormones, dextran charcoal separation and inverse Scatchard analysis. Similar kinetic parameters in cytosolic binding sites for both hormones were found in all cervical and uterine samples, indicating that the binding protein in both tissues is of the same nature. Receptor concentrations (fmol/mg cytosolic protein) in the cervix of early (1 to 5 d, n = 6) and late (17 to 30 d, n = 4) postpartum ewes were 348 +/- 66 vs 994 +/- 145 (P < 0.05) for E and 618 +/- 126 vs 1170 +/- 201 (P < 0.05) for P, respectively. These data suggest an increased synthesis of receptors, probably due to the presence of ovarian estrogen-active follicles. Cervical E and P receptor concentrations were similar or higher than those in the uterus (1.40 +/- 0.15, n = 10 and 1.51 +/- 0.19, n = 10; for E and P respectively), and these receptor ratios did not differ between the early and late postpartum period. The high ratio between cervical/uterine receptors suggests that the ovine cervix may be a very sensitive to steroid action. In conclusion, it was shown that restoration of steroid receptors during the postpartum period in the ovine cervix is similar to receptor dynamics in the uterus, and is probably associated with the recovery of ovarian cyclicity. PMID- 10735041 TI - Effect of alpha-tocopherol on plasma testosterone and plasminogen activator activity or inhibition in ram spermatozoa. AB - The objective of this study was to evaluate the effect of alpha-tocopherol on blood testosterone and specific proteolytic enzymes in spermatozoa and seminal plasma, with final aim the increase of sperm fertilizing ability with a nutritional supplement. The effect of alpha-tocopherol supplementation on testosterone parameters (mean value, basal level, peak number, mean concentration value during peaks, peak amplitude, peak duration) and plasminogen activator activity (PAA), plasminogen activator inhibition (PAI) and plasmin inhibition (PI) of spermatozoa and seminal plasma was studied in the ram during autumn (estrous period for the sheep in Greece) and spring (anestrous period). Treated animals showed a marked increase in serum alpha-tocopherol. Testosterone parameters were not affected by the alpha-tocopherol in either autumn or spring, however, the spermatozoal PAA and PAI (anti-tPA) were increased in the spring but not in autumn. These enzymes and their inhibitors are normally increased in autumn (the breeding season) and low in spring. If PAA can improve the fertilizing ability of spermatozoa in the spring, our finding may mean that a nutritional supplement, such as alpha-tocopherol, could provide rams for an accelerated onset of the breeding season in the ewe. PMID- 10735042 TI - The importance of having high glutathione (GSH) level after bovine in vitro maturation on embryo development effect of beta-mercaptoethanol, cysteine and cystine. AB - Supplementation of IVM medium with cysteamine, beta-mercaptoethanol, cysteine and cystine induced bovine oocyte glutathione (GSH) synthesis, but only the effect of cysteamine on the developmental competence of these oocytes was tested. During IVM of sheep oocytes, cysteamine but not beta-mercaptoethanol increased embryo development. However, it is not known how long the high intracellular oocyte GSH levels obtained after IVM with thiol compounds, can be maintained. Thus, the present study was carried out to evaluate the effects of supplementing maturation medium with 100 microM beta-mercaptoethanol, 0.6 mM cysteine and 0.6 mM cystine on 1) intracellular GSH level after IVM, 2) after IVF, 3) in 6 to 8-cell embryos and 4) on embryo development. In oocytes after IVM and in presumptive zygotes after IVF, intracellular GSH levels were significantly higher in the treated groups (P < 0.05). While, GSH content in 6 to 8-cell embryos was similar among treatment groups (P > 0.05). Differences in cleavage rates and the percentage of embryos that developed to morula and blastocyst stages were significantly higher (P < 0.05) for treated oocytes than for those matured in the control medium. We conclude from the results that the high intracellular GSH levels after induction of GSH synthesis in bovine IVM by thiol compounds remain during IVF and are still present at the beginning of IVC, improving developmental rates. Moreover, the results indicate that this metabolic pathway is an important component of the cytoplasmic maturation process that affects the subsequent steps of in vitro embryo production. PMID- 10735043 TI - Immunohistochemical detection of progesterone receptors in the canine uterus and their relation to sex steroid hormone levels. AB - The aim of this immunohistochemical study is to describe the normal distribution of progesterone receptors in the various cell types of the canine uterine horns, body and cervix. The results can be used for research on uterine and endocrinological pathology, since the impact of progesterone on different uterine cell types is partly determined by the receptor availability. Nuclear staining for progesterone receptors was observed in epithelial cells of the surface epithelium, glandular ducts and basal glands of the endometrium, in endometrial stroma cells and in myometrial smooth muscle cells. This staining was positively correlated with the estradiol-17 beta:progesterone ratio, and reflects the positive effect of estradiol-17 beta and the negative influence of progesterone on the receptors. Staining scores were high during proestrus and decreased through estrus to early metestrus. In late metestrus, staining scores of the stromal and smooth muscle cells increased again. In anestrus, high scores of the surface-epithelial cells contrasted with minimal scores of the basal glands. This finding suggests a different hormonal regulation of the progesterone receptor expression in both epithelial cell groups. The higher staining intensities for progesterone receptors in stromal cells compared with epithelial cells might be explained by the fact that stromal cells mediate some effects of steroid hormones on the epithelial cells in the genital tract. Therefore, the role of stromal cells in regulation of the cyclic endometrial changes and in pathologic changes of uterine tissue should not be underestimated. PMID- 10735044 TI - Effect of sperm diluents on the acrosome reaction in canine sperm. AB - In this study we investigated the influence of sperm diluting media and temperature on the incidence of the acrosome reaction in dog sperm. Ejaculates were collected from 5 dogs, diluted with six different media and then incubated at 37 degrees C and 20 degrees C. Fluorescein isothiocynate conjugated peanut agglutinin (FITC-PNA) and ethidium homodimer as a vital stain were used in combination to determine the acrosomal status of viable spermatozoa, the technique was validated using electron microscopy. The outer acrosomal membrane of dog spermatozoa was shown to be the specific binding site for FITC-PNA. After 6 h of incubation, ejaculates diluted in media with a high Ca2+ concentration showed a significantly higher percentage (means +/- SD) of acrosome reacted spermatozoa [64 +/- 7 and 58 +/- 9 in sperm capacitation medium with (SP-TALP-1) and without BSA (SP-TALP-2), respectively] than those diluted in media with a low Ca2+ concentration [36 +/- 5, 39 +/- 4, 18 +/- 2 and 20 +/- 4 in Canine Capacitation Medium (CCM), Egg Yolk Tris dog semen extender (EXT-1), Modified Egg Yolk Tris extender (EXT-2) and Modified CCM (MCCM), respectively]. The increase in the percentage of acrosome reaction (AR) was slower at 20 degrees C than at 37 degrees C. In addition, the percentage of viable acrosome reacted spermatozoa increased significantly from 19 +/- 5 and 22 +/- 3 in non-bound sperm to 27 +/- 4 and 30 +/- 6 in zona pellucida bound sperm (diluted in EXT-2 and MCCM, respectively). We conclude that the composition of the spermatozoa diluent has a marked effect on the incidence of the acrosome reaction. Therefore, both the media used to dilute dog sperm and the temperature at which the spermatozoa are handled are important factors to consider when processing spermatozoa for artificial insemination, IVF procedures or preservation. PMID- 10735045 TI - Effects of severity of dystocia on cold tolerance and serum concentrations of glucose and cortisol in neonatal beef calves. AB - Effects of dystocia on rectal temperature and serum cortisol and glucose concentrations, were studied in neonatal calves exposed to 0 degree C. Primiparous dams were observed continuously during parturition and if Stage II (labor) was not completed within 2 h after appearance of the allantochorion, delivery was completed with obstetrical assistance. Parturitions were scored (CDS) for difficulty and obstetric assistance required: CDS 1, no assistance (n = 8); CDS 2, minor manual assistance (n = 7); CDS 3, use of a mechanical calf puller (n = 5); CDS 4, cesarean section (n = 6). A blood sample, rectal temperature, and body weight were obtained within 30 min after birth. Calves were then fed 38 degrees C pooled colostrum, muzzled to prevent suckling, and placed back with their dam in a heated (22 degrees C) barn. At 4 h of age an indwelling jugular catheter was inserted. At 5 h of age calves were placed in a 0 degree C room for 140 min and blood samples and rectal temperatures were obtained every 10 or 20 min. A shivering score (1 = no shivering; 2 = moderate shivering; 3 = intense shivering) was assigned at each sampling time. Rectal temperatures were higher (P < 0.01) in CDS 1, 2 and 4 calves (39.0, 39.3, and 39.0 +/- .02 degrees C, respectively) than in calves with CDS 3 (38.3 +/- 0.02 degrees C) and were affected by duration of cold exposure (time; P < 0.01). Shivering was not affected by CDS but was affected by time (P < 0.01). Glucose concentrations were higher (P < 0.01) in CDS 3 calves (110.1 +/- 1.6 mg/dL) than in CDS 1, 2, or 4 calves (77.2, 86.4, and 89.0 +/- 1.3 mg/dL, respectively) and changed over time (P < 0.01). Cortisol concentrations were higher in CDS 1 calves (80.0 +/- 1.7 ng/mL) than in CDS 2, 3 or 4 calves (62.7, 58.2, and 57.7 +/- 2.0 ng/mL, respectively) and were affected by time (P < 0.01). We conclude that severe dystocia (CDS 3) resulted in lower calf rectal temperature, reduced serum cortisol, and increased serum glucose which could affect the ability of the calf to withstand cold stress. Minor dystocia did not cause and timely cesarean delivery prevented, the physiological aberrations encountered in severe dystocia. PMID- 10735046 TI - Comparison of synchronization of ovulation and induction of estrus as therapeutic strategies for bovine ovarian cysts in the dairy cow. AB - The benefit of using timed-insemination in lactating dairy cows for the treatment of ovarian cysts lies in the fact that cows do not have to be detected in estrus for insemination and achieving pregnancy. We compared the effectiveness of synchronization of ovulation with timed-insemination and induction of estrus with insemination at estrus in the treatment of bovine ovarian cysts in lactating dairy cows. After Day 65 post partum, a total of 368 lactating dairy cows was divided into 3 groups. Cows in Group 1 (n = 209, normal, noncystic) were treated with 100 ug, i.m. GnRH on Day 0; 25 mg, i.m. PGF2 alpha on Day 7; and 100 ug, i.m. GnRH on Day 9 and then were time-inseminated 16 h later. Cows in Group 2 (n = 76, abnormal, cystic) were treated with 100 ug, i.m. GnRH on Day 0; 25 mg, i.m. PGF2 alpha on Day 7; and 100 ug, i.m. GnRH on Day 9 and time-inseminated 16 h later. Cows in Group 3 (n = 83, abnormal, cystic) were treated with 100 ug, i.m. GnRH on Day 0; 25 mg, i.m. PGF2 alpha on Day 7; and inseminated at induced estrus within 7 d after treatment with PGF2 alpha. Day 0 was the day of initiation of the study. Conception and pregnancy rates among groups were compared using logistic regression and adjusted for parity, time of year and days in milk. Conception and pregnancy rates of Group 1 cows (31.5%) were not significantly different from those of Group 2 cows (23.6%). However, the pregnancy rate in normal cows (Group 1) was higher (P < 0.01) than in cystic cows (Groups 2 and 3). Cows in Group 3 had a higher conception rate than cows in Group 2 (51.7% > 23.6%; P < 0.01). However, pregnancy rates for cows in Groups 2 (23.6%) and 3 (18%) were not significantly different. The finding indicated that synchronization of ovulation and timed-insemination resulted in pregnancy rates similar to those of synchronization of estrus and insemination at an induced estrus within 7 d for the treatment of ovarian cysts in lactating dairy cows. PMID- 10735047 TI - Quality controls for bovine viral diarrhea virus-free IVF embryos. AB - Introduction of bovine viral diarrhea virus (BVDV) with cumulus-oocyte-complexes (COCs) from the abattoir is a concern in the production of bovine embryos in vitro. Further, International Embryo Transfer Society (IETS) guidelines for washing and trypsin treatment of in-vivo-derived bovine embryos ensure freedom from a variety of pathogens, but these procedures appear to be less effective when applied to IVF embryos. In this study, COCs were exposed to virus prior to IVM, IVF and IVC. Then, virus isolations from cumulus cells and washed or trypsin treated nonfertile and degenerated ova were evaluated as quality controls for IVF embryo production. The effect of BVDV on rates of cleavage and development was also examined. All media were analyzed prior to the study for anti-BVDV antibody. Two approximately equal groups of COCs from abattoir-origin ovaries were washed and incubated for 1 h in minimum essential medium (MEM) with 10% equine serum. One group was incubated in 10(7) cell culture infective doses (50% endpoint) of BVDV for 1 h, while the other was incubated without virus. Subsequently, the groups were processed separately with cumulus cells, which were present throughout IVM, IVF and IVC. Cleavage was evaluated at 4 d and development to morulae and blastocysts at 7 d of IVC. After IVC, groups of nonfertile and degenerated ova or morulae and blastocysts were washed or trypsin-treated, sonicated and assayed for virus. Cumulus cells collected at 4 and 7 d were also assayed for virus. Anti-BVDV antibody was found in serum used in IVM and IVC but not in other media. A total of 1,656 unexposed COCs was used to produce 1,284 cleaved embryos (78%), 960 embryos > or = 5 cells (58%), and 194 morulae and blastocysts (12%). A total of 1,820 virus-exposed COCs was used to produce 1,350 cleaved embryos (74%), 987 embryos > or = 5 cells (54%), and 161 morulae and blastocysts (9%). Rates of cleavage (P = 0.021), cleavage to > or = 5 cells (P = 0.026) and development to morula and blastocyst (P = 0.005) were lower in the virus-exposed group (Chi-square test for heterogeneity). No virus was isolated from any samples from the unexposed group. For the exposed group, virus was always isolated from 4- and 7-d cumulus cells, from all washed nonfertile and degenerated ova (n = 40) and morulae and blastocysts (n = 57) and from all trypsin-treated nonfertile and degenerated ova (n = 80) and morulae and blastocysts (n = 91). Thus, virus persisted in the system despite the presence of neutralizing antibody in IVM and IVC media, and both washing and trypsin treatment were ineffective for removal of the virus. Presence of virus in 4- and 7-d cumulus cells as well as in nonfertile and degenerated ova were good indicators of virus being associated with morulae and blastocysts. PMID- 10735048 TI - Embryonic origins of health: long-term effects of IVF in human and livestock. AB - Assisted reproduction technologies have been introduced 1) to overcome reproductive failures in the human, 2) to increase the number of offspring from selected females and 3) to reduce generation intervals in livestock in farm animals. The successful introduction of these technologies in clinical practice and in livestock breeding programs is the result of enormous scientific efforts. In general, the offspring generated by IVF (human) and IVP (cattle) is normal, but as numbers increase the restraints and drawbacks of new reproductive technologies become visible with respect to the overall efficiency as well as to the occurrence of abnormalities and/or anomalies in the offspring. The objective of the present symposium on "Embryonic Origins of Health" is to present "the state-of-the-art" and to discuss the restraints and possible long term effects of the application of assisted reproduction technology in both human and livestock. This introduction to the symposium focuses on the relation between early embryonic development and post-natal health. We hypothesize that IVF in the human and IVP in cattle influence the timing of cell-cell interactions during the early stages of embryogenesis which finally result in a incorrect timing of gene expression during the phylotypic stage and subsequent organogenesis. These deviations in embryonic timing might have consequence for the postnatal homeostasis and health. PMID- 10735049 TI - Postnatal health and welfare of offspring conceived in vitro: a case for epidemiological studies. AB - In vitro fertilization (IVF) has established itself as an important technique in human assisted reproduction and in livestock improvement. In both humans and livestock the possible long-term effects on health and welfare of offspring born after IVF and in vitro culture to the blastocyst stage are still largely unknown. Epidemiological studies in humans, using data collected for individuals born after normal (i.e. non-assisted) pregnancies, have provided evidence for associations between prenatal life events and adult-life disease. Due to the relatively short time that elapsed since the first IVF baby was born, comparable studies for IVF offspring are not yet possible. However, animal experiments and epidemiological studies with the available data from the livestock industry (mainly dairy cattle) may contribute to a better understanding of the risks involved. PMID- 10735050 TI - In utero programming of cardiovascular disease. AB - Low birth weight, thinness and short body length at birth are now known to be associated with increased rates of cardiovascular disease and non-insulin dependent diabetes in adult life. The fetal origins hypothesis proposes that these diseases originate through adaptations which the fetus makes when it is undernourished. These adaptations may be cardiovascular, metabolic or endocrine. They permanently change the structure and function of the body. Prevention of the diseases may depend on prevention of imbalances in fetal growth or imbalances between pre- and post-natal growth, or imbalances in nutrient supply to the fetus. PMID- 10735051 TI - Effects of different reproduction techniques: AI MOET or IVP, on health and welfare of bovine offspring. AB - Since the introduction of in vitro production (IVP) of bovine and sheep pre implantation embryos, increased birth weights and other deviations of IVP calves and lambs compared with AI or MOET offspring have been reported. Study 1 of the present paper, a comparison between AI, MOET and IVP (co-culture/serum) calves with respect to calf and calving characteristics in large-scale field conditions, confirms these reports. In addition, it is shown that MOET calves tend towards higher birth weights and have significantly longer gestations and more difficult calvings than AI calves. It is presently unknown if the effect of IVP is also observed later in life. In this paper, data on reproduction characteristics of bovine IVP co-culture/serum offspring are presented. Semen production--and non return data of one year old IVP bulls and superovulation-, AI- and OPU/IVP results of one year old IVP heifers are compared with those of one year old AI and MOET animals producing semen or embryos in the same time period. So far, there are no indications that the use of IVP is reflected in deviate reproduction characteristics of bovine IVP offspring. It has been suggested that use of co culture cells and serum during in vitro culture of bovine (and sheep) embryos may partially explain the increased birth weights and other deviations of bovine and sheep IVP offspring. Deletion of these factors in semi-defined culture media, e.g. Synthetic Oviductal Fluid (SOF), could result in more normal offspring. Study 2 investigates this hypothesis in both field conditions (Study 2a, comparing AI, IVP co-culture/serum and IVP SOF calves) and in semi-standardized conditions (Study 2b, comparing MOET, IVP co-culture/serum and IVP SOF calves at one herd). In Study 2a, although IVP SOF calves showed (non-significant) shorter gestations, easier calvings and lower percentages of perinatal mortality and congenital malformations than IVP co-culture calves, birth weights were not decreased. In Study 2b however, the difference between IVP co-culture and IVP SOF calves in birth weight and ease of calving was significant (P < 0.05), IVP SOF calves resembling MOET calves more. IVP calves differed significantly from MOET calves with respect to several physiological parameters, such as blood oxygen saturation level, heart beat frequency and some measures of the heart. In addition, in Study 2b, recipients receiving an IVP SOF embryo showed a more regular return to estrus than those receiving an IVP co-culture embryo. From Study 2 it can be concluded that using a semi-defined medium for in vitro culture (SOF) may improve characteristics of IVP calves born. PMID- 10735052 TI - Assisted reproductive technology (ART) in humans: facts and uncertainties. AB - Since the first in vitro fertilization (IVF) in human, the number of patients using Assisted Reproductive Technologies (ART) has increased tremendously. ART technologies have increased in number and their spectrum has also widened. The first IVF babies are now more than 20 years old. All the retrospective analyses have demonstrated that the obstetrical and pediatrical impact has not really affected single births. The main problems observed occur with multiple pregnancies, including high costs for the couples and for society. The decrease in the number of embryos transferred has improved the situation and moreover does not impair the final results. IntraCytoplasmic Sperm Injection (ICSI) is a more debatable and questionable technique with a real negative genetic impact. The main problem is chromosome abnormalities more specifically related to the sex chromosomes. The question of a systematic genetic work-up on the patients entering ICSI programs is discussed. No negative impact of cryopreservation has been demonstrated even though some controversy arises from time to time. Pre implantation Genetic Diagnosis (PGD) is now a interesting tool for patients carrying genetic defects. Blastocyst biopsy now has a future role in reproductive medicine. Gender selection through sperm sorting is also now a reality. As with the other developing bio-technologies related to reproduction, there are ethical questions. The decisions concerning these technologies do not belong solely to scientists but are rather a matter for society to decide. PMID- 10735053 TI - Environment of oocyte and embryo determines health of IVP offspring. AB - In vitro embryo production (IVP) enhances the number of offspring from a single female and offers the possibility of accelerated genetic progress in animal husbandry. However, it also leads to a low but unacceptable percentage of anomalies in the offspring. The aim of this paper is to introduce the three speakers at this afternoon session who will speak about the demands of culture conditions and the endometrial environment to support normal embryonic development without effects on the embryonic genome. It will be argued that the in vitro conditions should mimic precisely the oviductal contributions to homeostatic mechanisms within the embryo. The further normal development can be guaranteed at synchrony in development of both endometrium and embryo. If that is not the case one can expect disturbances of gene expression, in particular of imprinted genes. However, it cannot be excluded that some processes might have started already in the cytoplasm of the oocyte. Since the oocyte was not planned to be a separate subject in this symposium, this introduction is also aimed to ask attention for the selection of cumulus-oocyte-complexes (COCs) and the conditions around oocyte maturation in vitro. The optimal quality of both the oocyte and maturation medium are prerequisites for an undisturbed cytoplasmic maturation. It has been argued that the exclusion of COCs from atretic follicles, the abjuration of the use of serum and high O2 tension in the gas phase might help to reduce the proportion anomalies in the offspring after synchronous transfers. In human IVF, in vivo matured oocytes are used with no great problems. But before IVP, including oocyte maturation in vitro (IVM) and a longer lasting embryo culture (IVC), will be introduced into the human assisted reproduction, it is important to think about the ins and outs of the potential causes for deviations. PMID- 10735055 TI - Improving the safety of embryo technologies: possible role of genomic imprinting. AB - Although developments in mammalian in vitro embryo technologies have allowed many new clinical and agricultural achievements, their application has been hindered by limitations in the developmental potential of resulting embryos. Low efficiencies of development to the pre-implantation blastocyst stage have been consistently observed in most species, including humans, rabbits, pigs and ruminants. Furthermore, in cattle and sheep a wide range of congenital abnormalities currently termed "Large Offspring syndrome" (LOS) are commonly observed as a result of several embryo culture and manipulation procedures. This paper reviews the hypothesis that at least some of the problems associated with embryo technologies may result from disruptions in imprinted genes. Several imprinted genes (i.e. genes which express only the maternal or paternal allele) are known to have significant effects on fetal size and survival in other species and are possible candidates for involvement in livestock LOS. Major changes in putative imprinting mechanisms such as DNA methylation of imprinted genes occur in the mouse embryo during pre-implantation development. Alterations in DNA methylation are stabley transmitted through repeated cell cycles such that changes in the embryo may still act at the fetal stages. Thus any disruption in establishment and/or maintenance of imprinting during the vulnerable periods of embryo culture or manipulation is a plausible candidate mechanism for inducing fetal loss and Large Offspring Syndrome. Identification of these disruptions may provide crucial means to improve the success of current procedures. PMID- 10735054 TI - Interactions between embryos and the culture milieu. AB - Although in vitro production of embryos up to the blastocyst stage is now possible in numerous species, the quality and quantity of embryos are still not satisfactory. Clearly, culture conditions do not yet replace all of the benefits of development within the female reproductive tract. Analysis of the interactions between embryos and the components of culture media provides insights into regulatory mechanisms and how they are perturbed in vitro, and also offers some clues about the nature of the support provided to early embryos by the female tract. Further elucidation of these events and their underlying regulation will be helpful for improving culture media formulations to support normal embryo development in vitro. PMID- 10735056 TI - The effects of the early uterine environment on the subsequent development of embryo and fetus. AB - Synchrony between the embryo and the uterine endometrium is essential for the establishment of pregnancy and birth in people and livestock. When asynchronous conditions occur a variety of complication result that include failure of the embryo to implant, early embryonic mortality, retarded development and growth, and accelerated development and growth. These complications all appear to be induced within the first week of embryo development and not withstanding the immediate endpoint of large or small size at birth, may alter the course of development throughout the life of the animal. Progesterone appears to play a causative role in establishing the abnormal growth of the fetus by decelerating or accelerating embryonic development. This may act through increasing the transport of blood born growth factors into the uterine lumen or by stimulating the release of growth factors from the endometrium directly. It can not be ruled out that progesterone mediated abundance of, or absence of, appropriate nutrition may bring about the same lifelong outcome. In vitro culture situations that include serum and/or co-culture can also bring about these abnormalities of growth. It is hypothesized that exposure to growth factors "out of phase" may result in an irreversible induction of abnormal development. The described abnormalities that occur in sheep and cattle have not yet been described for children resulting from IVF. PMID- 10735057 TI - Reflections on the golden anniversary of the first embryo transfer to produce a calf. AB - In the belief that the vitality of a discipline benefits from a knowledge of its roots, events surrounding the first embryo transfer to produce a live calf (by Willett and his colleagues in Wisconsin fifty years ago) have been reviewed. That first success is discussed in the context of those times (including a brief review of the first international conference on embryo transfer, held in Texas in 1949) and in relation to subsequent events that led to the founding of the International Embryo Transfer Society in 1974. PMID- 10735058 TI - Bovine follicular development and its effect on the in vitro competence of oocytes. AB - The current knowledge is reviewed concerning correlations between follicular development in the cow and the competence of matured oocytes to develop into an embryo following IVF and IVC. At the follicular size of 3 mm, some oocytes become competent and the proportion of competent oocytes does not increase during development up to 7 mm. The proportion of competent oocytes increases greatly in follicles > 8 mm in both untreated and gonadotropin-stimulated cows. The competence of in vitro-matured oocytes from these large follicles is lower than the competence of in vivo-matured oocytes. These observations lead to the following concept. Oocytes have acquired an intrinsic capacity to develop into an embryo after IVM-IVF-IVC at the follicular stage of 3 mm, but require an additional "prematuration" to express this competence. In vivo, this prematuration occurs during preovulatory development before the occurrence of the LH surge. In follicles of 3-7 mm, a low level of atresia appears to improve the in vitro competence of oocytes which may act via a prematuration-like effect. A thorough understanding, however, of the effect of atresia and other factors on the competence of this highly heterogeneous oocyte population is still missing. Two routes to improve the embryo yield in ovum pick-up (OPU) practice are discussed. PMID- 10735059 TI - Alterations of expression of developmentally important genes in preimplantation bovine embryos by in vitro culture conditions: implications for subsequent development. AB - Recent advances in molecular technology and in vitro production of bovine embryos have enabled studies of gene transcription in preimplantation embryos. On the basis of knowledge of the sequence of the selected gene, various modifications of Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) technology have been employed. Several lines of evidence in mouse and cattle indicate that expression patterns of genes from in vitro-produced embryos are not necessarily representative of those of in vivo embryos. An important gene that has been found to be expressed by in vivo-derived bovine blastocysts, but not in their in vitro produced counterparts, is the Connexin43 gene that is crucial for maintenance of compaction. The bovine leukemia inhibitory factor (bLIF) and LIF-receptor-beta (LR-beta) genes were expressed by in vitro-produced embryos, but not in their in vivo counterparts. The heat shock protein gene 70.1 (Hsp 70.1) was upregulated by blastocysts produced in vitro compared to in vivo embryos, while the glucose transporter-1 mRNA (Glut-1) was downregulated by morulae produced in vitro as compared to in vivo-derived morulae. Furthermore, mRNA expression levels of a set of "marker genes" were shown to be affected by the presence or absence of serum in the culture medium. Most embryos grown under serum-free conditions had higher mRNA abundances than those cultured in serum-enriched medium. It is hypothesized that persistent alterations of the normal gene expression patterns may be responsible for the large offspring syndrome that is observed in approximately one third of the calves resulting from the transfer of in vitro-produced embryos. A primary candidate for such deviations may be an altered methylation pattern that can either lead to silencing or induction of a specific gene. Messenger RNA phenotyping of genes essential in early development provides a useful tool to assess the normality of the produced embryos and a tool to optimize in vitro culture conditions for bovine embryos. PMID- 10735060 TI - Mitochondrial genotype segregation and effects during mammalian development: applications to biotechnology. AB - Mitochondria are endosymbiotic organelles responsible for energy production in practically every eukaryotic cell. Their uniparental fashion of inheritance, maternally inherited in mammals, and the homogeneity of mitochondrial DNA (mtDNA) within individuals and matrilineages, are biological phenomena that remain unexplained. This paper reviews some of the recent findings on mitochondrial influences on the manner in which embryos develop and how their genotypes are inherited in mammals, with particular emphasis on the genetic "bottleneck" effect. Animal models carrying a mix of mtDNAs (heteroplasmic) have been produced by karyoplast and cytoplast transplantation to analyze the segregation patterns at different stages during embryogenesis, in fetuses and offspring. Comparisons performed between murine and bovine reveal interesting changes in segregation and replication of transplanted mtDNAs. We have recently obtained Bos indicus and Bos taurus fetuses and calves from embryos reconstructed using enucleated polymorphic oocytes of Bos taurus origin. These and other findings on mitochondrial biology will have important implications in determining the cytoplasmic genotype of clones and in the preservation of endangered breeds and species. PMID- 10735061 TI - Fundamental aspects of sperm cryobiology: the importance of species and individual differences. AB - While semen cryopreservation is successfully used for a few species, application to other species can be problematic. Here, I argue that species differences in female tract anatomy, subtle differences in sperm transport mechanisms, ability to time inseminations and deliver spermatozoa effectively are powerful determinants of fertility with cryopreserved spermatozoa. Poor sperm survival represents one major aspects of the problem and determining biophysical characteristics of the sperm plasma membrane is an established approach to solving it. However, this approach is unable to account for the consistent differences in post-cryopreservation sperm quality between individual males, an effect that is recognized is many species although only documented in a few. Searching for genetic differences between these individuals might offer a genomically-based direction in semen cryopreservation research. Cryopreservation of spermatozoa and spermatogenic cells for intracytoplasmic sperm injection has been developed primarily to deliver an intact genome and presents a very different set of technical problems. PMID- 10735062 TI - Fundamental cryobiology of mammalian oocytes and ovarian tissue. AB - Embryo cryopreservation is a widely used and relatively well-established procedure. By contrast, ovarian tissue and unfertilized oocytes are only rarely cryopreserved, even though for germ line storage these often would be preferable to embryo cryopreservation. There are many reasons for this discrepancy. Unfertilized mature (MII) stage oocytes are more difficult to cryopreserve than cleavage stage embryos of the same species. Many factors contribute to this including the oocyte's surface to volume ratio, single membrane, temperature sensitive metaphase spindle and zona, and its susceptibility to parthenogenetic activation and chill-injury. A completely different set of problems applies to primordial follicles. Oocytes in primordial follicles are very small and tolerate cryopreservation by slow cooling very well. The problem lies in the difficulty in producing mature oocytes from these primordial follicles. Better and/or more convenient cryopreservation procedures for both oocytes and ovarian tissue are being developed. This paper describes some of the advances in this area and outlines the relative merits and limitations of several currently available egg and ovarian tissue cryopreservation procedures. PMID- 10735063 TI - Ultrasonography as an important tool for the development and application of reproductive technologies in non-domestic species. AB - Ultrasound imaging in reproductive sciences offers new opportunities regarding optimization of the induction of the sexual cycle and ovulation, superovulation regimes, contraception programs, semen collection and testicular sperm extraction techniques, ovum pick up and ovarian transplantation procedures, as well as the application of artificial insemination, embryo collection and transfer. In non domestic species, most of which lack basic data, ultrasonography is an ideal tool to study reproductive biology in both captive and wild populations. The use of this imaging modality led us to develop new, or modify established, reproductive technologies. Ultrasonography has been an integral part of over 200 assisted reproduction procedures in 17 mammalian species performed by our research team between 1992 and 1999. These procedures included the initial characterization of sexual cycles, hormonal cycle induction, semen collection by electroejaculation or manual stimulation, non-surgical artificial insemination (AI), non-surgical embryo transfer and temporary hormonal contraception. For these investigations, a variety of newly developed equipment was applied and species-specific hormonal treatments designed. We used several commercial and customized ultrasound systems with a variety of technical features. Some relevant improvements of these applications will be described and the role of ultrasonography elucidated to. PMID- 10735064 TI - Infectious agents in bovine embryo production: hazards and solutions. AB - Infectious agents in systems for producing bovine embryos might reduce the number and quality of embryos generated, result in transmission of disease to recipients and offspring, or confound findings of research. Embryo-associated pathogens might also jeopardize human health when the goal of embryo production is creating transgenic animals intended to be a source of pharmaceuticals or organs. This paper addresses risks and resulting hazards of pathogen and microbial contaminant introduction into in vivo or in vitro embryo production systems. Additionally, methods for prevention and quality control are discussed. PMID- 10735066 TI - Targeted modification of the domestic animal genome. AB - While the technique of homologous recombination, or gene targeting, has led to the generation of transgenic mice of great value to biomedical research, similar approaches are only being developed in other species. With the exception of recent reports on the generation of gene-targeted sheep, the technology in domestic animals is still in its infancy (45). The development of techniques for generating large animals with deleted or modified genes will result in the generation of animals of great value to society. While the technical difficulties to achieve gene targeting in domestic species are significant, they are not insurmountable. Potential applications in both the bovine and porcine species are described with particular emphasis on the generation of cattle resistant to bovine spongiform encephalopathy (BSE) and pigs that can be of use in xenotransplantation. PMID- 10735065 TI - Cellular and molecular events after in vitro fertilization and intracytoplasmic sperm injection. AB - Intracytoplasmic sperm injection (ICSI) has heralded an era of tremendous improvements in treating male infertility leading to the births of thousands of babies. However, recent concerns over possible long-term effects of ICSI on offspring has prompted the development of a preclinical, nonhuman primate model to assess the safety of ICSI. Fluorescent imaging of rhesus macaque IVF zygotes revealed that this species shares many similarities with humans in terms of cytoskeletal and chromatin dynamics during fertilization. However, rhesus monkey zygotes fertilized by ICSI resulted in abnormal nuclear remodeling leading to asynchronous chromatin decondensation in the apical region of the sperm head, delaying the onset of DNA synthesis. The persistence of the acrosome and perinuclear theca on the apex of sperm introduced into the oocyte by ICSI may constrict the DNA in this region. Despite these differences, normal rhesus monkey ICSI embryos have been produced and have lead to several births after transfer. The irregularities described in this paper raise concerns that the ICSI procedure may result in chromatin damage during DNA decondensation and further highlight the need for devising improved pre-clinical assessment prior to global acceptance of this, and other, novel methods of assisted reproduction. PMID- 10735067 TI - New advances in somatic cell nuclear transfer: application in transgenesis. AB - The ability to produce live offspring by nuclear transfer from cultured somatic cells provides a route for the precise genetic manipulation of large animal species. Such modifications include the addition, or "knock-in", and the removal or inactivation, "knock-out", of genes or their control sequences. This paper will review some of the factors which affect the development of embryos produced by nuclear transfer, the advantages of using cultured cells as donors of genetic material, and methods that have been developed to enrich gene targeting frequency. Commercial applications of this technology in biomedicine and agriculture will also be addressed. PMID- 10735068 TI - Sperm-mediated transgenesis. AB - The idea of using a sperm cell for introducing exogenous DNA into an oocyte at the time of fertilization would be brilliant if only we were sure that it can be done. Since 1989, contradictory reports have appeared in the literature and, at present, no consensus has been reached on the topic. Given the potential impact of this method for the generation of transgenic animals, for both mammalian and non-mammalian species, this review summarizes what has been achieved in this field. While some aspects, such as the binding of DNA molecules to spermatozoa, have now a solid experimental base, others, such as the generation of real transgenic individuals, are still based on disputed evidence. A critical analysis of the most relevant data will be presented in order to provide the tools for an objective evaluation of the efficiency of this method. PMID- 10735069 TI - Developing efficient strategies for the generation of transgenic cattle which produce biopharmaceuticals in milk. AB - At the close of the millennium, a revolution in the treatment of disease is taking shape due to the emergence of new therapies based on human recombinant proteins. The ever-growing demand for such pharmaceutical proteins is an important driving force for the development of safe and large-scale production platforms. Since the efficacy of a human protein is generally dependent on both its amino acid composition as well as various post-translational modifications, many recombinant human proteins can only be obtained in a biologically active conformation when produced in mammalian cells. Hence, mammalian cell culture systems are often used for expression. However, this approach is generally known for limited production capacity and high costs. In contrast, the production of (human) recombinant proteins in milk of transgenic farm animals, particularly cattle, presents a safe alternative without the constraint of limited protein output. Moreover, compared to cell culture, production in milk is very cost effective. Although transgenic farm animal technology was still in its infancy a decade ago, today it is on the verge of fulfilling its potential of providing therapeutic proteins that can not be produced otherwise in sufficient quantities or at affordable cost. Since 1989, we have been at the forefront of this development, as illustrated by the birth of Herman, the first transgenic bull. In this communication, we will present an overview of approaches we have taken over the years to generate transgenic founder animals and production herds. Our initial strategies were based on microinjection; at the time the only viable option to generate transgenic cattle. Recently, we have adopted a more powerful approach founded on the application of nuclear transfer. As we will illustrate, this strategy presents a breakthrough in the overall efficiency of generating transgenic animals, product consistency, and time of product development. PMID- 10735070 TI - Economical and ecological importance of indigenous livestock and the application of assisted reroduction to their preservation. AB - Among the many mammalian species that are threatened as the result of habitat destruction are numerous species of rare or little-known native livestock that possess features that render them ideally adapted to their environment. Because of the vital and valuable role many of these species play both to the ecology and economy of their native countries, attention is being directed towards initiating breeding programs that might insure their continued survival. This review introduces and highlights the importance of some of these indigenous species and outlines efforts currently underway to apply assisted reproductive technologies to their conservation. PMID- 10735071 TI - Embryo technology in conservation efforts for endangered felids. AB - Most of the 36 species of wild cats are classified as threatened, vulnerable or endangered due to poaching and habitat loss. The important role of assisted reproduction techniques (ART) as part of a multifaceted captive breeding program for selected wild cat species is gradually gaining acceptance. This recognition is a result of the progress made during the last decade in which the feasibility of oocyte recovery from gonadotropin-treated females, in vitro fertilization, embryo cryopreservation and embryo transfer (ET) was demonstrated in the domestic cat (Felis catus). Additionally, embryos have been produced in vitro from oocytes matured in vitro after recovery from ex situ ovaries of both domestic and non domestic cat species and domestic kittens have been born following transfer of these embryos. In vitro fertilization has been successful in at least one-third of wild cat species and kittens were born after transfer of Indian desert cat (Felis sylvestris ornata) embryos into a domestic cat and con-specific transfer of tiger (Panthera tigris) embryos. The domestic cat is not only a valuable model for development of in vitro techniques but may serve as a recipient of embryos from several species of small wild cats. PMID- 10735072 TI - Assisted reproductive technology in canid species. AB - Assisted reproductive technologies in dogs began as early as the 18th century. The first scientifically recorded artificial insemination (AI) was performed in Italy by Spallanzani and lead to the birth of three pups. Progress in the area was slow, and subsequent development included AI equipment and methods for short term preservation of fresh, and later, for frozen semen which led to the world's first litter produced from frozen semen in 1969. Improvement of freezing methods and AI equipment from 1970 onwards has rendered AI useful as a breeding technique for dogs. In parallel, AI in foxes was developed in Scandinavia in the early 1980's; this resulted in the economically valuable crossbreeding of silver and blue foxes for the production of bluefrost pelts. Unfortunately, due to the particular physiology of the canine female, progress in other artificial breeding techniques has lagged behind. Only in the last few years have these techniques been successfully applied in basic research to study oocyte maturation, in vitro fertilization, embryo cryopreservation and embryo transfer in canids. PMID- 10735073 TI - Reproductive biotechnology and "big" biological questions. AB - In recent decades, scientists have learned to manipulate that cardinal characteristic of life, reproduction, with powerful techniques like artificial insemination, contraception, embryo transfer, cryopreservation, and cloning by nuclear transfer. While these technologies often are used for practical applications and basic research, they have another profound intrinsic quality, which is to engender deep-seated thinking about important biological questions. Examples that stimulate such thinking include a goat's giving birth to her identical twin sister via splitting embryos, cryopreservation, and embryo transfer; that a parthenogenetic embryo can never become an animal but can become a genetic mother via an aggregation chimera; or that a somatic cell can become the sole genetic parent of a calf via cloning. In this paper, I illustrate this thought-stimulating quality by considering contributions of reproductive technologies to understanding, if not completely answering, several important biological questions. PMID- 10735074 TI - Sexing mammalian sperm--overview. PMID- 10735075 TI - Effect of timing of artificial insemination on sex ratio. AB - For a number of years, the time of insemination or mating during estrus has been believed to influence the sex ratio of offspring, with early insemination resulting in more females and late insemination, more males. Possible mechanisms of altering the sex ratio include facilitating or inhibiting the transport of either X- or Y-chromosome-bearing sperm through the reproductive tract, preferential selection of sperm at fertilization, or sex-specific death of embryos after fertilization. In livestock species, there is evidence for preferential selection of X- or Y-bearing sperm, based on the maturational state of the oocyte at fertilization. In deer and sheep, early and late insemination appears to skew the sex ratio toward females and males, respectively. In cattle, conflicting reports on the effect of time of insemination on sex ratio make the premise less clear. Many of the published studies lack adequate observations for definitive conclusions and/or are based on infrequent observations of estrus, making it difficult to assess the effect of time of insemination on sex ratio. It is likely that any effect of time of insemination on sex ratio in cattle is relatively small. Evidence is accumulating that treatments used for synchronization of estrus or ovulation in cattle may influence the sex ratio. PMID- 10735076 TI - Difference in sperm head volume as a theoretical basis for sorting X- and Y bearing spermatozoa: potentials and limitations. AB - Volume-based sorting of X- and Y-chromosome-bearing sperm cells could be an interesting alternative to the existing technique based on DNA content. Advantages would be that DNA staining and ultraviolet excitation, used in the existing technique, could be avoided. To assess the possibilities and limitations of sperm-head volume as sorting criterion, achievable purity and yield are determined for bull sperm. Two important parameters in this respect are the magnitude of the volume difference and the biological variation within each (X or Y) population. Earlier, we established a difference in volume matching the difference in DNA content (3.8%) between X- and Y-bearing bull sperm heads by comparing thicknesses and areas of high numbers of pre-sorted X- and Y-bearing bull sperm heads by interference microscopy and subsequent image analysis. Unfortunately, despite the high number of measurements, a direct determination of biological variations was not possible due to an unknown contribution of instrumental variations. In this paper, we determine the contribution of instrumental errors by measuring a single sperm head, varying parameters such as location in the image, orientation angle, focusing etc., simulating the behavior of the measuring system. After correction, both for the instrumental variation, and for the fact that the original samples were not pure, biological variations in volume of 5.9 +/- 0.8% were found. Our results indicate that when 10% of the bull sperm are sorted on basis of their head volume, a theoretical enrichment of 80% could be achieved. Expected purity and yield are lower than what is standard for the existing technique. At the moment, a technique to physically separate X- and Y-bearing sperm cells based on volume is not available. However, for applications for which the potential hazards of DNA staining and UV excitation are problematic, the development of such technique should be considered. PMID- 10735077 TI - Do X and Y spermatozoa differ in proteins? AB - This article reviews the current knowledge about X- and Y-chromosomal gene expression during spermatogenesis and possible differences between X- and Y chromosome-bearing spermatozoa (X and Y sperm) in relation to whether an immunological method of separation of X and Y spermatozoa might some day be feasible. Recent studies demonstrated that X- and Y-chromosome-bearing spermatids do express X- and Y-chromosomal genes that might theoretically result in protein differences between X and Y sperm. Most, if not all, of these gene products, however, are expected to be shared among X and Y spermatids via intercellular bridges. Studies on aberrant mouse strains indicate that complete sharing might not occur for all gene products. This keeps open the possibility that X and Y sperm may differ in proteins, but until now, this has not been confirmed by comparative studies between flow-cytometrically sorted X and Y sperm for H-Y antigen or other membrane proteins. PMID- 10735078 TI - A new approach to immunological sexing of sperm. AB - A non-invasive, immunological method for sexing mammalian sperm would be of benefit to agricultural industries. This paper presents a new approach, based on the hypothesis that sex-specific proteins (SSPs) are evolutionarily more highly conserved than non-SSPs. Antibodies to non-SSPs were raised and used in an affinity procedure to remove non-SSPs and enrich for SSPs. Thereafter, using column chromatography, purified SSPs were obtained. Sex-specific antibodies (SSAbs) raised against these SSPs appear to bind to sex-chromosome-specific proteins (SCSPs) on the sperm membrane and make possible a sperm-sexing procedure. Antibodies to SCSPs were raised and used to identify putative SCSPs by affinity chromatography. The preliminary results presented here suggest that a viable immunological sperm sexing procedure can be developed. PMID- 10735079 TI - Sex preselection: high-speed flow cytometric sorting of X and Y sperm for maximum efficiency. AB - Sex preselection that is based on flow-cytometric measurement of sperm DNA content to enable sorting of X- from Y-chromosome-bearing sperm has proven reproducible at various locations and with many species at greater than 90% purity. Offspring of the predetermined sex in both domestic animals and human beings have been born using this technology since its introduction in 1989. The method involves treating sperm with the fluorescent dye, Hoechst 33342, which binds to the DNA and then sorting them into X- and Y-bearing-sperm populations with a flow cytometer/cell sorter modified specifically for sperm. Sexed sperm are then used with differing semen delivery routes such as intra-uterine, intra tubal, artificial insemination (deep-uterine and cervical), in vitro fertilization and embryo transfer, and intra-cytoplasmic sperm injection (ICSI). Offspring produced at all locations using the technology have been morphologically normal and reproductively capable in succeeding generations. With the advent of high-speed cell sorting technology and improved efficiency of sorting by a new sperm orienting nozzle, the efficiency of sexed sperm production is significantly enhanced. This paper describes development of the these technological improvements in the Beltsville Sexing Technology that has brought sexed sperm to a new level of application. Under typical conditions the high speed sperm sorter with the orienting nozzle (HiSON) results in purities of 90% of X- and Y-bearing sperm at 6 million sperm per h for each population. Taken to its highest performance level, the HiSON has produced X-bearing-sperm populations at 85 to 90% purity in the production of up to 11 million X-bearing-sperm per h of sorting. In addition if one accepts a lower purity (75 to 80%) of X, nearly 20 million sperm can be sorted per h. The latter represents a 30 to 60-fold improvement over the 1989 sorting technology using rabbit sperm. It is anticipated that with instrument refinements the production capacity can be improved even further. The application of the current technology has led to much wider potential for practical usage through conventional and deep-uterine artificial insemination of many species, especially cattle. It also opens the possibility of utilizing sexed sperm for artificial insemination in swine once low-sperm-dose methods are perfected. Sexed sperm on demand has become a reality through the development of the HiSON system. PMID- 10735080 TI - Sex preselection: laboratory validation of the sperm sex ratio of flow sorted X- and Y-sperm by sort reanalysis for DNA. AB - Laboratory validation is essential in developing an effective method for separating X and Y sperm to preselect sex. Utilizing sexed sperm from a particular experiment to test fertility and achieve the subsequent phenotypic sex without knowing the likely outcome at conception is too costly for most applications. Further, research advances need to be built on an ongoing assessment with respect to the collection of data to continue progress towards achieving a successful outcome. The Beltsville Sperm Sexing Technology, which is based on the sorting of X- and Y-bearing sperm through the process of flow cytometric sperm sorting, is also well suited for validation in the laboratory by "sort reanalysis" of the sperm X- and Y-bearing fractions for DNA content. Since the sexing technology is based on the use of Hoechst 33342, a permeant nuclear DNA stain for sorting X- and Y-bearing sperm, it also can be the marker for determining the proportions of X and Y populations by sort reanalysis. The process consists of using an aliquot of the sorted sperm and sonicating to obtain sperm nuclei. The uniformity of the nuclear staining is re-established through the addition of more Hoechst 33342. Separate analysis of each aliquot produces a histogram that is fitted to a double gaussian curve to determine proportions of X and Y populations. The relative breadths of the distributions of DNA of X- and Y bearing sperm within a species affects interpretations of the histogram. Sort reanalysis is consistently repeatable with differences in X/Y DNA equal to or greater than 3.0%. This information on sex ratio of the sperm then provides the precise tool by which one can predict the outcome in terms of sex, from a particular sample of semen. Simple analysis of unsorted sperm to determine the proportions of X- and Y-bearing sperm based on DNA content is also an effective tool for validating sperm-sex ratio, whether it is in a sample assumed to be 50:50 or predicted to be something other than 50:50. This simple analysis provides for a check on the potential sex ratio of any sample of semen. PMID- 10735081 TI - Physiology of spermatozoa at high dilution rates: the influence of seminal plasma. AB - Extensive dilution of spermatozoa, as occurs during flow-cytometric sperm sorting, can reduce their motility and viability. These effects may be minimized by the use of appropriate dilution and collection media, containing balanced salts, energy sources, egg yolk and some protein. Dilution and flow-cytometric sorting of spermatozoa, which involves the removal of seminal plasma, also destabilizes sperm membranes leading to functional capacitation. This membrane destabilization renders the spermatozoa immediately capable of fertilization in vitro, or in vivo after deposition close to the site of fertilization, but shortens their lifespan, resulting in premature death if the cells are deposited in the female tract distant from the site of fertilization or are held in vitro at standard storage temperatures. This functional capacitation can be reversed in boar spermatozoa by inclusion of seminal plasma in the medium used to collect the cells from the cell sorter and, consequently, reduces their in vitro fertility. It has yet to be determined whether seminal plasma would have similar effects on flow cytometrically sorted spermatozoa of other species, and what its effects might be on the in vivo fertility of flow sorted boar. PMID- 10735082 TI - Low dose insemination in synchronized gilts. AB - Conventional insemination techniques in pigs require 2 to 3 x 10(9) sperm/dose. When using the latest high-speed sperm-sorting technology, one can still sort only about 5 to 6 million sperm of each sex per hour. The objective of the present study was to find the minimal sperm concentration at a low-insemination volume in pigs without diminishing fertilization rate and litter size using surgical deep intra-uterine insemination (IUI). Semen from 3 boars was collected and diluted with Androhep to 5 x 10(8), 1 x 10(8), 1 x 10(7), 5 x 10(6) or 1 x 10(6) sperm/0.5 ml. In trial 1, 109 prepuberal gilts were synchronized and surgically inseminated into the tip of each uterine horn 32 h or 38 h after hCG treatment or at the time of ovulation, respectively. Pregnant gilts were allowed to go to term. Pregnancy and farrowing rates did not differ significantly except at the lowest sperm concentration if inseminated 32 h or 38 h after hCG treatment (p < 0.05). No differences were found among insemination groups for the total number of piglets, number of piglets born alive, stillborn piglets, and mummified fetuses. In trial 2, 34 gilts were inseminated as described above 32 h after hCG. Additionally, 9 gilts were inseminated once nonsurgically with 1 x 10(9) sperm as controls. Gilts were slaughtered 48 h after insemination, and embryos were recovered. Embryos were cultured in NCSU 23 (120 h), evaluated morphologically and stained with fluorescent dye (Hoechst 33342) to visualize nuclei. Recovery rates varied between 71.4% and 84.4%. Fertilization rate of the lowest sperm concentration (1 x 10(6) sperm/horn) differed significantly (p < 0.05) from all other groups. Cleavage rates at specific developmental stages did not differ. After 5 days of in vitro culture, embryos developed to morulae and blastocysts. No differences were found for these stages. In conclusion, no major differences were found between insemination groups as long as the sperm dosage was at least 10 million sperm per gilt. The low volume was sufficient for successful deep intra-uterine insemination. Embryo development was comparable to the controls. PMID- 10735083 TI - Cryopreservation of flow-sorted bovine spermatozoa. AB - Experiments were designed to maximize sperm viability after sorting by flow cytometry and cryopreservation. Experiments concerned staining sperm with Hoechst 33342 dye, subsequent dilution, interrogation with laser light, and postsort concentration of sperm. Concentrating sorted sperm by centrifugation to 10 to 20 x 10(6) sperm/ml reduced adverse effects of dilution. Exposing sperm to 150 mW of laser light resulted in lower percentages of progressively motile sperm after thawing than did 100 mW. Sorted sperm extended in a TRIS-based medium had higher postthaw sperm motility after incubation for 1 or 2 h than sperm extended in egg yolk citrate (EYC) or TEST media, and equilibrating sperm at 5 degrees C for 3 or 6 h prior to freezing was superior to an equilibration time of 18 h. For sorting sperm 4 to 7 h postcollection, it was best to hold semen at 22 degrees C neat instead of at 400 x 10(6)/ml in a TALP buffer with Hoechst 33342. Current procedures for sexing sperm using flow cytometry result in slightly lower postthaw motility and acrosomal integrity compared to control sperm. However, this damage is minor compared to that caused by routine cryopreservation. Fertilizing capacity of flow-sorted sperm is quite acceptable as predicted by simple laboratory assays, and sexed bovine sperm for commercial AI may be available within 2 years. PMID- 10735084 TI - In vitro fertilization with flow-cytometrically-sorted bovine sperm. AB - An attractive feature of IVF is that fewer sexed sperm are needed than for artificial insemination. However, sperm sexed by flow cytometry/cell sorting are probably pre-capacitated, necessitating modifications to standard IVF systems for optimal success. With current procedures, the percentages of oocytes fertilized with sorted and unsorted frozen bovine sperm are similar, and events during the first cell cycle are timed similarly for sorted and unsorted sperm. However, in most cases, blastocyst production with sorted sperm was approximately 70% of controls produced with unsorted sperm. In some early studies, there appeared to be an unexplained delay of about half a day in blastocyst development. Nevertheless, some dozens of apparently normal calves, pre-sexed with 90% accuracy, have resulted from frozen embryos produced via IVF with sexed sperm. IVF also has proven useful as a bioassay for improving sperm-sorting procedures such as determining potential detrimental effects of laser power. It is likely that use of IVF in cattle breeding programs will increase considerably when sexed, frozen sperm become commercially available. PMID- 10735085 TI - Insemination of heifers with sexed sperm. AB - Data from inseminating 1,000 heifers consecutively with sexed sperm and 370 heifers with control sperm in 11 small field trials are summarized. Semen was from 22 bulls of unknown fertility of various beef and dairy breeds, and 6 inseminators participated. Freshly collected sperm were sexed using a MoFlo flow cytometer/cell sorter after staining sperm with the DNA-binding dye Hoechst 33342; the principle is that the bovine X chromosome has 3.8% more DNA than the Y chromosome. Accuracy approaching 90% males or females was achieved. There was little difference in pregnancy rates between sexed, unfrozen and sexed, frozen sperm. In 5 of 6 field trials, there was little difference in pregnancy rates between insemination doses of 1.0 to 1.5 x 10(6) versus 3.0 x 10(6) sexed, frozen sperm. In the most recent trials, pregnancy rates with sexed, frozen sperm were within 90% of unsexed, frozen controls that had 7 to 20 times more sperm/insemination dose; however, in a few trials, control pregnancy rates were substantially higher than with low doses of sexed sperm. There were too few inseminations per bull to test bull differences in pregnancy rates rigorously. Insemination of sexed, frozen sperm bilaterally into the uterine horns produced pregnancy rates similar to insemination into the uterine body in 4 of 5 field trials. Pregnancy rates among inseminators did not differ significantly. There was no excess embryonic death between 1 and 2 months of gestation with pregnancies from sexed sperm, and very few abortions occurred between 2 months of gestation and term. Although rigorous epidemiological studies remain to be done, calves resulting from sexed sperm appear to exhibit no more abnormalities than controls. PMID- 10735086 TI - Applications of sexed semen in cattle production. AB - Sexed semen will contribute to increased profitability of dairy and beef cattle production in a variety of ways. It could be used to produce offspring of the desired sex from a particular mating to take advantage of differences in value of males and females for specific marketing purposes. Commercial dairy farmers, those who produce and market milk, could use sexed semen to produce replacement daughters from genetically superior cows and beef crossbred sons from the remainder of their cow population. To increase the rate of response to selection, seedstock dairy cattle breeders could produce bulls for progeny testing from a smaller number of elite dams by using sexed semen to ensure that all of them produced a son. Using sexed semen could then reduce the cost of progeny testing those bulls, because fewer matings would be necessary to produce any required number of daughters. Commercial beef cattle farmers, producing animals for eventual slaughter, could use sexed semen to capitalize on the higher value of male than female offspring for meat production. They could also use sexed semen to produce specialized, genetically superior replacement heifers from as small a proportion of the herd as possible. This would allow the remainder of the herd to produce male calves from bulls or breeds with superior genetic merit for growth, feed conversion efficiency, and carcass merit. Single-sex, bred-heifer systems, in which each female is sold for slaughter soon after weaning her replacement daughter, would be possible with the use of X-chromosome-sorted semen. Use of sexed semen would make terminal crossbreeding systems more efficient and sustainable in beef cattle. Fewer females would be required to produce specialized maternal crossbred daughters, and more could be devoted to producing highly efficient, terminal crossbred sons. PMID- 10735087 TI - Clinical experience with flow cytometric separation of human X- and Y-chromosome bearing sperm. AB - Numerous methods to separate human X- and Y-bearing sperm have been reported with unconfirmed separation after DNA analysis and inconsistent birth results. Successful flow cytometric separation of sperm resulting in alteration of the sex ratio of young born has been demonstrated in several animal species. Flow cytometric separation of human X- and Y-bearing sperm (MicroSort) has been confirmed after DNA analysis by fluorescence in situ hybridization (FISH). Pregnancies and births have resulted from the use of MicroSort after intrauterine insemination (IUI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). PMID- 10735088 TI - Issues affecting commercialization of sexed sperm. AB - A decision tree for genetics or sperm-sexing entities considering sales of sexed sperm is discussed in terms of: (a) how best to avoid harm; (b) how best to do good; (c) needed synergy with other assisted reproductive technologies; (d) constraints on biotechnology; and (e) costs with current and likely technologies versus potential benefits to producers. The sexed-sperm industry might wish to take a pro-active stance on societal issues potentially affecting use of sexed sperm. For most sales in animal agriculture, cost of added value must be < 50% of benefit. Cost is less important for emotionally-driven uses with horses and human beings. Current procedures for flow-sorting allow most sperm to retain their fertilizing potential. Added cost to produce and package 2 x 10(6) sperm is estimated at US $30 to US $46 with flow sorted sperm. Estimating cost of any alternative technology is premature. For IVF/embryo transfer (ET), cost and numbers of flow-sorted sexed sperm are appropriate for commercial use. For use in low-dose AI, however, added cost to supply one insemination dose must be near US $12. Flow-sorting instruments with higher throughput and lower purchase and operating costs are obligatory for economic application in most AI situations. Developers of antibody-based separations also will face issues of retention of fertilizing potential while minimizing cell loss, separation of living from dead sperm concurrent with sperm sexing, output, and cost. To benefit producers and consumers in a changing world, genetics and sperm-sexing companies will have to collaborate and interface to provide funding for needed research and development and to recover these costs, using mechanisms not yet obvious. PMID- 10735089 TI - Effect of clavulanic acid-potentiated amoxycillin on semen quality in dogs. AB - Two different doses of clavulanic acid-potentiated amoxycillin were evaluated for their effect upon semen quality and libido in dogs. There was no significant difference in either parameter between control dogs and dogs treated with clavulanic acid-potentiated amoxycillin at either 12.5 mgkg-1 or 25 mgkg-1 orally twice daily for 28 d. Despite the marked effect of certain antibiotic agents upon spermatogenesis, it appears that clavulanic acid-potentiated amoxycillin can be used in dogs at up to twice the therapeutic dosage recommended by the manufacturer without a deleterious effect upon semen quality. PMID- 10735090 TI - Effects of induced endometritis on the life-span of corpora lutea in pseudopregnant rabbits and incidence of spontaneous uterine infections related to fertility of breeding does. AB - A significant percentage of rabbit does fail to become pregnant after AI. We hypothesized that uterine infections induced by the insemination procedure are related to delayed luteolysis and high progesterone concentrations noted to present at the time of AI. The rabbits, randomly assigned to 4 groups (3 animals/group), were given 0.8 microgram GnRH analogue (Day 0) just prior to infusing the uterus with sterile extender (control group) or with extender inoculated with 0.5, 1, and 2 x 10(6) Pasteurella multocida (treated groups). The effects of treatments on functional life-span of CL were assessed by evaluating plasma progesterone from Day 0 to Day 23 of pseudopregnancy. In treated rabbits, the progesterone profiles closely overlapped those found in controls until approximately Day 14. Thereafter, they varied greatly between animals, but luteolysis was delayed by at least 5-6 d and developed less rapidly than in controls. On Day 21, progesterone concentrations were higher than normal in 4 treated does. In a field survey, vaginal swabs were collected at the time of the second AI from 114 non-pregnant rabbits and those positive to bacteriological culture, were killed humanely 16 d later to collect uterine swabs. Positive uterine swabs were found only in 19 of the 34 does having a positive vaginal swabs and all of them were not pregnant. The most frequent pathogen isolated was S. aureus (50%), followed by E. coli (37.5%) and P. multocida (12.5%). We demonstrated that uterine infection increases the life-span of CL in non-pregnant does and that infections of the genital tract system are quite common among does on breeding farms, probably related to using AI. PMID- 10735091 TI - Effect of milk production on the incidence of double ovulation in dairy cows. AB - To determine the effect of parity and milk production on the incidence of double ovulation, the synchronization of ovulation, using GnRH and prostaglandin F2 alpha followed by timed AI (Ovsynch), was initiated at a random stage of the estrous cycle in lactating Holstein cows (n = 237). Ovulatory response at 48 h after the second GnRH injection and conception rate at 28 d post AI were determined by transrectal ultrasonography. Ovulation was synchronized in 84% of cows receiving the Ovsynch protocol. Of the synchronized cows, 14.1% exhibited a double ovulation and 47.6% conceived. Conception rate tended to be greater (P = 0.08) for cows exhibiting double (64.0%) rather than single ovulation (45.2%). To determine the effect of milk production on the incidence of double ovulation, cows were classified into low (< or = 40 kg/d) or high (> 40 kg/d) milk production groups based on the average milk production of 40.5 +/- 0.8 kg/d collected 2 d before AI. Although the incidence of double ovulation tended to increase linearly (P = 0.09) with increasing parity, the incidence of double ovulation was nearly 3-fold greater (P < 0.05) for cows in the high (20.2%) than the low (6.9%) milk production group. Furthermore, the increase in the incidence of double ovulation with parity apparently occurred because, within a parity group, the proportion of cows with high milk production was greater for the older cows. Twinning rate of cows that calved (n = 58) was 5.2%. In a secondary objective, cows were retrospectively classified as cystic or normal based on ultrasonographic ovarian morphology at the time of the second GnRH injection. Incidence of ovarian cysts was 11%, and the synchronization and conception rate of cows classified as cystic was 73.1 and 36.8%, respectively, which did not differ from that of normal cows. We conclude that milk production is the primary factor affecting the incidence of double ovulation in lactating dairy cows and may explain the effect of parity on twinning rate. In addition, Ovsynch appears to be an effective method for establishing pregnancy in lactating dairy cows with ovarian cysts. PMID- 10735092 TI - Growth hormone gene polymorphism and reproductive performance of AI bulls. AB - Relationships between the growth hormone gene RFLP polymorphism and bull sperm characteristics were the objects of the present study. DNA was extracted from blood or sperm samples collected from 113 AI bulls and submitted for polymerase chain reaction (PCR) followed by digestion with Alu I restriction enzyme. The bGH genotypes were visualized on 10% polyacrylamide gel. The analyzed population of AI bulls consisted of dairy (Holstein Fresian [HF] crossbred [HF x Polish Black and White]) and beef breeds (Limousine, Charolaise, Piemontese, Angus and Hereford). The frequency of the Leu allele was 0.86 among dairy bulls and 0.38 in beef bulls (0.14 and 0.62 for the Val allele, respectively). Eight sperm characteristics and Day 60 non-return rates (NRR) were analyzed. The 3 genotype groups (LL, VV and LV) and the effect of production type (dairy or beef) on sperm characteristics were considered. None of the traits showed significant variability in relation to the bGH genotype, although a tendency was observed for LL bulls to have a lower ejaculate volume and VV bulls higher NRR. Moreover some statistically significant associations with production type were noticed: beef bulls were superior in sperm concentration and non-return rate, whereas dairy bulls excelled in individual fresh sperm motility. PMID- 10735093 TI - Evaluation of sperm tail membrane integrity by light microscopy. AB - During routine evaluation of trypan blue-Giemsa stained semen smears, sperm cells can be found with unstained heads and with stained tails. It was hypothesized that these cells were immotile and should not be considered as live. Sperm motility was determined in isoosmotic, and presumably isotonic trypan blue stained wet preparations. Bull, ram and boar semen smears were stained with hypoosmotic trypan blue-Giemsa to compare the relationship between the percentage of stained sperm tails and the percentage of sperm tails remaining straight under hypoosmotic conditions. Actively moving spermatozoa with unstained heads, but with stained tails were never observed in wet preparations. The correlation coefficient found between the percentage of sperm with stained tails and the percentage with straight tails was 0.81, 0.94 and 0.85 for bull, ram and boar spermatozoa, respectively. Results of this study show that sperm cells with an intact head membrane, but a stained and presumably membrane-damaged tail are not motile. Therefore these cells should be included in the dead category rather than alive in the usual live-dead studies with vital stains. PMID- 10735094 TI - Conception rates after artificial insemination or embryo transfer in lactating dairy cows during summer in Florida. AB - The objective was to compare conception rates to embryo transfer relative to AI, during summer heat stress, in lactating dairy cows. Holstein cows (n = 180; 50 to 120 d postpartum) were allocated randomly to 1 of 3 groups: artificial insemination (AI, n = 84), embryo transfer using either embryos collected from superovulated donors (ET-DON, n = 48), or embryos produced in vitro (ET-IVF, n = 48). Embryos from superovulated donors were frozen in 10% glycerol and were rehydrated in a 3-step procedure, in decreasing concentrations of glycerol in a sucrose medium before transfer. Embryos produced in vitro were frozen in 1.5 M ethylene glycol, thawed and transferred without rehydration. Blood samples were collected from AI and ET recipients on Days 0, 7 and 22 for measurement of progesterone in plasma. Conception rate was estimated for the three groups at Day 22 (progesterone > 1 ng/mL) and confirmed at Day 42 by palpation per rectum. Conception rate estimates at Day 22 did not differ among groups (AI, 60.7%; ET DON, 60.4%; ET-IVF, 54.2%), but conception rates at Day 42 differed (AI, 21.4%; ET-DON, 35.4%; ET-IVF, 18.8%; AI versus ET: P > 0.10 and ET-DON versus ET-IVF: P < 0.05). In cows considered pregnant at 22 d but diagnosed open at 42 d, the interestrous intervals were 28.8 +/- 2.2, 35.2 +/- 3.5 and 31.6 +/- 2.9 d, respectively, for AI, ET-DON and ET-IVF groups. Transfer of embryos collected from nonheat-stressed superovulated donors significantly increased conception rates in heat stressed dairy cattle. However, transfer of IVF-derived embryos had no advantage over AI. Where appropriate mechanisms are in place to attenuate the effects of heat stress, embryo transfer using frozen-thawed donor embryos increases conception rates. PMID- 10735095 TI - Embryo production by ovum pick up in unstimulated calves before and after puberty. AB - The possibility of producing embryos from oocytes repeatedly collected from unstimulated calves was tested, and results obtained before and after puberty were compared in the same animals. Ovum pick-up (OPU) coupled with in vitro embryo production was used on 2 sets of 7 and 9 calves, aged 7 to 10 m.o. at the start of the experiment. The oocytes were collected twice a week during a 2-m.o. period before puberty and a 1-m.o. period after puberty. Oocytes were fertilized and co-cultured with cumulus cells in modified synthetic oviduct fluid (SOF) up to Day 7 post insemination. Some Day 7 blastocysts were vitrified and transferred to recipient heifers. An average of 3.8 to 6.8 follicles was punctured per OPU session; 1.9 to 3.1 oocytes were collected, of which more than 60% were of good quality. The number of punctured follicles and collected oocytes varied between donors. Blastocyst rates of 19 to 27% were obtained for the 2 sets. Three normal calves were born from the transfer of 20 vitrified embryos. While no significant difference was observed for the first set of calves, a significant decrease in the number of punctured follicles was observed after puberty in the second set. A direct correlation was also obtained between the number of follicles punctured before and after puberty in the same animal. In conclusion, oocytes can be collected by repeated OPU in calves 7 to 10 m.o. old without affecting their growth or the onset of puberty. An average of 5 to 11 (range 0 to 16) blastocysts per donor was produced over 2 month. However, important variations were found between donors. The correlation observed for the number of follicles punctured before and after puberty suggests that this parameter is determined before puberty. PMID- 10735096 TI - Dynamics of prostaglandin secretion, intrauterine fluid and uterine clearance in reproductively normal mares and mares with delayed uterine clearance. AB - Two experiments were performed to investigate relationships between oxytocin, prostaglandin release, uterine emptying and fluid accumulation in the uterus. In Experiment 1, the effect of oxytocin on the pattern of prostaglandin release during uterine clearance of radiocolloid was measured in 5 normal mares and 5 mares with delayed uterine clearance. Uterine clearance was measured during estrus by scintigraphy at 0, 60 and 120 min after colloid infusion. After the 120 min reading, 20 IU, i.v., oxytocin were given, and the amount of colloid cleared was measured at 135, 150 and 180 min. Plasma was obtained prior to and during scintigraphy at 5- and 15-min intervals to measure concentrations of 15-keto 13,14-dihydro-PGF2 alpha metabolite (PGFM) by RIA. In Experiment 2, plasma PGFM levels were compared after administration of oxytocin in 8 normal mares and 6 mares with delayed uterine clearance to determine if intrauterine fluid stimulated prostaglandin release. Mares received 2 treatments in a cross-over design. Treatment 1 consisted of 20 IU, i.v., oxytocin during estrus. Treatment 2 consisted of an infusion of 10 mL, i.u., saline 15 min prior to oxytocin administration. Treatments were performed 4 to 6 h apart. Blood was collected and PGFM was measured as in experiment 1. Data were analyzed by least squares analysis of variance. In Experiment 1, regression analysis of scintigraphy and PGFM profiles indicated that time response curves differed between groups (P < 0.01). At 120 min, normal mares retained 40.4 +/- 4.9% (mean +/- SEM) of the radiocolloid while mares with delayed clearance retained 88 +/- 5%. Fifteen minutes after oxytocin administration (135 min), all normal mares and 4 of 5 mares with delayed clearance retained only < 6% of the colloid. During the first 120 min, plasma PGFM concentrations did not differ between the 2 groups. After oxytocin was given, plasma PGFM concentrations increased in 4 of 5 mares with delayed uterine clearance (80 to 3,096 pg/mL) but not in normal mares (13 to 46 pg/mL). In Experiment 2, plasma PGFM concentrations did not rise in normal mares but rose in 3 of 6 mares with delayed clearance (135 to 483 pg/mL) independent of treatment or period. The results suggest that intrauterine clearance of radiocolloid after oxytocin administration appears to be independent of PGF2 alpha release in normal mares during estrus. The difference in prostaglandin release response after oxytocin administration between the 2 groups was unrelated to the presence of intrauterine fluid. PMID- 10735097 TI - Effect of lecithin on in vitro and in vivo survival of in vitro produced bovine blastocysts after cryopreservation. AB - The use of soybean lecithin in an glycerol-based solution for slow freezing of in vitro matured, fertilized and cultured (IVMFC) bovine embryos was examined. Embryos were developed in vitro in INRA Menezo's B2 medium supplemented with 10% fetal calf serum (FCS) on Vero cells monolayers. Day 7 blastocysts were frozen in a two-step protocol consisting of exposure to 5% glycerol and 9% glycerol containing 0.2 M sucrose in F1 medium + 20% FCS. Soybean lecithin was either added or not to the freezing solutions at a final concentration of 0.1% (w/v). In Experiment 1, blastocysts were equilibrated in cryoprotectant solutions without cooling. Cryoprotectant was diluted from embryos with 0.5 M and 0.2 M sucrose. The percentages of fully expanded and hatched blastocysts treated with or without lecithin after 24 and 48 h in culture were not significantly different (100 versus 100% and 93.3 versus 100%, respectively). In Experiment 2, the in vitro survival of frozen-thawed IVMFC blastocysts was compared when cryoprotectant solutions were either supplemented or not with lecithin. No significant effect of lecithin was found on the ability of frozen-thawed blastocysts to re-expand after 48 h in culture (65.6 and 54.2%, respectively). However, the post-thaw hatching rate of embryos cryopreserved in the presence of 0.1% lecithin was significantly higher after 72 h in culture (52 and 31.8%, respectively). In Experiment 3, the ability of frozen-thawed IVMFC blastocysts to establish pregnancy following single embryo transfer was determined. Transfers of 58 and 66 frozen-thawed embryos cryopreserved with or without lecithin resulted in 6 and 10 (10.3 and 15.1%, respectively) confirmed pregnancies at Day 60. Addition of lecithin to cryoprotectants did not improve the in vivo development rate of cryopreserved IVMFC bovine blastocysts. PMID- 10735098 TI - A preliminary report on the effect of dietary energy on prostaglandin F2 alpha production in vitro, interferon-tau synthesis by the conceptus, endometrial progesterone concentration on days 9 and 15 of pregnancy and associated rates of embryo wastage in ewes. AB - Two groups of ewes were fed to provide 1.70 x (high energy group; n = 15) or 0.56 x (low energy group; n = 15) energy requirements for maintenance of liveweight from 14 d before a synchronized mating in November until slaughter at 9 or 15 d after mating. We investigated the effects on interferon-tau (IFN tau) secretion by the conceptuses, prostaglandin F2 alpha (PG) production in vitro by endometrial tissue, and associated rates of embryo mortality, endometrial progesterone content and progesterone production by luteal tissue. No differences between groups in pregnancy rate were detected on Day 9 between the 2 groups. Proportionately (6/6 vs 2/5), there were more pregnant ewes in the high energy group on Day 15, although this difference did not reach significance (P = 0.06). The proportion of corpora lutea represented by embryos was significantly lower in undernourished ewes (P < 0.05). Secretion in vitro of PG was lower in the 2 pregnant ewes of the low energy group on Day 15, and it was accompanied by higher IFN tau secretion by conceptuses recovered from these ewes. However, the limited number of pregnant ewes recorded on Day 15 prevented any statistical comparison. Neither mean endometrial content of progesterone nor ovarian venous progesterone concentrations and production of progesterone by luteal were affected by nutrition. The provisional results of the present experiment indicate that undernutrition may induce a reduction in the rate of secretion of IFN tau and can therefore increase production of PG from the endometrium. This could initiate luteolysis. The lower pregnancy rates observed in underfed ewes could be mediated through this alteration in the signal of maternal recognition of pregnancy. However, these findings remain to be shown in further experiments including a larger number of animals, as they only represent data from 2 undernourished animals. PMID- 10735099 TI - Efficient injection of bull spermatozoa into oocytes using a Piezo-driven pipette. AB - Recently, mouse and human offspring have been successfully obtained from embryos developed after intracytoplasmic sperm injection(ICSI), using a Piezo micromanipulator. In this study, the Piezo-ICSI procedure was used with in vitro matured bovine oocytes known to be difficult to fertilize microsurgically. The efficacy of Piezo-ICSI versus conventional ICSI was examined after oocytes were activated and fertilized with or without calcium ionophore (A23187) exposure. In conventional ICSI, the rate of fertilization was 19% (11/59) with A23187 and 5% (2/38) without it. However, when the Piezo-ICSI procedure was performed, the fertilization rate was 72% (47/65) with A23187 and 72% (28/39) without it. The rate of oocyte survival after microinjection was nearly similar for both methods. We suggest that the bovine oocyte is successfully activated and fertilized when an immobilized spermatozoon is injected exactly into the ooplasm through the oolemma, perforated easily by the pulsation of the Piezo. Moreover, an activating procedure such as exposure of oocytes to A23187 is not necessary, because the so called sperm factor (oocyte activating substances) is incorporated into the ooplasm along with a spermatozoon. In this respect, the Piezo-ICSI was more efficient than the conventional ICSI method for fertilizing and thus obtaining more bovine embryos. PMID- 10735100 TI - Humoral pathway for transfer of the boar pheromone, androstenol, from the nasal mucosa to the brain and hypophysis of gilts. AB - Signaling and priming pheromones play an important role in intraspecies behavioral and sexual interactions and in the control of reproduction. It is generally accepted that pheromones act by stimulating the dendritic receptors in the mucus-imbedded cilia of olfactory neurons massed in the olfactory epithelium. The boar pheromone androstenol, known to induce sexual behavior in pigs, is 1 of 2 pheromones that have been chemically defined, tritiated and thus made available for use in studies. In Experiment 1, sexually mature cyclic gilts at Days 16 to 21 of the estrous cycle were humanely killed and the heads separated from the bodies. The heads were attached to a perfusion system using heated, oxygenated, heparinized, autologous blood. A total amount of 10(8) dpm (758 ng) of 3H-5 alpha androstenol (3HA) was either infused into the angularis oculi veins that drain the nasal cavities (n = 7) over a 5-min period or applied through intranasal catheters onto the mucose surface (n = 16) for 2 min. In both groups frequent blood samples were collected from the carotid rete and from venous effluent. Concentration of 3HA in the arterial blood of the carotid rete after direct (into angularis oculi veins) or indirect (onto the nasal mucosa) administration of 3HA into veins draining the nasal cavities was significantly higher than background radioactivity before 3HA administration (P < 0.0001 and P < 0.05, respectively). The 3HA was selectively accumulated (compared with the respective control tissue) in the neurohypophysis (P < 0.001), adenohypophysis (P < 0.01), ventromedial hypothalamus (P < 0.05), corpus mammillare (P < 0.01), and perihypophyseal vascular complex (P < 0.001). In a second in vitro experiment, active uptake of 3HA into the nasal mucosa of the proximal, respiratory segment of the nasal cavity was observed. These results demonstrate a humoral pathway for the transfer of pheromones from the nasal cavity to the hypophysis and brain. Androstenol was taken up by the respiratory part of the nasal mucosa, resorbed into blood, transported to the cavernous sinus and transferred into the arterial blood of the carotid rete (supplying the hypophysis and brain), and then selectively accumulated in the hypophysis and certain brain structures. PMID- 10735101 TI - The effect of cryopreservation on the survivability, viability and motility of epididymal African buffalo (Syncerus caffer) spermatozoa. AB - The effect of cryopreservation on the viability and motility of epididymal African buffalo spermatozoa was studied in samples obtained from 17 and 13 animals in 1995 and 1996, respectively. Cryopreservation significantly reduced the viability and motility of the epididymal spermatozoa. The average percentage of live (+/- SE) spermatozoa declined significantly from 90.4 +/- 2.0% (1995) and 84.4 +/- 1.1% (1996) in fresh epididymal samples, to 57.0 +/- 2.0% and 56.3 +/- 1.1%, respectively, in frozen-thawed samples. The acrosomal integrity (+/- SE) of spermatozoa declined from 89.3 +/- 2.3% (1995) and 93.3 +/- 2.2% (1996) to 50.2 +/- 2.3% and 37.5 +/- 2.2%, respectively. In 1995, this effect was largely associated with the thermal equilibration prior to cryopreservation. PMID- 10735102 TI - Correlation between reproductive efficiency, as determined by new mathematical indexes, and the body condition score in dairy cows. AB - The aim of this work was to study a new mathematical model based on dynamic indexes designed to evaluate reproductive efficiency in dairy herds and to correlate the new index with the body condition score (BCS) in order to evaluate the reproductive state of the cows post partum. Four groups of dairy cows were used: 1) loose-housed Italian Friesian (loose Friesian, n = 190); 2) stanchioned Italian Friesian (stanchioned Friesian, n = 121); 3) stanchioned Italian Simmental (stanchioned Simmental, n = 120); and 4) loose-housed selected Italian Friesian cows (BCS test, n = 117). The first 3 herds were used to develop the new mathematical model while the fourth was used to correlate the method with the BCS. The new model was developed from the analysis of progesterone (P4) concentrations in whey and the frequency distribution of the cows in 3 reproductive states: cyclicity, acyclicity and pregnancy. The frequency distribution generated 3 curves, the intersections of which form a closed area. The barycenter of this closed area gives a simple static representation of the reproductive efficiency of each herd. We also studied the movement of the barycenter with time (dynamic index) for each reproductive status curve. The dynamic index allowed for evaluation of the reproductive efficiency of a group of cows at 40 d after calving, by analyzing the evolution of the different reproductive states post partum. A reproductive index called Cycle Time was characterized in a 240-d period of observation as the interval needed to bring all the animals from acyclic to pregnant status. The loose Friesian cows had the best reproductive efficiency. The BCS test was used to divide cows into 3 groups depending on the percentage loss of BCS due to the negative energy balance at 30 d post partum. Cows which lost more than 20% in BCS had the lowest reproductive efficiency. The following protocol was devised to monitor herds in order to identify cows that were likely to have reproductive problems: 1) measure BCS 10 d before calving; 2) monitor progesterone in whey starting 5 d after calving; 3) measure BCS 30 d after calving; 4) isolate cows that lost more than 20% of BCS; 5) measure progesterone only in the cows that lose more than 20% of BCS; 6) activate appropriate feeding strategies to help prevent excessive mobilization of body fat reserves. PMID- 10735103 TI - Modification of follicular dynamics by exogenous FSH and progesterone, and the induction of ovulation using hCG in postpartum beef cows. AB - Follicular growth and ovulation in response to FSH, progesterone and hCG were evaluated in postpartum beef cows. In Experiment 1, on Day 21 post partum, cows received an injection of either saline (control; n = 6), FSH (200 mg; n = 6), or a PRID (n = 5) for 10 d. Both FSH and PRID prolonged maintenance of a dominant follicle (15.5 +/- 1.16 and 14.4 +/- 1.29 d, respectively, vs 8.4 +/- 1.22 d in control; P < 0.01), and increased the maximum diameter of the dominant follicle (14.0 +/- 0.91 and 16.4 +/- 1.01 mm, respectively, vs 10.9 +/- 0.95 mm in control; P < 0.05). The PRID-maintained dominant follicle ovulated in 60% of cows, followed by normal estrous cycles (vs 0% in control; P = 0.01), whereas the dominant follicle ovulated in 33% of FSH-treated cows (P = 0.08). The PRID regimen shortened the interval to first ovulation preceding a normal cycle and continued cyclicity (44 +/- 4.1 vs 60 +/- 4.4 d in control; P = 0.02). In Experiment 2, on Day 21 post partum, cows received either saline (control), saline + PRID, or FSH + PRID (n = 16/group). Sixty hours after PRID withdrawal, cows received either saline or hCG (1,500 IU, n = 8/treatment). The FSH + PRID regimen increased the number of large (> 10 mm in diameter) follicles (3.6 +/- 0.43 vs 1.9 +/- 0.39 in control; P = 0.005). Both PRID and FSH + PRID prolonged maintenance of the largest follicle (11.0 +/- 0.82 and 11.2 +/- 0.91 d, respectively, vs 8.7 +/- 0.81 d in control; P < 0.05). The PRID-maintained dominant follicle ovulated in 50% of cows, followed by normal estrous cycles. The FSH + PRID-maintained largest follicle had become atretic at PRID withdrawal and was anovulatory. The FSH + PRID + hCG regimen increased the incidence of ovulation preceding a cycle of normal duration and continued cyclicity (100 vs 50% in PRID; P = 0.03), and reduced the interval to first ovulation preceding a cycle of normal duration and continued cyclicity (38 +/- 6.5 vs 58 +/- 6.3 d in control; P = 0.04). The area under the progesterone curve during the induced cycle was reduced after (PRID +/- FSH) + hCG than after PRID +/- FSH (P = 0.002). These results indicate that PRID alone or with FSH/hCG has the potential to modify the dominant follicle and initiate cyclicity in postpartum beef cows. PMID- 10735104 TI - Post-thaw evaluation of dog spermatozoa using new triple fluorescent staining and flow cytometry. AB - A new triple fluorescent staining method was developed to evaluate frozen-thawed dog spermatozoa. This method was used to compare functional parameters of canine spermatozoa cryopreserved using 2 different freezing-thawing protocols. One ejaculate from each of 10 dogs was split into 2 aliquots and processed using the Andersen method or the CLONE method. Semen samples were evaluated immediately after thawing and after 3 h of incubation at 37 degrees C. Plasma membrane integrity and acrosomal status of the spermatozoa were evaluated simultaneously by flow cytometry using a combination of 3 fluorescent dyes: Carboxy-SNARF-1 (SNARF), to identify the live spermatozoa; propidium iodide (PI), which only stains dead cells or cells with damaged membranes; and fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA), which binds to the acrosomal content of spermatozoa with damaged plasma and outer acrosomal membranes. The accuracy of this new staining method in quantifying the proportions of live and dead spermatozoa by flow cytometry was evaluated by comparing it with the staining technique using carboxyfluorescein diacetate and propidium iodide (CFDA PI), which yielded high correlation coefficients. The triple-stained sperm samples were also analyzed by epifluorescence microscopy, and both methods proved to be highly correlated. Post-thaw progressive motility and plasma membrane integrity were similar for the 2 freezing procedures, but the proportion of damaged acrosomes after thawing was lower using the Andersen method and the spermatozoa had a higher thermoresistance. This new triple staining method for assessing canine sperm viability and acrosomal integrity provides an efficient procedure for evaluating frozen-thawed dog semen samples either by flow cytometry or fluorescence microscopy. PMID- 10735105 TI - Semen quality in dogs and the influence of a short-interval second ejaculation. AB - Semen quality was examined in each of 65 known fertile dogs. Values were found to be similar to those previously published, although an apparent breed influence was demonstrated, with German shepherd dogs producing ejaculates of larger volume and greater total spermatozoal output than other breeds. A second ejaculate was collected from each dog with a mean interval of 63 min. The second samples had significantly lower values for the volume of the second fraction, the spermatozoal concentration and the total spermatozoal output. There were no differences for the percentage motility or the percentage of morphologically normal live spermatozoa. While there was no increase in semen quality of the second ejaculate, the technique may be useful since it results in the collection of approximately 70% more spermatozoa than a single ejaculate. These spermatozoa also had normal motility and morphology and could, therefore, be used for insemination or cryopreservation. PMID- 10735106 TI - Transvaginal, ultrasound-guided biopsy of the corpus luteum in cattle. AB - An ultrasound-guided transvaginal technique for corpus luteum biopsy was developed and tested in cattle. The biopsy needle set consisted of an inner needle (o.d. 1 mm) with a 20-mm long specimen notch, an outer cannula (o.d. 1.2 mm) with a cutting edge, and an automated spring-loaded handle with trigger. The biopsy needle set was inserted into the channel guide of the handle of a convex array transvaginal ultrasound probe. The transducer was positioned in the vaginal fornix, and the ovary was manipulated transrectally against the vaginal wall and transducer face. During monitoring on the ultrasound screen, the inner needle was pushed through the vaginal wall into the corpus luteum, and the cutting cannula was fired, cutting and trapping luteal tissue in the specimen notch. Three luteal biopsies at each of Hours 0 and 4 were taken 10 d after ovulation in 6 heifers; 6 other heifers served as controls. A biopsy core was obtained in 36 of 39 attempts (92%). The tissue specimens seemed normal based on gross evaluation. The effect of biopsy on luteal function was assessed by daily ultrasound monitoring of luteal area, by assay of progesterone concentrations in blood samples obtained daily, and by the length of the interval from biopsy to ovulation. No significant differences were found for post-biopsy function for any of the 3 end points. The results indicated repeated transvaginal, ultrasound-guided biopsy of the corpus luteum in cattle is a practical procedure and may be useful for experimental and diagnostic purposes. PMID- 10735107 TI - Plasma estradiol FSH and LH concentration after dominant follicle aspiration in the cow. AB - This work investigates the estrogenic role of the dominant follicle with regard to regulation of plasma FSH and LH concentration. Eight Holstein-Friesian cows were used for aspiration of the dominant follicle using ultrasound guidance during the early, mid and late stages of the luteal phase. Blood samples were collected at 15-min intervals from 4 h before until 7 h after aspiration. Plasma progesterone concentration increased from 0.7 to 7.2 ng mL-1 from early to mid luteal phase and then fell slightly to 5.9 ng mL-1 in the late luteal phase, but remained unaffected by follicle puncture. The follicular aspirate contained a thousandfold higher estradiol, than plasma concentration but its estradiol:progesterone ratio remained at around 2 at each stage of the luteal phase. Aspiration caused plasma estradiol concentration to fall from 1.4 to 0.7, 1.8 to 1.0 and 1.7 to 0.8 pg mL-1 in the early, mid and late stages of the luteal phase, respectively (P < 0.05). At the same time, mean plasma FSH concentration was increased from 1.1 to 1.8, 1.7 to 2.9 and 0.8 to 1.9 ng mL-1 (P < 0.05), respectively. The results suggest that estradiol secreted from dominant follicles selectively regulates gonadotropin secretion, since aspiration of the dominant follicle at any stage of the cycle affected circulating FSH but did not appear to influence the mean LH concentration. PMID- 10735108 TI - Follicular and FSH dynamics in ewes with a history of high and low ovulation rates. AB - Daily transrectal ultrasound scanning and twice-daily blood sampling were used to monitor the temporal relationships between FSH concentrations and follicle development during complete interovulatory intervals for ewes in which the ovulation rate in each of the 2 previous years was high or low (> or = 3 and < or = 2 ovulations, respectively). Follicles that reached > or = 5 mm were used to define a follicular wave and were tracked retrospectively to 3 mm (emergence). The hypothesis that FSH surges (identified with a computer program) and follicular waves (retrospectively determined based on ultrasound scanning) are temporally associated was supported in both groups by the emergence of an anovulatory or ovulatory follicular wave near the peak of an FSH surge. Further support for the hypothesis was a significant increase in FSH concentrations before and a significant decrease after follicular-wave emergence in both groups independent of the identification of FSH surges. Ewes with a history of high ovulation rates had smaller follicles (anovulatory and ovulatory) and more ovulations, but the 2 groups were similar in the number of ovulatory follicular waves and associated FSH surges, number and characteristics of the FSH surges, and mean FSH concentrations per interovulatory interval. Surges of FSH were periodic (every 3 or 4 d) regardless of the ovulation-rate group or follicle response. In ewes with a low ovulation rate, the nonovulatory FSH surges were most frequently associated with emergence of detected anovulatory follicular waves. In ewes with a high ovulation rate, more FSH surges were not associated with a detected follicular wave, as defined, presumably because the largest follicle did not reach 5 mm. The results indicated that the factors resulting in a high ovulation rate were not exerted through circulatory patterns or concentrations of FSH but involved a shorter growth phase and smaller maximal diameter of follicles. PMID- 10735109 TI - Bovine spermatozoal head size variation and evaluation of a separation technique based on this size. AB - The objectives of this study were to determine the variation of head areas of normal spermatozoa attributable to breed, individual bull and ejaculate and to verify separation of X and Y chromosome-bearing spermatozoa and separation effectiveness. Spermatozoa were evaluated using video enhanced contrast microscopy combined with video intensified fluorescent microscopy and the polymerase chain reaction (PCR). In Experiment 1, spermatozoal head areas were measured from 2 ejaculates collected from bulls of 3 beef and 2 dairy breeds. No differences in head areas were found between breeds or between bulls within breeds; variation was observed among ejaculates from individual bulls across breeds. In Experiment 2, spermatozoa from 5 ejaculates were separated on individual SEPDEVICEs (Patented). Head area, fluorescent intensity and PCR of spermatozoa retained in the SEPDEVICEs suggested a separation based on size in 1 of 5 samples. Ejaculate variation in head areas affected separation efficiency. PMID- 10735110 TI - Effect of timing of artificial insemination on gender ratio in beef cattle. AB - It was recently reported that cows inseminated at approximately 10 or 20 h before an expected ovulation deliver predominately a bull or heifer calf, respectively. The objective of this study was to further investigate the effect of timing of insemination on the gender of offspring in cattle. Angus heifers (n = 41) and cows (n = 98) were used in the study. Heifers were synchronized with a 16-d treatment of melengestrol acetate followed 17 d later with an injection of PGF2alpha. Cows were synchronized with GnRH followed 7 d later with PGF2alpha. A HeatWatch electronic estrus detection system was used to determine the onset of estrus. Based on previous studies, it was assumed that ovulation occurs approximately 32 h after the onset of estrus. Therefore, animals were artificially inseminated at either 8 to 10 h (early; > or = 20 h before expected ovulation) or 20 to 25 h (late; < or = 10 h before expected ovulation) after the onset of estrus. Sixty to 80 d after insemination, ultrasonography was used to confirm pregnancy status and to determine the gender of fetuses. Gender of calves was subsequently confirmed at calving. Data were analyzed for effects of time of insemination and sire or semen batch on gender ratio, as well as any effect of length and/or intensity of estrus on conception rate and gender ratio. Twenty nine of 41 heifers and 69 of 98 cows were detected in estrus after synchronization and were inseminated; 20 of 29 heifers and 48 of 69 cows were subsequently confirmed pregnant. Neither the length of estrus nor its intensity (number of mounts) had an effect on pregnancy rate or gender ratio (P > or = 0.418). Timing of insemination (early versus late) had no effect on gender ratio (P = 0.887). Semen from 13 sires representing 17 lots was used to inseminate the cows and heifers. No differences (P = 0.494) were detected in the gender ratios resulting from different sires or semen batches. In contrast to previous findings, our results indicate that inseminating beef cattle at approximately 20 or 10 h before an expected ovulation does not alter the gender ratio of the resultant calves. PMID- 10735111 TI - Fluorescence in situ hybridization with Y chromosome-specific probe in decondensed bovine spermatozoa. AB - This study was carried out to demonstrate bovine Y chromosome-bearing spermatozoa by rapid fluorescence in situ hybridization (FISH), using a digoxigenin (Dig) labeled DNA probe specific to bovine Y chromosome. Before the FISH procedure, sperm heads were treated for decondensation with dithiothreitol (DTT) and glutathione (GSH) with or without heparin supplementation. Concentrations of either above 2 mM DTT or above 100 mM GSH induced swelling of the sperm head, which resulted in sufficient detection of the Y chromosome signal in sperm nuclei by rapid FISH (49.8 to 53.4%). When FISH was used with 2 mM DTT or 100 mM GSH on specimens from 7 sires, the rate of detection of the Y chromosome signal varied among sires (5.4 to 49.6%), especially that of the GSH treatment. Supplementation of GSH with heparin (100 U/mL), however, could induce reliable, repeatable detection of the Y chromosome signal in sperm nuclei of all the 7 sires (48.4 to 50.3%). These results show that in bovine spermatozoa decondensed with GSH and heparin, rapid FISH can detect Y chromosome-bearing spermatozoa. PMID- 10735112 TI - Serum luteinizing hormone, testosterone, and thyroxine and growth responses of ram lambs fed locoweed (Oxytropis sericea) and treated with vitamin E/selenium. AB - Sixteen ram lambs (5 m.o. old, 45 +/- 1.5 kg) received a control diet (50% concentrate, no locoweed, n = 4), locoweed (20% locoweed for 21 d, n = 4), MUSE (2 mL i.m. of MUSE containing 5 mg selenium and 50 mg vitamin E/mL, n = 4) on Days 21 and 35([Day 0 = first day of trial]), or locoweed + MUSE (n = 4). The rams were maintained in individual pens (3 x 9 m) with free access to feed, water, salt and shade. On Day 7 after initiating locoweed, serum alkaline phosphatase (AP) increased (P < 0.01), and serum thyroxine (T4) decreased (P < 0.01) in locoweed-fed rams. Effects on serum AP and T4 remained constant in rams during the 21 d of locoweed feeding. Treatment with MUSE did not influence (P > 0.10) AP or T4. Locoweed-fed rams had reduced (P < 0.05) intake and body weight for the 2-wk period after locoweed feeding ended. The MUSE regimen or diet had no effect on intake or body weight (P > 0.50). Neither locoweed nor MUSE affected serum LH before or after GnRH administration on Day 22 (P > 0.10). On Day 50, however, area under the LH curve (AUC) was 966 units in locoweed-fed rams and 1,373 units (+/- 154) in controls (P = 0.09). Serum testosterone (T) was reduced in locoweed-fed rams before and after (P < 0.05) GnRH on Day 22. On Day 50, the T AUC was numerically lower (P = 0.14) in locoweed-fed rams (1,252 units) than in controls (1,539 +/- 130 units). Conversely, MUSE treatment resulted in increased (P = 0.02) T AUC on Day 50 (1,148 and 1,643 +/- 130 units in control and MUSE treated ram lambs, respectively). During the 6-wk period after locoweed feeding, serum immunoglobulin G averaged 14.0 and 18.6 (+/- 1.1) mg/mL in control and locoweed-fed rams (P < 0.01), respectively. Twenty percent dietary locoweed for 21 d exerts adverse effects on feed intake, growth, and reproduction in young ram lambs and MUSE was not effective in reversing these effects. PMID- 10735113 TI - Synchronization rate, size of the ovulatory follicle, and pregnancy rate after synchronization of ovulation beginning on different days of the estrous cycle in lactating dairy cows. AB - Recently a protocol was developed that precisely synchronizes the time of ovulation in lactating dairy cows (Ovsynch; GnRH-7d-PGF2 alpha-2d-GnRH). We evaluated whether initiation of Ovsynch on different days of the estrous cycle altered the effectiveness of this protocol. The percentage of cows (n = 156) ovulating to the first GnRH was 64% and varied (P < 0.01) by stage of estrous cycle. Treatment with PGF2 alpha was effective, with 93% of cows having low progesterone at second GnRH. The overall percentage of cows that ovulated after second GnRH (synchronization rate) was 87% and varied by response to first GnRH (92% if ovulation to first GnRH vs 79% if no ovulation; P < 0.05). There were 6% of cows that ovulated before the second injection of GnRH and 7% with no detectable ovulation by 48 h after second GnRH. Maximal diameter of the ovulatory follicle varied by stage of estrous cycle, with cows in which Ovsynch was initiated at midcycle having the smallest follicles. In addition, milk production and serum progesterone concentration on the day of PGF2 alpha affected (P < 0.05) size of the ovulatory follicle. Using these results we analyzed pregnancy rate at Days 28 and 98 after AI for cows (n = 404) in which Ovsynch was initiated on known days of the estrous cycle. Pregnancy rate was lower for cows expected to ovulate larger follicles than those expected to ovulate smaller follicles (P < 0.05; 32 vs 42%). Thus, although overall synchronization rate with Ovsynch was above 85%, there were clear differences in response according to day of protocol initiation. Cows in which Ovsynch was initiated near midcycle had smaller ovulatory follicles and greater pregnancy rates. PMID- 10735114 TI - Selection of the dominant follicle in cattle: establishment of follicle deviation in less than 8 hours through depression of FSH concentrations. AB - Deviation in follicle diameter in cattle is characterized by continued growth of the largest follicle of a follicular wave and a reduction or cessation of growth of the smaller follicles. Deviation begins when the largest follicle reaches about 8.5 mm. Two experiments were done to test the hypothesis that the deviation mechanism is established in < 8 h, as indicated by the temporal relationships between follicle removal and an increase in FSH concentrations (Experiment 1) and between a decrease in FSH concentrations and follicle inhibition (Experiment 2). In Experiment 1, the role of the first follicle to reach 8.5 mm was studied by follicle ablation (Hour 0). The combined mean FSH concentrations for the control group (n = 8) and ablation group before ablation (n = 7) progressively decreased (P < 0.02) over two 8-h intervals before the largest follicle reached > or = 8.5 mm (Hour-16, 1.77 +/- 0.11 ng/mL; Hour 0, 1.49 +/- 0.08 ng/mL). In controls, the concentrations continued to decrease (P < 0.02) until Hour 10 (1.21 +/- 0.09 ng/mL). Ablation of the largest follicle at > or = 8.5 mm resulted in increased (P < 0.02) circulating FSH concentrations between Hours 5 (1.34 +/- 0.04 ng/mL) and 8 (1.61 +/- 0.09 ng/mL). Growth rate of the second-largest follicle between Hours 0 and 8 was greater (P < 0.05) in the ablation group than in the controls, and the second largest follicle became dominant in 7 of 7 heifers following ablation of the largest follicle. In Experiment 2, a minimal single injection of a depressant of FSH concentrations (4.4 mL of steroid-reduced follicular fluid) was given when the largest follicle was a mean of 8.4 mm (Hour 0; controls, n = 4; treated, n = 4). An interaction of group and hour (P < 0.005) for FSH concentrations was attributable to an FSH decrease (P < 0.002) by Hour 6 and an increase (P < 0.002) between Hours 9 and 12 in the treated group. The growth rate of the largest follicle between Hours 0 and 12 was less (P < 0.05) in the treated group (0.2 +/- 0.2 mm/12 h) than in the control group (1.2 +/- 0.4 mm/12 h). The reduced diameter was recorded within 6 h after suppression of FSH concentrations, supporting the hypothesis. Our preferred interpretation is that when the largest follicle reaches a critical diameter of about > or = 8.5 mm, FSH concentrations continue to decrease and become lower than required by the smaller follicles but not the largest follicle. The results further indicate that a close temporal coupling between a change in FSH concentrations and the follicular response could establish the deviation mechanism in < 8 h or before the second largest follicle reaches a similar critical diameter. PMID- 10735115 TI - Pregnancy-associated glycoprotein levels in pregnant goats inoculated with Toxoplasma gondii or Listeria monocytogenes: a retrospective study. AB - The pregnancy-associated glycoprotein (PAG) concentration profiles of goats that had been experimentally inoculated with either Toxoplasma gondii or Listeria monocytogenes are described. All goats were examined regularly by ultrasonography. In T. gondii-infected females (n = 5), a slow decrease of PAG was observed throughout a period of 55 to 74 d after inoculation. Afterwards, the goats either aborted (n = 4) or kidded 1 dead and 1 weak fetus (n = 1). In L. monocytogenes-infected females (n = 8), a marked decrease of PAG was observed from the day of inoculation. Abortion occurred within 9 to 11 d post inoculation (n = 7). Only 1 goat kidded a healthy fetus. PMID- 10735116 TI - Ovum pick-up in sheep: efficiency of in vitro embryo production, vitrification and birth of offspring. AB - The production of offspring involving available technologies like ovum pick-up, in vitro embryo production and cryopreservation has not been fully described in the sheep. We tested the overall efficiency of these procedures on 20 Sarda dairy ewes that were twice stimulated for recovery of follicular oocytes. In total, 415 oocytes were aspirated from 522 follicles (11.5 oocytes/ewe), and 328 of them (9.1 oocytes/ewe) were selected for in vitro embryo production procedure. Development into blastocysts occurred in 98 embryos (2.7 blastocysts/ewe), of which 64 were vitrified and 34 were transferred, in pairs, directly to recipients. The pregnancy rate, diagnosed at 80 d for fresh and vitrified embryos, did not differ significantly (47.1 vs 42.8%, respectively), but there were significant differences in lambing rates between the 2 groups (41.2 vs 23.8%, respectively). Overall, 24 lambs were born; all weighed within the range for the breed, but head deformities were observed in 2 cases. The results of this study show that with application of the above techniques, it is possible to obtain repeatedly embryos and viable offspring. PMID- 10735117 TI - Effect of time during transport of excised mare ovaries on oocyte recovery rate and quality after in vitro maturation. AB - In the mare only a limited number of oocytes can be successfully collected in vivo, so that when large numbers of oocytes are needed for experimentation, ovaries harvested from slaughtered mares must be used. The resulting temperature changes and time intervals mandated by handling and transport of ovaries from the slaughterhouse to the laboratory adversely affect the rate of oocyte recovery and their quality after IVF and maturation. We chose to study the effect of temperature and time in transit of excised ovaries by evaluating rate of oocyte recovery, nuclear maturation stage reached before, and cleavage rate reached after IVF, following short (1.5 to 4 h) and long (6 to 8 h) storage. Temperatures in the storage container decreased from 37-C to 32 degrees and 27.5 degrees C during the short and long interval, respectively. The cumulus-oocytes complexes (COCs) were classified as having a compact cumulus, completely or partially surrounding the oocyte (compact); those having only a corona radiata surrounding the oocyte (corona); those having a completely or partially expanded cumulus, showing a cellular or sparsely cellular, gelatinous cloud around the oocyte (expanded); and those that were completely denuded of both cumulus and corona cells (denuded). All COCs, except the denuded ones, which were discarded, were matured in vitro for 30 h at 38.5 degrees C in 5% CO2. The recovery rate of oocytes was significantly higher after long vs short storage (48 vs 35%; P < 0.01), but the distribution of the collected COCs into the 4 classes was not affected by the storage time. After in vitro maturation nuclear maturity was not affected by the storage time, but oocytes with intact cytoplasmic membranes were more frequently found after short than after long storage (54 vs 34%; P = 0.07), and fully matured oocytes were more often seen with intact membrane (P < 0.01). Moreover, oocytes with intact membranes in metaphase II (MII) were associated with short storage intervals and the corona COC class, while damaged membranes and incomplete maturation were associated with the long storage and the compact COC class. PMID- 10735118 TI - Morphology and motility of spermatozoa from different regions of the epididymal duct in the domestic cat. AB - Spermatozoa undergo important maturational changes as they pass through the epididymal duct. Some domestic cats and many species of wild felids have high proportions of abnormal spermatozoa in their ejaculates. The epididymis has been shown to be able to remove certain abnormal sperm forms in some species while other sperm abnormalities originate in the epididymis. So far, it has not been shown how the epididymis affects sperm morphology in the domestic cat. Therefore, motility and sperm morphology were studied in spermatozoa from the efferent ducts and from the 6 regions of the epididymal duct. There were significant decreases in the proportions of spermatozoa with abnormalities of the sperm head, acrosomal defects, acrosomal abnormalities and in the proportion of midpiece abnormalities. In contrast, there was a small but significant increase in the proportion of spermatozoa with abnormalities of the tail. Spermatozoa acquired the capacity for motility in Region 4, where the cytoplasmic droplet also moved from a proximal to a distal position, indicating that important maturational changes take place in this region. The results of this study demonstrate that the proportions of sperm abnormalities originating in the testes decrease during epididymal transport, while some sperm tail abnormalities may actually originate in the epididymis. PMID- 10735119 TI - Relationships between calf birth weight, prepartum concentrations of plasma energy metabolites and resumption of ovulation postpartum in Limousine suckled beef cows. AB - The aim of this study was to determine the relationship between energy status before calving and calf birth weight and their potential effects on interval between calving and first ovulation. Sixty-nine Limousine, suckled beef cows were sampled weekly over a 3-yr period during the last 2 m.o. of pregnancy to determine the concentrations of nonesterified fatty acids (NEFA), beta-3 hydroxybutyrate (beta-OHB), glucose and glycerol. After parturition, progesterone concentrations were measured weekly to determine time of resumption of ovulation. Cows were allotted to 3 groups according to calf birth weight (Heavy: > 44 kg, n = 37; Medium: 39 to 43 kg, n = 56; and Light: < 38 kg, n = 45) and to postpartum ovarian resumption of cyclicity (Late: > 11 wk, n = 41; Mid: 7 to 10 wk, n = 57; and Early: < 6 wk, n = 40). Puerperium glycaemia of the dams was steady state (0.66 +/- 0.03 g/L) and was not related to calf birth weight. Plasma NEFA, beta OHB and glycerol values were higher (P < 0.05) in Heavy than in Medium and Light group dams during the last 4 wk of pregnancy. Interval between calving and first ovulation was significantly longer for primiparous than for multiparous cows (respectively, 9.9 +/- 2.0 and 7.7 +/- 1.4 wk; P < 0.05). Calf birth weight was not related to time of first ovulation. Late primiparous cows had higher NEFA plasma concentrations than Mid and Early group primiparous cows during the last 4 wk of pregnancy, whereas NEFA plasma concentrations were not related to interval between calving and first ovulation in multiparous cows. Thus, lipomobilization increased with calf birth weight during the last 4 wk of pregnancy. High level of body reserves mobilization was associated with delayed first ovulation in primiparous but not in multiparous cows. PMID- 10735120 TI - Prostaglandin E2, prostaglandin F2 alpha and endothelin-1 production by cow oviductal epithelial cell monolayers: effect of progesterone, estradiol 17 beta, oxytocin and luteinizing hormone. AB - The optimal oviductal environment, including contractile activity for gamete transport, fertilization and early embryonic development, is mediated by physiological and anatomical changes in the oviduct during the estrous cycle. Oviductal epithelial cell culture was utilized to investigate the effect of ovarian steroids (progesterone [P4] and estradiol 17 beta [E2]), oxytocin (OT) and luteinizing hormone (LH) on the local production of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha) and endothelin-1 (ET-1) in the cow oviduct. Epithelial cells isolated from oviducts collected during the follicular phase were cultured in M199 under standard culture conditions until monolayer formation. Then the cells were trypsinized and plated at a density of 3 x 10(4)/mL/well and cultured again until subconfluency, at which time the cells were incubated for 4 or 24 h with M199 only (control), high P4 (H-P4; 1 microgram/mL), low P4 (L-P4; 10 ng/mL), E2 (1 ng/mL), LH (10 ng/mL), OT (10(-9) M) ET-1 (10(-9) M), PGE2 (10(-8) M) PGF2 alpha (10(-9) M) or their combination (H P4 + E2, L-P4 + E2, LH + E2, ET-1 + E2, L-P4 + E2 + LH and H-P4 + E2 + LH). The production of both PG and ET-1 was increased by E2 + low P4 and LH + E2 + low P4 (P < 0.05), while LH + E2 enhanced the production of PGF2 alpha and ET-1 (P < 0.05). Moreover, E2 + ET-1 stimulated PG production (P < 0.05). However, OT had no effect on the production of any of these substances. These results suggest that the preovulatory LH surge, together with locally re-circulated high levels of E2 from the Graafian follicle and basal P4 from regressing corpus luteum (CL), induces the maximum stimulatory effect on oviductal PGE2, PGF2 alpha and ET-1 production during the periovulatory period. Consequently, the elevated local ET-1 concentration during periovulatory period may induce the high contractile activity of the oviduct and, at the same time, the stimulation of PG production. Thus, ET-1 may act as a local amplifier for oviductal PG production stimulated by LH and ovarian steroids. PMID- 10735121 TI - Echotextural changes in the follicular wall during follicle deviation in mares. AB - The echotextural changes occurring in the follicular wall in association with deviation in diameters were studied in 8 pony mares. Echotextural changes could be useful as a reference point for future studies of the follicle-selection phenomenon. Follicles were examined daily by transrectal ultrasound from 3 d before to 3 d after the beginning of deviation (Day 0). The following echotextural end points were recorded based on a scoring or percentage system: 1) thickness of granulosa, 2) echogenicity of granulosa, 3) prominence of an anechoic layer located beneath the granulosa, and 4) extent of involvement of the circumference of the follicle by the anechoic layer. Significant main effects indicated increasing values over Days -3 to 3 and a higher mean value for the dominant-follicle status for each end point. The interaction approached significance for thickness of granulosa and was significant for each of the other 3 end points. The interaction seemed due primarily to lower values in the subordinate follicle on Days 2 and 3. The first day of an overall echotextural difference between the 2 largest follicles (15.9 +/- 0.5 d after ovulation) or between days within the largest follicle (16.1 +/- 0.5 d) was about 1 d earlier (P < 0.05) than the beginning of diameter deviation (17.4 +/- 0.8). The effects of follicle diameter versus future follicle status were compared for Days -3 to 0 by grouping follicles for each status into 2 diameter categories (16.0 to 18.9 mm and 19.0 to 22.5 mm). Increasing echotextural values for the granulosa (thickness and echogenicity) were more attributable to follicle growth, whereas the increasing values for the anechoic layer were more attributable to status as a future dominant follicle. Results indicated that development of an anechoic layer beneath the granulosa distinguished the future dominant follicle from the largest future subordinate follicle before the beginning of diameter deviation. PMID- 10735122 TI - Birth of a Holstein freemartin calf co-twinned to a schistosomus reflexus fetus. AB - An unusual case of a live Holstein freemartin calf co-twinned with schistosomus reflexus fetus is presented here. Delivery of the schistosomus reflexus was achieved by fetotomy 24 h after manual delivery of a live heifer calf. The dam subsequently experienced concurrent metritis and left displacement of the abomasum; however, she conceived following insemination 85 d post partum. Cytogenetic examination of the live heifer using lymphocyte culture and cutaneous fibroblast cell culture failed to demonstrate chromosomal chimerism, whereas polymerase chain reaction (PCR) detected the presence of the bovine Y-chromosome marker BRY-1. Low concentrations of testosterone, estradiol and progesterone at 3, 6, 24 and 48 h after administration of hCG were detected in the serum of the freemartin heifer. Genetic, hormonal, histological and clinical findings established the live female co-twin calf was a freemartin, an abnormality of phenotypic sex. Failure to detect any significant peripheral concentrations of androgen supports the hypothesis that masculinization of the freemartin reproductive tract arises from diffusion of androgen and possibly other substances from the male co-twin rather than from endogenous production of androgen within the freemartin. This report documents that the freemartin condition can be induced by a male fetus with severe developmental abnormalities. PMID- 10735123 TI - Estrous cycle characteristics and response to estrus synchronization in mammoth asses (Equus asinus americanus). AB - Breeding records from a herd of mammoth asses (Equus asinus americanus) maintained on pasture in southeast Texas from 1990 to 1998 were reviewed. Jennies were pasture or hand mated, and estrus was either observed while the jennies were on pasture or when exposed to a jack after being penned. Eighty-one estrus periods and 43 diestrus intervals were recorded in 33 jennies over 4 seasons of the year (January-March, April-June, July-September, and October-December). Estrous cycle length and the duration of estrus were similar among seasons. Over all seasons, estrous cycle length was 23.3 +/- 2.6 d, duration of estrus was 5.9 +/- 2.1 d, and diestrus length was 17.4 +/- 2.6 d (mean +/- SD). During these same 9 yr, 58 injections of PGF2 alpha (5 mg, i.m.) were administered to 38 jennies without regard to stage of estrous cycle. Seventy-six percent (44/58) of the jennies showed signs of estrus after PGF2 alpha treatment, with an interval to estrus of 4.4 +/- 1.6 d and a duration of estrus of 5.6 +/- 1.7 d. Two estrus synchronization schemes were also assessed. Trial 1 was performed in October to November 1996, and Trial 2 was performed in February to March 1998. In Trial 1 (Group PE + PGF, n = 10), each jenny was injected intramuscularly once daily for 10 d with 150 mg progesterone and 10 mg estradiol-17 beta in sesame oil, and PGF2 alpha (10 mg) was injected intramuscularly on the last day of treatment. In Trial 2 (Group PGF-2X, n = 11), each jenny was injected intramuscularly twice, 16 d apart, with 10 mg PGF2 alpha. All Group PE + PGF jennies responded to treatment. One jenny in Group PGF-2X did not respond to either injection of PGF2 alpha, while 2 jennies responded to the first but not the second PGF2 alpha injection (8 of 11 jennies returned to estrus and ovulated after the second PGF2 alpha injection). Duration of estrus was 6.8 +/- 1.9 d for Group PE + PGF and 7.1 +/- 1.8 d for Group PGF-2X jennies. Interval to estrus and interval to ovulation following the last treatment were 9.0 +/- 0.9 d and 14.5 +/- 1.7 d, respectively, in Group PE + PGF jennies, and 4.5 +/- 0.9 d and 10.4 +/- 1.8 d, respectively, for Group PGF-2X jennies. In summary, estrous cycle characteristics of mammoth asses are similar to those reported for standard jennies, and estrus synchronization schemes used in horses are effective in mammoth asses. PMID- 10735124 TI - Contraceptive potential of the porcine zona pellucida vaccine in the African elephant (Loxodonta africana). AB - Immunocontraception has been successful in controlling free-roaming equids; however, what is the potential for the immunocontraceptive control of the African elephant (Loxodonta africana)? The porcine zona pellucida (pZP) glycoproteins share antigenic domains with the African elephant zona pellucida (elZP) glycoproteins, and anti-zona pellucida serum antibodies have been successfully stimulated. To determine the cross-reactivity of the pZP and elZP, immunocytochemistry was evaluated by light and electron microscopy. Specifically, the binding of polyclonal antibodies against total heat-solubilized-porcine zona pellucida to fixed elephant ovary sections was evaluated. The elZP of primary, secondary and tertiary follicles was recognized by the rabbit-anti-pZP serum, but there was no apparent recognition of the primordial follicles. The ability of anti-pZP antibodies to recognize the elZP demonstrates that there is molecular homology between the pZP and elZP glycoproteins. This homology makes the African elephant a candidate for pZP immunocontraception. Three captive elephants were vaccinated with 400 micrograms pZP with a synthetic trehalose dicorynomycolate (S TDCM) adjuvant. The elephants received 2 boosters of 600 micrograms pZP at 4 wk and 10 m.o. after the primary vaccination. The vaccinated female elephants developed significant (P < 0.05) titers to pZP over prevaccination levels. These levels persisted for 12 to 14 m.o. after the third vaccination. This preliminary evidence shows that the female elephant can develop significant serum antibody levels to pZP. These levels of antibodies are comparable to those required in horses for successful immunocontraception. Thus, porcine zona pellucida immunocontraception might be used to control elephant populations. PMID- 10735125 TI - Effect of culture media on embryo development from prepubertal goat IVM-IVF oocytes. AB - Experiments were carried out to develop and improve in vitro culture systems for IVM-IVF prepubertal goat oocytes. Cumulus oocyte complexes (COC) were obtained by slicing ovaries from slaughtered prepubertal goats. Oocytes were matured in TCM 199 supplemented with 20% estrous goat serum (EGS) + 10 micrograms/mL FSH + 10 micrograms/mL LH + 1 microgram/mL estradiol 17 beta for 27 h at 38.5 degrees C in 5% CO2 in air. Matured oocytes were placed in drops of TALP- fert medium supplemented with hypotaurine (1 microgram/mL) and inseminated with freshly ejaculated spermatozoa following capacitation as described by Younis et al. (69) but with 100 micrograms/mL heparin. At 24 h post insemination the ova were transferred to various in vitro culture media, and early embryo development was evaluated until Day 8 post insemination. Specifically, in the studies described here, we have compared the effects of (Experiment 1) co-culture systems using oviductal ephitelial cells (OEC) and cumulus cells (CC), both caprine and bovine; (Experiment 2) the presence of serum and/or OEC; (Experiment 3) 4 culture media (TCM199, Ham's F10, CZB abd SOF) for co-culture with OEC; and (Experiment 4) conditioned medium with OEC. In Experiment 1, the percentage of morulae plus blastocysts was higher in culture with OEC, both caprine and bovine (15.1 and 14.8%, respectively) than with CC (4.1 and 6.7%, respectively). In Experiment 2, the OEC with EGS did not improve the percentage of morulae and blastocysts obtained with OEC alone (14.3 and 23.1% respectively). In Experiment 3, this percentage was higher using OEC with TCM-199 compared to CZB medium (21.3 and 12.3%, respectively) and in Experiment 4, the results were 3.7, 11.2 and 21.3% for TCM-199 without cells, Conditioned Medium and co-culture with OEC, respectively. PMID- 10735126 TI - Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their correlation with fertility. AB - The objectives of this study were to 1) identify proteins found in stallion seminal plasma utilizing two-dimensional polyacrylamide gel electrophoresis (2D PAGE) in conjunction with Western blot analysis; and 2) to determine if any of these individual proteins were correlated with stallion fertility utilizing regression analysis. Fertility was quantified by assigning a breeding score for each stallion. Each score was calculated by dividing the number of conceptions by the number of breedings for each stallion for four successive breeding seasons (1992-1995). Ejaculates from stallions of known fertility (n = 6) were collected with a Missouri-style artificial vagina. Immediately after collection, the semen sample was filtered and the gel fraction removed. The resultant sperm-rich fraction was centrifuged in a Beckman Microfuge E at 10,000 x g and the seminal plasma aspirated from the pelleted sperm cells. Two-dimensional PAGE of the seminal plasma was performed under denaturing conditions which revealed that 14 proteins were common in all stallions in the research population. Four of these proteins (SP-1, SP-2, SP-3, and SP-4) were found to be significantly (P < 0.05) correlated with the breeding score assigned for each stallion. Regression analysis of protein optical densities with breeding score indicated that SP-1 (72 kDa, pI 5.6) was positively correlated with fertility (P < 0.05, r2 = 0.706), while SP-2 (75 kDa, pI 6.0), SP-3 (18 kDa, pI 4.3), and SP-4 (16 kDa, pI 6.5) were found to be negatively correlated (P < 0.05, r2 = 0.762, 0.730, 0.775 respectively) with fertility. Western blot analysis of SP-1 indicated there was an antigenic homology with a bovine 55 kDa fertility-associated seminal plasma protein identified in a study by Killian et al. (19). This suggests that the two proteins may have a similar physiological role and therefore common biological properties. These results indicate that analysis of stallion seminal plasma proteins can be used as an indicator of fertilizing capacity. Identification of such proteins in stallion seminal plasma could lead to better insight into the nature of subfertility or infertility in the horse, as well as to indicate better cryopreservation strategies. PMID- 10735127 TI - Generation of an equine oviductal epithelial cell line for the study of sperm oviduct interactions. AB - Equine oviductal epithelial cells (OEC) were transformed with simian virus 40 large T antigen (SV 40 T-ag) to create a cell line for the study of the interaction of equine spermatozoa with oviductal epithelium. One cell line was established based on the expression of the SV 40 T-ag and extended lifespan in culture. Immortalized equine OEC retained the characteristics of differentiated OEC such as the formation of monolayers with characteristic epithelial morphology and cell polarization as well as expression of cytokeratin and equine major histocompatibility complex I. Monolayers of immortalized equine OEC retained their functional competence to bind equine spermatozoa in a dose-dependent manner comparable to that of primary equine OEC cultures. This immortalized cell line of equine OEC provides a uniform, readily available system for sperm-OEC co cultures, and may be a useful model for the study of sperm-oviduct interactions in the horse. PMID- 10735128 TI - Quantity and infectivity of embryo-associated bovine viral diarrhea virus and antiviral influence of a blastocyst impede in vitro infection of uterine tubal cells. AB - In previous studies, bovine viral diarrhea virus (BVDV) remained associated with IVF embryos after viral exposure and washing. However, uterine tubal cells (UTC) were not infected when exposed embryos were washed and individually co-cultured with them. The objective of this study was to evaluate quantity and infectivity of embryo-associated virus and antiviral influence of a blastocyst as possible explanations for failure to infect the UTC in vitro. Morulae and blastocysts were produced in vitro and washed. A portion of the embryos were incubated for 2 h in medium containing 10(6) to 10(8) cell culture infective doses (50%, CCID50) of a genotype I, noncytopathic BVDV per milliliter and then washed again. Virus isolation was attempted on sonicated negative (virus unexposed) and positive (virus exposed) control embryo groups after washing. The influence of quantity and infectivity of embryo-associated virus was evaluated by transferring exposed, washed embryo groups (2, 5, and 10 embryos/group) or sonicate fluid of exposed, washed, sonicated embryo groups (2, 5, and 10 embryos/group) to cultures containing bovine UTC in IVC medium that was free of BVDV neutralizing activity. The antiviral influence of an embryo was evaluated by adding 1 to 10(5) CCID50 of BVDV to UTC in the presence or absence of a single unexposed blastocyst in IVC medium. After 2 d in co-culture, the UTC, IVC medium and washed embryos (when present) were tested separately for the presence of BVDV using virus isolation. Virus was isolated from sonicate fluids of all positive but no negative controls. Virus was not isolated from any UTC following 2 d of culture with virally exposed groups of intact embryos. However, virus was isolated from UTC cultured with sonicate fluids from some groups of 5 (60%) and 10 (40%) embryos. Infective virus also remained associated with some groups of 2 (20%), 5 (40%) and 10 (60%) intact embryos after 48 h of post-exposure culture. Finally, primary cultures of UTC were more susceptible to infection with BVDV in the absence of a blastocyst (P = 0.01). Results indicate that insufficient quantity and reduced infectivity of embryo-associated virus as well as an antiviral influence of intact IVF blastocysts may all contribute to failure of embryo-associated virus to infect UTC in vitro. PMID- 10735129 TI - Postpartum subestrus in dairy cows: comparison of treatment with prostaglandin F2 alpha or GnRH + prostaglandin F2 alpha + GnRH. AB - Two experiments (Experiment 1, 185 cows in 1996/97; Experiment 2, 168 cows in 1997/98) were conducted with Prim Holstein dairy cattle in the Mayenne region of France to investigate subestrus. Cows which had not been observed in estrus since calving were allocated alternately to treatment groups between 60 and 90 d post partum as follows: Experiment 1-Group 1: GnRH (Day 0, 100 micrograms i.m.), PGF2 alpha (Day 7, 25 mg i.m.), GnRH (Day 9, 100 micrograms i.m.) and AI (Day 10); Group 2: PGF2 alpha (Day 0, 25 mg i.m.), AI at estrus, or, if estrus was not observed, a second PGF2 alpha injection on Day 13, and AI on Day 16 and Day 17. Treatments in Experiment 2 were as follows: Group 1: as Experiment 1-Group 1 but AI at the observed estrus after Day 0, or at Day 10 if estrus was not observed; Group 2: as Experiment 1--Group 2, however, if a second PGF2 alpha injection was given on Day 13, AI at the observed estrus. Progesterone was measured in serum at Day 0 and in milk at AI. Pregnancy diagnosis was performed by measuring bovine pregnancy-specific protein B (bPSPB; Day 50 +/- 3) and confirmed by ultrasonography when the result was doubtful. In Experiment 1, farmers observed 47/101 (46.9%) Group 1 cows in estrus, 33/91 cows on Day 10 and 10 cows before Day 10. The progesterone concentrations were compatible with estrus in 69/86 (80%) cows on Day 10. In Group 2, 36/83 (43.4%) cows were inseminated after the first PGF2 alpha injection. After the second PGF2 alpha injection, only 29/43 (67%) cows had a low progesterone concentration at AI. Pregnancy rates were 36.1 and 32.5% for Groups 1 and 2, respectively. In Experiment 2, estrus was observed in 31/93 (33.7%) Group 1 cows. In Group 2, 51/75 (66%) cows were inseminated after the first injection of PGF2 alpha, 13/75 (17.3%) cows after the second injection, while 11/75 (14.7%) were not observed in estrus. Pregnancy rates were 53.7 and 53.3% in Groups 1 and 2, respectively. In conclusion, it is recommended that subestrus be treated with PGF2 alpha followed by AI at the observed estrus when estrus detection is good, while the use of GnRH + PGF2 alpha + GnRH is recommended when estrus detection is poor. PMID- 10735130 TI - Follicular and hormonal dynamics during the first follicular wave in heifers. AB - A few days after the first follicular wave emerges as 4-mm follicles, follicular deviation occurs wherein 1 follicle of the wave continues to grow (dominant follicle) while the others regress. The objectives of this study were to characterize follicle growth and associated changes in systemic concentrations of gonadotropins and estradiol at 8-h intervals encompassing the time of follicle deviation. Blood samples from heifers (n = 11) were collected and the ovaries scanned by ultrasound every 8 h from 48 h before to 112 h after the maximal value for the preovulatory LH surge. The follicular wave emerged at 5.8 +/- 5.5 h (mean +/- SEM) after the LH surge, and at this time the future dominant follicle (4.2 +/- 0.8 mm) was larger (P < 0.001) than the future largest subordinate follicle (3.6 +/- 0.1 mm). There was no difference in growth rates between the 2 follicles from emergence to the beginning of the deviation (0.5 mm/8 h for each follicle), indicating that, on average, the future dominant follicle maintained a size advantage over the future subordinate follicle. Deviation occurred when the 2 largest follicles were 8.3 +/- 0.2 and 7.8 +/- 0.2 mm in diameter, at 61.0 +/- 3.7 h after wave emergence. Diameter deviation was manifested between 2 adjacent examinations at 8-h intervals. Mean concentrations of FSH decreased, while mean concentrations of LH increased 24 and 32 h before deviation, respectively, and remained constant (no significant differences) for several 8-h intervals encompassing deviation. In addition to the increase and decrease in circulating estradiol concentrations associated with the preovulatory LH surge, an increase (P < 0.05) occurred between the beginning of deviation and 32 h after deviation. The results supported the hypotheses that deviation occurs rapidly (within 8 h), that elevated systemic LH concentrations are present during deviation, and that deviation is not preceded by an increase in systemic estradiol. PMID- 10735131 TI - Evaluation of the effect of GnRH on follicular ovarian cysts in dairy cows using trans-rectal ultrasonography. AB - The objectives of this study were to evaluate ovarian changes in cows with follicular ovarian cysts following treatment with either GnRH or saline. The parameters determined were the intervals from treatment to observation of a CL and from treatment to disappearance of the cyst, and the association between serum concentrations of LH, FSH and the LH/FSH ratio, before and after treatment, with the test intervals. Thirty-nine cows were identified as having follicular cysts. The GnRH treatment induced a significant increase in LH and the LH/FSH ratio. The gonadotropin response was not associated with the intervals from treatment to CL detection or to disappearance of the cyst. Survival curves for the intervals from treatment to CL detection and cyst disappearance indicate that treatment with GnRH or saline did not yield significantly different results for either parameter. The results question the efficacy of treating cystic ovarian disease with GnRH. PMID- 10735132 TI - The effect of media, serum and temperature on in vitro survival of bovine blastocysts after Open Pulled Straw (OPS) vitrification. AB - The recently introduced Open Pulled Straw (OPS) vitrification technique has successfully been used for cryopreserving porcine embryos as well as for bovine embryos and oocytes. The aim of this work is to investigate several factors on the in vitro survival of bovine blastocysts. In 5 experiments, a total of 862 in vitro produced blastocysts and expanded blastocysts was vitrified and warmed using the OPS technology, then cultured in vitro for an additional 3 days. The culture medium in Experiments 1 to 4 was SOFaa with supplements and 5% calf serum (CS). In Experiment 1, the replacement of TCM-199 + 20% CS with PBS + 20% CS in the holding medium during vitrification and warming did not result in significant differences in the re-expansion (92 vs 95%) and hatching rates (79 vs 72%). In Experiment 2, the PBS holding medium was supplemented with either 20% CS, 5 mg/mL bovine serum albumin (BSA) or 3 mg/mL polyvinylalcohol (PVA). Although the re expansion rates did not differ (98, 95 and 93%, respectively), there was a decrease in the hatching rate after vitrification with PVA (77 and 78 vs 51%, respectively). In Experiment 3, the influence of temperature of equilibration media prior to and rehydration media after the vitrification was investigated. When the temperature of these media was adjusted to 20 degrees C instead of the standard 35 degrees C, both the re-expansion and the hatching rates decreased markedly. However, increasing the time of equilibration with the diluted cryoprotectant solution at 20 degrees C eliminated these differences. In Experiment 4, the ethylene-glycol and dimethyl sulfoxide cryoprotectant mixture was replaced with ethylene glycol-ficoll-trehalose solution. No difference in the re-expansion (89 vs 96%, respectively) or hatching rate (79 vs 84%, respectively) was detected. In Experiment 5, the vitrified-warmed blastocysts were cultured in SOFaa medium supplemented with 5% CS or 5 mg/mL BSA. Although the re-expansion rates were identical in the 2 groups (95%), the hatching rates were lower when embryos were cultured in BSA (71 and 47%, respectively). These findings indicated the possible broader application for OPS, as they demonstrated that the physical advantages of rapid cooling and warming may be accompanied by different chemical composition (holding media, cryoprotective additives) according to the requirements of the biological structure. Our study also shows the need for serum supplementation of the medium for hatching to occur after OPS vitrification. PMID- 10735133 TI - [Von Hippel-Lindau disease]. AB - Von Hippel-Lindau disease (VHL) is an autosomal dominant inherited cancer syndrome. The disease was diagnosed in three patients: a 22-year-old woman who presented with decreased vision due to retinal angiomatosis and in whom a renal carcinoma was diagnosed five years later at a routine VHL analysis, a man aged 44 who died with bilateral renal cysts and clear-cell carcinomas with metastases, who had a positive family history but in whom no regular control investigations had been performed, and a 31-year-old man who also had a positive family history, but who presented with epididymal cystadenoma and who in the subsequent decades developed cerebral and renal neoplasms that all were detected at routine examinations and subsequently removed. These case histories emphasize the importance of: (a) early recognition of patients presenting with a 'sporadic VHL like' tumour, (b) frequent screening and early treatment of VHL patients and (c) nephron sparing surgery in VHL patients. The prognosis and quality of life of patients with VHL disease has significantly increased by early detection and treatment of asymptomatic tumours, in particular in the eye and kidney. PMID- 10735134 TI - [Insufficient scientific evidence for efficacy of widely used electrotherapy, laser therapy, and ultrasound treatment in physiotherapy]. AB - The Dutch Health Council recently published a report on the efficacy of electrotherapy, laser therapy and ultrasound treatment for musculoskeletal disorders. The assessment was based on three systematic reviews, including 169 randomized clinical trials, and focused on a best-evidence synthesis. Virtually no conclusive clinically relevant effects of the three forms of physical therapy were found. Possible exceptions are electrotherapy for osteoarthrosis of the hip or knee, laser therapy for pain treatment and rheumatoid arthritis, and ultrasound treatment for epicondylitis lateralis. But even for these putative indications, further research is clearly needed before implementation in practice is justifiable. It is strongly recommended that the current widespread use of electrotherapy, laser therapy and ultrasound treatment should be reduced, preferably by self-regulation within the profession itself. PMID- 10735135 TI - [Von Hippel-Lindau disease: protocols for diagnosis and periodical clinical monitoring. National Von Hippel-Lindau Disease Working Group]. AB - Von Hippel-Lindau disease (VHL) is an autosomal dominantly inherited syndrome with high penetrance, characterised by tumours in various organs. The Dutch VHL working group presents guidelines for DNA testing and clinical monitoring, to enhance early detection and treatment of VHL patients in the Netherlands. Diagnosis of VHL is justified in patients presenting with a typical VHL tumour with a positive family history, but patients with a VHL tumour and a negative family history may also have VHL. Diagnosis of VHL can be confirmed by molecular genetic analysis of the VHL gene which is informative in virtually all VHL families. In a patient with (suspicion for) VHL there is an indication for genetic counselling. A protocol for clinical monitoring of VHL is presented and is recommended for: carriers of a VHL germline mutation; members of VHL families with an unknown familial mutation; members of VHL families who decline testing of the familial mutation; patients suspected for VHL, but without a detectable VHL gene mutation. PMID- 10735136 TI - [Heat stroke: pathophysiology and pathogenesis]. AB - The pathophysiology of heat stroke is not fully understood. Endotoxins and cytokines appear to play a major part in the central nervous system and in the peripheral tissues. Use of specific receptor blockers prior to heat exposure can diminish cerebral damage in animal experiments. The energy depletion model of Hubbard gives a possible explanation for the rhabdomyolysis observed in some heat stroke patients. Failure of the ATP dependent Na(+)-K+ pump is essential in this theory. PMID- 10735137 TI - [Benefit of prehospital thrombolysis in the treatment of acute myocardial infarction]. AB - Randomized studies comparing early and late thrombolysis in the treatment of acute myocardial infarction show that mortality is lower if therapy is administered at an early stage, and especially if it is administered during the first hour of symptoms. Since only few patients can actually be treated within one or two hours, a search was made for time gaining strategies, including prehospital thrombolysis. In the region of Nijmegen, the Netherlands, it proved possible with prehospital treatment to treat 25% of the patients in the first hour after onset of ischaemic symptoms. The group given prehospital treatment included almost three times as many aborted infarctions as the group treated in hospital. In clinical practice, the proportion of aborted infarctions and more pronounced ECG abnormalities, namely, increase the probability of early prehospital treatment as well as the risk of death. Conditions of the organization of prehospital thrombolysis in a region are equipment for ECG transmission by telephone, good co-operation between cardiologists and cardiologic care units and special training of paramedics and general practitioners. PMID- 10735138 TI - [Angina pectoris and myocardial infarction: sex differences in risk profile, prognosis and referral by family physician]. AB - OBJECTIVE: To detect possible sex differences in risk factors, referrals and prognosis after angina pectoris (AP) and myocardial infarction (MI). DESIGN: Retrospective. METHOD: From 4 general practices in/around Nijmegen, the Netherlands, patients were selected who in 1985-1989 had a first episode of AP or a first MI. Sex differences regarding risk factors, referrals and prognosis were studied by means of patients' files. RESULTS: There were 71 men and 55 women with AP and 97 men and 58 women with MI. Mean age in AP was 69.6 year in women and 63.6 year in men, mean ages in MI were 74.9 and 64.4 year, respectively. Women with MI more frequently suffered from hypertension, AP or CVA/transient ischaemic attack (TIA). Men with MI were referred more often to a specialist than women, but not sooner. Development of cardiovascular co-morbidity after AP and after recovery from a MI were the same for both sexes. Of all patients with a MI 36% suffered a sudden unexpected death, mostly women. CONCLUSION: Women with a first episode of AP and a MI were 6-10 years older than men. Women with a MI suffered more frequently from hypertension, AP and CVA/TIA. The prognosis after angina pectoris showed no sex differences. The prognosis after MI was worse in women than in men. PMID- 10735139 TI - [Angina pectoris and normal coronary arteries: prevalence and prognosis in men and women]. AB - OBJECTIVE: To determine whether women with anamnestic suspicion of angina pectoris more often than men with suspicion of angina pectoris have normal coronary arteries on coronary arteriograms. Second, to compare the prognosis of these patients with that of the mean population. DESIGN: Literature study. METHOD: Articles were selected from Medline over the period 1966 to May 1998 with the use of search terms, and in De geinformeerde huisarts (The informed GP) over 1992 to November 1998 following which the relevance of the articles was assessed on the basis of title and abstract. Data on the prognosis of the general population were obtained from a WHO project. RESULTS: Three articles were selected about sex differences in the presence of angiographically normal coronary arteries in patients with suspected angina pectoris. In 2 articles normal coronary arteries were found more often in women (40-41%) than in men (8 12%). In 1 article women were found to have less severe and extensive coronary artery disease then men. Eight articles were selected about the prognosis of patients with suspected angina pectoris and angiographically normal coronary arteries. Per 1000 patient years the total number of deaths ranged from 0 to 6.59 (weighted mean: 4.05), the number of deaths due to coronary disease from 0 to 0.92 (0.47) and the number of patients with non fatal myocardial infarctions from 0 to 1.83 (0.94). These figures were similar to those in an average population. CONCLUSION: Women with anamnestic suspicion of angina pectoris have normal coronary angiograms more often than mean with anamnestic suspicion of angina pectoris. The prognosis of patients with suspected angina pectoris and normal coronary arteries in terms of mortality and number of non fatal myocardial infarctions is the same as in the general population. PMID- 10735140 TI - [Malignant intracranial germ cell tumor treated with chemotherapy and radiotherapy without histopathological confirmation]. AB - In two men aged 19 and 24 years, a rare malignant intracranial germ cell tumour was diagnosed in the pineal gland region and in the second patient in a suprasellar position as well. Although histological confirmation is required in general to diagnose a malignancy, obtaining it can be difficult and potentially dangerous in case of intracranial neoplasms. The clinical picture of a young patient with an intracerebral tumour localised in the midline of the brain and increased levels of the tumour markers alpha-foetoprotein and/or human chorion gonadotrophin (beta-HCG) in blood and/or CSF makes any other diagnosis highly unlikely. There is no place for radical surgery in the first-line treatment of malignant intracerebral germ cell tumours because of the sensitivity to radio- and chemotherapy. Also, the sensitivity to chemotherapy makes it possible to reduce radiation volume and dose in an effort to avoid the serious complications of craniospinal irradiation. Both patients responded well to chemotherapy based on cisplatin followed by radiotherapy. PMID- 10735141 TI - [Pharmacotherapy of patients with (early) rheumatoid arthritis]. PMID- 10735142 TI - [Physical diagnosis--percussion and palpation of spleen]. PMID- 10735144 TI - Revised guidelines for the control of MRSA in hospitals. PMID- 10735143 TI - [Endovascular stent implantation as a treatment of iliac artery disease]. PMID- 10735146 TI - Stigma of anxiety. PMID- 10735145 TI - The use of probiotics in paediatrics. PMID- 10735147 TI - Atypical neuroleptics. AB - The discovery that clozapine alleviates both positive and negative symptoms of schizophrenia has resulted in the emergence of newer atypical neuroleptics. Even though they are more expensive than traditional antipsychotics they have distinct and important advantages, which may influence the extent of the patient's rehabilitation and compliance. PMID- 10735149 TI - Clinical efficacy and antimicrobial pharmacodynamics. AB - Changes in the susceptibility of bacterial pathogens and the availability of new antimicrobial drugs mean that physicians need to understand the underlying pharmacodynamics of each antimicrobial therapy. Antimicrobial pharmacodynamics determine clinical efficacy and should therefore be carefully considered when selecting appropriate antibiotic agents in the therapeutic setting. PMID- 10735148 TI - Antiplatelet therapy. AB - Within the last few years antiplatelet therapy has developed exponentially, with new agents being tested in an increasing number of clinical scenarios. The mechanism of action of these newer agents and evidence of benefit is prevented in this review. PMID- 10735150 TI - Stalking (obsessive pursuit). AB - Stalking behaviour has long been recognized as causing great distress and prolonged psychological problems in many victims. Individuals subjected to stalking often feel powerless and helpless in the face of such unwelcome intrusions into their daily lives. There now seems to be a greater awareness and understanding of the phenomenon which, in turn, may encourage individuals affected to seek help. PMID- 10735151 TI - Stenting in the oesophagus. AB - Self-expanding metallic stents have assumed increasing importance in the palliative treatment of malignant dysphagia in recent years. This is most commonly the result of inoperable oesophageal cancer, but may also be the result of extrinsic compression on the oesophagus by other malignant masses. Stents are also occasionally used as a last resort in benign disease. PMID- 10735152 TI - Movicol in treatment of constipation and faecal impaction. AB - To resolve severe constipation or faecal impaction requires the induction of a large increase in faecal water content that hitherto has only been achieved with enemas. A novel iso-osmotic laxative has been shown to achieve a similar effect from oral dosing. PMID- 10735153 TI - Spiral computed tomography for pulmonary embolism. AB - Spiral computed tomography pulmonary angiography is increasingly used in the non invasive investigation of patients with suspected pulmonary embolism. This review will focus on current techniques and indications with an explanation of the computed tomography signs of pulmonary embolism, possible pitfalls in interpretation and limitations of the technique. PMID- 10735154 TI - Pulmonary embolism: clinical features and management. AB - Pulmonary embolism (PE) often presents diagnostic difficulties, as its presentation is varied and non-specific. This article attempts a logical approach to the management of a patient with suspected PE, starting with how it may occur, the assessment of clinical probability of PE and subsequent investigations and treatment. PMID- 10735155 TI - Clinical governance: what it is, what it isn't and what it should be. AB - Clinical governance is as much about setting standards and putting in place management systems to achieve them as it is about poorly performing doctors. Medical Royal Colleges are well placed to support individual hospital doctors in setting and monitoring clinical standards appropriate to their specialty. PMID- 10735156 TI - Computerized evaluations of their education and training by senior house officers in the West Midlands. AB - A computerized monitoring system of senior house officers' evaluations of their education and training has been developed in the West Midlands. Four successive surveys over 2 years show some significant improvements in their education and training. PMID- 10735157 TI - Measuring satisfaction with hospital training: why we need a standardized national questionnaire. AB - Trusts, deaneries and Royal colleges use different questionnaires to measure satisfaction with hospital training. A case is made for a standardized national questionnaire. This could be the first step towards a more coordinated system of training evaluation and approval. PMID- 10735158 TI - Sudden visual loss and headache: important symptoms of pituitary disease. PMID- 10735159 TI - An unusual cause of iron deficiency anaemia in an intravenous drug user. PMID- 10735160 TI - The mysterious case of the lost pituitary: amiodarone-induced hypothyroidism. PMID- 10735161 TI - MRSA control guidelines. PMID- 10735163 TI - Suction failure or potential for disaster? PMID- 10735164 TI - Web watch. PMID- 10735162 TI - Discrimination of staff grade doctors. PMID- 10735165 TI - Jargon busters: investments and savings. Part 2. PMID- 10735166 TI - Anaesthesia for Pierre-Robin syndrome. PMID- 10735167 TI - Managing the needle-phobic obstetric patient. PMID- 10735168 TI - Issue dedicated to the celebration of the 70th birthday of Professor Gyorgy Csaba. PMID- 10735169 TI - Study of histamine effects on phagocytosis and enzyme secretion of Tetrahymena pyriformis. AB - 1. The biogenic amine histamine develops effects not only in mammalian cells and tissues but in ciliated unicellular Tetrahymena as well. In addition to binding and internalization of labelled histamine, low concentrations can stimulate the phagocytosis of cells in inorganic salt solution. 2. In inorganic solution Tetrahymena cells secrete acid hydrolases to the medium. High concentration of histamine (10 mM) decreases the secretion of three investigated acid hydrolases in a different manner. We think that in this process the primary determinant is the alkaline character of histamine. 3. The effect of histamine on phagocytosis differs from the effect on secretion since the low, physiological concentration of histamine stimulates phagocytosis, the higher concentrations inhibit it. In the background of these effects possibly the hormone character is dominant. It is supported by the fact that histamine antagonists influence the process differently. PMID- 10735170 TI - Investigation on the formation of the hormone reception mechanism. AB - The formation of the hormone receptor mechanism is part of the embryonic development. The perinatal age seems to be critical for the maturation of this system, i.e. the development of the proper hormone selectivity, the number of the receptors, their sensitivity to the adequate hormone. Our team tried to get a closer view on this developmental period examining different parameters in different model systems. We were able to show that during the maturation of the hormonal system similar responses could be elicited by the later specific hormones or by the non-specific ones but structurally similar compounds. Also it became evident that one single hormonal treatment, applied during in the perinatal age, could influence the responsiveness to hormones of the adult animals. PMID- 10735171 TI - Non-conventional locations of hormone receptors (binding sites). A review. AB - Recent findings on the noncanonical positions of some well-known extracellular mediators and their receptors are reviewed. Peptide hormones (insulin) and/or their binding sites (cell membrane insulin receptor, nuclear insulin receptor); steroid hormones (corticosteroids and estrogens) and their putative membrane receptors are in the scope of this paper. The possible roles of these unusually located receptors in the intracellular signal propagation and physiological responses are also discussed. PMID- 10735172 TI - The environmental pollutant aromatic hydrocarbon, benzpyrene has deleterious effect on hormone receptor development. AB - In this review, works to clear the effect of perinatal and pubertal benzpyrene imprinting to the steroid hormone receptors of adult animals are summarized. There is a comparison between the perinatal and adolescent effects and between the effects on the receptors of males and females. On the basis of the experiments benzpyrene is a general and dangerous imprinter which can influence durably the development of different steroid receptors given directly to the animals studied (perinatally) of given to the nursing mothers or influencing the offspring generations of the perinatally treated parents or grandparents. The results call attention to the outstanding deleterious effects of benzpyrene pollution. PMID- 10735173 TI - The effect of beta-chloro D-alanine and L-cycloserine on the serine, phosphorus and palmitic acid uptake and metabolism of Tetrahymena lipids. AB - The serine palmitoyltransferase inhibitors beta-chloro-D-alanine and L cycloserine resulted in the uptake and metabolism of 3H-serine, 3H-palmitic acid and 32P significant alterations in the unicellular Tetrahymena pyriformis GL as compared to the untreated cells. In contrast with the higher eukariotic cells, by these treatments - except 5 mM L-cycloserine - the ceramide formation were not inhibited in Tetrahymena. L-cycloserine inhibited the conversion of phosphatidylserine (PS) to phosphatidyl-ethanolamine (PE) by decarboxylation, and the conversion of PE to phosphatidylcoline (PC) by methylation. The shorter L cycloserine treatments caused lower, and the longer treatments higher label in glycerophospholipids. beta-chloro-D-alanine resulted in the glycerophospholids higher lipid precursor incorporation both in the shorter and longer treatments. Presumably beta-chloro-D-alanine treatments inhibit the transaminase activity, and the higher concentration (5 and 10 mM) proved to be toxic for Tetrahymena. We found differences between the metabolism of serine and palmitic acid labeled lipids in the beta-chloro-D-alanine and L-cycloserine treated groups. This phenomenon is probably due to a difference in the uptake of phospholipid head group component serine and hydrophobic tail precursor palmitic acid: the incorporation of palmitic acid in Tetrahymena is extremely quick, on the other hand, the uptake of serine is slower, a clear time dependence was measured. PMID- 10735174 TI - Chemotaxis: the proper physiological response to evaluate phylogeny of signal molecules. AB - In this review we summarize our results gained on the investigations focused to characterize ligand and signaling mechanisms required for the chemotaxis in the unicellular model Tetrahymena. Our data show that short chain signal molecules (amino acids, oligopeptides) are distinguished upon their physicochemical characteristics - lipophilicity, residual volumes and statistical distribution of side-chain distances (e.g. in proline containing dipeptides), while the vertebrate hormones have also specific attractant or repellent effects in the model (FSH vs. TSH). Hormonal imprinting developed by pretreatments has also special, signal molecule dependent effect (histamine vs. serotonin). It is shown that "chemotactic selection" of cells, by the new probe developed by us is a suitable tool to provide subpopulations possessing enhanced chemotactic receptor effector mechanisms with respect to the selector signal molecules (IL-8, TNF alpha). PMID- 10735175 TI - Receptor tyrosine kinase, an autapomorphic character of metazoa: identification in marine sponges. AB - In the present review we summarize sequence data obtained from cloning of sponge receptor tyrosine kinases [RTK]. The cDNA sequences were mainly obtained from the marine sponge Geodia cydonium. RTKs (i) with immunoglobulin [Ig]-like domains in the extracellular region, (ii) of the type of insulin-like receptors, as well as (iii) RTKs with one extracellular speract domain, have been identified. The analyses revealed that the RTK genes are constructed in blocks [domains], suggesting a blockwise evolution. The phylogenetic relationships of the sequences obtained revealed that all sponge sequences fall into one branch of the evolutionary tree, while related sequences from higher Metazoa, human, mouse and rat, including also invertebrate sequences, together form a second branch. It is concluded that the RTK molecules have evolved in sponges prior to the "Cambrian Explosion" and have contributed to the rapid appearance of the higher metazoan phyla and that sponges are, as a taxon, also monophyletic. Due to the fact that protein tyrosine kinases in general and RTKs in particular have only been identified in Metazoa, they are, as a group qualified, to be considered as an autapomorphic character of all metazoan phyla. PMID- 10735176 TI - Stress-responsive gene expression in Tetrahymena. AB - Cells properly respond to extracellular stimuli and circumstantial environment. The unicellular eukaryotic protozoan Tetrahymena is a potentially useful animal cell model system for studying the molecular mechanism of adaptation to environment. Tetrahymena is exposed to fluctuations in temperature, pH, amounts of nutrients and concentration of dissolved gases in natural habitat. For example, the cells adapt to cold environment by increase in unsaturated fatty acids in membrane phospholipids to maintain proper membrane fluidity. To accomplish this modification, the activity of fatty acid desaturase is increased upon a down-shift in temperature. We have cloned delta9 fatty acid desaturase which is involved in this process and shown evidence that its mRNA level increased in response to cold environment. Moreover, in order to examine other genes responsive to clod stress, we have adopted mRNA differential display technique to temperature shift-down of T. thermophila. We have cloned two kinase genes, NIMA (never-in-mitosis in Aspergillus nidulans)-related protein kinase (TpNrk) and MAP kinase-related kinase (MRK). Interestingly, these genes were also shown to be expressed by the osmotic stress. PMID- 10735177 TI - Imprinting: perinatal exposures cause the development of diseases during the adult age. AB - Since the early reports linking the development of clear cell cervicovaginal adenocarcinoma in young women with diethylstilbestrol treatment of their mothers during pregnancy, it became clear that perinatal exposure to several substances may induce irreversible alterations, that can be detected later in life. Current evidence suggests that these substances induce, by the mechanism of imprinting, alterations of the differentiation of several cell-types, resulting in the development of disease during the adult age. The most known delayed effects to prenatal exposure to agents displaying hormone action, pollutants, food additives and natural food components, substances of abuse and stress by the mechanism of imprinting are described. Among them, estrogens, androgens, progestins, lead, benzopyrenes, ozone, dioxins, DDT, DDE, methoxychlor, chlordecone, parathion, malathion, polychlorobiphenyls, pyrethroids, paraquat, food additives, normal food constituents, tetrahydrocannabinol, cocaine and opiates. It is concluded that perinatal exposure to several agents causes irreversible changes that determine health conditions during adulthood. Several diseases developing during adulthood probably were determined during early stages of life, under the effect of exposure or preferential mother's diet during pregnancy. Regulations to avoid these early exposures may contribute to an important improvement of health conditions of humankind. PMID- 10735178 TI - Origins of signalling and memory: matters of life versus death. AB - This review focuses on the principles in cell-cell communication and cellular ability to respond to external chemical changes which have been so crucial for the development of life on planet Earth. We now know that the capacity of free living organisms which evolved more than a billion years ago to respond to intercellular signal molecules, originating either from themselves or from other sources in their vicinity, is so similar possibly even more sophisticated - to that of the cells in our own body, and these findings have had a major impact on our struggle to understand how life has evolved and how it can be maintained. Attention is drawn to the very important topic of mechanisms in cell death, being seen as an aggressive and very powerful instrument in the continuance of life and ability of life to proliferate into a plethora of new species, and use insulin related material as our paradigm. Such signal molecules (hormones) may have played a major role in cellular maintenance throughout evolution. PMID- 10735179 TI - Gender and racial correlates of personal control among emotionally disturbed older adults. AB - Black and white community-dwelling women and men over sixty-five years of age, all of whom had been referred for psychotherapy, were interviewed to determine their perceived control (internal vs. external) over the causes and solutions to their problems. Personal control orientations were found to vary both by race and by gender. Men perceived themselves as having more control over causes than did women, but women perceived themselves as having more control over solutions than did men. Blacks perceived themselves as more responsible both for causes and solutions than did whites. Of the four groups studied, white females saw themselves as having the least control over the causes of their problems, but those perceiving higher levels of control over causes were most depressed. White males saw themselves as having the least control over solutions, but those with higher levels of perceived control had lower levels of depressive symptomatology. PMID- 10735180 TI - Generativity in midlife and young adults: links to agency, communion and subjective well-being. AB - Three questions stimulated by Erik Erikson's theory of generativity were addressed: 1) Is generativity associated with greater subjective well-being? 2) Are agency and communion additive or interactive predictors of generativity? 3) Does generativity play a distinct role during the midlife period? Among ninety eight midlife adults, generativity was positively related to positive affectivity, satisfaction with life, and work satisfaction. Generativity was independently predicted by agentic (masculine) and communal (feminine) traits. Among fifty-eight young adults, generativity predicted positive affect at home. Generativity was independently predicted by agentic (power) and communal (love) interpersonal orientations. Using event-contingent recording of agentic and communal behavior at work, agency was a stronger predictor of generativity for young adult men, and communion was a stronger predictor for young adult women. The studies demonstrate that generativity has similar relations to agency and communion in young and midlife adults; however, generativity may be a stronger predictor of subjective well-being in midlife adults. PMID- 10735181 TI - Appraisal of clinical, psychosocial, and environmental health of elderly in Saudi Arabia: a household survey. AB - This was a cross-sectional national survey of the elderly population of Saudi Arabia conducted between January 1994 and December 1995. The participants' physical, mental, social, and environmental health status were assessed in a personal interview during which a structured data form was completed by a trained research team. Data on 6,139 elderly participants have been analyzed: 64.2 percent were males mean age: 69.2 +/- 7.8 years which was higher than females 68.0 +/- 7.6 years (P < 0.0001). The widowhood (46.7%) status was also higher than the widowerhood (4.1%) and 9.3 percent of the participants were never married. The proportion of female illiterates (95.8%) was higher than the males (71.4%). The proportion of participants with definite psychopathology was 33.8 percent and this increased with age and higher among females than males. Overall, 18.8 percent were dependent on others for ADL. Other clinical findings included visual impairment (46.1%) and difficulty in hearing (19.9%). However, a sizeable majority (72.2%) enjoyed good health. Only 3.7 percent lived alone. The proportion of the elderly taking part in any recreational exercise was 14.7 percent and only 4.3 percent had any experience of physical therapy. There were no special hospital wards for the elderly patients, and they were treated by general internists. There is a need for a national cost-effective program for the care of the elderly. The present widespread primary health care centers can be upgraded to coordinate the proposed services. PMID- 10735182 TI - Worry in pre- and post-retirement persons. AB - With two cohorts, elderly persons who were retired and older persons who were not retired, amount of worry as assessed by Wisocki's broadly designed Worry Scale Revised (WSR) and the Penn State Worry Questionnaire (PSWQ) was significantly greater for the pre-retirement group. Women were found to worry more than men on both the WSR and the PSWQ. Significant correlations between WSR and Worry Diary, PSWQ, and SCL-90-R (anxiety, depression, and obsessive-compulsiveness) scores strengthened claims for the WSR as a valid measure of worry in older/elderly persons. PMID- 10735183 TI - Perception of "appropriate" age for retirement among young adults, middle-aged adults, and elderly people. AB - The "appropriate" age for retirement as it is perceived by young adults, middle aged adults, and elderly people has been studied. No respondents were surprised or had trouble expressing an opinion about the minimum and maximum "appropriate" ages for retirement. Representations of the "appropriate" retirement age vary primarily as a function of the perceived physical constraints involved in the occupation, and also depend on the age of the person being questioned; the younger the respondent, and lower the perceived "appropriate" minimum age. There was no tendency among the young adults to prolong the work life of older individuals. Nor was there a tendency to associate aging with the loss of intellectual capacities likely to lead to early retirement. PMID- 10735184 TI - "Difficult to treat infections" pharmacokinetic and pharmacodynamic factors--a review. AB - "Difficult to cure infections" are characterized by poor penetration of antibiotics into infected vegetations, altered metabolic state of bacteria within the vegetation, absence of adequate host defense/cellular response. These infections typically include endocarditis, urinary tract infections (infected urinary tract stones), abscesses, infected fibrin clots (septic thromboemboli, haematomas, catheter-related infections) and foreign body infections. Four main aspects are discussed for the influence on human therapy: 1. the kinetics of antibiotic diffusion into vegetations 2. the specificity of some pharmacodynamic aspects and pharmacokinetic regimes 3. fibrin as one of the main constituents associated with infectious processes and 4. synergistical activities of antibiotic combinations on bacterial vegetations. PMID- 10735185 TI - Isolation of rare opportunistic pathogens in Hungary: case report and short review of the literature. Rhodococcus equi. AB - Rhodococcus equi is a well-established pathogen in foal pneumonia and is increasingly recognized as a pathogen in immunocompromised humans. We have isolated a Gram-positive coccobacillus from 8 blood samples and lung tissues of a renal transplant patient. Colony morphology, growth in Lowenstein-Jensen medium, 21 biochemical reactions, the characteristic morphological cycle (coccus-rod coccus) and the CAMP test established the R. equi diagnosis. Histological studies of 2 lung biopsy specimens revealed numerous microabscesses with aggregates of polymorphonuclear leukocytes surrounded by abundant foamy macrophages. Our isolates proved to be sensitive to majority of antibacterial drugs. The appropriate therapy (amoxicillin-clavulanate) proved to be effective, however six months later a relapse was observed. Data show that in spite of its rare occurrence, R. equi infection represents a diagnostic and therapeutic challenge. The taxonomical, epidemiological, clinico-pathological, diagnostic and therapeutic data of R. equi are discussed. PMID- 10735186 TI - Effect of aminoglycosides on surface hydrophobicity of Acinetobacter baumannii. AB - Effects of amikacin, gentamicin, netilmicin and tobramycin at subinhibitory concentrations (sub-MICs) (11/4, 1/8, 1/16 or 1/32 of their MICs) on the cell surface hydrophobicity of two Acinetobacter baumannii strains (7194 and 16265) were evaluated. Hydrophobicity was determined by two different methods - by adherence of bacteria to hydrocarbon (xylene) and by aggregation of bacteria in ammonium sulphate solutions at various concentrations. The adherence of A. baumannii strains to xylene decreased, mainly, after treatment with netilmicin at 1/4, 1/8 or 1/16 of the MIC (to 6.4%, 17.0% or 24.5% of the control value) (strain 7194) and after treatment with amikacin and gentamicin at 1/4 of their MICs (to 58.4% or 54.4%) (strain 16265). A decrease in surface hydrophobicity of exposed strains under these conditions was shown in salting-out test, too. Tobramycin reduced hydrophobic properties of A. baumannii strains at all tested sub-MICs to only a small extent. PMID- 10735187 TI - A new powerful antibacterial synergistic combination of trimethoprim and trimeprazine. AB - The antihistaminic phenothiazine trimeprazine (Tz) was found to exhibit significant antibacterial activity on the basis of in vitro and in vivo tests. For the study of synergism due to a combination between Tz and trimethoprim (Tm), drug soaked filter paper discs were placed on young culture lawns of sensitive bacteria on nutrient agar plates. Calculation of the area of inhibition zones for determining the degree of synergism between Tz and Tm showed the increase to be statistically significant (p<0.01) when compared with their individual effects. By the checkerboard assessment procedure, the fractional inhibitory concentration (FIC) index was found to be 0.18, confirming synergism. The protective capacity of this combination was then assessed in Swiss white mice using S. typhimurium as the challenge bacterium, and the level of bacterial load was determined from infected autopsied animals. Statistical analysis of the data by students 't' test finally proved that a combination of Tz+Tm was highly synergistic. PMID- 10735188 TI - Purification and characterization of extracellular beta-amylase of Bacillus megaterium B(6). AB - The extracellular beta-amylase from starch induced Bacillus megaterium B6 was purified to homogeneity in a very convenient way; through molecular sieving as demonstrated by the presence of a single band of protein in SDS-PAGE and single peak in gel scanning. The molecular mass of the purified enzyme (monomer) was found to be unusually high, around 105,000 Da. The pH and temperature optima of the purified beta-amylase were at 6.9 and 60 degrees C, respectively. Mn2+ and exogenous thiols were found to play a remarkable role in reactivation of thermally and chemically denatured enzyme. The purified enzyme could saccharify both pure and low quality starches, where maltose could be detected as the major end product. PMID- 10735189 TI - Growth behaviour and indole acetic acid (IAA) production by a Rhizobium isolated from root nodules of Alysicarpus vaginalis DC. AB - From the root nodules of Alysicarpus vaginalis DC, the symbiont was isolated and identified as a Rhizobium sp. The bacteria produced a high amount (107 microg/ml) of indole acetic acid (IAA) in culture from tryptophan supplemented yeast extract mannitol medium. The isolate preferred L-isomer of tryptophan for maximum IAA production. The production was maximum when the bacteria reached its stationary phase of growth. The production of IAA could be increased up to 70% over yeast extract glucose medium by supplementing ZnSO4, 7H2O (0.5 microg/ml). L-asparagine (0.2%) and sodium dodecyl sulfate (1.0 microg/ml). The possible relationship between the rhizobial IAA production and legume-rhizobia symbiosis is discussed. PMID- 10735190 TI - Microbial study of farmhouse ewe cheese during storage in olive oil. AB - The effect of storing farmhouse ewe cheese in oil and in vacuo over long periods of time on physicochemical properties (water activity and pH) and the microbiota of the cheese was investigated. The storage conditions were found to scarcely influence the sample pH. Also, the initial water activity (a(w) = 0.961) and its value after 9 months of storage (0.927) were both very similar to those for naturally ripened cheese. The incidence of pathogenic microbial groups was found to decrease with storage time (counts fell below 1 log CFU/g). The flora that effects proteolytic changes in cheese consisted of lactic microorganisms (viz. lactococci and lactobacilli), in addition, after 6 months of storage, of enterococci. The last are responsible for the formation of large amounts of soluble nitrogen (SN), non-protein nitrogen (NPN) and aminoacid nitrogen (NH2-N), which provide this type of cheese with very special sensory features while preventing dehydration and thus lengthening its shelf life. PMID- 10735191 TI - Some evidences for the involvement of plasmid in diuron herbicide degradation. AB - Pseudomonas sp. strain Bk8 was isolated from field soil contaminated with different urea-herbicides. This strain is a plasmid (pBkB)-harbouring organism capable of complete degradation of diuron herbicide. Plasmid-cured strain Bk8M was obtained by treatment of Pseudomonas sp. Bk8 with Mitomycin C. This cured strain is capable of only partial degradation of diuron side chain and accumulated a phenolic compound in the medium during growing on diuron as a sole source of carbon and energy. Conjugation experiment was carried out using Bk8M as a recipient and Bk8 as a donor of pBk8 plasmid. The transconjugant was able to degrade a diuron without accumulation of phenolic compound. It was proposed that plasmid pBk8 is self-transmissible and involved in the degradation of diuron aromatic ring but it is not connected with the transformation of diuron into diuron phenol compound. PMID- 10735192 TI - Further tests for genetic linkages of three morphological traits, three blood groups, and break points of two chromosome translocations on chromosome one in the chicken. AB - Two matings were conducted to further test the locations of the pea comb (P*), blue egg shell color (O*), and tardy feathering (T*) loci. In each mating a different chromosome rearrangement break point (R(B)) was tested against the three loci. Independent segregation was noted between the traits and the R(B) when the R(B) was on the long arm of chromosome 1. Significant linkage was noted when an R(B) on the short arm was tested against the three markers, indicating that the loci for P*, O*, and T* are on the short arm. Three blood group loci, EAD*, EAI*, and EAP*, were simultaneously tested against the short arm R(B). Independent segregation was noted in each instance, indicating that these blood group loci are not on the short arm of chromosome 1. PMID- 10735193 TI - Genetic effects of aging on egg production traits in the first laying cycle of White Leghorn strains and strain crosses. AB - Three White Leghorn strains, their two-way crosses, and two commercial lines were used to evaluate the effects of aging on mean heterosis, reciprocal, additive, Z chromosome, and heterotic effects and their variances in two egg production traits during the first laying cycle. Egg number of the survivors (EPF) and egg number including mortality and morbidity (EPM) were evaluated from hens housed one per cage in a randomized block design. For analyses, egg number was divided into 12 periods of 28 d each. Synchronization of the records was achieved by starting recording at age at first egg. The mean heterosis was significant over time and increased with age for both traits (P<0.05). Reciprocal effects were not significant across periods but increased in magnitude with age for EPF. The pattern of age changes in additive, Z-chromosome, and heterotic effects varied among strains, indicating genotypic differences in response to aging. These differences increased, on average, with age. The additive, heterotic, environmental, and phenotypic variances increased with age for both traits. A decline with physiological age was observed in heritabilities, which was due to a faster increase in environmental variance than to an increase in additive variance. Genetic variance of egg production increased with age, indicating that improvement of lifetime performance of layers is possible. This improvement could be achieved by selecting animals at older ages or by favoring individuals with better DNA repair or those who had more genes turned on or off during the course of aging. PMID- 10735194 TI - Utility of three restriction fragment length polymorphism probes for genotyping of the chicken major histocompatibility complex class IV region. AB - Three chicken B-G cDNA probes (gene 8.5, bg28, and bg32.1) were used to detect restriction fragment length polymorphisms (RFLP) in the chicken MHC class IV (B G). By using inbred and selected chicken lines with different B haplotypes identified by hemagglutination, we identified B haplotypes (B2, B9, B11, B12, B15, B19, B21, B31, and B32) by RFLP using the three probes following digestion of genomic DNA with four restriction endonucleases (BglII, EcoRI, HaeIII, and PvuII). The GSP inbred line, previously shown to contain B-F21 by the use of a monoclonal antibody, did not contain B-G21, based on RFLP tests, whereas line N had B-F21 and B-G21. Consequently, the RFLP typing with the clone of B-G cDNA was able to determine the B haplotype in more detail than typing by hemagglutination. In inbred and selected lines, three B-G cDNA are useful DNA probes for RFLP to identify B genotypes. Two families of chickens with segregating B haplotypes were analyzed by RFLP using these probes; however, identification of the B genotype by this method was difficult in the randomly bred population. Genotypic comparisons of RFLP with gene 8.5 and BglII and bg 28 as probes and digestion by the endonucleases EcoRI, HaeIII, and PvuII between the parents and their offspring were generally compatible within the expectations of Mendelian inheritance. PMID- 10735195 TI - Does excess dietary protein improve growth performance and carcass characteristics in heat-exposed chickens? AB - The effects of two environmental temperatures (22 and 32 C, constant) and five dietary protein contents (10 to 33% CP) were investigated in 4- to 6-wk-old broiler chickens. High ambient temperature reduced growth rate, feed efficiency, and breast muscle proportion and increased abdominal fat proportion. Irrespective of ambient temperature, increasing dietary protein content improved growth performance and carcass characteristics. At 32 C, there was a greater heterogeneity of the data, and bird responses were lower than at 22 C. We concluded that under conditions of chronic heat exposure, diets containing the highest protein levels, 28% and 33% compared with 20% CP, slightly improved chick performance. However, the effect was low and, in our experimental conditions, modifying dietary protein supply (variations in the total quantity of protein) is not sufficient to help broilers to withstand hot conditions. PMID- 10735196 TI - Ascites syndrome and related pathologies in feed restricted broilers raised in a hypobaric chamber. AB - It has been demonstrated that the incidence of ascites can be significantly reduced through feed restriction. This method is thought to have an effect by slowing the growth rate of the birds. Interestingly, when birds are grown in a hypobaric chamber, ascites incidence increases while the overall growth rate of the birds is decreased. Unfortunately, the restriction programs practiced also have a detrimental effect on growth characteristics. An experiment was conducted to determine if the timing and duration of feed restriction can be used to reduce the incidence of ascites for broilers reared under high altitude and local elevation without having a negative impact on growth. A total of 600 commercial broiler males were used. Birds were divided, placing 360 birds in the hypobaric chamber at a simulated 2900 m (9,500 ft) above sea level, and 240 birds were placed at local elevation [390 m (1,300 ft) above sea level]. At each altitude there were four treatments: 1) fully fed controls; 2) feed available for 8 h/d for 6 wk (the duration of the study); 3) feed available for 8 h/d during the first 3 wk, then full feed for the remaining 3 wk; and 4) full feed for the first wk, then 3 wk of 8 h of feed availability, then 2 wk of full feed. Birds and feed were weighed weekly, and mortalities were necropsied to determine the cause of death. At the end of 6 wk, blood samples were taken, and the birds were weighed, necropsied, and scored for ascites, and organ weights were recorded. All feed restriction treatments significantly reduced ascites incidence, when compared with the fully fed controls. Treatment 2 birds were significantly lighter than any other group at both altitudes. The fully fed controls at local elevation were heavier than the fully fed controls at simulated high altitude, as seen in past experiments. PMID- 10735197 TI - Growth, carcass characteristics, and incidence of ascites in broilers exposed to environmental fluctuations and oiled litter. AB - The effects of diurnal temperature fluctuations and removal of respirable dust, by application of canola oil to straw litter, on growth, carcass traits, and the degree of ascites was evaluated with 1,200 male broilers studied in two replicated 6-wk trials. Each trial used four pens of 150 birds. The temperature treatment consisted of a fluctuation of 3 C in temperature above the required temperature during the day (0600 to 1800 h) and 3 C below the required temperature at night (1800 to 0600 h) for a 6 C change in daily temperature. The control temperature was constant. All pens had the same mean daily temperature. In each trial, one control temperature pen and one fluctuation temperature pen received bi-weekly applications of canola oil to the litter (1.1 L/m2 of oil over 6 wk). At 6 wk of age, 30 birds from each pen were killed for determination of breast muscle, fatpad, and heart weights. All birds were scored for lesions of ascites at time of processing. A score of 0 or 1 represented slight pericardial effusion, slight pulmonary congestion, and edema. A score of 4 represented birds with marked accumulation of ascitic fluid in one or more ceolomic cavities (other than the pericardium) and advanced liver lesions. A cross-sectional image of each 4-mm heart slice (cross-section of the ventricles) was digitally recorded, and with image analysis we determined the right ventricular area (RVA), left ventricular area (LVA), and total heart area (HA). The final BW of the broilers were significantly different, the oiled-litter treatment (2,249 g) had lower weight gain compared with the nonoiled litter treatment (2,293 g). There were no differences in fatpad weight, shank length, lung weight, and percentage breast muscle between the main treatments. The Pectoralis minor and Pectoralis major weight were significantly heavier in the temperature fluctuation treatment than in the control temperature treatment by 3.0 and 12.0 g, respectively. The birds subjected to the control temperature treatment had a lower RVW than the birds subjected to the fluctuating temperature treatment. Temperature fluctuations also resulted in a 1.4% increase in the incidence of mortality. Temperature fluctuations negatively impact broiler growth due to heat loss when litter oiling was excessive. PMID- 10735198 TI - Comparison of the effects of dietary selenium, zinc, and selenium and zinc supplementation on growth and immune response between chick groups that were inoculated with Salmonella and aflatoxin or Salmonella. AB - The effects of four diets (basal diet, Se, Zn, and Se- and Zn-enriched diets) fed to chicks that were administered one of three treatments [Salmonella and aflatoxin inoculation (T1), Salmonella inoculation (T2), or uninoculated (T3)] were investigated for growth and immune responses. We found a significant improvement in growth performance represented by relative body gain (RBG) and feed efficiency (FE), for the Zn- and Se + Zn-enriched diets fed to the T1 and T2 groups. The antibody immune response was significantly improved for the Se enrichment diet in the T1 and T2 groups. The weight of the bursa and thymus, which relate to the level of the immune response, showed significant decreases, whereas the spleen had a significantly increased relative weight (RW) in the T1 group. The variable dietary trace elements supplement increased the thymic RW in the T2 group. PMID- 10735199 TI - Responses of chickens to a recombinant refractile body antigen of Eimeria tenella administered using various immunizing strategies. AB - A refractile body antigen (designated SO7') found in sporozoites of Eimeria tenella was administered to chickens in various immunizing forms to assess its ability to protect against virulent challenge. These included native antigen from the parasite (viable oocysts; per os), recombinant antigen (CheY-SO7'; s.c.), and naked DNA immunization (pcDNA3-SO7'; i.m.). Male White Leghorn chicks were inoculated with oocysts or injected with one of the forms of SO7' antigen at 1 wk of age, boosted at 3 wk of age with the same treatment, and subsequently challenged at 5 wk of age with oocysts of Eimeria tenella USDA Strain 80. Seven days postchallenge, chicks were killed, and weight gains and lesion scores were determined and compared with those of the controls. Significant protection against cecal lesions and weight loss was noted in birds inoculated with oocysts or injected with pcDNA3-SO7' (25 microg). Birds injected with other doses of pcDNA3-SO7' (12.5, 50, 60, and 100 microg) showed no reduction in cecal lesions and maintained poor rates of growth similar to controls. The recombinant antigen (CheY-SO7') was also nonprotective. These results offer promise to the use of DNA immunization strategies for controlling avian coccidiosis and show for the first time naked DNA vaccination with a single coccidial antigen that protects chickens (as measured by reduced lesion scores and high rate of growth) against cecal coccidiosis. PMID- 10735200 TI - Nonmajor histocompatibility complex alloantigen effects on the fate of Rous sarcomas. AB - Rous sarcoma virus-induced tumor outcome is controlled by the MHC (B). Additional data, using controlled segregation in families, has indicated non-MHC effects as well, but few studies have focused on blood groups other than the B complex. Segregating combinations of genes encoding erythrocyte (Ea) alloantigen systems A, C, D, E, H, I, P, and L in B2B5 and B5B5 MHC (B) backgrounds were examined for their effects on Rous sarcomas. Six-week-old chickens were inoculated in the wing web with 30 pfu of Rous sarcoma virus (RSV). Tumors were scored six times over a 10-wk period. A tumor profile index (TPI) was assigned to each chicken based on the six tumor size scores. Response was evaluated using tumor size at each measurement period, TPI, and mortality. The genotypes of Ea systems A, C, D, E, H, I, and P had no significant effect on any parameter in either B complex population. The Ea-L system had an effect on Rous sarcomas in the B2B5 intermediate responders and B5B5 progressors. Tumor size, TPI, and mortality were all significantly lower in B2B5 L1L1 chickens than in B2B5 L1L2 chickens. Mortality was lower in the B5B5 L1L1 birds than in B5B5 L1L2 chickens. It appears that the Ea-L system, or one closely linked, is acting in a manner independent of the B complex in response to RSV challenge. PMID- 10735201 TI - Active immunization with vasoactive intestinal peptide in turkey hens. AB - Active immunization of turkey hens against vasoactive intestinal peptide (VIP) has been shown to inhibit incubation behavior and to increase egg production in second-cycle hens. The objective of this study was to compare the effect of VIP immunization on first- and second-cycle turkey hens during a 27-wk production period. First- (25-wk-old) and second- (54-wk-old) cycle hens were intermixed, distributed among 16 pens, and subjected to a photoperiod of 6 h of light and 18 h of darkness for 10 wk. The first-cycle hens were divided into two groups: keyhole limpet hemocyanin (KLH)-immunized controls (n = 16) and VIP-immunized (n = 18). Second-cycle hens were divided into four groups: 1) unimmunized controls (n = 19), 2) KLH-immunized controls (n = 18), 3) VIP-immunized (n = 19), and 4) VIP-preimmunized (immunized during first cycle; n = 16). Each hen received four antigen injections beginning the day of photostimulation (4-wk intervals), except for the preimmunized hens, which received three injections beginning 4 wk after photostimulation. The maximum titer of VIP antibodies in first-cycle, second cycle, and preimmunized hens was 17.2+/-2.2, 20.9+/-2.9, and 21.7+/-3.2%, respectively. After photostimulation, plasma prolactin of first- and second-cycle control hens peaked between 484 +/-105 and 630+/-118 ng/mL. In contrast, prolactin changed very little in VIP-immunized turkeys. The average number of daily nest visits was less in first- and second-cycle VIP-immunized hens (1.68+/ 0.23 and 1.09+/-0.15 visits per hen per day, respectively) than in their respective KLH-immunized controls (2.47+/-0.36 and 2.65+/-0.45 visits per hen per day). Expression of incubation behavior was 50.0 and 52.6% in first- and second cycle control hens, respectively, upon termination of the study. In contrast, only 11.1% first-cycle and 5.2% second-cycle VIP-immunized turkeys exhibited the hormonal and behavioral characteristics of incubating hens. Average weekly egg production of first- and second-cycle VIP-immunized turkeys was similar (3.58+/ 0.19 vs. 3.63+/-0.14 eggs per hen per wk). First- and second-cycle control hens laid 2.63+/-0.25 and 2.41+/-0.20 eggs per hen per wk, respectively. The present results show that comparable egg production was attained in first- and second cycle hens by active immunization with VIP. PMID- 10735202 TI - Evidence for multiple prolactin receptor transcripts in the turkey. AB - Multiple prolactin receptor (PRL-R) mRNA transcript isoforms have been identified in mammals, but there are conflicting reports concerning the number of avian PRL R isoforms. We hypothesized that multiple turkey PRL-R transcript isoforms exist and that PRL-R mRNA abundance may be related to reproductive status. Two turkey PRL-R cDNA fragments were generated using reverse transcriptase polymerase chain reaction (RT-PCR) that displayed a high degree of similarity to mammalian and avian PRL-R. Northern blot analysis of poly A+ mRNA hybridized to a turkey PRL-R riboprobe revealed a 3.1-kb band in the liver, oviduct, and testes. Additional 1.5- and 10.7-kb transcripts were found in the liver and testes, respectively. Hybridization of the same Northern blot to a chicken PRL-R probe verified the presence of a 3.1-kb transcript in all three tissues. A Northern blot was used to examine turkey PRL-R transcript isoform expression in laying hens. A 3.1-kb band was found in the pineal, infundibulum, magnum, isthmus, kidney, and intestine. In addition, 10.7- and 7.3-kb bands were detected in the pineal, magnum, isthmus, and intestine. Turkey PRL-R transcript isoforms were also examined throughout the reproductive cycle. The 10.7-, 7.3-, and 3.1-kb isoforms were detected in the oviduct, intestine, and pineal during each reproductive state. Turkey PRL-R mRNA levels were also compared during the reproductive cycle. Turkey PRL-R mRNA levels were greatest in laying hen pineal glands (P<0.05) and in incubating hen oviducts. This study provides the first evidence for multiple PRL-R mRNA transcript isoforms in turkeys. PMID- 10735203 TI - Model of physiological stress in chickens 1. Response parameters. AB - A model was developed to study stress in chickens. Continuous administration of adrenocorticotropic hormone was employed (ACTH) using physiological mini-osmotic pumps. A validation of controls for this procedure showed that nonhandled (NHCON), sham surgical procedure for pump implantation (SMCON) and surgical implantation of a pump delivering saline (SALCON) were all acceptable controls. Continuous delivery of ACTH at 8 IU/kg BW/d for 7 d caused increases in plasma corticosterone (CS), glucose (GLU), cholesterol (CHOL), triglycerides (TRI), high density lipoprotein (HDL), total protein (TP), and the heterophil/lymphocyte (H/L) ratio. Body weight, as well as relative weights of the major immunobiological organs (i.e., spleen, thymus, and bursa of Fabricius) were decreased. Finally, liver was increased due to lipid and moisture accumulation. This model is the first to show in a single experiment all the major adaptive stress responses of chickens. PMID- 10735204 TI - Model of physiological stress in chickens 2. Dosimetry of adrenocorticotropin. AB - Continuous adrenocorticotropin (ACTH) delivery by mini-osmotic pumps is a reliable method to induce stress in broilers. The present study was conducted to determine the minimum effective dose of ACTH that will evoke physiological stress. Chicks, which had been reared under floor-pen conditions until 5 wk of age received mini-osmotic pumps by surgical implantation. The mini-osmotic pumps delivered 2, 4, 8, or 16 IU of porcine ACTH/kg BW/d for 7 consecutive d. Controls (CON) were untreated chicks. Continuous infusion of ACTH caused dose-related stress responses. Chicks that received 8 or 16 IU showed the greatest responses, whereas those that received 2 or 4 IU were intermediate to the birds dosed with high ACTH and the CON birds. Responses included decreased BW; decreased relative weights of the bursa of Fabricius, thymus, and spleen; and increased relative liver weight. Concomitantly, increased liver lipid and decreased liver moisture were recorded. Plasma concentrations of corticosterone (CS), glucose (GLU), total protein (TP), cholesterol (CHOL), and triglycerides (TRI) and the ratio of heterophils to lymphocytes were increased during ACTH infusion. ACTH infusion at 8 IU/kg BW/d for 7 d was determined to be the minimum effective dose that caused physiological stress in broilers. PMID- 10735205 TI - Model of physiological stress in chickens 3. Temporal patterns of response. AB - The temporal pattern of stress responses following continuous infusion of adrenocorticotropic hormone (ACTH; 8 IU/kg BW/d for 7 d) in broilers was determined in two experiments. Results indicate that the order of responses were elevated plasma corticosterone (CS) by 2 h, elevated plasma glucose (GLU) by 12 h, increased liver weight concomitant with increased hepatic lipid and decreased liver moisture by 18 h, decreased relative weight of the spleen by 24 h, elevated heterophil/lymphocyte (H/L) ratio by 2 d, decreased body weight and relative weights of the bursa of Fabricius and thymus by Day 4, and decreased liver soluble protein content by 12 d. PMID- 10735206 TI - Model of physiological stress in chickens 4. Digestion and metabolism. AB - Two similar experiments were conducted to evaluate the effects of stress on growth and feed utilization of broiler chicks. Stress was induced by continuous delivery of adrenocorticotropin (ACTH) at 8 IU/kg BW/d for 7 consecutive d. During the 7-d stress period, ACTH-treated chicks did not exhibit increases in feed intake, but increases in water intake concomitant with an increase in excreta output were recorded. The ACTH caused decreases in digestion of dry matter, proteins, gross energy, and carbohydrates, whereas fat digestibility was unaffected. Digestion of these nutrients was affected more than absorption during the stress period. By 1 wk poststress, all parameters were comparable with those of the control, except for feed intake, which was less than that of CON. The BW of ACTH-treated chicks were lower than CON through 1 wk after cessation of infusion of ACTH. Contrasted to effects during the stress period, after cessation of ACTH-treatment, absorption of all nutrients was reduced, and, during this poststress recovery period, digestion appeared to return to the range of CON values. Results indicate that all physiological parameters with the exceptions of reductions in BW and thymus weight returned to the range of CON values within 1 wk after cessation of ACTH infusion. However, feed intake and absorption of energy dry matter and nitrogenous compounds remained lower than CON at that time. Also, losses in skeletal muscle caused by prolonged gluconeogenesis apparently required extended periods for complete recovery, as evidenced by stressed chicks that did not gain comparably to CON after a 1 wk cessation of ACTH delivery. PMID- 10735207 TI - Model of physiological stress in chickens 5. Quantitative evaluation. AB - Physiological stress was studied in broilers using a new model employing continuous release of adrenocorticotropin (ACTH). A total of 42 adaptive response parameters were evaluated. A treatment response ratio, P-value transformation, and stress score were determined for each parameter. The treatment response ratio was calculated by dividing the maximum response of the ACTH-treated birds for each parameter by the average response of control (CON) birds in all cases in which maximum response of ACTH-treated birds exceeded the average response of CON. This treatment response ratio is designated hereafter as ACTH/CON ratio. In those cases in which average response of CON exceeded maximum response of ACTH treated birds, the reciprocal equation was employed (i.e., CON/ACTH ratio). Probability value transformations were determined by transforming P-values as follows: NS P-values or those exhibiting significance at values less than probability of 5% were expressed as P<0.05 = 1, P-values exhibiting significance between 0.05 and 0.02 were expressed as P at 0.05 to 0.02 = 2, and P-values exhibiting significance at probabilities of 1% or less were P<0.01 = 4. Each stress score was calculated as treatment response ratio times P-value transformation. The 42 response parameters were assigned to one of four adaptive categories, and these were morphologic, endocrine, and blood metabolites; digestion and metabolism; and reticuloendothelial responses. The endocrine and blood metabolites category had the highest average of the four stress response categories, and five of the top ten parameters were found in this category. PMID- 10735208 TI - Ascites resistance of progeny from broiler breeders selected for two generations using chronic unilateral pulmonary artery occlusion. AB - Broilers that survived unilateral pulmonary artery occlusion and lived to maturity comprised the first generation (GEN1) of an ascites-resistant line. Progeny from the GEN1 line previously were shown to tolerate fast growth and cool temperatures with a 50% lower incidence of ascites than chicks from the breeder pullet line serving as the base population for the resistant line. In the present study, progeny from the GEN1 line were subjected to unilateral pulmonary artery occlusion, and survivors were reared to breeding age to serve as the parent stock for the second generation (GEN2) ascites-resistant line. In two experiments (EXP1 and 2), chicks were reared separately by sex but were mixed by line within environmental chambers, where they were grown as rapidly as possible and exposed to cool (14 C) temperatures from 17 to 49 d of age. In EXP1, the ascites incidences in the base population, GEN1, and GEN2 lines, respectively, were 31% (48/157), 15% (8/52), and 4% (3/69) for males and 10% (13/128), 11% (5/46), and 3% (1/36) for females. In EXP2, the ascites incidences in the base and GEN2 lines, respectively, were 44% (71/163) and 6% (7/110) for males and 12% (19/155) and 0% (0/92) for females. The final BW for nonascitic broilers did not differ across lines in EXP1. In EXP2, the final BW was lighter for nonascitic GEN2 males (2,915+/-43 g) and females (2,382+/-17 g) than for nonascitic base population males (3,088+/-42 g) and females (2,493+/-22), respectively. Right:total ventricular weight ratios were higher for ascitic than nonascitic broilers, confirming the primary role for pulmonary hypertension in the pathogenesis of ascites. These experiments demonstrate ongoing improvement in the ascites resistance of progeny from broiler breeders that, for two consecutive generations, have survived the rigorous selection pressure imposed by unilateral pulmonary artery occlusion. PMID- 10735209 TI - Heterophil to lymphocyte ratio and tonic immobility reactions to preslaughter handling in broiler chickens treated with ascorbic acid. AB - Stress and fear responses were evaluated in broiler chicks that were pretreated for 24 h with 0 ppm (control) or 1,200 ppm of L-ascorbic acid (AA) in their drinking water. The birds were subsequently subjected to either upright handling (UH) or inverted (IH) handling for about 45 s. Heterophil (H) counts, lymphocyte (L) counts, and H/L ratios (H/L) ratios were determined immediately (T0) and at 20 h (T20) following the handling treatment. The H/L ratios were similar for both groups at T0, whereas 20 h after the handling treatment, AA-supplemented birds had lower ratios than controls, resulting in a significant water treatment x time of blood sampling interaction. Inverted handling had negligible effect on H/L ratios but augmented tonic immobility (TI) durations as compared with UH. Irrespective of handling procedure, supplemental AA reduced underlying fearfulness, as measured by TI reaction. Neither water treatment nor handling method had significant effect on number of attempts to induce TI. PMID- 10735211 TI - Cardio-pulmonary function in preascitic (hypoxemic) or normal broilers inhaling ambient air or 100% oxygen. AB - We evaluated the influence of the percentage saturation of hemoglobin with oxygen (HbO2) on the pulmonary arterial pressure in normal and preascitic (hypoxemic) broilers breathing ambient air or 100% O2. In Experiment 1, unanesthetized preascitic broilers (right:total ventricular weight ratios [RV:TV] = 0.32+/-0.02) breathing ambient air had initial values of 67% for HbO2 and 32 mm Hg for pulmonary arterial pressure. The HbO2 increased to > or =96.6% during inhalation of 100% O2; however, pulmonary arterial pressure was not reduced. In Experiment 2, anesthetized normal (RV:TV = 0.23; HbO2 = 88%) and preascitic broilers (RV:TV = 0.28; HbO2 = 76%) were compared. The groups did not differ in body weight or respiratory rate, but preascitic broilers had lower values for mean arterial pressure, total peripheral resistance, and partial pressure of O2 in arterial blood and had higher values for pulmonary arterial pressure. Inhaling 100% O2 increased HbO2 to 99.9% in both groups; however, pulmonary arterial pressure remained higher in preascitic than in normal broilers, and the pulmonary vascular resistance was not reduced during 100% O2 inhalation. Cardiac output was higher in preascitic than in normal broilers before and after, but not during, 100% O2 inhalation. Mean arterial pressure and total peripheral resistance increased in the preascitic but not in the normal group during 100% O2 inhalation. Low coefficients of determination (R2) were obtained for linear regression comparisons of HbO2 vs. pulmonary arterial pressure in both experiments. Overall, acute reversal of the systemic hypoxemia in preascitic broilers had little direct impact on pulmonary hypertension, providing no evidence of hypoxemic or hypoxic pulmonary vasoconstriction. Instead, acute reversal of the systemic hypoxemia primarily increased the total peripheral resistance and normalized the mean arterial pressure and cardiac output. A sustained reduction in cardiac output theoretically should attenuate pulmonary hypertension, but this was not observed because of the overriding influence of sustained pulmonary vascular resistance. PMID- 10735210 TI - Ultrastructure of spermatozoa from Japanese quail. AB - A monoclonal antibody (mAb) to epitopes on mitochondria from turkey spermatozoa cross-reacted with Japanese quail spermatozoal mitochondria. However, the pattern of binding was different from that observed for turkey sperm. The ultrastructure of quail spermatozoa was examined to determine the reason for this difference in antibody binding pattern. Light microscopy, as well as scanning (SEM) and transmission (TEM) electron microscopy, were used to study the morphology of spermatozoa from Japanese quail. Japanese quail had a sauropsid type of sperm cell, which is typical of nonpasserine birds. The spermatozoa were vermiform in shape, with a maximum width of 0.6 microm and an overall length between 230 and 250 microm. An acrosome (3.7 to 4.5 microm), nucleus (20.8 to 23.8 microm), midpiece (160 to 170 microm), and tail (40 to 60 microm) were observed. The TEM showed an acrosomal cap surrounding a perforatorium that inserted into the nucleus at the posterior end. Only a distal centriole was observed, which gave rise to a central axoneme with a 9+2 microtubular structure. The axoneme was encased by a spiraled mitochondrial sheath in the midpiece region (64 to 74% of the overall length of the sperm), and mitochondria numbers were estimated to be greater than 1,400 per sperm. In contrast, turkey sperm contain short midpieces with only 20 to 30 mitochondria per sperm. Differences in binding patterns of the mAb to turkey mitochondria between quail and turkey sperm were due to the presence of mitochondria on the exceptionally long midpieces of quail sperm. PMID- 10735212 TI - Exposure of turkey sperm to a synthetic peptide before insemination increases fertility. AB - Effects on fertility and hatch of eggs laid by hens inseminated with sperm exposed to a synthetic peptide were studied. Pooled semen from 40 randomly selected toms was split and held in vitro for 0 or 24 h before use. Just before insemination, sperm (at 8.33x10(9) sperm/mL) were exposed for 5 min to 0.0, 0.05, 0.25, or 0.50 microM peptide. Hens (28 per group) were inseminated within less than 30 min with 250x10(6) in 30 microL. Two inseminations 24 h before onset of lay were followed by weekly inseminations through 22 or 20 wk. For sperm that was fresh or held 24 h, peptide treatment (P<0.02 or 0.01) and week of lay (P<0.01) affected fertility and hatch of total eggs set. There was no effect of peptide treatment on hatchability of fertile eggs. For fresh sperm, use of 0.25 microM peptide, but not 0.05 or 5.0 microM peptide, increased (P<0.05) fertility and hatch of total eggs set compared with the control (0 microM). Values for fertility were 94 vs. 90% and for hatch were 84 vs. 80%. Increases in hatch were especially evident for fresh sperm after approximately 13 wk of lay. PMID- 10735213 TI - The development of pale, exudative meat in two genetic lines of turkeys subjected to heat stress and its prediction by halothane screening. AB - Previous research has indicated that seasonal-type heat stress (HS) can contribute to the development of pale, soft, exudative (PSE) meat in fast-growing turkeys and that halothane exposure may identify stress-susceptible animals. This study evaluated the ability of halothane screening to identify stress-susceptible birds prone to developing pale, exudative meat when reared to market age. Two lines of turkeys (n = 292), one selected for rapid overall growth (BODY) and the other for large breast muscle yield (BREAST), were exposed to 3% halothane for 5 min at 2 to 4 wk of age and were raised together until 16 wk of age. Approximately 10% of both BODY and BREAST birds were sensitive to halothane. Between 16 and 20 wk, all of the halothane sensitive (HAL+) and half of the halothane nonresponders (HAL-) were exposed to an HS environment of 30 to 36 C (night/day), whereas the other half of the HAL- birds were kept at an ambient temperature of 13 to 21 C (night/day). All birds were slaughtered at 20 wk of age, and samples were collected for pH, L* value, drip loss, cooking loss, and shear value. The BREAST strain had 5% greater breast percentage than the BODY strain, and there were no differences in ready-to-cook yields between any treatments. The HAL+ HS birds had significantly lower muscle pH (0 h) and significantly higher L* values at 2 h postmortem compared with HAL- HS birds in the BREAST strain; however, there was no difference in L* value at 24 h postmortem. The HAL- HS birds had significantly lower muscle pH (0 h and 2 h) and significantly higher L* values at 2 h postmortem compared with HAL- controls in the BODY strain. The HAL- HS BREAST birds had significantly higher drip loss than HAL- controls. No differences in shear value were found among any treatments. The incidence of PSE (2-h L* values >52) was significantly higher in HAL+ HS birds (34.7%) compared with HAL- HS birds (13.4%). These results suggest that halothane sensitivity early in life is associated with HS susceptibility and the development of pale meat when birds are slaughtered at market age. These results also suggest that halothane screening may be better at predicting the development of PSE meat during HS in the strain selected for large breast yield rather than rapid overall growth. PMID- 10735214 TI - Influence of flooring type during transport and holding on bacteria recovery from broiler carcass rinses before and after defeathering. AB - Four trials were conducted to determine whether conventional solid or elevated wire mesh flooring, during transport and holding of broilers prior to slaughter, influenced the number of bacteria recovered from feathered and defeathered carcasses. After 4 h off feed, 7-wk-old broilers were placed at commercial density into a modified commercial transport dump-coop on either fiberglass sheeting or 2.54x2.54 cm wire mesh flooring that allowed feces to fall through. Broilers were transported for 1 h and then held for 13 h under a covered shed before processing. Broilers were killed by electrocution, and the vents were plugged to prevent escape of feces. External carcass rinses were obtained twice (from the same carcass) from eight broilers per flooring treatment per trial, before scalding and defeathering and again after defeathering and removal of the head and feet. Greater numbers of total aerobes, coliforms, and Escherichia coli were recovered from feathered carcasses than from defeathered carcasses. Campylobacter count was also less for defeathered than feathered carcasses from the solid flooring treatment but did not significantly decrease following defeathering of carcasses from the wire flooring. The incidence of Campylobacter positive carcasses was reduced following defeathering for both flooring treatments, but the percentage of Salmonellae-positive carcasses remained constant. Coliform (log10 6.20 vs. 5.63 cfu/mL of rinse) and E. coli (log10 5.93 vs. 5.36) counts in the feathered rinses were significantly higher for the solid flooring compared with wire flooring, respectively. After defeathering, the number of coliforms (log10 3.12) and E. coli (log10 2.91) recovered did not differ between flooring treatments. Aerobic plate count (log10 7.06 and 4.02), Campylobacter count (log10 2.49 and 1.80), and the incidence of Campylobacter positive (44 and 11%) and Salmonellae-positive (52 and 50%) carcasses for feathered and defeathered rinses, respectively, did not differ between flooring treatments. These results indicate that although broilers transported and held on solid flooring had noticeably dirtier breast feathers and higher coliform and E. coli counts prior to scalding and defeathering, bacteria recovery from external carcass rinses did not differ between the solid and wire flooring treatments after defeathering. PMID- 10735215 TI - Endoscope-assisted correction of primary varicose veins. AB - The purpose of this study is to evaluate the possibilities and results of application of endoscopic surgery in the operation of primary varicose veins. With good illumination and magnified viewing, the varicosities, incompetent perforating veins, and healthy veins were clearly visualized. The incompetent perforating veins were clipped and divided securely. The varicose veins and tributaries were dissected and removed completely after being clipped and divided. The healthy veins were preserved if possible. Primary varicose veins in 43 limbs of 37 patients were operated. The operation was conducted through one access incision in 31 limbs (72%), two incisions in 11 limbs (25%), and three incisions in 1 limb. The mean number of incisions was 1.3 in each limb. Hematoma formation occurred in 1 patient due to the slipping of one clip on the third postoperative day. The other patients had uneventful postoperative courses. All 37 patients were reviewed 4 to 30 months postoperatively. Only one limb had recurrent varices at a new site. Ninety-seven percent of limbs (42 of 43) had no recurrence of varicose veins. Seventy-eight percent of patients (29 of 37) were extremely pleased with this operation. Although the other 22% of patients (8 of 37) appreciated this operation, they were not completely satisfied because some preoperative complaints persisted. In this series, the recurrent rate of varicose veins was low (1 in 43 limbs) and postoperative scarring was minimized, in addition to the advantages of endoscopic surgery. These results demonstrate that endoscopic surgery is a worthy alternative procedure for correcting primary varicose veins. PMID- 10735216 TI - Reconstruction of the high-risk chest wall with endoscopically assisted latissimus dorsi harvest and expander placement. AB - Certain patients requesting breast reconstruction may be described as having a high-risk chest wall with regard to implant loss and well-documented high complication rates. Such patients have a combination of one or more of the following: previous chest wall radiotherapy, heavy smoking, and thin, tethered chest wall flaps. If autologous transfer is not appropriate for such patients then reconstruction may be difficult. In this specific patient group the assistance of endoscopy has been used to raise the latissimus dorsi muscle to cover an expander placed within an endoscopically created chest wall pocket. The first 12 consecutive cases using this technique are discussed, showing an expander loss rate of 8% for the primary implant placement. PMID- 10735217 TI - Soft-tissue reconstruction of sole and heel defects with free tissue transfers. AB - Reconstruction of the weight-bearing surface of the foot is a challenging problem for the reconstructive surgeon. Because local tissues are not usually available for reconstruction, distant tissue transfers are often necessary. The authors report 20 patients with sole and heel defects that were reconstructed with free flaps. Two patients had bilateral reconstruction. Three patients were younger than 10 years. Etiological causes were burn scar (N = 7), trauma (N = 7), chronic wound (N = 3), and tumor resection (N = 5). All defects were located at a weight bearing area. Gracilis muscle (N = 11), neurosensorial radial forearm (N = 7), latissimus dorsi muscle (N = 2), rectus abdominis muscle (N = 1), and posterolateral thigh flaps (N = 1) were used for reconstruction. Muscle flaps were preferred for the deep and irregular defects or chronic, open infected wounds. All flaps survived except for one total and two partial complications of necrosis. Recurrence of ulceration was observed in 1 patient with spinal cord trauma. The mean follow-up period was 33.7 months (range, 1-84 months). Patients were evaluated by direct gait observation, footprints, pedograms, and the Semmes Weinstein monofilament test. All patients returned to normal daily activity with individual gait patterns. Functional outcomes of both muscle and fasciocutaneous flaps were satisfactory. Presence of deep sensation, preservation of musculoskeletal integrity, and patient compliance are the main factors for durability of reconstruction. PMID- 10735218 TI - Immediate Camitz opponensplasty in acute thenar muscle injury. AB - Thenar function impairment or destruction is not uncommon in hand crush injury or thumb avulsion injury. Although complete evaluation of thenar muscle function is difficult during an emergency setting because of skeletal instability and pain, if left untreated the injured hand may result in deficiency of opposition and may require secondary reconstruction. When the palmaris longus tendon is available from the open wound, a Camitz opponensplasty can be performed in the emergency operation setting even as an augmentation procedure for opposition if there is any suspicion of thenar muscle impairment. Seven patients with such injuries who underwent immediate Camitz opponensplasty were assessed to have an 80% angle of separation, a 90% angle of circumduction, and an 88% Kapandji test of the normal opposite hand. An emergency Camitz procedure provided a reliable, immediate one stage reconstruction of thenar function. PMID- 10735219 TI - Nonsurgical delay of cutaneous flaps using the flashlamp pumped pulsed dye laser. AB - The surgical delay procedure is an effective method for improving skin flap survival. However, it has many disadvantages, such as bleeding, infection, and pain. It also requires an additional operation and a long time for wound management. Nonsurgical delay has a great importance, but pharmacological efforts for delay phenomena have never found clinical application. In this study, the authors attempted to reproduce the delay phenomena via the flashlamp pumped dye laser. Forty Sprague-Dawley rats were studied in four groups of 10 rats each. The experimental and control flaps were on the same rat. Flaps were planned on the ventral skin of the rats, and were symmetrical, three sided, based caudally, and were 2 cm in width, extended to the measured midpoint between the xiphoid and the sternal notch. The laser was applied to the different areas of the experimental flaps 15 days before flap elevation. A week later flap survival was determined by overlaying millimeter-grid acetate paper. Full-surface and perimeter "lased" experimental flaps showed a marked increase in surviving areas when compared with the control flaps. This kind of flap delay has a great potential in clinic application. PMID- 10735220 TI - A prospective study of the anatomic variations of the median nerve in the carpal tunnel in Asians. AB - A prospective study of anatomic variations of the median nerve and its relationship to the surrounding structures was performed of 354 consecutive operations in 192 patients with carpal tunnel syndrome at Korea University Anam Hospital from July 1995 to September 1997. A total of 184 patients were women and 8 patients were men. A total of 162 patients were bilateral and 30 were unilateral. Regarding the course of the motor branch, the extraligamentous type was most common (96.1%, 340 patients). A total of 81.1% of patients (N = 287) had the origin of the motor branch at the radial side of the median nerve (or radial one third), and 17.2% of patients (N = 61) had the origin of the motor branch at the anterior portion of the median nerve. Of these 61 patients, 30 patients (49.1%) were of the preligamentous type. Only one motor nerve branch was found in 89.5% of patients (N = 317), and multiple branches were found in 10.5% of patients. As a result of the comparison between two hands when both hands were operated, the origin and number of motor branches and their courses were identical in most patients (>60%). Hypertrophy of the flexor pollicis brevis was found in 8.5% of patients (N = 30), hypertrophy of the palmaris brevis was found in 4.2% of patients (N = 15), absence of the palmaris longus was discovered in 0.6% of patients (N = 2), existence of the median artery was found in 0.6% of patients (N = 2), and high division of the median nerve rejoining with the thenar motor nerve was found in 0.3% of patients (N = 1). Clearly, the anatomy of the carpal tunnel in Koreans is somewhat different, in part, from the results obtained from studies of whites, but overall results are not significantly different. These results can help obtain a better surgical outcome and complete decompression of the median nerve during operation while preventing inappropriate or inadvertent injury to the motor branch of the median nerve in Koreans. PMID- 10735221 TI - The effects of the number and ratio of repaired arteries and veins on the survival rate in digital replantation. AB - From January 1990 to December 1998, 631 consecutive single-digit replantations were reviewed retrospectively to determine the essential number of vascular anastomoses and the artery-to-vein ratio for successful finger replantation. The correlations between the number and the ratio of anastomosed vessels and the survival rate were examined according to the amputated digital levels, and all of the correlated results were compared with each other statistically. In zone I, the survival rate of the digits with a vein repaired was higher than that of digits treated with the external bleeding method. In zone II, repairing more arteries than veins led to venous congestion and resulted in a failure of replantation. Thus, the ratio of an equal or greater number of veins repaired to the arteries repaired was an important factor in successful replantation in zone II, which may be due to the large amount of arterial input relative to the small volume of venous drainage via small veins in the replanted digit. In zone III, the equal number of arteries and veins repaired was also an important factor in successful replantation. However, unlike zone II, even when more arteries were repaired than veins, venous congestion occurred rarely because the vein in this area was larger, providing sufficient venous drainage. In zone IV, repair of two veins was needed to yield good results. Because the volume of the amputated part in zone IV was larger than in other zones, it was desirable to repair both arteries, even though repairing one artery yielded good results. In conclusion, it is desirable to repair as many vessels as possible to increase the possibility of a good result. However, digital amputations and their conditions for replantation differ. Therefore, the number and ratio of vessels that need to be repaired should be modified and adjusted in each case. PMID- 10735222 TI - Effects of hyperbaric oxygen treatment and heparin on the survival of unipedicled venous flaps: an experimental study in rats. AB - The effects of hyperbaric oxygen (HBO) and heparin on the survival of the rat inferior epigastric venous flap were investigated. Preliminary transcutaneous oxygen measurements showed that partial oxygen pressure values of venous flaps increased at 2.5 ATA pressure while inhaling 100% oxygen. During the experiment, 128 venous flaps of two different sizes and 50 composite grafts were prepared bilaterally in 89 rats. Perivenous areolar tissue was removed from the pedicle vein in all flaps. Half of the venous flaps were isolated from the wound bed. Initial flap perfusion was tested by fluorescein injection during flap elevation. Four treatment groups were created: control, heparin, HBO, and HBO+heparin. After 6 days of treatment, the mean surviving flap area was calculated for each group. Surviving flaps were reelevated, final flap perfusion was tested by fluorescein injection, and flaps were harvested for histological examination. The mean survival rates of the HBO (26.56%) and the HBO+heparin (36.87%) groups were significantly higher than the control (0%) and the heparin (0%) groups (p<0.01). None of the composite grafts survived. Smaller flaps and nonisolated flaps survived better, although not significantly (p>0.05). Veins were enlarged both clinically and histologically. Fluorescein uptake was delayed during initial flap elevation but was normal during reelevation. These findings imply that the rat inferior epigastric venous flap may be an ischemic flap with capillary circulation through a single venous pedicle, but it needs HBO treatment to survive, especially during the acute period. Heparin treatment, reducing the flap size, and presence of a vascular wound bed also improve survival rates. PMID- 10735223 TI - The beneficial effect of heparin in preischemic perfusion solutions for cold stored skin flaps. AB - Storage of skin flaps in the cold before replantation increases their tolerance to ischemic damage. Rat epigastric skin flaps were perfused immediately before 2 days of cold ischemia with 3 ml of normal saline containing either 10 U per milliliter of heparin (group 1, N = 11) or normal saline (group 2, N = 10), or stored without perfusion (group 3, N = 6), and replanted. Flap viability was assessed 7 days later. The mean flap survival in groups 1, 2, and 3 was 73% (p<0.01 compared with group 2), 10%, and 33% respectively. Intravascular fibrin deposits were detected histochemically 5 minutes before reperfusion in nonperfused flaps and 5 minutes after reperfusion in saline-perfused flaps, but not in flaps perfused with heparinized saline. Angiography revealed evidence of no reflow in the first 5 minutes of reperfusion in saline-perfused flaps, but normal blood flow in heparinized saline-perfused flaps. Tissue water content, myeloperoxidase activity, and hydroperoxide levels after 1 and 24 hours of reperfusion were not significantly different in flaps perfused with heparinized saline and normal saline. These findings indicate that in skin flaps perfused before ischemia, flaps perfused with heparinized saline survive significantly better than flaps perfused with normal saline. They also survive better than nonperfused flaps but the improvement is not significant. PMID- 10735224 TI - Penile lengthening for traumatic penile amputation due to ritual circumcision: a case report. AB - Some tribes in South Africa still practice ritual circumcision in adolescent boys. A traditional healer performs the procedure and, not uncommonly, amputation occurs. The authors present a case of a 20-year-old man who sustained a midshaft penile amputation as a result of a ritual circumcision. Treatment was with a modified penile-lengthening technique that incorporates gracilis muscle transposition to fill the dead space created after detaching the penile suspensory ligament. This may help prevent retraction. Almost 3 cm of lengthening was obtained. This is a useful method of treatment for penile amputation. PMID- 10735225 TI - The use of the prepuce for reconstruction of an intraoral burn. AB - Intraoral and commissural burns present a complex challenge for the reconstructive surgeon, with contractures being the main sequela encountered. Various reconstructive techniques have been described, none of which offers an ideal solution. A case of a severe intraoral contracture due to a caustic burn, and the modality of treatment is described. Once the contractures were released, a full-thickness preputial graft was used to cover the resultant buccal mucosal defect, with a satisfactory result. Due to its unique properties, the prepuce should be included as an additional tool in the reconstructive surgeon's armamentarium. PMID- 10735226 TI - Correction of scar contracture deformities of the big toe with a multiplanar distraction device. AB - A multiplanar distraction device was used in a 65-year-old woman for correction of multiplanar deformities of her right big toe. These deformities were caused by long-standing scar contracture after a crushing injury to the right foot. Without the necessity of other complicated procedures, the dorsal contracture and lateral deviation were corrected from 43 deg to 0 deg and from 22 deg to 0 deg respectively 3 weeks postoperatively. Kirschner wires were inserted temporarily for prevention of recurrence after removal of the frame, and were removed 6 weeks later. In follow-up after 8 months, the position of the big toe was stable and without recurrence of contracture. Application of the multiplanar distraction device simplified the surgical procedure to achieve this correction. PMID- 10735227 TI - Supratrochlear artery flap for the repair of lower eyelid defects. AB - An island pattern flap in the treatment of lower eyelid cicatricial ectropion is described. The flap is based on the supratrochlear artery and its associated veins. The donor site can be closed easily primarily. The very thin thickness of the pedicle increases the rotational arc of the flap. However, this procedure requires two stages and there is a slight bulkiness of the flap, both of which are major drawbacks of this method. This flap was used to restore missing supporting soft tissue of the lower eyelid in the clinical cases described herein. PMID- 10735228 TI - A prosthetic breast implant infected with Mycobacterium fortuitum. AB - Augmentation mammaplasty is a common operation performed in the United States. Postoperative wound infections are rare, but can be devastating. Most often, bacteria from the normal skin flora cause these infections, but more atypical organisms can lead to similar situations. The authors present a case of a prosthetic breast implant infected with Mycobacterium fortuitum after augmentation mammaplasty. The patient, diagnosis, and treatment are discussed so that others may recognize and treat this entity successfully before encountering major complications. Although it is an infrequent occurrence, plastic surgeons, infectious disease specialists, and primary care doctors who may see postoperative wound infections should be aware of this potential pathogen. It is important in any postimplant infection and especially crucial in cases of unresolving or recurrent infections with unusual or even clear drainage. With proper identification through acid-fast smear and culture, multiagent therapy can be initiated early. Additional complications, including implant removal, may thus be avoided. PMID- 10735230 TI - Treatment of painful neuromas: a case report. AB - The authors report a 15-year history of management of multiple recurrent neuromas in a patient with an amputated arm. Various surgical modalities were employed, including burying the nerve ends in muscle and bone. In addition, they also treated successfully one of the neuromas in this patient by capping the transected nerve with an extended autologous vein graft. This application of the extended autologous venous nerve conduit may be a novel alternative in the treatment of this challenging problem. PMID- 10735229 TI - Salvage of the exposed irradiated knee joint with free tissue transfer. AB - Extremity radiation results in substantial complications in 6% to 10% of patients and includes fracture, edema, pain, fibrosis, neuropathy, arterial thrombosis, joint immobility, soft-tissue necrosis, and chronic infection. Chronic ulceration and infection of an irradiated joint is considered a particularly challenging problem for the reconstructive surgeon, and results of surgical management of these complications have not been reported previously in the medical literature. Two patients are presented with large ulcerated and necrotic radiation wounds of the knee, with chronic contamination, osteomyelitis, and involvement of the joint space. Both patients were treated successfully with debridement and coverage with free tissue transfer. They obtained stable, healed wounds, became pain free, and were able to ambulate on long-term follow-up. Adherence to principles established previously for the management of radiation-induced ulcers on other parts of the body not involving joint spaces (namely, thorough wound debridement and coverage with nonirradiated, well-vascularized tissue) can allow successful extremity salvage even in the presence of joint exposure, contamination, and osteomyelitis. PMID- 10735231 TI - A nasal shaver for nasal surgery. AB - Provision of a nasal field in nasal surgery is hampered by nasal hairs. We recommend the Remington NE4 nasal shaver as a quick, painless, safe, inexpensive method of nasal hair removal, providing a user-friendly surgical field. PMID- 10735232 TI - Please Czech spelling. PMID- 10735233 TI - Alkali burns to the hand. PMID- 10735234 TI - A case of hemangioma of the buccal fat pad. PMID- 10735235 TI - Pigmented, superficial multicentric basal cell carcinoma. PMID- 10735236 TI - A large, superficial lipoma and three deep-seated lipomas. PMID- 10735237 TI - A case of rapidly recurring cheek Merkel cell carcinoma. PMID- 10735238 TI - Bacteriophages as indicators of enteric viruses and public health risk in groundwaters. AB - Low concentrations of all types of bacteriophages in groundwater limit their power to predict the presence of enteric viruses. There is little concordance in the literature regarding phage detection methods, thus making comparisons extremely difficult. Different authors have used different hosts, phage concentration methods, and end-point determinations. Also, markedly different volumes of sample have been employed, varying from 1 litre to 400 l. Bacteriophage concentration methods are not reproducible. There has been marked variability among groups in the natural substrates used (for example, beef extract), the type of adsorbing filter used, centrifugation instruments and conditions, and the delivery of the concentrate to the host cells. There is no consensus on the best bacterial host strain. Currently, several are employed with each showing differential sensitivities and specificities. In particular, host stability must be considered. Host stability has two components: the ability of the host to continue to be receptive to the bacteriophage after continued sub culture, and the lack of lysogenic or temperate bacteriophage in the host cell line which may be randomly and unpredictably activated. There is a lack of consistent recovery of bacteriophages from individual faecal specimens. In particular, only approximately 3% of individual humans carry the FRNA phages. While there is some evidence to indicate that the phages multiply in sewage, it is not clear how they do so since the host pili should not be produced at lower temperatures. These ecological factors need to be understood. Of all the phages thus far studied, Bacteroides fragilis HSP40 has the highest recovery rate from individual people. However, Bacteroides, being an anaerobe, is a difficult host for routine laboratory analysis. Methods for the enumeration of F(+)-specific phages and Bacteroides phages are complex, time-consuming, costly and not reproducible. Conversely, somatic coliphage methods are simpler and results can be available in 4-6 h. The occurrence of phages and viruses in groundwater depends on physicochemical characteristics that control their fate and transport in the groundwater/aquifer environment. There are very little actual data taken from the field that allow an understanding of the ecology and life span of phages in their natural environment. Moreover, the ability of phages to serve as a source of food for other microbes needs to be understood. There has been a lack of association of bacteriophage recovery with gastroenteritis outbreaks due to enteric viruses. There is only a small epidemiological database concerning the occurrence of enteric viruses in groundwater. PMID- 10735239 TI - Reduced susceptibility of thin Pseudomonas aeruginosa biofilms to hydrogen peroxide and monochloramine. AB - Pseudomonas aeruginosa attached to alginate gel beads in sparse, thin biofilms exhibited reduced susceptibility to monochloramine and hydrogen peroxide compared with planktonic cells of the same micro-organism. Disinfection rate coefficients for planktonic bacteria averaged 0.551 mg(-1)min(-1) for monochloramine and 3.1 x 10(-4)l mg(-1) min(-1) for hydrogen peroxide. The corresponding values for 24-h old biofilm cells were 0.291 mg min(-1) and 9.2 x 10(-5) 1 mg(-1) min(-1) for monochloramine and hydrogen peroxide, respectively. Several pieces of evidence support the interpretation that the reduced susceptibility of biofilm was not due simply to inadequate delivery of the antimicrobial agent to the local environment of the attached cells. No correlation between biofilm susceptibility and biofilm initial areal cell density was observed. Rapid delivery of hydrogen peroxide to the attachment surface, and subsequently to the interior, of the alginate gel beads was visualized by a direct experimental technique. Theoretical analysis of unsteady diffusion and diffusion reaction interactions also argued against any significant delay or barrier to antimicrobial or oxygen delivery. It was hypothesized that new genes are expressed when bacteria attach to a surface and begin to form a biofilm and that some of the resulting gene products reduce the susceptibility of the cell to antimicrobial agents including oxidative biocides such as monochloramine and hydrogen peroxide. PMID- 10735240 TI - Growth, pigment production and protease activity of Monascus purpureus as affected by salt, sodium nitrite, polyphosphate and various sugars. AB - The effect of different levels of salt, sodium nitrite, polyphosphate and various sugars on growth, pigment production, protease activity and culture pH caused by Monascus purpureus was studied in broth medium and ground meat. The addition of sodium chloride (> 50.0 g l(-1)) and polyphosphate (> 3.0g l(-1)) to broth medium decreased mycelial growth, pigment production and protease activity of M. purpureus, whereas low concentrations of sodium nitrite (< 0.2 g l(-1)) promoted mycelial growth and pigment production. When the basal medium and ground meat contained salt, 150.0 g l(-1), the mould growth was stopped. The medium with fructose as carbon source proved to be the most suitable for mycelium growth and pigment production, with maltose and glucose being the second most productive. When sucrose and lactose were used as carbon sources, mycelium growth and pigment production were inhibited but the protease activity increased significantly. The mould showed more tolerance to salt and polyphosphate in ground meat than in broth medium and used sucrose as a carbon source as well as glucose for growth and pigment production in the meat mixture. PMID- 10735241 TI - Growth of Listeria monocytogenes and Yersinia enterocolitica colonies under modified atmospheres at 4 and 8 degrees C using a model food system. AB - The growth of Listeria monocytogenes and Yersinia enterocolitica colonies was studied on solid media at 4 and 8 degrees C under modified atmospheres (MAs) of 5% O2: 10% CO2: 85% N2 (MA1), 30% CO2: 70% N2 (MA2) and air (control). Colony radius, determined using computer image analysis, allowed specific growth rates (mu) and the time taken to detect bacterial colonies to be estimated, after colonies became visible. At 4 degrees C both MAs decreased the growth rates of L. monocytogenes by 1.5- and 3.0-fold under MA1 (mu = 0.02 h(-1)) and MA2 (mu = 0.01 h(-1)), respectively, as compared with the control (mu = 0.03 h(-1)). The time to detection of bacterial colonies was increased from 15 d (control) to 24 (MA1) and 29 d (MA2). At 8 degrees C MA2 decreased the growth rate by 1.5-fold (mu = 0.04 h(-1)) as compared with the control (mu = 0.06 h(-1)) and detection of colonies increased from 7 (control) to 9 d (MA2). At 4 degrees C both MAs decreased the growth rates of Y. enterocolitica by 1.5- and 2.5-fold under MA1 (mu = 0.03 h( 1)) and MA2 (mu = 0.02 h(-1)), respectively, as compared with the control (mu = 0.05 h(-1)). At 8 degrees C identical growth rates were obtained under MA1 and the control (mu = 0.07 h(-1)) whilst a decrease in the growth rate was obtained under MA2 (mu = 0.04 h(-1)). The detection of colonies varied from 6 (8 degrees C, aerobic) to 19 d (4 degrees C, MA2). Refrigerated modified atmosphere packaged foods should be maintained at 4 degrees C and below to ensure product safety. PMID- 10735242 TI - Bacteriocin production by lactic acid bacteria isolated from Rioja red wines. AB - Forty-two lactic acid bacteria (LAB) of the genera Lactobacillus (32), Leuconostoc (6), Pediococcus (3) and Lactococcus (1), isolated from Rioja red wines, were tested for antimicrobial activity. All these strains, as well as 18 Leuconostoc oenos and 19 yeast strains were used as indicators. Only nine strains showed antimicrobial activity, and all were of the species Lactobacillus plantarum, which constitutes the predominant microflora in Rioja red wines after alcoholic fermentation. Lact. plantarum strain J-51 showed the widest range of action, inhibiting the growth of 31 strains of the four studied LAB genera. Lact. plantarum J-51 antimicrobial activity was lost after treatment with proteases, suggesting a proteinaceous nature for this activity. It was found to be stable between pH 3 and 9 and under strong heating conditions (100 degrees C for 60 min). Polymerase chain reaction (PCR) analysis of Lact. plantarum J-51 genome revealed the presence of the plnA gene that encodes the plantaricin precursor PlnA. A 366-bp fragment was sequenced and showed 95% identity with pln locus of Lact. plantarum C-11. The deduced precursor peptide sequence showed one mutation (Gly7 to Ser7) at the double glycine leader peptide, and the three putative 26-, 23- and 22-residue active peptides remain identical to those of Lact. plantarum C 11. Therefore, antimicrobial peptides constitute a potent adaptation advantage for those strains that dominate in a medium such as wine, and can play an important role in the ecology of wine microflora. PMID- 10735243 TI - The growth of Listeria monocytogenes in cheese packed under a modified atmosphere. AB - The effect of modified atmosphere Packaging (MAP) on the growth of Listeria monocytogenes in mould ripened cheeses was studied at refrigeration temperatures (2-8.3 degrees C) over a storage period of 6 weeks. Control experiments in cling film with no atmospheric modification produced a lag time before growth of up to 1 week and rapid subsequent growth. MAP with a CO2 concentration of less than 20% allowed growth to occur but when O2 was incorporated; the lag time was reduced from 3 to 2 weeks and subsequent growth was also faster, producing an increase in cell numbers of 1.4 log cycles over the incubation period. N2-MAP in the absence of O2 increased the lag time to 3 weeks and slowed growth, while the inclusion of CO2 extended the lag to 3 weeks and slowed subsequent growth even more. In MAP with 80:10:10 (v/v/v) N2:CO2:O2, there was a lag period of 2-3 weeks before growth of L. monocytogenes occurred, while the total viable aerobic count (TVAC) decreased by 2-3 log cycles and the total Lactobacillus count showed little change. It was concluded that MAP was not suitable for preventing the growth of L. monocytogenes in such cheeses. PMID- 10735244 TI - The microbial association of Greek taverna sausage stored at 4 and 10 degrees C in air, vacuum or 100% carbon dioxide, and its spoilage potential. AB - Strains of the Lactobacillus sakei/curvatus group, mainly non-slime-producing Lact. sakei, dominated the microbial flora of industrially manufactured taverna sausage, a traditional Greek cooked meat, stored at 4 degrees C and 10 degrees C in air, vacuum and 100% CO2. Atypical, arginine-positive and melibiose-negative strains of this group were isolated. The isolation frequency of Lact. sakei/curvatus from sausages stored anaerobically was as high as 92-96%, while other meat spoilage organisms were practically absent. Conversely, in air-stored sausages, leuconostocs, mainly Leuconostoc mesenteroides ssp. mesenteroides, had a considerable presence (14-21%), whereas Brochothrix thermosphacta, pseudomonads and Micrococcaceae grew, but failed to increase above 10(5) cfu g(-1) in all samples during storage. Only yeasts were able to compete against LAB and reached almost 10(7) cfu g(-1) after 30 d of aerobic storage at 10 degrees C. The great dominance (> 10(8) cfu g(-1)) of LAB caused a progressive decrease of pH and an increase of the concentration of L-lactate, D-lactate and acetate in all sausage packs. The growth of LAB and its associated chemical changes were more pronounced at 10 degrees C than 4 degrees C. At both storage temperatures, L-lactate and acetate increased more rapidly and to a higher concentration aerobically, unlike D-lactate, which formed in higher amounts anaerobically. Storage in air was the worst packaging method, resulting in greening and unpleasant off-odours associated with the high acetate content of the sausages. Carbon dioxide had no significant effect on extending shelf-life. The factors affecting the natural selection of Lact. sakei/curvatus in taverna sausage are discussed. Moreover, it was attempted to correlate the metabolic activity of this group with the physicochemical changes and the spoilage phenomena occurring in taverna sausage under the different storage conditions. PMID- 10735245 TI - Differentiation of Brucella species by random amplified polymorphic DNA analysis. AB - Random amplification of polymorphic DNA (RAPD) was used for discrimination between 46 Brucella strains and 14 representatives of the alpha-2 and alpha-1 subgroups of Proteobacteria. To evaluate a relatively quick and exact method for Brucella identification, the authors specified the most suitable conditions for RAPD amplification of Brucella DNA with two 10-mer primers, containing lower and higher percentages of G and C. The software package PHYLIP 3.1 was used for cluster analysis of the RAPD fingerprints. The optimization of RAPD conditions resulted in PCR mixes suitable for reliable typing of Brucellae. The distance based methods (Fitch-Margoliash, UPGMA and Neighbour-joining) gave clear discrimination between Brucella species. The constructed dendrograms put Br. canis and Br. suis bv. 1 in the same cluster and differentiated Brucella strains according to their host preferences. RAPD can be useful method to distinguish related bacterial species, and under strictly established conditions the reaction appears to be a simple, quick and sensitive technique for the epidemiological investigation of brucellosis. PMID- 10735246 TI - Purification and some characteristics of enterocin ON-157, a bacteriocin produced by Enterococcus faecium NIAI 157. AB - Bacteriocin-like activity (BLA) was screened in 690 strains of lactic acid bacteria isolated from plant materials such as silages and fermented vegetables. Among them, a strain identified as Enterococcus faecium NIAI 157 showed a clear BLA against the indicator strain, Ent. faecium IFO 13712. The proteinaceous nature and antimicrobial activity against closely related species strongly indicated that this BLA was a bacteriocin and was designated enterocin ON-157. The bacteriocin activity of this strain was extracellularly produced in the logarithmic growth phase in MRS broth and purified by ultrafiltration, ammonium sulphate precipitation and cation-exchange chromatography. Purified enterocin ON 157 had a molecular weight of approximately 2500 Da in SDS-PAGE analysis and was easily inhibited by treatment with alpha-amylase and proteolytic enzymes. Enterocin ON-157 had a bactericidal mode of action and inhibited the growth of the enterococci, Lactobacillus sake and Listeria monocytogenes. Enterococcus faecium NIAI 157 harboured two plasmids, 49.0 kb and 43.7 kb, and a variant missing a larger plasmid by curing with novobiocin lost the bactriocin activity. PMID- 10735247 TI - Biological control of an insect pest by gut-colonizing Enterobacter cloacae transformed with ice nucleation gene. AB - The ice nucleation (IN) gene inaA of epiphytic Erwinia (Pantoea) ananas IN10 was transformed into Enterobacter cloacae WBMH-3-CMr originated from the faeces of silkworms. The transformant designated as Ent. cloacae WBMH-3-CMr(pICE6S13) exhibited IN activity, unlike the parent strain. The transgenic strain was ingested by mulberry pyralid larvae, fed on detached mulberry leaves, and the supercooling capacity and cold hardiness of these larvae were examined. The mean supercooling point (SCP) of the larvae ingesting the transgenic strain was - 3.3 degrees C, 8 degrees C higher than that of larvae treated with distilled water (control) and 1.5 C higher than an ice nucleation active (INA) strain of Erw. ananas. The SCPs of the larvae were stably maintained over the 9 d after ingestion. The maintenance of these high SCPs was due to transgenic Ent. cloacae having a more stable and efficient gut colonization than Erw. ananas, which is identified by the distribution of a narrower range of SCPs (-2 to -5 degrees C) in larvae treated with the transgenic stain. Furthermore, most of the larvae ingesting the transgenic strain froze and died when they were exposed to cold conditions of -5 degrees C for 18 h, 3 or 7 d after ingestion. In contrast, most of the larvae ingesting no bacterium did not die under similar conditions. On the other hand, the growth ability of Ent. cloacae WBMH-3-CMr on mulberry leaves tended to be lower than that of epiphytic Erw. ananas, as assayed by pot tests. These findings would expand the possibility of biological control using INA bacteria since Ent. cloacae would harbour a broader host (insect) range for gut colonization and a smaller affinity to plants to benefit from prevention of plant frost injury. PMID- 10735248 TI - Chromium(VI) reductase activity is associated with the cytoplasmic membrane of anaerobically grown Shewanella putrefaciens MR-1. AB - Shewanella putrefaciens MR-1 can reduce a diverse array of compounds under anaerobic conditions, including manganese and iron oxides, fumarate, nitrate, and many other compounds. These reductive processes are apparently linked to a complex electron transport system. Chromium (Cr) is a toxic and mutagenic metal and bacteria could potentially be utilized to immobilize Cr by reducing the soluble and bioavailable state, Cr(VI), to the insoluble and less bioavailable state, Cr(III). Formate-dependent Cr(VI) reductase activity was detected in anaerobically grown cells of S. putrefaciens MR-1, with highest specific activity in the cytoplasmic membrane. Both formate and NADH served as electron donors for Cr(VI) reductase, whereas L-lactate or NADPH did not support any activity. The addition of 10 micromol l(-1) FMN markedly stimulated formate-dependent Cr(VI) reductase, and the activity was almost completely inhibited by diphenyliodonium chloride, an inhibitor of flavoproteins. Cr(VI) reductase activity was also inhibited by p-chloromercuriphenylsulphonate, azide, 2-heptyl-4-hydroxyquinolone N-oxide, and antimycin A, suggesting involvement of a multi-component electron transport chain which could include cytochromes and quinones. Cr(V) was detected by electron paramagnetic resonance (EPR) spectroscopy, suggesting a one-electron reduction as the first step. PMID- 10735249 TI - Use of restriction fragment length polymorphism analysis to differentiate strains of psychrophilic and psychrotropic clostridia associated with blown pack' spoilage of vacuum-packed meats. AB - Reference and meat strains of psychrophilic and psychrotrophic clostridia were differentiated using restriction fragment length polymorphism (RFLP) analysis of genomic DNA (DNA-RFLP) and the polymerase chain reaction-amplified 16S rDNA gene (PCR-RFLP). Groupings obtained with PCR-RFLP were confirmed with 16S rDNA gene sequencing. DNA-RFLP resolved 19 of the 22 meat strains into 11 groups. Three meat strains were untypable using this method. All reference strains representing different genotypic species could be distinguished by the restriction patterns of 16S rDNA genes. With PCR-RFLP, the 22 meat strains produced eight distinct genotypes. 16S rDNA gene sequencing confirmed that each genotype was represented by a distinct sequence. PCR-RFLP restriction patterns of 15 meat strains matched those of one of two of the seven reference strains used. Seven meat strains whose RFLP restriction patterns of 16S rDNA genes differed from those of any reference strains probably represent four previously undescribed species. Although RFLP analysis of the amplified 16S rDNA gene allowed differentiation of psychrophilic and psychrotrophic clostridia at the genotypic species level and below, comparison of PCR-RFLP patterns and 16S rDNA sequences of unknown clostridial isolates with patterns and sequences of reference strains may not effect ready identification of these micro-organisms. The results of this study will be useful in diagnosis of the cause of premature spoilage of chilled vacuum-packed meats and in tracing spoilage-causing clostridia to their source(s) in the abattoir. PMID- 10735250 TI - Phenotypic and genotypic diversity of yeasts isolated from water-buffalo Mozzarella cheese. AB - Water-buffalo Mozzarella (WBM) cheese is one of the several 'pasta filata' or stretched curd cheeses that originated in southern Italy, traditionally manufactured from raw milk employing natural whey starter cultures. Lactose- and galactose-fermenting yeasts isolated from WBM were studied to evaluate their role in the ripening of this cheese. The kinetic parameters of the growth of the yeasts as well as their principal metabolic end-products showed a great variability depending on the species. Moreover, the genetic polymorphism of the yeasts was studied for their differentiation at species level by means of the polymerase chain reaction (PCR) fingerprinting and mitochondrial DNA (mtDNA) restriction analysis. While the differentiation based on metabolic traits was not able to discriminate Kluyveromyces marxianus, Candida kefyr and C. sphaerica, the PCR analysis with primers M13 and RF2 resulted in a reliable and rapid method for differentiating at species level Saccharomyces cerevisiae, K. marxianus, K. lactis and their anamorphic species. Furthermore, mtDNA analysis proved to be more discriminating at strain level. PMID- 10735251 TI - The production of freeze-dried immobilized cultures of Streptococcus thermophilus and their acidification properties in milk. AB - The aim of this study was to prepare alginate-immobilized freeze-dried cultures of Streptococcus thermophilus and to compare the acidifying activities of these rehydrated cultures with classical free cell liquid inoculants. Streptococcus thermophilus BT1 grew in alginate beads and the population reached 10(10) cfu g( 1) after 6 h incubation. Re-inoculation of the beads in fresh medium with a further 6 h incubation did not improve the biomass level, but extending the incubation at 42 degrees C to 24 h caused significant death. The rehydrated immobilized cell technology (ICT) starter contained 13% free cells. In acidifying activity tests, the ICT culture had a similar acidification curve to that of a classical milk-grown free cell culture, except that it reached lower final pH values. Although the differences between the ICT and liquid cultures were not important, there were significant effects of inoculation level on lag time, maximum acidification rate and on the pH and time at which the acidification rate was at its highest. PMID- 10735252 TI - Characteristics of the biologically active 35-kDa metalloprotease virulence factor from Listeria monocytogenes. AB - Listeria monocytogenes, a facultative intracellular pathogen, synthesizes an extracellular protease which is responsible for the maturation of phosphatidylcholine phospholipase C (lecithinase), a virulence factor involved in cell-to-cell spread. This work describes the environmental parameters necessary for increased production of mature, 35-kDa active protease in strains of L. monocytogenes, and its detection using polyclonal antibodies raised against Bacillus subtilis neutral protease. High performance liquid affinity chromatography was exploited to isolate the biologically active form of the mature protease, which was then subjected to biochemical characterization using casein as a substrate. The protease is a zinc-dependent metalloprotease which degrades casein over a wide range of temperatures and pH values. It can also degrade actin, the most abundant protein in many eukaryotic cells. The Listeria protease was shown to exhibit a high thermal stability and a relatively narrow substrate specificity. A three-dimensional model built on the basis of the homology with thermolysin was used to understand the structural basis of these characteristics. PMID- 10735253 TI - Effect of some nutritional and environmental parameters on the production of diacetyl and on starch consumption by Pediococcus pentosaceus and Lactobacillus acidophilus in submerged cultures. AB - Three series of 5-day submerged cultures with Pediococcus pentosaceus MITJ-10 and Lactobacillus acidophilus Hansen 1748 were carried out in starch-based media, and the effect of cultural factors on the changes of starch, diacetyl and amylase activity determined. In axenic cultures, Ped. pentosaceus MITJ-10 produced more diacetyl (63.27 mg l(-1)) by adding glucose, yeast extract and CaCO3 (P < 0.01), at 28 degrees C (P < 0.05); but more starch was consumed (18.4 g l(-1)) in the absence of glucose (P < 0.01). Lact. acidophilus Hansen 1748 consumed more starch (26.56 g l(-1)) at 28 degrees C, with CaCO3, glucose (P < 0.01) and yeast extract (P < 0.05); however, the amylolytic activity (10077U l(-1)) was favoured at 35 degrees C (P < 0.01). Little starch was consumed in mixed cultures due to the low pH; nevertheless, diacetyl content rose to 135.76 mg l(-1) at 32 degrees C (P < 0.01). Therefore, both studied strains might be useful to produce aromatic extensors from starchy substrates. These natural aromatic extensors are of interest to the food industry. PMID- 10735254 TI - Use of oligotrophic bacteria for the biological monitoring of heavy metals. AB - Oligotrophic bacteria exhibited active growth even in nutritionally deficient medium made with nutrient broth that had been diluted with distilled water, 1 : 10 000. The oligotrophic bacteria, Sphingomonas paucimobilis KPS01 and Burkholderia cepacia KPC01 and KPC02 were found to be highly susceptible to heavy metals and to be potentially useful as sensors for the assessment of toxicity. The susceptibility of the bacteria to metals was measured by incubating the bacteria with metals of varying concentrations in the nutritionally deficient medium at 30 degrees C for 24 h. Bacteria were considered susceptible when the growth inhibition rate (EC50 was more than 50% of the control. The EC50 value of Ag+, Pb2+ and Cd2+ was 10(5)mmol l(-1) and Zn2+, Cr3+, Cr6+, Cu2+ and Hg2+ was 10(-4) mmol l(-1) in S. paucimobilis KPS01. Other strains also showed similar results. No difference in the EC50 was found using either the chloride or sulphate forms of these metals. The optimum incubation time was 24 h and a longer incubation time did not necessarily lead to more inhibition. The EC50 value rose in proportion to the concentration of nutrition in media. Environmental samples were tested and 14 out of 88 samples inhibited the growth of S. paucimobilis KPS01. PMID- 10735255 TI - Effect of biocontrol strains of Trichoderma on plant growth, Pythium ultimum polulations, soil microbial communities and soil enzyme activities. AB - Five strains of Trichoderma with known biocontrol activities were assessed for their effect upon pea growth and their antagonistic activity against large Pythium ultimum inocula. The effect of Trichoderma inocula upon the indigenous soil microflora and soil enzyme activities in the presence and absence of Pythium is assessed. In the absence of Pythium, Trichoderma strain N47 significantly increased the wet shoot weight by 15% but did not significantly affect the dry weight, whilst strains T4 and N47 significantly increased the root weights by 22% and 80%) respectively. Strains TH1 and N47 resulted in significantly greater root lengths. Pythium inoculation significantly reduced the root length and the number of lateral roots and nodules, and significantly increased the root and rhizosphere soil fungal populations. Pythium inoculation significantly reduced the plant wet and dry shoot weights and significantly increased the wet and the dry shoot/root ratio. All the Trichoderma strains reduced the number of lesions caused by Pythium and increased the number of lateral roots. The effect of the Pythium on emergence and shoot growth was significantly reduced by all the Trichoderma strains except strain To10. Inoculation with Trichoderma strains TH1 and T4 resulted in significantly greater wet root weights (62% and 57%, respectively) in the presence of Pythium compared to the Pythium control. Strain N47 significantly increased the shoot/root ratio compared to the Pythium control. Inoculation with Trichoderma strains T4, T12 and N47 significantly reduced Pythium populations. Pythium increased the activity of C, N and P cycle enzymes, whilst four Trichoderma strains reduced this effect, indicating reduced plant damage and C leakage. Overall, strains T4 and N47 had the greatest beneficial characteristics, as both these strains improved plant growth in the absence of Pythium and reduced plant damage in the presence of Pythium. The dual properties of these strains improve the commercial application, giving them an advantage over single action inocula, especially in the absence of plant pathogens. PMID- 10735256 TI - The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil). AB - The essential oil of Melaleuca alternifolia (tea tree) exhibits broad-spectrum antimicrobial activity. Its mode of action against the Gram-negative bacterium Escherichia coli AG100, the Gram-positive bacterium Staphylococcus aureus NCTC 8325, and the yeast Candida albicans has been investigated using a range of methods. We report that exposing these organisms to minimum inhibitory and minimum bactericidal/fungicidal concentrations of tea tree oil inhibited respiration and increased the permeability of bacterial cytoplasmic and yeast plasma membranes as indicated by uptake of propidium iodide. In the case of E. coli and Staph. aureus, tea tree oil also caused potassium ion leakage. Differences in the susceptibility of the test organisms to tea tree oil were also observed and these are interpreted in terms of variations in the rate of monoterpene penetration through cell wall and cell membrane structures. The ability of tea tree oil to disrupt the permeability barrier of cell membrane structures and the accompanying loss of chemiosmotic control is the most likely source of its lethal action at minimum inhibitory levels. PMID- 10735257 TI - Effect of pH control on lactic acid fermentation of starch by Lactobacillus manihotivorans LMG 18010T. AB - Lactic acid fermentation of starch by Lactobacillus manihotivorans LMG 18010T, a new amylolytic L(+) lactic acid producer, was investigated and compared with starch fermentation by Lact. plantarum A6. At non-controlled pH, growth and lactic acid production from starch by Lact. manihotivorans LMG 18010T lasted 25 h. Specific growth and lactic acid production rates continuously decreased from the onset of the fermentation, unlike Lact. plantarum A6 which was able to grow and convert starch product hydrolysis into lactic acid more rapidly and efficiently at a constant rate up to pH 4.5. In spite of complete and rapid starch hydrolysis by Lact. manihotivorans LMG 18010T during the first 6 h, only 45% of starch hydrolysis products were converted to lactic acid. When pH was maintained at 6.0, lactic acid, amylase and final biomass production by Lact. manihotivorans LMG 18010T increased markedly and the fermentation time was reduced by half. Under the same conditions, an increase only in amylase production was observed with Lact. plantarum A6. When grown on glucose or starch at pH 6.0, Lact. manihotivorans LMG 18010T had an identical maximum specific growth rate (0.35 h(-1)), whereas the maximum rate of specific lactic acid production was three times higher with glucose as substrate. Lactobacillus manihotivorans LMG 18010T did not produce amylase when grown on glucose. Based on the differences in the physiology between the two species and other amylolytic lactic acid bacteria, different applications may be expected. PMID- 10735258 TI - A bioluminescence assay for screening thermoregulated genes in a psychrotrophic bacterium Pseudomonas fluorescens. AB - Random transcription fusion delivery, with bacterial luciferase genes as reporter, was performed in the psychrotrophic bacterium Pseudomonas fluorescens. Direct screening on plates of the insertions allowed the isolation of fusions into thermoregulated genes with good accuracy, either in a library of insertion fusions, or after genetic transfer of a putative regulatory mutation. Using this method, it was shown that in Ps. fluorescens, nearly 40% of the genes are thermoregulated and belong to at least three classes according to the maximal temperature of expression of the fused genes. This is more than had been estimated by a previous method, and demonstrates the importance of thermoregulation in psychrotrophic bacteria. As this reporter is the first to be used for direct screening for genes regulated by temperature, it should be of great value in the study of mechanisms involved in adaptation to this environmental factor. PMID- 10735259 TI - Research on guidelines implementation in primary care. Introduction. PMID- 10735260 TI - Guidelines for evaluation. AB - Before implementing a guideline, reassurance is needed that the guideline is valid, reproducible and reliable. A method for assessing guideline validity has now been developed for general use. Guideline producers need to consider suitable strategies for implementing their guidelines. When designing and evaluating a guideline implementation strategy, three key questions need to be addressed: what are the most suitable intervention techniques; what are the desired effects of the guideline; and what will be the relevance of changes achieved? The time scale for the evaluation should also be determined carefully. PMID- 10735261 TI - Implementation strategies. AB - There is increasing information on the effectiveness of different interventions intended to reduce the gap between clinical practice and what is indicated by evidence on the effectiveness and cost-effectiveness of clinical interventions. Interventions include behavioural and financial/organizational. Unfortunately, none of the interventions have a substantial impact upon clinical practice. Many interventions that are evaluated are poorly developed, and there are several common methodological weaknesses in many of the evaluations conducted. The paper concludes with a comment on the nature of the market place for health technologies, and suggests that 'implementation interventions' may not be the solution to the problems of the poor uptake of medical research. PMID- 10735262 TI - Experimental and quasi-experimental designs for evaluating guideline implementation strategies. AB - The choice of study design for guideline implementation studies will determine the confidence with which the observed effects can be attributed to the interventions under study. In general, cluster randomized trials, of which there are different types, provide the most robust design. However, the use of these designs has implications for the power, conduct and analysis of studies. Wherever possible, designs allowing head-to-head comparisons, which incorporate baseline measures of performance, should be used. PMID- 10735263 TI - Sample size in guidelines trials. AB - In clinical trials, the statistical concepts of significance and power are used in the determination of sample size for trials. The trialist must provide an estimate of standard deviation and a hypothetical population difference to be detected. This must be modified to deal with the designs encountered in guideline research. These are cluster randomized trials, because the patients of a single doctor or practice form a cluster. The trialist must be able to provide information about the effects of clustering, in the form of an intraclass correlation coefficient. PMID- 10735264 TI - Measuring process and outcomes: professional compliance, professional opinions and patients' well-being. AB - This paper examines how professional and patient behavioural variables are conceptualized, their application in relation to guideline implementation and the different methods that may be employed in their assessment. The type of guideline and the process of development of guidelines are shown to influence the choice of indicators, with important issues for the assessment of professional compliance being raised by the development of patient-centred guidelines which require that clinical management takes account of patients' beliefs and preferences. Methodological problems relating to the use of routine data as indicators of professional compliance are discussed. The differing roles and contributions of quantitative and qualitative research in the assessment of professional and patient opinions are also examined. The paper advocates the development of guides to good practice in the assessment of these behavioural variables rather than specifying a uniform approach, and identifies questions requiring further research, especially in relation to patients' evaluations and satisfaction. PMID- 10735265 TI - Evaluating the cost-effectiveness of interventions designed to increase the utilization of evidence-based guidelines. AB - Evidence-based medicine has highlighted examples of where clinicians' treatment decisions do not accord with 'best practice' defined by high-quality research. This has resulted in the development of implementation interventions which attempt to change professional practice, such as educational programmes and audit with feedback. As these behavioural interventions themselves require a share of the health service's finite resources, it is important to evaluate their cost effectiveness in terms of their effect on health care and hence on health outcomes. This paper considers the economic characteristics of implementation interventions and introduces methods by which their cost-effectiveness can be estimated in advance of significant investment. The paper emphasizes that implementation interventions cannot be good value for money unless the 'good practice' for which increased utilization is considered important is itself cost effective. Furthermore, the likelihood that an implementation strategy will be cost-effective will depend on a number of factors including its cost, its effectiveness in terms of increasing utilization of 'good practice' and the costs and benefits of 'good practice' relative to an appropriate comparator. PMID- 10735266 TI - Twenty years of implementation research. PMID- 10735267 TI - Guidelines implementation and research in the future. PMID- 10735268 TI - A model for susceptibility polymorphisms for complex diseases: apolipoprotein E and Alzheimer disease. AB - Apolipoprotein E (APOE) polymorphisms are associated with variable risk and age of onset distributions for the common form of Alzheimer disease. Clinical genetic applications of APOE genotyping differ from those of family-specific mutations because the APOE genotypes are universally distributed in all populations, with ethnic and racial variations in relative allele frequencies. The association of a common disease affecting millions of people with genetic risk factors provides a new paradigm for understanding disease pathogenesis and an opportunity to focus on relevant genetic mechanisms. Clinical applications of APOE genotype information are based on epidemiological principles, rather than family-specific genetic counselling. At the present time it is not possible to predict when or if any individual will develop AD. Likewise, no APOE genotype provides escape from risk. However, an impending ethical and social dilemma is on the near horizon. When additional susceptibility polymorphisms are found, for example within a recently reported linkage to a chromosome 12 region, disease prediction may be possible for millions of unaffected individuals at a time when preventive therapies are not yet available. This impending capability should be a wake-up call for anticipating social, ethical, and legal problems. It differs from the current model of family specific mutations because, rather than affecting a relatively small population at risk, millions of people will be capable of receiving favorable or unfavorable prognostic information. The association of common polymorphisms with complex diseases finds its first example in APOE and AD. Just as the linkage search for other susceptibility genes can be modelled after AD, the medical, ethical, social, and legal implications of this paradigm can serve as a template for other complex disease loci being actively sought by industry and in academic laboratories. PMID- 10735269 TI - Oxidative phosphorylation defects and Alzheimer's disease. AB - Abnormalities in cellular bioenergetics have been identified in patients with Alzheimer's disease (AD) as well as in patients with other neurodegenerative diseases. The most commonly reported enzyme abnormalities are in the pyruvate dehydrogenase complex, the alpha-ketoglutarate dehydrogenase complex, and oxidative phosphorylation (OXPHOS). Although genetic evidence supporting primary OXPHOS defects as a cause for AD is weak, functionally important reductions in OXPHOS enzyme activities appear to occur in AD and may be related to beta-amyloid accumulation or other neurodegenerative processes. Since reduced neuronal ATP may enhance susceptibility to glutamate toxicity, OXPHOS defects could play an important role in the pathophysiology of AD. PMID- 10735270 TI - Multiple sclerosis: a polygenic disease involving epistatic interactions, germline rearrangements and environmental effects. AB - Several regions of the human genome are associated with susceptibility to multiple sclerosis (MS). We review studies of linkage of MS to germline genes using microsatellites. A modest effect on susceptibility was seen with markers in the vicinity of 6p21 (HLA) and 17q22. The influence of epistatic interactions between these genes is considered. The impact of genetic rearrangements of certain germline genes on susceptibility to MS is described. Analysis of TCR gene rearrangements has established some of the target antigens of the immune response in MS. Environmental influences on MS are described with particular attention given to how microbes might trigger demyelinating disease. PMID- 10735271 TI - A possible role of NAIP gene deletions in sex-related spinal muscular atrophy phenotype variation. AB - Childhood SMAs are common neuromuscular disorders, due to the occurrence of large genomic deletions encompassing the SMN gene and often extending to involve the NAIP gene. Although NAIP deletions are more frequently observed in patients affected by the acute form of the disease, it is not possible to establish an unambiguous correlation between deletion size and clinical severity. We have investigated the effects of gender on the association between NAIP gene deletion and disease severity. NAIP deletions were screened in 197 Italian SMA patients lacking SMN; the results obtained were correlated with disease severity in male and female samples separately. No significant relationship between deletion size and clinical phenotype was observed among male subjects, whereas in females the absence of NAIP was strongly associated with a severe phenotype (p <0.0001). These results provide a possible molecular explanation for the sex-dependent phenotype variation observed in SMA patients. PMID- 10735272 TI - ANOVA, a putative astrocytic RNA-binding protein gene that maps to chromosome 19q13.3. AB - Exon amplification from cosmids mapping to the glioma tumor suppressor gene candidate region on chromosome 19q13.3 yielded an exon with high homology to a portion of the NOVA1 gene, which encodes a neuron-specific RNA-binding protein recognized by the paraneoplastic syndrome antibody anti-Ri. Screening of a human brain cDNA library with this exon identified a 1.9 kb cDNA with extensive homology to NOVA1, including three nearly identical KH domains characteristic of a subtype of RNA-binding proteins. Northern blots demonstrated expression of a 2.5 kb mRNA in brain, but in no other tissues. In situ hybridization on human cerebral cortex showed mRNA expression restricted to astrocytes. We have therefore named the gene ANOVA, for astrocytic NOVA1-like gene. Southern blotting and single strand conformation polymorphism analyses did not show tumor-specific alterations of this gene in gliomas and RT-PCR studies showed expression in glioma cell lines, suggesting that ANOVA is not the chromosome 19q glioma tumor suppressor gene. Given that two cloned paraneoplastic antigens are neuronal RNA binding proteins and that glial proteins may act as paraneoplastic antigens, the ANOVA product may be a target antigen in one of the undefined human paraneoplastic syndromes. PMID- 10735273 TI - The genomic organization of human dystrobrevin. AB - Dystrophin-related and dystrophin-associated proteins (DAPs) are thought to play an important role in the stability and maintenance of the plasma membrane during muscle contraction and relaxation. Studies conducted on the electric organ of Torpedo californica have shown that some of the DAPs are also involved in the formation and maintenance of neuromuscular junctions (NMJs). In addition, dystrophin and several DAPs have been shown to be the primary genetic defect in a number of phenotypically similar muscular dystrophies. We previously reported the identification and characterization of human dystrobrevin, a protein which is unique in being both homologous to dystrophin and a dystrophin-associated protein. Here we describe the genomic organization of the human dystrobrevin gene. It is encoded by 23 exons spanning at least 180 kb of chromosome 18q12. Three different C-termini of dystrobrevin are generated by the mutually exclusive mRNA splicing of three exons. Two alternatively spliced exons (exons 11A and 12) are utilized exclusively in striated muscles. A comparison between the genomic organization of dystrophin and human dystrobrevin shows that the two genes have significant similarities in their genomic structure, implying an ancestral or evolutionary relationship. Based on intronic sequence, a primer set was designed to specifically amplify each exon of dystrobrevin to screen for mutations by SSCP in patients with neuromuscular diseases for which dystrobrevin could be a candidate. PMID- 10735274 TI - Locus heterogeneity in Friedreich ataxia. AB - Friedreich ataxia (FRDA) is the most common form of autosomal recessive ataxia. The disease locus was assigned to chromosome 9 and the disease gene, STM7/X25, has been isolated. To date most data suggest locus homogeneity in FRDA. We now provide strong evidence of a second FRDA locus. Studying two siblings with FRDA from two families we did not detect a mutation in STM7/X25. Haplotype analysis of the STM7/X25 region of chromosome 9 demonstrated that the relevant portion of chromosome 9 differs in the patients. Although the patients studied had typical FRDA, one sibpair had the uncommon symptom of retained tendon reflexes. In order to investigate whether retained tendon reflexes are characteristic of FRDA caused by the second locus, FRDA2, we studied an unrelated FRDA patient with retained tendon reflexes. The observation of typical mutations in STM7/X25 (GAA expansions) in this patient demonstrates that the two genetically different forms of FRDA cannot be distinguished clinically. PMID- 10735275 TI - Mutations in the delta-sarcoglycan gene are a rare cause of autosomal recessive limb-girdle muscular dystrophy (LGMD2). AB - The dystrophin-based membrane cytoskeleton of muscle fibers has emerged as a critical multi-protein complex which seems to impart structural integrity on the muscle fiber plasma membrane. Deficiency of dystrophin causes the most common types of muscular dystrophy, Duchenne and Becker muscular dystrophies. Muscular dystrophy patients showing normal dystrophin protein and gene analysis are generally isolated cases with a presumed autosomal recessive inheritance pattern (limb-girdle muscular dystrophy). Recently, linkage and candidate gene analyses have shown that some cases of limb-girdle muscular dystrophy can be caused by deficiency of other components of the dystrophin membrane cytoskeleton. The most recently identified component, delta-sarcoglycan, has been found to show mutations in a series of Brazilian muscular dystrophy patients. All patients were homozygous for a protein-truncating carboxy-terminal mutation, and showed a deficiency of the four sarcoglycan proteins. To determine if delta-sarcoglycan deficiency occurred in other world populations, to identify the range of mutations and clinical phenotypes, and to test for the biochemical consequences of delta-sarcoglycan gene mutations, we studied Duchenne-like and limb-girdle muscular dystrophy patients who we had previously shown not to exhibit gene mutations of dystrophin, alpha-, beta-, or gamma-sarcoglycan for delta sarcoglycan mutations (n = 54). We identified two American patients with novel nonsense mutations of delta-sarcoglycan (W30X, R165X). One was apparently homozygous, and we show likely consanguinity through homozygosity for 13 microsatellite loci covering a 38 cM region of chromosome 5. The second was heterozygous. Both were girls who showed clinical symptoms consistent with Duchenne muscular dystrophy in males. Our data shows that delta-sarcoglycan deficiency occurs in other world populations, and that most or all patients show a deficiency of the entire sarcoglycan complex, adding support to the hypothesis that these proteins function as a tetrameric unit. PMID- 10735276 TI - SCA2 trinucleotide expansion in German SCA patients. AB - Autosomal dominant spinocerebellar ataxias (SCA) are a group of clinically and genetically heterogeneous neurodegenerative disorders which lead to progressive cerebellar ataxia. A gene responsible for SCA type 2 has been mapped to human chromosome 12 and the disease causing mutation has been identified as an unstable and expanded (CAG)n trinucleotide repeat. We investigated the (CAG)n repeat length of the SCA2 gene in 842 patients with sporadic ataxia and in 96 German families with dominantly inherited SCA which do not harbor the SCA1 or MJD1/SCA3 mutation, respectively. The SCA2 (CAG)n expansion was identified in 71 patients from 54 families. The (CAG)n stretch of the affected allele varied between 36 and 64 trinucleotide units. Significant repeat expansions occurred most commonly during paternal transmission. Analysis of the (CAG)n repeat lengths with the age of onset in 41 patients revealed an inverse correlation. Two hundred and forty one apparently healthy octogenerians carried alleles between 16 and 31 repeats. One 50-year old, healthy individual had 34 repeats; she had transmitted an expanded allele to her child. The small difference between 'normal' and disease alleles makes it necessary to define the extreme values of their ranges. With one exception, the trinucleotide expansion was not observed in 842 ataxia patients without a family history of the disease. The SCA2 mutation causes the disease in nearly 14% of autosomal dominant SCA in Germany. PMID- 10735277 TI - Exon 5 encoded domain is not required for the toxic function of mutant SOD1 but essential for the dismutase activity: identification and characterization of two new SOD1 mutations associated with familial amyotrophic lateral sclerosis. AB - Two new mutations in the gene encoding cytoplasmic Cu,Zn superoxide dismutase (SOD1) have been discovered in patients with familial amyotrophic lateral sclerosis (FALS). These mutations result in the truncation of most of the polypeptide segment encoded by exon 5, one by the formation of a stop codon in codon 126 (L126Z) and the other by inducing alternative splicing in the mRNA (splicing junction mutation). These two mutants of SOD1 result in a FALS phenotype similar to that observed in patients with missense mutations in the SOD1 gene, establishing that exon 5 is not required for the novel toxic functions of mutant SOD1 associated with ALS. These mutant enzymes are present at very low levels in FALS patients, suggesting elevated toxicity compared to mutant enzymes with single site substitutions. This increased toxicity likely arises from the extreme structural and functional changes in the active site channel, beta-barrel fold, and dimer interface observed in the mutant enzymes, including the loss of native dismutase activity. In particular, the truncation of the polypeptide chain dramatically opens the active site channel, resulting in a marked increase in the accessibility and flexibility of the metal ions and side chain ligands of the enzyme active site. These structural changes are proposed to cause a decrease in substrate specificity and an increase in the catalysis of harmful chemical reactions such as peroxidation. PMID- 10735278 TI - Rediscovery of the case described by Alois Alzheimer in 1911: historical, histological and molecular genetic analysis. AB - In 1911, Alois Alzheimer published a detailed report (Zbl. ges. Neurol. Psych. 4: 356-385) on a peculiar case of the disease that had been named after him by Emil Kraepelin in 1910. Alzheimer describes a 56-year-old male patient (Johann F.) who suffered from presenile dementia and who was hospitalized in Kraepelin's clinic for more than 3 years. Post-mortem examination of the patient's brain revealed numerous amyloid plaques but no neurofibrillary tangles in the cerebral cortex, corresponding to a less common form of Alzheimer disease which may be referred to as 'plaque only'. We have identified well-preserved histological sections of this case and performed mutational screening of exon 17 of the amyloid precursor protein gene and genotyping for apolipoprotein E alleles. The patient was shown to be homozygous for apolipoprotein allele epsilon3 and lacked APP mutations at codons 692, 693, 713 and 717. This case is of historical importance as it may have convinced Kraepelin to name the disease after his co-worker, Alois Alzheimer. PMID- 10735279 TI - New fluorimetric method of liquid chromatography for the determination of the neurotoxin domoic acid in seafood and marine phytoplankton. AB - Domoic acid (DA) is a neurotoxic amino acid that is responsible for the human toxic syndrome, amnesic shellfish poisoning (ASP). A new rapid, sensitive liquid chromatographic (LC) method has been developed for the determination of DA in various marine samples. DA in marine biological materials was derivatised with 4 fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and analysed using isocratic reversed phase LC with fluorimetric detection. The calibration, based on standard DA solutions, was linear in the range 0.04-2 microg/ml (r2=0.998) and the detection limit (3:1, signal/noise) was better than 1 ng/ml. Using the certified reference material (MUS-1B), recoveries of DA from shellfish tissue were >95% (n=5). When a strong anion exchange SPE cartridge was used for sample clean-up the detection limit was 6 ng DA/g mussel tissue. Good reproducibility was achieved with RSD values ranging from 3% for 8 microg DA/g (n=5), to 5% for 0.04 microg DA/g (n=5). This new method was successfully applied to the determination of DA in naturally contaminated shellfish and in marine phytoplankton cultures of Pseudonitzschia sp. PMID- 10735280 TI - Anion-exchange high-performance liquid chromatography with conductivity detection for the analysis of phytic acid in food. AB - A sensitive method for the accurate determination of phytic acid in food samples is described. The proposed procedure involves the anion-exchange liquid chromatography with conductivity detection. Initially, two methods of determination of phytic acid were compared: absorptiometry and high-performance ion chromatography (HPIC) with chemically suppressed conductivity detector. Unlike most conventional methods involving precipitation by FeCl3, the simpler and more reliable HPIC assay avoids the numerous assumptions inherent in the iron precipitation and the accuracy is independent of the phytate content. The protocol was also applied to a survey of phytic acid concentration in some cereal, oil and legume seeds. PMID- 10735281 TI - Development of a method for the identification of azaspiracid in shellfish by liquid chromatography-tandem mass spectrometry. AB - Azaspiracid is the main toxin responsible for a number of recent human intoxications in Europe resulting from shellfish consumption. The first micro liquid chromatography-tandem mass spectrometry (micro-LC-MS-MS) method was developed for the determination of this novel shellfish poisoning toxin in mussels. The analyte was extracted from whole mussel meat with acetone and chromatographed on a C18 reversed-phase column (1.0 mm I.D.) by isocratic elution at 30 microl/min with acetonitrile-water (85:15, v/v), containing 0.03% trifluoroacetic acid. The toxin was ionised in an ionspray interface operating in the positive ion mode, where only the intact protonated molecule, [M+H]+, was generated at m/z 842. This served as precursor ion for collision-induced dissociation and three product ions, [M+H-nH2O]- with n=1-3, were identified for the unambiguous toxin confirmation by selected reaction monitoring LC-MS-MS analysis. A detection limit of 20 pg, based on a 3:1 signal-to-noise ratio, was achieved for the analyte. This LC-MS-MS method was successfully applied to determine azaspiracid in toxic cultivated shellfish from two regions of Ireland. PMID- 10735282 TI - Analysis of tetrahydro-beta-carboline-3-carboxylic acids in foods by solid-phase extraction and reversed-phase high-performance liquid chromatography combined with fluorescence detection. AB - The presence and analysis of two tetrahydro-beta-carboline-3-carboxylic acids in foods are studied. Sample preparation with benzenesulfonic acid strong cation exchange columns followed by RP-HPLC-fluorescence allowed a reliable analysis and spectral characterization of 1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (THCA) and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (MTCA). Experimental data showed that upon oxidation tetrahydro-beta-carboline-3 carboxylic acids gave rise to beta-carbolines (norharman and harman) that were also chromatographically separated and their fluorescent profile monitored. This approach was useful to confirm identification of tetrahydro-beta-carboline-3 carboxylic acids in foods. Several foods and beverages contained THCA and MTCA in varying proportions. Their occurrence in foods implies that diet is a source of these compounds in humans. PMID- 10735283 TI - Determination of quinolones in animal tissues and eggs by high-performance liquid chromatography with photodiode-array detection. AB - A rapid, specific reversed-phase HPLC method is described, with solid-phase extraction, for assaying five quinolones (ciprofloxacin, difloxacin, enrofloxacin, norfloxacin and marbofloxacin) with confirmative diode-array detection in samples of bovine kidney, muscle and eggs. The least efficient extraction was marbofloxacin from kidney tissue (64%). The lower detection limit for each quinolone was: enrofloxacin and ciprofloxacin, 1 ng; norfloxacin and difloxacin, 2 ng; marbofloxacin, 4 ng injected. The intra-day relative standard deviations were lower than 7.9% and lower than 8.6% for inter-day assays. These results indicate that the developed method had an acceptable precision. PMID- 10735284 TI - Quantitative determination of sulfonamide residues in foods of animal origin by high-performance liquid chromatography with fluorescence detection. AB - An HPLC method with fluorescence detection is proposed for the quantitative determination of residues of ten of the most used sulfonamides as their derivatives. Sulfonamides were isolated from meat, mix meat and kidney with ethyl acetate (first extraction) and acetone (second extraction) and further purified by partitioning three times with water-methylene chloride. The recovery for mix meat spiked with 1, 5 and 10 microg/kg of sulfonamides averaged 64%, 68% and 75%, respectively. Limits of quantitation were 1 microg/kg for sulfaquinoxaline and 0.5 microg/kg for the remaining sulfonamides. PMID- 10735285 TI - Determination of carbamate residues in fruits and vegetables by matrix solid phase dispersion and liquid chromatography-mass spectrometry. AB - Thirteen carbamates were analysed in orange, grape, onion and tomatoes by matrix solid-phase dispersion (MSPD) followed by liquid chromatography-mass spectrometry (LC-MS). Electrospray (ES) and atmospheric pressure chemical ionisation (APCI) were compared and both gave similar results in terms of sensitivity and structural information because at 20 V fragmentor voltages the fragmentation is minimal. The efficiency of different solid-phases (C18, C8, cyano, amine and phenyl) for the MSPD was compared. Mean recoveries using C8 varied from 64 to 106% with relative standard deviations of 5-15% in the concentration range of 0.01-10 mg kg(-1). Matrix constituents did not interfere significantly with the ionisation process of carbamates. The limits of detection were typically in the 0.001-0.01 mg kg(-1) range, which were between 10 and 100 times lower than the maximum residue levels (MRLs) established by the European Union (EU). The method was applied to residue detection in fruit and vegetable samples taken from Valencian markets, in which carbamates were detected at low concentrations. PMID- 10735286 TI - Determination of abamectin in citrus fruits by liquid chromatography-electrospray ionization mass spectrometry. AB - Liquid chromatography coupled to electrospray mass spectrometry (LC-ES-MS) with positive ion detection was used to determine abamectin in oranges. MS conditions were optimized to achieve maximum sensitivity. The main ion for abamectin was [M+Na]+ at a fragmentor voltage of 180 V. Abundant structural information can be obtained at different fragmentor voltages. The detection limit for the standard solution was 12 pg injected, and good linearity and reproducibility were observed. Abamectin residues were extracted using matrix solid-phase dispersion. Orange samples were homogenized with C18 bonded silica placed onto a glass column and eluted with dichloromethane. Recoveries of the abamectin from oranges fortified with approximately 0.01-10 mg/kg ranged from 94 to 99% with an overall average recovery of 96%. The quantification limit is 0.0025 mg/kg, which means detection limit for this analyte could be set at a few hundred picograms per gram of fruit. The presence in the electrosprayed solution of numerous citrus constituents did not interfere significantly with the ionization process of abamectin. The assay procedure provides a simple, rapid, and sensitive method for monitoring residues in oranges. The method was applied to field treatment orange samples. PMID- 10735287 TI - Determination of rotenone residues in raw honey by solid-phase extraction and high-performance liquid chromatography. AB - A method for determining residues of the insecticide rotenone in raw-honey by high-performance liquid chromatography (HPLC) is described. To extract the residues, organic solvents such as ethyl acetate, n-hexane/dichloromethane and solid-phase extraction with octadecylsilane cartridges or Florisil packed columns were tested. Determination was carried out by reversed-phase HPLC using acetonitrile-buffer phosphate (pH 7) (60:40, v/v) as mobile phase and detection at 210 nm. Although the data showed that the two extraction methods were able to isolate the pesticide residues, the extraction on octadecylsilane cartridges was preferred due to its simplicity and higher recovery. Recoveries depended strongly on the fortification level for the two extraction procedures. Practical determination limits of 0.015 mg/kg were obtained. In the analysis of honeys, from beehives treated with rotenone at therapeutical doses for 1 month, residual amounts below 0.2 mg/kg were found. PMID- 10735288 TI - Optimization of chromatographic parameters for the determination of biogenic amines in wines by reversed-phase high-performance liquid chromatography. AB - A method suitable for the determination of eight biogenic amines (histamine, tyramine, phenylethylamine, tryptamine, cadaverine, putrescine, spermidine and spermine) in wines has been developed. The method involves derivatization of the amines by treatment with dabsyl chloride, after which the derivates were analysed by reversed-phase liquid chromatography with gradient elution and spectrophotometric detection at 446 nm. Different variables affecting separation were optimized. Validation of the method included calibration experiments, the addition of standards amines for the determination of recovery and repeatability tests. Good linearity of the responses was obtained up to 500 microg l(-1), except for putrescine (up to 2100 microg l(-1)). The detection limits ranged between 10 and 60 microg l(-1) for standard solutions. The method was successfully applied to the analysis of five Spanish wines. PMID- 10735289 TI - Determination of phenols in wines by liquid chromatography with photodiode array and fluorescence detection. AB - A reversed-phase LC method, optimised for the separation of trans- and cis resveratrol, catechin, epicatechin, quercetin and rutin, is reported. Analyses were performed on a reversed-phase column by gradient elution. Detection was carried out by photodiode array, although the use of a fluorimetric detector considerably lowered the detection limits for catechin, epicatechin and both resveratrol isomers. Identification by the two different detection systems was based on retention characteristics, UV spectra and peak purity index were compared with commercial standards. The procedures were applied to the determination of the phenolic compounds in different types of wines and musts. PMID- 10735290 TI - Method development for the determination of anthocyanins in red wines by high performance liquid chromatography and classification of German red wines by means of multivariate statistical methods. AB - A simple and fast HPLC method without sample pretreatment is described for the separation of anthocyanins in red wines using a new pH-stable stationary phase. The linearity between peak area and concentration and ruggedness of the method were checked. Investigations were made about the safekeeping of red wine samples concerning anthocyanins. Classification of 52 different wine samples was performed by multivariate statistical methods. PMID- 10735291 TI - High-performance liquid chromatographic separation of the enantiomers of unusual alpha-amino acid analogues. AB - The direct and indirect stereochemical resolution of the enantiomers of ring- and alpha-methyl-substituted phenylalanines and phenylalanine amides was attempted by high-performance liquid chromatographic methods. The direct separation was carried out on two chiral stationary phases, the crown-ether-based Crownpak CR(+), and the teicoplanin-based Chirobiotic T, while the indirect resolution was performed by applying pre-column derivatization with 2,3,4,6-tetra-O-acetyl-beta D-glucopyranosyl isothiocyanate (GITC) and Nalpha-(2,4-dinitro-5-fluorophenyl)-L alanine amide (Marfey's reagent, FDAA). The Chirobiotic T column was efficient in the separation of ring- and alpha-methyl-substituted phenylalanine analogues, but was ineffective for the amides of these analogues. The Crownpak CR(+) column separated the ring-substituted phenylalanines and amides, whereas the alpha methylated analogues were coeluted. Of the two indirect methods, GITC derivatization seemed more effective than FDAA derivatization. PMID- 10735292 TI - Application of (1S,2S)- and (1R,2R)-1,3-diacetoxy-1-(4-nitrophenyl)-2 propylisothiocyanate to the indirect enantioseparation of racemic proteinogenic amino acids. AB - The application of (1S,2S)- or (1R,2R)-1,3-diacetoxy-1-(4-nitrophenyl)-2 propylisothiocyanate as a new chiral derivatizing agent for the resolution of compounds possessing an amino group is described. The reagent is easily accessible in both enantiomeric forms after a simple two-step synthesis. Its applicability was demonstrated on the example of the resolution of a series of alpha-amino acids. The diastereomeric thiourea derivatives produced were separated by reversed-phase high-performance liquid chromatography. The effects of pH, temperature and reagent excess on the derivatization kinetics were investigated, as were the effects of pH and organic modifier on the separation. PMID- 10735293 TI - Chiral separation of four 1,3-dioxolane derivatives by supercritical fluid chromatography on an amylose-based column. AB - The chiral separation of four 1,3-dioxolane derivatives by supercritical fluid chromatography on an amylose-based column is described. The effects of mobile phase composition, temperature and pressure have been investigated. The nature of the modifier is the parameter which has the highest impact on the chiral resolution and it is more important than the polarity of the mobile phase. The organic modifier used for the best enantiomeric separation was different for each compound, because it depends strongly on the molecular structure of the compound. PMID- 10735294 TI - Separation of isomeric naphthalenesulphonic acids by micro high-performance liquid chromatography with mobile phases containing cyclodextrin. AB - Aromatic sulphonic acids are important dye intermediates and the determination of the individual isomers after their preparation by sulphonation of the parent aromatic hydrocarbon is important for the monitoring of the dye production process. For this purpose, either reversed-phase chromatography with mobile phases containing strong electrolytes as additives or capillary zone electrophoresis with working electrolytes containing cyclodextrins can be used to separate and determine not only individual sulphonation products with various numbers of sulphonic groups, but also various isomeric di- and trisulphonic acids. However, the separation of some isomers using either of the two techniques is not fully satisfactory. In the present work, HPLC with mobile phases containing cyclodextrins was employed to improve previously achieved separations of aromatic sulphonic acids. Because of the high cost of cyclodextrin, microcolumn HPLC with diode-array detection on the columns prepared in laboratory by supercritical fluid packing technique was employed for this purpose. Capillary columns packed with various octadecyl silica gel materials were compared and their stability and efficiency were found suitable for the separation of the compounds tested. The selectivity of separation of some isomers improved significantly with respect to the previous methods. Procedures were designed for separation and analytical control of technological processes producing dye intermediates. PMID- 10735295 TI - Application of the restricted-access precolumn packing material alkyl-diol silica in a column-switching system for the determination of ketoprofen enantiomers in horse plasma. AB - The group of LiChrospher ADS (alkyl-diol silica) sorbents that make part of a unique family of restricted-access materials, have been developed as special packings for precolumns used in the LC-integrated sample processing of biofluids. The advantage of these sorbents lies in the direct injection of untreated biological fluids, that is without sample clean-up, the elimination of the protein matrix with a quantitative recovery together with an on-column enrichment. The present method is based on previous work applying UV detection at 260 nm for ketoprofen determinations. Plasma samples introduced to the ADS precolumn using a 0.1 M phosphate buffer, pH 7.0. After washing with the buffer the ADS column was backflushed with the mobile phase 0.01 M phosphate buffer-6% (v/v) 2-propanol-5 mM octanoic acid at a pH of 5.5, thus transporting the analytes to the chiral-HSA (human serum albumin) (100x4.0 mm) column where the separation of the ketoprofen enantiomers was achieved with a resolution factor of 1.4. The developed column-switching method was fully applicable to plasma injections. PMID- 10735296 TI - Enantioselective analysis of methadone in saliva by liquid chromatography-mass spectrometry. AB - Saliva was tested and evaluated as a biological matrix for methadone (Mtd) monitoring. Conventional method using a narrow bore C18 column, and an enantioselective method using a narrow bore alpha1-acid glycoprotein column, were developed using liquid chromatography coupled with a mass spectromeric (MS) detector. After optimisation of MS conditions by flow injection analysis, selected ion monitoring detection was used to enhance sensitivity. The total Mtd concentration and the enantiomeric ratio in saliva were validated using an experimental design. The methods were applied to samples provided by heroin addicts undergoing a Mtd treatment. Results on total Mtd determination showed a very poor correlation between saliva and serum, whereas the enantiomeric ratios of Mtd gave a very good one. PMID- 10735297 TI - Enantiomeric separation of clenbuterol by transient isotachophoresis-capillary zone electrophoresis-UV detection new optimization technique for transient isotachophoresis. AB - A method for the in-line preconcentration and enantioseparation of clenbuterol by transient isotachophoresis-capillary zone electrophoresis-UV absorbance detection (transient ITP-CZE-UV) has been developed. It implies the use of dimethyl-beta cyclodextrin as chiral selector and the application of a hydrodynamic counterflow during the ITP step. ITP is used to focus the sample constituents prior to CE whereas a counterpressure counterbalances the electrophoretic migration of the compounds. The sample is then focused and kept stationary in the proximity of the capillary inlet before CZE separation, leading to an extended-volume ITP-CZE system. A new strategy for the fast optimization of the counterpressure has been developed which implies the measurement of the hydrodynamic and electrophoretic velocities of the analyte during ITP. The in-line preconcentration and enantioseparation of clenbuterol selected as model compound was optimized using this method. Salbutamol was chosen as internal reference in order to check the reproducibility of the method. A 173-nl volume of aqueous ample solution was injected which implies an improvement of the injection volume of about 16 and a resolution of 4.8 was obtained for the clenbuterol enantiomers. A concentration detection limit of 10(-6) mol/l was readily achieved for clenbuterol and salbutamol using only 3 min ITP preconcentration in in-line counterflow transient ITP-CZE-UV. Thanks to its fast optimization, the method is applicable to any enantioseparation by means of only five very short preliminary measurements. PMID- 10735298 TI - Stereospecific analysis of lorazepam in plasma by chiral column chromatography with a circular dichroism-based detector. AB - The chiral separation of lorazepam was achieved on a chiral column with UV and circular dichroism (CD) detection. The good resolution of lorazepam enantiomers was obtained on the column of beta-cyclodextrin derivative immobilized silica gel under reversed-phase conditions. The enantiomeric separation and identification of lorazepam were succeeded by CD detector. The method described allows the quantitation of the stereoisomers of lorazepam in human plasma following the administration of a therapeutic dose of the racemic drug. Chiroptical detection is useful for the pharmacokinetic study of chiral drugs. PMID- 10735299 TI - Study of the cis-trans isomerization of enalapril by reversed-phase liquid chromatography. AB - Enalapril is a dipeptidic angiotensin converting enzyme inhibitor. It exists as a mixture of two conformers in solution with respect to the peptide bond involving the proline amino group. The RPLC of such products may yield peak splitting or multiple peaks as a result of the slow kinetics of the conformation change. In this study, the influence of the flow-rate, pH, temperature, organic modifier and counter ion on the peak shape and the separation of the cis and trans conformers are examined qualitatively by HPLC. It appears that decrease of relaxation time for isomerization with concomitant improvement in peak shape is favoured by a decrease in pH and flow-rate, increase of temperature, choice of organic solvent (nature, amount) and cationic counter ion concentration in the mobile phase. The elution order of the isomers was dependent on the nature of the organic modifier whereas the separation selectivity was improved by an increase of pH or the addition of a negatively charged counter ion. In addition, an NMR investigation on enalapril is described. PMID- 10735300 TI - Enantiospecific determination of PNU-83894 and its major metabolite, PNU-83892, in plasma, and its application to the characterization of the enantioselective pharmacokinetics of PNU-83894 in the dog. AB - A chiral method for the simultaneous analysis of the (+)- and (-)-enantiomers of PNU-83894 and its metabolite, PNU-83892, in plasma was developed to characterize the enantioselective pharmacokinetics of PNU-83894, a potential anticonvulsant candidate. The method involves solid-phase extraction (phenyl column) of the enantiomers from plasma followed by direct enantioselective separation on a beta cyclodextrin HPLC chiral column and UV detection at 230 nm. The linear range for this method was found to be 12.5 ng/ml to 5.00 microg/ml and the intra- and inter assay precision and accuracy for each enantiomer were <11% in all cases. The validity of this assay was also demonstrated by its application to the pharmacokinetic evaluation of PNU-83894 in the dog. PMID- 10735301 TI - Analysis of cis-trans isomers and enantiomers of sertraline by cyclodextrin modified micellar electrokinetic chromatography. AB - In this work development, optimization and validation of a cyclodextrin-modified micellar electrokinetic chromatography (CD-modified MEKC) method is proposed to resolve separation of the sertraline hydrochloride and synthesis-related substances. Sertraline hydrochloride, the cis-(1S,4S) enantiomer form, is used as an antidepressant therapeutic agent. A buffer concentration composed of 20 mM sodium borate, pH 9.0 with 50 mM sodium cholate, 15 mM sulfated beta-cyclodextrin and 5 mM hydroxypropyl-beta-cyclodextrin was found to be the most suitable background electrolyte. Quantitation of the impurities at levels of 0.1% in different samples of the bulk drug was determined. A comparison of the results with those obtained by HPLC methodology was also accomplished. The method proved appropriate for testing the purity of sertraline hydrochloride in bulk drug. PMID- 10735302 TI - Separation of some platinum group metal chelates with 8-hydroxyquinoline by various high-performance liquid chromatographic methods. AB - Different HPLC methodologies are employed to evaluate the separation and determination of some platinum metals (Pt, Pd, Ir and Rh) after the formation of 8-hydroxyquinolate chelates. With the aim of reducing the number of steps in treating the samples, the method developed did not include the elimination of excess chelating reagent before the analysis of metal chelates. Reversed-phase (RP), non-aqueous reversed-phase (NARP) and normal-phase (NP) HPLC are compared. The RP-HPLC method only permits the quantitative separation of Rh and Pd from the excess reagent. A silica column can be used to separate Ir and Rh by NP-HPLC. The NARP-HPLC method allows for the effective separation of the four elements tested, but the high detection limit (90 ng) for platinum and the peak width do not favour its application for quantitative measurement. Platinum group metals can be quantitatively separated and determined by NP-HPLC using a cyano column in less than 15 min. The broad linear range of all the elements (between 1 and 500 ng) is superior to that which has been previously reported and the detection limits (1.0 ng for Pt, 0.3 ng for Pd, 1.0 ng for Ir and 0.3 ng for Rh) are slightly lower. PMID- 10735303 TI - Determination of sulphonate dyes in water by ion-interaction high-performance liquid chromatography. AB - An ion-interaction high-performance liquid chromatography method for quick separation and determination of the sulphonated dyeAcid Yellow 1, and the sulphonated azo dyes Acid Orange 7, Acid Orange 12, Acid Orange 52, Acid Red 2, Acid Red 26, Acid Red 27 and Acid Red 88 has been developed. An RP-ODS stationary phase is used, and the mobile phase contains an acetonitrile-phosphate buffer (27:73, v/v) mixture at pH 6.7, containing 2.4 mM butylamine as ion-interaction reagent. Good separations were obtained using isocratic elution and spectrophotometric detection at 460 nm. The detection limits for the eight dyes ranged from 7 to 28 microg/l for an injection volume of 100 microl. Spiked tap water samples (100 ml), containing different concentration levels (0.3-1.2 microg/l) of the dyes were analyzed after acidification (pH 3) and preconcentration in disposable solid-phase extraction C18 cartridges. PMID- 10735304 TI - Determination of phenolic antioxidants in aviation jet fuel. AB - The world-wide aviation jet fuel used for civil and military aircraft is of a kerosene type. To avoid peroxide production after the refinery process a specific antioxidant additive should be added on fuel. The antioxidants generally used are based on hindered phenols in a range of concentration 10-20 microg/ml. In the present work a specific method to measure the concentration of phenolic antioxidants is shown. The method is based on a liquid chromatographic technique with electrochemical detection. The technique, because of its selectivity, does not require sample pre-treatments. The analysis of a 5-10 ml fuel sample can be performed in less than 10 min with a sensitivity of 0.1 microg/ml and a RSD=2.5%. A comparison with another highly selective gas chromatographic technique with mass spectrometric detection with selected ion monitoring (GC-MS-SIM) is reported. The sensitivity of GC-MS-SIM method was 2 microg/ml with a RSD=3.1%. PMID- 10735305 TI - Separation of polyesters by gradient reversed-phase high-performance liquid chromatography on a 1.5 microm non-porous column. AB - Efficient separation of polyesters composed of a large number of individual oligomers was achieved on a 1.5 microm "non-porous" octadecylsilyl (ODS) silica support by gradient high-performance reversed-phase liquid chromatography (gRP HPLC) with a mobile phase of acetonitrile, aqueous trifluoroacetic acid (0.2%) and tetrahydrofuran at ambient temperature and signal monitoring by UV absorption at 280 nm. Substantial signal splitting of oligomers in the low molecular weight (Mr) region is indicative that separation not only occurs with respect to molecular weight distribution (MWD) but also to chemical composition distribution (CCD) and functionality type distribution (FTD). Although separation according to CCD and FTD decreases with increasing number of oligomers, co-elution of species with identical number of repeat units but differing in either structure of repeat units or end-groups can be assumed from the relatively broad signals succeeding the aforementioned peaks showing at least partial resolution. Despite the observation that high Mr oligomers elute as sharp signals, the preceding observations suggest that each of these peaks presumably composes of more than one individual component. The polyester oligomers are eluted in the range of increasing Mr and therefore, either separation according to MWD or CCD/FTD was at least achieved for the low Mr sample constituents. Some principal mechanistic aspects of separation are discussed and adsorption seems to play the dominant role. The detection limit, defined as that sample amount yielding an unequivocal recognition on the base of its characteristic chromatographic fingerprint pattern was about 5,000 ppm for the pair Alftalat 3258 - Alftalat 3352 and 10,000 ppm for the pair Crylcoat 430 - Crylcoat 801. PMID- 10735306 TI - Analysis of higher polyamide-6 oligomers on a silica-based reversed-phase column with a gradient of formic acid as compared with hexafluoroisopropanol. AB - The analysis of polyamide-6 oligomers and polymer is usually performed with expensive fluorinated alcohols like 2,2,2-trifluoroethanol (TFE) or 1,1,1,3,3,3 hexafluoroisopropanol (HFIP). Formic acid is well known as a mobile phase additive to adjust pH in reversed-phase high-performance liquid chromatography. However, formic acid is seldom used as a modifier to perform gradient elution chromatography on octadecyl-modified silica-based columns. Here we demonstrate the determination of cyclic and linear polyamide-6 oligomers using formic acid as a modifier on an octadecyl-modified silica-based column. This column was shown to be stable for more than 5000 column volumes, even when a mobile phase of 65-95% formic acid in water at a flow of 1 ml/min is applied. With formic acid under the conditions used (65-95% formic acid in water) the oligomers are retained on the column, while the polymer does not precipitate. In comparison, during adsorption and separation with a HFIP gradient, precipitation of the polymer occurs. The implications of the different separation mechanisms, i.e., adsorption vs. precipitation chromatography are discussed. Loadability is shown to be much better with the formic acid system. However, with formic acid as a modifier UV detection below 250 nm is not feasible. The less sensitive evaporative light scattering detector is used to detect the polyamide oligomers in the formic acid phase. In addition it is shown that capillary zone electrophoresis (CZE) with UV absorbance detection using HFIP is an attractive combination as HFIP is UV transparent and CZE allows low modifier consumption. PMID- 10735307 TI - Analysis of organosilicone copolymers by gradient polymer elution chromatography with evaporative light scattering detection. AB - Organosilicone copolymers have found numerous applications in the cosmetics, detergent and coating industries. Coupling a polar polymer (like polyglycols) to a non-polar silicone gives anchorage and emulsification capabilities to the polymer. When coupling a silicone polymer to a polyglycol, the copolymer formed differs from the starting polymers by a single bond which is often difficult to evidence using spectroscopic techniques such as NMR or infrared, especially when the polymers have a high molecular mass. Gradient polymer elution chromatography (GPEC) coupled to an evaporative light scattering detector was developed for the characterization of copolymers based on their chemical composition distribution. Different block and graft polyglycol-silicone copolymers were successfully characterized by GPEC and residual homopolymers have been easily quantified. PMID- 10735308 TI - Hydrophobic-phase-modified fused-silica columns for capillary electrophoresis. AB - Coated capillaries modified with a hydrophobic layer were developed. Linear hydrocarbons and ethylbenzene modified surfaces greatly improved the electrophoretic performance of the capillaries. The column efficiency for organic compounds reached as high as 327 000 theoretical plate numbers per meter on a 50 microm I.D. linear hydrocarbon (C6) surface treated fused-silica capillary column. This value did not change during 50 repeated analyses and the columns showed strong stability against 0.1 M NaOH and 0.1 M HCl. The relative standard deviation of the run-to-run, day-to-day, and capillary-to-capillary coating with hydrophobic layer showed values of < or =2.5%, and good reproducibility. The separations of four aromatic amines and six pharmacological amines at pH 2.5 is reported. PMID- 10735309 TI - Ultra-thin-layer agarose gel electrophoresis II. Separation of DNA fragments on composite agarose-linear polymer matrices. AB - The effect of hydrophilic linear polymer additives (non-cross-linked polyacrylamide, hydroxyethyl cellulose and polyethylene oxide) on the migration behavior of double stranded DNA molecules, ranging from 200-1000 base pairs, were studied in ultra-thin-layer agarose gel electrophoresis. The detection sensitivity was found to be less than 0.1 ng/band using To-Pro-3 fluorophore labeling and fiber optic bundle-based scanning detection system with a 640 nm red diode laser. Among the various polymers investigated, addition of linear polyacrylamide resulted in the best separation performance (steepest Ferguson plots), while composite gels with hydroxyethylcellulose and polyethylene oxide still exhibited adequate resolving power. Using the composite matrices of 1% agarose-linear polyacrylamide (0.5-3%), 1% agarose-hydroxyethylcellulose (0.2-1%) and 1% agarose-polyethylene oxide (0.2-1%), the mechanism of the separation was found to be in the Ogston sieving regime. Activation energy curves were also plotted based on the slopes of the Arrhenius plots of the various composite matrices, and exhibited decreasing characteristics for the agarose-linear polyacrylamide composite matrix and increasing characteristics for the agarose hydroxyethylcellulose and agarose-polyethylene oxide composite matrices. PMID- 10735310 TI - Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments. AB - The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries. PMID- 10735311 TI - Determination of oligonucleotide ISIS 2922 in nanoparticulate delivery systems by capillary zone electrophoresis. AB - ISIS 2922 is an antisense oligonucleotide with antiviral activity against cytomegalovirus. However, its rapid degradation in biological fluids and its low capacity for diffusion across cell membranes limit its therapeutical use. One possibility to overcome these drawbacks consists of using nanoparticles as drug carriers. The aim of this study was to develop an analytical method for determining the amount of ISIS 2922 loaded into albumin nanoparticles. For this purpose, capillary zone electrophoresis (CZE) was performed on a fused-silica capillary filled with borate buffer (12.5 mM, pH 9.5). Paracetamol was used as an internal standard and a diode-array detection system was set at 270 nm. Under these conditions, the limit of quantitation of ISIS 2922 was 1.27 microg and the precision and accuracy of the method did not exceed 7%. Moreover, the use of paracetamol as internal standard and the quantification by means of a 'corrected area' procedure enabled us to reduce the peak variability and accurately determine the amount of oligonucleotide loaded in the albumin nanoparticles. In summary, this assay is a selective and sensitive CZE method for the accurate quantitation of ISIS 2922 oligonucleotide in albumin nanoparticles. PMID- 10735312 TI - Capillary electrophoresis of DNA damage after irradiation: apoptosis and necrosis. AB - Apoptosis is a type of cellular death but also directly regulates tumorigenesis through different gene expression. This phenomenon is often used as end-point in studies of radio- and chemosensitivity of cancer cells. Restriction DNA fragments have been separated quickly, efficiently and successfully by capillary gel electrophoresis (CGE). In this study CGE has been applied to distinguish between the discrete pattern of degraded DNA produced by apoptosis and randomized DNA breaks produced by ionizing radiation. The influence of different variables has been discussed and an example of fast separation by CGE of the apoptotic fragments produced by UV light treatment is shown. PMID- 10735313 TI - Determination of amino acids in overlapped capillary electrophoresis peaks by means of partial least-squares regression. AB - Amino acid derivatives of 1,2-naphthoquinone-4-sulfonate (NQS) can be separated by capillary electrophoresis at 30 kV in a fused-silica capillary by using a 40 mM sodium tetraborate-isopropanol (3:1, v/v) solution as background electrolyte. This procedure was suitable for the most common amino acids. However, the peaks of three amino acids (phenylalanine, isoleucine and tyrosine) were only partially resolved and peaks of histidine and leucine derivatives overlapped completely. Partial least-squares regression (PLS) may overcome the lack of selectivity for these amino acids. Spectroelectropherograms of the corresponding amino acid derivative peaks were monitored with a diode-array spectrophotometer in the range 225 to 540 nm. Both spectra and electropherograms can be used as multivariate data for further analysis. In general, the best predictions were obtained using the time domain. PMID- 10735314 TI - Direct measurement of homovanillic, vanillylmandelic and 5-hydroxyindoleacetic acids in urine by capillary electrophoresis. AB - Separation conditions in CE, with a neutral coated capillary and reversed polarity, have been optimised to make direct measurement of vanillylmandelic acid, homovanillic acid and 5-hydroxyindoleacetic acid possible in urine samples without pre-treatment. The method developed has been validated, presenting adequate parameters for linearity, accuracy and precision. Detection limits range from 0.03 to 2.5 microM. Finally the method has been applied to urine samples taken from patients, both adults and children, in hospital. Some of them were also measured by immunoassay and HPLC-electrochemical detection and results have been compared. PMID- 10735315 TI - Thiamine analysis in biological media by capillary zone electrophoresis with a high-sensitivity cell. AB - A capillary zone electrophoresis method with high-sensitivity cell (Z-cell) has been developed for the determination of thiamine in biological media (plasma, urine, saliva). The urine samples were diluted (1:1, v/v) in water and were directly injected into the apparatus. For the quantitative assay of thiamine in plasma it is necessary to precipitate the protein component. Good results were achieved by treating the sample with acetonitrile (1:3, v/v). Using a capillary with high sensitivity cell led to an approximately nine-fold improvement of the detection limit compared to standard capillaries and four-fold improvement compared to capillary with bubble cell. The samples in the biological media were analysed using a calibration curve for thiamine concentrations between 0.1 and 200 microg ml(-1). The detection limit, the effective mobility and the relative standard deviation of the migration times and of the peak areas were determined. PMID- 10735316 TI - Migration behavior and separation of benzenediamines, aminophenols and benzenediols by capillary zone electrophoresis. AB - The migration behavior and separation of five benzendiamines, five aminophenols and three benzenediols were investigated in capillary zone electrophoresis. The results indicate that benzendiamines and aminophenols are optimally separated with a phosphate buffer at pH 5, whereas benzenediol isomers are best separated at pH about 12. The addition of surfactant monomers of tetradecyltrimethylammonium bromide to a phosphate buffer at pH 5 under the conditions of reversed electroosmotic flow is effective for separating these dye intermediates, except for the separation of 1,2-benzenediol from 1,3-benzenediol. The addition of sodium tetraborate as an electrolyte modifier is effective in the separation of 1,2-benzenediol from 1,3-benzenediol, but the latter comigrates with the 1,4-benzenediol isomer at pH 5.0. The electrophoretic mobility of ionized analytes can be described with Offord's equation, and the migration order depends on their ratios of charge to mass. In addition, the pKa values of these analytes in 50 mM phosphate buffer are reported. PMID- 10735317 TI - Prediction of electrophoretic mobilities in non-aqueous capillary electrophoresis. Optimal separation of quinolones in acetonitrile-water media. AB - A model of electrophoretic behaviour is used to predict the optimum conditions for the separation of a series of quinolones zwitterionic substances in mixtures of acetonitrile-water up to 30% (w/w) of acetonitrile. The effect of pH, pKa, the electrophoretic mobility of protonated and anionic species, and activity coefficients on the electrophoretic behaviour of quinolones is considered. The model proposed allows the resolution between substances in acetonitrile-water mixtures to be predicted from a few experimental data and thus permits one to obtain the best experimental conditions for separation methodologies. PMID- 10735318 TI - Influence of pH and pKa values on electrophoretic behaviour of quinolones in aqueous and hydro-organic media. AB - Through correct pH measurements, pKa and activity coefficient values, a model describing their effect on electrophoretic behaviour of substances is established. The suggested model uses the pH values in the acetonitrile-water mixtures used and takes into account the effect of activity coefficients. The model permits the calculation of acidity constants of analytes in hydro-organic media and also the prediction of the effect of pH on the electrophoretic mobility. The model is tested by determining the dissociation constants of a series of nine quinolones in acetonitrile-water mixtures of 0, 5.5, 10 and 30% (w/w) acetonitrile. PMID- 10735319 TI - Analysis of size-classified ice crystals by capillary electrophoresis. AB - In order to analyse the main inorganic cations (NH4+, K+, Na+, Ca2+, Mg2+) and anions (Cl-, NO3-, SO4(2-)) as well as carboxylic and dicarboxylic acids in ice crystals by capillary electrophoresis, electrolyte systems were developed and optimised with respect to limits of detection, resolution, reproducibility and analysis time. We applied indirect UV detection, which enables the simultaneous detection of multiple components. Salicylic acid and 4-methylaminophenolsulfate were used as UV-active co-ions for analysis of anions and cations, respectively. The special features of these systems were low limits of detection in the range 0.3-0.9 micromol L(-1), i.e. absolute limits of detection were in the fmol range, and short analyses times. Separations of cations as well as anions including carboxylic and dicarboxylic acids were completed within 4 min allowing a high sample throughput. Furthermore, the applicability of the newly developed electrolyte systems was demonstrated by comparative analyses with ion chromatography and by first field experimental studies. PMID- 10735320 TI - Capillary zone electrophoretic separation and determination of imidazolic antifungal drugs. AB - Capillary zone electrophoresis (CZE) was adapted to the simultaneous determination of a mixture of three imidazolic antifungal drugs. Separation was achieved by using a fused-silica capillary column with an acetic acid-Tris buffer at pH 5.18 and UV detection at 196 nm. Several electrophoretic parameters were investigated: pH and buffer concentration, applied voltage, temperature and injection conditions. The optimized CZE method was applied to the individual determination of ketoconazole, clotrimazole and econazole in pharmaceutical forms, after a previous single extraction step in methanol, with recoveries of 98.00, 99.96 and 99.58% respectively. The antifungal drugs can be determined at a concentration level lower than 1.0 x 10(-7) M. PMID- 10735322 TI - Macromolecular surfactant as a pseudo-stationary phase in micellar electrokinetic capillary chromatography. AB - We examined polymers of sodium 11-acrylamidoundecanoate [poly(Na 11-AAU)] with a very high molecular mass (>10(6)) for their potential use as a pseudo-stationary phase in micellar electrokinetic capillary chromatography (MEKC). Size-exclusion chromatography and capillary electrophoresis studies reveal that the polymers are highly charged, and have a densely packed chain structure. For aromatic compounds, the polymeric surfactant showed significantly different selectivity than sodium dodecyl sulfate (SDS). It was suggested that one molecule of poly(Na 11-AAU) forms one micelle. The structural stability of this pseudo-stationary phase permitted its use with relatively high percentages of organic modifiers in the buffer medium, allowing the separation of highly hydrophobic compounds which are difficult to analyze by conventional MEKC with SDS. PMID- 10735321 TI - Micellar electrokinetic chromatography with bis(2-ethylhexyl)sodium sulfosuccinate vesicles determination of synthetic food antioxidants. AB - Capillary electrokinetic chromatography is suitable for the separation of mixtures of uncharged and charged solutes. In the present work the behavior of six synthetic food antioxidants--2[3]-tert.-butyl-4-hydroxyanisole, 2,6-di-tert. butyl-p-cresol, tercbutylhydroquinone, 3,4,5-trihydroxybenzoic acid propyl ester, 3,4,5-trihydroxybenzoic acid octyl ester and 3,4,5-trihydroxybenzoic acid dodecyl ester--was studied in a capillary electrophoresis system using capillary electrokinetic chromatography with vesicles of the surfactant bis(2 ethylhexyl)sodium sulfosuccinate (AOT). Several studies aimed at calculating the critical aggregation concentration of the surfactant were conducted to check that under the conditions used the AOT was in a state of aggregation. Having checked the association shown by the surfactant, we then explored the greater or lesser capacity of the antioxidants to interact with this compound. We followed the evolution of the molecular absorption spectra of each of the antioxidants in the presence of the surfactant at different concentrations and the retention factors were calculated at different pH values. Additionally, in order to determine which species--anionic or neutral--was present at the pH of the buffer used (boric/borate), the pKa values in acetonitrile-water (20:80) were obtained. Resolution and quantification of the antioxidants demand optimization of the variables involved in the system, such as the percentage of acetonitrile, the concentration of AOT and boric/borate buffer, pH, voltage, etc. When this part of the study had been completed, calibrations were obtained for each of the antioxidants, obtaining good linear correlation coefficients in all cases. Finally, we propose a method that allows the resolution of the six most employed antioxidants in a capillary electrophoretic system in 15 min, using electrokinetic chromatography with AOT as the pseudostationary phase. PMID- 10735323 TI - Optimization of the separation of phenolic compounds by micellar electrokinetic capillary chromatography. AB - A group of phenolic compounds including phenolic aldehydes, acids and flavonoids are separated by micellar electrokinetic chromatography (MECC). The influence of buffer (concentration and pH), concentration of sodium dodecylsulphate (SDS) and applied voltage were studied. To increase the selectivity of the separation and the resolution of the solutes organic solvents are added to the separation buffer, the best results were obtained when methanol was used at lower percentages. An optimized buffer (150 mM boric acid (pH 8.5)-50 mM SDS-5% methanol) provides the optimum separation with regard to resolution and migration time. This method was applied to the determination of these compounds in wine samples with good results. PMID- 10735324 TI - Characterization of cephalosporin transfer between aqueous and colloidal phases by micellar electrokinetic chromatography. AB - A new electrokinetic chromatographic method was applied to the determination of the partition coefficient between water and micelle for a group of cephalosporins (cefmetazol, cephradin, cefaclor, ceftazidim, cefodizim, cephapirin, cephalothin and ceftriaxon) using sodium dodecyl sulphate as an anionic surfactant in microemulsion and in micellar systems. In the new method, the running buffer contains both the micelles and the drug, and the injected solution contains the same concentration of micelles as the running buffer but not the drug. The mobility of the drug can be measured from a negative peak recorded the chromatogram. The required parameters for the determination of the capacity factor (mu(aq) and /mu(me) are the electrophoretic mobilities of the solutes in the aqueous and the micelle phases, mu(eff) is the effective mobility in the micellar system or in the microemulsion) were measured by the new micellar and microemulsion electrokinetic chromatography technique. Linear log-log relationships were found between both the micelle-water partition coefficient and the capacity factor and the n-octanol-water partition coefficient. PMID- 10735325 TI - Continuous split-flow thin cell and gravitational field-flow fractionation of wheat starch particles. AB - The combined employment of the SPLITT (split-flow thin) cell--a relatively new system for fast, continuous binary separation--and of gravitational field-flow fractionation (GrFFF)--a fractionation technique suitable for micron particle size distribution determination--was investigated for starch separation and characterization. Emphasis is placed on the main advantages of both techniques: operating under gentle earth gravity field, low cost and ease of maintenance. The reproducibility of GrFFF is demonstrated. Both the SPLITT separation and GrFFF fractionation results were checked by optical microscopy. Application examples of typical starch fractionation experiments are reported and discussed. PMID- 10735326 TI - ECT in treatment algorithms: no need to save the best for last. PMID- 10735328 TI - Relapse of depression after ECT: a review. AB - Relapse of severe depression after successful treatment with electroconvulsive therapy (ECT) continues to be a major problem. We review the literature on relapse after ECT and factors that predict relapse. Early studies showed that the relapse rate was approximately 50% without follow-up treatment and that the majority of these relapses occurred in the first 6 months. More recent studies have found even higher rates in delusional depression and possibly in "double depression." Studies of biological markers as predictors of relapse were examined. Six of nine studies of the dexamethasone suppression test and one study of cortisol hypersecretion show that post-ECT nonsuppressors are at higher risk; although insensitive for diagnostic purposes, this test may be useful, when persistently abnormal, as a predictor of relapse. Studies of the thyrotropin releasing hormone stimulation test and shortened rapid eye movement sleep latency are inconclusive. Medication resistance pre-ECT has been shown to predict relapse in two studies and highlights the need for more aggressive and effective treatment in this group. Further research into the prediction and prevention of depressive relapse after ECT is needed, and the field anxiously awaits current trials comparing ECT with combination lithium and nortriptyline. PMID- 10735327 TI - Determinants of seizure threshold in ECT: benzodiazepine use, anesthetic dosage, and other factors. AB - The electrical dosage of the ECT stimulus impacts on efficacy and cognitive side effects, yet seizure threshold (ST) may vary as much as 50-fold across patients. It would be desirable to predict ST on the basis of patient and treatment characteristics. In particular, concerns have been raised that benzodiazepine use and higher dosage of barbiturate anesthetics elevate ST. In a three-site study, ST was quantified at the first ECT session using an identical empirical titration procedure in 294 patients who met RDC and DSM-IIIR criteria for a major depressive episode. ST varied over a 35-fold range across patients treated with right unilateral (RUL) (n = 267) and bilateral (BL) (n = 27) ECT. Higher ST was associated with BL electrode placement (p = 0.001). Among patients treated with RUL ECT, univariate analyses indicated that higher ST was associated with advanced age (p < 0.001), male gender (p < 0.001), greater burden of medical illness (p < 0.001), weight (p < 0.01), duration of mood disorder (p < 0.01), and history of previous ECT (p < 0.05). Average lorazepam dose in the 48 hours prior to ECT was not associated with ST, but was associated with decreased seizure duration (p < 0.01). Absolute, but not weight-adjusted, methohexital dose was associated with ST (p < 0.01). Multivariate analyses in patients treated with unilateral ECT showed that only 27.6% of the variance in ST (p < 0.0001) could be predicted. In the multivariate analyses, only age (p = 0.0001), gender (p = 0.01), and methohexital dose (p = 0.0001) were independently related to ST. Low dosage of lorazepam and methohexital dosage below 1 mg/kg are unlikely to impact on ST. Given the limited capacity to predict ST, empirical titration remains the only accurate method to determine electrical dosage in RUL ECT. PMID- 10735329 TI - The efficacy of ECT in mixed affective states. AB - ECT is efficacious in the treatment of both the depressed and manic phases of bipolar disorder. While ECT is believed to be equally efficacious in the treatment of mixed affective states, to our knowledge there are no empirical studies on this issue. A chart review study was conducted to compare treatment response and clinical course in three groups of patients who received ECT in a general hospital service: bipolar depressed (n = 38), bipolar manic (n = 5), and bipolar mixed (n = 10), diagnosed by DSM-IV criteria. All three groups showed robust response rates, but the number of days of hospitalization was significantly longer in the mixed group (mean 30, SD 19.0 days) compared with the depressed group (mean 19.0, SD 10.9 days, t = 2.4, p < 0.03). There was a trend for the number of ECT treatments to be greater in the mixed compared with the depressed group. These findings indicate that patients with mixed affective states do respond well to a course of ECT, but the longer hospital stays and greater number of ECT treatments suggest that they may be more difficult to treat with ECT than patients with pure bipolar depression or bipolar mania. PMID- 10735330 TI - Seizure activity and safety in combined treatment with venlafaxine and ECT: a pilot study. AB - Some authors have described the combined use of ECT and psychotropic drugs, emphasizing possible interactions and synergisms of this combined therapy. We are unaware of reports of the concurrent use of the new antidepressant venlafaxine with ECT. The goals of our study were to assess the possible effects of venlafaxine on seizure length during ECT and the possible cardiovascular effects of this combined treatment. Nine severely ill, depressed patients were treated simultaneously with bilateral ECT and venlafaxine 150 mg/day and were compared with nine control, depressed subjects taking tricyclic antidepressants (TCA) and ECT. No patients had prolonged seizures and no spontaneous/tardive seizures outside ECT were observed. With regard to mean seizure length, no statistically significant differences were observed between the control group and the venlafaxine group. Neither significant increases in arterial blood pressure nor electrocardiographic recording abnormalities were found in venlafaxine patients when compared with the tricyclic group. Even though the small number of patients used is a significant limitation of this study, we found that combined venlafaxine and ECT appears to be safe when used in depression. PMID- 10735331 TI - The impact of clinically diagnosed personality disorders on acute and one-year outcomes of electroconvulsive therapy. AB - Clinical experience suggests that patients with depression and a comorbid personality disorder (PD) may have a poorer response to electroconvulsive therapy (ECT). Only a few published studies have examined the relationship between comorbid personality disorders and response of major depression to ECT. These studies have used relatively small numbers of patients. The present study is a retrospective review of 107 inpatients with a major depressive episode referred for ECT. Patients with a clinically diagnosed PD, especially a cluster B PD, had a significantly poorer acute response to ECT than those without a PD. During the first year after treatment, ECT responders with a comorbid PD had a higher rate of relapse of depression. The retrospective study design limits the strength of conclusions that can be drawn. Nevertheless, it appears that clinically diagnosed PDs may be predictive of poor outcome in patients receiving ECT for depression. Further prospective study of the relationship between both clinically diagnosed PDs and structured interview based PD diagnoses and ECT treatment response is warranted. PMID- 10735332 TI - Hemodynamic responses to ECT in a patient with critical aortic stenosis. AB - We present a case study of a 46-year-old woman with a psychotic depressive illness of 2 months' duration with the coexisting medical diagnoses of critical aortic stenosis, severe labile hypertension, renal failure necessitating hemodialysis of 7-years' duration, and systemic lupus. Because of unresponsiveness to an antidepressant drug regimen, severe motor retardation, mutism, and refusal of food and fluids by mouth, an urgent indication for electroconvulsive therapy (ECT) was established. However, the patient refused ECT, and to allow its initiation, a court order was obtained. In view of the coexisting diagnoses of critical aortic stenosis, labile hypertension, and renal failure, ECT represented a substantially increased risk in this patient because of severe arterial hypertension and tachycardia. The patient was successfully managed during each ECT, using a combination of metoprolol by mouth, which was supplemented by i.v. esmolol immediately prior to the application of the ECT stimulus, and sodium nitroprusside, which was infused for several minutes prior to the seizure and thereafter to attenuate arterial hypertension. Nevertheless, sudden death, a well-known complication of critical aortic stenosis, occurred 96 hours after the fourth ECT. PMID- 10735333 TI - Successful ECT in a case of Leonhard's cycloid psychosis. AB - In Leonhard's nosological system, acute, episodic psychoses with good short-term and long-term prognoses, characterized by mixed affective and schizophrenic features, confusion, and alternating psychomotor retardation and excitement are called cycloid psychoses. According to clinical lore, patients with cycloid psychoses show an excellent and prompt response to electroconvulsive therapy (ECT). We describe a patient with typical motility psychosis, a subtype of cycloid psychoses, who failed to respond to a combination of antipsychotic and benzodiazepine medication but quickly recovered after the administration of ECT. PMID- 10735334 TI - Successful electroconvulsive therapy given to a patient with von Willebrand's disease. PMID- 10735335 TI - ECT in patients with intracranial aneurysm. PMID- 10735336 TI - Raloxifene therapy in the reduction of fractures. PMID- 10735337 TI - Use of the word 'accident' in injury-causing events. PMID- 10735338 TI - Early diagnosis and empathy in managing somatization. PMID- 10735339 TI - Options and issues in managing menopause. PMID- 10735340 TI - Essentials of the diagnosis of heart failure. AB - Although heart failure is a common clinical syndrome, especially in the elderly, its diagnosis is often missed. A detailed clinical history is crucial and should address not only current signs and symptoms of heart failure but also signs and symptoms that point to a specific cause of the syndrome, such as coronary artery disease, hypertension or valvular heart disease. It is important to determine whether the patient has had a previous cardiac event, in particular a myocardial infarction. The physical examination should include Valsalva's maneuver, a test that is highly specific and sensitive for the detection of left ventricular systolic and diastolic dysfunction in patients with heart failure. An electrocardiograph and a chest radiograph should also be obtained. Two dimensional echocardiography of the heart helps differentiate systolic from diastolic dysfunction. Coronary angiography is indicated in patients with heart failure and anginal chest pain and should be strongly considered in patients with an electrocardiogram suggestive of ischemia or myocardial infarction. PMID- 10735341 TI - Treatment of nonmalignant chronic pain. AB - Nonmalignant, chronic pain is associated with physical, emotional and financial disability. Recent animal studies have shown that remodeling within the central nervous system causes the physical pathogenesis of chronic pain. This central neural plasticity results in persistent pain after correction of pathology, hyperalgesia, allodynia, and the spread of pain to areas other than those involved with the initial pathology. Patient evaluation and management focus on pain symptoms, functional disabilities, contributory comorbid illnesses, and medication use or overuse. Treatment of chronic pain involves a comprehensive approach using medication and functional rehabilitation. Functional rehabilitation includes patient education, the identification and management of contributing illnesses, the determination of reachable treatment goals and regular reassessment. PMID- 10735342 TI - Approach to the vaso-occlusive crisis in adults with sickle cell disease. AB - The vaso-occlusive crisis, or sickle cell crisis, is a common painful complication of sickle cell disease in adolescents and adults. Acute episodes of severe pain (crises) are the primary reason that these patients seek medical care in hospital emergency departments. Frequently, however, the pain is incompletely treated. Despite advances in pain management, physicians are often reluctant to give patients adequate dosages of narcotic analgesics because of concerns about addiction, tolerance and side effects. It is important to recognize a pain crisis early, correct the inciting causes, control pain, maintain euvolemia and, when necessary, administer adequate hemoglobin to decrease the hemoglobin S level. The family physician and the hematologist must work together to treat acute pain episodes promptly and effectively, manage the long-term sequelae of chronic pain and prevent future vaso-occlusive crises. PMID- 10735343 TI - Cervical cancer. AB - Cervical cancer is the second most common type of cancer in women worldwide, after breast cancer. A preponderance of evidence supports a causal link between human papillomavirus infection and cervical neoplasia. The presence of high-risk human papillomavirus genital subtypes increases the risk of malignant transformation. Widespread use of the Papanicolaou smear has dramatically reduced the incidence of cervical cancer in developed countries. Accurate and early recognition of abnormal cytologic changes prevents progression of the disease from preinvasive to invasive. Research is under way to determine if efforts to reduce the false-negative rate of the Papanicolaou smear should include rescreening programs and fluid-based technology. Once cervical cancer is diagnosed, clinical staging takes place. Early-stage tumors can be managed with cone biopsy or simple hysterectomy. Higher stage tumors can be treated surgically or with radiotherapy. Advanced metastatic disease may respond to radiation therapy and concurrent chemotherapy. Protein markers for detection of recurrence and vaccines for prevention of cervical cancer are under investigation. PMID- 10735344 TI - Using tissue adhesive for wound repair: a practical guide to dermabond. AB - Dermabond is a cyanoacrylate tissue adhesive that forms a strong bond across apposed wound edges, allowing normal healing to occur below. It is marketed to replace sutures that are 5-0 or smaller in diameter for incisional or laceration repair. This adhesive has been shown to save time during wound repair, to provide a flexible water-resistant protective coating and to eliminate the need for suture removal. The long-term cosmetic outcome with Dermabond is comparable to that of traditional methods of repair. Best suited for small, superficial lacerations, it may also be used with confidence on larger wounds where subcutaneous sutures are needed. This adhesive is relatively easy to use following appropriate wound preparation. Patients, especially children, readily accept the idea of being "glued" over traditional methods of repair. PMID- 10735345 TI - Managing menopause. AB - Many women will spend one third of their lifetime after menopause. A growing number of options are available for the treatment of menopausal symptoms like vasomotor instability and vaginal atrophy, as well as the long-term health risks such as cardiovascular disease and osteoporosis that are associated with menopause. Currently, hormone replacement therapy (estrogen with or without progestin) is the primary treatment for the symptoms and long-term risks associated with menopause. However, recent evidence calls into question the protective effect of estrogen on cardiovascular disease risk. The association of risk for breast cancer with estrogen replacement therapy also has not been fully clarified. In addition, many women cannot or choose not to take hormones. For treatment of osteoporosis and heart disease, pharmacologic choices include antiresorptive agents such as bisphosphonates and calcitonin, and estrogens or selective estrogen receptor modulators such as raloxifene. In addition, complementary options that include vitamins, herbal treatments, exercise and other lifestyle adaptations are gaining increased interest. The growing number of choices and questions in this area emphasizes the need to individualize a treatment plan for each woman to meet her specific needs. PMID- 10735346 TI - A 'stages of change' approach to helping patients change behavior. AB - Helping patients change behavior is an important role for family physicians. Change interventions are especially useful in addressing lifestyle modification for disease prevention, long-term disease management and addictions. The concepts of "patient noncompliance" and motivation often focus on patient failure. Understanding patient readiness to make change, appreciating barriers to change and helping patients anticipate relapse can improve patient satisfaction and lower physician frustration during the change process. In this article, we review the Transtheoretical Model of Change, also known as the Stages of Change model, and discuss its application to the family practice setting. The Readiness to Change Ruler and the Agenda-Setting Chart are two simple tools that can be used in the office to promote discussion. PMID- 10735347 TI - Somatizing patients: part II. Practical management. AB - Somatization is the experiencing of physical symptoms in response to emotional distress. It is a common and costly disorder that is frustrating to patients and physicians. Successful treatment of somatization requires giving an acceptable explanation of the symptoms to the patient, avoiding unwarranted interventions and arranging brief but regular office visits so that the patient does not need to develop new symptoms in order to receive medical attention. Antidepressants may be helpful in many patients, as well as cognitive psychotherapy when patients are willing to participate in it. Typical problems in managing such patients can be addressed by relying on the continuity established through regular visits to the same primary care physician. PMID- 10735348 TI - Appropriate use of psychotropic drugs in nursing homes. AB - The Omnibus Budget Reconciliation Act (OBRA) of 1987 limited the use of psychotropic medications in residents of long-term care facilities. Updates of OBRA guidelines have liberalized some dosing restrictions, but documentation of necessity and periodic trials of medication withdrawal are still emphasized. Antidepressant drugs are typically underutilized in nursing homes. Tricyclic antidepressants have many side effects and thus are not preferred medications in elderly patients. Anxiety and insomnia are common problems in the institutionalized elderly. If behavioral measures are not successful, antidepressant medications with shorter half-lives may avoid drug accumulation, which can lead to excessive sedation, cognitive impairment and an increased risk for falls. In the elderly, antipsychotic medications can cause serious side effects, such as extrapyramidal symptoms and tardive dyskinesia. Newer antipsychotic drugs are less often associated with these side effects, but they should be used only for specific diagnoses and when behavioral and environmental measures are unsuccessful. PMID- 10735349 TI - Photo quiz. Purple pubis syndrome. PMID- 10735350 TI - Management of pain in sickle cell disease. PMID- 10735351 TI - American thoracic Society updates statement on pulmonary rehabilitation. PMID- 10735352 TI - Risk of HIV infection in the athletic setting. PMID- 10735353 TI - Bending the rules to get a medication. PMID- 10735354 TI - Cardiac markers into the new millennium. PMID- 10735355 TI - Thermodynamic considerations of arteriovenous gradients of hydrogen ion concentration and carbon dioxide tension. AB - It is shown that, in a multicompartmental homeostatic system, the extent of interaction between any two compartments can be assessed by determination of the difference in free energy change of one particular reaction, or a series of coupled reactions, operative in both of the compartments under consideration. Hydrogen ion concentration and carbon dioxide tension have been used to determine free energy change difference relationships between the venous and arterial compartments (-deltadeltaG(a-v)) of the circulatory system. Data from the literature (from two studies of congestive heart failure and one study of experimentally induced cardiac arrest) are used to calculate -deltadeltaG(a-v). It was found that in control subjects -deltadeltaG(a-v) is close to zero, whereas in congestive heart failure or cardiac arrest, the value rises to 150 cal mol(-1) or more, whereas in blood, the approach towards equilibrium between hydrogen and bicarbonate ions and dissolved carbon dioxide (aqueous CO2) is known to be only moderately rapid. It is concluded that, in the system under study, and with respect to the reaction H+ + HCO3- = CO2 + H2O, a high value for the free energy change difference between the two compartments (high -deltadeltaG(a-v)) must be due to an insufficient blood circulation rate. Accordingly, -deltadeltaG(a-v) is probably a quantitative measure of cardiac insufficiency. PMID- 10735356 TI - Analytical aspects of biosensors. PMID- 10735357 TI - Detection of poisoning by substances other than drugs: a neglected art. PMID- 10735358 TI - Thyroid function tests: often justified in the acutely ill. AB - It is claimed that inappropriate requesting of thyroid function tests (TFTs) is common in acutely ill patients. Consecutive inpatient TFTs (n = 129) were assessed in relation to clinical history and common symptoms and signs of thyroid disease. Requests were justified in 69% of cases, most commonly on the basis of atrial fibrillation and/or tachycardia. There were no clear reasons for requesting TFTs in the remaining cases, although the yield of abnormal results in these patients was similar to that in those with justified requests. Thyroid stimulating hormone (TSH) concentration was increased (median 7.5 mU/L, range 4.8 38.6 mU/L) in 22 patients, six of whom had biochemical and/or clinical evidence of hypothyroidism (previously undiagnosed) and five of whom had pre-existing hypothyroidism. Of the remaining 11 patients with increased TSH levels, three were confirmed to have compensated hypothyroidism; non-thyroidal illness (NTI) (including the effect of drugs) accounted for four cases. In four patients (one of whom died during the admission) follow-up was not possible. Of six patients with reduced TSH concentration (range <0.05-0.35 mU/L), one was thyrotoxic on carbimazole, one was receiving thyroxine for hypothyroidism, one had NTI and three were lost to follow-up (two of whom died during their admission). Manifestations of thyroid disease are protean and often subtle, and TFTs are thus clinically justified in many unwell inpatients. Although NTI contributes to some cases of abnormal TSH levels, a significant number of TFT abnormalities are consistent with underlying thyroid abnormality requiring investigation/treatment. PMID- 10735359 TI - Effect of screening algorithm, parameter values and median smoothing on patient specific risk estimates for Down's syndrome screening. AB - The efficiency of a screening programme for Down's syndrome is usually expressed in terms of the detection rate for a given false positive rate. Two programmes with approximately equal detection rates and false positive rates would then be regarded as being broadly equivalent. While this might be the case at a population level, we show in this paper that it may be far from true at the individual patient level. Different algorithms, or even different implementations of the same algorithm can lead to calculation of very different individual risks and can result in different decisions for individual patients. Even though two programmes might lead to the same number of referrals, they could be referring quite different women. We consider the effect of using different parameter values within an algorithm, different algorithms and alternative ways of estimating the gestational age-dependent medians of the marker values. We show that the combined effects of these factors could explain much of the range of risks reported in the UK National External Quality Assessment Scheme for Down's syndrome screening. PMID- 10735360 TI - Auto-antibodies against oxidized LDL as a marker of coronary artery disease in patients with familial hypercholesterolaemia. AB - Auto-antibodies to oxidized low-density lipoprotein (ox-LDL) are thought to play a pivotal role in the pathogenesis of atherosclerosis. This study investigates the value of auto-antibodies to ox-LDL as a predictive marker of atherosclerosis in patients with both homozygous and heterozygous familial hypercholesterolaemia (FH), who are known to suffer from severe premature atherosclerosis. The influences of well-established risk factors for atherosclerosis such as age, LDL cholesterol levels and smoking on the results were also determined. Auto-antibody titres to ox-LDL and fasting lipid profiles were measured in 26 homozygous FH patients, 20 heterozygous FH patients without documented coronary artery disease (CAD), 24 heterozygotes with overt CAD and 10 healthy normocholesterolaemic controls. Carotid intima media thickness, used as an in vivo assessment of atherosclerosis, was also measured in the homozygous FH patients. Ox-LDL titres did not differ between the groups. There was also no association between ox-LDL titres and the LDL-cholesterol level (P=0.14), presence or absence of CAD (P=0.69), age (P=0.50), carotid intima-media thickness (P=0.51) or smoking (P=1.0). In conclusion, antibody titres against ox-LDL cannot be used as a predictive marker of the presence or severity of atherosclerosis in patients with FH. PMID- 10735361 TI - Localization of oxidized HDL in atheromatous plaques and oxidized HDL binding sites on human aortic endothelial cells. AB - We examined the localization of oxidized high-density lipoprotein (HDL) in atheromatous plaques and the oxidized HDL binding sites on endothelial cells. Histochemical analysis using CuSO4-oxidized HDL-specific 9F5-3a antibody indicated the presence of oxidized HDL in the intima of atheromatous plaques in human abdominal aortae. The cell surface binding of 125I-oxidized HDL to cultured human aortic endothelial cells (HAEC) was saturable, with an apparent dissociation constant (Kd) of 1.43 micromol/L. Competition for 125I-oxidized HDL binding was strong for oxidized HDL, moderate for native HDL and low for acetylated low-density lipoprotein (LDL) or oxidized LDL. Using oxidized HDL as a ligand for blotting, a major 130-kDa band was detected in HAEC. These results suggest that oxidized HDL and its putative binding protein are present in atheromatous plaques and endothelial cells, respectively. PMID- 10735363 TI - Evaluation of the Abbott IMx fluorescence polarization immunoassay and the bio rad enzyme immunoassay for homocysteine: comparison with high-performance liquid chromatography. AB - We evaluated the precision, linearity and accuracy of the Abbott IMx and Bio-Rad (Axis) homocysteine assays. Both assays make use of S-adenosyl-homocysteine hydrolase and excess adenosine, to convert homocysteine to S-adenosylhomocysteine (SAH). A monoclonal anti-SAH antibody is then used to quantify SAH. The IMx assay measures the fluorescence polarization of a conjugated SAH analogue for the final analytical step, whereas the Bio-Rad method uses a microplate enzyme immunoassay (EIA) employing an anti-mouse antibody peroxidase conjugate. The Abbott procedure is completely automated whereas the Bio-Rad EIA is performed manually. Between run coefficient of variation using commercial controls was 2.6% at 7 micromol/L, 2.5% at 13 micromol/L and 1.7% at 24 micromol/L for the Abbott method, and 19.7% at 6.4 micromol/L, 15.9% at 11.0 micromol/L and 14.5% at 23.4 micromol/L for the Bio-Rad method. Both assays correlated well with a high-performance liquid chromatography (HPLC) procedure for homocysteine: Bio-Rad EIA = 1.03HPLC + 1.0 micromol/L, r=0.98, s(y/x)=0.51; Abbott IMx = 1.02HPLC + 0.7 micromol/L, r=0.99, s(y/x) = 0.33. Both methods were linear up to 50 micromol/L homocysteine. The IMx assay had superior precision as well as the technological advantage of being completely automated. Both immunoassays exhibited greatly improved throughput compared with our existing HPLC method. PMID- 10735362 TI - Accumulated lipids, aberrant fatty acid composition and defective cholesterol ester hydrolase activity in cholesterol ester storage disease. AB - We confirmed accumulation of glycogen and lipids, particularly cholesterol esters, in the liver of a patient with cholesterol ester storage disease (CESD). Hepatic cholesterol ester concentration was 100-200 times that found in normal livers. Analysis of the fatty acid composition indicated a higher proportion (41%) of cholesterol linoleate (C18-2), a slightly lower proportion (33%) of cholesterol oleate (C18-1) and normal proportions (14%) of cholesterol palmitate (C16-0) in the CESD patient compared with the control. This fatty acid composition of cholesterol esters and the fatty acid composition of other classes of lipids in the patient's liver resembled that of LDL. We also found that acid cholesterol ester hydrolase activity in the CESD liver was reduced to 5% of that in the control liver, while neutral cholesterol ester hydrolase activity remained at the control level. These results suggest that accumulated cholesterol esters were derived mainly from serum LDL and that the accumulation resulted from lack of acid cholesterol ester hydrolase. PMID- 10735364 TI - Frontline immunochromatographic device for on-site urine testing of amphetamines: laboratory validation using authentic specimens. AB - We evaluated a new test device for amphetamines and methamphetamines (Frontline, cut-off limit 300 ng/mL) using authentic clinical and forensic specimens. The device is based on immunochromatography and is dipped into urine and read visually by comparison with a colour scale after a few minutes. A total of 658 specimens were tested by comparing results of the screening procedure with established immunoassays. Discordant results were further investigated by gas chromatography mass spectrometry or gas chromatography (with flame ionization detector). The Frontline device had a sensitivity of 93% and a specificity of 98%. When specimens were classified by urine amphetamine concentration, close agreement was obtained at concentrations below 150 ng/mL and above 1000 ng/mL. A small number of specimens with amphetamine concentrations between 300 and 1000 ng/mL tested negative in the Frontline test. This finding could to some extent be explained by the enantioselectivity of the antibodies in the Frontline test to d amphetamine. We conclude that the performance of the Frontline test device for amphetamines is adequate for presumptive clinical and forensic screening. PMID- 10735365 TI - Distribution of haptoglobin phenotypes in a Korean population, using the semi automated PhastSystem. AB - We have established a new phenotyping method for haptoglobin, based on sodium dodecyl sulphate-polyacrylamide gel electrophoresis using the PhastSystem (Pharmacia Biotech, Uppsala, Sweden), followed by immunoblotting for detection. We measured haptoglobin concentrations and determined the haptoglobin phenotypes of 316 healthy Koreans using this method: 31 (9.8%) were of Hp 1-1 type, 140 (44.3%) of Hp 2-1 type and 145 (45.9%) of Hp 2-2 type. The haptoglobin allele frequencies were calculated to be 0.32 for Hp1 and 0.68 for Hp2. We were able to visualize up to 12 bands from the human Hp 2-2 polymeric series, with molecular weights in the range 171.9 x 10(3) to 802.2 x 10(3). The reference range of serum haptoglobin concentrations obtained by the IFCC (International Federation of Clinical Chemistry) standard method was 0.27-2.14 g/L. The serum haptoglobin concentration in Koreans was similar to that of Caucasians, but the Hp1 allele frequency was lower in Koreans. Our method could be used in clinical laboratories as a simple and practical method of haptoglobin phenotyping. In addition, the Hp 2-2 polymeric series could be used as high molecular weight standards. PMID- 10735366 TI - Monitoring of carbamazepine and carbamazepine 10,11-epoxide in breast milk and plasma by high-performance liquid chromatography. AB - A high-performance liquid chromatographic method was developed for the measurement of carbamazepine (CBZ) and carbamazepine 10,11-epoxide (CBZE) in human breast milk and plasma. The method involves rapid C18 solid-phase extraction of CBZ and CBZE. Chromatographic separation was achieved with a reversed-phase C8 column using a mobile phase of potassium dihydrogenphosphate (pH 2.5) and acetonitrile (67:33 v/v), with ultraviolet detection at 254 nm. 2 Methyl CBZ was used as the internal standard. Determination of both CBZ and CBZE was possible in the range of 0.01-6.0 mg/L and 0.02-6.0 mg/L in milk and plasma, respectively. The recoveries of CBZ and CBZE added to the milk and plasma were 90.6-98.0% and 88.9-104.0%, respectively, with coefficients of variation less than 8.3% and 10.5%, respectively. The method has been used for drug level monitoring in milk and plasma samples obtained from CBZ-treated patients. The mean (SD) levels for CBZ in milk and plasma samples were 3.50 (0.4) mg/L and 6.18 (2.9) mg/L, and for CBZE were 1.28 (0.3) mg/L and 1.85 (1.0) mg/L, respectively. The mean (SD) milk/plasma ratios of CBZ and CBZE were 0.64 (0.2) and 0.79 (0.3), respectively. The milk/plasma ratio of CBZE was slightly higher than that of CBZ. PMID- 10735367 TI - Screening for autoantibodies to SS-A/RO by indirect immunofluorescence using HEp 2000 cells. AB - We evaluated indirect immunofluorescence (IF) using HEp-2000 slides, which are transfected with SS-A cDNA, for screening for anti-SS-A antibodies, by comparing it with counterimmunoelectrophoresis (CIE). A total of 2427 specimens were screened for IF reactivity and for SS-A precipitins, of which 1033 (43%) were negative on both IF and CIE. There were 1271 SS-A precipitin-negative specimens (52%) which were IF-positive but lacked the distinctive SS-A staining pattern. One precipitin-negative serum was IF-positive with the distinctive SS-A pattern in the HEp-2000 system. One hundred and twenty-two specimens (5%) were positive for anti-SS-A precipitins on CIE, 107 showed the distinctive SS-A fluorescence staining pattern, whereas 15 of these precipitin-positive samples (12%) were IF positive but did not display the distinctive SS-A pattern on the transfected cells. Fourteen of the 15 samples in which the distinctive SS-A pattern was not observed displayed other significant antinuclear antibody (titre equal or >1:320) patterns. In conclusion, the presence of the typical 'distinctive' SS-A pattern on IF using the HEp-2000 slides is highly specific for the presence of autoantibodies to SS-A and has a sensitivity of 88% for detecting these antibodies. PMID- 10735368 TI - Urine dipstick testing: comparison of results obtained by visual reading and with the Bayer CLINITEK 50. PMID- 10735369 TI - Could salivary phenylalanine concentrations replace blood concentrations? PMID- 10735370 TI - A patient with juvenile chronic arthritis and Henoch-Schonlein purpura. PMID- 10735371 TI - Urinary catecholamine excretion in relation to renal function. PMID- 10735372 TI - External quality assurance--a personal view. PMID- 10735373 TI - The impact of the polymerase chain reaction in clinical medicine. PMID- 10735375 TI - Lords of the fly decode Drosophila genome. PMID- 10735374 TI - International plan focuses on eradication of polio and containment of the virus. PMID- 10735376 TI - US effort to eliminate syphilis moving forward. PMID- 10735377 TI - From the Health Care Financing Administration. PMID- 10735378 TI - From the Centers for Disease Control and Prevention. Role of victims' services in improving intimate partner violence screening by trained maternal and child health-care providers--Boston, Massachusetts, 1994-1995. PMID- 10735379 TI - From the Centers for Disease Control and Prevention. Storm-related mortality- central Texas, October 17-31, 1998. PMID- 10735380 TI - Tube feeding in patients with advanced dementia. PMID- 10735381 TI - Tube feeding in patients with advanced dementia. PMID- 10735382 TI - Tube feeding in patients with advanced dementia. PMID- 10735383 TI - Tube feeding in patients with advanced dementia. PMID- 10735384 TI - Privacy of electronic medical information. PMID- 10735385 TI - Abortion and unplanned pregnancy. PMID- 10735386 TI - Abortion and unplanned pregnancy. PMID- 10735387 TI - Abortion and unplanned pregnancy. PMID- 10735388 TI - Abortion and unplanned pregnancy. PMID- 10735389 TI - Chronobiology of recombinant leptin therapy. PMID- 10735390 TI - Prostacyclin and thromboxane and the development of preeclampsia. PMID- 10735391 TI - Prostacyclin and thromboxane and the development of preeclampsia. PMID- 10735392 TI - Is Miss America an undernourished role model? PMID- 10735393 TI - Correlation between elevated levels of amyloid beta-peptide in the brain and cognitive decline. AB - CONTEXT: Alzheimer disease (AD) is characterized neuropathologically by the presence of amyloid beta-peptide (Abeta)-containing plaques and neurofibrillary tangles composed of abnormal tau protein. Considerable controversy exists as to whether the extent of accumulation of Abeta correlates with dementia and whether Abeta alterations precede or follow changes in tau. OBJECTIVES: To determine whether accumulation of Abeta correlates with the earliest signs of cognitive deterioration and to define the relationship between Abeta accumulation and early tau changes. DESIGN, SETTING, AND PATIENTS: Postmortem cross-sectional study of 79 nursing home residents with Clinical Dementia Rating (CDR) scale scores of 0.0 to 5.0 who died between 1986 and 1997, comparing the levels of Abeta variants in the cortices of the subjects with no (CDR score, 0.0 [n = 16]), questionable (CDR score, 0.5 [n = 11]), mild (CDR score, 1.0 [n = 22]), moderate (CDR score, 2.0 [n = 15]), or severe (CDR score, 4.0 or 5.0 [n = 15]) dementia. MAIN OUTCOME MEASURES: Levels of total Abeta peptides with intact or truncated amino termini and ending in either amino acid 40 (A(beta)x-40) or 42 (A(beta)x-42) in 5 neocortical brain regions as well as levels of tau protein undergoing early conformational changes in frontal cortex, as a function of CDR score. RESULTS: The levels of both A(beta)x-40 and A(beta)x-42 were elevated even in cases classified as having questionable dementia (CDR score = 0.5), and increases of both peptides correlated with progression of dementia. Levels of the more fibril prone A(beta)x-42 peptide were higher than those of A(beta)x-40 in nondemented cases and remained higher throughout progression of disease in all regions examined. Finally, increases in A(beta)x-40 and A(beta)x-42 precede significant tau pathology at least in the frontal cortex, an area chosen for examination because of the absence of neuritic changes in the absence of disease. CONCLUSIONS: In this study, levels of total A(beta)x-40 and A(beta)x-42 were elevated early in dementia and levels of both peptides were strongly correlated with cognitive decline. Of particular interest, in the frontal cortex, Abeta was elevated before the occurrence of significant tau pathology. These results support an important role for Abeta in mediating initial pathogenic events in AD dementia and suggest that treatment strategies targeting the formation, accumulation, or cytotoxic effects of Abeta should be pursued. PMID- 10735394 TI - Carrying passengers as a risk factor for crashes fatal to 16- and 17-year-old drivers. AB - CONTEXT: Injuries from motor vehicle crashes are the leading cause of death among teenagers. Carrying passengers has been identified as a possible risk factor for these crashes. OBJECTIVE: To determine whether the presence of passengers is associated with an increased risk of crashes fatal to 16- and 17-year-old drivers and whether the risk varies by time of day and age and sex of drivers and passengers. DESIGN AND SETTING INCIDENCE: study of data from the Fatality Analysis Reporting System and General Estimates System (1992-1997), as well as the Nationwide Personal Transportation Survey (1995). SUBJECTS: Drivers aged 16 and 17 years who drove passenger cars, vans, or pickup trucks. MAIN OUTCOME MEASURE: Driver deaths per 10 million trips by number of passengers, driver age and sex, and time of day; and driver deaths per 1000 crashes by passenger age and sex. RESULTS: Compared with drivers of the same age without passengers, the relative risk of death per 10 million trips was 1.39 (95% confidence interval [CI], 1.24-1.55) for 16-year-old drivers with 1 passenger, 1.86 (95% CI, 1.56 2.20) for those with 2 passengers, and 2.82 (95% CI, 2.27-3.50) for those with 3 or more passengers. The relative risk of death was 1.48 (95% CI, 1.35-1.62) for 17-year-old drivers with 1 passenger, 2.58 (95% CI, 2.24-2.95) for those with 2 passengers, and 3.07 (95% CI, 2.50-3.77) for those with 3 or more passengers. The risk of death increased significantly for drivers transporting passengers irrespective of the time of day or sex of the driver, although male drivers were at greater risk. Driver deaths per 1000 crashes increased for 16- and 17-year olds transporting male passengers or passengers younger than 30 years. CONCLUSION: Our data indicate that the risk of fatal injury for a 16- or 17-year old driver increases with the number of passengers. This result supports inclusion of restrictions on carrying passengers in graduated licensing systems for young drivers. PMID- 10735395 TI - Comparison of ciprofloxacin (7 days) and trimethoprim-sulfamethoxazole (14 days) for acute uncomplicated pyelonephritis pyelonephritis in women: a randomized trial. AB - CONTEXT: The optimal antimicrobial regimen and treatment duration for acute uncomplicated pyelonephritis are unknown. OBJECTIVE: To compare the efficacy and safety of a 7-day ciprofloxacin regimen and a 14-day trimethoprim sulfamethoxazole regimen for the treatment of acute pyelonephritis in women. DESIGN: Randomized, double-blind comparative trial conducted from October 1994 through January 1997. SETTING: Twenty-five outpatient centers in the United States. PATIENTS: Of 378 enrolled premenopausal women aged at least 18 years with clinical diagnosis of acute uncomplicated pyelonephritis, 255 were included in the analysis. Other individuals were excluded for no baseline causative organism, inadequate receipt of study drug, loss to follow-up, no appropriate cultures, and other reasons. INTERVENTIONS: Patients were randomized to oral ciprofloxacin, 500 mg twice per day for 7 days (with or without an initial 400-mg intravenous dose) followed by placebo for 7 days (n = 128 included in analysis) vs trimethoprim sulfamethoxazole, 160/800 mg twice per day for 14 days (with or without intravenous ceftriaxone, 1 g) (n = 127 included in the analysis). MAIN OUTCOME MEASURE: Continued bacteriologic and clinical cure, such that alternative antimicrobial drugs were not required, among evaluable patients through the 4- to 11-day posttherapy visit, compared by treatment group. RESULTS: At 4 to 11 days posttherapy, bacteriologic cure rates were 99% (112 of 113) for the ciprofloxacin regimen and 89% (90 of 101) for the trimethoprim-sulfamethoxazole regimen (95% confidence interval [CI] for difference, 0.04-0.16; P = .004). Clinical cure rates were 96% (109 of 113) for the ciprofloxacin regimen and 83% (92 of 111) for the trimethoprim-sulfamethoxazole regimen (95% CI, 0.06-0.22; P = .002). Escherichia coli, which caused more than 90% of infections, was more frequently resistant to trimethoprim-sulfamethoxazole (18%) than to ciprofloxacin (0%; P<.001). Among trimethoprim-sulfamethoxazole-treated patients, drug resistance was associated with greater bacteriologic and clinical failure rates (P<.001 for both). Drug-related adverse events occurred in 24% of 191 ciprofloxacin-treated patients and in 33% of 187 trimethoprim-sulfamethoxazole-treated patients, respectively (95% CI, -0.001 to 0.2). CONCLUSIONS: In our study of outpatient treatment of acute uncomplicated pyelonephritis in women, a 7-day ciprofloxacin regimen was associated with greater bacteriologic and clinical cure rates than a 14-day trimethoprim-sulfamethoxazole regimen, especially in patients infected with trimethoprim-sulfamethoxazole-resistant strains. PMID- 10735396 TI - Rates of and factors associated with recurrence of preterm delivery. AB - CONTEXT: Information about risk of recurrent preterm delivery is useful to clinicians, researchers, and policy makers for counseling, generating etiologic leads, and measuring the related public health burden. OBJECTIVES: To identify the rate of recurrence of preterm delivery in second pregnancies, factors associated with recurrence, and the percentage of preterm deliveries in women with a history of preterm delivery. DESIGN AND SETTING: Population-based cohort study of data from birth and fetal death certificates from the state of Georgia between 1980 and 1995. SUBJECTS: A total of 122 722 white and 56174 black women with first and second singleton deliveries at 20 to 44 weeks' gestation. MAIN OUTCOME MEASURE: Length of gestation (categorized as 20-31, 32-36, or > or =37 weeks) at second delivery compared with length of gestation at first delivery, by age and race. RESULTS: Most women whose first delivery was preterm subsequently had term deliveries. Of 1023 white women whose first delivery occurred at 20 to 31 weeks, 8.2% (95% confidence interval [CI], 6.6%-10.1%) delivered their second birth at 20 to 31 weeks and 20.1% (95% CI, 17.7%-22.8%) at 32 to 36 weeks. Of 1084 comparable black women, 13.4% (95 % CI, 11.4%-15.6%) delivered at 20 to 31 weeks and 23.4% (95% CI, 20.9%-26.1%) delivered at 32 to 36 weeks. Among women whose first delivery occurred at 32 to 36 weeks, all corresponding rates were lower than those whose first birth was at 20 to 31 weeks; the rates of second birth at 20 to 31 weeks were substantially lower (for white women, 1.9% [95% CI, 1.7%-2.2%]; for black women, 3.8% [95% CI, 3.4%-4.2%]). Compared with women aged 20 to 49 years at their second delivery, women younger than 18 years had twice the risk of recurrence of delivery at 20 to 31 weeks. Of all second deliveries at 20 to 31 weeks, 29.4% for white women and 37.8% for black women were preceded by a preterm delivery. CONCLUSIONS: Our data suggest that recurrence of preterm delivery contributes a notable portion of all preterm deliveries, especially at the shortest gestations. PMID- 10735397 TI - Preparticipation cardiovascular screening for US collegiate student-athletes. AB - CONTEXT: Sudden death in young competitive athletes due to unsuspected cardiovascular disease has heightened interest in preparticipation screening. OBJECTIVE: To assess screening practices for detecting potentially lethal cardiovascular diseases in college-aged student-athletes. DESIGN, SETTING, AND PARTICIPANTS: A total of 1110 National Collegiate Athletic Association member colleges and universities were surveyed between 1995 and 1997, with 879 (79%) responding to the questionnaire. MAIN OUTCOME MEASURES: Information on the administration and scope of the preparticipation screening process was obtained from the team physician or athletic director; preparticipation screening forms were evaluated for content and compared with 12 items recommended by the 1996 American Heart Association (AHA) consensus panel screening guidelines. RESULTS: Preparticipation screening was a requirement at 855 (97%) of 879 schools, was performed on campus at 713 schools (81 %), and was required annually by 446 schools (51 %). Team physicians were responsible for examinations at 603 (85%) of 713 schools with on-campus screening, although 135 of these schools (19%) also approved nurse practitioners and 244 schools (34%) allowed athletic trainers to perform examinations. Of the history and physical examination screening forms analyzed from 625 institutions, only 163 schools (26%) had forms that contained at least 9 of the recommended 12 AHA screening guidelines and were judged to be adequate, whereas 150 (24%) contained 4 or fewer of these parameters and were considered to be inadequate. Smaller Division III schools were more likely than larger Division I schools to have inadequate screening forms (30% vs 14%; P<.001). Relevant items that were omitted from more than 40% of the screening forms included history of exertional chest pain, dyspnea, or fatigue; familial heart disease or premature sudden death; and physical stigmata or family history of Marfan syndrome. CONCLUSION: The preparticipation screening process used by many US colleges and universities may have limited potential to detect (or raise the suspicion of) cardiovascular abnormalities capable of causing sudden death in competitive student-athletes. PMID- 10735398 TI - Guidelines for medical and health information sites on the internet: principles governing AMA web sites. American Medical Association. AB - Access to medical information via the Internet has the potential to speed the transformation of the patient-physician relationship from that of physician authority ministering advice and treatment to that of shared decision making between patient and physician. However, barriers impeding this transformation include wide variations in quality of content on the Web, potential for commercial interests to influence online content, and uncertain preservation of personal privacy. To address these issues, the American Medical Association (AMA) has developed principles to guide development and posting of Web site content, govern acquisition and posting of online advertising and sponsorship, ensure site visitors' and patients' rights to privacy and confidentiality, and provide effective and secure means of e-commerce. While these guidelines were developed for the AMA Web sites and visitors to these sites, they also may be useful to other providers and users of medical information on the Web. These principles have been developed with the understanding that they will require frequent revision to keep pace with evolving technology and practices on the Internet. The AMA encourages review and feedback from readers, Web site visitors, policymakers, and all others interested in providing reliable quality information via the Web. PMID- 10735399 TI - An 82-year-old woman with mood changes following a stroke. PMID- 10735400 TI - A 43-year-old woman coping with cancer, 1 year later. PMID- 10735401 TI - The origins of Alzheimer disease: a is for amyloid. PMID- 10735403 TI - JAMA Patient Page: urinary tract infections. PMID- 10735402 TI - Reducing fatal crash risk among teenaged drivers: structuring an effective graduated licensing system. PMID- 10735404 TI - Surgical treatment of 'short stump' bronchial fistula. AB - OBJECTIVE: The optimal management of bronchial fistulae remains a surgical challenge. To assess the relative efficacy of the transsternal approach in the treatment of short stump bronchial fistula we analyzed a cohort of patients who underwent this type of surgery in our department during an almost 19 year period. METHODS: Of a series of 49 patients with short stump bronchial fistula who were treated via the transsternal approach, 15 underwent bifurcational sleeve resection and 34 had tracheal wedge resections. In 19 cases the tracheal defects was up to 30 mm in diameter, in the remaining 30 cases the length was less than 10 mm. In all cases tracheobronchial fistulae were associated with concomitant empyema. Surgical debridement of the empyema cavity was achieved by VATS application. In five patients who underwent primary surgery for lung cancer tumor recurrence was seen in distal margins of the resected defect. RESULTS: There was no intraoperative mortality. Two patients died from acute pneumonia at postoperative day 3 and adult respiratory distress syndrome (ARDS) at postoperative day 7, respectively. Two further patients died due to anastomotic dehiscence from sepsis and respiratory failure the overall hospital mortality being 8 (16%). Major complications included one right pulmonary artery injury (2%), two healing disturbances after tracheal wedge resection and five after sleeve resection of the bifurcation. Late complication occurred in six patients (13.3%) with delayed healing at the suture site who later required treatment, two of these required additional stent applications. CONCLUSIONS: Surgical treatment of patients with short stump tracheobronchial fistulae by means of a transsternal approach allows reliable closure with low mortality and morbidity. PMID- 10735405 TI - Experimental study on the in vivo behaviour of a new collagen glue in lung surgery. AB - OBJECTIVE: To study the pneumostatic ability of a collagen polymerised with a polysaccharide (GAO) glue in lung surgery; its influence in pleuro-pulmonary adhesion formation; the pulmonary tissue reaction to it, its biodegradability, and the eventual alterations of pulmonary compliance induced by the glue. METHODS: Two groups of ten rabbits (controls and treated) were operated under ventilatory assistance by thoracotomy to promote pleural adhesions, and injury to the lung. Repeated chest X-rays were carried out postoperatively. Lungs were examined histologically at day 40. In vitro tests were performed to study glue effects on pulmonary compliance. RESULTS: Air leaks stopped 2 min after glue application. Persistent pneumothorax were likely seen in treated rabbits (ns). Glue induces a temporary reduction of pulmonary compliance. Glue did not increase adhesion formation, or interfere with the healing process. CONCLUSIONS: For its properties, GAO seems to be a good and well-tolerated tool to reduce air leaks from the lung, without inducing residual pleural symphysis. PMID- 10735406 TI - Paravalvular leakage after mitral valve replacement: improved long-term survival with aggressive surgery? AB - BACKGROUND: Following mitral valve replacement, surgical closure of paravalvular leaks is usually advised in severely symptomatic patients and in those requiring blood transfusions for persisting haemolysis. However, the long-term prognosis of less symptomatic patients or those not needing blood transfusions is unknown. METHODS: Between 1987 and 1997, we observed 96 patients with mitral paravalvular leakage. A paraprosthetic leak was diagnosed after a median time of 119 days (range: 1 day-23 years) after primary mitral valve replacement. During an average follow-up of 5 years (range: 1-23 years), 50/96 patients were referred for surgical closure. RESULTS: Compared with patients who received conservative treatment, those referred for surgery had a significantly lower mean preoperative haematocrit (P = 0.002) with a higher proportion of patients being in the NYHA class III/IV (P = 0.03). Age, gender, left ventricular function and number and size of leaks did not differ between the groups. The 30-day postoperative mortality for valve reoperation was 6% (3/50); during follow-up three further patients died, resulting in an overall mortality rate of 12%. In the group treated conservatively there was a mortality rate of 26% (12/46). Thus, the actuarial survival for patients referred for surgery was 98, 90 and 88% after 1, 5 and 10 years, compared with 90, 75 and 68% for patients treated conservatively (long-rank P = 0.03). In addition, there was a significant increase in mean haematocrit levels (P = 0.0001) and an improvement in NYHA class III/IV symptoms (P = 0.002), vertigo (P = 0.001) and fatigue (P = 0.001) after surgery. CONCLUSIONS: Following mitral valve replacement, a more aggressive surgical treatment is recommended for patients with paraprosthetic leaks. Surgery should be offered to less symptomatic patients, as well as those not requiring blood transfusion. PMID- 10735407 TI - Downstream turbulence and high intensity transient signals (HITS) following aortic valve replacement with Medtronic Hall or St. Jude Medical valve substitutes. AB - OBJECTIVE: High intensity transient signals (HITS) representing microembolization to the brain have been found to contribute to cognitive impairment and psychoneurological dysfunction in patients carrying a mechanical aortic valve. It is unknown, whether HITS represent gaseous or solid emboli. This animal study evaluates the impact of valve orientation on HITS for two different mechanical valves with both valves implanted in their best and worst orientation, which has been defined in previous studies with respect to downstream turbulence. METHODS: In four pigs a rotation device carrying either a Medtronic Hall (MH) or St. Jude Medical (SJM) valve size 23 mm was implanted. The device allowed rotation of the implanted valves without reopening of the aorta. Approximately 30 min after weaning from extracorporeal circulation, a Doppler probe was placed on both common carotid arteries. In different orientations of the implanted valves (best and worst position), HITS were detected by the Doppler probe and recorded for ten min by a transcranial Doppler sonography device (Medilab Inc., Estenfeld, Germany). RESULTS: HITS showed significant change with rotation for both valve designs. With the major orifice of the MH oriented towards the non-coronary leaflet (optimum position) very low HITS-counts (0.8-1.7/min) were observed. In the worst orientation HITS rose to 43-66/min. For the SJM the HITS count in the optimum position was 23.4-24/min and in the worst orientation 38-48/min. CONCLUSIONS: Valve orientation has an important impact on microembolization to the brain. In the optimum orientation (large orifice facing the non-coronary leaflet) the Medtronic Hall valve showed negligible incidence of HITS. The St. Jude Medical bileaflet valve showed less variation but demonstrated significant HITS counts at any orientation. As the MH in the worst position shows significantly higher turbulent stresses than the SJM but no higher incidence of HITS, a strong correlation between turbulence and HITS was not demonstrated by this study. PMID- 10735408 TI - Medium-term results after the modified Cox/Maze procedure combined with other cardiac surgery. AB - OBJECTIVE: Long-term results after the modified Cox/Maze III procedure combined with other cardiac procedure for the treatment of organic heart disease and chronic atrial fibrillation (AF) has not been clarified. This report describes our medium-term results after such operation. METHODS: Between March 1993 and August 1995, 104 consecutive patients with chronic AF underwent the modified Cox/Maze III procedure combined with other cardiac procedure. There were 100 long term survivors. There were 45 men and 55 women, with ages ranging from 21 to 77 years (mean 59.7). Patients were followed up and changes in rhythm, need for pacemaker implantation, and the incidence of CNS (central nervous system) complications were retrospectively studied. RESULTS: The follow-up was complete in 103 patients and 99 long-term survivors (99%). The mean follow-up period was 44.6 +/- 1.1 months. In the immediate postoperative period, 73 patients regained sinus rhythm (SR group), 21 patients were in AF (AF group), and six patients underwent pacemaker implantation because of sick sinus syndrome (SSS). During the follow-up period, eight patients died. One- and 5-year survival rates (Kaplan Meier) after surgery was 95.1 +/- 2.3 and 87.8 +/- 3.4% for the entire group. Preoperative NYHA class was 2.5 +/- 0.7 and medium-term NYHA class was 1.5 +/- 0.5. (P < 0.001) Changes in rhythm for the SR group were followed. Fifty-two patients of the SR group stayed in SR (72%), 16 patients converted back to AF (22%), and four patients had newly-developed SSS (6%) at follow-up period. Probability in SR maintenance for SR group at 1 year was 88.8 +/- 3.7% and at 5 years was 64.8 +/- 7.5%. Five patients experienced the CNS complication during the follow-up period. Two of the AF group and two of the SR group patients developed cerebral/cerebellar infarction. One of the SR group patients experienced small cerebral bleeding. CONCLUSIONS: The medium-term results after the modified Cox/Maze III procedure concomitant with other cardiac procedure are good with improved functional status and good survival rate. However, there seems to be gradual but constant attrition in the rate of SR maintenance in SR group. PMID- 10735409 TI - Improved outcome of APACHE II score-defined escalating systemic inflammatory response syndrome in patients post cardiac surgery in 1996 compared to 1988-1990: the ESSICS-study pilot project. AB - OBJECTIVE: Cardiac surgery using extracorporeal circulation leads to the release of cytokines and subsequently to a systemic inflammatory response syndrome, which is thought to be a negative prognostic factor for patients' outcome. A stratification for the risk of an escalating systemic inflammatory response syndrome had been achieved in a monocenter study carried out in 1988-1990, using APACHE II scoring on the morning of the 1st postoperative day. We now re evaluated this concept prospectively in three independent centers. METHODS: The APACHE II based risk stratification was put to test in three independent heart surgery centers in the period from June to December 1996. Nine hundred and forty five patients after elective cardiac surgery (excluding heart transplantation) with the assistance of the cardiopulmonary bypass were prospectively monitored. RESULTS: We found an increase in mortality with higher APACHE II score values determined on the 1st postoperative day. The mortality rose to nearly 50% with an APACHE II score of > or = 28. Patients at high risk for the development of a systemic inflammatory response syndrome (APACHE II score > or = 24) significantly differed from patients at lower risk (APACHE II score < 19) in the duration of mechanical ventilation and extracorporeal circulation, age and New York Heart Association (NYHA) classification (P < 0.05). CONCLUSION: The APACHE II score determined on the morning of the 1st postoperative day helps identifying the subgroup of patients with escalating systemic inflammatory response syndrome. Comparison with the data obtained in the years 1988-1990, suggests a better prognosis in the current trial for patients at high risk with a similar degree of escalating systemic inflammatory response syndrome. PMID- 10735410 TI - Total endoscopic computer enhanced coronary artery bypass grafting. AB - OBJECTIVE: In an effort to minimize access in coronary artery bypass (CAB) surgery, a total endoscopic approach using computer enhanced technology was developed. METHODS: By July 1999 the da Vinci telemanipulation system (Intuitive Surgical, Mountain View, CA) was used in 66 patients with coronary artery disease. In 12 patients undergoing routine coronary artery bypass grafting (CABG) (group 1) the internal thoracic artery (ITA) to left anterior descending artery (LAD) anastomosis was performed remotely using the system. In 32 patients (group 2) endoscopic dissection of the ITA was performed followed by a conventional minimally invasive direct coronary artery bypass (MIDCAB) operation. In 22 patients (group 3) the complete operation was performed endoscopically through 4 ports (total endoscopic coronary artery bypass, TECAB). Port-Access cardiopulmonary bypass with cardioplegic arrest was used for TECAB. RESULTS: In group 1 the time for performing the ITA to LAD anastomosis was 17 +/- 10 min. Mean graft flow was 38 +/- 25 ml/min. One anastomosis leaked and was repaired manually. In group 2 in 31/32 patients (96%) the ITA harvest was successfully performed with the system at mean of 61 +/- 27 min. There was a substantial learning curve associated with ITA take-down. In one patient a dissection caused insufficient free ITA graft flow which necessated additional vein grafting. Postoperative angiography demonstrated graft patency in all cases. In the TECAB group, the operation could be completed through four ports in 18 of the 22 patients (82%) with operating times in the range 220-507 min. In four patients, elective conversion to a minithoracotomy was required due to failure to identify the LAD (1), bleeding from the anastomosis (1), grafting of a diagonal branch (1) and torsion of the pedicle (1). One patient required reoperation for bleeding from an ITA side-branch. Median intubation time was 13 h and stay on ICU and hospitalization were 20 h and 7 days, respectively. A 3-month follow-up angiography revealed patent grafts in all TECAB patients. CONCLUSION: Endoscopic ITA harvesting and performing of arterial anastomoses can be safely performed with the da Vinci system. TECAB is possible on the arrested heart with good functional results. However, a substantial learning curve has to be overcome which is reflected in long operation times and an initial significant conversion rate. PMID- 10735411 TI - Difficulties in the interpretation of coronary angiogram early after coronary artery bypass surgery on the beating heart. AB - OBJECTIVE: The major objective of this study was to evaluate the findings in early postoperative coronary angiography in patients who underwent coronary revascularization on the beating heart without cardiopulmonary bypass. METHODS: Eighty-four consecutive patients receiving 113 grafts were studied. A coronary angiography was performed 0 to 5 days postoperatively. All the grafts were reviewed and classified in the following way: grade A (unimpaired run-off); grade B1 (<50 stenosis); grade B2 (>50% stenosis); grade O (occlusion). A second coronary angiography was performed in patients with a stenosis grade B2, 4 to 30 months postoperatively. An exercise test was performed by patients with B1 stenosis. RESULTS: Overall graft patency was 96% in the 113 grafts. None of the 14 patients with B1 stenosis in the early coronary angiography had any clinical signs of ischemia. Eight of the 12 patients who exhibited B2 stenosis either at the anastomotic site, in the graft or in the distal coronary artery at the first coronary angiography had a normal angiogram at the re-angiography. CONCLUSION: A majority of stenoses visualized at the early coronary angiography could not be seen at a later coronary angiography, which makes the interpretation of the angiogram unreliable as a tool for the decision as to redo-procedure in the early postoperative period. PMID- 10735412 TI - Batista procedure: elliptical modeling against spherical distention. AB - OBJECTIVE: Batista's cardio-reduction of mass and diameter changes the geometry of the left ventricle (LV). This in vivo study explores the LV changing from spherical distention to elliptic modeling. METHODS: Nineteen pigs were connected to cardio-pulmonary bypass (CPB), five of them without cardiac alteration (controls). The LV of the other fourteen pigs was incised between the left anterior descending and the circumflex arteries. Myocardial protection with the beating open method was used. In seven pigs, the LV incision was closed by direct suture to assess the surgical trauma of the Batista procedure (incision). In the other seven pigs a pericardial patch was placed for spherical distention of the LV as a model of heart failure (sphericalization). Patch removal and LV closure restored the normal cardiac geometry (elliptical modeling). Ventricular function was evaluated with Frank-Starling curves (stroke work index, SWI), with endsystolic elastance (EES) and diastolic compliance (beta(-1)) by impedance catheter, and with ejection fraction (EF) by transesophageal echocardiogram. Data were recorded after ventriculotomy, after sphericalization and after elliptical modeling (before and 30 min after discontinuation of CPB). RESULTS: CPB did not significantly alter controls' hemodynamic. Ventriculotomy decreased cardiac function (as % vs. post CPB-controls: SWI* 63 +/- 4; EES 93 +/- 2; beta(-1)* 86 +/- 5). Sphericalization additionally impaired the function (as % vs. ventriculotomy: SWI* 57 +/- 4; EES* 60 +/- 7; beta(-1)* 45 +/- 8). The elliptical modeling greatly improved ventricular performance (as % vs. sphericalization: SWI** 156 +/- 5; EES** 162 +/- 8; beta(-1)** 177 +/- 7; EF** 216 +/- 5) (P < 0.05 for Student's unpaired* and paired** t-test). CONCLUSIONS: Spherical distention of the left ventricular dimensions causes cardiac decompensation. The surgical trauma of the Batista procedure impairs the LV performance. However, the spherically distended LV benefits from Batista's cardio-reduction by elliptical modeling. PMID- 10735413 TI - Change of sternal perfusion following preparation of the internal thoracic artery -a scintigraphical study. AB - BACKGROUND: Today the internal thoracic artery (ITA) is the bypass graft of choice due to its superior long-term patency rate. It was the aim of this present prospective study to investigate possible perfusion disturbances and consecutive impaired wound healing induced by the ITA preparation. The sternal perfusion was assessed by bone scintigraphy. METHODS: Forty-four patients were included in the study. There were three groups: group I (control, no ITA preparation; n = 12); group II (preparation of the left ITA; n = 21); group III (preparation of both ITAs; n = 11). In all patients a median sternotomy was performed. A bone scintigraphy was performed 4 days before and 12 days after the bypass procedure. Scintigraphical pictures of all patients were assessed visually (one specialized investigator) and the impulse rate was counted by the aid of a computer program. RESULTS: Results of both evaluation methods showed congruently that neither the use of the left nor of both ITAs causes a statistically significant impairment of sternal perfusion. The percentage of postoperative increase of the rate of impulses was in group I: total sternum 55%; right side 56%; left side 55%. The respective numbers for group II were 58, 63 and 53%, and for group III 54, 52 and 56%. Surprisingly, perfusion scans in group II revealed an increase in the right sternal half after left ITA preparation. This may be due to the additional blood flow demand of collaterals branching between the right ITA and contralateral intercostal arteries representing a compensatory mechanism of the loss of the left ITA. During the observed postoperative time frame (mean 15 days) no healing disturbance of the sternal wound occurred in any patient. CONCLUSION: According to the present data the use of one or both ITAs does not cause an increase of healing disturbances, consecutive to a postoperatively decreased sternal blood perfusion. PMID- 10735414 TI - Myocardial protection with high-dose beta-blockade in acute myocardial ischemia. AB - OBJECTIVE: The risk of postoperative cardiac dysfunction is markedly increased by emergency coronary artery bypass grafting in the presence of acute myocardial ischemia. High dose beta-blockade during continuous coronary perfusion has been suggested as an alternative to conventional cardioplegia and this technique has been applied successfully in high risk patients for coronary artery bypass grafting (CABG) surgery. This study compared high dose beta-blockade with esmolol to continuous warm blood cardioplegia in a clinically oriented model of acute left ventricular (LV) ischemia and reperfusion. METHODS: Twelve dogs were subjected to 60 min of regional LV ischemia by left anterior descending branch (LAD) ligation. Cardiopulmonary bypass (CPB) and aortic crossclamp were applied after 45 min of ischemia. Thereafter, high dose beta-blockade during continuous coronary perfusion (ESMO, n = 6) or antegrade continuous warm blood cardioplegia (WBC, n = 6) were maintained for 60 min. Myocardial water content (measured from endomyocardial biopsies using a microgravimetric technique), global LV function (preload recruitable stroke work: PRSW), and regional LV function (echocardiographic wall motion score) were determined at baseline and after weaning from CPB. RESULTS: During aortic crossclamp interstitial edema formation was significantly higher in the WBC group with an average water gain of 2.2 +/- 0.49 vs. 0.76 +/- 0.12% in the ESMO group. Thereafter, edema resolved in both groups, but myocardial water gain remained significantly higher in the WBC group at 60 and 120 min post CPB (0.98 +/- 0.19 and 1.13 +/- 0.32% vs. 0.07 +/- 0.25 and 0.04 +/- 0.08%). Global LV function was significantly higher in the ESMO group at 60 and 120 min post CPB (PRSW 103 +/- 6 and 94.7 +/- 4.6% of baseline vs. 85.3 +/- 4.9 and 74.7 +/- 7.6% of baseline). However, regional LV function showed no significant difference between groups. CONCLUSIONS: High-dose beta blockade during continuous coronary perfusion may allow the surgeon to utilize the advantages of warm heart surgery, while avoiding the interstitial edema formation and temporary cardiac dysfunction associated with continuous warm blood cardioplegia. In high risk patients such as patients with unstable angina or after failed PTCA, high-dose beta-blockade may be an applicable alternative to cardioplegic arrest. PMID- 10735415 TI - Coronary oxygen persufflation combined with HTK cardioplegia prolongs the preservation time in heart transplantation. AB - BACKGROUND: One of the most restricting factors remaining in heart transplantation is the limited myocardial ischemia time. A new approach towards the prolongation of this time is the combination of primary cardioplegic arrest followed by continuous coronary oxygen persufflation (COP) with gaseous oxygen. METHODS: This technique was applied in pig hearts, which we transplanted orthotopically after cardioplegic arrest by original (n = 5) and modified (addition of hyaluronidase: n = 11) Bretschneider HTK solution and 14 h of hypothermic preservation. Depending on the different preservation techniques, we created four groups: (1), original HTK (HTK), n = 5; (2), modified HTK (mHTK), n = 5; (3), modified HTK solution plus COP (mHTK + COP), n = 6; and (4), as a control five hearts were transplanted after cardioplegic arrest by the original HTK solution and a cold ischemia time of 3 h comparable to clinical routine procedure. RESULTS: After 14 h of preservation and orthotopic transplantation, cardiac functional recovery in mHTK + COP hearts was similar to control hearts, and improved compared to hearts of both other groups. Hemodynamics were significantly better in hearts preserved by mHTK + COP and in the control group compared to the HTK-hearts (P < 0.05), not significant compared to mHTK hearts (dp/dt(max) in % of preoperative +/- standard error of mean (SEM): mHTK + COP, 85 +/- 9; control, 85 +/- 10.5; mHTK, 59 +/- 14; HTK, 50 +/- 4). The cardiac output (CO) in % of preoperative was: mHTK + COP, 68 +/- 5.4; control, 64 +/- 4; mHTK, 44 +/- 2.7; HTK, 25 +/- 11. The ATP of left ventricular myocardium in mHTK + COP hearts at 14.7 +/- 1 micromol/g dry weight (DW) and in the control at 14.59 +/- 1.8 was higher compared to that in mHTK at 12.2 +/- 2.8 (P is non-significant (n.s.) versus mHTK + COP and control) and in HTK-hearts at 7.0 +/- 0.5 (P < 0.05 versus mHTK + COP and control). CK-MB in percent of CK showed no increase in either group. CONCLUSIONS: These data show that COP combined with a mHTK solution represents a potential alternative to complement currently used cold storage techniques for prolonged preservation periods. PMID- 10735416 TI - Modified ultrafiltration lowers adhesion molecule and cytokine levels after cardiopulmonary bypass without clinical relevance in adults. AB - OBJECTIVE: Cardiac surgery with cardiopulmonary bypass (CPB) results in expression of cytokines and adhesion molecules (AM) with subsequent inflammatory response. The purpose of the study was to evaluate the clinical impact of modified ultrafiltration (MUF) and its efficacy in reducing cytokines and AM following coronary artery bypass grafting (CABG) in adults. METHODS: A prospective randomized study of 97 patients undergoing elective CABG was designed. Fifty patients were operated on using normothermic and 47 patients using hypothermic CPB. The normothermic group was subdivided into a group with modified ultrafiltration (n = 30) and a group without MUF (n = 20). In the hypothermic group 30 patients received MUF compared to 17 patients serving as controls. MUF was instituted after CPB for 15 min through the arterial and venous bypass circuit lines. Cytokines (IL-6, IL-8, TNF-alpha, IL-2R) and adhesion molecules (sE-selectin, sICAM-1) were measured preoperatively, pre-MUF, in the ultrafiltrate, 24 h, 48 h and 6 days after surgery by chemiluminescent enzyme immunometric assay or enzyme-linked immunosorbent assay (ELISA). Clinical parameters were collected prospectively until discharge. RESULTS: In all patients AM and cytokines were significantly elevated after normothermic and hypothemic CPB. AM and cytokines were significantly higher in hypothermia compared to normothermia. In hypothermic CPB sE-selectin was decreased after 24 h by 37% (P < 0.0063) and by 40% (P < 0.0027) after 48 h postoperatively. ICAM-1 was reduced by 43% (P < 0.0001) after 24 h and by 60% (P < 0.0001) after 6 days. Similar results were seen in cytokines with reduction up to 60% after 24 h. Changes after 48 h were noticeable but not significant. Reduction of AM and cytokines after normothermic CPB was minimal. Neither in normothermia, nor in hypothermia has sIL 2R been effectively removed from the circulation. There were no significant differences in the clinical variables between the patients with or without MUF. CONCLUSION: AM and cytokines are significantly elevated after hypothermic CPB compared to normothermic CPB. MUF led to a significant reduction in cytokine and AM levels after hypothermic CPB, except for IL-2R. MUF showed minimal effect in normothermia. We conclude that MUF is an efficient way to remove cytokines and AM. However, we were unable to demonstrate any significant impact of MUF in outcome of adults after elective CABG. PMID- 10735417 TI - Images in cardio-thoracic surgery. Giant chondrosarcoma of the anterior chest wall. PMID- 10735418 TI - Repair of aortic atresia and hypoplastic left heart syndrome without using graft material. AB - We present a modification of the Norwood stage 1 operation, where the neo-aorta was reconstructed without using graft material. After extensive mobilization of the descending aorta the ductus arteriosus was excised and an end-to-end anastomosis was created between the main pulmonary artery and the distal ductal aortic junction. The proximal ascending aorta was anastomosed side-to-side to the neo-aorta. Finally either a direct side-to-side anastomosis was created or a 3.5 mm Gore-Tex graft was implanted between the innominate artery and the right pulmonary artery. The first three patients had a favourable outcome: echocardiography showed good ventricular function and acceptable saturation (85%) at the follow up. PMID- 10735419 TI - In vivo model for reversal of pulmonary blood flow in canines. PMID- 10735420 TI - Alteplase as an alternative to urokinase. Advisory Panel on Catheter-Directed Thrombolytic Therapy. PMID- 10735421 TI - Initial results of reteplase in the treatment of acute lower extremity arterial occlusions. AB - PURPOSE: To assess the feasibility and efficacy of reteplase in transcatheter arterial thrombolysis for lower extremity occlusive disease. MATERIALS AND METHODS: Fifteen consecutive patients with acute lower extremity ischemia due to occlusive disease were treated by means of catheter-directed thrombolysis with reteplase. Following diagnostic angiography, thrombolysis was started either from an antegrade puncture site in cases of femoropopliteal occlusions, or from the contralateral groin in cases of thrombosis of the iliac artery, common femoral artery, or infrainguinal bypass grafts. Reteplase was infused at a rate of either 0.5 U/h (six patients) or 1.0 U/h (nine patients). RESULTS: Complete thrombolysis was achieved in all of the nine (100%) patients in the 1.0 U/h group and in four of six (66%) patients in the 0.5 U/h group for a combined success rate of 13 of 15 (87%). Clinical success was achieved in 11 of 15 patients overall (73%). Major bleeding complications occurred in none of the 9 patients in the 1.0 U/h group and in one (16%) of the six patients in the 0.5 U/h group for an overall rate of 6%. CONCLUSIONS: Reteplase shows promise as an alternative to urokinase in the treatment of lower extremity ischemia due to arterial occlusive disease. PMID- 10735422 TI - Complications associated with the use of urokinase and recombinant tissue plasminogen activator for catheter-directed peripheral arterial and venous thrombolysis. AB - PURPOSE: Catheter-directed thrombolytic dissolution of peripheral arterial and venous thrombus is in widespread use, yet the frequency and nature of associated complications remain ill defined. In an effort to better characterize the complications associated with urokinase (UK) and recombinant tissue plasminogen activator (rt-PA), the clinical course of patients treated for lower extremity vascular occlusions at a single institution was reviewed. MATERIALS AND METHODS: Over a 9-year period, 653 consecutive patients were treated for lower extremity arterial (527 patients) or venous (126 patients) occlusions with catheter directed UK (483 patients), rt-PA (144 patients), or both (26 patients). Decisions regarding the choice of thrombolytic agent were made by the clinician. In-hospital complications were subcategorized into hemorrhagic and nonhemorrhagic events and the rate of intracranial hemorrhage was specifically tabulated. RESULTS: There were no significant differences in the demographics or clinical presentation of patients treated with either UK or rt-PA. Bleeding complications occurred less often in the patients treated with UK (insertion site hematoma 21.9% vs. 43.8%, P<.0001, any bleeding necessitating transfusion 12.4% vs. 22.2%, P = .004, and intracranial hemorrhage 0.6% vs. 2.8%, P = .031). Cardiopulmonary complications necessitating transfer to the intensive care unit occurred more frequently in the patients treated with rt-PA (4.9% vs. 1.5%, P = .015). The risk of mortality was not statistically different between the UK and rt-PA treated patients (2.7% vs. 6.2%, P = .221). CONCLUSIONS: Thrombolysis appears safer with UK than with rt-PA, with a lower incidence of hemorrhagic complications. It is possible that this finding is related to differential dosing regimens or intrinsic pharmacologic differences between the agents. The observations of this retrospective analysis require confirmation with a prospective, randomized evaluation. PMID- 10735423 TI - Augmented experimental pulse-spray thrombolysis with tissue plasminogen activator, enabling dose reduction by one or more orders of magnitude. AB - PURPOSE: To critically evaluate and optimize methodologic details of pulse-spray thrombolysis with tissue plasminogen activator (tPA) in a subacute rabbit inferior vena cava thrombosis model. MATERIALS AND METHODS: Occlusive inferior vena cava thrombi were produced in 104 rabbits and 2 days later were treated for 1 hour with pulse-spray thrombolysis using tPA. Methodologic variables included pulse frequency, concentration and amount of tPA, infusion versus pulse therapy, and admixture of heparin. After the rabbits were killed, residual thrombus was weighed. RESULTS: The authors' earlier standard regimen (3 mg of tPA in 6 mL of heparinized saline, 0.2-mL hand pulses, frequency 1 pulse per 2 minutes) produced 60% mean lysis. Optimization involved increasing the pulse frequency to two per minute and decreasing tPA concentration by 98% to 0.01 mg/mL, modifications that produced 22% more thrombolysis, despite 92% reduction in amount of tPA to 0.25 mg. CONCLUSION: Consistent with the in vitro work of other investigators, a roughly bell-shaped dose-response curve was elicited in vivo for pulse-spray with tPA. By diluting tPA to an optimal level, and increasing pulse frequency to two per minute, thrombolysis was markedly augmented. These results suggest that the conventional dose of tPA used for clinical pulse-spray thrombolysis can be reduced by one to two orders of magnitude, possibly markedly reducing procedural risk. PMID- 10735424 TI - Outpatient uterine artery embolization for symptomatic uterine fibroids: experience in 49 patients. AB - PURPOSE: To assess the feasibility of performing uterine artery embolization as an outpatient treatment for symptomatic uterine fibroids. MATERIALS AND METHODS: Forty-nine consecutive patients (mean age, 44.5 years; range, 28-54 years) underwent uterine artery embolization during a 12-month period. Embolization was performed with 350-500 microm polyvinyl alcohol particles (44 of 49) or Gelfoam pledgets (five of 49). At discharge, patients were given instructions regarding the constitutional symptoms to expect after embolization. A specific medication regimen consisting of prochlorperazine, ketorolac, meperidine, and hydrocodone was prescribed for relief of these symptoms. All patients were telephoned within 24 hours of discharge. During long-term follow-up, a questionnaire was administered to all patients to evaluate the periprocedural experience. Three month clinical follow-up was available in 26 patients and 6-month imaging follow up was available in 16 patients. RESULTS: Fourteen patients presented with menorrhagia, six had bulk-related symptoms (abdominal distension, stress incontinence, pelvic pain), and 29 had both. Technical success for bilateral embolization was 98%. Forty-seven of 49 patients were discharged to home 6-8 hours after the procedure; two patients required overnight observation in an ambulatory unit (one because of postprocedure hypertension and one because of a late procedure completion time). At the first follow-up phone call, reported symptoms included pelvic pain/cramping in 83.7% (41 of 49), fatigue in 75.5% (37 of 49), nausea/vomiting in 46.9% (23 of 49), and a nonpurulent vaginal discharge in 18.4% (nine of 49). These symptoms were satisfactorily controlled with discharge medications in 48 of 49 patients. No patients returned to the hospital or visited an emergency room during the first 48 hours after discharge. Forty-six of 49 patients were satisfied with the decision for home discharge. The average uterine volume reduction in 16 patients with 6-month imaging follow-up was 47.5%; 88.5%, of patients (23 of 26) with 3-month clinical follow-up reported improvement or elimination of symptoms. CONCLUSION: With defined telephone follow up, staff availability, and a protocol designed to alleviate the postprocedure constitutional symptoms, uterine artery embolization is both safe and effective when performed as an outpatient procedure. PMID- 10735426 TI - External beam irradiation as an adjunctive treatment in failing dialysis shunts. AB - PURPOSE: To evaluate the utility of low-dose irradiation as adjunctive treatment for failing dialysis shunts related to stenoses. MATERIALS AND METHODS: Thirty one patients with 41 lesions in their dialysis shunts were successfully enrolled for this study. After imaging of the shunt and calculation of venous stenoses, each patient was randomized into one of two segments of the protocol: (i) angioplasty and/or stent placement alone, and (ii) angioplasty and/or stent placement followed by external beam irradiation. All patients with significant venous stenoses (> or =50%) were treated with appropriately sized PTA (percutaneous transluminal angioplasty) and Wallstents. Patients randomized to the external irradiation segment underwent localized irradiation via a Theratron cobalt unit of 7 Gy 0-24 hours and 24-48 hours after intervention. Those patients randomized to the control group received no additional treatment. Clinical follow up included resumption of successful dialysis with appropriate hemodynamic parameters. Two follow-up shunt images were obtained, follow-up 1 (fu-1) from 90 to 179 days and follow-up 2 (fu-2) from 180 to 365 days. Percentages of significant recurrent stenoses, defined as greater than 50%, were recorded and re treated as needed. RESULTS: Sixteen of the 31 patients underwent external beam irradiation. There were 21 lesions in the test group that underwent irradiation after intervention, and 20 lesions were treated with intervention alone. There were seven native arteriovenous fistulas and 24 Gore-tex grafts. All stenoses were either venous outflow stenoses (68%) or central stenoses (32%). The authors utilized chi2 analysis to compare restenoses rates between the control and irradiated groups at fu-1 (P<.99) and fu-2 (P<.10). CONCLUSIONS: Although the results show that external beam irradiation has minimal effects on the restenoses of dialysis grafts when used in conjunction with PTA and stent placement, further studies with a larger, more homogenous population are needed to assess the trend of improving patency rates after external beam irradiation. PMID- 10735425 TI - Neoadjuvant chemotherapy by transcatheter arterial infusion method for uterine cervical cancer. AB - PURPOSE: The effects of transcatheter intraarterial infusion of anticancer drugs on the prognosis of cervical cancer were retrospectively studied. MATERIALS AND METHODS: Two or three sessions of transcatheter arterial infusion therapy were performed in 97 patients with primary uterine cervical cancer. The number of patients in stage I, II, III, or IV were 15, 37, 34, and 11, respectively. The drugs infused were cisplatin (60-70 mg/m2), doxorubicin hydrochloride (30-40 mg/m2), mitomycin (15 mg/m2), and 5-fluorouracil (500 mg/body). They were infused via the bilateral internal iliac arteries. Seventy-five patients of the 97 (78%) underwent radical hysterectomy after arterial infusion: stage I (14 of 15), stage II (31 of 37), stage III (25 of 34), and stage IV (5 of 11). Three stage II and nine stage III patients received radical radiation therapy. The other 10 patients (one with stage I, three with stage II, and six with stage IV) did not receive an operation and radiation therapy after arterial infusion because of their distant metastases at the time of operation. Thirty-eight patients of 75 (51%) received postoperative radiation therapy. RESULTS: The complete histologic response rates (no active cancer cells) after arterial infusion were four of 14, four of 31, six of 25, and one of five for stages I, II, III, and IV. The histologic response rates, which showed no parametrial invasion after arterial infusion, were 12 of 14, 24 of 31, and 15 of 25 for stages I, II, and III. The histologic response rates, which demonstrated no lymph node metastases after arterial infusion, were 12 of 14, 24 of 31, and 19 of 25 for stages I, II, and III. The overall 5-year survival rates of the patients with stages I, II, and III were 93.3%, 58.7%, and 69.7%, respectively. The 5-year survival rates of the operated patients with stages I, II, and III were 100%, 60.5%, and 63.6%, respectively. The 5-year survival rates of the irradiated patients with stage III was 87.5%. Leukocytopenia and thrombocytopenia occurred as an acute complication in 75% and 79% of the patients, respectively. As a late complication, ileus occurred in 7%. CONCLUSION: Arterial infusion may improve the prognosis of patients with cervical cancer without increasing the incidence of late complications. PMID- 10735428 TI - Early failure of aortoiliac kissing stents: histopathologic correlation. PMID- 10735427 TI - Outpatient percutaneous transluminal renal artery angioplasty: a Canadian experience. AB - PURPOSE: The authors performed a retrospective study of their experience and complication rate while performing outpatient percutaneous transluminal renal artery angioplasty (PTRA) during a 5-year period. MATERIALS AND METHODS: From July 1992 to July 1997, 87 PTRAs were performed. Of these, 62 PTRA procedures were performed on 53 outpatients. In total, 66 arteries were dilated in 62 PTRA sessions using standard, established techniques. During the same period, only 25 PTRAs were performed on inpatients. Angioplasties were performed on those patients with demonstrated renal artery stenosis and poorly controlled hypertension and/or renal failure. Patients chosen for PTRA were picked by a team that included a vascular surgeon, a nephrologist, and a radiologist. Patients who were deemed suitable for an outpatient procedure were recommended by a nephrologist. Radiological input was sought at that time. Specific guidelines were used to select these patients who were otherwise healthy, well-orientated, and able to respond to an emergency situation. None of the subjects had significant risk factors. All were accompanied by an adult for the first 24 hours and all lived no more than an hour's travel time from a hospital. All were stable on discharge and were seen within 24 hours by a nephrologist. RESULTS: The technical success rate, defined as a residual stenosis on imaging of less than 30% and/or by a pressure gradient of less than 10 mm Hg across the stenosis, was 85%. The early complication rate was 5.6%, including two patients who developed a localized hematoma. In all, four patients were admitted to the hospital rather than being discharged to home after an average of 4.2 hours of observation. The late complication rate was 3% and involved two patients. One patient, who reported pain after balloon deflation, was readmitted 6 hours after discharge with hypotension, and a diagnosis of renal artery rupture was confirmed with computed tomography. Another patient developed peripheral atheroemboli 20 days after the procedure. CONCLUSION: Outpatient PTRA can be performed on selected patients. In this study, late complications occurred in only 3% of patients. Early complications were readily recognized in 5.6% of patients, and these patients were admitted for observation after the procedure. PMID- 10735429 TI - Bilateral renal artery stent infection and pseudoaneurysm formation. PMID- 10735430 TI - Nitinol properties affecting uses in interventional radiology. PMID- 10735431 TI - Relative ultrasonographic echogenicity of standard, dimpled, and polymeric-coated needles. AB - PURPOSE: To use quantitative ultrasonographic measurements to compare the effect of a polymeric coating designed to increase needle echogenicity to commercially available needles. MATERIALS AND METHODS: Commercially available standard smooth and dimpled echogenic tip 21-gauge needles established reference levels of echogenicity in gelatin-based and turkey breast phantoms. Examples of both types of needles were coated with a thin polymeric film that utilizes entrapped microbubbles of air on its surface to increase echogenicity. Samples of each type in both coated and noncoated versions were placed in phantoms in matched positions and imaged with clinical ultrasound machines. Similar numbers of each category were evaluated at various angles of insonation for a total of 109 images. Similar numbers of each category were imaged at 5-minute intervals for up to 38 minutes for a total of 226 images. Images were recorded, digitized, and evaluated for relative echo strength in arbitrary echogenic brightness units. RESULTS: Coating increased peak echogenicity over the entire needle to a level that closely approximates the peak echogenicity of dimpled needle tips (means: dimpled = 834, coated smooth = 803, coated dimpled = 823; P = .54). Smooth is lower than this group at 468 (P = .0001). Representative area echogenicity increased with coating or dimpling (smooth = 377 vs coated smooth = 778, coated dimpled = 690, dimpled = 775; P = .0001). Coating increased peak values 74% and area values 95% compared to smooth. Decreased angles of insonation moderately reduced echogenicity on coated smooth, coated dimpled, and dimpled, while it decreased to below good visibility threshold on standard smooth needles. The echogenicity of the coated needles fades in saline with time (1%/min). CONCLUSION: Objective measurements show that this coating significantly increases echogenicity of entire needles to match that obtained with a dimpled tip. PMID- 10735432 TI - CT fluoroscopy for lung nodule biopsy: a new device for needle placement and a phantom study. PMID- 10735433 TI - Transabdominal ultrasound guidance for transvenous biopsy of focal hepatic masses. PMID- 10735434 TI - Percutaneous aspiration of an intradural/extradural thoracic arachnoid cyst: use of MR imaging guidance. PMID- 10735435 TI - MR-guided angioplasty of renal artery stenosis in a pig model: a feasibility study. AB - PURPOSE: To test the hypothesis that magnetic resonance (MR) imaging can guide the percutaneous treatment of renal artery stenosis in a pig model. MATERIALS AND METHODS: Ameroid constrictors were surgically placed around six renal arteries in four pigs. After 30-36 days, all stenoses were documented by conventional x-ray aortograms. MR-guided renal angioplasty was attempted for three stenoses. For these pigs, MR angiography was performed with use of contrast-enhanced three dimensional (3D) techniques. The authors visualized catheters by filling them with dilute 4% gadolinium and imaging with two-dimensional (2D) and 3D MR fast spoiled gradient recalled echo techniques. Under MR guidance, the authors advanced a selective catheter into the affected renal artery and crossed the stenosis with a nitinol guide wire. Angioplasty was performed with a balloon catheter filled with dilute gadolinium. Stenosis and luminal diameter measurements were compared before and after angioplasty. RESULTS: After ameroid constrictor placement, four significant stenoses, one mild stenosis, and one occlusion developed. Under MR guidance, the authors achieved technical success in performing three of three (100%) attempted dilations. After MR-guided angioplasty, the mean reduction in stenosis was 35% and the mean increase in luminal diameter was 1.6 mm. CONCLUSION: Use of MR guidance for the angioplasty of renal artery stenosis in pigs is feasible. PMID- 10735436 TI - Percutaneous transmyocardial intracardiac retroperfusion shunts: technical feasibility in a canine model. AB - PURPOSE: To test the technical feasibility of creating a left ventricle to coronary sinus shunt using endovascular techniques. MATERIALS AND METHODS: By means of a right jugular vein approach, a needle puncture was made from the coronary sinus to the left ventricle in 10 dogs. The tracts were balloon dilated and lined with 6-mm Wallstents. Shunt patencies, immediate and 4-hours later, were fluoroscopically assessed by contrast material injection into the left ventricle. Blood pressure, pulse, oxygen saturation, and cardiac rhythm were monitored. The dogs were then euthanized. Thoracic cavities and hearts were dissected and inspected. RESULTS: Technical success and immediate shunt patency were 100%. No cardiac dysrhythmias, electrocardiographic changes, or reduction in voltage potential were seen. Eight (80%) of the shunts were patent at 4 hours, one (10%) had thrombosed, and one dog died. Nine (90%) dogs had no pericardial hematoma and one (10%) had minimal pericardial blood from needle passes into the pericardial sac. The coronary sinuses were intact and no injuries to the valve leaflets or chordae tendineae were seen. The puncture sites were from the coronary sinus, 1-2 mm (mean, 1.3) from its auricular orifice, into the left ventricle, just below the inferior margin of the posterior leaflet of the mitral valve. One dog died at 3 hours with no preceding electrocardiographic evidence of impending demise. Autopsy showed no pericardial hematoma and the heart findings were no different from the other nine dogs. CONCLUSIONS: Creation of a left ventricle to coronary sinus shunt with use of endovascular techniques is technically feasible. Study of a transmyocardial intracardiac coronary retroperfusion shunt to deliver oxygenated blood to the ischemic myocardium is warranted. PMID- 10735437 TI - Reporting standards for central venous access. Technology Assessment Committee. PMID- 10735438 TI - Neutron powder diffraction study of orthorhombic and monoclinic defective silicalite AB - The crystal structure of silicalite (SiO2) with a substantial amount of structural hydroxyl groups [Si(1-x)O(2-4x)(OH)4x, with 0.0855%, N= 96) the mean difference between venous RST and plasma glucose was significantly more than the mean difference between capillary RST and plasma glucose (0.018 versus 0.295. P = 0.002). Hence the higher the haematocrit the more inaccurate the venous RST. This study confirms the limited value of reagent strip tests in the assessment of blood glucose in the neonatal period. It suggests that venous RST may be more inaccurate in comparison to capillary and that high haematocrits have a greater effect on venous RST than capillary RST. PMID- 10735459 TI - The role of cortical evoked responses in predicting neuromotor outcome in very preterm infants. AB - To explore the role of posterior tibial somatosensory evoked potentials (SSEP) and flash visual evoked potentials (VEP) in the early prediction of functional brain injury, 93 infants of less than 32 weeks gestation at birth were studied. Neuromotor outcome was defined by clinical examination at the age of 2 years. SSEP, VEP and cerebral ultrasound were compared as predictors of neuromotor outcome. Posterior tibial SSEP were the most accurate single method of predicting neuromotor abnormalities with a 83% positive predictive power for cerebral palsy and a 100% predictive power for abnormal neurology, compared to 17% and 40% for cerebral ultrasound and 38% and 85% for VEP respectively. When SSEP and VEP results were concordant, the positive predictive power for cerebral palsy and abnormal neurology was 100%. Our results suggest that posterior tibial SSEP and VEP are accurate tools in the prediction of outcome in very preterm infants. PMID- 10735460 TI - Maternal cigarette smoking during pregnancy, low birth weight and subsequent blood pressure in early childhood. AB - Given the widely acknowledged inverse relationship between birth weight and blood pressure, a raised blood pressure in the offspring of smoking mothers as compared to those whose mothers did not smoke, would be anticipated by virtue of the reduction in birth weight associated with smoking during pregnancy. The objective of the present study was to test the hypothesis that maternal cigarette smoking during pregnancy has an effect on blood pressure in childhood independent of its effect on birth weight. Data was obtained from a prospective cohort study of 1708 pregnant women and their singleton offspring, delivered live at term, in Perth, Western Australia, commenced at 16 weeks gestation with serial blood pressure measurements through early childhood. Statistically significant associations were found between maternal smoking during pregnancy and systolic blood pressure at age six, between birth weight and systolic blood pressure at ages three and six, and between maternal smoking during pregnancy and birth weight. The relationship between birth weight and blood pressure in early childhood differed significantly on the basis of maternal cigarette smoking or not during pregnancy. This differential relationship persisted after adjustment for the child's current weight and socio-economic status. We concluded that intra-uterine exposure to maternal cigarette smoking increased children's blood pressure at age one through to age six. This was not wholly attributable to an effect on birth weight or confounding of the association between birth weight and subsequent blood pressure by the child's current weight or socio-economic factors. Furthermore, maternal smoking during pregnancy does not account for the acknowledged elevation in blood pressure associated with low birth weight. The present study is an exploration of a possible causal pathway underlying the birth weight/blood pressure association rather than simply a confirmation of such an association which has been detailed in many other papers. PMID- 10735461 TI - Maximal airway pressures during crying in healthy preterm and term neonates. AB - Respiratory muscle strength can be assessed by measurement of maximal inspiratory (PIMAX) and maximal expiratory pressure (P(EMAX)) during crying. There are, however, relatively few data on P(IMAX) and P(EMAX) in infancy, particularly from those born preterm. Our aim was to investigate which factors influenced P(IMAX) and P(EMAX) in preterm and term infants. Forty infants, median gestational age 37 weeks (range 26-43) and birthweight 2.579 kg (range 0.956-5.180) were studied at a postconceptional age (PCA) of 38 weeks (range 32-44). None had respiratory problems. A facemask was placed firmly over the infant's mouth and nose and the infant studied during spontaneous crying. A pneumotachograph fitted snugly into the facemask and from a sideport airway pressure changes were measured. During crying, the distal end of the pneumotachograph was occluded for five breaths and at least three separate occlusions were made. The highest P(EMAX) value sustained for at least 1 s and the highest peak inspiratory pressure P(IMAX) were recorded. The mean P(IMAX) and P(EMAX) were higher in the term compared to the preterm infants (70 cmH2O +/-S.D. 19 versus 58 cmH2O +/-S.D. 17 P(IMAX) and 53 cmH2O +/ S.D. 13 versus 44 cmH2O +/-S.D. 19 P(EMAX), P< 0.05). Both P(IMAX) and P(EMAX) related significantly with postconceptional age, gestational age and weight, but not postnatal age. Stepwise regression analysis demonstrated P(IMAX) related independently with PCA and P(EMAX) with weight. These results suggest respiratory muscle strength is influenced by maturation at birth. PMID- 10735462 TI - The haemodynamic effects of dopamine and volume expansion in sick preterm infants. AB - AIM: To determine the haemodynamic effects of dopamine and volume expansion in preterm neonates. Effect parameters were mean arterial blood pressure (MABP), left ventricular output (LVO) and global cerebral blood flow (CBF). METHODS: In a randomised, clinical control trial 36 preterm neonates were randomised to receive either dopamine 5 microg/kg per min, volume expansion with albumin 20% 15 ml/kg or no treatment. Parameters were measured before and 2 h after initiation of treatment. RESULTS: Dopamine was effective in increasing MABP; both treatments increased LVO, whereas no significant difference between the treatment groups and the control group could be demonstrated with regard to CBF. CONCLUSION: No effect on global cerebral blood flow could be demonstrated in this study, despite significant effects on systemic circulatory parameters. However, the variance on the measurement of cerebral blood flow indicates that a small but clinically significant effect may have been overlooked. PMID- 10735463 TI - Effects of complexation between liposome and poly(malic acid) on aggregation and leakage behaviour. AB - The design and development of novel pH-sensitive liposomes were investigated to improve the release of liposome-encapsulated chemicals. Stable liposomes comprising of L-alpha-dipalmitoylphosphatidylcholine (DPPC) and poly(carboxylic acid) were prepared and characterized. Poly(malic acid) (PMLA) was chosen as a fusogen, because of its excellent biodegradability in physiological regions. Octyl groups introduced in the poly(malic acid) worked as anchors at the surface of the liposomes and made a remarkable contribution to complexing. The interaction between the liposomes and the polyacids was studied in terms of the change in size of the liposomes. The influences of molecular weight and amounts of polymer upon their characteristics, especially fusion, were discussed. The influences of pH change with respect to the association behavior of the liposomes such as aggregation and fusion were estimated by the particle size of the liposomes, turbidimetry of the solution and resonance energy transfer assay. From the results of these studies, it was shown that more tightly complexed liposomes aggregated and fused more positively with increasing acidity of the solution. The leakage of calcein entrapped in the inner aqueous phase of the liposomes increased with decreasing pH. The effect of pH on the liposome aggregation in a solution qualitatively paralleled that found in the leakage behavior. PMID- 10735464 TI - Application of membrane-based dendrimer/DNA complexes for solid phase transfection in vitro and in vivo. AB - In this study a general description of the use of solid support membranes as the device for DNA delivery mediated by PAMAM dendrimers is presented. In contrast to the other DNA carriers, dendrimer/DNA complexes retain the ability to transfect after drying, which enabled coating or incorporation of complexes into poly(DL lactide-co-glycolide) or collagen-based bioerodable membranes. These studies provide support for the use of this technology for in vitro and in vivo transfection of skin cells. Expression of luciferase or green fluorescent protein from pCF1-Luc and pEGFP1 plasmids indicated that dendrimer/DNA complexes can mediate transfection after dissociation from the solid support and/or when retained on the surface of the membranes. Modification of the membranes by incorporation of an anionic lipid, phosphatidyl glycerol (PG) at 1-5% concentrations, resulted in more efficient in situ transfection, particularly with dendrimer/DNA complexes formed at the low charge ratios (1-5). We also report data supporting the feasibility of membrane-based dendrimer/DNA complexes, particularly formed at lower than neutralizing conditions, for topical in vivo delivery of DNA to hairless mouse skin. PMID- 10735466 TI - Structural aspects of fish skin collagen which forms ordered arrays via liquid crystalline states. AB - The ability of acid-soluble type I collagen extracts from Soleidae flat fish to form ordered arrays in condensed phases has been compared with data for calf skin collagen. Liquid crystalline assemblies in vitro are optimized by preliminary treatment of the molecular population with ultrasounds. This treatment requires the stability of the fish collagen triple helicity to be controlled by X-ray diffraction and differential scanning calorimetry and the effect of sonication to be evaluated by viscosity measurements and gel electrophoresis. The collagen solution in concentrations of at least 40 mg ml(-1) showed in polarized light microscopy birefringent patterns typical of precholesteric phases indicating long range order within the fluid collagen phase. Ultrastructural data, obtained after stabilization of the liquid crystalline collagen into a gelated matrix, showed that neutralized acid-soluble fish collagen forms cross-striated fibrils, typical of type I collagen, following sine wave-like undulations in precholesteric domains. These ordered geometries, approximating in vivo situations, give interesting mechanical properties to the material. PMID- 10735465 TI - Biodegradation behavior of ultra-high-strength hydroxyapatite/poly (L-lactide) composite rods for internal fixation of bone fractures. AB - The purpose of this study was to investigate the biodegradation behavior of the ultra-high-strength hydroxyapatite/poly(L-lactide) (HA/PLLA) composite rods for fracture repair. Two kinds of composite materials were used in this study: u HA/PLLA. which contained 30% by weight of uncalcined HA as reinforcing particles, and c-HA/PLLA, which contained 30% by weight of calcined HA as reinforcing particles. These composite rods were implanted in the subcutis and in the medullary cavities of rabbits. The specimens were removed at specific intervals between 2 and 52 weeks and the mechanical strength was measured for the rods in the subcutis, and the molecular weight and crystallinity were measured for the rods in both the subcutis and medullary cavities. The rod surfaces were examined using a scanning electron microscope (SEM). The specimens were examined histologically by light microscopy. The bending strength of the composites implanted in the subcutis was maintained at more than 200 M Pa at 25 weeks and at 150 MPa at 52 weeks. The molecular weight dropped to 45% of the initial values at 8 weeks and to approximately 10% at 52 weeks. Significant differences in the molecular weight were seen between c-HA/PLLA and u-HA/PLLA, with u-HA/PLLA showing a faster rate of decrease than c-HA/PLLA after 8 weeks. SEM demonstrated that HA particles disappeared increasingly from the rod surfaces over time and that the spaces left by these HA particles formed many pores in the composite surfaces at 52 weeks. Histologically, a fibrous tissue layer was formed around the composite rod from 4 weeks in the subcutis and in the diaphyseal area of the medullary canal. This became more mature over time. Bony tissue contact to the composites without fibrous tissue layers was seen in the metaphyseal area of the medullary canal. During the experimental period, there were no inflammatory cells such as mono- or multi-nuclear phagocytes. Although further long-term studies for degradation are needed, the composites have promising mechanical strength and no adverse tissue reaction for use as fracture-fixation devices during the experimental periods. PMID- 10735467 TI - The in-vivo wear performance of prosthetic femoral heads with titanium nitride coating. AB - This paper reports the study performed on four titanium nitride (TiN) coated prosthetic femoral heads collected at revision surgery together with patient data. Surface topology has been examined using Scanning Electron Microscopy (SEM) and elemental analysis of both coating and substrate have been evaluated using energy-dispersive X-ray spectrometry. Quantitative assessment of the surface topography is achieved using contacting profilometry. The average Ra roughness value is calculated at five different locations for each femoral head. The UHMWPE counterface worn volume has been measured directly on the acetabular components. TiN fretting and coating breakthrough occurred in two of the four components examined. In the damaged coating areas the surface profile is macroscopically saw toothed with average tooth height 1.5 microm. The average Ra value is 0.02 microm on the undamaged surfaces and 0.37 microm on the damaged ones. Failure of the coating adhesion resulted in the release of TiN fragments and of metallic particulate from the substrate fretting corrosion and in the increase of the head surface roughness affecting counterface debris production. Our results suggest that TiN-coated titanium alloy femoral heads are inadequate in the task of resisting third body wear mechanisms in vivo. PMID- 10735468 TI - Controlled release of cyclosporine from VP-HEMA copolymer systems of adjustable resorption monitorized by MEKC. AB - Soluble, uncrosslinked and high molecular weight copolymers of vinylpyrrolidone, VP, with 2-hydroxyethyl methacrylate, HEMA, prepared by free radical copolymerization, are proposed as supports for the modulated release of drugs, taking cyclosporine as a model system. The copolymerization parameters described as reactivity ratios, rVP = 0.08 and rHEMA = 7.97, indicate that the copolymer systems prepared at high conversion have two main components with a microstructural arrangement which depends on the average composition, i.e., an initial HEMA-rich copolymer and a final PVP homopolymer or VP-rich copolymer. This microstructural distribution controls the resorption rate of the polymeric support and therefore the release process of cyclosporine which is demonstrated experimentally by the application of a modern technique known as micellar electrokinetic capillary chromatography (MEKC). PMID- 10735469 TI - Temperature-dependent modulation of blood platelet movement and morphology on poly(N-isopropylacrylamide)-grafted surfaces. AB - Poly(N-isopropylacrylamide) (PIPAAm) exhibits a reversible, temperature-dependent soluble/insoluble transition at its lower critical solution temperature (LCST) of 32 degrees C in aqueous media. The temperature-responsive PIPAAm was grafted onto tissue culture polystyrene (TCPS) dish surfaces by electron beam irradiation. Blood platelet behaviors on PIPAAm-grafted surface were examined by computerized image analysis and scanning electron microscopy. Platelet behaviors on this surface were dramatically dependent upon temperature, but those on poly(ethylene glycol)(PEG)-grafted or polystyrene remained unchanged. Below the 32 degrees C (LCST), platelets on PIPAAm-grafted surfaces retained a rounded shape and an oscillating vibratory microbrownian motion for extended times, similarly to those on PEG-grafted surfaces. Above the LCST, platelets readily adhered, spread and developed characteristic pseudopodia on PIPAAm-grafted surface similarly to those on TCPS. An ATP synthesis inhibitor failed to hinder prevention of platelet adhesion onto PIPAAm-grafted surface (below the LCST) suggesting that the preventive mechanism is ATP-independent similarly to that of PEG-grafted surfaces. These results correlate platelet surface activation state with the hydration and structure of polymer surfaces, and demonstrate the ability to modulate such reactions by a small temperature change in situ. PMID- 10735470 TI - The degradation, swelling and erosion properties of biodegradable implants prepared by extrusion or compression moulding of poly(lactide-co-glycolide) and ABA triblock copolymers. AB - In the design of parenteral delivery systems the modulation of the biodegradation of a polymer matrix represents a promising strategy to control drug release. We have investigated the degradation of ABA triblock copolymers, consisting of poly(lactide-co-glycolide) A-blocks and poly(oxyethylene) B-blocks, and PLG, poly(lactide-co-glycolide), with respect to swelling behaviour, molecular weight loss and polymer erosion. Implants were prepared by either compression moulding or extrusion using a laboratory ram extruder. Insertion of an elastoplastic B block did not lower the processing temperature, but the entanglement of the polymer chains was significantly reduced as can be seen from the diameters of the extruded rods. The swelling of the rods showed a volume extension of 130% for an ABA containing 50% PEO and 20% for an ABA containing 20% PEO. Using 1H-NMR it was found that protons in the B-blocks of the swollen ABA copolymers were mobile, while the A-blocks remained rigid during incubation. The analysis of the pH inside ABA rods using electron paramagnetic resonance, EPR, gave a pH of 5.2 after incubation with a subsequent increase to pH 6.0 during the first day, approaching the pH of the medium after nearly 33 d. Acidic degradation products did not accumulate inside the ABA rods. Degradation and erosion started immediately upon incubation. By contrast, PLG rods showed the typical profile of degradation and erosion. In this case, the influence of the geometry of the device was insignificant. Consequently, ABA triblock copolymers may widen the spectrum of parenteral drug delivery with regard to release of pH-sensitive drugs as well as erosion-controlled release kinetics. PMID- 10735471 TI - The induction of micronuclei in V79 cells by the root canal filling material AH plus. AB - The in vitro micronucleus test (MNT) is an alternative to the chromosomal aberration assay for the detection of chromosomal mutations. Here, the epoxy resin-based root canal filling material AH Plus, the compounds paste A and paste B were tested for cytotoxicity and the induction of micronuclei in V79 cells. Paste A is the epoxy-resin-containing compound. AH Plus was tested immediately after mixing and after a setting time of 24 h. The materials were eluted in dimethyl sulfoxide (DMSO) and physiologic saline for 24 h. DMSO eluates of the mixed material, paste A and paste B clearly reduced the viability of V79 cells, but eluates prepared with physiological saline were at least 10-100-fold less toxic. Likewise, freshly mixed AH Plus was mutagenic in a dose-dependent manner in V79 cells after elution in dimethyl sulfoxide only. The numbers of micronuclei were about 7-fold higher in treated cell cultures compared with untreated controls. No mutagenicity was observed with DMSO and physiological saline eluates of mixed AH Plus set for 24 h. DMSO eluates of paste A induced effects similar to the freshly mixed AH Plus, and paste B was not effective. Therefore, we provide evidence for the induction of chromosomal mutations by freshly mixed AH Plus under experimental conditions. PMID- 10735472 TI - Sterilization, storage stability and in vivo biocompatibility of poly(trimethylene carbonate)/poly(adipic anhydride) blends. AB - Biodegradable blends of poly(trimethylene carbonate) (PTMC) and poly(adipic anhydride) (PAA) have been proven to be strong candidates for controlled drug delivery polymers in vitro. We now report on the stability, sterilizability and in vivo local tissue response of these matrices. Blend matrices were sterilized by beta-radiation or ethylene oxide gas treatment, stored at different times and temperatures, and analyzed for changes in physicochemical properties. Moisture uptake at different relative humidities and storage times was determined. Sterilization procedures induced hydrolysis of the matrices. Ethylene oxide gas sterilization had a significantly more marked effect upon the matrix properties than radiation treatment. The onset of degradation was reflected in a decrease of crystallinity and molecular weight along with a change of blend composition. A similar onset of matrix degradation was observed upon storage in air. The physicochemical properties of the blends were well preserved upon storage under argon atmosphere. Biocompatibility of PTMC/PAA implants was assessed in the anterior chamber of rabbits eyes for 1 month. At selected post-operative time points, aqueous humor was analyzed for white blood cells and the corneal thickness was measured. The results suggest good biocompatability of PTMC-rich matrices, whereas fast eroding PAA-rich matrices caused inflammatory responses, due to a burst release of degradation products. PMID- 10735473 TI - Characterization of protein-resistant dextran monolayers. AB - A range of synthetic thiolated dextrans of varying molecular weights and degrees of thiol substitution have been investigated as well-defined monolayer coatings for the reduction of nonspecific protein adsorption. Atomic force microscopy and surface plasmon resonance (SPR) analysis revealed that the surface coverage of the dextran monolayers increased with an increasing degree of thiol substitution, but conversely decreased with increasing molecular weight. SPR was then employed to monitor bovine serum albumin protein adsorption to thiolated dextran monolayers from a flowing buffered solution. Whilst a significant reduction of protein adsorption to a thiolated dextran layer coated surface compared to an uncoated surface was observed, the degree of conversion of hydroxyls to thiol groups and molecular weight was shown to affect the protein-resistant performance of the dextran layer. PMID- 10735474 TI - Mechanical properties of calcium phosphate coatings deposited by laser ablation. AB - Amorphous calcium phosphate and crystalline hydroxyapatite coatings with different morphologies were deposited onto Ti-6Al-4V substrates by means of the laser ablation technique. The strength of adhesion of the coatings to the substrate and their mode of fracture were evaluated through the scratch test technique and scanning electron microscopy. The effect of wet immersion on the adhesion was also assessed. The mechanisms of failure and the critical load of delamination differ significantly depending on the phase and structure of the coatings. The HA coatings with granular morphology have higher resistance to delamination as compared to HA coatings with columnar morphology. This fact has been related to the absence of stresses for the granular morphology. PMID- 10735475 TI - Bisoprolol prevents mortality and myocardial infarction after vascular surgery. PMID- 10735476 TI - Caffeine consumption and the risk of spontaneous abortions. PMID- 10735477 TI - Finger-stick vs. laboratory serological testing for H. pylori antibody. PMID- 10735478 TI - Primary care research: revisiting its definition and rationale. PMID- 10735479 TI - Two physician styles of focusing on the family. AB - BACKGROUND: Previous research has identified 2 styles of family physicians' focus on the patient's family: (1) using the family history as the context of care of the patient; and (2) maintaining a family orientation with the family as the unit of care. The purpose of our study was to determine whether these styles affect patient outcomes and time use during outpatient visits. METHODS: In a cross sectional study, data on 4454 outpatient visits to 138 community family physicians were collected using direct observation, patient and physician questionnaires, and medical record review. We computed partial correlations between the physician's family practice style score and patient outcomes for delivery of preventive services, patient visit satisfaction, and patient-reported delivery of specific components of primary care. We controlled for relevant patient characteristics. RESULTS: The patients of the physicians using either practice style had similar levels of satisfaction with coordination of care and interpersonal communication, and their value of continuity of care was comparable. Patients of physicians with a family-history style, however, rated their physicians lower on a measure of in-depth knowledge of the patient and family but higher on preventive services delivery. Differences in time use during the visit reflected how these styles were manifested during the outpatient visit. CONCLUSIONS: The different styles physicians use to focus on the family affect the process and outcomes of patient care. This difference may be explained by the developmental life cycle of family physicians, as younger physicians may be more focused on family history and older physicians may have a more family-oriented focus. Physicians may need to find alternate ways of meeting those patient needs not well met by their predominant practice style. PMID- 10735480 TI - Herbs, prayer, and insulin. Use of medical and alternative treatments by a group of Mexican American diabetes patients. AB - BACKGROUND: Clinicians are often concerned that use of alternative treatments by Mexican American patients with diabetes competes with medical treatment. We examined the use and evaluation of alternative treatments for diabetes by a sample of these patients. METHODS: Following a descriptive qualitative design, a convenience sample of 43 low-income Mexican Americans with type 2 diabetes were interviewed. We analyzed interview transcripts for alternative treatments named, patterns of use, evaluation of those treatments, and the use of biomedical approaches. We crosschecked the results for interrater reliability. RESULTS: Herbs were mentioned as possible alternative treatments for diabetes by 84% of the patients interviewed. However, most had never or rarely tried herbs and viewed them as supplemental to medical treatments. Most said prayer influences health by reducing stress and bringing healing power to medicines. None used curanderos (traditional healers) for diabetes. Most actively used biomedical treatments and were less actively involved in alternative approaches. Statistical tests of association showed no competition between biomedical and alternative treatments, and alternative treatment activity tended to be significantly lower than biomedical. Most study participants emphasized medical treatment and only used alternative treatments as secondary strategies. Those patients very actively using alternative approaches also tended to be very actively using biomedical methods; they were using all resources they encountered. CONCLUSIONS: Traditional attitudes and beliefs were not especially important to the patients in this study and presented no barriers to medical care. For these patients, it also cannot be assumed that belief in alternative treatments and God's intervention indicate fatalism or noncompliance but instead require consideration of individual treatment behaviors. PMID- 10735481 TI - Does identifying a discharge as "against medical advice" confer legal protection? AB - BACKGROUND: One in every 65 to 120 discharges from general hospitals are against medical advice and have an expected increased risk of adverse consequences and subsequent litigation. Does the term "against medical advice" confer legal protection? METHODS: We searched the MEDLINE and PsychInfo databases for relevant articles. We also searched the national medico-legal databases of LEXIS-NEXIS. Additional case law was obtained through a search for "against medical advice" in the West Premise CD-ROM database of New York State cases. RESULTS: We found 8 relevant cases. There was no case in which "against medical advice" was entirely protective, though partial protection existed in some cases. CONCLUSIONS: Since patients are admitted voluntarily to a general hospital, a discharge against medical advice is merely a withdrawal of the original consent. PMID- 10735482 TI - The high prevalence of sexual concerns among women seeking routine gynecological care. AB - BACKGROUND: Sexuality is an important part of health, quality of life, and general wellbeing. Studies indicate that less than half of patients' sexual concerns are known by their physicians, and physicians are unaware of how common these sexual concerns are in their practices. Our objective was to determine the prevalence and type of sexual concerns among women seeking routine gynecological care. METHODS: We mailed the survey in waves. Of 1480 women seeking routine gynecological care from the departments of Family Practice and Obstetrics and Gynecology at Madigan Army Medical Center between August 1992 and January 1993, 964 responded. The main outcome measures were self-reported sexual concerns and their experiences with discussing these concerns with a physician. RESULTS: A A total of 98.8% of the women we surveyed reported one or more sexual concerns. The most frequently reported concerns were lack of interest (87.2%), difficulty with orgasm (83.3%), inadequate lubrication (74.7%), dyspareunia (71.7%), body image concerns (68.5%), unmet sexual needs (67.2%), and needing information about sexual issues (63.4%). More than half reported concerns about physical or sexual abuse, and more than 40% reported sexual coercion at some point in their lives. CONCLUSIONS: Our results suggest that sexual health concerns are prevalent for women seeking routine gynecological care. Sexual health inquiry should be a regular and important part of health care maintenance. PMID- 10735483 TI - Bridging the gap between conventional and alternative medicine. AB - BACKGROUND: The use of various forms of complementary and alternative medicine (CAM) has become widespread. We investigated this use in Madison, Wisconsin. METHODS: We conducted semistructured indepth interviews focused on the knowledge, attitudes, and behaviors of a random sample of 17 patients who had used both CAM and conventional therapies during the past year. Participants were recruited using telephone listings. Twenty alternative practitioners were selected to represent the major modalities. The topics discussed included healing philosophy, choices of therapeutic methods, and ideas concerning concurrent use of differing therapeutic modalities. An 8-member multidisciplinary team analyzed the transcripts individually and in group meetings. RESULTS: Four major themes emerged from the interview data: (1) holism, (2) empowerment, (3) access, and (4) legitimization. Both patients and providers distinguished between the socially legitimized and widely accessible but disempowering and mechanistic attributes of conventional medicine and the holistic and empowering but relatively less accessible and less legitimate nature of alternative healing. There was a strong call for integrating the best aspects of both. CONCLUSIONS: Practitioners and users of alternative therapies in the Madison area confirmed both the alternative and complementary natures of unconventional health care, called for more integrated and accessible health care, and provided insights that could be useful in bridging the gap between conventional and alternative medicine. PMID- 10735484 TI - Information in the palm of your hand. AB - Physicians need a way to access and organize personal, patient-related, and medical reference information. Hand-held computers are a practical solution that can make this information available at the point of care. We review the major types of hand-held computers and their software and recommend specific models based on physicians' needs. We also describe software for hand-held computers, with special attention to medical record and medical reference software. PMID- 10735485 TI - Does treatment of acute herpes zoster prevent or shorten postherpetic neuralgia? AB - OBJECTIVE: Our goal was to determine if any treatment of acute herpes zoster alters the incidence or duration of postherpetic neuralgia (PHN), a common sequela in elderly patients. SEARCH STRATEGY: We systematically searched MEDLINE and The Cochrane Library. We also examined the reference lists of identified trials and reviews. SELECTION CRITERIA: We included all randomized controlled trials of treatments of zoster published in English that included assessment of pain at any time after rash healing. DATA COLLECTION/ANALYSIS: Forty-two trials met inclusion criteria, and 2 reviewers independently evaluated them for methodologic quality and the statistical and clinical significance of results. MAIN RESULTS: Four placebo-controlled trials of oral acyclovir with 692 patients provided marginal evidence for reduction in pain incidence at 1 to 3 months following zoster onset. Famciclovir significantly reduced duration but not incidence of PHN in one placebo-controlled trial of 419 patients. Valacyclovir significantly reduced duration but not incidence of PHN in one acyclovir controlled trial of 1141 patients. Steroids had no effect on PHN. Amitriptyline for 90 days reduced pain incidence at 6 months in one placebo-controlled trial of 80 patients. A single trial of percutaneous electrical nerve stimulation (PENS) in 50 patients suggested a decrease in pain incidence at 3 and 6 months compared with famciclovir. CONCLUSIONS: There is limited evidence that current interventions prevent or shorten PHN. Famciclovir and valacyclovir have been shown to reduce the duration of PHN in single published trials. Well-designed and larger trials of amitriptyline and PENS should be conducted. PMID- 10735486 TI - Stool antigen immunoassay for detection of H. pylori infection. PMID- 10735487 TI - Screening mammography in women aged 70 to 79 years. PMID- 10735488 TI - Antihistamines for atopic dermatitis. PMID- 10735489 TI - Intra-arterial prourokinase effective for acute stroke therapy. PMID- 10735491 TI - Vitamin C prevents reflex sympathetic dystrophy. PMID- 10735492 TI - Proper preparation for flexible sigmoidoscopy. PMID- 10735493 TI - Intervention for information-giving. PMID- 10735494 TI - Hevin is down-regulated in many cancers and is a negative regulator of cell growth and proliferation. AB - We have cloned a human Hevin cDNA from omental adipose tissue of different patients by reverse transcription polymerase chain reaction and shown a sequence variation due to a possible polymorphism at amino acid position 161 (E/G). Hevin protein expressed in vitro showed molecular weights of approximately 75 kDa and 150 kDa, suggesting that Hevin may form a homodimer in vitro. Using Northern blots and a human expressed sequence tAg database analysis, Hevin was shown to be widely expressed in human normal or non-neoplastic diseased tissues with various levels. In contrast to this, its expression was strongly down-regulated in most neoplastic cells or tissues tested. However, neither the mechanism nor the physiological meaning of this down-regulation is known. As an initial step towards investigating the functional role of Hevin in cell growth and differentiation, we transiently or stably expressed this gene in cancer cells (HeLa 3S) that are devoid of endogenous Hevin and measured DNA synthesis (cell proliferation) by 5-bromo-2'-deoxyuridine incorporation. Hevin was shown to be a negative regulator of cell proliferation. Furthermore, we have shown that Hevin can inhibit progression of cells from G1 to S phase or prolong G1 phase. This is the first report which describes the function of Hevin in cell growth and proliferation. Through database analysis, Hevin was found to be located on chromosome 4 which contains loss of heterozygosity of many tumour suppressor genes. Taken together, these results suggest that Hevin may be a candidate for a tumour suppressor gene and a potential target for cancer diagnosis/therapy. PMID- 10735495 TI - Sphincter-sparing surgery after preoperative radiotherapy for low rectal cancers: feasibility, oncologic results and quality of life outcomes. AB - The present study assesses the choice of surgical procedure, oncologic results and quality of life (QOL) outcomes in a retrospective cohort of 53 patients with low-lying rectal cancers (within 6 cm of the anal verge) treated surgically following preoperative radiotherapy (RT, median dose 45Gy) with or without concomitant 5-fluorouracil. QOL was assessed in 23 patients by using two questionnaires developed by the QOL Study Group of the European Organization for Research and Treatment of Cancer: EORTC QLQ-C30 and EORTC QLQ-CR38. After a median interval of 29 days from completion of RT, abdominoperineal resection (APR) was performed in 29 patients (55%), low anterior resection in 23 patients (20 with coloanal anastomosis) and transrectal excision in one patient. The 3 year actuarial overall survival and locoregional control rates were 71.4% and 77.5% respectively, with no differences observed between patients operated by APR or restorative procedures. For all scales of EORTC QLQ-C30 and EORTC QLQ-CR38, no significant differences in median scores were observed between the two surgical groups. Although patients having had APR tended to report a lower body image score (P = 0.12) and more sexual dysfunction in male patients, all APR patients tended to report better physical function, future perspective and global QOL. In conclusion, sphincter-sparing surgery after preoperative RT seems to be feasible, in routine practice, in a significant proportion of low rectal cancers without compromising the oncologic results. However, prospective studies are mandatory to confirm this finding and to clarify the putative QOL advantages of sphincter conserving approaches. PMID- 10735496 TI - Interferon-gamma in the first-line therapy of ovarian cancer: a randomized phase III trial. AB - Intraperitoneal treatment with interferon-gamma (IFN-gamma) has been shown to achieve surgically documented responses in the second-line therapy of ovarian cancer. To assess its efficacy in the first-line therapy, we conducted a randomized controlled trial with 148 patients who had undergone primary surgery for FIGO stage Ic-Illc ovarian cancer. In the control arm women received 100 mg/m(-2) cisplatin and 600 mg/m(-2) cyclophosphamide, the experimental arm included the above regimen with IFN-gamma 0.1 mg subcutaneously on days 1, 3, 5, 15, 17 and 19 of each 28-day cycle. Progression-free survival at 3 years was improved from 38% in controls to 51% in the treatment group corresponding to median times to progression of 17 and 48 months (P= 0.031, relative risk of progression 0.48, confidence interval 0.28-0.82). Three-year overall survival was 58% and 74% accordingly (n.s., median not yet reached). Complete clinical responses were observed in 68% with IFN-gamma versus 56% in controls (n.s.). Toxicity was comparable in both groups except for a mild flu-like syndrome, experienced by most patients after administration of IFN-gamma. Thus, with acceptable toxicity, the inclusion of IFN-gamm in the first-line chemotherapy of ovarian cancer yielded a benefit in prolonging progression-free survival. PMID- 10735497 TI - The value of rapid functional assays of germline p53 status in LFS and LFL families. AB - We have tested two rapid assays of p53 function, namely the apoptotic assay and the FASAY as means of detecting germline p53 mutations in members of Li-Fraumeni and Li-Fraumeni-like families. Results of the functional assays have been compared with direct sequencing of all 11 exons of the p53 gene. The results show good agreement between the two functional assays and between them and sequencing. No false-positives or negatives were seen with either functional assay although the apoptotic assay gave one borderline result for an individual without a mutation. As an initial screen the apoptotic assay is not only rapid but inexpensive and very simple to perform. It would be expected to detect any germline defect that leads to loss of p53 function. The apoptotic assay could be ideal as a means of prescreening large numbers of samples and identifying those that require further investigation. The FASAY detects mutations in exons 4-10, is rapid and distinguishes between functionally important and silent mutations. PMID- 10735498 TI - Specifically regulated genes in malignant melanoma tissues identified by subtractive hybridization. AB - A polymerase chain reaction (PCR)-based subtractive hybridization technique was used to identify transformation-related genes in malignant melanoma. Melanoma biopsies were compared with tissues of benign melanocytic naevi and 549 gene fragments were screened using arrayed filters. Thirty-eight clones were confirmed to be differentially expressed representing 30 different genes (18 melanoma specific and 12 naevus-specific genes). To further confirm differential gene expression, Northern blot analyses with six of the 30 genes as probes were performed. All six were differentially expressed in benign and malignant melanocytic lesions, specifically dbpB/YB-1, 67-kDa laminin receptor, CAGH-3, 71 kDa heat shock protein and two unknown genes. The expression levels of these genes were then analysed in 50 different tissues to determine their overall expression profile. In conclusion, the technique of PCR-based subtractive hybridization in combination with arrayed filters allows detection of differences in gene expression even in tissues from which high-quality RNA is hard to isolate. The genes identified in this study are of interest because of their potential role in the pathogenesis of malignant melanoma. PMID- 10735499 TI - A randomized phase III study comparing dacarbazine, BCNU, cisplatin and tamoxifen with dacarbazine and interferon in advanced melanoma. AB - The purpose of this study was to compare the response rate, overall and 1-year survival in patients with advanced melanoma treated with a standard therapy, dacarbazine and interferon-alpha (DTIC/IFN), or combination chemotherapy, consisting of dacarbazine, BCNU, cisplatin and tamoxifen (DBCT). Treatment toxicity and time spent in hospital were secondary end points. One hundred and five patients (of whom 100 were eligible) were randomized to receive either DTIC/IFN or DBCT. The trial was designed to detect a 25% absolute difference in response rate or in 1-year survival with 80% power. There was no significant difference in response rate: this was 17.3% with DTIC/IFN and 26.4% with DBCT. Median overall survival was similar at 199 and 202 days respectively. One-year survival rate favoured standard treatment (30.6 vs 22.6%), but did not differ significantly between arms. DBCT was associated with significantly greater haematological toxicity, and a greater need for time spent in hospital (5.75 days/treatment cycle vs 2.29 with dacarbazine and interferon). DBCT combination therapy cannot be recommended as standard treatment for advanced melanoma. Dacarbazine remains the standard chemotherapy for this condition. PMID- 10735500 TI - c-erbB-4 protein expression in human breast cancer. AB - The Type 1 family of growth factor receptors includes epidermal growth factor receptor (EGFR), c-erbB-2, c-erbB-3 and c-erbB-4. Overexpression of the first two members is associated with poorer prognosis in patients with breast carcinoma. In this study we examined the expression of c-erbB-4 protein using the monoclonal antibody HFR-1. A total of 127 consecutive cases of primary operable invasive breast carcinoma presenting between 1975 and 1977 were studied. All patients were managed by simple mastectomy or conservation surgery with radiotherapy and no adjuvant therapy given. Long-term follow-up was maintained. Routine, formalin fixed, paraffin-embedded tumour samples were used and sections were stained immunohistochemically using the Duet StreptABC method. Immunoreactivity was classified using a simple semi-quantitative scoring method. Protein expression was generally low but definite positive cytoplasmic, membranous and nuclear reactivity was identified in 58%, 41% and 25% of cases respectively. Expression at all three sites demonstrated significant inverse associations were histological grade. In addition, membrane accentuation correlated inversely with the Nottingham Prognostic Index (NPI), while cytoplasmic reactivity showed a positive association with c-erbB-3 expression. No significant associations were found with disease-free interval or survival. The results of this study demonstrate that higher levels of c-erbB-4 protein expression are associated with a more differentiated histological phenotype in contrast to the other members of the Type 1 family. Larger series with extended follow-up will be required to ascertain definitively the prognostic value of c-erbB-4 expression in breast carcinoma. PMID- 10735501 TI - Reduced expression of transforming growth factor-beta receptor type III in high stage neuroblastomas. AB - Transforming growth factor beta (TGF-beta) is a powerful inhibitor of cell proliferation and a potent inducer of differentiation. Resistance to TGF-beta action is a characteristic of many malignancies and has been attributed to alterations of TGF-beta receptors as well as disturbance of downstream transduction pathways. To analyse the TGF-beta response in neuroblastoma, the expression of TGF-beta1 and TGF-beta type I, II and III receptor genes was investigated in 61 cancer samples by means of reverse transcription polymerase chain reaction. The specimens analysed belong to different stages, namely nine samples of stage 1, ten of stage 2, nine of stage 3 and 28 of stage 4. Moreover, five samples were of stage 4S, which represents a tumour form undergoing spontaneous regression. The results obtained show that TGF-beta1 and TGF-beta type I and II receptor genes appear to be almost equally expressed in neuroblastomas of all stages. Conversely, TGF-beta type III receptor gene expression, which is required for an efficacious TGF-beta binding and function, is strongly reduced exclusively in neuroblastomas of stages 3 and 4. These findings were directly confirmed by immunohistochemical analyses of ten neuroblastoma specimens. Our results suggest the occurrence of an altered TGF beta response in advanced neuroblastomas which might be an important mechanism for escaping growth control and for developing invasiveness. Moreover, our findings allow the proposal of a novel mechanism, namely down-regulation of TGF beta type III receptor gene expression, to avoid TGF-beta inhibitory activity. PMID- 10735503 TI - Mutational status of K-ras and TP53 genes in primary sarcomas of the heart. AB - We investigated three patients with cardiac angiosarcomas and two with cardiac rhabdomyosarcomas, all for mutations at exons 5, 6, 7 and 8 of the p53 gene and at exon 1 of K-ras. No point mutations were observed in the p53 gene in any of the five cases; however, at exon 1 of K-ras, three patients (60%) presented the same mutation at the first base of codon 13 (G to A transition). Interestingly, this mutation was detected in both rhabdomyosarcoma and angiosarcoma histologic sarcoma types. PMID- 10735502 TI - Pretreatment apoptosis in carcinoma of the cervix correlates with changes in tumour oxygenation during radiotherapy. AB - A relationship between hypoxia and apoptosis has been identified in vitro and in experimental tumours. The aim of this study was to investigate the relationship between apoptosis, hypoxia and the change in oxygenation during radiotherapy in human squamous cell carcinoma of the cervix. Forty-two patients with locally advanced disease underwent pretreatment evaluation of tumour oxygenation using an Eppendorf computerized microneedle electrode. Twenty-two of these patients also had a second evaluation of tumour oxygenation after receiving 40-45 Gy external beam radiotherapy. Paraffin-embedded histological sections were obtained from random pretreatment biopsies for all 42 patients. Apoptotic index (AI) was quantified by morphology on TUNEL stained sections. No correlation was found between pretreatment measures of AI and either the median pO2 (r = 0.12, P = 0.44) or percentage of values < 5 mmHg (r = -0.02, P = 0.89). A significant positive correlation was found between AI and the change in tumour oxygenation (ratio of pre:post-treatment % values < 5 mmHg) following radiotherapy (r = 0.61, P = 0.002). The lack of correlation between apoptosis and hypoxia may occur because the Eppendorf measures both acute and chronic hypoxia, and the relative ability of acute hypoxia to induce apoptosis is unknown. These results indicate that cell death via apoptosis may be a mechanism of tumour reoxygenation during radiotherapy. PMID- 10735504 TI - Placental site trophoblastic tumour: a rare but potentially curable cancer. AB - Placental site trophoblastic tumour (PSTT) is a rare form of gestational trophoblastic disease (GTD). We have conducted an analysis of all cases of PSTT managed at the Trophoblastic Disease Screening and Treatment Centre, Sheffield, from 1984 to 1996. During this time we received 4,988 registrations for GTD and managed seven cases of PSTT. A large range of interval between antecedent pregnancy and presentation was observed - the most common presenting symptoms being irregular vaginal bleeding with or without preceding amenorrhoea. Three out of seven patients had disease confined to the uterus at diagnosis and were successfully treated by hysterectomy alone. Two out of seven patients had pulmonary metastases in addition to uterine tumour and were treated with combination chemotherapy--both are alive and well. Of the remaining two patients one had distant and the other loco-regional metastases and both died despite numerous therapeutic interventions. PMID- 10735505 TI - Loss of Fhit expression in non-small-cell lung cancer: correlation with molecular genetic abnormalities and clinicopathological features. AB - The FHIT gene is located at a chromosomal site (3p14.2) which is commonly affected by translocations and deletions in human neoplasia. Although FHIT alterations at the DNA and RNA level are frequent in many types of tumours, the biological and clinical significance of these changes is not clear. In this study we aimed at correlating loss of Fhit protein expression with a large number of molecular genetic and clinical parameters in a well-characterized cohort of non small-cell lung cancers (NSCLCs). Paraffin sections of 99 non-small-cell carcinomas were reacted with an anti-Fhit polyclonal antibody in a standard immunohistochemical reaction. Abnormal cases were characterized by complete loss of cytoplasmic Fhit staining. The Fhit staining results were then correlated with previously obtained clinical and molecular data. Fifty-two of 99 tumours lacked cytoplasmic Fhit staining, with preserved reactivity in adjacent normal cells. Lack of Fhit staining correlated with: loss of heterozygosity (LOH) at the FHIT 3p14.2 locus, but not at other loci on 3p; squamous histology; LOH at 17p13 and 5q but not with LOH at multiple other suspected tumour suppressor gene loci; and was inversely correlated with codon 12 mutations in K-ras. Fhit expression was not correlated overall with a variety of clinical parameters including survival and was not associated with abnormalities of immunohistochemical expression of p53, RB, and p16. All of these findings are consistent with loss of Fhit protein expression being as frequent an abnormality in lung cancer pathogenesis as are p53 and p16 protein abnormalities and that such loss occurs independently of the commitment to the metastatic state and of most other molecular abnormalities. PMID- 10735506 TI - Correlation of metallothionein expression with apoptosis in nasopharyngeal carcinoma. AB - The expression of metallothionein (MT), an intracellular ubiquitous low molecular weight protein thiol with antioxidant properties, was studied in nasopharyngeal cancer (NPC) and correlated with the apoptotic index. Immunohistochemical staining of randomly selected, formalin-fixed and paraffin-embedded normal and malignant nasopharyngeal tissues were analysed for the expression of MT using the commercially available E9 antibody directed against MT I and MT II isoforms. The corresponding apoptosis labelling indices were evaluated by the TUNEL method. Localization of MT at the ultrastructural level was studied by immunogold labelling. All the tumour sections (17 specimens) showed MT-immunopositivity. A direct correlation between the percentage of MT-positive cells and the staining intensity was noted (P < 0.001; Pearson's r = 0.95). There was absence of cytoplasmic staining and only nuclear staining (with localization in the nucleoplasm) was demonstrated in the tumour cells. In normal epithelium of the nasopharynx, the basal layer was stained. An inverse relationship was observed between the level of MT expression and the apoptotic index in the NPC tissues (P = 0.0059; Pearson's r = -0.6380). The results suggest that overexpression of MT in NPC may protect the tumour cells from entering into the apoptotic process and thereby contribute to tumour expansion. Preferential localization of MT in the nuclei of NPC cells may possibly enhance radioresistance since radiotherapy is known to eradicate tumour cells by free radical-induced apoptosis. PMID- 10735507 TI - Increasing chromosome 1 copy number parallels histological progression in breast carcinogenesis. AB - Chromosome 1 copy number in the benign breast lesions hyperplasia and atypical duct hyperplasia (ADH) was investigated using fluorescence in situ hybridization on paraffin sections. Progression of chromosome 1 changes occurring in parallel with histological progression from normal through hyperplasia and ADH to ductal carcinoma in situ (DCIS) and invasive carcinoma was also assessed, both overall and within individual patients. The mean signal number for normal cells was 1.14, while that for hyperplasia was 1.56 and ADH was 1.5, while values for DCIS of 1.95 and invasive duct carcinoma of 1.79, were higher (P < 0.001). Six of the seven cases also showed a significant trend towards an increasing proportion of cells with greater than 2 signals per nucleus occurring with histological progression (P < 0.001). These results support the concept that benign proliferative breast disease is a biological precursor of in-situ and invasive ductal carcinoma, the early histological changes possibly indicating a field effect with further genetic changes required for the development of a malignant phenotype. PMID- 10735508 TI - The status of Fas and Fas ligand expression can predict recurrence of hepatocellular carcinoma. AB - The status of Fas and Fas ligand (Fas L) expression was investigated in this study for 103 hepatocellular carcinomas (HCC). We studied the expression of the following three factors, Fas and Fas L expression in carcinoma cells and Fas L expression in stromal mononuclear cells (defined as stromal Fas L index). Fas expression in HCC cells was significantly decreased in cases with poor differentiation (P < 0.0001) and of larger size (P = 0.0058). Fas L expression in carcinoma cells was observed exclusively in moderately or poorly differentiated cases. Furthermore, each factor had prognostic significance for disease-free survival (DFS) (P< 0.0001, P = 0.0222 and 0.0027 respectively). We then scored the results of each factor and defined the total score as 'Fas-Fas L risk score'. The P-value of the score for DFS was even lower than that of the clinical stage by multivariate analysis. These results suggest that the evaluation of Fas and Fas ligand expression potentially has a significant prognostic value for DFS of HCC patients, in addition to the clinical stage, and can be regarded as a new prognostic marker. PMID- 10735509 TI - Analysis of pilocytic astrocytoma by comparative genomic hybridization. AB - Very little is known about genetic abnormalities involved in the development of pilocytic astrocytoma, the most frequently occurring brain tumour of childhood. We have analysed 48 pilocytic astrocytoma specimens using comparative genomic hybridization. Only five of 41 tumours from children showed abnormalities detectable by comparative genomic hybridization, and in each case this represented gain of a single chromosome. Interestingly, two of seven tumours from adults showed abnormalities, which were multiple and relatively complex. Six of the seven tumours showing abnormalities were from female patients (two adults and four children). The most frequently detectable abnormality was gain of 9q34.1 qter, which was present in three cases (two adult and one paediatric). PMID- 10735510 TI - Sigma-2 receptors as a biomarker of proliferation in solid tumours. AB - Over the past several years, our group has provided considerable evidence that the expression of sigma-2 (sigma2) receptors may serve as a biomarker of tumour cell proliferation. In these in vitro studies, sigma2 receptors were expressed 8 10 times more in proliferative (P) tumour cells than in quiescent (Q) tumour cells, and the extent and kinetics of their expression were independent of a number of biological, physiological and environmental factors often found in solid tumours. Moreover, the expression of sigma2 receptors followed both the population growth kinetics when Q-cells were recruited into the P-cell compartment and the proliferative status of human breast tumour cells treated with cytostatic concentrations of tamoxifen. However, these in vitro studies may or may not be indicative of what might occur in solid tumours. In the present study, the sigma2 receptor P:Q ratio was determined for the cells from subcutaneous 66 (diploid) and 67 (aneuploid) tumours grown in female nude mice. The sigma2 receptor P:Q ratio of the 66 tumours was 10.6 compared to the sigma2 receptor P:Q ratio of 9.5 measured for the 66 tissue culture model. The sigma2 receptor P:Q ratio of the 67 tumours was 4.5 compared to the sigma2 receptor P:Q ratio of approximately equal 8 measured for the 67 tissue culture model. The agreement between the solid tumour and tissue culture data indicates that: (1) the expression of sigma2 receptors may be a reliable biomarker of the proliferative status of solid tumours and (2) radioligands with both high affinity and high selectivity for sigma2 receptors may have the potential to non invasively assess the proliferative status of human solid tumours using imaging techniques such as positron emission tomography or single-photon emission computerized tomography. PMID- 10735511 TI - Activated mesothelial cells produce heparin-binding growth factors: implications for tumour metastases. AB - Curative surgery for gastrointestinal malignancy is commonly thwarted by local tumour recurrence. The heparin-binding growth factors, basic fibroblast growth factor (bFGF), heparin-binding epidermal growth factor-like growth factor (HB EGF) and vascular epidermal growth factor (VEGF) are all implicated in the metastatic process, but whether or not these essential growth factors are produced by the activated peritoneum is unknown. This study reveals that peritoneal mesothelial cells constitutively express mRNA for bFGF, HB-EGF and two VEGF spliced variants, VEGF121 and VEGF165. Mesothelial activation with interleukin (IL)-1b or tumour necrosis factor (TNF)-a produced an up-regulation of mRNA for HB-EGF and VEGF, but not bFGF expression. IL-6 failed to stimulate growth factor expression, whereas IL-2 produced a marked suppression in HB-EGF and bFGF, but not VEGF expression. Mesothelial cells were shown to predominantly express mRNA for the intermediate affinity (bg(c)) IL-2 receptor. Cytokine induced growth factor up-regulation was confirmed at the protein level using Western blotting of mesothelial cell lysates for HB-EGF and culture supernatant enzyme-linked immunosorbent assay for VEGF. The production of these growth factors by human mesothelial cells may play a significant role in post-operative peritoneal tumour recurrence. Their common heparin-binding property offers a potential therapeutic target for manipulating the growth factor environment of the human peritoneum. PMID- 10735512 TI - Molecular features of a human rhabdomyosarcoma cell line with spontaneous metastatic progression. AB - A novel human cell line was established from a primary botryoid rhabdomyosarcoma. Reverse transcription polymerase chain reaction investigations of this cell line, called RUCH-2, demonstrated expression of the regulatory factors PAX3, Myf3 and Myf5. After 3.5 months in culture, cells underwent a crisis after which Myf3 and Myf5 could no longer be detected, whereas PAX3 expression remained constant over the entire period. Karyotype analysis revealed breakpoints in regions similar to previously described alterations in primary rhabdomyosarcoma tumour samples. Interestingly, cells progressed to a metastatic phenotype, as observed by enhanced invasiveness in vitro and tumour growth in nude mice in vivo. On the molecular level, microarray analysis before and after progression identified extensive changes in the composition of the extracellular matrix. As expected, down-regulation of tissue inhibitors of metalloproteinases and up-regulation of matrix metalloproteinases were observed. Extensive down-regulation of several death receptors of the tumour necrosis factor family suggests that these cells might have an altered response to appropriate apoptotic stimuli. The RUCH-2 cell line represents a cellular model to study multistep tumorigenesis in human rhabdomyosarcoma, allowing molecular comparison of tumorigenic versus metastatic cancer cells. PMID- 10735513 TI - Risk of hepatitis A virus infection among sewage workers in Israel. AB - Sewage workers are exposed to a wide range of chemicals and biological agents, including the hepatitis A virus. Inasmuch as Israel is an endemic area for hepatitis A, it is unclear if sewage workers are at increased risk for hepatitis A or which factors contribute to such risk. The authors compared seropositivity of hepatitis A in 100 sewage workers with that in 100 blue-collar worker controls. Hepatitis A seropositivity was highly prevalent, but nonsignificant, in both sewage workers and controls (82% and 91%, respectively). In sewage workers, the major risk for serological positivity was age (odds ratio = 4.5, 95% confidence interval = 1.6, 12.4 for every 10 y). The factors associated negatively with seropositivity were years of education and years of seniority. The authors concluded that exposure to sewage is not a risk factor for hepatitis A infection in Israel, and, therefore, sewage workers do not require special attention in this regard. PMID- 10735514 TI - Effects of diesel exhaust on neurobehavioral and pulmonary functions. AB - Ten railroad workers and 6 electricians referred for shortness of breath also had slowness of response, memory loss, and disordered sleep, all of which suggested neurobehavioral impairment. The hypothesis was that diesel exhaust causes central nervous system impairment. Six electricians worked within enclosed concrete walls and roofs that trapped diesel exhaust from trucks. Seven railroad mechanics had tuned diesel engines indoors for 15-50 y, and 3 crewmen rode in locomotives. Neurobehavioral and visual functions were measured with a 26-test battery. Compared with unexposed men, the 16 in this study had significantly impaired reaction time, balance, blink reflex latency R-1, Culture Fair, peg placement, trail making, and verbal recall. Thirteen men had abnormal visual fields, and 11 had abnormal color confusion indices. Nine men had airways obstruction. The author could not attribute abnormalities to confounding factors or bias. Severe neurobehavioral impairment was associated with exposure to confined diesel exhaust. In additional studies of diesel-exposed workers, especially drivers of locomotives and trucks, investigators should use sensitive neurobehavioral methods. PMID- 10735515 TI - New ventilation systems at select schools in Sweden--effects on asthma and exposure. AB - The air-exchange rate is often low in schools. The authors studied the possible impact of improving school ventilation on health and exposure of pupils. Questionnaire data on allergies, asthma, and asthmatic symptoms were obtained in 1993 and 1995 for 1,476 primary- and secondary-school pupils in 39 randomly selected schools. Various exposure factors were measured in 1993 and 1995 in approximately 100 classrooms. In 12% of the classrooms, new ventilation systems were installed between 1993 and 1995; the subsequent air-exchange rate increased and the relative humidity and concentration of several airborne pollutants were reduced compared with classrooms in nonimproved buildings. The reporting of at least one asthmatic symptom and the reporting of more asthmatic symptoms in 1995 than in 1993 were less common among the 143 pupils who attended schools with new ventilation systems. PMID- 10735516 TI - Perinatal and infant mortality and low birth weight among residents near cokeworks in Great Britain. AB - With growing evidence of the adverse health effects of air pollution--especially fine particulates--investigators must concentrate on the fetus, neonate, and infant as potentially vulnerable groups. Cokeworks are a major source of smoke and sulfur dioxide. In the current study, the authors investigated whether populations residing near cokeworks had a higher risk of adverse perinatal and infant outcomes. Zones of 7.5-km radius around 22 cokeworks in Great Britain were studied, within which the authors assumed that exposure declined from highest levels within 2 km to background levels. Routinely recorded birth and death data for Great Britain during the period 1981-1992 were analyzed. Each individual record had a postcode that referred to a small geographical area of typically 15 17 addresses. The authors calculated expected numbers on the basis of regional rates, stratified by year, sex, and a small-area socioeconomic deprivation score. For all cokeworks combined, the observed/expected ratio (95% confidence intervals within parentheses) within 2 km of cokeworks was 1.00 (0.95, 1.06) for low-birth weight (i.e., < 2,500 g) infants; 0.94 (0.78, 1.12) for still births; 0.95 (0.83, 1.09) for infant mortality; 0.86 (0.72, 1.03) for neonatal mortality; 1.10 (0.90, 1.33) for postneonatal mortality; 0.79 (0.30, 1.46) for respiratory postneonatal mortality; and 1.07 (0.77, 1.43) for postneonatal Sudden Infant Death Syndrome. Respiratory postneonatal mortality was low throughout the entire 0-7.5-km study area (observed/expected = 0.74 [0.61, 0.88]). There was no statistically significant decline in risk with distance from cokeworks for any of the outcomes studied. The authors concluded that there was no evidence of an increased risk of low birth weight, stillbirths, and/or neonatal mortality near cokeworks, and there was no strong evidence for any association between residence near cokeworks and postneonatal mortality. One must remember, however, the limited statistical power of the study to detect small risks. PMID- 10735517 TI - Chlorine-induced damage documented by neurophysiological, neuropsychological, and pulmonary testing. AB - Chlorine causes acute pulmonary edema and damages airways, thus producing obliterative bronchiolitis. In the case series in this study, its adverse effects were extended to visual and central nervous system impairment. Twenty-two patients exposed briefly to undiluted chlorine at home or work were evaluated with a battery of neurobehavioral and visual tests. Their test scores, expressed as percentage predicted, were compared with those of unexposed subjects. Chlorine exposed subjects had impaired balance (with eyes open and eyes closed), delayed simple and choice reaction times, impaired color discrimination, impaired visual field performance, decreased hearing, and decreased grip strength. Blink reflex latency was delayed on the right. Cognitive performance (i.e., digit symbol and vocabulary), peg placement, trail making A and B, and verbal recall were significantly below predicted levels. Well-learned memory functions were not impaired. Adverse mood states scores were elevated as were the frequencies of 28 of 35 common symptoms. Forced vital capacities were reduced. The duration of chlorine exposures was from a breath or two to several hours, and exposures were associated with impaired neurophysiologic and neuropsychologic functions. Impairments appeared insidiously, were noted 1 to 48 mo after exposure, and persisted. Such functional losses must be prevented. Additional chlorine-exposed patients should be evaluated for neurological and pulmonary damage. PMID- 10735518 TI - Respiratory toxicity of mattress emissions in mice. AB - Groups of male Swiss-Webster mice breathed emissions of several brands of crib mattresses for two 1-hr periods. The authors used a computerized version of ASTM E-981 test method to monitor respiratory frequency, pattern, and airflow velocity and to diagnose abnormalities when statistically significant changes appeared. The emissions of four mattresses caused various combinations of upper-airways irritation (i.e., sensory irritation), lower-airways irritation (pulmonary irritation), and decreases in mid-expiratory airflow velocity. At the peak effect, a traditional mattress (wire springs with fiber padding) caused sensory irritation in 57% of breaths, pulmonary irritation in 23% of breaths, and airflow decrease in 11% of breaths. All mattresses caused pulmonary irritation, as shown by 17-23% of breaths at peak. The largest airflow decrease (i.e., affecting 26% of the breaths) occurred with a polyurethane foam pad covered with vinyl. Sham exposures produced less than 6% sensory irritation, pulmonary irritation, or airflow limitation. Organic cotton padding caused very different effects, evidenced by increases in both respiratory rate and tidal volume. The authors used gas chromatography/mass spectrometry to identify respiratory irritants (e.g., styrene, isopropylbenzene, limonene) in the emissions of one of the polyurethane foam mattresses. Some mattresses emitted mixtures of volatile chemicals that had the potential to cause respiratory-tract irritation and decrease airflow velocity in mice. PMID- 10735519 TI - Excretion of methyl mercury in human feces. AB - Fecal excretion of methyl mercury was confirmed in four Japanese male subjects. Perhaps the methyl mercury detected in feces is dependent on (a) the unabsorbed methyl mercury in diet, (b) exfoliation of intestinal cells, (c) hepatic bile, and (d) intestinal methylation of inorganic mercury. The calculated amounts of methyl mercury excreted daily in feces were similar to those found in urine. PMID- 10735520 TI - Phytosorbent prepared from sunflower seed husks prevents mercuric chloride accumulation in kidney and muscle of adult rabbits. AB - In the present study, the effect of a melanin-containing phytosorbent, "Victoria," on mercury accumulation in rabbits' tissues was studied. This phytosorbent is derived from black sunflower seed husks. Domestic rabbits were administered either one single nontoxic low-level dose of mercuric chloride (i.e., 50 microg/1 kg body weight [control group]) or combinations of mercury and the phytosorbent "Victoria" (i.e., experimental group). Mercury and phytosorbent were administered per os daily for 12 d. Mercury in tissues was determined by cold-vapor atomic absorption spectroscopy. Mercury in kidney and muscle of the experiment group was, on average, 25.8 and 4.7 times less, respectively, than in the control group. The authors concluded that the phytosorbent prevented accumulation of mercury in the kidney and muscle tissues and exerted a protective effect against mercury toxicity. PMID- 10735521 TI - Assessment of water use and sanitation behavior of a rural area in Bangladesh. AB - A health development project was established in a rural area of Bangladesh that entailed training village health promoters to provide health education and to motivate families to install tubewells and sanitary latrines. Following a 2-y period of project implementation, the authors sought to assess knowledge and practice of mothers and family members about use of safe water and household tubewells. A household survey in the project area was compared with a similar one in a nearby control area. Three-hundred households in each area were selected, and mothers were interviewed with a standard questionnaire. Significantly more mothers in the health development project area (45.7%) used tubewell water for domestic purposes than in the control area (32.8%). However, hygienic practices of mothers were inadequate. Sanitary latrines were present in less than 20% of households in both areas. Approximately 97% of mothers and 78% of adult family members always used household latrines. However, the use of household latrines by children was low (26.7%). There was no statistically significant difference in the use of household latrines between the project and control areas. The results showed an improvement in use of household tubewells in the project area; however, there was no improvement in sanitation practices of families in the project area. Health education alone, without improvement of socioeconomic status, is not effective in changing behavior. PMID- 10735522 TI - Factors influencing tetrachloroethylene concentrations in residences above dry cleaning establishments. AB - Indoor air quality has been increasingly recognized as a significant public health problem. Proximity to industrial or commercial sources contributes to contamination in homes. Air sampling was conducted at 12 residences in 8 buildings that housed dry cleaners and 6 residential control sites. The authors found that concentrations of tetrachloroethylene, a dry-cleaning solvent, were elevated significantly in residences located in buildings that also housed dry cleaning establishments (mean = 2.0 mg/m3) relative to their concentrations in control residences (mean = < 0.07 mg/m3 [p < .001]). Tetrachloroethylene concentrations remained elevated when the cleaners were closed on weekends (p < .01). The authors verified that colorimetric detector tubes were a useful screening tool for residences. Also identified were factors that could affect tetrachloroethylene concentrations. The use of exhaust fans and the implementation of required inspection and maintenance requirements by dry cleaners were associated significantly with reduced tetrachloroethylene concentrations in residences (p < .01). In all cases, tetrachloroethylene concentrations exceeded "minimal risk levels" posited by the Agency for Toxic Substances Disease Registry for chronic exposure to tetrachloroethylene. Residents who live in buildings that house dry cleaners may be exposed at concentrations that are of public health concern. Measures that might reduce this exposure were also identified. PMID- 10735523 TI - Is urban asthma caused by methyl tertiary butyl ether (MTBE)? PMID- 10735524 TI - Misclassification of carcinogenic methyl tertiary butyl ether (MTBE) by the National Toxicology Program Board: smokescreen in, science out? PMID- 10735525 TI - Corruption of previously published asbestos research. PMID- 10735526 TI - Robust gamma oscillations in networks of inhibitory hippocampal interneurons. AB - Recent experiments suggest that inhibitory networks of interneurons can synchronize the neuronal discharge in in vitro hippocampal slices. Subsequent theoretical work has shown that strong synchronization by mutual inhibition is only moderately robust against neuronal heterogeneities in the current drive, provided by activation of metabotropic glutamate receptors. In vivo neurons display greater variability in the interspike intervals due to the presence of synaptic noise. Noise and heterogeneity affect synchronization properties differently. In this paper we study, using model simulations, how robust synchronization can be in the presence of synaptic noise and neuronal heterogeneity. We find that stochastic weak synchronization (SWS) (i.e. when neurons spike within a short interval from each other, but not necessarily at each period) is produced with at least a minimum amount of noise and that it is much more robust than strong synchronization (i.e. when neurons spike at each period). The statistics produced by the SWS population discharge are consistent with previous experimental data. We also find robust SWS in the gamma-frequency range (20-80 Hz) for a stronger synaptic coupling compared with previous models and for networks with 10-1000 neurons. PMID- 10735527 TI - Learning with two sites of synaptic integration. AB - Since the classical work of D O Hebb 1949 The Organization of Behaviour (New York: Wiley) it is assumed that synaptic plasticity solely depends on the activity of the pre- and the postsynaptic cells. Synapses influence the plasticity of other synapses exclusively via the post-synaptic activity. This confounds effects on synaptic plasticity and neuronal activation and, thus, makes it difficult to implement networks which optimize global measures of performance. Exploring solutions to this problem, inspired by recent research on the properties of apical dendrites, we examine a network of neurons with two sites of synaptic integration. These communicate in such a way that one set of synapses mainly influences the neurons' activity; the other set gates synaptic plasticity. Analysing the system with a constant set of parameters reveals: (1) the afferents that gate plasticity act as supervisors, individual to every cell. (2) While the neurons acquire specific receptive fields the net activity remains constant for different stimuli. This ensures that all stimuli are represented and, thus, contributes to information maximization. (3) Mechanisms for maximization of coherent information can easily be implemented. Neurons with non-overlapping receptive fields learn to fire correlated and preferentially transmit information that is correlated over space. (4) We demonstrate how a new measure of performance can be implemented: cells learn to represent only the part of the input that is relevant to the processing at higher stages. This criterion is termed 'relevant infomax'. PMID- 10735528 TI - Generalized and partial synchronization of coupled neural networks. AB - Synchronization of neural signals has been proposed as a temporal coding scheme representing cooperated computation in distributed cortical networks. Previous theoretical studies in that direction mainly focused on the synchronization of coupled oscillatory subsystems and neglected more complex dynamical modes, that already exist on the single-unit level. In this paper we study the parametrized time-discrete dynamics of two coupled recurrent networks of graded neurons. Conditions for the existence of partially synchronized dynamics of these systems are derived, referring to a situation where only subsets of neurons in each sub network are synchronous. The coupled networks can have different architectures and even a different number of neurons. Periodic as well as quasiperiodic and chaotic attractors constrained to a manifold M of synchronized components are observed. Examples are discussed for coupled 3-neuron networks having different architectures, and for coupled 2-neuron and 3-neuron networks. Partial synchronization of different degrees is demonstrated by numerical results for selected sets of parameters. In conclusion, the results show that synchronization phenomena far beyond completely synchronized oscillations can occur even in simple coupled networks. The type of the synchronization depends in an intricate way on stimuli, history and connectivity as well as other parameters of the network. Specific inputs can further switch between different operational modes in a complex way, suggesting a similarly rich spatio-temporal behaviour in real neural systems. PMID- 10735529 TI - Controlling activity fluctuations in large, sparsely connected random networks. AB - Controlling activity in recurrent neural network models of brain regions is essential both to enable effective learning and to reproduce the low activities that exist in some cortical regions such as hippocampal region CA3. Previous studies of sparse, random, recurrent networks constructed with McCulloch-Pitts neurons used probabilistic arguments to set the parameters that control activity. Here, we extend this work by adding an additional, biologically appropriate, parameter to control the magnitude and stability of activity oscillations. The new constant can be considered to be the rest conductance in a shunting model or the threshold when subtractive inhibition is used. This new parameter is critical for large networks run at low activity levels. Importantly, extreme activity fluctuations that act to turn large networks totally on or totally off can now be avoided. We also show how the size of external input activity interacts with this parameter to affect network activity. Then the model based on fixed weights is extended to estimate activities in networks with distributed weights. Because the theory provides accurate control of activity fluctuations, the approach can be used to design a predictable amount of pseudorandomness into deterministic networks. Such nonminimal fluctuations improve learning in simulations trained on the transitive inference problem. PMID- 10735530 TI - Odour recognition and segmentation by a model olfactory bulb and cortex. AB - We present a model of an olfactory system that performs odour segmentation. Based on the anatomy and physiology of natural olfactory systems, it consists of a pair of coupled modules, bulb and cortex. The bulb encodes the odour inputs as oscillating patterns. The cortex functions as an associative memory: when the input from the bulb matches a pattern stored in the connections between its units, the cortical units resonate in an oscillatory pattern characteristic of that odour. Further circuitry transforms this oscillatory signal to a slowly varying feedback to the bulb. This feedback implements olfactory segmentation by suppressing the bulbar response to the pre-existing odour, thereby allowing subsequent odours to be singled out for recognition. PMID- 10735531 TI - Lateral cell movement driven by dendritic interactions is sufficient to form retinal mosaics. AB - The formation of retinal mosaics is thought to involve lateral movement of retinal cells from their clonal column of origin. The forces underlying this lateral cell movement are currently unknown. We have used a model of neurite outgrowth combined with cell movement to investigate the hypothesis that lateral cell movement is guided by dendritic interactions. We have assumed that cells repel each other in proportion to the degree of dendritic overlap between neighbouring cells. Our results first show that small cell movements are sufficient to transform random cell distributions into regular mosaics, and that all cells within the population move. When dendritic fields are allowed to grow, the model produces regular mosaics across all cell densities tested. We also find that the model can produce constant coverage of visual space over varying cell densities. However, if dendritic field sizes are fixed, mosaic regularity is proportional to the cell density and dendritic field size. Our model suggests that dendritic mechanisms may therefore provide sufficient information for rearrangement of cells into regular mosaics. We conclude by mentioning possible future experiments that might suggest whether dendritic interactions are adaptive or fixed during mosaic formation. PMID- 10735532 TI - Membrane-bound, ATP-dependent energy systems and tissue hypoxia in human gastric mucosa. AB - 1. The biochemical measurements of gastric mucosal ATP, ADP, AMP and cAMP offer excellent information on the extent of tissue oxygenation, when the observations are taken simultaneously from the same tissue sample. 2. Adenosine triphosphate is split by membrane-bound enzymes in two directions (to ADP and cAMP), while ADP and cAMP transform to AMP, supplying further energy liberation. 3. The liberated free energy modifies physiologically or pathophysiologically the functions of target organ cells. 4. Different drugs, hormones and mediators, have different effects on the regulation of ATP breakdown (into ADP vs cAMP). 5. The actual tissue level of ATP is a consequence of the equilibrium between the directions of ATP-ADP and ATP-cAMP breakdown, as well as of ATP resynthesis. 6. The biochemical measurement of the reduction in gastric mucosal ATP alone is not enough proof for the existence of tissue hypoxia, because demonstration of the simultaneous elevation of tissue lactate is also necessary. PMID- 10735533 TI - Apoptosis in the intestinal epithelium: its relevance in normal and pathophysiological conditions. AB - Apoptosis is now recognized as an important process responsible for maintenance of the cellular balance between proliferation and death. Apoptosis is distinct from necrosis in that it is a programmed form of cell death and occurs without any accompanying inflammation. This form of cell death can be induced by a wide range of cellular signals, which leads to activation of cell death machinery within the cell and is characterized by distinct morphological changes. Apoptosis is especially relevant in the gastrointestinal tract, as the mammalian intestinal mucosa undergoes a process of continual cell turnover that is essential for maintenance of normal function. Cell proliferation is confined to the crypts, while differentiation occurs during a rapid, orderly migration up to the villus. The differentiated enterocytes, which make up the majority of the cells, then undergo a process of programmed cell death (apoptosis). Although apoptosis is essential for the maintenance of normal gut epithelial function, dysregulated apoptosis is seen in a number of pathological conditions in the gastrointestinal tract. The cellular mechanisms regulating this tightly regimented process have not been clearly defined and this topic represents an area of active investigation as delineation of this process will lead to a better understanding of normal gut mucosal growth. PMID- 10735534 TI - Evaluation of lower oesophageal sphincter pressure using endoscopic manometric sleeve assembly. AB - BACKGROUND: The purpose of this study was to establish a method to determine lower oesophageal sphincter (LOS) pressure using an endoscopic manometric sleeve assembly. METHODS: We used a 250-cm, three-lumen, 2.2-mm external diameter silicon catheter with a 3-cm sleeve sensor that was passed easily through the biopsy channel (2.8 mm diameter) of the gastroscope. Each lumen was perfused with distilled water using a low-compliance, pneumohydraulic capillary infusion system. Forty-seven healthy subjects and 35 patients with oesophageal disorders underwent study during routine diagnostic endoscopy. In 27 of the subjects, standard transnasal manometry with a three-lumen, 4.5-mm diameter polyvinyl catheter with a 5-cm sleeve sensor was also performed. RESULTS: Lower oesophageal sphincter pressure (LOS) was evaluated in all subjects (median pressure 16 mmHg, range 0-55 mmHg) for 6 min during routine endoscopic examination. The LOS pressure readings between standard and endoscopic manometry correlated well. The LOS pressure was significantly lower in healthy subjects with hiatal hernia than in those without hernia and was also significantly lower in patients with reflux oesophagitis than in healthy subjects. CONCLUSIONS: Endoscopic manometric sleeve assembly was used during routine endoscopic examination and was useful for easily determining LOS pressure. It could be used for evaluation of oesophageal motility disorders, thus avoiding the use of a more invasive and time-consuming method. PMID- 10735536 TI - Mast cell stabilization prevents ethanol-induced rat gastric mucosal injury: mechanisms of protection. AB - INTRODUCTION: We previously demonstrated that 60% ethanol increased macromolecular leakage and induced lesion formation in areas of permanent flow stasis within gastric mucosal vessels. Mast cells and their mediators have been implicated in acute mucosal injury. Fluorescent in vivo microscopy was used to assess the effects of ketotifen, a mast cell stabilizer, and pyrilamine, a histamine (H1)-receptor antagonist, on ethanol-induced rat gastric mucosal injury. METHODS: Experiments were carried out on anaesthetized rats pretreated orally with ketotifen (1 mg/kg) or pyrilamine (30 mg/kg). Fluorescein isothiocyanate-bovine serum albumin (FITC-BSA; 0.2 mL/100 g), a marker for quantitating macromolecular leakage was administered intra-arterially. Ethanol (60%) or distilled water was applied topically to the gastric mucosa. Macromolecular leakage of FITC-BSA, vessel diameters and leucocyte activity were quantified using image analysis. RESULTS: Pretreatment with ketotifen or pyrilamine, followed by ethanol, caused no change in macromolecular leakage compared with controls. Both compounds prevented blood flow stasis in all areas and no lesion formation was observed. However, increased leucocyte activity and increases in vessel diameter were observed following pretreatment with ketotifen and pyrilamine, respectively. CONCLUSIONS: The data suggest that vasoactive substances released from mast cells may be involved in the aetiology of ethanol induced gastric mucosal damage. The prevention of these normal physiological responses to injury may lead to the employment of other microcirculatory mechanisms of defence. PMID- 10735535 TI - Role of energy metabolism in drug-induced acute gastric mucosal injuries in humans. AB - BACKGROUND: In various drug-induced gastric mucosal injuries, it has been speculated that changes in gastric mucosal energy metabolism differ according to the cause of injury. This study was conducted to investigate the role of energy metabolism in two drug-induced gastric mucosal injuries in humans. METHODS: The subjects were patients with acute gastric mucosal injury due to non-steroidal anti-inflammatory drugs (NSAID) or steroids, and normal controls. Two sets of tissue specimens from the antrum and corpus of the stomach were obtained. One specimen from each area was used for histological analysis and the other was stored in liquid nitrogen. The stored tissues were homogenized. Adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) were measured by the luciferin-luciferase method. RESULTS AND CONCLUSIONS: In the NSAID group, the ATP levels and the total adenine nucleotide (TAN) levels in both the antrum and corpus were significantly lower than in the control group. In the steroid group, no significant differences were observed in either the ATP or TAN levels. The NSAID decreased energy metabolism in the entire stomach while the steroid had a negligible effect on energy metabolism. PMID- 10735537 TI - Role of endothelin-1 in congestive gastropathy in portal hypertensive rats. AB - BACKGROUND: The aim of this study was to determine the role of endothelin (ET)-1 in portal hypertensive gastropathy (PHG) under portal hypertension, in order to investigate whether the ET(A/B) receptor inhibitor improves the permeability of gastric mucosal microvessels in PHG. METHODS AND RESULTS: Portal hypertensive rats (PVL) and sham-operated rats (CTR) were prepared and then the concentration of plasma ET-1 was measured and the vasopressor response to ET-1 was compared between the two groups. The plasma ET-1 levels in PVL increased significantly compared with CTR; however, the vasopressor response to ET-1 in PVL decreased more than in CTR. Next, the portal venous pressure was measured in both CTR and PVL pretreated with or without a nitric oxide (NO) synthase inhibitor, N(G)-nitro L-arginine methyl ester (L-NAME), before the injection of ET-1. The portal venous pressure of PVL after receiving ET-1 and being pretreated with L-NAME significantly increased in comparison to the pressure of PVL treated with ET-1 alone (without L-NAME). Moreover, Evans-Blue was injected into each rat and the absorbancy of the gastric contents was measured. The absorbancy of Evans-Blue in PVL increased significantly compared with CTR; however, the absorbancy in PVL+ ET(A/B) receptor inhibitor (Ro47-0203) decreased significantly more than in PVL. CONCLUSIONS: This study showed that ET-1 is a potent vasoconstrictive substance that also has a transitory vasodilative response through NO induced by ET-1 in portal hypertension. In addition, it was found that the vascular permeability of the gastric mucosa increased in portal hypertension and that Ro47-0203 inhibited the hyper-permeability. Accordingly, ET-1 may, thus, play an important role in the development of PHG through NO induced by ET-1. Ro47-0203 may, therefore, be a useful substance for improving PHG in portal hypertension. PMID- 10735538 TI - Effect of intragastric pH on control of peptic ulcer bleeding. AB - BACKGROUND: We have performed series studies to investigate the effect of intragastric pH on control of peptic ulcer bleeding. In laboratory and animal studies, both platelet aggregation and gastric mucosal bleeding time were shown to be extremely sensitive to different pH levels. Platelet aggregation decreased significantly at pH > or = 6.8 and gastric mucosal bleeding time fell significantly at pH > or = 6.4. In a prospective clinical trial, primed infusions of different dosages of omeprazole (8 or 4 mg/h) after a bolus (40 mg) produced consistently high intragastric pH values in patients with bleeding duodenal ulcer. These results were not significantly different from that obtained from omeprazole 40 mg bolus treatment every 12 h (P > 0.05). However, primed injection with cimetidine (800 mg/12 h) was less effective (P < 0.05). METHODS: In a retrospective analysis, 303 patients with bleeding peptic ulcer who were treated with cimetidine and 326 patients who were treated with omeprazole were compared. RESULTS: The emergency surgery (4.91%) and mortality rates (1.84%) in the omeprazole group were not significantly different (P > 0.05) from those (7.28 and 1.99%) in the cimetidine group. However, the standardized emergency surgery rate of the omeprazole group (3.28%) was significantly lower than that (9.28%) of the cimetidine group (P < 0.05). CONCLUSION: We conclude that increased intragastric pH to at least 6.4 with omeprazole is helpful in controlling peptic ulcer bleeding. Chinese patients require a lower dose of omeprazole than their Western counterparts to control ulcer bleeding. PMID- 10735539 TI - Determination of the optimal cut-off value for the [13C]-urea breath test based on a Helicobacter pylori-specific polymerase chain reaction assay. AB - BACKGROUND: This study was conducted to determine the optimal cut-off value and breath sample collection time for the [13C]-urea breath test based on the assessment of Helicobacter pylori status with a gastric juice-based polymerase chain reaction (PCR) assay. METHODS AND RESULTS: A total of 104 patients took 100 mg [13C]-urea orally and breath samples were collected at 5, 10, 20, 30 and 60 min. The increment of 13CO2:12CO, ratio from the baseline (delta13C) was measured using a laser spectroanalyser. The PCR assay was positive in 63 and negative in 41 patients. The optimal cut-off value of delta13C was calculated for each sample collection time so that the distance from the geometric mean value among Helicobacter pylori-positive patients and that from the arithmetic mean value among negative patients were simultaneously maximized. The cut-off value of 2.7% at 20 min had the longest distance, being separated by 3.16 SD from the two mean values. Using this cut-off value, the urea breath test showed 100% specificity and 98% sensitivity for the diagnosis of Helicobacter pylori infection. PMID- 10735540 TI - Endoscopic [13C]-urea breath test for quantification of Helicobacter pylori infection. AB - BACKGROUND: We previously developed a new diagnostic method for Helicobacter pylori infection and called it the endoscopic [13C]-urea breath test (EUBT). Here we evaluate the relationship between the EUBT results and the histological findings. METHODS: The EUBT was performed on 137 patients with gastroduodenal diseases. After the collection of a baseline breath sample, gastroduodenal endoscopy was performed. Twenty milliliters of 0.05% phenol red solution containing 100 mg of [13C]-urea was sprayed over the entire gastric mucosa under endoscopic observation. A breath sample was collected 15 min after spraying. The content of 13CO2 in the breath samples was measured by ratio mass spectrometry. Two biopsy specimens each from the antrum and the middle corpus were obtained for culture and histology. Helicobacter pylori colonization, activity, inflammation, atrophy and intestinal metaplasia were classified on a four-point scale according to the Updated Sydney System. RESULTS: We found positive correlations between the EUBT values and the H. pylori colonization and activity score in the antrum and corpus, and negative correlations between the EUBT values and the atrophy and intestinal metaplasia scores in the corpus. CONCLUSIONS: The EUBT can be an indicator of the intragastric bacterial load and the histological findings for H. pylori. PMID- 10735541 TI - Helicobacter pylori induces apoptosis in gastric epithelial cells through inducible nitric oxide. AB - BACKGROUND: Gastric mucosal injury by Helicobacter pylori has been suggested to be mediated by various cytokines induced by this organism. Nitric oxide (NO) is an important effector molecule involved in immune regulation and defence. To clarify the mechanisms by which H. pylori induces gastric mucosal cell injury, we examined whether H. pylori induces gastric epithelial death via NO production. METHODS: Cytotoxic and non-cytotoxic strains of H. pylori were used. The death of MKN45 cells caused by H. pylori was examined by the 3-(4,5-dimethyl-thiazole-2yl) 2,5-diphenyl tetrazolium bromide (MTT) assays. Aminoguanidine was used to inhibit inducible nitric oxide synthase (iNOS) activity. Expression of iNOS mRNA was determined by the reverse transcriptase-polymerase chain reaction and the DNA fragmentation analysis was performed by using agarose gel electrophoresis. RESULTS: The MTT assay revealed that neither viable H. pylori nor other components of the microorganism induced cell death. Both preincubation of MKN45 cells with interferon-gamma for 6 h and coculture with TNF-alpha significantly increased the cytotoxicity of H. pylori. Both cytotoxic and non-cytotoxic strains of H. pylori induced cell death. Expression of iNOS mRNA was observed in MKN45 cells at 6, 8 and 12 h after H. pylori inoculation. The cytotoxicity of H. pylori was inhibited by aminoguanidine and DNA fragmentation analysis showed that H. pylori induced apoptosis. CONCLUSIONS: These findings suggested that viable H. pylori induces apoptosis of gastric epithelial cells via nitric oxide. Our study indicated that iNOS expression plays an important role in gastric cell injury. PMID- 10735542 TI - Hepatitis C in injecting drug-using women during and after pregnancy. AB - BACKGROUND: A high proportion of female injecting drug users (IDU) have evidence of hepatitis C virus (HCV) infection. We undertook a prospective study of patients attending a clinic for pregnant IDU to determine the impact of pregnancy on the course of HCV infection and whether pregnancy is affected by HCV infection. METHODS: One hundred and thirty-one IDU were recruited and followed up with liver function tests, HCV serology and HCV-RNA tests. RESULTS: Of 131 patients, 125 had HCV antibodies (anti-HCV positive) at delivery, and of these 62% were HCV-RNA positive. The anti-HCV-negative women were younger and had a shorter duration of drug use than the anti-HCV-positive women. There were no differences between viraemic and non-viraemic women with respect to age, ethnicity, duration of injecting drug use, methadone maintenance dose, hepatitis B exposure or reported high-risk behaviour. Alanine aminotransferase (ALT) levels were higher and the proportion with ALT > 55 IU/L higher in viraemic women. Viraemia persisted in all 55 women who were viraemic at term. Eleven had an ALT flare post-partum that was unrelated to viral load and was clinically unsuspected. Four had concurrent elevated gamma-glutamyltranspeptidase and were considered to be drinking alcohol at hazardous levels. Four of 23 women who were HCV-RNA negative at term became positive during follow up. CONCLUSIONS: Pregnancy does not adversely affect the course of hepatitis C. A modest rebound in ALT levels, but not HCV-RNA, occurs after delivery in some viraemic women. This supports the theory that immune mechanisms rather than direct viral cytopathology are involved in hepatocyte injury during HCV infection. Hepatitis C infection did not influence pregnancy complications and outcomes. PMID- 10735543 TI - Amino acid substitutions in codons 9-11 of hepatitis C virus core protein lead to the synthesis of a short core protein product. AB - BACKGROUND: Previous in vitro experiments have indicated that if the ninth codon of the hepatitis C virus (HCV) core gene is mutated from arginine to lysine, a short 16-kDa (P16) instead of a 21-kDa (P21) core protein will be produced. In this study, we aimed to investigate whether similar mutations existed in patients with chronic HCV infection and whether such mutations led to the expression of P16. METHODS: The core gene was isolated from patients' sera by reverse transcription-polymerase chain reaction and sequenced. RESULTS: Three of 10 patients with hepatocellular carcinoma were found to have mutant viruses with missense mutations at codons 9-11: arginine-to-glycine mutation at codon 9 (case 1); lysine-to-glutamine mutation at codon 10 (case 5); and lysine-to asparagine/threonine-to-alanine double mutations at codons 10 and 11 (case 8). Site-directed mutagenesis and in vitro translation experiments revealed that P16 was expressed by all three mutants. Using gel-purified P21 and P16 proteins obtained from transformed Escherichia coli, the serum titres of anti-P21 and anti P16 were assayed. Unequal titres of anti-P16 and anti-P21 were found in only cases 1, 5 and 8. A rabbit antibody directed against P16 but not P21 was thus generated for immunohistochemical analysis. P16 was detected in the nuclei of hepatocytes in the peri-hepatoma tissue of a single case (case 1). CONCLUSIONS: These data indicate that missense mutations at codons 9-11 can occur during chronic HCV infection, which results in the expression of P16 core protein. PMID- 10735544 TI - Effect of hepatobiliary disease, chronic hepatitis C and hepatitis B virus infections and interferon-alpha on porphyrin profiles in plasma, urine and faeces. AB - BACKGROUND: Documentation of the profiles of porphyrins in hepatobiliary disease is limited. Strong associations of hepatitis B and C virus infections with porphyria cutanea tarda have suggested causal relationships. This study aimed to determine the nature of porphyrin abnormalities in hepatobiliary disease and the effect of interferon-alpha on porphyrin profiles. METHODS: Total porphyrins were measured in the plasma, urine and faeces of 83 patients with hepatobiliary disease (37 hepatitis C, 20 hepatitis B, 26 other causes) and 12 clinical controls, and porphyrin profiles were determined by high-performance liquid chromatography. RESULTS: Porphyrins were elevated in the plasma of 11 and urine of 23 patients with hepatobiliary disease, as a result of elevated coproporphyrin I. This was reflected in increased coproporphyrin I:III ratios. Abnormal total porphyrin levels had a significant negative correlation with plasma albumin, and a positive correlation with bilirubin and alkaline phosphatase, but not with aminotransferases. Total urinary porphyrins were elevated in three control patients, but coproporphyrin I:III ratios were normal. Although not seen in plasma or urine, porphyrins that are specific for porphyria cutanea tarda were found in the faeces of six patients, but this occurred with similar frequency in hepatitis B or C infection (four of 50) as in the clinical controls (two of 12). Interferon-alpha had no effect during or after therapy in six patients with hepatitis C. CONCLUSIONS: Reduced biliary excretion of coproporphyrin I occurs in more severe cholestasis and/or hepatic dysfunction. A causal relationship between viral liver disease and porphyria cutanea tarda which is unlikely to be precipitated by interferon-alpha, is not supported. PMID- 10735545 TI - Cavographic study of an early stage of obstruction of the hepatic portion of the inferior vena cava. AB - BACKGROUND: Liver disease caused by a chronic lesion of the hepatic portion of the inferior vena cava (IVC) is clinically characterized by dilated superficial veins in the body trunk with cephalad flow, hepatomegaly and splenomegaly. Cavography shows stenosis or complete obstruction near the cava-atrial junction. METHODS: Early (acute and subacute) forms of the disease were recognized. The early stage of the disease manifested as jaundice, hepatomegaly or ascites and fever. Patients with acute and subacute onset of the illness with no past history of liver disease were studied with inferior vena cavography. Some of the patients had repeat cavography at 6 months and at 1 year after the initial investigations. RESULTS: Three types of cavographic lesions were observed in the early stages of the disease: type 1, linear lucent area in the IVC close to cava-atrial junction; type 2, a smooth or irregular narrowing of almost the whole segment of the hepatic portion of the IVC; and type 3, a constriction or narrowing of a segment of the IVC near the cava-atrial junction. The first two types were associated with the acute stage of the disease and type 3 with the subacute stage. Type 2 and 3 lesions were associated with post-stenotic dilatation (PSD) close to the atrium. Lucent areas resulting from thrombosis are common in PSD. The acute and subacute hepatic IVC diseases in Nepal are commonly associated with bacterial infection. CONCLUSIONS: It is postulated that the early cavographic lesions are consistent with thrombosis and thrombophlebitis of the hepatic portion of the IVC, and that resolution of the lesions leads to the development of stenosis and to complete obstruction. PMID- 10735546 TI - Hepatobiliary and pancreatic: recurrent right upper quadrant pain. PMID- 10735547 TI - Gastrointestinal: colonic pseudopolyposis. PMID- 10735548 TI - Oesophageal cavernous haemangioma diagnosed histologically, not by endoscopic procedures. AB - Haemangioma of the oesophagus is uncommon in patients with benign oesophageal tumours. We present a patient with an oesophageal haemangioma detected during mass screening of the upper gastrointestinal tract. The patient, a 59-year-old man, had neither abdominal complaints nor a history of gastrointestinal diseases. Endoscopic examination revealed a blue-coloured submucosal tumour (approximately 3 cm in diameter) at the middle portion of oesophagus. Endoscopic Doppler ultrasonography showed an homogeneous and hypoechoic mass without blood flow in the submucosal layer of the oesophagus. However, a magnetic resonance imaging scan did not give a typical image for oesophageal haemangioma. Therefore, partial resection of the tumour was performed to obtain a differential diagnosis using the procedures of endoscopic ligation and polypectomy. Histological examination of the resected tissue showed a cavernous haemangioma in the oesophagus. This endoscopic technique may be useful for the differential diagnosis of oesophageal haemangioma. PMID- 10735549 TI - Endoscopic pancreatic sphincter balloon dilation for effective retrieval of pancreatic duct stone. AB - To facilitate pancreatic stone retrieval, four patients with chronic pancreatitis and pancreatic stones underwent endoscopic pancreatic sphincter balloon dilation (EPSBD) rather than pancreatic sphincterotomy. Extracorporeal shock wave lithotripsy combined with endoscopic removal was carried out in three patients. Stone removal following EPSBD was completely successful in all four patients. Patients showed no severe complications during the dilation procedure. In one patient, to prevent pancreatitis, an endoscopic nasopancreatic drain was placed for 1 week after EPSBD. Compared with pancreatic sphincterotomy, EPSBD can be performed safely in patients with chronic pancreatitis to assist in the extraction of pancreatic duct stones. Use of the EPSBD procedure in cases of chronic pancreatitis provides a useful approach to improve endoscopic clearance of pancreatic duct stones. PMID- 10735550 TI - Activity of platelet-activating-factor-acetylhydrolase and the nitric oxide metabolite level in the plasma of pregnant women who develop transient hypertension during later pregnancy. AB - It is reported that plasma platelet-activating-factor-acetylhydrolase (PAF-AH) is elevated in patients with essential hypertension. In this study, plasma PAF-AH activity was measured during pregnancy and after delivery to examine the relationship between plasma PAF-AH activity and the development of transient hypertension (TH) during pregnancy. Moreover, in order to examine the involvement of endothelial injury in TH, the plasma level of nitric oxide metabolite (NOx; NO2+NO3) was measured. The plasma PAF-AH activity in 51 pregnant women was consecutively measured in the 1st, 2nd and 3rd trimesters of gestation, and after delivery. Forty-one cases were normal pregnancies and 10 cases were complicated by TH later during pregnancy. The PAF-AH activity in the normal pregnancy group decreased in the 2nd trimester of gestation compared with the 1st trimester, but was elevated in the TH group. The incidence of elevation of PAF-AH in the TH group was significantly (7/10; 70.0%; P<0.01, Chi-squared test) higher than in the normal pregnancy group (9/41; 22.0%). The plasma NOx levels in the 2nd trimester were higher than those in the 1st trimester in both the normotensive and TH group (P<0.05 for both comparisons). The 51 patients were classified into two groups according to the change in the PAF-AH in the 2nd trimester: group A consisted of 35 patients whose PAF-AH activity did not increase, and group B consisted of 16 patients whose PAF-AH activity increased. The incidence of development of TH during later pregnancy in group B was significantly (7/16; 43.8%; P<0.01, Chi-squared test) higher than in group A (3/35; 8.6%). Hypertension developed after 36 weeks' gestation in all patients in the TH group. The results of the present study suggest that changes in PAF metabolism may relate to regulation of blood pressure in pregnant women whose pregnancy is complicated with TH, whereas NO metabolism does not differ between women with TH and those having a normal pregnancy. PMID- 10735551 TI - CAMP inhibits mitogen-activated protein (MAP) kinase activation and resumption of meiosis, but exerts no effects after spontaneous germinal vesicle breakdown (GVBD) in mouse oocytes. AB - Various signaling molecules have been implicated in the oocyte G2/MII transition, including protein kinase C (PKC), cAMP and mitogen-activated protein (MAP) kinases. However, the cross-talk among these signaling pathways has not been elucidated. The present study demonstrates that both germinal vesicle break down (GVBD) and MAP kinase phosphorylation (activation) are inhibited when intraoocyte cAMP is increased by treating the GV-intact oocytes with dibutyryl cyclic AMP (dbcAMP), forskolin, or isobutylmethylxanthine (IBMX). Okadaic acid, a specific inhibitor of protein phosphatase-1 and -2A, completely overcame this effect. Calphostin C, a specific inhibitor of PKC, accelerated both GVBD and MAP kinase phosphorylation, and this effect was attenuated by increased intraoocyte cAMP, whereas PKC activation inhibited these events. Once GVBD occurred, the progression of oocyte maturation and MAP kinase phosphorylation were independent of cAMP These results indicate that an increase in intraoocyte cAMP, in synergy with PKC activation, initiates a cascade of events resulting in inhibition of MAP kinase phosphorylation and GVBD in the mouse oocyte. PMID- 10735552 TI - Relationship between motility and oxygen consumption of sperm from the cauda epididymides of the rat. AB - The oxygen consumption of rat sperm was low (2.7 microL O2 10(8) sperm(-1) h(-1)) in caudal epididymal semen (CES) when stimulation of motility was avoided. The addition of 1 microL of Krebs Ringer phosphate buffer (KRP) to 40 microL of CES (CES:KRP = 40:1) did not activate motility, but stimulated oxygen consumption 2 fold. Inclusion of 1-5 mM glucose, acetate, pyruvate or lactate in the KRP further stimulated respiration rate (up to 4.3-fold) without activating motility, but respiration was reduced when 2-deoxyglucose replaced energy substrates. Inclusion of dibutyryl cAMP (1 mM) activated sperm motility in all samples and stimulated oxygen consumption 2.9-fold. Dilution of CES at the ratio of CES:KRP = 40:1000 also activated sperm motility and stimulated respiration rate 2.9-fold. The combined effect of dibutyryl cAMP and glucose in stimulating respiration was greater than their individual effects. However, the response to cAMP or substrates was not altered by incubation in KRP containing either 0 or 0.5 mM Ca2+. It was concluded that the motility and metabolism of rat epididymal sperm are suppressed in vivo. Respiration can be stimulated by a small (1.025-fold) dilution and further stimulated by the inclusion of energy substrate, without activating motility. However, a larger dilution or inclusion of cAMP activated motility and simultaneously stimulated metabolism, with exogenous substrate being required to stimulate respiration to the maximum rate. This suggests that prior to activation, the rate of oxygen consumption and sperm motility are not coupled. PMID- 10735553 TI - Involvement of serotoninergic pathways in the control of luteinizing hormone secretion in red deer hinds. AB - Two experiments were conducted to determine whether serotoninergic pathways, which are implicated in the neuroendocrine regulation of luteininzing hormone (LH) secretion in domestic animals, have a similar action in red deer hinds. In the non-breeding season (August), ovariectomized (n = 5) and ovariectomized thyroidectomized (n = 5) hinds received a vehicle solution followed 4 h later by either serotonin (66 microg kg(-1) i.v.) every 10 min for a further 4 h or the serotonin antagonist, cyproheptadine (3 mg kg(-1) i.v.) as a single injection. This procedure was repeated in the breeding season (June). In the non-breeding season serotonin was without effect, but cyproheptadine reduced LH pulse frequency and amplitude in ovariectomized-thyroidectomized hinds (P<0.01). During the breeding season, serotonin reduced LH pulse amplitude in ovariectomized hinds (P<0.05) and cyproheptadine reduced LH pulse frequency in both ovariectomized and ovariectomized-thyroidectomized hinds (P<0.05 and P<0.01, respectively). On each occasion, cyproheptadine increased (P<0.01) plasma prolactin concentration, whereas serotonin had no effect. These results indicate a stimulatory role for serotoninergic neurons on the hypothalamic GnRH pulse generator mechanism of red deer hinds during the breeding season. In a second experiment, the LH response to GnRH (5 microg i.v.) was examined in ovariectomized hinds (n = 5) following administration of a serotonin infusion (6.6 microg kg(-1) min(-1) i.v. for 15 min), cyproheptadine (3 mg kg(-1) i.v. as a single dose) or vehicle, in the breeding season (July) after induction of halothane anaesthesia and in the non breeding season (December) without anaesthesia. Halothane anaesthesia eliminated endogenous pulses of LH. In comparison with the vehicle-treated controls, the response of plasma LH to exogenous GnRH was not altered by serotonin or cyproheptadine in either season, which shows that serotonin has no effect on LH release at the pituitary gland level in these animals. It was concluded that in the regulation of LH release in red deer hinds, serotoninergic pathways have a stimulatory role operating at the hypothalamic level. PMID- 10735554 TI - Diminished levels of prostaglandin E in type I diabetic oocyte-cumulus complexes. Influence of nitric oxide and superoxide dismutase. AB - In the present work the prostaglandin E (PGE) production by ovulated, immature and in vitro matured oocyte-cumulus complexes (OCC) was evaluated in a rat model of type I diabetes induced by streptozotocin (60 mg kg(-1)). A diminished number of ovulated OCC were found in the type I diabetic rat. In contrast to the increment in PGE generation found previously in OCC and embryos from type II diabetic rats, it was found that PGE production by type I diabetic OCC was diminished in comparison with the controls. Nitric oxide synthase (NOS) activity is enhanced in proestrous ovaries from type I diabetic rats, but cGMP levels are diminished. SIN-1 (300 microM), a nitric oxide donor, significantly enhanced PGE generation by control OCC, but was unable to modify the PGE levels in type I diabetic OCC. L-NMMA, a nitric oxide inhibitor that diminished PGE values in type II diabetic OCC, did not modify PGE generation in either control and type I diabetic OCC. Superoxide dismutase (SOD, 1000 U mL(-1)), and SOD (1000 U mL(-1)) plus SIN-1 (300 microM), enhanced PGE generation by both control and diabetic OCC. The present results suggest that even when nitric oxide (NO) is overproduced in diabetic ovaries, the NO-PGE pathway is impaired in type I diabetic OCC. As SOD additions are able to increase PGE generation by diabetic OCC, high concentrations of free oxygen radicals might be quenching the NO, impairing its physiological functions. PMID- 10735555 TI - Apoptosis and cell proliferation in the seminal vesicles and coagulating glands of male Syrian hamsters exposed to diethylstilboestrol (DES). AB - Regression of the accessory sex glands was induced in male Syrian hamsters by chronic exposure to diethylstilboestrol (DES), an agonist of 17beta-oestradiol. Experimental groups (n = 4-5) were killed at increasing time intervals up to 6 months after initiation of treatment. Organ atrophy was evaluated by morphological examination. Apoptosis in the seminal vesicles and coagulating glands was visualized by in situ analysis of DNA fragmentation. Cell proliferation was monitored by immunostaining nuclei of S-phase cells after pulse labelling with BrdU. The volume of the seminal vesicles decreased drastically after 2 weeks of DES administration due to a marked reduction of secretions in the lumen of the glands. Cell proliferation in the seminal vesicles was stimulated by chronic administration of DES. Mitotic activity mostly increased during the first month of treatment, leading to epithelial hyperplasia associated with progressive hyperplasia of the fibromuscular stroma. Evidence of epithelial dysplasia and metaplasia, often associated with an infiltration of polymorphonuclear leukocytes, was observed in animals exposed to DES for 4 months or more. Regression of the seminal vesicles was also associated with apoptosis in the gland epithelium. Apoptosis appeared 3 days after starting DES administration and culminated at 1 month. Thereafter the number of apoptotic cells decreased progressively, but apoptosis remained present until the end of treatment. In contrast, coagulating glands were less sensitive to DES. No major morphological changes were observed in these glands except for a moderate reduction of protein secretion. The levels of the apoptotic and proliferating cells indices were very low in the coagulating glands during DES treatment. In conclusion, these data point to different sensitivities of the accessory sex glands to DES exposure because DES induces extensive alterations of the normal morphology of the seminal vesicles, but shows only a moderate impact on the coagulating glands. PMID- 10735556 TI - Normal fertility in ewes after cervical insemination with frozen-thawed spermatozoa supplemented with seminal plasma. AB - The effect of seminal plasma (SP) on the motility, capacitation status, penetration through cervical mucus and fertility of frozen-thawed ram spermatozoa was examined. In the presence of SP, motility of frozen-thawed spermatozoa was better (P<0.001) and there were more uncapacitated and less acrosome-reacted cells in comparison with controls (P<0.001). Frozen thawed spermatozoa were also better able to penetrate cervical mucus after addition of SP. Addition of SP increased the percentage of ewes pregnant after insemination of frozen-thawed (39/94, 41.5% v. 51/92, 55.4%; P<0.05) but not fresh spermatozoa (34/55, 61.8% v. 42/58, 72.4% for 0 v. 30% SP in the resuspension medium). Moreover, SP improved pregnancy rates after cervical (14/50; 28% v. 25/49; 51%; P<0.05) but not intrauterine insemination (25/44; 56.8 v. 26/43; 60.5%) with frozen-thawed spermatozoa. In a second experiment, pregnancy rates were 30/45 (66.7%), 9/37 (24.3%) and 24/40 (60.0%) for ewes inseminated with frozen-thawed spermatozoa in the uterus (control), cervix without SP and cervix after supplementation with SP, respectively (P<0.01 for unsupplemented v. supplemented spermatozoa). These experiments demonstrate that impaired function of cryopreserved spermatozoa can be overcome by addition of SP, resulting in normal fertility after cervical AI. PMID- 10735557 TI - In vitro maturation and in vitro fertilization of bovine oocytes cultured in a chemically defined, protein-free medium: effects of carbohydrates and amino acids. AB - This study was conducted to examine the effects of carbohydrates and amino acids on the maturation and fertilization of bovine oocytes. To evaluate the effect of each treatment without any unpredictable interference, oocytes were cultured in a simply defined medium (modified Tyrode's medium; mT) without the addition of hormones and proteins. In Experiment 1, oocyte maturation to the metaphase-II stage was significantly (P<0.0001) enhanced after the addition of glucose (5.6 mM), lactate (10 mM) and/or pyruvate (0.5 mM) to mT (37-74%) than after no addition (0%). In mT supplemented with glucose, the addition of 19 essential and non-essential amino acids (aa; 0, 0.01, 0.1, 1, 5 or 10%) did not further improve in vitro maturation (Experiment 2) or in vitro fertilization (Experiment 3) of oocytes. However, more (P<0.05) pronuclear formation after in vitro-insemination was found in oocytes matured in mT with 1% aa and glucose than in oocytes matured in mT with glucose alone (56% vs. 35%). Penetration of spermatozoa into the ooplasm was initiated at 3 h after insemination and pronuclear formation from 8 h (Experiment 4). When cultured inseminated oocytes were examined up to 192 h post insemination, a significant (P<0.05) increase in the number of 2-cell (18 v. 38%) and 8-cell embryos, (7 v. 20%) and morulae (0 v. 8%) was found after the addition of 1% aa to mT with glucose than after no addition (Experiment 5). A limited number of oocytes matured in mT with aa and glucose developed to the blastocyst stage (6%). These results indicate that exogenous carbohydrates and amino acids are prerequisites for the maturation and fertilization of bovine oocytes in vitro. Glucose alone promotes the nuclear maturation of oocytes, whereas amino acids aid the pronuclear formation of fertilized oocytes. PMID- 10735558 TI - Localization and partial characterization of a molecule found in the plasma membrane of starfish and sea urchin embryos using a novel monoclonal antibody. AB - A monoclonal antibody, P212, has been developed which reacts against 120 kD and 90 kD proteins, P212 antigens found in the plasma membranes of both starfish and sea urchin embryos. Immunocytochemical studies with colloidal gold tagged P212 showed that the P212 antigens are also found on the inner membranes of several different types of granules and vesicles located in the eggs and cytoplasm of embryonic cells, and in the trans region of the Golgi apparatus. One or both of the P212 antigens are also found in the cortical granules, granules which are exocytosed at fertilization. They are not found on intracellular membrane structures such as the rough endoplasmic reticulum, the nuclear membrane, or a number of deeper granular structures. The P212 antigens are probably located on the membranes of vesicles which are on the exocytosis-endocytosis pathway and may be involved in cell trafficking. PMID- 10735560 TI - Camel (camelus dromedarius) milk PP3: evidence for an insertion in the amino terminal sequence of the camel milk whey protein. AB - The camel (camelus dromedarius) milk proteose peptone 3 (PP3) was purified successively by size exclusion fast protein liquid chromatography and reversed phase high performance liquid chromatography and then characterized by amino acid residue composition determination and chemical microsequencing after CNBr or trypsin cleavages. In comparison with the previously reported structure of camel milk whey protein, the camel PP3 contains an insertion in the N-terminal region which has approximately 24 residues, whereas the remaining C-terminal regions of these two homologous proteins are essentially identical. The camel PP3 seems to contain a potential O-glycosylation site localized in this insertion and 2 or 3 phosphorylated serine residues. PP3 belongs to the glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) family and could therefore play an immunological role in the camel or its suckling young. PMID- 10735559 TI - Superoxide generation and tyrosine kinase. AB - NADPH oxidase is a multi-subunit enzyme complex responsible for superoxide generation in many cells, for example, B-lymphocytes and osteoclasts. NADPH oxidase is localized on the cell surface and generates superoxide extracellularly. After synthesis, components of this oxidase are transported to the cell membrane where the functional NADPH oxidase complex is assembled. The mechanism by which the membrane-bound components are transported to the cell surface of osteoclasts remains unclear. In this study, we examined the role of tyrosine kinase activity in the transport of NADPH oxidase components. When B lymphocytes and osteoclasts were treated with herbimycin A, a specific inhibitor of tyrosine kinase, superoxide production was significantly decreased. The amount of p91, the catalytic subunit of NADPH oxidase, was decreased in the cellular membrane of herbimycin A treated cells compared to untreated controls. Similar results were obtained for the movement of a regulatory subunit of the NADPH oxidase complex, p47, in B-lymphocytes. Thus, inhibition of tyrosine kinase decreases superoxide production by disrupting the translocation of the NADPH oxidase complex. PMID- 10735561 TI - Signal transduction pathways, and nuclear translocation of zinc and metallothionein during differentiation of myoblasts. AB - The changes in subcellular localization of metallothionein during differentiation were studied in two myoblast cell lines, L6 and H9C2. Addition of insulin like growth factor-I or lowering foetal bovine serum to 1% can induce differentiation of myoblasts to myotubes. Metallothionein and zinc were localized mainly in the cytoplasm in myoblasts but were translocated into the nucleus of newly formed myotubes during early differentiation. In fully differentiated myotubes, metallothionein content was decreased with a cytoplasmic localization. Addition of an inhibitor of mitogen-activated protein kinase, PD 98059, did not affect differentiation but blocked nuclear translocation of metallothionein. LY 294092, an inhibitor of PI3 kinase, and rapamycin, an inhibitor of p70S6 serine/threonine kinase, abolished insulin-like growth factor-I induced differentiation of myoblasts, retained metallothionein in the cytoplasm, and decreased metallothionein content. These results demonstrate that the cytoplasmic-nuclear translocation of metallothionein occurs during the early stage of differentiation of myoblasts to myotubes and can be blocked by inhibition of certain signal transduction pathways. The transient nuclear localization of metallothionein and zinc may be related to a high requirement for zinc for metabolic activities during the early stage of differentiation. PMID- 10735563 TI - An NH2-terminal deleted plasma membrane H+-ATPase is a dominant negative mutant and is sequestered in endoplasmic reticulum derived structures. AB - The NH2-terminus of the plasma membrane H+-ATPase is one of the least conserved segments of this protein among fungi. We constructed and expressed a mutant H+ ATPase from Saccharomyces cerevisiae deleted at an internal peptide within the cytoplasmic NH2-terminus (D44-F116). When the enzyme was subjected to limited trypsinolysis it was digested more rapidly than wild type H+-ATPase. Membrane fractionation experiments and immunofluorescence microscopy, using antibodies against H+-ATPase showed that the mutant ATPase is retained in the endoplasmic reticulum. The pattern observed in the immunofluorescence microscopy resembled structures similar to Russell bodies (modifications of the endoplasmic reticulum membranes) recently described in yeast. When the wild type H+-ATPase was co expressed with the mutant, wild type H+-ATPase was also retained in the endoplasmic reticulum. Co-expression of both ATPases in a wild type yeast strain was lethal, demonstrating that this is a dominant negative mutant. PMID- 10735562 TI - The phosphatase domains of LAR, CD45, and PTP1B: structural correlations with peptide-based inhibitors. AB - PTP1B is a cytosolic protein tyrosine phosphatase that is a regulator of the kinase activity of the insulin receptor; the two protein tyrosine phosphatases LAR and CD45 are receptor type phosphatases crucially important to cell function. LAR also is involved in regulation of the insulin receptor while CD45 is critical for T-cell activation. Although LAR and CD45 are both transmembrane phosphatases, these enzymes manifest their phosphatase activity through a catalytic cytosolic domain. We have utilized X-ray coordinates of related phosphatases (RPTPalpha and RPTmu) and comparative protein modeling to obtain molecular models of the D1 catalytic domains of CD45 and LAR. The models were tested using established protocols and found to be comparable to low resolution X-ray structures. The structure obtained for LAR was compared with the recently reported X-ray structure. Both the CD45-D1 and LAR-D1 structures were then compared to and contrasted with PTP1B. The active site of pockets of the three enzymes were found to be very uniform in structure and charge distribution. Also, the gross surface topology around the active site was found to be somewhat similar for the 3 phosphatases. However, there were significant differences in surface topology, and, more importantly, large changes in surface charge distribution. The differences between the surface features of these enzymes provide an explanation for the selectivity of inhibition by a number of peptides. PMID- 10735564 TI - Long chain arginine esters: a new class of cationic detergents for preparation of hydrophobic ion-paired complexes. AB - The ability of stoichiometric amounts (based on charged groups) of ionic detergents to bind to oppositely charged ionic compounds has been recently reviewed. These hydrophobic ion-paired (HIP) complexes display altered solubility properties. Most of the work to date on HIP compelxes has focused on basic drugs and anionic detergents. It would be extremely useful to extend this approach to acidic compounds, including DNA and RNA. However, most cationic detergents are relatively toxic. It is hypothesized that detergents constructed from naturally occurring or well tolerated components, coupled by labile linkages, will be less toxic and still able to form strong HIP complexes. This study describes the synthesis and characterization of long chain alkyl esters of arginine. This class of cationic detergents, which have not been reported previously, are less cytotoxic than alkyltrimethylammonium detergents, possibly making them more acceptable in drug delivery applications. These arginine esters exhibit detergent like properties. For example, the dodecyl ester of arginine has a critical micelle concentration of 0.07 mM, while being approximately 5-10 fold less toxic than tetradecyltrimethylammonium bromide. The arginine dodecyl ester forms stable HIP complexes with plasmid DNA. The complex is sufficiently stable to allow some modest level of transfection with Cos-7 cells in a time- and concentration dependent fashion. This work demonstrates that arginine-based cationic detergents are effective ion-pairing agents, appear to be less toxic than alkyltrimethylammonium compounds, and form stable complexes with DNA. PMID- 10735565 TI - There isn't an ideal smooth-surface material--yet: the history and future of urologic materials. AB - Indwelling and implantable urologic devices did not become commercially available until the late 1970s. The first such device was a ureteral stent made of silicone. Other materials-polyethylene, polyurethane, C-Flex, and Percuflex-have since been introduced. Each of these materials is rated for a particular indwell time, after which the material may begin to break down. Various methods of modifying the surface to make it more slippery or to prevent infection have been tested. Attempts have been made to create artificial organs and have not been entirely successful, and the future may lie with tissue-engineered materials such as small intestinal submucosa. PMID- 10735566 TI - A short history of the Foley catheter: from handmade instrument to infection prevention device. AB - Although it is one of the most frequently utilized devices in the hospitalized patient, the Foley catheter has often been taken for granted. This lack of attention is unfortunate, as the Foley catheter remains one of the primary sources of hospital-acquired infections, which increase morbidity, mortality, and the financial burden on the healthcare system. Although education on the appropriate techniques, proper use, and early removal of Foley catheters is important, such measures unfortunately result in transient benefits. Current technologic advancements have moved the coating technology to a state where bacterial adhesion and migration can be limited and the frequency of catheter associated urinary tract infections can be reduced. Future technological advances in the Foley catheter will help provide better care and comfort for the catheterized patient. PMID- 10735567 TI - Alloplasts and biointegration. AB - During the past 2000 years, medical science has embraced the use of alloplastic materials to improve and replace many bodily functions. During the past 50 years, there has been a virtual explosion of alloplastic implant and device technologies. Alloplasts have been borrowed from industry with few if any modifications. Experimental and clinical studies have shown that smooth, textured, and fabric-surfaced alloplasts excite a fibrous capsule around the implant and distance normal tissue and vascularity from the implant-tissue interface. In contrast, some porous alloplasts have been shown to bond with tissues, reducing the fibrous encapsulation and promoting vascularization. The physical and chemical stability of the implant in situ and its surface composition, texture, and pore size significantly influence the tissue response. Lessons learned during the past 50 years are now being translated into new medicine-specific alloplasts. PMID- 10735568 TI - Urinary encrustation of alloplastic materials. AB - Currently, all alloplastic materials placed within the urinary tract are susceptible to encrustation, and devices made from these biomaterials are prone to the associated complications. Encrustation is affected by such surface properties as roughness and irregularity, hydrophobicity and wetability, charge, polymer chemistry, and coatings. The composition of the surface may also influence reactivity at the interface. A raised urinary pH as a result of infection with urease-producing bacteria is an important mechanism of encrustation, but encrustation can take place in the absence of infection. Individual variations in the risk of encrustation have been identified. Better device designs are required, as are better models for examining biomaterials and biofilm formation. With a coordinated multidisciplinary approach and adequate resources, we may solve the problem of encrustation, which has such an adverse effect on patient quality of life and on healthcare costs. PMID- 10735569 TI - Soft porous PTFE-composite alloplasts: tissue-bonding characteristics. AB - Experimental and clinical investigations over the past three decades have validated numerous hypotheses about tissue bonding of prostheses. Highly porous PTFE composite has been demonstrated to be an effective, inert, scaffold-like matrix onto which other materials can become physically bonded to achieve a tissue-specific result. Hydrophilic agents such as carbon and aluminum oxide stimulate ingrowth of fibroblasts that secrete collagen. These additives promote a robust and durable alloplast-soft tissue union composed mostly of collagen that is watertight. Bone-growth stimulants-such as hydroxyapatite-when added to highly porous PTFE stimulate tissue ingrowth and maturation that is more bone like. PMID- 10735570 TI - Infection-resistant alloplasts. AB - Prosthesis-related infection accounts for nearly half of nosocomial infections, resulting in significant morbidity, mortality, prolonged hospitalization, and higher healthcare costs. Although numerous antimicrobial-coated surfaces have been suggested to guard against prosthesis-related infection, only a few, such as minocylcine plus rifampin, are clinically protective. The differences in clinical efficacy can be attributed at least in part to differences in the magnitude of leaching of the antimicrobial agent off the surface. There is a pressing need to explore the clinical efficacy of antimicrobial surfaces suitable for use in devices intended for long-term use. PMID- 10735571 TI - Bacteria, biofilms, and devices: the possible protective role of phosphorylcholine materials. AB - Phosphorylcholine (PC)-surfaced alloplastic materials improve the biocompatibility of medical devices in contact with blood or tears. Recent experimental studies support the contention that PC surfaces also retard bacterial and crystal adhesion in urine. PMID- 10735572 TI - A chemically intelligent antimicrobial coating for urologic devices. AB - Contact biocides are a relatively new form of infection-resistant materials. Surfacine is a silver based antimicrobial coating that can be immobilized on the surface of most alloplastic materials used to fabricate devices. It exhibits broad-spectrum antimicrobial activity exclusively at the surface without elution and does not induce antimicrobial activity in contacting fluids such as urine regardless of volume. Because it is permanently immobilized on the material surface, the coating is nontoxic to cells and is not anticipated to exhibit immunogenicity, teratogenicity, or carcinogenicity. PMID- 10735573 TI - Tissue engineering of artificial organs. AB - Tissue engineering efforts are currently being undertaken for every type of tissue and organ within the urinary system. Most of the effort expended to engineer genitourinary tissues has occurred within the last decade. Tissue engineering techniques require expertise in growth factor biology, a cell culture facility designed for human application, and personnel who have mastered the techniques of cell harvest, culture, and expansion. Polymer scaffold design and manufacturing resources are essential for the successful application of this technology. In order to apply these engineering techniques to humans, further studies need to be performed with many of the tissues described. The first human application of cell-based tissue engineering technology for urologic applications took place at our institution, with the injection of autologous cells for the correction of vesicoureteral reflux in children. The same technology has been expanded to treat adult patients with urinary incontinence. Trials of urethral tissue replacement with processed collagen matrices are in progress, and bladder replacement using tissue engineering techniques are currently being arranged. Recent progress suggests that engineered urologic tissues may have clinical applicability in the future. PMID- 10735574 TI - Bioengineering organs using small intestinal submucosa scaffolds: in vivo tissue engineering technology. AB - Tissue engineering is a promising field of research that has the potential to revolutionize urinary bladder reconstruction. Currently, two techniques for the induction of bladder regeneration are being researched. The first, the in vivo technique, involves the use of a biodegradable scaffold that the host bladder can use to remodel and regenerate. This technique takes advantage of the cell's natural ability to heal or regenerate itself back to a normal state. The second technology, the in vitro technique, involves establishment of primary cell cultures from the host's native bladder. These cells are seeded on a biodegradable scaffold to create a composite graft that is then transplanted back into the host for continuation of the regeneration process. Clearly, both techniques have advantages and disadvantages, and both will have some role in future urinary reconstruction. To date, the most successful results utilizing in vivo techniques have been with small intestinal submucosa (SIS). In this article, we discuss in vivo tissue engineering technology and the preclinical studies that have been performed utilizing SIS for urinary tract regeneration. PMID- 10735575 TI - The prosthetic ureter. AB - Ureteral replacement has always been a challenge. Two approaches have been explored: in situ augmentation or replacement and extra-anatomic passage of a conduit. An in-situ prosthetic ureter is basically a simple al. loplastic tube connected to the urinary tract by end-to-end sutures or by intubation and closure. Antireflux devices and peristaltic mechanisms are not necessary. Among the in-situ designs, only those composed of silicone and silicone rubber have performed at all well. Tissue engineering and acellular matrix grafts have produced impressive early results. Subcutaneous ureteral replacement with alloplasts, including a coaxial assembly of an inner silicone and outer expanded polytetrafluoroethylene tube, has produced good results. In the future, we are likely to see bioengineered neotissue combined with highly porous and infection resistant alloplasts to create better and more functional neo-organs. PMID- 10735576 TI - Urethral devices for managing stress urinary incontinence. AB - Although surgery remains the gold standard for managing the majority of patients with stress urinary incontinence or retention, not all patients are suitable candidates or desire surgery. For these patients, several urethral caps and inserts are available, which are described. In approximately 50% of patients, satisfactory control is achieved. Further investigation undoubtedly will yield better devices to serve the large number of patients with urinary control problems. PMID- 10735577 TI - Tissue-bonding cystostomy: potential alternative for neuropathic bladder dysfunction and potential model for other prostheses. AB - Neuropathic bladder dysfunction commonly results in urinary retention, incontinence, or both. At present, catheterization is commonly used in these patients to empty the bladder, but bacteriuria is a common result. Construction of a device that combines infection-resistant materials and a closed ecosystem has been attempted, yielding a prototype tissue-bonding cystostomy device. This device and the results of the early trials are described. PMID- 10735578 TI - Tissue expansion: a promising trend for reconstruction in urology. AB - To evaluate the role of the tissue expansion concept in reconstructive urology, the contemporary literature was reviewed, together with our own results with tissue expansion. The principle of tissue expansion has been applied in cases of bladder augmentation, ureteral dilation for the generation of indigenous tissue and subsequent ureterocystoplasties, ureteral elongation, and dilation of the renal pelvis, producing native tissue for the reconstruction of defects or strictures of the upper ureter. Even though the exact mechanism of action of mechanical strain in different tissues is not known, tissue expansion is a well accepted technique that can amplify the armamentarium of reconstructive urologists for the management of defects along the urinary tract. PMID- 10735579 TI - Intravesical neuromodulatory drugs: capsaicin and resiniferatoxin to treat the overactive bladder. AB - Current pharmacologic treatment of the overactive bladder relies on anticholinergic drugs. However, these drugs often have troublesome side effects and frequently are given in doses insufficient to restore continence in patients with detrusor instability. We present the background and basic and clinical research dealing with intravesical instillation of capsaicin and resinfferatoxin as treatments for the overactive bladder. Capsaicin is the main pungent ingredient in "hot" peppers of the genus Capsicum. It is a specific neurotoxin that desensitizes C-fiber afferent neurons, which may be responsible for the signals that trigger detrusor overactivity. Studies with capsaicin over the past 8 years have demonstrated clinical efficacy with minimal long-term complications. Most of these studies have also shown that the acute pain and irritation associated with capsaicin are a major deterrent to widespread use. Resiniferatoxin (RTX), an ultrapotent analog of capsaicin that appears to have similar efficacy but with much less acute side effects may be more useful. Intravesical instillation of capsaicin or resiniferatoxin is a promising treatment for the overactive bladder. PMID- 10735581 TI - Haplotype analysis of three polymorphisms of the COL2A1 gene and associations with generalised radiological osteoarthritis. AB - It was investigated whether radiographic osteoarthritis (ROA) is associated with specific haplotypes of the COL2A1 gene. Radiographs of knees, hips, hands, and spine were scored for the presence of ROA in subjects of 55-70 years from a population-based cohort study, the Rotterdam study. Cases had ROA in 3 or more joint groups; controls, from the same population, had ROA in less than 3 joint groups. Allele frequencies of 3 dimorphisms (HaeIII, HindIII, MaeII) and a VNTR polymorphism of the COL2A1 gene were determined. The VNTR allele 14R2 and the HindIII polymorphism showed a significant association. Haplotype analysis of the HaeIII, HindIII and VNTR polymorphisms showed that a specific haplotype (1-2 14R2) is strongly associated with ROA in 3 or more joint groups (OR = 5.3, 95% CI 2.3-12.7). Our results suggest that a specific haplotype of the COL2A1 locus may predispose to generalised ROA. PMID- 10735580 TI - Superiority of denaturing high performance liquid chromatography over single stranded conformation and conformation-sensitive gel electrophoresis for mutation detection in TSC2. AB - We evaluated denaturing high pressure liquid chromatography (DHPLC) as a scanning method for mutation detection in TSC2, and compared it to conformation-sensitive gel electrophoresis (CSGE) and single-stranded conformation polymorphism analysis (SSCP). The first 20 exons of TSC2 were amplified from 84 TSC patients and screened initially by CSGE and then by DHPLC. Optimization of DHPLC analysis of each exon was carried out by design of primers with minimum variation in the melting temperature of the amplicon, and titration of both elution gradient and temperature. CSGE analysis identified 40 shifts (21 unique) in the 84 patients and 20 exons. All of these variants were detected by DHPLC, and an additional 27 changes (14 unique) were identified. Overall 15 of 28 (54%) unique single base substitutions were detected by CSGE; all were detected by DHPLC. 25 definite or probable mutations were found in these 84 patients (30%) in exons 1-20 of TSC2. In a subsequent blinded analysis of 15 samples with 18 distinct TSC2 sequence variants originally detected by SSCP in another centre, all variants were detected by DHPLC except one where the variation occurred within the primer. Ten other (7 unique) sequence variants were detected in these samples which had not been detected by SSCP. Overall, 11 of 16 (69%) unique single base substitutions were detected by SSCP; all were detected by DHPLC. We conclude that DHPLC is superior to both CSGE and SSCP for detection of DNA sequence variation in TSC2, particularly for single base substitution mutations. PMID- 10735582 TI - Evidence for a major gene accounting for mild elevation in LDL cholesterol: the NHLBI Family Heart Study. AB - Studies of rare Mendelian disorders of low density lipoprotein cholesterol (LDL C) metabolism have identified specific genetic mutations in the LDL receptor and apolipoprotein B. Although these rare mutations account for a small proportion of LDL-C variation, twin and adoption studies indicate that at least 50% of the overall LDL-C observed variation is genetically determined. In a heterogeneous sample of 3227 subjects from the NHLBI Family Heart Study collected from four US centres, we find evidence for a common major gene accounting for mild elevations (1.25 standard deviations) in LDL-C. The analysis favored a recessive model with a frequency of 0.52 for the gene influencing elevated LDL-C, phenotypic means of 113 mg/dl for the normal genotypes and 146 mg/dl for the abnormal genotype, and a significant polygenic heritability. This statistically-inferred major gene accounted for 24% of the variation in LDL-C, with polygenes accounting for another 28% of the variation. Using parameters for major gene transmission estimated in the segregation analysis, LDL-C showed no linkage to the LDL receptor gene (LDLR), nor to the apolipoprotein E gene (APOE), nor to the cholesterol 7alpha-hydroxylase gene (CYP7A1), indicating the major gene effect influencing mild elevation in LDL-C is not explained by any of these candidate loci. PMID- 10735583 TI - Phylogeographic patterns of mtDNA reflecting the colonization of the Canary Islands. AB - Although the Canary Islands were settled by humans, possibly of Berber origin, as late as 2500 years ago, the precise course and numbers of early migrations to the archipelago remain controversial. We have therefore analysed mtDNA variation (HVS I as well as selected RFLP sites) in 300 individuals from the seven Canary Islands. The distribution and variation across the islands in a specific mtDNA clade of Northwest African ancestry suggest that there was one dominant initial settlement process that affected all the islands, from east to west. This indicates that a certain genetic affinity of present-day Canary Islanders to Northwest African Berbers mainly stems from the autochthonous population rather than slaves captured on the neighbouring African coast. The slave trade after the European conquest left measurable, though minor, traces in the mtDNA pool of the Canary Islands, which in its majority testifies to the European immigration. PMID- 10735584 TI - Statistical genetic analysis of plasma levels of vitamin D: familial study. AB - The present study attempted to evaluate the relative importance of familial/genetic factors in interindividual variation of plasma concentrations of vitamin D (calcidiol). Ninety-five nuclear pedigrees (187 males and 168 females, aged 18-91 and 18-86 years old respectively), from the Chuvasha Autonomy, Russia, were assessed for calcidiol and sex hormones. Age-adjusted 25-hydroxy-vitamin D (25OHD) measures showed very high pair-wise correlations between all members of families, which suggest that there may be a genetic factor transmitted within the families. To test this assumption, complex segregation analysis of non-adjusted 25OHD data was undertaken, in which genotype-sex specific dependence of the trait on age was incorporated. The analysis showed that some 44% of the 25OHD variance was attributable to all genetic and non-genetic factors incorporated into the model. About 22% of the variation was accounted for by a putative major gene effect. PMID- 10735585 TI - Significance testing for direct identity-by-descent mapping. AB - Direct identity-by-descent mapping is a technique for narrowing down the location of the gene or genes responsible for a given genetic disease to small segments of the genome. The technique involves DNA comparisons between pairs of affected individuals. The data generated are in the form of matching segments of the genome, representing regions likely to be identical-by-descent (IBD). Regions in the genome over which there are significantly more segments aligned than is expected by chance are taken as candidate regions for the disease gene or genes. Due to the complex geometric nature of the data, significance testing involves certain mathematical difficulties. We present here a new method for measuring this significance. This method introduces a novel statistic and is appropriate whether or not the relationships between the paired individuals are known. We give examples that we have calculated by implementing this method, including an application to real data. PMID- 10735586 TI - The additive genetic gamma frailty model for linkage analysis of age-of-onset variation. AB - Age of onset is a key factor in the linkage analysis of many complex diseases. Current methods in nonparametric linkage analysis are mainly concentrated on the affected relative pairs or affected family members with age of onset information either ignored or taken into account by specifying age-dependent penetrances for liability classes. On the other hand, gamma frailty models were developed in the biostatistics literature to model familial aggregation of age of onset. However, these frailty models cannot be used directly for linkage analysis. This paper extends the gamma frailty model by incorporating inheritance vector information and provides a semiparametric approach for linkage testing. For a given inheritance vector at the putative disease locus, we construct an additive genetic gamma frailty for each individual within a nuclear family and use the Cox proportional hazard model to model age of onset. We derive the conditional hazard ratio parameter for sib pairs and define a likelihood ratio based LOD score statistic under our model. The EM algorithm is used for estimating the parameters and the maximum likelihood functions. Simulated data sets are used to illustrate these new statistical methods. PMID- 10735587 TI - A correction to TDT statistics for mapping quantitative trait loci. PMID- 10735588 TI - Luteinizing hormone up-regulates the expression of interleukin-1 beta mRNA in human granulosa-luteal cells. AB - PROBLEM: Previously, we observed that follicular fluid obtained from patients with premature luteinization contained elevated interleukin-1 beta (IL-1 beta) levels. In this study. we aimed to examine the effects of luteinizing hormone (LH) on IL-1 beta expression and IL-1 beta on steroidogenesis in human granulosa luteal cells. METHOD OF STUDY: Human granulosa-luteal cells were obtained during oocyte retrieval. The cells were treated with either LH or IL-1 beta and subsequently were examined for the level of IL-1 beta transcript. The conditioned media were examined for IL-1 beta protein and steroid hormone levels. RESULTS: LH (250-500 mIU/mL) up-regulated the expression of IL-1 beta mRNA (up to a 4-fold increase over control; P<0.05) in the granulosa-luteal cells. IL-1 beta (5-50 ng/mL) increased the basal, but not LH-dependent, progesterone production from these cells in a dose-dependent manner after 96 and 144 hr of culture (P<0.05). However, an inhibitory effect of IL-1 beta on LH-dependent estradiol production was observed (up to 20% decrease, P<0.05). CONCLUSIONS: LH is capable of stimulating IL-1 beta transcript expression in human granulosa-luteal cells and may regulate ovarian steroidogenesis, at least partly through the activation of IL-1 beta. PMID- 10735589 TI - Anti-sperm antibodies from infertile patients and their cognate sperm antigens: a review. Identity between SAGA-1, the H6-3C4 antigen, and CD52. AB - PROBLEM: The correlation of anti-sperm antibodies (ASA) with some instances of unexplained infertility implicates a role for these antibodies in blocking fertilization. Improved diagnosis and treatment of immunologic infertility, as well as a more complete understanding of the mechanism behind this phenomenon, are dependent on the identification and characterization of relevant sperm antigens. METHOD OF STUDY: In this article, we review literature on methods employed to identify sperm antigens using anti-sperm polyclonal and monoclonal antibodies from infertile patients and vasectomized men. Particular focus is given to approaches using human and mouse monoclonal antibodies to define the SAGA-1 human sperm antigen. RESULTS: ASA present in sera and genital tract secretions from infertile patients and vasectomized men have been employed in a variety of methods to identify sperm antigens. In an alternate approach, a monoclonal antibody (mAb), H6-3C4, was immortalized from the lymphocytes of an infertile woman who exhibited sperm-immobilizing titers. Subsequently, the sperm agglutinating, murine S19 mAb was shown to react with the H6-3C4 cognate antigen. The H6-3C4 S19 cognate antigen, designated Sperm Agglutination Antigen-1 (SAGA 1), was characterized as a polymorphic, highly acidic, GPI-anchored glycoprotein on the surface of human spermatozoa. Purification with the S19 mAb followed by microsequencing demonstrated that the SAGA-1 core peptide is identical to CD52, a glycoprotein on the surface of human lymphocytes. Immunoblot analysis demonstrated that these two glycoproteins differed in carbohydrate composition. Thus, sperm SAGA-1 and lymphocyte CD52 represent glycoforms, glycoproteins with the same core peptide but with different carbohydrate structures. CONCLUSIONS: Autoimmunity to the SAGA-1 and/or CD52 glycoforms may lead to infertility. Structural and immunologic differences between these glycoproteins may be important factors in the etiology of immunologic infertility and other autoimmune disorders. PMID- 10735590 TI - Polyherbal formulations with wide spectrum antimicrobial activity against reproductive tract infections and sexually transmitted pathogens. AB - PROBLEM: Recent reports indicate high incidence of genital infections, most of which are sexually transmitted. Although specific drugs and antibiotics are available for some, a safe spermicidal formulation with wide spectrum antimicrobial action would be a desirable addition to the presently available spermicides. METHODS: Formulations at different dilutions were tested in culture systems on standard strains and clinical isolates including some isolates resistant to drugs. The effect on (HSV)-2 and Chlamydia trachomatis was determined in vivo in progestin sensitized mice. The effect on HIV-1 was investigated in two standardized systems. RESULTS: Polyherbal cream inhibited the growth in culture of clinical isolates of Candida albicans, Candida krusei and Candida tropicalis. Both the polyherbal cream and the Praneem polyherbal pessary inhibited urinary tract Escherichia coli (including multidrug resistant strains), and Neisseria gonorrhoeae (including 2 strains resistant to penicillin). Both formulations manifested virucidal activity against HIV-1 at >2 and 50% dilutions (in two different test systems) on contact for 1-2 min. Intravaginal inoculation of the cream and the pessary suspensions before inoculation of the pathogen prevented lesions and vaginal transmission of HSV-2 and C. trachomatis in progestin sensitized mice. CONCLUSIONS: Polyherbal formulations have wide spectrum antibacterial, antifungal and antiviral effect against the tested sexually transmitted pathogens. PMID- 10735591 TI - Interleukin-1 stimulates human uterine prostaglandin production through induction of cyclooxygenase-2 expression. AB - PROBLEM: Uterine infection occurs in as much as 20% of preterm labor and results in increased decidual cytokines. The objective of this study was to examine the effect of interleukin-1 (IL-1) and the cyclooxygenase-2 (COX-2) inhibitor, NS 398, on myometrial prostaglandin (PG) production and COX-2 expression. METHOD OF STUDY: Human uterine myocytes were stimulated with IL-1 (0-50 ng/mL) over 24 hr. PGE2, PGF2alpha, and 6-keto F1alpha were measured by enzyme-linked immunosorbent assay. Both COX-1 and COX-2 proteins and mRNA were measured by western and northern blot, respectively. RESULTS: IL-1 increased PG production beginning at 6 hr, COX-2 protein increased beginning at 4 hr and continued to increase at 24 hr. COX-2 mRNA increased at 2 hr and peaked at 4 hr. NS-398 blocked PG production but had no effect on COX-2 protein or mRNA. CONCLUSIONS: IL-1 increases PG production by myometrium by increased COX-2 expression. NS-398 completely blocks IL-1 induced PG production. With intrauterine infection, IL-1 may induce labor through the autocrine production of uterotonic PGs. PMID- 10735592 TI - Do soluble cell adhesion molecules play a role in endometriosis? AB - PROBLEM: Endometriosis is a chronic inflammatory disease associated with diverse immunologic disturbances. Cell adhesion molecules are essential for the development of immune and inflammatory reactions. This study was conducted to investigate whether or not serum and peritoneal levels of soluble cell adhesion molecules are altered in women with endometriosis. METHOD OF STUDY: The study group comprised five women with moderate-to-severe endometriosis. Eight healthy women with a normal diagnostic laparoscopy served as controls. Serum and peritoneal fluid samples from both groups were analyzed for the soluble isoform of intercellular cell adhesion molecule-1 (sICAM-1). vascular cell adhesion molecule-1 (sVCAM-1), endothelial selectin (sES), and platelet selectin (sPS). RESULTS: Serum levels of sICAM-1 were significantly increased in women with endometriosis (median levels: 410.4 ng/mL; range: 233.9 ng/mL 598.4 ng/mL vs. 235.7 ng/mL; range: 187.4 ng/mL -323.7 ng/mL; P = 0.02). Although the levels of sVCAM-1, sES, and sPS in both samples were higher in the study group, the differences did not reach significance. CONCLUSIONS: Our results suggest a role of ICAM-1 in the pathophysiology of endometriosis. However. the role of other investigated cell adhesion molecules should be confirmed by further studies. PMID- 10735593 TI - Establishment and epitope analysis of allo-specific cytotoxic T lymphocytes at a tumor site recognizing a spouse's HLA-A0206 molecule. AB - PROBLEM: The molecular basis of allo-reactivity in reproductive immunity has not been fully clarified. METHOD OF STUDY: Cytotoxic T lymphocytes (CTLs) were established from tumor-infiltrating lymphocytes (TILs). The allo-reactivity of the CTLs against various tumor cell lines or human leukocyte antigen (HLA)-A allele-transfected COS-7 cells was measured by 51Cr-release or interferon-gamma production assay. RESULTS AND CONCLUSIONS: We have established CTLs reacting to an HLA-A0206 molecule that matched a spouse's HLA-A allele from the TILs of a 68 year-old multiparous patient with gastric cancer. The amino acids at positions 66 and 88 in the alpha1 domain of HLA-A0206, both of which were common in the other HLA-A2 subtypes, were involved in the recognition by the CTLs. Endogenous peptides in the groove were not involved in the recognition. These results suggest the presence of long-lasting memory CTLs raised by the reproduction process, and may facilitate a better understanding of the molecular basis of allo recognition during reproduction. PMID- 10735594 TI - Study of early pregnancy factor (EPF) in equine (Equus caballus). AB - PROBLEM: Early pregnancy factor (EPF) is an immunosuppressive protein detected in the early pregnancy serum. We have already reported that we developed the rosette inhibition test for mare EPF and detected EPF in thoroughbreds. The aim of this study was to determine whether or not our method could be used clinically. METHODS OF STUDY: The rosette inhibition test for equine EPF was carried out on serum from six nonpregnant and six pregnant Shetland ponies, a female and a male Chinese pony, and four nonpregnant and 13 pregnant thoroughbred mares. In the thoroughbreds sera were collected during the pregnancy period. Furthermore, we measured progesterone and detected pregnant mare serum gonadotrophin (PMSG) in order to confirm pregnancy of the Chinese pony 3 and 6 months after mating. RESULTS: In the nonpregnant Shetland ponies, the rosette inhibition titre (RIT) was 6.0+/-1.0 and EPF was negative. In contrast, in the pregnant ponies, the RIT was 9.2+/-0.4 and EPF was positive. Based on these results, we diagnosed pregnancy of the Chinese pony. The RIT of the female Chinese pony (3 months after mating) was above 10 and EPF was positive. Furthermore, we detected PMSG and progesterone in the serum of this pony. EPF appeared in the maternal blood circulation at 24-72 hr after mating, it was detected until the second trimester, and after that it disappeared from the maternal serum. CONCLUSIONS: The pony's EPF was detected by using the same rosette inhibition test as in the thoroughbred and was present from 24 to 72 hr after mating until the second trimester. The results indicated that our method was useful for pregnancy diagnosis of Equine. PMID- 10735595 TI - Preventive effect of monoclonal antibodies to intercellular adhesion molecule-1 and leukocyte function-associate antigen-1 on murine spontaneous fetal resorption. AB - PROBLEM: Are cell adhesion molecules involved in the murine model of immunologically-mediated spontaneous abortion? METHOD OF STUDY: Pregnant CBA/J female mice mated with DBA/2 male mice were injected with monoclonal antibodies (MAbs) to intercellular adhesion molecule-1 (ICAM-1) and leukocyte function associate antigen-1 (LFA-1). On day 13 of gestation. viable and resorbed embryos were counted. Natural killer (NK) cell activity in the spleen, mixed lymphocyte reactions (MLR), mixed lymphocyte-placenta reactions (MLPR), and levels of interferon (IFN)-gamma were assayed. RESULTS: Significant suppression of fetal resorption was observed by the injection of MAb to ICAM-1 and LFA-1. NK cell activity and the MLR anti-(CBA/J x DBA/ 2)F1 were reduced in the antibody-treated CBA/J spleen. Moreover, the level of IFN-gamma was significantly lower in the MLPR supernatants from the antibody-treated group than those of the control group. CONCLUSIONS: One mechanism in the murine model of spontaneous abortion may be through the interaction of cell adhesion molecules, which may modulate NK cell activities and cytokine production. PMID- 10735596 TI - Inhibin A, inhibin B and activin A in follicular fluid of infertile women with tubal damage, unexplained infertility and emdometriosis. AB - PROBLEM: To measure and compare concentrations of inhibin A, inhibin B, activin A and oestradiol in the follicular fluid of women with endometriosis, tubal damage and unexplained infertility with oocyte quality and fertilising capacity. Also, to assess whether impaired follicular function in women with endometriosis might be related to altered inhibin or activin concentrations and whether this correlated. METHOD OF STUDY: Follicular fluids were collected from individual follicles during oocyte retrieval for in vitro fertilisation (IVF) in natural cycles. Inhibin A, inhibin B and activin A were measured using two-site enzyme immunoassay, and oestradiol was assayed by fluoro-immunometric method. RESULTS: Follicular fluid inhibin A levels were found to be significantly higher in women with endometriosis. Inhibin A was directly correlated with follicle size. There was no correlation between the levels of inhibin A, inhibin B, activin A and oocyte quality or fertilising capacity in the three groups of women. CONCLUSIONS: Follicular fluid concentration of inhibin A is elevated in follicles of women with endometriosis and is positively correlated with follicle maturation. However, we were unable to demonstrate any association between the follicular fluid concentrations of inhibin A, inhibin B, activin A or oestradiol and the quality and fertilisation capacity of oocytes in women with tubal damage, unexplained infertility or endometriosis. PMID- 10735597 TI - Localization of nitric oxide synthase in human trophoblast cells: role of nitric oxide in trophoblast proliferation and differentiation. AB - PROBLEM: There are conflicting reports about the isoform of nitric oxide synthase (NOS) present in trophoblast cells. In this study, we have examined the presence of different NOS isoforms in trophoblast cells. In addition, the role of nitric oxide (NO) in trophoblast function has also been studied by investigating the possible role of nitric oxide in trophoblast proliferation and differentiation. METHOD OF STUDY: NOS isoforms in primary-term trophoblast and JEG-3 cells were identified by immunocytochemistry. The intracellular localization of this enzyme was determined by confocal laser scanning microscopy. Trophoblast proliferation was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasolium bromide (MTT) conversion assay and cellular differentiation was monitored by human chorionic gonodotropin (hCG) and progesterone secretion, measured by radioimmunoassay. RESULTS: The immunoreactive NOS was present in human trophoblast cells of normal term placenta and JEG-3 cells (a choriocarcinoma cell line) maintained in culture. Nicotinamide adenine dinucleotide phosphate (NADPH) dependent diaphorase activity overlapped with the immunostaining of NOS. Specific antibodies against the different isoforms of NOS detected the presence of neuronal-type NOS (nNOS) only. The other two isoforms, i.e., eNOS (endothelial) and iNOS (macrophage specific) were completely absent. The nNOS was localized in cell cytoplasm. In culture, JEG-3 cells normally undergo proliferation and cytotrophoblast cells in primary culture differentiate to form hormone-secreting syncytial cells. Sodium nitroprusside (SNP), a nitric oxide donor, when added to the culture, significantly increased proliferation of JEG-3 cells and inhibited the differentiation of cytotrophoblast cells. The arrest by SNP in the formation of syncytial cells was further evidenced by the low secretion profile of hCG and progesterone. CONCLUSIONS: Our findings suggest for the first time the presence of nNOS in the human trophoblast cells and a previously unrecognized role of NO in trophoblast proliferation and differentiation. PMID- 10735598 TI - Interleukin-8 levels and granulocyte counts in cervical mucus during pregnancy. AB - PROBLEM: We measured interleukin-8 (IL-8) and granulocyte counts in cervical mucus to assess local immunity and parturition. METHOD OF STUDY: We detected IL-8 by enzyme linked immunosorbent assay (ELISA) and Western blotting. Granulocytes were counted on a slide glass containing mucus from the external cervical os. RESULTS: ELISA and Western blotting revealed IL-8 in cervical mucus from both nonpregnant and pregnant women. There were no significant differences in cervical mucus IL-8 levels or granulocyte counts between follicular, ovulatory, and luteal phases. However, IL-8 and granulocyte counts were significantly decreased after menopause. IL-8 and granulocyte counts were increased significantly during pregnancy, and were further increased after 38 weeks of gestation and at labor. IL-8 levels were significantly correlated with granulocyte counts, based on the study of 678 samples (P <0.0001). CONCLUSION: IL-8 is involved in the increase of cervical granulocytes and in the process of parturition. PMID- 10735599 TI - Interleukin-1 beta down-regulates the oxytocin receptor in cultured uterine smooth muscle cells. AB - PROBLEM: Intrauterine infection accounts for 20% of preterm labor and results in the production of decidual inflammatory cytokines, including interleukin-1 (IL 1). The oxytocin receptor plays a key role in the onset of preterm labor. Cytokines likely regulate oxytocin receptor expression through several cytokine induced DNA-binding proteins. METHOD OF STUDY: The objective of this study was to evaluate the effect of the IL-1 alone on oxytocin receptor number as measured by radioligand binding and immunocytochemistry, and oxytocin receptor mRNA as measured by reverse transcriptase-polymerase chain reaction (RT-PCR) in cultured uterine myocytes. RESULTS: Unexpectedly, IL-1 treatment decreased oxytocin receptor number from 111,067 to 23,941 receptors/cell. Loss of oxytocin receptor binding began after 8 hr of IL-1 treatment and was reversible after IL-1 removal. Immunocytochemistry confirmed a loss of cellular oxytocin receptors. Oxytocin receptor mRNA decreased beginning after 2 hr of IL-1 treatment. CONCLUSIONS: IL-1 down-regulates the uterine oxytocin receptor in a time- and dose-dependent fashion. PMID- 10735600 TI - Vascular cell adhesion molecule-1 in normal, failed, and ectopic pregnancy. AB - PROBLEM: Vascular cell adhesion molecule-1 (VCAM-1) is involved in early pregnancy establishment. This study sought to determine whether soluble VCAM-1 (sVCAM-1) serum levels differ among normal, failed, and ectopic pregnancy, its capacity to serve as a marker for pregnancy viability or ectopic pregnancy, and its correlation with serum progesterone and beta human chorionic gonadotrophin (PHCG) levels. METHOD OF STUDY: Maternal serum samples were obtained from 20 women with ectopic, 10 with normal, and 10 with failed intra-uterine pregnancy, all of comparable gestational age. Samples were assayed for sVCAM-1, progesterone, and betaHCG by specific assays. RESULTS: The median serum level of sVCAM-1 was comparable between the three pregnancy types (normal: 578.3 ng/mL, range 434.4-699.5 ng/mL; failed: 567.8 ng/mL, range 401.9 669.5 ng/mL; and ectopic: 470.7ng/mL range, 328.2-1151.1 ng/mL). Serum levels sVCAM-1 were not significantly correlated with betaHCG or progesterone levels. CONCLUSION: sVCAM-1 is not appropriate to serve as a marker for pregnancy viability or ectopic pregnancy. PMID- 10735601 TI - Immunotherapy prevents recurrent abortion without influencing natural killer receptor status. AB - PROBLEM: We assessed the expression of natural killer (NK) receptors in recurrent aborters before and after immunotherapy using their husbands' peripheral blood mononuclear cells (PBMCs). METHOD OF STUDY: Using stored PBMCs from recurrent aborters before and after the immunotherapy, the expression of NK receptors, CD158a, CD158b, CD159 and CD94, were analyzed using monoclonal antibodies for respective receptors. The diversity of killer activatory receptors (KARs) and killer inhibitory receptors (KIRs) was also examined using reverse transcriptase polymerase chain reaction (RT-PCR)-single strand conformation polymorphism (SSCP) method. RESULTS: In recurrent aborters, no apparent changes in NK receptor expression and the balance between KARs and KIRs were found before and after the immunotherapy. CONCLUSION: The allo-human leukocyte antigen (HLA)-stimulation caused by the immunotherapy for recurrent aborters did not affect the expression of NK receptors and the ratio of KARs to KIRs regardless of the outcome of subsequent pregnancies, suggesting that recurrent aborters may benefit from the immunotherapy through mechanisms unrelated to alteration in NK receptor status. PMID- 10735602 TI - Effects of paternal lymphocyte immunization on peripheral Th1/Th2 balance and TCR V beta and V gamma repertoire usage of patients with recurrent spontaneous abortions. AB - PROBLEM: The mechanism of immunotherapy for patients with recurrent spontaneous abortions is not well understood. In order to investigate the suppressor mechanism of paternal lymphocyte immunization, we examined peripheral blood lymphocyte subpopulations and the repertoire of T-cell receptor (TCR) gene segments. METHOD OF STUDY: Twelve patients with recurrent miscarriage were treated with immunization with paternal lymphocyte vaccinations three times during 12-14 weeks. Before and 2 weeks after the final inoculation, lymphocyte subsets and intra-cellular interferon (IFN)-gamma and/or interleukin (IL)-4 production were examined by flow cytometry. TCR V beta and V gamma repertoires were examined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: We found no significant difference in CD4/CD8 ratios, prevalence of CD56+CD3+ or CD57+CD3+ cells (possible extrathymic T cells), gamma(delta)T cells, and CD5+ CD19+ (B-1) cells. However, by in vitro activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, peripheral CD4 cells demonstrated a significant decrease of IFN-gamma-producing T helper 1 (Th1) cells and an increase of IL-4-producing T helper 2 (Th2) cells after immunotherapy. Seven of nine patients who exhibited remarkable decreases in Th1/Th2 ratios became pregnant within 6 months after three courses of immunotherapy, and four women delivered healthy babies, while none of the three patients who exhibited an increased or unchanged Th1/Th2 ratio had full-term pregnancies (chi2 < 0.0001). Further, changes in usage of TCR V beta and V gamma gene segments were observed after immunotherapy in six patients examined. CONCLUSION: Our findings suggest a shift of Th1-dominant to Th2-dominant status by vaccination might play important roles in maintaining successful pregnancies. Induction of some T cells that utilize different TCR repertoires possibly suppresses maternal rejection reactions. PMID- 10735603 TI - Effect of testicular macrophage conditioned media from rats with autoimmune orchitis on Leydig cell function. AB - PROBLEM: The aim of this study was to investigate the influence of immune activated testicular macrophages obtained from rats with autoimmune orchitis (EAO) on Leydig cell steroidogenesis. METHOD OF STUDY: EAO was induced in rats by active immunization with testis homogenate and adjuvants. Testicular and peritoneal macrophages from rats with EAO were isolated and cultured for 24 hr. Testosterone (T) production by purified Leydig cells incubated in vitro with macrophage-conditioned media (CM) from rats with EAO or control rats was measured. RESULTS: An increase in T production by Leydig cells incubated with CM from testicular, but not peritoneal, macrophages of rats with EAO was observed. This increase was dose-dependent up to a concentration of 30% CM; proportions higher than 35% exhibited an inhibitory effect. CONCLUSIONS: Immune-activated testicular macrophages obtained from rats with EAO induced both stimulatory and inhibiting steroidogenic effects on Leydig cells in vitro and not the exclusively inhibitory action that has widely been attributed to activated macrophages. This dual effect probably depends on the ability of these cells to synthesize different molecules that may exert opposite effects. PMID- 10735604 TI - Validation of a fatigue impact score in primary biliary cirrhosis: towards a standard for clinical and trial use. AB - BACKGROUND/AIMS: Fatigue is the commonest symptom of the chronic liver disease primary biliary cirrhosis (PBC). PBC is increasingly being diagnosed earlier in its natural history at a stage when concerns about progression to liver failure may be less prominent than current symptoms. The importance of symptomatic treatment is therefore increasingly being recognised. Research into the aetiology and treatment of fatigue in PBC has been hampered by the lack of relevant, reproducible measures of fatigue severity. The aim of this study was to validate the Fisk Fatigue Severity Score (FFSS), a measure of the impact of fatigue on daily living, for use in PBC and to use the FFSS to study the severity and correlates of fatigue in this condition. METHODS: Fifty-eight patients with PBC and 31 matched control patients attending hospital for nonhepatic disease were studied. The reproducibility of FFSS was assessed over periods from 1 hour to 1 month. Fatigue, as measured by FFSS, was compared in PBC patients and controls. The severity of fatigue in the patients with PBC was correlated with other symptoms and with established biochemical and histological markers of severity. RESULTS: The FFSS questionnaire was acceptable to patients and had reasonable intra-observer variation (coefficient of reproducibility 13% of mean value at 1 hour). The FFSS was more reproducible than a visual analogue scale. FFSSs varied over 4 weeks by up to 36%. Median FFSS's were 2.3 times higher in PBC patients than controls (p<0.005). There were no associations between FFSS and patient age, disease duration, or histological or biochemical markers of severity. CONCLUSIONS: The FFSS is a highly acceptable, internally consistent and reproducible measure of fatigue severity in PBC. We advocate its use in clinical assessment of patients, in studies of the aetiology of fatigue in PBC and, most importantly, in therapeutic trials of symptomatic treatment. PMID- 10735605 TI - Cholangiocarcinoma in primary sclerosing cholangitis: K-ras mutations and Tp53 dysfunction are implicated in the neoplastic development. AB - BACKGROUND/AIM: Cholangiocarcinoma is a feared complication of primary sclerosing cholangitis (PSC). Neoplastic bile duct strictures may be difficult to differentiate cholangiographically from the non-neoplastic bile duct irregularities characteristic of this disorder, and the diagnosis of cholangiocarcinoma may be difficult to establish with certainty, even in tissue samples. Thus, new methods which can improve the diagnostic accuracy of cholangiocarcinoma in PSC are needed. METHODS: We investigated the occurrence of K-ras codon 12 and 13 mutations, p53 protein accumulation, and Ki-67 expression in tumor tissue from PSC patients (n=33) who had developed cholangiocarcinoma, using bile duct specimens exised at liver transplantation of PSC patients without cholangiocarcinoma (n=15) as controls RESULTS: K-ras mutations were present in 11 (33%) of the cholangiocarcinoma samples and significantly more frequent in females. Nine tumors carried a codon 12 mutation, and 2 had a codon 13 mutation. The most frequent substitutions in codon 12 were GGT-->GAT (n=5) and GGT-->TGT (n=3). None of the control bile ducts had K-ras mutations. p53 protein was accumulated in 10 (31%) of the tumors, as opposed to negative findings in all the control samples. Sixteen (48%) tumors revealed either K-ras mutation or p53 accumulation. Ki-67 positivity was significantly higher in cholangiocarcinomas than in the non-neoplastic bile ducts (median 29% vs 12%, respectively; p=0.011). CONCLUSION: We conclude that K-ras mutations and p53 dysfunction are implicated in tumorigenesis of cholangiocarcinomas arising in PSC patients and that these abnormalities together with increased Ki-67 index may indicate neoplastic progression of bile ducts in these patients. PMID- 10735606 TI - Effects of extracellular matrix on the expression of peroxisomes in primary rat hepatocyte cultures. AB - BACKGROUND/AIMS: Peroxisomes in wild-type cells vary between tissues and developmental stages. In the liver of some peroxisomal deficiency disorder patients, rare parenchymal cells express normal peroxisomes (mosaics); the mechanism is unknown. Our aim was to find factors regulating peroxisome expression. METHODS: Liver-specific as well as peroxisome characteristics were studied in three types of primary rat hepatocyte cultures. RESULTS: Total glutathione S-transferase activity and albumin secretion both increased in the collagen I sandwich and immobilization gel cultures. In contrast, in monolayers cultured on plastic, total glutathione S-transferase activity decreased and albumin secretion was only 30-40% compared to the collagen cultures. Glycogen rosettes typical of liver parenchymal cells were always abundant. Laminin and collagen IV-producing stellate cells were numerous in the monolayer but almost absent in the sandwich cultures. In 6-day-monolayer cultures, the number of liver specific peroxisomes had decreased while atypical small or elongated peroxisomes appeared. Immunolabeling density for catalase and three beta-oxidation enzymes was decreased compared to adult rat liver; catalase specific activity in homogenates had dropped to 15% and 4% in the sandwich and monolayer cultures, respectively. In 17-day-sandwich cultures, some peroxisomes showed a very weak catalase reaction; total activity was 5%. Supplementation of the collagen type I cultures with several extracellular matrix factors could not prevent peroxisome dedifferentiation. CONCLUSION: The presence of these extracellular matrix components is not sufficient for normal peroxisome expression. It is suggested that hepatocyte-specific and peroxisomal features are regulated differently. The sandwich preserves hepatocyte differentiation better than the monolayer. PMID- 10735607 TI - Expression of cytokines and factors modulating apoptosis by human sinusoidal leucocytes. AB - BACKGROUND/AIMS: Liver sinusoids contain a large population of spontaneously cytotoxic cells (NK cells), CD8+ T cells and macrophages. The physiological role of these leucocytes remains unclear. They may participate in immune surveillance and peripheral tolerance by deleting tumour cells, virus-infected cells and activated T cells as they traffic through the liver. In order to gain further information about the function of these leucocytes within the hepatic sinusoids, we examined their production of immunomodulatory cytokines and apoptosis-related molecules. METHODS: Semi-quantitative polymerase chain reaction and immunohistochemistry were used to determine the spontaneous production of cytokines and apoptosis-related molecules by sinusoidal leucocytes isolated from donor liver preservation solution. RESULTS: In comparison with matched peripheral blood mononuclear cells, sinusoidal leucocytes produced more mRNA for IL-10, IL 15, TNF-alpha, IL-18, IFN-gamma, FasL, perforin and granzyme. IL-4 and IL-12 were not detected and IL-2 was only faintly detected in the liver-derived CD4+ population. Less bcl-2 was expressed in liver-derived CD4+ and CD8+ cells in comparison with matched peripheral blood cell populations. CONCLUSIONS: The cytokines produced spontaneously by sinusoidal leucocytes are consistent with their high level of activation and spontaneous cytotoxicity. Their strong expression of apoptosis-mediating molecules (FasL, perforin, granzyme and TNF alpha) support a role for these cells in immune surveillance and peripheral tolerance induction. PMID- 10735608 TI - High plasma levels of arginine and liver arginase in Kupffer-cell-depleted rats after partial hepatectomy. AB - BACKGROUND/AIMS: The remnant liver after partial hepatectomy releases arginase into the plasma, which is a reliable indicator of hepatocellular damage. Little information is available on how this release affects arginine plasma levels. We hypothesized that Kupffer cells after partial hepatectomy may prevent further hepatocellular damage, contributing to lower arginase release. The aim of the study was to evaluate the role of Kupffer cells in plasma arginase activity and arginine plasma levels after partial hepatectomy. METHODS: Wag/Rij rats (n=72, 250-275 g) were randomly assigned to receive 1 ml liposome-encapsulated dichloromethylene-diphosphonate in order to eliminate Kupffer cells (DMDP, n=24), 1 ml liposome encapsulated-phosphate buffered saline (PBS, n=24) or 1 ml NaCl 0.9% (NaCl, n=24) intravenously. Forty-eight hours later, all rats had a two third liver resection. Rats were killed at 0, 24, 48 and 96 h after partial hepatectomy. RESULTS: Arginase plasma activity was higher in the DMDP-treated group compared to NaCl and PBS (both p<0.01, p<0.05, p<0.01 and p<0.05 for 0, 24, 48 and 96 h after partial hepatectomy respectively). Arginine plasma levels increased, but were lower in the DMDP group compared to NaCl and PBS (both p<0.05, 24 h after hepatectomy). CONCLUSION: The study showed that Kupffer cell depletion results in a higher arginase release from the remnant liver after partial hepatectomy, indicating a hepatocellular protective function of Kupffer cells. Despite this arginase release, arginine plasma levels were increased after partial hepatectomy. PMID- 10735609 TI - Detection of alpha-1-antitrypsin PiZ individuals by SSCP and DNA sequencing in formalin-fixed and paraffin-embedded tissue: a comparison with immunohistochemical analysis. AB - BACKGROUND/AIM: The role of alpha-1-antitrypsin (AAT) deficiency in the development of cirrhosis and carcinoma of the liver can be investigated from the analysis of archival biopsy specimens. Immunohistochemistry can visualize the storage of defective protease inhibitor (Pi) variant Z, but does not allow differentation between homozygous and heterozygous patients. The aim of the study was to establish a method for the detection of the PiZ mutation on the gene level. METHODS: Liver biopsy and autopsy samples in which AAT deficiency was detected immunohistochemically by a monoclonal PiZ-antibody were analyzed by single-strand conformational polymorphism (SSCP) to reliably determine hetero- and homozygote carrier state in the absence of blood samples and to confirm the histological diagnosis. The accuracy of SSCP was verified by direct DNA sequencing. RESULTS: Tissue slices (>0.8 cm2) from 29 consecutive cases with immunohistochemically detected PiZ depositions and from ten PiZ-negative control cases were provided for extraction and amplification of DNA. In comparison to wild-type sequence of AAT exon V, all 29 cases showed band shifts on SSCP analysis, with a heterozygous pattern in 28 patients and a homozygous pattern in one patient. DNA sequence analysis revealed the same single-base mutation at position 342 of AAT exon V. CONCLUSIONS: SSCP analysis proved a sensitive and specific technique for the detection of the PiZ mutation at the gene level, which allowed unequivocal differentiation between heterozygous and homozygous PiZ status from paraffin-embedded archival tissue specimens. Besides a use in diagnostic pathology, this technique could be valuable for prenatal diagnosis and population screening purposes. PMID- 10735610 TI - Serum laminin and type IV collagen are accurate markers of histologically severe alcoholic hepatitis in patients with cirrhosis. AB - BACKGROUND/AIMS: Severe alcoholic hepatitis occurs mainly in patients with cirrhosis, and has a high death rate. Corticosteroid therapy has been particularly advocated as reducing mortality in patients with severe histologic lesions. However, identification of these patients is difficult, requiring transvenous liver biopsy. Extracellular matrix serum markers have been proposed as non-invasive diagnostic tools in alcoholic liver disease. The aim of this study was to determine the accuracy of 5 extracellular matrix serum markers, i.e. laminin (Lam), N-terminal peptide of type III procollagen (PIIINP), type I (CI), type III (CIII) and type IV (CIV) collagens in identifying patients with severe histologic alcoholic hepatitis from among those with cirrhosis and suspected alcoholic hepatitis. METHODS: We studied 80 consecutive patients with alcoholic cirrhosis and clinical suspicion of alcoholic hepatitis referred for transvenous liver biopsy. Clinical severity of alcoholic hepatitis was assessed according to Maddrey's score. Histological severity was scored using the sum of the 3 following items: polynuclear infiltration (0-3); hepatocytes alterations (0-3); Mallory bodies (0-2). According to this score, patients were divided into 3 groups: mild (1-3), moderate (4-6), and severe (7-8) alcoholic hepatitis. Serum levels of the 5 extracellular matrix serum markers were measured at the time of biopsy using radioimmunoassays. Diagnostic value for histologically severe alcoholic hepatitis of the 5 extracellular matrix serum markers was assessed using receiver operating characteristic curves. RESULTS: Histological alcoholic hepatitis was present in 67 patients (mean alcoholic hepatitis score: 3.4+/-2.3). Maddrey's score was 66% sensitive and 69% specific for the diagnosis of severe histologic alcoholic hepatitis. The serum Lam and CIV concentrations were the most accurate in identifying correctly patients with severe histologic alcoholic hepatitis. At a cut-off of 4.1 UI/ml, Lam was 90% sensitive and 77% specific, whereas at a cut-off of 150 ng/ml, CIV was 89% sensitive and 77% specific. Combination of markers did not result in improved diagnostic value. CONCLUSION: In patients with cirrhosis, determination of serum Lam or CIV could represent a simple and accurate non-invasive method for identification of patients with histologically severe alcoholic hepatitis eligible for corticosteroid treatment. PMID- 10735611 TI - Terlipressin is more effective in decreasing variceal pressure than portal pressure in cirrhotic patients. AB - BACKGROUND/AIMS: Terlipressin decreases portal pressure. However, its effects on variceal pressure have been poorly investigated. This study investigated the variceal, splanchnic and systemic hemodynamic effects of terlipressin. METHODS: Twenty cirrhotic patients with esophageal varices grade II-III, and portal pressure > or =12 mmHg were studied. Hepatic venous pressure gradient, variceal pressure and systemic hemodynamic parameters were obtained. After baseline measurements, in a double-blind administration, 14 patients received a 2mg/iv injection of terlipressin and six patients received placebo. The same measurements were repeated 60 min later. RESULTS: No demographic or biochemical differences were observed in basal condition between groups. Terlipressin produced significant decreases in intravariceal pressure from 20.9+4.9 to 16.3+/ 4.7 mmHg (p<0.01, -21+/- 16%), variceal pressure gradient from 18.9+/-4.8 to 13.5+/-6.0 mmHg (p<0.01, -28+/-27%), estimated variceal wall tension from 78+/-29 to 59+/-31 mmHg x mm (p<0.01, -27+/-22%), and hepatic venous pressure gradient from 19.4+/-4.5 to 16.8+/-5 mmHg (p<0.01, -14+/-12%) at 60 min. The change in variceal pressure after 60 min of terlipressin administration was greater than the change in wedge hepatic venous pressure (-4.7 mmHg vs -0.5 mmHg, respectively, p<0.0001). Terlipressin also caused significant decreases in heart rate and cardiac index and increases in mean arterial pressure and peripheral vascular resistance. CONCLUSIONS: Our results demonstrate that terlipressin produces significant and prolonged decreases in variceal pressure and variceal wall tension and has intrinsic effects on portal pressure and systemic hemodynamics. Variceal pressure provides a better assessment of the effects of terlipressin administration on esophageal varices than hepatic venous pressure gradient. PMID- 10735612 TI - High plasma cardiac natriuretic peptides associated with enhanced cyclic guanosine monophosphate production in preascitic cirrhosis. AB - BACKGROUND/AIMS: The initial abnormalities of renal sodium handling in cirrhosis remain unclear. The aim of this study was to characterize sodium metabolism in preascitic cirrhosis. METHODS: Ten patients with preascitic cirrhosis and ten controls were studied. All subjects ate a diet providing 120 mmol sodium during an equilibration period lasting 5 days and the study day. On the study day, after remaining in bed, plasma levels of atrial natriuretic peptide, brain natriuretic peptide, renin activity, aldosterone, noradrenaline, and cyclic guanosine monophosphate were measured at 7 am. Thereafter, they were instructed to maintain an upright posture until dinner and the measurements were repeated at 9 am and 6 pm. After having dinner, all subjects were asked to remain in bed and the measurements were repeated at 11 pm. To measure renal sodium and cyclic guanosine monophosphate excretion, 24-h urine collections were performed, starting from 7 pm on the day before the experimental day. RESULTS: Plasma levels of atrial natriuretic peptide, brain natriuretic peptide and cyclic guanosine monophosphate in patients with preascitic cirrhosis were significantly elevated compared with those in controls at every sampling time (p=0.03 or less, p= 0.04 or less, and p=0.01 or less). In contrast, plasma renin activities at every sampling time were significantly lower in patients than in controls (p= 0.04 or less). Plasma aldosterone and noradrenaline levels were not significantly different at every sampling time in the two groups. No significant differences in daily renal sodium excretion were found. However, urinary cyclic guanosine monophosphate excretion was significantly higher in patients than in controls (p<0.01). CONCLUSIONS: The initial abnormalities of sodium metabolism in cirrhosis might be characterized by blunted renal responsiveness to natriuretic peptides. The results of the study also provide indirect evidence that the impairment is mainly located at postreceptor levels of signal transduction pathway to the peptides, if the activation of antinatriuretic factors other than renin-angiotensin or sympathoadrenergic systems does not play a role. PMID- 10735613 TI - Spleno-renal shunt blood flow is an accurate index of collateral circulation in different models of portal hypertension and after pharmacological changes in rats. AB - BACKGROUND/AIMS: Recently, we developed a new method to measure collateral blood flow in rats: splenorenal shunt (SRS) blood flow (BF). The aims were to evaluate the reproducibility of SRSBF measurement in different models of portal hypertension, and to investigate the ability of SRSBF to disclose pharmacological changes. METHODS: Hemodynamics were determined in anesthetized rats with secondary biliary, CCl4 or DMNA cirrhosis and portal vein ligation (PVL) under baseline and pharmacological (octreotide, vapreotide) conditions. The main measurements performed were: SRSBF by the transit time ultrasound (TTU) method and % portosystemic shunts (PSS) by the microsphere method. RESULTS: SRSBF was 6 to 10 times higher in portal hypertensive rats and was similar in the different models of cirrhosis but was higher in portal vein ligated rats than in cirrhotic rats (1.1+/-0.7 vs 0.6+/-0.7 ml x min(-1) x 100 g(-1), p=0.01). SRSBF was correlated with mesenteric %PSS (r=0.61, p<0.01), splenic %PSS (r=0.54, p<0.05), portal pressure (r= 0.32, p<0.05) and the area of liver fibrosis (r=0.33, p<0.05). Octreotide significantly decreased SRSBF (-23+/-20%, p<0.01 vs placebo: 6+/-8%, NS). Vapreotide significantly decreased SRSBF but not mesenteric or splenic %PSS compared to placebo. The variations in SRSBF (-26+/-32%) and in splenic %PSS (0+/-15%) with vapreotide were significantly different (p<0.05) and not correlated (r=-0.1, NS). CONCLUSIONS: Determination of SRSBF by TTU is an accurate way to measure collateral blood flow in different models of intra- and extra-hepatic portal hypertension in rats. Its sensitivity provides accurate measurement of pharmacological changes, unlike the traditional estimation of %PSS by the microsphere method. PMID- 10735614 TI - The acid-labile subunit of the ternary insulin-like growth factor complex in cirrhosis: relation to liver dysfunction. AB - BACKGROUND/AIMS: In the circulation, insulin-like growth factor-I (IGF-I) is bound in a trimeric complex of 150 kDa with IGF binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS). Whereas circulating IGF-I and IGFBP-3 are reported to be low in patients with chronic liver failure, the level of ALS has not been described in relation to hepatic dysfunction. The aim of the present study was therefore to measure circulating and hepatic venous concentrations of ALS in relation to hepatic function and the IGF axis. METHODS: Twenty-five patients with cirrhosis (Child class A/B/C:5/10/10) and 30 controls with normal liver function were studied. During a haemodynamic investigation, blood samples were collected from the hepatic vein and femoral artery, and the plasma concentrations of ALS, IGF-I and IGFBP-3 were determined. RESULTS: Hepatic venous and arterial concentrations of ALS were significantly decreased in the cirrhotic patients compared with the controls (-69% and -68%, respectively, both p<0.001). IGF-I and IGFBP-3 were similarly decreased in the cirrhotic patients (-51%,p<0.001). A significant hepatic extraction of ALS was found in the controls (6%, p<0.01) and in the cirrhotic patients (8%, p=0.08). ALS correlated significantly with indicators of liver dysfunction, including the Child-Turcotte score (r=-0.69, p<0.0001), IGF-I (r=0.82, p<0.0001) and IGFBP-3 (r=0.74, p<0.0001). CONCLUSIONS: Circulating and hepatic venous ALS are decreased in patients with cirrhosis with significant relations to liver dysfunction and other components of the IGF complex. A small hepatic extraction was found in controls, which suggests extrahepatic production of ALS. Future studies should focus on organ-specific removal of ALS. PMID- 10735615 TI - Prognostic value of serum hyaluronan in patients with compensated HCV cirrhosis. AB - BACKGROUND/AIM: Serum hyaluronan (HA) levels increase according to the degree of liver fibrosis in patients with chronic viral hepatitis C. Patients with liver disease and markedly high serum HA levels have cirrhosis with typical signs of hepatic sinusoidal capillarization, a factor of aggravation of cirrhosis The aim of this study was to evaluate the prognostic value of serum HA for severe complications in asymptomatic patients with HCV cirrhosis. METHODS: Six hundred and sixty-eight patients with anti-HCV antibodies and increased serum alanine aminotransferase were referred to our hospital for evaluation, including liver biopsy. At entry, serum HA levels were measured in 91 patients (64 men, 27 women, 56 +/-11 years old) out of 103 who had asymptomatic, biopsy-proven cirrhosis According to the criteria of Child-Pugh, 82 were classified A and 9 B. The follow up period was 6 to 82 months (median: 38 months), and 51 of these patients received alpha-interferon therapy during the first year. Severe complications were defined as death or liver transplantation, ascites, bleeding from esophageal varices, encephalopathy, or hepatocellular carcinoma. RESULTS: Serum HA levels at entry were higher in the cirrhotic patients in whom severe complications occurred during the follow-up period (520+/-426 microg/l vs 197+/-146 microg/l, p<0.0001). The patients with serum hyaluronan levels >350 microg/l displayed higher probabilities of occurrence of severe complications (p<0.0001). Other factors associated with the occurrence of complications or death were: serum bilirubin >18mol/l (p = 0.03), platelet count <112x10(9)/l (p= 0.02), prothrombin time <63% (p<0.0001), serum albumin <36 g/l (p=0.002), alkaline phosphatase >81 IU/l (p=0.01), and no interferon treatment (p= 0.0003). Multivariate analysis identified five independent factors predictive of severe clinical complications, namely: hyaluronan (p=0.006), prothrombin time (p=0.04), bilirubin (p=0.04), albumin (p=0.04), and no therapy (p=0.03). CONCLUSION: Serum HA level is predictive for occurrence of severe complications in HCV cirrhosis, and can be used as a prognostic marker, in addition to the parameters of the Child-Pugh score, particularly in patients with compensated cirrhosis. PMID- 10735616 TI - Automatic quantification of liver fibrosis: design and validation of a new image analysis method: comparison with semi-quantitative indexes of fibrosis. AB - BACKGROUND/AIMS: Liver fibrosis is one of the most important and characteristic histologic alterations in progressive and chronic liver diseases. Thus, in both clinical and experimental practice, it is fundamental to have a reliable and objective method for its precise quantification. Several semi-quantitative scoring systems have been described. All are time-consuming and produce partially subjective fibrosis evaluations that are not very precise. This paper describes the design and validation of an original image analysis-based application, FibroQuant, for automatically and rapidly quantifying perisinusoidal, perivenular and portal-periportal and septal fibrosis and portal-periportal and septal morphology in liver histologic specimens. METHODS: The implemented image processing algorithms automatically segment interstitial fibrosis areas, while extraction of portal-periportal and septal region is carried out with an automatic algorithm and a simple interactive step. For validation, all automatically extracted areas were also manually segmented and quantified. RESULTS: Statistical analysis showed significant intra- and interoperator variability in manual segmentation of all areas. Automatic quantifications did not significantly differ from mean manual evaluations of the same areas. Comparison of our image analysis quantifications with staging histologic evaluations of liver fibrosis showed significant correlations (Spearman's, 0.72A, 313 + 1 G-->A and W23X) occurred with a relatively high frequency, accounting for 22.4% of the entire study cohort. Two of these common FH mutations (N543H-2393de19 and 1359 - 1G-->A) showed a preferential geographic distribution. Second, to further expand the array of LDL receptor gene mutations, we conducted mutation analysis by denaturing gradient gel electrophoresis (DGGE) in 141 children with definite FH. A mutation was identified in 111 patients, involving 16 new single base substitutions and four small deletions and insertions, which brings the number of different FH-causing mutations in our country up to 61. Our data indicate that an estimate of the prevalence of specific mutations, as well as the compilation of a database of all FH-causing mutations in a given country, can facilitate selection of the most appropriate molecular diagnostic approach. PMID- 10735633 TI - Long QT syndrome with a high mortality rate caused by a novel G572R missense mutation in KCNH2. AB - In a four-generation family with long QT syndrome, syncopes and torsades de pointes ventricular tachycardia (TdP) were elicited by abrupt awakening in the early morning hours. The syndrome was associated with a novel KCNH2 missense mutation, G572R, causing the substitution of a glycine residue at position 572, at the end of the S5 transmembrane segment of the HERG K(+)-channel, with an arginine residue. This segment is involved in the channel pore and the mutation may cause a reduction in the rapidly activating delayed rectifier K+ current (Ikr), or changed gating properties of the ion channel, leading to prolonged cardiac repolarization. The electrocardiograms of affected persons showed prolonged QT interval and notched T waves. Despite treatment with atenolol, 200 mg twice daily, the proband still experienced TdP episodes. Three untreated relatives of the proband died suddenly, and unexpectedly, at 18, 32, and 57 years of age. The G572R mutation is thus associated with a high mortality rate, and the clinical presentation illustrates that some mutations may not be controllable by just beta-blockade. PMID- 10735634 TI - Identification and characterization of -3c-g acceptor splice site mutation in human alpha-L-iduronidase associated with mucopolysaccharidosis type IH/S. AB - DNA screening for mutations in the alpha-L-iduronidase (IDUA) gene was performed in a Chinese mucopolysaccharidosis type IH/S patient. The patient had two different mutations: the maternal allele has L346R (t-g transversion in codon 346) and the paternal allele has 388-3c-g (c-g transversion at position -3 of the 3' splice site of intron 2). In transfected COS-7 cells, L346R showed no appreciable IDUA activity (0.4% of normal activity), although it did not cause an apparent reduction in IDUA mRNA or protein level. The 388-3c-g mutation profoundly affects normal splicing leading to a very unstable mRNA. Expression of the IDUA cDNA containing the mutated acceptor splice site showed trace amounts of enzyme activity (1.6% of normal activity). The results provide further support for the importance of cytosine at the -3 position in RNA processing. PMID- 10735635 TI - Genotype-phenotype analysis in Apert syndrome suggests opposite effects of the two recurrent mutations on syndactyly and outcome of craniofacial surgery. AB - Apert syndrome is an autosomal dominant condition characterized by craniosynostosis and severe syndactyly, caused by two recurrent mutations in the fibroblast growth factor receptor 2 gene (FGFR2). The genotype-phenotype correlations of 21 patients with Apert syndrome were analysed as to the craniofacial appearance following surgery and the degree of syndactlyly. The craniofacial appearance following craniofacial surgery was better in patients with the P253R mutation, whereas these patients showed a more pronounced severity of the syndactyly. PMID- 10735636 TI - Pseudodominance of lipoprotein lipase (LPL) deficiency due to a nonsense mutation (Tyr302>Term) in exon 6 of LPL gene in an Italian family from Sardinia (LPL(Olbia)). AB - We analyzed the molecular defect in the lipoprotein lipase (LPL) gene of a young boy from Sardinia who had primary hyperchylomicronemia, pancreatitis, and a complete LPL deficiency in post-heparin plasma. Analysis of LPL gene was performed by using single strand conformation polymorphism (SSCP) and direct sequencing of SSCP-positive region. The proband was homozygous for a C > A transversion in exon 6, which converts the codon for tyrosine at position 302 into a termination codon and eliminates an RsaI restriction site; this allowed the rapid screening of the proband's family members, among whom nine heterozygotes and one additional homozygote were identified. The homozygote was the proband's paternal grandmother who had shown the first clinical manifestation (recurrent pancreatitis) of LPL deficiency at the age of 54 years. LPL mutation carriers showed a mild dyslipidemic phenotype characterized by a reduction of high density lipoprotein-cholesterol (HDL-C) levels, HDL-C/total cholesterol ratio, and low density lipoprotein (LDL) size, associated with a variable increase of triglyceride levels. Five of these carriers were also heterozygotes for beta-thalassemia (Q39X mutation). In these double mutation carriers, plasma HDL-C levels were higher and plasma triglycerides tended to be lower than in carriers of LPL mutation alone. The Tyr302 > Term mutation encodes a truncated protein of 301 amino acids that is probably not secreted by the LPL producing cells. This is the first mutation of LPL gene found in Sardinians. PMID- 10735637 TI - Clinical variability of Larsen syndrome: diagnosis in a father after sonographic detection of a severely affected fetus. AB - Larsen syndrome shows a broad spectrum of clinical manifestation ranging from a lethal form of the disorder to a mild clinical expression with absence of major diagnostic features. Here we show that even intrafamilial manifestation may vary extremely to the point that Larsen syndrome in a father has been diagnosed only by typical sonographic features in an affected fetus. PMID- 10735638 TI - Mutations in a 10-bp polyadenine repeat of transforming growth factor beta receptor type II gene is an infrequent event in human epithelial ovarian cancer. PMID- 10735639 TI - The X-linked IAP gene does not contribute to the clinical phenotype of spinal muscular atrophy. PMID- 10735640 TI - Differentiation of Ambras syndrome from Hypertrichosis Universalis. PMID- 10735641 TI - Drug allergy: relationship between immunogenicity and clinical symptoms. PMID- 10735642 TI - In vitro methods for quantifying IgE antibodies to betalactams. PMID- 10735643 TI - Anaphylactoid and anaphylactic reactions to iodinated contrast material. AB - Some adverse reactions to iodinated contrast material (ICM) are considered allergy-like, with cutaneous, cardiovascular, respiratory, and digestive symptoms. Allergy-like reactions are usually unpredictable. Reactions are more frequent with ionic than with nonionic material, but the frequency of deaths is almost identical. In a recent study, 20 severe unexpected reactions to ICM, including 10 life-threatening reactions and one death, were investigated by measuring mediators in blood, within the first minutes or hours of reaction. The responsible ICMs were mostly ionic materials. Histamine and tryptase release correlated with the severity of the reaction. Specific IgE against the responsible ICM was significantly higher in reactors than in controls. A few patients had positive skin tests to the administered ICM, suggesting type-I allergic reaction. Only 2.4% and 3.1% of the cases yielded a positive IgE-RIA, in a second retrospective study which included 165 patients recruited during a 4 year period. In conclusion, IgE-mediated anaphylaxis is rare, but it may be one of the possible mechanisms of severe adverse reactions to ICM. PMID- 10735644 TI - Criteria of evaluation and of interpretation of Sepharose drug IgE-RIA to anaesthetic drugs. AB - The detection of antidrug specific IgE in serum is usually performed by a sandwich-type immunoassay in which the serum IgE is first adsorbed to a reactive phase and subsequently quantified via the binding of an anti-IgE tracer. The preparation of a new drug-reactive phase requires one to establish carefully different steps of validation: 1) criteria of positivity of control sera 2) competitive inhibition assays with the soluble drug, which should include the determination of the inhibition constant rather than estimation of a single inhibition percentage, especially when the assay is performed for the identification of determinants 3) estimation of nonspecific binding of IgE to the solid phase, including hydrophobic binding. The competitive inhibition depends on the concentration of the competitor and of IgE in the test-tube and the concentration of reactive drug bound to the solid phase. We have improved the inhibition assay by performing the Dixon test for calculating the inhibition constant (Ki) of the competitor. The Ki of six different muscle relaxants was determined in 12 patients who experienced an anaphylactic reaction to muscle relaxants. The values ranged between 1.5 nM and 2.5 microM. This confirmed the great heterogeneity of drug IgE cross-reactivity among patients. The Ki value of the incriminated drug was the lowest (affinity, the highest) in eight of the 12 patients. It was better correlated to clinical data than the classical inhibition assay. A hydrophobic environment seemed to be necessary, close to the quaternary ion, to allow IgE binding to the muscle relaxant. By contrast, in tiemonium, a hydroxyl group present at a distance of about 3 A from the quaternary ion may explain why this molecule had a high Ki (microM). In conclusion, it should be recommended, in molecular-recognition studies, that the inhibition constant of the soluble drug and of the related compounds be determined to complement the experiments based only on hapten inhibition assays. PMID- 10735645 TI - Diagnostic evaluation of delayed hypersensitivity to systematically administered drugs. PMID- 10735646 TI - Diagnosis of allergic reactions to sulfonamides. PMID- 10735647 TI - Reactive airways dysfunction syndrome (RADS): fact or fantasy? AB - Most evidence suggests that RADS is a distinct syndrome and not simply an exacerbation of pre-existing hyperresponsiveness. There is no doubt that much remains to be explained regarding pathogenesis. On the other hand, I urge caution about the claims of "low-level RADS". Here the data are much more anecdotal and leave much to be desired. PMID- 10735648 TI - Cross-talk between mast cells and eosinophils. PMID- 10735649 TI - Therapeutic perspectives in atopic dermatitis. PMID- 10735650 TI - Food allergy: an update. PMID- 10735652 TI - Clinical aspects of Hymenoptera venom allergy. PMID- 10735651 TI - Therapeutic perspectives in food allergy. AB - The therapeutic perspectives in food allergy are thus based upon known principles and mechanisms of tissue injury mediated by immunologic reactions. A thorough knowledge of these, together with new approaches to the identification, elimination, and modification of these reactions, is essential in the management of the patient with food allergy. PMID- 10735653 TI - Intranasal immunotherapy. PMID- 10735654 TI - Immunologic effect of immunotherapy. PMID- 10735655 TI - Reassessing our core values. PMID- 10735656 TI - Ethics gap in surgery. AB - BACKGROUND: Discussion of ethical issues occurs much less often in the surgical than in the medical literature. The reasons for this "ethics gap" are unknown. METHODS: Our clinical faculty ranked the ethical and legal acceptability of four treatment options in two cases of surrogate decision making. Only one option in each case was ethically and legally unacceptable (treating despite objection by the surrogate decision maker). RESULTS: Surprisingly often, faculty mistakenly believed the ethically unacceptable option to be acceptable, and the legally unacceptable option to be acceptable. Surgeons were not ethically different from other physicians. Surgeons (19 of 31, 62%), however, were significantly (p < 0.05) more likely than internists (18 of 51, 35%) or pediatricians (4 of 18, 22%) to believe, mistakenly, that operating on the baby without parental consent was legally acceptable. CONCLUSIONS: This pilot study did not identify why the surgical literature contains a relative dearth of ethics discussion. Broader investigations are needed, because it is important that we understand the reasons for the gap. Surgeons' strong ethic of personal responsibility for patients' welfare should be transmitted to young trainees, a goal best achieved by discussing and writing about ethics. Moreover, our legal data suggest that a gap may also exist between surgeons and other physicians in understanding health law. PMID- 10735657 TI - Thoracic surgery education. PMID- 10735658 TI - Substrate enriched warm blood cardioplegia reperfusion: an alternate view. PMID- 10735659 TI - Key outcomes '99: gone west? PMID- 10735660 TI - Intensive chemotherapy and radical resections for primary nonseminomatous mediastinal germ cell tumors. AB - BACKGROUND: Primary nonseminomatous germ cell tumors of the mediastinum (PNSGM), unlike malignancies of gonadal origin, have a poor prognosis. We report a single institutional experience over a 5-year period of PNSGM treated with intensive chemotherapy, followed by radical operation in those who responded to this neoadjuvant regimen. METHODS: From 1993 to 1998, 20 patients were referred for the management of PNSGM. All were male, with a median age of 30.5 years (range 18 to 48). Eighteen of 20 (90%) presented with symptoms. Most tumors were large, with a median diameter of 10 cm (range 3 to 20 cm). Thirteen patients (65%) had metastatic disease at the time of presentation. Eleven patients had received no prior treatment (initial group) and 9 were referred for salvage therapy after progression of their tumors, following treatment at other facilities (salvage group). All had elevated serum tumor markers (beta hCG and alpha-fetoprotein). Preoperative chemotherapy included alternating cycles of combinations of 3 or more drugs, including cisplatin, bleomycin, etoposide, vincristine, methotrexate, actinomycin, cyclophosphamide, and doxorubicin. An average of 10 cycles of chemotherapy was given to each patient in the initial group, and six to those in the salvage group. Five patients (25%) developed transient renal insufficiency, and 35% developed pulmonary infiltrates related to bleomycin. There were 3 chemotherapy related deaths. RESULTS: After chemotherapy, 11 patients underwent operation, with 10 complete resections of the residual mediastinal tumors. There were no perioperative deaths. The 2-year survival in the initial group is 72%, and 42% for the salvage group. CONCLUSIONS: An aggressive, multidisciplinary approach of alternating cycles of chemotherapy, followed by complete surgical resection of all remaining disease in patients whose markers normalize, can be associated with prolonged survival in patients with PNSGM. PMID- 10735661 TI - Effects of donor bone marrow infusion in clinical lung transplantation. AB - BACKGROUND: We have demonstrated that donor cell chimerism is associated with a lower incidence of obliterative bronchiolitis (OB) in lung recipients, and that donor chimerism is augmented by the infusion of donor bone marrow (BM). We herein report the intermediate results of a trial combining the infusion of donor BM and lung transplantation. METHODS: Clinical and in vitro data of 26 lung recipients receiving concurrent infusion of donor bone marrow (3.0 to 6.0 x 10(8) cells/kg) were compared with those of 13 patients receiving lung transplant alone. RESULTS: Patient survival and freedom from acute rejection were similar between groups. Of the patients whose graft survived greater than 4 months, 5% (1 of 22) of BM and 33% (4 of 12) of control patients, developed histologic evidence of OB (p = 0.04). A higher proportion (but not statistically significant) of BM recipients (7 of 10, 70%) exhibited donor-specific hyporeactivity by mixed lymphocyte reaction assays as compared with the controls (2 of 7, 28%). CONCLUSIONS: Infusion of donor BM at the time of lung transplantation is safe, and is associated with recipients' immune modulation and a lower rate of obliterative bronchiolitis. PMID- 10735662 TI - Hemodynamic effect of a low-resistance artificial lung in series with the native lungs of sheep. AB - BACKGROUND: An artificial lung with 1 to 6 month work life could act as a bridge to transplantation. A pumpless artificial lung has been developed. METHODS: The artificial lung was placed in series with the native lungs of adult sheep. Hemodynamics were observed, as the right ventricle generated flow through the device. Through a left thoracotomy, two 20-mm grafts were anastomosed in an end to-side fashion to the pulmonary artery. The grafts were externalized, and directed flow through the chest wall, to the extracorporeal lung. The animals were recovered, weaned from the ventilator, and when standing, flow was diverted through the device. RESULTS: Five of 7 animals survived 24 hours with 75% to 100% of the cardiac output diverted through the device. All animals were active, with interest in food and water, and able to stand. CONCLUSIONS: The right ventricle perfused the artificial lung with 75% to 100% of the cardiac output for 24 hours. This device demonstrates the feasibility of a pumpless pulmonary assist device relying on the right ventricle for perfusion. PMID- 10735663 TI - Videothoracoscopic treatment of primary spontaneous pneumothorax: a 6-year experience. AB - BACKGROUND: Timing and surgical approach in the treatment of primary spontaneous pneumothorax (PSP) are not well defined. The objective of this study is to propose a treatment protocol by videothoracoscopy (VATS) in PSP. METHODS: From July 1992 to May 1998, 432 patients underwent VATS treatment of PSP. Indications were: recurrent ipsilateral pneumothorax: 322 cases; persistent air leak following a first episode: 93 patients; recurrence following VATS: 16 cases; recurrence following thoracotomy: 1 patient. Vanderschueren's classification was used for staging. Surgical indications were: stages I and II, subtotal pleurectomy or talc poudrage; stages III and IV, stapling or ligation of the bullae and subtotal pleurectomy or talc poudrage. Differences in recurrence rates were calculated to compare the specific procedures. RESULTS: No postoperative deaths occurred. Complication rate was 4.16%. Conversion rate was 2.3%. Mean follow-up was 38 months (2 to 72 months). Overall recurrence rate was 4.4%. Specific recurrence rates following stapling and talc poudrage were, respectively, 1.27% and 1.79%. Talc poudrage and stapling of the bullae are respectively superior to subtotal pleurectomy (p < 0.0001) and ligation (p < 0.0001). CONCLUSIONS: Stapling of the bullae and talc poudrage by VATS represent the treatment of choice of PSP. PMID- 10735664 TI - Aggressive multimodality therapy for stage III esophageal cancer: a phase I/II study. AB - BACKGROUND: Stage III advanced locoregional esophageal carcinoma is frequently unresectable and inconsistently represented in therapeutic trials of esophageal cancer. METHODS: From 1992 to 1998, 34 of 131 total esophageal cancer patients were designated stage III (16 T3N1, 9 T4N0, 9 T4N1) and medically fit to enter a combined modality protocol with continuous infusion 5-fluorouracil (CIS-FU, 300 to 600 mg/m2/day), high-dose external beam irradiation (60 Gy), and interval esophagectomy. Staging before and after induction therapy included computed tomography; endoscopy, and endoscopic ultrasound. RESULTS: Significant toxicity from induction therapy included death (5/34; 14.7%), pneumonitis (5/34; 14.7%), mucositis (13/34; 38%), and hand-foot syndrome (3/34; 8.8%). In addition to the five deaths, 11 patients did not proceed to operation because of development of esophagorespiratory fistula in 3, distant disease in 2, persistence of T4 stage in 2, progression of comorbidities in 2, and patient refusal in 2. There was a discrepancy between clinical complete response (cCR) at restaging 56% (19/34) and pathologic CR (pCR) noted at the time of operation (8/34; 23.5%). Complete resections were possible in 16 of 18 patients explored. Complications in 4 patients included: death (1), airway injury (1), chylothorax requiring reoperation (1), anastomotic leak (1), recurrent nerve injury with vocal cord paresis (2), and ascaris infection (1). Actuarial survival analysis using the Kaplan-Meier method and log-rank testing showed a 36-month survival of 20% for the group as a whole and 27% for patients restaged cCR (cCR vs PR, p = 0.0046). Treatment failure is predominantly distant, with good local control in resected patients. N0 node status was strongly associated with survival (N0 vs N1 p = 0.0024). There is a trend towards improved survival in the resected group (resected 22% vs nonresected 10% at 3 years, p = 0.17). CONCLUSIONS: Response rates and survival are commensurate with multiple completed phase II and III trials. These are attained at a higher treatment-related mortality. T4 patients can be successfully resected in selected patients. Even in advanced disease, nodal status is a significant predictor of survival. PMID- 10735665 TI - Outpatient management of malignant pleural effusion by a chronic indwelling pleural catheter. AB - BACKGROUND: Previous studies have shown that a chronic indwelling pleural catheter (PC) safely and effectively relieved dyspnea, maintained quality of life, and reduced hospitalization in patients with malignant pleural effusions. Outpatient management of malignant pleural effusion with a PC may reduce length of stay and early (7-day) charges compared with inpatient management with chest tube and sclerosis. METHODS: A retrospective review of consecutive PC patients (n = 100; 60 outpatient, 40 inpatient) were treated from July 1, 1994 to September 2, 1998 and compared with 68 consecutive inpatients treated with chest tube and sclerosis between January 1, 1994 and December 31, 1997. Hospital charges were obtained from date of insertion (day 0) through day 7. RESULTS: Demographics were similar in both groups. Pretreatment cytology was positive in 126 of 168 patients (75%), negative in 21 (12.5%), and unknown in 21 (12.5%). Primary histology included lung (n = 61, 36%), breast (n = 39, 23%), lymphoma (n = 12, 7%), or other (n = 56, 34%). Median survival was 3.4 months and did not differ significantly between treatment groups. Overall median length of stay was 7.0 days for inpatient chest tube and inpatient PC versus 0.0 days for outpatient Pleurx. No mortality occurred related to the PC. Eighty-one percent (81/100) of PC patients had no complications. One or more complications occurred in 19 patients (19%). Patients treated with outpatient PC (n = 60) had early (7-day) mean charges of $3,391 +/- $1,753 compared with inpatient PC (n = 40, $11,188 +/- $7,964) or inpatient chest tube (n = 68, $7,830 +/- $4,497, SD) (p < 0.001). CONCLUSIONS: Outpatient PC may be used effectively and safely to treat malignant pleural effusions. Hospitalization is not required in selected patients. Early (7 day) charges for malignant pleural effusion are reduced in outpatient PC patients compared with inpatient PC patients or chest tube plus sclerosis patients. PMID- 10735666 TI - Acute lung injury and acute respiratory distress syndrome after pulmonary resection. AB - BACKGROUND: In this study we investigate the frequency and mortality of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) after pulmonary resection. METHODS: Patients that underwent pulmonary resection at the Royal Brompton Hospital between 1991 and 1997 were included. The case notes of all patients developing postoperative complications were retrospectively reviewed. RESULTS: The overall combined frequency of ALI and ARDS was 3.9%. The frequency was higher in patients over 60 years of age, males and those undergoing resection for lung cancer. ALI/ARDS caused 72.5% of the total mortality after resection in this series. CONCLUSIONS: In our experience ALI and ARDS are major causes of mortality after lung resection. PMID- 10735668 TI - Relationship between amount of lung resected and outcome after lung volume reduction surgery. AB - BACKGROUND: Lung volume reduction surgery (LVRS) is being actively investigated for palliative treatment of severe emphysema. Considerable focus is directed toward patient selection and outcomes of LVRS. However, there is little information available regarding surgical methods to guide optimal extent of resection. We hypothesized that acute improvement and long-term survival after bilateral staple LVRS would be related to the extent of tissue resected. METHODS: The relationship between acute improvement in forced expiratory volume in 1 second and forced vital capacity was examined as a function of the total grams of lung tissue resected in 237 patients who underwent bilateral staple LVRS by a single group of surgeons. Overall survival was assessed based on extent of resection by quartiles of tissue weight resected using Kaplan-Meier survival methods. RESULTS: Improvement in forced expiratory volume in 1 second and forced vital capacity correlated with extent of tissue resected (p < 0.01), although there was considerable variability to individual response (r = 0.3). In contrast, there was no apparent relationship between the amount of tissue resected and overall postoperative survival (p = 0.7). CONCLUSIONS: There is a correlation between the amount of tissue resected and improvement in forced expiratory volume in 1 second and forced vital capacity after bilateral staple LVRS, with generally greater postoperative improvement after larger volume resections. However, there does not appear to be greater long-term survival with larger volume resections despite greater improvement in spirometry. This study suggests that factors other than improvement in spirometric variables may govern optimal LVRS resection volumes and long-term outcome. Future studies will clearly be needed in this important area of LVRS emphysema research. PMID- 10735667 TI - Bilateral sequential single lung transplantation for pulmonary hypertension and Eisenmenger's syndrome. AB - BACKGROUND: Lung transplantation, with and without intracardiac repair for pulmonary hypertension (PH) and Eisenmenger's syndrome (EIS), has become an alternative transplant strategy to combined heart and lung transplantation (HLT). METHODS: Thirty-five patients with PH or EIS underwent either bilateral sequential single lung transplantation (BSSLT, group I, n = 13) or HLT (group II, n = 22). Another 74 patients, who underwent BSSLT for other indications, served as controls (group III). Immediate allograft function, early and medium-term outcomes, lung function, and 2-year survival were compared between the groups. RESULTS: Comparisons between groups I and II showed no significant difference in any variables except percent predicted forced vital capacity. Immediate allograft function was significantly inferior (p < 0.05) and the blood loss was greater (p < 0.01) in group I when compared with those in group III. However, this resulted in no significant difference in early and medium-term outcomes, and 2-year survival between the 2 groups. CONCLUSIONS: BSSLT for PH and EIS can be performed as an alternative procedure to HLT without an increase in early and medium-term morbidity and mortality. Results are comparable with BSSLT performed for other indications. PMID- 10735669 TI - Transsternal transpericardial approach for the repair of bronchopleural fistula with empyema. AB - BACKGROUND: Numerous surgical approaches have been reported for the repair of bronchopleural fistula. Recently the transsternal transpericardial approach has shown great promise with its positive results in cases of bronchopleural fistula complicated with empyema. The aim of this retrospective study was to assess the results of bronchopleural fistula treatment using the transsternal transpericardial approach. METHODS: Bronchopleural fistula developed in 16 of the 172 patients who had pneumonectomy between 1982 and 1996. In one case closure with fibrin sealant by bronchoscopy was tried. In the remaining cases fistula was closed by the transsternal transpericardial approach. RESULTS: The interval between pneumonectomy and fistula occurrence was 10 days or less in 5 patients and 10 days to 1 month in 11 patients. In all patients the empyema space was treated by continued drainage through the thoracostomy tube. Fibrin sealant was tried unsuccessfully for closure of moderate-sized bronchopleural fistula in one case. In three cases of right bronchopleural fistula, carinal resection and anastomosis of the trachea to the left main stem bronchus were performed. In the remaining cases bronchopleural fistula was closed using a hand suture technique. One patient died within 30 days after operation (6.25%) because of renal insufficiency. There was no recurrence of bronchopleural fistula. CONCLUSIONS: Transsternal transpericardial approach seems to be a safe and effective method with an easier technique in cases of bronchopleural fistula complicated with empyema. It has the added advantage of less recurrent fistula formation and enables resection in cases without sufficient bronchial stump. PMID- 10735670 TI - Flexible bronchoscopy: a safe method for metal stent implantation in bronchial disease. AB - BACKGROUND: Endoscopic bronchoplastic procedures, such as metal stent implantation, are for safety reasons and mainly performed using rigid bronchoscopy. Major complications, such as bleeding and accidental airway occlusion, are thought to be better managed with the rigid device. An increasing number of pneumologists, however, use the flexible fiberscope for endobronchial stenting. METHODS: Sixty-five stent implantations were performed in 51 patients with flexible fiberoptic bronchoscopy. We implanted 27 Tantalum Strecker stents (Boston Scientific Co, Watertown, MA), 20 Nitinol Accuflex stents (Boston Scientific Co) and 18 Wallstents (Schneider, Zurich, Switzerland). Underlying conditions were malignant disease in 84% and benign bronchial collapse in 16%. Sites of implantation were the trachea (45%), the main bronchi (35%), and other locations (20%). In 47 cases the patients received intravenous sedation combined with high frequency jet ventilation, and in 18 cases the patients were treated with topical anesthesia alone. RESULTS: Mean examination time was 58.3 (standard deviation 29.1) minutes. Eighty percent of patients experienced immediate clinical improvement in respiratory symptoms. Spirometric parameters (forced expiratory volume in one second, peak expiratory flow rate, forced vital capacity) increased. Complications included hypertension (17%), hypotension (12%), hypoxia (5%), bronchospasm (4%), initial displacement of the prosthesis (11%), and diameter mismatch between stent and bronchus (5%). All complications were managed safely. Relevant bleeding or asphyxia during the procedure has not been observed. Late stent migration was observed in 12% of cases. There were 3 fatalities within 30 days of stent placement which, however, were not attributed to the implantation technique. CONCLUSIONS: Flexible fiberoptic bronchoscopy is a safe and suitable method to perform endobronchial metal stent implantation. Complications were rare and not serious. Initial misplacement of the prosthesis occurred in some cases and necessitated removal and replacement within the same procedure. PMID- 10735672 TI - Mortality and paraplegia after thoracoabdominal aortic aneurysm repair: a risk factor analysis. AB - BACKGROUND: Recent recommendations regarding thoracoabdominal aortic aneurysm (TAAA) management have emphasized individualized treatment based on balancing a patient's calculated risk of rupture with their anticipated risk of postoperative death or paraplegia. The purpose of this study was to enhance this risk-benefit decision by providing contemporary results and determining which preoperative risk factors currently predict mortality and paraplegia after TAAA surgery. METHODS: Risk factor analyses based on data regarding 1,220 consecutive patients undergoing TAAA repair from 1986 through 1998 were performed using multiple logistic regression with step-wise model selection. RESULTS: The 30-day mortality rate was 4.8% (58 of 1,220) and the incidence of paraplegia was 4.6% (56 of 1,206). For elective cases, predictors of operative mortality included renal insufficiency (p = 0.0001), increasing age (p = 0.0005), symptomatic aneurysms (p = 0.0059), and extent II aneurysms (p = 0.0054). Extent II aneurysms (p = 0.0023) and diabetes (p = 0.0402) were predictors of paraplegia. CONCLUSIONS: These risk models may assist in decisions regarding elective TAAA operations. For patients who are acceptable candidates, contemporary surgical management provides favorable results. PMID- 10735671 TI - Superoxide possibly produced in endothelial cells mediates the neutrophil-induced lung injury. AB - BACKGROUND: The mechanism by which stimulated neutrophils (polymorphonuclear leukocytes [PMNs]) damage pulmonary vascular endothelium was investigated. METHODS: The ability of unstimulated and mechanically stimulated PMNs to adhere to pulmonary endothelial cells and, thereby, alter pulmonary vascular permeability was tested. Each series was conducted on 6 rats. To stimulate PMNs, they were agitated gently in a glass vial for 10 seconds. RESULTS: Perfusing lungs with the stimulated PMNs elicited a fivefold increase in permeability compared with lungs perfused with the unstimulated cells. This increase in permeability was blocked completely by preincubation of stimulated PMNs with CD18 monoclonal antibody. This increase in permeability was also blocked completely by superoxide dismutase (SOD) or the xanthine oxidase (XO) inhibitor allopurinol. Pulmonary vascular hemodynamics were unaffected by any treatment protocol. The accumulation of stimulated PMNs within the lungs was not inhibited by SOD but was partially blocked by allopurinol. CONCLUSIONS: These findings suggest that stimulated PMN-induced increases in pulmonary vascular filtration resulted from endothelial cell injury caused by superoxide anion possibly generated by XO, exclusively present in the endothelial cells. PMID- 10735673 TI - Effect of temperature and PaCO2 on cerebral embolization during cardiopulmonary bypass in swine. AB - BACKGROUND: Patients experience cerebral embolization during cardiopulmonary bypass (CPB). This study determined if alterations in temperature and/or PaCO2 can reduce cerebral and ocular embolization. METHODS AND RESULTS: Forty-four pigs underwent CPB: 24 animals at 28 degrees C, and 20 at 38 degrees C. The two temperature groups were randomized to undergo embolization (67-microm fluorescent microspheres) at either hypercarbia or hypocarbia. Before and after embolization, cerebral and ocular blood flow were determined at normocarbia. Reducing temperature or PaCO2 reduced cerebral and ocular embolization. Hypocarbia reduced cerebral embolization by 60% and 45% in normothermic and hypothermic groups, respectively (p < 0.0001 and p < 0.05). Relative to normothermic animals, hypothermia reduced cerebral embolization by 37% under an elevated CO2 condition (p < 0.05), but not under hypocarbic conditions. Similarly, regardless of temperature, fewer emboli were delivered to the eye in hypocarbic animals (p < 0.05), but hypothermia did not reduce ocular embolization. CONCLUSIONS: Cerebral embolization is determined by both temperature and PaCO2 at the time of embolization. In CPB practice, reductions in temperature and/or PaCO2 during periods of embolic risk may reduce brain injury. PMID- 10735674 TI - Simultaneous carotid endarterectomy and coronary revascularization. AB - BACKGROUND: Combined cardiac operation and carotid endarterectomy using our technique is an acceptable approach to simultaneous correction of both carotid and cardiac disease. METHODS: From August 1989 to March 1998, 121 consecutive patients underwent combined operations. Of these patients, 112 had coronary artery bypass grafting and carotid endarterectomy, and 9 had coronary artery bypass grafting, carotid endarterectomy, and valve repair or replacement. All patients had a critical stenosis of 85% or more of the carotid artery. Mean age of the patients was 69.2 years; 80 patients were 65 years old or older. There were 88 men and 33 women. Notable risk factors included chronic obstructive pulmonary disease (19.8%), congestive heart failure (28%), preoperative myocardial infarction and unstable angina (66.9%). Of the patients, 20.7% had a stenosis of greater than 50% of the left main coronary artery. The technique used was correction of both the carotid and coronary lesions during a single aortic cross-clamp period using retrograde continuous blood cardioplegia for myocardial protection. Systemic hypothermia to 25 degrees C was used for cerebral protection. RESULTS: Mean cross-clamp time was 118 minutes. Seven patients (5.8%) sustained perioperative cerebrovascular accidents. Two patients had transient ischemic attacks. The procedure-related mortality rate was 5.8%. CONCLUSIONS: The described technique is a good method for simultaneous repair of coronary and carotid lesions in a high-risk group of patients with concomitant disease. We will continue to use it. PMID- 10735675 TI - Surgical management of unstable patients in the evolving phase of acute myocardial infarction. AB - BACKGROUND: Acute myocardial infarction (AMI) can be treated with thrombolysis or coronary catheter intervention; surgical treatment--coronary artery bypass grafting (CABG)--is reserved for the patients in whom other procedures have failed. We performed CABG in 47 patients during the evolving phase of AMI, and analyzed their short-term and long-term results. METHODS: Preoperative, intraoperative, and postoperative data were analyzed in patients who underwent emergency CABGs for AMI between January 1, 1992, and July 31, 1998. CABGs performed more than 7 days after AMI were excluded from this study. RESULTS: The subjects were 47 patients (33 males and 14 females) with AMI who were treated by emergency CABG. Intraaortic balloon pumping was used in 44 cases and percutaneous circulatory pulmonary support was used in 3 cases. The mean interval between the onset of AMI and surgery was 27.4 +/- 27.9 hours. The mean number of bypass grafts was 3.0 +/- 1.1, and at least 1 arterial conduit was used in 45 cases (95.7%). Aortic clamp time, pump time, and operative time were 64.7 +/- 31.7, 117.3 +/- 55.2, and 313.2 +/- 84.8 minutes, respectively. IABP or percutaneous cardiopulmonary support were removed in the intensive care unit (ICU) 30.0 +/- 28.9 hours after CABG. The patients were extubated 41.4 +/- 40.5 hours after surgery, remained in ICU for 4.7 +/- 2.7 days, and were discharged from the hospital after 27.0 +/- 22.5 days. Three patients died from multiorgan failure related to postoperative sepsis, and 8 cases of major complications were observed. The actuarial 5-year survival rate of the patients treated with CABG was 83.0%. CONCLUSIONS: Surgical treatment in the unstable patients after AMI can be performed with acceptable risk. Arterial revascularization may contribute to improvement in long-term results. PMID- 10735676 TI - Composite cardiac binding in experimental heart failure. AB - BACKGROUND: Composite cardiac binding consists of wrapping the heart with a synthetic membrane and a pericardial interposition. The goal of the present study was to apply composite cardiac binding to a canine model of heart failure. METHODS: Twenty dogs were randomized to 2 groups: untreated heart failure (group 1, n = 13) and heart failure pretreated by composite cardiac binding (group 2, n = 7). They received a total dose of 1 mg x kg(-1) of intracoronary doxorubicin over 4 weeks. Hemodynamic data were obtained at weeks 0, 7, and 12. All animals were followed up with weekly echocardiography for 12 weeks. RESULTS: Survival in group 1 was 54% and in group 2 was 100% at week 12 (p = 0.0438). Left ventricular end-diastolic pressure increased by 153% in group 1 and by 59% in group 2 (p = 0.0395) at week 12. Ejection fraction decreased by 27% in group 1 and by 19% in group 2 (p = 0.4401) at week 12. CONCLUSIONS: Composite cardiac binding significantly prolongs survival and attenuates left ventricular dilatation and the increase in left ventricular end-diastolic pressure associated to chronic heart failure. Further evaluation in established heart failure is needed. Composite cardiac binding may be used for the prevention of recurrent dilatation following reduction ventriculoplasty. PMID- 10735677 TI - Troponin levels in patients with myocardial infarction after coronary artery bypass grafting. AB - BACKGROUND: The objective of this study was to evaluate serum cardiac troponin T and I levels in patients in whom electrocardiogram, myocardial scan, and serum CK MB levels of the MB isoenzyme of creatine kinase indicated perioperative myocardial infarction (MI) after coronary artery bypass grafting (CABG). METHODS: We studied 590 patients who underwent CABG at the Montreal Heart Institute between 1992 and 1996. Postoperative cardiac troponin T levels (493 patients), troponin I levels (97 patients), and activity of the MB isoenzyme of creatine kinase, electrocardiograms, clinical data, and clinical events were recorded prospectively. The diagnosis of perioperative PMI was defined by a new Q wave on the electrocardiogram, by serum levels of the MB isoenzyme of creatine kinase higher than 100 IU/L within 48 hours after operation, or both. RESULTS: After CABG, 22 patients in whom troponin T levels (22/493, 4.5%) and 6 patients in whom troponin I levels (6/97, 6.2%) were measured had sustained a perioperative MI according to current diagnostic criteria. In these patients, troponin T levels higher than 3.4 microg/L 48 hours after CABG best detected the presence of perioperative MI, with an area under the receiver operating characteristic curve of 0.95, a sensitivity of 90%, a specificity of 94%, a positive predictive value of 41%, a negative predictive value of 99%, and a likelihood ratio of 15. Serum troponin I levels higher than 3.9 microg/L 24 hours after CABG confirmed the perioperative MI with an area under the receiver operating curve of 0.86, a sensitivity of 80%, a specificity of 85%, a positive predictive value of 24%, a negative predictive value of 99%, and a likelihood ratio of 5. CONCLUSIONS: Serum troponin T levels higher than 3.4 microg/L 48 hours after CABG correlated best with the diagnosis of perioperative MI. Serum troponin T levels greater than 3.9 microg/L 24 hours after CABG also correlated with the diagnosis of perioperative MI, although a larger experience is needed to confirm the validity of the chosen cutoff value. PMID- 10735678 TI - Thoracic aortic aneurysms: treatment with endovascular self-expandable stent grafts. AB - BACKGROUND: This study was performed to evaluate the safety and feasibility of endovascular stent graft placement in the treatment of descending thoracic aortic aneurysms. METHODS: Between November 1996 and February 1999, endovascular stent graft repair was used in 21 patients. There were 5 women and 16 men with a mean age of 67 years (range, 41 to 87 years). An atherosclerotic aneurysm with a diameter of more than 6 cm was the indication for intervention in 19 patients (90.5%). In 2 patients (9.5%), a localized aortic dissection with a diameter of more than 6 cm was treated. In 71.4% (15 of 21) of patients, multiple stents were necessary for aneurysm exclusion. To allow safe deployment of the stent graft, preliminary subclavian-carotid artery transposition was performed in 9 patients (42.9%). Vascular access was achieved through a small incision in the abdominal aorta (n = 6), an iliac artery (n = 8), or a femoral artery (n = 7). Talent and Prograft stent grafts were used. RESULTS: Successful deployment of the endovascular stent grafts was achieved in all patients. Two patients died postoperatively (mortality rate, 9.5%), 1 of aneurysmal rupture and the other of impaired perfusion of the celiac axis. Repeat stenting was done in 3 patients because of intraoperative leakage. CONCLUSIONS: Endovascular stent graft repair is a promising and less invasive alternative to exclude the aneurysm from blood flow. This technique allows treatment of patients who are unsuitable for conventional surgical procedures. An exact definition of inclusion criteria and technical development of stent grafts should contribute to further improvements in clinical results. PMID- 10735679 TI - Intraoperative map guided operation for atrial fibrillation due to mitral valve disease. AB - BACKGROUND: This study was designed to determine if intraoperative atrial activation mapping facilitates operations for chronic atrial fibrillation associated with mitral valve disease. METHODS: Surgical treatment guided by intraoperative electrophysiologic mapping was performed in 12 patients with chronic atrial fibrillation associated with isolated mitral valve disease. In 10 of 12 patients, regular and repetitive activation (cycle length ranged from 118 to 210 msec) originated in the left atrial appendage and/or orifice of the left pulmonary vein. In the remaining 2 patients, dominant repetitive activation and sporadic complex activation were alternately observed in the left atrium. However, the activation sequence of the right atrium was extremely complex and chaotic. RESULTS: On the basis of intraoperative mapping, surgical procedures, including resection of the left atrial appendage and/or cryoablation of the orifice of the left pulmonary vein, were applied on the breakthrough site of the repetitive activation. No surgical procedure was performed on the right atrium in 11 patients. Ten of 12 patients (83%) have maintained sinus rhythm for 6 to 40 months (average 24.8 months) after operation. CONCLUSIONS: In the majority of the patients with isolated mitral valve disease, the left atrium acts as an electrical driving chamber for chronic atrial fibrillation. Computerized intraoperative mapping should guide surgeons in determining the appropriate surgical procedure for chronic atrial fibrillation. PMID- 10735680 TI - Low-dose aprotinin is ineffective to treat excessive bleeding after cardiopulmonary bypass. AB - BACKGROUND: Uncontrolled clinical experience at our institution suggested that low-dose aprotinin could control excessive bleeding after cardiopulmonary bypass (CPB). A randomized clinical trial was conducted to determine the efficacy of low dose aprotinin in the treatment of hemorrhage after cardiac surgery. METHODS: One hundred seventy-one patients undergoing cardiac surgery with CPB were included. Forty-four patients (26%) bled significantly in the intensive care unit (>100 mL/h) and received either aprotinin (200,000 KIU bolus + 100,000 KIU/h for 8 hours) or placebo in addition to our standard management of excessive bleeding. RESULTS: Median bleeding before study drug administration was not different between aprotinin (200 mL) and placebo (212.5 mL) groups. Bleeding decreased significantly with time and similarly in both groups. Ninety-five percent of patients required transfusions in both groups. Median blood products transfused were 13 and 8 units per patient in the aprotinin and placebo groups respectively (p = NS). CONCLUSIONS: Routine administration of low-dose aprotinin as part of the treatment protocol to control hemorrhage after CPB does not reduce bleeding or transfusion requirements and, therefore, cannot be recommended. PMID- 10735681 TI - A single-center experience with 1,378 CarboMedics mechanical valve implants. AB - BACKGROUND: The CarboMedics bileaflet prosthetic heart valve was introduced in 1986. We first implanted it in March 1991. The purpose of this study was to analyze our clinical experience with this valve. METHODS: Between March 1991 and December 1997, 1,378 valves were implanted in 1,247 patients, 705 men (56.5%) and 542 (43.5%) women with a mean age of 62 +/- 11.9 years (+/- the standard deviation). Follow-up is 99% complete and totals 3,978 patient-years. RESULTS: The early mortality rate was 4.4% (55/1,247). The survival rates at 1 year and 5 years were 91.8% +/- 0.8% (+/- the standard error of mean) (n = 1,062) and 79.2% +/- 1.4% (n = 281), respectively. Freedom from valve-related complications (linearized rate, 4.9% per patient-year) at 1 year and 5 years was 90.6% +/- 0.8% (+/- the standard error of the mean) (n = 996) and 80.6% +/- 1.4% (n = 243), respectively. Linearized rates for various complications were as follows: bleeding events, 1.73% per patient-year; embolic events, 1.76% per patient-year; operated valvular endocarditis, 0.18% per patient-year; valve thrombosis, 0.10% per patient year; and nonstructural dysfunction, 1.21% per patient-year. Freedom from reoperation at 1 year and 5 years was 98.6% +/- 0.3% (+/- the standard error of the mean) (n = 1,070) and 97.7% +/- 0.5% (n = 285), respectively. CONCLUSIONS: Midterm results demonstrate that the CarboMedics prosthetic heart valve exhibits a low incidence of valve-related complications. PMID- 10735682 TI - Risk factors and outcomes after coronary reoperation in 739 elderly patients. AB - BACKGROUND: As second coronary artery bypass graft (CABG) operations are becoming more common in elderly patients, we conducted a retrospective analysis of risk factors for in-hospital and late outcome in patients aged 70 and over. METHODS: We reviewed records of 739 patients who underwent second CABG at age 70 or older at our institution between 1983 and 1993. Preoperative, operative, and postoperative variables were analyzed to identify predictors of in-hospital and long-term mortality. RESULTS: The mean age (+/- standard deviation) at reoperation was 74 +/- 3 years and the mean interval after primary operation was 130 +/- 55 months. In-hospital mortality was 7.6% (n = 56). Preoperative factors associated with increased in-hospital mortality were preoperative creatinine greater than 1.6 mg/dL (p < 0.001), emergency operation (p < 0.001), female sex (p = 0.012), moderate or severe left ventricular dysfunction (p = 0.049), and left main coronary disease (p = 0.045). In-hospital, actuarial survival was 75% at 5 years and 49% at 10 years. Cardiac event-free survival was 60% at 5 years and 27% at 10 years. The factors independently associated with increased late death were hematocrit (p = 0.046), diabetes (p = 0.011), peripheral vascular disease (p < 0.001), left ventricular function (p < 0.001), history of cancer (p = 0.016), preoperative nonsinus rhythm (p = 0.003), anticoagulation or antiplatelet therapy (p = 0.018), postoperative encephalopathy (p = 0.001), and postoperative stroke (p = 0.014). CONCLUSIONS: CABG reoperation can have excellent results for many elderly patients, but mortality is markedly higher when elderly patients have certain risk factors and comorbidities, alone or in combination. This information should be helpful in educating patients before they decide whether to choose reoperation. PMID- 10735683 TI - Ischemic preconditioning protects against paraplegia after transient aortic occlusion in the rat. AB - BACKGROUND: Paraplegia can result from operations requiring transient occlusion of the thoracic aorta. A rat model of paraplegia with the characteristics of delayed paraplegia and transient ischemic dysfunction was developed to determine whether ischemic preconditioning (IPC) improved neurologic outcome. METHODS: Rats underwent balloon occlusion of the upper descending thoracic aorta. One group (2 minute IPC, n = 19) underwent 2 minutes of IPC and a second group (5 minute IPC, n = 19) had 5 minutes of IPC 48 hours before 10 minutes of occlusion. The control group (n = 31) had no IPC prior to 10 minutes of occlusion. RESULTS: Paraplegia occurred in 68% of the control animals (21 of 31 paraplegic: 6 delayed and 15 immediate paraplegia). Both the 2-minute IPC and 5-minute IPC groups had a decreased incidence of paraplegia when compared to controls (32%, p = 0.011 and 26%, p = 0.009, respectively). CONCLUSIONS: A rat model of spinal cord ischemia demonstrating both delayed paraplegia and transient ischemic dysfunction was characterized. Both 2-minute and 5-minute periods of IPC were found to protect against paraplegia. PMID- 10735684 TI - Human aortocoronary grafts and nitric oxide release: relationship to pulsatile pressure. AB - BACKGROUND: Short and long-term failure of saphenous vein grafts continues to be a significant problem for cardiac surgeons. The purpose of this study was to elucidate the early adaptive changes of human artery and vein conduits with respect to nitric oxide (NO) production under various pressure and pulsatile distention conditions. METHODS: Real-time amperometric NO determinations were made in an in vitro model using human saphenous vein segments (n = 12) and internal thoracic artery segments (n = 8) between 70 and 170 mm Hg, under static conditions recorded with a pressure transducer. Exposing the tissue to morphine (10(-6) M) also stimulated NO release. Under conditions in which the conduits were exposed to the respective pressures for 1 hour, they were then examined for their granulocyte-adhering potential using computer-assisted imaging techniques. RESULTS: A pressure-dependent decrease of NO release was found after 32 minutes of pulsatile pressure (170 mm Hg) in artery and vein, the latter of which appeared to be affected more negatively (p < 0.05; because many more observation points differed significantly after 32 minutes compared to 110 mm Hg values). In vessels maintained for 1 hour at these different pressures and then exposed to morphine (1 microM), stimulated NO release significantly diminished in the veins (artery 37.4 nM NO versus vein 18.1 nM NO; p < 0.05). Increased pressures also correlated with an increase in granulocyte adhesion to veins that could not be reduced following morphine exposure. CONCLUSIONS: Increased pressure and cyclic distention lead to loss of NO release and increased immunocyte adhesion, which are significantly more pronounced in saphenous vein than in internal thoracic artery, suggesting that in the long term this may contribute to the failure of saphenous vein conduits in coronary revascularization. PMID- 10735685 TI - Risk of dysphagia after transesophageal echocardiography during cardiac operations. AB - BACKGROUND: Dysphagia can be a significant complication following cardiac operations. This study evaluates its incidence and relationship to intraoperative transesophageal echocardiography (TEE) for specific indications versus known factors such as stroke or prolonged intubation. METHODS: Records of 838 consecutive cardiac surgical patients were reviewed, and categorized into those who received TEE for specific indications versus those who did not (nonTEE). Dysphagia was recorded when symptoms were confirmed by barium cineradiography. Multiple logistic regression identified significant factors causing dysphagia. RESULTS: TEE was significantly related to the development of postoperative dysphagia by multiple logistic regression (p < 0.001). After controlling for other significant factors (stroke, left ventricular ejection fraction, intubation time, duration of operation), the odds of dysphagia for TEE patients was 7.8 times greater than for nonTEE patients. CONCLUSIONS: TEE may be an independent risk factor for dysphagia following cardiac operations. PMID- 10735686 TI - Biologically variable pulsation improves jugular venous oxygen saturation during rewarming. AB - BACKGROUND: Conventional pulsatile (CP) roller pump cardiopulmonary bypass (CPB) was compared to computer controlled biologically variable pulsatile (BVP) bypass designed to return beat-to-beat variability in rate and pressure with superimposed respiratory rhythms. Jugular venous O2 saturation (SjvO2) below 50% during rewarming from hypothermia was compared for the two bypass techniques. A SjvO2 less than 50% during rewarming is correlated with cognitive dysfunction in humans. METHODS: Pigs were placed on CPB for 3 hours using a membrane oxygenator with alpha-stat acid base management and arterial filtration. After apulsatile normothermic CPB was initiated, animals were randomized to CP (n = 8) or BVP (roller pump speed adjusted by an average of 2.9 voltage output modulations/second; n = 8), then cooled to a nasopharyngeal temperature of 28 degrees C. During rewarming to stable normothermia, SjvO2 was measured at 5 minute intervals. The mean and cumulative area for SjvO2 less than 50% was determined. RESULTS: No between group difference in temperature existed during hypothermic CPB or during rewarming. Mean arterial pressure, arterial partial pressure O2, and arterial partial pressure CO2 did not differ between groups. The hemoglobin concentration was within 0.4 g/dL between groups at all time periods. The range of systolic pressure was greater with BVP (41 +/- 18 mm Hg) than with CP (12 +/- 4 mm Hg). A greater mean and cumulative area under the curve for SjvO2 less than 50% was seen with CP (82 +/- 96 versus 3.6% +/- 7.3% x min, p = 0.004; and 983 +/- 1158 versus 42% +/- 87% x min; p = 0.004, Wilcoxon 2-sample test). CONCLUSIONS: Computer-controlled BVP resulted in significantly greater SjvO2 during rewarming from hypothermic CPB. Both mean and cumulative area under the curve for SjvO2 less than 50% exceeded a ratio of 20 to 1 for CP versus BVP. Cerebral oxygenation is better preserved during rewarming from moderate hypothermia with bypass that returns biological variability to the flow pattern. PMID- 10735687 TI - Complete myocardial revascularization with bilateral internal thoracic artery T graft. AB - BACKGROUND: The internal thoracic artery is widely recognized as the ideal graft for coronary artery bypass procedures. However, because of the inadequate length of the conduit, use of bilateral internal thoracic artery grafting was not suitable for complete revascularization. To overcome this limitation, the T graft was introduced in the 1990s. We decided to prospectively assess the safety of this technique. METHODS: One hundred six patients with a mean age of 51.5 years underwent complete revascularization with an internal thoracic artery T graft. Mean left ventricular ejection fraction was 0.60 (range, 0.22 to 0.85). RESULTS: No patient required reexploration for bleeding, and no patient died within 30 days after operation. On the basis of electrocardiographic changes, 3 patients sustained a perioperative myocardial infarction. One patient had a sternal wound infection. Mean follow-up was 35 months (range, 15 to 61 months). The actuarial survival rate was 99% +/- 1% at 5 years. No myocardial infarctions were reported during the follow-up. Seven patients had recurrent angina. Eighty patients (76%) underwent postoperative stress tests, and 90% had negative results. CONCLUSIONS: Complete myocardial revascularization with the T graft is a safe and reliable technique with excellent midterm results. PMID- 10735688 TI - Mannitol, furosemide, and dopamine infusion in postoperative renal failure complicating cardiac surgery. AB - BACKGROUND: Acute renal failure occurring in the postoperative period, requiring dialysis after cardiac surgery is an important risk factor for an early mortality, and the overall mortality of this complication is as high as 40% to 60%. Dialysis in the early postoperative period is often complicated by acute hemodynamic, metabolic, and hematologic effects that adversely affect cardiopulmonary function in patients stabilizing from recent surgery. The purpose of this study was to avoid the need for dialysis by infusion of the solution of mannitol, furosemide, and dopamine in the early postoperative period in oliguric renal failure. METHODS: One hundred patients with postoperative oliguric or anuric renal failure despite adequate postoperative cardiac output and hemodynamic function were randomized. Forty patients (group A) were given intermittent doses of diuretics (furosemide, bumetadine, and ethracrynic acid) and fluids. Sixty patients (group B) were given continuous infusion of the solution of mannitol, furosemide, and dopamine; the infusion was started within 6 hours (mean 3.5 hours) in subgroup B1 (n = 30), and later than 6 hours (mean 7.5 hours) in subgroup B2 (n = 30) after the onset of renal failure. RESULTS: Diuresis occurred in 93.3% of group B (n = 56) versus 10% in group A (n = 4; patients with preop normal renal function). Ninety percent of group A (n = 36) required dialysis versus only 6.7% of group B (n = 4; patients with preexisting renal disease of subgroup B2). Renal function returned to preoperative normal (serum creatinine 0.9 +/- 0.05, p < 0.0001) or baseline value (serum creatinine 2.5 +/- 0.01, p < 0.0001) after first postoperative week in subgroup B1 and third postoperative week in subgroup B2. CONCLUSIONS: Infusion of solution of mannitol, furosemide, and dopamine promoted diuresis in patients with acute postoperative renal failure with adequate postoperative cardiac output and had decreased the need for dialysis in the majority of patients. Early administration of this solution in acute renal failure caused early restoration of renal function to normal or baseline status. It remains to be determined whether routine administration of this solution in the early postoperative period for oliguric renal failure influences the long-term mortality and morbidity in those patients who do require dialysis. PMID- 10735689 TI - Evaluation of valve sound and its effects on ATS prosthetic valves in patients' quality of life. AB - BACKGROUND: We interviewed patients and carried out frequency analyses to compare the closing sounds of ATS and St. Jude Medical (SJM) prosthetic valves. METHODS: Forty-five patients undergoing valve replacements using ATS valves were investigated. We interviewed patients at 1 month and 1 year after the operation, and carried out frequency analysis to investigate the prosthetic valve's closing sound. RESULTS: According to the results of the interviews, 84.4% of patients with ATS valves were not aware of the valve sounds. ATS valves scored significantly lower than SJM valves on audibility of the valve sound, disturbance during daytime, sleep disturbance, request for replacement with a soundless prosthetic valve, audibility to others, and noise index. According to the frequency analysis on the prosthetic valve's closing sound, the sound peak of the ATS valves was around 1.2 kHz, and the sound pressure of the ATS valves was significantly lower than that of the SJM valves. CONCLUSIONS: Though a further long-term observation on thromboembolism and hemolysis is needed for evaluation of prosthetic valves, the ATS valve is presently considered to impart a better quality of life. PMID- 10735690 TI - Platelets and prostacyclin in arterial bypasses: implications for coronary artery surgery. AB - BACKGROUND: We investigated effects of platelets and prostacyclin formation in human internal mammary (IMA) and radial (RA) arteries. METHODS: IMA and RA segments were suspended in organ bath with increasing concentrations of platelets. Experiments were applied with and without ketanserin, a 5HT2 receptor antagonist, or U3405, a TXA2 receptor antagonist. The release of prostacyclin (PGI2) was assessed by enzyme immunoassay in vessels without endothelium, before and after contraction with angiotensin (AT) I-II. RESULTS: In IMA and RA with endothelium, platelets caused contractions, significantly enhanced in arteries without endothelium. Contractions to platelets were higher in RA than in IMA. U3405 reduced the platelet induced contractions in RA but not in IMA. Ketanserin inhibited the platelet induced contractions in IMA and RA. The basal release of PGI2 was more important in IMA than in RA. Addition of AT/I-II significantly reduced the release of PGI2 in IMA but not in RA. CONCLUSIONS: The RA responds more powerfully to platelets than IMA. Protective system with PGI2 seems to be more powerless in RA than in IMA. This accentuates the importance of antispastic and antiplatelet drugs when arteries are used for coronary artery bypass surgery. PMID- 10735691 TI - Endoscopic and traditional saphenous vein harvest: a histologic comparison. AB - BACKGROUND: Vein trauma after saphenectomy by endoscopic or longitudinal techniques may influence the progression of medial and intimal hyperplasia and ultimately affect graft patency. This study compared the histologic characteristics of saphenous veins after endoscopic and longitudinal harvest. METHODS: One hundred seventy patients who underwent elective coronary artery bypass grafting had saphenectomy performed endoscopically (n = 88) or by a longitudinal incision (n = 82). Cross-sectional specimens from endoscopically (n = 151) and longitudinally (n = 158) harvested veins were submitted for hematoxylin-eosin, trichrome, and elastin staining. Blinded histologic evaluation involved graded analysis of endothelial, smooth muscle, and elastic lamina continuity in addition to medial and adventitial connective tissue uniformity. RESULTS: Regardless of harvest technique, endothelial, elastic lamina, and smooth muscle continuity as well as medial and adventitial connective tissue uniformity were not significantly different. CONCLUSIONS: Minor histologic alterations occur during saphenectomy, however, endoscopically and longitudinally harvested saphenous veins are histologically similar. PMID- 10735693 TI - Regression of hypertrophy after Carpentier-Edwards pericardial aortic valve replacement. AB - BACKGROUND: The purpose of this study was to determine whether significant regression of left ventricular hypertrophy is seen after implantation of small sizes (19 to 23 mm) of the Carpentier-Edwards (CE) pericardial valve, a stented pericardial valve. METHODS: Echocardiograms and electrocardiograms (ECGs) were performed at least 1 year after surgery (mean 18 months) in patients with 19-, 21 , and 23-mm CE pericardial aortic valves and compared with preoperative echocardiograms and ECGs. RESULTS: A total of 41 patients, mean age 79 +/- 9 years (range 46 to 93 years), were studied, including 7 19-mm, 22 21-mm, and 12 23-mm patients. The mean postoperative gradient was 22 +/- 7 mm Hg for 19-mm valves, 18 +/- 5 mm Hg for 21-mm valves, and 16 +/- 4 mm Hg for 23-mm valves. The postoperative valve areas were 1.1 +/- 0.3 cm2 for the 19-mm, 1.3 +/- 0.3 cm2 for the 21-mm, and 1.5 +/- 0.4 cm2 for the 23-mm valves. Left ventricular end diastolic diameter, end systolic diameter, septal thickness, and posterior wall thickness all decreased significantly (p <0.05) postoperatively. The proportion of patients with significant left ventricular hypertrophy on ECG decreased from 63% to 47% (p = 0.001). Left ventricular mass decreased significantly by echocardiography from 265 g preoperatively to 208 g postoperatively (p = 0.004). Left ventricular mass decreased for each valve size, and the greatest absolute reduction in mass occurred in the 19-mm valve recipients. CONCLUSIONS: Implantation of the 19-, 21-, and 23-mm CE pericardial valves results in significant reductions in left ventricular mass. These findings suggest that stented pericardial valves can be used in the small aortic root without the need for aortic root enlargement procedures. PMID- 10735692 TI - Efficacy of the internal mammary artery in combined aortic valve replacement coronary artery bypass grafting. AB - BACKGROUND: While internal mammary artery (IMA) use predicts improved survival after coronary bypass grafting (CABG), it remains unknown whether patients undergoing concomitant aortic valve replacement (AVR) realize a similar benefit. METHODS: All patients at a single teaching institution, undergoing combined AVR CABG, which included a graft to the left anterior descending coronary artery (LAD) from 1984 to 1994 (n = 227) were examined retrospectively. RESULTS: Patients receiving an IMA graft (yesIMA, n = 135) and patients receiving only saphenous vein grafts (nonIMA, n = 92) were not different in their presenting symptoms, or in their incidence of preoperative risk factors. The patients with IMA were more likely to be male, have a later year of operation, be younger, and have a greater body surface. Morbidity was not different between groups. IMA use did not affect 30-day mortality. Long-term actuarial survival was greater in the group with IMA (63% +/- 7% vs 42% +/- 6% at 5 years, p < 0.01). A multivariate Cox proportional hazards model demonstrated that use of an IMA graft improved survival, while recent myocardial infarction, diabetes, earlier year of operation, and lower ejection fraction diminished long-term survival. The relative risk of IMA grafting was 0.570. CONCLUSIONS: Within the limits of a retrospective analysis, patients in a modern era of cardiac operation, who undergo combined AVR-CABG, do not suffer increased morbidity from IMA use, and may realize a survival benefit from use of the IMA as a conduit for bypass of the LAD coronary artery. PMID- 10735694 TI - Influence of biopump with and without intraaortic balloon on the coronary and carotid flow. AB - BACKGROUND: The objective of this study was to evaluate the influence of biopump used for left ventricular assistance on the coronary and carotid flows in dogs with normal heart. The efficacy of the simultaneous use of an intraaortic balloon pump to compensate the possible deleterious effects of the circulatory assistance with continuous flow was also analyzed. METHODS: Fifteen dogs were studied. The hemodynamic evaluation included serial measurements of the classic parameters. Carotid and coronary blood flows were obtained by electromagnetic transducers. RESULTS: The hemodynamic evaluation did not show significant statistical changes. The use of circulatory-isolated assistance with biopump shows reduction (24.6% +/ 6.1%) in coronary flow, in relation to the control situation and the concomitant use of biopump and intraaortic balloon pump showed similar coronary flow. Regarding carotid flow, a similar trend was observed in relation to the positive influence of the pulsatile flow with an intraaortic balloon pump without statistical significance (p = 0.0582). CONCLUSIONS: The biopump reduces the coronary flow in dogs. The use of intraaortic balloon pump with the biopump increases the coronary flow significantly, reaching similar values to those observed without the circulatory assistance. PMID- 10735695 TI - Amelioration of the bleeding tendency of preoperative aspirin after aortocoronary bypass grafting. AB - BACKGROUND: Aspirin therapy is widely used in the treatment of cardiac disease. It has been recognized as a causative factor for increased bleeding and blood loss after open heart operations. METHODS: To determine whether high-dose aprotinin maintained its efficacy in reducing blood loss in the presence of aspirin pretreatment in patients undergoing aortocoronary bypass, we performed a double blind study on 60 adult patients. Half received high-dose aprotinin (Trasylol) and half placebo. RESULTS: Total hemoglobin loss, the primary efficacy variable was reduced from 36.1 +/- 31.4 g (mean +/- SD) to 14.1 +/- 16.0 g (p = 0.002). Blood loss was reduced intraoperatively and total loss was reduced from 837.3 mL +/- 404.9 mL to 368.7 mL +/- 164.3 mL (p < 0.001). The number of patients who did not receive any donor blood products was significantly higher in the aprotinin-treated patients (56.7% versus 23.3%, p = 0.008). Activation of the clotting cascade was significantly less in the treated patients toward the end of cardiopulmonary bypass both by measurement of thrombin-antithrombin III complex (p < 0.0001) and prothrombin fragment 1 + 2 (p < 0.0001). D-Dimer generation was significantly less from the onset of bypass and after reversal of heparin in the aprotinin-treated patients (p < 0.0001). CONCLUSIONS: High-dose aprotinin was highly effective in reducing bleeding in this high-risk group of patients. Biochemical analyses suggest the mechanism by which aspirin increases blood loss after cardiopulmonary bypass is different from the blood-preserving effects of aprotinin, which is acting as an antifibrinolytic agent. PMID- 10735696 TI - Evaluation of the practice of routine culturing of native valves during valve replacement surgery. AB - BACKGROUND: We routinely cultured native heart valves removed during valve replacement surgery even when infected carditis (IE) was not suspected. Several probable contaminated cultures prompted us to evaluate this practice. METHODS: The medical records of all patients who had positive valve cultures from 1995 to 1997 were reviewed for admission diagnoses, operative surgery, pathology and microbiology report, postoperative infections, and antibiotic use. Cases were excluded only for incomplete medical records or preoperative suspicion of IE. Long-term outcome for the cases was obtained from review of outpatient records and phone contact with the patient or physician. RESULTS: Thirty-two of 222 (14.4%) evaluable patients had positive valve cultures. Coagulase-negative Staphylococcus was the most common isolate. IE was not suggested in any of these cases based upon the surgical or the pathology report. Only 1 of 32 (3%) developed postoperative prosthetic valve endocarditis (PVE). Three patients died of unrelated causes, and the 28 surviving patients showed no sign of PVE, with a mean follow-up of 23 months. CONCLUSIONS: The incidence of false-positive native valve cultures is high. Positive cultures did not predict the occurrence of PVE sufficiently to justify obtaining them. Treating patients who had positive native valve cultures would have been unwarranted and poses an unnecessary risk. PMID- 10735697 TI - A controlled trial of substrate-enhanced, warm reperfusion ("hot shot") versus simple reperfusion. AB - BACKGROUND: Modified reperfusion after aortic cross-clamping is claimed to reduce myocardial injury, thus improving postoperative myocardial performance. METHODS: We measured perioperative release of creatine kinase-MB and troponin-T in 40 patients undergoing valve replacement (combined with coronary grafts in 12 cases) to determine whether infusion of a modified reperfusate before cross-clamp removal reduced myocardial injury. Patients were randomly allocated to one of two groups with minimization for age, surgeon, operation, and ventricular function. The control group received unmodified reperfusion, while the study group received a normothermic reperfusate, enhanced with glutamate and aspartate, for 5 minutes before removal of the cross-clamp. Serial determinations of troponin-T, creatine kinase-MB isoforms, and total creatine kinase-MB activity were made up to 5 days postoperatively. Requirements for inotropic support and evidence of myocardial infarction were documented. RESULTS: Creatine kinase-MB activity, creatine kinase MB isoforms, and troponin-T were not significantly different between the two groups. There were no differences in the incidence of postoperative myocardial infarction or in inotrope requirement. CONCLUSIONS: Our study did not demonstrate any advantage in using modified reperfusion in this group of patients. PMID- 10735698 TI - Anatomy of the muscular subpulmonary infundibulum with regard to the Ross procedure. AB - BACKGROUND: To clarify the precise anatomical relationship of the muscular subpulmonary infundibulum. METHODS: Eleven hearts were dissected, and microscopic sections taken through the arterial trunks of a 37-week-old fetus and of a neonate. The anatomy was also investigated during operative Ross procedures. RESULTS: The sinotubular junctions of the pulmonary and aortic roots cross obliquely. The leaflets of the pulmonary valve are lifted away from the ventricular septum by the free-standing subpulmonary infundibulum, whereas the aortic valve is deeply wedged between the atrioventricular junctions. The muscular infundibulum spirals around the aortic root, being longest below the right-facing aortic sinus and shortest below the left. The first septal perforating artery pierces the septum below the shortest part of the infundibulum, sometimes within a millimeter of the pulmonary valvar hinge, but a muscular sleeve lifts the pulmonary leaflets from the septal musculature. CONCLUSIONS: The pulmonary valvar leaflets are supported entirely by free standing musculature, having no direct relationship with the ventricular septum. This makes possible the Ross procedure. PMID- 10735699 TI - Issues and outcomes in the management of supravalvar aortic stenosis. AB - BACKGROUND: Supravalvar stenosis of the aorta is an uncommon congenital cardiac anomaly that involves not only the supravalvar aorta but the entire aortic root. Despite considerable attention to the importance of maintaining the integrity of the aortic root during supravalvar reconstruction, there has been little focus on the management of other components of the aortic root and left ventricular outflow tract, including the aortic valve, subvalvar region, and coronary arteries. METHODS: We reviewed the records of 36 consecutive patients with supravalvar aortic stenosis who underwent repair from 1992-1998 (median age, 4 years). Discrete stenosis was present in 29 patients, whereas the remaining 7 had the diffuse form of the disease. Associated anomalies of the aortic root and adjacent structures were present in 23 patients. The median pressure gradient across the left ventricular outflow tract was 70 mm Hg. Supravalvar stenosis was relieved by extended aortoplasty with a Y-shaped patch in 18 patients, resection of the stenotic segment of ascending aorta at the sinotubular junction with end to-end anastomosis of the ascending aorta in 7, the Ross procedure in 4, and other techniques in 7. Additional procedures included aortic valvuloplasty in 10 patients, resection of subvalvar stenosis in 11, and procedures on the coronary arteries in 2. RESULTS: There was 1 perioperative death, and no reoperations or other significant complications. During follow-up (median 33 months), there were no deaths and 3 reoperations for replacement of the aortic valve with a pulmonary autograft (n = 1) or mechanical prosthesis (n = 2). The median pressure gradient across the left ventricular outflow tract was 10 mm Hg. CONCLUSIONS: In patients with supravalvar aortic stenosis, abnormalities of the aortic valve, subaortic region, and coronary arteries are frequently present as well. Management of these issues is as critical to the long-term outcome of these patients as reconstruction of the supravalvar aorta. Aggressive valvuloplasty may help decrease the incidence of late aortic valve replacement, whereas the Ross procedure may be a preferable approach in some patients with complex outflow tract obstruction. PMID- 10735700 TI - Single coronary artery and complete transposition of the great arteries: a technical challenge resolved? AB - BACKGROUND: The origin of the coronary arteries from a single aortic sinus remains a rare congenital anomaly, once regarded as having little clinical significance. Contemporary surgical practice, however, frequently demands precise coronary reimplantation. In this article we emphasize a prophylactic surgical technique found especially helpful in the repair of D-transposition of the great arteries (D-TGA)/single coronary artery. METHODS: We reviewed the institutional cardiac registry. RESULTS: Since 1985, 398 neonates with D-TGA were repaired with the arterial switch procedure. A mortality rate of 38% was encountered in "simple" D-TGA (n = 174)/single coronary (2.9% left facing sinus (IRLCx), 7.5% right facing sinus (IIRLCx)) and 41% in neonates with D-TGA (n = 224)/single coronary (3.6% IRLCx, 12% IIRLCx). During the past 3.5 years the surgical mortality rate of neonates (n = 6) treated with origin of the coronary arteries from a single aortic sinus has dropped to 0%. CONCLUSIONS: The surgical repair of D-TGA/single coronary artery continues to trouble surgeons. The implantation of a well-mobilized coronary "button" into a previously anastomosed neoaorta remains a key prophylactic technique in the achievement of good technical results. PMID- 10735701 TI - Pectoralis major muscle flap for deep sternal wound infection in neonates. AB - BACKGROUND: Deep sternotomy wound infections during the neonatal period, their management utilizing the pectoralis major muscle flap (PMF), and their follow-up are reported. METHODS: Seven hundred-twenty consecutive pediatric cardiac operations performed from 1995 to mid 1998 in 108 neonates and 612 infants are reviewed. Nine children (1.25%), 6 neonates and 3 infants, developed deep sternotomy wound infections and underwent PMF reconstruction. The 6 neonates are reviewed. Their follow-up includes growth and development reports, physical examination, and computerized tomographic scans of the chest. RESULTS: The incidence of sternal wound complications in our neonatal patients (5.5%, 6 of 108) was significantly higher than in the infantile group (0.5%, 3 of 612), (p = 0.0001, odds ratio = 11.94). Five neonates were treated with a unilateral, turnover PMF reconstruction. One patient was treated by a bilateral rotational PMF. All sternal wounds healed successfully, and all patients survived. In a follow-up period, ranging from 6 to 31 months (mean 16.5 months), the growth and development of all operated neonates was as expected for their age. There were no signs of chronic sternal infection in any of them. CONCLUSIONS: Early recognition of sternal wound complications should facilitate surgical treatment. Utilizing the PMF promotes rapid wound healing and preservation of life in these severely ill neonates, with minimal developmental problems. PMID- 10735702 TI - Utility of near-infrared spectroscopic measurements during deep hypothermic circulatory arrest. AB - BACKGROUND: Near-infrared spectroscopy (NIRS) is used to monitor cerebral oxygenation during cardiac surgery. However, interpretation of the signals is controversial. The aim of the study was to determine which NIRS variable best correlated with brain damage as assessed by animal behavior and neurohistologic score and to compare the accuracy of NIRS and magnetic resonance spectroscopy (MRS) in predicting brain injury. METHODS: Forty 5-week-old piglets underwent 60 minutes of deep hypothermic circulatory arrest (DHCA) at 15 degrees C. Changes in brain adenosine triphosphate (ATP), phosphocreatine (PCr), and intracellular pH (pHi) were determined by MRS and correlated to changes in oxygenated hemoglobin (HbO2), deoxygenated hemoglobin (Hb), and oxidized cytochrome a,a3 (CytOx) NIRS signals. Brains were fixed on day 4 and examined using a neurohistologic score. RESULTS: Reductions in CytOx and HbO2 values were correlated closely with decreases in ATP, PCr, and pHi. The changes in CytOx and PCr showed the strongest correlation (r = 0.623). Maximal CytOx reduction during DHCA of more than -25 microM * differential pathlength factor (DPF) predicted brain damage with a sensitivity of 100% and a specificity of 75%. The histologic score was also correlated with a decrease in ATP (r = -0.52 for CytOx; r = -0.32 for ATP); HbO2, PCr, and pHi showed no correlations. CONCLUSIONS: Reduction in CytOx correlates with decreased brain energy state and predicts histologic brain injury after DHCA with a high sensitivity. These data suggest that the level of CytOx could be a very important predictor of brain damage during DHCA. PMID- 10735703 TI - Clinical efficacy of heparin-bonded bypass circuits related to cytokine responses in children. AB - BACKGROUND: Cardiopulmonary bypass (CPB) induces numerous systemic reactions. This study examined the efficacy of heparin-bonded CPB circuits on inflammatory responses and postoperative status in children. METHODS: Thirty-four infants undergoing elective cardiac surgery were randomly divided into two groups: a heparin-bonded CPB group (n = 17) and a non-heparin-bonded group (n = 17). Plasma levels of the inflammatory cytokines were measured before, during, and after CPB, and postoperative status was determined by examining the respiratory index, blood loss, and the post- and preoperative body weight percent ratio. RESULTS: Significant differences in tumor necrosis factor-alpha, interleukin-6, and interleukin-8 patterns were observed during and after CPB between the two groups (p < 0.01, p < 0.01, p < 0.05, respectively). All cytokines measured were significantly lower in the heparin-bonded group just after CPB (p < 0.05). There were no differences in duration of intubation, intensive care unit or hospital stay, or postoperative blood loss, but the respiratory index 3 hours after CPB and body weight percent ratio 24 and 48 hours after CPB were significantly reduced in the bonded group (p < 0.05, p < 0.01, p < 0.05, respectively). CONCLUSIONS: Our findings suggest that heparin bonding of the bypass circuits affects early postoperative status and reduces cytokine responses in pediatric cardiac surgery. PMID- 10735704 TI - Postoperative recovery in children after minimum versus full-length sternotomy. AB - BACKGROUND: Minimal access incisions for pediatric cardiac surgery have been reported to hasten postoperative recovery. This prospective study compared recovery after a minimum versus full-length sternotomy for repair of atrial septal defects in children. METHODS: We studied 35 children undergoing atrial septal defect repair using a full-length sternotomy (n = 18) or ministernotomy (n = 17) according to the surgeon's preference. All children were managed according to an established clinical practice guideline. Intraoperative comparisons included patient demographics, bypass and cross-clamp times, and, as a measure of stress response, epinephrine, norepinephrine, and lactate levels at six time intervals throughout the surgical procedure. Postoperative comparisons included pain scores at 6, 12, and 24 hours, frequency of emesis, analgesic requirements, respiratory rate and gas exchange, and length of intensive care unit and total hospital stay. Nurse and parent assessment scores of overall recovery were constructed using visual analog and Likert scales. RESULTS: No significant differences between mini- versus full-length sternotomy were detected for the measured outcome variables. No adverse outcomes were detected. CONCLUSIONS: In this prospective study, a ministernotomy did not enhance postoperative recovery, and the primary advantage appears to be an improved cosmetic result. PMID- 10735705 TI - Surgical closure of apical ventricular septal defects through a right ventricular apical infundibulotomy. AB - BACKGROUND: We present a new understanding of the anatomic position of apical ventricular septal defects and its surgical relevance. These defects occur between the left ventricular apex and the infundibular apex, rather than between the left and right ventricular apices. Often a sizable apical recess, the infundibular apex lies anteriorly and inferiorly to the moderator band and is the most leftward part of the right ventricle. METHODS: Four patients (2 boys and 2 girls) with a mean age of 109 days (range, 48 to 217 days) underwent patch closure through an apical infundibulotomy, which allowed complete visualization of the muscular apical ventricular septal defect. RESULTS: There were no early or late deaths at operation. No significant residual shunt at ventricular level was detected by postoperative two-dimensional and Doppler echocardiography. Intraoperative comparison of right atrial and pulmonary arterial blood samples showed a difference of less than 5%. At a mean follow-up of 18 months, all the patients are asymptomatic and growing well. CONCLUSIONS: The successful outcome of these 4 patients indicates that surgical closure of apical ventricular septal defects can be achieved safely and completely in early infancy through a limited right ventricular apical infundibulotomy. Long-term follow-up of these and similar patients is needed to provide further evaluation of this approach. PMID- 10735706 TI - Continuous pulmonary perfusion during cardiopulmonary bypass prevents lung injury in infants. AB - BACKGROUND: Lung injury after cardiopulmonary bypass is a serious complication for infants with congenital heart disease and pulmonary hypertension. Excessive neutrophil sequestration in the lung occurring after reestablishment of pulmonary circulation implies that interaction between neutrophils and pulmonary endothelium is the major cause of lung injury. METHODS: Thirty infants with either ventricular septal defect or atrioventricular septal defect and with pulmonary hypertension were enrolled in this study. We performed continuous pulmonary perfusion during total cardiopulmonary bypass on 16 patients (perfused group) and conventional cardiopulmonary bypass on 14 patients (control group). PaO2/FiO2 and neutrophil counts were assessed from immediately before surgery to 24 hours after termination of cardiopulmonary bypass. RESULTS: PaO2/FiO2 was higher in the perfused group than in the control group, and the difference was significant throughout the study period. Neutrophil counts decreased below prebypass values in both groups at 30 minutes after aortic unclamping, and the difference was significant in the control group but was not in the perfused group. Duration of postoperative ventilatory support was significantly less in the perfused group. CONCLUSIONS: Our study demonstrates that arrested pulmonary circulation during cardiopulmonary bypass is the major risk factor of lung injury and that continuous pulmonary perfusion is effective in preventing lung injury. PMID- 10735707 TI - Aortic valve repair after arterial switch operation. AB - A patient with transposition of the great arteries and a ventricular septal defect underwent an arterial switch operation 15 months after pulmonary artery banding. At 12 years of age, severe neoaortic valve regurgitation, due to dilated aortic sinuses and poor leaflet coaptation, developed. Aortic valve repair involved placement of subcommissural sutures, elliptical excision and tailored reduction of two anterior aortic sinuses, with triangular patch expansion of the proximal ascending aorta. A good result was obtained. PMID- 10735708 TI - Transjugular intrahepatic portosystemic shunt for recurrent hepatic hydrothorax. AB - For many years, pleural effusions have been recognized as a complication of cirrhosis, occurring in approximately 5.5% of patients. Recent studies have confirmed that small defects in the diaphragm allow for passage of ascitic fluid into the pleural space. Successful management of these patients is challenging, as many of the treatment options can be associated with increased morbidity. The initial treatment should focus on eliminating and preventing the recurrence of ascites with diuretics and water and salt restriction. For those patients who do not respond medically, more invasive techniques have been used including serial thoracentesis, chest tube placement, chemical pleurodesis, and peritoneovenous shunts. We present a patient with recurrent pleural effusions secondary to hepatic cirrhosis who was unsuccessfully treated medically, and subsequently treated with thoracentesis, chest tube drainage and pleurodesis, with ultimate resolution after transjugular intrahepatic portosystemic shunt placement. PMID- 10735709 TI - Thrombosed giant coronary artery aneurysm presenting as an intracardiac mass. AB - Giant coronary artery aneurysms are rare in adults and are usually found in association with Kawasaki's disease arising in childhood. We report a case of a thrombosed giant right coronary artery aneurysm presenting as an intracardiac mass detected after inferior wall myocardial infarction. Histologic analysis indicated that fibromuscular dysplasia was the underlying cause of the aneurysm. PMID- 10735710 TI - Pericardiectomy using an oscillating saw. AB - We describe the use of a microoscillating saw on the heart to remove calcific pericardium. This microoscillating saw proved indispensable to achieve a safe pericardiectomy in a 55-year-old woman with thick, severe idiopathic calcific constrictive pericarditis. PMID- 10735711 TI - Rhinolalia: an underappreciated sign of pneumomediastinum. AB - Thoracic surgeons are involved in the diagnosis and management of patients with mediastinal emphysema because of the potentially life-threatening conditions that either must be treated emergently or excluded. Although the classic findings of pneumomediastinum have been described for nearly 60 years, an underappreciated finding is the presence of rhinolalia. Case reports of this finding are sporadic and absent from the thoracic surgical literature. The presence of rhinolalia in association with spontaneous pneumomediastinum is highlighted in the following case presentation and prior reports are reviewed. PMID- 10735712 TI - Mitral and tricuspid valve rupture after moderate blunt chest trauma. AB - We present a patient with rupture of both atrioventricular valves in a previously healthy adult man who sustained a 5-foot fall. The mechanism of injury was such that it would not necessarily raise an adequate index of suspicion for valvular damage had valvular rupture not occurred. The usefulness of perioperative echocardiography is highlighted. PMID- 10735713 TI - Surgical management of massive atrial size mismatch in heart transplantation. AB - We describe a surgical technique that we successfully used in a case involving severe size mismatch between the recipient's atrial remnants and the donor heart atria. Complete recipient left atrial excision, extensive pulmonary vein mobilization, and plication of the right atrial remnant achieved this goal. PMID- 10735714 TI - Ectopic thyroid tissue in the left ventricular outflow tract. AB - Ectopic thyroid tissue in the heart (struma cordis) is rare. The only report from a hemodynamically significant obstruction of the left ventricular outflow tract (LVOT) by a heterotopic thyroid gland was published in 1988. In our patient, a 42 year-old woman with recurrent chest pain, two spheric cardiac tumors were diagnosed by transthoracal echocardiography. One of the tumors, obstructing the LVOT, could successfully be resected under cardiopulmonary bypass. The pathologic examination showed a colloid-filled ectopic thyroid gland. The second tumor, which was entirely located in the submembraneous part of the interventricular septum, had no hemodynamic influence, and was left in situ to avoid surgical damage of adjacent intraseptal structures. The midterm follow-up showed no recurrence. PMID- 10735715 TI - Traumatic rupture of an aberrant right subclavian artery. AB - We describe a patient who sustained a traumatic rupture of an aberrant right subclavian artery. An interposition graft was used to restore continuity of the artery to the descending thoracic aorta. PMID- 10735716 TI - Thoracoabdominal aortic aneurysm combined with aortic occlusion. AB - The case of a 73-year-old woman with aneurysms of the thoracoabdominal aorta and distal arch, combined with aortic occlusion, is reported. Cannulation from the femoral artery was not possible because of the aortic occlusion. Blood supply to the abdominal viscera and lower extremities was achieved only by selective perfusion from the celiac artery, superior mesenteric artery, and bilateral renal arteries. A unique choice of selective perfusion for distal circulatory support is described. PMID- 10735717 TI - Injection of vasodilators into arterial grafts through cardiac catheter to relieve spasm. AB - Both native coronary artery and coronary bypass grafts may develop vasospasm after coronary artery bypass grafting. We recommend that whenever there is a high suspicion of vasospasm in arterial grafts and the native coronary artery unresponsive to or not suitable for usual vasodilator therapy, prompt selective graft arteriogram should be performed. Intraluminal injection of vasodilators such as calcium antagonists in combination with nitroglycerin may provide an effective antispastic therapy and this procedure could be lifesaving as demonstrated in the present report. PMID- 10735718 TI - Weaning from mechanical support in a patient with acute heart failure and multiple sclerosis. AB - We describe a 19-year-old woman developing acute left ventricular heart failure during her first exacerbation of multiple sclerosis. Histopathologic examination of myocardial tissue showed extensive myocytolysis. A left ventricular assist device was implanted. Three months later the cardiac function was restored and the left ventricular assist device was explanted. After 1 year the patient still remains well and her cardiac function is normal. PMID- 10735719 TI - Aberrant right pulmonary artery and double outlet ventricle: one-stage repair. AB - We report a 13-month-old male child with anomalous origin of the right pulmonary artery from the ascending aorta and a double outlet right ventricle. Aortic wall was used for elongation of the pulmonary artery and side-to-end connection to the pulmonary trunk. Special emphasis is made on this particular operative technique for strictly laterally originating right pulmonary artery that requires no prosthetic material, avoids extreme stretching, and may enable normal growth potential. PMID- 10735720 TI - Redo lung volume reduction surgery in a patient with alpha1-antitrypsin deficiency. AB - Lung volume reduction surgery is a palliative procedure that improves dyspnea and pulmonary function in selected patients with advanced emphysema. Postoperative benefit is sustained for an individual period and depends on the emphysema morphology, the surgical technique, and other not yet well-defined factors. The question whether lung volume reduction surgery can be performed a second time on the same thoracic cavity is often raised but experience in this regard is lacking. We describe a patient who has undergone a successful redo operation 2 years after the initial lung volume reduction surgery. PMID- 10735722 TI - Aorticoright atrial tunnel. AB - Two unusual cases of aorticoright atrial tunnel are described. Both patients were referred to our institution for evaluation of a continuous heart murmur best heard along the right upper sternal border. Ascending aortography showed the tunnel taking its origin from the aortic root and entering the right atrium through a tortuous link. Both patients underwent surgical closure. In addition, a review of similar cases in the literature is presented. PMID- 10735721 TI - Pulmonary arteriovenous malformations after cavopulmonary connection. AB - We report the case of a child with hypoplastic left heart syndrome who developed pulmonary arteriovenous (AV) malformations after superior cavopulmonary anastomoses. Resolution of the pulmonary AV malformations occurred following a completion Fontan procedure. This phenomenon has been reported previously, but only in patients with heterotaxy and polysplenia. PMID- 10735723 TI - Occlusion of left coronary artery ostium by an aortic valve cusp. AB - Congenital anomalies of the aortic valve can be associated with other cardiac anomalies. In this report, we present a patient with an aortic valve anomaly associated with occlusion of left coronary ostia. In addition, we reviewed the literature and found 10 similar cases. Although compatible with life, this anomaly can lead to significant symptoms. Preoperative diagnosis as well as proper therapeutic planning should be tailored to correct valvular competence and restore coronary blood flow. PMID- 10735724 TI - Images in cardiothoracic surgery. Aortobronchial fistula. PMID- 10735725 TI - Deep circumflex iliac artery as a free arterial graft for myocardial revascularization. AB - When complete revascularization cannot be obtained with the internal thoracic artery and the other arterial grafts, the deep circumflex iliac artery (DCIA) may be an excellent alternative conduit. The deep circumflex iliac artery was used as a free graft for direct myocardial revascularization in 4 patients from January to July 1999. We describe our experience with this arterial conduit, review the anatomy of the artery, and present our harvesting technique. PMID- 10735726 TI - Pulmonary root translocation in transposition of great arteries repair. AB - Anterior pulmonary root translocation is used as a new approach for anatomic repair of transposition complexes with ventricular septal defect and pulmonary stenosis. It is performed to construct the right ventricle outflow tract, after patch diversion of left ventricle to aorta. Since 1994, 3 infants underwent this procedure. The preliminary results indicate some growth of the pulmonary root and suggest that this technique could diminish reoperations in this group of patients. PMID- 10735727 TI - Exposure of the mitral valve by transecting the ascending aorta during aortic and mitral valve replacement. AB - Combined aortic and mitral valve operations are still considered major cardiac surgical procedures. The duration of aortic cross-clamping and cardiopulmonary bypass is longer, which increases morbidity and mortality for these combined, complicated operations. Aortic valve exposure is generally satisfactory, but mitral valve exposure may be difficult and add to the length of the aortic cross clamping time. We have exposed the mitral valve by transecting the ascending aorta, and retracting both ends apart, to give direct access through the dome of the left atrium in 7 patients. This approach gave good exposure and did not increase the risk of complications. Exposure of the mitral valve through the left atrium using a superior approach, by transecting the ascending aorta, is a good option for patients with multiple cardiac conditions who are undergoing combined aortic and mitral valve operations. PMID- 10735728 TI - Methods for graft size selection in aortic valve-sparing operations. AB - Simple and reproducible methods for accurate restoration of aortic root dimensions during aortic valve-sparing operations are described. The methods are based on choice of an appropriate size vascular graft based on the measured or desired diameter of the aortic annulus. PMID- 10735729 TI - Rudolf Nissen's years in Bosphorus and the pioneers of thoracic surgery in Turkey. AB - Rudolph Nissen is one of the most distinguished pioneers in thoracic surgery, well known for his pneumonectomy operation performed for the first time in surgical history. He migrated to Istanbul like most of his contemporary colleagues, avoiding the fascist regime in Germany in the 1930s. He was enthusiastically welcomed in Turkey and appointed as the Chief of the First Surgery Clinic in Istanbul University in 1933. He worked with discipline and in cooperation with his Turkish colleagues, such as Ahmed Burhaneddin Toker, Fahri Arel, Dervis Manizade, and others, who were to become the leading authorities of general thoracic surgery in Turkey. During his 6 years of residence and working in Istanbul, he contributed highly to the practice of general and thoracic surgery. He had to leave for the United States in 1939 for treatment of his lung abscess due to a retained bullet from World War I. He stayed in New York and later in Basel until his death. He was presented an honorary professorship from Hacettepe University, Ankara in 1973. PMID- 10735730 TI - Myocardial laser revascularization: the controversy and the data. AB - The clinical and experimental data relevant to the theoretical mechanisms and clinical results of laser myocardial revascularization are reviewed. Both transmyocardial and percutaneous approaches are considered. Both types result in a reduction in anginal symptoms in patients refractory to conventional therapy and are likely to act through common pathways. The proximate mechanisms for the transmyocardial revascularization effect most likely relate to myocardial inflammation, secondary stimulation of growth factors, and denervation of the myocardium. PMID- 10735731 TI - Joint statement on redundant (duplicate) publication by the editors of the undersigned cardiothoracic journals. PMID- 10735732 TI - Acute upper limb ischemia: a complication of coronary artery bypass grafting. PMID- 10735733 TI - The lower plexus innervates the opponens pollicis and abductor pollicis brevis. PMID- 10735734 TI - Intrinsic hand muscles are innervated by C8 and T1, not C6 and C7, roots. PMID- 10735735 TI - Median-innervated intrinsic hand muscles. PMID- 10735736 TI - Aortobronchial fistula after coarctation repair. PMID- 10735737 TI - Continuous intravenous insulin infusion reduces infections in diabetics after CABG. PMID- 10735738 TI - Pectoralis major muscle flap reconstruction after clavicular-manubrial resection. PMID- 10735739 TI - "Mini-sternotomy" for resection of anterior mediastinal masses. PMID- 10735740 TI - Research and development in 2000: directions and priorities for the world's poultry science community. AB - The challenges and targets facing the world's poultry science community in the immediate future are reviewed in the context of meeting the dietary needs for animal protein of the world population. The prior need to provide for the increasing demand for cereals, oil seeds, and grain legumes for human consumption is assessed at having a reasonable chance of success. If this need is met, the requirement for extra feed resources for increased poultry production targets is also assessed as having a reasonable chance of success. A major component of this equation is the prediction of improved efficiency of poultry production of a similar order to that of the last 50 yr arising from 1) extension of the 20th century revolution in poultry technology to over 50% of the world population compared with the present 20 to 25%; 2) recent advances in genetics, nutrition, health, housing, and husbandry still awaiting application in industry; 3) future applications from current and future research in molecular biotechnology, nutrition, health, and reproduction; and 4) the development of efficient, small scale, extensive poultry production systems especially in countries where over 25% of the world population will still not be able to afford the products of a modern, intensive poultry industry, even in 50 yr. These challenges, targets, and predictions simply cannot be met unless the world's poultry science community increases its own efficiency, its professional initiatives to deal with the real challenges, and its social initiatives to influence socio-economic decisions on national and world stages. PMID- 10735741 TI - Antibody responses to different dosages of sheep red blood cells in lines of chickens selected for high and low antibody response to sheep red blood cells. AB - Temporal patterns of SRBC antibody response after primary and secondary inoculations were measured in White Leghorn males from lines selected 24 generations for high (HA) or low (LA) 5-d antibody titers to an i.v. inoculation with 0.1 mL of a 0.25% suspension of SRBC. Primary i.v. inoculations were administered at 50 d of age as 0.1 mL of either 0.025 or 0.25% suspension of SRBC. Antibody levels of SRBC were measured 3, 5, 7, 9, 11, 13, and 20 d after inoculation. Also, IgG levels were measured on samples obtained 3, 7, 13, and 20 d after inoculation. At 70 d of age, half of the cockerels in each line-dosage subclass were given a booster inoculation of 0.1 mL of 0.25% SRBC. The SRBC antibody was measured 3, 6, 9, and 13 d later in chicks both receiving and not receiving the second inoculation. Patterns of antibody response to SRBC dosage differed according to line, resulting in interactions of line by dosage by day. Concentrations of IgG were greater for Line HA than for LA at 7 and 11 d after inoculation with SRBC but not at 3 and 20 d. Antibody responses to the booster inoculation differed between lines with a dosage effect present for LA but not for HA chicks. The greater anamnestic response observed in LA than in HA chicks is explained in the context of the resource allocation paradigm. PMID- 10735742 TI - Serum immunoglobulin G and M concentrations did not appear to be associated with resistance to Pasteurella multocida in a large-bodied turkey line and a randombred control population. AB - Serum IgG and IgM concentrations in a randombred control line (RBC2) of turkeys and its subline (F), selected long term for increased 16-wk BW were compared. Six week-old poults were challenged with virulent Pasteurella multocida (1.2 x 10(7) bacteria per bird of serogroup A and serotype 3,4). Sera were collected prior to challenge, and concentrations of IgG and IgM in turkey sera were quantitatively estimated with a Sandwich ELISA using the cross-reactive polyclonal antibodies specific for the chicken Ig isotypes. The F-line turkeys had a higher serum IgM concentration than turkeys from the RBC2 line. There was no significant line difference in IgG concentration. The F line had higher mortality and a shorter number of days to death following challenge with P. multocida than did the RBC2 line. No significant correlation coefficient was found between immunoglobulin isotype concentration in serum and days to death following challenge with P. multocida. The present results suggested that prechallenge IgG and IgM concentrations in serum did not appear to be associated with resistance to P. multocida in a large-bodied turkey line and a randombred control population. PMID- 10735743 TI - The effect of intermittent lighting on metabolizable energy intake and heat production of male broilers. AB - Experiments were conducted to compare the effects of an intermittent lighting (IL) schedule with repeated cycles of 1 h light and 2 h darkness with a continuous lighting (CL) schedule on the performance, ME intake, and heat production of male broiler chickens. Body weight gain and feed intake were temporarily reduced after the changing from CL to IL; however, they were significantly higher in IL vs CL chickens during the subsequent period of 3 to 6 wk of age. The IL chickens exhibited a higher ME intake at 6 and 8 wk of age than did CL chickens. Total heat production in IL chickens was higher than for CL chickens, although heat production during the dark period was less than that during the light period for IL chickens. The higher feed intake observed in IL chickens appears to explain the superior body weight gain in IL broilers in simple terms. PMID- 10735744 TI - Nutrient flows for poultry production in The Netherlands. AB - Government targets for ammonia emission and for N and P loss per hectare (ha) of agricultural land were used to assess carrying capacity for poultry production in The Netherlands with data from 1990. In addition, the effect of alternative management strategies on carrying capacity was determined. Ammonia emission from poultry production in 1990 [20.5 gigagrams (Gg) N] exceeded the target for 2000 (i.e., 6.9 Gg N). Targets defined for 2000 and 2010 (i.e., 4.6 Gg N) can be achieved, however, without reducing poultry numbers, assuming national introduction of measurements studied. Measures that reduced ammonia emission directly, i.e., introduction of low-emission housing or manure application techniques, were most effective. In 1990, N and P losses equalled 215 kg/ha for N and 31 kg/ha for P. The N loss was slightly lower than the target for 2000 (219 kg N/ha) but exceeded the target for 2010 (144 kg N/ha). Reduction of application of artificial N fertilizer, however, reduced N loss effectively from 215 to 22 kg/ha. National P loss in 1990 exceeded the target for 2000 (15.3 kg P/ha). Reduction of application of artificial P fertilizer reduced P loss most effectively from 31 to 14 kg/ha. To achieve the target for 2010 (8.7 kg P/ha), additional reduction in P excretion by poultry is required. This reduction can be achieved by use of phytase in layer and broiler feed and by use of a coarse Ca source in layer feed. Unlike pig production, carrying capacity for poultry production in The Netherlands is not limited by governmental targets for acidification, eutrophication, or drinking water contamination. PMID- 10735745 TI - Evaluation of broiler growth velocity and acceleration in relation to pulmonary hypertension syndrome. AB - An evaluation was made of the relationship between individual daily growth patterns and susceptibility of broiler chickens to pulmonary hypertension syndrome (PHS). In the first experiment, 46 male broilers were weighed for each of 50 d, during which time 13 developed PHS. Three temporal phases (0 to 15, 16 to 35, and 36 to 50 d) of broiler growth velocity and acceleration were examined. Correlation dimensions and Lyapunov exponents suggested evidence of chaos in growth velocity and acceleration, but the absence of detectable differences between broilers in the normal and PHS categories led us to reject the hypotheses that growth is more chaotic in normal broilers than in broilers susceptible to PHS. Growth velocity and acceleration values for mean and SD were statistically evaluated as response variables for each growth phase. Mean values for velocity during the third phase were different between broilers in the normal and PHS categories (velocity: 68.8 vs 48.9 g/d, P = 0.03, respectively) and (acceleration: 0.3 vs -1.4 g/d2, P = 0.07, respectively). The third phase SD (reflecting oscillation for velocity and acceleration) was greater for normal than for PHS birds (velocity: 26.1 vs 21.3 g/d, P = 0.13, respectively; acceleration: 39.7 vs 28.2 g/d2, P = 0.03, respectively). The hypothesis was accepted that normal birds have greater oscillations in growth velocity and acceleration than birds susceptible to PHS. A general regression neural network (GRNN) with genetic adaptive calibration was trained to predict PHS based on individual growth phases and their combinations. Data representing the first, first two, and all three phases of growth were determined to have potential for computerized diagnostic weighing. With the GRNN, birds in all three data sets were successfully classified (100%) with or without PHS. A third hypothesis, therefore, was accepted that artificial neural networks could be used to distinguish the difference between normal broilers and those susceptible to PHS. In the second experiment, only one bird was diagnosed with PHS. Velocity and acceleration neural networks from Phase 1 and Phases 1 and 2 in the first experiment were applied to the growth velocity and acceleration data of Experiment 2. The Phase 1 neural networks were the most promising in that they correctly identified 71.6 and 72.4% of the birds as normal for velocity and acceleration data, respectively. In general, data in the second experiment exceeded the neural network range of training for both velocity and acceleration, which reflected increased oscillation during the second phase of growth. PMID- 10735746 TI - Behavior of white leghorn laying hens after withdrawal of feed. AB - To approximate a commercially induced molt, feed was withdrawn (FW) from Hy-Line W-36 hens (65-wk-old) until they lost 35% of their initial body weight. Behaviors of 36 FW hens and 36 control hens were videorecorded on Days 1 to 3, 8 to 10, and 19 to 21 of FW, when FW hens reached 15, 25, and 35% body weight loss. The FW hens then were fed a pullet grower ration until Day 28 after the start of FW, whereupon they were provided a layer ration. Second cycle production of FW hens to 40 wk postmolt initiation averaged 15.5 dozen eggs/hen housed. The FW hens manifested increased aggression on the first d of FW, perhaps indicative of frustration, and then exhibited increased standing, head movement, and nonnutritive pecking on Day 2, followed by reduction of these actions on Day 3. Resting behavior was observed 24 and 40% of the time for FW hens on Days 8 to 10 and 19 to 21 of the FW period, respectively. Nonnutritive pecking was higher for FW hens than for control hens throughout the FW period. Preening was more frequent for FW hens on Days 8 to 10, probably due to skin sensitivity near the start of feather push out, which began on Day 11. Behavior during the feed withdrawal period was consistent with conservation of bodily reserves, but FW hens never lost their capability for alertness and reactivity. The FW hens had significantly lower mortality during the study than control hens (2 vs 12%, respectively). PMID- 10735747 TI - Influence of the stocking density and rearing season on incidence of sudden death syndrome in broiler chickens. AB - Influences of the stocking density and rearing season on the incidence of sudden death syndrome (SDS) were investigated in commercial broiler chickens, which were reared until 63 d after birth. The SDS mortality and total mortality were measured at three different stocking densities of 12, 15, and 18 birds/m2 in rearing seasons of summer, autumn, and winter. At stocking densities of 15 and 12 birds/m2, neither SDS mortality nor total mortality was significantly different throughout the rearing season. However, at 18 birds/m2, SDS mortality significantly increased in summer and winter. The increased SDS mortality was accompanied by a significant increase in total mortality in summer but not in winter. Irrespective of the stocking density, body weight gain and feed intake were lower in summer with no significant change in feed efficiency. The data suggest that stocking density can act as a factor affecting the incidence of SDS in summer and winter, independent of feed efficiency. PMID- 10735748 TI - The effects of dietary mannaoligosaccharides on cecal parameters and the concentrations of enteric bacteria in the ceca of salmonella-challenged broiler chicks. AB - The ability of different enteric pathogens and coliforms to trigger agglutination of yeast cells (Saccharomyces cerevisiae, NCYC 1026) and a yeast cell wall preparation (MOS) was examined. Five of seven strains of Escherichia coli and 7 of 10 strains of Salmonella typhimurium and Salmonella enteritidis agglutinated MOS and Sac. cerevisiae cells. Strains of Salmonella choleraesuis, Salmonella pullorum, and Campylobacter did not lead to agglutination. Two strains that agglutinated MOS (S. typhimurium 29E and Salmonella dublin) and one nonagglutinating strain (S. typhimurium 27A) were selected as challenge organisms for in vivo studies in chicks under controlled conditions. In a series of three trials in which 3-d-old chicks were orally challenged with 10(4) cfu of S. typhimurium 29E, birds receiving 4,000 ppm of dietary MOS had reduced cecal S. typhimurium 29E concentrations (5.40 vs 4.01 log cfu/ g; P < 0.05) at Day 10. In a second series of three trials with S. dublin as challenge organism, the number of birds that tested salmonella positive in the ceca at Day 10 was less when MOS was part of the diet (90 vs 56%; P < 0.05). To test the effect of MOS on concentrations of bacteria that do not express Type 1 fimbriae, a challenge trial was conducted with S. typhimurium 27A. However, strain 27A did not colonize the birds sufficiently to evaluate whether MOS affected its cecal concentration. Mannanoligosaccharide did not significantly reduce the concentrations of cecal coliforms (P < 0.10) although they were numerically lower. It had no effect on cecal concentrations of lactobacilli, enterococci, anaerobic bacteria, lactate, volatile fatty acid, or cecal pH. PMID- 10735749 TI - Physical, chemical, and microbiological changes in the crop of broiler chickens subjected to incremental feed withdrawal. AB - Trials were conducted to determine the effect of feed withdrawal on the weight, pH, native bacterial flora, and Salmonella typhimurium persistence in crops of broilers. Six-week-old broilers were provided medicated or unmedicated feed and were subjected to feed withdrawal for 0 to 24 h in transportation crates or on litter. After feed withdrawal, broilers were stunned, bled, scalded, and picked. Crops were aseptically removed and weighed. Crops were then blended in 20 mL distilled water, and pH values of the suspensions were measured. Aerobic bacteria, Enterobacteriaceae, S. typhimurium, and lactic acid bacteria in the suspension were enumerated on appropriate bacteriological media. Feed withdrawal produced significant decreases in the crop weights. Crop pH increased by 1.0 unit within 6 h of feed withdrawal. Enterobacteriaceae and S. typhimurium populations generally decreased during the first 12 h of feed withdrawal and remained unchanged or increased during the final 12 h of the 24-h feed withdrawal period. The number of lactic acid bacteria recovered from the crop usually decreased as the duration of feed withdrawal increased. Changes in crop weights, pH, native microflora, or the recovery of S. typhimurium from the crop were similar in broilers subjected to feed withdrawal on litter or in crates and in broilers provided medicated or unmedicated feed before initiating feed withdrawal. Findings indicate that a decrease in the number of lactic acid producing bacteria in the crop and an increase in crop pH may be related to the reduction of anti Enterobacteriaceae activity in the crop during extended feed withdrawal. PMID- 10735750 TI - Flow cytometric analysis of T lymphocyte subpopulations in large-bodied turkey lines and a randombred control population. AB - To investigate the effect of BW selection on immune cell populations of turkeys, T lymphocyte subpopulation analyses were conducted using peripheral blood from lines selected for increased BW and a randombred control population. The lines used included an experimental line (F) selected long-term for increased 16 wk BW, a randombred control line (RBC2) that served as the base population of the F line, and sire lines (A and B) from each of two major commercial turkey breeders. The peripheral blood lymphocytes were isolated and stained with mouse anti chicken CD4 and CD8alpha antibodies in flow cytometric analysis. The polymorphism of CD8alpha in the F and A lines detected with the CT8 monoclonal antibody (mouse anti-chicken CD8alpha antibody) did not appear to be associated with BW and shank parameters. The present results showed that the F line had a significantly larger CD4+CD8- T cell subpopulation than did the RBC2 line at both ages, and this population proportion in the F line was also larger than that for one commercial sire line at 24 wk of age. There were no differences in other T cell subsets. The BW selection may have resulted in changes in T lymphocyte subpopulations and, therefore, may have affected disease resistance. The increased susceptibility to infectious diseases in the F line may be associated with the higher CD4+CD8- T cell subpopulation and the CD8alpha polymorphism. PMID- 10735751 TI - The effects of dietary available phosphorus levels and phytase on performance of young and older laying hens. AB - In the first of two experiments (20 to 70 wk of age), eight treatments consisted of corn-soybean meal diets (0.34% total P, 3.8% Ca, 17% CP, 2,758 kcal ME/kg) containing 0.10, 0.15, 0.20, 0.25, or 0.45% available P (AP), with the three lowest AP diets supplemented with 300 units of phytase/kg of diet. A second experiment evaluated the effect of feeding the 0.10% AP diet with and without phytase on performance and time required for onset of P deficiency in older hens (70 to 76 wk of age). In Experiment 1, the 0.10% AP diet with no supplemental phytase depressed performance by 28 wk of age. No other significant differences in performance were observed among treatments for the entire 20- to 70-wk period except that the 0.15% AP diet with no phytase resulted in body weights and tibia ash that were lower (P < 0.05) than those of hens fed the 0.45% AP diet during the last 30 wk. The mean daily AP intake of hens fed the 0.10% AP, 0.15% AP, 0.45% AP, or 0.10% AP + phytase diets was 94,159, 499, or 108 mg, respectively. Excreta P concentration was decreased by approximately 50% in birds consuming 0.10% AP + 300 U/kg phytase compared with those consuming 0.45% AP. In the second experiment, P deficiency signs occurred within 3 wk of consuming the unsupplemented corn-soybean meal diet (0.10% AP) compared with 8 wk in Experiment 1. The results of this study indicate that phytase improves P utilization in corn soybean meal diets for laying hens and that a corn-soybean meal diet containing 0.15% AP (159 mg AP/d) or containing 0.10% AP + 300 units of phytase/kg (108 mg AP/d) supported optimal egg production from 20 to 70 wk of age. Additionally, results suggested that older hens may exhibit P deficiency symptoms sooner than younger hens. PMID- 10735752 TI - Effect of dietary phosphorus, calcium, and phytase on performance of growing turkeys. AB - Female and male turkeys were fed 110, 73, 52, and 30% of the NRC (1994) nonphytate P (NPP) requirement without and with 500 phytase units (FTU)/kg during 4 to 14 or 16 wk of age, respectively. At 110% P (control; also 110% of NRC Ca), phytase was without effect. At 73% of NPP (100% Ca), without phytase, performance was similar to the control; with phytase, performance was equivalent, and in some stages, superior to the control. At 52% of NPP (90% Ca), performance was inferior without phytase and was variably similar or poorer than the control with phytase. At 30% NPP without phytase, poults gained poorly and showed a high incidence of leg disorder at 8 wk when they were removed from experiment; poults gained better with 80% NRC Ca compared with 110%. At 30% NPP with phytase, turkeys performed remarkably well, although suboptimally, at 80 or 110% NRC Ca. Phytase at 400, 300, and 200 FTU/kg with increasing age periods performed as well as 500 FTU/kg with 73% of NRC NPP (100% Ca) and 52% NRC NPP (90% Ca). These lower phytase levels were not as sufficient as 500 FTU/kg with 30% of NRC NPP; this inadequacy was more severe with higher dietary calcium. Phytase was effective in reducing dietary P requirements of growing turkeys when the NPP levels were below NRC (1994) requirements. PMID- 10735753 TI - Effect of selective gastric intrinsic denervation on gastric motility in turkeys. AB - The effect of selectively denervating portions of the myenteric plexus on gastroduodenal (GD) motility was examined in domestic turkeys (Meleagris gallopavo). The neural network beneath the lateral or medial side of the isthmus between the glandular stomach and the muscular stomach was denervated by application of 1% benzalkonium chloride (BC). Image intensification radiology (IIR) was performed postsurgically on the research subjects to observe GD contractions and to analyze aberrations. Two trials were performed. The first employed temporal controls by checking GD motility in all treated birds before and after BC had taken effect. The second trial controlled for the trauma associated with the surgical procedure by applying 0.9% saline solution in lieu of BC in four turkeys. The normal GD contractions sequence as confirmed by these controls was thin muscle contraction, duodenal contraction and outflow, thick muscle contraction, and, finally, glandular stomach contraction. Ablation of the nerves beneath the medial or lateral side of the isthmus resulted in the same abnormality in GD motility, namely a lack of thick muscle contractions. These results indicate that the entire myenteric plexus associated with the isthmus must remain intact for proper GD motility to take place. Furthermore, it can be inferred that initiation and regulation of the thick muscle pair acts via nerves encircling the isthmus. Secondary regulatory mechanisms controlling GD motility may exist and should be tested through future investigation. PMID- 10735754 TI - Immunization of chickens with quail and turkey perivitelline membrane proteins: production of antibodies and their effects on fertility. AB - The cross-reactivity of antibodies developed against zona pellucida proteins and their subsequent deleterious effect on fertility in heterologous species is well documented. However, similar investigations have not been undertaken in avian species. In Experiment 1, White Leghorn hens were immunized with intact germinal discs (GD) of quail and chickens. Chicken GD proteins did not elicit an immune response in chicken hens, whereas quail GD proteins were highly immunogenic. Anti quail GD antibodies did not bind with chicken inner perivitelline membrane (IPM) proteins as determined by immunoblot analysis. To examine the fertility of immunized hens, artificial insemination was performed at weekly intervals for 4 wk following the booster immunization. No significant differences were detected in fertility or hatchability of immunized hens when compared with unimmunized control hens. In Experiment 2, White Leghorn hens were immunized with intact turkey GD, solubilized turkey perivitelline membrane (PM) modified with dinitrophenol (DNP), and solubilized chicken IPM modified with DNP. High titers of antibodies were detected against the turkey GD and the DNP-modified turkey PM proteins. A weak immune response was observed in hens immunized with modified chicken IPM proteins. The fertility and hatchability of eggs laid by immunized hens, however, were not significantly different from those of unimmunized hens. Antibodies from immunized hens were further analyzed using an in vitro assay that assesses sperm penetration of intact IPM. Sperm penetration of intact IPM was inhibited to the same extent when IPM was preincubated with preimmune as well as anti-PM immunoglobulins. Collectively, these results suggested that the antibodies developed in these hens did not cross-react with the chicken IPM proteins involved in sperm-egg interaction and thus did not influence the fertility. PMID- 10735755 TI - Cardio-pulmonary function in broilers reared at warm or cool temperatures: effect of acute inhalation of 100% oxygen. AB - Fast growth and cool temperatures are the primary triggers for pulmonary hypertension syndrome (PHS, ascites) during commercial broiler growout. We evaluated cardio-pulmonary function in male broilers that initially were reared together at normal brooding temperatures and then were randomly assigned to chambers maintained at 28 C (warm group) or 16 C (cool group). Cardio-pulmonary evaluations were conducted between 35 and 42 d of age. The groups initially did not differ in body weight, right:total ventricular weight ratio, respiratory rate, heart rate, total peripheral resistance, or pulmonary vascular resistance, nor did their arterial blood gas values differ for the partial pressure of carbon dioxide, bicarbonate concentration, or hydrogen ion concentration. When compared with the warm group, the cool group had heavier total ventricular weights and higher values for pulmonary arterial pressure, cardiac output, stroke volume, mean systemic arterial pressure, and hematocrit and a marginally lower (P = 0.06) partial pressure of oxygen. Inhaling 100% O2 for 20 min caused equivalent increases in the arterial partial pressure of oxygen (> or =388 mm Hg) and the percentage saturation of hemoglobin with oxygen (99.9%) in both groups. The respiratory rate was reduced and total peripheral resistance in both groups was increased; the pulmonary arterial pressure, cardiac output, and heart rate in the cool group were also reduced. Cool temperatures contributed to the increase in pulmonary arterial pressure primarily by increasing the metabolic demand for oxygen, as reflected by incipient hypoxemia (reduced blood oxygen and elevated hematocrit), generalized ventricular hypertrophy, and an elevated cardiac output. It was the elevated cardiac output rather than hypoxemic pulmonary vasoconstriction that increased the pulmonary arterial pressure in the cool group when compared with the warm group. PMID- 10735756 TI - Economic feasibility analysis for an automated on-line poultry inspection technology. AB - On-line carcass inspection of chickens in the United States is currently done using visual (organoleptic) methods. Inspectors from the USDA Food Safety and Inspection Service (FSIS) inspect the viscera and carcass and, for older birds, the heads using a sequence of observations and palpations at a postmortem inspection station. The streamlined inspection system (SIS) and the new line speed inspection system (NELS) are the most prevalent visual inspection methods. The former has a line speed of 70 birds/min with two inspectors per line, and the latter has a line speed of 91 birds/min requiring three inspectors per line. Both inspection methods are labor intensive and prone to human error. In addition, the speed of the slaughter line is dictated by the number of birds per minute that can be inspected by FSIS inspectors. Ninety-one birds/min is currently the maximum visual inspection line speed allowed under current Federal regulations. This study evaluates the economic benefits of using automated inspection in place of visual inspection from the perspective of both the slaughter plant and FSIS. The results indicate that FSIS and slaughter plants would gain economic benefits by using automated inspection in place of visual inspection. The economic benefits to FSIS would accrue from labor savings, whereas the economic benefits to slaughter plants would accrue primarily from increased throughput from faster inspection line speeds. PMID- 10735757 TI - Gas exchange into shell eggs from cryogenic cooling. AB - The gas composition of the air cell in a shell egg is influenced by heating from egg washing and candling and the method of cooling and storage. This study found that N2 gas (-122 C), CO2 gas (-45 C), and cold air (-15 C) could be used to rapidly cool shell eggs from 47.7 C to 7 C in 30 min or less. These results suggest that the gas composition of the air cell in shell eggs can be significantly modified using N2 cooling and CO2 cooling. Commercial field studies have shown that these modifications, which take place during cryogenic cooling, can significantly reduce microbial levels and increase shelf life of shell eggs. Storage in a modified atmosphere environment further enhanced these changes. It was found that the CO2 concentration in the air cell of a shell egg can be increased from 0.04 to 48% by CO2 cooling and storage in a CO2 environment. PMID- 10735758 TI - A dark, firm dry-like condition in turkeys condemned for cyanosis. AB - A case-control study (n = 130) was conducted on toms condemned for cyanosis. Color (CIE L*a*b*), pH, and physical characteristics were measured on the Pectoralis major at slaughter and after 24 h. Meat from carcasses condemned for cyanosis had dark, firm, dry-like traits. It was darker and redder and showed higher water-holding capacity, lower cooking loss, and higher gel strength than did controls. Perimortem pH was negatively correlated with the lightness (L*) of meat at the time of slaughter (r = -0.58) and at 24 h postmortem (r = -0.64), positively correlated with water-holding capacity (r = 0.73) and gel strength (r = 0.43) and negatively correlated with cooking loss (r = -0.50). Ultimate pH was negatively correlated with lightness (L*) of meat at slaughter time (r = -0.62) and at 24 h postmortem (r = 0.79) was positively correlated with water-holding capacity (r = 0.87) and gel strength (r = 0.61) and negatively correlated with cooking loss (r = -0.52). Tests based on pH and L* of the P. major were also assessed; tests based on pH had a sensitivity in the range of 0.79 to 0.89 and specificity (Sp) of 0.60 to 0.94. Tests based on L* showed sensitivity of 0.75 to 0.92, and specificity of 0.79 to 0.97. The repeatability of measurements varied from good (L*: rho = 0.6) to excellent (pH: rho = 0.92). Overall, turkey breast condemned for cyanosis showed dark, firm, dry-like traits. Tests based on color and pH are described as a means of identifying turkeys condemned for cyanosis. PMID- 10735759 TI - Campylobacter recovery from external and internal organs of commercial broiler carcass prior to scalding. AB - Campylobacter is a human pathogen commonly found on live broilers and processed carcasses. To plan effective intervention strategies, it would be helpful to know which Campylobacter populations are associated with the external and internal organs of broilers. Six carcasses were collected after exiting the bleed tunnel at a commercial broiler plant on each of three visits (n = 18). Carcasses were placed individually into sterile plastic bags, sealed, and covered with ice for transport to the laboratory. Five locations were sampled aseptically from each carcass: breast feathers (hand picked from the sternal tracts); breast skin, including the sternal tracts; crop; ceca; and colon. Samples included adhering contamination or lumen contents and were covered with phosphate-buffered saline and blended. Serial dilutions were made for examination of Campylobacter, coliform, Escherichia coli, and total aerobic bacterial populations. Average sample weights (grams) were as follows: feathers, 1.5; skin, 6.5; crop, 5.1; ceca, 7.8; and colon, 3.1. Campylobacter populations (mean log10 colony-forming units per gram of sample) found were feathers, 5.4; skin, 3.8; crop, 4.7; ceca, 7.3; and colon, 7.2. Coliform/E. coli populations observed were feathers, 6.4/6.0; skin, 5.3/4.9; crop, 4.3/3.7; ceca, 6.6/6.2; and colon, 5.8/5.3. Total aerobic bacterial populations found were feathers, 7.9; skin, 7.1; crop, 5.8; ceca, 6.8; and colon, 6.4. On a per gram basis, ceca and colon are the internal organs that if ruptured could cause the highest number of Campylobacter to be leaked onto the carcass. The crop also contained more Campylobacter per gram than did the skin, and if compromised may increase the numbers on the surface of the carcass. However, even with no contamination from an internal organ, a substantial population of Campylobacter is already resident on broiler skin as the carcass enters the early stages of processing. PMID- 10735760 TI - [Guide to correct clinical practice in neurosurgery. World Federation of Societies of Neurosurgery. European Association of Societies of Neurosurgery]. PMID- 10735761 TI - Endoscopic endonasal transsphenoidal approach to the sellar region: technical evolution of the methodology and refinement of a dedicated instrumentation. AB - BACKGROUND: Endoscopic treatment of hydrocephalus has in time acquired a primary position in Neurosurgery. Today endoscopy can be employed in the vast majority of neurosurgical procedures in the realization of the so-called endoscope-assisted microneurosurgery. On the basis of the encouraging results obtained by Jho and Carrau, the authors report the pros and cons of the new endoscopic endonasal technique. METHODS: An endoscopic endonasal transsphenoidal approach to the sella was performed in 50 patients: 33 females and 17 males, of age included between 16 and 76 years; 45 were affected by pituitary adenomas (9 micro and 36 macro; 10 were recurrences), 3 by craniopharyngiomas, 2 by clival chordomas. The new instrumentation was realized through straight devices with a handle shifted 10-30 degrees horizontally. The possible danger to the nasal structures is reduced not only by the elimination of the bayonet-like shape but also by hiding the cutting extremity in the handle. RESULTS: The advantages of this technique have been represented by an easier access to the lesion, especially in recurrent tumors, a more complete excision of voluminous neoplasms, a decrease of ENT post-surgical complications and a reduction of hospitalization times and costs. The main limits have been the reduction of field depth and the lack of adequate instrumentation. CONCLUSIONS: In this peculiar surgery the absence of proper instruments has to be overcome because: a) there is less room to work, b) a conflict between the hands of the surgeon and the endoscope may occur, c) the blind introduction of the instruments in the nostril may cause damage to the nasal structures. The secret for the success of the endoscopic endonasal transsphenoidal technique is to create the optimal conditions, in order to avoid improper manoeuvres that may cause potential dangers. PMID- 10735762 TI - Inconsistent MRI findings in acute pre-Willisian brain ischemia in rabbit: a useful model? AB - BACKGROUND: We have investigated a model of "preterminal ischemia" in order A) to assess the MRI findings and the repeatability of the anatomical changes as showed by MRI, and B) to compare the data so obtained with the humans. METHODS: Twenty rabbits were used. Under general anesthesia, the occlusion of 1) the two common carotid arteries (from 2 hours up to 24 hours) was performed in 8 cases, and of 2) the epi-aortic vessels at the aortic arch (from 2 hours up to 4 hours) was performed in 4 cases in general anesthesia. RESULTS: In the vessels occlusion group MRI study showed inconsistent, inconstant, predominantly ill defined, linear and/or spotlike hyperintense deep cerebral alterations, variable in location, unilateral and bilateral in 8 cases. Although in carotid occlusion the damage was already evident within the first two hours, aortic arch occlusion was unable to produce global brain lesional pattern even up to 4 hours. MRI study of the sham operated animals was negative. CONCLUSIONS: The selective vulnerability of mesial deep cerebral structures to hypoxia, different in location from the human's, and the more effective pre-Willisian compensation, suggests to look for different animals when dealing with preterminal ischemic models comparable to the humans' as studied with MRI. PMID- 10735763 TI - Autoregulation of cortical blood flow during surgery for ruptured intracranial aneurysms. AB - BACKGROUND: Little information is available about the extent and the time course of possible impairment of cerebral circulation occurring after aneurysmal subarachnoid haemorrhage (SAH). The aim of this study was to correlate cerebral autoregulation, neurological impairment at surgery and timing of surgery in patients with ruptured intracerebral aneurysms. METHODS: Cortical blood flow (CoBF) was measured intraoperatively by a thermal diffusion probe in 77 patients during surgery for ruptured supratentorial aneurysms, who were operated on at different time intervals after bleeding. An autoregulation index (AI), expressed as the ratio between the change in CoBF and the change of mean arterial blood pressure at the time of rising the systemic blood pressure after occlusion of the aneurysm(s), was determined in each case. RESULTS: Among good-grade patients (WFNS grade I-II), those operated on days 0-2 after SAH had a significantly better autoregulatory response, compared either with patients who underwent surgery on days 3-7 after bleeding (p<0.01), or with those whose aneurysm was occluded more than 7 days after rupture (p<0.03). The mean AI of poor-grade patients (WFNS grade IV-V), who received surgery on days 0-2 after SAH, was significantly higher (p<0.01) compared with the corresponding value of good-grade patients. No significant difference was found between the mean AIs of patients who subsequently did, or did not, develop symptomatic vasospasm. CONCLUSIONS: It is concluded that good-grade patients operated on within 48 hours after bleeding take advantage of a preserved autoregulatory function during controlled hypotension. PMID- 10735764 TI - Basal meningiomas. A retrospective study of 139 surgical cases. AB - BACKGROUND: Basal meningiomas represent a significant group of intracranial tumours. Their surgical treatment presents still today several difficulties since these tumours, as it is well known, may involve critical neurovascular structures. METHODS: This is a retrospective study of 139 consecutive cases of basal meningioma operated on in our institution during the last two decades. Meningiomas location: olfactory groove 20 cases; suprasellar region 22; anterior cranial fossa 6; sphenoid wing 37; cerebellopontine angle/clivus 21; tentorium 26. In most cases the meningioma was larger than 5 cm. The tumour was completely removed (grade I-II of Simpson classification) in 111 cases (79.9%). The authors dwell upon some clinical and surgical aspects of prevailing groups (olfactory, suprasellar, sphenoidal, petroclival and tentorial meningiomas); regarding to operative procedures, the authors in particular emphasise the usefulness of the pterional approach to remove olfactory groove meningiomas and of the combined supra- and infratentorial approach, with preservation of transverse and sigmoid sinuses, to remove petroclival meningiomas. RESULTS: The surgery was followed by excellent or good results in 115 cases (82.7%): patients having a normal life with a score 80-100 of Karnofsky scale; poor outcome occurred in 5.8% of cases, postoperatively died 16 patients (mortality rate 11.5%). Tumour recurrence occurred in 7 cases. CONCLUSIONS: Based on their experience and on analysis of the reviewed literature, the authors conclude that advances in microsurgical techniques and in neuroradiological imaging have radically improved the outcome so that today basal meningiomas can be successfully treated in most cases. PMID- 10735765 TI - Presentation and management of supratentorial and infratentorial arachnoid cysts. Review of 25 cases. AB - BACKGROUND: To discuss the presentation, diagnosis, management modalities and outcomes of the arachnoid cysts. METHODS: EXPERIMENTAL DESIGN: Retrospective study with a mean follow-up period of 31 months (ranging between 9 months and 5 years). SETTING: Institutional practice (The Military Medical Faculty Hospital). Patients and the participants: 25 patients with arachnoid cysts treated surgically. INTERVENTION: Fenestration and drainage of the cyst into cisterns in 13 cases, cyst-peritoneal shunting in 8 cases and cyst excision in 4 cases. MEASURE: The arachnoid cysts were followed-up by Computed Tomography or/and Magnetic Resonance Imaging. RESULTS: The results are excellent in 21 cases, moderate in 2 cases and poor in 2 cases. CONCLUSIONS: Regardless of the procedure used for arachnoid cyst treatment, the outcomes are somewhat similar if the surgical indications and techniques are properly selected. PMID- 10735766 TI - Infections and re-infections in long-term external ventricular drainage. A variation upon a theme. AB - BACKGROUND: The infection of the external ventricular drainage (EDV) remains the main morbidity and mortality associated with this procedure, in the setting of the treatment of hydrocephalus and its complications, leading to excess of hospitalization with annual economic burden. METHODS. In this 3-year retrospective study we selected and reviewed the records of 15 of 143 patients (mean age 34 years with range from 1 months to 70 years; 12 males and 3 females) undergone to prolonged EVD in the setting of management of hydrocephalus (5 patients because of acute ventricular dilation post-intraventricular hemorrhage or post-hemorrhagic HCP, 8 because of V-P shunt infection, 1 because of post traumatic HCP and 1 because of shunt malfunction by elevated CSF protein) and developing a shunt infection or one or more superinfection. RESULTS: There was a 26% mortality and a 13% morbidity (1 patient had GOS score of 2, 1 score of 3 and 3 score of 5). The pathogens yielded by CSF culture were normal or transient flora of the patient's skin. The causes of infection were carefully analyzed. CONCLUSIONS: Based on our experience the management of infection in long-term EVD includes: the standardization of the environment of the surgery achieved with a) use of prophylactic antibiotics; b) preparation of the patient and sterile field; c) no touch technique. After implantation of EVD the risk of infection must be minimized by carefully nursing care of EVD, and administration of above prophylactic antibiotics. CSF must be collected for culture and cell count, glucose and protein when clinically indicated. When infection o reinfection is demonstrated by CSF culture then it is advisable to remove the entire hardware and start the antibiotic therapy intravenously and intraventricularly basing on susceptibility tests, CSF penetration of antibiotics, their bactericidal action, toxicity, specificity and cost. Regard to the duration of the therapy, a practical guide is treating for 10-14 days after three consecutive CSF sterile cultures. Thus, convention of EVD to a shunt can be performed within 3 weeks from admission, in the best favourable cases, decreasing the duration of hospital stay and the overall cost of neurosurgical management of the cerebral pathology requiring as therapeutic adjunct and EVD. PMID- 10735767 TI - Intramedullary increased MR signal intensity and its relation to clinical features in cervical myelopathy. AB - BACKGROUND: Areas of intramedullary hyperintensity in patients with cervical spondylogenetic myelopathy (CSM) have been described and studied by several authors. METHODS: In the present study, 100 patients were reappraised and divided into 2 groups according to whether or not MRI detected areas of hyperintensity on T2-weighted images. RESULTS: Statistical analysis demonstrated that intramedullary hyperintensity is most frequently associated with severe impairment of deambulation, muscular hypotonus-hypotrophy and hypoesthesias of the upper limbs. CONCLUSIONS: These radiological findings probably correspond to various types of lesions which, when irreversible, may influence postoperative neurological recovery. PMID- 10735768 TI - CBF changes during headache-free periods and spontaneous/induced attacks in migraine with and without aura: a TCD and SPECT comparison study. AB - BACKGROUND: The aim of the present study was to compare cranial arteries blood flow velocity as measured by means of transcranial Doppler sonography (TCD) with mean regional cerebral blood flow (rCBF) as measured by means of single photon emission computed tomography (SPECT) in migraine with and without aura during headache-free periods and spontaneous and/or induced attacks. METHODS: Regional cerebral blood flow (rCBF) and systematic ultrasonic Doppler flow were studied by Technetium-99m hexamethylpropilaminoxime (99mTc-HM-PAO) single photon emission computed tomography (SPECT) and transcranial Doppler sonography (TCD) respectively in controls (n=14) and in migraine with (n=13) and without aura (n=35) during headache free-intervals and spontaneous/histamine-induced attacks. RESULTS: In the migraine without aura group, Doppler flow examinations of the common carotid artery, external and internal carotid artery, ophthalmic artery and middle cerebral artery bilaterally did not reveal significant changes as compared with controls. During attacks, TCD examinations showed a moderate, although not statistically significant, reduction of blood flow velocity in the middle cerebral artery and in the internal carotid artery bilaterally as related to the interictal phase, concomitant with an increase of the flow velocity in the ophthalmic and external carotid artery. SPECT of these patients did not show, on the average, rCBF asymmetries during pain-free periods, although positive findings (i.e., focal hypoperfusion) were found in approximately half of the cases. During attacks, 74% of patients displayed a unilateral hypoperfusion, mainly in the occipital region. Low-flow areas were generally but not always consistent with the site of pain. In the migraine with aura group, significant reduction of blood flow velocity in middle cerebral artery was recorded by TCD on the affected side during attacks, as compared with the pain-free side. Hypoperfusion was registered between attacks by SPECT in approximately 2/3 of the patients. During attacks, a marked reduction of rCBF occurred in most patients (85%), mainly in the parieto-occipital region. The posterior rCBF asymmetries revealed at the SPECT and consistent with the general reduction of blood flow velocity documented by TCD may be related to cerebrovascular tone instability. CONCLUSIONS: Our findings do not support the paradigm that migraine with and without aura are two different entities. PMID- 10735769 TI - Intradiploic meningioma of the skull. Case report and review of literature. AB - A case of a patient with an accidentally discovered scalp mass is presented. Radiological investigation documented an osteolytic intradiploic lesion of the cranial vault. The lesion was surgically removed and histological examination revealed a meningioma. The literature concerning these uncommon tumours of the skull is reviewed, and the differential diagnosis is discussed. PMID- 10735771 TI - Intradural cervical chordoma. Case report. AB - We describe a rare case of an intradural spinal chordoma. Only two cases have been previously reported and it is the second case diagnosed with MRI. A 65-year old man presented with progressive disturbance of gait and weakness in the lower extremities. MRI revealed a intradural C6-C7 isointense mass, on T1- and T2 weighted images. The lesion enhanced after injection of gadolinium. The lesion was totally removed without difficulty by a C6-D1 laminectomy. Microscopic examination of the tumor revealed a chordoma. This is the third case in the literature of an intradural spinal chordoma. The appearance of this tumor with MRI may be similar to the chordomas of other locations. PMID- 10735770 TI - Hypertensive pneumocephalus and nasal fistula in ventriculo-peritoneal shunt: case report and review of literature. AB - A case of hypertensive endoventricular pneumocephalus associated with rhinoliquorrea raised after about 2 years from the application of ventriculo peritoneal shunt for hydrocephalus by stenosis of aqueduct is presented. The causes of such complication and diagnostic possibilities to point out the fistula by CT and ventricular cisternography are to talk over. Even the indication of external CSF drainage or to the repairing treatment will be discussed. Finally, literature is reviewed. PMID- 10735772 TI - Intramedullary schwannoma of the spinal cord. A case report and review of the literature. AB - A 9-year-old boy presented with the numbness in both arms and hands, and neck stiffness. On examination, he had a slight quadriparesis and restricted neck movements. There were no signs of von Recklinghausen's disease. Magnetic resonance imaging (MRI) scan disclosed a gadolinium enhanced intramedullary tumor located at C6-T1 associated with syringomyelia. C6-T1 laminectomies were performed and the intramedullary tumor was totally removed by a microsurgical technique. Postoperative course was uneventful. The pathological examination revealed an intramedullary schwannoma. The occurrence of intramedullary schwannoma in a patient without signs of von Recklinghausen's disease is extremely rare. We have been able to find 57 cases of intramedullary schwannoma reported in the literature. Intramedullary schwannomas are usually seen in males. The ages of the patients ranged from 9 to 75 years (mean 40.44 years). Only 4 cases in the pediatric age group have been reported. The duration of symptoms ranged from 3 months to 20 years (mean 31.03 months). Symptoms and signs varied with the location of tumor. The vertebral levels of intramedullary schwannomas were usually cervical (61%). MRI has been the choice of diagnostic tool in the cases reported since 1986. The majority of the cases showed either a partial or complete recovery in the postoperative period. PMID- 10735774 TI - Epidurals and cesarean deliveries: a new look at an old problem. PMID- 10735773 TI - Paraganglioma of the cauda equina. Case report with 33-month recurrence free follow-up and review of the literature. AB - Paraganglioma of the cauda equina is an unusual tumor and do not have the secretory properties of the same tumors arising outside the nervous system. In none of the few cases reported in literature a preoperative diagnosis was possible, and the surgical findings raised questions in the differential diagnosis with ependymomas. A rare case of paraganglioma of the cauda equina studied both pre- and postoperatively by MRI, and treated with subtotal excision combined with radiotherapy is described. Results and recurrence rates of the cases reported in literature are reviewed. Though MRI imaging has proven to be more sensitive than other radiological procedures, we stress the difficulties of preoperative diagnosis of paragangliomas in this site. The correct diagnosis of the paraganglioma of the cauda equina still relies on immunochemistry and electron microscopy. Total excision is often very difficult owing the tendency of these neoplasms to infiltrate cauda's roots. A 33-month recurrence free follow-up of our patient confirms that successful treatment is achieved by subtotal resection combined with radiotherapy. PMID- 10735775 TI - Epithelium-dependent bronchodilatory activity is preserved in pig bronchioles after normothermic cardiopulmonary bypass. AB - Analogous to vascular endothelium, bronchial epithelium modulates bronchomotor activity by releasing epithelium-derived relaxing factors. Cardiopulmonary bypass (CPB) is associated with endothelial dysfunction. We examined whether CPB may be associated with bronchiolar epithelial dysfunction in pigs. Pigs were exposed to normothermic CPB for 1.5 h and then separated from CPB. Lung tissues were biopsied before and 30 min after CPB. For time control, lung tissues were biopsied at baseline and after 2 hr of anesthesia. Bronchioles measuring about 100 microm were dissected, and the epithelium was either left intact or denuded. Each bronchiolar segment was preconstricted with 10 microM 5-hydroxytryptamine and relaxation responses to nitroprusside 10(-9)-10(-4) M, isoproterenol 10(-9) 10(-4) M, or the inhaled anesthetics halothane or isoflurane 0-2.5 minimum alveolar anesthetic concentration were examined in vitro by videomicroscopy. Bronchiolar segments demonstrated concentration-dependent relaxation responses to each of the dilators examined. Epithelial denudation reduced bronchodilation to isoproterenol, isoflurane, and halothane, but not to nitroprusside. Bronchodilation was not significantly affected by CPB. We conclude that, unlike vascular endothelial function, porcine bronchiolar epithelium-modulated bronchomotor activity is not significantly affected by normothermic CPB. IMPLICATIONS: Normothermic cardiopulmonary bypass does not result in epithelial dysfunction in pigs. Epithelium-dependent and epithelium-independent bronchodilators may be equally effective before and after cardiopulmonary bypass. PMID- 10735776 TI - Safe and efficient emergency transvenous ventricular pacing via the right supraclavicular route. AB - Infraclavicular and internal jugular central venous access are techniques commonly used for temporary transvenous pacing. However, the procedure still has a considerable complication rate, with a high risk/benefit ratio because of insertion difficulties and pacemaker malfunction. To enlarge the spectrum of alternative access sites, we prospectively evaluated the right supraclavicular route to the subclavian/innominate vein for emergency ventricular pacing with a transvenous flow-directed pacemaker as a bedside procedure. For 19 mo, 17 consecutive patients with symptomatic bradycardia, cardiac arrest, or torsade de pointes requiring immediate bedside transvenous pacing were enrolled in the study. The success rate, insertional complications, pacemaker performance, and patients' outcomes were recorded. Supraclavicular venipuncture was successful in all patients, in 16 of 17 at the first attempt. Adequate ventricular pacing was achieved within 1 to 5 min (median, 2 min) after venipuncture and within 10 s to 4 min (median, 30 s) after lead insertion (10:4. Whereas gelatin solutions have less intrinsic effect on blood coagulation, 10% dextran 40 has the strongest effect on coagulation. IMPLICATIONS: Blood coagulation may be compromised when the dilution ratio of blood volume to colloid solution volume is >10:4. Whereas gelatin solutions have less intrinsic effect on blood coagulation than hydroxyethyl starch or dextran, 10% dextran 40 has the strongest effect on coagulation. PMID- 10735779 TI - Does intraoperative hetastarch administration increase blood loss and transfusion requirements after cardiac surgery? AB - Hetastarch is used for intravascular volume expansion in cardiac surgery. Studies show conflicting effects of intraoperative hetastarch administration on postoperative bleeding. Hetastarch was routinely used for volume expansion during cardiovascular surgeries at our institution until its use was discontinued intraoperatively. We performed a retrospective chart review on patients undergoing primary coronary artery bypass grafting, valve repair or replacement requiring cardiopulmonary bypass (n = 444), 234 of which received intraoperative hetastarch and 210 did not. There was no difference in demographics, cardiac surgery, or cardiopulmonary bypass duration between the two groups. Blood loss for 0-4 h postoperatively was 377 +/- 244 mL in the group not receiving hetastarch compared with 515 +/- 336 mL in the group that received hetastarch (P < 0.001). For 0-24 h postoperatively, blood loss was 923 +/- 473 mL versus 1,283 +/- 686 mL in the absence and presence of hetastarch, respectively (P < 0.001). Allogeneic transfusion requirements (cryoprecipitate, fresh frozen plasma, and platelets) were larger in the hetastarch group (all P < 0.001). Nearly all (99%) patients in the hetastarch group received less than the manufacturer's recommended dose (20 mL/kg) of hetastarch. IMPLICATIONS: Our large retrospective study suggests that intraoperative use of hetastarch in primary cardiac surgery with cardiopulmonary bypass may increase bleeding and transfusion requirements. A large prospective study is needed to determine if intraoperative administration of hetastarch should be avoided during cardiovascular surgery. PMID- 10735780 TI - The effects of heparin, protamine, and heparinase 1 on platelets in vitro using whole blood flow cytometry. AB - The effects of heparinization and the reversal of heparin activity on platelet function after cardiopulmonary bypass have not been well defined. Flow cytometry has become a convenient and powerful technique for characterizing platelets. We examined the expression of a secretion marker (P-selectin) and an aggregation marker (activated fibrinogen receptor GP IIb-IIIa) on normal platelets in response to heparin, heparinase 1, and protamine in vitro using whole blood flow cytometry. Unfractionated heparin increased adenosine diphosphate-induced expression of P-selectin and GP IIb-IIIa in a dose-dependent manner. Heparinase 1 alone decreased both markers of platelet activation. Protamine alone increased P selectin expression but had no effect on GP IIb-IIIa expression. Heparinase 1 antagonized the stimulatory effect of heparin on both markers. In contrast, protamine antagonized the effect of heparin on GP IIb-IIIa expression but potentiated the effect of heparin on P-selectin expression. These in vitro observations suggest that 1) both heparin and its reversal agents affect platelet secretion and aggregation, and 2) heparinase 1 reverses heparin-induced platelet preactivation more effectively than protamine. IMPLICATIONS: This experimental in vitro study demonstrates that heparin and its reversal agents affect platelet secretion and aggregation. PMID- 10735781 TI - Anticoagulation for cardiac surgery in patients receiving preoperative heparin: use of the high-dose thrombin time. AB - Patients receiving heparin infusions have an attenuated activated clotting time (ACT) response to heparin given for cardiopulmonary bypass (CPB). We compared patients receiving preoperative heparin (Group H) to those not receiving heparin (REF group) with respect to ACT, high-dose thrombin time (HiTT), and markers of thrombin generation during CPB. Sixty-five consecutive patients (33 Group H, 32 REF group) undergoing elective CPB were evaluated. ACT and HiTT were measured at multiple time points. Plasma levels of thrombin-antithrombin III complex and fibrin monomer were determined at baseline, during CPB, and after protamine administration. Transfusion requirements and postoperative blood loss were measured and compared. ACT values after heparinization increased less in Group H and were significantly lower than those in the REF group (P < 0.01). HiTT values did not differ significantly between the two groups. Blood loss and transfusion requirements were not significantly different between the two groups. Plasma levels of thrombin-antithrombin III complexes and fibrin monomer also did not differ between groups at any time, despite a lower ACT in Group H after heparinization and during CPB. Our data suggest that thrombin formation and activity are not enhanced in patients receiving heparin therapy, despite a diminished ACT response to heparin. The utility of ACT and the threshold values indicative of adequate anticoagulation for CPB are relatively undefined in patients receiving preoperative heparin. HiTT should be investigated as a safe and accurate monitor of anticoagulation for CPB in patients receiving preoperative heparin therapy. IMPLICATIONS: The diminished activated clotting time response to heparin, in patients receiving preoperative heparin therapy, poses difficulties when attempting to provide adequate anticoagulation for cardiopulmonary bypass. Current data suggest that heparin resistance is not observed when high-dose thrombin time is used to monitor anticoagulation and that a lower activated clotting time value in these patients may be safe. PMID- 10735782 TI - Exposure to bacteroides fragilis endotoxin during cardiac surgery. AB - Although endotoxemia has been observed during cardiac surgery, the identity of endotoxins to which patients are exposed is unknown. We tested the hypothesis that antibodies to Bacteroides fragilis (an anaerobic gut commensal and a common pathogen) decrease during cardiac surgery, thereby reflecting systemic exposure to this type of endotoxin. Serum antiendotoxin antibody levels were measured in 55 patients during routine cardiac surgery at the following times: Preoperatively, Pre-CPB (immediately before initiation of cardiopulmonary bypass [CPB]), Pre-CPB+5 (5 min after initiation of CPB), and End (end of surgery). Antiendotoxin antibody levels were determined by using enzyme-linked immunosorbent assay. Total immunoglobulin M (IgM) levels were measured by using laser nephelometry and decreases in total IgM levels were used to control changes in antiendotoxin antibody levels attributable to hemodilution. Median (interquartile range) hemodilution corrected IgM anti-B fragilis antibody levels decreased by 12% (5%-20%) from Preoperatively to End of surgery (P < 0.001). In contrast, median hemodilution corrected anti-B fragilis antibody levels did not change significantly from Pre-CPB to Pre-CPB+5, validating the correction for hemodilution. Immunoglobulin G anti-B fragilis antibody levels and IgM and immunoglobulin G anticore antibody levels decreased similarly during surgery. Intraoperatively, levels of anti-B fragilis endotoxin antibodies decreased significantly out of proportion to hemodilution. These results suggest that cardiac surgical patients are exposed to B fragilis endotoxin. IMPLICATIONS: We prospectively measured hemodilution-corrected antiendotoxin antibody levels in 55 cardiac surgical patients. We observed significant decreases in hemodilution corrected levels of antibody to both Bacteroides fragilis and the core of endotoxin. PMID- 10735783 TI - Modulation of cardiac inward rectifier K(+)current by halothane and isoflurane. AB - The cellular mechanisms that underlie general anesthetic actions on the inward rectifier K(+) current (IKir), a determinant of the resting potential in myocardium, are not fully understood. Using the whole-cell patch clamp technique, therefore, we investigated the effects of halothane and isoflurane on IKir in guinea pig ventricular myocytes. At membrane potentials negative to the equilibrium potential for potassium both anesthetics decreased amplitude of the steady-state inward IKir in a concentration- and voltage-dependent manner. The slope conductance was reduced, but the activation kinetics of the inward current were not altered. At potentials positive to the equilibrium potential for potassium, the outward current was increased by both anesthetics, which also caused small depolarizing shifts in the activation curve. With high internal magnesium concentration, the outward current increase by isoflurane was abolished, and the inward current block by halothane was attenuated. Spermine prevented the effects of both anesthetics on IKir at all membrane potentials tested. The results show voltage-dependent modulation of cardiac IKir channel by volatile anesthetics. Distinct modification of anesthetic effects by inward rectification gating agents, magnesium and spermine, suggests anesthetic interactions with the IKir channel protein. IMPLICATIONS: Differential modulation of myocardial inward rectifier potassium current by volatile anesthetics under normal and altered rectification may contribute to the mechanism of dysrhythmic actions by these anesthetics. PMID- 10735784 TI - The hemodynamic and adrenergic effects of perioperative dexmedetomidine infusion after vascular surgery. AB - We tested dexmedetomidine, an alpha(2) agonist that decreases heart rate, blood pressure, and plasma norepinephrine concentration, for its ability to attenuate stress responses during emergence from anesthesia after major vascular operations. Patients scheduled for vascular surgery received either dexmedetomidine (n = 22) or placebo (n = 19) IV beginning 20 min before the induction of anesthesia and continuing until 48 h after the end of surgery. All patients received standardized anesthesia. Heart rate and arterial blood pressure were kept within predetermined limits by varying anesthetic level and using vasoactive medications. Heart rate, arterial blood pressure, and inhaled anesthetic concentration were monitored continuously; additional measurements included plasma and urine catecholamines. During emergence from anesthesia, heart rate was slower with dexmedetomidine (73 +/- 11 bpm) than placebo (83 +/- 20 bpm) (P = 0.006), and the percentage of time the heart rate was within the predetermined hemodynamic limits was more frequent with dexmedetomidine (P < 0.05). Plasma norepinephrine levels increased only in the placebo group and were significantly lower for the dexmedetomidine group during the immediate postoperative period (P = 0.0002). We conclude that dexmedetomidine attenuates increases in heart rate and plasma norepinephrine concentrations during emergence from anesthesia. IMPLICATIONS: The alpha(2) agonist, dexmedetomidine, attenuates increases in heart rate and plasma norepinephrine concentrations during emergence from anesthesia in vascular surgery patients. PMID- 10735785 TI - Interaction of L-arginine and phosphodiesterase inhibitors in vasodilation of the porcine internal mammary artery. AB - We tested the hypothesis that L-arginine (the substrate for nitric oxide production)-combined with amrinone, milrinone (Type III phosphodiesterase [PDE] inhibitors), zaprinast, or sildenafil (Type V PDE inhibitors)-would vasodilate synergistically. Internal mammary artery segments were excised from anesthetized swine, divided into rings, and suspended in a tissue bath at 37 degrees C. Force of contraction was measured during dose-response testing of combinations of L arginine and amrinone, milrinone, zaprinast, or sildenafil. Amrinone and milrinone were additive to L-arginine. N(G)-methyl-L-arginine (L-NMA) inhibited the effects of milrinone but not amrinone. The effective concentration of amrinone eliciting 50% relaxation (EC(50)) was 3.8E-05M (n = 6) when given alone and 4. 4E-05M (n = 6) with L-NMA. Milrinone had EC(50) = 6.0E-06M alone (n = 6) and 2.8E-05M (n = 6) with L-NMA. Zaprinast (EC(50) = 6.5E-05M, n = 6) and sildenafil (EC(30) = 1.8E-05M, n = 6) were synergistic with L-arginine. L-NMA blocked their effects, increasing the EC(50) for zaprinast to 9.9E-03M and the EC(30) for sildenafil to 6.1E+02M. In conclusion, L-arginine is additive to the vasodilation of the type III PDE inhibitors, amrinone and milrinone, but synergistic with the type V PDE inhibitors, zaprinast and sildenafil. IMPLICATIONS: Amrinone and milrinone, Type III cAMP-dependent phosphodiesterase inhibitors, are additive to L-arginine-dependent vasodilation. Zaprinast and sildenafil, Type V cGMP-dependent phosphodiesterase inhibitors, are synergistic with L-arginine. PMID- 10735786 TI - Localized airway anesthesia with lidocaine partially suppresses cardiovascular responses To lung inflation. AB - Lung inflation causes cardiovascular suppression via an increase in intrathoracic pressure and neural mechanisms. To examine the mechanisms involved, we mea-sured the heart rate (HR) and arterial blood pressure (AP) responses to lung inflation before and after spraying the bronchi with lidocaine to suppress airway reflex. Thirty women participated in the study. One group (n = 20, Group BT) had their tracheas intubated by using double-lumen tubes. The other group (n = 10, Group TT) received an ordinary endotracheal tube. They were all studied under general anesthesia by using nitrous oxide, isoflurane, and muscle relaxation after a thiopental induction. In each patient, airway pressure was increased for 3 s, and changes in HR and AP were measured. Lung inflation was repeated after 5 mL of 4% lidocaine had been sprayed into the main bronchi unilaterally in Group BT or bilaterally in Group TT. There were no significant differences in cardiovascular responses between left and right lung inflation with the pressure at 20 and 30 cm H(2)O. Both lungs inflated at 20 cm H(2)O caused an increase in HR with a significantly greater decrease in AP than with unilateral inflation. Anesthesia of the bronchi abolished the HR increase, but not the AP decrease. Lung inflation at 30 cm H(2)O caused significant decreases in HR and AP which were not affected with topical anesthesia. These results indicate that the cardiovascular responses elicited by lung inflation in anesthetized humans are predominantly the direct effect of the increase in intrathoracic pressure, although sympathetic afferent activity induced via stimulation of mechanoreceptors in the airways contributes. IMPLICATIONS: Localized airway anesthesia with lidocaine is unlikely to suppress the cardiovascular responses to lung inflation. This suggests that a limited number of neurogenic mechanisms are involved in the cardiovascular responses to lung inflation in anesthetized humans. PMID- 10735787 TI - Four cases of radical hysterectomy with acute normovolemic hemodilution despite low preoperative hematocrit values. PMID- 10735788 TI - Electrocardiographic artifacts during shoulder arthroscopy using a pressure controlled irrigation pump. PMID- 10735789 TI - The use of a ketamine-propofol combination during monitored anesthesia care. AB - Supplemental analgesics are commonly used to enhance analgesia and improve patient comfort during procedures performed under local anesthesia and sedation. Because the use of ketamine as an analgesic adjunct to propofol sedation has not been well established, we evaluated its impact on analgesia, sedation, and recovery after ambulatory surgery. One hundred female outpatients undergoing breast biopsy procedures under local anesthesia participated in this randomized, double-blinded, placebo-controlled study. After premedication with midazolam, 2 mg IV, patients received an infusion of a solution containing propofol (9.4 mg/mL) in combination with either placebo (saline) (Group 1) or ketamine, 0.94 mg/mL (Group 2), 1.88 mg/mL (Group 3), or 2.83 mg/mL (Group 4). The sedative infusion rate was varied to maintain a deep level of sedation (Observer Assessment of Alertness/Sedation score 4) and normal respiratory and hemodynamic functions. Sufentanil, 2.5 microg IV, "rescue" boluses were used as needed to treat patients' responses (if any) to local anesthetic infiltration or surgical stimulation. Ketamine produced a dose-dependent reduction in the "rescue" opioid requirements. However, there was an increase in postoperative nausea and vomiting, psychomimetic side effects, and delay in discharge times with the largest ketamine dosage (Group 4). The adjunctive use of ketamine during propofol sedation provides significant analgesia and minimizes the need for supplemental opioids. The combination of propofol (9.4 mg/mL)/ketamine (0.94-1.88 mg/mL) provides effective sedation/analgesia during monitored anesthesia care. IMPLICATIONS: Ketamine, when used in subhypnotic dosages, may be an useful adjuvant to propofol sedation. PMID- 10735790 TI - A randomized, double-blinded study of remifentanil versus fentanyl for tonsillectomy and adenoidectomy surgery in pediatric ambulatory surgical patients. AB - We compared, in a double-blinded manner, the anesthetic maintenance and recovery properties of remifentanil with a clinically comparable fentanyl-based anesthetic technique in pediatric ambulatory surgical patients. Anesthesia was induced with either halothane or sevoflurane and nitrous oxide and oxygen. Patients were randomized (computer generated) to receive either remifentanil or fentanyl in a blinded syringe with nitrous oxide and oxygen in one of four possibilities: halothane/remifentanil, halothane/fentanyl, sevoflurane/remifentanil or sevoflurane/fentanyl. In patients receiving remifentanil, a placebo bolus was administered, and a continuous infusion (0.25 microg. kg(-1). min(-1)) was begun. In patients receiving fentanyl, a bolus (2 microg/kg) was administered followed by a placebo continuous infusion. The time from discontinuation of the anesthetic to extubation, discharge from the postanesthesia care unit (PACU), and discharge to home, as well as pain scores, were assessed by a blinded nurse observer. Systolic blood pressure and heart rate were noted at selected times, and adverse events were recorded. Remifentanil provided faster extubation times and higher pain-discomfort scores. PACU and hospital discharge times were similar. There were no statistical differences among the groups for adverse events. There were statistically, but not clinically, significant differences in hemodynamic variables. We noted that continuous infusions of remifentanil were intraoperatively as effective as bolus fentanyl. Although patients could be tracheally extubated earlier with remifentanil, this did not translate to earlier PACU or hospital discharge times. In addition, remifentanil was associated with higher postoperative pain scores. The frequent incidence of postoperative pain observed in the postoperative recovery room suggests that better intraoperative prophylactic analgesic regimens for postoperative pain control are necessary to optimize remifentanil's use as an anesthetic for children. IMPLICATIONS: This is a study designed to examine the efficacy and safety of a short-acting opioid, remifentanil, when used in pediatric patients. The frequent incidence of postoperative pain observed in the postoperative recovery room suggests that better intraoperative prophylactic analgesic regimens for postoperative pain control are necessary to optimize remifentanil's use as an anesthetic for children. PMID- 10735791 TI - Pediatric evaluation of the bispectral index (BIS) monitor and correlation of BIS with end-tidal sevoflurane concentration in infants and children. AB - The bispectral index (BIS) has been developed in adults and correlates well with clinical hypnotic effects of anesthetics. We investigated whether BIS reflects clinical markers of hypnosis and demonstrates agent dose-responsiveness in infants and children. In an observational arm of this study, BIS values in children undergoing general anesthesia were observed and compared with similar data collected previously in a study of adults. In a second arm of the study, a range of steady-state end-tidal concentrations of sevoflurane was administered and corresponding BIS documented. Data were examined for differences between infants (0-2 yr) and children (2-12 yr). No difference was seen in BIS values in children before induction, during maintenance, and on emergence compared with adult values. There was no difference in BIS between infants and children at similar clinical levels of anesthesia. In children and infants, BIS was inversely proportional to the end-tidal concentration of sevoflurane. The sevoflurane concentration for a BIS = 50 (95% confidence interval) was significantly different: 1. 55% (1.40-1.70) for infants versus 1.25% (1.12-1.37) for children. Although validation with specific behavioral end points was not possible, BIS correlated with clinical indicators of anesthesia in children as it did in adults: as depth of anesthesia increased, BIS diminished. BIS correlated with sevoflurane concentration in infants and children. The concentration-response difference between infants and children was consistent with data showing that minimum alveolar concentration is higher in children less than 1 yr of age. IMPLICATIONS: The use of bispectral index (BIS) during general anesthesia improves the titration of anesthetics in adults. The data from this study suggest that the same equipment and method of electroencephalogram analysis may be applied to infants and children. PMID- 10735792 TI - Additional techniques for managing the difficult pediatric airway. PMID- 10735793 TI - An association between severe labor pain and cesarean delivery. AB - The relationship between epidural analgesia and cesarean delivery remains controversial. Several studies have documented an association, although others have not. This inconsistency may result from an association between severe labor pain and dystocia. We hypothesized that dystocia causes severe labor pain, such that more epidural medication is required to maintain comfort. We examined the relationship between labor outcome and severe pain, defined by the number of supplemental epidural boluses. We retrospectively reviewed the anesthesia records of 4493 parturients who received small-dose labor epidural analgesia. An independent association was found between operative delivery and maternal age, body mass index, nulliparity, fetal weight, induction of labor, and the number of boluses required during labor. By using multivariate analysis, the odds ratio of cesarean delivery among women who required at least three boluses was 2.3 compared with those who required two boluses or less. No association was found between the concentration of bupivacaine in the epidural infusion and operative delivery. Because women with cesarean deliveries appeared to have more pain, degree of labor pain may be a confounding factor in studies examining epidural analgesia and outcome. IMPLICATIONS: This is a retrospective observational study demonstrating an association between labor pain and cesarean delivery. Our results provide an alternative explanation of why epidural analgesia is associated with cesarean delivery. PMID- 10735794 TI - Postcesarean epidural morphine: a dose-response study. AB - The purpose of this study was to describe the dose-response relationship of epidural morphine for postcesarean analgesia for quality of analgesia and relation to the side effects of pruritus, nausea, and vomiting. Sixty term parturients undergoing nonurgent cesarean delivery were enrolled and randomized to receive a single dose of epidural morphine after delivery (0,1.25, 2.5, 3.75, or 5 mg). A patient-controlled analgesia (PCA) device provided free access to additional analgesics. PCA morphine use and the incidence and severity of side effects were recorded for 24 h. Data were analyzed with analysis of variance, Student's t-tests, and chi(2) analysis. Nonlinear regression was used to describe a dose-response curve. PCA use differed significantly among groups (P < 0.001); PCA use was significantly greater in Group 0 mg than Groups 2.5, 3.75, and 5 mg (P < 0.05). PCA use was also significantly greater in Group 1.25 mg than Groups 3.75 and 5 mg (P < 0.05). Pruritus scores were significantly higher in all groups given epidural morphine than the control group (0 mg) (P < 0.05), but did not differ among the treatment groups (1.25-5 mg), although pruritus scores were significantly higher in treatment groups than in the control (P < 0. 05). No relation was found between epidural morphine dose and incidence or severity of nausea and vomiting. We concluded that, for optimal analgesia, augmentation of epidural morphine with systemic analgesics or other epidural medications may be necessary. IMPLICATIONS: Quality of analgesia increases as the dose of epidural morphine increases to at least 3.75 mg; increasing the dose further to 5 mg did not improve analgesia. Side effects were not dose related. For optimal analgesia, augmentation of epidural morphine with systemic analgesics or other epidural medications may be necessary. PMID- 10735795 TI - Severe hypoglycemia after labor epidural analgesia. PMID- 10735796 TI - Prophylactic percutaneous sealing of lumbar postdural puncture hole with fibrin glue to prevent cerebrospinal fluid leakage in swine. AB - We explored the effect of fibrin glue injection at the site of dural puncture on cerebrospinal fluid (CSF) leakage in a swine model. Pigs were subjected to a lumbar dural CSF puncture in the sitting position with a 17-gauge Tuohy needle. Fibrin glue 1.4 mL was injected through the same needle into the epidural space. Evans blue dye was infused through the cisterna magna 15 min later, and the appearance of dyed CSF through the skin puncture and along the needle trajectory to the dura was inspected and categorized. In seven of eight animals, the CSF leak was sealed with fibrin glue. Control animals were injected with 1.4 mL saline. A sham operation group of animals underwent cisternal dye infusion without a lumbar puncture. CSF pressure at the cisterna magna was recorded throughout the procedure. No significant differences in the leakage indicators were found between the fibrin glue-injected and sham-operated group, whereas both groups showed significant differences with respect to the control group. The fibrin glue seal was effective against CSF pressures of 24.5 [17-31] cm H(2)O. We conclude that percutaneously injected fibrin glue is effective in stopping CSF leaks after dural puncture in this animal model. IMPLICATIONS: In this swine study, we repaired a cerebrospinal fluid leak after a dural puncture by percutaneously injecting tissue adhesive. The technique of percutaneous injection of fibrin glue seems promising for the prophylaxis of headache associated with cerebrospinal fluid leakage, and may be an alternative to an epidural blood patch. PMID- 10735797 TI - Local anesthetic infiltration for postoperative pain relief after laparoscopy: a qualitative and quantitative systematic review of intraperitoneal, port-site infiltration and mesosalpinx block. AB - In a systematic review, we evaluated randomized controlled trials (RCTs) of peripheral local anesthetics (LA) compared with placebo or no treatment in the control of postoperative pain after laparoscopic surgery. A total of 41 trials with data from 2794 patients were considered appropriate for analysis. Of these 41 RCTs, 13 evaluated intraperitoneal LA after cholecystectomy, four RCTs assessed intraperitoneal LA after other procedures, eight RCTs evaluated port site infiltration after various procedures, 12 RCTs evaluated mesosalpinx or fallopian tube block after sterilization, and four RCTs considered combined LA regimens. Outcome measures were pain scores, analgesic consumption, and time to first analgesic request. Efficacy was estimated by significant difference (P < 0.05), as reported in the original reports, and by calculation of the weighted mean difference of visual analog scale pain scores between treatment groups. Improved pain relief was observed in seven of the 13 RCTs of intraperitoneal LA after cholecystectomy and in four RCTs of other procedures. A statistically significant weighted mean difference of -13 mm visual analog scale (95% confidence intervals [CI]: -20 to -6) in favor of the treatment groups was observed after cholecystectomy. Three of eight trials of port-site infiltration showed significant differences but questionable clinical importance and validity in two; weighted mean difference was not statistically significant between treatment groups (95% CI -9 to 1). All RCTs of mesosalpinx or fallopian tube block after sterilization showed improved pain relief with a statistically significant weighted mean difference of -19 mm (95% CI -25 to -14) in favor of treatment groups. Data of combined regimens were positive, however, sparse. We conclude that there was evidence for a statistically significant but clinically questionable, important effect of intraperitoneal LA for postoperative pain control. There was evidence for a significant but short-lasting effect of mesosalpinx/fallopian tube block after sterilization, but there was a lack of evidence for any important effect of port-site infiltration. Data from combined regimens were too sparse for conclusions. IMPLICATIONS: A systematic review summarizes, through transparent methodology, available information from randomized, controlled trials to produce the best available evidence-based estimate of a "true" clinical effect of an intervention. This systematic review confirms intraperitoneal and mesosalpinx local anesthetic block, not port-site infiltration, to have some impact on postoperative pain after laparoscopy. PMID- 10735798 TI - Magnesium sulfate potentiates morphine antinociception at the spinal level. AB - Intrathecal magnesium sulfate coinfusion with morphine increases antinociception in normal rats; however, because magnesium also delays the onset of tolerance, it is not clear whether this additional antinociception is a result of potentiated analgesia or tolerance abatement. We examined the antinociceptive interaction of intrathecal (IT) bolus magnesium sulfate and morphine in morphine naive rats and those with mechanical allodynia after a surgical incision. After intrathecal catheter implantation, rats were given preinjections of magnesium or saline, followed by injections of morphine or saline. In morphine naive rats, IT bolus magnesium sulfate 281 and 375 microg followed by IT morphine 0.25 or 0.5 nmol enhanced peak antinociception and area under the response versus time curve two to-three-fold in the tail-flick test as compared with morphine alone. Likewise, in rats with incisional pain, IT bolus magnesium sulfate 188 and 375 microg followed by morphine 0.5 nmol reduced mechanical allodynia, whereas morphine 0.5 nmol alone did not. This study suggests that IT magnesium sulfate potentiates morphine at a spinal site of action. IMPLICATIONS: Magnesium sulfate potentiates morphine analgesia when coadministered intrathecally in normal rats, and in an animal model of mechanical allodynia after a surgical incision. These results suggest that intrathecal administration of magnesium sulfate may be a useful adjunct to spinal morphine analgesia. PMID- 10735799 TI - A comparison of intrathecal analgesia with fentanyl or sufentanil after total hip replacement. AB - We designed this study to compare the postoperative analgesic effects of intrathecal fentanyl and sufentanil, the end points being onset, quality, and duration of action. A total of 42 geriatric patients, scheduled for elective total hip replacement under continuous spinal anesthesia, were randomized in two double-blinded groups as soon as they experienced a pain score higher than 3 of 10 on the visual analog scale in the recovery room. Either 7.5 microg sufentanil or 40 microg fentanyl in 2 mL normal saline were intrathecally administered. Pain scores, rescue analgesia (ketorolac and morphine), and adverse effects (respiratory depression, postoperative nausea and vomiting, and itching) were recorded for 24 h after surgery. In both groups, comparing sufentanil to fentanyl, the time to a pain score <3 (9 +/- 9 vs 11 +/- 8 min), the time to the lowest pain score (18 +/- 6 vs 20 +/- 15 min), and the time to the first systemic analgesic intervention for a pain score >3 (241 +/- 102 vs 214 +/- 120 min) were comparable as were the analgesic requirements during the first 24 h. We conclude that, after total hip replacement, both lipid soluble opioids produce excellent analgesia with comparable onset, duration of action, and low incidence of minor adverse effects. IMPLICATIONS: We compared the postoperative analgesic properties of 40 microg intrathecal fentanyl and 7.5 microg sufentanil after total hip replacement. Both opioids provided satisfactory analgesia, with comparable onset (11 +/- 8 vs 9 +/- 9 min) and duration of action (214 +/- 120 vs 241 +/- 102 min), as well as low incidence of minor side effects. PMID- 10735800 TI - The effect of peripheral opioid block and body cooling on sensitivity to heat in capsaicin-treated skin. AB - We sought to determine whether stimulation of opioid receptors during body cooling would alter sensitivity to heat in the heat-sensitized, inflamed skin of 14 healthy volunteers. To investigate the contribution of opioid receptors to nociception, the opioid antagonist naloxone was introduced into the skin by iontophoresis after the topical application of capsaicin. For comparison, the same iontophoretic dose of saline was also administered. Shortly after the iontophoreses, sensitivity to heat was greater at the naloxone and saline sites than at iontophoresis-control sites in the capsaicin-treated skin, indicating that nonspecific aspects of the iontophoreses enhanced thermal hyperalgesia. The hyperalgesic effect of saline persisted during body cooling, whereas the naloxone site was less sensitive to heat (heat pain threshold 43.6 degrees +/- 1.0 degrees C) than either the saline site (40.8 degrees +/- 0.9 degrees C) or iontophoresis control sites (41.7 degrees +/- 1.0 degrees C) (P < 0. 05). We conclude that activation of opioid receptors contributed to thermal hyperalgesia in inflamed skin during body cooling. IMPLICATIONS: This study shows that opiate receptor block paradoxically inhibits sensitivity to heat-pain in inflamed skin during body cooling. The findings suggest that endogenous opioids release substances from nerves or other cells during inflammation, which heighten pain. Thus, opioids may fine-tune pain and the inflammatory response while healing takes place. PMID- 10735801 TI - N-methyl-D-aspartate receptor channel block by meperidine is dependent on extracellular pH. AB - Large concentrations of meperidine inhibit N-methyl-D-aspartate-(NMDA) receptor channels by channel block mechanisms. Extracellular pH regulates the activity and drug sensitivity of NMDA-receptor channels. We examined the influence of extracellular pH on sensitivity to meperidine of epsilon/zeta heteromeric NMDA receptor channels expressed in Xenopus oocytes. Inhibition of epsilon1/zeta1, epsilon2/zeta1, epsilon3/zeta1, and epsilon4/zeta1 channels by meperidine was dependent on pH, with more inhibition at acidic pH and less inhibition at alkaline pH. The degree of voltage-dependence of meperidine block was only slightly affected by changes in pH, whereas affinity for meperidine was greatly reduced at alkaline pH. Furthermore, interaction of meperidine with Mg(2+) block was reduced at alkaline pH. Because the percentage of the protonated form of meperidine is only slightly affected by pH, changes in properties of the meperidine binding site may be involved in mechanisms of alteration of meperidine potency by pH. IMPLICATIONS: At acidic pH the potency of meperidine for N-methyl D-aspartate-receptor channels was increased. Any antinociceptive and neuroprotective benefit from the N-methyl-D-aspartate-receptor antagonist property of meperidine may be pH dependent. PMID- 10735802 TI - Opioid substitution to improve the effectiveness of chronic noncancer pain control: a chart review. AB - We evaluated the efficacy and tolerability of opioids in the long-term management of chronic noncancer pain. This retrospective chart review included 86 outpatients who started receiving, between 1994 and 1998, long-acting opioids. For each patient, the number of different opioids used and the efficacy and tolerability of each opioid prescribed were noted. During a mean follow-up of 8.8 +/- 6.3 mo, the number of opioids used by each patient was 2.3 +/- 1.4. Patient diagnoses were: back pain (31), neuropathy (20), joint pain (13), visceral pain (7), reflex sympathetic dystrophy (7), headache (5), fibromyalgia (3). The first opioid prescribed was effective for 36% of patients, was stopped because of side effects in 30%, and was stopped for ineffectiveness in 34%. Of the remaining patients, the second opioid prescribed after the failure of the first was effective in 31%, the third in 40%, the fourth in 56%, and the fifth in 14%. There was one case of addiction and no case of tolerance. We conclude that if it is necessary to change the opioid prescription because of intolerable side effects or ineffectiveness, the cumulative percentage of efficacy increases with each new opioid tested. Failure of one opioid cannot predict the patient's response to another. IMPLICATIONS: This study showed that if a patient receiving chronic opioid therapy experiences an intolerable side effect or if the drug is ineffective, changing to a different opioid may result in a lessening or elimination of the side effect and/or improved analgesia. PMID- 10735803 TI - The accuracy and precision of body temperature monitoring methods during regional and general anesthesia. AB - We tested the hypotheses that accuracy and precision of available temperature monitoring methods are different between spinal anesthesia (SA) and general anesthesia (GA), and that patients receiving SA are at equal risk for hypothermia as those receiving GA. Patients scheduled for radical retropubic prostatectomy were enrolled. Either GA (n = 16) or SA (n = 16) was given according to patient and clinician preference. Temperatures were monitored with thermocouple probes at the tympanic membrane, axilla, rectum, and forehead skin surface. Tympanic temperatures were also measured with an infrared device, and forehead skin temperatures were monitored with two brands of liquid crystal thermometer strips. Accuracy and precision of these monitoring methods were determined by using tympanic membrane temperature, measured by thermocouple, as the reference core temperature (T(c)). At the end of surgery, T(c) was similar between SA (35.0 +/- 0.1 degrees C) and GA (35.2 +/- 0.1 degrees C) (P = 0.44). Accuracy and precision of each temperature monitoring method were similar between SA and GA. Rectal temperature monitoring offered the greatest combination of accuracy and precision. All other methods underestimated T(c). These findings suggest that patients receiving SA or GA are at equal and significant risk for hypothermia, and should have their temperatures carefully monitored, recognizing that most monitoring methods underestimate T(c). IMPLICATIONS: Body temperature should be monitored during spinal anesthesia because patients are at significant risk for hypothermia. Rectal temperature is a valid method of measuring core temperature, whereas other methods tend to underestimate true core temperature. PMID- 10735804 TI - Are there acid base changes during transurethral resection of the prostate (TURP)? AB - Acid base status during transurethral resection of the prostate (TURP) has been almost neglected. We therefore measured the acid base status and interpreted the observed changes according to the Stewart approach. The Stewart model focuses more on the influence of serum electrolyte concentrations on acid base changes than does the conventional Henderson-Hasselbalch approach. In 20 patients undergoing TURP, the following variables were determined: PaO(2), PaCO(2), pH(a), actual bicarbonate, standard base excess, serum concentration of sodium, potassium, chloride, lactate, and total protein. A study group (n = 11) and a control group (n = 9) were built, depending on the maximal amount of fluid absorption estimated with the aid of ethanol concentration monitoring in the expired gas. The study group developed a mild acidosis with a decrease in pH from 7.41 to 7.37 (P = 0.037), compared with a very discrete pH decrease from 7.44 to 7.42 in the control group. We found that moderate irrigant absorption during TURP leads to a specific metabolic acidosis. We speculate that larger amounts of irrigant absorption may cause a more severe metabolic acidosis. As the constellation of independently pH regulating variables appears to be typical for TURP, this acidosis could be named "TURP-acidosis." IMPLICATIONS: We measured acid base status in 20 patients undergoing transurethral resection of the prostate comparing a larger fluid absorption group with a minor or no fluid absorption group. We postulate the development of a typical metabolic transurethral resection of the prostate-acidosis caused by irrigant absorption. PMID- 10735805 TI - Nitrous oxide fraction in the carbon dioxide pneumoperitoneum during laparoscopy under general inhaled anesthesia in pigs. AB - During prolonged laparoscopy, the diffusion of other gases in the carbon dioxide (CO(2)) pneumoperitoneum may lessen its safety. Nitrous oxide (N(2)O)/CO(2) gas mixtures may become hazardous with regard to gas embolization and fire risk. We therefore evaluated the kinetics of pneumoperitoneal intrusion of N(2)O. In five anesthetized domestic pigs, controlled ventilation, with an initial fraction of inspired oxygen = 1.0, was adjusted to keep ETCO(2) pressure between 35 and 45 mm Hg. The peritoneum was insufflated with CO(2) to a pressure of 12 mm Hg, which was maintained throughout the procedure. T0 was defined as the time when N(2)O was introduced in the breathing circuit (N(2)O end-tidal fraction = 66%). Gas samples (10 mL) from the pneumoperitoneum were analyzed every 10 min after T0. The N(2)O concentration was measured by using capillary gas chromatography coupled with mass spectrometry. Percentages of N(2)O in the CO(2) increased with time (t) according to the ideal equation: N(2)O((t)) = 66 (1 - exp(-0.005t)). In the peritoneal cavity, <2 h were required for the N(2)O to reach the concentration of 29%, which can support combustion. Eight hours to 10 h after T0, the intraperitoneal N(2)O fraction approaches the level of the N(2)O end-tidal fraction. Options to prevent accumulation of N(2)O are suggested. IMPLICATIONS: Pig models were used to evaluate the time course of nitrous oxide (N(2)O) diffusion in the pneumoperitoneum during nitrous oxide/oxygen anesthesia. Although peritoneal N(2)O concentration approaches the end-expiratory value after 8-10 h, it reaches 29% within 2 h. At this level, N(2)O is known to support combustion. This N(2)O pollution should be prevented. PMID- 10735806 TI - A comparison of urapidil, clonidine, meperidine and placebo in preventing postanesthetic shivering. AB - This placebo-controlled study was performed to evaluate the efficacy of urapidil compared with clonidine and meperidine in preventing postanesthetic shivering, which is common after anesthesia administration and may be very distressing. We studied 120 patients undergoing elective abdominal or orthopedic surgery under standardized general anesthesia. After surgery, patients were randomly assigned to one of four groups (each group n = 30) using a double-blinded protocol: Group A received 0.2 mg/kg urapidil; Group B, 3 microg/kg clonidine; Group C, 0.4 mg/kg meperidine; and Group D, saline 0.9% as placebo. Postanesthetic shivering was scored by using a five-point scale. Clonidine and meperidine significantly reduced the incidence and the severity of shivering in comparison with placebo, whereas there were no significant differences between the urapidil and placebo groups. Both clonidine and meperidine caused a significantly prolonged emergence time (13.4 +/- 5.8 and 13. 3 +/- 5.0 min, respectively) compared with placebo (10.4 +/- 5.3 min) and urapidil (11.4 +/- 2.9 min). We confirmed that both clonidine and meperidine are effective in preventing postanesthetic shivering, whereas urapidil, in our setting and dosage, was not effective. Patients who received clonidine or meperidine had a prolonged emergence time. In the dosage used, urapidil seems to be unable to prevent postanesthetic shivering. IMPLICATIONS: Shivering (irregular muscle activity) is common after surgery and anesthesia. This study compared urapidil (an antihypertensive drug) as a prophylaxis with two established antishivering drugs (meperidine and clonidine) and placebo. In the dosage used, we were unable to show a significant benefit of urapidil. PMID- 10735807 TI - Combitube: a study for proper use. AB - The esophageal-tracheal Combitube((R)) (ETC; Kendall-Sheridan Catheter Corp., Argyle, NY) is a new device designed for difficult airways and emergency intubation. The manufacturer recommends that the ETC model 37F SA be used in patients with a height of between 122 and 152 cm. The aim of this study was to evaluate whether ventilation is effective and reliable in patients taller than 152 cm by using the ETC 37F SA in the esophageal position. Also, we evaluated whether the airway protection is adequate and whether direct intubation of the trachea with the ETC inserted in the esophagus is possible. We studied 25 anesthetized, paralyzed adult patients, 150 to 180 cm in height. Methylene blue was given orally to all patients before anesthesia induction. Under direct vision, a ETC 37F SA was inserted in the esophagus of all patients. The pharyngeal balloon inflation volume was titrated to air leak and cuff pressures were measured. During surgery, a laryngoscope was inserted into the pharynx with the pharyngeal balloon deflated and the laryngoscopic view was evaluated by using the Cormack-Lehane scale. The presence of methylene blue in the hypopharynx was investigated by direct laryngoscopic vision. Ventilation was effective and reliable in all 25 patients who were 150 to 180 cm in height (average 169 +/- 7 cm). In addition, a direct relationship between the pharyngeal balloon volume and patient height was established (P < 0.05), by using linear regression models. The laryngoscopic view of the glottis was adequate to allow direct tracheal intubation. No trace of methylene blue was detected in the hypopharynx. The ETC Model 37F SA may be used in patients from 122 to 185 cm in height. The trachea could be directly intubated with the ETC in the esophageal position in patients with normal airways. The airway protection appears to be adequate. IMPLICATIONS: The esophageal-tracheal Combitube((R)) Model 37F SA (Kendall-Sheridan Catheter Corp., Argyle, NY) may be used in patients from 122 to 185 cm in height. The trachea could be directly intubated with the Combitube((R)) in esophageal position in patients with normal airways. The airway protection appears to be adequate. PMID- 10735808 TI - Prevention of pain on injection with propofol: a quantitative systematic review. AB - The best intervention to prevent pain on injection with propofol is unknown. We conducted a systematic literature search (Medline, Embase, Cochrane Library, bibliographies, hand searching, any language, up to September 1999) for full reports of randomized comparisons of analgesic interventions with placebo to prevent that pain. We analyzed data from 6264 patients (mostly adults) of 56 reports. On average, 70% of the patients reported pain on injection. Fifteen drugs, 12 physical measurements, and combinations were tested. With IV lidocaine 40 mg, given with a tourniquet 30 to 120 s before the injection of propofol, the number of patients needed to be treated (NNT) to prevent pain in one who would have had pain had they received placebo was 1.6. The closest to this came meperidine 40 mg with tourniquet (NNT 1.9) and metoclopramide 10 mg with tourniquet (NNT 2.2). With lidocaine mixed with propofol, the best NNT was 2.4; with IV alfentanil or fentanyl, it was 3 to 4. IV lidocaine before the injection of propofol was less analgesic. Temperature had no significant effect. There was a lack of data for all other interventions to allow meaningful conclusions. The diameter of venous catheters and speed of injection had no impact on pain. IMPLICATIONS: IV lidocaine (0.5 mg/kg) should be given with a rubber tourniquet on the forearm, 30 to 120 s before the injection of propofol; lidocaine will prevent pain in approximately 60% of the patients treated in this manner. PMID- 10735809 TI - Pentobarbital has curare-like effects on adult-type nicotinic acetylcholine receptor channel currents. AB - Pentobarbital (PB) is widely used as a short-term sedative and anticonvulsive drug with a side-effect of relaxing muscle tone. We investigated block of nicotinic acetylcholine receptor (nAChR) channel currents by PB using the patch clamp technique in combination with an ultrafast system for solution exchange. As a preparation, recombinant rat adult-type nAChR channels transiently expressed in HEK293 cells were used. Appli-cation of 1 mM acetylcholine to small cells or outside-out patches showed a transient current with fast activation and desensitization kinetics. Adding PB to the acetylcholine-containing solution resulted in a decrease of the time constant of current decay and of the peak current amplitude starting at concentrations >0.01 mM PB. Preincubation of nAChR channels with PB led to a decrease of the peak current amplitude without alteration of activation and desensitization kinetics caused by competitive block of nAChR channels. In conclusion, similar to the effect of d-Tubocurarine, block of nAChR channel currents by PB can be explained by a combination of open-channel and competitive block. IMPLICATIONS: The interaction between adult-type nicotinic acetylcholine receptors, acetylcholine, and pentobarbital was biophysically investigated by using the patch-clamp technique in combination with tools for ultrafast solution exchange. PB elicited open-channel block and competitive block of nicotinic acetylcholine receptor channel currents, whereas the latter seems to be effective in clinically relevant concentrations. PMID- 10735810 TI - Sequencing cases in the operating room: predicting whether one surgical case will last longer than another. AB - A microscope will be used for the first case of the day in operating room (OR) 1 and then may be used in the second case of the day by a different surgeon in a different OR, OR 2. Provided that the probability is reasonably high that the first case of the day in OR 2 will last longer than the first case in OR 1, the OR manager can be confident in scheduling the microscope to be used by both surgeons on the same day. The OR manager can use statistical decision theory to sequence cases to decrease the impact of limitations in equipment or personnel on case scheduling. This increases utilization of both the capital equipment and OR time. In this study, we derived equations that can be programmed into a surgical services information system to reliably estimate the probability that one case will have a longer duration than another. We confirmed the accuracy of our method by using actual case duration data. IMPLICATIONS: Our statistical method uses historical case duration data from an operating room information system to estimate the actual probability to within 1.5% that the second case of a pair will last longer than the first case of a pair. PMID- 10735811 TI - Scheduling surgical cases into overflow block time- computer simulation of the effects of scheduling strategies on operating room labor costs. AB - "Overflow" block time is operating room (OR) time for a surgical group's cases that cannot be completed in the regular block time allocated to each surgeon in the surgical group. Having such overflow block time increases OR utilization. The optimal way to schedule patients into a surgical group's overflow block time is unknown. In this study, we developed a scheduling strategy that balances the OR manager's need to reduce staffing costs and the needs of patients and surgeons for flexibility in choosing the dates and times of cases. We used computer simulation to evaluate our scheduling strategy. Surgeons and patients (i) can schedule the case into any overflow block within 2 wk; (ii) can only schedule the case into a "first case of the day" start time more than 2 wk in the future if there is not enough open time for the case within 2 wk; (iii) must schedule the case to be done within 4 wk; and (iv) are encouraged to perform the case on the earliest possible date. Staffing costs were lowest when the OR manager did not incorporate surgeon and patient preferences when scheduling cases into overflow block time. The strategy we developed provides surgeons and patients with some flexibility in scheduling, while only increasing OR staffing costs slightly over the minimum achieved when the OR manager controls scheduling. IMPLICATIONS: The strategy we developed provides surgeons and patients with some flexibility in scheduling, while increasing OR staffing costs only slightly over the minimum achieved when the OR manager controls scheduling. Staffing costs were lowest when the operating room (OR) manager did not incorporate surgeon and patient preferences when scheduling cases into overflow block time. PMID- 10735812 TI - Changes in propofol concentration in a propofol-lidocaine 9:1 volume mixture. PMID- 10735814 TI - Thirteenth annual meeting of the society for pediatric anesthesia, dallas, texas, october 8, 1999 PMID- 10735813 TI - Removal of an aspirated prosthetic tooth by tracheal backflow air. PMID- 10735815 TI - One drop of morphine added to local anesthetics by means of a 23-gauge injection needle can relieve postoperative pain under spinal anesthesia. PMID- 10735816 TI - One-lung ventilation in the patient with laryngeal or tracheal stenosis. PMID- 10735817 TI - Anticholinesterases and the transplanted heart. PMID- 10735818 TI - Assessment of LV diastolic function. PMID- 10735820 TI - A structural deficiency of TCI syringes. PMID- 10735819 TI - Detection of a tracheal obstruction caused by a retrosternal goiter by the use of a cuff leak test. PMID- 10735821 TI - A real eye-opener! PMID- 10735822 TI - Atrioventricular dissociation. PMID- 10735823 TI - Reported data on granisetron and postoperative nausea and vomiting by Fujii et al. Are incredibly nice! PMID- 10735824 TI - Nonpharmacologic sedation in a deaf child. PMID- 10735825 TI - Which intravenous sodium channel blocker for neuropathic pain? PMID- 10735826 TI - Fiberoptic endotracheal intubation through an ultra-thin bronchoscope with suction channel in a newborn with difficult airway. PMID- 10735827 TI - How a rigid stylet can make an endotracheal tube move. PMID- 10735828 TI - Evidence based medicine. PMID- 10735829 TI - Take it with a pillar of salt or "ambitious but achievable targets". PMID- 10735830 TI - The District Immunisation Coordinator. PMID- 10735831 TI - Stamps in paediatrics. Immunisation. PMID- 10735832 TI - ChildLine--the first twelve years. AB - This brief article aims to give a history and overview of ChildLine's work in the first 12 years of its existence and to provide references to more detailed information on a range of issues that children and young people themselves have identified as important. All callers' names and identifying details have been changed to preserve confidentiality. PMID- 10735833 TI - Active surveillance of hepatitis C infection in the UK and Ireland. AB - AIM: To investigate the prevalence, distribution, and clinical details of paediatric hepatitis C virus (HCV) infection in the UK and Ireland. METHODS: Active monthly surveillance questionnaire study coordinated through the British Paediatric Surveillance Unit, to all consultant paediatricians in 1997 and 1998. RESULTS: A total of 182 HCV infected children were reported from 54 centres and by paediatricians from eight different specialties. In 40 children HCV was acquired through mother to child transmission (MTC children); 142 were infected by contaminated blood products (n = 134), organ transplantation (n = 2), needles (n = 4), or unknown risk factor (n = 2). Intravenous drug use was the risk factor for 35 mothers of MTC children. Twelve children were coinfected with HIV and four with HBV. Recent serum aspartate aminotransferase or alanine aminotransferase values were at least twofold greater than the upper limit of normal in 24 of 152 children; this occurred in five of 11 HIV coinfected children. Liver histology, available in 53 children, showed normal (7%), mild (74%), moderate (17%), or severe (2%) hepatitis. Twenty eight children had received therapy with interferon alfa. CONCLUSION: Most current paediatric HCV infection in UK and Ireland has been acquired from contaminated blood products, and most children are asymptomatic. There is a need for multicentre trials to inform clinical practice and development of good practice guidelines in this area. Long term follow up of this cohort of HCV infected children is planned to help determine the natural history over the long term of HCV acquired during infancy and childhood. PMID- 10735834 TI - Hereditary dyslipidemias and combined risk factors in children with a family history of premature coronary artery disease. AB - AIM: Schoolchildren aged 10-11 with a family history of premature coronary artery disease (CAD), were examined in order to identify children with genetically determined dyslipidemias and a combination of risk factors. METHODS: A total of 4000 questionnaires were distributed by the school; 55% of the families answered and returned the questionnaire. Blood lipids, apolipoprotein B, and Lp(a) lipoprotein were analysed in high risk children and their parents. RESULTS: A family history of premature CAD in parents or grandparents was identified in 208 families; 175 agreed to take part in a clinical examination and laboratory tests. Normal blood lipid tests were found in 89 children. Another 48 had an isolated increase of Lp(a) lipoprotein of minor clinical importance. Of the remaining 38 children, 23 had non-hereditary abnormalities of low (LDL) or high density lipoprotein (HDL) cholesterol or apolipoprotein B. Fifteen children were suspected to have genetically determined dyslipidemias or a combination of risk factors: in four, possible familial hypercholesterolaemia (FH); in five, possible familial combined hyperlipidaemia; in three, hereditary low HDL cholesterol; and in three a combination of high LDL cholesterol and Lp(a) lipoprotein concentrations. In addition, possible FH was detected in eight of the parents. CONCLUSION: It is worthwhile asking parents about the occurrence of premature CAD among their child's closest relatives. PMID- 10735835 TI - Does height influence progression through primary school grades? AB - AIM: To examine the relation between a child's height and grade progression in primary school. METHODS: Height was measured in a representative cross sectional sample of children from 24 primary schools in Victoria in late 1997. Height measurements were transformed to standardised scores using Cole's "LMS" method to allow for comparison across ages and genders. Children within each grade were divided into three equal groups based on age (youngest third, middle third, oldest third), again to allow for cross age comparison. RESULTS: A total of 2848 children aged 5-12 years (51% male) were included, with approximately 400 children in each of the seven grades from preparatory to grade 6. Analysis of variance showed a significant relation overall between age and height, with a sequential decrease in height from the youngest to the oldest third. When genders were considered separately, the relation remained significant for boys but not for girls. A total of 133 children (66% male) repeated a grade in primary school. When this group of grade repeaters was removed from the sample, analysis of variance showed no significant relation between standardised height score and age tertile for boys. Although birth weight category and maternal education were independent predictors of height scores overall, they did not appear to influence decisions to retain pupils in grades. CONCLUSIONS: Older boys within grades, notably those who have repeated a grade, are shorter than their peers. Decisions to retain pupils, particularly boys who are experiencing school difficulties, may be influenced by their height. PMID- 10735836 TI - Improved clinical practice but continuing service deficiencies following a regional audit of childhood diabetes mellitus. AB - AIM: To assess the changes in services for children with diabetes in the south west of England between two region-wide audits performed in 1994 and 1998. METHODS: Questionnaires were sent to consultant paediatricians, specialist diabetes nurses, dietitians, and Local Diabetes Service Advisory Groups. Information was gathered on consultant and nursing caseload, clinic structure, dietetic and psychological services, glycated haemoglobin use, and screening services. RESULTS: In 1994 there were 21 consultant paediatricians caring for children with diabetes, only seven of whom fulfilled the British Paediatric Association definition of a specialist. By 1998 there were 14, 12 of whom fulfilled this definition. In 1994 a significant number of children were being seen in general paediatric clinics; by 1998 all centres stated that children were being seen in designated diabetes clinics. Between the two audits, despite a decrease in the average caseload of specialist diabetes nurses, nursing services in many centres remained deficient, as did dietetic and psychology services. Glycated haemoglobin use increased from 16 of 21 consultants to all consultants. In 1998 there was still patchy paediatric representation on Local Diabetes Service Advisory Groups. CONCLUSIONS: The 1994 audit was followed by a change in clinical practice, in contrast to continuing deficiencies in resources, despite the availability of national recommendations and the widespread distribution of the audit report to those in a position of influence. PMID- 10735837 TI - Sputum induction for the diagnosis of pulmonary tuberculosis in infants and young children in an urban setting in South Africa. AB - BACKGROUND: Bacteriological confirmation of pulmonary tuberculosis is difficult in infants and young children. In adults and older children, sputum induction has been successfully used; this technique has not been tested in younger children. AIMS: To investigate whether sputum induction can be successfully performed in infants and young children and to determine the utility of induced sputum compared to gastric lavage (GL) for the diagnosis of pulmonary tuberculosis in HIV infected and uninfected children. SUBJECTS AND METHODS: 149 children (median age 9 months) admitted to hospital with acute pneumonia who were known to be HIV infected, suspected to have HIV infection, or required intensive care unit support. Sputum induction was performed on enrollment. Early morning GL was performed after a minimum four hour fast. Induced sputum and stomach contents were stained for acid fast bacilli and cultured for Mycobacterium tuberculosis. RESULTS: Sputum induction was successfully performed in 142 of 149 children. M tuberculosis, cultured in 16 children, grew from induced sputum in 15. GL, performed in 142 children, was positive in nine; in eight of these M tuberculosis also grew from induced sputum. The difference between yields from induced sputum compared to GL was 4.3% (p = 0.08). M tuberculosis was cultured in 10 of 100 HIV infected children compared to six of 42 HIV uninfected children (p = 0.46). CONCLUSION: Sputum induction can be safely and effectively performed in infants and young children. Induced sputum provides a satisfactory and more convenient specimen for bacteriological confirmation of pulmonary tuberculosis in HIV infected and uninfected children. PMID- 10735838 TI - A comparison of local anaesthetics for venepuncture. AB - AIM: To compare the effectiveness of EMLA cream and Ametop gel in providing analgesia for venous cannulation. METHODS: Single blind study in 120 children. RESULTS: Both anaesthetic agents produced adequate analgesia. However, Ametop gel was more effective, with a statistically significant difference in the pain scores of the two groups (p < 0.05). PMID- 10735839 TI - Sex preference PMID- 10735840 TI - Neurovascular compression of the trigeminal and glossopharyngeal nerve: three case reports. AB - Trigeminal neuralgia (TN) is a frequent cause of paroxysmal facial pain and headache in adults. Glossopharyngeal neuralgia (GPN) is less common, but can cause severe episodic pain in the ear and throat. Neurovascular compression of the appropriate cranial nerve as it leaves the brain stem is responsible for the symptoms in many patients, and neurosurgical decompression of the nerve is now a well accepted treatment in adults with both TN and GPN who fail to respond to drug therapy. Neither TN nor GPN are routinely considered in the differential diagnosis when assessing children with paroxysmal facial or head pain, as they are not reported to occur in childhood. Case reports of three children with documented neurovascular compression causing severe neuralgic pain and disability are presented. The fact that these conditions do occur in the paediatric population, albeit rarely, is highlighted, and appropriate investigation and management are discussed. PMID- 10735841 TI - Sclerosing treatment of lymphangiomas with OK-432. AB - Over a period of seven years, 15 patients (aged from birth to 15 years; median 22 months) with lymphangioma were treated with OK-432; they received a mean of three injections each. Ten received OK-432 as first line treatment; five were treated after surgery (three had a residual lymphangioma after incomplete removal and two had a late recurrence). OK-432 proved to be effective for primitive as well as for residual and recurrent lymphangioma. Seven cases were macrocystic; complete regression was obtained in all. Five cases were microcystic: two had more than 50% regression, and three less than 50%. Three cases were mixed, with both large and microscopic cysts: one had more than 50% regression, and two less than 50%. These last two cases underwent surgery after the sclerosing treatment. The results obtained were excellent in 100% of macrocystic cases; a shrinkage in size was obtained in all microcystic cases. OK-432 is therefore proposed as a first line option for treatment of lymphangiomas. PMID- 10735842 TI - Inherited deafness in childhood--the genetic revolution unmasks the clinical challenge. PMID- 10735843 TI - Urinary oxalate excretion in urolithiasis and nephrocalcinosis. AB - AIMS: To investigate urinary oxalate excretion in children with urolithiasis and/or nephrocalcinosis and to classify hyperoxaluria (HyOx). METHODS: A total of 106 patients were screened. In those in whom the oxalate: creatinine ratio was increased, 24 hour urinary oxalate excretion was measured. Liver biopsy and/or genomic analysis was performed if primary hyperoxaluria (PH) was suspected. Stool specimens were examined for Oxalobacter formigenes in HyOx not related to PH type 1 or 2 (PH1, PH2) and in controls. RESULTS: A total of 21 patients screened had HyOx (>0.5 mmol/24 h per 1.73 m(2)); they were classified into five groups. Eleven had PH (PH1 in nine and neither PH1 nor PH2 in two). Six had secondary HyOx: two enteric and four dietary. Four could not be classified. Seven patients had concomitant hypercalciuria. Only one of 12 patients was colonised with O formigenes compared to six of 13 controls. CONCLUSIONS: HyOx is an important risk factor for urolithiasis and nephrocalcinosis in children, and can coexist with hypercalciuria. A novel type of PH is proposed. Absence of O formigenes may contribute to HyOx not related to PH1. PMID- 10735844 TI - What do parents of wheezy children understand by "wheeze"? AB - BACKGROUND: Reported wheeze is the cornerstone of asthma diagnosis. AIMS: To determine what parents understand by wheeze. METHODS: Two studies were undertaken: (1) Parents of clinic attendees with reported wheeze (n=160) were asked by questionnaire what they understood by "wheeze" and how they knew their child was wheezy. Responses were compared to definitions of wheeze in 12 epidemiology studies and their response options. (2) The extent of agreement of parents' reports (n=139) of acute wheezing in their children and clinicians' findings of "wheeze" and "asthma" was examined. RESULTS: (1) "Sound" and "difficulty in breathing" were perceived central to "wheeze". "What you hear" was not selected by 23% (95% confidence interval (CI) 16-30%). "Whistling" was mentioned by 11% (CI 6-15%) but featured in 11 of 12 epidemiology questionnaires. (2) There was les than 50% agreement between parents' and clinicians' reports of wheeze and asthma. CONCLUSIONS: Conceptual understandings of "wheeze" for parents of children with reported wheeze are different from epidemiology definitions. Parents' reports of acute wheeze and clinicians' findings also differ. PMID- 10735845 TI - Imaging the less seriously head injured child. PMID- 10735846 TI - Biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons by bacteria. PMID- 10735847 TI - Molecular characterization of two-component systems of Helicobacter pylori. AB - Two-component systems are frequently involved in the adaptation of bacteria to changing environmental conditions at the level of transcriptional regulation. Here we report the characterization of members of the two-component systems of the gastric pathogen Helicobacter pylori deduced from the genome sequence of strain 26695. We demonstrate that the response regulators HP166, HP1043, and HP1021 have essential functions, as disruption of the corresponding genes is lethal for the bacteria, irrespective of the fact that HP1043 and HP1021 have nonconserved substitutions in crucial amino acids of their receiver domains. An analysis of the in vitro phosphorylation properties of the two-component proteins demonstrates that HP244-HP703 and HP165-HP166 are cognate histidine kinase response regulator pairs. Furthermore, we provide evidence that the variability of the histidine kinase HP165 caused by a poly(C) tract of variable length close to the 3' end of open reading frame 165/164 does not interfere with the kinase activity of the transmitter domain of HP165. PMID- 10735848 TI - RegA, iron, and growth phase regulate expression of the Pseudomonas aeruginosa tol-oprL gene cluster. AB - The tol-oprL region in Pseudomonas aeruginosa appears to be involved in pyocin uptake and required for cell viability. The complete nucleotide sequences of the tolQRA and oprL genes as well as the incomplete sequences of tolB and orf2 have been previously reported. In addition, the sequence of a P. aeruginosa iron regulated gene (pig6) has been described and found to share homology with an open reading frame located upstream of the Escherichia coli tolQRA genes (U. A. Ochsner and M. L. Vasil, Proc. Natl. Acad. Sci. USA 93:4409-4414, 1996). In this study, we cloned the remainder of the P. aeruginosa tol-oprL gene cluster and determined its nucleotide sequence. This cluster was found to consist of seven genes in the order orf1 tolQ tolR tolA tolB oprL orf2. Transcriptional analysis of this gene cluster was performed by detecting the presence of mRNAs spanning adjacent genes as well as by using a promoterless lacZ reporter gene fused to each of the seven genes contained in the tol-oprL locus. The results show that there are three major transcriptional units or operons in this region, orf1 tolQRA, tolB, and oprL-orf2, in contrast to the E. coli tol-pal region, where there are only two operons, orf1-tolQRA and tolB-pal-orf2. Analysis of gene expression indicated that the tol-oprL genes of P. aeruginosa are both iron and growth phase modulated. The first operon, orf1-tolQRA, is iron regulated throughout growth, but iron-regulated expression of tolB and oprL fusions occurs only in late log phase. The expression of the three operons was significantly less repressed by iron in fur mutants than in the wild-type strain, suggesting the involvement of Fur in the iron regulation of all three operons. RegA is a positive yet nonessential regulator of tol-oprL expression. PMID- 10735849 TI - Two types of Bacillus subtilis tetA(L) deletion strains reveal the physiological importance of TetA(L) in K(+) acquisition as well as in Na(+), alkali, and tetracycline resistance. AB - The chromosomally encoded TetA(L) protein of Bacillus subtilis is a multifunctional tetracycline-metal/H(+) antiporter that also exhibits monovalent cation/H(+) antiport activity and a net K(+) uptake mode. In this study, B. subtilis mutant strains JC112 and JC112C were found to be representative of two phenotypic types of tetA(L) deletion strains that are generated in the same selection. Both strains exhibited increased sensitivity to low tetracycline concentrations as expected. The mutants also had significantly reduced ability to grow in media containing low concentrations of K(+), indicating that the net K(+) uptake mode is of physiological consequence; the deficit in JC112 was greater than in JC112C. JC112 also exhibited (i) greater impairment of Na(+)- or K(+) dependent growth at pH 8.3 than JC112C and (ii) a greater degree of Co(+2) as well as Na(+) sensitivity. Studies were initiated to explore the possibility of two different patterns of compensatory changes in other ion-translocating transporters in these mutants. Increased expression of two loci has thus far been shown. Increased expression of czcD-trkA, a locus with a proposed involvement in K(+) uptake, occurred in both mutants. The increase was highest in the presence of Co(2+) and was higher in JC112 than in JC112C. Deletion of czcD-trkA resulted in diminished growth of the wild-type and both mutant strains at low [K(+)], supporting a significant role for this locus in K(+) uptake. Expression of yheL, which is a homologue of the Na(+)/H(+) antiporter-encoding nhaC gene from Bacillus firmus OF4, was also increased in both tetA(L) deletion strains, again with higher up-regulation in JC112. The phenotypes resulting from deletion of yheL were consistent with a modest role for YheL in Na(+)-dependent pH homeostasis in the wild type. No major role for YheL was indicated in the mutants in spite of the overexpression. The studies underscore the multiple physiological functions of TetA(L), including tetracycline, Na(+), and alkali resistance and K(+) acquisition. The studies also reveal and begin to detail the complexity of the response to mutational loss of these functions. PMID- 10735850 TI - Characterization of the enzymatic component of Clostridium perfringens iota toxin. AB - The iota(a) component (i(a)) of Clostridium perfringens ADP ribosylates nonmuscle beta/gamma actin and skeletal muscle alpha-actin. Replacement of Arg-295 in i(a) with alanine led to a complete loss of NAD(+)-glycohydrolase (NADase) and ADP ribosyltransferase (ARTase); that of the residue with lysine caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them. Substitution of alanine for Glu-378 and Glu-380 caused a complete loss of NADase and ARTase. However, exchange of Glu-378 to aspartic acid or glutamine resulted in little effect on NADase activity but a drastic reduction in ARTase activity (<0.1% of the wild-type activity). Exchange of Glu-380 to aspartic acid caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them; that of the residue to glutamine caused a complete loss of ARTase activity. Replacement of Ser-338 with alanine resulted in 0.7 to 2.3% wild-type activities, and that of Ser-340 and Thr-339 caused a reduction in these activities of 5 to 30% wild-type activities. The kinetic analysis showed that Arg-295 and Ser-338 also play an important role in the binding of NAD(+) to i(a), that Arg-295, Glu 380, and Ser-338 play a crucial role in the catalytic rate of NADase activity, and that these three amino acid residues and Glu-378 are essential for ARTase activity. The effect of amino acid replacement in i(a) on ARTase activity was similar to that on lethal and cytotoxic activities, suggesting that lethal and cytotoxic activities in i(a) are dependent on ARTase activity. PMID- 10735851 TI - Contribution of base excision repair, nucleotide excision repair, and DNA recombination to alkylation resistance of the fission yeast Schizosaccharomyces pombe. AB - DNA damage is unavoidable, and organisms across the evolutionary spectrum possess DNA repair pathways that are critical for cell viability and genomic stability. To understand the role of base excision repair (BER) in protecting eukaryotic cells against alkylating agents, we generated Schizosaccharomyces pombe strains mutant for the mag1 3-methyladenine DNA glycosylase gene. We report that S. pombe mag1 mutants have only a slightly increased sensitivity to methylation damage, suggesting that Mag1-initiated BER plays a surprisingly minor role in alkylation resistance in this organism. We go on to show that other DNA repair pathways play a larger role than BER in alkylation resistance. Mutations in genes involved in nucleotide excision repair (rad13) and recombinational repair (rhp51) are much more alkylation sensitive than mag1 mutants. In addition, S. pombe mutant for the flap endonuclease rad2 gene, whose precise function in DNA repair is unclear, were also more alkylation sensitive than mag1 mutants. Further, mag1 and rad13 interact synergistically for alkylation resistance, and mag1 and rhp51 display a surprisingly complex genetic interaction. A model for the role of BER in the generation of alkylation-induced DNA strand breaks in S. pombe is discussed. PMID- 10735852 TI - Requirement for the enzymes acetoacetyl coenzyme A synthetase and poly-3 hydroxybutyrate (PHB) synthase for growth of Sinorhizobium meliloti on PHB cycle intermediates. AB - We have identified two Sinorhizobium meliloti chromosomal loci affecting the poly 3-hydroxybutyrate degradation pathway. One locus was identified as the gene acsA, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase. Analysis of the acsA nucleotide sequence revealed that this gene encodes a putative protein with a molecular weight of 72,000 that shows similarity to acetyl-CoA synthetase in other organisms. Acetyl-CoA synthetase activity was not affected in cell extracts of glucose-grown acsA::Tn5 mutants; instead, acetoacetyl-CoA synthetase activity was drastically reduced. These findings suggest that acetoacetyl-CoA synthetase, rather than CoA transferase, activates acetoacetate to acetoacetyl-CoA in the S. meliloti poly-3-hydroxybutyrate cycle. The second locus was identified as phbC, encoding poly-3-hydroxybutyrate synthase, and was found to be required for synthesis of poly-3-hydroxybutyrate deposits. PMID- 10735853 TI - Probable identification of a membrane-associated repressor of Bacillus subtilis DNA replication as the E2 subunit of the pyruvate dehydrogenase complex. AB - Two Bacillus subtilis lysogenic libraries were probed by an antibody specific for a previously described membrane-associated inhibitor of B. subtilis DNA replication (J. Laffan and W. Firshein, Proc. Natl. Acad. Sci. USA 85:7452-7456, 1988). Three clones that reacted strongly with the antibody contained an entire open reading frame. Sequencing identified one of the clones (R1-2) as containing the E2 subunit of the pyruvate dehydrogenase complex, dihydrolipoamide acetyltransferase. An AT-rich sequence in the origin region was identified initially as the site to which extracts from the R1-2 clone were bound. This sequence was almost identical to one detected in Bacillus thuringiensis that also bound the E2 subunit but which was involved in activating the Cry1 protoxin gene of the organism, not in inhibiting DNA replication (T. Walter and A. Aronson, J. Biol. Chem., 274:7901-7906, 1999). However, the exact sequence was not as important in B. subtilis as the AT-rich core region. Binding would occur as long as most of the AT character of the core remained. Purified E2 protein obtained by use of PCR and an expression vector reacted strongly with antibody prepared against the repressor protein and the protein in the R1-2 clone, but its specificity for the AT-rich region was altered. The purified E2 protein was capable of inhibiting membrane-associated DNA replication in vitro, but anti-E2 antibody was variable in its ability to rescue repression when added to the assay. PMID- 10735854 TI - Autophagy and the cvt pathway both depend on AUT9. AB - In growing cells of the yeast Saccharomyces cerevisiae, proaminopeptidase I reaches the vacuole via the selective cytoplasm-to-vacuole targeting (cvt) pathway. During nutrient limitation, autophagy is also responsible for the transport of proaminopeptidase I. These two nonclassical protein transport pathways to the vacuole are distinct in their characteristics but in large part use identical components. We expanded our initial screen for aut(-) mutants and isolated aut9-1 cells, which show a defect in both pathways, the vacuolar targeting of proaminopeptidase I and autophagy. By complementation of the sporulation defect of homocygous diploid aut9-1 mutant cells with a genomic library, in this study we identified and characterized the AUT9 gene, which is allelic with CVT7. aut9-deficient cells have no obvious defects in growth on rich media, vacuolar biogenesis, and acidification, but like other mutant cells with a defect in autophagy, they exhibit a reduced survival rate and reduced total protein turnover during starvation. Aut9p is the first putative integral membrane protein essential for autophagy. A biologically active green fluorescent protein Aut9 fusion protein was visualized at punctate structures in the cytosol of growing cells. PMID- 10735855 TI - A novel phenanthrene dioxygenase from Nocardioides sp. Strain KP7: expression in Escherichia coli. AB - Nocardioides sp. strain KP7 grows on phenanthrene but not on naphthalene. This organism degrades phenanthrene via 1-hydroxy-2-naphthoate, o-phthalate, and protocatechuate. The genes responsible for the degradation of phenanthrene to o phthalate (phd) were found by Southern hybridization to reside on the chromosome. A 10.6-kb DNA fragment containing eight phd genes was cloned and sequenced. The phdA, phdB, phdC, and phdD genes, which encode the alpha and beta subunits of the oxygenase component, a ferredoxin, and a ferredoxin reductase, respectively, of phenanthrene dioxygenase were identified. The gene cluster, phdAB, was located 8. 3 kb downstream of the previously characterized phdK gene, which encodes 2 carboxybenzaldehyde dehydrogenase. The phdCD gene cluster was located 2.9 kb downstream of the phdB gene. PhdA and PhdB exhibited moderate (less than 60%) sequence identity to the alpha and beta subunits of other ring-hydroxylating dioxygenases. The PhdC sequence showed features of a [3Fe-4S] or [4Fe-4S] type of ferredoxin, not of the [2Fe-2S] type of ferredoxin that has been found in most of the reported ring-hydroxylating dioxygenases. PhdD also showed moderate (less than 40%) sequence identity to known reductases. The phdABCD genes were expressed poorly in Escherichia coli, even when placed under the control of strong promoters. The introduction of a Shine-Dalgarno sequence upstream of each initiation codon of the phdABCD genes improved their expression in E. coli. E. coli cells carrying phdBCD or phdACD exhibited no phenanthrene-degrading activity, and those carrying phdABD or phdABC exhibited phenanthrene-degrading activity which was significantly less than that in cells carrying the phdABCD genes. It was thus concluded that all of the phdABCD genes are necessary for the efficient expression of phenanthrene-degrading activity. The genetic organization of the phd genes, the phylogenetically diverged positions of these genes, and an unusual type of ferredoxin component suggest phenanthrene dioxygenase in Nocardioides sp. strain KP7 to be a new class of aromatic ring-hydroxylating dioxygenases. PMID- 10735856 TI - Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE. AB - We report attempts to analyze interactions between components of the pullulanase (Pul) secreton (type II secretion machinery) from Klebsiella oxytoca encoded by a multiple-copy-number plasmid in Escherichia coli. Three of the 15 Pul proteins (B, H, and N) were found to be dispensable for pullulanase secretion. The following evidence leads us to propose that PulE, PulL, and PulM form a subcomplex with which PulC and PulG interact. The integral cytoplasmic membrane protein PulL prevented proteolysis and/or aggregation of PulE and mediated its association with the cytoplasmic membrane. The cytoplasmic, N-terminal domain of PulL interacted directly with PulE, and both PulC and PulM were required to prevent proteolysis of PulL. PulM and PulL could be cross-linked as a heterodimer whose formation in a strain producing the secreton required PulG. However, PulL and PulM produced alone could also be cross-linked in a 52-kDa complex, indicating that the secreton exerts subtle effects on the interaction between PulE and PulL. Antibodies against PulM coimmunoprecipitated PulL, PulC, and PulE from detergent-solubilized cell extracts, confirming the existence of a complex containing these four proteins. Overproduction of PulG, which blocks secretion, drastically reduced the cellular levels of PulC, PulE, PulL, and PulM as well as PulD (secretin), which probably interacts with PulC. The Pul secreton components E, F, G, I, J, K, L, and M could all be replaced by the corresponding components of the Out secretons of Erwinia chrysanthemi and Erwinia carotovora, showing that they do not play a role in secretory protein recognition and secretion specificity. PMID- 10735857 TI - Multiple hexose transporters of Schizosaccharomyces pombe. AB - We have identified a family of six hexose transporter genes (Ght1 to Ght6) in the fission yeast Schizosaccharomyces pombe. Sequence homology to Saccharomyces cerevisiae and mammalian hexose transporters (Hxtp and GLUTp, respectively) and secondary-structure predictions of 12 transmembrane domains for each of the Ght proteins place them into the sugar porter subfamily within the major facilitator superfamily. Interestingly, among this sugar porter family, the emerging S. pombe hexose transporter family clusters are separate from monosaccharide transporters of other yeasts (S. cerevisiae, Kluyveromyces lactis, and Candida albicans) and of humans, suggesting that these proteins form a distinct structural family of hexose transporters. Expression of the Ght1, Ght2, Ght5, and Ght6 genes in the S. cerevisiae mutant RE700A may functionally complement its D-glucose uptake deficient phenotype. Northern blot analysis and reverse transcription-PCR showed that among all Ght's of S. pombe, Ght5 is the most prominently expressed hexose transporter. Ght1p, Ght2p, and Ght5p displayed significantly higher specificities for D-glucose than for D-fructose. Analysis of the previously described S. pombe D-glucose transport-deficient mutant YGS-5 revealed that this strain is defective in the Ght1, Ght5, and Ght6 genes. Based on an analysis of three S. pombe strains bearing single or double mutations in Ght3 and Ght4, we conclude that the Ght3p function is required for D-gluconate transport in S. pombe. The function of Ght4p remains to be clarified. Ght6p exhibited a slightly higher affinity to D-fructose than to D-glucose, and among the Ght's it is the transporter with the highest specificity for D-fructose. PMID- 10735858 TI - The net charge of the first 18 residues of the mature sequence affects protein translocation across the cytoplasmic membrane of gram-negative bacteria. AB - This statistical study shows that in proteins of gram-negative bacteria exported by the Sec-dependent pathway, the first 14 to 18 residues of the mature sequences have the highest deviation between the observed and expected net charge distributions. Moreover, almost all sequences have either neutral or negative net charge in this region. This rule is restricted to gram-negative bacteria, since neither eukaryotic nor gram-positive bacterial exported proteins have this charge bias. Subsequent experiments performed with a series of Escherichia coli alkaline phosphatase mutants confirmed that this charge bias is associated with protein translocation across the cytoplasmic membrane. Two consecutive basic residues inhibit translocation effectively when placed within the first 14 residues of the mature protein but not when placed in positions 19 and 20. The sensitivity to arginine partially reappeared again 30 residues away from the signal sequence. These data provide new insight into the mechanism of protein export in gram negative bacteria and lead to practical recommendations for successful secretion of hybrid proteins. PMID- 10735860 TI - Two nitrate/nitrite transporters are encoded within the mobilizable plasmid for nitrate respiration of Thermus thermophilus HB8. AB - Thermus thermophilus HB8 can grow anaerobically by using a membrane-bound nitrate reductase to catalyze the reduction of nitrate as a final electron acceptor in respiration. In contrast to other denitrifiers, the nitrite produced does not continue the reduction pathway but accumulates in the growth medium after its active extrusion from the cell. We describe the presence of two genes, narK1 and narK2, downstream of the nitrate reductase-encoding gene cluster (nar) that code for two homologues to the major facilitator superfamily of transporters. The sequences of NarK1 and NarK2 are 30% identical to each other, but whereas NarK1 clusters in an average-distance tree with putative nitrate transporters, NarK2 does so with putative nitrite exporters. To analyze whether this differential clustering was actually related to functional differences, we isolated derivatives with mutations of one or both genes. Analysis revealed that single mutations had minor effects on growth by nitrate respiration, whereas a double narK1 narK2 mutation abolished this capability. Further analysis allowed us to confirm that the double mutant is completely unable to excrete nitrite, while single mutants have a limitation in the excretion rates compared with the wild type. These data allow us to propose that both proteins are implicated in the transport of nitrate and nitrite, probably acting as nitrate/nitrite antiporters. The possible differential roles of these proteins in vivo are discussed. PMID- 10735859 TI - Replication of staphylococcal multiresistance plasmids. AB - Based on structural and functional properties, three groups of large staphylococcal multiresistance plasmids have been recognized, viz., the pSK1 family, pSK41-like conjugative plasmids, and beta-lactamase-heavy-metal resistance plasmids. Here we describe an analysis of the replication functions of a representative of each of these plasmid groups. The replication initiation genes from the Staphylococcus aureus plasmids pSK1, pSK41, and pI9789::Tn552 were found to be related to each other and to the Staphylococcus xylosus plasmid pSX267 and are also related to rep genes of several plasmids from other gram positive genera. Nucleotide sequence similarity between pSK1 and pI9789::Tn552 extended beyond their rep genes, encompassing upstream divergently transcribed genes, orf245 and orf256, respectively. Our analyses revealed that genes encoding proteins related to the deduced orf245 product are variously represented, in several types of organization, on plasmids possessing six seemingly evolutionarily distinct types of replication initiation genes and including both theta-mode and rolling-circle replicons. Construction of minireplicons and subsequent functional analysis demonstrated that orf245 is required for the segregational stability of the pSK1 replicon. In contrast, no gene equivalent to orf245 is evident on the conjugative plasmid pSK41, and a minireplicon encoding only the pSK41 rep gene was found to exhibit a segregational stability approaching that of the parent plasmid. Significantly, the results described establish that many of the large multiresistance plasmids that have been identified in clinical staphylococci, which were formerly presumed to be unrelated, actually utilize an evolutionarily related theta-mode replication system. PMID- 10735861 TI - Type IV pilus genes pilA and pilC of Pseudomonas stutzeri are required for natural genetic transformation, and pilA can be replaced by corresponding genes from nontransformable species. AB - Pseudomonas stutzeri lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. After transposon mutagenesis, transformation deficient mutants were isolated from a P. stutzeri JM300 strain. In one of them a gene which coded for a protein with 75% amino acid sequence identity to PilC of Pseudomonas aeruginosa, an accessory protein for type IV pilus biogenesis, was inactivated. The presence of type IV pili was demonstrated by susceptibility to the type IV pilus-dependent phage PO4, by occurrence of twitching motility, and by electron microscopy. The pilC mutant had no pili and was defective in twitching motility. Further sequencing revealed that pilC is clustered in an operon with genes homologous to pilB and pilD of P. aeruginosa, which are also involved in pilus formation. Next to these genes but transcribed in the opposite orientation a pilA gene encoding a protein with high amino acid sequence identity to pilin, the structural component of type IV pili, was identified. Insertional inactivation of pilA abolished pilus formation, PO4 plating, twitching motility, and natural transformation. The amounts of (3)H-labeled P. stutzeri DNA that were bound to competent parental cells and taken up were strongly reduced in the pilC and pilA mutants. Remarkably, the cloned pilA genes from nontransformable organisms like Dichelobacter nodosus and the PAK and PAO strains of P. aeruginosa fully restored pilus formation and transformability of the P. stutzeri pilA mutant (along with PO4 plating and twitching motility). It is concluded that the type IV pili of the soil bacterium P. stutzeri function in DNA uptake for transformation and that their role in this process is not confined to the species specific pilin. PMID- 10735862 TI - Roles of horizontal gene transfer and gene integration in evolution of 1,3 dichloropropene- and 1,2-dibromoethane-degradative pathways. AB - The haloalkane-degrading bacteria Rhodococcus rhodochrous NCIMB13064, Pseudomonas pavonaceae 170, and Mycobacterium sp. strain GP1 share a highly conserved haloalkane dehalogenase gene (dhaA). Here, we describe the extent of the conserved dhaA segments in these three phylogenetically distinct bacteria and an analysis of their flanking sequences. The dhaA gene of the 1-chlorobutane degrading strain NCIMB13064 was found to reside within a 1-chlorobutane catabolic gene cluster, which also encodes a putative invertase (invA), a regulatory protein (dhaR), an alcohol dehydrogenase (adhA), and an aldehyde dehydrogenase (aldA). The latter two enzymes may catalyze the oxidative conversion of n butanol, the hydrolytic product of 1-chlorobutane, to n-butyric acid, a growth substrate for many bacteria. The activity of the dhaR gene product was analyzed in Pseudomonas sp. strain GJ1, in which it appeared to function as a repressor of dhaA expression. The 1,2-dibromoethane-degrading strain GP1 contained a conserved DNA segment of 2.7 kb, which included dhaR, dhaA, and part of invA. A 12 nucleotide deletion in dhaR led to constitutive expression of dhaA in strain GP1, in contrast to the inducible expression of dhaA in strain NCIMB13064. The 1, 3 dichloropropene-degrading strain 170 possessed a conserved DNA segment of 1.3 kb harboring little more than the coding region of the dhaA gene. In strains 170 and GP1, a putative integrase gene was found next to the conserved dhaA segment, which suggests that integration events were responsible for the acquisition of these DNA segments. The data indicate that horizontal gene transfer and integrase dependent gene acquisition were the key mechanisms for the evolution of catabolic pathways for the man-made chemicals 1, 3-dichloropropene and 1,2-dibromoethane. PMID- 10735863 TI - Evidence for the presence of an F-type ATP synthase involved in sulfate respiration in Desulfovibrio vulgaris. AB - Using a library of genomic DNA from Desulfovibrio vulgaris Miyazaki F, a strict anaerobe, and two synthetic deoxyoligonucleotide probes designed for F-type ATPases, the genes for open reading frames (ORFs) 1 to 5 were cloned and sequenced. The predicted protein sequences of the gene products indicate that they are composed of 172, 488, 294, 471, and 134 amino acids, respectively, and that they share considerable identity at the amino acid level with delta, alpha, gamma, beta, and epsilon subunits found in other F-type ATPases, respectively. Furthermore, a component carrying ATPase activity was partially purified from the cytoplasmic membrane fraction of the D. vulgaris Miyazaki F cells. The N-terminal amino acid sequences of three major polypeptides separated by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis were identical to those of the products predicted by the sequences of ORF-2, ORF-3, and ORF-4, suggesting that an F-type ATPase is functioning in the D. vulgaris Miyazaki F cytoplasmic membrane. The amount of the F-type ATPase produced in the D. vulgaris Miyazaki F cells is similar to that in the Escherichia coli cells cultured aerobically. It indicates that the enzyme works as an ATP synthase in the D. vulgaris Miyazaki F cells in connection with sulfate respiration. PMID- 10735864 TI - A study of AroP-PheP chimeric proteins and identification of a residue involved in tryptophan transport. AB - In vivo recombination has been used to make a series of AroP-PheP chimeric proteins. Analysis of their respective substrate profiles and activities has identified a small region within span III of AroP which can confer on a predominantly PheP protein the ability to transport tryptophan. Site-directed mutagenesis of the AroP-PheP chimera, PheP, and AroP has established that a key residue involved in tryptophan transport is tyrosine at position 103 in AroP. Phenylalanine is the residue at the corresponding position in PheP. The use of PheP-specific antisera has shown that the inability of certain chimeras to transport any of the aromatic amino acids is not a result of instability or a failure to be inserted into the membrane. Site-directed mutagenesis has identified two significant AroP-specific residues, alanine 107 and valine 114, which are the direct cause of loss of transport activity in chimeras such as A152P. These residues replace a glycine and an alanine in PheP and flank a highly conserved glutamate at position 110. Some suggestions are made as to the possible functions of these residues in the tertiary structure of the proteins. PMID- 10735865 TI - Cellular responses to postsegregational killing by restriction-modification genes. AB - Plasmids that carry one of several type II restriction modification gene complexes are known to show increased stability. The underlying mechanism was proposed to be the lethal attack by restriction enzyme at chromosomal recognition sites in cells that had lost the restriction modification gene complex. In order to examine bacterial responses to this postsegregational cell killing, we analyzed the cellular processes following loss of the EcoRI restriction modification gene complex carried by a temperature-sensitive plasmid in an Escherichia coli strain that is wild type with respect to DNA repair. A shift to the nonpermissive temperature blocked plasmid replication, reduced the increase in viable cell counts and resulted in loss of cell viability. Many cells formed long filaments, some of which were multinucleated and others anucleated. In a mutant defective in RecBCD exonuclease/recombinase, these cell death symptoms were more severe and cleaved chromosomes accumulated. Growth inhibition was also more severe in recA, ruvAB, ruvC, recG, and recN mutants. The cells induced the SOS response in a RecBC-dependent manner. These observations strongly suggest that bacterial cells die as a result of chromosome cleavage after loss of a restriction modification gene complex and that the bacterial RecBCD/RecA machinery helps the cells to survive, at least to some extent, by repairing the cleaved chromosomes. These and previous results have led us to hypothesize that the RecBCD/Chi/RecA system serves to destroy restricted "nonself" DNA and repair restricted "self" DNA. PMID- 10735866 TI - Genetic and biochemical characterization of the pathway in Pantoea citrea leading to pink disease of pineapple. AB - Pink disease of pineapple, caused by Pantoea citrea, is characterized by a dark coloration on fruit slices after autoclaving. This coloration is initiated by the oxidation of glucose to gluconate, which is followed by further oxidation of gluconate to as yet unknown chromogenic compounds. To elucidate the biochemical pathway leading to pink disease, we generated six coloration-defective mutants of P. citrea that were still able to oxidize glucose into gluconate. Three mutants were found to be affected in genes involved in the biogenesis of c-type cytochromes, which are known for their role as specific electron acceptors linked to dehydrogenase activities. Three additional mutants were affected in different genes within an operon that probably encodes a 2-ketogluconate dehydrogenase protein. These six mutants were found to be unable to oxidize gluconate or 2 ketogluconate, resulting in an inability to produce the compound 2,5 diketogluconate (2,5-DKG). Thus, the production of 2,5-DKG by P. citrea appears to be responsible for the dark color characteristic of the pink disease of pineapple. PMID- 10735867 TI - A second [2Fe-2S] ferredoxin from Sphingomonas sp. Strain RW1 can function as an electron donor for the dioxin dioxygenase. AB - The first step in the degradation of dibenzofuran and dibenzo-p-dioxin by Sphingomonas sp. strain RW1 is carried out by dioxin dioxygenase (DxnA1A2), a ring-dihydroxylating enzyme. An open reading frame (fdx3) that could potentially specify a new ferredoxin has been identified downstream of dxnA1A2, a two-cistron gene (J. Armengaud, B. Happe, and K. N. Timmis, J. Bacteriol. 180:3954-3966, 1998). In the present study, we report a biochemical analysis of Fdx3 produced in Escherichia coli. This third ferredoxin thus far identified in Sphingomonas sp. strain RW1 contained a putidaredoxin-type [2Fe-2S] cluster which was characterized by UV-visible absorption spectrophotometry and electron paramagnetic resonance spectroscopy. The midpoint redox potential of this ferredoxin (E'(0) = -247 +/- 10 mV versus normal hydrogen electrode at pH 8.0) is similar to that exhibited by Fdx1 (-245 mV), a homologous ferredoxin previously characterized in Sphingomonas sp. strain RW1. In in vitro assays, Fdx3 can be reduced by RedA2 (a reductase similar to class I cytochrome P-450 reductases), previously isolated from Sphingomonas sp. strain RW1. RedA2 exhibits a K(m) value of 3.2 +/- 0.3 microM for Fdx3. In vivo coexpression of fdx3 and redA2 with dxnA1A2 confirmed that Fdx3 can serve as an electron donor for the dioxin dioxygenase. PMID- 10735868 TI - OmpR regulates the stationary-phase acid tolerance response of Salmonella enterica serovar typhimurium. AB - Tolerance to acidic environments is an important property of free-living and pathogenic enteric bacteria. Salmonella enterica serovar Typhimurium possesses two general forms of inducible acid tolerance. One is evident in exponentially growing cells exposed to a sudden acid shock. The other is induced when stationary-phase cells are subjected to a similar shock. These log-phase and stationary-phase acid tolerance responses (ATRs) are distinct in that genes identified as participating in log-phase ATR have little to no effect on the stationary-phase ATR (I. S. Lee, J. L. Slouczewski, and J. W. Foster, J. Bacteriol. 176:1422-1426, 1994). An insertion mutagenesis strategy designed to reveal genes associated with acid-inducible stationary-phase acid tolerance (stationary-phase ATR) yielded two insertions in the response regulator gene ompR. The ompR mutants were defective in stationary-phase ATR but not log-phase ATR. EnvZ, the known cognate sensor kinase, and the porin genes known to be controlled by OmpR, ompC and ompF, were not required for stationary-phase ATR. However, the alternate phosphodonor acetyl phosphate appears to play a crucial role in OmpR-mediated stationary-phase ATR and in the OmpR-dependent acid induction of ompC. This conclusion was based on finding that a mutant form of OmpR, which is active even though it cannot be phosphorylated, was able to suppress the acid-sensitive phenotype of an ack pta mutant lacking acetyl phosphate. The data also revealed that acid shock increases the level of ompR message and protein in stationary-phase cells. Thus, it appears that acid shock induces the production of OmpR, which in its phosphorylated state can trigger expression of genes needed for acid-induced stationary-phase acid tolerance. PMID- 10735869 TI - Domain structure, oligomeric state, and mutational analysis of PpsR, the Rhodobacter sphaeroides repressor of photosystem gene expression. AB - The transcription factor PpsR from the facultative photoheterotroph Rhodobacter sphaeroides is involved in repression of photosystem gene expression under aerobic growth conditions. We have isolated a number of spontaneous mutations as well as constructed directed mutations and deletions in ppsR. Repressor activities and the oligomeric state of the wild-type and mutant proteins were assayed. Our results suggest that the wild-type PpsR exists in cell extracts as a tetramer. Analysis of the PpsR mutants confirmed that the carboxy-terminal region of PpsR (residues 400 to 464) is involved in DNA binding. The central region of the protein (residues 150 to 400) was found to contain two PAS domains (residues 161 to 259 and 279 to 367). PAS domains are ubiquitous protein modules involved in sensory transduction as well as in protein-protein interactions. All spontaneously isolated mutations, which significantly impaired repressor activity and which mapped outside the DNA binding region, were positioned in the PAS domains. None of these, however, affected the overall oligomeric state. This implies that the conformation of the PAS domains within the tetramer is critical for repressor activity. Upstream of the first PAS domain resides a putative glutamine-rich hinge (residues 127 to 136) that connects the first PAS domain to the amino-terminal region (residues 1 to 135). The role of the amino terminus of PpsR is not obvious; however, extended deletions within this region abolish repressor activity, thus suggesting that the amino terminus is essential for structural integrity of the protein. We present a model of the domain architecture of the PpsR protein according to which PpsR is comprised of three regions: the carboxy terminus responsible for DNA binding, the central region primarily involved in protein oligomerization and possibly signal sensing, and the amino terminus of unknown function. This model may prove useful for determining the mode of PpsR action. PMID- 10735870 TI - Complex function for SicA, a Salmonella enterica serovar typhimurium type III secretion-associated chaperone. AB - Salmonella enterica encodes a type III secretion system within a pathogenicity island located at centisome 63 that is essential for virulence. All type III secretion systems require the function of a family of low-molecular-weight proteins that aid the secretion process by acting as partitioning factors and/or secretion pilots. One such protein is SicA, which is encoded immediately upstream of the type III secreted proteins SipB and SipC. We found that the absence of SicA results in the degradation of both SipB and SipC. Interestingly, in the absence of SipC, SipB was not only stable but also secreted at wild-type levels in a sicA mutant background, indicating that SicA is not required for SipB secretion. We also found that SicA is capable of binding both SipB and SipC. These results are consistent with a SicA role as a partitioning factor for SipB and SipC, thereby preventing their premature association and degradation. We also found that introduction of a sicA null mutation results in the lack of expression of SopE, another type III-secreted protein. Such an effect was shown to be transcriptional. Introduction of a loss-of-function sipC mutation into the sicA mutant background rescued sopE expression. These results indicate that the effect of sicA on sopE expression is indirect and most likely exerted through a regulatory factor(s) partitioned by SicA from SipC. These studies therefore describe a surprisingly complex function for the Salmonella enterica type III secretion-associated chaperone SicA. PMID- 10735871 TI - Construction and characterization of a recA mutant of Thiobacillus ferrooxidans by marker exchange mutagenesis. AB - To construct Thiobacillus ferrooxidans mutants by marker exchange mutagenesis, a genetic transfer system is required. The transfer of broad-host-range plasmids belonging to the incompatibility groups IncQ (pKT240 and pJRD215), IncP (pJB3Km1), and IncW (pUFR034) from Escherichia coli to two private T. ferrooxidans strains (BRGM1 and Tf-49) and to two collection strains (ATCC 33020 and ATCC 19859) by conjugation was analyzed. To knock out the T. ferrooxidans recA gene, a mobilizable suicide plasmid carrying the ATCC 33020 recA gene disrupted by a kanamycin resistance gene was transferred from E. coli to T. ferrooxidans ATCC 33020 by conjugation under the best conditions determined. The two kanamycin-resistant clones, which have retained the kanamycin-resistant phenotype after growth for several generations in nonselective medium, were shown to have the kanamycin resistance gene inserted within the recA gene, indicating that the recA::Omega-Km mutated allele was transferred from the suicide plasmid to the chromosome by homologous recombination. These mutants exhibited a slightly reduced growth rate and an increased sensitivity to UV and gamma irradiation compared to the wild-type strain. However, the T. ferrooxidans recA mutants are less sensitive to these physical DNA-damaging agents than the recA mutants described in other bacterial species, suggesting that RecA plays a minor role in DNA repair in T. ferrooxidans. PMID- 10735872 TI - Characterization of a 12-kilodalton rhodanese encoded by glpE of Escherichia coli and its interaction with thioredoxin. AB - Rhodaneses catalyze the transfer of the sulfane sulfur from thiosulfate or thiosulfonates to thiophilic acceptors such as cyanide and dithiols. In this work, we define for the first time the gene, and hence the amino acid sequence, of a 12-kDa rhodanese from Escherichia coli. Well-characterized rhodaneses are comprised of two structurally similar ca. 15-kDa domains. Hence, it is thought that duplication of an ancestral rhodanese gene gave rise to the genes that encode the two-domain rhodaneses. The glpE gene, a member of the sn-glycerol 3 phosphate (glp) regulon of E. coli, encodes the 12-kDa rhodanese. As for other characterized rhodaneses, kinetic analysis revealed that catalysis by purified GlpE occurs by way of an enzyme-sulfur intermediate utilizing a double displacement mechanism requiring an active-site cysteine. The K(m)s for SSO(3)(2 ) and CN(-) were 78 and 17 mM, respectively. The apparent molecular mass of GlpE under nondenaturing conditions was 22.5 kDa, indicating that GlpE functions as a dimer. GlpE exhibited a k(cat) of 230 s(-1). Thioredoxin 1 from E. coli, a small multifunctional dithiol protein, served as a sulfur acceptor substrate for GlpE with an apparent K(m) of 34 microM when thiosulfate was near its K(m), suggesting that thioredoxin 1 or related dithiol proteins could be physiological substrates for sulfurtransferases. The overall degree of amino acid sequence identity between GlpE and the active-site domain of mammalian rhodaneses is limited ( approximately 17%). This work is significant because it begins to reveal the variation in amino acid sequences present in the sulfurtransferases. GlpE is the first among the 41 proteins in COG0607 (rhodanese-related sulfurtransferases) of the database Clusters of Orthologous Groups of proteins (http://www.ncbi.nlm.nih.gov/COG/) for which sulfurtransferase activity has been confirmed. PMID- 10735873 TI - Intrinsic polymerase activities of UmuD'(2)C and MucA'(2)B are responsible for their different mutagenic properties during bypass of a T-T cis-syn cyclobutane dimer. AB - In wild-type Escherichia coli, translesion replication is largely dependent upon the UmuD'(2)C complex (DNA polymerase V [polV]) or its plasmid-encoded homologs, such as MucA'(2)B. Interestingly, both the efficiency of translesion replication of a T-T cis-syn dimer and the spectra of mutations observed are different in Umu and Muc-expressing strains. We have investigated whether the polIII core is responsible for these differences by measuring the frequency of dimer bypass, the error rate of bypass, and the resulting mutation spectrum in mutants carrying a deletion of dnaQ (epsilon subunit) or holE (theta subunit) or carrying the dnaQ allele mutD5, which is deficient in proofreading but is competent in the structural function of epsilon, or the dnaE antimutator allele spq-2. The chromosomal copy of the umuDC operon was deleted in each strain, and the UmuDC, UmuD'C, MucAB, or MucA'B proteins were expressed from a low-copy-number plasmid. With only few exceptions, we found that the characteristically different mutation spectra resulting from Umu- and Muc-mediated bypass are maintained in all of the strains investigated, indicating that differences in the activity or structure of the polIII core are not responsible for the observed phenotype. We also demonstrate that the MucA'(2)B complex is more efficient in promoting translesion replication than the UmuD'(2)C proteins and show that, contrary to expectation, the T-T dimer is bypassed more accurately by MucA'(2)B than by UmuD'(2)C. These results are consistent with the view that in a wild-type cell, the polV-like enzymes are responsible for the spectra of mutations generated during translesion replication and that polIII may simply be required to fix the misincorporations as mutations by completing chromosomal replication. Our observations also show that the mutagenic properties of a lesion can depend strongly on the particular enzyme employed in bypass. PMID- 10735874 TI - Cloning and characterization of the lipooligosaccharide galactosyltransferase II gene of Haemophilus ducreyi. AB - Haemophilus ducreyi is the etiologic agent of chancroid, a genital ulcer disease. The lipooligosaccharide (LOS) is considered to be a major virulence determinant and has been implicated in the adherence of H. ducreyi to keratinocytes. Strain A77, an isolate from the Paris collection, is serum sensitive, poorly adherent to fibroblasts, and deficient in microcolony formation. Structural analysis indicates that the LOS of strain A77 lacks the galactose residue found in the N acetyllactosamine portion of the strain 35000HP LOS as well as the sialic acid substitution. From an H. ducreyi 35000HP genomic DNA library, a clone complementing the defect in A77 was identified by immunologic screening with monoclonal antibody (MAb) 3F11, a MAb which recognizes the N-acetyllactosamine portion of strain 35000HP LOS. The clone contained a 4-kb insert that was sequenced. One open reading frame which encodes a protein with a molecular weight of 33,400 was identified. This protein has homology to glycosyltransferases of Haemophilus influenzae, Haemophilus somnus, Neisseria species, and Pasteurella haemolytica. The putative H. ducreyi glycosyltransferase gene was insertionally inactivated, and an isogenic mutant of strain 35000HP was constructed. The most complex LOS glycoform produced by the mutant has a mobility on sodium dodecyl sulfate-polyacrylamide gel identical to that of the LOS of strain A77 and lacks the 3F11-binding epitope. Structural studies confirm that the most complex glycoform of the LOS isolated from the mutant lacks the galactose residue found in the N-acetyllactosamine portion of the strain 35000HP LOS. Although previously published data suggested that the serum-sensitive phenotype of A77 was due to the LOS mutation, we observed that the complemented A77 strain retained its serum sensitive phenotype and that the galactosyltransferase mutant retained its serum resistant phenotype. Thus, the serum sensitivity of strain A77 cannot be attributed to the galactosyltransferase mutation in strain A77. PMID- 10735875 TI - Heterologous inducible expression of Enterococcus faecalis pCF10 aggregation substance asc10 in Lactococcus lactis and Streptococcus gordonii contributes to cell hydrophobicity and adhesion to fibrin. AB - Aggregation substance proteins encoded by the sex pheromone plasmid family of Enterococcus faecalis have been shown previously to contribute to the formation of a stable mating complex between donor and recipient cells and have been implicated in the virulence of this increasingly important nosocomial pathogen. In an effort to characterize the protein further, prgB, the gene encoding the aggregation substance Asc10 on pCF10, was cloned in a vector containing the nisin inducible nisA promoter and its two-component regulatory system. Expression of aggregation substance after nisin addition to cultures of E. faecalis and the heterologous bacteria Lactococcus lactis and Streptococcus gordonii was demonstrated. Electron microscopy revealed that Asc10 was presented on the cell surfaces of E. faecalis and L. lactis but not on that of S. gordonii. The protein was also found in the cell culture supernatants of all three species. Characterization of Asc10 on the cell surfaces of E. faecalis and L. lactis revealed a significant increase in cell surface hydrophobicity upon expression of the protein. Heterologous expression of Asc10 on L. lactis also allowed the recognition of its binding ligand (EBS) on the enterococcal cell surface, as indicated by increased transfer of a conjugative transposon. We also found that adhesion of Asc10-expressing bacterial cells to fibrin was elevated, consistent with a role for the protein in the pathogenesis of enterococcal endocarditis. The data demonstrate that Asc10 expressed under the control of the nisA promoter in heterologous species will be an useful tool in the detailed characterization of this important enterococcal conjugation protein and virulence factor. PMID- 10735876 TI - A two-component multidrug efflux pump, EbrAB, in Bacillus subtilis. AB - Genes (ebrAB) responsible for ethidium resistance were cloned from chromosomal DNA of Bacillus subtilis ATCC 9372. The recombinant plasmid produced elevated resistance against ethidium bromide, acriflavine, pyronine Y, and safranin O not only in Escherichia coli but also in B. subtilis. It also caused an elevated energy-dependent efflux of ethidium in E. coli. EbrA and EbrB showed high sequence similarity with members of the small multidrug resistance (SMR) family of multidrug efflux pumps. Neither ebrA nor ebrB was sufficient for resistance, but introduction of the two genes carried on different plasmids conferred drug resistance. Thus, both EbrA and EbrB appear to be necessary for activity of the multidrug efflux pump. In known members of the SMR family, only one gene produces drug efflux. Thus, EbrAB is a novel SMR family multidrug efflux pump with two components. PMID- 10735877 TI - A broad-specificity multidrug efflux pump requiring a pair of homologous SMR-type proteins. AB - The Bacillus subtilis genome encodes seven homologues of the small multidrug resistance (SMR) family of drug efflux pumps. Six of these homologues are paired in three distinct operons, and coexpression in Escherichia coli of one such operon, ykkCD, but not expression of either ykkC or ykkD alone, gives rise to a broad specificity, multidrug-resistant phenotype including resistance to cationic, anionic, and neutral drugs. PMID- 10735878 TI - The Yersinia enterocolitica phospholipase gene yplA is part of the flagellar regulon. AB - Yersinia enterocolitica yplA encodes a phospholipase required for virulence. Virulence genes are often regulated in response to environmental signals; therefore, yplA expression was examined using a yplA::lacZY transcriptional fusion. Maximal yplA expression occurred between pH 6.5 and pH 7.5 and was induced in the mid-logarithmic growth phase. Potential Fnr, cyclic AMP (cAMP) cAMP receptor protein (Crp), and sigma(F) regulatory sites were identified in the nucleotide sequence. Reduction of yplA expression by aeration, addition of glucose and sucrose, and application of high temperature and salt is consistent with Fnr-, cAMP-Crp-, and sigma(F)-mediated regulation, respectively. Expression of yplA was reduced in flhDC and fliA null strains, indicating that yplA is part of the flagellar regulon. PMID- 10735879 TI - Characterization of the promoter elements for the staphylococcal enterotoxin D gene. AB - Deletion analysis of the promoter for the Staphylococcus aureus enterotoxin D determinant indicated that a 52-bp sequence, from -34 to +18, was sufficient for sed promoter function and agr regulation. A consensus -10 Pribnow box sequence, a less conserved -35 sequence, and a TG dinucleotide motif were present. Transcribed sequences (+1 to +18) are essential for promoter activity. PMID- 10735880 TI - Role of motility in adherence to and invasion of a fish cell line by Vibrio anguillarum. AB - To understand further the role of the flagellum of Vibrio anguillarum in virulence, invasive and adhesive properties of isogenic motility mutants were analyzed by using a chinook salmon embryo cell line. Adhesion was unaffected but invasion of the cell line was significantly decreased in nonmotile or partially motile mutants, and the chemotactic mutant was hyperinvasive. These results suggest that active motility aids invasion by V. anguillarum, both in vivo and in vitro. PMID- 10735881 TI - A Bacillus subtilis gene of previously unknown function, yhaG, is translationally regulated by tryptophan-activated TRAP and appears to be involved in tryptophan transport. AB - Computer analysis of the Bacillus subtilis genome sequence revealed a gene with no previously attributed function, yhaG, specifying a transcript containing a presumptive binding site for the tryptophan-activated regulatory protein, TRAP. The presumptive TRAP binding site overlaps the yhaG Shine-Dalgarno sequence and translation initiation region. TRAP was shown to regulate expression of yhaG translationally. Production of the yhaG transcript in vivo was found to compete for the binding of TRAP to other known TRAP binding sites. YhaG is likely to be a transmembrane protein involved in tryptophan transport. PMID- 10735882 TI - Multiple alleles of Treponema pallidum repeat gene D in Treponema pallidum isolates. AB - Two new tprD alleles have been identified in Treponema pallidum: tprD2 is found in 7 of 12 T. pallidum subsp. pallidum isolates and 7 of 8 non-pallidum isolates, and tprD3 is found in one T. pallidum subsp. pertenue isolate. Antibodies against TprD2 are found in persons with syphilis, demonstrating that tprD2 is expressed during infection. PMID- 10735883 TI - Genetic requirements of phage lambda red-mediated gene replacement in Escherichia coli K-12. AB - Recombination between short linear double-stranded DNA molecules and Escherichia coli chromosomes bearing the red genes of bacteriophage lambda in place of recBCD was tested in strains bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell; formation of Lac(-) chloramphenicol resistant bacterial progeny was measured. Recombinant formation was found to be reduced in ruvAB and recQ strains. In this genetic background, mutations in recF, recO, and recR had large effects on both cell viability and on recombination. In these cases, deletion of the sulA gene improved viability and strain stability, without improving recombination ability. Expression of a gene(s) from the nin region of phage lambda partially complemented both the viability and recombination defects of the recF, recO, and recR mutants and the recombination defect of ruvC but not of ruvAB or recQ mutants. PMID- 10735884 TI - Identification of SopE2, a Salmonella secreted protein which is highly homologous to SopE and involved in bacterial invasion of epithelial cells. AB - Type III secreted Sop protein effectors are delivered into target eukaryotic cells and elicit cellular responses underlying Salmonella pathogenicity. In this work, we have identified another secreted protein, SopE2, and showed that SopE2 is an important invasion-associated effector. SopE2 is encoded by the sopE2 gene which is present and conserved in pathogenic strains of Salmonella. SopE2 is highly homologous to SopE, a protein encoded by a gene within a temperate bacteriophage and present in only some pathogenic strains. PMID- 10735885 TI - Cloning and sequence analysis of the mercury resistance operon of Streptomyces sp. Strain CHR28 reveals a novel putative second regulatory gene. AB - A DNA library of pRJ28, a large linear plasmid encoding mercury resistance, was constructed, and the mercury resistance genes were cloned. The 5,921-bp sequence was analyzed and showed a high degree of similarity to the Streptomyces lividans 1326 mercury resistance operon. Genes merR, merT, merP, and orfIV were found in a similar order and in a single transcription unit. merA and merB were found to be transcribed in the opposite direction to genes merR, merT, merP, and orfIV, as in S. lividans 1326. A novel putative regulatory gene, orfX, was found 22 bp downstream of merA. orfX encodes a 137-amino acid protein with a potential helix turn-helix motif in the N-terminal domain, characteristic of the MerR family of transcriptional regulators. Transcriptional studies showed that orfX is cotranscribed with merA and merB. It is hypothesized that orfX plays a role in the regulation of the mercury resistance operon, probably by binding at the MerR operator site. PMID- 10735886 TI - Identification of an operon required for ferrichrome iron utilization in Vibrio cholerae. AB - Mutagenesis of Vibrio cholerae with TnphoA, followed by screening for fusions that were activated under low-iron conditions, led to the identification of seven independent fusion strains, each of which was deficient in the ability to utilize ferrichrome as a sole iron source for growth in a plate bioassay and had an insertion in genes encoding products homologous to Escherichia coli FhuA or FhuD. Expression of the gene fusions was independent of IrgB but regulated by Fur. We report here a map of the operon and the predicted amino acid sequence of FhuA, based on the nucleotide sequence. Unlike those of the E. coli fhu operon, the V. cholerae ferrichrome utilization genes are located adjacent and opposite in orientation to a gene encoding an ATP-binding cassette transporter homolog, but this gene, if disrupted, does not affect the utilization of ferrichrome in vitro. PMID- 10735887 TI - Exemestane is superior to megestrol acetate after tamoxifen failure in postmenopausal women with advanced breast cancer: results of a phase III randomized double-blind trial. The Exemestane Study Group. AB - PURPOSE: This phase III, double-blind, randomized, multicenter study evaluated the efficacy, pharmacodynamics, and safety of the oral aromatase inactivator exemestane (EXE) versus megestrol acetate (MA) in postmenopausal women with progressive advanced breast cancer who experienced failure of tamoxifen. PATIENTS AND METHODS: A total of 769 patients were randomized to EXE 25 mg/d (n = 366) or MA (n = 403) 40 mg four times daily. Tumor response, duration of tumor control, tumor-related signs and symptoms (TRSS), quality of life (QOL), survival, and tolerability were evaluated. RESULTS: Overall objective response (OR) rates were higher in patients treated with EXE than in those treated with MA (15.0% v 12.4%); a similar trend was noted in patients with visceral metastases (13.5% v 10.5%). Median survival time was significantly longer with EXE (median not reached) than with MA (123.4 weeks; P =.039), as were the median duration of overall success (OR or stable disease > or = 24 weeks; 60.1 v 49.1 weeks; P =.025), time to tumor progression (20.3 v 16.6 weeks; P =.037), and time to treatment failure (16.3 v 15.7 weeks; P =.042). Compared with MA, there were similar or greater improvements in pain, TRSS, and QOL with EXE. Both drugs were well tolerated. Grade 3 or 4 weight changes were more common with MA (17.1% v 7.6%; P =.001). CONCLUSION: EXE prolongs survival time, time to tumor progression, and time to treatment failure compared with MA and offers a well tolerated treatment option for postmenopausal women with progressive advanced breast cancer who experienced failure of tamoxifen. PMID- 10735888 TI - Burdens and benefits of adjuvant cyclophosphamide, methotrexate, and fluorouracil and tamoxifen for elderly patients with breast cancer: the International Breast Cancer Study Group Trial VII. AB - PURPOSE: Information on the tolerability and efficacy of adjuvant chemoendocrine therapy for older women is limited. We studied these issues using the data collected as part of the International Breast Cancer Study Group Trial VII. PATIENTS AND METHODS: Postmenopausal women with operable, node-positive breast cancer were randomized to receive either tamoxifen alone for 5 years (306 patients) or tamoxifen plus three consecutive cycles of classical cyclophosphamide (100 mg/m(2) orally days 1 to 14), methotrexate (40 mg/m(2) intravenous days 1 and 8), and fluorouracil (600 mg/m(2) intravenous days 1 and 8) every 28 days (CMF; 302 patients). The median follow-up was 8.0 years. RESULTS: Among the 299 patients who received at least one dose of CMF, women 65 years of age or older (n = 76) had higher grades of toxicity compared with women less than 65 years old (n = 223) (P =.004). More women in the older age group compared with the younger women experienced grade 3 toxicity of any type (17% v 7%, respectively), grade 3 hematologic toxicity (9% v 5%, respectively), and grade 3 mucosal toxicity (4% v 1%, respectively). Older patients also received less than their expected CMF dose compared with younger postmenopausal women (P =.0008). The subjective burdens of treatment, however, were similar for younger and older patients based on quality-of-life measures (performance status, coping, physical well-being, mood, and appetite). For older patients, the 5-year disease free survival (DFS) rates were 63% for CMF plus tamoxifen and 61% for tamoxifen alone (hazards ratio [HR], 1.00; 95% confidence interval [CI], 0.65 to 1.52; P =.99). For younger patients, the corresponding 5-year DFS rates were 61% and 53% (HR, 0.70; 95% CI, 0.53 to 0.91; P =.008), but the test for heterogeneity of CMF effect according to age group was not statistically significant. The reduced effectiveness of CMF among older women could not be attributed to dose reductions according to dose received. CONCLUSION: CMF tolerability and effectiveness were both reduced for older patients compared with younger postmenopausal node positive breast cancer patients who received tamoxifen for 5 years. The development and evaluation of less toxic and more effective chemotherapy regimens are required for high-risk elderly patients. PMID- 10735889 TI - Correlation of vascular endothelial growth factor content with recurrences, survival, and first relapse site in primary node-positive breast carcinoma after adjuvant treatment. AB - PURPOSE: To determine the predictive value of vascular endothelial growth factor (VEGF) for relapse-free survival (RFS) and overall survival (OS) in primary node positive breast cancer (NPBC) after adjuvant endocrine treatment or adjuvant chemotherapy. MATERIALS AND METHODS: VEGF was quantitatively measured in tumor cytosols from 362 consecutive patients with primary NPBC using an enzyme immunoassay for human VEGF(165). Adjuvant treatment was given to all patients, either as endocrine therapy (n = 250) or chemotherapy (n = 112). The median follow-up time was 56 months. RESULTS: Univariate analysis showed VEGF to be a significant predictor of RFS (P =.0289) and OS (P =.0004) in the total patient population and in patients who received adjuvant endocrine treatment (RFS, P =.0238; OS, P =.0121). In the group of patients who received adjuvant chemotherapy, no significant difference was seen in RFS, but a difference was seen in OS (P =.0235). Patients with bone recurrences tended to have lower VEGF expression (median, 2.17 pg/microg DNA) than patients with visceral metastasis (4.41 pg/microg), brain metastasis (8.29 pg/microg), or soft tissue recurrences (3.16 pg/microg). Multivariate analysis showed nodal status (P =.0004), estrogen receptor (ER) status (P <.0001), and tumor size (P =.0085) to be independent predictors of RFS. VEGF was found to be an independent predictor of OS (P =.0170; relative risk [RR] = 1.82), as were ER (P <.0001; RR = 5.19) and nodal status (P =.0002; RR = 2.58). For patients receiving adjuvant endocrine treatment, multivariate analysis showed VEGF content to be an independent predictor of OS (P =.0420; RR = 1.90) but not of RFS. CONCLUSION: The results suggest that VEGF(165) content in tumor cytosols is a predictor of RFS and OS in primary NPBC. VEGF content might also predict outcome after adjuvant endocrine treatment, but further studies in a prospective setting with homologous treatments are required. PMID- 10735890 TI - Response of circulating tumor cells to systemic therapy in patients with metastatic breast cancer: comparison of quantitative polymerase chain reaction and immunocytochemical techniques. AB - PURPOSE: We previously developed a quantitative system for the detection of cytokeratin 19 (CK-19) transcripts using reverse transcriptase polymerase chain reaction (PCR) to detect breast carcinoma cells in blood and bone marrow. The aim of this study was to determine the value of this system in monitoring patients with metastatic disease and to compare it with an established immunocytochemical method. PATIENTS AND METHODS: Patients with progressive, locally advanced, and metastatic breast cancer (all stage IV) who were due to start systemic treatment were recruited. Blood samples were analyzed for CK-19 transcripts using quantitative PCR (QPCR) and immunocytochemistry (ICC) throughout their course of treatment. RESULTS: One hundred forty-five blood samples were obtained from 22 patients over 13 months. Seventy-two (49.6%) of these samples were positive by QPCR, and 56 (42%) of 133 were positive by ICC. Of the 133 specimens analyzed by both techniques, 95 (71.4%) had the same results for each, and of the 71 samples that were positive, 40 (56%) were positive by both methods. The relationship between the number of cells detected and the QPCR values was statistically significant (P <.0001). Of the 25 courses of assessable treatment, 17 (68%) of 25 treatment outcomes (either response or disease progression) were reflected by QPCR measurements, and 12 (57%) of 21 were reflected by ICC. During the course of the study, five patients showed a response, and of these, ICC was in agreement in four cases (80%) and QPCR in three cases (60%). Eighteen courses of treatment resulted in progression of the disease; however, only 15 of these were assessable by ICC. ICC was in agreement in eight (53%) of 15 of these cases, and QPCR in 15 (83%) of 18 cases. CONCLUSION: Circulating carcinoma cells are frequently found in patients with metastatic breast cancer. In the majority of patients, cancer cell numbers as evaluated by QPCR or ICC reflected the outcome of systemic treatment. PMID- 10735891 TI - Suramin therapy for patients with symptomatic hormone-refractory prostate cancer: results of a randomized phase III trial comparing suramin plus hydrocortisone to placebo plus hydrocortisone. AB - PURPOSE: Suramin is a novel agent that has demonstrated preliminary evidence of antitumor activity in hormone-refractory prostate cancer (HRPC). A prospective randomized clinical trial was designed to evaluate pain and opioid analgesic intake as surrogates for antitumor response in HRPC patients with significant, opioid analgesic-dependent pain. PATIENTS AND METHODS: A double-blind, placebo controlled trial randomized patients to receive a 78-day, outpatient regimen of either suramin plus hydrocortisone (HC, 40 mg/d) or placebo plus HC. Treatment assignment was unblinded when either disease progression or dose-limiting toxicity occurred; placebo patients were allowed to cross-over to open-label suramin plus HC. In addition to pain and opioid analgesic intake, prostate specific antigen (PSA) response, time to disease progression, quality of life, performance status, and survival were compared. RESULTS: Overall mean reductions in combined pain and opioid analgesic intake were greater for suramin plus HC (rank sum P =.0001). Pain response was achieved in a higher proportion of patients receiving suramin than placebo (43% v 28%; P =.001), and duration of response was longer for suramin responders (median, 240 v 69 days; P =.0027). Time to disease progression was longer (relative risk = 1.5; 95% confidence interval, 1.2 to 1.9) and the proportion of patients with a greater than 50% decline in PSA was higher (33% v 16%; P =.01) in patients who received suramin. Neither quality of life nor performance status was decreased by suramin treatment, and overall survival was similar. Most adverse events were of mild or moderate intensity and were easily managed medically. CONCLUSION: Outpatient treatment with suramin plus HC is well tolerated and provides moderate palliative benefit and delay in disease progression for patients with symptomatic HRPC. PMID- 10735893 TI - Hyperfractionated radiation therapy with or without concurrent low-dose daily cisplatin in locally advanced squamous cell carcinoma of the head and neck: a prospective randomized trial. AB - PURPOSE: To investigate whether the addition of cisplatin (CDDP) to hyperfractionation (Hfx) radiation therapy (RT) offers an advantage over the same Hfx RT given alone in locally advanced (stages III and IV) squamous cell carcinoma of the head and neck. PATIENTS AND METHODS: One hundred thirty patients were randomized to receive either Hfx RT alone to a tumor dose of 77 Gy in 70 fractions in 35 treatment days over 7 weeks (group I, n = 65) or the same Hfx RT and concurrent low-dose (6 mg/m(2)) daily CDDP (group II, n = 65). RESULTS: Hfx RT/chemotherapy offered significantly higher survival rates than Hfx RT alone (68% v 49% at 2 years and 46% v 25% at 5 years; P =.0075). It also offered higher progression-free survival (46% v 25% at 5 years; P =.0068), higher locoregional progression-free survival (LRPFS) (50% v 36% at 5 years; P =.041), and higher distant metastasis-free survival (DMFS) (86% v 57% at 5 years; P =.0013). However, there was no difference between the two treatment groups in the incidence of either acute or late high-grade RT-induced toxicity. Hematologic high-grade toxicity was more frequent in group II patients. CONCLUSION: As compared with Hfx RT alone, Hfx RT and concurrent low-dose daily CDDP offered a survival advantage, as well as improved LRPFS and DMFS. PMID- 10735892 TI - Randomized trial comparing cisplatin, gemcitabine, and vinorelbine with either cisplatin and gemcitabine or cisplatin and vinorelbine in advanced non-small-cell lung cancer: interim analysis of a phase III trial of the Southern Italy Cooperative Oncology Group. AB - PURPOSE: In our previous phase II study, the cisplatin, gemcitabine, and vinorelbine (PGV) regimen produced a median survival time (MST) of approximately 1 year in advanced non-small-cell lung cancer (NSCLC) patients. The present study was aimed at comparing the MST of patients treated with this triplet regimen with the MSTs of patients receiving cisplatin and vinorelbine (PV) or cisplatin and gemcitabine (PG). PATIENTS AND METHODS: From April 1997, patients with locally advanced or metastatic NSCLC, an age of < or = 70 years, and an Eastern Cooperative Oncology Group performance status < or = 1 were randomized to receive one of the following regimens: cisplatin 50 mg/m(2), gemcitabine 1,000 mg/m(2), and vinorelbine 25 mg/m(2) on days 1 and 8 every 3 weeks (arm A); cisplatin 100 mg/m(2) on day 1 and gemcitabine 1,000 mg/m(2) on days 1, 8, and 15 every 4 weeks (arm B); or cisplatin 120 mg/m(2) on days 1 and 29 and vinorelbine 30 mg/m(2)/wk (arm C). According to the two-stage design for phase III trials, an interim analysis was planned when the first 60 patients per arm were assessable for survival. RESULTS: The survival data of 180 NSCLC patients (stage IIIB, 76 patients; stage IV, 104 patients) were analyzed in April 1999. Overall, 128 patients had died (PGV, n = 33; PG, n = 42; and PV, n = 53). The MST of patients in the PGV, PG, and PV arms was 51, 42, and 35 weeks, respectively, and the corresponding 1-year projected survival rates were 45%, 40%, and 34%, respectively. When only patients with stage IV disease were considered, an even stronger difference was seen between PGV (MST = 47 weeks) and both PG (34 weeks) and PV (27 weeks). At multivariate Cox analysis, the estimate hazard of death for patients receiving PGV compared with those receiving PV was 0.35 (95% confidence interval, 0.16 to 0.77; P <.01). The response rates were 47% in the PGV arm, 30% in the PG arm, 25% in the PV arm. Both hematologic and nonhematologic toxicities were not substantially worse in patients who received the PGV regimen. CONCLUSION: The PGV regimen is associated with a substantial survival gain (MST > 3 months longer) when compared with the PV combination. Because this difference in survival met one of the early stopping rules, the accrual in the PV arm has been stopped (null hypothesis rejected). Enrollment still continues in the PGV and PG arm to ascertain whether the PGV regimen can also produce a significantly longer survival than that obtained with the PG regimen. PMID- 10735894 TI - p53 alterations predict tumor response to neoadjuvant chemotherapy in head and neck squamous cell carcinoma: a prospective series. AB - PURPOSE: The tumor suppressor gene p53 plays a crucial role in cell cycle control and apoptosis in response to DNA damages. p53 gene mutations and allelic losses at 17p are one of the most common genetic alterations in primary head and neck squamous cell carcinoma (HNSCC). Alterations of the p53 gene have been shown to contribute to carcinogenesis and drug resistance. PATIENTS AND METHODS: In this prospective series, patients with HNSCC were treated with cisplatin-fluorouracil neoadjuvant chemotherapy. p53 status was characterized in 106 patients with HNSCC (p53 mutations, allelic losses at p53 locus, and plasma anti-p53 antibodies) to determine the existence of a relationship between p53 gene status and response to neoadjuvant chemotherapy. RESULTS: Exons 4 to 9 of the p53 gene were analyzed, and mutations were found in 72 of 106 patients with HNSCC. p53 mutations were associated with loss of heterozygosity at chromosome 17p (P <.001). The prevalence of p53-mutated tumors was higher in the group of patients with nonresponse to neoadjuvant chemotherapy than in the group of responders (81% v 61%, respectively; P <.04). When compiling p53 mutations and anti-p53 antibodies in plasma, the correlation between p53 status and response to chemotherapy was significant (87% v 57%, respectively; P =.003). A multivariate analysis showed that p53 status is an independent predictive factor of response to chemotherapy. CONCLUSION: This prospective study suggests that p53 status may be a useful indicator of response to neoadjuvant chemotherapy in HNSCC. PMID- 10735895 TI - Post hoc economic analysis of temozolomide versus dacarbazine in the treatment of advanced metastatic melanoma. AB - PURPOSE: To determine the potential economic implications resulting from oral temozolomide (TEM) compared with intravenous (IV) dacarbazine (DTIC) for metastatic melanoma. PATIENTS AND METHODS: We performed a cost-effectiveness (CE) analysis using hazard ratios (HRs) from the phase III (Schering I95-018) trial comparing TEM 200 mg/m(2)/d orally for 5 days every 28 days with DTIC 250 mg/m(2)/d IV for 5 days every 21 days. Sensitivity analyses assessed a range of TEM's efficacy and costs, direct nonmedical costs, and the DTIC schedule. RESULTS: The trial found an overall survival trend favoring TEM; median survival times of patients treated with DTIC and TEM were 6.4 and 7.7 months, respectively (HR = 1.18; 95% confidence interval [CI], 0.92 to 1.52; intention to treat, P =.20). The mean increase in survival of TEM over DTIC was 1.1 months. The projected average costs per patient were greater with TEM than DTIC ($6,902 v $3,697, respectively). The incremental CE ratio using TEM was $36,990 per life year or $101 per day of life gained. The CE ratio's 95% CI ranged from -$65,180 (DTIC is more effective) to $18, 670 per year of life gained. The CE ratios decreased 50% if direct nonmedical costs were included and increased 50% if DTIC's efficacy was unchanged if given as a single daily dosage. Sixty percent of simulations found TEM with a CE threshold of less than $50,000 per life-year gained. CONCLUSION: Although the base-case efficacy of TEM compared with DTIC was not statistically significant, its associated incremental CE would be comparable with many interventions. TEM for metastatic melanoma illustrates the tension confronting providers choosing between similar agents that markedly differ in convenience and costs. PMID- 10735896 TI - Health-related quality of life in patients treated with temozolomide versus procarbazine for recurrent glioblastoma multiforme. AB - PURPOSE: To determine whether chemotherapy with temozolomide (TMZ) versus procarbazine (PCB) for recurrent glioblastoma multiforme (GBM) was associated with improvement in health-related quality of life (HRQOL). PATIENTS AND METHODS: HRQOL was assessed at baseline and during treatment using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30 and a Brain Cancer Module (BCM20) in two clinical trials that enrolled a total of 366 patients. Two hundred eighty-eight patients provided HRQOL data that could be used for analysis; 109 patients received TMZ in a phase II study, whereas 89 patients received TMZ and 90 received PCB in a randomized phase III study. Changes from baseline in the scores of seven preselected HRQOL domains (role and social functioning, global quality of life [QOL], visual disorders, motor dysfunction, communication deficit, and drowsiness) were calculated for all groups. Statistical significance, effect sizes, and proportions of patients with improved HRQOL scores (changes of > or = 10 points) were calculated. RESULTS: Before disease progression, patients treated with TMZ were found to have an improvement in most of the preselected HRQOL domain scores compared with their baseline (pretreatment) scores. Those who were progression-free on TMZ at 6 months had improvement in all the preselected HRQOL domains. Conversely, patients treated with PCB reported deterioration in HRQOL that was independent of whether or not the disease had progressed by 6 months. Patients with disease progression, regardless of treatment, experienced a sharp decline in all domains at the time of progression. CONCLUSION: Treatment with TMZ was associated with improvement in HRQOL scores compared with treatment with PCB. The deterioration reported by PCB treated patients was likely because of toxicity. Delaying disease progression by treatment with TMZ is beneficial to the HRQOL status of patients with recurrent GBM. PMID- 10735897 TI - Second malignant neoplasms after treatment for Hodgkin's disease in childhood or adolescence. AB - PURPOSE: To determine the frequency of and risk factors for second malignant neoplasms (SMNs) after treatment for Hodgkin's disease diagnosed in children and adolescents. PATIENTS AND METHODS: One hundred eighty-two consecutive, previously untreated patients with Hodgkin's disease who were younger than 20 years of age at diagnosis and who were referred to Roswell Park Cancer Institute (Buffalo, NY) for treatment between January 1, 1960, and December 31, 1989, were studied. Sex specific standardized incidence ratios (SIRs) were calculated. Kaplan-Meier survival estimates and Cox regression analyses were performed to determine the relationship of several demographic and treatment variables to SMN incidence. RESULTS: Twenty-eight patients developed an SMN at a mean of 14.93 +/- 8.09 years (range, 2.65 to 29.88 years) after diagnosis of Hodgkin's disease. The cumulative percentage of patients who developed an SMN was 26.27 +/- 6.75% at 30 years after diagnosis. The SIR was 9.39 (95% confidence interval [CI], 4.05 to 18.49) for male patients and 10.16 (95% CI, 5.56 to 17.05) for female patients. The most frequent SMNs were thyroid cancer, breast cancer, nonmelanoma skin cancer, non Hodgkin's lymphoma, and acute leukemia. Multivariate analysis of sex, treatment with any alkylating agent, treatment with doxorubicin, splenectomy, and relapse (as a time-dependent covariate) with time to SMN onset gave nonsignificant results. CONCLUSION: Successfully treated children and adolescents with Hodgkin's disease have a substantial risk for the occurrence of subsequent neoplasms. The most frequent SMNs (skin, thyroid, and breast) are readily detected by physical examination and available screening procedures. PMID- 10735898 TI - Localized childhood Hodgkin's disease: response-adapted chemotherapy with etoposide, bleomycin, vinblastine, and prednisone before low-dose radiation therapy-results of the French Society of Pediatric Oncology Study MDH90. AB - PURPOSE: The French Society of Pediatric Oncology MDH82 study demonstrated the effectiveness of 20 Gy irradiation of involved fields after doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) or mechlorethamine, vincristine, procarbazine, and prednisone/ABVD chemotherapy in children with localized Hodgkin's disease (HD). The response to primary chemotherapy was the only predictor of survival. To reduce long-term treatment complications without compromising efficacy, the MDH90 study was based on a new chemotherapy regimen devoid of both alkylating agents and anthracycline, followed by 20 Gy of radiotherapy (RT) for good responders. PATIENTS AND METHODS: From January 1990 to July 1996, 202 children were enrolled from 30 institutions. Good responders to four cycles of vinblastine, bleomycin, etoposide (VP16), and prednisone (VBVP) were given 20 Gy of RT and no further therapy. Poor responders were given vincristine, procarbazine, prednisone, and doxorubicin. After a second evaluation, good responders were given 20 Gy of RT, and poor responders were given 40 Gy of RT. RESULTS: One hundred seventy-one patients (85%) were good responders to VBVP, 27 (15%) were poor responders, and four did not respond. With a median follow-up of 74 months (range, 25 to 117 months), the 5-year overall survival rate (mean +/- SD) is 97.5% +/- 2.1%, and the event-free survival rate (mean +/- SD) is 91.1% +/- 1.8%. Significant predictors of worse event-free survival in multivariate analysis were hemoglobin < 10.5 g/L, "b" biologic class, and nodular sclerosis. CONCLUSION: These results suggest that most children with clinical stage I and II HD can be treated with chemotherapy devoid of alkylating agents and anthracycline, followed by low-dose RT. PMID- 10735899 TI - Six months of maintenance chemotherapy after intensified treatment for acute lymphoblastic leukemia of childhood. AB - PURPOSE: We postulated that intensification of chemotherapy immediately after remission induction might reduce the leukemic cell burden sufficiently to allow an abbreviated period of antimetabolite therapy. PATIENTS AND METHODS: Three hundred forty-seven children (ages 1 to 15 years) with previously untreated acute lymphoblastic leukemia (ALL) were enrolled onto the Tokyo L92-13 study, which excluded patients with mature B-cell ALL and patients less than 1 year old. One hundred twenty-four patients were classified as standard risk, 122 as high risk, and 101 as extremely high risk, according to age, peripheral-blood leukocyte count, selected genetic abnormalities, and immunophenotype. All subjects received four drugs for remission induction, followed by a risk-directed multidrug intensification phase and therapy for presymptomatic leukemia in the CNS. Maintenance chemotherapy with oral mercaptopurine and methotrexate was administered for 6 months, with all treatment stopped by 1 year after diagnosis. RESULTS: The mean (+/- SD) event-free survival (EFS) and overall survival rates for all patients were 59.5% +/- 3.4% and 81.5% +/- 2.2%, respectively, at 5. 5 years after diagnosis. EFS rates by risk category were similar (60. 2% +/- 6.0% for standard risk, 57.7% +/- 5.6% for high risk, and 62. 5% +/- 5.7% for extremely high risk), whereas overall survival rates differed significantly (91.2% +/- 2.7%, 80.0% +/- 4.1%, and 72.1% +/- 4.5%, respectively, P <.0001 by the log-rank test). There were 107 relapses. Eighty-five (79.4%) of these 107 patients achieved second complete remissions, with subsequent EFS rates of 61.5% +/- 7. 9% (standard risk), 42.6% +/- 8.1% (high risk), and 9.6% +/- 6.4% (extremely high risk). Of the five risk factors analyzed, only the response to prednisolone monotherapy among extremely high-risk patients proved important. CONCLUSION: Early treatment intensification did not compensate for a truncated phase of maintenance chemotherapy in children with standard- or high-risk ALL. However, 6 months of antimetabolite treatment seemed adequate for extremely high risk patients who were good responders to prednisolone and received intensified chemotherapy that included high-dose cytarabine early in the clinical course. PMID- 10735900 TI - Use of recombinant human granulocyte colony-stimulating factor to increase chemotherapy dose-intensity: a randomized trial in very high-risk childhood acute lymphoblastic leukemia. AB - PURPOSE: To determine whether the use of a recombinant human granulocyte colony stimulating factor ([G-CSF] lenogastrim) can increase the chemotherapy dose intensity (CDI) delivered during consolidation chemotherapy of childhood acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Sixty-seven children with very high-risk ALL were randomized (slow early response to therapy, 55 patients; translocation t(9;22) or t(4;11), 12 patients). Consolidation consisted of six courses of chemotherapy; the first, third, and fifth courses were a combination of high-dose cytarabine, etoposide, and dexamethasone (R3), whereas the second, fourth, and sixth courses included vincristine, prednisone, cyclophosphamide, doxorubicin, and methotrexate (COPADM). G-CSF was given after each course, and the next scheduled course was started as soon as neutrophil count was > 1 x 10(9)/L and platelet count was > 100 x 10(9)/L. CDI was calculated using the interval from day 1 of the first course to hematologic recovery after the fifth course (100% CDI = 105-day interval). RESULTS: CDI was significantly increased in the G-CSF group compared with the non-G-CSF group (mean +/- 95% confidence interval, 105 +/- 5% v 91 +/- 4%; P <.001). This higher intensity was a result of shorter post-R3 intervals in the G-CSF group, whereas the post-COPADM intervals were not statistically reduced. After the R3 courses, the number of days with fever and intravenous antibiotics and duration of hospitalization were significantly decreased by G-CSF, whereas reductions observed after COPADM were not statistically significant. Duration of granulocytopenia was reduced in the G CSF group, but thrombocytopenia was prolonged, and the number of platelet transfusions was increased. Finally, the 3-year probability of event-free survival was not different between the two groups. CONCLUSION: G-CSF can increase CDI in high-risk childhood ALL. Its effects depend on the chemotherapy regimen given before G-CSF administration. In our study, a higher CDI did not improve disease control. PMID- 10735901 TI - Hypersensitivity or development of antibodies to asparaginase does not impact treatment outcome of childhood acute lymphoblastic leukemia. AB - PURPOSE: Development of antibodies and hypersensitivity to asparaginase are common and may attenuate asparaginase effect. Our aim was to determine the relationship between antiasparaginase antibodies or hypersensitivity reactions and event-free survival (EFS). PATIENTS AND METHODS: One hundred fifty-four children with acute lymphoblastic leukemia received Escherichia coli asparaginase 10,000 IU/m(2) intramuscularly three times weekly for nine doses during multiagent induction and reinduction phases and for seven monthly doses during continuation treatment. Erwinia asparaginase was used in case of clinical hypersensitivity to E coli but not for subclinical development of antibodies. Plasma antiasparaginase antibody concentrations were measured on day 29 of induction in 152 patients. RESULTS: Antibodies were detectable in 54 patients (35.5%), of whom 30 (55.6%) exhibited hypersensitivity to asparaginase. Of the 98 patients who had no detectable antibodies, 18 (18.4%) had allergic reactions. Patients with antibodies were more likely to have a reaction than those without antibodies (P <.001). Among the 50 patients who experienced allergic reactions (including two for whom antibodies were not measured), 36 (72.0%) were subsequently given Erwinia asparaginase; seven (19.4%) reacted to this preparation. EFS did not differ among patients who did and did not have antibodies (P =.54), with 4-year EFS (+/- 1 SE) of 83% +/- 6% and 76% +/- 5%, respectively. Similarly, EFS did not differ among patients who did and did not develop allergic reactions (P =.68), with 4-year estimates of 82% +/- 6% and 78% +/- 5%, respectively. CONCLUSION: In this setting, in which most patients with allergy were switched to another preparation, there was no adverse prognostic impact of clinical or subclinical allergy to asparaginase. PMID- 10735902 TI - Fluorescence in situ hybridization on peripheral-blood specimens is a reliable method to evaluate cytogenetic response in chronic myeloid leukemia. AB - PURPOSE: To evaluate the usefulness of fluorescence in situ hybridization (FISH) on peripheral-blood specimens to evaluate the cytogenetic response to treatment in patients with chronic myeloid leukemia (CML). PATIENTS AND METHODS: In a first attempt, we analyzed 62 bone marrow specimens using interphase FISH and compared the results with those of conventional cytogenetics. In a second step, we analyzed 60 paired sets of bone marrow and peripheral-blood specimens with interphase FISH. RESULTS: The results of interphase FISH agreed with conventional cytogenetics on bone marrow for most patients, and only minor differences were found (r =.98). The comparison of interphase FISH on bone marrow versus peripheral-blood specimens showed a strong correlation between these two specimen sources (r =.97). CONCLUSION: Our results confirmed that FISH is a sensitive technique for the evaluation of response to treatment in patients with CML. Moreover, our study suggests that follow-up of cytogenetic response to therapy can be evaluated on peripheral-blood specimens, thus enabling an easier and more frequent evaluation of patients. The next step will be to evaluate this technique in a large prospective trial to define the prognostic value of complete remissions evaluated by FISH. PMID- 10735903 TI - Longitudinal study of adaptation to the stress of bone marrow transplantation. AB - PURPOSE: This prospective longitudinal study of adaptation to bone marrow transplantation (BMT) addressed three questions: (1) When during BMT do individuals experience the greatest distress? (2) What factors are associated with this distress? (3) Are there variables that could be potential clinical indicators of persons in greatest need of preventive intervention? PATIENTS AND METHODS: One hundred one participants undergoing either an autologous or allogeneic BMT completed questionnaires before hospitalization, before bone marrow infusion, 7 days and 14 days after transplantation, and then 1 month, 3 months, and 12 months after hospitalization. Adaptation was indicated by the degree of emotional distress. Independent variables were personal control, social support from specific sources, cognitive response, self-perception, and coping strategies, controlling for symptomatology. RESULTS: The greatest emotional distress occurred after admission to the hospital and before the bone marrow infusion. Anxiety and depression decreased 1 week after the transplant, although symptomatology increased during this time. The periods of least emotional distress were 3 months and 1 year after transplantation. Factors that accounted for the greatest variance in emotional distress/adaptation were the degree of emotional distress at baseline, personal control, cognitive response, and symptomatology. CONCLUSION: According to this longitudinal study, which includes pretransplant data, data from in-hospital transplantation, and posttransplant data, (1) psychosocial vulnerability of these BMT recipients was greatest during hospitalization before the transplant, (2) perceived personal control may be a potential indicator of vulnerability to secondary psychosocial morbidity, and (3) the demonstrated significance of psychosocial well-being before BMT indicates the importance of obtaining prospective data for both research and clinical purposes. PMID- 10735904 TI - Vinorelbine is an effective and safe drug for AIDS-related Kaposi's sarcoma: results of a phase II study. AB - PURPOSE: To assess the safety and efficacy of vinorelbine in patients with AIDS related Kaposi's sarcoma (KS). PATIENTS AND METHODS: From December 1994 to May 1997, within the Italian Cooperative Group on AIDS and Tumors, we enrolled 36 patients with AIDS-related KS who experienced disease progression after one or more regimens of systemic chemotherapy. Patients were treated with vinorelbine 30 mg/m(2) every 2 weeks by intravenous bolus. RESULTS: Of 35 assessable patients, three (9%) had a clinical complete response and 12 (34%) had a partial remission, for an overall objective response rate of 43% (95% confidence interval, 26% to 61%). For the 15 patients with objective responses, the median duration of response from the beginning of therapy until the development of progression was 176 days, whereas the median progression-free survival and the median survival durations for 35 assessable patients were 151 days and 216 days, respectively. Vinorelbine also induced responses in patients who had become resistant to regimens that included other vinca alkaloids. Overall, vinorelbine was well tolerated. Toxicity, including neurologic toxicity, was mild and reversible. Neutropenia was the most frequent dose-limiting toxicity. CONCLUSION: Vinorelbine is safe and effective in the treatment of patients with advanced KS who have been previously treated with one or more chemotherapy regimens. PMID- 10735905 TI - Primary systemic therapy in operable breast cancer. AB - PURPOSE: Laboratory studies suggest that primary systemic therapy (PST) could improve control of micrometastatic disease and impact on overall survival (OS). This article examines the rationale for and preclinical and clinical data of PST in operable breast cancer and the potential role of intermediate biomarkers as predictive and/or prognostic factors for response and survival. DESIGN AND METHOD: We conducted an extensive literative review (including MEDLINE) on preclinical studies, single-arm feasibility studies, large randomized single- and multi-institutional trials, and laboratory correlate studies of PST in breast cancer. RESULTS: Small trials in locally advanced disease showed high initial rates of response and local control. Six randomized clinical trials (RCTs) of PST for palpable, operable breast cancer have been reported since 1991 (from 204 to 1,523 patients each). These data clearly show a small but significant (less than 10%) absolute increase in the use of breast-conservation treatment (BCT) with similar rates of local control. Although one study showed better disease-free survival (DFS) and another showed better OS, most studies did not show any survival advantage of primary versus adjuvant systemic therapy. Thus far, pathologic complete response seems to be the best predictor of survival, but clinical response assessment correlates poorly with pathologic response. Pilot studies demonstrated feasibility of procuring tissue at diagnosis and after treatment for assays of potential intermediate biomarkers. Initial data suggest a potential correlation between markers of proliferation and apoptosis and in vivo chemotherapy sensitivity. CONCLUSION: Thus far, RCTs of PST versus standard adjuvant therapy have not shown any clear benefit for DFS or OS in early breast cancer. Ongoing trials should determine if specific subsets of patients at risk would benefit from additional systemic therapy and the potential role of intermediate biomarkers in identifying such women. Although PST results in a small increase in the rate of BCT with similar rates of local control, current PST strategies should not replace standard adjuvant approaches. Rather, they represent an acceptable alternative to women with palpable, operable tumors and an excellent arena for clinical trials. PMID- 10735906 TI - Osteoporosis due to cancer treatment: pathogenesis and management. AB - Many therapeutic regimens in cancer treatment carry the risk of causing or favoring the development of osteoporosis. Therapies in which hypogonadism may occur are most relevant in this respect. Prompt hormone replacement therapy is indicated in these patients. In patients in whom this is undesirable because of a hormone-dependent tumor, the risk of osteoporosis should be assessed by means of osteodensitometry, and prophylactic or therapeutic measures should be instituted if necessary. Early intervention improves outcome because osteoporosis therapy is most effective in preventing deterioration of bone mass. There remains much uncertainty in assessing the risk of combination chemotherapy with regard to the development of osteoporosis. Negative effects on the skeleton have, however, been demonstrated for individual drugs, such as methotrexate and ifosfamide. Negative effects of the tumor itself on bone metabolism may aggravate the degree of osteoporosis. Detailed data and long-term experience to assess the risk are urgently needed in this area and constitute an important research topic for the coming years and decades. This review discusses the most prevalent mechanisms of osteoporosis caused by cancer treatment and outlines therapeutic strategies for the prevention and treatment of therapy-induced bone loss. PMID- 10735908 TI - Case 2: myocardial metastases from a carcinoid tumor. PMID- 10735907 TI - Case 1: hypoglycemic syncope due to a peripheral neuroectodermal tumor. PMID- 10735909 TI - Interventional pain therapy for intractable abdominal cancer pain. PMID- 10735911 TI - Preface PMID- 10735910 TI - Predictors of response to chemotherapy. PMID- 10735912 TI - Minimal access esophagectomy: where are we up to? AB - Endoscopic techniques for esophagectomy are disparate. The aim of this article is to describe the main surgical endoscopic techniques applied to esophagectomy and to report their results. In most published series, the benefit in terms of postoperative morbidity cannot be demonstrated. This reflects the fact that postoperative morbidity after esophagectomy is related not only with the type of surgical approach but also with other factors related to the patient's status. Finally, the lack of long-term follow-up in most series does not permit to draw conclusion about the relevance of endoscopic esophagectomy. Contrary to other advanced surgical endoscopic procedures, endoscopic esophagectomy has not yet been convincing. PMID- 10735913 TI - Self-expanding metallic stents in the management of advanced esophageal cancer: a review. AB - Palliation of advanced esophageal cancer continues to be a challenge to clinicians. Self expanding metal stents have been used in the esophagus for palliation of advanced esophageal cancer since 1983. They are relatively easy to insert by practicing endoscopists and have low rates of early complications. Delayed complications necessitating reintervention can arise in as many as a third of patients. The majority of stents are placed under sedation using endoscopy and fluoroscopy. Once deployed, they expand in the esophagus causing pressure necrosis on the wall of the esophagus. Several stents are available on the market with newer designs continuing to emerge. Choice of stent seems random among clinicians. Stents have been used for the management of esophageal obstruction including cervical esophageal obstruction and obstruction at the esophagogastric junction, tracheopulmonary fistulae, and mediastinal esophageal compression. Complications include chest pain, deployment and expansion problems, stent migration, tumor overgrowth and ingrowth, gastroesophageal reflux, and stent-related hemorrhage. Despite their high cost, stenting produce better palliation and some cost savings in comparison to conventional methods of palliation. Combination therapy using stenting followed by chemo/radio therapy may increase quality survival. PMID- 10735914 TI - Endogastric surgery. AB - Endogastric surgery is a branch of minimally invasive surgery that combines flexible endoscopy and laparoscopy. By placing trocars directly into the stomach, quite a number of procedures may be performed. Leiomyomas and other benign gastric tumors are readily removed, and in Asia early gastric cancers are removed with these techniques. Large pancreatic pseudocysts abutting the posterior wall of the stomach may be drained through the stomach using an endogastric approach. Lastly, intragastric bleeding in areas not reachable with a conventional endoscope may be approached with an endogastric approach. Although the indications for these procedures are-in general-rare, they are not difficult to perform, and outcomes have been superb. PMID- 10735915 TI - Laparoscopic gastric resections. AB - The impressive breakthrough in laparoscopic surgery has pushed surgeons to perform gastric resection through such an approach. Laparoscopy reduces the surgical stress and the postoperative pain and has a positive impact on the rehabilitation time, the hospital stay, and return to work and social activities. Laparoscopic partial gastrectomy for benign diseases and for palliation has been accepted as an effective surgical option: they are reproducible operations performed worldwide at a more and more rapid pace. Laparoscopic gastric resections and laparoscopically assisted gastric resections for malignancy deserve a word of caution. Nevertheless, the investigators report their series of laparoscopic subtotal and distal gastrectomies for cancer with medium and long term results comparable with those of open surgery. Furthermore, new and less invasive surgical options have been recently introduced. Full and partial thickness local resections may be accomplished through intragastric procedures, for treatment of small benign tumors and early stage gastric cancer. PMID- 10735916 TI - Laparoscopic gastroplasty (adjustable silicone gastric banding). AB - Until now, for treatment of morbid obesity in the long term, surgery remained as the final option. For 40 years, surgeons looked at the best procedure. Among the restrictive procedures (gastroplasty), the laparoscopic adjustable silicone banding is the least invasive surgical treatment of morbid obesity. Between October 1992 and January 1998, we performed this procedure on 652 patients. Median body mass index was 45 (range, 35-65). Median hospital stay was 3 days (range, 2-10 days). The mean operative time was 80 minutes (range, 40-240 minutes). Four patients (0.6%) presented early complications: bleeding (1 patient), gastric perforation (2 patients), and pneumonia (1 patient). Forty seven (7.2%) patients presented late complications and needed to be reoperated. There is one case of mortality. Loss of mass body weight was 62% in 2 years. According to these results, laparoscopic adjustable silicone gastric banding seems to be a safe and efficient technique. PMID- 10735917 TI - Review article: proteinase-activated receptors - novel signals for gastrointestinal pathophysiology. AB - Proteinase-activated receptors (PARs) have the common property of being activated by the proteolytic cleavage of their extracellular N-terminal domain. The new NH2 terminus acts as a 'tethered ligand' binding and activating the receptor itself. Four members of this family have been cloned, three of which are activated by thrombin (PAR-1, PAR-3 and PAR-4) while the fourth (PAR-2) is activated by trypsin or mast cell tryptase. In physiological or pathophysiological conditions, the gastrointestinal tract is exposed more than other tissues to proteinases (digestive enzymes, proteinases from pathogens or proteinases from inflammatory cells) that can activate PARs. Since PARs are highly expressed throughout the gastrointestinal tract, the study of the role of PARs in these tissues appears to be particularly important. It has already been shown that PAR-2 activation induces calcium mobilization and eicosanoid production in enterocytes as well as changes in ion transport in jejunal tissue segments. PAR-2 activation also causes calcium mobilization and stimulates amylase release from pancreatic acini. Moreover, both PAR-1 and PAR-2 activation can alter the gastrointestinal motility. In inflammatory or allergic conditions, the proteinases that constitute the major agonists for PARs (thrombin, trypsin and mast cell tryptase) are usually released. The activation of PARs by these proteinases might contribute to the gastrointestinal disorders associated with these pathologies. A complete understanding of the role of PARs in the gastrointestinal tract will require the development of selective receptor antagonists that are not yet available. Nonetheless, the use of PAR agonists has already highlighted new potential functions for proteinases in the gastrointestinal tract, thus the control of PAR activation might represent a promising therapeutic target. PMID- 10735918 TI - Personal review: alarmism or legitimate concerns about long-term suppression of gastric acid secretion? AB - This article responds to controversial issues about the long-term use of acid suppression raised in a recent article in this journal by Waldum & Brenna. Although rebound acid secretion occurs following proton pump inhibitor therapy, the clinical significance of this is unclear, but the proposal that this is a major driver of acid-related diseases is considered implausible. The polypoid deformity of the gastric corpus that can occur with long-term proton pump inhibitor therapy is not neoplastic, and therefore has no bearing on other issues raised about proton pump inhibitor therapy and gastric malignancy. Current data in humans suggest that the magnitude of serum gastrin elevation from proton pump inhibitor treatment of up to 10 years, and any theoretical risks from this, have been overstated by Waldum & Brenna. Pernicious anaemia is a model of very doubtful validity for the risks of proton pump inhibitor therapy on several grounds. The proposal that diffuse gastric carcinoma arises from acid suppression induced stimulation of enterochromaffin-like cells is challenged vigorously, because this is based on an implausible and substantially criticized interpretation of histopathology. It is agreed that it is appropriate to be cautious about the safety of long-term acid suppression, because no data are available for lifelong treatment in humans. Such caution should be tempered by a critical assessment of the benefits of this treatment in relation to any possible risks. The substantial data that now exist from long-term treatment of humans with proton pump inhibitors has not thus far revealed any definite risks. The risk of death from anti-reflux surgery, although small, would seem to far exceed any possible risks associated with long-term proton pump inhibitor use. Available data suggest that denial of the benefits of effective acid suppressant therapy to patients with clear-cut troublesome acid related disorders is an overreaction to concerns about the biological effects of inhibiting acid secretion with proton pump inhibitors. PMID- 10735919 TI - Predictive factors of outcome of intensive intravenous treatment for attacks of ulcerative colitis. AB - BACKGROUND: Intensive intravenous treatment remains the first line therapy of severe, uncomplicated attacks of ulcerative colitis. AIM: To predict the failure of intensive intravenous treatment by combining clinical and laboratory parameters with endoscopy findings. METHODS: Retrospective study conducted in a tertiary care referral centre. Failure of intensive intravenous treatment was defined as colectomy before day 30, intravenous cyclosporin, or death. Predictive factors of outcome were assessed using univariate and multivariate prognostic analysis. RESULTS: Between January 1990 and May 1997, 85 consecutive patients were treated with intensive intravenous treatment for non-response to oral corticosteroids (n=59) and/or severe attack of ulcerative colitis (n=26). There were 41 successes and 44 failures (including 1 death, 13 cyclosporin and 30 colectomies before day 30). Multivariate prognostic analysis found that the presence of Truelove and Witts' criteria (P=0.018), an attack that had lasted more than 6 weeks (P=0.001), and severe endoscopic lesions (P=0.007) were associated with an increased risk of failure. Patients with severe endoscopic lesions and Truelove and Witts' criteria, or an attack of more than 6 weeks had a failure rate of 85-86%. CONCLUSION: Clinical, laboratory and endoscopic findings can predict the risk of failure of intensive intravenous treatment. A prospective study is required to confirm these results. PMID- 10735920 TI - Mucosal healing and a fall in mucosal pro-inflammatory cytokine mRNA induced by a specific oral polymeric diet in paediatric Crohn's disease. AB - BACKGROUND: Although enteral nutrition is a recognized form of treatment for intestinal Crohn's disease, there are persisting problems with feed palatability and only limited data as to its mode of action. AIM: To assess the effects of a specific oral polymeric diet (CT3211; Nestle, Vevey, Switzerland), which is rich in transforming growth factor beta2, on the mucosal inflammatory process. METHODS: Twenty-nine consecutive children with active intestinal Crohn's disease were treated with CT3211 as the sole source of nutrition for 8 weeks. Patients were assessed clinically, and endoscopically, whilst cytokine mRNA was measured in mucosal biopsies before and after treatment by quantitative reverse transcriptase polymerase chain reaction. RESULTS: After 8 weeks 79% of children were in complete clinical remission. Macroscopic and histological healing in the terminal ileum and colon was associated with a decline in ileal and colonic interleukin-1beta mRNA (pre-treatment to post-treatment ratio 0.008 and 0.06: P < 0.001, P = 0.006). In the ileum there was also a fall in interferon gamma mRNA (ratio 0.15, P < 0.001) with a rise in transforming growth factor beta1 mRNA (ratio 10, P = 0.04), whilst in the colon interleukin-8 mRNA fell with treatment (ratio 0.06, P < 0.05). CONCLUSIONS: The clinical response to oral polymeric diet CT3211 is associated with mucosal healing and a down regulation of mucosal pro inflammatory cytokine mRNA in both the terminal ileum and colon. In the ileum there was also an increase in transforming growth factor beta1 mRNA. PMID- 10735921 TI - Effect of gastric acid suppression on 13C-urea breath test: comparison of ranitidine with omeprazole. AB - BACKGROUND: The assessment of the effect of H2 antagonists on the results of the urea breath test has produced controversial results. AIM: To assess whether standard doses of both omeprazole and H2 blockers can adversely influence the accuracy of the urea breath test. METHODS: Sixty dyspeptic patients with ascertained Helicobacter pylori infection were recruited for this prospective, open study. They were randomized to receive either omeprazole 20 mg at 08:00 hours (n = 30) or ranitidine 300 mg at 22:00 hours (n = 30) for 14 days. The urea breath test was performed at baseline, on day 14, while patients were still taking the antisecretory drugs, and on day 21, 1 week after their cessation. Duplicate breath samples were collected after ingestion of 75 mg 13C-urea + citric acid. A delta value > 5 per thousand was considered positive. RESULTS: On day 14 the median delta values had declined, but not significantly (P = 0. 07) compared to baseline (13.79 vs. 22.39) with omeprazole, while they had increased (P = 0.27) with ranitidine (27.21 vs. 19.46). On the same day there were five out of 30 (17%) and five out of 28 (18%) false-negative results in the omeprazole and ranitidine groups, respectively. All these cases became positive again on day 21. However, in eight cases treated with omeprazole and 13 treated with ranitidine, there was an increase of 14-day delta values compared to baseline. CONCLUSIONS: Our study shows that both omeprazole and ranitidine at standard doses are able to negatively affect the results of the urea breath test. Their adverse effect resolves within 7 days of drug cessation and therefore the withdrawal of these drugs 7 days before testing seems to be sufficient to avoid false-negative results. The surprising finding that both antisecretory drugs reduce delta values in one group and increase them in another group of patients deserves further study. PMID- 10735922 TI - Furazolidone versus metronidazole in quadruple therapy for eradication of Helicobacter pylori in duodenal ulcer disease. AB - OBJECTIVE: Furazolidone, an old but cheap antibiotic, was shown to be a good alternative to metronidazole in triple therapy for Helicobacter pylori eradication in areas where metronidazole resistant bacteria are common, but randomized studies are lacking. AIM: A randomized controlled trial to determine the efficacy and safety of furazolidone compared to metronidazole in classic quadruple therapy for eradication of H. pylori infection in duodenal ulcer patients. METHODS: Patients with endoscopically proven duodenal ulcer and positive urease test were randomized to receive ranitidine 300 mg, amoxycillin 1000 mg and bismuth subcitrate 240 mg b.d, with either furazolidone 200 mg b.d (RABF), or metronidazole 500 mg b.d. (RABM) for 2 weeks. Compliance and side effects were monitored and recorded by table diary. H. pylori eradication was assessed at least 4 weeks after the completion of therapy with 14C-urea breath test. RESULTS: A total of 106 patients were enrolled and 101 (59 male, 42 female, mean age=40 +/- 11 years) completed the study. Endoscopic findings and demographic data were comparable in both groups. Intention-to-treat eradication rates were 75% and 55% (P=0.03) and per protocol eradication rates were 82 and 56% (P=0. 006) in the RABF and RABM groups, respectively. Side-effects were reported by 13 patients (27%) in the RABF group (one stopped treatment) compared to five patients (10%) in the RABM group (P=0. 04). CONCLUSION: Quadruple therapy containing furazolidone, instead of metronidazole, results in a significantly higher H. pylori eradication rate in Iranian duodenal ulcer patients. PMID- 10735923 TI - Effect of ornidazole and clarithromycin resistance on eradication of Helicobacter pylori in peptic ulcer disease. AB - BACKGROUND: Clarithromycin and nitroimidazoles such as metronidazole and ornidazole are among the most frequently used antibiotics for curing Helicobacter pylori infection. However, controversial data exist on whether their in vitro resistance has a negative impact on treatment outcome. METHODS: Patients with H. pylori positive active peptic ulcer disease were randomly assigned to receive lansoprazole 30 mg o.d., amoxycillin 1 g b.d. and ornidazole 500 mg b.d. (LAO) or lansoprazole 30 mg o.d., amoxycillin 1 g b.d. and clarithromycin 500 mg b.d. (LAC) for 2 weeks. Pre-treatment resistance to ornidazole and clarithromycin was assessed by Epsilometer (E-) test. Four weeks after completion of treatment, patients underwent a 13C urea breath test to assess H. pylori status. RESULTS: Data from 80 patients with active peptic ulcer disease and positive H. pylori status were analysed. The prevalence of primary drug resistance was 25% for metronidazole and 7.5% for clarithromycin. In patients treated with LAO, effective treatment was achieved in 87% of metronidazole-susceptible, but only 30% of metronidazole-resistant strains (P < 0.01). In the LAC group, therapy was successful in 81% of clarithromycin-susceptible strains, whereas treatment failed in all patients with primary clarithromycin resistance (n = 3). CONCLUSION: Resistance against nitroimidazoles significantly affects treatment outcome in H. pylori eradication therapy. PMID- 10735924 TI - Rifabutin-based 'rescue therapy' for Helicobacter pylori infected patients after failure of standard regimens. AB - BACKGROUND: The ideal treatment for patients who have failed eradication of Helicobacter pylori infection after standard proton pump inhibitor-based triple therapies has still to be determined. Although either a second course of triple therapy or a quadruple therapy (proton pump inhibitor plus bismuth-based triple therapy) has been proposed, the efficacy of these second-line therapies is relatively unknown. Therefore, alternative strategies are needed. AIM: To assess the efficacy and tolerability of rifabutin, a derivative of rifamycin-S, in patients who were still H. pylori infected after two or more courses of 1-week triple therapies. METHODS: Patients were given a 1-week regimen of pantoprazole 40 mg b.d. + amoxycillin 1 g b.d. + rifabutin 300 mg daily. Side-effects and compliance were determined at the end of therapy. Eradication rate was assessed with a 13C-urea breath test performed at 4 and 12 weeks after treatment. RESULTS: Forty-one patients (mean age 47 +/- 15 years) were studied. All patients took medications according to the proposed schedule. Side-effects were infrequent and mild. The eradication rates were 71% (95% CI: 57-85%) on intention-to-treat analysis and 74% (95% CI: 61-88%) on per protocol analysis. CONCLUSIONS: Rifabutin, in combination with pantoprazole and amoxycillin, is an effective and well tolerated regimen in patients who failed standard eradication treatments. PMID- 10735925 TI - Cure of Helicobacter pylori infection does not improve symptoms in non-ulcer dyspepsia patients-a double-blind placebo-controlled study. AB - BACKGROUND: It remains controversial whether the cure of H. pylori infection improves NUD symptoms. AIM: To conduct a double-blind placebo-controlled single centre study with concealed allocation to investigate this question. PATIENTS AND METHODS: Ninety NUD patients with H. pylori infection were randomly assigned to either the treatment group (50 patients) or placebo group (40 patients). The treatment group received omeprazole, amoxycillin, clarithromycin and the placebo group received omeprazole and placebos for 7 days. Symptoms were assessed every week for up to 12 weeks after completion of medication by a symptom questionnaire. Alteration of histological parameters for gastritis was also evaluated. RESULTS: The infection was cured in 41 out of 48 patients in the treatment group and none in the placebo group. There was no significant difference in the mean symptom scores at any assessment point up to 12 weeks between the treatment and placebo groups. Regarding histological parameters, activity and inflammation, not atrophy or intestinal metaplasia, were significantly improved in the treatment group. CONCLUSION: Although histological parameters were significantly improved in the treatment group, there was no significant improvement in symptoms of NUD in the treatment group compared to placebo. PMID- 10735927 TI - An evaluation of whole blood testing for Helicobacter pylori infection in the Chinese population. AB - BACKGROUND: Near patient tests for Helicobacter pylori were developed to assist in the management of dyspepsia patients in general practice. Most studies were performed in western populations. AIM: To evaluate the rapid whole blood test (Flexpack HP) for H. pylori in the Chinese population. METHODS: Consecutive dyspeptic patients referred for upper endoscopy were recruited. During upper endoscopy, biopsies were taken from the antrum and corpus for rapid urease test (CLO test) and histological examination. After endoscopy, the whole blood test (FlexPack HP) was performed according to the manufacturer's instruction. Patients then received a 13C-urea breath test. Results of the whole blood test were compared with the gold standard (CLO test, histology and 13C-urea breath test). RESULTS: A total of 294 consecutive patients gave a valid Flexpack HP result for interpretation. The mean age of patients was 47.7 (range 15-85) years. Analysis showed a sensitivity, specificity, positive predictive value, negative predictive value and accuracy of 58%, 92%, 91%, 63% and 73% respectively. CONCLUSION: The FlexPack HP whole blood test showed good specificity but lacked sensitivity. It is not sensitive enough to be used in a general practice setting for the test-and treat approach in the Chinese population. PMID- 10735926 TI - Seven-day triple therapy with ranitidine bismuth citrate or omeprazole and two antibiotics for eradication of Helicobacter pylori in duodenal ulcer: a multicentre, randomized, single-blind study. AB - AIM: To investigate the efficacy of a 1-week triple therapy with amoxycillin, clarithromycin, and omeprazole or ranitidine bismuth citrate (RBC) in curing Helicobacter pylori infection and healing duodenal ulcers. METHODS: One hundred and ninety-two consecutive out-patients with duodenal ulcer, in whom H. pylori infection was confirmed by histology and a urease biopsy test, were randomly assigned to a 1-week treatment with either 400 mg b.d. ranitidine bismuth citrate (RAC group) or 20 mg omeprazole b.d. (OAC group) in combination with 1 g amoxycillin b.d. and 500 mg clarithromycin b.d. RESULTS: Eradication of H. pylori was successful in 77% (per protocol) and 61% (intention-to-treat) of the patients in the RAC group and in 79% (per protocol) and 70% (intention-to-treat) of those in the OAC group. The difference was not significant. Per protocol analysis showed ulcers were healed in 97% of patients in the RAC group and 96% in the OAC group. Adverse effects were seen in four patients in each group: they caused discontinuation of the therapy in one patient of the OAC group. CONCLUSIONS: Eradication rates obtained in this study were lower than those expected on the basis of previously reported studies. The two 1-week treatment regimens were equally effective in healing H. pylori associated duodenal ulcer disease. PMID- 10735928 TI - Gastric myoelectric activity in older adults treated with cisapride for gastro oesophageal reflux disease. AB - BACKGROUND: The incidence of both gastro-oesophageal reflux disease (GERD) and upper gastrointestinal motility disorders appears to increase with age. However, there is a dearth of data concerning the utility of a prokinetic agent such as cisapride in the treatment of older adults with symptomatic GERD. AIM: To investigate the incidence of electrogastrographic abnormalities in older adults with and without GERD symptoms, as well as the effect of cisapride therapy on symptoms of GERD and electrogastrographic responses. METHODS: We report on 18 older adults with symptomatic GERD and 10 older adult controls (mean ages 71 and 75 years, respectively). Subjects underwent electrogastrographic evaluation pre- and postprandially under baseline conditions and after 1 month of treatment with 10 mg q.d.s. of cisapride. RESULTS: Heartburn frequency and postprandial fullness were both significantly (P < 0. 05) reduced after cisapride treatment. Acid contact time was not significantly changed. The percentage of 2-4 cpm activity in the electrogastrographic analysis was significantly (P < 0.05) increased with cisapride treatment, and the instability coefficient was significantly (P < 0.05) diminished with cisapride treatment. CONCLUSIONS: Enhanced gastric functioning and reduction in heartburn suggest that cisapride is efficacious in the treatment of older adults with symptomatic GERD, and that gastric dysrhythmias and postprandial fullness are resolved with cisapride treatment. PMID- 10735929 TI - Lack of a pharmacokinetic interaction between lansoprazole or pantoprazole and theophylline. AB - AIM: To study the potential pharmacokinetic interaction between lansoprazole or pantoprazole and theophylline at steady state. METHODS: Theophylline 200 mg extended-release formulation was administered twice daily on days 1-11 to 30 healthy, non-smoking males. On days 5-11, 15 subjects received concomitant lansoprazole 30 mg once daily (o.d.) and 15 subjects received concomitant pantoprazole 40 mg o.d. RESULTS: No significant changes in the steady-state theophylline maximum plasma concentration (Cmax), time to Cmax (Tmax), minimum plasma concentration (Cmin), area under the plasma concentration-time curve over the 12-h dosing interval (AUC0-12), or apparent total oral clearance (CL/F) were observed within the two treatment groups when theophylline was administered alone or in combination with lansoprazole or pantoprazole. In addition, no significant differences in the changes of steady-state theophylline pharmacokinetics from day 4 to day 11 were noted between the two treatment groups. Treatment with theophylline in combination with either lansoprazole or pantoprazole was well tolerated. All adverse events were transient and rated mild to moderate in severity. CONCLUSION: Co-administration of either lansoprazole or pantoprazole in healthy subjects does not significantly affect the steady-state pharmacokinetics of theophylline at the therapeutic doses tested. PMID- 10735930 TI - Plasma antioxidant levels in chronic cholestatic liver diseases. AB - BACKGROUND: [corrected] A predictable consequence of cholestasis is malabsorption of fat-soluble factors, (vitamins A, D, E, K) and other free radical scavengers, such as carotenoids. It has been suggested that oxygen-derived free radicals may be involved in the pathogenesis of chronic liver damage. AIMS: (i) To evaluate retinol, alpha-tocopherol and carotenoid plasma levels in two groups of patients with chronic cholestatic liver disease (primary biliary cirrhosis and primary sclerosing cholangitis); (ii) to compare the respective plasma levels with those of the general population; (iii) to correlate the plasma levels with disease severity. METHODS: A total of 105 patients with chronic cholestasis were included in the study: 86 with primary biliary cirrhosis (81 female, five male, mean age 55.5 +/- 11 years), 19 with primary sclerosing cholangitis (seven female, 12 male, mean age 35 +/- 11 years; six patients had associated inflammatory bowel disease); 105 sex- and age-matched subjects from the general population in the same geographical area (88 female, 17 male, mean age 51.3.5 +/- 10 years) served as controls. Carotenoids (lutein zeaxanthin, lycopene, beta-carotene, alpha carotene, beta-cryptoxanthin), retinol and alpha-tocopherol were assayed by high pressure liquid chromatography. A food frequency questionnaire was administered to each subject to evaluate the quality and the quantity of dietary compounds. Data were processed by analysis of variance and linear regression analysis, as appropriate. RESULTS: Both primary biliary cirrhosis and primary sclerosing cholangitis patients had significantly lower levels of retinol, alpha-tocopherol, total carotenoids, lutein, zeaxanthin, lycopene, alpha- and beta-carotene than controls (P < 0.0001). Among the cholestatic patients, no significant difference in the concentration of antioxidants was observed between primary biliary cirrhosis and primary sclerosing cholangitis subjects. Anti-oxidant plasma levels were not affected by the severity of the histological stage in primary biliary cirrhosis, but a negative correlation was found between total carotenoids and both alkaline phosphatase (ALP) and gammaglutamyl transpeptidase (GGT) (P < 0.013 and P < 0.018, respectively). Within the primary sclerosing cholangitis group, no correlation was found between total carotenoids and cholestatic enzymes. Nutritional intake in cholestatic patients was comparable to controls, including fruit and vegetable intake. CONCLUSIONS: Although no clinical sign of deficiency is evident, plasma levels of antioxidants are low in cholestatic patients even in early stages of the disease. This is probably due to malabsorption of fat-soluble vitamins, as well as other mechanisms of hepatic release, suggesting the need for dietary supplementation. PMID- 10735931 TI - Fasting induces impairment of gastric mucosal integrity in non-insulin-dependent diabetic (db/db) mice. AB - BACKGROUND: Although diabetic patients often have gastrointestinal complications, the gastric mucosal function in diabetes has not been well documented. AIM: To investigate the effect of fasting on the gastric mucosa in C57BL/KsJ-db +/+ db (db/db) mice, genetically non-insulin-dependent diabetic animals. METHODS: Blood glucose levels, gastric mucosal morphology, and the amount of gastric mucin were examined before and after 18 h of fasting with free access to water in db/db mice and their non-diabetic littermates (db/m). RESULTS: Although 18 h of fasting reduced the blood glucose levels of both db/db and db/m mice, fasting decreased the amount of gastric adherent mucin and caused haemorrhagic gastric lesions only in db/db mice. After fasting, oral administration of ethanol induced much more severe gastric damage in db/db than in db/m mice. The above fasting-induced gastric damage such as haemorrhagic lesions, loss of the mucin, and the increased sensitivity to ethanol worsened as the duration of diabetes became longer. Glucose ingestion in drinking water during the fasting counteracted the fall in blood glucose and prevented the decrease in the amount of gastric mucin and the formation of gastric mucosal lesions in db/db mice. CONCLUSION: These findings indicate that fasting-induced glucose deficit causes gastric mucosal lesions and increases the susceptibility of gastric mucosa to noxious agents owing to the loss of mucus glycoprotein in db/db mice. Prolonged diabetes is likely to augment the severity of fasting-induced impairment of the gastric mucosal function. PMID- 10735932 TI - Nitric oxide in the lower urinary tract: physiological and pathological implications. PMID- 10735933 TI - The assessment of female pelvic floor dysfunction. PMID- 10735935 TI - Chemotherapy and management of bladder tumours. PMID- 10735934 TI - Hereditary prostate cancer. PMID- 10735937 TI - The assessment of female pelvic floor dysfunction PMID- 10735936 TI - Nitric oxide in the lower urinary tract: physiology and pathological implications PMID- 10735938 TI - Hereditary prostate cancer PMID- 10735939 TI - Chemotherapy, management of bladder tumours PMID- 10735940 TI - Correct answers to multiple choice questions appearing in the european urology update series 1999 PMID- 10735941 TI - Non-melanoma skin cancer and the 'new National Health Service': implications for U.K. dermatology? PMID- 10735942 TI - Measuring the activity of disease in cutaneous lupus erythematosus. PMID- 10735943 TI - Cytokines and chemokines in the initiation and regulation of epidermal Langerhans cell mobilization. AB - Langerhans cells (LC) are members of the wider family of dendritic cells. LC reside in the epidermis where they serve as sentinels of the immune system, their responsibilities being to sample the external environment for changes and challenges and to deliver information (antigen) to responsive T lymphocytes within skin draining lymph nodes. The ability of LC to migrate from the epidermis to regional lymph nodes is therefore of pivotal importance to the induction of cutaneous immune responses. The journey that LC have to make from the skin has a number of requirements. Initially it is necessary that LC disassociate themselves from surrounding keratinocytes and are liberated from other influences that encourage their retention in the epidermis. Subsequently, migrating LC must successfully traverse the basement membrane of the dermal-epidermal junction and make their way, via afferent lymphatics, to draining lymph nodes. Effective entry into lymph nodes is necessary, as is correct positioning of cells within the paracortex. There is increasing evidence that both cytokines and chemokines, and their interaction with appropriate receptors expressed by LC, orchestrate the mobilization and movement of these cells. We here consider the parts played by these molecules, and how collectively they induce and direct LC migration. PMID- 10735944 TI - Laser resurfacing of the skin for the improvement of facial acne scarring: a systematic review of the evidence. AB - This review presents and evaluates the evidence of the effectiveness of laser resurfacing for facial acne scars. Primary studies of all types of design in any language were identified from MEDLINE, EMBASE, the Cochrane database, Science Citation Index and various internet sites. Studies were accepted if they included patients treated by any laser for atrophic or ice-pick acne scars. The quality of the studies was assessed and data extracted by two independent researchers. There were no controlled trials but 14 case series were found which reported the effects of either the carbon dioxide or erbium:YAG laser. All of the studies were of poor quality. The types and severity of scarring were poorly described and there was no standard scale used to measure scar improvement. There was no reliable or validated measure of patient satisfaction; most improvement was based on visual clinical judgement, in many cases without blinded assessment. The inaccurate use of ordinal scales meant that any improvement was impossible to quantify with any validity, although the evidence suggested that laser treatment had some efficacy (a range in individual patients of 25-90% for both the carbon dioxide laser and the erbium:YAG laser). Changes in pigmentation as a side-effect were common (in up to 44% of patients), although lasting only a few weeks. Laser resurfacing technology is increasingly used in clinical practice to treat acne scars. Despite the poor quality evidence, it is plausible that there is some improvement of acne scarring; there is insufficient information, however, for patients to make informed decisions on whether to opt for treatment and there is not enough evidence to compare the two types of laser. There is a particular lack of information about the psychological effects of acne scar improvement. Good quality randomized controlled trials are needed with standardized scarring scales and validated patient outcome measures in order to assess the effectiveness of laser resurfacing in this group of patients. PMID- 10735945 TI - Identification of pancreatic type I secreted phospholipase A2 in human epidermis and its determination by tape stripping. AB - Phospholipases A2 (PLA2) catalyse the release of fatty acids from the sn-2 position of phospholipids and have been suggested to play a key part in permeability barrier homeostasis. Using a sensitive and versatile fluorometric method, significant PLA2 activity has been detected in both human skin homogenates and tape strippings of stratum corneum. Based on various properties (resistance to heat and sulphuric acid treatment, neutral optimal pH, absolute requirement for millimolar calcium concentrations, inhibition by dithiothreitol and p-bromophenacyl bromide, and resistance to a trifluoromethyl ketone derivative of arachidonic acid, AACOCF3, a specific inhibitor of cytosolic PLA2), this enzyme was characterized as a secretory PLA2 (sPLA2). Immunohistochemistry revealed strong labelling of type I pancreatic sPLA2 at the stratum corneum stratum granulosum junction, type II sPLA2 being undetectable. An increase in PLA2 activity in tape-stripped material from the deepest level of the stratum corneum was correlated with partial morphological disappearance of type I sPLA2 immunolabelling. Our data thus provide the first convincing evidence that pancreatic sPLA2 is significantly expressed in human epidermis, where it might participate in the accumulation of free fatty acids contributing to the permeability barrier. In addition, our method for determining PLA2 activity in easily available tape strippings should allow further clinical studies aimed to explore possible PLA2 abnormalities in various dermatoses. PMID- 10735946 TI - Expression of the inducible isoform of nitric oxide synthase in pigment cell lesions of the skin. AB - Nitric oxide (NO) is a small molecule produced during the conversion of L arginine to L-citrulline by NO synthase (NOS). Several isoforms of NOS exist, of which the Ca2+-independent, inducible NOS (iNOS or NOS2) is most prominently expressed by macrophages. iNOS activity and increased levels of iNOS have been found in various tumours and tumour cell lines but not in normal tissues; however, the precise role of NO in tumour progression has yet to be elucidated. We studied the expression of iNOS in paraffin sections of 41 benign naevi and 52 primary malignant melanomas (MM) of the skin, as well as in 13 metastatic MM. In addition, nitrotyrosine, indicative of NO production and formation of peroxynitrite, was studied in frozen sections of 13 naevi and 30 MM. Virtually all naevi expressed iNOS, but very few expressed nitrotyrosine, indicating either that iNOS in naevi is functionally inactive, or that naevus cells lack reactive oxygen radicals and thus do not form peroxynitrite. Normal melanocytes in adjacent uninvolved skin were unreactive for both markers. In MM, iNOS was most frequently expressed in the 'pure' and 'invasive' radial growth phase (RGP), whereas expression in the vertical growth phase (VGP) and metastatic phase occurred only in 76% of cases; moreover, in these latest phases of tumour progression, iNOS staining was weak and focal. We conclude that iNOS is expressed de novo in most benign pigment cell lesions. In MM (iNOS-generated) NO appears to play an important part in the early steps of invasion (i.e. the 'invasive' RGP), where it may stimulate neo-angiogenesis and may be a prerequisite for further tumour progression; this view is also supported by the finding of iNOS in the associated blood vessels in the papillary dermis. Finally, our data suggest that (iNOS-generated) NO plays a less significant part in the VGP and in metastatic melanoma. PMID- 10735947 TI - Preliminary evidence of an association of tumour necrosis factor microsatellites with increased risk of multiple basal cell carcinomas. AB - Tumour necrosis factor alpha (TNF-alpha) appears important in ultraviolet-induced immunosuppression, suggesting that it is a susceptibility candidate for cutaneous basal cell carcinoma (BCC). We now describe data on the association between TNF microsatellite polymorphisms, first on susceptibility in 202 controls and 133 cases each having two to 30 BCCs, and secondly, within the cases, on BCC numbers. The data show that the proportions of individuals with TNF a1- and a7-containing genotypes were significantly different (P = 0. 0271, P = 0.0393, respectively) between cases and controls. Secondly, within the cases, TNF alleles d4 (P = 0.023) and d6 (P = 0.006) alone, and the TNF a2-b4-d5 haplotype (P = 0.007), were significantly associated with the number of BCC lesions. These preliminary data provide the first evidence that TNF microsatellite polymorphism may influence the pathogenesis of multiple BCC. PMID- 10735948 TI - Basal cell carcinoma: are early appointments justifiable? AB - In the U.K., patients with suspected skin tumours are usually referred from a general practitioner to a hospital-based dermatologist for treatment. The urgency of such referrals is currently a topic of political importance. We have studied case record data from 162 patients with basal cell carcinoma (BCC) to establish the relevance of referrals routinely being considered urgent. At presentation, mean tumour size (maximum dimension) was 7. 4 mm (median 10) and mean duration of BCC was 20.5 months (median 12). There was no correlation (all P > 0.05) between tumour size and patient age (r = 0.1325), tumour size and duration (r = 0.4433), or tumour size and interval between referral and hospital consultation (r = 0.0695). If the slow growth rate of the average BCC is assumed to be linear, a reduction in referral interval from the mean value of 10.7 weeks in our patients down to the U.K. government target of 2 weeks would equate to a size difference in BCC of 0.7 mm, which is not therapeutically significant. Individual BCCs with rapid growth or other features of concern may require urgent referral, but evidence from our patients and from other studies is that this is not routinely necessary. PMID- 10735949 TI - Autocytotoxic T-cell clones in lichen planus. AB - We examined the in vitro cytotoxic activity of cutaneous T-cell lines and clones from lichen planus (LP) patients against autologous epidermal keratinocytes. T cells were cultured from LP lesions and adjacent clinically normal skin and cloned by limiting dilution. Keratinocytes were cultured from LP lesions and adjacent clinically normal skin and immortalized by transfection with the E6 and E7 genes from human papillomavirus 16 (HPV16). The lesional T-cell line from one LP patient contained 27% gammadelta+ T cells and was significantly more cytotoxic against autologous lesional keratinocytes than the T-cell line from clinically normal skin. Clones isolated from the lesional T-cell line were significantly more cytotoxic against autologous lesional keratinocytes than clones isolated from the non-lesional T-cell line. Most cytotoxic clones from LP lesions were CD8+ and most non-cytotoxic clones from LP lesions were CD4+. One cytotoxic clone was CD4- and CD8- and expressed the gammadelta T-cell receptor. Two CD8+ LP lesional T-cell clones showed dose-dependent killing of HPV16 E6/E7-immortalized autologous lesional and normal keratinocytes, but no cytotoxic activity against Epstein-Barr virus-transformed autologous B-cell blasts. The cytotoxic activity of CD8+ lesional T-cell clones against autologous lesional keratinocytes was partially blocked with anti-major histocompatibility complex (MHC) class I monoclonal antibodies. These data support the hypothesis that CD8+ lesional T cells recognize an antigen associated with MHC class I on lesional keratinocytes and that CD8+ cytotoxic T cells lyse keratinocytes in LP lesions. PMID- 10735950 TI - Measuring the activity of the disease in patients with cutaneous lupus erythematosus. AB - The Systemic Lupus Activity Measure (SLAM) is a system proposed by rheumatologists to measure disease activity in their patients with systemic lupus erythematosus (LE). It involves scoring a group of clinical symptoms and laboratory findings, the maximum possible score being 84. In systemic LE, the mid point is between 9 and 12. We applied SLAM to 176 patients with cutaneous LE. Ninety-seven had localized discoid LE (L-DLE), 59 had disseminated discoid LE (D DLE) and 20 had subacute cutaneous LE (SCLE). Eighty-five patients had low activity disease (0-4 points), 72 mildly active disease (5-9 points), 15 moderately active disease (10-14 points) and only four had very active disease (>/= 15 points). The most frequent lesions in patients who scored more than 10 points were photosensitivity, cicatricial alopecia, Raynaud's phenomenon and oral ulcers. Fifty patients were followed up for more than 5 years (mean follow-up 9 years). Nine of these had an increased SLAM score. Seven had L-DLE, one D-DLE and one SCLE. Seven of the 50 patients had photosensitivity, five cicatricial alopecia, five non-cicatricial alopecia, two Raynaud's phenomenon and two oral ulcers. Three patients who started with L-DLE evolved to D-DLE. The SLAM system is useful in the monitoring of disease activity in patients with cutaneous LE. Over time, even L-DLE patients may develop active disease. Photosensitivity, alopecia, oral ulcers and Raynaud's phenomenon seem to herald a worse prognosis. PMID- 10735951 TI - Antineutrophil cytoplasmic antibodies and HLA class II alleles in minocycline induced lupus-like syndrome. AB - We report 14 patients with minocycline-induced lupus-like syndrome (four men, 10 women; mean age 27.8 years) who developed a lupus-like illness after chronic use of minocycline for acne (1-10 years, median 3.8). Clinical features resolved completely on drug withdrawal (mean follow-up 11 months) and reappeared in two patients who were rechallenged. Sera from all 14 patients contained antineutrophil cytoplasmic antibodies (ANCA) giving a perinuclear pattern on indirect immunofluorescence on ethanol-fixed human neutrophils (p-ANCA), whereas 14 control asymptomatic individuals taking minocycline for acne were ANCA negative. Eleven of the 14 patients had elevated antimyeloperoxidase antibodies and 10 had antielastase antibodies on enzyme-linked immunosorbent assay, which diminished on extended follow-up, as did other serological abnormalities. Major histocompatibility complex class II typing demonstrated that all of the 13 patients tested were either HLA-DR4 (nine of 13) or HLA-DR2 (four of 13) positive, and all had an HLA-DQB1 allele encoding for tyrosine at position 30 of the first domain. Our findings suggest a model whereby the presence of p-ANCA may be a marker for the development of lupus-like symptoms in genetically susceptible individuals taking minocycline for acne. PMID- 10735952 TI - The clinical spectrum of epidermolysis bullosa simplex. AB - As part of the U.K. National Epidermolysis Bullosa Register, we have systematically recorded clinical information on 130 (77%) of the 168 known Scottish epidermolysis bullosa simplex (EBS) sufferers. Three subtypes of EBS were recognized: Dowling-Meara (EBS-DM), Weber-Cockayne (EBS-WC) and Kobner (EBS Kb), seen in 5%, 42% and 53% of patients, respectively. As there is considerable overlap between EBS-WC and EBS-Kb, with both phenotypes frequently seen within the same pedigree, EBS-WC is best regarded as a milder variant of EBS-Kb rather than a separate disorder. Improvement with age is common in all variants of EBS, but is not invariable. Pain due to acral blistering in EBS-Kb/EBS-WC has a more marked impact on life-style than the blisters of EBS-DM. Oral blistering, nail involvement and aplasia cutis congenita occur in all EBS subtypes and laryngeal involvement is a feature of EBS-DM. Seasonal variation is not seen in EBS-DM but is common in EBS-Kb/EBS-WC. PMID- 10735953 TI - Changes in collagen I and collagen III metabolism in patients with generalized atopic eczema undergoing medium-dose ultraviolet A1 phototherapy. AB - Fourteen patients suffering from acute, exacerbated atopic eczema were screened for changes in collagen I and collagen III metabolism in serum (n = 11), urine (n = 11) and skin biopsies (n = 9) before and after medium-dose ultraviolet (UV) A1 phototherapy (15 exposures of 50 J/cm2 over a 3-week period, total dose 750 J/cm2). Mature collagen I and, to a lesser extent, mature collagen III were found to be decreased after the therapy in skin samples from the irradiated patients. As markers of collagen I degradation, the cross-links pyridoline and deoxypyridoline were analysed in urine using high-performance liquid chromatography. Both cross-links were found to be mildly increased after UVA1 phototherapy, without reaching statistical significance. As markers of de novo collagen synthesis we screened for the procollagen I-carboxyterminal peptide (PICP) and procollagen III-aminoterminal peptide (PIIINP) levels in serum and skin. The ratio of PICP to PIIINP in serum dropped significantly after the UVA1 phototherapy, suggesting a different impact of UVA1 on the two collagens. These findings were paralleled by a diminished ratio of PICP to PIIINP in tissue samples. Staining for matrix metalloproteinase 1 (MMP-1) and its specific counterpart, tissue inhibitor of MMP-1 (TIMP-1), showed slight increases for both proteins by therapeutic UVA1; this was also seen in serum for TIMP-1 but not MMP 1. In our study, high-energy UVA1 doses induced changes of the skin collagens in patients with atopic eczema which are measurable by their metabolites in serum and urine. PMID- 10735954 TI - Lichen sclerosus premenarche: autoimmunity and immunogenetics. AB - Lichen sclerosus is among the most frequently seen paediatric vulval disorders. In adults a strong association between lichen sclerosus and autoimmune diseases, and also with HLA class II locus DQ7, has been well demonstrated in women and a weaker association in men. These associations have not previously been studied in children, although in other autoimmune diseases, the HLA associations have been strongest in children. We performed HLA tissue typing and looked for autoimmune associations in a group of 30 children with vulval lichen sclerosus. HLA DQ7 was present in 66% of female children with lichen sclerosus compared with 31% in controls. Previous studies reported DQ7 in 51% of adult female patients and 45% of male patients. Sixteen per cent of the children were homozygous for DQ7 as opposed to 5% of controls. In the childhood group, only 4% had another autoimmune disease, but 56% of their parents or grandparents did. Age differences make comparison difficult, but the family history of autoimmunity appears to be strong in the early-onset group, in addition to the stronger association with DQ7. PMID- 10735955 TI - Hand dermatosis in upper secondary school pupils: 2-year comparison and follow up. AB - The purpose of this comparative study was to record the prevalence and to estimate the incidence of self-reported hand dermatosis in a cohort of upper secondary school pupils on two different occasions, in 1995 and 1997. A previously validated questionnaire was used. Of the 1273 pupils invited to participate, 1136 (89.2%) responded to the questionnaire. The 1-year prevalence increased, although not significantly, from 9.6% in 1995 to 10.5% in 1997. For girls, the 1-year prevalence increased significantly, from 12.4% to 15.7%, while for boys the 1-year prevalence decreased, but not significantly, from 6.7% to 5.1%. The cumulative incidence was significantly higher (P < 0.001) among the girls (10.5%) than among the boys (2.8%). Multiple logistic regressions showed that the highest risk factors for 1-year prevalence in 1995 were having self reported childhood eczema, and a family history of atopic eczema or asthma. In 1997, the highest risk factors were self-reported childhood eczema, female gender and a family history of atopic eczema. This study has identified the point at which the genders diverge with regard to hand dermatosis, with boys tending to recover, while girls show a steady or even increasing prevalence. PMID- 10735956 TI - Changes in the pattern of sensitization to common contact allergens in denmark between 1985-86 and 1997-98, with a special view to the effect of preventive strategies. AB - The objective of the present study is to describe any changes in the prevalence of sensitization to common contact allergens in patch-tested patients over a 12 year period. Attention is given to possible effects of preventive strategies introduced in Denmark regarding nickel and chromate sensitization during that period, and particular areas of concern are identified. Members of the Danish Contact Dermatitis Group collected patch-test results from consecutive eczema patients as well as information about exposures and demographic variables over a 6-month period in 1985-86. The investigation was repeated in 1997-98 in the same clinics, at the same time of year, using identical methods and patch-test substances, including nickel sulphate 5%, potassium dichromate 0.5% and fragrance mix 8%. Nickel was the most common contact allergen in both study periods, followed by the fragrance mix. In children 0-18 years of age, the frequency of nickel allergy decreased from 24.8% in the first study period to 9.2% in the second study period (P < 0. 0008). Fragrance mix allergy doubled in frequency from 4.1% in 1985-86 to 9.9% in 1997-98, an increase that affected all age groups. Contact allergy to potassium dichromate decreased significantly from 3.0% in the first period to 1.2% in the second period (P = 0. 001). The decrease was seen in both sexes and was most pronounced among those of working age. No other significant changes were found in the frequency of sensitization to common allergens over the 12-year observation period. PMID- 10735957 TI - Lipodystrophy associated with protease inhibitors. AB - Lipodystrophies, characterized by reduction of subcutaneous fat over part or all of the body surface, are uncommon. Their causes are unknown. Recently, lipodystrophy has been reported in human immunodeficiency virus (HIV)-infected patients taking protease inhibitors, which have been recommended since 1996 as standard therapy for HIV disease in combination with nucleoside analogues. In these cases, lipodystrophy consists of an association of peripheral lipoatrophy with central adiposity. We report four HIV-infected men on protease inhibitors who developed a disfiguring lipodystrophy. In three of them, the protease inhibitor was administered for a mean duration of 21.5 months (range 19-23) with good immunological and virological responses. Patient 4 had been treated for 2 years with successive combinations of protease inhibitors with nucleoside analogues without success. The four patients progressively developed an increase in abdominal girth associated with fat wasting of the face and legs. Two of them had recurrent paronychia of the great toes. Triglyceride levels were moderately increased in all patients, and one had a slightly increased cholesterol level. One patient had elevated glucose and insulin plasma levels during a glucose tolerance test. In two patients, a deep biopsy taken from the thigh showed thinning of the subcutaneous fat without other morphological changes. Computed tomographic scans of the face and abdomen confirmed the loss of almost all subcutaneous fat of the cheek and temporal regions, and abdominal perivisceral fat accumulation. For patients 1-3, the protease inhibitor was replaced by a non nucleoside reverse transcriptase inhibitor. Nine months later, dysmorphic changes had not regressed, but lipid abnormalities had returned to normal and the paronychia had disappeared. PMID- 10735958 TI - Acquired immune deficiency syndrome-related hyperkeratotic Kaposi's sarcoma with severe lymphoedema: report of five cases. AB - Kaposi's sarcoma (KS) is the most frequent neoplasm in acquired immune deficiency syndrome (AIDS) patients. Whereas typical cases present as erythematous, plaque or nodular lesions, hyperkeratotic variants of AIDS-associated KS are rare. We describe five patients with AIDS-associated KS with hyperkeratosis and lymphoedema as prominent features. We also speculate on its pathogenesis. PMID- 10735959 TI - Use of mycophenolate mofetil in the treatment of paraneoplastic pemphigus. AB - Paraneoplastic pemphigus (PNP) is a rare autoimmune blistering disease with circulating antibodies that bind the cell surface of the epidermis and other non stratifying epithelia, and immunoprecipitate a complex of four or five proteins (250 kDa, 230 kDa, 210 kDa, 190 kDa and occasionally 170 kDa).1,2 Combinations of immunosuppressive agents are usually required to obtain even partial control of the skin lesions.3 Mucous membrane lesions are refractory to treatment. We describe a patient with PNP whose skin and oral lesions are quiescent following treatment with oral mycophenolate mofetil. PMID- 10735960 TI - PUVA-induced lichen planus pemphigoides. AB - A 72-year-old woman had suffered from parapsoriasis en plaque (large plaque type) controlled by topically applied psoralen ultraviolet A (PUVA) therapy. The parapsoriasis lesions gradually disappeared, but numerous tiny red papules with pruritus appeared over the forearms and lower legs 120 days after starting PUVA therapy. These papules developed to form violaceous plaques. Histological findings demonstrated the characteristics of lichen planus. Two months later, tense bullae developed on the plaques and on uninvolved skin of the limbs. These were subepidermal, with linear deposits of IgG and C3 along the basement membrane zone (BMZ) in immunofluorescence of peribullous skin, and immunodeposits of type IV collagen along the floor of the bullae. We therefore, diagnosed lichen planus pemphigoides (LPP). Using systemic and topical steroid therapy, the lesions rapidly resolved and there has been no recurrence. This case suggests that the combination of basal cell injuries caused by chronic inflammation and PUVA therapy could expose BMZ components to autoreactive lymphocytes and induce LPP. PMID- 10735961 TI - Coexistence of psoriasis and linear IgA bullous dermatosis. AB - Linear IgA bullous dermatosis (LABD) is characterized by IgA autoantibodies against components of the basement membrane zone (BMZ). A 97-kDa protein is one of the major autoantigens associated with this disease. We report a 68-year-old man who developed LABD after a 3-year history of psoriasis and in the context of active hepatitis C virus infection. He had been treated with cyclosporin for psoriasis for about 9 months. Histologically, there was a subepidermal blister containing neutrophils and eosinophils with lymphocytes infiltrating predominantly in the dermis. Direct immunofluorescent staining showed linear IgA deposition at the BMZ. The patient's IgA autoantibodies bound exclusively to the epidermal side of 1 mol/L salt-split normal human skin. Immunoblot analysis identified a 97-kDa autoantigen in epidermal extracts. This appears to be the first case of LABD with IgA autoantibodies against a 97-kDa autoantigen, associated with psoriasis and hepatitis C virus infection. PMID- 10735962 TI - Epidermolysis bullosa acquisita with ultraviolet radiationsensitivity. AB - A 37-year-old male patient developed a bullous eruption and erythematous plaques mainly in exposed areas following prolonged sun exposure. In addition, blisters were noted on oral and nasal mucous membranes. Histopathological examination of a lesional skin biopsy revealed a subepidermal blister. Linear deposition of IgG and C3 at the epidermal basement membrane zone was revealed by direct immunofluorescence microscopy of a perilesional skin biopsy. Indirect immunofluorescence on 1 mol/L salt-split skin showed binding of autoantibodies to the dermal side of the split. Immunoblot analysis of dermal extracts demonstrated that the patient's serum contained IgG antibodies against type VII collagen, whereas no reaction was seen with epidermal extracts or by enzyme-linked immunosorbent assay using a recombinant form of bullous pemphigoid 180. Standardized ultraviolet (UV) radiation provocation induced blistering with both UVA (13.5 J/cm2) and UVB (0. 04 J/cm2) within 24 h clinically and histologically. External and systemic UV-protective medication and nine cycles of high dosage immunoglobulins given intravenously (1.2 g/kg body weight over 2-3 days every 4 weeks) resulted in the cessation of blister formation. To the best of our knowledge, this is the first report of a case of epidermolysis bullosa acquisita with sensitivity to UV. PMID- 10735963 TI - Systemic lupus erythematosus with C1q deficiency. AB - We report a case of systemic lupus erythematosus associated with C1q deficiency. Our patient presented at the age of 6 years with cutaneous lupus. She later developed Raynaud's phenomenon, non-scarring alopecia, oral ulceration and grand mal seizures due to cerebral vasculitis. Complement C3 and C4 levels were consistently normal during flares of her lupus and haemolytic activity of her complement was absent, suggesting a deficiency of an early component of the complement cascade. No C1q could be detected. PMID- 10735964 TI - Two cases of merkel cell tumour arising in patients with chronic lymphocytic leukaemia. AB - Chronic lymphocytic leukaemia (CLL) has been associated with an increased incidence of second neoplasms, especially skin cancers such as basal and squamous cell carcinomas. No association with the rarer skin cancer, the Merkel cell tumour (MCT), has previously been reported. Two patients with MCT had a previous diagnosis of CLL. MCT is an aggressive skin cancer, as up to 45% of patients have lymph node involvement at presentation and 5-year disease-free survival is as low as 30%. It is most commonly found on sun-exposed areas of the body, and ultraviolet radiation together with drug-induced or CLL-induced immunosuppression may be the underlying mechanism in the observed relationship between CLL and other skin cancers. PMID- 10735965 TI - Macroscopic and microscopic mucinosis in chronic sclerodermoid graft-versus-host disease. AB - Secondary cutaneous mucinosis is a well-recognized feature of connective tissue diseases such as lupus erythematosus and dermatomyositis. We report the first three cases of dermal mucinosis in association with severe chronic cutaneous graft-versus-host disease of the sclerodermoid variety. One patient had clinical changes due to abundant mucin accumulation within the papillary dermis (mucinoma). In the other two patients histological examination revealed extensive deposits of mucin predominantly within the reticular dermis. The microscopic appearances were striking, with numerous vacuolated spaces interspersed between collagen bundles. We speculate that this appearance is the result of ground substance becoming trapped within grossly sclerodermoid connective tissue. PMID- 10735966 TI - Cerebriform nodular amelanotic metastases of malignant melanoma: a challenge in differential diagnosis of a rare variant. AB - High variability of the clinical appearance of malignant melanoma (MM) and its metastases render the differential diagnosis of solid amelanotic tumours difficult. We report a 71-year-old woman with several unusual cutaneous tumours of cerebriform morphology, suggesting skin metastases from occult internal cancer. Histopathological findings and thorough investigations, however, revealed a late-stage metastatic MM. We discuss the differential diagnosis of skin metastases of various origin and underline the difficulties for early detection of MM. PMID- 10735967 TI - Angiomatoid (malignant) fibrous histiocytoma as a second tumour in a child with neuroblastoma. AB - Neuroblastoma occurring as a disseminated disease in children has a poor prognosis. Haematogenous metastases usually involve the marrow, bone, liver and skin. A second neoplasm may also develop. We describe a child with retroperitoneal neuroblastoma (stage 3) who developed a nodular mass in the inguinal area which was suspected to be a metastasis. Histopathology disclosed an angiomatoid (malignant) fibrous histiocytoma, and excision was curative. The occurrence of angiomatoid (malignant) fibrous histiocytoma as a second tumour in a patient with neuroblastoma has not previously been reported. PMID- 10735968 TI - Squamous cell carcinoma arising in Hailey-Hailey disease of the vulva. AB - A 61-year-old woman, who was known to have Hailey-Hailey disease, presented with increasing vulval soreness. Biopsy showed vulval intraepithelial neoplasia (VIN) 3 and subsequent histology from a vulvectomy specimen showed extensive VIN with early invasive squamous cell carcinoma. This may be another example of chronic inflammation of the vulval area leading to the development of squamous cell carcinoma. However, in this case, chronic human papillomavirus may also have played a part, leading to VIN and reactivation of the Hailey-Hailey disease. We can find no previous reports of squamous cell carcinoma developing in the setting of Hailey-Hailey disease. PMID- 10735969 TI - Apocrine acrosyringeal keratosis in association with syringocystoadenoma papilliferum. AB - We report the clinical and histopathological features of a keratosis that developed in association with syringocystadenoma papilliferum. This tumour shows a pinkish, pedunculated, spherical nodule with a cerebriform surface and visible keratinous plugs. In addition to the typical features of syringocystadenoma papilliferum, the tumour shows many hyperkeratotic columns surrounded by acanthotic epidermis with the characteristics of trichilemmal keratinization and keratohyalin granules. This keratosis seems to be derived from the middle to lower portion of the apocrine acrosyringium, based on the distribution of keratohyalin granules and the direct connection with the apocrine acrosyringium in an early lesion. Accordingly, we propose to identify this rare keratosis as apocrine acrosyringeal keratosis. PMID- 10735970 TI - A case of guttate psoriasis following Kawasaki disease. AB - We report a case of guttate psoriasis following Kawasaki disease, in a patient with Staphylococcus aureus demonstrated in a throat swab. We suggest that preceding staphylococcal infection can play a key role in the pathogenesis of some cases of guttate psoriasis, possibly by the production of superantigens. PMID- 10735971 TI - The treatment of atopic dermatitis with adjunctive high-dose intravenous immunoglobulin: a report of three patients and review of the literature. AB - There are few reports of the use of high-dose intravenous immunoglobulin (hdIVIg) in the treatment of atopic dermatitis (AD). We describe our experience using this therapy in three patients with severe AD, all of whom had steroid-related side effects. These patients received either Alphaglobin(R) or Sandoglobulin(R) 2 g/kg monthly: all had improved skin scores, allowing reduction of their steroid dose. Total IgE fell in one of three patients. We discuss the side-effects of hdIVIg and their management, and detail the differences between the available immunoglobulin products available in the U.K. There are several proposed mechanisms of action of this therapy which may be operative, and those most important in AD are discussed. In view of the time and expense involved in the treatment of patients with hdIVIg, careful patient assessment is vital. We describe dose reduction strategies and methods for cost containment. In addition, one of the patients has embarked on IVIg home therapy training. This will be the first time this has been attempted for a dermatological indication. Training of this type may be available through an immunotherapy service such as exists for patients with primary immunodeficiencies. PMID- 10735972 TI - Viral folliculitis on the face. AB - The common clinical presentations of herpes simplex virus (HSV) and molluscum contagiosum (MC) are well known to dermatologists. However, folliculitis due to these viruses is an infrequently reported entity and might be considered a sign of immunosuppression [such as infection with human immunodeficiency virus, (HIV)], especially in cases of folliculitis due to MC. The purpose of this study was to describe the clinical and histopathological characteristics of viral folliculitis due to HSV and MC. We retrospectively collected all our cases with histologically proven folliculitis due to HSV and MC between 1994 and 1999. A total of seven patients aged 7-54 years was identified. Prior to establishment of the diagnosis of folliculitis due to HSV and MC, they were treated with topical antibiotics or topical steroids, without improvement. Tentative diagnoses were bacterial folliculitis, syringoma, perifollicular fibrosis, contact dermatitis or pseudolymphoma. Biopsy of the lesions revealed multiple molluscum bodies in the follicular epithelium with sparing of the epidermis in four patients, and ballooning degeneration and intranuclear viral inclusions in the follicular epithelium in the other three. Three patients had evidence of underlying immune suppression, such as pregnancy, chronic viral hepatitis B and nasopharyngeal carcinoma. One patient had suffered from epilepsy for 4 years. Testing for HIV by enzyme-linked immunosorbent assay was negative in the four patients in whom this was performed, and T-cell subsets were normal in the three patients in whom these were quantified. In cases of molluscum folliculitis treated with simple curettage, the lesions cleared without scarring or recurrence. In cases of herpetic folliculitis, the lesions improved with antihistamines or acyclovir. MC or HSV should be considered in cases that present with folliculitis-like dermatoses but which are refractory to anti-infective and anti-inflammatory treatment. PMID- 10735973 TI - Pacing-induced palmar sweating evaluated by unique hygrometer: possible implications of sympathetic activation during tachycardia. AB - Although reflex sympathetic activation is a major determinant of the haemodynamic tolerability of ventricular tachycardia (VT), the methods for evaluating this aspect during on-going VT remain invasive and complicated. Palmar sweating as an indirect but non-invasive measure of sympathetic activity was estimated by means of a unique hygrometer under right ventricular (RV) rapid pacing (up to 150 beats min-1) replicating VT, and concurrent monitoring of aortic blood pressure in five patients with various kinds of cardiac arrhythmias in our electrophysiological laboratory. The peak palmar sweating rate in arbitrary units was augmented as the RV pacing rate increased and was proportional to the pacing-induced fall in systolic blood pressure (SBP), with a correlation coefficient of more than 0.903 (P<0.006). The slope of linearity between the sweating rate and the fall in SBP varied among individual patients, with greater sweating amplitude in the younger patients even with the same extent of fall in SBP. This preliminary study suggests sympathetic acceleration caused by haemodynamic deterioration under simulated VT, and therefore this protocol may be able to predict the haemodynamic tolerability of sustained monomorphic VT. PMID- 10735974 TI - Distribution of ventilation-to-perfusion ratios analysed in planar scintigrams of emphysematous patients. AB - The aim of the present study was to develop and evaluate a method for semi quantification of ventilation-to-perfusion ratios in small regions in planar pulmonary scintigrams using 99mTc-Technegas (Tetley Manufacturing Ltd, Sydney, Australia) and 99mTc-macro-aggregated human albumin. Eight emphysematous patients and eight healthy volunteers were investigated in the supine position. Anterio posterior and posterio-anterior images of ventilation and ventilation-and perfusion were acquired consecutively. Perfusion images were obtained by subtracting the corrected ventilation images from ventilation-and-perfusion images. Lung regions of interest were divided into 2.03 x 2.03 cm regions. The ratio of normalized counts of ventilation-to-perfusion (V/Q) was calculated in each square region. The log (V/Q)s of healthy volunteers were distributed symmetrically and similarly around 0, whereas the corresponding ratios in patients distributed unevenly. The standard deviations of log (V/Q)s were significantly higher in patients than in healthy volunteers. Parametric images of V/Q distribution were constructed to visualize the magnitude and location of 'waste ventilation' and 'shunt' effects. In conclusion, the present semi quantitative method is validated by confirming the expected dispersed V/Q distribution in emphysematous patients compared to healthy volunteers. PMID- 10735975 TI - Efficient lung scintigraphy. AB - Lung scintigraphy is a first-choice method to diagnose lung embolism. The clinical routine in most centres is a perfusion study complemented with a ventilation study when judged necessary. We describe a routine with ventilation scintigraphy preceding perfusion scintigraphy, which is completed within one hour. Furthermore, the data acquired allow the determination of lung clearance of the tracer 99mTc-DTPA (diethylene triamine penta-acetate) used for the ventilation scintigraphy. An aerosol generator charged once a day with 99mTc-DTPA solution is used for all inhalations during the day. Inhalation is monitored with a counter and interrupted when the count rate corresponds to about 20 MBq. The ventilation imaging starts and ends with posterior projections. This allows calculation of lung clearance of 99mTc-DTPA. Perfusion scintigraphy is performed in a standard fashion with 100 MBq of 99mTc-MAA (macro-aggregated albumin). The ventilation study was considered to give some diagnostic information in the majority of the patients. The clearance determination allows detection of inflammatory lung disease. The background activity caused by the ventilation study comprised only 13% of the activity in the perfusion scintigraphy and did not significantly interfere with interpretation of the perfusion scan. The cost for the investigation is low because of the rational system for aerosol administration and the short time for a complete study. PMID- 10735976 TI - Cardiac output measured by electric bioimpedance compared with the CO2 rebreathing technique at different exercise levels. AB - In the lower range of cardiac output (CO, up to 15 l min-1), we found an excellent agreement between CO measured by bioimpedance and carbon dioxide rebreathing techniques. CO estimated by bioimpedance was generally lower. The bioimpedance method had better reproducibility. Both methods seem valuable for non-invasive studies in healthy subjects at work. PMID- 10735977 TI - Segmental composition of whole-body impedance cardiogram estimated by computer simulations and clinical experiments. AB - Whole-body impedance cardiography (ICGWB) has been proposed as a feasible means of measuring cardiac output (CO). However, the source distribution of heart related impedance variations in the whole body is not known. To establish how much of a signal originates in each segment of the body and what the contribution of each is to stroke volume (SV) in ICGWB, impedance in the extremities and trunk were investigated in 15 healthy volunteers. In addition, the theoretical measurement properties of ICGWB were studied using a computer model of the whole body anatomy as a volume conductor. The model confirmed the expected result that most of the basal impedance originates from the extremities. Clinical experiments revealed that the heart-related amplitude variations in the ICGWB signal originate more evenly from various body segments, the trunk slightly more than the arms or legs. The heart-related ICGWB signal represents a weighted sum of segmental pulsatile events in the body yielding physiologically meaningful data on almost the whole circulatory system. PMID- 10735978 TI - Adaptation related to cytokines in man: effects of regular swimming in ice-cold water. AB - The cytokine response after thermal stress (sauna + swimming in ice-cold water) was investigated in subjectively healthy persons. Two groups were studied at the end of the winter season: habitual and inexperienced winter swimmers. Blood was collected at rest, after a sauna bath and after a short swim in ice-cold water. Conventional methods and ELISA kits were used to determined the blood picture, serum cortisol and dehydroepiandrosterone sulphate, plasma anti-diuretic hormone (ADH) levels, and the levels of several cytokines in plasma and in the supernatants of blood cell cultures which were stimulated with lipopolysaccharide (LPS). In regular winter swimmers, the concentrations of plasma interleukin 6 (IL 6), leukocytes, and monocytes at rest were significantly higher than in inexperienced subjects. In experienced female winter swimmers, the plasma concentration of the soluble receptor for IL-6 was significantly lower than in inexperienced female swimmers. In both groups, granulocytosis, haemoconcentration and significant increases in the concentrations of ADH, cortisol and IL-6 were observed after the stimuli. However, the changes in the cortisol concentration were dramatically larger in habitual winter swimmers. A significant correlation was found between the delta values of cortisol and the basal concentrations of IL 6. In cell cultures, the LPS-induced release of IL-1beta and IL-6 was higher at rest in the inexperienced winter swimmers. This release was dramatically suppressed after exposure to the stimuli in the inexperienced winter swimmers but tended to increase in the regular winter swimmers. These stresses appear to challenge both the neuro-endocrine and the immune systems and the results indicate that adaptive mechanisms occur in habitual winter swimmers. PMID- 10735979 TI - Osteocalcin metabolism in the pulmonary circulation. AB - The aim of this study was to evaluate the role of the pulmonary vessel endothelium in the removal of circulating osteocalcin, by measuring the osteocalcin levels in serum from pulmonary and radial artery blood from 39 patients undergoing aorto-coronary bypass. Because of the discrepancies between methods of measurement, two methods were used. Significant differences were observed in group A (n = 18), tested with heterologous radioimmunoassay (2.85 +/- 0.67 microg l-1 in the pulmonary versus 2.69 +/- 0.67 microg l-1 in the radial artery serum, P<0.001) and in group B (n = 21), tested with a two-site immunoradiometric assay (5.22 +/- 1.46 versus 4.93 +/- 1.36 microg l-1, P<0.01). The percentage differences were -5.54 +/- 4.76% (P<0.001) in group A and -4.99 +/ 8.13% (P<0.01) in group B; the comparison between the percentage differences was not significant. These different osteocalcin concentrations between the two vascular compartments were considered a marker of osteocalcin degradation. Therefore, the study seems to demonstrate that, as well as kidney, liver and bone, the lung is a relevant site of osteocalcin catabolism. The proteolytic activity of pulmonary vessel endothelium seems to involve about 5% of the circulating peptide. PMID- 10735980 TI - Muscle glycogen does not interfere with a 13CO2 breath test to monitor liver glycogen oxidation. AB - Naturally 13C-enriched carbohydrate has been used to label the liver glycogen pool for metabolic studies. The utilization of this glycogen was then monitored by the appearance of 13CO2 in breath. Using this method, it is assumed that during sedentary fasting the contribution of muscle glycogen towards oxidation is negligible. We investigated the influence of a different level of 13C enrichment of muscle glycogen on the 13C enrichment of breath CO2 while the breath test was carried out. In six healthy volunteers, the muscle glycogen stores were grossly depleted by a cycling exercise prior to consumption of the 13C-enriched diet which was given over a 10 h period. The oxidation of liver glycogen was measured during an 18 h sedentary fast. The results were compared with a control group who had not depleted their muscle glycogen before labelling. A higher 13C enrichment of muscle glycogen did not interfere with two parameters of liver glycogen oxidation, i.e. the duration of the plateau phase of 13CO2 and the return to baseline time. It was also shown that the 13C-labelled muscle glycogen was still available after the 18 h fast because a strenuous exercise led to a rapid 13CO2 enrichment. It is concluded that muscle glycogen 13C enrichment does not invalidate a 13CO2 breath test to measure liver glycogen oxidation during a sedentary fast. PMID- 10735981 TI - Acute physiological effects of exhaustive whole-body vibration exercise in man. AB - Vibration exercise (VE) is a new neuromuscular training method which is applied in athletes as well as in prevention and therapy of osteoporosis. The present study explored the physiological mechanisms of fatigue by VE in 37 young healthy subjects. Exercise and cardiovascular data were compared to progressive bicycle ergometry until exhaustion. VE was performed in two sessions, with a 26 Hz vibration on a ground plate, in combination with squatting plus additional load (40% of body weight). After VE, subjectively perceived exertion on Borg's scale was 18, and thus as high as after bicycle ergometry. Heart rate after VE increased to 128 min-1, blood pressure to 132/52 mmHg, and lactate to 3.5 mM. Oxygen uptake in VE was 48.8% of VO2max in bicycle ergometry. After VE, voluntary force in knee extension was reduced by 9.2%, jump height by 9.1%, and the decrease of EMG median frequency during maximal voluntary contraction was attenuated. The reproducibility in the two VE sessions was quite good: for heart rate, oxygen uptake and reduction in jump height, correlation coefficients of values from session 1 and from session 2 were between 0.67 and 0.7. Thus, VE can be well controlled in terms of these parameters. Surprisingly, an itching erythema was found in about half of the individuals, and an increase in cutaneous blood flow. It follows that exhaustive whole-body VE elicits a mild cardiovascular exertion, and that neural as well as muscular mechanisms of fatigue may play a role. PMID- 10735982 TI - Circulatory response to single circuit weight and walking training sessions of similar energy cost in middle-aged overweight females. AB - The aim of this study was to compare circulatory responses to circuit weight (CWT) and aerobic walking training sessions of similar energy cost in middle-aged overweight females. Thirty-three middle-aged pre-menopausal females participated in the experiment. They were divided into overweight (n=18, 36.2 +/- 6.3 years, 166.3 +/- 8.0 cm, 83.5 +/- 9.7 kg, BMI 30.2 +/- 3.1 kg m-2) and non-overweight control (n=15, 34.1 +/- 6.3 years, 165.0 +/- 5.6 cm, 61.6 +/- 5.0 kg, BMI 22.7 +/ 1.5 kg m-2) groups. Individual physical working capacity (PWC) was measured using the cycle ergometer test (calculated at the level of predicted HRmax (205 - (1/2) age). A CWT session consisted of leg extension, bench press, sit-ups and leg press exercises. The subjects performed four circuits at the maximal possible speed, using a work-to-rest ratio of 60 s. Blood pressure (BP) was measured during every rest period between the exercises, and the heart rate (HR) was recorded continuously during the whole CWT programme. During the walking training session, the subjects walked as fast as possible on the indoor track. The total energy cost of the walking training session was the same as during the CWT session, approximately 270 kcal, and was controlled by a CALTRAC accelerometer. HR and BP were measured every 5 min during the walking training session. The PWC index was significantly (P<0.05) higher in the overweight group in comparison with the control females (215.4 +/- 76.1 and 187.9 +/- 42.4 W, respectively). The resting BP was normal in both groups (<140/90 mmHg). HR was between 120 and 140 beats min-1 during CWT and walking sessions. There were no differences in BP during both training sessions in overweight and control subjects. It was concluded that both CWT and walking training sessions were acceptable forms of physical activity to increase cardiovascular fitness in middle-aged overweight and normal body weight females. PMID- 10735983 TI - The effect of high- and low-frequency transcutaneous electrical nerve stimulation upon cutaneous blood flow and skin temperature in healthy subjects. AB - The reported non-analgesic effects of transcutaneous electrical nerve stimulation (TENS) include alterations to the local circulation; however, research in this area has produced equivocal findings. In the present study, the effect of low- (4 Hz) and high-frequency (110 Hz) TENS on forearm skin blood perfusion was assessed using laser Doppler flowmetry. The effect on skin temperature was also assessed using a skin thermistor. Thirty healthy human volunteers were recruited and randomly assigned to a control or one of the two treatment groups. TENS was applied to the skin overlying the median nerve under double-blind conditions for 15 min. Blood flow and skin temperature readings were recorded pre-TENS, during TENS application and continued for 15 min post-TENS application. Analysis of results showed significant increases in blood perfusion during the treatment period in the low-frequency group when compared to the other two groups (P = 0.0106; ANOVA). No significant changes in skin temperature were observed. The results of this study demonstrate that low-frequency TENS produces a local increase in cutaneous blood flow. PMID- 10735984 TI - Well-being and cerebral oxygen saturation during acute heart failure in humans. AB - Cerebral symptoms and near-infrared spectrophotometry-determined cerebral oxygen saturation (ScO2) were followed in patients treated for normotensive acute congestive heart failure. The reproducibility and normal range for ScO2 were established from 39 resting subjects without cardio-respiratory disease: the ScO2 ranged from 55 to 78% with a coefficient of variation for triple determination of 6%. Patients rated cerebral symptoms on a scale with end-points of 0 (best) and 10 (worst). In eight patients with acute heart failure, arterial oxygen tension increased during decongestive treatment, from 9.1 (4.9-10) to 10.4 kPa (7.3-17); median with range, as did arterial oxygen saturation, from 94 (48-97) to 97% (87 99) (P<0.02), whereas the mean arterial pressure, heart rate and arterial carbon dioxide tension remained unchanged. The cerebral symptom score improved from 8 (3 10) to 1 (1-9) and the ScO2 increased from 34 (20-58) to 50% (19-91) (P<0.02). A ninth patient presented with a silent but massive myocardial infarction: she was cerebrally obtunded with a ScO2 of 18% and soon died. In patients with normotensive acute heart failure and cerebral symptoms, cerebral oxygen saturation is low, and during successful treatment ScO2 increases with the well being of the patient. PMID- 10735986 TI - Growth kinetics of Acetobacterium sp. on methanol-formate in continuous culture. AB - The fermentative metabolism of Acetobacterium sp. grown on methanol-formate in continuous culture is described. The reaction stoichiometry of methanol-formate, including cells, were as follows: CH3OH + 1.13HCOOH --> 0.87CH3COOH + 0.47 cell C. Formate enhanced growth yields by approximately 60% compared with methanol-CO2 grown cultures. Comparison of yields on methanol-formate allowed calculation of an energy yield of 1.3 mol ATP per mol acetate formed during homoacetate fermentation. The magnitudes of YEG,the theoretical maximum yield of YE, and m, the maintenance coefficient, were determined by growing the organism in methanol formate and resulted in 16.5 g cell (mol methanol catabolized)-1 and 0.674 mmol methanol catabolized (g cell)-1 h-1, respectively. It is concluded that formate might replace CO2 as a source of carboxyl donor. PMID- 10735985 TI - The effect of dietary restriction and menstrual cycle on excess post-exercise oxygen consumption (EPOC) in young women. AB - The purpose of this study was to evaluate the effect of acute dietary restriction on excess post-exercise oxygen consumption (EPOC) in young women at two different phases of the menstrual cycle. Five young sedentary women (age 21-22 years) participated in this study. Each subject visited the laboratory eight times for measurement of EPOC. They performed cycle ergometer exercise for 60 min at a work rate corresponding to approximately 70% of VO2max under each four different conditions (i.e. standard diet/follicular phase (SF), standard diet/luteal phase (SL), restricted diet/follicular phase (RF) and restricted diet/luteal phase (RL)). The exercise was performed in the morning and VO2 was measured for the last 15 min of each hour for 7 h after the exercise. As a control, VO2 was also measured with an identical time schedule under the same four conditions but without exercise. EPOC was calculated as the difference of the VO2-time integral for 7 h between the exercise and control trial days in each of the four conditions (i.e. SL, SF, RL and RF). The diet was precisely controlled during 2 days (i.e. the test day and the day preceding it). The standard diet was 1600 kcal day-1 and the restricted diet was half of the standard diet. A two-way (dietary and menstrual cycle factors) ANOVA indicated that EPOC was significantly affected only by the dietary factor. The dietary restriction decreased EPOC compared to the standard dietary condition (SF 8.6 +/- 2.1, RF 5.3 +/- 1.6, SL 8.9 +/- 4.8, RL 4.0 +/- 1.2 l). These data indicate that for young sedentary women, EPOC is significantly lowered by prior acute dietary restriction but is not influenced by different phases of the menstrual cycle. PMID- 10735988 TI - Fluorometric assessment of gram-negative bacterial permeabilization. AB - Uptake of the fluorescent probe 1-N-phenylnaphthylamine (NPN), as adapted to an automated spectrofluorometer enabling multiwell reading of microtitre plates, was applied to determine permeability changes in Gram-negative bacteria. An intact outer membrane is a permeability barrier, and excludes hydrophobic substances such as NPN but, once damaged, it can allow the entry of NPN to the phospholipid layer, resulting in prominent fluorescence. With Escherichia coli O157, Pseudomonas aeruginosa, and Salmonella typhimurium as test organisms and ethylenediaminetetraacetic acid and sodium hexametaphosphate as the model permeabilizers, quantitative and highly reproducible NPN uptake levels were obtained that differed characteristically between the test bacteria. Furthermore, citric acid was shown to be a potent permeabilizer at millimolar concentrations, its effect being partly (Ps. aeruginosa, Salm. typhimurium) or almost totally (E. coli O157) abolished by MgCl2, suggesting that part of the action occurs by chelation. Sodium citrate induced weak NPN uptake, which was totally abolished by MgCl2. In conclusion, the NPN uptake assay with the automated spectrofluorometer serves as a convenient method in analysing and quantifying the effects of external agents, including potential food preservatives, on Gram-negative bacteria. PMID- 10735987 TI - Physiological effects of high hydrostatic pressure treatments on Listeria monocytogenes and Salmonella typhimurium. AB - The effect of a high hydrostatic pressure treatment on the Gram-positive Listeria monocytogenes strain Scott A and the Gram-negative Salmonella typhimurium strain Mutton (ATCC13 311) has been determined in stationary phase cell suspensions. Pressure treatments were done at room temperature for 10 min in sodium citrate (pH 5.6) and sodium phosphate (pH 7.0) suspension buffers. Increasing pressure treatments resulted in an exponential decrease of cell counts. Salmonella typhimurium suspended at low pH was more sensitive to pressure treatments. Progressive morphological changes were evident with the pressure increase. Cell lysis only appeared with the highest pressure treatments. Cell volume was not affected by pressure treatment. A progressive decrease of deltapH (pHin - pHout), intracellular potassium and ATP contents was demonstrated with the pressure increase. A parallel lowering of membrane potentials was measured. PMID- 10735989 TI - Electrotransformation of Clostridium acetobutylicum ATCC 824 using high-voltage radio frequency modulated square pulses. AB - Molecular biological improvement of industrial solventogenic clostridia could be enhanced by a higher efficiency of electrotransformation. In this research, we used a new approach to determine the frequency spontaneously generated by Clostridium acetobutylicum ATCC 824 cells during the application of a square high voltage pulse. Once the frequency of 100 kHz was determined we transformed clostridial cells with pSOS84 plasmid DNA using radio-frequency modulated high voltage square pulses (electric field strength 12 kVcm-1; pulse duration 22.5 ms; frequency of pulse modulation 100 kHz) to reach an efficiency exceeding 106 transformants microg-1 of plasmid DNA. We propose a possible role for cellular membrane structures in affecting the transformation yield. PMID- 10735990 TI - Viability of Escherichia coli O157:H7 in natural river water determined by the use of flow cytometry. AB - The enzymatic activity and viability of Escherichia coli O157:H7 in natural river water was determined by flow cytometry. River water was collected at two sites (an agricultural area and an industrial area) on the Aigawa River (Osaka, Japan). To facilitate estimation of the physiology of E. coli O157 in natural river water, bacterial cells in the water were stained with 6-carboxyfluorescein diacetate (6CFDA) and propidium iodide (PI). The cells were sorted into two populations, using a flow cytometer, based on their esterase activity. Each population was stained with E. coli O157:H7 fluorescent antibody (FA), and E. coli O157:H7 cells were observed in the esterase-active population. River water samples collected at the same points were incubated with yeast extract containing antibiotics to prevent cell division, and bacterial cells in the incubated samples were stained with PI and FA. Escherichia coli O157:H7 existed in both the viable (elongated and/or fattened) and inactive bacterial population determined by flow cytometry. These results indicate that E. coli O157:H7 may retain metabolic activity and growth potential in the natural aquatic environment. PMID- 10735991 TI - One explanation for the variability of the bacterial suspension test. AB - Disinfection kinetic studies of sodium dodecyl sulphate, benzalkonium chloride and sodium hypochlorite against Staphylococcus aureus revealed that when a higher inoculum level of Staph. aureus than normal was used (approximately 1 log higher), the efficacy of disinfection was severely attenuated. Kinetic analysis using the Hom model for experiments carried out on tests using 3 x 108 organisms ml-1 were unable to account for the large increase in disinfection power observed when smaller inoculum levels were used. Since the inoculum was the same in every way except for the numbers used, the large variations in the log reduction/time curves could not be explained by a variation in the resistance of the population to the biocide, as identical log reduction-time curves should have resulted. The level of disinfection achieved for a given concentration of biocide was found to be approximately linearly related to the cell number ml-1 of test solution and not to the log number. The variation observed is believed to occur due to intrinsic self-quenching of the biocide by the microbes during the course of the disinfection test. As the level of free biocide decreases, the rate of reaction decreases, giving the tails of the log reduction/time curves. Such intrinsic self quenching could explain the large variations known to occur in the legally required disinfection suspension tests. PMID- 10735992 TI - Expression of the multiple antibiotic resistance operon (mar) during growth of Escherichia coli as a biofilm. AB - The multiple antibiotic resistance (mar) operon is a global regulator controlling the expression of various genes in Escherichia coli which constitutes the mar regulon. Upregulation of mar leads to a multi-drug resistant phenotype, which includes resistance towards structurally unrelated antibiotics, organic solvents and the disinfectant pine oil. Biofilms also display similar decreases in susceptibility to antimicrobial agents. A marOII-lacZ fusion strain (SPC105) of E. coli was used to monitor mar expression under various growth conditions including batch, continuous and biofilm culture. In chemically-defined media (CDM), mar expression was maximal in mid-log and declined in the stationary phase. Conversely, in rich media (Luria-Bertani broth), minimal expression in mid log was followed by an increase in the stationary phase. In continuous culture, expression was inversely related to specific growth rate (mu = 0.05-0.4 h-1). LacZ expression by the marOII-lacZ fusion was generally low within the total biofilm population and equivalent to that of stationary phase cultures grown in batch culture. When the expression of mar in CDM batch culture was compared with that in biofilm populations, beta-galactosidase activity was generally higher throughout batch culture than in the attached population. Overall, these results suggest that while mar expression will be greatest within the depths of a biofilm where growth rates are suppressed, its probable induction within biofilms cannot explain the elevated levels of antibiotic resistance observed. PMID- 10735993 TI - Spore-forming, Desulfosporosinus-like sulphate-reducing bacteria from a shallow aquifer contaminated with gasoline. AB - Previous studies on the geochemistry of a shallow unconfined aquifer contaminated with hydrocarbons suggested that the degradation of some hydrocarbons was linked to bacterial sulphate reduction. There was attenuation of naphthalene, 1,3,5 trimethylbenzene (TMB), toluene, p-xylene and ethylbenzene in the groundwater with concomitant loss of sulphate. Here, the recovery of eight strains of sulphate-reducing bacteria (SRB) from the contaminated site is reported. All were straight or curved rod-shaped cells which formed endospores. Amplification and sequencing of the 16S rDNA indicated that the strains were all sulphate reducers of the Gram-positive line of descent, and were most closely related to Desulfosporosinus (previously Desulfotomaculum) orientis DSM 8344 (97-98.9% sequence similarity). The strains clustered in three phylogenetic groups based on 16S rRNA sequences. Whole cell fatty acid compositions were similar to those of D. orientis DSM 8344, and were consistent with previous studies of fatty acids in soil and groundwater from the site. Microcosms containing groundwater from this aquifer indicated a role for sulphate reduction in the degradation of [ring-UL 14C]toluene, but not for the degradation of [UL-14C]benzene which could also be degraded by the microcosms. Adding one of the strains that was isolated from the groundwater (strain T2) to sulphate-enriched microcosms increased the rate of toluene degradation four- to 10-fold but had no effect on the rate of benzene degradation. The addition of molybdate, an inhibitor of sulphate reduction, to the groundwater samples decreased the rate of toluene mineralization. There was no evidence to support the mineralization of [UL-14C]benzene, [ring-UL 14C]toluene or unlabelled m-xylene, p-xylene, ethylbenzene, TMB or naphthalene by any of the strains in pure culture. Growth of all the strains was completely inhibited by 100 micromol l-1 TMB. PMID- 10735994 TI - Identification of pediococci by ribotyping. AB - Pediococci are among the most prevalent microbial contaminants in breweries and they can cause ropiness and the accumulation of high levels of diacetyl in beer. The accurate identification of pediococci is important, because different species do not possess equal spoilage potential. In this study, 18 Pediococcus strains, mainly of brewery origin, were first identified using phenotypical characterization (API 50 CHL and SDS-PAGE profiling), and then ribotyped using a RiboPrinterR System. Six Pediococcus type strains and three other Pediococcus strains were used as references. Ribotyping showed higher discriminative capacity than phenotypical identification methods. Strains could be identified to species level and in many cases, differentiated even at strain level using this genetic fingerprinting method. The identifications performed by ribotyping were confirmed by 16S rDNA sequencing of selected strains. Automated ribotyping was found to be a rapid and reliable method for identifying pediococci, but requires the construction of a comprehensive fingerprint library. PMID- 10735995 TI - Serovars of Mycobacterium avium complex isolated from AIDS and non-AIDS patients in Spain. AB - Antigen fingerprinting based on surface glycolipid antigens was applied to the epidemiology of clinical isolates of the Mycobacterium avium complex from 128 acquired immunodeficiency syndrome (AIDS) and 31 non-AIDS patients from several different regions of Spain. The application of thin-layer chromatography, gas chromatography-mass spectrometry and monoclonal antibodies, combined with ELISA, allowed a facile identification, differentiation and classification of the isolates. The cumulative results demonstrate that, among the clinical isolates, serovar 4 was predominant in both AIDS (33.6%) and non-AIDS (22.6%) isolates. In general, the results demonstrate geographical as well as disease-related differences in the distribution of Myco. avium complex serovars of clinical importance. PMID- 10735996 TI - Quantitative determination of total coliforms and Escherichia coli in marine waters with chromogenic and fluorogenic media. AB - This study compared the performance of LMX(R) broth (LMX), Chromocult Coliform(R) agar (CC) and Chromocult Coliform agar plus cefsulodin (10 microg ml-1) (CC-CFS), with standard methods multiple tube fermentation (MTF), for the enumeration of total coliforms and Escherichia coli from marine recreational waters. LMX and CC are two media designed to concurrently detect total coliform (TC) bacteria and E. coli by the specific action of beta-galactosidase (total coliforms) and beta glucuronidase (E. coli). Overall results for the TC test showed that LMX, CC and MTF recovered 2.63, 1.95 and 1.90 times as many TCs as CC-CFS, respectively. Data from the multiple range test showed significant differences (P < 0.05) between TC counts on CC-CFS and LMX. The traditional MTF was less sensitive for E. coli enumeration. However, there was no statistically significant differences between LMX, CC, CC-CFS and the MTF method for E. coli enumeration. Background interference was reduced on CC-CFS and the counts obtained reflected more accurately the number of TCs. Therefore, the contribution of beta-galactosidase positive, non coliform bacteria (Aeromonas spp. and Vibrio spp.) to TC counts should not be neglected. PMID- 10735997 TI - Identification and enumeration of oleic acid and linoleic acid hydrating bacteria in the rumen of sheep and cows. AB - The diversity and population densities of facultative anaerobic bacteria with the capacity to hydrate oleic acid and linoleic acid in the rumen of sheep and dairy cows were determined. The screening of representative colonies, from rumen fluid plated aerobically on a range of agar media, revealed that sheep rumen fluid contained hydration-positive strains of Streptococcus, Staphylococcus, Enterococcus, Lactobacillus and Pediococcus, whereas cow rumen fluid contained hydration-positive strains of Streptococcus, Lactobacillus and Staphylococcus. Mean counts of facultative anaerobic bacteria in sheep and cattle rumen were log10 7.29 and log10 6.40, respectively, and were independent of diet. Approximately 56% of facultative anaerobic bacteria were able to hydrate oleic and/or linoleic acid in anaerobic broth culture. For both sheep and cows, the most numerous hydration-positive isolates were strains of Strep. bovis. The results, which are the first to show that pediococci have the capacity to hydrate unsaturated fatty acids, suggest that lactic acid bacteria are the major unsaturated fatty acid hydrating bacteria in the rumen. PMID- 10735998 TI - Determination of enteroviruses, hepatitis A virus, bacteriophages and Escherichia coli in Adriatic Sea mussels. AB - The aim of the present study was to evaluate the incidence of enteric viruses in mussels and to verify the possibility of using phages as indirect indicators of mussel viral contamination. Mussels (36 samples) collected from three different areas of the Adriatic Sea were analysed to determine the following parameters: Escherichia coli, somatic coliphage (T6 phage), F-Plus (MS2 phage), B40-8 (phage of Bacteroides fragilis), enteroviruses and hepatitis A virus. Most of the results of the bacteriological analysis (most probable number (MPN) ml-1) were in accordance with the bacteriological limits established by European law, with the exception of seven samples. The bacteriophage analyses were always negative for F Plus and B40-8, with the exception of a few samples, whereas the somatic coliphages were generally between 0 and 20 MPN g-1, with the exception of two samples (110 MPN g-1). The virological analysis showed five samples positive for the presence of enteroviruses and 13 for the presence of hepatitis A virus (in three samples both viruses were present). Most of these samples presented acceptable bacteriological parameters and the bacteriophages were absent or their value was generally very low. The results show that the detection of E. coli and phages does not seem to be a good indicator of viral contamination. PMID- 10735999 TI - Detection of Flavobacterium psychrophilum from fish tissue and water samples by PCR amplification. AB - Rainbow trout fry syndrome and cold-water disease, caused by Flavobacterium psychrophilum, are important diseases in farmed salmonids. Some of the presently available techniques for the detection of Fl. psychrophilum are either time consuming or lack sufficient sensitivity. In the present investigation, the possible detection of Fl. psychrophilum from fish tissue and water samples was examined using nested PCR with DNA probes against a sequence of the 16S rRNA genes. The DNA was extracted using Chelex(R) 100 chelating resin. The primers, which were tested against strains isolated from diseased fish, healthy fish, fish farm environments and reference strains, proved to be specific for Fl. psychrophilum. The obtained detection limit of Fl. psychrophilum seeded into rainbow trout brain tissue was 0.4 cfu in the PCR tube, corresponding to 17 cfu mg-1 brain tissue. The PCR-assay proved to be more sensitive than agar cultivation of tissue samples from the brain of rainbow trout injected with Fl. psychrophilum. In non-sterile fresh water seeded with Fl. psychrophilum the detection limit of the PCR-assay was 1.7 cfu in the PCR tube, corresponding to 110 cfu ml-1 water. The PCR-assay detected Fl. psychrophilum in water samples taken from a rainbow trout farm, but Fl. psychrophilum could not be isolated using inoculation on selective agar. The method presented here has the potential to detect low levels of Fl. psychrophilum in fish tissue and in water samples, and the technique can be a useful tool for understanding the epidemiology of Fl. psychrophilum. PMID- 10736000 TI - Antimicrobial agents from plants: antibacterial activity of plant volatile oils. AB - The volatile oils of black pepper [Piper nigrum L. (Piperaceae)], clove [Syzygium aromaticum (L.) Merr. & Perry (Myrtaceae)], geranium [Pelargonium graveolens L'Herit (Geraniaceae)], nutmeg [Myristica fragrans Houtt. (Myristicaceae), oregano [Origanum vulgare ssp. hirtum (Link) Letsw. (Lamiaceae)] and thyme [Thymus vulgaris L. (Lamiaceae)] were assessed for antibacterial activity against 25 different genera of bacteria. These included animal and plant pathogens, food poisoning and spoilage bacteria. The volatile oils exhibited considerable inhibitory effects against all the organisms under test while their major components demonstrated various degrees of growth inhibition. PMID- 10736001 TI - Arabinose fermentation by Lactobacillus plantarum in sourdough with added pentosans and alphaalpha-L-arabinofuranosidase: a tool to increase the production of acetic acid. AB - Sixty-five strains of obligately and facultatively heterofermentative sourdough lactic acid bacteria were screened for their capacity to grow optimally in the presence of arabinose, ribose and xylose as carbon sources. Lactobacillus alimentarius 15F, Lact. brevis 10A, Lact. fermentum 1F and Lact. plantarum 20B showed higher growth rate, cell yield, acidification rate and production of acetic acid when some pentoses instead of maltose were added to the SDB medium. Lactobacillus plantarum 20B used arabinose also in a synthetic medium where complex growth factors such as yeast extract were omitted. Other Lact. plantarum strains did not show the same property. Pentosan extract was treated with alpha-L arabinofuranosidase from Aspergillus niger or endo-xylanase from Bacillus subtilis to produce hydrolysates containing mainly arabinose and xylose, respectively. In particular, the hydrolysate containing arabinose substantiated the growth and the production of lactic acid and, especially, of acetic acid by Lact. plantarum 20B. Sourdough fermentation by Lact. plantarum 20B with addition of pentosan extract and alpha-L-arabinofuranosidase increased the acidification rate, titratable acidity and acetic acid content compared with traditional sourdough. A facultatively heterofermentative strain, Lact. plantarum 20B, also produced a sourdough with an optimal fermentation quotient. PMID- 10736002 TI - Isolation, purification and characterization of xylanasefrom Staphylococcus sp. SG-13 and its application in biobleaching of kraft pulp. AB - A haloalkalophilic Staphylococcus sp. SG-13 produced an alkalistable xylanase in wheat bran medium. A 12-fold purification was achieved by using standard purification techniques. The purified xylanase exhibited a dual pH optima of 7.5 and 9.2. The optimum temperature for enzyme activity was 50 degrees C. The enzyme was stable at 50 degrees C for more than 4 h. The xylanase exhibited Km and Vmax values of 4 mg ml-1, 90 micromol min-1 per mg for birchwood xylan and 7 mg ml-1, 55 micromol min-1 per mg for oatspelt xylan, respectively. The substrate binding affinity of xylanase was more for oatspelt xylan but birchwood xylan was hydrolysed more rapidly. The xylanase activity was stimulated by Fe2+, Ni2+, Cu2+ and dithiothreitol up to 60% and was strongly inhibited in the presence of Co2+, Hg2+, Pb2+, phenyl methane sulphonyl fluoride, ethylenediaminetetraacetic acid, and acetic anhydride up to 100%. The xylanase dose of 1.8 U g-1 moisture free pulp, exhibited bleach boosting of kraft pulps optimally at pH 9.5-10.0 and 50 degrees C after 4 h of reaction time. Pretreatment of pulp with xylanase and its subsequent treatment with 8% hypochlorite, reduced the kappa number by 30%, enhanced the brightness and viscosity by 11% and 1.8%, respectively, and improved the paper properties such as tensile strength and burst factor up to 10% and 17%, respectively. PMID- 10736003 TI - Prevalence and characteristics of verotoxin-producing Escherichia coli (VTEC) in stool samples from asymptomatic human carriers working in the meat processing industry in Switzerland. AB - A total of 5590 stool samples from healthy employees in the meat industry were screened by PCR for verotoxin-producing Escherichia coli (VTEC). The PCR product of VT-encoding genes was detected in 3. 5% of the samples. Phenotypic and genotypic traits of 47 VTEC strains isolated from asymptomatic carriers were characterized. A variety of serotypes was found; one strain belonged to the serotype O157:H7. The majority of the isolates proved to be VT2-positive. Fifty seven percent of the verotoxin-producing strains harboured the genes for one or several additional virulence associated factors, including intimin (eae, 8.5%), the 60 MDa plasmid (42.5%), enterohaemolysin (EHEC-hlyA, 38.3%), the heat-stable enterotoxin (astA, 6.4%), a serin protease (espP, 6.4%), colicin production (col D157, 12.8%) and a secretion system II (etpD, 10.6%). None of the strains was positive for a specific enzyme with catalase-peroxidase activity (katP). PMID- 10736004 TI - Influence of growth temperature on cryotolerance and lipid composition of Lactobacillus acidophilus. AB - In order to correlate the lipid composition of the membrane of Lactobacillus acidophilus CRL 640 with the freeze-thaw behaviour of the cultures grown at different temperatures, fatty acid methyl esters (FAMEs) from extracts grown at 25, 30, 37 and 40 degrees C were obtained and compared. Cultures grown at 25 degrees C (M25) exhibited more resistance to the freeze-thaw process probably because of an increase in C18:2 and C16:0 fatty acids. This culture also exhibited a lesser amount of phospholipids as shown by the sugar: phosphorus ratio. In all cases, the presence of the uncommon 10-hydroxyoctadecanoic acid was determined. From the extracts of the M25 and M37 cultures, diacylphosphatidylglycerol, cardiolipin, diglycosyldiglycerides, triglycosyldiglycerides and neutral lipids were isolated and identified. The structural elucidation was carried out by FAMEs and sugar analysis and by mass spectrometry using fast atom bombardment ionization. The changes in lipid composition due to different growth temperatures could be indicative of the resistance of the bacteria to freeze-thaw processes. PMID- 10736005 TI - Enterocin 012, a bacteriocin produced by Enterococcus gallinarum isolated from the intestinal tract of ostrich. AB - Enterococcus gallinarum strain 012, isolated from the duodenum of ostrich, produced enterocin 012 which is active against Ent. faecalis, Lactobacillus acidophilus, Lact. sake, Listeria innocua, Propionibacterium acidipropionici, Propionibacterium sp., Clostridium perfringens, Pseudomonas aeruginosa and Salmonella typhimurium. One of the four pathogenic strains of Escherichia coli isolated from the intestinal tract of ostrich was inhibited by enterocin 012. No antimicrobial activity was recorded against Bacillus cereus, Cl. sporogenes, Cl. tyrobutyricum, Leuconostoc cremoris, Pediococcus pentosaceus, Staphylococcus carnosus and Streptococcus thermophilus. Enterocin 012 was resistant to treatment with lysozyme, catalase, lipase and papain, but sensitive to Proteinase K, alpha chymotrypsin, trypsin and pepsin. Treatment of enterocin 012 with gastric juice from the duodenum resulted in a 50% loss of antibacterial activity. Half of the activity was lost when incubated at 80 degrees C for 30 min, or when kept overnight at a pH of 1.0-5.0 and pH 11.0 and 12.0, respectively. Enterocin 012 production started in mid-logarithmic growth and reached a maximum of 800 AU ml 1, but increased further to 1600 AU ml-1 in the stationary growth phase. The peptide is approximately 3.4 kDa in size, as determined after partial purification with Amberlite XAD-1180 and ammonium sulphate precipitation, followed by tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The mechanism of antimicrobial activity against Lact. sake LMG 13558 is bactericidal and caused cell lysis of active growing cells. PMID- 10736006 TI - Influence of physiological factors on the lysis effect of Cytophaga on the red microalga Rhodella reticulata. AB - The influence of different factors on the lysis of the red microalga, Rhodella reticulata, by Cytophaga sp. LR2 was studied. The pathogenic bacterial strain was more resistant than the alga to the physiological parameters studied, which assured long-term survival of bacteria in algal cultures. Cytophaga sp. LR2 infected R. reticulata at temperatures between 15 and 30 degrees C, in the illuminated as well as the non-illuminated cultures, at pH values between 5.0 and 9.0, and in the presence of NaCl and CaCl2 in the culture medium. SEM showed a different morphology of the bacteria in algal cultures from those of axenic cultures of Cytophaga. Observations of specific associations between algal and bacterial cells revealed that the role of the slime extrusions on the bacterial surface was attachment of Cytophaga to algal cells, and that their clumping leads to rapid lysis. PMID- 10736007 TI - Quantal microbiology. AB - Quantal microbiology describes a similarity between physics and microbiology. In both sciences there is an apparent dichotomy between the certainty and stability of the macro-subject and the uncertainty/complexity of the individual atom/cell. Classical physics is to quantum mechanics as classical microbiology is to quantal microbiology. PMID- 10736008 TI - An evaluation of potential interferences in a fluorimetric assay for the rapid detection of thermotolerant coliforms in sewage. AB - A 1-h fluorimetric assay of beta-D-galactosidase activity was evaluated for determining thermotolerant coliforms (TTC) in sewage samples. Above TTC concentrations of 2.3 x 103 colony-forming units (cfu) 100 ml-1, the assay response was related to TTC concentration. However, below this concentration, a large background signal was observed which was independent of TTC concentration. A separation scheme involving various filtration treatments and additions of a beta-D-galactosidase inhibitor was devised and used to quantify the sources of this anomalous assay response. The interferences encountered were largely due to the presence in sewage of non-specific cell-free enzymes or other cell-free substances that were capable of hydrolysing the fluorogenic substrate. Despite this apparent limitation, the fluorimetric enzyme assay has potential as an 'early warning' indicator of treatment process failure and gross sewage contamination and leakage in situations where TTC concentrations exceed 2.3 x 103 cfu 100 ml-1 PMID- 10736009 TI - Antimicrobial activity of Mitracarpus scaber extract and isolated constituents. AB - The antimicrobial activity of a methanol extract and isolated constituents of Mitracarpus scaber, a species used in folk medicine by West African native people, was evaluated against Staphylococcus aureus and Candida albicans strains. The mitracarpus methanol extract possesses both antibacterial and antimycotic activities (minimum inhibitory concentration-MIC 31.25 and 62.50 microg ml-, respectively). This extract was subsequently fractioned and monitored by bioassays leading to the isolation of seven compounds screened for antibacterial and antimycotic activities. Among these compounds, gallic acid and 3,4,5 trimethoxybenzoic acid inhibited the growth of Staph. aureus (MIC 3.90 and 0.97 microg ml-). 4-Methoxyacetophenone and 3,4,5-trimethoxyacetophenone effectively inhibited C. albicans (MIC 1.95 microg ml-). The other compounds (kaempferol-3-O rutinoside, rutin and psoralen) which were also isolated showed low antibacterial and antimycotic activities (125-500 microg ml-). PMID- 10736010 TI - Isolation of shiga toxin-producing Escherichia coli (STEC) from foods using EHEC agar. AB - Enterohaemorrhagic Escherichia coli (EHEC) agar was evaluated for its ability to recover one isolate of each of three serotypes (O157:H7, O26 and O113:H21) of shiga toxin-producing E. coli (STEC) from raw mince, pasteurized milk and salami after enrichment. The method detected around one colony-forming unit (cfu) in 25 ml in milk, but was less sensitive with salami, requiring 10-1000 cfu 25 g-1 (depending on serotype) for detection. In raw minced beef any enterohaemolysin producing colonies were outnumbered by other colonies and only one of 12 enrichments yielded the inoculum serotype. Additional tests were conducted on 15 retail meat products. One 25-g sample of each product was processed as purchased, while another was inoculated with 157-185 cfu of a cocktail of E. coli O157, O113 and O26 cultures. Recovery was easily achieved with cooked meat products and salami. Recovery from raw minced meat was again difficult, but sometimes possible. Testing more suspect colonies than were tested in this study would presumably increase the sensitivity of the method. PMID- 10736011 TI - Response of Micromonospora echinospora (NCIMB 12744) spores to heat treatment with evidence of a heat activation phenomenon. AB - The effects of heat treatment on spores of the actinomycete Micromonospora echinospora were investigated. The percentage of culturable spores in untreated spore stocks was found to be approximately 20%. A 60 degrees C treatment of spores in phosphate buffer for 10 min led to an approximately five-fold increase in the number of culturable units. This indicated that a large proportion of the spores were constitutively dormant. Within 10 min and in the absence of an external energy-yielding substrate, the heat treatment was found to stimulate spore respiration suggesting that endogenous storage compounds were being utilized. Heating spores at 70 degrees C shortened the time period required for activation; holding times greater than 10 min, however, resulted in a reduction of culturable cells. Classic thermal death characteristics were seen at temperatures of 80 degrees C and above with D-values of 21.43, 2.67, 0.45 and 0.09 min being recorded at 70, 80, 90 and 100 degrees C, respectively. Spores of this organism, while being weakly heat resistant in comparison with bacterial endospores, are significantly more resistant than vegetative cells. PMID- 10736012 TI - Growth of Escherichia coli O157 in poorly fermented laboratory silage: a possible environmental dimension in the epidemiology of E. coli O157. AB - Laboratory silages, inoculated with either c. 1000 cfu g-1, an atoxigenic Escherichia coli O157 or a toxigenic E. coli O157 isolate, were made in plastic bags which permitted limited aerobic spoilage. Replicate bags of each treatment were opened at weekly intervals after incubation at 20 degrees C. In all silages the fermentation was slow and aerobic spoilage with visible moulding ocurred at the tie ends after 7 d. In all the aerobically spoiled silages Enterobacteriaceae reached over 107 cfu g-1 within 1 week. The E. coli in control silages increased from barely detectable levels to 104 cfu g-1 within 13 d; over the same period both strains of E. coli O157 increased from 103 to 107 cfu g-1. The increases in the poorly fermented interior of the silage bags were initially similar but declined slightly as the pH fell. It is suggested that faecal contamination of grass followed by poor silage management may be a factor in the persistence of E. coli O157 carriage in ruminants. PMID- 10736013 TI - Simultaneous molecular subtyping and shiga toxin gene detection in Escherichia coli using multiplex polymerase chain reaction. AB - A robust random amplification of polymorphic DNA (RAPD)-polymerase chain reaction (PCR) protocol was developed for the combined epidemiological typing and shiga toxin detection of clinical shiga toxin-producing O157 and non-O157 Escherichia coli isolates. Using shiga toxin gene-specific primers, combined with two short 10-mer primers, in a multiplex shiga toxin/RAPD-PCR the fingerprints generated allowed differentiation between epidemiologically unrelated strains and allowed identification of a band amplified from the shiga toxin gene(s). Hybridization with a digoxigenin-labelled probe specific for stx1 and stx2 confirmed its identity. The combination of primers in this way allows valuable additional information to be gained from discriminatory RAPD profiles, with further benefits of time and cost savings over tests performed individually. PMID- 10736014 TI - Development of a rapid method for the identification of Lactobacillus spp. isolated from naturally fermented italian sausages using a polymerase chain reaction-temperature gradient gel electrophoresis. AB - A rapid method for the identification of Lactobacillus spp. isolated from naturally fermented Italian sausages was developed. It is based on the amplification of a small fragment from the 16S rRNA gene followed by temperature gradient gel electrophoresis (TGGE). Lactobacillus sakei, L. curvatus, L. alimentarius, L. casei, L. plantarum and L. brevis, obtained from International Collections, were used to optimize the method. Thiry-nine strains of Lactobacillus spp. were isolated from naturally fermented sausages and, after traditional identification, were tested by the PCR-TGGE protocol developed. No differences were observed comparing the results obtained, apart from five strains identified as L. curvatus that showed a PCR-TGGE profile identical to L. sakei. PMID- 10736015 TI - Development of a rapid positive/absent test for coliforms using sensitive bioluminescence assay. AB - We have developed a sensitive bioluminescence assay for beta-galactosidase using a luminescent substrate, D-luciferin-O-beta-galactopyranoside (LuGal). The detection limit for beta-galactosidase was 3 x 10-20 mol per assay, which was approximately 50-fold more sensitive than the test using a fluorescent substrate. This assay was applied to a positive/absent (P/A) test for coliforms. Observations made after 7 h of culture followed by a 10-min enzyme assay using LuGal were comparable to those made after a 22-24-h culture by the current method. Therefore, the LuGal method allows a rapid P/A test for coliforms. PMID- 10736016 TI - The viable but non-culturable state of wine micro-organisms during storage. AB - Colony counting and DEFT did not give the same results when wine micro-organisms were enumerated. Both methods were used to monitor the population of acetic acid bacteria (AAB) and lactic acid bacteria (LAB) during wine storage. Results suggest that part of the populations had reached a viable but non-culturable (VBNC) state. These cells were unable to produce colonies but could hydrolyse fluorescent esters and could be counted by DEFT. For AAB, O2 deprivation quickly induced this state. Recovery from this state was very rapid as soon as O2 was available. The response was not so clear for LAB during wine storage. However, a similar state was induced by sulfiting. Moreover, filtration of wine stored in barrels and contaminated by Brettanomyces, AAB and LAB demonstrated that cell size was not homogeneous. Cells which remained in wine after several weeks could pass through a 0.45-microm membrane. However, when they re-entered a growing phase, they were again retained by membrane filtration. During and after the decline phase, wine micro-organisms might survive as smaller cells in a VBNC state. PMID- 10736017 TI - The effects of preslaughter washing on the reduction of Escherichia coli O157:H7 transfer from cattle hides to carcasses during slaughter. AB - Fresh bovine faeces were inoculated with a non-toxigenic, antibiotic resistant strain of Escherichia coli O157:H7, spread on the rump areas of 30 heifers and allowed to dry for 24 h. Ten of the cattle then entered the normal slaughter process without further treatment. The remaining cattle were washed with a powerhose for 1 min (10 animals) and 3 min (10 animals) before entering the normal slaughter process. Both washing treatments removed all visible faecal materials on the live animals although a significant reduction (P < 0.05) in E. coli O157:H7 levels on the hides was only observed on those animals which were powerhosed for 3 min. After slaughter, E. coli O157:H7 was detected on carcasses and on the knives and hands of operatives. Preslaughter washing for 3 min did not statistically reduce the numbers of E. coli O157:H7 transferred from the hide to the carcass during slaughter. However, the organism was not detected on three of the four areas of the carcass sampled, indicating that washing may be a suitable method of decontamination animal hides before slaughter and as such deserves further investigation. PMID- 10736018 TI - Chitinolytic activity of actinomycetes from a cerrado soil and their potential in biocontrol. AB - The crude enzyme extracts from five actinomycetes selected from a cerrado soil presented very good endochitinolytic activity when compared to a commercial chitinase. Exochitinase and chitobiase activities were also detected. They were identified as Streptomyces, but could not be characterized to species level, probably corresponding to new ones. The crude extracts, obtained from growth on fungal mycelium plus chitin of three of the strains, have shown a very pronounced activity against phytopathogenic fungi. In tests using growing cells, all five strains were active. These data suggest that these strains are potential biocontrol agents. PMID- 10736019 TI - Escherichia coli O26 detection from foods using an enrichment procedure and an immunomagnetic separation method. AB - We found effective enrichment procedures for detecting Escherichia coli O26 in foods using methods that are used for E. coli O157. Ground beef or radish sprouts inoculated with approximately 6 colony-forming units of E. coli O26 were homogenized in 225 ml of various broths. After static incubation at 37 degrees C or 42 degrees C for 6 h or 18 h, we isolated the inoculated bacterium by plating onto Rainbow Agar O157 with novobiocin. In combination with the immunomagnetic separation method, E. coli O26 was isolated from all samples by using enrichment in tryptone soy broth at 37 degrees C for 6 h and in modified E. coli broth with novobiocin (mEC + n) at 42 degrees C for 18 h in ground beef and radish sprouts, respectively. Enrichment in mEC + n at 42 degrees C for 18 h was effective for isolating both E. coli O26 and E. coli O157 from both ground beef and radish sprouts. PMID- 10736020 TI - Low surfactant concentration increases fungal mineralization of a polychlorinated biphenyl congener but has no effect on overall metabolism. AB - Three white rot fungi were compared for their ability to attack polychlorinated biphenyl (PCB) congeners in the presence and absence of the non-ionic Triton X 100 or the anionic Dowfax 8390 surfactants at half their critical micelle concentrations. Neither surfactant affected PCB biodegradation monitored by gas chromatography but the release of 14CO2 from 2,4',5-[U-14C]trichlorobiphenyl by Trametes versicolor was stimulated 12% by Triton X-100. Since mineralization is the complete metabolism of the congener and biodegradation was measured as substrate disappearance, Triton X-100 is proposed to aid intracellular solubilization of 2,4',5-trichlorobiphenyl for complete oxidation by T. versicolor. PMID- 10736021 TI - Construction and expression of functional multi-domain polypeptides in Escherichia coli: expression of the Neurospora crassa metallothionein gene. AB - A system for the construction of polymeric peptides in Escherichia coli was utilized to prepare a library of plasmids coding for tandem repeats of the Neurospora crassa metallothionein gene. Selected oligomeric metallothionein clones were expressed and targeted to the periplasm as a fusion with the maltose binding protein. Bacterial cells harbouring the expressed oligopeptides were characterized for their ability to bind 109Cd2+. The metal-binding ability was enhanced for all the oligomeric constructs tested and, in the best case, a 6.5 fold increased capacity for metal uptake was achieved with cells expressing a tandem 9-mer in comparison with cells expressing a monomer. Plateauing of the metal uptake ability occurred at between six and nine tandem repeats, possibly due to a combination of lowered translation levels, inefficient export and prematurely terminated translation products. The overall enhancement of the heavy metal removal capacity was approximately 65-fold relative to non-recombinant cells. The use of this strategy for the design and expression of de novo polypeptides containing multiple functional domains for use in bioremediation is discussed. PMID- 10736022 TI - Purification and characterization of acetyl esterase from Candida guilliermondii. AB - An extracellular acetyl esterase (EC 3.1.1.6) from Candida guilliermondii NRRL Y 17257 was purified to homogeneity by acetone precipitation and QAE sepharose anion-exchange chromatography. The enzyme was a monomer with an apparent molecular weight of 67 kDa and a pI of 7.6. It had maximum activity at pH 7.5 and at 50-60 degrees C. It was relatively stable over a pH range of 5.8-8.0 and exhibited thermal stability up to 60 degrees C. The Km and Vmax values on alpha naphthylacetate were 2.63 mM and 213.3 micromol alpha- naphthol min-1 mg-1 protein, respectively. PMID- 10736023 TI - The genetics of amphibian declines: population substructure and molecular differentiation in the yosemite toad, Bufo canorus (Anura, bufonidae) based on single-strand conformation polymorphism analysis (SSCP) and mitochondrial DNA sequence data. AB - We present a comprehensive survey of genetic variation across the range of the narrowly distributed endemic Yosemite toad Bufo canorus, a declining amphibian restricted to the Sierra Nevada of California. Based on 322 bp of mitochondrial cytochrome b sequence data, we found limited support for the monophyly of B. canorus and its closely related congener B. exsul to the exclusion of the widespread western toad B. boreas. However, B. exsul was always phylogenetically nested within B. canorus, suggesting that the latter may not be monophyletic. SSCP (single-strand conformation polymorphism) analysis of 372 individual B. canorus from 28 localities in Yosemite and Kings Canyon National Parks revealed no shared haplotypes among these two regions and lead us to interpret these two parks as distinct management units for B. canorus. Within Yosemite, we found significant genetic substructure both at the level of major drainages and among breeding ponds. Kings Canyon samples show a different pattern, with substantial variation among breeding sites, but no substructure among drainages. Across the range of B. canorus as well as among Yosemite ponds, we found an isolation-by distance pattern suggestive of a stepping stone model of migration. However, in Kings Canyon we found no hint of such a pattern, suggesting that movement patterns of toads may be quite different in these nearby parklands. Our data imply that management for B. canorus should focus at the individual pond level, and effective management may necessitate reintroductions if local extirpations occur. A brief review of other pond-breeding anurans suggests that highly structured populations are often the case, and thus that our results for B. canorus may be general for other species of frogs and toads. PMID- 10736024 TI - Molecular systematics, biogeography and population structure of neotropical freshwater needlefishes of the genus Potamorrhaphis. AB - Phylogenetic relationships of populations and species within Potamorrhaphis, a genus of freshwater South American needlefishes, were assessed using mitochondrial cytochrome b sequences. Samples were obtained from eight widely distributed localities in the Amazon and Orinoco rivers, and represented all three currently recognized species of Potamorrhaphis. The phylogeny of haplotypes corresponded imperfectly to current morphological species identities: haplotypes from P. guianensis, the most widespread species, did not make up a monophyletic clade. Geography played a strong role in structuring genetic variation: no haplotypes were shared between any localities, indicating restricted gene flow. Possible causes of this pattern include limited dispersal and the effects of current and past geographical barriers. The haplotype phylogeny also showed a complex relationship between fishes from different river basins. Based on the geographical distribution of clades, we hypothesize a connection between the middle Orinoco and Amazon via rivers of the Guianas. More ancient divergence events may have resulted from Miocene alterations of river drainage patterns. We also present limited data for two other Neotropical freshwater needlefish genera: Belonion and Pseudotylosurus. Pseudotylosurus showed evidence of substantial gene flow between distant localities, indicating ecological differences from Potamorrhaphis. PMID- 10736025 TI - Mitochondrial DNA sequences support allozyme evidence for cryptic radiation of New Zealand Peripatoides (Onychophora). AB - A combination of single-strand conformation polymorphism analysis (SSCP) and sequencing were used to survey cytochrome oxidase I (COI) mitochondrial DNA (mtDNA) diversity among New Zealand ovoviviparous Onychophora. Most of the sites and individuals had previously been analysed using allozyme electrophoresis. A total of 157 peripatus collected at 54 sites throughout New Zealand were screened yielding 62 different haplotypes. Comparison of 540-bp COI sequences from Peripatoides revealed mean among-clade genetic distances of up to 11. 4% using Kimura 2-parameter (K2P) analysis or 17.5% using general time-reversible (GTR + I + Gamma) analysis. Phylogenetic analysis revealed eight well-supported clades that were consistent with the allozyme analysis. Five of the six cryptic peripatus species distinguished by allozymes were confirmed by mtDNA analysis. The sixth taxon appeared to be paraphyletic, but genetic and geographical evidence suggested recent speciation. Two additional taxa were evident from the mtDNA data but neither occurred within the areas surveyed using allozymes. Among the peripatus surveyed with both mtDNA and allozymes, only one clear instance of recent introgression was evident, even though several taxa occurred in sympatry. This suggests well-developed mate recognition despite minimal morphological variation and low overall genetic diversity. PMID- 10736026 TI - Patterns of paternal relatedness in British grey seal colonies. AB - Previous studies of breeding behaviour in the grey seal, Halichoerus grypus, have painted conflicting pictures. Behavioural observations suggest a classical polygynous system with a small number of dominant males fathering most of the offspring. However, genetic analysis suggests that many potential fathers spend little time ashore, that some pairs of seals show partner fidelity and that the dominant males are not as successful as their behaviour would suggest. Here we used paternal relatedness between pups with known mothers, sampled over an 11 year period, to show that behavioural dominance leading to enhanced fitness is a feature of only a handful of males located near the centre of the breeding colony. The vast majority of pups are fathered by any of a large number of males who all share approximately equal success, including virtually all those males who have previously escaped our best sampling efforts. As expected, the frequency of full-sibs is reduced in this longer time series relative to the original study. However, absolute estimates of the frequency of full-sibs seem to be confounded by a tendency for females who produce paternally unrelated pups to have conceived to males who are more genetically dissimilar from each other than expected by chance alone. Together, these elements of breeding behaviour would help to maintain maximum genetic diversity and to minimize the effects of inbreeding. PMID- 10736027 TI - Multiple paternity increases effective size of southern Atlantic salmon populations. AB - Genetic analyses were performed on the progeny of Atlantic salmon (Salmo salar L.) sampled in natural redds of three rivers flowing into the Bay of Biscay, the Nivelle, the Mandeo and the Sella. These rivers are at the southern limit of the European distribution of the species and their populations are small and endangered by human activities. Nine variable number of tandem repeat (VNTR) loci (five minisatellites and four microsatellites) were used for parentage analysis. Multiple male participation was recognized in the fertilization of eggs. A large proportion was fertilized by precociously mature parr. We demonstrate that multiple paternity derived from mature parr is crucial for the conservation of genetic variability in small populations of Atlantic salmon. PMID- 10736028 TI - Postsealing genetic variation and population structure of two species of fur seal (Arctocephalus gazella and A. tropicalis). AB - Commercial sealing in the 18th and 19th centuries had a major impact on the Antarctic and subantarctic fur seal populations (Arctocephalus gazella and A. tropicalis) in the Southern Ocean. The intensive and unrestricted nature of the industry ensured substantial reductions in population sizes and resulted in both species becoming locally extinct at some sites. However, both species are continuing to recover, through the recolonization of islands across their former range and increasing population size. This study investigated the extent and pattern of genetic variation in each species to examine the hypothesis that higher levels of historic sealing in A. gazella have resulted in a greater loss of genetic variability and population structure compared with A. tropicalis. A 316-bp section of the mitochondrial control region was sequenced and revealed nucleotide diversities of 3.2% and 4.8% for A. gazella and A. tropicalis, respectively. There was no geographical distribution of lineages observed within either species, although the respective PhiST values of 0.074 and 0.19 were significantly greater than zero. These data indicate low levels of population structure in A. gazella and relatively high levels in A. tropicalis. Additional samples screened with restriction endonucleases were incorporated, and the distribution of restriction fragment length polymorphism (RFLP) and sequence haplotypes were examined to identify the main source populations of newly recolonized islands. For A. tropicalis, the data suggest that Macquarie Island and Iles Crozet were probably recolonized by females from Marion Island, and to a lesser extent Ile Amsterdam. Although there was less population structure within A. gazella, there were two geographical regions identified: a western region containing the populations of South Georgia and Bouvetoya, which were the probable sources for populations at Marion, the South Shetland and Heard Islands; and an eastern region containing the panmictic populations of Iles Kerguelen and Macquarie Island. The latter region may be a result of a pronounced founder effect, or represent a remnant population that survived sealing at Iles Kerguelen. PMID- 10736029 TI - Patterns of microsatellite polymorphism in the range-restricted bonobo (Pan paniscus): considerations for interspecific comparison with chimpanzees (P. troglodytes). AB - The endangered great ape, Pan paniscus (bonobo) has the smallest range of the African apes. Virtually nothing is known about the genetic diversity or genetic structure of this species, while substantial amounts of polymorphism have been reported for the bonobo's widespread congener, the chimpanzee (P. troglodytes). Given its restricted range, what is the extent of genetic variation in the bonobo relative to the chimpanzee, and is the bonobo genetically depauperate? To investigate patterns of genetic polymorphism, bonobos of wild origin were genotyped for 28 microsatellite loci. The mean number of alleles per locus (5.2) and the mean observed heterozygosity (0.52) in bonobos were similar to variation observed in a wild chimpanzee community (P. t. schweinfurthii). The rarer bonobo is not genetically depauperate and may have genetic diversity comparable to the eastern chimpanzee subspecies. Bonobos have approximately 55% of the allelic diversity and 66% of the observed heterozygosity exhibited by all three chimpanzee subspecies sampled across equatorial Africa. Resampling techniques were used to quantify the effects of sample size differences and number and choice of loci between bonobos and chimpanzees. The examination of these variables underscores their importance in accurately interpreting interspecific comparisons of diversity estimates. PMID- 10736030 TI - Phylogeography and mitochondrial DNA (mtDNA) diversity in North American collared lemmings (Dicrostonyx groenlandicus). AB - Variation in the nucleotide sequence of the mitochondrial control region (250 bp) and the cytochrome b region (870 bp) was examined in collared lemmings (Dicrostonyx groenlandicus) from 19 localities in northern Alaska and the Canadian Arctic. The division of D. groenlandicus in two phylogeographical groups with limited divergence across the Mackenzie River is consistent with the separation of this species in more than one refugial area located to the northwest of the Laurentide ice sheet during the last glaciation. Populations of D.groenlandicus from formerly glaciated areas are no less variable than those in nonglaciated areas. Instead, the low intrapopulation and intraregional diversity estimates in D. groenlandicus are probably a result of regional bottleneck events due to range contractions during Holocene warming events. These results are consistent with findings previously reported on collared lemmings (D. torquatus) from the Eurasian Arctic. PMID- 10736031 TI - Microsatellite DNA analysis of genetic effects of harvesting in old-growth eastern white pine (Pinus strobus) in Ontario, Canada. AB - Microsatellite DNA markers from 13 simple sequence repeat (SSR) loci were used to compare genetic diversity between preharvest pristine and postharvest residual gene pools of two adjacent virgin, old-growth ( approximately 250 years) stands of eastern white pine (Pinus strobus L.) in Ontario. There was concurrence in genetic diversity changes in the postharvest gene pools of the two stands. The total and mean numbers of alleles detected in each stand were reduced by approximately 26% after tree density reductions of approximately 75%. Approximately 18 and 21% of the low-frequency (0. 25 > P > or = 0.01) alleles and 76 and 92% of the rare (P < 0.01) alleles were lost from residual stands A and B, respectively, after harvesting. Multilocus gametic diversity was reduced by 38 and 85% and genotype additivity by approximately 50% in the residual stands after harvesting. Latent genetic potential of each stand was reduced by approximately 40%. Although heterozygosity was reduced (1-5%) in the postharvest residual stands, the reductions were not substantial and not comparable to those using other genetic diversity measures. The reductions in genetic diversity measures were slightly higher than those theoretically expected in postbottleneck populations according to drift theory. In the absence of substantial gene migration that could ameliorate the genetic losses, the ability of the postharvest white pine gene pools to adapt to changing environmental and disease conditions may have been compromised. The microsatellite DNA results for genetic effects of harvesting in old-growth eastern white pine stands were similar to those that we reported earlier from allozyme analysis (Buchert et al. 1997). The results indicate that silvicultural practices should ensure that the gene pools of remaining pristine old-growth stands are reconstituted in the regenerating stands. PMID- 10736032 TI - Intraspecific phylogeography in the sedge frog Litoria fallax (Hylidae) indicates pre-Pleistocene vicariance of an open forest species from eastern Australia. AB - The eastern sedge frog Litoria fallax (Anura: Hylidae) is common throughout the open forests and coastal wetlands along the eastern coast of Australia. Its range spans four biogeographical zones from northern Queensland to central New South Wales. Phylogenetic analysis of mitochondrial DNA (mtDNA) haplotypes of 87 L. fallax individuals from 22 populations identified two major mtDNA lineages, differing by 11-12% sequence divergence. The two clades of haplotypes were separated by the McPherson Range, indicating that this mesic upland area has acted as a major long-term barrier to gene flow for this open forest species. Slight isolation by distance was observed within both the northern and southern lineages but was insufficient to explain the large sequence divergence between lineages. Within the northern lineage, additional phylogeographical structure was observed across the relatively dry Burdekin Gap which separates Atherton populations from all populations in the central and eastern Queensland biogeographical zones. There was less phylogeographical structure in the southern lineage suggesting historical gene flow across the drier portions of the Great Dividing Range. These data, together with recent observations of deep phylogeographical divergences in rainforest-restricted Litoria suggest that the east coast hylids of Australia represent an old (Tertiary) radiation. Individual species of Litoria have been strongly affected by climatic and ecological barriers to gene flow during the Quaternary. PMID- 10736033 TI - Chloroplast DNA variation in a rainforest tree (Aucoumea klaineana, burseraceae) in Gabon. AB - One of the dominant savannah colonists in Gabon is Aucoumea klaineana or Okoume (Burseraceae), an endemic species which belongs to a monotypic genus. Chloroplast DNA (cpDNA) variation was studied in this species by means of PCR amplification of 40 kb of cpDNA sequences, followed by restriction analysis of the resulting fragments. No insertion/deletion events were noted, and a single point mutation was found. The level of differentiation among the 19 populations studied was relatively low (GST = 0.54) compared to other plant species (mean of 0.76), in agreement with the pioneer status of the species. However, cpDNA diversity was geographically structured, with the less frequent haplotype occurring only in populations from southern Gabon. This distribution might suggest either that there were two ancient source populations of Okoume, one in the north and the other in the south, from which the colonizing process of the savannah began after the last ice age, or alternatively that there was one polymorphic source in the south. The low level of cpDNA diversity could indicate that Okoume populations in these refugia were quite small. PMID- 10736034 TI - Polymorphic microsatellite markers in the ornate dragon lizard, Ctenophorus ornatus. PMID- 10736035 TI - Polymorphic DNA microsatellites in the domestic pigeon, Columba livia var. domestica. PMID- 10736036 TI - A suite of highly polymorphic microsatellite markers in turbot (Scophthalmus maximus L.) with potential for use across several flatfish species. PMID- 10736037 TI - Five microsatellite loci in eelgrass Zostera marina and a test of cross-species amplification in Z. noltii and Z. japonica. PMID- 10736038 TI - Isolation and characterization of microsatellite loci in the Japanese marsh warbler Locustella pryeri. PMID- 10736039 TI - Development of primer sets to amplify fragments of conserved genes for use in population studies of the fungus Daldinia loculata. PMID- 10736040 TI - Novel microsatellite loci isolated from the northern krill, Meganyctiphanes norvegica (Crustacea, Euphausiacea). PMID- 10736041 TI - Spatial and temporal aspects of the genetic structure of Juniperus communis populations. AB - Juniperus communis is a dioecious, wind pollinated shrub or small tree that produces 'berries' (female cones) containing a small number of seeds that are thought to be dispersed by birds. The expectation, therefore, would be that populations of Juniper are genetically diverse with little structuring between them. In Britain, the species has two main centres of distribution: a highland zone in the north and west, in which populations are still large and sexually reproducing, and a southern zone on chalk downlands in which populations are small and fragmented and individuals suffer from a decline in fertility. Thus, one would expect the large sexually viable populations in the north to exhibit high levels of within-population genetic variation, while the declining southern populations would be genetically depauperate. The analysis of amplified fragment length polymorphisms (AFLPs) was used to test this hypothesis. Surprisingly, all populations studied showed high levels of genetic variation although there was clear structuring between populations. On the basis of the geographical structuring of the populations it was hypothesized that J. communis colonized Britain via three separate routes. PMID- 10736042 TI - Geographic distribution and frequency of a taurine Bos taurus and an indicine Bos indicus Y specific allele amongst sub-saharan African cattle breeds. AB - We report for the first time, and for the whole of sub-Saharan Africa, the geographical distribution and the frequency of an indicine and a taurine Y specific allele amongst African cattle breeds. A total of 984 males from 69 indigenous African populations from 22 countries were analysed at the microsatellite locus INRA 124. The taurine allele is probably the oldest one on the continent. However, the taurine and the indicine alleles were present in 291 males (30%), and 693 males (70%), respectively. More particularly, 96% of zebu males (n = 470), 50% of taurine males (n = 263), 29% of sanga males (crossbreed Bos taurus x Bos indicus, n = 263) and 95% of zebu x sanga crossbred males (n = 56) had the indicine allele. The Borgou, a breed classified as zebu x taurine cross showed only the zebu allele (n = 12). The indicine allele dominates today in the Abyssinian region, a large part of the Lake Victoria region and the sahelian belt of West Africa. All the sanga males (n = 64) but only one from the Abyssinian region had the indicine allele. The taurine allele is the commonest only among the sanga breeds of the southern African region and the trypanotolerant taurine breeds of West Africa. In West Africa and in the southern Africa regions, zones of introgression were detected with breeds showing both Y chromosome alleles. Our data also reveal a pattern of male zebu introgression in Mozambique and Zimbabwe, probably originating from the Mozambique coast. The sanga cattle from the Lake Victoria region and the Kuri cattle of Lake Chad, cattle populations surrounded by zebu breeds were, surprisingly, completely devoid of the indicine allele. Human migration, phenotypic preferences by the pastoralists, adaptation to specific habitats and to specific diseases are the main factors explaining the present-day distribution of the alleles in sub Saharan Africa. PMID- 10736043 TI - Mitochondrial sequence analysis of Salamandra taxa suggests old splits of major lineages and postglacial recolonizations of central Europe from distinct source populations of Salamandra salamandra. AB - Representatives of the genus Salamandra occur in Europe, Northern Africa and the Near East. Many local variants are known but species and subspecies status of these is still a matter of dispute. We have analysed samples from locations covering the whole expansion range of Salamandra by sequence analysis of mitochondrial D-loop regions. In addition, we have calibrated the rate of divergence of the D-loop on the basis of geologically dated splits of the closely related genus Euproctus. Phylogenetic analysis of the sequences suggests that six major monophyletic groups exist (S. salamandra, S. algira, S. infraimmaculata, S. corsica, S. atra and S. lanzai) which have split between 5 and 13 million years ago (Ma). We find that each of the Salamandra species occupies a distinct geographical area, with the exception of S. salamandra. This species occurs all over Europe from Spain to Greece, suggesting that it was the only species that has recolonized Central Europe after the last glaciation. The occurrence of specific east and west European haplotypes, as well as allozyme alleles in the S. salamandra populations suggests that this recolonization has started from at least two source populations, possibly originating in the Iberian peninsula and the Balkans. Two subpopulations of S. salamandra were found that are genetically very distinct from the other populations. One lives in northern Spain (S. s. bernardezi) and one in southern Italy (S. s. gigliolii). Surprisingly, the mitochondrial lineages of these subpopulations group closer together than the remainder S. salamandra lineages. We suggest that these populations are remnants of a large homogeneous population that had colonized Central Europe in a previous interglacial period, approximately 500 000 years ago. Animals from these populations were apparently not successful in later recolonizations. Still, they have maintained their separate genetic identity in their areas, although they are not separated by geographical barriers from very closely related neighbouring populations. PMID- 10736044 TI - Phylogeography of the pitviper clade Agkistrodon: historical ecology, species status, and conservation of cantils. AB - We used mitochondrial DNA sequences from three gene regions and two tRNAs (ND4, tRNA-HIS-SER, 12S, and 16S rDNA) to investigate the historical ecology of the New World pitviper clade Agkistrodon, with emphasis on the disjunct subspecies of the cantil, A. bilineatus. We found strong evidence that the copperhead (A. contortrix) is basal to its congeners, and that the cottonmouth (A. piscivorus) is basal to cantils. Phylogeography and natural history of the living terminal taxa imply that Agkistrodon primitively occupied relatively temperate habitats, with subsequent evolution of tropicality in ancestral A. bilineatus. Our best supported phylogeny rejects three gulf arc scenarios for the biogeography of A. bilineatus. We find significant statistical support for an initial divergence between populations on the east and west coasts of Mexico and subsequent occupancy of the Yucatan Peninsula, by way of subhumid corridors in northern Central America. Based on phylogenetic relationships, morphological and molecular divergence, and allopatry we elevate A. b. taylori of northeastern Mexico to species status. Taylor's cantil is likely threatened by habitat destruction and small geographical range, and we offer recommendations for its conservation and management. PMID- 10736045 TI - Nuclear DNA microsatellite analysis of genetic diversity and gene flow in the Scandinavian brown bear (Ursus arctos). AB - In the 1930s, the Scandinavian brown bear was close to extinction due to vigorous extermination programmes in Norway and Sweden. Increased protection of the brown bear in Scandinavia has resulted in the recovery of four subpopulations, which currently contain close to 1000 individuals. Effective conservation and management of the Scandinavian brown bear requires knowledge of the current levels of genetic diversity and gene flow among the four subpopulations. Earlier studies of mitochondrial DNA (mtDNA) diversity revealed extremely low levels of genetic variation, and population structure that grouped the three northern subpopulations in one genetic clade and the southernmost subpopulation in a second highly divergent clade. In this study, we extended the analysis of genetic diversity and gene flow in the Scandinavian brown bear using data from 19 nuclear DNA microsatellite loci. Results from the nuclear loci were strikingly different than the mtDNA results. Genetic diversity levels in the four subpopulations were equivalent to diversity levels in nonbottlenecked populations from North America, and significantly higher than levels in other bottlenecked and isolated brown bear populations. Gene flow levels between subpopulations ranged from low to moderate and were correlated with geographical distance. The substantial difference in results obtained using mtDNA and nuclear DNA markers stresses the importance of collecting data from both types of genetic markers before interpreting data and making recommendations for the conservation and management of natural populations. Based on the results from the mtDNA and nuclear DNA data sets, we propose one evolutionarily significant unit and four management units for the brown bear in Scandinavia. PMID- 10736046 TI - Molecular tracking of mountain lions in the Yosemite valley region in California: genetic analysis using microsatellites and faecal DNA. AB - Twelve microsatellite loci were characterized in California mountain lions (Puma concolor) and sufficient polymorphism was found to uniquely genotype 62 animals sampled at necropsy. Microsatellite genotypes obtained using mountain lion faecal DNA matched those from muscle for all of 15 individuals examined. DNA from potential prey species and animals whose faeces could be misidentified as mountain lion faeces were reliably distinguished from mountain lions using this microsatellite panel. In a field application of this technique, 32 faecal samples were collected from hiking trails in the Yosemite Valley region where seven mountain lions previously had been captured, sampled, and released. Twelve samples yielded characteristic mountain lion genotypes, three displayed bobcat type genotypes, and 17 did not amplify. The genotype of one of the 12 mountain lion faecal samples was identical to one of the mountain lions that previously had been captured. Three of the 12 faecal samples yielded identical genotypes, and eight new genotypes were detected in the remaining samples. This analysis provided a minimum estimate of 16 mountain lions (seven identified by capture and nine identified by faecal DNA) living in or travelling through Yosemite Valley from March 1997 to August 1998. Match probabilities (probabilities that identical DNA genotypes would be drawn at random a second time from the population) indicated that the samples with identical genotypes probably came from the same mountain lion. Our results demonstrate that faecal DNA analysis is an effective method for detecting and identifying individual mountain lions. PMID- 10736047 TI - Comparison of genetic diversity of the invasive weed Rubus alceifolius poir. (Rosaceae) in its native range and in areas of introduction, using amplified fragment length polymorphism (AFLP) markers. AB - Theory predicts that colonization of new areas will be associated with population bottlenecks that reduce within-population genetic diversity and increase genetic differentiation among populations. This should be especially true for weedy plant species, which are often characterized by self-compatible breeding systems and vegetative propagation. To test this prediction, and to evaluate alternative scenarios for the history of introduction, the genetic diversity of Rubus alceifolius was studied with amplified fragment length polymorphism (AFLP) markers in its native range in southeast Asia and in several areas where this plant has been introduced and is now a serious weed (Indian Ocean islands, Australia). In its native range, R. alceifolius showed great genetic variability within populations and among geographically close populations (populations sampled ranging from northern Vietnam to Java). In Madagascar, genetic variability was somewhat lower than in its native range, but still considerable. Each population sampled in the other Indian Ocean islands (Mayotte, La Reunion, Mauritius) was characterized by a single different genotype of R. alceifolius for the markers studied, and closely related to individuals from Madagascar. Queensland populations also included only a single genotype, identical to that found in Mauritius. These results suggest that R. alceifolius was first introduced into Madagascar, perhaps on multiple occasions, and that Madagascan individuals were the immediate source of plants that colonized other areas of introduction. Successive nested founder events appear to have resulted in cumulative reduction in genetic diversity. Possible explanations for the monoclonality of R. alceifolius in many areas of introduction are discussed. PMID- 10736048 TI - Do discrepancies between microsatellite and allozyme variation reveal differential selection between sea and lagoon in the sea bass (Dicentrarchus labrax)? AB - In the present study the genetic structure of Dicentrarchus labrax (14 samples from the Mediterranean) was analysed at six microsatellite loci, in order to test the hypothesis that some enzymatic loci undergo selection between marine and lagoon habitat. Eight of the 14 samples were analysed at both microsatellite and allozyme markers. The analysis of the genetic variation among the Mediterranean samples showed that (i) &Fcirc;ST values obtained with the six microsatellite loci were much smaller than those obtained with the 28 allozymes and (ii) microsatellite loci seemed to reflect more the geographical proximity than an ecological one. Thirteen enzymatic loci exhibited moderate to high values compared with microsatellites. This was interpreted as evidence that these allozymes are non-neutral. However, only six loci seemed to be implicated in differentiation between marine and lagoon samples, the causes of selection being unknown for the others. A possible scenario of population dynamics of the sea bass between marine and lagoon habitat is suggested. PMID- 10736049 TI - Random amplified polymorphic DNA analysis of southern brown bandicoot (Isoodon obesulus) populations in Western Australia reveals genetic differentiation related to environmental variables. AB - Random amplified polymorphic DNA (RAPD) markers were used to analyse genetic variation within and between populations of Isoodon obesulus in Western Australia. Genetically controlled geographical variation in body size associated with habitat type and rainfall exists in this species, raising the question of whether local conditions may influence gene flow in I. obesulus. The RAPD markers displayed substantial genetic variation, with all animals possessing unique RAPD phenotypes over 39 polymorphic bands produced by three primers. Significant geographical subdivision was apparent (PhiST = 0.208) with southwest locations being divergent from all others, despite there being no physical barriers to gene flow. The pattern of subdivision was unrelated to physical distance between the locations, but was related to both annual rainfall and habitat type. Therefore, the most reasonable explanation for this pattern of subdivision appears to be that gene flow is restricted by selection against migrants between local populations with substantially different habitat type or rainfall. Restriction of gene flow through selection against migrants is rarely investigated, and the results of this study suggest that the importance of this process in the formation of population structure may be underestimated. PMID- 10736050 TI - Comparative genetic diversity of parasites and their hosts: population structure of an urban cockroach and its haplo-diploid parasite (oxyuroid nematode). AB - Few studies have investigated the genetic structure of both host and parasite populations at a level of populations and at a level of individuals. We investigated the genetic structure of the urban cockroach Blattella germanica and its oxyuroid parasite Blatticola blattae. Random amplified polymorphic DNA (RAPD) markers were used to quantify genetic diversity between and within four populations (from two cities in France) of the host and its parasite. Diversity based on phenotypic frequencies was calculated for each RAPD marker using Shannon Wiener's index. We used multivariate analyses to test the significance of genetic differentiation between host and parasite populations. Analysis of molecular variance was also used. Both methods gave similar results. Diversity between pairs of individuals was estimated by Nei & Li's index. Genetic diversity was higher within host or parasite populations (80% and 82%, respectively, of explained diversity) than between host or parasite populations (20% and 18%, respectively, explained diversity). The genetic distances between pairs of parasite populations (or individuals) were not correlated with the genetic distances between the corresponding pairs of host populations (or individuals). PMID- 10736051 TI - GeoDis: a program for the cladistic nested analysis of the geographical distribution of genetic haplotypes. PMID- 10736052 TI - Microsatellite markers for the fossa (Cryptoprocta ferox). PMID- 10736053 TI - Isolation of polymorphic microsatellite loci from the malaria vector Anopheles funestus. PMID- 10736054 TI - Characterization of highly polymorphic microsatellite markers in the marsupial honey possum (Tarsipes rostratus). PMID- 10736055 TI - Characterization of microsatellite loci in the redclaw crayfish, Cherax quadricarinatus. PMID- 10736056 TI - Novel microsatellite markers for the European oyster Ostrea edulis. PMID- 10736057 TI - Novel polymorphic microsatellite loci isolated from the yellow waterlily, Nuphar lutea. PMID- 10736058 TI - Characterization of novel microsatellite loci isolated from the tropical dioecious tree Simarouba amara. PMID- 10736059 TI - Isolation and characterization of microsatellite loci in the land snail Helicella itala, and cross-species amplification within the family Helicidae. PMID- 10736060 TI - Microsatellite loci in Macrotermes michaelseni (Isoptera: termitidae). PMID- 10736061 TI - Editorial PMID- 10736062 TI - Staging of multiple sclerosis (MS) lesions: pathology of the time frame of MS. AB - Several processes take place during an attack of demyelination in multiple sclerosis (MS). The timing of these various processes, and thus of the attack in its entirety, is important if therapeutic stratagies are to be planned. Attempts have been made to introduce and investigate variables relevant to timing the disease processes, leading to staging systems for MS. Here, the terminology and the various parameters used are reviewed, including inflammatory cells, glial cells, axonal loss and myelin staining; then the different systems are compared, including the system put forward by Bo and Trapp, our own modification of that, the Bruck and Lassmann system and the recent consensus reached at a Vienna meeting. It is concluded that an ideal staging system does not yet exist, and that, more than anything else, the material dictates the choice for a staging system. The terminology of the Vienna consensus could be used as a reference to facilitate international comparison. PMID- 10736063 TI - Increased expression of gelatinases and alteration of basement membrane in rat soleus muscle following femoral artery ligation. AB - Acute severe muscle ischaemia is characterized by significant remodelling of basement membranes of myofibres. It was hypothesized that peripheral artery insufficiency is accompanied by similar muscle extracellular matrix (ECM) changes involving matrix metalloproteinase gelatinases. Using a model of femoral artery ligation, both gelatinase activity and basement membrane component degradation were studied in hindlimb skeletal muscles. SDS-PAGE zymography of muscle homogenates showed that acute moderate ischaemia was followed by a significant transient increase in expression of 72- and 92-kDa gelatinases during 48 h; the latter probably originated from inflammatory cells. In situ zymography showed that this increase occurred chiefly at the periphery of myofibres. Immunolocalization demonstrated 72-kDa gelatinase in interspaces and at the periphery of myofibres, and suggested that this enzyme may explain the gelatinolytic activities found by in situ zymography. Type IV collagen and laminin staining showed that the gelatinase expression increase correlated with dramatic basement membrane component alterations. Our data show that even moderate ischaemia results in significant muscle basement membrane remodelling due to matrix degrading enzymes matrix metalloproteinases (MMP) gelatinases. PMID- 10736064 TI - Establishment and characterization of a human cell line from paediatric cerebellar glioblastoma multiforme. AB - Permanent glioma cell lines are invaluable tools in understanding the biology of glioblastomas. The present study reports the establishment of a clonal human cell line, GBM6840, derived from a biopsy of paediatric cerebellar glioblastoma multiforme. GBM6840 had a doubling time of 32 h and grew as a monolayer of large round cells that retained immunopositivity for glial fibrillary acidic protein and vimentin. Karyotypic analysis revealed a modal chromosome number of 68 and polysomies of chromosomes 3, 5 and 20, as well as the presence of 3-4 marker chromosomes. GBM6840 also showed anchorage-independent growth in soft agar and tumour formation in nude mice. The p16(CDKN2A) gene was transcriptionally silenced by hypermethylation, consistent with the lack of protein expression observed in the original tumour and cultured cells. Western blot analysis revealed normal protein expression of pRb and CDK4. It appears that p16 is the major component altered in the cell cycle pathway and may confer these cells unrestrained proliferation potential. Neither EGFR gene amplification nor over expression of the protein was detected in the cultured cells. Over-expression of the p53 protein was observed in the majority of cells, despite undetectable mutation (exons 5-8) in the gene. One allele of the PTEN gene was found to be mutated during in vitro cultivation. Telomerase activity was demonstrated in the cultured cells but not in the original tumour, supporting the hypothesis that telomerase is required for the in vitro immortalization process. PMID- 10736065 TI - Possible role of the superoxide anion in the development of neuronal tolerance following ischaemic preconditioning in rats. AB - There is a large body of evidence that reactive oxygen species play a major role in the pathogenesis of ischaemic brain damage. On the other hand, it has recently been suggested that superoxide anions participate in the development of neuronal tolerance against lethal ischaemia following ischaemic preconditioning (PC). The present study aimed to examine whether or not the intravenous administration of human recombinant Cu/Zn superoxide dismutase (hr SOD) prior to PC would affect the subsequent development of neuronal tolerance. Animals were randomly assigned to the following three groups: group 1, sham PC treated with vehicle; group 2, PC treated with hr SOD and group 3, PC treated with vehicle. For PC, 10 min occlusion of the middle cerebral artery (MCA) by a modified intraluminal suture method was followed by 60 min recirculation and this procedure was successively repeated three times. The procedures were similar for sham PC except that the MCA was kept unoccluded. Just prior to PC or sham PC, a bolus of hr SOD (6 x 103 IU/2 ml/kg) was administered intravenously. Seventy-two hours thereafter, rats were subjected to lethal ischaemia, i.e. MCA occlusion for 100 min followed by recirculation for 48 h. The infarct area and volume were assessed with the 2,3,5 triphenyltetrazolium stain. A significant difference in the infarct volume was revealed between the sham PC/vehicle and the PC/vehicle groups (total and cortex P < 0.01; striatum P < 0.05), showing that PC induced a marked neuronal tolerance against lethal ischaemia. The infarct volume in the PC/SOD group was close to that in the sham PC/vehicle group, being significantly greater than that in the PC/vehicle group (total and cortex P < 0.01) and showing that the administration of hr SOD suppressed the development of neuronal tolerance induced by PC. In a parallel experiment, expression of 72-kDa heat-shock protein (hsp 72) at 72 h after PC was considerably reduced in rats treated with hr SOD compared with those treated with vehicle. These results suggest that superoxide anions intraluminally generated within cerebral microvessels participate in the development of neuronal tolerance as well as the induction of hsp 72 following PC. PMID- 10736066 TI - Synapse loss associated with abnormal PrP precedes neuronal degeneration in the scrapie-infected murine hippocampus. AB - Numbers of neurones, synapses and axon terminals were quantified in a murine scrapie model with severe hippocampal pyramidal cell loss, in which definite clinical scrapie is evident from 226 days post-infection (dpi) and death occurs around 250 dpi. Disease-specific PrP accumulations were first seen at 70 dpi (28% of the incubation period (IP)) in thalamus and as sparse foci within the stratum pyramidale of CA1. By 98 dpi (39% IP), PrP was seen in the stratum radiatum and was found at later stages throughout all levels of the hippocampus. At the ultrastructural level in the stratum radiatum of CA1, a decrease in the numbers of simple synapses from 84 dpi (34% IP) and in perforated synapses from 98 dpi (42% IP) was found using an unbiased stereological method, the disector analysis. Degeneration of axon terminals was found from 98 dpi (39% IP) onwards. Neuronal loss was detected in CA1 from 180 dpi (72% IP). The results suggest that the fundamental lesion in the hippocampus of ME7-infected mice is associated with PrP release from CA1 pyramidal neurones, which perturbs synaptic function and leads to degeneration of preterminal axons, and that subsequent pathological changes including neurone loss are sequelae to this initial insult. PMID- 10736067 TI - Neuronal death in brain infarcts in man. AB - The mechanism of neuronal death in brain ischaemia remains unclear. Morphology, terminal transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) and immunohistochemistry for the pro-apoptotic enzyme caspase-3 (CASP3), for its substrates poly(ADP-ribose) polymerase (PARP) and the DNA-dependent protein kinase catalytic subunit (DNA-PKCS) and for poly(ADP-ribose) (PAR), an end product of PARP activity, were used to investigate neuronal death in brain infarcts from 15 men and 20 women, aged 46-95 years. The infarcts varied in age from 18 h to several months. Neuronal death was characterized morphologically by cell shrinkage, cytoplasmic hypereosinophilia and moderate nuclear pyknosis with later chromatin dispersal and disintegration, but not features of apoptosis. Occasional apoptotic bodies were seen but these appeared to be related to inflammatory cells, endothelial cells and occasional glia, including satellite cells. Neurones within infarcts showed strong nuclear and cytoplasmic labelling for CASP3 during the first 2 days after infarction. Neuronal DNA-PKCS, PARP and poly(ADP-ribose) immunoreactivity was demonstrable in scattered neurones in and adjacent to infarcts for 18-24 h but thereafter declined to below detectable levels in most cases. TUNEL labelled cells towards the edge of the infarcts, particularly at 2-4 days, but most of the labelling could be prevented by preincubation of the sections in diethyl pyrocarbonate to inactivate endogenous nucleases. Between 3 days and 3 weeks, CASP3 and DNA-PKCS were detected in proliferating capillaries and CASP3, PARP and poly(ADP-ribose) in infiltrating macrophages. Our findings indicate that neuronal death in human brain infarcts has some of the early biochemical features of programmed cell death, with upregulation of CASP3 and rapid disappearance of DNA-PKCS and PARP. However, the morphological changes are not those of apoptosis, the DNA cleavage occurs relatively late, and some of the TUNEL is probably mediated by the release of endogenous endonucleases during protease or microwave pretreatment of the damaged tissue. PMID- 10736068 TI - Molecular analysis of alterations of the p18INK4c gene in human meningiomas. AB - Meningiomas are common primary brain tumours frequently presenting with deleted and/or mutated NF2 gene located on 22q.1p has been reported as the second most commonly deleted chromosomal region in these neoplasms. A new member of the INK4 family of CDK inhibitors, the p18INK4c gene, has recently been mapped to this chromosomal arm. By virtue of its structural and functional similarities with the p16 gene, p18 has been implicated as a tumour suppressor gene in a variety of cancers. In this paper 40 human meningiomas were analysed for loss of heterozygosity (LOH) at the p18 locus, mutations and inactivating methylation of the p18 gene. LOH at D1S193, D1S463 and D1S211 microsatellite marker loci mapped to 1p32 was detected in 13 of 35 (37%), four of 20 (20%), and six of 24 (25%) tumour samples, respectively. One sample presented with homozygous deletion at D1S193. Mutational analysis using single stranded conformational polymorphism (SSCP) and direct sequencing did not detect any missense mutation but revealed a novel silent mutation, G to T, at coding nucleotide 435. Analysis of HgaI, BsaHI, ScrFI and Eco0109I restriction sites of p18 exon 1 revealed absence of inactivating methylation. Immunohistochemistry with p18 monoclonal antibody detected presence of cytoplasmic p18 staining in 21 of 22 examined samples. One sample did not stain and was shown to carry homozygous deletion at D1S193. Despite the high frequency of LOH at 1p32 microsatellite markers, the lack of genetic and epigenetic aberrations in the p18 gene together with the presence of p18 protein in all but one meningioma samples argues against the role of p18 as a tumour suppressor gene important for meningioma development. PMID- 10736069 TI - Differential expression of S100 calcium-binding proteins characterizes distinct clinical entities in both WHO grade II and III astrocytic tumours. AB - The computer-assisted microscopic analysis of Feulgen-stained nuclei enabled us to identify two subgroups of astrocytomas (WHO grade II) and two subgroups of anaplastic astrocytomas (WHO grade III) with significantly distinct clinical outcomes (Decaestecker et al. Brain Pathol 1998; 8: 29-38). The astrocytomas labelled in the present study as typical (TYP-ASTs) behaved clinically like real astrocytomas while atypical astrocytomas (ATYP-ASTs) behaved similarly to anaplastic astrocytomas. The anaplastic astrocytomas that we labelled as typical (TYP-ANAs) behaved clinically like anaplastic astrocytomas while atypical ones (ATYP-ANAs) behaved like glioblastomas. In the present study, we investigate whether some biological characteristics could be evidenced across these four groups of TYP- and ATYP-ASTs and TYP- and ATYP-ANAs. The data show that the levels of expression (immunohistochemically assayed and quantitatively determined by means of computer-assisted microscopy) of vimentin, the glial fibrillary acidic protein and the platelet-derived growth factor-alpha did not differ significantly across these four groups of astrocytic tumours. The level of cell proliferation (determined by means of both the anti-proliferating cell nuclear antigen and the anti-MIB-1 antibodies; P < 0.001 to P < 0.0001) differed very significantly between the astrocytomas and anaplastic astrocytomas, but not between the typical and atypical variants identified in each group. In sharp contrast, the levels of expression of the S100A3 and S100A5 proteins differed markedly in the solid tumour tissue in relation to the astrocytic tumour types and grades. In addition, while the levels of expression of S100A6 did not change in the astrocytic tumour tissue in relation to histopathological grade, the levels of expression of this S100 protein (but not those of S100A3 and S100A5) differed markedly in the blood vessel walls according to whether these vessels originated from low- or high-grade astrocytic tumours. PMID- 10736070 TI - Membranous expression of glucose transporter-1 protein (GLUT-1) in embryonal neoplasms of the central nervous system. AB - The human erythrocyte GLUT-1 is a transmembrane protein which facilitates transport of glucose in the cell in an energy-independent fashion. Neuroectodermal stem cells show strong membrane immunoreactivitry with this marker at early developmental stages in rodents. Membranous expression by undifferentiated neuroectodermal cells gradually decreases while GLUT-1 becomes confined to the endothelial cells, when these acquire blood-brain barrier function. We thus sought to determine whether GLUT-1 expression was limited to embryonal neoplasms of the central nervous system (CNS) which are presumably derived from developmentally arrested neuroectodermal stem cells. Archival material of 40 primary CNS neoplasms were examined for immunoreactivity with anti GLUT-1. This included both non-embryonal neoplasms (18 astrocytic tumours, one ependymoma and three oligodendroglioma) and embryonal neoplasms (12 cerebellar medulloblastomas, four supratentorial PNETs and two atypical teratoid/rhabdoid tumours (AT/RhT)). In addition, cell lines and nude mice xenografts derived from both undifferentiated and differentiated tumours were assessed for GLUT-1 immunoreactivity by both immunohistochemistry and Western blotting. All embryonal tumours, MBs and PNET xenografts consistently showed GLUT-1 membrane staining. Non-embryonal neoplasms were negative except for vascular staining. Membrane protein fraction of embryonal tumours cell lines immunoreacted by immunoblot with GLUT-1, whereas the glioblastoma cell line was negative. Expression of GLUT-1 supports the stem cell nature of the cells of origin of MBs, supratentorial PNET and AT/RhTs. As a result, GLUT-1 is a useful marker to define the embryonal nature of CNS neoplasms. PMID- 10736071 TI - Humidification in paediatric anaesthesia. PMID- 10736072 TI - Perioperative management of children with congenital phakomatoses. PMID- 10736073 TI - Propofol and pain on induction: the effect of injectate temperature in children. AB - A prospective, randomized, double-blind study was conducted to determine whether cooled intravenous propofol reduces the incidence of pain on induction of anaesthesia in children. Seventy patients aged 3-10 years, ASA I or II, were randomized to receive 1% propofol plus 0.05% lidocaine either at room temperature (20-23 degrees C) or cooled to 4C. Pain was assessed by a blinded observer using a behavioural scale. Data from 69 patients were analysed. The incidence of pain was 9/34 (26%, 95% confidence intervals 11-41%) in the room temperature injectate group and 5/35 (14%, 95% confidence intervals 3-26%) in the cold injectate group. These differences were not statistically significant (P=0.21, chi-squared test). Pain scores were similar in both groups. PMID- 10736074 TI - Onset and duration of action of rocuronium in children receiving chronic anticonvulsant therapy. AB - The onset and time course of action of rocuronium in normal children and children receiving anticonvulsant drugs for prolonged periods was characterized. A single bolus dose of 0.6 mg.kg-1 rocuronium was administered i.v. to seven nonepileptic patients on no medication, and eight patients on chronic anticonvulsant therapy consisting of either phenytoin, carbamazepine, or both who were age and weight matched. Neuromuscular transmission was monitored by the evoked compound electromyography of the thenar muscles using train of four stimulation every 20 s. Recovery times of the first twitch to 10%, 25%, 50%, 75% and 100% of baseline values and recovery index were obtained. The onset times were 1.05+/-0.5 and 1.41+/-0.5 min for the control and anticonvulsant groups respectively and were not significantly different. Children receiving chronic anticonvulsant therapy had significantly shorter recovery index than the control group (control 10.4+/ 5.1 min, anticonvulsant 4.8+/-1.7 min, P<0.05). Furthermore, the duration of recovery to 10%, 50%, 75% and 100% of baseline T1 values was less in the anticonvulsant drug group. Our data confirm resistance to rocuronium in children on chronic anticonvulsant drugs. PMID- 10736075 TI - The caudal canal in children: a study using magnetic resonance imaging. AB - The anatomy of the caudal (sacral extradural) space was studied in 41 children, using magnetic resonance imaging. The distance from the upper margin of the sacrococcygeal membrane to the dural sac, the length of the membrane and the maximum depth of the caudal space were each measured. Age, height, weight and body surface area were recorded and, using multiple linear regression (stepwise technique), equations predicting the length of the membrane and the distance between its upper margin and the lower limit of the dural sac were obtained. Wide variability limits the clinical usefulness of these equations. In all patients, the maximum depth of the caudal space was found to be at the upper margin of the sacrococcygeal membrane. No correlation was found between this maximum depth and the age, height, weight or body surface area of the child. PMID- 10736076 TI - Pharmacokinetics of ropivacaine following caudal analgesia in children. AB - Ropivacaine has a favourable toxicity profile for epidural anaesthesia in adults, so it may also be an appropriate agent for epidural analgesia in children. We therefore designed this study to determine the pharmacokinetic variables of ropivacaine relevant to the risk of toxicity, after caudal administration in children. We studied nine healthy children, aged 1-6 years who received 1 ml.kg-1 of ropivacaine 0.25% for caudal analgesia. Venous blood samples were collected at intervals for 12 h after injection. Total plasma concentration of ropivacaine was assayed by high performance liquid chromatography, and pharmacokinetic descriptors were estimated from the plasma concentration-time data. The median peak venous plasma concentration was 799 microg.l-1 [interquartile range (IQR) 707-1044 microg.l-1], and was reached at a median time of 1.5 h (IQR 0.5-2 h). The mean elimination half-life was 3.9 h (95% CI 2.7-5.0 h), and the mean apparent clearance and volume of distribution were 7.6+/-1. 6 ml.min-1.kg-1 (95% CI 6.1-9.1 ml.min-1.kg-1) and 2.4+/-0.6 l.kg-1 (95% CI 1.9-3.0 l.kg-1), respectively. Analgesia was satisfactory in all cases and no systemic ropivacaine toxicity was observed. Caudal administration of weight-adjusted doses of ropivacaine to children resulted in systemic exposure similar to that reported for adults. No systemic toxicity was observed. The findings strengthen predictions that the relative systemic safety of epidural ropivacaine in adults will apply to children. However, the pharmacokinetics and safety of epidural ropivacaine need to be studied further in children with circumstances that affect drug disposition and systemic tolerance. PMID- 10736077 TI - Tunnelling of caudal epidural catheters in infants. AB - The working conditions in the developing world necessitate the development of many adaptations and improvizations of accepted anaesthetic techniques to improve patient care. Subcutaneous tunnelling of caudally placed epidural catheters is one such improvization to prevent the soiling of the catheter by urine and faeces. This study compares the duration of retention of catheter in a group with tunnelled catheters with an untunnelled group. The absence of a catheter emerging through skin at the site of catheter insertion hastens healing thus prolonging the retention of catheter in the tunnelled group and soiling ceases to be a major problem. PMID- 10736078 TI - An assessment of desflurane for use during cardiac electrophysiological study and radiofrequency ablation of supraventricular dysrhythmias in children. AB - Desflurane has several properties making it a desirable agent for use in electrophysiological studies (EPS) for diagnosis and treatment of cardiac dysrhythmias. We studied 47 children, mean age 12.8+/-4.6 years, mean weight 52.9+/-24.0 kg, with clinical history of supra- ventricular tachycardia (SVT) during EPS using desflurane in a crossover comparison with fentanyl. The patients served as their own controls. All received oral premedication with lorazepam, and intravenous induction with thiopentone, rocuronium, and oxygen. Group 1 (n=24) were administered fentanyl 10 microg.kg-1 bolus i.v. with an infusion of 3 microg.kg-1.h-1 during initial EPS. Fentanyl was discontinued and desflurane, 6% endtidal, was administered and the EPS repeated. Group 2 (n=23) were initially administered 6% desflurane after induction, and following EPS the desflurane was discontinued and the patients administered fentanyl 3 microg.kg-1 bolus and EPS repeated (explanations of EPS abbreviations are provided). Desflurane reduced the mean arterial pressure (MAP) in all patients. In Group 1, desflurane shortened the sinus cycle length (SCL), i.e. increasing the heart rate, and atrial effective refractory period (AERP) while Group 2 demonstrated no such effect on AERP. There were no other significant differences between fentanyl or desflurane techniques in terms of EPS measurements. SVT was inducible with both agents in both groups. Desflurane seems an acceptable agent for use during EPS procedures. PMID- 10736079 TI - Continuous monitoring of oesophageal pH during general anaesthesia with laryngeal mask airway in children. AB - We studied the incidence of gastro-oesophageal reflux (GOR) during general anaesthesia with the laryngeal mask airway (LMA) in a paediatric population with two ventilatory regimes: spontaneous breathing and controlled mechanical ventilation (CMV). Thirty children between 6 months and 15 years, ASA I-II, for routine surgery, were randomly assigned in two groups: spontaneous ventilation (n=14), and CMV (n=16). A pH probe was situated in the central third of the oesophagus. Some 66% of the patients breathing spontaneously had GOR episodes vs. 92% of the patients with CMV (P < 0,01). Reflux took place mainly after LMA removal (21% vs. 68%; P < 0,01) and in the Postanaesthetic Care Unit (PACU) (29% vs. 43%; P < 0,05). There was a high incidence of GOR during general anaesthesia and in the PACU in paediatric patients anaesthetized with the LMA. GOR episodes were significantly more evident in the CMV group, mainly after LMA removal, but without clinical significance. PMID- 10736080 TI - Continuous epidural butorphanol relieves pruritus associated with epidural morphine infusions in children. AB - We examined the efficacy of epidural butorphanol to either prevent or relieve pruritus associated with epidural morphine infusion in children. Forty-six children were randomized to receive either epidural morphine (M) or epidural M with butorphanol (B) for postoperative analgesia. They received bupivacaine and either M 50 microg.kg-1 or the same dose of M plus B 10 microg.kg-1. Following surgery, a continuous infusion of 0.1% bupivacaine with either M 20 microg.ml-1 or M 20 microg.ml-1 + B 4 microg.ml-1 was given at a rate of 0.3 ml.kg-1.h-1. Pain scores and pruritus scores were recorded every 4 h during epidural infusion. Subjects with a pruritus score=2 received diphenhydramine 0.5 mg.kg-1 i.v. and were switched to an alternate epidural infusion; subjects receiving M (group M) were switched to M+B while subjects receiving M+B (group B) were switched to hydromorphone (H) 4 microg.ml-1. There was no difference in the initial incidence of pruritus (group M 11/18; group B 13/28). No subject in group M required a second change of epidural infusion because of continued pruritus after being switched to M+B; five of 13 subjects in group B continued to experience pruritus after being switched to H and required a second change of epidural infusion or an alternate analgesic modality (P=0.038). The median pruritus score in the first 24 h after changing epidural infusions was 0 in subjects in group MDelta (changed from M to M+B) and 1 in subjects in group BDelta (changed from M+B to H; P=0.012). While the median sedation score in the first 24 h was 1 in both groups, there was a greater incidence of sedation scores of 2 in group B than group M (28% vs 12.3%; P=0.021). B 10 microg.kg-1 was not effective in preventing pruritus associated with bolus epidural administration of M 50 microg.kg-1 in children. B 1.2 microg.kg-1. h-1 was effective in relieving pruritus associated with continuous epidural infusion of M 6 microg.kg-1.h-1. PMID- 10736082 TI - Tropisetron plus dexamethasone is more effective than tropisetron alone for the prevention of postoperative nausea and vomiting in children undergoing tonsillectomy. AB - The 5-HT3 antagonists are effective in reducing postoperative nausea and vomiting (PONV) associated with paediatric tonsillectomy. Although prophylactic tropisetron can reduce the incidence of PONV by half, the resulting level of over 40% is still unacceptably high. The aim of this study was to evaluate the effect of adding dexamethasone to tropisetron. In a blinded study, 59 children (mean age 6.1 years) were administered 0.1 mg.kg-1 up to 2 mg of tropisetron and 66 children (mean age 5.7 years) received the same dose of tropisetron plus 0.5 mg.kg-1 up to 8 mg of dexamethasone. Both drugs were given intravenously during induction of anaesthesia for tonsillectomy. During the inpatient stay of 24 h, the incidence of postoperative vomiting in the tropisetron alone group was 53% compared with 26% in the combination group (P=0.002, chi-squared). A significant reduction in nausea from 53% to 30% was also observed (P=0.02). Parents completed a daily diary for 5 days following discharge. Delayed vomiting occurred in 27% and 11% of the tropisetron and combination therapy groups, respectively (P=0.025) Sixteen percent and 9%, respectively, required medical attention (P=0.27). Tropisetron plus dexamethasone is more effective than tropisetron alone in reducing the incidence of PONV following paediatric tonsillectomy. PMID- 10736081 TI - Haemodynamic, acid-base and electrolyte changes during plasma replacement with hydroxyethyl starch or crystalloid solution in young pigs. AB - We investigated haemodynamic, acid-base and electrolyte changes during almost total plasma replacement with hydroxyethyl starch (HES) and physiological balanced electrolyte solution (PBE) by using a cell saver in ten young pigs. In the PBE group an additional 3550 (444) ml crystalloid solution [Mean (SD)] was infused over the course of the study in order to maintain pulmonary capillary wedge pressure. Plasma protein levels decreased in both groups and the colloid osmotic pressure increased in HES and decreased in PBE. At the end of the study, body weight [HES 10.4 (1), PBE 13.1 (1.4) kg, P < 0.01] and lactic acid concentration [HES 0.9 (0.3), PBE 2.9 (1. 3) mmol.l -1, P < 0.01] was higher and tissue oxygen delivery [HES 327 (22), PBE 89 (29) ml.min.m2, P < 0.01] was lower in the PBE group. There were only moderate acid-base changes in both groups, but at the end, anion gap was significant lower in HES. In conclusion, maintenance of colloid osmotic pressure close to the physiological range of infants seems to be advantageous during major paediatric surgery. PMID- 10736083 TI - A comparison of rectal and intramuscular codeine phosphate in children following neurosurgery. AB - Codeine is frequently used for postoperative analgesia in children. Intramuscular injections are not ideal and the rectal route may be preferable. We compared rectal and intramuscular codeine administered following neurosurgery. 20 children (over 3 months) undergoing elective neurosurgical procedures, were randomized to receive either rectal or intramuscular codeine phospate (1 mg.kg-1) at the end of the procedure. Serum levels of codeine and morphine were assayed at intervals following administration (0, 30, 60, 120, 240 min). Fentanyl was the intraoperative analgesic and postoperative rescue analgesia was paracetamol, diclofenac and intramuscular codeine. The Children's Hospital of Eastern Ontario Pain Scale was used to assess analgesia. Peak codeine levels in both groups were observed at 30 min and morphine levels were consistently low. The plasma codeine levels were significantly greater at 30 and 60 min following intramuscular injection, and were associated with slightly better analgesia scores, but did not reach statistical significance. However, the peak plasma level occurred at similar times in both groups. Codeine is absorbed as rapidly via the rectal route compared with the intramuscular route but the peak levels are lower. PMID- 10736084 TI - Quality of sedation during mechanical ventilation. AB - The aim of the study was to determine the quality of sedation in ventilated patients on a general paediatric intensive care unit (PICU), including those treated with infusions of neuromuscular blocking agents. Twenty-eight ventilated children on a PICU had their level of sedation determined using an arousability scale dependent upon the response to tracheal suction. Observed levels of sedation were then compared to a predetermined desired level of sedation. A total of 81 assessments were performed and 90% were considered satisfactory. Thirty-two of these assessments were performed in 15 children following the temporary discontinuation of infusions of neuromuscular blocking agents; 97% of these assessments were considered satisfactory. A regime of continuous intravenous midazolam and morphine with additional oral sedation using chloral hydrate and antihistamines when required provides a satisfactory level of sedation for the majority of children ventilated on a PICU, including those treated with infusions of neuromuscular blocking agents. PMID- 10736085 TI - Traumatic pharyngo-oesophageal perforation in the newborn: a condition mimicking oesophageal atresia. AB - Two newborn infants with traumatic perforation of the pharyngo-oesophageal region are presented. This injury was induced by pharyngeal suction catheters and/or vigorous attempts at nasogastric or tracheal intubation during resuscitation of the newborn. The true nature of this condition remained unrecognized and the babies were thus referred with a tentative diagnosis of oesophageal atresia. The perforation itself could be treated successfully without surgery, despite a severe complication in one infant resulting from inadvertent use of barium sulphate contrast medium. Raising awareness of the possibility of this injury should help in avoiding this complication by gentle and skilful action during newborn resuscitation, particularly in the premature infant. PMID- 10736086 TI - Superior vena cava obstruction and liver transplantation in a child. AB - We report a case of superior vena cava obstruction in a child, which was probably secondary to long-term central venous cannulation. The obstruction was asymptomatic preoperatively, but became evident during liver transplantation, and complicated the intraoperative management. There is one other case report of this occurring in an adult in similar circumstances, and we believe that ours is the first report of such a presentation in the paediatric age group. PMID- 10736087 TI - Anaesthetic management for a left pneumonectomy in a child with bronchopleural fistula. AB - The anaesthetic management of a left pneumonectomy in a 18-month-old girl with a bronchopleural fistula is described. An ordinary tracheal tube was slit at the bevel to ensure upper lobe ventilation on right endobronchial intubation. A combination of a bronchial blocker, endobronchial intubation with a slit tube, and nerve blocks for these manoeuvres was used. Pain relief by a thoracic epidural block ensured good physiotherapy and a comfortable postoperative period. PMID- 10736088 TI - Anaesthesia for liver transplantation in a patient with methylmalonic acidaemia. AB - Liver transplantation is an accepted modality of treatment in some patients with inborn errors of metabolism. We present a child with methylmalonic acidaemia who had successful liver transplantation. The pathophysiology, medical and anaesthetic managements are discussed. PMID- 10736089 TI - Beta 2-microglobulin/CD8 -/- mice reveal significant role for CD8+ T cells in graft rejection responses in beta 2-microglobulin -/- mice. AB - Beta 2-microglobulin (beta 2m) -/- mice have often been used as a model to investigate host resistance to grafted tissues in the absence of CD8+ T cells. However, the realization that beta 2m -/- mice have a small pool of CD8+ T cells imply that these cells may take part in immune responses in vivo. To directly address the role of CD8+ T cell responses in beta 2m -/- mice, we introduced a CD8 null mutation into these mice. The beta 2m/CD8 -/- mice and the corresponding control mice were primed, and challenged with syngeneic tumour grafts. While beta 2m -/- mice readily cleared such tumour grafts, similar tumour grafts grew progressively in a dose dependent manner in the beta 2m/CD8 -/- mice. The present results imply that residual CD8+ T cells in beta 2m -/- mice may carry out significant biological functions, and suggest that studies using beta 2m -/- mice as a model for CD8+ T cell deficiency must be regarded with some caution. PMID- 10736090 TI - Blockade of the B7-CD28 pathway by CTLA4-Ig counteracts rejection and prolongs survival in small bowel transplantation. AB - Allograft rejection involves T-cell activation, requiring T-cell receptor interactions with major histocompatibility complex (MHC) molecules and costimulatory signals delivered through the B7-CD28 pathway. We evaluated the effect of blocking this pathway on graft rejection and survival, in a rat experimental model of small bowel transplantation. Heterotopic small bowel transplantation was performed between PVG donor rats and DA recipient rats. The recipient animals were treated with CTLA4-Ig or irrelevant immunoglobulin (Ig)G as control and followed for 18, 30 or 90 days. The survival rate and degree of inflammation and accumulation of CD4+ T cells and macrophages were determined in the transplanted bowels. We found that administration of CTLA4-Ig significantly improved the survival rate compared to control rats: after 30 days 73% of the treated rats had survived and at 90 days 5/8 rats were still living, whereas in the control group only 2/8 rats had survived. The grafts showed preserved mucosal structure with only a mild degree of subacute inflammation and the accumulation of CD4+ T cells and macrophages was noticeably reduced in treated animals as compared to control rats. Necrosis was extensive in control rats, whereas CTLA4 Ig treated animals had grafts with at least some preserved villus morphology and no necrotic tissue. Although small bowel transplantation has proven exceptionally difficult, in this study we have shown that CTLA4-Ig treatment may provide a promising strategy to prevent rejection and induce long term tolerance and graft survival. PMID- 10736091 TI - Apoptotic cells actively inhibit the expression of CD69 on Con A activated T lymphocytes. AB - Although apoptosis is commonly viewed as a silent cell death without damage to adjacent tissues, the effect of apoptosis on immunity has been unclear. We have investigated the influence of apoptotic cells on T-cell activation. The K562 or HL-60 human leukemia cell lines that had been induced apoptosis by FTY720 or cycloheximide (CHX) were added into the culture of mouse spleen cells stimulated with Con A. Six to 20 h later, the expression of CD69, an early T-cell activation antigen, was detected using flowcytometry. Living cells and necrotic cells served as control groups. Apoptotic K562 or HL-60 cells induced by either FTY720 or CHX unanimously inhibited CD69 expression on the CD3+ mouse T cells while living and necrotic cells did not. The inhibition was proportional to the number of apoptotic cells and was different in the T-cell subsets, showing a rapid and transient inhibition on the CD3+CD8+ T-cell activation but with a slow and continuous inhibition on CD3+CD8- T-cell activation. In conclusion, the apoptotic cells actively inhibit a T-cell activation that is independent of the cell lines or the apoptotic inducers, indicating that the apoptotic cells dominantly regulate T-cell immunity. PMID- 10736092 TI - Schistosoma mansoni in mice: the pattern of primary cercarial exposure determines whether a secondary infection post-chemotherapy elicits a T helper 1- or a T helper 2-associated immune response. AB - Reinfection with Schistosoma mansoni following chemotherapy often results in an ameliorated granulomatous reaction and hence a mild disease. This study examined some of the immunological mechanisms that could be associated with this residual protection. BALB/c mice were infected with either a single dose (group A) of 100 S. mansoni cercariae or with 10 doses of 10 cercariae each (group B) given at 3 day intervals. The mice were treated with praziquantel 8 weeks postinfection and, 2 weeks later, together with another group of naive mice (group C), they were infected with a single dose of 100 cercariae each. All the animals were killed 8 weeks later and schistosome egg antigen (SEA)- and soluble adult worm antigen preparation (SWAP)-induced cytokine recall responses in splenocytes, as well as serum immunoglobulin levels, were quantified and hepatic granuloma sizes measured. Group A animals had higher levels of SEA-induced interferon-gamma (IFN gamma) but lower levels of interleukin (IL)-5 than groups B and C (P < 0.01). Group B animals had low SEA-induced IFN-gamma levels and elevated IL-5 levels, although these were lower than group C. SEA-induced IL-10 was low in both groups A and B as compared to group C (P < 0.01). SWAP was less effective as an inducer of splenocyte cytokine production than SEA but both SWAP-induced IFN-gamma and IL 5 were detected in groups A and C. SEA- and SWAP-specific immunoglobulin E (IgE) and immunoglobulin G (IgG) titres were not significantly different between the three groups. Granuloma diameters were larger in group C (mean 297 +/- 51.3 microm) as compared to groups A (174 +/- 49 microm, P < 0.01) and B (247.5 +/- 44 microm, P < 0.05). Taken together, these results demonstrate that granuloma size is reduced during a reinfection exposure compared with a primary infection. This reduction is associated with a T helper 1 response in mice exposed to a single large dose of cercariae in the primary infection and with a predominantly T helper 2 response in those infected with multiple small doses. PMID- 10736093 TI - Lipopolysaccharide induces expression of APO2 ligand/TRAIL in human monocytes and macrophages. AB - Monocytes express cytotoxic factors of the tumour necrosis factor (TNF) ligand superfamily, including TNF and Fas ligand, both on the cell surface and in secreted form. In this report, we show that human monocytes and monocyte-derived macrophages stimulated with lipopolysaccharide (LPS) express APO2 ligand (APO2L, TRAIL), a recently discovered cytotoxic member of the TNF ligand superfamily. LPS increased the transcription of APO2L mRNA in monocytes and macrophages. Flow cytometric analysis showed low surface and high intracellular levels of APO2L, and LPS increased the expression of both. In addition, LPS increased the monocyte and macrophage-mediated cytotoxicity against the APO2L-sensitive Jurkat and RPMI 8226 cells. Addition of the APO2L-binding decoy receptor 1 (DcR1)-Fc fusion protein inhibited the cytotoxicity by 30-70%. LPS also stimulated the release of soluble APO2L from the monocytes and macrophages. Monocytic phagocytosis of target cells was increased by LPS and partially inhibited by DcR1-Fc. Taken together, these data demonstrate a novel mechanism of cytotoxicity mediated by LPS-activated human monocytes and macrophages. PMID- 10736094 TI - Naive monocytes can trigger transendothelial migration of peripheral blood cells through the induction of endothelial tumour necrosis factor-alpha. AB - In this manuscript we describe a potentially new mechanism by which unstimulated human monocytes activate endothelial cells (EC) through the secondary induction of endothelial tumour necrosis factor alpha (TNF-alpha). Serum free supernatants (SN) of peripheral blood mononuclear cells (PBMC) strongly induce the expression of intercellular adhesion molecule 1 (ICAM-1, CD54), vascular cell adhesion molecule 1 (VCAM-1, CD106), and endothelial-leukocyte adhesion molecule 1 (ELAM 1, CD62E) on human EC 24 and 4 h post treatment, respectively. Further characterization of the responsible subpopulation revealed the CD14+ monocytes and a monocytic cell line (MM6) to produce an endothelial activating factor (EAF). The EAF also triggers an adhesion and a transendothelial migration (TEM) of peripheral blood cells. Using neutralization with an anti TNF-alpha MoAb MAK195, EAF is not identical with TNF-alpha, but induces the expression of endothelial TNF-alpha, since MAK195 blocked TEM only when coincubated with EC, not with monocytes. Furthermore, intracellular TNF-alpha was significantly upregulated in EC after treatment with SN-MM6. Another evidence for a secondary autocrine mechanism was provided by culturing the EC with a conditioned medium of SN-MM6 treated EC. This conditioned medium induces an adhesion molecule expression and TEM in a similar way to SN-MM6 and can completely be inactivated by anti TNF-alpha. Taken together, these data may have an impact for, e.g. transplantational settings that donor monocytes may trigger an inflammatory response in the absence of further activation signals by eliciting an endogenous TNF-alpha response in the host. PMID- 10736095 TI - Interleukin-12 amplifies the M. leprae hsp65-cytotoxic response in the presence of tumour necrosis factor-alpha and interferon-gamma generating CD56+ effector cells: interleukin-4 downregulates this effect. AB - Interleukin-12 (IL-12) is a major immunomodulatory cytokine that represents a functional bridge between the early resistance and the subsequent antigen specific adaptive immunity. TNF-alpha and IFN-gamma have an important role in the generation of hsp65 specific cytotoxic T lymphocytes (CTL) that lyse hsp65-pulsed autologous macrophages (hsp65 CTL). Since a positive feedback mechanism between TNF-alpha, IFN-gamma and IL-12 has been described, we undertook to evaluate the role of IL-12 on the hsp65 CTL generation in leprosy patients. Our results show that the presence of IL-12 during the first 24 h of the in vitro antigen stimulation amplifies the hsp65 cytotoxic response whenever both IFN-gamma and TNF-alpha are present. The addition of these three cytokines (CKs) was able to abrogate the inhibitory effect of IL-10 on hsp65 CTL in cells from paucibacillary patients (PB) but not that of IL-4 in PB and normal controls (N). Both IL-12 or anti IL-4 enhanced the cytotoxic activity in cells from multibacillary patients (MB). Anti IL-4 upregulated the binding of IFN-gamma and did not modify that of TNF-alpha so the low CTL activity could be as a result of IL-4 by a decrease of the IFN-gamma binding on MB cells. Cells from those MB patients taking thalidomide (MB-T) did neither bind IFN-gamma nor TNF-alpha even when antigen or anti-IL-4 were added, demonstrating that thalidomide inhibits either the in vitro binding or receptor expression of both TNF-alpha and IFN-gamma. Development of CD56 effector cells during the hsp65 stimulation was observed in PB and N by the addition of IL-12 plus TNF-alpha and IFN-gamma, while in MB and MB-T anti IL-4 was also required. So, the inhibitory effect of IL-4 on either production of IFN gamma, TNF-alpha and/or IL-12 or their receptors could be the mechanism underlying the lack of the hsp65 CTL generation in cells from MB. PMID- 10736096 TI - Monocytes secrete interleukin-6 when co-cultured in vitro with benign or malignant autologous fragment spheroids from squamous cell carcinoma patients. AB - Biopsies from tumour and benign mucosa were removed from patients with head and neck squamous-cell carcinoma (HNSCC), chopped into cubes and transferred to a nonadhesive culture system where in vitro fragment (F)-spheroids were established. The F-spheroids stabilized within 14 days of culture in vitro with epithelial cells and fibroblasts on the surface. F-spheroids were co-cultured with freshly isolated autologous monocytes. The monocytes of 10 of 11 patients secreted interleukin (IL)-6 at a level similar to that of the average monocyte endotoxin-stimulated response. Secreted IL-1beta or tumour necrosis factor-alpha (TNF-alpha) levels greater than 0.1 times the endotoxin-stimulated secretion were determined in one and two of the 11 co-culture experiments, respectively. This different monocyte response to F-spheroids compared with endotoxin stimulation was also present at the mRNA expression level. HNSCC monocytes secreted no IL-6 after co-culture with autologous fibroblasts. When monocytes and F-spheroids were cultured separated by a semipermeable membrane, the IL-6 supernatant level was only approximately 25% of that observed during co-culture with direct contact. F spheroids secreted only trace amounts of IL-6. In conclusion, monocytes of HNSCC patients generally secrete IL-6, but not IL-1beta or TNF-alpha, after stimulation with epithelial-associated components of F-spheroids upon direct contact and in part by a soluble substance. PMID- 10736097 TI - Immature B cells in bone marrow express Fas/FasL. AB - Negative selection is a process by which autoreactive lymphocytes are eliminated from the developing antigen receptor repertoire. The mechanisms regulating negative selection of immature B lymphocytes in the bone marrow are poorly elucidated. Human bone marrow cells were examined in order to investigate the presence of the members of the Fas (APO-1/CD95) system. Here we demonstrate the expression of Fas in immature B lymphocytes (CD10/CD19+/CD40+/sIg+), and the presence of Fas natural ligand (FasL) in CD19+ bone marrow cells. The observed expression of apoptosis-related molecules might indicate how negative selection of autoreactive B cells can occur in human bone marrow. PMID- 10736098 TI - Resistance of extrathymic T cells to stress and the role of endogenous glucocorticoids in stress associated immunosuppression. AB - When mice were exposed to restraint stress for 12 or 24 h, severe lymphopenia was induced in all immune system organs, including the liver and the thymus. However, in adrenalectomized mice, this response was completely absent. Phenotypic characterization revealed that interleukin (IL)-2Rbeta+CD3int cells (i.e. extrathymic T cells) with CD4+ phenotype and the NK1.1+ subset of CD3int cells (i.e. NKT cells) in the liver as well as the mature conventional T cells in the thymus were resistant to such stress. In adrenalectomized mice, there was no significant change in the distribution of lymphocyte subsets in all tested organs before stress. Interestingly, the number of lymphocytes in the liver and spleen and the proportion of NKT cells in the liver rather increased after stress in these adrenalectomized mice. Therefore, endogenous steroid hormones were indicated to be important in the induction of immunosuppressive states after stress. Among stress associated cytokines, the secretion of tumour necrosis factor (TNF)-alpha was completely suppressed while that of IL-6 was partially suppressed in adrenalectomized mice. These results suggest that endogenous steroid hormones are important for the induction of the stress associated immunosuppression and that NKT cells are resistant to stress, namely, resistant to exposure to endogenous steroid hormones. PMID- 10736099 TI - Diminished expression of CD59 on activated CD8+ T cells undergoing apoptosis in systemic lupus erythematosus and Sjogren's syndrome. AB - The present study was undertaken to examine the phenotype of T cells undergoing in vitro apoptosis in patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and primary Sjogren's syndrome (SS). Compared with normal controls, we found diminished expression of CD59 antigen (one of the cell-surface complement-regulatory proteins) on CD8+ T cells, but not on CD4+ T cells, from patients with SLE and SS. Three-colour immunofluorescence analysis revealed that these CD59dim CD8+ T cells were activated T cells, expressing both human leucocyte antigen (HLA)-DR and CD45RO antigens. In addition, these CD59dim CD8+ T cells were more susceptible to in vitro apoptosis than CD59bright CD8+ T cells. In two patients with active lupus, the percentage of CD59dim CD8+ T cells was significantly decreased after steroid therapy. These findings suggest that decreased expression of CD59 antigen on in vivo-activated CD8+ T cells may be correlated with disease activity and may be involved in activation-induced apoptosis in patients with SLE and SS. PMID- 10736100 TI - Increased N-linked glycosylation leading to oversialylation of monomeric immunoglobulin A1 from patients with Sjogren's syndrome. AB - Increased serum immunoglobulin A (IgA) level is a common finding in primary Sjogren's syndrome (pSS). IgA might not be properly eliminated because of an abnormal glycosylation. We reported previously that IgA1 from patients with pSS was oversialylated. We extend this finding by showing that monomeric IgA1 contains more sialic acid (SA) in patients than in controls, as determined by enzyme-linked immunosorbent assay (ELISA) and Western blot with Sambucus nigra agglutinin (SNA), a lectin specific for SA. To localize this excess of SA on the N- and/or O-linked oligosaccharides, we analysed them separately, using N- and O linked oligosaccharide profiling kits based on fluorophore-assisted carbohydrate electrophoresis. N-linked, but not O-linked, oligosaccharides of patients' IgA1 were oversialylated, and this seemed to be linked to an excess of SA on the same number of polysaccharides as normal IgA1. To localize the abnormality to the Fab and/or Fc fragments, monomeric IgA1 was digested with protease, separated and transferred to nitrocellulose, where SA was identified by SNA. Both Fab and Fc fragments appeared to be oversialylated. Oversialylation of N-linked oligosaccharides of IgA1 from patients with pSS might prevent the recognition of IgA by receptors that are responsible for their clearance, resulting in an excess of serum IgA and related immune complexes. PMID- 10736101 TI - Enhanced sialyltransferase activity in B lymphocytes from patients with primary Sjogren's syndrome. AB - Despite the indisputable role of immunoglobulin (Ig)A in the pathogenesis of primary Sjogren syndrome (pSS), the causative abnormality remains largely unknown. As an extension of our report that IgA is oversialylated in this disease, the thrust of the present study was to measure the sialyltransferase (ST) activity in B lymphocytes. ST containing lysates of B cells from 17 pSS patients and 10 controls, were obtained using a combination of detergents, and incubated with affinity purified IgA that had been previously desialylated. The deposition of cytidine 5' monophosphate sialic acid (SA) by ST from B cells onto IgA was detected by two ELISA based upon the use of biotinylated lectins (Sambucus nigra agglutinin which is specific for alpha2-6 SA and Maackia amurensis which is specific for alpha2-3 SA). In parallel, the amount of SA on IgA from ten of the 17 patients and eight of the 10 controls was assayed using the same method. An excess of alpha2-3 and alpha2-6 SA on IgA was found in those patients with excessive activity of alpha2-3 and alpha2-6 ST. Thus, IgA hypersialylation in pSS patients may result from undue activity of ST. PMID- 10736102 TI - Elevated CD40 ligand expressing blood T-cell levels in multiple sclerosis are reversed by interferon-beta treatment. AB - Myelin protein reactive CD4+ T cells are considered to be involved in the proposed immunopathogenesis of multiple sclerosis (MS). One particularly important molecule for T-cell activation is the CD40L (gp39) that is expressed on the surface of T cells. This study focuses on the CD40 and the CD40L expression on mononuclear cells prepared from blood from patients with MS, other neurological diseases (OND) and healthy subjects. Immunostaining followed by a three channel flow cytometry was adopted. Patients with MS had higher levels of CD3+CD40L+, CD4+CD40L+ and CD8+CD40L+ T cells compared to patients with OND and healthy subjects. Cross-sectional comparisons revealed that the elevation of CD40L+ T cell subtypes was confined to the patients with untreated MS and not observed in the patients with MS treated with interferon-beta (IFN-beta). Follow up studies showed that levels of CD3+CD40L+ and CD4+CD40L+ T cells decreased in individual patients after the initiation of the IFN-beta treatment. The enhanced expression of CD40L on CD3+, CD4+ and CD8+ T cells in patients with MS may implicate a role for this molecule in disease immunopathogenesis. PMID- 10736103 TI - In vitro interleukin-13 production by peripheral blood in patients with newly diagnosed insulin-dependent diabetes mellitus and their first degree relatives. AB - It is generally accepted that proinflammatory cytokines secreted by macrophages/monocytes as well as cytotoxic T cells are responsible for pancreatic B-cell destruction in animal models of autoimmune diabetes and presumably in insulin-dependent diabetes mellitus (IDDM) in humans. The aim of the present study was to evaluate the production of interleukin (IL)-13-a Th2 cells derived anti-inflammatory cytokine, by peripheral blood of newly diagnosed IDDM patients and their first degree relatives with a low or high risk of IDDM development. The study was carried out in 20 patients with a recent onset of type 1 diabetes, their first degree relatives with high (with DRB1*03 and/or DRB1*04 HLA class II alleles and two or more autoantibodies directed against pancreatic B-cell antigens) (n = 20) or a low (with DQB1*0602 allele) risk of type 1 diabetes development (n = 10) and a control age matched group of healthy volunteers (n = 18). IL-13 concentrations in supernatant of 72 h cultures of peripheral blood after incubation with phytohemagglutinin (PHA) or PHA+ insulin were quantified by enzyme-linked immunosorbent assay (ELISA). The levels of IL-13 in the supernatants were significantly lower in at high risk of IDDM first degree relatives of diabetic patients (P < 0.02), higher in subjects with low genetic risk of diabetes type 1 (P < 0.02), and normal in IDDM patients in comparison to the control group. We have also observed that the adding of human insulin to the cultures resulted in a significant increase of in vitro IL-13 production in prediabetics, but not in the other studied groups. In conclusion our findings suggest that the IL-13 alterations could play an important role in the pathogenesis of type 1 diabetes. We would speculate that IL-13 as an anti inflammatory cytokine and a mediator of the Th2 pathway could be the potential therapeutic approach in the prevention of type 1 diabetes. PMID- 10736104 TI - Evidence that human immunoglobulin M rheumatoid factors can Be derived from the natural autoantibody pool and undergo an antigen driven immune response in which somatically mutated rheumatoid factors have lower affinities for immunoglobulin G Fc than their germline counterparts. AB - The question of whether immunoglobulin (Ig)M rheumatoid factors (RF) arise as the result of an abnormal expansion of already existing clones producing natural autoantibodies or emerge as new clones that are somatically mutated owing to an antigen driven immune response has never been conclusively answered. In this study, an inhibition ELISA was utilized to measure the affinities of recombinant antibodies using VH segments reverted back to their closest germline counterparts (germline revertants). In all cases, the somatically mutated parental RFs had a decreased affinity for immunoglobulin (Ig)G Fc compared to the germline revertant, indicating that the antibodies in the germline configuration had the higher affinities. This demonstrates that somatic mutation is not a prerequisite to generate disease associated antibodies. The presence of mutations in the parental IgM RFS suggests that these cells had been involved in a germinal centre reaction. As the germinal centre is the conventional site of the acquisition of mutations during an antigen driven response, these data suggest a role for germinal centres in the generation of the antibody diversity in addition to the selection of higher affinity antibodies. Assuming that only antigen selected cells survive deletion, these data support the hypothesis that IgM RFS can be derived from the natural autoantibody repertoire and result from an antigen driven response. Mechanisms controlling the survival of B cells based on the affinity/avidity of the immunoglobulin receptor are shown to be functional in patients with rheumatoid arthritis. PMID- 10736105 TI - Neutralizing human anti crotoxin scFv isolated from a nonimmunized phage library. AB - Combinatorial phage display technology offers a new possibility for making human antibodies which could be used in immune therapy. We explored the use of this technology to make human scFvs specific for crotoxin, the main toxic component of the venom of the South-American rattlesnake Crotalus durissus terrificus. Crotoxin, a phospholipase A2 neurotoxin constituted by the association of two subunits, exerts its lethal action by blocking neuromuscular transmission. This is the first report of human anticrotoxin scFvs (scFv 1, scFv 6 and scFv 8) isolated from a naive library of more than 1010 scFv clones with in vivo neutralizing activity. Nevertheless, differences are observed at the level of biological and immunological effects. Only scFv 8 is able to reduce the myotoxicity induced by crotoxin and scFv 1 is capable of altering the in vitro enzymatic activity of this toxin. All three scFvs recognize a region of one subunit located at the junction with the other one. Moreover these scFvs share strong amino acid homologies at the level of either the heavy or the light chain. Taken together, our results suggest that the use of human anticrotoxin scFvs may lead to a new and less aggressive passive immune therapy against poisoning by the venom of Crotalus durissus terrificus. PMID- 10736106 TI - Protective DNA immunization against Chlamydia pneumoniae. AB - We have investigated the efficacy of the DNA vaccination using the heat shock protein 60 (HSP-60) gene of C. pneumoniae, for protection of mice against infection with the bacteria. C57Bl/6 mice had a 5-20-fold reduction of C. pneumoniae numbers in lungs when immunized intranasally (i.n.) with plasmids (p) encoding pHSP-60. The reduction of the bacterial load coincided with a decreased severity of disease. No specific antibodies were detected after protective i. n. immunization. In contrast, mice immunized intradermally (i.d.) were not protected against challenge with C. pneumoniae, although specific humoral Immunoglobulin (Ig)G responses were generated. Co-inoculation i.n. of pHSP-60 with pIL-12 but not with pGM-CSF further increased protection of mice against infection with C. pneumoniae. Lungs from pHSP-60 i.n. immunized and infected mice showed higher levels of interferon (IFN)-gamma mRNA, and spleen cells from these mice co cultured with r-HSP-60 released higher levels of IFN-gamma and displayed higher proliferative responses than nonimmunized and infected controls. pHSP-60 immunized IFN-gamma receptor (R)-/- mice were not protected against infection with C. pneumoniae. Likewise, i.n. administration of pIFN-gamma alone induced significant protection. DNA vaccine-induced protection was CD4+ and CD8+ T-cell dependent, as shown by DNA-vaccination of MHC class II-/-, CD4-/-, CD8-/- and CD4 /-CD8-/-mice. Interestingly, DNA vaccine induced CD4+ T cells, in the absence of CD8+ T cells, were involved in worsening the outcome of infection. This worsening was linked with a shift towards a Th2 cytokine pattern. PMID- 10736107 TI - Human intestinal endothelium shows high susceptibility to cytomegalovirus and altered expression of adhesion molecules after infection. AB - Human cytomegalovirus (HCMV) causes gastro intestinal disease with ulcerations, apparently as a consequence of cytopathic damage to endothelial cells (EC) and subsequent microvascular obliteration. In this study we showed that cultured human intestinal microvascular endothelial cells (HIMEC) are much more susceptible to HCMV infection than human umbilical vein endothelial cells (HUVEC). When both cell types were challenged with a clinical isolate of HCMV (10 pfu per cell), 30% of HIMEC expressed HCMV immediate early proteins, but only 10% of HUVEC. Enhanced susceptibility was also reflected in the expression of early and late HCMV proteins. In addition, the interleukin-1beta (IL-1beta)-induced cellular expression of adhesion molecules differed between HIMEC and HUVEC after HCMV-infection. E-selectin was unaffected in HUVEC but increased in HIMEC, whereas vascular cell adhesion molecule (VCAM)-1 was increased in HUVEC but decreased in HIMEC. Furthermore, HCMV-infection enhanced the expression of intercellular adhesion molecule (ICAM)-1 in both cell types. In conclusion, the enhanced susceptibility to HCMV infection observed in HIMEC and the elevated expression of E-selectin and ICAM-1 observed in these cells may provide an indication to the liability of developing gastrointestinal HCMV disease and may have a possible relevance to the survival of intestinal transplants. PMID- 10736108 TI - Effect of interleukin-7 on the in vitro development and maturation of monocyte derived human dendritic cells. AB - We have compared the cell phenotype and functional properties of monocyte/macrophage derived dendritic cells (DCs) obtained by culture of human adherent peripheral blood mononuclear cells (PBMCs) in medium containing granulocyte macrophage colony stimulating factor (GM-CSF) either alone (GM-CSF DCs), or in combination with interleukin (IL)-4 (IL4-DCs) or IL-7 (IL7-DCs). The cell surface phenotype of GM-CSF-DCs and IL-7-DCs was characterized by a high expression of major histocompatibility complex (MHC) class I and II, CD80, CD86 and CD40. In contrast to 'classical' IL-4-DCs, these two types of DCs expressed CD14 and a CD21-like molecule detected by two out of four CD21-specific monoclonal antibodies (MoAb) tested. The same pattern of reactivity with CD21 specific antibodies was observed in freshly isolated adherent PBMCs but not in B lymphocytes. This reactivity was upregulated by IL-7 in a dose dependent manner. Lipopolysaccharide (LPS) treatment induced the upregulation of CD40, CD80, CD86 and the T-cell stimulatory capacity in IL-4-DCs and, to a lesser extent, in the IL-7-DCs whereas GM-CSF-DCs responded very poorly to such treatment. Our data indicate that, together with GM-CSF, the IL-7 drives macrophage precursors to a differentiation stage that is close to but distinct from the phenotype of IL-4 DCs. Comparison of DC development in the presence of IL-7 or IL-4 may help in dissecting signalling pathways that regulate the expression of functionally relevant DC markers. PMID- 10736109 TI - Differences in lymphocyte subsets in the wall of high endothelial venules and the lymphatics of human palatine tonsils. AB - Lymphocyte trafficking plays a critical role in disseminating specifically primed lymphocytes all over the body. Most concepts on the interaction of adhesion molecules on lymphocyte subsets and specialized endothelia such as those in high endothelial venules (HEV) are based on animal experiments as kinetic studies cannot be performed in humans. We therefore characterized lymphocyte subsets in the wall of HEV and in the lumen of lymphatics of 18 human palatine tonsils by immunohistology. All subsets studied were found in the wall of HEV (% of lymphocytes): 32% CD20+, 50% CD3+, 14% CD4+, 32% CD8+ and also 21% CD45RA+ and 39% CD45RO+. In the lymphatics, used to indicate lymphocytes emigrating from the tonsils, a different composition was found; e.g. many more T cells and three times more CD45RA+ than RO+ lymphocytes. Thus, HEV are not a selective entry site nor lymphatics an exit for specific lymphocyte subsets only, at least in these tonsils with chronic stimulation. PMID- 10736110 TI - Functional and phenotypic characteristics of dendritic cells generated in human plasma supplemented medium. AB - One successful approach to generate dendritic cells (DC) is to cultivate peripheral blood monocytes in fetal calf serum (FCS)-containing medium in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-4. Because the use of xenogenic proteins has to be strictly avoided for clinical applications, alternative protocols use human plasma instead of FCS. The aim of our study was to characterize DC generated in the presence of human plasma; moreover, we describe a novel protocol to generate DC directly from peripheral blood mononuclear cells (PBMC). DC generated from purified monocytes in the presence of 1% human plasma (HP-DC) and GM-CSF and IL-4 both in the allogenic mixed leukocyte reaction (MLR) and in the tetanus presentation assay were potent stimulators of T-cell proliferation. DC generated from PBMC were equally effective stimulators in the allogenic MLR as those generated from purified monocytes. When the immunophenotype of DC generated from FCS containing medium (FCS-DC) was compared to that of HP-DC, the surface expression of CD1a and CD80 was significantly lower in HP-DC. In contrast, the expression of CD83 and CD86 was significantly higher in HP-DC than in FCS-DC. The capacity of receptor mediated endocytosis and macropinocytosis was found to be significantly lower in HP-DC when compared to FCS-DC. The differences in the immunophenotype, macropinocytosis and endocytosis between the HP-DC and the FCS-DC were observed independently of the generation of the cells from PBMC or purified monocytes. Our data indicate that HP-DC are potent stimulators of T-cell proliferation and exhibit a characteristic phenotype of intermediate maturity. Moreover, DC can be directly generated from PBMC preparations. PMID- 10736111 TI - Cryptococcus neoformans neutralizes macrophage and astrocyte derived nitric oxide without interfering with inducible nitric oxide synthase induction or catalytic activity - possible involvement of nitric oxide consumption. AB - The effect of Cryptococcus neoformans on the accumulation of nitrite, an indicator of nitric oxide (NO) synthesis, was investigated in cytokine (interferon-gamma [IFN-gamma] and interleukin [IL]-1)-stimulated cultures of rat peritoneal macrophages and C6 astrocytoma cells. Cytokine-induced nitrite generation in cultures of both cell types was inhibited in a dose-dependent manner by live C. neoformans, but not by heat-killed cryptococcal cells or conditioned medium from yeast cultures. C. neoformans-mediated reduction of nitrite formation coincided with impairment of NO-dependent macrophage tumoricidal activity. Cytokine-triggered induction of inducible NO synthase (iNOS) was unaffected in C6 cells, and only marginally reduced in macrophages. When cells were pretreated with cytokines for 24 h to induce iNOS, and any further induction was prevented by inhibition of protein synthesis, C. neoformans was still able to reduce nitrite accumulation in cultures of both cell types. Finally, live C. neoformans, but not heat-killed yeast cells or yeast culture supernatant, significantly reduced nitrite production in a culture solution of NO releasing compound S-nitrosoglutathione (GSNO). Thus, it appears that cryptococcal reduction of nitrite formation in macrophage and C6 cultures was caused by the consumption of NO by some yeast molecule, rather than by the inhibition of cellular NO synthesis. PMID- 10736112 TI - CD14-dependent and independent pathways in lipopolysaccharide-induced activation of a murine B-cell line, CH12. LX. AB - Using a lipopolysaccharide (LPS)-responsive murine B-cell line, CH12. LX, we assessed the possible role of CD14 in LPS-induced activation of B cells. Flow cytometric analysis indicated that CH12.LX cells expressed the CD14 molecule with a lower intensity than did the macrophage cell line J774.1. A reverse transcription-polymerase chain reaction and Northern blot analysis revealed low, but significant, levels of CD14 mRNA in CH12.LX cells, whose cDNA was identical to that of the mouse macrophage CD14 gene. After stimulation with LPS, CH12.LX cells proliferated, accompanied by up-regulations of CD14, transforming growth factor (TGF)-beta and interleukin (IL)-6 mRNA, and increased IgM and IgA secretion. In the absence of serum or with the addition of anti-CD14 monoclonal antibodies, however, LPS-stimulation induced neither the up-regulation of CD14 and TGF-beta mRNA nor an increase in IgA secretion. These findings indicate that CD14 expression is not restricted to myeloid cells, but is involved in some cellular activation events of murine B cells elicited by LPS. Furthermore, a CD14 independent pathway may also exist in the LPS-induced activation of B cells that leads to proliferation, IL-6 production and the enhancement of IgM (but not IgA) secretion. PMID- 10736113 TI - Immune abnormalities in aneurysmal subarachnoid haemorrhage patients: relation to delayed cerebral vasospasm. AB - Peripheral blood CD3+, CD19+, CD4+, CD8+ and CD45RO+ mononuclear cell subsets, T cell proliferative responses to combinations of coimmobilized OKT3 antibody and an ECM protein (collagen I, collagen IV, fibronectin or elastin), and T-cell adhesion to collagen IV, fibronectin and elastin were studied in patients with aneurysmal subarachnoid haemorrhage. No significant difference was found in the major lymphocyte subsets between subarachnoid haemorrhage patients receiving no dexamethasone for brain oedema treatment and healthy blood donors. Compared with the latter, both the dexamethasone-untreated and -treated subarachnoid haemorrhage patients showed decreased relative proliferative responses of circulating T cells to OKT3 combinations with collagen IV and fibronectin, and an increased PHA-activated T-cell adhesion to elastin. CD45RO+, CD4+ and CD19+ peripheral blood cell subsets, CD4+/CD8+ cell ratio, PHA-activated T-cell adhesion to fibronectin and collagen IV, and OKT3-triggered T-cell costimulatory responses to elastin, collagen IV and fibronectin were significantly higher in subarachnoid haemorrhage patients presenting with delayed cerebral vasospasm (DCV) than in their DCV-free counterparts. The DCV-related differences in circulating lymphocyte subsets showed no apparent relationship to the glucocorticoid treatment, whereas the differences in the other indices were confined to the dexamethasone-untreated subarachnoid haemorrhage patients. The above results suggest that the CD4+/CD8+ ratio and T cell-ECM interactions play a role in the emergence of subarachnoid haemorrhage/DCV and may represent potential targets for subarachnoid haemorrhage therapy. PMID- 10736114 TI - Neutralization of disease associated autoantibodies by an immunoglobulin M- and immunoglobulin A-enriched human intravenous immunoglobulin preparation. AB - Immunoglobulin preparations enriched with IgM and IgA are used in the therapy of severe bacterial infections and for the treatment of acute graft-versus-host disease, but not as yet, in the treatment of autoimmune diseases. We investigated the potential of an IgM- and IgA-enriched immunoglobulin preparation to neutralize activity autoantibodies from patients with autoimmune diseases. We demonstrate that Pentaglobin(R) was at least as effective as intravenous immunoglobulin (Sandoglobulin(R)) in inhibiting autoantibody activity. Each of the immunoglobulin isotypes present in Pentaglobin(R) may be responsible for the inhibitory effect. Pentaglobin(R) immobilized on an affinity matrix retained the disease associated autoantibodies and interacted with F(ab')2 fragments of IgG autoantibodies. Suppression of autoantibody activity is dependent, at least in part, on idiotypic interactions. The present findings provide a rationale for considering these preparations for the immunomodulation of autoimmune disease. PMID- 10736115 TI - Serum immunoglobulinD in infants and children. AB - Serum immunoglobulinD (IgD) concentration is usually low in healthy individuals as compared to other immunoglobulin classes. Most studies on serum IgD are concerned with serum levels in healthy adults but reference values for young children and infants are not easily available. In order to establish age specific reference values we measured IgD levels in serum of 184 healthy Icelandic children, age 0-14 years and 60 healthy blood donors age 18-63, using the ELISA technique. Special attention was paid to the youngest age groups. Results showed low IgD values in infants and young children, gradually increasing until the age of 10 but then decreasing with age. We conclude that IgD gradually increases with age in childhood as other immunoglobulin classes but later declines. These findings can be of importance in revealing the function of IgD in the immune system as well as in the diagnosis of the hyper-IgD syndrome. PMID- 10736117 TI - Ex vivo interferon-gamma response to human immunodefiency virus-1 derived peptides in human immunodeficiency virus-1 infected patients. AB - The pattern of human immunodeficiency virus (HIV)-1 antigen-activated production of interferon (IFN)-gamma by immunocompetent cells of HIV-1 infected patients has been studied using a simplified assay combining a small volume (25 microliter) of whole blood stimulation with various HIV-1 antigens, and cytokine measurement in the same wells of microtitre plates (enzyme-linked immunotrapping assay, ELITA). The levels of IFN-gamma were higher using this assay than in the supernatant from stimulated whole blood cultures, therefore ELITA was used in the rest of the study. Specific immune responses to HIV-1 proteins (gp120, p24) and synthetic peptides derived from these proteins and from gp41 were detected in patients, but not in healthy controls. Decreased levels of IFN-gamma were observed in CDC class B (n = 5) and C (n = 4), compared with CDC class A (n = 5), following HIV-1 antigen-specific challenge. The positive response of cells from different patients to overlapping peptides of p25 (amino acids 329-344 and 335-351) was suggestive of a new epitope of HIV-1 gag recognized by T cells in the overlap region. In conclusion, the difference in in vitro antigen-specific T-cell responses of HIV-1-infected patients was shown using the ELITA method. Our results raise the possibility of using this method in screening specific antigens in HIV-1 infection. PMID- 10736116 TI - Immunological cytokine correlates of protective immunity and pathogenesis in leprosy. AB - The in vitro production of interferon (IFN)-gamma, interleukin (IL)-5, tumour necrosis factor (TNF)-alpha and IL-10 by blood mononuclear cells in response to whole Mycobacterium leprae and polyclonal stimulii of 23 individuals, representing a variety of conditions in relation to exposure/susceptibility to M. leprae, was assayed. In most cases, healthy household contacts of newly diagnosed multibacillary leprosy patients, designated exposed household contacts (EC), showed low-to-undetectable in vitro IFN-gamma production in addition to substantial TNF-alpha production in response to M. leprae. In contrast, peripheral blood mononuclear cells from previously exposed contacts (R) regarded as resistant-to-leprosy released low-to-moderate levels of IFN-gamma together with a mixed cytokine profile resembling a T helper (Th)0-type response. TNF alpha/IL-10 ratios in response to M. leprae and Concanavalin A were significantly higher in EC than in R contacts suggesting a role for the TNF-alpha/IL-10 ratio in restraining mycobacteria proliferation and spreading early in infection. The cytokine profiles of leprosy patients were taken as reference points. Post treatment lepromatous leprosy patients secreted relatively high levels of IL-10 in response to M. leprae, whereas one self-cured tuberculoid leprosy patient produced simultaneously high levels of IFN-gamma and TNF-alpha. In addition, the quantitative changes in the cytokines released by peripheral blood mononuclear cells in EC contacts after Bacille Calmette-Guerin (BCG) vaccination were investigated. Vaccination induced amplification of IFN-gamma production with a concomitant decrease in TNF-alpha/IL-10 ratios that resembled the cytokine pattern observed in R contacts. IFN-gamma production was observed in response to both a cross-reactive antigen (Ag 85) and a M. leprae-specific protein (MMP-I), which attests to a BCG nonspecific stimulation of the immune system, thereby casting these antigens as likely candidates for inclusion in a subunit vaccine against leprosy. Finally, a model for protective x pathologic response to mycobacteria is presented. PMID- 10736118 TI - Implementation of an emergency department triage procedure for the detection and isolation of patients with active pulmonary tuberculosis. AB - STUDY OBJECTIVES: To investigate the ability of an emergency department screening protocol to initiate respiratory isolation of patients with pulmonary tuberculosis at ED triage before chest radiography. METHODS: We conducted a prospective cohort study with retrospective medical record review of adult patients who presented for care to an urban, university-affiliated hospital in Los Angeles County over a 4-month period. Ambulatory patients were administered a triage screening protocol that used patient-reported tuberculosis risk factors and symptoms in combination with selective chest radiography to screen patients at ED triage for active pulmonary tuberculosis. RESULTS: A total of 10,674 patients were screened; 2, 218 were isolated at triage and underwent chest radiography, and 378 were kept in isolation in the ED. The respiratory isolation of pulmonary tuberculosis (RIPT) protocol detected 17 of 27 visits made by patients with unsuspected pulmonary tuberculosis, yielding a sensitivity of 63% (95% confidence interval [CI] 42% to 81%). The estimated specificity was 78%. For each patient with tuberculosis who was detected by the RIPT protocol, 624 patients were screened at triage, 130 chest radiographs were taken, and 22 patients were placed in respiratory isolation in the ED. Patients with undetected pulmonary tuberculosis more commonly had nonpulmonary chief complaints (76% versus 20%; odds ratio [OR] 13, 95% CI 2.1 to 78.3), and only 60% (95% CI 26% to 88%) were ultimately isolated in the hospital. Among RIPT screen-positive patients, radiographic findings predictive of pulmonary tuberculosis were cavitary lesions (OR 84.3, 95% CI 22.6 to 315), upper lobe infiltrates (OR 24.2, 95% CI 9.1 to 64.4), pleural effusions (OR 8.9, 95% CI 2.5 to 31.8), diffuse/interstitial infiltrates (OR 5.7, 95% CI 1.8 to 17.9), and non-upper lobe infiltrates (OR 3.1, 95% CI 1.0 to 9.5). CONCLUSION: The RIPT screening protocol was only moderately sensitive for isolating patients with pulmonary tuberculosis at ED triage. Future studies should evaluate modified and abridged screening protocols, as well as the cost-effectiveness of triage screening. PMID- 10736119 TI - Resistive heating is more effective than metallic-foil insulation in an experimental model of accidental hypothermia: A randomized controlled trial. AB - STUDY OBJECTIVE: We study a resistive-heating blanket in a volunteer model of severe accidental hypothermia to evaluate differences in rates of rewarming, core temperature afterdrop, and body heat content and distribution during active and passive rewarming. METHODS: Eight volunteers participated in a crossover design on 2 days. The volunteers were anesthetized and cooled to 33 degrees C (91.4 degrees F); anesthesia was subsequently discontinued, and shivering was prevented with meperidine. On one randomly assigned day, the volunteers were rewarmed passively with reflective foil (passive insulation), whereas on the other they were covered with a carbon fiber-resistive heating blanket set to 42 degrees C (107.6 degrees F; active rewarming). Trunk and head temperature and heat content were calculated from core (tympanic membrane) temperature. Peripheral (arm and leg) tissue temperature and heat content were estimated by using fourth-order regressions and integration over volume from 30 tissue and skin temperatures. RESULTS: Core heat content increased 73+/-14 kcal (mean+/-SD) during 3 hours of active warming, but only 31+/-24 kcal with passive insulation, a difference of 41+/-20 kcal (95% confidence interval [CI] 27 to 55 kcal; P <. 001). Peripheral tissue heat content increased linearly by 111+/-16 kcal during active warming but only by 38+/-31 kcal during passive warming, a difference of 74+/-34 kcal (95% CI 50 to 97; P <.001). Consequently, total body heat increased 183+/-22 kcal during active warming but only 68+/-54 kcal with passive insulation, a difference of 115+/-42 kcal (95% CI 86 to 144 kcal; P <.001). Core temperature increased from 32.9 degrees C+/-0.2 degrees C to 35.2 degrees C+/-0. 4 degrees C during 3 hours of active warming, a difference of 2.3 degrees C+/-0.4 degrees C. In contrast, core temperature with foil insulation only increased from 32.9 degrees C+/-0.2 degrees C to 33. 8 degrees C+/-0.5 degrees C, a difference of only 0.8 degrees C+/-0. 4 degrees C. The difference in the core temperature increase between the two treatments was thus 1.5 degrees C+/-0.4 degrees C (95% CI 1. 2 degrees C to 1.7 degrees C; P <.001 between treatments). Active warming was not associated with an afterdrop, whereas the afterdrop was 0.2 degrees C+/-0.2 degrees C and lasted a median of 45 minutes (interquartile range, 41 to 64 minutes) with passive insulation. CONCLUSION: Resistive heating more than doubles the rewarming rate compared with that produced by reflective metal foil and does so without producing an afterdrop. It is therefore likely to be useful in the prehospital setting. PMID- 10736120 TI - Parents as direct contributors to the medical record: validation of their electronic input. AB - STUDY OBJECTIVES: We assessed the validity and completeness of data in the past medical history (PMH) obtained electronically from parents and examined effects of the human-computer interface and sociodemographic variables on electronic parental report. METHODS: We compared parents' electronic report of PMH data with a criterion standard, structured face-to-face interview by a pediatrician blinded to the electronic data. The electronic medical record interface enabled parents to provide 5 elements of the PMH: birth status, allergies, current medications, immunization status, and previous hospitalizations. The setting was the emergency department waiting room in an academic, urban children's hospital; parents of infants up to 12 months old participated. Outcome measures were validity of the PMH data obtained using the electronic medical record interface and odds of having an invalid or incomplete response using the electronic medical record interface. RESULTS: One hundred parents were enrolled (69.4% of eligible subjects). Study subjects did not differ from nonenrollees on demographic variables and visit characteristics. The validity of the electronic medical record interface data was high across the PMH elements (94% to 99%). Two demographic features predicted invalid response: parental primary language other than English or Spanish (odds ratio [OR] 11.4, 95% confidence interval CI 1.7 to 76.3), and Asian ethnicity (OR 14. 6, 95% CI 1.2 to 182.4). Incomplete responses were predicted by limited previous experience with computers; computer-naive subjects had an eightfold increased odds of skipping a question (OR 7.9, 95% CI 1.8 to 34.6). CONCLUSION: Parents are accurate independent reporters of their infants' general PMH using the electronic medical record interface. Their participation in care may be enhanced by allowing them to contribute medical information directly to the electronic medical record. PMID- 10736121 TI - Sexual Assault Nurse Examiner programs in the United States. AB - STUDY OBJECTIVE: We sought to provide a descriptive study of the Sexual Assault Nurse Examiner (SANE) programs and their characteristics in the United States. METHODS: A confidential survey addressing patient and staff demographics, administration attributes, examination procedures, and medical and legal issues was mailed to SANE programs in the United States. RESULTS: Sixty-one (66%) of 92 programs responded. More than half of the programs (32/58 [55%]) had been in operation for less than 5 years. Thirty (52%) of the 58 programs performed the initial sexual assault examination in hospital emergency departments. Written consent (57/59 [97%]) was obtained for the initial examination, and most (51/59 [86%]) programs used preprepared commercial sexual assault kits. Program directors were predominately registered nurses. All but one program mandated specific training requirements for their staff, with a median requirement of 80 hours. Procedures used for initial examinations varied; most offered pregnancy testing (56/58 [97%]), pregnancy prophylaxis (57/59 [97%]), and sexually transmitted disease (STD) prophylaxis (53/59 [90%]). HIV testing was not offered in 32 (54%) of 59 programs. Almost all programs used Wood's lamp (51/59 [86%]), colposcopes (42/59 [71%]), and photographs (46/59 [78%]) for documentation. Median time required per patient for initial examination and evidence collection was 3 hours (range, 1 to 8 hours). Follow-up is consistently offered to the survivor. Most programs (45/61 [74%]) could report the number of survivors treated, but few could provide information on survivor medical follow-up or the number of prosecutions by survivors and their outcomes. CONCLUSION: This survey provided an overview of SANE programs. SANE programs are similar across the country with regard to staffing, training, STD and pregnancy prophylaxis, and documentation techniques. They are inconsistent in the use of STD cultures, HIV testing, and alcohol and drug screening. SANE programs were unable to provide data regarding survivor follow-up and legal outcomes. This information is essential to evaluate the programs' effectiveness and to improve performance. The need for better outcome data should be addressed to define success or failure of SANE programs. PMID- 10736122 TI - Analysis of 1,076 cases of sexual assault. AB - STUDY OBJECTIVE: Rates of sexual assault are increasing, and evidence exists that its demographics and characteristics are changing. The purpose of our study was to describe victim, assailant, assault, and treatment characteristics for sexual assault victims and to provide descriptive data on the evidentiary examination. METHODS: Prospective data were collected on all sexual assault victims presenting to an urban Level I trauma center from January 1992 to December 1995 for treatment and evidentiary examination. Data from crime laboratory records were retrospectively reviewed. RESULTS: One thousand one hundred twelve patients presented after a sexual assault. A total of 1,076 (97%) patients consented to the medical and evidentiary examination and were enrolled in the study. Age ranged from 1 to 85 years (mean, 25 years; median, 23 years), with 96% (1,036/1,076) female and 4% (41/1,076) male victims. The number of assailants was greater than 1 in 20% (208/1,044) of cases, and the assailant was a stranger only 39% (409/1,094) of the time. Force was used in 80% (817/1,027) of reported assaults, and in 27% (275/1,014) of cases a weapon was present. Vaginal intercourse was involved in 83% (851/1,023) of female victims. Oral assault was involved in 25% (271/1,053) of all cases, and anal penetration was involved in 17% (178/1,058) of all cases. Overall, general body trauma was seen 67% (621/927) of the time, and genital trauma occurred in 53% (388/736) of cases. Twenty percent (147/1,712) of patients had no trauma noted on examination. Sperm were noted on the emergency department wet mount in only 13% (93/716) of the victims, and of the 612 cases with both ED sperm data and crime laboratory semen data available, evidence of sperm and semen were found 48% (296/612) of the time by either. CONCLUSION: Health care professionals should be aware that general body trauma is common, that the assailant is often someone known to the victim, and that evidence of semen is commonly found by the crime laboratory even when it is not found in the ED analysis of a wet mount. PMID- 10736123 TI - Shorter duration of oral N-acetylcysteine therapy for acute acetaminophen overdose. AB - STUDY OBJECTIVE: We sought to evaluate the safety and efficacy of a shorter N acetylcysteine (NAC) regimen in the treatment of acute acetaminophen overdose. METHODS: We performed a retrospective case series in a large urban county hospital. Of 305 patients identified through the emergency department, 75 patients met the criteria inclusion: an acute overdose ingestion, serum acetaminophen concentration in toxic range according to the Rumack-Matthew nomogram, and oral NAC treatment initiated within 24 hours of the ingestion. The regional poison control center recommended oral treatment with NAC 140 mg/kg, followed by maintenance doses of 70 mg/kg every 4 hours until the serum acetaminophen level was no longer detectable, rather than the standard 72-hour treatment regimen. RESULTS: The primary outcome measure was the development of hepatotoxicity. Twenty-five (33.3%) patients were treated for a period of less than 24 hours, 25 (33.3%) were treated for 24 to 36 hours, and 25 (33.3%) were treated for 37 to 64 hours; the mean and median duration of treatment was 31 hours. None of the patients treated for less than 24 hours had evidence of hepatotoxicity (aspartate aminotransferase [AST] or alanine aminotransferase [ALT] level >1,000 IU/L); hepatotoxicity developed in 2 (8%) patients treated for 24 to 36 hours and 4 (16%) patients treated for 37 to 64 hours. There were no deaths or patients who received liver transplantation. The overall incidence of hepatotoxicity in our patients was similar to that found in other protocols with administration of oral NAC for 72 hours or intravenous NAC for 20 or 48 hours. CONCLUSION: This observational study suggests that a shorter course of oral NAC therapy in patients who do not show evidence of hepatotoxicity within 36 hours of an acute acetaminophen overdose is safe and effective. PMID- 10736124 TI - Failure of intravenous N-acetylcysteine to reduce methemoglobin produced by sodium nitrite in human volunteers: A randomized controlled trial. AB - STUDY OBJECTIVE: To determine whether intravenous N -acetylcysteine (NAC) produces a clinically significant decline in sodium nitrite-induced methemoglobinemia in human volunteers. METHODS: We conducted a randomized, control crossover trial with each subject serving as his own control. Methemoglobinemia was induced with intravenous sodium nitrite (4 mg/kg) administered over 10 minutes starting at time 0. At time 30 minutes, subjects were randomly assigned to treatment with intravenous NAC for 100 minutes (150 mg/kg over 1 hour followed by 14 mg/kg per hour for 40 minutes) or administration of an equal volume of 5% dextrose in water. Each subject received the alternative treatment after an interval of at least 1 week. Blood methemoglobin concentrations were measured by multiwavelength co-oximetry at time 0, 15, 30, 50, 70, 90, 110, and 130 minutes. Area under the methemoglobin concentration-time curve (AUC) between 30 and 130 minutes was compared between groups using a 2 tailed, paired t test. RESULTS: There were no statistically significant differences in the control and treatment groups with respect to baseline hemoglobin or methemoglobin concentrations, as well as nitrite-induced methemoglobin concentrations at the initiation of treatment (0.85+/-0.06 g/dL, 0.88+/-0.04 g/dL; mean+/-SEM; P =.31). Mean AUC for the control group (77.1+/-5.7 g x min/dL) was significantly lower than the mean AUC for the treatment group (84.5+/-4.7 g x min/dL); P =.01). CONCLUSION: Intravenous NAC failed to enhance methemoglobin reduction in this model. PMID- 10736125 TI - A comparison of physostigmine and benzodiazepines for the treatment of anticholinergic poisoning. AB - STUDY OBJECTIVE: To compare the efficacy and safety of physostigmine with benzodiazepines for the treatment of agitation and delirium associated with anticholinergic poisoning. METHODS: We conducted a retrospective study of 52 consecutive patients referred to a university hospital toxicology consultation service who were treated with physostigmine, benzo-diazepines, or both for anticholinergic agitation and delirium. Patients treated with physostigmine were compared with those treated with benzodiazepines with respect to demographics, severity of poisoning, response to treatment, side effects of treatment, and complications. RESULTS: Physostigmine controlled agitation and reversed delirium in 96% and 87% of patients, respectively. Benzodiazepines controlled agitation in 24% of patients but were ineffective in reversing delirium. Initial treatment with physostigmine (n=30) resulted in a significant decrease in the incidence of agitation (P <.001) and level of central nervous system stimulation (P <.001), whereas initial treatment with benzodiazepines (n=22) did not (P =.03 and P =.05, respectively). Patients treated initially with physostigmine had a significantly lower incidence of complications (7% versus 46%; P <. 002) and a shorter time to recovery (median, 12 versus 24 hours; P =. 004) than those treated initially with benzodiazepines. There were no significant differences between these groups in the incidence of side effects (7% versus 14%; P =0.6) and length of stay (median, 32 versus 39 hours; P =.15). CONCLUSION: Results suggest that physostigmine is more effective and safer than benzodiazepines for the treatment of anticholinergic agitation and delirium. A prospective controlled study is necessary to confirm such findings. PMID- 10736126 TI - Unusual cause of chest pain in a 60-year-old woman. AB - Chest pain is a common emergency department complaint. Many patients presenting with this complaint are discharged with the nonspecific diagnosis of musculoskeletal chest pain. That was the diagnosis given to the patient in this case at the time of her initial presentation to the ED, a diagnosis that was correct but incomplete. This case illustrates the importance of taking a complete history, including a thorough review of systems in every patient, even when the diagnosis seems obvious. PMID- 10736127 TI - Priapism after androstenedione intake for athletic performance enhancement. AB - Priapism is a relatively rare condition characterized by persistent painful erection of the corpora cavernosum that occurs unrelated to sexual stimulation or desire. It has been associated with cocaine exposure, sickle cell anemia, anticoagulant therapy, neoplastic syndromes, diabetes, and the use of psychotropic medications. Androstenedione is a testosterone precursor used by athletes to enhance performance; the lay press has recently touted this substance as an over-the-counter sexual stimulant. We describe an otherwise healthy man with 2 episodes of priapism after the use of androstenedione. The patient did not have any factors known to precipitate priapism. PMID- 10736128 TI - Wellness issues and the emergency medicine resident. PMID- 10736129 TI - National Highway Traffic Safety Administration (NHTSA) notes. Marijuana and alcohol combined severely impede driving performance. PMID- 10736130 TI - Change of shift. Thank you, Melissa. PMID- 10736131 TI - Mandatory reporting laws. PMID- 10736132 TI - Mandatory reporting laws PMID- 10736133 TI - EMRA's 25 years. Emergency Medical Residents' Association. PMID- 10736135 TI - Improving the prospects of the world's 6 billionth child. PMID- 10736134 TI - Generalized seizures associated with low-calorie dieting. PMID- 10736136 TI - Anticipatory guidance--it's worth the effort! PMID- 10736137 TI - Do pediatric nurse practitioners recognize sexual abuse? AB - INTRODUCTION: This study was undertaken to describe pediatric nurse practitioners' (PNPs') knowledge of genital anatomy and their ability to recognize sexual abuse. METHOD: A 33-item questionnaire developed by Ladson, Johnson, and Doty (1987) was revised, and PNPs were asked questions about demographics, their knowledge of normal and abnormal prepubescent genital anatomy, and their recognition of the association between sexually transmitted diseases and sexual abuse in prepubescent girls. The questionnaire was mailed to 213 PNPs within the state of Ohio, all of whom were members of the Ohio Chapter of NAPNAP. RESULTS: No statistically significant correlation was noted between any demographic characteristics and correct responses to questionnaire items. Two anatomical parts--hymen and vaginal opening--were correctly identified by less than 60% of the participants. Only 67.1% of the PNPs stated that a child can have a normal genital examination after penile penetration of the vagina. CONCLUSIONS: PNPs need to increase their knowledge of normal prepubescent genital anatomy and physical findings of sexual abuse and report these findings if abuse is suspected. PMID- 10736138 TI - Ability of caregivers to read delayed hypersensitivity skin tests in children exposed to and infected by HIV. The Pediatric Pulmonary and Cardiovascular Complications of Vertically Transmitted HIV Study Group. AB - OBJECTIVES: To identify whether nonmedical home caregivers (parents, guardians) could accurately interpret the results of delayed hypersensitivity skin tests in children, thereby obviating the need for a return visit for a professional reading. METHODS: Patients who were enrolled in the Pediatric Pulmonary and Cardiac Complications of HIV (P2C2HIV) study were given annual skin tests to tuberculin purified protein derivative, Candida, and tetanus as part of the study protocol. Caregivers were instructed verbally about how to read and measure erythema and induration. They were given written instructions to take home as well and were asked to return in 48 to 72 hours for the professional reading. On the morning of their return visit, they were to read the skin tests and record the results before coming to the clinic. RESULTS: When compared with readings by professional staff, caregivers had a high percentage of false-negative readings for both Candida (24%) and tetanus (41%) skin tests. These false-negative readings did not correlate with age, gender, race, or educational level of the caregivers. CONCLUSIONS: The high percentage of false-negative readings by the caregivers emphasizes the need for professional reading of delayed hypersensitivity skin tests. PMID- 10736139 TI - The specialist role of the music therapist in developmental programs for hospitalized children. AB - Music therapy is used to address the needs of hospitalized children in the attainment of their normal growth and development milestones. Clinical practice literature proposes that the achievement of musical milestones parallels the developmental competence achieved in the areas of gross motor and fine motor skills; speech, language, and communication skills; sensory motor skills; and social and interactive skills. Pediatric health care workers are encouraged to use music appropriately in their work settings and, where possible, consult and liaise with a registered music therapist, that is, a trained and qualified practitioner, to achieve best practice outcomes in this field. PMID- 10736140 TI - Youth perceptions of comprehensive adolescent health services through the Boston HAPPENS program. AB - INTRODUCTION: The Boston HAPPENS (HIV Adolescent Provider and Peer Education Network for Services) program is a collaborative network of care made up of 8 organizations that serve youth and provide coordinated care for human immunodeficiency virus (HIV)-positive, homeless, and at-risk youth aged 12 to 24 years. Learning youth perceptions about the program is essential to determine if the program is meeting their needs. METHODS: In this qualitative evaluation, 18 youth served by the network met in 4 focus groups to provide their view of the program. Services within 5 categories were assessed: (a) medical care, (b) mental health and substance abuse care, (c) HIV prevention and care, (d) case management, and (e) allocation of finances. RESULTS: Boston HAPPENS has achieved name recognition and provides many needed services for youth from a wide variety of backgrounds. The youth were comfortable receiving care and were appreciative of the comprehensive services available. They provided suggestions for how mental health services could be offered as one-on-one counseling as part of "wellness care." Young participants also requested more recreational and support opportunities for young people living with HIV. DISCUSSION: Qualitative evaluations such as this give a voice to youth to advocate for services they need. By including youth ideas and perspectives during program development and implementation, services can be more attractive to groups of at-risk youth who historically have been less likely to seek care. PMID- 10736141 TI - Comparing two methods of topical anesthesia used before intravenous cannulation in pediatric patients. AB - INTRODUCTION: The purpose of this study was to compare the efficacy of two methods of topical anesthesia before venipuncture or intravenous cannulation of pediatric patients to assess which type of topical anesthetic would better alleviate pain in the least amount of time with the greatest success rate of cannulation. METHODS: This study was a randomly controlled trial of 100 preoperative pediatric patients aged 5 to 21 years. The following methods of topical anesthesia were compared: (a) iontophoresis of a topical solution of 2% lidocaine with epinephrine 1:100,000 using a Phoresor Dose Controller (PDC) with Numby Stuff electrodes and a eutectic mixture of local anesthetic and (b) 2.5% lidocaine and 2.5% prilocaine (eutectic mixture of local anesthetics [EMLA] cream). RESULTS: Children reported less pain with iontophoresis (M = 0.08) compared with EMLA cream (M = 1.88, P < .001). Time to accomplish topical anesthesia was shorter with iontophoresis (13 minutes) compared with EMLA cream (60 minutes, P < .001). Failure to accomplish venipuncture occurred 5 times with the iontophoresis method and 8 times with the EMLA method (not significant). No dermal burns resulted from use of iontophoresis. DISCUSSION: We conclude that use of iontophoresis in pediatric patients is safe, rapid, and significantly more effective than is EMLA cream in reducing pain associated with venipuncture or intravenous cannulation. PMID- 10736142 TI - Depression in children. PMID- 10736143 TI - Child with incontinence. PMID- 10736144 TI - "To err is human": a report from the Institute of Medicine. PMID- 10736145 TI - Nurturing the social and emotional development of children, a.k.a. discipline. PMID- 10736146 TI - Expert witness testimony in medical liability cases. PMID- 10736147 TI - Letter & response. PMID- 10736148 TI - Questions & answers. PMID- 10736149 TI - Proton translocation by cytochrome c oxidase: a rejoinder to recent criticism. AB - Ten years ago, intermediate reaction steps in the catalytic cycle of cytochrome c oxidase were titrated with phosphorylation potential in isolated mitochondria, and the results were interpreted as evidence for thermodynamic linkage of proton translocation exclusively to the oxidative reaction steps of the catalytic cycle [Wikstrom, M. (1989) Nature 338, 776-778]. Michel has recently argued that this work was flawed, and proposed a mechanism in which one of the four steps of proton translocation is linked to the reductive phase of the catalytic cycle [Michel, H. (1999) Biochemistry 38, 15129-15140]. Here, the original data are scrutinized and related to information that has accumulated since this work was published. The analysis shows that the main conclusions from this work still hold. Michel's mechanism of proton translocation is briefly discussed, and found to be at odds with some experimental observations. PMID- 10736150 TI - Topological effects of the TATA box binding protein on minicircle DNA and a possible thermodynamic linkage to chromatin remodeling. AB - DNA ring closure experiments on short restriction fragments ( approximately 160 bp) bound by the TATA box binding protein (TBP) have demonstrated the formation of negative topoisomers, consistent with crystallographically observed TBP induced DNA untwisting but in contrast to most previous results on topological effects in plasmid DNA. The difference may be due to the high free energy cost of substantial writhe in minicircles. A speculative mechanism for the loss of TBP induced writhe suggests that TBP is capable of inducing DeltaTw between 0 and 0.3 in minicircles, via loss of out-of-plane bending upon retraction of intercalating Phe stirrups, and that TBP can thus act as a "supercoil shock absorber". The proposed biological relevance of these observations is that they may model the behavior of DNA in constrained chromatin environments. Irrespective of the detailed mechanism of TBP-induced supercoiling, its existence suggests that chromatin remodeling and enhanced TBP binding are thermodynamically linked. Remodeling ATPases or histone acetylases release some of the negative supercoiling previously restrained by the nucleosome. When TBP takes up the supercoiling, its binding should be enhanced transiently until the unrestrained supercoiling is removed by diffusion or topoisomerases. The effect is predicted to be independent of local remodeling-induced changes in TATA box accessibility. PMID- 10736151 TI - Structural basis of cleavage by RNase H of hybrids of arabinonucleic acids and RNA. AB - The origins of the substrate specificity of Escherichia coli RNase H1 (termed RNase H here), an enzyme that hydrolyzes the RNA strand of DNA-RNA hybrids, are not understood at present. Although the enzyme binds double-stranded RNA, no cleavage occurs with such duplexes [Lima, W. F., and Crooke, S. T. (1997) Biochemistry 36, 390]. Therefore, the hybrid substrates may not adopt a canonical A-form geometry. Furthermore, RNase H is exquisitely sensitive to chemical modification of the DNA strands in hybrid duplexes. This is particularly relevant to the RNase H-dependent pathway of antisense action. Thus, only very few of the modifications currently being evaluated as antisense therapeutics are tolerated by the enzyme, among them phosphorothioate DNA (PS-DNA). Recently, hybrids of RNA and arabinonucleic acid (ANA) as well as the 2'F-ANA analogue were shown to be substrates of RNase H [Damha, M. J., et al. (1998) J. Am. Chem. Soc. 120, 12976]. Using X-ray crystallography, we demonstrate here that ANA analogues, such as 2'F ANA [Berger, I., et al. (1998) Nucleic Acids Res. 26, 2473] and [3.3.0]bicyclo ANA (bc-ANA), may not be able to adopt sugar puckers that are compatible with pure A- or a B-form duplex geometries, but rather prefer the intermediate O4' endo conformation. On the basis of the observed conformations of these ANA analogues in a DNA dodecamer duplex, we have modeled a duplex of an all-C3'-endo RNA strand and an all-O4'-endo 2'F-ANA strand. This duplex exhibits a minor groove width that is intermediate between that of A-form RNA and B-form DNA, a feature that may be exploited by the enzyme in differentiating between RNA duplexes and DNA-RNA hybrids. Therefore, the combination of the established structural and functional properties of ANA analogues helps settle existing controversies concerning the discrimination of substrates by RNase H. Knowlegde of the structure of an analogue that exhibits enhanced RNA affinity while not interfering with RNase H activity may prove helpful in the design of future antisense modifications. PMID- 10736152 TI - Purification, cloning, and characterization of the CEL I nuclease. AB - CEL I, isolated from celery, is the first eukaryotic nuclease known that cleaves DNA with high specificity at sites of base-substitution mismatch and DNA distortion. The enzyme requires Mg(2+) and Zn(2+) for activity, with a pH optimum at neutral pH. We have purified CEL I 33 000-fold to apparent homogeneity. A key improvement is the use of alpha-methyl-mannoside in the purification buffers to overcome the aggregation of glycoproteins with endogenous lectins. The SDS gel electrophoresis band for the homogeneous CEL I, with and without the removal of its carbohydrate moieties, was extracted, renatured, and shown to have mismatch cutting specificity. After determination of the amino acid sequence of 28% of the CEL I polypeptide, we cloned the CEL I cDNA. Potential orthologs are nucleases putatively encoded by the genes BFN1 of Arabidopsis, ZEN1 of Zinnia, and DSA6 of daylily. Homologies of CEL I with S1 and P1 nucleases are much lower. We propose that CEL I exemplifies a new family of neutral pH optimum, magnesium-stimulated, mismatch duplex-recognizing nucleases, within the S1 superfamily. PMID- 10736153 TI - An NMR and molecular modeling study of the site-specific binding of histamine by heparin, chemically modified heparin, and heparin-derived oligosaccharides. AB - The diprotonated form of histamine binds site-specifically to heparin, a highly sulfated 1-->4 linked repeating copolymer comprised predominantly of 2-O-sulfo alpha-L-iduronic acid (the I ring) and 2-deoxy-2-sulfamido-6-O-sulfo-alpha-D glucopyranosyl (the A ring). The binding is mediated by electrostatic interactions. The structural features of histamine and heparin, which are required for the site-specific binding, have been identified from the results of (1)H NMR studies of the binding of histamine by six heparin-derived oligosaccharides and four chemically modified heparins and molecular modeling studies. The results indicate that the imidazolium ring of diprotonated histamine is critical for directing site-specific binding, while the ammonium group increases the binding affinity. The imidazolium ring binds within a cleft, with the A ring of an IAI triad at the top of the cleft, and the I rings forming the two sides. The H3 proton of the A ring is in the shielding cone of the imidazolium ring. The carboxylate group of the I-ring at the reducing end of the IAI triad and possibly the sulfamido group of the A-ring are essential for site specific binding, whereas the 2-O-sulfate group of the I ring and the 6-O-sulfate group of the A ring are not. The results indicate that histamine binds to the IAI triad with the I rings in the (1)C(4) conformation. Also, the configuration of the carboxylate group is critical, as indicated by the absence of site-specific binding of histamine by the related IAG sequence, where G is alpha-D-glucuronic acid. The molecular modeling results indicate that the N1H and N3H protons of the imidazolium ring of site-specifically bound histamine are hydrogen bonded to the carboxylates of the I rings at the nonreducing and reducing ends of the IAI trisaccharide sequence. PMID- 10736154 TI - Structural studies on bioactive compounds. 30. Crystal structure and molecular modeling studies on the Pneumocystis carinii dihydrofolate reductase cofactor complex with TAB, a highly selective antifolate. AB - The crystal structure of the ternary complex of NADPH, the potent antifolate [2, 4-diamino-5-?3-[3-(2-acetyloxyethyl)-3-benzyltriazen-1-yl]-4 -chloroph enyl?-6 ethylpyrimidine] (TAB, 1) and Pneumocystis carinii dihydrofolate reductase (pcDHFR), refined to 2.1 A resolution, reveals that TAB binds similar to the antifolates trimethoprim and methotrexate. These data also reveal multiple conformations for the binding geometry of TAB with two preferred orientations of the acetyloxy and benzyl groups that results from a 180 degrees rotation about the N2-N3 triazenyl bond. The methyl of the acetyloxy and benzyl ring of TAB probes large hydrophobic regions of the p-aminobenzoyl folate binding pocket of the active site, in particular the region near Phe69, which is unique to the pcDHFR sequence. These results confirm prior molecular modeling investigations of the binding of TAB to pcDHFR that identified four low-energy binding geometries, two involving rotations about the terminal N(2)-N(3) triazenyl linkage and two involving atropisomerism about the pivotal pyrimethamine-phenyl bond. The primary differences in the molecular dynamics (MD) models and those observed in this crystal complex result from small conformational changes in active-site residues on energy minimization. However, two MD models place the acetyloxy and benzyl ring groups in a region of the active site between the cofactor-binding region and the p-aminobenzoyl folate pocket; an orientation never observed in any DHFR crystal structure to date. These conformers interact with solvent near the enzyme surface and are probably not observed due to the loss of specific hydrogen bonds with the enzyme. The high species pcDHFR selectivity of TAB could be the result of ligand flexibility that enables multiple binding orientations at the enzyme active site. Further modification of the acetyloxy region of TAB could increase its potency and selectivity for pcDHFR. PMID- 10736155 TI - Structural changes accompanying pH-induced dissociation of the beta-lactoglobulin dimer. AB - We have used NMR spectroscopy to determine the three-dimensional (3D) structure, and to characterize the backbone dynamics, of a recombinant version of bovine beta-lactoglobulin (variant A) at pH 2. 6, where the protein is a monomer. The structure of this low-pH form of beta-lactoglobulin is very similar to that of a subunit within the dimer at pH 6.2. The root-mean-square deviation from the pH 6.2 (crystal) structure, calculated for backbone atoms of residues 6-160, is approximately 1.3 A. Differences arise from the orientation, with respect to the calyx, of the A-B and C-D loops, and of the flanking three-turn alpha-helix. The hydrophobic cavity within the calyx is retained at low pH. The E-F loop (residues 85-90), which moves to occlude the opening of the cavity over the pH range 7.2 6.2, is in the "closed" position at pH 2.6, and the side chain of Glu89 is buried. We also carried out measurements of (15)N T(1)s and T(2)s and (1)H-(15)N heteronuclear NOEs at pH 2.6 and 37 degrees C. Although the residues of the E-F loop (residues 86-89) have the highest crystallographic B-factors, the conformation of this loop is reasonably well defined by the NMR data, and its backbone is not especially mobile on the pico- to nanosecond time scale. Several residues (Ser21, Lys60, Ala67, Leu87, and Glu112) exhibit large ratios of T(1) to T(2), consistent with conformational exchange on a micro- to millisecond time scale. The positions of these residues in the 3D structure of beta-lactoglobulin are consistent with a role in modulating access to the hydrophobic cavity. PMID- 10736156 TI - Deciphering the role of the electrostatic interactions involving Gly70 in eglin C by total chemical protein synthesis. AB - Eglin c from the leech Hirudo medicinalis is a potent protein inhibitor of many serine proteinases including chymotrypsin and subtilisins. Unlike most small protein inhibitors whose solvent-exposed enzyme-binding loop is stabilized primarily by disulfide bridges flanking the reactive-site peptide bond, eglin c possesses an enzyme-binding loop supported predominantly by extensive electrostatic/H-bonding interactions involving three Arg residues (Arg48, Arg51, and Arg53) projecting from the scaffold of the inhibitor. As an adjacent residue, the C-terminal Gly70 participates in these interactions via its alpha-carboxyl group interacting with the side chain of Arg51 and the main chain of Arg48. In addition, the amide NH group of Gly70 donates an H-bond to the carbonyl C=O groups of Arg48 and Arg51. To understand the structural and functional relevance of the electrostatic/H-bonding network, we chemically synthesized wild-type eglin c and three analogues in which Gly70 was either deleted or replaced by glycine amide (NH(2)CH(2)CONH(2)) or by alpha-hydroxylacetamide (HOCH(2)CONH(2)). NMR analysis indicated that the core structure of eglin c was maintained in the analogues, but that the binding loop was significantly perturbed. It was found that deletion or replacement of Gly70 destabilized eglin c by an average of 2.7 kcal/mol or 20 degrees C in melting temperature. As a result, these inhibitors become substrates for their target enzymes. Binding assays on these analogues with a catalytically incompetent subtilisin BPN' mutant indicated that loss or weakening of the interactions involving the carboxylate of Gly70 caused a decrease in binding by approximately 2 orders of magnitude. Notably, for all four synthetic inhibitors, the relative free energy changes (DeltaDeltaG) associated with protein destabilization are strongly correlated (slope = 0.94, r(2) = 0. 9996) with the DeltaDeltaG values derived from a decreased binding to the enzyme. PMID- 10736157 TI - In vitro evolution of a dimeric variant of human pancreatic ribonuclease. AB - Site-directed mutagenesis of human pancreatic RNase (HP-RNase) was used as a model system for investigating the genetic events underlying the evolutionary origins of protein oligomers. HP-RNase is a monomeric enzyme with no natural tendency to oligomerize (K(d) for any dimers in solution of >280 mM). Nevertheless, deletion of five amino acid residues in the loop linking the N terminal helix of HP-RNase to the rest of the protein was found to drive polypeptide chains to fold into dimers. These dimers could not be dissociated by heating at 70 degrees C, and small amounts of monomer were detected only in highly diluted samples. Measurement of dimer and monomer concentrations under equilibrium conditions yielded a K(d) of 1.5 microM. This implies that the deletion increases the protein propensity to dimerize at least 5.2 orders of magnitude. Moreover, the HP-RNase dimers were found to be over 4.6 orders of magnitude more stable than the dimers of bovine pancreatic RNase A obtained by lyophilization from acetic acid (K(d) > 73 mM). Cross-linking experiments with divinyl sulfone indicated that the HP-RNase dimers are stabilized by the exchange between subunits of their N-terminal helices. This generates composite active sites, i.e., each contributed by two subunit chains, that retain full enzymatic activity. Overall, these results show that a deletion of few residues in a key region of a monomeric protein can be the primary event irreversibly leading to oligomerization of the protein through the swap of a secondary structure element between protomers. PMID- 10736158 TI - Uphill electron transfer in the tetraheme cytochrome subunit of the Rhodopseudomonas viridis photosynthetic reaction center: evidence from site directed mutagenesis. AB - The cytochrome (cyt) subunit of the photosynthetic reaction center from Rhodopseudomonas viridis contains four heme groups in a linear arrangement in the spatial order heme1, heme2, heme4, and heme3. Heme3 is the direct electron donor to the photooxidized primary electron donor (special pair, P(+)). This heme has the highest redox potential (E(m)) among the hemes in the cyt subunit. The E(m) of heme3 has been specifically lowered by site-directed mutagenesis in which the Arg residue at the position of 264 of the cyt was replaced by Lys. The mutation decreases the E(m) of heme3 from +380 to +270 mV, i.e., below that of heme2 (+320 mV). In addition, a blue shift of the alpha-band was found to accompany the mutation. The assignment of the lowered E(m) and the shifted alpha-band to heme3 was confirmed by spectroscopic measurements on RC crystals. The structure of the mutant RC has been determined by X-ray crystallography. No remarkable differences were found in the structure apart from the mutated residue itself. The velocity of the electron transfer (ET) from the tetraheme cyt to P(+) was measured under several redox conditions by following the rereduction of P(+) at 1283 nm after a laser flash. Heme3 donates an electron to P(+) with t(1/2) = 105 ns, i.e., faster than in the wild-type reaction center (t(1/2) = 190 ns), as expected from the larger driving force. The main feature is that a phase with t(1/2) approximately 2 micros dominates when heme3 is oxidized but heme2 is reduced. We conclude that the ET from heme2 to heme3 has a t(1/2) of approximately 2 micros, i.e., the same as in the WT, despite the fact that the reaction is endergonic by 50 meV instead of exergonic by 60 meV. We propose that the reaction kinetics is limited by the very uphill ET from heme2 to heme4, the DeltaG degrees of which is about the same (+230 meV) in both cases. The interpretation is further supported by measurements of the activation energy (216 meV in the wild-type, 236 meV in the mutant) and by approximate calculations of ET rates. Altogether these results demonstrate that the ET from heme2 to heme3 is stepwise, starting with a first very endergonic step from heme2 to heme4. PMID- 10736159 TI - Analysis of affinity and specificity in an EF-hand site using double mutant cycles. AB - The effects of three mutations on the EF-hand Ca(2+)/Mg(2+) binding site of smooth muscle myosin regulatory light chain (RLC) were studied: D5S, in which an aspartate is replaced by a serine in position 5 of the loop; D9E, in which an aspartate is replaced by a glutamate in position 9; and D12E, in which the aspartate in position 12 is replaced by a glutamate. All possible combinations of the three mutations were produced. The single mutants D5S and D9E and the double mutant D5S/D9E have low affinity for Ca(2+). All the mutants containing mutation D12E are Ca(2+)-specific and have higher affinities than wild type, even when containing mutations D5S or D9E. All of the mutants studied have lower affinity for Mg(2+) than the wild-type protein. As expected, the changes in binding free energy that each mutant produces depend on the residues present at the other positions of the site, since the mutated positions are very close in the protein structure. Coupling energies are about the same for all pairs of mutants when binding Ca(2+), but can have different values when binding Mg(2+). D5S and D9E have a large negative coupling energy for Mg(2+) binding which suggests an interaction between these two positions. When mutation D12E is present, the coupling energy for Mg(2+) binding between D5S and D9E is much lower, suggesting that this interaction occurs only if an aspartate is in position 12. Glutamate in position 9 may be able to coordinate Mg(2+) directly in the double mutant D5S/D9E. PMID- 10736160 TI - X-ray crystallographic and analytical ultracentrifugation analyses of truncated and full-length yeast copper chaperones for SOD (LYS7): a dimer-dimer model of LYS7-SOD association and copper delivery. AB - Copper-zinc superoxide dismutase (CuZnSOD) acquires its catalytic copper ion through interaction with another polypeptide termed the copper chaperone for SOD. Here, we combine X-ray crystallographic and analytical ultracentrifugation methods to characterize rigorously both truncated and full-length forms of apo LYS7, the yeast copper chaperone for SOD. The 1.55 A crystal structure of LYS7 domain 2 alone (L7D2) was determined by multiple-isomorphous replacement (MIR) methods. The monomeric structure reveals an eight-stranded Greek key beta-barrel similar to that found in yeast CuZnSOD, but it is substantially elongated at one end where the loop regions of the beta-barrel come together to bind a calcium ion. In agreement with the crystal structure, sedimentation velocity experiments indicate that L7D2 is monomeric in solution under all conditions and concentrations that were tested. In contrast, sedimentation velocity and sedimentation equilibrium experiments show that full-length apo-LYS7 exists in a monomer-dimer equilibrium under nonreducing conditions. This equilibrium is shifted toward the dimer by approximately 1 order of magnitude in the presence of phosphate anion. Although the basis for the specificity of the LYS7-SOD interaction as well as the exact mechanism of copper insertion into SOD is unknown, it has been suggested that a monomer of LYS7 and a monomer of SOD may associate to form a heterodimer via L7D2. The data presented here, however, taken together with previously published crystallographic and analytical gel filtration data on full-length LYS7, suggest an alternative model wherein a dimer of LYS7 interacts with a dimer of yeast CuZnSOD. The advantages of the dimer-dimer model over the heterodimer model are enumerated. PMID- 10736162 TI - Structural characterization of adenine nucleotides bound to Escherichia coli adenylate kinase. 1. Adenosine conformations by proton two-dimensional transferred nuclear Overhauser effect spectroscopy. AB - Adenosine conformations of adenosine 5'-triphosphate (ATP) and adenosine 5' monophosphate (AMP), and of an ATP analogue, adenylyl imidodiphosphate (AMPPNP), bound to Escherichia coliadenylate kinase (AKe) in the complexes of AKe.Mg(II)ATP, AKe.AMP.Mg(II)GDP, AKe. AMPPNP, and AKe.Mg(II)AMPPNP were determined by transferred two-dimensional nuclear Overhauser effect spectroscopy (TRNOESY) measurements and molecular dynamics simulations. The glycosidic torsion angles, chi, deduced for the adenine nucleotides in these complexes are 51 degrees, 37 degrees, 49 degrees, and 47 degrees, respectively, with an experimental error of about +/-5 degrees. These values are in general agreement with those previously measured for other ATP-utilizing enzymes, suggesting a possible common motif for adenosine recognition and binding. The pseudorotational phase angle, P, of the sugar puckers for the bound nucleotides varied between 50 degrees and 103 degrees. These solution-state conformations are significantly different from those in published data from X-ray crystallography. A computation of the ligand NOEs, made by using the program CORCEMA [Moseley, H. N. B., Curto, E. V., and Krishna, N. R. (1995) J. Magn. Reson. B108, 243-261] with the protein protons in the vicinity of nucleotide included, on the basis of the X-ray structure of the AKe.AMP.AMPPNP complex [Berry, M. B., Meador, B., Bilderback, T., Liang, P., Glaser, M., and Philips, G. N. , Jr. (1994) Proteins: Struct., Funct., Genet. 19, 183-198], showed that polarization transfer to the protein protons does not produce significant errors in the structures determined by considering the ligand NOEs alone. PMID- 10736161 TI - Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system. In vitro intragenic complementation: the roles of Arg126 in phosphoryl transfer and the C terminal domain in dimerization. AB - Enzyme I mutants of the Salmonella typhimurium phosphoenolpyruvate:sugar phosphotransferase system (PTS), which show in vitro intragenic complementation, have been identified as Arg126Cys (strain SB1690 ptsI34), Gly356Ser (strain SB1681 ptsI16), and Arg375Cys (strain SB1476 ptsI17). The mutation Arg126Cys is in the N-terminal HPr-binding domain, and complements Gly356Ser and Arg375Cys enzyme I mutations located in the C-terminal phosphoenolpyruvate(PEP)-binding domain. Complementation results in the formation of unstable heterodimers. None of the mutations alters the K(m) for HPr, which is phosphorylated by enzyme I. Arg126 is a conserved residue; the Arg126Cys mutation gives a V(max) of 0.04% wild-type, establishing a role in phosphoryl transfer. The Gly356Ser and Arg375Cys mutations reduce enzyme I V(max) to 4 and 2%, respectively, and for both, the PEP K(m) is increased from 0.1 to 3 mM. It is concluded that this activity was from the monomer, rather than the dimer normally found in assays of wild-type. In the presence of Arg126Cys enzyme, V(max) for Gly356Ser and Arg375Cys enzymes I increased 6- and 2-fold, respectively; the K(m) for PEP decreased to <10 microM, but the K(m) became dependent upon the stability of the heterodimer in the assay. Gly356 is conserved in enzyme I and pyruvate phosphate dikinase, which is a homologue of enzyme I, and this residue is part of a conserved sequence in the subunit interaction site. Gly356Ser mutation impairs enzyme I dimerization. The mutation Arg375Cys also impairs dimerization, but the equivalent residue in pyruvate phosphate dikinase is not associated with the subunit interaction site. A 37 000 Da, C-terminal domain of enzyme I has been expressed and purified; it dimerizes and complements Gly356Ser and Arg375Cys enzymes I proving that the association/dissociation properties of enzyme I are a function of the C-terminal domain. PMID- 10736163 TI - Structural characterization of adenine nucleotides bound to Escherichia coli adenylate kinase. 2. 31P and 13C relaxation measurements in the presence of cobalt(II) and manganese(II). AB - 13C spin-lattice relaxation rates have been measured for two complexes of Escherichia coli adenylate kinase (AKe), viz., AKe. [U-(13)C]ATP and AKe.[U (13)C]AMP.GDP in the presence of the substituent activating paramagnetic cation Mn(II) for the purpose of determination of the enzyme-bound conformations of ATP and AMP. (GDP has been added to the AMP complex with the enzyme in order to hold the cation in the bound complex.) Measurements of relaxation times at three different (13)C frequencies, 181.0, 125.7, and 75.4 MHz, indicate that the relaxation times in the enzyme-nucleotide complexes with the paramagnetic cation are not exchange-limited; i.e. , they are larger than the effective lifetimes of cation binding to these complexes and are, therefore, dependent on the cation (13)C distances. An analysis of the frequency-dependent relaxation data allowed all of the ten Mn(II)-(13)C distances to be determined in each of the complexes. Similar measurements of the (31)P relaxation rate made on AKe.ATP and AKe.AMP.GDP complexes in the presence of Co(II) as the activating cation yielded Co(II)-(31)P distances for each adenine nucleotide. These distances, together with the interproton distances determined previously from TRNOESY experiments [Lin, Y., and Nageswara Rao, B. D. (2000) Biochemistry 39, 3636-3646], led to a complete characterization of both ATP and AMP conformations in AKe-bound complexes. These conformations differ significantly from the nucleotide conformations in crystals of AKe. AP(5)A and AKe.AMP.AMPPNP as determined by X-ray crystallography. PMID- 10736164 TI - Improving low-temperature catalysis in the hyperthermostable Pyrococcus furiosus beta-glucosidase CelB by directed evolution. AB - The beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus (CelB) is the most thermostable and thermoactive family 1 glycosylhydrolase described to date. To obtain more insight in the molecular determinants of adaptations to high temperatures and study the possibility of optimizing low temperature activity of a hyperthermostable enzyme, we generated a library of random CelB mutants in Escherichia coli. This library was screened for increased activity on p-nitrophenyl-beta-D-glucopyranoside at room temperature. Multiple CelB variants were identified with up to 3-fold increased rates of hydrolysis of this aryl glucoside, and 10 of them were characterized in detail. Amino acid substitutions were identified in the active-site region, at subunit interfaces, at the enzyme surface, and buried in the interior of the monomers. Characterization of the mutants revealed that the increase in low-temperature activity was achieved in different ways, including altered substrate specificity and increased flexibility by an apparent overall destabilization of the enzyme. Kinetic characterization of the active-site mutants showed that in all cases the catalytic efficiency at 20 degrees C on p-nitrophenyl-beta-D-glucose, as well as on the disaccharide cellobiose, was increased up to 2-fold. In most cases, this was achieved at the expense of beta-galactosidase activity at 20 degrees C and total catalytic efficiency at 90 degrees C. Substrate specificity was found to be affected by many of the observed amino acid substitutions, of which only some are located in the vicinity of the active site. The largest effect on substrate specificity was observed with the CelB variant N415S that showed a 7.5-fold increase in the ratio of p-nitrophenyl-beta-D-glucopyranoside/p-nitrophenyl-beta D-galactopyra noside hydrolysis. This asparagine at position 415 is predicted to interact with active-site residues that stabilize the hydroxyl group at the C4 position of the substrate, the conformation of which is equatorial in glucose containing substrates and axial in galactose-containing substrates. PMID- 10736165 TI - Evaluation of biosynthetic pathways to delta-aminolevulinic acid in Propionibacterium shermanii based on biosynthesis of vitamin B12 from D-[1 13C]glucose. AB - Analysis of the (13)C nuclear magnetic resonance (NMR) spectrum of (13)C-labeled vitamin B(12) biosynthesized from D-[1-(13)C]glucose by Propionibacterium shermanii provided evidence suggesting that delta-aminolevulinic acid (ALA) incorporated in the (13)C-labeled vitamin B(12) may have been synthesized via both the Shemin pathway and the C5 pathway under anaerobic conditions in the ratio of 1 < [(ratio of ALA biosynthesis from the Shemin pathway)/(that from the C5 pathway)] < 1.8. The D-ribose moiety of vitamin B(12) was labeled with (13)C at R-1, R-3, and R-5. The aminopropanol moiety of vitamin B(12) was labeled on Pr 1 and Pr-2, but not Pr-3. PMID- 10736166 TI - Identification of essential arginines in the acetate kinase from Methanosarcina thermophila. AB - Site-directed mutagenesis is a powerful tool for identifying active-site residues essential for catalysis; however, this approach has only recently become available for acetate kinase. The enzyme from Methanosarcina thermophila has been cloned and hyper-produced in a highly active form in Escherichia coli (recombinant wild-type). The role of arginines in this acetate kinase was investigated. Five arginines (R91, R175, R241, R285, and R340) in the M. thermophila enzyme were selected for individual replacement based on their high conservation among sequences of acetate kinase homologues. Replacement of R91 or R241 with alanine or leucine produced variants with specific activities less than 0.1% of the recombinant wild-type enzyme. The circular dichroism spectra and other properties of these variants were comparable to those of recombinant wild type, indicating no global conformational changes. These results indicate that R91 and R241 are essential for activity, consistent with roles in catalysis. The variant produced by conservative replacement of R91 with lysine had approximately 2% of recombinant wild-type activity, suggesting a positive charge is important in this position. The K(m) value for acetate of the R91K variant increased greater than 10-fold relative to recombinant wild-type, suggesting an additional role for R91 in binding this substrate. Activities of both the R91A and R241A variants were rescued 20-fold when guanidine or derivatives were added to the reaction mixture. The K(m) values for ATP of the rescued variants were similar to those of recombinant wild-type, suggesting that the rescued activities are the consequence of replacement of important functional groups and not changes in the catalytic mechanism. These results further support roles for R91 and R241 in catalysis. Replacement of R285 with alanine, leucine, or lysine had no significant effect on activity; however, the K(m) values for acetate increased 6 10-fold, suggesting R285 influences the binding of this substrate. Phenylglyoxal inhibition and substrate protection experiments with the recombinant wild-type enzyme and variants were consistent with the presence of one or more essential arginine residues in the active site as well as with roles for R91 and R241 in catalysis. It is proposed that R91 and R241 function to stabilize the previously proposed pentacoordinate transition state during direct in-line transfer of the gamma-phosphate of ATP to acetate. The kinetic characterization of variants produced by replacement of R175 and R340 with alanine, leucine, or lysine indicated that these residues are not involved in catalysis but fulfill important structural roles. PMID- 10736167 TI - Effects of single-tryptophan mutations on R67 dihydrofolate reductase. AB - R67 dihydrofolate reductase (DHFR) is an R-plasmid-encoded enzyme that confers clinical resistance to the antibacterial drug trimethoprim. This enzyme shows no sequence or structural homology to the chromosomal DHFRs. The active form of the protein is a homotetramer possessing D(2) symmetry and a single active-site pore. Two tryptophans occur per monomer: W38 and its symmetry-related residues (W138, W238, and W338) occur at the dimer-dimer interfaces, while W45 and its symmetry related partners (W145, W245, and W345) occur at the monomer-monomer interfaces. Two single-tryptophan mutant genes were constructed to determine the structural and functional consequences of four mutations per tetramer. The W45F mutant retains full enzyme activity and the fluorescence environment of the unmutated W38 residues clearly monitors ligand binding and a pH dependent tetramer right harpoon over left harpoon 2 dimers equilibrium. In contrast, four simultaneous W38F mutations at the dimer-dimer interfaces result in tetramer destabilization. The ensuing dimer is relatively inactive, as is dimeric wild-type R67 DHFR. A comparison of emission spectra indicates the fluorescent signal of wild-type R67 DHFR is dominated by the contribution from W38. Equilibrium unfolding/folding curves at pH 5.0, where all protein variants are dimeric, indicate the environment monitored by the W38 residue is slightly less stable than the environment monitored by the W45 residue. PMID- 10736168 TI - Investigation of spectroscopic intermediates during copper-binding and TPQ formation in wild-type and active-site mutants of a copper-containing amine oxidase from yeast. AB - Copper amine oxidases possess the unusual ability to generate autocatalytically their organic cofactor, which is subsequently utilized in turnover. This cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), is formed within the active site of these enzymes by the oxidation of a single tyrosine residue. In vitro, copper(II) and oxygen are both necessary and sufficient for the conversion of tyrosine to TPQ. In this study, the biogenesis of TPQ has been characterized in an amine oxidase from Hansenula polymorpha expressed as the apo-enzyme in Escherichia coli. With the WT enzyme, optical absorbances which are copper or oxygen dependent are observed and characterized. Active-site mutants are used to investigate further the nature of these spectral species. Evidence is presented which suggests that tyrosine is activated for reaction with oxygen by liganding to Cu(II). In the following paper in this issue [Schwartz, B., Dove, J. E., and Klinman, J. P. (2000) Biochemistry 39, 3699-3707], the initial reaction of precursor protein with oxygen is characterized kinetically. Taken together, the available data suggest a mechanism for the oxidation of tyrosine to TPQ where the role of the copper is to activate substrate. PMID- 10736170 TI - Kinetic and mechanistic analysis of the E. coli panE-encoded ketopantoate reductase. AB - Ketopantoate reductase (EC 1.1.1.169) catalyzes the NADPH-dependent reduction of alpha-ketopantoate to form D-(-)-pantoate in the pantothenate/coenzyme A biosynthetic pathway. The enzyme encoded by the panE gene from E. coli K12 was overexpressed and purified to homogeneity. The native enzyme exists in solution as a monomer with a molecular mass of 34 000 Da. The steady-state initial velocity and product inhibition patterns are consistent with an ordered sequential kinetic mechanism in which NADPH binding is followed by ketopantoate binding, and pantoate release precedes NADP(+) release. The pH dependence of the kinetic parameters V and V/K for substrates in both the forward and reverse reactions suggests the involvement of a single general acid/base in the catalytic mechanism. An enzyme group exhibiting a pK value of 8.4 +/- 0.2 functions as a general acid in the direction of the ketopantoate reduction, while an enzyme group exhibiting a pK value of 7.8 +/- 0.2 serves as a general base in the direction of pantoate oxidation. The stereospecific transfer of the pro-S hydrogen atom of NADPH to the C-2 position of ketopantoate was demonstrated by (1)H NMR spectroscopy. Primary deuterium kinetic isotope effects of 1.3 and 1.5 on V(for) and V/K(NADPH), respectively, and 2.1 and 1.3 on V(rev) and V/K(HP), respectively, suggest that hydride transfer is not rate-limiting in catalysis. Solvent kinetic isotope effects of 1.3 on both V(for) and V/K(KP), and 1.4 and 1.5 on V(rev) and V/K(HP), respectively, support this conclusion. The apparent equilibrium constant, K(eq)', of 676 at pH 7.5 and the standard free energy change, DeltaG, of -14 kcal/mol suggest that ketopantoate reductase reaction is very favorable in the physiologically important direction of pantoate formation. PMID- 10736169 TI - Kinetic analysis of oxygen utilization during cofactor biogenesis in a copper containing amine oxidase from yeast. AB - A detailed kinetic analysis of oxygen consumption during TPQ biogenesis has been carried out on a yeast copper amine oxidase. O(2) is consumed in a single, exponential phase, the rate of which responds linearly to dissolved oxygen concentration. This behavior is observed up to conditions of maximally obtainable oxygen concentrations. In contrast, no viscosity effect is observed on rate, implicating a high K(m) for O(2). Binding of oxygen appears to occur faster than its consumption and to result in displacement of the precursor tyrosine onto copper to form a charge-transfer species, described in the the preceding paper of this issue [Dove, J. E., Schwartz, B., Williams, N. K., and Klinman, J. P. (2000) Biochemistry 39, 3690-3698). Reaction between this intermediate and O(2) is proposed to occur in a rate-limiting step, and to proceed more rapidly when the tyrosine is deprotonated. This rate-limiting step in cofactor biogenesis does not display a solvent isotope effect and is, thus, uncoupled from proton transfer. Comparisons are drawn between the proposed biogenesis mechanism and that for the oxidation of reduced cofactor during catalytic turnover in the mature enzyme. PMID- 10736171 TI - The structural unit of the secretory Na+-K+-2Cl- cotransporter (NKCC1) is a homodimer. AB - The oligomeric state of the secretory Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) in rat parotid plasma membranes was studied using the reversible chemical cross linker DTSSP [3, 3'-dithiobis(sulfosuccinimidyl propionate)]. The monomeric apparent molecular mass of NKCC1 is approximately 170 kDa. However, we show here that this protein migrates as a approximately 355 kDa complex on SDS-PAGE gels after membrane treatment with DTSSP, indicating that NKCC1 exists as an oligomer in the plasma membrane. The stability of this oligomer is such that it is not disrupted by solubilization of the membrane by low concentrations of the nonionic detergent Triton X-100 (0.3%) or the mild ionic detergent deoxycholate (20 mM); however, higher concentrations of Triton X-100 or treatment with the denaturing detergent SDS do result in destabilization of the NKCC1 complex. In additional experiments, we immunoprecipitated the 355 kDa cross-linked complex from biotinylated membranes, then cleaved the cross-linking bonds and analyzed the resulting components of the NKCC1 oligomer by avidin blotting, silver staining, and 2D electrophoresis. In these studies, we were unable to detect the presence of any proteins other than NKCC1 itself in the 355 kDa oligomer, suggesting that this complex is an NKCC1 dimer. Strong evidence for this conclusion was provided by a quantitative analysis of the molecular sizes of oligomers formed by full length NKCC1 and an N-terminally truncated version of NKCC1 expressed in HEK293 cells. Taken together, our data provide convincing evidence that the dominant structural unit of NKCC1 in the plasma membrane is a homodimer. PMID- 10736172 TI - Anchor structure of cell wall surface proteins in Listeria monocytogenes. AB - Many surface proteins of Gram-positive bacteria are anchored to the cell wall by a mechanism requiring a COOH-terminal sorting signal with a conserved LPXTG motif. In Staphylococcus aureus, surface proteins are cleaved between the threonine and the glycine of the LPXTG motif. The carboxyl of threonine is subsequently amide linked to the amino group of the pentaglycine cell wall crossbridge. Here we investigated the anchor structure of surface proteins in Listeria monocytogenes. A methionine and six histidines (MH(6)) were inserted upstream of the LPXTG motif of internalin A (InlA), a cell-wall-anchored surface protein of L. monocytogenes. The engineered protein InlA-MH(6)-Cws was found anchored in the bacterial cell wall. After peptidoglycan digestion with phage endolysin, InlA-MH(6)-Cws was purified by affinity chromatography. COOH-terminal peptides of InlA-MH(6)-Cws were obtained by cyanogen bromide cleavage followed by purification on a nickel-nitriloacetic acid column. Analysis of COOH-terminal peptides with Edman degradation and mass spectrometry revealed an amide linkage between the threonine of the cleaved LPXTG motif and the amino group of the m diaminopimelic acid crossbridge within the listerial peptidoglycan. These results reveal that the cell wall anchoring of surface proteins in Gram-positive bacteria such as S. aureus and L. monocytogenes occurs by a universal mechanism. PMID- 10736173 TI - Selective reconstitution of human D4 dopamine receptor variants with Gi alpha subtypes. AB - G protein-coupled receptors (GPCRs) are seven-transmembrane (TM) helical proteins that bind extracellular molecules and transduce signals by coupling to heterotrimeric G proteins in the cytoplasm. The human D4 dopamine receptor is a particularly interesting GPCR because the polypeptide loop linking TM helices 5 and 6 (loop i3) may contain from 2 to 10 similar direct hexadecapeptide repeats. The precise role of loop i3 in D4 receptor function is not known. To clarify the role of loop i3 in G protein coupling, we constructed synthetic genes for the three main D4 receptor variants. D4-2, D4-4, and D4-7 receptors contain 2, 4, and 7 imperfect hexadecapeptide repeats in loop i3, respectively. We expressed and characterized the synthetic genes and found no significant effect of the D4 receptor polymorphisms on antagonist or agonist binding. We developed a cell based assay where activated D4 receptors coupled to a Pertussis toxin-sensitive pathway to increase intracellular calcium concentration. Studies using receptor mutants showed that the regions of loop i3 near TM helices 5 and 6 were required for G protein coupling. The hexadecapeptide repeats were not required for G protein-mediated calcium flux. Cell membranes containing expressed D4 receptors and receptor mutants were reconstituted with purified recombinant G protein alpha subunits. The results show that each D4 receptor variant is capable of coupling to several G(i)alpha subtypes. Furthermore, there is no evidence of any quantitative difference in G protein coupling related to the number of hexadecapeptide repeats in loop i3. Thus, loop i3 is required for D4 receptors to activate G proteins. However, the polymorphic region of the loop does not appear to affect the specificity or efficiency of G(i)alpha coupling. PMID- 10736174 TI - The glucose transporter of the Escherichia coli phosphotransferase system: linker insertion mutants and split variants. AB - The IICB(Glc) subunit of the glucose transporter acts by a mechanism which couples vectorial translocation with phosphorylation of the substrate. It contains 8 transmembrane segments connected by 4 periplasmic, 2 short, 1 long (80 residues), cytoplasmic loops and an independently folding cytoplasmic domain at the C-terminus. Random DNase I cleavage, EcoRI linker insertion, and screening for transport-active mutants afforded 12 variants with between 46% and 116% of wild-type sugar phosphorylation activity. They carried inserts of up to 29 residues and short deletions in periplasmic loops 1, 2, and 3, in the long cytoplasmic loop 3, and in the linker region between the membrane spanning IIC(Glc) and the cytoplasmic IIB(Glc) domains. Disruption of the gene at the sites of linker insertion decreased the expression level and diminished phosphotransferase activity to between 7% and 32%. IICB(Glc) with a discontinuity in the cytoplasmic loop was purified to homogeneity as a stable complex. It was active only if encoded by a dicistronic operon but not if encoded by two genes on two different replicons, suggesting that spatial proximity of the nascent polypeptide chains is important for folding and membrane assembly. PMID- 10736175 TI - A three-step kinetic mechanism for peptide binding to MHC class II proteins. AB - Peptide binding reactions of class II MHC proteins exhibit unusual kinetics, with extremely slow apparent rate constants for the overall association (<100 M(-)(1) s(-)(1)) and dissociation (<10(-)(5) s(-)(1)) processes. Various linear and branched pathways have been proposed to account for these data. Using fluorescence resonance energy transfer between tryptophan residues in the MHC peptide binding site and aminocoumarin-labeled peptides, we measured real-time kinetics of peptide binding to empty class II MHC proteins. Our experiments identified an obligate intermediate in the binding reaction. The observed kinetics were consistent with a binding mechanism that involves an initial bimolecular binding step followed by a slow unimolecular conformational change. The same mechanism is observed for different peptide antigens. In addition, we noted a reversible inactivation of the empty MHC protein that competes with productive binding. The implications of this kinetic mechanism for intracellular antigen presentation pathways are discussed. PMID- 10736176 TI - Interaction of heparin with a synthetic pentadecapeptide from the C-terminal heparin-binding domain of fibronectin. AB - The synthetic pentadecapeptide FN-C/H II (KNNQKSEPLIGRKKT-NH(2)) has the sequence of the carboxy-terminal heparin-binding domain of module III(14) of fibronectin. Interaction of FN-C/H II with bovine lung heparin has been studied by (1)H and (23)Na NMR spectroscopy and by heparin affinity chromatography. FN-C/H II binds to heparin from pD <2 up to pD approximately 10; at higher pD, the binding decreases as the lysine side-chain ammonium groups are titrated. Na(+) counterions are displaced from the counterion condensation volume that surrounds sodium heparinate by FN-C/H II, which provides direct evidence that the binding involves electrostatic interactions. The pK(A) values for each of the five ammonium groups of FN-C/H II increase upon binding to heparin which, together with chemical shift data, indicates that the binding involves both delocalized and direct electrostatic interactions between ammonium groups of FN-C/H II and carboxylate and/or sulfate groups of heparin. NMR data also provide evidence for the direct interaction of the guanidinium group of the arginine side chain with anionic sites on heparin. The affinity of heparin for FN-C/H II and for 13 analogue peptides in which lysine and arginine residues were systematically substituted with alanine increases as the number of basic residues increases. The relative contribution of each lysine and arginine to the affinity of heparin for FN-C/H II is R(12) > K(13) > K(14) > K(1) > K(5). Nuclear Overhauser enhancement (NOE) data indicate that, while FN-C/H II is largely unstructured in aqueous solution, the bound peptide interconverts among overlapping, turn-like conformations over the L(9) - T(15) segment of the peptide. NOE data for the interaction of FN-C/H II with a heparin-derived hexasaccharide, together with the number of Na(+) ions displaced from heparin by FN-C/H II as determined by (23)Na NMR, indicates that the peptide binds to a hexasaccharide segment of heparin. Identical NMR and heparin affinity chromatography results were obtained for the interaction of FN-C/H II and its D-amino acid analogue peptide with heparin, which is of interest for the potential use of peptides as therapeutic agents for diseases in which cell adhesion plays a critical role. PMID- 10736177 TI - Transposing sequences between fetal and adult hemoglobins indicates which subunits and regulatory molecule interfaces are functionally related. AB - To correlate amino acid sequence changes with hemoglobin function we are carrying out a detailed recombinant analysis of the adult hemoglobin/fetal hemoglobin (HbA/HbF) systems. The important physiological differences between these two tetramers lie at unspecified sites in the 39 sequence substitutions of the 146 amino acids in their beta and gamma chains. In this paper, significant differences in the tetramer-dimer dissociation constants (referred to as tetramer "strength" or "stability") of adult (HbA) and fetal (HbF) hemoglobin tetramers have been used to probe the relationship between the allosteric, sliding interface and the effects of the allosteric regulator, 2,3-DPG, in promoting oxygen release. The single amino acid difference at the allosteric interfaces of these two hemoglobins, Glu-43(beta) --> Asp-43(gamma), which is not near the DPG binding site, leads to a significantly lower DPG response, approaching that of HbF. The results are inconsistent with the long-held idea that the replacement of His-143(beta) in HbA to Ser-143(gamma) in HbF is solely responsible for the lowered DPG response in HbF. On the other hand, the Val-1(beta) --> Gly-1(gamma) replacement near the DPG binding site has no effect on the DPG response. The replacement of His-116(beta) by the hydrophobic Ile-116(gamma) at the rigid alpha(1)beta(1) interface has a marginal yet detectable effect on the allosteric alpha(1)beta(2) interface. The results, overall, are interpreted using a model involving electrostatic coupling between certain side chains and extend the concept of a long-range relationship between some distant regions of the tetramer that are likely mediated through the central cavity. PMID- 10736178 TI - A novel peptide antagonist of CXCR4 derived from the N-terminus of viral chemokine vMIP-II. AB - The viral macrophage inflammatory protein-II (vMIP-II) encoded by Kaposi's sarcoma-associated herpesvirus is unique among all known chemokines in that vMIP II shows a broad-spectrum interaction with both CC and CXC chemokine receptors including CCR5 and CXCR4, two principal coreceptors for the cell entry of human immunodeficiency virus type 1 (HIV-1). To elucidate the mechanism of the promiscuous receptor interaction of vMIP-II, synthetic peptides derived from the N-terminus of vMIP-II were studied. In contrast to the full-length protein that recognizes both CXCR4 and CCR5, a peptide corresponding to residues 1-21 of vMIP II (LGASWHRPDKCCLGYQKRPLP) was shown to strongly bind CXCR4, but not CCR5. The IC(50) of this peptide in competing with CXCR4 binding of (125)I-SDF-1alpha is 190 nM as compared to the IC(50) of 14.8 nM of native vMIP-II in the same assay. The peptide selectively prevented CXCR4 signal transduction and coreceptor function in mediating the entry of T- and dual-tropic HIV-1 isolates, but not those of CCR5. Further analysis of truncated peptide analogues revealed the importance of the first five residues for the activity with CXCR4. These results suggest that the N-terminus of vMIP-II is essential for its function via CXCR4. In addition, they reveal a possible mechanism for the distinctive interactions of vMIP-II with different chemokine receptors, a notion that may be further exploited to dissect the structural basis of its promiscuous biological function. Finally, the potent CXCR4 peptide antagonist shown here could serve as a lead for the development of new therapeutic agents for HIV infection and other immune system diseases. PMID- 10736179 TI - Mechanisms of fibroblast growth factor 2 intracellular processing: a kinetic analysis of the role of heparan sulfate proteoglycans. AB - The interaction of fibroblast growth factor 2 (FGF-2) with heparan sulfate proteoglycans (HSPG) has been demonstrated to enhance receptor binding and alter the intracellular distribution of internalized FGF-2. In the present study, the intracellular fate of FGF-2 was analyzed in vascular smooth muscle cells (VSMC) under native and HSPG-deficient conditions. HSPG-deficient cells were generated by treatment with sodium chlorate. Cells were incubated with FGF-2 at 37 degrees C for prolonged periods (0-48 h) to allow for FGF-2 uptake and processing. Processing of FGF-2 occurred in stages. Initially a family of low molecular weight (LMW) fragments (4-10 kDa) were detected that accumulated to much higher ( approximately 10-fold) levels in native compared to heparan sulfate-deficient cells. Pulse-chase experiments revealed that the half-life of these LMW intermediates was significantly greater in native ( approximately 18 h) compared to HSPG-deficient cells ( approximately 4 h). Rate constants for FGF-2 processing were derived by modeling the uptake and processing of FGF-2 as a set of first order differential equations. The kinetic analysis indicated that the greatest differences between native and HSPG-deficient VSMC was in the formation of LMW and further suggested that these FGF-2 products appear to represent a stable subpool of internal FGF-2 that is favored in cells that contain HSPG. Thus, HSPG might function as a cellular switch between immediate and prolonged signal activation by heparin-binding growth factors such as FGF-2. In the absence of HSPG, FGF-2 can interact with and activate its receptor, yet in the presence of HSPG, FGF-2 might be able to mediate prolonged or unique biological responses through intracellular processes. PMID- 10736180 TI - Cystic fibrosis transmembrane conductance regulator has an altered structure when its maturation is inhibited. AB - Inefficient maturation and trafficking to the cell surface of the cystic fibrosis transmembrane conductance regulator (CFTR) is the primary cause of cystic fibrosis. CFTR protein that fails to mature accumulates as an immature core glycosylated protein and is rapidly degraded. To determine how the structures of mature and immature CFTR are different, we compared the properties of CFTR that had been expressed in the presence or absence of the proteasome inhibitor, MG-132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal). Transient expression of wild-type CFTR in the presence of submicromolar concentrations of MG-132 blocks maturation of the protein. We found that expression of CFTR in the presence of MG-132 trapped the protein in a trypsin-sensitive conformation. In addition, the structure of the carboxyl-terminus of immature and mature CFTR differed as histidine-tagged mature CFTR was preferentially recovered by metal-chelate chromatography. No chloride channel activity was detected when membranes containing immature CFTR were fused with planar lipid bilayers. These results show that expression of CFTR in the presence of MG-132 traps the protein in an altered conformation that may be inactive. PMID- 10736181 TI - Nuclear magnetic resonance studies of the N-terminal fragment of adenosine diphosphate ribosylation factor 1 in micelles and bicelles: influence of N myristoylation. AB - The N-terminal fragment of adenosine diphosphate (ADP) ribosylation factor 1 (ARF1) is proposed to be involved in the guanosine triphosphate- (GTP-) dependent, reversible association of the protein with membranes through the interaction of not only the N-linked myristoyl chain but also its highly conserved N-terminal hydrophobic residues. Based on the N-terminal sequence of this protein, specifically (13)C- and (15)N-labeled peptides were synthesized with and without an N-myristoyl anchor. The behavior, including structure, dynamics, and orientation, of these peptides in a lipid environment was then studied through a combination of solution (1)H nuclear magnetic resonance (NMR) techniques in micelles and heteronuclear solid-state NMR experiments in magnetically aligned bicelles. The work presented is an extension of the previously reported characterization of the myristoylated N-terminal fragment of ARF1 [Losonczi and Prestegard (1998) Biochemistry 37, 706-716] to include a comparison to a nonmyristoylated analogue. Results indicate that both myristoylated and nonmyristoylated peptides are alpha-helical in a lipid environment and that N-myristoylation does not greatly influence the structure of the peptides. Evidence is presented suggesting association of the peptides with bilayer disks through a combination of edge and surface interactions. PMID- 10736182 TI - Divalent metal binding properties of the methionyl aminopeptidase from Escherichia coli. AB - The metal-binding properties of the methionyl aminopeptidase from Escherichia coli (MetAP) were investigated. Measurements of catalytic activity as a function of added Co(II) and Fe(II) revealed that maximal enzymatic activity is observed after the addition of only 1 equiv of divalent metal ion. Based on these studies, metal binding constants for the first metal binding event were found to be 0.3 +/ 0.2 microM and 0.2 +/- 0.2 microM for Co(II)- and Fe(II)-substituted MetAP, respectively. Binding of excess metal ions (>50 equiv) resulted in the loss of approximately 50% of the catalytic activity. Electronic absorption spectral titration of a 1 mM sample of MetAP with Co(II) provided a binding constant of 2.5 +/- 0.5 mM for the second metal binding site. Furthermore, the electronic absorption spectra of Co(II)-loaded MetAP indicated that both metal ions reside in a pentacoordinate geometry. Consistent with the absorption data, electron paramagnetic resonance (EPR) spectra of [CoCo(MetAP)] also indicated that the Co(II) geometries are not highly constrained, suggesting that each Co(II) ion in MetAP resides in a pentacoordinate geometry. EPR studies on [CoCo(MetAP)] also revealed that at pH 7.5 there is no significant spin-coupling between the two Co(II) ions, though a small proportion ( approximately 5%) of the sample exhibited detectable spin-spin interactions at pH values > 9.6. EPR studies on [Fe(III)_(MetAP)] and [Fe(III)Fe(III)(MetAP)] also suggested no spin-coupling between the two metal ions. (1)H nuclear magnetic resonance (NMR) spectra of [Co(II)_(MetAP)] in both H(2)O and D(2)O buffer indicated that the first metal binding site contains the only active-site histidine residue, His171. Mechanistic implications of the observed binding properties of divalent metal ions to the MetAP from E. coli are discussed. PMID- 10736183 TI - Calcium binding properties of recombinant calcium binding protein 40, a major calcium binding protein of lower eukaryote Physarum polycephalum. AB - Calcium binding protein 40 (CBP40) is a Ca(2+)-binding protein abundant in the plasmodia of Physarum polycephalum. CBP40 consists four EF-hand domains in the COOH-terminal half and a putative alpha-helix domain in the NH(2)-terminal half. We expressed recombinant proteins of CBP40 in Escherichia coli to investigate its Ca(2+)-binding properties. Recombinant proteins of CBP40 bound 4 mol of Ca(2+) with much higher affinity (pCa(1/2) = 6.5) than that of calmodulin. When residues 1-196 of the alpha-helix domain were deleted, the affinity for Ca(2+) decreased to pCa(1/2) = 4.6. A chimeric calmodulin was generated by conjugating the alpha helix domain of CBP40 with calmodulin. The affinity of Ca(2+) for the chimeric calmodulin was higher than that for calmodulin, suggesting that the alpha-helix domain is responsible for the high affinity of CBP40 for Ca(2+). CBP40 forms large aggregates reversibly in a Ca(2+)-dependent manner. A mutant protein with a deletion of NH(2)-terminal 32 residues, however, could not aggregate, indicating the importance of these residues for the aggregation. The aggregation occurs above micromolar levels of Ca(2+) concentration, so it may only occur when CBP40 is secreted out of the plasmodial cells. PMID- 10736184 TI - DNA sequence-dependent contributions of core histone tails to nucleosome stability: differential effects of acetylation and proteolytic tail removal. AB - Modulation of nucleosome stability in chromatin plays an important role in eukaryotic gene expression. The core histone N-terminal tail domains are believed to modulate the stability of wrapping nucleosomal DNA and the stability of the chromatin filament. We analyzed the contribution of the tail domains to the stability of nucleosomes containing selected DNA sequences that are intrinsically straight, curved, flexible, or inflexible. We find that the presence of the histone tail domains stabilizes nucleosomes containing DNA sequences that are intrinsically straight or curved. However, the tails do not significantly contribute to the free energy of nucleosome formation with flexible DNA. Interestingly, hyperacetylation of the core histone tail domains does not recapitulate the effect of tail removal by limited proteolysis with regard to nucleosome stability. We find that acetylation of the tails has the same minor effect on nucleosome stability for all the selected DNA sequences. A comparison of histone partitioning between long donor chromatin, acceptor DNA, and free histones in solution shows that the core histone tails mediate internucleosomal interactions within an H1-depleted chromatin fiber amounting to an average free energy of about 1 kcal/mol. Thus, such interactions would be significant with regard to the free energies of sequence-dependent nucleosome positioning. Last, we analyzed the contribution of the H2A/H2B dimers to nucleosome stability. We find that the intact nucleosome is stabilized by 900 cal/mol by the presence of the dimers regardless of sequence. The biological implications of these observations are discussed. PMID- 10736185 TI - Estrogen receptor alpha interaction with estrogen response element half-sites from the rat prolactin gene. AB - Estrogen regulation of the rat prolactin gene requires sequences within the DNase I hypersensitive site II (HSII). We have used overexpressed mouse estrogen receptor alpha (ERalpha) protein to study interactions of ERalpha with an imperfect estrogen response element (ERE) and four ERE half-site sequences from HSII. We confirmed that ERalpha has higher affinity for ERE half-sites than for the imperfect ERE. As expected, the imperfect ERE formed a complex with ERalpha similar to that between mERalpha and a consensus ERE in gel shift assays. The ERalpha complex with half-sites, however, had faster mobility on a 4% polyacrylamide gel than the ERalpha complex with a consensus ERE, indicating that the complexes had different compositions. Ferguson analysis revealed that the ERalpha/half-site complex had a larger molecular weight and higher negative charge than the ERalpha/consensus ERE complex. Similar results were observed with purified human ERalpha, showing that the ERalpha/half-site complex contained only ERalpha and oligonucleotides. These results are best explained by a model in which a dimer of ERalpha is bound to two half-site oligonucleotides. We propose that two ERalpha dimers may interact with the four ERE half-sites in HSII to influence estrogen regulation of this gene. PMID- 10736186 TI - Formate-induced inhibition of the water-oxidizing complex of photosystem II studied by EPR. AB - The effects of various formate concentrations on both the donor and the acceptor sides in oxygen-evolving PS II membranes (BBY particles) were examined. EPR, oxygen evolution and variable chlorophyll fluorescence have been observed. It was found that formate inhibits the formation of the S(2) state multiline signal concomitant with stimulation of the Q(A)(-)Fe(2+) signal at g = 1.82. The decrease and the increase in intensities of the multiline and Q(A)(-)Fe(2+) signals, respectively, had a linear relation for formate concentrations between 5 and 500 mM. The g = 4.1 signal formation measured in the absence of methanol was not inhibited by formate up to 250 mM in the buffer. In the presence of 3% methanol the g = 4.1 signal evolved as formate concentration increased. The evolved signal could be ascribed to the inhibited centers. Oxygen evolution measured in the presence of an electron acceptor, phenyl-p-benzoquinone, was also inhibited by formate proportionally to the decrease in the multiline signal intensity. The inhibition seemed to be due to a retarded electron transfer from the water-oxidizing complex to Y(Z)(+), which was observed in the decay kinetics of the Y(Z)(+) signal induced by illumination above 250 K. These results show that formate induces inhibition of water oxidation reactions as well as electron transfer on the PS II acceptor side. The inhibition effects of formate in PS II were found to be reversible, indicating no destructive effect on the reaction center induced by formate. PMID- 10736187 TI - Site-directed spin-labeling of the catalytic sites yields insight into structural changes within the F0F1-ATP synthase of Escherichia coli. AB - Electron spin resonance (ESR) spectroscopy using site-specific cysteine spin labeling of the catalytic nucleotide binding sites of F(1)-ATPase was employed to investigate conformational changes within the nucleotide binding sites of the enzyme. Mutant Escherichia coli F(1) that had been modified at position beta Y331C with a spin label showed almost normal catalytic activity and enabled us to study the effects of binding of different nucleotides and of the F(o) subunit b on the conformation of the catalytic binding sites. The ESR spectra of the spin labeled, nucleotide-depleted F(1) indicate asymmetry within the sites as is expected from the structural models of the enzyme. Nucleotide binding to the enzyme clearly affects the conformation of the sites; the most pronounced feature upon nucleotide binding is the formation of catalytic site(s) in a very open conformation. Using the same beta-331 spin-labeled F(1) and a truncated form of F(o) subunit b, b(24)(-)(156), we found that binding of b(24)(-)(156) to spin labeled F(1) significantly changes the conformation of the catalytic sites. In this paper we present data that for the first time directly show that a conformational binding change takes place upon binding of nucleotides to the nucleotide binding sites and that also show that binding of b(24)(-)(156) strongly affects the conformation of the catalytic sites, most likely by increasing the population of binding sites that are in the open conformation. PMID- 10736188 TI - Cell-to-cell communication via plant endomembranes. AB - Cell-to-cell communication was investigated in epidermal cells cut from stem internodal tissue of Nicotiana tabacum and Torenia fournieri. Fluorescently labelled peptides and dextrans were microinjected using iontophoresis into the cytoplasm andcortical endomembrane network of these cells. The microinjected endomembrane network was similar in location and structure to the endoplasmic reticulum (ER) as revealed by staining with 3, 3'-dihexyloxacarbocyanine iodide (DiOC(6)). No cell-to-cell movement of dextrans was observed following cytoplasmic injections but injection of dextrans into the endomembrane network resulted in rapid diffusion of the probes to neighbouring cells. It is proposed that the ER acts as a pathway for intercellular communication via the desmotubule through plasmodesmata. PMID- 10736189 TI - Dna-end binding activity of Ku in synchronized cells. AB - Three different types of cells were synchronized by various methods and DNA-end binding (DEB) activities of Ku were compared with asynchronous controls. In CHO K1 cells synchronized in G1 phase by serum starvation and in S phase by serum refeeding, DEB activity was reduced in S cells but remained unchanged in G1 cells. However, the same type of cells synchronized in G1/S phase by double thymidine block and in S phase by releasing the blockage, have the same DEB activity as asynchronous controls. A similar result was found in RKO and HeLa cells synchronized by the latter method. Arresting cells in mitosis with nocodazole also generated different cell cycle effects. Ku activity was reduced in CHO K1 and RKO cells, but not in HeLa cells after treatment with nocodazole. In phase-enriched cells separated by centrifugal elutriation, DEB activities were similar at different stages of the cell cycle in all three types of cells. Thus, different synchronization procedures can give very different values of Ku activity in a cell type-dependent manner. Results from elutriated cells are consistent, and suggest DEB activity of Ku does not change with the cell cycle. PMID- 10736190 TI - Developmental regulation of granule size and numbers in larval salivary glands of drosophila by steroid hormone ecdysone. AB - The size and number of secretory granules in late larval salivary glands of Drosophila melanogaster have been related to interecdysial and early metamorphic development represented by well-known puffs in polytene chromosomes. Interecdysial period (puff stage 1 (PS1)) is characterized by presence of numerous small granules (11,000 per cell). The transition from PSI to early metamorphic phase (PS2 and upwards), induced by rapid elevation in endogenous steroid hormone ecdysone, is accompanied by continuous growth of granule diameter with concomitant reduction in their number per cell. In the PS4, just prior to secretion, approximately 3000 mature granules occur per cell. The mature state is associated with the change from hyperbolic to Gaussian distribution of granule number over their size range. Similar changes in secretory granule parameters were observed in interecdysial salivary glands explanted from 3rd instar larvae and cultured in vitro in medium containing 5x10(-6) m ecdysone. PMID- 10736191 TI - Differences in rat rbc cytosol induced after in vivo phenylhydrazine treatment. AB - Analysis of rat red blood cell (RBC) cytosol protein fraction after in vivo phenylhydrazine treatment revealed increased amounts of 68-kDa protein. This protein is present in trace amounts in normal rat RBC cytosol. We also present data that 68-kDa protein from RBC cytosol has an identical isoelectric point, partial proteolytic map and immunological determinants as a protein of the same molecular mass from rat exosomes. These data indicate that 68-kDa protein is normally present in rat RBC cytosol, is exported via exosomes during reticulocyte maturation, and is increased in induced haemolytic anaemia. PMID- 10736192 TI - Permeabilization by streptolysin-o reveals a role for calcium-dependent protein kinase c isoforms alpha and beta in the response of cultured cardiomyocytes to hyposmotic challenge. AB - Immunocytochemical techniques indicate that the uninhibited activity of protein kinase C alpha and protein kinase C beta are necessary for a normal regulatory volume decrease (RVD) response of cultured chick embryo cardiomyocytes subjected to a hyposmotic environment. Antibodies against protein kinase C isoforms alpha, beta, gamma and epsilon were introduced into the cultured myocytes using a developed streptolysin-O (SLO) permeabilization technique that allows the targeted cells to accumulate large biomolecules without perturbing their normal physiological state. The loaded cells were then tested for their ability to RVD when submitted to hypo-osmotic stimulus. Results show that exposing the cultured cells to SLO in the presence of antibodies against protein kinase C alpha and beta, prior to volume challenge, significantly slows the RVD rate. Additional experiments that combined anti-alpha and anti-beta antibodies in the same exposure media did not result in a significantly different rate than the anti alpha or anti-beta rates alone. The evidence gained in this study is in agreement with previous work in the cultured chick embryo cardiomyocyte that report the involvement of a calcium dependent protein kinase C in the signal transduction pathway of the RVD. PMID- 10736193 TI - Localization of beta-endorphin in tetrahymena by confocal microscopy. Induction of the prolonged production of the hormone by hormonal imprinting. AB - The unicellular Tetrahymena has hormone receptors and hormones which are characteristic of higher vertebrates, as well as similar signal transduction pathways. In the present experiments, immunocytochemistry and confocal microscopy were used to study the presence and localization of beta-endorphin in Tetrahymena pyriformis GL. Endorphin (or endorphin-like material) was localized in the cortical structures, oral field, cilia and nuclear envelope. One-hour treatment with beta-endorphin ('hormonal imprinting') increased the presence of immunocytochemically demonstrable endorphin immediately and after 24 h, and was especially strong after 96 h of treatment. Simultaneous treatment with naloxone, an opioid antagonist, did not inhibit endorphin effect, but had an additive effect on endorphin production. Naloxone alone induced a very intensive accumulation of endorphin 96 h after treatment. The results support the possibility of a hormone production being induced by the imprinting procedure, but the imprinter-like effect of naloxone also points to the importance in this case of non-discriminatory receptors also being involved in the process. PMID- 10736194 TI - Inhibition of early DNA-damage and chromosomal aberrations by Trianthema portulacastruml. In carbon tetrachloride-induced mouse liver damage. AB - The underlying molecular mechanisms of the antihepatotoxic activity of Trianthema portulacastrum by monitoring its effect on mouse liver DNA-chain break, sugar base damage and chromosomal aberrations, during chronic or acute treatment with carbon tetrachloride (CCl(4)) have been studied. Daily oral feeding with the ethanolic extract (150 mg/kg basal diet, per os) was given 2 weeks before CCl(4)treatment and continued until the end of the experiment (13 weeks). T. portulacastrum extract offer unique protection (P< 0.05-0. 001) against the induction of liver-specific structural-type chromosomal anomalies 15, 30 or 45 days after the last CCl(4)insult, compared to control mice. This was further evidenced by extract-mediated protection (15 days prior feeding following a single necrogenic dose of CCl(4)) of the generation of DNA chain-break and Fe sugar-base damage assays. The observed hepatoprotective mechanism could be due to its ability to counteract oxidative injury to DNA in the liver of mouse. PMID- 10736195 TI - Helical extrusions at the nuclear envelope: possible involvement in nucleocytoplasmic trafficking. AB - The existence of intracytoplasmic helical inclusion 24 h after serum stimulation of 3T3 cells which had previously been serum deprived (0. 2%) for 2 days has been previously reported. Further evidence of their relationship with nuclear pores has been found, and their involvement in nucleocytoplasmic transport seems more probable. Helical formations resembling those originally seen have been found seemingly extruding from nuclear pores into the cytoplasm. However, it is unlikely that these structures contribute significantly to the translocation of material from the nucleus to the cytoplasm on a regular basis because their occurrence is rare. An association of the helical complexes with microfilaments has also been observed. Their relationship to annulate lamellae remain enigmatic. PMID- 10736196 TI - The Tetrahymena homolog of bacterial and mammalian 4-hydroxyphenylpyruvate dioxygenases localizes to membranes of the endoplasmic reticulum. AB - The expression and intracellular localization of the Tetrahymena homolog of 4 hydroxyphenylpyruvate dioxygenase (HPPD) were investigated in wild-type Tetrahymena thermophila strain B1868 VII and the mutant strains IIG8, defective in food vacuole formation, MS-1, blocked in secretion of lysosomal enzymes, and SB 281, defective in mucocyst maturation. Immunoelectron microscopy and confocal laser scanning microscopy demonstrated that Tetrahymena HPPD primarily localized to membranes of the endoplasmic reticulum. In addition, Tetrahymena HPPD was detected in association with membranes of the Golgi apparatus, and transport vesicles in exponentially growing wild-type and mutant strains. In starved cells, Tetrahymena HPPD localized exclusively to membranes of small vesicles. Since no de novo synthesis of Tetrahymena HPPD takes place in cells starved for more than 30 min, these results suggest that there is a flow of Tetrahymena HPPD from the endoplasmic reticulum to small vesicles, possibly via the Golgi apparatus, and that Tetrahymena HPPD contains a signal for vesicle membrane retrieval or retention. PMID- 10736197 TI - Ribosome synthesis in Tetrahymena: a quantitative analysis. AB - We have performed a detailed quantitative analysis of the transcription and accumulation of ribosomal RNA and ribosomal protein mRNA in the ciliated protozoan Tetrahymena thermophila during changes in growth conditions, and found that: (1) nutritional downshifts lead to a rapid decrease in transcriptional activity whereas nutritional upshifts lead to rapid restoration of transcriptional activity, (2) starvation leads to decreased translation of ribosomal protein mRNA and (3) the rate of ribosomal protein mRNA degradation decreases after a nutritional upshift. We present evidence that the proximal promoters of two ribosomal protein genes and the ribosomal RNA gene compete for binding of nuclear factor(s) in vitro, suggesting that the coordinated regulation of these genes may involve a common set of transcriptional regulators. PMID- 10736198 TI - Effects on protein kinase C and gene expression in a human mast cell line, HMC-1, following microwave exposure. AB - We used a resonant cavity which delivered a continuous wave exposure at 864.3 MHz at an average specific absorption rate (SAR) of 7 W/kg to determine non-thermal biological effects of microwave exposure. A human mast cell line, HMC-1, was used as the biological target. Cells were given three exposures each of 20-min duration daily for 7 days. The temperature of the cell culture medium during the exposure fell to 26.5 degrees C. Effects were seen on localization of protein kinase C, and expression of three genes of 588 screened. The affected genes included the proto-oncogene c-kit, the transcription factor Nucleoside diphosphate kinase B and the apoptosis-associated gene DAD-1. Stress response genes were variably upregulated. No significant effect on morphology or on F actin distribution was detected. We conclude that low-power microwave exposure may act on HMC-1 cells by altering gene expression via a mechanism involving activation of protein kinase C, and at temperatures well below those known to induce a heat shock response. PMID- 10736199 TI - The effects of exhaustive exercise on the activity levels of catalase in various tissues of male and female rats. AB - The effects of exhaustive exercise on the activity levels of catalase (EC 1.11.1.6) in various tissues of male and female Sprague-Dawley rats (Rattus norvegicus) were investigated. Both the male and female rats were subdivided into an experimental group and a control group consisting of eight rats each. One group of each sex was subjected to a swimming session of 1 h (experimental group) while the other group of each sex served as sedentary control groups. The tissues investigated were liver, heart, kidney and lung. The activity levels of catalase in all the tissues investigated were significantly (P< 0.05) elevated in both male and female rats as a result of exercise. The average increase in the activity levels of catalase in the various tissues investigated for both male and female rats was 417% (males 404%; females 430%). The male and female rats exhibited comparable activity levels of catalase in all the tissues investigated. The higher activity levels of catalase as a result of exercise might be indicative of a compensatory measure to counteract the possible detrimental effects associated with oxidative stress. PMID- 10736200 TI - Ultrastructural identification of storage compartments and localization of activity-dependent secretion of neurotrophin 6 in hippocampal neurons. AB - A modulatory role of neurotrophins (NTs) in activity-dependent neuronal plasticity by pre- and postsynaptic mechanisms is now well established. In this context, it is important to identify the storage compartments and to localize the precise site(s) and mechanism of NT secretion in order to deduce the spatial and temporal availability of NTs. We approached these questions at the ultrastructural level, exploiting the unique property of NT6 to bind tightly to heparan sulfate proteoglycans at the neuronal surface (R. Gotz et al., 1994, Nature 372, 266-269), permitting the localization of secretion sites excluding diffusion artifacts. The myc tagging of NT6 permitted glutaraldehyde fixation and hence good preservation of the membrane structure, permitting immunogold labeling of NT6myc at the neuronal surface. NT6myc is preferentially secreted from neurites compared to neuronal cell bodies. In agreement with light-microscopic observations, the ultrastructural localization of NT6myc by postembedding procedures showed a predominant localization in ER-like membrane-confined compartments, partially associated with microtubules. PMID- 10736201 TI - Specification of the central noradrenergic phenotype by the homeobox gene Phox2b. AB - The closely related homeobox genes Phox2a and Phox2b are expressed in all central and peripheral noradrenergic neurons. Our previous results have shown that Phox2a controls the differentiation of the main noradrenergic center of the brain, the locus coeruleus, but leaves unaffected the other noradrenergic centers. Here, we report that Phox2b has a wider and overlapping role, in that it is required for the differentiation of all noradrenergic centers in the brain, including the locus coeruleus. Together with the previously reported lack of dopamine-b hydroxylase and tyrosine hydroxylase expression in the peripheral nervous system of Phox2b knock-out embryos, our present findings make Phox2b a master regulator of all central and peripheral noradrenergic differentiation. We discuss the nonredundancy of Phox2 genes and their complex partnership with the bHLH transcription factor Mash1, which is also required for the differentiation of most noradrenergic cell types. PMID- 10736202 TI - Ectopic expression of NCAM in skeletal muscle of transgenic mice results in terminal sprouting at the neuromuscular junction and altered structure but not function. AB - The neuromuscular system provides an excellent model for the analysis of molecular interactions involved in the development and plasticity of synaptic contacts. The neural cell adhesion molecule (NCAM) is believed to be involved in the development and plasticity of the neuromuscular junction, in particular the axonal sprouting response observed in paralyzed and denervated muscle. In order to explore the role of myofiber NCAM in modulating the differentiation of motor neurons, we generated transgenic mice expressing a GPI-anchored NCAM isoform that is normally found in developing and denervated muscle, under the control of a skeletal muscle-specific promoter. This results in the constitutive expression of NCAM at postnatal ages, a time when the endogenous mouse NCAM is absent from the myofiber. We found that a significant number of neuromuscular junctions in adult transgenic animals displayed terminal sprouting (>20%) reminiscent of that elicited in response to cessation of neuromuscular activity. Additionally, a significant increase in the size and complexity of neuromuscular synapses as a result of extensive intraterminal sprouting was detected. Electrophysiological studies, however, revealed no significant alterations of neuromuscular transmission at this highly efficient synapse. Sprouting in response to paralysis or following nerve crush was also significantly enhanced in transgenic animals. These results suggest that in this ectopic expression model NCAM can directly modulate synaptic structure and motor neuron-muscle interactions. The results contrast with knockout experiments of the NCAM gene, where very limited changes in the neuromuscular system were observed. PMID- 10736203 TI - Syntrophin isoforms at the neuromuscular junction: developmental time course and differential localization. AB - The syntrophins are a family of cytoplasmic adapter proteins that associate with dystrophin family proteins and have putative signaling and structural roles at the neuromuscular junction. We have localized the syntrophin family members within the rodent junction from birth to adulthood. Alpha-syntrophin is the only isoform on the postsynaptic membrane at birth. In the adult, it occurs on the crests of the junctional folds, with utrophin, and in the troughs, with dystrophin. Surprisingly, neuronal nitric oxide synthase (nNOS) does not accompany alpha-syntrophin onto the crests. Beta2-syntrophin, a junction-specific form, is not present at birth and occurs mainly in the troughs in the adult. Beta1-syntrophin is a sarcolemmal form at birth, not concentrated at the junction, and disappears entirely from most fibers by 6 weeks. In positive fibers, junctional beta1-syntrophin occurs exclusively in the troughs. These results suggest that the syntrophin isoforms have distinct functions at the junction and show that the known protein-protein associations of the syntrophins and nNOS in skeletal muscle are not sufficient to explain their localizations. PMID- 10736204 TI - Changing subcellular distribution and activity-dependent utilization of a dendritically localized mRNA in developing Purkinje cells. AB - In cerebellar Purkinje neurons, the degree of dendritic segregation of the Purkinje cell-specific mRNA L7/pcp-2 is correlated with their development and synaptic investment. This developmental pattern is also observed in Purkinje cells in primary dissociated culture. Short-term (12-48 h) stimulation of cultured Purkinje cells by potassium-induced depolarization or blockade of their inhibitory GABAergic input results in an increased incidence of Purkinje cells with L7/pcp-2 mRNA-positive dendrites and increased levels of L7 protein expression, the latter by a posttranscriptional mechanism. None of these treatments affected the localization of the mRNA encoding calbindin D28k nor the level of this protein in Purkinje neurons. Protracted exposure to depolarizing levels of potassium or elimination of GABAergic transmission resulted in conspicuous changes of Purkinje cell dendritic morphology. These data suggest a scenario in which activity-driven translation of subcellularly segregated mRNAs may contribute to the developmental and functional plasticity of nerve cells. PMID- 10736205 TI - N-cadherin influences migration of oligodendrocytes on astrocyte monolayers. AB - Oligodendrocyte cell migration is required for the development of the nervous system and the repopulation of demyelinated lesions in the adult central nervous system. We have investigated the role of the calcium-dependent adhesion molecules, the cadherins, in oligodendrocyte-astrocyte interaction and oligodendrocyte progenitor migration. Immunostaining demonstrated the expression of N-cadherin on the surfaces of both oligodendrocytes and astrocytes, and oligodendrocyte-like cells adhered to and spread on N-cadherin substrates. The blocking of cadherin function by antisera or specific peptides reduced adhesion of oligodendroglia to astrocyte monolayers, diminished contact time between oligodendrocyte processes and individual astrocytes, and significantly increased the migration of oligodendrocyte-like cells on astrocyte monolayers. Furthermore, a soluble cadherin molecule without adhesive properties increased oligodendroglial proliferation on various extracellular matrix substrates. These data suggest that cadherins are at least partially responsible for the poor migration-promoting properties of astrocytes and that decreasing cell-cell adhesion might effect repopulation of demyelinated multiple sclerosis lesions by oligodendrocyte progenitors. PMID- 10736206 TI - Bundling of microtubules in the growth cone induced by laminin. AB - Axons growing in the developing nervous system are guided by cues in the environment which act at the growth cone. So far, the initial cytoskeletal target of these cues has been found to be the network of actin filaments in the peripheral region of the growth cone. Laminins are constituents of the extracellular matrix which promote axonal growth. They exert effects on the actin network. Here, laminin 1 is shown to affect microtubules as well. Acute addition of laminin 1 to rat sympathetic neurons quickly caused the advance of microtubules and their bundling within the initial widely spread growth cone and then the outgrowth of thin, rapidly growing nascent axons. The bundling was pharmacologically separable from the advance of microtubules caused by laminin, as the former but not the latter was blocked by lithium. The bundling did not depend on the peripheral network of actin filaments, as it was unimpaired by the removal of this network with cytochalasin D. Thus, microtubules seem to be a direct cytoskeletal target for laminin 1 in the growth cone, with important consequences for axonal outgrowth. PMID- 10736208 TI - EDITORIAL REPORT. PMID- 10736210 TI - Worldwide publication of the EJSO: the IDEAL way forward. PMID- 10736207 TI - PDGF and FGF-2 signaling in oligodendrocyte progenitor cells: regulation of proliferation and differentiation by multiple intracellular signaling pathways. AB - In this paper we address the linking of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (FGF-2) to intracellular signaling molecules in oligodendrocyte progenitors. It is demonstrated that both growth factors activate downstream targets similar to those shown for protein kinase C (PKC) activation. Yet, neither the arrest of terminal oligodendrocyte differentiation nor the proliferation induced by PDGF or FGF-2 can be antagonized by inhibition of PKC. Rather, p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, and pp70 S6 kinase were found to be necessary for the mitogenic activity of PDGF and FGF-2. Paradoxically, these kinases were also necessary for the onset of oligodendrocyte differentiation in control cells. In addition, cAMP-dependent kinase A (PKA) activation inhibited the mitogenic response of oligodendrocyte progenitors to FGF 2. Taken together, the molecular mechanism that controls oligodendrocyte lineage progression is operated by at least two signal pathways, which interfere either with proliferation and/or differentiation of oligodendrocyte progenitors. PMID- 10736211 TI - Evolving mechanisms of morphogenesis: on the interplay between differential adhesion and cell differentiation. AB - Differential cell adhesion, mediated by e.g. integrin and cadherins/catenines, plays an important role in morphogenesis and it has been shown that there is intimate cross-talk between their expression and modification, and inter-cellular signalling, cell differentiation, cell growth and apoptosis. In this paper, we introduce and use a formal model to explore the morphogenetic potential of the interplay between these processes. We demonstrate the formation of interesting morphologies. Initiated by cell differentiation, differential cell adhesion leads to a long transient of cell migrations, e.g. engulfing and intercalation of cells and cell layers. This transient can be sustained dynamically by further cell differentiation, and by cell growth/division and cell death which are triggered by the (also long range) forces (stretching and squeezing) generated by the cell adhesion. We study the interrelation between modes of cell differentiation and modes of morphogenesis. We use an evolutionary process to zoom in on gene regulation networks which lead to cell differentiation. Morphogenesis is not selected for but appears as a side-effect. The evolutionary dynamics shows the hallmarks of evolution on a rugged landscape, including long neutral paths. We show that a combinatorially large set of morphologies occurs in the vicinity of a neutral path which sustains cell differentiation. Thus, an almost linear molecular phylogeny gives rise to mosaic evolution on the morphological level. PMID- 10736212 TI - Natural selection of memory-one strategies for the iterated prisoner's dilemma. AB - In the iterated Prisoner's Dilemma, mutually cooperative behavior can become established through Darwinian natural selection. In simulated interactions of stochastic memory-one strategies for the Iterated Prisoner's Dilemma, Nowak and Sigmund discovered that cooperative agents using a Pavlov (Win-Stay Lose-Switch) type strategy eventually dominate a random population. This emergence follows more directly from a deterministic dynamical system based on differential reproductive success or natural selection. When restricted to an environment of memory-one agents interacting in iterated Prisoner's Dilemma games with a 1% noise level, the Pavlov agent is the only cooperative strategy and one of very few others that cannot be invaded by a similar strategy. Pavlov agents are trusting but no suckers. They will exploit weakness but repent if punished for cheating. PMID- 10736213 TI - Control strategies for the polarotactic orientation of the microorganism Euglena gracilis. AB - A simple mathematical model for the signal received by the dichroic photoreceptor molecules in the motile alga, Euglena gracilis, when irradiated by polarized light, is described and used to test hypotheses for the control strategies employed by the microorganism during negative phototaxis. The model is used to analyse and explain the experimental results of Hader (1987. Arch. Microbiol.147, 179-183). PMID- 10736214 TI - Simulated brain tumor growth dynamics using a three-dimensional cellular automaton. AB - We have developed a novel and versatile three-dimensional cellular automaton model of brain tumor growth. We show that macroscopic tumor behavior can be realistically modeled using microscopic parameters. Using only four parameters, this model simulates Gompertzian growth for a tumor growing over nearly three orders of magnitude in radius. It also predicts the composition and dynamics of the tumor at selected time points in agreement with medical literature. We also demonstrate the flexibility of the model by showing the emergence, and eventual dominance, of a second tumor clone with a different genotype. The model incorporates several important and novel features, both in the rules governing the model and in the underlying structure of the model. Among these are a new definition of how to model proliferative and non-proliferative cells, an isotropic lattice, and an adaptive grid lattice. PMID- 10736215 TI - Transition-state ensemble in enzyme catalysis: possibility, reality, or necessity? AB - Proteins are not rigid structures; they are dynamic entities, with numerous conformational isomers (substates). The dynamic nature of protein structures amplifies the structural variation of the transition state for chemical reactions performed by proteins. This suggests that utilizing a transition state ensemble to describe chemical reactions involving proteins may be a useful representation. Here we re-examine the nature of the transition state of protein chemical reactions (enzyme catalysis), considering both recent developments in chemical reaction theory (Marcus theory for SN2 reactions), and protein dynamics effects. The classical theory of chemical reactions relies on the assumption that a reaction must pass through an obligatory transition-state structure. The widely accepted view of enzymatic catalysis holds that there is tight binding of the substrate to the transition-state structure, lowering the activation energy. This picture, may, however, be oversimplified. The real meaning of a transition state is a surface, not a single saddle point on the potential energy surface. In a reaction with a "loose" transition-state structure, the entire transition-state region, rather than a single saddle point, contributes to reaction kinetics. Consequently, here we explore the validity of such a model, namely, the enzymatic modulation of the transition-state surface. We examine its utility in explaining enzyme catalysis. We analyse the possibility that instead of optimizing binding to a well-defined transition-state structure, enzymes are optimized by evolution to bind efficiently with a transition-state ensemble, with a broad range of activated conformations. For enzyme catalysis, the key issue is still transition state (ensemble) stabilization. The source of the catalytic power is the modulation of the transition state. However, our definition of the transition state is the entire transition-state surface rather just than a single well defined structure. This view of the transition-state ensemble is consistent with the nature of the protein molecule, as embodied and depicted in the protein energy landscape of folding, and binding, funnels. PMID- 10736216 TI - A condition for successful escape of a mutant after primary HIV infection. AB - Cytotoxic T lymphocytes (CTLs) vigorously restrict primary human immunodeficiency virus (HIV) infection. However, the frequently erroneous process of viral replication favors the creation of mutants not recognizable by primary CTLs. Variants that tolerate the mutations may have selective advantage and may increase in abundance, until the immune system reacts against them. Therefore, such variants represent a way of propagating the viremia. With the aid of a simple mathematical model, here we estimate the intensity of CTL cross-reactivity against different strains of HIV in a typical progressor. We show that below a critical intensity of cross-reactivity, the concentration of a mutant created at primary peak grows and causes a secondary peak in viremia. Above this critical intensity, such a mutant strain is prevented from reaching a detectable level. We speculate about how this result may contribute to the design of an anti-HIV vaccine. PMID- 10736217 TI - Consequences of the non-specific binding of a protein to a linear polymer: reconciliation of stoichiometric and equilibrium titration data for the thrombin heparin interaction. AB - Theoretical aspects of the thermodynamic characterization of cooperative protein interactions with non-specific segments of a linear polymer lattice have been re examined. This reconsideration has not only provided an alternative derivation of recursive expressions for the stoichiometry of random ligand binding prior to elimination of the parking problem but also extended that treatment to include binding with overlap of additional lattice units. The major obstacle to thermodynamic characterization of non-specific protein-polymer interactions is determination of the lattice capacity for ligand, which in turn defines the length of the polymer segment to which the protein binds. Although these parameters are most readily obtained from studies under conditions that ensure essentially stoichiometric interaction, the endpoint of such a titration is likely to reflect the irreversible rather than the equilibrium binding capacity of the lattice for ligand. Consideration of published results for spectrofluorometric titrations of the thrombin-heparin system under stoichiometric conditions in such terms has permitted their reconciliation with results of a later publication on the interaction under equilibrium conditions. PMID- 10736218 TI - A population genetics model for multiple quantitative traits exhibiting pleiotropy and epistasis. AB - We study a population genetics model of an organism with a genome of L(tot)loci that determine the values of T quantitative traits. Each trait is controlled by a subset of L loci assigned randomly from the genome. There is an optimum value for each trait, and stabilizing selection acts on the phenotype as a whole to maintain actual trait values close to their optima. The model contains pleiotropic effects (loci can affect more than one trait) and epistasis in fitness. We use adaptive walk simulations to find high-fitness genotypes and to study the way these genotypes are distributed in sequence space. We then simulate the evolution of haploid and diploid populations on these fitness landscapes and show that the genotypes of populations are able to drift through sequence space despite stabilizing selection on the phenotype. We study the way the rate of drift and the extent of the accessible region of sequence space is affected by mutation rate, selection strength, population size, recombination rate, and the parameters L and T that control the landscape shape. There are three regimes of the model. If LT<>L(tot), there are many small peaks that can be spread over a wide region of sequence space. Compensatory neutral mutations are important in the population dynamics in this case. PMID- 10736219 TI - Evolution of specificity in an immune network. AB - A dynamic antigen response of the immune network is discussed, based on shape space modelling. The present model extends the shape-space modelling by introducing the evolution of specificity of idiotypes. When the amount of external antigen increases, a measure of stability of the immune network is lost and thus the network can respond to the antigen. It is shown that specific and non-specific responses emerge as a function of antigen amounts. A specific response is observed with a fixed-point attractor, and a non-specific response is observed with a chaotic attractor for the lymphocyte population dynamics. The network topology also changes between fixed-point and chaotic attractors. For some antigen amounts, chaotic attractors will vanish or become long-lived super transient states. A dynamic bell-shaped response function will thus emerge. The relevance of long-lived chaotic transient states embedded in fixed-point attractors is discussed with respect to immune functions. PMID- 10736220 TI - Chaotic or periodic variation? Looking at Crustacea hearts. AB - A simple relationship between the burst length of the trigger neurons of the Crustacea cardiac ganglion and the length of the heartbeat, is shown to lead to chaotic heart rate. Interestingly, the same type of relationship is also capable of generating complex periodic deviations from steady heart rhythm. Real Crustacea hearts are not likely to follow strictly either suite. The general procedure of analysis, however, is applicable to various biological rhythms, and explains how variation around the mean value can occasionally show up as intricate patterns which repeat themselves with accuracy. PMID- 10736221 TI - Experimental analysis and computer prediction of CTF/NFI transcription factor DNA binding sites. AB - Accurate prediction of transcription factor binding sites is needed to unravel the function and regulation of genes discovered in genome sequencing projects. To evaluate current computer prediction tools, we have begun a systematic study of the sequence-specific DNA-binding of a transcription factor belonging to the CTF/NFI family. Using a systematic collection of rationally designed oligonucleotides combined with an in vitro DNA binding assay, we found that the sequence specificity of this protein cannot be represented by a simple consensus sequence or weight matrix. For instance, CTF/NFI uses a flexible DNA binding mode that allows for variations of the binding site length. From the experimental data, we derived a novel prediction method using a generalised profile as a binding site predictor. Experimental evaluation of the generalised profile indicated that it accurately predicts the binding affinity of the transcription factor to natural or synthetic DNA sequences. Furthermore, the in vitro measured binding affinities of a subset of oligonucleotides were found to correlate with their transcriptional activities in transfected cells. The combined computational experimental approach exemplified in this work thus resulted in an accurate prediction method for CTF/NFI binding sites potentially functioning as regulatory regions in vivo. PMID- 10736222 TI - Sequences in sigma(54) region I required for binding to early melted DNA and their involvement in sigma-DNA isomerisation. AB - The bacterial sigma(54) RNA polymerase functions in a transcription activation mechanism that fully relies upon nucleotide hydrolysis by an enhancer binding activator protein to stimulate open complex formation. Here, we describe results of DNA-binding assays used to probe the role of the sigma(54) amino terminal region I in activation. Of the 15 region I alanine substitution mutants assayed, several specifically failed to bind to a DNA structure representing an early conformation in DNA melting. The same mutants are defective in activated transcription and in forming an isomerised sigma-DNA complex on the early opened DNA. The mechanism of activation may therefore require tight binding of sigma(54) to particular early melted DNA structures. Where mutant sigma(54) binding to early melted DNA was detected, activator-dependent isomerisation generally occurred as efficiently as with the wild-type protein, suggesting that certain region I sequences are largely uninvolved in sigma isomerisation. DNA-binding, sigma isomerisation and transcription activation assays allow formulation of a functional map of region I. PMID- 10736223 TI - Analysis of expression of prgX, a key negative regulator of the transfer of the Enterococcus faecalis pheromone-inducible plasmid pCF10. AB - Conjugative transfer of pCF10, a plasmid found in Enterococcus faecalis, is induced by the peptide pheromone cCF10 and the donor-recipient aggregation is mediated by PrgB. Expression of prgB in pCF10 is negatively regulated by PrgX. The prgX gene is adjacent to prgQ which provides the promoter for prgB expression. The prgX and prgQ genes are transcribed in opposite directions. A deletion spanning nucleotides+259 to +398 from the prgQ transcription initiation site abolished the prgX gene products at both RNA and protein levels. An RNA, named Qa, was found to be transcribed in the antisense direction in the prgQ region. The transcription start site and the promoter were found to be almost identical with those of the traD determinant in pAD1, another pheromone responsive plasmid. The first inverted repeat sequence in prgQ, IRS1, was required for the full activity of the Qa promoter. Although the size of the major Qa RNA detected by Northern blot analysis was too short (ca 120 nt) to be an mRNA for PrgX protein, the transcription from the Qa promoter was shown to proceed through to prgX. Transcriptional fusion analyses showed that the transcription of prgX requires one or more trans elements. Moreover, deletion of prgX greatly reduced the level of the Qa RNA and abolished transcription of prgX. Although transcription initiation from the Qa promoter was not increased by PrgX, transcriptional readthrough to prgX was increased by the protein. Based on these data, we conclude that transcription of prgX is initiated from the Qa promoter in prgQ, and PrgX autoregulates its transcription either by mediating transcriptional readthrough or increasing mRNA stability. The PrgX protein, prgX RNA, and the Qa RNA were downregulated by cCF10 induction; however, prgX gene products were still detected for at least 40 minutes after induction. PMID- 10736224 TI - The major HIV-1 packaging signal is an extended bulged stem loop whose structure is altered on interaction with the Gag polyprotein. AB - The major packaging signal of human immunodeficiency virus type 1 (HIV-1) RNA has been localised to the region 3' to the major splice donor within the leader sequence. Secondary structural studies for this region of the HIV-1 genome have shown the existence of a stem-loop structure capped by a purine-rich tetraloop. Extensive mapping data presented here lead to the complete characterisation of the structure of the stem-loop, including a new purine-rich internal loop in the lower part of the structure and the previously established GGAG tetraloop at its tip. Biochemical analysis reveals that both internal loop and tetraloop are primary sites for interaction with Gag polyprotein, and that binding of Gag protein leads to a conformational change which alters the RNA structure. NMR spectroscopy has been used to determine the three-dimensional structure of this complete stem-loop structure. The structural analysis reveals a significant difference between the apical part of the stem-loop structure, which adopts a well-defined conformation, and the purine-rich internal loop, which is instead very flexible. In contrast to what is generally observed for internal loop structures in RNA, this region of the encapsidation signal adopts a structure lacking stable interstrand interactions capable of stabilising a unique conformation. We suggest that the stem-loop structure represents a nucleation site for Gag protein binding, and that the protein exploits the flexibility of the internal loop to initiate the unwinding of the structure with successive addition of Gag molecules interacting with the RNA and each other through conserved I (interaction) domains. PMID- 10736225 TI - Archaeal homologs of eukaryotic methylation guide small nucleolar RNAs: lessons from the Pyrococcus genomes. AB - Ribose methylation is a prevalent type of nucleotide modification in rRNA. Eukaryotic rRNAs display a complex pattern of ribose methylations, amounting to 55 in yeast Saccharomyces cerevisiae and about 100 in vertebrates. Ribose methylations of eukaryotic rRNAs are each guided by a cognate small RNA, belonging to the family of box C/D antisense snoRNAs, through transient formation of a specific base-pairing at the rRNA modification site. In prokaryotes, the pattern of rRNA ribose methylations has been fully characterized in a single species so far, Escherichia coli, which contains only four ribose methylated rRNA nucleotides. However, the hyperthermophile archaeon Sulfolobus solfataricus contains, like eukaryotes, a large number of (yet unmapped) rRNA ribose methylations and homologs of eukaryotic box C/D small nucleolar ribonuclear proteins have been identified in archaeal genomes. We have therefore searched archaeal genomes for potential homologs of eukaryotic methylation guide small nucleolar RNAs, by combining searches for structured motifs with homology searches. We have identified a family of 46 small RNAs, conserved in the genomes of three hyperthermophile Pyrococcus species, which we have experimentally characterized in Pyrococcus abyssi. The Pyrococcus small RNAs, the first reported homologs of methylation guide small nucleolar RNAs in organisms devoid of a nucleus, appear as a paradigm of minimalist box C/D antisense RNAs. They differ from their eukaryotic homologs by their outstanding structural homogeneity, extended consensus box motifs and the quasi-systematic presence of two (instead of one) rRNA antisense elements. Remarkably, for each small RNA the two antisense elements always match rRNA sequences close to each other in rRNA structure, suggesting an important role in rRNA folding. Only a few of the predicted P. abyssi rRNA ribose methylations have been detected so far. Further analysis of these archaeal small RNAs could provide new insights into the origin and functions of methylation guide small nucleolar RNAs and illuminate the still elusive role of rRNA ribose methylations. PMID- 10736226 TI - An A-type double helix of DNA having B-type puckering of the deoxyribose rings. AB - DNA usually adopts structure B in aqueous solution, while structure A is preferred in mixtures of trifluoroethanol (TFE) with water. However, the octamer d(CCCCGGGG) and other d(C(n)G(n)) fragments of DNA provide CD spectra that suggest that the base-pairs are stacked in an A-like fashion even in aqueous solution. Yet, d(CCCCGGGG) undergoes a cooperative TFE-induced transition into structure A, indicating that an important part of the aqueous duplex retains structure B. NMR spectroscopy shows that puckering of the deoxyribose rings is of the B-type. Hence, combination of the information provided by CD spectroscopy and NMR spectroscopy suggests an unprecedented double helix of DNA in which A-like base stacking is combined with B-type puckering of the deoxyribose rings. In order to determine whether this combination is possible, we used molecular dynamics to simulate the duplex of d(CCCCGGGG). Remarkably, the simulations, completely unrestrained by the experimental data, provided a very stable double helix of DNA, exhibiting just the intermediate B/A features described above. The double helix contained well-stacked guanine bases but almost unstacked cytosine bases. This generated a hole in the double helix center, which is a property characteristic for A-DNA, but absent from B-DNA. The minor groove was narrow at the double helix ends but wide at the central CG step where the Watson-Crick base pairs were buckled in opposite directions. The base-pairs stacked tightly at the ends but stacking was loose in the duplex center. The present double helix, in which A-like base stacking is combined with B-type sugar puckering, is relevant to replication and transcription because both of these phenomena involve a local B-to-A transition. PMID- 10736227 TI - Two Holliday junction resolving enzymes in Sulfolobus solfataricus. AB - Holliday junction resolving enzymes bind specifically to four-way DNA junctions created by the process of homologous recombination, cleaving them to yield recombinant duplex DNA products. Homologous recombination is known to occur in the third domain of life, the archaea, and may constitute a simplified model for the corresponding eucaryal pathway, but has not been well characterised. Identification of a gene encoding an archaeal Holliday junction resolving enzyme, Hjc, has recently been reported in the euryarchaea, and an activity has been observed in the hyperthermophilic crenarchaeote Sulfolobus solfataricus. Here we report the identification, heterologous expression and characterisation of the Hjc protein from Sulfolobus. We demonstrate that Sulfolobus has two distinct junction resolving enzymes, Hjc and Hje, with differing substrate specificities. PMID- 10736228 TI - Molecular basis for the polyamine-ompF porin interactions: inhibitor and mutant studies. AB - By testing the sensitivity of Escherichia coli OmpF porin to various natural and synthetic polyamines of different lengths, charge and other molecular characteristics, we were able to identify the molecular properties required for compounds to act as inhibitors of OmpF in the nanomolar range. Inhibitors require at least two amine groups to be effective. For diamines, the optimum length of the hydrocarbon spacer was found to be of eight to ten methylene groups. Triamine molecules based on a 12-carbon motif were found to be more effective that spermidine, an eight-carbon trivalent derivative. But differences in inhibition efficiencies were also found for trivalent compounds depending on the relative position of the internal secondary amine group with respect to the terminal groups. Finally, quaternary ammonium derivatives had no effect, suggesting that the nature of the terminal amine is important for the interaction. From these observations, we deduce that inhibition efficiency in the nanomolar range requires a 12-carbon chain triamine with terminal primary amine groups and replacement of the eighth methylene by a secondary amine. The need for this type of molecular architecture suggests that inhibition is governed by interactions between specific amine groups and protein residues, and that this is not simply due to the accumulation of charges into the pore. Together with previous observations from site-directed mutagenesis studies and inspection of the crystal structure of OmpF, these results allowed us to propose three residues (D113, D121 and Y294) as putative sites of interaction between the channel and spermine. Alanine substitution at each of these three residues resulted in a loss of inhibition by spermine, while mutations of only D113 and D121 affected inhibition by spermidine. Based on these observations, we suggest a model for the molecular determinants involved in the porin-polyamine interaction. PMID- 10736229 TI - Crystallization of the yeast MATalpha2/MCM1/DNA ternary complex: general methods and principles for protein/DNA cocrystallization. AB - We describe our efforts to crystallize binary MCM1/DNA and ternary MATalpha2/MCM1/DNA complexes, including the unsuccessful attempts to crystallize MCM1/DNA complexes and the successful design of DNA crystal packing that resulted in high-resolution crystals of the MATalpha2/MCM1/DNA complex. We detail general procedures useful for preparing protein/DNA cocrystals, including improved methods for producing and purifying DNA-binding proteins and DNA fragments, for purifying protein/DNA complexes, and for controlling pH conditions during crystallization. We also describe the rational design of DNA for protein/DNA cocrystallization attempts, based on our analysis of how straight and bent DNA with single base-pair overhangs can pack end-to-end in a crystal. PMID- 10736230 TI - X-ray structure of the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa: basis of substrate specificity. AB - The homodimeric enzyme form of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa ATCC 17933 crystallizes readily with the space group R3. The X-ray structure was solved at 2.6 A resolution by molecular replacement. Aside from differences in some loops, the folding of the enzyme is very similar to the large subunit of the quinoprotein methanol dehydrogenases from Methylobacterium extorquens or Methylophilus W3A1. Eight W-shaped beta-sheet motifs are arranged circularly in a propeller-like fashion forming a disk-shaped superbarrel. No electron density for a small subunit like that in methanol dehydrogenase could be found. The prosthetic group is located in the centre of the superbarrel and is coordinated to a calcium ion. Most amino acid residues found in close contact with the prosthetic group pyrroloquinoline quinone and the Ca(2+) are conserved between the quinoprotein ethanol dehydrogenase structure and that of the methanol dehydrogenases. The main differences in the active-site region are a bulky tryptophan residue in the active-site cavity of methanol dehydrogenase, which is replaced by a phenylalanine and a leucine side-chain in the ethanol dehydrogenase structure and a leucine residue right above the pyrrolquinoline quinone group in methanol dehydrogenase which is replaced by a tryptophan side-chain. Both amino acid exchanges appear to have an important influence, causing different substrate specificities of these otherwise very similar enzymes. In addition to the Ca(2+) in the active-site cavity found also in methanol dehydrogenase, ethanol dehydrogenase contains a second Ca(2+)-binding site at the N terminus, which contributes to the stability of the native enzyme. PMID- 10736231 TI - Thermal stability and atomic-resolution crystal structure of the Bacillus caldolyticus cold shock protein. AB - The bacterial cold shock proteins are small compact beta-barrel proteins without disulfide bonds, cis-proline residues or tightly bound cofactors. Bc-Csp, the cold shock protein from the thermophile Bacillus caldolyticus shows a twofold increase in the free energy of stabilization relative to its homolog Bs-CspB from the mesophile Bacillus subtilis, although the two proteins differ by only 12 out of 67 amino acid residues. This pair of cold shock proteins thus represents a good system to study the atomic determinants of protein thermostability. Bs-CspB and Bc-Csp both unfold reversibly in cooperative transitions with T(M) values of 49.0 degrees C and 77.3 degrees C, respectively, at pH 7.0. Addition of 0.5 M salt stabilizes Bs-CspB but destabilizes Bc-Csp. To understand these differences at the structural level, the crystal structure of Bc-Csp was determined at 1.17 A resolution and refined to R=12.5% (R(free)=17.9%). The molecular structures of Bc Csp and Bs-CspB are virtually identical in the central beta-sheet and in the binding region for nucleic acids. Significant differences are found in the distribution of surface charges including a sodium ion binding site present in Bc Csp, which was not observed in the crystal structure of the Bs-CspB. Electrostatic interactions are overall favorable for Bc-Csp, but unfavorable for Bs-CspB. They provide the major source for the increased thermostability of Bc Csp. This can be explained based on the atomic-resolution crystal structure of Bc Csp. It identifies a number of potentially stabilizing ionic interactions including a cation-binding site and reveals significant changes in the electrostatic surface potential. PMID- 10736232 TI - The solution structure of the cytokine-binding domain of the common beta-chain of the receptors for granulocyte-macrophage colony-stimulating factor, interleukin-3 and interleukin-5. AB - The haemopoietic cytokines, granulocyte-macrophage colony-stimulating factor, interleukin-3 and interleukin-5 bind to cell-surface receptors comprising ligand specific alpha-chains and a shared beta-chain. The beta-chain is the critical signalling subunit of the receptor and its fourth domain not only plays a critical role in interactions with ligands, hence in receptor activation, but also contains residues whose mutation can lead to ligand-independent activation of the receptor. We have determined the NMR solution structure of the isolated human fourth domain of the beta-chain. The protein has a fibronectin type III fold with a well-defined hydrophobic core and is stabilised by an extensive network of pi-cation interactions involving Trp and Arg side-chains, including two Trp residues outside the highly conserved Trp-Ser-Xaa-Trp-Ser motif (where Xaa is any amino acid) that is found in many cytokine receptors. Most of the residues implicated in factor-independent mutants localise to the rigid core of the domain or the pi-cation stack. The loops between the B and C, and the F and G strands, that contain residues important for interactions with cytokines, lie adjacent at the membrane-distal end of the domain, consistent with their being involved cooperatively in binding cytokines. The elucidation of the structure of the cytokine-binding domain of the beta-chain provides insight into the cytokine dependent and factor-independent activation of the receptor. PMID- 10736233 TI - Structure-based evaluation of sequence comparison and fold recognition alignment accuracy. AB - The biological role, biochemical function, and structure of uncharacterized protein sequences is often inferred from their similarity to known proteins. A constant goal is to increase the reliability, sensitivity, and accuracy of alignment techniques to enable the detection of increasingly distant relationships. Development, tuning, and testing of these methods benefit from appropriate benchmarks for the assessment of alignment accuracy.Here, we describe a benchmark protocol to estimate sequence-to-sequence and sequence-to-structure alignment accuracy. The protocol consists of structurally related pairs of proteins and procedures to evaluate alignment accuracy over the whole set. The set of protein pairs covers all the currently known fold types. The benchmark is challenging in the sense that it consists of proteins lacking clear sequence similarity. Correct target alignments are derived from the three-dimensional structures of these pairs by rigid body superposition. An evaluation engine computes the accuracy of alignments obtained from a particular algorithm in terms of alignment shifts with respect to the structure derived alignments. Using this benchmark we estimate that the best results can be obtained from a combination of amino acid residue substitution matrices and knowledge-based potentials. PMID- 10736234 TI - Directed evolution study of temperature adaptation in a psychrophilic enzyme. AB - We have used laboratory evolution methods to enhance the thermostability and activity of the psychrophilic protease subtilisin S41, with the goal of investigating the mechanisms by which this enzyme can adapt to different selection pressures. A combined strategy of random mutagenesis, saturation mutagenesis and in vitro recombination (DNA shuffling) was used to generate mutant libraries, which were screened to identify enzymes that acquired greater thermostability without sacrificing low-temperature activity. The half-life of seven-amino acid substitution variant 3-2G7 at 60 degrees C is approximately 500 times that of wild-type and far surpasses those of homologous mesophilic subtilisins. The dependence of half-life on calcium concentration indicates that enhanced calcium binding is largely responsible for the increased stability. The temperature optimum of the activity of 3-2G7 is shifted upward by approximately 10 degrees C. Unlike natural thermophilic enzymes, however, the activity of 3-2G7 at low temperatures was not compromised. The catalytic efficiency, k(cat)/K(M), was enhanced approximately threefold over a wide temperature range (10 to 60 degrees C). The activation energy for catalysis, determined by the temperature dependence of k(cat)/K(M) in the range 15 to 35 degrees C, is nearly identical to wild-type and close to half that of its highly similar mesophilic homolog, subtilisin SSII, indicating that the evolved S41 enzyme retained its psychrophilic character in spite of its dramatically increased thermostability. These results demonstrate that it is possible to increase activity at low temperatures and stability at high temperatures simultaneously. The fact that enzymes displaying both properties are not found in nature most likely reflects the effects of evolution, rather than any intrinsic physical-chemical limitations on proteins. PMID- 10736236 TI - Normal expression of cell adhesion molecules in placentae from women with systemic lupus erythematosus and the antiphospholipid syndrome. AB - Pregnant women with active systemic lupus erythematosus (SLE) and/or the antiphospholipid syndrome (APS) are prone to recurrent miscarriage, pre eclampsia, intrauterine growth restriction and premature delivery. Placental dysfunction may account for these complications yet the mechanisms remain uncertain. Amongst these, an inflammatory response in the placental vasculature could play a role, involving recruitment of neutrophils and platelets and the increased endothelial expression of cell adhesion molecules (CAM), central to the recruitment process. The aim of this study was primarily to investigate CAM expression in the fetoplacental vasculature in women with SLE/APS. Circulating maternal concentrations of soluble CAM were also elucidated. There were no differences in CAM immunostaining in placentae from patients with SLE and/or APS compared with controls. In both patients and controls moderate immunostaining for the intercellular adhesion molecule-1 (ICAM-1) was observed in placental vascular endothelium and mild immunostaining was present in the placental villous stroma. Strong immunostaining for platelet endothelial CAM (PECAM) occured in the placental vascular endothelium whereas P-selectin was mildly expressed in the stem vessel endothelium only. Vascular CAM-1 (VCAM-1) and E-selectin were undetectable in either study or control placentae. In contrast, ICAM-1 and VCAM-1 but not E-selectin, as assessed by immunoassay (ELISA), were elevated in maternal serum from SLE/APS patients compared with controls. This study suggests that upregulation of CAM expression and subsequent activation of neutrophil and/or platelet activity within the placental villous tree is unlikely to be a mechanism by which the adverse pregnancy outcome arises in SLE/APS pregnancies. However, maternal endothelial cell activation (ECA) may play a more important role. PMID- 10736235 TI - Pre-eclampsia does not change the adhesion molecule status in the placental bed. AB - During normal placentation trophoblast cells invade maternal tissues and remodel the uterine arteries into low-resistance channels. In pre-eclampsia, trophoblast invasion is impaired and this, along with endothelial dysfunction, has been suggested to play a role in the pathogenesis of pre-eclampsia. We studied the expression of adhesion molecules important for leukocyte extravasation in the placental bed with immunohistochemistry and compared the expression in pre eclampsia to that in normal pregnancy. Our major finding was that only invasive trophoblasts expressed cutaneous lymphocyte antigen-1 (CLA-1) in the third trimester of pregnancy, whereas villous trophoblasts did not. In the first trimester both villous trophoblasts and invasive trophoblast cells in decidua remained negative for CLA-1. Pre-eclampsia did not change the expression of leukocyte-endothelium adhesion or lymphocyte homing-associated antigens, ICAM-1, ICAM-2, VCAM, P-selectin, E-selectin, L-selectin, CLA-1, CD73, VAP-1 and alphaEbeta7 in the placental bed. Furthermore, pre-eclampsia was not associated with an aberrant accumulation of lymphocytes carrying antigens of any particular known organ-specific homing systems. The results on the unchanged pattern of adhesion molecule expression in pre-eclampsia suggests that there is no major change in the adhesive properties of the endothelium of the placental bed in pre eclampsia. PMID- 10736237 TI - Adhesion molecules of syncytiotrophoblast microvillous membranes inhibit proliferation of human umbilical vein endothelial cells. AB - It has been shown previously that syncytiotrophoblast microvillous membranes (STBM), isolated from normal or pre-eclampsia placentae, specifically inhibit the proliferation of cultured human umbilical vein endothelial cells (HUVEC) and disrupt the cell monolayer without causing cell death. We have previously shown that this anti-proliferative activity resides in a self-aggregating complex in which eight proteins, namely integrins alpha(5)(CD49e) and alpha(V)(CD51), dipeptidyl peptidase IV (DPP IV, CD26), alpha-actinin, transferrin receptor (TfR, CD71), transferrin, placental alkaline phosphatase (PLAP) and monoamine oxidase A (MAO-A) were identified. In the present study, we investigated which of these components causes the anti-proliferative activity of STBM. Antibodies against integrin alpha(5)and alpha(V)and DPP IV all reduced the STBM-induced inhibition of proliferation of HUVEC, which was also reversed by added fibronectin. A preparation of PLAP inhibited endothelial proliferation, but this was not due to enzymatic activity. The preparation was shown to be impure with more than 12 bands present on Coomassie blue stained SDS-PAGE gels. These included integrins alpha(5)and alpha(V), which could account, at least in part, for the inhibitory activity. We could not exclude, however, the possibility of other unidentified factors being involved. We conclude that adhesion molecules account for a major part of the anti-proliferative activity of STBM; these appear to compete for ligands in the extracellular matrix or serum with the appropriate receptors on HUVEC. PMID- 10736238 TI - Lateral placental growth occurs by trophoblast cell invasion of decidual veins. AB - During human pregnancy, growth of the placenta is proportionally greater than the growth of the decidual surface, suggesting that trophoblast cells invade the decidua at the placenta's margin. We hypothesized that a method of lateral placental growth was trophoblast cell invasion of decidual veins. This was investigated in two in situ pregnancies and in tissues from 100 women undergoing elective termination at 8-12 weeks of gestation. Decidua was compared to normal secretory endometrium. Histological sections were stained by immunohistochemistry to identify trophoblast cell and vascular antigens, as well as vascular cell adhesion molecule (VCAM), integrin subunits beta(1)and beta(4), and oncofetal fibronectin. Dilated veins were observed in all decidua but not in the secretory endometrium. Decidual and myometrial veins contained villi, trophoblast cell islands and syncytial elements. Decidual endothelial cells expressed vascular cell adhesion molecule (VCAM). Villous trophoblast cells were integrin subunits beta(4)positive and beta(1)negative. Trophoblast cell islands in the placenta and within decidual veins were integrin subunits beta(1)positive and beta(4)negative. Trophoblast cell islands and villi attached to veins, and mononuclear cells, invaded decidual stroma. Oncofetal fibronectin was present at sites of trophoblast invasion. These findings suggest that a method of lateral placental growth is trophoblast cell invasion of veins. PMID- 10736239 TI - Messenger RNA for membrane-type 2 matrix metalloproteinase, MT2-MMP, is expressed in human placenta of first trimester. AB - An intimately regulated cell surface activation of matrix metalloproteinases (MMPs) is believed to be of critical importance for the control of trophoblast invasion. A histological investigation of the expression and localization of three different MMPs, the membrane-type matrix metalloproteinases 1 and 2 (MT1 MMP, MT2-MMP) and matrix metalloproteinase 2 (MMP-2/gelatinase A) was performed by in situ hybridization on consecutive sections from human placentae of first trimester pregnancies. Cytokeratin immunostaining identified trophoblast cells. Both normal and tubal implantation sites were studied. We observed a high degree of coexpression of MT2-MMP, MT1-MMP and MMP-2 mRNAs in single extravillous cytotrophoblasts that had invaded the endometrium and tubal wall. Furthermore, mRNAs for all three genes were also seen in cytotrophoblasts of cell islands. In contrast to this coexpression pattern, MT2-MMP expression was absent from cell columns and decidual cells, in which signals for MT1-MMP and MMP-2 mRNAs were seen. The present data on the cellular expression of MT2-MMP mRNA in placenta extend our knowledge of the proteolytic events that take place during early pregnancy. The data suggest that MT2-MMP, capable of activating MMP-2 in vitro, is involved in the invasion of extravillous cytotrophoblast, possibly related to the physiological activation of MMP-2. PMID- 10736240 TI - Involvement of EAT/mcl-1, a bcl-2 related gene, in the apoptotic mechanisms underlying human placental development and maintenance. AB - The EAT/mcl-1 gene was isolated during the early differentiation of a retinoic acid-induced human embryonal carcinoma cell line to the trophectoderm lineage. EAT/mcl-1, a bcl-2 related gene, is involved in the genetic pathway of apoptosis; this suggests a role for apoptosis and the involvement of this gene in early placental development. In the current investigation, we analysed expression of EAT/mcl-1 at the mRNA level by Northern blot analysis and in situ hybridization, as well as at the protein level, by immunoblot analysis and immunohistochemistry. Our results identified constant expression of this gene in the placenta throughout pregnancy as well as a shift in its localization from the cytotrophoblast in the first trimester to the syncytiotrophoblast in the third trimester. In addition, there was an inverse correlation between EAT/mcl-1 expression and TaT-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) reactivity in trophoblasts in the first trimester. These results suggest a role for EAT/mcl-1 in both early placental development in regulating trophoblast differentiation as well as a role for this gene in placental maintenance in regulating the process of trophoblast turnover. PMID- 10736241 TI - Expression of urokinase, plasminogen activator inhibitors and urokinase receptor in pregnant rhesus monkey uterus during early placentation. AB - We have investigated plasmin mediated proteolysis associated with trophoblast invasion during early stages of pregnancy in the rhesus monkey. In situ hybridization and immunocytochemical localization were used to define the cellular and tissue distribution of urokinase plasminogen activator (uPA), plasminogen activator inhibitor type 1 (PAI-1) and 2 (PAI-2) and urokinase receptor in early monkey placenta and uterus. Our results indicate: (1) uPA is expressed in proliferating and invasive cytotrophoblast located in chorionic villi as well as in extravillous trophoblast associated with uterine arterioles. This raises the possibility that urokinase may play an important role in trophoblast invasion. (2) PAI-1 mRNA is specifically localized in two areas where invasive trophoblast cells encounter maternal tissue directly. The extravillous cytotrophoblast cells at the maternofetal junction express PAI-1 mRNA. The invasive endovascular trophoblast cells within the uterine arterioles also express PAI-1 mRNA. The location sensitive expression of PAI-1 mRNA at the maternofetal junction may imply a protective function of this protease inhibitor that might be induced through interaction with decidual cells. (3) Urokinase receptor antigen has also been found at the maternofetal junction and in endovascular trophoblast cells of the invaded maternal blood vessel. (4) PAI-2 immunoreactivity is found in association with cytotrophoblast cells in anchoring choronic villi suggesting its association with early placentation. In conclusion, we propose that the plasmin/plasminogen activator system may not only regulate extracellular matrix degradation, but also modify migration and invasive behaviour of extravillous trophoblast cells, during early placentation. PMID- 10736242 TI - Temporal and regional regulation of major histocompatibility complex class I expression at the bovine uterine/placental interface. AB - In most mammals trophoblast cells do not express major histocompatibility complex (MHC) antigens. This probably protects the placenta from immune attack. We have used immunohistochemistry to study the ontogeny of MHC class I expression by bovine trophoblast and endometrial epithelial cells. The interplacentomal, placentomal arcade and placentomal villous/crypt regions were studied. In the interplacentomal region a substantial proportion of trophoblast cells were class I positive from the sixth month on and about half of the endometrial epithelium was class I positive throughout pregnancy. In the arcade region trophoblast class I expression was first observed in the sixth month, increased slowly and peaked at term. Here there was no endometrial epithelial class I expression until term and then only a small percentage of cells were positive. In contrast, in the placentomal villous/crypt region both trophoblast and endometrial epithelium were class I negative throughout gestation. This study shows that cattle have extensive trophoblast class I expression. Moreover class I expression on placentomal, cryptal endometrial epithelium is shut down. Because binucleate trophoblast cells migrate and fuse with endometrial epithelial cells, total shut down of class I expression in areas of intimate interdigitation may be critical for avoidance of immunological rejection. PMID- 10736243 TI - Cellular localization of vascular endothelial growth factor in ovine placenta and fetal membranes. AB - To further understand the role of vascular endothelial growth factor (VEGF) in mediating angiogenesis and vascular permeability during development in the sheep placenta and fetal membranes, we examined the localization of VEGF mRNA and protein in placental, chorionic and amniotic tissues by in situ hybridization and immunohistochemistry in ovine fetuses at 62, 102 and 141 days gestation (term=150 days). In the placenta, VEGF mRNA expression and VEGF protein immunostaining were strong in cytotrophoblasts surrounding the villi. In addition, VEGF protein was localized in smooth muscle cells around fetal and maternal blood vessels and in the maternal epithelium. There was no apparent difference in placental VEGF mRNA or protein levels associated with advancing gestation. In the fetal membranes, VEGF mRNA was detected in the amniotic epithelium and the chorionic cytotrophoblastic cell layer. The intensity of the hybridization signals in both amnion and chorion appeared low at 62 days, moderate at 102 days and high at 141 days gestation. VEGF protein was detected in amniotic epithelium and chorionic cytotrophoblasts at all gestational ages studied. The increase in VEGF gene expression in fetal membranes as term approaches suggests that during fetal development VEGF may promote the vascularity and permeability of the microvessels which perfuse the fetal membranes, as well as permeability of the amniotic membrane itself. Thus VEGF may participate in the regulation of amniotic fluid volume. PMID- 10736244 TI - Differential regulation in human amnion epithelial and fibroblast cells of prostaglandin E(2) production and prostaglandin H synthase-2 mRNA expression by dexamethasone but not tumour necrosis factor-alpha. AB - Previous studies have identified both pro-inflammatory cytokines and glucocorticoids as positive regulators of amnion prostaglandin (PG) biosynthesis. The stimulatory effects of dexamethasone (Dex), a glucocorticoid agonist, on prostaglandin endoperoxide H synthase (PGHS)-2 mRNA expression and PG biosynthesis in amnion have been attributed to an atypical response by the mesenchymal cells of the amnion. The objective of this study was to confirm previous findings concerning cell type-dependant Dex-induced upregulation of PGHS 2 mRNA expression and PG production using separated amnion cell populations, in comparison with the effects of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha). Amnion cells from placentae delivered at term by caesarian section were isolated by tryptic digestion and epithelial cells were then separated from mesenchymal cells by differential absorption onto plastic. After 24-72 h, the two cell populations were passaged and sub-cultured. Cells were treated with Dex (10(-9)-10(-6) m) or TNF-alpha (0.1-50 ng/ml) or media alone. Thereafter, PGE(2)production was determined and PGHS-2 mRNA content analysed by a competitive quantitative RT-PCR method established and validated for this study. PGE(2)production in fibroblast-enriched cultures was increased to 310+/-41 per cent (mean+/-sem, n=4 wells per treatment point) of control in the presence of 10(-8) m Dex. Conversely, PGE(2)production in Dex-treated amnion epithelial cells was decreased to 67+/-24 per cent of control. Altered PGE(2)biosynthesis was accompanied by the upregulation of PGHS-2 mRNA in amnion fibroblasts but not in epithelial cells. TNF-alpha increased PG output and PGHS-2 expression independent of cell type. Glucocorticoids therefore appear to have opposing effects on PG biosynthesis in the two major cellular components of the human amnion. PMID- 10736245 TI - Detection of N-terminal pro-corticotrophin releasing hormone (CRH) and a 'novel' CRH in human maternal plasma and placenta. AB - Corticotrophin-releasing hormone (CRH) is released from the human placenta in high concentrations towards the end of the third trimester of pregnancy, but relative concentrations of other cleavage products derived from the CRH prohormone are unknown. We have measured CRH and N-terminal (1-100) and (1-127) proCRH peptides in maternal plasma in the second and third trimesters of pregnancy and in term placental extract using immunoradiometric assays (IRMAs) specific to different regions of the CRH precursor. Levels of N-terminal proCRH (amino acid residues 1-100) rose from 24+/-4 pmol/l (mean+/-s.e.) in the second trimester to 378.8+/-65 pmol/l (mean+/-s.e.) at term. Levels of intact proCRH and/or (1-127) proCRH remained relatively constant throughout the second and third trimesters, with a concentration of 29.3+/-3.8 pmol/l (mean+/-s.e.). In the course of this work a novel form of CRH that cross-reacts within specific CRH immunoassays was observed. The use of two IRMAs developed for CRH (1-41) having different C-terminal epitope specificities provided evidence for two types of CRH coexisting in maternal plasma. Separation of term placental extract by HPLC and application of the two CRH IRMAs revealed two peaks of immunoreactivity one of which coeluted with synthetic CRH (1-41). PMID- 10736247 TI - Oxidation of eugenol by purified human term placental peroxidase. AB - The oxidation of eugenol by purified human term placental peroxidase (HTPP) was examined. Spectral analyses indicated that, similar to horseradish peroxidase, HTPP is capable of catalyzing the oxidation of eugenol. The accumulated stable product in the reaction medium due to eugenol oxidation by HTPP was tentatively identified as quinone methide of eugenol (EQM). The EQM formation exhibited a pH optimum of 8.0 and was dependent on incubation time, amount of HTPP and the concentration of both eugenol and hydrogen peroxide. The specific activity of approx 2.8 micromoles of EQM/min/mg protein was observed with different preparations of HTPP. The EQM formation was significantly suppressed by glutathione and ascorbic acid. The classical peroxidase inhibitors viz. potassium cyanide and sodium azide blocked the reaction in a concentration manner. Collectively, the results suggest that eugenol may undergo peroxidative metabolism in human placenta. PMID- 10736246 TI - Immunohistochemical localization of ECE-1 in the human placenta. AB - Over the last several years, endothelin (ET-1) has emerged as an important mediator in the pathogenesis of pre-eclampsia and preterm labour, as well as in the normal function of gestational tissues. While the distribution of ET and its binding sites in the human placenta have been well studied, much less has so far been reported about the distribution of placental ET-1 processing enzymes. By immunohistochemical analysis and immunofluorescence, endothelin-converting enzyme 1 (ECE-1), the enzyme that synthesizes ET-1, is localized to five distinct cell populations in the human placenta: (1) the endothelial cells lining the maternal basal plate blood vessels, (2) the intermediate trophoblasts, (3) the endothelial cells lining the chorionic villous blood vessels, (4) the chorionic villous stromal cells and (5) the chorionic villous trophoblasts. The localization of ECE 1 corresponds with the previously reported distribution of ET-1 in the human placenta and is in accordance with the function of this enzyme in regulating vascular tone through synthesis of ET-1. The abundance of ECE-1 in the basal plate is consistent with a second possible function of this enzyme in affecting uterine contractions. ECE-1 may serve as a target for prognosis and therapy in states of pathologically altered vascular tone and/or altered myometrial smooth muscle tone in gestation. PMID- 10736249 TI - A quantitative study on the effects of maternal smoking on placental morphology and cadmium concentration. AB - The aim of this study was to quantify the effects of maternal cigarette smoking on placental morphology, paying particular attention to variables known to be influential in facilitating oxygen diffusion. Structural quantities were estimated by stereological analyses of placental samples drawn from non-smoking and smoking women whose smoking habits were assessed both subjectively (from volunteered cigarette consumption) and objectively (by determining levels of plasma cotinine, a major metabolite of nicotine). Concentrations of placental cadmium were also measured. In the smoking group, maternal and fetal haematocrits were elevated and mean birthweight was reduced. Within placentae, the most significant alterations were increases in cadmium levels, the relative volumes of maternal intervillous space, the relative surface areas of fetal capillaries and decreases in the relative and absolute volumes of fetal capillaries. Findings indicate that changes in capillary volume are the result of a decrease in mean capillary diameter rather than total length. The mean thickness of the trophoblast component of the villous membrane was also increased in the smoking group. Although increased haematocrits suggest that fetuses of smoking mothers suffer hypoxic stress, these morphological changes are likely to compromise, rather than assist, transplacental oxygen transfer. This is in marked contrast to the adaptive changes seen in pregnancies associated with preplacental hypoxia and suggests that other factors might be compromising the fetoplacental unit. Finally, although the morphological changes associated with maternal smoking seem to be the result of an all-or-none, rather than dose-dependent, effect, the available evidence is not conclusive. PMID- 10736248 TI - Antepartum glucocorticoid therapy suppresses human placental xenobiotic and steroid metabolizing enzymes. AB - We investigated the effects of maternal gestational corticosteroid therapy on placental xenobiotic and steroid metabolizing enzymes at term in 20 glucocorticoid/betamethasone treated (with various doses) and control (n=10) women. A single dose of betamethasone (12 mg i.m. twice at a 24-h interval) was given to 15 mothers at risk of preterm delivery to prevent respiratory syndrome in their premature newborns. Five mothers were treated more than once. The gestation time in mothers receiving the glucocorticoid therapy varied from 22-38 gestational weeks. Compared with controls, a significant decrease in placental aromatase activity (53.6+/-18.0 pmol/mg/min versus 119+/-30 pmol/mg/min, P=0.0007) and placental CYP19 mRNA content (by 50 per cent ) was observed in mothers treated with glucocorticoids. Also the formation of androstenedione (13.2+/-8.1 pmol/mg/min, steroids versus 30.03+/-5.2 pmol/mg/min, controls, P< 0.001), using testosterone as the substrate, and 7-ethoxycoumarin O-deethylase (P< 0.05) and 7-ethoxyresorufin O-deethylase (P< 0.09) were slightly decreased in the glucocorticoid treated compared to control patients' values. The changes were not dependent on the number of treatments or the time between treatment and delivery. Our results demonstrate that even a single dose of glucocorticoid given to expectant mothers is associated with diminished placental steroid hormone and xenobiotic metabolizing enzymes at term. Further studies are needed to assess whether these changes affect the well-being of the fetus and its later development. PMID- 10736250 TI - Placenta of idiopathic fetal growth restriction: cytochemically detectable enzyme activities do not change at a subcellular level. AB - The present study was designed to localize some important enzymes, such as adenosine diphospate-degrading enzyme (ADP-degrading enzyme) (plasma membrane enzyme), cytochrome c oxidase (mitochondrial enzyme) and glucose-6-phosphatase (endoplasmic reticulum enzyme), in placentae from patients with idiopathic fetal growth restriction (FGR) associated with absent end-diastolic flow velocity in the fetal umbilical artery. We compared these enzyme activities and their localization patterns to those in placentae both from pre-eclampsia with FGR and normal pregnancy with appropriate for their gestational age infants. In idiopathic FGR placentae, the intensity and localization patterns of these three enzymes did not differ from those seen in the placentae from normal pregnancy. Decreased ADP-degrading enzyme activity and cytochrome c oxidase negative mitochondria, which were characteristic features of pre-eclamptic trophoblasts, were absent from trophoblasts of the idiopathic FGR placentae. These observations indicated that enzyme-cytochemically detectable trophoblastic cell dysfunction may be absent in idiopathic FGR, or if present, there is less functional impairment of each trophoblast in this disease than in pre-eclampsia. Though both idiopathic FGR and pre-eclampsia lead to placental insufficiency, and finally to restricted fetal growth, a different mechanism and pathophysiology may work at the cellular and subcellular levels in these two diseases. PMID- 10736251 TI - Placental synthesis of oestriol in Down's syndrome pregnancies. AB - In the second trimester, oestriol is synthesized in the placenta and secreted into the maternal circulation. 16alpha-hydroxy dehydroepiandrosterone sulphate (16alpha-OH-DHEAS) is formed in the fetal liver by hydroxylation of dehydroepiandrosterone sulphate (DHEAS) and transported to the placenta where it undergoes desulphation by steroid sulphatase (STS) and aromatization to oestriol. Maternal serum levels of unconjugated oestriol (UE3) are lower in Down's syndrome pregnancies than in unaffected pregnancies in the second trimester. The underlying cause of this variation was investigated in placenta, fetal liver, maternal serum and amniotic fluid from Down's syndrome pregnancies by measuring the levels of UE3, DHEAS and STS in appropriate tissues and in corresponding samples from unaffected pregnancies. UE3 levels, expressed as multiples of the control median at the appropriate gestation (MOM), were lower in placental tissue (0.52 MOM), maternal serum (0.65 MOM) and amniotic fluid (0.61 MOM) than in unaffected pregnancies. There was a significant correlation between placental and maternal serum levels of UE3 in the Down's syndrome cases. The median STS activity in placental tissue from Down's syndrome pregnancies (1.14 MOM) was not significantly different from that of the control pregnancies (1. 01 MOM), suggesting that placental turnover of the fetal precursor DHEAS is not reduced. However, levels of DHEAS were reduced in maternal serum (0.69 MOM), placental tissue (0.54 MOM) and fetal liver (0.65 MOM) from Down's syndrome pregnancies. Thus, a diminished supply of the fetal precursor DHEAS may be the cause of the decreased placental production of UE3 in Down's syndrome pregnancies in the second trimester. PMID- 10736252 TI - Sonographically thick placenta: a marker for increased perinatal risk--a prospective cross-sectional study. AB - The aim of this study was to determine placental thickness by ultrasound examination throughout pregnancy and establish the correlation of sonographically thick placenta with perinatal mortality and morbidity. Placental thickness was determined by routine sonographic examination throughout pregnancy in 561 normal singleton pregnancies. Thick placenta was determined as placenta that was above the 90th percentile. Gravidae between 20-22 weeks' gestation (n=193) and 32-34 weeks (n=73) were then divided into two groups according to placental thickness. The study group consisted of 44 gravidae with thick placenta. The control group included 151 gravidae with placental thickness between the 10th and 90th percentile. A comparison of perinatal mortality and morbidity rates as well as the incidence of small and large for gestational age neonates was conducted.A linear increase of placental thickness was found to correlate with gestational age throughout pregnancy. No statistical differences were observed between the two groups with regard to obstetrical variables such as maternal age, parity and gestational age at delivery. No correlation was found between placental thickness and maternal age or parity. The incidence of perinatal mortality was significantly higher among gravidae with thick placentae (6.82% versus 0.66 per cent, P=0.037, 95 per cent confidence interval 1.71-70.29). Birthweight at term was found to be above 4000 g in 20.45 per cent of the thick-placenta group as compared to 5.3 per cent in the control group (P=0.001, 95 per cent CI 2.08 13.85), and birthweight of less than 2500 g was found in 15. 9 per cent of the thick-placenta group as compared to 7.3 per cent in the control group (P=0.03, 95 per cent CI 1.11-8.14). The incidence of fetal anomalies was 9.1 per cent in the thick-placenta group and 3.97 per cent in the control group (not significant). Sonographically thick placenta is associated with increased perinatal risk with increased mortality related to fetal anomalies and higher rates of both small for gestational age and large for gestational age infants at term. PMID- 10736253 TI - Phagocytosis of chorion laeve trophoblasts in patients with chorioamnionitis associated preterm delivery: ultrastructural and enzyme-histochemical observations. AB - The purpose of the present study was to examine morphological evidence for the presence of phagocytosing chorion laeve trophoblasts in fetal membranes from patients who had undergone chorioamnionitis-related preterm delivery. Chorion laeve trophoblasts from six patients, who underwent preterm delivery (28-34 weeks of gestation) complicated with chorioamnionitis, were analysed using transmission electron microscopy and ultrastructural enzyme-histochemistry for acid phosphatase, and compared with those from gestational age-matched chorioamnionitis-negative controls. Cytosomal cell projections, phagosomes, phagocytosis of degenerated cells and cell debris, attachment or fusion of lysosomes to the phagosomal membranes and phagosomal membranes positive for acid phosphatase staining were characteristically observed much more frequently in trophoblasts with chorioamnionitis than those without. The results indicated that chorion laeve trophoblasts in fetal membranes from patients having undergone chorioamnionitis-related preterm delivery underwent phagocytosis. Such phagocytosing trophoblasts may play a role in the pathophysiology or pathogenesis of infection-related preterm delivery. PMID- 10736254 TI - Image analysis of nucleomegalic cells in Chagas' disease placentitis. AB - Villous stroma nucleomegalic cells harbouring intracytoplasmic parasites are an unusual finding which appears to be restricted to Chagas' disease placentitis. Ploidy analysis of these cells in two cases (twelve nuclei in Case 1 and 26 in Case 2) showed these nuclei to contain quantitatively abnormal DNA. Only a few were diploid, the rest proving to be tetraploid, hypertetraploid and even aneuploid. Little is known about the pathogenesis of these macronuclei. However, since these cells contain large numbers of parasites it is possible that a parasite-induced derangement of the cell cycle at G2 phase may be operative. PMID- 10736255 TI - Lack of membrane transport of l-thyroxine sulphate in the human choriocarcinoma cell line, JAr. AB - We examined uptake of l -thyroxine sulphate (T(4)S) and possible interactions between T(4)S and thyroxine (T(4)) uptake in the choriocarcinoma cell line JAr. Cells were incubated with 50 p m(125)I-T(4)S in the absence (total uptake) and in the presence (non-specific uptake) of 10 microm T(4)S. Cells were also incubated at 37 degrees C for 2 min with 50 p m(125)I-T(4)in the presence of an increasing amount of unlabelled T(4)(0-10 microm) or T(4)S (0-30 microm). There was negligible total uptake of(125)I-T(4)S (1.14+/-0. 05 fmol/mg cellular protein, mean+/-sem) and no specific uptake after 120 min incubation. Minor inhibition of(125)I-T(4)uptake by T(4)S could be explained entirely by a low level of residual T(4)(0. 2 per cent) in the T(4)S preparation. These findings indicate that T(4)S does not share the T(4)membrane transporter. PMID- 10736256 TI - Re: Apoptotic and proliferative activities in first trimester placentae (Placenta (1999), 20, 223-227, C. C. W. Chan, T. T. Lao and A. N. Y. Cheung) PMID- 10736257 TI - LETTER: response PMID- 10736258 TI - Moledular chaperones ten years. Introduction. PMID- 10736259 TI - Nuclear chaperones. AB - We discuss nuclear chaperones that bind correctly folded protein subunits and mediate molecular interactions, particularly between proteins and nucleic acids. The charge of these chaperones helps to prevent non-specific electrostatic interactions between the components. Thus, an ordered assembly of macromolecular complexes is mediated, most notably in the formation and maintenance of chromatin, though similar principles are likely to apply in ribonucleoprotein assembly. Here, we discuss roles for nuclear chaperones in mediating nucleosome assembly and remodelling during DNA replication and transcription, and upon fertilisation. PMID- 10736260 TI - Roles of molecular chaperones in cytoplasmic protein folding. AB - Newly synthesized polypeptide chains must fold and assemble into unique three dimensional structures in order to become functionally active. In many cases productive folding depends on assistance from molecular chaperones, which act in preventing off-pathway reactions during folding that lead to aggregation. The inherent tendency of incompletely folded polypeptide chains to aggregate is thought to be strongly enhanced$L in vivo *I$Lby the high macromolecular concentration of the cellular solution, resulting in crowding effects, and by the close proximity of nascent polypeptide chains during synthesis on polyribosomes. The major classes of chaperones acting in cytoplasmic protein folding are the Hsp70s and the chaperonins. Hsp70 chaperones shield the hydrophobic regions of nascent and incompletely folded chains, whereas the chaperonins provide a sequestered environment in which folding can proceed unimpaired by intermolecular interactions between non-native polypeptides. These two principles of chaperone action can function in a coordinated manner to ensure the efficient folding of a subset of cytoplasmic proteins. PMID- 10736262 TI - Intramolecular chaperones: polypeptide extensions that modulate protein folding. AB - Several prokaryotic and eukaryotic proteins are synthesized as precursors in the form of pre-pro-proteins. While the pre-regions function as signal peptides that are involved in transport, the propeptides can often catalyze correct folding of their associated proteins. Such propeptides have been termed intramolecular chaperones. In cases where propeptides may not directly catalyze the folding reaction, it appears that they can facilitate processes such as structural organization and oligomerization, localization, sorting and modulation of enzymatic activity and stability of proteins. Based on the available literature it appears that propeptides may actually function as 'post-translational modulators' of protein structure and function. Propeptides can be classified into two broad categories: Class I propeptides that function as intramolecular chaperones and directly catalyze the folding reaction; and Class II propeptides that are not directly involved in folding. PMID- 10736261 TI - PapD-like chaperones and pilus biogenesis. AB - The assembly of adhesive pili from individual subunits by periplasmic PapD-like chaperones in Gram-negative bacteria offers insight into the complex process of organelle biogenesis. PapD-like chaperones bind, stabilize, and cap interactive surfaces of subunits until they are assembled into the pilus. Subunits lack the seventh *gb-strand necessary to complete their immunoglobulin-like folds; the chaperone supplies this missing strand. Indeed, the chaperone may act as a template, providing steric information to facilitate subunit folding. In the mature pilus, each subunit is thought to supply the missing strand to complete the fold of its neighbor. Thus, one general function of chaperones in organelle biogenesis may be to cap highly interactive surfaces of subunits until they reach the proper assembly site. PMID- 10736263 TI - Chaperones in progesterone receptor complexes. AB - Studying the components, pathways, and dynamics of progesterone receptor (PR) assembly with chaperones has provided a highly valuable model system for understanding the coordinate actions of chaperones. Chaperones are primarily adapted to facilitate protein folding processes, and the actions of chaperones toward PR and other steroid receptors probably remain within this general functional boundary. Unlike a typical misfolded protein substrate, PR's folding is effectively arrested prior to hormone binding, thus extending indefinitely the chaperone-interaction phase that normally would be transitory during progressive protein folding. While one could consider this a limitation in PR's ability to fold properly, perhaps a more accurate view is that PR is specially adapted to remain 'misfolded', and thus extend chaperone interactions that function efficiently in repressing PR's transcriptional activity while the receptor awaits an activating signal. PMID- 10736264 TI - The function of alpha-crystallin in vision. AB - The alpha-crystallins account for approximately one-third of the total soluble protein in the lens, contributing to its refractive power. In addition, alpha crystallin also has a chaperone-like function and thus can bind unfolding lens proteins. Alpha B-crystallin is also found outside the lens, having an extensive tissue distribution. It is over-expressed in response to stresses of all kinds, where it is thought to serve a general protective function. Recently, it has been shown in humans that naturally occurring point mutations in the alpha-crystallins result in a deficit in chaperone-like function, and cause cataracts as well as a desmin-related myopathy. This review summarizes much of the past and current knowledge concerning the structure and functions of alpha-crystallin. PMID- 10736266 TI - Associations of Vertebrate Skeletal Concentrations and Discontinuity Surfaces in Terrestrial and Shallow Marine Records: A Test in the Cretaceous of Montana. AB - Although lags of bones and teeth are commonly cited criteria for marine unconformities, the consistency of the association of vertebrate fossils and discontinuity surfaces, as well as the taphonomic (postmortem) controls on this relationship, are poorly understood. A field test across fluvial, paralic, and shallow marine facies in the Campanian Two Medicine and Judith River formations of Montana indicates that the distribution of vertebrate skeletal concentrations is poorly correlated with the inferred durations of erosional and omissional hiatuses. Instead, vertebrate concentrations associated with discontinuities of all durations tend to be patchy and closely track the abundance of fossil material in underlying and lateral facies. Based on the analysis of 83 measured sections, we found first that erosional bases of channels and minor scours within channels yield vertebrate lags; tidally influenced fluvial deposits are more productive than are "upland" fluvial deposits. Second, erosional shoreface ravinements and their correlative transgressive marine flooding surfaces (fourth order sequence boundaries) have well-developed vertebrate lags only along segments that cut across older shoreface deposits. Third, a nonerosional, widely traceable discontinuity, which is interpreted as the nonmarine extension of a 75.4-Ma third-order transgressive surface, is completely lacking in vertebrate concentrations. Despite being unfossiliferous itself, this discontinuity does mark a regional change in the richness of the vertebrate fossil record, with overlying beds characterized by a much greater abundance of skeletal material. Fourth, a laterally extensive set of erosional surfaces, embedded within multistory fluvial sandstone sheets, is the nonmarine extension of an 80-Ma third order sequence boundary in the marine basin and lacks vertebrate concentrations. The strong dependence of vertebrate lag development on preexisting local sources of skeletal material rather than on the magnitude of the erosional vacuity or the duration of the hiatus contrasts with skeletal concentrations of invertebrates in marine successions, where exhumation is generally much less important than the production of new elements during the hiatus. These findings provide a guide to prospecting productive fossil horizons in terrestrial records and underscore fundamental differences in the ways in which bioclastic material accumulates in terrestrial and shallow marine settings, the qualities of paleobiologic data derived from such concentrations, and the relative reliabilities of skeletal material as cues to stratigraphically significant discontinuities. PMID- 10736265 TI - Glycerol as a correlate of impaired glucose tolerance: dissection of a complex system by use of a simple genetic trait. AB - Glycerol kinase (GK) represents the primary entry of glycerol into glucose and triglyceride metabolism. Impaired glucose tolerance (IGT) and hypertriglyceridemia are associated with an increased risk of diabetes mellitus and cardiovascular disease. The relationship between glycerol and the risk of IGT, however, is poorly understood. We therefore undertook the study of fasting plasma glycerol levels in a cohort of 1,056 unrelated men and women of French Canadian descent. Family screening in the initial cohort identified 18 men from five families with severe hyperglycerolemia (values above 2.0 mmol/liter) and demonstrated an X-linked pattern of inheritance. Linkage analysis of the data from 12 microsatellite markers surrounding the Xp21.3 GK gene resulted in a peak LOD score of 3.46, centered around marker DXS8039. In addition, since all of the families originated in a population with a proven founder effect-the Saguenay Lac St.-Jean region of Quebec-a common disease haplotype was sought. Indeed, a six marker haplotype extending over a region of 5.5 cM was observed in all families. Resequencing of the GK gene in family members led to the discovery of a N288D missense mutation in exon 10, which resulted in the substitution of a highly conserved asparagine residue by a negatively charged aspartic acid. Although patients with the N288D mutation suffered from severe hyperglycerolemia, they were apparently otherwise healthy. The phenotypic analysis of the family members, however, showed that glycerol levels correlated with impaired glucose metabolism and body-fat distribution. We subsequently noted a substantial variation in glycerolemia in subjects of the initial cohort with normal plasma glycerol levels and demonstrated that this variance showed significant family resemblance. These results suggest a potentially important genetic connection between fasting glycerolemia and glucose homeostasis, not only in this X-linked deficiency but, potentially, in individuals within the "normal" range of plasma glycerol concentrations. PMID- 10736267 TI - Evidence for Differential Unroofing in the Adirondack Mountains, New York State, Determined by Apatite Fission-Track Thermochronology. AB - Apatite fission-track ages of 168-83 Ma for 39 samples of Proterozoic crystalline rocks, three samples of Cambrian Potsdam sandstone, and one Cretaceous lamprophyre dike from the Adirondack Mountains in New York State indicate that unroofing in this region occurred from Late Jurassic through Early Cretaceous. Samples from the High Peaks section of the Adirondack massif yielded the oldest apatite fission-track ages (168-135 Ma), indicating that it was exhumed first. Unroofing along the northern, northwestern, and southwestern margins of the Adirondacks began slightly later, as shown by younger apatite fission-track ages (146-114 Ma) determined for these rocks. This delay in exhumation may have resulted from burial of the peripheral regions by sediment shed from the High Peaks. Apatite fission-track ages for samples from the southeastern Adirondacks are distinctly younger (112-83 Ma) than those determined for the rest of the Adirondack region. These younger apatite fission-track ages are from a section of the Adirondacks dissected by shear zones and post-Ordovician north-northeast trending normal faults. Differential unroofing may have been accommodated by reactivation of the faults in a reverse sense of motion with maximum compressive stress, sigma1, oriented west-northwest. A change in the orientation of the post Early Cretaceous paleostress field is supported by a change in the trend of Cretaceous lamprophyre dikes from east-west to west-northwest. PMID- 10736268 TI - Microbialite morphostratigraphy as a tool for correlating Late Cambrian-Early Ordovician sequences. AB - Microbialite morphostratigraphy is a new tool for intrabasinal correlation using diverse microbialite structures (morphotypes). The recognition of the succession of morphotypes over constrained temporal intervals and broad areas is a function of the complex interactions that operate to create the structure. Because so many nonlinked variables (e.g., biotic, sedimentological, physicochemical) are involved, similar morphotypes do not reoccur over long temporal intervals. To demonstrate the technique, the upper Cambrian-lowermost Ordovician shelf strata of the Great Basin, United States, were correlated using both morphostratigraphy and standard lithostratigraphy. Six morphozones and one morphosubzone were recognized, as were four main lithologic successions. Because the boundaries between the morphozones and lithologic successions did not coincide, it is inferred that the characteristics of the various microbialite structures are not solely controlled by physical factors. The principles for establishing a morphostratigraphy outlined in this article allow for the potential to correlate along other ancient marine margins in both the same Cambrian and Ordovician interval, as well as any interval in the Phanerozoic in which diverse microbialite structures occur. PMID- 10736269 TI - Geochemistry of the Archean Kam Group, Yellowknife Greenstone Belt, Slave Province, Canada. AB - The geochemistry and isotope systematics of Archean greenstone belts provide important constraints on the origin of the volcanic rocks and tectonic models for the evolution of Archean cratons. The Kam Group is a approximately 10-km-thick pile of submarine, tholeiitic mafic, and subordinate felsic volcanic rocks erupted between 2712 and 2701 Ma that forms the bulk of the Yellowknife greenstone belt in the dominantly granite-metasedimentary Slave Province. Mafic rocks range from Normal-mid-ocean range basalt-like basalts to slightly light rare-earth-element-enriched (LREE-enriched) but Nb-depleted basaltic andesites and andesites, whereas dacitic to rhyodacitic felsic rocks are strongly LREE enriched and highly depleted in Nb. The varepsilonTNd range from +5 to -3 in the mafic to intermediate rocks and from 0 to -5.5 in the felsic rocks. The varepsilonTNd decreases with increasing La/Sm, SiO2 and decreasing Nb/La, suggesting that as the mafic magmas evolved they were contaminated by older basement rocks. Gneissic granitoids >2.9 Ga in age, found at the base of the Kam Group, have varepsilonTNd between -6 and -9 and are excellent candidates for the contaminant. The geochemical and isotopic data, combined with the submarine eruptive setting and field evidence for existing continental basement, support a continental margin rift model for the Kam Group. Similar geochemical-isotopic studies are required on other Slave greenstone belts in order to test evolutionary models for the Slave Province. PMID- 10736270 TI - Actualistic Ophiolite Provenance: The Cyprus Case. AB - The island of Cyprus represents an excellent site to assess quantitatively petrologic clastic response to actively obducting oceanic sources in order to define an actualistic reference for ophiolite provenance, in terms of framework composition and heavy mineral suites. An improved methodology, an extension of the classic ternary QFL logic to include a wider spectrum of key indexes and ratios, provides an accurate synthesis of modal data and allows differentiation of three main petrographic provinces and at least seven subprovinces. Diagnostic signatures of detritus from various levels of an oceanic lithospheric source, and criteria for distinguishing provenance from suprasubduction versus mid-oceanic ophiolites are also outlined. Modern sands derived from the Troodos Ophiolite contain variable proportions of largely pelagic carbonate to chert, boninite to basalt, diabase to metabasite, plagiogranite to gabbroic, and cumulate grains supplied from progressively deeper-seated levels of the multilayered oceanic crust. Dense minerals are mainly clinopyroxenes (diopside), prevailing over orthopyroxenes (enstatite, hypersthene, clinoenstatite), hornblende, tremolite/actinolite, and epidote. Where serpentinized mantle harzburgites have been unroofed, detritus is markedly enriched in cellular serpentinite grains and enstatite, with still negligible olivine and spinel. Sedimentaclastic sands dominated by chert (Mamonia Province) or carbonate grains (Kyrenia Province) are deposited along the southern and northern shores of the island, respectively. Compositions of Cyprus sands are virtually unaffected by climatic, sedimentary, or anthropic processes; recycling of sandstones from foreign sources is a major process only in the Karpaz Peninsula. Petrographic analysis also provides an independent mean to identify prevalent directions of longshore sand transport. PMID- 10736271 TI - Timing of the Acadian Orogeny in Northern New Hampshire. AB - New U-Pb geochronology constrains the timing of the Acadian orogeny in the Central Maine Terrane of northern New Hampshire. Sixteen fractions of one to six grains each of zircon or monazite have been analyzed from six samples: (1) an early syntectonic diorite that records the onset of the Acadian; (2) a schist, a migmatite, and two granites that together record the peak of the Acadian; and (3) a postkinematic pluton that records the end of the Acadian. Zircon from the syntectonic Wamsutta Diorite gives a 207Pb/206Pb age of circa 408 Ma, the time at which the boundary between the deforming orogenic wedge and the foreland basin was in the vicinity of the Presidential Range. This age agrees well with the Emsian position of the northwest migrating Acadian orogenic front and records the beginning of the Acadian in this part of the Central Maine Terrane. We propose a possible Acadian tectonic model that incorporates the geochronologic, structural, and stratigraphic data. Monazite from the schist, migmatite, Bigelow Lawn Granite, and Slide Peak Granite gives 207Pb/206U ages, suggesting the peak of Acadian metamorphism and intrusion of two-mica granites occurred at circa 402-405 Ma, the main pulse of Acadian orogenesis. Previously reported monazite ages from schists that likely record the peak metamorphism in the Central Maine Terrane of New Hampshire and western Maine range from circa 406-384 Ma, with younger ages in southeastern New Hampshire and progressively older ages to the west, north, and northeast. Acadian orogenesis in the Presidential Range had ended by circa 355 Ma, the 207Pb/235U age of monazite from the Peabody River Granite. From 408 to perhaps at least 394 Ma, Acadian orogenesis in the Presidential Range was typical of the tectonic style, dominated by synkinematic metamorphism, seen in central and southern New Hampshire, Massachusetts, and Connecticut. From no earlier than 394 Ma to as late as 355 Ma, the orogenesis was typical of the style in parts of Maine dominated by postkinematic metamorphism. PMID- 10736273 TI - Anomalously Low Temperature Orthopyroxene, Spinel, and Sapphirine Occurrences in Metasediments from the Bamble Amphibolite-to-Granulite Facies Transition Zone (South Norway): Possible Evidence for Localized Action of Saline Fluids: A Discussion. PMID- 10736272 TI - Pb-Nd Isotopes Indicate the Origin of Island Arc Terranes in the Early Paleozoic Pacific. AB - The Takaka Terrane in New Zealand is one of the best exposed arc fragments of the early Paleozoic Australian-Antarctic convergent margin and constitutes one of the most outboard terranes of this margin in paleogeographic reconstructions. Pb-Nd isotope compositions of clinopyroxenes from the Cambrian Devil River Volcanics of the Takaka Terrane enable identification of the location of the terrane in the Paleo-Pacific Ocean. The Devil River Volcanics, a suite of primitive arc and back arc rocks, are interbedded with the partly continent-derived Haupiri Group sediments. Extremely radiogenic Pb and unradiogenic Nd compositions in the arc rocks cannot be explained by assimilation of the Haupiri Group sediments or a continental basement of such a composition. Pb isotope compositions of the Takaka Terrane sediments are much less radiogenic and overlap with crustal compositions of the Lachlan Fold Belt in Australia, suggesting that both units are derived from one source, the Australian-Antarctic Pacific margin. Pb-Nd isotope compositions in the Devil River Volcanics reflect contamination of their mantle sources by subducted sediments derived from Archean provinces in either Antarctica or Laurentia. Both provinces show characteristically high 207Pb/204Pb500 and were located at the Pacific rim in the Cambrian. Mixing between mantle and Proterozoic continental material from present western South America or eastern Laurentia cannot explain the high 207Pb/204Pb500 in the New Zealand rocks. As in New Zealand, extreme spreads in Pb-Nd isotope compositions in other Cambrian volcano-sedimentary sequences in southeast Australia and Tasmania can be explained by the same model, suggesting that all these fragments originated along the Australian-Antarctic Gondwana margin. Pb isotope compositions of arc rocks, therefore, provide a new tool for terrane analysis in the early Paleozoic Pacific ocean. PMID- 10736274 TI - Anomalously Low Temperature Orthopyroxene, Spinel, and Sapphirine Occurrences in Metasediments from the Bamble Amphibolite-to-Granulite Facies Transition Zone (South Norway): Possible Evidence for Localized Action of Saline Fluids: A Reply. PMID- 10736275 TI - Circulation online only : march 28, 2000 PMID- 10736276 TI - L-NMMA (a nitric oxide synthase inhibitor) is effective in the treatment of cardiogenic shock. AB - BACKGROUND: The objective was to assess the safety and efficacy of L-NMMA in the treatment of cardiogenic shock. METHODS: We enrolled 11 consecutive patients with cardiogenic shock that persisted after >24 hours from admission, despite coronary catheterization and primary percutaneous transluminal coronary revascularization, when feasible, and treatment with mechanical ventilation, intraaortic balloon pump (IABP), and high doses of catecholamines. L-NMMA was administered as an IV bolus of 1 mg/kg and continuous drip of 1 mg. kg(-1). h(-1) for 5 hours. Treatment with catecholamines, mechanical ventilation, and IABP was kept constant throughout the study. RESULTS: Within 10 minutes of L-NMMA administration, mean arterial blood pressure (MAP) increased from 76+/-9 to 109+/-22 mm Hg (+43%). Urine output increased within 5 hours from 63+/-25 to 156+/-63 cc/h (+148%). Cardiac index decreased during the steep increase in MAP from 2. 0+/-0.5 to 1.7+/ 0.4 L/(min. m(2)) (-15%); however, it gradually increased to 1.85+/-0.4 L/(min. m(2)) after 5 hours. The heart rate and the wedge pressure remained stable. Twenty-four hours after L-NMMA discontinuation, MAP (+36%) and urine output (+189%) remained increased; however, cardiac index returned to pretreatment level. No adverse events were detected. Ten out of eleven patients could be weaned off mechanical ventilation and IABP. Eight patients were discharged from the coronary intensive care unit, and seven (64%) were alive at 1-month follow up. CONCLUSIONS: L-NMMA administration in patients with cardiogenic shock is safe and has favorable clinical and hemodynamic effects. PMID- 10736277 TI - Direct in vivo evidence demonstrating neointimal migration of adventitial fibroblasts after balloon injury of rat carotid arteries. AB - BACKGROUND: Clinical and experimental evidence suggest that the adventitia participates in the response to endoluminal vascular injury. The current study used a direct approach to test the hypothesis that, after balloon injury of the rat carotid artery, adventitial fibroblasts migrate in a luminal direction and contribute to neointima formation. METHODS AND RESULTS: Primary syngeneic adventitial fibroblasts were stably transduced with retroviral particles coordinating expression of beta-galactosidase (LacZ) and introduced into the adventitia of right carotid arteries of rats immediately after balloon injury. At defined times after injury and fibroblast implantation, rats were euthanized, and arterial tissue was examined for detection of LacZ mRNA (reverse transcription polymerase chain reaction), DNA (polymerase chain reaction), and in situ enzymatic activity. LacZ expression was detected in the media 5 days postinjury and in both media and neointima at 7, 10, and 14 days postinjury. LacZ was undetectable in injured vessels that had not been seeded with transduced fibroblasts and was restricted to the adventitia in seeded vessels that were not injured. CONCLUSIONS: These observations provide direct demonstration of adventitial fibroblast migration into neointima of arteries after endoluminal injury. PMID- 10736278 TI - The apolipoprotein epsilon4 allele determines prognosis and the effect on prognosis of simvastatin in survivors of myocardial infarction : a substudy of the Scandinavian simvastatin survival study. AB - BACKGROUND: Carriers of the epsilon4 allele of the apolipoprotein E gene are at a higher risk of coronary heart disease than individuals with other genotypes. We examined whether the risk of death or a major coronary event in survivors of myocardial infarction depended on apolipoprotein E genotype and whether the benefits of treatment with simvastatin differed between genotypes. METHODS AND RESULTS: Cox proportional hazards models were used to analyze 5.5 years of follow up data from 966 Danish and Finnish myocardial infarction survivors enrolled in the Scandinavian Simvastatin Survival Study. A total of 16% of the 166 epsilon4 carriers in the placebo group died compared with 9% of the 312 patients without the allele, which corresponds to a mortality risk ratio of 1.8 (95% confidence interval, 1.1 to 3.1). The risk ratio was unaffected by considerations of sex, age, concurrent angina, diabetes, smoking, and serum lipids in multivariate analyses. Simvastatin treatment reduced the mortality risk to 0.33 (95% confidence interval, 0.16 to 0.69) in epsilon4 carriers and to 0.66 (95% confidence interval, 0. 35 to 1.24) in other patients (P=0.23 for treatment by genotype interaction). Apolipoprotein E genotype did not predict the risk of a major coronary event. Baseline serum levels of lipoprotein(a) also predicted mortality risk and could be combined with epsilon4-carrier status to define 3 groups of patients with different prognoses and benefits from treatment. CONCLUSIONS: Myocardial infarction survivors with the epsilon4 allele have a nearly 2-fold increased risk of dying compared with other patients, and the excess mortality can be abolished by treatment with simvastatin. PMID- 10736279 TI - Expression of angiotensin II and interleukin 6 in human coronary atherosclerotic plaques: potential implications for inflammation and plaque instability. AB - BACKGROUND: Patients with an activated renin-angiotensin system (RAS) or genetic alterations of the RAS are at increased risk of myocardial infarction (MI). Administration of ACE inhibitors reduces the risk of MI, and acute coronary syndromes are associated with increased interleukin 6 (IL-6) serum levels. Accordingly, the present study evaluated the expression of angiotensin II (Ang II) in human coronary atherosclerotic plaques and its influence on IL-6 expression in patients with coronary artery disease. METHODS AND RESULTS: Immunohistochemical colocalization of Ang II, ACE, Ang II type 1 (AT(1)) receptor, and IL-6 was examined in coronary arteries from patients with ischemic or dilated cardiomyopathy undergoing heart transplantation (n=12), in atherectomy samples from patients with unstable angina (culprit lesion; n=8), and in ruptured coronary arteries from patients who died of MI (n=13). Synthesis and release of IL-6 was investigated in smooth muscle cells and macrophages after Ang II stimulation. Colocalization of ACE, Ang II, AT(1) receptor, and IL-6 with CD68 positive macrophages was observed at the shoulder region of coronary atherosclerotic plaques and in atherectomy tissue of patients with unstable angina. Ang II was identified in close proximity to the presumed rupture site of human coronary arteries in acute MI. Ang II induced synthesis and release of IL-6 shortly after stimulation in vitro in macrophages and rat smooth muscle cells. CONCLUSIONS: Ang II, AT(1) receptor, and ACE are expressed at strategic sites of human atherosclerotic coronary arteries, suggesting that Ang II is produced primarily by ACE within coronary plaques. The observation that Ang II induces IL 6 and their colocalization with the AT(1) receptor and ACE is consistent with the notion that the RAS may contribute to inflammatory processes within the vascular wall and to the development of acute coronary syndromes. PMID- 10736280 TI - Noninvasive detection of myocardial ischemia from perfusion reserve based on cardiovascular magnetic resonance. AB - BACKGROUND: Myocardial perfusion reserve can be noninvasively assessed with cardiovascular MR. In this study, the diagnostic accuracy of this technique for the detection of significant coronary artery stenosis was evaluated. METHODS AND RESULTS: In 15 patients with single-vessel coronary artery disease and 5 patients without significant coronary artery disease, the signal intensity-time curves of the first pass of a gadolinium-DTPA bolus injected through a central vein catheter were evaluated before and after dipyridamole infusion to validate the technique. A linear fit was used to determine the upslope, and a cutoff value for the differentiation between the myocardium supplied by stenotic and nonstenotic coronary arteries was defined. The diagnostic accuracy was then examined prospectively in 34 patients with coronary artery disease and was compared with coronary angiography. A significant difference in myocardial perfusion reserve between ischemic and normal myocardial segments (1.08+/-0.23 and 2.33+/-0.41; P<0.001) was found that resulted in a cutoff value of 1.5 (mean minus 2 SD of normal segments). In the prospective analysis, sensitivity, specificity, and diagnostic accuracy for the detection of coronary artery stenosis (> or =75%) were 90%, 83%, and 87%, respectively. Interobserver and intraobserver variabilities for the linear fit were low (r=0.96 and 0.99). CONCLUSIONS: MR first-pass perfusion measurements yielded a high diagnostic accuracy for the detection of coronary artery disease. Myocardial perfusion reserve can be easily and reproducibly determined by a linear fit of the upslope of the signal intensity-time curves. PMID- 10736281 TI - Determinants of coronary remodeling in transplant coronary disease: a simultaneous intravascular ultrasound and Doppler flow study. AB - BACKGROUND: Coronary remodeling plays a significant role in lumen loss in transplant allograft vasculopathy (TxCAD), but the determinants of remodeling are unknown. We assessed the relationship between remodeling and plaque topography, coronary compliance, and blood flow in TxCAD. METHODS AND RESULTS: One artery in each of 27 transplant patients was investigated with simultaneous intravascular ultrasound and coronary flow measurements (basal and hyperemic by Doppler flow wire). At 4 to 8 different cross sections (mean 5.1+/-1. 2), plaque topography (concentric or eccentric) was determined, and total vessel area, lumen area, and intimal/medial area (IMA) were measured. Mean remodeling ratio (vessel area/IMA) in eccentric lesions (E, n=28) was significantly larger than that in concentric lesions (C, n=70) (E 5.87+/-0.93 versus C 3.58+/-0.62; P<0.001), despite similar IMA (E 3.89+/-0.68 versus C 3.90+/-0.41; P=NS) and distribution of imaged segments. Remodeling ratio was consistently larger in eccentric lesions in all 3 vessel segments when analyzed separately, and mean remodeling ratio for each artery was larger in vessels with predominantly eccentric lesions. Coronary compliance ([Delta lumen area/diastolic lumen area]/Delta mean arterial pressure x 10(3)) was also significantly greater in eccentric lesions versus concentric lesions (proximal 1.00+/-0.39 versus 0.22+/-0.04; mid 0.71+/-0.17 versus 0.21+/ 0.10; distal 0.43+/-0.13 versus 0. 01+/-0.08; all P<0.01). Coronary flow reserve was also significantly higher in coronary arteries with primarily eccentric lesions (E 2. 49+/-0.64 versus C 1.87+/-0.28; P<0.01). CONCLUSIONS: Vessel remodeling in transplant vasculopathy is significantly greater in eccentric lesions than in concentric lesions, possibly due to greater coronary compliance and resistive vessel function. PMID- 10736282 TI - Assessment of nonuniformity of transmural myocardial velocities by color-coded tissue Doppler imaging: characterization of normal, ischemic, and stunned myocardium. AB - BACKGROUND: Transmural myocardial contractile performance is nonuniform across the different layers of the left ventricular wall. We evaluated the accuracy of color M-mode tissue Doppler imaging (TDI) to assess the transmural distribution of myocardial velocities and to quantify the severity of dysfunction induced by acute ischemia and reperfusion in the inner and outer myocardial layers. METHODS AND RESULTS: Thirteen open-chest dogs underwent 15 minutes of left anterior descending coronary artery occlusion followed by 120 minutes of reperfusion. M mode TDI was obtained from an epicardial short-axis view. Systolic velocities were calculated within endocardium and epicardium of the anterior and posterior walls. Regional myocardial blood flow was assessed by radioactive microspheres. Segment shortening was measured by sonomicrometry in endocardium and epicardium of both the anterior and posterior walls. At baseline, endocardial velocities were higher than epicardial velocities, resulting in an inner/outer myocardial velocity gradient. Ischemia caused a significant and comparable reduction in endocardial and epicardial systolic velocities in the anterior wall with the disappearance of the velocity gradient. Systolic velocities significantly correlated with segment shortening in both endocardium and epicardium during ischemia and reperfusion. In the first minutes after reflow, endocardial velocities showed a greater improvement than epicardial velocities, and the velocity gradient resumed although to a limited extent, indicative of stunning. CONCLUSIONS: TDI is an accurate method to assess the nonuniformity of transmural velocities and may be a promising new tool for quantifying ischemia-induced regional myocardial dysfunction. PMID- 10736283 TI - A newly created splice donor site in exon 25 of the MyBP-C gene is responsible for inherited hypertrophic cardiomyopathy with incomplete disease penetrance. AB - BACKGROUND: Hypertrophic cardiomyopathy is a myocardial disorder resulting from inherited sarcomeric dysfunction. We report a mutation in the myosin-binding protein-C (MyBP-C) gene, its clinical consequences in a large family, and myocardial tissue findings that may provide insight into the mechanism of disease. METHODS AND RESULTS: History and clinical status (examination, ECG, and echocardiography) were assessed in 49 members of a multigeneration family. Linkage analysis implicated the MyBP-C gene on chromosome 11. Myocardial mRNA, genomic MyBP-C DNA, and the myocardial proteins of patients and healthy relatives were analyzed. A single guanine nucleotide insertion in exon 25 of the MyBP-C gene resulted in the loss of 40 bases in abnormally processed mRNA. A 30-kDa truncation at the C-terminus of the protein was predicted, but a polypeptide of the expected size ( approximately 95 kDa) was not detected by immunoblot testing. The disease phenotype in this family was characterized in detail: only 10 of 27 gene carriers fulfilled diagnostic criteria. Five carriers showed borderline hypertrophic cardiomyopathy, and 12 carriers were asymptomatic, with normal ECG and echocardiograms. The age of onset in symptomatic patients was late (29 to 68 years). In 2 patients, outflow obstruction required surgery. Two family members experienced premature sudden cardiac death, but survival at 50 years was 95%. CONCLUSIONS: Penetrance of this mutation was incomplete and age-dependent. The large number of asymptomatic carriers and the good prognosis support the interpretation of benign disease. PMID- 10736284 TI - Atrial fibrillation after coronary artery bypass surgery: a model for preoperative risk stratification. AB - BACKGROUND: Atrial fibrillation (AF) occurs in 20% to 40% of patients after CABG. Identification of patients vulnerable for arrhythmia will allow targeting of those most likely to benefit from prophylactic therapy. The aim of the present study was to evaluate accuracy of a prospectively defined signal-averaged P-wave duration (SAPD) cutoff and additional preoperative characteristics for the prediction of AF after CABG. METHODS AND RESULTS: Patients undergoing elective isolated CABG were recruited to the present prospective study. SAPD was recorded in all patients. Filtered signals from 3 orthogonal leads were combined in a vector analysis, and total SAPD was measured preoperatively. Postoperative in hospital AF occurred in 92 (28.2%) of 326 patients. Patients who developed AF were older (65.9 versus 61.7 years of age; P<0.0005) and had longer SAPD (158 versus 145 ms; P<0.0005) than non-AF patients. Incidence of AF increased in patients > or =75 years of age and increased progressively throughout the range of SAPD. Stepwise logistic regression analysis of preoperative variables identified that SAPD >155 ms (odds ratio, 5.37; 95% CI, 3.10 to 9.30; P<0.0005), advanced age (odds ratio, 1. 53; 95% CI, 1.26 to 1.86 per 5-year increase in age; P<0.0005), and male sex (odds ratio, 2.88; 95% CI, 1.30 to 6.40; P<0.01) independently predicted AF. Prospectively defined SAPD >155 ms predicted AF with positive and negative predictive accuracy of 49% and 84%, respectively. CONCLUSIONS: A combination of prolonged SAPD, advanced age, and male sex identifies patients at high risk for development of AF after CABG. PMID- 10736285 TI - Electrophysiological end point for catheter ablation of atrial fibrillation initiated from multiple pulmonary venous foci. AB - BACKGROUND: The end point for catheter ablation of pulmonary vein (PV) foci initiating atrial fibrillation (AF) has not been determined. METHODS AND RESULTS: Ninety patients underwent mapping during spontaneous or induced ectopy and/or AF initiation. Ostial PV ablation was performed by use of angiograms to precisely define targeted sites. Success defined by elimination of AF without drugs was correlated with the procedural end point of the abolition of distal PV potentials. A total of 197 arrhythmogenic PV foci (97%)-single in 31% and multiple in 69%-and 6 atrial foci were identified. A discrete radiofrequency (RF) application eliminated the PV potentials in 9 PV foci, whereas 2 foci from the same PV required RF applications at separate sites in 19 cases. In others, a wider region was targeted with progressive elimination of ectopy. In 49 patients, multiple sessions were necessary owing to recurrent or new ectopy. The clinical success rates were 93%, 73%, and 55% in patients with 1, 2, and > or =3 arrhythmogenic PV foci. Recovery of local PV potential and the inability to abolish it were significantly associated with AF recurrences (90% success rate with versus 55% without PV potential abolition). PV stenosis was noted acutely in 5 of 6 cases, remained unchanged at restudy, and was associated with RF power >45 W. CONCLUSIONS: Multiple PV foci are involved in initiation of AF, and elimination of PV muscle conduction is associated with clinical success. PMID- 10736286 TI - Hypoxia-induced pulmonary blood redistribution in subjects with a history of high altitude pulmonary edema. AB - BACKGROUND: Pulmonary hypertension has been suggested to play an important role in development of high-altitude pulmonary edema (HAPE), and individual susceptibility has been suggested to be associated with enhanced pulmonary vascular response to hypoxia. We hypothesized that much greater pulmonary vasoconstriction would be induced by acute alveolar hypoxia in HAPE-susceptible (HAPE-s) subjects and that changes in pulmonary blood flow distribution could be demonstrated by radionuclide study. METHODS AND RESULTS: We performed ventilation perfusion scintigraphy in 8 HAPE-s subjects and 5 control subjects while each was in the supine position and acquired functional images of pulmonary blood flow and ventilation under separate normoxic and hypoxic (arterial oxygen saturation, 70%) conditions. We also measured acceleration time/right ventricular ejection time (AcT/RVET) with Doppler echocardiography under each condition in both groups. Moreover, we assayed human leukocyte antigen (HLA) alleles serologically in the HAPE-s group. Pulmonary blood flow was significantly shifted from the basal lung region to the apical lung region under hypoxia in HAPE-s subjects, although no significant change in regional ventilation was observed. With Doppler echocardiography, HAPE-s subjects showed increased pulmonary arterial pressure during hypoxia compared with control subjects. The magnitude of cephalad redistribution of lung blood flow was significantly higher in the HLA-DR6 positive than in HLA-DR6-negative HAPE-s subjects. CONCLUSIONS: These findings suggest that acute hypoxia induces much greater cephalad redistribution of pulmonary blood flow that results from exaggerated vasoconstriction in the basal lung in HAPE-s subjects. Furthermore, pulmonary vascular hyperreactivity to hypoxia may be associated with HLA-DR6. PMID- 10736287 TI - Pharmacokinetic evaluation of triiodothyronine supplementation in children after modified Fontan procedure. AB - BACKGROUND: Triiodothyronine (T(3)) supplementation may be a useful adjunct in the management of patients after cardiopulmonary bypass. Limited data are available regarding the use and pharmacokinetics of T(3) in children. The present study was performed to evaluate T(3) pharmacokinetics in a cohort of children undergoing the modified Fontan procedure. METHODS AND RESULTS: A total of 28 patients were enrolled in this randomized, prospective study. The patients were divided into 4 groups: 1 group received a placebo and 3 groups received intravenous T(3) at dosages of 0.4, 0.6, and 0.8 microg/kg, respectively. All 28 patients survived their operative procedures. Two patients developed low cardiac output, and 3 patients had pleural effusions. The median length of hospital stay was 7 days. The mean free T(3) level was 316+/-67 pg/dL after then administration of a placebo. Patients who received T(3) had mean peak free T(3) levels of 972+/ 88, 1351+/-299, and 1869+/-281 pg/dL for the dosages of 0.4, 0.6, and 0.8 microg/kg, respectively. The calculated half-life of T(3) was 7 hours. CONCLUSIONS: The half-life of intravenous T(3) in children is approximately one third of that reported for adults. These results provide a framework for studying the efficacy of T(3) supplementation in children undergoing open-heart surgery. PMID- 10736288 TI - Influence of postangioplasty beta-irradiation on endothelial function in porcine coronary arteries. AB - BACKGROUND: Postangioplasty (PTCA) intracoronary radiation therapy (ICRT) has been demonstrated to limit restenosis. The consequences of these procedures on coronary reactivity are unknown. METHODS AND RESULTS: Porcine coronary arteries were studied after PTCA immediately (n=5) and 6 weeks (n=5) after ICRT (n=5 and 5, respectively), after combined PTCA+ICRT (n=5 and 7, respectively), and after no intervention (n=11). A 3-cm-long source train of Sr/Y(90) was used in vivo to deliver 16 Gy at a depth of 2 mm from the source center, as used in clinical trials. Arterial rings were mounted on myographs to record isometric tension. After achieving steady-state contraction to depolarizing physiological solution containing 40 mmol/L KCl, measured baseline tension was significantly elevated immediately after all interventions. It returned to normal levels 6 weeks after PTCA and ICRT alone but was significantly reduced if combined. Active contractions induced by 40 mmol/L KCl were maintained after combined therapy both immediately after and at 6 weeks. In these depolarizing conditions, nitric oxide dependent relaxation to substance P was trivial after PTCA+ICRT and reduced after ICRT, whereas in the presence of physiological solution and N(omega)-nitro-L arginine, substance P-induced relaxation was reduced after PTCA and abolished after PTCA+ICRT 6 weeks after intervention. In rings without endothelium, the relaxation mediated by sodium nitroprusside (0.1 micromol/L) was reduced immediately after PTCA and at 6 weeks. CONCLUSIONS: PTCA+ICRT altered the passive mechanical properties of porcine coronary arterial wall. Furthermore, at 6 weeks, receptor-operated release of endothelium-derived nitric oxide and endothelium derived hyperpolarizing factor was reduced by ICRT and PTCA alone, respectively, and was prevented by their combination. PMID- 10736289 TI - Monoclonal anti-CD18 antibody prevents transcellular biosynthesis of cysteinyl leukotrienes in vitro and in vivo and protects against leukotriene-dependent increase in coronary vascular resistance and myocardial stiffness. AB - BACKGROUND: Cysteinyl leukotrienes (cys-LT) can constrict small and large vessels and increase vascular permeability. Formation of cys-LT arising from polymorphonuclear leukocytes (PMNL) and endothelial cell cooperation (transcellular synthesis) led to the hypothesis that PMNL-endothelial cell adhesion may represent a key step toward the formation of vasoactive cys-LT. METHODS AND RESULTS: We studied the effect of pretreatment with a monoclonal antibody directed against the CD18 subunit of PMNL beta(2)-integrin on the synthesis of cys-LT in a PMNL-perfused isolated rabbit heart in vitro and in a model of permanent ligature of the left descending coronary artery in the rabbit in vivo. Challenge of PMNL-perfused rabbit hearts with formyl-met-leu-phe (0.3 micromol/L) caused synthesis of cys-LT and increase in coronary perfusion pressure that were prevented by the anti-CD18 antibody. Similar results were obtained with the use of A-23187 (0.5 micromol/L) as a challenge. Persistence of PMNL-associated myeloperoxidase activity in the perfusion buffer was observed in the presence of the anti-CD18 antibody, indicating decreased PMNL infiltration. Coronary artery ligature in vivo increased urinary excretion of leukotriene E(4), supporting the activation of the 5-lipoxygenase pathway during experimental acute myocardial infarction. Pretreatment with the anti-CD18 antibody (1 mg/kg) prevented the increase in leukotriene E(4) excretion. CONCLUSIONS: These data support the importance of adhesion in promoting cys-LT formation, originating from PMNL-endothelial cell cooperation, and contributing to myocardial stiffness and increased coronary resistance. PMID- 10736290 TI - KB-R7943 block of Ca(2+) influx via Na(+)/Ca(2+) exchange does not alter twitches or glycoside inotropy but prevents Ca(2+) overload in rat ventricular myocytes. AB - BACKGROUND: The Na(+)/Ca(2+) exchange (NCX) extrudes Ca(2+) from cardiac myocytes, but it can also mediate Ca(2+) influx, load the sarcoplasmic reticulum with Ca(2+), and trigger Ca(2+) release from the sarcoplasmic reticulum. In ischemia/reperfusion or digitalis toxicity, increased levels of intracellular [Na(+)] ([Na(+)](i)) may raise levels of intracellular [Ca(2+)] ([Ca(2+)](i)) via NCX, leading to cell injury and arrhythmia. METHODS AND RESULTS: We used KB-R7943 (KBR) to selectively block Ca(2+) influx via NCX to study the role of NCX mediated Ca(2+) influx in intact rat ventricular myocytes. Removing extracellular Na(+) caused [Ca(2+)](i) to rise, due to Ca(2+) influx via NCX, and this was blocked by 90% with 5 micromol/L KBR. However, KBR did not alter [Ca(2+)](i) decline due to NCX. Thus, we used 5 micromol/L KBR to selectively block Ca(2+) entry but not efflux via NCX. Under control conditions, 5 micromol/L KBR did not alter steady-state twitches, Ca(2+) transients, Ca(2+) load in the sarcoplasmic reticulum, or rest potentiation, but it did prolong the late low plateau of the rat action potential. When Na(+)/K(+) ATPase was inhibited by strophanthidin, KBR reduced diastolic [Ca(2+)](i) and abolished the spontaneous Ca(2+) oscillations, but it did not prevent inotropy. CONCLUSIONS: In rat ventricular myocytes, Ca(2+) influx via NCX is not important for normal excitation-contraction coupling. Furthermore, the inhibition of Ca(2+) efflux alone (as [Na(+)](i) rises) may be sufficient to cause glycoside inotropy. In contrast, Ca(2+) overload and spontaneous activity at high [Na(+)](i) was blocked by KBR, suggesting that net Ca(2+) influx (not merely reduced efflux) via NCX is involved in potentially arrhythmogenic Ca(2+) overload. PMID- 10736291 TI - Transfection of antisense p53 tumor suppressor gene oligodeoxynucleotides into rat carotid artery results in abnormal growth of vascular smooth muscle cells. AB - BACKGROUND: Although loss of activity of an antioncogene, the p53 tumor suppressor gene product, has been postulated in the pathogenesis of human restenosis, little is known about the role of p53 in the regulation of vascular smooth muscle cell (VSMC) growth. In this study, to clarify the role of p53 in the pathogenesis of restenosis, we examined transfection of antisense p53 oligodeoxynucleotides (ODN) into VSMC in vitro and rat carotid artery in vivo. METHODS AND RESULTS: The specificity of antisense p53 ODN was confirmed by a significant decrease in p53 protein. Transfection of antisense p53 ODN into VSMC resulted in a significant increase in DNA synthesis and cell number as compared with sense and scrambled ODN (P<0.01). Importantly, transfection of antisense p53 ODN into rat intact carotid artery resulted in a significant increase in the ratio of neointima to medial area at 2 and 4 weeks after transfection, accompanied by a significant decrease in p53 protein (P<0.01). Moreover, cotransfection of wild-type p53 plasmid completely abolished neointimal formation induced by antisense p53 ODN. The sustained effect of a single antisense ODN administration was confirmed by the kinetics of ODN in the vessel wall with the use of FITC-labeled ODN. CONCLUSIONS: Overall, the present study demonstrated that loss of p53 by antisense p53 ODN resulted in an abnormal VSMC growth in vitro and in vivo. These results demonstrated the potential contribution of p53 to the pathogenesis of restenosis. PMID- 10736292 TI - Inhibition of neointima formation after experimental coronary artery stenting: a new biodegradable stent coating releasing hirudin and the prostacyclin analogue iloprost. AB - BACKGROUND: To minimize acute stent thrombosis and development of restenosis, stents coated with biodegradable and nonbiodegradable polymers have been proposed to serve as sustained-release drug carriers. METHODS AND RESULTS: In both a sheep and a pig model, we examined the vascular response to standard and high-pressure implantation of coronary Palmaz-Schatz stents coated with a 10-microm layer of polylactic acid (MW 30 kDa) releasing recombinant polyethylene glycol (r-PEG) hirudin and the prostacyclin analogue iloprost, both drugs with antithrombotic and potentially antiproliferative effects. Study observation time was 28 days. Between the corresponding stent groups, no differences were observed with regard to preplacement and postplacement implantation parameters. The morphometric analysis demonstrated that the coating was associated with a greater lumen diameter through a reduction in the mean restenosis area by 22.9% (P<0.02) in the standard-pressure model (sheep) and by 24.8% (P<0.02) in the overstretch pig model compared with uncoated control stents without inducing a local inflammatory response. CONCLUSIONS: The results from this study demonstrate beneficial effects of a polymeric stent coating with polylactic acid releasing r-PEG-hirudin and iloprost on the development of restenosis after coronary stent placement at 4 weeks, independent of the extent of vascular injury. Future studies are proposed to investigate the integration of other substances to further enhance the potential of the stent coating on reducing neointimal formation. PMID- 10736293 TI - Regulation of PKA binding to AKAPs in the heart: alterations in human heart failure. AB - BACKGROUND: cAMP-dependent protein kinase (PKA) regulates a broad range of cellular responses in the cardiac myocyte. Downstream regulation of the PKA pathway is mediated by a class of scaffolding proteins called A-kinase anchoring proteins (AKAPs), which sequester PKA to specific subcellular locations through binding to its regulatory subunit (R). However, the effect of RII autophosphorylation on AKAP binding and the degree of RII autophosphorylation in failing and nonfailing human hearts remains unknown. METHODS AND RESULTS: We investigated AKAP-RII binding by overlay analysis and surface plasmon resonance spectroscopy and measured RII autophosphorylation in human hearts by backphosphorylation. Binding of Ht31 peptide (representing the RII-binding region of AKAPs) to cardiac RII was increased approximately 145% (P<0.01) for autophosphorylated RII relative to unphosphorylated control. By surface plasmon resonance, RII autophosphorylation significantly increased binding affinity to Ht31 by approximately 200% (P<0.01). Baseline PKA-dependent phosphorylation of RII was significantly decreased approximately 30% (P<0.05) in human hearts with dilated cardiomyopathy compared with nonfailing controls. CONCLUSIONS: These results suggest that AKAP binding of PKA in the heart is regulated by RII autophosphorylation. Therefore AKAP targeting of PKA may be reduced in patients with end-stage heart failure. This mechanism may be responsible for the decreased cAMP-dependent phosphorylation of proteins in dilated cardiomyopathy that we and others have previously observed. PMID- 10736294 TI - Contributions of nuclear cardiology to diagnosis and prognosis of patients with coronary artery disease. PMID- 10736295 TI - Images in cardiovascular medicine. Biventricular dysplasia. PMID- 10736296 TI - Measuring and improving quality of care: a report from the American Heart Association/American College of Cardiology First Scientific Forum on Assessment of Healthcare Quality in Cardiovascular Disease and Stroke. PMID- 10736297 TI - Longitudinal visualization of spontaneous coronary plaque rupture by 3D intravascular ultrasound. PMID- 10736298 TI - Hyperhomocyst(e)inemia is a risk factor for arterial endothelial dysfunction in humans. PMID- 10736299 TI - Doppler echocardiographic index of global right ventricular function. PMID- 10736300 TI - Chlamydia pneumoniae in coronary artery disease. PMID- 10736301 TI - Is acquired activated protein C resistance a cardiovascular risk? PMID- 10736302 TI - Differences in cardiac energetics between patients with familial and nonfamilial hypertrophic cardiomyopathy. PMID- 10736304 TI - Human gene therapy: science under fire. PMID- 10736303 TI - Evaluating quality of care for patients with heart failure. PMID- 10736306 TI - Gap between death rates for blacks and whites remains as large as in 1950. PMID- 10736305 TI - National Heart, Lung, and Blood Institute halts part of antihypertensive and lipid-lowering treatment to prevent heart attack trial (ALLHAT) PMID- 10736308 TI - Membrane cholesterol content modulates activation of volume-regulated anion current in bovine endothelial cells. AB - Activation of volume-regulated anion current (VRAC) plays a key role in the maintenance of cellular volume homeostasis. The mechanisms, however, that regulate VRAC activity are not fully understood. We have examined whether VRAC activation is modulated by the cholesterol content of the membrane bilayer. The cholesterol content of bovine aortic endothelial cells was increased by two independent methods: (a) exposure to a methyl-beta-cyclodextrin saturated with cholesterol, or (b) exposure to cholesterol-enriched lipid dispersions. Enrichment of bovine aortic endothelial cells with cholesterol resulted in a suppression of VRAC activation in response to a mild osmotic gradient, but not to a strong osmotic gradient. Depletion of membrane cholesterol by exposing the cells to methyl-beta-cyclodextrin not complexed with cholesterol resulted in an enhancement of VRAC activation when the cells were challenged with a mild osmotic gradient. VRAC activity in cells challenged with a strong osmotic gradient were unaffected by depletion of membrane cholesterol. These observations show that changes in membrane cholesterol content shift VRAC sensitivity to osmotic gradients. Changes in VRAC activation were not accompanied by changes in anion permeability ratios, indicating that channel selectivity was not affected by the changes in membrane cholesterol. This suggests that membrane cholesterol content affects the equilibrium between the closed and open states of VRAC channel rather than the basic pore properties of the channel. We hypothesize that changes in membrane cholesterol modulate VRAC activity by affecting the membrane deformation energy associated with channel opening. PMID- 10736307 TI - Permeation properties of inward-rectifier potassium channels and their molecular determinants. AB - The structural domains contributing to ion permeation and selectivity in K channels were examined in inward-rectifier K(+) channels ROMK2 (Kir1.1b), IRK1 (Kir2.1), and their chimeras using heterologous expression in Xenopus oocytes. Patch-clamp recordings of single channels were obtained in the cell-attached mode with different permeant cations in the pipette. For inward K(+) conduction, replacing the extracellular loop of ROMK2 with that of IRK1 increased single channel conductance by 25 pS (from 39 to 63 pS), whereas replacing the COOH terminus of ROMK2 with that of IRK1 decreased conductance by 16 pS (from 39 to 22 pS). These effects were additive and independent of the origin of the NH(2) terminus or transmembrane domains, suggesting that the two domains form two resistors in series. The larger conductance of the extracellular loop of IRK1 was attributable to a single amino acid difference (Thr versus Val) at the 3P position, three residues in front of the GYG motif. Permeability sequences for the conducted ions were similar for the two channels: Tl(+) > K(+) > Rb(+) > NH(4)(+). The ion selectivity sequence for ROMK2 based on conductance ratios was NH(4)(+) (1.6) > K(+) (1) > Tl(+) (0.5) > Rb(+) (0.4). For IRK1, the sequence was K(+) (1) > Tl(+) (0.8) > NH(4)(+) (0.6) >> Rb(+) (0.1). The difference in the NH(4)(+)/ K(+) conductance (1.6) and permeability (0.09) ratios can be explained if NH(4)(+) binds with lower affinity than K(+) to sites within the pore. The relatively low conductances of NH(4)(+) and Rb(+) through IRK1 were again attributable to the 3P position within the P region. Site-directed mutagenesis showed that the IRK1 selectivity pattern required either Thr or Ser at this position. In contrast, the COOH-terminal domain conferred the relatively high Tl(+) conductance in IRK1. We propose that the P-region and the COOH terminus contribute independently to the conductance and selectivity properties of the pore. PMID- 10736309 TI - Probing pores with peptide plugs. Topology of membrane-inserted diphtheria toxin. PMID- 10736310 TI - Topography of diphtheria Toxin's T domain in the open channel state. AB - When diphtheria toxin encounters a low pH environment, the channel-forming T domain undergoes a poorly understood conformational change that allows for both its own membrane insertion and the translocation of the toxin's catalytic domain across the membrane. From the crystallographic structure of the water-soluble form of diphtheria toxin, a "double dagger" model was proposed in which two transmembrane helical hairpins, TH5-7 and TH8-9, anchor the T domain in the membrane. In this paper, we report the topography of the T domain in the open channel state. This topography was derived from experiments in which either a hexahistidine (H6) tag or biotin moiety was attached at residues that were mutated to cysteines. From the sign of the voltage gating induced by the H6 tag and the accessibility of the biotinylated residues to streptavidin added to the cis or trans side of the membrane, we determined which segments of the T domain are on the cis or trans side of the membrane and, consequently, which segments span the membrane. We find that there are three membrane-spanning segments. Two of them are in the channel-forming piece of the T domain, near its carboxy terminal end, and correspond to one of the proposed "daggers," TH8-9. The other membrane-spanning segment roughly corresponds to only TH5 of the TH5-7 dagger, with the rest of that region lying on or near the cis surface. We also find that, in association with channel formation, the amino terminal third of the T domain, a hydrophilic stretch of approximately 70 residues, is translocated across the membrane to the trans side. PMID- 10736311 TI - Permeation of large tetra-alkylammonium cations through mutant and wild-type voltage-gated sodium channels as revealed by relief of block at high voltage. AB - Many large organic cations are potent blockers of K(+) channels and other cation selective channels belonging to the P-region superfamily. However, the mechanism by which large hydrophobic cations enter and exit the narrow pores of these proteins is obscure. Previous work has shown that a conserved Lys residue in the DEKA locus of voltage-gated Na(+) channels is an important determinant of Na(+)/K(+) discrimination, exclusion of Ca(2+), and molecular sieving of organic cations. In this study, we sought to determine whether the Lys(III) residue of the DEKA locus interacts with internal tetra-alkylammonium cations (TAA(+)) that block Na(+) channels in a voltage-dependent fashion. We investigated block by a series of TAA(+) cations of the wild-type rat muscle Na(+) channel (DEKA) and two different mutants of the DEKA locus, DEAA and DERA, using whole-cell recording. TEA(+) and larger TAA(+) cations block both wild-type and DEAA channels. However, DEAA exhibits dramatic relief of block by large TAA(+) cations as revealed by a positive inflection in the macroscopic I-V curve at voltages greater than +140 mV. Paradoxically, relief of block at high positive voltage is observed for large (e.g., tetrapentylammonium) but not small (e.g., TEA(+)) symmetrical TAA(+) cations. The DEKA wild-type channel and the DERA mutant exhibit a similar relief of-block phenomenon superimposed on background current rectification. The results indicate: (a) hydrophobic TAA(+) cations with a molecular diameter as large as 15 A can permeate Na(+) channels from inside to outside when driven by high positive voltage, and (b) the Lys(III) residue of the DEKA locus is an important determinant of inward rectification and internal block in Na(+) channels. From these observations, we suggest that hydrophobic interfaces between subunits, pseudosubunits, or packed helices of P-region channel proteins may function in facilitating blocker access to the pore, and may thus play an important role in the blocking and permeation behavior of large TAA(+) cations and potentially other kinds of local anesthetic molecules. PMID- 10736312 TI - Transduction ion channels directly gated by sugars on the insect taste cell. AB - Insects detect sugars and amino acids by a specialized taste cell, the sugar receptor cell, in the taste hairs located on their labela and tarsi. We patch clamped sensory processes of taste cells regenerated from the cut end of the taste hairs on the labelum of the flashfly isolated from the pupa approximately 20 h before emergence. We recorded both single channel and ensemble currents of novel ion channels located on the distal membrane of the sensory process of the sugar receptor cell. In the stable outside-out patch membrane excised from the sensory processes, we could repeatedly record sucrose-induced currents for tens of minutes without appreciable decrease. An inhibitor of G-protein activation, GDP-beta-S, did not significantly decrease the sucrose response. These results strongly suggested that the channel is an ionotropic receptor (a receptor/channel complex), activated directly by sucrose without mediation by second messengers or G protein. The channel was shown to be a nonselective cation channel. Analyses of single channel currents showed that the sucrose-gated channel has a single channel conductance of approximately 30 pS and has a very short mean open time of approximately 0.23 ms. It is inhibited by external Ca(2+) and the dose-current amplitude relation could be described by a Michaelis-Menten curve with an apparent dissociation constant of approximately 270 mM. We also report transduction ion channels of the receptor/channel complex type directly gated by fructose and those gated by L-valine located on the sensory process. PMID- 10736313 TI - Functional impact of the ryanodine receptor on the skeletal muscle L-type Ca(2+) channel. AB - L-type Ca(2+) channel (L-channel) activity of the skeletal muscle dihydropyridine receptor is markedly enhanced by the skeletal muscle isoform of the ryanodine receptor (RyR1) (Nakai, J., R.T. Dirksen, H. T. Nguyen, I.N. Pessah, K.G. Beam, and P.D. Allen. 1996. Nature. 380:72-75.). However, the dependence of the biophysical and pharmacological properties of skeletal L-current on RyR1 has yet to be fully elucidated. Thus, we have evaluated the influence of RyR1 on the properties of macroscopic L-currents and intracellular charge movements in cultured skeletal myotubes derived from normal and "RyR1-knockout" (dyspedic) mice. Compared with normal myotubes, dyspedic myotubes exhibited a 40% reduction in the amount of maximal immobilization-resistant charge movement (Q(max), 7.5 +/ 0.8 and 4.5 +/- 0.4 nC/muF for normal and dyspedic myotubes, respectively) and an approximately fivefold reduction in the ratio of maximal L-channel conductance to charge movement (G(max)/Q(max)). Thus, RyR1 enhances both the expression level and Ca(2+) conducting activity of the skeletal L-channel. For both normal and dyspedic myotubes, the sum of two exponentials was required to fit L-current activation and resulted in extraction of the amplitudes (A(fast) and A(slow)) and time constants (tau(slow) and tau(fast)) for each component of the macroscopic current. In spite of a >10-fold in difference current density, L-currents in normal and dyspedic myotubes exhibited similar relative contributions of fast and slow components (at +40 mV; A(fast)/[A(fast) + A(slow)] approximately 0.25). However, both tau(fast) and tau(slow) were significantly (P < 0.02) faster for myotubes lacking the RyR1 protein (tau(fast), 8.5 +/- 1.2 and 4.4 +/- 0.5 ms; tau(slow), 79.5 +/- 10.5 and 34.6 +/- 3.7 ms at +40 mV for normal and dyspedic myotubes, respectively). In both normal and dyspedic myotubes, (-) Bay K 8644 (5 microM) caused a hyperpolarizing shift (approximately 10 mV) in the voltage dependence of channel activation and an 80% increase in peak L-current. However, the increase in peak L-current correlated with moderate increases in both A(slow) and A(fast) in normal myotubes, but a large increase in only A(fast) in dyspedic myotubes. Equimolar substitution of Ba(2+) for extracellular Ca(2+) increased both A(fast) and A(slow) in normal myotubes. The identical substitution in dyspedic myotubes failed to significantly alter the magnitude of either A(fast) or A(slow). These results demonstrate that RyR1 influences essential properties of skeletal L-channels (expression level, activation kinetics, modulation by dihydropyridine agonist, and divalent conductance) and supports the notion that RyR1 acts as an important allosteric modulator of the skeletal L-channel, analogous to that of a Ca(2+) channel accessory subunit. PMID- 10736314 TI - Dual regulation of calcium mobilization by inositol 1,4, 5-trisphosphate in a living cell. AB - Changes in cytosolic free calcium ([Ca(2+)](i)) often take the form of a sustained response or repetitive oscillations. The frequency and amplitude of [Ca(2+)](i) oscillations are essential for the selective stimulation of gene expression and for enzyme activation. However, the mechanism that determines whether [Ca(2+)](i) oscillates at a particular frequency or becomes a sustained response is poorly understood. We find that [Ca(2+)](i) oscillations in rat megakaryocytes, as in other cells, results from a Ca(2+)-dependent inhibition of inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release. Moreover, we find that this inhibition becomes progressively less effective with higher IP(3) concentrations. We suggest that disinhibition, by increasing IP(3) concentration, of Ca(2+)-dependent inhibition is a common mechanism for the regulation of [Ca(2+)](i) oscillations in cells containing IP(3)-sensitive Ca(2+) stores. PMID- 10736315 TI - An intermediate state of the gamma-aminobutyric acid transporter GAT1 revealed by simultaneous voltage clamp and fluorescence. AB - The rat gamma-aminobutyric acid transporter GAT1 expressed in Xenopus oocytes was labeled at Cys74, and at one or more other sites, by tetramethylrhodamine-5 maleimide, without significantly altering GAT1 function. Voltage-jump relaxation analysis showed that fluorescence increased slightly and monotonically with hyperpolarization; the fluorescence at -140 mV was approximately 0. 8% greater than at +60 mV. The time course of the fluorescence relaxations was mostly described by a single exponential with voltage-dependent but history-independent time constants ranging from approximately 20 ms at +60 mV to approximately 150 ms at -140 mV. The fluorescence did not saturate at the most negative potentials tested, and the midpoint of the fluorescence-voltage relation was at least 50 mV more negative than the midpoint of the charge-voltage relation previously identified with Na(+) binding to GAT1. The presence of gamma-aminobutyric acid did not noticeably affect the fluorescence waveforms. The fluorescence signal depended on Na(+) concentration with a Hill coefficient approaching 2. Increasing Cl(-) concentration modestly increased and accelerated the fluorescence relaxations for hyperpolarizing jumps. The fluorescence change was blocked by the GAT1 inhibitor, NO-711. For the W68L mutant of GAT1, the fluorescence relaxations occurred only during jumps to high positive potentials, in agreement with previous suggestions that this mutant is trapped in one conformational state except at these potentials. These observations suggest that the fluorescence signals monitor a novel state of GAT1, intermediate between the E*(out) and E(out) states of Hilgemann, D.W., and C.-C. Lu (1999. J. Gen. Physiol. 114:459 476). Therefore, the study provides verification that conformational changes occur during GAT1 function. PMID- 10736316 TI - Ion-Ion interactions at the selectivity filter. Evidence from K(+)-dependent modulation of tetraethylammonium efficacy in Kv2.1 potassium channels. AB - In the Kv2.1 potassium channel, binding of K(+) to a high-affinity site associated with the selectivity filter modulates channel sensitivity to external TEA. In channels carrying Na(+) current, K(+) interacts with the TEA modulation site at concentrations 1 min results in its slow depriming. We now report here that the activation of this unique CICR markedly enhances impulse-evoked exocytosis of transmitter. The conditioning nerve stimulation (10-20 Hz, 2-10 min) that primes the CICR mechanism produced the marked enhancement of the amplitude and quantal content of end-plate potentials (EPPs) that decayed double exponentially with time constants of 1.85 and 10 min. The enhancement was blocked by inhibitors of ryanodine receptors and was accompanied by a slight prolongation of the peak times of EPP and the end-plate currents estimated from deconvolution of EPP. The conditioning nerve stimulation also enhanced single impulse- and tetanus-induced rises in intracellular Ca(2+) in the terminals with little change in time course. There was no change in the rate of growth of the amplitudes of EPPs in a short train after the conditioning stimulation. On the other hand, the augmentation and potentiation of EPP were enhanced, and then decreased in parallel with changes in intraterminal Ca(2+) during repetition of tetani. The results suggest that ryanodine receptors exist close to voltage-gated Ca(2+) channels in the presynaptic terminals and amplify the impulse-evoked exocytosis and its plasticity via CICR after Ca(2+)-dependent priming. PMID- 10736318 TI - Uncoupling protein homologs: emerging views of physiological function. AB - The widespread occurrence of excess weight and related diseases demands that efforts be made to understand energy expenditure from the gene to the whole animal. For some time, it has been understood that mitochondrial oxidation of fuels generates an electrochemical gradient via outward pumping of protons by the electron transport chain. ATP production via F(1)F(0) ATP synthase is then facilitated by the inward flux of protons down the gradient. There is a growing appreciation that a significant portion of the metabolic rate of endotherms is attributable to counteracting "proton leak" (uncoupling), wherein a flux of protons down the electrochemical gradient generates heat independently of ATP production. Proton leak is especially apparent in thermogenic brown adipose tissue, which expresses a tissue-specific uncoupling protein (UCP1). The recent discovery of widely expressed putative UCP1 homologs [UCP2, UCP3, UCP4, UCP5/brain mitochondrial carrier protein-1 (BMCP1)] raised the possibility that innate proton leak and metabolic rate are regulated by UCP1-like proteins. On the basis of current published data, one may not exclude the possibility that UCP homologs influence metabolic rate. PMID- 10736319 TI - The biochemistry of chromium. AB - Chromium has been known to be a micronutrient for mammals for four decades, but progress in elucidating the role of chromium has proceeded slowly. However, recent studies have shed light on a potential role of chromium in maintaining proper carbohydrate and lipid metabolism at a molecular level. The oligopeptide chromodulin binds chromic ions in response to an insulin-mediated chromic ion flux, and the metal-saturated oligopeptide can bind to an insulin-stimulated insulin receptor, activating the receptor's tyrosine kinase activity. Thus, chromodulin appears to play a role in an autoamplification mechanism in insulin signaling. The molecular agent responsible for transporting chromium from mobile pools to insulin-sensitive cells is probably the metal transport protein transferrin. Chromium from the popular dietary supplement chromium picolinate enters cells via a different mechanism. Release of chromium from chromium picolinate for use in cells requires reduction of the chromic center, a process that can lead potentially to the production of harmful hydroxyl radicals. PMID- 10736320 TI - Physiological importance of quinoenzymes and the O-quinone family of cofactors. AB - O-quinone cofactors derived from tyrosine and tryptophan are involved in novel biological reactions that range from oxidative deaminations to free-radical redox reactions. The formation of each of these cofactors appears to involve post translational modifications of either tyrosine or tryptophan residues. The modifications result in cofactors, such as topaquinone (TPQ), tryptophan tryptophylquinone (TTQ), lysine tyrosylquinone (LTQ) or the copper-complexed cysteinyl-tyrosyl radical from metal-catalyzed reactions. Pyrroloquinoline quinone (PQQ) appears to be formed from the annulation of peptidyl glutamic acid and tyrosine residues stemming from their modification as components of a precursor peptide substrate. PQQ, a primary focus of this review, has invoked considerable interest because of its presence in foods, antioxidant properties and role as a growth-promoting factor. Although no enzymes in animals have been identified that exclusively utilize PQQ, oral supplementation of PQQ in nanomolar amounts increases the responsiveness of B- and T-cells to mitogens and improves neurologic function and reproductive outcome in rodents. Regarding TPQ and LTQ, a case may be made that the formation of TPQ and LTQ is also influenced by nutritional status, specifically dietary copper. For at least one of the amine oxidases, lysyl oxidase, enzymatic activity correlates directly with copper intake. TPQ and LTQ are generated following the incorporation of copper by a process that involves the two-step oxidation of a specified tyrosyl residue to first peptidyl dopa and then peptidyl topaquinone to generate active enzymes, generally classed as "quinoenzymes." Limited attention is also paid to TTQ and the copper-complexed cysteinyl-tyrosyl radical, cofactors important to fungal and bacterial redox processes. PMID- 10736321 TI - Beta-carotene modulates human prostate cancer cell growth and may undergo intracellular metabolism to retinol. AB - Epidemiologic and animal studies provide support for a relationship between high intakes of carotenoids from fruits and vegetables with reduced risk of several malignancies including prostate cancer. The highly controlled environments of in vitro systems provide an opportunity to investigate the cellular and molecular effects of carotenoids. The effects of beta-carotene (BC) on in vitro growth rates, p21(WAF1) and p53 gene expression, as well as the conversion of BC to retinol were investigated in three human prostate adenocarcinoma cell lines: PC 3, DU 145 and LNCaP. In these experiments, media concentrations of 30 micromol BC/L for 72 h significantly (P < 0.05) slowed in vitro growth rates in all three cell lines, independently of p53 or p21(WAF1) status or expression. (14)C-labeled retinol was detected in prostate tumor cells incubated with (14)C-labeled BC, suggesting metabolic conversion of BC to retinol. Conversely, no (14)C-labeled retinol was detected in media incubated without prostate cancer cells. These studies support a hypothesis that in vitro biological effects of BC on prostate cells may result in part from the conversion of BC to retinol or other metabolites. The possibility that prostate cancer cells in vivo locally metabolize provitamin A carotenoids to retinol and other related metabolites may have implications for our understanding of prostate cancer etiology and the design of future prevention studies. PMID- 10736322 TI - Dietary selenium and vitamin E intakes alter beta-adrenergic response of L-type Ca-current and beta-adrenoceptor-adenylate cyclase coupling in rat heart. AB - Previously we have shown that both insufficient (combined with vitamin E deficiency) and excess intake of selenium (Se) impairs isoproterenol (ISO) induced contractions of rat papillary muscle. In the present study, we used patch clamp and biochemical techniques to investigate mechanisms of this effect in rats fed a Se- and vitamin E-deficient, a Se-excess or a normal diet. Whole-cell configuration of patch-clamp technique was used to investigate L-type Ca(2+) currents (I(Ca,L)) and their regulation by beta-adrenergic receptor stimulation in enzymatically isolated single rat ventricular myocytes. Alteration of Se and vitamin E intake did not affect peak I(Ca,L), but the threshold potential of activation was significantly different among groups. Maximal I(Ca,L) responses to ISO were depressed in both experimental groups, but the EC(50) values were not affected. In the Se-deficient group, basal, ISO- or forskolin-induced adenylate cyclase (AC) activity, measured in cardiac membrane preparations, was reduced when compared to the control, whereas 5' guanylyimidodphosphate (GppNHp) stimulated activity was unaffected. Decreased beta-adrenoceptor density and reduced GppNHp-induced affinity shift in ISO binding were also observed in the deficient group. No such differences were present in the excess group. These results suggest that combined Se and vitamin E deficiency interferes with beta adrenoceptor-AC coupling, whereas excess intake of Se does not affect it. Thus, in the deficient group, the impairment of I(Ca) responses to ISO may be a result of a defect in beta-adrenoceptor-AC pathway. Impairment of I(Ca) response in the excess group, however, appears to have a different underlying mechanism. PMID- 10736323 TI - Nutritional origins of insulin resistance: a rat model for diabetes-prone human populations. AB - While there has been little success identifying the genetic bases of noninsulin dependent (type-2) diabetes, current epidemiological data and animal models implicate fetal undernutrition in the development of type-2 diabetes. We examined the effects of fetal undernutrition on insulin responses and glucose tolerance in adulthood in genetically normal rats. Control rats were adequately nourished in utero and consumed nutritionally adequate (N) diets throughout life. Experimental rats (F1 generation) were undernourished in utero and consumed either N or high energy, high-fat (HF) diets postweaning. The offspring of the experimental rats (F2 generation) received the respective diets of their parent. Body weights of experimental F1 rats at d 4 were 40% less than that of control pups, and they remained significantly smaller than controls throughout adulthood. The experimental F1 rats consuming N diets postweaning had a reduced insulin response (-30%) at 30-min postglucose challenge in adulthood (P > 0.05). However, their offspring (F2 generation) displayed a markedly elevated insulin response [+80% at 30 min (P < 0.05) and + 230% at 120 min (P < 0.001) postglucose challenge]. The insulin response of the F2 generation rats fed the high-energy, HF diet was even more pronounced [+130% at 30 min (P < 0.003) and + 250% at 120 min (P < 0.001) postglucose challenge]. Thus, undernourishment in utero produces striking insulin resistance in genetically normal, well-nourished second-generation rats. PMID- 10736324 TI - Soy protein increases glomerular filtration rate in dogs with normal or reduced renal function. AB - In mammals, protein ingestion increases the glomerular filtration rate (GFR), an effect which has been incriminated as a risk factor in progression of renal disease. Some studies suggest that a postprandial increase in GFR is absent or mild with vegetable proteins compared to animal proteins. The objective of this experiment was to determine whether vegetable (soy) protein had different effects than animal protein on GFR in dogs with normal or reduced renal function. A trial was conducted in which GFR was measured in four dogs with normal kidney function and seven dogs with reduced renal mass before and after administering protein. Normal dogs were fed four protein sources (casein, soy meal, soy flakes and purified soy protein). Dogs with reduced renal mass were fed three protein sources (casein, purified soy protein and pork liver). All proteins significantly (P < 0.05) increased the GFR in both groups except for casein (P = 0.066) in normal dogs. Proteins did not differ significantly in the magnitude of the increase in GFR that was induced. This study indicates that soy proteins in dogs have the same effect on GFR as animal-source proteins, which is contrary to reports of effects in humans. PMID- 10736325 TI - Lipoprotein lipase activity and mRNA are up-regulated by refeeding in adipose tissue and cardiac muscle of sheep. AB - Previous studies in rodents have shown that the lipoprotein lipase (LPL) regulation is complex and often opposite in adipose tissue (AT) and muscle in response to the same nutritional treatment. However, neither LPL responses nor the molecular mechanisms involved in the nutritional regulation have been studied in both AT and muscle of ruminant species. To explore this, we measured the LPL activity and mRNA levels in perirenal AT and cardiac muscle (CM) of control, 7-d underfed or 14-d-refed ewes. Underfeeding decreased (P < 0.01) LPL activity both in AT (-59%) and CM (-31%), and these activities were restored (P < 0.01) by refeeding (AT, +248%; CM, +34%). Variations of LPL mRNA level measured by real time reverse transcription-polymerase chain reaction or by Northern blot followed variations of LPL activity: underfeeding decreased AT- and CM-LPL mRNA levels ( 58 and -53%, respectively), and refeeding restored (P < 0.01) them in CM (+117%) and increased them over the baseline in AT (+640%). Quantification of either 3.4- or 3.8-kb LPL mRNA levels revealed a predominant (P < 0.001) expression of the 3.4-kb mRNA in AT (60%) and of the 3.8-kb mRNA in CM (56%), without any preferential regulation of one of these mRNA species by the nutritional status. This work reveals a tissue-specific expression pattern of the ovine LPL gene and a pretranslational nutritional regulation of its expression, which is achieved in the same direction in perirenal AT and CM. The different regulation of CM-LPL between ewes and rats probably arises from peculiarities of ruminant species for nutrient digestion and absorption and liver lipogenesis. PMID- 10736326 TI - Liver insulin-like growth factor-I mRNA is not affected by diet composition or ration size but shows diurnal variations in regularly-fed gilthead sea bream (Sparus aurata). AB - Nutritional regulation of insulin-like growth factor-I (IGF-I) mRNA was assessed in liver of gilthead sea bream (Sparus aurata). As in mammals, starvation lowered the IGF-I mRNA content, which was recovered by refeeding. However, in contrast to previous observations in rats, neither diet composition nor ration size significantly affected hepatic IGF-I mRNA. Although fish growth depended on the quantity of diet supplied, no relationship was found between growth and liver IGF I mRNA levels, a fact that challenges the importance, at least in fish, of liver derived IGF-I on body growth attributed by the classical somatomedin hypothesis. In addition, diurnal modulation of mRNA levels occurred following food intake, suggesting that the intake of food may play a key role in the regulation of the short-term anabolic effects of IGF-I. PMID- 10736327 TI - Rates of urea production and hydrolysis and leucine oxidation change linearly over widely varying protein intakes in healthy adults. AB - The quantitative relationships between nitrogen (N) intake, urea production, excretion and amino acid oxidation are currently a matter of debate. Some investigators have proposed that urea production is essentially constant over a wide range of N intakes and that urea hydrolysis is regulated according to the N needs of the organism. We have assessed this proposal by compiling results from four separate experiments in healthy young adults (n = 34) carried out in our laboratories and all at the end of the respective diet periods using an identical 24-h continuous intravenous infusion of [(15)N, (15)N]urea and L-[1 (13)C]leucine. The N intakes were: expt. 1; protein-free diet for 5 d; expt. 2; N at 44 mg N. kg(-1). d(-1) from a balanced L-amino acid mixture for 13 d; expt. 3; N at 161 mg. kg(-1). d(-1) from egg protein for 6 d; expt. 4 -one group received 157 mg. kg(-1). d(-1) and the other 392 mg. kg(-1). d(-1) from milk-protein-based diets for 6 d. Urea production and excretion were linearly correlated with N intake (r = 0.98 and 0.94, respectively; P < 0.01). Urea hydrolysis increased linearly with N intake (r = 0.7; P < 0.05), with considerable variation in the rate among individuals, especially at the N intake of approximately 160 mg N. kg( 1)d(-1). These findings are consistent with the generally accepted view that a control of body N balance is via a regulation of urea production. They do not support the concept that urea hydrolysis is the more important site in the control of body N loss. PMID- 10736328 TI - Plant stanol esters affect serum cholesterol concentrations of hypercholesterolemic men and women in a dose-dependent manner. AB - The effect of plant stanol ester on serum cholesterol is dose-dependent. However, it is not clear what the dose is beyond which no additional benefit can be obtained. Therefore, we determined the dose-response relationship for serum cholesterol with different doses of plant stanol ester in hypercholesterolemic subjects. In a single-blind design each of 22 men or women consumed five different doses of plant stanol [target (actual) intake 0 (0), 0.8 (0.8), 1.6 (1.6), 2.4 (2.3), 3.2 (3.0) g/d] added as plant stanol esters to margarine for 4 wk. The order of dose periods was randomly determined. Serum total cholesterol concentration decreased (calculated in reference to control) by 2.8% (P = 0.384), 6.8% (P < 0.001), 10.3% (P < 0.001) and 11.3% (P < 0.001) by doses from 0.8 to 3.2 g. The respective decreases for LDL cholesterol were 1.7% (P = 0. 892), 5.6% (P < 0.05), 9.7% (P < 0.001) and 10.4% (P < 0.001). Although the decreases were numerically greater with 2.4 and 3.2 g doses than with the 1.6 g dose, these differences were not significant (P = 0.054-0.516). Serum plant stanols rose slightly, but significantly with the dose (P < 0.001). Apolipoprotein B concentration was decreased significantly already at the dose of 0.8 g (8.7%, P < 0.001). Apolipoprotein E genotype did not affect the lipid responses. We conclude that significant reduction of serum total and LDL cholesterol concentrations is reached with the 1.6-g stanol dose, and increasing the dose from 2.4 to 3.2 g does not provide clinically important additional effect. PMID- 10736329 TI - Lactation delays postpartum bone mineral accretion and temporarily alters its regional distribution in women. AB - The objective of this work was to compare long-term changes in bone mineral in lactating (L) and nonlactating (NL) women for 2 y postpartum. The 40 L women (mean duration of breastfeeding 345 +/- 177 d) and 36 NL women were enrolled during late pregnancy. Subjects were healthy and nonsmoking with a mean age of 28.8 +/- 4.1 y. Bone mineral content (BMC) was measured at 0.5, 3, 6, 12, 18 and 24 mo by dual-energy X-ray absorptiometry set for total body scan with regional analysis. BMC adjusted for bone area, weight and height (adj-BMC) decreased in L women at the lumbar spine (-3.1%, P < 0. 001) and pelvis (-0.9%, P = 0.03) by 3 mo, and at the total body (-0. 9%, P = 0.05) by 6 mo. Losses were recovered following onset of menses. Adj-BMC at the lumbar spine, pelvis, thoracic spine and total body increased over baseline by 24 mo in L women. In NL women, adj-BMC increased over baseline within 3 mo and continued to increase thereafter. Net total-body gains were greater in the 27 NL women who completed the final measurement than in their 26 L counterparts (+2.3% vs. +0.6%, P = 0.001). Net regional gains differed at the head, legs, and ribs, but not at the lumber spine, pelvis or thoracic spine. Duration of breastfeeding, parity, onset of menses and maternal age affected bone changes in L women. These results indicate that lactation delays bone mineral accretion and temporarily alters its regional distribution in postpartum women. PMID- 10736330 TI - Distribution of protein turnover changes with age in humans as assessed by whole body magnetic resonance image analysis to quantify tissue volumes. AB - We tested the hypothesis that nonmuscle lean tissue mass and its rate of protein catabolism remain constant with aging despite changes in the proportional contribution of these tissues to whole-body protein metabolism. Whole-body protein kinetics, using the 60-h oral [(15) N]glycine method, and muscle and nonmuscle protein catabolism, based on protein kinetic data, urinary N(tau) methylhistine excretion and lean tissue volumes defined by whole-body magnetic resonance imaging, from eight healthy elderly subjects (5 females and 3 males, mean age 71.5 y) were compared with those of seven young persons (3 females and 4 males, mean age 28 y). There were no significant age or gender effects on rates of protein kinetics per L total lean tissue. There was a lower (P < 0.004) rate of muscle protein catabolism in the elderly (1.8 +/- 0.2 vs. 2.6 +/- 0.1 g. L( 1). d(-1)) and a trend (P = 0.08) for lower muscle volume (19.7 +/- 1.5 vs. 25.0 +/- 2.4 L). This contrasted with intraabdominal lean tissue, where the rate of protein catabolism (13. 8 +/- 0.6 vs. 13.2 +/- 0.9 g. L(-1 ). d(-1)) and volume (7.5 +/- 0.3 vs 8.0 +/- 0.5 L) did not differ between age groups. Thus, the decrease in the contribution by muscle to whole-body protein metabolism with age is associated with an increase from 62 to 74% (P < 0.001) in the contribution by nonmuscle lean tissues. These findings have potential implications for the nutrition of both normal and sick elderly persons. PMID- 10736331 TI - Dietary diacylglycerol suppresses accumulation of body fat compared to triacylglycerol in men in a double-blind controlled trial. AB - We examined the effects of the long-term ingestion of dietary diacylglycerols (DG) in a double-blind controlled study of human lipid metabolism. Healthy men (n = 38; aged from 27 to 49 y, body mass index (BMI) ranging from 21.8 to 27.4 kg/m(2)) completed the study. To accustom the subjects to the test diets prior to the experiment, they were supplied with test diets of triacylglycerol (TG) oil for 4 wk (control period). The test oils (10 g/d) were included in bread, mayonnaise or shortbread and served for the breakfast. The target for total lipid intake was 50 g/d (15 g for breakfast, 15 g for lunch and 20 g for dinner) throughout the study. The subjects were then divided into two groups so that mean BMI and the hepatic fat content, determined by computed tomography, for each group were not different. One group (DG group) consumed test meals containing DG rich oil (10 g/d) while the other group (TG group) consumed the same meal as during the control period. Ten grams of the DG-rich oil contained 5.5 g 1,3-DG, 2.5 g 1,2-DG and 2 g TG. The actual lipid intake during the study was 43 g/d. Body weight, BMI and waist circumference decreased in both groups at the end of the test period of 16 wk. However, the magnitudes of decreases in these variables were significantly greater in the DG group than in the TG group. Decreases of total fat, visceral fat area and subcutaneous fat area of the abdominal traverse images of computed tomography in the DG group were also significantly greater than those in the TG group. Hepatic fat content decreased significantly in the DG group while no change was observed in the TG group. Serum lipid concentrations (TG, total cholesterol, free fatty acid) and related metabolites (glucose, insulin, total ketone body) did not change significantly in either group. Thus, in contrast to TG, DG apparently suppressed accumulation of fat and possibly reduces the risk of diseases associated with visceral fat obesity. PMID- 10736332 TI - Neither background diet nor type of soy food affects short-term isoflavone bioavailability in women. AB - To characterize bioavailability of soybean isoflavones, proposed anticarcinogenic food components, eight women, ages 20-41 y, were fed 0.9 mg isoflavones/kg body wt from soymilk at 0730, 1230 and 1730 h for 1 d. Subjects consumed three background diets in random order: a diet prepared for them (basic foods diet) or a self-selected diet at the specified times, or a self-selected diet eaten ad libitum. In a second study, women were fed single isoflavone doses of 0.8-1.4 mg/kg in breakfast casseroles containing tofu, tempeh, cooked soybeans or texturized vegetable protein. Both studies were conducted in randomized, cross over designs. Plasma, urine and fecal isoflavones were measured by reverse-phase HPLC. After consumption of background diets, 48-h urinary recovery of daidzein (D) was 26-27%, and of genistein (G), 18-20% of the dose given with each diet. At 24 h after consumption of different background diets, plasma D and G concentrations were similar (1.4 +/- 0.7 mmol/L) and were not affected by diet selection. Urinary recoveries of D over 24 h from the various soy foods were 38 51%, and of G, 9-16% of the dose given. In both studies, urinary recovery of D was significantly greater than that of G. Only a few percentage of the total isoflavone dose was recovered in feces, probably due to bacterial breakdown of these compounds. Therefore, isoflavone bioavailability may not be affected by choice of background diet or food source of isoflavones. PMID- 10736333 TI - Four-day multimedia diet records underestimate energy needs in middle-aged and elderly women as determined by doubly-labeled water. AB - Systematic problems exist in the quantification of food intake in populations using traditional self-reported measures. The objective of this study was to determine the effectiveness of an innovative multimedia diet record (MMDR) for dietary energy intake assessment. Dietary intake was estimated by combining the use of a microcassette tape recorder and 35-mm camera in 53 women whose ages ranged from 50 to 93 y (64.9 +/- 11.3 y), with body weights of 62.4 +/- 12.2 kg and body mass indexes (BMI) of 24.4 +/- 4.0 kg/m(2). Using household measures, subjects voice-recorded and photographed all food and beverages consumed for four consecutive days. A two-point doubly-labeled water (DLW) method was used over 13 d to calculate carbon dioxide production, total body water, and subsequently, total energy expenditure (TEE) through the use of a food quotient. Mean body weight did not change between d 1 and 14. TEE and reported energy intake were compared using MMDR. Mean reported energy intakes 7.5 +/- 1.9 MJ/d (1774 +/- 476 kcal/d) were lower (P < 0.01) than TEE by 10.4 +/- 3.1 MJ/d (2477 +/- 736 kcal/d), indicating underreporting of food intake. Reporting accuracy (reported energy intake/TEE' 100%) was 76.0 +/- 22.9%. Mean energy expenditure (MJ/d), as determined by doubly-labeled water, was higher (P < 0.01) in each stratified age range when compared to reported energy intake by MMDR. There were no significant differences in reporting accuracy among the stratified age groups. Using the MMDR method, this population of weight-stable women underreported their food intakes compared to their determined energy expenditure estimated by DLW. PMID- 10736334 TI - High rates of infant macrosomia: a comparison of a Canadian native and a non native population. AB - The Cree of James Bay have the highest ever reported mean birth weight and a high prevalence of infant macrosomia. This study was designed to examine independent risk factors for infant macrosomia among the Cree, to compare these to risk factors among non-Native Canadians and to determine if ethnic differences persist after adjusting for differences in the distribution of other risk factors. Macrosomia was defined as birth weight >90(th) percentile for gestational age of a reference population. Independent determinants of macrosomia were examined in 385 Cree and 5644 non-Native women. The potential effect of ethnicity (Cree vs. non-Native) was determined after statistically adjusting for age, parity, pregravid weight, height, net rate of weight gain, gestational diabetes mellitus (GDM) and smoking status. The prevalence of macrosomia among the Cree was 34.3% vs. 11.1% among non-Natives. Although GDM significantly increased the risk for macrosomia among the Cree (odds ratio: 4.46, 95% CI: 2.24-9.26), it was not a significant risk factor among non-Natives (odds ratio: 1.15, 95% CI: 0.79-1.65). The risk for infant macrosomia remained elevated among the Cree compared with non Natives after adjusting for other risk factors (odds ratio: 3.64, 95% CI: 2.69 4.90). In conclusion, the Cree have a high prevalence of macrosomia despite controlling for important differences in pregravid weight and GDM. Some of this variation may be due to genetic differences in fetal growth. The differential impact of GDM on macrosomia in the two ethnic groups may be due to differences in treatment strategies for GDM. PMID- 10736335 TI - Chronic heat exposure alters protein turnover of three different skeletal muscles in finishing broiler chickens fed 20 or 25% protein diets. AB - Heat-exposed chickens exhibit a lower growth rate and a depressed protein retention which may result from an alteration in protein metabolism. A high protein diet seems to be beneficial under hot conditions because it tends to improve growth. Effects of high ambient temperature (32 vs. 22 degrees C) and dietary crude protein (25 vs. 20%) on muscle protein turnover were investigated in finishing broiler chickens. At 5-6 wk of age, protein synthesis was measured in vivo in the Pectoralis major, Sartorius and Gastrocnemius muscles (flooding dose of [(3)H]-phenylalanine). Protein breakdown was determined in the same muscles as the difference between protein synthesis and deposition. Chronic heat stress markedly reduced protein synthesis, irrespective of muscle type (P < 0.05). This was mainly related to the lower capacity for protein synthesis (muscle RNA/Protein) (P < 0.01). Chronic heat exposure also decreased protein breakdown in the P. major and Sartorius; this effect was not observed in the GASTROCNEMIUS: Protein synthesis was more affected than breakdown, leading to reduced protein deposition, at least in the P. major and Gastrocnemius muscles. Increasing dietary protein content had no significant impact on muscle protein turnover. Particularly at 32 degrees C, the high-protein diet did not significantly modify either protein synthesis, ribosomal capacity or translational efficiency. However, it favored muscle protein deposition, which was probably related to reduced proteolysis. In conclusion, we showed that chronic heat exposure decreased muscle protein deposition, mainly by reducing protein synthesis. Under these conditions, the impaired protein synthesis was not restored by a 5% higher protein intake. PMID- 10736336 TI - Consumption of soy protein reduces cholesterol absorption compared to casein protein alone or supplemented with an isoflavone extract or conjugated equine estrogen in ovariectomized cynomolgus monkeys. AB - Dietary intake of soy protein is associated with reductions in plasma cholesterol. Isoflavones are thought to be active components of soy and responsible for the beneficial effects because of their structural similarities to estrogen. The purposes of this study were to determine if i) soy protein or a semipurified soy extract, rich in isoflavones, is responsible for improving the lipid profile and ii) altered intestinal cholesterol metabolism is one mechanism for hypocholesterolemic effects. Ovariectomized adult female cynomolgus monkeys (40) were assigned to groups fed diets containing i) casein-lactalbumin (CAS) ii) intact soy protein (SOY), iii) CAS plus an isoflavone-rich semipurified soy extract similar in isoflavone content as SOY (ISO) or iv) CAS plus conjugated equine estrogen (CEE) for 20 wk. Cholesterol absorption was determined using the fecal isotope ratio method. Bile acid excretion was measured using the 3alpha hydroxysteroid dehydrogenase assay. The SOY group had significantly lower total- and VLDL + LDL-cholesterol compared to the other three groups and significantly higher HDL-cholesterol compared to the CAS and CEE groups. Cholesterol absorption was significantly lower in the SOY group compared to the other groups, but bile acid excretion was not significantly affected. The hypocholesterolemic effect of soy protein appears to be mediated in part by decreased cholesterol absorption. The semipurified soy extract, rich in isoflavones, added to casein protein did not have lipid-lowering effects. Other components of soy such as saponins, phytic acid or the amino acid composition may be involved in the hypocholesterolemic effects seen in this study. PMID- 10736337 TI - Replacement of soybean oil with tallow in rye-based diets without xylanase increases protein synthesis in small intestine of broilers. AB - We examined the effects of dietary fat type (10% of either soybean oil, S, or beef tallow, T)(3) and xylanase supplementation (-, without; +, with 1 g of Avizyme 1300 per kg diet) in rye- based diets (56%) on tissue protein synthesis in male broilers. Birds were injected with a large flooding dose of a phenylalanine solution (150 mmol/L, 38 atom percentage excess [(15) N] phenylalanine) and tissues were obtained after a 10-min incorporation period. [(15) N]-enrichment in tissue free phenylalanine and tissue protein bound phenylalanine were measured by gas-chromatography mass-spectrometry and by gas chromatography combustion isotope ratio mass-spectrometry, respectively in order to calculate tissue specific fractional rates of protein synthesis (k(s)). The k(s) (%/d) in (S-), (S+), (T-) and (T+)-fed birds were 56, 64, 84 and 61 (SEM = 3.7) in duodenum, 51, 52, 75 and 58 in jejunum (SEM = 3.1), 66, 67, 105 and 68 (SEM =7.0) in jejunal mucosa cells, 53, 56, 68 and 50 (SEM = 3.7) in ileum and 52, 45, 118 and 39 (SEM = 20.2) in pancreas, respectively. Significant fat, enzyme or interaction effects in these tissues were mainly caused by the elevated k(s) in (T-)-fed birds which was closely associated with intestinal viscosity. We conclude that the effect of soluble nonstarch polysaccharides (NSP) and of NSP hydrolyzing enzymes may be explained partially by modification in tissue protein synthesis of the intestinal tract. PMID- 10736338 TI - Zn-depleted mice absorb more of an intragastric Zn solution by a metallothionein enhanced process than do Zn-replete mice. AB - The influence of metallothionein (MT)(2) on Zn absorption was investigated in MT null (MT-/-) and normal (MT+/+) mice fed Zn-depleted (ZnD) diets for 7 d and compared with those fed Zn-replete (ZnR) diets in a previous study. Mice were starved for 20 h, then administered an oral gavage of aqueous (65)ZnSO(4) solution at doses of 154, 770 or 1540 nmol of Zn, and the amount transferred into nongut tissues was determined 4 h later. (65)Zn transfer did not differ between genotypes in ZnR mice. However ZnD MT+/+ mice had a 30-40% greater transfer from the 154 and 770 Zn doses compared to ZnR MT+/+ mice. This was not observed in MT /- mice. In MT+/+ mice, Zn depletion enhanced the induction of MT by Zn in the intestine and pancreas. (65)Zn uptakes in the liver and pancreas were greater in MT+/+ than MT-/- mice, and this was greater (50%) at the 154 and 770 doses in mice fed ZnD diets. Plasma Zn concentrations were raised to a similar extent in ZnR and ZnD MT-/- mice. ZnR MT+/+ mice had significantly lower plasma Zn levels than MT-/-mice; this difference was less marked in the ZnD mice. We conclude that a MT-facilitated enhancement in Zn absorption occurs in response to dietary Zn deficiency. PMID- 10736339 TI - Absorption and metabolism of genistein in isolated rat small intestine. AB - Studies suggest a variety of biological effects for the isoflavonoid genistein, but there is little information regarding small intestinal absorption and metabolism. The aim of this study was to investigate the absorption and metabolism of luminally administered genistein in an isolated preparation of luminally and vascularly perfused rat small intestine. A synthetic perfusate free from blood components was used as vascular medium, with a perfluorocarbon as oxygen carrier. Luminal media consisted of a bicarbonate buffered sodium chloride solution spiked with genistein (12 micromol/L). Viability was maintained during the entire perfusion as indicated by no significant differences between genistein and control perfusions for perfusion pressure, lactate-pyruvate ratio, oxygen uptake and acid-base homeostasis. Luminal disappearance rate of genistein did not change throughout the entire perfusion time. After a significant increase until about 30 min, vascular appearance rate of total genistein reached an equilibrium. The intestinal absorption of luminally administered genistein was 40.6%, corresponding to an average uptake of 2.9 nmol. min(-1). g dry intestine(-1). The majority (31.3%) of the absorbed genistein appeared as genistein glucuronide, also recovered as the main metabolite on the luminal side (13.3%). Only small amounts of genistein (2.6%) and genistein glucuronide (2.9%) were found in the intestinal tissue. The results demonstrate a favorable uptake of genistein, a pertinent addition to the ongoing discussion about health benefits of isoflavones. PMID- 10736340 TI - Dietary trans fatty acids affect the essential fatty-acid concentration of rat milk. AB - Increasing efforts have been made to determine the distribution and concentration of trans fatty acids in milk, due to the importance of lipids in infant growth and development. In general, trans fatty acid concentration of milk reflects trans fatty acid intake, but insufficient data are available to assess the effects of dietary trans fatty acids on maternal milk. Thus, controlled studies are needed to establish whether there is a dose-response relationship and whether trans fatty acids could affect the concentration of essential fatty acids (EFA), long-chain polyunsaturated fatty acids (PUFA) and the (n-6)/(n-3) ratio in milk. Three groups of six rats each were fed for 10 wk one of three diets differing in trans fatty acid concentration (Control, 0 mol/100 mol; high trans concentration (H), 14.5 mol/100 mol; very high trans concentration (VH), 30 mol/100 mol), but containing the same proportions of linoleic and alpha-linolenic acids and a ratio of 18:2(n-6)/18:3(n-3) of about 7:1. Trans fatty acids were incorporated into maternal milk in a dose-dependent manner. In addition, rats fed trans isomers had greater linoleic acid levels than controls. The proportion of alpha-linolenic acid in milk was lower in the VH group, and the (n-6)/(n-3) cis PUFA ratio in milk of the VH group was greater than that in controls. Total long-chain PUFA levels did not differ among groups. These results suggest that high intakes of trans fatty acids affect the EFA concentration but not that of long-chain PUFA of rat milk, provided that EFA are supplied in sufficient amounts. PMID- 10736341 TI - Digestion of fat does not differ in growing pigs fed diets containing fish oil, rapeseed oil or coconut oil. AB - We studied the digestion of fat and fatty acids in diets containing oils with different fatty acid composition. Four barrows (initial weight 35 kg) were fitted with a simple T-cannula at the terminal ileum. Three wheat starch and fish meal based diets were formulated to contain either 150 g fish oil, rapeseed oil or coconut oil/kg. A basal diet, which did not contain oil, was also prepared. The diets were fed according to a 4 x 4 Latin square design. Each experimental period comprised 5 d adaptation to the diets, 3 d fecal collection and 2 d digesta collection. The apparent ileal and fecal digestibilities of fat were relatively high (88 - 93%). The ileal digestibilities of total, saturated and monounsaturated fatty acids did not differ among the diets. However, the digestibilities of polyunsaturated fatty acids (PUFA) in the fish and rapeseed oil diets were higher (P < 0.05) than in the coconut oil diet. The ileal digestibilities of 18:1, 18:2 and 18:3 in the rapeseed oil diet ranged from 94 to 97%. The ileal digestion of the unsaturated long-chain fatty acids 20:5(n-3) and 22:6(n-3) in the fish oil diet was nearly complete (97 - 98%). Apparent fecal digestibilities of saturated fatty acids (76 - 89%) were lower than apparent ileal digestibilities (89 - 94%). The digestibilities of fat and fatty acids were relatively high when pigs were fed diets containing fish oil, rapeseed oil or coconut oil. There were few differences in the digestibilities of saturated, monounsaturated and PUFA in the fish oil, rapeseed oil or coconut oil diets. PMID- 10736342 TI - Early posthatch starvation decreases satellite cell proliferation and skeletal muscle growth in chicks. AB - The effect of posthatch starvation on skeletal muscle growth and satellite cell proliferation was examined in chicks. Chicks were either fed or starved for 48 h posthatch (d 0-d 2, d 2-d 4 or d 4-d 6) and then refed for 41 d. Body and breast muscle weights were significantly lower in starved chicks than in fed controls throughout the experiment. Histochemical staining revealed that skeletal muscle fiber development in the starved group lagged behind that of the fed group. Starvation from d 2 to 4 and d 4 to 6 posthatch had a progressively lesser effect than did immediate posthatch starvation (P < 0.05). In vitro culturing of breast muscle satellite cells revealed that DNA synthesis and number of cells per gram of muscle in the fed chicks peaked on d 2 and d 3, and then declined. In contrast, DNA synthesis in the cells of starved chicks declined on d 2 and increased on d 3 when chicks were refed. A similar pattern was seen for the number of cells per gram muscle; however, in general cell numbers tended to be higher in the starved group than in controls (P < 0.1). The results obtained with cultured cells were parallel with in situ immunostaining with 5-bromo-2' deoxyuridine and proliferating cell nuclear antigen in breast muscle from experimental chicks, and with growth hormone receptor expression. These results suggest that satellite cell cultures are a reliable tool for evaluating muscle growth in postnatal chickens. We conclude that sufficient feed in the immediate postnatal period is critical for satellite cell proliferation and skeletal muscle development and is thus important for optimal muscle growth. PMID- 10736343 TI - A new approach to estimating the minimum dietary requirement of phosphorus for large rainbow trout based on nonfecal excretions of phosphorus and nitrogen. AB - A new method was developed to estimate the minimum dietary requirement of phosphorus (P) for large fish for which conventional methods are not suitable. The method is based upon nonfecal (mainly urinary) excretion of inorganic P and total nitrogen from fish placed in a metabolic tank. In the first experiment, small and large rainbow trout (body wt 203 and 400 g, respectively) and, in the second experiment, P-sufficient, P-deficient and starved rainbow trout (different in diet history; body wt 349-390 g) were fed a constant amount (standard feeding rate) of semipurified diets with incremental P concentrations once daily at 15 degrees C. In all cases, there was no measurable excretion of P when dietary P concentration was low; however, beyond a specific dietary concentration, excretion of P increased rapidly. The point where the fish started to excrete P was assumed to be the minimum dietary requirement. By d 3 of consuming the experimental diets, the response of the fish to dietary P concentration stabilized, and excretion of P remained constant within dietary treatment groups for the subsequent sampling days (d 6, 9 and 12). The minimum dietary requirement of available P for fish having body wt of 203 and 400 g was estimated to be 6.62 and 5.54 g/kg dry diet, respectively, and that for P-sufficient, P-deficient and starved fish was estimated to be 4.06, 5.83 and 4.72 g/kg dry diet, respectively, when feed efficiency is 1. PMID- 10736344 TI - Dietary taurine alters ascorbic acid metabolism in rats fed diets containing polychlorinated biphenyls. AB - The effect of dietary taurine on ascorbic acid metabolism and hepatic drug metabolizing enzymes was investigated in rats fed diets containing polychlorinated biphenyls (PCB) to determine whether taurine has an adaptive and protective function in xenobiotic-treated animals. Young male Wistar rats (60 g) were fed diets containing 0 or 0.2 g/kg diet PCB with or without 30 g/kg diet of taurine for 14 d. The rats fed the PCB-containing diets had greater liver weight, higher ascorbic acid concentrations in the liver and spleen and greater hepatic cytochrome P-450 contents than control rats that were not treated with PCB (P < 0.01). In PCB-fed rats, urinary ascorbic acid excretion was enhanced, and serum cholesterol concentration (especially HDL-cholesterol) was significantly elevated compared with those in control rats. Dietary taurine significantly potentiated the increases in the urinary excretion of ascorbic acid and the rise in the levels of cytochrome P-450 which were caused by PCB treatment. On the other hand, the supplementation of taurine to control diet did not alter these variables. Taurine may enhance the hepatic drug-metabolizing systems, leading to the stimulation of the ascorbic acid metabolism in rats fed diets containing PCB. PMID- 10736345 TI - Food intake abolishes the response of rat jejunal Na(+),K(+)-ATPase to dopamine. AB - The aim of the present study was to evaluate whether the sensitivity of jejunal Na(+),K(+)-ATPase to inhibition by dopamine (DA) in young rats is related to the type of food (breast milk vs. solid) or reflects a developmental adaptation. When 18-d-old rats were separated from their dams and fed solid food (the same used to feed adult rats) for 2 d, intestinal Na(+),K(+)-ATPase activity was significantly greater than that of breast-fed pups of the same age (20 d) (127 +/- 8 vs. 52 +/- 4 nmol Pi. mg protein(-1). min(-1); P < 0.05). Activity in rats fed solid food was insensitive to inhibition by 1 micromol/L DA. Na(+),K(+)-ATPase activity in 60-d-old rats (117. 4 +/- 4.2 nmol Pi. mg protein(-1). min(-1)) was also higher (P < 0. 05) than in breast-fed rats, and DA (1 micromol/L) did not inhibit enzyme activity. The B(max) value for binding of [(3)H]-Sch 23390 in 20-d-old breast-fed rats did not differ from that in age-matched rats fed a solid food for 2 d and or that in 60-d-old rats. Levels of DA, but not L-3,4-dihydroxyphenylalanine and amine metabolites, in the jejunal mucosa of 20-d-old rats that had eaten solid food for 2 d were 60% lower than in age-matched rats, breast-fed rats, and not different from those in the jejunal mucosa of 60-d-old rats fed the solid food. We conclude that in adult rats, in contrast to in young rats, DA does not inhibit jejunal Na(+),K(+)-ATPase activity, and food intake in young rats plays an important role in the development of the insensitivity of Na(+),K(+)-ATPase activity to DA. PMID- 10736346 TI - Retinol analysis in dried blood spots by HPLC. AB - There are many advantages to measuring vitamin A in dried blood spots (DBS) from a finger prick as compared to plasma collected by venipuncture. The advantages include easier collection, transport and storage; accessibility to younger and more remote populations; and decreased risk of disease transmission. We describe a method for the extraction of retinol from DBS for analysis by HPLC and initial comparison to plasma retinol. The effects of various buffers, detergents, antioxidants and chelators were evaluated to establish the most effective approach to elute the retinol: retinol binding protein (holo-RBP) complex from the blood collection cards. The process involves ultrasonic agitation to elute holo-RBP into a phosphate buffer containing an antioxidant and metal chelator. The holo-RBP complex was denatured by the addition of ethanol containing additional antioxidants permitting the extraction of free retinol into hexane. Following solvent evaporation, the extract was dissolved in methanol for HPLC analysis. The initial measured retinol levels in freshly collected DBS declined for 6-10 d whether stored at 25, 4 or -20 degrees C, but remained consistent thereafter (homeostatic). By incorporating a "recovery/volume adjustment" factor, measured retinol values in homeostatic DBS were adjusted to the equivalent of plasma retinol. For 17 normal adults, the correlation coefficient was 0.90 between plasma retinol and adjusted DBS retinol in samples that had been stored at -70 degrees C for < 9 mo. The use of this new sample matrix for vitamin A assessment will allow access to previously unavailable populations. PMID- 10736347 TI - Metabolic consequences of a high dietary-protein intake in adulthood: assessment of the available evidence. PMID- 10736348 TI - International Symposium on Glutamate. Introduction to the symposium proceedings. PMID- 10736349 TI - Glutamate: an amino acid of particular distinction. AB - In this introductory paper to the symposium, we consider why L-glutamate (GLU) is such an abundant biomolecule. We begin with a brief discussion of the prebiotic dawn of events and some evolutionary features of GLU in the biological and metabolic world. The properties of GLU are then examined with reference to its overall structural motif and to the reactivity of the molecule at the tautomeric 2 carbon and at the 4- and 5-C positions. This chemical viewpoint reveals that the GLU molecule offers a number of features/properties not shared by its homologs (amino adipic and aspartic acids). These properties make GLU a favorable choice for facilitating its involvement in multiple metabolic processes that play major roles in the nitrogen economy of the host, as well as serving as a nutrient, an energy-yielding substrate, a structural determinant and an excitatory molecule. PMID- 10736350 TI - Glutamic acid, twenty years later. AB - This review examines progress in understanding the physiologic functions of glutamic acid in the body since the first symposium on glutamic acid physiology and biochemistry was held at the Mario Negri Institute in Milan in 1978. The topics reviewed, although not exhaustive, include the metabolism of glutamic acid, umami taste, the role of glutamic acid as a neurotransmitter, glutamate safety and the development of new drugs resulting from the knowledge of the neurodegeneration induced by high doses of glutamic acid. PMID- 10736351 TI - Glutamate and the flavor of foods. AB - Investigations of the effects of glutamic acid or its salts on the flavor, hedonic characteristics or acceptance of foods have emphasized a sodium salt of L glutamic acid, monosodium glutamate (MSG). Food palatability increased with appropriate concentrations of MSG, and could be represented f(MSG and NaCl) as points on the surface of an elliptic paraboloid. Hedonic effects differed between foods, were a function of concentrations of MSG and other ingredients and did not necessarily become positive when only MSG was added. For example, with boiled or fried rice, ratings were neutral or negative with MSG alone, positive for fried rice with MSG and NaCl, but positive for boiled rice only if soy sauce was also added. A one-dish meal, Chinese noodle, became positive with an appropriate concentration of NaCl plus a MSG-5'ribonucleotide mixture, but not with NaCl alone. Flavor of meat, fish or vegetable stocks containing natural glutamates differed from that of quinine, sucrose, NaCl or acid solutions. Glutamates and free amino acids plus nucleotides were necessary for full boiled potato flavor. Overall, the effects of MSG on foods were different from those of NaCl but often interacted with NaCl, and positive effects were facilitated by low concentrations of 5'-ribonucleotides. PMID- 10736352 TI - Function and importance of glutamate for savory foods. AB - Flavoring systems are of vital importance in savory food manufacturing. Many industrially prepared foods are particularly attractive to potential consumers primarily because of their typical flavors. Therefore, it is no surprise that the food industry dealing with these product segments shows great interest in the use of food or food ingredients carrying the typical umami taste and flavor enhancement systems. Figures are provided showing the importance of glutamate in traditional cuisines and also in meals prepared by industrial manufacturing. It is also interesting to see how food intake patterns of glutamate differ from one cultural group to another. The ever-growing importance of balanced food formulations (carbohydrates, fats, proteins and minerals) brings special challenges to the use of different ingredients, requiring development of appropriate flavor delivery systems. Again flavor enhancement is of great importance. Questions about the addition of glutamate or the total glutamate content of foods are of little importance, from a scientific point of view. However, in a given legal framework, important business opportunities can be realized. One of the main concerns of manufacturers of savory food is how to provide the consumer with tasty foods while complying with increasingly severe local legal constraints concerning the use of many potent flavoring systems. PMID- 10736353 TI - Umami and food palatability. AB - Umami is the term that identifies the taste of substances such as L-glutamate salts, which were discovered by Ikeda in 1908. Umami is an important taste element in natural foods; it is the main taste in the Japanese stock "dashi," and in bouillon and other stocks in the West. The umami taste has characteristic qualities that differentiate it from other tastes, including a taste-enhancing synergism between two umami compounds, L-glutamate and 5'-ribonucleotides, and a prolonged aftertaste. The key qualitative and quantitative features of umami are reviewed in this paper. The continued study of the umami taste will help to further our general understanding of the taste process and improve our knowledge of how the taste properties of foods contribute to appropriate food selection and good nutrition. PMID- 10736354 TI - Intensification of sensory properties of foods for the elderly. AB - Taste and smell losses in the elderly can reduce appetite and lead to inadequate dietary intake. Although these chemosensory deficits are generally not reversible, sensory interventions including intensification of taste and odor can compensate for perceptual losses. One method for "treatment" of chemosensory losses involves sensory enhancement of foods with flavors and monosodium glutamate (MSG). Amplification of flavor and taste can improve food palatability and acceptance, increase salivary flow and immunity, and reduce oral complaints in both sick and healthy elderly. PMID- 10736355 TI - Physiological studies on umami taste. AB - The first electrophysiological studies on umami taste were conducted with rats and cats. Unlike humans, these animals did not show a large synergism between monosodium glutamate (MSG) and disodium guanylate (GMP) or disodium inosinate (IMP). The taste nerve responses of these animals to umami substances were not differentiated from the salt responses. The canine taste system was sensitive to umami substances and showed a large synergism between MSG and GMP or IMP. The umami substances showed no enhancing effects on other basic tastes. Amiloride, an inhibitor for the response to NaCl, did not inhibit the large response induced by the synergism between MSG and the nucleotides, indicating that the response to the umami substances is independent of the response to salt. Single-fiber analysis on the responses of mouse glossopharyngeal nerve and monkey primary taste cortex neurons also showed that the responses to umami substances are independent of other basic tastes. On the basis of these results, it was proposed that the umami taste is a fifth basic taste, and that there is a unique receptor for umami substances. Hence, we compared the taste of agonists for brain glutamate receptors. In humans, the order of intensity of umami taste induced by a mixture of 0.5 mmol/L GMP and 1.5 mmol/L of various agonists was glutamate > ibotenate > L(+)-2-amino-4-phosphonobutyric acid (L-AP4) = (+/-)1 aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD). Kainate, N-methyl-D aspartic acid (NMDA) and (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), which are agonists for ionotropic receptors, had no umami taste. It was concluded that the umami receptor is not identical to any known glutamate receptors; there seems, therefore, to be a unique receptor for umami. PMID- 10736357 TI - Receptor and transduction processes for umami taste. AB - The unique taste of umami argues for a specific receptor at the taste cell level. The taste synergism between monosodium glutamate (MSG) and certain 5' ribonucleotides provides a pharmacologic test for hypothetical mechanisms of umami taste. Early neurophysiologic and biochemical studies demonstrated specific recognition of L-glutamate by taste tissue and suggested that the synergism found with certain 5'-ribonucleotides was due to a peripheral event. The search for a receptor for umami relies at present on the data in the literature on central nervous system (CNS) glutamate receptors. These data distinguish several classes of receptors on the bases of pharmacologic properties and mode of action. Two hypotheses now seek to explain umami taste transduction. One states that umami is transduced by an N-methyl-D-aspartate (NMDA)-type glutamate ion channel receptor, the other that this taste is transduced via a metabotropic-type glutamate receptor. Evidence for the first hypothesis derives from earlier reconstitution studies, revealing a glutamate-stimulated ion channel conductance whose kinetics were affected by 5'-ribonucleotides. Additional evidence is provided from more recent calcium-imaging and patch-clamp studies, both showing that an ionotropic type receptor on rodent taste cells mediates glutamate-induced depolarization. Evidence for the second mechanism derives from studies that located the message for an metabotropic-type (mGluR4) receptor to rat taste buds, and from whole-cell patch-clamp recordings that revealed sustained cellular conductances to glutamate and an mGluR4 agonist. It appears likely that both mechanisms are involved in umami taste transduction, suggesting the possibility that reception and transduction of the umami signal constitute a collective property of a number of cells within the taste bud. PMID- 10736356 TI - Intake of umami-tasting solutions by mice: a genetic analysis. AB - In two-bottle preference tests with water and solutions of monosodium glutamate (MSG) and inosine-5'-monophosphate (IMP), mice from the C57BL/6ByJ inbred strain consumed more and had higher preferences for these solutions compared with mice from the 129/J strain. The C57BL/6ByJ mice consumed 300 mmol/L MSG in large amounts, which were comparable to intakes of highly preferred solutions of sweeteners. The strain differences in voluntary consumption of 300 mmol/L MSG depended at least in part on postingestive effects because prior experience with MSG influenced the expression of the strain difference in MSG acceptance. The strain difference in MSG acceptance was in the opposite direction to the strain difference in NaCl acceptance and was not affected by previous consumption of saccharin. Although the C57BL/6ByJ mice had higher avidity for both MSG and sweeteners than did the 129/J mice, there was no correlation between preferences for these solutions in the second hybrid generation (F(2)) derived from these two strains. Thus, the strain differences in MSG acceptance are not related to the strain differences in salty or sweet taste responsiveness and most likely represent specific umami taste responsiveness. High acceptance of MSG solutions by the C57BL/6ByJ mice was inherited as a recessive trait in the F(2) generation. Further genetic linkage analyses using the F(2) hybrids are being conducted to map chromosomal locations of genes determining the strain difference in MSG acceptance. PMID- 10736358 TI - Chemosensory signal transduction in paramecium. AB - Paramecia are ciliated single-cell eukaryotic organisms that can respond to chemical cues in their environment. Glutamate is among those cues, which attract cells. We describe briefly here the following attributes of glutamate chemoresponse: 1) Cells are attracted to L-glutamate relative to KCl at high concentrations of glutamate. 2) There are at least two specific, relatively low affinity glutamate binding sites on the cell surface. Glutamate can be displaced from only one of the binding sites by inosine monophosphate (IMP), and quisqualate displaces glutamate from the second site, which is likely to be the glutamate receptor involved in attraction to glutamate. 3) IMP is a repellent and does not act synergistically with glutamate, whereas guanosine monophosphate (GMP) does. 4) Similarly, glutathione is an attractant, but glutamate and glutathione appear to use different transduction pathways. 5) Glutamate hyperpolarizes the cell. The ionic mechanism is not yet verified, but is likely to involve a K conductance. 6) Glutamate induces a rapid and robust increase in cAMP in the cell. Protein kinase A (PKA) is possibly involved in the transduction pathway because kinase inhibitors such as H7 and H8 inhibit glutamate response, but do not affect responses to other attractants, such as acetate and ammonium. Activation of PKA by the rapid rise in cAMP may sustain the hyperpolarization phosphorylation and activation of the plasma membrane calcium pump. 7) Candidate glutamate binding proteins are being identified among the cell surface proteins with the use of affinity chromatography. PMID- 10736359 TI - Responses to umami substances in taste bud cells innervated by the chorda tympani and glossopharyngeal nerves. AB - The chorda tympani (CT) and glossopharyngeal (GL) nerves of several mammalian species respond differently to umami substances (US) such as monosodium glutamate (MSG), disodium 5'-inosinate (IMP) and disodium 5'-guanylate (GMP). In mice and rhesus monkeys, responses to US are greater in the GL than the CT nerve, with the GL nerve containing larger numbers of MSG-sensitive fibers. Gurmarin, a sweet response inhibitor, suppresses the mouse CT responses to the mixture of MSG and IMP to approximately 65% of control levels but not to the metabotropic and ionotropic glutamate agonists 2-amino-4-phophonobutyrate and N-methyl-D aspartate. Gurmarin does not inhibit any taste responses in the GL. In mice, CT responses to MSG may be masked by their greater sensitivity to sodium ions. Calcium imaging studies demonstrate that some mouse taste cells isolated from the fungiform papilla innervated by the CT respond selectively (as indicated by a rise in intracellular Ca(2+) concentrations) to MSG and/or IMP or GMP. These MSG responses are not suppressed notably by reducing the Ca(2+) concentration of the stimulus solution, suggesting that the observed Ca(2+) release is from intracellular stores. Measurements of second messengers in the mouse fungiform papilla have revealed consistently that MSG elicits increases in both inositol 1,4,5-trisphosphate and adenosine 3', 5'-cyclic monophosphate levels. Together, these results suggest that US may stimulate two different transduction mechanisms in the fungiform papilla. They also suggest that gurmarin-insensitive components of receptors for US, including metabotropic and ionotropic glutamate receptors, may be commonly involved in transduction for umami taste in taste cells on both anterior and posterior parts of the tongue. PMID- 10736360 TI - Hypothalamic and amygdalar neuronal responses to various tastant solutions during ingestive behavior in rats. AB - The forebrain, including the amygdala (AM) and hypothalamus, may be a higher brain center that modulates the activity of a brainstem neural system that influences ingestive behavior via descending projections. In this study, to elucidate the characteristics of sensory information processing in the forebrain in relation to this putative connection, we recorded neuronal activity in the AM and hypothalamus [lateral hypothalamic area (LHA), medial hypothalamic area (MHA)] of rats during discrimination of conditioned sensory stimuli and the ingestion of various tastant solutions. Of 420 responsive AM neurons identified, 24 were taste responsive and located mainly in the central nucleus of the AM. Multivariate analyses of these taste neurons suggested that in the AM, taste quality is processed on the basis of palatability. In the hypothalamus, of 282 LHA and MHA neurons recorded, 144 responded to one or more conditioned auditory stimuli and/or licking of one or more solutions. Stress, which is known to influence feeding behavior, increased the mean spontaneous activity of LHA neurons but decreased the mean spontaneous neuronal activity of MHA neurons. This pattern of changes in spontaneous neuronal activity correlated with alterations in feeding behavior during stress. Furthermore, the activity of both AM and LHA neurons was modulated flexibly during conditioned associative learning. Together, the data suggest that the activity of the AM and hypothalamic neurons is altered when animals must modulate ingestive behavior by learning a new stimulus associated with food and by being exposed to stress, suggesting that these forebrain areas are important modulators of the activity of a basic neural system in the brainstem that influences ingestive behavior. PMID- 10736361 TI - The representation of umami taste in the taste cortex. AB - To investigate the neural encoding of glutamate (umami) taste in the primate, recordings were made from taste-responsive neurons in the cortical taste areas in macaques. Most of the neurons were in the orbitofrontal cortex (secondary) taste area. First, it was shown that there is a representation of the taste of glutamate that is separate from the representation of the other prototypical tastants, sweet (glucose), salt (NaCl), bitter (quinine) and sour (HCl). Second, it was shown that single neurons that had their best responses to sodium glutamate also had good responses to glutamic acid. Third, it was shown that the responses of these neurons to the nucleotide umami tastant inosine 5' monophosphate were more correlated with their responses to monosodium glutamate than to any prototypical tastant. Fourth, concentration-response curves showed that concentrations of monosodium glutamate as low as 0.001 mol/L were just above threshold for some of these neurons. Fifth, some neurons in the orbitofrontal region which responded to monosodium glutamate and other food tastes, decreased their responses after feeding with monosodium glutamate to behavioral satiety, revealing a mechanism of satiety. In some cases, this reduction was sensory specific. Sixth, it was shown in psychophysical experiments in humans that the flavor of umami is strongest with a combination of corresponding taste and olfactory stimuli (e.g., monosodium glutamate and garlic odor). The hypothesis is proposed that part of the way in which glutamate works as a flavor enhancer is by acting in combination with corresponding food odors. The appropriate associations between the odor and the glutamate taste may be learned at least in part by olfactory to taste association learning in the primate orbitofrontal cortex. PMID- 10736362 TI - Mechanisms of umami taste preference and aversion in rats. AB - The influence of glutamate intake on growth and appetite, and the mechanisms of preference and aversion for monosodium L-glutamate (MSG) solutions were investigated in rats. Food intake, but not weight gain, was reduced significantly in rats fed a glutamate + glutamine (Glx)-deficient diet compared with those fed a control diet. Increase in the voluntary intake of Glx solutions was more rapid in rats fed the Glx-deficient diet. The preference and aversion for MSG solutions were distinctly different in 14 rat strains tested. Brown-Norway rats showed a strong preference for 60 mmol/L MSG and did not show aversive behavior toward solutions containing up to 600 mmol/L MSG. Sprague-Dawley (SD) rats showed a moderate preference for 60 mmol/L MSG and a weak aversion for MSG concentrations higher than 240 mmol/L; Long-Evans Agouti rats showed a moderate preference for 60 mmol/L MSG and a marked aversion for MSG concentrations higher than 120 mmol/L. Aversion was not due to nonspecific hyperosmotic effects. After section of gastric branches of the vagus nerve, MSG became aversive to SD rats. Aversion to 240 mmol/L MSG was reduced by 23-39% when combined with proline, alanine, glycine and glucose. These results show that the preference and aversion for MSG are determined by genetic factors, as well as vagus nerve function, and that the aversion to high MSG concentrations is reduced by the presence of other glucogenic amino acids and sugars. PMID- 10736363 TI - Reflex effects of oral, gastrointestinal and hepatoportal glutamate sensors on vagal nerve activity. AB - Glutamate sensors in the oral cavity, gastrointestinal canal and hepatoportal region are thought to function in the reflex regulation of vagal activity to the gastrointestinal tract and pancreas. In support of this notion, the findings summarized in this report demonstrate that the infusion of monosodium glutamate (MSG) into the stomach (150 mmol/L, 3 mL), duodenum (150 mmol/L, 3 mL) and portal vein (10 mmol/L, 0.1 mL) increases afferent activity in the vagal gastric, celiac and hepatic nerves, suggesting the existence of glutamate sensors in the gastric wall, intestinal wall and hepatoportal region. Further, oral, gastric and intestinal infusions of MSG (150 mmol/L, isotonic solution) and the infusion of MSG (10 mmol/L, 0.1 mL) into the portal vein resulted in reflex activation of the efferent gastric and pancreatic branches of the vagus. The intravenous injection of 10 mmol/L MSG (0.1 mL) also induced a reflex activation of the efferent discharges of the gastric branch of the vagus; however, in hepatic and celiac vagotomized rats, the intravenous injection of MSG (1 or 3mol/L, 1 mL) produced no effect on gastric vagal activity. The results of these experiments demonstrate the importance of the afferent nerve signals from visceral glutamate sensors in generating the reflex activation of gastrointestinal and pancreatic functions in response to MSG administration. PMID- 10736364 TI - Glutamine and glutamate exchange between the fetal liver and the placenta. AB - The transport and metabolism of glutamine (GLN) and glutamate (GLU) during fetal development exhibit unique characteristics that clearly emphasize the importance of the interaction between the placenta and the fetal liver. GLN is delivered into the fetal circulation at a rate that is the highest of all the amino acids. In contrast, approximately 90% of fetal plasma GLU is extracted by the placenta. Conversely, the fetal liver has a large net output of GLU and a net uptake of GLN. We have studied the fluxes of GLU and GLN into and out of the placenta and fetal liver, as well as their interconversion in these organs, during late gestation in sheep. In the fetus, 45% of GLN carbon taken up by the liver exits as GLU; indeed, the production of GLU from GLN is large, approximately 3.7 micromol/(min.kg fetus), and accounts for virtually all of the GLU produced in the fetus. In contrast, only 6% of GLU carbon is converted to GLN in the placenta; most of the fetal plasma GLU taken up by this organ is converted to CO(2). Remarkably, placental GLU uptake accounts for >60% of the fetal plasma GLU disposal rate. In some respects, the net output of GLU from the liver in fetuses replaces the net hepatic glucose output that is characteristic of postnatal life. We also examined GLN and GLU fluxes in pregnant sheep during either dexamethasone induced or spontaneous parturition. At parturition, a striking reduction in GLU output from the fetal liver occurred, leading to a fall in fetal arterial GLU concentrations and a marked decrease in placental GLU uptake. These changes were progressive as parturition advanced and correlated with a marked decrease in progesterone output from the pregnant uterus. PMID- 10736365 TI - Intestinal glutamate metabolism. AB - Although it is well known that the intestinal tract has a high metabolic rate, the substrates that are used to generate the necessary energy remain poorly established, especially in fed animals. Under fed conditions, the quantification of substrate used by the gut is complicated by the fact that potential oxidative precursors are supplied from both the diet and the arterial circulation. To circumvent this problem, and to approach the question of the compounds used to generate ATP in the gut, we combined measurements of portal nutrient balance with enteral and intravenous infusions of [U-(13)C]substrates. We studied rapidly growing piglets that were consuming diets based on whole-milk proteins. The results revealed that 95% of the dietary glutamate presented to the mucosa was metabolized in first pass and that of this, 50% was metabolized to CO(2). Dietary glucose was oxidized to a very limited extent, and arterial glutamine supplied no >15% of the CO(2) production by the portal-drained viscera. Glutamate was the single largest contributor to intestinal energy generation. The results also suggested that dietary glutamate appeared to be a specific precursor for the biosynthesis of glutathione, arginine and proline by the small intestinal mucosa. These studies imply that dietary glutamate has an important functional role in the gut. Furthermore, these functions are apparently different from those of arterial glutamine, the substrate that has received the most attention. PMID- 10736366 TI - Glutamine and glutamate metabolism across the liver sinusoid. AB - The liver shows net glutamine uptake after a protein-containing meal, during uncontrolled diabetes, sepsis and short-term starvation, but changes to net release during long-term starvation and metabolic acidosis. Some studies report a small net release of glutamate by the liver. The differential expression of glutamine synthetase (perivenous) and glutaminase (periportal) within the liver indicates that glutamine is used for urea synthesis in periportal cells, whereas glutamine synthesis serves to detoxify any residual ammonia in perivenous cells. Experiments in vivo suggest that changes in net hepatic glutamine balance are due predominantly to regulation of glutaminase activity, with the flux through glutamine synthetase being relatively constant. PMID- 10736367 TI - Glutamate, at the interface between amino acid and carbohydrate metabolism. AB - The liver is the major site of gluconeogenesis, the major organ of amino acid catabolism and the only organ with a complete urea cycle. These metabolic capabilities are related, and these relationships are best exemplified by an examination of the disposal of the daily protein load. Adults, ingesting a typical Western diet, will consume approximately 100 g protein/d; the great bulk of this is metabolized by the liver. Although textbooks suggest that these amino acids are oxidized in the liver, total oxidation cannot occur within the confines of hepatic oxygen uptake and ATP homeostasis. Rather, most amino acids are oxidized only partially in the liver, with the bulk of their carbon skeleton being converted to glucose. The nitrogen is converted to urea and, to a lesser extent, to glutamine. The integration of the urea cycle with gluconeogenesis ensures that the bulk of the reducing power (NADH) required in the cytosol for gluconeogenesis can be provided by ancillary reactions of the urea cycle. Glutamate is at the center of these metabolic events for three reasons. First, through the well-described transdeamination system involving aminotransferases and glutamate dehydrogenase, glutamate plays a key catalytic role in the removal of alpha-amino nitrogen from amino acids. Second, the "glutamate family" of amino acids (arginine, ornithine, proline, histidine and glutamine) require the conversion of these amino acids to glutamate for their metabolic disposal. Third, glutamate serves as substrate for the synthesis of N-acetylglutamate, an essential allosteric activator of carbamyl phosphate synthetase I, a key regulatory enzyme in the urea cycle. PMID- 10736368 TI - Glutamate, a window on liver intermediary metabolism. AB - In isotopic experiments, the labeling pattern of glutamate opens a window on hepatic metabolism, particularly the citric acid cycle, gluconeogenesis and fatty acid oxidation. This is because glutamate is in isotopic equilibrium with alpha ketoglutarate, whose labeling pattern is influenced by the following: 1) the contributions of glucose and fatty acids to acetyl-CoA, 2) the relative contributions of pyruvate carboxylase and pyruvate dehydrogenase to the entry of pyruvate carbon into the citric acid cycle, and 3) the rate of gluconeogenesis in relation to citric acid cycle activity. In humans and primates, hepatic glutamate can be sampled noninvasively via urinary phenylacetylglutamine, which is formed in liver from phenylacetate (a side product of phenylalanine catabolism) and glutamine (which equilibrates with liver glutamate and alpha-ketoglutarate). The (14)C- or (13)C-labeling pattern of the glutamate moiety of phenylacetylglutamine can be measured by sequential degradations to (14)CO(2), gas chromatography-mass spectrometry or nuclear magnetic resonance (NMR). When phenylacetylglutamine is labeled from singly labeled [(14)C]- or [(13)C]substrates, relative metabolic rates can be computed from the labeling pattern using Landau's model. In diabetic patients infused with [3-(13)C]pyruvate, the noninvasive sampling of hepatic glutamate via phenylacetylglutamine allows one to test the degree of liver insulinization via the (pyruvate carboxylase)/(pyruvate dehydrogenase) activity ratio. This ratio regulates gluconeogenesis in part. Its measurement may allow the identification of patients who might benefit from the intraperitoneal administration of insulin, or from recently developed antidiabetic drugs. PMID- 10736369 TI - Hormonal control of renal and systemic glutamine metabolism. AB - Glutamine is the most abundant free amino acid in the human body. Recent studies indicate that it may be an important vehicle for interorgan nitrogen and carbon transport. However, relatively little is known about hormonal factors regulating its metabolism in humans. We review here our recent work on the effects of insulin, glucagon and epinephrine on plasma glutamine kinetics and its conversion to glucose by liver and kidney. PMID- 10736370 TI - Circadian variations in plasma and erythrocyte glutamate concentrations in adult men consuming a diet with and without added monosodium glutamate. AB - This study evaluated the effect of monosodium glutamate (MSG) ingestion as a component of the diet on the 24-h variations in plasma and whole-blood glutamate (GLU) concentrations in healthy adult men. In the first arm of the study, subjects were given test meals without added MSG for 3 d. Protein and energy intakes of the subjects were 1.5 g and 40 kcal/(kg body weight.d), respectively. On d 3, blood samples were collected over the 24-h period. One week later, the same protocol was repeated, except that 100 mg/(kg body weight.d) MSG was added to the meals (15, 40 and 45 mg/kg body weight to breakfast, lunch and dinner, respectively). Both plasma and whole-blood samples were analyzed for free amino acids. Unlike large neutral amino acids, which experienced high peak plasma concentrations at 2100-2300 h, the circadian variations in plasma GLU concentrations were small, varying between 33 and 48 micromol/L on days in which no MSG was fed, and between 32 and 53 micromol/L on days in which MSG was added to the meals. In both trials, plasma GLU concentration increased (P < 0.01) after lunch and dinner, and decreased early in the morning (P < 0.05). Calculated erythrocyte GLU concentrations varied between 500 and 640 micromol/L, with or without MSG addition to the meals. The rather low plasma GLU concentrations over the 24-h period, despite high dietary intake of MSG, indicate that dietary MSG is metabolized very rapidly. PMID- 10736371 TI - Plasma amino acid patterns and visceral protein status in users and nonusers of monosodium glutamate. AB - Free amino acids in plasma and total protein, albumin, transferrin and retinol binding protein (RBP) in serum were determined in two groups of healthy adults consisting of 10 female nonusers and 10 female users of monosodium glutamate (MSG). Users or nonusers of MSG were those consuming or not consuming MSG regularly at their homes for at least 1 y. On the bases of body mass index and serum protein concentrations, each of the two groups appeared to have an adequate protein-energy status. Fasting plasma glutamate concentrations in female nonusers and users of MSG were 22.4 +/- 3.2 and 21.8 +/- 2.0 nmol/mL (means +/- SEM), respectively; these values were not significantly different. These findings indicate that there is no glutamate accumulation in the plasma of MSG users and imply the safety of long-term MSG intake. PMID- 10736372 TI - Glutamate as a neurotransmitter in the brain: review of physiology and pathology. AB - Glutamate is the principal excitatory neurotransmitter in brain. Our knowledge of the glutamatergic synapse has advanced enormously in the last 10 years, primarily through application of molecular biological techniques to the study of glutamate receptors and transporters. There are three families of ionotropic receptors with intrinsic cation permeable channels [N-methyl-D-aspartate (NMDA), alpha-amino-3 hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate]. There are three groups of metabotropic, G protein-coupled glutamate receptors (mGluR) that modify neuronal and glial excitability through G protein subunits acting on membrane ion channels and second messengers such as diacylglycerol and cAMP. There are also two glial glutamate transporters and three neuronal transporters in the brain. Glutamate is the most abundant amino acid in the diet. There is no evidence for brain damage in humans resulting from dietary glutamate. A kainate analog, domoate, is sometimes ingested accidentally in blue mussels; this potent toxin causes limbic seizures, which can lead to hippocampal and related pathology and amnesia. Endogenous glutamate, by activating NMDA, AMPA or mGluR1 receptors, may contribute to the brain damage occurring acutely after status epilepticus, cerebral ischemia or traumatic brain injury. It may also contribute to chronic neurodegeneration in such disorders as amyotrophic lateral sclerosis and Huntington's chorea. In animal models of cerebral ischemia and traumatic brain injury, NMDA and AMPA receptor antagonists protect against acute brain damage and delayed behavioral deficits. Such compounds are undergoing testing in humans, but therapeutic efficacy has yet to be established. Other clinical conditions that may respond to drugs acting on glutamatergic transmission include epilepsy, amnesia, anxiety, hyperalgesia and psychosis. PMID- 10736373 TI - Transport of glutamate and other amino acids at the blood-brain barrier. AB - In most regions of the brain, the uptake of glutamate and other anionic excitatory amino acids from the circulation is limited by the blood-brain barrier (BBB). In most animals, the BBB is formed by the brain vascular endothelium, which contains cells that are joined by multiple bands of tight junctions. These junctions effectively close off diffusion through intercellular pores; as a result, most solutes cross the BBB either by diffusing across the lipoid endothelial cell membranes or by being transported across by specific carriers. Glutamate transport at the BBB has been studied by both in vitro cell uptake assays and in vivo perfusion methods. The results demonstrate that at physiologic plasma concentrations, glutamate flux from plasma into brain is mediated by a high affinity transport system at the BBB. Efflux from brain back into plasma appears to be driven in large part by a sodium-dependent active transport system at the capillary abluminal membrane. Glutamate concentration in brain interstitial fluid is only a fraction of that of plasma and is maintained fairly independently of small fluctuations in plasma concentration. Restricted brain passage is also observed for several excitatory glutamate analogs, including domoic acid and kynurenic acid. In summary, the BBB is one component of a regulatory system that helps maintain brain interstitial fluid glutamate concentration independently of the circulation. PMID- 10736374 TI - Brain uptake of glutamate: food for thought. AB - Glutamate transporters in cells of the central nervous system play a key role, not only in providing glutamate for metabolic and protein synthesis purposes, but also in terminating glutamate's synaptic actions and keeping the extracellular glutamate concentration below levels that cause neuronal death. Recent advances in our understanding of how glutamate transport is powered allow a prediction of how glutamate transport will fail in stroke, releasing excess glutamate that triggers the death of neurons, thereby causing mental and physical handicap. PMID- 10736375 TI - Compartmentation of brain glutamate metabolism in neurons and glia. AB - Intrasynaptic [glutamate] must be kept low in order to maximize the signal-to noise ratio after the release of transmitter glutamate. This is accomplished by rapid uptake of glutamate into astrocytes, which convert glutamate into glutamine. The latter then is released to neurons, which, via mitochondrial glutaminase, form the glutamate that is used for neurotransmission. This pattern of metabolic compartmentation is the "glutamate-glutamine cycle." This model is subject to the following two important qualifications: 1) brain avidly oxidizes glutamate via aspartate aminotransferase; and 2) because almost no glutamate crosses from blood to brain, it must be synthesized in the central nervous system (CNS). The primary source of glutamate carbon is glucose, and a major source of glutamate nitrogen is the branched-chain amino acids, which are transported rapidly into the CNS. This arrangement accomplishes the following: 1) maintenance of low external [glutamate], thereby maximizing signal-to-noise ratio upon depolarization; 2) the replenishing of the neuronal glutamate pool; 3) the "trafficking" of glutamate through the extracellular fluid in a nonneuroactive form (glutamine); 4) the importation of amino groups from blood, thus maintaining brain nitrogen homeostasis; and 5) the oxidation of glutamate/glutamine, a process that confers an additional level of control in terms of the regulation of brain glutamate, aspartate and gamma-aminobutyric acid. PMID- 10736376 TI - Neuroglial responses to elevated glutamate in the medial basal hypothalamus of the infant mouse. AB - Elevated plasma glutamate can cause selective loss of neurons in the brains of infant mice. The arcuate nucleus-median eminence region exhibits the greatest sensitivity to glutamate while it undergoes developmental maturation during early postnatal life. To investigate glutamate-induced cellular responses, groups of nursing 7-d-old mice (n = 31-93) were given single subcutaneous injections of 0.1 0.5 mg monosodium glutamate (MSG)/g body wt or an equivalent volume (30-50 microL) of water vehicle (n = 93). Injection of 0.2 mg MSG/g body wt produced a 16-fold rise in plasma glutamate after 15 min (2.10 vs. 0. 122 mmol/L control) and was the lowest harmful dose tested. It not only induced injury of small bilateral groups of medial basal hypothalamic neurons at 5 h postinjection, but also enhanced their expression of the N-methyl-D-aspartate (NMDA)R1 glutamate receptor subunit. Higher dosages of 0.3-0.5 mg MSG/g body wt yielded dose-related increases in NMDAR1 staining intensity and larger numbers of damaged neurons within the ventromedial arcuate nucleus. Administration of the live-cell nuclear stain bis-benzimide (0.95 micromol/L) indicated that MSG accessed the entire brain (n = 20) and methylene blue (1.0 g/L) permeated extracellular spaces by 15 min postinjection (n = 19), before cell death was evident (0.75 mmol/L propidium iodide) from co-injected MSG. Immunostaining, which mimicked that for glial fibrillary acidic protein, suggested that glutamate was retained in tanycytes. We conclude that elevated plasma glutamate induces glutamate receptor expression during selective injury of ventromedial arcuate neurons and propose that by sequestering glutamate, tanycytes may amplify local concentrations and promote neuronal damage in infant mice. PMID- 10736377 TI - Peripheral glutamate receptors: molecular biology and role in taste sensation. AB - Glutamate is the most widespread excitatory neurotransmitter in the mammalian brain. Two classes of glutamate receptor have been cloned, the ionotropic (ligand gated ion channels) and the metabotropic (G protein-coupled receptors). Three subclasses of ionotropic glutamate receptors are known; they are named after selective agonists, i.e., alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), N-methyl-D-aspartate (NMDA) and kainate receptors. Fifteen functional subunits assemble together in heteromultimeric complexes to form these receptors as follows: GluR1-GluR4 for AMPA; GluR5-GluR7 and KA1-KA2 for kainate; and NR1, NR2A-NR2D and NR3 for NMDA receptors. Within a subclass, the subunit composition strongly influences the pharmacologic and biophysical properties of the receptors. The metabotropic glutamate receptors fall into the following three groups, each containing two or more individual receptor proteins: group I (mGluR1, mGluR5), group II (mGluR2, mGluR3), and group III (mGluR4, mGluR6, mGluR7 and mGluR8). In contrast to the ionotropic receptors, the metabotropic glutamate receptors appear to act as monomers or homodimers rather than heteromers. Messenger RNAs encoding several ionotropic subunits and a mGluR4-like receptor have been identified in taste buds. Although controversial, the evidence is consistent with an NMDA receptor serving as a primary taste transducer for monosodium glutamate (MSG), and a metabotropic glutamate receptor modulating the flavor-enhancing effect of MSG. Thus the neurotransmitter glutamate is intimately involved in the central processing of taste information. PMID- 10736378 TI - Glutamate and epilepsy. AB - Epileptic syndromes have very diverse primary causes, which may be genetic, developmental or acquired. In rodent models, altering glutamate receptor or glutamate transporter expression by knockout or knockdown procedures can induce or suppress epileptic seizures. Regardless of the primary cause, synaptically released glutamate acting on ionotropic and metabotropic receptors appears to play a major role in the initiation and spread of seizure activity. In rodent models of acquired epilepsy and in human temporal lobe epilepsy, there is evidence for enhanced functional efficacy of ionotropic N-methyl-D-aspartate (NMDA) and metabotropic (Group I) receptors. In animal models of epilepsy, antagonists acting at NMDA receptors or at Group I metabotropic receptors have potent anticonvulsant actions. PMID- 10736379 TI - Modulatory role of neuropeptides in seizures induced in rats by stimulation of glutamate receptors. AB - Stimulation of glutamate receptors has been reported to modulate the expression of neuropeptides and their receptors in neurons. On the other hand, neuropeptides are known to regulate the presynaptic glutamate release and neuronal responses to excitatory neurotransmission. This evidence indicates a functional interaction between glutamatergic and neuropeptidergic transmission in the central nervous system (CNS). In this report, we provide pharmacologic evidence in experimental models of seizures, suggesting that somatostatin (SRIF) and neuropeptide Y (NPY) are endogenous modulators of glutamate-mediated hyperexcitability in the CNS. Electroencephalographic (EEG) and behavioral seizures were induced in rats by intrahippocampal or systemic injection of kainic acid, a glutamate analog. The number of EEG seizures and their total duration were inhibited significantly by intracerebral application of a SRIF(1) receptor agonist. Similarly, kainate seizures were reduced by N[-2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl-D arginamide++ +] (BIBP 3226), a NPY Y(1) receptor antagonist. Enhanced seizure susceptibility to pentylentetrazol, ensuing in rats after a systemic administration of kainic acid, was reduced significantly by intracerebral application of RC 160, a SRIF(1) receptor agonist, or NPY 13-36, a Y(2)/Y(5) receptor agonist. This evidence suggests that neuropeptide analogs may be of value for controlling seizures and possibly in other pathologic conditions associated with excessive glutamate function. PMID- 10736380 TI - The safety evaluation of monosodium glutamate. AB - L-Glutamic acid and its ammonium, calcium, monosodium and potassium salts were evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1988. The Committee noted that intestinal and hepatic metabolism results in elevation of levels in systemic circulation only after extremely high doses given by gavage (>30mg/kg body weight). Ingestion of monosodium glutamate (MSG) was not associated with elevated levels in maternal milk, and glutamate did not readily pass the placental barrier. Human infants metabolized glutamate similarly to adults. Conventional toxicity studies using dietary administration of MSG in several species did not reveal any specific toxic or carcinogenic effects nor were there any adverse outcomes in reproduction and teratology studies. Attention was paid to central nervous system lesions produced in several species after parenteral administration of MSG or as a consequence of very high doses by gavage. Comparative studies indicated that the neonatal mouse was most sensitive to neuronal injury; older animals and other species (including primates) were less so. Blood levels of glutamate associated with lesions of the hypothalamus in the neonatal mouse were not approached in humans even after bolus doses of 10 g MSG in drinking water. Because human studies failed to confirm an involvement of MSG in "Chinese Restaurant Syndrome" or other idiosyncratic intolerance, the JECFA allocated an "acceptable daily intake (ADI) not specified" to glutamic acid and its salts. No additional risk to infants was indicated. The Scientific Committee for Food (SCF) of the European Commission reached a similar evaluation in 1991. The conclusions of a subsequent review by the Federation of American Societies for Experimental Biology (FASEB) and the Federal Drug Administration (FDA) did not discount the existence of a sensitive subpopulation but otherwise concurred with the safety evaluation of JECFA and the SCF. PMID- 10736381 TI - Pituitary hormone secretion in normal male humans: acute responses to a large, oral dose of monosodium glutamate. AB - Numerous studies have shown that the administration of a glutamate receptor agonist or a high dose of glutamate stimulates pituitary hormone secretion in animals. However, only a single human study has reported that an oral load of glutamic acid induced the secretion of prolactin and probably adrenocorticotropic hormone (ACTH) (but not other pituitary hormones). Because of glutamate's use in foods as monosodium glutamate (MSG), a flavoring agent, and the limited amount of human data, we studied the effect of a large oral dose of MSG in humans on the secretion of prolactin and other pituitary hormones. Fasting male subjects bearing venous catheters received on separate days each of the following four treatments: a vehicle, MSG (12.7 g), a high protein meal (a physiologic stimulus of prolactin secretion) by mouth, or an intravenous infusion of thyrotropin releasing hormone (TRH, a pharmacologic stimulus of prolactin secretion). Plasma hormone responses were quantitated by RIA at 20-min intervals for 4 h. The protein meal induced a modest increase and TRH infusion a substantial increase in plasma prolactin, whereas MSG ingestion did not. MSG ingestion also did not raise the plasma concentrations of any of the other pituitary hormones measured (luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone, growth hormone) or of cortisol. Ingestion of MSG raised plasma glutamate concentrations 11-fold; the protein meal did not raise plasma glutamate. The results demonstrate that MSG ingestion in humans does not modify anterior pituitary hormone secretion. One implication is that diet-derived glutamate may not penetrate into hypothalamic regions controlling anterior pituitary function. PMID- 10736382 TI - Review of alleged reaction to monosodium glutamate and outcome of a multicenter double-blind placebo-controlled study. AB - Monosodium glutamate (MSG) has a long history of use in foods as a flavor enhancer. In the United States, the Food and Drug Administration has classified MSG as generally recognized as safe (GRAS). Nevertheless, there is an ongoing debate exists concerning whether MSG causes any of the alleged reactions. A complex of symptoms after ingestion of a Chinese meal was first described in 1968. MSG was suggested to trigger these symptoms, which were referred to collectively as Chinese Restaurant Syndrome. Numerous reports, most of them anecdotal, were published after the original observation. Since then, clinical studies have been performed by many groups, with varying degrees of rigor in experimental design ranging from uncontrolled open challenges to double-blind, placebo controlled (DBPC) studies. Challenges in subjects who reported adverse reactions to MSG have included relatively few subjects and have failed to show significant reactions to MSG. Results of surveys and of clinical challenges with MSG in the general population reveal no evidence of untoward effects. We recently conducted a multicenter DBPC challenge study in 130 subjects (the largest to date) to analyze the response of subjects who report symptoms from ingesting MSG. The results suggest that large doses of MSG given without food may elicit more symptoms than a placebo in individuals who believe that they react adversely to MSG. However, the frequency of the responses was low and the responses reported were inconsistent and were not reproducible. The responses were not observed when MSG was given with food. PMID- 10736383 TI - Additive-induced urticaria: experience with monosodium glutamate (MSG). AB - In patients with chronic urticaria, the incidence of reactions to any additives, including monosodium glutamate (MSG), is unknown. Although many studies have investigated the association of additives and urticaria, most have been poorly designed. This study sought to determine the prevalence of reactions to additives, including MSG, in patients with chronic urticaria using a rigorous protocol. We studied 65 subjects (44 women, 21 men; ages 14-67). All had urticaria for >6 wk without discernible etiology. Subjects with active urticaria were studied while they were taking the lowest effective dose of antihistamine. Screening challenges to the 11 additives most commonly associated with exacerbations of chronic idiopathic urticaria were performed in a single-blind fashion. The dose of MSG given was 2500 mg. Skin scores were obtained to determine a positive reaction in an objective manner. Subjects with a positive screening challenge were rechallenged (at least 2 wk later) with a double-blind, placebo-controlled protocol as in-patients in our General Clinical Research Center. Two subjects had positive single-blind, placebo-controlled challenges, but neither had a positive double-blind, placebo-controlled challenge. We conclude, with 95% confidence, that MSG is an unusual (<3% at most) exacerbant of chronic idiopathic urticaria. PMID- 10736384 TI - Monosodium glutamate and asthma. AB - Allen et al. (1987) conducted oral monosodium glutamate (MSG) challenges with 32 asthmatic volunteers and reported that 14 reacted to MSG. Another study by Moneret-Vautrin (1987) also reported MSG-induced asthma attacks in 2 of 30 asthmatic patients. Four additional studies have been conducted and none has confirmed the results of the above authors. These studies, by Schwartzstein et al. (1987), Germano (1991), Woods et al. (1998) and Woessner et al. (1999), challenged a total of 45 patients who gave a history of asthma attacks in oriental restaurants. None of these patients experienced asthmatic reactions after ingesting MSG (one-sided confidence interval of 0-0.066). Another 109 asthmatic patients, without a history of asthma in oriental restaurants, also did not react to ingestion of MSG (one-sided confidence interval of 0-0.027). With a confidence interval < 0.05 there is a >95% probability that MSG history-negative asthmatic patients are not sensitive to MSG. For the MSG history-positive asthmatics, 45 patients, in well-performed studies, underwent negative challenges to MSG, contrasting with two studies reporting positive challenges. Allen et al. (1987) and Moneret-Vautrin (1987), who reported positive MSG challenge results, performed studies with the following characteristics: 1) single blinded, conducted after discontinuing essential antiasthma medications; 2) used effort dependent peak expiratory flow rate measurement of lung function; 3) added AM bronchodilators in some patients; 4) ignored wandering baselines on the placebo challenge days; and 5) conducted some challenges in the AM and some at night. In summary, the existence of MSG-induced asthma, even in history-positive patients, has not been established conclusively. PMID- 10736386 TI - Second International Conference on Glutamate: conference summary. PMID- 10736385 TI - The administration to Indonesians of monosodium L-glutamate in Indonesian foods: an assessment of adverse reactions in a randomized double-blind, crossover, placebo-controlled study. AB - Monosodium L-glutamate (MSG) has been suggested to cause postprandial symptoms after the ingestion of Chinese or oriental meals. Therefore, we examined whether such symptoms could be elicited in Indonesians ingesting levels of MSG typically found in Indonesian cuisine. Healthy volunteers (n = 52) were treated with capsules of placebo or MSG (1.5 and 3.0 g/person) as part of a standardized Indonesian breakfast. The study used a rigorous, randomized, double-blind, crossover design. The occurrence of symptoms after MSG ingestion did not differ from that after consumption of the placebo. PMID- 10736387 TI - Anatomy and biomechanics of the shoulder. AB - The anatomies and biomechanics of the glenohumeral joint and the scapulothoracic articulation are the subjects of this article. The anatomies of bones, joints, ligaments, and muscles are described in detail, and current biomechanical concepts concerning motion, stability, and force are presented. Morphologic and biomechanical changes in pathologic conditions briefly are described. PMID- 10736389 TI - Conservative management of sternoclavicular injuries. AB - Injuries to the sternoclavicular joint are rare because of its strong ligamentous support. Because of the strong forces involved and the proximity of the joint to the great vessels and other mediastinal structures, however, sternoclavicular injuries can be very serious and potentially life threatening. Sternoclavicular injuries include traumatic sprains and dislocations, atraumatic spontaneous dislocations, and epiphyseal fractures in patients under 25 years of age. Diagnosis is made by history, physical examination, and radiographic studies such as the CT scan. Conservative management consists of benign neglect and closed or percutaneous reduction and immobilization. If the injury is treated acutely, conservative management often produces good long-term results. PMID- 10736388 TI - Acromioclavicular joint injuries. AB - Injuries to the acromioclavicular joint are common, and most can be treated nonoperatively. Appropriate treatment depends on accurate diagnosis and appreciation of the classification of these various injuries. Descriptions of the commoner acromioclavicular joint injuries, details of the nonoperative treatment, and indications for surgical treatment are reviewed. PMID- 10736390 TI - Fractures about the shoulder: conservative management. AB - Fractures about the shoulder are a frequent occurrence. The clavicle and the proximal humerus are fractured much more often than the scapula. Appropriate diagnosis of these injuries and any associated injuries is essential. The vast majority of these fractures may be treated conservatively with good functional results. PMID- 10736391 TI - Shoulder dislocations in the young patient. AB - Shoulder dislocations are often seen in young patients, particularly those patients involved in collision sports. A conservative approach to treating these injuries includes protection from early re-injury, rehabilitation, and gradual return to athletics and use. It is important to recognize associated injuries such as fractures and nerve injuries. Surgical management is considered early for the small subgroup with the highest risk of recurrence: young athletes suffering a first traumatic dislocation returning to competitive athletics. Late operative repair is reserved for those who fail extensive nonoperative management. PMID- 10736393 TI - The painful shoulder in the throwing athlete. AB - Successful nonoperative treatment of shoulder injuries in the throwing athlete requires a team approach. The physician, physical therapist, and athletic trainer work together with the athlete to determine a diagnosis and design a treatment protocol. Injury recovery requires restoration of muscle strength and balance followed by a careful return to a throwing program. PMID- 10736392 TI - Shoulder dislocations in patients older than 40 years of age. AB - Shoulder dislocations in patients over 40 years of age are common and are frequently associated with serious injuries to adjacent structures. Associated injuries such as brachial plexus injury, rotator cuff tear, axillary artery injury, fractures about the shoulder, and recurrent dislocation can make shoulder dislocations challenging problems for the clinician. Early diagnosis and treatment of associated injuries and treatment complications are the mainstays in prevention of morbidity and, even, mortality. PMID- 10736394 TI - The painful shoulder in the swimming athlete. AB - Given the popularity of swimming and the high risk of injury associated with the sport, many clinicians come into contact with the swimmer's shoulder. This article describes the mechanism of injury, diagnostic tools, and subtle signs of injury for swimmer's shoulder. It focuses on conservative treatment for the injury, including methods for stretching and strengthening and eliminating acute inflammation. PMID- 10736395 TI - Posterior shoulder instability. AB - Understanding the anatomic restraints to posterior shoulder instability and the resulting pathophysiology helps the treating physician make a correct diagnosis and formulate an appropriate treatment plan. A nonoperative program directed at reducing pain and increasing stability through comprehensive shoulder strengthening methods has generally been successful in treating recurrent posterior shoulder subluxation. Surgical options for treatment are reserved for those patients who fail to recognize improvement after six months of therapy and have no evidence of a psychological disturbance as the cause of their posterior instability. PMID- 10736396 TI - Multidirectional instability of the glenohumeral joint. AB - The concept of multidirectional instability (MDI) was introduced as an important clinical entity in 1980. Previously, it had received little mention in the literature and was not considered to be clinically relevant. MDI is a symptomatic glenohumeral subluxation or dislocation occurring in more than one direction. The basic pathology of this condition is a loose and redundant joint capsule. Most patients with MDI can be treated successfully by conservative methods, such as patient education, a shoulder girdle strengthening program, or modification of the patient's routine activity. PMID- 10736397 TI - Shoulder impingement. AB - Understanding the pathophysiology and treatment of rotator cuff disorders is the key to understanding all other aspects of shoulder rehabilitation. Impingement rehabilitation focuses on strengthening the humeral head depressors, while ignoring the deltoid and supraspinatus muscles. Later treatment includes specific retraining of scapular balancing muscles. The final phase of treatment includes strengthening the prime humeral movers in positions that avoid further stress to the injured rotator cuff tendons and, last of all, specifically strengthening the supraspinatus muscle. PMID- 10736398 TI - Nonoperative treatment of rotator cuff tears. AB - Rotator cuff tears and subacromial impingement are second only to acromioclavicular joint disorders as the most common causes of shoulder pain. Although most orthopedic surgeons are willing to initially treat shoulder impingement syndrome conservatively, they are reluctant to manage rotator cuff tears-especially full-thickness tears-nonoperatively. The purpose of this article is to explain the biomechanical rationale of nonoperative treatment, review the literature pertaining to nonoperative treatment of full-thickness rotator cuff tears, and describe a nonoperative treatment program. PMID- 10736399 TI - Tendon ruptures about the shoulder. AB - Although degeneration and strain of musculotendinous structures are frequent causes of shoulder pain, sudden violent injuries in young individuals can lead to complete musculotendinous ruptures. Some of these conditions lend themselves well to nonoperative treatment. This article will enable the physician to determine the clinical diagnosis and formulate a treatment plan for each patient as an individual. Some patients may be satisfied with an accurate diagnosis only, while others demand more aggressive operative care. PMID- 10736400 TI - Nerve lesions about the shoulder. AB - The shoulder is the most mobile joint in the body. Because it serves as a way station for the nerves supplying the upper limb, it creates a potential for nerve lesions that may be caused or significantly influenced by the complex dynamics of the shoulder girdle. This article presents the most commonly encountered lesions as well as an algorithm for their diagnosis and treatment. PMID- 10736401 TI - Definitions of fungal rhinosinusitis. AB - Classification of fungal rhinosinusitis is important for the accurate prediction of prognosis and direct therapy. The most important distinction is between invasive and noninvasive fungal rhinosinusitis. Within the invasive division are acute invasive and chronic invasive (granulomatous and nongranulomatous forms) rhinosinusitis. Within the noninvasive division are saprophytic colonization, fungus balls, and allergic fungal rhinosinusitis. This article briefly outlines the definition and management of each of these manifestations. PMID- 10736402 TI - Overview of basic medical mycology. AB - To introduce the general mycologic aspects of fungal rhinosinusitis, this article reviews, in brief, the biology of fungi and the principles of fungal pathogenesis. A glossary of frequently used mycologic terms is provided. The basis of fungal classification and strategies for the diagnosis of mycotic infections are summarized. The morphologic criteria for the identification of the common etiologic agents of rhinosinusitis are presented. PMID- 10736403 TI - Histopathology of fungal rhinosinusitis. AB - Treatments for the various kinds of fungal rhinosinusitis are fundamentally different, and it is essential to obtain an accurate diagnosis of the particular syndrome in a given case. Microscopic examination of specimens is the most definite and rapid means of establishing a diagnosis of fungal rhinosinusitis. This article compares the utility of various staining and microscopy methods, presents two keys for the algorithmic evaluation of fungal cells as seen in clinical specimens, discusses practical aspects of fungal spore formation within clinical specimens, and reevaluates the role of Candida albicans in rhinosinus disease. PMID- 10736404 TI - Agents for treatment of invasive fungal infections. AB - The incidence of fungal infections continues to rise as the population of immunocompromised individuals increases. Despite the enlarging numbers of infections, there are only a few antifungal agents for treatment of deep-seated, invasive infections. These agents include amphotericin B, flucytosine, terbinafine, and several azoles. Progress has been made in understanding the role of these agents in a variety of infections and this article examines in detail these agents and their prophylactic, empiric, and therapeutic uses in invasive mycoses. This article focuses on general concepts of antifungal therapies and provides a detailed review of each antifungal agent available for treatment of deep-seated mycoses. PMID- 10736405 TI - Prevention and prophylaxis of invasive fungal sinusitis in the immunocompromised patient. AB - Fungal infections are a leading cause of morbidity and mortality among immunocompromised patients. Invasive fungal sinusitis is a devastating complication of immunosuppression. Treatment options are limited and often ineffective, making prevention important. Measures to decrease environmental exposure, indications for antifungal prophylaxis, and limitations of current regimens are discussed. PMID- 10736406 TI - Diagnosis and management of rhinosinusitis before scheduled immunosuppression: a schematic approach to the prevention of acute fungal rhinosinusitis. AB - Acute (invasive) fungal rhinosinusitis is an aggressive and potentially lethal complication of insulin dependent diabetes, as well as immunosuppression from chemotherapy and bone marrow transplant. The goal of this article is to describe methods to help with the diagnosis and treatment of sinusitis prior to developing this state of immune-compromise. This will help eliminate a significant risk factor for these patients that can lead to serious morbidity and ultimately mortality. PMID- 10736407 TI - An algorithmic approach to the diagnosis and management of invasive fungal rhinosinusitis in the immunocompromised patient. AB - Acute invasive fungal rhinosinusitis is a difficult disorder to diagnose and treat. A systematic approach to the susceptible patient, however, leads to an earlier diagnosis with improved survival. Early evaluation with rigid nasal endoscopy with frozen section biopsy of suspicious lesions or the middle turbinate should be considered in the high-risk population. Complete surgical resection and the reversal of neutropenia appear to be critical elements in achieving a successful outcome in patients with invasive fungal rhinosinusitis. PMID- 10736408 TI - Invasive fungal sinusitis in the acquired immunodeficiency syndrome. AB - Invasive fungal sinusitis can present as either an indolent or fulminant process that primarily affects immunocompromised individuals. In this article, the clinical characteristics of four cases of invasive fungal sinusitis in patients with AIDS are analyzed and 22 additional previously reported cases in the literature are reviewed. In addition to HIV infection, other variables common to these cases include facial pain or headache out of proportion to clinical or radiographic findings, CD4 lymphocyte count less than 50 cells/mm(3), absolute neutrophil count less than 1,000 cells/mm(3), subtle radiographic evidence suggesting invasion and an indolent clinical course of the invasive infection. The most common pathogen detected was Aspergillus fumigatus. Maintaining a high index of suspicion, critically assessing these clinical findings, and prudently reviewing CT scans may facilitate early diagnosis and prompt intervention in these patients. PMID- 10736409 TI - Mucormycosis of the nose and paranasal sinuses. AB - Rhinocerebral mucormycosis is an invasive fungal infection initiated in the paranasal sinuses that frequently progresses to orbital and brain involvement. If recognized early, involvement is limited to the nasal cavity and paranasal sinuses. Diabetics in poor control are at greatest risk, however, any immunocompromised individual may be infected. The mainstays of therapy are reversal of immunosuppression, systemic amphortericin B, and surgical debridement. Survival has improved dramatically, yet deaths still occur if the infection is not recognized and not treated early in its course or if the source of immunocompromise is not reversible. Several case examples illustrate the clinical course of this unusual, but potentially fatal, fungal infection. Taxonomy, clinical presentation, diagnosis, and management of mucormycosis of the paranasal sinuses are reviewed in detail. PMID- 10736410 TI - Unusual fungal pathogens in fungal rhinosinusitis. AB - Approximately 300 fungal species are known to cause mycotic disease in humans and other animals. More than 50 of these species are documented as agents of rhinosinusitis. Most such infections are caused by species of Aspergillus, Rhizopus, Alternaria, Bipolaris, and Curvularia. A growing number, however, has been attributed to lesser known fungi. Here, 38 fungi that are unusual causes of rhinosinusitis are tabulated and referenced in conjunction with their associated symptoms. PMID- 10736411 TI - Chronic invasive fungal rhinosinusitis. AB - Chronic invasive fungal rhinosinusitis is an increasingly recognized, but inadequately characterized, disease entity which is separate and distinct from acute fulminant invasive fungal sinusitis and allergic fungal sinusitis. Chronic invasive fungal rhinosinusitis is divided into granulomatous and nongranulomatous subtypes based on histopathology, but the clinical distinction between the two subtypes is not clear. Current management includes varying degrees of surgical debridement and a prolonged course of antifungal agents. A protracted clinical course with recurrence after treatment is common. PMID- 10736412 TI - Fungus balls of the paranasal sinuses. AB - Fungus balls of the paranasal sinuses represent a noninvasive manifestation of fungal sinusitis. Patients are immunocompetent but no more allergic than the general population. There is little tissue reaction to the tangled mat of hyphae. If the patient becomes immunocompromised, then the fungus ball may become invasive, as illustrated by an included case report. One hundred sixty-three additional cases of patients with paranasal sinus fungus balls are reviewed from the literature. PMID- 10736413 TI - Allergic fungal rhinosinusitis: pathophysiology, epidemiology, and diagnosis. AB - Allergic fungal rhinosinusitis (AFRS) is believed to have a cause similar to allergic bronchopulmonary aspergillosis (ABPA). Both are thought to be mediated by both type I (IgE) and type III (IgE-antigen immune complexes) Gell and Coombs reactions. ABPA patients also exhibit unique characteristics, such as HLA-DR2 or HLA-DR5 genotypes, and elevated suppressor T cell activity. While the pathophysiology of AFRS is similar histopathologically, similar immunologic studies have not been as well documented. Most cases of AFRS involve dematiaceous fungi, rather than Aspergillus. A suggested laboratory work-up for the disease is presented. PMID- 10736414 TI - Allergic fungal rhinosinusitis: surgical management. AB - Allergic fungal sinusitis is a newly characterized disease entity that has commanded a great deal of interest over the past 2 decades. As more information is gathered about its underlying etiology, clinical presentation, and response to therapy, the treatment of allergic fungal sinusitis is becoming more refined. Most current treatment protocols for allergic fungal sinusitis are based upon a combined surgical and medical approach. This article addresses pertinent surgical aspects as related to the management of allergic fungal sinusitis. PMID- 10736415 TI - Allergic fungal rhinosinusitis: perioperative management, prevention of recurrence, and role of steroids and antifungal agents. AB - Allergic fungal sinusitis can best be thought of as chronic fungal affectation (not an infection) of the sinuses to which the body's immune system hyperreacts, thereby creating significant inflammation, edema, obstruction, and polyposis. Currently it is felt that allergic fungal sinusitis requires both surgical and medical management. The effectiveness and required duration of the various medical treatments remain unknown and are under active investigation. Both surgical intervention and the use of systemic steroids are recommended in the treatment of allergic fungal sinusitis. Experience with surgical and medical management, follow-up of patients, and proposed treatment protocols are discussed. PMID- 10736416 TI - Allergic fungal sinusitis: the role of immunotherapy. AB - The role of surgery and anti-inflammatory therapy (such as corticosteroids) in the management of allergic fungal sinusitis is accepted fairly universally. Although once thought to be contraindicated in the treatment of allergic fungal sinusitis, specific immunotherapy with fungal antigens has been shown to be extremely beneficial to these patients, when combined with surgery and adjunctive medical management. Recurrences have been prevented and systemic corticosteroid requirements virtually eliminated in an experience that now spans over 4 years. PMID- 10736417 TI - Geographic variation in allergic fungal rhinosinusitis. AB - Allergic fungal rhinosinusitis (AFRS) has a worldwide distribution. This survey of 20 otolaryngologic practices throughout the United States confirmed a variation in the frequency of AFRS relative to endoscopic sinus procedures performed for all other diagnoses. The highest incidence occurred in Memphis, Tennessee at 23%, with three other southern practices reporting a frequency of at least 10%. In the northern locations the frequency ranged from 0 to 4%. No correlation with mould counts was demonstrated, possibly because of incomplete mould data relative to most of the surgical locations. PMID- 10736418 TI - Purinergic and pyrimidinergic receptors as potential drug targets. AB - In the last decade, the field of purinergic pharmacology has continued to grow as the complexity of the receptor families and the various enzymes involved in purine metabolism have been defined in molecular terms. A major theme that has emerged from these studies is the functional complexity of the interactions between P1 and P2 receptors, based upon the dynamic interrelationship between ATP and adenosine as extracellular signaling molecules. It is now clear that ATP and its degradation products (particularly ADP and adenosine) form a complex cascade for the regulation of cell-to-cell communication that can function to attenuate the consequences of tissue trauma (e.g. ischemia) that involve alterations in cellular energy charge and depletion of ATP stores. In addition to the P2 receptor family, alterations in cellular ATP stores can also affect the function of other receptors, e.g. K(ATP) channels, and mitochondrial function. The discovery of pyrimidine-preferring (UTP/UDP) P2Y receptors has also raised the possibility that the corresponding nucleoside, uracil, may function as a signaling molecule. PMID- 10736419 TI - Transport of cyclic AMP and synthetic analogs in the perfused rat liver. AB - The purpose of the present work was to investigate the transport of cyclic AMP (cAMP) and analogs in the rat liver. The experimental system was the isolated once-through perfused liver. Transport was measured by employing the multiple indicator dilution technique. The single-pass recovery of tracer [(32)P]cAMP was equal to 94.4 +/- 1. 4%; no significant extracellular transformation of cAMP occurred during a single passage. The unidirectional influx rates of dibutyryl cAMP were a saturable function of its concentration, with K(m) = 72.75 +/- 9.24 microM and V(max) = 0.464 +/- 0.026 micromol min(-1) (mL cellular space)(-1). The unidirectional influx rates of cAMP were much lower than those of dibutyryl-cAMP and were a linear function of the concentration (up to 100 microM). The transfer coefficient for influx (k(in)) was equal to 0.860 +/- 0.058 mL min(-1) (mL extracellular space)(-1). cAMP inhibited the influx of dibutyryl-cAMP; the IC(50) was 0.83 mM. The following series of increasing unidirectional influx rates was found: cAMP < monobutyryl-cAMP approximately 2-aza-epsilon-cAMP < rp-cAMPS approximately sp-cAMPS < 8-Br-cAMP approximately dibutyryl-cGMP approximately 8 Cl-cAMP < O-dibutyryl-cAMP. There was no precise correlation between the rates of influx of the various cyclic nucleotides and their lipophilicity. It was concluded that the penetration of cAMP and its analogs into the liver cells was a facilitated process. Lipophilicity was not the only factor determining the rate of transport. The transformation of dibutyryl-cAMP was limited by both transport and activity of the intracellular enzymic systems. The intracellular transformation of exogenous cAMP, however, was limited by the transport process. PMID- 10736420 TI - Changes in isoprenoid lipid synthesis by gemfibrozil and clofibric acid in rat hepatocytes. AB - We studied whether gemfibrozil and clofibric acid alter isoprenoid lipid synthesis in rat hepatocytes. After incubation of the cells with the agent for 74 hr, [(14)C]acetate or [(3)H]mevalonate was added, and the cells were further incubated for 4 hr. Gemfibrozil and clofibric acid increased ubiquinone synthesis from [(14)C]acetate and [(3)H]mevalonate. The effect of gemfibrozil was greater than that of clofibric acid. Also, gemfibrozil decreased dolichol synthesis from [(14)C]acetate and [(3)H]mevalonate. However, clofibric acid increased dolichol synthesis from [(3)H]mevalonate. Gemfibrozil decreased cholesterol synthesis from [(14)C]acetate and [(3)H]mevalonate. Clofibric acid decreased cholesterol synthesis from [(14)C]acetate, but did not affect synthesis from [(3)H]mevalonate. These results suggest that both agents, at different rates, activate the synthetic pathway of ubiquinone, at least from mevalonate. Gemfibrozil may inhibit the synthetic pathway of dolichol, at least from mevalonate. Contrary to gemfibrozil, clofibric acid may activate the synthetic pathway of dolichol from mevalonate. Gemfibrozil may inhibit the synthetic pathway of cholesterol from mevalonate in addition to the pathway from acetate to mevalonate inhibited by both agents. PMID- 10736421 TI - Specificity of inhibitors of serine palmitoyltransferase (SPT), a key enzyme in sphingolipid biosynthesis, in intact cells. A novel evaluation system using an SPT-defective mammalian cell mutant. AB - In the present study, we demonstrate a model cell system for evaluating the specificity of inhibitors of serine palmitoyltransferase (SPT), the enzyme that catalyzes the first step of sphingolipid biosynthesis. The LY-B strain is a Chinese hamster ovary (CHO) cell mutant defective in SPT, and the LY-B/cLCB1 strain is a genetically corrected revertant of the mutant. Although LY-B cells grew only slightly in sphingolipid-deficient medium, their growth was restored to the level of LY-B/cLCB1 cells under sphingosine-supplied conditions, indicating that, in CHO cells, the growth inhibition caused by SPT inactivation was rescued almost fully by the metabolic complementation of sphingolipids. Cultivation of LY B/cLCB1 cells in sphingolipid-deficient medium in the presence of 10 microM sphingofungin B and ISP-1 (myriocin, thermozymocidin), potent inhibitors of SPT activity, caused severe growth inhibition with approximately 95% inhibition of de novo sphingolipid synthesis. The growth inhibition by sphingofungin B and ISP-1 was rescued substantially by exogenous sphingosine, whereas the cytotoxicity of two other types of SPT inhibitor, L-cycloserine and beta-chloro-L-alanine, was hardly rescued. Similar cytotoxic patterns of these inhibitors also were observed on the growth of SPT-defective LY-B cells cultured under sphingosine-supplied conditions. The SPT inhibitors did not affect metabolic conversion of exogenous [(3)H]sphingosine to complex sphingolipids. Thus, the cytotoxicity of sphingofungin B and ISP-1, but not L-cycloserine or beta-chloro-L-alanine, is due largely to inhibition of sphingolipid synthesis by inhibiting the SPT activity. PMID- 10736422 TI - Differential cytotoxicity and induction of apoptosis in tumor and normal cells by hydroxymethylacylfulvene (HMAF). AB - This investigation compared the effects of hydroxymethylacylfulvene (HMAF), a novel antitumor drug with alkylating properties, in eight human tumor (prostate, colon, and leukemia) cell lines, and five human normal (prostate and renal proximal tubule epithelial, colon mucosa, fibroblasts, and endothelial) cell lines. Drug-induced growth inhibition paralleled the uptake of HMAF into both tumor and normal cells, although normal cells were 3- to 4-fold more tolerant to the accumulated drug. In both tumor and normal cells, approximately two-thirds of internalized [(14)C]HMAF-derived radioactivity was bound covalently to macromolecules. Trypan blue exclusion and cell counts indicated that HMAF was cytotoxic in tumor but cytostatic in normal cells. Correspondingly, profound apoptosis was detected in all tumor cell lines examined. A 4-hr treatment with HMAF followed by 20-hr post-incubation induced a potent DNA fragmentation in nearly all tumor lines. Apoptosis-resistant PC-3 and HT-29 cells underwent significant DNA fragmentation after 24 hr of continuous treatment with HMAF. In contrast to tumor cell lines, marginal or very low levels of apoptosis were detected in the normal cells even after prolonged treatments with HMAF at concentrations that exceeded 15- to 800-fold the GI(50) values in tumor cells. This resistance of normal cells to apoptosis could not be accounted for by differences in drug accumulation or drug covalent binding to macromolecules. The qualitatively different responses of the tumor and normal cells studied suggest a greater tolerance of normal cells to HMAF-macromolecular adducts. The demonstrated differential cytotoxic/cytostatic and apoptotic effects of HMAF can be of significance for the clinical use of this promising new agent. PMID- 10736424 TI - Metabolism and cytotoxic effects of 2-chloroadenine, the major catabolite of 2 chloro-2'-deoxyadenosine. AB - EHEB cells, a continuous cell line derived from a patient with B cell chronic lymphocytic leukemia (B-CLL), synthesized, when incubated with tritiated 2-chloro 2'-deoxyadenosine (CdA), labeled mono-, di-, and triphosphate ribonucleosides at a much higher rate than CdA deoxyribonucleotides. Further analysis revealed that these ribonucleotides were formed from labeled 2-chloroadenine (CAde), which contaminated commercial tritiated CdA at a proportion of 2-3%. Since CAde is the major catabolite of CdA measured in plasma after oral or intravenous administration of CdA to patients, its metabolism and in particular its potential cytotoxicity were investigated both in EHEB cells and in B-CLL lymphocytes. Phosphorylation of CAde was inhibited by adenine, indicating that its initial metabolism most probably proceeds via adenine phosphoribosyltransferase (EC 2.4.2.7). In both cell types, chloro-ATP was the major metabolite formed from CAde and its concentration increased proportionally at least up to 50 microM CAde. At high concentration, CAde metabolism was accompanied by a decrease in intracellular ATP. Cytotoxicity of CAde, evaluated by 3-(4,5-dimethylthiazol-2 yl)-2,5-diphenyltetrazolium bromide (MTT) assay, showed an IC(50) of 16 microM in EHEB cells and 5 microM in B-CLL lymphocytes. At cytotoxic concentrations, apopain/caspase-3 activation and high molecular weight DNA fragmentation were observed, indicating that CAde cytotoxicity results from induction of apoptosis. However, since CAde cytotoxicity requires higher concentrations than CdA, it probably does not play a role in the therapeutic effect of CdA in the treatment of hematologic malignancies. PMID- 10736423 TI - Structure and activity of specific inhibitors of thymidine phosphorylase to potentiate the function of antitumor 2'-deoxyribonucleosides. AB - A new class of 5-halogenated pyrimidine analogs substituted at the 6-position was evaluated as competitive inhibitors of thymidine phosphorylase (TPase). The most potent member of the series was 5-chloro-6-(2-iminopyrrolidin-1-yl)methyl 2,4(1H,3H)-pyrimidine dio ne hydrochloride (TPI), which has an apparent K(i) value of 1.7 x 10(-8) M. TPI selectively inhibited the activity of TPase, but not that of uridine phosphorylase, thymidine kinase, orotate phosphoribosyltransferase, or dihydropyrimidine dehydrogenase. In vitro inhibition studies of TPI using a thymidine analogue, 5-trifluoromethyl-2' deoxyuridine (F(3)dThd), as the substrate demonstrated that F(3)dThd phosphorolytic activity was inhibited markedly by TPI (1 x 10(-6) M) in extracts from the liver, small intestine, and tumors of humans, from the liver and small intestine of cynomolgus monkeys, and from the liver of rodents, but not from the liver or small intestine of dogs or the small intestine of rodents, suggesting that the distribution of TPase differs between humans and animal species, and that TPI could contribute to the modulation of TPase in humans. When F(3)dThd or 5-iodo-2'-deoxyuridine (IdUrd) was coadministered to mice with TPI at a molar ratio of 1:1, the blood levels of F(3)dThd (or IdUrd) were about 2-fold higher than when F(3)dThd (or IdUrd) was administered alone. In monkeys, the maximum concentration (C(max)) and the area under the concentration-time curve (AUC) after oral F(3)dThd alone were 0.23 microg/mL and 0.28 microg. hr/mL, respectively, but markedly increased to 15.18 microg/mL (approximately 70-fold) and 28.47 microg. hr/mL (approximately 100-fold), respectively, when combined with equimolar TPI. Combined oral administration of TPI significantly potentiated the antitumor activity of F(3)dThd on AZ-521 human stomach cancer xenografts in nude mice. In conclusion, TPI may contribute not only to inhibition of TPase mediated biological functions but also to potentiation of the biological activity of various 2'-deoxyuridine and thymidine derivatives by combining with them. PMID- 10736425 TI - Reversal of MRP-mediated doxorubicin resistance with quinoline-based drugs. AB - The overexpression of P-glycoprotein (P-gp) and the multidrug resistance associated protein (MRP) have been shown to confer broad drug resistance in tumor cells. We have demonstrated previously direct binding between MRP and a quinoline based photoreactive drug (iodo-azido-amino quinoline, IAAQ) (Vezmar et al., Biochem Biophys Res Commun 241: 104-111, 1997). In this report, we show the reversal of multidrug resistance in two MRP-overexpressing cell lines, HL60/AR and H69/AR, with four quinoline-based drugs. Non-toxic concentrations (5-20 microM) of chloroquine, quinine, quinidine, and primaquine potentiated the toxicity of doxorubicin in a concentration-dependent manner. These quinoline based drugs showed a 5- to 10-fold decrease in the IC(50) of doxorubicin in H69/AR and HL60/AR cells. Primaquine was the most active, with modulation ratios of 10- and 5-fold versus 8- and 3-fold with MK-571 for H69/AR and HL60/AR, respectively. Moreover, using IAAQ, we showed that molar excesses of chloroquine, quinine, quinidine, and MK-571 inhibit the photoaffinity labeling of MRP. Primaquine and vinblastine showed lesser inhibition of MRP photoaffinity labeling by IAAQ. Taken together, the results of this study demonstrated the reversal of doxorubicin resistance with several quinoline-based drugs. Moreover, these drugs have been shown to reverse P-gp-mediated MDR and are clinically well tolerated. PMID- 10736426 TI - Time-dependent changes in brain monoamine oxidase activity and in brain levels of monoamines and amino acids following acute administration of the antidepressant/antipanic drug phenelzine. AB - Phenelzine (PLZ) is a non-selective monoamine oxidase (MAO) inhibitor commonly used to treat depression and panic disorder. Acute administration of PLZ produces several neurochemical changes, including an increase in brain levels of the catecholamines norepinephrine (NE) and dopamine (DA), of 5-hydroxytryptamine (5 HT), and of the amino acids alanine and gamma-aminobutyric acid (GABA). The goal of the present series of experiments was to characterize the time course of these PLZ-induced changes. Male Sprague-Dawley rats were sacrificed 6, 24, 48, 96, 168, or 336 hr after acute PLZ administration (15 or 30 mg/kg, i.p., based on free base weight). Whole brain levels of monoamines and amino acids were determined using HPLC, and MAO A and B activities were determined using a radiochemical procedure. The results indicated that PLZ changed amino acid levels 6 and 24 hr after injection, but not 48 hr later. In contrast, the effects of PLZ on MAO activity and monoamines were longer-lasting. For example, PLZ-induced increases in dopamine and 5-HT were observed 1 week after injection, and PLZ-induced inhibition of MAO activity persisted for 2 weeks. Thus, in addition to demonstrating that the effects of PLZ on MAO activity and monoamines were long lasting, these results indicate that the effects of PLZ on MAO activity and on brain levels of monoamines and amino acids are temporally dissociated. These findings regarding the long-term effects of PLZ on neurochemistry will have considerable critical implications for the design and interpretation of behavioral studies of the acute effects of PLZ. PMID- 10736428 TI - Growth hormone regulation and developmental expression of rat hepatic CYP3A18, CYP3A9, and CYP3A2. AB - The present study investigated the role of growth hormone (GH) in hepatic CYP3A18 and CYP3A9 expression in prepubertal and adult male rats. For comparison, the effects of GH on CYP3A2 expression were also measured. Initial experiments demonstrated that CYP3A18 mRNA levels were greater during puberty and adulthood than during the prepubertal period, CYP3A9 mRNA was not expressed until puberty and its expression increased in adulthood, and CYP3A2 mRNA levels were relatively constant from prepuberty to adult life. Hypophysectomy, which results in the loss of multiple pituitary factors including GH, increased CYP3A2 and CYP3A18 mRNA expression 3- to 4-fold, but it did not affect CYP3A9 mRNA levels or CYP3A mediated testosterone 2beta- or 6beta-hydroxylase activity in adult rats. GH administered as twice daily s.c. injections (0.12 microg/g body weight) to hypophysectomized or intact adult rats did not affect CYP3A18 or CYP3A9 mRNA expression. The same treatment decreased CYP3A2 mRNA and protein and testosterone 2beta- and 6beta-hydroxylase activity levels in intact but not hypophysectomized rats. However, in intact prepubertal rats, intermittent GH administration decreased CYP3A18 and CYP3A2 mRNA levels, but a higher dosage (3.6 microg/g) was required to suppress CYP3A2. Overall, the present study demonstrated that: (a) the constitutive expression of CYP3A18, CYP3A9, and CYP3A2 does not require the presence of GH, (b) CYP3A18 is more sensitive than CYP3A9 to GH modulation in adult rats; and (c) CYP3A2 is less sensitive to the suppressive influence of GH during the prepubertal period than during adult life. PMID- 10736427 TI - Effect of receptor-selective retinoids on growth and differentiation pathways in mouse melanoma cells. AB - Treatment of B16 mouse melanoma cells with all-trans-retinoic acid (ATRA) results in inhibition of cell proliferation and induction of differentiation. Accompanying these events is an induction of retinoic acid receptor beta (RARbeta) expression, an increase in protein kinase Calpha (PKCalpha) expression, and enhanced activator protein-1 (AP-1) transcriptional activity. These cells express nuclear RARalpha and RARgamma and nuclear retinoid X receptors (RXR) alpha and beta constitutively. We tested the ability of receptor-selective retinoids to induce the biochemical changes found in ATRA-treated melanoma cells and also tested their effectiveness in decreasing anchorage-dependent and independent growth. The RXR-selective ligand (2E,4E)-6-(5,6,7,8-tetrahydro 3,5,5,8, 8-pentamethyl-2-naphthalenyl)-3,7-dimethyl-2,4,6-octatrienoic acid (SR11246) was most effective at inhibiting anchorage-dependent growth, whereas the RARgamma-selective ligand 6-[(5,6,7, 8-tetrahydro-5,5,8, 8-tetramethyl-2 naphthalenyl)(hydroxyimino)methyl]-2-naphthalen ecarbo xylic acid (SR11254) was most potent at inhibiting anchorage-independent growth. In contrast, 4-(5,6,7,8 tetrahydro-5,5, 8,8-tetramethyl-2-naphthalenecarboxamido)-benzoic acid (Am580), an RARalpha-selective ligand, was the most effective receptor-selective agonist for inducing RARbeta mRNA and increasing the amount of PKCalpha protein. All of the retinoids induced a concentration-dependent increase in AP-1 transcriptional activity, with little difference in effectiveness among the receptor-selective retinoids. A synergistic increase in the amount of PKCalpha was found when an RAR selective agonist was combined with an RXR-selective agonist. One possible explanation for this result is that an RXR-RAR heterodimer in which both receptors are liganded is required for maximum expression of this critical component of the ATRA-induced differentiation pathway. Our data suggest that synthetic retinoids can activate different growth and differentiation pathways preferentially in B16 melanoma cells, due, most likely, to their ability to activate a different subset of receptors. PMID- 10736429 TI - Inflammatory oedema induced by the lys-49 phospholipase A(2) homologue piratoxin i in the rat and rabbit. Effect of polyanions and p-bromophenacyl bromide. AB - Piratoxin-I (PrTX-I) is a Lys-49 phospholipase (PLA(2)) homologue, isolated from Bothrops pirajai snake venom, that has no phospholipase activity. In this study, we investigated the in vivo oedematogenic activity of PrTX-I in both the rat and the rabbit as well as the ability of PrTX-I to activate rat mast cells in vitro. In the rat paw and skin, PrTX-I (3-100 microg/paw) induced a dose-dependent oedema that was associated with extensive mast cell degranulation. The involvement of mast cells in PrTX-I-mediated oedema formation in the rat was further confirmed by the findings that this protein significantly activated rat peritoneal mast cells in vitro, causing the release of [(14)C]5-hydroxytryptamine ([(14)C]5-HT; 51 +/- 1%). In the rabbit, PrTX-I (10-100 microg/site) also induced dose-dependent skin oedema formation that was not affected by either mepyramine (a histamine H(1) receptor antagonist) or cyproheptadine (1.0 microg/site), indicating that mast cells do not play a role in this animal species. The bradykinin B(2) receptor antagonist Hoe 140 (0.5 microg/site) and the platelet activating factor (PAF) receptor antagonist WEB 2086 (200 microg/site) also failed to affect the PrTX-I-induced rabbit skin oedema, ruling out the involvement of kinins and PAF. The PLA(2) inhibitor p-bromophenacyl bromide greatly reduced the PrTX-I-induced oedema in both the rat and the rabbit, and also inhibited the rat in vitro mast cell activation induced by this PLA(2) homologue. The polyanions heparin and dermatan sulphate efficiently prevented oedema formation in both species, and heparin inhibited PrTX-I-induced rat mast cell degranulation. Our results are consistent with the suggestion that the cationic charge of PrTX-I plays a major role in the inflammatory responses induced by this PLA(2) homologue. PMID- 10736430 TI - Depression of constitutive murine cytochromes P450 by staphylococcal enterotoxin B. AB - Most in vivo studies demonstrating decreased activities of hepatic cytochromes P450 with inflammation have used Gram-negative bacterial lipopolysaccharide (LPS) as the inflammatory stimulant. But products of Gram-positive bacteria, such as staphylococcal enterotoxin B (SEB), also stimulate inflammatory mediators, albeit with a different pattern than LPS. Therefore, effects of SEB on the regulation of murine constitutive P450s were determined in this study and compared with those of LPS. LPS-responsive C3H/HeN and LPS-unresponsive C3H/HeJ mice were injected with either LPS (0.5 mg/kg) or SEB (0.66 to 6.6 mg/kg), and hepatic cytochromes P450 and serum tumor necrosis factor-alpha, interleukin-6, nitrate/nitrite, and serum amyloid A concentrations were determined up to 24 hr. HeJ mice were generally less responsive than HeN mice to both stimuli, with lower cytokine, nitrate/nitrite, and serum amyloid A responses. However, in both mouse strains SEB caused more prolonged cytokine, higher nitrate/nitrite, and lower serum amyloid A concentrations than LPS. Despite these differences, in HeN mice, after both SEB and LPS administration, total P450 concentrations were equally depressed by 40%. Both SEB and LPS depressed CYP1A1 and 1A2 microsomal protein concentrations by 45 and 30%, respectively; CYP2E1 by 64%; and CYP3A by 70%. There was comparable inhibition of enzymatic activities. In HeJ mice, SEB was only slightly more effective in depressing P450s than LPS, as might be expected. These data showed that the Gram-positive bacterial inflammatory stimulant SEB caused effects on murine hepatic cytochromes P450 similar to those of LPS, even though the pattern of inflammatory mediators induced after SEB exposure was different. PMID- 10736431 TI - Inhibitory action of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3 isoquinolinecarboxam ide (PK 11195) on some mononuclear phagocyte functions. AB - Peripheral benzodiazepine receptors (PBRs) are widely distributed throughout the body, but their functions are unknown. They are found on mononuclear phagocytes, and they are up-regulated in a number of neurological and other disease states. We explored the functional consequences of PBR ligand binding to mononuclear derived cells using the high-affinity ligands 1-(2-chlorophenyl)-N-methyl-N-(1 methylpropyl)-3-isoquinolinecarboxam ide (PK 11195) and 4'-chlorodiazepam (7 chloro-5-(4'-chlorophenyl)-1, 3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one; Ro 5 4864). The functions were the following: respiratory burst; secretion of glutamate, interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF alpha); toxicity of culture supernatants towards SH-SY5Y human neuroblastoma cells; and expression of the inflammatory surface markers HLA-DR and Fcgamma RII (CDw32). PK 11195 inhibited the respiratory burst response, reduced release of glutamate and IL-1beta, and suppressed secretion of products cytotoxic to neuronal cells. Selectivity was suggested by the failure of PK 11195 to influence TNF-alpha secretion or expression of HLA-DR and CDw32. Powerful ligands of PBRs, such as PK 11195, may be useful inhibitors of selective macrophage functions, retarding both local and systemic inflammation. Since PK 11195 readily enters the brain, it may be beneficial in treating central as well as peripheral inflammatory diseases. PMID- 10736432 TI - Prevention of renal ischemia-reperfusion-induced injury in rats by picroliv. AB - Picroliv is a potent antioxidant extracted from the roots and rhizome of Picrorhiza kurrooa. It has been shown to impart significant hepatoprotective activities, partly by modulation of free radical-induced lipid peroxidation. Lipid peroxidation and reactive oxygen species are associated with tissue injury in post-ischemic acute renal failure. The efficacy of picroliv was assessed in an in vivo model of renal ischemia-reperfusion injury (IRI) in rats at a dose of 12 mg/kg orally for 7 days. The animals were killed at various times after reperfusion. Increased lipid peroxidation and apoptotic cell number reflected the oxidative damage following renal IRI. Picroliv-pretreated rats exhibited lower lipid peroxidation, improved antioxidant status, and reduced apoptosis, indicating better viability of renal cells. Immunohistochemical studies revealed that picroliv pretreatment attenuated the expression of intercellular adhesion molecule-1 in the glomerular region. These results suggested that picroliv pretreatment protects rat kidneys from IRI, perhaps by modulation of free radical damage and adhesion molecules. PMID- 10736473 TI - Biotransformation of lantadenes, the pentacyclic triterpenoid hepatotoxins of lantana plant, in guinea pig. AB - Oral administration of lantana (Lantana camara var. aculeata) leaf powder to guinea pigs at a dose of 6 g/ kg body weight elicited cholestasis. The animals were euthanized 48 h after dosing. Liver homogenates, bile, gall bladder, blood, urine, contents of gastrointestinal tract (GIT) and faeces were analysed for the principal hepatotoxin in lantana leaves viz. lantadene A (LA), its congeners and biotransformation products, using high performance liquid chromatographic technique. Lantadenes could not be detected in liver, bile, gall bladder, blood and urine samples. LA and lantadene B (LB), their derivatives reduced lantadene A (RLA), reduced lantadene B (RLB) and two unidentified metabolites could be detected in the contents of lower GIT and faeces. In vitro incubation of lantana leaf powder with guinea pig caecal contents under anaerobic conditions elicited biotransformation of LA and LB to RLA and RLB, respectively. On the other hand, incubation of lantana leaf powder with cattle rumen liquor under anaerobic conditions did not elicit biotransformation of lantadenes. PMID- 10736472 TI - The venomous hair structure, venom and life cycle of Lagoa crispata, a puss caterpillar of Oklahoma. AB - The presence of a unique population of Lagoa crispata, puss caterpillar, in western Oklahoma is reported. A detailed microscopic examination shows the structure of the L. crispata spines resemble the type 4 spines described by [Kawamoto, F., Kumada, N., 1984. Biology and venoms of lepidoptera. In: Tu, A.T. (Ed.), Handbook of Natural Toxins, Insect Poisons, Allergens and other invertebrate venoms, vol. 2, pp. 291-332 (ch. 9)]. The major food source of L. crispata are the leaves of oak (shin oak). The high tannin content of this food source results in spine extracts high in oak tannins. These extracts have activity but enzyme and toxin activity is lost with time. The gel filtration protein fractions are colored from brown to yellow and are inactive as enzymes or toxins. No hyaluronidase, protease or phosphohydrolase activity is detected in these protein fractions. The life cycle shows these caterpillars have 6 instars. Characterizations and annual emerging times of each instar are included. PMID- 10736474 TI - Degradation of the cyanobacterial toxin cylindrospermopsin, from Cylindrospermopsis raciborskii, by chlorination. AB - Cylindrospermopsin, a potent cyanobacterial toxin produced by Cylindrospermopsis raciborskii and other cyanobacteria, is regularly found in water supplies of Queensland, Australia. This study focussed on the effectiveness of chlorination as a water treatment procedure for cylindrospermopsin degradation. The results demonstrate that relatively low chlorine doses (<1 mg l(-1)) are sufficient for degradation of cylindrospermopsin, when the dissolved organic carbon content is low. However, if organic matter other than cylindrospermopsin is present in the solution, the effectiveness of chlorine for cylindrospermopsin degradation is reduced as other organic matter present consumes chlorine. Under the experimental conditions using samples with a solution pH of 6-9, a residual chlorine concentration of 0.5 mg l(-1)99% of cylindrospermopsin. Toxin degradation via chlorination occurs within the first minute and no difference was observable between degradation in an open system and in a closed system. With a decrease of the pH from 6 to 4 a reduction in the efficiency of chlorine for degradation of cylindrospermopsin was observable, a possible indication that cylindrospermopsin is more stable to chlorine degradation at lower pH. However, in normal water treatment this is not relevant since the pH is consistently higher than 6. PMID- 10736475 TI - Effects of volatile aldehydes from Aspergillus-resistant varieties of corn on Aspergillus parasiticus growth and aflatoxin biosynthesis. AB - The fungi Aspergillus flavus and Aspergillus parasiticus produce a potent class of hepatocarcinogens known as aflatoxins. Corn-derived volatile compounds have been previously found to affect growth and aflatoxin production in A. flavus. In this study, the effects on A. parasiticus of three corn-derived volatile compounds, n-decyl aldehyde, hexanal and octanal, were measured. These three compounds were previously found to be variably expressed in five Aspergillus resistant maize strains and three susceptible strains. In this study, A. parasiticus radial growth was restricted least by n-decyl aldehyde and most by octanal. Treatments of 100 microl of both hexanal and octanal were found to completely inhibit radial growth of the fungus using an agar plate assay method. While the volatile compound n-decyl aldehyde had less of an effect on radial growth than the other volatiles, the n-decyl aldehyde treated colonies had a predominance of uniquely aerial hyphae. These colony structures were found to have more complex hyphae and significantly fewer conidiophores than the control and other aldehyde treatments. Furthermore, aflatoxin production by the fungus was reduced by n-decyl aldehyde and hexanal, but was stimulated by octanal. The results presented here indicate that all three volatile compounds reduce radial growth but only n-decyl aldehyde significantly inhibits aflatoxin biosynthesis in A. parasiticus. PMID- 10736476 TI - Characterization of a thrombin-like enzyme from the venom of Trimeresurus jerdonii. AB - From the venom of Trimeresurus jerdonii, a distinct thrombin-like enzyme, called jerdonobin, was purified by DEAE A-25 ion-exchange chromatography, Sephadex G-75 gel filtration, and fast protein liquid chromatography (FPLC). SDS-PAGE analysis of this enzyme shows that it consists of a single polypeptide chain with a molecular weight of 38,000. The NH(2)-terminal amino acid sequence of jerdonobin has great homology with venom thrombin-like enzymes documented. Jerdonobin is able to hydrolyze several chromogenic substrates. The enzyme directly clots fibrinogen with an activity of 217 NIH units/mg. The fibrinopeptides released, identified by HPLC, consisted of fibrinopeptide A and a small amount of fibrinopepide B. The activities of the enzyme were inhibited by phenylmethylsulfonyl fluoride (PMSF) and p-nitrophenyl-p-guanidinobenzoate (NPGB). However, metal chelator (EDTA) had no effect on it, indicating it is venom serine protease. PMID- 10736477 TI - The presynaptic activity of huwentoxin-I, a neurotoxin from the venom of the chinese bird spider Selenocosmia huwena. AB - Three different types of isolated nerve-synapse preparations, guinea pig ileum, rat vas deferens and toad heart, were used to investigate the physiological activity of Huwentoxin-I, a neurotoxin from the venom of the spider Selenocosmia huwena. The twitch response of isolated guinea pig ileum induced by electrical stimulus can be inhibited by HWTX-I. After blockage, contraction of the ileum can be induced by exogenously applied acetylcholine. HWTX-I caused the inhibition of the twitch response to electrical nerve stimulation in the rat vas deferens. After the twitch was completely inhibited, noradrenaline triggered rhythmic contraction of the vas deferens. The inhibitory effect on heart of toad induced by stimulating sympathetic-vagus nerve can be reversed by HWTX-I, although exogenously applied acetylcholine still acts as an effective inhibitor. All of these results support the conclusion that HWTX-I has the presynaptic activity that effects the release of neurotransmitter from the nerve endings of both the cholinergic synapse and the adrenergic synapse. PMID- 10736478 TI - Difference in hydrophobicity between botulinum type B activated and non-activated neurotoxins under low pH conditions. AB - We showed that botulinum type B activated neurotoxin with a di-chain structure became hydrophobic more quickly and extensively than did the non-activated toxin with a single-chain structure on low pH exposure. The activated toxin possessed 50-fold higher toxicity than did the non-activated type. The difference in the susceptibility to hydrophobic change may be one clue to answering the question of why the activated toxin possesses a higher toxicity than does the non-activated type. PMID- 10736479 TI - Increments in serum cytokine and nitric oxide levels in mice injected with Bothrops asper and Bothrops jararaca snake venoms. AB - Changes in serum levels of several cytokines and nitric oxide were studied in BALB/c mice injected intraperitoneally with one median lethal dose (LD(50)) of the venoms of Bothrops asper and Bothrops jararaca, two of the medically most important poisonous snakes of Latin America. Despite differences observed in the time-course of cytokine increments and in serum cytokine levels, both venoms induced prominent elevations of TNF-alpha, IL-1, IL-6, IL-10 and IFN-gamma. There was an early increase in TNF-alpha and IL-1, followed by a more pronounced increment by 18 h. IL-6 levels peaked between 4 and 6 h, and this cytokine probably modulates the secretion of TNF-alpha and IL-1 and the synthesis of acute phase proteins. Both venoms induced an early increment in serum IL-10, whereas IFN-gamma levels reached higher values in mice injected with B. jararaca venom than in those receiving B. asper venom. Serum nitric oxide concentration increased in mice injected with both venoms rapidly after envenomation, remaining elevated for 24 h. It is concluded that a complex pattern of cytokine and nitric oxide synthesis and secretion occurs in severe experimental envenomation by B. asper and B. jararaca venoms. Furthermore, it is suggested that some of these mediators, particularly TNF-alpha, IL-1 and nitric oxide, might play a relevant role in the pathophysiology of systemic alterations induced by these venoms. PMID- 10736480 TI - A sleep inducing factor from common Indian toad (Bufo melanostictus, Schneider) skin extract. AB - Bufo melanostictus (common Indian toad) acquire different bioactive substances in their skin during their life-time in wide ecological habitat. Earlier investigation from this laboratory revealed that toad (B. melanostictus) skin extract (TSE) posses different bioactive compounds of different diversity (Das, M., Auddy, B. and Gomes, A., 1996. Pharmacological study of the toad skin extract on experimental animals. Indian J. Pharmacol. 28, 72-76). Among these sleep induction and sleep potentiation indicated the possibility of sleep inducing factor(s) in TSE. One such sleep inducing factor (SIF) was isolated and purified by neutral alumina column chromatography followed by HPLC. Spectroscopy (UV, IR, FAB-MASS) study indicated that the sleep inducing factor was a 880 Dalton conjugated aromatic compound with a hydroxyl and carbonyl functional group. Biological study showed that SIF produced no lethality in male albino mice upto the dose of 8 mg/kg, i.v. Cyproheptadine antagonised SIF induced contraction of isolated smooth muscle indicating histamine/serotonin receptor mediated action of SIF. EEG studies showed that SIF increased sleep and decreased awakening condition of freely moving rats. Biochemical studies showed that SIF produced significant alteration of brain biogenic amine levels, monoamine oxidase (MAO) and tryptophan hydroxylase (TH) activity. This may be the reason of SIF induced sleep, although the SIF induced sleep mechanism needs further detail investigation. PMID- 10736482 TI - Extended field prostate biopsy enhances cancer detection. PMID- 10736481 TI - High sensitivity bioassay of paralytic (PSP) and amnesic (ASP) algal toxins based on the fluorimetric detection of [Ca(2+)](i) in rat cortical primary cultures. AB - A high sensitivity bioassay able to recognise small amounts of paralytic and amnesic toxins in algal acetic extracts is described. The method is based on the measure of intracellular [Ca(2+)](i) in primary cultures of rat cortical neurones preloaded with Fura-2 and submitted to electrical field stimulation. Under normal conditions the basal [Ca(2+)](i) level was about 50-100 nM and was nearly doubled during the peaks induced by trains of electrical pulses at 10 Hz for 10 s. Saxitoxin (STX) 3.5 nM and tetrodotoxin (TTX) 24 nM halved the peaks height without affecting basal [Ca(2+)](i). Conversely, domoic acid increased the basal [Ca(2+)](i) (EC(50)=3. 7 microM) and decreased the calcium peaks (EC(50)=7.3 microM). CNQX (a competitive antagonist of AMPA/KA receptors) at 10 microM shifted to the right by a factor of 3 the concentration-response curves of domoic acid. The extracts of non-toxic algae were well tolerated by up to 10 microg protein/ml, whereas extracts of Alexandrium lusitanicum at 1-4 microg protein/ml reduced [Ca(2+)](i) peaks and increased basal calcium levels. This toxic effect of A. lusitanicum was unexpected since parallel HPLC analysis showed only the presence of gonyautoxins, known to act like saxitoxin. Therefore, the bioassay on rat cortical neurones revealed a complex composition of the toxins present in A. lusitanicum. The relevance of fluorimetric detection of [Ca(2+)](i) in primary neuronal cultures in the evaluation of algal risk is stressed. PMID- 10736483 TI - Extended field prostate biopsies: too much of a good thing? PMID- 10736484 TI - Blood use strategies in urologic surgery. PMID- 10736485 TI - Clinical study designs in the urologic literature: a review for the practicing urologist. PMID- 10736486 TI - Intracavernous alprostadil alfadex (EDEX/VIRIDAL) is effective and safe in patients with erectile dysfunction after failing sildenafil (Viagra). AB - OBJECTIVES: Sildenafil (Viagra), an oral treatment for erectile dysfunction, has proved popular since its introduction in 1998. However, not all patients respond to this form of therapy. Consequently, this study investigated the efficacy of intracavernous alprostadil alfadex (EDEX/VIRIDAL) treatment in patients not responding to sildenafil. METHODS: In an open-label, multicenter study, patients with erectile dysfunction were treated with sildenafil for 4 weeks. The initial dose was 50 mg, which was increased to 100 mg if no response was achieved. Patients not responding to treatment, measured using the International Index of Erectile Function (IIEF) questionnaire, entered an alprostadil alfadex in-office titration phase, to determine the optimal dose, up to 40 microgram. A 6-week alprostadil alfadex at-home treatment phase followed. RESULTS: In 67 patients who did not respond satisfactorily to sildenafil, the alprostadil alfadex at-home therapy resulted in improvements in questions 3 and 4 of the IIEF in 60 (89.6%) and 57 (85.1%) patients, respectively. The mean improvement in IIEF score for these patients was 2.75 and 2.63 for questions 3 and 4, respectively. The most common side effect was penile pain in 25 (29. 4%) of 85 patients treated with alprostadil alfadex in-office and at home. CONCLUSIONS: Alprostadil alfadex therapy can be used effectively and safely in men who fail initial therapy with sildenafil. PMID- 10736487 TI - Human glandular kallikrein as a tool to improve discrimination of poorly differentiated and non-organ-confined prostate cancer compared with prostate specific antigen. AB - OBJECTIVES: Human glandular kallikrein (hK2) possesses 80% structure identity with prostate-specific antigen (PSA) and is secreted by identical prostate epithelial cells. Although increasing with pathologic stage, PSA is not clinically sufficient to predict histologic grade and pathologic stage of prostate cancer (PCa) in individual cases. To address this issue, serum hK2 in various PCa grades was investigated. METHODS: Sera from 122 consecutive patients with PCa, graded as well-differentiated (G1, n = 35); moderately differentiated (G2, n = 61), and poorly differentiated (G3, n = 26) PCa, was studied. In patients who underwent radical prostatectomy (n = 42), 24 had organ-confined (pT2a-b) and 18 extracapsular (pT3a or greater) disease. hK2 was measured by an indirect immunofluorometric assay with a functional sensitivity of 0.03 ng/mL. Total PSA (tPSA), free PSA (fPSA), and PSA bound to alpha(1)-antichymotrypsin (PSA-ACT) were also measured. Multivariate logistic regression analysis was used for evaluation of the best combinations of tumor markers. RESULTS: Median hK2 and tPSA increased twofold from G1 to G2 tumors (hK2 0.07 versus 0.14 ng/mL, P <0.002; tPSA 6.1 versus 12.1 ng/mL, P <0.0002). Between G2 and G3 tumors, hK2 increased threefold (0.14 versus 0.43 ng/mL, P <0.02), and tPSA showed no significant increase (12.1 versus 26.5 ng/mL, P <0.18). The f/t PSA ratio decreased between G1 and G2 cancers (0.15 vs. 010, P <0.001); no difference was found between G2 and G3 tumors (0.10 versus 0.11, P = 0.93). However, the hK2/fPSA ratio distinguished between G1 and G3 tumors and G2 and G3 tumors (0.085 [G1] and 0.11 [G2] versus 0.22 [G3], P <0.0002 and P <0.002, respectively). Using multivariate regression analysis, the fPSA/(tPSA x hK2) ratio differentiated G2 and G3 tumors (P <0.01). In the tPSA range of 3 to 15 ng/mL, hK2, the hK2/fPSA ratio, and the fPSA/(tPSA x hK2) ratio differentiated between the G1/G2 and G3 tumors, and tPSA, the f/t PSA ratio, and PSA-ACT did not. In radical prostatectomy cases, hK2 (0.06 versus 0. 156, P <0.005) and the fPSA/(tPSA x hK2) ratio (2.104 versus 0.828, P <0.005) discriminated between pT2a-b and pT3a or greater PCa. CONCLUSIONS: hK2 significantly improved the identification of poorly differentiated (G3) tumors compared with PSA. By multivariate logistic regression analysis, the hK2/fPSA and fPSA/(tPSA x hK2) ratios further improved the detection of PCa grade. This improvement was also seen with the intermediate range of tPSA. hK2 was also helpful in the prediction of organ-confined disease. Thus, hK2 may be a useful tool for more accurate prediction of tumor grade or stage and allow better clinical decision-making. PMID- 10736488 TI - Association of chronic urinary symptoms in women and Ureaplasma urealyticum. AB - OBJECTIVES. To determine the incidence of Ureaplasma urealyticum in women experiencing chronic urinary symptoms and to determine whether antibiotic therapy targeting these organisms is effective. METHODS: Forty-eight consecutive women referred to our academic medical center for chronic voiding symptoms and possible interstitial cystitis underwent urologic evaluation, including culture screening for U. urealyticum and Mycoplasma hominis. Patients with positive cultures were treated with a 1-g dose of azithromycin; persistent infection was treated with 7 days of doxycycline, ofloxacin, or erythromycin. Patients reported symptom severity (0, mild; 3, severe) and voiding frequency before and 6 months after treatment. RESULTS: Positive cultures were obtained in 23 (48%) of 48 patients; 22 had U. urealyticum and 1 had M. hominis. All had negative cultures after treatment. The mean symptom severity score improved with treatment (2.2 to 0.7, P <0.001), and the mean urinary frequency decreased (9.2 daily to 6.8 daily, P <0.001). Two of the 23 patients experienced no improvement; one had detrusor instability and the other had medically related urinary frequency. Of the 25 patients with negative cultures, interstitial cystitis was established in only 9 (19% of the total sample). CONCLUSIONS: Although often overlooked or improperly treated, U. urealyticum and M. hominis infections may account for a large proportion of unexplained chronic voiding symptoms. Culture and treatment should be considered before pursuing more costly and invasive tests. PMID- 10736489 TI - Association of level of injury and bladder behavior in patients with post traumatic spinal cord injury. AB - OBJECTIVES: The expected urodynamic findings of patients with suprasacral and sacral spinal cord injury have previously been reported. However, the associations between the radiographically determined level or levels of injury and urodynamic findings are ill defined. This study investigated these relationships, specifically the bladder behavior of patients with post-traumatic spinal cord injury with combined suprasacral and sacral injuries. METHODS: A retrospective review of the patient records, spinal imaging studies, and video urodynamic studies of 316 patients with post-traumatic spinal cord injury was performed. Of these patients, 243 had complete spinal computed tomography or magnetic resonance imaging studies and constitute the study population. Patients were categorized by the radiographically determined level or levels of injury, clinical neurologic level and completeness of injury, and urodynamic findings. RESULTS: Of the 196 patients with suprasacral injuries, 186 (94.9%) demonstrated hyperreflexia and/or detrusor sphincter dyssynergia, 82 (41.8%) had low bladder compliance (less than 12.5 mL/cm H(2)O), and 79 (40.3%) had high detrusor leak point pressures (greater than 40 cm H(2)O). Of the 14 patients with sacral injuries, 12 (85.7%) manifested areflexia, 11 (78.6%) had low compliance, and 12 (85.7%) had high leak point pressures. Of the 33 patients with combined suprasacral and sacral injuries, urodynamic studies showed 23 with hyperreflexia and/or detrusor sphincter dyssynergia (67.7%), 9 with areflexia (27.3%), 19 (57.6%) with low compliance, and 20 (60.6%) with high leak point pressures. CONCLUSIONS: In patients with a single level of spinal cord injury, this study revealed a significant association between the level of injury and the type of voiding dysfunction. Patients with combined suprasacral and sacral injuries, as identified with precise spinal imaging techniques, had relatively unpredictable urodynamic findings. Management of the urinary tract in patients with spinal cord injury must be based on urodynamic findings rather than inferences from the neurologic evaluation. PMID- 10736490 TI - Tumor-targeted chemotherapy with SMANCS in lipiodol for renal cell carcinoma: longer survival with larger size tumors. AB - OBJECTIVES: To evaluate the anticancer effects of a lipophilic macromolecular anticancer agent, poly(styrene-co-maleic acid)-conjugated neocarzinostatin (SMANCS), dissolved in a lipid contrast medium (Lipiodol) given via the renal artery to patients with renal cell carcinoma. METHODS: Among 467 patients with renal cell carcinoma treated between April 1984 and March 1993, 191 were treated with SMANCS dissolved in a lipid contrast medium (a 3:2 mixture of Lipiodol F and Lipiodol Ultrafluid; Lpd). Selective arterial infusion of SMANCS/Lpd was performed at a dose of 1.0 or 1. 5 mg/mL. The infusion was repeated at intervals of about 2 weeks or longer, but the doses and the total number of infusions varied among patients, according to results of computed tomography analysis. RESULTS: Statistical analysis was performed for 415 patients who met the criteria of this study. Twenty-six surgical patients with metastases who underwent infusion therapy of SMANCS/Lpd for primary lesions showed 3 and 5-year survival rates of 23.0% and 12.8%, respectively; the rates were 19.3% and 9.7% in 31 patients who did not receive SMANCS infusion therapy. In 125 surgical patients without metastases who underwent SMANCS/Lpd infusion, the 5 and 10-year survival rates were 83.0% and 75.2%, respectively, whereas rates of 84.6% and 78.9% were observed in 199 surgical patients whose median tumor size was significantly smaller, however, than the SMANCS/Lpd infusion group. The maximal tumor diameter at the beginning of treatment was significantly larger (mean diameter 70.8 mm) in the SMANCS/Lpd infusion group than in the noninfusion group (59.1 mm). The survival rate was statistically better for patients with tumors of 100 mm diameter or larger in the SMANCS/Lpd infusion group (P <0.05): 5 and 10-year survival rates were 70.4% and 61.6%, respectively, for the infusion group and 64.6% and 50.9% for the group receiving no drug. In patients with larger tumor (greater than 110 mm), the survival rate at 13 years was 75% in the SMANCS/Lpd infusion group and 0% in the surgery group. CONCLUSIONS: Arterial infusion therapy with SMANCS/Lpd appears to be effective for large renal cell carcinoma without metastases in conjunction with surgery. PMID- 10736492 TI - Identifying patients who require urodynamic testing before surgery for stress incontinence based on questionnaire information and surgical history. AB - OBJECTIVES: To determine whether knowledge about previous surgical history for incontinence and responses to a validated lower urinary tract symptom questionnaire for women could identify those who should undergo urodynamic testing before surgery for stress urinary incontinence (SUI). METHODS: A retrospective analysis of all women who completed the Urogenital Distress Inventory-6 questionnaire (UDI-6) and then underwent complete urodynamic studies was conducted. The critical urodynamic diagnoses to determine before anti incontinence surgery were defined as coexisting SUI and detrusor instability (DI), Valsalva leak point pressure less than 60 cm H(2)O, and DI alone in women suspected clinically of having SUI. Models were established for deciding who should undergo urodynamic studies on the basis of questionnaire responses and information about previous surgical history. Cost savings and the ability of the various models to identify patients with critical urodynamic diagnoses were calculated. RESULTS: A total of 174 women completed the UDI-6 and underwent urodynamics studies. Sixty-two had SUI (36%), 54 had DI (31%), 18 had both SUI and DI (10%), and 19 women suspected of having SUI were found instead to have DI (11%). Among women with SUI, 39 had a Valsalva leak point pressure less than 60 cm H(2)O (63%). No group of question responses or combination of question responses and presence of previous incontinence surgery was able to identify all three critical urodynamic diagnoses with statistical significance. Still, the combination of a response of "2" or "3" to question 3 on the UDI-6 and a positive history of previous surgery would have identified 91% of the critical diagnoses, and a substantial cost savings would have been realized. CONCLUSIONS: Using the UDI-6 and information obtained from the patient's history to determine who should undergo urodynamic testing before surgery for SUI can result in substantial cost savings without sacrificing patient care. PMID- 10736491 TI - Value of immunohistochemical Ki-67 and p53 determinations as predictive factors of outcome in renal cell carcinoma. AB - OBJECTIVES: Nuclear grade and tumor stage are important prognostic factors in renal cell carcinoma, but tumors of similar stage and grade can exhibit a wide variation in biologic behavior and clinical outcome. In this retrospective study, we evaluated the immunologic markers, Ki-67 (MIB1) and p53, in 73 cases of conventional (clear cell) renal cell carcinoma and compared these markers with the accepted prognostic features of grade, stage, and tumor size in predicting outcome. METHODS: Specimens of 73 renal cell carcinomas of different nuclear grade (20 Furhman I/II, 32 Fuhrman III, and 21 Fuhrman IV) and different stage (10 pT1, 23 pT2, 36 pT3, and 4 pT4) were immunostained with monoclonal antibodies against Ki-67 and p53. RESULTS: Univariate statistical analysis showed that tumor size (P <0. 001), nuclear grade (P <0.01), tumor stage (P <0.01), Ki-67 index (P <0.001), and p53 immunostaining (P <0.03) correlated significantly with a poor prognosis. A Ki-67 index of 20% was a powerful predictor of survival in all patients (P <0.00001), with strong predictive values. On multivariate analysis, the Ki-67 index and metastases were significant independent prognostic factors (P <0.02 and <0.01, respectively). CONCLUSIONS: Ki-67 immunostaining appeared to be an additional prognostic indicator of biologic aggressiveness in renal cell carcinoma. Immunohistochemical assessment of tumor antigens could be used to identify patients at high risk of tumor progression in addition to conventional prognostic factors. PMID- 10736493 TI - The tension-free vaginal tape procedure: correction of stress incontinence with minimal alteration in proximal urethral mobility. AB - OBJECTIVES: To prospectively assess the degree of urethral hypermobility in the preoperative and postoperative periods after the tension-free vaginal tape (TVT) procedure and correlate our findings with surgical outcome. METHODS: Twenty patients with stress incontinence underwent the TVT procedure. A Q-tip test was performed before the procedure and at the 3-week postoperative follow-up visit. Cure was defined as the absence of the subjective complaint of urine leakage and the absence of stress incontinence on stress testing at cystometric capacity. RESULTS: Seventeen patients (85%) were cured by the TVT procedure, 2 patients (10%) were improved, and 1 patient (5%) was unchanged. The mean preoperative and postoperative Q-tip values were 42.75 degrees and 31.75 degrees, respectively. Twelve patients had a Q-tip test result of 30 degrees or greater after surgery and 11 (92%) of these 12 patients were cured by the procedure. CONCLUSIONS: On the basis of these results, we propose that the cure of stress incontinence does not require the correction of proximal urethral hypermobility. PMID- 10736494 TI - Antibacterial mesh sling: a prospective outcome analysis. AB - OBJECTIVES: To prospectively assess the outcome results of the Gore-Tex antibacterial mesh sling in women with pelvic prolapse and stress incontinence. METHODS: Between July 1997 and November 1998, 30 women with stress incontinence and vaginal prolapse underwent vaginal reconstruction with the antibacterial mesh sling. All patients had documented stress incontinence by preoperative urodynamic evaluation. Postoperatively, we conducted an outcome analysis on the basis of the results from a self-reported satisfaction questionnaire, a detailed pelvic examination, a cough stress test, and the Q-tip test. Urodynamic studies were performed on patients with persistent stress incontinence or urge symptoms. Cystoscopy was performed on patients suspected of having urethral erosion. RESULTS: Complete follow-up was available for all 30 patients. Of the group, 12 (40.0%) of 30 underwent implantation of the antibacterial mesh sling alone and 18 (60.0%) underwent additional prolapse repair. The mean follow-up was 18 months (range 9 to 24); mean age was 58 years (range 29 to 87). Stress incontinence was cured in 28 (93.3%) of 30 patients. De novo urge incontinence developed in 1 (5.2%) of 18 patients; urge symptoms persisted in 5 (41.7%) of 12 patients. The postoperative mean Valsalva Q-tip angle was 15 degrees (range 0 degrees to 25 degrees ). The incidence of urethral obstruction and erosion was zero. The mean postoperative pain score was 3 of 10 (range 0 to 6), and the mean satisfaction score was 9 of 10 (range 7 to 10). The mean time to discharge was 21 hours (range 6 to 29). All 30 women (100%) were willing to undergo surgery again. CONCLUSIONS: The safety profile of the Gore-Tex antibacterial mesh is better than that of the conventional Gore-Tex sling. When meticulous surgical technique is used, excellent short-term results can be achieved in the correction of female stress incontinence. PMID- 10736495 TI - Phase I/II study of paclitaxel, carboplatin, and methotrexate in advanced transitional cell carcinoma: a well-tolerated regimen with activity independent of p53 mutation. AB - OBJECTIVES: To evaluate the feasibility and activity of paclitaxel, carboplatin, and methotrexate in advanced transitional cell carcinoma (TCC) of the urothelium and to relate the activity of this combination to the mutational status of p53. METHODS: In the Phase I portion, paclitaxel 200 mg/m(2) (3-hour infusion), carboplatin dosed to an area under the curve (AUC) of 6 mg/mL. min, and methotrexate 10 mg/m(2), increasing in 10-mg/m(2) increments, were administered on day 1 and every 21 days thereafter with granulocyte colony-stimulating factor (G-CSF) and leucovorin support. Subsequently, a Phase II study was initiated in which the carboplatin dose was lowered to an AUC of 5 to allow treatment without G-CSF. p53 expression was evaluated using immunohistochemistry. RESULTS: Thirty three patients were accrued. Median age was 66 years. No dose-limiting toxicities were seen in the Phase I portion despite escalation of the methotrexate to 60 mg/m(2). Principal toxicities were myelosuppression and neuropathy. The overall response rate (Phase I and II) was 56% (95% confidence interval 38% to 74%). Median survival was 15.5 months; 88% of patients overexpressed p53 at the primary site. CONCLUSIONS: Paclitaxel, carboplatin, and methotrexate were well tolerated and active in advanced TCC. The high response rate to this regimen despite frequent p53 mutation is consistent with the p53-independent mechanism of paclitaxel. Whether this regimen is superior to methotrexate/vinblastine/doxorubicin/cisplatin, other paclitaxel-based regimens, or to paclitaxel alone will require comparative trials. PMID- 10736496 TI - Urinary tissue polypeptide-specific antigen for the diagnosis of bladder cancer. AB - OBJECTIVES: To evaluate the diagnostic characteristics of the urinary measurement of cytokeratin tissue polypeptide-specific antigen (TPS) for the detection of bladder cancer. METHODS: Three hundred thirty-five individuals in five groups were studied: group 1, subjects with microhematuria under suspicion for primary bladder cancer; group 2, patients being followed up with scheduled cystoscopic examinations; group 3, patients in follow-up receiving chemotherapy instillations; group 4, patients with other urologic diseases; and group 5, healthy subjects. Urine samples belonging to subjects from groups 1, 2, and 3 were collected immediately before cystoscopy. Additionally, patients from groups 2 and 3 were monitored with urinary TPS for a minimum period between two cystoscopies. TPS was measured by an enzyme immunosorbent assay. RESULTS: Receiver operating characteristic analysis gave a sensitivity of 64% and a specificity of 84% at a threshold value of 279 U/L. The positive and negative predictive value was 66% and 82%, respectively; accuracy was 77%. TPS could discriminate the presence of bladder tumor sooner than the scheduled cystoscopies in 9 of 19 follow-up patients with recurrence. False-positive results during follow-up were found in 112 urine samples, one third of which were associated with urinary tract infections. TPS did not appear to be specific for bladder cancer, with elevated results in 45% of patients from group 4, which might lead to clinical misinterpretation of urinary TPS results. CONCLUSIONS: Urinary TPS might provide additional information for the detection of bladder cancer as an adjunct to cystoscopy. Considering the false-positive rates, different urologic diseases should be ruled out before making clinical decisions on the basis of elevated urinary TPS results. PMID- 10736497 TI - Meta-analysis of clinical trials of permixon in the treatment of symptomatic benign prostatic hyperplasia. AB - OBJECTIVES: Permixon is a compound extracted from the fruit of the American dwarf palm tree, Serenoa repens. Controversy regarding the use of phytotherapeutic agents in men with lower urinary tract symptoms suggestive of benign prostatic hyperplasia is significant. We analyzed all available clinical trial data of the Permixon preparation to determine its clinical efficacy compared with placebo. METHODS: All published clinical trial data on Permixon (11 randomized clinical trials and 2 open label trials), involving 2859 patients, were used. These trials were disparate in size (from 22 to 592 patients) and duration (from 21 to 180 days). Peak urinary flow rate and nocturia were the two common end points. The statistical analysis was based on a random effects meta-analysis. RESULTS: The average +/- SE placebo effect on the peak urinary flow rate was an increase of 0.51 +/- 0.51 mL/s. The estimated effect of Permixon was a further increase of 2.20 +/- 0.51 mL/s (P <0.001). Placebo was associated with a reduction in the mean number +/- SE of nocturnal urinations of 0.69 +/- 0.15. A further reduction of 0.50 +/- 0.01 episodes of urination (P <0.001) occurred that was attributable to Permixon. Some heterogeneity was found among the studies. Treatment duration did not appear to impact either of these effects. CONCLUSIONS: This meta-analysis of all available published trials of Permixon in the treatment of men with benign prostatic hyperplasia revealed a significant improvement in peak flow rate and reduction in nocturia greater than with placebo. PMID- 10736498 TI - One-year follow-up of 2829 patients with moderate to severe lower urinary tract symptoms treated with alfuzosin in general practice according to IPSS and a health-related quality-of-life questionnaire. BPM Group in General Practice. AB - OBJECTIVES: To determine the effectiveness of alfuzosin on symptom reduction, patients' perceived health-related quality of life (HRQL) improvement, adverse outcomes, treatment failure, and progression to acute urinary retention and prostate surgery in patients with lower urinary tract symptoms suggestive of benign prostatic hyperplasia (BPH) in a 1-year prospective, open-labeled study. METHODS: A total of 2829 patients (mean age 65.9 years) were included in the study and received either alfuzosin 2.5 mg three times daily or alfuzosin slow release 5 mg twice daily. The evaluation was based on the International Prostate Symptom Score (IPSS), the eighth IPSS question, and a nine-item BPH HRQL questionnaire (BPHQL9) exploring well-being, the patient's perceived sexual life, and BPH-specific interferences with activities. RESULTS: A total of 2442 patients (86. 3%) completed the study; the main reasons for noncompletion were adverse events (n = 141, 5.0%), lack of efficacy (n = 136, 4.8%), and death (n = 48, 1.7%); 121 patients (4.3%) underwent prostate surgery, and 33 patients (1.2%) experienced acute urinary retention. No correlation was found between noncompletion and prostate volume or baseline severity. The distribution of patients (in percentages) according to the IPSS, IPSS question 8, and BPHQL9 classes of severity (mild/moderate/severe) at baseline was 1.9/49.0/49.1, 0. 7/65.5/33.8, and 7.7/50.4/41.9, respectively, and at 1 year was 47. 4/50.3/2.4, 34.1/64.9/1.0, and 39.0/50.9/10.1, respectively. The IPSS (19.5 +/- 0.1) was reduced by 49.6% (9.9 +/- 0.1) at 6 months and by 53.8% (11.1 +/- 0.1) at 12 months. Symptom reduction strongly correlated with the initial symptom severity (P <0.0001). The BPHQL9 score (34.6 +/- 0.3) gradually improved up to 12 months (52 +/- 0.4; +93.3%), and this improvement involved all three dimensions. Vertigo (n = 53, 1.9%), hypotension (n = 47, 1.6%), and dizziness (n = 16, 0. 6%) were the most frequent adverse events. CONCLUSIONS: This study confirms the effectiveness of alfuzosin and the need to include HRQL measurement in the decision-making process when assessing patients with lower urinary tract symptoms. PMID- 10736499 TI - In vivo interstitial temperature mapping of the human prostate during cryosurgery with correlation to histopathologic outcomes. AB - OBJECTIVES: To determine the critical temperatures below which human prostatic tissue can be cryoablated in situ and the comparative cryoablative efficacy of single versus double-freeze cryosurgery. METHODS: Six patients with prostate cancer previously scheduled for prostatectomy underwent unilateral or bilateral cryosurgery using a single cryosurgery probe per hemiprostate. Intraprocedural interstitial prostatic temperatures were measured by thermocouple junctions placed at various radial distances from the probe. After subsequent prostatectomy, whole-mount sections of the prostate gland were subjected to histopathologic evaluation. RESULTS: Uniform coagulative necrosis was observed in proximity to the cryosurgery probe. The percentage of the prostate volume falling within the zone of necrosis produced by a single probe was significantly greater (P = 0.048) after a double freeze (median 13%; range 8% to 20%) than a single freeze (median 4%; range 0% to 12%). The critical temperature for cryoablation with a double freeze was -41.4 degrees C (95% confidence interval -49.9 degrees to -33.0 degrees C) compared with -61.7 degrees C (95% confidence interval -74.5 degrees to -48.9 degrees C) for a single freeze (P <0.0005). CONCLUSIONS: Uniform coagulative necrosis of human prostatic tissue in vivo can be accomplished throughout a significantly larger zone with a double freeze than with a single freeze. A double freeze at temperatures below approximately -40 degrees C results in necrosis. These findings provide a basis for more optimal use of temperature monitoring during cryosurgery, which is essential to ensure effective treatment of the entire prostate gland with minimum risk of damage to adjacent tissues such as the rectum and external sphincter. PMID- 10736500 TI - Analysis of repeated biopsy results within 1 year after a noncancer diagnosis. AB - OBJECTIVES: A prostate biopsy data base derived from patients referred to private practice urologists was analyzed for the cancer diagnosis rates of the "initial" biopsy and the repeated biopsy performed within 1 year for those patients with a noncancer diagnosis. METHODS: A retrospective analysis assessed 132,426 prostate biopsies received and processed by a single pathology laboratory between March 1994 and September 1998; none had had a previous biopsy processed at this laboratory. Prostate cancer was diagnosed in 50,521 of the patients (38.2%). The remaining 81,905 patients (61.8%) had a noncancer diagnosis of either no evidence of malignancy (NEM), high-grade prostatic intraepithelial neoplasia (HGPIN), small acinar glands suspicious for cancer (suspicious), or suspicious with HGPIN (Susp-HGPIN). We identified 6380 (7.8%) of these "noncancer" patients who underwent a repeated biopsy within 1 year. RESULTS: The incidence of NEM, HGPIN, suspicious, and Susp-HGPIN biopsy diagnoses in the "noncancer" patients (81,905) was 55.3%, 3.7%, 2.5%, and 0.3%, respectively. The rate at which these "noncancer" patients (81,905) underwent a repeated biopsy was 4.8% for patients with a diagnosis of NEM, 26.6% for HGPIN, 40.4% for suspicious, and 47.5% for Susp-HGPIN. The overall cancer diagnosis rate in the repeated biopsy patient sample (6380) was 25.7%. When stratified by the initial biopsy diagnosis, the cancer diagnosis rate for the repeated biopsies was 19.8%, 22.6%, 40.0%, and 53.1%, for the patients with NEM, HGPIN, suspicious, and Susp-HGPIN, respectively. The repeated biopsy diagnosis rates did not vary dramatically when analyzed at 3-month intervals during the 1-year period. Also, a strong correlation (79%) was observed between the number of tissue samples obtained at the initial and repeated biopsy procedures. In a subset of patients with free and total prostate-specific antigen (PSA) results obtained before the repeated biopsy (n = 813), we were able to construct a multivariate logistic regression algorithm using the patients' age, initial biopsy diagnosis, total PSA, and free/total PSA ratio that could predict the likelihood of cancer on the repeated biopsy with an accuracy of 70%. CONCLUSIONS: Men who have an initial noncancerous biopsy diagnosis remain at risk of prostate cancer, especially if the initial diagnosis was suspicious or Susp-HGPIN. These data suggest that the initial biopsy strategy needs to be improved and/or expanded to increase the overall cancer detection rate in the primary biopsy. In addition, combining factors such as the initial biopsy diagnosis, family history, digital rectal examination results, prostate gland volume, age, total PSA, and free/total PSA ratio could provide valuable information for predicting the likelihood of cancer. PMID- 10736502 TI - Reply by the authors PMID- 10736501 TI - Editorial comment PMID- 10736503 TI - ACT-PSA and complexed PSA elimination kinetics in serum after radical retropubic prostatectomy: proof of new complex forming of PSA after release into circulation. AB - OBJECTIVES: To study the elimination kinetics of alpha(1)-antichymotrypsin (ACT) prostate-specific antigen (PSA) and complexed PSA (cPSA) in comparison to the biexponential decrease of total PSA and free PSA after radical prostatectomy. METHODS: Serum total PSA, free PSA, ACT-PSA, and cPSA values and the corresponding ratios were determined in venous blood from 12 patients with prostate cancer. The samples were taken before surgery, immediately after surgery, 1, 2, 3, 4, 5, and 6 hours after surgery, and then once daily for the next 10 days. Total PSA and cPSA were analyzed by using Immuno 1 PSA assays (Bayer Corporation); free PSA was measured using the AxSym test kit (Abbott Diagnostics). For ACT-PSA, the ES analyzer system was used (Roche Diagnostics). Statistical calculations were performed with the analysis of variance and Wilcoxon tests. RESULTS: During the first 6 hours after radical retropubic prostatectomy, we found nearly constant levels of ACT-PSA and cPSA, in contrast to the rapid elimination of free PSA and significant decrease in total PSA. From days 1 to 10, a continuous and nearly identical decrease of ACT-PSA and cPSA occurred compared with total PSA; free PSA was eliminated more rapidly. CONCLUSIONS: In addition to the opinion that the first PSA decrease might be an effect of the operation itself or caused by renal elimination alone, our findings indicate that the initial rapid decrease of free PSA immediately after surgery could be caused by new complex forming of PSA with ACT and other serum protease inhibitors. PMID- 10736504 TI - Transverse prostate measurement obtained using transabdominal ultrasound: possible role in transurethral needle ablation of the prostate. AB - OBJECTIVES: To correlate the maximal transverse prostate measurement determined using transabdominal ultrasound with the maximal transverse prostate measurement determined using transrectal ultrasound. METHODS: The records of 287 patients who had undergone both transabdominal pelvic ultrasound for determination of postvoid residual urine volume and transrectal ultrasound for evaluation of elevated serum prostate-specific antigen levels or abnormal digital rectal examinations were reviewed. For all patients, the maximal transverse prostate dimension had been recorded at each study. Linear regression analysis was used to correlate these two data sets. RESULTS: There was a strong correlation between the maximal transverse prostate dimension determined using transabdominal ultrasound and that determined using transrectal ultrasound. The R(2) for the regression analysis was 0.448. From the regression analysis, the equation that converts the maximal transabdominal transverse prostate measurement into its equivalent transrectal counterpart was determined: transrectal measurement (cm)=0.5109 [transabdominal measurement (cm)]+2.2935. CONCLUSIONS: A strong mathematical correlation was found between the maximal transverse prostate dimension determined using transabdominal ultrasound and that obtained using transrectal ultrasound. This may have clinical significance when performing transurethral needle ablation of the prostate. PMID- 10736505 TI - Sampling, submission, and report format for multiple prostate biopsies: a 1999 survey. AB - OBJECTIVES: Much variation occurs in sampling, submission, and reporting of prostate biopsies. Current practice standards among physicians across the United States are uncertain. METHODS: We surveyed predominantly nonacademic urologists and pathologists. The response rate was 57 (21%) of 271 urologists and 47 (55%) of 85 pathologists. RESULTS: Fifty-five percent of urologists performed six (or more) site-designated biopsies; 41% used unspecified bilateral biopsies. More than one half of urologists and pathologists reported submitting or receiving six or more separate, site-designated containers. The remainder of physicians (less than one half) reported the submission of all left needle cores in one container and all right cores in the other. Most pathologists (70%) stated that billing depended on the number of containers; 15% were unsure. One hundred percent of academic and 68% of nonacademic urologists deemed the report format therapeutically relevant (P <0.03), as did 57% of pathologists. Physicians submitting or receiving sextant needle biopsies in separate containers shared a 3:1 preference for issuance of a separate line diagnosis for each sextant site instead of condensing all diagnoses into one line with one Gleason score. Similarly, for each biopsy site, 61% of urologists wanted a separate Gleason score, and 68% wanted a separate designation for the percentage of tissue with tumor. Fifty-six percent and 64% of urologists and pathologists, respectively, deemed it relevant to specify the site(s) of high-grade prostatic intraepithelial neoplasia, even if cancer were present. CONCLUSIONS: Much variation persists in prostate biopsy sampling and reporting, hindering communication among physicians from different institutions. However, similar percentages of urologists and pathologists reported separate versus combined site sampling and separate versus combined site report format preferences. PMID- 10736506 TI - Combination of the preoperative PSA level, biopsy gleason score, percentage of positive biopsies, and MRI T-stage to predict early PSA failure in men with clinically localized prostate cancer. AB - OBJECTIVES: Early (2 years or less) prostate-specific antigen (PSA) failure has been shown to predict for distant failure. The independent clinical predictors of time to postoperative PSA failure were used to identify prostate cancer patients at high risk for early PSA failure. METHODS: A Cox regression multivariable analysis was used to determine whether additional predictive information was provided by the endorectal coil magnetic resonance imaging (erMRI) T-stage when controlling for the established prognostic factors in predicting the time to postoperative PSA failure in 977 men with palpable (T2) or PSA-detected (T1c) prostate cancer. RESULTS: Preoperative PSA (P = 0.0001), percentage of positive prostate biopsies (P= 0.0001), erMRI T-stage (P = 0.0001), biopsy Gleason score (P = 0.0015), and clinical stage T2c disease (P = 0.004) were independent predictors of time to postoperative PSA failure. Two-year PSA failure rates derived from the Cox regression model and bootstrap estimates of the 95% confidence intervals are presented in nomogram format stratified by the preoperative PSA, percentage of positive prostate biopsies, erMRI T-stage, and the biopsy Gleason score. CONCLUSIONS: Patients at high risk for early PSA failure and subsequent distant progression include men with erMRI T3 disease and 3 or more of 6 cores positive for a Gleason score 6 or higher disease when the PSA is more than 10 but not more than 20 ng/mL and any Gleason score when the PSA is more than 20 ng/mL. Men with erMRI T2 disease and 3 or more of 6 cores positive for a Gleason score 8 or higher disease and who have a PSA more than 20 ng/mL are also at high risk. Neoadjuvant therapy trials in these select patients may be justified. PMID- 10736507 TI - Complete duplication of the bladder, urethra, vagina, and uterus in girls. AB - OBJECTIVES. To present our experience with 3 girls with complete duplication of the bladder, urethra, vagina, and uterus, a very rare anomaly, and review published reports. Associated anomalies in these cases represent a wide spectrum. METHODS: We report 3 girls with complete duplication of the bladder, urethra, vagina, and uterus. The associated anomalies were different in each child. The first patient had a symphysis diastasis creating an abdominal hernia with two bladders dislocated into this space. The second patient had anal atresia with colon duplication. The third had cloacal exstrophy with one opened bladder and a second closed, with persistent urogenital sinus. Each patient was carefully evaluated, but the anatomy in 2 patients was not completely defined until surgical exploration. RESULTS: Each girl underwent surgical repair. In the first patient, iliac osteotomy, approximation of the symphysis pubis, and excision of the hernia was done. In the second, excision of the common wall between the two descending colons and posterior sagittal anorectoplasty were performed. In the third patient, staged reconstruction of the cloacal exstrophy with anastomosis of the bladders and vaginas was done. CONCLUSIONS: Complete duplication of the urogenital system in girls is associated with different anomalies. Which surgical procedures will be required depends on the multiple anatomic variables. PMID- 10736508 TI - Lymphatic mapping and intraoperative lymphoscintigraphy for identifying the sentinel node in penile tumors. AB - Lymph node mapping has become an integral part of the management of melanoma and breast cancer with regard to both staging and treatment. We report our technique for lymphatic mapping and intraoperative lymphoscintigraphy applied to a patient with penile melanoma. This technique may improve the sensitivity of identifying the sentinel lymph node in patients with malignant penile lesions. PMID- 10736509 TI - Resection of a posterior pubic symphyseal protuberance using the electrocautery device. AB - A posterior protuberance of the pubic symphysis can make a radical retropubic prostatectomy difficult. We describe a technique using electrocautery to remove this protuberance, which can be safely and easily performed. PMID- 10736510 TI - Carcinoma in a urethral diverticulum: magnetic resonance imaging and sonographic appearance. PMID- 10736516 TI - Communicating hematocele in children following splenic rupture: diagnosis and management AB - Acute hematocele is commonly associated with direct testicular trauma. Blood within the tunica vaginalis may infrequently accompany blunt abdominal injury in the presence of a communicating hydrocele. Optimal management involves early recognition and treatment of the abdominal source of bleeding. Elective repair of the communicating hydrocele/hematocele should follow. We report 2 cases of boys with scrotal swelling due to communicating hematoceles. Both cases were associated with a patent processus vaginalis and splenic laceration secondary to blunt trauma. PMID- 10736519 TI - Effect of a lipidic extract from lepidium meyenii on sexual behavior in mice and rats. AB - OBJECTIVES: To determine the effect of oral administration of a purified lipidic extract from Lepidium meyenii (MacaPure M-01 and M-02) on the number of complete intromissions and mating in normal mice, and on the latent period of erection (LPE) in rats with erectile dysfunction. METHODS: Mice and rats were randomly divided into several experimental and control groups. A 10% ethanol suspension of M-01 and M-02 was orally administered for 22 days to the experimental groups according to the dosage specified by the experimental design. On day 22, 30 minutes after the dose was administered to the male mice, 2 virgin female mice were placed with 1 male mouse. The number of complete intromissions of each male mouse in 3 hours was recorded. In an assessment of 1 day of mating, each male mouse was cohabited with 5 estrous female mice overnight. The number of sperm positive females was recorded. The LPE was measured to assess the sexual function in rats with erectile dysfunction. By using a YSD-4G multifunction instrument, an electric pulse at 20 V was applied to stimulate the rat's penis, and the duration from the start of the stimulus to full erection was measured in seconds as the LPE. RESULTS: In the normal male mice, the number of complete intromissions during the 3-hour period was 16.33 +/- 1.78, 46.67 +/- 2.39, and 67.01 +/- 2.55 for the control group, M-01 group, and M-02 group, respectively. In the assessment of mating, the number of sperm-positive females increased from 0.6 +/- 0.7 in the control group to 1.5 +/- 0.5 in the M-01 experimental group. The LPE of male rats with erectile dysfunction was 112 +/- 13 seconds with a regular diet (control group). The oral administration of M-01 at a dose of 180 or 1800 mg/kg body weight and M-02 at a dose of 45, 180, or 1800 mg/kg body weight reduced the LPE to 54 +/- 12 seconds, 54 +/- 13 seconds, 71 +/- 12 seconds, 73 +/- 12 seconds, and 41 +/- 13 seconds, respectively. The LPE of the surgical rats treated with M-01 at the lowest dose (45 mg/kg) was 121 +/- 12 seconds; thus, the change was not significant. CONCLUSIONS: Oral administration of M-01 and M-02 enhanced the sexual function of the mice and rats, as evidenced by an increase in the number of complete intromissions and the number of sperm-positive females in normal mice, and a decrease in the LPE in male rats with erectile dysfunction. The present study reveals for the first time an aphrodisiac activity of L. meyenii, an Andean Mountain herb. PMID- 10736518 TI - Effects of antidepressants in adrenergic neurotransmission of human vas deferens. AB - OBJECTIVES: To evaluate the effects of sertraline, fluoxetine, and amitriptyline on the contractile responses of the human vas deferens muscle elicited by norepinephrine, electrical field stimulation, and KCl, because the therapeutic action of antidepressants may be accompanied by sexual dysfunction related to the contractility of the vas deferens smooth muscle. METHODS: Ring segments of the epididymal part of the vas deferens were taken from 32 elective vasectomies and mounted in organ baths for isometric recording of tension. We then studied the effects of sertraline, fluoxetine, and amitriptyline on the neurogenic and agonist-induced contractile responses. RESULTS: Amitriptyline caused concentration-dependent inhibition of neurogenic and norepinephrine-induced contractions. In contrast, only the highest concentration (10(-5) M) of sertraline and fluoxetine reduced the adrenergic contractions. The dihydropyridine calcium antagonist nifedipine (10(-6) M) completely prevented the inhibitory effect of sertraline and fluoxetine on neurogenic and norepinephrine induced contractions but did not change the inhibition caused by amitriptyline. Sertraline, fluoxetine, and amitriptyline (all at 10(-5) M) attenuated contractions elicited by KCl and reduced contractions induced by CaCl(2) in KCl depolarized preparations. CONCLUSIONS: The results indicate that sertraline and fluoxetine inhibit vas deferens motility through inhibition of Ca(2+) entry, with no effect on the adrenergic receptors, and amitriptyline acts as an adrenoceptor antagonist and inhibitor of the entry of calcium. PMID- 10736520 TI - Mitochondrial DNA deletion of the human detrusor after partial bladder outlet obstruction-correlation with urodynamic analysis. AB - OBJECTIVES: To investigate mitochondrial DNA (mtDNA) mutations in human detrusor after partial bladder outlet obstruction (BOO) and correlate the findings with the results of urodynamic studies. METHODS: Sixty-two male patients with and without BOO were recruited and assessed by the International Prostate Symptom Score, a quality-of-life assessment index, and sonography. The severity of partial BOO was determined by pressure-flow study with an International Continence Society (ICS) nomogram. Random detrusor biopsies obtained cystoscopically were analyzed by polymerase chain reaction (PCR) techniques to detect possible mtDNA deletions. Primer-shift PCR and DNA sequencing were then performed to characterize specific mtDNA deletions. A semiquantitative PCR method was used to determine the proportion of the deleted mtDNA in detrusor. Finally, the mtDNA deletion and the urodynamic results were compared statistically. RESULTS: A 4977-bp mtDNA deletion was identified in the human detrusor. Its incidence and proportion were found to increase after partial BOO (P = 0.005 and 0.012, respectively). The incidence of the mtDNA deletion was 4.2% (1 of 24) in the unobstructed group, 27.8% (5 of 18) in the equivocal group, and 40% (8 of 20) in the obstructed group. The mean proportion of the 4977-bp deleted mtDNA was 23.7 and 12.7 times higher in the obstructed and equivocal groups, respectively, compared with that of the unobstructed group. CONCLUSIONS: We found mtDNA with the 4977-bp deletion in human detrusor and an increase of this deletion after partial BOO. This molecular change might account for the previous observations of mitochondrial functional impairment and voiding dysfunction after partial BOO. PMID- 10736522 TI - Mytilin B and MGD2, two antimicrobial peptides of marine mussels: gene structure and expression analysis. AB - Previous research has shown that mytilins and MGDs are two types of 4-kDa, cysteine-rich, cationic antimicrobial peptides, which are abundant in hemocytes of the mussels, Mytilus galloprovincialis and M. edulis. The expression of the genes encoding these peptides has been analyzed in the hemocytes of animals subjected to various stress factors, as well as during larval development. Variations in gene expression in adult mussels have been tested under conditions of physical stress, bacterial challenge and heat shock. The results suggest that in adult mussels, the MGD2 gene may be over-expressed with physical and temperature stress, but that reduced expression occurs with bacterial challenge. Gene expression during development has been analyzed using different larval and post-larval stages, ranging from 4-day-old veliger larvae to 32-day-old post larvae. The results show that the expression of both mytilin B and MGD2 is developmentally regulated, but neither gene is expressed in mussels until after larval settlement and metamorphosis. Finally, the genes encoding two isoforms of these peptides have been cloned and sequenced, revealing that both genes contain four exons and three introns. PMID- 10736521 TI - Interaction of hemocytes and prophenoloxidase system of fifth instar nymphs of Acheta domesticus with bacteria. AB - The hemocytes to which bacteria adhere were defined and the contribution of the prophenoloxidase system of fifth instar nymphs of Acheta domesticus to adhesion were examined. The physicochemical parameters affecting hemocyte and phenoloxidase activity were determined. Both plasmatocytes and granular cells responded to bacteria, the latter cells entrapping the microorganisms on filopodial extensions. The optimum pH for hemocyte adhesion to glass slides was 6.5, the granular cells being the most sensitive hemocyte type. Although hydrophobic resin beads and positively-charged beads favoured hemocyte attachment, these parameters did not contribute to differential bacterial adhesion to hemocytes. Activation of phenoloxidase was neither enhanced nor inhibited by 0.1 and 1 mg/ml of laminarin or zymosan nor by dead Bacillus subtilis. However, live B. subtilis activated the enzyme and dead Xenorhabdus nematophilus inhibited enzyme activation. Serine protease components of the prophenoloxidase system had opsonic properties for B. subtilis but not for X. nematophilus. Phenoloxidase activity was enhanced by Ca(2+) and Mg(2+) and inhibited by SO(2-)(4). PMID- 10736523 TI - Flow cytometric analysis of PKH26-labeled goldfish kidney-derived macrophages. AB - We recently demonstrated that a goldfish macrophage cell line (GMCL) and primary in vitro-derived kidney macrophage (IVDKM) cultures contain three distinct macrophage subpopulations. Morphological, cytochemical, functional, and flow cytometric characterization of these sub-populations suggested that they may represent cells of the macrophage lineage temporally arrested at distinct differentiation junctures of fish macrophage development (putative early progenitors, monocytes, and macrophages). In this study, we examined the proliferation and differentiation events leading to the generation of mature macrophage-like cells from goldfish kidney hematopoietic tissues. The flow cytometric studies were done after labeling macrophages with PKH26 fluorescent dye and analysis of the data using the MODFIT software. Our results showed that IVDKM cultures proliferated non-synchronously, suggesting the presence of a temporal control mechanism regulating the number of cells entering the paths towards maturation. Such control is most evident during early progenitor proliferation and differentiation events. Our results showed that proliferation may not be a requirement for differentiation of early progenitors to putative monocyte and macrophage subsets. Detailed observation of the mature macrophage like subpopulation indicated that: 1) they appear to develop from both, the differentiation of monocyte-like cells, and direct differentiation of early progenitors in the absence of a monocyte-like stage; and (2) mature macrophage like cells appeared to be capable of self-proliferation. Our results suggest the presence of alternate pathways of fish macrophage development other than the classical hematopoietic pathway. PMID- 10736524 TI - Cloning and analysis of expression of Mx cDNA in Japanese flounder, Paralichthys olivaceus. AB - Myxovirus resistance (Mx) is an interferon (IFN)-induced protein that specifically prevents growth, in vivo and in vitro, of certain classes of virus. We isolated the Mx cDNA from a leukocyte cDNA library obtained from a Japanese flounder infected with Hirame rhabdovirus (HRV). The Mx cDNA was found to contain 2385 bp coding for 620 amino acids. The amino acid sequence had approximately 51 and 78% identities to the sequences of human and rainbow trout Mx cDNAs, respectively. We propose that the amino acid sequence encoded by this cDNA clone contains a tripartite guanosine-5'-triphosphate (GTP)-binding motif, GXXXSGKS/T, DXXG and T/NKXD, and a signature of the dynamin family, LPRG(S/K)GIVTR, which are conserved in all IFN-induced Mx proteins of vertebrates. In addition, the C terminal region of the Japanese flounder Mx protein, like those of other Mx proteins, contains a highly conserved leucine zipper motif. Japanese flounder infected with the HRV showed increased expression of Mx mRNA in the leukocytes starting at 48 h and peaking at 72 h. An RT-PCR (reverse transcription-polymerase chain-reaction) analysis of Mx mRNA from a healthy fish showed that the Mx mRNA was predominantly expressed in the kidney, spleen, intestine, brain, peritoneal cavity fluid and gill, and only minimally expressed in leukocytes, liver, muscle and mucus. PMID- 10736525 TI - Interaction between turkey monocytes and avian Chlamydia psittaci in the presence of Mycoplasma sp.: the importance of nitric oxide. AB - The interaction between Chlamydia psittaci and turkey monocytes was studied in vitro. Purified monocytes were inoculated with C. psittaci, in the presence or absence of Mycoplasma hyorhinis. Whereas turkey monocytes produced high amounts of nitric oxide (NO) following the inoculation with M. hyorhinis, inoculation with C. psittaci did not induce NO production in these phagocytes. The monocytes strongly supported chlamydial growth, as demonstrated by the presence of inclusion forming units, the positive direct immunofluorescence staining and transmission electron microscopy. In contrast, upon co-inoculation of the monocytes with C. psittaci and M. hyorhinis, a reduced replication rate of C. psittaci was observed. N(G)-monomethyl-L-Arginine, a competitive inhibitor for the enzyme NO-synthase, inhibited the NO production and reversed the antichlamydial activity of the M. hyorhinis co-inoculated turkey monocytes. These results imply two considerations. First, as chlamydiae are obligate intracellular bacteria, special care should be taken to guard chlamydial cultures from mycoplasmal contamination, in order to prevent false results when investigating the response of immunomodulating cells to chlamydial infection. Secondly, as a mycoplasmal co-infection in vitro has the capacity of inducing antichlamydial activity in turkey monocytes, through the action of NO, it could be suggested that a similar interaction might take place in vivo. Moreover, it was shown that avian M. gallisepticum strains were also able to induce NO in turkey monocytes. Considering the high prevalence of both C. psittaci and Mycoplasma sp. in turkeys, this interaction, through the pivotal role of NO, might influence the outcome of respiratory diseases in turkeys. PMID- 10736526 TI - Cyclosporin A, but not bursectomy, abolishes the protective immunity of chickens against Leucocytozoon caulleryi. AB - Chickens that survive primary infection with Leucocytozoon caulleryi show strong resistance to reinfection. Using bursectomized (BX) or cyclosporin A (CsA) treated chickens, we performed experiments to determine which type of immunity, humoral or cellular immunity, plays an important role in the resistance of chickens against reinfection with L. caulleryi. BX chickens were inoculated with 2, 20 or 200 sporozoites of L. caulleryi intravenously at 3 weeks of age. Some BX chickens which were inoculated with 2 or 20 sporozoites survived the primary infection. These birds had no parasites in their peripheral blood after challenge infection with 5 x 10(3) sporozoites, even though they had no antibody to L. caulleryi. In contrast, CsA-treated chickens had parasitemia, serum-soluble antigen and antibodies, as did untreated chickens during primary infection. After secondary infection, CsA-treated chickens had parasitemia and serum-soluble antigen, even though they had specific antibodies to L. caulleryi whereas untreated chickens showed no parasitemia. The number of CD4(+), CD8(+), T cell receptor (TCR) alpha ss-bearing cells and TCRgamma delta-bearing cells decreased markedly in the peripheral blood of CsA-treated chickens compared to those of untreated chickens. Lymphocyte proliferation in response to concanavalin A, and T cell growth factor production, were also markedly suppressed in CsA-treated chickens. These results suggest that cell-mediated immunity plays an important role in the development of resistance of chickens against reinfection with L. caulleryi. PMID- 10736527 TI - Effects of density on displacement, falls, injuries, and orientation during horse transportation(1). AB - Three groups of slaughter-type horses, totaling 30 mares and 29 geldings, were used to determine density effects on displacement (distance moved during a stop), falls, injuries, and orientation using a single-deck, open-topped commercial semi trailer. Each horse was assigned to one of two treatments: high density (1.28 m(2)/horse with 14 horses) or low density (2.23 m(2)/horse with eight horses). Both treatments occurred sequentially on the same day (treatment order was alternated each trial), using the middle 2.44x7.32 m compartment of a large semi tractor trailer. The horses were transported for two laps around a 7.28-km course, averaging 25 min and 36+/-89 s. Each lap consisted of two 60 degrees turns, four 90 degrees turns, two 120 degrees turns, one 180 degrees turn, six hard brakes, and six rapid accelerations, which were more severe than conditions usually experienced in commercial transport. Displacement, falls, and orientation were recorded for each horse using overhead video cameras. Average displacement between the two densities was not different (P=0.47). The proportion of horses that fell in the high-density treatment (40%) was greater (P=0.046) than the low density treatment (17%). The proportion of horses injured was greater (P=0.006) in high density (64%) than low density (29%). However, there was not a significant difference in the average severity of injury for the high-density treatment (1.77) versus the low-density treatment (0.92), P=0.48. Overall, the horses did not show a preference (P=0.38) for facing toward (47.5%) or away (40.7%) from the direction of travel and orientation did not differ (P0.18) between the high and low-density treatments. High stocking density of horses during transport increases the incidence of falls and injuries, and made it more difficult to get up when a subject was floored. PMID- 10736528 TI - The number of farm mates influences social and maintenance behaviours of Japanese Black cows in a communal pasture. AB - The objective of the present study was to investigate optimal group size in cattle in communal pastures where cattle from various farms are turned out: (1) to ascertain if cows derived from each farm form an affiliative group and (2) to investigate the effect of the number of farm mates on social and maintenance behaviours when farm mates form an affiliative group. Farm mates were defined as cows from the same farm. A total of 27 cows having zero, one, two to four, or sixteen farm mates were selected as focal animals in two communal pastures. Each focal animal was followed by an individual observer for 8 h from sunrise. The nearest neighbour and the distance to the nearest neighbour were recorded at 10 min intervals, and maintenance behaviours at 1-min intervals; social behaviours and participants were recorded continuously. Most focal animals frequently choose cows from the same farm as their nearest neighbours allogroomed with these farm mates significantly more (P<0.001). Two focal animals did not form affiliative groups with farm mates. The mean distance to the nearest neighbours, whether farm mates or non-farm mates from focal animals having one and two to four farm mates was shorter than cows turned out with no farm mate (1.4 vs. 1.9 times as long as a cow's body length, P<0.05). Cows having two to four farm mates performed (7.8 s/h/cow, P=0.06) and received allogrooming more (9.8 s/h/cow) and escaped less during agonistic encounters (0.1 frequencies/h/cow, P<0.01) than ones having other numbers of farm mates. The mean duration of grazing behaviour per bout tended to increase with the number of farm mates (5.2 to 10.3 min, P<0.05). The mean duration of recumbency behaviour of cows having two to four farm mates (44.0 min/bout) was longer than the ones having other numbers of farm mates. Social and maintenance behaviours of cows having 16 farm mates were like those having zero or one farm mates, which suggested that the social bond among them was weak. It is concluded that a group of cows having two to four farm mates, that is, a group size of three to five cows, may be optimal for a stable life in a communal pasture. PMID- 10736529 TI - The spontaneous qualitative assessment of behavioural expressions in pigs: first explorations of a novel methodology for integrative animal welfare measurement. AB - Qualitative assessments of behaviour integrate and summarize the different aspects of an animal's dynamic style of interaction with the environment, using descriptors such as 'timid' or 'confident'. Although such qualitative terms are widely used in the study of animal temperament and personality, their use in relation to questions of animal welfare has yet to be formally explored. The terms used in integrative assessment (e.g., content, distressed) tend to have expressive, welfare-related connotations, and lie at the heart of the lay public's concern for animal suffering. For this reason they are frequently dismissed as 'anthropomorphic' and unscientific. However, in theory it is possible that these terminologies reflect observable aspects of behavioural organization. They may therefore be liable to scientific analysis, and be of use as integrative welfare measurements. A first step in investigating this hypothesis is to examine the inter-observer reliability of assessments of behavioural expression. This study investigated the extent to which 18 naive observers showed agreement when given the opportunity to qualitatively describe, independently and in their own words, the behavioural expressions of 20 individual growing pigs. Pigs were brought singly into a test pen and given the opportunity to interact with a human squatting in the centre of the test pen. Observers were instructed to first observe each pig and then to write down terms which adequately summed up the emergent qualities of that pig's behaviour. Data thus consisted of 18 sets of individually generated descriptive terms, attributed to 20 pigs. This procedure was repeated a month later with the same observers but using a new group of 20 pigs. To analyze the resulting 36 sets of descriptive terms, pigs in each set were given a score for each term. This score was either 0 (term not used for that pig) or 1 (term used for that pig). These data were analyzed with Generalized Procrustes Analysis (GPA), a multivariate statistical technique which finds a consensus between observer assessment patterns (the 'pig consensus profile'), and provides a measure of observer agreement. Results show that for each group of 20 pigs, the 'pig consensus profile' differed significantly from an analysis of the same data in randomized form (p<0.001), indicating that the consensus profiles were not artifacts of the GPA procedures. It can therefore be concluded that observers showed significant agreement in their spontaneous assessment of pig expressions, which suggests that these assessments were based on commonly perceived and systematically applied criteria. The extent to which these shared criteria reflect observable aspects of behaviour now requires further study. PMID- 10736530 TI - Rearing without early access to perches impairs the spatial skills of laying hens. AB - The effect of rearing with and without perches on the spatial ability of domestic hens (Gallus gallus domesticus) was investigated. No access or late access to perches during rearing has been shown to increase the later prevalence of floor eggs and cloacal cannibalism in loose-housed laying hens. This may be explained by either the birds reared without perches have difficulty using perches due to low muscle strength, lack of motor skills, and inability to keep balance, or they have impaired spatial skills necessary for moving around in three-dimensional space. These alternative explanations are not mutually exclusive.Thirty, day-old chicks were randomly allocated into two equal groups and reared in litter pens, one with access to perches (P+) and one without (P-). At 8 weeks of age, all birds were given access to perches, and by 15 weeks, all birds were using perches for roosting at night. At 16 weeks, 10 birds from each group were tested in pens where food was presented on a wire mesh tier 40 cm above the ground (T40). Three consecutive tests, with increasing difficulty for the bird to reach the food, were then performed. Firstly, the food was presented at 80 cm above the ground but with the tier at 40 cm still present; secondly, food was presented on the tier at 80 cm; and then, finally, with the food on a 160 cm high tier with the tier at 80 cm still present. All birds were food deprived for 15 h before each test and the time from the bird entering the pen until reaching the food was recorded. There was no difference in the time to reach the food between P+ and P- birds in the T40 test. But as the difficulty of the task increased, the difference between the P+ and P- birds became significant, with the P- birds taking a longer time to reach the food or not reaching it at all. Since there was no difference between P+ and P- in the T40 test, it seems reasonable to suppose that the later differences did not depend on differences in physical ability. Therefore, the results may imply that rearing without early access to perches, in some ways, impairs the spatial cognitive skills of the domestic hen. PMID- 10736531 TI - A comparative assessment of potential conditioned taste aversion agents for vertebrate management. AB - A conditioned taste aversion (CTA) is acquired through an association between the taste of a food and a feeling of illness experienced after ingestion. It can be induced deliberately by the addition of an undetectable illness-inducing chemical to food. Harnessing the CTA response could provide humane and effective means of controlling vertebrate pest problems. For field use, the ideal illness-inducing chemical should induce a robust CTA after a single oral dose, at which it must cause neither chronic illness nor persistent detrimental effects in the target or any non-target species at risk of exposure; it must also be undetectable and physically stable in the bait substrate. At present, no compound that satisfactorily meets all of these criteria has been identified. 17alpha ethinyl oestradiol meets most but, as a synthetic oestrogenic hormone, it can affect reproductive processes. The ability of two potentially safe compounds, cinnamamide (160 mg/kg) and thiabendazole (100 and 200 mg/kg) to generate a CTA in the laboratory rat Rattus norvegicus11 post-treatment tests (6 months). Thiabendazole at 200 mg/kg induced the next best CTA, persisting for five post treatment tests. Cinnamamide and thiabendazole could provide safe alternative CTA agents to 17alpha ethinyl oestradiol for field use; the use of a second dose of these compounds to improve longevity of the CTA warrants further study. PMID- 10736532 TI - Short-term social memory in the laboratory rat: its susceptibility to disturbance. AB - Adult rats presented with a juvenile conspecific for 5 min on two occasions, separated by a 15-min inter-exposure interval (IEI), investigated the reintroduced juvenile significantly less in the second encounter. It is suggested that this was because the adult rats remembered the identity of the juvenile, because when a novel juvenile was introduced for the second encounter, no such reduction in investigation was observed. When the rats were either handled, placed in a smaller, novel, cage, or introduced to a new juvenile midway through the IEI, investigation of the reintroduced juvenile did not decrease. This indicated that memory of that juvenile had been disrupted. However, a simple change of cage during the IEI had no disruptive effect on memory. These results suggest that routine husbandry procedures can disrupt short-term social memory, which may lead to an increase in aggression due to a failure of recognition. This has implications for the welfare of captive social animals. PMID- 10736533 TI - Military injuries and public health. An introduction. PMID- 10736534 TI - Reflections on injuries in the military: the hidden epidemic. PMID- 10736535 TI - There's safety in numbers. PMID- 10736536 TI - The use of existing military administrative and health databases for injury surveillance and research. PMID- 10736537 TI - An armed forces epidemiological board evaluation of injuries in the military. PMID- 10736538 TI - Deaths due to injury in the military. AB - INTRODUCTION: More military personnel die of injuries each year than any other cause. This paper provides a basic epidemiologic description of injury deaths in the military. METHODS: Using fatality data from the Department of Defense Directorate of Information and Operations Reports and population data from the Defense Manpower Data Center, death rates of men and women in the military services for unintentional injury, suicide, homicide, and illness were calculated for the 1980-1992 period. RESULTS: From 1980 to 1992, injuries (unintentional injuries, suicides, and homicides combined) accounted for 81% of all nonhostile deaths among active duty personnel in the Armed Services. The overall death rate due to unintentional injuries was 62.3 per 100,000 person-years. The suicide rate was 12.5, the homicide rate 5.0, and the death rate due to illness 18.4. From 1980 to 1992 mortality from unintentional injuries declined about 4% per year. The rates for suicide and homicide were stable. Men in the services die from unintentional injuries at about 2.5 times the rate of women and from suicides at about twice the rate of women. Women in the military, however, have a slightly higher homicide rate than men. CONCLUSION: Injuries (unintentional injuries, suicides, and homicides) are the leading cause of death among active duty members of the U.S. Armed Forces, accounting for about four out of five deaths. The downward trend for fatal unintentional injuries indicates the success that can be achieved when attention is focused on preventing injuries. Further reduction in injury mortality would be facilitated if collection and coding of data were standardized across the military services. PMID- 10736539 TI - Disabilities due to injury in the military. AB - INTRODUCTION: Disability is a major health and economic issue in the Armed Forces associated with increased use of medical care, the loss of active duty time, and substantial compensation costs. METHODS: The role of injuries in physical disability from the early 1980s to 1994 was assessed by reviewing administrative data from the U.S. Army Physical Disability Agency, the Naval Disability Evaluation Board, and the Air Force Physical Disability Division. Information on the number of disability cases reviewed in 1994, the leading causes of disability, and the disposition of each case were examined most closely. Also, information from the Department of Defense on the cost of compensating disability cases was reviewed. RESULTS: Disability generally appears to be significant across the services, ranging from 10 to 30 events per 1000 personnel per year depending on the service. Evidence from the data reviewed indicates that 30% to 50% of disability cases may be due to injury. The leading conditions that bring about board reviews and lifetime compensation appear to be lower back and knee conditions, both commonly thought to be due to injuries. Total direct costs of compensation reached $1. 5 billion for fiscal year 1990. CONCLUSIONS: While current disability data systems are maintained for administrative and not research purposes, the information available may be valuable for injury surveillance and research and suggests that injury-related disability is a major health and economic burden for the Armed Forces. PMID- 10736540 TI - Hospitalization due to injuries in the military. Evaluation of current data and recommendations on their use for injury prevention. AB - INTRODUCTION: Injuries inflict the largest health impact on military populations in terms of hospitalization. Hospitalized injuries result in the largest direct costs of medical care and the most lost workdays, include the largest proportion of disabling injuries, and have the largest impact on troop readiness. Efforts are now beginning to focus on how injury surveillance data can be used to reduce the burden of injuries. This article examines the value of administrative hospital discharge databases in the military for routine injury surveillance, as well as investigation of specific injury problems, including musculoskeletal conditions that are frequently sequelae of old injuries. METHODS: Data on hospitalizations for injuries and musculoskeletal conditions were obtained from separate administrative agencies for the Army, Navy, and Air Force. Since 1989, a Standard Inpatient Data Record (SIDR) has been used to ensure uniformity in data collection across the services utilizing standard ICD-9 codes. Cause of injury was coded using special military cause codes (STANAG codes) developed by NATO. Data were analyzed on both nature and cause of injury. Denominator data on troop strength were obtained from the Defense Manpower Data Center (DMDC). RESULTS: Hospital records data indicate that injuries and musculoskeletal conditions have a bigger impact on the health of service members and military/combat readiness than any other ICD-9 Principal Diagnostic Group (higher incidence and higher noneffective rate or days not available for duty). Hospitalization rates for injury appeared to decline for all services from 1980 to 1992. In 1992, service specific injury hospitalization rates per 1000 person-years were 15.6 for the Army, 8.3 for the Navy (enlisted only), and 7.7 for the Air Force, while the corresponding hospitalization rate for musculoskeletal conditions was higher in all three services: 28.1, 9.7, and 12.0, respectively. CONCLUSIONS: Military hospital discharge databases are an important source of information on severe injuries and are more comprehensive than civilian databases. They include detailed injury information that can be useful for injury prevention and surveillance purposes. Specifically, it can be used to identify high-risk groups or hazards for targeting prevention resources. These may vary widely by service, rank, and job tasks. Hospital discharge data can also be used to evaluate the effectiveness of interventions for reducing injury rates. Recommendations were submitted to further improve data collection and the use of hospital data for research and injury prevention. PMID- 10736541 TI - Military training-related injuries: surveillance, research, and prevention. AB - BACKGROUND: Musculoskeletal physical training-related injuries are a major problem in military populations. Injuries are important in terms of loss of time from work and training and decreased military readiness. The implications of these injuries in terms of patient morbidity, attrition rates, and training costs for military personnel are staggering. This article reviews: (1) pertinent epidemiologic literature on musculoskeletal injury rates; (2) injury type and location; and (3) risk factors for military populations. Suggestions for injury surveillance and prevention are also offered. METHODS: Existing military and civilian epidemiologic studies were used to estimate and compare the size of the injury problem, identify risk factors, and test preventive measures. Most of the military research data obtained was from Marine and Army recruits, Army Infantry soldiers, and Naval Special Warfare candidates. Additional studies conducted in operational forces provided documentation of the injury problem in these populations as well. RESULTS: Injury rates during military training are high, ranging from 6 to 12 per 100 male recruits per month during basic training to as high as 30 per 100 per month for Naval Special Warfare training. Data collected show a wide variation in injury rates that are dependent largely on the following risk factors: low levels of current physical fitness, low levels of previous occupational and leisure time physical activity, previous injury history, high running mileage, high amount of weekly exercise, smoking, age, and biomechanical factors. (Data are contradictory with respect to age.) CONCLUSION: Considering the magnitude of training injuries in military populations, there is a substantial amount of work that remains to be performed, especially in the areas of surveillance, prevention, and treatment. Modifiable risk factors have been identified suggesting that overuse and other training injuries could be decreased with proper interventions. Outpatient surveillance systems are available to capture musculoskeletal injury data but need to be refined. Given the size of the problem, a systematic process of prevention should be initiated starting with routine surveillance to identify high-risk populations for the purpose of prioritizing research and prevention. Properly planned interventions should then be implemented with the expectation of dramatically reduced lost work/training time, attrition, and medical costs, while increasing military readiness. PMID- 10736542 TI - Non-battle injury casualties during the Persian Gulf War and other deployments. AB - OBJECTIVE: To review injury occurrence and to evaluate various injury surveillance systems used on recent deployments of U.S. military personnel. BACKGROUND: Injuries that occur in a deployed military force are more likely to have an immediate and detrimental effect on the military mission than those in garrison or training. These injuries have a direct impact on deployed personnel and unit readiness and consume limited field medical resources. METHODS: Data collected during four recent deployments were evaluated. Administrative databases established for the routine collection of death and hospital admissions were used to characterize mortality and morbidity in the Persian Gulf War. Surveillance teams deployed to Haiti, Somalia, and Egypt provided inpatient and outpatient data for those missions. RESULTS: Data collected by these surveillance systems are presented. Unintentional trauma accounted for 81% of deaths during the Persian Gulf War and 25% of hospital admissions. During operations in Somalia and Haiti, 2.5% to 3.5% of about 20,000 troops in each deployment sought medical treatment for an injury or orthopedic problem each week. In Egypt, injuries accounted for about 25% of all outpatient visits to medical treatment facilities. CONCLUSIONS: Injuries were the leading cause of death and a leading cause of morbidity during recent deployments of U.S. troops. Comprehensive injury surveillance systems are needed during deployments to provide complete and accurate information to commanders responsible for the safety of the force. Recommendations for establishing such systems are made in this article. PMID- 10736543 TI - Injuries in the military: a review and commentary focused on prevention. AB - BACKGROUND: In November 1996, the Armed Forces Epidemiological Board (AFEB) Injury Prevention and Control Work Group issued a report that cited injuries as the leading cause of morbidity and mortality among military service members. This article reviews the types and categories of military morbidity and mortality data examined by the AFEB work group and the companion Department of Defense (DoD) Injury Surveillance and Prevention Work Group. This article further uses the injury data reviewed to illustrate the role of surveillance and research in injury prevention. The review provides the context for discussion of the implications of the AFEB work group's findings for the prevention of injuries in the military. METHODS: The AFEB work group consisted of 11 civilian injury epidemiologists, health professionals and scientists from academia, and other non DoD government agencies, plus six military liaison officers. Injury data from medical databases were provided to the civilian experts on the AFEB work group by the all-military DoD Injury Surveillance and Prevention Work Group. The AFEB work group assessed the value of each database to the process of prevention and made recommendations for improvement and use of each data source. RESULTS: Both work groups found that injuries were the single leading cause of deaths, disabilities, hospitalizations, outpatient visits, and manpower losses among military service members. They also identified numerous data sources useful for determining the causes and risk factors for injuries. Those data sources indicate that training injuries, sports, falls, and motor vehicle crashes are among the most important causes of morbidity for military personnel. CONCLUSIONS: While the work group recommends ways to prevent injuries, they felt the top priority for injury prevention must be the formation of a comprehensive medical surveillance system. Data from this surveillance system must be used routinely to prioritize and monitor injury and disease prevention and research programs. The success of injury prevention will depend not just on use of surveillance but also partnerships among the medical, surveillance, and safety agencies of the military services as well as the military commanders, other decision makers, and service members whose direct actions can prevent injuries and disease. PMID- 10736544 TI - Self-reported risk-taking behaviors and hospitalization for motor vehicle injury among active duty army personnel. AB - BACKGROUND: Motor vehicle crashes are a leading cause of injury in the Army. Behaviors increasing risk for motor vehicle crashes are also prevalent, but research has not linked these behaviors directly to injury outcomes (e.g., hospitalizations). METHODS: To evaluate the relationship between behavior and motor vehicle crash injuries, 99, 981 Army personnel who completed Health Risk Appraisal surveys in 1992 were followed for up to 6 years. Cox proportional hazards modeling was used to evaluate speeding, seat belt use, drinking patterns, and demographics. RESULTS: A total of 429 soldiers were hospitalized for motor vehicle injury. Unadjusted analyses revealed that heavy drinking, drinking and driving, speeding, low seat belt use, younger age, minority race/ethnicity, and enlisted rank were significantly associated with motor vehicle injury, but neither smoking nor gender was. Multivariate models showed a significant trend of increasing injury risk with younger ages. Soldiers under age 21 were injured almost five times more often than those over age 40 (HR 4.89, 2.56-9.33). Also associated with risk for hospitalizations were minority race (HR 1.78, 1.46 2.18), heaviest drinkers versus abstainers (HR 1.81, 1.11-2.94), and seat belt use of 50% or less versus 100% (HR 1.40, 1.07-1.85). Although nonsignificant, there was evidence of an age-drinking interaction where the difference in injury risk between those older and those younger than 21 was greatest at low alcohol consumption levels. CONCLUSIONS: Modifiable risk factors associated with motor vehicle injuries include heavy drinking and low seat belt use. Programs targeting these behaviors that meet the needs of young and minority soldiers are needed. The high density of young, at-risk soldiers residing in base housing may provide a unique opportunity for a residential intervention program. PMID- 10736545 TI - Cigarette smoking and exercise-related injuries among young men and women. AB - BACKGROUND: We evaluate whether a recent history of cigarette smoking is a risk factor for exercise-related injuries sustained during Army basic training, controlling for factors such as demographic, physical fitness, and health variables. METHODS: We conducted an observational cohort study in 1087 male and 915 female Army recruits undergoing 8-week basic military training. Data were collected from questionnaires, anthropometric measurements, physical fitness tests, company training logs, and medical records of all clinic visits. RESULTS: During the 8-week training period, 33% of men and 50% of women had at least one clinic visit for injury, including 14% of men and 25% of women who lost more than 5 days of training due to injury. Recruits who reported smoking at least one cigarette in the month prior to beginning basic training (which was conducted in a smoke-free environment) had significantly higher injury rates during training than those who did not report smoking (40% versus 29% for men, and 56% versus 46% for women). The relationship with smoking history was present most strongly for overuse injuries (32% versus 24% in men and 51% versus 40% in women). Multiple logistic regression analyses controlling for all other factors consistently showed adjusted odds ratios of about 1.5 for injury rate in those with a history of smoking compared to those without. CONCLUSIONS: The association of history of cigarette smoking with injury occurrence was consistent throughout the analyses, with very little confounding by other factors. The detrimental effects of smoking on injuries appears to persist at least several weeks after cessation of smoking. PMID- 10736546 TI - Case-control study of discharge from the U.S. Army for disabling occupational knee injury: the role of gender, race/ethnicity, and age. AB - BACKGROUND: Occupational injuries are responsible for more lost time from work, productivity, and working years of life than any other health condition in either civilian or noncombat military sectors. Injuries, not illnesses, are the leading cause of morbidity and mortality among U.S. Army personnel. We examined the separate and joint roles of gender, race/ethnicity, and age in the odds of discharge from the Army for disabling knee injury. METHODS: A total of 860 women and 7868 men were discharged from the Army between 1980 and 1995 for knee-related disability and met all inclusion criteria for this study. All women and a subsample of 1005 men were included in these analyses, along with a simple random sample of three controls per case, stratified by gender, drawn from the population of all active-duty enlisted soldiers in each year from 1980 to 1995. We identified predictors of the occurrence or nonoccurrence of discharge from the Army for disabling knee injury using unconditional multiple logistic regression analyses. RESULTS: We found relations between the risk of knee-related disability and age and race, with marked effect modification by gender. Non-Caucasian men and women were at lower risk than Caucasians at all ages. At most ages, Caucasian women were at higher risk than Caucasian men, and non-Caucasian women were at lower risk than non-Caucasian men. Within race/ethnicity and gender, the risks for men showed an inverted "U" shape with increasing age, and the risks for women showed a "J" shape with increasing age. CONCLUSIONS: Age, race/ethnicity, and gender interactions are important in occupational injury. Differences in risk may be related to differences in work assignments, leisure activities, physical or physiological differences, or the ways in which disability compensation is granted. PMID- 10736547 TI - Prior knee injury and risk of future hospitalization and discharge from military service. AB - BACKGROUND: Athletic capability is paramount for survival in military basic training and successful service. Orthopedic conditions are common reasons for hospitalization and premature discharge of military recruits. Medical fitness for military service is determined through a medical examination. Individuals medically disqualified may receive a waiver to enter the service on a case-by case basis. This study was carried out to determine how individuals with a medical waiver for knee problems compared to recruits without a history of knee injury regarding hospitalization and military discharge. METHODS: Two hundred eighty-one enlisted recruits with a history of a waiver for a knee condition were considered high risk. The comparison group was 843 recruits without prior knee pathology. Comparisons were made using frequency and chi-square analyses, relative risk estimates, and survival analyses. RESULTS: Individuals in the high risk group were 1.4 (CI 1.0, 2.1) times more likely to be hospitalized for any diagnoses and 8.0 (CI 2. 1, 29.9) times more likely to be hospitalized for a knee condition than those in the comparison group. Individuals with a knee waiver were 2.1 (CI 1.3, 3.5) times more likely to be prematurely discharged, and 14.0 (CI 4.6, 39.6) times more likely to be discharged for a knee-related condition than those in the comparison group. CONCLUSION: Unfavorable outcomes were more likely in recruits disqualified initially and granted a waiver than in recruits without a history of knee injury. Military service requires intense physical activity; therefore, further research should be conducted to limit knee-related morbidity, especially in those with a prior history of knee injury. PMID- 10736548 TI - Sports and physical training injury hospitalizations in the army. AB - INTRODUCTION: Injuries are the leading health problem in the military services. Sports and physical training activities are an area in which a substantial number of injuries can occur. Although athletic injuries are not often investigated in military populations, the Armed Forces database provides a unique opportunity to investigate sports injuries. METHODS: An Army database of all hospital admissions for active duty Army personnel in the 1989-1994 period was used to study injuries resulting from sports and Army physical training. RESULTS: For the 6-year time period reviewed, there were 13,861 hospital admissions for injuries resulting from sports or Army physical training: 94% (13,020) of these admissions were men and 6% (841) were women. The rates of sports injuries were 38 and 18 per 10,000 person-years for men and women, respectively. Sports injuries accounted for an average of 29,435 lost duty days each year: Men lost an average of 13 days per injury and women lost an average of 11 days per injury. Acute musculoskeletal injuries in the categories of fractures, sprains/strains, and dislocations accounted for 82% of all injuries. The knee was the most often injured body area in both genders, with the anterior cruciate ligament (ACL) identified as the most frequently injured body part overall. The top seven injuries were virtually identical for men and women, with only slight variations in order. Although the rates of all hospitalized sports injuries were higher for men than women, women had a higher proportion of ACL injuries from basketball and softball, ankle fractures from softball and head injuries from basketball. For men, football and basketball contributed to the highest rates of injuries. The highest injury rates for women were from Army physical training and basketball. For both men and women, Army physical training was the leading cause of lumbosacral strains. CONCLUSIONS: Sports and Army physical training injuries account for a significant amount of lost duty time and impact military readiness. PMID- 10736549 TI - U.S. Air force recruit injury and health study. AB - OBJECTIVES: To assess the types, rates, and risks of injury for male and female USAF recruits. DESIGN: Outpatient visits for female (5250) and male recruits (8656) were collected and analyzed for rate of injury, types of injuries, and risk of injury throughout a 6-week training period. RESULTS: One third of female recruits and one sixth of male recruits were injured at least once during recruit training. The overall rate for injuries in women and men was 63.0 and 27.8 per 1000 person-weeks, respectively. The adjusted relative risk for women versus men for all injuries was 2.22, and was consistent (1.67 to 3.27) across injury sites. Despite declining absolute rates of injury by week (106.1-13.4 for women and 53.7 13.2 for men), relative risk of injury for women versus men remained fairly constant throughout each training week. The relative risk for injury serious enough to result in medical hold was 1.69 for women vis-a-vis men. Approximately half of all medical discharges for women and men were for injuries. CONCLUSIONS: Female recruits were injured twice as often as male recruits, and were 1.5 times more likely to be removed from their training cohort for injury. Relative risk for injuries to specific body areas remained fairly consistent, indicating that no gender-specific injuries were occurring. Further efforts to determine the cause of injuries should be undertaken, and interventions aimed at reducing the disparate risk of injuries in women should be developed and evaluated. PMID- 10736550 TI - High injury rates among female army trainees: a function of gender? AB - BACKGROUND: Studies suggest that women are at greater risk than men for sports and training injuries. This study investigated the association between gender and risk of exercise-related injuries among Army basic trainees while controlling for physical fitness and demographics. METHODS: Eight hundred and sixty-one trainees were followed during their 8-week basic training course. Demographic characteristics, body composition, and physical fitness were measured at the beginning of training. Physical fitness measures were taken again at the end of training. Multivariate logistic regression analysis was used to evaluate the association between gender and risk of injury while controlling for potential confounders. RESULTS: Women experienced twice as many injuries as men (relative risk [RR] = 2.1, 1.78-2.5) and experienced serious time-loss injuries almost 2.5 times more often than men (RR = 2.4, 1. 92-3.05). Women entered training at significantly lower levels of physical fitness than men, but made much greater improvements in fitness over the training period.In multivariate analyses, where demographics, body composition, and initial physical fitness were controlled, female gender was no longer a significant predictor of injuries (RR = 1.14, 0.48 2.72). Physical fitness, particularly aerobic fitness, remained significant. CONCLUSIONS: The key risk factor for training injuries appears to be physical fitness, particularly cardiovascular fitness. The significant improvement in endurance attained by women suggests that women enter training less physically fit relative to their own fitness potential, as well as to men. Remedial training for less fit soldiers is likely to reduce injuries and decrease the gender differential in risk of injuries. PMID- 10736552 TI - Evaluating risk of re-injury among 1214 army airborne soldiers using a stratified survival model. AB - BACKGROUND: Many factors interact to influence an injured individual's risk of sustaining a second injury. However, the quantitative assessment of subsequent injury risk has been limited, primarily due to methodologic constraints. The purpose of this study is to present analytical methodology not previously employed in injury epidemiology to identify risk factors for subsequent injury. METHODS: Data were collected from a retrospective cohort of 1214 U.S. Army Airborne soldiers. Lower extremity and low-back musculoskeletal injuries were identified from outpatient medical records. The Prentice, Williams, and Peterson (PWP) model, stratified by injury event, was used to identify risk factors for initial and subsequent injuries. A Cox proportional hazards model to the time of last injury was used to determine the magnitude of the increased risk associated with having a previous injury history. RESULTS: Risk factors for initial injuries were similar to those seen in other epidemiologic studies of military populations. However, this study found that race/ethnicity, physical fitness, medical provider training, and initial injury types (traumatic versus other) were associated with subsequent injury risk. Additionally, the observed risk of injury was seven times greater among previously injured individuals. CONCLUSIONS: In this population, the risk factors for injury differed by event (initial or subsequent injury), and prior injury history was a risk factor for subsequent injury. The associations between demographic characteristics, the nature of the initial injury, and risk of subsequent injury suggest that changes in the evaluation and medical management of injured individuals may decrease the risk of subsequent injury. PMID- 10736551 TI - Effect of rest from running on overuse injuries in army basic training. AB - BACKGROUND: It has been hypothesized that a period of rest from running in the early weeks of basic military training will prevent stress fractures among recruits. DESIGN: Modification of running schedules in companies of Army recruits undergoing basic military training was assigned. SETTING/PARTICIPANTS: Six male training companies were enrolled and followed during their 8 weeks of basic military training at Fort Bliss, Texas, in summer/fall 1989. INTERVENTION: Intervention companies were asked to rest from running during the second, third, or fourth week of basic military training. MAIN OUTCOME MEASURES: Data were collected from questionnaires, anthropometric measurements, Army physical fitness tests, company training logs, and medical record abstraction of all clinic visits. RESULTS: Among the 1357 enrolled male recruits, there were 236 (17%) with overuse injury and 144 (11%) with traumatic injury, resulting in 535 clinic visits and 1927 training days lost. Stress fracture/reaction rates varied from 3 to 8 per 100 recruits among the intervention companies and 2 to 7 per 100 recruits among the non-intervention companies. Total injury rates were 18 to 35 per 100 recruits in the intervention companies and 18 to 29 per 100 recruits in the non-intervention companies. CONCLUSIONS: The study provided no evidence for a protective effect on overuse injuries of resting from running for 1 week early in basic military training. There was varied physical training among the companies, however, with variation of injury rates that likely related to factors other than the intervention. PMID- 10736553 TI - Viewpoint: a comparison of cause-of-injury coding in U.S. military and civilian hospitals. AB - INTRODUCTION: Complete and accurate coding of injury causes is essential to the understanding of injury etiology and to the development and evaluation of injury prevention strategies. While civilian hospitals use ICD-9-CM external cause-of injury codes, military hospitals use codes derived from the NATO Standardization Agreement (STANAG) 2050. DISCUSSION: The STANAG uses two separate variables to code injury cause. The Trauma code uses a single digit with 10 possible values to identify the general class of injury as battle injury, intentionally inflicted nonbattle injury, or unintentional injury. The Injury code is used to identify cause or activity at the time of the injury. For a subset of the Injury codes, the last digit is modified to indicate place of occurrence. This simple system contains fewer than 300 basic codes, including many that are specific to battle- and sports-related injuries not coded well by either the ICD-9-CM or the draft ICD-10-CM. However, while falls, poisonings, and injuries due to machinery and tools are common causes of injury hospitalizations in the military, few STANAG codes correspond to these events. Intentional injuries in general and sexual assaults in particular are also not well represented in the STANAG. Because the STANAG does not map directly to the ICD-9-CM system, quantitative comparisons between military and civilian data are difficult. CONCLUSIONS: The ICD-10-CM, which will be implemented in the United States sometime after 2001, expands considerably on its predecessor, ICD-9-CM, and provides more specificity and detail than the STANAG. With slight modification, it might become a suitable replacement for the STANAG. PMID- 10736554 TI - Qualitative assessment of cause-of-injury coding in U.S. military hospitals: NATO standardization agreement (STANAG) 2050. AB - BACKGROUND: Accurate injury cause data are essential for injury prevention research. U.S. military hospitals, unlike civilian hospitals, use the NATO STANAG system for cause-of-injury coding. Reported deficiencies in civilian injury cause data suggested a need to specifically evaluate the STANAG. METHODS: The Total Army Injury and Health Outcomes Database (TAIHOD) was used to evaluate worldwide Army injury hospitalizations, especially STANAG Trauma, Injury, and Place of Occurrence coding. We conducted a review of hospital procedures at Tripler Army Medical Center (TAMC) including injury cause and intent coding, potential crossover between acute injuries and musculoskeletal conditions, and data for certain hospital patients who are not true admissions. We also evaluated the use of free-text injury comment fields in three hospitals. RESULTS: Army-wide review of injury records coding revealed full compliance with cause coding, although nonspecific codes appeared to be overused. A small but intensive single hospital records review revealed relatively poor intent coding but good activity and cause coding. Data on specific injury history were present on most acute injury records and 75% of musculoskeletal conditions. Place of Occurrence coding, although inherently nonspecific, was over 80% accurate. Review of text fields produced additional details of the injuries in over 80% of cases. CONCLUSIONS: STANAG intent coding specificity was poor, while coding of cause of injury was at least comparable to civilian systems. The strengths of military hospital data systems are an exceptionally high compliance with injury cause coding, the availability of free text, and capture of all population hospital records without regard to work-relatedness. Simple changes in procedures could greatly improve data quality. PMID- 10736555 TI - Older women's health and access to care. PMID- 10736556 TI - Medicare and drug coverage: a women's health issue. AB - The lack of prescription drug coverage under the Medicare program translates into high out-of-pocket drug costs for seniors. This nationwide study of older Americans with hypertension ("high blood pressure") demonstrates that women bear the disproportionate burden of this gap in Medicare coverage. Women form the majority of older people with hypertension, and are less likely to have supplemental policies to cover the cost of the prescription drugs that are needed to treat the disease. Moreover, women have substantially lower incomes. Despite their economic vulnerability, older women with hypertension spend substantially more on prescription drugs than men. PMID- 10736557 TI - Discussion of papers by Karen Scott Collins and Jan Blustein. PMID- 10736558 TI - Discussion of papers by Karen Scott Collins and Jan Blustein. PMID- 10736559 TI - Making capitated Medicare work for women: policy and research challenges. AB - Growth in capitated Medicare has special ramifications for older women who comprise the majority of Medicare beneficiaries. Older women are more likely than men to have chronic conditions that lead to illness and disability, and they often have fewer financial and social resources to cope with these problems. Gender differences in health status have a number of important implications for the financing and delivery of care for older women under both traditional fee-for service Medicare and capitation. The utilization of effective preventive interventions, new therapeutic interventions for the management of common chronic disorders, and more cost-effective models of chronic disease management could potentially extend the active life expectancy of older women. However, there are financial and delivery system barriers to achieving these objectives. Traditional FFS Medicare has gaps in coverage of care for chronic illness and disability that disproportionately impact women. Managed care potentially offers flexibility to allocate resources creatively, to develop new models of care, and offer enhanced benefits with lower out-of-pocket costs. However, challenges to realizing this potential under Medicare managed care with unique implications for older women include: possible gender bias in capitation payments, risk selection, inadequacy of risk adjustment models, benefit and market instability, and disenrollment patterns. PMID- 10736560 TI - Sex differences in assessments of the quality of Medicare managed care. AB - Women rate their care slightly more positively than men on the Medicare managed care Consumer Assessments of Health Plans Study (CAHPS) survey. On four of five composites, women have comparable or slightly more positive composite scores than men. Responses to individual questions in 1997 indicate that women may have slightly more problems getting referrals, equipment, and assistance. In 1998, there were no differences in difficulty getting a referral or assistance, but women were less likely to say their plan provided help, services, and equipment. Women were less likely to get a flu shot in both years. Further monitoring of CAHPS data on sex differences in difficulty getting needed medical equipment are warranted. PMID- 10736561 TI - Discussion of papers by Paul Cleary and Arlene Bierman. PMID- 10736562 TI - MARCKS: a case of molecular exaptation? AB - MARCKS (myristoylated alanine-rich C kinase substrate, 32 kDa) and its 20 kDa brother MARCKS-related protein (MRP) are abundant, widely distributed proteins unusually rich in alanine and glutamic acid, and with lysines, serines and phenylalanines concentrated in a compact "effector domain" (ED) near the middle of the sequence. Its conformation in solution appears to be labile, with little evidence for definite secondary structure. MARCKS (and MRP) interact inter alia with lipid bilayer membranes (via the myristoyl group and the ED), with protein kinases (which phosphorylate the serines in the ED), and with calmodulin (via the ED); synergies between these diverse interactions present an unusually rich array of possibilities for a variety of regulatory roles. The proteins appear to be essential for controlling cell shape changes, possibly via involvement in cytoskeleton-membrane linkage. MRP deficiency leads to neural tube defects in brain development; MARCKS overexpression strongly depresses the proliferation of cancer cells. PMID- 10736563 TI - Determination of interactions between human thrombopoietin and its receptor MPL by yeast two-hybrid system and affinity biosensor. AB - The binding of human thrombopoietin to the extracellular domain of its receptor MPL prompts a cascade transduction of intracellular signals, leading to the development of megakaryocyte precursors and the production of circulating platelets. We have used a yeast two-hybrid system to reveal, via in vivo interactions between different deletion constructs of MPL and thrombopoietin, that the extracellular subunit 1 of MPL is the ligand binding site and the N terminal domain of thrombopoietin alone is sufficient for the binding. The extracellular portion of MPL was heterologously expressed in E. coli and its specific affinity with thrombopoietin was visualized in vitro by resonance mirror biosensor technique. PMID- 10736564 TI - Production and characterization of the extracellular domain of recombinant human fibroblast growth factor receptor 4. AB - Among the members of the fibroblast growth factor receptor family the FGFR4 has demonstrated strong dependence on heparin-like material for its activation by fibroblast growth factors. We have produced and characterized a recombinant human FGFR4 extracellular domain (FGFR4ed), in order to study its biochemical properties in isolated conditions. The FGFR4ed was expressed in an insect cell system and purified from the culture medium by Ni(2+)-affinity and gel filtration chromatography. Pure FGFR4ed was tested for FGF- and heparin-binding by covalent crosslinking experiments and by biosensor analysis. In solution, FGFR4ed formed complexes with acidic FGF (FGF-1) and basic FGF (FGF-2), both in the presence and absence of heparin. Immobilized FGFR4 also bound FGF-8 besides FGF-1 and FGF-2. Furthermore, heparin alone induced receptor oligomerization on the surface of the receptor coupled chip. Thus, the recombinant FGFR4ed revealed properties described for the cellular form of this receptor and can be used for interaction studies. PMID- 10736565 TI - Interaction of mitochondrial phosphate carrier with fatty acids and hydrophobic phosphate analogs. AB - Mitochondrial transporters, in particular uncoupling proteins and the ADP/ATP carrier, are known to mediate uniport of anionic fatty acids (FAs), allowing FA cycling which is completed by the passive movement of FAs across the membrane in their protonated form. This study investigated the ability of the mitochondrial phosphate carrier to catalyze such a mechanism and, furthermore, how this putative activity is related to the previously observed HgCl(2)-induced uniport mode. The yeast mitochondrial phosphate carrier was expressed in Escherichia coli and then reconstituted into lipid vesicles. The FA-induced H(+) uniport or Cl(-) uniport were monitored fluorometrically after HgCl(2) addition. These transport activities were further characterized by testing various inhibitors of the two different transport modes. The phosphate carrier was found to mediate FA cycling, which led to H(+) efflux in proteoliposomes. This activity was insensitive to ATP, mersalyl or N-ethylmaleimide and was inhibited by methylenediphosphonate and iminodi(methylenephosphonate), which are new inhibitors of mitochondrial phosphate transport. Also, the HgCl(2) induced Cl(-) uniport mediated by the reconstituted yeast PIC, was found to be inhibited by these reagents. Both methylenediphosphonate and iminodi(methylenephosphonate) blocked unidirectional Cl(-) uptake, whereas Cl(-) efflux was inhibited by iminodi(methylenephosphonate) and phosphonoformic acid only. These results suggest that a hydrophobic domain, interacting with FAs, exists in the mitochondrial phosphate carrier, which is distinct from the phosphate transport pathway. This domain allows for FA anion uniport via the phosphate carrier and consequently, FA cycling that should lead to uncoupling in mitochondria. This might be considered as a side function of this carrier. PMID- 10736566 TI - Karyotypic abnormalities associated with haemopoietic lineage switching are not linked with mutations to p53. AB - Leukemic cells can undergo lineage switching to display the phenotypic features of another haemopoietic pathway, as exemplified by B lymphoma and erythroleukemic cell lines generating variants with a monocytic appearance. Unlike the diploid parental lines, the vast majority of myeloid derivative lines examined (12 of 13 lines) were aneuploid. As p53 is involved in the maintenance of chromosomal stability, we investigated the role of p53 in the emergence of abnormal karyotypes in cells which had undergone lineage switching. Single strand conformation polymorphism and sequence analysis of cDNA, together with protein immunoprecipitations, were used to assess the p53 status of parental and variant cell lines. Unexpectedly, four or five monocytic lines with chromosomal alterations contained wild type p53. Conversely, a p53 point mutation found in one aneuploid monocytic line was also present in the diploid parental pre-B cell. These results provide strong evidence that mechanisms other than p53 mutations are responsible for karyotypic abnormalities seen in cells that have undergone lineage switching. PMID- 10736567 TI - Effects of 4,4'-diisothyocyanatostilbene-2,2'-disulfonic acid on Trypanosoma cruzi proliferation and Ca(2+) homeostasis. AB - Cell viability requires the perfect functioning of the processes controlling ATP and Ca(2+) homeostasis. It is known that cell death caused by a variety of toxins or pathological conditions is associated with a disruption of ATP and Ca(2+) homeostasis. This study shows that 4,4'-diisothyocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibits Trypanosoma cruzi epimastigote cell growth. This thiol reagent thiocyanate derivative was able to inhibit two ecto-enzymes present in this parasite. The ecto-ATPase and ecto-phosphatase activities were inhibited in a dose-dependent manner (K(i)=47.7 and 472.5 microM, respectively), but the 5'nucleotidase and 3'nucleotidase activities were not. DIDS uptake was approached by fluorescence microscopy. Pulse-chase experiments revealed the DIDS accumulation in compartments, presumably endocytic, in the posterior region of epimastigotes. In addition, we show that the T. cruzi mitochondria studied in permeabilized cells are able to accumulate and retain medium Ca(2+) in the absence of DIDS. However, in the presence of increasing concentrations of DIDS (50-200 microM), Ca(2+) transport was inhibited in a dose-dependent manner. DIDS also caused a disruption of the mitochondrial membrane potential, in the same concentration range, thus explaining its effect on Ca(2+) uptake. The presence of EGTA prevented the elimination of the mitochondrial membrane potential (DeltaPsi), supporting previous data suggesting that the binding of Ca(2+) to the mitochondrial membrane exposes buried thiols to react with DIDS. This thiocyanate derivative was also able to inhibit Ca(2+) uptake by the endoplasmic reticulum in a dose-dependent manner. Taken together, the data presented here provide further insights into the mechanisms underlying the antiproliferative actions of DIDS in T. cruzi. PMID- 10736568 TI - Isolation and partial characterization of the N-terminal functional unit of subunit RtH1 from Rapana thomasiana grosse hemocyanin. AB - Two different structural subunits were identified in Rapana thomasiana hemocyanin: RtH1 and RtH2. RtH1-a is the N-terminal functional unit in the subunit RtH1 and its stability toward temperature and chemical denaturation by guanidinium hydrochloride (Gdn.HCl) are studied and compared with the structural subunit RtH1 and the whole Rapana hemocyanin molecule. The conformational changes, induced by the various treatments, were monitored by CD and fluorescence spectroscopy. The critical temperatures (T(c)) for RtH1-a, the structural subunits and the native Hc, determined by fluorescence spectroscopy, coincide closely with the melting temperatures (T(m)), determined by CD spectroscopy. The free energy of stabilization in water, DeltaG(D)(H(2)O), determined from (Gdn. HCl) denaturation studies, is about two times higher for the structural subunit RtH1 and the whole hemocyanin molecule as compared to the functional unit RtH1-a. The oligomerization between the structural subunits or the eight functional units, assembled in subunit RtH1, has a stabilizing effect on the whole molecule as well as the structural subunits. PMID- 10736569 TI - Iron-induced lipid peroxidation and protein modification in endoplasmic reticulum membranes. Protection by stobadine. AB - Treatment with FeSO(4)/EDTA (0.2 micromol Fe(II) per mg of protein) was used to study the effect of oxidative stress on lipid peroxidation and structural properties of endoplasmic reticulum (ER) membranes isolated from rabbit brain. Oxidative stress resulted in conjugated diene formation and a decrease of 1 anilino-8-naphthalenesulfonate (ANS) fluorescence in a time-dependent manner. In contrast, fluorescence anisotropy of 1, 6-diphenyl-1,3,5-hexatriene was increased early after the initiation of lipid peroxidation and no further increase was observed after 1, 2 and 3 h of peroxidation. FeSO(4)/EDTA treatment was accompanied by formation of conjugates of lipid peroxidation products with membrane proteins, as detected by the increase in fluorescence excitation (350 360 nm) and emission (440-450 nm) maximum. Oxidative stress also induced a marked decrease of the intrinsic fluorescence of aromatic amino acids, suggesting modification or changes in the environment of these amino acid residue(s). The lipid antioxidant, stobadine, completely prevented the changes of ANS fluorescence and production of peroxidized lipid-protein conjugates whereas tryptophan fluorescence was only partially protected. These results suggest that Fe(II) induces both lipid-mediated- and lipid peroxidation independent modification of ER membrane proteins. The study also demonstrates that stobadine is a potent inhibitor of Fe(II)-induced protein modification. PMID- 10736570 TI - Neurotoxin 6-aminonicotinamide affects levels of soluble proteins and enzyme activities in various tissues of golden hamsters. AB - The effects of neurotoxin 6-aminonicotinamide (6-AN) on the levels of soluble proteins and enzyme activities in various tissues of golden hamsters were investigated. SDS-polyacrylamide gel electrophoresis showed that a soluble spinal cord protein with molecular mass 75.0 kDa was present at a higher concentration in the treated group compared to that in the control while that of a molecular mass 64.8 kDa appeared to be missing. However, there were no noticeable differences in protein concentrations observed with the cerebrum, brain stem, and cerebellum. Similarly, treatment with 6-AN decreased the concentration of a soluble protein in pectoral muscle having molecular mass 97.2 kDa and increased those having molecular masses 207.4 and 32.1 kDa. In the kidney, soluble proteins with molecular masses 176.6 kDa was missing and those of molecular masses 97.6, 49, 43.3, and 33.8 kDa were decreased whereas those of molecular masses 64.7 and 33.1 kDa were increased. In the testis the soluble proteins with molecular masses 125.4, 88.7, 69.0, 31.2, 19.1, and 17.4 kDa were missing and those of molecular masses 97.0, 51.3, 42.0, 33.0, 27.2, and 22.6 kDa were present in lower amounts whereas those of molecular masses 311.5, 75.0, 64.0, 54.1, and 53.2 kDa were present in higher amounts. The specific activity of 6-phosphogluconate dehydrogenase was markedly increased in the liver but that of other tissues was not affected. Acetylcholinesterase activity was markedly reduced in the spleen but was enhanced in the intestine. Monoamine oxidase activity was markedly reduced in the brain stem, cerebrum, kidney, and liver. The results suggest that the changes in levels of soluble proteins and enzyme activities shown with golden hamster tissues by 6-AN administration were quite different from those shown with quail tissues. PMID- 10736571 TI - Glucose-mediated Ca(2+) signalling in single clonal insulin-secreting cells: evidence for a mixed model of cellular activation. AB - Using clonal insulin-secreting BRIN-BD11 cells, we have assessed whether the graded response of the whole cell population to glucose can be accounted for by a dose-dependent recruitment of individual cells, an amplification of the response of the recruited cells or both. Cytosolic free Ca(2+) concentration ([Ca(2+)](i)) is an established index of beta-cell function. We used fura-2 microfluorescence techniques to assess the [Ca(2+)](i) responsiveness of single BRIN-BD11 cells to glucose and other secretagogues. Glucose (1-16.7 mM) evoked oscillatory [Ca(2+)](i) rises in these cells resembling those found in parental rat pancreatic beta-cells. The percentage of glucose-responsive cells was 11% at 1 mM and increased to 40-70% at 3-16.7 mM glucose, as assessed by a single-stimulation protocol. This profile was unrelated to possible differences in the cell cycle, as inferred from experiments where the cultured cells were synchronized by a double thymidine block protocol. Individual cells exhibited variable sensitivities to glucose (threshold range: 1-5 mM) and a variable dose-dependent amplification of the [Ca(2+)](i) responses (EC(50) range: 2-10 mM), as assessed by a multiple-stimulation protocol. Glyceraldehyde and alpha-ketoisocaproic acid had glucose-like effects on [Ca(2+)](i). The data support a mixed model for the activation of insulin-secreting cells. Specifically, the graded secretory response of the whole cell population is likely to reflect both a recruitment of individual cells with different sensitivities to glucose and a dose-dependent amplification of the response of the recruited cells. PMID- 10736572 TI - Intrinsic ribonuclease activities in ribonuclease and ribosome-inactivating proteins from the seeds of bitter gourd. AB - Alpha- and beta-momorcharins are ribosome-inactivating proteins present in the seeds of the bitter gourd (Momordica charantia). Both of them possess ribonuclease activity which may account for some of their biological properties. However, the activity is weak and hence it is important to confirm that the ribonuclease activity observed is not due to any contamination. To this end, the ribonuclease from the seeds of M. charantia (RNase-MC) was purified and compared with the ribonuclease activity of the momorcharins. Purification was achieved by ion-exchange chromatographies on DEAE-cellulose, SP-Sepharose and Mono-S. RNase MC had a molecular mass of 22 kDa. It acted on tRNA to release acid-soluble UV absorbing species with a pH optimum around 6.0-6.5. When polyhomoribonucleotides were used as substrates, it was found that RNase-MC acted preferentially on polyU but exerted much weaker activity on polyC, polyG and polyA. Chromatographic analysis of the reaction product indicated that mono- and oligo-ribonucleotides, but not free base, were generated from polyU, suggesting that the enzymatic action involved ribonucleolytic cleavage. RNase-MC exhibited a much more potent (at least 1000-fold higher) ribonuclease activity than alpha- and beta momorcharins. RNase-MC, alpha-momorcharin and beta-momorcharin were separable on Mono-S, indicating that the ribonuclease activities present in the three proteins were distinct entities. PMID- 10736573 TI - Introduction to this issue: sex offenders part one PMID- 10736574 TI - Sexual predator commitment laws: lessons for law and the behavioral sciences. AB - Sex offender commitment laws use a mental health commitment model to lock up the "most dangerous" sex offenders after their prison sentences expire. In Kansas v. Hendricks, the United States Supreme Court rejected the major constitutional challenges to these laws. The Hendricks case clarifies important ambiguities about the use of civil commitment to enforce "police power" interests, as opposed to "parens patriae" interests. Hendricks also clarifies the role of "treatment" in justifying civil commitment. While there remain some important legal issues to be resolved, the future direction of sex offender commitment schemes will turn most significantly on policy decisions. The behavioral sciences can play an important role in shaping these decisions. The most significant questions concern whether expensive commitment programs are the most effective use of scarce treatment and supervision dollars. Additional research should be directed to improving dynamic predictors of recidivism, operationalizing "inability to control" standards, judging the "social meaning" of commitment laws, and assessing the potential treatment disincentives of these laws. PMID- 10736575 TI - Psychological assessment, treatment, and outcome with sex offenders. AB - The frequency and impact of sexual offenses have led to the recent enactment of sexual "predator" laws. Such laws are intended to reduce sexual violence through treatment and involuntary confinement. Sixty years ago, similar laws identifying "sexual psychopaths" were enacted and, in many states, eventually repealed for multiple reasons; among those reasons was an inability to demonstrate that treatment had any significant impact on recidivism. That inability forced us to reexamine, among other issues, the population(s) which undergo treatment, the outcomes that are used to measure treatment effectiveness, and the processes that constitute treatment itself. Those issues are considered in this paper through a review of treatment programs based on psychodynamic, behavioral, and cognitive behavioral theories. Although the evidence is sparse, it is fair to conclude that the latter have been found to be effective. We call for dynamic measures, effective treatments, and the resources necessary for both developments. PMID- 10736576 TI - Pharmacotherapy of paraphilias in the next millennium. AB - Paraphilias are psychiatric disorders of abnormal sexual behavior whose prevalence has markedly increased during the last decade. Treatment modalities currently used fall into three categories: surgical castration, psychotherapy, and pharmacotherapy. The pharmacological interventions consist of antiandrogens that either completely reduce testosterone secretion and/or antagonize the action of testosterone at the level of the receptor, and psychotropic drugs, namely antidepressants. Cyproterone and medroxyprogesterone acetates are the two antiandrogens more commonly used. They are only effective in relatively high doses, but have a substantial number of severe side effects which has considerably limited their use. Psychotropic drugs may be effective solely in men with a definite obsessive-compulsive disorder component. Because of the erratic results and lack of permanent eradication of the paraphilic manifestations their use in paraphilias is highly controversial. Long-acting gonadotropin-releasing hormone (GnRH) agonist analogues are the most potent antiandrogens, and selectively abolish testosterone secretion in a totally reversible fashion. They are administered parenterally once every 1 to 3 months, and have the fewest side effects. Long-acting GnRH analogues, together with psychotherapy, are highly effective in controlling selected paraphilias (pedophilia, exhibitionism, and voyeurism), and are the most promising mode of therapy in the next millennium. There is an urgent need for good methodological research; carefully designed double-blind controlled studies with a large number of subjects in order to validate or not the use of the various pharmacotherapies. PMID- 10736577 TI - Correctional institution based sex offender treatment: a lapse behavior study. AB - The correctional community has engaged in a surge of sex offender treatment programs, both within the confines of the correctional institutions as well as at the community level. While there has been an increase in efforts to assess the effectiveness of these programs the research has been hampered by inconsistency in appropriate outcome measures. The present study addresses these shortfalls, while focusing on the early behavioral lapse indicators. The study focuses on the premise that not all sex offenders lapse in similar manners, and that those with adult and statutory victims would show more impulsive-criminal trends in lapse behavior when compared to those with child victims. The results support the premise on several variables, but not on sexual deviancy. PMID- 10736578 TI - Women accused of sexual offenses. AB - Few studies exist on female sexual offenders or women accused of sexual offenses. In some instances, conclusions from existing studies conflict. In an effort to better understand the phenomenon of sexual abuse by females, we gathered data on all women charged with a sexual offense referred to our facility (William S. Hall Psychiatric Institute in Columbia, South Carolina) for a criminal responsibility/competence to stand trial evaluation from 1987 to 1997. Alleged sexual offenses included criminal sexual conduct one, two, and three and performing a lewd act upon a minor. Fifteen women were referred. This study examines characteristics of the accused women and their victims. Patterns of the alleged offenses and outcome of forensic evaluation are also examined. We found women accused of committing sexual offenses to have a high likelihood of past sexual and physical victimization as well as ongoing physical victimization. Borderline intellectual function and mental retardation were common and women acted frequently with co-defendants. The victims knew the perpetrator in every instance. PMID- 10736579 TI - Sex offenders, sentencing laws and pharmaceutical treatment: a prescription for failure. AB - The phenomenon of adults who have sexual interests involving children as partners, or pedophiles, is considered among the most sociopathological of human conditions. Considerable literature is devoted to issues and problems associated with or related to pedophilia, including prevalence, etiology, treatment, and outcome studies. The sexual victimization of children, based upon data gathered from a number of sources, suggests an intractable problem that is national in scope. Recent manifestations of society's efforts to deal with the sexual victimization of children include the enactment of criminal sentencing laws that mandate the treatment of offenders with certain pharmaceutical agents, such as medroxy-progesterone acetate ("MPA"). Because sentencing laws as a rule vary widely from state to state, there is considerable variation as to who is subject to MPA treatment laws, and how such laws-including specific provisions, such as clinical criteria, if any, required for treatment; type and period of treatment plans; informed consent; etc-are implemented. Most important, these sentencing laws may have remarkably little relation to what is widely considered effective treatment for pedophilia disorders. We examine in detail the most recent sentencing laws pertaining to treatment of persons who have been convicted of sex offenses involving children as victims. Our critique may offer insight and suggestions as to how such sentencing laws can be more suitably tailored to the treatment needs of persons with pedophilia disorders. PMID- 10736580 TI - Sexual predator evaluations and phrenology: considering issues of evidentiary reliability. AB - This article reviews six assessment procedures used for assessing the recidivism risk of previously convicted sexual offenders. The review of these procedures examines whether they comply with generally accepted ethical and practice standards. With few exceptions, most risk assessment instruments fail to comply with these standards. Currently used instruments for risk assessment continue to rely excessively on clinical judgment; and, as a result, they remain at a preliminary stage of development. Consequently, these instruments amount to experimental procedures; and, therefore, they cannot support expert testimony in a legal proceeding. PMID- 10736581 TI - Complementary feeding of infants and young children. PMID- 10736582 TI - Lot quality assurance sampling for monitoring immunization coverage in Madras City. AB - OBJECTIVE: To explore the usefulness of Lot Quality Assurance Sampling (LQAS) to identify divisions in a city that had immunization coverage levels of 80% for any of the four EPI vaccines. METHODS: Only 43 divisions were considered for the study, the stratification factor being the death rate. The hypothesis that 80% coverage is 'unacceptable' was stipulated. Critical value (the number of unimmunized children) was chosen as 3. A simple random sample of 36 children in the age-group 12-23 months was taken from each selected division. Since sampling frames of children were not available, a simple random sample of 36 households was selected. Immunization status of each child was assessed by interviewing the child's mother/guardian. If the number of unimmunized children exceeded 3, then the division was regarded having coverage level 80% and rejected. RESULTS: The coverage was classified as unacceptable(i. e., below 80%) in 19 divisions for Polio and DPT vaccines, in 26 divisions for Measles vaccine and in 4 divisions for BCG vaccine. The average time spent for undertaking the LQAS survey was 6 man days per division. CONCLUSION: This study demonstrated the utility of the LQAS technique in identifying 'unsatisfactory' pockets in Madras City, when the overall coverage was satisfactory. The technique will have greater application with an increase in the number of large units (cities/districts) having an overall coverage of 90% or more. PMID- 10736583 TI - Status of lipid peroxidation and antioxidant enzymes in hypoxic ischemic encephalopathy. AB - OBJECTIVE: To compare the activities of key antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase] and the level of malonyl dialdehyde (MDA) in neonates with hypoxic ischemic encephalopathy (HIE) and controls. DESIGN: Prospective cross sectional study. SETTING: Tertiary care level II neonatal unit of teaching hospital. METHODS: Fifteen term new borns with HIE were registered for the study whereas normal term appropriate for gestational age babies were enrolled as controls. Clinical features were recorded. Activities of SOD, GPx, catalase and the content of MDA were estimated at 24 hours of age. The results obtained were statistically analyzed. RESULTS: Activities of SOD and Catalase and the level of MDA were significantly higher in cases as compared to the controls. There was however no significant alternation in the activity of GPx levels in both the groups. CONCLUSION: The increased activities of antioxidant enzymes are unable to counteract the excessively generated oxidants in vivo, as is evident from the increased MDA levels. Hence, oxygen free radicals may play a significant role in the pathogenesis of HIE. PMID- 10736584 TI - Update immunization policies, guidelines and recommendations. PMID- 10736585 TI - Screening young children for delayed development. PMID- 10736586 TI - Prevalence of malaria in East Delhi--a hospital based study. PMID- 10736587 TI - Immunogenicity of a low dose of indigenously developed recombinant hepatitis-B vaccine in neonates and infants. PMID- 10736589 TI - Umbilical cord fall in preterm and term newborns in vaginal and caesarean deliveries. PMID- 10736588 TI - Attention deficit hyperactivity disorder among psychiatric outpatients. PMID- 10736590 TI - Nebulised salbutamol: an antidote for beta blockers. PMID- 10736591 TI - Chronic granulomatous disease. PMID- 10736592 TI - Minimal change disease and Hodgkin's disease: a rare association. PMID- 10736593 TI - CHILD syndrome. PMID- 10736594 TI - Prune Belly syndrome. PMID- 10736596 TI - How to avoid multiple injections? - reply PMID- 10736595 TI - How to avoid multiple injections? PMID- 10736597 TI - Intervals between different vaccines. PMID- 10736598 TI - Intervals between different vaccines - reply PMID- 10736599 TI - Hepatotoxicity of salicylate therapy in acute rheumatic fever. PMID- 10736601 TI - Body mass index: An emerging age-independent anthropometric criteria - reply PMID- 10736600 TI - Body mass index: an emerging age-independent anthropometric criteria. PMID- 10736602 TI - Spectrum of colonic polyps. PMID- 10736603 TI - Spectrum of colonic polyps - reply PMID- 10736604 TI - Lymphangiectasia of small intestine. PMID- 10736605 TI - Need for strengthening immunization services in villages. PMID- 10736606 TI - Response: alpha-2-mu-Globulin Nephropathy, Posed Mechanisms, and White Ravens. PMID- 10736607 TI - Circadian rhythms of arterial pressure: basic regulatory mechanisms and clinical value. AB - The circadian rhythm of arterial pressure (AP) is not a passive consequence of the impact of exogenous factors. Endogenous mechanisms play an important role in the generation and maintenance of AP rhythm. The adaptation of the exogenous components of AP rhythm to the demands of the environment is modulated by the circadian-time-dependent responsiveness of the biologic oscillator. A neuronal network in the rostral hypothalamus including the suprachiasmatic nucleus is implicated in the generation of AP rhythm, in the modification of the rhythm amplitude (possibly due to homeostatic constraints), and in the regulation of its phase. The central sympathoexcitatory pathway to the upper thoracic cord plays a crucial role in the maintenance of normal circadian AP rhythm. The circadian pattern of AP is influenced also by hormonal factors such as the hypothalamic pituitary-adrenal and the hypothalamic-pituitary-thyroid axes, the renin angiotensin-aldosterone system, opioids, and various vasoactive peptides. The circadian variations of AP depend on physiological state--sleep and wakefulness, pregnancy, work, and senescence (primary aging). In some essential hypertensive patients and in patients with secondary hypertension the nocturnal fall in AP is reduced or absent (nondippers). Target-organ damage is more advanced in nondippers than in dippers. The occurrence of cardiovascular events exhibits a prominent circadian pattern, with events more frequent in the morning (06:00 12:00 h). PMID- 10736608 TI - Diagnostic thresholds for the clinical use of ambulatory blood pressure monitoring. AB - The technique of noninvasive ambulatory blood pressure monitoring is now well established as an instrument in clinical research and as a diagnostic tool in clinical practice. Diagnostic thresholds for ambulatory monitoring were derived by averaging the 95th percentiles of the ambulatory blood pressure measurements in the normotensive subjects enrolled in various large-scale studies. Subjects normotensive on conventional sphygmomanometry, in the absence of other risk factors, have a low cardiovascular risk profile in comparison with their hypertensive counterparts. The evidence from prospective outcome studies also supports the proposed thresholds. More outcome studies are still ongoing. However, their results are unlikely to change the proposed thresholds, which are in line with the recommendations of the JNC VI (the Sixth Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure) and those of many other national expert committees. PMID- 10736609 TI - Circadian clocks and hypertension: genetics and interactions. AB - Recent advances in molecular genetics of circadian rhythms and hypertension led to the discovery of separate groups of genes implicated in their regulation. Importantly, the identification in both mammals and flies of 6 homologous circadian clock genes strongly indicates that the circadian period is controlled by an evolutionary conserved set of genes. Studies in familial and experimental hypertension reveal that elevated blood pressure is due to mutations in genes implicated in the function of the renin-angiotensin-aldosterone system. A chronobiologic approach to experimental hypertension indicates that hypertension can be associated with selectively inverted circadian rhythm of arterial pressure. Several lines of evidence suggest that the rostral hypothalamus is an area of central integration of the endogenous rhythmic and other regulatory influences that modulate the phase and amplitude of circadian arterial pressure rhythmicity. The combination of advanced molecular genetics and continuous blood pressure monitoring with chronobiologic assessment emerges as a fruitful approach in better understanding the pathogenesis of hypertension. PMID- 10736610 TI - Chronopharmacology and its impact on antihypertensive treatment. AB - Circadian rhythms have been documented throughout the plant and animal kingdom at every level of eukariotic organization. Circadian rhythms are endogenous in nature, driven by oscillators or clocks, and persist under free-running (e.g. constant darkness) conditions. The genes expressing the biological clock have been identified in various species. The important feature of endogeneous biological rhythms is their anticipatory character. Rhythmicity inherent to all living systems, allows them to adapt more easily and to better survive under changing environmental conditions during the 24 hours of a day as well as during changing seasons. Having this in mind it is easy to conceive that not only must the right amount of the right substance be at the right place, but also this must occur at the right time. Also in man nearly all functions of the body including those influencing pharmacokinetic parameters such as drug absorption and distribution, drug metabolism and renal elimination display significant daily variations. Also the onset and symptoms of diseases such as coronary infarction, angina pectoris, stroke, ventricular tachycardia are circadian phase dependent. Myocardial infarction and angina attacks as well as silent ischemias (ST-segment depression) in stable angina pectoris have an early morning peak between 8-12 h. In contrast, ECG abnormalities and angina attacks in variant angina mainly occur at night. Blood pressure and heart rate in normotensives and essential (primary) hypertensive patients display highest values during daytime followed by a nightly drop and an early morning rise. In about 70% of forms of secondary hypertension (e.g. renal disease, hyperthyroidisms, hormonal diseases, gestational hypertension), however, this rhythmic pattern is abolished or even reversed exhibiting nightly peaks in blood pressure. This form of hypertension is accompanied by increased end organ damages. Thus, different subtypes of a disease (angina pectoris, hypertension) can display different circadian patterns in symptoms. These observations are a challenge for basic and clinical research to get a better understanding on the underlying mechanisms of regulation. Moreover, they call for a circadian time-specified drug treatment. From above it is evident that pharmacokinetics may also not be constant within a day. Chronopharmacokinetics have been shown for several cardiovascular active drugs (propranolol, nifedipine, verapamil, enalapril, isosorbide-5-mononitrate, digoxin, etc.). Far more drugs were shown to display significant daily variations in their effects (chronopharmacodynamics, chronotoxicology) even after chronic application or constant infusion. In conclusion, there is clear evidence that the dose/concentration-response relationship of drugs can be significantly dependent on the time of day. Thus, circadian time has to be taken into account as an important variable influencing a drug's pharmacokinetics and/or its effects or side effects. PMID- 10736611 TI - Diet and cancer. Perspectives of prevention. PMID- 10736612 TI - EPIC-Italy. A molecular epidemiology project on diet and cancer. PMID- 10736613 TI - Nutritional factors in human cancers. AB - A variety of external factors interacting with genetic susceptibility influence the carcinogenesis process. External factors including oxidative compounds, electrophilic agents, and chronic infections may enhance genetic damage. In addition, various hormonal factors which influence growth and differentiation are critically important in the carcinogenic process. Diet and nutrition can influence these processes directly in the gastrointestinal tract by providing bioactive compounds to specific tissues via the circulatory system, or by modulating hormone levels. Differences in certain dietary patterns among populations explain a substantial proportion of cancers of the colon, prostate and breast. These malignancies are largely influenced by a combination of factors related to diet and nutrition. Their causes are multifactorial and complex, but a major influence is the widespread availability of energy-dense, highly processed and refined foods that are also deplete in fiber. These dietary patterns in combination with physical inactivity contribute to obesity and metabolic consequences such as increased levels of IGF-1, insulin, estrogen, and possibly testosterone. These hormones tend to promote cellular growth. For prostate cancer, epidemiologic studies consistently show a positive association with high consumption of milk, dairy products, and meats. These dietary factors tend to decrease 1.25(OH)2 vitamin D, a cell differentiator, and low levels of this hormone may enhance prostate carcinogenesis. While the nutritional modulation of growth-enhancing and differentiating hormones is likely to contribute to the high prevalence of breast, colorectal, prostate, and several other cancers in the Western world, these cancers are relatively rare in less economically developed countries, where malignancies of the upper gastrointestinal tract are quite common. The major causes of upper gastrointestinal tract cancers are likely related to various food practices or preservation methods other than refrigeration, which increase mucosal exposure to irritants or carcinogens. PMID- 10736614 TI - Alcohol and cancer. PMID- 10736615 TI - Energy sources and risk of cancer of the breast and colon-rectum in Italy. AB - Dietary habits are thought to be involved as determinant of breast and colorectal cancer. Nevertheless results of epidemiological studies on diet show several inconsistencies. This is true for the findings related to energy and its sources. Between 1991 and 1996, 2569 women with incident breast cancer (median age: 55 years) and 2588 controls (median age: 56 years), and 1953 subjects with cancer of the colon-rectum (median age: 62 years) and 4154 controls (median age: 58 years) were interviewed in the hospitals of six Italian areas. The validated food frequency questionnaire included questions on 78 foods and recipes and specific questions on individual fat intake pattern. Significant risks for breast and colorectal cancer emerged with increasing intake of energy (odds ratios in highest vs. lowest quintile were 1.32 and 1.49 respectively). Due to the high interrelations existing among the various sources of energy, the separated analysis of each macronutrient didn't achieve the independent estimates of the effects. In order to overcome this situation, we used a completely partitioned model in which all the main sources of energy were entered simultaneously as continuous variables in the regression. High intake of starch led to an increase of cancer risk (odds ratios for an addition of 100 kcal/day were 1.08 and 1.10 for breast and colorectal cancer respectively). A positive association was also found for saturated fat (odds ratios 1.16 for breast and 1.12 for colorectal cancer). High intakes of polyunsaturated fatty acids (chiefly derived from olive and seed oils) were protective more markedly for breast cancer. A possible interpretation of the risk for starch, implies the glycemic overload and hyperinsulinemia due to the high grade of refinement of cereals (the main source of starch) eaten in Italy. PMID- 10736616 TI - Organochlorines and breast cancer. A study on Neapolitan women. PMID- 10736617 TI - Olive oil consumption and cancer mortality in Italy. A correlation study. PMID- 10736618 TI - Cell division cycle alterations and human tumors. PMID- 10736619 TI - Regulation of p53 function in normal and malignant cells. PMID- 10736620 TI - The role of micronutrients in DNA synthesis and maintenance. PMID- 10736621 TI - Biological effects of hydroxytyrosol, a polyphenol from olive oil endowed with antioxidant activity. PMID- 10736622 TI - Protective effects of butyric acid in colon cancer. PMID- 10736623 TI - Short-chain fatty acid in the human colon. Relation to inflammatory bowel diseases and colon cancer. AB - Short chain fatty acids (SCFAs) are the end products of anaerobic bacteria break down of carbohydrates in the large bowel. This process, namely fermentation, is an important function of the large bowel; SCFAs, mainly acetate, propionate and butyrate account for approximately 80% of the colonic anion concentration and are produced in nearly constant molar ratio 60:25:15. Among their various properties, SCFAs are readily absorbed by intestinal mucosa, are relatively high in caloric content, are metabolized by colonocytes and epatocytes, stimulate sodium and water absorption in the colon and are trophic to the intestinal mucosa. While the fermentative production of SCFAs has been acknowledged as a principal mechanism of intestinal digestion in ruminants, the interest in the effects of SCFAs production on the human organism has been raising in the last ten years. SCFAs are of major importance in understanding the physiological function of dietary fibers and their possible role in intestinal neoplasia. SCFAs production and absorption are closely related to the nourishment of colonic mucosa, its production from dietary carbohydrates is a mechanism whereby considerable amounts of calories can be produced in short-bowel patients with remaining colonic function and kept on an appropriate dietary regimen. SCFAs enemas or oral probiotics are a new and promising treatment for ulcerative colitis. The effects have been attributed to the oxidation of SCFAs in the colonocytes and to the ability of butyrate to induce enzymes (i.e. transglutaminase) promoting mucosal restitution. Evidence is mounting regarding the effects of butyrate on various cell functions the significance of which needs further considerations. Up until now, attention has been related especially to cancer prophylaxis and treatment. This article briefly reviews the role of SCFAs, particularly butyrate, in intestinal mucosal growth and potential clinical applications in inflammatory and neoplastic processes of the large bowel. PMID- 10736624 TI - Brassica vegetables and cancer prevention. Epidemiology and mechanisms. AB - This paper first gives an overview of the epidemiological data concerning the cancer-preventive effect of brassica vegetables, including cabbages, kale, broccoli, Brussels sprouts, and cauliflower. A protective effect of brassicas against cancer may be plausible due to their relatively high content of glucosinolates. Certain hydrolysis products of glucosinolates have shown anticarcinogenic properties. The results of six cohort studies and 74 case control studies on the association between brassica consumption and cancer risk are summarized. The cohort studies showed inverse associations between the consumption of brassica's and risk of lung cancer, stomach cancer, all cancers taken together. Of the case-control studies 64% showed an inverse association between consumption of one or more brassica vegetables and risk of cancer at various sites. Although the measured effects might have been distorted by various types of bias, it is concluded that a high consumption of brassica vegetables is associated with a decreased risk of cancer. This association appears to be most consistent for lung, stomach, colon and rectal cancer, and least consistent for prostatic, endometrial and ovarian cancer. It is not yet possible to resolve whether associations are to be attributed to brassica vegetables per se or to vegetables in general. Further epidemiological research should separate the anticarcinogenic effect of brassica vegetables from the effect of vegetables in general. The mechanisms by which brassica vegetables might decrease the risk of cancer are reviewed in the second part of this paper. Brassicas, including all types of cabbages, broccoli, cauliflower, and Brussels sprouts, may be protective against cancer due to their glucosinolate content. Glucosinolates are usually broken down through hydrolysis catalysed by myrosinase, an enzyme that is released from damaged plant cells. Some of the hydrolysis products, viz. indoles, and isothiocyanates, are able to influence phase 1 and phase 2 biotransformation enzyme activities, thereby possibly influencing several processes related to chemical carcinogenesis, e.g. the metabolism, DNA-binding, and mutagenic activity of promutagens. Most evidence concerning anticarcinogenic effects of glucosinolate hydrolysis products and brassica vegetables has come from studies in animals. In addition, studies carried out in humans using high but still realistic human consumption levels of indoles and brassica vegetables have shown putative positive effects on health. The combination of epidemiological and experimental data provide suggestive evidence for a cancer preventive effect of a high intake of brassica vegetables. PMID- 10736625 TI - Stilbenes and bibenzyls with potential anticancer or chemopreventive activity. PMID- 10736627 TI - Diet, fibers, and colon cancer. PMID- 10736626 TI - Post-translational modifications of eukaryotic initiation factor-5A (eIF-5A) as a new target for anti-cancer therapy. AB - Eukaryotic translation initiation factor 5A (eIF-5A) is the only cell protein that contains the unusual basic amino acid hypusine [N epsilon-(4-amino-2 hydroxybutyl)lysine]. Hypusine is formed by the transfer of the butylamine portion from spermidine to the epsilon-amino group of a specific lysine residue of eIF-5A precursor and the subsequent hydroxylation at carbon 2 of the incoming 4-aminobutyl moiety. Agents that reduce cell hypusine levels inhibit the growth of mammalian cells. These observations suggest that hypusine is crucial for proliferation and transformation of eukaryotic cells. Here we have studied whether the inhibition of hypusine synthesis can potentiate the anti-cancer activity of the anti-tumour agents interferon-alpha (IFN alpha) and cytosine arabinoside (ara-C). We have found that IFN alpha increased epidermal growth factor receptor (EGF-R) expression, but reduced S phase and proliferative marker expression in human epidermoid KB cells and that this effect was antagonised by epidermal growth factor (EGF). Growth inhibition induced by IFN alpha was paralleled by decreased hypusine synthesis and, when EGF counteracted anti proliferative effects, a reconstitution of hypusine levels was recorded. We also studied the effects of IFN alpha on the cytotoxicity of the recombinant toxin TP40 which inhibits elongation factor 2, another step of protein synthesis, through EGF-R binding and internalisation; IFN alpha induced an about 27-fold increase of TP40 cytotoxicity in KB cells. Ara-C, another antineoplastic agent commonly used in haematologic malignancies, induced both apoptosis and iron depletion in human acute myeloid leukaemic cells. The combination of ara-C and of the iron chelator desferioxamine, a strong inhibitor of hypusine synthesis, had a synergistic activity on apoptosis in these cells. The data strongly suggest that the post-translational modifications of eIF-5A could be a suitable target for the potentiation of the activity of anti-cancer agents. PMID- 10736628 TI - Phytochemicals as modulators of cancer risk. AB - These results, describing antitumor activity of some of the phytochemicals that have been actively studied, suggest that dietary changes could play a role in decreasing the incidence of a variety of tumors. 13C and the other compounds discussed may well be only prototypes for other as yet unexplored phytochemicals present in the diet. There have been no attempts to explore the possibilities of synergistic action among the various phytochemicals, 13C, limonene, curcumin, epigallocatechin gallate, sulforaphene, or genistein. Mixtures of these compounds might well show potency at lower doses for each of the compounds and show even greater promise than that already demonstrated. PMID- 10736629 TI - Low dose exposure to carcinogens and metabolic gene polymorphisms. PMID- 10736630 TI - Carcinogen-DNA adducts as tools in risk assessment. AB - Genotoxic chemicals are known to react with DNA either directly or after metabolic activation to form adducts, a step thought to be relevant with respect to chemical carcinogenesis. Evaluation of cancer risk due to exposure to chemicals requires information about the internal dose which depends on individual variation in rates of metabolic activation and detoxification. The presence and the amount of specific DNA adducts provide a good indication of chemical exposure and genetic damage resulting from exposure to carcinogens and account for some of the factors affecting individual susceptibility to cancer. Analysis of DNA adducts requires that the sensitivity of the methods be sufficiently high to allow the detection of about 1 adduct/109 normal nucleotides. Most suitable methods are based on 32P-postlabelling, immunoassays or physico-chemical techniques such as HPLC coupled to synchronous fluorescence spectroscopy or gas chromatography-mass spectrometry. These methods have been used to assess human exposure to a variety of chemical carcinogens including polycyclic aromatic hydrocarbons, aromatic amines, heterocyclic aromatic amines or aflatoxins. In some instances, the use of DNA-adducts has given accurate estimates of risk. PMID- 10736631 TI - Significance of genetic polymorphisms in cancer susceptibility. PMID- 10736632 TI - DNA repair pathways and cancer prevention. AB - This article describes the five main classes of DNA repair processes that occur in humans with respect to their mechanism of action, major substrates, and role in protection against endogenous and environmental DNA damaging agents. The importance of all of these processes in protection from the initiation of neoplastic growth has been established either in studies of inheritable diseases affecting DNA repair or experiments with transgenic animals or both. The capacity of DNA repair pathways to deal with DNA damage is therefore a critical factor in the cellular response to environmental, and dietary carcinogens. DNA repair activity and factors affecting this activity either directly or indirectly must be taken into account in risk assessment. PMID- 10736633 TI - Cereals, fiber, and cancer prevention. ECP Consensus Panel. PMID- 10736634 TI - Carnitine system and tumor. PMID- 10736635 TI - History in the making. PMID- 10736636 TI - Pediatric septic arthritis. PMID- 10736637 TI - Surgical management of vesicoureteral reflux. AB - Vesicoureteral reflux (VUR) is a urologic condition in which there is a retrograde flow of urine from the bladder through the ureter back up to the upper urinary tract. The condition may be classified as primary or secondary and is more often identified in Caucasian females. The primary goal in the management of VUR is the prevention of pyelonephritis and subsequent renal scarring. PMID- 10736638 TI - Diagnosis, treatment, and lifestyle changes of interstitial cystitis. AB - Interstitial cystitis (IC) is a chronic inflammation of all the layers of the bladder wall. As it is difficult to diagnose and it affects mostly women, many physicians misdiagnose it as a psychological problem. Almost half a million individuals in the United States suffer from this cureless disorder. This article will review the anatomy, physiology, etiology, diagnosis, treatments, and current research foci of IC, along with the effects of the disease on lifestyle and personal issues from the patient's perspective. PMID- 10736639 TI - Structural data elements--standardized terms and definitions. AB - Perioperative nurses must have an organized approach to collect, organize, classify, and capture clinical nursing data to communicate nursing practice. Standardized nursing language provides a systematic method of collecting basic elements of perioperative nursing care. This article examines how structural elements were developed and validated for this standardized language. PMID- 10736640 TI - Surgical treatment of nonmelanoma skin cancer. AB - Nonmelanoma skin cancer is the most common human malignancy. An estimated 2.75 million patients worldwide are diagnosed with skin cancer each year, with more than one million in the United States alone. Treatment for skin cancer includes methods such as cryosurgery, curettage and electrodessication, local excision, and Mohs micrographic surgery. Regardless of the method used, the goal is to provide the patient with the safest, most cost-effective and curative treatment. PMID- 10736641 TI - Reducing blood exposures during orthopedic surgical procedures. AB - Surgical team members constantly are exposed to blood during procedures. Inadvertent injuries (e.g., needle sticks, cuts) and contaminations expose team members and patients to the risk of transmission of bloodborne pathogens. Injuries and contaminations can be decreased significantly for scrub people and first assistants, however, by introducing new working methods (i.e., no-touch instrument passing technique, instrument neutral zone). PMID- 10736642 TI - Moisturizing alcohol hand gels for surgical hand preparation. AB - With the use of novel formulary technology, unique moisturizing hand gels have been developed that offer significant advantages in perioperative and other health care settings. These advantages include the time-saving capabilities of a waterless formulation, the persistence and effectiveness of a surgical scrub, and the moisturization and protective properties of a lotion. Extensive laboratory and clinical studies, involving in vivo antimicrobial activity against resident and transient flora, skin moisturization on normal and dry skin, and compatibility with latex gloves, have supported these advantages. Nondrying alcohol hand gels can be used for antiseptic hand washing, hand scrubs between procedures (i.e., reentry scrubs), brushless surgical scrubs, moisturizers, and glove-donning aids. PMID- 10736643 TI - Student interest in perioperative nursing practice as a career. AB - Perioperative nursing is in crisis because of the difficulty recruiting sufficient numbers of graduate nurses and the threat of nonnursing employees performing traditional nursing duties. The relative unpopularity of perioperative nursing as a career choice for graduate nurses is commonly attributed to little exposure to the specialty's theory and practice in undergraduate nursing education programs. This article discusses results of a longitudinal study's first phase to ascertain career preferences of undergraduate nursing students. Findings suggest that when students begin nursing programs, they consider perioperative nursing a favorable career option. Few studies regarding student nurses' attitudes toward perioperative nursing exist, making these results significant. PMID- 10736644 TI - A historical review of the AORN Journal. AB - The purpose of this project was to critically review 35 years of AORN Journal content from 1963 to 1998 to determine if the original mission and goal of the Journal were being met. Content analysis revealed a diverse mixture of feature articles and regular columns that typically targeted perioperative nurses. Feature articles addressed multiple perioperative specialty areas, along with other areas of interest to perioperative nurses. Regular columns provided a means of keeping AORN members informed of educational offerings, policy changes, AORN business, and clinical practice issues. PMID- 10736645 TI - Understanding patients' needs is the foundation of perioperative nursing. AB - As I reflected on Mrs L's procedure, I realized the importance of my role. I coordinated the efforts of the radiologist, radiological technologist, surgeon, pathologist, and nurses to provide Mrs L with a positive surgical experience, and I collaborated with Mrs L and her husband to make them as comfortable and relaxed as possible. I connected with Mrs L and her husband--they knew I would help them in any way to make this a positive experience. In addition, I collected data about Mrs L's perception and satisfaction with her experience to explore her response to her stereotactic surgical biopsy procedure experience. Perioperative nurses are important to patients and their family members. By listening to patients' needs, planning for their individual care, and being present, perioperative nurses help patients through difficult situations. I use my experience and knowledge to serve as a role model for breast imaging personnel, nurses, and other coworkers, and to improve each patient's perioperative experience. PMID- 10736646 TI - Shared governance and teamwork--myth or reality. AB - Taking time to develop key strategies and implement our model enabled team members to deliver excellent patient care through shared governance and team empowerment. The benefits of using this model are medical and nursing staff members working together and having ownership in what they do. A sense of accomplishment will be evident, and team members likely will achieve the team's goals and those of the organization. Nurses frequently practice traditional shared governance. To achieve our goals, however, we must take the opportunity that working with medical team members offers. Ensuring that we remain interested and committed to our team model is the next major challenge the team members face. We must continue to be vibrant and open to change. Educational programs, seminars, journal clubs, social activities, and challenging team members for ideas help maintain interest and focus. As managers, we must allow the team to grow and develop by promoting a climate in which learning and mastery is valued, making it clear that there is no failure, only mistakes that provide feedback. We can facilitate this by trusting, believing in, and supporting team members to provide excellent patient care while achieving their goals and those of the organization. PMID- 10736647 TI - A guide to preoperative pregnancy testing for the nurse practitioner. PMID- 10736648 TI - Expanding your perioperative practice into the health care industry. AB - Perioperative nurses who pursue nontraditional roles with medical products companies must assess their personal and professional skills to determine if they would fill that role satisfactorily. They must consider the risks involved and determine if the job is appealing. Working as a health care industry nurse can positively affect patient outcomes while providing a company with unique skills and insights. This career choice can be very rewarding. PMID- 10736649 TI - Internet resources for researching nonmelanoma skin cancers. PMID- 10736650 TI - Decision analysis--putting it all together. PMID- 10736651 TI - Obesity--Helping patients avoid the epidemic. PMID- 10736652 TI - Baby fat is cute, but chubby kids may be in danger. AB - An increasing percent of children are overweight. A review of the prevalence of overweight children in different demographic groups is provided in an effort to alert dentists to the role they could play in preventing medical complications. PMID- 10736653 TI - More than "just" food: what are our youngsters eating? AB - More than one in ten children in poor families experience hunger. The vast majority of children of all ages and family incomes do not have a good diet. A review of these realities is provided in an effort to alert dentists to the magnitude of these problems. PMID- 10736654 TI - Enamel caries formation and lesion progression with a fluoride dentifrice and a calcium-phosphate containing fluoride dentifrice: a polarized light microscopic study. AB - The effects of a fluoride dentifrice (Colgate Total) and a calcium-phosphate containing fluoride dentifrice (Enamelon) on caries-like enamel lesion formation and progression were evaluated in vitro. One quarter from each tooth (n = 12) was assigned to one of the study groups: 1) control group [artificial saliva only]; 2) Colgate Total; and 3) Enamelon. The dentifrices were applied to the enamel windows (3 minutes, 3 time per day for 14 days). Following treatment, enamel lesions were created with an acidified gel. After lesion initiation, sections were obtained for polarized light microscopy. To evaluate lesion progression, enamel windows with caries-like lesions in each test group were treated again for 14 days, returned to acidified gels for lesion progression, and then sections were obtained for polarized light microscopy. Mean lesion depths (polarized light, water imbibition) were as follows: 1) Lesion Initiation: Control 109 +/- 19 microns, Colgate Total 76 +/- 14 microns, Enamelon 63 +/- 17 microns; 2) First Lesion Progression: Control 164; +/- 24 microns, Colgate Total 124 +/- 22 microns, Enamelon 110 +/- 19 microns; 3) Second Lesion Progression: Control 235 +/- 23 microns; Colgate Total 172 +/- 27 microns, Enamelon 153 +/- 18 microns. Both Colgate Total and Enamelon enhanced the resistance of sound enamel and caries-like enamel lesions to a continuous in vitro cariogenic challenge when compared with matched controls (P < .05, ANOVA, DMR). Enamelon provided a further reduction in lesion depth for all periods when compared with Colgate Total (P > .05, ANOVA, DMR). Addition of bioavailable calcium and phosphate ions to a fluoride dentifrice may improve the ability of enamel to resist caries initiation and subsequent lesion progression. PMID- 10736655 TI - Compomers adaptation to Class I and V cavities in permanent teeth. AB - This study evaluated the adaptation of Compoglass, Dyract and Hytac to Class I and V cavities and the effect of etching on that adaptation. Sixty molars were used, divided for the three compomers used, which were subdivided for Class I and V and redivided into etched and nonetched cavities (5 each). Standard Class I and V cavities were prepared. The adhesives were applied and the cavities restored with the compomers. The restoration was finished and the cavosurface margins examined under the light microscope for any gap formation. The restored cavity was sectioned in two halves, which were fixed in glutaraldehyde and polished. The restoration-tooth interface was examined under the light microscope for any existent gap. Then one half from each tooth was prepared for SEM examination. The results revealed good adaptation of the compomers at the cavosurface margins except nonetched Class V cavities restored with Dyract. Restorations in Class V were more adapted than Class 1. Dyract and Compoglass showed better adaptation inside the cavity than Hytac. Cementum wall displayed good adaptation with all restorative materials. Etched cavities showed consistent hybrid layer formation whereas nonetched cavities showed interrupted hybrid layer with poor resin penetration. PMID- 10736656 TI - Ketac Molar Versus Dyract Class II restorations in primary molars: twelve month clinical results. AB - The aim of the present clinical study was an in vivo evaluation of an improved conventional glass ionomer cement Ketac Molar (ESPE), compared to a polyacid modified composite resin, Dyract (Dentsply/De Trey), used in primary molars. Fifty-three Ketac Molar and fifty-two Dyract restorations were placed in box-only preparations in primary molars. The application time for the chemically cured Ketac Molar, was longer compared to the light-cured Dyract. In comparing the materials, no differences were found, comparing both materials regarding secondary caries, marginal adaptation, wear and fracture toughness. One case of recurrent caries adjacent to a Ketac Molar restoration and two cases in the Dyract group were reported. Two Ketac Molar restorations and one Dyract showed bulk fracture at the time of evaluation. At the twelve-month evaluation, no difference between the investigated materials was registered, which indicates that Ketac Molar can be used as a proximal restoration in the primary dentition. It should be emphasized, however, that one-year data should not be extrapolated to indicate the long-term success of restorations. PMID- 10736657 TI - Cariogenic oral flora and its relation to dental caries. AB - Many attempts have been made to establish the risk profile of dental caries; however, no diagnostic procedures are yet available to reliably predict such risk particularly among the pediatric and adolescent population. Age of subject, a history of caries affecting the primary dentition, the prevalence of Streptococcus mutans, pH values, salivary flow, and the frequency and amount of sugar consumption have been the factors most studied. A cross-sectional study is made of schoolchildren in the 12-13-year age-range to evaluate the relationship between dental caries and CFU/ml of S. mutans and Lactobacillus, salivary buffer capacity, and salivary flow. Likewise, an evaluation is made of the predictive value of the variables, bacterial count and salivary pH with respect to caries. A total of 167 children were subjected to oral examination to establish the DMFT and DMFS indices, followed by the collection of saliva for quantitating S. mutans, Lactobacillus, pH and salivary flow, by the Dentocult (SM)(LB) and Dentobuff systems (Vivadent). The data obtained were subjected to descriptive analysis, comparisons among variables were made by nonparametric testing with a confidence level of 95 percent. Test predictive value was studied in terms of sensitivity, specificity, positive and negative predictive values, and probability ratio. Statistically significant (p < 0.001) correlations were observed between the caries indices and bacterial counts. No significant association was recorded with the rest of the variables studied. Bacterial counts as well as salivary buffer capacities exhibited greater negative than positive predictive values, i.e., they were more effective in identifying healthy individuals than patients who required treatment. PMID- 10736658 TI - Survey of emergency preparedness of pediatric dentists from the Southeastern United States. AB - The fact that 91 percent of respondents to this survey said they could benefit from more emergency training indicates an awareness on the part of clinicians of the importance of emergency preparedness. The average number of hours of continuing education courses in emergency management over the last ten years of the responding dentists was less than six hours. This, coupled with the fact that one third of the responding dentists were not comfortable with their staff's emergency preparedness, should serve as a wake up call to all practicing pediatric dentists, to our institutions who train these dentists, and to the Associations who regulate and govern the practice of dentistry. More continuing education courses on this topic would be very timely and helpful to these respondents. PMID- 10736659 TI - Dental health behavior of children with BBTD treated using general anesthesia or sedation, and of their parents in a recall examination. AB - The aim of the study was to assess the dental status and dental health behavior of children with Baby Bottle tooth Decay treated using general anesthesia or sedation, and the dental health behavior of their parents in a recall examination. The study population consisted of sixty-five children, among whom thirty-four were treated using general anesthesia and thirty-one using sedation. The recall examination included a full dental examination from which the children's dif index could be drawn. Loe's plaque index was used to assess the amount of plaque on the teeth. Sociodemographic information and the dental health behavior of the parents and children were obtained. Plaque index was similar in the general anesthesia and sedation groups. The parents of the general anesthesia group were younger than the parents of the sedation group (35.0 +/- 6.7 and 38.8 +/- 6.2 for the fathers, and 32.4 +/- 5.9 and 34.9 +/- 5.3 for the mother, respectively). More firstborn children were treated using general anesthesia than using sedation. Significantly more siblings were treated in the sedation group. Children treated using sedation had significantly more siblings treated in the same mode. Parents of the children in the general anesthesia group were significantly more involved in brushing their children's teeth than the other group. In the sedation group, more children brush their teeth without parental help. Significantly more children in the general anesthesia group reduced their sweet consumption than in the sedation group. We conclude that preventive behaviors were more frequently adopted among the families of children treated using general anesthesia. PMID- 10736660 TI - Management of the crying child during dental treatment. AB - The purposes of this study were: (1) To present parents' attitudes toward their child's crying in the dental environment. (2) To suggest a classification of crying children, and discuss its management implications. One hundred and four parents accompanying their children to dental treatment completed a questionnaire assessing the following: the tendency of the child to cry, the preferred approach of the operator to the crying child, and how the parents perceive their own role in such a case. Also the operator's assessment of the child's cooperation and of the child's tendency to cry were recorded. The sociodemographic variables of the parent were recorded, but no association was found with the dependent variables. Fifty-three percent of the parents assessed their children as having a tendency to cry, while only 25 percent children were assessed as such by the operators. Seventy-three percent of the parents preferred that the operator cease the treatment and calm the crying child before resuming. Sixty-nine percent of the parents stated that they should always help to manage the child, when crying takes place. Eleven children who were assessed by the operators as having a tendency to cry cooperated well during the treatment. The successful completion of dental treatment of a crying child is viewed as a partnership of the dentist and the parent. It is important that the dentist informs the parent about the method to be used, and have their consent. A new classification of the crying child is suggested. PMID- 10736662 TI - The treatment of mandibular cysts associated with osteogenesis imperfecta. AB - A fifteen-year-old boy with osteogenesis imperfecta (OI) and dentinogenesis imperfecta also had a big cyst in the mandible and needed surgical therapy. Six months postoperatively we saw a complete regeneration of the bone-structures. We came to the conclusion that cysts which appear independently from the disease of OI can heal after surgical intervention. PMID- 10736661 TI - Use of pediatric dental services in the 1990s: some continuing difficulties. AB - Despite continuing need for dental services, the use of dental services has not increased uniformly throughout the population of children. Minority group children, children in families with other than two parents present, children in lower income families and families without insurance, and children with special needs, receive reduced levels of dental services than their counterparts in the general population. PMID- 10736663 TI - Four million chickens get Newcastle disease vaccine. PMID- 10736664 TI - The use of fluoroquinolones for companion animal antimicrobial therapy. PMID- 10736665 TI - Anastomosis of right ventral colon to descending colon to bypass a non-functional descending colon anastomosis in a miniature pony. PMID- 10736666 TI - Chiari 1/syringomyelia complex in a King Charles Spaniel. AB - A 9-year-old King Charles Spaniel presented with a history of progressive forelimb weakness and paroxysmal involuntary flank scratching over a 2-year period. Neurological examination suggested a myelopathy of C1 to C4 spinal cord segments. Advanced imaging studies revealed hydrocephalus, caudal herniation of part of the caudal lobe of the cerebellum through the foramen magnum and marked syrinx formation to the level of the caudal thoracic spine, resembling Arnold Chiari malformation with secondary hydromyelia in humans. Mechanical obstruction at the craniocervical junction, altering CSF flow dynamics, may lead to syrinx formation. Response to diuretic therapy was moderate but surgical decompression may offer better long term prognosis. PMID- 10736667 TI - Surgical salvage from comminuted metatarsal fracture using a weight-bearing pin putty apparatus in a dog. AB - Comminuted metatarsal fractures in a dog were treated using intramedullary pins externally connected and stabilized by epoxy resin putty and application of a fibreglass cast. Two weeks later the cast was removed. After further 2 weeks application of a Robert-Jones bandage the dog could stand and walk with the pin putty apparatus in position. The pin-putty apparatus was removed 3 months postoperatively, and the dog resumed normal activity. This fixation method could be an easy, economical and effective alternative treatment for managing comminuted fractures of the metatarsal and possibly metacarpal bones in selected cases. PMID- 10736668 TI - Lufenuron and tick paralysis. PMID- 10736669 TI - Pancreatic pseudocyst causing extrahepatic biliary obstruction in a dog. AB - A 3-year-old Rhodesian Ridgeback was examined because of recurrent pancreatitis of 2 months duration. The dog had signs of abdominal pain and jaundice. Blood biochemical findings were consistent with extrahepatic bile duct obstruction, but on abdominal ultrasonography no cause of obstruction was identified. At surgery a pancreatic pseudocyst was found in the body of the pancreas. Cystoduodenostomy, cystic omentalization and biliary diversion resulted in excellent long-term recovery. PMID- 10736670 TI - Sorghum ergot (Claviceps africana) associated with agalactia and feed refusal in pigs and dairy cattle. AB - OBJECTIVE: To establish the aetiology and define the main clinical features of a syndrome characterised by severe feed refusal, death of piglets and reduced milk production in pigs and dairy cattle. DESIGN: Clinical, pathological, toxicological and epidemiological examination of clinical cases in 10 piggeries and 4 dairies, located between 50 and 150 km south-west of Rockhampton. RESULTS: All cases were associated with the feeding of sorghum grain infected with sorghum ergot (Claviceps africana). There was mild to severe feed refusal when the sorghum was first offered. Sows fed ergot before farrowing had shrunken udders, produced no colostrum, and displayed signs of oestrus. All of their piglets died, apparently from starvation: necropsy of a few piglets showed that they were born alive and walked, but had ingested no milk. Sows fed the grain after farrowing had severe reductions in milk production despite aggressive sucking by piglets, leading to very poor growth of piglets. There were no signs of infectious disease. Ergot in sorghum samples ranged from 1 to 31% ergot sclerotes by weight. Total alkaloid concentrations in mixed feeds ranged from 5 to 40 mg/kg, with dihydroergosine accounting for approximately 90%. At the same time, in the same districts, there were reports of feed refusal and reduced milk production from 4 dairy farms. Grain samples from these farms contained up to 17% C africana ergot sclerotia. CONCLUSION: Agalactia and feed refusal are classical signs of poisoning by rye ergot (C purpurea), but this is the first time that sorghum ergot has been associated with a similar syndrome. PMID- 10736671 TI - Synchronising ovulation in dairy cows with either two treatments of gonadotropin releasing hormone and one of prostaglandin, or two treatments of prostaglandin. AB - OBJECTIVE: To compare oestrus synchronisation using two treatments of gonadotropin-releasing hormone (GnRH) and one of prostaglandin F2 alpha (PG) with a double prostaglandin synchronisation protocol under southern Australian conditions. DESIGN: A clinical trial. PROCEDURE: Eight hundred and forty, seasonally calving, lactating dairy cows within nine herds in the Tallangatta district of northeast Victoria were randomly allocated to treatment and control groups. The treatment (GnRH) group received gonadotropin-releasing hormone followed by prostaglandin F2 alpha and then a second treatment with gonadotropin releasing hormone. These cows were inseminated at a fixed time after the second gonadotropin-releasing hormone treatment. Cows in the control (PG) group received two injections of prostaglandin F2 alpha, 14 days apart, and were inseminated according to detected oestrus. RESULTS: The effect of GnRH treatment on first service conception rate (CRS1) and 30 day pregnancy rate (PR30) varied between herd (P < 0.001 and P < 0.02, respectively). A significant difference in CRS1 between treatment (GnRH) and control (PG) groups existed in pooled data from eight of the nine herds (38.1% vs 65.9%, P < 0.001). A significant difference also existed in PR30 between treatment (GnRH) and control (PG) groups in pooled data from eight of the nine herds (64.1% vs 72.4%, P = 0.03). Pregnancy rates after 56 days of mating for both groups were not significantly different (79.8% vs 84.1%, P = 0.13 for treatment (GnRH) and control (PG) groups, respectively). Submission rates (proportion of cows submitted for insemination) for the treatment (GnRH) groups were 100%. There was significant variation in submission rates in the control (PG) groups. CONCLUSION: The GnRH protocol may be of benefit in herds where a poor response to the double prostaglandin program is anticipated. However, in the majority of herds in this trial, the double prostaglandin program achieved better results with fewer inseminations. PMID- 10736672 TI - The relationship between the rate of intake of trichostrongylid larvae and the occurrence of diarrhoea and breech soiling in adult merino sheep. AB - BACKGROUND AND OBJECTIVE: A hypersensitive inflammatory response, associated with the ingestion of trichostrongylid larvae, is a major cause of diarrhoea and soiling of the breech with faeces in adult Merino sheep ('hypersensitivity scours'). This response is characterised by an infiltration of eosinophils and altered lymphocyte populations in the gut of affected sheep. The objective of this study was to investigate the numbers of Ostertagia circumcincta and Trichostrongylus vitrinus infective larvae that were needed to induce diarrhoea in adult Merino sheep grazing improved pastures. METHOD: Sheep from three farms were categorised as either being susceptible, or not susceptible, to hypersensitivity scours, then transported to an experimental farm and grazed on pastures with low numbers of trichostrongylid infective larvae. They were ranked by farm of origin and previous worm egg count, then systematically allocated to one of five dose-rate groups and infected with 0, 2, 4, 10 or 20 x 10(3) trichostrongylid infective larvae per week for 13 weeks. RESULTS: In sheep selected as being susceptible to hypersensitivity scours, the lowest dose rate of infective larvae (2000 per week) was as effective in inducing diarrhoea as the highest dose rate (20,000 per week). In contrast, even the highest larval dose did not induce diarrhoea in sheep selected as not susceptible to hypersensitivity scours. Sheep selected with an increased susceptibility to hypersensitivity scours also had a significantly higher amount of moisture in their faeces at all times except during summer. CONCLUSIONS: The apparent lack of a threshold dose of trichostrongylid infective larvae needed to induce hypersensitivity scours suggests that even improved worm control programs, which substantially decrease the ingestion of larvae, may not be able to reduce the prevalence of hypersensitivity scours or the severity of breech soiling. Controlled-release anthelmintic capsules will control breech soiling but are often not a cost effective strategy. Thus, genetic selection of less susceptible sheep is probably the best long-term option for the control of hypersensitivity scours, and the assessment of faecal moisture may be a convenient marker of susceptibility to this syndrome. PMID- 10736673 TI - Evaluation of a novel antimicrobial polymer for the control of porcine postweaning colibacillosis. AB - OBJECTIVE: To determine whether CHEMYDERTM polymer has potential for use in the control of porcine postweaning colibacillosis (PWC). PROCEDURE: Two experiments were conducted in which 50 young pigs, either receiving CHEMYDERTM polymer in their food or not, were challenged orally with cultures of beta-haemolytic Escherichia coli immediately after weaning. Their response in terms of development of diarrhoea, and the extent of colonisation of the intestinal tract by the bacteria was monitored. In a third experiment CHEMYDERTM polymer was added to the water supply of a group of 15 pigs on a piggery where PWC was an ongoing clinical problem. The response of these pigs was compared with that of pigs vaccinated against PWC or left unmedicated. RESULTS: In both experimental infection trials the pigs receiving CHEMYDERTM polymer showed significantly reduced intestinal colonisation with the challenge strain of E coli, and, in trial 2, significantly less diarrhoea after weaning compared to pigs not receiving CHEMYDERTM polymer. In the field trial the pigs receiving CHEMYDERTM polymer had significantly less diarrhoea and required significantly less antibiotic treatment than the other two groups of pigs. CONCLUSION: CHEMYDERTM polymer has potential for use in the control of PWC. PMID- 10736674 TI - Effect on systemic antibody concentrations of topical application of choleratoxin to skin of sheep. AB - OBJECTIVE: To examine the ability of a vaccine formulation combining choleratoxin with an experimental antigen to induce a systemic antibody response when applied topically on unbroken skin of sheep. DESIGN: Seven treatment groups of five adult sheep received systemic or topical priming followed 4 weeks later by systemic or topical boosting with choleratoxin and/or bovine serum albumin. Topical vaccines were administered to clipped skin on the ventral abdomen for 2 h. Booster immunisations were repeated 8 weeks after initial boosting. Serum antibody titres to choleratoxin and bovine serum albumin were determined by ELISA. RESULTS: An antibody response to choleratoxin was observed in serum, but no antibody response to bovine serum albumin was detected. CONCLUSION: Transdermal delivery may be feasible for livestock vaccines, however, further work is necessary to develop formulations that induce protective immunity by this route. PMID- 10736675 TI - Effect of grazing sorghum (Sorghum bicolor) infected with ergot (Claviceps africana) on beef cattle. PMID- 10736676 TI - Collars 'harmless'. PMID- 10736677 TI - Diagnosis and treatment of an osteochondroma of the distal tibia in a 3-year-old horse. PMID- 10736678 TI - Effect of phenobarbitone on the low-dose dexamethasone suppression test and the urinary corticoid: creatinine ratio in dogs. AB - OBJECTIVES: To investigate potential effects of phenobarbitone on the low-dose dexamethasone suppression (LDDS) test and urinary corticoid to creatinine ratio in dogs in a controlled prospective study and in a clinical setting. ANIMALS: Ten crossbreed experimental dogs and 10 client-owned dogs of mixed breeds treated chronically with phenobarbitone to control seizures. PROCEDURES: Experimental dogs were allocated to treatment (6 mg/kg oral phenobarbitone, n = 6) and control (n = 4) groups. LDDS tests (dexamethasone 0.01 mg/kg intravenously, cortisol concentration determined at 0, 2, 4, 6 and 8 h) were conducted repeatedly over a 3-month period. Urinary corticoid to creatinine ratios were measured before LDDS tests. A single LDDS test was performed on 10 epileptic dogs. RESULTS: LDDS and urinary corticoid to creatinine ratios in dogs were not affected by treatment with phenobarbitone. CONCLUSIONS: Phenobarbitone does not interfere with LDDS testing regardless of dosage or treatment time. Urinary corticoid to creatinine ratios are also unaffected. PMID- 10736679 TI - Effects of phenobarbitone on serum biochemical tests in dogs. AB - OBJECTIVES: To investigate effects of phenobarbitone on serum activities of alanine aminotransferase, alkaline phosphatase and gamma-glutamyl transferase and concentrations of bilirubin, albumin, cholesterol and total protein in dogs. ANIMALS: Ten crossbreed experimental dogs and 10 client-owned dogs of mixed breeds treated chronically with phenobarbitone to control seizures. PROCEDURES: Experimental dogs were allocated to treatment (6 mg/kg oral phenobarbitone, n = 6) and control (no treatment, n = 4) groups in which serum biochemical tests were performed at intervals during a 3-month period. Biochemical tests were performed once on the 10 epileptic dogs. RESULTS: Phenobarbitone caused increased serum alkaline phosphatase activity but did not affect gamma-glutamyl transferase activity or bilirubin, cholesterol, albumin and total protein concentrations. Phenobarbitone had minimal effect on alanine aminotransferase activity. CONCLUSIONS: Individual dogs treated with phenobarbitone may have small increases in serum alanine aminotransferase activity and variable increases in alkaline phosphatase activity but are unlikely to have alterations in gamma-glutamyl transferase activity or bilirubin, cholesterol, albumin or total protein concentrations. PMID- 10736680 TI - Diarrhoea associated with cryptosporidial oocyst shedding in a quarterhorse stallion. AB - Cryptosporidial oocyst shedding was detected in a 2.5-year-old Quarterhorse stallion with diarrhoea. Based on the detection of cryptosporidial oocysts, and the absence of other aetiological agents or enteropathogens, cryptosporidiosis was presumptively diagnosed. This case was unusual because cryptosporidial oocyst shedding is primarily found in some immunocompromised or immature horses, and has not been reported in healthy mature horses. PMID- 10736681 TI - Yersiniosis and trace element deficiency in a dairy herd. PMID- 10736682 TI - Contamination of food is a public health, public perception and trade issue. PMID- 10736683 TI - Temporal patterns and quantification of excretion of Mycobacterium avium subsp paratuberculosis in sheep with Johne's disease. AB - OBJECTIVES: To determine the frequency of excretion of Mycobacterium avium subsp paratuberculosis in Merino sheep with Johne's disease and to quantify excretion in a group of Merino sheep. DESIGN: A pen and laboratory experiment. PROCEDURE: Seven sheep selected from an affected flock on the basis of acid-fast bacilli in the sheep's faeces were housed and total daily faecal output was collected, weighed and subjected to culture for M avium subsp paratuberculosis. An end-point titration method was used to enumerate viable M avium subsp paratuberculosis in a 15 day pooled sample from five sheep that had acid-fast bacilli in their faeces while housed. RESULTS: Four sheep with subclinical multibacillary Johne's disease excreted M avium subsp paratuberculosis each day for 11 days of cultural observation. A further three sheep were intermittent excreters but lacked other evidence of infection with M avium subsp paratuberculosis. The average number of viable bacteria excreted was 1.09 x 10(8) per gram of faeces while total daily excretion was 8.36 x 10(10) viable M avium subsp paratuberculosis per sheep. Examination of faecal smears stained with Ziehl Neelsen was an unreliable means of assessing daily excretion in individual animals except in those with severe lesions. CONCLUSION: Excretion of M avium subsp paratuberculosis in Merino sheep with multibacillary Johne's disease occurred daily, proving that environmental contamination can be continuous on farms with endemic ovine Johne's disease. Faecal culture is a useful method for detecting infection as it does not appear to be affected by the timing of collection of a sample from sheep with multibacillary disease however, to maximise the sensitivity of disease surveillance using faecal culture, sampling rates should be adjusted to take account of the proportions of multibacillary and paucibacillary cases. PMID- 10736684 TI - A bovine stress syndrome associated with exercise-induced hyperthermia. AB - OBJECTIVE: To investigate an exercise-induced bovine stress syndrome under field and controlled experimental conditions. DESIGN AND PROCEDURE: In the field study, cattle affected with the stress syndrome were observed while they were grazing and during normal mustering using horses. This study served to define the clinical nature of the syndrome. The experimental study utilised three affected and five normal unaffected cattle. These animals were compared on the basis of their response to a defined exercise program, which consisted of walking 3.6 km in 2 h. Blood samples and measurements of respiratory rate, ambient temperature and rectal temperature were taken immediately before exercise, and at 0.5, 1.0, 1.5 and 2.0 h during the exercise and 24 h later. Clinical and blood constituent data were subjected to standard analysis of variance and repeated measures analysis. RESULTS: In the field study, affected cattle were observed to show abnormally anxious and hyperactive behaviour. This behaviour was exhibited by affected cattle during the experimental exercise program where it was shown to be accompanied by hyperthermia and hyperventilation. The experimental study showed that affected cattle developed metabolic acidosis and became hyperglycaemic. Their plasma creatine kinase activity remained markedly increased at 24 h after exercise but other clinical and blood constituent variables had returned to normal values. CONCLUSION: The clinical and biochemical changes detected in affected cattle were consistent with exercise-induced malignant hyperthermia. PMID- 10736685 TI - Vaccinating chickens against avian influenza with fowlpox recombinants expressing the H7 haemagglutinin. AB - OBJECTIVE: To evaluate the vaccine efficacy of a fowlpox virus recombinant expressing the H7 haemagglutinin of avian influenza virus in poultry. PROCEDURE: Specific-pathogen-free poultry were vaccinated with fowlpox recombinants expressing H7 or H1 haemagglutinins of influenza virus. Chickens were vaccinated at 2 or 7 days of age and challenged with virulent Australian avian influenza virus at 10 and 21 days later, respectively. Morbidity and mortality, body weight change and the development of immune responses to influenza haemagglutinin and nucleoprotein were recorded. RESULTS: Vaccination of poultry with fowlpox H7 avian influenza virus recombinants induced protective immune responses. All chickens vaccinated at 7 days of age and challenged 21 days later were protected from death. Few clinical signs of infection developed. In contrast, unvaccinated or chickens vaccinated with a non-recombinant fowlpox or a fowlpox expressing the H1 haemagglutinin of human influenza were highly susceptible to avian influenza. All those chickens died within 72 h of challenge. In younger chickens, vaccinated at 2 days of age and challenged 10 days later the protection was lower with 80% of chickens protected from death. Chickens surviving vaccination and challenge had high antibody responses to haemagglutinin and primary antibody responses to nucleoprotein suggesting that although vaccination protected substantially against disease it failed to completely prevent replication of the challenge avian influenza virus. CONCLUSION: Vaccination of chickens with fowlpox virus expressing the avian influenza H7 haemagglutinin provided good protection against experimental challenge with virulent avian influenza of H7 type. Although eradication will remain the method of first choice for control of avian influenza, in the circumstances of a continuing and widespread outbreak the availability of vaccines based upon fowlpox recombinants provides an additional method for disease control. PMID- 10736686 TI - Echocardiographic measurements in greyhounds, whippets and Italian greyhounds- dogs with a similar conformation but different size. AB - OBJECTIVE: To determine the effect of body size on various echocardiographic measurements in dogs of widely differing size, but identical body conformation. DESIGN: A randomised echocardiographic study of healthy sighthounds. ANIMALS: 60 dogs comprising an equal number (20) of racing Greyhounds, Whippets and Italian Greyhounds. PROCEDURE: Following sedation with acepromazine and morphine, and acclimatization, a thorough echocardiographic examination was performed on each dog using standard methods. RESULTS: Dimensions measured echocardiographically were highly correlated with body size. These data were subsequently examined using analyses of variance and regression. Body surface area was the best overall predictor of dimensional measurements. In comparison to previous studies using dogs of differing size and conformation, the spread of values for measurements plotted against body surface area showed substantially narrower ranges. Thus, the relationship between echocardiographic measurements and body surface area was much closer for dogs with an identical somatotype than for dogs of differing size and conformation. Commonly used ejection phase indices (fractional shortening, ejection fraction and velocity of circumferential fibre shortening) were negatively correlated with body size. In contrast, the thickening fraction of the left ventricular posterior wall, another ejection phase index, was independent of body weight and body surface area for all three breeds and when the data were pooled. CONCLUSION: Taken in consideration with previous work, this study demonstrates that body conformation and body size both influence canine echocardiographic measurements. Commonly used ejection phase indices are significantly affected by body size, with larger sighthounds having lower values. A more appropriate method of quantitating left ventricular function may be the determination of the thickening fraction of the left ventricular posterior wall. PMID- 10736687 TI - Detection of heterogeneous genotypes among Australian strains of Taylorella equigenitalis. PMID- 10736688 TI - Update on porcine reproductive and respiratory syndrome. PMID- 10736689 TI - One dog--two chips! PMID- 10736690 TI - Heterologous expression of bovine opsin in Pichia pastoris. PMID- 10736691 TI - Baculovirus expression system for expression and characterization of functional recombinant visual pigments. PMID- 10736692 TI - Development of stable cell lines expressing high levels of point mutants of human opsin for biochemical and biophysical studies. AB - Stable HEK293S cell lines expressing high levels of normal and mutant human rod opsins were generated. Cellular expression is uniform across a population. Secondary overexpression of the same opsin transgene linked to a different drug selection marker (hygro(R)) yielded expression clones with increased opsin levels compared to the neo(R) parent strain. Wild-type and mutant human opsins regenerate with native chromophore and demonstrate spectroscopic properties consistent with previous reports of bovine opsin mutants. HEK293S cells can be grown in larger scale suspension culture (10(9) cells/liter) or in roller bottles (10(8) cells/bottle) to facilitate milligram-order preparations of purified pigments. These cell lines should be useful in any time-resolved spectroscopic or biophysical experiments that require either uniform cellular levels of opsin protein or regenerable pigment, or large amounts of purified visual pigment. They should also be useful in experiments where uniform constitutive levels of a given mutant human visual pigment are needed in each cell. These and similar types of constitutive or inducible cell lines may also be useful for studying mechanisms of human cell death that occur by mutations in the human rod opsin gene. PMID- 10736693 TI - Folding and assembly of rhodopsin from expressed fragments. PMID- 10736694 TI - Isolation of isoelectric species of phosphorylated rhodopsin. PMID- 10736695 TI - Rhodopsin trafficking in photoreceptors using retinal cell-free system. PMID- 10736696 TI - Preparation and analysis of two-dimensional crystals of rhodopsin. PMID- 10736697 TI - Domain approach to three-dimensional structure of rhodopsin using high-resolution nuclear magnetic resonance. PMID- 10736698 TI - Analysis of functional microdomains of rhodopsin. PMID- 10736699 TI - Mapping tertiary contacts between amino acid residues within rhodopsin. PMID- 10736700 TI - Energetics of rhodopsin photobleaching: photocalorimetric studies of energy storage in early and later intermediates. PMID- 10736701 TI - Absorption spectroscopy in studies of visual pigments: spectral and kinetic characterization of intermediates. PMID- 10736702 TI - Structural determinants of active state conformation of rhodopsin: molecular biophysics approaches. PMID- 10736703 TI - pKa of the protonated Schiff base of visual pigments. AB - The pKa of bovine rhodopsin is greater than 15; that of the long-wave-length sensitive gecko P521 pigment ranges from 8.4 to 10.5 depending on chloride concentration; and that of octopus, an invertebrate, is 10.5. These pKa values are much higher than are needed just to maintain the Schiff base in its protonated state in the photoreceptor cell. The high pKa of the Schiff base may be at least partially related to a low pKa of its counterion, which would lower the frequency of thermal isomerization of the chromophore and thus lower the dark noise in the photoreceptor cell. After light absorption, the high pKa of the protonated Schiff base of a vertebrate visual pigment must get lowered enough to allow it to deprotonate, a required step in vertebrate visual excitation. This deprotonation step is not required in invertebrate visual excitation. PMID- 10736704 TI - Assays for detection of constitutively active opsins. PMID- 10736705 TI - Synthetic retinals: convenient probes of rhodopsin and visual transduction process. PMID- 10736706 TI - Assays for activation of opsin by all-trans-retinal. AB - The data collected with the techniques discussed in this chapter suggest significant differences between the active conformation(s) of the opsin/atr complex, which are reversibly formed in the dark, and the active conformation (R*) of the meta-II photoproduct. First, there is good evidence for noncovalent opsin/atr complexes with considerable activity (although covalent binding of atr is found in mutant opsins. Even more intriguing, all-trans-retinal in an amount that saturates the activity of the opsin/atr complex toward Gt does not measurably inhibit the access of 11-cis-retinal to the light-sensitive binding site during regeneration (Fig. 2C). On the other hand, forced protonation at or near Glu-134 appears to be an integral mechanism for both the meta-II and the opsin-like activities (Fig. 4). Thus, it is not inconceivable that these two activities of the receptor arise from two fundamentally different conformations, one meta-II-like and one opsin-like. They would be similar with respect to the Gt (or RK) protein-protein interaction but different in their mode of retinal protein interaction. PMID- 10736707 TI - Assays for activation of recombinant expressed opsins by all-trans-retinals. PMID- 10736708 TI - Electrical approach to study rhodopsin activation in single cells with early receptor current assay. AB - The ERC is a conformation-dependent charge motion similar to the gating currents of ionic channels. Both the waveforms and bandwidth of ERCs and ionic channel gating currents are similar, providing support to the initial suggestion that the ERP was a kind of gating current. In ionic channels the electrostatic field promotes motion of alpha-helical elements that stimulate large-scale molecular events that promote opening of the ionic pore. In ionic channels gating currents of expressed channel mutants has contributed significantly to understanding the mechanism of activation. Given the known role of electrical processes to rhodopsin activation, the ERC approach applied to mutant and wild-type visual pigments is likely to lead to a fuller understanding of the mechanism of conformational activation. This method is currently well suited to investigate the later phases of rhodopsin activation that are thought to be electrostatic in nature. We anticipate that ERC studies will make significant contributions to understanding how the breakdown of the electrostatic interaction between the PSB and its counterion is initiated and propagated to induce the proton uptake on the cytoplasmic surface of the pigment and the shaping of the transducin docking domain. We encourage collaboration to apply the ERC methodology to interesting mutant pigments and retinal analogs. We expect that the ERC methodology can soon be applied to understand rapid charge displacements associated with photochemistry (i.e., R1), the effects of transduction proteins on R2, and the measurement of electrical processes during cone visual pigment activation. PMID- 10736709 TI - Analysis of amino acid residues in rhodopsin and cone visual pigments that determine their molecular properties. PMID- 10736710 TI - Phylogenetic analysis and experimental approaches to study color vision in vertebrates. AB - To elucidate the molecular mechanisms of vertebrate color vision, it is essential to establish associations between amino acid substitutions and the directions of lambda max shifts of visual pigments. In this way, we can identify critical amino acid changes that may be responsible for lambda max shifts of visual pigments. In this process, we may consider only highly conserved residues, simply because the evolutionary conservation often implies functional importance. Using such an "evolutionary model" as a convenient tool in designing mutagenesis experiments, we can test specific hypotheses on the molecular mechanisms that are responsible for color vision in vertebrates. Virtually any vertebrate opsin cDNA can be expressed in COS cells, reconstituted with 11-cis-retinal, and the lambda max values of the regenerated pigments can be measured rather easily. By constructing mutant pigments with desired amino acid changes and conducting the in vitro assay and comparing their lambda max values with those of corresponding wild-type pigments, we can elucidate the molecular mechanisms of lambda max shifts--and color vision--of vertebrates rigorously. PMID- 10736711 TI - Light scattering methods to monitor interactions between rhodopsin-containing membranes and soluble proteins. PMID- 10736712 TI - Heterogeneity of rhodopsin intermediate state interacting with transducin. PMID- 10736713 TI - Limited proteolytic digestion studies of G protein-receptor interactions. PMID- 10736714 TI - Monitoring proton uptake from aqueous phase during rhodopsin activation. PMID- 10736715 TI - Use of peptides-on-plasmids combinatorial library to identify high-affinity peptides that bind rhodopsin. PMID- 10736716 TI - Purification of rhodopsin kinase by recoverin affinity chromatography. PMID- 10736717 TI - Heterologous expression and reconstitution of rhodopsin with rhodopsin kinase and arrestin. PMID- 10736718 TI - Arrestin: mutagenesis, expression, purification, and functional characterization. PMID- 10736719 TI - Mapping interaction sites between rhodopsin and arrestin by phage display and synthetic peptides. PMID- 10736720 TI - Characterization of RanBP2-associated molecular components in neuroretina. PMID- 10736721 TI - Intrinsic biophysical monitors of transducin activation: fluorescence, UV-visible spectroscopy, light scattering, and evanescent field techniques. PMID- 10736722 TI - Fluorescent probes as indicators of conformation changes in transducin on activation. PMID- 10736723 TI - G alpha t/G alpha i1 chimeras used to define structural basis of specific functions of G alpha t. PMID- 10736724 TI - Enzymology of GTPase acceleration in phototransduction. PMID- 10736725 TI - Mutational analysis of functional interfaces of transducin. PMID- 10736726 TI - Isolation and properties of protein phosphatase type 2A in photoreceptors. PMID- 10736727 TI - Purification and characterization of protein phosphatase type 2C in photoreceptors. PMID- 10736728 TI - Photoreceptor serine/threonine protein phosphatase type 7: cloning, expression, and functional analysis. PMID- 10736729 TI - Purification and assay of bovine type 6 photoreceptor phosphodiesterase and its subunits. PMID- 10736730 TI - Transcriptional regulation of the cGMP phosphodiesterase beta-subunit gene. PMID- 10736731 TI - Inhibition of photoreceptor cGMP phosphodiesterase by its gamma subunit. PMID- 10736732 TI - Kinetics and regulation of cGMP binding to noncatalytic binding sites on photoreceptor phosphodiesterase. PMID- 10736733 TI - Purification and autophosphorylation of retinal guanylate cyclase. PMID- 10736734 TI - Use of nucleoside alpha-phosphorothioates in studies of photoreceptor guanylyl cyclase: purification of guanylyl cyclase activating proteins. PMID- 10736735 TI - Heterologous expression and assays for photoreceptor guanylyl cyclases and guanylyl cyclase activating proteins. PMID- 10736736 TI - Spectrophotometric determination of retinal rod guanylyl cyclase. PMID- 10736737 TI - Calcium-dependent activation of membrane guanylate cyclase by S100 proteins. PMID- 10736738 TI - Characterization of guanylyl cyclase and phosphodiesterase activities in single rod outer segments. PMID- 10736739 TI - Covalent tethering of ligands to retinal rod cyclic nucleotide-gated channels: binding site structure and allosteric mechanism. PMID- 10736740 TI - Using state-specific modifiers to study rod cGMP-activated ion channels expressed in Xenopus oocytes. PMID- 10736741 TI - Identification and characterization of calmodulin binding sites in cGMP-gated channel using surface plasmon resonance spectroscopy. PMID- 10736742 TI - Determination of fractional calcium ion current in cyclic nucleotide-gated channels. PMID- 10736743 TI - Modulation of rod cGMP-gated cation channel by calmodulin. PMID- 10736744 TI - Purification and biochemical analysis of cGMP-gated channel and Na+/Ca(2+)-K+ exchanger of rod photoreceptors. PMID- 10736745 TI - Spectrofluorometric detection of Na+/Ca(2+)-K+ exchange. PMID- 10736746 TI - Purification and characterization of ABCR from bovine rod outer segments. PMID- 10736747 TI - ABCR: rod photoreceptor-specific ABC transporter responsible for Stargardt disease. PMID- 10736749 TI - Factors affecting yield and survival of cells when suspensions are subjected to centrifugation. Influence of centrifugal acceleration, time of centrifugation, and length of the suspension column in quasi-homogeneous centrifugal fields. AB - The goals of the centrifugation of cell suspensions are to obtain the maximum yield of cells with minimum adverse effects of centrifugation. In the case of mechanically sensitive cells such as mouse sperm, the two goals are somewhat contradictory in that g-forces sufficient to achieve high yields are damaging, and g-forces that yield high viability produce low yields. This paper mathematically analyzes the factors contributing to each goal. The total yield of pelleted cells is determined by the sedimentation rate governed by Stokes' Law, and depends on the relative centrifugal force, centrifugation time, size and shape of the cells, density of the cells and medium, viscosity of the medium, and the length of the column of suspension. Because in the situation analyzed the column is short relative to the rotor radius, the analysis considers the centrifugal field to be quasi-homogeneous. The assumption is that cells are not damaged during sedimentation, but that they become injured at an exponential rate once they are pelleted, a rate that will depend on the specific cell type. The behavior is modeled by the solution of coupled differential equations. The predictions of the analysis are in good agreement with experimental data on the centrifugation of mouse sperm. PMID- 10736748 TI - Developmental regulation of cell migration. Insight from a genetic approach in Drosophila. AB - Cell movements are fascinating and dramatic features of normal animal development. Moreover, failures in cell migration can lead to birth defects, and inappropriate cell migration can lead to cancer metastasis. Genetic approaches are beginning to provide some insights into the molecular basis for the developmental regulation of cell migration. This review discusses the progress that has been made in understanding the regulation of cell migration during Drosophila development, using a molecular genetic approach. In particular, these studies have implicated signaling through a receptor tyrosine kinase in the spatial control of migration. Reorganization of the cytoskeleton, under the control of the guanosine triphosphatase, Rac, is also critical for cell migration. Finally, genetic studies have demonstrated that the timing of cell migration is under transcriptional control. PMID- 10736750 TI - Principles of macromolecular organization and cell function in bacteria and archaea. AB - Structural organization of the cytoplasm by compartmentation is a well established fact for the eukaryotic cell. In prokaryotes, compartmentation is less obvious. Most prokaryotes do not need intracytoplasmic membranes to maintain their vital functions. This review, especially dealing with prokaryotes, will point out that compartmentation in prokaryotes is present, but not only achieved by membranes. Besides membranes, the nucleoid, multienzyme complexes and metabolons, storage granules, and cytoskeletal elements are involved in compartmentation. In this respect, the organization of the cytoplasm of prokaryotes is similar to that in the eukaryotic cell. Compartmentation influences properties of water in cells. PMID- 10736751 TI - The adenine pocket of a single catalytic site is derivatized when the bovine heart mitochondrial F1-ATPase is photoinactivated with 4-amino-1 octylquinaldinium. AB - The bovine heart mitochondrial F1-ATPase (MF1) is reversibly inhibited in the dark by 4-amino-1-octylquinaldinium (AOQ) with an I0.5 value of 48 microM. When irradiated in the presence of AOQ, MF1 is photoinactivated with an apparent Kd of 12 microM. About 1.1 mol of [3H]AOQ were incorporated per mol of MF1 on complete photoinactivation. Fractionation of a cyanogen bromide digest of MF1 photolabeled with [3H]AOQ followed by fractionation of peptic digests of partially purified cyanogen bromide fragments led to isolation of two CNBr/peptic fragments labeled with 3H. Sequence analysis of the labeled peptides revealed that one contained residues 423-441 of the beta subunit. A gap in position 2 of the sequence indicates that beta Phe424 is derivatized. The phenyl side-chain of this residue is part of a pocket that binds the adenine moiety of ATP or ADP at catalytic sites. The other peptide, which was labeled to a greater extent, contained residues 342-358 of the beta subunit, but in this case, no gap was found in the sequence indicating that the derivatized amino-acid side-chain might not have survived the conditions of automatic Edman degradation. This peptide contains beta Tyr345, the side-chain of which is also a component of the pocket that binds the adenine moiety of ATP or ADP to catalytic sites. However, for the reason stated, there is no direct evidence that beta Tyr345 is labeled in this peptide. PMID- 10736752 TI - Evidence for a slow time-scale of interaction for magnetic fields inhibiting tamoxifen's antiproliferative action in human breast cancer cells. AB - One critical biophysical feature of environmental-level magnetic field (MF) interactions with biological systems is the time-scale of interaction. A recently proposed fast/slow hypothesis states that a fast mechanism can only sense the instantaneous absolute value of the MF, and that a slow mechanism is potentially capable of sensing features such as frequency and relative orientation and magnitude of the field components. Here we applied the fast/slow hypothesis to a breast cancer model system: A 1.2 microT (rms), 60-Hz field inhibits tamoxifen's (TAM's) cytostatic action in MCF-7 cells via a MF interaction. We measured the growth of MCF-7 cells treated with TAM over 7 d, within different MFs: a sinusoidal, 60-Hz, 0.2-microT(rms) field; a sinusoidal, 60-Hz, 1.2-microT(rms) field; and a full-wave rectified version of the 1.2-microT(rms) sinusoidal field. A fast mechanism should not be able to distinguish between the latter two exposures. We observe that the rectified 1.2-microT field does not inhibit TAM's action, but that the 1.2-microT sinusoidal field does. Therefore, the 1.2-microT MF inhibition of TAM's cytostatic action operates via a relatively slow mechanism, and we predict that there exists a biologically dynamic complex capable of sensing a 1.2-microT, 60-Hz sinusoidal MF with an intrinsic time-scale of 17 ms or longer, the period of the 60-Hz applied field. PMID- 10736754 TI - Beta-adrenergic receptor desensitization in cardiac hypertrophy and heart failure. PMID- 10736753 TI - Mechanisms of nuclear translocation of insulin. AB - Insulin (Ins) and various other hormones and growth factors have been shown to be rapidly internalized and translocated to the cell nucleus. This review summarizes the mechanisms that are involved in the translocation of Ins to the nucleus, and discusses its possible role in Ins action, based on observations by the authors and others. Ins is internalized to endosomes by both receptor-mediated and fluid phase endocytosis, the latter occurring only at high Ins concentrations. The authors recently demonstrated the caveolae are the primary cell membrane locations responsible for initiating the signal transduction cascade induced by Ins. Once Ins is internalized, Ins dissociates from the Ins receptor in the endosome, and is translocated to the cytoplasm, where most Ins is degraded by Ins degrading enzyme (IDE), although how the polypeptides cross the lipid bilayer is unknown. Some Ins escapes the degradation and binds to cytosolic Ins-binding proteins (CIBPs), in addition to IDE. IDE and some CIBPs are known to be binding proteins for other hormones or their receptors, and are involved in gene regulation, suggesting physiological relevance of CIBPs in the signaling of Ins and other hormones. Ins is eventually translocated through the nuclear pore to the nucleus, where Ins tightly associates with nuclear matrix. The role of Ins internalization and translocation to the nucleus is still controversial, although there is substantial evidence to support its role in cellular responses caused by Ins. Many studies indicate that nuclear translocation of various growth factors and hormones plays an important role in cell proliferation or DNA synthesis. It would be reasonable to suggest that Ins internalization, its association with CIBPs, and its translocation to the nucleus may be essential for the regulation of nuclear events by Ins. PMID- 10736755 TI - [Evidence-based medicine: present status and problems in our country]. AB - Present status and problems of evidence-based medicine (EBM) in our country are discussed in this paper based on the report from the committee on the EBM of the Ministry of Health and Welfare of our country. Before introducing the content of the report from the above-mentioned governmental committee, a short history of the development of EBM and necessity of introducing the EBM in our country are discussed. In this report, 3 factors necessary to conduct the EBM; 1) clinical practice guidelines which are going to be developed in our country, 2) a strategy to conduct EBM in our country, and 3) problems of EBM in clinical practice have been discussed with a proposal of strategies for the future development of EBM in our country. PMID- 10736756 TI - [Insulin resistance syndrome and hypertension]. AB - Insulin resistance syndrome is the theory that glucose intolerance, hyperinsulinemia, increased very low density lipoprotein triglyceride level, decreased high density lipoprotein cholesterol level, and hypertension are proposed consequences of insulin resistance. These metabolic disturbances have been shown to increase the risk of coronary artery disease. In this theory, insulin resistance and the resultant hyperinsulinemia are considered to raise blood pressure through 1) sympathetic nervous system activation, 2) renal sodium retention, 3) renin-angiotensin system stimulation, and 4) intracellular calcium accumulation in vascular smooth muscle. Indeed, metabolic disturbance and insulin resistance have been pointed out in essential hypertensives. Leptin is a recently discovered hormone produced by an adipocyte-specific ob gene, that contributes to the regulation of energy balance by informing the hypothalamus of the amount of adipose tissue in the body. As a result, the hypothalamus adjusts food intake, thermogenesis, and energy expenditure appropriately. It was clarified that ob gene expression and plasma leptin level in humans were highly correlated with the body mass index, insulin sensitivity and blood pressure. Thus, leptin could play a role in the pathophysiology of insulin-resistant hypertension. PMID- 10736757 TI - [Development of artificial implant bone]. AB - This study tested the hypothesis that a significant amount of the new bone produced by heterotopic periosteal grafts is derived osteoinductively because proliferating periosteal cells express the bone morphogenetic protein (BMP). Rabbit ulnar and radial periosteum were autografted as free grafts (FGs) to the forelimb musculature, and as millipore diffusion chambers grafts (MDCGs) to the rectus abdominus muscle. The sections were immunostained with monoclonal antibody against recombinant human (rh) BMP-2. Sections from FGs recovered 5 to 28 days postoperatively exhibited cartilage and bone; fibrous tissue, cartilage, bone, and osteochondroid differentiated within MDCGs. Although BMP-2 was expressed by mesenchymal cells, osteoblasts, osteocytes, and osteoclasts, none of the MDCGs produced the osteoinductive signature of transmembrane bone formation. These observations indicated that the larger fraction of the new bone produced by heterotopic periosteal autografts is derived from the graft cells. As the artificial implant bone, 1) the freeze-dried bone, 2) autoclaved bone, 3) sintered bone, 4) hydroxyapatite, and 5) titanium metal were used. Sprague-Dawley rats (aged 8 weeks) were used, the parietal bone was cut off, they were treated to freeze-dried bone, autoclaved bone and sintered bone. Then each treated bone was reimplanted. The grafts were harvested at 1, 2, 3, and 5 weeks postoperatively, the specimens were observed with a scanning electron microscope and a conventional light microscope. All of the treated bone were able to bone union, especially in the sintered bone and autoclaved bone, the blood vessels and the bone formation were produced at extremely early stage. In implantation of the non-living body materials, bone formation and revascularization was inhibited. We think that bone transplantation after irradiation should be done after recovery from the radiation damage to the periosteal cells and the blood vessels. PMID- 10736758 TI - [Correlation between laminin and fibronectin on the basement membrane and tumor progression in early gastric cancer: an immunohistochemical study]. AB - Using an immunohistochemical procedure, I studied the possible relationship between the distribution of the basement membrane glycoprotein laminin (LN) or fibronection (FN) and the histological type or the pattern of infiltrating growth into surrounding tissue of the superficial depressed-type early gastric cancer in 78 cases (mucosal cancer: 36 cases, submucosal cancer: 42 cases). The staining patterns of the basement membrane LN and FN were divided into continuous (C), discontinuous (D), and negative (N). The patterns of infiltrating growth into surrounding tissue were classified into expanding growth and infiltrating growth. In regard to the relationship between LN staining patterns and histological types, the frequency of C pattern was significantly high in tubular adenocarcinoma (51.9%) but none in poorly differentiated adenocarcinoma (0%). On the other hand, the frequency of N pattern was significantly lower in tubular adenocarcinoma (3.9%) than in poorly differentiated adenocarcinoma (61.5%). Also in regard to the relationship between FN staining patterns and histological types, similar results were observed. There was no significant relationship between the mucosal staining patterns of LN or FN and the depth of invasion. I compared the staining patterns between the mucosal lesions and the submucosal lesions in submucosal carcinoma. In regard to the relationship of LN staining, the frequency of N pattern was significantly lower in mucosal lesions than in submucosal lesions. There was no difference in patterns of FN staining between the mucosal lesions and the submucosal lesions. In regard to the relationship between the LN staining pattern and the pattern of infiltrating growth into surrounding tissue, the frequency of N pattern was significantly lower in expanding growth (23.5%) than in infiltrating growth (68.0%). There was no significant relationship between the FN staining pattern and the infiltrating growth pattern. These findings suggest that the disappearance of LN in gastric cancer is an important factor in the invasion of the submucosa or in determining the pattern of infiltrating growth of tumors into surrounding tissue. PMID- 10736759 TI - [Investigation of binding capacity of beta 2-glycoprotein I with anti-beta 2 glycoprotein I antibodies in lymphocytes undergoing apoptosis]. AB - Apoptosis (programmed cell death) is accompanied by loss of phospholipid asymmetry, i.e., translocation of aminophospholipids such as phosphatidylserine to the outer leaflet of the plasma membranes, which results in recognition of these cells by macrophages. In the present study, I studied on the involvement of beta 2-glycoprotein I (beta 2-GPI) and anti-beta 2-GPI antibodies in apoptotic lymphocytes. By flowcytometric analysis, I demonstrated that beta 2-GPI could bind to a human T cell line, Jurkat cells, treated with an anti-Fas antibody, CH11. beta 2-GPI-bound cells were detected 2 hr later after anti-Fas treatment and the most cells were bound to beta 2-GPI by 6 hr later. The accumulation of beta 2-GPI-bound cells paralleled with morphological changes and DNA fragmentation of the cells. I also demonstrated that anti-beta 2-GPI antibodies and a beta 2-GPI-dependent anti-cardiolipin antibody, established from a patient with antiphospholipid syndrome, could recognize beta 2-GPI-bound Jurkat cells treated with anti-Fas-antibody. These results imply that beta 2-GPI and anticardiolipin antibody may have a role in the clearance of apoptotic cells from the blood stream. PMID- 10736760 TI - [A study on development of enzyme immunoassay of human osteopontin: molecular fragility of serum osteopontin]. AB - Osteopontin, a secreted acidic phosphoglycoprotein with calcium binding activity, has been reported to have diverse functions, including cell adhesion, calcium remodeling, cell signaling and cellular transformation. This recalls us to a concept that the osteopontin can be a marker for certain clinicopathological states. However, a method for analysis of osteopontin has not successfully been developed to be utilized. We have isolated osteopontin from human milk, prepared antibodies specific to it, and tried to establish an enzyme immunoassay (EIA) system to determine osteopontin in serum. It was found that serum osteopontin but not milk osteopontin showed paradoxical phenomena in reaction with the antibodies, i.e., no detectable reaction in immunoelectrophoresis and in EIA, but showing positive reaction in Western blot and in inhibition tests. Taking these phenomena and other circumstantial evidence into account, it was concluded that the serum osteopontin had high molecular fragility, fragmenting into small peptides with paucity of epitopes during incubation time. These findings indicate the possible presence of osteopontins with different molecular characteristics among tissues, and that these characteristics of osteopontin will interfere with the development of quantitative assay. PMID- 10736761 TI - [Bruise-like discolorations can appear after death]. AB - It is not sufficiently emphasized in our country that bruising can also appear post-mortem. We report two cases in which we observed discolorations which looked like ante-mortem bruising. Case 1: A 37-year-old man was found prone on a river shore and taken out of the water by a rescuer by grasping at the right upper arm approximately one hour and 30 minutes after death. At inspection, two thumb-sized discolorations resembling ante-mortem bruising were observed on the lateral and frontal surfaces of the right upper arm. Case 2: A 40-year-old woman was found prone immersed in a moat and taken out of the water in the above-mentioned manner approximately one hour and 45 minutes after death. At inspection, two thumb-sized discolorations appearing to be ante-mortem bruising were observed on the inner surface of the right upper arm. The cause of death in both instances was drowning. Bibliographic investigation revealed that bruising of significant size can appear after death. We speculate generally on conditions for generation of post-mortem bruising. Additionally, we believe that post-mortem bruising should be sufficiently considered, because it can be important whether a person was grasped when he or she was alive or dead. PMID- 10736763 TI - [The 11th Meeting of Hokkaido Society for Study of Osteoporosis] [In Process Citation] PMID- 10736762 TI - [In view of insurance for care and assistance for the elderly]. PMID- 10736764 TI - Laboratory study on leachability of five herbicides in South Australian soils. AB - Norflurazon, oxadiazon, oxyfluorfen, trifluralin and simazine are herbicides widely used in the vineyards of the Barossa Valley, South Australia. The leaching behaviour of norflurazon, oxadiazon, oxyfluorfen and trifluralin was investigated on four key soils in the Barossa Valley. Leaching potential on packed soil columns and actual mobility using intact soil columns were investigated. On the packed soil columns, norflurazon was the most leachable herbicide. More of the herbicides were detected in the leachates from the sandy soils (Mountadam and Nuriootpa) than from the clayey soils (Lyndoch and Tanunda). Organic matter is generally low in soils in the Barossa region. Porosity and saturated conductivity significantly affect herbicide movement and in the sandy Mountadam and Nuriootpa soils, the water flux is greater than for the higher clay content Lyndoch and Tanunda soils. Increasing the time interval between herbicide application and the incidence of "rainfall" reduced the amounts of herbicides found in the leachates. The use of intact soil columns and including simazine for comparison showed that both norflurazon and simazine were present in the leachates. Simazine was the first herbicide to appear in leachates. Sectioning of the intact soil columns after leaching clearly demonstrated that norflurazon and simazine reached the bottom of the soil columns for all soils studied. Greater amounts of norflurazon were retained in the soil columns compared with simazine. The other herbicides were mostly retained in the initial sections of the soil columns. PMID- 10736765 TI - Effects of five insecticides used in apple orchards on Hyaliodes vitripennis (Say) (Hemiptera: Miridae). AB - Azinphos-methyl, carbaryl, dimethoate, phosmet and phosalone were used in apple orchards to manage apple aphid, apple maggot, woolly apple aphid and leaf eating caterpillars. Among the five insecticides evaluated, dimethoate, carbaryl and azinphosmethyl were the most toxic to the nymphs and adults of Hyaliodes vitripennis (Say) from two regions. Phosalone was the least toxic. Nymphs were more resistant than the adults. While the LC50 for dimethoate was 130 ppm for nymphs, it was 3 ppm for adults from St. Jean-Baptiste-de-Rouville. There were also significant differences in the level of resistance between the two regions where the H. vitripennis were collected. At St. Alexandre the LC50 for phosalone on nymphs was 19,250 ppm whereas, at St. Jean-Baptiste-de-Rouville it was 160,000 ppm. PMID- 10736766 TI - Sorption of atrazine and metolachlor by earthworm surface castings and soil. AB - Atrazine and metolachlor were more strongly retained on earthworm (Lumbricus terrestris L.) castings than on soil, suggesting that earthworm castings at the surface or at depth can reduce herbicide movement in soil. Herbicide sorption by castings was related to the food source available to the earthworms. Both atrazine and metolachlor sorption increased with increasing organic carbon (C) content in castings, and Freundlich constants (Kf values) generally decreased in the order: soybean-fed > corn-fed > not-fed-earthworm-castings. The amount of atrazine or metolachlor sorbed per unit organic carbon (Koc values) was significantly greater for corn-castings compared with other castings, or soil, suggesting that the composition of organic matter in castings is also an important factor in determining the retention of herbicides in soils. Herbicide desorption was dependent on both the initial herbicide concentration, and the type of absorbent. At small equilibrium herbicide concentrations, atrazine desorption was significantly greater from soil than from any of the three casting treatments. At large equilibrium herbicide concentrations, however, the greater organic C content in castings had no significant effect on atrazine desorption, relative to soil. For metolachlor, regardless of the equilibrium herbicide concentration, desorption from soybean- and corn-castings treatments was always less than desorption from soil and not-fed earthworm castings treatments. The results of this study indicate that, under field conditions, the extent of herbicide retention on earthworm castings will tend to be related to crop and crop residue management practices. PMID- 10736767 TI - Storm flow export of metolachlor from a coastal plain watershed. AB - During an 18-month (1994-1995) survey of the surface water in an Atlantic Coastal Plain watershed, metolachlor was most frequently detected during storm flow events. Therefore, a sampling procedure, focused on storm flow, was implemented in June of 1996. During 1996, three tropical cyclones made landfall within 150 km of the watershed. These storms, as well as several summer thunderstorms, produced six distinct storm flow events within the watershed. Metolachlor was detected leaving the watershed during each event. In early September, Hurricane Fran produced the largest storm flow event and accounted for the majority of the metolachlor exports. During the storm event triggered by Hurricane Fran, the highest daily average flow (7.5 m2 s-1) and highest concentration (5.1 micrograms L-1) ever measured at the watershed outlet were recorded. Storm flow exports leaving the watershed represented 0.1 g ha-1 or about 0.04% of active ingredient applied. PMID- 10736768 TI - Removal of 2,4-D from aqueous solution by the adsorbents from spent bleaching earth. AB - The removal of 2,4-D (2,4-dichlorophenoxyacetic acid) from aqueous solutions by activated spent bleaching earths (SBE) was studied at 20 degrees C. Experiments were performed as a function of time, initial concentration, dose and particle size of the adsorbent. The Langmuir and Freundlich adsorption equations were fitted by the adsorption data obtained. The values of Langmuir and Freundlich constants were determined. The adsorption kinetic was found to follow Lagergren equation. Both the boundary layer and intraparticle diffusion played important roles in the adsorption rate of 2,4-D. As the size of the adsorbent increased, the time to reach equilibrium increased but adsorption capacity decreased. PMID- 10736769 TI - Comparison of residue levels of persistent organochlorine compounds in butter from Spain and from other European countries. AB - Commercially available butter from Spain (n = 36) and from other European countries (n = 20) has been analyzed by high resolution gas chromatography with an electron capture detector for organochlorine contaminants. The results showed that both groups of samples have a similar pattern of these pollutants, but in all cases where significant differences in concentrations were found, Spanish samples presented higher values. This was especially notable for lindane (median values 11.6 vs. 3.0 ng/g wet weight), hexachlorobenzene (6.4 vs. 0.5) and beta hexachlorocyclohexane (3.1 vs. 1.2). Total PCBs showed no differences (5.4 vs. 6.6), but detailed analysis of congener composition indicated a higher presence in Spanish samples of the most persistent chlorinated PCBs. No sample presented levels of concern for any single organochlorine compound. It is interesting to note that consumption of butter in Spain is low so, although levels of organochlorines are higher than those found in other countries, consumers are less exposed through this foodstuff than in other European countries. These results also confirm the fact that environmental pollution by organochlorines is more important in Spain than in the rest of Europe. PMID- 10736771 TI - Interpreting the relationship between pheromone component emission from commercial lures and captures of Helicoverpa zea (Boddie) in bucket and cone traps. AB - Male corn earworm moths, Helicoverpa zea (Boddie), were captured in conical Texas pheromone traps (cone traps) and bucket traps baited with four different commercial lures manufactured by three different manufacturers. Because significant numbers were captured in bucket traps baited with some of the lures, and none with others, the volatile emissions from all of the lures were sampled and analyzed by gas chromatographic methods. The numbers of males captured in two types of trap were compared with bait emissions in an endeavor to define a more effective lure for bucket traps. The lure from one manufacturer captured the same numbers of males in both trap types; one captured more in bucket traps than in cone traps, and another captured only a small number in bucket traps. The emission rate of all active compounds from each of the different lures was approximately linear for the duration of the assays. A gas-liquid chromatographic peak associated with a third compound, (Z)-9-tetradecenal, which reduces behavioral responses, was observed in the emissions from all lures evaluated. The effectiveness of the Hercon (Emmigsville, PA) lure in capturing males in both types of trap was associated with a lower emission of (Z)-11-hexadecenal, (Z)-9 hexadecenal and (Z)-9-tetradecenal than from the other lures. PMID- 10736770 TI - Population dynamics in Daphnia magna as modified by chronic tetradifon stress. AB - Two Daphnia magna offsprings (animals from the first and third brood) whose parentals (F0-generation) were exposed during 21 days to different tetradifon (4 chlorophenyl 2,4,5-trichlophenyl sulfone) concentrations, were transferred to a pesticide free medium during 21 days (recovery period). The algae Nannochloris oculata (5 x 10(5) cells/mL) was used as food. In this recovery study, survival, growth and reproduction (mean total young per female, mean brood size, onset of reproduction and mean number broods per female) were assessed as individual parameters and the intrinsic rate of natural increase (r) as population parameter, for F1 generation (1st and 3rd broods). Reproduction was still reduced in F1 (1st and 3rd broods) generation daphnids from parentals (F0) exposed to 0.18 mg/L tetradifon and higher concentrations even after 21 days in clean water. However, survival was not significantly different (p > 0.05) in those F1 offsprings from parentals pre-exposed to the acaricide. Growth was still reduced in F1 daphnids from parentals pre-exposed to 0.10-0.44 mg/L tetradifon. The intrinsic rate of natural increase (r) was still affected in F1 generation daphnids, specially in those from the third brood. Therefore, F1 generation D. magna offsprings from a parental generation (F0) previously exposed to the acaricide tetradifon were not able to recuperate completely when a recovery period of 21 days was allowed. PMID- 10736772 TI - A field study on downwind odor transport from swine facilities. AB - This paper presents field data to illustrate the characteristics of downwind odor dispersion. A total of eight swine facilities were studied in this project and power functions were found to be an appropriate description of the pattern of downwind odor transport for both swine buildings and manure storage lagoons and tanks. The coefficients of correlation for the power regressions ranged from 0.75 0.87. It was found that swine buildings have the potential of generating more odor than manure storage facilities such as lagoons and tanks and thus could be the major odor sources causing downwind odor nuisance. Therefore, to maintain the building clean should be highly recommended as swine producers' best management practice. For earthen basins, the odor strength was reduced by 80% at about 250 meter distance from the source. While for the swine building, a 50% of reduction in odor strength was observed at the same distance. The study did not show any advantages of using lagoons over concrete (or steel) manure tanks in terms of reducing downwind odor concentrations. Land application of manure did not cause persistent downwind odor problems. PMID- 10736773 TI - Combined quantum mechanical and molecular mechanical reaction pathway calculation for aromatic hydroxylation by p-hydroxybenzoate-3-hydroxylase. AB - The reaction pathway for the aromatic 3-hydroxylation of p-hydroxybenzoate by the reactive C4a-hydroperoxyflavin cofactor intermediate in p-hydroxybenzoate hydroxylase (PHBH) has been investigated by a combined quantum mechanical and molecular mechanical (QM/MM) method. A structural model for the C4a hydroperoxyflavin intermediate in the PHBH reaction cycle was built on the basis of the crystal structure coordinates of the enzyme-substrate complex. A reaction pathway for the subsequent hydroxylation step was calculated by imposing a reaction coordinate that involves cleavage of the peroxide oxygen-oxygen bond and formation of the carbon-oxygen bond between the C3 atom of the substrate and the distal oxygen of the peroxide moiety of the cofactor. The geometric changes and the Mulliken charge distributions along the calculated reaction pathway are in line with an electrophilic aromatic substitution type of mechanism. The energy barrier of the calculated reaction is considerably lower when the substrate hydroxyl moiety is deprotonated, in comparison with the barrier found with a protonated hydroxyl moiety. This effect of the protonation state of the substrate on the calculated energy barrier supports experimental observations that deprotonation is required for hydroxylation of the substrate. A notable event in the calculated reaction pathway is a lengthening of the peroxide oxygen-oxygen bond at an intermediate stage. Further analysis of the reaction pathway indicates that this oxygen-oxygen bond elongation is accompanied by an increase in electrophilic reactivity on the distal oxygen of the peroxide moiety, which may assist the C-O bond formation in the reaction of the C4a-hydroperoxyflavin intermediate with the substrate. Analysis of the effect of individual active site residues on the reaction reveals a specific transition state stabilization by the backbone carbonyl moiety of Pro293. The crystal water 717 appears to drive the hydroxylation step through a stabilizing hydrogen bond interaction to the proximal oxygen of the C4a-hydroperoxyflavin intermediate, which increases in strength as the hydroperoxyflavin cofactor converts to the anionic (deprotonated) hydroxyflavin. PMID- 10736774 TI - XCrySDen--a new program for displaying crystalline structures and electron densities. AB - XCrySDen is a molecular and crystalline-structure visualization program, but its main function is as a property analyzer program. It can run on most UNIX platforms, without any special hardware or software requirements. Special efforts were made to allow for appropriate display of 3D isosurfaces and 2D contours, which can be superimposed on crystalline structure and interactively rotated and manipulated. XCrySDen is also a graphical user interface for the CRYSTAL95/98 (Saunders, V. R., et al. CRYSTAL98--User's Manual. University of Torino, Turin, Italy, 1999) ab initio program and a visualization system for the WIEN97 (Blaha, P., et al. Comput. Phys. Commun. 1990, 59, 399) ab initio program. In this article the program functions are presented with a short description of the algorithms. PMID- 10736775 TI - VRDD: applying virtual reality visualization to protein docking and design. AB - We have developed an interactive docking program called VRDD. It offers various modes of displaying molecules in an immersive, three-dimensional virtual reality (VR) environment. It allows a user to interactively perform molecular docking aided by automatic docking and side chain conformational search. Binding free energies are computed in real time, and the program enables the user to explore only clash-free orientations of a ligand. VRDD also supplies visual and auditory feedback during docking and side chain search, indicating the levels of atomic overlap and interaction energy. The stunning VR graphics immerse users in the scene and can maximally stimulate their design intuition. We have tested VRDD on three cases with increasing complexity: a nine-residue-long peptide bound to a major histocompatibility complex (MHC) molecule, barstar bound to barnase, and an antibody bound to a hemagglutinin. Without prior knowledge, combinations of hand docking and automatic refinement led to accurate complex structures for the first two complexes. The third case, for which all automatic docking algorithms failed to identify the correct complex in a previous blind test, also failed for VRDD. Our results show that the combination of VR docking and automatic docking can make unique contributions to molecular modeling. PMID- 10736776 TI - Investigation and modification of molecular structures with the nanoManipulator. AB - The nanoManipulator system adds a virtual reality interface to an atomic force microscope (AFM), thus providing a tool that enables the user not only to image but also to manipulate nanometer-sized molecular structures. As the AFM tip scans the surface of these structures, the tip-sample interaction forces are monitored, which in turn provide information about the frictional, mechanical, and topological properties of the sample. Computer graphics are used to reconstruct the surface for the user, with color or contours overlaid to indicate additional data sets. Moreover, by means of a force-feedback pen, which is connected to the scanning tip via software, the user can touch the surface under investigation to feel it and to manipulate objects on it. This system has been used to investigate carbon nanotubes, fibrin, DNA, adenovirus, and tobacco mosaic virus. Nanotubes have been bent, translated, and rotated to understand their mechanical properties and to investigate friction on the molecular level. AFM lithography is being combined with the nanoManipulator to investigate the electromechanical properties of carbon nanotubes. The rupture forces of fibrin and DNA have been measured. This article discusses how some of the graphics and interface features of the nanoManipulator made these novel investigations possible. Visitors have used the system to examine chromosomes, bacterial pili fibers, and nanochain aggregates (NCAs). Investigators are invited to apply to use the system as described on the web at http:@www.cs.unc.edu/Research/nano/doc/biovis it.html. PMID- 10736777 TI - Three-dimensional molecular field analyses of octopaminergic agonists and antagonists for the locust neuronal octopamine receptor class 3. AB - The quantitative structure-activity relationship (QSAR) of a set of 70 octopaminergic agonists and 20 antagonists against octopamine receptor class 3 (OAR3) in locust nervous tissue was analyzed by molecular field analysis (MFA). MFA of these compounds evaluated effectively the energy between a probe and a molecular model at a series of points defined by a rectangular grid. Contour surfaces for the molecular fields are presented. These results provide useful information in the characterization and differentiation of octopaminergic receptor types and subtypes. PMID- 10736778 TI - Schematic representation of residue-based protein context-dependent data: an application to transmembrane proteins. AB - An algorithmic method for drawing residue-based schematic diagrams of proteins on a 2D page is presented and illustrated. The method allows the creation of rendering engines dedicated to a given family of sequences, or fold. The initial implementation provides an engine that can produce a 2D diagram representing secondary structure for any transmembrane protein sequence. We present the details of the strategy for automating the drawing of these diagrams. The most important part of this strategy is the development of an algorithm for laying out residues of a loop that connects to arbitrary points of a 2D plane. As implemented, this algorithm is suitable for real-time modification of the loop layout. This work is of interest for the representation and analysis of data from (1) protein databases, (2) mutagenesis results, or (3) various kinds of protein context-dependent annotations or data. PMID- 10736779 TI - The art of molecular graphics. What does a molecule look like? PMID- 10736780 TI - Specificity of the tumor necrosis factor receptor superfamily. PMID- 10736781 TI - Calculation of concerted motions in biomolecules. CECA, Workshop, Lyon, France. October 11-13, 1999. PMID- 10736782 TI - Rotational superposition: a review of methods. AB - Rotational superposition is one of the most commonly used algorithms in molecular modelling. Many different methods of solving superposition have been suggested. Of these, methods based on the quaternion parameterization of rotation are fast, accurate, and robust. Quaternion parameterization-based methods cannot result in rotation inversion and do not have special cases such as co-linearity or co planarity of points. Thus, quaternion parameterization-based methods are the best choice for rotational superposition applications. PMID- 10736783 TI - Teaching computational chemistry in the undergraduate and graduate chemistry curriculum. PMID- 10736784 TI - Auto-induction of E5110 metabolism, a novel non-steroidal anti-inflammatory drug, during toxicokinetic studies in beagle dogs. AB - Following single-dose intravenous and oral studies to determine the absolute bioavailability of E5110 in beagle dogs, repeated dose pharmacokinetic studies were conducted as toxicokinetics in a subacute toxicity test. E5110 was administered orally once a day for 91 days at doses of 0, 1, 10, 50 and 250 mg/kg/day to dogs. On the first day during repeated administration, the Cmax and AUC values of E5110 in females were lower than those for males, and it seems that this may be due to a sex-related difference in the activity of cytochrome P450 (CYP) 3A. During repeated administration of E5110, the plasma levels of E5110 on day 15 and day 91 were markedly lower than those on the first day. On day 91, the AUCs for E5110 were 55.9%, 38.8%, 10.9% and 7.8% in males, and 76.2%, 80.3%, 10.5% and 11.9% in females on the first day, for doses of 1, 10, 50 and 250 mg/kg/day, respectively. Liver microsomes prepared after the last dose of E5110 showed increased activities of benzphetamine N-demethylase, p-nitroanisole O demethylase, and aminopyrine N-demethylase, at doses of 1 mg/kg/day or more. A dose-dependent increase in P450 content was also observed. Furthermore, the capacity for 5-hydroxylate E5110 was increased, and Western blot analysis indicated an induction of CYP2B and 3A; therefore CYP3A may contribute to a main metabolic pathway of E5110. These results suggested that the decrease in plasma concentrations of E5110 that were observed during repeated administration represents a typical case of auto-induction of the phenobarbital type. PMID- 10736785 TI - Inhibitory effects of repeated intravenous injections of dexamethasone on pulmonary toxicity of a new mitomycin C analogue, KW-2149, in a novel rat model. AB - An animal model for testing pulmonary toxicity of KW-2149, a new mitomycin C analogue, was developed (by intravenously injecting 3.28 mg/kg of the drug into male SD rats 3 times at weekly intervals), and exhibits pleural effusion from 1 week after the last injection. In this animal model, repeated intravenous injections of dexamethasone (DM), following any of three different schedules examined, were more or less effective of reducing the amount of effusion. The optimal results were obtained with 4 administrations a week (i.e. twice before and twice after KW-2149 treatment). The results of the present experiment suggest possible clinical application of DM in protecting patients from pulmonary toxicity of KW-2149. PMID- 10736786 TI - Induction of unscheduled DNA synthesis in hairless mouse epidermis by ultraviolet light. AB - The activity of ultraviolet (UV) light to induce unscheduled DNA synthesis (UDS) was investigated in hairless mouse epidermis by means of an in vivo-in vitro assay using a liquid scintillation counting method. Groups of three to five 8 week-old female hairless mice were irradiated with UV-B or UV-A, then skin samples were taken and cultured individually in medium containing [3H]thymidine with or without hydroxyurea (HU) for 2 hr. DNA of the epidermis was extracted, and incorporation of [3H]thymidine and the DNA content were determined with a liquid scintillation counter and a fluorescence spectrophotometer, respectively. Induction of UDS was judged in terms of the UDS index [(the ratio of DNA synthesis in the presence of HU to that in its absence) x 100]. UV-B increased the UDS index 1 hr after irradiation of 500 J/m2, which corresponds to approximately 1 minimal erythema dose or 1 minimal edema dose, and showed a dose dependent increase up to 17-fold in the UDS index at irradiation doses of 500 to 2,000 J/m2. In a time-course study, UV-B also increased replicative DNA synthesis (RDS) 48 hr after irradiation at 1,000 J/m2. On the other hand, UV-A did not increase the UDS index at irradiation doses of 2 x 10(5) to 8 x 10(5) J/m2. These results show that induction of UDS by UV irradiation depends on wavelength and an increase of RDS in the epidermis exposed to UV-B irradiation appears after induction of UDS. PMID- 10736787 TI - Effects of an agent inducing dominant lethals on rat sperm--examination with ethyl methanesulfonate. AB - Ethyl methanesulfonate (EMS), an alkylating agent which induces dominant lethals, was administered in oral doses of 100 mg/kg to Crj:CD(SD)IGS male rats for 5 consecutive days. At the termination of treatment and after a 28-day withdrawal, mating with untreated females and sperm analysis (motion, number, and morphology) were performed. The copulated females were sacrificed at 20 days of gestation. At the termination of treatment, no clinical signs related to EMS were observed except for a decrease in body weight. Gross pathology and sperm analysis revealed no abnormalities in treated males. However, females mated at the termination of treatment had a clearly higher fetal mortality. Females mated after the 28-day withdrawal exhibited lower fetal mortality than females mated at the termination of treatment. On the other hand, females mated after the 28-day withdrawal exhibited a lower implantation rate that was not observed in females mated at the termination of treatment. For males after a 28-day withdrawal, sperm analysis revealed both a decrease in sperm motion and number and an increase in morphological change. These findings indicate that two types of male reproductive toxicity induced by EMS can be distinguished. One induces a low implantation rate that can be detected by sperm analysis, while the other induces fetal lethals that could not be detected by sperm analysis in this study. PMID- 10736788 TI - Effect of food restriction on hepatotoxicity of carbon tetrachloride in rats. AB - Five-week-old Sprague-Dawley rats of both sexes were assigned to two types of feeding condition. One was fed ad libitum (AL) on commercial chow and another was fed a restricted amount of the chow (FR), approximately 75% of that fed in the AL condition. In each feeding condition, animals were orally administered carbon tetrachloride (CCl4) at levels of 0 (control), 0.1 or 0.2 ml/kg 6 days a week for 8 weeks. Lesions of the liver (hepatic cellular degeneration and fibrosis) and of the kidney (proximal tubular vacuolation and glomerular sclerosis) induced by CCl4 were aggravated in the FR group. The FR-control showed a higher metabolic activity of aniline in the liver than the AL-control group. Plasma lipid-peroxide (LPO) level was higher in the AL-control group than in the FR-control group. With CCl4 0.2 ml/kg treatment, however, the plasma LPO level was reversed between the AL and the FR groups. Taking together these somewhat unexpected results, it was concluded that (1) 25% food restriction increases toxicity of repeatedly administered CCl4 in rats, and (2) aggravation of CCl4 toxicity may be partly due to enhanced metabolic activation of CCl4 resulting from food restriction. PMID- 10736789 TI - Flow cytometric detection and analysis of tailless sperm caused by sonication or a chemical agent. AB - Flow cytometric analysis has been developed to detect tailless sperm with heads detached from the tails at the neck position. When isolated tailless sperm suspension was subjected to flow cytometry, a second sperm population appeared alongside the normal sperm population on light scatter-histogram. The percentage of this second sperm population (85.2%) was in good agreement with that for the tailless sperm (88.7%) determined microscopically, indicating that the second sperm population would correspond to tailless sperm population in the light scatter-histogram. Rates for tailless sperm determined by flow cytometry significantly correlated with those estimated microscopically following exposure of sperm to either sonication (r = 0.94, P < 0.01), or nitrobenzene (r = 0.80, P < 0.01). The results indicated the utility of the light scatter-histogram in flow cytometry as a simple and convenient procedure for the detection of tailless sperm induced by chemical compounds. PMID- 10736790 TI - Histopathological study of the effects of a single intratracheal instillation of surface active agents on lung in rats. AB - Pulmonary drug administration of most peptide/protein drugs is characterized by low bioavailability due to low permeability. Surface active agents have been tested as an absorption enhancer, but few studies have been carried out on the local toxicity of these additives. In the present study, to clarify the toxic effects of surface active agents on the lung, a relatively high concentration (1%) of polyoxyethylene 9 lauryl ether (Laureth-9) and sodium glycocholate (SGC) was given to rats in a single intratracheal instillation (100 microliters/rat), and the lung was evaluated histopathologically. In the rats treated with Laureth 9, lung lesions were observed in the bronchi to alveoli. At 1 day after administration, edema, hemorrhage and inflammatory cell infiltration due to degeneration and desquamation of epithelium were observed. At 3 and 7 days after administration, the wound healing process resulting from the lung injury, such as hyperplasia of epithelium and sporadic fibrosis, was noted. SGC also induced lung lesions with a similar histopathological nature, whereas the lesions were mostly confined to the alveoli. These results suggest that surface active agents induce acute inflammation of the lung by intratracheal instillation, that the distribution of lesions is different among surface active agents, and moreover that pathological examination is indispensable for clarifying local toxicity of absorption enhancers in pulmonary drug-delivery studies. PMID- 10736791 TI - Induction of hepatic CYP2B1/2 by a teratogenic compound cis-1-[-4-(p-menthane-8 yloxy)phenyl]piperadine (YM9429) in rats. AB - A teratogenic compound cis-1-[-4-(p-menthane-8-yloxy)phenyl]piperadine (YM9429) selectively induces skeletal malformations characterized by cleft palate in rat fetuses. In the present study, we investigated the effect of YM9429 on hepatic cytochrome P-450s and their activities in rats. Oral administrations of YM9429 at a dose of 250, 500 or 750 mg/kg daily for 3 days induced cytochrome P-450 contents in a dose-dependent manner. Concomitant induction of enzyme activities of benzphetamine N-demethylase, erythromycin N-demethylase and, to a lesser extent, aminopyrine N-demethylase was observed. Immunoblot analysis revealed that YM9429 up-regulated hepatic levels of CYP2B1/2 and CYP3A1/2 proteins. A single dose of YM9429 at 250 mg/kg induced CYP2B1/2 protein levels significantly. These results suggest that YM9429 is a strong inducer of cytochrome P-450 with characteristics resembling those of phenobarbital. PMID- 10736792 TI - [Frozen section biopsy in the assessment of organs for transplantation]. AB - BACKGROUND: The success of transplantation and the increase in its indications has caused organ donations to be unable to meet the global demand. By broadening the donor selection criteria and accepting so-called suboptimal donors in an attempt to solve this problem, a careful individualized assessment is required of the validity of each donor. This often implies the need for a pretransplantation biopsy study. MATERIAL AND METHODS: Evaluation of frozen-section biopsy studies in the assessment of graft and donor validness in the the transplantation program of the La Fe University Hospital (Valencia, Spain) during 1998, when a total of 301 solid organ transplantations were performed. RESULTS: Sixty pretransplantation frozen-section biopsies were performed (29 of the liver, 21 renal, 3 lymph nodes, 3 brain, 2 prostatic, 1 myometrial and 1 pericardial). As a result of the bioptic study, 18 transplantations were invalidated (30% of all biopsies). The most frequent causes of invalidation were liver macroesteatosis (7 cases), malignancies (5 cases) and renal parenchymal lesions (5 cases). Among the tumor diagnosis there were one false-positive and one false-negative results of malignancy. CONCLUSION: Frozen-section biopsies evaluation in suboptimal organ donors or donors suspected of suffering disease integrates morphological analysis in the transplantation team decision process, and affords a quality factor in the selection of donors. PMID- 10736793 TI - [Nocturnal arterial pressure and the cardiovascular risk factors in patients with arterial hypertension]. AB - BACKGROUND: Blood pressure (BP) has a circadian rhythm. Most of the people presents a BP fall between 10-20% during nighttime hours (dipper condition). Measurement of these circadian variations is performed by continuous blood pressure ambulatory monitoring. We have studied the possible relation between blood pressure nocturnal fall and cardiovascular risk factors in hypertensive patients. METHODS: We selected 100 hypertensive patients from the Hypertension and Lipids Unit of San Cecilio University Hospital of Granada, Spain. They were divided into two groups: dippers group and non-dippers, depending of whether or not the fall of nocturnal systolic and diastolic BP was > 10%. All patients included in the study underwent complete clinical history, exhaustive physical examination, complementary examinations, urine and hematology tests, and continuous blood pressure ambulatory monitoring by the Space Labs system. RESULTS: The heart rate was found to be significantly higher (p = 0.0253) in the hypertensive dippers group than in the non-dippers. The latter showed higher plasma creatinine values (p = 0.0343) and lower potassemia values (p = 0.0140) than the dippers group. The dippers group presented concentrations of HDL cholesterol significantly higher (p = 0.008) and diurnal diastolic BP values (p = 0.0211) also higher than the other group. CONCLUSIONS: Non dippers hypertensive patients present a higher number of cardiovascular risk factors as well as a higher tendency to renal lesions, and worse prognosis than the dippers group. PMID- 10736794 TI - [The evaluation of unnecessary venous access ports in an emergency service]. AB - BACKGROUND: To determine the incidence of unnecessary cannulations of peripheral veins in an emergency department (ED). METHODS: We retrospectively analyzed a sample of 1,113 patients from a total of 24,637 who were attended at ED. We reviewed the cannulations of peripheral veins and if they was used or not. RESULTS: In 202 (18.5%) cases, venous access was obtained. In 84 (41.6%) the venous access was not used. CONCLUSION: The unnecessary use of venous cannulations is a common practice and generates a substantial added cost of poor quality. PMID- 10736795 TI - [Arterial pressure, the moon, the stars...]. PMID- 10736796 TI - [Primary immunodeficiencies. The current prospects in diagnosis and treatment]. PMID- 10736797 TI - [Primary immunodeficiencies in Spain. Data of the Spanish Registry of Primary Immunodeficiencies. REDIP 1980-1999 (Registro Espanol de Immunodeficiencias Primarias)]. PMID- 10736798 TI - [The multidisciplinary care of acute cerebral stroke]. PMID- 10736799 TI - [A 66-year-old man with fever of 5 months evolution]. PMID- 10736800 TI - [Antithrombotic treatment with the coronary stent]. PMID- 10736801 TI - [Participation in epidemiological studies]. PMID- 10736802 TI - [The value of the electroencephalogram in assessing patients with suspected spongiform encephalopathy]. PMID- 10736804 TI - [Psychosis, epilepsy and celiac disease]. PMID- 10736803 TI - [EDTA-dependent pseudoleukocytopenia]. PMID- 10736805 TI - [Tuberculin 5 UT-PPD RT-23. Is it being used correctly?]. PMID- 10736806 TI - [Empyema due to Hafnia alvei]. PMID- 10736807 TI - High-dose chemotherapy with autologous stem cell rescue in the outpatient setting. AB - Intensive outpatient care is rapidly becoming the primary mode of care for selected patients undergoing high-dose chemotherapy with autologous peripheral blood stem cell (PBSC) transplantation. Although the traditional inpatient model of care may still be necessary for high-risk patients, published data suggest that outpatient care is safe and feasible during or after administration of high dose chemotherapy and autologous PBSC transplant. Blood and marrow transplant (BMT) centers have developed programs to provide more outpatient care under three basic models: an early discharge model, a delayed admission model, and a comprehensive, or total, outpatient model. This review will describe these models of care and address the elements necessary for the development of an outpatient BMT program, including patient selection, staff development, and patient and caregiver education. Available supportive care strategies to facilitate outpatient care will also be highlighted. Clinical outcome data and pharmacoeconomic analyses evaluating various outpatient BMT programs, as well as limited quality-of-life evaluations, will be reviewed. PMID- 10736808 TI - Cancer and male sexual dysfunction. AB - Many men with cancer develop erectile dysfunction during treatment of their malignancy. Erectile dysfunction in these patients often has multiple causes, both physiologic and psychological. Various treatment options, including mechanical/surgical approaches and pharmacologic agents, are available to treat erectile dysfunction. Recent elucidation of the mechanism of erection at the organ, cellular, and molecular level have led to effective new local and systemic treatments. The successful treatment of erectile dysfunction depends on the provider's thorough knowledge of the mechanism of erection and the various available therapies. This article provides an overview of the pathophysiology and evaluation of erectile dysfunction, as well as useful treatment regimens. PMID- 10736809 TI - Clinical trials referral resource. Clinical trials with arsenic trioxide. PMID- 10736810 TI - Update on the management of primary CNS lymphoma. AB - Primary central nervous system (CNS) lymphoma is a non-Hodgkin's lymphoma restricted to the nervous system. The incidence of this lymphoma is rising in the immunocompetent population but may be decreasing in patients with the acquired immune deficiency syndrome (AIDS) due to the introduction of highly active antiretroviral therapy. A periventricular, diffusely enhancing lesion on magnetic resonance imaging (MRI) is suggestive of primary CNS lymphoma, but a stereotactic biopsy is needed to make a definitive diagnosis. Concurrent leptomeningeal and ocular involvement is common in this brain tumor. Because primary CNS lymphoma is exquisitely sensitive to steroids, these drugs should be withheld until tissue is obtained for diagnosis. Age and performance status are important prognostic factors, regardless of specific treatment. Methotrexate in high doses is the single most effective chemotherapeutic agent for primary CNS lymphoma. It substantially improves survival when combined with radiation therapy and is better than radiotherapy alone as a single agent. Multimodality treatment results in delayed cognitive neurotoxicity, particularly in older patients. New treatment protocols have focused on the use of chemotherapy alone. PMID- 10736811 TI - Potential role of tumor vaccines in GI malignancies. AB - Although surgery remains the only curative option for patients with gastrointestinal (GI) malignancies, the use of adjuvant chemotherapy and/or localized radiation is considered standard therapy for patients who present with locoregional disease. Even with adjuvant therapy, however, the 5-year survival rate for such patients ranges from 2% to 50%, depending on the specific cancer type and stage. As a result, more effective interventions are necessary for all but the earliest stages of GI malignancies. Colon cancer represents the paradigm for the management of GI malignancies, not only because it is, by far, the most common cancer in this group, but also because the biological progression to disease is well characterized. Immunotherapy is an alternative approach for treating GI malignancies that can either: (1) activate tumor-specific T-cells; or (2) use monoclonal antibodies derived from tumor-specific antigens. Monoclonal antibodies act by a mechanism that is distinct from that of chemotherapy and, thus, represent a non-cross-resistant treatment with an entirely different spectrum of toxicities. Thanks to an improved understanding of tumor immunology, as well as the events needed to generate an optimal immune response, the possibility of designing an effective colon cancer vaccine approach that induces both humoral and cellular responses has become even more realistic. PMID- 10736812 TI - Prostate-specific antigen (PSA) best practice policy. American Urological Association (AUA). AB - Prostate cancer is one of the most common forms of noncutaneous cancer in men in the United States. Despite its prevalence, the natural history of this disease is remarkably heterogeneous. In many patients, the cancer progresses slowly, resulting in moderately or poorly differentiated tumors that remain localized to the prostate gland. Although potentially life-threatening, such cancers are often curable. In other patients, however, tumor growth is rapid and can spread beyond the confines of the prostate. In such cases, the cancer is not curable, and long term survival is considerably diminished. Strategies for managing prostate cancer have therefore been aimed at early detection and local treatment of the cancer. Prostate-specific antigen (PSA) is a tumor marker currently used for early detection of prostate cancer. Measurement of serum PSA levels has significant clinical application in other areas of prostate disease management. The purpose of this report is to provide current information on the use of PSA testing for: (1) the evaluation of men at risk for prostate cancer, (2) assistance in pretreatment staging, and (3) the posttreatment monitoring and management of men with this disease. The following summary is based on a review of the literature and the expert opinions of a multispecialty panel convened by the American Urological Association (AUA). It is intended to serve as a resource for urologists and primary care physicians. PMID- 10736813 TI - The use of advance directives by persons with serious mental illness for psychiatric treatment. AB - OBJECTIVE: A health care proxy is an advance directive that allows an individual to indicate in writing who can act on his behalf when he lacks the capacity to make health care decisions, and what limitations he is placing on this authority. Of great interest in medical settings, health care proxies are beginning to receive more attention in psychiatric settings. Are these proxies useful when applied to psychiatric treatment decisions? This paper examines health care proxies in Massachusetts and their potential use for decisions about psychiatric interventions at one Massachusetts state hospital. METHOD: A point in time study of a state hospital's entire census was done by reviewing all patients' records for demographic, diagnostic, and legal data, and for the presence and content of the state required health care proxy form. RESULTS: Of the 161 patients in the hospital, 71 (44%) had full guardians and were ineligible to complete their own proxies. Of the remaining 90 patients, 53 (33% of the total population) had a proxy form in their chart, but 34 (21%) of these patients refused to sign them. Of the 19 (12%) signed proxies, 16 (10%) of the patients were deemed competent to have executed meaningful health care proxies. CONCLUSIONS: This preliminary analysis reveals that only 10% of a state hospital population had meaningful health care proxies. Further outcome studies are needed to determine if the process of offering health care proxies and the presence of properly executed proxies are meaningful and beneficial to chronically ill psychiatric patients. PMID- 10736814 TI - The integration of primary care and behavioral healthcare in northern California Kaiser-Permanente. AB - Integration of behavioral healthcare and primary care has a number of presumed benefits, including better communication between providers and systems, leading to improved patient care. There are studies showing medical cost offsets, although they tend to be in circumscribed research settings. Northern California Kaiser-Permanente has designed a new primary care system providing mental health clinicians on a primary care team. Those clinicians evaluate patients, create treatment plans, provide brief interventions, coordinate care with specialty behavioral healthcare, and consult with primary care physicians. Those physicians also have an increased role in the detection and treatment of behavioral health problems via guidelines developed with behavioral health. Structural changes within the overall system, including regional call centers and computerized clinical information systems, support the integration. Quality programs also support the ongoing improvement of the integration process. There are investment expenses in this type of re-design, but also expected cost savings. An infrastructure is now in place to measure both clinical outcomes, and cost effects of the new model. PMID- 10736815 TI - The treatment of anxiety disorders in a primary care HMO setting. AB - Anxiety disorders are common, yet under diagnosed, in primary care settings. Many patients with anxiety and other psychiatric disorders do not seek care in mental health care settings. An integrated primary care/mental health model offers one approach to improving outcomes for patients with anxiety disorders. This model has been researched for the treatment of depression with positive results but has not been well studied for the treatment of anxiety disorders. We describe the results of care for a cohort of adult patients with Generalized Anxiety Disorder (GAD) and clinically significant anxiety secondary to Major Depressive Disorder (MDD) treated in an integrated model. Compared to a matched cohort of adults treated in a primary care setting with usual care, the intervention cohort experienced significantly improved reduction in symptoms of anxiety at 6 months. The intervention cohort also was significantly more satisfied with care. PMID- 10736816 TI - Mind-body innovations--an integrative care approach. AB - Integration of behavioral health and medicine has gained increased support recently within the new field of complementary medicine. Providers from both disciplines are acknowledging the "mind-body" connection and recognizing the value of treating the "whole" patient through working within an integrative delivery model. This paper describes two treatment programs which were developed using the principles of the mind-body connection and implemented within an integrative setting at a large HMO. The results of research studies are presented and discussed to demonstrate the efficacy of these programs. PMID- 10736817 TI - Development of a model for the detection and treatment of depression in primary care. AB - Development and implementation of a protocol for identifying and treating depressed patients in a busy, group model, HMO primary care practice is described. Initial results and barriers to implementation are reported. A simple depression screening tool was adapted and a protocol for using available primary care staff and resources was developed to efficiently integrate screening and treatment for depression into daily practice. Important components of the program included specification of the target population, adaptation of a patient self report diagnostic and rating tool, a nurse-educator protocol for patient follow up and telephone support, a database to track patient progress, training for primary care staff, and collaboration with specialty Behavioral Health Care. After piloting, plans were made for transferring the model to other practices. Although the protocol was seen as beneficial to the PCP, Nurses, and patients involved, the scarcity of resources in other primary care practices proved a major barrier to full implementation. PMID- 10736819 TI - [Healing in the intestinal anastomosis--comparison of the Albert-Lembert and Gambee methods]. AB - An experimental comparative histomorphological study was made on intestinal healing processes following an Albert-Lembert suture with approximation of the serosal surface and Gambee's layer to layer anastomosis of a dog. There was no obvious complications such as postoperative hemorrhage, anastomotic stenosis or anastomotic leakage. Although both anastomoses resulted in a good healing process, Gambee's layer to layer suture, which caused the submucosal layers to face each other, showed better wound healing at the anastomosis in terms of layer to layer attachment. As a conclusion, Gambee's layer to layer anastomosis seemed to be a better anastomotic technique in terms of wound healing for the intestinal anastomosis. PMID- 10736818 TI - A look to the past, directions for the future. AB - This article represents the history of primary care and behavioral health integration at Group Health Cooperative (GHC) over the last decade, and foreshadows probable futures for this work into the next decade. To build from a logical progression, the article responds to a series of questions: 1. Why integrate primary care and behavioral health? 2. What has been done so far and how well has it worked? 3. Keeping the end in mind, what's the idealized picture of integration for the future? 4. How to get from here to there? What will help or hinder the effort? and 5. Again, why make these efforts to integrate? PMID- 10736820 TI - A case of angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) together with total atelectasis of a lung caused by lymphadenopathy and pleural effusion. AB - Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is a clinicopathologic entity established by Frizzera in 1974. Reported cases of AILD with pleuropulmonary involvement have been increasing recently. At Nogata Central Hospital, an 82-year-old male inpatient with brain infarctions and chronic bronchitis showed a rapid growth of systemic lymphadenopathy and various other symptoms. We diagnosed the case as AILD by histopathologic examination of a lymph node. Chest radiography and computed tomography demonstrated a loss of volume of the right lung caused by intrathoracic lymphadenopathy and a pleural effusion. Although cases of AILD with pleuropulmonary involvement have been increasing, no cases with almost total atelectasis of a lung have been reported as yet. AILD should be taken into account as a disease which may cause atelectasis of a lung. PMID- 10736821 TI - Enzymatic determination of sn-glycerol-1-phosphate. AB - A sensitive and positive colorimetric method for quantification of sn-glycerol-1 phosphate (G-1-P) is described. The use of G-1-P-specific dehydrogenase from Methanobacterium thermoautotrophicum and a tetrazolium salt (an NAD-recycling system) allowed a positive measurement of G-1-P, and caused an increase in sensitivity. Because G-1-P is not only the backbone of the ether glycerolipids in Archaea (formerly archaebacteria) but also the component of lipoteichoic acid polymers from some Gram-positive bacteria, membrane-derived oligosaccharide from Escherichia coli, unacylated moiety of phosphatidylglycerol from bacteria and mitochondria, and some phosphoglycolipids of lactic acid bacteria, this method may be useful in the structural and metabolic studies of these materials. PMID- 10736822 TI - [Syndromes of autonomic dysfunction associated with orthostatic intolerance]. AB - Stimulated by the wide-spread use of tilt table testing, disorders of autonomic function with orthostatic hypotension have recently gained attention by clinical cardiologists. At the same time, improved characterization of the underlying circulatory responses have led to a reclassification of these syndromes. In particular, three subgroups of chronic primary dysautonomia have been defined: pure autonomic dysfunction, multiple system atrophy, and the postural orthostatic tachycardia syndrome. On the other hand, acute dysautonomias represent a rare yet clinically sometimes dramatic form of autonomic disorders. Several diseases as well as enzymatic disorders, and pharmacological drugs may cause secondary dysautonomia. The clinical correlate of all these forms of dysautonomia is orthostatic hypotension and syncope. Thus, a careful history forms the basis of a successful diagnostic workup of the underlying cause of syncope. This review summarizes the current knowledge of autonomic disorders, their classification and diagnostic and therapeutic strategies. PMID- 10736823 TI - [Intraocular pressure during hemodialysis]. AB - Hemodialysis, a useful treatment for patients with severely compromised renal function, has also unfavorable side effects. In the ophthalmologic area, a rise in intraocular pressure (IOP) during dialysis accompanied by ocular pain has been reported. In our study when measuring IOP, as well as serum osmolality and plasma CO2 pressure every 30 minutes during routine hemodialysis in renal failure patients with a normal aqueous outflow facility, the mean percent changes of IOP to the initial value showed no significant difference at any time, although the changes in serum osmolality decreased significantly. The mean percent changes of plasma CO2 pressure also did not show any significant difference during dialysis. In patients with a poor aqueous outflow facility, the mean percent changes of IOP increased significantly after 90 minutes, with the exception of the change at 180 minutes. The mean percent changes of serum osmolality decreased significantly after starting dialysis. A negative correlation in the mean percent change of IOP with that of serum osmolality was detected. The administration of an intravenous hyperosmotic agent prevented significant changes not only in serum osmolality but also in IOP. Therefore, it is considered that hemodialysis causes a decrease in serum osmolality, resulting in an osmotic gradient between the plasma and the intraocular fluids due to the presence of the blood-ocular barrier. Although the osmotic gradient draws water from the plasma into the eye, if there is no abnormal obstruction in the aqueous outflow pathway, an amount of aqueous humor matching the increase in intraocular fluid goes through the pathway out of the eye to maintain the normal level of IOP. In eyes with an obstructed aqueous outflow pathway, however, this compensatory mechanism of aqueous humor drainage does not work well, and results in an IOP elevation. PMID- 10736824 TI - [Physical characteristics of medical students at UOEH (1979-1996)]. AB - The purpose of this study is to explore future directions for health and physical education at UOEH. To do so, the authors first analyzed data taken from physical fitness tests conducted for newly incoming medical students every year since UOEH was founded more than twenty years ago. It was seen that male students tended to be overweight, but that the level of their physical fitness was about the same as the national average. We then managed to obtain data from a physical fitness test conducted for about 50 members of the 1997 graduating class just after their graduation. The results from comparing these data with those taken six years before showed a remarkable tend: that is, the males who had been already over weight when they became freshmen, tended to gain more weight over the six years spent at UOEH. This paper examines the causes of these results, and an ideal future Japanese university health and physical education plan. PMID- 10736826 TI - Hydrogen for airships? PMID- 10736825 TI - [Report from the Sixth UOEH meeting of gastrointestinal diagnostic imaging]. PMID- 10736827 TI - Granting immunity. PMID- 10736828 TI - Brain terrain. PMID- 10736829 TI - Brain invaders. PMID- 10736830 TI - Working under pressure. PMID- 10736831 TI - Bioagent chip. PMID- 10736832 TI - Mobilizing the Internet. PMID- 10736833 TI - Why go to Mars? PMID- 10736834 TI - How to go to Mars. PMID- 10736835 TI - The Mars direct plan. PMID- 10736836 TI - To Mars by way of its moons. PMID- 10736837 TI - A bus between the planets. PMID- 10736838 TI - Staying sane in space. PMID- 10736839 TI - Invaders from Hollywood. PMID- 10736840 TI - The tick-tock of the biological clock. PMID- 10736842 TI - Dissecting a hurricane. PMID- 10736841 TI - Swarm smarts. PMID- 10736843 TI - The Bromeliads of the Atlantic Forest. PMID- 10736844 TI - An automated precision magnetometer. PMID- 10736845 TI - High density oligonucleotide and DNA probe arrays for the analysis of target DNA. AB - The acquisition of sequence, expression and other information concerning genetic material constitutes a crucial component of the modern revolution in molecular biology. One important advance in this area is the development of high density oligonucleotide/DNA microarrays which allows the rapid sequence analysis of genomic target samples in addition to diagnostic possibilities with respect to genetic and infectious disease. In the present article we review protocols for the design of such microarrays and their principles of operation. Together with a look at some recent applications we include brief remarks as to the possibilities for the future. PMID- 10736846 TI - Multiresidue analysis of pesticides in vegetables and fruits using a high capacity absorbent polymer for water. AB - A single extraction and a single clean-up procedure was developed for multi residue analysis of pesticides in non-fatty vegetables and fruits. The method involves the use of a high capacity absorbent polymer for water as a drying agent in extraction from wet food samples and of a graphitized carbon column for clean up. A homogeneously chopped food sample (20 g) and polymer (3 g) were mixed to absorb water from the sample and then 10 min later the mixture was vigorously extracted with ethyl acetate (100 ml). The extract (50 ml), separated by filtration, was loaded on a graphitized carbon column without concentration. Additional ethyl acetate (50 ml) was also eluted and both eluates were concentrated to 5 ml for analysis. The procedure for sample preparation was completed within 2 h. In a recovery test, 107 pesticides were spiked and average recoveries were more than 80% from asparagus, orange, potato and strawberry. Most pesticides were recovered in the range 70-120% with usually less than a 10% RSD for six experiments. The results indicated that a single extraction with ethyl acetate in the presence of polymer can be applied to the monitoring of pesticide residues in foods. PMID- 10736847 TI - Experimental demonstration and simulation of electrochemical non-linear responses to glucose and its interferents with an amperometric sensor. AB - A novel sensing system based on the multi-dimensional information contained in a dynamic non-linear response is proposed. A sinusoidal potential was applied to an amperometric-type glucose sensor and the resulting current of the sensor was analyzed by fast Fourier transformation (FFT). The amplitudes of the higher harmonics of FFT characterize the non-linear properties of the response. The amplitudes of the higher harmonics of FFT exhibit characteristic changes which depend on the concentration and the kinetics of the reactions of glucose and its interferents at the sensor surface. The essential features of the current potential curve were reproduced by a computer simulation based on the kinetics of electrochemical reactions. PMID- 10736848 TI - Modified gas-permeable silicone rubber membranes for covalent immobilisation of enzymes and their use in biosensor development. AB - Novel enzyme membranes are introduced. Modified polymeric gas-permeable layers were developed enabling biological components which have available reactive groups (-NH2, -OH, -SH, -COOH) to couple covalently on to their surfaces. Therefore, gas-permeable two component room temperature vulcanizing (2K-RTV) silicone rubber was modified using additional cross-linking agents. Triethoxysilanes with functional groups on their side chains such as epoxy or amino groups were used. A special attribute of the resulting gas-permeable membranes is that their formation and modification occur simultaneously during one reaction step. IR spectroscopy was used to observe the changes in the polymeric structure due to the reaction with the additional cross-linking agents. Sensors equipped with these layers are suitable to measure dissolved gases such as O2, CO2 and NH3 consumed or produced by enzymes converting their substrates. Determination of glucose, a well investigated enzymatic detection process, was chosen to demonstrate the applicability of the enzyme immobilisation. Glucose oxidase was immobilised on the membranes and glucose was detected by amperometric measurement of oxygen consumption. It is expected that this immobilisation method will also be useful for miniaturised planar biosensors. PMID- 10736849 TI - Synthesis of meso-tetra-(3,5-dibromo-4-hydroxylphenyl)-porphyrin and its application to second-derivative spectrophotometric determination of lead in clinical samples. AB - A new very sensitive and selective chromogenic reagent, meso-tetra-(3,5-dibromo-4 hydroxylphenyl)porphyrin [T(DBHP)P], was synthesized and studied for the determination of trace lead in detail. In 0.10 mol l-1 NaOH medium, lead reacts with T(DBHP)P to form a 1:2 yellow complex, which gives a maximum absorption at 479 nm; 0-0.48 microgram ml-1 Pb(II) obeyed Beer's law. The molar absorptivity of the complex and Sandell's sensitivity are 2.5 x 10(5) 1 mol-1 cm-1 and 0.000812 microgram cm-2, respectively. Second-derivative spectrophotometry is better than conventional spectrophotometry in sensitivity and selectivity, and its limit of quantification, limit of detection and relative standard deviation are 0.70 ng ml 1, 0.21 ng ml-1 and 1.0%, respectively. Ca (3250-fold), Mg (2000-fold), Sr (1000 fold), Ba (750-fold), Al (1000-fold), Bi (500-fold), Fe (2000-fold), Co (750 fold), Ni (1000-fold), Cu (750-fold), Zn (1250-fold), Cd (2500-fold) and Ag (550 fold) do not interfere with the determination of lead. The chromogenic system is remarkably superior to other reagents, especially porphyrin compounds. The influence caused by oxygen in air or in solution can be easily eliminated by adding Na2SO3. The reaction is very stable, the stability constant of the complex being 1.2 x 10(45). The chromogenic reaction is completed within 1 min at room temperature when 8-hydroxylquinoline is used as catalyst. The proposed method has been applied to the direct determination of trace lead in clinical samples. The accuracy and precision are both very satisfactory. PMID- 10736850 TI - Speciation of metabolites of selenate in rats by HPLC-ICP-MS. AB - The metabolic pathway for and metabolites of selenium (Se) administered intravenously to rats in the form of selenate at a dose of 0.3 mg Se kg-1 body weight were studied by speciating Se in the bloodstream, liver and urine by HPLC inductively coupled argon plasma mass spectrometry. Selenate was not taken up by red blood cells (RBCs) and disappeared from the bloodstream much faster than selenite, without any change in its chemical form before it disappeared from the plasma. Selenium excreted into the urine after the administration of selenate showed different patterns from those of selenite in both amounts and chemical forms. With the selenate group, the concentration of Se in urine was highest at 0 6 h and the chemical species of Se was selenate at 0-6 h; thereafter a monomethylselenol-related Se compound (MMSe*) and trimethylselenonium ions (TMSe) appeared, selenate not being excreted after 6 h. On the other hand, in the selenite group, the concentration of Se peaked at 6-12 h, and the chemical species of Se were MMSe* and TMSe. Selenate was reduced in vitro on incubation in either a liver homogenate or supernatant fraction, although much more slowly than in the whole body. Selenate was not reduced by glutathione or dithiothreitol. The results suggest that in contrast to selenite, which is taken up by and reduced in RBCs, and then transferred to the liver, approximately 20% of the selenate administered to rats was excreted into the urine without any change in its chemical form with the present dose, and the major portion of selenate was taken up by the liver, reduced and then utilized for the synthesis of selenoproteins or excreted into the urine after being methylated. PMID- 10736851 TI - Solid-phase microextraction for the determination of pethidine and methadone in human urine using gas chromatography with nitrogen-phosphorus detection. AB - A simple and rapid analytical method is presented for the determination of pethidine (meperidine) and methadone in human urine using solid-phase microextraction (SPME) and gas chromatography with nitrogen-phosphorus detection (GC-NPD). After the analytes had been partitioned between an extracting phase and the aqueous sample matrix, the needle of the coating fiber assembly was injected directly into the GC injector. The analytes were thermally desorbed in the heated injector (240 degrees C) and subsequently separated and detected by the GC-NPD system. The factors influencing the SPME method, such as the salt (NaCl) effect (15%), pH (pH 11), and equilibration time (30 min), were optimized. The calibration graphs for urine samples showed a good linearity. The detection limit was below 1 ng ml-1 for both drugs. PMID- 10736852 TI - Determination of organochlorine pesticide residues in foods using solid-phase extraction clean-up cartridges. AB - Fourteen organochlorine pesticide residues in fatty foods were determined using a simple and rapid procedure based on solid-phase extraction (SPE) clean-up cartridges with octadecyl (C18)-bonded porous silica, a tandem C18 and Florisil column, Alumina-N and Florisil. A Florisil cartridge eluted with 12 ml petroleum ether-ethyl ether (95 + 5) was the most efficient clean-up procedure capable of eliminating the matrix interference and satisfying the agreed acceptable recovery for the large numbers of organochlorine pesticides in nine kinds of foods having different fat contents. Average recoveries of organochlorine pesticides in shellfish, fish and meats ranged from 77 to 105%, 84 to 98% and 85 to 107%, respectively. In addition, analysis of a certified Standard Reference Material (SRM 1945) verified the satisfactory performance of Florisil clean-up cartridge. This SPE method not only yielded comparable results for nonfatty foods, but also provided a reliable separation and quantification of organochlorine pesticides for analyzing a large number of foods with a wide range of fat content. PMID- 10736853 TI - GC-MS determination of steroids related to androgen biosynthesis in human hair with pentafluorophenyldimethylsilyl-trimethylsilyl derivatisation. AB - An efficient method for the simultaneous determination of eight steroids, androstenedione, dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), testosterone, androsterone, etiocholanolone, progesterone and pregnenolone, in human hair by gas chromatography-mass spectrometry (GC-MS) using d3-testosterone as internal standard is described. The method involves alkaline digestion, liquid liquid extraction and subsequent conversion to mixed pentafluorophenyldimethylsilyl-trimethylsilyl (flophemesyl-TMS) derivatives for sensitive analysis in the selected ion monitoring (SIM) mode. This method showed good overall repeatability and reproducibility of 4.88-11.24 and 3.19-9.58%, respectively. For the first time, the quantification of DHT, DHEA and pregnenolone in human hair has been achieved by GC-MS, testosterone was also quantified. The detection of four steroids in hair samples was possible in the concentration range 0.12-8.45 ng g-1. The other four steroids, androstenedione, androsterone, etiocholanolone and progesterone, were not detected. The detection limits for SIM of the steroids varied in the range 0.02-0.5 ng g-1, and the SIM responses were linear with correlation coefficients varying from 0.991 to 0.996 for most of the steroids studied. The concentrations of the four steroids detected were different in male and female hair samples. PMID- 10736854 TI - Evaluation of an immunobiosensor for the on-site testing of veterinary drug residues at an abattoir. Screening for sulfamethazine in pigs. AB - A study was conducted to determine the feasibility of performing "on-site" screening for sulfamethazine (SMT), at an abattoir, using a rapid immunobiosensor method. This involved transfer of the biosensor technology and an assay developed in the laboratory, to the cold, humid conditions of a modern pig-processing factory. A pre-determined threshold limit of 0.4 microgram ml-1 SMT in bile was used to identify the likelihood that corresponding tissue samples contained SMT concentrations in excess of the European maximum permissible residue limit of 0.1 mg kg-1. Bile samples containing SMT concentrations above the threshold limit were deemed positive and the corresponding kidney and muscle samples were sent to the laboratory for HPLC analysis. The robustness of the biosensor instrumentation in the harsh operating conditions was monitored throughout the project. The performance of the assay, on-site, was assessed by the regular inclusion of QA samples and by the submission of control 'SMT-positive' pigs to the abattoir. Sampling procedures, identification and traceability were also under scrutiny. During the project, 337 (9.35%) of the total kill were tested for SMT residues, representing 75% of all producers submitting pigs for slaughter. Twelve animals, including the ten controls, gave positive bile results. HPLC analysis confirmed SMT residues in all 12 kidneys (11 in excess of the permissible level). Ten muscle samples also contained violative SMT levels. Throughout the project, the biosensor performed reliably, with no adverse reaction of any mechanical or electrical components. The SMT assay also performed reliably. This is the first report of a biosensor being used for 'on-site' drug screening. PMID- 10736855 TI - Determination of low concentrations of potentially toxic elements in human liver from newborns and infants. AB - One hundred and fifty-seven liver samples from newborns and infants who had died from sudden infant death syndrome (SIDS) or other known causes have been analysed by ICP-MS for Ag, Cd, Co, Pb and Sb. The median concentrations found were: 15.4 (Ag), 2.9 (Cd), 15.9 (Co), 65.2 (Pb) and 1.8 (Sb) ng g-1 wet mass. There was no measurable difference in the concentrations of any of these elements between the SIDS and non-SIDS groups. The validity of the results was assessed by analysis of appropriate reference materials, interlaboratory comparison and isotope dilution analysis. The instrumental limits of detection were 0.25 (Ag), 0.14 (Cd), 0.21 (Co), 3.8 (Pb) and 0.38 (Sb) ng g-1 wet mass. The limits of detection of the method depend on the reagent blank and the extent of background contamination. PMID- 10736857 TI - Chiral determination of various adrenergic drugs by thin-layer chromatography using molecularly imprinted chiral stationary phases prepared with alpha agonists. AB - Thin-layer chromatography (TLC) based on molecularly imprinted polymers (MIPs) of alpha-agonists as chiral stationary phases was applied to the determination of enantiomers of various adrenergic drugs including alpha- and beta-agonists and beta-antagonists (beta-blockers). In this study, three MIPs imprinted with (+) ephedrine, (+)-pseudoephedrine and (+)-norephedrine plus a non-imprinted polymer (non-MIP) were prepared, processed and coated on a glass support as thin layers. then enantiomeric determination of adrenergic drugs was carried out by development of their racemates on the TLC plates, using established conditions. From the results, the racemates of the compounds used as print molecules were well separated into two isomers on the MIP-plates, except on the plate based on MIP of (+)-norephedrine. Most adrenergic drugs structurally related to print molecules were completely resolved into two spots with the MIP plates. In general the retention of (+)-isomers (or 1S-isomers) was greater than that of (-)-isomers (or 1R-isomers), indicating the stereoselectivity of the MIPs with the former isomers. Moreover, the role between the chemical structures of the analytes with chiral recognition of the MIPs has been investigated. The proposed method enables rapid determination of enantiomers and screening of large numbers for optical purity of adrenergic drugs. PMID- 10736856 TI - Supercritical fluid extraction (SFE) as a multi-residue extraction procedure for beta-agonists in bovine liver tissue. AB - A supercritical fluid extraction procedure has been developed for the extraction of beta-agonists in bovine liver samples. The method is suitable for compounds of different beta-agonist classes: the substituted aniline-type compounds (e.g. clenbuterol) and the phenolic-type compounds (e.g. salbutamol), including conjugated forms of the latter. The developed procedure involves a combination of supercritical fluid extraction with enzyme immunoassay for the determination of clenbuterol and salbutamol residues at the low ppb level. Addition of methanol modifier and removal of sample moisture are necessary for the extraction of more polar analytes such as salbutamol. Method validation incorporating intra- and inter-assays was carried out on fortified liver tissue and showed good recovery and low variation (RSD < 15%). An enzyme hydrolysis procedure was incorporated into the method for the deconjugation of conjugated residues. The developed procedure was shown to be successful for the determination of both clenbuterol and salbutamol in incurred liver tissue. PMID- 10736858 TI - Determination of residues of the plant growth regulator chlormequat in pears by ion-exchange high performance liquid chromatography-electrospray mass spectrometry. AB - We report a method which we have used routinely for the determination of chlormequat residues in pears. After extraction with methanol, determination was performed, without clean-up, by ion-exchange HPLC using an SCX column eluted with aqueous ammonium formate-methanol, and HPLC-MS with an electrospray interface. MS and MS-MS were employed concurrently, using selected ion monitoring and selected reaction monitoring, respectively, of the 35Cl and 37Cl isotopes of the chlormequat cation and the CID transitions of each of these precursors to the common product ion at m/z 58. The method was suitable for determinations at concentrations of chlormequat cation of 0.04 mg kg-1. Concentrations determined using the four signals were in good agreement (mean RSD 3%). The mean recovery of chlormequat cation at 0.16 mg kg-1, measured using the m/z 122-->58 signal, was 86% (RSD 7%) under repeatability conditions and 88% (RSD 15%) in routine application of the method over a 3 month period. Analysis of an in-house reference sample of pears, similarly analysed over the 3 month period, gave an RSD of 10% with a mean of 0.14 mg kg-1. Mean recovery at 0.016 mg kg-1, under repeatability conditions on two occasions, was 101% (RSD 6%) and 56% (RSD 12%). PMID- 10736859 TI - A method for the separation of residues of nine compounds in cattle liver related to treatment with oxfendazole. AB - A method for the determination of nine compounds closely related to oxfendazole has been developed for the monitoring of residues in food. The method is based on a multi-residue procedure for basic drug residues and used strong cation exchange solid phase extraction for sample clean-up. These nine compounds include fenbendazole, which is itself a licensed veterinary product. The pro-drug febantel converts quickly to fenbendazole or oxfendazole soon after administration. The method is therefore suitable for monitoring residues following the use of any of these compounds. Some of these analytes have been shown to be present as residues following the treatment of farm animals with oxfendazole. Average recoveries for the nine compounds from tissue fortified with 100 micrograms kg-1 were between 34% and 96% with relative standard deviations between 3% and 22%. PMID- 10736860 TI - Enhanced microdialysis recovery of some tricyclic antidepressants and structurally related drugs by cyclodextrin-mediated transport. AB - The enhanced microdialysis relative recovery (RR) of some hydrophobic tricyclic drugs (imipramine, desipramine, amitriptyline, carbamazepine and promethazine) is discussed. Enhanced RR was achieved by including a binding agent [beta cyclodextrin (beta-CD) or 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD)] in the microdialysis perfusion fluid to form inclusion complexes with the drugs, which increases the analyte flux through the membrane material. The maximum effect of the RR increase for all the drugs studied was observed using a commercially available polycarbonate-polyether (PC) membrane. With a 4 mm PC membrane and 4.41 mmol l-1 (0.5% w/v) beta-CD included in the microdialysis perfusion fluid (0.9% saline, pH 7.4) at a flow rate of 0.5 microliter min-1, RR enhancements over controls were as follows: carbamazepine 136, imipramine 268, desipramine 298, amitriptyline 634, and promethazine 987%. Increasing beta-CD [up to 17.63 mmol l 1 (2% w/v)] or HP-beta-CD [up to 32.5 mmol l-1 (5% w/v)] concentration in the microdialysis perfusion fluid enhanced carbamazepine RR three (beta-CD) to four (HP-beta-CD) times compared to controls through PC microdialysis membranes. The PC membrane gave enhanced RR values that were twice those for cuprophan or AN-69 membranes. Enhanced RR with cyclodextrins was successfully applied to sampling from a protein solution containing desipramine in a 4% w/v bovine serum albumin solution. These results suggest that addition of cyclodextrins to microdialysis perfusion fluids may be used to increase microdialysis RR during blood sampling. PMID- 10736861 TI - Polarization-based oxygen sensor. AB - A new approach to oxygen sensing based on the luminescence polarization observed from a novel type of sensor is described. The oxygen sensor consists of an oxygen sensitive silicone film containing tris(4,7-diphenyl-1,10 phenanthroline)ruthenium(II) chloride [Ru(dpp)3Cl2] and an oxygen-insensitive film of Styryl 7 in poly(vinyl alcohol). Polarizers are used to select orthogonally polarized emission components from Ru(dpp)3Cl2 and Styryl 7. The polarization of the combined emission was found to be highly sensitive to the partial pressure of oxygen. This method of polarization sensing is generic and can be used with any fluorophore which displays an analyte-dependent change in intensity. PMID- 10736862 TI - Improved detection of salicylic acids using terbium-sensitized luminescence in aqueous micellar solutions of cetyltrimethylammonium chloride. AB - The determination of salicylic, p-aminosalicylic and 5-fluorosalicylic acids was investigated using terbium-sensitized luminescence in aqueous solutions. Formation of a ternary chelate between terbium, EDTA and the salicylic acid requires dissociation of the phenol group which is adjacent to the dissociated carboxylic group. The reaction is obtained in alkaline solutions and is enhanced in the presence of cetyltrimethylammonium chloride. As evidenced by absorbance and fluorescence measurements, the cationic surfactant plays an important role in the formation of the ternary chelate and then terbium luminescence depends mainly on the extent of chelate formation. Linearity is found over more than four orders of magnitude and detection limits are in the range (2-4) x 10(-10) mol l-1 for the three acids. PMID- 10736863 TI - Simultaneous determination of benzoic acid and saccharin in soft drinks by using lanthanide-sensitized luminescence. AB - A simple and fast approach is used for the first time to develop a time resolved lanthanide-sensitized luminescence method for the simultaneous determination of a preservative and a sweetener, namely benzoic acid (BZ) and saccharin (SC), respectively, in food samples. The method involves the formation of the corresponding ternary chelates with terbium(III) and trioctylphosphine oxide (TOPO) in the presence of Triton X-100, and the measurement of the initial rate and equilibrium signal of this system, which were obtained in 0.1 and 5 s, respectively. The dynamic ranges of the calibration graphs, obtained by using kinetic and equilibrium measurements, were 0.2-36 micrograms ml-1 and 0.15-30 micrograms ml-1, respectively, for BZ, and 3.3-24 micrograms ml-1 and 4-36 micrograms ml-1 for SC and the detection limits were 0.07 and 0.04 microgram ml 1, respectively, for BZ, and 1.1 and 1.2 micrograms ml-1, respectively, for sodium SC. The relative standard deviation ranged between 2.3 and 3.0%. Both compounds were determined simultaneously by using a system of two equations which were resolved by using the calibration data obtained individually for each analyte and by multiple linear regression. Mixtures of BZ and SC in ratios between 3:1 and 1:9 were satisfactorily resolved by using both approaches. The method was applied to the direct analysis of several soft drinks. Analytical recoveries ranged between 89.3 and 108.5%. PMID- 10736864 TI - Development and validation of methods for the determination of miokamycin in various pharmaceutical preparations by use of near infrared reflectance spectroscopy. AB - New methods for the determination of the nominal content of miokamycin in three commercial pharmaceutical preparations available in many different forms are proposed. Solid samples, grinding of which is the sole pretreatment required, are analysed by near infrared (NIR) spectroscopy, using a fibre-optic probe. The active principle is quantified by partial least-squares regression (PLSR). The three proposed methods were validated with a view to their use as control methods; the selectivity of the method, and the repeatability, intermediate precision, accuracy, linearity and robustness of each PLSR calibration model used were determined. The relative standard error of prediction (RSEP) was < 1.5% and the validation results testify to the suitability of the proposed methods. PMID- 10736865 TI - Evaluation of the 'antioxidant power' of olive oils based on a FIA system with amperometric detection. AB - A new method for the evaluation of the 'total antioxidant power' of olive oils, based on a flow injection analysis system with electrochemical detection, is described. It represents a attractive alternative to the mostly used Rancimat method since it is based on the chemical structure of antioxidants and does not require the manipulation of several parameters, such as temperature and oxygen pressure, to accelerate oil oxidation. The proposed procedure is simple, rapid, allows a throughput of 90 samples h-1 and provides a good precision: an RSD of 3.5% was obtained for caffeic acid at the concentration level of 5 mg L-1 (n = 12). A comparison of the proposed was obtained for caffeic acid at the concentration level of 5 mg L-1 (n = 12). A comparison of the proposed procedure with two other methods (Rancimat method and ABTS.+ decoloration assay) was performed to investigate the applicability and limitations of the proposed method. PMID- 10736866 TI - Determination of formaldehyde in liquid, solid and semisolid pharmaceuticals and cosmetics by flow injection-pervaporation. AB - A spectrophotometric method is proposed for the determination of formaldehyde in liquid, solid and semisolid cosmetic and pharmaceutical samples, employing, for the first time in this field, the coupling of a continuous flow configuration to a pervaporation unit. The method is based on the reaction of the analyte with pararosaniline in acidic medium and subsequent formation of a coloured product (alkylsulfonic acid chromophore) with sodium sulfite, which was monitored spectrophotometrically. The method was applied to samples in which the formaldehyde content is regulated by law. PMID- 10736867 TI - The analysis of beta-agonists by packed-column supercritical fluid chromatography with ultra-violet and atmospheric pressure chemical ionisation mass spectrometric detection. AB - Packed-column supercritical fluid chromatography (pSFC) using ultra-violet (UV) and atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS) provides a versatile method for the identification and quantification of beta agonists. We have achieved good separation of clenbuterol, salbutamol, terbutaline and fenoterol with good resolution and reasonable retention times using a high concentration of methanol modifier in the supercritical CO2, together with small amounts of both acidic (trifluoroacetic acid, TFAA) and basic (triethylamine, TEA, or diethylamine, DEA) additives. APCI-MS gave unambiguous identification of the 4 analytes, and increasing cone voltage provided informative fragmentation patterns. The pSFC-MS technique was shown to be linear (R2 > or = 0.996) over the concentration range 1-50 micrograms ml-1. Single ion monitoring (SIM) gave detection limits (on-column) of 2.5 ng (clenbuterol), 0.83 ng (terbutaline), 7.6 ng (salbutamol) and 2.7 ng (fenoterol). The pSFC-MS system was shown to be reproducible within a day, between days, and between restrictors. Analysis of milk samples 'spiked' with beta-agonists showed that the matrix caused no interference, with detection limits of approximately 500 micrograms l-1 of beta-agonists. More dilute solutions could be analysed by pre-concentration before the SFC stage. PMID- 10736868 TI - Simultaneous determination of inulin and p-aminohippuric acid (PAH) in human plasma and urine by high-performance liquid chromatography. AB - Inulin and p-aminohippuric acid (PAH) clearances are used for the estimation of glomerular filtration rate (GFR) and effective renal plasma flow (ERPF). A simple and rapid high-performance liquid chromatography (HPLC) method with UV detection is described for the simultaneous determination of inulin and PAH in the same chromatogram in the plasma and urine of humans. Plasma and urine samples were hydrolyzed with perchloric acid (0.7%) in boiling water. The mobile phase consisted of 0.01 M potassium dihydrogenphosphate with 0.02 M tetramethylammonium chloride and o-phosphoric acid (pH 3)-acetonitrile (94:6, v/v), pumped at a rate of 1.2 ml min-1 on a C8 reversed-phase column. Tannic acid was used as the internal standard and UV detection at 285 nm was employed. The calibration curves were linear over the concentration range of 12.5-100 mg l-1 for inulin and 6.25 50 mg l-1 for PAH with determination coefficients greater than 0.997. The method is accurate (bias < 13%) and reproducible (intra- and inter-day relative standard deviation less than 11%), with a limit of quantitation of 12.5 mg l-1 and 6.25 mg l-1 for inulin and PAH, respectively. Analytical recoveries from urine and plasma were ranged from 81 to 108% for both compounds. This fully validated method, which allows the simultaneous determination of inulin and PAH clearances, is simple, rapid (total run time < 10 min) and requires only a 200 microliters plasma or urine sample. PMID- 10736869 TI - Determination of malondialdehyde in traditional fish products by HPLC. AB - The oxidation state of traditional fish products was measured by determining the malondialdehyde (MDA) level by HPLC and the results were compared to those given by a spectrophotometric method. The procedure involves oxidation of the products by incubation at 40 degrees C for 3 d. Samples were steam distilled in a Kjeldahl distillation apparatus and the MDA was determined in the aqueous distillates by HPLC, using a micro-Bondapak C18 column, with mixed mobile phase of 1% acetic acid-acetonitrile (85 + 15; v/v). A total time of 2 min was necessary to assay each distillate and only MDA was detected. MDA can be determined at a level of 1.5 x 10(-8) mol l-1. The highest rate of oxidation of the samples, as shown by the changes in the TBA test and MDA concentration determined by HPLC, was observed in smoked fish and the lowest in dried-salted fish. PMID- 10736870 TI - Development of an ELISA for paraquat; improvement of antibody characteristics by reversed affinity chromatography. AB - An enzyme-linked immunosorbent assay (ELISA) for paraquat is described. The microtitration plate-based assay has a limit of detection of 10 pg per well, and was specific for paraquat and monoquat. Reversed affinity chromatography was used to refine the polyclonal antibody preparation and eliminate interfering antibodies. There were consequent and significant improvements in assay sensitivity and performance. The potential for application of the assay to a variety of matrices is discussed. PMID- 10736871 TI - Screen-printed amperometric biosensors for the rapid measurement of L- and D amino acids. AB - Screen-printed three-electrode amperometric sensors incorporating L- and/or D amino acid oxidase for the general purpose measurement of L- or D-amino acids is described. The working electrode incorporates rhodinized carbon, to facilitate hydrogen peroxide oxidation at a decreased operating potential, and immobilized enzyme. The devices responded to all 20 common L-amino acids and all of the D amino acids examined, the exceptions being L- and D-proline. Linear response profiles were observed for L-leucine, L-glycine and L-phenylalanine with limits of detection of 0.47, 0.15 and 0.20 mM respectively. The devices were reproducible and exhibited stability over a 56 d test period. The biosensor compares favourably with a standard photometric amino acid test and was used to monitor milk ageing effects. The assay is cheap, simple to perform and rapid, requiring only buffer-electrolyte and a small sample volume. PMID- 10736872 TI - Fluorimetric determination of phytic acid based on the activation of the oxidation of 2,2'-dipyridyl ketone hydrazone catalysed by Cu(II). AB - Phytic acid exerts an activation effect on the oxidation of 2,2'-dipyridyl ketone hydrazone catalysed by Cu(II) ion and the oxidation product is highly fluorescent. A fixed time method for the fluorimetric determination of phytic acid based on this effect is described. The calibration graph is linear over the range 0.05-0.6 mg l-1 phytic acid, resulting in a limit of detection of 0.03 mg l 1 phytic acid. The relative standard deviation is in the range 1.4-1.8%, depending on the sample analysed. The method was successfully applied to the determination of phytic acid in human urine (20 samples) and food samples (nine different products). The results obtained for urine samples ranged from 0.31 to 3.6 mg l-1 phytic acid and for food samples from 3.8 to 22 mg g-1 phytic acid. This is the first procedure to be reported for the determination of phytic acid based on fluorimetric measurements. PMID- 10736873 TI - Interaction of a novel red-region fluorescent probe, Nile blue, with DNA and its application to nucleic acids assay. AB - A novel fluorimetric method was developed for the rapid determination of DNA and RNA based on their quenching effect on the cationic red-region fluorescent dye Nile Blue (NB). In the investigation of the interaction of NB with DNA by steady state polarization measurements, thermal denaturing study, determination of absorption and fluorescence characteristics, salt effect study and electrophoresis experiments, the results supported the suggestion that NB served as an intercalator to the stack base pairs of nucleic acids. Further evidence showed that the quenching could be ascribed to the static quenching mode. A binding constant of about 10(6) M-1 and a binding site size of about three base pairs were obtained by spectral methods. Under optimum conditions, the calibration curves for the determination of calf thymus DNA (CT DNA) and yeast RNA were linear over the ranges 3.0 ng mL-1-2.0 micrograms mL-1 and 27 ng mL-1-10 micrograms mL-1, respectively. The detection limits were 3.0 ng mL-1 for CT DNA and 27 ng mL-1 for RNA. The relative standard deviation (n = 6) was within 2.1% in the middle of the linear range. Interferences from some interesting co existing substances in the determination of DNA were also examined. PMID- 10736874 TI - Nephelometric determination of micro amounts of nucleic acids with protamine sulfate. AB - Nucleic acids can form large particle complexes with protamine sulfate by electrostatic forces, which results in strong light scattering. Based on this, a nephelometric method is described for sensitive and convenient determination of nucleic acids with protamine sulfate by using a common spectrofluorimeter. Maximum light scattering is produced in the range of pH 2.2-4.4 with the same excitation and emission wavelengths at 365 nm. Under optimal conditions, the calibration curves are linear in the range 0.05-60.0 micrograms cm-3 for nucleic acids. The corresponding detection limits are 12.5 ng cm-3 for calf thymus DNA, 9.0 ng cm-3 for fish sperm DNA, and 18.0 ng cm-3 for yeast RNA, respectively. Six synthetic samples are determined with satisfactory results. The relative standard deviation of five replicate measurements is 3.2% for 2.0 micrograms cm-3 calf thymus DNA. PMID- 10736875 TI - Kinetic spectrophotometric determination of hydrocortisone acetate in a pharmaceutical preparation by use of partial least-squares regression. AB - A kinetic spectrophotometric method for the determination of hydrocortisone acetate based on its condensation with isonicotinic acid hydrazide is proposed. The method is applied to the determination of hydrocortisone acetate in a commercially available pharmaceutical preparation, presented as a pomade, that also contains another corticosteroid and additional active compounds. The operating procedure involves dissolving the pomade in chloroform and the addition of the reagent solution directly to the cuvette, in this way avoiding the previous extraction of analytes from the insoluble pomade matrix required by the alternative HPLC procedure. Calibration is performed by partial least-squares regression, using absorbance or first derivative spectra values recorded each minute during the first 30 min of reaction. Use of first derivative spectra overcomes possible scattered light problems produced by excipients precipitating, and produced slightly better results than absorbance data. The relative standard deviation obtained for 11 replicates analysed on different days was approx. 1.5%. The proposed method improves both accuracy and precision of the classical initial rate method and the precision of the HPLC procedure. PMID- 10736876 TI - Sequential injection technique for automated titration: spectrophotometric assay of vitamin C in pharmaceutical products using cerium(IV) in sulfuric acid. AB - For the first time sequential injection analysis (SIA) technique has been employed for titrimetry. A new SI titrimetric spectrophotometric method for the assay of vitamin C in drug formulations was explored. The method is based on the oxidation reaction of vitamin C with cerium(IV) in sulfuric acid media using a spectrophotometer as a detector with the wavelength monitored at 410 nm. A 2(3) factorial design chemometric approach was employed to study the interaction effect of the chemical and system variables, mainly cerium(IV), sulfuric acid concentrations and the flow rate. The results of the chemometric optimization revealed that the optimum operating conditions for the SI titrimetric analysis of vitamin C were 7.0 x 10(-3) M cerium(IV), 0.455 M sulfuric acid and 28.9 microL s 1 flow rate. A linear calibration plot for the determination of vitamin C was obtained in the concentration range between 30 to 200 ppm. The method was applied to the determination of vitamin C in pharmaceutical preparations and no excipient was found to pose any interference, thus rendering the method suitable for the determination of the drug in pharmaceutical preparations. The SIA method is found to be accurate when the results were statistically compared with the results obtained by the BP standard method. The SIA method is superior when compared to the conventional titration method, the BP standard method and previous methods with respect to precision and automation in solution handling. PMID- 10736878 TI - Recovery of some common solvents from protective clothing breakthrough indicator pads by microwave-solvent extraction and gas chromatography. AB - The efficiency of solvent adsorption using Permea-Tec general solvent pads, used for the detection of chemical breakthrough of protective clothing, was determined for methanol, acetone, ethyl methyl ketone, trichloroethylene (TriCE), tetrachloroethylene (TetCE), toluene, m-xylene, and D-limonene. Known volumes of single or mixed solvents were added to pads in the range 0.2-5.0 microliters (0.16-8.13 micrograms). After microwave-solvent extraction (ME) into hexan-1-ol, the samples (0.5-3.0 microliters) of the filtered and extracted solutions were analyzed by gas chromatography. All solvents exhibited > 97% adsorption on the pads at spiking levels of 0.48-0.98 microgram for each solvent. The solvent recovery for the system was calculated for each solvent, with solvents with boiling points below 110 degrees C showing recoveries of > 90%, and with solvents with boiling points above 110 degrees C showing recoveries from 80 to 90%. The recovery precision was good (RSD < or = 4%) for all solvents over the range 1.0 2.5 microliters of applied solvents to pads for ME and 1.0 microliter of extracted solutions for GC analysis. PMID- 10736877 TI - Optimized procedure for the determination of antimony in lipid-rich environmental matrices by flow injection hydride generation atomic absorption spectrometry. AB - An analytical procedure for the reliable determination of Sb in digests of lipid rich environmental matrices in the low ng l-1-range based on flow injection hydride generation atomic absorption spectrometry (FI-HG-AAS) has been developed. Prior to HG-AAS, aliquots (250 to 320 mg) of dry samples were mineralized with 3 ml nitric acid and 0.5 ml of each sulfuric and perchloric acids in open digestion vessels made of glassy carbon in a heating block. Procedure detection and quantification limits of a previously developed procedure for the determination of Sb in plant materials by FI-HG-AAS were decreased with respect to the lower Sb concentrations in animal tissues, the sensitivity of the instrumental response was increased, and the composition of the acid digestion mixture was re-optimized for lipid-rich samples. The accuracy and precision of the developed procedure was evaluated by the analysis of the two reference materials Bovine Liver 1577a and Pig Kidney CRM 186. These reference materials have been additionally spiked with appropriate amounts of Sb to obtain recovery data. The solution detection limit (3 sigma) in digested samples was 0.021 microgram l-1, the detection limit for the whole procedure based on the dry powders was 7 pg g-1, the method quantification limit for a reliable determination of Sb was 23 pg g-1. The reproducibility of repetitive measurements was 6.0% at 0.1 microgram Sb l-1 and 2.2% at 0.5 microgram Sb l-1. Calibration curves were linear from 0.05 to 3 micrograms Sb l-1. To demonstrate the suitability of the developed method, concentrations of Sb have been determined in pigeon eggs (approximately 2 ng Sb g 1), as well as in bream livers (approximately 4 ng g-1) and in deer livers (approximately 5 to 8 ng g-1) from animals living in remote and urban industrialized areas of Germany, respectively. PMID- 10736879 TI - Guidelines for in-house validation of analytical methods for pesticide residues in food and animal feeds. AB - Criteria are presented by which analytical methods may be judged to have been validated for the determination of pesticide residues. All stages of analysis are addressed, from initial preparation of samples to the production of results, but with a focus on simplicity and cost-effectiveness of the requirements. Criteria are provided for both quantitative and qualitative (screening) methods and they may be applied to single- or multi-residue methods. PMID- 10736880 TI - Clinical governance--a new label for old ingredients: quality or quantity? PMID- 10736881 TI - Partnerships or power struggles? The 'Crown' review of prescribing. PMID- 10736882 TI - Repeat prescribing management--a cause for concern? AB - BACKGROUND: No existing studies of repeat prescribing management have been carried out on statistically adequate samples permitting an extrapolation of results with regard to the population of general practitioners (GPs). AIM: To provide adequate regional evidence of the quality of repeat prescribing management for the profession and its administrators, and to test a scoring system for quality assurance in repeat prescribing practice. METHOD: A semi structured questionnaire was administered by one observer to a statistically representative population sample of Northern Ireland's general practices to investigate the extent to which they adopted recommended procedures for the management of repeat prescribing. Responses to 26 of these questions were used to score the quality of management. The subjects were a random sample of 57 practices stratified for number of partners, geographical area, and fundholding status. RESULTS: The main outcome measures were the percentage adoption of recommended procedures at the time of repeat prescription issue and at the review consultation, use of computing for repeat prescribing and the effects of fundholding; and quality assurance scores. During issue of repeats, essential checks are often omitted; the potential of computerization for improving management is often not realized. At review consultation, the opportunities for quality assurance are often missed. Fundholders manage repeat prescribing significantly better than non-fundholders, but in neither group is the mean management score exemplary. CONCLUSION: We have identified and quantified serious deficiencies in repeat prescribing management in a representative sample large enough to permit extrapolation to the regional population of GPs. In response, we have devised guidelines that GPs might use to address this problem. We have tested and proved a scoring system for repeat prescribing evaluation. PMID- 10736883 TI - Anxiety among women with mild dyskaryosis: a randomized trial of an educational intervention. AB - BACKGROUND: Women with mild dyskaryosis are currently managed by six-month cytological surveillance. While there is good evidence that women suffer psychological distress on receipt of an abnormal test, and that this is amenable to educational intervention, it remains uncertain whether this distress is prolonged and, if so, how it should best be managed. AIM: To investigate whether a structured educational intervention containing a risk communication package impacts upon psychological sequelae associated with this surveillance. METHOD: A pragmatic cluster-randomized controlled trial during 14 months in 1995 and 1996, based in general practices in Avon and South Glamorgan, that compared the intervention with standard care. Follow-up was by postal questionnaire at six weeks and four months after the screening laboratory had reported the test result. The intervention was an invitation to attend the general practice to consult with a practice nurse trained to deliver the package. The main outcome measures were Spielberger state-anxiety, SF-36 Mental Health dimension, four condition-specific questions regarding concerns about gynaecological health and timing of the repeat smear test, and attendance for the repeat test. RESULTS: Of 514 eligible women, 270 were recruited, of whom 240 returned the six-week questionnaire and 181 returned the four-month questionnaire. On all but one outcome measure, the differences between the groups were not statistically significant. At six-week follow-up, the proportion who preferred the repeat test to be sooner than six months was statistically significantly higher among controls (74% versus 53%; 95% CI = 9% to 33%). At the four-month follow-up, the difference was 7% (95% CI = -7% to 21%). CONCLUSION: There appear to be high levels of anxiety during surveillance for mild dyskaryosis that were not reduced by the intervention. Given that a personally delivered educational intervention designed to reduce anxiety could be viewed as the best available practice, it is of concern that women in the intervention group demonstrated sustained anxiety over a four-month period. The research agenda therefore seems to return to the fundamental question of whether surveillance should be the management of choice. PMID- 10736884 TI - Barriers to optimum management of heart failure by general practitioners. AB - BACKGROUND: Published research offers clear pointers to the management of heart failure; however, the evidence for implementation into practice is sub-optimal. AIM: To identify the salient barriers to adopting evidence-based management of heart failure in the community. METHOD: Structured interviews were used to elicit the views of a stratified sample of 100 general practitioners (GPs) about the diagnosis and treatment of heart failure. Responses to three heart failure case scenarios provided an indication of the degree to which GPs' knowledge of heart failure and trial results might be applied to diagnosis and treatment intentions. RESULTS: Participants were generally well aware of clinical trials that showed that prognosis could be improved by treatment, but trial results appeared to have little influence on treatment intentions in the three case scenarios. The major barriers to optimum management were the difficulties of differential diagnosis and the perceived properties of angiotensin-converting enzyme inhibitors (ACE-I) relative to diuretics. In the case scenarios, less than 30% reported that they would undertake basic investigations, such as chest X-ray or haemoglobin, or prescribe ACE-I. Over 70% perceived diuretics to be a useful diagnostic tool. The most frequent reasons for not prescribing ACE-I were the perceived inconvenience and risks of adverse effects (41%) and the view that most patients can be managed successfully on diuretics alone (27%). Over two-thirds of the sample were dissatisfied with the quality of information accompanying heart failure patients discharged from hospital. CONCLUSION: Facilitating evidence-based management of heart failure in the community requires further support for GPs in the form of additional training in the diagnosis of heart failure and the optimum use of both ACE-I and diuretics, and by improved communication between GPs and hospital doctors on a case-by-case basis. PMID- 10736885 TI - Is chronic non-specific low back pain chronic? Definitions of a problem and problems of a definition. AB - BACKGROUND: Chronic low back pain (LBP) accounts for the majority of the disability and costs for LBP. However, the definition of chronicity is unclear. AIM: To elicit practitioners' definitions of chronic LBP patients, both in general and in the patients they were treating; to assess the most common characteristics of these practitioners' chronic LBP patients; and to assess the stability of chronicity in a sample of the general population. METHOD: Semi structured interviews were conducted with 33 practitioners working in private practice, 71 LBP patients and their therapists, and 252 employees of a chain store who were assessed yearly in a prospective study. RESULTS: The therapists' definitions of chronic LBP patients generally included psychosocial aspects. Only physical symptoms and signs were stressed in the patients they were treating. These patients displayed common characteristics with reference to pain, functional problems, and contact with health care services. Duration of symptoms was not sufficient to define chronicity. In the employee population, chronicity defined according to pain duration was unstable. However, the same was true when chronicity was measured according to the criteria defined in the patient population. CONCLUSION: There is a discrepancy between theory and practice regarding the definition of chronic LBP. This discrepancy concerns not only the literature but also clinical practice itself. The term 'chronic' LBP as currently used is therefore equivocal. PMID- 10736886 TI - Someone to talk to? The role of loneliness as a factor in the frequency of GP consultations. AB - BACKGROUND: There are many reasons why people frequently consult their general practitioner (GP). Although loneliness is increasingly recognized as a problem affecting well-being for elderly people, it has rarely been addressed as a predictor of frequency of consultation. AIM: To examine whether loneliness is associated with rates of GP consultations (home and surgery visits). METHOD: Analysis of data collected in face-to-face interviews at the second wave of a longitudinal health survey of two adult age cohorts living in four socially contrasting urban localities in Glasgow City. There were a total of 691 subjects: 142 males and 176 females aged 40 years at interview; and 167 males and 206 females aged 60 years at interview. Frequency of reported GP consultations in the past 12 months at home or in the surgery was examined. RESULTS: After controlling for sociodemographic and socioeconomic variables and health, loneliness was significantly associated with frequency of consultation at the surgery but not with the frequency of home visits. CONCLUSION: Loneliness may still be underestimated as a factor related independently to frequency of consultations with a GP at the surgery. PMID- 10736887 TI - Attitudes towards, and utility of, an integrated medical-dental patient-held record in primary care. AB - BACKGROUND: The need for closer coordination between primary care medical and dental services has been recognized. AIM: To assess the attitudes of general medical practitioners (GMPs), general dental practitioners (GDPs), and patients to an integrated medical-dental patient-held record (integrated medical-dental PHR); to examine patients' use of these records, and the utility of the records for doctors and dentists. METHOD: A three-phase study was carried out: (1) postal survey of GMPs and GDPs; (2) randomized trial of patients, using postal questionnaires before and one year after the issue of integrated medical-dental PHRs to cases; (3) assessment by doctors and dentists of anonymized integrated medical-dental PHRs from this trial. The study was carried out in medical and dental practices in affluent and deprived areas in Greater Glasgow Health Board. Two hundred and thirteen GMPs, 183 GDPs, and 369 patients registered with GMPs and GDPs were surveyed. Eighteen GDPs and GMPs assessed the integrated medical dental PHRs. RESULTS: Eighty per cent of dentists had contacted a doctor and 16% of doctors had contacted a dentist in the previous three months; 87% of dentists and 68% of doctors thought an integrated medical-dental PHR would be of some use. Twenty-one per cent of dentists and 85% of doctors had practice computers. Most patients wanted to be able to see and read their own records. Twenty-four per cent of patients said there were mistakes and 30% noticed omissions in the integrated medical-dental PHR issued. Experience of having an integrated medical dental PHR made patients more positive towards the idea of having a patient-held record and being able to check the accuracy of records. Integrated medical-dental PHRs contained important information for half the GDPs and one-third of the GMPs. CONCLUSION: Both professionals and patients have reasonably positive attitudes towards the use of patient-held records. Among patients, the experience of having the integrated medical-dental PHR led to greater enthusiasm towards the idea. Dentists in particular would benefit from the transfer of information from doctors, but better methods are needed to ensure that patients take the integrated medical-dental PHR with them. Given the current lack of ability to easily produce an integrated medical-dental PHR, further examination of the routine issue of a copy of their medical summary, by GMPs, to all patients would be worthwhile. PMID- 10736888 TI - Collation and comparison of multi-practice audit data: prevalence and treatment of known diabetes mellitus. AB - BACKGROUND: Different methods have been used to determine the prevalence and treatment of diabetes. Despite the large number of studies, previous estimations of prevalence and treatment have been carried out on relatively small numbers of patients, and then in only a few practices in single geographical regions. AIM: To investigate the feasibility of collating data from multi-practice audits organized by primary care audit groups in order to estimate the prevalence and treatment of patients with known diabetes, and to discuss the methodological issues and reasons for variation. METHOD: A postal questionnaire survey of all primary care audit groups in England and Wales that had conducted a multi practice audit of diabetes between 1993-1995. Prevalence rates and patterns of diabetic care were compared with other community-based surveys of known diabetes from 1986-1996 identified on MEDLINE. RESULTS: Twenty-five (43%) audit groups supplied data from multi-practice audits of diabetes. Seven (28%) multi-practice audits involving 259 practices fulfilled the inclusion criteria for prevalence estimation. The overall prevalence of diabetes based on a population of 1,475,512 patients was 1.46% (range between audit groups = 1.18% to 1.66%; chi 2 = 308; df = 6; P < 0.0001). Male to female ratio was 1.15:1. Treatment of diabetes could be ascertained for 10 (40%) audit groups comprising 319 practices. Of these, 23.4% (range = 16.5%-27.4%) were controlled by diet, 48.5% (range = 43.6%-55.8%) were prescribed oral hypoglycaemic drugs, and 28.2% (range = 25.0%-32.4%) were treated with insulin. There were significant variations between audit groups in treatment pattern (chi 2 = 250; df = 18; P < 0.0001). CONCLUSION: Prevalence and treatment rates of diabetes and other chronic diseases can be assessed and compared using data from multi-practice audits. Collation of audit data could improve the precision of quantitative estimates of health status in populations. A standard method of data recording and collection may provide a new approach that could considerably improve our ability to monitor disease and its management. PMID- 10736889 TI - General practitioners' use of computers during the consultation. AB - This study documents the extent of reported computer use by general practitioners (GPs) in consultations with patients, and identifies barriers to their use. There was a 65% response rate from a random sample of 600 GPs in the South and West National Health Service (NHS) region who were sent a questionnaire. Ninety-one per cent (357) had a desktop computer terminal in their consulting rooms. Of these, 98% used the computer to look up information or prescribe medication, 75% entered details about selected problems presented by patients, and 36% entered information about the patient's presenting problem at every consultation. Only 18% used computers to access reference information. Use of the computer for anything other than looking up patient information or prescribing was positively associated with fundholding status and use of a personal computer at home, and was independent of number of years in practice. Sixty-five per cent of responders had positive attitudes to the inclusion of management guidelines on the computer software, and 45% of responders held positive views towards the idea of integrating management guidelines with the patient's personal computerized medical record. Consideration should be given to targeting training at those GPs who appear to be reluctant to use computers during the consultation. PMID- 10736890 TI - The effect of the 1996 'beef crisis' on depression and anxiety in farmers and non farming controls. AB - This paper looks at the effect of the 1996 'BSE crisis' on the mental health of farmers from one semi-ural practice in North Yorkshire. In 1996, Hospital Anxiety and Depression (HAD) scales were sent to farmers and controls who had participated in a previous study in 1994. Comparative data for the two groups for the two years were obtained and analysed. The data showed that, despite fears raised as a result of the 'BSE crisis', the overall rates of depression and anxiety fell in both groups between 1994 and 1996, with the rates falling significantly more in the control group. However, the farmers were still more depressed and anxious than the controls, and those farmers that had been depressed or anxious in 1994 were more likely to be depressed or anxious in 1996. A longer period of time may be needed to determine the effect of the beef crisis on the mental health of farmers. PMID- 10736891 TI - What's the 'best buy' for treatment of constipation? Results of a systematic review of the efficacy and comparative efficacy of laxatives in the elderly. AB - Constipation is a common cause of general practice consultations in elderly people, and laxative use is common among this group of patients. However, there appears to be little evidence to guide laxative treatment in this population. This paper reports the results of a systematic review of randomized controlled trials of the efficacy of laxatives in the treatment of constipation in the elderly. While the results of the review suggest that laxatives can improve bowel movement frequency, stool consistency, and symptoms of constipation, with a few exceptions, the relevant trials have serious methodological shortcomings. However, the review finds little evidence of marked differences in effectiveness between laxatives and, in particular, there appears to be no evidence to support the current National Health Service (NHS) trend towards prescribing the more expensive stimulant laxatives. This is an area where good quality trial evidence is now needed. PMID- 10736892 TI - Community-based teaching: the challenges. AB - The amount of undergraduate medical education delivered in general practice is expanding rapidly, both in the United Kingdom and internationally. There are a number of challenges facing general practice as well as medical schools, health authorities and primary care groups, which must be met for this development to be sustainable. These include: impact on service general practice; resources; difficulties with integrating basic sciences with clinical teaching; recruitment, training and maintenance of GP tutors; quality control; impact on academic departments of primary care; and the importance of rigorous evaluation of educational initiatives. Possible solutions are discussed, such as development of university linked practices and the move toward a culture of 'evidence-based education', where all medical education is scrutinized for effectiveness. PMID- 10736893 TI - Mental health services--primary concerns for the future. PMID- 10736894 TI - Management of painful joints. PMID- 10736895 TI - Fortress general practice. PMID- 10736896 TI - Reducing antibiotics for respiratory tract symptoms in primary care: 'why' only sore throat, 'how' about coughing? PMID- 10736897 TI - Chickenpox in pregnancy. PMID- 10736898 TI - Fatigue. PMID- 10736899 TI - Revalidation. PMID- 10736900 TI - Expanding the role of the family history in primary care. PMID- 10736901 TI - Clinical and patient satisfaction outcomes of a new treatment for somatized mental disorder taught to general practitioners. AB - BACKGROUND: Patients with mental disorder presenting with medically unexplained symptoms (somatized mental disorder) are common in primary care, difficult to treat, and function poorly in their daily lives. AIM: To examine the effects on patient outcome and satisfaction of a training package for somatized mental disorder delivered to general practitioners (GPs). METHOD: A prospective study of a before-and-after training study of different cohorts of patients attending eight GPs who acted as their own controls. Patients were stratified according to their belief that the presenting medical symptom had either a partial or completely physical cause. RESULTS: One hundred and three patients in the cohort before training, and 112 patients in the cohort after training, were diagnosed with somatized mental disorder by the study GPs. After training there were significant improvements in interview-rated psychiatric disorder (P = 0.032) at one month, self-rated psychiatric disorder (P = 0.024), and global function (P = 0.020) at three months in patients who believed their symptoms to have a partial physical cause. Training at one-month follow-up reduced depressive symptoms in patients with major depression but did not significantly change any other outcome in patients who believed their symptoms had only a physical cause. There was no overall change in patient satisfaction. CONCLUSION: Training GPs clinically benefited patients with somatized mental disorder who believe that their symptoms have a partial physical cause. PMID- 10736902 TI - The simulated surgery--an alternative to videotape submission for the consulting skills component of the MRCGP examination: the first year's experience. AB - BACKGROUND: The simulated surgery was developed to examine the consulting skills of general practice (GP) registrars by observing their consultations with standardized patients. It was introduced in 1997 as an alternative to videotape submission in the consulting skills component of the Membership of the Royal College of General Practitioners (MRCGP) examination for those candidates who are unable to prepare a videotape. AIM: To describe the methodology of the examination and to report on the first year's experience. METHOD: The development of the cases and the techniques of marking and standard setting in the simulated surgery are described. RESULTS: Thirty-eight GP registrars took part in pilot examinations and 37 candidates were examined for the MRCGP. The distribution of their marks and the resulting pass/fail decisions are reported. The reliability of the 20-case simulated surgery, using Cronbach's alpha coefficient, is greater than 0.85. CONCLUSION: The simulated surgery is a feasible, valid, and reliable examination of consulting skills. Cost and manpower requirements remain a problem, but these are being addressed by current plans. PMID- 10736903 TI - How many surgery appointments should be offered to avoid undesirable numbers of 'extras'? AB - BACKGROUND: Patients seen as 'extras' (or 'fit-ins') are usually given less time for their problems than those in pre-booked appointments. Consequently, long queues of 'extras' should be avoided. AIM: To determine whether a predictable relationship exists between the number of available appointments at the start of the day and the number of extra patients who must be fitted in. This might be used to help plan a practice appointment system. METHOD: Numbers of available appointments at the start of the day and numbers of 'extras' seen were recorded prospectively in 1995 and 1997 in one group general practice. Minimum numbers of available appointments at the start of the day, below which undesirably large numbers of extra patients could be predicted, were determined using logistic regression applied to the 1995 data. Predictive values of the minimum numbers calculated for 1995, in terms of predicting undesirable numbers of 'extras', were then determined when applied to the 1997 data. RESULTS: Numbers of extra patients seen correlated negatively with available appointments at the start of the day for all days of the week, with coefficients ranging from -0.66 to -0.80. Minimum numbers of available appointments below which undesirably large numbers of extras could be predicted were 26 for Mondays and four for the other week-days. When applied to 1997 data, these minimum numbers gave positive and negative predictive values of 76% and 82% respectively, similar to their values for 1995, despite increases in patient attendance and changes in the day-to-day pattern of surgery provision between the two years. CONCLUSION: A predictable relationship exists between the number of available appointments at the start of the day and the number of extras who must be fitted in, which may be used to help plan the appointment system for some years ahead, at least in this relatively stable suburban practice. PMID- 10736904 TI - Retention of young general practitioners entering the NHS from 1991-1992. AB - BACKGROUND: The supply of general practitioners (GPs) in the National Health Service (NHS) is dynamic and there are fears that there will be an inadequate number of doctors to meet the needs of the NHS. There are particular concerns about changes in the career trajectory of young GPs and what they mean for overall supply. AIM: To identify predictors of retention among young, new entrant GPs entering the NHS between 1 October 1991 and 1 October 1992. METHOD: Two-year retention rates of young (35 years of age or less) new entrant GPs have been modelled using a multilevel logit model. Retention is defined as young, new entrant GPs remaining in their initial health authority for two years or more. RESULTS: Two hundred and fifty-two (13.0%) members of the study group left general practice within two years of entry (i.e. were not retained). Sex (females had lower retention [95% CI = 0.43-0.75]), practice size (young GPs in larger practices had higher retention [95% CI = 1.10-1.29]), and belonging to a practice in one of 16 Greater London Health Authorities (which had lower retention [95% CI = 0.39-0.82]) were identified as major predictors of retention. Deprivation, measured at the individual GP patient list level, had a very slight association with retention (P = 0.097; 95% CI = 1.00-1.02). Deprivation measured at the health authority level (95% CI = 0.99-1.01) was not found to be a statistically significant predictor of retention (P = 0.83). CONCLUSION: None of the statistically significant predictors of retention suggest any policy panacea to end this phenomenon. The challenge for policy is to learn to deal with the dynamic nature of the GP workforce with a non-crisis mentality. PMID- 10736905 TI - Elderly people's beliefs about influenza vaccination. AB - BACKGROUND: Influenza is an important cause of death in the elderly. The uptake of influenza vaccination, despite its effectiveness, is low. AIM: To examine beliefs about influenza vaccination in elderly patients at risk from influenza. METHOD: A qualitative study using semi-structured interviews with 50 patients aged over 75 years at risk from influenza, equally divided between vaccinated and non-vaccinated groups. RESULTS: Although they acknowledged their medical diseases, the patients regarded themselves as healthy in the sense of being independent and active. Few in either the vaccinated or non-vaccinated groups believed themselves at risk of dying from influenza even though they recognized it could be fatal for particular groups of people. Decisions to have the vaccination were based on other considerations, including whether it was thought likely to reduce (or increase) the number and severity of colds and influenza like illnesses. Although the group with negative views towards vaccination placed more emphasis on the 'side-effects' from the vaccination (including colds and influenza), this was also common in the group who were more positive towards vaccination; however, the side-effects were interpreted in different ways. CONCLUSION: Recommendations to vaccinate according to individual risk status are not in keeping with lay beliefs. The policy to include all people aged 75 years and older as a group requiring influenza vaccination is supported by this study. The evidence that vaccination reduces morbidity from influenza and does not cause colds and influenza needs stressing. PMID- 10736906 TI - The effect of a special nurse on patients' knowledge of epilepsy and their emotional state. Epilepsy Evaluation Care Group. AB - BACKGROUND: People with epilepsy often report being given insufficient information and support. However, there is little evidence from general practice about how much they know and how they feel. AIM: To describe social differences in the knowledge of epilepsy of people with the condition and test the potential effect of a nurse intervention in general practice on patients' knowledge and depression levels. METHOD: A questionnaire that included measures of knowledge, anxiety, and depression was sent to people with epilepsy aged over 15 years who were registered with 37 general practitioners. Responders were randomized to a controlled trial, offering either two appointments with an epilepsy nurse or usual care. Six months later they were reassessed. RESULTS: Two hundred and fifty one out of 283 (89%) of the patients with epilepsy completed questionnaires and entered the study at Stage 1. One hundred and ninety-six out of 232 (84%) of those who entered the study, who remained in the practices and were eligible, returned questionnaires at Stage 2. The average duration of epilepsy was 23 years (range 2-79 years). There were significant differences in patients' levels of knowledge of epilepsy at Stage 1. Younger people, those who had left school after 16 years of age, those with GCSEs, and people who belonged to self-help groups had higher knowledge levels, and these were independent effects. Older people and those with a recent epilepsy attack had significantly higher depression scores. Knowledge scores did not differ significantly after the nurse intervention (Stage 2). At Stage 2, the risk of depression was less in the group randomized to be offered nurse input; the effect was mainly in a subgroup of patients with no recent epilepsy attack; their risk of depression was a third of the risk in the control group. CONCLUSIONS: Knowledge of epilepsy differs significantly, with social factors and self-help group membership having independent effects. A nurse run clinic reduced the risk of depression for people with no recent epilepsy attack, but knowledge levels were not affected. This does not exclude the potential for patients learning more about epilepsy; it may be useful to suggest that patients join self-help groups early on. PMID- 10736907 TI - Can primary care groups learn how to manage demand from fundholders? A study of fundholders in Nottingham. AB - BACKGROUND: Primary care groups (PCGs) will commission care for their patients and may be increasingly required to manage clearly defined resources. Existing general practice fundholders already operate in this environment, but can PCGs learn from the experience of fundholders in managing demand? AIM: To explore how general practice fundholders manage demand for hospital and community health services, and for prescribing. METHOD: A general practitioner (GP), and a fundholding manager from each of 26 practices were invited to take part. Questionnaires were developed, with structured and semi-structured components, and piloted in three practices. Interviews were conducted between October 1996 and February 1997 by the same interviewer (MDT). RESULTS: All practices stated that they were monitoring their waiting lists and giving priority to patients whose problems had become worse, but eight of the 23 GPs felt that they were unable to manage demand. Eight of the 15 fundholders who had developed in-house services actively managed the waiting list for these clinics. All fundholders had identified areas of unmet demand. Widely differing methods for increasing supply to meet demand were identified, and are described. Formularies were used by 12 out of the 23 fundholders. Guidelines were only considered useful by eight of the 23 practices; fundholders from later waves were less likely to find guidelines useful than fundholders from earlier waves (odds ratio [OR] = 0.11; 95% confidence interval [CI] = 0 to 0.96). Private specialist surgery was less likely to be accessed by later wave fundholders using the fund than by early wave fundholders (OR = 0.10; 95% CI = 0.09 to 0.97). CONCLUSION: Fundholders in Nottingham had not developed consistent approaches to managing demand within limited resources. Given the apparent diversity of attitudes and practices, the larger PCGs will require strong support to develop the intended commissioning function. PMID- 10736908 TI - Risk stratification in the management of atrial fibrillation in the community. AB - This study assessed whether risk stratification in patients with atrial fibrillation (AF) in the community had a bearing on the likelihood of receiving aspirin or warfarin therapy. Seven hundred and fifty patients were identified from 14 practices by means of diagnostic READ codes or repeat prescriptions for digoxin from practice computers. The study demonstrates that general practitioners appreciate the importance of antithrombotic therapy in patients who have suffered stroke, but take poor account of increasing age and other independent risk factors. A more proactive approach to risk identification and treatment seems justified. PMID- 10736909 TI - The role of automated external defibrillators in rural general practice. AB - In a questionnaire survey (100% response rate) investigating the availability and use of automated external defibrillators (AEDs), it was found that the success rate (number discharged alive) compared favourably with pre-hospital defibrillation by other providers, and that AEDs aided the management of dysrhythmias not commonly seen in general practice. With appropriate training they are useful in rural general practice. PMID- 10736910 TI - Women with angina pectoris receive less antiplatelet treatment than men. AB - In a study investigating the prevalence of underprescription of platelet therapy for women with angina pectoris, the complete medication histories of patients were examined and indicators of possible comorbidity and comedication were recorded. A higher percentage of women than men were not treated with any form of antithrombotic treatment (37% versus 18%), suggesting a serious, and possibly hazardous, undertreatment with acetylsalicylic acid (ASA) in women compared with men. PMID- 10736911 TI - Driving after severe head injury: the need for assessment. AB - This paper reports on a survey of return to driving after severe head injury. It highlights the lack of information provision, low rates of Driver and Vehicle Licensing Agency (DVLA) notification, and poor uptake of driving assessments. The findings highlight the need for liaison between head injury services and general practitioners (GPs) when assessing driving fitness. PMID- 10736912 TI - The objective assessment of general practitioners' educational needs: an under researched area? AB - A systematic literature review of the educational needs assessment of general practitioners (GPs) is described. A total of 36 studies, based on the subject of educational needs in primary care, were identified. The methods of searching and the difficulties in identifying papers on the chosen subject are discussed. Although there is an extensive literature on the reporting of the perception of GPs' learning needs, there have been relatively few studies describing their objective evaluation. Seventeen papers on this subject are described, and it was found that questionnaires and structured interviews were the most usual method adopted. There is a case for developing and evaluating a wider range of methods for assessing educational needs in primary care, particularly if the move towards portfolio-based learning is to gain momentum. PMID- 10736913 TI - The physician healer: ancient magic or modern science? AB - The therapeutic role of general practitioners (GPs) is one that, over the years, has slowly diminished with the growing fashion for evidence-based medicine. However, it is clear that the art of healing and the strength of the doctor patient relationship play a vital role in improving the well-being of patients. This is exemplified by the placebo effect, where the attitude of the doctor can make an appreciable difference to the psychological response of the patient who feels the need to be understood and listened to empathically. By maximizing the role of the physician healer, there is considerable scope for bridging the gap left by the impersonality of medical science, while at the same time increasing the GP's effectiveness. PMID- 10736914 TI - Health visitors and child health surveillance. PMID- 10736916 TI - Epidural steroid injections for sciatica. PMID- 10736915 TI - Health visitors and child health surveillance. PMID- 10736917 TI - Dutch GPs acknowledge the need for preconceptual health care. PMID- 10736918 TI - A bridge across the no-man's land. PMID- 10736919 TI - Relationship between new and return consultations and workload in general practice. PMID- 10736920 TI - Awareness of BACUP in primary health care: the potential of voluntary services. PMID- 10736921 TI - Stigmatization of sufferers of mental disorders. PMID- 10736923 TI - Domestic violence: the general practitioner's role. PMID- 10736922 TI - Consulting with a cough. PMID- 10736924 TI - Medical students in GP consultations. PMID- 10736925 TI - [Audioscan automatic audiometry: theoretical basis and normative data]. AB - Audioscan automatic audiometry is a high resolution method to explore hearing thresholds in the 125 Hz-16 KHz range offering a resolution of 64 points per octave. This procedure makes it possible to study hearing threshold levels at intermediate frequencies which are not measured by conventional audiometry. As suggested in the literature, it is possible to identify hearing threshold notches, considered indicators of mild or subclinical cochlear pathology. In clinical practice, the use of a new audiometric technique requires affirmation of reliability, sensitivity and replicability in time. It also requires defining standard criteria for use in various otologic pathologies. The present study examined 50 normal hearing subjects with conventional audiometry and Audioscan (Essilor model) measurements. A standard acquisition protocol was followed where three Audioscan measurements were conducted per subject. Subjects with a history of acute otologic pathology and familiarity for genetic deafness were ruled out of the study. Multiple, pairwise correlation analyses of the audiometric and Audioscan responses indicate that: (1) there are no significant differences between threshold levels measured by Audioscan and conventional audiometry; (2) there are no significant variations in the hearing levels measured by three Audioscan retests. Three mean indicators of the threshold level have been defined, expressing Audioscan normality in three different frequency ranges. The data suggest that there are some difficulties in the subjectivity of the Audioscan method and some technical problems regarding the lower octave frequencies and these need to be addressed. The results obtained in this study confirmed reliability of the Audioscan in the mid and high frequencies. The definition of the standard mi criteria makes it easier to interpret the Audioscan measurements from cases presenting various otologic pathologies. Therefore, the plasticity and high detection sensitivity of hearing loss make the automatic Audioscan audiometry a useful clinical tool for diagnostic and/or preventive purposes. PMID- 10736926 TI - [Effectiveness indices in "Particle Repositioning Maneuver" by Parnes and Price Jones]. AB - The Parnes and Price-Jones "Particle Repositioning Maneuver" (PRM) was used to treat Paroxysmal Positional Vertigo (VPP) in 36 consecutive cases, studying both any induced secondary nystagmus and any persistent Paroxysmal Positional Nystagmus (PPNy) upon repetition of the Hallpike positioning after 15 minutes (re test). The purpose of the study was to evaluate the ability to use secondary Ny and the retest to predict maneuver effectiveness. Although the PRM was quite effective (PPNy disappeared in 86% of the cases after a single session), the low observation frequency reduced the feasibility of using secondary Ny. On the other hand the systematic introduction of the Hallpike re-test into clinical practice does not offer the desired cost/benefit ratio because of the time required to prevent false negatives due to PPNy fatigue. In fact, secondary Ny was only evoked in 12 out of 36 cases (33%) and of these only 9 out of 36 (25%) presented direction congruous with canalolith release (liberating Ny). The predictive value of evoked liberating Ny appeared further reduced by the persistence of PPNy upon subsequent re-testing in two cases and in one case by the persistence of the disorder upon follow-up. The Hallpike test appears a more reliable indicator of effectiveness as it proved in agreement with subsequent clinical findings in 80% of the cases. The present results lead one to conclude that, when using PRM in cases where no secondary liberating Ny was found (75%), we should rely on the high effectiveness of this procedure (86% of the cases resolved in a single session) rather than perform a Hallpike retest. Thus it is possible to select a small number of patients (22%) still presenting PPNy (positive retest) and requiring repetition of the therapeutic maneuver. PMID- 10736927 TI - [Rhinosinusal inverted papilloma: diagnostic and therapeutic difficulties in a series of 26 cases]. AB - Inverted papilloma (IP) is a rare and yet complex pathology, particularly due to the risk of recurrence (approximately 50%) and the onset of carcinoma (approximately 15%). Moreover, in clinical practice it is difficult to diagnose and treat. The present work reviews a personal case study in order to make an essentially clinical evaluation of the problems of diagnosis and treatment. From 1989 to 1997, among approximately 1200 cases of surgery for phlogistic or neoplastic nasal-sinus pathologies, 26 cases of IP were treated. Of these 22 underwent a minimum follow-up of 12 months. In 5 out of the 22, it was a recurrence of 'nasal polyps' which had been surgically treated at another site (with histology performed for only 2 of the patients). In 8 of the 22, imaging did not indicate IP, resulting in an underestimation of the disorder and most likely affecting treatment. The following procedures were performed: 6 sphenoethmoidotomies through an endonasal approach (with and without endoscopy); 19 radical spheno-ethmoidomaxillotomies extended to varying degrees to the demolition of bone and, in 3 cases, using a paralatero-nasal approach (because of invasion of the anterior ethmoid); 4 endonasal exereses for IP arising in the septum (1 with reconstructive naso-genieno flap); 1 maxillectomy. Of the patients (22) who had undergone surgery in our Division, 5 showed recurrences and 3 of these had undergone spheno-ethmoidotomy through the endonasal approach. Moreover, all the cases of recurrence involved underestimated preoperative diagnosis (i.e. the 8 patients for whom IP was only recognized after histology). Among the 5 recurrences, for 2 carcinoma fields were diagnosed; for 3 additional surgery was required: one patient underwent additional surgery twice while another 3 times (lastly with a maxillectomy). The surgery did not lead to serious complications and at the present time all patients are alive and macroscopically disease free. Analysis of the literature and personal experience has lead to the following considerations: routine diagnostic evaluation of "polyps" is most likely still inadequate; it appears that the risk of recurrences can be controlled only with adequately aggressive surgery; endonasal forms (above all septal) could be disconnected, normally leading to peculiar histological and clinical features. PMID- 10736928 TI - [Study on cell proliferation in nasal polyposis as predictive index of recurrence]. AB - The purpose of the present research has been to identify a cellular parameter to be used as an index of increased cell proliferation and, therefore, the likelihood of recurrence. A total of 114 patients with bilateral nasal polyposis underwent naso-ethmoid dissection under endoscopy. Of these, 36 were affected by allergic rhinopathy, 6 bronchial asthma, 9 ASA syndrome and 9 diabetes mellitus; in addition 33 cases were classified as recurrences. The polyp material from each patient was analyzed using a Coulter XL cytofluorimeter, evaluating the cycles of approximately 10-20 thousand cells. The objective was to identify the nasal polyp activity status in the various patients, evaluating cell proliferation indicated by the percentage of cells in phase S and G2 + M. Of the 114 patients, 21 were ruled out of the study because of a high degree of cell debris. Cytofluorimetry made it possible to identify 3 groups based on the different percentage of cells in the genomic synthesis phase. In the first group (A) of 42 patients, the number of cells in the active phase ranged from 2.1% to 10%. The second group (B) of 21 patients showed a percentage in the 14.7-41.9% range. The third group (C) of 30 patients--all affected by recurrent polyps--showed a high percentage (25%-49.8%) of cells in the active phase. It is interesting to note that: 1. A full 91% of the allergic patients fell within group A. 2. Of the 9 patients with ASA, 7 were in the group with recurrences. 3. The 30 patients affected by recurrent polyps were all in group C and that of these 12 had more than one recurrence, expressing a higher percentage of active cells. The above appears to indicate that the cell parameter evaluated here cytofluorimetrically may be a new, simple, reliable index to predict nasal polyps recurrences. PMID- 10736929 TI - [Modified type I thyroplasty in the surgical treatment of unilateral laryngeal paralysis]. AB - Subsequent to previous experience in the surgical treatment of vocal cord atrophy using various materials (Teflon, Hydron Gel, Gax Collagene) the authors treated severe glottic insufficiency using the Isshiki Type I Thyroplasty modified in terms of shape and thickness of the silicone prosthetic implant. This procedure provides several important advantages: it does not modify the mass, volume and stiffness of the operated vocal cord; it can be reversed or modified; it permits intraoperative control of the results. The first three cases operated with this technique are analyzed and videostroboscopic and electroacoustic evaluation are presented. PMID- 10736930 TI - [Surgical treatment of benign thyroid nodules]. AB - Benign thyroid nodules is a significant clinical problem since it involves more than 90% of all thyroid surgery. This has led the authors to perform a retrospective study of 190 patients suffering from this pathology out of 299 patients who underwent surgery from 1979 to 1995. 89 total thyroidectomies and 101 radical hemithyroidectomies were performed in patients suffering from benign thyroid nodules. Two cases of recurrent monolateral paralysis were found as well as 3 cases of permanent hypoparathyroidism in those patients who underwent total thyroidectomies. The results of numerous authors as well as the present case review indicate that more radical surgery does not significantly affect the degree of permanent complications. For this reason the authors are convinced that such radical surgery should be the treatment of choice, particularly in young patients whose life expectancy is long and where the likelihood of recurrence is greater. PMID- 10736931 TI - [Alessandro Volta and first attempts at electrotherapy of deafness]. PMID- 10736932 TI - [Otorhinolaryngology and women]. PMID- 10736933 TI - [Effects of sulphur-arsenic-ferrous water treatment on specific chronic phloglosis of the upper respiratory tract]. AB - The aim of the present study was to demonstrate the therapeutic effect sulphurous arsenical-ferruginous waters from had on aspecific phlogosis of the upper respiratory tract (URT). This double-blind study involved treating 37 adults of both sexes with sulphurous-arsenical-ferrignous water (group A) and another 14 subjects, again suffering from the same aspecific, chronic catarrhal disorder and selected with the same criteria, with the aerosol vapor inhalation of drinking water from the city mains (group B). All subjects included in the study underwent the following tests both before and after the cycle of crenotherapy: Objective E.N.T. examination Anterior Active Computerized Rhinomanometry (AACR) Mucociliary transport time (MTT) Nasal cytology Physical and immunochemical examination of the nasal mucous. The post crenotherapy variations achieved indicate that the sulphurous-ferruginous waters have a beneficial therapeutic effect: indeed, the mucosa was able to perform its complex functions, particularly specific and aspecific defense of the organism. In fact, among other things, group A showed decreased resistance and increased nasal respiratory flow, normalized of mucociliary transport, decreased bacterial layer and increased plasma cells in the rhinocytogram, and an increase in albumin, non secretory immunoglobulin and the secretory portion of secretory immunoglobulin A in the nasal mucosa. None of these variations--many of which were statistically significant--were seen in the controls group treated with drinking water from the public mains. PMID- 10736934 TI - [Introduction of family medicine in the university field. Dream or reality?]. PMID- 10736936 TI - [Cases of heart failure requiring hospitalization]. AB - OBJECTIVE: To find the characteristics of patients with heart failure requiring admittance to the referral hospital for the A Coruna Health Area. DESIGN: Descriptive, crossover and retrospective study. SETTING: Health area of 500,000 inhabitants. PATIENTS AND OTHER PARTICIPANTS: Patients admitted with diagnosis of heart failure (1995; n = 636). A simple randomised sampling was undertaken, stratified by sex (n = 225; alpha = 0.05; accuracy = 6%). Sample size was increased by 25% due to possible losses of information. MEASUREMENTS AND MAIN RESULTS: The most common underlying causes were: ischaemic cardiopathy 36.4%, dilated myocardiopathy 23.6% and hypertension 16.5%. The most common catalysing factors were: infection 32.6% and tachycardia 24.6%. The commonest symptoms were dyspnoea 90.6% and orthopnoea 63.9%. The most frequent sign was crepitus 79.8%, followed by oedemas 58.3%. An echocardiogram was performed on 42.6% and 22% had digoxinaemia. Most common linked pathology was: hypertension 33.5% and COPD 25.0%. After admission the use of diuretics (30%), digoxin (9%) and ACE inhibitors (27%) was increased. 7.7% of patients died. Variables most commonly linked to death were myocardial infarction (OR = 21.8), hyponatraemia (OR = 12.2) and kidney failure (OR = 7.04). CONCLUSIONS: Given the underlying causes and catalysing factors seen in the results of this study, family doctors play a decisive role in prevention and control of heart failure. PMID- 10736935 TI - [Reliability of blood pressure measurement: the patient versus primary care workers]. AB - OBJECTIVE: To evaluate the degree of inter-observer concordance in the blood pressure figures taken by a nurse and a doctor with a mercury sphygmomanometer and by the patient with a semi-automatic device. DESIGN: Observational, crossover study. SETTING: A rural health centre. PATIENTS: 318 people selected by systematic sampling. INTERVENTIONS: Pressure was taken in different ways by 3 different "observers" (0): the patient with a semi-automatic device (01), a nurse (02) and a family doctor (03). Inter-observer concordance was assessed through the Intra-class Correlation Coefficient (ICC) and the kappa index. MEASUREMENTS AND MAIN RESULTS: 42.1%, 41.8% and 44.3% of patients had high blood pressure (> or = 140/90 mmHg) according to 01, 02 and 03, respectively. 33% were known to be hypertense. The ICC for systolic pressure was 0.81 (95% CI, 0.75-0.87) between 01 and 02; 0.84 (CI, 0.78-0.90) between 01 and 03; and 0.87 (CI, 0.82-0.92) between 02 and 03. The ICC for diastolic pressure was 0.67 (CI, 0.59-0.75), 0.72 (CI, 0.64-0.79) and 0.79 (CI, 0.72-0.86) for 01-02, 01-03 and 02-03, respectively. The kappa index was 0.53 (CI, 0.43-0.62) for 01-02; 0.60 (CI, 0.51-0.68) for 01-03; and 0.67 (CI, 0.58-0.75) for 02-03. CONCLUSIONS: Inter-observer concordance was good, with no substantial differences between the measurements made by the different observers. As the figures determined by the semi-automatic device were reliable, this is a good option for the follow-up and monitoring of hypertense patients. PMID- 10736937 TI - [Diagnosis of dyslipidemia in primary care: a service to be improved. Results of a multicenter evaluation]. AB - OBJECTIVE: The variability of the quality of care in the primary care procedures to diagnose lipaemia was evaluated. DESIGN: Observation, multi-centred and retrospective study. SETTING: Random sample of 10 primary care centres in the Murcia region. PATIENTS AND OTHER PARTICIPANTS: Patients over 19 with lipaemia detected over the previous twelve months in the general medical clinics at health centres. 500 cases in all were studied (50 per centre). MEASUREMENTS AND MAIN RESULTS: Compliance with criteria of good clinical practice was evaluated. These criteria were formulated by health centre doctors and included: adequate diagnosis, classification into primary or secondary, phenotype, recording of cardiovascular risk factors and assessment of cardiovascular (CV) risk. The differences between the centres studied and the effect of the centre's characteristics (teaching, location, existence of lipaemia programme) and of the patients' (age and gender) were also examined. We found that none of the cases evaluated satisfied the five quality criteria at the same time. Assessment of CV risk and the aetiopathogenic classification were the criteria least complied with (1.5% +/- 1.0). Centres varied considerably. Their characteristics affected the quality of the procedure evaluated, which in all centres had a lot of room for improvement. CONCLUSIONS: The quality of the procedures for diagnosing lipaemia can be considerably improved. It varies a lot from centre to centre. PMID- 10736938 TI - [Complete nutritional status in school children. Opinions and attitudes]. AB - OBJECTIVES: To discover the cooking habits of school students' families and the full nutritional picture of the school population of Huercal de Almeria. To gather the views and attitudes of school students and mothers on the low amount of milk and vegetables consumed. DESIGN: Crossover, descriptive and technical study with focus group. SETTING: Rural area. METHODS: The study had three stages: 1. A questionnaire for all the school students on their food habits (675). 2. A sample of 101 students aged between 4 and 6 and between 10 and 14 was chosen for the nutritional study. Students at nutritional risk in 1996 (95% CI; p.q = 0.25). Intake was analysed through the "memory of 24 hours", measurement of physical details and biochemical determinations. 3. We gathered views and attitudes by the focus group technique in 1997, with 60 girls between 10 and 14 and mothers in 6 groups. MAIN RESULTS: Habits: the most commonly eaten meat was chicken, followed by pork, stewed if possible. Blue and fried fish, full-cream milk, white bread, fresh green vegetables and olive oil. Nutritional study: the students' weight and height were above average. Denutrition was rare: we found three students with a percentile less than three. There was tendency to overweight in both sexes, with cholesterol levels above 200 mgr in the 4-6 group. Hyper-protein and hyper-fatty diet. The Ca/P quotient was P < 1, especially in the 10-14 girls. Consumption of pulses, fibre, cereals and root vegetables was less than the recommended amounts. Micronutrients were sufficient except for less iron in 10 to 14 girls. Focal groups: the four groups of girls coincided in rejection of green vegetables, repeating to some extent habits acquired in their families. Usually vegetables are rejected without having been tried, due to smell, texture and look. Positive knowledge about milk and vegetables was not shown in practice. CONCLUSIONS: Hyper protein and hyper-fat diet with high cholesterol levels, weight and height above the average and tendency to overweight. They all rejected green vegetables. They repeated family eating habits. PMID- 10736939 TI - [Evaluation and validation of Omron Hem 705 CP and Hem 706/711 monitors for self measurement of blood pressure]. AB - OBJECTIVE: To validate two devices for self-measurement of blood pressure--The Omron Hem-705CP and the Omron Hem 706/711--according to the revised protocol of the British Hypertension Society (BHS). The results were also analysed according to the criteria for accuracy of the revised standard of the Association for the Advancement of Medical Instrumentation (AAMI). DESIGN: The British Hypertension protocol for the evaluation of blood pressure measuring devices. SETTING: Primare care. Zone III Health Care in Albacete (Spain). PARTICIPANTS: 95 subjects to validate the Omron 706/711 and 100 subjets to Omron 705. RESULTS: Two monitors surpassed the validation according to the standars set out by the BHS protocol (705 with a A grading for SBP and DBP, 706/711 obtained a B grading for SBP and an A grading for DBP and a PASS for SBP and DBP. Upon analyzing the behavior of the appliances by subgroups of BP measures (high, mid, and low), 705 for SBP > 160 mmHg obtained a B grading for the BHS protocol, but it did not pass the accuracy AAMI criteria (SD, 8.5, but difference between observers-device is -0.1 mmHg). The rest of subgroups of BP obtained a A grading for the BHS protocol and a PASS (AAMI). 706/711 surpassed in all BP subgroups BHS protocol, for DBP 80 100, SBP > 160 and DBP > 100 with a B grading, for the rest of BP subgroups obtained a B grading and a PASS (AAMI). CONCLUSIONS: On the basis of these results, we conclude both monitors Omron HEM 706/711 and the Omron 705CP surpassed the accuracy criteria required for BHS and AAMI, and can be recommended for clinical use. PMID- 10736940 TI - [Prevalence of vaginal candidiasis in a low-risk obstetric population in Santander]. AB - OBJECTIVES: To find the prevalence of vaginal candidiasis in pregnant women monitored through primary care and to see whether it is on the increase. DESIGN: Descriptive and retrospective study of pregnant women. SETTING: Cazona Health District (34,783 inhabitants). SUBJECTS: The entire obstetric population monitored by our health centre between 1992 and 1997 inclusive. MEASUREMENTS AND RESULTS: Retrospective study of clinical histories of all the pregnant women seen at our health centre (878 pregnancies). After discounting those referred to obstetricians, abortions/miscarriages and premature births, 549 normal low-risk pregnancies (62.30% of the total) were monitored to completion in primary care. The women's age range was 16 to 40. The mean prevalence of candidiasis was 18% (CI, 15.0-21.6). Its frequency increased as the number of pregnancies increased and dropped as the age of the pregnant women increased. CONCLUSIONS: Prevalence found in our study was 18% and did not increase as the years passed. PMID- 10736941 TI - [Benzodiazepine prescription at a health center: prevalence, its use and user characteristics]. AB - OBJECTIVES: To find the prevalence of prescription of benzodiazepines (BDZ) in 1997 at a health centre (HC), and the characteristics of both their consumption and the takers. DESIGN: Crossover, observational study. SETTING: Primary care urban centre. PATIENTS: From a total of 7356 patients over 14 with clinical records and belonging to four lists, a random sample stratified by lists was selected. INTERVENTION: A form was used to gather social and demographic data, educational level, family context, linked pathologies, number of visits to HC per year, BDZ prescription and variables defining the kind of consumption. MAIN RESULTS: The prevalence of BDZ prescription was 7.7% (CI, 6-10%). Consumption profile: 33% long BDZ, 31% intermediate and 33% short. 44% consumed BDZ occasionally or for less than 2 weeks, and 42% had been taking it for over a year. For 56% (95% CI, 40-70) their G.P. was the origin of the prescription. The reason for the prescription was not specified in 42% of cases. The variables which defined the profile of the consumers, included in the logistic regression, were: sex, number of visits and linked pathologies, whose OR were: 1.57 (CI, 1.08 2.03), 1.11 (CI, 1.06-1.17) and 1.61 (CI, 1.04-2.05). CONCLUSIONS: The prevalence of BDZ prescription during 1997 in the population seen at our clinics was very similar to the figures found in other studies, with higher annual consumption averages and without the reason for taking it being specified in half the cases. The profile of BDZ takers was: mainly women, people with linked pathologies, and as a function of the number of attendances. PMID- 10736942 TI - [Epidemiology resources in the Internet for the primary care physician]. PMID- 10736943 TI - [Temporal disability: management improvements]. PMID- 10736944 TI - [Health education: reducing falls in the elderly]. PMID- 10736945 TI - [15 years of family medicine in Spain]. PMID- 10736947 TI - [Podiatry in primary care, when?]. PMID- 10736946 TI - [Novelties in the management of hypertension according to the new 1999 guide of the WHO]. PMID- 10736948 TI - [Internet and research. Postal mail versus electronic mail]. PMID- 10736949 TI - Antenatal screening for HIV infection is improving in London. PMID- 10736950 TI - Report calls for 15% cut in hospital acquired infections. PMID- 10736951 TI - New policy on the control and prevention of rabies. PMID- 10736952 TI - AIDS and HIV infection in the United Kingdom: monthly report. PMID- 10736954 TI - Meningococcal disease and the law: does non-notification really happen? AB - In Victoria, legislation clearly makes the notification of clinical or confirmed cases of meningococcal disease mandatory. Statistical modelling suggests that meningococcal disease is significantly under-notified, and that incorrect codes might be being ascribed to some in-patient episodes. The aims of this study were (i) to test the assumption that cases identified as non-notified cases were true cases, and (ii) to identify the reasons for non-detection on the hospital separation database and non-notification to the infectious diseases unit. Of 26 cases not identified on the in-patient dataset, the main causes were either being given completely incorrect ICD-9-CM codes (11 cases) or being given codes for a different type of meningitis (8 cases). Of 29 non-notified admissions, most were clinically (17) or microbiologically (6) confirmed cases, although 5 were coded in error and were not cases of meningococcal disease. Therefore, although the allocation of incorrect ICD-9-CM codes at separation was a major reason for discrepancy, non-notification was a real and recent problem. It is also possible that some clinical staff did not understand the relationship between Neisseria meningitidis and meningococcal disease, the public health implications of this infection, or the law relating to it. PMID- 10736953 TI - Rubella infection in pregnancy. AB - It is over 50 years since a syndrome of congenital abnormalities following maternal rubella infection was first recognised. Despite the potentially devastating effects of the congenital rubella syndrome, immunisation rates are not optimal and infections in pregnancy still occur. Four cases of rubella infection occurring in pregnancy are presented. Laboratory diagnosis of primary infection and reinfection is discussed, and the need for full immunisation in childhood, and of women of child-bearing age is reiterated. PMID- 10736955 TI - An outbreak of Salmonella typhimurium RDNC A045 at a wedding feast in South Australia. AB - In April 1998 an outbreak of salmonellosis amongst guests at a wedding feast was investigated. Of the 58 attendees interviewed 38 (66%) subsequently developed gastrointestinal symptoms. Stool cultures from 7 cases grew Salmonella Typhimurium RDNC A045. Food samples were culture-negative for Salmonella spp. A cohort study implicated spatchcock (RR 2.5, 95% CI 1.09-5.77) and scampi (RR 2.0, 95% CI 1.05-3.89). Temperature abuse and cross-contamination within the kitchen during preparation and cooking are likely to have been the main contributing factors to this outbreak. Control measures included staff education in safe food handling and improvements in poultry processing methods to minimise carcass contamination. PMID- 10736956 TI - Communicable diseases surveillance. PMID- 10736957 TI - [Insulin-like growth factor binding protein 1 (IGFBP-1) in vaginal secretion as a marker of premature rupture of amniotic membranes]. AB - Diagnosis of premature rupture of amniotic membranes in premature pregnancy allows to estimate dangers which threaten foetus and pregnant woman and helps to put into practice the most accurate therapeutics procedures. The aim of this study was to prove the clinical usefulness of test detecting insulin-like growth factor binding protein-1 in vaginal secretion for diagnose of premature rupture of amniotic membranes and compare the sensiviteness and specificity the above test with Gram stained vaginal smears. We examined group of 50 pregnant women between 25 and 42 week of pregnancy treated in Department of Fertility and Obstetrics Medical University of Wroclaw. Pregnant women were divided into 3 groups. In each of them both tests were performed to diagnose premature rupture of amniotic membranes. Achieved scores shows that Amni Check is specific and sensitive test detecting even insignificant premature rupture of amniotic membranes and has higher sensitivity comparing with common method used until now for detection of premature rupture of amniotic membranes based on Gramm stained vaginal secretion. PMID- 10736958 TI - [Rubella: the problem of women in procreative age]. AB - The only effective method of preventing rubella virus infection is by vaccinating women who lack the antibodies IgG-RUB, which have been proven to prevent rubella. In these study we analyze the natural seroconversion against rubella virus among women in the procreative ages of 23-30 with ELFA assay (enzymimmunofluorescence assay), using a mini-VIDAS instrument. 7.8% of 148 women tested lacked IgG-RUB. Such seronegative women should be vaccinated before pregnancy. PMID- 10736959 TI - [Bone marrow involvement in ovarian cancer by immunohistochemical assessment]. AB - In the study below we introduced certain conclusions from bone marrow analysis in patient with ovarian cancer in the presence of epithelial cells. The study included cytological bone marrow slides taken from 31 patients. We analyzed the cellular antigen expression using immuno-histological chemistry technic with NCL CA 125, NCL-EMA and NCL-C11 antibodies. In the material drived from 3 patients with advanced neoplastic changes, we detected the presence of cells that were stained positively. In order to define the actual frequency of cell presence with tested antigen expression in bone marrow as well as to signify the diagnostic and prognostic value of their detection requires a larger study group as well as using other examination technics such as PCR or transluscent photometry. PMID- 10736960 TI - [Methods of treatment of ureteral stenosis after radiotherapy in patients with gynecological tumors]. AB - Authors present methods of treatments at the Clinic of Urology Medical University of Lodz in patients after gynecological tumors with ureteral strictures caused by radiotherapy or progression of cancer. PMID- 10736961 TI - [Giant trichobezoar in the stomach coexisting with pregnancy: a case report]. AB - OBJECTIVES: Bezoars are conglomerates of undigested material in the stomach. Gastrointestinal obstructions caused by bezoars are uncommon but are encountered with increasing frequency in general surgery. STUDY DESIGN: In this case report, authors describe a trichobezoar in the stomach in 23 years old woman which was found and removed during caesarean section. The caesarean section was made in 30 week of pregnancy on account of placental abruption. RESULTS: The patient was admitted to Department of Gynecology & Obstetrics in Hospital of Slupsk in emergency service with diagnosis in second pregnancy. Doctor on duty decided immediate caesarean section on account of haemorrhage from genital tract caused by placental abruption. Female newborn weight 850 g received 5 points in Apgar scale was delivered. Next, during laparotomy was found tumor inside stomach and gastrotomy was done. The giant trichobezoar could be removed from the stomach only by surgical management. In anamnesis we found out that the woman has suffered from trichotillomania and trichophagia from childhood. The chronically swallowed hair and nail pieces an enormous trichobezoar which nearly filled the whole stomach. The possible causes of bezoar formation, the diagnostic and therapeutic problems are discussed. PMID- 10736962 TI - [Pregnancy and delivery after the transfer of one blastocyst with enzymatically removed zona pellucida]. AB - OBJECTIVES: The embryonic development up to the blastocyst stage and hatching from zona pellucida are prerequisites for implantation and successful pregnancy. It is suggested that one of the possibilities limiting the implantation rate is impaired hatching. To overcome this problem an artificial alteration of the zona pellucida have been carried out in many laboratories. DESIGN: The report of the first pregnancy in Poland obtained after the transfer of enzymatically zona removed blastocyst. MATERIALS AND RESULTS: The patient was previously treated three times in in vitro fertilisation program without success. In the fourth program the embryos were cultured in co-culture of Vero cells to the blastocyst stadium. On the day 5 after insemination the zona of cavitating and expanding blastocyst was removed by pronase. The zona free blastocyst was transferred to the uterus. As a result of implanted blastocyst the ongoing pregnancy developed normally and the patient delivered healthy baby. CONCLUSION: Enzymatic zona pellucida hatching probably increases the rate of implantation. It is simple, safe and economic techniques. PMID- 10736964 TI - [The role of adhesive molecules in pathogenesis of endometriosis. Laboratory findings and clinical aspects]. AB - Adhesive molecules have played an important and well-established role in cell-to cell communication for a long time. They reveal specific functions in an ongoing disturbed immunological reactions, including endometriosis. This review is to describe the role of topical factors involved in the pathogenesis of arising and course of endometriosis, taking adhesive molecules into special consideration. It does not only include the mechanisms and the effects of their action but also some advantages that can be gained from the knowledge of their function. The attention is also drawn to the putative role of certain proteolitical enzymes and their inhibitors which when more active can intensify or reduce the symptoms of the disease. PMID- 10736963 TI - [Aggressive clinical behavior of ovarian tumor of low malignant potential: a case report]. AB - We report a case of ovarian tumor of low malignant potential with very aggressive clinical behaviour reminded of invasive carcinoma and very advanced stage. The patient was dead in short term after radical operation although ovarian tumors of low malignant potential have excellent prognosis. PMID- 10736965 TI - [Premature rupture of amniotic membranes: a new view of pathomechanism: the role of plasmin, cytokines and metalloproteinases]. AB - The paper surveys current studies on fibrinolytic components, plasmin, cytokines and metalloproteinases in human foetal membranes, and, on the basis of new findings, reviews the mechanism of premature rupture of foetal membranes. Plasmin, which can be formed in foetal membranes, causes a transformation of procollageneses into collageneses, and thus initiates a cascade enzymatic process called a proteolytic (collagenolytic) cascade of plasmin. In the case of foetal membranes inflammation at the lower pole (ascending inflammation from the vagina and cervical canal of uterus), the so-called inflammatory cytokines can be created, initiating a local collagenolytic process, forming in that place punctum minoris resistentiae. PMID- 10736966 TI - Inclusion of piroxicam into beta-cyclodextrin by means of supercritical carbon dioxide: thermal, spectroscopic and physicochemical studies. AB - The preparation of a piroxicam-beta-cyclodextrin inclusion compound using supercritical CO2 was investigated. The solubility piroxicam in supercritical CO2 was determined. The influence of the temperature, the pressure and the time of exposure on the inclusion rate were studied and a complete inclusion was achieved by keeping a physical mixture of piroxicam and beta-cyclodextrin (1:2.5 mol/mol) for 6 hours at 150 degrees C and 150 bar of CO2. This complex was characterised by Differential Scanning Calorimetry and Fourier Transform Infrared Spectrometry. Supercritical carbon dioxide may prove to be a novel useful complexation method of non-polar drugs into beta-cyclodextrin. PMID- 10736967 TI - NIH releases draft of stem cell research guidelines. PMID- 10736968 TI - Pathogen analysis and genetic predisposition testing using microelectronic arrays and isothermal amplification. AB - BACKGROUND: A simple yet powerful tool for providing for rapid gene identification in the clinic would be the combination of isothermal gene amplification with electronic microchip analysis. This is a first report of such a union of these technologies. METHODS: The first assay demonstrates discrimination between four bacterial pathogens. For this, one portion of the bacterial 16S rRNA gene encompassing a microheterogeneous region was isothermally amplified using Strand Displacement Amplification (SDA). Type identification was then made by "sandwich" assay format either using selective electronic hybridization of amplicons to sequence-specific capture oligonucleotides and a universal, fluorescently labeled reporter oligonucleotide, or, alternatively, sequence-specific reporters and a universal capture oligonucleotide. The second assay tested for the presence or absence of the Factor V Leiden point mutation using DNA obtained from 18 patients in a blind assay. For this, allele-specific SDA was developed. Following amplification using a sense-biotinylated primer and either the corresponding antisense wild type or mutant primer, multiple patient amplicons were targeted to specified locations on the microarray and visualized using a fluorescently labeled reporter oligonucleotide. Positive signals were scored as greater than or equal to two times the background. RESULTS: Bacterial type-specific signals were between 3- to 10-fold greater than nonspecific in both assay formats. Using allele-specific SDA, 100% agreement was observed between PAGE analysis, microarray results, and clinical diagnosis in Factor V mutation analysis. CONCLUSIONS: We demonstrated two model clinical assays combining amplified materials and microelectronic arrays, one potentially suitable for pathogen screening and the other for a deleterious genetic mutation. PMID- 10736969 TI - CD2 (OKT11) augments CD3-mediated intracellular signaling events in human T lymphocytes. AB - CD2 (LFA-2) is expressed on thymocytes, natural killer cells, and virtually all peripheral T cells. CD2 binds to its primary ligand CD58 (LFA-3) on antigen presenting cells (APC) and stabilizes the T cell-APC interaction; this stable interaction then optimizes Ag-specific T-cell activation. We assessed whether CD2 cross-linking by mAb augments the process of T-cell stimulation through the TCR/CD3 complex. Plate-bound anti-CD2 or anti-CD3 mAb alone had no measurable effect on any of the assessed activation parameters of resting T cells. However, concomitant signaling through both CD2 and CD3 by plate-bound antibodies resulted in marked increases in CD69 expression on the T-cell surface and T-cell-cellular metabolism, as assessed by the ability of the cell to reduce 3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxylmethoxyphenyl)-2-( 4-sulphophenyl)- 2H tetrazolium (MTS) to formazen. In addition, simultaneous cross-linking of CD2 and CD3 caused a significant (P < 0.001) increase in phosphatidylinositol hydrolysis in resting T cells compared to stimulation with anti-CD3 mAb alone and anti-CD3 mAb plus anti-CD2 isotype control antibody. These results indicate that CD2 augments signaling through CD3, and consequently functions as a costimulatory molecule for resting T cells in the initial activation step. PMID- 10736970 TI - Vasoactive agents modulate migration of monocytes across glomerular endothelial cells. AB - BACKGROUND: Macrophages seem to play an important role in the development of glomerulosclerosis. In both human and experimental animal models of focal glomerulosclerosis (FSGS), infiltration of macrophages in the mesangium has been considered key in the development of FSGS. METHODS: In the present study, we evaluated the effect of vasoactive agents on the migration of monocytes across a filter in a modified Boyden chamber as well as across a cultured glomerular endothelial cell layer (in vitro model of glomerular mesangium). Both light as well as scanning electron microscopic studies were performed. We evaluated the effect of vasoactive agents including histamine, prostaglandin (PG) E2, angiotensin II, endothelin-1, platelet-activating factor, and interleukin-1 (IL) on the migration of monocytes/macrophages across an endothelial cell layer as well as a gelatin-coated filter. In addition, we evaluated the effect of cyclic adenosine 3',5' cyclic monophosphate (cAMP) and PGE2 on vasoactive-induced migration of monocytes. RESULTS: Histamine increased (P < 0.003) the migration of monocytes across the filter. This effect of histamine was dose-dependent. Histamine at concentrations of 10(-8) to 10(-5) mol/L induced optimal migration across the filter (control, 16.6 +/- 1.1 vs histamine, 10(-8) mol/L, 40.9 +/- 0.9 monocytes/high power field). Cimetidine, an H2 receptor blocker, attenuated (P < 0.001) the effect of histamine on the migration of monocytes. PGE2 inhibited the migration of monocytes in a dose-dependent manner. Histamine increased (P < 0.001) the passage of monocytes across the glomerular endothelial cell layer (control, 1012 +/- 37 vs 1711 +/- 163 cpm/well). Histamine also increased the migration of murine macrophages across the glomerular endothelial cell layer. PGE2 inhibited the migration of monocytes across the endothelial cell layer under basal as well as histamine-stimulated states. Dibutyryl cyclic (DBc) AMP also attenuated the migration of monocytes under basal as well as histamine-stimulated states. Both PGE2 and DBcAMP also attenuated the IL-1 beta-stimulated migration of monocytes. Angiotensin II, endothelin-1, and platelet-activating factor did not modulate the migration of monocytes. CONCLUSIONS: Vasoactive agents directly modulate the transmigration of monocytes. The present in vitro study provides a basis for a hypothesis that vasoactive agents may also be modulating the migration of monocytes across the glomerular endothelial cell layer (into the mesangium). PMID- 10736971 TI - Biotechnology stock prices before public announcements: evidence of insider trading? AB - BACKGROUND: Unique financial challenges faced by biotechnology companies developing therapeutics have contributed to the creation of a highly sensitive market, where stock prices are capable of great fluctuation. The potential for significant financial reward and the nature of the scientific review process make this industry susceptible to illegal share trading on nonpublic information. We examined stock prices of biotechnology products before and after announcement of Phase III clinical trial and Food and Drug Administration (FDA) Advisory Panel results for indirect evidence of insider trading. METHODS: Biotechnology stock prices were recorded for 98 products undergoing Phase III clinical trials and 49 products undergoing FDA Advisory Panel review between 1990 and 1998. Prices were recorded for 120 consecutive trading days before and after public announcement of these two events. We compared the average change in stock price of successful products ('winners') with unsuccessful products ('losers') before the public announcement of results for both critical events. RESULTS: The difference between average stock price change from 120 to 3 days before public announcement of results of Phase III clinical trial winners (+27%) and losers (-4%) was highly significant (P = 0.0007). A similar but non-significant difference was observed between the average stock price of winning (+27%) and losing products (+13%) before FDA Advisory Panel review announcements (P = 0.25). CONCLUSIONS: Our results provide indirect evidence that insider trading may be common in the biotechnology industry. Clinical investigators may wish to consider this issue before participating in any equity position in the biotechnology industry, especially if they are going to perform research for those companies. PMID- 10736972 TI - Role of endogenous nitric oxide in circadian blood pressure regulation in healthy humans and in patients with hypertension or atherosclerosis. AB - BACKGROUND: Nitric oxide (NO) is involved in the regulation of blood pressure and local blood flow. Its biological activity is impaired in hypertension and atherosclerosis. Because blood pressure undergoes a circadian rhythm, we investigated whether systemic NO production is dependent on a circadian variability, and whether the phasing of diurnal rhythm in NO production corresponds to the one in blood pressure in humans. METHODS: We studied three groups of human subjects: 8 healthy volunteers (HV), 8 patients with essential hypertension (HT), and 8 patients with peripheral arterial occlusive disease (PAOD). Twenty-four-hour ambulatory blood pressure monitoring was performed simultaneously with eight consecutive 3-hour urine collection periods. Urinary nitrate excretion was measured by gas chromatography-mass spectrometry; urinary cyclic GMP excretion was assessed by RIA. RESULTS: Twenty-four-hour mean arterial blood pressure was 119.8 +/- 2.0/75.8 +/- 1.5 mm Hg in HV, 145.0 +/- 6.4/94.9 +/- 2.8 mm Hg in HT (P < 0.05 vs HV), and 137.0 +/- 7.3/81.5 +/- 1.9 mm Hg in PAOD (P = NS vs HV). There was significant circadian variation in blood pressure in all groups, but daily amplitude was lower in HT and PAOD than in HV (P < 0.05); 24 hour mean urinary nitrate excretion was 183.4 +/- 27.2 mumol/mmol creatinine in HV, 102.9 +/- 18.1 mumol/mmol creatinine in HT, and 162.1 +/- 22.2 mumol/mmol creatinine in PAOD (P < 0.05 vs HV and HT). Urinary cyclic GMP excretion was 211.8 +/- 19.0 nmol/mmol creatinine in HV, 108.6 +/- 12.4 nmol/mmol creatinine in HT, and 97.9 +/- 13.4 nmol/mmol creatinine in PAOD (P < 0.05 for HT and PAOD vs HV). Circadian variation was present in urinary nitrate and cyclic GMP excretion in HV but was significantly diminished in HT and PAOD, respectively; 24-hour mean nitrate-to-cyclic GMP ratio was 0.89 +/- 0.05 in HV and 1.10 +/- 0.10 in HT (P = NS). It was increased to 2.02 +/- 0.17 in PAOD (P < 0.05 vs HV and HT). CONCLUSIONS: There is significant circadian variation in urinary nitrate and cyclic GMP excretion rates, two marker molecules for systemic NO production, in healthy humans. NO production is increased in the morning, concomitantly with the morning increase in blood pressure, indicating that NO may buffer blood pressure increase. Diurnal variation in nitrate and cyclic GMP excretion is absent in HT, pointing to impaired NO formation. The major change in PAOD is increased nitrate/cyclic GMP ratio, which points to increased oxidative inactivation of NO in this disease. Disturbed formation and activity of NO may contribute to blood pressure alterations in cardiovascular disease. PMID- 10736973 TI - Dopaminergic and cholinergic involvement in the inhibitory effect of dexamethasone on the TSH response to TRH. AB - BACKGROUND: Glucocorticoid administration is associated with reduced basal thyroid-stimulating hormone (TSH) levels and a blunted TSH response to thyrotropin-releasing hormone (TRH), despite thyroid hormone levels within the normal range. In light of the inhibitory effect of somatostatin and dopamine on TSH secretion, we examined whether this condition is caused by glucocorticoids through an increased hypothalamic somatostatinergic and/or dopaminergic inhibitory control of TSH. We measured the TSH response to TRH and serum-free T4 and T3 levels. The study group comprised 18 normal men (age 24-35) within 10% of the ideal body weight, randomly divided into 3 groups of six. METHODS: We used the antidopaminergic agent metoclopramide (MCP) and the acetylcholinesterase inhibitor pyridostigmine, which enhances acetylcholine and thus inhibits hypothalamic somatostatin release. Subjects from group 1 were tested with TRH (20 micrograms in an intravenous bolus) after placebo, dexamethasone (dex) (2 mg/day in 4 divided doses for 3 days before the experimental day), or dex plus pyridostigmine (120 mg p.o.). Subjects from group 2 were tested with TRH after placebo, dex, or dex plus MCP (2.5 mg in an i.v. bolus injection). Subjects from group 3 were tested with TRH after placebo, dex, or dex plus pyridostigmine plus MCP. RESULTS: In all subjects from groups 1, 2, and 3, TRH-induced TSH rise was significantly lower after dex than after placebo treatment. Neither pyridostigmine nor MCP, given alone, changed the TSH response to TRH after dex treatment. In contrast, the concomitant administration of MCP and pyridostigmine significantly enhanced the TRH-induced TSH rise in dex-treated subjects and made the TSH response to TRH similar to that observed in the TRH plus placebo test. CONCLUSIONS: These data indicate that enhanced-hypothalamic somatostatinergic and dopaminergic inhibitory activities are involved in the mechanism underlying the reduced TSH response to TRH induced by glucocorticoid treatment. PMID- 10736974 TI - Erythropoietin-inducible immediate-early genes in human vascular endothelial cells. AB - BACKGROUND: Patients receiving recombinant human erythropoietin (rHuEPO) may experience side effects arising from the vascular system. The underlying mechanisms, however, are largely unknown. METHODS: To elucidate downstream events following erythropoietin receptor triggering, a differential display analysis of human vascular endothelial cell mRNA was performed. RESULTS: We identified eight genes that were upregulated by rHuEPO as confirmed in two further independent cell culture experiments using a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) protocol. The genes coded for proteins that may be assigned to four different groups: 1) proteins implicated in the regulation of vascular functions (thrombospondin-1, 20 kDa myosin regulatory light chain; relative increase of rHuEPO induced mRNA levels: 155.2%, P = 0.043; 137.6%, P = 0.046, respectively); 2) gene products involved in gene transcription and/or translation (c-myc purine-binding transcription factor PuF, tryptophanyl tRNA synthetase, S19 ribosomal protein; increase of mRNA levels: 126.4%, P = 0.032; 150.9%, P = 0.012; 134.9%, P = 0.038); 3) subunits of mitochondrial enzymes related to energy transfer (NADH dehydrogenase subunit 6, cytochrome C oxidase subunit 1; increase of mRNA concentrations: 141.7%, P = 0.007; 140.3%, P = 0.01); and 4) regulators of signal transduction (protein tyrosine phosphatase G1, increase of transcript level: 160.3%, P = 0.016). CONCLUSIONS: We report on novel molecular downstream events following rHuEPO receptor triggering of human vascular endothelial cells. We identified EPO-responsive immediate-early genes, coding for proteins involved in vascular functions, gene transcription, and/or translation, energy transfer, and signal transduction. Thus, our data provide new insights into the molecular changes induced by EPO in human vascular endothelial cells. PMID- 10736975 TI - [The bioethical approach in anesthesia and resuscitation: a new resource for emerging questions]. PMID- 10736976 TI - The role of the anesthesiologist in transplantation. PMID- 10736977 TI - [Protocol for preoperative chest X-rays in elective surgery]. AB - BACKGROUND: To evaluate the effects of a protocol designed to optimize the use of preoperative chest X-rays (CXRs) in the evaluation of patients undergoing anesthesia for elective surgery. DESIGN: Observational prospective study. SETTING: General 350 bedded hospital, with main surgical branches. METHODS: a) PROTOCOL: routine CXRs can be avoided in patients aged less than 60 years, nonsmokers, without acute-chronic respiratory, cardiovascular symptoms, free from neoplastic diseases, not candidates to major vascular, abdominal or thoracic surgery, not treated with immunosuppressive therapy, nor immigrants from areas of endemic TB. b) All out patient subjects admitted to anesthesiology service for evaluation prior to elective surgery. RESULTS: Out of 5198 patients, 3795 were enrolled in the protocol; in 152 cases, preoperative CXRs were performed, 3456 patients (57.2% ASA 1; 42% ASA 2; 0.8% ASA 3) underwent surgery without CXRs. Thirty-four percent of patients had general anesthesia, 54.5% regional anesthesia, 20.6% regional-peripheral anesthesia with/without MAC. No critical events nor major complications were observed in the perioperative period in these subjects. Preoperative CXRs (performed in 152 cases) yelded useful informations with effect on the clinical management in 20 instances. CONCLUSIONS: In a context of adequate preoperative anesthesiologic evaluation, this protocol proved to be effective in reducing the number of routine preoperative CXRs in patients undergoing elective surgery. This resulted in a substantial reduction of radiation exposure both to the subject and to the general population, and costs saving, without evident negative side-effects. PMID- 10736978 TI - [The effect of antithrombin iii concentrations during cardiopulmonary surgery]. AB - BACKGROUND: Evaluation of influence of pre-op continuous e.v. heparin infusion in patients undergoing urgent myocardial surgical revascularization, on the anticoagulation threshold needed for cardiopulmonary bypass. Analysis of the efficacy of ATIII substitutional therapy to allow best ACT values during extracorporeal circulation, and to reduce intra and post-op bleeding and need for homologus transfusion. SETTING: Operative room and ICU of a cardiac surgery unit in a regional hospital. METHODS: Two groups of coronary patients in preoperative treatment with heparin were randomized in a prospective double blind study for an intraoperative treatment with heparin and ATIII (Group A) and heparin plus placebo (Group B). An investigation was made on the influence of preoperative heparin treatment regarding extracorporeal circulation, the variation of the coagulation parameters in CEC with substitutive therapy of ATIII and the reduction of the therapeutic strength of heparin during perfusion, the problem of bleeding and the incidence of blood transfusions and lastly the economic questions of the two procedures. RESULTS: The study showed the necessity of repeated bolus of heparin during CEC and the rapid loss of its effect in the group not subjected ATIII therapy. A less incidence of bleeding in Group A was observed; for this reason the patients received significantly less packed red cells and FFP and a discrete number of patients of this group were not transfused. Surely the method of using the ATIII is much more expensive from the economic point of view, but the benefits of avoiding the problems of a blood transfusion (infections, immunodepression etc.), of the reduced stay in the Intensive Care Unit, of the riduced risk involved with problems of bleeding and the need of repeated operative procedures make this method fundamental in patients with reduced plasma levels of ATIII such as coronary patients who are under heparin treatment for several days. CONCLUSIONS: Intraoperative administration of ATIII can reduce most problems due to heparinization of the extracorporeal circuit, such as onset of fibrinolysis, CID and platelets depletion or inactivation causing intra and post-op massive bleeding. PMID- 10736979 TI - [Emergence and postoperative course after anesthesia with sevoflurane versus propofol]. AB - BACKGROUND: To compare the postoperative period, with particular regard to occurrence of adverse effects at the time of emergence from anaesthesia, recovery parameters, any post-surgical analgesia requirements and laboratory tests changes in 80 ASA I and II patients undergoing sevoflurane or propofol anaesthesia for elective extracavity surgery. DESIGN: A prospective randomized clinical trial. METHODS: Patients were randomly allocated into two groups: in the first group, thiopentone was administered for induction of anaesthesia and sevoflurane for maintenance; the second group received propofol either for induction of anaesthesia and maintenance. All patients received vecuronium for neuromuscular blockade and fentanyl as needed. At the end of surgery, the occurrence of adverse effects, recovery parameters, time of discharge from recovery area, any post surgical analgesia requirements, time of walking resumption and any laboratory tests changes were recorded. RESULTS: In the sevoflurane group times of discharges from recovery area were significantly faster. In both groups total bilirubin increase was recorded until 72 hours after the end of anaesthesia. CONCLUSIONS: Significant differences of postoperative adverse effects and laboratory tests changes were not recorded in both groups of anaesthetics. PMID- 10736980 TI - [Laryngeal mask vs tracheal intubation during mechanical ventilation in pediatric anesthesia]. AB - AIM: The aim of this study was to evaluate and compare the parameters for mechanical respiration in pediatric patients undergoing controlled ventilation with a laryngeal mask (LM) and an uncuffed orotracheal tube. METHODS: The study examined 100 ASA 1 patients undergoing general anesthesia with myoresolution and mechanical ventilation using a Servo Ventilator 900 (constant flow, 25% insufflation, 10% teleinspiratory pause, tidal volume 10 ml/kg). All patients were divided into 2 groups matched for age and weight. An uncuffed orotracheal tube was used in one group (Group T) and a laryngeal mask (LM) in the other (Group M). The following parameters were measured: peak and pause pressure in the respiratory passage (Paw), total inspiratory resistance (R tot), compliance (C) and air loss expressed as a fraction of inspired volume (Vi-Ve/Vi). The statistical analysis of results was performed using Student's "t"-test and the level of significance was p < 0.05. RESULTS: Peak pressures were comparable in the two groups and were lower than the pressure needed to open the lower esophageal sphincter. These values could be further reduced by the extension of insufflation time achieved by abolishing the teleinspiratory pause included in the study to measure air resistances and compliance. Air losses were also similar in both groups, being respectively 13 and 11%. This means that environmental pollution using LM was not greater than with the uncuffed tube and confirms that, even with the latter, the protection of the airways cannot be regarded as absolute. Total inspiratory resistances were respectively 16.1 cm H2O/l/sec in group T and 15.1 cm H2O/l/sec in group M. This occurred in spite of the fact that the latter showed an in vitro capacity to oppose lower resistances compared to the corresponding tubes given that it was shorter with a larger diameter. Studies using the mechanical model did not include the laryngeal mask--larynx connection which may cause increased resistance owing to the variable position of the epiglottis, although this cannot be identified clinically. CONCLUSIONS: The laryngeal mask allows mechanical ventilation with low pressure in the respiratory passage and reduced air losses compared to the uncuffed tracheal tube. The risk of gastroesophageal insufflation is therefore minimal and artificial ventilation is reliable, if correctly performed. Lower levels of inspiratory resistance might be an advantage in spontaneous breathing owing to the consequent reduction of respiratory effort, but they do not appear to be significantly lower than with the tracheal tube. PMID- 10736981 TI - Interscalene brachial plexus anesthesia with either 0.5% ropivacaine or 0.5% bupivacaine. AB - BACKGROUND: To compare intra- and postoperative clinical properties of interscalene brachial plexus block performed with either 0.5% ropivacaine or 0.5% bupivacaine. METHODS: EXPERIMENTAL DESIGN: prospective, randomized, double-blind study. SETTING: in patient at the University Hospital, Department of Orthopedic Surgery. PATIENTS: 30 ASA physical status I-II patients scheduled for elective shoulder surgery. INTERVENTIONS: interscalene brachial plexus block was performed using the multiple injection technique and a nerve stimulator by injecting 20 ml of either 0.5% ropivacaine (n = 15) or 0.5% bupivacaine (n = 15). Postoperative analgesia consisted of 100 mg intravenous ketoprofen, if required. A blind observer evaluated hemodynamic variables as well as sensory and motor blocks from the end of injection to achieve a surgical anesthesia (readiness for surgery: loss of pinprick sensation from C4 to C7 with the inability to elevate the operated limb against gravity). The time lasting from block placement to first requirement for postoperative pain medication was also recorded. RESULTS: No differences in anthropometric parameters and hemodynamic variables were observed throughout the study, and no signs of central nervous system (CNS) and cardiovascular toxicity, or other untoward events were reported in any patients. Readiness for surgery was obtained after 28 +/- 15 min with 0.5% bupivacaine and 22 +/- 8 min after 0.5% ropivacaine (p = NS). No differences in postoperative pain relief was observed between the two groups (11.1 +/- 5 hrs after 0.5% ropivacaine and 10.9 +/- 3.9 hrs after 0.5% bupivacaine, respectively). CONCLUSIONS: This study confirmed that 0.5% ropivacaine has clinical properties similar to those of 0.5% bupivacaine, when used for interscalene brachial plexus block, providing similarly long duration in postoperative pain relief. Compared with bupivacaine, ropivacaine has the further advantage of a lower potential for central nervous system and cardiovascular toxicity. PMID- 10736982 TI - Biphasic waveforms for automatic external defibrillation in human: a review. AB - Ventricular fibrillation is the principal cause of sudden cardiac arrest and the electrical defibrillation is often the only effective therapy. A very interesting question is represented by the electric parameters of defibrillation shock. Today, monophasic waveform is widely used in Europe and in the United States, but, recently, the Food and Drug Administration grants approval for an automatic external defibrillator (AED) producing a biphasic pulse. In this review we discuss about the effectiveness and the safety of biphasic waveform, by examining a series of human studies between 1982 and 1999. We have found that available data are often incomplete, unclear, dishomogeneous and, consequently, difficult to compare. Furthermore, among the authors there is no concordance about the meaning of "safety", "effectiveness", "success", "equivalence" and "superiority" of biphasic versus monophasic shock: however, biphasic shock, that uses a lower energy level, seems to reduce post-defibrillation heart damage. Due to the lack of homogeneous studies it is not possible to state which kind of signal is more reliable, even if some clinical reports and experimental data seem to tribute to the biphasic waveform a better therapeutic effectiveness and safety. By examining the current scientific literature, we conclude that further studies have to be performed to definitively validate the use of biphasic shock. PMID- 10736984 TI - Presumptive delayed gas embolism after laparoscopic cholecystectomy. AB - A 50-year-old woman, with a history of arterial hypertension treated with beta blocker and Ca-antagonist, presented cardiac arrest 6 hours after elective laparoscopic cholecystectomy. During surgical intervention, arterial hypotension without any respiratory change was observed. Dyspnea, asthenia and anxiety were the clinical signs appearing approximately 2 hours before cardiac arrest. After resuscitation, myocardial infarction, dissecting thoracic aortic aneurysm and major pulmonary thromboembolism were excluded. The signs of increased resistance to the right ventricular outflow and the relevant alteration of coagulation tests, lasting only a few hours, suggested venous gas embolism. Subsequently, the patient presented a cortical blindness, persisting at hospital discharge. The anesthetists should be aware about the complication that we observed after laparoscopic surgery. The least sign of cardiorespiratory instability appearing in the postoperative period must be taken into account and signal the need for increased monitoring. PMID- 10736983 TI - [Fluoxetine versus fluvoxamine for treatment of chronic pain]. AB - AIM: This study aimed: 1) to compare the analgesic efficacy and profile of two antidepressants with the same mechanism of action (SSRI: selective serotonine reuptake inhibitors): fluoxetine vs fluvoxamine; 2) to investigate the relationship between analgesic efficacy and antidepressant effects in both drugs; 3) to evaluate the relationship between the analgesic profile and the quality (global or neuropathic) of pain. METHODS: Fifty-three depressed patients were randomly treated with 20 mg/die of fluoxetine and 100 mg/die of fluvoxamine for chronic pain. Forty subjects (20 with fluoxetine and 20 with fluvoxamine) completed the 2-month study and were followed up on day 14, 28 and 56 of treatment. The intensity and quality of pain was assessed using Quid and depression-anxiety symptoms with the Hamilton Rating Scale (HAMD). RESULTS: The intensity and overall quality of pain deteriorated at day 14 in the fluvoxamine group and improved in those treated with fluoxetine. However, a comparable level of analgesia was achieved with both drugs at 2 months. After 2 weeks the neuropathic component of pain improved in patients treated with fluvoxamine. The improvement in pain observed in patients treated with fluoxetine depended on an improvement in depressive symptoms, whereas this relationship was not observed in the fluvoxamine group. CONCLUSIONS: Although a comparable level of analgesia was observed after two months of treatment, the two drugs show different analgesic profiles. Their analgesic action appears to depend on different mechanisms. PMID- 10736985 TI - [A case of tetanus: the problem of differential diagnosis]. AB - A clinical case concerning differential diagnosis between tetanus, atropine poisoning and acute hypocalcemia is reported. A 51 year-old man has been hospitalized in ICU, coming from the emergency service of another hospital, with a diagnosis of suspected atropine poisoning (he had been under treatment with atropine collyrium 1% for same days). The patient at the moment of hospitalization presented: preserved coscience with good orientation in time and space, thrismus, slight nuchal rigidity, hypertonia to the inferior limbs, accentuated osteotendinous reflex to the four limbs, asthenia, intense perspiration, tachycardia, apyrexia and not appreciable ocular signs for previus pathology. At observation the patient showed to have had a thyroidectomy (presence of surgical scar), and he didn't remember to have been vaccinated against tetanus. Several small scars to the hands were observed (particularly a recent felon to the first finger of the rigth hand) all referable to his activity as agriculture laborer. The hematochemical examinations were performed and the slight hypocalcemia slightly laver than normal, the leukocytosis neutrophilia, apyrexia, abundant perspiration and preserved conscience in presence of thrismus and hypertonia to the inferior limbs led to the diagnosis of a possible case of tetanus. PMID- 10736986 TI - [The medical deontological code and ethical questions regarding the end of life. A contribution to clarity from anesthesiologists and intensive care personnel]. AB - The Medical Deontological Code (MDC) discusses ethical questions regarding the end of life, which often require anesthetists and intensive care operators to take decisions regarding patients with terminal diseases in Article 14: Intensity of diagnostic-therapeutic procedures under heading IV (Diagnostic and therapeutic procedures) and Article 37: Caring for the terminally ill under heading V (Caring for the terminally ill). The original formulation of Article 37 prompted immediate dissent among numerous anesthetists-IC operators and bioethics experts who signed a petition addressed to the Permanent Commission for the Revision of the Deontological Code in which they asked of Article 37 and proposed a reformulation. In this paper the authors outline the arguments used to back up this requests and its broad acceptance by the Commission, as shown by the amendments made to Articles 37 and 38 of the MDC and the clarifications given un the Commentary to the MDC approved on 1/9/99. These amendments correct a deontological regulation whose original formulation appeared to be contradictory and inapplicable to the terminally ill patients. This matter clearly shows the importance of bioethical questions facing. Anesthetists and Intensive Care operators and underlines the need for reflection on these themes within the profession and a more active participation in the general debate on ethical and deontological aspects of the medical profession. PMID- 10736987 TI - [Palliative surgical treatment of thoracic esophageal cancer]. AB - BACKGROUND: Esophageal carcinoma is frequently diagnosed at an advanced stage, therefore most patients may only benefit from surgical or endoscopic palliation. METHODS: From 1982 to 1998, out of 247 patients who underwent palliation for thoracic esophageal carcinoma, 29 (11.7%) underwent surgical palliation. Eight received a palliative resection, 10 a bypass, 5 a jejunostomy and 6 a gastrostomy. A retrosternal transposition of the stomach (17 patients) and colon (1 patient) was performed. In 15 out of 29 patients palliation was decided during surgical exploration. All resected patients underwent postoperative radiotherapy (400 Gy) while 3 received also preoperative chemotherapy (PDD and % FU). RESULTS: Seven temporary neck fistulas and 9 cardiorespiratory complications were recorded. Two patients (11%) died of severe cardiac and respiratory insufficiency. Mean survival for resected patients was 12.5 months (range 3-21), higher than for bypass (11.5 months; range 3-18) and for jejunostomy or gastrostomy (5 months; range 2-12). CONCLUSIONS: Palliative resection, when technically feasible, is the treatment of choice in advanced thoracic esophageal carcinoma in selected patients. The type of procedure to be performed depends on site, extent of the disease and surgeon's experience together with the quality of life expected by the patient. PMID- 10736988 TI - [Acute biliary pancreatitis. Therapeutic trends]. AB - BACKGROUND: Treatment of biliary pancreatitis includes suppression of the biliary cause by cholecystectomy and common bile duct clearance. Endoscopic retrograde cholangiopancreatography (ERCP) and endoscopic sphincterotomy for eradication of biliary stones and laparoscopic cholecystectomy (L.C.) for residual gallbladder stones would be ideal but were once considered to be contraindicated by most surgeons. The timing of definitive biliary tract surgery and the role of ERCP have been the focus of discussion in recent years. METHODS: During a two-year study period 51 patients with acute biliary pancreatitis were studied. Seven patients (14%) underwent emergency laparotomy, necrosectomy, cholecystectomy, exploration of the common bile duct and T-tube insertion, because unstable clinical conditions, with evidence of pancreatic and peripancreatic necrosis on CT-scan. Elective open cholecystectomy and CBD exploration were performed in 7 patients after the resolution of acute pancreatitis during the same hospital admission. RESULTS: Early ERCP and L.C. were associated with favourable outcomes. 33 patients underwent ERCP preoperatively: 17 within 72 hours of admission and 16 after signs of clinical improvement. Laparoscopic cholecystectomy performed 3-25 days after admission was successful in 27 of 29 patients. Postsphincterectomy bleeding occurred in one patient and was treated successfully by endoscopic epinephrine injection. For median hospital stay and recurrence there were statistical differences between early and delayed ERCP. CONCLUSIONS: ERCP and sphincterectomy have a certain role in conjunction with laparoscopic cholecystectomy in the management of patients with acute biliary pancreatitis, particularly in institutions where there is easy access to expert interventional endoscopic techniques. This policy should reduce the risk of cholangitis and recurrent pancreatitis. PMID- 10736989 TI - [Perforation of the small intestine]. AB - BACKGROUND: Small bowel perforation is a major problem in abdominal typhi disease, but is seldom observed in Italy, as Salmonella typhi infections are rare in this Nation. The cause of perforation varies greatly. The reported mortality is high and varies from 23 up to 42%. A retrospective study has been performed in order to find how to improve the outcome. METHODS: A series spanning 10 years is reviewed, from January 1, 1987 to December 31, 1997, comprising 60 patients with small bowel perforation, operated in a urgency setting in the Operating Room of the Emergency Department of the Molinette Hospital in Torino. Resection and primary anastomosis were utilized in 33 patients, 27 underwent oversewing. In 3 patients a colostomy was felt necessary because of a concomitant damage of the colon. RESULTS: No leakages occurred. Hospital stay varies from 1 day to 76 days (24 days mean). Mortality is consistent with literature: 20 patients (33%) but the cause is related to the primary diseases of the patients. Delay in diagnosis did not affect the patient's outcome. CONCLUSIONS: In conclusion, it is confirmed the one-time surgery as the choice treatment in small bowel perforations from causes other then S. typhi infection. Mortality is not directly related to the consequences of surgical repair. PMID- 10736990 TI - [Meconium ileus. A clinical contribution]. AB - BACKGROUND: Meconium ileus (MI) is a form of neonatal intestinal obstruction due to an abnormal thickened meconium within the terminal ileum. The aim of this retrospective paper is to review our experience with neonates affected from MI treated at our institute over a twenty year period. METHODS: This report deals with 23 neonates with MI: 14 newborns had uncomplicated MI due to obstruction of the terminal ileum with meconium pellets, while 9 presented with complications (intestinal atresia, volvulus, pseudocyst, peritonitis). RESULTS: A water soluble contrast enema (Gastrografin) was attempted in 12 cases with a success rate of 7/12 (58%). The remaining sixteen neonates underwent laparotomy, with 4 treated by resection and primary anastomosis, 7 by enterostomy (chimney or double barrelled) and 5 managed with T-tube enterostomy. Survival rate was 93% in uncomplicated MI and 67% in complicated forms. CONCLUSIONS: On the basis of personal experience the authors suggest that the treatment of choice for uncomplicated MI is Gastrografin enema, with T-tube enterostomy to be reserved for enema failure. The surgical treatment of the complicated forms depends on the intra-abdominal findings; nowadays chimney or double barrelled enterostomy is to be reserved in cases where peritonitis, late diagnosis, prematurity or associated anomalies complicate the disease. PMID- 10736991 TI - [Total thyroidectomy in surgical treatment of euthyroid goiter]. AB - BACKGROUND AND AIMS: The aim of this study was to compare the experiences and results achieved by a single surgeon using total thyroidectomy and partial exeresis in the treatment of uni- and multinodular euthyroid goiter. METHODS: The results of two groups of operations performed at two different periods were analysed: a more recent group (1995-97) in which management tended to prefer total thyroidectomy, and a retrospective group (1984-1994) in which a more conservative attitude was adopted to benign thyroid pathology. RESULTS: An analysis of the results and complications in each group showed that there were no statistically significant differences between the percentage of recurrent and parathyroid lesions during total thyroidectomy and partial exeresis. In practice, the risk of these lesions is higher during redo surgery for cancer and/or recurrence. The recidivation of goitrogenic pathology is high, amounting to around 29% of total cases. The most significant findings, however, which further justifies the use of total thyroidectomy is the increasingly frequent observation of "occult carcinomas" within the benign pathology, registered in the retrospective group (13.27%) and in the more recent one (12.35%). CONCLUSIONS: On the basis of this analysis, the authors confirm their support for the use of total thyroidectomy on principle as the correct and rational treatment for euthyroid goiter, and reserve the use of conservative treatment for single nodular lesions where it is possible to perform a correct hemithyroidectomy and isthmectomy following the patient's informed choice. PMID- 10736992 TI - [Role of ultrasonography in the control of patients receiving perioperative total parenteral nutrition]. AB - BACKGROUND: On the basis of personal experience and literature, this study has been carried out in order to clarify the impact of ultrasonography on the diagnosis and prevention of hepatobiliary complications Total Parenteral Nutrition (TPN)-related. METHODS: Between April 1995-April 1997 in the Department of General Surgery-University of Catanzaro, 18 adult patients submitted to major thoracic or abdominal surgery, underwent to TPN for perioperative time (range: 9 55 days). In these patients ultrasonography was performed at pre-established days in pre- and postoperative time in order to diagnose precociously the possibility of hepatobiliary complications. RESULTS: Ultrasonography showed: 3 cases of hepatic steatosis, 5 cases of steatosis and gallbladder biliary sludge, 2 cases of gallbladder biliary sludge alone, 1 case of gallbladder biliary sludge with microlithiasis, 1 case of acute acalculous cholecystitis. The other 6 patients presented only little laboratory tests alterations, but normal ultrasonographic findings. CONCLUSIONS: Therefore it is underlined the importance of systematic ultrasonographic follow-up of patients on TPN as a non invasive method in the early diagnosis of hepatobiliary complications TPN-related. The good compliance of the patients to this instrumental investigation and the not excessive increase of their management's costs are also underlined. PMID- 10736993 TI - [Surgery of dysphagic diseases. Personal experience (349 surgical interventions)]. AB - The authors report their surgical experience relating to dysphagic diseases of the esophagus (349 cases). In the light of these results, they describe the different surgical techniques used in the various pathologies: 1) Esophageal diverticula: The value of a careful evaluation of subdiverticular spasm is emphasised using preoperative manometry in cervical and epiphrenic diverticula, leading to subdiverticular myotomy when present. 2) esophageal achalasia and intermediate motor disorder: A clear difference must be drawn between these two diseases owing to the different motor behaviour of the esophagus. Dilatation of the LES is only useful in intermediate motor disorder and should be avoided in esophageal achalasia where a gastroesophageal reflux is produced if dilatation fails. Intraoperative manometry is very useful during the extramucosa myotomy phases as an indication of the complete removal of the sphincteric barrier, thus avoiding the risk of persisting disease. 3) Non-neoplastic stenosis. In primary stenosis (caused by caustic agents, primary GER or associated with JE) a conservative approach is advisable, whereas in iatrogenic stenosis (mainly linked to dilatation or cardiac surgery), owing to the anatomic complexity of the esophagogastric junction, a more radical approach is often required in the form of esophagogastric resection or even sub-total esophagectomy. 4) Neoplastic stenosis: Leiomyomas, although unusual, represent a clear indication for thoracoscopic access, provided that the dimensions allow it. Esophageal cancers represent a major surgical problem. A radical approach is represented by TE and the subsequent use of the stomach, or more rarely, the colon to reconstruct the alimentary tract. In spite of the very low resectability rate owing to locoregional spreading, until recently palliative surgery was essential to allow patients to eat. The introduction of autoexpanding prostheses, positioned using endoscopic methods, has provided a better solution to this problem. PMID- 10736994 TI - Pruritus ani. AB - The possible dermatological and proctologic causes, of Pruritus Ani that often, due to its persistence and for its consequent lesions of scratching, becomes a real disease, are analysed. Particular attention is given to therapeutical treatment, describing which hygienic-dietetic rules must be followed and which treatments can be utilised, if the cause of the symptom is known and if it is possible to remove it or not. Moreover, personal experience on 312 patients examined in twelve years is reported. PMID- 10736995 TI - [Hurthle cell carcinoma and "insular" carcinoma: uncommon varieties of thyroid carcinoma]. AB - The Hurthle cell carcinoma and "insular" carcinoma are two uncommon types of thyroid carcinoma. These neoplasms do not present accurate characteristics as to incidence, natural history, pathophysiology and therapies. Two cases are presented: the first was submitted to near-total thyroidectomy for Hurthle cell carcinoma, the second to total thyroidectomy for "insular" carcinoma with bone and lung metastases. Some literature previous studies on pathophysiology and natural history of both tumors are discussed. At the end, personal trend on the surgical treatment of these two thyroid neoplasms is presented: total thyroidectomy for CCh and "insular" carcinoma, and near-total thyroidectomy for Hurthle cell adenoma. PMID- 10736996 TI - [Use of vascular endostapler in surgery of the lower rectum]. AB - A practical and effective method for rapid and bloodless preparation of the rectum using endovascular stapler devices during low anterior resection, or abdominal-perineal excision, is described. This method is presented as an effective means for easily dividing the anterior and lateral attachment of the rectum. The application of this technique is the absence of intraoperative bleeding related to injury of middle hemorrhoidal vessels, with minimal risk of autonomic pelvic nerve damage. An additional factor relevant in the choice of this technique, is the easier possibility to perform rectal dissection of an oncologically adequate tumor clearance from the margin of rectal tumor and with complete radical transection of the lateral ligaments fastly proceeding with the downward mobilization of the rectum close to the pelvic side walls, between the parietal and visceral layer of the pelvic fascia. PMID- 10736997 TI - [Cicatricial stenosis of colorectal anastomosis. Transanal treatment with circular stapler]. AB - Anastomotic strictures complicating colorectal anastomoses can be difficult to treat. This condition must not be considered as an uncommon complication. In 20% of patients it may be a serious state that may require a therapy. Two patients treated successfully without complication with the transanal use of an CEEA stapler are presented. The staple cutter is safe and easy to use, and except for a conventional anoscope, no special equipment, including fluoroscope, is needed. On the basis of the successful results obtained, the procedure using staple cutter is recommended for the treatment of anastomotic stricture of the rectum. PMID- 10736998 TI - [Hemorrhoids and conservative treatment. Review of the literature on the use of diosmin and micronized hesperidin]. PMID- 10736999 TI - [History of surgery in Preci better known as surgeons of Norcia]. AB - This paper makes a small contribution to a more detailed knowledge of the history of Italian surgery. Few know that Italy can boast a category of surgeons who should be regarded as the world forebears of modern surgery. These skilled individuals are better known as "Norcini", a term which is often regarded as negative and contemptible. However, in practice, they came from Preci and were only called "Norcini" because their village formed part of the "contado" of Norcia. Preci still possesses a large collection of surgical instruments and other items which testify how the art of surgery developed from this small centre. Extensive research has led to the reconstruction of a general picture of this phenomenon and its influence in Italy and throughout the world. These surgeons were the only ones capable of operating gallstones, cataracts and inguinal hernia. They developed specific instruments and were the first to introduce cauterization and narcosis. The latter enabled them to overcome almost all the factors which limited surgery at the time, namely infection, pain and hemorrhage. This study attempts to explain how and why such skillful surgeons were to be found in such a small and isolated area of Umbria. PMID- 10737000 TI - [Alcohol intake and nutrition]. AB - This article contains summaries of our studies carried out at the society of the research "alcohol and health" with discussions on some related studies. Items included: (1) Discussions on "could alcoholic beverage regard as a nutrient?". (2) Nutrients in alcoholic bevarages. Distilled alcoholic beverages contain little nutrients except energy, while brewered alcoholic beverages contains nutritionally significant amounts of magnesium, niacin, and vitamin B2. (3) Dietary habits and alcoholics. Survey studies on researchers working at a brewing industry revieled that positive correlations were observed between intakes amounts of alcoholic beverages and intakes amounts of pulses, fishes, eggs, and seasonings and spices. While, negative correlations were observed between intake amount of confectioneries, fruits and daily products, and intake amount of alcohol. As nutrients, intakes of energy and sodium increased and intakes of dietary fibers, niacin, vitamin C, carotene, and zinc decreased in proportion to increase in alcoholic intakes. (4) Effect of alcohol intake on metabolism of nutrients. To clarify the influence of alcohol intake on nutrients metabolism, our research group carried out several animal experiments. Thiamin status evaluated by blood thiamin level and erythrocyte transketolase activity a thiamin dependent enzyme, decreased significantly by excess administration of alcohol. Effect of alcohol on metabolism of zinc, a cofactor of alcohol dehydrogenase, was not significant in our experiments, although other researchers reported that zinc metabolism was influenced by alcohol intake. In addition, we found that copper concentration in liver decreased significantly in alcohol administered rats as compared to control rats. The mechanisms concerning alcohol intakes on copper metabolism remains to be clarified. PMID- 10737002 TI - [Treatment of Bell's palsy with acyclovir and prednisolone]. AB - Many current studies have suggested that herpes simplex virus is a probable cause of Bell's palsy, and that treatment with antiviral agents such as acyclovir might benefit the patients. In the present study, 69 patients with Bell's palsy were treated with oral administration of acyclovir (2000 mg/day) and prednisolone (60 40 mg/day) at Ehime University Hospital between Oct. 1995 and Dec. 1998. Patients enrolled in this study met the following criteria: 1) severe or complete paralysis with a score lower than 20 by the 40-point Japanese grading system, and 2) treatment started within 7 days of onset. The overall recovery rate was 95.7% (66/69). The rate in patients who started this treatment within 3 days after disease onset was 100%, and this early treatment was highly efficacious in the prevention of nerve degeneration and resulted in a significantly better recovery. By comparison, the recovery rate in patients whose treatment was started 4 days or more after onset was only 84.2%. All patients who were given a diagnosis of zoster sine herpete and treated with acyclovir-prednisolone had a good outcome. These results suggest that early treatment, within 3 days after palsy onset, is necessary for effective acyclovir-prednisolone therapy of Bell's palsy. PMID- 10737001 TI - [Simultaneous administration of ethanol emphasizes the effect of methamphetamine on central nervous system in rat with high alcohol preference]. AB - We studied about influence of alcohol preference and of simultaneously administered ethanol (EtOH) on the effect of methamphetamine (MA) on the central nervous system neurochemically and behavioral pharmacologically using two strains of rat with high or low preference for alcohol (HAP and LAP). In the neurochemical study, we determined dopamine (DA) and serotonin (5-HT) level in striatum and nucleus accumbens (N.Acc) by means of brain microdialysis after administration of MA 1 mg/kg alone (LAP-MA group, HAP-MA group) or EtOH 2 g/kg + MA 1 mg/kg (LAP-EtOH/MA group, HAP-EtOH/MA group) every 20 minutes for 10 sessions. In the behavioral pharmacological study, we observed rat's behavior in the open field and performed scoring according to Locomotion-Stereotypy (L-S) rating scale by Ellinwood every five minutes, and counted locomotion and rearing by means of infrared beam cutting every 20 minutes after administration of MA 1 mg/kg alone (LAP-MA group, HAP-MA group) or EtOH 2 g/kg + MA 1 mg/kg (LAP-EtOH/MA group, HAP-EtOH/MA group). When MA was given alone, HAP rat showed no significant difference in locomotion, rearing and L-S score compared to LAP rat, although HAP rat showed significantly lower elevation of DA and 5-HT in both striatum and N.Acc. When EtOH was simultaneously administered with MA, in LAP rat, DA and 5-HT elevation in the striatum and N.Acc showed no significant differences between LAP EtOH/MA group and LAP-MA group. Locomotion and rearing reduced and L-S score temporarily reduced significantly in LAP-EtOH/MA group compared to LAP-MA group. However in HAP rat, HAP-EtOH/MA group showed significantly higher DA and 5-HT elevation in both striatum and N.Acc, and also showed significantly higher L-S score and higher locomotion count compared to HAP-MA group. Our results indicate that effect of MA may be influenced by alcohol preference, and that simultaneous administration of EtOH emphasizes the effect of MA on central nervous system in rat with high alcohol preference. PMID- 10737003 TI - [Simple evaluation of CT findings in the paranasal sinuses for chronic sinusitis]. AB - The CT scores and scoring for improvement based on them, which we proposed previously, is a simple and highly reproducible method of evaluation of sinus units before and after an operation for chronic sinusitis. We compared this evaluation method with the results of quantitative assay and showed its advantages and disadvantages. The subjects were 258 sinuses in patients who underwent endonasal sinus surgery (ESS) in the department of otolaryngology, Kyoto Prefectural University of Medicine Hospital from April 1996 to April 1997. The subjects were evaluated according to the following 4 grades negligible shadow in the paranasal sinus CT scored 0, less than 50% shadow scored 1, more than 50% of shadow scored 2, and mostly filled with shadow scored 3. Furthermore, the preoperative and postoperative CT scores were compared and the rate of improvement was rated in the following 3 grades: score 0 for unchanged or aggravated subjects, score 1 for subjects showing improved CT score by 1 grade, and score 2 for those showing improved CT score by 2 grades or a postoperative CT score of 0. Quantitative image analysis was input into a personal computer and the ratio occupied by the shadow was calculated, as the shadow ratio. While some discrepancies were seen in parts in the comparison of the quantitative image analysis and CT scores as the former captures minute shadows, a positive correlation was obtained overall. Attention is needed to accurately evaluate small paranasal sinuses such as the frontal sinus, and small amounts of shadow, which are areas where errors may occur. A satisfactory correlation was obtained between the score for the improvement rate and the difference in the shadow ratios before and after surgery. The CT scores and the scores for the improvement rate showed no difference from the results of other evaluation methods reported in the past, and evaluation of similar precision was possible. It was thought that this simple evaluation method of CT findings in the paranasal sinuses, which we examined in the present study, was quite useful as a simple stage classification method that could be utilized in everyday practice considering its facility, reproducibility and satisfactory precision. PMID- 10737004 TI - [Laser arytenoidectomy for bilateral vocal fold paralysis]. AB - Laser arytenoidectomy can be performed via an intralaryngeal approach which preserves airway and voice quality without aspiration. Laser arytenoidectomy is minimally invasive surgery, and a useful surgical procedure for bilateral vocal fold paralysis. CO2 laser arytenoidectomy was performed for 12 cases of bilateral vocal fold paralysis. Recommended methods for this surgical procedure are: 1) Submucous laser arytenoidectomy should be done. 2) To widen the posterior glottis, not only the arytenoid cartilage but also the posterior part of the thyroarytenoid muscle should be removed. 3) Membranous portions of the vocal folds should not be vaporized. 4) The wound should be covered with mucosa with fibrin glue. PMID- 10737005 TI - [Tyrosine kinase expression in the inner ear of mice]. AB - Tyrosine-specific phosphorylation events serve to modulate cell to cell interactions during the normal growth and development of many vertebrate tissues. Numerous studies have been performed on the involvement of growth factor receptor tyrosine kinases (RTKs) in inner ear function. Many RTKs, however, have only been recently identified, and their expression patterns have not yet been systematically examined in all tissues. In this study, we surveyed mouse inner ear transcript sequences for the presence of RTKs using a degenerate-PCR based methodology. Isolates encoding RTKs that had and had not been previously identified in the inner ear were obtained. Our data reveals the presence of numerous RTKs in the inner ear that have not yet been studied in the context of inner ear and demonstrates the usefulness of RT-PCR analysis in identifying genes involved in inner ear function. PMID- 10737006 TI - [Parathyroid function after total or subtotal thyroidectomy]. AB - Postoperative parathyroid function was evaluated in 24 total thyroidectomy and 8 subtotal thyroidectomy patients seen by our department between January 1995 and July 1997. Parathyroid function was assessed by measuring the level of serum intact parathyroid hormon (intact-PTH). Hypoparathyroidism was avoided in 23 patients (95.8%) who received a total thyroidectomy and in 7 patients (87.5%) who received a subtotal thyroidectomy. Supplementary therapy for hypoparathyroidism was not required as long as the blood supply to more than two parathyroid glands was preserved. Half of the patients in this study did not require any postoperative supplementary therapy. Thus, the preservation of more than two parathyroid glands is essential for the prevention of hypoparathyroidism. In cases where the parathyroid glands had been resected, parathyroid gland transplantation were performed. In all cases, supplementary therapy was eventually no longer required. In two cases requiring supplementary therapy, a normal range of parathyroid activity was observed 30 months after surgery. The administration of vitamin D3 may suppress the recovery of parathyroid function in patients recieving parathyroid transplantations. PMID- 10737007 TI - [The possible role of adhesion molecule, alpha 3 integrin, in the synthesis of intracrescentic extracellular matrix in accelerated anti-GBM nephritis]. AB - Studies were performed on the behavior of crescent-composing cells of crescent glomerulonephritis induced by the injection of anti-GBM antibody to WKY rats. alpha 3 integrin, podocalyxin and ED1 were stained immunohistochemically as markers of parietal, visceral epithelial cells, and macrophages, respectively. Cellular fibronectin (cFn) and type I collagen were also stained in the same manner. Fluoresceine- and Rhodamine-conjugated IgGs were used as second antibodies for double staining to show the geographic relation of the two target molecules. Morphological examinations were performed at day 7, 14 and 28 after disease induction. Periodic acid-methenamine-silver (PAM) and Azan staining showed time-dependent increase in extension and cellularity. PAM-positive intracrescentic fibers were likewise increased, which was related to an increased frequency of ruptured capsules. Intracrescentic cFn increasing in parallel with PAM-positive fibers and type I collagen, was remarkably stained at day 28. Intracrescentic ED1-positive cells showed also a significant increase at day 28. Podocalyxin was not stained in the crescent, while ED1 and alpha 3 integrin were remarkably stained. Double staining showed apposition of cFn and alpha 3 integrin, indicating close contact of these molecules, but such apposition was not observed between ED1 and cFn. These findings indicate that ED1 and alpha 3 integrin--(possibly visceral cell derived) positive cells occupy significant cellular components of the crescent and that the latter plays a part in synthesis of the intracrescentic extracellular matrix. PMID- 10737008 TI - [Natriuresis and blood pressure in patients with chronic renal failure following L-arginine infusion]. AB - Nitric oxide (NO) is known to be generated from L-arginine and may regulate glomerular filtration, tubular sodium reabsorption, and renin secretion. Impairment of renal function might influence NO production secondary to endothelial dysfunction, decreased NO synthesis and increased activity of arginine analogues inhibiting NO synthase. In this study, we evaluated the effect of L-arginine on the blood pressure and urinary sodium excretion in patients with chronic renal failure. A 300-ml dose of 10% L-arginine solution was administered intravenously over 30 min and blood pressure was monitored every 10 min under basal conditions and for 120 min after infusion. The patients were divided into two groups based on the reduction in mean blood pressure (dMBP) following infusion, namely non-responders (dMBP < 10 mmHg) and responders (dMBP > 10 mmHg). Urine and blood samples were collected to determine electrolytes, urinary NO2 + NO3 by the Griess method, urinary cGMP, plasma renin activity (PRA), and the plasma aldosterone concentration (PAC). L-arginine significantly decreased MBP in 8 patients and caused no significant change in 10 patients. Urinary sodium excretion and the NO2 + NO3 level were significantly increased following L arginine infusion and the increment of fractional excretion of sodium was higher in responders. However, there were no significant changes in PRA, PAC, and cGMP. Our findings suggest that a vasodilator effect of NO induced by L-arginine loading may, at least in part, be associated with increased renal sodium excretion in patients with chronic renal failure. PMID- 10737009 TI - [Analysis of glomerular anionic charge status in renal biopsy specimens of childhood minimal change nephrotic syndrome using confocal laser scanning microscopy]. AB - Analysis of glomerular anionic charge in human renal biopsy specimens has been restricted previously to staining of sites at the electron microscopic level, which is a product that needs skills and precludes a wide observable area. The introduction of a new tool, confocal laser scanning microscopy together with FITC conjugated poly-L-lysine as a cationic tracer, which demonstrates fixed anionic sites in thin sections from routinely formalin-fixed and paraffin-embedded renal biopsy tissue, has now enabled glomerular charge at light microscopic level. In this method, the patterns of staining in tissue showing minimal change nephrotic syndrome (MCNS) indicate that the intensity of anionic charge in 4 children with heavy proteinuria was significantly less than that in 7 children without proteinuria at remission, supporting previous observations using electron microscopy. Furthermore, staining the serial sections after methylation or saponification revealed that carboxyl components such as sialic acid may be responsible for proteinuria. We anticipate that this method may facilitate the investigation of the participation of charged components in the pathogenesis of MCNS and their role in relation to glomerular proteinuria. PMID- 10737010 TI - [Usefulness of the low protein rice on the diet therapy in patients with chronic renal failure]. AB - Diet therapy for patients with chronic renal failure is based on low protein and high energy. To achieve strict diet therapy, it is necessary to use specially formulated low protein foods. Previously, rice with a low content of protein was not available, but recently, a low protein rice (LGC-1: low glutelin content-1) has been developed. LGC-1 was found to have a low content of glutelin in its seed protein compared to other ordinary rice. Glutelin is the major digestive protein in the rice grain. We studied the usefulness of LGC-1 in the diet therapy of patients with chronic renal failure. Twenty-three patients were placed on the low protein diet (0.6-0.9 g/kg/day) during the pre-study period (a mean of 10 months). Subsequently they were followed with the same diet using LGC-1 for the staple foods during the study period (mean of 7 months). Protein intake and the slope of the reciprocal of serum creatinine did not differ between each study period in all patients. Among the 23 patients, 9 consumed rice mainly as the staple food (120-180 g/day as polished rice: rice group), according to the results of a questionnaire. In the rice group, protein intake decreased (from 47 +/- 9 to 42 +/- 9 g/day, p < 0.05), and the slope of the reciprocal of serum creatinine reduced (from -4.59 +/- 4.33 to -1.47 +/- 3.51 x 10(-4) dl/mg/day, p < 0.05) during the study period, compared with the pre-study period. We conclude that LGC-1 is a useful and effective food for a low protein diet in patients with chronic renal failure, especially, for those who consume rice mainly as their staple food. PMID- 10737011 TI - [Phosphate kinetics in patients on chronic hemodialysis]. AB - To investigate the phosphate kinetics in hemodialysis (HD), 8 patients in a stable condition, who were receiving HD three times a week for 4 hours per session, were investigated. Plasma phosphate was under 7 mg/dl, and residual renal function had almost disappeared. Dialysate containing phosphate was prepared by adding Na2HPO4 using a micro-infusion pump from the inlet of single pass dialysate in the individual dialysate delivery system. In the first week, Na2HPO4 was not added as the control period. In the second session of the second and third week, Na2HPO4 was added to give a phosphate concentration of 1.0 and 2.0 mmol/l in the dialysate, respectively. Total phosphate mass removal was 777 +/- 46.64 mg in the control period, 403 +/- 67.21 mg in the second week, and -230 +/- 214.8 mg in the third week. Total phosphate mass removal in the second and third week was significantly lower than that of the control period. Plasma phosphate concentration was significantly decreased after the HD compared with before the HD in the control and second week. There was no significant difference in plasma phosphate concentration between the period before HD and at 48 hours in the control and the second week. Plasma phosphate concentration before HD not only depended on phosphate mass removal by HD, but also on other factors. We suggest that dialysate containing phosphate might prevent excessive phosphate removal from non-extracellular compartments during HD. PMID- 10737012 TI - [Henoch-Schonlein purpura nephritis (HSPN) in children: comparison of the incidence and severity between two 12-year groups]. AB - From 1971 to 1982, we treated 286 patients of HSP in Kitasato University Hospital. In these 286 patients, 137 developed purpura nephritis (47.9%). During the second 12-year period (from 1985 to 1996), we treated 34 HSPN patients among 189 HSP patients (18.0%). The clinico-pathological evaluations and comparisons in 30 cases from 1971 to 1982 and in 11 cases from 1985 to 1996 were performed, using the ISKDC grading. The numbers of patients of HSP and the incidence of HSPN both decreased in the more recent 12-year group. Furthermore, the severity of the renal histopathological findings decreased in the more recent group as well. PMID- 10737014 TI - [The long-term care insurance for elderly]. PMID- 10737013 TI - [High PTHrP level induced hypercalcemia and acute renal failure in a multiple myeloma patient]. AB - Multiple myeloma causes various renal injuries by direct invasion of myeloma cells, AL amyloidosis and hypercalcemia. Hypercalcemia induced by myeloma has been thought to be a result of local osteolysis. Recently, however, it was noted that no significant difference existed in the degree of bone-destruction between hypercalcemic and normocalcemic multiple myeloma. The exact mechanisms of hypercalcemia induced by multiple myeloma remain unconfirmed. In the present study, we report a 70-year-old man, suffering from acute renal failure due to multiple myeloma and severe hypercalcemia. While the serum PTH level was low, PTHrP was markedly increased. Bone scintigraphy implied systemic increase in bone turnover in addition to cold spots corresponding to punched out lesions on bone Xp. After the intravenous administration of bisphosphonate, hypercalcemia and hot accumulation on bone scintigraphy were improved while the PTHrp level and bone destruction by myeloma cells were not improved. The present case suggests involvement of PTHrP in hypercalcemia of multiple myeloma. PMID- 10737015 TI - [The regulation of bone metabolism and the pathogenesis of osteoporosis]. PMID- 10737016 TI - [A perspective for atherosclerosis research]. PMID- 10737017 TI - [Cellular senescence and chromosome telomeres]. PMID- 10737018 TI - [Oxidative stress and age-related genes]. PMID- 10737019 TI - [Animal model for studying molecular mechanism of aging]. PMID- 10737020 TI - [Premature aging and genome instability syndromes caused by Rec Q helicase mutations]. PMID- 10737021 TI - [Alcohol consumption and change of activity of daily living among the elderly living in an urban community]. AB - Alcohol consumption has both adverse and beneficial effects on survival. We examined the baseline status of alcohol consumption and the changes of instrumental activity of daily living (I-ADL) by a prospective survey for the elderly living in an urban community who were participants in the "Longitudinal and Interdisciplinary Study on Aging by the Tokyo Metropolitan Institute of Gerontology (TMIG-LISA)". Detailed information on alcohol intake was collected during a structured interview. At a baseline survey in 1991, 1) the frequency of current drinkers decreased lineally by age. 2) the most popular alcohol beverage was beer for both sexes and average ethanol intake per day were 41 (10-45) g in men and 24 (8-28) g in women. Current drinkers had significantly stronger grip strength, higher velocity in maximum walking and higher score on the TMIG Index of Competence, designed to measure the higher-level competence in elderly community residents. After 4 years follow-up, in 1995, 73.3 percent of the participants completed the examination. They were classified into five categories as "current drinkers", "lifetime abstainers", "beginning to drink during follow up period", "quit drinking during follow-up period" and "quit drinking at baseline". The decline of I-ADL was significantly greater among the elderly who "quit drinking during follow-up period" than "current drinkers". Further "current drinkers" had significantly lower decline of I-ADL than "lifetime abstainers". Our findings show beneficial effects of moderate alcohol intake in the maintenance of a higher level of competence in the elderly living in a community. PMID- 10737022 TI - [Effects of healthy aging on the regional cerebral blood flow measurements using 99mTc-ECD SPECT assessed with statistical parametric mapping]. AB - To clarify the effects of normal aging on the cerebral blood flow, regional cerebral blood flow measurements++ using a Patlak Plot method of 99mTc-ECD were performed in 53 normal volunteers aged 18 to 87 years old (mean = 47.9 years). The subjects, 29 men and 24 women, were normal on clinical examination, and had neither a history of neurological nor psychiatric disease and no abnormal CT or MRI images. Statistical parametric mapping (SPM) was used for automatching and an objective approach to analysis of SPECT image data. Global cerebral blood flow showed a significant decline with age (r = 0.406). The SPM analysis (voxel height; p < 0.001, Bonferroni correction; p < 0.05) demonstrated significant age related decrease of relative rCBF in prefrontal cortices, anterior cingulate giri, and insular cortices-temporal poles bilaterally, these affected areas belonged to limbic or association cortices. Therefore, these decreases in rCBF may suggest cognitive changes that occur during normal aging. PMID- 10737023 TI - [Cognitive dysfunction among the elderly evaluated by the cognitive abilities screening instrument (CASI)]. AB - The Cognitive Abilities Screening Instrument (CASI) was developed by Japanese and American researchers, including the authors, to examine cognitive functions, based on previous neuropsychological tests, such as Hasegawa's Dementia Scale and the MMSE. From the Adult Health Study (AHS) population of the Radiation Effects Research Foundation, 2052 men and women aged 60 or over and living in Hiroshima were evaluated for their cognitive functions using the CASI. Dementia was diagnosed in 93 and 1,959 were considered not to suffer from dementia based on the DSM-III-R criteria using neurological examination and the Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE) completed by caregivers. Multivariate regression analysis not only indicated that total score was higher with lower age and higher education but that the slope of its decrease is steeper with higher age and fewer years of education. The slope of score decrease with increase of age was steeper for women than for men. Furthermore, CASI items were categorized by domains of cognitive function. Multivariate regression analysis for each cognitive domain showed clear effects of age and level of education. The effect of age for the nondementia subjects was large in cognitive domains dealing with temporal orientation, short-term memory, and list generating fluency. The most useful cognitive domains for diagnosing dementia were temporal orientation, short-term memory, and list-generating fluency, as was indicated by high sensitivity and specificity. PMID- 10737024 TI - [A swallowing study, based on clinico-pathological evaluation, performed by video fluoroscopy]. AB - A clinico-pathological evaluation was performed on patients requiring nasogastric nutritional support. As a result, it was found that nasogastric tube feeding was common in patients with cerebrovascular diseases (CVD) and senile dementia of Alzheimer's type (SDAT). Pneumonia was anamnestic in many CVD patients, which was frequently the direct indication for nasogastric tube feeding and the major cause of death in these patients. On the other hand, pneumonia was not common in SDAT in which the major indication of nasogastric tube feeding was abnormal appetite. However, pneumonia was an infrequent cause of death in SDAT compared to CVD patients. The mean age in which nasogastric tube feeding was started was 8 years older in SDAT than CVD patients, however, there was no significant difference in the duration of nasogastric tube feeding ranging from initiation to death. A swallowing study, based on a clinico-pathological evaluation, was performed by video-fluoroscopy on healthy seniors and senior patients neurological diseases. There was no abnormal finding in the healthy seniors. Findings in CVD patients with single-sided neurological diseases indicated that 27.3% had moderate abnormalities and 18.2% had severe abnormalities. In CVD with bilateral defects, 35.7% had moderate abnormalities and 42.9% had severe abnormalities. Though even single-sided CVD defects can frequently cause swallowing disorder, oral food intake was maintained in nearly half of the patients with bilateral CVD, despite high incidence of severe swallowing disorder. In the mild SDAT group, rated on a scale from 0.5 to 1.0 according to the Clinical Dementia Rating (CDR), 11.1% had moderate swallowing disorder. In the CDR 2-3 group, 23.1% had moderate disability and 15.4% had severe disability. It appears that SDAT patients do not suffer from rapid deterioration in swallowing ability, which was relatively retained in this disease group. In Parkinson's disease patients with a Yahr grade of I-II, 55.6% had normal findings and 44.4% had mild abnormalities. In Yahr grade III-IV patients, 28.6% had mild and 28.6% had severe disability. Patients with severe dysfunction had a high incidence of silent aspiration. The swallowing function was maintained in the early course of mild Parkinson's disease patients, however the ability rapidly deteriorated with the course of the disease. The radiological findings of the swallowing study supported the clinico-pathological characteristics of each disease. PMID- 10737025 TI - [Coping with stress in the elderly]. AB - In terms of adaptability, unlike inanimate objects, living organisms exist in a dynamic balance between "wear and tear" and "repair and recovery". We regarded 17 hydroxycorticosteroids (17-OHCS) as a compound related to tissue "wear and tear" (Hans Selye) and sought for a compound related to tissue "repair and recovery". This led us to the discovery of 17-ketosteroid sulfates (17-KS-S) in urine. Elderly persons, unlike young adults, show low levels in 17-KS-S and little diurnal changes. In an elderly person 17-KS-S can decrease on significant life events (e.g. a spouse's death) and remain at low levels for a long time. Elderly persons with frailty need to improve their lifestyle (meals, exercise, rest, sleep, etc.) qualitatively and quantitatively to adapt themselves to stress adequately. Increased 17-KS-S levels were to be related to improvement in lifestyle. PMID- 10737026 TI - [A case of very late onset overlap syndrome of systemic sclerosis and systemic lupus erythematosus]. AB - A 88-year-old woman was admitted with appetite loss and dehydration in April 1999. She first noticed finger swelling in May 1996 and systemic sclerosis (SSc) was diagnosed in February 1997 on the basis of a clinical picture of low-grade fever, diffuse skin thickening, Raynaud's phenomenon, and pulmonary fibrosis. Retrospectively pulmonary fibrosis could have been identified on chest X ray film in September 1995. Although her appetite loss and dehydration were improved by hydration, pleural effusion continued. After detailed examinations, anti topoisomerase 1 (Scl-70) antibody, anti-Sm antibody, and an anti-double-stranded DNA antibody (dsDNA) were found in her serum. However, the other antibodies to anti-SS-A, SS-B, Jo-1, and RNP were not detected. These results suggest that this elderly case was a very late onset overlap syndrome of systemic lupus erythematosus (SLE) and SSc. Thus, the pleural effusion in this case may have been caused by SLE-associated pleuritis. Although the late onset elderly cases with overlap syndrome are very rare, SLE, SSc, and their combination can be found in aged patients over 80 years old. SLE and SSc should be carefully considered for the differential diagnosis of pleural effusion and skin thickening even in elderly patients. PMID- 10737027 TI - Outpatient cardiac rehabilitation in North Carolina. PMID- 10737028 TI - Slowing the progression of chronic renal insufficiency. PMID- 10737029 TI - Ozone. The good, the bad, and the ugly. AB - Sensitive individuals are at risk of adverse health effects from exposure to even modest levels of ozone. Such individuals are likely to develop respiratory symptoms and are more likely to be hospitalized when exposed to ozone at currently accepted levels. A review of scientific documents regarding the health effects of ozone has led the DHHS to agree with the EPA that the primary ambient air standard for ozone should be changed from 0.12 ppm (1-hour average) to 0.08 ppm (8-hour average). Achieving the proposed 0.08 ppm standard should decrease physician visits, hospitalization, and use of respiratory medications. In addition, because very susceptible healthy individuals may experience adverse health effects of the current standard, it is clear that the current standard provides little or no margin of safety, particularly for sensitive individuals. In order to avoid exposure to unhealthy levels of ozone, people should take precautions to minimize exposure from 2:00 to 6:00 pm on days when ozone levels are high. This is especially so for children, adults who are active outdoors, those with respiratory disease such as asthma, and people with unusual susceptibility to ozone. PMID- 10737030 TI - My father, my life, and chlorpromazine. PMID- 10737031 TI - Assuring access to dental care for low-income families in North Carolina. The NC Institute of Medicine Task Force Study. AB - Following publication of the Task Force's recommendations for improving dental care access among low-income populations, North Carolina has taken several steps forward. The Division of Medical Assistance and the NC Dental Society are forming an advisory committee (comprising Medicaid patients, providers, and representatives from all elements of organized dentistry in the state) to review dental coverage and reimbursement rates. Using existing state funds, the NC Office of Research, Demonstrations and Rural Health Development has recruited 15 additional dentists and 1 dental hygienist to practice in community facilities serving low-income and uninsured patients. In 1999, the NC General Assembly revised the NC Dental Practice Act. Now, under the general direction of a licensed public health dentist, specially trained public health dental hygienists can perform oral health screenings and preventive and educational services outside the public school setting. The NC Institute of Medicine has begun exploring how to use dental hygienists to expand preventive dental services to underserved populations in federally-funded community or migrant health centers, state-funded health clinics, and the not-for-profit clinics that serve predominantly Medicaid, low-income or uninsured populations. A report is to be sent to the Governor and the Joint Legislative Commission on Governmental Operations no later than May 1, 2000. In 1999, the General Assembly directed the NC State Board of Dental Examiners to establish a procedure for streamlined licensing of dentists and dental hygienists who have been practicing in other states. This should increase the number of qualified dental practitioners in the state. The proposed rules governing the new licensing pathway are to be prepared by May 15, 2000. The Board of Dental Examiners will determine which new procedures will be needed to allow less burdensome and more timely entry of qualified out-of-state licensed applicants, while still affording the public the same protection as under current law and procedures. The NC Institute of Medicine is organizing a work group to study the feasibility of new residency programs in pediatric dentistry in addition to the current program located in Chapel Hill. The Institute will present a report to the General Assembly, no later than May 1, 2000. On April 1, 1999, the state Medicaid program authorized use of ADA Procedure Code 1203, which allows reimbursement for the application of dental fluoride varnishes without a full prophylaxis. It also authorized pediatricians, nurse practitioners, or physician's assistants to apply these varnishes to the teeth of young children, allowing more rapid dissemination of this proven preventive procedure among the state's low-income children. Implementation began in Carolina Access II and III project sites in the fall, 1999, and should spread statewide in 2000. Furthermore, the General Assembly's 1999 session expanded NC Health Choice to cover dental sealants, fluoride treatment, simple extractions, stainless steel crowns, and pulpotomies. Since publication of the Task Force Report in May 1999, considerable forward movement has taken place. It was apparent that the problems associated with poor dental care were severe, of immediate concern, and needed a broad, nonpolitical analysis followed by action from public and private-sector policy makers and shapers. The key recommendation of the Task Force (to increase the level of payment to dentists for services provided to Medicaid beneficiaries) was not acted on in the 1999 session of the General Assembly, but it was seriously discussed in legislative hearings and will be considered further in the year 2000 legislative session. Given the number of problems surrounding adequate health care for North Carolina's low-income populations, inquiries such as that described here can point the way to the concrete and feasible steps that need to be taken. (ABSTRACT TRUNCATED) PMID- 10737032 TI - A community-based pharmaceutical care program for the elderly reduces emergency room and hospital use. PMID- 10737033 TI - Prehospital ECG transmission speeds reperfusion for patients with acute myocardial infarction. PMID- 10737034 TI - Cancer data. PMID- 10737035 TI - Is physician-assisted suicide ethical? Is euthanasia? PMID- 10737036 TI - A woman with diarrhea, weight loss, and a gassy liver. PMID- 10737037 TI - The role of episiotomy in modern obstetrics. PMID- 10737038 TI - [[Chimerism and tolerance]. AB - The goal of transplant immunology since its beginnings has been, and continues to be, the induction of long lasting unresponsiveness to a foreign graft. Although the transplantation of solid organs and cellular grafts is already a clinical routine, life-long nonspecific immunosuppression is still required to maintain the graft. Malignancies, opportunistic infections and organ toxicity are the main limitations of the antirejection therapy. One approach that finally could make the dream of tolerance induction come true, is hematopoietic chimerism. Hematopoietic chimerism is defined as the coexistence of donor and host hematopoietic cells in the recipient's system. This coexistence might induce the mechanisms of central or peripheral tolerance to foreign, donor's antigens. The aim of this paper is to present current knowledge about relationship between the state of macro- or microchimerism and transplantation tolerance in bone marrow and organ transplantations. PMID- 10737039 TI - [Nitric oxide--metabolism, function and biological properties]. AB - This paper reviews results on studies on nitric oxide synthesis, signalling and its positive and negative role in organism. PMID- 10737040 TI - [Infection as a likely risk factor for ischemic stroke]. AB - This review presents recent informations concerning the role of viral and bacterial (especially Helicobacter pylori and Chlamydia pneumoniae) infections in the development of ischaemic stroke. Among possible pathogenic pathways that link infections and stroke, the special attention is paid to the coagulation abnormalities and immunological reactions. PMID- 10737041 TI - [Macrophage receptors]. AB - Macrophages play a very important role in specific and nonspecific immune response. They are involved in different processes such as phagocytosis and killing of microorganisms, antigen presentation and production of cytokines etc. Their biological activity, ability to cooperate with other cells and to receive signals from extracellular environment are determined by their surface membrane receptors. The paper describes the structure and role of Fc receptors (FcR), adhesion receptors, complement receptors (CR), CD14 (LPS receptors) and receptors for chemotactic agents, present on the surface of macrophages. PMID- 10737042 TI - [Experimental hepatocarcinogenesis--evaluation of the significance of experimental modeling. Classical models]. AB - There are an increasing number of models for the study of liver cancer development with such agents as chemicals, hormones, and viruses. This process is almost always a multistep and during the long period of cancer development, discrete cells or cell populations acquire step-by-step the various properties that go to make up a cancer. The hepatocarcinogenesis models are useful in identification and analysis of the preneoplastic and neoplastic alterations, as well HCC. This article presents a review of theoretical foundation and mechanisms of various models of experimental hepatocarcinogenesis as well contain a short presentation of animals practical in those experiments, principally rats. The remarkable similarities between many models with different carcinogens in animals and humans suggest the importance of such studies in understanding of molecular basis of liver cancer development. PMID- 10737043 TI - [The role of proteolytic enzymes in skin neoplasm progression and development of metastasis]. AB - Proteases are reported to play an essential role in the proliferative, invasive, and metastatic potential of malignant tumor cells. Metastasis is characterized by a complex series of interactions between tumor cells and their environment. Tumor expansion and invasion are associated with the destruction of normal tissues around the tumor by a variety of classes of tumor and host-derived extracelullar matrix-degrading proteinases. Increased levels of the major lysosomal proteinases cathespin B, D, L, metalloproteinases and plasminogen activators, are correlated with tumor cell invasion and metastasis in human melanoma and other skin tumors, breast and colorectal cancer. PMID- 10737044 TI - [[Stressful increase in body temperature--hyperthermia or fever]. AB - Psychological factors are recognized to influence immune responses and susceptibility to various disease processes. Exposure to psychological stress produces rise in body temperature in animals and human beings. Ther are considerable evidence that support the hypothesis that stress "hyperthermia" is actually a fever (i.e. an elevation in thermoregulatory set-point). The stress induced rise in body temperature is mediated by endogenous pyrogens and prostaglandin E2 produced inside the blood-brain barrier. Moreover, it has been shown that beta-adrenoceptors in the central nervous system are involved in the psychological stress-induced elevation in body temperature. Since adrenalectomized animals develop larger stress-induced fevers, it appears that glucocorticoids may act in negative feedback on stress-induced fever. PMID- 10737046 TI - Minding the mind. PMID- 10737045 TI - [Metabolism of ethyl alcohol in the human body]. AB - More than 90% of ingested ethanol is metabolized in the body to acetaldehyde and acetate. Ethanol is metabolized in the liver via three distinct enzymatic pathways: alcohol dehydrogenase (ADH), the microsomal ethanol oxidizing system (MEOS) and catalase. It is generally accepted that alcohol dehydrogenase is the predominant pathway for hepatic ethanol oxidation. Acetaldehyde is metabolized to acetate by a group of dehydrogenase enzymes called aldehyde dehydrogenase. PMID- 10737047 TI - Topography and relationships of mind and brain. PMID- 10737048 TI - Protective and damaging effects of stress mediators: central role of the brain. PMID- 10737049 TI - Interactions between the immune and neuroendocrine systems. PMID- 10737050 TI - Depression really does hurt your heart: stress, depression, and cardiovascular disease. PMID- 10737051 TI - Circuits and mechanisms governing hypothalamic responses to stress: a tale of two paradigms. AB - The results of recent studies support a partitioning of stress models into at least two basic classes. While these have been referred to as 'systemic' and 'neurogenic', we would suggest that the terms interoceptive and exteroceptive, respectively, are more apt descriptors. This is based on the similarities in the overall patterns of activational responses seen as a consequence of exposure to a range of perturbations in the internal versus external environments. While stressors of each class may share in common such fundamental features as a capacity to enlist certain PVH effector populations and medullary catecholamine containing neurons, both the capacity to involve specific output neuron classes and the dependence of hypothalamic effects on the integrity of aminergic afferents in at least some interoceptive and exteroceptive models, are clearly differential. The available evidence suggests that interoceptive stress effects on PVH effector populations may be conceived essentially as simple reflex responses, mediated at a subcortical level by cell groups and associated circumventricular organs that comprise the core of a system involved in the processing of visceral sensory information. Based on the general pattern of acute footshock-induced Fos expression and commonalities of cellular activation profiles seen in this and other acute exteroceptive paradigms, it seems a reasonable assumption that pathways that convey somatosensory/nociceptive information to the PVH are apt to mediate adaptive visceromotor responses in these models. Multiple candidates for such roles have been identified at various levels of what may be viewed as the ascent of the spinothalamic pathway through the brainstem and thalamus, and on through the limbic forebrain and hypothalamus. Dissecting the relative contributions of these in determining PVH output will speak to important conceptual issues concerning the extent to which the affective and visceromotor responses to exteroceptive stressors are organized, and the level(s) at which these different avenues of emotional expression may be integrated. PMID- 10737052 TI - Long-term behavioral and neuroendocrine adaptations to adverse early experience. PMID- 10737053 TI - Neurobiological correlates of defensive behaviors. PMID- 10737054 TI - Effects of perinatal pain and stress. AB - Neonatal intensive care exposes preterm neonates to a series of repeated, randomly occurring invasive procedures and handling, resulting in acute pain, chronic pain, and prolonged stress during a critical window associated with epochal brain development. Characteristics of the immature pain system in preterm neonates (such as a low pain threshold, prolonged periods of windup, overlapping receptive fields, immature descending inhibition) predisposes them to greater clinical and behavioral sequelae from inadequately treated pain than older age groups. Evidence for developmental plasticity in the neonatal brain suggests that repetitive painful experiences during this period or prolonged exposure to analgesic drugs may alter neuronal and synaptic organization permanently. Traditionally, clinicians have chosen the perspective that routine use of analgesic or sedative drugs in preterm neonates may create more problems than minimal therapy. However, the immediate and long-term consequences of inadequately treated pain have forced them to reconsider the risk-benefit ratios for such therapy. Whereas the short-term consequences of prolonged analgesic therapy in human neonates are well-known (tolerance, withdrawal, ventilator dependency), long-term consequences are relatively unknown. Advances in the study of repetitive pain associated with routine NICU care have challenged the perspective that prolonged pain and stress were inevitable consequences of premature birth. PMID- 10737055 TI - Early life abuses in the past history of patients with gastrointestinal tract and pelvic floor dysfunctions. PMID- 10737056 TI - Responses of afferent neurons to the contents of the digestive tract, and their relation to endocrine and immune responses. PMID- 10737057 TI - The controls of eating: brain meanings of food stimuli. PMID- 10737058 TI - Effects of nutrients on brain function. AB - While many of the above examples support a role of these dietary components in modifying the synthesis, storage, release and actions of various neurotransmitter molecules in the central nervous system, most of the responses to eating everyday foods are expected to produce subtle changes in physiological and/or behavioral parameters. However, the observed subtle changes may have significant consequences when present in individuals with altered homeostasis as might be present in various disease states or certain environmental situations (e.g. depression, PMS, stress). Studies in the future should investigate the effects of various diets, e.g., vegetarian, macrobiotic, traditional Eastern, etc. on physiological and psychological functioning. Care should be taken to differentiate between the responses of subgroups of subjects, e.g. male vs. female, old vs. young, and lean vs. obese, as some differences in the rate of neurotransmitter synthesis and receptor dynamics have been reported in some studies. Chronic consumption of these diets may lead to long-term alterations in the neurotransmitter systems' dynamics, or as is often the situation with long term pharmacological treatments, may result in adaptive changes to minimize the acute effects of such treatments. To date, no such studies have been performed that have systematically addressed many of these issues. Future studies will require careful design so as to enhance the chances of detecting such alterations in function. However, the most significant alterations in function occur when a dietary component is administered in a purified form, separate from the normal diet. In this case the compound should be treated more like a pharmacological agent than a nutrient since adverse (i.e. antinutritive) effects may result. The most difficult studies however will use everyday foods with the aim of detecting changes based on the underlying biochemical changes. PMID- 10737059 TI - The evolving neurobiology of gut feelings. AB - The bi-directional communication between limbic regions and the viscera play a central role in the generation and expression of emotional responses and associated emotional feelings. The response of different viscera to distinct, emotion-specific patterns of autonomic output is fed back to the brain, in particular to the cingulofrontal convergence region. Even though this process unfolds largely without conscious awareness, it plays an important role in emotional function and may influence rational decision making in the healthy individual. Alterations in this bi-directional process such as peripheral pathologies within the gut or alterations at the brain level may explain the close association between certain affective disorders and functional visceral syndromes. PMID- 10737060 TI - Integration of viscerosomatic sensory input at the spinal level. AB - The major point of this chapter is that there is evidence to support the idea that cervical headache might not only result from injured somatic structures in the neck but also occur because of interactions with visceral organs. The complex arrangement of convergent inputs from somatic and visceral afferent fibers and of the propriospinal pathways in the upper cervical segments may create an environment to precipitate such headaches (Fig. 8). It is possible that the soreness experienced in the muscles innervating the neck may not be due to direct injury but may occur as muscle hyperalgesia that is often associated with visceral pain (Giamberardino, et al., 1993). Much more research is required to understand these complex interactions before patients who suffer pain of cervical headache can be treated satisfactorily. PMID- 10737061 TI - The medial pain system, cingulate cortex, and parallel processing of nociceptive information. PMID- 10737062 TI - Pain as a visceral sensation. PMID- 10737064 TI - Mechanisms of analgesia produced by hypnosis and placebo suggestions. PMID- 10737063 TI - Pain modulation: expectation, opioid analgesia and virtual pain. AB - To summarize, although there are multiple potential target nuclei for modulating pain transmission and several candidate efferent pathways that exert modulatory control, the most completely described pain modulating circuit includes the amygdala, PAG, DLPT and RVM in the brainstem. Through descending projections, this circuit controls both spinal and trigeminal dorsal horn pain transmission neurons and mediates both opioid and stimulation produced analgesia. Several different neurotransmitters are involved in the modulatory actions of this circuit, which exerts bi-directional control of pain through On cells that facilitate and Off cells that inhibit dorsal horn nociceptive neurons. There is evidence that this circuit contributes to analgesia in humans and may be activated by acute stress or the expectation of relief. Conversely, through the facilitating effect of On cells, this circuit is theoretically capable of generating or enhancing perceived pain intensity. Such an effect could provide a physiological mechanism for the pain enhancing actions of mood, attention and expectation. PMID- 10737066 TI - Affect, cognition, the immune system and health. PMID- 10737065 TI - The role of vagal visceral afferents in the control of nociception. AB - We have shown that activity in subdiaphragmatic vagal afferents modulates mechanical hyperalgesic behavior in the rat. Subdiaphragmatic vagotomy decreases paw-withdrawal threshold to mechanical stimulation (baseline and after intradermal injection of bradykinin), thus enhancing mechanical hyperalgesic behavior. Most of this decrease is generated by an endocrine signal released by the adrenal medullae because denervation or removal of the adrenal medullae prevents or reverses these changes. This novel mechanism may imply that: (a) the brain is able to regulate sensitivity of nociceptors all over the body by a neuroendocrine mechanisms, (b) sensitivity of nociceptors can be influenced by changes in parts of the body which are remote from the location of the sensitized nociceptors and (c) circulating catecholamines can influence nociceptors in a way which is different from those reported so far (see Janig and McLachlan, 1994; Janig, 1996a; Janig et al., 1996). PMID- 10737067 TI - Memory networks in the prefrontal cortex. PMID- 10737068 TI - Non-conscious brain processing indexed by psychophysiological measures. PMID- 10737069 TI - Brain mediation of active and passive emotional coping. PMID- 10737070 TI - Specificity in the organization of the autonomic nervous system: a basis for precise neural regulation of homeostatic and protective body functions. AB - Experimental investigations of the lumbar sympathetic outflow to skin, skeletal muscle and viscera and the thoracic sympathetic outflow to the head and neck have shown that each target organ and tissue is supplied by one or two separate pathways which consists of sets of pre- and postganglionic neurons with distinct patterns of reflex activity. This probably applies to all sympathetic and parasympathetic systems. The specificity of the messages that these peripheral pathways transmit from the central nervous system arises from integration within precisely organized pathways in the neuraxis. The messages in these discrete functional pathways are transmitted to the target tissues often via organized neuroeffector junctions. Modulation in the periphery can occur within each pathway, both in ganglia and at the level of the effector organs. This organization is the basis not only for precise neural regulations of all homeostatic body functions in which the autonomic nervous system is involved but also the basis of one main component in the regulation of protective body functions: (a) Elementary defense behaviors which are organized in the mesencephalon (confrontational defense, flight, quiescence), (b) regulation of the immune system by the sympathetic nervous system, and (c) adaptive autonomic motor responses during basic emotions require precisely working autonomic, in particular sympathetic, systems. In this sense, the concept of the functioning of the sympathetic nervous system in an "all-or-none" fashion, without distinction between different effector organs, and of simple functional antagonistic organization between sympathetic and parasympathetic nervous system is misleading, inadequate and untenable. PMID- 10737071 TI - The impact of emotions on the heart. PMID- 10737072 TI - Neural influence on immune responses: underlying suppositions and basic principles of neural-immune signaling. PMID- 10737073 TI - The cost-effectiveness of mind-body medicine interventions. AB - Evidence is mounting that addressing the psychosocial needs of patients makes economic and health sense. If there were a drug or surgical procedure that could reduce ambulatory care visits, decrease postsurgical length of stay, reduce c section rates, or decrease death rates from cancer, this medical intervention would be widely accepted and utilized with little hesitation. The beliefs and biases that delay and retard the use of psychosocial interventions need to be challenged (Engel, 1977; Williamson et al., 1991). This brief review of mind-body interventions suggests that health care providers can ill afford to treat patients simply as disordered machines whose health can be restored with physical or chemical interventions alone. Indeed, a burgeoning interest in alternative and complementary medicine with a focus on non-drug, non-surgical interventions as well as the exploding field of lay literature and self-help groups suggests that many patients are ready, willing, and even demanding that mind-body health techniques be considered as part of health care (Friedman et al., 1997). While the health care system cannot be expected to address all the psychosocial needs of people, clinical intervention can be brought into better alignment with the emerging evidence on the health and cost-effectiveness of mind-body interventions. Mind-body medicine is not something separate or peripheral to the main tasks of medical care but should be an integral part of evidence-based, cost effective, quality health care. PMID- 10737074 TI - Towards an integrative model of irritable bowel syndrome. PMID- 10737075 TI - Bridging the gap between mind and body: do cultural and psychoanalytic concepts of visceral disease have an explanation in contemporary neuroscience? PMID- 10737076 TI - Complementary and alternative medicine (CAM): epidemiology and implications for research. PMID- 10737077 TI - Biological mechanisms of acupuncture. PMID- 10737078 TI - Intricate tactile sensitivity: a key variable in western integrative bodywork. PMID- 10737079 TI - The science of breathing--the yogic view. PMID- 10737080 TI - Exploring the nature and functions of the mind: a Tibetan Buddhist meditative perspective. AB - The Tibetan Buddhist classification of mind, its divisions and the system of meditation practices aimed at achieving ideal states of being are a complex presentation of psychological, behavioral and spiritual concepts and processes that demand thorough understanding before assessing their value and outcome (Yeshe, 1995). It is the authors' belief that with the rapid advances in the neuroscience of mental processes, both scientist and Tibetan practitioner can benefit from each other's knowledge and experience to enhance our search for greater understanding of the relationship between mind, body and spirit. PMID- 10737081 TI - [Victims' rights and civil valuation]. PMID- 10737082 TI - [A medical enigma: Ludwig van Beethoven]. PMID- 10737083 TI - [The mechanisms of hearing and hearing loss]. AB - The ear is constituted of an apparatus for transmission, the external ear and the middle ear, and of an apparatus for perception, the inner ear or cochlea and the acoustic nerves. A disorder can occur at each of these levels, leading to different types of hearing loss: defective transmission through a disorder of the external canal and/or by disorder of the tympanic-middle ear bone apparatus; endocochlear hearing loss through disorder of the ciliary cells of the Corti ganglion and/or by metabolic disorder or by pressure modification (such as Meniere's disease) of the endolymph compartment of the inner ear; retrocochlear hearing loss through disorders of the 8th nerve; central hearing loss through disorder of the temporal cortex. PMID- 10737084 TI - [Investigations and diagnosis of deafness]. AB - Otoscopic examination and audiometric tests complement each other for the diagnosis of hearing loss. Objective tests measure the hearing loss at some specific frequencies. Endo- or retrocochlear origin of the deafness is identified thanks to these tests, leading to selection of the most appropriate therapeutic option. In children, behavioral assessment techniques are to be adapted to the patient's age. Information from tympanometry, otoacoustic emissions and possibly auditory brainstem responses, is used to complete the diagnosis. Language skills are also assessed in order to adapt the child's deafness management to his/her actual communication ability. PMID- 10737085 TI - [Hereditary sensorineural deafness]. AB - Deafness is the most common sensory defect. The investigation of the cause of deafness is critical for genetic counselling, and sometimes for appropriate management of associated pathologies. About two thirds of cases of congenital deafness are genetic forms, and the proportion is probably similar concerning the forms of deafness that appears during childhood. Some of the genetic forms are syndromic and the associated signs are sometimes inapparent or may appear during childhood. Consequently, a systematic search for the most frequent syndromes is necessary in each deaf individual. In the majority of genetic cases, deafness is the sole defect (non-syndromic deafness) and the major mode of transmission is autosomal recessive. The DFNB1 form of deafness, due to connexin 26 gene mutations, underlies half of the cases of non syndromic congenital deafness cases. The hearing loss has a prelingual onset, and it is most frequently severe or profound. There is no associated pathologies or radiological anomalies of the inner ear, and the vestibular tests are normal. The possibility of offering molecular diagnosis of connexin 26 gene defects is profoundly modifying daily medical practice in the investigation of the cause of deafness. PMID- 10737086 TI - [Deafness of infectious origin]. AB - A large number of cases of deafness is due to infection. Both viral and bacterial agents can induce lesions of the middle ear, the inner ear and/or of the cochlear vestibular nerve. The ear is reached through the Eustachian tube, the outer ear or the subarachnoid spaces. The characteristics of infection-induced hearing loss depend on the infectious agent and on the site of infection. We discuss successively the characteristics of deafness caused by infection of the middle ear, represented by acute and chronic otitis of common or mycobacterial origin, by infection of the inner ear with bacterial labyrinthitis and otosyphilis, and lastly, neurolabyrinthitis of viral origin or due to Lyme disease. PMID- 10737087 TI - [Traumatic and occupational deafness]. AB - The frequency of accidental, traumatic hearing loss is increasing due to a sometimes violently noisy environment and to the development of sports as leisure activities. The diagnosis is based on knowledge of the circumstances of the trauma and on otoscopic examination. Total audiometry localises the damage. Occupational hearing loss forms a special subset of traumatic deafness. This trauma is usually due to intense noise occurring at the work-place. It is of insidious onset, irreversibly progressive and without treatment once under way; Prevention is based on knowledge of the deleterious effects of noise and on the individual factors of the subject at risk. PMID- 10737088 TI - [Aging and deafness]. AB - The hearing loss that appears with ageing or presbyacusis should not lead to withdrawal from communication an elderly person. The disorder should be recognised early. The first warning sign is difficulty for understanding in a noisy environment. When an individual indicates this problem, measurements of auditory functions should first be made and regular followed-up should be scheduled or therapeutic measures taken. Such measures generally include a hearing aid that amplifies the affected frequencies and speech therapy to learn lip reading. Some patients may profit from psychological follow-up during this new learning process. The family physician is crucial in the rehabilitation in encouraging the patient to seek medical help as soon as hearing loss begins. PMID- 10737089 TI - [Tinnitus and deafness]. AB - The relationships between tinnitus and hearing loss are studied from a clinical prospect. Five critical points are discussed. 1. Some degree of hearing loss is found in the vast majority of tinnitus patients; but an individual may well have a sensorineural hearing loss and no tinnitus at all. 2. A minor adjunction to the neurophysiological model of Jastreboff is proposed to take account of the association between tinnitus and hearing loss. 3. Tinnitus appears to cause more distress when hearing loss is marked. 4. Self-reported hearing loss should be considered when implementing habituation sound therapy. 5. According to McKinney, the rate of success on tinnitus that can be expected with habituation sound therapy is not significantly affected by hearing level. PMID- 10737090 TI - [Therapy techniques of deafness]. AB - Techniques for treating hearing loss comprise surgery, hearing aids and rehabilitation. Medical treatments are devolved to the correction of the cause of the loss of hearing, essentially infection. In hearing loss, surgery is routinely used to repair the consequences of chronic infection and of trauma. It is also used for treatment of otospongiosis. It is used to place vibratory or electric prostheses, which will increase the quality of indications for surgery. For instance, cochlear implants can be used in profound or total, bilateral hearing loss. In parallel, the quality and the miniaturisation of external auditory devices is improving. Rehabilitation, by helping to reconstitute missing factors, comprises a third technique of treatment of hearing loss. PMID- 10737091 TI - [Medical demography: 1 table, 2 figures, 3 points]. PMID- 10737092 TI - [Pruritus (other than anal and vulvar pruritus). Diagnostic orientation]. PMID- 10737093 TI - [Multiple sclerosis. Epidemiology, physiopathology, diagnosis, evolution]. PMID- 10737094 TI - [Acute salpingitis. Etiology, diagnosis, development, treatment]. PMID- 10737095 TI - [Indications for chromosome and DNA analysis for diagnosis of genetic diseases]. PMID- 10737096 TI - [Alcoholism]. PMID- 10737097 TI - [Pulmonary pneumocystosis in an HIV-infected subject. Diagnosis, development, treatment and prevention]. PMID- 10737098 TI - [Schizophrenic syndromes. Diagnosis, development, treatment]. PMID- 10737099 TI - [Signal transduction of cytokine receptors and its regulation]. PMID- 10737100 TI - [Blood group antigens and cell adhesion]. PMID- 10737101 TI - [Sterol carrier protein-2 (SCP2): the review of its physiological role]. PMID- 10737102 TI - [Genetic analysis of the function of a Drosophila integral membrane proteoglycan]. PMID- 10737104 TI - [Regulation of body weight by leptin]. PMID- 10737103 TI - [Ubiquitin-dependent proteolysis by SCF complex]. PMID- 10737105 TI - [SRP-independent targeting pathway to endoplasmic reticulum: histidine decarboxylase as a model protein]. PMID- 10737106 TI - [Low-molecular-compound mimetic of cytokine]. PMID- 10737107 TI - [Mechanisms of bromovirus cell-to-cell movement and adaptation to plants]. PMID- 10737108 TI - [Movement proteins of plant viruses]. PMID- 10737110 TI - [Phytoreoviral proteins with affinities to insect vector cells]. PMID- 10737109 TI - [Tomato spotted wilt tospovirus: plant-infecting bunyaviridae]. PMID- 10737111 TI - [Molecular biology of the viroid]. PMID- 10737112 TI - [Structure and molecular variability of capillovirus gemones]. PMID- 10737113 TI - [Tick-borne encephalitis]. PMID- 10737114 TI - [How should taxonomic confusions of Caliciviridae, particularly of caliciviruses causing gastroenteritis in humans, be resolved? A personal perspective]. PMID- 10737115 TI - [Persistentent infection and demyelination induced by Theiler's murine encephalomyelitis virus (TMEV)]. PMID- 10737116 TI - [Biological and epidemiological characteristics of Aichi virus, as a new member of Picornaviridae]. PMID- 10737117 TI - [Molecular biology of prion diseases]. PMID- 10737118 TI - Recent progress in the genetics of human epilepsies. AB - Despite several lines of evidence indicating a strong genetic influence in the etiology of idiopathic epilepsies, progress in the mapping and identification of human epilepsy genes has been limited until recently. In addition to the localisation and/or isolation of several genes causing progressive epilepsies associated with cerebral degeneration, at least seven human genomic regions (6p, 8q, 10q, 15q, 16p, 19q, 20q) are now known to harbour genes implicated in idiopathic epilepsies. In the case of nocturnal frontal lobe epilepsy, mutations in a nicotinic acetylcholine receptor subunit gene have been identified. Systematic studies of rare epileptic disorders inherited as monogenic Mendelian traits, as well as studies on more complex polygenic idiopathic epilepsies, are still needed in order to identify all the epilepsy genes. This will allow better diagnosis and genetic counseling in families of affected individuals, a better understanding of both the pathophysiology of epilepsies and normal brain functioning, and the design of new pharmacological and genetic therapies. PMID- 10737119 TI - Clinical and molecular genetics of primary dystonias. AB - Primary dystonias are movement disorders with dystonia as a major symptom. They are frequently inherited as Mendelian traits. There are at least eight clinically distinct autosomal dominant and two X-linked recessive forms. In addition, pedigree analyses suggest the occurrence of an autosomal recessive variant. The clinical classification is increasingly being replaced by a genetic one. To date gene loci have been identified in at least six autosomal dominant forms, i.e., in idiopathic torsion dystonia (9q34), focal dystonia (18p), adult-onset idiopathic torsion dystonia of mixed type (8p21-q22), dopa-responsive dystonia (14q22.1 q22.2), and paroxysmal dystonic choreoathetosis (2q25-q33; 1p21-p13.3). Gene loci in the X-linked recessive forms have been assigned to Xq13.1 in the X-linked dystonia parkinsonism syndrome and to Xq22 in X-linked sensorineural deafness, dystonia, and mental retardation. The disease genes have been identified in two autosomal dominant forms and in one X-linked recessive form. Mutations in a gene coding for an ATP-binding protein were detected in idiopathic torsion dystonia (DYT1), and the GTP cyclohydrolase 1 gene is mutated in dopa-responsive dystonia (DYT5). In sensorineural deafness, dystonia, and mental retardation, mutations were found in the gene DDP coding for a polypeptide of unknown function. This article reviews the clinical and molecular genetics of primary dystonias, critically discusses present findings, and proposes referring to the known forms, most of which can be distinguished by genetic criteria, as dystonias 1-12. PMID- 10737120 TI - No genetic association between the LRP receptor and sporadic or late-onset familial Alzheimer disease. AB - The low-density lipoprotein receptor-related protein gene (LRP1) is often mentioned as a candidate gene for Alzheimer disease (AD) because of its role as a receptor for apolipoprotein E (apoE), a major genetic risk factor for late-onset familial and sporadic AD. A recent association study of a tetranucleotide repeat polymorphism located 5' to the LRP1 gene detected an increase in the 87 base pair allele in AD cases compared to unaffected controls. Additionally, an independent study involving a genomic screen for genes associated with late-onset AD identified a region as a possible location of a late-onset AD gene on chromosome 12p between D12S373 and D12S390, about 10 cM proximal to LRP1. We examined 144 late-onset multiplex AD families, 436 sporadic AD cases, and 240 controls and found no evidence of linkage or association of LRP1 and AD. Our data indicate that genetic variation of the LRP1 gene is not a major risk factor in the etiology of AD. PMID- 10737121 TI - Identification of two mutations and a polymorphism in the chloride channel CLCN-1 in patients with Becker's generalized myotonia. AB - Myotonia congenita is an inherited muscle disorder characterized by muscle stiffness and hypertrophy. Its clinical phenotype depends, in part, on whether it is inherited as a dominant or recessive trait, respectively designated Thomsen's disease or Becker's generalized myotonia (BGM). In either case, it is associated with abnormalities in the muscle currents that are linked to the gene (CLCN-1) on human chromosome 7q35 encoding the skeletal muscle chloride channel. Single strand conformation polymorphism analysis was used to screen two families with the BGM for mutations in the CLCN-1 gene. Two new mutations were found (G 201ins and A317Q). The latter mutation has been previously described in Thomsen's disease. PMID- 10737122 TI - Genetic fine mapping of the Miyoshi myopathy locus and exclusion of eight candidate genes. AB - Miyoshi myopathy (MM) is an early adult-onset, autosomal recessive disorder characterized by weakness and muscular atrophy starting in the distal muscles. The disease locus has been previously mapped by linkage analysis to chromosome 2p using the microsatellite marker D2S291. Initial haplotype analysis of markers in families from three different origins (North American, Japanese, and Tunisian) suggested that the MM gene is located in a 4-cM region flanked by markers D2S292 on the telomeric side and D2S286 on the centromeric side. To delineate critical recombination events revealing a more refined localization of the MM gene, we have determined the pattern of segregation of 12 marker loci in two consanguineous families of Tunisian origin. In this study we have: (1) detected recombination events with the disease locus in one family, placing the MM gene most likely between markers D2S443 (CHLC.GGAA4D07.1876) and D2S2109; (2) generated a yeast artificial chromosome contig that spans approximately 3.8 megabases and extends from marker D2S358 to marker D2S286; (3) physically mapped 21 polymorphic markers, 5 genes, 3 STSs, and 1 EST within this contig; (4) detected and mapped a new polymorphism within this interval, allowing us to further reduce the MM locus to a 360-kilobase segment; (5) mapped the gene for the cytoskeletal protein beta-adducin within the MM candidate region, failing to find a consistent pattern of mutation of this gene in our MM patients; (6) excluded seven other candidate myopathy genes from the Miyoshi locus. PMID- 10737123 TI - Novel mutations of mitochondrial complex I in pathologically proven Parkinson disease. AB - Complete sequence analysis of all mitochondrial complex I genes was performed in 22 cases of neuropathologically confirmed idiopathic Parkinson disease (PD). DNA from the substantia nigra was used as a template for polymerase chain reaction based genomic sequencing. Seven novel mutations causing the exchange of amino acids were detected in subunit genes ND1 (3992 C/ T, 4024 A/G), ND4 (11253 T/C, 12084 C/T), ND5 (13711 G/A, 13768 T/C), and ND6 (14582 T/C). In addition, five known missense mutations affecting the ND1 (3335 T/C, 3338 T/C), ND2 (5460 G/A), ND3 (10398 A/G), and ND5 (13966 A/G) genes as well as three secondary LHON mutations (4216 T/C, 4917 A/ G, 13708 G/A) were found in the PD group. Among the novel mutations, the 11253 T/C transition which changes a conserved isoleucine residue into threonine is most likely to be of functional relevance. Furthermore, 43 synonymous polymorphisms were detected in PD brains, including 20 novel sequence variants. Haplogroup analysis revealed that most unique missense mutations were found in PD cases belonging to the D(c) haplogroup. Our data are in line with the view that PD is not a single disease entity but comprises a genetically heterogeneous group of disorders. The results of our study further suggest that 90% or more of all idiopathic PD cases are not due to sequence variation of mitochondrial complex I, but that mitochondrial mutations may play a pathogenic role in a subset of PD patients. PMID- 10737124 TI - Adult-onset glycogen storage disease type II: phenotypic and allelic heterogeneity in German patients. AB - Glycogen storage disease type II (GSDII, Pompe's disease) is an autosomal recessive inherited deficiency of lysosomal alpha-glucosidase (GAA). Clinical as well as biochemical and allelic heterogeneity have been described in GSDII. We identified mutations within the GAA gene in seven unrelated German patients, six with adult- and one with juvenile-onset GSDII. Beside previously described mutations [IVS1 (-13T --> G), delta(exon) 18, C1634T], we characterized four new mutations of GSDII: IVS6 (-22T --> G), 271delG, G1912T (Gly638Trp), and 2432insC. The IVS6 (-22T --> G) mutation gives rise to aberrant splicing, causing inframe deletions of 25 or 40 amino acids within the GAA coding sequence and the insertion of a sequence of seven missense amino acids. Two affected siblings and an unrelated patient with adult GSDII are apparently homozygous for the exon 18 deletion. Both siblings are also heteroallelic for IVS1 (-13T --> G). In conclusion, we observed pronounced allelic heterogeneity and an unexpectedly high frequency of homozygosity for larger in-frame deletions within the GAA coding sequence in German adult-onset GSDII patients. PMID- 10737125 TI - Lack of association between apolipoprotein E genotype and sporadic amyotrophic lateral sclerosis. AB - Amyotrophic lateral sclerosis (ALS) is a neuro-degenerative disorder with both sporadic and familial forms. Approximately 20% of autosomal dominant ALS is caused by mutations in the Cu/Zn superoxide dismutase (SOD1) gene. The causes of the remaining forms of ALS are unknown. The apolipoprotein E (APOE) gene is a known genetic risk factor for Alzheimer disease (AD), another neuro-degenerative disease. The APOE-4 allele increases risk and decreases age at onset in AD. Studies examining ALS and APOE have failed to show a significant effect of APOE on overall risk in ALS. Studies examining the effect of APOE-4 on site of onset in ALS (bulbar or limb) have been contradictory, with some studies showing an APOE association with bulbar onset and others showing no effect. Sample size was limited in these previous reports, particularly with respect to the number of bulbar onset cases (n = 33, 34 and 53). The present study examines a large collaborative data set of ALS patients (n = 363; 95 with bulbar onset) and age matched neurologically normal controls. The results for these data showed no significant differences in the percentage of subjects with the APOE-4/4 and APOE 4/X genotypes (X = APOE-2 or APOE-3) when comparing cases and controls in both the overall data set or in the data set stratified by site of onset. Similarly, logistic regression analysis in the overall and stratified data set while controlling for sex showed no increase or decrease in risk of ALS associated with the APOE-4 allele. In addition, there were no significant differences in age at onset between patients with APOE-X/X, and APOE-4/4 or APOE-4/X genotypes, overall or stratified by site of onset. We conclude based on these data that the APOE gene is not a major genetic risk factor for site of onset in ALS. PMID- 10737126 TI - Chromosomal localization of two genes underlying late-infantile neuronal ceroid lipofuscinosis. AB - Classical late-infantile neuronal ceroid lipofuscinosis (LINCL; CLN2) is an inherited neurodegenerative disorder of childhood characterized by seizures, loss of vision, and progressive motor and mental deterioration. The hallmark of this disease is the accumulation of enlarged, secondary lysosomes packed with curvilinear bodies in cells of affected individuals. The biochemical basis of LINCL remains unknown and there is no treatment effective in delaying the progression of this fatal disorder. During a genome-wide search using a set of highly polymorphic markers and 15 affected individuals from 7 multi-affected families, we obtained evidence for linkage of the LINCL gene CLN2 with markers on chromosome 11p15.5. We then genotyped patients and all available family members, including 8 single-affected families, for markers spanning 15 cM of 11p15.5. We obtained a maximum two-point LOD score of 6.16 at 0 = 0.00 at the marker locus D11S2362. Multipoint analysis yielded a maximum LOD score of 6.90 localized to the same marker. Using haplotype analysis, we localized CLN2 to a minimum candidate region of 11 cM flanked by marker loci D11S4046 on the telomeric side and D11S1996 on the centromeric side. Additionally, we present data suggesting that the gene underlying a variant LINCL subtype found in Costa Rica maps to the region defined by the CLN6 locus on chromosome 15q21-23. The mapping of these two LINCL loci provides a genetic basis for understanding the clinical heterogeneity observed in this group of diseases. PMID- 10737127 TI - Histopathology and APOE genotype of the first Alzheimer disease patient, Auguste D. AB - Alois Alzheimer published two papers on the disease which was named after him by Emil Kraepelin in 1910. Each of these papers contains clinical and pathological data on a patient Alzheimer had seen at the hospital. We have previously reported on the rediscovery of tissue sections from Alzheimer's second published case of Alzheimer disease, Johann F., which probably gave the disease its name (Neurogenetics 1997; 1:73-80). Here, we describe the histopathology and APOE genotype of Alois Alzheimer's first patient, Auguste D. As in the case of Johann F., a large number of tissue sections belonging to Alzheimer's laboratory, which was later headed by Spielmeyer, were found among material kept at the Institute of Neuropathology of the University of Munich. As described by Alzheimer in his original report (Allg Zeitschr Psychiatr 1907; 64:146-148), there were numerous neurofibrillary tangles and many amyloid plaques, especially in the upper cortical layers of this patient. Yet, there was no microscopic evidence for vascular, i.e., arteriosclerotic, lesions. Interestingly, Alzheimer's histological preparations did not include the hippocampus or entorhinal region. The APOE genotype of this patient was shown to be epsilon3/epsilon3 by PCR-based restriction enzyme analysis, indicating that mutational screening of the tissue is feasible. The historical importance of the case of Auguste D. lies in the fact that it marks the beginning of research into Alzheimer disease. In addition, neurofibrillary tangles were first described in this brain. PMID- 10737128 TI - Vessel wall alterations in patients with renal failure. AB - Cardiovascular complications are a major cause of morbidity and mortality in patients with renal failure. Death due to myocardial infarction and to stroke is more frequent in hemodialysis patients than in the total population. These cardiovascular diseases are mainly the consequence of atherosclerosis and cause decreased life expectancy in patients with renal failure. Ultrasound techniques now make it possible to measure atherosclerotic lesions in big and medium-sized arteries. Thickening of the intima-media-complex is an early sign of atherosclerosis in these vessels. It reduces the distensibility of the arteries during systole. The distensibility of big and medium-sized arteries can be determined using ultrasound-doppler-techniques. In our studies, the intima-media thickness of the carotid artery was significantly (p< 0.01) increased in patients with chronic renal failure (1.32+/-0.49 mm, n=28) as compared with aged-matched healthy control subjects (0.75 +/- 0.20, n= 29). The distensibility coefficient was higher (p< 0.05) in healthy controls (26 +/- 1.8 10(-3)/kPa, n= 12) than in patients with renal insufficiency (19 +/- 1.7 10(-3)/kPa, n = 12). This demonstrates increased stiffness of the vessel wall resulting in loss of Windkessel function and increased work load of the heart. PMID- 10737129 TI - Is there any difference between intermediate-acting and long-acting calcium antagonists in diurnal blood pressure and autonomic nervous activity in hypertensive coronary artery disease patients? AB - Recently, there have been some reports indicating that calcium antagonists induce a reflex increase in sympathetic activity, triggering cardiac events, especially in coronary artery disease (CAD) patients. In this study, we assessed heart rate (HR) variability (HRV) using power spectral analysis of the 24-h RR interval in 25 hypertensive outpatients with CAD treated with nifedipine. We compared blood pressure (BP), HR, and HRV variation in the same patients substituting benidipine (long-acting) for nifedipine (intermediate-acting). There were no significant differences in 24-h, daytime, nighttime, and morning BP between the nifedipine phase and the benidipine phase. HRV parameters (LF: low frequency power, HF: high frequency power, LF/HF ratio) also showed no significant differences in 24-h, daytime, nighttime, and morning LF, HF, and LF/HF ratio between the nifedipine phase and the benidipine phase. Blood pressure, HR, and HRV parameters, except the LF component from 2 to 4 h after nifedipine administration (the most effective duration), showed no differences compared to before administration. The LF component after the nifedipine administration was lower than before administration. In conclusion, in hypertensive patients with CAD, whose BP levels were well-controlled by twice-daily use of intermediate-acting nifedipine, switching from nifedipine to a long-acting calcium antagonist, benidipine, maintained well-controlled BP levels to a similar degree, but it may not have additional benefit in sympatho-vagal balance. PMID- 10737130 TI - Noninvasive, continuous evaluation of peripheral vascular resistance in humans. AB - We sought a noninvasive alternative method of monitoring peripheral vascular resistance continuously in humans, based on the analysis of arterial pressure waveforms. Radial arterial pressure waveforms were recorded noninvasively with a tonometer and analysed using a neural network method. To test the accuracy of this method, the peripheral vascular resistance was also determined by an invasive thermodilution method using a Swan-Ganz catheter in 20 subjects. To test the method in a clinical application, peripheral vascular resistance was determined by the noninvasive method before and after administration of nifedipine in 6 patients with essential hypertension. Neural network analysis of waveforms reliably yielded values between 0.00 and 1.00. Peripheral vascular resistance determined by neural network analysis and according to the invasive method showed a significant (p< 0.005) positive linear correlation. The peripheral vascular resistance measured by neural network analysis showed a significant (p< 0.05) decrease 30 min after administration of nifedipine, paralleling a decrease in blood pressure. Neural network analysis of tonometric radial artery waveforms provides an accurate, noninvasive, and continuous index of peripheral vascular resistance in human subjects. This simple method should permit more extensive homodynamic studies and larger epidemiological surveys in contrast to those undertaken using invasive techniques. PMID- 10737131 TI - Relationship between home blood pressure measurement and medication compliance and name recognition of antihypertensive drugs. AB - This study examined the relationship of home blood pressure measurement to medication compliance and name recognition of antihypertensive drugs in outpatients with hypertension. A total of 1,452 consecutive outpatients (842 males, 610 females; mean age 65+/-11 yr) seeking care at our institute answered questions at our cardiovascular outpatient clinic such as whether they had a sphygmomanometer at home, how often they measured their blood pressure at home, and how often they missed taking their medication. Among a total of 777 patients on antihypertensive drugs who had a sphygmomanometer at home, 16 of the 242 patients (6.5%) who measured their home blood pressure every day occasionally missed taking their medication, whereas this number was 22 for the 216 patients (10.1%) who measured their home blood pressure several times a week, 16 for the 146 patients (11.0%) who measured their home blood pressure several times a month, and 25 for the 173 patients (14.5%) who never measured their home blood pressure (p< 0.01 between patients who measured their home blood pressure every day and those who did not measure their home blood pressure). Among a total of 271 patients taking one or two antihypertensive drugs, the number of patients who could name their antihypertensive drugs was 47 of the 86 patients (55%) who measured their home blood pressure every day, 43 of the 78 patients (55%) who measured their home blood pressure several times a week, 24 of the 41 patients (58%) who measured their home blood pressure several times a month, and 22 of the 66 patients (33%) who never measured their home blood pressure (p< 0.02). In conclusion, medication compliance and antihypertensive drug name recognition were better in patients who measured their home blood pressure than in patients who did not measure their home blood pressure. From these results, we conclude that physicians should recommend home blood pressure measurement to patients being treated with antihypertensive drugs, because there is a possibility that home blood pressure measurement might improve medication compliance. PMID- 10737132 TI - Plasma aldosterone concentrations are not related to the degree of angiotensin converting enzyme inhibition in essential hypertensive patients. AB - There is increasing evidence of important cardiovascular effects of aldosterone via classical mineralocorticoid receptors in the heart. Aldosterone plus excess salt administration has been shown to produce both cardiac hypertrophy and cardiac fibrosis in rats. Various clinical studies have reported that aldosterone plays an important role in cardiac hypertrophy; however, the factors that control plasma aldosterone concentrations during angiotensin-converting enzyme (ACE) inhibitor treatment have still not been established. In the present study, we examined the relationship between plasma aldosterone concentrations and the degree of ACE inhibition in 25 essential hypertensive patients treated with an ACE inhibitor. Blood pressure decreased with treatment and plasma ACE activity, estimated in vitro (by a colorimetric method) and in vivo (by plasma angiotensin II/angiotensin I ratio) assay, was suppressed compared with that of hypertensive patients treated with medication other than ACE inhibitors. No relationship was found between the level of ACE inhibition and plasma aldosterone concentrations, which rose in parallel with the duration of ACE inhibitor treatment. The present study demonstrates that continuous ACE inhibitor therapy produces significant suppression of plasma ACE activity in essential hypertensive patients, but that no relationship exists between plasma aldosterone concentrations and levels of ACE inhibition. Plasma aldosterone concentrations tend to increase with the duration of ACE inhibitor treatment, although this increase did not reflect a reduced inhibition of ACE activity. PMID- 10737133 TI - Effect of long-term treatment with antihypertensive drugs on quality of life of elderly patients with hypertension: a double-blind comparative study between a calcium antagonist and a diuretic. NICS-EH Study Group. National Intervention Cooperative Study in Elderly Hypertensives. AB - We investigated the effect of long-term treatment with a calcium antagonist (nicardipine hydrochloride retard tablet) and a diuretic (trichlormethiazide) on quality of life (QOL) in elderly hypertensives in a multicenter, randomized, double-blind, comparative study (National Intervention Cooperative Study in Elderly Hypertensives Study Group). The percentage of patients who experienced side effects was 17.2% in the nicardipine group and 18.1% in the trichlormethiazide group and 2.9% and 4.3% of participants, respectively, withdrew due to those side effects. These results suggested that nicardipine was tolerated slightly better than trichlormethiazide. There were no significant differences between the two groups in terms of total QOL score or in degree of change (delta score) before and after calcium antagonist or diuretic administration. Lower score was seen in 3 categories (general symptoms, sleep scale, and sexual function) in the trichlormethiazide group (p< 0.05) and in one category (cognitive function) in the nicardipine group, but there was no significant difference in delta score in any of the individual items. In conclusion, the two anti-hypertensive agents had nearly equivalent effects on QOL in the long-term treatment of hypertension in the elderly and that neither resulted in a deterioration in QOL. PMID- 10737134 TI - Sodium-dependent calcium release from vascular smooth muscle mitochondria. AB - Interest in mitochondrial calcium (Ca2+) uptake and release waned as it became apparent that sarcoplasmic reticulum calcium stores dominate the control of cytoplasmic calcium concentration. Our recent demonstration of a very large rise in vascular smooth muscle (VSM) cytoplasmic sodium (Na+) concentration after inhibition of the sodium, potassium-ATPase (sodium pump) led us to several questions. Do VSM mitochondria show Na(+)-dependent Ca2+ release? Are the documented changes in cytoplasmic Na+ concentration sufficient to cause Ca2+ release? Do features of the cardiac mitochondrial exchange system, including differential sensitivity to a number of calcium antagonists and cation specificity, apply to VSM? We isolated mitochondria from bovine aorta and mesenteric arteries and employed arsenazo III as the Ca2+ indicator. Mitochondria from arterial vessels accumulated added calcium (up to 50 nmol Ca2+/mg protein) and released Ca2+ on exposure to Na+. This concentration-dependent relationship was linear from 0 to 10 mM of Na+, and it plateaued between 20 mM and 40 mM of Na+. VSM mitochondria exposed to 20 mM Na+ released 118 +/- 25 nmol Ca2+ per mg mitochondrial protein in 20 min, when a new equilibrium was reached. Lithium (Li+), in contrast to Na+, produced much smaller amounts of Ca2+ release from the VSM mitochondria. Na+-dependent Ca2+ release was antagonized in a concentration dependent manner by diltiazem (0-320 microM) with a Ki of 10.2 microM. Nifedipine had a lesser effect, and verapamil produced almost no inhibition. VSM mitochondria responses resemble those from heart mitochondria in that Na+ dependent Ca2+ release is present with a similar range of sensitivity to Na+ and a similar pattern of influence of diltiazem, nifedipine and verapamil. However, the influence of Li+ on Ca2+ release was much smaller and the amount of the Ca2+ released was much greater for VSM mitochondria compared with that reported for heart mitochondria. The large amount of Ca2+ released and the range of Na+ concentration that provoked Ca2+ release being within the physiologically achievable range raise the interesting possibility that these mechanisms may modify intramitochondrial cytosolic Ca2+ concentration, and hence could potentially contribute to the contractile response that follows inhibition of the sodium pump. PMID- 10737135 TI - Mapping of four simple sequence repeat (SSR) markers on rat chromosome 4. AB - We previously reported that several markers on rat chromosome (Chr) 4 cosegregated with the occurrence of cerebral stroke and brain edema in stroke prone spontaneously hypertensive rats (SHRSP). To obtain insights into the positional candidate genes for stroke susceptibility in this region, we mapped four genes, Taurine transporter (Tau), tumor necrosis factor receptor (Tnfr), GABA transporter (Gat1) and glucose transporter-3 (Glut3) genes, using newly developed simple sequence repeat (SSR) markers on rat Chr 4. We isolated the SSRs for the genes either by screening a rat genomic library or by searching the GenBank database. By linkage analysis using two sets of backcrosses, Gat1 and Tnfr were mapped in the region associated with stroke, while Taut was located distant from the region. The Glut3 locus was also assigned to rat Chr 4 using a rat x mouse hybrid clone panel. These results indicated that the Tnfr, Gat1 and Glut3 genes were good positional candidates for the stroke susceptibility in SHRSP, suggesting that further evaluation of these genes by functional studies could prove useful. PMID- 10737136 TI - Higher-order chromatin structure: looping long molecules. AB - Chromatin structure plays a variety of roles in eukaryotes, ranging from the structural organization of the genome to the facilitation of transcription factors and remodeling of individual gene promoters. Higher-order chromatin structure typically refers to those structural features of the genome that serve to facilitate large-scale condensation and packaging. It is becoming increasingly clear, however, that large-scale features that create loop domains play an important role in the management and functional organization of the genome as well. Recently, plant models have made significant contributions to our understanding of higher-order chromatin structures in eukaryotes. PMID- 10737137 TI - Magnesium chelatase subunit D from pea: characterization of the cDNA, heterologous expression of an enzymatically active protein and immunoassay of the native protein. AB - Mg-chelatase catalyzes the insertion of Mg into protoporphyrin and lies at the branchpoint of heme and (bacterio)chlorophyll synthesis. In prokaryotes, three genes--BchI, D and H--encode subunits for Mg-chelatase. In higher plants, homologous cDNAs for the I, D and H subunits have been characterized. Since the N terminal half of the D subunit is homologous to the I subunit, the C-terminal portion of the pea D was used for antigen production. The antibody recognized the chloroplast D subunit and was used to demonstrate that this subunit associated with the membranes in the presence of MgCl2. The antibody immunoprecipitated the native protein and inhibited Mg-chelatase activity. Expression in Escherichia coli with a construct for the full-length protein (minus the putative transit peptide) resulted in induction of 24.5 kDa (major) and 89 kDa (minor) proteins which could only be solubilized in 6 M urea. However, when host cells were co transformed with expression vectors for the full-length D subunit and for the 70 kDa HSP chaperonin protein, a substantial portion of the 89 kDa protein was expressed in a soluble form which was active in a Mg-chelatase reconstitution assay. PMID- 10737138 TI - Mapping and expression of a bifunctional thymidylate synthase, dihydrofolate reductase gene from maize. AB - A bifunctional gene (ZmDHFR-TS) encoding dihydrofolate reductase (DHFR) and thymidylate synthase (TS) was cloned from a Zea mays cDNA library. Both of these enzymes are involved in nucleotide biosynthesis, specifically in the formation of thymidine monophosphate (TMP). Comparison of the deduced amino acid sequence with DHFR-TS sequences from three other plant sources revealed over 75% similarity and motifs typical of DHFR-TS proteins. Two copies of the gene were mapped to chromosomes 2 and 4. This represents the first DHFR-TS gene cloned from a monocotyledonous plant. Expression of ZmDHFR-TS was examined in developing kernels and various tissues of maize by RNA gel blot hybridization analysis in order to determine the relationship between expression of this gene and DNA synthesis. RNA transcripts for ZmDHFR-TS accumulated to high levels in developing maize kernels when endosperm cells were undergoing endoreduplication and cell division. Meristematic maize tissues had high levels of ZmDHFR-TS mRNA, but transcripts were barely detectable in RNA isolated from the root elongation zone and from mature leaf tissues. PMID- 10737140 TI - Rice calcium-dependent protein kinase isoforms OsCDPK2 and OsCDPK11 show different responses to light and different expression patterns during seed development. AB - We investigated the spatial and temporal expression patterns of two rice calcium dependent protein kinases (CDPKs), OsCDPK2 and OSCDPK11, using isoform-specific antisera. Bands of the expected molecular sizes for OsCDPK2 (59 kDa) and OsCDPK11 (61 kDa) were detected on western blots. OsCDPK2 and OsCDPK11 mRNA and protein levels increased in unison during flower development. However, at the onset of seed development, the protein expression profiles diverged significantly. OsCDPK2 protein was expressed at low levels during early seed development, but increased to high levels that were maintained in later stages (20 days after fertilisation, DAF). Conversely, OsCDPK11 protein levels were high at the beginning of seed development, but fell rapidly from 10 DAF onwards. This decrease in the level of OsCDPK11 protein was associated with the abundant synthesis of a truncated mRNA species. OsCDPK2 expression was also closely associated with light perception. OsCDPK2 protein was barely detectable in green leaves exposed to light, but levels increased sharply when plants were shifted to darkness. Initially, this increase reflected a rapid elevation in the levels of OsCDPK2 mRNA, which was normally located in the mesophyll. Conversely, OsCDPK11 mRNA and protein levels were unaffected by light. These data strongly indicate that two rice CDPK isoforms have different functions in seed development and in response to light in leaves. PMID- 10737139 TI - Expression of tandem invertase genes associated with sexual and vegetative growth cycles in potato. AB - The organisation of two invertase genes (invGE and invGF) linked in direct tandem repeat within the potato genome is detailed. The genes exhibit a similar intron/exon structure which differs from previously described plant invertase genes; while intron locations are conserved between the genes, minor differences in exon length are seen. Both genes encode enzymes with putative extracellular location. Biochemical analysis of gene expression showed expression in floral tissues for both genes, with expression of the upstream gene (invGE) also detected in leaf tissue. Promoter sequences from both genes have been fused to the beta-glucuronidase (GUS) reporter gene (uidA) and transformed into potato. One promoter-GUS reporter construct was also transformed into tobacco. Histochemical analysis of transgenic lines defined specific expression from the downstream (invGF) promoter in potato and tobacco pollen, with expression first detected in the late uninucleate stage of tobacco microspore development. The invGE promoter determined expression in pollen and other floral tissues, but also at lateral nodes in stem, root and tuber. An association of invertase expression with generative tissue, both in vegetative and sexual modes of growth, is indicated. PMID- 10737141 TI - Sequence and functional analysis of the left-hand part of the T-region from the nopaline-type Ti plasmid, pTiC58. AB - The Agrobacterium tumefaciens nopaline strain C58 transfers a large, 29 kb T-DNA into plant cells during infection. Part of this DNA (the 'common DNA') is also found on the T-DNA of octopine strains, the remaining DNA is nopaline strain specific. Up to now, only parts of the C58 T-DNA and related T37 T-DNA have been sequenced. We have sequenced the remainder of the nopaline-specific T-DNA (containing genes a to d) and acs to iaaM. Gene c codes for a new unknown T-DNA protein. Gene a is homologous to the agrocinopine synthase gene. Genes b, c', d and e are part of a larger family: they are related to the T-DNA genes 5, rolB, lso and 3'. Genes 5, rolB and lso induce or modify plant growth and have been called T-DNA oncogenes. Our studies show that gene 3' (located on the TR-DNA of octopine strains) is also oncogenic. Although the b-e T-DNA fragment from C58 and its individual genes lack growth-inducing activity, an a-acs deletion mutant was distinctly less virulent on Kalanchoe daigremontiana and showed reduced shoot formation on Kalanchoe tubiflora. Shoot formation could be restored by genes c and c' in co-infection experiments. Contrary to an earlier report, a C58 e gene deletion mutant was fully virulent on all plants tested. PMID- 10737142 TI - Expression of fungal thermotolerant endo-1,4-beta-glucanase in transgenic barley seeds during germination. AB - The malting quality of two barley cultivars, Kymppi and Golden Promise, was modified to better meet the requirements of the brewing process. The egl1 gene, coding for fungal thermotolerant endo-1,4-beta-glucanase (EGI, cellulase), was transferred to the cultivars using particle bombardment, and transgenic plants were regenerated on bialaphos selection. Integration of the egl1 gene was confirmed by Southern blot hybridization. The transgenic seeds were screened for the expression of the heterologous EGI. Under the high-pI alpha-amylase promoter, the egl1 gene was expressed during germination. The heterologous enzyme was thermotolerant at 65 degrees C for 2 h, thus being suitable for mashing conditions. The amount of heterologous EGI produced by the seeds (ca. 0.025% of soluble seed protein), has been shown to be sufficient to reduce wort viscosity by decreasing the soluble beta-glucan content. A decrease in the soluble beta glucan content in the wort improves the filtration rate of beer. PMID- 10737143 TI - Aaknox1, a kn1-like homeobox gene in Acetabularia acetabulum, undergoes developmentally regulated subcellular localization. AB - Homeobox-containing genes play developmentally important roles in a wide variety of plants, animals and fungi. As a way of studying how development is controlled in the unicellular green macroalga Acetabularia acetabulum, we used degenerate PCR to clone a knotted1-like (kn1-like) homeobox gene, Aaknox1 (Acetabularia acetabulum kn1-like homeobox 1). Aaknorx1 is the first knotted1-like homeobox gene to be cloned from a non-vascular plant and shows strong conservation with kn1-like genes from the vascular plants (ca. 56% amino acid identity within the homeodomain). Sequencing of cDNA clones indicates that Aaknor1 possesses at least two distinct polyadenylation sites spaced ca. 600 bp apart. Southern analysis suggests that several other kn1-like homeobox genes exist in the Acetabularia genome. Northern analyses demonstrate that expression of Aaknox1 is developmentally regulated, with peak levels of expression during early reproductive phase. Northern analyses further demonstrate that Aaknox1 mRNA undergoes a change in its subcellular localization pattern during the progression from late vegetative to early reproductive phase. In late adult phase, Aaknox1 is distributed uniformly throughout the alga; in early reproductive phase, Aaknox1 is present in a gradient with the highest concentration of the mRNA at the base of the stalk, near the single nucleus. These data suggest that Aaknox1 may have a role during early reproductive development and that mRNA localization may be one mechanism by which A. acetabulum regulates gene expression posttranscriptionally. PMID- 10737144 TI - RAPD polymorphisms detected among the flax genotrophs. AB - The occurrence of environmentally induced heritable changes in certain flax varieties has been shown to be accompanied by changes in the genomic DNA. A large difference in nuclear DNA contents has been characterized between the extreme types, termed genotrophs. The genomic variation between a series of genotrophs has been studied by the polymerase chain reaction using random arbitrary oligonucleotide primers. A total of 320 primers were used in the reactions and 253 polymorphic bands observed. The polymorphic bands were derived from all parts of the genome, namely the highly repetitive. middle-repetitive and low-copy number sequences. They were also shown to be distributed throughout the genome. In one group of genotrophs, all of which were induced by temperature treatment, there was a clustering of the polymorphisms with a high degree of shared polymorphisms. These results are in agreement with earlier studies showing that a dispersed fraction of the genome is susceptible to variation when environmentally induced heritable changes occur. PMID- 10737145 TI - The eEFIA gene family is differentially expressed in maize endosperm. AB - eEF1A appears to be a multifunctional protein in eukaryotes, where it serves as a protein synthesis factor as well as a cytoskeletal protein. In maize endosperm, the eEF1A concentration is highly correlated with lysine content, and eEF1A synthesis is increased in opaque2 mutants compared to wild type. To investigate the basis for the increased synthesis of eEF1A in opaque2, we characterized the genes encoding this protein and measured their relative level of expression in endosperm and other tissues. Maize contains 10 to 15 eEF1A genes that are nearly identical in nucleotide and amino acid sequences. However, these genes can be distinguished based on their 3' non-coding sequences, which are less conserved. By screening endosperm and seedling cDNA libraries, we show that most of the maize eEF1A genes are expressed, and the relative level of their transcripts varies in different tissues. At least five genes are transcribed in the endosperm, and two account for ca. 80% of the RNA transcripts. The expression of several genes is enhanced in opaque2 endosperm, although the significance of this is unclear. PMID- 10737146 TI - The Arabidopsis thaliana RER1 gene family: its potential role in the endoplasmic reticulum localization of membrane proteins. AB - Many endoplasmic reticulum (ER) proteins are known to be localized to the ER by a mechanism called retrieval, which returns the molecules that are exported from the ER to the Golgi apparatus back to the ER. Signals are required to be recognized by this retrieval system. In the work on yeast Saccharomyces cerevisiae, we have demonstrated that transmembrane domains of a subset of ER membrane proteins including Sec12p, Sec71p and Sec63p contain novel ER retrieval signals. For the retrieval of these proteins, a Golgi membrane protein, Rer1p, is essential (Sato et al., Mol. Biol. Cell 6 (1995) 1459-1477; Proc. Natl. Acad. Sci. USA 94 (1997) 9693-9698). To address the role of Rer1p in higher eukaryotes, we searched for homologues of yeast RER1 from Arabidopsis thaliana. We identified three cDNAs encoding Arabidopsis counterparts of Rer1p with an amino acid sequence identity of 39-46% to yeast Rer1p and named AtRER1A, AtRER1B, and AtRER1C1. AtRer1Ap and AtRer1Bp are homologous to each other (85% identity), whereas AtRer1C1p is less similar to AtRer1Ap and AtRer1Bp (about 50%). Genomic DNA gel blot analysis indicates that there are several other AtRER1-related genes, implying that Arabidopsis RER1 constitutes a large gene family. The expression of these three AtRER1 genes is ubiquitous in various tissues but is significantly higher in roots, floral buds and a suspension culture in which secretory activity is probably high. All the three AtRER1 cDNAs complement the yeast rer1 mutant and remedy the defect of Sec12p mislocalization. However, the degree of complementation differs among the three with that of AtRER1C1 being the lowest, again suggesting a divergent role of AtRer1C1p. PMID- 10737147 TI - Large alkyl side-chains of isoleucine and leucine in the NPIRL region constitute the core of the vacuolar sorting determinant of sporamin precursor. AB - The N-terminal propeptide of the sporamin precursor contains vacuolar targeting information within the Asn-26/Pro-27/Ile-28/Arg-29/Leu-30 (NPIRL) sequence. An Agrobacterium-mediated transient expression assay with tobacco BY-2 cells was employed to investigate the role of each amino acid of the NPIRL region in vacuolar targeting. Replacement of Asn-26, Pro-27, Ile-28 and Leu-30 with several amino acids caused secretion of the mutant prosporamin. Leu was the only amino acid that could be substituted for Ile-28 without affecting transport. Exchange of Leu-30 for amino acids with small side-chains abolished vacuolar delivery. These results indicate that the consensus composition of the NPIRL sequence is [preferably Asn]-[not acidic]-[Ile or Leu]-[any amino acid]-[large and hydrophobic] and suggest that the large alkyl side-chains of Ile-28 and Leu-30 constitute the core of the vacuolar sorting determinant. PMID- 10737148 TI - Effects of antisense repression of an Arabidopsis thaliana pyruvate dehydrogenase kinase cDNA on plant development. AB - Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase (PDH) complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. This report describes the cloning of a pyruvate dehydrogenase kinase cDNA (AtPDHK) from Arabidopsis thaliana and focuses on the effects of antisense down-regulation of its expression on plant growth and development. The deduced amino acid sequence of AtPDHK exhibits extensive similarity to other plant and mammalian PDHKs, containing conserved domains typical of two-component histidine protein kinases. The Escherichia coli expressed AtPDHK specifically phosphorylated mammalian PDH E1 in a time-dependent manner. Antisense expression of the AtPDHK cDNA led to marked elevation of mtPDC activity in transgenic plants with increases ranging from 137% to 330% compared to control plants. Immunoblot analyses performed with a monoclonal antibody to the E1alpha mtPDH component (the subunit phosphorylated by PDHK) indicated that the increased mtPDC activity was not the result of an increase in the level of PDH protein. MtPDC from transgenic plants showed a reduced sensitivity to ATP-dependent inactivation compared to that observed in wild-type plants. Collectively, these data suggest that the antisense partial silencing of the negative regulator, PDHK, was responsible for the increased mtPDC activity observed in the antisense PDHK plants. Transgenic plants with partially repressed AtPDHK also displayed altered vegetative growth with reduced accumulation of vegetative tissues, early flower development and shorter generation time. The potential role for AtPDHK gene manipulation in crop improvement is discussed. PMID- 10737149 TI - Is preoperative cardiac testing necessary among vascular surgery patients? PMID- 10737150 TI - Negative impact of cardiac evaluation before vascular surgery. AB - The optimal preoperative evaluation of cardiac risk in patients with peripheral vascular disease is controversial. In developing a paradigm for preoperative cardiac workup, potential adverse effects of evaluation and cardiac intervention must be considered. This study analyzed the deleterious outcomes of extensive, comprehensive cardiac evaluation and intervention before planned vascular surgery in patients treated at the Denver Department of Veterans Affairs Medical Center. Over a 12-month period between 1994 and 1995, 161 patients were scheduled to undergo major vascular operations; 153 patients came to operation. The decision to pursue a cardiac evaluation was variously made by a combination of surgeons, cardiologists, and anesthesiologists. No defined protocol was followed. Cardiac history, chest X-rays and ECGs were obtained for all patients. Extended cardiac evaluation included these studies plus special tests, including echocardiography (echo), radionuclide ventriculography (RNVG), dipyridamole thallium scintigraphy (DTS), and cardiac catheterization (CC). Extended cardiac evaluations were undertaken in 42 patients. Complications related to percutaneous transluminal coronary angioplasty (PTCA) and coronary artery bypass grafting (CABG) were also recorded. Cardiac mortality and morbidity after vascular interventions were itemized in all 153 patients. Forty-two male patients, aged 68 +/- 9 years, underwent extended cardiac evaluations before planned vascular operations. The median elapsed time for cardiac workup was 14 days (mean 30 +/- 59 days). The median and mean times from cardiac workup to vascular surgery were 25 days and 76 +/- 142 days, respectively. Eighteen (43%) patients had echo or RNVG; 22 (52%) patients had DTS; 27 (64%) had CC; 9 (21%) had PTCA; 7 (17%) had CABG. Sixteen (38%) patients had untoward events related to cardiac evaluation. Eight patients (19%: one with cerebrovascular disease, and seven with aortic aneurysms) refused vascular surgery after extended cardiac workup. Complications attributable to CC, PTCA, and CABG included prosthetic graft infection, pseudoaneurysms (two), sternal wound infections (two), renal failure and brain anoxia. Two patients with severe limb ischemia who were candidates for revascularization ultimately required amputations because of delay due to cardiac evaluations. Extensive cardiac evaluation prior to vascular operations can result in morbidity, delays, and refusal to undergo vascular surgery. The underlying indication for vascular operations and the local iatrogenic cardiac complication rates must be considered before ordering special studies. PMID- 10737151 TI - Nutritional therapy for peripheral arterial disease: a double-blind, placebo controlled, randomized trial of HeartBar. AB - We investigated the clinical effects of a food bar enriched with L-arginine and a combination of other nutrients known to enhance the activity of endothelium derived nitric oxide (EDNO) in individuals with claudication from atherosclerotic peripheral arterial disease. The study was a 2-week, double-blind, placebo controlled trial of subjects randomized to three groups (two active bars, one active and one placebo bar, and two placebo bars per day) followed by an 8-week open-label period. Subjects (n=41) were outpatient volunteers with intermittent claudication. Pain-free and total walking distances were measured by variable grade, treadmill exercise testing. Quality of life was assessed using the Medical Outcome Survey (SF-36). After 2 weeks of treatment, the pain-free walking distance increased 66% while the total walking distance increased 23% in the group taking two active bars/day. The general and emotional/social functioning components of the SF-36 also improved. These effects were not observed in the one active bar/day and placebo groups. The effects were maintained after 10 weeks and, in addition, an improvement in walking distance was observed in the group taking one active bar. These findings reveal that use of a nutrient bar designed to enhance EDNO activity improves pain-free and total walking distance as well as quality of life in individuals with intermittent claudication. PMID- 10737153 TI - Pulmonary embolism and intracardiac thrombi--individual therapeutic procedures. AB - Mobile right heart thrombus is a severe but rare presentation of thromboembolic disease and usually coexists with an already massive pulmonary embolism (PE). But looking at the literature there is no clear consensus on therapeutic management. We therefore tried to find possible therapeutic guidelines and to evaluate an optimal diagnostic procedure looking at three patients who presented at our department with mobile right heart thrombus in the last year. The first patient with a small (diameter = 1 cm) thrombus in the right ventricle and peripheral pulmonary embolism underwent successful thrombolytic therapy without any complications. Patients II and III showed large intracardiac masses, in patient III extending into the superior vena cava, with central PE. These two patients underwent pulmonary arteriotomy. The diagnostic line in each case was transthoracal echocardiography followed by a helix lung CT scan. Only patients with small intracardiac thrombi and thrombotic masses in the peripheral pulmonary arteries but with hemodynamically significant PE should be treated with thrombolytic agents. PMID- 10737152 TI - Circulator boot therapy alters the natural history of ischemic limb ulceration. AB - Despite numerous advances in interventional radiology and vascular surgery, the clinician continues to be confronted with inoperable vascular disease. Previous studies have shown that ulceration associated with a transcutaneous oxygen pressure (tcPO2) of <20 mmHg is refractory to all attempts at healing. External pneumatic compression for the treatment of peripheral vascular disease has been available for several years, although there is a relative paucity of data regarding its role in clinical practice as well as its efficacy. The objective of this study was to examine the experience with circulator boot therapy in the treatment of ischemic ulcers in the absence of osteomyelitis, and specifically to determine whether such therapy can be of benefit in ischemic limb ulceration associated with a tcPO2 of <20 mmHg. A retrospective chart review was undertaken of ail patients with a lower limb ulcer who, in the absence of osteomyelitis, underwent circulator boot therapy at the Gonda Vascular Center. A total of 98 patients was identified. Two patients died within 1 month of commencing therapy and were not included in further analysis. The tcPO2 data were unavailable in five patients. Outcome in the patient population was classified as favorable if (1)healing was achieved, (2)the ulcer decreased in size, or (3) the affected limb improved sufficiently to allow successful revascularization. An unfavorable outcome was one where a major amputation was performed or where the ulcer increased in size. Out of a total of 29 patients with a tcPO2 <20 mmHg at the area of ulceration, 19 had a favorable outcome following circulator boot therapy. Of the remaining 62 patients with a tcPO2 >20 mmHg, 54 had a favorable outcome. Circulator boot therapy is associated with improved outcomes in limb ulceration due to peripheral vascular disease. Complete ulcer healing as well as preservation of the affected limb can be achieved in most cases. PMID- 10737154 TI - Vascular endothelial growth factor is efficiently synthesized in spite of low transfection efficiency of pSG5VEGF plasmids in vascular smooth muscle cells. AB - The limitation of lipotransfection with plasmid vectors is its low efficiency and the short-term expression of introduced genes. This is particularly important when the synthesis of high amounts of therapeutic products is required. However, growth factors with paracrine action overcome this problem. The aim of our study was to check whether the amounts of vascular endothelial growth factor (VEGF) generated after plasmid lipotransfection into vascular smooth muscle cells (VSMC) can be sufficient to stimulate endothelial cell proliferation. Two plasmids, pSG5 VEGF121 and pSG5-VEGF165, harboring human VEGF121 and VEGF165 isoforms were constructed and lipotransfected into COS-7 cells or to rat VSMC. The transfection efficiency, estimated by the expression of control, beta-galactosidase gene, was about 50% in COS-7 but rarely exceeded 5% in VSMC. However, despite this, the smooth muscle cells generated high amounts of VEGF protein, up to 3 ng/ml medium. The biological activity of this VEGF was confirmed by enhanced proliferation of human umbilical vein and coronary artery endothelial cells, stimulated with conditioned media of pSG5-VEGF transfected cells. Thus, the low transfection efficiency does not preclude the generation of high amounts of VEGF by VSMC. After reaching the maximum at about 48 h after transfection, the generation of VEGF decreased in the following days. Such a situation may be sufficient for the gene therapy of restenosis when the long-term expression of therapeutic gene(s) is not necessary. Thus, we suggest that the pSG5-VEGF121, and pSG5-VEGF165 plasmids can be used for therapeutic application. PMID- 10737155 TI - Insights into the molecular pathogenesis of atherosclerosis and therapeutic strategies using gene transfer. AB - Gene therapy for the treatment of atherosclerosis and related diseases has shown its potential in animal models and in the first human trials. Gene transfer to the vascular system can be performed both via intravascular and extravascular periadventitial routes. Intravascular gene transfer can be done with several types of catheters under fluoroscopic control. Extravascular gene transfer, on the other hand, provides a well-targeted gene delivery route available during vascular surgery. It can be done with direct injection or by using perivascular cuffs or surgical collagen sheets. Ex vivo gene delivery via transfected smooth muscle cells or endothelial cells might be useful for the production of secreted therapeutic compounds. Gene transfer to the liver has been used for the treatment of hyperlipidemia. The first clinical trials for the induction of therapeutic angiogenesis in ischemic myocardium or peripheral muscles with VEGF or FGF gene transfer are under way and preliminary results are promising. VEGF has also been used for the prevention of postangioplasty restenosis because of its capability to induce endothelial repair and production of NO and prostacyclin. However, further basic research is needed to fully understand the pathophysiological mechanisms involved in conditions related to atherosclerosis. Also, further development of gene transfer vectors and gene delivery techniques will improve the efficacy and safety of human gene therapy. PMID- 10737156 TI - The endothelium: a new target for therapy. AB - At one time considered merely a monolayer of cells lining the vascular conduit, the endothelium has emerged recently as an organ with functions as complex as any in the body. A highly active regulatory organ, the endothelium senses and assesses the hemodynamic, humoral, and inflammatory signals to which it is constantly exposed by the blood and responds by secreting factors that affect vessel tone and structure. These interactions are not merely of academic interest. It has been increasingly recognized that endothelial dysfunction plays a pivotal role in the development and progression of atherosclerosis and coronary artery disease. PMID- 10737157 TI - Acquired skeletal muscle metabolic myopathy in atherosclerotic peripheral arterial disease. AB - Peripheral arterial disease (PAD) is associated with an increased risk of overall cardiovascular mortality, and substantial morbidity resulting from claudication. While the initial disease process is clearly the result of atherosclerosis in the arterial circulation of the limb, altered hemodynamics do not completely explain the pathophysiology of claudication. Work from several laboratories has demonstrated secondary changes in the skeletal muscle of patients with PAD which are consistent with the presence of an acquired metabolic myopathy in these patients. Key findings include an alteration in the expression of mitochondrial enzymes, the accumulation of metabolic intermediates, altered regulation of mitochondrial respiration, increased oxidative stress, and the presence of somatic mutations in the mitochondrial genome. Understanding the metabolic changes associated with PAD is important in understanding the pathophysiology of claudication and in the development of novel therapeutic strategies. PMID- 10737158 TI - Images in vascular medicine. Limited cutaneous scleroderma. PMID- 10737159 TI - Solute fluxes in different treatment modalities. AB - The principles governing solute flux or transport in different artificial kidney treatment modalities are reviewed. Solute clearance profiles were calculated for identical artificial kidney membranes during haemodialysis, haemofiltration and haemodiafiltration. It was shown that the clearance of small solutes depends largely on the dialysate flow rate and is similar when using either haemodialysis or haemodiafiltration. In contrast, clearance of middle molecules, especially low molecular-weight proteins, depends largely on convective transport induced by high ultrafiltration rates and is maximized when using either haemofiltration or haemodiafiltration. Optimal fluxes for both small solutes and middle molecules can be achieved by using postdilution haemodiafiltration. Recent work has shown that use of the reduction in plasma concentration, even after normalization for changes in extracellular volume during therapy, is not an exact measure of beta2 microglobulin (and other low-molecular-weight proteins) clearance. It is proposed that beta2-microglobulin clearance be reported in future studies instead of the normalized reduction in beta2-microglobulin plasma concentration. Additional studies are necessary to determine the effects of postdialysis rebound on the calculated clearance for beta2-microglobulin and other high-molecular-mass uraemic toxins. PMID- 10737160 TI - Protein permeability in dialysis. PMID- 10737161 TI - Clinical significance of granulocyte-inhibiting proteins. PMID- 10737162 TI - Beta2-microglobulin and amyloidosis. AB - Dialysis-associated amyloidosis is a serious complication in chronic dialysis patients. Its clinical expression in terms of arthralgias, destructive arthopathies and carpal tunnel syndrome is often associated with amyloid deposits, which are mainly composed of beta2-microglobulin (beta2-M) fibrils, but in addition contain a number of other compounds. It is probable that beta2-M amyloid deposition is related, at least in part, to the elevated plasma beta2-M that is characteristic of chronic renal failure. The latter can decrease with high-performance dialysis techniques but cannot be reduced to the normal range. Almost certainly, several other systemic and local factors are involved, including beta2-M transformed by advanced glycation end products and advanced oxidation protein products, serum P component, ubiquitin, calcium crystals, cytokines, immunoglobulin light chains, proteases and antiproteases, as well as modified collagen and glucosaminoglycans. It is also possible that the beta2-M protein, in its native or modified form, exerts noxious effects on bone and joint tissues, in addition to its mere 'passive' presence as amyloid fibrils. Several retrospective studies and one prospective study suggest that dialysis strategies with highly permeable, synthetic membranes and/or ultrapure dialysate may be partially protective or at least delay the onset of dialysis amyloidosis. Successful kidney transplantation generally halts the disease process and leads to rapid relief of osteoarticular pain although regression of beta2-M-amyloid deposits probably does not occur. PMID- 10737163 TI - 'Carbonyl stress' and dialysis-related amyloidosis. AB - Advanced glycation end products (AGEs) are formed by nonenzymatic glycation and oxidation (glycoxidation) reactions. As AGE formation is related to hyperglycaemia, they have been implicated in the pathogenesis of diabetic complications. They also increase in normoglycaemic uraemic patients: AGEs, such as pentosidine and carboxymethyllysine (CML), are elevated in both the plasma proteins and skin collagen of uraemic patients, being several times greater than in normal subjects and nonuraemic diabetic patients. However, AGE concentrations do not differ between diabetics and non-diabetics in uraemia. AGE accumulation in uraemia, therefore, cannot be attributed to hyperglycaemia, or simply to the decreased removal by glomerular filtration of AGE-modified proteins as over 90% of plasma pentosidine and CML are linked to albumin. Recently, evidence has suggested that, in uraemia, the increased carbonyl compounds, derived from both carbohydrates and lipids, modify proteins both by the glycoxidation reaction (leading to augmented AGE production) and also by the lipoxidation reaction (leading to the augmentation of the advanced lipoxidation end product, ALE, production). Thus, uraemia might be a state of carbonyl overload with potentially damaging proteins ('carbonyl stress'). Carbonyl stress in uraemia appears to be relevant to long-term complications associated with chronic renal failure and dialysis, such as dialysis-related amyloidosis. Immunohistochemical studies, with specific antibodies to AGEs and ALEs, identified carbonyl stress in long-lived beta2-microglobulin amyloid deposits. Furthermore, proteins modified with carbonyl stress exhibit several biological activities through interactions with several types of cell, e.g. monocytes/macrophages, synovial cells and osteoclasts/osteoblasts, which might partially account for dialysis arthropathies. PMID- 10737164 TI - Treatment modalities in comparison: when do clinical differences emerge? AB - BACKGROUND: Despite technological advances in dialysis equipment and modalities, the survival, morbidity and quality of life of uraemic patients undergoing regular haemodialysis treatment are still severely affected by acute intradialysis and long-term complications, possibly related to the treatment itself. Convective treatment modalities, such as haemodiafiltration and haemofiltration, are thought to be further improvements over standard diffusive haemodialysis. Moreover, several of the pathways activated in patients during dialysis have the potential to produce many side-effects. These occur three times a week, and are particularly intense in patients dialysed with so-called 'bio incompatible' membranes. Thus the biocompatibility of dialysis membranes is increasingly recognized as one of the main factors in the improvement of dialysis treatment. METHODS: The main clinical studies to date are reviewed to highlight the clinical effects of different treatment modalities and membranes on the most important acute and long-term haemodialysis-related complications. RESULTS: Epidemiological studies suggest that semisynthetic and synthetic membranes may reduce morbidity and mortality in dialysis patients. Despite the proven biological superiority of biocompatible membranes, we lack definitive evidence that thrice-weekly complement and cell activation over a period of years is detrimental to patients, because the results of prospective randomized studies are conflicting. However, it is important to remember that factors other than the dialysis membrane could influence the 'biocompatibility' of dialysis, including the dialysate, dialyser geometry, the distribution of blood in the dialyser, reuse, the sterilizant and materials used in reprocessing. CONCLUSIONS: Further large-scale prospective and randomized trials with a long follow-up are needed in order to better clarify the clinical effect of different treatment modalities on the morbidity and mortality of patients on chronic renal replacement therapy. In particular, it must be clarified whether the possible clinical differences in treatment modalities are based on differences in the clearance of middle molecules or on biocompatibility, or, more generally, on the increasingly recognized clinical importance of high-flux treatments, and the possible interaction between membrane flux and biocompatibility. PMID- 10737165 TI - Improved outcome for haemodialysis patients treated with high-flux membranes. AB - Improved survival for haemodialysis patients has been reported for synthetic, high-flux biocompatible membranes. The reported data fail to answer the question whether improved survival is related to an effect of enhanced biocompatibility or to increased clearance of larger molecular species of putative uraemic toxins. A retrospective analysis of 715 patients treated by continuous haemodialysis for up to 5 years was undertaken. Low-flux polysulfone dialysis was used exclusively for 252 patients and 463 patients were exposed for at least 3 months to high-flux polysulfone dialysis. Patients treated with high-flux dialysis had a lower mortality (21 vs 36 per 1000 years) and significantly lower standardized mortality ratio. For non-diabetic patients the 5-year probability of survival was significantly greater for high-flux patients (Kaplan-Meier: 92% vs 69%; P=0.036). High-flux dialysis significantly reduced the adverse effect of age on survival. In a Cox proportional hazard model membrane flux (high vs low) was one of the covariates with strong predictor value for reduction of death risk in non diabetic patients. Although other variables may explain the better survival of patients exposed to high-flux dialysis the data reported here suggest that higher membrane flux, implying higher clearance of larger molecular species and independent of biocompatibility, is associated with improved survival for haemodialysis patients. PMID- 10737166 TI - On-line haemodiafiltration versus low-flux haemodialysis. A prospective randomized study. AB - BACKGROUND: Current methods of renal replacement therapy lead only to an insignificant removal of larger, potentially toxic, substances, which are excreted by healthy kidneys. On-line preparation of substituate from dialysate and the use of high-flux membranes allow substantial convective removal of such substances. A modified on-line haemodiafiltration method with the use of a large membrane surface and a high convective part was chosen to test whether the elimination of larger substances, such as low-molecular-mass proteins, has a clinical impact. METHODS: In a prospective, controlled study over 24 months, 44 unselected chronic dialysis patients were randomized to undergo either low-flux haemodialysis (HD; n = 21) or haemodiafiltration (HDF; n = 23). To eliminate confounding factors, low-molecular efficacy was matched (Kt/V 1.8), and the same membrane material (polysulfone), ultrapure dialysate and the same treatment duration (4.5 h) were applied to each group. RESULTS: Morbidity, mortality, blood pressure, dialysis-associated hypotensive episodes, haematocrit and erythropoietin dose did not differ between the groups. The same was true for body weight and, accordingly, bioimpedance values, clinical hydration score, skinfold thickness, plasma albumin, prealbumin and transferrin. beta2-Microglobulin in the plasma did not change in the HD group and varied between 32 and 43 mg/l throughout the 2 years. In HDF, beta2 microglobulin decreased from similar values to 18 mg/l predialysis (P<0.01) in the first 6 months of HDF treatment and then remained constant during the remaining 18 months. CONCLUSION: In the absence of any clinical marker of uraemic toxicity the removal of larger molecules over the time-span of 2 years during HDF had no clinical implication compared with extremely (and for routine practice unrealistically) well-dialysed patients with low-flux HD. In the absence of any side-effects of on-line HDF and supposing that plasma beta2-microglobulin is a marker of morbidity, on-line HDF ensures an excellent dialysis quality which apparently takes time to translate into measurable clinical sequelae. PMID- 10737167 TI - On-line haemodiafiltration. Remarkable removal of beta2-microglobulin. Long-term clinical observations. AB - BACKGROUND: The accumulation of beta2-microglobulin (beta2-M) in long-term dialysis patients may lead to dialysis amyloidosis. In this respect, the removal with different modes of on-line haemodiafiltration (HDF) of beta2-M was studied. Long-term clinical observations in patients with more than 10 years of dialysis, treated mainly with biocompatible and highly permeable membranes and in the last years with on-line HDF are also reported. METHODS: In the first part of this report, the reduction ratios and clearances of beta2-M, blood urea nitrogen, creatinine and phosphorus (P) of on-line HDF with 40 to 120 ml/min replacement fluid are compared with bicarbonate haemodialysis (HD). In the second part, we investigated 16 patients with more than 10 years of dialysis treatment. The prevalence of dialysis amyloidosis and the mean values for serum albumin, serum total cholesterol, HDL and LDL cholesterol and parathyroid hormone are reported, as well as the mean dose of erythropoietin. RESULTS: In the first part with on line HDF, starting from HDF 60 ml/min a significantly higher beta2-M reduction ratio and clearance vs HD is noted. In HDF100 (i.e. with 241 replacement volume per 4-h treatment) vs HD, a beta2-M reduction ratio of 72.7% vs 49.7% (P= 0.0000) and a beta2-M clearance of 116.8 vs 63.8 ml/min (P=0.0000) was obtained. Comparing HDF120 with HDF100, there is a significantly higher beta2-M clearance with the former (P<0.005), although the beta2-M reduction ratio was not significantly better. In the HDF120 session the amount of beta2-M in the total dialysate was 292 mg per session. If one adds the known 17% adsorption on the polysulfone membrane, a total of 341.6 mg beta2-M per session is removed, which adds up to 1024.8 mg a week. Concerning the small molecules, our results with HDF100 also show a higher creatinine and especially P clearance vs HD. In the second part with 16 patients with more than 10 years of dialysis treatment (mean 14 years 1 month), the mean time on HDF amounted to 39.5% of the total treatment time. In four patients only biocompatible and highly permeable membranes were used, AN69 and mainly polysulfone, and in four other patients these membranes were used for more than 95% of the treatment time. Therefore, it is not surprising that the prevalence of carpal tunnel syndrome was only 12.5% in the patients after 10 years of dialysis. Twenty-five percent of these patients met the criteria for diagnosis of beta2-M bone-amyloidosis, proposed by van Ypersele de Strihou et al., but without a retrospective X-ray analysis. The mean predialysis beta2-M value was 29.6 mg/l. The mean values for serum albumin, serum total cholesterol, HDL and LDL cholesterol were within normal limits. For the parathyroid hormone a mean of 287.5 pg/ml was found. Subtotal parathyroidectomy was performed in five patients. The mean dose of 43 U erythropoietin/kg per session is comparable with those reported in the literature. Conclusions. Like Canaud, in our renal unit, treatment with on-line HDF with a highly permeable and biocompatible membrane has proven to be an efficient, well-tolerated and safe technique. Furthermore it leads to a low prevalence of dialysis amyloidosis and a superior P clearance. However, continuous attention must be paid to an on-line sterile and apyrogenic dialysate. Although on-line HDF is undoubtedly a more optimal approach of chronic dialytic treatment, it also carries a higher cost, which is currently evaluated to be nearly US$11 per session. PMID- 10737168 TI - Should ultra-pure dialysate be mandatory? PMID- 10737169 TI - On-line haemodiafiltration. Safety and efficacy in long-term clinical practice. PMID- 10737170 TI - Cardiovascular stability during haemodialysis, haemofiltration and haemodiafiltration. AB - Several comparative studies have claimed that procedures based substantially or exclusively on pressure-driven water-solute transport, such as haemodiafiltration or haemofiltration, afford better protection of the cardiovascular tolerance to fluid removal than conventional haemodialysis. During each depurative modality, several factors are set in motion that might impact, each in its own right, upon the haemodynamic response to fluid withdrawal. To explore the haemodynamic effect of each of them singularly, one needs to keep all other components unvaried. However, this is very difficult to accomplish. For instance, to confirm the alleged greater protection of cardiovascular stability by pure convection vs diffusion, one needs to keep unvaried all the other factors potentially affecting haemodynamic tolerance, i.e. the rate of body fluid removal, the membrane, the buffer, the blood temperature in the extracorporeal circuit, depuration efficiency, the sodium balance, the fluid sterility and so on. Such studies are still awaited. However, clinical trials published to date have not resolved the question of whether haemofiltration and haemodiafiltration provide a better haemodynamic tolerance to fluid removal. If we limit our consideration to controlled trials only, most prospective studies have adopted a cross-over design implemented on very small patient samples and for very short periods. Such an approach is liable to generate misleading results because the incidence of dialysis hypotension often fluctuates from time to time. Owing to such fluctuations, results can be strongly affected by the 'order effect' of the cross over from one technique to the other. The negative results provided by parallel comparisons of procedures should be taken with caution because patients samples did not include a suitable proportion of unstable patients. PMID- 10737171 TI - Can sterile and pyrogen-free on-line substitution fluid be routinely delivered? A multicentric study on the microbiological safety of on-line haemodiafiltration. AB - BACKGROUND: Microbial contamination is characterized not only by the presence of bacteria, but also by high concentrations of biologically active by-products. They are potentially able to cross ultrafiltration and dialysis membranes and stimulate immunocompetent blood cells to synthesize cytokines. In turn, cytokine induction causes acute symptoms and has been incriminated in the long-term complications of haemodialysis patients. Infusion of large volumes of substitution fluids following ultrafiltration of microbially contaminated dialysis fluids may place patients on on-line therapies at particular risk. METHODS: In this study we evaluated 30 machines with a two-stage ultrafiltration system in routine clinical haemodiafiltration settings in six centres for 6 months. Microbiological safety was assessed monthly and at the last use of the filters by determining microbial counts, endotoxin concentration and cytokine inducing activity. RESULTS: No pyrogenic episodes were observed during the study period. Double-filtration of standard dialysis fluid (range, <1-895 cfu/ml, 0.0028-4.6822 IU/ml) resulted in sterile substitution fluids with endotoxin concentrations well below the Ph.Eur. standard for haemofiltration solutions (range, 0.0014-0.0281 vs 0.25 IU/ml). Moreover, they did not differ from commercial haemofiltration solutions and depyrogenated saline. Likewise, there was no difference in the cytokine-inducing activity between the solutions tested. The high microbiological quality of the ultrafiltered dialysis fluid, which was in the same range as substitution fluid, translates into both the absence of cytokine induction by dialyser back-transport and a redundant safety mode of the on-line system by a second filtration step. CONCLUSION: On-line HDF treatment can routinely be provided with ultra-pure dialysis fluids and sterile substitution fluids at pyrogen-free levels. The online preparation of substitution fluids thus can be considered microbiologically safe. PMID- 10737172 TI - Production of extracellular lipases by Penicillium cyclopium purification and characterization of a partial acylglycerol lipase. AB - Penicillium cyclopium, grown in stationary culture, produces a type I lipase specific for triacylglycerols while, in shaken culture, it produces a type II lipase only active on partial acylglycerols. Lipase II has been purified by ammonium sulfate precipitation and chromatographies on Sephadex G-75 and DEAE Sephadex. The enzyme exists in several glycosylated forms of 40-43 kDa, which can be converted to a single protein of 37 kDa by enzymatic deglycosylation. Activity of lipase II is maximal at pH 7.0 and 40 degrees C. The enzyme is stable from pH 4.5 to 7.0. Activity is rapidly lost at temperatures above 50 degrees C. The enzyme specifically hydrolyzes monoacylglycerols and diacylglycerols, especially of medium chain fatty acids. The sequence of the 20 first amino acid residues is similar to the N-terminal region of P. camembertii lipase and partially similar to lipases from Humicola lanuginosa and Aspergillus oryzae, but is different from Penicillium cyclopium lipase I. However, it can be observed that residues of valine and serine at positions 2 and 5 in Penicillium cyclopium lipase II are conserved in Penicillium expansum lipase, of which 16 out of the 20 first amino acid residues are similar to Penicillium cyclopium lipase I. PMID- 10737173 TI - Purification and partial characterization of a novel glucanhydrolase from Lipomyces starkeyi KSM 22 and its use for inhibition of insoluble glucan formation. AB - A novel glucanhydrolase from a mutant of Lipomyces starkeyi ATCC 74054 was purified. The single protein (100 kDa) showed either dextranolytic or amylolytic activity. We referred to the glucanhydrolase as a DXAMase. The DXAMase was produced in a starch medium and it was 3.75-fold more active for hydrolysis of the purified insoluble-glucan of Streptococcus mutans than Penicillium funiculosum dextranase. Aggregation of S. mutans cells with dextran and adherence to glass were eliminated by incubating with the DXAMase. The addition of DXAMase (0.1 IU/ml) to the mutansucrase reaction digest with sucrose reduced the formation of insoluble-glucan about 80%. Also the DXAMase (0.5 IU/ml) removed 80% of the pre-formed sucrose-dependent adherent film. These in vitro properties of L. starkeyi KSM 22 DXAMase are desirable for its application as a dental plaque control agent. PMID- 10737174 TI - Cloning and characterization of a gene complementing the mutation of an ethanol sensitive mutant of sake yeast. AB - Ethanol-sensitive mutants (esl to es10) were isolated from sake yeast, Saccharomyces cerevisiae SY-32. These mutants were unable to grow at 7% ethanol at which the wild type strain SY-32 does grow. The mutants had a variety of fermentation rates and viabilities in the presence of ethanol. The gene ERG6, complementing the ethanol-sensitive mutation of es5, was cloned from an SY-32 gene library. ERG6 encodes S-adenosylmethionine: delta 24-sterol-C methyltransferase (EC 2.1.1.41) in the ergosterol synthetic pathway. Mutant es5 had a reduced ability to synthesize ergosterol. An erg6 disruptant was also ethanol-sensitive. These results suggested that ERG6 plays an important role in the ethanol tolerance of S. cerevisiae. PMID- 10737175 TI - Serum glucose and insulin response in rats administered with sucrose or starch containing adenosine, inosine or cytosine. AB - Blood glucose and insulin responses and gastric emptying were examined in rats intubated with sucrose or soluble starch that contained adenosine, inosine and cytosine. The increase in serum glucose and insulin levels in the rats following loading with sucrose (2.5 g/kg of body weight) or soluble starch (1.875 g/kg of body weight) was significantly reduced by the administration of adenosine, inosine and cytosine (0.0625-0.125 g/kg of body weight). The gastric emptying rates were only marginally affected by the nucleoside administration. The activities of sucrase, maltase, isomaltase and glucoamylase in a crude preparation from the small intestinal mucosa of rats were mildly inhibited by the nucleosides. The decrease in blood glucose and insulin levels may have been in response to a decrease in glucose absorption caused by the inhibiting effect of the nucleosides on the mucosal enzymes that digest sucrose, maltose, and malto- and isomalto-oligosaccharides. PMID- 10737176 TI - Biosynthesis of depudecin, a metabolite of Nimbya scirpicola. AB - Feeding experiments of labeled acetates to Nimbya scirpicola proved the carbon origin of the straight-chain polyketide, depudecin. Differential hydrogen exchange of the 2H label originating from 2H labeled acetate along the polyketide chain occurred. In particular, the deuterium of an epoxide methine at C-3 was lost to a substantial extent in the formation of depudecin. PMID- 10737177 TI - Effects of Ethanolamine as a nitrogen source on hydrogen production by Rhodobacter capsulatus. AB - Ethanolamine was examined as a nitrogen source in the production of hydrogen by Rhodobacter capsulatus ST-410, a hydrogenase-deficient mutant of the strain B 100. It was found that ethanolamine supports cell growth as the sole nitrogen source and permits a large amount of hydrogen evolution, detected at 138 micromol/ml-culture from 3.5 mM ethanolamine and 30 mM DL-malate. The amount corresponded to a stoichiometric yield of 77% and was close to that obtained from 7.0 mM L-glutamate and 30 mM DL-malate. The hydrogen evolution rate per unit biomass (cells) was higher than that with L-glutamate, and the cells grown with ethanolamine had higher nitrogenase activity than the cells grown with L glutamate. In terms of bioconversion of cellulosic and hemicellulosic biomass to hydrogen, D-glucose, D-xylose, and D-cellobiose were tested as substrates. The results indicated that those sugars permit a large evolution of hydrogen through cultivation with ethanolamine as a nitrogen source. For instance, the cells grown with 3.5 mM ethanolamine evolved hydrogen of 289 micromol/ml-culture (80% yield) from 30 mM D-glucose under a controlled pH of 6.4 to 6.9. PMID- 10737178 TI - Characterization of the cellulolytic complex (cellulosome) from Ruminococcus albus. AB - The cellulolytic complex was isolated from the culture supernatant of Ruminococcus albus strain F-40 grown on cellulose by a Sephacryl S-300HR column chromatography. The molecular mass of the cellulolytic complex was found to be larger than 1.5 x 10(6) Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the cellulolytic complex contained at least 15 proteins with molecular weights from 40kDa to 250 kDa. Among them, 11 proteins showed endoglucanase and/or xylanase activities on the zymograms. Immunological analysis using an antiserum raised against the dockerin domain of endoglucanase VII of R. albus (DocVII) suggested that at least 7 proteins in the cellulolytic complex contained a dockerin domain immunoreactive with the anti-Doc-VII antiserum. Furthermore, DocVII was shown to specifically interact with a 40-kDa protein of the cellulolytic complex by Far-Western blot analysis. These results strongly suggest that the cellulolytic complex produced by R. albus resembles the cellulosome specified for the cellulolytic complex of several clostridia such as Clostridium thermocellum and respective components are assembled into the cellulosome by the mechanism common in all of the cellulolytic clostridia, i.e., the cellulosome is formed by the interaction between a dockerin domain of catalytic components and a cohesin domain of a scaffolding protein. PMID- 10737179 TI - An organic solvent resistant tyrosinase from Streptomyces sp. REN-21: purification and characterization. AB - We found a tyrosinase, which has high activity in the presence of organic solvents, in the culture filtrate of Streptomyces sp. REN-21. The organic solvent resistant tyrosinase (OSRT) was purified from the culture filtrate by three column chromatographies. About 1.2 mg of purified OSRT was obtained from 5.6 liters of the culture filtrate with a yield of 26.0%. The purified enzyme had a single polypeptide chain with a molecular mass of about 32,000 Da. The optimum pH and temperature of OSRT were pH 7.0 and 35 degrees C using L-beta-(3,4 dihydroxyphenyl)alanine (L-DOPA) as substrate. OSRT showed stereospecificity toward L-, DL-, and D-enantiomers of DOPA or tyrosine. OSRT had 44% of the activity of the control even in the presence of 50% ethanol, while a mushroom tyrosinase showed only 6% activity under the same conditions. Moreover, OSRT retained its original activity even after 20 h of incubation at 30 degrees C in the presence of 30% ethanol. PMID- 10737181 TI - Interspecific transformation of Bacillus subtilis competent cells by chromosomal DNA in lysates of protoplasts of Bacillus amyloliquefaciens. AB - Competent cells of Bacillus subtilis were transformed with chromosomal DNA in lysates of protoplasts of B. subtilis or B. amyloliquefaciens. The interspecific transformation frequency of B. subtilis by cysA in a conserved region was 3.1 x 10(4) transformants per microg DNA, 60 times higher than that for conventional transformation using purified DNA. Increased interspecific transformation frequencies of B. subtilis were also observed for arg-1, lys-1, leuB, aroG, thr 5, hisH, or metC markers outside the conserved region (3.1 x 10 approximately 5.2 x 10(2) transformants per microg DNA). An interspecific cotransformation ratio (33-50%) as high as an intraspecific one (46%) using purified DNA was also detected between cysA and rpsL markers, which are separated by 16 kb on the B. subtilis chromosome. Interspecific double transformation of the cysA-arg-1 or cysA-metC marker was observed, which have not been detected for conventional transformation. The involvement of mutS in the interspecific transformation was not significant. PMID- 10737180 TI - Activation of maturation promoting factor and 26S proteasome assembly accelerated by a high concentration of 1-methyladenine in starfish oocytes. AB - In the oocyte maturation process of the starfish Asterina pectinifera, the extent of inhibition of germinal vesicle breakdown (GVBD) by the proteasome inhibitor MG115 (benzyloxycarbonyl-leucyl-leucyl-norvalinal), as well as the timing of activation of pre-MPF (inactive maturation promoting factor) and 26S proteasome assembly, were found to be dependent on the concentration of the maturation inducing hormone 1-methyladenine (1-MeAde). Activation of pre-MPF was accelerated by increasing the concentration of 1-MeAde, while there was little effect on the time required for GVBD. Assembly of the 26S proteasome was also accelerated by increasing the concentration of 1-MeAde. These results indicate that a higher concentration of 1-MeAde triggers acceleration of the assembly and increase in the activity of the 26S proteasome, which results in activation of pre-MPF, although there is little effect on the timing of GVBD. It was also clarified that the timing of GVBD is controlled by a rate-liming step after MPF-activation. PMID- 10737182 TI - Thermostable lipase of Bacillus Stearothermophilus: high-level production, purification, and calcium-dependent thermostability. AB - An efficient expression system was developed for the production of the thermostable lipase from Bacillus stearothermophilus L1 in an Escherichia coli system. A structural gene corresponding to mature lipase was subcloned in the pET 22b(+) expression vector and its expression was induced by IPTG at 30 degrees C in E. coli cells. The lipase activity in a cell-free extract was as high as 448,000 units/g protein, which corresponds to as much as 26% of the total cellular protein and is 77 times higher than that of E. coli RR1/pLIP1. Based on its pI (7.4) and pH stability data reported previously, the L1 lipase was efficiently purified to homogeneity with CM (at pH 6.0) and DEAE (at pH 8.8) column chromatographies with a recovery yield of 62%. The specific activity of the purified enzyme was 1700 units/mg protein when olive oil emulsion was used as a substrate. Its optimum temperature for the hydrolysis of olive oil was 68 degrees C and it was stable up to 55 degrees C for 30 min-incubation. The thermostability increased by about 8-10 degrees in the presence of calcium ions. This calcium-dependent thermostability was confirmed by the tryptophan fluorescence emission kinetics showing that the enzyme starts to unfold at 66 degrees C in the presence of calcium ions but at 58 degrees C in the absence of calcium ions, implying that the calcium ions bind to the thermostable enzyme and stabilize the protein tertiary structure even at such high temperatures. PMID- 10737183 TI - Inhibiting effects of theanine on caffeine stimulation evaluated by EEG in the rat. AB - In this study, the inhibiting action of theanine on the excitation by caffeine at the concentration regularly associated with drinking tea was investigated using electroencephalography (EEG) in rats. First, the stimulatory action by caffeine i.v. administration at a level higher than 5 micromol/kg (0.970 mg/kg) b.w. was shown by means of brain wave analysis, and this level was suggested as the minimum dose of caffeine as a stimulant. Next, the stimulatory effects of caffeine were inhibited by an i.v. administration of theanine at a level higher than 5 micromol/kg (0.781 mg/kg) b.w., and the results suggested that theanine has an antagonistic effect on caffeine's stimulatory action at an almost equivalent molar concentration. On the other hand, the excitatory effects were shown in the rat i.v. administered 1 and 2 micromol/kg (0.174 and 0.348 mg/kg) b.w. of theanine alone. These results suggested two effects of theanine, depending on its concentration. PMID- 10737184 TI - Alpha-glucosidase inhibitors from clove (Syzgium aromaticum). AB - The ellagitannins, casuarictin and eugeniin, were isolated as rat intestinal maltase inhibitors from methanol extracts of clove (Syzgium aromaticum). Eugeniin showed inhibitory activity with an IC50 value of 10(-3) M. A structure-activity relationship study among the isolates and their related compound, penta-O-galloyl beta-D-glucose, indicates that an increasing number of galloyl units in the molecule might lead to an increase in the inhibitory activity. Eugeniin also inhibited maltase activity toward the human intestinal epithelial cell line, Caco 2. PMID- 10737185 TI - 1-aminocyclopropane-1-carboxylate (ACC) deaminase induced by ACC synthesized and accumulated in Penicillium citrinum intracellular spaces. AB - We have already described how 1-aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of the plant hormone ethylene, is synthesized in Penicillium citrinum through the same reaction by the catalysis of ACC synthase [EC 4.4.1.14] as in higher plants. In addition, ACC deaminase [EC 4.1.99.4], which degrades ACC to 2-oxobutyrate and ammonia, was also purified from this strain. To study control of induction of ACC deaminase in this organism, we have isolated and analyzed the cDNA of P. citrinum ACC deaminase and studied the expression of ACC deaminase mRNA in P. citrinum cells. By the analysis of peptides from the digests of the purified and modified ACC deaminase with lysylendopeptidase, 70 % of its amino acid sequences were obtained. These amino acid sequences were used to identify a cDNA, consisting of 1,233 bp with an open reading frame of 1,080 bp encoding ACC deaminase with 360 amino acids. The deduced amino acids from the cDNA are identical by 52% and 45% to those of enzymes of Pseudomonas sp. ACP and Hansenula saturnus. Through Northern blot analysis, we found that the mRNA of ACC deaminase was expressed in P. citrinum cells grown in a medium containing 0.05% L methionine. These findings suggest that ACC synthesized by ACC synthase and accumulated in P. citrinum intracellular spaces can induce the ACC deaminase that degrades the ACC. PMID- 10737186 TI - Studies on the 1,1-diphenyl-2-picrylhydrazyl radical scavenging mechanism for a 2 pyrone compound. AB - The radical scavenging mechanisms for the 2-pyrone compound, 4-hydroxy-3,6 dimethyl-2H-pyrane-2-one (1), and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (4) in several solvent systems were evaluated by the quantitative change in compounds detected at 270 nm and subsequent HPLC analyses. The HPLC profile for each condition suggested that the reaction proceeded by a different mechanism in each solvent system. In organic solvents (CHCl3, iso-propanol, and EtOH), 1-[4 (3,4-dihydro-3,6-dimethyl-2,4-dioxo-2H-pyran-3-yl) phenyl]-1-phenyl-2 picrylhydrazine (2) was produced as an adduct of the DPPH radical and 1. On the other hand, the reaction in a buffer solution (an acetate buffer at pH 5.5) gave several degradation products with 1[4-(2,3-dihydro-2,5-dimethyl-3-oxo-fur-2-yl) phenyl]-1-phenyl-2-picrylhydrazine (5), this being structurally elucidated by spectroscopic analyses. The decrease of the DPPH radical in each reaction system suggests that compound 1 could scavenge about 1.5-1.8 equivalents of the radical in organic solvents and about 3.5-3.9 in the buffer solution. PMID- 10737187 TI - Role of basic residues of human lactoferrin in the interaction with B lymphocytes. AB - We have previously demonstrated that lactoferrin was incorporated into B lymphocytes and that a trypsin treatment for a short period reduced the number of lactoferrin molecules incorporated into B lymphocytes. An N-terminal sequence analysis revealed that the mild trypsin treatment had cleaved the three N terminal amino acids, Gly1-Arg2-Arg3. Chemical conjugation of lost sequence analogue Gly-Arg-Arg-Gly with the mildly digested lactoferrin recovered the interaction with B lymphocytes, while conjugation of acetyl-Arg-Arg-Gly, a deamino analogue of Gly-Arg-Arg-Gly, did not recover the interaction. This shows that the N-terminal basic region containing N-terminal Gly played an important role in the interaction with B lymphocytes. Acylation of the amino groups of lactoferrin also significantly reduced the interaction with B lymphocytes, and an O-methylisourea treatment of the amino groups, which preserved the positive charge, hardly affected the interaction. These results suggest that both the N terminal basic region and the basic characteristics of the whole molecule contributed to its interaction with B lymphocytes. PMID- 10737188 TI - Subsite structure and catalytic mechanism of a new glycosyltrehalose-producing enzyme isolated from the hyperthermophilic archaeum, Sulfolobus solfataricus KM1. AB - A glycosyltrehalose-producing enzyme from Sulfolobus solfataricus KM1 catalyzes a conversion of maltooligosaccharides to glycosyltrehaloses and also hydrolyzes maltooligosaccharides to liberate glucose, as a side reaction. From the sum of the conversion and hydrolysis reaction rates, the rate parameters involved in the "splitting" of the alpha-1,4 glucosidic linkage were calculated. From the data obtained, the subsite structure for maltooligosaccharides was identified. From the analysis of the hydrolysate of maltotriose in [18O labeled H2O, the hypothesis of the C1-O bond splitting and the formation of a glycosyl (maltosyl) enzyme intermediate was strongly supported. From the analysis of the reaction product in the presence of [3H] labeled glucose, the occurrence of intermolecular transglycosylation was confirmed. These data strongly support the suggestion that the catalytic mechanism of this enzyme is a transglycosylation. PMID- 10737189 TI - Apoptosis induced by nicotinamide-related compounds and quinolinic acid in HL-60 cells. AB - In our previous paper, we have reported that niacin-related compounds, particularly picolinic acid, dipicolinic acid, and isonicotinamide, induced apoptosis in HL-60 cells but that niacin did not. Moreover, picolinamide, N1 methylnicotinamide, 6-aminonicotinamide, quinolinic acid, and cinchomeronic acid also had the function of DNA fragmentation in HL-60 cells analyzed by flow cytometry, the ratio of DNA fragmentation finally being about 40% after treatment with these compounds at 10 mM for 24 h. In this study, we found that these compounds also induced apoptosis in HL-60 cells. The wide-spectrum caspase inhibitors prevented DNA fragmentation induced by these compounds. Interestingly, 6-aminonicotinamide induced apoptosis at a comparatively low concentration, while picolinic acid, dipicolinic acid, and isonicotinamide did not at 1 mM. Our results suggest that both NAD metabolism and NAD biosynthesis may be related to the process of apoptosis induced by niacin-related compounds. PMID- 10737190 TI - Purification and some properties of a beta-glucosidase from Flavobacterium johnsonae. AB - Flavobacterium johnsonae was isolated as a microorganism that produced a beta glucosidase with hydrolytic activity of beta-glucosyl ester linkages in steviol glycosides. The enzyme was purified to homogeneity from a cell-free extract by streptomycin treatment, ammonium sulfate fractionation, and column chromatographies on S-Sepharose and phenyl-Toyopearl. The molecular mass of the purified enzyme was about 72 kDa by SDS-PAGE. An isoelectric point of pI 8.8 was estimated by isoelectric focusing. The enzyme was most active at pH 7.0, and was stable between pH 3.0 and 9.0. The optimum temperature was 45 degrees C, and the enzyme was stable below 35 degrees C. The enzyme hydrolyzed glucosyl ester linkages at site 19 of rebaudioside A, stevioside, and rubusoside, although it could not degrad beta-glucosidic linkages at site 13 of rebaudioside B or steviol bioside. The enzyme acted on aryl beta-glucosides such as p-nitrophenyl beta glucoside, phenyl betaglucoside, and salicin, and glucobioses such as sophorose and laminaribiose. The enzyme activity on Rub was inactivated completely by Hg2+, and reduced by Fe3+, Cu2+, p-chloromercuric benzoate, and phenylmethylsulfonyl fluoride (residual activity; 67.9-84.8%). The pNPG hydrolysis was also inactivated to almost the same degrees. Kinetic behaviors in the mixed substrate reactions of rebaudioside A and steviol monoside, and of steviol monoglucosyl ester and phenyl beta-glucoside suggested the glucosidic and glucosyl ester linkages were hydrolyzed at a single active site of the enzyme. PMID- 10737191 TI - Synthesis of four stereoisomers of 1,4-thiazane-3-carboxylic acid 1-oxide via the asymmetric transformation (combined isomerization-preferential crystallization) of 1,4-thiazane-3-carboxylic acid. AB - In order to synthesize four stereoisomers of 1,4-thiazane-3-carboxylic acid 1 oxide (TCA SO), (S)-1,4thiazane-3-carboxylic acid [(S)-TCA], which is one of the precursors, was prepared by the asymmetric transformation (combined isomerization preferential crystallization) of (RS)-TCA. This asymmetric transformation was used (2R, 3R)-tartaric acid [(R)-TA] as a resolving agent and salicylaldehyde as the epimerization catalyst in propanoic acid at 110 degrees C to afford a salt of (S)-TCA with (R)-TA in 100% de with a yield of over 90%. Optically pure (S)-TCA was obtained by treating the salt with triethylamine in methanol in a yield of over 80%, based on (RS)-TCA as the starting material. In addition, asymmetric transformation of (R)-TCA gave (S)-TCA in a yield of 60-70%. (S)-TCA was oxidized by hydrogen peroxide in dilute hydrochloric acid to selectively crystallize (1S, 3S)-TCA.SO. (1R, 3S)-TCA SO of 70% de from the filtrate was allowed to form a salt with (R)-TA after a treatment with triethylamine to give (1R, 3S)-TCA SO as a single diastereoisomer. (1R, 3R)- and (1S, 3R)-TCA.SO were also prepared by starting from (R)-TCA that had been synthesized from L-cysteine. PMID- 10737192 TI - Inhibitory effects of bovine lactoferrin on the adherence of enterotoxigenic Escherichia coli to host cells. AB - Adherence is an essential and prerequisite step for the colonization of mucosal surfaces by enterotoxigenic Escherichia coli (ETEC). We studied the effect of bovine lactoferrin (BLF) on the adherence of ETEC to human epithelial cells in vitro, and to intestinal mucosa of ICR germfree mice in vivo. In the in vitro study, BLF was found to inhibit the adherence of ETEC. This adhesion-inhibiting activity of BLF was found to lessen with decreasing BLF concentration, but the data obtained suggest a positive inhibitory effect of BLF against the adhesion of ETEC cells. In the in vivo study, the counts of adherent bacteria in various sections of the intestinal tract (duodenum, jejunoileum, and large intestine) were lower in the BLF group than in the control group, suggesting the possible action of BLF as an intestinal tract adherence-blocking agent with regards to ETEC. PMID- 10737193 TI - Isolation of a cDNA encoding a 31-kDa, pathogenesis-related 5/thaumatin-like (PR5/TL) protein abundantly expressed in apple fruit (Nalus domestica cv. Fuji). AB - A fruit-specific and pathogenesis-related 5/thaumatin-like (PR5/TL), 31-kDa protein was isolated by 2D-PAGE from fully-grown apples (Malus domestica cv. Fuji) and named Mdtl1 (Malus domestica thaumatin-like protein 1). Using the N terminal sequence of the protein, the full-length cDNA encoding Mdtll was isolated. The cDNA clone (Mdtl1) consists of 944 bp with an open reading frame (ORF) of 744 bp encoding a protein of 247 amino acids. The deduced amino acid sequence of Mdtl1 shows high similarity to the sequences of PR5/TL proteins. Mdtl1 is a slightly acidic protein with a putative signal peptide and a putative N-glycosylation site, and lacks a C-terminal extension. This suggests that Mdtl1 is an apoplastic glycoprotein. Results of northern blotting indicated that expressions of Mdtl1 are developmentally regulated. Southern blot analysis showed that Mdtl1 may be present as a single copy, and there exist other genes closely related to Mdtl1 in the apple genome. PMID- 10737194 TI - Synthesis of regioselectively protected forms of cytidine based on enzyme catalyzed deacetylation as the key step. AB - N4-Acetylcytidine (77%) and 2',3'-O, N4-triacetylcytidine (95%) were obtained from the hydrolysis of a common precursor, the peracetylated form of cytidine with Aspergillus niger lipase (Amano A) and Burkholderia cepacia esterase (SC esterase S), respectively, under very mild conditions. The experimental procedure for the conversion of triacetylcytidine to a corresponding phosphoramidite (82%), an intermediate for sugar nucleotide synthesis, is also elaborated. PMID- 10737195 TI - Cloning and characterization of EPD2, a gene required for efficient pseudohyphal formation of a dimorphic yeast, Candida maltosa. AB - Candida maltosa is a dimorphic fungus and its pseudohyphal growth is partly induced by additional copies of the centromeric DNA (CEN) region1) or n-alkane. In the course of analyzing the induction mechanism of pseudohyphal growth, we isolated EPD1, which is similar in sequence to PHR1 and PHR2 of Candida albicans. Epdlp could be involved in cell wall maintenance and is essential for pseudohyphal growth induced by CEN and n-hexadecane at pH 4 and by n-hexadecane at pH 7.) In this paper, we cloned EPD2 of C. maltosa, which is highly similar to EPD1, PHR1, and PHR2. The transcription of EPD2 is induced strongly when cells are grown in SD medium of higher pH (pH 7), but not in SD medium of lower pH (pH 4). This pattern of expression was an inverse of that of EPD1. This alternate expression is similar to that between PHR1 and PHR2. The expression of EPD2 was much higher when C. maltosa was grown on the n-hexadecane solid medium than grown in the n-hexadecane liquid medium. The efficiency of pseudohyphal formation of an epd2 null mutant on n-hexadecane medium at pH 7 or 7.5 was lower than that of the wild-type strain. These results suggest that Epd2p is required for efficient pseudohyphal formation induced by n-hexadecane in the medium at pH 7. PMID- 10737196 TI - Identification of alpha-D-glucosylglycerol in sake. AB - alpha-D-Glucosylglycerol (GG) was found for the first time in sake (Japanese rice wine) in an amount of about 0.5%. GG was also found in miso and mirin which had been brewed by using koji. GG was hydrolyzed into glucose and glycerol in an equimolar ratio with maltase (EC 3.2.1.20, alpha-glucosidase from yeast), but not with emulsin (EC 3.2.1.21, beta-glucosidase from almond). The retention times and mass spectra of trimethylsilyl derivatives by a GC-MS analysis of GG in sake were comparable to those of various GG samples synthesized by glycol cleavage. It was proven that GG in sake consisted of three components, viz., 2-O-alpha-D-glucosyl glycerol (GG-II), (2R)-1-O-alpha-D-glucosylglycerol (R-GG-I) and (2S)-1-O-alpha-D glucosylglycerol (S-GG-I). The ratio of the three components in GG was 6:66:28 for sake. It is considered that GG was formed by transglucosylation of the glucosyl groups to glycerol by alpha-glucosidase from koji in the sake mash. PMID- 10737197 TI - An efficient method for production of uridine 5'-diphospho-N-acetylglucosamine. AB - Uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) has been synthesized by a yeast-based method from 5'-UMP and glucosamine, in which yeast cells catalyze the conversion of 5'-UMP to 5'-UTP and provide enzymes involved in UDP-GlcNAc synthesis using 5'-UTP and glucosamine as substrates. However, this conventional method is not suitable for practical production of UDP-GlcNAc because of the low yield of the product. We found that the yqgR gene product of Bacillus subtilis, which has been identified as a glucokinase, can catalyze the phosphorylation of N acetylglucosamine (GlcNAc) to give GlcNAc-6-phosphate, an intermediate of UDP GlcNAc biosynthesis. The addition of the yqgR gene product to the yeast-based reaction system enabled us to synthesize UDP-GlcNAc using GlcNAc in place of glucosamine. The addition of two enzymes, GlcNAc-phosphate mutase and UDP-GlcNAc pyrophosphorylase, increased the yield of UDP-GlcNAc. Using this novel method, UDP-GlcNAc was produced at an amount of 78 mM from 100 mM 5'-UMP and 100 mM GlcNAc. PMID- 10737198 TI - Cloning and expression of the Momordica charantia trypsin inhibitor II gene in silkworm by using a baculovirus vector. AB - MCTI-II (Momordica charantia trypsin inhibitor II) isolated from bitter gourd (Momordica charantia LINN.) seeds is one of the serine protease inhibitors of the squash family. We cloned cDNA that encodes MCTI-II and constructed an expression system for MCTI-II by using a baculovirus vector. The recombinant baculovirus was inoculated to early fifth-instar larvae of the silkworm (strain: Shunrei x Shougetsu). Four days after infection, the hemolymph of silkworm larvae was collected and the recombinant protein was purified. Two kinds of expressed MCTI II protein were obtained. An amino acid sequence analysis of the two proteins indicates that both were similar to the authentic inhibitor, except for the addition of a tripeptide derived from the vector at the N-terminus. One of the two inhibitors (MCTI-II A) resulted in a single PTH-amino acid in each Edman degradation cycle, while the other (MCTI-II B) resulted in two PTH-amino acids, suggesting the occurrence of cleavage of the reactive site. The inhibitory activities of MCTI-II expressed toward trypsin are examined in terms of the Ki value, these being 6.4 x 10(-10)M for MCTI-II A and 5.2 x 10(-10) M for MCTI-II B. PMID- 10737199 TI - Production of isopropyl cis-6-hexadecenoate by regiospecific desaturation of isopropyl palmitate by a double mutant of a Rhodococcus strain. AB - Resting cells of a double mutant noted as KSM-MT66, derived from Rhodococcus sp. strain KSM-B-3 by UV irradiation, were found to cis-desaturate isopropyl hexadecanoate, yielding isopropyl cis-6-hexadecenoate. Addition of sodium glutamate (1.0%), Mg SO4 (2 mM), and thiamine (2 mM) increased the productivity of the unsaturated product in phosphate buffer. Optimal temperature and pH for the reaction were around 26 degrees C and 7, respectively. Under the optimized conditions, more than 50 g/l of isopropyl cis-6-hexadecenoate was produced after a 3-day incubation by resting cells of the mutant. Thus, cis-6-hexadecenoic acid, the main component of human sebaceous lipids, can be manufactured economically by the rhodococcal bioconversion. PMID- 10737200 TI - Amplifying effect of dietary taurine on the induction of cytochrome P-450 and on the urinary excretion of ascorbic acid in rats fed on phenobarbital-containing diets. AB - Dietary taurine amplified the induction of cytochrome P-450 and the urinary excretion of ascorbic acid in rats fed on phenobarbital (PB)-containing diets. These facts suggest that taurine could influence the hepatic metabolism of xenobiotics via the induction of drug-metabolizing enzymes (DME) and the ascorbic acid metabolism. Taurine might improve the function of DME exposed by some xenobiotics. PMID- 10737201 TI - Purification and some properties of high-molecular-weight xylanases, the xylanases 4 and 5 of Aeromonas caviae W-61. AB - Aeromonas caviae W-61 produces multiple extracellular xylanases, the xylanases 1, 2, 3, 4, and 5 [Nguyen, V. D. et al., Biosci. Biotechnol. Biochem., 56, 1708-1712 (1993)]. Here we purified and characterized high-molecular-weight xylanases, the xylanases 4 and 5 from the culture fluids of the bacterium. The purified xylanases 4 and 5, which had molecular masses of 120 and 140 kDa, respectively, were endo-beta-1,4-xylanases with similar enzymatic properties except for trans xylosidase activity. The xylanase 4 showed a prominent transxylosidase activity when xylotriose and xylotetraose were used as the substrates, while the xylanase 5 had little transxylosidase activity under the same conditions. Protein sequencing indicated that the xylanase 4 was a C-terminally-truncated xylanase 5, suggesting that the C-terminal truncation of the xylanase 5 may endow the enzyme with transxylosidase activity. PMID- 10737202 TI - Antimutagenicity of the purple pigment, hordeumin, from uncooked barley bran fermented broth. AB - The novel purple pigment hordeumin, an anthocyanin-tannin pigment, was produced from barley bran-fermented broth. The mutagenicity or antimutagenicity of hordeumin was investigated according to the Ames method, an indication of the safety of food, using Salmonella typhimurium TA98. Despite the presence of S-9 mix, hordeumin was not mutagenic. On the other hand, hordeumin effectively decreased a reverse mutation from Trp-P-1, Trp-P-2, IQ, and B[a]P. Furthermore, hordeumin also decreased the reverse mutation from dimethyl sulfoxide extracts of grilled beef. PMID- 10737203 TI - Effect of a polysaccharide (TAP) from the fruiting bodies of Tremella aurantia on glucose metabolism in mouse liver. AB - An acidic polysaccharide (TAP) obtained from the fruiting bodies of Tremella aurantia significantly increased the activities of glucokinase, hexokinase, and glucose-6-phosphate dehydrogenase, and decreased the activity of glucose-6 phosphatase in normal and diabetic mouse liver after intraperitoneal administration, while the glycogen content in the liver was reduced. Furthermore, TAP lowered the plasma cholesterol level in normal and diabetic mice. PMID- 10737204 TI - Coumarin-related compounds as plant growth inhibitors from two rutaceous plants in Thailand. AB - Chemical investigation of naturally occurring plant growth inhibitors from Rutaceous plants in Thailand led us to identify five 7-methoxycoumarins and one 5,7-dimethoxycoumarin from Murraya paniculata, and six furanocoumarins from Citrus aurantifolia. Of these compounds, murranganon senecioate (1) is a new natural compound found in M. paniculata. Minumicrolin (6) was found to be highly active against the 2nd leaf sheath elongation of rice seedlings. PMID- 10737205 TI - A new type of glycoglycerolipids from Corynebacterium aquaticum. AB - A new type of glycoglycerolipids, S361A and S365A, were obtained from Corynebacterium aquaticum strains, S361 and S365, newly isolated from soils, and were identified as (2R)-1-[alpha-glucopyranosyl-(1alpha-3)-(6O-acyl-alpha-manno pyranosyl)]-3-O-acylglycerol and (2R)-1-[alpha-mannopyranosyl-(1alpha-3)-(6-O acyl-alpha-mannopyran osyl)]-3-O-acylglycerol, respectively. S365A was identical to a novel glycoglycerolipid recently isolated from some bacteria, but S361A was a new analog having a glucosylmannosyl in place of the dimannosyl group. Our results indicate that this sn-2 lysotype of glyceroglycolipids may be widely distributed in bacteria. PMID- 10737206 TI - Cloning and sequencing of the maltohexaose-producing amylase gene of Klebsiella pneumoniae. AB - The molecular characterization of the maltohexaose-producing amylase gene of Klebsiella pneumoniae revealed an open reading frame in which 2,031 base pairs encode a protein of 677 amino acids with a calculated molecular weight of 75,921. The amylase gene had high similarities of 73.6% in DNA sequence and 79.3% in deduced amino acid sequence with the periplasmic alpha-amylase MalS gene of Escherichia coli. PMID- 10737207 TI - Lysyl-tRNA synthetase of Bacillus stearothermophilus molecular cloning and expression of the gene. AB - The gene of the lysyl-tRNA synthetase of Bacillus stearothermophilus NCA1503 was cloned and sequenced. The gene consists of 1485 bp nucleotides commencing with an ATG start codon and ending with a TAA stop codon, and encodes a polypeptide of 493 amino acids. The recombinant enzymes were expressed in E. coli using an expression plasmid containing the T7 RNA polymerase/promoter. PMID- 10737208 TI - Basidiomycete fungal gene encoding a regulatory subunit A homologue of protein phosphatase 2A. AB - A gene, Le.paa, encoding a regulatory subunit A (PR65) homologue of protein phosphatase 2A was isolated from the basidiomycete mushroom Lentinus edodes. The deduced Le.paa gene product (Le.PR65) had the highest sequence similarity to the Schizosaccharomyces pombe PR65 protein (54.1% similarity). The Le.paa gene was shown to be transcribed more actively during the late stages of fruiting development of the fungus. Gill tissue in which basidiospores are formed contained abundant Le.paa transcript as compared with gill-depleted pileus and stipe. PMID- 10737209 TI - Isolation and identification of the probing stimulants in the rice plant for the white-back planthopper, Sogatella furcifera (homoptera: delphacidae). AB - Adult females of the white-back planthopper, Sogatella furcifera, showed characteristic behavior of stylet sheath deposit on a parafilm membrane when fed on a 2% aqueous crude rice leaf and stem extract containing 15% sucrose. Subsequent bioassays revealed that the butanol-soluble fraction of the extract was highly active against the insects. When the butanol fraction was chromatographed on an ODS open column and eluted in sequence with a mixture of an increasing concentration of methanol in water, the 40 % methanol fraction was separated as the most active. A further bioassay of the HPLC components in the active fraction revealed that two major components (1 and 3) stimulated the high probing activity of the white-back planthopper only when they were combined. Of the active components, one component (3) was identified to be tricin 5-O glucoside by spectroscopic analyses. PMID- 10737210 TI - Purification and characterization of a family G/11 beta-xylanase from Streptomyces olivaceoviridis E-86. AB - A beta-xylanase (GXYN) was purified from the culture filtrate of Streptomyces olivaceoviridis E-86 by successive chromatography on DE-52, CM-Sepharose and Superose 12. The molecular mass of the xylanase was estimated to be 23 kDa, indicating that the enzyme consists of a catalytic domain only. The enzyme displayed an optimum pH of 6, a temperature optimum of 60 degrees C, a pH stability range from 2 to 11 and thermal stability up to 40 degrees C. The N terminal amino acid sequence of GXYN was A-T-V-I-T-T-N-Q-T-G-T-N-N-G-I-Y-Y-S-F-W , and sharing a high degree of similarity with the N-terminal sequence of xylanases B and C from Streptomyces lividans, indicating GXYN belongs to family G/11 of glycoside hydrolases. GXYN was inferior to xylanase B from Streptomyces lividans in the hydrolysis of insoluble xylan because of its lack of a xylan binding domain. PMID- 10737211 TI - Identification of a methylated tea catechin as an inhibitor of degranulation in human basophilic KU812 cells. AB - We examined the constituents of tea that had an inhibitory effect on the degranulation process in the human basophilic cell line, KU812. Among the constituents purified from a extract of 'Benihomare' oolong tea by column chromatography, a methylated (-)-epigallocatechin gallate ((-)-epigallocatechin 3 O-(3-O-methyl) gallate) was found to inhibit the degranulation of KU812 cells that had been stimulated with calcium ionophore A23187. The inhibitory effect of this methylated (-)-epigallocatechin gallate on degranulation was greater than that of (-)-epigallocatechin gallate. This result indicates that methylation of ( )-epigallocatechin gallate may be an effective modification for the catechin to inhibit degranulation from human basophils. PMID- 10737212 TI - A simple and rapid method for the preparation of a cell-free extract with CCAAT binding activity from filamentous fungi. AB - A simple and rapid method for the preparation of a cell-free extract with the CCAAT-binding activity was established with Aspergillus nidulans as a model fungus. Proteins were extracted with 6 M guanidine hydrochloride directly from mycelia and renatured by dialysis. This method was found applicable to other filamentous fungi such as Aspergillus oryzae and Trichoderma viride. PMID- 10737213 TI - Deconvolution can be used in electrooptic studies to correct for non-ideal electric excitation pulses only when the electric dipole moment of the studied molecules is predominantly induced. AB - The electric field pulses used for most measurements of transient electrooptic properties such as birefringence and dichroism, are rectangular and assumed to be ideal, but in practice do all such pulses have non-zero rise and fall times. Claims have been made that this non-ideality may be taken into account by employing standard deconvolution techniques. We find that this approach yields exact results in the zero electric field limit when the electric dipole moment of the studied macromolecules is predominantly induced. However, for finite electric field strengths and/or macromolecules with partly or fully permanent electric dipole moments, we find that the deconvolution method yields erroneous estimates of the electrooptic relaxation times. When the decay time of the electric pulse and the electrooptic decay time are equal, and the system is operated in the Kerr domain, this systematic error for macromolecules with purely permanent electric dipole moment equals 37%. In a research field where the uncertainty of the reported relaxation times normally is assumed to be only a few percent this is an error that may seriously mislead unsuspecting users. We find that this systematic error can readily be avoided by employing standard numerical integration of a set of coupled first-order differential equations instead of the standard deconvolution techniques. PMID- 10737214 TI - Cryosolvents useful for protein and enzyme studies below -100 degrees C. AB - For the study of protein structure, dynamics, and function, at very low temperatures it is desirable to use cryosolvents that resist phase separation and crystallisation. We have examined these properties in a variety of cryosolvents. Using visual and X-ray diffraction criteria, methanol:ethanediol (70%:10%), methanol:glycerol (70%:10%), acetone:methoxy-ethanol:ethanediol (35%:35%:10%), dimethylformamide:ethanediol (70%:10%), dimethylformamide (80%), methoxyethanol (80%), and methoxyethanol:ethanediol (70%:10%) were all found to be free of phase changes down to at least -160 degrees C. The least viscous of these, methanol:ethanediol (70%:10%), was miscible down to -125 degrees C and showed no exo or endothermic transitions when examined using DSC. It is therefore potentially particularly suitable for very low temperature cryoenzymology. PMID- 10737215 TI - DNA-printing: utilization of a standard inkjet printer for the transfer of nucleic acids to solid supports. AB - The use of total cDNA as a probe for hybridization enables the transcription level of a large number of genes to be analyzed at the same time. Some effort has been spent to develop high density gene arrays on different solid supports to facilitate this hybridization. We achieved a high resolution by utilizing inkjet printer technology as a useful alternative to blotting the target genes onto a membrane. By the use of an ordinary inkjet printer model we show that it is possible to print DNA onto hybridization membranes and hybridize using either specific genes or total cDNA as probes. The high resolution of these prints (300 dpi) might be used in the future to construct complex micro-arrays to analyze simultaneously large numbers of genes. PMID- 10737216 TI - Miniaturized direct on air sampling filter quantification of pollen allergens. AB - A recently in this journal reported luminescence immunoassay for the direct quantification of birch and grass pollen allergens on air sampling filters. DOSIS, has been miniaturized. By means of a commercially available chlorinated analogue of the previously used 1,2 dioxetane phosphate derivative as enzyme substrate, the air sampling filter diameter could be reduced from 25 mm to 13 mm. The procedure leads to a more than twenty times reduction of the previously reported limit of quantification for the grass pollen allergen. PMID- 10737217 TI - Determination of rate constants for the reaction of hydroxyl radicals with some purines and pyrimidines using sunlight. AB - Sunlight mediated hydroxyl radical production from aqueous ferric perchlorate at low pH has been investigated using deoxyribose-thiobarbituric acid assay. The rate of production of hydroxyl radical was found to be dependent on the time of irradiation. Hydroxyl radical scavengers can compete with deoxyribose for hydroxyl radicals produced in the system leading to a decreased yield of thiobarbituric acid chromogen. The second-order rate constants of the added scavengers can be determined using a simple competition kinetic method. The rate constants for the reaction of hydroxyl radical with a number of purine and pyrimidine derivatives were determined using this method. The rate constants obtained (1-7 x 10(9) dm(3) mol(-1) s(-1)) were found to be in good agreement with those reported using pulse radiolysis technique. The rate constants of dimethyluracil, xanthosine, amino and methyl substituted pyrimidines, cytidine monophosphate and uridine monophosphate were also determined by this method. It is proposed that sunlight mediated production of hydroxyl radical coupled with deoxyribose-thiobarbituric acid assay is a simple and efficient method for the determination of rate constants for the reaction of hydroxyl radical with a wide range of biomolecules. PMID- 10737218 TI - Estimation of submicrogram quantities of protein using the dye eosin Y. AB - Eosin B is used to estimate proteins above 1 microg/ml concentration [Waheed AA, Gupta, PD. Anal. Biochem. 1996:233:249-256; Waheed AA, Gupta PD. J. Biochem. Biophys. Meth. 1996;33:187-196]. In the present report we describe a method for estimating submicrogram quantities of proteins using the dye eosin Y. The increase in sensitivity of this assay is approximately two fold under optimal assay condition. The optimum concentration of eosin Y and citric acid for submicrogram assay is 0.01 and 0.05%; (final concentration) respectively. The protein-dye complex formation is completed within 2 min and its absorbance is stable up to 60 main with a variation of +/-4.0%. The interference due to sugars, reducing agents, glycerol and some neutral detergents like Triton X-100, NP-40 and Tween-20 is less than 12% whereas Brij-35, ethanol, acetone and chelators like EGTA and EDTA suppress the absorbance by about 12-18%. However, basic buffers like Tris, urea, CHAPS and NaN, interfere with the formation of the protein-dye complex. The increase in absorbance of protein-eosin Y complex compared to that of protein-eosin B complex is due to the higher extinction co efficient of eosin Y compared to eosin B. PMID- 10737219 TI - A simple approach for the simultaneous isolation and immobilization of invertase using crude extracts of yeast and Jack bean meal. AB - Crude cell-free extract of yeast cells was mixed with sufficient amount of Jack bean meal extract so as to precipitate all the invertase. The precipitate was then cross-linked using 2% glutaraldehyde retaining over 60% of the activity. The immobilized invertase could be reused for over ten batches without loss in activity. PMID- 10737220 TI - Probing the cathepsin D using a BODIPY FL-pepstatin A: applications in fluorescence polarization and microscopy. AB - Redistribution of cathepsin D, a major lysosomal aspartic endopeptidase, has been related to various pathological progressions during tumor formation and oxidation stress. We have synthesized a fluorescent probe for cathepsin D, where the pepstatin A was covalently conjugated with the BODIPY (Boron dipyrromethene difluoride) fluorophore. In vitro, BODIPY FL-pepstatin A inhibits cathepsin D activity with an IC50 of 10 nM. The nature of its binding to cathepsin D was further characterized using a fluorescence polarization measurement. Results showed that BODIPY FL-pepstatin A selectively binds to cathepsin D at pH 4.5. In fixed cells, BODIPY FL-pepstatin A stained lysosomes, where it co-localized with cathepsin D. This staining was depleted when cells were co-incubated with unlabeled pepstatin A in acidic buffer. In live cells, BODIPY FL-pepstatin A is internalized and transported to lysosomes. The staining in the lysosomes can be competed with unlabeled pepstatin A. These properties, along with the good photostability of the BODIPY FL fluorophore, make this probe a novel tool for the study of the secretion and trafficking of cathepsin D. PMID- 10737221 TI - The identification of the A-type RNA helices in a 55mer RNA by selective incorporation of deuterium-labelled nucleotide residues (Uppsala NMR-window concept) AB - The 55-nt long RNA, modelling a three-way junction, with non-uniformly incorporated deuterated nucleotides has been synthesised in a pure form. The NMR window part in this partially deuterated 55mer RNA consists of natural non enriched nucleotide blocks at the three-way junction (shown in a square box in Fig. 2), whereas all other nucleotides of the rest of the molecule are partially deuterated (> 97 atom% 2H at C2', C3', C5', C5, and approximately 50 atom% 2H at C4'). The secondary structure of this 55mer RNA was determined by 2D 1H NOESY spectroscopy in D2O or in 10% D2O-H2O mixture. The use of deuterated building blocks in the specific region of the 55mer RNA allowed us to identify two distinct A-type RNA helices in a straightforward manner by observing connectivities of H1' with the basepaired imino and the aromatic H2 of all adenosine nucleotides as the first step for the determination of its tertiary structure in a cost- and time-effective manner without employing any 13C/15N labelling. These two decameric helices involve 40 nucleotides, for which all non exchangeable H1', H6, H2, H8 and H5 protons (all 40 H1', all 40 H6 or H8 aromatics, all seven H2 of adenine nucleotide and all four non-deuterated H5 of cytosines) as well as all 16 exchangeable imino protons (with the exception of four terminal basepairs) and 16 amino protons of cytosines have been assigned. Since all aromatic-H2', H3' as well as H5'/5'' crosspeaks from partially deuterated residues have been eliminated from the NMR spectra, the observation of natural nucleotide residues in the NMR window part has essentially been simplified. It has been found that the crosspeaks from the natural nucleotides located at the three-way junction in the NMR-window part show different degrees of line-broadening, thereby indicating that the various nucleotide residues have very different mobilities with respect to themselves as well as compared to other nucleotides in the helices. The assignment of H2' and H3' in the NMR-window part has been made based on NOESY and DQF-COSY crosspeaks. It is noteworthy that, even in this preliminary study, it has been possible to identify 10 H2' out of total 14 and 9 H3' out of 14. The data show that expanded AU containing a tract of 55mer RNA does not self-organise into a tight third helix, as the two decameric A type helices, across the three-way junction which is evident from the absence of any additional imino protons, except those that already have been assigned for the two decameric helices. PMID- 10737222 TI - Vitamin E suppresses the induction of reactive oxygen species release by lipopolysaccharide, interleukin-1beta and tumor necrosis factor-alpha in rat alveolar macrophages. AB - Over the last decade, although investigations have suggested that vitamin E affects the immune response, not much is known about its affect on the alveolar macrophage functions. In the present study, we have investigated the effect of high vitamin E (DL-alpha-tocopheryl acetate, alpha-TA) supplementation for 10 d on the activation state of rat alveolar macrophages induced by lipopolysaccharide (LPS), interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha on the basis of their ability to produce reactive oxygen species (ROS), such as superoxide (O2 *) and H2O2. LPS treatment (1 and 10 microg/mL) caused 2.44 and 2.54-fold increases in O2-*, and 2.1 and 2.3-fold increases in H2O2, respectively, from alveolar macrophages (AMs) in the diet group fed 50 mg alpha-TA/kg. However, this enhancement was not observed for the AMs of the diet groups fed 250 or 1,250 mg alpha-TA/kg. Similar results were obtained on treating the AMs with proinflammatory cytokines IL-1beta or TNF-alpha. The observed suppression in ROS release in response to various stimulants may be due to the direct and/or indirect effect of high vitamin E (250 and 1,250 mg alpha-TA/kg diet) supplementation. It may therefore, be concluded that high alpha-TA supplementation in the diet modulates the activation of AMs in rats. PMID- 10737223 TI - Effects of ascorbic acid on peroxidation of human erythrocyte membranes by lipoxygenase. AB - The effects of ascorbic acid (AsA) on membrane phospholipids (PLs) and tocopherols (Tocs) of human erythrocyte during peroxidation by soybean lipoxygenase (LOX) were investigated. After extraction of the membrane lipids, alpha-tocopherol (alpha-Toc), gamma-tocopherol (gamma-Toc) and cholesterol were simultaneously measured by high-performance liquid chromatography (HPLC), and the changes of Tocs were expressed on the basis of cholesterol. The phospholipid classes and corresponding hydroperoxides (PL-OOHs) were detected simultaneously by HPLC, and the changes were calculated on the basis of sphingomyelin. These methods are sensitive to the changes of membrane Tocs and PLs by peroxidation. Control incubation without LOX and AsA was done for 45 min at 30 degrees C. After the incubation with LOX, alpha- and gamma-Tocs were exhausted, PLs decreased, and PL-OOHs and malondialdehyde (MDA) increased. Incubation with both AsA and LOX further increased MDA significantly, but it preserved about 30% of alpha-Toc and 45% of gamma-Toc of the control levels and did not decrease PLs or PL-OOHs from the levels after the incubation with LOX. Subsequent incubation with AsA for 45 min after the incubation with LOX (after Tocs were exhausted) showed a 240% increase in MDA, but it decreased PLs by only about 15% of the preincubation values and recovered gamma-Toc to about 13% of the control. The subsequent incubation with AsA after the control incubation increased PLs to higher than that of the control reaction. These results show that AsA protects and regenerates the membrane Tocs against enzymatic peroxidation. The results also indicate that repair of the membrane PLs is promoted in the presence of AsA. PMID- 10737224 TI - Gender and exercise influence on tissue antioxidant vitamin status in rats. AB - Although gender differences in antioxidant status based largely on differing estrogen levels have been postulated, it is not known if other gender based differences in tissue antioxidants exist. This experiment examined whether gender based differences in tissue vitamin C and vitamin E concentration exist, and investigated the possibility of gender based differences in indices of tissue oxidative stress following an acute exercise bout. It was determined that female rats had significantly higher levels of vitamin E in liver and heart tissues than males and that males had significantly more vitamin C in the plantaris muscle than females. However, female rats also had less liver glutathione than males. Acute exercise resulted in significant and equal tissue oxidative stress in both genders as indicated by tissue glutathione status. With some exceptions, tissue vitamin C and vitamin E concentrations were generally unaffected by acute exercise in either gender. Hence, while some gender differences in tissue antioxidant status in rats are evident, these differences do not affect tissue indices of oxidative stress following acute exercise. PMID- 10737225 TI - Vitamin K2 (menatetrenone) induces iNOS in bovine vascular smooth muscle cells: no relationship between nitric oxide production and gamma-carboxylation. AB - It has been recently reported that vitamin K2 (menaquinone-4: menatetrenone, VK2) has an anti-atherogenic effect as well as the ability to produce clotting factors and improve osteoporosis. However, the mechanism by which VK2 acts on atherosclerosis is still unclear. In this paper, we investigated the effects of vitamin K and its side chain on NO production as an anti-atherogenic substance in a cultured vascular system. Treatment of bovine vascular smooth muscle cells (SMC) with VK2 (30 microM) caused a time-dependent (24-72 h) increase in the nitrite (NO2) level in the conditioned medium, but not in bovine vascular endothelial cells. Classical NOS inhibitor (L-nitro arginine) and iNOS-specific inhibitors completely blocked the increased nitrite level induced by VK2 treatment, but D-nitro arginine could not it. Immunostaining and Western blotting analysis showed that VK2 induced iNOS protein in the SMC. VK2 has a naphtoquinone nucleus, which is identical in menadione (VK3), and an unsaturated side chain, which is called geranylgeraniol (GGO). To determine whether the structure of VK2 was related to an increasing nitrite level, we investigated the nitrite level in conditioned medium treated with VK3 or GGO. Neither VK3 nor GGO treatment of SMC increased the nitrite level. In addition, warfarin, an inhibitor of VK2-dependent gamma-carboxylation, did not affect the increased nitrite level induced by VK2 in SMC. In conclusion, VK2 caused NO production through iNOS induction in bovine SMC, that was not related to the structure of VK2, naphtoquinone nucleus or its side chain, independently of gamma-carboxylation. PMID- 10737226 TI - Developmental induction and villus-crypt distribution of retinol esterifying enzyme activities in chick duodenum. AB - Retinol absorbed and generated from dietary beta-carotene can be esterified by retinol esterifying enzyme(s) in intestinal absorptive cells. In this study, we observed the developmental changes and villus-crypt distribution of the activities of two retinol esterifying enzymes (lecithin-retinol acyltransferase (LRAT); and acyl-CoA-retinol acyltransferase (ARAT) in chick duodenum) to seek the possibility that these enzymes play distinct roles in retinol absorption and metabolism. Intestinal LRAT activity was barely expressed in embryonic stages until 2-3 d before hatching, when its activity becomes detectable; thereafter it abruptly increased to the maximal level at the third day of the posthatch period. In contrast, ARAT activity was present in the duodenum at the earliest stage examined, the 15th day of embryogenesis, and was elevated to the maximal level 3 4 d after hatching. An assay of LRAT and ARAT activities along the villus-crypt axis of the duodenum by a cryostat sectioning technique revealed that between the day of hatching and 1 d posthatch, an abrupt induction of LRAT activity occurred only in the villus region of the duodenum, where a coordinated induction of cellular retinol-binding protein, type II (CRBPII), was observed. In contrast, the rise in ARAT activity observed around the hatching period occurred at the broader portions of the villi including the area of villus-crypt junction. These observations in the developmental changes and distribution of LRAT and ARAT activities suggest that LRAT activity but not ARAT activity is closely related to the induction of CRBPII in the duodenum of developing chicks. PMID- 10737227 TI - Effects of taste stimulation on the behavior of serum amino acid concentrations and amylase and trypsin activities in fasting rats. AB - The effect of taste stimulation on serum free-amino acid concentrations and amylase and trypsin activities in fasting rats was studied. Following an acclimation period of 5 d, male Sprague-Dawley rats were fasted for 4 d and sacrificed after taste stimulation with a palatable sodium saccharin or unpalatable quinine sulfate flavored diet. Blood was collected from the portal vein and inferior vena cava at 0, 5, 10, 20 and 30 min after taste stimulation. Intestinal contents were also collected at the same time intervals as the blood collections. Total amino acid concentrations in the saccharin stimulated group increased significantly at 5 and 20 min following taste stimulation in comparison with the control of 0 time in the portal vein, and a significant difference between the saccharin and quinine stimulated groups was also observed at 5 min. No difference was found in the inferior vena cava. A high level of alanine and low level of glutamine were depicted in the portal vein as compared to that of the inferior vena cava. The elevation of alanine that is gluconeogenic amino acid was remarkable in the saccharin group at 20 min in the portal vein. Moreover, amylase and trypsin activities in the saccharin group reached peak values promptly and kept constant throughout the experiment as compared to the quinine group. The results suggest that taste stimulation originates changes in the cephalic phase amino acid concentrations in the portal vein and that taste information, overcoming a hunger, plays an important role in amino acid metabolism and digestive enzyme activities. Therefore, eating with gusto is significant for the maintenance of body functions even under starvation conditions. PMID- 10737228 TI - Vitamin A deficiency and low prevalence of anemia in Yaqui Indian children in northwest Mexico. AB - A study of 296 school-age Yaqui Indian children (6-10 y) was conducted in 26 rural communities. Vitamin A status was determined by retinol and carotenoid serum levels according to a method described previously (IVACG, 1982). Serum retinol and carotenoids in children were analyzed according to community size. Vitamin A intake was assessed in a sub-sample by means of a 24 h recall questionnaire. Serum retinol distribution showed that 6.3% of the children were below 10 microg/100 mL (0.35 micromol/L) and 40% were in the range of 10-20 microg/100 mL (0.35-0.70 micromol/L). Differences (p < 0.02) were found between small and large communities (Median, 95% CI): 19.2 (17.1, 20.9) microg/100 mL and 22.9 (20.3, 24.1) microg/100 mL. Serum carotenoid levels were significantly higher in large than in small and medium communities: 72 (68.2, 77.8) microg/100 mL versus 62.4 (53.3, 68.2) and 62.4 (55.7, 69.6) microg/100 mL, respectively. Food staples were wheat flour tortillas, pinto beans, corn tortillas, few animal products and scarce fresh vegetables. Mean vitamin A consumption was 244+/-29 microg RE (34.9% of the US RDA). Iron status showed that only 4 children were classified as anemic, with two of them having iron deficiency anemia. Iron deficient erythropoiesis was observed in 7.8% of the children and iron depletion only in 4.4%. The Yaqui diet seems to provide adequate amounts of iron but not of vitamin A or its precursors, which renders a vitamin A status of sub-clinical deficiency that could be considered a public health problem. PMID- 10737229 TI - Long-term effects of dietary alpha-linolenic acid from perilla oil on serum fatty acids composition and on the risk factors of coronary heart disease in Japanese elderly subjects. AB - Although important roles of dietary n-3 fatty acids in the prevention of coronary heart disease (CHD) have been suggested, long-term effects of dietary alpha linolenic acid (ALA, 18:3n-3) have not yet been established under controlled conditions. We tested whether a moderate increase of dietary ALA affects fatty acids composition in serum and the risk factors of CHD. Oxidized LDL (OxLDL) was directly measured by ELISA using antibody specific to OxLDL. By merely replacing soybean cooking oil (SO) with perilla oil (PO) (i.e., increasing 3 g/d of ALA), the n-6/n-3 ratio in the diet was changed from 4:1 to 1:1. Twenty Japanese elderly subjects were initially given a SO diet for at least 6 mo (baseline period), a PO diet for 10 mo (intervention period), and then returned to the previous SO diet (washout period). ALA in the total serum lipid increased from 0.8 to 1.6% after 3 mo on the PO diet, but EPA and DHA increased in a later time, at 10 mo after the PO diet, from 2.5 to 3.6% and 5.3 to 6.4%, respectively (p<0.05), and then returned to baseline in the washout period. In spite of increases of serum n-3 fatty acids, the OxLDL concentration did not change significantly when given the PO diet. Body weight, total serum cholesterol, triacylglycerol, glucose, insulin and HbA1c concentrations, platelet count and aggregation function, prothrombin time, partial thromboplastin time, fibrinogen and PAI-1 concentration, and other routine blood analysis did not change significantly when given the PO diet. These data indicate that, even in elderly subjects, a 3 g/d increase of dietary ALA could increase serum EPA and DHA in 10 mo without any major adverse effects. PMID- 10737231 TI - Antibacterial activity of garlic powder against Escherichia coli O-157. AB - The antibacterial activity of garlic powder against O-157 was tested by using garlic bulbs post-harvested 1 y. O-157 at 10(6-7) cfu/mL perished after incubation for 24 h with a 1% solution of garlic powder. The use of powder from fresh garlic was more effective for antibacterial activity than that from old garlic; the 1% solution of fresh garlic powder eradicating the O-157 in 6 h. The antibacterial activity was resistant to heat treatment of 100 degrees C for 20 min. The water-soluble components of garlic powder were fractionated into three fractions (Fr. 1-3) by Sephadex G-100 column chromatography, among which Fr. 3 showed antibacterial activity against O-157 but the other fractions were scarce in activity. The antibacterial activity was also shown against other types of pathogenic bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), Salmonella enteritidis, and Candida albicans. Thus, the practical use of garlic powder is expected to prevent bacteria-caused food poisoning. PMID- 10737230 TI - Lipid alterations in the liver and serum of rats in histidine-excess and copper deficiency. AB - To obtain further information on lipid metabolism in the histidine-excess and copper-deficiency, rats were fed basal, histidine-excess (the addition of 50 g L histidine/kg diet) or copper-deficient diets for 0, 7, 21 and 42 d ad libitum. Liver triacylglycerol accumulated and the serum triacylglycerol level decreased after feeding of the histidine-excess diet for 21 or 42 d, but not after feeding of the copper-deficient diet. Serum cholesterol level increased in rats fed the histidine-excess diet for 7, 21 and 42 d, but not in rats fed the copper deficient diet. Copper content in the liver and serum significantly decreased in rats fed the histidine-excess diet. Copper content in the liver and serum was markedly decreased in rats fed the copper-deficient diet. Liver zinc content was constant, but the serum zinc level decreased in rats fed the histidine-excess diet. Feeding of the copper-deficient diet hardly affected zinc content in the liver and serum. Urinary copper and zinc increased in rats fed the histidine excess diet, and decreased or showed a decreasing tendency in rats fed the copper deficient diet. Overall results indicated that feeding the histidine-excess diet caused copper deficiency, whereas hypercholesterolemia was not shown in rats fed the copper-deficient diet although the livers of rats fed the copper-deficient diet contained less copper than those of rats fed the histidine-excess diet. Thus, the responses on liver triacylglycerol and serum cholesterol to copper deficiency induced by the feeding of a histidine-excess diet are different from those to copper deficiency induced by feeding of a copper-deficient diet. PMID- 10737232 TI - Antiobesity activity of extracts from Lagerstroemia speciosa L. leaves on female KK-Ay mice. AB - Banaba in the Tagalog name, Lagerstroemia speciosa L., has been used as a folk medicine for a long time among diabetics in the Philippines. Extracts from banaba leaves have been reported to reduce diabetic symptoms in genetically diabetic mice (Type II, KK-Ay). In the present study, female mice of the same strain showing remarkable body weight gain were used to examine the antiobesity effect of dietary banaba extract. Five-week-old female KK-Ay mice were fed a control diet or test diet containing 5% of a hot-water extract from banaba leaves instead of cellulose for 12 wk. Neither group showed any changes in diet intake during the experimental period. Body weight gain and parametrial adipose tissue weight were lowered significantly in the banaba diet group. Blood glucose levels were not suppressed in the banaba diet group, but hemoglobin A1C was found to be suppressed at the end of the experiment. No effects on the serum lipids were observed, but the mice fed banaba extract showed a significant decrease, to 65% of the control level in total hepatic lipid contents. This decrease was due to a reduction in the accumulation of triglyceride. These results suggest that banaba had a beneficial effect on obese female KK-Ay mice. PMID- 10737233 TI - Characterization of kintoki bean (Phaseolus vulgaris) alpha-amylase inhibitor: inhibitory activities against human salivary and porcine pancreatic alpha amylases and activity changes by proteolytic digestion. AB - The effects of some experimental parameters on alpha-amylase inhibition by an alpha-amylase inhibitor from kintoki bean were examined. The rate of inhibition against pancreatic alpha-amylase increased with a rise in temperature to 50 degrees C, but the inhibition of salivary alpha-amylase reached a maximum above 35 degrees C. Although an increase in NaCl concentration to 1.5 M caused an increase in the inhibitory activities against both amylases, these inhibitory activities tended to decrease above 1.5 M NaCl. The effects of proteolytic digestion on the amylase inhibitory activity were also studied. The inhibitor was slightly inactivated by pepsin digestion for 2 h. Although the inhibitor rapidly lost the inhibitory activity by chymotrypsin digestion within 2 h, it was quite resistant to proteolytic digestion by trypsin. PMID- 10737234 TI - Screening for domestic violence. PMID- 10737235 TI - Persistent tachycardia before back surgery. PMID- 10737236 TI - Abdominal pain and diarrhea after minor trauma. PMID- 10737237 TI - Is it Mobitz 1 or 2? PMID- 10737238 TI - Empowering the caregiver. PMID- 10737239 TI - Immunotherapy for dust mite sensitivity. PMID- 10737240 TI - New insights into causes and treatments of kidney stones. AB - Recent findings have provided insight into the molecular basis of kidney stone formation and entirely changed our approach to management of calcium stones. Understanding the role of genetic factors and the various promotors and inhibitors of stone formation should lead to more effective prophylaxis and treatment of other types of stones as well. PMID- 10737241 TI - Cancer in the elderly: tailoring treatment. AB - The body's ability to meet the stresses of cancer therapy declines in later life. Because the decline may vary from minimal to profound and is only weakly linked to chronologic age, a thorough functional assessment should be undertaken in all elderly cancer patients. The results can guide decisions about how to treat the cancer. PMID- 10737242 TI - Management of the asymptomatic HIV-infected patient: an update. AB - Guidelines for initiating antiretroviral therapy in asymptomatic patients continue to be debated. Physicians and patients should decide whether drug treatment is advisable after jointly considering psychosocial issues as well as measurements of immune function and HIV burden. Other components of care include appropriate immunizations, screening for other sexually transmitted infections, safer-sex counseling, and referral for substance abuse treatment, if indicated. PMID- 10737243 TI - Integrins: the molecular glue of life. AB - The molecular interactions responsible for cellular adhesion, either to other cells or to extracellular matrix, are now known to be complex, well orchestrated, and under sophisticated control. Among the participating molecules, integrins serve a notably broad range of biologic processes, including platelet aggregation and leukocyte extravasation. The loss of an adhesion interaction may result in disease, as may the stimulation of excessive adhesiveness. PMID- 10737245 TI - The docs behind the dot-coms. PMID- 10737244 TI - Case in point. Sarcoidosis. PMID- 10737246 TI - Culture clash on medical errors. PMID- 10737247 TI - You've got mail. PMID- 10737248 TI - Will the Internet change our approach to disease management? PMID- 10737249 TI - To err is inevitable--human and otherwise. PMID- 10737250 TI - Fasting or postprandial glucose targets? PMID- 10737251 TI - Coping with change: 5. Self-talk. PMID- 10737252 TI - Acute chest pain after laparotomy. PMID- 10737253 TI - In vivo effects of recombinant-interferon-beta1b treatment on polymorphonuclear cell and monocyte functions and on T-cell-mediated antibacterial activity in patients with relapsing-remitting multiple sclerosis. AB - Treatment with Interferon (IFN)-beta has been proposed as a therapeutic approach in multiple sclerosis (MS) patients, mostly in view of its immunomodulating actions. At the same time, evidence has been provided that MS patients exhibit polymorphonuclear cell (PMN) deficits, which can explain the increased susceptibility to infections in these subjects. Here, in 28 patients with relapsing-remitting (RR) MS under treatment with recombinant (r)-IFN-beta PMN polarization and PMN and monocyte (MO) phagocytosis and killing, as well as T cell mediated antibacterial activity, were evaluated before treatment and over a period of nine months of treatment. Our results point out an enhanced rate of polarization (both "spontaneous" or N-formyl-methionyl-leucyl-phenylalanine induced) in MS patients. After r-IFN-beta1b treatment the polarization rate was further increased. On the contrary, PMN and MO phagocytosis and killing were depressed in comparison to controls and values were further reduced by r-IFN beta1b treatment. In patients T-cell mediated antibacterial activity was decreased at T0 and dramatically dropped in the course of r-IFN-beta1b therapy. PMID- 10737254 TI - sICAM-1, sCD95 and sCD95L levels in chronic liver diseases of different etiology. AB - The release of soluble circulating molecules represents a prominent feature during the course of immune-mediated clinical conditions. To further assess the relationship between serum concentrations of adhesion or apoptotic-related soluble structures and liver diseases, we evaluated the levels of intercellular adhesion molecule-1 (sICAM-1), Fas receptor (CD95) and Fas ligand (sCD95L) in a group of patients affected by Hepatitis C Virus (HCV)-induced chronic hepatitis (CH-C), HCV-positive liver cirrhosis with superimposed hepatocellular carcinoma (HCC), autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and alcoholic liver cirrhosis (ALC). Results show that sICAM-1 values were in all instances significantly elevated when compared to those seen in healthy donors. Similar findings were noted in subjects with liver diseases in terms of sCD95 concentrations, even if to a different degree of statistical significance. Finally, sCD95L amounts were augmented in AIH, PBC, ALC and CH-C in comparison to controls, while in the HCC counterpart sCD95L levels fell within normal range. All together, these findings emphasize the occurrence of circulating soluble molecules in patients with various chronic liver diseases, likely reflecting the involvement of several pathogenetic mechanisms. PMID- 10737255 TI - Cytokine pattern secretion by murine spleen cells after inactivated Candida albicans immunization. Effect of cocaine and morphine treatment. AB - In the present work we have analyzed: i) the effect of heat-inactivated Candida albicans immunization on the cytokine production by murine spleen cells; ii) the effect of a subchronic cocaine and morphine treatment on this production. The treatment with a single dose of inactivated Candida blastospores induced interleukin-2(IL-2), interferon-gamma (IFNgamma) and interleukin-4 (IL-4) production at 24 h after in vitro restimulation of splenocytes. In this model, the exposure to morphine (25 mg/kg, 5 days before, during and 5 days after inoculation with the yeast) significant decreased IL-2 and IL-4 levels, while secretion of IFN-gamma was unaltered. The same cocaine treatment (10 mg/kg) resulted in unchanged levels of the three cytokines tested. The results showed that non-viable Candida cells of this strain induce a predominant Th0 response. This immune effect is in part impaired only by a subchronic administration of morphine. PMID- 10737257 TI - 12-O-tetradecanoyl phorbol 13-acetate, protein kinase C (PKC) activator, protects human leukemia HL-60 cells from taxol-induced apoptosis: possible role for extracellular signal-regulated kinase. AB - The purpose of this study was to evaluate whether the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) signaling pathway contributes to 12-O-tertadecanoyl phorbol 13-acetate (TPA)-mediated protection from taxol induced apoptosis of human leukemia HL-60 cells. Treatment of cells with taxol for 12 h resulted in apoptosis of HL-60 cells. TPA was protective against taxol induced apoptosis and this anti-apoptotic effect was reversible when TPA was used in conjunction with staurosporine and H-7, PKC inhibitors, suggesting that TPA may protect HL-60 cells against taxol-induced apoptosis via the PKC-dependent pathway. Since TPA stimulates MEK signal transduction pathway in HL-60 cells, we postulated that MEK pathway may be playing a role in the ability of TPA to inhibit taxol-induced apoptosis. PD098059, a specific MEK kinase inhibitor, abolished the ability of TPA to inhibit taxol-induced apoptosis. These results suggest that activation of PKC in HL-60 cells confers protection against taxol induced apoptosis and that MEK mediates anti-apoptotic signaling of PKC. PMID- 10737256 TI - Protein kinase C independent activation of inducible nitric oxide synthase by tumor necrosis factor-alpha in TM4 Sertoli cells. AB - To investigate the nitric oxide (NO) production and its signalling mechanism in TM4 Sertoli cells, the cells were treated with recombinant tumor necrosis factor alpha (rTNF-alpha), recombinant interleukin-1 alpha (rIL-1alpha), or lipopolysaccharide (LPS), either alone or in combination with recombinant interferon-gamma (rIFN-gamma), and NO production was measured by using the Griess method. TM4 Sertoli cells produced a small amount of NO upon treatment with rIFN gamma. The effect of rIFN-gamma was drastically increased by cotreatment with rTNF-alpha in a dose-dependent manner. However, combination of rIL-1alpha or LPS with rIFN-gamma did not synergize to activate cells. RIFN-gamma in combination with rTNF-alpha showed marked increase of the expression of iNOS protein. Protein kinase C inhibitors did not inhibit the production of NO induced by rIFN-gamma plus rTNF-alpha. These results suggest that the role of TNF-alpha is to provide TM4 Sertoli cells with the active cofactor for NO production and TNF-alpha induced signaling for induction of NO synthesis is not dependent on protein kinase C activation. PMID- 10737258 TI - Protein A-activated rat splenic lymphocyte proliferation involves tyrosine kinase phospholipase C-protein kinase C pathway. AB - Protein A (PA) of Staphylococcus aureus was long been known for its affinity towards the Fc domain of immunoglobulin G. It is now well established that PA is a potent biological response modifier showing simultaneously antitumor, antitoxic, and anticarcinogenic properties. This bacterial protein was also observed to stimulate production of cytokines. But the molecular mechanism(s) of immunocyte activation by PA still remained essentially unknown. In this report, we demonstrate a hitherto undescribed role of PA as a signal inducer in rat splenic lymphocytes. Our studies describe that PA induces transition of G0/G1 to S and G2/M phases of cell cycle, thus ultimately stimulating splenic lymphocyte proliferation. It has also been revealed that PA binds to rat splenic lymphocytes in a dose dependent manner and stimulates proliferation via tyrosine kinase phospholipase C (PLC)-Ca2+-dependent protein kinase C (PKC) pathway. These observations will be of valid help in correlating the immunostimulatory activities of PA with the molecular mechanism(s) of its action. PMID- 10737259 TI - Expression of protein kinase C genes in normal (+/+) and W mutant alleles (Wsh/Wsh, W/Wv) mice testes. AB - In the present study, we investigated the expression of mRNA of protein kinase C (PKC) isoenzymes (alpha, beta, gamma, delta, epsilon, zeta, eta, and theta) in normal (+/+) and W mutant alleles mice testes. In +/+ mice testes, abundant expression of PKCdelta and PKCtheta was observed, while other PKCs (alpha, beta, gamma, epsilon, zeta, and eta) generally were not detected by Northern blotting. The PKCdelta and PKCtheta isoenzymes demonstrated a distinctive cellular distribution when evaluated by in situ hybridization. We have previously shown that PKCdelta gene was selectively expressed in spermatid of +/+ testes. Here we show that PKCdelta gene is also present in spermatid of Wsh/Wsh mice testes and PKCtheta gene was present in interstitial cells of +/+, Wsh/Wsh, and W/Wv mice testes. These studies provide the evidence of selective cell distributions of the PKC isoenzymes and suggest that PKC has the functional significance in testes. PMID- 10737261 TI - Polyamine metabolism in prostaglandin E2-treated human T lymphocytes. AB - The effects of Prostaglandin (PG) E2 treatment of human T lymphocytes on polyamine metabolism were investigated. PGE2 is known to inhibit lymphocyte proliferation, while polyamines play an important role in several biochemical processes leading to increased cell growth. Preincubation of T lymphocytes with PGE2 (10(-6) M) for 10 min was able to increase ornithine decarboxylase (ODC) activity and putrescine as well as spermine levels, while spermidine concentration was drastically reduced. After 30 and 60 min of treatment, a decrease in ODC activity and putrescine concentration was observed. On the contrary, the initial inhibition of spermine-N1-acetyltransferase (SAT) activity was followed by a progressive increase of this catabolic enzyme. These changes were related to modifications of cAMP concentrations. Our data may help clarify the mechanisms underlying the biphasic effect of PGE2, which ultimately leads to inhibition of cell proliferation. PMID- 10737260 TI - Immunomodulatory effect of the homoeopathic drug Engystol-N on some activities of isolated human leukocytes and in whole blood. AB - Engystol-N at the doses of 10(-4) and 10(-8) in isolated human leukocytes stimulates the superoxide anion generation by neutrophils and the cytokine(s) production by T lymphocytes. In whole blood the same concentrations of the drug produce the decrease of the superoxide anion generation of neutrophils, this inhibiting activity appears 6 h after the administration of the drug and persists only in presence of lymphocytes. Culture media of T lymphocytes treated with Engystol-N show the same inhibiting effect on superoxide anion generation by neutrophils. From these data it is possible to conclude that the drug stimulates the secretion of lymphokine(s) with inhibiting action on superoxide anion generation of neutrophils that prevail over the direct stimulating effect, confirming and extending the immunomodulatory ability of the drug. PMID- 10737262 TI - Effect of exopolysaccharide V2-7, isolated from Halomonas eurihalina, on the proliferation in vitro of human peripheral blood lymphocytes. AB - The immunomodulatory activity of the exopolysaccharide V2-7, a sulfated polymer excreted by the moderately halophilic bacteria Halomonas eurihalina, was studied in vitro. [3H]thymidine incorporation and flow-cytometry measurements showed that this exopolysaccharide enhanced the unspecific proliferation of human lymphocytes in response to the presence of anti-CD3 monoclonal antibody. It was effective at concentrations of less than 1 microg/ml, maximum activity being achieved at 0.2 microg/ml. PMID- 10737263 TI - Effect of San-Ao-Tang on immediate and late airway response and leukocyte infiltration in asthmatic guinea pigs. AB - San-Ao-Tang (SAT), a traditional Chinese medicines, has been used to treat patients with the bronchial asthma for several centuries. However, the therapeutic mechanisms of this Chinese medicine are still far from clear. To understand the mechanism of antiasthmatic property of SAT, a guinea pig model of allergic asthma was used to investigate the effects of SAT on Dermatophagoides pteronyssinus-induced immediate and late asthmatic responses and airway inflammation. Our results showed that administration of SAT (10 g/kg) extracts significantly inhibited the antigen induced immediate asthmatic responses (IAR) in actively sensitized guinea pig. Examination of bronchoalveolar lavage fluid (BALF) revealed that SAT significantly inhibited the increase in neutrophil in the airway at 1, 2, 4, 6, 8 hr after antigen challenge. Histopathologic examination showed SAT suppressed the neutrophil infiltration into lung tissue. These results suggest that the antiasthmatic effect of SAT be mainly due to its bronchodilator effect and its ability to inhibit the neutrophil into the airway. The precise mechanism of action of SAT in asthma remains to be elucidated. PMID- 10737265 TI - Muscle properties and coordination during voluntary movement. AB - The aims of this review were to provide some insight into the theoretical problems and questions associated with muscle coordination and control of movement, to consider some of the experimental findings on force-sharing among synergistic muscles, and to discuss some of the practical implications of force sharing and movement control for optimal performance in sports. Theoretically, the distribution problem is introduced and the problems and difficulties of this approach in making quantitative predictions of force-sharing among muscles is discussed. Experimentally, the force-sharing research in the cat ankle extensor muscles is critically evaluated, and a complete force-sharing picture is given for the soleus and (medial) gastrocnemius for tasks covering a large range of speeds and intensities. In the application section, optimal performance criteria in jumping and cycling are considered. Furthermore, the plasticity of skeletal muscle properties is illustrated using examples from high-performance sports. The review ends with a summary of the topics discussed and suggestions for future research. PMID- 10737264 TI - Garlic extracts stimulate proliferation of rat lymphocytes in vitro by increasing IL-2 and IL-4 production. AB - Garlic components are known to modulate certain immune functions. However, mechanisms of their action are not sufficiently elucidated. This study was, therefore, undertaken to examine the effects of aqueous and ethanolic extracts prepared from a garlic powder sample on proliferation of rat spleen lymphocytes in culture. Cells were stimulated with the combination of phorbol myristate acetate (PMA) and a Ca ionophore (A23187) or R73 monoclonal antibody (mAb) directed to the alphabeta chain of T cell receptor. It has been shown that both extracts significantly stimulated proliferation of lymphocytes. The effect correlated with upregulation of the Interleukin 2 receptor alpha (IL-2R alpha) expression and the increase in IL-2 production. Stimulation of IL-2 production by the extracts was higher in cultures with PMA/Ca ionophore than in cultures with R73 mAb. In contrast, both extracts stimulated production of IL-4 by splenocytes triggered by R73 mAb. The complete dependence of lymphocyte proliferation in cultures with R73 mAb and garlic extracts on IL-2 and IL-4 was demonstrated using neutralising mAbs to IL-2R alpha and IL-4. These results suggest that the potentiating effect of garlic extracts on lymphocyte proliferation in vitro differs depending on specific stimulators of cell proliferation and probably on the type of responding cells. PMID- 10737266 TI - The effect of pedal crank arm length on joint angle and power production in upright cycle ergometry. AB - The aim of this study was to determine the effect of five pedal crank arm lengths (110, 145, 180, 230 and 265 mm) on hip, knee and ankle angles and on the peak, mean and minimum power production of 11 males (26.6+/-3.8 years, 179+/-8 cm, 79.6+/-9.5 kg) during upright cycle ergometry. Computerized 30 s Wingate power tests were performed on a free weight Monark cycle ergometer against a resistance of 8.5% body weight. Joint angles were determined, with an Ariel Performance Analysis System, from videotape recorded at 100 Hz. Repeated-measures analysis of variance and contrast comparisons revealed that, with increasing crank arm lengths, there was a significant decrement in the minimum hip and knee angles, a significant increment in the ranges of motion of the joints, and a parabolic curve to describe power production. The largest peak and mean powers occurred with a crank arm length of 180 mm. We conclude that 35 mm changes in pedal crank arm length significantly alter both hip and knee joint angles and thus affect cycling performance. PMID- 10737267 TI - Electromyographic analysis of exercise resulting in symptoms of muscle damage. AB - Surface electromyographic (EMG) signals were recorded from the hamstring muscles during six sets of submaximal isokinetic (2.6 rad x s(-1)) eccentric (11 men, 9 women) or concentric (6 men, 4 women) contractions. The EMG per unit torque increased during eccentric (P < 0.01) but not during concentric exercise. Similarly, the median frequency increased during eccentric (P < 0.01) but not during concentric exercise. The EMG per unit torque was lower for submaximal eccentric than maximum isometric contractions (P < 0.001), and lower for submaximal concentric than maximum isometric contractions (P < 0.01). The EMG per unit torque was lower for eccentric than concentric contractions (P < 0.05). The median frequency was higher for submaximal eccentric than maximum isometric contractions (P < 0.001); it was similar, however, between submaximal concentric and maximum isometric contractions (P = 0.07). Eccentric exercise resulted in significant isometric strength loss (P < 0.01), pain (P < 0.01) and muscle tenderness (P < 0.05). The greatest strength loss was seen 1 day after eccentric exercise, while the most severe pain and muscle tenderness occurred 2 days after eccentric exercise. A lower EMG per unit torque is consistent with the selective recruitment of a small number of motor units during eccentric exercise. A higher median frequency during eccentric contractions may be explained by selective recruitment of fast-twitch motor units. The present results are consistent with the theory that muscle damage results from excessive stress on a small number of active fibres during eccentric contractions. PMID- 10737268 TI - The influence of cadence and power output on force application and in-shoe pressure distribution during cycling by competitive and recreational cyclists. AB - The aim of this study was to determine the response of cyclists to manipulations of cadence and power output in terms of force application and plantar pressure distribution. Two groups of cyclists, 17 recreational and 12 competitive, rode at three nominal cadences (60, 80, 100 rev x min(-1)) and four power outputs (100, 200, 300, 400 W) while simultaneous force and in-shoe pressure data were collected. Two piezoelectric triaxial force transducers mounted in the right pedal measured components of the pedal force and orientation, and a discrete transducer system with 12 transducers recorded the in-shoe pressures. Force application was characterized by calculating peak resultant and peak effective pedal forces and positive and negative impulses. In-shoe pressures were analysed as peak pressures and as the percent relative load. The force data showed no significant group effect but there was a cadence and power main effect. The impulse data showed a significant three-way interaction. Increased cadence resulted in a decreased positive impulse, while increased power output resulted in an increased impulse. The competitive group produced less positive impulse but the difference became less at higher cadences. Few between-group differences were found in pressure, notable only in the pressure under the first metatarsal region. This showed a consistent pattern of in-shoe pressure distribution, where the primary loading structures were the first metatarsal and hallux. There was no indication that pressure at specific sites influenced the pedal force application. The absence of group differences indicated that pressure distribution was not the result of training, but reflected the intrinsic relationship between the foot, the shoe and the pedal. PMID- 10737269 TI - Longitudinal changes in submaximal oxygen uptake in 11- to 13-year-olds. AB - The aim of this study was to monitor longitudinal changes in young people's submaximal oxygen uptake (VO2) responses during horizontal treadmill running at 8 km x h(-1). The 236 participants (118 boys, 118 girls) were aged 11.2+/-0.4 years (mean +/- s) at the onset of the study. Submaximal VO2, peak VO2 and anthropometry were recorded annually for three consecutive years. The data were analysed using multi-level regression modelling within a multiplicative, allometric framework. The initial model examined sex, age and maturity-related changes in submaximal VO2 relative to body mass as the sole anthropometric covariate. Our results demonstrate that the conventional ratio standard ml x kg( 1) x min(-1) does not adequately describe the true relationship between body mass and submaximal VO2 during this period of growth. The effects of maturity and age were non-significant, but girls consumed significantly less VO2 than boys running at 8 km x h(-1). In subsequent models, stature was shown to be a significant explanatory variable, but this effect became non-significant when the sum of two skinfolds was added. Thus, within this population, submaximal VO2 responses were explained predominantly by changes in body mass and skinfold thicknesses, with no additional maturity-related increments. When differences in body mass and skinfolds were controlled for, there was still a difference between the sexes in submaximal VO2, with girls becoming increasingly more economical with age. PMID- 10737270 TI - The relationship between motivational climate, perceived ability and sources of distress among elite athletes. AB - Situational factors as well as individual differences are assumed to play an important role in perceptions of stress. One factor that may affect an elite athlete's perception of stress may be his or her perceived motivational climate. To examine the relative importance of dispositional and situational factors on the perceptions of distress (i.e. negative stress), we assessed goal orientations, perception of motivational climate and sources of distress among Norwegian participants at the 1994 Winter Olympic Games in Lillehammer. Perception of a performance climate was associated with cognitive sources of distress, the coach, and team aspects as a source of distress. Furthermore, a performance climate was a significant predictor of high total distress. The athletes with lower perceptions of ability perceived the coach to be more a source of distress than athletes with a high perception of ability. A perception of a mastery climate was negatively associated with the coach as a source of distress. These findings indicate that, to reduce the perception of distress, the coach should focus on creating a mastery climate for elite athletes. PMID- 10737271 TI - The effects of required amplitude and practice on frequency stability and efficiency in a cyclical task. AB - From an ecological point of view, motor learning emerges from the interplay of constraints on action, which shape behaviour towards the optimal solution, and practice, conceived as an active exploration of the work-space, to search this optimal solution. In the experiment reported here, we studied this interplay for a cyclical task performed on a ski-simulator. Our aim was to assess the respective effects of amplitude and practice on frequency variability and efficiency. On the basis of previous empirical findings, amplitude was expected, beyond a critical value, to constrain and stabilize the frequency of the movement. Three groups of participants practised during four sessions at three different amplitudes (15, 22.5 and 30 cm). The results showed that participants moving at large amplitude displayed more stable and more consistent frequencies. Nevertheless, there was no interaction effect between target amplitude and practice. On the other hand, movement economy and harmonicity increased with practice, but were not affected by amplitude. Finally, the results of transfer tests showed that the effects of large amplitude on frequency variability were not resistant to a subsequent decrease in target amplitude. These results suggest that constraints and practice act independently on motor behaviour, and that a high constraint could be detrimental to the development of effective search strategies. PMID- 10737272 TI - Changes in blood lactate and pyruvate concentrations and the lactate-to-pyruvate ratio during the lactate minimum speed test. AB - The aim of this study was to assess the responses of blood lactate and pyruvate during the lactate minimum speed test. Ten participants (5 males, 5 females; mean +/- s: age 27.1+/-6.7 years, VO2max 52.0+/-7.9 ml x kg(-1) x min(-1)) completed: (1) the lactate minimum speed test, which involved supramaximal sprint exercise to invoke a metabolic acidosis before the completion of an incremental treadmill test (this results in a 'U-shaped' blood lactate profile with the lactate minimum speed being defined as the minimum point on the curve); (2) a standard incremental exercise test without prior sprint exercise for determination of the lactate threshold; and (3) the sprint exercise followed by a passive recovery. The lactate minimum speed (12.0+/-1.4 km x h(-1)) was significantly slower than running speed at the lactate threshold (12.4+/-1.7 km x h(-1)) (P < 0.05), but there were no significant differences in VO2, heart rate or blood lactate concentration between the lactate minimum speed and running speed at the lactate threshold. During the standard incremental test, blood lactate and the lactate-to pyruvate ratio increased above baseline values at the same time, with pyruvate increasing above baseline at a higher running speed. The rate of lactate, but not pyruvate, disappearance was increased during exercising recovery (early stages of the lactate minimum speed incremental test) compared with passive recovery. This caused the lactate-to-pyruvate ratio to fall during the early stages of the lactate minimum speed test, to reach a minimum point at a running speed that coincided with the lactate minimum speed and that was similar to the point at which the lactate-to-pyruvate ratio increased above baseline in the standard incremental test. Although these results suggest that the mechanism for blood lactate accumulation at the lactate minimum speed and the lactate threshold may be the same, disruption to normal submaximal exercise metabolism as a result of the preceding sprint exercise, including a three- to five-fold elevation of plasma pyruvate concentration, makes it difficult to interpret the blood lactate response to the lactate minimum speed test. Caution should be exercised in the use of this test for the assessment of endurance capacity. PMID- 10737273 TI - The hard task of improving the quality of care at the end of life. PMID- 10737274 TI - Beyond heparin and aspirin: new treatments for unstable angina and non-Q-wave myocardial infarction. AB - The goals of therapy for unstable angina and non-Q-wave myocardial infarction (MI) are to maintain myocardial perfusion by inhibiting platelet aggregation and fibrin deposition at sites of plaque rupture, thereby preventing ongoing or new myocardial ischemia and cardiac death. Although aspirin and heparin sodium are cornerstones in the management of unstable angina and non-Q-wave MI, both have significant limitations that have prompted the development of new agents. The thienopyridines, ticlopidine hydrochloride and clopidogrel, appear to be at least as effective as aspirin in the management of unstable angina. Glycoprotein IIb/IIIa receptor antagonists are a new class of platelet inhibitors that are more potent than aspirin, because they target the final common pathway of platelet aggregation. Low-molecular-weight heparins provide a more stable pharmacodynamic response and are more convenient to use than unfractionated heparin. Direct thrombin inhibitors show promise for inhibiting thrombin-mediated platelet aggregation and fibrin deposition. We focus on the opportunities presented by these agents, detailing mechanisms of action, advantages over aspirin and heparin, and performance in recent clinical trials. PMID- 10737275 TI - Predictors of recurrence after deep vein thrombosis and pulmonary embolism: a population-based cohort study. AB - BACKGROUND: The appropriate duration of oral anticoagulation after a first episode of venous thromboembolism (VTE) is uncertain and depends upon VTE recurrence rates. OBJECTIVE: To estimate VTE recurrence rates and determine predictors of recurrence. METHODS: Patients in Olmsted County, Minnesota, with a first lifetime deep vein thrombosis or pulmonary embolism diagnosed during the 25 year period from 1966 through 1990 (N = 1,719) were followed forward in time through their complete medical records in the community for first VTE recurrence. RESULTS: Four hundred four patients developed recurrent VTE during 10,198 person years of follow-up. The overall (probable/definite) cumulative percentages of VTE recurrence at 7, 30, and 180 days and 1 and 10 years were 1.6% (0.2%), 5.2% (1.4%), 10.1% (4.1%), 12.9% (5.6%), and 30.4% (17.6%), respectively. The risk of recurrence was greatest in the first 6 to 12 months after the initial event but never fell to zero. Independent predictors of first overall VTE recurrence included increasing age and body mass index, neurologic disease with paresis, malignant neoplasm, and neurosurgery during the period from 1966 through 1980. Independent predictors of first probable/definite recurrence included diagnostic certainty of the incident event and neurologic disease in patients with hospital acquired VTE. Recurrence risk was increased by malignant neoplasm but varied with concomitant chemotherapy, patient age and sex, and study year. CONCLUSIONS: Venous thromboembolism recurs frequently, especially within the first 6 to 12 months, and continues to recur for at least 10 years after the initial VTE. Patients with VTE with neurologic disease and paresis or with malignant neoplasm are at increased risk for recurrence, while VTE patients with transient or reversible risk factors are at less risk. PMID- 10737276 TI - Recurrent venous thromboembolism after deep vein thrombosis: incidence and risk factors. AB - BACKGROUND: The recurrence rate after deep vein thrombosis (DVT) is high and the risk factors for recurrent thromboembolic events have only been investigated on a small scale. OBJECTIVES: To estimate the cumulative incidence of recurrent venous thromboembolic events after a first or a second DVT and to identify possible risk factors for recurrent venous thromboembolism. METHODS: We prospectively followed up 738 consecutive patients with an objectively verified symptomatic DVT for 3.7 to 8.8 years. Medical records and death certificates for all patients were reviewed during follow-up and recurrent DVT and pulmonary embolism were registered. RESULTS: The 5-year cumulative incidence of recurrent venous thromboembolic events was 21.5% (95% confidence interval [CI], 17.7%-25.4%) after a first DVT and 27.9% (95% CI, 19.7%-36.1%) after a second DVT. The 5-year cumulative incidence of fatal pulmonary embolism was 2.6% (95% CI, 1.1%-4.1%) after a first DVT. Proximal DVT (relative risk [RR], 2.40; 95% CI, 1.48-3.88; P<.001), cancer (RR, 1.97; 95% CI, 1.20-3.23; P<.001), and history of a venous thromboembolism (RR, 1.71; 95% CI, 1.16-2.52; P<.01) predicted an independently increased risk of recurrent events in multivariate survival analysis. Postoperative DVT (RR, 0.27; 95% CI, 0.13-0.55; P<.001) and a long duration of oral anticoagulation therapy (RR, 0.95; 95% CI, 0.92-0.98; P<.01) involved a smaller risk of recurrent events. Sex, age, initial antithrombotic therapy, or immobilization did not affect the risk of a recurrent event. CONCLUSIONS: The recurrence rate after a symptomatic DVT is high. Patients with proximal DVT, diagnosed cancer, short duration of oral anticoagulation therapy, or a history of thromboembolic events had a higher risk of recurrent events, while patients with postoperative DVT had a lower recurrence rate. This knowledge could help identify patients who might benefit most from prolonged prophylactic treatment in various risk situations. PMID- 10737277 TI - Consumption of NSAIDs and the development of congestive heart failure in elderly patients: an underrecognized public health problem. AB - BACKGROUND: Experimental studies have shown that administration of nonsteroidal anti-inflammatory drugs (NSAIDs) to susceptible individuals can lead to the development of congestive heart failure (CHF). There have been few epidemiological investigations of the importance of this adverse effect. OBJECTIVE: To estimate the relative risk of first admission to a hospital with CHF in recent users of NSAIDs, compared with nonusers, and to determine whether the estimated relative risk was increased in those with a history of heart disease and the extent to which the level of risk varied with the dose and half life of the drugs consumed. METHODS: We conducted a matched case-control study of the relationship between recent use of NSAIDs and hospitalization with CHF. Cases (n = 365) were patients admitted to hospitals with a primary diagnosis of CHF. Controls (n = 658) were patients without CHF who were admitted to the same hospitals as case patients. Structured interviews were used to obtain information on several study factors, including recent use of aspirin and other NSAIDs. RESULTS: Use of NSAIDs (other than low-dose aspirin) in the previous week was associated with a doubling of the odds of a hospital admission with CHF (adjusted odds ratio, 2.1; 95% confidence interval, 1.2-3.3). Use of NSAIDs by patients with a history of heart disease was associated with an odds ratio of 10.5 (95% confidence interval, 2.5-44.9) for first admission with heart failure, compared with 1.6 (95% confidence interval, 0.7-3.7) in those without such a history. The odds of a first admission to a hospital with CHF was positively related to the dose of NSAID consumed in the previous week, and was increased to a greater extent with long half-life than with short half-life drugs. Assuming these relationships are causal, NSAIDs were responsible for approximately 19% of hospital admissions with CHF. CONCLUSIONS: The burden of illness resulting from NSAID-related CHF may exceed that resulting from gastrointestinal tract damage. NSAIDs should be used with caution in patients with a history of cardiovascular disease. PMID- 10737278 TI - Occurrence, causes, and outcome of delirium in patients with advanced cancer: a prospective study. AB - CONTEXT: Delirium impedes communication and contributes to symptom distress in patients with advanced cancer. There are few prospective data on the reversal of delirium in this population. OBJECTIVES: To evaluate the occurrence, precipitating factors, and reversibility of delirium in patients with advanced cancer. DESIGN: Prospective serial assessment in a consecutive cohort of 113 patients with advanced cancer. Precipitating factors were examined using standardized criteria; 104 patients met eligibility criteria. SETTING: Acute palliative care unit in a university-affiliated teaching hospital. MAIN OUTCOME MEASURES: Delirium occurrence and reversal rates, duration, and patient survival. Strengths of association of various precipitating factors with reversal were expressed as hazard ratios (HRs) in univariate and multivariate analyses. RESULTS: On admission, delirium was diagnosed in 44 patients (42%), and of the remaining 60, delirium developed in 27 (45%). Reversal of delirium occurred in 46 (49%) of 94 episodes in 71 patients. Terminal delirium occurred in 46 (88%) of the 52 deaths. In univariate analysis, psychoactive medications, predominantly opioids (HR, 8.85; 95% confidence interval [CI], 2.13-36.74), and dehydration (HR, 2.35; 95% CI, 1.20-4.62) were associated with reversibility. Hypoxic encephalopathy (HR, 0.39; 95% CI, 0.19-0.80) and metabolic factors (HR, 0.44; 95% CI, 0.21-0.91) were associated with nonreversibility. In mulitivariate analysis, psychoactive medications (HR, 6.65; 95% CI, 1.49-29.62), hypoxic encephalopathy (HR, 0.32; 95% CI, 0.15-0.70), and nonrespiratory infection (HR, 0.23; 95% CI, 0.08-0.64) had independent associations. Patients with delirium had poorer survival rates than controls (P<.001). CONCLUSIONS: Delirium is a frequent, multifactorial complication in advanced cancer. Despite its terminal presentation in most patients, delirium is reversible in approximately 50% of episodes. Delirium precipitated by opioids and other psychoactive medications and dehydration is frequently reversible with change of opioid or dose reduction, discontinuation of unnecessary psychoactive medication, or hydration, respectively. PMID- 10737279 TI - Emphysematous pyelonephritis: clinicoradiological classification, management, prognosis, and pathogenesis. AB - BACKGROUND: Emphysematous pyelonephritis (EPN) is a rare, severe gas-forming infection of renal parenchyma and its surrounding areas. The radiological classification and adequate therapeutic regimen are controversial and the prognostic factors and pathogenesis remain uncertain. OBJECTIVES: To elucidate the clinical features, radiological classification, and prognostic factors of EPN; to compare the modalities of management (ie, antibiotic treatment alone, percutaneous catheter drainage combined with antibiotic treatment, or nephrectomy) and outcome among the various radiological classes of EPN; and to clarify the gas-forming mechanism and pathogenesis of EPN by gas analysis and pathological findings. PATIENTS AND METHODS: Forty-eight EPN cases from our institution were enrolled between August 1,1989, and November 30, 1997. According to the radiological findings on computed tomographic scan, they were classified into the following classes: (1) class 1: gas in the collecting system only; (2) class 2: gas in the renal parenchyma without extension to extrarenal space; (3) class 3A: extension of gas or abscess to perinephric space; class 3B: extension of gas or abscess to pararenal space; and (4) class 4: bilateral EPN or solitary kidney with EPN. The clinical manifestations, management, and outcome were compared. The gas contents of specimens from 6 patients were analyzed. The pathological findings from 8 patients who received nephrectomy were reviewed. The statistical methods consisted of the Fisher exact test (2 tailed) for categorical variables and Wilcoxon rank sum test for continuous variables to test the predictors of poor prognosis. RESULTS: Forty-six patients (96%) had diabetes mellitus, and 10 (22%) of the 46 also had urinary tract obstruction in the corresponding renoureteral unit. The other 2 nondiabetic patients (4%) had severe hydronephrosis. Twenty-one (72%) of the 29 patients with diabetes mellitus also had a glycosylated hemoglobin A(1c) level higher than 0.08. Escherichia coli (69%) and Klebsiella pneumoniae (29%) were the most common pathogens. The mortality rate in patients who received antibiotic treatment alone was 40% (2 of 5 patients). The success rate of management by percutaneous catheter drainage (PCD) combined with antibiotic treatment was 66% (27 of 41 patients). In classes 1 and 2 EPN, all the patients who were treated using a PCD or ureteral catheter combined with antibiotic treatment survived. In extensive EPN (classes 3 and 4), 17 (85%) of the 20 patients with fewer than 2 risk factors (ie, thrombocytopenia, acute renal function impairment, disturbance of consciousness, or shock) were successfully treated using PCD combined with antibiotic treatment; and the patients with 2 or more risk factors had a significantly higher failure rate than those with no or only 1 risk factors (92% vs 15%, P<.001). Eight of the 14 patients who had an unsuccessful treatment using a PCD underwent subsequent nephrectomy, 7 of whom survived. Only 2 patients were managed by direct nephrectomy and survived. The overall success rate of nephrectomy was 90% (9 of 10 patients). The total mortality was 18.8% (9 of 48 patients). Five of the 6 gas samples contained hydrogen (average, 12.8%), and all had carbon dioxide (average, 14.4%). The pathological findings from 8 of 10 who underwent nephrectomy revealed poor perfusion in most cases (ie, infarction, 5 patients; vascular thrombosis, 3 patients; and arteriosclerosis and/or glomerulosclerosis, 4 patients). CONCLUSION: Acute renal infection with E coli or K pneumoniae in patients with diabetes mellitus and/or urinary tract obstruction is the cornerstone for the development of EPN. Mixed acid fermentation of glucose by Enterobacteriaceae is the major pathway of gas formation. For localized EPN (classes 1 and 2), PCD combined with antibiotic treatment can provide a good outcome. (ABSTRACT TRUNCATED) PMID- 10737280 TI - Risk factors for deep vein thrombosis and pulmonary embolism: a population-based case-control study. AB - BACKGROUND: Reported risk factors for venous thromboembolism (VTE) vary widely, and the magnitude and independence of each are uncertain. OBJECTIVES: To identify independent risk factors for deep vein thrombosis and pulmonary embolism and to estimate the magnitude of risk for each. PATIENTS AND METHODS: We performed a population-based, nested, case-control study of 625 Olmsted County, Minnesota, patients with a first lifetime VTE diagnosed during the 15-year period from January 1, 1976, through December 31, 1990, and 625 Olmsted County patients without VTE. The 2 groups were matched on age, sex, calendar year, and medical record number. RESULTS: Independent risk factors for VTE included surgery (odds ratio [OR], 21.7; 95% confidence interval [CI], 9.4-49.9), trauma (OR, 12.7; 95% CI, 4.1-39.7), hospital or nursing home confinement (OR, 8.0; 95% CI, 4.5-14.2), malignant neoplasm with (OR, 6.5; 95% CI, 2.1-20.2) or without (OR, 4.1; 95% CI, 1.9-8.5) chemotherapy, central venous catheter or pacemaker (OR, 5.6; 95% CI, 1.6 19.6), superficial vein thrombosis (OR, 4.3; 95% CI, 1.8-10.6), and neurological disease with extremity paresis (OR, 3.0; 95% CI, 1.3-7.4). The risk associated with varicose veins diminished with age (for age 45 years: OR, 4.2; 95% CI, 1.6 11.3; for age 60 years: OR, 1.9; 95% CI, 1.0-3.6; for age 75 years: OR, 0.9; 95% CI, 0.6-1.4), while patients with liver disease had a reduced risk (OR, 0.1; 95% CI, 0.0-0.7). CONCLUSION: Hospital or nursing home confinement, surgery, trauma, malignant neoplasm, chemotherapy, neurologic disease with paresis, central venous catheter or pacemaker, varicose veins, and superficial vein thrombosis are independent and important risk factors for VTE. PMID- 10737281 TI - Payer status and the utilization of hospital resources in acute myocardial infarction: a report from the National Registry of Myocardial Infarction 2. AB - BACKGROUND: Prior studies have suggested that payer status may be an important determinant of medical resource utilization and outcome in acute myocardial infarction (AMI). METHODS: A national cohort of 332,221 patients with AMI enrolled from June 1994 to July 1996 were compared within 5 payer groups to ascertain the influence of payer status on hospital resource allocation for AMI in the United States. RESULTS: Medicare comprised the largest proportion (56%), followed by commercial insurance (25%), health maintenance organization (HMO) (10%), uninsured (6%), and Medicaid (3%). Compared with commercially insured patients, Medicare and Medicaid patients received fewer reperfusion therapies, underwent fewer invasive cardiac procedures, and had longer hospitalizations. After adjusting for differences in clinical characteristics, Medicare recipients were as likely as commercially insured patients to receive acute reperfusion therapies or any invasive cardiac procedure. Uninsured and HMO patients tended to utilize hospital resources with intermediate frequency. Medicare recipients aged 65 years or older and the HMO group had similar hospital mortality rates compared with the commercial group (odds ratio [OR], 1.07; 95% confidence interval [CI], 0.96-1.20 and OR, 0.93; 95% CI, 0.83-1.04, respectively), but Medicaid and uninsured groups had higher hospital mortality rates compared with the commercial group (OR, 1.30; 95% CI, 1.14-1.48 and OR, 1.29; 95% CI, 1.12-1.48, respectively). CONCLUSION: This report suggests significant variation by payer status in the management of AMI throughout the United States, but no important differences in mortality among the 3 largest payer groups. PMID- 10737282 TI - Regional and racial differences in response to antihypertensive medication use in a randomized controlled trial of men with hypertension in the United States. Department of Veterans Affairs Cooperative Study Group on Antihypertensive Agents. AB - BACKGROUND: Stroke incidence and mortality rates are higher in the southeastern region of the United States, which is called the "Stroke Belt." We compared the response to antihypertensive medication use in patients from different US regions. METHODS: The short-term and 1-year efficacy of the antihypertensive medications hydrochlorothiazide, atenolol, diltiazem hydrochloride (sustained release), captopril, prazosin hydrochloride, and clonidine was compared by US region in a randomized controlled trial of 1,105 men with hypertension from 15 US Veterans Affairs medical centers. RESULTS: Compared with patients outside the Stroke Belt, patients inside the Stroke Belt achieved significantly lower treatment success rates of diastolic blood pressure control at 1 year with hydrochlorothiazide (63% vs 41%), atenolol (62% vs 46%), captopril (60% vs 30%), and clonidine (69% vs 43%); there were no differences in treatment success rates with diltiazem (70% vs 71%) or prazosin (54% vs 53%). When controlling for race, patients inside the Stroke Belt had significantly lower treatment success rates with hydrochlorothiazide (P = .003) and clonidine (P = .003), and the lower success rate with atenolol approached significance (P = .15). Regardless of region, blacks were less likely than whites to achieve treatment success with atenolol (P = .02) or prazosin (P = .03) and more likely with diltiazem (P = .05). There was a trend for blacks residing inside the Stroke Belt to have a lower treatment success rate than other race-region groups when treated with captopril (P = .07). Many regional and racial differences in diet, lifestyle, and other characteristics were observed. After adjustment for these characteristics by regression analysis, the effect of residing inside the Stroke Belt remained for captopril (P = .01) and clonidine (P = .01) and approached significance for hydrochlorothiazide (P = .10). CONCLUSIONS: Hypertension in patients residing inside the Stroke Belt responded less to the use of several antihypertensive medications and important differences were shown in a number of characteristics that may affect the control of blood pressure, compared with patients residing outside the Stroke Belt. PMID- 10737283 TI - The accuracy of physical examination to detect abdominal aortic aneurysm. AB - BACKGROUND: Abdominal palpation during physical examination is an important means of detecting abdominal aortic aneurysm (AAA), but limited information is available on its accuracy. METHODS: Two hundred subjects (aged 51-88 years), 99 with and 101 without AAA as determined by previous ultrasound, each underwent physical examination of the abdomen by 2 internists who were blinded to each other's findings and to the ultrasound diagnosis. RESULTS: The overall accuracy of abdominal palpation for detecting AAA was as follows: sensitivity, 68% (95% confidence interval [CI], 60%-76%); specificity, 75% (95% CI, 68%-82%); positive likelihood ratio, 2.7 (95% CI, 2.0-3.6); negative likelihood ratio 0.43 (95% CI, 0.33-0.56). Interobserver pair agreement for AAA vs no AAA between the first and second examinations was 77% (kappa = 0.53). Sensitivity increased with AAA diameter, from 61% for AAAs of 3.0 to 3.9 cm, to 69% for AAAs of 4.0 to 4.9 cm, 72% for AAAs of 4.0 cm or larger, and 82% for AAAs of 5.0 cm or larger. Sensitivity in subjects with an abdominal girth less than 100 cm (40-in waistline) was 91% vs 53% for girth of 100 cm or greater (P<.001). When girth was 100 cm or greater and the aorta was palpable, sensitivity was 82%. When girth was less than 100 cm and the AAA was 5.0 cm or larger, sensitivity was 100% (12 examinations). Factors independently associated with correct examination findings included AAA diameter (odds ratio [OR], 1.95 per centimeter increase; 95% CI, 1.06-3.58); abdominal girth (OR, 0.90 per centimeter increase; 95% CI, 0.87 0.94); and the examiner's assessment that the abdomen was not tight (OR, 2.68; 95% CI, 1.17-6.13). CONCLUSIONS: Abdominal palpation has only moderate overall sensitivity for detecting AAA, but appears to be highly sensitive for diagnosis of AAAs large enough to warrant elective intervention in patients who do not have a large girth. Abdominal palpation has good sensitivity even in patients with a large girth if the aorta is palpable. PMID- 10737284 TI - Olive oil and reduced need for antihypertensive medications. AB - BACKGROUND: The blood pressure (BP) effects of changing the total fat intake and saturated-unsaturated fat ratio are still controversial, despite evidence that saturated fat-enriched diets are associated with higher BP levels. This double blind, randomized crossover study evaluated a possible difference between antihypertensive effects of monounsaturated (MUFA) (extra-virgin olive oil) and polyunsaturated fatty acids (PUFA) (sunflower oil). METHODS: Twenty-three hypertensive patients were assigned randomly to MUFA or PUFA diet for 6 months and then crossed over to the other diet; effects were evaluated on the basis of daily antihypertensives needed. RESULTS: Diets high in MUFA and PUFA differed from the habitual diet for reduced total and saturated fats, whereas they differed from each other for MUFA (17.2% vs 10.5%) and PUFA content (3.8% vs 10.5%). Resting BP was significantly lower (P = .05 for systolic BP; P = .01 for diastolic BP) at the end of the MUFA diet compared with the PUFA diet. Blood pressure responses during sympathetic stimulation with the cold pressor test and isometric exercise were similar. Daily drug dosage was significantly reduced during the MUFA but not the PUFA diet (-48% vs - 4%, P<.005). All patients receiving the PUFA diet required antihypertensive treatment, whereas 8 of those receiving the MUFA diet needed no drug therapy. CONCLUSIONS: A slight reduction in saturated fat intake, along with the use of extra-virgin olive oil, markedly lowers daily antihypertensive dosage requirement, possibly through enhanced nitric oxide levels stimulated by polyphenols. PMID- 10737285 TI - Should noncardiac chest pain be treated empirically? A cost-effectiveness analysis. AB - BACKGROUND: Chest pain is a common clinical problem, but up to 30% of patients who present with chest pain lack coronary disease. Subsequent investigation often reveals an esophageal source for the pain, with gastroesophageal reflux disease identified most frequently. Controversy exists regarding whether to establish the cause or to empirically treat as reflux. OBJECTIVE: To assess the cost effectiveness of empirical treatment in patients with noncardiac chest pain. METHODS: Decision analysis was used to compare a strategy of empirical treatment as reflux using an H-blocker or proton pump inhibitor with initial investigation for gastrointestinal causes over a period of up to 16 weeks and over a period of more than a year. The prototype patient was an outpatient with chest pain and a normal coronary angiogram. Gastrointestinal investigations included an upper gastrointestinal tract series, endoscopy, manometry, 24-hour pH monitoring, and provocation tests. The main outcome measure was direct medical costs per case treated from a third-party payer perspective. RESULTS: Total medical costs were $2,187 per case treated for the initial investigation arm and $849 for the empirical treatment arm in the 8- to 16-week model. One-way sensitivity analyses revealed that the model was robust; the treatment arm was less expensive in all cases. At just over a year empirical treatment remained dominant. CONCLUSIONS: An initial therapeutic trial with antisecretory agents for patients with noncardiac chest pain is cost-effective compared with investigation for gastrointestinal causes in the short term of weeks, with cost savings persisting beyond a year. PMID- 10737286 TI - Around-the-clock, controlled-release oxycodone therapy for osteoarthritis-related pain: placebo-controlled trial and long-term evaluation. AB - BACKGROUND: Although opioid analgesics have well-defined efficacy and safety in treatment of chronic cancer pain, further research is needed to define their role in treatment of chronic noncancer pain. OBJECTIVE: To evaluate the effects of controlled-release oxycodone (OxyContin tablets) treatment on pain and function and its safety vs placebo and in long-term use in patients with moderate to severe osteoarthritis pain. METHODS: One hundred thirty-three patients experiencing persistent osteoarthritis-related pain for at least 1 month were randomized to double-blind treatment with placebo (n = 45) or 10 mg (n = 44) or 20 mg (n = 44) of controlled-release oxycodone every 12 hours for 14 days. One hundred six patients enrolled in an open-label, 6-month extension trial; treatment for an additional 12 months was optional. RESULTS: Use of controlled release oxycodone, 20 mg, was superior (P<.05) to placebo in reducing pain intensity and the interference of pain with mood, sleep, and enjoyment of life. During long-term treatment, the mean dose remained stable at approximately 40 mg/d after titration, and pain intensity was stable. Fifty-eight patients completed 6 months of treatment, 41 completed 12 months, and 15 completed 18 months. Common opioid side effects were reported, several of which decreased in duration as therapy continued. CONCLUSIONS: Around-the-clock controlled-release oxycodone therapy seemed to be effective and safe for patients with chronic, moderate to severe, osteo-arthritis-related pain. Effective analgesia was accompanied by a reduction in the interference of pain with mood, sleep, and enjoyment of life. Analgesia was maintained during long-term treatment, and the daily dose remained stable after titration. Typical opioid side effects were reported during short- and long-term therapy. PMID- 10737287 TI - Clinical survival predictors in patients with advanced cancer. AB - BACKGROUND: The clinical and epidemiological relevance of different prognostic factors for survival in patients with advanced or terminal cancer remains controversial. PURPOSES: To establish the survival of patients with cancer after diagnosis of terminal disease and to determine the predictors of survival. METHODS: An inception cohort of 227 consecutive patients aged 18 years or older with terminal cancer of the lung, breast, and gastrointestinal tract were observed from July 1, 1996, through December 31, 1998. Tumor- and treatment specific, clinical, laboratory, demographic, and socioeconomic variables were recorded at baseline. The relationships between these characteristics and survival time were examined using univariate Kaplan-Meier and multivariate Cox regression analyses. RESULTS: At the time of data analysis, 208 patients (91.6%) had died; the overall median survival for the sample was 15.3 weeks. Shorter survival was independently associated (P< or =.05) with a primary tumor of the lung (vs breast and gastrointestinal tract combined), liver metastases, moderate to-severe comorbidity levels (vs absent-to-mild levels), weight loss of greater than 8.1 kg in the previous 6 months, serum albumin levels of less than 35 g/L, lymphocyte counts of less than 1 X 10(9)/L, serum lactate dehydrogenase levels of greater than 618 U/L, and clinical estimation of survival by the treating physician of less than 2 months (vs 2-6 and >6 months). Performance status, symptoms other than nausea and vomiting, tumor burden, and socioeconomic characteristics such as social support and education and income levels did not appear to be independently associated with survival after adjusting for the effect of prognostic factors. CONCLUSIONS: Simple clinical and laboratory assessments are useful aids in the prediction of survival in patients with solid malignant neoplasms at the onset of terminal stages. Methodological improvements in the design and implementation of survival studies may reduce prognostic uncertainty and ultimately provide better care for the terminally ill patients and their families. PMID- 10737288 TI - Eligibility for outpatient treatment with low-molecular-weight heparin in venous thromboembolism. PMID- 10737289 TI - Herbal interactions with cardiac drugs. PMID- 10737290 TI - Troglitazone-induced pulmonary edema. PMID- 10737291 TI - Ruptured solitary iliac artery aneurysm presenting as a seizure disorder. PMID- 10737292 TI - Pathogenesis and diagnosis of infections with Mycobacterium bovis in cattle. Independent Scientific Group on Cattle TB. AB - In last week's Veterinary Record, members of the Independent Scientific Group on Cattle TB discussed the approach they are adopting in attempting to develop sustainable strategies for controlling bovine tuberculosis in cattle (VR, February 19, pp 207-210). In this second, complementary article, they consider the extent to which efforts to control the disease may be constrained by limitations in current testing procedures. PMID- 10737293 TI - Comparison of in vitro activity of danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin against Mycoplasma mycoides subspecies mycoides small colony type. AB - Minimum inhibitory concentrations (MIC) and minimum mycoplasmacidal concentrations (MMC) of the antimicrobials danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin were determined in vitro for 20 isolates of Mycoplasma mycoides subspecies mycoides small colony type (MmmSC), the causative agent of contagious bovine pleuropneumonia (CBPP). The majority of strains were most susceptible to tilmicosin, followed by danofloxacin, oxytetracycline, florfenicol and spectinomycin with MIC50 values of 0.015, 0.25, 0.5, 1 and 8 microg/ml, and MMC50 values of 0.06, 0.5, 8, 8 and 16 microg/ml, respectively. However, tilmicosin had poor mycoplasmacidal activity against two recent strains from Portugal. There was no evidence of resistance to danofloxacin in any of the strains. PMID- 10737294 TI - Functional and histopathological evidence of cardiac parasympathetic dysautonomia in equine grass sickness. AB - The parasympathetic terminal cardiac ganglia were examined in three normal horses and in five horses with grass sickness. Histopathological changes, consistent with those observed in other ganglia of horses with grass sickness, were identified in the terminal cardiac ganglia of the affected horses. A functional analysis of cardiac autonomic control by time domain analysis (TDA) of heart rate variability was applied to eight horses with grass sickness, and double-paired to 16 normal horses on the basis of their age, sex and breed, and the time of day at which the electrocardiographs were obtained. There were statistically significant differences between the normal and the affected horses in terms of the triangular index (P=0.01), a geometric method of TDA, the sNN100 index (P=0.009), a statistical method of TDA, and the R to R interval (P<0.0005). These results indicate that there is a pathological and functional parasympathetic decentralisation to the heart in equine grass sickness. PMID- 10737295 TI - Field immobilisation of southern elephant seals with intravenous tiletamine and zolazepam. AB - Southern elephant seals (Miroungo leonina) were immobilised with a mixture of tiletamine and zolazepam administered intravenously at a mean (sd) dose rate of 0.46 (0.08) mg/kg. This dose provided a satisfactory degree of anaesthesia with no side effects, and the induction, duration and recovery times were short. The mean (sd) induction time was 26 (9) seconds and the mean level of anaesthesia was 4.4 units on an eight-point scale. Male seals were given less drug than female seals, remained immobilised for shorter periods and recovered sooner. The mean (sd) dose of drug administered to males was 0.44 (0.06) mg/kg and to females 0.48 (0.08) mg/kg, and the mean (sd) duration times were 14.9 (4.5) minutes and 16.1 (5.3) minutes. The mean (sd) time taken to recover from immobilisation was 14.5 (4.6) minutes for males and 15.7 (5.3) minutes for females. Physiological condition and size significantly affected the duration of anaesthesia. Thin seals remained immobilised for 18 (7) minutes whereas fatter seals remained immobilised for 15 (4) minutes (P<0.0001). PMID- 10737296 TI - Effect of bovine leukaemia virus on embryonic development and association with in vitro fertilised embryos. PMID- 10737297 TI - Occurrence of Salmonella in tortoises in a rescue centre in Italy. PMID- 10737298 TI - Thermography of a septic metatarsophalangeal joint in a heifer. PMID- 10737299 TI - Obstructive urolithiasis in a reticulated giraffe (Giraffa camelopardalis reticulata). PMID- 10737300 TI - Pet Travel Scheme. PMID- 10737301 TI - Pet Travel Scheme. PMID- 10737302 TI - Finding of disgraceful professional conduct. PMID- 10737303 TI - Payments for quality produce. PMID- 10737304 TI - Vertigo: analysis by magnetic resonance imaging and angiography. AB - The relationship of vascular disease of the vertebrobasilar artery system to isolated vertigo was examined by magnetic resonance imaging and angiography. Eighty-nine individuals complaining of vertigo were evaluated by standard otoneurologic investigations, and the data were correlated with the vascular patterns of the cervical region and posterior fossa. The age distribution extended from the fourth decade to the ninth decade; the peak occurrence was observed in the eighth. Approximately 85% of the group experienced numerous episodes of vestibular dysfunction from months to years before examination; the remaining segment was examined following the first episode due to severity or persistence of symptoms. The criteria for vascular abnormality proposed by the authors are based upon comparison with previous normal findings. Approximately 52% of the cohort demonstrated abnormal configurations or evidence of diminished flow within the vertebrobasilar artery system. Of this segment, a vertebral artery was most frequently abnormal, in 76%; the basilar artery was judged pathological in 32%, and combined disease of several arteries was evident in 20%. PMID- 10737305 TI - Validation of outcomes survey for adults with chronic suppurative otitis media. AB - Currently, there is no valid, disease-specific outcomes measure to evaluate health impact and treatment effectiveness for patients with chronic suppurative otitis media (CSOM). The Chronic Ear Survey (CES) is a new, disease-specific outcomes measure for CSOM that was administered in a prospective manner to 91 patients with CSOM. It was then validated according to established criteria for reliability, validity, and sensitivity to clinical change by correlation with objective data and self-assessment questionnaires such as the Hearing Handicap Inventory for Adults (HHIA) and the generic 36-Item Short-Form Health Survey (SF 36). Significant correlations between subscale scores of the CES and audiometric data and between subscale scores of the HHIA and SF-36 were found. The standardized response mean for the CES total score was 0.42, indicating moderate sensitivity to clinical change. Overall, results demonstrated that the CES is a reliable and valid instrument for investigation of health status and health related quality-of-life outcomes. PMID- 10737306 TI - Microsurgical anatomy of the perigeniculate ganglion area as seen from a translabyrinthine approach. AB - Detailed anatomic knowledge of the microsurgical anatomy of the perigeniculate ganglion area is essential to probing adjacent to the facial nerve by a translabyrinthine approach. This study was designed to investigate the surgical anatomy of the perigeniculate ganglion area of the facial nerve from a translabyrinthine point of view. We dissected 15 human temporal bones under a microscope, measured the lengths of the tympanic segment and the labyrinthine segment by a middle cranial fossa approach, and measured the angle between the tympanic and labyrinthine segments by a translabyrinthine approach. The distance of the facial nerve from the cochleariform process to the geniculate ganglion was 3.8+/-0.7 mm. The length of the labyrinthine segment of the facial nerve was 4 +/ 0.8 mm. The angle between the tympanic and labyrinthine segments from a translabyrinthine point of view was 26 degrees +/-5 degrees. Precise knowledge about the microsurgical anatomy of the perigeniculate ganglion area of the facial nerve from a translabyrinthine viewpoint is imperative for facial nerve decompression by a translabyrinthine approach. PMID- 10737307 TI - Distribution and location of immunoreactive atrial natriuretic peptides in middle ear mucosa of guinea pig. AB - The distribution and location of atrial natriuretic peptides (ANP) in the middle ear mucosa (MEM) of guinea pigs were studied with immunocytochemistry (avidin biotin-peroxidase complex method) and immuno-electron microscopy. A great quantity of ANP-immunoreactive (ANP-IR) cells, including oval small cells and irregular large cells, was observed in the tissue of the MEM. The strong ANP-IR product was observed in the inner membrane of the partial capillary walls. Under immuno-electron microscopy, the ANP-IR product was found to be spheres of 250 to 350 nm in diameter. The results of this study suggest that the MEM has the function of secreting ANP. This paper provides the morphological basis for the study of ANP's function in the MEM. PMID- 10737308 TI - Effect of pentoxifylline on the healing of guinea pig tympanic membrane. AB - Although most tympanic membrane perforations heal spontaneously, persistent perforations frequently require treatment by otolaryngologists. Initial management strategies include keeping the ear dry, ensuring aural hygiene, and using topical antibiotics. For persistent perforations, paper patching or myringoplasty may be required. Recently, agents such as hyaluronic acid and epidermal growth factor have been used to promote tympanic membrane healing. Similarly, pentoxifylline, a pharmaceutical agent with hemorrheological and antithrombotic properties, has been shown to increase perfusion and accelerate wound healing. This double-blinded prospective study attempts to examine the effect of pentoxifylline on tympanic membrane healing of 50 guinea pigs subjected to myringotomy. Serial examinations and histopathologic sectioning of the tympanic membranes revealed no significant difference in rate of healing or quality of repair between the pentoxifylline and control groups. PMID- 10737309 TI - Overexpression of Fas-ligand in human nasal polyps. AB - Apoptosis mediated through the Fas/Fas-L system is essential in regulating immune function, developing organs, and conferring immune privilege. To illustrate the role of the Fas/Fas-L system in the pathogenesis of human nasal polyps, we investigated the transcripts and protein level of the Fas-L gene in 8 human nasal polyp tissues and 7 nasal turbinate mucosa specimens using reverse transcription polymerase chain reaction and Western blotting. Localization of Fas-L was performed with immunohistochemistry. The transcripts of the Fas-L gene were detected at similar levels in both polyps and nasal mucosa. There was a significant overexpression of Fas-L protein on nasal polyps compared to nasal mucosa. Fas-L-positive cells were localized on the epithelial layers of cystically dilated glands and the down-growing epithelium of nasal polyps. Fas-L may play an important role in the pathogenesis of human nasal polyps, including cystic degeneration of submucosal glands and conferring of immune privilege to nasal polyp formation. PMID- 10737310 TI - Secretory cell differentiation and mucus secretion in cultures of human nasal epithelial cells: use of a monoclonal antibody to study human nasal mucin. AB - We have developed an air-liquid interface culture system for human nasal epithelial cells that differentiate into mucociliary phenotypes in a defined serum-free medium. Dissociated cells obtained from nasal polyps were cultured on a collagen gel substrate. At confluence, the cells lost characteristics of differentiated cells, and secretory cell and ciliated cell differentiation appeared after 7 days in an air-liquid interface. After 21 days, about half of the epithelial cells were stained with Alcian blue-periodic acid-Schiff stain or monoclonal antibody HCS18, which was directed against human nasal mucin specific for epithelial secretory (goblet) cells. The quantitative examination using the antibody HCS18 revealed that the antibody-reactive nasal mucin was secreted only on the apical side of the cultures, and interleukin-1beta and tumor necrosis factor alpha stimulated these mucus secretions. The culture system with an antimucin monoclonal antibody developed in this study should be useful for studying polarized mucus secretion from human nasal epithelial cells. PMID- 10737311 TI - Pneumosinus dilatans of the maxillary sinus. Case report. AB - A case of the rare condition pneumosinus dilatans of the maxillary sinus is presented. This is a rare differential diagnosis of a maxillary disease. The diagnostic imaging features of this condition are described, and a recommendation of views for adequately delineating this disorder is given. Only a few cases of pneumosinus dilatans of the maxillary sinus are described in the English-language literature, and the precise cause and pathogenesis remain obscure. We performed an enlargement of the maxillary ostium in order to increase the ventilation of the paranasal sinus. In the short-term follow-up, this patient has been asymptomatic. The postulated mechanism of air trapping in the sinus cavity seems to be logical in this case, because the symptoms were eliminated by surgically decompressing the sinus to overcome this valve mechanism. PMID- 10737312 TI - Laryngeal blastomycosis: a commonly missed diagnosis. Report of two cases and review of the literature. AB - Blastomycosis is a relatively uncommon fungal disease that most commonly affects the lungs. Other organs may be involved, usually secondary to dissemination of the organism. Laryngeal blastomycosis may occur in isolation from active pulmonary disease. The signs, symptoms, clinical features, and pathological findings of laryngeal blastomycosis mimic those of squamous cell carcinoma. Misdiagnosis may result in inappropriate treatment with potential morbidity. Proper understanding of the clinical presentation and familiarity with the histopathologic features of this disease are therefore imperative. In this paper, we report 2 cases of laryngeal blastomycosis, 1 of which was misdiagnosed as squamous cell carcinoma, clinically and microscopically, with consequent radiotherapy and laryngectomy. In the other case, a clinical diagnosis of glottic squamous cell carcinoma was rendered. However, blastomycosis was identified in a biopsy specimen. We also review cases of isolated laryngeal blastomycosis that have been reported in the English-language literature during the last 80 years. A number of those cases were misdiagnosed clinically and microscopically as squamous cell carcinoma. PMID- 10737313 TI - Glottographic phase difference in recurrent nerve paralysis. AB - Phase measures with simultaneously recorded electroglottography (EGG) and photoglottography (PGG) signals have been studied in canine models and found to be sensitive to the effect of recurrent laryngeal nerve (RLN) paralysis on vocal fold vibration. This study examined the usefulness of this type of measure in clinical application. The combined glottographic signals were obtained from 5 men with a diagnosis of unilateral RLN paralysis and 5 age-matched controls. In the patient group, EGG waveforms were found to have a consistent phase delay in relation to PGG. A measure of the overall glottographic phase difference (GPD) was found to significantly distinguish the control group (mean GPD = 0.2371 ms) from the patient group (mean GPD = -0.2765 ms). A validity analysis performed on 19 subjects with or without unilateral RLN paralysis confirmed that the GPD test had a relatively high efficiency (91.7%) in detecting unilateral RLN paralysis in the male population. PMID- 10737314 TI - Doxorubicin chemomyectomy: effects on evoked vocal fold tension and mucosal wave. AB - Chemomyectomy of the thyroarytenoid muscle is a potential alternative approach to the management of spasmodic dysphonia (laryngeal dystonia) that could provide a prolonged response. To be useful, chemomyectomy should produce weakening of vocal fold closure without disruption of the mucosal wave. Sixteen dogs were studied. In 8 animals, doxorubicin hydrochloride (3 mg) and verapamil hydrochloride (0.5 mg) were injected unilaterally into the thyroarytenoid muscle 2 months before evaluation. The remaining animals served as noninjected controls. Injection of doxorubicin and verapamil decreased the average evoked tension of the vocal fold by 74.7%, compared to an average side-to-side difference of 12.7% in the control group (p = .001). A mucosal wave was recognized bilaterally with videostroboscopy in all dogs. Doxorubicin did not significantly change the vocal fold appearance or mucosal wave amplitude. These results support further laboratory study of chemomyectomy as a potential alternative treatment for laryngeal dystonia. PMID- 10737315 TI - Rare benign tumors: laryngeal and hypopharyngeal lipomata. AB - Lipomata of the larynx and the hypopharynx are very rare benign lesions; as they look macroscopically like retention cysts, their diagnosis is usually made after surgery. Sometimes, lipomata of the hypopharynx become very large and life threatening. Plain fluoroscopy and fluoroscopy aided with barium often fail to depict the lesion if it is localized subepiglottically; standard barium swallow examination and computed tomography allow an accurate diagnosis to be made in many cases, although magnetic resonance imaging is still more accurate and allows not only a more specific diagnosis of the lesion, but also a better depiction of the origin of the frequently pedunculated tumor and its extension into the parapharyngeal space. This paper adds 6 cases of hypopharyngeal and 1 case of laryngeal lipoma to the literature and discusses modern diagnostic and therapeutic strategies. PMID- 10737316 TI - Scleroma of the larynx and associated squamous cell carcinoma: a causal relationship? AB - The known risk factors for development of laryngeal carcinoma are smoking, alcohol drinking, air pollution, laryngeal keratosis, single adult papillomas, and previous irradiation. Laryngeal scleroma as a risk factor for the development of laryngeal carcinoma has not been mentioned in the English-language literature, to my knowledge. This is a report of 2 cases of proven long-term rhinolaryngoscleroma that have changed to laryngeal carcinoma. PMID- 10737317 TI - Cineradiography of the pharyngoesophageal segment in postlaryngectomy patients. AB - The use of tracheoesophageal voice prostheses has gained wide acceptance in the field of vocal rehabilitation after total laryngectomy. In a randomized study with 3 arms, alaryngeal speech proficiency was assessed in 60 postlaryngectomy patients: 20 patients underwent primary unilateral pharyngeal myotomy, 21 patients underwent neurectomy of the pharyngeal plexus in addition to pharyngeal myotomy, and 19 patients did not undergo an additional surgical procedure. Pharyngoesophageal (PE) dynamics were examined during esophageal and tracheoesophageal speech. A single vibrating PE segment was seen in good alaryngeal speakers. Hypertonicity, spasm, strictures, and hypotonicity of the PE segment were correlated significantly with poor or moderate alaryngeal speech. Unilateral myotomy with or without unilateral neurectomy prevented hypertonicity or spasm of the PE segment. The acquisition of alaryngeal speech did not differ significantly between the 2 groups who had undergone an additional surgical procedure. Evaluation of anatomic and physiological factors may be helpful in subsequent clinical management to achieve effective alaryngeal speech. PMID- 10737318 TI - Dysphagia after radiotherapy: endoscopic examination of swallowing in patients with nasopharyngeal carcinoma. AB - Dysphagia is a late sequela compromising the lives of more than one fourth of patients with nasopharyngeal carcinoma (NPC) who survive long after radiotherapy. By using fiberoptic endoscopic examination of swallowing as a modality for dysphagia evaluation, we were able to easily recognize the functional and anatomic changes in 31 dysphagic NPC patients. The majority of patients were found to aspirate after the act of swallowing (77.4%). Seventeen (54.8%) had tongue atrophy, and 9 (29%) had vocal cord palsy. Dysfunctions such as dry mouth (45.2%), velopharyngeal incompetence (58%), premature leakage (41.9%), delay or absence of swallow reflex (87.1%), poor pharyngeal constriction (80.6%), pharyngeal residue retention (83.9%), penetration or aspiration (93.5%), and silent aspiration (41.9%) were noted in these patients. Multiple dysfunctions were demonstrated in each patient. Abnormality of pharyngeal constriction and/or aberrant upper esophageal sphincter function played the major role in the postswallow aspiration of these irradiated NPC patients. Clinically compromised patients (weight loss of > or =5 kg in 1 year or pneumonia) had more of the above anatomic and functional impairments. The radiation dosage and volume bore no correlation to the time of symptom onset, or to the occurrence of functional changes. PMID- 10737319 TI - Osteomyelitis of the skull base with atypical onset and evolution. AB - Skull base osteomyelitis arises as a complication of malignant external otitis, but it can be also due to middle ear and/or mastoid infection. Other causes can be infections of the paranasal sinuses or of the mandible or maxilla due to odontic caries. Generally, osteomyelitis involves elderly patients affected by diabetic immunodeficiency or microvascular disease. In this paper, we present 3 new cases of skull base osteomyelitis with atypical onset and evolution. The difficulties of diagnosis and details of the management are discussed. PMID- 10737320 TI - Trigeminal trophic syndrome mimicking Wegener's granulomatosis. A case report with a review of the literature. AB - Trigeminal neuropathy with nasal ulceration, called trigeminal trophic syndrome, is an unusual complication of anesthesia in the trigeminal area. We present a case to illustrate the diagnostic and management problems that this syndrome presents. PMID- 10737321 TI - Transverse lag screw fixation in midline mandibulotomy. A case series. AB - Vertical midline mandibulotomy has provided a relatively simple and efficient means of obtaining access to intraoral tumors that are too large or too posterior to be removed transorally. Midline mandibulotomy has had the advantage of nerve and muscle preservation and places the osteotomy outside the typical field of radiotherapy, in contrast to lateral and paramedian osteotomies. Plate and screw fixation has been the usual means of osteosynthesis for these mandibulotomies; however, plate contouring over the symphyseal surface has been a time-consuming process. Unless the plate was contoured exactly, mandibular malalignment and malocclusion in dentulous patients has occurred. Use of parallel transverse lag screws has become a popular method of osteosynthesis for parasymphyseal fractures, and we have extended their use for mandibulotomy fixation. This paper reports our clinical experience with transverse lag screw fixation of midline mandibulotomies in 9 patients from 1994 to 1997. There were 7 men and 2 women with a mean age of 56 (range 35 to 71 years). The pathological diagnosis in all patients was squamous cell carcinoma; 8 cases were primary, and 1 patient presented with recurrent tumor. No tumors involved the mandibular periosteum. One patient had had previous radiotherapy, and 3 patients underwent postoperative radiotherapy. The mean follow-up has been 17 months (range 9 to 27). There was 1 minor complication and 1 major complication related to our technique. The major complication was a delayed nonunion of the mandibulotomy. This occurred because the 2 parallel screws were placed too close to one another, and this placement resulted in a delayed sagittal fracture of the anterior cortex and subsequent nonunion. Transverse lag screw fixation has not affected occlusion in our dentulous patients. Speech and diet were normal in the majority of our patients. Transverse lag screw fixation of the midline mandibulotomy has been a relatively safe, rapid, and reliable method for tumor access and postextirpation mandibular stabilization and has significant advantages over other current methods of mandibulotomy and fixation. PMID- 10737322 TI - Multiple cranial nerve palsies as a complication of infectious mononucleosis due to inflammatory lesion in jugular foramen. PMID- 10737323 TI - A new technique distinguishing alpha2-3 sialyl linkage from alpha2-6 linkage in sialyllactoses and sialyl-N-acetyllactosamines by post-source decay fragmentation method of MALDI-TOF mass spectrometry. AB - Alpha2-3 and alpha2-6 sialyl linkage types of sialyllactoses and sialyl-N acetyllactosamines were analyzed by post-source decay (PSD) fragmentation method using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. A new matrix of norharmane was suited for the MALDI-TOF measurements of sialyl oligosaccharides. The fragment ions B1 produced by the cleavage of alpha2-3 sialyl linkages indicate much higher intensity than those produced by the cleavage of alpha2-6 sialyl linkages in sialyllactoses and sialyl N-acetyllactosamines. Thus, alpha2-3 sialyl linkages cleave much easier than alpha2-6 sialyl linkages in MALDI-PSD fragmentation method. These results suggest that the new techniques using PSD fragmentation of MALDI-TOF mass spectrometry enables us to distinguish alpha2-3 sialyl linkage from alpha2-6 linkage in sialyl oligosaccharides. PMID- 10737324 TI - Synthesis of hexose-related imidazolidinones: novel glycation products in the Maillard reaction. AB - Carbohydrate-peptide esters which mimic the reactivity of sugar 6-phosphates in nonenzymatic glycations were used as model compounds for the study of the Maillard reaction in vitro. We found that intramolecular cyclization of the monosaccharide ester in which the sugar moiety (D-glucose or D-galactose) is linked, through the C-6 hydroxy group, to the C-terminal carboxy group of the endogenous opioid pentapeptide leucine-enkephalin, in methanol as the solvent, resulted in the formation of imidazolidinone diastereoisomers having cis or trans relative geometry of the substituents at the imidazolidinone ring moiety. The diastereoisomeric imidazolidinones were separated and each transformed by hydrolysis into the corresponding D-gluco- and D-galacto-related imidazolidinone products of leucine-enkephalin. Along with the previous evidence that, from the same sugar-peptide esters by changing the reaction conditions Amadori rearrangement products could be obtained [Horvat et al. (1998) J Chem Soc Perkin Trans 1:909-13], the presented results point to the possibility that similar carbohydrate-related imidazolidinones may also be generated in the early stage of the Maillard reaction in vivo. PMID- 10737325 TI - A highly convergent approach to O- and N-linked glycopeptide analogues. AB - Deprotected C-glycopyranosyl-ketones have been conjugated by a chemoselective approach to a peptide or an amino acid bearing an aminooxy group on the N terminus or on the side-chain, respectively. The coupling reaction, performed in aqueous media, does not require protecting groups on the peptide or saccharide moieties, nor auxiliary coupling reagents. PMID- 10737326 TI - N-glycan structures of murine hippocampus serine protease, neuropsin, produced in Trichoplusia ni cells. AB - N-glycans of neuropsin (serine protease in the murine hippocampus) expressed in Trichoplusia ni cells were released from the glycopeptides by digestion with glycoamidase A (from sweet almond), and the reducing ends of the oligosaccharides were reductively aminated with 2-aminopyridine. The derivatized N-glycans were separated and structurally identified by a two dimensional high-performance liquid chromatography (HPLC) mapping technique on two kinds of HPLC columns. Fourteen different major N-glycan structures were identified, of which 6 were high-mannose type (9.1%), and the remaining 8 were paucimannosidic type. The presence of insect specific N-glycan structures containing both alpha1,3- and alpha1,6- di-fucosylated innermost N-acetylglucosamine residue (23.3%), as below, was also confirmed by 600 MHz 1H-NMR spectroscopy. PMID- 10737327 TI - Structural analyses of new tri- and tetrasaccharides produced from disaccharides by transglycosylation of purified Trichoderma viride beta-glucosidase. AB - A new beta-glucosidase was partially purified from Trichoderma viride cellulase. This beta-glucosidase catalyzed a transglycosylation reaction of cellobiose to give beta-D-Glc-(1-->6)-beta-D-Glc-(1-->4)-D-Glc (1, yield: 18.8%) and beta-D-Glc (1-->6)-beta-D-Glc-(1-->6)-beta-D-Glc-(1-->4)-D-Glc (2, 3.7%), regioselectively. Furthermore, the enzyme regioselectively converted laminaribiose and gentiobiose into beta-D-Glc-(1-->6)-beta-D-Glc-(1-->3)-D-Glc (3, 15.3%) and beta-D-Glc-(1- >6)-beta-D-Glc-(1-->6)-D-Glc (4, 20.2%), respectively. The structures (1-4) of the products were determined by 1H and 13C NMR spectroscopies. This high regio- and stereoselectively of the beta-glucosidase could be applied for oligosaccharide synthesis. PMID- 10737328 TI - The sialate pyruvate-lyase from pig kidney: purification, properties and genetic relationship. AB - For further insight into the structural relationship between mammalian and microbial sialate pyruvate-lyases, the enzyme from pig kidney was purified to homogeneity from the tissue homogenate by a heat precipitation step followed by anion exchange and Hydrophobic Interaction Chromatography or native gel electrophoresis, respectively. The pure enzyme preparation exhibited an about 1000-fold increase of specific activity compared to the supernatant after the first centrifugation and revealed a single band at 34-37 kDa after SDS-PAGE, which represents the monomeric form of the protein. While the native enzyme seems to be a trimer according to the molecular weight obtained by gel filtration (108 kDa), crosslinking with dimethylpimelimidate suggests it to be a tetramer. The lyase is optimally active at about 75 degrees C and in the pH range of 7.6 to 8.0 and belongs to the class I-aldolases, due to its non-requirement of metal ions and the presence of lysine as the main functional residue in its catalytic centre. These data are similar to those obtained with bacterial lyases. However, peptide fragments of this enzyme show less similarity to primary lyase structures of microbia than to those derived from expressed sequence tags of mammals. PMID- 10737330 TI - Sulfated sialic acid-polymers inhibit the cytotoxic action of bee and snake venom. AB - Colominic acid is an alpha2,8-linked sialic acid polymer produced by Escherichia coli. We found that synthetic sulfated-colominic acids (SC) remarkably inhibited the cytotoxicity of bee and snake venom toward mouse fibroblast cells, but colominic acids showed no inhibition themselves, indicating the important role of sulfate groups in the inhibitory activity of SC. Other sulfated carbohydrates such as chondroitin sulfates, heparin and heparan sulfate showed no inhibition. SC also exhibited potent inhibition of melittin, a highly basic peptide, which is a major cytotoxic component of bee venom. SC did not inhibit phospholipase A2 activity in bee venom. This suggests that the inhibition of bee and snake venom by SC is due to inhibition of melittin and cardiotoxin, which is a cytolytic peptide in snake venom, respectively. SC with a higher sulfur content and a larger molecular mass showed more potent activity. The interaction between SC and melittin basically seems an ionic one, however, the conformation of SC is also likely important. For the binding of SC to melittin leading loss of its cytotoxic activity, the sulfate groups of SC must be properly arranged to interact with lysine and arginine residues of melittin molecules, which play an important role in the cytolytic activity. A higher molecular mass of SC substituted with more sulfate groups is required for more obvious inhibition of the cytotoxic activity. PMID- 10737331 TI - What actually is an obstructive sleep hypopnea? PMID- 10737329 TI - Characterisation of tissue-specific oligosaccharides from rat brain and kidney membrane preparations enriched in Na+,K+-ATPase. AB - The organ-specific nature of the glycosylation of Na+,K+-ATPase-enriched preparations from kidney and brain tissues has earlier been indicated by the use of lectin-staining techniques. Na+,K+-ATPase is ubiquitous and abundant, and subject to upregulation during cell-division and in certain pathological conditions. Lectins specific for the different carbohydrates displayed by the Na+,K+-ATPases may, therefore, be useful carriers/mediators in tissue-specific targeting. N-linked oligosaccharides purified from Na+,K+-ATPase-enriched preparations from rat brain and kidney were consequently characterised in detail in this study using weak anion exchange and normal phase HPLC (combined with serial glycosidase digestions) and matrix-assisted laser desorption/ionisation mass spectrometry. The oligomannose series of glycans were most abundant in the brain tissue preparation and this contrasted with the renal-associated oligosaccharides that were dominated by families of tetra-antennary glycans (with/without a core fucose) with up to four lactosaminylglycan residues in either branched or linear formation. PMID- 10737332 TI - Differential effects of permanent and rotating shifts on self-report sleep length: a meta-analytic review. AB - STUDY OBJECTIVES: The current study used the meta-analytic technique to quantitatively assess the effects of permanent and rotating shift-work schedules on sleep length. DESIGN: A meta-analysis was completed on 36 primary studies resulting in 165 effect sizes. Effect sizes comparing shift-workers to a permanent day shift control group were calculated for permanent evening shifts, permanent night shifts, and morning, evening, and night shifts worked as part of slowly and rapidly rotating shift systems. SETTING: NA PATIENTS OR PARTICIPANTS: NA INTERVENTIONS: NA RESULTS: Permanent night shifts resulted in a decrease, whereas permanent evening shifts resulted in an increase in sleep length. The shifts within rotating schedules followed the same pattern, with the addition of morning shifts having a moderate detrimental effect on sleep length. Furthermore, the speed of shift rotation had an impact. Slowly rotating shifts, in general, had the least detrimental effect on sleep length of the permanent and rotating shift-work schedules studied here. The pattern of effects among morning, evening, and night shifts was the same for rapidly and slowly rotating shifts, with night shifts having the greatest detrimental effect, morning shifts having a moderate detrimental effect and evening shifts having a positive effect on sleep length. In addition, nights on rotating shifts had a greater negative effect on sleep length than permanent night shifts. CONCLUSIONS: Slowly rotating shifts have the least negative impact on sleep length of shift-work schedules including a night shift. However, permanent night shifts could be an alternative shift-work schedule in operational settings that require many workers at night. PMID- 10737333 TI - Influence of sex and age on duration and frequency of sleep apnea events. AB - OBJECTIVES: Differences between men and women potentially provide insight into the regulation sleep apnea events. This study, therefore, examined how apnea frequency and duration varied according to age, sex, and sleep stage in a clinical population. DESIGN: NA SETTING: NA PATIENTS: Patients were 215 women and 215 men referred to a sleep disorders center with symptoms of obstructive sleep apnea and matched for BMI. Apnea events were compared across three age groups (18 39, 40-59, and 60-88 years) in stage 2 and in REM sleep. INTERVENTIONS: NA RESULTS: In stage 2 sleep, young and middle aged women were similar averaging 15 and 13 apnea events per hour respectively. Men had significantly more events averaging 27 and 30 events per hour for the corresponding age groups. The apnea frequency doubled from middle age to older women, and the sex difference narrowed between the older males and females to a non significant difference (26 events per hour for women versus 34 events per hour for men). Apnea duration was significantly longer in men than in women. Stage 2 apnea duration increased significantly with age for men (20.1, 21.5, 23.8 s) and women (16.7, 18.3, 20.6 s) across the three age groups. This also occurred in REM sleep in for men (22.8, 26.5, 29.8 s) and women (19.3, 22.4, 26.6 s). CONCLUSIONS: Duration did not demonstrate the marked "menopausal effect" that there was for apnea frequency. Female gender and younger age conferred benefit primarily by preventing airway collapse (reduced apnea frequency) with less of an effect on apnea duration, i.e., the ability to end the apnea. Compared to stage 2 sleep, REM sleep reduced the differences between men and women in apnea frequency. One explanation may be that differences in muscle tone of the upper airway account for the sex differences in apnea frequency. PMID- 10737334 TI - An instrumental method for long-term continuous REM sleep deprivation of neonatal rats. AB - STUDY OBJECTIVES: The present study describes a new method for instrumental REM sleep deprivation (RSD) of neonatal rats. DESIGN: In the new method, an experimental neonatal rat and a yoked control neonatal rat were singly housed in a small plexiglass chamber which was divided into two separate units by a vertical wall. The floor of the housing chamber was attached to the platform of a standard laboratory test tube shaker. EEG and EMG electrodes were implanted by the soft head plug method which permitted continuous, long-term polysomnography. EEG and EMG signals were sent to a computer that was programmed to turn on the shaker for 5 seconds whenever the experimental rat entered REM sleep. SETTING: NA PATIENTS: NA INTERVENTIONS: NA RESULTS: The shaking of the chamber usually terminated REM sleep by entry to slow-wave sleep or wake. Amount of RSD depended on the shaker's oscillation speed. At higher speed the method reduced REM sleep by more than 80%. CONCLUSIONS: Thus, the new instrumental method of RSD can be used to study developmental functions of neonatal REM sleep. In particular, the instrumental method can test the hypothesis that in rats neonatal RSD produces the adult depressogenic effect of neonatally administered clomipramine. PMID- 10737335 TI - The relationship of lymphocytes in blood and in lymph to sleep/wake states in sheep. AB - Based on evidence of a role for immune-associated cytokines in sleep induction, we investigated the possibility that lymphocyte distribution between blood and lymphatics could be altered as a function of sleep/wakefulness. Blood and lymph sample were obtained from 5 sheep during periods of slow-wave sleep and wake. Blood and lymph lymphocytes were phenotyped using monoclonal antibodies against CD4, CD8, gd T-cell receptors and a surface marker on ovine B cells. Lymph flow rates and efferent lymph cell output were measured. Lymph flow and prescapular efferent lymphocyte output were reduced during sleep compared to wakefulness (p<0.0005). There were no differences in lymphocyte subsets in the blood and in the lymph during sleep/wake brain states. These data indicate that migration of cells in the peripheral lymphatic system is altered during sleep compared to wakefulness. PMID- 10737336 TI - Characteristics of spontaneous sleep with varying NREMS Episodes in healthy men: implication for delta activity homeostasis. AB - STUDY OBJECTIVES: We investigated the effects of the variation in the number of NREMS episodes on human sleep, in particular on delta activity distribution. PARTICIPANTS: Forty-one medication-free healthy men without personal or family history of psychiatric disorders. INTERVENTIONS: Subjects slept four consecutive nights, the last three were analyzed. Sleep was undisturbed and uninterrupted following self-selected schedule. RESULTS: With the exception of one night out of 123, subjects slept at least four NREMS episodes with 80% presenting five or six episodes per night. Yet, only 24% slept the same number of NREMS episodes across three nights. Shorter first NREMS episode was associated with greater number of NREMS episodes, and the total number of NREMS episodes was significantly predicted by the duration of the first one. Whether subjects slept four, five or six NREMS episodes, the proportion of TST spent in REMS, total delta power, total spectral power and the rate of delta power in the first NREMS episode did not differ between subjects. In each night, the distribution of delta activity across the first four NREMS episodes was not modified by the total number of NREMS episodes. CONCLUSION: When spontaneous sleep is allowed, healthy men mostly sleep beyond the fourth NREMS episode. Based on delta activity distribution across the first four episodes, we have demonstrated that the number of NREMS episodes does not modify delta sleep homeostasis. Only duration of first NREMS episode predicts the number of NREMS episodes slept. These results suggest that sleep homeostasis and NREMS-REMS alternation may be two independent processes. PMID- 10737337 TI - Activity, arousal, and the MSLT in patients with insomnia. AB - OBJECTIVES: It has recently been shown that physiological arousal following walking increased sleep latencies during daytime naps as compared to sleep latencies following TV viewing. Patients with insomnia have been shown to have increased physiological arousal and to also have longer MSLT latencies. It was hypothesized that insomnia patients, who are at a higher state of physiological arousal, would be unable to relax while lying in bed and watching TV and therefore would have relatively longer sleep latencies in naps following TV watching (due to inability to relax) as compared to walking. DESIGN: Twelve patients with psychophysiological insomnia took Multiple Sleep Latency Tests after either watching television for 15 minutes or after a 5-minute walk following baseline, sleep deprivation, and recovery sleep conditions. SETTING: Sleep Laboratory PATIENTS: Twelve patients with psychophysiological insomnia INTERVENTIONS: Manipulation of state arousal and sleep deprivation RESULTS: Sleep latencies were significantly longer following the walk as compared to watching TV (11.9 vs. 6.9 min. respectively). Sleep latencies were 13.4 and 3.8 min. following baseline and sleep deprivation conditions. Heart period, used as a measure of physiological arousal, was significantly elevated throughout naps following the walk as compared to naps following TV viewing. Heart period was also significantly correlated with nap sleep latency. CONCLUSIONS: The insomnia patients in this study had significantly increased arousal, as measured by heart rate, and significantly longer sleep latencies after walking as compared to resting. The magnitude of these changes was similar to that seen in normal subjects in a previous study. These data, in concert with previous work, support the contention that measured sleep tendency is a combination of sleep drive and level of central nervous system arousal, where arousal has both state and trait components. PMID- 10737338 TI - Effects of sleep deprivation on daytime sleepiness in primary insomnia. AB - STUDY OBJECTIVES: This study investigated changes in MSLT scores and recovery sleep following total sleep deprivation in subjects with insomnia as compared to normal sleepers. DESIGN: Matched-groups design. SETTING: A sleep disorders center in a large medical center. PARTICIPANTS: Ten individuals with psychophysiological insomnia and ten age- and sex-matched normal sleepers served as subjects. INTERVENTIONS: Subjects underwent total sleep deprivation after baseline polysomnography and MSLT. A post-deprivation MSLT was obtained, as well as polysomnography on the recovery night and an MSLT after the recovery night. MEASUREMENTS AND RESULTS: Both groups showed significant decreases in MSLT scores following total sleep deprivation, as compared to baseline. Both groups had significantly shorter scores on a nighttime MSLT compared to a daytime MSLT. The insomnia group also showed a significant increase in total sleep time on the recovery night compared to baseline. CONCLUSIONS: The MSLT is sensitive to changes in sleepiness associated with total sleep deprivation in individuals with primary insomnia. PMID- 10737339 TI - Identifying montages that best detect electrographic seizure activity during polysomnography. AB - STUDY OBJECTIVES: Recognizing epileptic seizures during video polysomnography (VPSG) can be challenging, particularly when using standard, limited EEG montages and paper speed. Few sleep laboratories have PSG equipment that allows for the recording of 18 channels of EEG without compromising the ability to detect sleep apnea, periodic limb movements, and parasomnias. We studied the ability of sleep medicine- and EEG-trained polysomnographers to correctly identify epileptic seizures during PSG using 4, 7, and 18 channels of simultaneous EEG, recording at conventional PSG and EEG paper speeds. The purpose of this study was to determine the value of limited EEG montages viewed with EEG reformatting capability in the identification of seizures during PSG. DESIGN: Blinded EEG analysis of seizures and arousals during VPSG. SETTING: Tertiary care hospital with sleep laboratory and epilepsy monitoring unit. PATIENTS: Subjects with focal (partial) epilepsy that underwent video-EEG monitoring. INTERVENTIONS: We designed two 7-channel EEG montages that might facilitate the identification of seizures arising from the frontal and temporal lobes. Sleep medicine- and EEG-trained polysomnographers were asked to review tracings containing frontal or temporal lobe epileptic seizures and arousals from sleep. Utilizing the capability of our digital recording equipment to reformat EEG channels and change paper speeds, we asked the readers to classify events recorded with 4, 7, and 18 channels of simultaneous EEG, at paper speeds of 10 and 30 mm/sec. MEASUREMENTS AND RESULTS: 6 readers viewed 32 sleep-related events (13 frontal lobe seizures, 11 temporal lobe seizures, and 8 arousals). The following factors were analyzed for their influence on accuracy of event detection: 1) the type of training of the reader (EEG vs. sleep medicine); 2) the number of EEG channels (4, 7, or 18); and 3) paper speed (10 vs. 30 mm/sec). Pair-wise comparisons and generalized estimating equations were used to identify factors leading to more accurate detection of seizures and arousals. 77% of events were correctly identified: 74% of seizures and 88% of arousals. Seizure detection was better using 7 and 18 channels (sensitivity of 82% and 86%, respectively) than 4 EEG channels (sensitivity of 67%) for temporal lobe seizures only. The number of EEG channels did not affect the accuracy of frontal lobe seizure detection. For EEG-trained readers, accuracy was greater using 30 mm/sec than 10 mm/sec paper speed (85% vs. 78% correct, respectively). CONCLUSIONS: Adding EEG channels and EEG reformatting capabilities to PSG interpretation improves the detection of some types of epileptic seizures. Accuracy of temporal lobe seizure detection using an abbreviated 7-channel montage approximates that of an 18-channel EEG recording. However, the same is not true of frontal lobe seizures in which accuracy was similar regardless of the number of EEG channel available. Further studies are needed to identify specific EEG montages that would best detect epileptiform activity during VPSG. PMID- 10737340 TI - Relationship of temporal lobe seizures to sleep and arousal: a combined scalp intracranial electrode study. AB - STUDY OBJECTIVES: The role of arousal from sleep in promoting epileptic seizures is controversial. To examine the question of whether seizures precede or follow arousals from sleep, we defined the timing of temporal lobe seizures in relation to sleep and arousal using combined scalp-intracranial electrodes. DESIGN: Retrospective review of 67 sleep-related mesial temporal lobe seizures in 14 subjects. SETTING: Inpatient epilepsy monitoring laboratory. PATIENTS: Subjects with medically refractory mesial temporal lobe seizures undergoing epilepsy surgery evaluations. INTERVENTIONS: None MEASUREMENTS AND RESULTS: Electroencephalographic (EEG) and/or polygraphic recordings and videotapes were independently reviewed to determine intracranial electrode seizure onset times and time of initial arousal from sleep. In 60 seizures in 13 subjects, intracranial ictal onsets always preceded clinical arousals from sleep. Electrographic signs of arousal in the scalp EEG, defined by the presence of sustained alpha or theta activity, either coincided with or followed, but never preceded, intracranial ictal onsets. In seven seizures in one subject with known seizures upon awakening, intracranial ictal onsets always followed clinical arousals and electrographic signs of arousal from sleep. Seven of the 14 subjects had electrooculogram and chin electromyogram monitoring; in these subjects, no seizures occurred during REM sleep with the majority occurring during NREM stage 2 sleep. CONCLUSIONS: Most sleep-related temporal lobe seizures occurred during NREM sleep and preceded arousals, supporting the premise that processes involved in the initiation and maintenance of NREM sleep play a greater role in facilitating temporal seizures than those involved in promoting REM sleep and arousal. However, arousal from sleep may provoke seizures in exceptional cases. PMID- 10737341 TI - Practice parameters for the evaluation of chronic insomnia. An American Academy of Sleep Medicine report. Standards of Practice Committee of the American Academy of Sleep Medicine. AB - Chronic insomnia is the most common sleep complaint which health care practitioners must confront. Most insomnia patients are not, however, seen by sleep physicians but rather by a variety of primary care physicians. There is little agreement concerning methods for effective assessment and subsequent differential diagnosis of this pervasive problem. The most common basis for diagnosis and subsequent treatment has been the practitioner's clinical impression from an unstructured interview. No systematic, evidence-based guidelines for diagnosis exist for chronic insomnia. This practice parameter paper presents recommendations for the evaluation of chronic insomnia based on the evidence in the accompanying review paper. We recommend use of these parameters by the sleep community, but even more importantly, hope the large number of primary care physicians providing this care can benefit from their use. Conclusions reached in these practice parameters include the following recommendations for the evaluation of chronic insomnia. Since the complaint of insomnia is so widespread and since patients may overlook the impact of poor sleep quality on daily functioning, the health care practitioner should screen for a history of sleep difficulty. This evaluation should include a sleep history focused on common sleep disorders to identify primary and secondary insomnias. Polysomnography, and the Multiple Sleep Latency Test (MSLT) should not be routinely used to screen or diagnose patients with insomnia complaints. However, the complaint of insomnia does not preclude the appropriate use of these tests for diagnosis of specific sleep disorders such as obstructive sleep apnea, periodic limb movement disorder, and narcolepsy that may be present in patients with insomnia. There is insufficient evidence to suggest whether portable sleep studies, actigraphy, or other alternative assessment measures including static charge beds are effective in the evaluation of insomnia complaints. Instruments such as sleep logs, self-administered questionnaires, symptom checklist, or psychological screening tests may be of benefit to discriminate insomnia patients from normals, but these instruments have not been shown to differentiate subtypes of insomnia complaints. PMID- 10737342 TI - Evaluation of chronic insomnia. An American Academy of Sleep Medicine review. AB - Insomnia is a condition which affects millions of individuals, giving rise to emotional distress, daytime fatigue, and loss of productivity. Despite its prevalence, it has received scant clinical attention. An adequate evaluation of persistent insomnia requires detailed historical information as well as medical, psychological and psychiatric assessment. Use of a classification system for sleep disorders and familiarity with major diagnostic groups will facilitate the clinician's evaluation and treatment. Thorough assessment also requires attention to the unique aspects of presentation and specific set of etiologies which are associated with particular age groups. PMID- 10737343 TI - Harmonization of vascular surgical training in Europe. A task for the European Board of Vascular Surgery (EBVS). AB - The competence of vascular surgeons defined as the level of skill, knowledge and experience necessary to safely perform vascular surgical procedures is determined to a high degree by the quality of the preceding training. In Europe, quality assurance of vascular surgical training, unlike in the USA, is not uniform and is not the responsibility of a centralized European authority, but is a matter in which the different countries have autonomous regulations. Consequently, different targets for duration, contents and general principles for training in vascular surgery have been set. Although in the past this may not have been a problem, the unification of countries in the European Community (EC), at present known as the European Union (EU), has changed this perspective because there is increasing impetus towards a mutual recognition of trade and education between member states. In 1975, EC directive 75/362 was adopted, which insured 'freedom of migration' for medical doctors along with many other professional trades (Publications of the European Communities no. L167, 30-6-1975, p. 1). This directive implicated that certificates, diplomas and other documents issued by the national competent authorities proving medical qualification allowed physicians to practice in any EU country. In order to make this law practical it seems essential that specialist training programmes throughout the EU should conform to certain agreed basic standards. The objective of this article is to present an overview on the current pattern of vascular surgical training in Europe. In addition, the structures that were established during the recent years to promote uniformly high standards of training in vascular surgery throughout the EU will be discussed. PMID- 10737344 TI - The Australian experience in training and certification of vascular surgery. AB - Training in vascular surgery in Australia and New Zealand has been closely related to the policies and politics of the Royal Australasian College of Surgeons. It has evolved from being part of the training for General Surgery through a two year post Fellowship training program in Vascular Surgery commencing in 1985 to the establishment in 1996, by the College, of a Board in Vascular Surgery responsible for training and examining in Vascular Surgery as specialty independent of the Board in General Surgery. PMID- 10737345 TI - Training and certification of vascular surgeons in the United States. PMID- 10737346 TI - The influence of gender on outcome following peripheral vascular surgery: a review. AB - There is considerable evidence that outcome following intervention for coronary artery disease in women may be worse than it is for men. The influence of gender on outcome following peripheral vascular surgery has received less attention and is the subject of this review. The incidence of most vascular procedures in women is 20-40% that of men. Women appear to have a greater risk of dying following surgery for abdominal aortic aneurysm, a higher risk of stroke and death following carotid endarterectomy, and poorer patency rates following infrainguinal bypass. The causes may include delayed diagnosis and referral, increased co-morbidity and possibly the effect of smaller arteries in women. PMID- 10737347 TI - Early carotid endarterectomy after stroke. AB - The authors sought to determine if patients with stroke and a high-grade carotid stenosis benefited from a delay before carotid endarterectomy. A retrospective study of 45 patients undergoing carotid endarterectomy after stroke is presented. The patients were divided into two groups: group I (early group, n = 20), composed of patients who had carotid endarterectomy less than 6 weeks after stroke, and group II (late group, n = 25), comprised of patients who had carotid endarterectomy more than 6 weeks after stroke. As assessed by cerebral angiography, 100% of patients in group I and 64% of patients in group II had carotid artery stenoses > 76% (P < 0.001). The median interval from stroke to carotid endarterectomy was 14 days in group I and 129 days in group II. There was no mortality in either group. No patients in either group demonstrated any neurological deterioration. The authors conclude that, in select patients, carotid endarterectomy may be done safely less than 6 weeks after stroke in order to avoid new events or carotid occlusion while awaiting surgery. PMID- 10737348 TI - The association of platelet-derived growth factor receptor expression, plaque morphology and histological features with symptoms in carotid atherosclerosis. AB - Platelet-derived growth factor may influence smooth muscle cell migration and proliferation and, therefore, carotid plaque composition and stenosis. Platelet derived growth factor receptor expression and histological features were compared in carotid plaques from symptomatic and asymptomatic patients. Immunocytochemistry and histology determined platelet-derived growth factor-alpha and -beta receptor expression, white blood cell infiltration, smooth muscle cell, elastin, cholesterol, collagen and intraplaque haemorrhage in carotid artery plaques removed at surgery or the post-mortem. Plaques with > 70% stenosis from asymptomatic (n = 10) and symptomatic patients (n = 27) had higher expression of platelet-derived growth factor and beta receptors and higher scores for macrophages and intraplaque haemorrhage than plaques with < 70% stenosis from asymptomatic patients (n = 33). Plaques with > 70% stenosis from symptomatic patients had significantly lower alpha receptor expression than plaques with > 70% stenosis from asymptomatic patients. The reduction of alpha receptor expression, which may inhibit smooth muscle cell migration, suggests that differential expression of platelet-derived growth factor receptor subunits in plaques may be related to symptoms. PMID- 10737349 TI - Venous physiology in the different patterns of recurrent varicose veins and the relationship to clinical severity. AB - Venous function measured by air-plethysmography (APG) was compared to anatomical patterns of reflux assessed by duplex scanning and associated clinical features in 253 limbs with recurrent varicose veins following previous superficial venous surgery. The results showed that a previous history of deep venous thrombosis, previous procedure with preservation of the long saphenous vein, and a history of healed ulcer or current ulcer were each associated with worse venous function. Patterns of reflux which included multiple sites of reflux and presence of deep incompetence were also associated with worse venous function. Where there was reflux in the groin, limbs with a wide recurrent saphenofemoral junction presented the worst venous filling time and venous filling index, whereas those with reflux unrelated to the common femoral vein had nearly normal venous physiology and occurred almost exclusively in females. The other patterns of recurrence in the groin were physiologically indistinguishable from each other. In conclusion, certain patterns of reflux, clinical and operative features are associated with worse venous physiology in limbs with recurrent varicose veins. These features of recurrence with more severe physiological disturbance may necessitate a higher priority for surgical intervention. PMID- 10737350 TI - The effect of intraoperative autotransfusion on antibiotic pharmacokinetics during elective abdominal aortic aneurysm repair. AB - AIM: Blood loss during aortic surgery has been reduced by the use of cell-salvage techniques (CS). Antibiotics are used routinely to prevent prosthetic graft infection. The influence of CS on antibiotic levels is unknown. This study measured antibiotic levels in serum and cell-salvage fluid during aortic reconstruction. METHODS: Teicoplanin, a glycopeptide with activity against gram positive bacteria was the antibiotic studied. Serial blood levels were measured after a single intravenous dose (400 mg) in five patients undergoing elective aortic aneurysm repair. Patient ages ranged from 67 to 82 yr. Cell-salvage (Dideco compact A75171) fluid was also assayed. SETTING: District General Hospital RESULTS: Serum teicoplanin levels peaked at mean 67.8 mg/l (SD 8.9 mg/l) 5 min after administration, and fell to mean 2.88 mg/l (SD 0.4 mg/l) at 720 min. This is less than levels in healthy volunteers but above the MIC90 for most Gram positive bacterial pathogens encountered in aortic surgery. Teicoplanin levels in discarded CS fluid at the end of the procedure were 0.56 mg/l (SD 0.71 mg/l). CONCLUSIONS: Teicoplanin blood levels are reduced during aortic surgery. Levels remain adequate for antibacterial prophylaxis for 12 h postoperatively other than for methicillin-resistant staphylococcus epidermidis (MRSE). Compared with patients undergoing arterial reconstruction without the use of a cell-salvage device there is no significant loss due to CS use. PMID- 10737351 TI - Immunophenotypic analysis of the aortic wall in Takayasu's arteritis: involvement of lymphocytes, dendritic cells and granulocytes in immuno-inflammatory reactions. AB - The present study was undertaken to examine the cellular composition of the aortic wall in Takayasu's arteritis and to investigate the association of different cell types in the immuno-inflammatory reactions of this disease. Specimens of aortic wall affected by Takayasu's arteritis were obtained from 10 patients (five male, five female), aged 32 to 68 years (mean 49.5 years) at elective operation. The mean duration of disease was 6.5 years (range 2 months to 13 years). Specimens were embedded in paraffin and the sections stained with antibodies to CD3 (to identify T cells), CD20 (B cells), S-100 (dendritic cells), CD15 (granulocytes), CD68 (macrophages), alpha-SMA (smooth muscle cells) and von Willebrand factor (endothelial cells). Immunohistochemical examination demonstrated that all specimens showed histological alteration with the replacement of the muscular and elastic layers of the media and adventitia by dense fibrous tissue, and were characterized by varying degrees of inflammatory cell infiltration. In five cases, inflammatory nodules consisting of numerous T cells and B cells were observed in the adventitia. Within the inflammatory nodules, as well as around areas of neovascularization in the deep portion of the intima, lymphocytes were co-localized with dendritic cells. In addition, in the adventitia, the accumulation of a large number of granulocytes was observed. The present study demonstrates that immune inflammation is a typical feature of Takayasu's disease, and that the interactions between dendritic cells and lymphocytes may be important in the control of the immune reactions in this vascular pathology. PMID- 10737352 TI - Impact of malignant hematological disorders on cardiac surgery. AB - BACKGROUND: Patients suffering from malignant hematological disorders may be at increased risk, when undergoing cardiac surgery. We report on our experiences with ten cardiac procedures in nine patients (3 males, 6 females, 19-85 yr old, mean age 61 yr). METHODS AND RESULTS: There were two patients with Hodgkin's lymphoma and one patient each with Waldenstrom's syndrome, multiple myeloma, polycythemia, myelodysplasia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma and idiopathic aplastic anemia. Cardiac diseases included coronary artery disease in six, aortic stenosis in two, and mitral insufficiency in one patient. Consecutively, cardiac procedures were coronary artery bypass grafting in six, aortic valve replacement in two, and mitral valve replacement in one patient. No patient died. Postoperatively, one patient suffered from a pericardial tamponade requiring surgical removal and 5 months later from a prosthetic endocarditis necessitating change of the bioprosthesis. One patient developed a superficial wound infection, which was treated conservatively. Four patients received no blood products. Altogether, we transfused 32 packed red blood cells, seven units of fresh frozen plasma and 16 platelet concentrates. Total drainage loss was 883 ml (250-1510 ml). CONCLUSIONS: Cardiac surgery in patients suffering from malignant hematological disorders may be performed, but carries an increased morbidity. Therefore, indications for cardiac procedures must be carefully considered. PMID- 10737353 TI - Three-channeled aortic dissection in Marfan syndrome. AB - Among the various types of aortic dissection, three-channeled aortic dissection, in which two adjacent false lumens are present, is rarely observed. The authors surgically treated 20 Marfan syndrome patients with this type of dissection, who accounted for 29.4% of 68 patients with aortic dissection in Marfan syndrome and who were surgically treated in the authors' institution prior to July 1999. Patient age ranged from 24 to 51 years (mean of 40 years). The male:female ratio was 13:7. Valved conduit procedure had been performed previously in 14 patients. Pain was observed at various times in 13 patients. Computed tomography scanning and magnetic resonance imaging were useful for preoperative diagnosis. The morphology of the first and second false lumens was Stanford type A + type B in 10 patients and type B + type B in 10 patients. Re-entry of the second false lumen was observed in five patients. The descending aorta was replaced in 13 patients, thoracoabdominal aortic replacement was performed in five, extra anatomic bypass in one patient and iliac artery-superior mesenteric artery bypass in one. Four patients died in hospital. There were three late deaths during a follow-up of 1-208 months (mean of 51 months). When pain recurs in Marfan patients with aortic dissection, three-channeled dissection should be suspected and subsequent surgery is required. The incidence of this dissection is high in patients with Marfan syndrome. PMID- 10737354 TI - Congenital division of the left atrium: the influence of associated congenital lesions on the timing and mode of presentation. AB - PURPOSE OF STUDY: Congenital division of an atrium is commonly associated with other cardiac malformations, which may influence presentation. BASIC METHODS: Cases diagnosed at our institution between 1980 and 1997 were reviewed, to determine whether associated lesions influence presentation. PRINCIPLE FINDINGS: 52 patients were diagnosed with atrial division (49 left, 3 right), mean age 4.8 yr. Patients could be divided according to age at diagnosis. Group 1: 32 patients aged < or = 1 yr. 14 patients had associated cardiac malformations other than atrial septal defect (ASD). Presentation was with pulmonary oedema. Group 2: 20 patients > 1 yr. 5 patients had associated major malformations other than ASD. Commonest presentation--isolated shortness of breath. Fifty patients underwent membrane resection and correction of associated anomalies (4% mortality). At follow-up (mean 12 yr) all patients were in NYHA class I or II. CONCLUSIONS: Patients with division of the atrium present early in infancy and with more severe symptoms when associated with other cardiac malformations. PMID- 10737355 TI - Massive renal arteriovenous fistula presenting as a pulsatile abdominal mass. PMID- 10737356 TI - Intracellular trafficking of GABA(A) receptors. AB - Some of the mechanisms that control the intracellular trafficking of GABA(A) receptors have recently been described. Following the synthesis of alpha, beta, and gamma subunits in the endoplasmic reticulum, ternary receptor complexes assemble slowly and are inefficiently inserted into surface membranes of heterologous cells. While beta3, beta4, and gamma2S subunits appear to contain polypeptide sequences that alone are sufficient for surface targeting, these sequences are neither conserved nor essential for surface expression of heteromeric GABA(A) receptors formed from alpha1beta or alpha1betagamma subunits. At the neuronal surface, native GABA(A) receptor clustering and synaptic targeting require a gamma2 subunit and the participation of gephyrin, a clustering protein for glycine receptors. A linker protein, such as the GABA(A) receptor associated protein (GABARAP), may be necessary for the formation of GABA(A) receptor aggregates containing gephyrin. A substantial fraction of surface receptors are sequestered by endocytosis, another process which apparently requires a GABA(A) receptor gamma2 subunit. In heterologous cells, constitutive endocytosis seems to predominate while, in cortical neurons, internalization is evoked when receptors are occupied by GABA(A) agonists. After constitutive endocytosis, receptors are relatively stable and can be rapidly recycled to the cell surface, a process that may be regulated by protein kinase C. On the other hand, a portion of the intracellular GABA(A) receptors derived from ligand-dependent endocytosis is apparently degraded. The clustering of GABA(A) receptors at synapses and at coated pits are two mechanisms that may compete for a pool of diffusable receptors, providing a model for plasticity at inhibitory synapses. PMID- 10737357 TI - Dizocilpine-induced neuropathological changes in rat retrosplenial cortex are reversed by subsequent clozapine treatment. AB - In this study, we examined the effect of post-treatment with clozapine on the neuropathological changes in the rat retrosplenial cortex induced by the administration of non-competitive NMDA receptor antagonist dizocilpine ((+)-MK 801). The maximal increase in vacuolized neurons, which are representative of neuropathology, was observed 4 hours after a single injection of dizocilpine (0.5 mg/kg s.c.), with a complete reversal of the neuropathology after 16-24 hours. The administration of clozapine (10 mg/kg, i.p.,) 4 hours after the administration of dizocilpine significantly decreased the number of vacuolized neurons in the retrosplenial cortex 6, 8 or 10 hours after administration of dizocilpine, compared to vehicle-treated animals. Furthermore, the administration of clozapine (5, 10 or 20 mg/kg i.p.) 4 hours after the administration of dizocilpine produced a significant decrease in the number of vacuolized neurons in the retrosplenial cortex in a dose-dependent manner when measure 6 hours post dizocilpine. These results show that neuropathological changes in the rat retrosplenial cortex produced by dizocilpine can be attenuated by post-treatment with clozapine. PMID- 10737358 TI - No interaction of memantine with acetylcholinesterase inhibitors approved for clinical use. AB - The loss of cholinergic neurons within the basal forebrain of patients with Alzheimer's disease (AD) may underlie aspects of the dementia. Excessive activation of N-methyl-D-aspartate (NMDA) receptors may underlie the degeneration of cholinergic cells. New drug therapies have been designed to either enhance cholinergic function by inhibition acetylcholinesterase (AChE), e.g. galanthamine, tetrahydroaminoacridine or donepezil, or by attenuation of NMDA receptor function, e.g. memantine. A combination of these two therapeutic approaches may be more beneficial at slowing the progression of the AD. The current study investigated whether memantine would attenuate the inhibition of AChE produced by these three drugs. The results indicate that these AChE inhibitors do not lose their therapeutic efficacy in combination with memantine. Our in vitro data suggest that the clinical combination of memantine with a reversible AChE inhibitor should be a valuable pharmacotherapeutic approach to dementia. PMID- 10737359 TI - Bile acid secretion during rat liver carcinogenesis. AB - Retro-differentiation of liver parenchyma during neoplastic processes is characterized by the expression of tumor antigens, such as alpha-fetoprotein and the placental isoenzyme of glutathione-S-transferase (GST-P). To investigate whether this may also affect a typical liver function such as bile acid secretion was the aim of this work. Rat hepatocarcinogenesis was induced by diethylnitrosamine (i.p., 200 mg/Kg body weight at day 0) and promoted by two thirds partial hepatectomy (at day 21) plus 2-acetamidofluorene administration (50 mg/Kg body weight, subcutaneously, twice a week from day 14 to day 35). In order to carry out planimetric measurements of neoplastic tissue after immunohistochemical staining, a novel monoclonal antibody (MAb 14.1.3) against GST-P with no cross-reactivity against the major liver isoform of GST (GST-H) was raised. Analysis of total biliary bile acid output using the 3alpha hydroxysteroid dehydrogenase method indicated that a significant reduction (-26%) occurred during the formation of GST-P-positive foci (12 wk). This was restored to normal values during adenoma formation (16-20 wk), but decreased again during carcinoma transformation (32 wk). These changes were not parallel to that observed in bile flow, which was progressively but slightly decreased throughout the whole period under study. HPLC analysis of bile samples collected for 1 h at different time points during hepatocarcinogenesis revealed that in contrast to what happens during cholestatic disease, a continuous and progressive increase in the cholic acid-to-chenodeoxycholic acid ratio (from 4.4+/-0.5 in control animals to 15.1+/-1.9 in rats with hepatocellular carcinoma) occurs. A significant and transient increase at 16 wk (+120%) in the proportion of bile acids amidated with glycine as compared to those conjugated with taurine was also observed. These results indicate that the mechanisms accounting for the secretion of major bile acids are modified differently at various steps of rat liver tumor development. PMID- 10737360 TI - Possible role of P-450-derived metabolites in endothelium-dependent relaxation of rat small mesenteric arteries. AB - We reported previously that acetylcholine (ACh)-induced endothelium-dependent relaxation of rat mesenteric microvessels depended both on nitric oxide (NO) and on a charybdotoxin (CTX)-sensitive endothelium-derived hyperpolarizing vasodilator. Cytochrome P450 (CYP)-dependent arachidonic acid metabolites act in some systems as hyperpolarizing vasodilators. We sought to quantitate contributions of such metabolites to the CTX-sensitive component of ACh-induced vasodilation in isolated rat mesenteric resistance arteries. ACh relaxed these vessels nearly completely (93.3+/-1.2%, n = 71); cyclooxygenase inhibition with indomethacin did not diminish this response (94.3+/-11.4%, n = 9). NO synthase inhibition with Nitro-L-arginine (NNLA) reduced relaxation by 30% (n = 54, p<0.05). Pretreatment of vessels with CYP inhibitors, either clotrimazole (CTM) or 17-octadecynoic acid (17-ODYA), or with selective K+ channel inhibitors, either tetraethyammonium acetate (TEA) or CTX, each led to similar small reductions in maximal relaxation (17%, 22%, 16%, and 9% respectively, n = 3-6). Combined pretreatment with NNLA + either (CTM or 17-ODYA) or (TEA or CTX) each led to similar maximal relaxations (52.2+/-4.8%, 50.6+/-9.2, 37.6+/-8.6%, and 44.1+/-11.5%, respectively, n = 6-35; p<0.05 for NNLA+[CTM or TEA or CTX] vs NNLA alone). Combined pretreatment with NNLA+CTM+(CTX or TEA) led to similar maximal relaxations (43.0+/-7.3%, 43.7+/-15%, n = 6-11) that did not differ from values in vessels pretreated with either NNLA+CTM or NNLA+(CTX or TEA). We conclude that the ACh-induced vasodilation was insensitive to cyclooxygenase inhibition, partially sensitive to NO synthase inhibition, and that the K+ channel blockers TEA and CTX identified the same minor component of ACh relaxation as did the CYP inhibitor CTM. PMID- 10737361 TI - Dietary protein content affects the profiles of extracellular amino acids in the medial preoptic area of freely moving rats. AB - This study examined the effect of protein consumption on extracellular amino acid concentrations in the medial preoptic area (MPOA) of rats. Rats were given free access to diets containing 0, 25 or 50 % protein for 3-h duration, starting from the onset of dark cycle (1800 h). The microdialysis probe was implanted into the MPOA at 1500 h. Dialysates were collected every 20 min from 1700 h to 2100 h. Amino acid concentrations in dialysate samples were determined by reverse phase HPLC. Extracellular amino acid concentrations in the MPOA were elevated by protein consumption within 20 to 40 min following the start of the meal. The 50 % protein diet resulted in increased (p<0.05) alanine, glutamine, isoleucine, leucine, methionine, tyrosine and valine concentrations, when compared with both baseline and the 0% protein diet. When the 25 % protein diet was fed, amino acid concentrations in the MPOA were between those after the 0 and 50% protein diets. The ratio of tryptophan to the total branched-chain amino acids in extracellular fluid was highest after the 0% protein diet and increased with time. We conclude that extracellular amino acid profiles in the MPOA are affected by dietary protein content. PMID- 10737362 TI - Expression of thyrotropin-releasing hormone (TRH) receptor subtype 1 in mouse pancreatic islets and HIT-T15, an insulin-secreting clonal beta cell line. AB - Thyrotropin-releasing hormone (TRH), originally isolated as a hypothalamic hormone, has been reported to be present and released from the pancreatic beta cells, affecting pancreatic functions. However, it still remains unclear whether TRH receptor is expressed in the pancreas. In the present study, we characterized TRH receptors (TRHR) in mouse pancreatic islets and HIT-T15 cells, a hamster clonal beta cell line. RT-PCR study showed significant expression of TRHR subtype 1 (TRHR1) mRNA in both mouse pancreatic islets and HIT-T15 (HIT) cells. In contrast, there was no expression of TRHR2 mRNA, a novel subtype of TRHR which is expressed predominantly in the central nervous system. Sequencing analysis demonstrated that TRHR1 of the islets was identical to that in the pituitary, and cloned hamster TRHR1 shared 93.3 % homology with that of the mouse at the nucleic acid level. Northern blot analysis of TRHR 1 mRNA in HIT-T15 cells showed a single strong hybridization signal approximately 3.7 kb in length. Furthermore, Scatchard plot analysis in HIT-T15 cells revealed that the Kd value for MeTRH was 0.63 nM. Significant elevation of intracellular calcium concentration was observed in response to as little as 10 nM TRH , and this was not affected by removal of extracellular calcium. This is the first description indicating the presence of functional TRH receptor subtype 1 in the pancreatic beta cells, and our observations suggested the regulation of pancreatic function by TRH through autocrine or paracrine mechanisms. PMID- 10737363 TI - Quantitative analysis of matrix metalloproteinases-2 and -9, and their tissue inhibitors-1 and -2 in human placenta throughout gestation. AB - To elucidate the implication of type IV collagenases(MMP-2 and MMP-9) and their tissue inhibitors (TIMP-1 and TIMP-2) for placental development, we quantified their levels in the conditioned media of placental organ culture and primary culture of the trophoblast as well as in the tissue extracts of placentas from different stages of gestation using specific enzyme-linked immunosorbent assays. First trimester villous tissue secreted about 10 times more pro-MMP-2 than pro MMP-9, and pro-MMP-2 levels dramatically decreased in the second trimester. On the other hand, pro-MMP-9 levels were more than 10 times higher than those of pro MMP-2 in the primary culture of the first trimester trophoblast, indicating the involvement of stromal cells for prominent pro-MMP-2 secretion from first trimester villous tissue described above. Levels of TIMPs, especially those of TIMP-2, remained constant throughout gestation both in the culture media and tissue extracts. Gelatin zymography revealed abundant secretion of the active form of MMP-2 as well as pro-MMP-2 from first trimester villous tissue. Western immunoblot analysis confirmed the presence of both TIMP-1 and TIMP-2 in placental tissue. These results suggest that active secretion of MMP-2 from villous tissue in the first trimester and constant production of TIMPs throughout gestation are characteristic of placental development. PMID- 10737364 TI - NADPH dependent superoxide generation in the ovary and uterus of mice during estrous cycle and early pregnancy. AB - NADPH dependent O2- production in the ovary and uterus of cycling and pregnant mice was estimated employing a novel technique of time-lapse electron spin resonance enhancement of a superoxide-trapping spin trap. The NADPH dependent O2- generation in the mouse ovary increased during the early pre-ovulatory phase in cycling females and during extended luteal phase in pregnant animals. A peak of uterine NADPH dependent O2- production at proestrous in the cycling animals at pre-implantation phase in pregnant animals suggested a contribution of this enzyme towards generating high levels of superoxide anion radical during the respective stages. Both ovarian and uterine NADPH dependent O2- production appeared to be LH-inducible. PMID- 10737365 TI - The regulation of prostaglandin and arachidonoyl-CoA formation from arachidonic acid in rabbit kidney medulla microsomes by palmitoyl-CoA. AB - Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). In the present study, we investigated the effects of palmitic acid (PA) and palmitoyl-CoA (PA-CoA) on the PG and AA-CoA formation from high and low concentrations of AA (60 and 5 microM) in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 60 or 5 microM [14C]-AA in 0.1 M-Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl2 and CoA). After incubation, PG (as total PGs), AA-CoA and residual AA were separated by selective extraction using petroleum ether and ethyl acetate. PA (10-100 microM) had no effect on the PG and AA-CoA formation from either 60 or 5 microM AA. PA-CoA (10-100 microM) was without effect on the PG and AA-CoA formation from 60 microM AA, whereas it markedly decreased the PG formation (6-40%) and increased the AA-CoA formation (1.1-2.3-fold) from 5 microM AA, showing that the effects of PA-CoA on the PG and AA-CoA formation change depending on the AA concentration. These results suggest that PA-CoA, but not PA, may regulate the PG and AA-CoA formation at low substrate concentrations (close to the physiological concentration of AA), and that this in-vitro method using 5 microM AA may be useful for clarifying the homeostatic control of the metabolic fate of AA into these two enzymatic pathways. PMID- 10737367 TI - Intrinsic prostacyclin contributes to exudation induced by bradykinin or carrageenin: a study on the paw edema induced in IP-receptor-deficient mice. AB - To prove that prostaglandin I2 (PGI2) is a major prostaglandin involved in bradykinin-induced exudation, we examined carrageenin- or bradykinin-induced paw edema in prostacyclin receptor-deficient mice (IPKO). Paw volume of wild-type mice (IPWT) increased gradually 5-6 hr after the carrageenin injection in a similar manner as in ICR mice, but the swelling in IPKO mice was significantly smaller (about 60% of the IPWT volume). Indomethacin, at 10 mg/kg, suppressed the swelling of the IPWT paw to the level of the non-pretreated IPKO, which was not affected by indomethacin, confirming the previous result that PGI2 is a major prostaglandin involved in the swelling. The paw edema of IPWT and IPKO was significantly attenuated by the nonpeptide bradykinin B2-receptor antagonist FR173657, at 30 mg/kg, to the same level of swelling, indicating kinin involvement. Injection of bradykinin (1.2 nmole) into the paw caused rapid edema, which peaked around 15 min in both mice. However, the edema induced in IPKO was smaller and almost at the same level as that elicited in the indomethacin-treated IPWT, suggesting that edema induced by bradykinin includes the intrinsic effect of PGI2. Concomitant injection of carbacyclin with bradykinin caused enhancement of edema in IPWT mice but not in IPKO mice, indicating that intrinsic PGI2 could cause enhancement of bradykinin- or even carrageenin-induced edema formation. These results clearly demonstrate that bradykinin released by carrageenin may be a key mediator to induce PGI2 formation, and both autacoids work together to induce enhanced inflammatory exudation. PMID- 10737366 TI - The effect of pentoxifiline on post-injury hyperalgesia in rats and postoperative pain in patients. AB - Recent studies demonstrate that activation of proinflammatory cytokines following injury intensifies the process of nociception. The present investigation assessed the influence of pre-injury pentoxifiline (PTFL, a non-specific cytokine inhibitor) on the development of post-injury nociception in animals and patients. It was established that intrathecal or intraperitoneal PTFL, elevated the nociceptive threshold for mechanical stimuli in the formalin test in rats. Pre injury PTFL also inhibited pain-related behaviour. These findings correlate with a lower TNFalpha level in the serum of animals receiving pre-injury PTFL. In clinical investigations PTFL was administered intravenously before elective cholecystectomy. Patients who received preoperative PTFL had lower opioid requirements in the early postoperative period than control. At the same time, serum levels of TNFalpha and IL6 were lower in the PTFL group. Our results confirm the hypothesis as to the possibility of modulating of nociception through preemptive administration of a cytokine inhibitor. PMID- 10737368 TI - Cocaine abusers show a blunted response to alcohol intoxication in limbic brain regions. AB - Cocaine and alcohol are frequently used simultaneously and this combination is associated with enhanced toxicity. We recently showed that active cocaine abusers have a markedly enhanced sensitivity to benzodiazepines. Because both benzodiazepines and alcohol facilitate GABAergic neurotransmission we questioned whether cocaine abusers would also have an enhanced sensitivity to alcohol that could contribute to the toxicity. In this study we compared the effects of alcohol (0.75 g/kg) on regional brain glucose metabolism between cocaine abusers (n = 9) and controls (n = 10) using PET and FDG. Alcohol significantly decreased whole brain metabolism and this effect was greater in controls (26+/-6%) than in abusers (17+/-10%) even though they had equivalent levels of alcohol in plasma. Analysis of the regional measures showed that cocaine abusers had a blunted response to alcohol in limbic regions, cingulate gyrus, medial frontal and orbitofrontal cortices. CONCLUSIONS: The blunted response to alcohol in cocaine abusers contrasts with their enhanced sensitivity to benzodiazepines suggesting that targets other than GABA-benzodiazepine receptors are involved in the blunted sensitivity to alcohol and that the toxicity from combined cocaine-alcohol use is not due to an enhanced sensitivity to alcohol in cocaine abusers. The blunted response to alcohol in limbic regions and in cortical regions connected to limbic areas could result from a decreased sensitivity of reward circuits in cocaine abusers. PMID- 10737369 TI - Molecular biology of esophageal squamous cell carcinoma. AB - Esophageal squamous cell carcinoma (ESCC) is the predominant histologic subtype of esophageal cancer and characterized by high mortality rate and geographic differences in incidence. With the advances in the field of molecular biology, our understanding of the pathogenesis, epidemiology and behavior of ESCC continues to evolve. The recent development includes research in etiopathogenesis (viruses and cancer susceptibility genes), keratins, tumor related genes (oncogenes, tumor suppressor genes, genes involved in metastasis and apoptosis genes), proliferation-related factors (nuclear proteins, flow cytometry/morphometry, argyrophilic nucleolar organizer region) and factors related to metastases (cell adhesion molecules and enzymes related to degradation of extracellular matrix). There are ranges of molecular techniques potentially available to complement the traditional approaches in the management of ESCC. On the other hand, critics are needed in the interpretation and translation of these research findings from laboratories to clinics. Further investigations, education and collaborations between the various scientific and clinical disciplines are important to successful application of these molecular findings aiming at improving management of patients with ESCC. PMID- 10737370 TI - Salvage surgery for recurrent primary nasopharyngeal carcinoma. AB - Recurrent primary nasopharyngeal carcinoma after radiotherapy exhibits wide local extension [Uttley D, Moore A, Archer DJ. J Neurosurg 1989;71:705-10]. This wide exposure of the nasopharynx and its vicinity is essential for an oncological radical resection to be carried out. The resection should embrace the tumor with an adequate mucosal margin including the cartilaginous portion of the Eustachian tube. Paranasopharyngeal tissues should be included in the resection whenever indicated. The anterolateral approach to the nasopharynx and the paranasopharyngeal space provides adequate exposure required for an oncological resection. Morbidity associated with the operation is low and this is recommended for resection of most recurrent primary nasopharyngeal carcinoma after radiotherapy. PMID- 10737371 TI - Assessing the relative costs of standard open surgery and laparoscopic surgery in colorectal cancer in a randomised controlled trial in the United Kingdom. AB - The role of laparoscopic surgery for the treatment of colorectal cancer is being explored in a multi-centre, randomised clinical trial in the UK, the MRC CLASICC Trial (Conventional versus Laparoscopic-assisted Surgery in Colorectal Cancer). An important end-point of the trial is the cost-effectiveness of laparoscopic surgery compared with that of conventional open surgery. The economic evaluation of this trial has been modelled on that in a similar trial being conducted in the USA in colon cancer. The aim of this paper is to discuss the rationale for modelling the UK trial on the US trial, and to describe the adaptations necessary for the UK trial. The parallel design of the economic evaluation in both trials will provide a unique opportunity to compare the cost implications of incorporating laparoscopic surgery in the UK and the USA, and to determine any cross-cultural differences. The UK trial will also provide information about the cost-effectiveness of laparoscopic surgery in rectal cancer. PMID- 10737372 TI - New trends in the treatment of beta-thalassemia. AB - Thalassemia is the world's most common hereditary disease, and is a paradigm of monogenic genetic diseases. Because of increased population mobility, the disease is found today throughout the world, even in places far from the tropical areas in which it arose. Therapy of thalassemia has in the past been confined to transfusion and chelation. Recently, novel modes of therapy have been developed for thalassemia, based on the pathophysiology and molecular pathology of the disease, both of which have been extensively studied. This review will discuss the therapeutic modalities currently in use for the supportive treatment of thalassemia, both those that are standard therapy and those that are in clinical trials. We will include transfusion, chelation (intravenous and oral), antioxidants and various inducers of fetal hemoglobin (hydroxyurea, erythropoietin, butyrates, hemin). Most of the newer therapies are suitable primarily for thalassemia intermedia patients. In addition, the treatment modalities currently in use for the curative treatment of thalassemia major will be discussed, including bone marrow transplantation in its various forms. Experimental therapeutic methods, such as intrauterine bone marrow transplantation and gene therapy, are included. Physicians caring for thalassemia patients have an increasing variety of treatment options available. Future clinical studies will determine the place of newer agents and modalities in improving the quality of life as well as the life expectancy of thalassemia patients. PMID- 10737373 TI - Aggressive lymphoma in the elderly. AB - Persons 65 years of age and older are the fastest growing segment of the United States population. Over the next 30 years they will comprise approximately 20% of the population. There will be a parallel rise in the number of patients with non Hodgkin's lymphoma. Age has long been known to be an adverse prognostic factor. Clinical trials of older patients are complicated by the effect of comorbid illness, particularly its effect on overall survival. CHOP (cyclophosphamide, Adriamycin, vincristine, prednisone) remains the standard therapy for all patients with aggressive non-Hodgkin's lymphoma. There are a number of regimens which may be beneficial for older patients with significant comorbidity and poor performance status. The randomized trials in the elderly has reaffirmed CHOP and emphasize the need for adequate dosing, maintaining schedule and anthracyclines. Relapsed patients have a poor prognosis but selected fit older patients may benefit from aggressive reinduction regimens and possibly bone marrow transplantation. Future research should include defining the role of comorbidity, measurement of organ dysfunction and assessment of performance status with geriatric functional scales. New drug treatments should also be explored. PMID- 10737374 TI - Ifosfamide in the elderly: clinical considerations for a better drug management. AB - The therapeutic strategy used for elderly affected by malignant neoplasms should take into account the common variables present in oncological patients, and the specific alteration of the elderly metabolism and their possible morbidity. Ifosfamide is an active drug for various tumors with elevated incidence in the elderly. We analyze the principle criteria in the selection of these patients and the alterations of metabolism which correlates to aging with their consequences on the pharmacokinetics of ifosfamide. Finally, we propose clinical guidelines to optimize the use of ifosfamide in elderly patients affected by malignant tumors. PMID- 10737376 TI - Management of the frail person with advanced cancer. AB - The frail population is increasing: currently, approximately 400,000 frail persons have cancer in the USA. Although the frail person is not a candidate for aggressive life-prolonging antineoplastic treatment, he/she is a candidate for aggressive symptom palliation. Most common symptoms include pain, especially bone pain, anemia, and fatigue. Destruction of cancer with antineoplastic treatment is pivotal to symptom palliation. A number of cytotoxic agents including gemcitabine, taxanes in low doses, vinorelbine, oral fluorinated pyrimidine, appear suitable for the management of metastatic cancer in the frail patient and should be tested in clinical trials. PMID- 10737375 TI - Steroidal aromatase inhibitors in elderly patients. AB - The choice of treatment for elderly breast cancer patients needs particular care because the presence of physiological functional impairments can modify the drug bioavailability in an unpredictable manner. Hormonal treatment remains one of the choices and, although tamoxifen has proved to be effective in any setting, the use of selective aromatase inhibitors is arousing. Depending on their chemical structure, aromatase inhibitors are either steroidal (such as exemestane and formestane) or non-steroidal (such as letrozole, vorozole and anastrozole). Formestane has been studied in elderly patients with breast cancer and has been found to induce an overall response rate of 51% (95% CI, 35-67%). The drug suppresses estradiol (E2) levels, and changes in other hormones (FSH, LH and SHBG) are observed, but with poor clinical significance, thus confirming its selectivity and potency. Formestane has also been demonstrated to be as effective as tamoxifen. Exemestane and non-steroidal aromatase inhibitors appear to be very promising drugs. PMID- 10737378 TI - The United Kingdom Childhood Cancer Study PMID- 10737377 TI - Elderly patients with cancer: an ethical dilemma. AB - Longevity is the greatest risk factor for developing cancer. However, despite the increasing focus placed on cancer in the elderly, a prominent discrepancy relating to the age of the patient persists, with elderly people often receiving less than optimal treatment and care. There is no doubt that ageism has a profound impact on health care received by elderly people. Incorporating ethical principles into their clinical practice could facilitate health professionals in the provision of optimal treatment and care for elderly patients with cancer. This article explores some basic ethical assumptions regarding the needs of this patient group in relation to the ethical dilemmas of truth telling, consent and relationships, and power. PMID- 10737379 TI - Tumour necrosis factor alpha increases melphalan concentration in tumour tissue after isolated limb perfusion. AB - Several possible mechanisms for the synergistic anti-tumour effects between tumour necrosis factor alpha (TNF-alpha) and melphalan after isolated limb perfusion (ILP) have been presented. We found a significant sixfold increase in melphalan tumour tissue concentration after ILP when TNF-alpha was added to the perfusate, which provides a straightforward explanation for the observed synergism between melphalan and TNF-alpha in ILP. PMID- 10737380 TI - Plasma vascular endothelial but not fibroblast growth factor levels correlate with colorectal liver mestastasis vascularity and volume. AB - The extent to which plasma levels of angiogenic factors in healthy individuals and tumour volume-related variations in colorectal cancer affect the accuracy of circulating angiogenic factors as predictors of colorectal cancer vascularity is unknown. We used enzyme-linked immunosorbant assay to measure plasma vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) levels in colorectal liver metastasis (CLM) patients, and 'no cancer' controls. CLM volume was determined from computerized tomography scans, and tumour vessel count and vessel volume from anti-endothelial antibody-stained biopsies. There was a significant (P= 0.03) increase in plasma VEGF level in 29 CLM patients (median 180.3 pg/ml(-1), iqr 132.5-284.8 pg/ml(-1) compared with 19 controls (median 125.8 pg/ml(-1), iqr 58.2-235.9 pg/ml(-1). There were significant correlations between plasma VEGF and tumour vessel count (r = 0.66, P = 0.03), tumour vessel volume (r= 0.59, P = 0.03), and CLM volume (r= 0.53, P = 0.03). A VEGF level in the upper quartile of the plasma VEGF distribution had a 70% sensitivity and 75% specificity in predicting an upper quartile liver metastasis tumour vessel count. No relation was identified between CLM and plasma bFGF levels. Plasma VEGF level predicted CLM vascularity, despite an overlap with normal levels and tumour volume-related variations. PMID- 10737381 TI - Reduction in cytokine production in colorectal cancer patients: association with stage and reversal by resection. AB - The aim of this study was to assess monocyte/macrophage function, as defined by lipopolysaccharide (LPS)-induced production of tumour necrosis factor (TNF) alpha, interleukin (IL)-10 and interferon (IFN)-gamma by stimulated whole blood cultures in patients with colorectal carcinoma before and after surgical resection. Forty colorectal cancer patients prior to surgery and 31 healthy controls were studied. Heparinized venous blood was taken from colorectal cancer patients prior to surgery and from healthy controls. Serial samples were obtained at least 3-6 weeks post-operatively. Blood was stimulated with LPS for 24 h and supernatants were assayed for TNF-alpha, IFN-gamma and IL-10 by enzyme-linked immunosorbent assay. LPS-induced production of TNF-alpha and of IFN-gamma was reduced in patients with colorectal carcinoma compared to controls (TNF-alpha, 11,269 pg/ml(-1) ?12,598?; IFN-gamma, 0.00 pg/ml(-1) ?226?; median ?IQR?) (TNF alpha, 20,576 pg/m(-1) ?11,637?, P < 0.0001; IFN-gamma, 1,048 ?2,428?, P = 0.0051, Mann-Whitney U-test). Production in patients after surgery had increased (TNF-alpha: 17,620 pg/ml(-1) ?7,986?; IFN-gamma. 410 pg/ml(-1) ?2,696?; mean ?s.d.?) and were no longer significantly reduced when compared to controls (TNF alpha, P = 0.28; IFN-gamma, P = 0.76). Production of TNF-alpha and IFN-gamma prior to surgery were reduced to a greater extent in patients with Dukes' stage C tumours compared to those with Dukes' stage A and B stage. There was no difference in IL-10 production between any group. Monocytes/macrophages from patients with colorectal carcinoma are refractory to LPS stimulation as reflected by reduction in TNF-alpha and IFN-gamma production and this is more pronounced in patients with advanced stage tumours. This suppression is not mediated by IL-10 and disappears following surgical resection of the tumour. This provides evidence for tumour induced suppression of immune function in patients with colorectal cancer and identifies a potential therapeutic avenue. PMID- 10737382 TI - Decrease of CA 19-9 during chemotherapy with gemcitabine predicts survival time in patients with advanced pancreatic cancer. AB - Chemotherapy with gemcitabine has been shown to be an effective regimen in advanced or metastatic pancreatic cancer with improvement of both quality of life and survival time. The response of the tumour marker CA 19-9 to chemotherapy with gemcitabine was studied in order to find out whether it is related to survival time of patients. Forty-three consecutive patients (median age 61 years, range 39 76 years; 20 males, 23 females) suffering from histologically proven locally advanced or metastatic pancreatic adenocarcinoma and a baseline Karnofsky-index > or = 60 were treated with gemcitabine in a dose of 1,000 mg/m(-2) weekly x 7 followed by 1 week of rest during the first cycle and thereafter 1,000 mg/m(-2) weekly x 3 followed by 1 week of rest until progression. In 36 of 43 patients serial measurements of CA 19-9 could be performed. Patients with a decrease of > 20% of the baseline CA 19-9 level after 8 weeks of treatment (n = 25) had a significantly better median survival than patients with a rise or a decrease < or = 20% (n = 11) (268 vs 110 days; P < 0.001). The response of CA 19-9 was the strongest independent predictor of survival (P < 0.001) in the multivariate analysis. In conclusion, a decrease of CA 19-9 > 20% during the first weeks of chemotherapy with gemcitabine is associated with a better survival of patients with locally advanced or metastatic pancreatic cancer. Serial measurements of CA 19-9 are useful to decide whether further chemotherapy after the first weeks of treatment is indicated. PMID- 10737383 TI - Diagnostic accuracy of large-core needle biopsy for nonpalpable breast disease: a meta-analysis. AB - For the evaluation of non-palpable lesions of the breast, image-guided large-core needle biopsies are increasingly replacing needle-localized open breast biopsies. In this study, the diagnostic accuracy of this minimally invasive technique was evaluated by reviewing the available literature. Five cohort studies were included in a meta-analysis. Sensitivity rate, histological agreement between needle biopsy and subsequent surgery or long-term mammographic follow-up and clinical consequences for different disease prevalences were assessed. The sensitivity rate of large-core needle biopsy for the diagnosis of breast cancer was high (97%). The reclassified agreement rate between core biopsy and subsequent surgical biopsy or long-term mammographic follow-up was also high (94%). In case of 20% breast cancer prevalence among women referred after screening (as in the US), the risk of breast cancer despite benign large-core needle biopsy result is less than 1%. In European countries, however, prevalence of breast cancer among referred women is 60-70%. This would result in a risk of breast cancer despite benign large-core needle biopsy result of 4-6%. The results of this meta-analysis indicate that the image guided large-core needle biopsy is a promising alternative for the needle localized breast biopsy. However, additional research is needed to explore the limiting factors of the technique. Without such detailed knowledge, a benign histological diagnosis on large-core needle biopsy in countries with high prevalence of malignancy among referred women should be interpreted with caution. PMID- 10737384 TI - Manganese superoxide dismutase as a diagnostic marker for malignant pleural mesothelioma. AB - Although several immunohistochemical markers are available, differential diagnosis between mesothelioma and metastatic adenocarcinoma of the pleura is difficult. We have found that the immunoreactivity of manganese superoxide dismutase (MnSOD), an important antioxidant enzyme, is high in mesothelioma compared to healthy pleural mesothelium. The aim of the present study was to investigate whether MnSOD can be used in the differential diagnosis of malignant mesothelioma and metastatic adenocarcinoma of the pleura. MnSOD expression was assessed by using immunohistochemistry in biopsies of malignant mesothelioma (n = 35) and metastatic adenocarcinoma of the pleura (n = 21). MnSOD immunoreactivity was assessed semiquantitatively with and without microwave pretreatment. Fifteen of the 35 malignant mesotheliomas showed moderate or strong MnSOD expression without and 23 with microwave pretreatment, the corresponding figures for metastatic adenocarcinoma of the pleura being 1 and 2 out of 21 (P = 0.002 and P < 0.001, respectively by Fisher's exact test). Only mesothelioma biopsies showed strong MnSOD reactivity, and it was never negative in mesothelioma, whereas one third of the adenocarcinomas showed no MnSOD reactivity. In conclusion, MnSOD immunoreactivity can, combined with other markers, aid the differential diagnosis between malignant mesothelioma and metastatic adenocarcinoma of the pleura. PMID- 10737385 TI - Expression of oestrogen and progesterone receptors in low-grade endometrial stromal sarcomas. AB - We analysed oestrogen receptor (ER) and progesterone receptor (PR) expression in a retrospective series of 21 low-grade endometrial stromal sarcomas (LGSSs). Archival formalin-fixed and paraffin-embedded material was analysed by immunohistochemistry. ER and PR were measured with monoclonal antibodies and the peroxidase-antiperoxidase method and a score was calculated as for breast carcinoma based on both the percentage of positive tumour cell nuclei and the staining intensity. ER were seen in 15 (71%) and PR in 20 (95%) of tumours respectively. ER expression was scored as high in three (14%), moderate in four (19%), and low in eight (38%) tumours. Six (29%) tumours did not stain for ER and all of these were positive for PR. PR expression was scored as high in eight (38%), moderate in ten (47%) and weak in two (10%) LGSSs. Only one (5%) LGSS did not stain for PR (this tumour was positive for ER). ER and PR expression in LGSS is heterogeneous. This may have implications for hormone therapy in the management of these tumours. These results suggest that ER and PR should be routinely quantified in LGSSs by immunohistochemical methods. PMID- 10737386 TI - Decreased GTPase activity of K-ras mutants deriving from human functional adrenocortical tumours. AB - Our previous studies have shown that seven out of 15 patients with adrenocortical tumours contained K-ras gene mutation. In addition, the mutation type was a multiple-site mutation, and the hot spots were located at codons 15, 16, 18 and 31, which were different from those reported before (codons 12, 13 and 61). To understand whether the mutation hot spots in human adrenocortical tumours were associated with activation of K-Ras oncogene and the alterations of its biocharacteristics, mutant K-Ras genes were cloned from tumour tissues and then constructed with expression vector pBKCMV. Mutant K-Ras genes were expressed at high levels in Escherichia coli and the resultant K-Ras proteins were shown to be functional with respect to their well-known specific, high-affinity, GDP/GTP binding. The purified K-Ras protein from E. coli were then measured for their intrinsic GTPase activity and the GTPase activity in the presence of GTPase activating protein for Ras. The results showed that the wild-type cellular K-Ras protein (p21BN) exhibits about ten times higher intrinsic GTPase activity than the activated protein (p21BM3) encoded by mutant K-Ras gene, which mutated at codon 60. With regards to the codon 15, 16, 18 and 31 mutant K-Ras proteins (p21BM2), the GTPase activity in the presence of GAP is much lower than that of the normal K-Ras protein, whereas the intrinsic GTPase activity is nearly the same as that of the normal K-Ras protein. These results indicated that mutations at these hot spots of K-Ras gene were indeed activated K-Ras oncogene in adrenocortical tumours; however, their association with tumors needs further experiments to prove. PMID- 10737387 TI - Increased activity of MAP, p70S6 and p90rs kinases is associated with AP-1 activation in spontaneous liver tumours, but not in adjacent tissue in mice. AB - Growth factor-responsive protein kinases regulate expression of genes involved in cell cycle control, cell proliferation and differentiation. To better understand the role of these kinases in the abnormal proliferation of malignant cells, we examined basal and epidermal growth factor (EGF)-inducible mitogen-activated protein kinase (MAPK), p70S6k and p90rsk activities in spontaneous hepatocellular neoplasms (adenomas and carcinomas) from CBA-T6 mice and in L1 sarcoma tumours implanted in livers of BALB/c mice. In spontaneous and implanted hepatic tumours, basal cytoplasmic and nuclear MAPK, p70S6k and p90rsk activities were significantly higher compared to the activities found in the part of the liver uninvolved by the tumour. Interestingly, the activities of these enzymes in the uninvolved tissue of the livers harbouring the tumour were higher compared to the livers from control mice. Basal kinase activities correlated with tumour morphology; they were lower in adenomas than in carcinomas and sarcomas. In contrast to basal activities, EGF-triggered kinase responses in normal livers and hepatic tumours were indistinguishable. Activating protein-1 (AP-1) DNA-binding activity was detected in tumours but not in the adjacent tissues. Constitutively activated kinases and AP-1 transcription factor found in hepatic malignancies are reminiscent of cells activated by EGF, suggesting that EGF and its intracellular effectors play a role in these malignancies. PMID- 10737388 TI - Absence of telomerase activity and telemorase catalytic subunit mRNA in melanocyte cultures. AB - The classic model of activation of telomerase, for which activity has been found in most cancers including cutaneous malignant melanoma (CMM), dictates that enzyme activity is generated by pathological reactivation of telomerase in telomerase-negative somatic cells. However, recent data demonstrated physiological up-regulation in some normal cell types when established as proliferating cultures, indicating that, in some cancer types, telomerase is expressed by the process of up-regulation in telomerase-competent precursor cells. In this study, cultures of epidermal melanocytes, progenitor cells of CMM, were established and harvested in the logarithmic phase of growth. Telomerase activity was looked for using a non-isotopic variant of the telomeric repeat amplification protocol, and transcript expression of the hTERT gene, the rate limiting catalytic telomerase subunit, was investigated by the reverse transcription polymerase chain reaction. Neither telomerase activity nor hTERT mRNA could be detected in proliferating melanocyte cultures. Our in vitro data argue against the model of telomerase as a common biomarker of cell proliferation. The results further suggest that telomerase is tightly controlled in normal melanocytes, and that telomerase is reactivated rather than up regulated in melanocytic precursors during melanoma initiation or progression. PMID- 10737389 TI - Sensitization of tumour cells to lysis by virus-specific CTL using antibody targeted MHC class I/peptide complexes. AB - A number of cell surface molecules with specificity to tumour cells have been identified and monoclonal antibodies (mAb) to some of these antigens have been used for targeting tumour cells in vivo. We have sought to link the powerful effector mechanisms of cytotoxic T-cells with the specificity of mAb, by targeting recombinant HLA class I molecules to tumour cells using an antibody delivery system. Soluble recombinant MHC class I/peptide complexes including HLA A2.1 refolded around an immunodominant peptide from the HIV gag protein (HLA A2/gag) were synthesized, and the stability of these complexes at 37 degrees C was confirmed by enzyme-linked immunosorbent assay using a conformation-specific antibody. MHC class I-negative lymphoma cells (Daudi) were labelled with a biotinylated mAb specific for a cell surface protein (anti-CD20) then linked to soluble biotinylated HLA-A2/gag complexes using an avidin bridge. Flow cytometry revealed strong labelling of lymphoma cells with HLA-A2/gag complexes (80-fold increase in mean channel fluorescence). CTL specific for HLA-A2/gag efficiently lysed complex-targeted cells, while control CTL (specific for an HLA-A2.1 restricted epitope of melan-A) did not. Similarly, SK-mel-29 melanoma cells were also efficiently lysed by HLA-A2/gag-specific CTL when HLA-A2/gag complexes were linked to their surface via the HMW-MAA specific anti-melanoma antibody 225.28s. With further consideration to the in vivo stability of the MHC class I/peptide complexes, this system could prove a new strategy for the immunological therapy of cancer. PMID- 10737390 TI - Protein kinase C in human renal cell carcinomas: role in invasion and differential isoenzyme expression. AB - The role of protein kinase C (PKC) in in vitro invasiveness of four different human renal cell carcinoma (RCC) cell lines of the clear cell type was investigated. Different PKC-inhibitors markedly inhibited invasiveness of the highly invasive cell lines, suggesting an invasion-promoting role of PKC in human RCC. Analysis of PKC-isoenzyme expression by protein fractionation and immunoblotting revealed that all cell lines expressed PKC-alpha, -epsilon, -zeta, -mu and -iota as known from normal kidney tissue. Interestingly, PKC-delta, known to be expressed by normal kidney epithelial cells of the rat, was absent on protein and RNA levels in all RCC cell lines investigated and in normal human kidney epithelial cells. PKC-epsilon expression levels correlated positively with a high proliferation activity, but no obvious correlation between expression levels of distinct PKC-isoenzymes and in vitro invasiveness was observed. However, by immunofluorescence microscopy, membrane localisation of PKC-alpha and PKC-epsilon reflecting activation of the enzymes, was associated with a highly invasive potential. In conclusion, our results suggest a role for PKC in invasion of human RCCs and might argue in favour of a particular role of PKC-alpha and PKC epsilon. Our results further suggest that organ-specific expression patterns of PKC-isoenzymes are not necessarily conserved during evolution. PMID- 10737391 TI - Gender of offspring and long-term maternal breast cancer risk. AB - Gender of offspring is influenced by maternal hormonal level during pregnancy, which is believed to influence the subsequent maternal breast cancer risk. However, analysing national birth and cancer registrations in a cohort of 998,499 women, we found no association between gender of offspring and subsequent breast cancer risk. PMID- 10737392 TI - The United Kingdom Childhood Cancer Study: objectives, materials and methods. UK Childhood Cancer Study Investigators. AB - An investigation into the possible causes of childhood cancer has been carried out throughout England, Scotland and Wales over the period 1991-1998. All children known to be suffering from one or other type of the disease over periods of 4-5 years have been included, and control children matched for sex, age and area of residence have been selected at random from population registers. Information about both groups of children (with and without cancer) has been obtained from parental questionnaires, general practitioners' and hospital records, and from measurement of the extent of exposure to radon gas, terrestrial gamma radiation, and electric and magnetic fields. Samples of blood have also been obtained from the affected children and their parents and stored. Altogether 3,838 children with cancer, including 1,736 with leukaemia, and 7,629 unaffected children have been studied. Detailed accounts are given of the nature of the information obtained in sections describing the general methodology of the study, the measurement of exposure to ionizing and non-ionizing radiation, the classification of solid tumours and leukaemias, and the biological material available for genetic analysis. PMID- 10737393 TI - Cancer incidence near municipal solid waste incinerators in Great Britain. Part 2: histopathological and case-note review of primary liver cancer cases. AB - We reported previously a 37% excess risk of liver cancer within 1 km of municipal incinerators. Of 119/235 (51%) cases reviewed, primary liver cancer was confirmed in 66 (55%) with 21 (18%) definite secondary cancers. The proportions of true primaries ranging between 55% and 82% (i.e. excluding secondary cancers) give revised estimates of between 0.53 and 0.78 excess cases per 10(5) per year within 1 km. PMID- 10737394 TI - Does antibacterial treatment for urinary tract infection contribute to the risk of breast cancer? AB - Low lignan status has been reported to be related to an elevated risk of breast cancer. Since lignan status is reduced by antibacterial medications, it is plausible to hypothesize that repeated use of antibiotics may also be a risk factor for breast cancer. History of treatment for urinary tract infection was studied for its prediction of breast cancer among 9,461 Finnish women 19-89 years of age and initially cancer-free. During a follow-up in 1973-1991, a total of 157 breast cancer cases were diagnosed. Women reporting previous or present medication for urinary tract infection at baseline showed an elevated breast cancer risk in comparison with other women. The age-adjusted relative risk was 1.34 (95% confidence interval (CI) = 0.98-1.83). The association was concentrated to women under 50 years of age. The relative risk for these women was 1.74 (95% CI 1.13-2.68), whereas it was 0.97 (95% CI 0.59-1.58) for older women. The relative risk in the younger age-group was 1.47 (95% CI 0.73-2.97) during the first 10 years of follow-up, and 1.93 (95% CI 1.11-3.37) for follow-up times longer than 10 years. These data suggest that premenopausal women using long-term medication for urinary tract infections show a possible elevated risk of future breast cancer. The results are, however, still inconclusive and the hypothesis needs to be tested by other studies. PMID- 10737395 TI - Completeness of cancer registration: a new method for routine use. AB - We report a new method of estimating the completeness of cancer registration, in which the proportions of unregistered patients are derived from the time distributions of three probabilities, each of which can be directly estimated from the registry's own data--the probabilities of survival, of registration of the cancer during the patient's life, and of the mention of cancer on the death certificate of a cancer patient who dies. This method allows completeness to be assessed routinely by factors such as age, sex, geographical area and tumour type. PMID- 10737396 TI - Risk factors for Hodgkin's disease by Epstein-Barr virus (EBV) status: prior infection by EBV and other agents. AB - A UK population-based case-control study of Hodgkin's disease (HD) in young adults (16-24 years) included 118 cases and 237 controls matched on year of birth, gender and county of residence. The majority (103) of the cases were classified by Epstein-Barr virus (EBV) status (EBV present in Reed-Stenberg cells), with 19 being EBV-positive. Analyses using conditional logistic regression are presented of subject reports of prior infectious disease (infectious mononucleosis (IM), chicken pox, measles, mumps, pertussis and rubella). In these analyses HD cases are compared with matched controls, EBV positive cases and EBV-negative cases are compared separately with their controls and formal tests of differences of association by EBV status are applied. A prior history of IM was positively associated with HD (odds ratio (OR) = 2.43, 95% confidence interval (CI) = 1.10-5.33) and with EBV-positive HD (OR = 9.16, 95% CI = 1.07-78.31) and the difference between EBV-positive and EBV-negative HD was statistically significant (P = 0.013). The remaining infectious illnesses (combined) were negatively associated with HD, EBV-positive HD and EBV-negative HD (in the total series, for > or =2 episodes compared with < or =1, OR = 0.45, 95% CI = 0.25-0.83). These results support previous evidence that early exposure to infection protects against HD and that IM increases subsequent risk; the comparisons of EBV-positive and EBV-negative HD are new and generate hypotheses for further study. PMID- 10737397 TI - Independence between developmental stability and canalization in the skull of the house mouse. AB - The relationship between the two components of developmental homeostasis, that is canalization and developmental stability (DS), is currently debated. To appraise this relationship, the levels and morphological patterns of interindividual variation and fluctuating asymmetry were assessed using a geometric morphometric approach applied to the skulls of laboratory samples of the house mouse. These three samples correspond to two random-bred strains of the two European subspecies of the house mouse and their F1 hybrids. The inter- and intraindividual variation levels were found to be smaller in the hybrid group compared to the parental ones, suggesting a common heterotic effect on skull canalization and DS. Both buffering mechanisms might then depend on the same genetic condition, i.e. the level of heterozygosity. However, related morphological patterns did not exhibit any congruence. In contradiction with previous studies on insect wing traits, we therefore suggest that canalization and DS may not act on the same morphological characters. The fact that this discrepancy could be related to the functional importance of the symmetry of the characters under consideration is discussed in the light of our knowledge of the genetic bases of both components of developmental homeostasis. PMID- 10737398 TI - The oculomotor distractor effect in normal and hemianopic vision. AB - The present study investigated the inhibitory effect of visual distractors on the latency of saccades made by hemianopic and normal human subjects. The latency of saccades made by hemianopic subjects to stimuli in their intact visual field was not affected by visual distractors presented within their hemianopic field. In contrast, the latency of saccades made by normal subjects was increased significantly under distractor conditions. The latency increase was larger for temporal than nasal distractors. The results are inconsistent with previous proposals that the crossed retinotectal pathway from the nasal hemiretina to the superior colliculus may mediate a blindsight inhibitory effect when distractors appear within a hemianopic temporal visual field. Instead, the distractor effect appears to reflect the normal processes involved in saccade target selection which may be mediated by a circuit involving both cortical and subcortical structures. PMID- 10737399 TI - Demonstration of a foraging advantage for trichromatic marmosets (Callithrix geoffroyi) dependent on food colour. AB - It has been suggested that the major advantage of trichromatic over dichromatic colour vision in primates is enhanced detection of red/yellow food items such as fruit against the dappled foliage of the forest. This hypothesis was tested by comparing the foraging ability of dichromatic and trichromatic Geoffroy's marmosets (Callithrix geoffroyi) for orange- and green-coloured cereal balls (Kix) in a naturalized captive setting. Trichromatic marmosets found a significantly greater number of orange, but not green, Kix than dichromatic marmosets when the food items were scattered on the floor of the cage (at a potential detection distance of up to 6 m from the animals). Under these conditions, trichromats but not dichromats found significantly more orange than green Kix, an effect that was also evident when separately examining the data from the end of the trials, when the least conspicuous Kix were left. In contrast, no significant differences among trichromats and dichromats were seen when the Kix were placed in trays among green wood shavings (detection distance < 0.5 m). These results support an advantage for trichromats in detecting orange coloured food items against foliage, and also suggest that this advantage may be less important at shorter distances. If such a foraging advantage for trichromats is present in the wild it might be sufficient to maintain the colour vision polymorphism seen in the majority of New World monkeys. PMID- 10737400 TI - The role of colour in signalling and male choice in the agamid lizard Ctenophorus ornatus. AB - Bright coloration and complex visual displays are frequent and well described in many lizard families. Reflectance spectrometry which extends into the ultraviolet (UV) allows measurement of such coloration independent of our visual system. We examined the role of colour in signalling and mate choice in the agamid lizard Ctenophorus ornatus. We found that throat reflectance strongly contrasted against the granite background of the lizards' habitat. The throat may act as a signal via the head-bobbing and push-up displays of C. ornatus. Dorsal coloration provided camouflage against the granite background, particularly in females. C. ornatus was sexually dichromatic for all traits examined including throat UV reflectance which is beyond human visual perception. Female throats were highly variable in spectral reflectance and males preferred females with higher throat chroma between 370 and 400 nm. However, female throat UV chroma is strongly correlated to both throat brightness and chest UV chroma and males may choose females on a combination of these colour variables. There was no evidence that female throat or chest coloration was an indicator of female quality. However, female brightness significantly predicted a female's laying date and, thus, may signal receptivity. One function of visual display in this species appears to be intersexual signalling, resulting in male choice of females. PMID- 10737401 TI - Nanometre-range acoustic sensitivity in male and female mosquitoes. AB - Johnston's sensory organ at the base of the antenna serves as a movement sound detector in male mosquitoes, sensing antennal vibrations induced by the flight sounds of conspecific females. Simultaneous examination of acoustically elicited antennal vibrations and neural responses in the mosquito species Toxorhynchites brevipalpis has now demonstrated the exquisite acoustic and mechanical sensitivity of Johnston's organ in males and, surprisingly, also in females. The female Johnston's organ is less sensitive than that of males. Yet it responds to antennal deflections of +/- 0.0005 degrees induced by +/- 11 nm air particle displacements in the sound field, thereby surpassing the other insect movement sound detectors in sensitivity. These findings strongly suggest that the reception of sounds plays a crucial role in the sensory ecology of both mosquito sexes. PMID- 10737402 TI - Barn owl (Tyto alba) siblings vocally negotiate resources. AB - Current theory proposes that nestlings beg to signal hunger level to parents honestly, or that siblings compete by escalating begging to attract the attention of parents. Although begging is assumed to be directed at parents, barn owl (Tyto alba) nestlings vocalize in the presence but also in the absence of the parents. Applying the theory of asymmetrical contests we experimentally tested three predictions of the novel hypothesis that in the absence of the parents siblings vocally settle contests over prey items to be delivered next by a parent. This 'sibling negotiation hypothesis' proposes that offspring use each others' begging vocalization as a source of information about their relative willingness to contest the next prey item delivered. In line with the hypothesis we found that (i) a nestling barn owl refrains from vocalization when a rival is more hungry, but (ii) escalates once the rival has been fed by a parent, and (iii) nestlings refrain from and escalate vocalization in experimentally enlarged and reduced broods, respectively. Thus, when parents are not at the nest a nestling vocally refrains when the value of the next delivered prey item will be higher for its nest-mates. These findings are the exact opposite of what current models predict for begging calls produced in the presence of the parents. PMID- 10737403 TI - Pre-dawn infidelity: females control extra-pair mating in superb fairy-wrens. AB - Despite great interest in the use of extra-pair mating as a tool for examining female choice and intersexual selection, the underlying assumption of female control has proved difficult to verify empirically. We combined microsatellite genotyping and radiotelemetry of fertile females in order to investigate mate choice in superb fairy-wrens Malurus cyaneus, the bird with the highest known rate of extra-pair fertilization. All five females radio tracked during the peak of fertility, two to four days before the first egg is laid, undertook pre-dawn forays. All extra-pair young produced by the female were sired by a male visited during their forays, indicating that females control extra-pair fertilizations. In a larger sample of paternity data, some broods were sired by two extra-group males. In virtually all the cases the territory of the two sires were on an identical linear trajectory from the female's territory. This again suggests that extra-group paternity in superb fairy-wrens is directly linked to female extra territorial forays. In other species mixed paternity has been taken to indicate that females attempt to insure against infertile pairings or try to maximize the genetic diversity of their brood. However, in fairy-wrens the likelihood of multiple extra-group paternity increased greatly as females traversed more territories in order to mate, perhaps suggesting that females which foray further are more likely to have difficulties locating the preferred male. PMID- 10737404 TI - Body mass and individual fitness in female ungulates: bigger is not always better. AB - In female vertebrates, differences in fitness often correspond to differences in phenotypic quality, suggesting that larger females have greater fitness. Variation in individual fitness can result from variation in life span and/or variation in yearly reproductive success, but no study has yet assessed the relationships between the components of fitness and phenotypic quality while controlling for life span. We tried to fill this gap using data from long-term monitoring (23 years) of marked roe deer and bighorn sheep, two ungulates with very different life histories. In both species, we found a strong positive relationship between an adult female's mass and her probability of reaching old age: over the long term, bigger is indeed better for ungulate females. On the other hand, we found no evidence in either species that heavier females had higher fitness when differences in life span were accounted for: over the short term, bigger is not necessarily better. Our results indicate that, while broad differences in phenotypic quality affect individual fitness, when differences in life span are accounted for phenotypic quality has no residual effect on fitness. Therefore, within a given range of phenotypic quality, bigger is not always better, for reasons which may differ between species. PMID- 10737405 TI - Energy assimilation, parental care and the evolution of endothermy. AB - The question of the selection forces which initiated the evolution of endothermy in birds and mammals is one of the most intriguing in the evolutionary physiology of vertebrates. Many students regard the aerobic capacity model as the most plausible hypothesis. This paper presents an alternative model, in which the evolution of endothermy in birds and mammals was driven by two factors: (i) a selection for intense post-hatching parental care, particularly feeding offspring, and (ii) the high cost of maintaining the increased capacity of the visceral organs necessary to support high rates of total daily energy expenditures. PMID- 10737406 TI - Three energy variables predict ant abundance at a geographical scale. AB - Energy theory posits three processes that link local abundance of ectotherms to geographical gradients in temperature. A survey of 49 New World habitats found a two order of magnitude span in the abundance (nests m(-2)) of ground nesting ants (Formicidae). Abundance increased with net primary productivity (r2=0.55), a measure of the baseline supply of harvestable energy. Abundance further increased with mean temperature (r2=0.056), a constraint on foraging activity for this thermophilic taxon. Finally for a given mean temperature, ants were more abundant in seasonal sites with longer, colder winters (r2 = 0.082) that help ectotherm taxa sequester harvested energy in non-productive months. All three variables are currently changing on a global scale. All should be useful in predicting biotic responses to climate change. PMID- 10737407 TI - Estimating ancestral geographical distributions: a Gondwanan origin for aphid parasitoids? AB - We tested the published hypothesis of a Gondwanan origin for the overwhelmingly northern hemisphere aphid parasitoids (Aphidiinae) as follows: (i) finding their sister group by a phylogenetic analysis of the entire Braconidae (Insecta: Hymenopterai using sequence data from approximately 500 bp fragments of both the nuclear 28S (D2 region) and mitochondrial 16S rDNA genes, (ii) using this sister group relationship and the more informative 28S D2 gene to estimate the phylogeny of the Aphidiinae and (iii) estimating the ancestral distribution for the Aphidiinae using maximum-likelihood and maximum-parsimony methods. Both methods indicated a Gondwanan origin. PMID- 10737408 TI - The shifting roles of dispersal and vicariance in biogeography. AB - Dispersal and vicariance are often contrasted as competing processes primarily responsible for spatial and temporal patterns of biotic diversity. Recent methods of biogeographical reconstruction recognize the potential of both processes, and the emerging question is about discovering their relative frequencies. Relatively few empirical studies, especially those employing molecular phylogenies that allow a temporal perspective, have attempted to estimate the relative roles of dispersal and vicariance. In this study, the frequencies of vicariance and dispersal were estimated in six lineages of birds that occur mostly in the aridlands of North America. Phylogenetic trees derived from mitochondrial DNA sequence data were compared for towhees (genus Pipilo), gnatcatchers (genus Polioptila), quail (genus Callipepla), warblers (genus Vermivora) and two groups of thrashers (genus Toxostoma). Different area cladograms were obtained depending on how widespread and missing taxa were coded. Nonetheless, no cladogram was obtained for which all lineages were congruent. Although vicariance was the dominant mode of evolution in these birds, approximately 25% of speciation events could have been derived from dispersal across a preexisting barrier. An expanded database is now needed to estimate the relative roles of each process. Applying a molecular clock calibration, nearly all speciation events are of the order of a million or more years old, much older than typically presumed. PMID- 10737409 TI - Complementarity as a biodiversity indicator strategy. AB - Richness, rarity, endemism and complementarity of indicator taxon species are often used to select conservation areas, which are then assumed to represent most regional biodiversity. Assessments of the degree to which these indicator conservation areas coincide across different taxa have been conducted on a variety of vertebrate, invertebrate and plant groups at a national scale in Britain, Canada, USA and South Africa and at a regional scale in Cameroon, Uganda and the USA. A low degree of spatial overlap among and within these selected indicator conservation areas has been demonstrated. These results tend to suggest that indicator conservation areas display little congruence across different taxa. However, some of these studies demonstrate that many conservation areas for indicator taxa capture a high proportion of non-target species. Thus it appears that indicator conservation areas might sample overall biodiversity efficiently. These indicator conservation areas may, however, exclude species essential for effective conservation, e.g. rare, endemic or endangered species. The present study investigated the value of indicator taxa as biodiversity surrogates using spatial congruence and representativeness of different indicator priority conservation areas. The conservation status of species excluded by the indicator approaches is also assessed. Indicator priority conservation areas demonstrate high land area requirements in order to fully represent non-target species. These results suggest that efficient priority area selection techniques must reach a compromise between maximizing non-target species gains and minimizing land-use requirements. Reserve selection procedures using indicator-based complementarity appear to be approaches which best satisfy this trade-off. PMID- 10737410 TI - Pervasive compensatory adaptation in Escherichia coli. AB - To investigate compensatory adaptation (CA), we used genotypes of Escherichia coli which were identical except for one or two deleterious mutations. We compared CA for (i) deleterious mutations with large versus small effects, (ii) genotypes carrying one versus two mutations, and (iii) pairs of deleterious mutations which interact in a multiplicative versus synergistic fashion. In all, we studied 14 different genotypes, plus a control strain which was not mutated. Most genotypes showed CA during 200 generations of experimental evolution, where we define CA as a fitness increase which is disproportionately large relative to that in evolving control lines, coupled with retention of the original deleterious mutation(s). We observed greater CA for mutations of large effect than for those of small effect, which can be explained by the greater benefit to recovery in severely handicapped genotypes given the dynamics of selection. The rates of CA were similar for double and single mutants whose initial fitnesses were approximately equal. CA was faster for synergistic than for multiplicative pairs, presumably because the marginal gain which results from CA for one of the component mutations is greater in that case. The most surprising result in our view, is that compensation should be so readily achieved in an organism which is haploid and has little genetic redundancy This finding suggests a degree of versatility in the E. coil genome which demands further study from both genetic and physiological perspectives. PMID- 10737411 TI - Meeting the photosynthetic demand for inorganic carbon in an alga-invertebrate association: preferential use of CO2 by symbionts in the giant clam Tridacna gigas. AB - Unlike most marine invertebrates which excrete respiratory CO2, giant clams (Tridacna gigas) must acquire inorganic carbon (Ci) in order to support their symbiotic population of photosynthetic dinoflagellates. Their capacity to meet this demand will be reflected in the Ci concentration of their haemolymph during periods of high photosynthesis. The Ci concentration in haemolymph was found to be inversely proportional to irradiance with a minimum Ci concentration of 0.75 mM at peak light levels increasing to 1.2 mM in the dark. The photosynthetic rate of isolated zooxanthellae under conditions that prevail in the haemolymph at peak light levels was significantly less than the potential Pmax (maximum photosynthetic rate) indicating that zooxanthellae are carbon limited in hospite. This is consistent with previous studies on the hermatypic coral symbiosis. The Pmax was not affected by pH but there was a dramatic increase in the half saturation constant for Ci (K0.5 (Ci)) with increasing pH (6.5-9.0) and only a small decrease in K0.5 (CO2) over the same range. These results indicate that zooxanthellae in giant clams use CO2 as the primary source of their Ci in contrast to symbionts in corals, which use bicarbonate. The physiological implications are discussed and comparison is made with the coral symbiosis. PMID- 10737412 TI - Effect of noise exposure on rat cardiac peripheral benzodiazepine receptors. AB - Noise is an environmental physical agent, which is regarded as a stressful stimulus: impairment and modifications in biological functions are reported, after loud noise exposure, at several levels in human and animal organs and apparatuses, as well as in the endocrine, cardiovascular and nervous system. In the present study equilibrium binding parameters of peripheral benzodiazepine receptors (PBRs) labelled by the specific radioligand [3H]PK 11195, were evaluated in cardiac tissue of rats submitted to 6 or 12 h noise exposure and of rats treated "in vivo" with PBR ligands such as PK 11195, Ro54864, diazepam and then noise-exposed. Results revealed a statistically significant decrease in the maximum number of binding sites (Bmax) of [3H]PK 11195 in atrial membranes of 6 or 12 h noise exposed rats, compared with sham-exposed animals, without any change in the dissociation constant (Kd). The "in vivo" PBR ligand pre-treatment counteracted the noise-induced modifications of PBR density. As PBRs are mainly located on mitochondria we also investigated whether noise exposure can affect the [3H]PK 11195 binding parameters in isolated cardiac mitochondrial fractions. Results indicated a significant Bmax value decrease in right atrial mitochondrial fractions of rats 6 or 12 h noise-exposed. Furthermore, as PBR has been suggested to be a supramolecular complex that might coincide with the not-yet-established structure of the mitochondrial permeability transition (MPT)-pore, the status of the MPT-pore in isolated heart mitochondria was investigated in noise- and sham exposed rats. The loss of absorbance associated with the calcium-induced MPT-pore opening was greater in mitochondria isolated from hearts of 6 h noise- than those of sham-exposed rats. In conclusion, these findings represent a further instance for PBR density decrease in response to a stressful stimulus, like noise; in addition they revealed that "in vivo" administration of PBR ligands significantly prevents this decrease. Finally, our data also suggest the involvement of MPT in the response of an organism to noise stress. PMID- 10737414 TI - Suppression of heat-induced HSP-70 by simultaneous exposure to 50 mT magnetic field. AB - Effect of extremely low frequency magnetic field (ELFMF) at 50 mT and 60 Hz on heat-induced expression of heat shock protein 70 (hsp-70) was examined in HL60RG cells. No increase in hsp-70 production was observed in the cells after exposure to 50 mT ELFMF alone. Simultaneous exposure to 50 mT ELFMF in combination with mild heat at 42 and 40 degrees C suppressed heat-induced hsp-70 expression. The suppression of hsp-70 occurred when cells were simultaneously exposed to both for longer periods of more than 5 h. However, the suppression of hsp-70 was not observed at a magnetic density of 5 and 0.5 mT. This result suggests that exposure to 50 mT ELFMF may act on a protection against the concomitant mild heat stress in HL60RG cells. PMID- 10737413 TI - Purification and characterization of glycolactin, a novel glycoprotein from bovine milk. AB - A novel glycoprotein designated glycolactin, with a molecular weight of 64 kDa, a sequence hitherto unknown in the literature and capable of inhibiting the hemagglutinating activities of soybean lectin and Ricinus communis agglutinin 120, was isolated from bovine milk. Its lectin-inhibiting activity differed from that of lactoferrin, another milk protein. Like other milk proteins, glycolactin inhibited superoxide formation in vitro. Glycolactin inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of about 31 nM. It exhibited ribonucleolytic (RNase) activity towards yeast transfer RNA with a pH optimum of 7.5, and specific RNase activity towards poly C. The purification protocol of glycolactin involved removal of globulin from the acid whey fraction of bovine milk by precipitation with 1.8 M (NH4)2SO4, and adsorption on the ion exchangers CM-Sepharose and Mono S. Deglycosylation of glycolactin using glycopeptidase F produced only a slight decrease of 4 kDa in the molecular weight of glycolactin. PMID- 10737415 TI - Influence of morphine treatment in pregnant rats on the mineralocorticoid activity of the adrenals in their neonates. AB - Exposure of pregnant rats to morphine, from day 11 to day 18 of gestation, was previously reported to induce both an adrenal atrophy and hypoactivity of the glucocorticoid function in newborns at term, but did not affect, in vitro, the responsiveness of those glands to adrenocorticotrophin hormone (ACTH) concerning corticosterone release. Moreover, these effects were mediated by maternal hormones from the adrenal glands. In the present work, we investigated the effects of a prenatal morphine exposure on the mineralocorticoid activity of the adrenals in neonates. The first aim of the present study was to determine in these newborns 1) the adrenal and plasma aldosterone concentrations at birth time and during the early postnatal period 2) the plasma levels of Na+ and K+ at birth time, 3) the in vitro responsiveness of the newborn adrenals to angiotensin II (A(II)) and ACTH. The second aim of our study was to investigate the mineralocorticoid activity of the adrenals in newborns from adrenalectomized mothers treated with morphine during gestation. According to present data morphine given to intact mothers induced in newborns a severe adrenal atrophy but increased adrenal aldosterone content and plasma aldosterone level. However, prenatal morphine was unable to affect significantly Na+/K+ ratio in both mothers and newborns. In vitro, the adrenals of neonates from morphine-treated mothers were unresponsive to An and ACTH for promoting aldosterone release; in contrast, aldosterone secretion was significantly stimulated by high potassium levels (55 mEq). Maternal adrenalectomy performed one day before the beginning of morphine treatment prevented morphine-induced adrenal atrophy but was unable to affect significantly the adrenal mineralocorticoid function of the offspring. Such data suggest that a prenatal morphine exposure stimulated both aldosterone synthesis and release in neonates. However, this basal hyperfunction did not appear to be coupled with an enhanced adrenal responsivity to AII or ACTH. Prenatal morphine induced hyperactivity of the mineralocorticoid function of the newborn adrenals, which drastically contrast with hypoactivity of the glucocorticoid one, was independent of adrenal factors from maternal origin. PMID- 10737416 TI - Involvement of adrenaline in diazepam-induced hyperglycemia in mice. AB - Diazepam-elicited hyperglycemia in mice was inhibited by adrenalectomy and the catecholamine synthesis inhibitor alpha-methyl-p-tyrosine, although it was unaffected by pretreatment with the corticosterone synthesis inhibitor dexamethasone. Diazepam-induced hyperglycemia was prevented by the alpha2 adrenoceptor antagonist idazoxan, while the beta1 and beta2 adrenoceptor antagonists, metoprolol and ICI 118 551, did not affect it. Furthermore, diazepam increased plasma adrenaline levels in mice. These results suggest that diazepam induced hyperglycemia is closely related to adrenaline release from the adrenal gland. PMID- 10737417 TI - Interleukin 12 and indomethacin exert a synergistic, angiogenesis-dependent antitumor activity in mice. AB - Nonsteroidal anti-inflammatory drugs have been shown to reduce the incidence and mortality from colorectal cancer. It has recently been demonstrated that these drugs are capable of suppressing the production of pro-angiogenic factors from tumor cells. The mechanisms of antitumor action of interleukin 12 include the enforced secretion of anti-angiogenic factors and stimulation of antitumor immunity. Therefore, we hypothesized that the combination of a model nonsteroidal anti-inflammatory drug--indomethacin and interleukin 12--would result in enhanced angiogenesis-dependent antitumor effects against a colon-26 carcinoma cells transplanted into syngeneic mice. As expected the combined administration of both agents simultaneously resulted in a strengthened antitumor activity that was manifested as a retardation of tumor growth and prolongation of mouse survival. Importantly some mice were completely cured after the combined treatment. As administration of interleukin 12 and indomethacin resulted in enhanced inhibition of angiogenesis it seems possible that prevention of new blood vessel formation is one of the mechanisms responsible for the observed antitumor effects. PMID- 10737418 TI - Enhancement of natural immunity seen after voluntary exercise in rats. Role of central opioid receptors. AB - Chronic voluntary exercise in wheels for 5 weeks in spontaneously hypertensive rats (SHR) augments in vivo natural killer (NK) cell cytotoxicity. Endogenous beta-endorphin is increased in cerebrospinal fluid after voluntary exercise in rats and we have recently shown that beta-endorphin administered i.c.v. augments NK cell mediated cytotoxicity in vivo in a similar way as chronic voluntary exercise. We have now further investigated the involvement of central opioid systems in the exercise-induced augmentation in natural immunity. Exercise consisted of voluntary running in wheels for 5 weeks. In vivo cytotoxicity was measured as clearance of injected 51Cr-labeled YAC-1 lymphoma cells from the lungs. The clearance of YAC-1 cells in vivo was significantly increased in runners as compared to sedentary controls. Selective delta, kappa, or mu-opioid receptor antagonists were administered i.c.v. with osmotic minipumps during the last 6 days of the 5 weeks of running. The delta-receptor antagonist naltrindole (40-50 microg/day) significantly but not completely inhibited the enhanced NK cell cytotoxicity seen after 5 weeks of exercise. Neither the kappa-receptor antagonist nor-BNI or the mu-receptor antagonist beta-FNA influenced the augmentation in NK cell cytotoxicity. Nor-BNI per se significantly augments in vivo cytotoxicity, indicating some inhibiting effect on natural immunity that could be mediated through the kappa-opioid receptor. Our data suggest the involvement of central delta-opioid receptors in the enhancement of natural cytotoxicity seen after chronic voluntary exercise. PMID- 10737419 TI - Receptor binding and G-protein activation by new Met5-enkephalin-Arg6-Phe7 derived peptides. AB - Met5-enkephalin-Arg6-Phe7 (Tyr-Gly-Gly-Phe-Met-Arg-Phe, MERF) is a naturally occurring heptapeptide that binds to opioid and non-opioid recognition sites in the central nervous system. Four synthetic analogs with single or double amino acid substitutions were prepared by solid phase peptide synthesis to achieve proteolytically more stable structures: Tyr-D-Ala-Gly-Phe-Met-Arg-Phe (I), Tyr-D Ala-Gly-Phe-D-Nle-Arg-Phe (II), Tyr-D-Ala-Gly-Phe-L-Nle-Arg-Phe (III) and Tyr-Gly Gly-Phe-L-Nle-Arg-Phe (IV). In this study receptor binding characteristics and G protein activation of MERF and its derivatives were compared in crude membrane fractions of frog and rat brain. Synthetic MERF-derived peptides were potent competitors for [3H]MERF and [3H]naloxone binding sites with the exception of analog (II) which turned to be substantially less active. The presence of 100 mM NaCl or 100 microM 5'-guanylylimidodiphosphate, Gpp(NH)p, decreased the affinity of the peptides in [3H]naloxone binding assays, suggesting that these ligands might act as agonists at the opioid receptors. Some of the compounds were also used to stimulate guanosine-5'-O-(3-[gamma-[35S]thio)triphosphate ([35S]GTPgammaS) binding in rat and frog brain membranes at concentrations of 10( 9)-10(-5) M. The EC50 values of analog (II) were the highest in both tissues. Analog (I) was as effective as MERF in rat brain membranes, but showed lower maximal stimulation in frog brain preparation. Again, analog (II) seemed to be the least efficacious peptide that stimulated [35S]GTPgammaS binding only by 59%. Specificity of the peptides was further investigated by the inhibition of agonist stimulated [35S]GTPgammaS binding in the presence of selective antagonists for the opioid receptor types. The mu-selective antagonist cyprodime displayed the lowest potency in inhibiting the effects of the peptides, whereas norbinaltorphimine (kappa-selective antagonist) and naltrindole (delta-selective antagonist) were quite potent in both tissues. We concluded that MERF and its derivatives are able to activate G-proteins mainly via kappa- and delta-opioid receptors. PMID- 10737421 TI - Altered ischemic induction of immediate early gene and heat shock protein 70 mRNAs after preconditioning in rat hearts. AB - Immediate early genes and heat shock protein (HSP) 70s, which may play a role in adaptation and cellular protection, respectively, are induced by ischemia in hearts. We examined if the induction of immediate early gene (c-fos, c-myc, c jun, and junB) and HSP70 mRNAs by ischemia is affected by ischemic preconditioning. Transient ischemia (5 or 10 minute) was applied to Wistar rat (n=75) hearts, by tightening a snare placed around left coronary arterial branches 7 days before applying ischemia. Rats without earlier ischemia (control group, C) and rats with 5-minute ischemia 12 or 24 hours earlier (EI12 or 24 group) were given 10-minute ischemia and sacrificed at 0, 0.5, 1, 2, and 4 hour. RNA was extracted from the ischemic region and Northern blot analysis was performed. The induction of c-fos and c-myc mRNAs was significantly increased in EI12 but not in EI24 compared with that in C. The induction of c-jun and junB mRNAs showed no change in both EI12 and EI24 compared with that in C. The induction of HSP72 and 73 mRNAs was decreased in EI12 and decreased further in EI24. Thus, ischemic preconditioning altered the induction of immediate early gene and HSP70 mRNAs by ischemia. The effect of preconditioning differed among genes studied and changed with time after preconditioning. Ischemic preconditioning alters protective and adaptive responses to ischemia at the gene level. PMID- 10737420 TI - Influence of adrenal cortex on testicular activity in the toad during the breeding season. AB - The aim of the present study was to determine the role of adrenals in gonadal activity in the male toad during the breeding season. Exogenous administration of corticosterone or metapyrone for 6 days inhibited adrenal delta5-3beta(delta 5-3 beta) hydroxysteroid (delta5-3beta-HSD) and testicular 17beta (17 beta) hydroxysteroid dehydrogenase (17beta-HSD) activities, decreased the serum levels of testosterone and inhibited spermatogenesis. When toads were treated with corticosterone a significant rise of serum corticosterone was noted while metapyrone treatment appeared to decrease serum corticosterone levels. It is concluded that adrenocortical hormone plays an indirect role in testicular activity in toads during the breeding season. PMID- 10737422 TI - Chronic electroconvulsive shock decreases (+/-) 1-(4-iodo-2,5-dimethoxyphenyl)-2 aminopropane hydrochloride (DOI)-induced wet-dog shake behaviors of dexamethasone treated rats. AB - Electroconvulsive shock (ECS) therapy is considered to be an effective treatment for depression, but its mechanism of action is still unknown. We investigated the effect of chronic ECS in rats treated for 14 days with dexamethasone (Dex), a glucocorticoid receptor agonist. Chronic injection of sesame oil decreased body weight change and increased serotonin (5-HT)-2A receptor number and DOI (5-HT-2A, 2C receptor agonist)-induced wet-dog shake (WDS) behaviors. Dex treatment for 14 days decreased body weight of rats, but repeated ECS did not reverse this decrease. Dex also abolished plasma corticosterone levels, and ECS failed to restore these levels. These results indicate that chronic ECS does not antagonize the effect of Dex. The treatment with Dex increased 5-HT-2A receptor binding density of rat frontal cortex and the number of DOI-induced WDS behaviors. Chronic ECS reduced the enhanced WDS behaviors by Dex but had little effect on receptor density. These results suggest that chronic ECS might suppress 5-HT-2A receptor function at the postreceptor signaling level rather than at the receptor itself, without changing HPA axis function in Dex-treated rats. PMID- 10737423 TI - Stereoselective inhibition of dopaminergic activity by gamma vinyl-GABA following a nicotine or cocaine challenge: a PET/microdialysis study. AB - Elevation of endogenous GABA by the racemic mixture of gamma vinyl-GABA (GVG, Vigabatrin) decreases extracellular nucleus accumbens (NAc) dopamine (DA) levels and diminishes the response to many drugs of abuse known to elevate DA in the mesocorticolimbic system. We investigated the effects of the individual enantiomers (S(+)-GVG, R(-)-GVG) on cocaine-induced NAc DA in rodents as well as the effects of nicotine-induced increases in primates. In a series of microdialysis experiments in freely moving animals, S(+)-GVG (150 mg/kg), R(-) GVG (150 mg/kg) or racemic (R, S) GVG (300 mg/kg) was administered 2.5 hours prior to cocaine (20 mg/kg) administration. When compared with cocaine alone, the R(-) enantiomer did not significantly inhibit cocaine induced NAc DA release. S(+)-GVG, at half the dose of the racemic mixture (150 mg/kg), inhibited cocaine induced DA elevation by 40%, while the racemic mixture (300 mg/kg) inhibited cocaine-induced DA release by 31%. In addition, our PET studies in primates demonstrated that S(+)-GVG completely inhibits nicotine-induced increases in the corpus striatum, again at half the dose of the racemic mixture. The R(-) enantiomer was ineffective. Although the S(+) enantiomer has been well established as the active compound in the treatment of epilepsy, the efficacy of this enantiomer with regard to mesolimbic DA inhibition generates a complex series of clinical and neurochemical issues. Further investigations will determine the locus of action and physiologic properties of each enantiomer. PMID- 10737424 TI - New treatment protocol including lympholytic and antiretroviral drugs to inhibit murine AIDS. AB - Highly active antiretroviral therapy (HAART), although very efficient in reducing viral load to undetectable levels within 2 weeks, does not eradicate HIV-1 infection and after the suspension of therapy, HIV RNA rebounds to pretherapy levels. This limited efficacy is mainly due to the existence of viral reservoirs such as CD4+ T cells, macrophages, and dendritic cells in which the virus can remain latent. Elimination of these latent reservoirs would be a possible solution to this problem and various efforts are now being directed to this end. With this goal in mind, we investigated a lympholytic drug with known activity against lymphoproliferative malignancies, 2-fluoro-ara-AMP (fludarabine). The murine model of AIDS was used to evaluate the efficacy of alternating administration of fludarabine and azidothymidine (AZT). The aim of this experiment was to eliminate infected cells with fludarabine and protect noninfected cells with AZT. LP-BM5-infected mice were treated with two different therapeutic protocols: one group was treated with two alternating 3-week cycles of fludarabine and AZT (treatment A), whereas the other was treated with three alternating 2-week cycles of fludarabine and AZT (treatment B); both treatments lasted 12 weeks and the animals in the two groups received the same amount of drug. At different times of infection, disease-related findings (i.e., splenomegaly, lymphadenopathy, hypergammaglobulinemia, T-cell and B-cell spleen cell proliferative index, and phenotypes of peripheral blood lymphocytes) were analyzed and the content of proviral DNA in the lymph nodes was quantified. The results obtained show that treatment B was more effective in inhibiting disease progression than treatment A. In fact, all parameters investigated were almost within control values. These results were also confirmed by the quantification of proviral DNA content in the lymph nodes, which after 12 weeks of treatment A declined by approximately 50%, whereas treatment B decreased proviral DNA content by approximately 85% with respect to infected/untreated mice. The data obtained suggest that a therapeutic protocol including three cycles rather than two of a lympholytic drug and antiretroviral drugs is more advantageous. The efficacy of the treatment could likely increase if other drugs were used in addition to AZT and more cycles of fludarabine were added. This approach appears to be of potential interest in an HIV-1 eradication protocol. PMID- 10737425 TI - Detection of infectious HIV in circulating monocytes from patients on prolonged highly active antiretroviral therapy. AB - The existence of a reservoir of resting CD4+ T cells harboring latent replication competent HIV has been demonstrated in patients on prolonged highly active antiretroviral therapy (HAART). Latently infected tissue macrophages may constitute a second HIV reservoir. The pool of these cells may be maintained by incoming infected monocytes from blood and/or by in situ viral replication. In this study, the presence of infectious HIV was investigated in highly purified monocytes from 5 patients receiving HAART with undetectable plasma viral load for up to 16 months. HIV was detected in freshly isolated monocytes and recovered following Staphylococcus aureus Cowan strain 1 (SAC) or lipopolysaccharide (LPS) activation. No new drug resistance-associated mutation was found in monocyte associated HIV. These results demonstrate the long-term persistence of infectious virus in cells of the monocyte-macrophage lineage in patients receiving HAART. These cells are capable of releasing infectious virus under appropriate stimulations. PMID- 10737426 TI - HIV-1 subtype C in commerical sex workers in Addis Ababa, Ethiopia. AB - In this study, we have investigated the diversity of the current HIV-1 strains circulating in Addis Ababa, Ethiopia; in addition, we have evaluated the applicability of peptide enzyme-linked immunosorbent assay (ELISA) and heteroduplex mobility assay (HMA) for HIV-1 subtyping. Previous studies have indicated that HIV-1 subtype C is the major subtype present in HIV-positive samples collected from various risk groups between 1988 and 1995 in Addis Ababa. To assess the possible influx of new HIV-1 subtypes, 150 commercial sex workers (CSW) reporting in 1997 to two Health Centers in Addis Ababa were enrolled in an unlinked anonymous cross-sectional study. Subtyping was performed according to the World Health Organization algorithm of peptide ELISA, followed by HMA and DNA sequencing. As a result, the HIV-1 prevalence among these CSWs was found to be 45% (67 of 150). Of the 67 samples, 66 contained HIV-1 of subtype C and only one was of subtype D. This confirms the persistent overall presence of HIV-1 subtype C in Addis Ababa and a low influx of other subtypes into this location. PMID- 10737427 TI - The SPICE study: 48-week activity of combinations of saquinavir soft gelatin and nelfinavir with and without nucleoside analogues. Study of Protease Inhibitor Combinations in Europe. AB - OBJECTIVES: To compare the efficacy and safety of saquinavir soft gelatin capsules (SQV-SGC) and nelfinavir (NFV), with or without two concomitant nucleoside reverse transcriptase inhibitors (NRTIs), in an exploratory objective to identify populations most likely to benefit from quadruple therapy. DESIGN: Phase II/III, open-label, randomized, parallel-arm, multicenter trial. PARTICIPANTS: Enrollment included 157 protease inhibitor-naive adults (> or = 13 years) with HIV-1 RNA > or = 10,000 copies/ml; 132 participants completed 48 weeks of therapy. INTERVENTIONS: SQV-SGC 1200 mg, NFV 750 mg, SQV-SGC 800 mg plus NFV 750 mg, all with two NRTIs, and SQV-SGC 800 mg plus NFV 750 mg alone, all three times daily for 48 weeks. MAIN OUTCOME MEASURES: Proportion of participants with HIV-1 RNA <50 copies/ ml (16 and 48 weeks); time to virologic relapse (48 weeks). RESULTS: Proportions of patients with HIV RNA <50 copies/ml were not statistically significantly different between arms at 16 or 48 weeks, although trends favored the quadruple-therapy arm. In patients experiencing virologic relapse, time to relapse was statistically significantly longer in the quadruple therapy arm than in the other three arms (p = .007). Quadruple therapy provided benefit in NRTI-experienced patients and those with viral loads above the median value at baseline. Adverse events were mainly mild gastrointestinal disorders in all treatment arms. CONCLUSIONS: Quadruple therapy, including SQV-SGC and NFV, gave a more durable response than triple therapy with either single protease inhibitor. Quadruple therapy might particularly benefit NRTI-experienced patients and those with high baseline viral loads. PMID- 10737428 TI - Performance of five different assays for the quantification of viral load in persons infected with various subtypes of HIV-1. Swiss HIV Cohort Study. AB - Five methods for the assessment of plasma viral load (VL) were evaluated in 103 seropositive patients infected with various subtypes of HIV-1. The methods included three RNA-based assays (Amplicor Monitor 1.5, Quantiplex version 2.0, NucliSens), one ultrasensitive reverse transcriptase (PERT) assay and one "boosted" p24 antigen (Ag) enzyme immunoassay (EIA). Subtyping was based on sequencing in env. The sensitivities were, in decreasing order, Amplicor > PERT > p24 Ag > NucliSens > Quantiplex. The low sensitivity of NucliSens was related to the missing of several non-B (A, E, F, G) or recombinant strains, whereas that of Quantiplex did not depend on subtype. In the 1 group O sample and 4 group M samples, only PERT assay or p24Ag EIA produced a positive result. In the quantitative range, correlation was best between Amplicor and Quantiplex (r = 0.8848), fair between Amplicor and NucliSens (r = 0.7064) or PERT assay (r = 0.7266), lowest between Amplicor and p24Ag EIA (r = 0.3989). Amplicor underestimated VL in 1 subtype E sample. Thus, Amplicor performed best in terms of sensitivity (compared with all other assays) and accuracy (compared with NucliSens, PERT assay, and p24Ag) for non-B subtypes in group M samples. PERT assay appears useful for VL assessment in infections by group O or other highly divergent viruses. PMID- 10737429 TI - HIV-associated bacteremia: how it has changed in the highly active antiretroviral therapy (HAART) era. AB - To evaluate the changing characteristics of HIV-associated bacteremia in the highly active antiretroviral therapy (HAART) era, we conducted a prospective case control study, comparing two periods of time, before (period A) and after (period B) the introduction of HAART. In total, 174 patients with bacteremia and 348 controls were studied. By comparing incidence in periods A and B, a statistically significant reduction of bacteremia, from 11.8 to 6.3/100 person-years (PY), was observed (p = .0001). Incidence of hospital-acquired bacteremia decreased from 5.8 episodes/100 PY in period A to 2.4/100 PY in period B (p = .0005). A similar trend was also observed for community-acquired episodes of bacteremia, with a value close to statistical significance. Logistic regression analysis indicated that intravenous drug abuse, central venous catheter (CVC) use, high value on APACHE III score, and neutropenia were independent risk factors for bacteremia in both the study periods. Interestingly, comparing the prevalence of bacteremia risk factors in the two study periods, we observed a significant reduction in the use of CVC (p = .04, period A versus period B) and in neutropenia (p = .04). The crude mortality rate was 31% in period A and 23% in period B (p = not significant [ns]). Logistic regression analysis indicated that an high value of Acute Physiology and Chronic Health Evaluation III (APACHE III) score (p < .001) predicted an increased risk of death. Analysis of prognostic factors of bacteremia did not significantly differ in both the study periods. We conclude that HAART has determined a significant reduction of the incidence and a modification of the characteristics of bacteremia. This reduced incidence may produce a substantial impact on future morbidity and health care costs of patients with HIV. PMID- 10737430 TI - Molecular and virologic characteristics of lymphoid malignancies in children with AIDS. AB - PURPOSE: To characterize AIDS-associated lymphoid malignancies in children. PATIENTS AND METHODS: We studied lymphomas and B-cell leukemias from 25 children with AIDS for immunoglobulin heavy chain gene clonality, c-myc oncogene abnormalities, and presence of HIV and Epstein-Barr virus. RESULTS: Monoclonal immunoglobulin gene rearrangements were identified in 22 of 23 cases tested, the single exception being one of mucosa-associated lymphoid tissue. Immunoglobulin gene/c-myc translocations were found in 3 of 4 cases of B (surface immunoglobulin positive)-acute lymphoblastic leukemia, 8 of 11 small noncleaved cell lymphomas, and 1 of 5 large cell lymphomas. Mutations of c-myc were found in 2 of 13 small noncleaved cell lymphomas, 1 of 2 Epstein-Barr virus-positive mucosa-associated lymphoid tissue neoplasms, and 1 of 4 Epstein-Barr virus-negative B-acute lymphoblastic leukemia. Six small noncleaved cell lymphomas, both mucosa associated lymphoid tissue neoplasms and one of large cell lymphoma had high levels of Epstein-Barr virus in tumor tissue. Hodgkin's disease tissue and B acute lymphoblastic leukemia tumors were negative for EBV. Proviral HIV-1 was not detected in any tumor. CONCLUSIONS: AIDS-associated lymphoid malignancies in children appear to have a different distribution of histologic subtypes than adult HIV-infected individuals, fewer large cell lymphomas occur in children. The small noncleaved cell lymphomas exhibit a lower frequency as well as different locations of c-myc mutations than AIDS-associated small noncleaved cell lymphomas in adults. PMID- 10737431 TI - Causal pathways for CCR5 genotype and HIV progression. AB - A homozygous 32-bp deletion in the gene encoding CCR5, a major coreceptor for HIV 1, leads to resistance to infection with HIV-1, and heterozygosity for the deletion is associated with delayed disease progression in persons infected with HIV-1. We investigated the effect of CCR5 heterozygosity on disease progression as measured by both CD4+ T-cell count decline and the occurrence of clinical AIDS symptoms. Using a unified statistical model for CD4 count progression and AIDS development, we examined whether the effect of CCR5 heterozygosity on clinical AIDS is direct or indirect through its effect on CD4 counts. Based on data from the Multicenter AIDS Cohort Study, we noted a protective effect of CCR5 heterozygosity on both CD4 cell count progression and on AIDS occurrence. Furthermore, we found that this protective effect on the occurrence of AIDS was completely mediated through an effect on the CD4 marker. Additional adjustment for the effect of an initial viral load measurement indicate that CCR5 heterozygosity did not have predictive value for either CD4 progression or the development of AIDS beyond its association with early viral load. PMID- 10737432 TI - Course of viral load throughout HIV-1 infection. AB - HIV-1 RNA levels are routinely monitored as part of patient management. However, little is known about the course of HIV-1 RNA levels over the entire period of infection. The aim of this study was to investigate the course of HIV-1 RNA levels in a cohort of men with hemophilia who were observed for up to 17 years after HIV-1 seroconversion, and to assess the risk of HIV disease progression at any HIV-1 RNA level. Viral loads were measured on annual stored serum samples in 107 men with hemophilia A using the Roche Amplicor Monitor assay with non-B primers. On average, HIV-1 RNA levels increased significantly by 0.11 log10 per year over the course of HIV infection. This rate of increase was significantly faster in those who developed AIDS or died over the subsequent 12 to 17 year period, and in those who were older at HIV- 1 seroconversion. The risk of developing AIDS and death remained low when the HIV-1 RNA level was below 4 log10 copies/ml, but increased rapidly thereafter, supporting current guidelines for the initiation of antiretroviral therapy after the viral load has exceeded this level. PMID- 10737433 TI - Trends in prescriptions for highly active antiretroviral therapy in four New York City HIV clinics. AB - OBJECTIVE: To describe trends in prescriptions for antiretroviral therapies and factors associated with prescriptions for highly active antiretroviral therapy (HAART). METHODS: Medical records of patients at four HIV clinics in New York City were reviewed every 6 months. For the four 6-month periods 1997 to 1998, we identified patients with a CD4+ nadir <500 cells/microl; sample sizes were 434, 432, 503, and 643, respectively. Trends in HAART prescriptions were tested by logistic regression using robust variance estimates because some patients contributed more than one time period. Associations between HAART prescriptions and patient characteristics were tested by chi2 and multiple logistic regression analysis. RESULTS: Patients were predominantly black or Hispanic (89%-90%) and male (66%-68%), and injection drug use was the most prevalent HIV risk (38%-49%). From 1997 to 1998, HAART prescriptions increased from 54% to 89% of antiretroviral prescriptions, and the proportion that included an nonnucleoside reverse transcriptase inhibitors (NNRTI) increased from 3% to 10%. HAART prescriptions were inversely associated with CD4+ nadir group during all time periods, and in the second half of 1998, patients with CD4+ nadir between 50 and 199 cells/microl were as likely to be prescribed HAART as the most immunosuppressed patients (CD4+ nadir <50 cells/microl; 91% versus 92%). HAART prescriptions were associated with clinic, HIV risk, and other patient characteristics in some time periods but not consistently. CONCLUSIONS: In these four HIV clinics, prescriptions for HAART increased significantly from 1997 to 1998, leveling off at 89% in the second half of 1998. Increasingly, HAART was prescribed for healthier patients and included an NNRTI. PMID- 10737435 TI - HIV incidence and HIV-associated mortality in a cohort of factory workers and their spouses in Tanzania, 1991 through 1996. AB - OBJECTIVE: To describe HIV incidence and HIV-associated mortality in a cohort of factory workers and their spouses with access to adequate sexually transmitted diseases (STD) treatment services and moderate exposure to sexual health interventions. METHODS: Follow-up visits at 4-month intervals and home follow-up of those who failed to keep appointments were used to estimate HIV incidence and mortality in a cohort of factory workers and their spouses in Mwanza, Tanzania, during 1991 to 1996. RESULTS: HIV prevalence at intake was 10.5% and 15.3% among 1,594 men and 880 women, respectively. HIV incidence was just over 1/100 person years (PY) in 1,427 men and 1.9/100 PY in 745 women. The main risk factors were related to sexual behavior and clinical evidence of an STD, although only one seventh of those with HIV seroconversions had had an STD during the period following the second-from-last follow-up visit. HIV incidence among discordant couples was 5.0 and 8.3/100 PY for 41 men and 37 women, respectively. Mortality rates were 9.0 and 7.8/1,000 PY for men and women, respectively, and 65% of male and 60% of female deaths were attributable to HIV infection. CONCLUSIONS: In this factory population with good access to and use of STD treatment and with a moderate level of exposure to HIV prevention education, HIV incidence among men and women is still over 1/100 PY, which suggests a relatively high level of program effort is needed to lower incidence. More than half of all adult deaths were attributable to HIV, but greater increases in HIV-associated mortality are likely. PMID- 10737436 TI - HIV and HTLV prevalences among women seen for sexually transmitted diseases or pregnancy follow-up in Maputo, Mozambique. PMID- 10737434 TI - HIV-1 diversity in Brazil: genetic, biologic, and immunologic characterization of HIV-1 strains in three potential HIV vaccine evaluation sites. Brazilian Network for HIV Isolation and Characterization. AB - The Brazilian Network for HIV Isolation and Characterization was established for the surveillance of HIV variability in Brazil. Here, we report characterization of HIV strains and virus-specific immune responses from 35 clinical samples collected from three potential HIV vaccine sites. Three genetic subtypes of HIV-1 were identified by heteroduplex mobility assay (HMA) B (in 82.9% of the samples), F (14.3%), and C (2.9%). Phylogenetic analysis based on the C2V3/env DNA sequence from all 25 specimens examined was 100% concordant with HMA results. Four variants of subtype B with different tetrapeptides at the tip of the V3 loop were found: the GPGR motif (North American), GWGR motif (Brazilian B"), and two minor variants, GFGR and GPGS, as previously detected. No significant association was found between HIV-1 subtypes and the mode of transmission or biologic properties of HIV-1 isolates (derived from 88.6% of the specimens). Only 5 of 16 isolates studied were neutralized by the autologous sera. Consistent with previous results, no relation between viral subtype and peptide enzyme-linked immunosorbent assay (ELISA) seroreactivity or neutralization was evident. This study also demonstrated the effectiveness of the collaborative approach followed by Brazilian scientists when addressing a complex subject such as HIV variability. PMID- 10737437 TI - Comparison of simultaneous active and passive AIDS case reporting in San Francisco. PMID- 10737438 TI - Evidence of HIV-1 genetic diversity among pregnant women with AIDS or infected with HIV-1 in Central Brazil. PMID- 10737439 TI - Inborn errors of mitochondrial fatty acid oxidation. AB - Inborn errors of the mitochondrial beta-oxidation of long-chain fatty acids represent an evolving field of inherited metabolic disease. Fatty acid oxidation defects demonstrate an abnormal response to the process of fasting adaptation and affect those tissues that utilize fatty acids as an energy source. These tissues include cardiac and skeletal muscle and liver. Muscle directly uses fatty acids as an energy source whilst hepatic metabolism of fatty acids is mostly directed toward the synthesis of ketone bodies for energy utilization by tissues such as brain. The clinical phenotypes of fatty acid oxidation disorders include disease of one or more of these fatty acid-metabolizing tissues. In this review, we provide an overview of the pathway, discuss the disorders that are well established, and describe recent advances in the field. Currently available diagnostic procedures are critically evaluated. PMID- 10737440 TI - Hepatitis delta virus: the molecular basis of laboratory diagnosis. AB - Infection with hepatitis delta virus (HDV), a satellite virus of hepatitis B virus (HBV), is associated with severe and sometimes fulminant hepatitis. The traditional methods for the diagnosis of HDV infection, such as detection of serum anti-HD antibodies, are sufficient for the clinical diagnosis of delta infection. However, such techniques lack the sensitivity and specificity required to more accurately characterize the nature of HDV infection and to assess the efficacy of therapies. Recent improvements in molecular techniques, such as HDV RNA hybridization and RT-PCR, have provided increased diagnostic precision and a more thorough understanding of the natural course of HDV infection. These advances have enhanced the clinician's ability to accurately evaluate the stage of HDV infection, response to therapy, and occurrence of reinfection after orthotopic liver transplant. This review focuses on the recent advances in the understanding of the molecular biology of HDV and in the laboratory diagnosis of HDV infection. PMID- 10737441 TI - Monitoring hospital-acquired infections to promote patient safety--United States, 1990-1999. AB - Hospital-acquired infections are adverse patient events that affect approximately 2 million persons annually. National Nosocomial Infections Surveillance (NNIS) is a voluntary, hospital-based reporting system established to monitor hospital acquired infections and to guide the prevention efforts of infection control practitioners (ICPs). The NNIS approach may be a model for future programs aimed at preventing other adverse patient events. This report describes the decrease in infection rates reported in NNIS hospitals during 1990-1999, presents the results of a survey of ICP responsibilities, and discusses the importance of NNIS for monitoring adverse patient events. PMID- 10737442 TI - Corporate action to reduce air pollution--Atlanta, Georgia, 1998-1999. AB - Ground-level ozone, a colorless gas, is a major constituent of smog. Since the early 1980s, controlled studies have demonstrated that exposure to elevated levels of ozone reduces inspiratory capacity in humans. In addition, ecologic analyses have indicated that daily emergency department visits for asthma exacerbations are elevated following days of high ozone pollution. The Partnership for a Smog-Free Georgia (PSG) is a state-sponsored program to reduce the number of days that ground-level ozone exceeds the national ambient air quality standard (NAAQS) in metropolitan Atlanta by providing federal and state subsidized commuting alternatives for local business employees. This report summarizes commuter data from three PSG partners to estimate reductions in emissions and monthly vehicle miles traveled that were associated with enrollment in PSG. PMID- 10737443 TI - Developing and expanding contributions of the global laboratory network for poliomyelitis eradication, 1997-1999. AB - In 1988, the World Health Assembly resolved to eradicate poliomyelitis globally by 2000. Substantial progress toward achieving this goal has been reported from all countries where polio is endemic, and three regions of the World Health Organization (WHO) (American Region, European Region, and Western Pacific Region) appear to be free of indigenous wild poliovirus transmission. One key strategy for polio eradication is establishing sensitive surveillance systems for polio (through notification of acute flaccid paralysis [AFP] cases) and poliovirus. To ensure that specimens from AFP cases undergo appropriate processing for viral isolation, WHO has established a global laboratory network. This report describes the proficiency of the network and provides updates on structure, accreditation, performance, expanding activities, and future plans. PMID- 10737444 TI - Does health systems thinking guide health services actions? PMID- 10737445 TI - Racial variation in treatment for transient ischemic attacks: impact of participation by neurologists. AB - OBJECTIVE: This study evaluates the role of neurologists in explaining African American-white differences in the use of diagnostic and therapeutic services for cerebrovascular disease. DATA SOURCES/STUDY SETTING: Medicare inpatient hospital records were used to identify a random 20 percent sample of patients age 65 and over hospitalized with a principal diagnosis of TIA between January 1, 1991 and November 30, 1991 (n = 17,437). STUDY DESIGN: Medicare administrative data were used to identify five outcome measures: noninvasive cerebrovascular tests, cerebral angiography, carotid endarterectomy, anticoagulant therapy (as proxied by outpatient prothrombin time tests), and the specialty of the attending physician (neurologist versus other specialist). DATA COLLECTION/EXTRACTION METHODS: All Medicare claims were extracted for a 30-day period beginning with the date of admission. PRINCIPAL FINDINGS: Even after adjusting for patient demographics, comorbidity, ability to pay, and provider characteristics, African American patients were significantly less likely to receive noninvasive cerebrovascular testing, cerebral angiography, or carotid endarterectomy, compared with white patients, and to have a neurologist as their attending physician. At the same time, patients treated by neurologists were more likely to undergo diagnostic testing and less likely to undergo carotid endarterectomy. CONCLUSIONS: The findings suggest that African American patients with TIA may have less access to services for cerebrovascular disease and that at least some of this may be attributed to less access to neurologists. More research is needed on how patients at risk for stroke are referred to specialists. PMID- 10737446 TI - Are nonspecific practice guidelines potentially harmful? A randomized comparison of the effect of nonspecific versus specific guidelines on physician decision making. AB - OBJECTIVE: To test the ability of two different clinical practice guideline formats to influence physician ordering of electrodiagnostic tests in low back pain. DATA SOURCES/STUDY DESIGN: Randomized controlled trial of the effect of practice guidelines on self-reported physician test ordering behavior in response to a series of 12 clinical vignettes. Data came from a national random sample of 900 U.S. neurologists, physical medicine physicians, and general internists. INTERVENTION: Two different versions of a practice guideline for the use of electrodiagnostic tests (EDT) were developed by the U.S. Agency for Health Care Policy and Research Low Back Problems Panel. The two guidelines were similar in content but varied in the specificity of their recommendations. DATA COLLECTION: The proportion of clinical vignettes for which EDTs were ordered for appropriate and inappropriate clinical indications in each of three physician groups were randomly assigned to receive vignettes alone, vignettes plus the nonspecific version of the guideline, or vignettes plus the specific version of the guideline. PRINCIPAL FINDINGS: The response rate to the survey was 71 percent. The proportion of appropriate vignettes for which EDTs were ordered averaged 77 percent for the no guideline group, 71 percent for the nonspecific guideline group, and 79 percent for the specific guideline group (p = .002). The corresponding values for the number of EDTs ordered for inappropriate vignettes were 32 percent, 32 percent, and 26 percent, respectively (p = .08). Pairwise comparisons showed that physicians receiving the nonspecific guidelines ordered fewer EDTs for appropriate clinical vignettes than did physicians receiving no guidelines (p = .02). Furthermore, compared to physicians receiving nonspecific guidelines, physicians receiving specific guidelines ordered significantly more EDTs for appropriate vignettes (p = .0007) and significantly fewer EDTs for inappropriate vignettes (p = .04). CONCLUSIONS: The clarity and clinical applicability of a guideline may be important attributes that contribute to the effects of practice guidelines. PMID- 10737447 TI - Relationships between in-hospital and 30-day standardized hospital mortality: implications for profiling hospitals. AB - OBJECTIVE: To examine the relationship of in-hospital and 30-day mortality rates and the association between in-hospital mortality and hospital discharge practices. DATA SOURCES/STUDY SETTING: A secondary analysis of data for 13,834 patients with congestive heart failure who were admitted to 30 hospitals in northeast Ohio in 1992-1994. DESIGN: A retrospective cohort study was conducted. DATA COLLECTION: Demographic and clinical data were collected from patients' medical records and were used to develop multivariable models that estimated the risk of in-hospital and 30-day (post-admission) mortality. Standardized mortality ratios (SMRs) for in-hospital and 30-day mortality were determined by dividing observed death rates by predicted death rates. PRINCIPAL FINDINGS: In-hospital SMRs ranged from 0.54 to 1.42, and six hospitals were classified as statistical outliers (p <.05); 30-day SMRs ranged from 0.63 to 1.73, and seven hospitals were outliers. Although the correlation between in-hospital SMRs and 30-day SMRs was substantial (R = 0.78, p < .001), outlier status changed for seven of the 30 hospitals. Nonetheless, changes in outlier status reflected relatively small differences between in-hospital and 30-day SMRs. Rates of discharge to nursing homes or other inpatient facilities varied from 5.4 percent to 34.2 percent across hospitals. However, relationships between discharge rates to such facilities and in-hospital SMRs (R = 0.08; p = .65) and early post-discharge mortality rates (R = 0.23; p = .21) were not significant. CONCLUSIONS: SMRs based on in-hospital and 30-day mortality were relatively similar, although classification of hospitals as statistical outliers often differed. However, there was no evidence that in-hospital SMRs were biased by differences in post discharge mortality or discharge practices. PMID- 10737449 TI - On "risk-adjusting acute myocardial infarction mortality: are APR-DRGs the right tool?". PMID- 10737448 TI - Risk-adjusting acute myocardial infarction mortality: are APR-DRGs the right tool? AB - OBJECTIVE: To determine if a widely used proprietary risk-adjustment system, APR DRGs, misadjusts for severity of illness and misclassifies provider performance. DATA SOURCES: (1) Discharge abstracts for 116,174 noninstitutionalized adults with acute myocardial infarction (AMI) admitted to nonfederal California hospitals in 1991-1993; (2) inpatient medical records for a stratified probability sample of 974 patients with AMIs admitted to 30 California hospitals between July 31, 1990 and May 31, 1991. STUDY DESIGN: Using the 1991-1993 data set, we evaluated the predictive performance of APR-DRGs Version 12. Using the 1990/1991 validation sample, we assessed the effect of assigning APR-DRGs based on different sources of ICD-9-CM data. DATA COLLECTION/EXTRACTION METHODS: Trained, blinded coders reabstracted all ICD-9-CM diagnoses and procedures, and established the timing of each diagnosis. APR-DRG Risk of Mortality and Severity of Illness classes were assigned based on (1) all hospital-reported diagnoses, (2) all reabstracted diagnoses, and (3) reabstracted diagnoses present at admission. The outcome variables were 30-day mortality in the 1991-1993 data set and 30-day inpatient mortality in the 1990/1991 validation sample. PRINCIPAL FINDINGS: The APR-DRG Risk of Mortality class was a strong predictor of death (c = .831-.847), but was further enhanced by adding age and sex. Reabstracting diagnoses improved the apparent performance of APR-DRGs (c = .93 versus c = .87), while using only the diagnoses present at admission decreased apparent performance (c = .74). Reabstracting diagnoses had less effect on hospitals' expected mortality rates (r = .83-.85) than using diagnoses present at admission instead of all reabstracted diagnoses (r = .72-.77). There was fair agreement in classifying hospital performance based on these three sets of diagnostic data (K = 0.35-0.38). CONCLUSIONS: The APR-DRG Risk of Mortality system is a powerful risk-adjustment tool, largely because it includes all relevant diagnoses, regardless of timing. Although some late diagnoses may not be preventable, APR DRGs appear suitable only if one assumes that none is preventable. PMID- 10737450 TI - Multiple approaches to assessing the effects of delays for hip fracture patients in the United States and Canada. AB - OBJECTIVE: To examine the determinants of postsurgery length of stay (LOS) and inpatient mortality in the United States (California and Massachusetts) and Canada (Manitoba and Quebec). DATA SOURCES/STUDY SETTING: Patient discharge abstracts from the Agency for Health Care Policy and Research Nationwide Inpatient Sample and from provincial health ministries. STUDY DESIGN: Descriptive statistics by state or province, pooled competing risks hazards models (which control for censoring of LOS and inpatient mortality data), and instrumental variables (which control for confounding in observational data) were used to analyze the effect of wait time for hip fracture surgery on postsurgery outcomes. DATA EXTRACTIONS: Data were extracted for patients admitted to an acute care hospital with a primary diagnosis of hip fracture who received hip fracture surgery, were admitted from home or the emergency room, were age 45 or older, stayed in the hospital 365 days or less, and were not trauma patients. PRINCIPAL FINDINGS: The descriptive data indicate that wait times for surgery are longer in the two Canadian provinces than in the two U.S. states. Canadians also have longer postsurgery LOS and higher inpatient mortality. Yet the competing risks hazards model indicates that the effect of wait time on postsurgery LOS is small in magnitude. Instrumental variables analysis reveals that wait time for surgery is not a significant predictor of postsurgery length of stay. The hazards model reveals significant differences in mortality across regions. However, both the regressions and the instrumental variables indicate that these differences are not attributable to wait time for surgery. CONCLUSIONS: Statistical models that account for censoring and confounding yield conclusions that differ from those implied by descriptive statistics in administrative data. Longer wait time for hip fracture surgery does not explain the difference in postsurgery outcomes across countries. PMID- 10737451 TI - A controlled time-series trial of clinical reminders: using computerized firm systems to make quality improvement research a routine part of mainstream practice. AB - OBJECTIVE: To explore the feasibility of conducting unobtrusive interventional research in community practice settings by integrating firm-system techniques with time-series analysis of relational-repository data. STUDY SETTING: A satellite teaching clinic divided into two similar, but geographically separated, primary care group practices called firms. One firm was selected by chance to receive the study intervention. Forty-two providers and 2,655 patients participated. STUDY DESIGN: A nonrandomized controlled trial of computer generated preventive reminders. Net effects were determined by quantitatively combining population-level data from parallel experimental and control interrupted time series extending over two-month baseline and intervention periods. DATA COLLECTION: Mean rates at which mammography, colorectal cancer screening, and cholesterol testing were performed on patients due to receive each maneuver at clinic visits were the trial's outcome measures. PRINCIPAL FINDINGS: Mammography performance increased on the experimental firm by 154 percent (0.24 versus 0.61, p = .03). No effect on fecal occult blood testing was observed. Cholesterol ordering decreased on both the experimental (0.18 versus 0.1 1, p = .02) and control firms (0.13 versus 0.07, p = .03) coincident with national guidelines retreating from recommending screening for young adults. A traditional uncontrolled interrupted time-series design would have incorrectly attributed the experimental-firm decrease to the introduction of reminders. The combined analysis properly indicated that no net prompting effect had occurred, as the difference between firms in cholesterol testing remained stochastically stable over time (0.05 versus 0.04, p = .75). A logistic-regression analysis applied to individual-level data produced equivalent findings. The trial incurred no supplementary data collection costs. CONCLUSIONS: The apparent validity and practicability of our reminder implementation study should encourage others to develop computerized firm systems capable of conducting controlled time-series trials. PMID- 10737452 TI - Variation in the use of alternative levels of hospital care for newborns in a managed care organization. AB - OBJECTIVE(S): To assess the extent to which variation in the use of neonatal intensive care resources in a managed care organization is a consequence of variation in neonatal health risks and/or variation in the organization and delivery of medical care to newborns. STUDY DESIGN: Data were collected on a cohort of all births from four sites in Kaiser Permanente by retrospective medical chart abstraction of the birth admission. Likelihood of admission into a neonatal intensive care unit (NICU) is estimated by logistic regression. Durations of NICU stays and of hospital stay following birth are estimated by Cox proportional hazards regression. RESULTS: The likelihood of admission into NICU and the duration of both NICU care and hospital stay are proportional to the degree of illness and complexity of diagnosis. Adjusting for variation in health risks across sites, however, does not fully account for observed variation in NICU admission rates or for length of hospital stay. One site has a distinct pattern of high rates of NICU admissions; another site has a distinct pattern of low rates of NICU admission but long durations of hospital stay for full-term newborns following NICU admission as well as for all newborns managed in normal care nurseries. CONCLUSIONS: Substantial variations exist among sites in the risk adjusted likelihood of NICU admission and in durations of NICU stay and hospital stay. Hospital and NICU affiliation (Kaiser Permanente versus contract) or affiliation of the neonatologists (Kaiser Permanente versus contract) could not explain the variation in use of alternative levels of hospital care. The best explanation for these variations in neonatal resource use appears to be the extent to which neonatology and pediatric practices differ in their policies with respect to the management of newborns of minimal to moderate illness. PMID- 10737453 TI - Molecular epidemiology of viral infections. How sequence information helps us understand the evolution and dissemination of viruses. AB - Viruses evolve much faster than cellular organisms. Together with recent advances in nucleic acid sequencing and biocomputing, this allows us to distinguish between related strains of viruses, and to deduce the relationships between viruses from different outbreaks or individual patients. Databases of nucleotide sequences contain a large number of viral sequences with which novel sequences from local outbreaks can be compared. In this way the dissemination of viruses can be followed both locally and globally. We here review the biological and technological background to the use of virus nucleic acid sequences in epidemiological studies, and provide examples of how this information can be used to monitor human viruses. Molecular studies are particularly valuable for understanding the dissemination and evolution of viruses. The knowledge obtained is useful in epidemiological reconstructions, in real-time surveillance, and may even enable us to make predictions about the future developments of viral diseases. PMID- 10737454 TI - MAP2 and neurogranin as markers for dendritic lesions in CNS injury. An immunohistochemical study in the rat. AB - We compared two staining methods for the demonstration of dendrites under normal and pathological conditions of the rat central nervous system. MAP2- and neurogranin immunohistochemistry was applied to samples from normal tissue, spinal cord subjected to graded compression trauma, cerebral cortex following contusion trauma, and brains with focal ischemic lesions induced by occlusion of the middle cerebral artery (MCAO). Normal rats showed MAP2 immunoreactivity in nerve cell bodies and dendrites of brain and spinal cord. However, neurogranin staining was present only in nerve cell bodies and dendrites of the normal brain, and not in the spinal cord. Reduction of MAP2 immunoreactivity was seen in lesions of spinal cords subjected to compression trauma. Neurogranin staining was of no value in this experimental condition since it was not present under normal conditions. The brain contusions showed loss of both MAP2- and neurogranin immunoreactivity at the site of the lesion. MCAO resulted in an extensive loss of MAP2- and neurogranin staining in the ipsilateral hemisphere. In conclusion, our study shows that MAP2 immunostaining is a sensitive method for identifying dendritic lesions of various CNS injuries in the rat. Neurogranin immunostaining is an alternative method for investigations of dendritic pathology in the brain but not in the spinal cord. PMID- 10737455 TI - Lack of endotoxin tolerance with respect to TNF alpha production in the subarachnoid space. AB - To study endotoxin tolerance in the subarachnoid space 0.1 mg of endotoxin derived from Neisseria meningitidis was injected intracisternally into rabbits on 2 consecutive days. On day 1 the maximum peak level of TNF alpha was 7 ng/ml 2 h after injection, whereas on day 2 the highest levels were 3.6 ng/ml and 3.7 ng/ml, respectively, 1 and 2 h after injection. Pretreatment with intravenous endotoxin 5 or 21 h before consecutive intracisternal endotoxin did not affect the cerebrospinal fluid (CSF) levels of TNF alpha. In contrast, there was a marked endotoxin tolerance with respect to TNF alpha in the systemic circulation. Cells appearing in the CSF 5, 12 and 20 h after intracisternal injection of endotoxin were harvested, cultured, and then stimulated with 0.1 mg/ml of endotoxin. In 10 experiments a marked TNF alpha production in the range 10-70 ng/ml was detected in the supernatants, whereas unstimulated cells did not produce TNF alpha. We conclude that tolerance to endotoxin does not develop in the subarachnoid space as evaluated by the present experimental design. The pattern of TNF alpha production and endotoxin tolerance is distinctly different in the subarachnoid space and systemic circulation. PMID- 10737456 TI - An in vivo replica study of microbial colonization in "rapid" and "slow" dental plaque formers. AB - The aim of the present study was to investigate the morphological features of supragingival plaque development in "rapid" and "slow" plaque formers using the replica technique. Forty-nine healthy volunteers were screened for their plaque formation rate after 3 days of oral hygiene abstinence using the plaque index (PI). Five subjects with the highest mean PI and six with the lowest mean PI were selected as "rapid" and "slow" plaque formers, respectively. After a series of thorough prophylaxis and oral hygiene instruction, all subjects went through a 14 day period of no oral hygiene to allow undisturbed plaque formation. Light body silicone elastomeric impressions were taken for the upper right central incisor of each subject on day-0, 1, 3, 7, and 14. Positive replicas were then poured from the impressions using epoxy resin and observed under a scanning electron microscope. A more complex supragingival plaque structure was seen in the day-1 and day-3 replicas of the "rapid" group compared to the "slow" group. From day-7 to day-14, during the maturation period of supragingival plaque, no discernible differences were noted between the two groups. Quantitatively, the percentage of plaque coverage on the tooth surfaces was higher in the "rapid" group than in the "slow" group in day-1 and day-3 replicas. These observations indicate that the morphological and topographical features of supragingival plaque in "slow" and "rapid" plaque formers differ, especially in the early developmental phase. PMID- 10737457 TI - Low prevalence of antibodies and other plasma factors binding to CC chemokines and IL-2 in HIV-positive patients. AB - Neutralizing cytokine antibodies are found in healthy and diseased individuals, including patients treated with recombinant cytokines. Identification of CCR-5 as co-receptor for HIV has focused interest on CC chemokines and their potential therapeutic use. Chemokine-binding components in plasma of HIV-infected patients were therefore assessed by radioimmunoassay and radioreceptor assay. IgG from 4/505 HIV patients and 9/2000 healthy controls (p>0.05) bound rMIP-1alpha and rMIP-1beta, but not rRANTES. No other plasma factors bound the chemokines. The antibodies inhibited receptor binding of both chemokines. There was no association between presence of antibodies and disease stage or HIV progression rate. Three of 11 patients treated with rIL-2 developed IgG antibodies suppressing cellular binding and growth promotion of rIL-2. Hence, circulating factors, including antibodies MIP-1alpha/MIP-1beta, are uncommon in healthy individuals and HIV patients, and are apparently without prognostic significance. In contrast to earlier reports, IL-2 antibodies were found only in HIV patients treated with rIL-2. PMID- 10737458 TI - Evaluation of a commercial test for antibodies to the chlamydial lipopolysaccharide (Medac) for serodiagnosis of acute infections by Chlamydia pneumoniae (TWAR) and Chlamydia psittaci. AB - A commercial test (rELISA) based on a recombinant chlamydial lipopolysaccharide (LPS) antigen has been evaluated for the diagnosis of acute infections caused by Chlamydia pneumoniae (TWAR) and Chlamydia psittaci. This test and a microimmunofluorescence test (MIF) were compared in 160 patients with community acquired pneumonia. Seventeen of nineteen cases with significant titre changes detected by rELISA were confirmed by MIF. The two remaining cases not confirmed by MIF were considered false-positive reactions. One case positive by MIF only was judged not to be a true-positive reaction. All three cases occurred in patients with Mycoplasma pneumoniae infection and may be the result of a mitogenic effect. High antibody titres have been used to indicate acute C. pneumoniae infection. We found high MIF or rELISA titres to be equally common in patients and controls; no association between the two tests was detected. An unexpected cross-reactivity between the rELISA antigen and parvovirus was observed, which might have diagnostic implications. Both MIF and rELISA detected acute C. pneumoniae and C. psittaci infection, and there was good agreement between the tests. Single serum diagnosis was generally not feasible with either MIF or rELISA. PMID- 10737459 TI - The resistance of human monocyte-derived macrophages to Chlamydia pneumoniae infection is enhanced by interferon-gamma. AB - Chlamydia pneumoniae is an intracellular bacterium which commonly causes respiratory infections. Chronic infections have been associated with atherosclerosis and the organism has been detected in macrophages in the disease lesions. Growth of chlamydiae in different epithelial cell lines is restricted by interferon-gamma (IFN-gamma), a monocyte activator produced by T cells. We have studied the influence of IFN-gamma on the growth and infectivity of C. pneumoniae in HL-cells and human monocyte-derived macrophages. Low concentrations of the cytokine significantly restricted the growth and productivity of C. pneumoniae in epithelial cells in vitro. In macrophages, however, no effect on the growth of the bacteria in infected cells was found, but high doses clearly restricted the production of infectious progeny. The results suggest that IFN-gamma participates in the resistance to C. pneumoniae. The bacterium is, however, still capable of infecting macrophages that are important in the pathogenesis of atherosclerosis and it may thus participate in the inflammatory process associated with the disease. PMID- 10737460 TI - The prevalence of gentamicin resistance among clinical isolates of enterobacteria in a Danish region. AB - The susceptibility of enterobacteria to gentamicin was studied in the County of North Jutland during 1993-1998. A total of 378 patients had a first-time gentamicin-resistant isolate. The annual number rose from 34 patients in 1993 to 89 in 1998. The prevalence of resistance per 1000 patients with isolates examined was 13.5 for Escherichia coli, 15.8 for Klebsiella pneumoniae, 26.1 for Klebsiella oxytoca, and 150.4 for Citrobacter freundii. E. coli accounted for 67% of gentamicin-resistant isolates. K. oxytoca was probably associated with a nosocomial epidemic, whereas a source for C. freundii remained unresolved. An analysis confined to E. coli identified specimens other than urine as risk factors for gentamicin resistance. Likewise, resistance to sulfonamides and to fluoroquinolones were strong risk factors for gentamicin resistance among urinary isolates of E. coli. Thus, it is likely that aminoglycoside resistance may be promoted by other antibiotic groups due to co-resistance. The therapeutic and prophylactic uses of aminoglycosides have as yet not been undermined, but continuous population-based surveillance and vigilance are recommended. PMID- 10737461 TI - Characterization of the genetic diversity in superficial and systemic human isolates of Candida parapsilosis by randomly amplified polymorphic DNA (RAPD). AB - The application of randomly amplified polymorphic DNA (RAPD) technology for strain delineation of medically important yeasts has proved to be a valuable tool in clinico-epidemiological studies of Candida species. Candida parapsilosis, a form species of the fungi imperfecti, is an emerging pathogen gaining recognition as an opportunistic agent, especially in the immunocompromised. Therefore, 15 clinical isolates of C. parapsilosis obtained from oral, cutaneous and systemic Candida infections were typed by RAPD analysis using four different primers. The primers RSD6 and RSD9 elicited 7 genotypes each, whereas primers RSD7 and RSD12 revealed 6 and 10 genotypes, respectively. When the data were correlated, a higher degree of genomic heterogeneity in systemic isolates was noted compared with the oral and cutaneous isolates, which shared somewhat similar RAPD profiles. However, a single oral isolate (P5) and two systemic isolates (P13 and P15) elicited radically divergent profiles, dissimilar to their counterparts. RAPD study of the latter two isolates with three additional primers (RSD8, RSD10 and RSD11) confirmed the observed genomic disparity. These data substantiate the previous observations on the genomic heterogeneity in C. parapsilosis and point to genetic shifts which may be associated with ecodiversity, as well as the possible existence of distinct genetic groups within this form species. PMID- 10737462 TI - Chronic pain in torture survivors. AB - According to Amnesty International government-sanctioned torture is verified in one third of the countries in the world. The physical and psychological sequelae are numerous. This study focuses on pain diagnosis, characterising pain types as nociceptive, visceral or neuropathic. Torture victims from the Middle East, treated at the Rehabilitation and Research Centre for Torture Victims (RCT) in Copenhagen, participated in the study. The patients were referred to a pain specialist for evaluation of unsolved pain problems. Eighteen male torture victims were examined. Twelve patients experienced pain at more than three locations. Nociceptive and neuropathic pain were demonstrated in all patients. Specific neuropathic pain conditions were related to the following four types of physical torture: Palestinian hanging, falanga, beating and kicking of the head, and positional torture. When treating torture victims, it is important to know about torture methods, to think differently than normal on etiological and pathogenetic factors and always consider the presence of neuropathic pain. PMID- 10737463 TI - Sex determination by discriminant function analysis of the right tibia in the prehispanic population of the Canary Islands. AB - This study has been performed in order to define standards usable to determine the sex of prehispanic individuals from the Canary Islands from their skeletal remains. Osteometric information at the right tibia was obtained from 59 complete skeletons from Gran Canaria, housed in the Museo Canario (Las Palmas), 45 males and 14 females (this constitutes the totality of complete prehispanic skeletons known from Gran Canaria). The parameters measured were: tibial length, proximal and distal epiphyseal breadth, transverse and anteroposterior diameter, perimeter at the nutrition foramen levels and minimum shaft perimeter. These parameters were subjected to different SPSS discriminant function analysis, combining all of them, or only the proximal or distal ones, without tibial length, etc., in order to obtain functions usable even if only bone fragments are available. Transverse diameter, proximal epiphyseal breadth and minimum shaft perimeter showed the highest discriminant power. The functions obtained showed high average accuracies, ranging from 94.9 to 98.3%, with female accuracies of 100%. The functions obtained were further applied to a test prehispanic population (ten males and ten females) from El Hierro. Overall accuracies of the functions when applied to this population ranged from 65 to 94.7%, with female accuracies ranging 80% to 100%. PMID- 10737464 TI - Drugs in prehistory: chemical analysis of ancient human hair. AB - Concern about drug abuse in modern populations has led to the development of specific methods for identification of cocaine, opiates and cannabis in human hair. Drug use in prehistory can provide indirect evidence of interpopulational contact and social stratification. This paper reports drug evaluation in nineteen ancient hair samples from archaeological sites in northern Chile. Each sample was tested for the presence of traces of cocaine, opiates and cannabis, in order to establish a standard methodology for studies of drug use among prehistoric groups. Although results are negative, this absence of evidence could be due to two main causes: (1) the individuals evaluated did not use any drugs, which does not mean that other members of their cultural group did, or (2) the wide range of known drugs studied did not consider some group specific drugs, derived from local or imported plants, thus meaning that a greater drug range must be tested. In any case, our study confirms that drug testing in prehistoric samples is viable. However, in order to determine what kind of substances were used in prehistoric times new patterns that incorporate all drugs which are not part of the western pharmacopeia must be created. Finally, a methodology for the study of drug use among prehistoric groups using ancient hair samples is described. PMID- 10737465 TI - Postmortem changes in cytochrome c oxidase activity in various organs of the rat and in human heart. AB - Cytochrome c oxidase (COX), a mitochondrial enzyme, is inactivated by cyanide or carbon monoxide (CO) intoxication. To test whether cytochrome c has potential as an indicator of these toxins in cadavers, we measured COX activity in the main organs of the rat, and in the human heart, at various times after death. Each tissue sample or organ was homogenized and the COX activity in the mitochondrial fraction was measured using ferrous cytochrome c as the substrate. COX activity was significantly higher in rat brain, heart and kidney than in lung and liver from 0 to 4 days after death. The loss of COX activity was significantly slower in the brain and heart than in the lung, liver and kidney. Most importantly, COX activity correlated with the time-since-death for each of the rat organs we tested (r2=0.70-0.95), but for the human heart (r2=0.47). It may be possible that COX activity is likely to be a useful indicator of the time-since-death, and is worth pursuing as an indicator of the tissue cyanide and CO content. PMID- 10737466 TI - Post-mortem analysis of apoptotic changes associated with human skin bruises. AB - The estimation of the age of skin bruises is of importance in forensic medicine, especially in child abuse cases. Time-dependent changes in bruise colour and/or associated histological features have been used with a limited degree of success. An increased rate of apoptosis in the injured tissue has been considered as a novel time-dependent marker of cell death, by injury inflicted in a rat model. The object of the present study was to apply the TUNEL method of DNA end labelling to identify and enumerate apoptotic cells in bruised and normal skin in order to study the relationship of apoptotic cell density with the age of the bruise. A commercially available DNA end labelling kit, TUNEL method, was standardised, validated and used for this purpose. Twenty unselected post-mortem cases with bruises due to a variety of causes were studied. The apoptotic cells stained with TUNEL reaction were counted in 10 high power fields in the epidermis, as well as in the dermis of formaldehyde-fixed paraffin-embedded skin specimens. The mean positive cell densities (+/- 1 S.E.) were compared with respect to the age of the bruise. In the epidermis, the mean apoptotic cell count was statistically significantly greater in the bruised skin compared to normal skin in 2- to 6-day-old bruises; whilst in the dermis the same was true in 3- to 8-day-old bruises. The overall findings suggest that there is a quiescent period prior to the increase in the apoptotic cell activity that is seen following skin bruising. This is so provided the post-mortem skin samples were collected within a lapse of 6 days or less between the time of death and formalin fixation and paraffin embedding to avoid the bias made by the difference of length of post mortem interval. PMID- 10737467 TI - Vitreous humor carbohydrate-deficient transferrin concentrations in the postmortem diagnosis of alcoholism. AB - Deaths from the effects of alcohol intoxication are encountered routinely in forensic practice. In an important number of cases difficulty may arise in interpreting the significance of results obtained in the autopsy. In clinical practice biochemical markers, particularly serum gamma-glutamyl-transpeptidase (GGT), alanine aminotransferase (ALT), aspartate transaminase (AST), carbohydrate deficient transferrin (CDT), and erythrocyte mean corpuscular volume are used to diagnose heavy alcohol consumption. CDT is used as a reliable and specific marker. In postmortem diagnosis, because of the difficulty in interpreting blood alcohol levels and relatively non-specific pathological features, biochemical compounds have been studied for use as possible markers. The aim of this study was to evaluate the usefulness of the postmortem determination of CDT in vitreous humor as a confirmation of antemortem alcoholism. CDT levels were studied in 66 male cadavers with a mean age of 55.9 years (S.D. 17.0, range 22-87 years) with a mean postmortem interval of 17.9 h (S.D. 11.4, range 4-72 h). Cases were assigned to two diagnostic groups according to the antemortem diagnosis of alcoholism. Statistically significant differences were found for CDT and ALT concentrations between the two diagnostic groups. The highest vitreous humor levels of CDT and ALT were obtained in the group of cases with a previous diagnosis of alcoholism. Our results suggest that vitreous humor CDT levels are useful in cases where the postmortem diagnosis of alcoholism is hindered by the non-specificity of data. PMID- 10737468 TI - Sudden death from massive pulmonary tumor embolism due to hepatocellular carcinoma. AB - Massive pulmonary embolism in cancer patients can be due to detached thrombi or tumor. Pulmonary tumor embolism is often undiagnosed antemortem. We report a 52 year-old Chinese man admitted for management of hepatocellular carcinoma (HCC). Computerized tomography showed tumor involvement of hepatic vein and inferior vena cava. He died suddenly on the day of admission. At autopsy the main pulmonary arteries of both lungs were blocked by large tumor emboli, the immediate cause of death. Although rapid death in patients with HCC is usually caused by intraperitoneal hemorrhage from spontaneous rupture of tumor, massive pulmonary tumor embolism should also be considered in these patients, especially when antemortem evidence of hepatic vein and/or inferior vena cava invasion is present. PMID- 10737469 TI - Sonographic staging of anterior urethral strictures. AB - PURPOSE: Although radiographic retrograde urethrography has traditionally been the gold standard for imaging the anterior urethra, sonourethrography has proved to be precise and effective for evaluating urethral strictures. We review the evolution of sonourethrography and demonstrate its practical contemporary applications. MATERIALS AND METHODS: We performed literature reviews on MEDLINE and chart reviews of our patient records from 1988 to 1998. RESULTS: Sonourethrography measures stricture length in the bulbar urethra more accurately than conventional retrograde urethrography. Spongiofibrosis is manifested sonographically by a lack of urethral distensibility during retrograde instillation of saline solution. Posterior shadowing is noted in severe posttraumatic cases. Sonographic staging before treatment of complex or reoperative anterior strictures elucidates complicating features, such as calculi, urethral hair, false passage and stent encrustation. CONCLUSIONS: Sonographic staging of anterior urethral strictures offers clinically important information that may be useful in guiding reconstructive therapy. PMID- 10737470 TI - Gentamicin for the practicing urologist: review of efficacy, single daily dosing and "switch" therapy. AB - PURPOSE: We review the literature on gentamicin, including single daily dosing and "switch" therapy. MATERIALS AND METHODS: We used MEDLINE to search the literature from 1966 to June 1997, and then manually searched bibliographies to identify studies that our initial search might have missed. RESULTS: Gentamicin has attractive characteristics, including wide spectrum, infrequent resistance, economy and familiarity. Although limited by well known toxicities, gentamicin remains a drug of choice for serious Gram-negative infections. Dosing strategies, such as single daily dosing and switch therapy, have renewed enthusiasm for this time-honored drug. CONCLUSIONS: Gentamicin remains a valuable drug in urology. Once daily dosing and switch therapy offer the potential to increase effectiveness and convenience while decreasing toxicity and costs. PMID- 10737471 TI - Prostate specific antigen bounce after radioactive seed implantation followed by external beam radiation for prostate cancer. AB - PURPOSE: Prostate specific antigen (PSA) may temporarily increase following radiotherapy for prostate cancer without signaling cancer recurrence. We describe this phenomenon which is called PSA bounce. MATERIALS AND METHODS: From 1984 to 1995, 779 stage T1T2N0 cancer cases were treated with simultaneous radiotherapy with a 125iodine prostate implant followed by external beam radiation. Median pretreatment PSA was 7.7 ng./ml. (range 0.3 to 188). PSA bounce was defined as an increase of 0.1 ng./ml. or greater above the preceding PSA level after simultaneous radiation followed by a subsequent decrease below that level. Disease-free status was defined as the ability to achieve and maintain posttreatment PSA 0.2 ng./ml. or less. RESULTS: PSA bounce was observed in 35% of men (273 of 779). Median time to PSA bounce was 18 months from the time of implant and 92% of bounces were observed within 36 months. Median pre-bounce PSA was 0.7 ng/ml. (range 0.1 to 8.9) and median bounce height (increase above the pre-bounce level) was 0.4 ng./ml. (range 0.1 to 15.8). No distinguishing characteristics were observed between men with PSA bounce and those with cancer recurrence, and bounce had no prognostic significance relative to recurrence. CONCLUSIONS: PSA bounce is common following seed implantation for prostate cancer. It produces anxiety in men previously treated for prostate cancer and confounds the diagnosis of recurrence. PMID- 10737472 TI - Prognostic indicators for renal cell carcinoma: a multivariate analysis of 643 patients using the revised 1997 TNM staging criteria. AB - PURPOSE: We determine independent prognostic indicators for renal cell carcinoma using the revised 1997 TNM staging criteria. MATERIALS AND METHODS: The records of 643 consecutive patients undergoing partial or radical nephrectomy at our institution between 1987 and 1998 were reviewed. Preoperative evaluation of functional status using the Eastern Cooperative Oncology Group (ECOG) criteria was performed in all cases. Renal cell carcinoma grade and stage were evaluated using the 1997 American Joint Committee on Cancer grading and TNM staging criteria, respectively. Patients were followed for a mean plus or minus standard deviation of 47+/-40 months (median 87). Kaplan-Meier survival curves were used to determine 5-year cancer specific survival for all patient groups. Univariate analysis using log rank sum tests was performed to evaluate the prognostic significance of overall TNM stage, tumor stage, disease grade and ECOG status. Multivariate analysis was performed to determine which factors had an independent impact on survival of patients with renal cell carcinoma. RESULTS: The 5-year cancer specific survival rate was 91%, 74%, 67% and 32% for TNM stages I, II, III and IV lesions, respectively (p<0.001). Analysis demonstrated a survival rate of 83% for stage T1, 57% for stage T2, 42% for stage T3 and 28% for stage T4 disease (p<0.001), and 89% for grade 1, 65% for grade 2, and 46% for grades 3 and 4 (p<0.001). Multivariate analysis revealed that overall TNM stage and grade of disease were the most important prognostic indicators for renal cell carcinoma (p<0.001). ECOG classification was a less significant predictor (p = 0.031) and tumor stage was not shown to have any independent impact on patient survival (p = 0.138). CONCLUSIONS: Better survival rates of patients with localized and advanced renal cell carcinoma can be demonstrated with recent advances in diagnosis and treatment. The revised 1997 TNM criteria manifest an appropriate adjustment in staging renal cell carcinoma based on these improvements, with overall stage correlating with cancer specific survival. In contrast, while effectively predicting survival, tumor stage did not demonstrate an independent impact on renal cell carcinoma prognosis under multivariate analysis. Instead, other factors, such as ECOG status and more importantly grade of disease, appeared to affect survival significantly as independent elements. Based on our recent experience with patients treated for renal cell carcinoma in the era of enhanced technology and improved survival, tumor grade and molecular markers may serve as useful adjuncts to TNM staging in guiding treatment and predicting survival outcomes. PMID- 10737473 TI - Laparoscopic lymphocelectomy: a multi-institutional analysis. AB - PURPOSE: Because symptomatic lymphoceles are infrequent, single center studies generally report small numbers of patients. We report a multi-institutional experience with and long-term outcome following laparoscopic lymphocelectomy in 81 patients. MATERIALS AND METHODS: Data were obtained from 9 institutions at which at least 5 cases of laparoscopic lymphocelectomy had been performed. Baseline patient demographics, operative time and blood loss, special operative adjunct techniques, postoperative course, convalescence, complications and lymphocele recurrence data were collected and analyzed. RESULTS: A total of 56 men and 25 women with a mean age of 41 years were included in the study. Lymphocele formed after renal transplantation in 78 patients (96%) and after pelvic lymph node dissection in 3 (4%). Average operating time was 123 minutes with a mean blood loss of 43 ml. Omentopexy was performed in 11 cases (13.6%). No intraoperative stenting of the transplant ureter was performed. Intraoperative complications consisted of laryngospasm, bladder injury, inferior epigastric artery injury and mild renal capsule hematoma in 1 patient each. Conversion to open surgery was required for repair of bladder injury in 1, repair of preexisting hernia in 1, unusually thickened lymphocele wall in 1 and inaccessible lymphocele location in 4 cases. Mean time to ambulation and resumption of regular diet was 1 day, and mean hospital stay was 1.5 days. Postoperative complications included trocar site hernia in 1 and urinary retention in 2. Convalescence averaged 2.5 weeks. During a mean followup of 27 months 5 patients (6%) had lymphocele recurrence. CONCLUSIONS: Laparoscopic lymphocelectomy is safe, minimally invasive and effective. It is an excellent alternative to the conventional open surgical approach. PMID- 10737474 TI - Laparoscopic nephroureterectomy for upper tract transitional cell cancer: the Washington University experience. AB - PURPOSE: Laparoscopic nephroureterectomy has only recently been done to treat patients with upper tract transitional cell carcinoma. We retrospectively evaluated our experience with and long-term followup of laparoscopic nephroureterectomy, compared our results to those of contemporary series of open nephroureterectomy and reviewed the literature. MATERIALS AND METHODS: We reviewed the charts of and followed up by telephone 25 patients who underwent laparoscopic nephroureterectomy between May 1991 and June 1998, and 17 who underwent open nephroureterectomy between March 1990 and January 1997. Demographic, perioperative and followup data were compared. We performed a MEDLINE search and reviewed the literature on laparoscopic nephroureterectomy for upper tract transitional cell carcinoma. RESULTS: Laparoscopic nephroureterectomy required twice the operating time of open nephroureterectomy (7.7 versus 3.9 hours). However, patients who underwent the laparoscopic procedure had a 74% decrease in analgesia requirements (37 versus 144 mg. morphine sulfate equivalent), a 63% shorter hospital stay (3.6 versus 9.6 days) and a 72% more rapid convalescence (2.8 versus 10 weeks). Subsequent bladder transitional cell carcinoma and overall cancer specific survival were similar at a mean followup of 2 years. There was no sign of trocar site or peritoneal seeding after laparoscopic nephroureterectomy. CONCLUSIONS: Although laparoscopic nephroureterectomy is a longer operation, it has the same efficacy and is better tolerated by patients than open nephroureterectomy for upper tract transitional cell carcinoma. As operating time decreases due to surgeon experience and the recent development of hand assisted laparoscopy, laparoscopic nephroureterectomy may soon become the procedure of choice for the ablative management of upper tract transitional cell carcinoma. PMID- 10737475 TI - Percutaneous management of grade II upper urinary tract transitional cell carcinoma: the long-term outcome. AB - PURPOSE: We report the long-term outcome of our experience with percutaneous treatment of grade II upper urinary tract transitional cell carcinoma. MATERIALS AND METHODS: A total of 61 patients with transitional cell carcinoma of the upper urinary tract were treated percutaneously between 1984 and 1998. Of the patients 24 (39%) had grade II disease. Immediate nephroureterectomy was performed due to muscle invasive disease in 2 patients, bleeding in 1 and inability to resect the whole tumor in 1. Percutaneous resection was the actual treatment in 15 patients with stage Ta and 5 with stage T1 disease. RESULTS: Recurrence was noted in 5 patients (25%), including 3 (20%) with stage Ta tumors and 2 (40%) with stage T1 disease after a median followup of 48 months (range 9 months to 12 years). All stage Ta disease recurrences were superficial. In 1 patient with a stage T1 tumor invasive and metastatic disease developed. Disease specific survival was 95% overall, and 100% for stage Ta and 80% for stage T1 disease. No tumor seeding was detected along the percutaneous tract. CONCLUSIONS: Percutaneous surgery has proved safe and effective in treating superficial grade II upper tract transitional cell carcinoma. Offering an endoscopic approach electively to healthy individuals with a normal contralateral kidney seems viable. PMID- 10737476 TI - Changes in calcium metabolism and bone demineralization after orthotopic intestinal neobladder creation. AB - PURPOSE: We evaluated calcium metabolism and bone demineralization by measuring specific markers for bone reabsorption and bone mineral density in patients with an intestinal neobladder. MATERIALS AND METHODS: We studied 33 men 55 to 72 years old who underwent creation of an orthotopic sigmoid (23), ileocolic (8) or ascending colon (2) neobladder after cystectomy. Mean followup plus or minus standard deviation (SD) was 28.4+/-30.1 months (range 4 to 114). Serum electrolytes and arterial blood gases were measured. As markers of bone absorption we assayed urinary pyridinoline, deoxypyridinoline and N-terminal pyridinoline cross-linked telopeptides, and serum pyridinoline cross-linked C terminal telopeptide of type I collagen. Bone mineral density of the spine and femur was determined by dual x-ray absorptiometry. RESULTS: Mean blood pH plus or minus SD was 7.38+/-0.04 (range 7.29 to 7.43). Mean plasma bicarbonate was 22.9+/ 3.4 mmol./l. and mean base excess was -1.63+/-3.61 mmol./l. Serum sodium, potassium, calcium, alkaline phosphatase and phosphate were normal in most patients. Mean serum chloride was 108.0+/-3.5 mEq./l., and was elevated in 9 of the 33 patients (27.3%). Serum intact parathyroid hormone was normal in all patients, osteocalcin was increased in 2 and 1alpha, 25-dihydroxyvitamin D3 was decreased in 2. Pyridinoline cross-linked C-terminal telopeptide of type I collagen was higher in 19 of 33 cases (57.6%) and N-terminal pyridinoline cross linked telopeptides were elevated in 6 (18.2%). Pyridinoline and deoxypyridinoline were higher than normal in 19 (57.6%) and 7 (21.2%) patients, respectively. C-terminal telopeptide of type I collagen and deoxypyridinoline significantly correlated with serum pH (p = 0.017 and 0.0418, respectively). Z score for the bone mineral density of L2 to L4, the femoral neck and Ward's triangle was -0.350+/-1.031, -0.82+/-0.99 and -0.94+/-1.01, respectively. CONCLUSIONS: In patients with a neobladder of intestinal segments metabolic acidosis results in increased bone absorption and decreased bone mass. Thus, attention should be given to bone metabolism in patients with even mild acidosis after orthotopic neobladder creation. PMID- 10737477 TI - Increased tyrosine hydroxylase immunoreactivity in bladder tissue from patients with classic and nonulcer interstitial cystitis. AB - PURPOSE: Interstitial cystitis is a chronic debilitating condition which mainly affects women. Accumulated evidence indicates that interstitial cystitis is a heterogeneous syndrome. The nonulcer subtype appears different than classic interstitial cystitis in regard to symptoms, and endoscopic and histological findings as well as response to various treatments. We further explore the neurogenic nature of this disease using indirect immunofluorescence to evaluate the presence and density of various autonomic and sensory nerve fibers. MATERIALS AND METHODS: Specimens from the bladder wall of 6 patients with classic interstitial cystitis, 7 with nonulcer interstitial cystitis and 6 controls were evaluated to determine the presence and density of nerve fibers containing tyrosine hydroxylase, calcitonin gene-related peptide, neuropeptide Y and substance P using specific antibodies, and the general presence of nerve fibers using a mixture of antibodies against nerve filament, neuron specific enolase and S-100 protein. RESULTS: Increased density and number of nerve fibers immunoreactive for tyrosine hydroxylase were noted in interstitial cystitis cases compared to controls. Furthermore, there was a difference between classic and nonulcer disease in the overall density of nerves using the antibody mixture. CONCLUSIONS: Our findings indicate an altered peripheral sympathetic innervation in interstitial cystitis cases, which may be an indication of primary neurogenic etiology. The difference in nerve density observed after incubation with the antibody mixture between classic and nonulcer interstitial cystitis supports the hypothesis that the 2 forms represent separate entities. PMID- 10737478 TI - Analytical and prospective evaluation of urinary cytokeratin 19 fragment in bladder cancer. AB - PURPOSE: We evaluated CYFRA 21-1, an immunoradiometric assay, developed to detect soluble cytokaratin 19 fragment, for its diagnostic performance in bladder transitional cell carcinoma as well as its analytical performance. MATERIALS AND METHODS: We assessed CYFRA 21-1 in the serum and urine of 182 patients, including 66 with bladder transitional cell carcinoma (group 1), 66 with another urological pathology (group 2) and 50 free of urothelial disease (group 3). The power of urinary CYFRA as a diagnostic test for bladder transitional cell carcinoma was evaluated by receiver operating characteristics curve analysis. Analytical performance was assessed by determining intra-assay and interassay precision, and accuracy by dilution testing and recovery of supplemented analyte. RESULTS: Mean urinary CYFRA plus or minus standard deviation was 154.39+/-49.00, 22.6+/-8.9 and 2.40+/-0.14 ng./ml. in groups 1 to 3, respectively (significantly different). Receiver operating characteristics curve analysis revealed optimal 96.9% sensitivity and 67.2% specificity for a threshold value of 4 ng./ml. Analytical determination showed that intra-assay and interassay precision provides a satisfactory coefficient of variation. The assay for accuracy had acceptable recovery in diluted samples as well as in those with supplemented analyte. CONCLUSIONS: The urinary immunoradiometric CYFRA 21-1 assay performs well analytically. Urinary CYFRA 21-1 is a useful marker for diagnosing transitional cell carcinoma and provides sensitivity in low grade disease. PMID- 10737479 TI - Outcomes after intravesical bacillus Calmette-Guerin are not affected by substaging of high grade T1 transitional cell carcinoma. AB - PURPOSE: Substaging of T1 bladder tumors into T1a and T1b based on invasion of the tumor superficial to and beyond the muscularis mucosa has been assigned prognostic significance. We determined whether outcomes after intravesical bacillus Calmette-Guerin (BCG) differ between stage T1a and T1b subcategories. MATERIALS AND METHODS: Retrospective pathological evaluation of the initial transurethral resection specimens of stage T1 bladder tumors was performed by 2 pathologists. Grade 1, 2 or 3 and stage T1a or T1b were assigned to each case. Followup was from the date of transurethral resection to date of death or the last visit. Kaplan-Meier probability and log rank test were used to evaluate recurrence and progression. RESULTS: Substaging was performed in 49 of the 55 patients (89%) with stage T1 disease. Disease was stage T1a in 32 (65%), stage T1b in 17 (35%), grade 3 in 45 (92%) and grade 2 in 4 (8%) cases. Maximum followup was 147 months (median 71) and 28 cases had a minimum of 5 years of followup. Recurrence was noted in 33 cases (67.3%), including 22 stage T1a (69%) and 11 stage T1b (65%), at a median followup of 11.3 and 8.6 months, respectively. Progression to a higher stage of disease was recorded in 12 cases (24.4%), including 7 (22%) stage T1a and 5 (29%) stage T1b, at a median followup of 108 and 120 months, respectively. The difference between T1a and T1b subcategories was not statistically significant in regard to recurrence-free (p = 0.7203) and progression-free (p = 0.574) outcomes. CONCLUSIONS: Substaging of T1 tumors did not affect response to BCG in regard to recurrence or progression. Therefore, intravesical BCG is effective for stages T1a and T1b disease. PMID- 10737480 TI - Maintenance bacillus Calmette-Guerin immunotherapy for recurrent TA, T1 and carcinoma in situ transitional cell carcinoma of the bladder: a randomized Southwest Oncology Group Study. AB - PURPOSE: Bacillus Calmette-Guerin (BCG) immunotherapy has been widely accepted as the optimal treatment for carcinoma in situ and high grade superficial transitional cell carcinoma. However, controversy remains regarding the role of maintenance therapy, and its long-term effect on recurrence and progression. MATERIALS AND METHODS: All patients in the study had transitional cell carcinoma of the bladder with carcinoma in situ or an increased risk of recurrence. The criteria for increased risk were 2 or more episodes of tumor within the most recent year, or 3 or more tumors within 6 months. At least 1 week following biopsy of carcinoma in situ and resection of any stage Ta or T1 transitional cell tumors 660 patients were started on a 6-week induction course of intravesical and percutaneous Connaught BCG. Three months following initiation of BCG induction therapy 550 consenting patients were stratified by purified protein derivative skin test and the presence of carcinoma in situ, and then randomized by central computer to receive BCG maintenance therapy (maintenance arm) or no BCG maintenance therapy (no maintenance arm). Maintenance therapy consisted of intravesical and percutaneous BCG each week for 3 weeks given 3, 6, 12, 18, 24, 30 and 36 months from initiation of induction therapy. The 384 eligible patients who were disease-free at randomization constitute the primary intent to treat analytic group because they could be followed for disease recurrence. All patients were followed for adverse effects of treatment, recurrence, disease worsening and survival. RESULTS: No toxicities above grade 3 were noted in the 243 maintenance arm patients. The policy of withholding maintenance BCG from patients with increased side effects may have diminished the opportunity to observe severe toxicity. Estimated median recurrence-free survival was 35.7 months (95% confidence interval 25.1 to 56.8) in the no maintenance and 76.8 months (64.3 to 93.2) in the maintenance arm (log rank p<0.0001). Estimated median time for worsening-free survival, defined as no evidence of progression including pathological stage T2 disease or greater, or the use of cystectomy, systemic chemotherapy or radiation therapy, was 111.5 months in the no maintenance and not estimable in the maintenance arm (log rank p = 0.04). Overall 5-year survival was 78% in the no maintenance compared to 83% in the maintenance arm. CONCLUSIONS: Compared to standard induction therapy maintenance BCG immunotherapy was beneficial in patients with carcinoma in situ and select patients with Ta, T1 bladder cancer. Median recurrence-free survival time was twice as long in the 3-week maintenance arm compared to the no maintenance arm, and patients had significantly longer worsening-free survival. PMID- 10737481 TI - Prostate blood flow characteristics in the chronic prostatitis/pelvic pain syndrome. AB - PURPOSE: We determine whether the chronic prostatitis/pelvic pain syndrome is associated with abnormal prostate blood flow. MATERIALS AND METHODS: We used color Doppler ultrasonography to examine 53 patients with inflammation, 80 men without inflammation and 22 healthy controls. Images were recorded and scored using standardized criteria to characterize the degree and distribution of prostatic vascularity. RESULTS: Flow was observed to the entire prostatic capsule in 77% of patients but only 18% of controls (p<0.0001). Parenchymal flow was evaluated using several criteria. On a 2-point scale flow was classified as grade 2 in 74% of patients compared to 27% of controls (p<0.0001). Similar findings were noted on a Doppler spot scale, with flow classified as grade 2 in 47% of patients compared to 14% of controls (p<0.004). Patients also had more parenchymal Doppler spots than controls (p<0.01). Diffuse blood flow throughout the prostatic parenchyma was observed in 63% of patients compared to 36% of controls (p<0.03). There was no significant difference in the amount or distribution of blood flow in patients with and without inflammation. CONCLUSIONS: The chronic prostatitis/pelvic pain syndrome was associated with increased blood flow to the prostatic capsule and diffuse flow throughout the prostatic parenchyma. Despite technical limitations, color Doppler ultrasonography may provide objective documentation of prostate blood flow abnormalities in patients with this syndrome. PMID- 10737482 TI - The mechanism of adverse events associated with terazosin: an analysis of the Veterans Affairs cooperative study. AB - PURPOSE: We determined the mechanism of adverse events associated with alpha1 blockers for treating benign prostatic hyperplasia (BPH). MATERIALS AND METHODS: We randomized 1,229 men with clinical BPH at 31 Veterans Affairs medical centers into equal treatment groups, including those who received placebo, terazosin, finasteride, and combined terazosin and finasteride therapy, respectively. Adverse events were captured at all study visits during our 1-year study. Our current review of adverse events is limited to patients randomized to the placebo and terazosin groups. We compared the incidence of orthostatic blood pressure change, postural symptoms and orthostatic hypotension in men who were normotensive and hypertensive at baseline, respectively. We also determined the association of changes in systolic blood pressure with the incidence of treatment related adverse events. RESULTS: The treatment related rates of dizziness, asthenia, postural hypotension and syncope were 19%, 6%, 6% and 1%, respectively. Of these adverse events only postural hypotension was associated with orthostatic blood pressure changes. The incidence of asthenia, dizziness and postural hypotension was not significantly greater in patients with a systolic blood pressure decrease of 5 or greater and less than 5 mm. Hg, respectively. CONCLUSIONS: Dizziness and asthenia are not associated with changes in blood pressure, suggesting that these treatment related adverse events associated with alpha1-blockers are not related to vascular events. Designing a subtype selective alpha1 antagonist that has less effect on blood pressure may not result in marked improvement in tolerability over commercially available alpha1-blockers. PMID- 10737483 TI - The positive yield of imaging studies in the evaluation of men with newly diagnosed prostate cancer: a population based analysis. AB - PURPOSE: We determine the positive yield of imaging studies performed on men with newly diagnosed prostate cancer. MATERIALS AND METHODS: A prospective, population based survey was conducted on 3,690 men with prostate cancer diagnosed between October 1, 1994 and October 31, 1995. Cases were identified by the rapid case ascertainment systems used in 6 geographic regions participating in the Surveillance, Epidemiology and End Results Program. Based on information captured in primary medical record reviews we estimated the positive yield of bone scans, computerized tomography (CT) and magnetic resonance imaging. RESULTS: The positive yield of bone scan and CT was less than 5% and 12%, respectively, for all men with prostate specific antigen (PSA) 4 to 20 ng./ml., and less than 2% and 9%, respectively, for those who also had a Gleason score of 6 or less. Only men with PSA greater than 50 ng./ml. and those with Gleason scores 8 to 10 and PSA greater than 20 ng./ml. had positive yields greater than 10% and 20% for bone scan and CT, respectively. CONCLUSIONS: Imaging studies designed to identify metastases and/or extracapsular extension in men with newly diagnosed prostate cancer frequently have a low positive yield. Wide variations exist in the use of imaging studies and are associated with tumor factors, such as Gleason score and serum PSA, and nontumor factors, such as state of residence. More extensive cost effectiveness analyses are needed to define appropriate guidelines for ordering imaging studies to optimize the positive yield among men with newly diagnosed prostate cancer. PMID- 10737484 TI - Optimal predictors of prostate cancer on repeat prostate biopsy: a prospective study of 1,051 men. AB - PURPOSE: We compare the ability of total prostate specific antigen (PSA), percent free PSA, PSA density and transition zone PSA density to predict the outcome of repeat prostatic biopsy in men with serum total PSA 4 to 10 ng./ml. who were diagnosed with benign prostatic hyperplasia after initial biopsy. MATERIALS AND METHODS: In this prospective study 1,051 men with total PSA 4 to 10 ng./ml. underwent transrectal ultrasound guided sextant biopsy with 2 additional transition zone biopsies. In 254 subjects biopsy specimens were also obtained from suspicious areas identified during transrectal ultrasound and digital rectal examination. All subjects with biopsy specimens negative for prostate cancer underwent repeat biopsy 6 weeks after initial biopsy. The ability of total PSA, percent free PSA, PSA density and transition zone PSA density to improve the diagnostic power of PSA testing was assessed with univariate and multivariate analyses as well as receiver operating characteristics (ROC) curves. RESULTS: Initial biopsy was positive (prostate cancer) in 231 and negative (benign prostatic hyperplasia) in 820 of the 1,051 subjects. Prostate cancer was detected on repeat biopsy in 10% of subjects (83 of 820) with negative initial biopsy. Percent free PSA and transition zone PSA density were the most accurate predictors of prostate cancer in these subjects. At a cutoff of 30% percent free PSA would have detected 90% of cancers (sensitivity) and eliminated 50% of unnecessary repeat biopsies (specificity). Sensitivity and specificity of transition zone PSA density at a cutoff of 0.26 ng./ml./cc was 78% and 52%, respectively. ROC curve analysis also showed that percent free PSA was a significantly better predictor of repeat biopsy results than total PSA, PSA density and transition zone PSA density. The area under the ROC curve was 74.5% for percent free PSA, 69.1% for transition zone PSA density, 61.8% for PSA density and 60.3% for total PSA. CONCLUSIONS: At least 10% of patients with negative initial prostatic biopsy results will be diagnosed with prostate cancer on repeat biopsy. Percent free PSA and transition zone PSA density enhance the specificity of PSA testing compared to total PSA or PSA density when determining which patients should undergo repeat biopsy. Repeat biopsy should be performed in patients with percent free PSA less than 30% or transition zone PSA density 0.26 ng./ml./cc or greater. In our study percent free PSA was the most accurate predictor of prostate cancer in repeat biopsy specimens. PMID- 10737485 TI - The need for hospital care of patients with clinically localized prostate cancer managed by noncurative intent: a population based registry study. AB - PURPOSE: We studied the need for hospital care of patients 74 years old or younger with clinically localized prostate cancer managed by deferred endocrine therapy. MATERIALS AND METHODS: Our series included all cases of newly diagnosed, clinically localized prostate cancer reported to the Danish Cancer Registry from 1977 to 1992. Information on the number of hospitalizations and operations performed from 1977 to 1994 was obtained from the Danish Hospital Discharge Registry. RESULTS: Our study included 4,790 men 37 to 74 years old with clinically localized prostate cancer. During the year of diagnosis and once a year of the following 9 years patients were hospitalized an average of 2.2 and 1 times and remained hospitalized an average of 22 and 10 days, and prostate cancer accounted for approximately 80% and 67% of hospitalizations the year of diagnosis and once a year subsequently. Nearly 90% of patients underwent transurethral prostatic resection within the year of diagnosis and in 30% repeat resection was necessary. A third of the patients underwent orchiectomy during the study period. Median time from the diagnosis to orchiectomy was 23 months. The estimated probability of surviving 5 and 10 years without orchiectomy was 39% and 17%, respectively. CONCLUSIONS: Patients diagnosed with clinically localized prostate cancer managed expectantly had a substantial need for hospital care in the years after the diagnosis. When evaluating outcome and quality of life after treatment of localized prostate cancer, the cost and impact of associated morbidity must be considered as well as patient survival. PMID- 10737486 TI - Prostate cancer is highly predictable: a prognostic equation based on all morphological variables in radical prostatectomy specimens. AB - PURPOSE: We determine whether biochemical prostate specific antigen (PSA) failure can be accurately predicted from preoperative serum PSA combined with 6 morphological variables from radical retropubic prostatectomy specimens in men with peripheral zone cancers. The unexpected limitation imposed by preoperative serum PSA on biochemical failure led us to compare peripheral zone to transition zone cancers. MATERIALS AND METHODS: A total of 326 peripheral zone and 46 transition zone cancers treated only with radical retropubic prostatectomy were followed for a minimum of 3 years (mean and median greater than 5). All prostates were sectioned at 3 mm. intervals and morphological variables were quantitated using the Stanford technique. Biochemical failure was defined as serum PSA 0.07 ng./ml. or greater and increasing. Multivariate logistic regression was used to identify variables with the most independent influence on biochemical failure and derive a clinical equation to predict failure in peripheral zone cancers. The validity of the predictive equation was assessed by out of sample validation and cross validation techniques. The 46 transition zone cancers were compared to the 326 peripheral zone cancers by Student's t and Wilcoxon tests. RESULTS: Of the peripheral zone failures 60% occurred in the first year after radical retropubic prostatectomy and 95% had occurred by the end of year 4. The highest preoperative serum PSA was 23 ng./ml. among the 181 men biochemically free of disease. Only 15.8% of 57 men with PSA greater than 15 ng./ml. were biochemically disease-free. For the 48 transition zone cancers cure rates were independent of serum PSA with 6 men having PSA greater than 50 ng./ml. Biochemical disease-free status was noted in 80% of transition zone compared to 56% of peripheral zone cancers (p = 0.0009). The most important variables predicting biochemical disease-free status for peripheral zone cancers were percent Gleason grade 4/5, cancer volume, serum PSA and prostate weight. Foci of vascular invasion, intraductal cancer and lymph nodes were less significant variables, and capsular penetration, positive surgical margins and seminal vesical invasion were insignificant. The multivariate logistic equation for predicting failure in peripheral zone cancers was highly accurate and requires only 2 to 3 minutes with a simple calculator. CONCLUSIONS: Failure of radical retropubic prostatectomy to cure peripheral zone prostate cancer is highly predictable based on 6 morphological variables from the prostatectomy specimen and serum PSA. The level of serum PSA profoundly limits biochemical cure rates in peripheral zone cancers. Transition zone cancers have a high cure rate, despite high serum PSA and adverse morphological variables. Men with serum PSA greater than 15 and perhaps even greater than 10 ng./ml. have such a low cure rate for peripheral zone cancer that re-biopsy attempts appear indicated to prove a transition zone location or else therapy other than radical retropubic prostatectomy should be sought. Pathologists should indicate whether the primary (largest) cancer is in the peripheral or transition zone to prevent overoptimistic reports of cure with radical prostatectomy procedures, as 85% of all tumors are in the peripheral zone. PMID- 10737487 TI - Elevated levels of serum secretoneurin in patients with therapy resistant carcinoma of the prostate. AB - PURPOSE: The majority of prostate cancers show some degree of neuroendocrine differentiation. It was previously demonstrated that chromogranin A, a constituent of large dense core vesicles of neuroendocrine cells, is frequently elevated in patients with metastatic prostate cancer. We evaluate the expression of secretoneurin, which is generated by proteolytic processing of secretogranin II (chromogranin C), in patients with prostate disease. MATERIALS AND METHODS: Secretoneurin was measured in sera of 16 healthy men whose blood was drawn for prostate cancer screening (controls), and in 9 patients with prostatitis, 19 with benign prostate hyperplasia and 54 with prostate cancer detected by radioimmunoassay. Therapy resistant disease (clinical stage D3) was noted in 20 prostate cancer cases. Serum prostate specific antigen was measured in all patients and controls. In addition, chromogranin A, prostate acid phosphatase and interleukin-6 were determined in patients with D3 prostate cancer. Molecular properties of secretoneurin immunoreactivity were analyzed by gel filtration chromatography followed by radioimmunoassay. RESULTS: Mean secretoneurin was 58.9+/-8 fmol./ml. in patients with therapy resistant prostate cancer. Levels were significantly higher than those measured in sera from controls and patients with prostatitis, benign prostatic hyperplasia and pT2 or pT3 prostate cancer. There was a statistically significant correlation between secretoneurin and chromogranin A in patients with endocrine therapy failure (r = 0.543, p<0.05). There was no correlation between serum secretoneurin and prostate specific antigen, prostate acid phosphatase or interleukin-6. Gel filtration chromatography analysis of sera of 3 patients with D3 prostate cancer revealed a peak of secretoneurin immunoreactivity where intact secretoneurin elutes, thus showing that the processed peptide is circulating in the blood. CONCLUSIONS: Secretoneurin is elevated in sera of patients with endocrine therapy refractory prostate cancer. Our results support the concept that neuroendocrine differentiation is associated with prostate cancer progression. PMID- 10737488 TI - Differences in sexual function and quality of life after nerve sparing and nonnerve sparing radical retropubic prostatectomy. AB - PURPOSE: We determine the impact of nerve sparing techniques on quality of life after radical retropubic prostatectomy for prostate cancer. MATERIALS AND METHODS: The RAND/UCLA Prostate Cancer Index and several questions about surgical outcomes, including sexual function, were sent to 170 consecutive patients at least 1 year after radical retropubic prostatectomy. Statistical analysis was performed on data for the entire group as well as subgroups of patients after nerve sparing and nonnerve sparing surgery. RESULTS: Nonnerve sparing surgery was performed in 83 of 129 responders (nonnerve sparing group) and the remaining 46 were treated with unilateral nerve sparing surgery (nerve sparing group). Scores for sexual function, sexual bother, physical function and physical limitation domains were significantly better in the nerve sparing group. Spontaneous erectile activity was reported by 50% of nerve sparing group patients. Nerve sparing status did not affect urinary function, bowel function or disease outcome. CONCLUSIONS: Nerve sparing techniques have positive effects on quality of life and sexual function for patients undergoing radical retropubic prostatectomy. PMID- 10737489 TI - Impact of positive surgical margins on prostate cancer recurrence and the use of secondary cancer treatment: data from the CaPSURE database. AB - PURPOSE: We determined the impact of positive surgical margins on prostate specific antigen (PSA) recurrence and secondary treatment in patients who underwent radical prostatectomy as definitive local treatment for prostate cancer. MATERIALS AND METHODS: We reviewed the pathology reports of 1,383 patients in the CaPSURE database, a longitudinal disease registry of men with prostate cancer, who underwent radical prostatectomy as definitive local treatment. Pathological stage, Gleason score, and the number and location of any positive surgical margins were determined in each patient. PSA recurrence was defined as PSA 0.2 ng./ml. or greater on 2 consecutive occasions after radical prostatectomy. Secondary cancer treatment consisted of radiation or androgen deprivation after radical prostatectomy. Adjuvant and nonadjuvant secondary treatment was given within and more than 6 months after radical prostatectomy, respectively. Kaplan-Meier event rates of PSA recurrence and secondary treatment were calculated for patients with positive and negative surgical margins. We performed multivariate Cox proportional hazards analysis to adjust for clinical differences in groups. RESULTS: Patients with positive surgical margins were significantly more likely to undergo secondary adjuvant or nonadjuvant cancer treatment and have PSA recurrence than those with negative margins. After adjusting for patient age, ethnicity, PSA at diagnosis, pathological stage and Gleason score, surgical margin status was an important independent predictor of PSA recurrence and secondary treatment (p = 0.06 and 0.0011, respectively). The number of positive margins and positive margin location had little impact on the outcomes measured. CONCLUSIONS: These data indicate that surgical margin status is an independent predictor of PSA recurrence and secondary cancer treatment in patients who underwent radical prostatectomy as definitive local therapy for prostate cancer. PMID- 10737490 TI - Benefit of adjuvant radiation therapy for localized prostate cancer with a positive surgical margin. AB - PURPOSE: Positive surgical margins are common after radical prostatectomy, and the role of adjuvant therapy in such cases is controversial. We determined the benefit of postoperative external beam radiation therapy in patients with margin positive prostate cancer with respect to biochemical progression or cancer recurrence. To decrease confounding factors that may affect the likelihood of biochemical progression our study was limited to men with organ confined cancer and a single positive margin. MATERIALS AND METHODS: We retrospectively evaluated the records of a nested matched cohort of 76 patients with pathological stage T2N0 prostate cancer and a single positive margin who underwent adjuvant radiation therapy within 3 months of radical prostatectomy. There was a positive margin at the prostatic apex in 35 cases, prostatic base in 18, posterior prostate in 11, urethra in 7, and prostatic apex and urethra in 5. These patients were matched 1:1 with 76 controls who did not receive adjuvant radiation therapy. Neither group received androgen deprivation therapy. Patients and controls were matched exactly for the margin positive site, age at surgery, preoperative serum prostate specific antigen, Gleason score and DNA ploidy. Biochemical relapse was defined as posttreatment PSA greater than 0.2 ng./ml. RESULTS: Overall there was significant estimated improvement plus or minus standard error in 5-year clinical and biochemical progression-free survival in 88%+/-5% versus 59%+/-11% of patients treated with adjuvant radiation therapy versus no radiation therapy (p = 0.005). No patient who received radiation therapy had local or distant recurrence, while 16% of controls had recurrence (p = 0.015). When stratified by site of margin positivity, the 5-year estimated clinical and biochemical progression-free rate in 18 cases and controls with a positive base margin was 95%+/-15% and 65%+/-13%, respectively (p = 0.02). The rate in 35 cases and cases with a positive apex margin was 95%+/-5% and 64%+/-15%, respectively (p = 0.07). Limited sample size precluded analysis of the other sites. CONCLUSIONS: Patients with localized prostate cancer and a singe positive surgical margin appear to have a lower rate of biochemical relapse at 5 years when adjuvant radiation therapy is administered. Definitive evidence of the beneficial effect of adjuvant radiation therapy for patients with involved surgical margins awaits conclusion of randomized clinical trials. PMID- 10737491 TI - Detection of micrometastatic prostate cancer cells in the lymph nodes by reverse transcriptase polymerase chain reaction is predictive of biochemical recurrence in pathological stage T2 prostate cancer. AB - PURPOSE: We evaluated whether detecting prostate cancer cells by the nested reverse transcriptase-polymerase chain reaction (RT-PCR) in lymph nodes has predictive value for serum prostate specific antigen (PSA) recurrence in patients undergoing radical prostatectomy. MATERIALS AND METHODS: We assessed the presence of prostate cancer cells by RT-PCR for prostate specific membrane antigen and PSA assay in lymph nodes dissected from 38 patients with localized prostate cancer treated with radical prostatectomy. The results of nested RT-PCR assay were compared with biochemical recurrence. RESULTS: Nested RT-PCR was positive in the lymph nodes of 2 of 18 patients (11%) with stage pT2a and 5 of 20 (25%) with stage pT2b disease. All 7 patients had biochemical recurrence. We noted a significant difference in the Kaplan-Meier recurrence-free actuarial probability curve in those with positive and negative nested RT-PCR results for prostate specific membrane antigen, PSA and prostate specific membrane antigen-PSA in the lymph nodes (p = 3.02x10(-7), 2.23x10(-7) and 3.02x10(-7), respectively). Multivariate analysis of serum PSA, Gleason score and preoperative RT-PCR assay in peripheral blood showed that nested RT-PCR for prostate specific membrane antigen, PSA and prostate specific membrane antigen-PSA in the lymph nodes were independent predictors of recurrence (p = 0.0089, 0.0075 and 0.0089, respectively). CONCLUSIONS: Nested RT-PCR of the lymph nodes may be a useful pretreatment prognostic test for patients undergoing radical prostatectomy. Further research is necessary using a much larger number of patients with a longer followup. PMID- 10737492 TI - More data on locally extensive prostate cancer--betting on the margin. PMID- 10737493 TI - Nonnephrotoxic, dynamic, contrast enhanced magnetic resonance urography: use in nephrology and urology. AB - PURPOSE: We assessed the value of breath-hold, gadolinium (Gd) enhanced magnetic resonance urography for diagnosing urinary tract disease. MATERIALS AND METHODS: Dynamic magnetic resonance urography was performed in 38 consecutive patients presenting with urological disease in whom excretory urography was contraindicated. Renal function was impaired in 18 cases with a serum creatinine of 140 to 400 micromol./l. Fast spoiled, gradient echo sequences were acquired during breath-holding 5, 10 and 15 minutes after intravenous injection of 10 ml. of a Gd chelate. Opacification of the collecting system was evaluated using a scale of 0 to 3. Diagnostic value of the examinations was also analyzed. RESULTS: Dynamic magnetic resonance urography was sufficient for making the diagnosis in 95% of cases. We identified 71 pathological findings and the surgical anastomosis was always well visualized. Mean image quality score was 2.34 points for renal calices, 2.86 for renal pelves and 2.48 for ureters. Image quality significantly improved after injecting a diuretic. CONCLUSIONS: Gd enhanced magnetic resonance urography provides quality dynamic imaging of the urinary tract. It is a new approach for diagnosing urinary tract disorders in patients in whom excretory urography should be avoided. PMID- 10737494 TI - Intraoperative ultrasound guided retroperitoneal laparoscopic renal biopsy in the morbidly obese patient. PMID- 10737495 TI - Laparoscopic ileal ureter. AB - PURPOSE: We describe the operative technique of laparoscopic ileal ureter replacement. MATERIALS AND METHODS: A transperitoneal 3-port approach was used. Ileovesical and pyeloileal anastomoses were performed with intracorporeal laparoscopic freehand suturing and knot tying techniques. RESULTS: Operating time was 8 hours and blood loss was 200 cc. Both anastomoses were immediately watertight. Hospital stay was 4 days and a cystogram at 14 days confirmed widely patent anastomoses without extravasation. CONCLUSIONS: Laparoscopic ileal ureter replacement satisfactorily duplicates established open surgical principles. The laparoscopic technique is efficient and technically straightforward. PMID- 10737497 TI - Microsurgical testis biopsy: a novel technique for retrieval of testicular tissue. AB - PURPOSE: In vitro fertilization with intracytoplasmic sperm injection has resulted in a dramatic increase in the need for diagnostic and therapeutic testis biopsies. We developed a microsurgical testis biopsy technique which allows identification of testicular vessels and individual seminiferous tubules. We compare the results of this technique to our prior series of nonmicroscopic biopsies. MATERIALS AND METHODS: A retrospective study of 226 consecutive patients who had undergone open testes biopsy with or without an operating microscope was performed. Between 1988 and 1994 standard open testis biopsy was performed without a microscope in 119 patients and a single sample of testicular tissue was taken. After 1994 microsurgical biopsy was performed under 6 to 25x magnification in 107 patients, nearly half of whom had multiple biopsies of each testis. The complication rates of the 2 procedures were compared. RESULTS: Scrotal hematoma required surgical drainage in 3 of the 119 standard testis biopsy cases and testis atrophy was noted in 1, for a total complication rate of 3.4%. There were no episodes of clinically detectable testicular atrophy or scrotal hematoma requiring surgical drainage in the 107 microsurgical biopsy cases (p<0.05). In 2 men the microscope allowed identification of larger tubules that contained sperm. CONCLUSIONS: Use of the operating microscope for testicular biopsy allows identification and avoidance of testicular vessels, minimizing complications. It also may allow selection of seminiferous tubules more likely to contain sperm. PMID- 10737496 TI - The plug and patch: a new technique for repair of corporal perforation during placement of penile prostheses. AB - PURPOSE: Proximal corporal perforation is an uncommon intraoperative complication during placement of a penile prosthetic device. Unrecognized corporal perforation can result in migration or malfunction of the prosthesis. A windsock repair using a nonabsorbable mesh plug has been described. We describe a new technique called the plug and patch to treat this complication. MATERIALS AND METHODS: From July 1994 to May 1999, 175 patients underwent placement of a penile prosthesis at our institution. Of the patients 8 (4.5%) had proximal corporal perforation. Each perforation was repaired using our plug and patch technique. A 5x7 cm. polyglycolic acid patch was folded on itself and passed into the area of the corporal perforation to act as a plug. A solitary absorbable suture was placed affixing the tail of the patch to the corpora. The corpus was subsequently re dilated and remeasured, and the prosthesis was placed as usual. RESULTS: All 8 patients were satisfied with the penile prosthesis. There were no infectious complications or proximal migration of the prosthetic cylinders. The plug repair added an average of 6 minutes to the operating time. Only 1 patient had discomfort in the area of the repair, which resolved spontaneously approximately 6 weeks postoperatingly. CONCLUSIONS: The plug and patch is a rapid and effective technique to repair intraoperating proximal corporal perforations. Potential advantages include shorter operating time, technical simplicity and the use of only absorbable materials. This technique may result in lower infection rates compared to the standard windsock repair. PMID- 10737498 TI - Evaluation of repeat daily voiding measures in the National Interstitial Cystitis Data Base Study. AB - PURPOSE: We address the importance of 3 consecutive daily frequency volume charts relative to a single day in research studies for interstitial cystitis. In addition, differences in voiding patterns between weekend days and weekdays were evaluated. MATERIALS AND METHODS: Longitudinal frequency volume chart data for 305 women enrolled in the National Interstitial Cystitis Data Base Study before December 31, 1995 and meeting protocol specifications were summarized for 3 outcome measures. Longitudinal data methods designed to accommodate different within patient correlation patterns for repeat measures within and among patient contacts were implemented in a generalized estimating equation modeling framework, permitting overall testing of day and weekend effects. RESULTS: Day 2 and day 3 measures were not statistically different (1% level) from day 1 for nocturnal voiding frequency and nocturia. Although statistically significant for 24-hour frequency, the mean difference of -0.36 void daily between days 1 and 3 was not likely to be clinically significant. Also, measures on weekdays were not significantly different from those on weekends for each of the 3 outcome measures. CONCLUSIONS: Our results suggest that the current clinical research requirement of 3 consecutive days for frequency volume charts can be reduced to a single day. Furthermore, day selection need not be affected by weekday versus weekend considerations. PMID- 10737499 TI - Use of rectus abdominis muscle flap for the treatment of complex and refractory urethrovaginal fistulas. AB - PURPOSE: Urethrovaginal fistulas are commonly repaired transvaginally with local tissue flaps, such as the Martius labial fat pad graft. Although this flap is ideal, if it fails and the fistula persists, subsequent treatment options are limited. We describe the use of a pedicled rectus abdominis muscle flap for the repair of complex and refractory urethrovaginal fistulas. MATERIALS AND METHODS: Six women with a mean age of 53 years (range 41 to 62) who had complex and refractory urethrovaginal fistulas were referred to our continence center. Mean number of prior attempted repairs was 1.3 and in all cases at least 1 Martius flap had failed. Transvaginal urethrovaginal fistula closure was performed followed by a pedicled rectus abdominis muscle flap interposed between the fistula closure and vaginal suture line. The muscle flap was based on the inferior epigastric vessels, and provided additional support to the urethra, bladder neck and bladder base. RESULTS: Urethrovaginal fistula repair with the rectus abdominis muscle flap was successful in all cases. No fistula recurred. Of the patients 5 (83%) were continent and able to void to completion at a mean followup of 23 months (range 2 to 66). CONCLUSIONS: The rectus abdominis muscle flap is a useful adjunct in the repair of complex and refractory urethrovaginal fistulas. It can be used with confidence to provide support to the bladder neck and proximal urethra in patients after failed prior repair with the Martius flap procedure. The pelvic surgeon may be able to recognize other applications for the rectus abdominis muscle flap in pelvic floor reconstruction. PMID- 10737500 TI - Voiding cystourethrography findings in elderly women with urge incontinence. AB - PURPOSE: We report voiding cystourethrography findings associated with urge incontinence in elderly women. MATERIALS AND METHODS: Two observers jointly reviewed voiding cystourethrograms of 50 cognitively intact and mobile elderly female participants in a drug trial for urge incontinence and 19 continent volunteers. Multichannel urodynamic testing was performed in all subjects. Bladder wall trabeculation and diverticula, cystocele and vesicoureteral reflux were noted. Maximal bladder capacity, post-void residual, and history of bladder suspension and hysterectomy were obtained from clinical records. RESULTS: Of the incontinent women 35 (70%) had trabeculation, which was mild in 30 (60%) and moderate in 5 (10%), and 41 (82%) had cystocele, which was mild in 23 (46%), moderate in 15 (30%) and severe in 3 (6%). Maximal bladder capacity ranged from less than 100 to more than 900 cc, and was greater than 500 in 37 incontinent women (74%). Of the continent women 16 (84%) had smooth bladders, 2 had mild (11%) and 1 had moderate (5%) trabeculation, and 11 (58%) had cystocele, which was mild in 8 (42%) and moderate in 3 (16%). In 7 continent women maximal bladder capacity was greater than 500 cc (37%). Differences between the 2 groups in regard to bladder wall trabeculation, maximal bladder capacity and presence of cystocele were significant at p<0.05. CONCLUSIONS: Large bladder capacity, bladder wall trabeculation and small to moderate cystocele on voiding cystourethrography are associated with urge incontinence in the elderly female population. PMID- 10737501 TI - Sacral nerve neuromodulation in the treatment of patients with refractory motor urge incontinence: long-term results of a prospective longitudinal study. AB - PURPOSE: Conservative treatment rarely results in a durable cure of patients with urge incontinence and bladder overactivity. Instrumental and surgical procedures often have significant side effects and less than optimal results. We developed a technique of sacral nerve neuromodulation using chronic unilateral electrical stimulation of the S3 sacral nerve to inhibit the micturition reflex to provide effective nondestructive alternative therapy for patients whose condition is refractory to conservative treatment. MATERIALS AND METHODS: Of 85 patients 45 who responded to a test with a temporary electrode underwent implantation of a permanent S3 sacral nerve electrode coupled to a pulse generator. Treatment results were evaluated by urodynamic studies and voiding/incontinence diaries documenting pad use, incontinence episodes, voiding frequency and voided volume. Partial success and cure were defined as 50% to 90% and more than 90% improvement, respectively, in pad use and/or incontinence episodes. RESULTS: Of 45 patients 18 (40%) were cured at an average followup of 47.1 months and 9 (20%) achieved partial success. Median number of pads used and median number of incontinence episodes daily had decreased from 5.4 to 1.2 (p = 0.0001) and 7.1 to 1.3 (p = 0.0001), respectively, 6 months after implantation. Subsequently these results remained almost constant for 5 years. Bladder overactivity disappeared in 19 of the 44 patients (43%). The repeat intervention rate was 37.7% and there was no permanent injury or nerve damage. CONCLUSIONS: Sacral nerve neuromodulation is safe, effective and durable in patients with urge incontinence refractory to conservative treatment. PMID- 10737502 TI - Bladder neck incompetence in patients with spinal cord injury: significance of sympathetic skin response. AB - PURPOSE: We investigated whether recording the perineal sympathetic skin response, which reflects the sympathetic function of the thoracolumbar spinal cord, represents a reliable and accurate diagnostic tool for assessing bladder neck competence and incompetence. MATERIALS AND METHODS: We compared the sympathetic skin response recorded from the hand, foot and perineal skin with urodynamic findings in 90 patients with neurogenic bladder dysfunction, including 66 with spinal cord injury and 24 with cauda equina lesions. RESULTS: Video urodynamics revealed an incompetent bladder neck in 11 of 32 patients (34%) with complete and 7 of 34 (21%) with incomplete spinal cord injury but in only 1 of 24 (4%) with the conus-cauda equina syndrome. This association significantly correlated with the lesion level at T10 to L2 in 12 of 26 cases (46%) as well as with the loss of perineal but preserved hand and foot sympathetic skin response in 13 of 18 (72%). CONCLUSIONS: Recording the perineal sympathetic skin response in addition to that of the hand and foot represents a sensitive diagnostic tool for assessing sympathetic nerve function within the thoracolumbar spinal cord. It is of diagnostic value for evaluating neurogenic bladder neck incompetence in spinal cord injured patients. PMID- 10737503 TI - Differences in bladder compliance with time and associations of bladder management with compliance in spinal cord injured patients. AB - PURPOSE: Controversy continues on the optimal method of bladder management in spinal cord injured patients. We investigated the effects of bladder management on bladder compliance and changes in compliance with time. MATERIALS AND METHODS: We retrospectively reviewed the charts, and video urodynamic and upper tract radiographic studies of 316 patients with spinal cord injury. Patients were categorized according to interval since injury and bladder management method, including clean intermittent catheterization, spontaneous voiding and chronic Foley catheterization. Those with upper tract complications were compared with asymptomatic controls at the bladder compliance threshold values of 10.0, 12.5, 15.0 and 20.0 cc/cm. water. RESULTS: No significant differences were noted among bladder management method groups for followup, level, completeness or mechanism of injury. A bladder compliance threshold of 12.5 cc/cm. water was selected for the remaining comparisons based on the frequency of complications compared with asymptomatic controls. Patients using intermittent catheterization had a significantly higher incidence of normal compliance than the Foley management group for suprasacral, complete and incomplete injury (p<0.01). Normal bladder compliance was more common in patients with suprasacral than sacral and incomplete than complete spinal cord injury for each bladder management type. Logistic regression analysis of compliance versus bladder management and age of injury (interval since injury) revealed that intermittent catheterization and spontaneous voiding were associated more with normal compliance than Foley catheterization (RR = 9.2, 5.4 and 1.0, respectively). Combined data showed that each successively older age of injury cohort was at 23% greater risk for loss of normal compliance than the preceding cohort. Low compliance was statistically associated with vesicoureteral reflux, radiographic upper tract abnormality, pyelonephritis and upper tract stones (p<0.01, <0.01, 0.04 and <0.01, respectively). CONCLUSIONS: Clean intermittent catheterization protects bladder compliance in spinal cord injured patients regardless of the level or completeness of injury and helps to prevent low compliance with time. Also, in the population studied low compliance was associated with upper tract complications. Therefore, clean intermittent catheterization is the superior method for preserving bladder compliance and preventing the upper tract complications associated with low compliance. PMID- 10737504 TI - New strategies in the diagnosis and management of voiding dysfunction. PMID- 10737505 TI - True mycotic arteritis by Candida albicans in 2 kidney transplant recipients from the same donor. PMID- 10737506 TI - Renal medullary carcinoma: can early diagnosis lead to long-term survival? PMID- 10737507 TI - Abnormal mammogram as the presenting sign of renal cell carcinoma. PMID- 10737508 TI - Sporadic bilateral papillary renal carcinoma exhibiting C-met mutation in the left kidney tumor. PMID- 10737509 TI - Laparoscopic radical nephrectomy for renal cell carcinoma in a patient with level I renal vein tumor thrombus. PMID- 10737510 TI - A solitary metastasis of renal cell carcinoma to the urethra. PMID- 10737511 TI - Idiopathic retroperitoneal hematoma mimicking cystic tumor associated with hemorrhagic renal cyst. PMID- 10737512 TI - Urinary cytology changes in protease inhibitor induced urolithiasis. PMID- 10737513 TI - Lymphoscintigraphy for penile cancer. PMID- 10737514 TI - Testicular calculus. PMID- 10737515 TI - Initiation of sperm production after bilateral orchiopexy: clinical and biological implications. PMID- 10737516 TI - Re: Left renal cell carcinoma communicating with inferior mesenteric vein. PMID- 10737517 TI - Re: Editorial: Interstitial cystitis--a light at the end of the tunnel? PMID- 10737518 TI - Re: The value of a second transurethral resection in evaluating patients with bladder tumors. PMID- 10737519 TI - Re: A remote controlled intraurethral insert for artificial voiding: a new concept for treating women with voiding dysfunction. PMID- 10737520 TI - Re: Pelvic fracture urethral injuries: the unresolved controversy. PMID- 10737521 TI - Re: Dynamic suburethral suspension with pedicled external oblique aponeurosis in the management of female urinary incontinence. PMID- 10737522 TI - Re: Prostate specific antigen: a decade of discovery--what we have learned and where we are going. PMID- 10737523 TI - Re: Intraoperative frozen section monitoring of nerve sparing radical retropubic prostatectomy. PMID- 10737524 TI - Re: Chemoprevention of urological cancer. PMID- 10737525 TI - Re: Estramustine phosphate withdrawal syndrome with dramatic pain relief. PMID- 10737526 TI - Evaluation of renal function in normal and hydronephrotic kidneys in rats using gadolinium diethylenetetramine-pentaacetic acid enhanced dynamic magnetic resonance imaging. AB - PURPOSE: Magnetic resonance imaging (MRI) is becoming established as a modality complementary to computerized tomography and ultrasound for evaluating kidney function. The evaluation of renal function during ureteral obstruction may be improved by contrast enhanced MRI. We evaluated dynamic changes in relative signal intensity in normal and hydronephrotic rat kidneys after intravenous injection of gadolinium (Gd) diethylenetetramine-pentaacetic acid (DTPA). MATERIALS AND METHODS: Using Gd-DTPA enhanced dynamic MRI we evaluated 35, 1-year old rats, including 10 with partial and complete unilateral ureteral obstruction, respectively, and 15 with sham operated nonobstructed kidneys. Partial obstruction was created in 2-day-old rats by embedding the left ureter into the psoas muscle. Complete obstruction was created 10 days before MRI by placing a ligature around the upper third of the left ureter. MRI was performed before and 0.1 to 60 minutes after and intravenous injection of Gd-DTPA. We calculated relative signal intensity per time interval in the renal cortex, medulla and pelvis. RESULTS: Dynamic relative signal intensity patterns differed significantly among normal, and partially and completely obstructed kidneys. In normal kidneys signal intensity changes were divided into 3 phases after Gd-DTPA administration. During phase 1 rapid parenchymal uptake of Gd-DTPA within the initial minute after injection produced a rapid signal intensity decrease in the cortex, which achieved a minimum of 28% of pre-injection intensity. During phase 2 signal intensity decreased in the medulla and pelvis to a minimum of 18% and 22%, respectively, of pre-injection intensity within 5 minutes. During phase 3 significant relative signal intensity recovery began in the cortex at 7 minutes, and in the medulla and pelvis at 10 to 15 minutes, and lasted 30 to 60 minutes. In partially obstructed kidneys the 3 phases were significantly slower than in controls. In completely obstructed kidneys only phase 1 occurred within 60 minutes. CONCLUSIONS: Gd-DTPA enhanced dynamic MRI provides useful information for distinguishing obstructed from nonobstructed and partially from completely obstructed kidneys. PMID- 10737527 TI - Collagen-to-smooth muscle ratio helps prediction of prognosis after pyeloplasty. AB - PURPOSE: We quantitatively evaluated the collagen-to-smooth muscle tissue matrix ratio in ureteropelvic junction obstruction, and compared the ratio with the degree of obstruction, patient age and postoperative renal recovery. MATERIALS AND METHODS: We analyzed histological sections from 65 patients with ureteropelvic junction obstruction and 6 normal controls. Morphological and functional grading systems were adapted to determine the degree of renal obstruction. To examine smooth muscle and collagen tissue, sections were stained using Masson's trichrome. Two distinct populations of collagen versus smooth muscle were identified and the tissue matrix ratio was calculated by color image analysis. RESULTS: The mean tissue matrix ratio plus or minus standard deviation was 1.32+/-0.79 in all patients with ureteropelvic junction obstruction but only 0.30+/-0.10 in normal controls (p <0.0001). It appeared that the lower the tissue matrix ratio, the better the improvement in postoperative hydronephrosis (r = 0.50, p = 0.0001). Better recovery of renal function after pyeloplasty was observed with a decrease in the tissue matrix ratio (r = -0.43, p = 0.0004). We divided patients according to the tissue matrix ratio into groups 1--ratio 1 or less, 2--greater than 1 to 1.5 and 3--greater than 1.5 to determine a more detailed and clinically applicable correlation of tissue matrix ratio with postoperative renal functional changes. Better improvement in postoperative renal function was observed in group 1 than in group 3 (p = 0.002). Also, the tissue matrix ratio increased with patient age (r = 0.33, p = 0.007). CONCLUSIONS: Since our data represent an objective and quantitative parameter associated with ureteropelvic junction obstruction, we believe that our findings may help to predict the prognosis after pyeloplasty. PMID- 10737528 TI - The role of nitric oxide in obstructive nephropathy. AB - PURPOSE: Ureteral obstruction leads to tubulointerstitial fibrosis and loss of renal function. Nitric oxide production ameliorates fibrosis due to obstructive uropathy. However, nitric oxide is produced by 3 isoforms of the enzyme, nitric oxide synthase. We evaluated the role of inducible nitric oxide synthase in obstructive uropathy using nitric oxide synthase knockout mice, and determined whether the administration of L-arginine to promote nitric oxide synthesis by alternative nitric oxide synthase isoforms modulates renal fibrosis in these animals. MATERIALS AND METHODS: Complete unilateral ureteral obstruction was created in wild-type C57 and inducible nitric oxide synthase knockout mice. Control animals of each strain underwent sham surgery. Throughout the experiment mice had free access to untreated tap water or water supplemented with 10 gm./l. L-arginine. Animals were sacrificed 1 and 2 weeks, respectively, after creation of unilateral ureteral obstruction. We obtained serum as well as bladder and obstructed renal pelvic urine, and determined the nitrite level in each fluid. Renal cortical thickness was measured in the normal and obstructed kidneys. The degree of tubulointerstitial fibrosis was evaluated by trichrome staining and type I collagen deposition in kidney tissue specimens. RESULTS: Nitrite was significantly decreased in the serum, bladder and renal pelvic urine of inducible nitric oxide synthase knockout mice with unilateral ureteral obstruction compared with that in wild-type C57 mice at 1 and 2 weeks (p<0.05). In knockout mice with unilateral ureteral obstruction 1 week in duration that drank tap or L-arginine supplemented water nitrite in serum and each urine sample was higher than in sham operated knockout controls. The level returned to baseline after 2 weeks of obstruction (p<0.05). After 2 weeks of obstruction there was significantly greater cortical thinning in knockout than in C57 mice (p<0.05). Moreover, knockout mice given L-arginine supplemented water for 2 weeks had even greater cortical thinning than after 1 week or than mice given tap water for 1 to 2 weeks (p<0.05). Decreased renal cortical thickness in knockout mice after 2 weeks of obstruction was associated with less intense trichrome staining and a virtual absence of type I collagen deposition compared with findings in the wild-type C57 strain. CONCLUSIONS: Inducible nitric oxide synthase knockout mice with unilateral ureteral obstruction have significantly lower nitrite in serum and urine than wild-type C57 mice. Knockout mice also have more severe renal cortical thinning than C57 animals after creation of unilateral ureteral obstruction. Providing L-arginine supplemented water to inducible nitric oxide synthase knockout mice exacerbates the loss of cortical thickness. Alterations in cortical thinning that we observed in knockout mice were associated with decreased tubulointerstitial fibrosis and a decreased net renal extracellular matrix accumulation. These data indicate that endothelial or neuronal nitric oxide synthase may be more important than inducible nitric oxide synthase for modulating renal fibrosis in obstructive uropathy. PMID- 10737529 TI - Role of the bladder in delayed failure of kidney transplants in boys with posterior urethral valves. AB - PURPOSE: There is a tendency toward less favorable long-term graft function in patients with posterior urethral valves than in controls. We studied the role of the bladder in boys who underwent transplantation by simultaneously evaluating renal graft and voiding function. MATERIALS AND METHODS: Between 1972 and 1994, 66 boys with posterior urethral valves underwent kidney transplantation. Of these boys 44 with a mean age of 9.7 years who retained a functional renal graft did not undergo any surgery on the lower urinary tract except for the initial treatment of posterior urethral valves. Long-term evaluation included a voiding questionnaire, radiological assessment and serum creatinine measurement. RESULTS: Average followup was 9.01 years (range 2.4 to 19.6). There was no voiding dysfunction symptomatology in 23 boys, while 3 (14.2%) and 8 (38.1%) of the remaining 21 had daytime and nighttime frequency, respectively. Dysuria and incontinence were present in 11 (52.4%) and 12 (57.1%) patients, respectively. Urodynamics in 11 cases revealed a mean bladder compliance plus or minus standard deviation of 11.3+/-2.8 ml./cm. water. In boys with a voiding disorder mean serum creatinine increased after 5 years of followup. At 10 years after kidney transplantation mean serum creatinine was 140.3+/-36.0 and 285.7+/-36.2 micromol./l. in asymptomatic boys and those with a voiding disorder, respectively (p<0.01). CONCLUSIONS: Valve bladder has a role in the deterioration of renal transplants in boys with posterior urethral valves. In those with a voiding disorder closer followup is needed, including urodynamic and radiological studies. Bladder dysfunction, such as hypocompliance and/or hyperreflexia, requires medical or surgical treatment. PMID- 10737530 TI - Comparison of flaps versus grafts in proximal hypospadias surgery. AB - PURPOSE: We analyzed the complication rate in patients who underwent proximal hypospadias repair using preputial skin as a flap or free graft. MATERIALS AND METHODS: We retrospectively reviewed the records of 142 patients who underwent proximal hypospadias repair. Repairs were subdivided into tubed and onlay repairs within the flap and free graft groups. We analyzed postoperative complications, including fistulas, proximal strictures, diverticula and meatal stenosis. RESULTS: Median patient age at followup was 11.3 months and median followup was 9.4 months. Two-thirds of the repairs were performed with free grafts. A proximal stricture developed in 8 and 0 patients who underwent free tubed graft and free onlay repair, respectively (p = 0.047). Otherwise there was no significant difference in the complication rate of the various types of repair. Of the 43 patients who had stricture, fistula or meatal stenosis 29 (67%) presented more than 1 year after surgery. CONCLUSIONS: In repairs performed with free grafts there is a significantly higher proximal stricture rate when a tube rather than an onlay is used. Otherwise we noted no significant difference in the complication rates of flaps and grafts used to repair proximal hypospadias. A significant number of complications presented more than 1 year postoperatively and they may even present as late as 4 years. This finding suggests that longer followup may be necessary to assess completely the outcome of proximal hypospadias surgery. PMID- 10737531 TI - Hormonal treatment may harm the germ cells in 1 to 3-year-old boys with cryptorchidism. AB - PURPOSE: Hormonal treatment with human chorionic gonadotropin (HCG) or gonadotropin releasing hormone may be given initially for cryptorchidism. We evaluated whether hormonal treatment is safe for the germ cells in boys with cryptorchidism 1 to 3 years old in whom follicle-stimulating hormone, luteinizing hormone and testosterone values are normally low. MATERIALS AND METHODS: We measured the number of spermatogonia per tubule at orchiopexy in 72 consecutive boys with cryptorchidism who underwent simultaneous testicular biopsy. In 19 patients gonadotropin releasing hormone was unsuccessful, while 8 received HCG and 45 underwent orchiopexy without hormonal therapy. Groups were otherwise equal. RESULTS: Patients who underwent surgery only had a higher number of spermatogonia per tubule than those in whom hormonal treatment was unsuccessful (p<0.05). Spermatogonia per tubule values were normal only after surgery alone (p = 0.06). Gonadotropin releasing hormone and HCG influenced germ cells equally. CONCLUSIONS: In 1 to 3-year-old boys with cryptorchidism gonadotropin releasing hormone or HCG given for testicular descent may suppress the number of germ cells. PMID- 10737532 TI - Endodermal sinus tumor of the vagina. PMID- 10737533 TI - Expression of transforming growth factor-beta1 and its receptors related to the ureteric fibrosis in a rat model of obstructive uropathy. AB - PURPOSE: Transforming growth factor (TGF-beta) is a multifunctional cytokine that may stimulate fibroblast proliferation and regulate the synthesis of matrix components. To elucidate the role of TGF-beta1 in the process of ureteric fibrosis in obstructive uropathy, we conducted this study. MATERIALS AND METHODS: The expression of TGF-beta1 and TGF-beta receptors I, II, III were studied in 54 rats by the method of immunohistochemistry. RESULTS: The degree of smooth muscle hypertrophy and serosa thickening aggravated gradually along the course of ureteric ligation. The expression of TGF-beta1 in the serosa layer was noticed from day 7 after ligation. The expression of TGF-beta1 and receptors in the serosa layer progressively increased during the period of ureteric obstruction, reached a peak on day 21 after ligation, and then declined. The expression of TGF beta1 and TGF-beta receptors (II and III) in the serosa layer was correlated significantly (r = 0.8048 and 0.7974, respectively; p values both <0.0001). The expression of TGF-beta1 also correlated significantly with the thickening of serosa layer and the severity of hydroureter (r = 0.6921 and 0.5394, respectively; p values both <0.005). The expression of TGF-beta1 and TGF-beta receptors in the smooth muscle layer was transient and weak, and the expression of TGF-beta1 and TGF-beta receptors was accompanied with the fibrosis in the muscle layer. CONCLUSIONS: We conclude that TGF-beta1 might participate in the progression ofureteric fibrosis in obstructed ureters. PMID- 10737534 TI - Distribution of collagen XII and XIV in the bladder wall of the male rat with outlet obstruction. AB - PURPOSE: Our previous studies indicate that neonatal estrogenization with diethylstilbestrol (neoDES) of male mice and rats causes partial outlet obstruction. In the present study, type XII and XIV collagens were localized in the bladder to study their role in the development of obstruction. MATERIALS AND METHODS: The bladder sections immunostained with smooth muscle specific a-actin antibody were double labeled either with collagen type XII or type XIV antibodies. The specimens were then analyzed with conventional and confocal fluorescence microscope. RESULTS: Type XII and XIV collagens were not evenly distributed in the bladder. Further, in neonatally estrogenized rats collagen XIV appeared inside smooth muscle fascicles. CONCLUSIONS: Non-overlapping distributions of collagen XII and XIV suggest their different roles in the urinary bladder. Penetration of collagen XIV inside smooth muscle fascicles may have a role in the development of DES-induced partial outlet obstruction. PMID- 10737535 TI - Analysis of bladder related nerve cuff electrode recordings from preganglionic pelvic nerve and sacral roots in pigs. AB - PURPOSE: Electrical stimulation of appropriate lower urinary tract (LUT) nerves may be used in bladder dysfunction to achieve continence and abolish hyper reflexic detrusor contractions. It can also be used for consequent emptying of the bladder. To control the time course of the described functional phases, knowledge of bladder sensory information is needed. We investigated if the latter could be extracted from the LUT nerve activity. MATERIALS AND METHODS: In acute experiments using 10 pigs, tripolar cuff electrodes were placed unilaterally around the pelvic nerve and the S3 and S2 roots. The cuff electrode signals, filling rate and the bladder and rectal pressures were recorded during slow and fast bladder fillings/emptyings. RESULTS: Two pigs were excluded from the analysis because of no observed changes in the nerve signals in one animal, and because of electrical noise problems in the other animal. Fast bladder pressure increases resulted in a sudden pelvic nerve signal rise in 6 out of 7 pigs (3 out of 6 for the S3 nerve signal). Slow bladder pressure increase was reflected in the recorded nerve activity only in 3 out of 8 and in 3 out of 7 pigs for the pelvic and S3 cuff signals respectively. In 2 animals small spontaneous bladder contractions were clearly reflected in the pelvic nerve signal (contractions were observed only in 3 pigs). Except in one pig, there were no slow/fast bladder filling responses recorded in the S2 roots. It is shown that the recorded responses were afferent. CONCLUSIONS: Cuff electrodes can be used to record bladder afferent information from the pelvic nerve and the sacral root S3 in pig. Pelvic nerve recordings were more selective than the sacral root recordings. Nerve activity increases were more distinct and repeatable during rapid bladder pressure changes and small spontaneous bladder contractions than during slow bladder fillings. PMID- 10737536 TI - Effects of an environmental anti-androgen on erectile function in an animal penile erection model. AB - PURPOSE: Erectile function is testosterone dependent. For example, interference with either the levels or receptor binding of this steroid hormone may induce erectile dysfunction. Several environmental contaminants can interfere with the actions of endogenous hormones and have been termed 'endocrine disrupters.' p,p DDE, a prominent and persistent metabolite of the insecticide DDT, has been shown to be an androgen receptor antagonist. The objective was to determine whether endocrine disrupters, as exemplified by p,p-DDE, are factors in the etiology of erectile dysfunction. MATERIALS AND METHODS: Using the established rat model of apomorphine-induced (80 microg./kg, s.c.) erections we assessed the dose-response effects of p,p-DDE in comparison to the known androgen receptor antagonist flutamide in acute (0.5 to 12 hours) and short-term (up to 8 weeks) experiments in both intact (Study 1) and castrated (Study 2) rats. As a follow up (Study 3), castrated rats treated with p,p-DDE were given increasing doses of testosterone (0.48 to 2.4 mg./kg., i.p.), eight weeks after p,p-DDE administration, to assess reversibility of p,p-DDE effect. RESULTS: A single dose of flutamide (50 mg./kg., i.p.) was found to significantly decrease apomorphine-induced erections to less than 50% over 12 hours following flutamide administration with recovery of erectile response within 48 hours. In comparison, a single dose of p,p-DDE (500 mg./kg., i.p.) decreased apomorphine-induced erections for at least two weeks (1.15+/-0.3 versus 2.5+/-1.1). Castration significantly decreased apomorphine induced erections to approximately 0.5 erections/30 minutes. Flutamide (50 mg./kg.; i.p.) or p,p-DDE (50 mg./kg.; i.p.) did not further suppress the apomorphine erections in castrated rats. Testosterone supplementation (480 microg./kg; s.c.) in vehicle treated castrated rats recovered erectile response to pre-castrated levels, whereas p,p-DDE treated castrated rats required 4 times the dose of testosterone (2 mg./kg.; s.c.) given to vehicle treated rats to recover erections. CONCLUSIONS: The endocrine disrupter p,p-DDE can markedly interfere with erectile function and demonstrates persistence after a single dose. This supports our novel concept that environmental hormones may cause erectile dysfunction. PMID- 10737537 TI - A randomized phase II trial comparing two different sequence combinations of autologous vaccine and human recombinant interferon gamma and human recombinant interferon alpha2B therapy in patients with metastatic renal cell carcinoma: clinical outcome and analysis of immunological parameters. AB - PURPOSE: The clinical observation of spontaneous regression in patients with renal cell carcinoma (RCC) and the response to various immunotherapeutic therapies strongly suggest a role for the host immune system in this disease. Prior studies showed that sequential administration of interferon (IFN) gamma and IFN alpha to RCC patients was safe. Clinical responses as well as immune changes in the peripheral blood mononuclear cell compartment were observed. Autologous tumor cell vaccines (AV) have also demonstrated activity in renal cell carcinoma. We hypothesize that the addition of AV to sequential IFN gamma and a therapy might improve the tumor-specific immune response by providing an appropriate source of antigen in the appropriate cytokine environment. To our knowledge, this is the first trial using AV combined with IFN alpha and IFN gamma. The purpose of this study was to evaluate the feasibility of manufacturing and administering (AV) from resected tumor samples, and administration of AV with combination IFN gamma and IFN alpha therapy. Finally, the impact on immunological parameters of these treatment options was assessed. MATERIALS AND METHODS: Patients with metastatic RCC were randomly assigned to receive AV plus bCG along with a sequential administration of IFN gamma and a either together or after initiation of vaccine. Toxicity and clinical responses were evaluated. Modulations of the immune system were investigated by analyzing phenotype, cytokine mRNA expression, T cell proliferation and cytotoxicity in the peripheral blood mononuclear cell compartment. RESULTS: Fourteen patients with metastatic renal cell carcinoma were enrolled in this study; 9 were available for response evaluation. In a 70 day period, 3 (33%) showed mixed responses, 5 (56%) stable disease and 1 (11%) progression of disease. Toxicities were consistent with previous clinical reports. In the flow-cytometry phenotype analysis, stimulation of distinct subsets of circulating T-lymphocytes and a decrease of CD8+ T cell subsets was demonstrated. T-cell proliferation to allogeneic tumor cell stimulation improved following treatment. IL-4 and IL-5 mRNA levels were reduced in all patients after treatment. Patients who responded to treatment did not produce any IL-4 mRNA at all, before or after treatment. CONCLUSIONS: AV with IFNgamma and IFNalpha therapy might induce a MHC class-mediated cytotoxic T lymphocyte (CTL) response. We suggest that adequate therapy might direct T cell response toward a Th1 type response. We hypothesize a state of improved immune readiness in patients who might eventually respond to immunotherapy. PMID- 10737538 TI - Early renal ischemia, with or without reperfusion, activates NFkappaB and increases TNF-alpha bioactivity in the kidney. AB - PURPOSE: Acute tubular necrosis (ATN) and the ensuing renal failure induced by ischemia and reperfusion injury (I/R) remain a major cause of morbidity and mortality among patients in the intensive care unit. Although it is well established that exogenous tumor necrosis factor-alpha (TNF) induces renal injury, it remains unknown whether ischemia and/or reperfusion activates the signaling mechanisms required for renal TNF production. We hypothesized that ischemia and/or reperfusion would activate the oxidant sensitive TNF transcription factor, nuclear factor kappa B (NFkappaB), and thereby lead to renal TNF production. MATERIALS AND METHODS: Male Sprague-Dawley rats were anesthetized with sodium pentobarbital, after which various periods of renal ischemia, with or without reperfusion, were induced in rats. At different time intervals, kidneys were harvested and NFkappaB activation (electrophoretic mobility shift assay), TNF mRNA content (RT-PCR), and TNF bioactivity (WEHI-164 cell clone cytotoxicity assay) were determined. RESULTS: Results indicate that 15 minutes of ischemia alone activates NFkappaB, whereas peak activation occurred at 30 minutes of ischemia alone. NFkappaB remained activated through 60 minutes reperfusion. Thirty minutes of ischemia is required to induce renal TNF mRNA production; however, renal TNF protein expression and bioactivity peaked following 1 hour of ischemia and 2 hours reperfusion. CONCLUSIONS: These results are the initial demonstration that renal ischemia, with or without reperfusion, activates the TNF transcription factor NFkappaB and increases TNF bioactivity in the kidney. PMID- 10737539 TI - Altered synaptic transmission in the hippocampus of the castrated male mouse is reversed by testosterone replacement. AB - PURPOSE: To determine the effect of castration on hippocampal function, we have investigated synaptic transmission in the castrated male mouse in vivo. We also examined whether administering testosterone can reverse the changes. MATERIALS AND METHODS: Male 12 weeks-old C57BL/6J mice were divided into three experimental groups; sham-castration (Control), the castration group (Cast), and the castration plus testosterone propionate group (Cast+TP). Field excitatory postsynaptic potentials (fEPSP) were evoked in the CA1 area of the hippocampus by stimulating the commissural fibers of the contralateral hippocampus. Field EPSPs were evoked in the granular cells of the dentate gyrus (DG) by stimulating the ipsilateral perforant path fibers. RESULTS: Laminar analysis of the fEPSPs in the hippocampal CA1 pyramidal cell layer did not differ significantly between the three experimental groups. However, paired pulse facilitation (PPF) of the fEPSP with short inter-stimulus intervals (30 to 100 msec) was significantly suppressed in Cast group. This suppression was reversed by testosterone injection (Cast+TP). Longterm potentiation (LTP) in the CA1 pyramidal neurons by high frequency stimulation (HFS) did not differ significantly between the three experimental groups, whereas potentiation evoked by primed burst stimulation (PBS) was much weaker in the Cast group compared with the Control group. Testosterone injection restored the PBS-induced potentiation to the control level. Synaptic transmission between perforant pathway and the granule cells in the dentate gyrus (DG) did not differ significantly among the three experimental groups. CONCLUSIONS: Suppression of PPF and impairment of the potentiation by PBS in CA1 hippocampal neurons was observed in castrated male mice and these changes were reversed by testosterone injection. These findings suggest that altered synaptic transmission in the castrated male mouse is caused by disturbance of inhibitory neuronal networks that are influenced by testosterone. PMID- 10737540 TI - Deletion of Y-chromosome specific genes in human prostate cancer. AB - PURPOSE: The present study is based on the hypothesis that deletion of Y chromosome-specific genes is associated with prostate cancer. To test this hypothesis, we analyzed the deletion of six Y-chromosome-specific genes in prostate cancer samples. MATERIALS AND METHODS: Fifty human prostate cancer specimens were processed for microdissection of pure epithelial cells. DNA was extracted from these cells and amplified using PCR and analyzed for loss of six different Y-chromosome-specific genes (SRY, ZFY, BPY1, SMCY, RBM1 and BPY2). We used D8S262 primer (chromosome 8p23) for internal control to assess the quality and loading of DNA for each sample. RESULTS: Deletion was observed in most of the prostate cancer specimens with at least one Y-chromosome-specific gene. The loss of SRY gene (Yp11.32) was shown in 38% of cases whereas the other genes show 18% loss in ZFY (Yp11.31), 14% in BPY1 (Yq11.2), 52% in SMCY (Yq11.22), 32% in RBM1 (Yq11.23) and 42% in BPY2 (Yq12.1). The loss of most genes analyzed is seen more frequent in advanced stages and grades of prostate cancer. CONCLUSION: There was a significant loss of Y-chromosome-specific genes in prostate cancer. The loss of SRY and BPY2 genes was more frequent in higher stages and grades of prostate cancer. This is the first report to demonstrate that the loss of Y-chromosome specific genes is associated with prostate cancer, suggesting their role in pathogenesis of this disease. PMID- 10737541 TI - Role of anionic proteins in kidney stone formation: interaction between model anionic polypeptides and calcium oxalate crystals. AB - PURPOSE: We tested the effect of molecular weight and amino acid composition (aspartate versus glutamate) in model peptides on calcium oxalate dihydrate (COD) formation to understand how known urinary inhibitor proteins might control spontaneous crystallization. MATERIALS AND METHODS: Supersaturated solutions of CaCl2 and Na2C2O4 in HEPES buffered saline solution were prepared at various calcium (Ca) to oxalate (Ox) ratios, but constant supersaturation, in the presence of protein inhibitors (polyaspartic acid molecular weight series or polyglutamic acid). The resulting crystals were collected and evaluated with optical microscopy. RESULTS: With no added inhibitors, the crystal size increased with Ca to Ox ratio, while the number of crystals decreased. With protein inhibitors at equivalent mass concentrations, intermediate molecular weight proteins produced a greater proportion of COD in Ca rich conditions than did either extreme. In Ox rich conditions, the proportion of COD was directly related to protein molecular weight. However, at equivalent molar concentrations, the proportion of COD produced was directly related to molecular weight under all conditions. Larger protein concentrations were required to produce COD at high Ox conditions, in proportion to the increased number of crystals produced. Polyglutamic acid had a much weaker effect on crystal structure, but it changed the COM morphology. CONCLUSIONS: The results suggest that a discrete number of protein molecules per crystal were required to direct crystallization toward COD, and that a characteristic size of polypeptide chain can be defined. The charge of the side group was not the sole determinant of this effect, as polyglutamic and polyaspartic acids behaved differently. Calcium oxalate crystal nucleation rates appeared to increase with Ox content. PMID- 10737542 TI - Correlation between the structure and function of the rabbit urinary bladder following partial outlet obstruction. AB - PURPOSE: To understand the relationship between contractile and structural changes in the obstructed bladder, rabbit bladder was partially obstructed for up to 70 days and alterations in tension response to field stimulation and carbachol were compared with alterations in ultrastructure and innervation of detrusor smooth muscle (SM). The effect of partial outlet obstruction on the physiological responses to field stimulation (FS) (nerve mediated contraction) and carbachol (receptor mediated contraction) were correlated with the structure and innervation of the detrusor smooth muscle (SM) of the same animal during a 70 day period. MATERIALS AND METHODS: 28 rabbits were subjected to 1 to 70 days of mild partial outlet obstruction. Sham operated rabbits were euthanized at 7, 14, 28, and 70 days post-obstruction. At each time period, isolated strips of bladder body were mounted in individual baths and the contractile response to FS and carbachol determined. Three additional strips from each bladder were fixed for electron microscopy. RESULTS: Bladder mass increased rapidly during the first 7 days after obstruction, was constant for the next 7 days, and then continued to increase gradually. Dysfunction of the contractile response to FS was noted as early as 3 days and progressively increased over the 70-day study period. The decrease in the response to FS increased at a significantly faster rate than the decrease in the contractile response to carbachol. In ultrastructure studies, at 3 and 7 days post-obstruction the majority of SM cells displayed the characteristics of hypertrophy. At 28 days some SM cells displayed loosely packed myofilaments and an irregular distribution of sarcoplasmic dense bodies. At 70 days swollen mitochondria were present in all cell types of the bladder wall. Evidence of axonal degeneration was first observed at 7 days post-obstruction and became more extensive thereafter. No evidence of mitotic figures, nerve growth cones or regenerating SM cells was observed. CONCLUSIONS: Prolonged partial bladder outflow obstruction is accompanied by a progressive decrease in contractility of SM. The present study describes the structural damage that occurs in the bladder wall in response to partial outlet obstruction and correlates these observations with the contractile dysfunction with which it is associated. Furthermore, mitochondrial damage in vessels and fibroblasts is suggestive of bladder wall ischemia. PMID- 10737543 TI - Effects of anoxia on force, intracellular calcium and lactate production of urinary bladder smooth muscle from control and diabetic rats. AB - PURPOSE: To examine the effects of inhibiting oxidative metabolism on lactate production (J(Lac)), force and [Ca2+]i in longitudinal smooth muscle from urinary bladders of control and diabetic rats. MATERIALS AND METHODS: Strips of longitudinal smooth muscle were isolated from urinary bladders of diabetic rats and their age-matched controls. Force and [Ca2+]i were measured simultaneously in muscle strips loaded with the calcium indicator, fura-2. Separate muscle strips were used to determine J(Lac) by standard enzymatic assay. The muscles were stimulated to contract with 65 mM K+ or 1 microM carbachol (CCh) in the presence of 2.5 mM Ca2+ and either 5, 10 or 25 mM glucose. Oxidative metabolism was inhibited either by replacing O2 in solution with N2, or by addition of 2 mM NaCN. RESULTS: J(Lac) was significantly less in diabetic muscles than control muscles under both normoxic and anoxic conditions. During stimulation under anoxic conditions, the diabetic muscles were less able to maintain force than the controls. Despite a marked decline in force in both diabetic and control muscles under anoxic conditions, [Ca2+]i remained elevated to levels that were in fact higher than those observed during stimulation under normoxic conditions. Increasing the glucose concentration had no significant effect during normoxia, however, under anoxic conditions, the higher concentration improved force maintenance in both control and diabetic muscles. There were no apparent effects of the glucose concentration on [Ca2+]i in either diabetic or control muscles. CONCLUSION: The results reveal that urinary bladder smooth muscle from diabetic rats has a reduced ability to maintain contraction under anoxic conditions. This most likely reflects a greater energy limitation as evidenced by the reduced J(Lac) in diabetic muscles. In both diabetic and control muscles there was a marked dissociation between force and [Ca2+]i when oxidative metabolism was inhibited. This may indicate preferential use of glycolytically produced ATP for maintenance of [Ca2+]i homeostasis under these conditions. PMID- 10737545 TI - A suspected case of trimethoprim-sulfamethoxazole-induced loss of fingernails and toenails. PMID- 10737544 TI - Sex differences in urethral pressure response to electrical stimulation of the hypogastric nerves in rats. AB - PURPOSE: This experiment was performed to study the pharmacology of transmitters mediating the response, and the characteristics of the hypogastric nerve (HGN) of female rats, because electrical stimulation of the HGN was found to unexpectedly reduce urethral pressure in female rats. MATERIALS AND METHODS: Male and female Wistar rats (weighing about 250 gm.), 10 weeks and 6 months old, respectively, were used under anesthesia. Fluid was infused from the bladder neck into the urethral lumen at a constant rate (0.5 ml./10 minutes), and infusion pressure signals were measured. Bilateral HGNs were electrically stimulated at 5 and 10 Hz for 30 s. RESULTS: Electrical stimulation of the HGN reduced urethral infusion pressure in about 80% of the female rats, and the introduction of N(omega)-nitro L-arginine methylester (L-NAME, 30 mg./rat, i.v.) elevated the urethral pressure response from a reduced state. Prazosin (0.1 mg./kg., i.v.) and hexamethonium (10 mg./kg., i.v.), which inhibited elevation of urethral pressure in male rats, also reversed and inhibited the elevation of urethral pressure in the female rats treated with L-NAME. CONCLUSION: The HGN in female rats contained nerve endings that released nitric oxide (NO) and norepinephrine (NE). NO released during HGN stimulation inhibited the release of (NE) and reduced urethral infusion pressure in female rats. Nerves with synapses in the pelvic ganglia released NE in both male and female rats, but nerves that released NO did not have synapses in the ganglia. Only NE was released from the HGN nerve endings in male rats. PMID- 10737546 TI - Mannitol enhances intracellular calcium diffusion in the rat ileum--a hypothesis. AB - The addition of 92 or 136 mM mannitol to a modified saline solution that contained 1.25 mM Ca2+ led to a mannitol concentration-dependent increase in the amount of calcium absorbed in 1 h from 8 cm long ileal loops prepared from fasted male Sprague-Dawley rats, with body weights of 190 +/- 10 g. It is argued that this mannitol-enhanced movement of calcium out of the loop cannot have utilized the paracellular pathway, inasmuch as the luminal calcium concentration of the mannitol instillate decreased during the experiment, with a negative calcium gradient between luminal and body fluids. Instead it is proposed that uncomplexed mannitol and the uncharged calcium complex of mannitol entered the ileal cells. The uncomplexed intracellular mannitol would bind additional calcium that had crossed the brush border down its gradient. The increase in total intracellular calcium will raise the effective intracellular gradient and thereby amplify intracellular calcium diffusion. This in turn increases calcium absorption. PMID- 10737547 TI - Regulation of inducible nitric oxide synthase by dietary phytoestrogen in MCF-7 human mammary cancer cells. AB - We examined the effects of the phytoestrogen biochanin A on the growth of the MCF 7 human breast cancer cell line. The results showed that biochanin A treatment induced dose- and time-dependent inhibition on MCF-7 cell growth at concentrations above 20 microg x mL(-1). An examination of treated MCF-7 cell morphology revealed condensation of the chromosome and dehydration of the cytoplasm, suggesting apoptosis as an important factor in biochanin A-related cell growth inhibition. The results also showed that at a concentration of 40 microg x mL(-1), biochanin A decreased the levels of inducible nitric oxide synthase, thus inhibiting the production of nitric oxide, a known second messenger and inducer of apoptosis, and affecting the overall cell protein pattern. No significant difference in superoxide dismutase protein levels were, however detected at concentrations of 40 or 100 microg x mL(-1) of biochanin A. The data suggest that the inhibitory effects of biochanin A on human breast cancer cell growth are linked to inducible nitric oxide synthase and the associated production of nitric oxide. PMID- 10737548 TI - Immediate postnatal sucking in the rabbit: its influence on pup survival and growth. AB - This study was aimed at characterising the impact of immediate postnatal sucking on pup survival and development. The interactive effects of postnatal success with the day 0 weight of pups, the nest-access regimen (controlled or free) or parity of females was investigated. Pups (n = 900) were categorised according to their initial ingestion of colostrum. In primiparous does: (1) pup mortality between d0-d10 was higher for unsuccessful than for successful early suckers; (2) lighter d0-weight reduced survival for unsuccessful but not for successful pups; (3) free nest-access of females annihilated the survival advantage fostered by the initial sucking success. In secondiparous does, these impacts waned. Finally, whatever the does' parity, only d0-weight influenced pup weight-gain between d0 21. Thus, pup survival seemed to depend (at least in primiparae) on their ability to suck right after birth, and to display a pattern of energy saving without being disturbed by the females' nest entries. PMID- 10737549 TI - Non-starch polysaccharides extracted from seaweed can modulate intestinal absorption of glucose and insulin response in the pig. AB - We have investigated the possible effects of algal polysaccharides on postprandial blood glucose and insulin responses in an animal model, the pig. Three seaweed fibres of different viscosities, extracted from Palmaria palmata (PP), Eucheuma cottonii (EC), or Laminaria digitata (LD), were compared to purified cellulose (CEL). Blood glucose and plasma insulin levels were monitored and intestinal absorption quantified for 8 h following a high carbohydrate test meal supplemented with 5% fibre. Digestive contents were also sampled, 5 h postprandial. As compared to CEL, PP had no effect on glucose and insulin responses. The latter decreased with EC, but glucose absorption balance was not modified. LD addition resulted in a dramatically reduced glucose absorption balance, accompanied by a higher amount of starch left in the small intestine. Among polysaccharides tested, only the highly viscous alginates could affect intestinal absorption of glucose and insulin response. PMID- 10737550 TI - Availability of calcium from skim milk, calcium sulfate and calcium carbonate for bone mineralization in pigs. AB - Dairy products provide abundant, accessible calcium for humans, while some calcium sulfate-rich mineral waters could provide appreciable amounts of calcium. But there is little evidence that this calcium is as available as milk calcium for making bone. The availability of calcium was studied by monitoring bone parameters in 2-month-old pigs fed restricted amounts of calcium (70% RDA) for 2.5 months. The 3 main (> or = 50% Ca intake) Ca sources were either CaCO3 or CaSO4 or skim milk powder (29% of the diet). The bones of the pigs fed the "milk" diet had higher (P < 0.01) ash contents, breaking strength and density (DEXA) than those of the two others groups, in which the bone values were similar. Thus, the calcium provided by a diet containing milk appears to ensure better bone mineralization than do calcium salts included in a non-milk diet. The calcium restriction may have enhanced some milk properties to stimulate calcium absorption in these young, rapidly growing pigs. PMID- 10737551 TI - The physiological role of beta-endorphin in porcine ovarian follicles. AB - Beta-endorphin-like immunoreactivity (beta-END-LI) was measured by radioimmunoassay in porcine ovarian follicular fluid (FF) from small, medium and large follicles throughout the oestrous cycle. The concentration of beta-END-LI in FF from small follicles collected on days 1-5 of the cycle was at least tenfold higher than in the fluid from any other follicles independently from their size and the period of the cycle. The level of beta-END-LI in small follicles on days 6-10 was drastically decreased. Subsequently, on days 11-16 its concentration was enhanced and reduced again in pre-ovulatory period of the cycle. Concentrations of beta-END-LI in FF from medium follicles were relatively equal throughout the cycle (days 6-21). No significant differences in beta-END-LI levels were found between small, medium and large follicles from days 17-21. However, beta-END-LI concentrations in medium follicles on days 11-13 and 14-16 were statistically lower than those in small follicles. Moreover, the effects of FSH, prolactin (PRL), progesterone (P4), testosterone (T) and 17 beta-oestradiol (E2) on beta-END-LI release by granulosa cells (GCs) from large follicles and, on the other hand, the effects of the opioid agonist FK 33-824 alone or in combination with FSH, PRL or naloxone (NAL) on follicular steroidogenesis were studied. FSH drastically increased beta-END-LI output in a dose-dependent fashion. This stimulatory effect of the gonadotrophin was inhibited by the highest dose of P4 (10(-5) M). The effect of PRL and the steroids added to the cultures on beta-END-LI release was negligible. FSH- or PRL-induced P4 secretion by GCs was essentially abolished by both FK 33-824 and NAL. However, androstenedione (A4) and testosterone output by the cells was greatly potentiated by FK 33-824. In the presence of NAL, FSH or PRL, A4 release stimulated by FK 33 824 was suppressed to the basal level. Secretion of E2 was completely free from the influence of FK 33-824 or NAL; only oestrone (E1) output was modulated by them in cultures where FSH or PRL was present. In conclusion, FSH appears to be the key regulator of beta-END-LI secretion by porcine granulosa cells. Moreover, steroidogenesis in pig granulosa cells is modulated by opioid peptides acting both alone and by way of interaction with FSH or PRL. PMID- 10737552 TI - Embryo survival, uterine fluids and tubal SEM in progesterone-asynchronized rabbits. AB - Survival of embryos exposed to several concentrations of uterine proteins and changes in tubal morphology in rabbits given low preovulatory doses of progesterone (P4) that had previously not affected ovulation or fertilization, but caused severe embryo mortality, were studied. In experiment 1, 332 morulae were cultured for 24 h in a control medium containing < 0.5 to > 3.0 mg x mL(-1) of Day 3 uterine fluid proteins. There was no difference in blastocyst development nor implantation to Day 12 following transfer of the blastocysts to recipients, except fewer implants developed in the BSA control. In experiment 2 the oviducts and uteri of control and P4-treated does were examined by SEM for 8 days following ovulation. Secretory cells in the oviducts and to a lesser extent in the uteri were stimulated by P4 treatment for 3 to 4 days after ovulation. Morphology of ciliated cells was unaffected. The subtle changes did not fully account for P4-induced embryo mortality in vivo. PMID- 10737553 TI - Subarachnoid hemorrhage and myocardial damage clinical and experimental studies. AB - Subarachnoid hemorrhage (SAH) due to aneurysmal rupture is frequently complicated by cardiopulmonary episodes, including sudden death. We investigated the pathogenesis of cardiopulmonary complications from clinical observation of 715 cases with SAH. There was transient left ventricular asynergy in 9.4% (67/715) of the cases, which consisted of mechanical heart failure and myocardial necrosis. Plasma catecholamine concentration was higher in these patients compared with those without left ventricular asynergy. Transient left ventricular asynergy was considered to result from myocardial derangement: "a panic myocardium," due to a sudden burst of catecholamine. Concerning arrhythmia in SAH, cases with life threatening arrhythmia, such as ventricular tachycardia or ventricular fibrillation, had higher concentrations not only of plasma catecholamine but also of serum CK-MB, myosin light chain and troponin T, compared with patients who had no ventricular arrhythmia. This implies that life-threatening arrhythmia in SAH would result from myocardial damage due to catecholamine. We devised a novel animal model of SAH in order to clarify the relation between sympathetic nervous activity and myocardial damage immediately after the onset of SAH. The animal experiments showed that sympathetic nervous activity as well as cardiac contractility were transiently elevated, but cardiac function subsequently declined. Serum CK-MB was increased from the onset of SAH and a high value was maintained throughout the entire experimental period. In conclusion, extraordinary transient enhancement of sympathetic nervous activity induces myocardial damage resulting from what is characterized by "a panic myocardium." PMID- 10737554 TI - Relation of plasma lipoprotein(a) with myocardial viability and left ventricular performance in survivors of myocardial infarction. AB - Previous studies have reported that high serum lipoprotein(a) levels may be responsible for total occlusion of the infarct-related artery via inhibition of intrinsic fibrinolysis during acute myocardial infarction. We evaluated whether this would result in a greater extent of myocardial necrosis and impaired left ventricular function in patients with high lipoprotein(a) levels. Sixty-eight patients with prior myocardial infarction, who were not receiving thrombolytic therapy underwent coronary angiography and stress-redistribution-reinjection Tl 201 scintigraphy. Antegrade TIMI flow in the infarct-related artery was lower (1.54 +/- 1.14 vs 2.15 +/- 1.05; p = 0.03) and the collateral index was higher (1.3 +/- 1.0 vs 0.8 +/- 0.9; p = 0.07) in patients with high lipoprotein(a) levels (> 30 mg/dl) compared to those with low lipoprotein(a) levels (< or = 30 mg/dl). Regional wall motion score index was lower (0.8 +/- 0.8 vs 1.4 +/- 0.5; p = 0.008) and global ejection fraction was higher (46 +/- 10% vs 40 +/- 11%; p = 0.03) in patients with low lipoprotein(a) levels. On SPECT images, the number of non-viable defects was higher in patients with high lipoprotein(a) levels (4.0 +/ 2.5 vs 1.9 +/- 1.3; p = 0.0002), whereas the number of viable defects was higher in those with low lipoprotein(a) levels (2.5 +/- 1.8 vs 1.5 +/- 1.3; p = 0.02). We conclude that high lipoprotein(a) levels may prolong the occlusion of infarct related artery during acute myocardial infarction and lead to a greater extent of myocardial necrosis and impaired left ventricular function. PMID- 10737556 TI - Plasma homocysteine levels in acute coronary syndromes. AB - Hyperhomocysteinemia is currently regarded as an independent and modifiable risk factor for ischemic vascular diseases and thrombosis. We measured fasting plasma total homocysteine levels by HPLC with fluorescence detection in 30 patients presenting with acute coronary syndromes and 30 age and sex-matched control subjects. Demographic data, classical risk factors (systolic blood pressure, diabetes mellitus, smoking, ethanol intake, family history of ischaemic heart disease) and life-style habits were recorded. Lipid fractions including total cholesterol, triglycerides, HDL-cholesterol, total cholesterol/HDL-cholesterol ratio, serum creatinine, LDL-cholesterol and vitamins involved in the metabolism of homocysteine, folic acid and vitamin B12 were also assessed. Total fasting homocysteine concentrations were significantly higher in the patient group (12.2 +/- 1.01 micromol/l) than in the control subjects (7.05 +/- 0.36 micromol/l; p < 0.0001). Homocysteine correlated positively with age (r = 0.617; p < 0.01) and serum creatinine (r = 0.457; p < 0.01) in the patient group. Hyperhomocysteinemia was not associated with vitamin B12 or folate deficiency states. Vitamin B12 concentration was 273 +/- 16.4 ng/l in the control group and 284.3 +/- 32.2 ng/l in the patient group (p = NS). Serum folate concentration also was not significantly different between controls and patients; 7.57 +/- 0.58 microg/l and 8.05 +/- 0.72 microg/l, respectively. Since no significant difference was observed in the lipid parameters between patients and controls, the hyperhomocysteinemia in the patient group supports the view that homocysteine is an independent risk factor for cardiovascular diseases. Our results strongly suggest that elevated homocysteine levels are among the interacting factors in the complex, multifactorial pathophysiology of ischemic heart disease. PMID- 10737555 TI - Comparison of exercise stress testing with dobutamine stress echocardiography and radionuclide ventriculography for diagnosis of coronary artery disease. AB - Dobutamine stress echocardiography, Tc-99m radionuclide ventriculography (RNVG), and exercise stress testing were performed prospectively in 63 patients with suspected coronary artery disease to compare the values of exercise testing, dobutamine stress echocardiography and RNVG in the non-invasive diagnosis of coronary artery disease. The sensitivities of dobutamine stress echocardiography and RNVG were found to be higher than that of exercise testing (93-62%, p < 0.001; 83-62%, p < 0.05). The sensitivities of dobutamine stress echocardiography and RNVG were similar (p > 0.05). There were no differences between the sensitivities of the three techniques in multiple vessel disease (p > 0.05). The specificities of dobutamine stress echocardiography and RNVG were higher than that of exercise testing (for both of the tests 86-62%, p < 0.05). The diagnostic accuracy of dobutamine stress echocardiography and RNVG were similar (p > 0.05). The results of dobutamine stress echocardiography RNVG were concordant with each other in 46 patients (76%, kappa = 65%) in sectional analysis. Dobutamine stress echocardiography and RNVG tests were comparable with each other in 85% of the 189 segments (kappa = 64%). The expected 5% decrease at peak doses of dobutamine was not detected in stress echocardiography in 25 patients and in RNVG in 26 of the patients. Dobutamine stress echocardiography and RNVG are superior to exercise testing in the diagnosis of single vessel disease and there is no significant difference between the two techniques. When the ejection fraction is considered in dobutamine stress echocardiography and RNVG, it does not make an additional contribution to the diagnosis of coronary artery disease. PMID- 10737557 TI - Concerns about sources of electromagnetic interference in patients with pacemakers. AB - Electromagnetic noise is rapidly increasing in our environment so electromagnetic interference (EMI) with pacemakers (PM) may become a more important problem despite technological improvements in PM. The aim of this study was to evaluate the kinds of EMI which affect the quality of life of PM patients. The participants (1,942 Japanese Association for Pacemaker Patients: Pacemaker Tomonokai) were asked to respond to a questionnaire about their major EMI troubles, and 1,567 patients (80.7%) responded by mail. The main concerns were from mobile telephones (MT) (39%), magnetic resonance imaging (MRI) (17%), electronic kitchen appliances, automobile engines and high voltage power lines. If possible, PM implantation sites should be carefully selected not only according to the physician's convenience but also considering information on each patient's habits and physical limitations. PMID- 10737558 TI - Clinical implication of left precordial T wave inversions in the presence of complete right bundle branch block. AB - This study was designed to elucidate whether left precordial negative T waves are electrocardiographic indicators for the diagnosis of hypertrophic cardiomyopathy (HCM) even in the presence of complete right bundle branch block (CRBBB). In 7 consecutive patients with CRBBB accompanied by negative T waves in at least one of the left precordial leads (V4, V5, V6, maximal negativity; 1.06 +/- 0.40 mVol) (left precordial negative T wave group) and in 15 randomly selected CRBBB patients without left precordial T wave inversions (control group), echocardiography was performed to rule out underlying diseases causing left ventricular overload and to identify candidates for magnetic resonance (MR) imaging. None had anginal pain indicating ischemic heart disease. When 2 dimensional echocardiography indicated left ventricular hypertrophy with wall thickness > or = 15 mm, the magnitude and distribution of hypertrophy were scrutinized on contiguous left ventricular MR short-axis images. The diagnostic criterion of HCM was the demonstration of hypertrophy with a wall thickness of 20 mm or more on the left ventricular MR short-axis images. All patients in the left precordial negative T wave group had negative T waves in both I (negativity; 0.27 +/- 0.17 mVol) and aVL (negativity; 0.23 +/- 0.14 mVol), whereas none in the control group did. The diagnostic criterion for HCM was fulfilled in six patients in the left precordial negative T wave group. However there were no patients who fulfilled the criterion in the control group. Negative T waves were recorded in the I (negativity; 0.30 +/- 0.17 mVol), aVL (negativity; 0.25 +/- 0.14 mVol), V4 (negativity; 1.03 +/- 0.46 mVol), V5 (negativity; 0.83 +/- 0.37 mVol) and V6 leads (negativity; 0.31 +/- 0.31 mVol) in all patients with HCM, while they were recorded in only 6% of the patients without HCM. In conclusion, the existence of left precordial negative T waves in the presence of CRBBB strongly indicates HCM. PMID- 10737559 TI - Regional left ventricular motion during early filling phase in patients with right ventricular pressure overload. AB - Global left ventricular (LV) diastolic function has been reported to be disturbed under conditions of right ventricular pressure overload (RVPO). However, from the standpoint of regional wall motion, only a little information related to the mechanism of LV diastolic dysfunction is available. Eight patients with RVPO and 7 healthy volunteers were investigated using tagged cine magnetic resonance imaging. Regional diastolic fraction (RDF) was determined in 4 segments (anterior, lateral, inferior, and septal) in the mid-ventricular short axis section and in 2 segments (septal and lateral) in the 4-chamber section. A heterogeneity index was obtained from the RDFs of the short axis section. In the RVPO group, in both short axis and 4-chamber sections, the RDF of the septal segment was depressed, and it showed an inverse correlation with the right-to left ventricular systolic pressure (RV/LV) ratio (r = -0.74, p < 0.05) in the short axis section. In the 4-chamber section, the RDF was lower in the septal segment than in the lateral segment (p < 0.05). The heterogeneity index in the RVPO group was greater than that in the control group (p < 0.01). The index correlated positively with the RV/LV ratio (r = 0.77, p < 0.05). The altered regional diastolic motion results in increased heterogeneity in regional diastolic motion. PMID- 10737560 TI - Immunoglobulin in atherosclerotic lesions of human aorta. AB - An augmented expression of mRNA for IgG light chain was demonstrated age dependently on atheromatous lesions of the aorta in Watanabe heritable hyperlipidemic (WHHL) rabbits. The present study was designed to determine factors related to inflammation in human vessels excised during surgery. We detected IgG mRNAs using RT-PCR in human atherosclerotic lesions but not in human umbilical arteries which have no atheromatous lesions. To determine the clonality of IgGs, cDNAs encoding variable regions of IgG heavy chain were examined using RT-PCR. Atherosclerotic lesions had several subtypes of IgG gene families' suggesting the involvement of polyclonal B-cells. mRNAs of interleukins-6 (IL-6), -1alpha (IL-1alpha), and -1beta (IL-1beta) were also detected in the same samples. In summary, inflammatory reactions were present in the atherosclerosis lesion. PMID- 10737561 TI - Aortic atherosclerosis is a marker for significant coronary artery disease. AB - Atherosclerosis is a generalized process that may involve the entire vasculature as well as the coronary arteries. Aortic atherosclerosis (AA) is associated with an increased risk for recurrent ischemic stroke and cardiovascular death and can be diagnosed by transesophageal echocardiography (TEE). We performed TEE in 60 patients (47 men and 13 women; age range 37-78, mean 53.5 +/- 9.9) who underwent coronary angiography, to assess whether atherosclerosis in the thoracic aorta correlates with coronary artery disease (CAD) or may be a marker for it. Significant CAD was defined as either > 50% reduction of internal diameter of the left main coronary artery or > 70% reduction of the internal diameter in the anterior descending, right coronary or circumflex artery. The number of diseased vessels was based on the Coronary Artery Surgery Study criteria. A grading system was used to detect AA. The thoracic aorta was considered to be normal and classified as grade I when the internal surface was smooth and without lumen irregularities or increased echo-intensity. Grade II changes consisted of increased echodensity of the intima without lumen irregularity or thickening. Grade III changes consisted of increased echodensity of intima with well defined atheroma extending < 3 mm in the aorta. Grade IV and V changes consisted of atheroma > 3 mm and protruding mobile plaques, respectively. Grades III-V were considered as AA. Twenty two of the 29 patients (75.9%) with CAD and 10 of the 31 patients (32.3%) without CAD had AA detected by TEE. There was a significant relationship between CAD and AA (r = 0.44, p < 0.001). The sensitivity and specificity of AA in detecting CAD were 75.9% and 67.7%, respectively. Our data suggest that AA is common in patients with significant CAD. Detection of AA by TEE may be a marker for CAD and early detection of aortic atherosclerosis may contribute to diagnostic and therapeutic interventions and thereby improve the prognosis. PMID- 10737562 TI - Relationship between coronary blood flow velocity waveform and transmural distribution of myocardial blood flow in coronary artery. AB - It is important to know the transmural distribution of myocardial blood flow in assessing the severity of ischemia in coronary heart disease. We analyzed the relation between phasic waveform of epicardial coronary flow velocity with a Doppler flow probe in the left anterior descending artery in dogs and regional myocardial blood flow using a colored microsphere technique. Time-velocity integral in an average of 5 cardiac cycles was measured as an index of coronary blood flow during diastole (TVId) and systole (TVIs). The diastolic fraction of coronary blood flow (%DF) was defined as TVId/(TVId + TVIs). Myocardial specimens were divided into inner (subendocardial), middle, and outer (subepicardial) layers, and the inner layer to outer layer myocardial blood flow ratio (endo/epi ratio) was used as an index of transmural distribution of myocardial perfusion. The mean endo/epi ratio and the mean %DF decreased as the pressure gradient increased. There was a moderate but significant correlation (r = 0.57) between the endo/epi ratio and the %DF. In conclusion, analysis of the phasic pattern of coronary blood flow velocity provides some information about the transmural distribution of blood flow in the myocardium. The %DF may be a useful index for evaluating subendocardial ischemia. PMID- 10737563 TI - Effects of the thromboxane A2 receptor antagonist on platelet deposition and intimal hyperplasia after balloon injury. AB - Thromboxane A2 (TXA2) after vascular injury plays an important role in the process of restenosis. S-1452, a potent and selective TXA2 receptor antagonist, blocks the receptors of vascular smooth muscle cells (VSMC) as well as platelets. The purpose of this study was to determine whether S-1452 could reduce platelet deposition and intimal hyperplasia in vascular injury models. New Zealand White Rabbits (n = 41) were fed a 0.5% cholesterol diet. For the short-term study, eighteen rabbits after balloon injury of iliac artery were assigned to 3 groups; systemic administration of S-1452, single local administration of S-1452 using a local delivery balloon, and single local administration of saline solution. Platelet deposition in injured artery using 111In-labeled platelets was reduced by 50% in systemic administration and by 60% in local administration compared to saline infusion. For the long-term study, balloon injury of the iliac artery was performed 4 weeks after starting the 0.5% cholesterol diet. Twenty-three rabbits were classified into 4 groups; systemic administration of S-1452, oral placebo administration, single local administration of S-1452, and local administration of saline solution (control group). The platelet aggregation induced by U-46619 was significantly lower in the S-1452 group than in the control group. Systemic administration of S-1452 significantly reduced the intimal area (152 +/- 33 vs 735 +/- 135 microm2, p < 0.001) and number of cells in the intima (513 +/- 57 vs 993 +/- 57, p < 0.01) compared to controls. In contrast, a single local administration failed to reduce neointimal thickness. Systemic administration of S-1452 reduced intimal hyperplasia as well as platelet deposition in a rabbit injury model, but its single local administration inhibited only platelet deposition. PMID- 10737564 TI - Embryonic smooth muscle myosin heavy chain SMemb is expressed in pressure overloaded cardiac fibroblasts. AB - Left ventricular hypertrophy (LVH) is a secondary adaptation to increased external load. Various qualitative and quantitative changes in myocytes and extracellular components occur during the development of LVH. It has recently been demonstrated that alpha-smooth muscle actin (alpha-SMA)-expressing myofibroblasts appear in the interstitium of the heart subjected to increased workload suggesting that cardiac fibroblasts as well as myocytes alter their phenotype in response to pressure overload. In the present study, to explore the load-induced response and phenotypic modulation of cardiac fibroblasts, the localization of embryonic smooth muscle myosin heavy chain (SMemb) and alpha-SMA in thoracic aorta-constricted rat hearts was investigated by immunohistochemistry, and the morphology of the SMemb-expressing cells was examined by electron microscopy. In addition, to clarify the mechanisms by which SMemb is induced in pressure-overloaded hearts, mRNA expression of SMemb in aorta constricted rat hearts and in transforming growth factor-beta1 (TGF-beta1) treated or mechanically-stretched cultured cardiac fibroblasts was investigated. Enhanced staining of SMemb and alpha-SMA was detected in the interstitial spindle shaped cells in the fibrotic lesions of the pressure-overloaded left ventricles by immunohistochemistry. These cells were demonstrated by electron microscopy to have features specific for activated fibroblasts such as serrated nuclei or prominent rough endoplasmic reticulum. These cells also had characteristic features of myofibroblasts, i.e. irregularly arranged actin filaments and scattered dense bodies. Northern blot analysis revealed increased mRNA levels of SMemb both in aorta-constricted rat hearts and in cultured cardiac fibroblasts stimulated by TGF-beta1 or by mechanical stretch. These results suggest that SMemb may be a molecular marker both for the detection of activated cardiac fibroblasts that may play important roles in the remodeling of pressure overloaded cardiac interstitium, and for the identification of the regu latory mechanisms that control the phenotypic modulation of cardiac fibroblasts in response to pressure overload. PMID- 10737565 TI - A computer model of myocardial disarray in simulating ECG features of hypertrophic cardiomyopathy. AB - Hypertrophic cardiomyopathy (HCM) was simulated with a computer heart model having a realistic shape and rotating fiber orientation in order to elucidate possible mechanisms for abnormal ECG findings. The disarray of myocardial muscle in HCM was simulated by assigning random fiber direction and isotropic electrophysiologic properties to abnormal hypertrophic regions, in contrast to the anisotropic modeling for normal myocardium. With these models, main ECG features including abnormal Q wave and QS pattern were reproduced and were comparable with clinical findings. This study suggests that the change in anisotropy in the hypertrophic myocardium is likely to be the main factor responsible to the ECG features of HCM. PMID- 10737566 TI - Atrial fibrillation and continuous hypotension induced by sildenafil in an intermittent WPW syndrome patient. AB - A 55-year-old Japanese man was hospitalized for palpitations and severe chest oppression one hour after he ingested about 1500 ml of beer and sildenafil (Viagra) 50 mg. At 43 years of age, he had been diagnosed with intermittent WPW syndrome following a paroxysmal supraventricular tachycardia (PSVT) attack. He took a 1 mg tablet of doxazosin daily for mild hypertension. On admission, his blood pressure was 90/54 mmHg and his heart beat was weak and irregular with a rate of about 220/min. Since atrial fibrillation (Af) was diagnosed on an electrocardiogram (minimum RR interval; 0.22 seconds), direct current shock was performed with 100 joules and 150 joules but conversion to sinus rhythm failed. Sinus rhythm returned spontaneously from Af four hours after taking sildenafil. Since blood pressure was 50/17 mmHg despite the return to sinus rhythm, blood pressure was maintained by dopamine for twelve hours after sinus rhythm returned. The patient underwent catheter ablation for curative therapy and thereafter has not had any further episodes of tachycardia. PMID- 10737567 TI - Dynamic left ventricular outflow tract obstruction in a patient with pheochromocytoma. AB - Symmetric left ventricular hypertrophy or asymmetric septal hypertrophy associated with pheochromocytoma simulating hypertrophic obstructive cardiomyopathy have been rarely reported. In this report, we present a case with pheochromocytoma that had dynamic left ventricular outflow tract obstruction without asymmetric septal hypertrophy. A surface echo revealed resolution of the systolic anterior motion of the mitral valve and all Doppler evidence of left ventricular outflow tract obstruction following removal of the tumor. Dynamic left ventricular outflow tract obstruction seen in this patient was probably due to excessive secretion of cathecolamines by the tumor. PMID- 10737568 TI - Improved patient outcome after surgical treatment for loculated empyema. AB - BACKGROUND: Empyemas complicate the hospital course of many patients. Advanced stages of empyema often require surgical intervention. METHODS: A retrospective review of 70 adult patients with empyema, hospitalized between the years of 1992 and 1997, was performed. Data on age, length of stay, comorbidities, diagnostic studies, and treatment was obtained. We compared patient outcome from patients with loculated empyemas who had surgical treatment and those who were managed nonsurgically. RESULTS: Seventy patient records were reviewed, 37 of which were of patients with loculated empyemas. Parapneumonic empyemas comprised 60% of all cases. Chest radiographs, computed tomography scan, and thoracentesis were the most common studies performed in both groups. Thirty-three patients with the radiographic finding of a loculated empyema were treated with either surgical decortication or tube thoracostomy. CONCLUSION: Empyemas at various stages of development require different forms of therapy; advanced empyemas treated early with decortication have a shorter duration of treatment, lower incidence of recurrence and fewer complications. PMID- 10737569 TI - Nonocclusive bowel necrosis occurring in critically ill trauma patients receiving enteral nutrition manifests no reliable clinical signs for early detection. AB - BACKGROUND: Nonocclusive bowel necrosis (NOBN) has been associated with early enteral nutrition (EN). The purpose of this study was to determine the incidence of this complication in our trauma intensive care unit population and to define a typical patient profile vulnerable to NOBN. METHODS: Thirteen cases of NOBN were identified among 4,311 patients (0.3%) over a 64-month period ending October 1998. Their charts were analyzed for a variety of clinical data, including prospective EN tolerance data in 4. RESULTS: Twelve (92%) patients were enterally fed prior to diagnosis for 10 +/- 8 days (range 3 to 21). Tachycardia (n = 12, 92%); fever/hypothermia, (n = 12, 92%), and an abnormal white blood cell count (n = 11, 85%) were consistently present. Abdominal distention was common but tended to be a late sign (n = 12). Seven (56%) survived. In 4 patients with tolerance data, 3 reached the goal rate of feeds prior to diagnosis. Two became distended at >12 hours from diagnosis. Gastric tonometry demonstrated a decreased NgpHi (<7.30) after starting EN in all 3 in whom it was monitored. CONCLUSIONS: NOBN developed in 0.3% of our trauma patients. Onset occurs in the second week in high acuity patients who have had a period of EN tolerance. Clinical findings resemble bacterial sepsis with tachycardia, fever, and leukocytosis. Gastrointestinal specific signs are not consistent or occur late. Thus, we could not identify an early, useful clinical indicator. Gastric carbon dioxide tonometry may detect a vulnerable subgroup of patients. PMID- 10737570 TI - The efficacy of X-rays after chest tube removal. AB - BACKGROUND: The insertion and subsequent removal of chest tubes are frequently performed procedures. We hypothesize that routine chest radiographs obtained after chest tube removal to confirm the absence of any post-procedure complications have little impact on clinical management. MATERIALS AND METHODS: A 5-year retrospective study of 73 patients with tube thoracotomies was performed in a level II trauma center's intensive care unit. Patients were identified from billing records for chest tube placement. Medical records and official chest x ray film reports, both before and after removal, were reviewed, and demographic data were collected. RESULTS: Of the 73 patients examined, only 8 had postprocedure reports that differed from the preprocedure reports. Two of these 8 patients required reinsertion of a chest tube to treat the recurrence of a significant pneumothorax. However, the decision to reinsert the chest tube was based on the patient's clinical appearance rather than on the x-ray findings. CONCLUSION: Chest radiography following the removal of chest tubes should not be a routinely performed procedure, but should preferably be based on the good clinical judgement and discrimination of the surgeon. PMID- 10737571 TI - Objective criteria may assist in distinguishing necrotizing fasciitis from nonnecrotizing soft tissue infection. AB - BACKGROUND: Optimal treatment of necrotizing fasciitis (NF) requires rapid diagnosis. The purpose of the study was to identify objective admission measurements that help differentiate NF from nonnecrotizing (non-NF) infection and, among NF patients, to identify admission factors that predict mortality. METHODS: Twenty-one NF cases were paired with matched non-NF controls. Statistical comparison of admission vital signs, laboratory values, and radiographic studies was performed. RESULTS: On multivariate analysis, admission white blood cell count (WBC) >14 x 10(9)/L, serum sodium <135 mmol/L, and blood urea nitrogen (BUN) >15 mg/dL separated NF from non-NF patients. Mortality for NF patients was predicted by admission WBC >30 x 10(9)/L. Mortality was also significantly increased for patients transferred from an outside institution prior to definitive therapy. CONCLUSIONS: Objective admission criteria (elevated WBC and BUN and decreased serum sodium) can assist in distinguishing NF from non NF infections. The best objective predictor of mortality in NF patients is marked elevation of admission WBC. PMID- 10737572 TI - Local recurrence following breast conservation therapy in African-American women with invasive breast cancer. AB - BACKGROUND: African-American women have a lower survival rate than white women following a diagnosis of invasive breast cancer. Limited information is available regarding the impact of race on results of breast conservation therapy (BCT). METHODS: Local recurrence rates were compared in 71 African-American patients (73 breasts) and 204 white patients (208 breasts) with stage I and II breast cancer treated with BCT. RESULTS: Overall 5-year actuarial recurrence rates were 13% in African-Americans and 4% in whites (P = 0.075). These rates were 9% and 4%, respectively, if patients with local skin/soft tissue recurrences were excluded (P = 0.587). Exclusion of these skin/soft tissue failures eliminated any significant difference seen in recurrence between stage II African-American and white patients (P = 0.163). African-American women had less favorable recurrences, including tumor in more than one quadrant or local skin/ soft tissue involvement (P = 0.001). CONCLUSIONS: Overall actuarial recurrence rates were slightly higher, but not significantly different, in African-American and white women following BCT. A much less favorable pattern of local recurrence was seen in the African-American patients (P = 0.001), which may represent the presence of more biologically aggressive tumors in these women. PMID- 10737573 TI - Video-assisted evacuation of empyema is the preferred procedure for management of pleural space infections. AB - BACKGROUND: Empyema remains a cause of morbidity and mortality. Thoracoscopy has proved its versatility in the management of pleural space disorders. The suitability of video-assisted thoracic surgery (VATS) for decortication in the management of the fibrotic stage of empyema is unclear. METHODS: VATS evacuation of empyema and decortication was performed on seventeen patients presenting with pleural space infections. A retrospective review was performed and constitutes the basis of this report. RESULTS: VATS evacuation of empyema and decortication was successfully performed in 13 of 17 patients. Blood loss was 325 +/- 331 cc. Mean hospital stay was 18 +/- 10 days. Postoperative hospitalization was 11 +/- 7 days. Chest tubes remained in place for 7 +/- 3 days. There were no operative mortalities. CONCLUSIONS: Video-assisted evacuation of empyema and decortication is an effective modality in the management of the exudative and fibrinopurulent stages of empyema. An organized empyema should be approached thoracoscopically, but may require open decortication. PMID- 10737574 TI - Surgical Internet at a glance: bariatric surgery. PMID- 10737575 TI - Transthoracic ultrasonography is an alternative to subxyphoid ultrasonography for the diagnosis of hemopericardium in penetrating precordial trauma. AB - BACKGROUND: Surgeon-performed ultrasonography is increasingly becoming part of the initial evaluation of patients after blunt or penetrating trauma. Currently, most institutions obtain a subxyphoid or subcostal view of the heart and pericardial space, and a three-view ultrasonogram of the abdomen to detect blood in the pericardial sac or in three dependent abdominal areas. METHODS: A left parastemal standard transverse transthoracic view is described in addition to the aforementioned views. This facilitates the visualization of the pericardial sac when a subxyphoid or subcostal view cannot be obtained because of anatomical reasons (narrow subxyphoid space) or local factors (pain, fractures, subcutaneous emphysema, or chest wall contusion). RESULTS: The transthoracic view can be useful in patients where the subxyphoid view is difficult to obtain through the conventional approach. In most patients an excellent view of the pericardial sac and ventricles can be obtained and, therefore, expedites the diagnosis and treatment of patients with hemopericardium. CONCLUSION: Surgeon-performed ultrasonography has become the diagnostic test of choice for patients suspected of having hemopericardium and cardiac tamponade. Transthoracic ultrasonography is an excellent alternative for those patients where a subxyphoid or subcostal view to visualize the pericardial sac and heart cannot be obtained owing to local or anatomical factors. PMID- 10737576 TI - Extent of resection in the management of duodenal adenocarcinoma. AB - BACKGROUND: It has been postulated that segmental duodenal resection (SR) is not an adequate operation for patients with adenocarcinoma of the duodenum and that pancreaticoduodenectomy (PD) is the procedure of choice, regardless of the tumor site. However, data from previous studies do not clearly support this position. METHODS: We reviewed the records of 63 patients treated for duodenal adenocarcinoma from 1979 through 1998. Perioperative outcome, patient survival, and extent of lymphadenectomy were compared in patients who underwent PD and SR. RESULTS: The overall morbidity for PD and SR was 27% and 18%, respectively (not significant [NS]). Patients who underwent SR had a 5-year survival of 60% versus 30% for patients who underwent PD (NS). Lymph node status was a prognostic factor for survival (P = 0.014). The mean number of lymph nodes in the specimens was 9.9 +/- 2.1 for PD and 8.3 +/- 4.4 for SR (NS). CONCLUSIONS: Segmental duodenal resection for patients with duodenal adenocarcinoma is associated with acceptable postoperative morbidity and long-term survival. The procedure is especially well suited for distal duodenal tumors. Clearance of lymph nodes and outcome are comparable to PD. PMID- 10737577 TI - Autologous blood transfusion in patients with hepatocellular carcinoma undergoing hepatectomy. AB - BACKGROUND: Homologous blood transfusion (HBT) has the risk of an immunosuppressive effect and may adversely affect the prognosis of patients with carcinomas. Autologous blood transfusion (ABT) has not yet become a standard procedure in gastroenteric cancer surgery. We investigated the usefulness and problems of ABT combined with the use of recombinant human erythropoietin (rh EPO). METHODS: An evaluation of autologous blood transfusion (ABT) combined with recombinant human erythropoietin (rh-EPO) treatment was conducted in 46 patients with hepatocellular carcinoma undergoing hepatectomy. Preoperative autologous blood donation (ABD) was accomplished for 25 of the 46 patients. The preoperative changes in hemoglobin and hematocrit in relation to route of administration of erythropoietin were studied. In addition, intraoperative blood requirements and the postoperative complications for patients who predonated were compared with those of patients who underwent surgery without autologous predonation. RESULTS: The proportion of patients not requiring additional homologous blood transfusions (HBT) during operation was significantly higher in the ABD group than in the non ABD group (88% versus 38%). The incidence of postoperative complications was significantly higher in patients receiving HBT than in nontransfused patients and in those receiving ABT. CONCLUSIONS: Preoperative autologous blood donation in combination with rh-EPO therapy markedly reduced the requirement for homologous blood transfusion during surgery in patients with hepatocellular carcinoma having hepatectomy. PMID- 10737578 TI - Influence of botulinum toxin site of injections on healing rate in patients with chronic anal fissure. AB - BACKGROUND: Botulinum toxin induces healing in patients with idiopathic anal fissure. METHODS: Fifty patients affected by posterior anal fissure were treated with 20 units of botulinum toxin, injection in the internal anal sphincter on each side of the posterior midline (group I) or on each side of the anterior midline (group II). RESULTS: At 2 months evaluation, a healing scar was observed in 15 patients of group I and in 22 patients of group II(P = 0.025). Resting anal pressure was significantly different from the baseline values at 1-month as well as at 2-month check-ups in both groups, but the values were significantly lower in patients of group II. CONCLUSIONS: The intersite comparison revealed that anterior injection of the internal anal sphincter resulted in improved lowering of resting anal pressure and produced an earlier healing scar. PMID- 10737579 TI - Extent of surgery for intermediate-risk well-differentiated thyroid cancer. AB - BACKGROUND: Methods of assigning patients with papillary or follicular thyroid cancer (well-differentiated thyroid cancer) to risk groups for the purpose of determining appropriate therapy have been developed. Despite these efforts, the optimal extent of surgery for intermediate-risk patients remains controversial. METHODS: A retrospective study was conducted of 208 patients with well differentiated thyroid cancer (DTC) from two institutions. Univariate and multivariate analysis of patient- and tumor-related variables was performed. A regression model was obtained, three risk groups (low, intermediate, and high) were defined, and survival curves were generated. RESULTS: Prognostic variables were age (P <0.001), distant metastases (P <0.001), tumor size (P <0.001) and an aggressive growth pattern (P = 0.03) by univariate analysis and age (P <0.001) and distant metastases (P <0.001) by multivariate analysis. Tumor size (P = 0.07) was included in the regression model. Total thyroidectomy appeared to provide a survival advantage for intermediate risk patients. High-risk patients treated by lobectomy had a poorer prognosis. CONCLUSIONS: Total thyroidectomy may provide a survival advantage for intermediate-risk patients with DTC. A prospective randomized trial with 200 such patients is required to confirm this finding. PMID- 10737580 TI - Major surgery increases serum levels of vascular endothelial growth factor only temporarily. AB - BACKGROUND: Vascular endothelial growth factor (VEGF) is a hypoxia-induced endothelial cell-specific mitogen, which is angiogenic in vivo and up-regulated in several malignancies. VEGF can be used as a prognostic marker, but the effect of surgical trauma on serum VEGF (S-VEGF) concentrations is unknown and might reduce the value of VEGF as a serum marker. METHODS: We monitored S-VEGF levels by enzyme-linked immunosorbent assay in patients undergoing surgery. RESULTS: Eighteen patients with major surgery had slightly elevated S-VEGF compared with the preoperative level (median 9.5 pg/mL) on the first (median 35 pg/mL; P = 0.0002) and third (median 19 pg/mL; P = 0.004) postoperative day, but not in later samples. The levels measured in 8 patients after minor surgery did not differ from the preoperative levels (P = 0.14). CONCLUSIONS: Even major surgery is associated only with a slight and transient increase in S-VEGF levels, and, therefore, is unlikely to interfere markedly with the use of VEGF as a prognostic marker. PMID- 10737581 TI - Ondansetron versus placebo for prophylaxis of nausea and vomiting in patients undergoing ambulatory laparoscopic cholecystectomy. AB - BACKGROUND: Postoperative nausea and vomiting is a common problem in patients undergoing laparoscopic cholecystectomy (LC). The purpose of this study was to evaluate the efficacy of ondansetron given at the induction of anesthesia in patients scheduled for ambulatory LC. METHODS: A total of 84 patients undergoing ambulatory LC were enrolled in a randomized, prospective, double-blinded study in which the subjects received either placebo or 4 mg ondansetron intravenously at induction of anesthesia. A nausea scoring system was employed utilizing a 5-point linear scale, with 1 point given for no nausea and a maximum of 5 points for an episode of emesis. Each patient received a total of four scores postoperatively. RESULTS: The patients receiving placebo had significantly more episodes of nausea (53 versus 32; P <0.009) and emesis (11 versus 2; P <0.02), higher mean total nausea scores (7.2 versus 5.4; P <0.006), and need for additional postoperative antiemetics (23 versus 14; P <0.05) than those receiving ondansetron. CONCLUSIONS: In patients undergoing ambulatory laparoscopic cholecystectomy, ondansetron at induction was highly effective in decreasing postoperative nausea and vomiting and should become the standard. PMID- 10737582 TI - The road to ambulatory laparoscopic management of perforated appendicitis. AB - BACKGROUND: This study examines the contribution of increased laparoscopic skills to ambulatory management of perforated appendicitis. METHODS: All 38 perforated appendicitides from 151 laparoscopic appendectomies done by one surgeon were studied and ambulatory management (discharge less than 24 hours after surgery) of the last 18 perforations reported. RESULTS: Over 4 years, rate of attempted laparoscopic appendectomy rose from 67% to 100% for perforations. Conversion rate fell from 100% to 22%. Ileus and pain control were not problems for most laparoscopic perforations, so by the end of 1997, experience suggested these patients might be discharged within 24 hours. Ambulatory rate was 57% (conversions excluded). There were no readmissions for wound infections or postoperative abdominal abscesses. CONCLUSIONS: Increasing laparoscopic skills allows laparoscopic treatment of complicated appendicitis with a low conversion rate and no infectious complications. Over one half of these patients can be managed as outpatients without jeopardy to outcome. PMID- 10737583 TI - Complications and recommended practices for electrosurgery in laparoscopy. AB - BACKGROUND: Electrosurgery is one of the most commonly used energy systems in laparoscopic surgery. Two major categories of potential complications related to electrosurgery in laparoscopy are mechanical trauma and electrothermal injury. The latter can result from unrecognized energy transfer in the operational field or, less commonly, to unnoticed stray current outside the laparoscopic field of view. Stray current can result from insulation failure, direct coupling, or capacitive coupling. METHODS: We reviewed the literature concerning essential biophysics of electrosurgery, including electrosurgical waveform differentiation, tissue effect, and variables that determine tissue effect. The incidence of electrosurgical injuries and possible mechanisms responsible for the injuries are discussed. Different types of injuries may result in different clinical manifestations and histopathological findings. Gross and microscopic pathological check-ups of the injury sites may distinguish between different mechanisms, and thus provide further clues postoperatively. RESULTS: Several recommended practices are proposed to avoid electrosurgical injury laparoscopically. To achieve electrosurgical safety and to prevent electrosurgical injuries, the surgical team should have a good understanding of the biophysics of electrosurgery, the basis of equipment and general tissue effects, as well as the surgeon's spatial orientation and hand-eye coordination. Some intraoperative adjuvant procedures and newly developed safety devices have become available may aid to improve electrosurgical safety. CONCLUSIONS: Knowledge of the biophysics of electrosurgery and the mechanisms of electrosurgical injury is important in recognizing potential complications of electrosurgery in laparoscopy. Procedures for prevention, intraoperative adjuvant maneuvers, early recognition of the injury with in-time salvage treatment, and alertness to postoperative warning signs can help reduce such complications. PMID- 10737584 TI - Using the World-Wide-Web to obtain feedback on the quality of surgical residency training. AB - BACKGROUND: Resident feedback can lead to an improvement in teaching performance. Compliance with our previous rotation evaluation system has been poor. METHODS: Rotation evaluation forms were integrated to the departmental World-Wide-Web site. Anonymously completed evaluations are automatically sent to an appointed surgery professor via electronic mail (e-mail). They are then collated and transmitted to program directors. RESULTS: Ninety-two percent (24 of 26) of our surgical residents have an e-mail account. During the 2-month trial, 18 residents completed 48 evaluations. This represents a 75% (18 of 24) compliance rate among residents with e-mail capabilities. The response rate was 82% (9 of 11) for core residents, 64% (7 of 11) for third- and fourth-year residents, and 50% (2 of 4) for chiefs. A second 2-month trial yielded a 75% (18 of 24) response rate and 43 evaluations. CONCLUSIONS: The World-Wide-Web is a user-friendly medium suitable to obtain valuable feedback on the quality of residency training. Compliance was improved using this new system. PMID- 10737585 TI - Inflammatory response to open tension-free inguinal hernioplasty versus conventional repair. PMID- 10737586 TI - Late infection after mesh-plug inguinal hernioplasty. PMID- 10737587 TI - Reconstruction of the hepatic and portal veins using a patch from the right ovarian vein. PMID- 10737588 TI - Expression of 15 glutamate receptor subunits and various splice variants in tissue slices and single neurons of brainstem nuclei and potential functional implications. AB - Brainstem nuclei serve a diverse array of functions in many of which ionotropic glutamate receptors are known to be involved. However, little detailed information is available on the expression of different glutamate receptor subunits in specific nuclei. We used RT-PCR in mice to analyze the glutamate receptor subunit composition of the pre-Botzinger complex, the hypoglossal nucleus, the nucleus of the solitary tract, and the inferior olive. Analyzing 15 receptor subunits and five variants, we found all four alpha-amino-3-hydroxy-5 methyl-4-propionic acid (AMPA) and six NMDA receptor (NR) subunits as well as three of five kainate (KA) receptors (GluR5, GluR6, and KA1) to be expressed in all nuclei. However, some distinct differences were observed: The inferior olive preferentially expresses flop variants of AMPA receptors, GluR7 is more abundant in the pre-Botzinger complex than in the other nuclei, and NR2C is most prominent in the nucleus of the solitary tract. In single hypoglossal motoneurons and interneurons of the pre-Botzinger complex investigation of GluR2 editing revealed strong expression of the GluR2-R editing variant, suggesting low Ca2+ permeability of AMPA receptors. Thus, Ca2+-permeable AMPA receptors are unlikely to be the cause for the reported selective vulnerability of hypoglossal motoneurons during excitotoxic events. PMID- 10737589 TI - Regulation of alpha3 nicotinic acetylcholine receptor subunit mRNA levels by nerve growth factor and cyclic AMP in PC12 cells. AB - To investigate the effects of nerve growth factor (NGF) and cyclic AMP (cAMP) on the level of the nicotinic acetylcholine receptor subunit alpha3 mRNA, we used PC12h cells, PC12 cells expressing dominant-negative Ras protein, and the parental PC12 cells. PC12h cells have NGF-responsive tyrosine hydroxylase activity. Expression of dominant-negative Ras protein prevents the signaling through the Ras-mitogen-activated protein kinase cascade. The morphological changes of the parental PC12 cells in response to NGF and 8-(4 chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPTcAMP), a cell penetrating cAMP analogue, were similar to those of PC12h cells. NGF up-regulated the alpha3 mRNA level in PC12h cells and down-regulated the alpha3 mRNA level in the parental PC12 cells. Expression of dominant-negative Ras protein and an inhibitor of mitogen-activated protein kinase kinase inhibited the effects of NGF on alpha3 mRNA level. CPTcAMP down-regulated the alpha3 mRNA level in all three PC12 cell lines. An inhibitor of protein kinase A inhibited the CPTcAMP-induced down-regulation of alpha3 mRNA. The alpha3 mRNA down-regulation required prolonged treatment with CPTcAMP even after cAMP response element binding protein phosphorylation was decreased. Membrane depolarization with high K+ had no effect on the alpha3 mRNA level in PC12h cells. Based on these results, we propose that at least two unknown effectors regulate alpha3 mRNA levels in PC12 cells. PMID- 10737590 TI - Regulation of mouse delta-opioid receptor gene transcription: involvement of the transcription factors AP-1 and AP-2. AB - The aim of this study was to examine the effects of the phorbol ester O tetradecanoylphorbol 13-acetate (TPA) and forskolin on delta-opioid receptor gene transcription. Treatment of NG108-15 cells with TPA (100 nM) for 48 h increased delta-opioid receptor mRNA levels, whereas different concentrations of forskolin induced a transient down-regulation of mRNA 5 h after treatment, followed by increased mRNA levels after 48 h. Reporter gene assays in transiently transfected NG108-15 cells in combination with electrophoretic mobility shift assays indicate that the increase of delta-opioid receptor mRNA after stimulation with TPA is mediated by transcription factor AP-1, which binds 355 bp upstream of the start codon within the gene promoter. The forskolin-induced mRNA increase is mediated neither by a cyclic AMP-response element nor indirectly by AP-1 up-regulation. Reporter gene assays, mutational analysis, and electrophoretic mobility shift assays revealed that delta-opioid receptor gene regulation by forskolin is mediated by transcription factor AP-2, which binds to an element 157 bp upstream of the start codon. PMID- 10737591 TI - Interaction between the serotoninergic and dopaminergic systems in d-fenfluramine induced activation of c-fos and jun B genes in rat striatal neurons. AB - To test for the relative contributions of the dopaminergic and serotoninergic systems in the striatum to the effects of d-fenfluramine, an indirect serotonin receptor agonist, we assessed the expression of Fos/Jun proteins induced by d fenfluramine given alone or in the presence of dopaminergic or serotoninergic agents. To determine the neuronal targets of d-fenfluramine in the striatum, we identified the phenotypes of striatal neurons in which d-fenfluramine induced Fos expression. Our results demonstrated that d-fenfluramine evokes nuclear expression of Fos/Jun B proteins in the striatum, and that the Fos expression was dose-dependent and accompanied by transient induction of c-fos mRNA. Fos expression was blocked by p-chloroamphetamine, a serotoninergic neurotoxin. Pretreatment with SCH 23390, a D1-dopamine receptor antagonist, led to a marked decrease in Fos/Jun B expression in the caudoputamen, but not in the cortex, whereas pretreatment with methiothepin, a nonselective serotonin 5-HT1 receptor antagonist, blocked Fos expression completely in the cortex and only partially in the caudoputamen. The expression of Fos/Jun B in the striatum occurred mainly in dynorphin-containing neurons and in a subpopulation of striatal interneurons that exhibited NADPH-diaphorase activity. Most of the enkephalin-containing neurons of the striatum did not show Fos/Jun B staining. These results suggest that the mechanism by which d-fenfluramine induces c-fos and jun B expression in the rat caudoputamen depends at least in part on activation of the dopaminergic system by serotonin. PMID- 10737592 TI - Uptake of lipoprotein-associated alpha-tocopherol by primary porcine brain capillary endothelial cells. AB - From the severe neurological syndromes resulting from vitamin E deficiency, it is evident that an adequate supply of the brain with alpha-tocopherol (alphaTocH), the biologically most active member of the vitamin E family, is of utmost importance. However, uptake mechanisms of alphaTocH in cells constituting the blood-brain barrier are obscure. Therefore, we studied the interaction of low (LDL) and high (HDL) density lipoproteins (the major carriers of alphaTocH in the circulation) with monolayers of primary porcine brain capillary endothelial cells (pBCECs) and compared the ability of these two lipoprotein classes to transfer lipoprotein-associated alphaTocH to pBCECs. With regard to potential binding proteins, we could identify the presence of the LDL receptor and a putative HDL3 binding protein with an apparent molecular mass of 100 kDa. At 4 degrees C, pBCECs bound LDL with high affinity (K(D) = 6 nM) and apolipoprotein E-free HDL3 with low affinity (98 nM). The binding capacity was 20,000 (LDL) and 200,000 (HDL3) lipoprotein particles per cell. alphaTocH uptake was approximately threefold higher from HDL3 than from LDL when [14C]alphaTocH-labeled lipoprotein preparations were used. The majority of HDL3-associated alphaTocH was taken up in a lipoprotein particle-independent manner, exceeding HDL3 holoparticle uptake 8- to 20-fold. This uptake route is less important for LDL-associated alphaTocH (alphaTocH uptake approximately 1.5-fold higher than holoparticle uptake). In line with tracer experiments, mass transfer studies with unlabeled lipoproteins revealed that alphaTocH uptake from HDL3 was almost fivefold more efficient than from LDL. Biodiscrimination studies indicated that uptake efficacy for the eight different stereoisomers of synthetic alphaTocH is nearly identical. Our findings indicate that HDL could play a major role in supplying the central nervous system with alphaTocH in vivo. PMID- 10737593 TI - Interaction among mitochondria, mitogen-activated protein kinases, and nuclear factor-kappaB in cellular models of Parkinson's disease. AB - Oxidative stress induced by acute complex I inhibition with 1-methyl-4 phenylpyridinium ion activated biphasically the stress-activated c-Jun N-terminal kinase (JNK) and the early transcription factor nuclear factor-kappaB (NF-kappaB) in SH-SY5Y neuroblastoma cells. Early JNK activation was dependent on mitochondrial adenine nucleotide translocator (ANT) activity, whereas late-phase JNK activation and the cleavage of signaling proteins Raf-1 and mitogen-activated protein kinase (MAPK) kinase (MEK) kinase (MEKK)-1 appeared to be ANT independent. Early NF-kappaB activation depended on MEK, later activation required an intact electron transport chain (ETC), and Parkinson's disease (PD) cybrid (mitochondrial transgenic cytoplasmic hybrid) cells had increased basal NF kappaB activation. Mitochondria appear capable of signaling ETC impairment through MAPK modules and inducing protective NF-kappaB responses, which are increased by PD mitochondrial genes amplified in cybrid cells. Irreversible commitment to apoptosis in this cell model may derive from loss of Raf-1 and cleavage/activation of MEKK-1, processes reported in other models to be caspase mediated. Therapeutic strategies that reduce mitochondrial activation of proapoptotic MAPK modules, i.e., JNK, and enhance survival pathways, i.e., NF kappaB, may offer neuroprotection in this debilitating disease. PMID- 10737594 TI - p27Kip1 expression by contact inhibition as a prognostic index of human glioma. AB - The clinical manifestations of human glioma are known to be diverse, ranging from aggressive growth and invasion to apparent dormancy; however, the molecular mechanism underlying this diversity has been largely unexplored. In the present study, we characterized four human glioma cell lines, T98G, A172, U251, and NAC6, each of which has distinct growth properties. A172 and U251 cells continue to grow after confluency, whereas the growth of T98G and NAC6 cells is contact inhibited. Northern and western blot analyses revealed that at high cell density, the expression of p27Kip1 cyclin-dependent kinase inhibitor was dramatically enhanced at both the RNA and the protein levels in T98G and NAC6 cells but not in A172 or U251. These facts together with the finding that overexpression of p27Kip1 caused G1 arrest in A172 and T98G cells suggest that the induction of p27Kip1 represents an important determinant of growth at high cell density. Immunohistochemical analyses of 42 primary gliomas revealed an inverse correlation between the level of p27 protein and the Ki-67 proliferative index. Kaplan-Meier plots demonstrated that a low level of p27 in tumors is associated with decreased overall survival. Thus, disrupted regulation of p27 expression at high cell density may play an important role in determining the clinical behavior of human gliomas as well as the prognosis for glioma patients. PMID- 10737595 TI - Negligible glucose-6-phosphatase activity in cultured astroglia. AB - 2-Deoxy[14C]glucose-6-phosphate (2-[14C]DG-6-P) dephosphorylation and glucose-6 phosphatase (G-6-Pase) activity were examined in cultured rat astrocytes under conditions similar to those generally used in assays of glucose utilization. Astrocytes were loaded with 2-[14C]DG-6-P by preincubation for 15 min in medium containing 2 mM glucose and 50 microM 2-deoxy[14C]glucose (2-[14C]DG). The medium was then replaced with identical medium including 2 mM glucose but lacking 2 [14C]DG, and incubation was resumed for 5 min to diminish residual free 2-[14C]DG levels in the cells by either efflux or phosphorylation. The medium was again replaced with fresh 2-[14C]DG-free medium, and the incubation was continued for 5, 15, or 30 min. Intracellular and extracellular 14C contents were measured at each time point, and the distribution of 14C between 2-[14C]DG and 2-[14C]DG-6-P was characterized by paper chromatography. The results showed little if any hydrolysis of 2-[14C]DG-6-P or export of free 2-[14C]DG from cells to medium; there were slightly increasing losses of 2-[14C]DG and 2-[14C]DG-6-P into the medium with increasing incubation time, but they were in the same proportions found in the cells, suggesting they were derived from nonadherent or broken cells. Experiments carried out with medium lacking glucose during the assay for 2 deoxyglucose-6-phosphatase activity yielded similar results. Evidence for G-6 Pase activity was also sought by following the selective detritiation of glucose from the 2-C position when astrocytes were incubated with [2-3H]glucose and [U 14C]glucose in the medium. No change in the 3H/14C ratio was found in incubations for as long as 15 min. These results indicate negligible G-6-Pase activity in cultured astrocytes. PMID- 10737596 TI - Possible role of CREB in the stimulation of oligodendrocyte precursor cell proliferation by neurotrophin-3. AB - We have previously shown that the transcription factor CREB (cyclic AMP-response element binding protein) could be a mediator of neuronal signals that, coupled to different signal transduction pathways, may play different regulatory roles at specific stages of oligodendrocyte (OLG) development. We have found before that in committed OLGs, CREB activation by phosphorylation can be triggered by beta adrenergic stimulation and appears to play a role in the induction of OLG differentiation by cyclic AMP. In contrast, in OLG precursor cells, CREB phosphorylation is stimulated by neuroligands that increase calcium levels by a process that involves a mitogen-activated protein kinase (MAPK)/protein kinase C (PKC) pathway. This observation suggested that at this early developmental stage, CREB could play a role in regulating cell proliferation. In support of this hypothesis, we have now found that a rapid and dramatic stimulation of CREB phosphorylation is one of the earliest events that precedes the increase in cell proliferation that is observed when OLG precursors are treated with neurotrophin 3 (NT-3). Experiments in which CREB phosphorylation was investigated in the presence of different kinase inhibitors indicated that the activation of this transcription factor in the presence of NT-3 is mediated by the concerted action of MAPK- and PKC-dependent signal transduction pathways. Moreover, our present results also showed that down-regulation of CREB expression in the OLG precursors abolished the increase in DNA synthesis that is observed when the cultures are treated with NT-3. Thus, these results support the idea that in immature OLG precursors, CREB plays an important role in transducing signals which, like NT-3, may regulate cell proliferation. PMID- 10737597 TI - Nuclear Ca2+/calmodulin translocation activated by mu-opioid (OP3) receptor. AB - Previous evidence has suggested a role for calmodulin (CaM) in opioid receptor signaling. We demonstrate here that morphine stimulation of the mu-opioid (OP3) receptor causes rapid CaM translocation to the nucleus in OP3-transfected human embryonic kidney (HEK)-293 cells and in SH-SY5Y human neuroblastoma cells. Ca2+ influx into the cells resulting from OP3 receptor activation was required for nuclear CaM translocation. Moreover, in HEK-OP3 and SH-SY5Y cells, increased nuclear CaM content was associated with enhanced phosphorylation of the nuclear transcription factor cyclic AMP-responsive element-binding protein. This appeared to be mediated by Ca2+/CaM kinases and also by a pathway involving protein kinase C. CaM was previously shown to bind directly to the OP3 receptor and to be released from the plasma membrane on agonist stimulation. To test whether OP3 mediated CaM release contributes to nuclear CaM signaling, we used a mutant OP3 receptor (K273A) with reduced affinity for CaM that fails to release CaM from the plasma membrane. K273A-OP3 activated Ca2+ influx to a similar extent as wild-type OP3; however, CaM translocation to the nucleus was attenuated. These results indicate that OP3-stimulated Ca2+ influx results in nuclear CaM translocation, which appears to be enhanced by simultaneous CaM release by OP3 wild-type receptor from plasma membranes. These results suggest a novel Ca2+/CaM signaling pathway of opioid receptors in the regulation of transcriptional activity. PMID- 10737598 TI - A mechanism for the neuroprotective effect of apolipoprotein E: isoform-specific modification by the lipid peroxidation product 4-hydroxynonenal. AB - Inheritance of the apolipoprotein E (apoE) epsilon4 allele increases the risk for Alzheimer's disease and may also influence the pathogenesis of other neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). The influence of apoE genotype on disease susceptibility must ultimately be explained by the fact that apoE proteins differ in only two amino acids: apoE2 has two cysteine residues, apoE3 has one cysteine residue, and apoE4 has none. We previously reported increased protein modification by the lipid peroxidation product 4-hydroxynonenal (HNE), which covalently binds to proteins on cysteine residues, in human ALS lumbar spinal cord. We now report increased levels of HNE modified apoE in lumbar spinal cord samples from mice expressing an ALS-linked mutation in Cu/Zn-superoxide dismutase relative to controls. Studies of interactions of pure apoE proteins with HNE showed that the isoforms differ in the amount of HNE they can bind, with the order E2 > E3 > E4. This correlated with the differential ability of apoE isoforms to protect against apoptosis induced by HNE in cultures of mouse spinal cord motor neurons and by the amyloid beta-peptide in cultures of rat hippocampal neurons. These data suggest that apoE plays a major role in detoxifying HNE, and the differential neuroprotective effect of its isoforms may help explain the relationship between apoE genotype and the susceptibility to neurodegenerative diseases. PMID- 10737599 TI - Astrocytes provide cysteine to neurons by releasing glutathione. AB - Cysteine is the rate-limiting precursor of glutathione synthesis. Evidence suggests that astrocytes can provide cysteine and/or glutathione to neurons. However, it is still unclear how cysteine is released and what the mechanisms of cysteine maintenance by astrocytes entail. In this report, we analyzed cysteine, glutathione, and related compounds in astrocyte conditioned medium using HPLC methods. In addition to cysteine and glutathione, cysteine-glutathione disulfide was found in the conditioned medium. In cystine-free conditioned medium, however, only glutathione was detected. These results suggest that glutathione is released by astrocytes directly and that cysteine is generated from the extracellular thiol/disulfide exchange reaction of cystine and glutathione: glutathione + cystine<-->cysteine + cysteine-glutathione disulfide. Conditioned medium from neuron-enriched cultures was also assayed in the same way as astrocyte conditioned medium, and no cysteine or glutathione was detected. This shows that neurons cannot themselves provide thiols but instead rely on astrocytes. We analyzed cysteine and related compounds in rat CSF and in plasma of the carotid artery and internal jugular vein. Our results indicate that cystine is transported from blood to the CNS and that the thiol/disulfide exchange reaction occurs in the brain in vivo. Cysteine and glutathione are unstable and oxidized to their disulfide forms under aerobic conditions. Therefore, constant release of glutathione by astrocytes is essential to maintain stable levels of thiols in the CNS. PMID- 10737601 TI - Microglial apoptosis induced by chromogranin A is mediated by mitochondrial depolarisation and the permeability transition but not by cytochrome c release. AB - Chromogranin A is up-regulated in the senile plaques of Alzheimer's brain and is a novel activator of microglia, transforming them to a neurotoxic phenotype. Treatment of primary cultures of rat brain microglia or the murine N9 microglial cell line with chromogranin A resulted in nitric oxide production, which triggered microglial apoptosis. Exposure of microglia to chromogranin A resulted in a fall in mitochondrial membrane potential. Mitochondrial depolarisation and apoptosis were reduced significantly by cyclosporin A, but not by the calcineurin inhibitor FK506. Cytochrome c did not translocate from the mitochondria to the cytosol, but its expression became significantly enhanced within the mitochondria. Inhibition of caspase 1 attenuated chromogranin A-induced microglial apoptosis, but did not prevent mitochondrial depolarisation, indicating that apoptosis occurred downstream of mitochondrial depolarisation. Conversely, staurosporine-induced microglial apoptosis led to mitochondrial cytochrome c release, but not caspase 1 activation. Our findings provide insight into the pathways controlling activation-triggered microglial apoptosis and may point to routes for the modulation of microglial evoked neurotoxicity. PMID- 10737600 TI - Brevetoxin-induced autocrine excitotoxicity is associated with manifold routes of Ca2+ influx. AB - Real-time alterations in intracellular Ca2+ ([Ca2+]i) were monitored in fluo-3 loaded cerebellar granule neurons (CGNs) exposed to the brevetoxin PbTx-1. [Ca2+]i was measured using a fluorescent plate reader (FLIPR), which measures simultaneously the mean intracellular Ca2+ change in a population of cultured cells in each well of a 96-well plate. PbTx-1 produced rapid and concentration dependent increases in neuronal [Ca2+]i with a potency nearly identical to that determined previously for PbTx-1-induced neurotoxicity. The NMDA receptor antagonists MK-801, dextrorphan, and D(-)-2-amino-5-phosphonopentanoic acid, and tetanus toxin, an inhibitor of Ca2+-dependent exocytotic neurotransmitter release, effected significant reductions in both the integrated fluo-3 fluorescence response and excitatory amino acid release and protected CGNs against PbTx-1 neurotoxicity. The L-type Ca2+ channel antagonist nifedipine produced a modest reduction in the fluo-3 response but reduced substantially the plateau phase of the PbTx-1 increment in [Ca2+]i when combined with MK-801. When nifedipine and MK-801 were combined with the Na+/Ca2+ exchanger (reversed mode) inhibitor KB-R7943, the PbTx-1 increment in [Ca2+]i was nearly completely attenuated. These data show that Ca2+ entry into PbTx-1-exposed CGNs occurs through three primary routes: NMDA receptor ion channels, L-type Ca2+ channels, and reversal of the Na+/Ca2+ exchanger. There was a close correlation between reduction of the integrated fluo-3 fluorescence response and the level of neuroprotection afforded by blockers of each Ca2+ entry pathway; however, simultaneous blockade of L-type Ca2+ channels and the Na+/Ca2+ exchanger, although reducing the integrated [Ca2+]i response to a level below that provided by NMDA receptor blockade alone, failed to completely attenuate PbTx-1 neurotoxicity. This finding suggests that in addition to total [Ca2+]i load, neuronal vulnerability is governed principally by the NMDA receptor Ca2+ influx pathway. PMID- 10737602 TI - Microtubule/MAP-affinity regulating kinase (MARK) is activated by phenylarsine oxide in situ and phosphorylates tau within its microtubule-binding domain. AB - Tau is a microtubule-associated protein (MAP) that is functionally modulated by phosphorylation and that is hyperphosphorylated in several neurodegenerative diseases. Because phosphorylation regulates both normal and pathological tau functioning, it is of interest to identify the signaling pathways and enzymes capable of modulating tau phosphorylation in vivo. Previously, it was demonstrated that in SH-SY5Y human neuroblastoma cells and rat primary cortical cultures tau is phosphorylated at Ser262/356, within its microtubule-binding domain, by a staurosporine-sensitive protein kinase in response to the vicinal thiol-directed agent phenylarsine oxide. The current study demonstrates the presence of a 100-kDa protein kinase activity in SH-SY5Y cells that associates with microtubules, phosphorylates tau at Ser262/356, is activated by phenylarsine oxide, and is inhibited by the protein kinase inhibitor staurosporine. Isolation of individual protein bands from a polyacrylamide gel revealed two closely spaced proteins containing Ser262/356-directed protein kinase activity. Mass spectrometry analysis indicated that these protein bands correspond to the 100 kDa microtubule/MAP-affinity regulating kinase (MARK), which has been shown previously to phosphorylate tau within its microtubule-binding domain. Immunoblot analysis of the protein kinase bands confirmed this finding, providing the first demonstration that activation of endogenous MARK results in increased tau phosphorylation within its microtubule-binding domain in situ. PMID- 10737603 TI - Novel cathepsin D inhibitors block the formation of hyperphosphorylated tau fragments in hippocampus. AB - Lysosomal disturbances may be a contributing factor to Alzheimer's disease. We used novel compounds to test if suppression of the lysosomal protease cathepsin D blocks production of known precursors to neurofibrillary tangles. Partial lysosomal dysfunction was induced in cultured hippocampal slices with a selective inhibitor of cathepsins B and L. This led within 48 h to hyperphosphorylated tau protein fragments recognized by antibodies against human tangles. Potent nonpeptidic cathepsin D inhibitors developed using combinatorial chemistry and structure-based design blocked production of the fragments in a dose-dependent fashion. Threshold was in the submicromolar range, with higher concentrations producing complete suppression. The effects were selective and not accompanied by pathophysiology. Comparable results were obtained with three structurally distinct inhibitors. These results support the hypothesis that cathepsin D links lysosomal dysfunction to the etiology of Alzheimer's disease and suggest a new approach to treating the disease. PMID- 10737605 TI - Expression of T-cadherin (CDH13, H-Cadherin) in human brain and its characteristics as a negative growth regulator of epidermal growth factor in neuroblastoma cells. AB - In the present study, we first examined the expression of T-cadherin in human CNS by northern blot analysis, immunohistochemical staining, and in situ hybridization. Northern blot analysis demonstrated expression of T-cadherin in human adult cerebral cortex, medulla, thalamus, and midbrain. Immunohistochemical staining with a newly generated monoclonal antibody, designated MA-511, revealed strong expression of T-cadherin in neural cell surface membrane and neurites in adult cerebral cortex, medulla oblongata, and nucleus olivaris. Little or no expression of T-cadherin was found in spinal cord. We further examined T-cadherin expression in various developing nervous systems, and found that T-cadherin expression was lower in developing brain than in adult brain. In situ hybridization revealed that neural cells in medulla oblongata and nucleus olivaris, but not in spinal cord, possessed T-cadherin molecules. We transfected T-cadherin-negative TGW and NH-12 neuroblastoma cells with a T-cadherin cDNA containing expression vector. T-cadherin-expressing neuroblastoma cells lost mitogenic proliferative response to epidermal growth factor. Epidermal growth factor is known to be required for proliferation of neural stem cells. This finding, together with those of the present study, suggests that T-cadherin functions as a negative regulator of neural cell growth. PMID- 10737604 TI - Palmitoyl protein thioesterase 1 protects against apoptosis mediated by Ras-Akt caspase pathway in neuroblastoma cells. AB - Palmitoyl protein thioesterase (PPT) 1 is an enzyme involved in deacylation of palmitoylated proteins. A deficiency in PPT1 results in a genetic disease, infantile neuronal ceroid lipofuscinosis, associated with massive death of cortical neurons. The role of PPT1 in neuronal survival and apoptosis was studied in human neuroblastoma (LA-N-5) cells overexpressing PPT1. Overexpression of PPT1 was shown both by the 200-350% increase in depalmitoylating activity over basal level (as determined by an in vitro PPT assay) and by western blot analysis of transiently expressed epitope-tagged PPT1. Overexpressed PPT1 showed the same acidic pH optimum (pH 4.0) as the endogenous enzyme, when assayed with a P0 derived octapeptide substrate, and reduced the growth rate by 30%. LA-N-5 cells underwent apoptosis, as evidenced by increased caspase 3-like activity and increased DNA fragmentation, when challenged with either C2-ceramide or a phosphatidylinositol 3-kinase inhibitor (LY294002). Overexpression of PPT1 inhibited this C2-ceramide- or LY294002-mediated activation of caspase-3 by 50%. There was also a concomitant decrease in DNA fragmentation and cell death. Consistent with increased resistance to apoptosis, we found increased phosphorylation of the antiapoptotic protein Akt (protein kinase B) in PPT1 overexpressing cells. p21Ras is known to be dynamically palmitoylated and depalmitoylated and is involved in both growth and cell death. The C2-ceramide induced membrane association of p21Ras was reduced by 30-50% in PPT1 overexpressing cells compared with control. PPT overexpression also led to reduced membrane association of another palmitoylated protein, GAP-43, a neuron specific protein. Our studies suggest that protein palmitoylation could be a physiological regulator of apoptosis. PMID- 10737606 TI - Astrocyte differentiation of fetal neuroepithelial cells by interleukin-11 via activation of a common cytokine signal transducer, gp130, and a transcription factor, STAT3. AB - The interleukin (IL)-6 family cytokines utilize membrane glycoprotein gp130 in common as a critical signal-transducing receptor component. IL-11, a cytokine initially identified as a plasmacytoma growth factor, belongs to this family. We show here that IL-11 and its cognate receptor components are expressed in fetal mouse neuroepithelial cells. We also show that after 4 days of culture with IL 11, cells with typical astrocytic morphologies expressing glial fibrillary acidic protein (GFAP; a marker for astrocytes) come out. This differentiation process is totally dependent on the gp130-mediated signal-transduction pathway involving activation of a latent cytoplasmic transcription factor, STAT3 (for signal transducer and activator of transcription 3), because (a) IL-11-induced astrocyte differentiation is not observed when neuroepithelial cells prepared from gp130 deficient mice were used, (b) stimulation of neuroepithelial cells by IL-11 rapidly induces tyrosine-phosphorylation of STAT3, and (c) transfection of neuroepithelial cells with a dominant-negative form of STAT3 inhibits IL-11 induced activation of the GFAP gene promoter. We have further identified, in the GFAP promoter region, a STAT3 site at which nucleotide substitutions almost completely abolished the IL-11-induced GFAP promoter activation. Taken together, it is suggested that IL-11 contributes to astrocytogenesis in fetal brain via activation of gp130 and STAT3. PMID- 10737607 TI - Sodium nitroprusside prevents chemical hypoxia-induced cell death through iron ions stimulating the activity of the Na+-Ca2+ exchanger in C6 glioma cells. AB - In C6 glioma cells exposed to chemical hypoxia, an increase of extracellular lactate dehydrogenase (LDH) activity, cell death, and intracellular Ca2+ concentration ([Ca2+]i) occurred. Sodium nitroprusside (SNP), a nitric oxide donor and an iron-containing molecule, reduced chemical hypoxia-induced LDH release and cell death. These effects were counteracted by bepridil and by 5-(N-4 chlorobenzyl)-2',4'-dimethylbenzamil (CB-DMB), two specific inhibitors of the Na+ Ca2+ exchanger. SNP also increased the activity of the Na+-Ca2+ exchanger as a Na+ efflux pathway, stimulated by Na+-free conditions and evaluated by monitoring [Ca2+]i in single cells. In addition, SNP produced a further increase of chemical hypoxia-elicited [Ca2+]i elevation, and this effect was blocked by bepridil. Chemical hypoxia-evoked cell death and LDH release were counteracted by the ferricyanide moiety of the SNP molecule, K3Fe(CN)6, and by ferric chloride (FeCl3), and this effect was counteracted by CB-DMB. In addition, the iron ion chelator deferoxamine reversed the protective effect exerted by SNP on cell injury. Collectively, these findings suggest that the protective effect of SNP on C6 glioma cells exposed to chemical hypoxia is due to the activation of the Na+ Ca2+ exchanger operating as a Na+ efflux-Ca2+ influx pathway induced by iron present in the SNP molecule. PMID- 10737609 TI - Chronic blockade of N-methyl-D-aspartate receptors alters gamma-aminobutyric acid type A receptor peptide expression and function in the rat. AB - Chronic in vivo or in vitro application of GABA(A) receptor agonists alters GABA(A) receptor peptide expression and function. Furthermore, chronic in vitro application of N-methyl-D-aspartate (NMDA) agonists and antagonists alters GABA(A) receptor function and mRNA expression. However, it is unknown if chronic in vivo blockade of NMDA receptors alters GABA(A) receptor function and peptide expression in brain. Male Sprague-Dawley rats were chronically administered the noncompetitive NMDA receptor antagonist MK-801 (0.40 mg/kg, twice daily) for 14 days. Chronic blockade of NMDA receptors significantly increased hippocampal GABA(A) receptor alpha4 and gamma2 subunit expression while significantly decreasing hippocampal GABA(A) receptor alpha2 and beta2/3 subunit expression. Hippocampal GABA(A) receptor alpha1 subunit peptide expression was not altered. In contrast, no significant alterations in GABA(A) receptor subunit expression were found in cerebral cortex. Chronic MK-801 administration also significantly decreased GABA(A) receptor-mediated hippocampal Cl- uptake, whereas no change was found in GABA(A) receptor-mediated cerebral cortical Cl- uptake. Finally, chronic MK-801 administration did not alter NMDA receptor NR1, NR2A, or NR2B subunit peptide expression in either the cerebral cortex or the hippocampus. These data demonstrate heterogeneous regulation of GABA(A) receptors by glutamatergic activity in rat hippocampus but not cerebral cortex, suggesting a new mechanism of GABA(A) receptor regulation in brain. PMID- 10737608 TI - Dopamine receptor-mediated regulation of striatal cholinergic activity: positron emission tomography studies with norchloro[18F]fluoroepibatidine. AB - Large numbers of in vitro studies and microdialysis studies suggest that dopaminergic regulation of striatal acetylcholine (ACh) output is via inhibitory dopamine D2 receptors and stimulatory dopamine D1 receptors. Questions remain as to the relative predominance of dopamine D2 versus D1 receptor modulation of striatal ACh output under physiological conditions. Using positron emission tomography, we first demonstrate that norchloro[18F]fluoroepibatidine ([18F]NFEP), a selective nicotinic ACh receptor (nAChR) ligand, was sensitive to changes of striatal ACh concentration. We then examined the effect of quinpirole (D2 agonist), raclopride (D2 antagonist), SKF38393 (D1 agonist), and SCH23390 (D1 antagonist) on striatal binding of [18F]NFEP in the baboon. Pretreatment with quinpirole increased the striatum (ST) to cerebellum (CB) ratio by 26+/-6%, whereas pretreatment with raclopride decreased the ST/CB ratio by 22+/-2%. The ratio of the distribution volume of [18F]NFEP in striatum to that in cerebellum, which corresponds to (Bmax/K(D)) + 1 (index for nAChR availability), also showed a significant increase (29 and 20%; n = 2) and decrease (20+/-3%; n = 3) after pretreatment with quinpirole and raclopride, respectively. However, both the D1 agonist and antagonist had no significant effect. This suggests that under physiological conditions the predominant influence of endogenous dopamine on striatal ACh output is dopamine D2, not D1, receptor-mediated. PMID- 10737610 TI - Zinc and copper modulate differentially the P2X4 receptor. AB - The rat ATP P2X4 receptor was expressed in Xenopus laevis oocytes to assess the effect of zinc and copper as possible regulators of purinergic mechanisms. ATP applied for 20 s evoked an inward cationic current with a median effective concentration (EC50) of 21.4+/-2.8 microM and a Hill coefficient (nH) of 1.5+/ 0.1. Coapplication of ATP plus 10 microM zinc displaced leftward, in a parallel fashion, the ATP concentration-response curve, reducing the EC50 to 8.4+/-1.8 microM (p < 0.01) without altering the receptor nH. The zinc potentiation was fast in onset, easily reversible, and voltage-independent and did not require metal preexposure. The zinc EC50 was 2-5 microM, with a bell-shaped curve. At concentrations of 100-300 microM, zinc produced less potentiation, and at 1 mM, it inhibited 50% the ATP current. The effect of zinc was mimicked by cadmium. In contrast, copper inhibited the ATP-evoked currents in a time- and concentration dependent fashion, reducing the maximal current (Imax) without altering the EC50. The copper-induced inhibition was slow in onset, slowly reversible, and voltage independent. Whereas coapplication of 300 microM copper plus ATP reduced Imax to 36.2+/-5%, the coapplication of, or 60-s preexposure by, 10 microM copper reduced Imax to 79+/-9.2% (p < 0.05) and 39.6+/-8.7% (p < 0.01), respectively. The inhibition was noncompetitive in nature and mimicked by mercury. Cobalt, barium, and manganese did not modify significantly the ATP-evoked current, demonstrating metal specificity. The simultaneous 1-min preapplication of both metals revealed that the 10 microM zinc-induced potentiation was obliterated by 10 microM copper, whereas 30 microM copper not only reduced the potentiation, but inhibited the ATP response. Following coapplication of both metals for 20 s with ATP, at least 100 microM copper was required to counteract the 10 microM zinc-induced potentiation. The simultaneous preincubation with both metals provided evidence for a noncompetitive interaction. We hypothesize the existence of metal binding site(s), which are most likely localized in the extracellular domain of the P2X4 receptor structure. These sites are selective and accessible to extracellular metal applications and bind micromolar concentrations of metals. The present results are compatible with the working hypothesis that trace metals, such as copper and zinc, are physiological modulators of the P2X4 receptor. The modulation of brain purinergic transmission by physiologically and toxicologically relevant trace metal cations is highlighted. PMID- 10737611 TI - Interaction of Na+, K+, and Cl- with the binding of amphetamine, octopamine, and tyramine to the human dopamine transporter. AB - Little information is available on the role of Na+, K+, and Cl- in the initial event of uptake of substrates by the dopamine transporter, i.e., the recognition step. In this study, substrate recognition was studied via the inhibition of binding of [3H]WIN 35,428 [2beta-carbomethoxy-3beta-(4-fluorophenyl)[3H]tropane], a cocaine analogue, to the human dopamine transporter in human embryonic kidney 293 cells. D-Amphetamine was the most potent inhibitor, followed by p-tyramine and, finally, dl-octopamine; respective affinities at 150 mM Na+ and 140 mM Cl- were 5.5, 26, and 220 microM. For each substrate, the decrease in the affinity with increasing [K+] could be fitted to a competitive model involving the same inhibitory cation site (site 1) overlapping with the substrate domain as reported by us previously for dopamine. K+ binds to this site with an apparent affinity, averaged across substrates, of 9, 24, 66, 99, and 134 mM at 2, 10, 60, 150, and 300 mM Na+, respectively. In general, increasing [Na+] attenuated the inhibitory effect of K+ in a manner that deviated from linearity, which could be modeled by a distal site for Na+, linked to site 1 by negative allosterism. The presence of Cl- did not affect the binding of K+ to site 1. Models assuming low binding of substrate in the absence of Na+ did not provide fits as good as models in which substrate binds in the absence of Na+ with appreciable affinity. The binding of dl-octopamine and p-tyramine was strongly inhibited by Na+, and stimulated by Cl- only at high [Na+] (300 mM), consonant with a stimulatory action of Cl- occurring through Na+ disinhibition. PMID- 10737612 TI - Modulation of the neurotoxic effects of methamphetamine by the selective kappa opioid receptor agonist U69593. AB - Kappa-opioid receptor agonists prevent alterations in dopamine neurotransmission that occur in response to repeated cocaine administration. The present microdialysis study examined whether administration of the selective kappa-opioid receptor agonist U69593 with methamphetamine prevents alterations in dopamine levels produced by neurotoxic doses of methamphetamine. Swiss Webster mice were injected intraperitoneally with methamphetamine (10.0 mg/kg) or saline, four times in 1 day, at 2-h intervals. Prior to the first and third injection, they received U69593 (0.32 mg/kg s.c.) or vehicle. Microdialysis was conducted 3, 7, or 21 days later. Basal and K+-evoked (60 and 100 mM) dopamine overflow were reduced 3 days after methamphetamine administration. These effects were long lasting in that they were still apparent 7 and 21 days after methamphetamine treatment. Intrastriatal (5.0 and 50 microM) or systemic (1.0-10.0 mg/kg) administration of methamphetamine increased dopamine concentrations in control animals. In mice preexposed to methamphetamine, methamphetamine-evoked dopamine overflow was reduced. In animals that had received methamphetamine with U69593, basal dopamine levels did not differ from those of vehicle-treated controls. U69593 treatment attenuated the decrease in K+-evoked dopamine produced by prior methamphetamine exposure. The reduction in methamphetamine-evoked dopamine levels was also attenuated. The administration of U69593 alone did not modify basal or stimulus-evoked dopamine levels. These data demonstrate that repeated methamphetamine administration reduces presynaptic dopamine neuronal function in mouse striatum and that co-administration of a selective kappa-opioid receptor agonist with methamphetamine attenuates these effects. U69593 treatment did not modify the hyperthermic effects of methamphetamine, indicating that this kappa opioid receptor agonist selectively attenuates methamphetamine-induced alterations in dopamine neurotransmission. PMID- 10737613 TI - Changes in the kinetics of dopamine release and uptake have differential effects on the spatial distribution of extracellular dopamine concentration in rat striatum. AB - The objective of this study was to examine whether the limited diffusion distance of dopamine in rat striatum produces spatial heterogeneity in the extracellular dopamine concentration on a dimensional scale of a few micrometers. Such heterogeneity would be significant because it would imply that the concentration of dopamine at a given receptor depends on the receptor's ultrastructural location. Spatially resolved measurements of extracellular dopamine were performed in the striatum of chloral hydrate-anesthetized rats with carbon fiber microdisk electrodes. Dopamine was monitored during electrical stimulation of the nigrostriatal pathway before and after administration of drugs that selectively affect the kinetics of evoked dopamine release and dopamine uptake. The effects of nomifensine (20 mg/kg), L-DOPA (250 mg/kg), and alpha-methyl-p-tyrosine (250 mg/kg) on the amplitude of the stimulation responses were examined. The outcome of these experiments was compared with predictions derived from a mathematical model that combines diffusion with the kinetics of release and uptake. The results demonstrate that the extracellular dopamine concentration is spatially heterogeneous on a micrometer scale and that changing the kinetics of dopamine release and uptake has different effects on this spatial distribution. The impact of these results on brain neurochemistry is considered. PMID- 10737614 TI - Norepinephrine-stimulated increase in Na+, K+-ATPase activity in the rat brain is mediated through alpha1A-adrenoceptor possibly by dephosphorylation of the enzyme. AB - Rapid eye movement sleep deprivation is reported to increase Na+,K+-ATPase activity. This increase was shown earlier to be stimulated by norepinephrine acting on alpha1-adrenoceptor. The involvement of a subtype of alpha1 adrenoceptor and the possible molecular mechanism of action of norepinephrine in increasing the enzyme activity were investigated using receptor agonists and antagonists, as well as stimulants and blockers of signal transduction pathway. It was observed that incubation of the homogenate with cyclic AMP, forskolin, A23187 (a calcium ionophore), or calmodulin alone did not stimulate the Na+,K+ ATPase activity. However, although the spontaneous activity of the Na+,K+-ATPase was not affected by prazosin, WB4101, heparin, W13, or cyclosporin A alone, each of them could prevent the norepinephrine-stimulated increase in the enzyme activity. Based on these results and our previous findings, it is proposed that norepinephrine acted on alpha1A-adrenoceptor and increased intracellular calcium, which in the presence of calmodulin activated a calmodulin-dependent phosphatase, calcineurin. This calcineurin possibly dephosphorylated Na+,K+-ATPase and increased its activity. The physiological significance especially in relation to rapid eye movement sleep deprivation is discussed. PMID- 10737615 TI - Amyloid beta-peptide effects on synaptosomes from apolipoprotein E-deficient mice. AB - Apolipoprotein E (apoE) is present in the brain and may contribute to neurophysiologic or neuropathologic events, depending on environmental and genetic influences. Recent studies indicate a role for apoE in synaptic plasticity and maintenance of synaptic membrane symmetry, suggesting that apoE may be involved in regulating synaptic homeostasis. In the present study, cerebrocortical synaptosomes were prepared from transgenic mice lacking apoE (apoE KO) to analyze the possible contribution of apoE toward maintaining homeostasis in synaptosomes. Synaptosomal preparations from apoE KO and wild-type mice exhibited similar basal levels of reactive oxygen species, mitochondrial function, and caspase activity; however, following application of amyloid beta peptide [Abeta(1-40)], apoE KO synaptosomes displayed increased levels of oxidative stress, mitochondrial dysfunction, and caspase activation compared with synaptosomes from wild-type mice. Synaptosomal membranes from apoE KO mice were more fluid than wild-type synaptosomes and contained higher levels of thiobarbituric acid-reactive substances, consistent with elevated levels of lipid peroxidation occurring in the synapses of apoE KO mice. Together, these data are consistent with a role for apoE in maintaining homeostasis by attenuating oxidative stress, caspase activation, and mitochondrial homeostasis in synapses. PMID- 10737616 TI - Phosphorylation sites on tau identified by nanoelectrospray mass spectrometry: differences in vitro between the mitogen-activated protein kinases ERK2, c-Jun N terminal kinase and P38, and glycogen synthase kinase-3beta. AB - The stress-activated kinases c-Jun N-terminal kinase (JNK) and p38 are members of the mitogen-activated protein (MAP) kinase family and take part in signalling cascades initiated by various forms of stress. Their targets include the microtubule-associated protein tau, which becomes hyperphosphorylated in Alzheimer's disease. It is necessary, as a forerunner for in vivo studies, to identify the protein kinases and phosphatases that are responsible for phosphate turnover at individual sites. Using nanoelectrospray mass spectrometry, we have undertaken an extensive comparison of phosphorylation in vitro by several candidate tau kinases, namely, JNK, p38, ERK2, and glycogen synthase kinase 3beta (GSK3beta). Between 10 and 15 sites were identified for each kinase. The three MAP kinases phosphorylated Ser202 and Thr205 but not detectably Ser199, whereas conversely GSK3beta phosphorylated Ser199 but not detectably Ser202 or Thr205. Phosphorylated Ser404 was found with all of these kinases except JNK. The MAP kinases may not be strictly proline specific: p38 phosphorylated the nonproline sites Ser185, Thr245, Ser305, and Ser356, whereas ERK2 was the most strict. All of the sites detected except Thr245 and Ser305 are known or suspected phosphorylation sites in paired helical filament-tau extracted from Alzheimer brains. Thus, the three MAP kinases and GSK3beta are importantly all strong candidates as tau kinases that may be involved in the pathogenic hyperphosphorylation of tau in Alzheimer's disease. PMID- 10737617 TI - N-methyl bases of ethanolamine prevent apoptotic cell death induced by oxidative stress in cells of oligodendroglia origin. AB - A major reason for brain tissue vulnerability to oxidative damage is the high content of polyunsaturated fatty acids (PUFAs). Oligodendroglia-like OLN 93 cells lack PUFAs and are relatively insensitive to oxidative stress. When grown in serum-free defined medium in the presence of 0.1 mM docosahexaenoic acid (DHA; 22:6 n-3) for 3 days, OLN 93 cells release in the medium 2.6-fold more thiobarbituric acid-reactive substances (TBARS) after a 30-min exposure to 0.1 mM H2O2 and 50 microM Fe2+. Release of TBARS was substantially decreased by approximately 20 and 30% on coincubation with either 1 mM N monomethylethanolamine or N,N'-dimethylethanolamine (dEa), respectively. The protective effect of dEa was concentration- and time-dependent and was still visible after dEa removal, suggesting a long-lasting mechanism of protection. After 24 h following H2O2-induced stress, cell death monitored by cell sorting showed 16% of the cells in the sub-G1 area, indicative of apoptotic cell death. DHA-supplemented cultures showed 35% cell death, whereas cosupplements with dEa reduced cell death to 12%, indicating cell rescue. Although the exact mechanism for this protection is not known, the nature of the polar head group and the degree of unsaturation may determine the ultimate resistance of nerve cells to oxidative stress. PMID- 10737618 TI - Autoxidation and neurotoxicity of 6-hydroxydopamine in the presence of some antioxidants: potential implication in relation to the pathogenesis of Parkinson's disease. AB - 6-Hydroxydopamine (6-OHDA) is a dopaminergic neurotoxin putatively involved in the pathogenesis of Parkinson's disease (PD). Its neurotoxicity has been related to the production of reactive oxygen species. In this study we examine the effects of the antioxidants ascorbic acid (AA), glutathione (GSH), cysteine (CySH), and N-acetyl-CySH (NAC) on the autoxidation and neurotoxicity of 6-OHDA. In vitro, the autoxidation of 6-OHDA proceeds rapidly with the formation of H2O2 and with the participation of the H2O2 produced in the reaction. The presence of AA induced a reduction in the consumption of O2 during the autoxidation of 6-OHDA and a negligible presence of the p-quinone, which demonstrates the efficiency of AA to act as a redox cycling agent. The presence of GSH, CySH, and NAC produced a significant reduction in the autoxidation of 6-OHDA. In vivo, the presence of sulfhydryl antioxidants protected against neuronal degeneration in the striatum, which was particularly remarkable in the case of CySH and was attributed to its capacity to remove the H2O2 produced in the autoxidation of 6-OHDA. These results corroborate the involvement of oxidative stress as the major mechanism in the neurotoxicity of 6-OHDA and the putative role of CySH as a scavenger in relation to PD. PMID- 10737619 TI - Human Bcl-2 protects against AMPA receptor-mediated apoptosis. AB - Dysfunctions of the (S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of ionotropic receptor for the brain's major excitatory neurotransmitter, L-glutamate, occur in various neurological conditions. We have previously demonstrated that AMPA receptor-mediated excitotoxicity occurs by apoptosis and here examined the influence of the expression of cell death repressor gene Bcl-2 on this excitotoxic insult. Using neuronal cortical cultures prepared from transgenic mice expressing the human Bcl-2 gene, the influence of Bcl-2 on AMPA receptor-mediated neuronal death was compared with that seen with staurosporine and H2O2. At day 6 cultures were exposed to AMPA (0.1-100 microM), and cellular injury was analyzed 48 h after insult using phase-contrast microscopy, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay, and DNA staining with 4,6-diamidino-2-phenylindole and Sytox Green. AMPA produced a concentration-dependent increase in cell death that was significantly attenuated by human Bcl-2. AMPA (3 microM) increased the number of apoptotic nuclei to 60% of control in wild-type cultures, and human Bcl-2 significantly decreased the number of apoptotic nuclei to 30% of AMPA-treated cultures. Human Bcl-2 only provided significant neuroprotection against neuronal injury induced by low concentrations of staurosporine (1-10 nM) and H2O2 (0.1-30 microM) and where neuronal death was by apoptosis, but not against H2O2-induced necrosis. Our findings indicate that overexpression of Bcl-2 in primary cultured neurons protects in an insult-dependent manner against AMPA receptor-mediated apoptosis, whereas protection was not seen against more traumatic insults. This study provides new insights into the molecular therapeutics of neurodegenerative conditions. PMID- 10737620 TI - Correlation between structure of Bcl-2 and its inhibitory function of JNK and caspase activity in dopaminergic neuronal apoptosis. AB - To examine the correlation between the structure of Bcl-2 and its inhibitory function of c-Jun N-terminal kinase (JNK) and caspase activity, we established a dopaminergic neuronal cell line, MN9D overexpressing Bcl-2 (MN9D/Bcl-2) or its structural mutants. The mutants comprised a point mutation in the BH1 (G145A; MN9D/BH1) or BH2 (W188A; MN9D/BH2) domain and a deletion mutation in the C terminal (MN9D/C22), BH3 (MN9D/BH3), or BH4 (MN9D/BH4) domain. As determined by the TUNEL (terminal deoxynucleotidyltransferase nick end-labeling) and MTT [3 (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay, apoptotic death of MN9D/Neo cells reached 80-90% within 24 h in response to 1 microM staurosporine. Upon staurosporine treatment, JNK activity increased six- to sevenfold over the basal level within 2-4 h. Treatment of MN9D/Neo with both staurosporine and a caspase inhibitor, Z-VAD, attenuated cell death without suppressing JNK activation. Both staurosporine-induced cell death and JNK activation were attenuated in MN9D/Bcl-2. As determined by cleavage of poly(ADP ribose) polymerase into 85 kDa, Bcl-2 blocked caspase activity as well. When cells overexpressing one of the Bcl-2 mutants were treated with staurosporine, death was attenuated in MN9D/BH1, MN9D/BH2, and MN9D/C22 but not in MN9D/BH3 and MN9D/BH4. Similarly, both JNK and caspase activation were blocked in MN9D/BH1, MN9D/BH2, and MN9D/C22, whereas they were not suppressed in MN9D/BH3 and MN9D/BH4. Taken together, our data indicate that there exists a close structural and functional correlation of Bcl-2 to JNK and caspase activity in staurosporine induced dopaminergic neuronal cell death. PMID- 10737622 TI - Activation of poly(ADP-ribose) polymerase in the rat hippocampus may contribute to cellular recovery following sublethal transient global ischemia. AB - We have investigated the role of poly(ADP-ribose) polymerase (PARP) activation in rat brain in a model of sublethal transient global ischemia. Adult male rats were subjected to 15 min of ischemia with brain temperature reduced to 34 degrees C, followed by 1, 2, 4, 8, 16, 24, and 72 h of reperfusion. PARP mRNA expression was examined in the hippocampus using quantitative RT-PCR, northern blot analysis, and in situ hybridization. Protein expression was assessed using western blot analysis. PARP enzymatic activity was investigated by measuring nuclear [3H]NAD incorporation. The presence of poly(ADP-ribose) polymers was assessed immunocytochemically. Although PARP mRNA and protein expressions were not altered after ischemia, enzymatic activity was increased 4.37-fold at 1 h (p < 0.05 vs. sham) and 1.73-fold (p < 0.05 vs. sham) at 24 h of reperfusion. Immunostaining demonstrated the presence of poly(ADP-ribose) polymers in CA1 neurons. Cellular NAD+ levels were not significantly altered at any time point. Furthermore, systemic administration of 3-aminobenzamide (30 mg/kg), a PARP inhibitor, prevented the increase in PARP activity at 1 and 24 h of reperfusion, significantly decreased the number of surviving neurons in the hippocampal CA1 region 72 h after ischemia (p < 0.01 vs. sham), and increased DNA single-strand breaks assessed as DNA polymerase I-mediated biotin-dATP nick-translation (PANT) positive cells (p < 0.01 vs. sham). Furthermore, using an in vitro DNA repair assay, 3-aminobenzamide (30 mg/kg) was shown to block DNA base excision repair activity. These data suggest that the activation of PARP, without subsequent NAD+ depletion, following mild transient ischemia may be neuroprotective in the brain. PMID- 10737621 TI - Enhancement of anandamide formation in the limbic forebrain and reduction of endocannabinoid contents in the striatum of delta9-tetrahydrocannabinol-tolerant rats. AB - Recent studies have shown that the pharmacological tolerance observed after prolonged exposure to synthetic or plant-derived cannabinoids in adult rats is accompanied by down-regulation/desensitization of brain cannabinoid receptors. However, no evidence exists on possible changes in the contents of the endogenous ligands of cannabinoid receptors in the brain of cannabinoid-tolerant rats. The present study was designed to elucidate this possibility by measuring, by means of isotope dilution gas chromatography/mass spectrometry, the contents of both anandamide (arachidonoylethanolamide; AEA) and its biosynthetic precursor, N arachidonoylphosphatidylethanolamine (NArPE), and 2-arachidonoylglycerol (2-AG) in several brain regions of adult male rats treated daily with delta9 tetrahydrocannabinol (delta9-THC) for a period of 8 days. The areas analyzed included cerebellum, striatum, limbic forebrain, hippocampus, cerebral cortex, and brainstem. The same regions were also analyzed for cannabinoid receptor binding and WIN-55,212-2-stimulated guanylyl-5'-O-(gamma-[35S]thio)-triphosphate ([35S]GTPgammaS) binding to test the development of the well known down regulation/desensitization phenomenon. Results were as follows: As expected, cannabinoid receptor binding and WIN-55,212-2-stimulated [35S]GTPgammaS binding decreased in most of the brain areas of delta9-THC-tolerant rats. The only region exhibiting no changes in both parameters was the limbic forebrain. This same region exhibited a marked (almost fourfold) increase in the content of AEA after 8 days of delta9-THC treatment. By contrast, the striatum exhibited a decrease in AEA contents, whereas no changes were found in the brainstem, hippocampus, cerebellum, or cerebral cortex. The increase in AEA contents observed in the limbic forebrain was accompanied by a tendency of NArPE levels to decrease, whereas in the striatum, no significant change in NArPE contents was found. The contents of 2-AG were unchanged in brain regions from delta9-THC-tolerant rats, except for the striatum where they dropped significantly. In summary, the present results show that prolonged activation of cannabinoid receptors leads to decreased endocannabinoid contents and signaling in the striatum and to increased AEA formation in the limbic forebrain. The pathophysiological implications of these findings are discussed in view of the proposed roles of endocannabinoids in the control of motor behavior and emotional states. PMID- 10737623 TI - Pretreatment with calpain inhibitor CEP-4143 inhibits calpain I activation and cytoskeletal degradation, improves neurological function, and enhances axonal survival after traumatic spinal cord injury. AB - The pathophysiology of traumatic spinal cord injury (SCI) involves abnormal activation of the neutral cysteine protease calpain I (EC 3.4.22.17). In the present study we examined the effect of the calpain inhibitor CEP-4143 on cytoskeletal protection and neurological recovery after SCI in adult rats. Microinjection of 50 mM CEP-4143 into the T7 vertebral segment 10 min before a 35 g clip compression injury resulted in inhibition of calpain activation at 2 and 4 h postinjury, as determined by western blotting for calpain I-mediated spectrin degradation, and significantly attenuated the degradation of dephosphorylated NF200 neurofilament protein at 4 and 8 h postinjury. To examine the in vivo chronic neuroprotective effects of CEP-4143, animals underwent microinjection with saline or 50 mM CEP-4143 10 min before injury, followed by weekly blinded behavioral assessments for 6 weeks. Animals receiving CEP-4143 treatment showed significant improvement over saline-treated controls on the Basso Beattie Bresnahan locomotor rating scale (p < 0.02) and inclined plane test (p < 0.05). Counts of neurons in the red nucleus retrogradely labeled by fluoro-gold after introduction distal to the injury site were significantly higher in CEP-4143 treated animals. Finally, morphometric assessment of the injury site by computer assisted image analysis revealed significant tissue preservation in CEP-4143 treated animals. We conclude that the calpain antagonist CEP-4143 exhibits biochemical, behavioral, and anatomical neuroprotection following traumatic SCI. PMID- 10737624 TI - Role for dopamine in malonate-induced damage in vivo in striatum and in vitro in mesencephalic cultures. AB - Defects in mitochondrial energy metabolism have been implicated in the pathology of several neurodegenerative disorders. In addition, the reactive metabolites generated from the metabolism and oxidation of the neurotransmitter dopamine (DA) are thought to contribute to the damage to neurons of the basal ganglia. We have previously demonstrated that infusions of the metabolic inhibitor malonate into the striata of mice or rats produce degeneration of DA nerve terminals. In the present studies, we demonstrate that an intrastriatal infusion of malonate induces a substantial increase in DA efflux in awake, behaving mice as measured by in vivo microdialysis. Furthermore, pretreatment of mice with tetrabenazine (TBZ) or the TBZ analogue Ro 4-1284 (Ro-4), compounds that reversibly inhibit the vesicular storage of DA, attenuates the malonate-induced DA efflux as well as the damage to DA nerve terminals. Consistent with these findings, the damage to both DA and GABA neurons in mesencephalic cultures by malonate exposure was attenuated by pretreatment with TBZ or Ro-4. Treatment with these compounds did not affect the formation of free radicals or the inhibition of oxidative phosphorylation resulting from malonate exposure alone. Our data suggest that DA plays an important role in the neurotoxicity produced by malonate. These findings provide direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular DA levels and indicate that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through a mechanism involving DA. PMID- 10737625 TI - Elevated cortical extracellular fluid glutamate in transgenic mice expressing human mutant (G93A) Cu/Zn superoxide dismutase. AB - Transgenic mice expressing a mutated (G93A) human Cu/Zn superoxide dismutase (SOD1) develop motor neuron pathology and clinical symptoms similar to those seen in patients with amyotrophic lateral sclerosis. Loss of motor neurons is most prominent in lumbar, followed by cervical cord and then brainstem. No significant cell death has been reported in motor cortex. The integrity of the cortical glutamate reuptake systems was evaluated using intracerebral microdialysis and western immunoblot assays for the glutamate transporters GLT-1, GLAST, and EAAC1. The basal extracellular fluid levels of aspartate, glutamate, glutamine, 3,4 dihydroxyphenylacetic acid, and 5-hydroxyindole-3-acetic acid were evaluated by HPLC. The extraction fraction of L-3H]glutamate, corrected with [14C]mannitol, was also evaluated. GLT-1, EAAC1, and GLAST protein levels were determined by semiquantitative chemiluminescence immunoblot of proteins from membrane-enriched fractions. The relative optical density of film was translated into relative protein level by comparison with a standard control mouse. The SOD1 mutant mice demonstrated a significant (p < 0.05) increase in basal levels of extracellular aspartate and glutamate. In addition, when the glutamate extraction fraction was challenged with exogenous unlabeled glutamate (500 microM) by reversed microdialysis, the glutamate extraction fraction in the mutant SOD1 mice was decreased significantly from control levels. The SOD1 mutant mice demonstrated no difference in the cortical protein levels of the glutamate transporter subtypes. This study demonstrates that in areas of no visible pathology and no loss of glutamate transporter proteins, SOD1 mutant mice have elevated extracellular fluid aspartate and glutamate levels and a decreased capacity to clear glutamate from the extracellular space. PMID- 10737626 TI - Protein kinase C-alpha and -epsilon down-regulate cell surface sodium channels via differential mechanisms in adrenal chromaffin cells. AB - In cultured bovine adrenal chromaffin cells, our [3H]saxitoxin ([3H]STX) binding, immunoblot, and northern blot analyses specified protein kinase C (PKC) isoform specific posttranscriptional and posttranslational mechanisms that direct down regulation of cell surface Na channels. Immunoblot analysis showed that among 11 PKC isoforms, adrenal chromaffin cells contained only conventional (c)PKC-alpha, novel (n)PKC-epsilon, and atypical (a)PKC-zeta. Treatment of adrenal chromaffin cells with 100 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) or 100 nM phorbol 12,13-dibutyrate (PDBu) caused a rapid (<15 min) and sustained (>15 h) translocation of PKC-alpha and -epsilon (but not -zeta) from cytosol to membranes, whereas a biologically inactive 4alpha-TPA had no effect. Thymeleatoxin (TMX), an activator of cPKC, produced similar membrane association of only PKC-alpha at 100 nM, with the potency of TMX being comparable with those of TPA and PDBu. Treatment with either 100 nM TPA or 100 nM TMX reduced cell surface [3H]STX binding to a comparable extent at 3, 6, and 12 h, whereas TPA lowered the binding to a greater extent than TMX at 15, 18, and 24 h; at 15 h, Go6976, a specific inhibitor of cPKC, completely blocked TMX-induced decrease of [3H]STX binding while preventing by merely 57% TPA-induced decrease of [3H]STX binding. Treatment with 100 nM TPA lowered the Na channel alpha-subunit mRNA level between 3 and 12 h, with its maximum 52% fall at 6 h, and it was accompanied by a subsequent 61 % rise of the beta1-subunit mRNA level at 24 h. Go6976 failed to prevent TPA-induced reduction of the alpha-subunit mRNA level; TMX did not change the alpha- and beta1-subunit mRNA levels throughout the 24-h treatment. Brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network, augmented TPA- and TMX-induced decrease of [3H]STX binding at 1 and 3 h. Our previous and present studies suggest that PKC down-regulates cell surface Na channels without altering the allosteric gating of Na channels via PKC isoform specific mechanisms; cPKC-alpha promotes Na channel internalization, whereas nPKC epsilon decreases the alpha-subunit mRNA level by shortening the half-life of alpha-subunit mRNA without changing its gene transcription. PMID- 10737627 TI - Regulation of adenylyl cyclase, ERK1/2, and CREB by Gz following acute and chronic activation of the delta-opioid receptor. AB - Opioid tolerance and physical dependence in mammals can be rapidly induced by chronic exposure to opioid agonists. Recently, opioid receptors have been shown to interact with the pertussis toxin (PTX)-insensitive Gz (a member of the Gi subfamily), which inhibits adenylyl cyclase and stimulates mitogen-activated protein kinases (MAPKs). Here, we established stable human embryonic kidney 293 cell lines expressing delta-opioid receptors with or without Gz to examine the role of Gz in opioid receptor-regulated signaling systems. Each cell line was acutely or chronically treated with [D-Pen2,D-Pen5]enkephalin (DPDPE), a delta selective agonist, in the absence or presence of PTX. Subsequently, the activities of adenylyl cyclase, cyclic AMP (cAMP)-dependent response element binding proteins (CREBs), and MAPKs were measured by determining cAMP accumulation and phosphorylation of CREBs and the extracellular signal-regulated protein kinases (ERKs) 1 and 2. In cells coexpressing Gz, DPDPE inhibited forskolin-stimulated cAMP accumulation in a PTX-insensitive manner, but Gz could not replace Gi to mediate adenylyl cyclase supersensitization upon chronic opioid treatment. DPDPE-induced adenylyl cyclase supersensitization was not associated with an increase in the phosphorylation of CREBs. Both Gi and Gz mediated DPDPE induced activation of ERK1/2, but these responses were abolished by chronic opioid treatment. Collectively, our results show that although Gz mediated opioid induced inhibition of adenylyl cyclase and activation of ERK1/2, Gz alone was insufficient to mediate opioid-induced adenylyl cyclase supersensitization. PMID- 10737628 TI - Cellular and subcellular localization of peripheral benzodiazepine receptors after trimethyltin neurotoxicity. AB - The peripheral benzodiazepine receptor (PBR) is currently used as a marker of inflammation and gliosis following brain injury. Previous reports suggest that elevated PBR levels in injured brain tissue are specific to activated microglia and infiltrating macrophages. We have produced hippocampal lesions using the neurotoxicant trimethyltin (TMT) to examine the cellular and subcellular nature of the PBR response. Degenerating, argyrophilic pyramidal neurons were observed in the hippocampus at 2 and 14 days after TMT exposure. Reactive microglia were also evident at both times with a maximal response observed at 14 days, subsiding by 6 weeks. Astrocytosis was observed at 14 days and 6 weeks, but not 2 days, after TMT administration, suggesting that the onset of the astroglia response is delayed, but more persistent, compared with microgliosis. Morphological evidence from [3H]PK11195 microautoradiography and PBR immunohistochemistry indicates that both astrocytes and microglia are capable of expressing high levels of PBR after injury. This was confirmed by double labeling of either Griffonia simplicifolia isolectin B4, a microglial-specific marker, or glial fibrillary acidic protein, an astrocyte-specific protein with PBR fluorescence immunohistochemistry. These results demonstrate that PBR expression is increased after brain injury in both activated microglia and astrocytes. Our findings also provide the first evidence for in situ nuclear localization of PBR in glial cells. PMID- 10737630 TI - GM3 alpha2,8-sialyltransferase (GD3 synthase): protein characterization and sub golgi location in CHO-K1 cells. AB - GD3 synthase (Sial-T2) is a key enzyme of ganglioside synthesis that, in concert with GM2 synthase (GalNAc-T), regulates the ratio of a- and b-pathway gangliosides. In this work, we study the sub-Golgi location of an epitope-tagged version of chicken Sial-T2 transfected to CHO-K1 cells. The expressed protein was enzymatically active both in vitro and in vivo and showed a molecular mass of approximately 47 or approximately 95 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence or absence of, respectively, beta mercaptoethanol. The 95-kDa form of Sial-T2 was also detected if the protein was retained in the endoplasmic reticulum (ER) due to impaired glycosylation, indicating that it was formed in the ER. Confocal immunofluorescence microscopy showed Sial-T2 localized to the Golgi complex and, within the organelle, partially co-localizing with the mannose-6-phosphate receptor, a marker of the trans-Golgi network (TGN). In cells treated with brefeldin A, a major fraction of Sial-T2 redistributed to the ER, even under controlled expression to control for mislocalization due to protein overloading. In experiments of incorporation of sugars into endogenous acceptors of Golgi membranes in vitro, GD3 molecules formed by incubation with CMP-NeuAc were converted to GD2 upon incubation with UDP-GalNAc. These results indicate that Sial-T2 localizes mainly to the proximal Golgi, although a fraction is located in the TGN functionally coupled to GalNAc T. Consistent with this, most of the enzyme was in an endoglycosidase H (Endo-H) sensitive, neuraminidase (NANase)-insensitive form. A minor secreted form lacking approximately 40 amino acids was Endo-H-resistant and NANase-sensitive, indicating that the cells were able to process N-glycans to Endo-H-resistant forms. Taken together, the results of these biochemical and immunocytochemical experiments indicate that in CHO-K1 cells, most Sial-T2 localizes in the proximal Golgi and that a functional fraction is also present in the TGN. PMID- 10737629 TI - Ligand recognition of serine-cysteine amino acid exchanges in transmembrane domain 5 of alpha2-adrenergic receptors by UK 14,304. AB - Ligand binding of UK 14,304 reveals notable species (i.e., human-rodent) and receptor-subtype differences of alpha2-adrenergic receptors (alpha2-ARs). To study the molecular basis of the selectivity of UK 14,304, we compared a series of conservative serine-cysteine exchange mutants at ligand-accessible positions in transmembrane domain 5 of the human and mouse alpha2A-ARs. UK 14,304 bound with approximately 200-fold higher affinity to the human alpha2A-AR wild-type receptor compared with the human alpha2A-ARSer201 mutant, but only an approximately fivefold difference was seen with the corresponding mouse alpha2A AR variant. These effects of cysteine-serine exchanges only involved the agonist low-affinity forms of the receptors, as the affinity of [3H]UK 14,304 for the agonist high-affinity receptor populations was not influenced. The apparent affinities of a set of eight structurally diverse alpha2-AR ligands (six agonists and two antagonists) were not influenced significantly by the cysteine-serine exchanges (except for oxymetazoline and yohimbine, with up to nine- and eightfold differences in affinity, respectively). We conclude that position 201 (a) plays a primary role in determining observed subtype/species selectivity of UK 14,304 in competitive antagonist radioligand binding assays and (b) does not determine the subtype selectivity of chlorpromazine. PMID- 10737631 TI - Distinct internalization of M2 muscarinic acetylcholine receptors confers selective and long-lasting desensitization of signaling to phospholipase C. AB - Although M1-M4 muscarinic acetylcholine receptors (mAChRs) in HEK-293 cells internalize on agonist stimulation, only M1, M3, and M4 but not M2 mAChRs recycle to the plasma membrane. To investigate the functional consequences of this phenomenon, we compared desensitization and resensitization of M2 versus M4 mAChRs. Treatment with 1 mM carbachol for 1 h at 37 degrees C reduced numbers of cell surface M2 and M4 mAChRs by 40-50% and M2 and M4 mAChR-mediated inhibition of adenylyl cyclase, intracellular Ca2+ concentration ([Ca2+]i) increases, and phospholipase C (PLC) activation by 60-70%. Receptor-mediated inhibition of adenylyl cyclase and [Ca2+]i increases significantly resensitized within 3 h. However, M4 but not M2 mAChR-mediated PLC activation resensitized. At 16 degrees C, M2 mAChR-mediated [Ca2+]i increases and PLC stimulation desensitized to a similar extent as at 37 degrees C. However, at 16 degrees C, where M2 mAChR internalization is negligible, both M2 mAChR responses resensitized, demonstrating that M2 mAChR resensitization proceeds at the plasma membrane. Examination of M2 mAChR responses following inactivation of cell surface mAChRs by quinuclidinyl benzilate revealed substantial receptor reserve for coupling to [Ca2+]i increases but not to PLC. We conclude that M2 mAChR internalization induces long-lasting PLC desensitization predominantly because receptor loss is not compensated for by receptor recycling or receptor reserve. PMID- 10737632 TI - Purification and characterization of a trypsin-like serine proteinase from rat brain slices that degrades laminin and type IV collagen and stimulates protease activated receptor-2. AB - A trypsin-like serine proteinase was purified from the incubation medium of rat brain slices by gelatin zymography. The purification consisted of ammonium sulfate precipitation, benzamidine-Sepharose 6B affinity chromatography, and carboxymethyl-cellulose and gel filtration chromatographies. The gelatinolytic activity, identified at 22 kDa (P22) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, was eluted as one active peak throughout the purification, and the final preparation gave a single protein peak on reverse-phase HPLC. Diisopropyl fluorophosphate, benzamidine, p toluenesulfonyl-L-lysine chloromethyl ketone, and aprotinin completely inhibited the activity of P22, whereas phenanthroline, p-toluene-sulfonyl-L-phenylalanine chloromethyl ketone, and elastinal did not. P22 efficiently digested the extracellular matrix proteins laminin and type IV collagen. P22 produced an increase in intracellular Ca2+ concentration in A172 glioblastoma, which was desensitized through prior stimulation with protease-activated receptor-2 agonist peptide SLIGKV, indicating that P22 can stimulate protease-activated receptor-2. Rat brain penetration injury induced gelatinolytic activity in the lesioned area whose molecular size was consistent with that of P22. These results indicated that on incubation of rat brain slices, a trypsin-like serine proteinase was secreted into the medium that was capable of digesting extracellular matrix and stimulating protease-activated receptor-2. It is suggested that the gelatinolytic activity induced by brain injury might be that of P22. PMID- 10737633 TI - Modification of cysteines reveals linkage to acetylcholine and vesamicol binding sites in the vesicular acetylcholine transporter of Torpedo californica. AB - Properties of cysteinyl residues in the vesicular acetylcholine transporter (VAChT) of synaptic vesicles isolated from Torpedo californica were probed. Cysteine-specific reagents of different size and polarity were used and the effects on [3H]vesamicol binding determined. The vesamicol dissociation constant increased 1,000-fold after reaction with p-chloromercuriphenylsulfonate or phenylmercury acetate, but only severalfold after reaction with relatively small methylmercury chloride or methylmethanethiosulfonate (MMTS). Methylmercury chloride, but not MMTS, protected binding from phenylmercury acetate. Thus, two classes of cysteines react to affect vesamicol binding. Class 1 reacts with only organomercurials, and class 2 reacts with both organomercurials and MMTS. Quantitative analysis of the competition between p-chloromercuriphenylsulfonate and VAChT ligands was possible after defining second-order reaction conditions. The results indicate that each cysteinyl class probably contains a single residue. Acetylcholine protects cysteine 1, but apparently does not protect cysteine 2. Vesamicol, which binds to a different site than acetylcholine does, apparently protects both cysteines, suggesting that it induces a conformational change. The relatively large reagent glutathione removes a substituent from cysteine 1, but not cysteine 2, suggesting that cysteine 2 is deeper in the transporter than cysteine 1 is. The complete sequence of T. californica VAChT is given, and possible identities of cysteines 1 and 2 are discussed. PMID- 10737634 TI - Differential assembly of human tau isoforms in the presence of arachidonic acid. AB - Six tau isoforms arise from the alternative splicing of a single gene in humans. Insoluble, filamentous deposits of tau protein occur in a number of neurodegenerative diseases, and in some of these diseases, the deposition of polymers enriched in certain tau isoforms has been documented. Because of these findings, we have undertaken studies on the efficacy of fatty acid-induced polymerization of the individual tau isoforms found in the adult human CNS. The polymerization of each tau isoform in the presence of two concentrations of arachidonic acid indicated that isoforms lacking N-terminal exons e2 and e3 formed small, globular oligomers that did not go on to elongate into straight (SF) or paired helical (PHF) filaments under our buffer conditions. The polymerization of all isoforms containing e2 or e2 and e3 occurred readily at a high arachidonic acid concentration. Conversely, at a lower arachidonic acid concentration, only tau isoforms containing four microtubule binding repeats assembled well. Under all buffer conditions employed, filaments formed from three of the isoforms containing e2 and e3 resembled SFs in morphology but began to form PHF-like structures following extended incubation at 37 degrees C. These results indicate that polymerization of the intact tau molecule may be facilitated by e2 and e3. Moreover, tau isoforms containing three versus four microtubule binding repeats display different assembly properties depending on the solvent conditions employed. PMID- 10737635 TI - Interaction of human substantia nigra neuromelanin with lipids and peptides. AB - Neuromelanin was isolated from human substantia nigra using different procedures. In the pigment isolated by any of these procedures a peptide component covalently bound to the melanic structure was found, as shown by treatment with reagents known to eliminate noncovalently bound proteins. The amino acid content of such a peptide component was reproducible and corresponded to approximately 15% of the neuromelanin weight. Neuromelanin also showed the ability to absorb specifically lipid molecules, approximately 20% of its weight, and among these lipids cholesterol was identified, constituting approximately 5% of the total lipid mixture. A synthetic melanin, incubated with putamen homogenate, bound tissue peptides with an amino acid content quite close to that of neuromelanin. The same synthetic melanin adsorbed a lower amount of lipids from the putamen homogenate compared with neuromelanin. The sulfur content of neuromelanin was also reproducible even using different isolation procedures. A nonpigmented tissue like corpus callosum was used as a control and extracted by the method used for neuromelanin isolation; a total elimination of tissue components was found, thus demonstrating the capability of the reported procedures to isolate neuromelanin alone. The presence of a peptide component in the neuromelanin structure and the selective affinity for lipid molecules suggest new aspects of the functional role and metabolic pathway of neuromelanin. PMID- 10737636 TI - Adrenomedullin receptor is found exclusively in noradrenaline-secreting cells of the rat adrenal medulla. AB - Adrenomedullin, originally identified in the adrenal medulla, has binding sites in the adrenal gland; however, its role in the adrenal medulla is unclear. This study was designed to characterise adrenomedullin binding sites in the rat adrenal medulla, using ligand binding studies, immunocytochemistry, and mRNA analysis. A single population of specific adrenomedullin receptors was identified in adrenal medullary homogenates. 125I-Adrenomedullin was displaced only by adrenomedullin1-50 and not by calcitonin gene-related peptide or amylin at concentrations up to 100 nmol/L. The receptor K(D) was 3.64 nmol/L with a receptor density of 570 fmol/mg of protein. Analysis of mRNA revealed that the genes encoding both the putative adrenomedullin receptors, termed calcitonin receptor-like receptor (CRLR) and L1, were expressed in the rat adrenal medulla. Dual-colour indirect-labelled immunofluorescence was used to localise phenylethanolamine N-methyltransferase (PNMT) and the adrenomedullin receptor in the same section. PNMT is the enzyme that converts noradrenaline to adrenaline and is not expressed in noradrenaline-secreting cells. These studies revealed that both CRLR and L1 were expressed only in cells that did not express PNMT, suggesting that adrenomedullin receptors are only found in noradrenaline secreting cells. Further evidence to support this conclusion was provided by the demonstration of colocalisation of adrenomedullin receptors with dopamine beta hydroxylase, confirming the presence of the receptors in medullary chromaffin cells. Taken together, these data suggest that adrenomedullin acts through a specific adrenomedullin receptor in the rat adrenal medulla. RT-PCR and northern blot analysis revealed greater abundance of mRNA for L1 than for CRLR, possibly suggesting that L1 may be the major adrenomedullin receptor expressed in this tissue. As it has been reported that adrenomedullin is synthesised predominantly by adrenaline-secreting cells, it appears likely that adrenomedullin is a paracrine regulator in the adrenal medulla. PMID- 10737637 TI - Cloning of rat parkin cDNA and distribution of parkin in rat brain. AB - The rat parkin cDNA sequence was characterized after screening a rat hypothalamus cDNA library with a 32P-labeled probe containing the entire open reading frame of the human parkin cDNA. This sequence encompasses 1,576 bp and contains a single open reading frame that encodes a 465-amino acid protein. The rat parkin amino acid sequence exhibits a very striking homology to the human and mouse parkin, with 85 and 95% identity, respectively. Both the N-terminal ubiquitin and the ring-IBR (in between ring)-ring finger domains appear to be highly conserved among rat, human, and mouse parkin. An affinity-purified polyclonal antibody (ASP5p) was generated with a synthetic peptide corresponding to amino acids 295 311 of the parkin sequence, which is identical in the three species. Western blotting revealed that ASP5p recognizes a single 52-kDa band, which corresponds to the molecular mass of the parkin protein. Immunostaining with ASP5p showed that parkin is principally located in the cytoplasm of neurons that are widely distributed in the rat brain. Parkin-immunoreactive neurons abound in structures that are specifically targeted in Parkinson's disease, e.g., subtantia nigra, but are also present in unaffected structures, e.g., cerebellum. Furthermore, parkin enriched glial cells can be detected in various nuclei of the rat brain. Thus, the role of parkin may be much more global than previously thought on the basis of genetic findings gathered in cases of early-onset parkinsonism. PMID- 10737638 TI - Efficacy evaluation of lasalocid plus roxarsone combination medication with different geographic field strains of Eimeria acervulina. AB - Performance of broiler chickens medicated with lasalocid alone (at 125 ppm) or in combination with roxarsone (at 50 ppm) was evaluated in battery and floorpen trials after challenge with geographically different field strains of coccidia containing predominately the upper intestinal species Eimeria acervulina. No significant difference in bird performance measured at 6 days postinfection (PI) was observed between lasalocid plus roxarsone-medicated (L+RM) or lasalocid medicated (LM) birds challenged in separate battery trials with mixed-species inocula from Alabama or Georgia containing 92% or 88% E. acervulina, respectively. In contrast, L+RM birds challenged in another battery trial with a Louisiana mixed-species inoculum containing 92% E. acervulina showed significant reduction in average weight gain at 6 days PI compared with LM-challenged birds. A floorpen trial done with the same Louisiana inoculum showed significant reduction in average bird weight gain at 27 and 35 days of age (6 and 14 days PI) for L+RM-challenged birds compared with both unmedicated-nonchallenged (UMNC) control and LM-challenged birds. The LM+R groups were significantly lower in average bird weight at 27 days of age than the unmedicated-challenged controls. Feed conversions (FCs) for L+RM birds were significantly higher than those for the UMNC control birds during time of challenge (21-27 days of age) and for the 1 to-27-day-of-age time period. No significant difference in FC was seen between the UMNC and LM groups. Results of this study showed that performance of broiler birds medicated with lasalocid plus roxarsone could vary for geographically different mixed-species challenge inocula that contained predominately E. acervulina. PMID- 10737639 TI - Effect of route of vaccination on the prevention of infectious laryngotracheitis in commercial egg-laying chickens. AB - Commercial egg-laying chickens were vaccinated for infectious laryngotracheitis (ILT) with one of five commercially available vaccines (designated A, B, C, D, and E) on five separate farms by either eyedrop (e), spray (s), or double dose in the water (w) method. Groups were identified by the vaccine designation and the method of vaccination. Birds from the test groups were transferred to an isolation facility and challenged intratracheally 3 wk after vaccination. The remaining birds were given a second vaccination with the original chicken embryo origin vaccine by spray or a chicken embryo origin vaccine if the first vaccine was of tissue culture origin. After challenge, birds were monitored for clinical signs. Those surviving were euthanatized on day 6 postchallenge, and tissues and blood were collected for histopathology, virus isolation, and serology. On the basis of histopathology and enzyme-linked immunosorbent assay (ELISA) results, after one vaccination, all chickens given vaccines by eyedrop were provided better protection than nonvaccinated controls (CTLs). Birds in groups Bs and Ds had lower microscopic lesion scores whereas only birds given Bs had higher ELISA titers than CTLs. Birds in groups As and Cs and groups Bw birds taken from the rear of the barn (r) had microscopic lesion scores that were no different from those of CTLs. These same birds in addition to vaccine Ds had ELISA titers no different from those of CTLs. Of all vaccines, only A given by eyedrop or spray produced higher virus isolation titers than those of CTLs. The remainder of the vaccines produced virus isolation titers that were no different from those of CTLs. After two vaccinations, all groups had lower microscopic lesion scores than CTLs. Only Bw birds from the middle of the barn Bs, EeDs, and AsAs had virus isolation results that were higher than those of CTLs. Only groups BwrBs, CsCs, and DsDs had ELISA titers no different from those of controls. These results suggest that a priming vaccination followed by a booster dose offers better protection against ILT than a single vaccination alone. Vaccine application by eyedrop provides more uniform protection if only one vaccination is given, whereas spray vaccination may serve as an alternative method of vaccination for birds receiving two doses of vaccine. PMID- 10737640 TI - Experimental and serologic observations on avian pneumovirus (APV/turkey/Colorado/97) infection in turkeys. AB - An avian pneumovirus (APV) was isolated from commercial turkeys in Colorado (APV/Colorado) showing clinical signs of a respiratory disease. The results of virus neutralization and indirect fluorescent antibody tests showed that the APV/Colorado was partially related to APV subgroup A but was unrelated to APV subgroup B. Turkeys experimentally inoculated with the APV/Colorado were observed for signs, lesions, seroconversion, and virus shedding. Thirty-six 7-wk-old turkeys were distributed into three groups. Eighteen turkeys were inoculated oculonasally with APV/Colorado, six were placed in contact at 1 day postinoculation (DPI), and 12 served as noninoculated controls. Tracheal swabs and blood samples were collected at 3, 5, 7, 10, 14, and 21 DPI. Tissues were collected from three inoculated and two control turkeys on aforementioned days for pathologic examination and APV isolation. Inoculated turkeys developed respiratory disease, yielded APV at 3, 5, and 7 DPI, and seroconverted at 10 DPI. Contact turkeys yielded APV at 7 and 10 DPI. No gross lesions were observed in the turbinates, infraorbital sinuses, and trachea. However, microscopic examination revealed acute rhinitis, sinusitis, and tracheitis manifested by congestion, edema, lymphocytic and heterophilic infiltration, and loss of ciliated epithelia. The inflammatory lesions were seen at 3 DPI and became extensive at 5 and 7 DPI. Active regenerative changes in the epithelia were seen at 10 and 14 DPI. Serologic survey for the presence of antibodies in commercial turkeys (24,504 sera from 18 states) and chickens (3,517 sera from 12 states) to APV/Colorado showed seropositive turkeys in Minnesota, North Dakota, and South Dakota and no seropositive chickens. This report is the first on the isolation of an APV and APV infection in the United States. PMID- 10737641 TI - Complement resistance-related traits among Escherichia coli isolates from apparently healthy birds and birds with colibacillosis. AB - In this study, 294 Escherichia coli isolates from birds with colibacillosis were collected from disease outbreaks throughout the United States and were compared with 75 fecal E. coli isolates of apparently healthy chickens by their possession of several purported virulence genes, resistance to rough-lipopolysaccharide specific bacteriophages (rLPSr), and elaboration of capsule. Traits were selected for study on the basis of their association with complement resistance. The genes targeted in this study included those encoding colicin V (cvaC) and the outer membrane proteins TraT (traT), OmpA (ompA), and Iss (iss). No significant differences were found between the two groups of isolates in the occurrence of cvaC-, traT-, or ompA-homologous sequences or in rLPSr. Only a few isolates were encapsulated, and the isolates of healthy birds were significantly more likely to be encapsulated than were the isolates of sick birds. However, iss, whether detected through hybridization or amplification, was found in more of the disease associated isolates than in those of healthy birds. This difference was highly significant. Further, iss sequences were widely distributed among isolates of different serotypes from various avian host species and sites within these hosts. Such results suggest that possession of the iss sequence by an avian E. coli isolate may be a good indicator of that isolate's potential to cause disease. This association warrants further study because iss and the protein it encodes may be useful targets of future colibacillosis control efforts. PMID- 10737642 TI - Nucleotide sequence and vaccinia expression of the nucleoprotein of a highly virulent, neurotropic strain of Newcastle disease virus. AB - The nucleoprotein (NP) of Newcastle disease virus (NDV) was selected to study the relative importance of an internal structural protein in the avian immune response. The NP gene of the virulent, neurotropic NDV Texas GB (TGB) strain was cloned and sequenced. Nucleotide sequence data for the NP gene allowed comparison of the deduced amino acid sequences for the NP genes of NDV-TGB and the avirulent duck isolate NDV-D26. These comparisons demonstrated an 89% nucleotide sequence homology and a 97% homology between the deduced amino acid sequences. The NDV-TGB NP expressed in recombinant vaccinia virus (rVAC) was electrophoretically and immunologically identical to the wild-type NDV-TGB. Although inoculation of chickens with the recombinant vaccinia virus expressing the NDV NP gene elicited anti-NDV antibodies in higher titers than in birds inoculated with live LaSota NDV, this strong anti-NDV response did not protect against lethal challenge with NDV-TGB. PMID- 10737643 TI - Characterization of the stunting syndrome agent: relatedness to known viruses. AB - An enteric disease of young turkeys, referred to as stunting syndrome (SS), causes reduced growth and impaired feed efficiency. A recently isolated virus, stunting syndrome agent, (SSA) has been found to be the etiologic agent of SS. The objective of the present study was to determine relatedness of the SSA with other viral agents. Serologic (viral neutralization and enzyme-linked immunosorbent assay [ELISA]) assays and a reverse transcriptase-polymerase chain reaction (RT-PCR) were used. The antisera against turkey enteric coronavirus (bluecomb agent), bovine coronavirus (BCV), bovine Breda-1 virus, bovine Breda-2 virus, avian infectious bronchitis virus (IBV), avian influenza virus, Newcastle disease virus (NDV), and transmissible gastroenteritis virus (TGEV) of swine were evaluated by dot-immunobinding avidin-biotin-enhanced ELISA and did not react with SSA. The homologous (anti-SSA) antiserum was positive by ELISA. Similarly, anti-SSA antiserum did not react when NDV, IBV, BCV, or TGEV was used as antigen but did react with the homologous (SSA) virus. The virus neutralization assay was performed by inoculating 24-to-25-day-old turkey embryos via the amniotic route and by assessing the embryo infectivity on the basis of gross intestinal lesions and intestinal maltase activity at 72 hr postinoculation. None of the aforementioned antisera neutralized SSA infectivity in embryos except for the homologous anti-SSA antiserum. A RT-PCR was performed with known primers specific for NDV, IBV, BCV, and TGEV. The known primers failed to amplify SSA genome but amplified their respective viral genomes. We concluded that the SSA was distinct from the viral agents that were evaluated. PMID- 10737644 TI - Chicken anemia virus and fowl adenoviruses: association to induce the inclusion body hepatitis/ hydropericardium syndrome. AB - The effects of a simultaneous and/or a subsequent coinfection with chicken anemia virus (CAV) isolate 10343 and fowl adenovirus (FAV) isolate 341 in specific pathogen-free light chickens were evaluated. The simultaneous coinfection was conducted by the intramuscular route, whereas the subsequent coinfection trial considered FAVs administered orally. In trial 1, 20-day-old chickens simultaneously coinfected with CAV (10343) and FAV (341) intramuscularly (i.m.) showed 55% mortality and characteristic signs and lesions of inclusion body hepatitis/hydropericardium (IBH/HPS). In contrast, birds singly infected with FAV i.m. showed 10% mortality due to IBH/HPS. Trial 2 showed that birds receiving FAV 341 orally at day 7 post-CAV intramuscular infection (group A) developed a mild form of IBH/HPS with presence of inclusion bodies (INIBs) in 60% of the group and virus-neutralizing antibodies against FAV 341. Group B (FAV orally 14 days after CAV) showed significant decreased weight gain, nonspecific microscopic lesions in the liver, spleen, bursa, and thymus, and an antibody response against FAV 341. However, no INIBs could be detected in the hepatocytes of these chickens. Group C (FAV orally 35 days after CAV) showed nonspecific histopathologic changes in the liver and no antibody response to FAV. The oral single infection with FAV isolate 341 induced neither mortality nor macroscopic lesions of IBH/HPS in the birds. The present results corroborate previous reports on pathogenicity of Chilean FAV isolates, which suggest that synergism with other viruses or prior immunosuppression is necessary to produce IBH/HPS in chickens. These results also suggest that the susceptibility of chickens to FAV oral infection resulting in IBH/HPS varies throughout the course of CAV infection. PMID- 10737645 TI - Alterations in macrophage-produced cytokines and nitrite associated with poult enteritis and mortality syndrome. AB - Poult enteritis and mortality syndrome (PEMS) is an acute, transmissible, infectious intestinal disease associated with high mortality and morbidity in turkey poults. Earlier studies demonstrated immune dysfunction, involving both humoral and cell-mediated immunity, associated with PEMS. The current study examined cytokines and metabolites produced by macrophages from poults exposed to PEMS agent(s). Six trials were conducted with six separate hatches of poults. Poults in the PEMS group were exposed to PEMS agent(s) via contact exposure at 7 days of age whereas uninfected poults served as control poults. Abdominal macrophages were harvested from control (uninfected) and PEMS poults at various times postexposure and cultured for 18-24 hr in the presence of Escherichia coli lipopolysaccharide. Interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) bioactivities and nitrite levels in macrophage culture supernatants were quantified. Macrophage supernatants from PEMS poults had greater IL-1-mediated stimulation index compared with the macrophage supernatants from uninfected control poults in both trials. However, this increase was significant only in trial 1. IL-6 activity tested in three separate trials was significantly higher in PEMS macrophage supernatants over the controls. On the contrary, TNF-alpha production by macrophages was decreased in PEMS macrophage culture supernatants. Nitrite levels in PEMS macrophage culture supernatants were significantly higher in two out of three trials. These findings suggest that the enhanced production of proinflammatory cytokine/metabolites by activated macrophages in PEMS poults may be responsible, at least in part, for the physiological intestinal inflammation, gut motility, and anorexia that characterize this disease. PMID- 10737646 TI - Genetic relationships of infectious bronchitis virus isolates from Mississippi broilers. AB - A 582-base pair segment located in the nucleocapsid protein terminal part of the S1 gene of 26 Arkansas (Ark)-type infectious bronchitis virus (IBV) isolates from Mississippi broilers was amplified and sequenced. Reverse transcription polymerase chain reaction and cycle sequencing techniques were used to elucidate the genetic and deduced amino acid relationships among the isolates. Analysis suggested that the nucleotide and amino acid sequences of the isolates were highly conserved, with greater than 99.4% nucleotide and 98.4% amino acid homology among the Mississippi isolates. The Mississippi isolates had less than 2.3% nucleotide and 5.2% amino acid variation when compared with the vaccine strain Ark DPI and less than 3.0% nucleotide and 5.2% amino acid variation when compared with the Ark-type Georgia variant. This study suggests that the 26 IBV isolates that are genetically closely related to the vaccine strain may be derived from the vaccine strain by point mutation. The results also indicate that within a 1-yr period Ark-type IBV in Mississippi was spread with little or no change in its genetic sequence. The 21 identical isolates appeared in different farms and companies, suggesting that poor biosecurity was practiced and contributed to the spreading of the disease. PMID- 10737647 TI - Echocardiographic study of pulmonary hypertension syndrome in broiler chickens. AB - Echocardiography was used to study cardiovascular structure and function during the development of pulmonary hypertension syndrome (PHS) in broiler chickens. Body weight-normalized right and left ventricular diameters at both end-diastole (RVDD, LVDD) and end-systole (RVDS, LVDS) were determined weekly in broilers reared under either normobaric (altitude, 96.7 m) or hypobaric conditions (simulated altitude, 2900 m) until 5 wk of age. Hypobaric-exposed broilers had larger RVDD at 3 and 4 wk of age and larger RVDS at 3, 4, and 5 wk of age. Hypobaric-exposed broilers also had larger LVDD at 2, 3, 4, and 5 wk of age and larger LVDS at 4 wk of age. Right (RVFS) and left ventricular fractional shortening (LVFS) were smaller in hypobaric- vs. normobaric-exposed broilers at 3, 4, and 5 wk of age and at 4 wk of age, respectively. Among hypobaric-exposed birds, PHS-positive (+) broilers had larger RVDD and RVDS than PHS-negative (-) broilers on week 3 and on weeks 1 and 3 after hypobaric exposure, respectively. PHS-positive (+) broilers also had smaller RVFS on week 1 after hypobaric exposure. Electrocardiographic and post-mortem data indicated that PHS+ broilers also developed right ventricular hypertrophy when compared with PHS-negative (-) broilers. These results are consistent with the hypothesis that PHS develops as a result of pulmonary hypertension and cardiac overload and suggest that PHS+ broilers have a greater and more persistent reaction to hypoxia than PHS- broilers. PMID- 10737648 TI - Minimum inhibitory concentration determinations for various antimicrobial agents against 1570 bacterial isolates from turkey poults. AB - Minimum inhibitory concentrations (MICs) were determined for 1570 bacteria from eight geographic locations (1204 Escherichia coli, 231 other enteric gram negative bacilli [including Citrobacter spp., Enterobacter spp., Klebsiella spp., Proteus spp., and Salmonella spp.], 31 Pseudomonas spp., 18 coagulase-positive staphylococci, 26 coagulase-negative staphylococci, and 55 streptococci and enterococci) by the National Committee for Clinical Laboratory Standards broth microdilution procedure. Antimicrobial agents tested included ampicillin, ceftiofur, enrofloxacin, erythromycin, florfenicol, gentamicin, neomycin, spectinomycin, sulfamethazine, tetracycline, and trimethoprim/sulfadiazine. Against the E. coli strains tested, ceftiofur, enrofloxacin, gentamicin, and trimethoprim/sulfadiazine were the most active compounds with MIC at which 50% of the strains are at or below (MIC50) = 0.5, < or = 0.03, 0.5, and 0.13 microg/ml, respectively, and MIC at which 90% of the strains are at or below (MIC90) = 1.0, 0.13, 32.0, and 2.0 microg/ml, respectively. Ampicillin, florfenicol, neomycin, and spectinomycin were the next most active compounds against the E. coli strains, with MIC50 = 4.0, 4.0, 16.0, and 16.0 microg/ml, respectively. MIC90 values for these compounds against E. coli strains were > 32.0, 8.0, 512.0, and > 128.0 microg/ml, respectively. The remaining compounds exhibited limited, strain dependent activity against the E. coli strains tested. As with the E. coli, enrofloxacin, ceftiofur, and trimethoprim/sulfadiazine were also the most active compounds against the 231 other enteric organisms tested, with MIC50 < or = 1.0 microg/ml for all of these genera. The remaining compounds exhibited limited activity against these genera. Against the gram-positive cocci tested, ampicillin, enrofloxacin, ceftiofur, and trimethoprim/sulfadiazine were most active, whereas the remaining compounds exhibited strain-dependent activity. When MIC data for E. coli were summarized separately, differences were observed between the geographic locations for the various antimicrobial agents. In conclusion, ceftiofur, enrofloxacin, and trimethoprim/sulfadiazine were the most active of the compounds tested against all of the bacterial strains. PMID- 10737649 TI - A rapid-plate hemagglutination assay for the detection of infectious bronchitis virus. AB - A rapid-plate hemagglutination (HA) test to detect infectious bronchitis virus (IBV) in allantoic fluid of embryonated eggs was introduced into routine procedures for IBV identification. This system was tested in 468 diagnostic cases received by the Poultry Diagnostic and Research Center at the University of Georgia. Allantoic fluids from inoculated embryos were harvested and treated with commercially available neuraminidase enzyme. IBV in neuraminidase-treated allantoic fluid was identified by clear and consistent HA of chicken red blood cells within 1 min of incubation. The specificity of the neuraminidase rapid plate HA assay was examined with other avian viruses in individual and dual embryonic infections. Sensitivity of this test was compared with embryo lesions and reverse transcriptase-polymerase chain reaction (RT-PCR). The rapid-plate HA assay of neuraminidase-treated allantoic fluid correlated with the RT-PCR during the early stages of IBV detection, identification, and isolation in embryonated eggs. PMID- 10737650 TI - High mortality and growth depression experimentally produced in young turkeys by dual infection with enteropathogenic Escherichia coli and turkey coronavirus. AB - Six-day-old turkeys were inoculated with turkey coronavirus (TCV) and an enteropathogenic Escherichia coli (EPEC) (isolate R98/5) that were isolated from poult enteritis and mortality syndrome (PEMS)-affected turkeys. Turkeys inoculated with only R98/5 did not develop clinically apparent disease, and only mild disease and moderate growth depression were observed in turkeys inoculated with only TCV. Turkeys dually inoculated with TCV and R98/5 developed severe enteritis with high mortality (38/48, 79%) and marked growth depression. R98/5 infection resulted in attaching/effacing (AE) intestinal lesions characteristic of EPEC: adherence of bacterial microcolonies to intestinal epithelium with degeneration and necrosis of epithelium at sites of bacterial attachment. AE lesions were more extensive and were detected for a prolonged duration in dually inoculated turkeys compared with turkeys inoculated with only R98/5. An apparent synergistic effect in dually inoculated turkeys was indicated by increased mortality, enhanced growth depression, and enhanced AE lesion development. The results suggest that TCV promoted intestinal colonization by R98/5; however, R98/5 did not appear to alter TCV infection. The present study provides a possible etiologic explanation for PEMS. PMID- 10737651 TI - Comparative susceptibility of Marek's disease cell lines to chicken infectious anemia virus. AB - Chicken infectious anemia virus (CIAV) is known to infect and replicate in various Marek's disease chicken cell lines (MDCCs) derived from Marek's disease (MD) tumors. One line, MDCC-MSB1, has been the substrate used in most studies. We compared a total of 26 MDCCs, including two sublines of MDCC-MSB1, MSB1 (L) and MSB1 (S), four other MD tumor-derived lines, and 20 lines derived from MD virus induced local lesions, for susceptibility to the Cux-1 and CIA-1 strains of CIAV. The cell lines represented six phenotypic groups of T cells based on the expression of CD4, CD8, and TCR-2 and -3 surface markers. Susceptibility was measured by the number of cells positive for viral antigen in immunofluorescence (IF) tests at 3-10 days postinfection. No clear-cut differences were found in susceptibility related to phenotype, although CD4-/8+ lines and CD4-/8- lines might be more susceptible than CD4+/8- lines. However, several individual lines were more susceptible to Cux-1 than the two MSB1 sublines tested. Contrary to an earlier report, cells of MDCC-CU147, a CD8+, TCR3+, local-lesion derived line, were found to be susceptible to CIA-1. In fact, CU147 was distinguished by very high susceptibility to both CIAV strains. In direct comparisons with MSB1, CU147 detected approximately 10-fold lower doses of virus. Also, virus spread was faster (P < 0.05) in CU147 than in MSB1 and other lines. Results from polymerase chain reaction (PCR) tests to detect infection in titrations were in general agreement with IF test results although PCR detected infection in a few terminal dilution cultures that were negative by IF. PMID- 10737652 TI - A homopolymer stretch composed of variable numbers of cytidine residues in the terminal repeats of infectious laryngotracheitis virus. AB - A homopolymer stretch composed of variable numbers of cytidine residues was found within the inverted terminal repeats of infectious laryngotracheitis virus (ILTV). A polymerase chain reaction procedure was developed to amplify a 750-bp fragment containing this homopolymer stretch. This fragment was then sequenced directly to determine the number of repeated cytidine residues in this homopolymer stretch, which could be used for strain differentiation. By this procedure, vaccine strains of tissue culture origin could be differentiated into two types: type I contains eight repeated cytidine residues, whereas type II contains 10 such residues. Vaccine strains of chicken embryo origin could also be divided into two types: type I contains mainly 11 repeated cytidine residues, whereas type II contains 15-21 such repeats. In comparison, two of the five field isolates examined contain 12-13 repeats; the other three field isolates contain 15-19 repeats, which were similar to the type II chicken-embryo-origin vaccines. The number of repeated cytidine residues described here could serve as a marker for the strain differentiation and epidemiologic study of ILTV. PMID- 10737653 TI - Failure of a recombinant fowl poxvirus vaccine containing an avian influenza hemagglutinin gene to provide consistent protection against influenza in chickens preimmunized with a fowl pox vaccine. AB - Vaccines against mildly pathogenic avian influenza (AI) have been used in turkeys within the United States as part of a comprehensive control strategy. Recently, AI vaccines have been used in control programs against highly pathogenic (HP) AI of chickens in Pakistan and Mexico. A recombinant fowl pox-AI hemagglutinin subtype (H) 5 gene insert vaccine has been shown to protect specific-pathogen free chickens from HP H5 AI virus (AIV) challenge and has been licensed by the USDA for emergency use. The ability of the recombinant fowl pox vaccine to protect chickens preimmunized against fowl pox is unknown. In the current study, broiler breeders (BB) and white leghorn (WL) pullets vaccinated with a control fowl poxvirus vaccine (FP-C) and/or a recombinant fowl poxvirus vaccine containing an H5 hemagglutinin gene insert (FP-HA) were challenged with a HP H5N2 AIV isolated from chickens in Mexico. When used alone, the FP-HA vaccine protected BB and WL chickens from lethal challenge, but when given as a secondary vaccine after a primary FP-C immunization, protection against a HP AIV challenge was inconsistent. Both vaccines protected against virulent fowl pox challenge. This lack of consistent protection against HPAI may limit use to chickens without previous fowl pox vaccinations. In addition, prior exposure to field fowl poxvirus could be expected to limit protection induced by this vaccine. PMID- 10737654 TI - Protective immunity against Newcastle disease: the role of antibodies specific to Newcastle disease virus polypeptides. AB - Studies were performed to determine if passive immunization with hyperimmune sera generated to specific Newcastle disease virus (NDV) proteins conferred protection against virus challenge. Six groups of 3-wk-old chickens were passively immunized with antiserum against either hemagglutinin-neuraminidase/fusion, (HN/F) protein, nucleoprotein/phosphoprotein (NP/P), Matrix (M) protein, a mixture of all NDV proteins (ALL), intact ultraviolet-inactivated NDV (UVNDV), or negative sera. Blood samples were collected 2 days postimmunization, and the birds were challenged with Texas GB strain of NDV. Antibody titers were detected from those recipient birds that had received the antisera against the HN/F, ALL, or UVNDV by a hemagglutination inhibition test, an enzyme-linked immunosorbent assay (ELISA), and a virus neutralization test. Antibodies were detected only by the ELISA from the birds that had received antisera against NP/P and M protein. Antibody titers in the recipient birds dropped by two dilutions (log2) after 2 days postinjection. Birds passively immunized with antisera against HN/F, ALL, and UVNDV were protected from challenge, whereas chickens passively immunized with antisera against NP/P and M protein and specific-pathogen-free sera developed clinical signs of Newcastle disease. The challenge virus was recovered from the tracheas of all passively immunized groups. The presence of neutralizing antibodies to NDV provided protection from clinical disease but was unable to prevent virus shedding from the trachea. PMID- 10737655 TI - Protective immunity against Newcastle disease: the role of cell-mediated immunity. AB - The role of cell-mediated immunity (CMI) in protection of birds from Newcastle disease was investigated by two different strategies in which only Newcastle disease virus (NDV)-specific CMI was conveyed without neutralizing antibodies. In the first strategy, selected 3-wk-old specific-pathogen-free (SPF) birds were vaccinated with either live NDV (LNDV), ultraviolet-inactivated NDV (UVNDV), sodium dodecyl sulfate-treated NDV (SDSNDV), or phosphate-buffered saline (PBS) (negative control) by the subcutaneous route. Birds were booster vaccinated 2 wk later and challenged with the velogenic Texas GB strain of NDV 1 wk after booster. All vaccinated birds had specific CMI responses to NDV as measured by a blastogenesis microassay. NDV neutralizing (VN) and hemagglutination inhibition (HI) antibody responses were detected in birds vaccinated with LNDV and UVNDV. However, birds vaccinated with SDSNDV developed antibodies that were detected by western blot analysis but not by the VN or HI test. Protection from challenge was observed only in those birds that had VN or HI antibody response. That is, birds with demonstrable CMI and VN or HI antibody response were protected, whereas birds with demonstrable CMI but no VN or HI antibody response were not protected. In the second strategy, birds from SPF embryos were treated in ovo with cyclophosphamide (CY) to deplete immune cells. The birds were monitored and, at 2 wk of age, were selected for the presence of T-cell activity and the absence of B cell activity. Birds that had a significant T-cell response, but not a B-cell response, were vaccinated with either LNDV, UVNDV, or PBS at 3 wk of age along with the corresponding CY-untreated control birds. The birds were booster vaccinated at 5 wk of age and were challenged with Texas GB strain of NDV at 6 wk of age. All birds vaccinated with LNDV or UVNDV had a specific CMI response to NDV, VN or HI NDV antibodies were detected in all CY-nontreated vaccinated birds and some of the CY-treated vaccinated birds that were found to have regenerated their B-cell function at 1 wk postbooster. The challenge results clearly revealed that CY-treated birds that had NDV-specific CMI and VN or HI antibody responses to LNDV or UVNDV were protected, as were the CY-nontreated vaccinated birds. However, birds that had NDV-specific CMI response but did not have VN or HI antibodies were not protected from challenge. The results from both strategies indicate that specific CMI to NDV by itself is not protective against virulent NDV challenge. The presence of VN or HI antibodies is necessary in providing protection from Newcastle disease. PMID- 10737656 TI - Retrospective study on Escherichia coli infection in broilers subjected to postmortem examination and antibiotic resistance of isolates in Trinidad. AB - An 8-yr retrospective study was conducted to evaluate the rate of Escherichia coli infection and antibiotic resistance of isolates from diseased broilers submitted for diagnosis in Trinidad from 1990 to 1997. Of a total of 906 cases of diseased birds subjected to postmortem examination, 603 (66.6%) had E. coli infection. The number of cases increased over the years from 16 in 1990 to a peak of 294 in 1996. For every year, at least 50% of all broiler cases had E. coli infection. The rate of infection was significantly higher during the rainy season (74.1 +/- 6.9%) than during the dry season (57.8 +/- 7.0%). Approximately 50% of all E. coli isolates were resistant to 9 out of a total of 11 antimicrobial drugs selected for the study. The isolates showed an increasing trend of resistance to amoxicillin, apramycin, gentamicin, nitrofurantoin, norfloxacin, and sulfamethoxazole-trimethoprim. However, only the trends of resistance to apramycin and norfloxacin were statistically significant. Overall, of the antimicrobial drugs selected, norfloxacin relatively appeared as the best choice for treatment. From this study, we conclude that the high rate of E. coli infection in broilers submitted for diagnosis along with the high resistance of isolates to antimicrobial drugs constitute a threat to the poultry industry on the island. PMID- 10737657 TI - In situ hybridization, immunohistochemistry, and in situ reverse transcription polymerase chain reaction for detection of infectious bursal disease virus. AB - Development of molecular techniques for the detection of infectious bursal disease virus (IBCV) is an important area of research. An in situ hybridization (ISH) test was developed with a 491-bp cDNA fragment derived from the VP2 gene of IBDV. The fragment was amplified and simultaneously labeled with incorporation of digoxigenin-11-dUTP in a nested polymerase chain reaction (PCR) assay. The resulting digoxigenin-labeled 491-bp nested PCR product was used as probe for ISH to detect and localize IBDV RNA in formalin-fixed, paraffin-embedded bursae of Fabricius from chickens both experimentally infected as well as commercially reared. Bursae from six clinically ill commercial broilers suspected to be IBDV infected were examined by ISH and immunohistochemistry. In two samples, IBDV infection was detected by both ISH and immunohistochemistry, whereas in the other two histologically normal bursae, IBDV was detected only by ISH. Two commercial chickens with atrophied bursae were negative by both ISH and immunohistochemistry. No positive IBDV stained cells were in RNase treated sections from infected birds, uninfected chickens, or reovirus-infected chickens. The ISH test developed herein resulted in important modifications, which makes it superior to other previously published procedures. We also described a direct in situ reverse transcription-polymerase chain reaction method for the amplification and detection of IBDV genome in formalin fixed, paraffin-embedded bursae of Fabricius with a single primer pair with direct incorporation of digoxigenin-11 deoxyuraciltriphosphate (dUTP) into the amplicon. Both molecular tests with their important modifications represent improved detection of IBDV. PMID- 10737658 TI - Cloning and expression of the VP2 gene of an infectious bursal disease virus. AB - A serotype I infectious bursal disease virus (IBDV) strain HZ96 was isolated in Hangzhou, China, in 1996 and attenuated by adaptation to chicken embryo fibroblast cells. The VP2 gene of strain HZ96 was amplified by reverse transcription-polymerase chain reaction, cloned, and sequenced. Compared with the VP2 sequences of other IBDV strains, HZ96 is most related to two attenuated strains, CU-1 and PBG98, and two attenuated Chinese strains, Harbin and CJ801bkf. HZ96 shares nucleotide sequence homology 98.9% with CU-1 and PBG98, 98.5% with Harbin, and 98.6% with CJ801bkf. Most of the sequence variations observed between HZ96 and other strains are located in the middle variable region from nucleotides 637 to 996. Similar to other attenuated IBDVs, HZ96 has unique substitutions at residues 279 (Asp to Asn) and 284 (Ala to Thr), suggesting that these two substitutions may be directly related to adaptation of the virus to cell culture and attenuation of its virulence. As part of our effort to develop a submit vaccine for IBDV, the VP2 gene of HZ96 was cloned into a heat-inducible expression vector and expressed in Escherichia coli system. A protein band with expected molecular weight of 52 kD was detected by direct protein staining and western blotting. PMID- 10737659 TI - Cloning and sequencing of the iss gene from a virulent avian Escherichia coli. AB - Control of colibacillosis is important to the poultry industry. We have found that the presence of a gene for increased serum survival, iss, is strongly correlated with Escherichia coli isolated from birds with colibacillosis. Therefore, the iss gene and its protein product, Iss, are potential targets for detection and control of avian colibacillosis. The iss gene was amplified from a virulent avian E. coli isolate and sequenced. The sequences of the gene and the predicted protein product were compared with those of iss from a human E. coli isolate and lambda bor. The iss gene from the avian E. coli isolate has 96.8% identity with the iss gene from the human E. coli isolate and 89.4% identity with lambda bor. The Iss protein from the avian isolate has 87% identity with Iss from the human isolate and 90% identity with Bor. The low identity between the two Iss proteins is because of a frame-shift in their respective coding sequences. In sum, iss from this avian E. coli isolate is very similar to iss from a human E. coli isolate, but because of a frameshift mutation in the coding sequence of iss from the human E. coli isolate, Iss proteins from avian and human E. coli isolates have only 87% identity. The strong association of iss with E. coli isolated from birds with colibacillosis, suggests that this sequence be studied for its value as a marker or target to be used in colibacillosis control. PMID- 10737660 TI - Iss from a virulent avian Escherichia coli. AB - No single characteristic of virulent avian Escherichia coli has been identified that can be exploited in colibacillosis detection protocols. Research in our lab suggests a strong association between the presence of an iss DNA sequence with an isolate's disease-causing ability. The study presented here focuses on the techniques used in the expression, purification, and characterization of avian E. coli Iss protein. In brief, iss was cloned into an expression vector, the construct was transformed into a protease-deficient E. coli, and expression was induced. The protein was expressed as a glutathione-S-transferase (GST) fusion and purified by affinity chromatography. The GST portion was cleaved from Iss, Iss was harvested by affinity chromatography, and the identity of Iss was confirmed by N-terminal sequencing. Currently, purified Iss is being used to prepare hybridomas for production of monoclonal antibodies with the goal of evaluating anti-Iss as a reagent for the detection of virulent avian E. coli. PMID- 10737661 TI - Induction of hydropericardium in one-day-old specific-pathogen-free chicks by adenoviruses from inclusion body hepatitis. AB - The pathogenicities of inclusion body hepatitis (IBH) and hydropericardium syndrome (HPS) strains of adenovirus for specific-pathogen-free (SPF) chicks were compared. One-day-old SPF chicks inoculated intramuscularly with the DPI-2 (serotype 2), S-PL1 (serotype 2), TR630 (serotype 8), and Saga97 (serotype 8) strains from IBH and with the LVP-1 strain (serotype 4) from HPS exhibited the mortality, liver enlargement, and hydropericardium characteristic of gross change found in HPS. The chicks inoculated with the IBH and HPS strains exhibited similar histologic and immunohistochemical changes. Neither mortality nor pathologic changes occurred in 3-wk-old SPF chicks inoculated with IBH strains, although HPS strain induced HPS lesions in them. This study indicates that IBH strains of adenovirus can also reproduce HPS lesions and mortality in 1-day-old SPF chicks and that IBH and HPS strains may have similar pathogenicities except for their different virulence for older chickens. PMID- 10737662 TI - Genetic variation in response of turkeys to experimental infection with Bordetella avium. AB - One hundred ninety-six male and female turkeys representing two genetic lines were experimentally infected with Bordetella avium. The lines of turkeys included a randombred control line (RBC2) and a subline (F) of RBC2 selected for increased 16-wk body weight. No difference was found between lines RBC2 and F in the number of days to onset of clinical signs, and no mortality due to B. avium infection was observed in either line. Interestingly, however, a significant depression (12%) occurred in body weight of F line poults infected with B. avium, but no significant depression occurred in body weight of RBC2 poults. PMID- 10737663 TI - DNA fingerprinting of plasmid-containing serotype A: 3,4 Pasteurella multocida isolated from cases of fowl cholera in chickens and turkeys. AB - The live, attenuated vaccine strains of Pasteurella multocida have been hypothesized to be responsible for homologous serotype outbreaks of fowl cholera on farms that use the commercial vaccines. We have further hypothesized that the naturally occurring Clemson University (CU) vaccine strain may be transformed to virulence by the acquisition of plasmid DNA. To test this hypothesis, we obtained seven homologous serotype (A:3,4) P. multocida isolates, all plasmid bearing, that were cultured from fowl cholera cases in vaccinated flocks and compared the isolates with the CU reference vaccine by molecular methods. Restriction fragment length polymorphisms (RFLPs) were detected by DNA/DNA hybridization with labeled probes specific for the cya, aroA, and rrn genes of P. multocida. The RFLPs obtained from BglII-digested genomic DNA probed with cya demonstrated no differences among the isolates. Although three isolates probed with aroA showed a RFLP identical to the vaccine strain, five isolates were distinctly different. Isolates probed with rrn grouped into three different restriction patterns that were dissimilar from that of the vaccine strain. Therefore, we have shown that these fowl cholera isolates are different from the CU vaccine strain and that these outbreaks were not vaccine related. PMID- 10737664 TI - Pyrogallol poisoning of pigeons caused by acorns. AB - Green acorns are known to contain high concentrations of pyrogallol. Here, we describe an extended case report of two pigeons found dead with a filled muscular stomach of acorns. The following pathologic findings were observed: irritation of mucosal membranes in the gastrointestinal tract, blackish discolored chyme, hyperemic organs, and general edemas. The muscular stomach (ventriculus) was filled with pieces of acorns, and the abdominal cavity contained bloody aqueous fluid. In order to uncover the cause of death, we determined pyrogallol in liver and kidney of one dead pigeon and in ventriculus contents of both pigeons by gas chromatography/mass spectrometry. A further aim of our study was to compare pathologic findings and pyrogallol concentrations in kidney, liver, and ventriculus of poisoned pigeons with those of healthy pigeons. The pyrogallol concentrations in samples of dead pigeons were 16-1200-fold higher than in control animals fed grass and maize-corn. Altogether, the acorn-filled ventriculus, the pathologic findings, the well nourished state, and the high pyrogallol concentrations in the dead pigeons suggest an acute pyrogallol poisoning by acorn. With respect to controls, we conclude that pyrogallol concentrations of 6 ng/g of kidney, 8 ng/g of liver, and 2 ng/g of gastric content do not affect the health of pigeons. PMID- 10737665 TI - Pharmacokinetics of indomethacin in poultry. AB - Indomethacin (INDO) is a nonsteroidal antiinflammatory drug widely used since the 1970s. The pharmacokinetic behavior of INDO (2 mg/kg) has been studied after intravenous (i.v.) and oral administration to broiler chickens. After i.v. administration, a first fast distribution phase and a later and slower elimination phase were observed. The elimination half-life and mean residence time (MRT) obtained were 1.0 hr and 0.8 hr, respectively. After oral administration, a flip-flop phenomenon was observed giving an elimination half life and MRT approximately three times and six times higher, respectively, than the i.v. administration. The plasma concentrations after oral administration were sustained during 8-10 hr, giving an antinflammatory cover over the dose producing 50% of maximal effect during this time period. PMID- 10737666 TI - Lesions of bone and bone marrow in myeloid leukosis occurring naturally in adult broiler breeders. AB - Lesions of bone and bone marrow in myeloid leukosis (ML) occurring naturally in adult broiler breeders were investigated pathologically. During gross examination, nodules and protrusions were commonly observed on the surface of the sternum, ribs, vertebrae, and synsacrum. The bone marrow of all the bones of the body was pale in color. Histologically, granulated myelocytes proliferated in the bone marrow of various bones and in the periosteum of the sternum, ribs, vertebrae, and synsacrum. The first proliferation of tumor cells occurred in the bone marrow of epiphysis. The myelocytes invaded through haversian and Volkmann's canals from the bone marrow to periosteal areas. Hematopoiesis was suppressed by marked proliferation of tumor cells in the bone marrow of the whole bone. Atrophy was also seen in the bones, including medullary bones of the chickens suffering from ML. Proliferation of myelocytes was seen in the bone marrow and periosteum of ossified cartilaginous rings of the trachea and larynx. Marked proliferation of myelocytes was seen in the dura mater of spinal cords, and it subsequently depressed the spinal cords. Bone formation with cartilage was seen in the periosteum of the sternum having marked proliferation of myelocytes in the bone marrow and periosteum. Ultrastructurally, tumor cells showed large nuclei and cytoplasm with large round electron-dense lysosomes. The virus particles were rarely detected in the cytoplasm of tumor cells. The polymerase chain reaction test of tumor samples showed positive for subgroup J avian leukosis virus. This study indicates that the myelocytes can invade through the compact bones to the periosteum in the sternum, ribs, vertebrae, synsarcum, and ossified cartilage of trachea and larynx having thinner compact bones. In addition, the periosteal osteogenesis with cartilage in the sternum may be reactive change against the bone atrophy because of the marked proliferation of myelocytes. PMID- 10737667 TI - Avian pneumovirus infection in Minnesota turkeys: experimental reproduction of the disease. AB - Avian pneumovirus (APV) is an emerging viral respiratory disease agent of turkeys in Minnesota. Clinical signs of APV infection include open mouth breathing, ocular and nasal discharge, and swelling of infraorbital sinuses. The virus spreads rapidly among flocks of susceptible turkeys and is associated with increased mortality rates. A flock of 11-wk-old turkeys experienced a respiratory problem characterized by coughing, sneezing, swollen sinuses, and nasal discharge. The reverse transcriptase-polymerase chain reaction (RT-PCR) performed on tissues from the nasal turbinates and tracheal tissues was positive for avian pneumovirus. Turbinate tissue was inoculated into chicken embryo fibroblasts, and cytopathic effect was observed after five blind passages. In an attempt to reproduce the disease, 50 microl of this cell culture-propagated virus was instilled into each conjunctival space and nostril of 23-day-old turkey poults. The poults were sacrificed at 2-day intervals for 12 days, and serum, tissues, and tracheal and cloacal swabs were collected. Between days 2 and 10 after exposure, the poults developed ocular and nasal discharge and swollen sinuses. The virus was detected by RT-PCR and virus isolation from the nasal turbinates of poults sacrificed on days 4 and 6 postinoculation. Antibodies to APV were detected by enzyme-linked immunosorbent assay. PMID- 10737668 TI - Mycobacterium infection in a captive-reared capercaillie (Tetrao urogallus). AB - One captive male capercaillie (Tetrao urogallus) was found dead on December 1993 at the breeding center of capercaillie in Catalonia, Spain. The bird was emaciated and, at necropsy, had numerous nodules of various sizes subcutaneously in the cervical region, pleura, lungs, liver, spleen, and mesentery. Microscopic examination revealed granulomatous lesions with central caseous necrosis, epithelioid cells, giant cells, and few lymphocytes in all affected organs. Numerous acid-fast bacilli were demonstrated in the tubercles with Ziehl-Nielsen stain. PMID- 10737669 TI - An occurrence of cardiac rupture in a capercaillie (Tetrao urogallus) probably induced by myocardial infarction. AB - A pair of capercaillies (Tetrao urogallus) were newly introduced from Austria into a museum in Japan, and the female bird died on the way to the museum. Autopsy revealed cardiac tamponade filled with a large quantity of blood coagulum in the pericardial cavity and cardiac rupture along the apex around 1.5 cm in length. Microscopically, myocardial necrosis associated with cellular infiltration was observed. The results indicated that the bird suffered from cardiac rupture on the way to the museum. Myocardial infarction was considered to be a predisposing factor for the incident. PMID- 10737670 TI - Neuropeptide Y phase advances the in vitro hamster circadian clock during the subjective day with no effect on phase during the subjective night. AB - The mammalian daily (circadian) clock is located in the suprachiasmatic nuclei of the hypothalamus. Clock function can be detected by the measurement of the circadian change in spontaneous firing rate of suprachiasmatic nuclei cells in a brain slice preparation in vitro. We investigated the effects of neuropeptide Y on this rhythm of firing rate in hamster suprachiasmatic nuclei neurons. Slices were prepared using standard techniques. On the 1st day in vitro, neuropeptide Y (200 ng/200 nL; 47 pmol) was applied as a microdrop to the suprachiasmatic nuclei region at various times. Spontaneous single-unit firing was measured for 6-12 h on the 2nd day in vitro. Peak firing rate in treated slices was compared with that of untreated control slices to measure phase shifts induced by the peptide. Neuropeptide Y induced phase advances of circa-3h when applied during the subjective day (ZT 2-10) but did not significantly alter phase when applied during the subjective night. The phase shifts to neuropeptide Y in the hamster tissue in vitro are similar in phase dependency and magnitude to shifts measured in vivo. PMID- 10737671 TI - Neuropeptide Y enhances potassium excretion by mechanisms distinct from those controlling sodium excretion. AB - Neuropeptide Y (NPY) is an established modulator of renal function. Although NPY reduces renal blood flow and does not alter glomerular filtration rate, it enhances diuresis and natriuresis. Although initial studies on natriuresis did not detect kaliuresis, we now report that a retrospective analysis of previous studies regarding natriuresis demonstrates NPY-induced kaliuresis under several experimental conditions. Kaliuresis was observed despite a marked reduction in urinary potassium concentrations, which may explain why it has not been noted in some initial studies. In a direct comparison of NPY-induced kaliuresis and natriuresis, both effects were slow in onset (requiring >45 min to develop fully) and blocked by the cyclooxygenase inhibitor indomethacin. While natriuresis occurred solely via a Y5 receptor, kaliuresis involved a Y1 receptor and an additional receptor subtype, possibly Y2. The L-type Ca2+ entry blocker nifedipine abolished natriuresis but did not inhibit kaliuresis. A combination of experiments with the bradykinin B2 receptor antagonist icatibant, the angiotensin II receptor antagonist losartan, and the converting enzyme inhibitor ramiprilat revealed that NPY-induced natriuresis involves bradykinin while kaliuresis involves angiotensin II. We conclude that NPY-induced kaliuresis is much less pronounced than natriuresis and is mediated by distinct mechanisms. PMID- 10737673 TI - Pharmacology and quantitative structure-activity relationships of imidazolylpropylguanidines with mepyramine-like substructures as non-peptide neuropeptide Y Y1 receptor antagonists. AB - The design of non-peptide, Y1-selective antagonists of neuropeptide Y (NPY) as pharmacological tools is in progress and is increasingly important as therapeutic applications are expected. Starting from the potent histamine H2 agonist and weak NPY Y1 antagonist arpromidine, 16 imidazolylpropylguanidine derivatives were synthesized and tested for Y1 antagonistic activity (inhibition of NPY-stimulated Ca2+ increase in human erythroleukemic cells), where the pheniramine-like moiety of arpromidine was replaced with 2-pyridylaminoalkyl, benzyl-(2 pyridyl)aminoalkyl, and phenyl-(2-pyridyl)alkylaminoalkyl partial structures derived from mepyramine. The pA2 values of the most active compounds are in the range of 6.2-6.5. Quantitative structure-activity relationships (QSAR) were investigated by fragment regression analysis. Results indicate that a tetramethylene spacer between the guanidino group and the amino nitrogen is optimal. For an at least moderate degree of Y1 antagonistic activity, a second benzyl or phenyl group must be present in addition to the 2-pyridyl ring. At this second group, hydrophobic substituents such as 3,4-di-CI and 4-Br further enhance Y1 antagonism. The most active derivative additionally bears a 5-Br substituent at the 2-pyridyl moiety. Structure-activity relationships suggest that the compounds might be able to partially imitate the role of NPY when interacting with Y1 receptors and thus behave as moderate non-peptide NPY Y1 antagonists. PMID- 10737672 TI - Plasma and cerebrospinal fluid concentration of neuropeptide Y, serotonin, and catecholamines in patients under propofol or isoflurane anesthesia. AB - Propofol is a widely used anesthetic for both induction and maintenance of anesthesia during surgery. A strong feeling of hunger has been reported during the early recovery period after propofol anesthesia. We have investigated the effect of propofol on appetite in 10 patients undergoing a craniotomy and in parallel measured neuropeptide Y (NPY), catecholamines, and serotonin levels in the cerebrospinal fluid and plasma during anesthesia. Ten patients anesthetized with a volatile agent (isoflurane) served as a control group. Plasma NPY and catecholamines levels were not affected by surgery at any time. We observed a strong increase in NPY concentrations in the cerebrospinal fluid independently of the anesthetic technique agent used, whereas catecholamines were unchanged. We found that serotonin concentrations decreased significantly in the plasma (but not in the cerebrospinal fluid) of patients treated by propofol when compared with the control group; this decrease was associated with an increase of hunger early postoperatively. We concluded that the proappetite effect of propofol is mediated through a decrease of serotonin at the peripheral level. PMID- 10737674 TI - Potent and selective tools to investigate neuropeptide Y receptors in the central and peripheral nervous systems: BIB03304 (Y1) and CGP71683A (Y5). AB - We have evaluated 3 newly developed neuropeptide Y receptor antagonists in various in vitro binding and bioassays: BIBO3304 (Y1), T4[NPY33-36]4 (Y2), and CGP71683A (Y5). In rat brain homogenates, BIBO3304 competes for the same population of [125I][Leu31,Pro34] peptide YY (PYY) binding sites (75%) as BIBP3226, but with a 10 fold greater affinity (IC50 of 0.2 +/- 0.04 nM for BIBO3304 vs. 2.4 +/- 0.07 nM for BIBP3226),while CGP71683A has high affinity for 25% of specific [125I][Leu31,Pro34]PYY binding sites. Both BIBO3304 and CGP71683A (at 1.0 microM) were unable to compete for a significant proportion of specific [125I]PYY3-36/Y2 sites. The purported Y2 antagonist T4[NPY33-36]4 competed against [125I]PYY3-36 binding sites with an affinity of 750 nM. These results were confirmed in HEK 293 cells transfected with either the rat Y1, Y2, Y4, or Y5 receptor cDNA. BIBO3304, but not CGP71683A, competed with high affinity for [125I][Leu31,Pro34]PYY binding sites in HEK 293 cells transfected with the rat Y1 receptor cDNA, whereas the reverse profile was observed upon transfection with the rat Y5 receptor cDNA. Additionally, both molecules were inactive at Y2 and Y4 receptor subtypes expressed in HEK 293 cells. Receptor autoradiographic studies revealed the presence of [125I][Leu31,Pro34]PYY/BIBO3304-insensitive sites in the rat brain as reported previously for BIBP3226. Finally, the selective antagonistic properties of BIBO3304 were demonstrated in a Y1 bioassay (rabbit saphenous vein; pA2 value of 9.04) while being inactive in Y2 (rat vas deferens) and Y4 (rat colon) bioassays. These results confirm the high affinity and selectivity of BIBO3304 and CGP71683A for the Y1 and Y5 receptor subtypes, respectively, while the purported Y2 antagonist, T4[NPY33-36]4 possesses rather low affinity for this receptor. PMID- 10737675 TI - PYY preference is a common characteristic of neuropeptide Y receptors expressed in human, rat, and mouse gastrointestinal epithelia. AB - This investigation describes the relative potencies of four peptide agonists, namely, peptide YY (PYY), [Leu3l,Pro34]PYY (Pro34pYY), neuropeptide Y (NPY), and [Leu31,Pro34]NPY (Pro34NPY), as antisecretory agents in human, rat, and mouse gastrointestinal preparations. The inhibition of agonist responses by the Y1 receptor antagonist BIBP 3226 was also tested in each preparation. An unexpectedly pronounced preference for PYY and Pro34PYY was observed in functional studies of two human epithelial lines stably transfected with the rat Y1 receptor (Y1-7 and C1Y1-6). NPY and Pro34NPY were at least an order of magnitude less effective than PYY in these functional studies but were only marginally less potent in displacement binding studies using membrane preparations of the same clonal lines. The orders of agonist potency obtained in Y1-7 and C1Y1-6 epithelia were compared with those obtained from a single human colonic adenocarcinoma cell line (Colony-6, which constitutively expresses Y1 receptors) and also from mucosal preparations of rat and mouse descending colon. Similar peptide orders of potency were obtained in rat and mouse colonic mucosae and Colony-6 epithelia, all of which exhibited PYY preference (although less pronounced than with Y1-7 and C1Y1-6 epithelia) and significant sensitivity to the Y1 receptor antagonist, BIBP 3226. We have compared the pharmacology of these five mammalian epithelial preparations and provide cautionary evidence against the reliance upon agonist concentration-response relationships alone, in the characterization of NPY receptor types. PMID- 10737676 TI - Cloning and functional expression of the hNPY Y5 receptor in human endometrial cancer (HEC-1B) cells. AB - Aiming to develop a functional assay for the human NPY Y5 receptor based on adenylyl cyclase activity, HEC-1B cells, in which cAMP synthesis can be efficiently stimulated with forskolin, were selected for the transfection with the pcDNA3-Y5-FLAG and the pcDEF3-Y5 vectors. After optimization of the transfection procedure, the binding of [3H]propionyl-NPY to transiently and stably expressed Y5 receptors was determined. The affinities of NPY, NPY derivatives, and rPP (pNPY > or = p(Leu31Pro34)NPY = p(2-36)NPY > or = p(D Trp32)NPY > p(13-36)NPY > rPP) were in accordance with the NPY Y5 receptor subtype. For [3H]propionyl-pNPY approximately 1.7 x 10(5) and 1 x 10(6) binding sites per transiently and stably transfected cell, respectively, were determined. The KD values were 2.4 +/- 0.4 and 1.7 +/-0.2 nM, respectively. Due to the high expression of the receptor protein, both stably and transiently transfected cells can be conveniently used in routine radioligand binding studies. By contrast, functional assays were only feasible with HEC-1B cells stably expressing the Y5 receptor. In these cells, 10 nM pNPY inhibited the forskolin-stimulated cAMP synthesis by 75%. This effect was partially antagonized by the Y5 antagonist N ?trans-[4-(2-naphthylmethylamino)-methyl]cyclohexylmethyl) naphthalene-2 sulfonamide. Although the genetic variability of cancer cells is in principle incompatible with a stable phenotype, both ligand binding characteristics and functionality of the Y5 receptor remained unchanged for more than 30 passages. PMID- 10737677 TI - Orexigenic effect of the melanocortin MC4 receptor antagonist HS014 is inhibited only partially by neuropeptide Y Y1 receptor selective antagonists. AB - Neuropeptide Y (NPY) and melanocortin (MC) peptides have opposite effects on food intake: NPY-like peptides and MC receptor antagonists stimulate feeding and increase body weight, whereas melanocortins and NPY antagonists inhibit food intake. In this study we tested whether the orexigenic effect of the selective MC4 receptor antagonist HS014 (1 nmol) could be inhibited by three different NPY antagonists, (R)-N2-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]D-argininam ide (BIBP3226), (R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N2(diphenyl acetyl)-argininamidetrifluoroacetate (BIBO3304), and decapeptide [D-Tyr(27,36)D Thr32]NPY(27-36), after icv administration in freely feeding male rats. All three NPY receptor antagonists inhibited the orexigenic effects of HS014 partially and with markedly different potency. [D-Tyr(27,36)D-Thr32]NPY(27-36) was active only in subconvulsive dose. The NPY Y1 selective antagonist BIBP3226 was more effective in inhibiting the effect of HS014 than BIBO3304 despite in vitro data indicating that BIBP3226 is about 10 times less potent than BIBO3304 at NPY Y1 receptor. An enantiomer of BIBO3304, BIBO3457, failed to inhibit HS014-induced feeding, indicating that the effects of BIBO3304 were stereoselective. These results suggest that stimulation of food intake caused by weakening of melanocortinergic tone at the MC4 receptor is partially but not exclusively related to NPY Y1 receptor activation. PMID- 10737678 TI - FMRFamides exert a unique modulation of rodent pancreatic polypeptide sensitive neuropeptide Y (NPY) receptors. AB - FMRFamide and related peptides (RFamides) were found to inhibit the association binding of iodinated human pancreatic polypeptide ([125I]hPP) to Y5-like neuropeptide Y (NPY) receptor in rodent tissues. An allosteric regulation of the activity of the rodent kidney PP-sensitive neuropeptide Y (NPY) receptor by RFamides was indicated by potency decrease with particle concentration in the inhibition of the association binding of 125I-labeled human pancreatic polypeptide (hPP) by RFamides at rabbit kidney membranes. The competition by C terminal hexapeptide of hPP (LTRPRY.NH2) did not show such affinity change. The steady-state binding of hPP showed little sensitivity to any of the RFamides tested. The Y1-selective binding of [125I][Leu31,Pro34]hPYY (at 2 nM hPP) was much less sensitive to RFamides than the binding of [125I]hPP, albeit with some differences across tissue or cell types. The binding of Y2-selective agonist 125I labeled human peptide YY (3-36) was quite insensitive to RFamides. The presence of a unique component in the inhibition of hPP binding by RFamides was further indicated by a degree of antagonism with phospholipase C inhibitor U-73122, and by an only limited cooperation with a N5-amiloride compound, and with alkylator chloroethylclonidine. Change of the chirality of individual residues in the FMRFamide molecule produced a significant reduction of inhibitory potency only with D-Phe in the C-terminal position. Substitution of the (C-3) L-Met by L-Leu greatly increased the inhibitory potency of RFamides relative to otherwise identical congeners. RFamides could act both as ligands of membrane neighbors of the PP receptor, and as competitors of Y5-like NPY receptor epitopes that accommodate the C-terminal aspects of agonist peptides. PMID- 10737679 TI - Neuropeptide Y induced increase of cytosolic and nuclear Ca2+ in heart and vascular smooth muscle cells. AB - It was reported that neuropeptide Y (NPY) affects cardiac and vascular smooth muscle (VSM) function probably by increasing intracellular Ca2+. In this study, using fura-2 microfluorometry and fluo-3 confocal microscopy techniques for intracellular Ca2+ measurement, we attempted to verify whether the action of NPY receptor's stimulation in heart and VSM cells modulates intracellular Ca2+ and whether this effect is mediated via the Y1 receptor type. Using spontaneously contracting single ventricular heart cells of 10-day-old embryonic chicks and the fluo-3 confocal microscopy Ca2+ measurement technique to localize cytosolic ([Ca]c) and nuclear ([Ca]n) free Ca2+ level and distribution, 10-10 M of human (h) NPY significantly (P < 0.05) increased the frequency of cytosolic and nuclear Ca2+ transients during spontaneous contraction. Increasing the concentration of hNPY (10(-9) M) did not further increase the frequency of Ca2+ transients. The L type Ca2+ channel blocker, nifedipine (10(-5) M), significantly (P < 0.001) blocked the spontaneous rise of intracellular Ca2+ in the absence and presence of hNPY (10(-10) and 10(-9) M). However, the selective Y1 receptor antagonist, BIBP3226 (10(-6) M), significantly decreased the hNPY-induced (10(-10) and 10(-9) M) increase in the frequency of Ca2+ transients back to near the control level (P < 0.05). In resting nonworking heart and human aortic VSM cells, hNPY induced a dose-dependent sustained increase of basal resting intracellular Ca2+ with an EC50 near 10(-9) M. This sustained increase was cytosolic and nuclear and was completely blocked by the Ca2+ chelator EGTA, and was significantly decreased by the Y1 receptor antagonist BIBP3226 in both heart (P < 0.05) and VSM (P < 0.01) cells. These results strongly suggest that NPY stimulates the resting basal steady-state Ca2+ influx through the sarcolemma and induces sustained increases of cytosolic and nuclear calcium, in good part, via the activation of the sarcolemma membrane Y1 receptor type in both resting heart and VSM cells. In addition, NPY also increased the frequency of Ca2+ transients during spontaneous contraction of heart cells mainly via the activation of the Y1 receptor type, which may explain in part the active cardiovascular action of this peptide. PMID- 10737680 TI - Food intake regulation in rodents: Y5 or Y1 NPY receptors or both? AB - Neuropeptide Y (NPY), one of the most abundant peptides in rat and human brains, appears to act in the hypothalamus to stimulate feeding. It was first suggested that the NPY Y1 receptor (Y1R) was involved in feeding stimulated by NPY. More recently a novel NPY receptor subtype (Y5R) was identified in rat and human as the NPY feeding receptor subtype. There is, however, no absolute consensus since selective Y1R antagonists also antagonize NPY-induced hyperphagia. Nevertheless, new anti-obesity drugs may emerge from further pharmacological characterization of the NPY receptors and their antagonists. A large panel of Y1R and Y5R antagonists (such as CGP71683A, BIBO3304, BIBP3226, 1229U91, and SYNAPTIC and BANYU derivatives but also patentable in-house-synthesized compounds) have been evaluated through in vitro and in vivo tests in an attempt to establish a predictive relationship between the binding selectivity for human receptors, the potency in isolated organs assays, and the inhibitory effect on food intake in both normal and obese hyperphagic rodents. Although these results do not allow one to conclude on the implication of a single receptor subtype at the molecular level, this approach is crucial for the design of novel NPY receptor antagonists with potential use as anti-obesity drugs and for evaluation of their possible adverse peripheral side effects, such as hypotension. PMID- 10737681 TI - Hemopoiesis in healthy old people and centenarians: well-maintained responsiveness of CD34+ cells to hemopoietic growth factors and remodeling of cytokine network. AB - In vitro hemopoiesis and hemopoietic cytokines production were evaluated in 9 centenarians (median age 100.5 years, age range: 100-104 years), 10 old people (median age: 71 years, age range: 66-73 years), and 10 young people (median age: 35 years, age range: 30-45 years), all carefully selected for their healthy status. The main findings were the following: (i) a trend towards a decreased absolute number of CD34+ progenitor cells in the peripheral blood of old people and centenarians, in comparison to young subjects; (ii) a well-preserved capability of CD34+ cells from old people and centenarians to respond to hemopoietic cytokines, and to form erythroid (BFU-E), granulocyte-macrophagic (CFU-GM), and mixed colonies (CFU-GEMM) in a way (number, size, and morphology) indistinguishable from that of young subjects; (iii) an age-related decreased in vitro production of granulocyte-macrophagic colony-stimulating factor (GM-CSF) and a decreased production of interleukin-3 (IL-3) in centenarians by phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC); (iv) a linear increase of the serum level of stem cell factor (SCF), measured in the above-mentioned subjects and in 65 additional subjects, including 4 centenarians. These data suggest that basal hematopoietic potential is well preserved in healthy centenarians, and that the hemopoietic cytokine network undergoes a complex remodeling with age. PMID- 10737682 TI - Restoration of bone mass in the severely osteopenic senescent rat. AB - Studies in humans and rats suggest that age impairs the ability to form bone. This impairment may be due to a depletion or deficit in osteoprogenitor stem cells. Such a deficit would be expected to reduce the ability of the skeleton to respond to therapy designed to restore lost bone. This study evaluated whether severely osteopenic senescent rats are capable of responding to a potent anabolic factor in bone, prostaglandin E2 (PGE). Growing female Sprague Dawley rats were ovariectomized at 3 months and aged until the start of treatment at 23 months. Rats were treated daily with PGE (3 mg/kg sc) or vehicle for 56 days. Tibiae were harvested for bone histomorphometry and femora were obtained for mRNA analysis of bone matrix proteins. The cancellous bone area was fivefold greater in PGE treated rats than in vehicle-treated controls and not different from age-matched ovary-intact rats. PGE approximately doubled the bone-forming surface and the mineral apposition rate and increased the bone formation rate fourfold. The increased cancellous bone area in PGE-treated rats was primarily due to an increase in osteoblasts over osteoclasts. One hundred percent of the endocortical surface and 72 +/- 9% of the periosteal surface of cortical bone was undergoing mineralization in PGE-treated rats, whereas no mineratization was evident in vehicle-treated rats. An architectural analysis of cancellous bone indicates that trabecular number and thickness were increased and separation decreased in the treated rats. Imaging by microcomputed tomography further revealed that with PGE treatment, trabeculae in the medial plane of the proximal tibial metaphysis were more robust and continuous with the endocortical surface. PGE also significantly induced message levels for the prepro-alpha (I) subunit of type I collagen (collagen), osteonectin, and osteocalcin. In summary, bone mass can be restored to severely osteopenic senescent rats, suggesting that aging does not necessarily diminish the capacity of the skeleton to form bone. PMID- 10737683 TI - Effect of aging on aortic expression of the vascular cell adhesion molecule-1 and atherosclerosis in murine models of atherosclerosis. AB - Although age is a strong risk factor for atherosclerosis, it is unclear whether age may directly influence the process of atherogenesis. We, therefore, performed several studies in young (2-4 months old), mature (10-14 months old), and old (20 22 months old) mice to determine if the rate of aortic lesion formation increases with age, and whether this is related to increases in oxidative stress or vascular cell adhesion molecule (VCAM-1) expression in the aortic wall. In chow fed low-density lipoprotein receptor-deficient (LDLR-/-) mice, plasma total cholesterol levels increased with age (250 +/- 52 mg/dl in young, 276 +/- 58 in mature, and 314 +/- 101 mg/dl in old mice). In contrast, the extent of atherosclerosis rose more rapidly, increasing from 3.6 +/- 2.7% of the aortic surface in mature mice to 18.2 +/- 8% in old mice. Plasma and tissue levels of antioxidant enzymes and molecules, as well as plasma thiobarbituric acid reactive substances and low-density lipoprotein susceptibility to oxidation, did not change with age. In a second study, VCAM-1 expression in the aortic arch and the extent of atherosclerosis in the aortic origin were significantly greater in old LDLR-/- mice than in young LDLR-/- mice. Additionally, after 1 month of a high fat diet, which induced equally elevated plasma cholesterol levels in both young and old LDLR-/- mice, VCAM-1 expression and aortic lesion formation were still greater in old mice. The extent of atherosclerosis correlated well (r = .65,p <.01) with the expression of VCAM-1 in the aortic origin. In a final study, we measured VCAM-1 expression and atherosclerosis in young, mature, and old C57BL/6 mice, which have low plasma cholesterol levels (< or =100 mg/dl) when fed a standard chow diet. Although plasma cholesterol levels did not increase with age, old C57BL/6 mice had significantly more VCAM-1 expression in the aortic arch than did young mice. However, no lesions were observed in the aortic origin in either group. These data demonstrate that plasma cholesterol levels and VCAM-1 expression increase with age and suggest that this may contribute to the increased rate of atherosclerotic lesion formation in LDLR-/- mice. Importantly, the age-dependent increase in VCAM-1 expression does not appear to be related to plasma cholesterol levels. This study also suggests that in the absence of elevated plasma cholesterol, an increased expression of VCAM-1 alone is not sufficient for lesion formation. PMID- 10737684 TI - Basal concentrations and acute responses of serum hormones and strength development during heavy resistance training in middle-aged and elderly men and women. AB - Effects of 6 months of heavy resistance training combined with explosive exercises on both basal concentrations and acute responses of total and free testosterone, growth hormone (GH), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), cortisol and sex hormone-binding globulin (SHBG), as well as voluntary neural activation and maximal strength of knee extensors were examined in 10 middle-aged men (M40; 42 +/- 2 years), 11 middle aged women (W40; 39 +/- 3 years), 11 elderly men (M70; 72 +/- 3 years), and in 10 elderly women (W70; 67 +/- 3 years). The maximal integrated electromyographic (iEMG) and 1 repetition maximum (RM) knee-extension values remained unaltered in all groups during a 1-month control period with no strength training. During the 6-month training the 1RM values increased in M40 by 27 +/- 9% (p < .001), in M70 by 16 +/- 6% (p < .001), in W40 by 28 +/- 11% (p < .001), and in W70 by 24 +/- 10% (p < .001). The iEMGs of the vastus lateralis and medialis muscles increased(p < .05-.001) in M40, M70, W40, and W70. No systematic changes occurred during the experimental period in the mean concentrations of serum total and free testosterone, DHEA, DHEAS, GH, cortisol, or SHBG. However, the mean levels of individual serum free testosterone in W70 and serum testosterone in the total group of women correlated with the individual changes recorded in strength during the training (r = .55,p <.05; and r = .43,p <.05). The single exercise session both before and after the training resulted in significant responses in serum total and free testosterone concentrations in both male groups (p <.05-.01), but not in the female groups, as well as in serum GH levels in all groups (p <.05 .01) except W70 (ns). In summary, the present strength training led to great increases in maximal strength not only in middle-aged but also in elderly men and women. The strength gains were accompanied by large increases in the maximal voluntary activation of the trained muscles. None of the groups showed systematic changes in the mean serum concentrations of hormones examined. However, a low level of testosterone, especially in older women, may be a limiting factor in strength development and testosterone could mediate interactions with the nervous system contributing to strength development. The physiological significance of the lack of acute responsiveness of serum GH to heavy resistance exercise in older women for their trainability during prolonged strength training requires further examination. PMID- 10737685 TI - Chronic [D-Ala2]-growth hormone-releasing hormone administration attenuates age related deficits in spatial memory. AB - The age-related decline in growth hormone is one of the most robust endocrine markers of biological aging and has been hypothesized to contribute to the physiological deficits observed in aged animals. However, there have been few studies of the impact of this hormonal decline on brain aging. In this study, the effect of long-term subcutaneous administration of [D-Ala2]-growth hormone releasing hormone (GHRH) on one measure of brain function, memory, was investigated. Animals were injected daily with 2.3 microg of [D-Ala2]-GHRH or saline from 9 to 30 months of age, and the spatial learning and reference memory of animals were assessed by using the Morris water maze and compared with those of 6-month-old animals. Results indicated that spatial memory decreased with age and that chronic [D-Ala2]-GHRH prevented this age-related decrement (24% improvement in the annulus-40 time and 23% improvement in the number of platform crossings compared with saline treated, age-matched controls; p < .05 each). No changes were noted in sensorimotor performance. [D-Ala2]-GHRH attenuated the age related decline in plasma concentrations of insulinlike growth factor-1 (IGF-1) (p <.05). These data suggest that growth hormone and IGF-1 have important effects on brain function, that the decline in growth hormone and IGF-1 with age contributes to impairments in reference memory, and that these changes can be reversed by the chronic administration of GHRH. PMID- 10737686 TI - Risk of malnutrition in retirement homes elderly persons measured by the "mini nutritional assessment". AB - BACKGROUND: The combined influence of age-associated factors such as general health, degree of dependency, diminished odor perception, and poor oral health on the risk for malnutrition was explored. METHODS: A total of 81 persons living in retirement homes took part in the study (mean age 83.4 years, SD = 6.6, range 61 98). The Mini-Nutritional Assessment (MNA) was used to evaluate the risk of malnutrition. Odor perception was measured by the detection threshold for isoamylacetate. The number of drugs taken by each person was counted. General health status was determined by the Medical Outcome Study (MOS) scores. Oral examinations were carried out to count the number of natural teeth and type of dentures. RESULTS: On average, women had slightly, but significantly, lower MNA scores than men (respectively, 23.4, SD = 2.8; and 24.6, SD = 2.6; p = .048). The correlations between age and MNA score and between odor perception and MNA score were not significant. Significant correlations were found between age and number of natural teeth (r = -.26, p = .001) and between MNA score and number of natural teeth (r = .27, p = .001). The mean MNA score of complete denture wearers (22.8, SD = 2.9) was significantly lower than that of partial denture wearers (25.8, SD = 2.9; p = .0005). The total MOS and MNA scores were not correlated, but a significant correlation was found with the subscales mental functioning (r = .29, p = .003), social functioning (r = . 19, p = .045), and perceived health (r = .19, p = .047). No relation was found between the activities of daily living (ADL) and MNA scores. A significant negative correlation was observed between number of drugs taken and the MNA score (r = -.34, p = .001). When participants without risk of malnutrition (MNA > or = 24) were compared with those at risk (MNA = 17-23.5), again, the number of drugs taken was significantly different (on average, respectively, 4.5, SD = 2.9; and 7.0, SD = 2.6; p < .0005). Using multiple regression to test the separate effects of the different independent variables, the number of drugs taken showed a significant negative regression coefficient (beta = -.31, p = .008), as did the mental health score (beta = .27, p =.02), giving a total R2 = .32. The other parameters did not contribute significantly. CONCLUSION: Among the elderly in retirement homes, the health state (as measured by the MOS subscale mental health and by the medication use) appears to be the most clinically relevant parameter to explain the risk for malnutrition. Loss of natural teeth and perceived health are less independently contributing, whereas no contribution derives from decline of odor perception, degree of dependency, and age itself. PMID- 10737687 TI - An estimate of long-term care needs and identification of risk factors for institutionalization among Hong Kong Chinese aged 70 years and over. AB - BACKGROUND: The goal of this study is to estimate the long-term care needs of the Hong Kong Chinese population age 70 years and older, and to identify risk factors for institutionalization. METHODS: A three-year prospective follow-up study was carried out in Hong Kong Special Administrative Region, China. 2,032 subjects aged 70 years and older were recruited territory-wide by stratified random sampling of the Old Age and Disability Allowance register, covering over 90% of the elderly population. A questionnaire was administered at baseline to obtain information on social, functional, physical, and mental health status, and place of residence. A repeat interview was carried out at 36 months. The number of subjects moving from home to institution or vice versa, and the number who had died, were noted. Univariate analysis was performed to determine risk factors for institutionalization, and backward stepwise multiple logistic regression was used to identify independent factors predisposing to institutionalization. RESULTS: The institutionalization rate per year is estimated to be 0.7% for the 70-79 age group, and 1.5% for the 80+ age group. Using population figures projected by the Hong Kong Census, the corresponding number of places required will be 24,150 and 42,000, respectively, for the two age groups by 2005, whereas the number of government subvented places projected to be available is unlikely to be more than 40,000 for the whole population. Age, being a woman, being single, not having a formal education, cognitive impairment, physical dependency, and the presence of depressive symptoms were factors predisposing to institutionalization. In multivariate analysis, age, marital status, and dependency were identified as independent factors. CONCLUSION: The requirement for institutional places is unlikely to be met by government, the shortfall likely to be met by the private sector. Maintenance of functional independence, good social support network, engagement in social activities, and good informal carer support may reduce demand for institutional care. PMID- 10737688 TI - Feasibility and measurement properties of the functional reach and the timed up and go tests in the Canadian study of health and aging. AB - BACKGROUND: Physical performance measures may offer advantages over self-report in the functional assessment of older people. Estimates of the feasibility, reliability, and construct validity of these measures in large, heterogeneous samples are necessary to establish their importance relative to traditional measures of function. METHODS: Analysis of clinical data from Phase 2 of the Canadian Study of Health and Aging, a nation-wide representative survey of elderly people in Canada (N = 2,305). RESULTS: Both physical performance measures proved infeasible in many subjects (29.3% for the Timed Up and Go [TUG], 35.9% for the Functional Reach [FR]). Cognitive impairment was the most important determinant of inability to complete the tests. For those able to complete the tests, cognitively unimpaired subjects could reach farther (median 29 cm) and complete the TUG in less time (median 12 seconds) than those cognitively impaired (25 cm for FR, 15 seconds for the TUG). Test-retest reliability between the screening and clinical administrations of the TUG was .56 for all participants (intra-class correlations), .50 for the cognitively unimpaired, and .56 for the cognitively impaired. Construct validity was substantial, and correlations between performance measures and self-report activities of daily living (ADL) measures ranged from .40 to .70. Compared with a global clinical measure of frailty, correlations were more modest (.38 to .60). CONCLUSIONS: The FR and the TUG were not feasible tools in this study. The TUG showed poor test-retest reliability. Our data support the observation that subsequent studies of measurement instruments typically reveal lower performance than the original reports. PMID- 10737689 TI - Comparative effects of two physical activity programs on measured and perceived physical functioning and other health-related quality of life outcomes in older adults. AB - BACKGROUND: Although inactivity is an important contributor to impaired functioning and disability with age, little is known concerning how improvements in physical functioning and well-being in older adults vary with the type of physical activity undertaken. METHODS: One hundred three adults age 65 years and older, recruited via population-based methods, were randomized to 12 months of community-based, moderate-intensity endurance and strengthening exercises (Fit & Firm) or stretching and flexibility exercises (Stretch & Flex). A combination of class- and home-based exercise formats was used. Measured and self-rated physical performance along with perceived functioning and well-being were assessed pre- and postintervention. RESULTS: Fit & Firm subjects showed greater 12-month improvements in both measured and self-rated endurance and strength compared to Stretch & Flex subjects. Stretch & Flex subjects reported greater improvements in bodily pain, and Stretch & Flex men evidenced greater improvements in flexibility relative to Fit & Firm subjects. Although overall exercise adherence was high in both exercise conditions (approximately 80%), subjects in both conditions showed better adherence to the home- versus class-based portions of their exercise prescriptions. CONCLUSIONS: Community-based programs focusing on moderate intensity endurance and strengthening exercises or flexibility exercises can be delivered through a combination of formats that result in improvement in important functional and well-being outcomes. This represents one of the first studies to report significant improvements in an important quality of life outcome-bodily pain-with a regular regimen of stretching and flexibility exercises in a community-based sample of older adults. PMID- 10737690 TI - Comorbidity of five chronic health conditions in elderly community residents: determinants and impact on mortality. AB - BACKGROUND: Comorbidity is common in elderly persons. Its extent, correlates, and life-threatening impact in representative community residents are unclear. METHODS: Self-reported information of physician-diagnosed coronary artery disease (CAD), cerebrovascular disease (CVD), diabetes, and cancer was obtained annually between 1986-87 and 1992-93, and hypertension was obtained triennially from the participants of the Duke Established Populations for Epidemiologic Studies of the Elderly, a stratified multistage sample of 4,126 Black and White community residents aged 65-100, living in a five-county area of North Carolina. Date of death was obtained from death certificates identified through search of the National Death Index. Statistical procedures included descriptive statistics, logistic regression, and survival analysis. RESULTS: Of this sample, 57% reported hypertension, 20% diabetes, 15% CAD, 9% cancer, and 9% CVD; 29% reported none of these conditions, whereas 29% reported two or more. Demographic characteristics were not related to comorbidity with CVD or cancer. Increased education tended to be protective. The effect of age, gender, and race varied with condition. At baseline there was substantial comorbidity among hypertension, CAD, CVD, and diabetes, but not with cancer. Hypertension, CVD, and diabetes were risk factors for CAD, whereas diabetes was a risk factor for CVD. After controlling for demographic characteristics, all health conditions except hypertension were predictive of 6-year mortality, as was the presence of comorbidity. CONCLUSION: We found significant comorbidity in older persons who have hypertension, CAD, CVD, or diabetes; particular risk of developing comorbidity, particularly CAD, among those with hypertension, CVD, and diabetes; and risk of CVD in those with diabetes. With the exception of hypertension, these conditions, and comorbidity per se, are life-threatening. PMID- 10737691 TI - Self-reported prevalence and health correlates of functional limitation among Massachusetts elderly Puerto Ricans, Dominicans, and non-Hispanic white neighborhood comparison group. AB - BACKGROUND: Limited data suggest that Puerto Ricans experience greater disability than other ethnic groups, but few studies have examined the factors associated with this apparent difference. METHODS: We describe the prevalence of functional limitation and disability in a representative sample of Puerto Rican and Dominican elders in Massachusetts, and in a neighborhood comparison group of non Hispanic whites (NHWs). We then relate disability scores, based on both prevalence and severity of ADL or IADL limitation, with self-reported history of diagnosed health conditions--adjusting for age, sex, body mass index (BMI; weight kg/height m(2)), income, education, living alone, smoking, and alcohol use. RESULTS: Seventy-five percent of Dominican women and 73% of Puerto Rican women reported difficulty with at least one ADL, compared with 64% of NHW women. Puerto Rican men reported significantly more limitation than did NHW or Dominican men. Conditions significantly associated with at least two disability measures among the NHW included smoking, former heavy alcohol use, arthritis, cataract, respiratory disease, and high BMI, but not stroke, diabetes, history of heart attack, or depression. The patterns for Puerto Ricans differed, with the strongest associations between disability and stroke, arthritis, diabetes, and depression, followed by history of heart attack, high BMI, cataract, poverty status, and respiratory disease. Only arthritis and depression were consistently significantly associated with disability among this smaller sample of Dominican elders. CONCLUSIONS: Functional limitation and disability are more prevalent among Puerto Ricans and among Dominican women than among neighborhood NHWs in Massachusetts. Greater disability is associated with the presence of chronic health conditions, which differ by ethnic group. Additional research is needed to further define the social and health factors that contribute to these ethnic differences. PMID- 10737692 TI - Correlates of delayed referral for the diagnosis of dementia in an outpatient population. AB - BACKGROUND: Early diagnosis of dementia is critical, but there is usually a time lag between onset of symptoms and referral for neuropsychological testing and dementia diagnosis. We aimed to identify factors correlated with this delayed referral. METHODS: We studied 140 patients with cognitive deterioration referred to the Memory Clinic of the Catholic University (Rome) between 1995 and 1996. Alzheimer's disease or multi-infarct dementia was diagnosed according to National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) criteria and Hachinski ischemic score. Global cognitive and neuropsychological functions were assessed with the Mini-Mental State Exam (MMSE) and the Mental Deterioration Battery. The performance on the Activities of Daily Living was used to measure physical function. The time between onset of signs of cognitive deterioration and referral for diagnosis (time to diagnosis: TTD) was estimated through a semistructured interview of the caregiver. Independent correlates of TTD were identified after adjustment for potential confounders and stratifying patients based on level of physical function. RESULTS: Of 127 eligible patients, 63% had Alzheimer's disease, 26% multi-infarct dementia, and 11% had dementia of other types. Mean age was 73.9 +/- 8.2 years, and 59% of patients were females. The mean TTD was 13.8 +/- 10.8 months and did not differ by gender, household composition, or type of dementia. For patients with normal physical function, increased age (beta = .50), female sex (beta = .51), and low MMSE score (beta = .36) were associated with longer TTD. Among patients with physical impairment, only MMSE score showed an association with TTD, but it was of opposite direction (beta = -.31). These associations were consistent by type of dementia. CONCLUSIONS: Age, gender, and degree of cognitive impairment are important correlates of the time between onset of signs/symptoms and referral for dementia diagnosis. These factors are independent of the type of dementia but are influenced by the level of physical function. PMID- 10737693 TI - The joint effect of apolipoprotein E epsilon4 and MRI findings on lower-extremity function and decline in cognitive function. AB - BACKGROUND: Cognitive decline and poor physical function are risk factors for disability in old age and may occur more often in subjects with the apolipoprotein E epsilon4 (ApoE-epsilon4) allele. The objective of this study was to investigate the joint effect of ApoE-epsilon4 and structural changes detected on MRI brain scans on cognitive decline and lower-extremity function. METHODS: Brain MRI (1.5 T), neuropsychological tests, and lower-extremity physical function tests were administered to World War II male veteran twins ages 69 to 80. Quantification of MRI scans used a previously published algorithm to segment brain images into total cerebral brain (TCB), cerebrospinal fluid (CSF), and white-matter hyperintensity (WMH) volumes. A short battery of physical performance tests was used to assess lower-extremity function. Ten-year changes in performance on the Mini-Mental State Exam (MMSE), the Benton Visual Retention Test (BVRT), and the Digit Symbol Substitution (DSS) test were used to assess cognitive decline. RESULTS: For the sample as a whole, the comparison of subjects by median split of total cerebral brain volume found that those with brain volumes below the median performed worse on tests of gait and balance (p < .01) and experienced greater cognitive decline on the MMSE and BVRT cognitive test batteries (both p < .01). In addition, subjects with WMH volumes above the median had poor performance on the standing balance tasks and experienced greater decline on the DSS test (p < .01). Stratified analyses revealed that the joint effect of radiological findings and the ApoE-epsilon4 allele on cognitive decline and lower-extremity function was often greater than that expected from the separate effects combined. CONCLUSIONS: We conclude that radiological findings in conjunction with ApoE-epsilon4 may single out a group at higher risk for dementia. We speculate that the observed interaction effect may be due to increased susceptibility to brain injury or impaired repair mechanisms in subjects with ApoE-epsilon4. PMID- 10737694 TI - 1999 Curt P. Richter award. Glucocorticoids and the regulation of memory consolidation. AB - This paper summarizes recent findings on the amygdala's role in mediating acute effects of glucocorticoids on memory consolidation in rats. Posttraining activation of glucocorticoid-sensitive pathways involving glucocorticoid receptors (GRs or type II) enhances memory consolidation in a dose-dependent inverted-U fashion. Selective lesions of the basolateral nucleus of the amygdala (BLA) or infusions of beta-adrenoceptor antagonists into the BLA block the memory modulatory effects of systemic injections of glucocorticoids. Additionally, posttraining infusions of a specific GR agonist administered directly into the BLA enhance memory consolidation, whereas those of a GR antagonist impair. These findings indicate that glucocorticoid effects on memory consolidation are mediated, in part, by an activation of GRs in the BLA and that the effects require beta-adrenergic activity in the BLA. Other findings indicate that the BLA interacts with the hippocampus in mediating glucocorticoid-induced modulatory influences on memory consolidation. Lesions of the BLA or inactivation of beta adrenoceptors within the BLA also block the memory-modulatory effects of intrahippocampal administration of a GR agonist or antagonist. These findings are in agreement with the general hypothesis that the BLA integrates hormonal and neuromodulatory influences on memory consolidation. However, the BLA is not a permanent locus of storage for this information, but modulates consolidation processes for explicit/associative memories in other brain regions, including the hippocampus. PMID- 10737695 TI - Relationship between depressive symptoms and diabetes among native Hawaiians. AB - Increased prevalence of depression has been reported among diabetes patients. We examined this association between diabetes and depressive symptoms in a population-based study where glucose tolerance status was determined with World Health Organization (WHO) criteria. Fasting plasma glucose (FPG) was determined from blood collected from 574 native Hawaiians. The Centers for Epidemiological Studies-Depression (CES-D) scale was used to assess depressive symptoms in association with diabetes history and hemoglobin A1c (HbA1c). A significant association was observed between depressive symptoms and HBA1c that persisted after adjusting for age, BMI, gender, education, and after exclusion of participants reporting a history of diabetes. Diabetes history was no longer associated with CES-D depressive symptoms after adjusting for HbA1c. These results support the hypothesis that depressive symptoms associated with diabetes may be partially explained by a shared neuroendocrinological disturbance. PMID- 10737696 TI - Effects of chronic mild stress (CMS) on thyroid hormone function in two rat strains. AB - This work was carried out to assess the effects of chronic mild stress (CMS) on thyroid function. The CMS model produced an anhedonic effect (reduced preference to sucrose) in Sprague-Dawley and Wistar rats and this effect was reversed by imipramine (IMI) treatment. The effects of CMS on thyroid function were assessed by measuring tT4 (total Thyroxine), tT3 (total Triiodothyronine), TSH (Thyroid Stimulating Hormone) and fT4 (free Thyroxine) serum levels with appropriate immunoassays. CMS increased tT4 and tT3 serum levels in Sprague-Dawley and Wistar rats, but not TSH and fT4 serum levels. Imipramine (IMI) treatment normalized tT4 values. Albumin which binds a fraction of peripheral tT4 and tT3 was also significantly increased in response to CMS, possibly contributing to tT4 and tT3 elevations. The above findings suggest an impact of CMS on thyroid function, especially in tT4 values the changes being reversed with IMI treatment. PMID- 10737697 TI - Influence of a natural stressor (predator odor) on locomotor activity in the meadow vole (Microtus pennsylvanicus): modulation by sex, reproductive condition and gonadal hormones. AB - Sex differences in a variety of non-reproductive behaviors have been indicated to occur in seasonally breeding polygynous promiscuous rodents such as the meadow vole, Microtus pennsylvanicus. The present study was designed to assess the effects of reproductive and hormonal status on the locomotor responses of meadow voles following brief exposure to the odors of a natural predator, the Red fox (Vulpes vulpes). Adult male and female meadow voles, which are seasonal photoperiodically-induced breeders, were housed in either mixed sex pairs under a long, reproductively stimulatory photoperiod (simulating breeding: long light cycle, paired: LLC + P) or in same-sex pairs under a short, reproductively inhibitory photoperiod (simulated non-breeding: short light cycle, non-paired: SLC-NP). On 2 consecutive days following 1 day of baseline activity monitoring, voles were exposed individually for 3 min to fox odor and a novel pungent control odor (extract of almond). The levels of various measures of activity that were displayed by the voles were assessed by an automated Digiscan activity monitoring system. LLC + P (simulated breeding) voles displayed higher basal levels of activity relative to SLC + NP (simulated non-breeding) voles, with males displaying greater activity than females. LLC + P (simulated breeding) males displayed a significant reduction in activity levels following exposure to fox odor relative to control odor. The reductions in activity following fox odor exposure were related to plasma testosterone levels such that a larger behavioral response (i.e. greater reduction) was associated with higher levels of testosterone. Furthermore, dividing males into high and low testosterone groups based on the median levels of testosterone revealed that high but not low testosterone males displayed reductions in activity following exposure to fox odor relative to control odor. No changes in activity levels following exposure to fox odor were noted in SLC-NP males, and either SLC-NP or LLC + P females. These results show that this sexually dimorphic non-reproductive behavior is significantly influenced by reproductive condition and gonadal hormone levels. PMID- 10737698 TI - Pentobarbital stimulates the activity of the GnRH pulse generator interacting with opioid neurons in rats in proestrus. AB - In order to investigate the possibility that i.p. injection of pentobarbital sodium (PB, 32 mg/kg bw) potentiates the GnRH pulse generator activity, effects of i.v. infusions of an opiate receptor antagonist naloxone (NAL, 2 mg/h) on the pulsatile LH secretion were compared in saline (SAL)- and PB-injected rats in proestrus and diestrus 1. In SAL-injected rats in proestrus, NAL infusions significantly increased both the frequency and amplitude of LH pulses, and also the overall mean LH concentration. In PB-injected rats in proestrus, all the parameters of the pulsatile LH secretion were similar to those in SAL-injected rats in proestrus. The NAL infusion in PB-injected rats caused an increase in the frequency, but it was similar to that in SAL-injected rats. But, increases in the amplitude and the overall mean LH observed during NAL infusions in PB-injected rats were greater than in SAL-injected rats. In SAL-injected rats in diestrus 1, NAL infusions increased all the parameters, as in rats in proestrus. In PB injected rats in diestrus 1, LH secretion was severely suppressed. NAL infusions recovered the pulsatile LH secretion, but the frequency and the overall mean LH of the secretion were smaller than those obtained during NAL infusions in SAL injected rats. In addition, characteristic increases in the MUA (volleys), which occur in association with the initiation of an LH pulse and thus are considered to represent an increased activity of the GnRH pulse generator, appeared more frequently during NAL infusions in PB-injected rats in proestrus than in SAL injected rats. These results suggest that the GnRH pulse generator in rats in proestrus, but not in rats in diestrus 1, is refractory to PB and further is potentiated by PB in the response to NAL. Together with the fact that this dosage of PB blocks the surge of LH secretion in rats in proestrus, the concept of the existence of separate neuronal mechanisms responsible for the surge and pulsatile secretion of LH are supported. PMID- 10737699 TI - Behavioral and electrophysiological effects of androstadienone, a human pheromone. AB - Androstadienone is the most prominent androstene present on male human axillary hair and on the male axillary skin surface. We have previously shown that this volatile steroid is able to stimulate [corrected] the human female vomeronasal organ in picogram (pg) quantities, resulting in changes in autonomic activity. These effects are gender-specific. The purpose of the present study was to ascertain whether androstadienone could be considered a human pheromone by altering behavior as well as autonomic function. Forty normal female subjects were randomized in a double-blind manner to receive either control or 100 pg of androstadienone directly to the vomeronasal organ. We report that administration of this steroid under these conditions results in a significant reduction of nervousness, tension and other negative feeling states. Concordant changes were observed in autonomic physiology. PMID- 10737701 TI - A novel rapid colorimetric assay of carcinoembryonic antigen levels in the abdominal cavity to detect peritoneal micrometastasis during gastric cancer surgery. AB - We showed that the carcinoembryonic antigen (CEA) level in a peritoneal washing is an indicator of the postoperative survival of gastric cancer patients. On the premise that the polyvinylidine difluoride membrane adsorbs a fixed quantity of protein, this study was designed to produce a rapid, colorimetric, semi quantitative assay of peritoneal CEA levels by using anti-CEA antibodies. At the time of laparotomy, peritoneal washings were collected from 60 gastric cancer patients, and CEA levels were determined by our assay and by an enzyme immunoassay (EIA) method. The accuracy of our method corresponded with the results of the EIA method. All the cases with high levels of CEA in the peritoneal washings showed positive color changes. Our new assay had no relation to protein concentrations of the samples. The assay makes use of diluted peritoneal washings without adaptation by protein concentration. The measurement can be completed in the operating room within 30 min. As a result, this assay can detect peritoneal microdissemination easily during surgery, and it can be used as an indication of intraoperative chemotherapy against peritoneal micrometastasis. PMID- 10737700 TI - Dopamine function in obsessive compulsive disorder: cortisol response to acute apomorphine stimulation. AB - Central dopaminergic dysfunction has been suggested to be involved in the pathogenesis of obsessive compulsive disorder (OCD). In 15 patients with OCD and in 15 age-sex matched controls we evaluated the dopamine (DA) function by measuring the cortisol (CORT) responses to stimulation with the DA agonist apomorphine (APO). The CORT response to acute saline administration was also measured, to exclude the existence of a pathology of the circadian secretion of the hormone which could obscure the significance of the CORT response to APO stimulation. Basal levels of CORT were the same in patients and controls, but the values after saline administration were significantly higher in patients than in controls. APO stimulation-induced CORT rises were significantly higher in patients than in controls, but when the data after APO were corrected for those after saline, there were no significant difference between the two groups of subjects. Our data suggest that there are no alterations of the central dopaminergic function connected with the regulation of the hypothalamo-pituitary adrenal axis in OCD. PMID- 10737702 TI - Metabolism and effect of platelet-activating factor on the growth of human myeloma cell lines. AB - In this study we have investigated the presence of PAF receptor (PAF-R) on 5 myeloma cell lines (U266, L363, IM9, OPM2 and XG1), their metabolism of PAF and lyso PAF, and the effect of PAF on their growth. All myeloma cell lines express a PAF acetylhydrolase activity and metabolize [3H]PAF and [3H]lyso PAF in 1-alkyl-2 acyl analogue of phosphatidylcholine. Polymerase chain reaction on reverse transcript (RT-PCR) experiments indicate that OPM2, U266, IM9, XG1 and L363 cells express the PAF-R transcript 1 but not the PAF-R transcript 2. Flow cytometry experiments reveal that PAF-R are present on these myeloma cell lines. PAF and the non-metabolizable PAF agonist 1-O-hexadecyl-2-N-methycarbamyl-glycero-3 phosphocholine have no effect on the growth of OPM2, U266, IM9, XG1 and L363 assessed by [3H]thymidine incorporation into DNA. As a positive control of PAF effect on myeloma cells, PAF (1 microM) enhances by 100% the immunoglobulin synthesis by IM9 cells cultured for 48 h. In conclusion the five myeloma cell lines used in this study metabolize PAF through the deacetylation/reacylation pathway. They express membrane PAF-R through the PAF-R mRNA transcript 1 but PAF does not affect their growth. PMID- 10737703 TI - Optimising methods for determining RER status in colorectal cancers. AB - Approximately 13% of colorectal cancers display microsatellite instability (MSI), a form of replication error repair. Colorectal cancers developing in individuals with constitutional defects in the mismatch repair (MMR) genes hMLH1, hMSH2, hPMS1 and hPMS2 consistently show evidence of this phenomenon. Since MSI is indicative of MMR deficiency, testing colorectal cancers for MSI provides a method of refining the identification of carriers of germline MMR mutations. To assess which microsatellites represent the best reporters of replication error (RER) status we have examined 116 early onset colorectal cancers for MSI. MSI was assessed using eight dinucleotide- and two mononucleotide-repeat fluorescently labelled polymerase chain reaction (PCR) markers. The two mononucleotide repeat markers (BAT25 and BAT26) were highly sensitive and typing of either represents an efficient strategy for defining RER status of colorectal cancers and obviates the requirement of typing numerous microsatellite markers. PMID- 10737704 TI - Prenylated chalcones and flavanones as inducers of quinone reductase in mouse Hepa 1c1c7 cells. AB - The objective of this study was to determine if prenylchalcones (open C-ring flavonoids) and prenylflavanones from hops and beer are inducers of quinone reductase (QR) in the mouse hepatoma Hepa 1c1c7 cell line. All the prenylchalcones and prenylflavanones tested were found to induce QR but not CYP1A1 in this cell line. In contrast, the synthetic chalcone, chalconaringenin, and the flavanone, naringenin, with no prenyl or geranyl groups, were ineffective in inducing QR. The hop chalcones, xanthohumol and dehydrocycloxanthohumol hydrate, also induced QR in the Ah-receptor-defective mutant cell line, Hepa 1c1c7 bp(r)c1. Thus, the prenylflavonoids represent a new class of monofunctional inducers of QR. PMID- 10737705 TI - SYT-SSX fusion proteins in synovial sarcomas: detection and characterization with new antibodies. AB - To identify and characterize the SYT-SSX fusion proteins in synovial sarcomas, we developed two polyclonal antibodies against the N-terminal part and for the C terminal part of the SYT-SSX2 protein. Specificity was demonstrated on COS-7 cells transfected with two subtypes of SYT-SSX fusion genes, SYT-SSX1 and SYT SSX2. Both antibodies recognized a single protein of 61 kDa in an immunoprecipitation of the transfected COS-7 cell lysates. These antibodies also detected the native protein of 61 kDa in the lysate of a human synovial sarcoma cell line (HS-SY41) with immunoprecipitation, and in extracts of human synovial sarcomas with western blot analysis. An immunohistochemical study, using human synovial sarcoma tissues, demonstrated that the SYT-SSX fusion proteins localized in the nucleus of the tumor cells. These antibodies provide a useful method for studying the expression of the SYT-SSX fusion proteins. PMID- 10737706 TI - Differences in the expression and effects of interleukin-1 and -2 on androgen sensitive and -insensitive human prostate cancer cell lines. AB - The presence of interleukin-1 (IL-1) and IL-2 mRNA in five human prostate cancer (PCa) cell lines and their effects on cellular proliferation and prostate specific antigen (PSA) levels were examined. IL-1 was found in androgen unresponsive PC-3 and DU-145 but not in the androgen-responsive LNCaP, MDA-PCA-2a and MDA-PCA-2b cell lines. IL-2 message was absent while that of GAPDH (positive control) was present in all five cell lines. IL-1 decreased while IL-2 increased PSA levels of near-confluent LNCaP cells after 24 h of treatment. IL-1 inhibited whereas IL-2 stimulated the growth of sub-confluent LNCaP cells (72 h). Neither cytokine affected the proliferation of DU-145 or PC-3 cells. PMID- 10737707 TI - Overexpression of fatty acid synthase in SKBR3 breast cancer cell line is mediated via a transcriptional mechanism. AB - Overexpression of fatty acid synthase (FAS) in certain breast, prostate and ovarian tumors has been correlated with aggressive cancer phenotype and poor prognosis. The objective of this study was to use a breast cancer-derived cell line, SKBR3, as a model to define the underlying mechanism for overexpression of FAS in cancer cells. Different stages of gene expression where overproduction of FAS could potentially be achieved were investigated. Whereas gross chromosomal rearrangement at the FAS locus, amplification of the FAS gene, increases in FAS message stability and longer half-life of the FAS protein were not detected, an increase in the rate of transcription of the FAS gene, and consequently a higher abundance of FAS-mRNA, was found to be primarily responsible for FAS overexpression in this cell line. PMID- 10737708 TI - Association between acetylator genotype and 2-amino-1-methyl-6-phenylimidazo[4,5 b]pyridine (PhIP) DNA adduct formation in colon and prostate of inbred Fischer 344 and Wistar Kyoto rats. AB - 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine (HCA) found in cooked meats, causes colon and prostate tumors in male rats. Polymorphic N-acetyltransferase metabolizes N-hydroxy-PhIP to a DNA-reactive form. Liver, colon, and prostate PhIP-DNA adduct levels were compared in male rapid-acetylator Fischer 344 (F344) and slow-acetylator Wistar-Kyoto (WKY) rats fed 0.01 or 0.04% PhIP. Liver PhIP-DNA adduct levels at both PhIP doses, and colon PhIP-DNA adduct levels at the 0.01% PhIP dose were unaffected by acetylator genotype. However, in rats fed 0.04% PhIP, colon PhIP-DNA adduct levels were higher in rapid acetylator F344 rats (P < 0.05). Similarly, prostate PhIP-DNA adduct levels were higher in rapid acetylator F344 rats at both PhIP doses (P < 0.05). The combination of the high-PhIP dose and rapid-acetylator genotype resulted in the highest level of PhIP-DNA adducts in rat colon and prostate. PMID- 10737709 TI - Licania michauxii Prance root extract induces hsp 70 mRNA and necrotic cell death in cultured human hepatoma and colon carcinoma cell lines. AB - An extract of Licania michauxii Prance root was found to be cytotoxic to cultured human hepatoma (HepG2) and colon carcinoma (Caco-2) cells. Morphological and nuclear characteristics of treated cells were consistent with necrotic death. Increases in the chaperone protein hsp 70 and hsp 70 mRNA were dose dependent reaching peak mRNA levels (40-fold above control) at 6 h. Increases in nuclear localization of hsp 70 was also observed with treatment. Heat treatment of cells for 45 min to induce hsp 70 prior to treatment with the extract provided transient protection from the necrotic response. PMID- 10737710 TI - Relationship between alcohol drinking, ADH2 and ALDH2 genotypes, and risk for hepatocellular carcinoma in Japanese. AB - The polymorphism in the ALDH2 gene plays a central role in Asian alcohol hypersensitivity and has been associated with the risk for esophageal cancer. In the present study, we attempted to examine associations between the ADH2 and ALDH2 polymorphisms, alcohol drinking and hepatocellular carcinoma (HCC) development in a case-control study in Japan. One hundred and two patients with HCC (85 males and 17 females) and 125 control subjects (101 males and 24 females) were enrolled in the study. Higher cumulative amounts of alcohol consumption (drink-years of > or = 40 drinks/day x year) showed a significant association with HCC development (odds ratio, OR = 2.7; 95% CI = 1.3-5.5, adjusted for age and smoking). By contrast, we could find no association of the ALDH2 genotypes with HCC development (adjusted OR for ALDH2*1/*2 = 1.1; 95% CI = 0.6-2.1). Likewise, the ADH2 genotypes were not associated with HCC development (adjusted OR for ADH2*2/*2 = 0.8; 95% CI = 0.5-1.5). The present results do not support a contribution of acetaldehyde, an active metabolite of ethanol, to HCC development and rather indicate a direct involvement of ethanol in hepatocarcinogenesis. PMID- 10737711 TI - Cuphiin D1, the macrocyclic hydrolyzable tannin induced apoptosis in HL-60 cell line. AB - Cuphiin D1 (CD1), a new macrocyclic hydrolyzable tannin isolated from Cuphea hyssopifolia, has been shown to exert antitumor activity both in vitro and in vivo. In this study, we explored the mechanism of the CD1-induced antitumor effect on human promyelocytic leukemia (HL-60) cells. The results showed that CD1 induced cytotoxicity in HL-60 cells and the IC50 was 16 microM after 36 h treatment. HL-60 cells treated with CD1 for 36 h decreased the uptake of [3H] labeled thymidine, uridine and leucine in a dose dependent manner. Electron micrographs demonstrated that HL-60 cells treated with 16 microM CD1 for 36 h exhibited chromatin condensation, indicating the apoptosis occurrence. Flow cytometric analysis demonstrated the presence of apoptotic cells with low DNA content, a decrease of cell population at G2/M phase, and a concomitant increase of cell population at G1 phase. CD1 also caused DNA fragmentation and inhibited Bcl-2 expression in the HL-60 cells. These results suggest that the inhibition of Bcl-2 expression in HL-60 cell might account for the mechanism of CD1-induced apoptosis. PMID- 10737712 TI - Diverse effects of 9-hydroxyellipticine on the chemosensitivity of human pancreatic cancer cells harboring p53 mutations. AB - Recently, it has been shown that 9-hydroxyellipticine (9-HE), an antitumor alkaloid has a unique property of restoring functional wild-type (wt) p53 activity via inhibition of mutant (mt) p53 protein phosphorylation. In the present study, we investigated the effect of 9-HE on the drug sensitivity of human pancreatic cancer cells. Exposure of cells to 9-HE at a relatively low concentration of 1 microM induced almost no cell death but was sufficient to restore wt p53 activity, as evidenced by an induction of endogenous p21WAF1/CIP1 concomitant with G1 and G2/M arrests in cell-cycle progression. Pretreatment with 1 microM 9-HE markedly enhanced cell killing when combined with cisplatin or mitomycin C. In contrast, 9-HE pretreatment protected cells from killing by 5 fluorouracil, VP-16, or vincristine. These effects of 9-HE were specific for several cell lines containing mt p53 and were not observed in p53-negative or wt p53 expressing cells. Taken together, these findings suggest that 9-HE may exert different effects on the drug sensitivity of pancreatic cancer cells displaying p53 mutations possibly through restoration of wt p53. PMID- 10737713 TI - Expression of thrombomodulin in squamous cell carcinoma of the lung: its relationship to lymph node metastasis and prognosis of the patients. AB - Thrombomodulin (TM) is a type of thrombin receptor that was identified originally on the endothelium and acts as a natural anticoagulant through converting thrombin from a procoagulant protease to an anticoagulant. We reported previously that TM was also expressed in the squamous epithelium mainly at the intercellular bridges. In this study, we examined TM expression in the primary lesions of 81 patients with squamous cell carcinomas (SCCs) of the lung and in the lymph node metastatic lesions of 39 patients using immunohistochemical methods. The carcinoma tissues expressed TM mainly at the cell-cell boundaries and also in the cytoplasm. When TM expression was compared between the primary and metastatic lesions in the 39 patients who had lymph node metastasis, 26 (67%) showed decreased TM expression, 13 (33%) showed no change, and none (0%) showed an increase in the metastatic lesions. Wilcoxon's signed-rank test indicated that tumor cells that were positive for TM expression were significantly rarer in the metastatic lesions than in the primary tumors (P < 0.0001). The present study also showed that the patients with TM-negative expression in the primary tumors showed significantly poorer survival than those with TM-positive expression, mainly due to distant metastases of poorly-differentiated SCCs with negative TM expression in the primary tumors. These results indicate that the reduction of TM expression seems to play an important role in the metastatic process of lung SCCs. PMID- 10737714 TI - Expression of MAT1/PEA-15 mRNA isoforms during physiological and neoplastic changes in the mouse mammary gland. AB - MAT1 is a novel transforming gene which was cloned from a mouse mammary tumor induced by N-methyl-N-nitrosourea in vitro in the presence of lithium as a mitogen. Later, it was found to be identical to the 3' untranslated region (UTR) of the 2.5 kb isoform of PEA-15 (phosphoprotein enriched in astrocytes-15 kDa). We re-cloned MAT1/PEA-15 cDNAs and showed 2.5, 2.0 and 1.8 kb isoforms and confirmed MAT1 localization as reported. The 2.0 and 1.8 kb isoforms were produced by alternative splicing and alternative polyadenylation at the 3' UTR, respectively. To analyze the role of MAT1/PEA-15, we examined the expression of MAT1/PEA-15 mRNA in normal mammary tissues and in mammary tumors. The mammary gland during pregnancy, lactation and weaning showed weak but stable expression. Compared with normal mammary gland, mammary tumors showed stronger expression. Aberrant expression of MAT1/PEA-15 isoforms was found in mouse mammary epithelial cell lines, FSK7 and TM6, which lost the 2.5/2.0 and 2.5 kb isoforms, respectively. In contrast to other oncogenes like c-myc, MAT1/PEA-15 mRNA was extremely stable after actinomycin D and cycloheximide treatments suggesting that other protein expression is prerequisite for degradation of MAT1/PEA-15 mRNA. It evoked the possibility of the 3' UTR of MAT1/PEA-15 (designated as MAT1-T) as a riboregulator in mammary tumorigenesis and necessity for further analysis of human breast cancers as well as mouse mammary tumors. PMID- 10737715 TI - Modifying effects of carotenoids on superoxide and nitric oxide generation from stimulated leukocytes. AB - Excessive and prolonged generation of superoxide (O2-) and nitric oxide (NO) from inflammatory leukocytes is associated with several lifestyle-related diseases, including cancer. In the present study, we screened 19 natural carotenoids for their modifying effects on O2- and NO generation from differentiated human promyelocytic HL-60 cells and mouse macrophage RAW 264.7 cells, respectively. Of the carotenoids tested, halocynthiaxanthin, isolated from oysters, showed the highest suppressive effect on the generation of both free radicals. The inhibitory potencies of certain carotenoids on radical generation markedly exceeded that of beta-carotene. In addition, some important structural moieties regulating radical generation suppression are discussed. PMID- 10737717 TI - Induction of S100A4 gene expression inhibits in vitro invasiveness of human squamous cell carcinoma, KOSC-3 cells. AB - S100A4 is considered functionally involved in metastasis and invasiveness of rodent and human mammary tumors. We screened the expression of S100A4 in human squamous cell carcinoma cell lines, and found 2 cell lines which were highly invasive, but did not express any noticeable extent of S100A4. To examine whether the expression of S100A4 regulated invasiveness of squamous cell carcinoma, we transfected S100A4 cDNA into KOCS-3 and HSC-4 squamous cell carcinoma cells. The transfectants from KOSC-3 cells expressing sense S100A4 decreased invasiveness by 80% compared with cells of the wild type or those with the vector only. PMID- 10737716 TI - DNA hypermethylation at the pS2 promoter region is associated with early stage of stomach carcinogenesis. AB - pS2, a member of the trefoil peptide family, has been suggested to be a gastric specific tumor suppressor. We examined the expression of pS2 in gastric carcinomas, adenomas and non-neoplastic mucosa and analyzed the DNA methylation in the pS2 promoter. Reduced expression of pS2 was frequently associated with well-differentiated adenocarcinomas. The CpG sites within the promoter region of the pS2 gene were methylated in pS2-negative gastric carcinoma cell lines whereas it was not in pS2-positive cell line. The promoter methylation was detected in gastric carcinoma tissues and intestinal metaplasia with reduced pS2 expression whereas none of the carcinomas with preserved pS2 expression showed the promoter methylation. These findings suggest that reduced expression of pS2 due to the promoter methylation may participate in an early stage of stomach carcinogenesis, especially of well differentiated type. PMID- 10737718 TI - SDZ PSC 833 the drug resistance modulator activates cellular ceramide formation by a pathway independent of P-glycoprotein. AB - SDZ PSC 833 (PSC 833) is a new multidrug resistance modulator. Recent studies have shown that the principal mechanism of action of PSC 833 is to bind P glycoprotein (P-gp) and prevent cellular efflux of chemotherapeutic drugs. We previously reported that PSC 833 increases cellular ceramide levels. The present study was conducted to determine whether the impact of PSC 833 on ceramide generation is dependent on P-gp. Work was carried out using the drug-sensitive P gp-deficient human breast adenocarcinoma cell line, MCF-7, and drug resistant MCF 7/MDR1 clone 10.3 cells (MCF-7/MDR1), which show a stable MDR1 P-gp phenotype. Overexpression of P-gp in MCF-7/MDR1 cells did not increase the levels of glucosylceramide, a characteristic which has been associated with multidrug resistant cells. Treatment of MCF-7 and MCF-7/MDR1 cells with PSC 833 caused similar ceramide elevation, in a dose-responsive manner. At 5.0 microM, PSC 833 increased ceramide levels 4- to 5-fold. The increase in ceramide levels correlated with a decrease in survival in both cell lines. The EC50 (concentration of drug that kills 50% of cells) for PSC 833 in MCF-7 and MCF 7/MDR1 cells was 7.2 +/- 0.6 and 11.0 +/- 1.0 microM, respectively. C6-Ceramide exposure diminished survival of MCF-7 cells; whereas, MCF-7/MDR1 cells were resistant to this short chain ceramide analog. Preincubation of cells with cyclosporine A, which has high affinity for P-gp, did not diminish the levels of ceramide generated upon exposure to PSC 833. These results demonstrate that PSC 833-induced cellular ceramide formation occurs independently of P-gp. As such, these data indicate that reversal of drug resistance by classical P-gp blockers may be modulated by factors unrelated to drug efflux parameters. PMID- 10737719 TI - Non-ionic detergent Tween 80 modulates VP-16 resistance in classical multidrug resistant K562 cells via enhancement of VP-16 influx. AB - The non-ionic detergent Tween 80, which is used as a solvent for lipophilic drugs such as VP-16 and Taxotere, was found to reverse VP-16 resistance of the P glycoprotein-associated multidrug resistance phenotype via increasing VP-16 influx. In adriamycin-resistant human chronic myelogenous leukemia K562 cells (K562/ADM), which overexpress mdr1 mRNA, the accumulation of VP-16 was only about 10% that in wild-type K562 cells. Tween 80 enhanced VP-16 accumulation in K562/ADM cells but did not influence VP-16 accumulation in parental K562 cells. VP-16 efflux was rapid and similar in both sensitive and resistant cell lines and was not blocked by Tween 80 or verapamil. Under glucose-free conditions, VP-16 accumulation in K562/ADM cells was only half of that in K562 cells. Tween 80 increased VP-16 accumulation in K562/ADM cells in glucose-free medium. In growth inhibition assay, Tween 80 reversed K562/ADM sensitivity to VP-16 without cell damage. Taken together, Tween 80 reverses VP-16 sensitivity in multidrug resistant K562 cells by increasing influx, which is considered to be the primary mechanism of VP-16 resistance in K562/ADM cells. PMID- 10737720 TI - Expression of matrix metalloproteinase-2 in glial and neuronal tumor cell lines: inverse correlation with proliferation rate. AB - The expression of matrix metalloproteinases (MMPs) has been found to be positively correlated to the degree of malignancy in gliomas, indicating that poorly differentiated brain tumor cells produce more MMPs than differentiated ones. We determined the production of active MMP-2 in five glial (U138MG, U373MG, A172, C6, GOS-3), two neuronal (SK-N-SH, SK-N-MC), and two pluripotent cell lines with facultative neuronal and glial differentiation (P19 and NT2) by gelatin zymography. The MMP-2 activity profiles were compared to the proliferative activities of the cell lines. MMP-2 expression varied from barely existent (P19 cells) to strong (U138MG and SK-N-SH). Interestingly, for the cell lines with high MMP-2 expression levels, low proliferative activities were recorded, and vice versa. Retinoic acid induced neuronal differentiation and a reduction of proliferation of P19 cells; the differentiated cells produced significantly more MMP-2 than untreated cells. Upon confluency, GOS-3 cells showed reduced proliferation, but increased MMP expression. Thus, proliferative activity was inversely correlated to MMP-2 expression in the tumor cell lines analyzed. PMID- 10737721 TI - Influence of perinatal genistein exposure on the development of MNU-induced mammary carcinoma in female Sprague-Dawley rats. AB - Genistein, a phytoestrogen, was subcutaneously (s.c.) injected to pregnant Sprague-Dawley CD rats on gestational days 16-20 at either 25 mg (Group 1) or 5 mg/day (Group 2). Female offspring of mothers not exposed to genistein during pregnancy received 12.5 mg genistein s.c. at neonatal days 15 and 18 (Group 3), or received vehicle only (Group 4). At 35 days of age, 4-9 female offspring from each group were autopsied to observe the influence of genistein, and remainder of female offspring received 50 mg/kg N-methyl-N-nitrosourea (MNU) intraperitoneally and were sacrificed when mammary tumors were larger than 1 cm in size or when they reached 35 weeks of age. Genistein treatment during the perinatal period resulted in lower body weight and lower relative uterine-ovarian weight at 35 days, and a prolonged estrus cycle with a long estrus phase at 12-16 weeks. However, at 35 days (time at MNU administration), mammary gland development, cell proliferation rate (PCNA labeling index), and the number of estrogen receptor (ER)- and progesterone receptor (PgR)-positive cells were similar between genistein-treated and untreated rats. Twenty-five or 5 mg genistein/day in utero (between days 16 and 20 of gestation) or 12.5 mg genistein/day on neonatal days 15 and 18 did not affect the incidence of mammary tumors > 1 cm or the latency but did increase the number of mammary cancer lesions when MNU was administered at the time when the mammary gland growth in genistein-treated and untreated rats was similar. Thus, perinatal genistein is an endocrine disrupter and increases the multiplicity of MNU-induced mammary carcinoma in rats. PMID- 10737722 TI - Colony formation of soft tissue sarcoma cells is inhibited by lipid-mediated antisense oligodeoxynucleotides targeting the human mdm2 oncogene. AB - More than one third of human soft tissue sarcoma (STS) have elevated levels of the MDM2 oncoprotein, resulting either from gene amplification or alternate mechanisms. MDM2 functions as a negative feedback regulator of the tumor suppressor p53. The aim of the present study was to investigate whether mdm2 antisense oligodeoxyribonucleotides (AS-ODNs) can influence the growth characteristics of two MDM2-overexpressing STS cell lines (US8-93, LMS6-93) where both have heterozygous p53 non-missense mutations. Cells were treated with lipofectamine-complexed mdm2 AS-ODNs complementary to a sequence of the mdm2 cDNA initiation site in comparison to sense control ODNs. After seeding and cultivation of a defined cell number the clonogenic survival was performed. The treatment of US8-93 cells with AS-ODNs, but not with sense ODNs, decreased the number of colonies up to > 80%. Western blot analysis demonstrated a significant decreasing of MDM2 protein level in AS-ODN transfected cells indicating an AS specific inhibition of mdm2 transcription in US8-93 cells. Additionally, an increase of the G2/M population was found. In contrast, in the LMS6-93 cells treated with AS-ODNs only a decrease in clonogenic survival up to 26%, no change in MDM2 protein level and no cell cycle alterations were seen. All these factors taken together into consideration can be suggest that lipid-mediated mdm2 AS-ODNs could be as an effective therapeutic strategy for STS with an abnormal mdm2 overexpression. PMID- 10737723 TI - Enhanced cytotoxic effect of Ara-C by low intensity ultrasound to HL-60 cells. AB - A clonogenic assay was tested in order to determine the effects of low intensity ultrasound on HL-60 cells in the presence of cytosine arabinoside (Ara-C). HL-60 cells were exposed to ultrasound at an intensity of 0.3 W/cm2 (48 kHz). Cells were then cultured for 8 days and the number of colonies was statistically analyzed (ANOVA). Ultrasound exposure alone for 120 s resulted in no significant decrease of colonies compared to non-treated cells (P0 = 0.1426). Significant differences (P0 < 0.005) were obtained between ultrasound treated and untreated cells in the presence of various concentrations of Ara-C (2 x 10(-9), 1 x 10(-8), 2 x 10(-8), 5 x 10(-8), 1 x 10(-7) M). Morphological evaluation of ultrasound irradiated cells with scanning electron microscopy showed minor disruption of cell surface and disappearance of microvilli. These observations suggests that low intensity ultrasound altered the cell membrane thus resulting in change in Ara-C uptake into HL-60 cells. PMID- 10737724 TI - Suppressive effects of dominant negative ras mutant N116Y on transformed phenotypes of human bladder cancer cells. AB - To investigate the suppressive effect of dominant negative H-ras mutant N116Y on transformed phenotypes, we established two N116Y ras mutant stable transfectant clones (C5, C13) of human bladder cancer cell line, UMUC-2. These N116Y ras mutant transfectants, especially the C5 cells, showed a dramatic change of cellular morphology and significantly reduced growth in soft agar compared to their control. Furthermore, phosphorylation of the Jun NH2-terminal kinase (JNK) was significantly decreased in these transfectants compared to the control. These results suggest that the N116Y-induced suppression of transformed phenotypes in UMUC-2 cells is associated with inhibition of JNK phosphorylation. PMID- 10737725 TI - Prevalence of the APC E1317Q variant in colorectal cancer patients. AB - The notion that some common variants of APC might confer an increased colorectal tumour risk is supported by studies of the I1307K polymorphism. Recently it has been proposed that the E1317Q variant is also associated with an increased risk. We have studied the prevalence of E1317Q in 364 colorectal cancer patients and in 290 controls. Two patients were shown to possess E1317Q. Neither had a family history of colorectal cancer or co-existent adenomatous polyps. Two controls also carried E1317Q. This finding suggests that E1317Q is unlikely to be associated with anything more than a moderate increase in risk of colorectal cancer. PMID- 10737726 TI - Enzyme-linked PNA lectin-binding assay of serum T-antigen in patients with SCC of the uterine cervix. AB - The expression of Thomsen-Friendenreich antigen (T-Ag) is associated with enhanced metastatic potential, poor prognosis and decreased survival rate in a variety of malignancies, and their detection and quantification can be used in serologic diagnosis. T-antigen expressions were measured by the enzyme-linked lectin assay (ELLA) with peanut agglutinin (PNA) in the sera of patients with squamous cell carcinoma (SCC) of the uterine cervix from 286 patients. This study has a sensitivity of 80%, specificity of 82% and a positive predictive value of 93%. Quantification of the T-antigen may provide useful biochemical indices for clinical assessment of the tumor spread and invasiveness of disease in SCC of the uterine cervix. Moreover, the ELLA assay is cheap, easy to perform and reproducible in the prognosis and diagnosis of SCC of the uterine cervix. PMID- 10737727 TI - Glutathione-related factors are not correlated with sensitivity of human tumour cells to actinomycin D. AB - Glutathione (GSH) contents and activities of glutathione S-transferases (GST), glutathione reductase (GSH-RD), glutathione peroxidase (GSHpx) and glutathione conjugate export pump (GS-X pump) were determined in eight human tumour cell lines with different sensitivities to melphalan, a substrate of glutathione conjugation, and actinomycin D which has not been shown to be detoxified by glutathione-related mechanisms. Chang liver cells with highest GSH content and highest activities of GST, GSH-RD, GSHpx and GS-X pump were found to be most resistant to melphalan. Statistical analysis showed significant correlations between sensitivities of the human tumour cells to melphalan and the glutathione related factors (r = 0.72-0.79; except for GST, r = 0.65, P = 0.08), while there were no significant correlations observed between sensitivities of the human tumour cells to actinomycin D and all the glutathione-related factors tested (r = -0.25-0.14). Significant correlations of the glutathione-related factors to resistance of human tumour cells to melphalan, a substrate of glutathione conjugation, but not to resistance of the human tumour cells to actinomycin D which has not been shown to be detoxified by glutathione-related mechanisms suggested that glutathione-related mechanisms contribute to drug resistance by increased detoxification of the drugs involved. PMID- 10737728 TI - Hemopexin as a carrier protein of tumor-localizing Ga-metalloporphyrin-ATN-2. AB - During size exclusion HPLC, ATN-2 binding protein separated from human and mouse sera, SCCVII and colon 26 tumor tissues were found in fraction 13 (A: estimated molecular weight 70,000). Fraction 13(A) of human sera was exclusively reactive to the human hemopexin antibody. During two-dimensional electrophoresis and amino acid sequence analysis, Fraction 13(A) of C3H/He mouse sera was found to have partial homology with the mouse hemopexin precursor. Glycoprotein with the same domain structure of hemopexin has been proposed to be an important carrier protein that forms the tumor-localizing activity of water-soluble porphyrin. PMID- 10737730 TI - Diapause induction in adults of three Rhipicephalus appendiculatus stocks. AB - Newly-moulted adults of three Rhipicephalus appendiculatus stocks, originating from Kenya, Zambia and Zimbabwe, were kept in different photoperiods (12h:12h, 13h:11h and 14h:10h light:dark). The stock originating from Kenya showed almost no behavioural diapause in any of these day lengths, whereas virtually all individuals of the Zimbabwean stock entered a behavioural diapause irrespective of day length. Adults from the Eastern Province of Zambia, a transition zone between the multivoltine phenology in equatorial Africa and the univoltine in southern Africa, exhibited a photoperiod-dependent diapause response. The possible mechanisms of diapause regulation and their origin are discussed. PMID- 10737732 TI - Models of temporal variation in questing activity in individuals of Ixodes ricinus (Acari: Ixodidae). AB - Intensive observations of the questing activity of Ixodes ricinus ticks in the field were made to provide data on the range of durations of periods of continuous questing activity, and of the variation in questing activity between individuals. Continuous periods of questing were observed to extend to a maximum of 28 hours. Substantial variation in questing activity between individuals was observed. Models fitted to the distribution of durations of bouts of questing activity provide insights into the questing ecology of I. ricinus. Results suggest that questing duration may not be solely dependent on the state of hydration of the tick. A function fitted to the frequency distribution of the proportions of active life that individuals spend on questing, provides an empirically-based model that can be used to generate a stochastic expression of variation of questing activity in individuals in a questing population. PMID- 10737731 TI - Molecular individuality: polymorphism of salivary gland proteins in three species of ixodid tick. AB - Pooled tissue samples are frequently used in biochemical studies involving parasites in order to ensure that there is sufficient material for experimentation. A pooled sample is considered to represent the overall phenotypic characteristics of the investigated population. However, this will not be the case if there is a significant degree of molecular polymorphism among individuals in the sampled population. Here we demonstrate marked differences in the protein profile of salivary glands among individuals from three species of ixodid tick (Rhipicephalus appendiculatus, Amblyomma variegatum, Ixodes ricinus), and show that pooling the tissue of several individuals masks substantial qualitative differences among the individuals. Our observations indicate that much greater caution is needed in general when using pooled samples if the molecular diversity within the population is not clearly defined. PMID- 10737733 TI - Seasonal abundance of Allothrombium monochaetum and Allothrombium pulvinum in Navarra-Nafarroa (northern Spain), with notes on larval host preference and rate of parasitism. AB - In this paper we report on the seasonal abundance of velvet mite larvae of Allothrombium pulvinum Ewing and Allothrombium monochaetum Goldarazena and Zhang in a meadow located in the mediterranean Navarra-Nafarroa region (northern Spain). Larvae of A. monochaetum peaked in numbers on Aphis fabae in mid-June while larvae of A. pulvinum peaked in the third week of July. Both species preferred the aphid thorax as attachment site. We also provide a list of aphid hosts of both mites and observed density of parasites on the hosts. PMID- 10737734 TI - Assessment of Amblyseius fallacis (Acari: Phytoseiidae) for biological control of tetranychid mites in an Ontario peach orchard. AB - We introduced a mass-reared pyrethroid-resistant strain of the predatory phytoseiid mite Amblyseius fallacis (Garman) into an Ontario peach orchard in an attempt to control populations of the phytophagous mites Panonychus ulmi Koch and Tetranychus urticae Koch (Acari: Tetranychidae). Releases of 1,000 and 2,000 mites per tree were made, at three different times. The release of 2,000 mites per tree in June and in July resulted in significantly higher phytoseiid densities than was observed on control trees. However, densities of P. ulmi or T. urticae were not significantly affected by any release rate or by timing. The release of 1,000 A. fallacis per tree, or of any density in August, did not significantly increase phytoseiid abundance. In the following year, population dynamics of both phytoseiid and phytophagous mites were not significantly affected by the previous year's release. Amblyseius fallacis can be a useful predator in some fruit orchards. However, further research is necessary into the timing and rate of release, modified spray programmes, and with different crops, in order to clarify the role of this species for biological control in Ontario peach orchards. PMID- 10737729 TI - Molecular detection of pathogen DNA in ticks (Acari: Ixodidae): a review. AB - Ticks play an important role in human and veterinary medicine, in particular due to their ability to transmit a wide spectrum of pathogenic micro-organisms of protozoal, rickettsial, bacterial and viral origin. Pathogens in ticks can be identified by conventional methods such as indirect immunofluorescence, isolation in cell culture or by using histological staining techniques. However, the advent of the polymerase chain reaction (PCR) has resulted in tremendous improvements in the specific and sensitive detection of pathogen DNA in ticks. In this paper, literature on DNA extraction methods, PCR protocols, primers and probes, which are in use for the successful detection and identification of pathogens in ticks, are critically reviewed. Some recommendations are also given towards the end of this review. PMID- 10737735 TI - Accumulation and turnover of 2-tridecanone in Tetranychus urticae and its consequences for resistance of wild and cultivated tomatoes. AB - In this study we assessed the dynamic changes of 2-tridecanone in a herbivorous mite (Tetranychus urticae) on tomato (Lycopersicon esculentum, cv. 'Moneymaker'), a plant with methyl ketones in the tetracellular tips of the glandular trichomes (Type VI). We showed that spider mites accumulate 2-tridecanone when foraging on cultivated tomato. Thus, the rate of mite-trichome contact multiplied by the amount of toxin per trichome tip exceeded the relative rate of toxin turnover multiplied by the amount of toxin per mite. The relative rate of toxin turnover was estimated to be 1.1 per day on cucumber, a plant without this toxin. The amount per trichome tip varied from 0.33 ng for middle-leaf trichomes to 1.26 ng for main-stem trichomes. Hence, to achieve a static level of 2-tridecanone equal to 8-17 ng per mite--representing the level we found in mites on middle leaves- the rate of mite-trichome contact should be 26-57 per day. Because methyl ketone apparently accumulates in the spider mites on tomato, the rate of mite-trichome contact is probably higher than that. We expect the accumulation of ketones to occur especially on the stems of cultivated tomato, since this is the area most densely occupied with glandular hairs and because here the hairs have higher levels of the methyl ketones. Using dose-response relationships assessed earlier (Chatzivasileiadis and Sabelis, 1997, 1998), we estimated that the number of mite trichome contacts causing 50% mortality per day is equal to 88 on a tomato stem, whereas it equals 70 for another strain of spider mites collected from cucumber. On wild tomato, L. hirsutum f. glabratum (PI 134417), just one to two contacts would suffice to cause 50% mortality per day. We suggest that methyl ketones from glandular hairs on tomato are an important mortality factor for spider mites on wild tomato and probably also on cultivated tomato. PMID- 10737736 TI - Pyrrolidine inhibitors of human cytosolic phospholipase A(2). PMID- 10737737 TI - Design, synthesis, and antimicrobial activity of 6-O-substituted ketolides active against resistant respiratory tract pathogens. PMID- 10737738 TI - Mapping the melatonin receptor. 6. Melatonin agonists and antagonists derived from 6H-isoindolo[2,1-a]indoles, 5,6-dihydroindolo[2,1-a]isoquinolines, and 6,7 dihydro-5H-benzo[c]azepino[2,1-a]indoles. AB - 6H-Isoindolo[2,1-a]indoles (5, 7, 10, 13), 5,6-dihydroindolo[2, 1-a]isoquinolines (20, 21), and 6,7-dihydro-5H-benzo[c]azepino[2, 1-a]indoles (23, 25, 27, 30) have been prepared as melatonin analogues to investigate the nature of the binding site of the melatonin receptor. The affinity of analogues was determined in a radioligand binding assay using cloned human mt(1) and MT(2) receptor subtypes expressed in NIH 3T3 cells. Agonist and antagonist potency was measured using the pigment aggregation response of a clonal line of Xenopus laevis melanophores. The 2-methoxyisoindolo[2, 1-a]indoles (7a-d) showed much higher binding affinities than the parent isoindoles (5a-e), and whereas 7a-c were agonists in the functional assay, 7d and 5a-e were antagonists. The 2-ethoxyisoindolo[2,1 a]indoles (10a-d) showed reduced binding affinities compared to their methoxy analogues, while the 5-chloro derivative 13 showed a considerable reduction in binding affinity and potency compared to 7a. The 10-methoxy-5,6-dihydroindolo[2, 1-a]isoquinolines (21a-c) had higher binding affinities than the corresponding parent indoloisoquinolines (20a-c) in the human receptor subtypes, and the parent compounds were antagonists whereas the 10-methoxy derivatives were agonists in the functional assay. The N-cyclobutanecarbonyl derivatives of both the parent (20d) and 10-methoxyl (21d) series had similar binding affinities and were both antagonists with similar potencies. The 11-methoxy-6, 7-5H-benzo[c]azepino[2,1 a]indoles (25a-d) had higher binding affinities than the corresponding parent compounds (23a-d) at the MT(2) receptor but similar affinities at the mt(1) site; all of the compounds were antagonists in the functional assay. Changing 11 methoxy for 11-ethoxy decreased the binding affinity slightly, and this was more evident at the MT(2) receptor. All of the derivatives investigated had either the same or a greater affinity for the human MT(2) receptor compared to the mt(1) receptor (range 1:1-1:132). This suggests that the mt(1) and MT(2) receptor pockets differ in their ability to accommodate alkyl groups in the indole nitrogen region of the melatonin molecule. Two compounds (7c and 25c) were tested in functional assays on recombinant mt(1) and MT(2) melatonin receptors. Compound 7c is a potent agonist with some selectivity (44-fold) for the MT(2) receptor, while 25c is an MT(2)-preferring antagonist. Increasing the carbon chain length between N-1 of indole and the 2-phenyl group from n = 1 through n = 3 leads to a fairly regular decrease in the binding affinity, but, remarkably, when n = 3, it converts the methoxy compounds from melatonin agonists to antagonists. The Xenopus melatonin receptor thus cannot accommodate an N-n-alkyl chain attached to a 2-phenyl substituent with n > 2 in the required orientation to induce or stabilize the active receptor conformation. PMID- 10737739 TI - Molecular modeling of the aldose reductase-inhibitor complex based on the X-ray crystal structure and studies with single-site-directed mutants. AB - Aldose reductase (AR) has been implicated in the etiology of the secondary complications of diabetes. This enzyme catalyzes the reduction of glucose to sorbitol using nicotinamide adenine dinucleotide phosphate as an essential cofactor. AR has been localized at the sites of tissue damage, and inhibitors of this enzyme prevent the development of neuropathy, nephropathy, retinopathy, and cataract formation in animal models of diabetes. The crystal structure of AR complexed with zopolrestat, a potent inhibitor of AR, has been described.(1) We have generated a model of the AR-inhibitor complex based on the reported Calpha coordinates of the protein and results of a structure-activity relationship study using four structurally distinct classes of inhibitors, recombinant human AR, and four single-site-directed mutants of this enzyme. The effects of the site directed mutations on residues within the active site of the enzyme were evaluated by average interaction energy calculations and by calculations of carbon atom surface area changes. These values correlated well with the IC(50) values for zopolrestat with the wild-type and mutant enzymes, validating the model. On the basis of the zopolrestat-binding model, we have proposed binding models for 10 other AR inhibitors. Our models have enabled us to gain a qualitative understanding of the binding domains of the enzyme and how different inhibitors impact the size and shape of the binding site. PMID- 10737740 TI - Histaprodifens: synthesis, pharmacological in vitro evaluation, and molecular modeling of a new class of highly active and selective histamine H(1)-receptor agonists. AB - A new class of histamine analogues characterized by a 3, 3-diphenylpropyl substituent at the 2-position of the imidazole nucleus has been prepared outgoing from 4,4-diphenylbutyronitrile (4b) via cyclization of the corresponding methyl imidate 5b with 2-oxo-4-phthalimido-1-butyl acetate or 2-oxo-1,4-butandiol in liquid ammonia, followed by standard reactions. The title compounds displayed partial agonism on contractile H(1) receptors of the guinea-pig ileum and endothelium-denuded aorta, respectively, except 10 (histaprodifen; 2-[2-(3, 3 diphenylpropyl)-1H-imidazol-4-yl]ethanamine) which was a full agonist in the ileum assay. While 10 was equipotent with histamine (1), methylhistaprodifen (13) and dimethylhistaprodifen (14) exceeded the functional potency of 1 by a factor of 3-5 (13) and 2-3 (14). Compounds 10 and 13-17 relaxed precontracted rat aortic rings (intact endothelium) with relative potencies of 3.3- up to 28-fold (compared with 1), displaying partial agonism as well. Agonist effects were sensitive to blockade by the selective H(1)-receptor antagonist mepyramine (pA(2) approximately 9 (guinea-pig) and pA(2) approximately 8 (rat aorta)). The affinity of 10 and 13-17 for guinea-pig H(1) receptors increased 20- to 100-fold compared with 1. Two lower homologues of 10 were weak partial H(1)-receptor agonists while two higher homologues of 10 were silent antagonists endowed with micromolar affinity for rat and guinea-pig H(1) receptors. In functional selectivity experiments, 10, 13, and 14 did not stimulate H(2), H(3), and several other neurotransmitter receptors. They displayed only low to moderate affinity for these sites (pA(2) < 6). For a better understanding of structure-activity relationships, the interaction of 1 and 10, 13 and 14 within the transmembrane (TM) domains of the human histamine H(1) receptor were studied using molecular dynamics simulations. Remarkable differences were found between the binding modes of 10, 13, and 14 and that of 1. The imidazole ring of 10, 13, and 14 was placed 'upside down' compared with 1, making the interaction of the N(pi)-atom with Tyr431 possible. This new orientation was mainly caused by the space filling substitution at the 2-position of the imidazole ring and influenced the location of the protonated N(alpha)-atom which was positioned more between TM III and TM VI. This orientation can explain both the increased relative potency and the maximum effect of 10, 13, and 14 compared with 1. Compound 13 (methylhistaprodifen; N(alpha)-methyl-2-[2-(3, 3-diphenylpropyl)-1H-imidazol-4 yl]ethanamine) is the most potent histamine H(1)-receptor agonist reported so far in the literature and may become a valuable tool for the study of physiological and pathophysiological H(1)-receptor-mediated effects. PMID- 10737741 TI - Highly selective chiral N-substituted 3alpha-[bis(4'-fluorophenyl)methoxy]tropane analogues for the dopamine transporter: synthesis and comparative molecular field analysis. AB - In a continuing effort to further characterize the role of the dopamine transporter in the pharmacological effects of cocaine, a series of chiral and achiral N-substituted analogues of 3alpha-[bis(4'-fluorophenyl)methoxy]tropane (5) has been prepared as potential selective dopamine transporter ligands. These novel compounds displaced [(3)H]WIN 35,428 binding from the dopamine transporter in rat caudate putamen with K(i) values ranging from 13. 9 to 477 nM. Previously, it was reported that 5 demonstrated a significantly higher affinity for the dopamine transporter than the parent drug, 3alpha-(diphenylmethoxy)tropane (3; benztropine). However, 5 remained nonselective over muscarinic m(1) receptors (dopamine transporter, K(i) = 11.8 nM; m(1), K(i) = 11.6 nM) which could potentially confound the interpretation of behavioral data, for this compound and other members of this series. Thus, significant effort has been directed toward developing analogues that retain high affinity at the dopamine transporter but have decreased affinity at muscarinic sites. Recently, it was discovered that by replacing the N-methyl group of 5 with the phenyl-n-butyl substituent (6) retention of high binding affinity at the dopamine transporter (K(i) = 8.51 nM) while decreasing affinity at muscarinic receptors (K(i) = 576 nM) was achieved, resulting in 68-fold selectivity. In the present series, a further improvement in the selectivity for the dopamine transporter was accomplished, with the chiral analogue (S)-N-(2-amino-3-methyl-n-butyl)-3alpha-[bis(4'-fluorophenyl)metho xy] tropane (10b) showing a 136-fold selectivity for the dopamine transporter versus muscarinic m(1) receptors (K(i) = 29.5 nM versus K(i) = 4020 nM, respectively). In addition, a comparative molecular field analysis (CoMFA) model was derived to correlate the binding affinities of all the N-substituted 3alpha-[bis(4' fluorophenyl)methoxy]tropane analogues that we have prepared with their 3D structural features. The best model (q(2) = 0. 746) was used to accurately predict binding affinities of compounds in the training set and in a test set. The CoMFA coefficient contour plot for this model, which provides a visual representation of the chemical environment of the binding domain of the dopamine transporter, can now be used to design and/or predict the binding affinities of novel drugs within this class of dopamine uptake inhibitors. PMID- 10737742 TI - 2',6'-Dimethylphenoxyacetyl: a new achiral high affinity P(3)-P(2) ligand for peptidomimetic-based HIV protease inhibitors. AB - Starting from palinavir (1), our lead HIV protease inhibitor, we have discovered a new series of truncated analogues in which the P(3)-P(2) quinaldic-valine portion of 1 was replaced by 2', 6'-dimethylphenoxyacetyl. With EC(50)'s in the 1 2 nM range, some of these compounds are among the most potent inhibitors of HIV replication in vitro, reported to date. One of the most promising members in this series (compound 27, BILA 2185 BS) exhibited a favorable overall pharmacokinetic profile, with 61% apparent oral bioavailability in rat. X-ray crystal structures and molecular modeling were used to rationalize the high potency resulting from incorporation of this structurally simple, achiral ligand into the P(3)-P(2) position of hydroxyethylamine-based HIV protease inhibitors. PMID- 10737743 TI - 3D-QSAR CoMFA study on imidazolinergic I(2) ligands: a significant model through a combined exploration of structural diversity and methodology. AB - Displaying an unprecedented structural diversity, 119 I(2) ligands, and their pK(i) values, were collected and submitted to a comparative molecular fields analysis (CoMFA) study. They were discerned into three structural subsets (A, B, C), to explore the I(2) 3D-QSARs from finite structural systems (A, B, C) to more complex ones (AB, AC, BC, ABC). In addition, various key steps of the CoMFA methology were explored. The applied method used two pharmacophore templates and seven molecular field combinations (electrostatic, lipophilic, steric), as well as eight alignment methods (two point-by-point and six similarity-based variations). That way, 644 CoMFA models were obtained and further selected according to their predictive ability through two filters. The first filter was mainly based on the q(2), which internally evaluates the predictive ability from the training set. For the second filter, the predictive ability was externally evaluated through the prediction of test sets. Finally, one model was extracted from the whole data as the best. Indeed, it combines three features of upmost importance for the further design of ligands endowed with high I(2) affinity: structural diversity (n = 73), robustness (N = 9, r(2) = 0.96, s = 0. 28, F = 148), and a great fully assessed predictive ability (q(2) = 0.50, r(2)(test set) = 0.81, n(test set) = 46). On the basis of structural data and CoMFA isocontours, some elements of the I(2) tridimensional pharmacophore are also suggested. PMID- 10737744 TI - Biarylcarbamoylindolines are novel and selective 5-HT(2C) receptor inverse agonists: identification of 5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]- 5 pyridyl]carbamoyl]-6-trifluoromethylindoline (SB-243213) as a potential antidepressant/anxiolytic agent. AB - The evolution, synthesis, and biological activity of a novel series of 5-HT(2C) receptor inverse agonists are reported. Biarylcarbamoylindolines have been identified with excellent 5-HT(2C) affinity and selectivity over 5-HT(2A) receptors. In addition, (pyridyloxypyridyl)carbamoylindolines have been discovered with additional selectivity over the closely related 5-HT(2B) receptor. Compounds from this series are inverse agonists at the human cloned 5 HT(2C) receptor, completely abolishing basal activity in a functional assay. The new series have reduced P450 inhibitory liability compared to a previously described series of 1-(3-pyridylcarbamoyl)indolines (Bromidge et al. J. Med. Chem. 1998, 41, 1598) from which they evolved. Compounds from this series showed excellent oral activity in a rat mCPP hypolocomotion model and in animal models of anxiety. On the basis of their favorable biological profile, 32 (SB-228357) and 40 (SB-243213) have been selected for further evaluation to determine their therapeutic potential for the treatment of CNS disorders such as depression and anxiety. PMID- 10737746 TI - Artificial neural network applied to prediction of fluorquinolone antibacterial activity by topological methods. AB - A new topological method that makes it possible to predict the properties of molecules on the basis of their chemical structures is applied in the present study to quinolone antimicrobial agents. This method uses neural networks in which training algorithms are used as well as different concepts and methods of artificial intelligence with a suitable set of topological descriptors. This makes it possible to determine the minimal inhibitory concentration (MIC) of quinolones. Analysis of the results shows that the experimental and calculated values are highly similar. It is possible to obtain a QSAR interpretation of the information contained in the network after the training has been carried out. PMID- 10737745 TI - Investigating protein-ligand interactions with a mutant FKBP possessing a designed specificity pocket. AB - Using structure-based design and protein mutagenesis we have remodeled the FKBP12 ligand binding site to include a sizable, hydrophobic specificity pocket. This mutant (F36V-FKBP) is capable of binding, with low or subnanomolar affinities, novel synthetic ligands possessing designed substituents that sterically prevent binding to the wild-type protein. Using binding and structural analysis of bumped compounds, we show here that the pocket is highly promiscuous-capable of binding a range of hydrophobic alkyl and aryl moieties with comparable affinity. Ligand affinity therefore appears largely insensitive to the degree of occupancy or quality of packing of the pocket. NMR spectroscopic analysis indicates that similar ligands can adopt radically different binding modes, thus complicating the interpretation of structure-activity relationships. PMID- 10737747 TI - 5-HT reuptake inhibitors with 5-HT(1B/1D) antagonistic activity: a new approach toward efficient antidepressants. AB - As part of our research program toward new, potential antidepressants, a series of unsymmetrical ureas has been prepared and evaluated as 5-HT reuptake inhibitors with 5-HT(1B/1D) antagonistic activities. The design of these compounds was based on coupling of various indole derivatives, previously shown to inhibit 5-HT reuptake, to three different aniline moieties, which are part of known 5-HT(1B/1D) ligands. Binding experiments in rat frontal cortex using [(125)I]iodocyanopindolol, in calf striatum using [(3)H]5-HT, and in rat hippocampus using [(3)H]8-OH-DPAT as radioligands, respectively, revealed significantly higher affinity at the 5-HT(1B) receptor as compared to the affinities for the 5-HT(1A) and 5-HT(1D) receptors for a number of compounds, among them 4-(5-fluoro-1H-indol-3-yl)piperidine-1-carboxylic acid [4-methoxy-3-(4 methylpiperazin-1-yl)phenyl]amide (5), the corresponding 4-fluoro-1H-indol-3-yl analogue 21a, and the corresponding 6-fluoro-1H-indol-3-yl analogue 21b. Conformational restriction of the aniline moiety in 5 only slightly enhanced the 5-HT(1B) affinity, whereas introduction of an aniline moiety with higher conformational flexibility resulted in a less potent 5-HT(1B) receptor ligand as compared to 5. The functional 5-HT(1B/1D) antagonistic activity was investigated using the rabbit saphenous vein model as well as the [(3)H]5-HT release from guinea pig cortical slices. All new compounds tested in the rabbit saphenous vein model were shown to antagonize the sumatriptan-evoked contractile responses with pA(2) values ranging from 7.3 to 8.7. These observations were consistent with the results of the cortical slice model, in which the ureas were found to block the sumatriptan-induced inhibition of potassium-evoked [(3)H]5-HT release. The 5-HT reuptake inhibition of the ureas determined in rat brain synaptosomes was found to be either increased or decreased as compared to the uncoupled indole derivatives indicating that the reuptake inhibition shown by the ureas is not only due to the indole part but also affected by the aniline moiety of the molecule. Among this series of compounds described the ureas 5, 21a, and 21b seem to be the most interesting candidates showing both 5-HT reuptake inhibition and 5 HT(1B/1D) antagonism in vitro. This dual pharmacological profile should in theory lead to a pronounced enhancement in serotonergic neurotransmission and consequently to a more efficient treatment of depression. PMID- 10737748 TI - 1,2,4-Triazolo[4,3-a]quinoxalin-1-one: a versatile tool for the synthesis of potent and selective adenosine receptor antagonists. AB - 4-Amino-6-benzylamino-1,2-dihydro-2-phenyl-1,2,4-triazolo[4, 3-a]quinoxalin-1-one (1) has been found to be an A(2A) versus A(1) selective antagonist (Colotta et al. Arch. Pharm. Pharm. Med. Chem. 1999, 332, 39-41). In this paper some novel triazoloquinoxalin-1-ones 4-25 bearing different substituents on the 2-phenyl and/or 4-amino moiety of the parent 4-amino-1, 2-dihydro-2-phenyl-1,2,4 triazolo[4,3-a]quinoxalin-1-one (3) have been synthesized and tested in radioligand binding assays at bovine A(1) and A(2A) and cloned human A(3) adenosine receptors (AR). Moreover, the binding activities at the above-mentioned AR subtypes of the 1,4-dione parent compounds 26-31 and their 5-N-alkyl derivatives 33-37 were also evaluated. The substituent on the 2-phenyl ring exerted a different effect on AR subtypes, while replacement of a hydrogen atom of the 4-amino group with suitable substituents yielded selective A(1) or A(3) antagonists. Replacement of a hydrogen atom of the 4-NH(2) with an acyl group, or replacement of the whole 4-NH(2) with a 4-oxo moiety, shifted the binding activity toward the A(3) AR. The binding results allowed elucidation of the structural requirements for the binding of these novel tricyclic derivatives at each receptor subtype. In particular, A(1) and A(2A) binding required the presence of a proton donor group at position-4, while for A(3) affinity the presence of a proton acceptor in this same region was of paramount importance. PMID- 10737749 TI - Anilide derivatives of an 8-phenylxanthine carboxylic congener are highly potent and selective antagonists at human A(2B) adenosine receptors. AB - No highly selective antagonists of the A(2B) adenosine receptor (AR) have been reported; however such antagonists have therapeutic potential as antiasthmatic agents. Here we report the synthesis of potent and selective A(2B) receptor antagonists. The structure-activity relationships (SAR) of 8-phenyl-1, 3-di-(n propyl)xanthine derivatives in binding to recombinant human A(2B) ARs in HEK-293 cells (HEK-A(2B)) and at other AR subtypes were explored. Various amide derivatives of 8-[4-[[carboxymethyl]oxy]phenyl]-1,3-di-(n-propyl)xanthine, 4a, were synthesized. A comparison of aryl, alkyl, and aralkyl amides demonstrated that simple anilides, particularly those substituted in the para-position with electron-withdrawing groups, such as nitro, cyano, and acetyl, bind selectively to human A(2B) receptors in the range of 1-3 nM. The unsubstituted anilide 12 had a K(i) value at A(2B) receptors of 1.48 nM but was only moderately selective versus human A(1)/A(2A) receptors and nonselective versus rat A(1) receptors. Highly potent and selective A(2B) antagonists were a p-aminoacetophenone derivative 20 (K(i) value 1.39 nM) and ap-cyanoanilide 27 (K(i) value 1.97 nM). Compound 27 was 400-, 245-, and 123-fold selective for human A(2B) receptors versus human A(1)/A(2A)/A(3) receptors, respectively, and 8.5- and 310-fold selective versus rat A(1)/A(2A) receptors, respectively. Substitution of the 1,3 dipropyl groups with 1,3-diethyl offered no disadvantage for selectivity, and high affinities at A(2B) receptors were maintained. Substitution of the p carboxymethyloxy group of 4a and its amides with acrylic acid decreased affinity at A(2B) receptors while increasing affinity at A(1) receptors. 1, 3 Di(cyclohexylmethyl) groups greatly reduced affinity at ARs, although the p carboxymethyloxy derivative 9 was moderately selective for A(2B) receptors. Several selective A(2B) antagonists inhibited NECA-stimulated calcium mobilization in HEK-A(2B) cells. PMID- 10737750 TI - SH2-directed ligands of the Lck tyrosine kinase. AB - Two separate libraries, prepared via parallel synthesis, were employed to identify low-molecular-weight SH2-targeted ligands for the Lck tyrosine protein kinase. These libraries were constructed to furnish non-amino acid analogues of the (1) Glu-Glu and (2) Ile residues of the Lck SH2 domain peptide ligand Ac-pTyr Glu-Glu-Ile-amide. The lead compound acquired in this study exhibits a dissociation constant for the Lck SH2 domain that is comparable to that displayed by Ac-pTyr-Glu-Glu-Ile-amide. These results demonstrate that the standard amino acid residues Glu-Glu-Ile can be completely replaced with non-amino acid moieties without loss of SH2 affinity. PMID- 10737751 TI - Doubly homologated dihalovinyl and acetylene analogues of adenosine: synthesis, interaction with S-adenosyl-L-homocysteine hydrolase, and antiviral and cytostatic effects. AB - Treatment of the 6-aldehyde derived by Moffatt oxidation of 3-O-benzoyl-1,2-O isopropylidene-alpha-D-ribo-hexofuranose (2c) with the dibromo- or bromofluoromethylene Wittig reagents generated in situ with tetrabromomethane or tribromofluoromethane, triphenylphosphine, and zinc gave the dihalomethyleneheptofuranose analogues 3b and 3d, respectively. Acetolysis, coupling with adenine, and deprotection gave 9-(7,7-dibromo-5,6, 7-trideoxy-beta D-ribo-hept-6-enofuranosyl)adenine (5a) or its bromofluoro analogue 5b. Treatment of 5a with excess butyllithium provided the acetylenic derivative 9-(5,6, 7 trideoxy-beta-D-ribo-hept-6-ynofuranosyl)adenine (6). The doubly homologated vinyl halides 5a and 5b and acetylenic 6 adenine nucleosides were designed as putative substrates of the "hydrolytic activity" of S-adenosyl-L-homocysteine (AdoHcy) hydrolase. Incubation of AdoHcy hydrolase with 5a, 5b, and 6 resulted in time- and concentration-dependent inactivation of the enzyme (K(i): 8.5 +/- 0.5, 17 +/- 2, and 8.6 +/- 0.5 microM, respectively), as well as partial reduction of enzyme-bound NAD(+) to E-NADH. However, no products of the "hydrolytic activity" were observed indicating these compounds are type I mechanism-based inhibitors. The compounds displayed minimal antiviral and cytostatic activity, except for 6, against vaccinia virus and vesicular stomatitis virus (IC(50): 15 and 7 microM, respectively). These viruses typically fall within the activity spectrum of AdoHcy hydrolase inhibitors. PMID- 10737753 TI - Design and SAR of novel potassium channel openers targeted for urge urinary incontinence. 2. Selective and potent benzylamino cyclobutenediones. AB - A novel series of benzylamine, potassium channel openers (KCOs) is presented as part of our program toward designing new, bladder-selective compounds for the treatment of urge urinary incontinence (UUI). We have found that the in vitro potency of (R)-4-[3,4-dioxo-2-(1,2, 2-trimethyl-propylamino)-cyclobut-1 enylamino]-3-ethyl-benzo nitrile 1 in the relaxation of precontracted rat detrusor strips can also be obtained with cyanobenzylamine derivative 4 (IC(50) = 0.29 microM) (Figure 3). Addition of a 2-Cl substituted benzylamine moiety and changing the alkylamino substituent of 4 to a t-Bu amine gives 31 (IC(50) = 0.14 microM)-a compound with similar in vitro potency as 4 as well as relaxant activity on bladder smooth muscle in vivo when administered orally (31, ED(50) = 3 mg/kg) in a rodent model of bladder instability. Further modifications, particularly the replacement of the t-Bu amino substituent with a tert-amylamine, gave a similarly active compound 60 (IC(50) = 0.10 microM) which shows excellent in vivo efficacy (ED(50) = 0.6 mg/kg). Moreover, 60, 3-(2,4-dichloro-6-methyl benzylamino)-4-(1, 1-dimethyl-propylamino)-cyclobut-3-ene-1,2-dione (WAY-151616), shows excellent tissue selectivity for bladder K channels over arterial tissue (60, MAP ED(20) = 100 mg/kg; selectivity: MAP ED(20)/bladder ED(50) = 166). Other manipulations of the benzylamino cyclobutenediones, acylation of the benzylamine, conversion of the benzylamine substituent to a benzamide, homologation of the benzylamine to a phenethylamine, and incorporation of a methyl group at the benzyl carbon, all led to substantial loss of in vitro activity, although some in vivo activity was maintained in the acylated analogues. Compound 60 represents an attractive candidate for development in the treatment of UUI. PMID- 10737752 TI - Design and SAR of novel potassium channel openers targeted for urge urinary incontinence. 1. N-Cyanoguanidine bioisosteres possessing in vivo bladder selectivity. AB - A structurally novel series of adenosine 5'-triphosphate-sensitive potassium (K(ATP)) channel openers is described. As part of our efforts directed toward identifying novel, bladder-selective potassium channel openers (KCOs) targeted for urge urinary incontinence (UUI), we found that bioisosteric replacement of the N-cyanoguanidine moiety of pinacidil (1, Figure 1) with a diaminocyclobutenedione template afforded squaric acid analogue 2, the prototype of a novel series of K(ATP) channel openers with unique selectivity for bladder smooth muscle in vivo. Further modification of the heterocyclic ring to give substituted aryl derivatives (3) afforded potent KCOs that possessed the desired detrusor selectivity when administered orally. The effects of these potassium channel agonists on bladder contractile function was studied in vitro using isolated rat detrusor strips. Potent relaxants were evaluated in vivo in a rat model of bladder instability. Lead compounds were evaluated concomitantly in normotensive rats for their effects on mean arterial blood pressure (MAP) and heart rate as a measure of in vivo bladder selectivity. (R)-4-[3,4-Dioxo-2-(1,2, 2-trimethyl-propylamino)-cyclobut-1-enylamino]-3-ethyl-benzo nitrile (79) met our potency and selectivity criteria and represents an attractive development candidate for the treatment of UUI. Electrophysiological studies using isolated rat bladder detrusor myocytes have demonstrated that compound 79 produces significant hyperpolarization which is glyburide-reversed, thus consistent with the activation of K(ATP). The design, synthesis, structure-activity relationships (SAR), and pharmacological activity associated with this series of novel KCOs will be discussed. PMID- 10737755 TI - Novel and selective calcitonin-inducing agents. AB - A series of xanthine sulfonamides is presented as a class of calcitonin (CT) inducers - a potentially new method for treating diseases associated with postmenopausal bone loss such as osteoporosis. We have found that certain di-n butylxanthine sulfonamides 4 upregulate CT transcription in a CT-luciferase reporter gene assay (CT-luci) and increase the production and release of CT in a CT secretion/RIA assay (CTS). In addition, these compounds do not have potent PDE4 inhibitory activity as do the related xanthine methylene ketones such as denbufyllene (2). One compound in particular (9) shows a transcription activation ratio (TAR) of 2.1 in CT-luci, a CTS increase of 3.6-fold, and a PDE4 (phosphodiesterase type IV) IC(50) = 4.1 microM. In addition, this compound showed a statistically significant 47% trabecular bone protection in ovariectomized-induced osteopenia (OVX) rats as determined by assay when administered for 4 weeks at 30 mg/kg/day, i. p. by quantitative computed tomography (QCT). When administered p.o., compound 9 shows 50% trabecular bone protection when administered for 3 weeks at 50 mg/kg/day, i.p. This compared with salmon CT which shows 62% trabecular bone protection when administered at 50 IU/kg/day for 4 weeks. PMID- 10737754 TI - Further SAR studies of piperidine-based analogues of cocaine. 2. Potent dopamine and serotonin reuptake inhibitors. AB - The synthesis and monoamine transporter activity of additional members of a series of 3,4-disubstituted piperidines (truncated analogues of the WIN series) are described. All members of this series were prepared from arecoline hydrobromide in optically pure form and were evaluated for their ability to inhibit high affinity uptake of dopamine (DA), serotonin (5-HT) and norepinephrine (NE) into rat brain nerve endings (synaptosomes). Most of the compounds prepared in this series are reasonably potent DAT inhibitors (K(i) values of 4-400 nM) and have selectivity for the 5-HT transporter relative to both the NE transporter (3-9-fold) and to the DAT ( approximately 25-fold). In the present series, (-)-methyl 1-methyl-4beta-(2-naphthyl)piperidine-3beta carboxylate (6) was found to be the most potent piperidine-based ligand, exhibiting K(i)'s of 21 nM and 7.6 nM at the DAT and 5-HTT, respectively. While the 5-HTT activity of compound 6 is comparable to that of the antidepressant medication fluoxetine, it is less selective. As is apparent from the data presented, the naphthyl substituted piperidines 6-9, which differ in their stereochemistry, show different degrees of selectivity for the three transporters. Consistent with results reported in the literature for the tropane analogues, removal of the methyl group from the nitrogen atom of 9 leads to a further enhancement in 5-HTT activity. To examine the in vivo effects of these piperidines, preliminary behavioral screening was carried out on piperidine 14. Despite its 2.5-fold greater DAT activity compared to cocaine, piperidine 14 was found to be about 2. 5-fold less potent in increasing distance traveled in mice. However, consistent with its DAT activity, piperidine 14 was found to be about 2.5-fold more potent than cocaine in enhancing stereotypic movements. Further studies of these piperidine-based ligands may provide valuable insights into the pharmacological mechanisms underlying the enhancement in distance traveled versus stereotypic movements. The present results have important implications for better understanding the structural motifs required in the design of agents with specific potency and selectivity at monoamine transporters. PMID- 10737756 TI - Phosphorylated morpholine acetal human neurokinin-1 receptor antagonists as water soluble prodrugs. AB - The regioselective dibenzylphosphorylation of 2 followed by catalytic reduction in the presence of N-methyl-D-glucamine afforded 2-(S)-(1-(R)-(3, 5 bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluoro)phenyl-4-(5-(2- phosphoryl-3 oxo-4H,-1,2,4-triazolo)methylmorpholine, bis(N-methyl-D-glucamine) salt, 11. Incubation of 11 in rat, dog, and human plasma and in human hepatic subcellular fractions in vitro indicated that conversion to 2 would be expected to occur in vivo most readily in humans during hepatic circulation. Conversion of 11 to 2 occurred rapidly in vivo in the rat and dog with the levels of 11 being undetectable within 5 min after 1 and 8 mg/kg doses iv in the rat and within 15 min after 0.5, 2, and 32 mg/kg doses iv in the dog. Compound 11 has a 10-fold lower affinity for the human NK-1 receptor as compared to 2, but it is functionally equivalent to 2 in preclinical models of NK-1-mediated inflammation in the guinea pig and cisplatin-induced emesis in the ferret, indicating that 11 acts as a prodrug of 2. Based in part on these data, 11 was identified as a novel, water-soluble prodrug of the clinical candidate 2 suitable for intravenous administration in humans. PMID- 10737757 TI - Active conformations of neotame and other high-potency sweeteners. AB - We carried out extensive conformational analysis of three high-potency sweeteners: neotame, superaspartame, and SC-45647. We then identified six possible pharmacophore features (carboxylate, two hydrophobic groups, and three NH groups) and wrote a computer program to exhaustively compare intramolecular distances among all possible sets of five-point pharmacophores (carboxylate + two hydrophobic groups + two NH groups) for the three compounds. The best pharmacophore model superimposes low-energy conformers of the three compounds in such a way that the five pharmacophore points match well both sterically and with respect to orientation of hydrogen bond donors and acceptors. PMID- 10737758 TI - Methyl-substituted dispiro-1,2,4,5-tetraoxanes: correlations of structural studies with antimalarial activity. AB - Two tetramethyl-substituted dispiro-1,2,4,5-tetraoxanes (7,8,15, 16 tetraoxadispiro[5.2.5.2]hexadecanes) 3 and 4 were designed as metabolically stable analogues of the dimethyl-substituted dispiro-1, 2,4,5-tetraoxane prototype WR 148999 (2). For a positive control we selected the sterically unhindered tetraoxane 5 (7,8,15, 16-tetraoxadispiro[5.2.5.2]hexadecane), devoid of any substituents. Tetraoxanes 3 and 4 were completely inactive in contrast to tetraoxanes 2 and 5. We hypothesize that the two inactive tetraoxanes possess sufficient steric hindrance about the tetraoxane ring due to the two additional axial methyl groups to prevent their activation to presumed parasiticidal carbon radicals by inhibiting electron transfer from heme or other iron(II) species. For each of the tetraoxanes 2-4, the tetraoxane and both spirocyclohexyl rings are in a chair conformation and the bond lengths and angles are all quite normal except for the C1-C2 bond which is slightly lengthened. Comparison of the modeled and X ray structures for tetraoxanes 2-5 reveals that molecular mechanics (MMX and MM3) and 3-21G calculations each gave accurate structural parameters such as bond lengths, bond angles, and dihedral angles. In contrast, semiempirical methods such as AM1 gave poor results. PMID- 10737759 TI - clag9: A cytoadherence gene in Plasmodium falciparum essential for binding of parasitized erythrocytes to CD36. AB - The propensity of isolates of the malaria parasite Plasmodium falciparum to delete a segment of chromosome 9 has provided positional information that has allowed us to identify a gene necessary for cytoadherence. It has been termed the cytoadherence-linked asexual gene (clag9). clag9 encodes at least nine exons and is expressed in blood stages. The hydrophobicity profile of the predicted CLAG9 protein identifies up to four transmembrane domains. We show here that targeted gene disruption of clag9 ablated cytoadherence to C32 melanoma cells and purified CD36. DNA-induced antibodies to the clag9 gene product reacted with a polypeptide of 220 kDa in the parental malaria clone but not in clones with a disrupted clag9 gene. PMID- 10737760 TI - Comparative organization of cattle chromosome 5 revealed by comparative mapping by annotation and sequence similarity and radiation hybrid mapping. AB - A whole genome cattle-hamster radiation hybrid cell panel was used to construct a map of 54 markers located on bovine chromosome 5 (BTA5). Of the 54 markers, 34 are microsatellites selected from the cattle linkage map and 20 are genes. Among the 20 mapped genes, 10 are new assignments that were made by using the comparative mapping by annotation and sequence similarity strategy. A LOD-3 radiation hybrid framework map consisting of 21 markers was constructed. The relatively low retention frequency of markers on this chromosome (19%) prevented unambiguous ordering of the other 33 markers. The length of the map is 398.7 cR, corresponding to a ratio of approximately 2.8 cR(5,000)/cM. Type I genes were binned for comparison of gene order among cattle, humans, and mice. Multiple internal rearrangements within conserved syntenic groups were apparent upon comparison of gene order on BTA5 and HSA12 and HSA22. A similarly high number of rearrangements were observed between BTA5 and MMU6, MMU10, and MMU15. The detailed comparative map of BTA5 should facilitate identification of genes affecting economically important traits that have been mapped to this chromosome and should contribute to our understanding of mammalian chromosome evolution. PMID- 10737761 TI - Carbon nanotube atomic force microscopy tips: direct growth by chemical vapor deposition and application to high-resolution imaging. AB - Carbon nanotubes are potentially ideal atomic force microscopy probes because they can have diameters as small as one nanometer, have robust mechanical properties, and can be specifically functionalized with chemical and biological probes at the tip ends. This communication describes methods for the direct growth of carbon nanotube tips by chemical vapor deposition (CVD) using ethylene and iron catalysts deposited on commercial silicon-cantilever-tip assemblies. Scanning electron microscopy and transmission electron microscopy measurements demonstrate that multiwalled nanotube and single-walled nanotube tips can be grown by predictable variations in the CVD growth conditions. Force-displacement measurements made on the tips show that they buckle elastically and have very small (1,200 milliosmol). After 1-desamino-8-d arginine-vasopressin administration or water deprivation, the AQP3 null mice were able to concentrate their urine partially to approximately 30% of that in wild type mice. Osmotic water permeability of cortical collecting-duct basolateral membrane, measured by a spatial filtering optics method, was >3-fold reduced by AQP3 deletion. To test the hypothesis that the residual concentrating ability of AQP3 null mice was due to the inner medullary collecting-duct water channel AQP4, AQP3/AQP4 double-knockout mice were generated. The double-knockout mice had greater impairment of urinary-concentrating ability than did the AQP3 single knockout mice. Our findings establish a form of nephrogenic diabetes insipidus produced by impaired water permeability in collecting-duct basolateral membrane. Basolateral membrane aquaporins may thus provide blood-accessible targets for drug discovery of aquaretic inhibitors. PMID- 10737775 TI - Escherichia coli O157:H7 in beef cattle presented for slaughter in the U.S.: higher prevalence rates than previously estimated. PMID- 10737774 TI - Rab geranylgeranyl transferase alpha mutation in the gunmetal mouse reduces Rab prenylation and platelet synthesis. AB - Few molecular events important to platelet biogenesis have been identified. Mice homozygous for the spontaneous, recessive mutation gunmetal (gm) have prolonged bleeding, thrombocytopenia, and reduced platelet alpha- and delta-granule contents. Here we show by positional cloning that gm results from a G-->A substitution mutation in a splice acceptor site within the alpha-subunit of Rab geranylgeranyl transferase (Rabggta), an enzyme that attaches geranylgeranyl groups to Rab proteins. Most Rabggta mRNAs from gm tissues skipped exon 1 and lacked a start codon. Rabggta protein and Rab geranylgeranyl transferase (GGTase) activity were reduced 4-fold in gm platelets. Geranylgeranylation and membrane association of Rab27, a Rab GGTase substrate, were significantly decreased in gm platelets. These findings indicate that geranylgeranylation of Rab GTPases is critical for hemostasis. Rab GGTase inhibition may represent a new treatment for thrombocytosis and clotting disorders. PMID- 10737776 TI - Do ultrastable proteins from hyperthermophiles have high or low conformational rigidity? PMID- 10737777 TI - Singing in the brain. PMID- 10737778 TI - Optimal prediction and the Mori-Zwanzig representation of irreversible processes. AB - Optimal prediction methods compensate for a lack of resolution in the numerical solution of complex problems through the use of prior statistical information. We point out a relation between optimal prediction and the statistical mechanics of irreversible processes, and use a version of the Mori-Zwanzig formalism to produce a higher-order optimal prediction method. PMID- 10737779 TI - Fragilities of liquids predicted from the random first order transition theory of glasses. AB - A microscopically motivated theory of glassy dynamics based on an underlying random first order transition is developed to explain the magnitude of free energy barriers for glassy relaxation. A variety of empirical correlations embodied in the concept of liquid "fragility" are shown to be quantitatively explained by such a model. The near universality of a Lindemann ratio characterizing the maximal amplitude of thermal vibrations within an amorphous minimum explains the variation of fragility with a liquid's configurational heat capacity density. Furthermore, the numerical prefactor of this correlation is well approximated by the microscopic calculation. The size of heterogeneous reconfiguring regions in a viscous liquid is inferred and the correlation of nonexponentiality of relaxation with fragility is qualitatively explained. Thus the wide variety of kinetic behavior in liquids of quite disparate chemical nature reflects quantitative rather than qualitative differences in their energy landscapes. PMID- 10737780 TI - A note on statistical analysis of shape through triangulation of landmarks. AB - In an earlier paper, the author jointly with S. Suryawanshi proposed statistical analysis of shape through triangulation of landmarks on objects. It was observed that the angles of the triangles are invariant to scaling, location, and rotation of objects. No distinction was made between an object and its reflection. The present paper provides the methodology of shape discrimination when reflection is also taken into account and makes suggestions for modifications to be made when some of the landmarks are collinear. PMID- 10737781 TI - Rhodopsin kinase: expression in mammalian cells and a two-step purification. AB - A suitable system for expression of the rhodopsin kinase (RK) gene and its mutants is needed for structure-function studies of RK. Previously, investigation of the baculovirus system showed satisfactory production of RK, but posttranslational isoprenylation was deficient. We now report on a comparative study of expression of the RK gene in yeast (Pichia pastoris), COS-1 cells and in an HEK293 stable cell line. Expression in COS-1 cells, by using pCMV5 vector, is the most satisfactory. A two-step procedure for purification of the expressed enzyme with an N-terminal histidine tag has been developed. The purified enzyme has correct posttranslational modifications and shows a somewhat broader pH vs. catalytic activity profile than the wild-type enzyme. PMID- 10737782 TI - Rhodopsin kinase: two mAbs binding near the carboxyl terminus cause time dependent inactivation. AB - Two mAbs generated against rhodopsin kinase (RK) were characterized for their epitopes. Both antibodies recognize short peptide sequences, overlapping but distinct, close to the carboxyl terminus. Binding of RK to the antibodies is slow. Attempts were made to use the antibodies immobilized on protein A-Sepharose beads to bind and purify the enzyme. Time-dependent inactivation of the enzyme occurred after its binding to the antibodies. Studies using different conditions to maintain the enzyme in the active form during binding or to reactivate the purified inactivated enzyme were unsuccessful. PMID- 10737783 TI - Structure and function in rhodopsin: destabilization of rhodopsin by the binding of an antibody at the N-terminal segment provides support for involvement of the latter in an intradiscal tertiary structure. AB - A monoclonal anti-rhodopsin antibody (B6-30N), characterized by Hargrave and coworkers [Adamus, G., Zam, Z. S., Arendt, A., Palczewski, K., McDowell, J. M. & Hargrave, P. (1991) Vision Res. 31, 17-31] as recognizing a short peptide sequence at the N terminus, failed to bind to rhodopsin when the latter was solubilized in dodecylmaltoside (DM). Of the detergents tested thus far, DM affords maximum stability to rhodopsin. Solubilization of rhodopsin in cholate allowed binding of the antibody, but the binding caused destabilization as evidenced by the accelerated loss of absorbance at 500 nm. The result provides support for the earlier conclusion that the N-terminal segment is an integral part of a tertiary structure in the intradiscal domain of native rhodopsin coupled to a tertiary structure in the transmembrane domain. Additional comparative studies on the stability of rhodopsin in different detergents were carried out after direct solubilization from rod outer segments and after extensive treatments to remove the endogenous phospholipids. Purification of rhodopsin in DM resulted in essentially quantitative removal of endogenous phospholipids. When rhodopsin thus purified was treated with the above antibody in DM and in cholate, enhanced destabilization (5-fold) was observed in the latter detergent. PMID- 10737784 TI - Crystal structure of the functional domain of the splicing factor Prp18. AB - The splicing factor Prp18 is required for the second step of pre-mRNA splicing. We have isolated and determined the crystal structure of a large fragment of the Saccharomyces cerevisiae Prp18 that lacks the N-terminal 79 amino acids. This fragment, called Prp18Delta79, is fully active in yeast splicing in vitro and includes the sequences of Prp18 that have been evolutionarily conserved. The core structure of Prp18Delta79 is compact and globular, consisting of five alpha helices that adopt a novel fold that we have designated the five-helix X-bundle. The structure suggests that one face of Prp18 interacts with the splicing factor Slu7, whereas the more evolutionarily conserved amino acids in Prp18 form the opposite face. The most highly conserved region of Prp18, a nearly invariant stretch of 19 aa, forms part of a loop between two alpha-helices and may interact with the U5 small nuclear ribonucleoprotein particles. The structure is consistent with a model in which Prp18 forms a bridge between Slu7 and the U5 small nuclear ribonucleoprotein particles. PMID- 10737785 TI - Impact of progesterone receptor on cell-fate decisions during mammary gland development. AB - Mammary epithelium contains lineage-limited progenitors that give rise to cells that form distinct morphological structures, ducts vs. lobules, depending on the endocrine status of the female. Progesterone signaling through progesterone receptor (PR) is essential for lobulo-alveolar development that accompanies pregnancy, but not for ductal growth accompanying puberty. PR exists in two molecular forms, A and B, and an imbalance in the native ratio of the two isoforms can lead to alterations in PR signaling. Indeed, as we reported previously, in transgenic mice carrying additional A form of PR, mammary development is abnormal, characterized by excessive lateral ductal branching. This suggests that alterations in PR signaling may have important consequences to mammary development, particularly with regard to ductal vs. alveolar growth. To test this further, we created transgenic mice carrying additional B form of PR and report that mammary development in these mice is also abnormal, characterized by inappropriate alveolar growth. More importantly, these mammary glands, on serial transplantation, undergo a premature arrest in ductal growth without any alteration in the potential for lobulo-alveolar growth. Such an arrest in ductal growth does not occur with transgenics carrying additional A form of PR. These studies, therefore, provide strong evidence to indicate that PR signaling may be of paramount importance for appropriate cell-fate decisions during normal mammary development, and also that this requires a regulated expression of the two isoforms. PMID- 10737786 TI - Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase. AB - We have examined amino acid substitutions at residues 115 and 116 in the reverse transcriptase (RT) of HIV-1. A number of properties were examined, including polymerization and processivity on both DNA and RNA templates, strand displacement, ribonucleotide misincorporation, and resistance to nucleoside analogs. The RT variants Tyr-115-Phe and Phe-116-Tyr are similar to wild-type HIV 1 RT in most, but not all, respects. In contrast, the RT variant Tyr-115-Val is significantly impaired in polymerase activity compared with wild-type RT; however, Tyr-115-Val is able to incorporate ribonucleotides as well as deoxyribonucleotides during polymerization and is resistant to a variety of nucleoside analogs. PMID- 10737788 TI - BH4 domain of antiapoptotic Bcl-2 family members closes voltage-dependent anion channel and inhibits apoptotic mitochondrial changes and cell death. AB - A change of mitochondrial membrane permeability is essential for apoptosis, leading to translocation of apoptogenic cytochrome c and apoptosis-inducing factor into the cytoplasm. We recently showed that the Bcl-2 family of proteins regulate cytochrome c release and the mitochondrial membrane potential (Deltapsi) by directly modulating the activity of the voltage-dependent anion channel (VDAC) through binding. Here we investigated the biochemical role of the conserved N terminal homology domain (BH4) of Bcl-x(L), which has been shown to be essential for inhibition of apoptosis, with respect to the regulation of mitochondrial membrane permeability and found that BH4 was required for Bcl-x(L) to prevent cytochrome c release and Deltapsi loss. A study using VDAC liposomes revealed that Bcl-x(L), but not Bcl-x(L) lacking the BH4 domain, inhibited VDAC activity. Furthermore, BH4 oligopeptides of Bcl-2 and Bcl-x(L), but not mutant peptides, were able to inhibit both VDAC activity on liposomes even in the presence of Bax and apoptotic Deltapsi loss in isolated mitochondria. It was also shown that the BH4 domain, fused to the protein transduction domain of HIV TAT protein (TAT BH4), efficiently prevented apoptotic cell death. These results indicate that the BH4 of Bcl-2/Bcl-x(L) is essential and sufficient for inhibiting VDAC activity, which in turn prevents apoptotic mitochondrial changes, and for preventing apoptotic cell death. Finally, the data suggest that the TAT-BH4 peptide is potentially useful as a therapeutic agent for diseases caused by accelerated apoptosis. PMID- 10737787 TI - HuR binding to cytoplasmic mRNA is perturbed by heat shock. AB - AU-rich elements (AREs) located in the 3' untranslated region target the mRNAs encoding many protooncoproteins, cytokines, and lymphokines for rapid degradation. HuR, a ubiquitously expressed member of the embryonic lethal abnormal vision (ELAV) family of RNA-binding proteins, binds ARE sequences and selectively stabilizes ARE-containing reporter mRNAs when overexpressed in transiently transfected cells. HuR appears predominantly nucleoplasmic but has been shown to shuttle between the nucleus and cytoplasm via a novel shuttling sequence HNS. We report generation of a mouse monoclonal antibody 3A2 that both immunoblots and immunoprecipitates HuR protein; it recognizes an epitope located in the first of HuR's three RNA recognition motifs. This antibody was used to probe HuR interactions with mRNA before and after heat shock, a condition that has been reported to stabilize ARE-containing mRNAs. At 37 degrees C, approximately one-third of the cytoplasmic HuR appears polysome associated, and in vivo UV crosslinking reveals that HuR interactions with poly(A)(+) RNA are predominantly cytoplasmic rather than nuclear. This comprises evidence that HuR directly interacts with mRNA in vivo. After heat shock, 12-15% of HuR accumulates in discrete foci in the cytoplasm, but surprisingly the majority of HuR crosslinks instead to nuclear poly(A)(+) RNA, whose levels are dramatically increased in the stressed cells. This behavior of HuR differs from that of another ARE-binding protein, hnRNP D, which has been implicated as an effector of mRNA decay rather than mRNA stabilization and of the general pre-RNA-binding protein hnRNP A1. We interpret these differences to mean that the temporal association of HuR with ARE-containing mRNAs is different from that of these other two proteins. PMID- 10737789 TI - The insertion of two amino acids into a transcriptional inducer converts it into a galactokinase. AB - The transcriptional induction of the GAL genes of Saccharomyces cerevisiae occurs when galactose and ATP interact with Gal3p. This protein-small molecule complex associates with Gal80p to relieve its inhibitory effect on the transcriptional activator Gal4p. Gal3p shares a high degree of sequence homology to galactokinase, Gal1p, but does not itself possess galactokinase activity. By constructing chimeric proteins in which regions of the GAL1 gene are inserted into the GAL3 coding sequence, we have been able to impart galactokinase activity upon Gal3p as judged in vivo and in vitro. Remarkably, the insertion of just two amino acids from Gal1p into the corresponding region of Gal3p confers galactokinase activity onto the resultant protein. The chimeric protein, termed Gal3p+SA, retains its ability to efficiently induce the GAL genes. Kinetic analysis of Gal3p+SA reveals that the K(m) for galactose is similar to that of Gal1p, but the K(m) for ATP is increased. The chimeric enzyme was found to have a decreased turnover number in comparison to Gal1p. These results are discussed in terms of both the mechanism of galactokinase function and that of transcriptional induction. PMID- 10737790 TI - Accurate protein crystallography at ultra-high resolution: valence electron distribution in crambin. AB - The charge density distribution of a protein has been refined experimentally. Diffraction data for a crambin crystal were measured to ultra-high resolution (0.54 A) at low temperature by using short-wavelength synchrotron radiation. The crystal structure was refined with a model for charged, nonspherical, multipolar atoms to accurately describe the molecular electron density distribution. The refined parameters agree within 25% with our transferable electron density library derived from accurate single crystal diffraction analyses of several amino acids and small peptides. The resulting electron density maps of redistributed valence electrons (deformation maps) compare quantitatively well with a high-level quantum mechanical calculation performed on a monopeptide. This study provides validation for experimentally derived parameters and a window into charge density analysis of biological macromolecules. PMID- 10737791 TI - Auditory sensitivity provided by self-tuned critical oscillations of hair cells. AB - We introduce the concept of self-tuned criticality as a general mechanism for signal detection in sensory systems. In the case of hearing, we argue that active amplification of faint sounds is provided by a dynamical system that is maintained at the threshold of an oscillatory instability. This concept can account for the exquisite sensitivity of the auditory system and its wide dynamic range as well as its capacity to respond selectively to different frequencies. A specific model of sound detection by the hair cells of the inner ear is discussed. We show that a collection of motor proteins within a hair bundle can generate oscillations at a frequency that depends on the elastic properties of the bundle. Simple variation of bundle geometry gives rise to hair cells with characteristic frequencies that span the range of audibility. Tension-gated transduction channels, which primarily serve to detect the motion of a hair bundle, also tune each cell by admitting ions that regulate the motor protein activity. By controlling the bundle's propensity to oscillate, this feedback automatically maintains the system in the operating regime where it is most sensitive to sinusoidal stimuli. The model explains how hair cells can detect sounds that carry less energy than the background noise. PMID- 10737793 TI - The hermaphrodite sperm/oocyte switch requires the Caenorhabditis elegans homologs of PRP2 and PRP22. AB - Sex determination in the hermaphrodite germ line of Caenorhabditis elegans is controlled posttranscriptionally. The switch from spermatogenesis to oogenesis relies on regulation of the fem-3 sex-determining gene via a regulatory element in the fem-3 3' untranslated region. Previous work showed that at least six mog genes are required for repression by the fem-3 3' untranslated region, and that one of those genes, mog-1, encodes a DEAH-box protein. In this paper, we report the cloning of mog-4 and mog-5 and the finding that mog-4 and mog-5 also encode DEAH-box proteins. Our molecular identification of mog-4 and mog-5 relied on genetic mapping and transformation rescue and was confirmed by a missense mutation in each gene. A phylogenetic analysis revealed that the C. elegans MOG 1, MOG-4, and MOG-5 proteins are closely related to the yeast proteins PRP16, PRP2, and PRP22, respectively. In view of their effect on fem-3 regulation and their homology to PRP16, PRP2, and PRP22, we propose that MOG-1, MOG-4, and MOG-5 are required for posttranscriptional regulation, perhaps by modifying the conformation of ribonucleoprotein complexes. PMID- 10737792 TI - Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion. AB - MYC affects normal and neoplastic cell proliferation by altering gene expression, but the precise pathways remain unclear. We used oligonucleotide microarray analysis of 6,416 genes and expressed sequence tags to determine changes in gene expression caused by activation of c-MYC in primary human fibroblasts. In these experiments, 27 genes were consistently induced, and 9 genes were repressed. The identity of the genes revealed that MYC may affect many aspects of cell physiology altered in transformed cells: cell growth, cell cycle, adhesion, and cytoskeletal organization. Identified targets possibly linked to MYC's effects on cell growth include the nucleolar proteins nucleolin and fibrillarin, as well as the eukaryotic initiation factor 5A. Among the cell cycle genes identified as targets, the G1 cyclin D2 and the cyclin-dependent kinase binding protein CksHs2 were induced whereas the cyclin-dependent kinase inhibitor p21(Cip1) was repressed. A role for MYC in regulating cell adhesion and structure is suggested by repression of genes encoding the extracellular matrix proteins fibronectin and collagen, and the cytoskeletal protein tropomyosin. A possible mechanism for MYC mediated apoptosis was revealed by identification of the tumor necrosis factor receptor associated protein TRAP1 as a MYC target. Finally, two immunophilins, peptidyl-prolyl cis-trans isomerase F and FKBP52, the latter of which plays a role in cell division in Arabidopsis, were up-regulated by MYC. We also explored pattern-matching methods as an alternative approach for identifying MYC target genes. The genes that displayed an expression profile most similar to endogenous Myc in microarray-based expression profiling of myeloid differentiation models were highly enriched for MYC target genes. PMID- 10737794 TI - The coevolution of cell senescence and diploid sexual reproduction in unicellular organisms. AB - In this paper, we investigate the coevolution of diploid sexual reproduction and cell senescence (i.e., cell aging). We use probability analysis, computer simulation, and exact numerical computation to analyze the impacts of deleterious recessive mutations on sexual and asexual reproduction. The main conclusion is that, without cell senescence, the evolutionary advantages of sexual reproduction cannot be realized in unicellular organisms that reproduce both sexually and asexually. Also, cell senescence is found to be useful in the maintenance of sexual reproduction. This result suggests that diploid sexual reproduction was unlikely to establish itself as a widespread reproduction mechanism without the complementary process of cell senescence. PMID- 10737795 TI - APC mutations in sporadic colorectal tumors: A mutational "hotspot" and interdependence of the "two hits". AB - Although APC mutations occur at a high frequency in colorectal cancers, few studies have performed a comprehensive analysis by screening the whole gene for mutations and assessing allelic loss. APC seems to act as a tumor-suppressor gene in a "nonclassical" fashion: data from familial adenomatous polyposis (FAP) show that the site of the germ-line mutation determines the type of "second hit" in FAP tumors, and simple protein inactivation is selected weakly, if at all. In this study, we screened the entire coding region of APC for mutations and assessed allelic loss in a set of 41 colorectal cancer cell lines. Of 41 cancers, 32 (83%) showed evidence of APC mutation and/or allelic loss. We identified several APC mutations and found a "hotspot" for somatic mutation in sporadic colorectal tumors at codon 1,554. Our results suggest that APC mutations occur in the great majority of colorectal cancers, the exceptions almost all being RER+ tumors, which may substitute for altered APC function by mutations in beta catenin and/or at other loci. When combined with previously published data, our results show that there is interdependence of the "two hits" at APC in sporadic colorectal tumors as well as in FAP. APC mutations in the "mutation cluster region," especially those close to codon 1,300, are associated with allelic loss, whereas tumors with mutations outside this region tend to harbor truncating mutations. The causes of this phenomenon are probably selection for retained N terminal and lost C-terminal APC functions, effects on beta-catenin levels, and APC protein stability. PMID- 10737796 TI - Early highly active antiretroviral therapy for acute HIV-1 infection preserves immune function of CD8+ and CD4+ T lymphocytes. AB - Highly active antiretroviral therapy (HAART) has been advocated for the management of primary HIV-1 infection without clear understanding of its immunological effects. Here, we demonstrate that early use of HAART during primary infection preserves HIV-specific CD8(+) T cells physically and functionally while HIV-specific T cell help is sustained. We also show that even transient administration of HAART at seroconversion can preserve HIV-specific immunity. In contrast, delayed initiation of HAART is associated with a progressive loss of HIV-specific CD8(+) T cells and absent HIV-specific T cell help. These results imply that HIV-specific T help is damaged during primary HIV 1 infection. Early drug treatment, which preserves this immunity, also preserves HIV-specific CD8(+) T cells. These results have implications for understanding the early pathogenesis of HIV-1 infection and suggest that acute HIV infection should be treated aggressively and as early as possible. PMID- 10737797 TI - Fulminant spontaneous autoimmunity of the central nervous system in mice transgenic for the myelin proteolipid protein-specific T cell receptor. AB - Proteolipid protein (PLP)-139-151 is the dominant encephalitogenic peptide that induces experimental autoimmune encephalomyelitis (EAE) in SJL (H-2(s)) mice. To examine the contribution of T cell receptor (TCR) specificity in the induction of EAE, we generated transgenic mice expressing the rearranged TCR genes from an encephalitogenic or a nonencephalitogenic PLP-139-151/I-A(s)-specific T cell clone. Both types of transgenic lines developed spontaneous EAE, but, remarkably, the lines expressing the TCR from the nonencephalitogenic clone showed increasingly higher frequencies of disease (60-83%) in progressive SJL backcrosses and could not be propagated on the susceptible background. The T cells from the transgenic mice were not tolerized, because they responded vigorously to the antigen in vitro and mediated EAE when the mice were immunized with antigen. Besides being the only description of a TCR transgenic mice for the PLP-139-151/I-A(s) epitope, the results demonstrate that the TCR from a nonencephalitogenic PLP-specific T cell clone can induce autoimmune disease when expressed appropriately in vivo. PMID- 10737798 TI - Deregulated expression of insulin-like growth factor 1 in prostate epithelium leads to neoplasia in transgenic mice. AB - Transgenic mice expressing human insulin-like growth factor 1 (IGF-1) in basal epithelial cells of prostate have been characterized. Transgene expression led to activation of the IGF-1 receptor and spontaneous tumorigenesis in prostate epithelium. Hyperplasia was evident in these mice by 2-3 months of age. Atypical hyperplasias and prostatic intraepithelial neoplasia were evident by 6-7 months of age. Well differentiated adenocarcinomas appeared in mice 6 months or older. Less differentiated tumors, diagnosed as small cell carcinomas, were also observed in two of the older mice. Both lobes of the mouse prostate gland (dorsolateral and ventral) presented preneoplastic and neoplastic changes. The incidence of tumors in mice >/=6 months of age (38 mice total) was 50%. The development of neoplasia in these transgenic mice appeared to follow a stepwise progression through early preneoplastic changes that ultimately culminated in frank neoplasia. These mice offer an animal model for prostate cancer that will allow study of the stepwise development of this disease and the mechanism(s) whereby IGF-1 mediates this process. PMID- 10737799 TI - Genetic modification of survival in tissue-specific knockout mice with mitochondrial cardiomyopathy. AB - We recently described a mouse model that reproduces important pathophysiological features of mitochondrial DNA (mtDNA) mutation diseases. The gene for mouse mitochondrial transcription factor A, Tfam (also called mtTFA), a nucleus-encoded key regulator of mtDNA expression, was targeted with loxP sites (Tfam(loxP)) and disrupted in vivo by transgenic expression of cre-recombinase from the muscle creatinine kinase (Ckmm) promoter. This promoter is active from embryonic day 13, and the knockouts had normal respiratory chain function in the heart at birth and developed mitochondrial cardiomyopathy postnatally. In this paper, we describe a heart-knockout strain obtained by mating Tfam(loxP) mice to animals expressing cre-recombinase from the alpha-myosin heavy chain (Myhca) promoter. This promoter is active from embryonic day 8, and the knockouts had onset of mitochondrial cardiomyopathy during embryogenesis. The age of onset of cardiac respiratory chain dysfunction can thus be controlled by temporal regulation of cre recombinase expression. Further characterization demonstrated that approximately 75% of the knockouts died in the neonatal period, whereas, surprisingly, approximately 25% survived for several months before dying from dilated cardiomyopathy with atrioventricular heart conduction blocks. Modifying gene(s) affect the life span of the knockouts, because approximately 95% of the knockout offspring from an intercross of the longer-living knockouts survived the neonatal period. Thus, the tissue-specific knockouts we describe here not only reproduce important pathophysiological features of mitochondrial cardiomyopathy but also provide a powerful system by which to identify modifying genes of potential therapeutic value. PMID- 10737800 TI - Shotgun sequencing of the human transcriptome with ORF expressed sequence tags. AB - Theoretical considerations predict that amplification of expressed gene transcripts by reverse transcription-PCR using arbitrarily chosen primers will result in the preferential amplification of the central portion of the transcript. Systematic, high-throughput sequencing of such products would result in an expressed sequence tag (EST) database consisting of central, generally coding regions of expressed genes. Such a database would add significant value to existing public EST databases, which consist mostly of sequences derived from the extremities of cDNAs, and facilitate the construction of contigs of transcript sequences. We tested our predictions, creating a database of 10,000 sequences from human breast tumors. The data confirmed the central distribution of the sequences, the significant normalization of the sequence population, the frequent extension of contigs composed of existing human ESTs, and the identification of a series of potentially important homologues of known genes. This approach should make a significant contribution to the early identification of important human genes, the deciphering of the draft human genome sequence currently being compiled, and the shotgun sequencing of the human transcriptome. PMID- 10737801 TI - The t(14;21)(q11.2;q22) chromosomal translocation associated with T-cell acute lymphoblastic leukemia activates the BHLHB1 gene. AB - We have cloned the genomic breakpoints for a balanced t(14;21)(q11. 2;q22) chromosomal translocation associated with T-cell acute lymphoblastic leukemia. Sequence analysis of the genomic breakpoints indicated that the translocation had been mediated by an illegitimate V(D)J recombination event that disrupted the T cell receptor (TCR) alpha locus and placed the TCR alpha locus enhancer on the derivative 21 chromosome. We identified a previously unreported transcript, designated BHLHB1 (for basic domain, helix-loop-helix protein, class B, 1) that had been activated by the translocation. BHLHB1 mapped to the region of chromosome 21 that has been proposed to be responsible, at least in part, for the learning deficits seen in children with Down's syndrome. Although BHLHB1 expression normally is restricted to neural tissues, T-cell lymphoblasts with the t(14;21)(q11.2;q22) also expressed high levels of BHLHB1 mRNA. Expression of BHLHB1 dramatically inhibited E2A-mediated transcription activation in NIH 3T3 fibroblasts and Jurkat T cells. This observation suggests that BHLHB1, similar to SCL/TAL1, may exert a leukemogenic effect through a functional inactivation of E2A or related proteins. PMID- 10737802 TI - Functional analysis of gapped microbial genomes: amino acid metabolism of Thiobacillus ferrooxidans. AB - A gapped genome sequence of the biomining bacterium Thiobacillus ferrooxidans strain ATCC23270 was assembled from sheared DNA fragments (3.2-times coverage) into 1,912 contigs. A total of 2,712 potential genes (ORFs) were identified in 2.6 Mbp (megabase pairs) of Thiobacillus genomic sequence. Of these genes, 2,159 could be assigned functions by using the WIT-Pro/EMP genome analysis system, most with a high degree of certainty. Nine hundred of the genes have been assigned roles in metabolic pathways, producing an overview of cellular biosynthesis, bioenergetics, and catabolism. Sequence similarities, relative gene positions on the chromosome, and metabolic reconstruction (placement of gene products in metabolic pathways) were all used to aid gene assignments and for development of a functional overview. Amino acid biosynthesis was chosen to demonstrate the analytical capabilities of this approach. Only 10 expected enzymatic activities, of the nearly 150 involved in the biosynthesis of all 20 amino acids, are currently unassigned in the Thiobacillus genome. This result compares favorably with 10 missing genes for amino acid biosynthesis in the complete Escherichia coli genome. Gapped genome analysis can therefore give a decent picture of the central metabolism of a microorganism, equivalent to that of a complete sequence, at significantly lower cost. PMID- 10737803 TI - The high copper tolerance of Candida albicans is mediated by a P-type ATPase. AB - The pathogenic yeast Candida albicans has higher resistance than the baker's yeast Saccharomyces cerevisiae to elevated concentrations of copper. To understand the basis of this differential resistance, we performed a functional screen for C. albicans genes involved in copper detoxification. Here, we report the isolation of two such genes: a metallothionein, CaCUP1, and a copper transporting P-type ATPase, CaCRP1. Both genes are induced by extracellular copper. Gene disruptions indicated that the copper extrusion pump is responsible for the unusual resistance of C. albicans to copper, whereas the metallothionein is responsible for the residual copper resistance of the Cacrp1Delta mutant. We show further that under acidic and anaerobic conditions, such as prevail in the natural niche of C. albicans, the digestive tract of animals, CaCRP1 function becomes essential for survival in the presence of even very low copper concentrations. These observations suggest that copper in the gastrointestinal tract may present a toxic challenge to which enteric organisms had to adapt. PMID- 10737804 TI - Pregnenolone binds to microtubule-associated protein 2 and stimulates microtubule assembly. AB - Fetal or adult rat-brain cytosol and fetal rat-brain microtubules contain a high affinity, low-capacity pregnenolone-binding protein. The equilibrium dissociation constant is in the 30-50 nM range. The best competitors (in decreasing order) are pregnenolone sulfate, progesterone, Delta5-pregnene-3beta,20alpha-diol, and 3beta hydroxy-5alpha-pregnan-20-one. It was hypothesized that the pregnenolone-binding protein pertained to microtubule-associated proteins (MAPs). Indeed, partial purification of fetal brain cytosol by fast pressure liquid chromatography with sequential ion-exchange and gel-filtration columns yielded two fractions, one of very high molecular mass, >200 kDa, and the other of 40-60 kDa, enriched in [(3)H]pregnenolone-binding activity and in proteins immunolabeled with monoclonal anti-tubulin and anti-MAP2 antibodies. Because many proteins are associated with microtubules, binding assays were repeated with purified calf-brain tubulin, MAP2, and Tau protein. Only the MAP2 fraction showed saturable [(3)H]pregnenolone binding with an affinity very close to that of rat-brain microtubules, but with a much larger concentration of binding sites (16 pmol/mg MAP2), which was increased more than 8-fold after copolymerization of MAP2 with tubulin. Finally, steroid effects on microtubule-assembly kinetics were assayed. Pregnenolone induced a large, dose-related increase of both the rate and extent of MAP2-induced tubulin assembly, whereas progesterone, inactive per se, counteracted the stimulatory effect of pregnenolone. Electron microscopic analysis confirmed that pregnenolone increased assembly of microtubules produced a completely normal structure. The stimulatory effect on MAP2-tubulin interaction was also observed in fetal rat brain neuron cultures. Therefore, we propose a mechanism of neurosteroid action, the control of microtubule or, more generally, of neural cytoskeleton dynamics, with potential roles in brain development, plasticity, and aging. PMID- 10737805 TI - Cost-benefit analysis potential in feeding behavior of a predatory snail by integration of hunger, taste, and pain. AB - Hunger/satiation state interacts with appetitive and noxious stimuli to determine feeding and avoidance responses. In the predatory marine snail Pleurobranchaea californica, food chemostimuli induced proboscis extension and biting at concentration thresholds that varied directly with satiation state. However, food stimuli also tended to elicit avoidance behavior (withdrawal and avoidance turns) at concentration thresholds that were relatively low and fixed. When the feeding threshold for active feeding (proboscis extension with biting) was exceeded, ongoing avoidance and locomotion were interrupted and suppressed. Noxious chemostimuli usually stimulated avoidance, but, in animals with lower feeding thresholds for food stimuli, they often elicited feeding behavior. Thus, sensory pathways mediating appetitive and noxious stimuli may have dual access to neural networks of feeding and avoidance behavior, but their final effects are regulated by satiation state. These observations suggest that a simple cost-benefit computation regulates behavioral switching in the animal's foraging behavior, where food stimuli above or below the incentive level for feeding tend to induce feeding or avoidance, respectively. This decision mechanism can weigh the animal's need for nutrients against the potential risk from other predators and the cost of relative energy outlay in an attack on prey. Stimulation of orienting and attack by low-level noxious stimuli in the hungriest animals may reflect risk taking that can enhance prey capture success. A simple, hedonically structured neural network model captures this computation. PMID- 10737806 TI - Optical probing of neuronal circuits with calcium indicators. AB - An experimental difficulty in unraveling circuits in the mammalian nervous system is the identification of postsynaptic targets of a given neuron. Besides ultrastructural reconstructions, simultaneous recordings from pairs of cells in brain slices have been used to identify connected neurons. We describe in this paper a technique using calcium imaging that allows rapid identification of potential postsynaptic targets. This method consists of stimulating one neuron ("trigger") while imaging a population of cells to detect which other neurons ("followers") are activated by the trigger. By using bulk-loading of calcium indicators in slices of mouse visual cortex, we demonstrate that neurons that display somatic calcium transients time-locked to the spikes of a trigger neuron can be monosynaptically connected to it. This technique could be applied to reconstruct and assay circuits in the central nervous system. PMID- 10737807 TI - Direct interaction between synaptotagmin and the intracellular loop I-II of neuronal voltage-sensitive sodium channels. AB - Synaptotagmin, a synaptic vesicle protein involved in Ca(2+)-regulated exocytosis, displayed direct high affinity interaction with neuronal sodium channels. Monoclonal antibodies directed against synaptotagmins I and II adsorbed in a concentration-dependent and -specific manner [(3)H]saxitoxin prelabeled sodium channels extracted with detergent from nerve endings. Conversely, co immunoprecipitation of synaptotagmin was achieved by antibodies against sodium channel subunits. Consistent with the co-immunoprecipitation assays, solubilized [(3)H]saxitoxin-prelabeled sodium channels were trapped on immobilized maltose binding protein (MBP)-synaptotagmin I. In vitro recombinant protein assays were employed to identify the interaction site of synaptotagmin I, which was located on the cytoplasmic loop between domains I and II of the sodium channel alphaIIA subunit. The co-immunoprecipitated synaptotagmin-sodium channel complexes were found to be Ca(2+)-dependent; this effect was mimicked by Ba(2+) and Sr(2+) but not Mg(2+). Finally the complex was shown to be distinct from the synaptotagmin SNARE protein complex that can selectively interact with presynaptic calcium channels (N and P/Q types). Thus, our findings demonstrate an unexpected and direct interaction between sodium channels and synaptotagmin. The Ca(2+) regulated association between sodium channels and a protein implicated in vesicular fusion may have intriguing consequences for the establishment and regulation of neuronal excitability. PMID- 10737808 TI - Protease-activated receptor-2 modulates myocardial ischemia-reperfusion injury in the rat heart. AB - Protease-activated receptor-2 (PAR-2) is a member of seven transmembrane domain G protein-coupled receptors activated by proteolytic cleavage whose better known member is the thrombin receptor. The pathophysiological role of PAR-2 remains poorly understood. Because PAR-2 is involved in inflammatory and injury response events, we investigated the role of PAR-2 in experimental myocardial ischemia reperfusion injury. We show for the first time that PAR-2 activation protects against reperfusion-injury. After PAR-2-activating peptide (2AP) infusion, we found a significant recovery of myocardial function and decrease in oxidation at reflow. Indeed, the glutathione cycle (glutathione and oxidized glutathione) and lipid peroxidation analysis showed a reduced oxidative reperfusion-injury. Moreover, ischemic risk zone and creatine kinase release were decreased after PAR 2AP treatment. These events were coupled to elevation of PAR-2 and tumor necrosis factor alpha (TNFalpha) expression in both nuclear extracts and whole heart homogenates. The recovery of coronary flow was not reverted by L-nitroarginine methylester, indicating a NO-independent pathway for this effect. Genistein, a tyrosine kinase inhibitor, did not revert the PAR-2AP effect. During early reperfusion injury in vivo not only oxygen radicals are produced but also numerous proinflammatory mediators promoting neutrophil and monocyte targeting. In this context, we show that TNFalpha and PAR-2 are involved in signaling in pathophysiological conditions, such as myocardial ischemia-reperfusion. At the same time, because TNFalpha may exert pro-inflammatory actions and PAR-2 may constitute one of the first protective mechanisms that signals a primary inflammatory response, our data support the concept that this network may regulate body responses to tissue injury. PMID- 10737809 TI - Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. AB - We describe a general approach for identifying components of subcellular structures in a multicellular organism by exploiting the ability to generate thousands of independent transformants in Arabidopsis thaliana. A library of Arabidopsis cDNAs was constructed so that the cDNAs were inserted at the 3' end of the green fluorescent protein (GFP) coding sequence. The library was introduced en masse into Arabidopsis by Agrobacterium-mediated transformation. Fluorescence imaging of 5,700 transgenic plants indicated that approximately 2% of lines expressed a fusion protein with a different subcellular distribution than that of soluble GFP. About half of the markers identified were targeted to peroxisomes or other subcellular destinations by non-native coding sequence (i.e., out-of-frame cDNAs). This observation suggests that some targeting signals are of sufficiently low information content that they can be generated frequently by chance. The potential of the approach for identifying markers with unique dynamic processes is demonstrated by the identification of a GFP fusion protein that displays a cell-cycle regulated change in subcellular distribution. Our results indicate that screening GFP-fusion protein libraries is a useful approach for identifying and visualizing components of subcellular structures and their associated dynamics in higher plant cells. PMID- 10737810 TI - Distorting the mental health market. PMID- 10737813 TI - Law & psychiatry: the changing face of juvenile justice. PMID- 10737814 TI - Personal accounts: a touch of dynamic psychiatry. PMID- 10737816 TI - Managed care: public-sector managed behavioral health care: V. redefining "medical necessity"-the Iowa experience. PMID- 10737817 TI - Datapoints: employer-based mental health benefits: implications for mental health parity. PMID- 10737818 TI - Economic grand rounds: the mismanagement of medication management. PMID- 10737819 TI - Conference report: APA conference of experts on medicare legislation for psychiatric disorders august 9-10, 1965, washington, D.C AB - Editor's Note: The report on an American Psychiatric Association conference on Medicare legislation reprinted below was published in the October 1965 issue of Mental Hospitals. The conference was convened to draft recommendations to be used in setting standards for care in psychiatric facilities that wished to participate in the Medicare program. Richard G. Frank, Ph.D., discusses the dramatic impact of the Medicare and Medicaid programs on mental health care in a commentary and analysis beginning on page 465. PMID- 10737820 TI - The creation of Medicare and Medicaid: the emergence of insurance and markets for mental health services. AB - Editor's Note: As a follow-up to the preceding article first published in the October 1965 issue (see page 461), Richard G. Frank, Ph.D., offers an analysis of the evolving Medicare and Medicaid programs and their impact on public mental health care. He shows that many of the themes raised at the 1965 APA conference on Medicare legislation for psychiatric disorders continue to dominate public debate. PMID- 10737821 TI - Patients' and providers' perceptions of outpatient treatment termination in a managed behavioral health organization. AB - OBJECTIVE: A common complaint about managed care is that treatment decisions of patients and providers are frequently altered by concurrent review of ongoing outpatient treatment. The objective of this study was to examine this perception from the perspectives of patients and providers. METHODS: A total of 190 patients and their providers were surveyed about the reason that outpatient treatment was terminated. The sample was randomly drawn from completed outpatient treatment episodes of a large national managed behavioral health organization. RESULTS: In more than three-quarters of the cases, outpatient treatment ended because patients and providers agreed that treatment goals were partially or completely met. Only 5 percent of patients and 3 percent of providers said that treatment ended because the managed care organization denied ongoing treatment. Agreement between patient-provider pairs was generally poor regarding the perceived reason for termination, except when termination was attributed to concurrent review by the managed behavioral health organization. CONCLUSIONS: In this study of a single large managed behavioral health organization, outpatient treatment was most likely to end based on the decisions of patients and providers rather than utilization review decisions. PMID- 10737822 TI - Utility of routine drug screening in a psychiatric emergency setting. AB - OBJECTIVE: The study determined whether dispositions from an urban psychiatric emergency service would differ between patients who received a mandatory urine drug test and those who may or may not have had a test based on the attending psychiatrist's clinical judgment. The accuracy of clinicians' suspicion of substance use among mandatorily screened patients was also examined. METHODS: A total of 392 consenting patients presenting to an urban psychiatric emergency service were randomly assigned to a mandatory-screen group (N=198) or a usual care group (N=194). Physicians ordered screens based on clinical judgment. Additional screens were performed without physicians' knowledge for patients in the mandatory-screen group for whom no screen was ordered. Demographic and clinical information, results of drug screens, and information about dispositions were collected from clinical charts or hospital databases. RESULTS: No difference in dispositions was found between the mandatory-screen group and the usual-care group. Survival analysis did not reveal a difference between the two groups in length of stay in inpatient psychiatric units. As for accuracy of physicians' suspicion of substance use, positive drug screens were recorded for 10.2 percent of the 198 patients in the mandatory-screen group who did not admit drug use or for whom physicians did not expect drug use. A total of 39.3 percent of the patients who were suspected of use and 88.2 percent of those who admitted use had positive drug screens. Only 20.8 percent of patients who denied substance use had positive screens. CONCLUSIONS: Routine urine drug screening in a psychiatric emergency service did not affect disposition or the subsequent length of inpatient stays. The results do not support routine use of drug screens in this setting. PMID- 10737823 TI - Tenure in supportive housing for homeless persons with severe mental illness. AB - OBJECTIVE: The study examined the long-term effectiveness of approaches to housing homeless persons with serious mental illness. METHODS: A total of 2,937 persons placed in high-, moderate, -and low-intensity housing were followed for up to five years. Intensity reflected on the amount of structure and degree of clients' independence. The outcome variable was tenure in housing. Cox stepwise regression was used to calculate risk ratios of becoming discontinuously housed. RESULTS: Thirty percent of the sample were initially placed in high-intensity settings, 18 percent in moderate-intensity settings, and 52 percent in low intensity settings. Those in high-intensity settings tended to be younger, to be referred from hospitals, and to have a history or diagnosis of substance abuse. Individuals in moderate-intensity settings were more likely to be female and were least likely to have substance abuse problems. Individuals in low-intensity settings were more likely to be referred by municipal shelters and to have lived in municipal shelters for four or more months. After one, two, and five years, 75 percent, 64 percent, and 50 percent, respectively, of the sample were continuously housed. Older age was associated with longer tenure, and having a history of substance abuse was associated with shorter tenure. Individuals referred from a state psychiatric center had a greater risk of shorter tenure than other types of referrals. CONCLUSIONS: Results show that homeless persons with serious mental illness can remain in stable housing for periods of up to five years, supporting the premise that long-term residential stability can be enhanced by providing access to safe and affordable supportive housing. PMID- 10737824 TI - Pathways to housing: supported housing for street-dwelling homeless individuals with psychiatric disabilities. AB - OBJECTIVE: This study examined the effectiveness of the Pathways to Housing supported housing program over a five-year period. Unlike most housing programs that offer services in a linear, step-by-step continuum, the Pathways program in New York City provides immediate access to independent scatter-site apartments for individuals with psychiatric disabilities who are homeless and living on the street. Support services are provided by a team that uses a modified assertive community treatment model. METHODS: Housing tenure for the Pathways sample of 242 individuals housed between January 1993 and September 1997 was compared with tenure for a citywide sample of 1, 600 persons who were housed through a linear residential treatment approach during the same period. Survival analyses examined housing tenure and controlled for differences in client characteristics before program entry. RESULTS: After five years, 88 percent of the program's tenants remained housed, whereas only 47 percent of the residents in the city's residential treatment system remained housed. When the analysis controlled for the effects of client characteristics, it showed that the supported housing program achieved better housing tenure than did the comparison group. CONCLUSIONS: The Pathways supported housing program provides a model for effectively housing individuals who are homeless and living on the streets. The program's housing retention rate over a five-year period challenges many widely held clinical assumptions about the relationship between the symptoms and the functional ability of an individual. Clients with severe psychiatric disabilities and addictions are capable of obtaining and maintaining independent housing when provided with the opportunity and necessary supports. PMID- 10737825 TI - Changes in services and structure in community residential treatment facilities for substance abuse patients. AB - OBJECTIVE: This study examined the extent to which community residential facilities that contract with the Department of Veterans Affairs (VA) to treat substance abuse patients are providing more services and structure to meet the needs of a client population with increasingly severe problems. METHODS: A nationwide sample of 249 community residential facilities with VA contracts was surveyed in 1995 and again in 1998. RESULTS: In 1998 facilities were more likely than they were in 1995 to have psychiatrists and psychologists available to patients as well as specialized counseling and psychoeducational, rehabilitation, and medical services. Facilities also provided more social and recreational activities, and more structure was provided by discouraging patients' choice of individual daily living patterns. In 1998 the facilities were more likely to admit dual diagnosis patients, those with substance use disorders and psychiatric disorders. Programs that changed toward accepting dual diagnosis patients had more services and structure than programs that consistently accepted only substance abuse patients. CONCLUSIONS: Community residential facilities that contract with VA are responding appropriately to an increasingly ill patient population by providing more services and structure. PMID- 10737826 TI - Therapeutic limit setting in an assertive community treatment program. AB - OBJECTIVE: The study examined the use of therapeutic limit-setting activities by members of assertive community treatment teams with clients who had serious mental illness. METHODS: Case managers from 40 Veterans Affairs intensive psychiatric community care teams reported their use of 25 limit-setting activities with 1,564 veterans during the first six months of treatment. The 25 item measurement scale was factor analyzed, and a standard multiple regression procedure was used to regress scale scores on clients' characteristics, the frequency of case managers' contact with service providers and others, and clients' and case managers' perceptions about the therapeutic alliance. RESULTS: Case managers relied most frequently on informal verbal approaches to limit setting and relied least on formal legal restrictions. Factor analysis of the instrument, the Therapeutic Limit Setting (TLS) scale, reduced the number of items to 20 and resulted in a five-factor solution. The limit-setting factors were verbal guidance, money management, contingent withholding of services or support, enforced hospitalization, and invocation of external authorities. The TLS and its subscales were characterized by high internal consistency, modest intercorrelation, and unique relationships with variables related to clients' characteristics, the treatment process, and the therapeutic alliance. Case managers were more likely to set limits with clients who had more extensive hospitalization histories, a representative payee, recent alcohol or drug use, more arrests, and more severe symptoms. CONCLUSIONS: Case managers used a range of limit-setting strategies in assertive community treatment. Limit setting is a frequent and potentially important aspect of assertive community treatment that may be useful for comparing levels of assertiveness in assertive community treatment teams and other community-based rehabilitation services. PMID- 10737827 TI - Use of community support services by middle-aged and older patients with psychotic disorders. AB - OBJECTIVE: The study sought to determine the degree to which use of community services is related to predisposing, enabling, and need factors among older patients with psychotic disorders who live in the community and to assess whether high use of community services is associated with improving or declining psychopathology. METHODS: The sample consisted of 89 middle-aged and elderly community-dwelling patients with schizophrenia or other psychotic disorders. Assessments at baseline and two follow-ups at six-month intervals included measures of psychopathology, well-being, and social adjustment, in addition to the frequency of use of 17 formal community services in three categories psychological, social, and daily living services. RESULTS: Ninety-two percent of patients reported use of community support services. The mean number of annual service contacts per patient was 36.6 for psychological services, 81 for social services, and 39.7 for daily living services. High users of psychological services were younger and experienced more severe positive psychotic symptoms and depressive symptoms. High users of social services were of higher socioeconomic status, more likely to be female, and had a longer history of psychosis, more cognitive deficits, and more severe negative psychotic and depressive symptoms. Patients who used daily living services were older, had poorer functional health status and more cognitive deficits, and had more severe negative psychotic and depressive symptoms. A trend was noted for high users of social services to experience relief from depressive symptoms over time. CONCLUSIONS: Use of community services is common among older outpatients with psychotic disorders, but its frequency varies as a function of patient characteristics. PMID- 10737828 TI - Management of negative symptoms among patients with schizophrenia attending multiple-family groups. AB - OBJECTIVE: Outcomes for negative symptoms over a one-year period were examined in two groups of patients, one receiving psychoeducational multiple-family group treatment and one receiving standard care. METHODS: A total of 63 outpatients, ages 18 to 45 years, with DSM-IV diagnoses of schizophrenic disorders were randomly assigned to standard care or multiple-family group psychoeducation treatment at a large mental health center in Spokane, Washington. Treatment assignment was stratified by whether patients were taking typical or atypical antipsychotic medications. Negative symptom status was monitored monthly for one year by raters blind to group assignment and measured as a composite of five symptoms using the Modified Scale for the Assessment of Negative Symptoms. RESULTS: When the analysis controlled for baseline negative symptoms, participants in the multiple-family group experienced significantly reduced negative symptoms compared with those receiving standard care. Taking atypical antipsychotic medication or having a diagnosis of substance abuse was not associated with the severity of negative symptoms. An additional analysis of the five individual negative symptoms indicated small but consistent group differences on all dimensions except inattention. Negative symptoms were significantly correlated with relapse to acute illness but not with outpatient or inpatient service use. CONCLUSIONS: The study demonstrated that a psychoeducational multiple-family group intervention was more effective than standard care in managing negative symptoms over a 12-month period. The results are particularly relevant because negative symptoms are associated with relapse, poor social and occupational functioning, cognitive impairment, and lower subjective quality of life. PMID- 10737829 TI - Prevalence of cognitive impairment among homeless people in inner Sydney. AB - The prevalence of cognitive impairment was assessed among a cohort of homeless men and women selected randomly from the dining rooms of the seven largest hostels offering emergency shelter in inner Sydney. They were interviewed using sections of the Composite International Diagnostic Interview 2.0. A total of 204 subjects (155 men and 49 women) were interviewed, of whom 20 (10 percent) showed evidence of cognitive impairment as assessed by the Mini Mental State Examination. Subjects with cognitive impairment were significantly older than those without impairment (mean ages of 57 and 41, respectively). Reasons for cognitive impairment among homeless individuals are complex and remain to be elucidated. PMID- 10737830 TI - Guardianship applications for elderly patients: why do they fail? AB - Variables associated with successful completion of guardianship applications for elderly patients were identified. Thirteen patients for whom applications were approved were compared with 26 whose applications did not reach the court. Patients for whom the process was successful scored significantly higher on the anergia-depression subscale of the Brief Psychiatric Rating Scale and had significantly more medical conditions in the past year. A survey of next of kin revealed that the process had a much better chance of success when the unit social worker made the guardianship recommendation and when family members were given more information about the taxing and time-consuming process of obtaining guardianship. PMID- 10737831 TI - Antipsychotic drug use patterns and the cost of treating schizophrenia. AB - This study investigated the relationships between antipsychotic drug use patterns and direct costs for 3,321 Medi-Cal patients with schizophrenia. Ordinary least squares regression models were used to estimate the impact on costs of receiving antipsychotic drug treatment, delays in treatment, changes in therapy, and continuous therapy. Average costs were $25,940 per year per patient. Having used an antipsychotic drug was correlated with lower psychiatric hospital costs ($2,846 less) but higher nursing home costs. Completing one year of uninterrupted drug therapy was correlated with higher nursing home costs. Delayed drug treatment and changes in therapy increased the cost by $9,418 and $9,719, respectively. PMID- 10737832 TI - Potential cost savings from pill splitting of newer psychotropic medications. AB - Many of the newer psychotropic medications have a pricing structure in which there are only small cost differences between pills of different strengths, affording an opportunity for cost saving by splitting pills. Twelve newer psychotropic medications were examined. Although savings varied for each drug, aggregate data indicated that splitting pills can produce an annual savings of up to $1.45 billion, which represents about 10 percent of the retail sales of these drugs. Such savings can benefit individuals, state Medicaid programs, community mental health centers, and managed care companies. The authors propose several strategies for encouraging the use of pill splitting. PMID- 10737834 TI - A social perspective in research and practice PMID- 10737833 TI - A social perspective in research and practice. PMID- 10737835 TI - Counting the mentally ill in jails and prisons. PMID- 10737837 TI - A question of abuse PMID- 10737836 TI - A question of abuse. PMID- 10737838 TI - Competency and the psychiatric workforce. PMID- 10737839 TI - Risk of transfusion-transmitted human T-cell lymphotropic virus-type I in Latin America. AB - In a recent publication, G. Schmunis et al presented data from Latin America on blood quality and on important approaches to preventing human immmunodeficiency virus (HIV), acquired immunodeficiency syndrome (AIDS), and other infectious diseases that may be transmitted by blood transfusion. Mass screening between 1987 and 1988 in Peru of blood from more than 90,000 donors highlighted potential problems in the donor population, and it also uncovered inappropriate practices related to the use of blood transfusions. Since then, adequate screening of the blood supply for HIV was implemented and new transfusion-related AIDS cases have been prevented during the past 8 years. PMID- 10737840 TI - Food irradiation: a public health opportunity. AB - Public health scientists have had an interest in food irradiation for a hundred years and more. The first investigations occurred within a few years of the discovery of x-ray and short wavelength by the German physicist Roentgen, in 1895. German and French scientists carried on studies on pasteurization of food by radiation until 1914 and the war years. The problem was an unacceptable taste following irradiation. In 1921, the x-ray was reported by the scientists of the United States Department of Agriculture (USDA) to be effective in killing Trichinella cysts in pork and that it could kill disease-causing organisms and halt food spoilage. PMID- 10737841 TI - Counterpoint on food irradiation. AB - Dr. Steele's extensive argument illustrates well one side of the food irradiation controversy. The proponents and opponents are involved in a heated debate. I am not opposed to the technology, but I am opposed to food irradiation as public policy until the proponents and the manufacturers are willing to answer some important questions. PMID- 10737842 TI - Amoxicillin for fever and sore throat due to non-exudative pharyngotonsillitis: beneficial or harmful? AB - OBJECTIVES: To determine duration of signs and symptoms and adverse reactions after treatment with amoxicillin of patients with fever and sore throat due to non-exudative pharyngotonsillitis. DESIGN: This was a randomized, double-blinded, placebo-controlled trial. Outpatients at four medical centers were enrolled. Patients over 5 years of age presented with fever and sore throat for less than 10 days due to non-exudative pharyngotonsillitis. Cases with any of the following symptoms or illness were excluded: earache, nasal discharge with foul smell, rheumatic fever, valvular heart disease, renal disease, and penicillin hypersensitivity. Amoxicillin or identical placebo at the dosage of 50 mg/ kg per day was given three or four times daily for 7 days. RESULTS: There were 1217 patients enrolled in this study. Some were lost to follow-up, which is the reason for the variability in number of cases in these analyses. After therapy, duration of fever was 2.46 and 2.48 days (P = 0.78) and of sore throat 3.01 and 3.04 days (P = 0.80) in amoxicillin (n = 431) and placebo (n = 436) groups, respectively. Complications were clinically documented in 13 (2.5%) and 16 (3.0%) cases in amoxicillin (n = 527) and placebo (n = 524) groups (P = 0.56). Two cases (0.46% and 0.46%) from each group (n= 433 and 431) were positive by antistreptolysin O antibody determination. The history of carditis and abnormal urinalysis after treatment were not obtained. CONCLUSIONS: Amoxicillin therapy for non-exudative pharyngotonsillitis conferred no beneficial or harmful effect. PMID- 10737843 TI - Two-year assessment of the pathogen frequency and antimicrobial resistance patterns among organisms isolated from skin and soft tissue infections in Latin American hospitals: results from the SENTRY antimicrobial surveillance program, 1997-98. SENTRY Study Group. AB - OBJECTIVES: This study was conducted to evaluate the frequency of occurrence and antimicrobial susceptibility of bacterial isolates collected from patients with skin and soft tissue infections (SSTI) in Latin American hospitals, as part of the SENTRY Antimicrobial Surveillance Program. The dissemination of multidrug resistant methicillin-resistant Staphylococcus aureus (MDR-MRSA) among the Latin American countries also was studied. MATERIAL AND METHODS: A total of 885 bacterial isolates were analyzed. At the monitoring laboratory, antimicrobial susceptibility testing utilizing the reference broth microdilution method and confirmation of species identification were performed. Enterobacteriaceae possibly producing extended-spectrum beta-lactamases (ESBL) and MDR-MRSA isolates were genotyped by ribotyping using the RiboPrinter and by pulsed-field gel electrophoresis. RESULTS: Staphylococcus aureus (31%) was the most common etiologic agent causing SSTI, followed by Escherichia coli (13.4%) and Pseudomonas aeruginosa (11%). Thirty-one percent of S. aureus isolates were resistant to oxacillin (methicillin). The presence of ESBL phenotypes was markedly higher among the Klebsiella pneumoniae (35.5%) than E. coli isolates (10. 2%). Meropenem was the compound with the highest susceptibility rate among the Enterobacteriaceae (100%) and P. aeruginosa (95%) isolates. A great genetic similarity was observed among the MDR-MRSA in Latin America. CONCLUSION: High resistance rates to antimicrobial drugs among the most frequent bacterial pathogens were observed in 10 medical centers in Latin America. This study also demonstrated a clonal dissemination of a MDR-MRSA strain in several nations. PMID- 10737844 TI - Three-year surveillance study of nosocomial bacterial resistance in Argentina. The Antimicrobial Committee; and the National Surveillance Program (SIR) Participants Group. AB - INTRODUCTION: A national surveillance program (SIR) was introduced in 1996 in Argentina by the Antimicrobial Committee of the Argentinean Society for Microbiology to assess bacterial resistance. The present study reports the rates of nosocomial bacterial resistance found by this program. METHODS: A 2-month point-prevalence study was conducted twice yearly (i.e., April-May and October November) from 1996 to 1998, by 27 Argentinean centers. Susceptibility testing was carried out by the disk diffusion method following the National Committee for Clinical Laboratory Standards guidelines. RESULTS: In all, 6343 isolates recovered from 5603 inpatients (> or =48-hr hospitalization) were included. Methicillin resistance was 58% and 56% in Staphylococcus aureus and coagulase negative staphylococci (CNS), respectively. Although no vancomycin resistance was found in staphylococci, 2% and 8% of the S. aureus and CNS strains, respectively, proved resistant to teicoplanin. No ampicillin resistance was displayed by Enterococcus faecalis. High-level gentamicin and streptomycin resistance in enterococci were 33% and 37%, respectively. Acquired glycopeptide resistance in enterococci emerged in 1997 (2%). Imipenem resistance in Acinetobacter spp and Pseudomonas aeruginosa was 9% and 21%, respectively. Among Enterobacteriaceae, 1% and 5% of the Klebsiella pneumoniae and Enterobacter cloacae isolates, respectively, proved resistant to imipenem. Ceftazidime and cefepime resistance was found in 63% and 33% of the E. cloacae strains. Resistance to extended spectrum cephalosporins was shown by 48%, 26%, and 8% of the K. pneumoniae, Proteus mirabilis, and Escherichia coli isolates, respectively. CONCLUSIONS: The alarming rates of resistance found in this study provide compelling evidence of the need for more rational use of antimicrobial agents in Argentina. PMID- 10737845 TI - Ten-year review of invasive pneumococcal diseases in children and adults from Uruguay: clinical spectrum, serotypes, and antimicrobial resistance. AB - OBJECTIVES: Since 1987, the Reference Laboratory of the Ministry of Health of Uruguay has been monitoring infections due to Streptococcus pneumoniae in patients under 5 years of age, in those between 5 to 14 years of age, and in adults. The purpose of the present study was to retrospectively analyze a 10-year collection of invasive S. pneumoniae isolates from children 5 to 14 years of age and adults. METHODS: The Reference Children's Hospital, Pasteur Hospital, and two private hospitals in Montevideo as well as four hospitals located in other representative areas of the country participated in the pneumococcal surveillance program. Based on the information available at the Microbiology Department of the Central Public Health Laboratory (demographic data, date and site of isolate, and clinical diagnosis), all patients with an invasive pneumococcal disease were recorded. Pneumonia was clinically and radiologically diagnosed and etiology was assessed by isolation of S. pneumoniae from blood or pleural fluid. All specimens were collected at the Emergency Service. Capsular serotyping and antimicrobial susceptibilities were determined for each isolate. RESULTS: During the 10-year period, 228 invasive S. pneumoniae were identified and included in the study (blood, n = 129; cerebrospinal fluid [CSF], n = 73; pleural fluid, n = 20; peritoneal fluid, n = 3; synovial fluid, n = 1; pericardic fluid, n = 1; abscess, n = 1). The most frequent clinical presentations were pneumonia (n = 71) and meningitis (n = 69). Thirty-five adults had an underlying condition including, four with malignancies, four with lupus, two with human immunodeficiency virus (HIV)-infected, and two patients in hemodialysis among others. Eighteen of the 228 patients died (7.9% fatality rate), but only four of these had an underlying condition. Eleven fatal cases were attributable to meningitis (2 children, 9 and 11 years old; 9 adults, mean age, 59 y). Four patients with pneumonia and three with sepsis died, including a splenectomized woman. Nine different capsular serotypes (1, 5, 7, 9, 12, 15, 19A, 20, and 23A) were identified among the 18 fatal cases. Resistance to penicillin, generally combined with trimethoprim sulfamethoxazole, fluctuated annually, not surpassing 10%. CONCLUSIONS: The study results indicated that 96% of the serotypes involved in severe pneumococcal diseases were included in the 23-valent vaccine and that S. pneumoniae resistance to penicillin was moderate. PMID- 10737846 TI - Surveillance of bacterial pathogens associated with acute diarrhea in Lima, Peru. AB - OBJECTIVES: A study was conducted in Lima, Peru, from January to April 1995, to determine the bacterial pathogens associated with acute diarrhea in adults, their susceptibility to common antimicrobials, the risk factors involved in cholera transmission, and the best clinical predictors of cholera. METHODS: A random sample of adult patients with acute diarrhea was studied. Epidemiologic and clinical data and risk factors to acquire diarrheal diseases were evaluated. Identification of bacteria and susceptibility to antimicrobials were determined. RESULTS: The study included 336 patients. Vibrio cholerae O1 (52.7%), Shigella spp. (4. 8%), and Salmonella spp (2.7%) were the pathogens most commonly isolated. No resistance to antimicrobials was observed. Patients with cholera had less access to municipal water (P = 0.0018) and were less likely to have homes connected to a sewage system (P = 0. 0003) or to have indoor toilet facilities (P = 0.0001) than those without cholera. Liquid stools (odds ratio [OR] = 16.51; confidence interval [CI] = 13.71-19.02; P = 0.003), severe dehydration (OR = 2. 48; CI = 1.57-3.38; P = 0.0083), generalized cramps (OR = 4.63; CI = 3.10-6.17, P < 0.0001), and washerwoman's hands (OR = 2.45; CI = 1. 55-3.34; P = 0.017) were the best clinical predictors of cholera in this setting. CONCLUSIONS: Cholera is still prevalent in Lima, and people living in environments with low sanitary conditions are especially at risk. Clinical signs of severe dehydration and liquid stools were the best predictors of cholera. PMID- 10737847 TI - Risk of giardiasis from consumption of wilderness water in North America: a systematic review of epidemiologic data. AB - OBJECTIVES: A meta-analytic study was conducted to test the hypothesis that consumption of water from North American backcountry sources poses a statistically significant risk for acquisition of giardiasis. METHODS: The biomedical literature was surveyed by accessing Medline, and identified studies were supplemented with references in current reviews, published dissertations, and prior communications with state health departments. Studies were classified by methodologic design and subjected to predetermined inclusion criteria. Odds ratios with 95% confidence intervals, chi-squares, and P-values for epidemiologic surveys were either computed from raw data or abstracted directly from the included studies. RESULTS: Of 104 articles identified in the initial screening, nine met the inclusion criteria. Neither of two case reports met the criteria of the Centers for Disease Control and Prevention (CDC) for waterborne disease outbreak. Two prospective studies were identified, but neither showed a significant association. Of four case-control studies providing data, three reported an odds ratio of greater than one. CONCLUSIONS: Published reports of confirmed giardiasis among outdoor recreationalists clearly demonstrate a high incidence among this population. However, the evidence for an association between drinking backcountry water and acquiring giardiasis is minimal. Education efforts aimed at outdoor recreationalists should place more emphasis on handwashing than on water purification. Further studies should attempt to separate the specific risk factor of drinking water from backcountry sources from other behaviors among this group that may contribute to the risk. PMID- 10737848 TI - Seroepidemiology of Hantavirus in the Philippines. AB - OBJECTIVE: This study was undertaken to determine the seroepidemiology of Hantavirus infection in the Philippines. METHODS: This is a cross-sectional study done in asymptomatic volunteers from various communities in the Philippines selected by a stratified multistage sampling design. Antibody to Hantavirus was detected by particle agglutination (PA) test using Hantadia high-density particle agglutination (HDPA) reagent kit. RESULTS: The prevalence of positive Hantavirus antibody among 461 subjects was the same in both males (6.1%) and females (6.1%) in rural (7.6%), urban (5.6%), and urban poor (5.1%) populations. CONCLUSIONS: The prevalence of Hantavirus infection in the Filipino population is comparable to that seen in other developing countries. The HDPA can be conveniently used as a rapid tool to detect the Hantavirus antibody for seroepidemiologic and diagnostic purposes. PMID- 10737849 TI - Spontaneous spleen rupture during typhoid fever. AB - Typhoid fever currently is an uncommon disease in western countries, and cases usually are imported from endemic areas.1 The most common complications are intestinal bleeding or perforation and necrotizing cholecystitis, although hepatitis, myocarditis, nephritis, and meningitis may occur. Spontaneous spleen rupture during typhoid fever is a known but rare complication. This report describes a new case and reviews the literature. PMID- 10737850 TI - HIV heterosexual transmission: a hypothesis about an additional potential determinant. AB - Transmission rates of human immunodeficiency virus (HIV) during heterosexual intercourse vary dramatically around the world. In Asia and South America, they are extraordinarily high, whereas in the United States and Europe, rates are much lower even after a large number of unprotected contacts. The transmission rates in Africa also probably are high, but the available studies unfortunately are weak. In Thailand, female-to-male transmission rates per contact were estimated at.056 (l in 18) compared to.0002 to.0015 (1/5000-1. 5/1000) for male-to-female transmission in the United States and Europe. Male-to-female transmission in Thailand appears to show, as expected, even greater transmission likelihood compared to female-to-male rates. In general, in the United States and Europe, transmission rates within heterosexual couples range from less than 10% to 22%, whereas in Thailand and Brazil, the rates exceed 40%. The much lower transmission rate per contact in the United States and Europe is based on an assumption that HIV transmitters are a homogeneous group. Wiley and colleagues argue that transmitters are likely to be a heterogeneous group with a large percentage of very low frequency transmitters and a small percentage of high frequency transmitters. That hypothesis is given some support by a cluster of cases in rural New York State in which one man appeared to infect 31% of his many contacts. PMID- 10737851 TI - Developmental patterning of the myocardium. AB - The heart in higher vertebrates develops from a simple tube into a complex organ with four chambers specialized for efficient pumping at pressure. During this period, there is a concomitant change in the level of myocardial organization. One important event is the emergence of trabeculations in the luminal layers of the ventricles, a feature which enables the myocardium to increase its mass in the absence of any discrete coronary circulation. In subsequent development, this trabecular layer becomes solidified in its deeper part, thus increasing the compact component of the ventricular myocardium. The remaining layer adjacent to the ventricular lumen retains its trabeculations, with patterns which are both ventricle- and species-specific. During ontogenesis, the compact layer is initially only a few cells thick, but gradually develops a multilayered spiral architecture. A similar process can be charted in the atrial myocardium, where the luminal trabeculations become the pectinate muscles. Their extent then provides the best guide for distinguishing intrinsically the morphologically right from the left atrium. We review the variations of these processes during the development of the human heart and hearts from commonly used laboratory species (chick, mouse, and rat). Comparison with hearts from lower vertebrates is also provided. Despite some variations, such as the final pattern of papillary or pectinate muscles, the hearts observe the same biomechanical rules, and thus share many common points. The functional importance of myocardial organization is demonstrated by lethality of mouse mutants with perturbed myocardial architecture. We conclude that experimental studies uncovering the rules of myocardial assembly are relevant for the full understanding of development of the human heart. PMID- 10737852 TI - Storage of lipid droplets in and production of extracellular matrix by hepatic stellate cells (vitamin A-storing cells) in Long-Evans cinnamon-like colored (LEC) rats. AB - LEC rats spontaneously develop hepatocellular carcinoma with cholangiofibrosis after chronic hepatitis, but the mechanism of development of the hepatic injury is not clear. To investigate the role of hepatic stellate cells in induction or suppression of hepatic fibrosis, we morphologically examined the liver of LEC rats. Accumulation of copper was analyzed by the Danscher-Timm's sulfide-silver method. Histopathological changes were evaluated by hematoxylin and eosin staining, and by Masson's trichrome method. Activated stellate cells were identified by immunostaining method for alpha-smooth muscle actin. Cytological alterations of the stellate cells were investigated by transmission electron microscopy. To evaluate the lipid content in the stellate cells, we analyzed the area of lipid droplets of the cells by morphometric analysis. Also for evaluation of the changes in the number of stellate cells, the numbers of nucleated stellate cells and parenchymal cells were counted and statistically analyzed. Hepatic parenchymal cells showed excessive accumulation of copper at 5 weeks of age. Submassive necrosis was observed at 19 weeks of age. The liver of LEC rats 1.5 years of age showed cholangiofibrosis and subcellular injury of hepatic parenchymal cells. However, no diffuse hepatic fibrosis was observed in the liver, and hepatic stellate cells around the regions of cholangiofibrosis were negative for alpha-smooth muscle actin. The area of lipid droplets of a stellate cell in the liver of LEC rats was 1.6 to 1.8 times as large as that of normal Wistar rats. The hepatic stellate cells did not participate in the accumulation of collagen fibers around themselves when the cells contained a large amount of vitamin A-lipid droplets, even though the development of hepatic lesions was in progress. Our present data are consistent with our previous hypothesis that there is an antagonistic relationship between the storage of vitamin A and the production of collagen in stellate cells. PMID- 10737853 TI - Cell death during development of intercalated ducts in the rat submandibular gland. AB - Programmed cell death, or apoptosis, occurs during the development of many tissues and organs in almost all multicellular organisms. Although apoptosis of salivary gland cells has been demonstrated in several pathological conditions, the role of apoptosis in the postnatal development of the salivary glands is unknown. We have studied the development of the rat submandibular gland (SMG) during its transition from the perinatal stage to the mature adult stage. Terminal tubule or Type I cells, which synthesize the secretory protein SMG-C, are prominent in the perinatal acini and are believed to form the intercalated ducts of the adult gland. Between 25 days and 30 days after birth, the number of Type I cells and their SMG-C immunoreactivity markedly decreased. Apoptotic cells in association with the developing intercalated ducts were labeled with the Terminal Deoxyribonucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) method. Between 25 and 40 days of age, from 50 to 80% of the apoptotic cells in cryostat sections of the SMG were closely associated with the intercalated ducts. Electron microscopy showed that the Type I cells became vacuolated, their secretory granules were reduced in size and number, and the amount of rough endoplasmic reticulum was decreased. Cellular debris resembling apoptotic bodies was phagocytosed by macrophages and adjacent intercalated duct cells. These observations suggest that the loss of Type I cells and reduction of SMG-C immunoreactivity during development of the intercalated ducts of the adult rat SMG is due, at least in part, to apoptosis. PMID- 10737854 TI - Presence and morphological variability of Golgi tendon organs in the distal portion of sheep extraocular muscle. AB - This study was undertaken to demonstrate the presence of Golgi tendon organs (GTOs) in the distal portion of sheep extraocular muscle (EOM) and to describe the morphological variability of these receptors. Extraocular muscles of a young and an old sheep were perfusion fixed and/or immersion fixed. Tissue was prepared for light microscopy and transmission electron microscopy. Immunohistochemistry was done to demonstrate the myosin pattern of the intracapsular muscle fibers of the GTOs. All GTOs in the distal portions of the sheep EOMs were located in a distinct muscle layer which was designated in a former investigation as the so called peripheral patch layer. Each EOM of the young sheep contained GTOs; between four and 15 GTOs were counted in the rectus EOMs. Eight GTOs were found in the superior rectus of the old sheep. Golgi tendon organs in EOMs of the young and the old sheep did not differ in their morphology. In the young sheep the mean length of the GTOs was 447 +/- 132 microm (n = 60) and their mean width 101 +/- 26 microm (n = 60). In the old sheep values were 576 +/- 188 microm (mean length, n = 8) and 103 +/- 18 microm (mean width, n = 8). The GTOs were encapsulated by perineurial cells. In 12 GTOs, only collagen bundles were inside. In the remaining GTOs (56), intracapsular muscle fibers were present. Muscle fibers entered the proximal poles of the GTOs and either terminated inside the receptors or muscle fibers left the GTOs at their distal poles. These intracapsular muscle fibers were of the multiply-innervated type. In the GTOs variably shaped nerve terminals were found which contained a high number of mitochondria. In two GTOs, additionally, nerve terminals with aggregates of densely packed vesicles were present. PMID- 10737855 TI - Increased in vivo levels of neurotransmitters to trigeminal motoneurons: effects on craniofacial bone and TMJ. AB - The results of chronic, in vivo delivery of excitatory and inhibitory neurotransmitter substances upon the craniofacial skeleton are of ongoing interest to clinician and basic scientist alike. Our purpose was to document and compare the effects of biodegradable glycine, glutamate, and thyrotropin releasing hormone (TRH) microspheres upon the craniofacial skeleton and TMJ of actively growing rats. Glycine, glutamate, TRH, and blank microspheres were stereotactically implanted in proximity to motoneurons within the trigeminal motor nucleus in order to test the following null hypotheses: (1) neurotransmitter microspheres implanted near trigeminal motoneurons of growing rats have no significant effect on the craniofacial skeleton and temporomandibular joints of implanted animals, and (2) there are no significant differences between the relative effects of glutamate, TRH (excitatory to trigeminal motoneurons), and glycine (inhibitory to trigeminal motoneurons) implants upon the craniofacial skeleton and temporomandibular joint. Fifty male Sprague-Dawley rats underwent stereotactic neurosurgery at 35 days; five rats each were killed at 14 and 21 days postoperative for data collection and comparison between glycine-, glutamate-, TRH-, blank-microsphere, and sham surgery rats. Glycine rats had significantly (P < or = 0.05, 0. 01) smaller implant-side cranial dimensions and mandibular condyles, all glycine rats showed increased gracility of implant-side bones, and deviation of their facial skeleton away from the implant-side; this was in contrast to the generally larger implant side bony structures in both glutamate and TRH rats. The two null hypotheses were both rejected. Due to their inhibitory and excitatory effects upon trigeminal motoneurons, masticatory muscles, and their neuromuscular generation of biomechanical forces that affect bone, the neurotransmitter substances glycine, glutamate, and TRH appear to play an important role in the growth and development of the mammalian craniofacial skeleton and TMJ. PMID- 10737856 TI - Calbindin D28k-like immunoreactivity during the formation of the enamel-free area in the rat molar teeth. AB - Previous studies have demonstrated the presence of calbindin D28k in the ameloblasts derived from the inner enamel epithelium. The occlusal surfaces of the rodent molars partly lack the enamel covering, which is referred to as enamel free area (EFA). In the present study, we compared the immunohistochemical localization of calbindin D28k-like immunoreactivity (CB-LI) in the cells at the EFA (EFA cells) and ameloblasts of the rat molar teeth at the light microscopic level. CB-LI was strong in the ameloblasts of the presecretory through the protective stages, while it was faint at the late secretory to transitional stages. However, some mature ameloblasts lacked the immunoreactivity. On the other hand, the majority of EFA cells showed distinct polarization and elongation that were absent in few cells at the early stage of EFA formation. At all stages, the EFA cells adjacent to the ameloblasts showed CB-LI, however, some cells adjacent to the mature ameloblasts lacked the reaction. Intensive CB-LI was demonstrated in EFA cells at the reduced enamel epithelium. These immunohistochemical findings suggest EFA cells have cytochemical properties similar to those of ameloblasts. PMID- 10737857 TI - Endocranial capacity in Sts 71 (Australopithecus africanus) by three-dimensional computed tomography. AB - In a recent report on early hominid endocranial capacity, it was predicted that future studies would show that: (1) "several key early hominid endocranial estimates may be inflated"; (2) "current views on the tempo and mode of early hominid brain evolution may need reevaluation"; and (3) endocranial capacity in one of these, Sts 71, was "probably closer to 370 cm(3), very near the mean value for female chimpanzees, and not the currently accepted 428 cm(3)" (Conroy et al., Science, 1998; 280: 1730-1731; Falk, Science 1998; 20:1714). Subsequent studies tend to support the first two predictions, but not the third (Culotta, Science, 1999; 284: 1109; Falk, Am. J. Phys. Anthropol. Suppl., 1999; 28: 126; Falk et al., J. Hum. Evol. [in press]). Here we detail the reasons for thinking the currently accepted endocranial value for Sts 71 is probably correct by providing the first quantitative details of endocranial reconstruction in Sts 71 using three-dimensional computed tomography. Relative brain expansion in the hominid lineage started some half-million years before the earliest appearance of the genus Homo, possibly coincident with enhanced tool-making skills and carnivory. PMID- 10737858 TI - Effects of fetal exposure to nicotine on dental development of the laboratory rat. AB - Nicotine is one of the most widely used toxins in the world today. Most addiction research relating to nicotine in particular, as well as opioids and alcohol, has concentrated on the cellular and molecular biology of the mammalian brain and on features of organ structure and physiology associated with substance abuse. Thus, while numerous studies have been conducted to examine nicotine's detrimental physiological effects in a variety of soft tissues, this investigation attempts to examine further the gross morphological consequences of this drug on a hard tissue, the first molar crown of the laboratory rat. It is hypothesised that by providing nicotine to rats during and after the fetal cycle, changes in dental structure will occur, owing to perturbations of development induced by this toxin. The dentitions of Fisher rats exposed to nicotine during and after the fetal cycle, and those of their non-treated controls, were examined. By carefully measuring the length, width and occlusal (chewing) areas of the first maxillary molars, it was possible to identify any gross morphological effects of nicotine on dental development. It was found that dental asymmetries (calculated as a size difference between a tooth and its antimere) were significantly increased while occlusal areas were significantly decreased in nicotine-exposed rats compared to control rats. In addition, significant differences were detected within the experimental group, females tending to exhibit the deleterious effects of nicotine more so than males. These results are in accordance with the predicted outcome; in similar studies of physiological systems and soft tissues, dental development is affected by the presence of nicotine. PMID- 10737859 TI - Sensory nerve supply of the human oro- and laryngopharynx: a preliminary study. AB - To date, the details of human sensory innervation to the pharynx and upper airway have not been demonstrated. In this study, a single human oro- and laryngopharynx obtained from autopsy was processed with a whole-mount nerve staining technique, Sihler's stain, to determine its entire sensory nerve supply. The Sihler's stain rendered all mucosa and soft tissue translucent while counterstaining nerves. The stained specimen was then dissected and the nerves were traced from their origins to the terminal branches. It was found that the sensory innervation of the human pharynx is organized into discrete primary branches that innervate specific areas, although these areas are often connected by small neural anastomoses. The density of innervation varied, with some areas receiving almost no identifiable nerve supply (e.g., posterior wall of the hypopharynx) and certain areas contained much higher density of sensory nerves: the posterior tonsillar pillars; the laryngeal surface of the epiglottis; and the postcricoid and arytenoid regions. The posterior tonsillar pillar was innervated by a dense plexus formed by the pharyngeal branches of the IX and X nerves. The epiglottis was densely innervated by the internal superior laryngeal nerve (ISLN) and IX nerve. Finally, the arytenoid and postcricoid regions were innervated by the ISLN. The postcricoid region had higher density of innervation than the arytenoid area. The use of the Sihler's stain allowed the entire sensory nerve supply of the pharyngeal areas in a human to be demonstrated for the first time. The areas of dense sensory innervation are the same areas that are known to be the most sensitive for triggering reflex swallowing or glottic protection. The data would be useful for further understanding swallowing reflex and guiding sensory reinnervation of the pharynx to treat neurogenic dysphagia and aspiration disorders. PMID- 10737860 TI - Transfer RNA recognition by aminoacyl-tRNA synthetases. AB - The aminoacyl-tRNA synthetases are an ancient group of enzymes that catalyze the covalent attachment of an amino acid to its cognate transfer RNA. The question of specificity, that is, how each synthetase selects the correct individual or isoacceptor set of tRNAs for each amino acid, has been referred to as the second genetic code. A wealth of structural, biochemical, and genetic data on this subject has accumulated over the past 40 years. Although there are now crystal structures of sixteen of the twenty synthetases from various species, there are only a few high resolution structures of synthetases complexed with cognate tRNAs. Here we review briefly the structural information available for synthetases, and focus on the structural features of tRNA that may be used for recognition. Finally, we explore in detail the insights into specific recognition gained from classical and atomic group mutagenesis experiments performed with tRNAs, tRNA fragments, and small RNAs mimicking portions of tRNAs. PMID- 10737861 TI - Studies of DNA dumbbells VII: evaluation of the next-nearest-neighbor sequence dependent interactions in duplex DNA. AB - Melting experiments were conducted on 22 DNA dumbbells as a function of solvent ionic strength from 25-115 mM Na(+). The dumbbell molecules have short duplex regions comprised of 16-20 base pairs linked on both ends by T(4) single-strand loops. Only the 4-8 central base pairs of the dumbbell stems differ for different molecules, and the six base pairs on both sides of the central sequence and adjoining loops on both ends are the same in every molecule. Results of melting analysis on the 22 new DNA dumbbells are combined with our previous results on 17 other DNA dumbbells, with stem lengths containing from 14-18 base pairs, reported in the first article of this series (Doktycz, Goldstein, Paner, Gallo, and Benight, Biopoly 32, 1992, 849-864). The combination of results comprises a database of optical melting parameters for 39 DNA dumbbells in ionic strengths from 25-115 mM Na(+). This database is employed to evaluate the thermodynamics of singlet, doublet, and triplet sequence-dependent interactions in duplex DNA. Analysis of the 25 mM Na(+) data reveals the existence of significant sequence dependent triplet or next-nearest-neighbor interactions. The enthalpy of these interactions is evaluated for all possible triplets. Some of the triplet enthalpy values are less than the uncertainty in their evaluation, indicating no measurable interaction for that particular sequence. This finding suggests that the thermodynamic stability of duplex DNA depends on solvent ionic strength in a sequence-dependent manner. As a part of the analysis, the nearest-neighbor (base pair doublet) interactions in 55, 85, and 115 mM Na(+) are also reevaluated from the larger database. PMID- 10737862 TI - Specificity of hydroxylmethyluracil-containing DNA for transcription factor 1: structural insights. AB - The genomic materials from some Bacillus subtilis bacteriophages are found to contain 5-(hydroxymethyl)-2'-deoxyuridine in place of thymine. Phage-encoded proteins such as transcription factor 1 specifically and preferentially bind to the minor grooves of these hmU-containing DNA but not to thymine-containing DNA. Data from electrophoretic mobility shift assays suggest that the inherent, localized flexibility of hmU-DNA, which is sequence-specific, is responsible for its discriminative binding. We discuss here, from the NMR-derived structural point of view, how differential DNA flexibility can contribute to specific binding of TF1 to hmU-DNA. PMID- 10737863 TI - Effects of experience on the parental responses of male Mongolian gerbils. AB - We examined responses of adult male Mongolian gerbils to nest site and young during the hours preceding and days following birth of a litter. We found that (a) male attendance at the nest site was markedly reduced for several hours following the birth of pups; (b) this lack of contact by males with nest and pups did not result from active exclusion of males from the nest by their mates; (c) males lacking previous experience of pups, but not those familiar with pups, avoided contact with pups on the day of their birth; and (4) 3-day-old gerbil pups were attractive even to males encountering young for the first time. PMID- 10737864 TI - A developmental analysis of clonidine's effects on cardiac rate and ultrasound production in infant rats. AB - Under controlled conditions, infant rats emit ultrasonic vocalizations during extreme cold exposure and after administration of the alpha(2) adrenoceptor agonist, clonidine. Previous investigations have determined that, in response to clonidine, ultrasound production increases through the 2nd-week postpartum and decreases thereafter. Given that sympathetic neural dominance exhibits a similar developmental pattern, and given that clonidine induces sympathetic withdrawal and bradycardia, we hypothesized that clonidine's developmental effects on cardiac rate and ultrasound production would mirror each other. Therefore, in the present experiment, the effects of clonidine administration (0.5 mg/kg) on cardiac rate and ultrasound production were examined in 2-, 8-, 15-, and 20-day old rats. Age-related changes in ultrasound production corresponded with changes in cardiovascular variables, including baseline cardiac rate and clonidine induced bradycardia. This experiment is discussed with regard to the hypothesis that ultrasound production is the acoustic by-product of a physiological maneuver that compensates for clonidine's detrimental effects on cardiovascular function. PMID- 10737865 TI - The effects of prenatal drug exposure, term status, and caregiving on arousal and arousal modulation in 8-week-old infants. AB - Prenatal exposure to cocaine, as well as other drugs, has been linked with "dysregulation," usually defined as problems in arousal and/or behavioral regulation. This study was designed to describe the physiological basis of dysregulation as a function of prenatal cocaine/polydrug exposure and term status. Eight-week-old infants were selected because they are just developing the ability to modulate arousal. One hundred-eighteen infants (23 preterm control, 27 preterm drug-exposed, 29 full-term control, and 39 full-term drug-exposed) completed a protocol during which heart rate (HR) and respiratory rate (RR) were measured. Drug group differences were found in baseline, arousal (response to stress), and arousal modulation (recovery from stress). A hierarchical multiple regression analysis was conducted to determine the portion of variance attributable to postnatal caregiving environment, term status, and specific drug exposure. Term status accounted for significant variance in arousal (both RR and HR), and in arousal modulation (only RR). Prenatal exposure to cocaine contributed a significant amount of unique variance in HR arousal whereas tobacco contributed significantly to HR arousal modulation. Prenatal drug exposure and preterm status contributed differently to dysregulation as measured by physiological responses. PMID- 10737866 TI - Handedness and cortisol in tufted capuchin monkey infants. AB - In this research, we examined the relationship between handedness and levels of plasma cortisol in infant monkeys (Cebus apella). Specifically, we sought to test the hypothesis that stress functioning is related to hemispheric specialization and is manifested in a positive correlation between cortisol levels and the frequency of right- versus left-hand use. We found a significant relationship between stress cortisol at age 6 months and lateral bias towards greater use of the right versus left hand at ages 6 and 12 months. These data suggest an early developmental influence of stress reactivity on the emergence of hemispheric specialization for manual control in infant monkeys through the 1st year of postnatal life. PMID- 10737867 TI - Deferred imitation of object-related actions in human-reared juvenile chimpanzees and orangutans. AB - Deferred imitation of object-related actions (e.g., picking up a cloth with a set of tongs) was assessed in 3 enculturated juvenile orangutans (Pongo pygmaeus) and 3 enculturated juvenile chimpanzees (Pan troglodytes). For each task, animals were given 4 min to explore the objects (baseline), followed by a demonstration of the target behavior, and 10 min later, were re-presented the objects (deferred phase). Each animal displayed deferred imitation on at least one trial, with each species demonstrating deferred imitation on approximately half of all possible trials. The findings were interpreted as reflecting cognitive abilities in juvenile great apes that permit deferred imitation under humanlike rearing conditions. PMID- 10737868 TI - Abstracts: international society for developmental psychobiology, 32nd annual meeting PMID- 10737869 TI - Adsorbed IgG: a potent adhesive substrate for human macrophages. AB - Previous reports from our laboratory have demonstrated qualitatively that preabsorbed IgG can enhance long-term macrophage adhesion in vitro. This investigation further characterizes and quantifies the biological effect of adsorbed human IgG on human macrophages and probes the potential mechanisms. Ten day human monocyte/macrophage cultures on Plastek M (PM), a normally poor cellular substrate for macrophages, confirmed the ability of preabsorbed IgG to dramatically enhance long-term macrophage adhesion. An adsorption solution concentration of 200 microg/mL of IgG was necessary to provide a consistent, optimal cellular response. (125)I adsorption studies indicated Langmuir-style IgG adsorption at low concentrations; however, no adsorption maximum was observed. Additional adsorption analysis revealed that the IgG fragments Fab, F(ab')(2), and Fc adsorb at levels only 20-40% that of the whole molecule. Despite the lower adsorption levels, both preabsorbed Fab and F(ab')(2) were shown to be as effective as whole molecule IgG at enhancing long-term macrophage adhesion. Surprisingly, the preabsorbed Fc fragment demonstrated no IgG-like activity, thereby eliminating the possibility of an Fc receptor-based mechanism. Other possible mechanisms, such as macrophage lectins, novel macrophage Fab receptors, and complement activation by adsorbed IgG and IgG fragments, are discussed. PMID- 10737870 TI - Leukocyte adhesion on model surfaces under flow: effects of surface chemistry, protein adsorption, and shear rate. AB - The effect of specific chemical functionalities on the adhesion of polymorphonuclear leukocytes (PMNs) under flow was investigated using a set of well-characterized, chemically functionalized surfaces prepared by self-assembly of alkanethiolate monolayers on gold surfaces. Terminal functionalities included CH(3), CH(2)OH, COOH, and (OCH(2)CH(2))(3)OH groups. A new surface modification was used to incorporate a phosphorylcholine moiety on the hydroxyl-terminated monolayer. Surface modification was verified using contact-angle measurements, ellipsometry, and X-ray photoelectron spectroscopy. Adhesion on the surfaces was studied in the presence and absence of pre-adsorbed fibrinogen. Fibrinogen adsorption on self-assembled monolayers (SAMs) was quantified using radioisotope detection. PMN adhesion was found to be dependent on the monolayer's terminal functionality. Adhesion was higher on the hydrophobic CH(3) surface and the polar COOH monolayer. Leukocyte adhesion was least on the phosphorylcholine-rich surface, followed by the ethylene-oxide-containing monolayer. Cell adhesion also was low on the hydrophilic OH monolayer. Attachment was decreased with increasing shear rate, exhibiting a three-fold decrease between 20 and 100 s(-1). Fibrinogen adsorption was higher on the CH(3) monolayer but comparable for the other four SAMs. Preincubation of the surfaces with fibrinogen decreased adhesion on all SAMs examined. PMID- 10737871 TI - Surfactant polymers designed to suppress bacterial (Staphylococcus epidermidis) adhesion on biomaterials. AB - We describe a series of surfactant polymers designed as surface-modifying agents for the suppression of bacterial adhesion on biomaterials. The surfactant polymers consist of a poly(vinyl amine) backbone with hydrophilic poly(ethylene oxide) (PEO) and hydrophobic hexanal (Hex) side chains (PVAm/PEO:Hex). Surface modification is accomplished by simple dip coating from aqueous solution, from which surfactant polymers undergo spontaneous surface-induced assembly on hydrophobic biomaterials. The stability of PVAm/PEO:Hex on pyrolytic graphite (HOPG) and polyethylene (PE) was demonstrated by the absence of detectable desorption under flow conditions of pure water over a 24-h period. PEO surfactant polymers with four different PEO:Hex ratios (1:1.4, 1:2.5, 1:4.6, and 1:10.7) and a dextran surfactant polymer were compared with respect to S. epidermidis adhesion under dynamic flow conditions. Suppression of S. epidermidis adhesion was achieved for all modified surfaces over the shear range 0-15 dyn/cm(2). The effectiveness depended on the surfactant polymer composition such that S. epidermidis adhesion to modified surfaces decreased significantly with increasing PEO packing density. Modified HOPG was more effective in reducing bacterial adhesion compared with the corresponding modification on PE, which we attribute to the presence of defects in surfactant polymer assembly on PE. Our results are discussed from the perspective of critical factors, such as optimal PEO packing density and hydration thickness, that contribute to the effectiveness of surfactant polymers to shield a biomaterial from adhesive bacterial interactions. PMID- 10737872 TI - Swelling and delamination of multi-electrode sensor arrays studied by variable pressure scanning electron microscopy. AB - Multi-electrode sensor arrays are made of soft and wet materials not easily examined by most microscopic techniques. In this paper, we have demonstrated that low-vacuum scanning electron microscopy (LVSEM) and energy-dispersive X-ray analysis (EDX) are adequate for studying the hydration, swelling, and possible delamination of multi-electrode sensor arrays. We found that the LVSEM environment had no detectable effect on the morphology of Na(+), K(+), and Ca(++) sensors, and EDX analysis indicated that all three membranes have similar compositions. However, once hydrated, the sensors exhibited different behaviors. The K(+) and Ca(++) sensors swelled more than the Na(+) sensor did. This swelling is due principally to water sorption in the membrane. We believe that the larger thickness of the K(+) and Ca(++) membrane is partly responsible for the observed swelling effect. A simple Griffith analysis of the interface rupture confirms the experimental evidence that these thicker membranes also are more prone to delamination failure. PMID- 10737873 TI - Third-body wear of cobalt-chromium-molybdenum implant alloys initiated by bone and poly(methyl methacrylate) particles. AB - The potential for bone and poly(methyl methacrylate) (PMMA) debris to initiate wear on ASTM-F75 and ASTM-F799 CoCrMo alloys articulating against ultrahigh molecular weight polyethylene (UHMWPE) was investigated. Third-body wear particles of bone and PMMA bone cement (with and without the radiopacifier, barium sulfate) were introduced between CoCrMo and UHMWPE in a reciprocating sliding wear test. A scanning electron microscope and a white light interference surface profilometer were used to study the surface damage and quantify the surface roughnesses of the worn alloys. The CoCrMo alloys, which are widely used as the femoral components in total artificial knees and hips, showed surface damage as the result of wear in the presence of bone or PMMA debris. Severe scratches were generated within 2700 cycles (94.5-m sliding distance) on the alloy's surface. Ploughing was the major wear mechanism. Carbides in the F75 alloy surface appeared to be unaffected by the debris. A quantitative study was performed on the surface roughness (average roughness, R(a), and root mean square roughness, RMS) of the alloy after wear testing. A nonparametric Wilcoxon rank sum test of wear severity (R(a) and RMS) was performed based on the surface roughness data. The surfaces of the specimens tested with the PMMA and bone particles were significantly rougher than those of the controls (p < 0.01). Small scratches also occurred on some of the control specimen surfaces and may have been second-body wear caused by defects and impurities in the UHMWPE. PMID- 10737874 TI - Polymer latexes for cell-resistant and cell-interactive surfaces. AB - Novel polymer latexes were prepared that can be applied in several ways for the control and study of cell behavior on surfaces. Acrylic latexes with glass transitions ranging from -30 to 100 degrees C were synthesized by dispersion polymerization in a water and alcohol solution using an amphiphilic comb copolymer as a stabilizing agent. The comb had a poly(methyl methacrylate) backbone and hydrophilic poly(ethylene glycol) (PEG) side chains, which served to stabilize the dispersion and create a robust hydrophilic coating on the final latex particles. The end groups of the comb stabilizer can be selectively functionalized to obtain latex particles with a controlled density of ligands tethered to their surfaces. Latexes were prepared with adhesion peptides (RGD) linked to the surface of the acrylic beads to induce attachment and spreading of cells. Coalesced films obtained from the RGD-bearing latex particles promoted attachment of WT NR6 fibroblasts, while films from unmodified latex particles were resistant to these cells. Additionally, RGD-linked beads were embedded in cell-resistant comb polymer films to create cell-interactive surfaces with discrete clustered-ligand domains. Cell attachment and morphology were seen to vary with the surface density of the RGD-bearing latex beads. PMID- 10737875 TI - Immobilization of PEO-PPO-PEO triblock copolymers on PTFE-like fluorocarbon surfaces. AB - Poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers were immobilized on thin fluorocarbon films with a structure very similar to poly(tetrafluoroethylene) (PTFE) using a microwave argon plasma. Fourier transform infrared attenuated total reflection and spectroscopic ellipsometry were used to study this process. After investigating the decomposition behavior of PEO-PPO-PEO triblock copolymers, we found that under appropriate plasma conditions, this material can be immobilized on a PTFE-like surface, while its general structure is preserved. PMID- 10737876 TI - Preparation and characterization of apatite deposited on silk fabric using an alternate soaking process. AB - Apatite-deposited silk fabric composite materials were developed using a new alternate soaking process. The characteristics of deposited apatite were studied using scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectrophotometry (FTIR), and X-ray photoelectron spectroscopy (XPS). Apatite weight increased with alternating soaking in a calcium solution [200 mM aqueous calcium chloride solution buffered with tris(hydroxymethyl) aminomethane and HCl (pH 7.4)] and a phosphate solution (120 mM aqueous disodium hydrogenphosphate) changed every hour. SEM showed that apatite deposited after 21 or more repeated soakings was over 20 microm thick. XRD showed that with alternate soakings, the apatite crystals deposited on silk fabric elongated along the c axis. FTIR and XPS indicated the existence of carbonate, HPO(4)(2-), and Na(+) ions in addition to constituent ions of hydroxyapatite. A loss of HPO(4)(2 ) and Na(+) ions in the deposit upon further soaking might be associated with an increasing apatite crystallinity. Apatite deposited on silk by the alternate soaking process was a deficient apatite containing carbonate, HPO(4)(2-), and Na(+) ions as in a natural bone tissue. Thus, this apatite-silk composite material might be potentially bioactive. PMID- 10737877 TI - Behavior of human osteoblastic cells on stoichiometric hydroxyapatite and type A carbonate apatite: role of surface energy. AB - To determine the role of physicochemical characteristics of the surface of dense ceramics on osteoconduction, we studied the proliferation and differentiation of human trabecular (HT) osteoblastic cells, extracellular collagenous matrix production, and biologic apatite formation on stoichiometric hydroxyapatite (HA) and type A carbonate apatite (CA). The surface physicochemical characteristics (composition, roughness) of HA and CA carefully were determined by Fourier transformed infrared, X-ray photoelectron, and Raman spectroscopies, and by FTIR microscopy, before and after cell culture. On both HA and CA substrates, HT cells attached, proliferated, and differentiated. Cell proliferation did not differ on HA and CA. However, the initial cell attachment and spreading of HT cells were much lower on CA compared to HA. Physicochemical and biologic analyses showed that collagenous synthesis by HT cells after 6 weeks of culture also was lower on CA than on HA. Quantitative histologic analysis confirmed that the collagenous matrix production was lower on CA than on HA. Measurement of wettability showed that the polar interaction energy with water was significantly lower on CA than on HA. The lower cell attachment and collagen production on CA compared to HA clearly were related to the low affinity of HT cells for the CA surface. This study shows that the surface energy of the biomaterial greatly influences the initial cell attachment and spreading of human osteoblastic cells at the surface and affects collagenous matrix deposition on the biomaterial. This suggests that the enhancement of polar components of the surface of dense biomaterials may improve osteoblastic cell attachment and, thereby, osteoconduction. PMID- 10737878 TI - Characterization of the inflammatory response to biomaterials using a rodent air pouch model. AB - Using a rodent air pouch, the inflammatory responses to biomaterials with distinct physical properties and chemical compositions were compared. The polymers examined were expanded poly(tetrafluoroethylene) (ePTFE), silicone, low density polyethylene (LDPE), poly(L-lactic acid) (PLLA), poly(desaminotyrosyl tyrosine ethyl carbonate) [poly(DTE carbonate)], and poly(desaminotyrosyl tyrosine benzyl carbonate) [poly(DTBzl carbonate)]. We found that implantation of disks (4.5-4.8 mm) of these materials into rodent air pouches for 2 days had no effect on the number or type of cells recovered relative to sham controls. With each of the materials, macrophages were the predominant cell type identified (60 75%), followed by granulocytes (20-25%) and lymphocytes (10%). Implantation of poly(DTE carbonate), ePTFE, LDPE, or poly(DTBzl carbonate) into the pouches for 2 days caused an increase in release of superoxide anion by the pouch cells. Cells from pouches containing poly(DTE carbonate) also released more hydrogen peroxide and were more phagocytic. In contrast, PLLA and silicone had no effect on the functional activity of cells recovered from the pouches. Prolonging the implantation time of poly(DTE carbonate) or PLLA to 7 days did not alter the number or type of cells isolated from the pouches. However, cells from pouches containing poly(DTE carbonate) for 7 days continued to produce increased quantities of superoxide anion relative to sham control pouch cells. These results suggest that the air pouch model is a highly sensitive method and therefore useful for evaluating the functional responses of inflammatory cells to biomaterials. PMID- 10737879 TI - Surface contact fatigue and flexural fatigue of dental restorative materials. AB - Antagonistic contact on a dental restoration may produce surface and subsurface stresses leading to fatigue wear as well as to bulk stressing, eventually causing catastrophic failure. It was the aim of the present work to study the outcome of two different approaches to fatigue testing of materials involving either surface contact fatigue or flexural fatigue mechanisms. A range of materials was tested, including conventional glass-ionomers, resin-modified glass-ionomers, poly-acid modified composites, and composites. Materials were prepared and tested using both surface contact and flexural fatigue. The results show that conventional glass-ionomers have the least resistance to fatigue under both regimes while composites have the longest fatigue lives and the highest values of flexural fatigue limit. However, the results also support the fact that catastrophic failure should be investigated separately from surface contact fatigue. Within the group of composite products tested, a hybrid composite material had a significantly greater flexural fatigue limit than a microfilled one, but the latter material had a significantly greater surface contact fatigue life, indicating that wear behavior cannot be predicted from bulk fracture characteristics and vice versa. The process of wear occurs by a combination of a number of fundamental processes, and the contribution fatigue makes will vary according to the environment and nature of the material. PMID- 10737880 TI - Enhanced wear performance of highly crosslinked UHMWPE for artificial joints. AB - It is well known that osteolysis induced by polyethylene wear debris is the main cause of long-term failure of hip and knee prostheses. We developed a treatment of medical-grade ultra-high molecular-weight polyethylene (UHMWPE) in order to improve its tribologic properties and reduce its wear. Medical-grade UHMWPE was irradiated with a 200 kGy dose of radiation, thermally stabilized at a temperature close to the melting point, and then sterilized with ethylene oxide. The irradiation treatment was performed to crosslink the UHMWPE. The thermal stabilization treatment, contributing to the reaction between the free radicals generated by the irradiation process, was chosen to enhance crosslinking and to prevent oxidation and the shortening of chains. The non-invasive sterilization process with ethylene oxide was chosen to prevent the re-formation of free radicals. The wear performance of this material was compared to UHMWPE, untreated or treated with different sterilization techniques, using gamma and beta irradiation. Insoluble crosslinked constituents were measured with an extraction method. Wear was evaluated using a flat-on-ring wear test machine. While small differences were found among the different sterilization processes, 200 kGy irradiated UHMWPE followed by thermal treatment and sterilization with ethylene oxide had the least wear and the greatest amount of crosslinking. PMID- 10737881 TI - Characterization of a polymeric PLGA-injectable implant delivery system for the controlled release of proteins. AB - Physico-chemical properties of injectable polymeric implant systems, based on the principle that a water-insoluble polymer dissolved in a biocompatible solvent will precipitate upon contact with water, were studied and utilized to predict the release of proteins from these systems. Polylactide-co-glycolide copolymer (PLGA) and glycofurol were chosen since they both have pharmaceutical precedence. Changes in polymer composition, its weight percent in solution, molecular weight, and protein loading level were assessed to provide formulations with the desired release rates and duration of release. PMID- 10737882 TI - In vivo biocompatibility of dextran-based hydrogels. AB - Dextran-based hydrogels were obtained by polymerization of aqueous solutions of methacrylated dextran (dex-MA) or lactate-hydroxyethyl methacrylate-derivatized dextran (dex-lactate-HEMA). Both nondegradable dex-MA and degradable dex-lactate HEMA disk-shaped hydrogels, varying in initial water content and degree of substitution (DS, the number of methacrylate groups per 100 glucose units), were implanted subcutaneously in rats. The tissue reaction was evaluated over a period of 6 weeks. The initial foreign-body reaction to the dex-MA hydrogels was characterized by infiltration of granulocytes and macrophages and the formation of fibrin, and exudate, as well as new blood vessels. This reaction depended on the initial water content as well as on the DS of the hydrogel and decreased within 10 days. The mildest tissue response was observed for the gel with the highest water content and intermediate DS. At day 21 all dex-MA hydrogels were surrounded by a fibrous capsule and no toxic effects on the surrounding tissue were found. No signs of degradation were observed. The initial foreign-body reaction to the degradable dex-lactate-HEMA hydrogels was less severe compared with the dex-MA gels. In general, the size of the dex-lactate-HEMA hydrogels increased progressively with time and finally the gels completely dissolved. Degradation of the dex-lactate-HEMA hydrogels was associated with infiltration of macrophages and the formation of giant cells, both of which phagocytosed pieces of the hydrogel. A good correlation between the in vitro and the in vivo degradation time was found. This suggests that extra-cellular degradation is not caused by enzymes but depends only on hydrolysis of the ester and/or carbonate bonds present in the crosslinks of the hydrogels. After 21 days, the degradable hydrogels, as such, could not be retrieved, but accumulation of macrophages and giant cells was observed, both of which contained particles of the gels intracellularly. As for the dex-MA hydrogels, no toxic effects on the surrounding tissue were found. The results presented in this study demonstrate that dextran based hydrogels can be considered as biocompatible materials, making these hydrogels attractive systems for drug delivery purposes. PMID- 10737883 TI - Alginate hydrogel linked with synthetic oligopeptide derived from BMP-2 allows ectopic osteoinduction in vivo. AB - Bone morphogenetic proteins (BMP) are unique molecules with a specific biological activity for inducing ectopic bone formation when implanted with a suitable carrier matrix. However, incorporation of BMP into the carrier has disadvantages, including early burst release and protein degradation in biological environments. Therefore, we considered that the next greatest challenge in achieving successful clinical use was the development of a carrier system for site-specific delivery of the morphogenetic signal of BMP. In this study, a novel BMP-2-derived oligopeptide, NSVNSKIPKACCVPTELSAI, was coupled covalently to alginate. Then NSVNSKIPKACCVPTELSAI-linked alginate hydrogel composites were implanted into the calf muscle of rats and harvested 3 or 8 weeks after surgery. Ectopic bone formation was observed in alginate hydrogel linked with BMP-2-derived peptide. It is suggested that alginate hydrogel linked with an oligopeptide derived from BMP 2 might provide an alternative system for topical delivery of the morphogenetic signal of BMP-2. PMID- 10737884 TI - Histomorphometric study on high-strength hydroxyapatite/poly(L-lactide) composite rods for internal fixation of bone fractures. AB - The purpose of this study was to investigate the bone-implant interface of high strength hydroxyapatite (HA)/poly(L-lactide) (PLLA) composite rods. As reinforcing particles, two types of HA particles-calcined HA (c-HA) and uncalcined HA (u-HA)-were applied to allow comparison of their suitability as bioactive fillers. Four types of composites (c-HA30, c-HA40, u-HA30, and u-HA40), which contained 30 or 40% by weight of each HA particle, were used. Unfilled PLLA rods were used as controls. A hole was drilled in the distal femora of 50 rabbits, and a composite or unfilled PLLA rod was implanted in a press-fit manner. Two, 4, 8, and 25 weeks after implantation, the samples were examined histologically by light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). An image analyzer was used for histomorphometric analysis of the bone-implant interface. An affinity index was calculated for each material; this was the length of bone directly apposed to the rods expressed as a percentage of the total length of the rod surface. In all the composites, histologic examination showed new bone formation at 2 weeks after implantation. The bone gradually grew along the composite surface. SEM showed direct bone contact with the composites without intervening fibrous tissue. During follow-up, the affinity indices of all the composite rods were significantly higher than those of the unfilled PLLA rods (p < 0.01; two-way ANOVA). The maximum affinity index (41%) was attained at 4 weeks in c-HA40 rods. In contrast, little bone contact was seen in unfilled PLLA rods. The only significant difference in affinity indices among the composites was that c-HA40 had a higher affinity index than u-HA40 (p < 0.05 at 4 weeks). No disintegration of rods or polymer debris, which could elicit inflammatory tissue reactions, was observed even at 25 weeks. Our results indicate that osteoconductive bone formation on composites could enhance the stability between bone and implant in fracture repair. PMID- 10737885 TI - Studies on small (<350 microm) alginate-poly-L-lysine microcapsules. V. Determination of carbohydrate and protein permeation through microcapsules by reverse-size exclusion chromatography. AB - Membrane molecular weight (MW) cut-off is a critical factor for immunoprotection of transplanted microencapsulated cells as well as for graft survival. Our goal was to study dextran and protein permeation through small (<350 microm in diameter) alginate-poly-L-lysine microcapsules made with an electrostatic system. Microcapsules were packed into a column, and gel-sieving chromatography was performed with proteins and dextrans of known MW. The objectives of this study were (1) to validate this approach for the assessment of the MW cut-off of <350 microm-in-diameter microcapsules and (2) to evaluate the effect on MW cut-off of changes in experimental conditions. Elution profiles of proteins suggest that the MW cut-off of our small microcapsules lies between 14,500 and 44,000 Da whereas dextrans > or =19,000 Da were excluded. The increase in poly-L-lysine (PLL) concentration from 0.02 to 0.08% reduced the MW cut-off. Increasing the PLL MW from 11.6 to 69.6 kDa induced no change in the MW cut-off. The results also show that the method can be used to discriminate between adsorption and absorption and that insulin diffuses freely across the microcapsule membrane. This method will be useful in establishing the ideal MW cut-off, in optimizing microcapsule characteristics, and in performing routine quality controls. PMID- 10737886 TI - Modulating fibroblast adhesion, spreading, and proliferation using self-assembled monolayer films of alkylthiolates on gold. AB - Ultrathin, highly organized functionalized alkylthiol monolayers were applied as model substrates for cell growth and protein adsorption studies. The aim of this approach was to improve the understanding of molecular surface determinants required for adhesion-dependent cell growth and proliferation using well controlled surface chemistry. Carboxyl- and methyl-terminated alkylthiol monolayers on gold were used to monitor Swiss 3T3 fibroblast adhesion, spreading, and growth. Stress fiber and focal contact formation were determined by immunostaining of actin filaments and paxillin. Fibronectin deposition and conformation on these surface chemistries in the presence and absence of competing proteins were also determined. The relative levels of adsorbed fibronectin were assessed using radiolabeled proteins. Exposure of the 10th type III cell integrin binding domain of fibronectin was assessed using a radiolabeled monoclonal antibody. Distinct alkylthiol substrate chemistry-dependent differences were observed in fibroblast adhesion, spreading, and growth. The formation of focal contacts and stress fibers was enhanced on the carboxyl terminated surface relative to the methyl surface. Relative deposition and conformations of adsorbed fibronectin were shown to be dependent on surface chemistry in both the presence and absence of competing proteins. The results indicated that well-controlled culture surfaces modulate differential cell adhesion, spreading, and growth through modulations of the amounts and conformations of adsorbed extracellular matrix molecules (e.g., fibronectin). PMID- 10737887 TI - Controlled release of transforming growth factor beta1 from biodegradable polymer microparticles. AB - Recombinant human transforming growth factor beta1 (TGF-beta1) was incorporated into biodegradable microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) at 6 ng/1 mg microparticles. Fluorescein isothiocynate labeled bovine serum albumin (FITC-BSA) was coencapsulated as a porogen at 4 microg/1 mg of microparticles. The effects of PEG content (0, 1, or 5 wt %) and buffer pH (3, 5, or 7.4) on the protein release kinetics and the degradation of PLGA were determined in vitro for up to 28 days. The entrapment yield of TGF-beta1 was 83.4 +/- 13.1 and 54.2 +/- 12.1% for PEG contents of 0 and 5%, respectively. The FITC-BSA and TGF-beta1 were both released in a multiphasic fashion including an initial burst effect. Increasing the PEG content resulted in the decreased cumulative mass of released proteins. By day 28, 3.8 +/- 0. 1 and 2.8 +/- 0.3 microg (based on 1 mg microparticles) of loaded FITC-BSA and 3.4 +/- 0.2 and 2.2 +/- 0.3 ng of loaded TGF-beta1 were released into pH 7.4 phosphate buffered saline (PBS) from microparticles with 0 and 5% PEG, respectively. Aggregation of FITC-BSA occurred at lower buffer pH, which led to decreased release rates of both proteins. For microparticles with 5% PEG, 2.3 +/- 0.1 microg of FITC-BSA and 2.0 +/- 0.2 ng of TGF-beta1 were released in pH 7.4 buffer after 28 days, while only 1.7 +/- 0.3 microg and 1.3 +/- 0.4 ng of the corresponding proteins were released in pH 3 buffer. The degradation of PLGA was also enhanced at 5% PEG content, which was significantly accelerated at acidic pH conditions. The calculated half-lives of PLGA were 20.3 +/- 0.9 and 15.9 +/- 1.2 days for PEG contents of 0 and 5%, respectively, in pH 7.4 PBS and 14.8 +/- 0.4 and 5.5 +/- 0.1 days for 5% PEG in pH 7.4 and 3 buffers, respectively. These results suggest that PLGA/PEG blend microparticles are useful as delivery vehicles for controlled release of growth factors. PMID- 10737888 TI - Effects of transforming growth factor beta1 released from biodegradable polymer microparticles on marrow stromal osteoblasts cultured on poly(propylene fumarate) substrates. AB - Recombinant human transforming growth factor beta1 (TGF-beta1) was incorporated into microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) to create a delivery vehicle for the growth factor. The entrapment efficiency of TGF-beta1 in the microparticles containing 5% PEG was 40.3 +/- 1.2% for a TGF-beta1 loading density of 6.0 ng/1 mg of microparticles. For the same loading, 17.9 +/- 0.6 and 32.1 +/- 2.5% of the loaded TGF-beta1 was released after 1 and 8 days, respectively, followed by a plateau for the remaining 3 weeks. Rat marrow stromal cells showed a dose response to TGF-beta1 released from the microparticles similar to that of added TGF-beta1, indicating the activity of TGF-beta1 was retained during microparticle fabrication and after TGF-beta1 release. An optimal TGF-beta1 dosage of 1.0 ng/mL was determined through a 3-day dose response study for maximal alkaline phosphatase (ALP) activity. The TGF-beta1 released from the microparticles loaded with 6.0 ng TGF-beta1/1 mg of microparticles for the optimal dosage of TGF-beta1 enhanced the proliferation and osteoblastic differentiation of marrow stromal cells cultured on poly(propylene fumarate) substrates. The cells showed significantly increased total cell number, ALP activity, and osteocalcin production with values reaching 138,700 +/- 3300 cells/cm(2), 22.8 +/- 1.5 x 10( 7) micromol/min/cell, and 15.9 +/- 1.5 x 10(-6) ng/cell, respectively, after 21 days as compared to cells cultured under control conditions without TGF-beta1. These results suggest that controlled release of TGF-beta1 from the PLGA/PEG blend microparticles may find applications in modulating cellular response during bone healing at a skeletal defect site. PMID- 10737889 TI - Limiting values for bacterial zeta potentials. PMID- 10737890 TI - DNA methylation and cancer. AB - The methylation of DNA is an epigenetic modification that can play an important role in the control of gene expression in mammalian cells. The enzyme involved in this process is DNA methyltransferase, which catalyzes the transfer of a methyl group from S-adenosyl-methionine to cytosine residues to form 5-methylcytosine, a modified base that is found mostly at CpG sites in the genome. The presence of methylated CpG islands in the promoter region of genes can suppress their expression. This process may be due to the presence of 5-methylcytosine that apparently interferes with the binding of transcription factors or other DNA binding proteins to block transcription. In different types of tumors, aberrant or accidental methylation of CpG islands in the promoter region has been observed for many cancer-related genes resulting in the silencing of their expression. How this aberrant hypermethylation takes place is not known. The genes involved include tumor suppressor genes, genes that suppress metastasis and angiogenesis, and genes that repair DNA suggesting that epigenetics plays an important role in tumorigenesis. The potent and specific inhibitor of DNA methylation, 5-aza-2' deoxycytidine (5-AZA-CdR) has been demonstrated to reactivate the expression most of these "malignancy" suppressor genes in human tumor cell lines. These genes may be interesting targets for chemotherapy with inhibitors of DNA methylation in patients with cancer and this may help clarify the importance of this epigenetic mechanism in tumorigenesis. PMID- 10737891 TI - Extracellular ATP-mediated phospholipase A(2) activation in rat thyroid FRTL-5 cells: regulation by a G(i)/G(o) protein, Ca(2+), and mitogen-activated protein kinase. AB - We investigated the mechanism of phospholipase A(2) (PLA(2)) activation in response to the P2 receptor agonist ATP in rat thyroid FRTL-5 cells. The PLA(2) activity was determined by measuring the release of [(3)H]-arachidonic acid (AA) from prelabeled cells. ATP evoked a dose- and time-dependent AA release. This release was totally inhibited by pertussis toxin (PTX) treatment, indicating the involvement of a G(i)/G(o) protein. The AA release was also diminished by chelating extracellular Ca(2+) with EGTA or by inhibiting influx of Ca(2+) using Ni(2+). Although the activation of protein kinase C (PKC) by 12-phorbol 13 myristate acetate (PMA) alone did not induce any AA release, the ATP-evoked AA release was significantly reduced when PKC was inhibited by GF109203X or by a long incubation with PMA to downregulate PKC. Both the ATP-evoked AA release and the mitogen-activated protein kinase (MAP kinase) phosphorylation were decreased by the MAP kinase kinase (MEK) inhibitor PD98059. Furthermore, the ATP-evoked MAP kinase phosphorylation was also inhibited by GF109203X and by downregulation of PKC, suggesting a PKC-mediated activation of MAP kinase. Inhibiting Src-like kinases by PP1 attenuated both the MAP kinase phosphorylation and the AA release. These results suggest that these kinases are involved in the regulation of MAP kinase and PLA(2) activation. Elevation of intracellular cAMP by TSH or by dBucAMP did not induce a phosphorylation of MAP kinase. Furthermore, neither the ATP-evoked AA release nor the MAP kinase phosphorylation were attenuated by TSH or dBucAMP. Taken together, our results suggest that ATP regulates the activation of PLA(2) by a G(i)/G(o) protein-dependent mechanism. Moreover, Ca(2+), PKC, MAP kinase, and Src-like kinases are also involved in this regulatory process. PMID- 10737892 TI - Hormonal regulation of [Ca(2+)](i) in periosteal-derived osteoblasts: effects of parathyroid hormone, 1,25(OH)(2)D(3) and prostaglandin E(2). AB - The effects of hormonal modulators of osteoblast function, parathyroid hormone, 1,25(OH)(2)D(3) and prostaglandins on [Ca(2+)](i) in periosteal-derived osteoblasts from rat femurs have been investigated. Our results show that application of parathyroid hormone PTH (10(-5) M) and prostaglandin E(2) (PGE(2)) (4 microM) result in a rapid heterogeneous elevation in [Ca(2+)](i) that, in the case of PTH, is dependent on both extracellular and intracellular sources of calcium. Variable responses to treatments have been found within populations of cells. The PGE(2) response is dose dependent. Treatment with 1,25(OH)(2)D(3) (10( 8) M) induces a brief (60-90 sec) elevation in [Ca(2+)](i) that is almost totally abolished in EGTA-buffered Ca(2+)-free medium. Interactive effects of multiple hormone treatments have been observed. Pretreatment with 1,25(OH)(2)D(3) results in near-total inhibition of the PTH and PGE(2) responses. In conclusion, modulation of [Ca(2+)](i) appears to play a role not only in the direct effects of osteotropic hormones on osteoblasts but also in the synergistic and antagonistic effects between circulating hormones. PMID- 10737893 TI - Expression and release of insulin-like growth factor binding proteins in isolated epiphyseal growth plate chondrocytes from the ovine fetus. AB - Insulin-like growth factor-II (IGF-II) is an autocrine modulator of epiphyseal chondrogenesis in the fetus. The cellular availability of IGFs are influenced by the IGF-binding proteins (IGFBPs). In this study, we investigated the control of expression and release of IGFBPs from isolated epiphyseal growth plate chondrocytes from the ovine fetus by hormones and growth factors implicated in the chondrogenic process. Chondrocytes were isolated from the proliferative zone of the fetal ovine proximal tibial growth plate and maintained in monolayer culture at early passage number. Culture media conditioned by chondrocytes under basal conditions released IGFBPs of 24, 34, and 29 kDa, and a less abundant species of 39-43 kDa that were identified immunologically as IGFBP-4, IGFBP-2, IGFBP-5, and IGFBP-3, respectively. Messenger RNAs encoding each species were identified by Northern blot analysis within chondrocytes, as was mRNA encoding IGFBP-6. Exposure to IGF-I or IGF-II (13 or 26 nM) caused an increase in expression and release of IGFBP-3. The release of IGFBP-2 and IGFBP-5 were also potentiated without changes to steady state mRNA, and for IGFBP-5 this was due in part to a release from the cell membrane in the presence of IGF-II. Insulin (16.7 or 167 nM) selectively increased mRNA and the release of IGFBP-3, while cortisol (1 or 5 microM) inhibited both mRNA and release of IGFBP-2 and IGFBP-5. Transforming growth factor-beta1 (TGF-beta1) (0.1 or 0.2 nM) increased the expression and release of IGFBP-3, and caused an increase in mRNAs encoding IGFBP 2 and IGFBP-5. Neither growth hormone (GH), fibroblast growth factor-2, nor thyroxine (T(4)) had any effect on IGFBP expression or release. The results suggest that IGFBP expression and release within the developing growth plate can be modulated by IGF-II and other trophic factors, thus controlling IGF availability and action. PMID- 10737894 TI - Interaction between CD44 and the repeat domain of ankyrin promotes hyaluronic acid-mediated ovarian tumor cell migration. AB - The adhesion molecule, CD44, interacts with ankyrin within its cytoplasmic domain and binds to hyaluronic acid (HA) at its extracellular domain. In this study, we focused on the functional domain in ankyrin (in particular, the ankyrin repeat domain [ARD]) responsible for CD44 binding and its role in regulating HA-mediated ovarian tumor cell function. Using recombinant fragments of ankyrin (e.g., ARD and subdomain 1 [S1, aa1-aa217], subdomain 2 [S2, aa218-aa381], subdomain 3 [S3, aa382-aa612], and subdomain 4 [S4, aa613-aa834]) and in vitro binding assays, we determined that the S2 but not S1, S3, or S4 of ARD is the primary ankyrin binding region for CD44. Microinjection of antiglutathione S-transferase (GST) tagged S2 or GST-tagged ARD fusion protein into CD44-positive ovarian tumor cells (e.g., SKOV3 cell line) promotes ankyrin association with CD44 in plaque-like structures and membrane projections. Additionally, we demonstrated that transfection of SKOV3 cells with S2cDNA or ARD cDNA results in an upregulation of HA-mediated tumor cell migration. Taken together, we believe that the S2 of the ARD plays a pivotal role in the direct binding to CD44 and promotes the cytoskeleton activation required for HA-mediated function such as ovarian tumor cell migration. PMID- 10737895 TI - CKbeta-8 [CCL23], a novel CC chemokine, is chemotactic for human osteoclast precursors and is expressed in bone tissues. AB - We have previously demonstrated that a tartrate-resistant acid phosphatase (TRAP) positive subpopulation of mononuclear cells isolated from collagenase digests of human osteoclastoma tissue exhibits an osteoclast phenotype and can be induced to resorb bone. Using these osteoclast precursors as a model system, we have assessed the chemotactic potential of 16 chemokines. Three CC chemokines, the recently described CKbeta-8, RANTES, and MIP-1alpha elicited significant chemotactic responses. In contrast, 10 other CC chemokines (MIP-1beta, MCP-1, MCP 2, MCP-3, MCP-4, HCC-1, eotaxin-2, PARC, SLC, ELC) and 3 CXC chemokines (IL-8, GROalpha, SDF-1) were inactive. None of these chemokines showed any chemotactic activity for either primary osteoblasts derived from human bone explants or the osteoblastic MG-63 cell line. The identity of the osteoclast receptor that mediates the chemotactic response remains to be established. However, all three active chemokines have been reported to bind to CCR1 and cross-desensitization studies demonstrate that RANTES and MIP-1alpha can partially inhibit the chemotactic response elicited by CKbeta-8. CKbeta-8, the most potent of the active CC chemokines (EC(max) 0.1-0.3 nM), was further characterized with regard to expression in human bone and cartilage. Although expression is not restricted to these tissues, CKbeta-8 mRNA was shown to be highly expressed in osteoblasts and chondrocytes in human fetal bone by in situ hybridization. In addition, CKbeta-8 protein was shown to be present in human osteophytic tissue by immunolocalization. These observations suggest that CKbeta-8, and perhaps other chemokines, may play a role in the recruitment of osteoclast precursors to sites of bone resorption. PMID- 10737896 TI - Tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin does not require extracellular signal-regulated kinase activation in Swiss 3T3 cells stimulated by bombesin or platelet-derived growth factor. AB - The experiments presented here were designed to examine the contribution of the extracellular signal-regulated mitogen-activated protein kinases (ERKs) to the tyrosine phosphorylation of the focal adhesion proteins p125(Fak), p130(Cas), and paxillin induced by G protein-coupled receptors (GPCRs) and tyrosine kinase receptors in Swiss 3T3 cells. Stimulation of these cells with bombesin, lysophosphatidic acid (LPA), endothelin, and platelet-derived growth factor (PDGF) led to a marked increase in the tyrosine phosphorylation of these focal adhesion proteins and in ERK activation. Exposure of the cells to two structurally unrelated mitogen-activated protein kinase or ERK kinase (MEK) inhibitors, PD98059 and U0126, completely abrogated ERK activation but did not prevent tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin. Furthermore, different dose-response relationships were obtained for tyrosine phosphorylation of focal adhesion proteins and for ERK activation in response to PDGF. Putative upstream events in the activation of focal adhesion proteins including actin cytoskeletal reorganization and myosin light chain (MLC) phosphorylation were also not prevented by inhibition of ERK activation. Thus, our results demonstrate that the activation of the ERK pathway is not necessary for the increase of the tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin induced by either GPCRs or tyrosine kinase receptors in Swiss 3T3 cells. PMID- 10737897 TI - Suppression of malignant growth potentials of v-Src-transformed human gallbladder epithelial cells by adenovirus-mediated dominant negative H-Ras. AB - Although Src transformation of NIH3T3 mouse fibroblasts has been shown to be dependent on Ras function, the signaling mechanism whereby Src induces malignant transformation of human epithelial cells still remains unclear. In the present study, we analyzed the functional role of Ras, which acts downstream of Src in intracellular signaling, in the acquisition of fully neoplastic potentials by v Src-transformed human gallbladder epithelial cells (HAG/src3-1) by infecting these cells with replication-defective adenovirus vector expressing dominant negative H-Ras (AdCARasY57). High efficiency of gene transduction was demonstrated with the adenovirus vector containing beta-gal gene insert (AdCALacZ). On infection with AdCARasY57, the activity of mitogen-activated protein (MAP) kinase, a major downstream event triggered by Ras, was markedly inhibited over 7 days, indicating that the inhibition of Ras function by AdCARasY57 remains active during this period. AdCARasY57 did not inhibit the monolayer growth of HAG-1 cells transfected with activated H-ras, but inhibited the HAG/src3-1 cells by 30%, as compared with cells infected with AdCALacZ as a control. This growth inhibition by AdCARasY57 was strengthened nearly twofold on surfaces coated with an antiadhesive polymer (poly 2-hydroxyethylmethacrylate) that can quantitate anchorage-independent growth, and was much more pronounced up to 95% when assayed in soft agar. The HAG/src3-1 cells transfected with beta-gal gene produced tumors in nude mice within 4 weeks after implantation, whereas cells infected with AdCARasY57 failed to form tumors during this period. These findings show that Ras function is essential for v-Src-induced anchorage independent growth in vitro as well as tumorigenesis in vivo, and that mitogenic activity driven by v-Src is not solely dependent on MAP kinase pathway. Because anchorage-independent growth correlates with tumor growth in vivo as well as metastatic potential, targeting Ras would be potentially useful for the treatment of human tumors with elevated Src tyrosine kinase activity. PMID- 10737898 TI - Developmental expression and assembly of connexins into homomeric and heteromeric gap junction hemichannels in the mouse mammary gland. AB - During the development of the mammary gland, duct-lining epithelial cells progress through a program of expansive proliferation, followed by a terminal differentiation that allows for the biosynthesis and secretion of milk during lactation. The role of gap junction proteins, connexins, in the development and function of this secretory epithelium was investigated. Connexins, Cx26 and Cx32, were differentially expressed throughout pregnancy and lactation in alveolar cells. Cx26 poly-(A)(+) RNA and protein levels increased from early pregnancy, whereas Cx32 was detectable only during lactation. At this time, immunolocalization of connexins by confocal microscopy and immunogold labeling of high-pressure frozen freeze-substituted tissue showed that both connexins colocalized to the same junctional plaque. Analysis of gap junction hemichannels (connexons) isolated from lactating mammary gland plasma membranes by a rate density centrifugation procedure, followed by immunoprecipitation and by size exclusion chromatography, showed that Cx26 and Cx32 were organized as homomeric and heteromeric connexons. Structural diversity in the assembly of gap junction hemichannels demonstrated between pregnant and lactating mammary gland may account for differences in ionic and molecular signaling that may physiologically influence the onset and/or maintenance of the secretory phenotype of alveolar epithelial cells. PMID- 10737899 TI - p53/56(lyn) antisense shifts the 1,25-dihydroxyvitamin D3-induced G1/S block in HL60 cells to S phase. AB - p53/56(lyn) is a member of the src family that is predominantly expressed in hematopoietic cells and is thought to play a role in cellular proliferation. In this study, we demonstrate the participation of p53/56(lyn) in 1,25 dihydroxyvitamin D(3) (1, 25D(3))-induced growth arrest in HL60 cells. We show that the mRNA and protein levels of p53/56(lyn) are markedly elevated after 1, 25D(3) treatment, which is accompanied by an increase of p53/56(lyn) kinase activity. We also demonstrate that treatment with p53/56(lyn) antisense oligodeoxynucleotides reverses the 1,25D(3)-induced G1/S block, and results in an accumulation of cells with S-phase DNA content. BrdU pulse-chase experiments reveal that this accumulation results from an increased proportion of cells actively synthesizing DNA, which are inhibited from exiting the S-phase compartment. These results indicate that upregulation of p53/56(lyn) contributes significantly to the G1/S growth arrest induced by 1,25D(3) in HL60 cells and thus its activation may be a desirable outcome of chemotherapeutic regimens. PMID- 10737900 TI - Delay of M-phase onset by aphidicolin can retain the nuclear localization of zinc and metallothionein in 3T3-L1 fibroblasts. AB - The transient nuclear localization of metallothionein during cell growth and differentiation may be related to the increased requirement of zinc for DNA synthesis, activation of metalloenzymes, and transcription factors. Treatment of 3T3-L1 fibroblasts with aphidicolin, an inhibitor of nuclear DNA synthesis, caused a cell-cycle block at G1/S phase and a delay in the onset of M phase. This also resulted in the accumulation of both zinc and metallothionein in the nucleus. After removal of aphidicolin, the cells rapidly reentered S phase, and during the G2/M phase of cell cycle both zinc and metallothionein began to relocate to the cytoplasm. Delaying the onset of M phase in 3T3-L1 cells could prevent the cytoplasmic relocation of metallothionein. The nuclear translocation of both zinc and metallothionein during the cell cycle can be considered as a normal process and this may be a general mechanism in response to mitogenic signals. PMID- 10737901 TI - Effect of platelet-activating factor receptor expression on CHO cell motility. AB - Tumor cell migration may favor local mass expansion and metastasis dissemination. Several tumors were found to express the receptor for platelet-activating factor (PAF), a potent mediator of leukocyte chemotaxis and endothelial cell migration. However, its functional role on tumor cells is largely unexplored. In the present study, we evaluated the motogenic effect of PAF on Chinese hamster ovarian (CHO) cancer cells transfected with the human PAF-receptor cDNA (CHO PAF-R). By using time-lapse recording, we detected a rapid motogenic response to PAF stimulation on CHO PAF-R, whereas no effect was evident on vector-only transfected cells. Such an effect was observed on scattered cell motility, on cells seeded on a fibronectin- or collagen-coated surface, and on migration of confluent monolayer cells. Cell speed increased at 1 h and was maximal 6-8 h after PAF stimulation on CHO PAF-R. Concomitantly, PAF induced marked changes in cytoskeleton actin distribution with cell contraction, assembling of stress fibers, and polar foci of adhesion. In conclusion, the present study demonstrates that PAF is a potent inducer of tumor cell motility, thus suggesting a role for this mediator in tumor growth and dissemination. PMID- 10737902 TI - Epidermal growth factor signaling pathway influences retinoid metabolism by reduction of retinyl ester hydrolase activities in normal and malignant keratinocytes. AB - The effects of EGFR signaling on retinol metabolism were evaluated in the squamous cell carcinoma cell lines defective in LRAT. In a 24-h incubation, the presence of EGF resulted in a 20-25% increase in retinyl ester accumulation. Assessment of retinol esterification and retinyl ester utilization (hydrolysis), in cell cultures and in cell homogenates, revealed that the increase in retinyl ester mass was the result of a reduction in retinyl ester hydrolysis. When grown in the absence of EGF, the cultures used about 40% of their retinyl esters, compared to about 21% in cultures grown with EGF. This effect of EGF was blocked by an EGF receptor-neutralizing antibody, an EGF receptor tyrosine-kinase inhibitor (PD153035), and a specific inhibitor of MEK kinase influencing the mitogen-activated protein kinase (MAPK) cascade (PD98059). Both transcription and translation were required, suggesting that signaling from the EGF receptor through the MAPK cascade controls the expression of modulators or inhibitors of the retinyl ester hydrolase(s). Thus EGFR signaling can alter the intracellular concentration of retinol by suppressing the access to the retinyl ester pool. Similar EGF effects were seen in cultures of normal keratinocytes. PMID- 10737903 TI - Insulin-like growth factor binding protein-3 mediates IGF-I action in a bovine mammary epithelial cell line independent of an IGF interaction. AB - IGF-I is mitogenic for the bovine mammary epithelial cell line MAC-T. In addition, IGF-I specifically upregulates IGFBP-3 synthesis in these cells. To investigate this effect on cell growth and IGF-I responsiveness, cell lines were developed that constitutively express IGFBP-3. MAC-T cells transfected with IGFBP 3 (+BP3) or vector alone (Mock) grew similarly over 7 days in 10 or 1% fetal calf serum. Basal DNA synthesis was lower (70%) in +BP3 cells compared to Mock cells. However, DNA synthesis was increased by IGF-I (1-50 ng/ml) relative to untreated controls to a greater extent in +BP3 cells compared to Mock cells. IGF-I (20 ng/ml) increased DNA synthesis 11- and threefold in +BP3 and Mock cells, respectively. Additionally, +BP3 cells were more sensitive to the lower concentrations of IGF-I (1-5 ng/ml). In contrast, preincubation of Mock cells with exogenous IGFBP-3 did not enhance responsiveness or sensitivity to IGF-I. Basal DNA synthesis was unaffected by either an IGF neutralizing antibody or exogenous IGFBP3, indicating the differences observed between +BP3 and Mock cells were not attributable to sequestration of endogenous IGF-I by IGFBP-3. There were no differences between +BP3 and Mock cells in IGF-I receptor number or affinity. DNA synthesis was also increased in +BP3 cells, compared to controls, in response to 5 microg/ml insulin and 2.5 ng/ml Long R(3)IGF-I, indicating that the potentiated response did not require an interaction with IGFBP-3. These results suggest that IGF-I regulation of IGFBP-3 represents a regulatory loop, the function of which is to increase IGF-I bioactivity, using a mechanism that does require an IGF-I-IGFBP-3 interaction. PMID- 10737904 TI - New dimensions in cancer biology and therapy. AB - The most successful and productive approach to defeat cancer relies on highly integrated and interchanging cooperation between basic research, diagnosis, and therapeutic innovation. Nevertheless, much remains to be done to achieve a consistent and continuous flow of theoretical and practical information among scientists actively involved in these equally relevant fields. The major objective of the International Conferences, "New Dimensions in Cancer Biology and Therapy," has been identified in gathering together basic scientists and clinicians who represent scientific leaders in their field, whose working efforts are focused on specific human malignancies. Thus, in pursuit of this well-defined goal, the third edition of the Conference has focused on human immunodeficiency virus (HIV) and cancer, cutaneous melanoma, and colorectal carcinoma, which are ideal clinical examples for innovative diagnostic and therapeutic intervention, as well as optimal models to unveil new mechanisms of tumor pathogenesis. PMID- 10737905 TI - Intestinal permeation enhancers. AB - This review addresses the field of improving oral bioavailability through the use of excipients that increase intestinal membrane permeability. The critical issues to consider in evaluating these approaches are 1) the extent of bioavailability enhancement achieved, 2) the influence of formulation and physiological variables, 3) toxicity associated with permeation enhancement, and 4) the mechanism of permeation enhancement. The categories of permeation enhancers discussed are surfactants, fatty acids, medium chain glycerides, steroidal detergents, acyl carnitine and alkanoylcholines, N-acetylated alpha-amino acids and N-acetylated non-alpha-amino acids, and chitosans and other mucoadhesive polymers. Some of these approaches have been developed to the stage of initial clinical trials. Several seem to have potential to improve oral bioavailabilities of poorly absorbed compounds without causing significant intestinal damage. In addition, the possible use of excipients that inhibit secretory transport is reviewed. PMID- 10737906 TI - Chemical stability of nucleic acid-derived drugs. AB - Nucleic acid-derived drugs exhibit both chemical and physical instability. This mini-review focuses on the prevalent hydrolytic and oxidative pathways of chemical degradation as they are affected by various endogenous (primary structure, chemical modifications in bases, sugars and phosphate residues) and exogenous (pH, buffer concentration, metal cation presence, oxygen presence) factors. PMID- 10737907 TI - Energy/temperature diagram and compression behavior of the polymorphs of D mannitol. AB - Three modifications of D-mannitol were produced and investigated: mod. I (mp 166.5 degrees C, heat of fusion 53.5 kJ mol(-1)), mod. II (mp 166 degrees C, heat of fusion 52.1 kJ mol(-1)), and mod. III (mp incongruent 150-158 degrees C, heat of transition, III to I 0.2 kJ mol(-1)). The measured densities are 1.490 +/- 0.000 g cm(-3) [95% confidence interval (CI)] for mod. I, 1.468 +/- 0.002 g cm( 3) (95% CI) for mod. II, and 1.499 +/- 0.004 g cm(-3) (95% CI) for mod. III. It was possible to relate the different modifications given in the literature to one of the three pure crystal forms or to mixtures of two or all three modifications. The thermodynamic relationship among the crystal forms is represented in a semi schematic energy/temperature diagram. From these data we can conclude that mod. III is thermodynamically stable at absolute zero. It is enantiotropically related to mod. I and mod. II. FTIR and Raman spectra, differential scanning calorimetry curves, and X-ray powder patterns of these crystal forms are depicted for doubtless assignment in the future. The water uptake of the three modifications at 92% relative humidity and 25 degrees C is less than 1%. The differences of the heat capacities and the heats of solution between mod. II and III are not significant, whereas mod. I shows small significant differences compared with the other modifications. In addition, compaction studies of these crystal forms were performed by means of an instrumented hydraulic press. The results show that mod. III should have the best tableting behavior under these conditions. PMID- 10737908 TI - Plasma pharmacokinetics and tissue biodistribution of boron following administration of a boronated porphyrin in dogs. AB - This study was undertaken to determine the plasma pharmacokinetics and tissue biodistribution of boron in dogs following the administration of a boronated porphyrin (BOPP) compound, a potential sensitizing agent for binary therapies of cancer. An intravenous dose of 35 mg/kg of BOPP was administered to a total of sixteen dogs and plasma samples obtained at multiple time points for up to 28 days after administration. Groups of four dogs each were studied for 25, 79, 240, and 672 h. At the end of each study period, subjects were sacrificed and tissue samples obtained. Boron concentrations were determined for all tissue and plasma samples, and pharmacokinetic parameters were determined using mixed effects modeling. Plasma boron levels displayed triexponential kinetics with a long terminal half-life and small volume of distribution. Liver, lymph node, adrenal, and kidney tissues accumulated the highest levels of boron, with very low levels associated with most tissues of the head. We conclude that BOPP has pharmacokinetic and tissue distribution properties that suggest that it may be a suitable compound for use as a sensitizing agent in binary therapy of cancer. PMID- 10737909 TI - Structure and solid-state chemistry of anhydrous and hydrated crystal forms of the trimethoprim-sulfamethoxypyridazine 1:1 molecular complex. AB - The crystal structure of the equimolar trimethoprim (TMP) and sulfamethoxypyridazine (SMPD) complex in the anhydrous form (TMP. SMPD) and that of the species with 1.5 molecules of water of crystallization (TMP.SMPD.W) are reported in this article. X-ray powder diffraction patterns (both computer generated and experimental) and thermal analytical data from differential scanning calorimetry (DSC) and thermogravimetry useful for the characterization of TMP.SMPD and TMP.SMPD.W are provided. The stability of TMP.SMPD.W, which retains its crystallographic order under 0% relative humidity (RH) conditions at room temperature (22 degrees C) and 20 mmHg, is accounted for in terms of crystal structure and hydrogen bonding. Transformation of TMP.SMPD to the hydrate complex by exposure to approximately 100% RH, suspension in water, and wet granulation, and dehydration of TMP.SMPD.W by thermal treatment and by desiccation with methanol were investigated and tentatively interpreted in terms of crystal properties. Interactions in the physical mixture of TMP and SMPD by grinding, compression, heating, and contact with water were also studied. Water-mediated formation of TMP.SMPD.W by wetting and metastable eutectic melting-mediated formation of TMP.SMPD by heating was demonstrated. Mechanical activation by milling makes the physical mixture prone to solid-state transformation into dimorphic anhydrous cocrystals by supply of thermal energy during a DSC scan. PMID- 10737910 TI - Effect of alpha(1)-acid glycoprotein on the pharmacokinetics of tamsulosin in rats treated with turpentine oil. AB - The pharmacokinetics of tamsulosin (TAM) was investigated using male Sprague Dawley rats in which plasma alpha(1)-acid glycoprotein (alpha(1)-AGP) levels were elevated by the subcutaneous injection of 0.2 mL/kg of turpentine oil. alpha(1) AGP levels increased about eight times after turpentine oil treatment, causing a threefold decrease in plasma unbound fraction (f(u)) of TAM. When 0.3 mg/kg of TAM was dosed intravenously, total and nonrenal clearances (CL(tot) and CL(nr)) in turpentine-treated rats were 47% and 44% lower than those in nontreated controls, respectively. The area under the concentration-time curve of plasma unbound TAM (AUC(inf,u)) was lower than that in the control. When 1 mg/kg of TAM was dosed orally, oral clearance (CL(oral)) in alpha1-AGP-induced rats was 65% lower than in the control. The AUC(inf,u) and unbound oral clearance (CL(oral,u)) were nearly equal in both groups. Moreover, a positive correlation was observed between fu and CL(oral) of TAM (r(2) = 0.603, P < 0.01), whereas no correlation was observed between f(u) and CL(oral,u). The absolute bioavailability (BA) increased from 19.2% to 46.9% by induction of alpha(1)-AGP. These results suggest that decreased f(u) caused by the elevation of plasma alpha(1)-AGP level affects the pharmacokinetics of TAM, but does not affect the CL(oral,u,) which represents the hepatic metabolism of TAM. PMID- 10737911 TI - Biodistribution, metabolism, and in vivo gene expression of low molecular weight glycopeptide polyethylene glycol peptide DNA co-condensates. AB - The biodistribution, metabolism, cellular targeting, and gene expression of a nonviral peptide DNA gene delivery system was examined. (125)I-labeled plasmid DNA was condensed with low molecular weight peptide conjugates and dosed i.v. in mice to determine the influence of peptide DNA formulation parameters on specific gene targeting to hepatocytes. Optimal targeting to hepatocytes required the combined use of a triantennary glycopeptide (Tri-CWK(18)) and a polyethylene glycol-peptide (PEG-CWK(18)) to mediate specific recognition by the asialoglycoprotein receptor and to reduce nonspecific uptake by Kupffer cells. Tri-CWK(18)/PEG-CWK(18) DNA co-condensates were stabilized and protected from metabolism by glutaraldehyde crosslinking. An optimized formulation targeted 60% of the dose to the liver with 80% of the liver targeted DNA localized to hepatocytes. Glutaraldehyde crosslinking of DNA condensates reduced the liver elimination rate from a t((1/2)) of 0.8 to 3.6 h. An optimized gene delivery formulation produced detectable levels of human alpha1-antitrypsin in mouse serum which peaked at day 7 compared to no expression using control formulations. The results demonstrate the application of formulation optimization to improve the targeting selectivity and gene expression of a peptide DNA delivery system. PMID- 10737912 TI - Absolute structure determination of the highly biologically active bisdehydrodoisynolic acids. AB - In a project designed to relate the unexpected in vivo and in vitro properties exhibited by (+)- and (-)-bisdehydrodoisynolic acid with their absolute stereochemical structure, an X-ray crystal-structure analysis was undertaken of the highly estrogenic, poorly binding (-) enantiomer. (1) and (13)C NMR spectra are also reported for the first time. The crystal structure shows the cis juxtaposition of the carboxyl and ethyl groups, which are separated by a large torsion angle, and that only the carbon atom holding the carboxyl group is out of the plane in which the remainder of the fused three-ring moiety lies. The crystal structure, which unequivocally characterizes the (-) enantiomer as cis 13(S),14(R) and, implicitly, the (+) enantiomer as cis-13(R),14(S), will be useful in continued studies aimed at explaining the selective estrogen receptor modulation (SERM) of these enantiomers which, in some cases, produces significantly different end-organ effects compared to those of estradiol, in both males and females, affording the promise of a variety of therapeutic and pharmacologic applications. PMID- 10737913 TI - Influence of cardiac output on dexmedetomidine pharmacokinetics. AB - Dexmedetomidine, a highly selective alpha(2)-adrenoceptor agonist, reduces the requirements for anesthetic, analgesic, sedative, and hypnotic drugs. Dexmedetomidine pharmacokinetics were characterized in healthy subjects after intravenous administration by means of a computer-controlled infusion pump. A series of seven stepwise increasing pseudo-steady-state plasma concentrations were targeted. The influence of cardiac output on the pharmacokinetics was investigated by use of a compartmental modeling approach in which the elimination clearance was characterized as being either cardiac output independent or dependent. At dexmedetomidine concentrations of 0, 0.6, and 1.2 ng/mL, mean (SD) estimated cardiac outputs were 5. 6 (0.85), 5.1 (0.67), and 4.5 (0.83) L/min, and mean (SD) clearances were 40 (10), 38 (9.0), and 35 (8.5) L/h, respectively. Dexmedetomidine V(SS) and elimination half-life were 72 (19) L and 1. 9 (0.62) h, respectively. The approximately 3 to 19% decrease in cardiac output observed within the anticipated therapeutic range of 0.3 to 1.2 ng/mL was similar to that observed for clonidine. The decrease in cardiac output with increasing plasma concentrations of dexmedetomidine resulted in a corresponding decrease in drug elimination clearance of < or =12% within the therapeutic range; however, this decrease in dexmedetomidine clearance is likely not clinically relevant. PMID- 10737914 TI - Transdermal drug delivery using electroporation. I. Factors influencing in vitro delivery of terazosin hydrochloride in hairless rats. AB - The use of electroporation pulses as a physical means of enhancing the permeability of skin to deliver drugs is in the early stages of development. In this article, a systematic study examining the parameters influencing electroporative transdermal delivery of terazosin hydrochloride to hairless rat skin are reported. It was found that voltage, pulse length (tau), and number of pulses were the three most important parameters, in that order. For creating a significant enhancement in drug delivery to the skin, without causing any apparent change in its external appearance, it was necessary to deliver five or more exponentially decaying electroporation pulses, at 88 +/- 2.5 V (voltage across the skin), with a decay time constant of 20 ms. Electrodes with larger area could attain the same voltages across the skin with a much lower applied voltage and possessed other advantages with regard to performance of the drug delivery system. PMID- 10737915 TI - Transdermal drug delivery using electroporation. II. Factors influencing skin reversibility in electroporative delivery of terazosin hydrochloride in hairless rats. AB - A previous study indicated that the parameters governing the performance of electroporative delivery to the skin, are voltage, pulse length, number of pulses and electrode area.1 This article describes a study in which the reversibility of the electroporation technique is evaluated with in vitro methods. The skin's reversal from an enhanced permeation mode as a result of electroporation to the base level was used as an index to understand the mechanism of drug delivery and also as a preliminary indicator of safety. Maximum delivery of the model drug, terazosin hydrochloride, occurred during the pulsing. Electroporative delivery with a wire electrode (small-area electrode, 0.56 cm(2)) using 20 pulses at U(skin,0) 88 V, and pulse length 20 ms, did not cause any damage to the skin. Increasing the pulse length to 60 ms, while keeping the rest of the parameters fixed, caused a visible change in the external appearance of the skin. However, with the use of a spiral electrode (large-area electrode, 2.74 cm(2)) at 60-ms pulse length, there was minimal damage to the skin. This may be attributed to the more uniform flow of current over the whole skin area. The large-area electrode required a smaller electrode voltage, U(electrode,0) for any given U(skin,0) and also delivered nearly double the instantaneous power density compared with the small-area electrode. These findings indicate that using shorter pulses and large area electrodes is a safer technique than large pulses and small-area electrodes when electroporation is used to enhance skin's permeability for drug delivery. PMID- 10737916 TI - The hexadentate hydroxypyridinonate TREN-(Me-3,2-HOPO) is a more orally active iron chelator than its bidentate analogue. AB - Bidentate hydroxypyridinone chelators effectively complex and facilitate excretion of trivalent iron. To test the hypothesis that hexadentate chelators are more effective than bidentate chelators at low concentrations, urinary and biliary Fe excretions were determined in Fe-loaded rats before and after administration of a bidentate chelator, Pr-(Me-3,2-HOPO), or its hexadentate analogue, TREN-(Me-3,2-HOPO). The bidentate chelator slightly increased biliary Fe excretion in Fe-loaded rats after IV (90 micromol/kg) and PO (90 or 270 micromol/kg) administration, but chelation efficiency did not exceed 1%. The hexadentate chelator markedly increased biliary Fe excretion, achieving overall chelation efficiencies of 14% after IV administration of 30 micromol/kg and 8 or 3% after PO (30 or 90 micromol/kg) administration. The hexadentate chelator was significantly more effective than the bidentate chelator after IV injection and oral dosing. In chelator-treated Fe-loaded or saline-injected rats, >90% of the excreted Fe was in the bile. Oral TREN-(Me-3,2-HOPO), given to non-Fe-loaded rats, did not appreciably change Fe output, indicating that there was little Fe depletion in the absence of Fe overload. These results support the hypothesis that greater Fe chelation efficiency can be achieved with hexadentate than with bidentate chelators at lower, and presumably safer, concentrations. The results also demonstrate that TREN-(Me-3, 2-HOPO) is a promising, orally effective, Fe chelator. PMID- 10737917 TI - Evaluation of skin damage caused by percutaneous absorption enhancers using fractal analysis. AB - Fractal analysis of the cross-sectional morphology of rat skin was conducted to evaluate pathologic changes evoked by percutaneous absorption enhancers. Male hairless rats (WBN/Ht-ILA), 8 weeks old, weighing 160 to 180 g were used. Under anesthetization, glass cells (10-mm inner diameter) were attached to the rats' abdomens, and test solutions containing various mixtures of the percutaneous absorption enhancers, sodium lauryl sulfate, isopropanol, 2-methyl-1-butanol, and sodium myristate were applied. Six hours after application, the solutions were removed and the abdominal skin was excised. Skin cross sections were analyzed with a charge-coupled device (CCD) camera. Image data taken by the CCD camera were fed into a desktop digital computer; then the fractal dimension of each skin cross section was determined on the basis of the box-counting algorithm. A pathologic study was also performed on the skin treated with the test solution. All sections of skin were examined with an optical photo microscope. Pathologic findings were classified into five levels. The total irritation score (TIS) was defined as the summation of damage levels in all regions. Only with the administration of hydrogel containing 2-methyl-1-butanol or sodium lauryl sulfate were positive values of TIS observed. However, the TIS values were independent of the concentration of these components. The most severe skin damage was evoked by application of sodium lauryl sulfate. Noticeable skin damage was also seen with 2 methyl-1-butanol. No irritation to the skin resulted from treatment with isopropanol or sodium myristate. When test solution containing sodium lauryl sulfate was applied to the skin, a remarkable increment in fractal dimensions was noted. This may suggest that the structure of the skin was greatly compromised as a result of sodium lauryl sulfate application. Although no change in fractal dimension was observed as a result of application of the test solution containing only 25% isopropanol, the fractal dimension of skin cross section gradually increased with increasing concentrations of isopropanol, 2-methyl-1-butanol or sodium myristate in the test solutions. The increment of fractal dimension was around 0.4. Thus, the skin structure was also altered by the application of high concentrations of these compounds. Although the relevance to humans is not known, fractal analysis of skin structure is thought to be useful as a novel method for detecting skin damage that is brought about by the application of percutaneous absorption enhancers. PMID- 10737919 TI - Hematopoiesis in larval Pseudoplusia includens and Spodoptera frugiperda. AB - Maintenance of circulating hemocytes in larval Lepidoptera has been attributed to both mitosis of hemocytes already in circulation and the release of hemocytes from hematopoietic organs. In this study, we compared hematopoiesis in the noctuids Pseudoplusia includens and Spodoptera frugiperda. For both species, hemocyte densities per microl of blood increased with instar. Differential hemocyte counts indicated that plasmatocytes were the most abundant hemocyte type during early instars but granular cells were the most abundant hemocyte type in the last instar. Hematopoietic organs were located in the meso- and metathorax of S. Frugiperda and P. Includens. These organs contained large numbers of hemocytes in S. Frugiperda, but contained few hemocytes in P. Includens. The majority of the hemocytes recovered from hematopoietic organs were identified as plasmatocytes. Using hemocyte type-specific markers and bromodeoxyuridine (BrdU) incorporation experiments, we determined that all hemocyte types with the exception of oenocytoids synthesize DNA. BrdU labeling indices for both species also fluctuated with the molting cycle. Ligation experiments suggested that hematopoietic organs are an important source of circulating plasmatocytes in S. Frugiperda but not in P. Includens. Injection of heat killed bacteria into larvae induced higher levels of BrdU labeling than injection of sterile saline, suggesting that infection and wounding induce different levels of hemocyte proliferation. Arch. PMID- 10737920 TI - Molecular cloning of a cDNA for a small GTP binding protein, BRho, from the embryo of Bombyx mori and its characterization after expression and purification. AB - A cDNA clone encoding a small GTP binding protein (Brho) was isolated from an embryonic cDNA library of Bombyx mori that encoded a polypeptide with 202 amino acids sharing 60-80% similarity with the Rho1 family of GTP binding proteins. The effector site and one of the guanine nucleotide binding sites differed from other members of the Rho family. To characterize the biochemical properties of Brho, the clone was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The fusion protein bound [(35)S] GTPgammaS and [(3)H] GDP with association constants of 11x10(6) M(-1) and 6.2x10(6) M(-1), respectively. The binding of [(35)S] GTPgammaS was inhibited by GTP and GDP, but by no other nucleotides. The calculated GTP-hydrolysis activity was 89.6 m mol/min/mol of Brho. Bound [(35)S] GTPgammaS and [(3)H] GDP were exchanged with GTPgammaS most efficiently in the presence of 6 mM MgCl(2). These results suggest that Brho has a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolytic activity, and returns to the GTP-bound state with the exchange of GDP with GTP. Arch. PMID- 10737921 TI - Molecular cloning and characterization of a midgut chymotrypsin-like enzyme from the lesser grain borer, Rhyzopertha dominica. AB - A cDNA encoding a chymotrypsinogen-like protein in midguts of the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) was cloned and sequenced. The 901 bp cDNA contains an 816-nucleotide open reading frame encoding 272-amino acids. The predicted molecular mass and pI of the mature enzyme are 23.7 kDa and 4.64, respectively. The encoded protein includes amino acid sequence motifs that are conserved with 5 homologous chymotrypsinogen proteins from other insects. Features of the putative chymotrypsin-like protein from R. dominica include the serine proteinase active site (His(90), Asp(133), Ser(226)), conserved cysteine residues for disulfide bridges, the residues (Gly(220), Gly(243), Asp(252)) that determine chymotrypsin specificity, and both zymogen activation and signal peptides. A TPCK-sensitive caseinolytic protein (P6) with an estimated molecular mass of 24 kDa is present in midgut extracts of R. dominica and can be resolved by electrophoresis on 4-16% polyacrylamide gels. The molecular mass of this caseinolytic enzyme is similar to that of the chymotrypsin deduced from cDNA. Midgut extracts of R. dominica readily hydrolyzed azocasein and N-succinyl-alanine-alanine-proline-phenylalanine-p- nitroanilide (SAAPFpNA), a chymotrypsin-specific substrate. Properties of the enzymes responsible for these activities were partially characterized with respect to distribution in the gut, optimum pH, and sensitivity toward selected proteinase inhibitors. Optimal activity against both azocasein and SAAPFpNA occurs in a broad pH range from about 7 to 10. Both azocasein and SAAPFpNA activities, located primarily in the anterior midgut region, are inhibited by aprotinin, phenylmethyl sulphonylfluoride (PMSF), and soybean trypsin inhibitor (STI). TPCK (N-alpha tosyl-L-phenylalanine chloromethyl ketone) and chymostatin inhibited more than 60% of SAAPFpNA but only about 10-20% of azocasein activity. These results provide additional evidence for the presence of serine proteinases, including chymotrypsin, in midguts of R. dominica. Arch. Insect Biochem. Physiol. 43:173 184, 2000.Published 2000 Wiley-Liss, Inc. PMID- 10737923 TI - Recognition and architecture of the framework structure of protein. AB - Based on the concept that the framework structure of a protein is determined by its secondary structure sequence, a new method for recognition and prediction of the structural class is suggested. By use of parameters N(alpha), N(beta), and N(beta(alpha)beta) (the number of alpha-helices, beta-strands, and beta(alpha)beta fragments), one can recognize the structural class with an accuracy higher than 90% when applied to the complete set (standard set) published in October 1995 and the structure data newly released before July 1998 (test set). Furthermore, the framework structures of beta, alpha, and alpha/beta protein are studied. It is found that these structures can be built from some basic units and that their architecture obeys some definite rules. Based on the packing of these basic units a set of rules for the recognition of topologies of the framework structure are worked out. When applied to the 1995 standard set and the 1998 test set the rates of correct recognition are higher than 77%. The simplicity and universality of framework structures are indicated which may be related to the evolutionary conservation of these folds. Proteins 2000;39:9-25. PMID- 10737922 TI - Homologs of the alpha- and beta-subunits of mammalian brain platelet-activating factor acetylhydrolase Ib in the Drosophila melanogaster genome. AB - The mammalian intracellular brain platelet-activating factor acetylhydrolase, implicated in the development of cerebral cortex, is a member of the phospholipase A2 superfamily. It is made up of a homodimer of the 45 kDa LIS1 protein (a product of the causative gene for type I lissencephaly) and a pair of homologous 26-kDa alpha-subunits which account for all the catalytic activity. LIS1 is hypothesized to regulate nuclear movement in migrating neurons through interactions with the cytoskeleton, while the alpha-subunits, whose structure is known, contain a trypsin-like triad within the framework of a unique tertiary fold. The physiological significance of the association of the two types of subunits is not known. In an effort to better understand the function of the complex we turned to genomic data mining in search of related proteins in lower eukaryotes. We found that the Drosophila melanogaster genome contains homologs of both alpha- and beta-subunits, and we cloned both genes. The alpha-subunit homolog has been overexpressed, purified and crystallized. It lacks two of the three active-site residues and, consequently, is catalytically inactive against PAF-AH (Ib) substrates. Our study shows that the beta-subunit homolog is highly conserved from Drosophila to mammals and is able to interact with the mammalian alpha-subunits but is unable to interact with the Drosophila alpha-subunit. Proteins 2000;39:1-8. PMID- 10737924 TI - Conformational flexibility of the catalytic Asp dyad in HIV-1 protease: An ab initio study on the free enzyme. AB - The enzyme protease from the human immunodeficiency virus type 1 (HIV-1 PR) is one of the main targets for therapeutic intervention in AIDS. Computer modeling is useful for probing the binding of novel ligands, yet empirical force field based methods have encountered problems in adequately describing interactions of the catalytic aspartyl pair. In this work we use ab initio dynamic methods to study the molecular interactions and the conformational flexibility of the Asp dyad in the free enzyme. Calculations are performed on model complexes that include, besides the Asp dyad, the conserved Thr26 and Gly27 residues and water molecules present in the active site channel. Our calculations provide proton location and binding mode of the active-site water molecule, which turn out to be different from those of the eukariotic isoenzyme. Furthermore, the calculations reproduce well the structural features of the aspartyl dyad in the protein. Finally, they allow the identification of both dipole/charge interactions and a low-barrier hydrogen bond as important stabilizing factors for the peculiar conformation of the active site. These findings are consistent with site-directed mutagenesis experiments on the 27, 27; positions (Bagossi et al., Protein Eng 1996;9:997-1003). The electric field of the protein frame (included in some of the calculations) does not affect significantly the chemical bonding at the cleavage site. Proteins 2000;39:26-36. PMID- 10737925 TI - Crystal structures of the complexes of trichosanthin with four substrate analogs and catalytic mechanism of RNA N-glycosidase. AB - Four substrate analogs-nicotinamide adenine dinucleotide, adenylyl (3', 5') guanosine, guanylyl (3',5') adenosine, and adenosine 2', 5'-diphosphate-have been used to prepare the complexes with trichosanthin (TCS), a type I ribosome inactivating protein that possesses the activity of N-glycosidase. The crystal structures of the complexes have been determined and refined at high resolution. The refined structures show that the N-glycosidic bonds of all the four substrate analogues are hydrolyzed and a common structure is shared by the four complexes, in which only adenine, the product of the enzymatic reaction, is bound in the active center. The structure is compared with those of native trichosanthin and a previously reported trichosanthin-NADPH complex in which the N-glycosidic bond is uncleaved. The structural comparison shows that the conformation of Tyr70 obviously differs from those in the latter two structures, i.e., the side chain of Tyr70 is rotated along its Cbeta-Cgamma bond by approximately 70 degrees. The water molecule found to be preassociated with the N-glycosidic bond in the TCS NADPH complex structure and proposed to be the water candidate responsible for hydrolyzing the N-glycosidic bond disappears in the trichosanthin-product complex structure. Based on the comparison of the three structures representing the different stages of the enzymatic reaction, the catalytic mechanism of RNA N glycosidase has been further elucidated. Proteins 2000;39:37-46. PMID- 10737926 TI - Structure and dynamics of the pore-lining helix of the nicotinic receptor: MD simulations in water, lipid bilayers, and transbilayer bundles. AB - Multiple nanosecond duration molecular dynamics simulations on the pore-lining M2 helix of the nicotinic acetylcholine receptor reveal how its structure and dynamics change as a function of environment. In water, the M2 helix partially unfolds to form a molecular hinge in the vicinity of a central Leu residue that has been implicated in the mechanism of ion channel gating. In a phospholipid bilayer, either as a single transmembrane helix, or as part of a pentameric helix bundle, the M2 helix shows less flexibility, but still exhibits a kink in the vicinity of the central Leu. The single M2 helix tilts relative to the bilayer normal by 12 degrees, in agreement with recent solid state NMR data (Opella et al., Nat Struct Biol 6:374-379, 1999). The pentameric helix bundle, a model for the pore domain of the nicotinic receptor and for channels formed by M2 peptides in a bilayer, is remarkably stable over a 2-ns MD simulation in a bilayer, provided one adjusts the pK(A)s of ionizable residues to their calculated values (when taking their environment into account) before starting the simulation. The resultant transbilayer pore shows fluctuations at either mouth which transiently close the channel. Proteins 2000;39:47-55. PMID- 10737927 TI - Molecular dynamics simulation of solvated azurin: correlation between surface solvent accessibility and water residence times. AB - A system containing the globular protein azurin and 3,658 water molecules has been simulated to investigate the influence on water dynamics exerted by a protein surface. Evaluation of water mean residence time for elements having different secondary structure did not show any correlation. Identically, comparison of solvent residence time for atoms having different charge and polarity did not show any clear trend. The main factor influencing water residence time in proximity to a specific site was found to be its solvent accessibility. In detail for atoms belonging to lateral chains and having solvent accessible surface lower than approximately 16 A(2)a relation is found for which charged and polar atoms are surrounded by water molecules characterized by residence times longer than the non polar ones. The involvement of the low accessible protein atom in an intraprotein hydrogen bond further modulates the length of the water residence time. On the other hand for surfaces having high solvent accessibility, all atoms, independently of their character, are surrounded by water molecules which rapidly exchange with the bulk solvent. Proteins 2000;39:56-67. PMID- 10737928 TI - Molecular modeling of substrate binding in wild-type and mutant Corynebacteria 2,5-diketo-D-gluconate reductases. AB - 2,5-diketo-D-gluconic acid reductase (2,5-DKGR; E.C. 1.1.1.-) catalyzes the Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent stereo-specific reduction of 2, 5-diketo-D-gluconate (2,5-DKG) to 2-keto-L-gulonate (2-KLG), a precursor in the industrial production of vitamin C (L-ascorbate). Microorganisms that naturally ferment D-glucose to 2,5-DKG can be genetically modified to express the gene for 2,5-DKGR, and thus directly produce vitamin C from D glucose. Two naturally occurring variants of DKGR (DKGR A and DKGR B) have been reported. DKGR B exhibits higher specific activity toward 2,5-DKG than DKGR A; however, DKGR A exhibits a greater selectivity for this substrate and significantly higher thermal stability. Thus, a modified form of DKGR, combining desirable properties from both enzymes, would be of substantial commercial interest. In the present study we use a molecular dynamics-based approach to understand the conformational changes in DKGR A as the active site is mutated to include two active site residue changes that occur in the B form. The results indicate that the enhanced kinetic properties of the B form are due, in part, to residue substitutions in the binding pocket. These substitutions augment interactions with the substrate or alter the alignment with respect to the putative proton donor group. Proteins 2000;39:68-75. PMID- 10737929 TI - Monte Carlo simulation of diffusion of adsorbed proteins. AB - We present the results of three-dimensional lattice Monte Carlo simulations of protein diffusion on the liquid-solid interface in a wide temperature range including the most interesting temperatures (from slightly below T(f) and up to T(c), where T(f) and T(c) are the folding and collapse temperatures). For the model under consideration (27 monomers of two types), the temperature dependence of the diffusion coefficient is found to obey the Arrhenius law with the normal value (approximately 10(-2)-10(-3) cm(2)/s) of the preexponential factor. Proteins 2000;39:76-81. PMID- 10737930 TI - Efficient sampling in collective coordinate space. AB - Collective motions in biological macromolecules have been shown to be important for function. The most important collective motions occur on slow time scales, which poses a sampling problem in dynamic simulation of biomolecules. We present a novel method for efficient conformational sampling. The method combines the simulation of an ensemble of concurrent trajectories with restraints acting on the ensemble of structures as a whole. Two properties of the ensemble may be restrained: (i) the variance of the ensemble and (ii) the average position of the ensemble. Both properties are defined in a subspace of collective coordinate space spanned by an arbitrary number of modes. We show that weak restraints on the ensemble variance suffice for an increase in sampling efficiency along soft modes by two orders of magnitudes. The resulting trajectories exhibit virtually the same structural quality as trajectories generated by restraint-free-molecular dynamics simulation, as judged by standard structure validation tools. The method is used to probe the resistance of a structure against conformational changes along collective modes and clearly distinguishes soft from stiff modes. Further applications are discussed. Proteins 2000;39:82-88. PMID- 10737931 TI - Plasticity and steric strain in a parallel beta-helix: rational mutations in the P22 tailspike protein. AB - By means of genetic screens, a great number of mutations that affect the folding and stability of the tailspike protein from Salmonella phage P22 have been identified. Temperature-sensitive folding (tsf) mutations decrease folding yields at high temperature, but hardly affect thermal stability of the native trimeric structure when assembled at low temperature. Global suppressor (su) mutations mitigate this phenotype. Virtually all of these mutations are located in the central domain of tailspike, a large parallel beta-helix. We modified tailspike by rational single amino acid replacements at three sites in order to investigate the influence of mutations of two types: (1) mutations expected to cause a tsf phenotype by increasing the side-chain volume of a core residue, and (2) mutations in a similar structural context as two of the four known su mutations, which have been suggested to stabilize folding intermediates and the native structure by the release of backbone strain, an effect well known for residues that are primarily evolved for function and not for stability or folding of the protein. Analysis of folding yields, refolding kinetics and thermal denaturation kinetics in vitro show that the tsf phenotype can indeed be produced rationally by increasing the volume of side chains in the beta-helix core. The high resolution crystal structure of mutant T326F proves that structural rearrangements only take place in the remarkably plastic lumen of the beta-helix, leaving the arrangement of the hydrogen-bonded backbone and thus the surface of the protein unaffected. This supports the notion that changes in the stability of an intermediate, in which the beta-helix domain is largely formed, are the essential mechanism by which tsf mutations affect tailspike folding. A rational design of su mutants, on the other hand, appears to be more difficult. The exchange of two residues in the active site expected to lead to a drastic release of steric strain neither enhanced the folding properties nor the stability of tailspike. Apparently, side-chain interactions in these cases overcompensate for backbone strain, illustrating the extreme optimization of the tailspike protein for conformational stability. The result exemplifies the view arising from the statistical analysis of the distribution of backbone dihedral angles in known three-dimensional protein structures that the adoption of straight phi/psi angles other than the most favorable ones is often caused by side-chain interactions. Proteins 2000;39:89-101. PMID- 10737932 TI - Molecular organization, structural features, and ligand binding characteristics of CD44, a highly variable cell surface glycoprotein with multiple functions. AB - CD44 is a type I transmembrane protein and member of the cartilage link protein family. It is involved in cell-cell and cell-matrix interactions and signal transduction. Several CD44 ligands have been identified. CD44 is a major cell surface receptor for hyaluronan, a component of the extracellular matrix. It is implicated in diseases such as cancer and inflammation and therefore intensely studied. A characteristic feature of CD44 is the occurrence of many isoforms that are expressed in a cell-specific manner and differentially glycosylated. Although a number of CD44 isoforms have been characterized, the structural diversity of CD44 makes it often challenging to study (isoform-specific) CD44-ligand interactions at the molecular level of detail. The structural organization and ligand binding characteristics of CD44 are focal points of this review. On the basis of recent structural and mutagenesis studies, details of the CD44 hyaluronan interaction are beginning to be understood. Proteins 2000;39:103-111. PMID- 10737933 TI - A fast method to sample real protein conformational space. AB - A fast computer program, FOLDTRAJ, to generate plausible random protein structures is reported. All-atom proteins are made directly in continuous three dimensional space starting from primary sequence with an N to C directed build-up method. The method uses a novel pipelined residue addition approach in which the leading edge of the protein is constructed three residues at a time for optimal protein geometry, including the placement of cis proline. Build-up methods represent a classic N-body problem, expected to scale as N(2). When proteins become more collapsed, build-up methods are susceptible to backtracking problems which can scale exponentially with the number of residues required to back out of a trapped walk. We have provided solutions to both these problems, using a multiway binary tree that makes the N-body problem of bump-checking scale as NlogN, and speeding up backtracking by varying the number of tries before backtracking based on available conformational space. FOLDTRAJ is independent of energy potentials, other than that implicit in the geometrical properties derived by statistical studies of known structures, and in atomic Van der Waals radii. WHAT-CHECK shows that the program generates chirally and physically valid proteins with all bond lengths, angles and dihedrals within allowable tolerances. Random structures built using sequences from PDB files 1SEM, 2HPR, and 1RTP typically have 5-15% alpha-helical content (according to DSSP) and on the order of 20% beta-strand/extended content. Ensembles of random structures are compared with polymer theory and with experimentally determined fluorescence resonance energy transfer distances. Reasonably sized structure ensembles do sample most of the conformational space available to proteins. The method is also capable of protein reconstruction using Calpha--Calpha direction vectors, and it compares favorably with methods that reconstruct protein backbones based on alpha-carbon coordinates, having an average backbone and Cbeta root mean square deviation of 0.63 A for nine different protein folds. Proteins 2000;39:112-131. PMID- 10737934 TI - The relative order of helical propensity of amino acids changes with solvent environment. AB - A model peptide of sequence Ac-Y-VAXAK-VAXAK-VAXAK-NH(2), where X is substituted with one of nineteen amino acids (P excluded), was synthesized and titrated with methanol to study helical propensity as a function of solvent environment. The CD spectra of these peptides are largely random coil in 2 mM sodium phosphate buffer (pH 5.5) and show a conformational change to alpha-helix with increasing methanol content. Singular value decomposition was used to correct the CD spectra for the absorbing side chains of W, Y, F, C, and M, and this correction can be substantial. With correction both W and F become good helix formers. The free energy for helix propagation was calculated using the Lifson-Roig statistical model for each of the nineteen amino acids at each point in their titration. The results show that the rank order of helical propensity for the nineteen amino acids changes with solvent environment. This result will be particularly important if proteins undergo hydrophobic collapse before secondary structures are formed, because amino acids can then see different solvent environments as the secondary structures are formed. Related amino acids are found to have interesting correlations in the shape of their titration curves. This finding provides one explanation for the limiting 70% accuracy in predicting secondary structure from sequence, since the helical propensities used are calculated for an average solvent environment. Proteins 2000;39:132-141. PMID- 10737935 TI - Sequence and structural features of the T-fold, an original tunnelling building unit. AB - A similar fold has been found in four archetype enzymes that perform different functions. This new fold has been named the T-fold because it is found in multimeric proteins crossed by a tunnel. The T-fold consists of an antiparallel beta-sheet of four sequential strands, and two antiparallel helices between the second and third strand, layered on the concave side of the beta-sheet. The presently known T-fold proteins share a high structural similarity (a mean of 1.4 A root mean square (r.m.s.) deviation on the common core) while they only exhibit a low level of sequence identity (a mean of 10.5% on the aligned regions). They bind to substrates belonging to the purine or pterin families, and share a fold related binding site with a glutamate or glutamine residue anchoring the substrate and a lot of conserved interactions. They also share a similar oligomerization mode: several T-folds join together to form a beta(2n)alpha(n) barrel, then two barrels join together in a head-to-head fashion to made up the native enzymes. The T-fold has the characteristics of a globular domain, with a hydrophobic core and a clearly defined topohydrophobic network. It defines a new class of common folds or recurrent domains found in distantly related proteins. However, it is likely not stable in monomeric form and until now is only observed in association with other T-folds through multimerization. Proteins 2000;39:142 154. PMID- 10737936 TI - Molecular modelling of Trematomus newnesi Hb 1: insights for a lowered oxygen affinity and lack of root effect. AB - Three-dimensional structural models of the hemoglobin (Hb 1) of the Antarctic fish Trematomus newnesi were built by homology modelling, using as template the X ray crystallographic structures of Trematomus (previously named Pagothenia) bernacchii Hb 1, both in R and T state. The Hbs of these two fishes, although showing remarkably different oxygen binding properties, differ only by 4 residues in the alpha chain (142 aa) and 10 residues in the beta chain (146 aa). T. newnesi Hb1 R-state model, essentially performed as a quality control of the adopted modelling procedure, showed a good correspondence with the crystallographic one. Modelling of T. newnesi Hb1 in the T state was performed taking into account that the proton uptake by aspartate residues, proposed to be responsible for half of the Root effect in T. bernacchii Hb 1 (showing sharp pH dependent oxygen affinity and T-state overstabilization at low pH, i. e. Bohr and Root effect), does not occur in T. newnesi Hb1 (having nearly pH-independent lower oxygen affinity). Comparison with the template structure (submitted to the same minimization procedure) indicates that, in T. newnesi Hb1 T-state model, the substitution of Ile for Thr in 41 C6, in central position of the switch region, induces at the alpha(1)beta(2) interface structural modifications able to hamper the protonation. Similar modifications are also found in T. bernacchii Hb 1 modelled in the T state with the single substitution Thr-->Ile in 41alpha. These models also suggest that the lower oxygen affinity observed in T. newnesi Hb1 is related to structural differences at the alpha(1)beta(2) interface leading to a more stable low-affinity T state. Proteins 2000;39:155-165. PMID- 10737937 TI - Adipyl crosslinked bovine hemoglobins as new models of allosteric systems. AB - As indicated by peptide analyses and mass spectrometry estimations, intramolecular crosslink with bis(3,5-dibromosalicyl)adipate of bovine hemoglobin results in the formation of two main components covalently bridged across the beta-cleft. In one component the crosslink joins the beta(1)V1-beta(2)K81 residues (XL-Peak-1), in the other the bridge is between the beta(1)K81 beta(2)K81 residues (XL-Peak-2). Both components are tetrameric with a mass near MW = 67 kDa as estimated by gel filtration, and a hydrodynamic radius near 3. 20 nm, estimated by dynamic light scattering. They have very low oxygen affinity with Pm near 100 mmHg (XL-Peak-1) and near 70 mmHg (XL-Peak-2) respectively at 37 degrees C, at neutral pH. The Bohr effect is almost absent in XL-Peak-1, while in XL-Peak-2 it is very near normal. Both systems show oxygen binding cooperativity with an index near n = 2.0. Flash photolysis kinetics of the recombination with CO could be resolved into a fast and a slow component. The amplitude of the fast rates were not concentration-dependent. The stopped-flow kinetics were autoaccelerating, consistent with their ligand-binding cooperativity. All rates were very similar to those of normal hemoglobin, suggesting that the oxy- rather than the deoxy-forms of the systems were affected by the crosslink. Proteins 2000;39:166-169. PMID- 10737938 TI - Cohesin-dockerin recognition in cellulosome assembly: experiment versus hypothesis. AB - The cohesin-dockerin interaction provides the basis for incorporation of the individual enzymatic subunits into the cellulosome complex. In a previous article (Pages et al., Proteins 1997;29:517-527) we predicted that four amino acid residues of the approximately 70-residue dockerin domain would serve as recognition codes for binding to the cohesin domain. The validity of the prediction was examined by site-directed mutagenesis of the suspected residues, whereby the species-specificity of the cohesin-dockerin interaction was altered. The results support the premise that the four residues indeed play a role in biorecognition, while additional residues may also contribute to the specificity of the interaction. Proteins 2000;39:170-177. PMID- 10737939 TI - Protein docking using spherical polar Fourier correlations. AB - We present a new computational method of docking pairs of proteins by using spherical polar Fourier correlations to accelerate the search for candidate low energy conformations. Interaction energies are estimated using a hydrophobic excluded volume model derived from the notion of "overlapping surface skins," augmented by a rigorous but "soft" model of electrostatic complementarity. This approach has several advantages over former three-dimensional grid-based fast Fourier transform (FFT) docking correlation methods even though there is no analogue to the FFT in a spherical polar representation. For example, a complete search over all six rigid-body degrees of freedom can be performed by rotating and translating only the initial expansion coefficients, many unfeasible orientations may be eliminated rapidly using only low-resolution terms, and the correlations are easily localized around known binding epitopes when this knowledge is available. Typical execution times on a single processor workstation range from 2 hours for a global search (5 x 10(8) trial orientations) to a few minutes for a local search (over 6 x 10(7) orientations). The method is illustrated with several domain dimer and enzyme-inhibitor complexes and 20 large antibody-antigen complexes, using both the bound and (when available) unbound subunits. The correct conformation of the complex is frequently identified when docking bound subunits, and a good docking orientation is ranked within the top 20 in 11 out of 18 cases when starting from unbound subunits. Proteins 2000;39:178-194. PMID- 10737940 TI - ATP-activated oligomerization as a mechanism for apoptosis regulation: fold and mechanism prediction for CED-4. AB - Fold recognition algorithm FFAS (Rychlewski et al., Protein Sci, 2000;9:232-241) was used to match the nucleotide-binding adaptor shared by APAF-1, certain R gene products and CED-4 (NB-ARC domain) to the structure of the D2 domain of N ethylemaleimide-Sensitive Fusion Protein and the delta; subunit of clamp loader of DNA polymerase III. The predicted structure consists of the p-loop ATP-binding domain, followed by two alpha-helical domains that regulate the oligomerization process. This prediction suggests a detailed molecular mechanism for the "induced proximity" hypothesis (Salvesen and Dixit, Proc Natl Acad Sci USA 1999;96:10964 10967) for CED3/caspase-9 activation by CED4/APAF-1 complex. According to this model, the ATP binding acts as a trigger in CED-4 oligomerization and the helical domain immediately following the ATP-binding domain provides additional mechanisms for regulation of the oligomerization process. This model explains most of known experimental data about CED-4-mediated caspase activation and, at the same time, suggest experiments that could test this hypothesis. PMID- 10737941 TI - Conversion of yeast phosphoglycerate kinase into amyloid-like structure. AB - Yeast phosphoglycerate kinase is a structurally well-characterized enzyme consisting of 415 amino acids without disulfide bonds. Anion-induced refolding from its acid-unfolded state gives rise to the formation of worm-like amyloid fibrils with a persistence length of 73 nm. Electron microscopy and small-angle X ray scattering data indicate that the fibrils have an elliptical cross-section with dimensions of 10.2 nm x 5.1 nm. About half of all amino acids are organized in form of cross-beta structure which gives rise to typical infrared spectra, X ray diffraction and yellow-green birefringence after Congo red staining. The kinetics of amyloid formation, monitored by infrared spectroscopy, dynamic light scattering and X-ray scattering, was found to be strongly dependent on protein concentration. The infrared data indicate that the formation of cross-beta structure practically comes to an end already after some hours, whereas the length-growth of the amyloid fibrils, monitored by small-angle X-ray scattering, was not yet completed after 1,300 hours. PMID- 10737942 TI - Molecular dynamics simulation accurately predicts the experimentally-observed distributions of the (C, N, O) protein atoms around water molecules and sodium ions. AB - A molecular dynamics simulation of the operator binding domain of the lambda repressor protein has been carried out. The protein was embedded in explicit waters, Na(+) and CL(-) ions. The Amber 4.1 computer package and the Cornell et al. Force field were used for energy-minimization and molecular dynamics simulation. We find that the atoms distributions in the environment of waters and Na(+) ions are in excellent agreement with those derived from the analysis of water molecules in crystal structures and ion-binding proteins. We also find that, on the whole, both distributions are similar to each other. PMID- 10737943 TI - Crystal structure of human ubiquitous mitochondrial creatine kinase. AB - Creatine kinase (CK), catalyzing the reversible trans-phosphorylation between ATP and creatine, plays a key role in the energy metabolism of cells with high and fluctuating energy requirements. We have solved the X-ray structure of octameric human ubiquitous mitochondrial CK (uMtCK) at 2.7 A resolution, representing the first human CK structure. The structure is very similar to the previously determined structure of sarcomeric mitochondrial CK (sMtCK). The cuboidal octamer has 422 point group symmetry with four dimers arranged along the fourfold axis and a central channel of approximately 20 A diameter, which extends through the whole octamer. Structural differences with respect to sMtCK are found in isoform specific regions important for octamer formation and membrane binding. Octameric uMtCK is stabilized by numerous additional polar interactions between the N termini of neighboring dimers, which extend into the central channel and form clamp-like structures, and by a pair of salt bridges in the hydrophobic interaction patch. The five C-terminal residues of uMtCK, carrying positive charges likely to be involved in phospholipid-binding, are poorly defined by electron density, indicating a more flexible region than the corresponding one in sMtCK. The structural differences between uMtCK and sMtCK are consistent with biochemical studies on octamer stability and membrane binding of the two isoforms. PMID- 10737944 TI - Enhancement of catalytic efficiency of enzymes through exposure to anhydrous organic solvent at 70 degrees C. Three-dimensional structure of a treated serine proteinase at 2.2 A resolution. AB - The enzyme behavior in anhydrous media has important applications in biotechnology. So far chemical modifications and protein engineering have been used to alter the catalytic power of the enzymes. For the first time, it is demonstrated that an exposure of enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes: proteinase K, wheat germ acid phosphatase, alpha amylase, beta-glucosidase, chymotrypsin and trypsin have been exposed to acetonitrile at 70 degrees C for three hours. The activities of these enzymes were found to be considerably enhanced. In order to understand the basis of this change in the activity of these enzymes, the structure of one of these treated enzymes, proteinase K has been analyzed in detail using X-ray diffraction method. The overall structure of the enzyme is similar to the native structure in aqueous environment. The hydrogen bonding system of the catalytic triad is intact after the treatment. However, the water structure in the substrate binding site undergoes some rearrangement as some of the water molecules are either displaced or completely absent. The most striking observation concerning the water structure pertains to the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules are located in the recognition site. The sites occupied by acetonitrile molecules are independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. All of them are interlinked through water molecules. The methyl group of one of the acetonitrile molecules (CCN1) interacts simultaneously with the hydrophobic side chains of Leu-96, Ile-107, and Leu-133. The development of such a hydrophobic environment at the recognition site introduces a striking conformation change in Ile-107 by rotating its side chain about C(alpha)--C(beta) bond by 180 degrees to bring about the delta-methyl group within the range of attractive van der Waals interactions with the methyl group of CCN1. A similar change has earlier been observed in proteinase K when it is complexed to a substrate analog lactoferrin fragment. PMID- 10737946 TI - Hiking in the energy landscape in sequence space: a bumpy road to good folders. AB - With the help of a simple 20-letter lattice model of heteropolymers, we investigated the energy landscape in the space of designed good-folder sequences. Low-energy sequences form clusters, interconnected via neutral networks, in the space of sequences. Residues that play a key role in the foldability of the chain and in the stability of the native state are highly conserved, even among the chains belonging to different clusters. If, according to the interaction matrix, some strong attractive interactions are almost degenerate (i.e., they can be realized by more than one type of amino acid contacts), sequence clusters group into a few superclusters. Sequences belonging to different superclusters are dissimilar, displaying very small ( approximately 10%) similarity, and residues in key sites are, as a rule, not conserved. Similar behavior is observed in the analysis of real protein sequences. PMID- 10737945 TI - Ab initio calculations on hidden modulators of theta class glutathione transferase activity. AB - The glutathione transferases decrease the pKa of glutathione, allowing its deprotonation and the formation of the more reactive thiolate anion. The thiolate is maintained in the active site through a weak conventional hydrogen bond first sphere interaction donated by a Tyr hydroxyl in the Alpha, Mu, Pi, and Sigma glutathione transferase classes that can be modified by other second sphere or indirect thiolate contacts. However, the Theta and Delta class isoforms use a Ser hydroxyl for stabilizing the GSH thiolate, and as such, have a different chemical system compared with that of the Tyr possessed by other classes. We have used high level ab initio methods to investigate this interaction by using a simple methanol methanethiol system as a model. The hydrogen bond strength of this initial first sphere interaction was calculated to be less than that of the Tyr interaction. A putative second sphere interaction exists in the Theta and Delta class structures between Cys or Ser-14 and Ser-11 in the mammalian Theta subclass 1 and 2, respectively. The effect of this interaction on the first sphere interaction has also been investigated and found to significantly increase the energy of the bond. PMID- 10737947 TI - Computer simulations of protein folding by targeted molecular dynamics. AB - We have performed 128 folding and 45 unfolding molecular dynamics runs of chymotrypsin inhibitor 2 (CI2) with an implicit solvation model for a total simulation time of 0.4 microseconds. Folding requires that the three-dimensional structure of the native state is known. It was simulated at 300 K by supplementing the force field with a harmonic restraint which acts on the root mean-square deviation and allows to decrease the distance to the target conformation. High temperature and/or the harmonic restraint were used to induce unfolding. Of the 62 folding simulations started from random conformations, 31 reached the native structure, while the success rate was 83% for the 66 trajectories which began from conformations unfolded by high-temperature dynamics. A funnel-like energy landscape is observed for unfolding at 475 K, while the unfolding runs at 300 K and 375 K as well as most of the folding trajectories have an almost flat energy landscape for conformations with less than about 50% of native contacts formed. The sequence of events, i.e., secondary and tertiary structure formation, is similar in all folding and unfolding simulations, despite the diversity of the pathways. Previous unfolding simulations of CI2 performed with different force fields showed a similar sequence of events. These results suggest that the topology of the native state plays an important role in the folding process. PMID- 10737948 TI - Side-chain flexibility in proteins upon ligand binding. AB - Ligand binding may involve a wide range of structural changes in the receptor protein, from hinge movement of entire domains to small side-chain rearrangements in the binding pocket residues. The analysis of side chain flexibility gives insights valuable to improve docking algorithms and can provide an index of amino acid side-chain flexibility potentially useful in molecular biology and protein engineering studies. In this study we analyzed side-chain rearrangements upon ligand binding. We constructed two non-redundant databases (980 and 353 entries) of "paired" protein structures in complexed (holo-protein) and uncomplexed (apo protein) forms from the PDB macromolecular structural database. The number and identity of binding pocket residues that undergo side-chain conformational changes were determined. We show that, in general, only a small number of residues in the pocket undergo such changes (e.g., approximately 85% of cases show changes in three residues or less). The flexibility scale has the following order: Lys > Arg, Gln, Met > Glu, Ile, Leu > Asn, Thr, Val, Tyr, Ser, His, Asp > Cys, Trp, Phe; thus, Lys side chains in binding pockets flex 25 times more often then do the Phe side chains. Normalizing for the number of flexible dihedral bonds in each amino acid attenuates the scale somewhat, however, the clear trend of large, polar amino acids being more flexible in the pocket than aromatic ones remains. We found no correlation between backbone movement of a residue upon ligand binding and the flexibility of its side chain. These results are relevant to 1. Reduction of search space in docking algorithms by inclusion of side-chain flexibility for a limited number of binding pocket residues; and 2. Utilization of the amino acid flexibility scale in protein engineering studies to alter the flexibility of binding pockets. PMID- 10737949 TI - Preface PMID- 10737950 TI - Summary of major conclusions from the International Workshop on Genotoxicity Test Procedures. PMID- 10737951 TI - Report from the In Vitro Micronucleus Assay Working Group. AB - At the Washington International Workshop on Genotoxicity Test Procedures (March 25-26, 1999), the current methodologies and data for the in vitro micronucleus test were reviewed. From this, guidelines for the conduct of specific aspects of the protocol were developed. Because there are a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at this time. Agreement was achieved on the following topics: Cells. The choice of cells is flexible, yet the choice of cell type should be justified and take into consideration doubling time, spontaneous frequency of micronuclei, and genetic background. Slide preparation. A fixation method that preserves the cytoplasm and cytoplasmic boundaries, and minimizes clumping should be used. Use of fluorescent DNA specific dyes is encouraged for better detection of small micronuclei. Analysis. Micronuclei should have a diameter less than one-third of the main nucleus, and should be clearly distinguishable from the main nucleus. In the cytokinesis-block method, binucleated cells selected for analysis should have two clearly distinguishable main nuclei. Cells where the main nucleus(ei) is undergoing apoptosis should not be scored for micronuclei because the assumed micronuclei may have been the result of nuclear fragmentation during the apoptotic process. Toxicity. Cytotoxicity can be measured by various methods including cell growth, cell counts, nucleation (i.e., percent binucleated), division/proliferation index, confluence. A majority of the group recommended that the highest concentration should induce at least 50% cytotoxicity (by whatever measure is selected). Cytochalasin B. There is much debate regarding the use of cytochalasin B. For human lymphocytes, the use of cytochalasin B (6 microg/ml [lymphocytes cultured from whole blood cells] and 3-6 microg/ml [isolated lymphocyte cultures]) is recommended. For cell lines, because there were no definitive data showing a clear advantage or disadvantage of the use of cytochalasin B for a variety of chemicals, the majority opinion of the group was that at this time, the use of cytochalasin B for cell lines is considered optional. Further studies (many chemicals of a variety of potencies, tested both with and without cytochalasin B) are clearly needed to resolve this issue. Number of doses. At least three concentrations should be scored for micronuclei. Treatment/harvest times. At this time, there are not enough data to define the most appropriate treatment/harvest times. Following the principles of the in vitro metaphase assay (with or without metabolic activation), it was agreed that there was a need for a short treatment followed by a recovery time in the absence of test chemical, there was a need for a long treatment (maybe with and without recovery time), and ideally, treatment should cover cells in different cell cycle stages. PMID- 10737952 TI - Considerations on photochemical genotoxicity: report of the International Workshop on Genotoxicity Test Procedures Working Group. AB - Recent toxicological observations have caused concern regarding the need to test, for example, pharmaceuticals and cosmetic products for photochemical genotoxicity. The objective of this report is to give assistance on how to adapt existing test methods to investigate the potential of light-absorbing compounds to induce genotoxic effects on photoactivation. In general, the Organization for Economic Co-Operation & Economic Development (OECD) draft guideline on in vitro phototoxicity testing served as a basis for consideration. Concomitant exposure of the cells to the test compound and solar simulated light was considered appropriate as the initial, basic test condition. Optimization of the exposure scheme, e.g., a change of the irradiation spectrum, might be indicated depending on the initial test results. Selection of test compound concentrations should be based on results obtained with the dark version of the respective test system but might have to be modified if phototoxic effects are observed. Selection of the irradiation dose has to be performed individually for each test system based on dose-effect studies. The irradiation should induce per se a small, reproducible toxic or genotoxic effect. The report includes a specification of necessary controls, discusses factors that might have an impact on the irradiation characteristics, and gives a rationale for the omission of an external metabolic activation system. It also addresses the question that physicochemical and pharmacokinetic properties might trigger the need to test a chemical for photochemical genotoxicity. Relevant experimental observations are presented to back up the recommendations. The working group did not reach a consensus as to whether a single, adequately perfomed in vitro test for clastogenicity would be sufficient to exclude a photogenotoxic liability or whether a test battery including a gene mutation assay would be needed for product safety testing regarding photochemical genotoxicity. PMID- 10737953 TI - Mouse lymphoma thymidine kinase locus gene mutation assay: International Workshop on Genotoxicity Test Procedures Workgroup Report. AB - The Mouse Lymphoma Assay (MLA) Workgroup addressed and reached consensus on a number of issues. Discussion focused on five areas: (1) acceptable assay versions; (2) cytotoxicity measure; (3) 24-hr treatment; (4) microwell colony counting and sizing; and (5) data acceptability/statistical analysis. Although the International Conference on Harmonisation (ICH) indicated a preference for the microwell over the soft agar method, all of the workgroup members agreed that both versions of the MLA are equally acceptable. The workgroup agreed that it is desirable for both assay versions to use the same measure of cytotoxicity to define the acceptable and required concentration range. Currently, laboratories using the microwell version use the relative survival (RS) determined by cloning immediately after the treatment. Laboratories using the soft agar method do not obtain an RS but use the relative total growth (RTG), a combination of the relative suspension growth (RSG) during the expression period and the relative cloning efficiency determined at the time of mutant selection. The workgroup agreed to investigate the RSG, the RS, and the RTG and to develop further guidance. In the interim, the workgroup reached consensus that the RTG be used as the standard measure of cytotoxicity. The ICH recommended a 24-hr treatment in the absence of S9 when negative results are obtained with short (3-4 hr) treatments. The workgroup agreed to retain this requirement but acknowledged that more data are needed prior to making final recommendations concerning the need for and the specific protocol for the 24-hr treatment. Environ. Mol. Mutagen. 35:185-190, 2000 Published 2000 Wiley-Liss, Inc. PMID- 10737954 TI - Cytotoxicity and chromosome aberrations in vitro: experience in industry and the case for an upper limit on toxicity in the aberration assay. AB - The chromosome aberration assay in vitro is a useful and sensitive test for detection of genotoxins. However, aberrations can occur secondary to toxicity, with compounds that do not react with DNA and are not genotoxic in vivo. Thus, some positive results in the in vitro aberration assay are not relevant to human risk. To help evaluate the influence of toxicity, data were collected from 27 pharmaceutical and chemical companies and contract laboratories. When cytotoxicity was measured by cell counts or confluence, compounds expected to damage DNA (Category 1) generally induced aberrations without severe concomitant cytotoxicity, i.e., at cell growth 60% or more of control. The more toxic nucleoside analogues, topoisomerase inhibitors, fluoroquinolone antibiotics, antifolates, and producers of reactive oxygen were still positive with cell growth 50% or more of control. In contrast, when there was evidence that the compounds were not DNA damaging (Category 2), there was a higher proportion of toxicity-associated clastogens, with positive results at less than 50% of control cell growth. When mitotic index (MI) was used as an indicator of cytotoxicity, the pattern was less clear, although there was a tendency to more mitotic suppression with the Category 2 compounds. Overall the data indicate that a limit on toxicity, and a more accurate way of estimating it, would increase the accuracy of the assay by reducing the frequency of nonrelevant positive results with a threshold-type of dose relation. The rationale for evaluating positive results in the in vitro aberration assay, especially those associated with toxicity, is discussed, as is the need for a harmonized regulatory approach. PMID- 10737955 TI - Appropriate levels of cytotoxicity for genotoxicity tests using mammalian cells in vitro. AB - Among standard battery genotoxicity assays, the in vitro chromosome aberration test and the mouse lymphoma tk assay (MLA) yield about fourfold higher incidences of positive test results than the bacterial reverse mutation test or in vivo bone marrow tests. This is a result of experience with submissions of 335 new pharmaceuticals to the German Federal Institute for Drugs and Medical Devices. While all of the standard systems have their value in detecting relevant genotoxins, there is no supportive evidence for DNA reactivity for a considerable number of in vitro clastogens and MLA positives. In particular the clastogenic response of such compounds is often associated with high cytotoxicity. This may invoke the need to change the approach to test for clastogenicity in vitro. A combination of measures such as (1) a change in the upper limits of cytotoxicity that are currently given in International Conference on Harmonisation (ICH) and Organization for Economic Co-Operation & Economic Development (OECD) guidelines, (2) the creation of a common ground of understanding for interpretation of in vitro (positive) test results, and (3) lowering the upper limits of test compound concentration irrespective of cytotoxicity may prove useful to ensure a sufficient reliability of genotoxicity testing with mammalian cells in vitro. PMID- 10737956 TI - Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. AB - Atthe International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA protein cross-linking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission. The expert panel used the current Organization for Economic Co-operation and Development (OECD) guidelines for in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data. The related methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS; (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies. PMID- 10737957 TI - Methods of DNA adduct determination and their application to testing compounds for genotoxicity. AB - At the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC (March 25-26, 1999), a working group considered the uses of DNA adduct determination methods for testing compounds for genotoxicity. When a drug or chemical displays an unusual or inconsistent combination of positive and negative results in in vitro and in vivo genotoxicity assays and/or in carcinogenicity experiments, investigations into whether or not DNA adducts are formed may be helpful in assessing whether or not the test compound is a genotoxin. DNA adduct determinations can be carried out using radiolabeled compounds and measuring radioactive decay (scintillation counting) or isotope ratios (accelerator mass spectrometry) in the isolated DNA. With unlabeled compounds adducts may be measured by (32)P-postlabeling analysis of the DNA, or by physicochemical methods including mass spectrometry, fluorescence spectroscopy, or electrochemical detection, or by immunochemical methods. Each of these approaches has different strengths and limitations, influenced by sensitivity, cost, time, and interpretation of results. The design of DNA binding studies needs to be on a case-by-case basis, depending on the compound's profile of activity. DNA purity becomes increasingly important the more sensitive, and less chemically specific, the assay. While there may be adduct levels at which there is no observable biological effect, there are at present insufficient data on which to set a threshold level for biological significance. PMID- 10737958 TI - In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring. AB - An expert working group on the in vivo micronucleus assay, formed as part of the International Workshop on Genotoxicity Test Procedures (IWGTP), discussed protocols for the conduct of established and proposed micronucleus assays at a meeting held March 25-26, 1999 in Washington, DC, in conjunction with the annual meeting of the Environmental Mutagen Society. The working group reached consensus on a number issues, including: (1) protocols using repeated dosing in mice and rats; (2) integration of the (rodent erythrocyte) micronucleus assay into general toxicology studies; (3) the possible omission of concurrently-treated positive control animals from the assay; (4) automation of micronucleus scoring by flow cytometry or image analysis; (5) criteria for regulatory acceptance; (6) detection of aneuploidy induction in the micronucleus assay; and (7) micronucleus assays in tissues (germ cells, other organs, neonatal tissue) other than bone marrow. This report summarizes the discussions and recommendations of this working group. In the classic rodent erythrocyte assay, treatment schedules using repeated dosing of mice or rats, and integration of assays using such schedules into short-term toxicology studies, were considered acceptable as long as certain study criteria were met. When the micronucleus assay is integrated into ongoing toxicology studies, relatively short-term repeated-dose studies should be used preferentially because there is not yet sufficient data to demonstrate that conservative dose selection in longer term studies (longer than 1 month) does not reduce the sensitivity of the assay. Additional validation data are needed to resolve this point. In studies with mice, either bone marrow or blood was considered acceptable as the tissue for assessing micronucleus induction, provided that the absence of spleen function has been verified in the animal strains used. In studies with rats, the principal endpoint should be the frequency of micronucleated immature erythrocytes in bone marrow, although scoring of peripheral blood samples gives important supplementary data about the time course of micronucleus induction. When dose concentration and stability are verified appropriately, concurrent treatment with a positive control agent is not necessary. Control of staining and scoring procedures can be obtained by including appropriate reference samples that have been obtained from a separate experiment. For studies in rats or mice, treatment/sampling regimens should include treatment at intervals of no more than 24 hr (unless the test article has a half-life of more than 24 hr) with sampling of bone marrow or blood, respectively, within 24 or 40 hr after the last treatment. The use of a DNA specific stain is recommended for the identification of micronuclei, especially for studies in the rat. In the case of a negative assay result with a non-toxic test article, it is desirable that systemic exposure to the test article is demonstrated. The group concluded that successful application of automated scoring by both flow cytometry and image analysis had been achieved, and defined criteria that should be met if automated scoring is employed. It was not felt appropriate to attempt to define specific recommended protocols for automated scoring at the present time. Other issues reviewed and discussed by the working group included micronucleus assays that have been developed in a number of tissues other than bone marrow. The group felt that these assays were useful research tools that could also be used to elucidate mechanisms in certain regulatory situations, but that these assays had not yet been standardized and validated for routine regulatory application. PMID- 10737959 TI - In vivo transgenic mutation assays. AB - Transgenic rodent gene mutation models provide quick and statistically reliable assays for mutations in the DNA from any tissue. For regulatory applications, assays should be based on neutral genes, be generally available in several laboratories, and be readily transferable. Five or fewer repeated treatments are inadequate to conclude that a compound is negative but more than 90 daily treatments may risk complications. A sampling time of 35 days is suitable for most tissues and chemicals, while shorter sampling times might be appropriate for highly proliferative tissues. For phage-based assays, 5 to 10 animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3 x 10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (PFU or CFU) per tissue. Data should be generated for two dose groups but three should be treated, at the maximum tolerated dose (MTD), two-thirds the MTD, and one third the MTD. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a block design and the total number of PFUs or CFUs and the MF for each tissue and animal reported. Sequencing data would not normally be required but might provide useful additional information in specific circumstances. Statistical tests used should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is statistically nonsignificant with all mean MF within two standard deviations of the control. PMID- 10737960 TI - Report and summary of the major conclusions from statistics in genotoxicity testing working group from the International Workshop on Genotoxicity Test Procedures (IWGTP), March 1999. AB - A working group of five statisticians experienced in the use of statistical methods in mutagenicity reviewed aspects of the statistical analysis of genotoxicity test procedures. Issues discussed included methods for integrating biological importance and statistical significance, the relationship of the experimental unit to the experimental design, and the impact of new developments in statistics and computing. Three major recommendations were made relating to the need for: (1) the effective use of statistical advice in designing interlaboratory and intralaboratory investigations; (2) the development of appropriate experimental designs for new assays; and (3) education and training in the use of statistical methodology in mutagenicity testing. Environ. Mol. Mutagen. 35:260-263, 2000 Published 2000 Wiley-Liss, Inc. PMID- 10737961 TI - Aromatase inhibitor and 17alpha-methyltestosterone cause sex-reversal from genetical females to phenotypic males and suppression of P450 aromatase gene expression in Japanese flounder (Paralichthys olivaceus). AB - The sex of Japanese flounder (Paralichthys olivaceus) is easily altered by water temperature or sex steroid hormone treatment during the period of sex determination. We have previously shown that rearing the genetically female larvae at high water temperature caused the suppression of P450 aromatase (P450arom) gene expression in the gonad and phenotypic sex-reversal of the individuals to males (Kitano et al. 1999. J Mol Endocrinol 23:167-176). In the present study, we show that treatment of genetically female larvae with fadrozole (aromatase inhibitor) or 17alpha-methyltestosterone induces sex-reversal as well as suppression of P450arom gene expression. The effect of fadrozole was counteracted by co-administration of estradiol-17beta. Effective periods for fadrozole treatment to induce sex-reversal were similar to those for high water temperature treatment. RT-PCR did not detect P450arom mRNA in gonad of the sex reversed, phenotypic males. These results indicate that sex-reversal of the genetically female larvae by aromatase inhibitor (or 17alpha-methyltestosterone) may be due to the suppression of P450arom gene expression and the resultant decrease in the amount of estrogen. PMID- 10737962 TI - Specific expression of soluble adenylyl cyclase in male germ cells. AB - The cAMP signaling pathway is an important mediator of extracellular signals in organisms from prokaryotes to higher eukaryotes. In mammals two types of adenylyl cyclase synthesize cAMP; a ubiquitous family of transmembrane isoforms regulated by G proteins in response to extracellular signals, and a recently isolated soluble enzyme insensitive to heterotrimeric G protein modulation. Using the very sensitive reverse transcription-polymerase chain reaction (RT-PCR), soluble adenylyl cyclase (sAC) expression is detectable in almost all tissues examined; however, Northern analysis and in situ hybridization indicate that high levels of sAC message are unique to male germ cells. Elevated levels of sAC mRNA are first observed in pachytene spermatocytes and expression increases through spermiogenesis. The accumulation of high levels of message in round spermatids suggests sAC protein plays an important role in the generation of cAMP in spermatozoa, implying possible roles in sperm maturation through the epididymis, capacitation, hypermotility, and/or the acrosome reaction. PMID- 10737963 TI - Differential regulation of the translation and the stability of two maternal transcripts in preimplantation rabbit embryos. AB - In most species, transcription is essentially silent during the first mitotic cell cycles that follow fertilization. This means that the regulation of gene expression in early embryos heavily relies on the translational activation or inactivation of maternal mRNAs. In mammals, the mechanisms that control the translation of maternal mRNAs have been mainly studied in the mouse when maternal to zygotic transition occurs after the first mitotic division. In other mammalian species, however, this transition occurs later after several cell cycles, and little is known concerning the regulation of maternal information during this period. To address this question, we have used rabbit pre-implantation embryos to analyze the translational activation and stability of two maternal mRNAs, mm 41 and mm61. During the cleavage period, these mRNAs exhibit distinct kinetics for both their translational activation and degradation. In addition, these mRNAs both undergo cytoplasmic polyadenylation but with different efficiencies. This polyadenylation was functionally correlated with the translational activation of these mRNAs; inhibiting polyadenylation prevented translational activation. The differential efficiency of cytoplasmic polyadenylation, driven by cis-elements in the 3' untranslated region of these mRNAs, was also observed in Xenopus laevis embryos, which emphasizes the high conservation of this mechanism between species. PMID- 10737964 TI - CD52 mRNA is modulated by androgens and temperature in epididymal cell cultures. AB - Cultured rat epididymal tissue explants formed >90% pure, adherent growing epithelial cell monolayers. Despite their flattened and apparently androgen receptor-negative phenotype, these cells for a short period kept characteristics of the epididymal duct epithelium, i.e., expression of the tissue-specific marker CD52 and responsiveness of its mRNA toward temperature elevation and androgen withdrawal. When cells were grown on permeable supports at 33 degrees C, androgen supplementation or withdrawal specifically modulated the levels as well as the length of the CD52 mRNA. Elevation of the culture temperature to a quasi abdominal milieu of 37 degrees C selectively reduced the CD52 mRNA levels under all culture conditions. This reduction was not affected by the presence of androgens and was not accompanied by changes in length, suggesting that the modulation of CD52 mRNA in epididymal cells by androgens and by temperature is synergic, but may involve different molecular mechanisms. CD52 mRNA levels, however, were not stable in the primary cultures but decreased rapidly to undetectable levels after 4-5 days at all culture conditions. GAPDH mRNA levels, on the other hand, were stable throughout the culture period. PMID- 10737965 TI - New approach to cell lineage analysis in mammals using the Cre-loxP system. AB - The Cre-loxP site-specific recombination system was used for cell lineage analysis in mammals. We constructed an expression plasmid, pCETZ-17, which consists of cytomegalovirus enhancer/chicken beta-actin promoter (CAG), a portion of the rabbit beta-globin gene, loxP-flanked DNA sequence (containing enhanced green fluorescent protein (EGFP) cDNA), and lacZ gene encoding E. coli beta galactosidase (beta-gal). When circular pCETZ-17 plasmid DNA was microinjected into the pronuclei of fertilized eggs and these eggs were allowed to develop to two-cell stage, 62.8% (59/94) of the two-cell embryos exhibited distinct fluorescence in one or both blastomeres, but never expressed lacZ protein, as evaluated by histochemical staining with X-Gal, a substrate for beta-gal. When both circular plasmids, pCETZ-17 and pCAG/NCre (containing CAG and DNA sequences encoding nuclear location signal and Cre), were co-injected into fertilized eggs, almost all (87.0%, 47/54) embryos exhibited low or no fluorescence, but 51.9% (27/52) exhibited positive staining for beta-gal activity. This indicates that transient expression of the Cre recombinase gene removed the loxP-flanked DNA sequence in pCETZ-17 and then caused expression of the downstream lacZ sequence. We next microinjected pCETZ-17 into the pronuclei of fertilized eggs, cultured these injected eggs for 1 day, and collected only two-cell embryos expressing EGFP in both blastomeres. One blastomere of the EGFP-expressing two-cell embryos was microinjected with pCAG/NCre, and these treated embryos were cultured for 1 day up to four-cell stage. When the developing four-cell embryos were subjected to staining with X-Gal, cell lineage-related staining pattern for beta-gal activity was observed in most (77.8%, 7/9) embryos. These findings were further confirmed using two-cell embryos derived from a transgenic mouse line carrying CETZ-17 transgene. Thus, our system, which is based on transient expression of the Cre recombinase gene directly introduced into nuclei of embryonic cells by microinjection, is a powerful means for cell lineage analysis in mammals. PMID- 10737966 TI - Stage-specific nuclear antigen is expressed in rat male germ cells during early meiotic prophase. AB - A germ cell nuclear antigen with approximately 44-kDa molecular weight was identified by a novel monoclonal antibody designated as Mab 2F2 from the library we have accumulated against rat testicular cells. In immature 20-day-old and adult rat testis the recognized antigen was expressed in the nuclei of early meiotic cells from preleptotene to early pachytene spermatocytes exhibiting a stage-specific appearance in the cycle of the seminiferous epithelium. The immunoreactivity was clearly associated with the meiotic chromosomes. The antigen was not detected in the late pachytene spermatocytes and more advanced stages of spermatogenesis. No labeling was observed in spermatogonia and somatic Sertoli and Leydig cells. The pattern of expression of the recognized antigen during early meiotic stages of spermatogenesis but not in mitotically dividing spermatogonia could strengthen its possible role in meiotic division. PMID- 10737967 TI - Specific regulation of CENP-E and kinetochores during meiosis I/meiosis II transition in pig oocytes. AB - To understand the mechanisms which regulate meiosis-specific cell cycle and chromosome distribution in mammalian oocytes, the level and the localization of CENP-E and the kinetochore number and direction on a half bivalent were examined during pig oocyte maturation. CENP-E is a kinetochore motor protein whose intracellular level and localization are strictly regulated in the somatic cell cycle. The localizations of CENP-E on meiotic chromosomes from diakinesis stage to anaphase I and at the spindle midzone at telophase I were shown by immunofluorescent confocal microscopy to be similar to those in somatic cells of pig and other species. Further, ultrastructural analysis revealed the presence of CENP-E on fibrous corona and outer plate of kinetochores of the meiotic chromosomes. However, unlike mitosis, CENP-E staining was continuously detected either at the spindle midzone or on the kinetochores of segregated chromosomes during the first polar body emission. Consistent with this, immunoblot analysis revealed that CENP-E level remained high during meiosis I/meiosis II (MI/MII) transition and that some of CENP-E survived through the transition even in cycloheximide-treated oocytes in which cyclin B1 was completely degraded. Furthermore, examinations of CENP-E signals in confocal microscopy and kinetochores in electron microscopy in MI and MII oocytes provide the cytological evidence in mammalian oocytes which suggests that each sister chromatid in a pair has its own kinetochore which localizes side-by-side so that two sister chromatids on a half bivalent are oriented toward and connected to the same pole in MI. PMID- 10737968 TI - Developmental regulation of effect of epidermal growth factor on porcine oocyte cumulus cell complexes: nuclear maturation, expansion, and F-actin remodeling. AB - Epidermal growth factor (EGF) efficiently stimulates expansion of mouse and rat oocyte-cumulus complexes (OCC). Contradictory data have been published by several laboratories about the ability of EGF to stimulate expansion of porcine OCC. We assumed that these contradictions may have resulted from heterogeneous conditions used for isolation, culture, and assessment of OCC. The present experiments were designed to test the hypothesis that porcine OCC acquire the ability to synthesize hyaluronic acid (HA) and undergo expansion following EGF-stimulation gradually during the growth of follicles. For this reason, we isolated OCC from follicles of different sizes and assessed quantity of produced HA and proportions of expanding OCC after stimulation by EGF. In addition, we assessed in those OCC changes in morphology of cumulus cells and assembly of F-actin microfilaments, which are necessary for expansion to occur. Finally, nuclear maturation of EGF stimulated OCC was assessed and its relationship with occurrence of expansion was evaluated. In all experiments, OCC stimulated with FSH were used as positive controls. The results showed that EGF did not stimulate production of HA, rearrangement of F-actin and expansion in OCC isolated from small follicles (<4 mm in diameter). OCC isolated from large preovulatory follicles (6-7 mm in diameter and PMSG-stimulated follicles) underwent efficient expansion when stimulated by EGF (93% and 100%, respectively). EGF dramatically stimulated total production of HA in these OCC and its retention in extracellular matrix of the expanding cumulus. Cumulus cells of the large OCC underwent essential changes of their morphology and extensive rearrangement of F-actin microfilaments following stimulation with EGF. Interestingly, EGF enhanced nuclear maturation of OCC isolated from both small and large follicles, which suggest diversity of signaling pathways controlling maturation and expansion. FSH caused cumulus expansion, F-actin remodeling, and enhancement of oocyte nuclear maturation in OCC originated from both small and large follicles. We conclude that EGF can stimulate expansion of porcine OCC in vitro; however, only of those isolated from large follicles. This indicates that EGF may have a physiological role in regulation of porcine cumulus expansion in preovulatory follicles, presumably as a mediator of signals elicited by the LH surge. PMID- 10737969 TI - Effect of dexamethasone on the expression of p34(cdc2) and cyclin B1 in pig oocytes in vitro. AB - The meiotic division in oocytes is arrested in the G2 phase of the cell cycle. Resumption of meiosis, also known as oocyte maturation, entails a G2 to M transition. At the G2-M boundary, maturation promoting factor (MPF) activation is usually induced via several ways, including tyrosine dephosphorylation of p34(cdc2) and synthesis of cyclin B according to cell type and species. Previous studies in our laboratory demonstrated that glucocorticoids directly inhibit the meiotic maturation of pig oocytes in vitro. The aim of this study was therefore to investigate the influence of glucocorticoids on the expression of p34(cdc2) and cyclin B1 in resumption of meiosis of pig oocytes. We detected the relative levels and association of p34(cdc2) and cyclin B1. Isolated cumulus-enclosed oocytes were cultured in Waymouth MB752/1 medium supplemented with sodium pyruvate (50 microgram/ml), LH (0.5 microgram/ml), FSH (0.5 microgram/ml), and estradiol-17beta (1 microgram/ml) in the presence or absence of dexamethasone (DEX) for 24 hr; they then were cultured without hormonal supplements in the presence or absence of DEX for an additional 24 hr. We found that cyclin B1, as well as p34(cdc2), was already present in fully grown G2-arrested pig oocytes when removed from the follicle. In these oocytes, cyclin B1 and p34(cdc2) were already associated in complex. Treatment with DEX at concentrations of 1 microgram/ml or above decreased the level of cyclin B1, but had no effect on the level of p34(cdc2). The exposure of oocytes to DEX also decreased the amount of complexed p34(cdc2)-cyclin B1. These findings suggest that the inhibitory action of DEX on meiotic maturation could be due, at least in part, to the reduced amount of p34(cdc2)-cyclin B1 complex. PMID- 10737970 TI - Vitrification of bovine oocytes with the open pulled straw method: ultrastructural consequences. AB - The objective of the present study was to investigate the ultrastructural consequences of vitrification of bovine oocytes at the metaphase II (MII) stage by the so-called "Open Pulled Straw" method. Oocytes were matured in vitro for 22 hr and cryopreserved by vitrification. After warming and additional 2 hr of culture, the oocytes were inseminated in vitro. Oocytes were fixed for transmission electron microscopy immediately after warming, at 4 hr after warming (i.e., 2 hr post insemination [hpi]), at 26 hr after warming (i.e., 24 hpi), and at 74 hr after warming (i.e., 72 hpi). Control oocytes (i.e., nonvitrified oocytes) were processed at 22 hr after in vitro maturation and at 2, 22, and 72 hpi. Compared to the controls, the vitrified oocytes fixed immediately after warming presented an additional category of small membrane-bound vesicles and lacked the typical compartment of solitary cortical granules aligned along the oolemma. Instead, they presented clusters of cortical granules that displayed varying degrees of degeneration. In vitrified oocytes fixed at 2 hpi, the small vesicles were less abundant, and more advanced degeneration of the cortical granule clusters was noted. In vitrified oocytes fixed at 24 hpi, the small vesicles were practically absent, and polyspermic penetration was observed as were vacuoles containing degraded cortical granule content. In vitrified oocytes fixed at 72 hpi, lack of cleavage as well as vacuolization and degeneration of blastomeres were noted. Moreover, the nucleolar ultrastructure signaled aberrant activation of the ribosomal RNA genes. In conclusion, vitrification of bovine oocytes at the MII stage resulted in cell biological alterations in the oocyte after warming that apparently were reflected in the subsequent fertilization and embryonic development. PMID- 10737971 TI - Microfilament stabilization by jasplakinolide arrests oocyte maturation, cortical granule exocytosis, sperm incorporation cone resorption, and cell-cycle progression, but not DNA replication, during fertilization in mice. AB - Jasplakinolide (JAS), which induces microfilament polymerization and stabilization, inhibits microfilament-mediated events in murine oocyte maturation and fertilization in a fashion unlike the effects of cytochalasin B (CCB) and latranculin A (LAT A). JAS prevents egg polar body emission at a much lower concentration than either CCB or LAT A. Microfilament bundles were detected on the entire egg cortex after JAS exposure. Conversely, microfilament patterns did not change after exposure to CCB, and few microfilaments were observed after exposure to LAT A. Eggs that were allowed to recover from JAS were unable to recover normal microfilament organization. During oocyte maturation, JAS prevented both spindle migration to the oocyte cortex and first polar body emission. During in vitro fertilization, sperm head entered the eggs and formed pronuclei, but sperm tail entry, pronuclear centration, and second polar body emission were not detected. DNA synthesis occurs in these JAS-treated zygotes. JAS inhibited not only the formation, but also the disassembly, of incorporation cones. JAS was also found to prevent cortical granule exocytosis following artificial activation, and cortical granules were still beneath the plasma membrane even after activation. Finally, incorporation of microinjected nonmuscle actin into the microfilament network of mice eggs was delayed by JAS. We conclude that JAS acts as a microfilament inhibitor during maturation and fertilization and is more powerful than other inhibitors. Its mechanism differs in that it promotes assembly and stabilization of microfilaments. JAS is a novel cell permeable tool for the investigation of microfilament-dependent events in early mammalian development. PMID- 10737972 TI - In vitro effects of beta-carotene for the motility, ATP, and intracellular free Ca(2+) concentrations of fowl spermatozoa. AB - The motility of intact fowl spermatozoa was vigorous at 25 degrees C, but decreased gradually following the addition of 0-100 microM beta-carotene in a dose-dependent manner. Even in the presence of stimulators of fowl sperm motility, such as Ca(2+) or calyculin A, the motility of intact spermatozoa at both 25 and 40 degrees C remained inhibited following the addition of beta carotene. Under all of these circumstances, sperm ATP concentrations were not reduced by the addition of beta-carotene. Moreover, the motility of demembranated spermatozoa was not inhibited by the addition of the same concentrations of beta carotene. No changes in intracellular free Ca(2+) concentrations, measured by means of a fluorescent Ca(2+) indicator, fura-2, were observed in intact beta carotene -treated spermatozoa. These results suggest that beta-carotene is involved in the inhibition of the flagellar movement of fowl spermatozoa without change in energy production, and that the target of beta-carotene might be present in the cytoplasmic matrix and/or the plasma membrane, but not retained in the axoneme and/or accessory cytoskeletal components. PMID- 10737973 TI - Chromatin condensation during spermiogenesis in the golden hamster (Mesocricetus aureus): a flow cytometric study. AB - DNA-staining of hamster testis cell suspensions followed by flow cytometry demonstrated appearance of the first haploid cells at 23 days post partum (dpp) and of condensed chromatin (in elongated spermatids and spermatozoa) at 33-34 dpp. Mature spermatozoa were first observed in the caput epididymis at 36-37 dpp, thus completing the first spermatogenic wave. Testicular cell suspensions from animals from 23 to 38 dpp were stained with acridine orange, and flow cytometer gating was adjusted to include only the haploid cells. Acridine orange intercalated into double-stranded DNA to produce green fluorescence. The decrease in green fluorescence intensity from 23 until 37 dpp was caused by changes in the binding of DNA to basic proteins in such a fashion as to impede the access of the dye to the DNA double helix. When the green fluorescence values (of the most advanced spermatids) were plotted against the age of the hamsters (in dpp) or the corresponding steps of spermiogenesis, the decrease in fluorescence could be seen to occur in three phases. The inflection point between the first and second phases was observed at about spermiogenesis step 7, consistent with the hypothesis that this represents removal of histone from the chromatin. The second phase presumably represents the period in which transition proteins are bound to the DNA. At approximately steps 15 or 16 a further inflection point was seen where protamines replaced the transition proteins. The red fluorescence produced when acridine orange bound to RNA in spermatids, increased early in spermiogenesis and decreased dramatically at 34 dpp, consistent with the fact that elongating spermatids discard the bulk of their cytoplasm during the maturation process. PMID- 10737974 TI - VMD2 mutations in vitelliform macular dystrophy (Best disease) and other maculopathies. AB - Mutations in the gene VMD2 are associated with autosomal dominant vitelliform macular dystrophy (Best disease). VMD2 is expressed in the retinal pigment epithelium and codes for a 585 amino acid putative transmembrane protein with undetermined functional properties. To date, 48 different mutations, predominantly missense, have been described in Best disease families. These mutations generally affect amino acids in the first 50% of the protein, and occur in four distinct clusters possibly representing regions of functional importance. VMD2 has also been investigated in other macular diseases. Mutations have been documented in a significant percentage of patients with adult vitelliform macular dystrophy (AVMD) and in a single case of "bull's-eye" maculopathy. Results of analysis in two large series of individuals with age-related macular degeneration (AMD) suggest that VMD2 does not play a major role in this prevalent disorder. PMID- 10737975 TI - Hypophosphatasia: the mutations in the tissue-nonspecific alkaline phosphatase gene. AB - Hypophosphatasia is an inborn error of metabolism caused by a deficiency of liver , bone- or kidney-type alkaline phosphatase due to mutations in the tissue nonspecific alkaline phosphatase (ALPL) gene. Most of the 65 distinct mutations described to date are missense mutations, a result which must be correlated with the great variability of clinical expression ranging from stillbirth without mineralized bone to pathologic fractures developing only late in adulthood. Correlations of genotype and phenotype have been established on the basis of clinical data exhibited by the patients, transfection studies, computer-assisted modeling, and examination of biochemical properties of ALP in cultured fibroblasts of patients. Screening for mutations in the TNSALP gene allows genetic counseling and prenatal diagnosis of the disease in families with severe forms of hypophosphatasia, and screening may also be helpful in confirming diagnosis of hypophosphatasia when biochemical and clinical data are not clear. Screening is also the necessary first step in further studies to elucidate dominant transmission of the disease and of liver-, bone- and kidney-type alkaline phosphatase activity mechanism. PMID- 10737976 TI - AluY insertion (IVS4-52ins316alu) in the glycerol kinase gene from an individual with benign glycerol kinase deficiency. AB - Glycerol kinase deficiency has three distinct forms: an isolated form which may be benign or symptomatic, and a complex form which is symptomatic and part of an Xp21 contiguous gene syndrome. Here we report the case of a male with benign isolated glycerol kinase deficiency who was incidentally identified after observation of pseudohypertriglyceridemia. DNA sequencing of this subject's glycerol kinase gene showed the insertion of an AluY sequence in intron 4 of the glycerol kinase gene. Although Alu insertions have been implicated in other diseases, and a closely related AluY element is found as an insert in the C1 inhibitor gene in patients with hereditary angioedema, this is the first case of glycerol kinase deficiency caused by an Alu insertion. PMID- 10737977 TI - Analysis of a 43.6 kb deletion in a patient with Hunter syndrome (MPSII): identification of a fusion transcript including sequences from the gene W and the IDS gene. AB - Mucopolysaccharidosis type II (Hunter syndrome) is an X-linked lysosomal storage disorder. A novel mutation is described in an MPS II patient in whom the disorder is caused by a 43.6 kb deletion. Southern blot analysis, PCR analysis and subsequent sequencing of the deletion junction revealed that the deletion spans exons 1-7 of the iduronate-2-sulfatase (IDS) gene, the IDS-2 locus and exons 3-5 of the recently identified gene W. Short direct repeats of 12 bp were identified at both deletion breakpoints, suggesting that the deletion is the result of an illegitimate recombination event. A sequence motif (TGAGGA) which is identical to a consensus sequence frequently associated with deletions in man was identified at both breakpoints. This further supports the notion that this motif is a hot spot for recombination. Gene expression studies by RT-PCR analysis of total RNA derived from fibroblasts of the patient revealed the presence of a novel fusion transcript. DNA sequence analysis of the cDNA demonstrated that it consists of exons derived from both the gene W and the IDS gene. A similar but longer fusion transcript containing exons 2-4 of the gene W and exons 4-9 of the IDS gene could also be detected in RNA of normal cell lines originating from different tissues. This result further demonstrates the complex gene expression profile of the IDS region, which may contribute to the observed genomic instability of this region. PMID- 10737978 TI - Mutation in the PAX6 gene in twenty patients with aniridia. AB - This is a report on the nature of the mutations in the PAX6 gene in twenty patients with aniridia. Five of the twenty patients had sporadic aniridia with deletions in chromosome 11p13. Three of the five had WAGR syndrome (Wilms tumor, aniridia, genitourinary anomalies, mental retardation), and the other two had deletions whose breakpoints occurred between the PAX6 and the WT1 genes. Allelic losses at PAX6 were of paternal origin. The remaining fifteen patients with aniridia had intragenic mutations in the PAX6 gene, with mutations found from exon 5 to exon 12. Twelve cases of dysfunctional PAX6 were due to premature termination of the protein by nonsense mutations (five cases), splicing defect (one case), deletion (two cases), deletion-insertions (two cases), and tandem repeat insertions (two cases). One patient (P2) had a PAX6 protein with de novo in-frame deletion of alanine, arginine, and proline at codon positions 37, 38, and 39. These codons are in the paired box region, and codon 38 is in contact with the phosphate group of the sugar-phosphate backbone of the target DNA. Another patient (P8) had a single nucleotide transition at c.1182 (nucleotide number, Genbank accession #M93650, used as in Glaser et al. [1992]), which generated both a missense mutation (Q255H) and a splicing defect. A missense mutation was found at G387E in a third patient (P10). All observed mutations support the notion that haploinsufficiency in PAX6 results in aniridia and associated eye anomalies. PMID- 10737979 TI - Screening for mutations in the peripheral myelin genes PMP22, MPZ and Cx32 (GJB1) in Russian Charcot-Marie-Tooth neuropathy patients. AB - Charcot-Marie-Tooth disease (CMT) and related inherited peripheral neuropathies, including Dejerine-Sottas syndrome, congenital hypomyelination, and hereditary neuropathy with liability to pressure palsies (HNPP), are caused by mutations in three myelin genes: PMP22, MPZ and Cx32 (GJB1). The most common mutations are the 1.5 Mb CMT1A tandem duplication on chromosome 17p11.2-p12 in CMT1 patients and the reciprocal 1.5 Mb deletion in HNPP patients. We performed a mutation screening in 174 unrelated CMT patients and three HNPP families of Russian origin. The unrelated CMT patients included 108 clinically and electrophysiologically diagnosed CMT1 cases, 32 CMT2 cases, and 34 cases with unspecified CMT. Fifty-nine CMT1A duplications were found, of which 58 belonged to the CMT1 patient group. We found twelve distinct mutations in Cx32, six mutations in MPZ, and two mutations in PMP22. Of these respectively, eight, five, and two lead to a CMT1 phenotype. Eight mutations (Cx32: Ile20Asn/Gly21Ser, Met34Lys, Leu90Val, and Phe193Leu; MPZ: Asp134Gly, Lys138Asn, and Thr139Asn; PMP22: ValSer25-26del) were not reported previously. Phenotype-genotype correlations were based on nerve conduction velocity studies and mutation type. PMID- 10737980 TI - Determination of 30 X-linked adrenoleukodystrophy mutations, including 15 not previously described. AB - X-linked Adrenoleukodystrophy (X-ALD) is the most frequent peroxisomal disease. It mainly involves the nervous system white matter, adrenal cortex and testes. Several distinct clinical phenotypes are known. The principal biochemical abnormality is the accumulation of saturated very-long-chain fatty acids (VLCFAs : > C22:0, mainly C26:0), which is due to impaired capacity for beta-oxidation in peroxisomes. Diagnosis is usually based on the VLCFA levels in plasma or cultured skin fibroblasts in both patients and carriers. In 0.1% of affected males, however, the plasma C26:0 level is borderline normal, and 15% of obligate female carriers have normal results. Effective mutation detection in these families is therefore fundamental to unambiguously determine the genetic status of each individual at risk. Of particular concern are female members of kindreds segregating X-ALD mutations, because normal VLCFA levels do not guarantee lack of carrier status. We describe a fast method for detection of X-ALD mutations. The method is based on SSCP analysis of nested PCR fragments followed by sequence determination reactions. Using this methodology we have found X-ALD mutations in 30 kindreds, including 15 not previously reported. PMID- 10737981 TI - beta-galactosidase gene mutations affecting the lysosomal enzyme and the elastin binding protein in GM1-gangliosidosis patients with cardiac involvement. AB - GM1-gangliosidosis is a lysosomal storage disorder caused by deficiency of acid beta-galactosidase (GLB1). We report five new beta-galactosidase gene mutations in nine Italian patients and one fetus, segregating in seven unrelated families. Six of the eight patients with the infantile, severe form of the disease presented cardiac involvement, a feature rarely associated with GM1 gangliosidosis. Molecular analysis of the patients' RNA and DNA identified two new RNA splicing defects, three new and three previously described amino acid substitutions. Interestingly, all patients with cardiac involvement were homozygous for one of these mutations: R59H, Y591C, Y591N, or IVS14-2A>G. In contrast, all other patients were compound heterozygous for one of the following mutations: R201H, R482H, G579D, IVS8+2T>C. Although we could not directly correlate the presence of cardiac abnormalities with specific genetic lesions, the mutations identified in patients with cardiomyopathy fell in the GLB1 cDNA region common to the lysosomal enzyme and the Hbeta-Gal-related protein, also known as the elastin binding protein (EBP). Consequently, both molecules are affected by the mutations, and they may contribute differently to the occurrence of specific clinical manifestations. PMID- 10737982 TI - SLC7A7 genomic structure and novel variants in three Japanese lysinuric protein intolerance families. AB - Lysinuric protein intolerance (LPI) is a rare inherited disease caused by defective transport of the dibasic amino acids at the basolateral membranes of epithelial cells in the renal tubules and small intestine. The metabolic defect leads to brain dysfunction caused by hyperammonemia with a functional impairment of the urea cycle. Recently, mutations in the human SLC7A7 cDNA coding for y(+)LAT-1, which express dibasic amino acid transport activity, were reported to be responsible for LPI. In the present study, we examined the genomic structure of SLC7A7 by DNA sequencing of PCR products, and determined that the gene had 11 exons and 10 introns spanning about 18 kb of genomic DNA. We also identified an alternative RNA splicing at the 5' untranslated region of the SLC7A7 mRNA in human peripheral blood leukocytes, cultured lymphoblasts, and fibroblasts. As a result of mutational analysis of SLC7A7 in three Japanese LPI families, we found a nonsense mutation (R410X), a splicing mutation(911+1G>A) in intron 4, and four silent polymorphisms (201C/T, 445A/G, 784C/T, 946T/C). Identification of the genomic structure of SLC7A7 may provide a molecular basis for a genetic survey for LPI. PMID- 10737983 TI - Detection of two novel large deletions in SLC3A1 by semi-quantitative fluorescent multiplex PCR. AB - Cystinuria is an autosomal recessive aminoaciduria in which two clinical types have been described (type I and non-type I). Cystinuria type I is caused by mutations in SLC3A1, a gene located in 2p16 coding for an amino acid transporter named rBAT. Using multiplex semi-quantitative fluorescent PCR, we amplified the ten exons of SLC3A1 together with exon 5 of DSCR1 (located on chromosome 21) as a double-dose control gene. We detected two large novel deletions in a Belgian family, one comprising exons 2-10 and another one at exon 10. The method described here can be used to detect a range of deletions from single-base differences in size to entire missing exons, making it useful for scanning genes with a small to medium number of exons. PMID- 10737984 TI - Geographical clustering of low density lipoprotein receptor gene mutations (C292X; Q363X; D365E & C660X) in Cyprus. AB - In Cyprus, no data are yet available on the frequencies of clinically diagnosed FH patients. Further, until now, familial hypercholesterolaemia in Cyprus had not been studied at the molecular level to determine the nature or frequency of LDLR gene mutations. Being a relatively homogeneous population, we anticipated that a few founder mutations would predominate on the island. In the present study, three previously identified LDLR gene mutations were found to cosegregate with high LDL cholesterol levels in 23 unrelated, clinically diagnosed families with FH. Geographical clustering of each of these LDLR gene mutations was indicated, a phenomenon arising from low migration rates and high inbreeding. The latter cultural practices account for the discovery of a homozygous FH sib pair whose parents are carriers of the same mutation. Microsatellite and intragenic haplotype analysis in this FH population, suggested that the families which shared the same LDLR gene mutation have a common origin. This is supported by their relative geographical distribution. Thirty young FH individuals were also offered presymptomatic diagnosis which should facilitate the prevention of premature coronary artery disease. Finally, results from this study support the suggestion that the formation of tendon xanthomata in FH patients may be under environmental influence. Hum Mutat 15:380, 2000. PMID- 10737985 TI - Lymphocyte mRNA analysis of the ornithine transcarbamylase gene in Italian OTCD male patients and manifesting carriers: identification of novel mutations. AB - A new simple, non-invasive method using ornithine transcarbamylase (OTC) mRNA isolated from peripheral blood (PBL) or lymphoblastoid cell lines has been performed. This approach based on reverse transcription and nested PCR to obtain a double strand PBL OTC cDNA allowed the identification of genetic lesions in five Italian families affected by OTC deficiency (OTCD). In the PBL OTC mRNA two new mutations, T262K and W265L, have been detected in three unrelated male OTCD patients with mild symptoms. One known mutation, T264A, has been identified in one manifesting carrier. The known mutation E310X, detected on genomic DNA of another manifesting carrier, failed to be detected in her PBL OTC mRNA because of the presence of a STOP codon. All mutations have been confirmed in the patients' and their relatives' genomic DNA. In three patients the mutations have also been confirmed in the mRNA isolated from frozen liver biopsy. The T262K amino acid substitution has been detected in a male's PBL OTC mRNA at homozygous state while a heterozygous pattern has been detected at the genomic DNA level, suggesting that the patient is a somatic mosaic for this mutation. Here we show that PBL OTC mRNA analysis is useful to detect genetic lesions in male and female OTCD patients. PMID- 10737986 TI - Identification of a novel mutation (867delA) in the glucose-6-phosphatase gene in two siblings with glycogen storage disease type Ia with different phenotypes. AB - We identified a novel mutation (867delA) in the glucose-6-phosphatase gene of two siblings with glycogen storage disease type Ia. Although both siblings share the same mutations, their phenotype regarding adult height and hepatomegaly differs. In glycogen storage disease type Ia, substantial heterogeneity in phenotype is observed. So far, no evidence for a clear genotype-phenotype correlation has been found. Hum Mutat 15:381, 2000. PMID- 10737987 TI - Frequency of germ-line BRCA1 mutations among Spanish families from a Mediterranean area. AB - We have carried out a study of breast cancer in Spanish families in which the entire coding region of the BRCA1 gene have been analyzed. To identify BRCA1 mutations, PTT and CSGE methods were used followed by direct sequencing. We investigated 51 breast cancer women with a family history. Among these we have identified 7 frameshifts mutations (15%), 185delAG (4 times), 1623del5 and 3450del4 (2 times), and 3 missense mutations, Ser1613Gly, Met1652Ile and Ala1708Glu, which are likely polymorphisms. These findings show that BRCA1 is implicated in a fraction of Spanish familial breast cancer similar to other countries. There was association between bilateral breast cancer and BRCA1 mutations. The CSGE technique has been demonstrated to be a highly reliable method for mutation screening because of its sensitivity and high throughput. PMID- 10737988 TI - Fatal hypertrophic cardiomyopathy associated with an A8296G mutation in the mitochondrial tRNA(Lys) gene. AB - We describe an 8-day-old baby girl presenting a fatal infantile form of hypertrophic obstructive cardiomyopathy, associated with an A8296G mutation in the mitochondrial tRNA(Lys) gene. She was born from a healthy unrelated couple, and was the first infant of dizygotic twins. Soon after birth, she was noted to have tachypnea and generalized hypotonia. She had high levels of lactate and pyruvate, and was diagnosed as having hypertrophic cardiomyopathy using echocardiography. She died by cardiac failure. Mitochondrial DNA analysis was performed by sequencing after PCR-subcloning methods, and the percentage of mutation was measured using PCR-RFLP methods. In various tissues obtained at autopsy, analysis showed a heteroplasmic population of A8296G mutation in the mitochondrial tRNA(Lys) gene in all the tissues examined. Maternal inheritance was demonstrated in the family members. Our data demonstrated that an A8296G mutation in the mitochondrial tRNA(Lys) gene showed clinical heterogeneity from a milder form previously reported as mitochondrial diabetes mellitus, to a more severe form as hypertrophic obstructive cardiomyopathy, according to the spatial distribution of this mutation. Hum Mutat 15:382, 2000. PMID- 10737989 TI - Novel de novo nonsense mutation of MECP2 in a patient with Rett syndrome. AB - Because of the recent identification of several mutations of methyl-CpG-binding protein 2 (MECP2) in patients with Rett syndrome (RTT), a patient with suspected RTT from an autism clinic was screened for mutations. She was found to have a novel heterozygous nonsense mutation, 129C>T (Q19X), which leads to the most severely truncated MECP2 protein reported to date. Sequencing of parental DNA revealed the mutation was de novo. The patient was not affected with microcephaly or hyperventilation, but had other features of Rett syndrome including severe mental retardation and symptoms of autistic disorder. Moderately skewed X chromosome inactivation (XCI) may have contributed to her relatively mild phenotype. PMID- 10737990 TI - Molecular basis of hypoxanthine-guanine phosphoribosyltransferase deficiency in thirteen Spanish families. AB - We have determined the molecular basis of hypoxanthine-guanine phosphoribosyltransferase (HPRT; HPRT1) deficiency in eight Lesch-Nyhan patients and in five partially HPRT deficient patients with mild to severe neurologic symptoms. Eight of these thirteen mutations have not been previously described. HPRT Zaragoza II (a GG insertion in exon 2), HPRT Murcia (an AG deletion in exon 4), HPRT Asturias (a A deletion in exon 4) and HPRT Cartagena (a A insertion in exon 6) cause a frame-shift resulting in a premature stop codon. HPRT Sevilla is a splice-site mutation resulting in exon 8 skipping in the HPRT mRNA. HPRT Huelva, Madrid II and Zaragoza I are point mutations that result in single amino acid changes in the mutated HPRT protein (118G-->A, G40R; 143G-->A, R 48 H; 397G- >A, V133 M, respectively). Three mutations have been previously described in unrelated families, and two mutations have been already published. All mutations that resulted in truncated proteins corresponded to patients with the Lesch-Nyhan phenotype. Characterization of the HPRT mutation allowed us to make carrier detection in 33 women and prenatal diagnosis in two fetuses. Hum Mutat 15:383, 2000. PMID- 10737991 TI - Identification of fifteen novel PHEX gene mutations in Finnish patients with hypophosphatemic rickets. AB - We have carried out a mutation screening of the PHEX gene in Finnish patients with hypophosphatemia. A total of 100% (5/5) of the familial HYP patients (X linked hypophosphatemia) and 93% (14/15) of the sporadic cases were found to carry a mutation in the PHEX gene. We identified 18 mutations, of which 15 were novel. We report also a new polymorphism 46bp upstream of exon 16. Two families were segregating the same nonsense mutation in exon 1 (R20X), but since this mutation has been previously reported in three independent studies, we consider it to be a mutational hotspot rather than a Finnish founder mutation. We did not find PHEX gene mutations in two additional hypophosphatemia families in which the mode of inheritance was other than X-linked dominant. Also, no mutation could be detected in a patient with suspected oncogenic osteomalacia (OHO). PMID- 10737992 TI - Genetic screening of fourteen mutations in Jordanian familial Mediterranean fever patients. AB - Familial Mediterranean fever is an autosomal recessive disorder characterised by episodic fever, abdominal and pleuritic pain, serositis and arthritis. The FMF gene (MEFV) has been mapped to chromosome 16p13.3 and generates a protein found exclusively in granulocytes. Seventeen mutations have been reported up to the present in FMF patients. This study involves the screening of 14 mutations in 42 Jordanian patients by two methods: RFLP and ARMS. The most frequent mutations were M694V and V726A (20% and 14% of the alleles respectively), followed by M680I and E148Q (9.5% and 7% of the alleles respectively). The A744S mutation accounts for 2.5% and the M694I, T267I and F479L mutations account each for 1% of the alleles. E167D, R761H, P369S, I692del and M694del mutations were not found in this population. Forty-four percent of the alleles did not have any of the 14 mutations. The results show the diversity and the frequency of the mutations in the Jordanian patients, and open the way for further investigations on patients diagnosed to have FMF and in whom no mutations were found. Hum Mutat 15:384, 2000. PMID- 10737993 TI - Partial deletion of the AGXT gene (EX1_EX7del): A new genotype in hyperoxaluria type 1. AB - Primary hyperoxaluria type 1 (PH1) is a rare autosomal (2q37.3) recessive metabolic disease caused by a deficiency of the hepatic peroxisomal enzyme alanine:glyoxylate amino transferase. Molecular heterogeneity is important in PH1 as most of the patients (if the parents are unrelated) are compound heterozygotes for rare mutations. We describe the first large deletion in the AGXT gene, removing exons 1 to 7 (EX1_EX7del) that was responsible for one case of severe PH1. This 10 kb deletion was identified by Southern blotting of genomic DNA digested by Xba I and hybridized with different exonic probes. Both parents (from Turkey) are first cousin and carry the deletion. It is of note that the presently reported patient did not exhibit any AGT catalytic activity and even so, he progressed towards end-stage renal disease only at 19 years old. PMID- 10737994 TI - Bruton's tyrosine kinase mutations in 8 Chinese families with X-linked agammaglobulinemia. AB - Bruton's tyrosine kinase (BTK) is involved in B-cell development. Mutation of BTK results in X-linked agammaglobulinemia (XLA). BTK is expressed in most haemopoietic lineages except mature T cells and plasma cells. We identified six novel and two known mutations of BTK in 11 Chinese XLA patients from 8 families. Family 1 had a novel point mutation at the start codon (135G-->T) in exon 2. Family 2 had known mutation of single A insertion in a stretch of 7 A residues (341-347insA) recognized as mutation hotspot in exon 3. Family 3 had a novel point mutation in exon 11 (1074A-->G) which led to aberrant splicing. Family 4 had known mutation in exon 19 (2053C-->T) in CpG mutation hotspot. The novel mutation of family 5 was an A deleted in a run of three As (1017-1019delA) in exon 10. In family 6, exons 2 and 3 were lost in BTK mRNA, a novel deletion. Family 7 had a novel substitution in exon 2 (227T-->C) which led to change of a conserved leucine to serine. Family 8 had a novel point mutation at beginning of intron 14 (IVS14+ 6 T-->G) resulting in aberrant splicing. Hum Mutat 15:385, 2000. PMID- 10737996 TI - Novel mutations of the RPGR gene in RP3 families. AB - X-linked retinitis pigmentosa is a severe retinal degeneration characterized by night blindness and visual field constriction, leading to complete blindness within the third decade of life. Mutations in the RPGR gene (retinitis pigmentosa GTPase regulator), located on Xp21.1 in the RP3 region, have been associated with an RP phenotype. Further to our previous mutation screening of RPGR in families segregating with the RP3 locus, we have expanded this study to include other 8 RP3 pedigrees. Here we report the results of this expanded study and the identification of five mutations in RPGR, four of which are novel (IVS6+5 G>A, 950-951delAA, 963 T>C, EX8del) and one of which occurs in the donor splice site of intron 1 (IVS1+1 G>A). These findings bring the proportion of "RP3 genotypes" with a mutation in this gene to 27% (10/37). Hum Mutat 15:386, 2000. PMID- 10737995 TI - The E148Q mutation in the MEFV gene: is it a disease-causing mutation or a sequence variant? AB - Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent attacks of serositis. To date more then 18 mutations responsible for the disease were identified in the MEFV gene, one such a mutation is E148Q in exon 2 of the gene. While screening FMF patients for mutations in the MEFV gene, we have identified 2 individuals parents of 2 unrelated FMF patients, who were homozygous for E148Q mutation. Upon clinical examination they were absolutely disease free and therefore raised the possibility that this mutation is a benign polymorphism rather than a mutation causing disease. To further investigate the role of the E148Q in FMF we analyzed 25 parents of FMF patients and a control group of 70 individuals, Jews of Moroccan extraction to match for ethnicity of the patients. The rate of E148Q in the control group was 6.4%, being 7.8% among the patient group. Among the parents group (obligatory carriers), in addition to the 2 parents that were homozygous E148Q, in 2 families one of the parents was heterozygote for E148Q but transmitted the other allele (apparently with unknown FMF mutation) to the affected child. Two healthy sibs of one of the E148Q homozygous were also homozygous E148Q. These observations are not in accordance to the notion that E148Q is a mutation causing disease. PMID- 10737997 TI - Identification of six novel WASP gene mutations in patients suffering from Wiskott-Aldrich syndrome. AB - Mutation in the gene encoding the Wiskott-Aldrich Syndrome protein (WASP) has been identified as the genetic defect responsible for WAS, an X-linked primary immunodeficiency disease characterized by eczema, thrombocytopenia, and recurrent infections. In this study, the WASP gene of 7 unrelated patients with classical WAS of Dutch descent was examined by single-strand conformation polymorphism and sequence analysis. We have identified 6 novel mutations that involve nonsense mutations (196C-->A, 344C-->T), or small deletions (553delG, 768del19, IVS8+1delGTGA, 911delT), all of which result in predicted truncation of WASP protein synthesis. PMID- 10737998 TI - Novel mutations of ubiquitin protein ligase 3A gene in Italian patients with Angelman syndrome. AB - Angelman syndrome is a neurobehavioral disorder caused by defects of imprinted gene(s) on chromosome 15q11-13. AS-specific DNA methylation is found in patients carrying 3-4 Mb deletions ( approximately 70%), paternal uniparental disomy (3 5%) or imprinting center mutations (2-9%), while normal methylation pattern with biparental inheritance characterizes the remaining approximately 20-25% AS patients (Stalker et al.,1998; Tsai et al.,1998). Mutations in the Ubiquitin protein ligase 3A gene (UBE3A) have been found in the latter group, but only preliminary figures are available on their frequencies. We selected a sample of 25 AS patients with a clinical diagnosis of AS and a normal methylation pattern in order to search for mutations of the UBE3A gene. Automated sequencing of exons 8, 9, 10, 11 and 12 performed on our 25 patients allowed us to identify three novel mutations: an 897insA in two unrelated familial cases, a 2544insA and an E167X in two sporadic cases. Mutation R482X previously reported in a sporadic patient was identified in a third familial case. Hum Mutat 15:387, 2000. PMID- 10737999 TI - Novel KCNQ1 mutations associated with recessive and dominant congenital long QT syndromes: evidence for variable hearing phenotype associated with R518X. AB - Congenital long QT syndrome may be transmitted as either an autosomal dominant or recessive trait. Two families with the autosomal recessive Jervell and Lange Nielsen syndrome (JLNS), and one family with the autosomal dominant Romano-Ward syndrome (RWS) were evaluated for mutations in KCNQ1. Two different novel frameshift mutations were discovered in one of the JLNS families (1188delC) and in the RWS family (504delG). A third allele (R518X) was observed in the second JLNS family. The R518X allele was previously associated with recessive long QT syndrome without deafness, but was present in a congenitally deaf proband in our study. These data extend the range of known KCNQ1 mutations associated with both recessive and dominant forms of congenital long QT syndrome, and demonstrate that the R518X allele may be associated with or without congenital deafness. PMID- 10738000 TI - Three novel mutations and twelve polymorphisms identified in the USH2A gene in Israeli USH2 families. AB - The Usher syndromes are autosomal recessive hereditary disorders characterized by hearing impairment and progressive visual loss due to Retinitis Pigmentosa (RP). Moderate to severe sensorineural hearing loss and progressive RP characterizes Usher syndrome type IIa (USH2A), which maps to the long arm of chromosome 1q41. Recently, three deletions carried by USH2 patients, which were found in a novel gene isolated from the critical 1q41 region, defined this gene as responsible for USH2A. The USH2A gene is predicted to encode a 1546 amino acid protein which possesses domains that are observed in basal lamina and extracellular matrix proteins and in cell adhesion molecules. Affected individuals and additional members from eleven USH2 Israeli families of diverse ethnic origin were screened for the presence of changes in all 20 coding exons of the USH2A gene. Three novel mutations (239-242insCGTA, R334W, T1515M) were identified in three families of Jewish Moroccan and Jewish Iranian origins. Twelve polymorphisms were found in the families, four of which are novel. None of the known USH2 mutations were identified in the families studied in this work. Hum Mutat 15:388, 2000. PMID- 10738001 TI - A novel polymorphism (219G>A) in the transferrin receptor gene. PMID- 10738002 TI - Novel WT1 exon 9 mutation (D396Y) in a patient with early onset Denys Drash syndrome. PMID- 10738003 TI - A novel mutation, Y103X, and exon skipping in a patient with Hunter disease. PMID- 10738004 TI - A novel mutation (Q239R) identified in a Taiwan Chinese patient with type VI mucopolysaccharidosis (Maroteaux-Lamy syndrome). PMID- 10738005 TI - Identification of a novel missense mutation (T16A) in the glucose-6-phosphatase gene in a Taiwan Chinese patient with glycogen storage disease Ia (Von Gierke disease). PMID- 10738006 TI - Identification of two novel mutations [P122S (364C>T) and 1601delAC] in the SLC3A1 gene in type I cystinurics. PMID- 10738007 TI - A novel missense mutation, S1159F, in exon 19 of the CFTR gene. PMID- 10738008 TI - An R223P mutation in EXT2 gene causes hereditary multiple exostoses. PMID- 10738010 TI - In memoriam PMID- 10738009 TI - From the editor PMID- 10738011 TI - Risk factors for exacerbations and hospital admissions in asthma of early childhood. AB - Hospital admissions and readmissions for asthma in early childhood remain causes for concern. The purpose of this study was to identify predisposing risk factors related to asthma exacerbations and precursors of hospital admissions in young children. Subjects were patients with doctor-diagnosed asthma from a clinical registration study, aged 0-4 years, and followed up for 2 years. Data from histories and laboratory tests for atopic status at initial presentation, and the patient's condition at visits over the 2-year follow-up period were evaluated. Exacerbation was defined as increases in cough and/or wheeze and/or breathlessness, increase in beta(2)-agonist use, and a clinical need for a short course of oral corticosteroids. Age groups 0-1 year and 2-4 years, based on age at initial presentation, were analyzed separately. In the age group 0-1 year, 71/113 (63%) patients had at least one exacerbation, and 20 experienced recurrent exacerbations (>/=3). Predisposing risk factors for exacerbation were damp housing (odds ratio (OR) 7.6 (2. 0-28.6)) and colds (OR 3.6 (1.4-9.6)), and for recurrent exacerbations sensitization to inhalant allergens (Phadiatop(R)) (OR 8.1 (1.6-40.5)) and damp housing (OR 3.8 (1.1-12.8)). Hospital admissions were significantly associated with number of exacerbations. In the age group 2-4 years, 58/144 (40%) patients had at least one exacerbation, and 21 experienced recurrent exacerbations (>/=2). Predisposing risk factors for exacerbation were mean age at initial presentation (OR 0.92 (0.88-0.97)) and level of total IgE (OR 2.3 (1.4-3.9)), whereas for recurrent exacerbations no predictor variables were found. Hospital admissions were significantly associated with damp housing. Results from this study may facilitate recognition of young asthmatic patients at risk of (recurrent) exacerbations, and help to identify those in whom early intervention with anti-inflammatory therapy may be necessary. We also emphasize the importance of preventive measures in decreasing damp housing. PMID- 10738013 TI - Randomized trial of salbutamol via metered-dose inhaler with spacer versus nebulizer for acute wheezing in children less than 2 years of age. AB - The aim of this study was to compare the efficacy of salbutamol delivered via a metered-dose inhaler with a spacer and facial mask (MDI-S) vs. a nebulizer (NEB) for the treatment of acute exacerbations of wheezing in children. In a single blind, prospective, randomized clinical trial, 123 outpatients (1-24 months of age), presenting with "moderate to severe" wheezing, were seen in the emergency department. Children were randomly assigned to one of two salbutamol treatment groups. In the first hour, the MDI-S group received 2 puffs (100 microg/puff) every 10 min for 5 doses, and the NEB group received 0.25 mg/kg every 13 min for 3 doses. If the clinical score was >5 at the end of the first hour, the patients received another hour of the same treatment and also betamethasone (0.5 mg/kg intramuscular). On enrollment and after the first and the second hour of treatment each child had a validated clinical score assigned by a blinded investigator. There were no differences at the time of admission to the emergency department between groups in clinical score or demographic data. Success (clinical score 0.05). We conclude that in this study population, children less than 2 years of age with moderate-severe exacerbations of wheezing responded faster to salbutamol delivered by MDI with a spacer and facial mask than to salbutamol delivered by nebulizer. PMID- 10738012 TI - Salivary cotinine levels in children presenting with wheezing to an emergency department. AB - We set out to evaluate salivary cotinine concentrations to judge tobacco smoke exposure among infants and children, and to examine the results in relation to age and wheezing. This was a case-control study of wheezing children (n = 165) and children without respiratory tract symptoms (n = 106) who were enrolled in the Pediatric Emergency Department at the University of Virginia. The age range of both wheezing and control patients was 2 months to 16 years. Questionnaires were combined with cotinine assays in saliva to evaluate exposure to environmental tobacco smoke (ETS) for each child. The prevalence of exposure to one or more smokers at home was high (68%); and 43% of the children enrolled were exposed to ETS from their mothers. According to the questionnaires, and after adjusting for age and race, a wheezing child in this study was more likely than a control to be exposed to at least one smoker at home (odds ratio = 1.9; 95% CI = 1.1-3.4). However, the odds of exposure to ETS from smoking mothers did not differ significantly between wheezing and control patients, and no significant association was found between the presence of wheezing and salivary cotinine levels. Among children exposed to ETS at home, cotinine levels were significantly higher in saliva from those under the age of two years, and from toddlers aged 2 and 3 years, compared to values from children over age 4 years. Moreover, the number of smokers in the home strongly influenced cotinine levels from children under age 4 years. In addition, higher cotinine levels were observed in saliva from children under age 2 years who were exposed to ETS from their mothers. Cotinine levels were similar and significantly correlated in paired samples of saliva and serum from children under 4 years of age (n = 54), (r = 0.92, P < 0.001). Based on information gathered from questionnaires, the results indicate that wheezing children were more likely than controls to be exposed to ETS at home. However, significant differences in ETS exposure between wheezing and control groups with respect to maternal smoke exposure or comparisons of salivary cotinine levels were not apparent. It was clear that determinations of salivary cotinine for monitoring the prevalence and intensity of household smoke exposure in this study were most valuable during the first 4 years of life. PMID- 10738014 TI - Effects of endotracheal suctioning in high-frequency oscillatory and conventionally ventilated low birth weight neonates on cerebral hemodynamics observed by near infrared spectroscopy (NIRS). AB - Adverse changes in cerebral hemodynamics during endotracheal suctioning have been reported in conventionally ventilated newborns, whereas observations on the effect of endotracheal suctioning during high-frequency ventilation have not been reported to date. The present study was designed to investigate the effect of endotracheal suctioning on cerebral hemodynamics in high-frequency and conventionally ventilated infants. Changes in cerebral concentration of oxygenated (cO(2)Hb) and deoxygenated hemoglobin (cHHb) and oxidized cytochrome aa3 (cCyt.aa3) were measured by noninvasive near-infrared spectroscopy. In an open prospective study, 26 suctioning periods in 9 high-frequency and in 6 conventionally ventilated newborn infants were investigated. Heart rate, arterial oxygen saturation (SaO(2)), mean blood pressure (MABP), and transcutaneous carbon dioxide tension (TcpCO(2)) were monitored continuously. In both groups, a marked decrease in heart rate, SaO(2) and in cO(2)Hb, an increase in cHHb, and a variable pattern in the concentration of total hemoglobin were noted during endotracheal suctioning. During suctioning, no statistically significant differences between the two methods of mechanical ventilation could be observed. We conclude that the mode of ventilation had no significant effect on changes in cerebral hemodynamics during endotracheal suctioning. PMID- 10738015 TI - Improvement in respiratory compliance after surfactant therapy evaluated by a new method. AB - Descriptions of the effects of intratracheally applied surfactant on respiratory system compliance (C(rs)) have been somewhat controversial because the commonly used methods for assessing pulmonary function were designed for a linear pressure/volume (P/V) relation of the respiratory system. In infants with lung disease a linear P/V relation cannot be expected. Therefore, a new method (APVNL) was employed which enabled us to calculate respiratory system compliance (C(rs)) and resistance (R(rs)) based on changes in volume (V). This method is independent of the P/V relation, and was used to assess the effects of intratracheal instillation of surfactant. Fourteen infants (gestational age, 24 to 30 weeks) with respiratory distress syndrome were treated with bovine surfactant intratracheally while the fractional inspired oxygen concentration (FiO(2)) exceeded 50%. C(rs) was evaluated for the infants using the APVNL method and the method of linear regression (LR) based on the equation of motion designed for linear P/V relationships. Two hours after surfactant treatment, the median reduction of FiO(2) was 33% (95% CI: 20-50%; P < 0.01). There was no correlation between the change in FiO(2) and the change in C(rs), using either the APVNL method or the LR method. Two hours after surfactant treatment, the median improvement in C(rs) was 0.37 mL/cmH(2)O/kg (95% CI: 0.07-1. 16 mL/cmH(2)O) at a change in V of 1 mL/kg (P < 0.02) and 0.23 mL/cmH(2)O/kg (95% CI: 0-0.57 mL/cmH(2)O) at a change in V of 2 mL/kg (P < 0.05) when the APVNL method was used. The LR method could not show a significant change in C(rs) after surfactant treatment. Further, R(rs) did not show significant changes 2 hr after surfactant administration. We conclude that the APVNL method is more appropriate for evaluating changes of C(rs) elicited by surfactant treatment than the LR method. The APVNL method demonstrated significant initial improvements in compliance as lung volumes were increased; there were no significant further decreases in C(rs) as peak inspiratory pressures and the upper limits of tidal volume were approached. PMID- 10738016 TI - Obstructive sleep apnea syndrome in obese Singapore children. AB - We set out to determine the prevalence of obstructive sleep apnea syndrome (OSAS) among obese Singapore school children and identify risk factors for OSAS. This study was designed as a prospective study in three phases. Parents completed a questionnaire with regards to sleep and daytime symptoms in Phase 1. Children suspected to have OSAS based on the questionnaire and all with a percent ideal body weight (IBW) >/=180 were called for clinic visits in Phase 2. All whose percent IBW >/=180 and those in whom the physicians strongly suspected OSAS were subjected to a polysomnogram in phase 3. The children were recruited from the School Health Nutritional Clinic for obese children. The investigations were carried out at Tan Tock Seng Hospital. In all, 3,671 children were screened with the questionnaire. Of these, 146 were selected to undergo polysomnography. Twenty six had abnormal sleep studies with apnea/hypoxia indices (AHIs) >5/hr. The significant clinical feature which correlated with OSAS was sleep sitting up (P = 0.005). The risk is higher in morbidly obese (IBW >/=180), with a prevalence of 13.3% (8/60), than in less obese children (IBW <180). One in eight (12.5%) of these children was asymptomatic and would have been missed based on the questionnaire. Presence of adenotonsillar hypertrophy led to increased risk of OSAS. The prevalence of OSAS was 0.7% (26/3,671) among the obese schoolchildren in Singapore, which is similar to the prevalence reported by others. Using discriminant analysis, the estimated prevalence increased to 5.7%. In the morbidly obese (IBW >/=180), the prevalence rate is higher at 13.3%. PMID- 10738017 TI - Inhaled mannitol identifies methacholine-responsive children with active asthma. AB - Inhaled mannitol has been developed for bronchial challenge testing in adults. This study determined if mannitol could identify children with active asthma and responsive to methacholine, and whether mannitol challenge was faster to complete than methacholine challenge. Twenty-five children (aged 6-13 years) responsive to methacholine and 10 nonasthmatic children unresponsive to methacholine were studied. The methacholine challenge (Cockcroft protocol) was followed by a mannitol challenge on separate days. Twenty-one asthmatic children were positive to mannitol. Three taking inhaled corticosteroids with borderline methacholine responsiveness did not respond to mannitol, and one could not complete the mannitol challenge due to cough. The geometric mean (GM) and 95% confidence interval (CI) for PD(15) for mannitol was 39 mg (19, 78), and PC(20) for methacholine was 0.6 mg/mL (0.35-1.02) (r(p) = 0.75, p < 0.001, n = 21). Responses to mannitol were repeatable: GM PD(15) for the first challenge was 29 mg (CI: 17,50), and for the second challenge, 33 mg (CI: 20, 55) (P = 0.44, n = 9). Mannitol was faster to administer than methacholine (median (range)) 14 min (5-32) vs. 29 min (19-49), respectively (P < 0.001). Time to recover to baseline FEV(1) spontaneously and after bronchodilator administration was similar for both challenges. There were no significant falls in arterial oxygen saturations. During mannitol challenge, the mean (SD) fall in FEV(1) in nonasthmatic children was 3.1% (2.9). We conclude that mannitol identifies children with airway hyperresponsiveness and is faster to perform than the methacholine challenge. PMID- 10738018 TI - A modified nasopharyngeal tube to relieve high upper airway obstruction. AB - Infants with high upper airway obstruction (UAO) are managed with a variety of techniques to relieve their UAO. Among these techniques, the least invasive and safest is the nasopharyngeal tube (NPT). However, the traditional NPT is not always satisfactory, and tracheostomies need to be done. We recently described a modified NPT technique that, in contrast to the traditional tube, does not add airway dead space and resistance, is easy to use, is well-tolerated, has proven highly successful, and allows the simultaneous use of oxygen nasal prongs. This modified NPT has many advantages over the traditional NPT as a temporary management of high UAO that resolves with growth of the infant. This report highlights the respiratory care of 10 infants with high UAO (Pierre Robin syndrome, Down syndrome, Goldenhar syndrome, isolated microngathia, and idiopathic hypotonia) who were managed with a modified NPT. The modified NPT described potentially reduces the need for surgical intervention to relieve high UAO in infants. PMID- 10738019 TI - Primary ciliary dyskinesia (PCD). AB - This article summarizes the current state of the scientific and clinical knowledge that relates to primary ciliary dyskinesia (PCD). Although PCD is a rare disease with a prevalence of 1 in 20,000 it has a well recognized morbidity. It is believed that an accurate diagnosis and the application of appropriate management can significantly reduce this morbidity. The cilia themselves are highly complicated organelles that perform important functions, particularly in the respiratory and reproductive tracts, and they have been the focus of many years of research. Our current knowledge of ciliary function and mucociliary clearance is summarized, and the relationship with laterality defects is discussed. A phenotype resembling PCD is also seen in animal models, and some of these are described before reviewing the clinical aspects of PCD in humans and new developments in the field that may have implications for the future investigation and management of affected individuals. PMID- 10738020 TI - Primary small cell bronchogenic carcinoma in a 14-year-old boy. AB - A 14-year-old Korean boy was admitted with cough, hemoptysis, and fever. A chest X-ray showed a solitary pulmonary mass and pneumonitis. Bronchial biopsy by fiberoptic bronchoscopy revealed a poorly differentiated small cell carcinoma. All of the staging information indicated that the patient had limited disease. During the 7 months following diagnosis, he received adjuvant chemotherapy in conjunction with radiotherapy. The size of the lesion was reduced by almost 50%. Small cell bronchogenic carcinoma has not been reported previously in childhood. PMID- 10738021 TI - Improvement of disseminated lymphangiomatosis with recombinant interferon therapy. AB - Disseminated lymphangiomatosis is a rare disorder with a poor prognosis. We present a case involving a 3-year-old boy who presented with pulmonary infiltrates, multiple lytic lesions of the ribcage, and small cystic lesions in the spleen. Open-lung and bone biopsies revealed disseminated lymphangiomatosis. Significant clinical and radiologic improvement were observed and persisted after 28 months of treatment with recombinant interferon alpha-2b (IFN alpha-2b). No significant toxicity has been observed. PMID- 10738022 TI - Selected abstracts PMID- 10738023 TI - What is a unit? PMID- 10738024 TI - Transfusion goes on line PMID- 10738025 TI - Acute anemic events in sickle cell disease. PMID- 10738026 TI - Transmission of tick-borne agents of disease by blood transfusion: a review of known and potential risks in the United States. PMID- 10738027 TI - Transfusion-associated transmission of babesiosis in New York State. AB - BACKGROUND: Babesiosis can be life-threatening in immunocompromised individuals. Although the disease is usually transmitted by tick bite, more than 20 cases have been reported of infection transmitted by transfusion of blood or blood components obtained from apparently healthy donors from endemic areas in the United States. This report describes several recent cases of transfusion transmitted babesiosis in New York State. STUDY DESIGN AND METHODS: Transfusion associated incidents of babesiosis infection were identified and investigated. Seroprevalence of babesiosis in healthy blood donors in a highly endemic area was ascertained. RESULTS: In three incidents, babesiosis was diagnosed in five of eight patients given infected blood: two premature infants, an elderly patient with gastrointestinal bleeding, and two patients with thalassemia. Seroprevalence in blood donors on Shelter Island (Suffolk County, eastern Long Island), a highly endemic area, was 4.3 percent in May 1998. CONCLUSIONS: Infected donors lived in endemic areas and were asymptomatic with no history of tick bite. Blood collected in January 1997 from one donor was infectious. Those transfusion recipients who were infected were neonatal, elderly, or chronically transfused patients. Babesiosis should be included in the differential diagnosis of febrile illness in immunocompromised recipients of blood transfusion, particularly in the Northeastern United States. PMID- 10738028 TI - Reducing the risk of transfusion-transmitted rickettsial disease by WBC filtration, using Orientia tsutsugamushiin a model system. AB - BACKGROUND: Careful donor screening and infectious disease marker testing have significantly reduced the incidence of transfusion-transmitted diseases and improved the safety of the blood supply. However, transfusion-transmitted diseases resulting from the use of asymptomatic yet infectious donors continue to put patients at risk. This study was undertaken to determine if third-generation WBC filters could remove Orientia tsutsugamushi-infected cells from contaminated blood. STUDY DESIGN AND METHODS: Packed RBCs were inoculated with human MNCs infected with O. tsutsugamushi at levels estimated to occur in asymptomatic infectious donors. WBC reduction was accomplished with a third-generation WBC filter. Prefiltration and postfiltration specimens were collected, serially diluted, and injected into mice to determine the infectivity of the samples. RESULTS: Mice receiving WBC-reduced packed RBCs showed no signs of illness or markers of infectivity, which suggested that a reduction of as much as 10(5) infectious rickettsiae could be achieved by filtration. CONCLUSION: The high efficiency, third-generation, WBC-reduction filters that were tested may provide protection against the transfusion transmission of scrub typhus rickettsiae by removing from contaminated blood cells that contain intracellular bacteria. PMID- 10738029 TI - GVHD after transfusion of stored RBC concentrates in a solution of mannitol, adenine, phosphate, citrate, glucose, and NaCl following trauma. AB - BACKGROUND: It has not previously been reported that WBC-reduced RBC preparations can cause transfusion-associated GVHD, even in an immunocompetent individual. CASE REPORT: A 74-year-old man suffered a hemorrhage from the mesentery of the transverse colon after a traffic accident. During surgery, he received 10 units of RBCs from 10 donors in a solution containing mannitol, adenine, phosphate, citrate, glucose and NaCl (MAP). MAP RBCs had been stored for 7 to 8 days before use. On the 27th day after surgery, an erythematous, pruritic rash appeared over the face, neck, and trunk, which was associated with low-grade fever and pancytopenia. Transfusion-associated GVHD was strongly suspected and was confirmed by skin biopsy. To determine the origin of lymphocytes causing GVHD, several microsatellite loci were amplified from DNA of the patient's nails and blood and from blood samples of all 10 RBC donors by using PCR. Amplified alleles derived from the patient's blood were identical to those from one of the 10 samples. CONCLUSION: These findings indicate that transfusions of MAP-RBCs can cause transfusion-associated GVHD in an elderly but immunocompetent host. PMID- 10738030 TI - An immediate, allergic skin reaction to aprotinin after reexposure to fibrin sealant. AB - BACKGROUND: The safety of fibrin tissue adhesives has been a concern since they entered wide clinical application. Most commercially available kits contain the proteolytic inhibitor, aprotinin, to stabilize the fibrin clot. A bovine protein, this substance has an allergenic potency. CASE REPORT: This case report presents a patient who had a generalized allergic skin reaction, probably triggered by aprotinin upon reexposure to fibrin sealant injected subgaleally to achieve closure of a liquor fistula after neurosurgical treatment. The serologic investigation revealed aprotinin-specific IgE and IgG. From 1990 through 1998, reports of five allergic reactions following 1 million exposures to fibrin sealant were made to the manufacturer. The clinical relevance of allergic reactions to aprotinin contained in fibrin sealants and measures to avoid them are discussed. CONCLUSION: These hypersensitivity reactions are extremely rare (incidence, 0.5/100,000 for all reactions and 0.3/100,000 for serious reactions), but they must be kept in mind as possible adverse events after repeated applications of fibrin sealants within a few weeks. PMID- 10738031 TI - TT virus infections among blood donors in Iceland: prevalence, genotypes, and lack of relationship to serum ALT levels. AB - BACKGROUND: The TT virus (TTV) is a newly identified blood-borne virus. Its association with disease is still unknown, and screening of blood donors has not been implemented. Several genotypes of the TTV have been identified. STUDY DESIGN AND METHODS: Three hundred seventy healthy blood donors were randomly selected and tested for TTV by the PCR method. Sequencing of a part of the genome was performed to identify various genotypes of the virus. ALT levels were determined in both infected and uninfected individuals. RESULTS: The TT virus (TTV), was detected in the sera of 23 (6.2%) of 370 healthy Icelandic blood donors; this prevalence is lower than that reported in Japan but higher than that in Scotland. The virus was found in all groups over the age of 19. Sequencing and phylogenetic analysis of 202 bp from open reading frame 1 demonstrated genotypes 1b and 2b 2c and genotype 4 isolates, with the latter bearing 89-percent nucleotide homology with other genotype 4 sequences deposited at GenBank. One sample showed a mixed genotype 1b/2c infection. Serum ALT levels were within normal limits in all infected individuals. CONCLUSION: The TTV carrier state does not cause significant liver injury. PMID- 10738032 TI - Molecular mechanisms that lead to reduced expression of duffy antigens. AB - BACKGROUND: In the Duffy blood group system, the null phenotype Fy(a-b-) has been classically associated with a mutated GATA box, while the Fy(x) phenotype weak Fy(b) is associated with Arg89Cys and Ala100Thr mutations. This report assesses the prevalence of the Duffy GATA box and the Fy(x)-associated mutations in white and African American (black) donors and investigates the molecular mechanism underlying the Fy(x) phenotype. STUDY DESIGN AND METHODS: PCR RFLP Duffy genotyping was performed on blood samples from blacks and whites. Duffy antigen expression (Fy(a), Fy(b), Fy6, Fy3) on RBCs was measured by flow cytometry. By site-directed mutagenesis, the relevance of each Fy(x)-associated mutation to Duffy (mRNA, antigen, and protein) expression was analyzed in transfectants by Northern blotting, flow cytometry, and immunoblotting. RESULTS: The mutated GATA box occurred at a high allele frequency (0.8) in blacks and was rare among whites. Conversely, the Fy(x)-associated mutations were absent in blacks, but present in 3.5 percent of whites. By flow cytometry, Duffy antigens (Fy(a) or Fy(b), Fy6 and Fy3) showed a dosage effect in RBC samples that were transcriptionally silenced by the GATA box mutation in one allele. By contrast, the reduced (10%) Duffy protein in Fy(x) RBCs was shown by heterologous expression analysis not to be due to reduced RNA levels, but to protein instability caused by Arg89Cys. CONCLUSIONS: Reduced Duffy expression can result from mutations affecting transcription (mutated GATA box in one allele) or instability of the translated protein (Arg89Cys). The frequencies of these mutations vary among populations. PMID- 10738033 TI - Evidence supporting the requirement for two proline residues for expression of c. AB - BACKGROUND: In humans, c antigen expression is associated with a proline residue at amino acid position 103 in the second extracellular loop of the CE protein. Comparison of nonhuman primate Rh proteins suggested that c reactivity might actually involve two proline residues. It has been shown that the RBCs of New World capuchin monkeys (Cebus apella) react with anti-c. To further define the amino acid residues involved in c expression, Rh cDNA from the capuchin was analyzed. STUDY DESIGN AND METHODS: Rh transcripts were amplified by reverse transcription PCR from RNA isolated from the reticulocytes of a capuchin monkey and were cloned and sequenced. RESULTS: Rh transcripts from the capuchin monkey, whose RBCs react with anti-c, were found to encode adjacent proline residues at 102 and 103. CONCLUSION: Sequencing of Rh transcripts from the capuchin monkey supports the hypothesis that the expression of c requires two adjacent proline residues. Proline causes bends or loops in proteins, which, in this case, might form a unique, stable structure resistant to perturbations induced by changes in upstream or downstream residues. This would explain the scarcity in humans of c variants as compared to the other major Rh antigen variants, and the preservation of c reactivity despite 24-percent divergence between the human and capuchin Rh proteins. PMID- 10738034 TI - Amino acid substitutions in human erythroid protein band 3 account for the low incidence antigens NFLD and BOW. AB - BACKGROUND: The low-incidence red cell antigens NFLD (700.37) and BOW (700.46) were first described in 1984 and 1988, respectively. Recent investigations showed that antigens of the Diego blood group system (including a number of low incidence antigens) are coded by SLC4A1 (solute carrier family 4, anion exchanger member 1 gene). Among these newly characterized Diego system antigens is Wu (designated DI9). Because a serologic relationship among Wu, NFLD, and BOW has been established, a series of genetic and molecular investigations of SLC4A1 in relation to NFLD and BOW were undertaken. STUDY DESIGN AND METHODS: By the use of exon-specific primers, single-strand conformational polymorphism (SSCP) analysis of SLC4A1 was performed on DNA isolated from an NFLD+ person from Japan, from the members of a Canadian kindred segregating for NFLD, and from two unrelated BOW+ persons. Exons displaying SSCPs were subjected to genetic linkage analysis (for NFLD only) and DNA sequencing. RESULTS: SSCPs in DNA amplified from exons 12 and 14 of SLC4A1 were observed for all NFLD+ subjects. Linkage between each of these polymorphisms and NFLD was established with peak lods = 4.82 at theta = 0.00 for combined paternal and maternal meiosis. DNA sequencing of exons 12 and 14 of SLC4A1 from NFLD+ persons identified A-->T and C-->G mutations that underlie Glu429Asp and Pro561Ala substitutions in human erythroid band 3 protein (band 3). DNA from the two unrelated BOW+ persons only exhibited an SSCP in exon 14 of SLC4A1. Subsequent DNA sequencing revealed a C-->T mutation that accounts for a Pro561Ser substitution in band 3. CONCLUSION: SLC4A1 codes for the low-incidence red cell antigens NFLD and BOW. In light of these findings, both antigens have been assigned to the Diego blood group system. PMID- 10738035 TI - Standardized units of RBCs: is it time for implementation? AB - BACKGROUND: Current practice for the preparation of RBCs from whole blood for transfusion results in poorly standardized contents of RBC Hb. The principle of apheresis, metering the anticoagulant into the collected blood, which is pumped into an empty container, allows variation in the collected volume according to properties of the donor. STUDY DESIGN AND METHODS: The total Hb mass of each person in a representative group of Swedish blood donors was evaluated by using Hb concentration and blood volume (BV), with the latter calculated from each donor's weight and height. The number of blood units that could be collected without exceeding 13 percent of the BV was estimated at a standardized content of RBC Hb set at 40, 45, and 50 g. RESULTS: With Hb standards of 45 and 50 g per unit of RBCs, it would be possible to collect 1 unit, but not more, from 93 female donors in the study; with 40 g of Hb as the standard, 2 units could be collected from 6 percent of the donors. Using a standard of 40 g of Hb, it would be possible to collect 2 units or more from 95 percent of 121 male donors. The corresponding figures at Hb standards of 45 and 50 g were 81 and 50 percent, respectively, of the male donors. The largest number of units that could be collected would thus be obtained at a 40-g Hb standard. However, the greatest total mass of RBC Hb would have been obtained at 45 g. Even the yield of plasma would reach a maximum at this RBC Hb standard. CONCLUSION: Depending on the donor's Hb and BV, it is possible to collect either 1 or 2 units of RBCs without exceeding 13 percent of any donor's BV, provided the collected volume of blood in each unit is less than the current standard. Such practice would allow better use of the donor population. Two-unit blood collections may reduce donor exposure in transfusions. Applying a standard at 45 g of RBC Hb per unit was found to permit the collection of maximum RBC Hb and plasma in the evaluated population of Scandinavian donors. Perhaps it is time to discuss a change in current rules for the preparation of RBCs for transfusion. PMID- 10738036 TI - Diversion of initial blood flow to prevent whole-blood contamination by skin surface bacteria: an in vitro model. AB - BACKGROUND: Sepsis arising from the transfusion of bacterially contaminated platelet components continues to be an infrequent, yet serious transfusion complication. Skin organisms are implicated in a number of these septic episodes. A model system was used to investigate if a skin organism's bioburden in blood components could be reduced by diverting the first few mL of whole blood away from the primary container. STUDY DESIGN AND METHODS: A sterile medication site was inserted into a bag containing sterile saline or whole blood; the site was deliberately contaminated with Staphylococcus aureus and allowed to dry. After needle puncture of the contaminated medication site, bacteria levels were measured 1) in successive 7-mL tubes of blood or saline drawn through a diversion arm, 2) in 40 mL of a connected transfer pack, and 3) in blood or saline from a needle puncture of the original container via another sterile medication port. RESULTS: Diverting the first 21 to 42 mL of saline or whole blood reduces the downstream bioburden of deliberately introduced surface S. aureus by approximately 1 log. CONCLUSION: Development of a diversion system for collection of whole blood in sample tubes before filling the primary container may reduce the bioburden of subsequently prepared components and thereby the frequency of sepsis due to skin organisms. PMID- 10738037 TI - Successful mobilization of peripheral blood HPCs with G-CSF alone in patients failing to achieve sufficient numbers of CD34+ cells and/or CFU-GM with chemotherapy and G-CSF. AB - BACKGROUND: Mobilization with chemotherapy and G-CSF may result in poor peripheral blood HPC collection, yielding <2 x 10(6) CD34+ cells per kg or <10 x 10(4) CFU-GM per kg in leukapheresis procedures. The best mobilization strategy for oncology patients remains unclear. STUDY DESIGN AND METHODS: In 27 patients who met either the CD34 (n = 3) or CFU-GM (n = 2) criteria or both (n = 22), the results obtained with two successive strategies-that is, chemotherapy and G-CSF at 10 microg per kg (Group 1, n = 7) and G-CSF at 10 microg per kg alone (Group 2, n = 20) used for a second mobilization course-were retrospectively analyzed. The patients had non-Hodgkin's lymphoma (5), Hodgkin's disease (3), multiple myeloma (5), chronic myeloid leukemia (1), acute myeloid leukemia (1), breast cancer (6), or other solid tumors (6). Previous therapy consisted of 10 (1-31) cycles of chemotherapy with additional chlorambucil (n = 3), interferon (n = 3), and radiotherapy (n = 7). RESULTS: The second collection was undertaken a median of 35 days after the first one. In Group 1, the results of the two mobilizations were identical. In Group 2, the number of CD34+ cells per kg per apheresis (0.17 [0.02-0.45] vs. 0.44 [0.11-0.45], p = 0. 00002), as well as the number of CFU-GM (0.88 [0.00-13.37] vs. 4.19 [0.96-21.61], p = 0.00003), BFU-E (0.83 [0.00-12.72] vs. 8.81 [1. 38-32.51], p = 0.00001), and CFU-MIX (0.10 [0.00-1.70] vs. 0.56 [0. 00-2.64], p = 0.001134) were significantly higher in the second peripheral blood HPC collection. However, yields per apheresis during the second collection did not significantly differ in the two groups. Six patients in Group 1 and 18 in Group 2 underwent transplantation, and all but one achieved engraftment, with a median of 15 versus 12 days to 1,000 neutrophils (NS), 22 versus 16 days to 1 percent reticulocytes (NS), and 26 versus 26 days to 20,000 platelets (NS), respectively. However, platelet engraftment was particularly delayed in many patients. CONCLUSION: G-CSF at 10 microg per kg alone may constitute a valid alternative to chemotherapy and G-CSF to obtain adequate numbers of peripheral blood HPCs in patients who previously failed to achieve mobilization with chemotherapy and G-CSF. This strategy should be tested in prospective randomized trials. PMID- 10738038 TI - Irradiation of platelet components: inhibition of lymphocyte proliferation assessed by limiting-dilution analysis. AB - BACKGROUND: Using a limiting-dilution analysis (LDA) assay that measures clonigenic T cells, it has been demonstrated that, with 2500 cGy, there is no T cell growth in red cell components irradiated in blood bags. In the current study, the LDA assay was used to investigate the effect of gamma radiation on the proliferative capacity of T cells in plateletpheresis components. STUDY DESIGN AND METHODS: Platelets were collected by using an apheresis instrument and settings that provided sufficient mononuclear cells for the LDA assay. Platelet components (n = 8) were irradiated in 1-L plastic bags 24 hours after collection with 500, 1500, and 2500 cGy of gamma radiation in a stepwise manner. Mononuclear cells were isolated after each irradiation dose by the use of ficoll-hypaque. A density separation medium was used to reduce the platelet numbers. T cells were enumerated by fluorescence-activated cell sorter and functionally assessed by LDA assay, which quantified T cells proliferating in the presence of polyclonal stimuli and cytokines. The frequency of T-cell growth (f) was visually scored after 4 weeks of incubation at 37 degrees C. Data were calculated as f(experimental)/f(control) and expressed as log(10) reduction. RESULTS: The T cell content of the mononuclear cell population was 17 +/- 10.5 percent, which was unaltered by irradiation. After 500-cGy irradiation, functional T cells were reduced by 2.09 log(10). Irradiation with 1500 cGy resulted in a 3. 96 log(10) reduction, but viable clonable T cells were detected in all experiments. With 2500-cGy irradiation, no T-cell growth was detected; this represented a greater than 4.86 log(10) reduction. CONCLUSION: As the dose of gamma radiation delivered to plateletpheresis components increased, the number of residual functional T cells decreased exponentially. Irradiation with 2500 cGy inactivates T cells in apheresis platelets, as measured by an LDA assay. PMID- 10738039 TI - Blood bank conditions and RBCs: the progressive loss of metabolic modulation. AB - BACKGROUND: Human RBC metabolism is modulated by the cell oxygenation state. Among other mechanisms, competition of deoxyhemoglobin and some glycolytic enzymes for the cytoplasmic domain of band 3 is probably involved in modulation. This metabolic modulation is connected to variations in intracellular NADPH and ATP levels as a function of the oxygenation state of the cell, and, consequently, it should have physiologic relevance. The present study investigates the effect of storage on this metabolic modulation and its relationship with the alteration of membrane protein composition. STUDY DESIGN AND METHODS: RBCs stored in CPD saline-adenine-glucose-mannitol were assayed for glucose uptake and partition between glycolysis and the pentose phosphate pathway at high and low oxygen saturation by nuclear magnetic resonance spectroscopy after 1, 14, 21, 35, and 42 days of storage. Membrane protein composition was determined by SDS-PAGE on Days 1, 14, 35, and 42. Metabolic values and 2,3 DPG concentration were also measured after rejuvenation for 1 hour at 37 degrees C with pyruvate-inosine-phosphate adenine solution on Day 21. RESULTS: Metabolic differences between RBCs incubated at high and low oxygen saturation decreased during storage, and, on Day 35, the two groups did not have significant differences (p = 0.111). SDS-PAGE showed that membrane protein composition was concurrently modified. The percentage of unmodified band 3 decreased during storage, principally between Days 14 and 35. In rejuvenated RBCs, oxygen-dependent modulation was not restored. CONCLUSIONS: RBCs stored in CPD-saline-adenine-glucose-mannitol do show a progressive loss of oxygen-dependent metabolic modulation, which is not restored after rejuvenation and which seems partly related to modifications in membrane proteins, mainly band 3. PMID- 10738040 TI - Effects of UVB radiation on cytokine generation, cell adhesion molecules, and cell activation markers in T-lymphocytes and peripheral blood HPCs. AB - BACKGROUND: Immunomodulatory effects of UV light have increasingly become a focus in transfusion medicine, BMT and transplantation immunology. In the transplant setting, the use of UVB radiation may reduce or abolish T-cell activation without compromising either bone marrow (BM) engraftment or graft-versus-leukemia effect. In this study, BM and apheresis-derived peripheral blood HPCs were used to investigate the effects of UVB on colony-forming ability, CD34+ cell viability, and growth potential, as well as on the secretion of MNC cytokines and the expression of cell surface markers and adhesion molecules. STUDY DESIGN AND METHODS: After UVB radiation, enriched populations of T cells and antigen presenting cells (APCs) were treated with PHA, and the MNC response was measured, as was colony-forming ability. CD34+ cells were quantified and their growth potential was determined in culture. Next, T-cell activation status, cell adhesion molecule and cell surface activation marker expression, and cytokine profiles were evaluated, and cytokine mRNA was quantitated. Parallel studies were done in unirradiated control cell populations. RESULTS: Low-dose (10 mJ/cm(2)) UVB mitigates MNC proliferative responses by 94 percent while maintaining 60 and 80 percent of colony-forming ability in peripheral blood HPC and BM preparations, respectively, and >50 percent of colony-forming ability in CD34+ cell-enriched samples. Low-dose UVB radiation also significantly reduces T-cell production of TNFalpha, TNFalpha mRNA, TNFbeta, IL-2, and IL-6 and downregulates T-cell expression of CD28, CD25, CD69, and intercellular adhesion molecule 1. CONCLUSION: These findings have shown that a "window" of low-dose UVB radiation (10 mJ/cm(2)) exists, at which BM- and peripheral blood-derived MNC proliferation is inactivated, while the HPCs are relatively spared. UVB light selectively affects T cells, while APCs are resistant to low doses of UVB. UVB radiation also alters the expression of some cell surface markers and cytokines that are important in T-cell activation pathways. Reduction of T-cell activation without cytocidal effect may allow UVB radiation to become an immunomodulating agent in BM or HPC transplantation. PMID- 10738041 TI - A prospective, randomized, sequential crossover trial of large-volume versus normal-volume leukapheresis procedures: effects on serum electrolytes, platelet counts, and other coagulation measures. AB - BACKGROUND: LVL procedures with the administration of heparin as an additional anticoagulant are increasingly performed because of the potentially higher yield of autologous peripheral blood HPCs. A prospective, randomized crossover trial was performed to evaluate the influence of leukapheresis volume-that is, large versus normal-on serum electrolytes, platelet count, and other coagulation measures in 25 patients with breast cancer and 14 patients with non-Hodgkin's lymphoma. STUDY DESIGN AND METHODS: Patients were randomly assigned to start either with an LVL on Day 1 followed by a normal-volume leukapheresis (NVL) on Day 2 or vice versa. In LVL, heparin was administered in addition to ACD-A. Bleeding complications, transfusion support, whole-blood counts, and several coagulation measures as well as plasma heparin levels were evaluated. RESULTS: Although the duration, the infused amount of ACD-A, the flow rate, the drop in platelet count, and the drop in potassium were significantly greater in LVL, and although LVL patients also received heparin, there was no significant difference in clinical tolerance or bleeding complications. After LVL, patients exhibited a significantly longer activated partial thromboplastin time (APTT), with a median of 70 seconds (range, 44-100 sec), and a median anti-factor Xa activity of 0.69 IU per mL (range, 0.10-1.29 IU/mL). The value of the APTT after LVL correlated with anti-factor Xa activity (r = 0.37, p<0.05), but not with platelet count or heparin infusion rate. Markers for coagulation activation did not increase during NVL or LVL. CONCLUSION: LVL with heparin as an additional anticoagulant seems to be a safe procedure in patients with low preleukapheresis platelet counts. No activation of coagulation occurred after NVL or LVL procedures. PMID- 10738042 TI - Fulminant babesiosis treated with clindamycin, quinine, and whole-blood exchange transfusion. AB - BACKGROUND: Babesiosis is an increasingly recognized parasitic infection with manifestations that range from a subclinical or mild flu-like illness to life threatening disease. Risk factors that may be associated with a more severe clinical course include immunosuppression, splenectomy, and advanced age. The most effective chemotherapeutic regimen, clindamycin plus quinine, is sometimes ineffective in cases of severe disease. CASE REPORT: A previously healthy, 58 year-old man was infected by Babesia microti, presumably through a tick bite. He developed fulminant disease characterized by severe hemolytic anemia, disseminated intravascular coagulation, acute renal failure, and respiratory failure. There was no history of splenectomy or immunodeficiency. He was given oral clindamycin (300 mg/4x/day) 2 days before admission. Oral quinine (650 mg/3x/day) was added upon hospitalization. There was no clinical improvement despite antibiotic therapy with clindamycin and quinine. On the second hospital day, a whole-blood exchange transfusion was performed to simultaneously lower the parasite load and replace the patient's plasma. With an automated blood cell separator, 87 percent of the patient's total blood volume was exchanged. As replacement fluid, 6.7 L of packed RBCs reconstituted with FFP (average Hct, 33%) was used. The patient's Hct increased from 26.9 percent before the exchange to 28.3 percent after the exchange. The percentage of parasitized RBCs decreased from 13.8 percent just before exchange to 4.2 percent immediately after exchange. There was rapid clinical improvement after the whole-blood exchange transfusion. The patient's subsequent clinical course was marked by a disappearance of the parasitemia and continued slow, general improvement. Therapy with clindamycin was continued for 14 days after the exchange transfusion and quinine for 17 days. CONCLUSION: In cases of severe babesiosis, prompt institution of whole-blood exchange transfusion, in combination with appropriate antimicrobial therapy, can be life-saving. PMID- 10738043 TI - Transfusion of buffy coat-depleted blood components and risk of postoperative infection in orthopedic patients. PMID- 10738044 TI - Clinical consequences of alterations in platelet transfusion dose: a prospective, randomized, double-blind trial. PMID- 10738045 TI - The cost-effectiveness of autologous transfusion revisited: implications of an increased risk of bacterial infection with allogeneic transfusion. PMID- 10738046 TI - Lack of difference in CMV transmission via the transfusion of filtered irradiated and nonfiltered irradiated blood to newborn infants in an endemic area. PMID- 10738047 TI - Rationale for universal WBC reduction of blood components? PMID- 10738048 TI - Hypertension and antihypertensive therapy as risk factors for type 2 diabetes mellitus. Atherosclerosis Risk in Communities Study. AB - BACKGROUND: Previous research has suggested that thiazide diuretics and beta blockers may promote the development of type 2 diabetes mellitus. However, the results of previous studies have been inconsistent, and many studies have been limited by inadequate data on outcomes and by potential confounding. METHODS: We conducted a prospective study of 12,550 adults 45 to 64 years old who did not have diabetes. An extensive health evaluation conducted at base line included assessment of medication use and measurement of blood pressure with a random-zero sphygmomanometer. The incidence of new cases of diabetes was assessed after three years and after six years by measurement of serum glucose concentrations while the subjects were fasting. RESULTS: After simultaneous adjustment for age, sex, race, education, adiposity, family history with respect to diabetes, physical activity level, other health-related behavior, and coexisting illnesses, subjects with hypertension who were taking thiazide diuretics were not at greater risk for the subsequent development of diabetes than were subjects with hypertension who were not receiving any antihypertensive therapy (relative hazard, 0.91; 95 percent confidence interval, 0.73 to 1.13). Likewise, subjects who were taking angiotensin-converting-enzyme inhibitors and calcium-channel antagonists were not at greater risk than those not taking any medication. In contrast, subjects with hypertension who were taking beta-blockers had a 28 percent higher risk of subsequent diabetes (relative hazard, 1.28; 95 percent confidence interval, 1.04 to 1.57). CONCLUSIONS: Concern about the risk of diabetes should not discourage physicians from prescribing thiazide diuretics to nondiabetic adults who have hypertension. The use of beta-blockers appears to increase the risk of diabetes, but this adverse effect must be weighed against the proven benefits of beta blockers in reducing the risk of cardiovascular events. PMID- 10738049 TI - Amiodarone to prevent recurrence of atrial fibrillation. Canadian Trial of Atrial Fibrillation Investigators. AB - BACKGROUND: The restoration and maintenance of sinus rhythm is a desirable goal in patients with atrial fibrillation, because the prevention of recurrences can improve cardiac function and relieve symptoms. Uncontrolled studies have suggested that amiodarone in low doses may be more effective and safer than other agents in preventing recurrence, but this agent has not been tested in a large, randomized trial. METHODS: We undertook a prospective, multicenter trial to test the hypothesis that low doses of amiodarone would be more efficacious in preventing recurrent atrial fibrillation than therapy with sotalol or propafenone. We randomly assigned patients who had had at least one episode of atrial fibrillation within the previous six months to amiodarone or to sotalol or propafenone, given in an open-label fashion. The patients in the group assigned to sotalol or propafenone underwent a second randomization to determine whether they would receive sotalol or propafenone first; if the first drug was unsuccessful the second agent was prescribed. Loading doses of the drugs were administered and electrical cardioversion was performed (if necessary) within 21 days after randomization for all patients in both groups. The follow-up period began 21 days after randomization. The primary end point was the length of time to a first recurrence of atrial fibrillation. RESULTS: Of the 403 patients in the study, 201 were assigned to amiodarone and 202 to either sotalol (101 patients) or propafenone (101 patients). After a mean of 16 months of follow-up, 71 of the patients who were assigned to amiodarone (35 percent) and 127 of those who were assigned to sotalol or propafenone (63 percent) had a recurrence of atrial fibrillation (P<0.001). Adverse events requiring the discontinuation of drug therapy occurred in 18 percent of the patients receiving amiodarone, as compared with 11 percent of those treated with sotalol or propafenone (P=0.06). CONCLUSIONS: Amiodarone is more effective than sotalol or propafenone for the prevention of recurrences of atrial fibrillation. PMID- 10738050 TI - Viral load and heterosexual transmission of human immunodeficiency virus type 1. Rakai Project Study Group. AB - BACKGROUND AND METHODS: We examined the influence of viral load in relation to other risk factors for the heterosexual transmission of human immunodeficiency virus type 1 (HIV-1). In a community-based study of 15,127 persons in a rural district of Uganda, we identified 415 couples in which one partner was HIV-1 positive and one was initially HIV-1-negative and followed them prospectively for up to 30 months. The incidence of HIV-1 infection per 100 person-years among the initially seronegative partners was examined in relation to behavioral and biologic variables. RESULTS: The male partner was HIV-1-positive in 228 couples, and the female partner was HIV-1-positive in 187 couples. Ninety of the 415 initially HIV-1-negative partners seroconverted (incidence, 11.8 per 100 person years). The rate of male-to-female transmission was not significantly different from the rate of female-to-male transmission (12.0 per 100 person-years vs. 11.6 per 100 person-years). The incidence of seroconversion was highest among the partners who were 15 to 19 years of age (15.3 per 100 person-years). The incidence was 16.7 per 100 person-years among 137 uncircumcised male partners, whereas there were no seroconversions among the 50 circumcised male partners (P<0.001). The mean serum HIV-1 RNA level was significantly higher among HIV-1 positive subjects whose partners seroconverted than among those whose partners did not seroconvert (90,254 copies per milliliter vs. 38,029 copies per milliliter, P=0.01). There were no instances of transmission among the 51 subjects with serum HIV-1 RNA levels of less than 1500 copies per milliliter; there was a significant dose-response relation of increased transmission with increasing viral load. In multivariate analyses of log-transformed HIV-1 RNA levels, each log increment in the viral load was associated with a rate ratio of 2.45 for seroconversion (95 percent confidence interval, 1.85 to 3.26). CONCLUSIONS: The viral load is the chief predictor of the risk of heterosexual transmission of HIV-1, and transmission is rare among persons with levels of less than 1500 copies of HIV-1 RNA per milliliter. PMID- 10738051 TI - HTLV-II-associated cutaneous T-cell lymphoma in a patient with HIV-1 infection. PMID- 10738052 TI - Images in clinical medicine. Furuncular myiasis. PMID- 10738053 TI - Sedation and analgesia for procedures in children. PMID- 10738054 TI - Medical termination of pregnancy. PMID- 10738055 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 10-2000. A 63-year-old man with changes in behavior and ataxia. PMID- 10738056 TI - Investigators' responsibilities for human subjects in developing countries. PMID- 10738057 TI - Antihypertensive therapy and the risk of type 2 diabetes mellitus. PMID- 10738058 TI - Preventing sexual transmission of HIV--new ideas from sub-Saharan Africa. PMID- 10738060 TI - Correction: Congenital Heart Disease in Adults. PMID- 10738059 TI - Correction: Looking Back on the Millennium in Medicine. PMID- 10738061 TI - Visual attention and metacontrast modify latency to perception in opposite directions. AB - In human observers, cue-induced visual attention ('bottom-up' transient focal attention) shortens the latency of perception. Metacontrast reduces the intensity of perception and can even obliterate it. We show that a close relationship exists between both, but that their effects are reversed: cue-induced visual attention not only shortens latency but also intensifies perception, and metacontrast not only lowers intensity of perception but also prolongs latency. A common neurophysiological mechanism for both is possible. Indirect evidence suggests that this could be a subthreshold modulation of neuronal thresholds by de- and hyperpolarization. PMID- 10738062 TI - Hyperacuity deficits in anisometropic and strabismic amblyopes with known ages of onset. AB - In order to evaluate the influence of etiology of amblyopia and of age at onset of amblyopia on the resulting constellation of spatial vision deficits, resolution/vernier and recognition/resolution acuity ratios were measured in groups of children with either strabismic amblyopia or anisometropic amblyopia with known ages of onset. Strabismic amblyopia with infantile onset (<9 months) and strabismic amblyopia with late onset (18-30 months) were both associated with abnormally low resolution/vernier and abnormally high recognition/resolution acuity ratios. Among amblyopes with infantile onset (<9 months), moderate amblyopia was associated with different resolution/vernier and recognition/resolution acuity ratios in anisometropic and strabismic groups. Infantile amblyopes with poor acuity outcomes included children who initially presented with anisometropia but later developed strabismus and children who initially presented with esotropia but later developed anisometropia; both subgroups with mixed amblyopia had poor resolution/vernier acuity ratios. Data from moderate amblyopes support the hypothesis that anisometropia and strabismus disrupt visual maturation in fundamentally different ways rather than simply at different stages in visual development. PMID- 10738063 TI - Attentional modulation of motion-in-depth processing. AB - I used a novel effect of adaptation to a moving stimulus to investigate the role of attention in the processing of motion-in-depth (MID). Following adaptation to expansion, the time required to detect an expanding target in the same location as the adaptation target was significantly longer than following viewing of a constant-sized target. Conversely, following adaptation to contraction, detection times (DTs) for an expanding target were significantly shorter than for a constant-sized adaptation target. Changes in DTs following adaptation to contraction were substantially larger than those following adaptation to expansion. Attentional modulation of MID processing was examined by adding an alphanumeric discrimination task that distracted the observer's attention away from the location of the adaptation target. I compared the magnitude of DT effects while (a) observers passively fixated the alphanumeric sequence (single task condition) and (b) observers performed the discrimination task (dual task condition). DT effects were significantly smaller in the dual task condition than in the single task condition indicating that MID processing is modulated by attention. PMID- 10738064 TI - Infant color vision: sharp chromatic edges are not required for chromatic discrimination in 4-month-olds. AB - In our previous demonstrations of chromatic discrimination in infants, we have used test and surround fields of different chromaticities that abutted each other at sharp chromatic edges. In order to see whether sharp chromatic edges are necessary for infants to make chromatic discriminations, 16-week-old infants were tested with three stimulus configurations in which sharp chromatic edges were eliminated. The three edge manipulations involved black borders, a dark surround, or blurred edges around the chromatic test field. In each case red, green, and violet test fields were used. Although performance decreased when sharp chromatic edges were eliminated, observers' percent correct scores remained clearly above chance for eight of the nine discriminations (three colors x three edge manipulations). We argue that all three edge manipulations reduce the likelihood of mediation of chromatic discrimination by M (magnocellular) cells. These data thus provide evidence that young infants have functional P (parvocellular) pathways, and use them for making chromatic discriminations. PMID- 10738065 TI - Apparent string shortening concomitant with letter crowding. AB - In our previous studies of the crowding effect, we have observed that human observers tend to underestimate the length of a letter string (the number of letters in the string) when the letters are close to visual acuity, and the interletter spacings are small. In this study, we asked our observers to identify letters in randomly presented four-letter and five-letter strings. We found that, when a priori knowledge of the lengths of letter strings was not available, the probability of underestimating string length increased with decreasing interletter spacing. The causes of underestimation errors appeared to be the omission of an interior letter and the merging of two neighboring letters. Since our experiments were conducted in the foveal region, neither spatial uncertainty nor split attention can explain the underestimation errors. The effect of the point spread function of the eye on closely packed letter strings is discussed. PMID- 10738066 TI - Global motion adaptation. AB - Image motion is initially detected locally. Local motion signals are then integrated across space in order to specify the global motion of objects or surfaces. It is well known that prolonged exposure to motion causes adaptation at the local motion level. We have investigated whether adaptation also occurs at the global motion level. We have devised a global motion stimulus (a random dot kinematogram) which has equal motion energy in opposite directions but nonetheless gives rise to global motion perception. At the local motion level, adaptation to this stimulus should cause equal adaptation in both directions and should not give rise to an aftereffect. Any aftereffect seen must therefore be attributable to adaptation at the global motion level. We find that following adaptation to this stimulus, judgements of the perceived direction of a test pattern are systematically biased towards the direction opposite to the adapting direction, suggesting that adaptation does occur at a level of visual processing at which global motion is represented. PMID- 10738067 TI - Luminance spatial frequency differences facilitate the segmentation of superimposed textures. AB - Do superimposed textures segregate on the basis of a difference in their luminance spatial frequency? We addressed this question using orientation gratings, which consist of dense arrays of Gabor micropatterns whose orientations vary sinusoidally across space. Two orientation gratings of the same texture spatial frequency were combined in anti-phase, to produce a 'dual-modulation' orientation grating. Thresholds for detecting the dual-modulation gratings were measured as a function of the difference in Gabor spatial frequency between the two grating components. When the two components were made from the same Gabors, thresholds were relatively high. However a one octave difference in Gabor spatial frequency between the components caused thresholds to fall close to those of single-modulation orientation gratings. The fall in threshold was accompanied by a change in appearance of the stimulus; to that of two transparent, interwoven, flow patterns. We show that these results are incompatible with current Filter Rectify-Filter models of 'second-order' pattern detection. Rather, they favour the idea that feature analysis precedes texture analysis, with the visual system encoding local orientation content prior to the texture stage. PMID- 10738068 TI - Attention and short-term memory in contrast detection. AB - Low-contrast visual stimuli have been found to produce a memory trace, enhancing subsequent target detection for as much as 16 s. Here we show that the memory trace depends on dynamic interactions between low-level stimulus properties and a higher-level gating process. Detection of vertical targets (Gabor signals) was enhanced by preceding vertical Gabor primes, but suppressed by preceding tilted primes--pointing to a competitive process of dynamic resource allocation. The priming effect was also dependent on a temporal cue, activating a sensory gating process with maximal effect at 300-500 ms delay. The results suggest a two-step process in which attention affects transition between perception and memory: a non-selective gating process followed by competition between overlapping representations. PMID- 10738069 TI - Asymmetries in the time-course of chromatic adaptation and the significance of contrast. AB - The time-course of chromatic adaptation was determined as a function of the spectral content of the adaptation-light and of image-contrast. The adaptation lights varied along the chromatic cardinal axes or one of their intermediate axes in an equiluminant plane in colour-space. We found an asymmetry in the initial time-course of adaptation (0.2-10 s) in that adaptation to middle-wavelength light was significantly faster than adaptation to short- and long-wavelength light. The asymmetry was only observed in the presence of a spatially complex pattern. It was fully supported by luminance and chromatic contrast and present under haploscopic stimulus conditions. PMID- 10738070 TI - Continuity-based and discontinuity-based segmentation in transparent and spatially segregated global motion. AB - The mechanisms underlying the parsing of a spatial distribution of velocity vectors into two adjacent (spatially segregated) or overlapping (transparent) motion surfaces were examined using random dot kinematograms. Parsing might occur using either of two principles. Surfaces might be defined on the basis of similarity of motion vectors and then sharp perceptual boundaries drawn between different surfaces (continuity-based segmentation). Alternatively, detection of a high gradient of direction or speed separating the motion surfaces might drive the process (discontinuity-based segmentation). To establish which method is used, we examined the effect of blurring the motion direction gradient. In the case of a sharp direction gradient, each dot had one of two directions differing by 135 degrees. With a shallow gradient, most dots had one of two directions but the directions of the remainder spanned the range between one motion-defined surface and the other. In the spatial segregation case the gradient defined a central boundary separating two regions. In the transparent version the dots were randomly positioned. In both cases all dots moved with the same speed and existed for only two frames before being randomly replaced. The ability of observers to parse the motion distribution was measured in terms of their ability to discriminate the direction of one of the two surfaces. Performance was hardly affected by spreading the gradient over at least 25% of the dots (corresponding to a 1 degrees strip in the segregation case). We conclude that detection of sharp velocity gradients is not necessary for distinguishing different motion surfaces. PMID- 10738071 TI - The role of the attention focus in the visual information processing underlying saccadic adaptation. AB - Three experiments were performed to determine how an error signal for driving saccadic adaptation is derived from visual information processing. The first experiment demonstrated that an intrasaccadic displacement of a visual background does not influence saccadic adaptation when a small foveal target is used. The second experiment showed that when a different type of target, a 4.8 deg annulus, is used an intrasaccadic background shift influences the adaptive process. The third experiment showed that the size of the saccade target determines the size of the attention focus around the time of a saccade. These findings suggest that the attention focus selects the visual information used for a trans-saccadic comparison in order to generate the error signal. PMID- 10738072 TI - Computational modelling of interleaved first- and second-order motion sequences and translating 3f+4f beat patterns. AB - Despite detailed psychophysical, neurophysiological and electrophysiological investigation, the number and nature of independent and parallel motion processing mechanisms in the visual cortex remains controversial. Here we use computational modelling to evaluate evidence from two psychophysical studies collectively thought to demonstrate the existence of three separate and independent motion processing channels. We show that the pattern of psychophysical results can largely be accounted for by a single mechanism. The results demonstrate that a low-level luminance based approach can potentially provide a wider account of human motion processing than generally thought possible. PMID- 10738073 TI - Weighted directional energy model of human stereo correspondence. AB - Previous work [Prince, S. J. D, & Eagle, R. A. (1999). Size-disparity correlation in human binocular depth perception. Proceedings of the Royal Society: Biological Sciences, 266, 1361-1365] has demonstrated that disparity sign discrimination performance in isolated bandpass patterns is supported at disparities much larger than a phase disparity model might predict. One possibility is that this extended performance relies on a separate second-order system [Hess, R. F., & Wilcox, L. M. (1994). Linear and non-linear filtering in stereopsis. Vision Research, 34, 2431-2438]. Here, a 'weighted directional energy' model is developed which explains a large body of crossed versus uncrossed disparity discrimination data with a single mechanism. This model assumes a population of binocular complex cells at every image point with a range of position disparity shifts. These cells sample a local energy function which is weighted so that energy at large disparities is relatively attenuated. Disparity sign is determined by summing and comparing energy at crossed and uncrossed disparities in the presence of noise. The model qualitatively predicts matching data for one-dimensional Gabor stimuli. This scheme also predicts DMax in Gabor stimuli and filtered noise. Moreover, a range of 'non-linear' phenomena, in which disparity is perceived from contrast envelope information alone, can be explained. The weighted directional energy model presents a biologically plausible, parsimonious explanation of matching behaviour in bandpass stimuli for both 'first-order' and 'second-order' stimuli which obviates the need for multiple mechanisms in stereo correspondence. PMID- 10738074 TI - Local luminance factors that determine the maximum disparity for seeing cyclopean surface shape. AB - We measured the maximum disparity grating amplitude (d(max)) for seeing cyclopean surface shape, using stereograms made from dense arrays of micropatterns, whose luminance characteristics were manipulated. In Experiment 1, we used disparity gratings made from Gabor micropatterns. D(max) was found to vary inversely both with luminance spatial frequency and with Gabor size, but was constant for a constant bandwidth (frequency times size). To test whether this was due to changes in bandwidth per se or to changes in the number of local features, in Experiment 2 we manipulated the local feature content with a range of micropatterns that we termed 'edgels'. The results supported neither hypothesis. In Experiment 3 we varied the phases of the Fourier components of square wave edgels, thereby introducing more features, and we found that this did not change d(max). Taken together, our results show that d(max) decreases with an increase in the number of local luminance cycles at each luminance scale. D(max) is mainly limited by false target matching between similar components of the micropatterns. Stereopis, in terms of surface shape perception, is served only by first order mechanisms, and only by luminance filters that are broadband. PMID- 10738076 TI - Radiation therapy for breast cancer at the millennium. PMID- 10738075 TI - Stereodeficient subjects demonstrate non-linear stereopsis. AB - There appear to be two modes of stereoscopic processing: a conventional linear operation that is dependent on correspondence between local luminance components in the two eyes' views, and a non-linear or second-order processing mode. This second mode may use disparity information provided by particular 'non-Fourier' features of the stimulus such as the contrast envelope. Preliminary results suggest that people who fail standard clinical stereotests are able to extract non-linear disparity information from Gabor stimuli [McColl & Mitchell, 1998. Vision Research, 38, 1889-1900]. Here we evaluate the status of the non-linear mechanism in such individuals by using two types of contrast enveloped stimuli, namely random line and Gabor micropatterns, in a task that requires near/far depth judgements [Ziegler & Hess, 1999. Vision Research, 39, 1491-1507]. Although our sample was small, three of our four subjects who had performed poorly on at least one standard clinical test of stereopsis could perform the task, as well as one 'stereoblind' subject who had failed all four standard clinical tests. The overall results suggest that individuals with stereoanomalies show a diversity of deficits, but some nevertheless can see depth using 'non-linear' mechanisms. PMID- 10738077 TI - Clinical outcomes of post-operative locoregional radiotherapy in pre-menopausal and post-menopausal Chinese women with breast cancer. AB - BACKGROUND AND PURPOSES: Recent randomized studies from the West show that post operative locoregional radiotherapy improves survival in lymph node (LN) positive pre-menopausal women with breast cancer but this benefit has not been established in the Chinese population. There is no published study on clinical outcomes (locoregional recurrence, survival and toxicities) of post-operative locoregional radiotherapy in Chinese women with breast cancer. MATERIALS AND METHODS: We conducted a retrospective study of 399 female Chinese patients with breast cancer who had received post-mastectomy locoregional radiotherapy in our center between 1984 and 1990. The patients were stratified according to tumor size, menopausal and LN status. We analyzed the incidence and pattern of locoregional recurrence, distant recurrence, survival rates and toxicity related to radiotherapy. RESULTS: Of the 399 patients 216 were pre-menopausal and 183 were post-menopausal. The mean age was 49.3 years (23-83). Distribution of tumor size and LN status of the two groups was similar. Median follow-up was 71.9 months. Locoregional recurrence occurred in 57 (14.3%) patients (pre-menopausal 24 (11.1%); post-menopausal 33 (18.3%) P=0.489). Recurrence was more common in LN positive patients (18.2%) than LN negative patients (9.2%). The pattern of locoregional recurrence was: chest wall 24, axilla LN 12, supraclavicular LN 10, chest plus LN 11. The distant recurrence rate was 39.6% for all patients and 75.4% for patients with locoregional recurrences. The overall 10-year survival rate for all patients was 54%. For LN positive patients the 10-year survival rate of pre-menopausal and post-menopausal women was 38 and 51%, respectively (P=0.207), and for LN negative patients the rate was 73 and 70%, respectively (P=0.603). Acute skin toxicity included redness (30.8%), dry desquamation (12.8%), and wet desquamation (6. 8%). Long-term toxicities included skin atrophy (0.3%), telangectasia (3.3%), pneumonitis (2.8%) and brachial plexus palsy (1.3%). CONCLUSIONS: In our series Chinese patients with node-positive breast cancer have a relatively high locoregional recurrence rate in spite of mastectomy and post-operative radiotherapy. Limited use of adjuvant system chemotherapy may account, at least in part, for this finding. Clinical outcomes of post-operative radiotherapy in pre-menopausal and post-menopausal breast cancer patients are similar. PMID- 10738078 TI - Optimized MLC-beam arrangements for tangential breast irradiation. AB - BACKGROUND AND PURPOSE: Very large numbers of women are treated with tangential breast irradiation after breast-conserving surgery due to mammary carcinoma. The aim of this study was to improve a conventional treatment plan by modifying the dose intensity in the beams to reduce the absorbed dose outside the planning target volume (PTV) and to reduce the absorbed dose variation inside the PTV diotherapy of mammary carcinoma. MATERIALS AND METHODS: Treatment planning was performed both with conventional technique and with a simple intensity modulation technique for 12 consecutive patients. RESULTS: In all cases a higher degree of dose conformity was obtained with the dose intensity modulation technique. The relative gain was found to be similar for all patients irrespective of the size of the target volume or the irradiated lung volume. CONCLUSION: Simple manual intensity modulation can be used to improve the dose distribution in tangential breast irradiation. With modern accelerators the increased time for this technique is less than 2 min per fraction. PMID- 10738079 TI - Evaluation of the dose uniformity for double-plane high dose rate interstitial breast implants with the use of dose reference points and dose non-uniformity ratio. AB - BACKGROUND AND PURPOSE: To investigate the influence of dwell time optimizations on dose uniformity characterized by dose values in dose points and dose non uniformity ratio (DNR) and to analyze which implant parameters have influence on the DNR. MATERIALS AND METHODS: Double-plane breast implants with catheters arranged in triangular pattern were used for the calculations. At a typical breast implant, dose values in dose reference points inside the target volume and volumes enclosed by given isodose surfaces were calculated and compared for non optimized and optimized implants. The same 6-cm treatment length was used for the comparisons. Using different optimizations plots of dose non-uniformity ratio as a function of catheter separation, source step size, number of catheters, length of active sections were drawn and the minimum DNR values were determined. RESULTS: Optimization resulted in less variation in dose values over dose points through the whole volume and in the central plane only compared to the non optimized case. At implant configurations consisting of seven catheters with 15 mm separation, 5-mm source step size and various active lengths adapted according to the type of optimization, the no optimization, geometrical (volume mode) and dose point (on dose points and geometry) optimization resulted in similar treatment volumes, but an increased high dose volume was observed due to the optimization. The dose non-uniformity ratio always had the minimum at average dose over dose normalization points, defined in the midpoints between the catheters through the implant volume. The minimum value of DNR depended on catheter separation, source step size, active length and number of catheters. The optimization had only a small influence on DNR. CONCLUSIONS: In addition to the reference points in the central plane only, dose points positioned in the whole implant volume can be used for evaluating the dose uniformity of interstitial implants. The dose optimization increases not only the dose uniformity within the implant but also the high dose volume. The optimization on dose points and geometry provides the most uniform dose distribution. The dose non-uniformity ratio can be minimized by selecting the isodose line of the midpoints between the catheters in the whole volume for the dose prescription, but the dose coverage may not be adequate. For a clinically acceptable plan, a compromise should be made between dose non-uniformity and coverage. PMID- 10738080 TI - A survey on staging and treatment in uterine cervical carcinoma in the Radiotherapy Cooperative Group of the European Organization for Research and Treatment of Cancer. AB - BACKGROUND: The treatment outcome of advanced stage uterine cervical carcinoma remains unsatisfactory. In order to elaborate a novel trial within The Radiotherapy Cooperative Group (RCG) of the European Organization for Research and Treatment of Cancer (EORTC), we conducted a survey in 1997-1998 to determine the variability of pre-treatment assessment and treatment options. The variability of choosing surgery, defined radiation therapy techniques and chemotherapy are investigated, as well as the center's choices of future treatment strategies. METHODS: Fifty two of 81 RCG centers from the RCG have participated in the survey. As one would expect, there is a large variation in the techniques used for pretreatment evaluation and treatment options. There is no 'standard' for reporting acute and late side effects. Chemotherapy is used neither systematically nor uniformly, and some centers continue to use neadjuvant chemotherapy modalities. RESULTS: Furthermore, the survey reveals that there is a strong demand for the reduction of overall treatment-time, for clinical investigation of novel combined modality treatment strategies, especially chemo radiation therapy, and also for the use of new radiation sensitizers. CONCLUSION: We conclude that a more homogeneous approach to the pretreatment evaluation as well as treatment techniques is required in order to allow adequate quality control in any future trial of the RCG in the EORTC. PMID- 10738081 TI - Are clinical parameters valuable prognostic factors in childhood primitive neuroectodermal tumors? A multivariate analysis of 105 cases. AB - BACKGROUND: Medulloblastoma is one of the most frequent brain tumors in children. Long-term survivors are often confronted with serious late sequelae, caused by the therapy. Therefore, prognostic markers must be identified that allow the children to be assigned to different treatment schedules according to their predicted outcome. PATIENTS AND METHODS: The medical data of 110 children with a medulloblastoma or central primitive neuroectodermal tumor (PNET), that were admitted to the Emma Kinderziekenhuis in Amsterdam were analyzed by univariate and multivariate analyses. RESULTS: In univariate analysis the following characteristics had a significant influence on progression free survival (PFS): (a) presence of meningeal metastases at the time of diagnosis, (b) presence of tumor cells in the cerebrospinal fluid before or after surgery, (c) extent of resection, (d) necessity for permanent cerebrospinal fluid (CSF) shunting and (e) radiation dose to the posterior fossa. On multivariate analysis only the presence of metastases and the radiation dose to the posterior fossa retained significance. CONCLUSION: At the time of diagnosis, no reliable clinical prognostic markers are available for the majority of patients. Further molecular studies must be undertaken to identify such prognostic factors. PMID- 10738082 TI - Radiation therapy of optico-hypothalamic gliomas (OHG)--radiographic response, vision and late toxicity. AB - BACKGROUND: Management strategies for optic pathway gliomas include observation, surgery, irradiation, chemotherapy and a combination of these modalities. It has been the policy of our University Hospital to consider radiation as the standard treatment for progressive optic pathway gliomas. This report describes the clinical presentation, treatment patterns and outcome with special emphasis on the long term functional status of patients with optico-hypothalamic gliomas (OHG). PATIENTS AND METHODS: Between 1975 and 1997, 25 patients with OHG were treated by radiation therapy (RT) following surgery or biopsy. All patients received a local RT with a 0.5-1 cm margin around the lesions as depicted on CT or MRI scans. Age adjusted radiation doses ranged from 45 to 60 Gy with a single fraction size of 1.6-2 Gy. Endpoints of the study were: radiographic response, survival, progression-free survival and time to endocrinologic toxicity as well as the visual function during follow-up. The median follow-up time was 9 years (range, 1.5-23 years). RESULTS: A partial response was noted in six (24%) of the patients, 13 (52%) patients had a stable tumour throughout the observation period and six (24%) patients had a tumour progression. Overall survival and progression free survival rates were 94 and 69% at 10 years, respectively. A significant influence on progression-free survival was noted for age at diagnosis (P=0.04) and total dose (P=0.05). Nine out of 13 (69%) patients aged below 10 years compared with 3/12 (25%) patients aged above 10 years experienced hypothalamic pituitary deficiency (P=0.008) during follow-up. As for visual acuity, nine patients had an improvement, another 13 patients a stable situation and three patients a measurable deterioration. Visual field deficits improved in three, remained unchanged in 16 patients and worsened in only one patient. CONCLUSION: Postoperative RT with a total dose above 45 Gy should be considered as standard treatment in OHG with documented progression. Close radiographic monitoring and lifelong yearly evaluation for the need of possible hormone replacement are strongly recommended. PMID- 10738084 TI - Telemedicine in radiotherapy treatment planning: requirements and applications. AB - BACKGROUND AND PURPOSE: Telemedicine facilitates decentralized radiotherapy services by allowing remote treatment planning and quality assurance of treatment delivery. A prerequisite is digital storage of relevant data and an efficient and reliable telecommunication system between satellite units and the main radiotherapy clinic. The requirements of a telemedicine system in radiotherapy is influenced by the level of support needed. In this paper we differentiate between three categories of telemedicine support in radiotherapy. RESULTS AND DISCUSSION: Level 1 features video conferencing and display of radiotherapy images and dose plans. Level 2 involves replication of selected data from the radiotherapy database - facilitating remote treatment planning and evaluation. Level 3 includes real-time, remote operations, e.g. target volume delineation and treatment planning performed by the team at the satellite unit under supervision and guidance from more experienced colleagues at the main clinic. PMID- 10738083 TI - The carcinogenic risk of high dose total body irradiation in non-human primates. AB - PURPOSE: High dose total body irradiation (TBI) in combination with chemotherapy, followed by rescue with bone marrow transplantation (BMT), is increasingly used for the treatment of haematological malignancies. With the increasing success of this treatment and its current introduction for treating refractory autoimmune diseases the risk of radiation carcinogenesis is of growing concern. Studies on tumour induction in non-human primates are of relevance in this context since the response of this species to radiation does not differ much from that in man. MATERIALS AND METHODS: Since the early sixties, studies have been performed on acute effects in Rhesus monkeys and the protective action of bone marrow transplantation after irradiation with X-rays (average total body dose 6.8 Gy) and fission neutrons (average dose 3.4 Gy). Of those monkeys, which were irradiated and reconstituted with autologous bone marrow, 20 animals in the X irradiated group and nine animals in the neutron group survived more than 3 years. A group of 21 non-irradiated Rhesus monkeys of a comparable age distribution served as controls. All animals were regularly screened for the occurrence of neoplasms. Complete necropsies were performed after natural death or euthanasia. RESULTS: At post-irradiation intervals of 4-21 years an appreciable number of tumours was observed. In the neutron irradiated group eight out of nine animals died with one or more malignant tumours. In the X-irradiated group this fraction was 10 out of 20. The tumours in the control group, in seven out of the 21 animals, appeared at much older age compared with those in the irradiated cohorts. The histogenesis of the tumours was diverse with a preponderance of renal carcinoma, sarcomas among which osteosarcomas, and malignant glomus tumours in the irradiated groups. CONCLUSIONS: When corrected for competing risks, the carcinogenic risk of TBI in the Rhesus monkeys is similar to that derived from the studies of the Japanese atomic bomb survivors. The increase of the risk by a factor of 8, observed in the monkeys, indicates that patients are likely to develop malignancies more frequently and much earlier in life after TBI than non-exposed individuals. This finding underlines the necessity of regular screening of long-term surviving patients subjected to TBI and BMT. PMID- 10738085 TI - Variation in sensitizing effect of caffeine in human tumour cell lines after gamma-irradiation. AB - BACKGROUND AND PURPOSE: We have investigated whether the protective role of the G2 checkpoint has increasing importance when the p53-dependent G1 checkpoint is inactivated. MATERIALS AND METHODS: We have studied the differential effect of caffeine by clonogenic assays and flow cytometry in three human tumour cell lines with different functionality of p53 protein. RESULTS: The radiosensitizing effect of caffeine (2 mM) expressed itself as a significant decrease in surviving fraction at 2 Gy and a significant increase in alpha-values in RT112 and TE671, both with non-functional p53. However, no radiosensitizing effect was seen in cells with a normal p53 function (MCF-7 BUS). Two millimoles of caffeine also caused important changes in the cell cycle progression after irradiation. MCF-7 BUS showed a G1 arrest after irradiation and an early G2 arrest but those cells that reached the second G2 did not arrest significantly. In contrast, TE671 exhibited radiosensitization by caffeine, no G1 arrest, a G2 arrest in those cells irradiated in G2, no significant accumulation in the second G2 but an overall delay in release from the first cell cycle, which could be abrogated by caffeine. RT112 was similar to TE671 except that the emphasis in a G2 arrest was shifted from the block in cells irradiated in G2 to those irradiated at other cell cycle phases. CONCLUSION: The data presented confirm that p53 status can be a significant determinant of the efficacy of caffeine as radiosensitizer in these tumour cell lines, and document the importance of the G2 checkpoint in this effect. PMID- 10738086 TI - Low-dose radiotherapy selectively reduces adhesion of peripheral blood mononuclear cells to endothelium in vitro. AB - BACKGROUND AND PURPOSE: The anti-inflammatory effect of low-dose radiotherapy (LD RT) still is not understood. The adhesion of leukocytes to endothelial cells (EC) of the vessel wall is the initial event of tissue invasion, and thus, crucially contributes to the regulation of inflammation. We investigated the influence of LD-RT on the adhesion process in vitro. MATERIALS AND METHODS: Isolated peripheral-blood-mononuclear-cells (PBMC) were incubated with an activated murine endothelioma cell-line under shear conditions at 4 degrees C after irradiation with single doses between 0.1 and 10.0 Gy. Adherent cells were counted microscopically and compared to a non-irradiated control. In parallel, viability and expression of adhesion molecules, especially of L-selectin, and lineage specific markers on the cell surface were determined by dye exclusion and cytofluorometry, respectively. Modulation of adhesion by soluble L-selectin was tested in the adhesion assay. RESULTS: Radiation doses of 0.1-0.5 Gy reduced the adhesion of viable PBMC to EC in vitro by 70% of the control level 4 h after irradiation. Leukocytes showed a marked reduction of L-selectin expression after LD-RT. Soluble L-selectin can inhibit the adhesion of PBMC to EC. CONCLUSION: The anti-inflammatory effect of LD-RT might, in part, be due to the reduction in the adhesion of PBMC to EC. This reduction in adhesion might be a consequence of the reduced expression of L-selectin on the surface of PBMC, and the inhibition of adherence by soluble L-selectin shed by PBMC in vitro. PMID- 10738087 TI - Induction of antigen-specific T cell responses in human volunteers after intranasal immunization with a whole-cell pertussis vaccine. AB - We have studied the ability of an intranasally administered whole-cell pertussis vaccine (WCP) without adjuvant to induce antigen-specific T cell responses in humans. Six adult volunteers were given a vaccine dose (corresponding to 250 microg protein) by nasal spray four times at weekly intervals, and peripheral blood mononuclear cells were assayed for antigen-specific proliferative T cell responses. All six vaccinees had a WCP-specific response, which in four of them remained elevated throughout the 2 month study. All participants also responded to the filamentous haemagglutinin (FHA) antigen, and four of them responded to inactivated pertussis toxin (PTd). A significant correlation between T cell proliferation against WCP and WCP-specific IgA antibody levels in nasal secretions was observed. This demonstrates that intranasal administration of a non-proliferating bacterial vaccine without any additional mucosal adjuvant can induce vaccine-specific T cell responses related to mucosal IgA secretion. PMID- 10738088 TI - Antibody responses of goldfish (Carassius auratus L.) to DNA-immunisation at different temperatures and feeding levels. AB - DNA vaccines which work in winter are needed for fish. At 18 weeks after intramuscular injection of a plasmid containing lacZ goldfish at 15 degrees C had five-fold more antibody to betagalactosidase than those at 25 degrees C or those at 100) but no CTL responses. The data indicate that antigens can be efficiently expressed under viral or eukaryotic promoter/enhancer control for immunogenic in vivo presentation, but that the technique, dose and/or route of DNA injection have a decisive role in determining the type of immune response elicited. PMID- 10738091 TI - Recombinant classical swine fever (CSF) viruses derived from the Chinese vaccine strain (C-strain) of CSF virus retain their avirulent and immunogenic characteristics. AB - Two recombinant classical swine fever (CSF) viruses (Flc2, Flc3) transcribed from a DNA copy of the genome of the Chinese (C) strain, a CSF virus vaccine strain, were characterized in vivo in rabbits and pigs. Rabbits were inoculated intravenously with Flc2 or Flc3, the parent C-strain virus, a biologically cloned C-strain or CSF virus strain Brescia (C.1.1.1). After 24-96 h fever was detected in the rabbits inoculated with the different C-strain viruses. Apart from those in the control group, all the C-strain inoculated rabbits had developed CSF virus neutralizing antibodies 4 weeks later and were protected against a parent C strain challenge. In the second experiment, pigs were inoculated with the parent C-strain or recombinant C-strain virus (Flc2 or Flc3) and then challenged after 4 weeks with the virulent CSF virus strain Brescia. None of the pigs showed clinical signs of classical swine fever after vaccination or challenge, whereas the control pigs developed clinical signs typical for acute CSF. Pigs inoculated with the different C-strain viruses were not viremic after inoculation or challenge, and CSF virus neutralizing antibodies were detected from day 14 onwards. The results from both experiments demonstrated that the two recombinant viruses had retained the biological and immunogenic properties of the parent C strain in rabbits and pigs. We conclude that the full-length cDNA of the C-strain can serve as a matrix for further development of a live recombinant CSF virus marker vaccine. PMID- 10738090 TI - Changes in the seroepidemiology of hepatitis B infection in Catalonia 1989-1996. AB - The objective of this study was to investigate the prevalence of hepatitis B markers in a representative sample of 2142 subjects in Catalonia, Spain, and to compare it with previous studies. Multiple logistical regression analysis was carried out to determine variables associated with the markers studied. The prevalence of anti-HBc and HBsAg was 9.1% and 1.2%, respectively. Male gender, urban habitat, birth place outside Catalonia and lower social class were associated with the presence of anti-HBc. Carrier status was only associated with male gender. Between 1989 and 1996 there was a decrease of 46% in the prevalence of serum HBV markers mainly in the 25-44 (P<0.0001) and 35-64 year (P=0.0002) age groups, in those born in Catalonia (P=0.003) and in those in the higher social classes (P<0.0001). These decreases can be explained by the improved socioeconomic conditions and, partially, by the routine pre-adolescent and risk group programmes of immunization. PMID- 10738092 TI - A comparison of multiple regimens of pneumococcal polysaccharide-meningococcal outer membrane protein complex conjugate vaccine and pneumococcal polysaccharide vaccine in toddlers. AB - Children who had been randomized to receive one dose of either heptavalent pneumococcal polysaccharide-meningococcal outer membrane protein complex conjugate vaccine (PCV) or 23-valent pneumococcal polysaccharide vaccine (PN23) at 12, 15, or 18 months of age were subsequently randomized to receive a booster injection of either PCV or PN23 12 months later. For those children who received a priming dose of PCV (N=75) compared to PN23 (N=48) at 12, 15, or 18 months of age, higher serum antibody concentrations were achieved 1 month following a booster injection of either PCV or PN23 for all serotypes tested (p<0.001). Within the group of children receiving a priming dose of PCV, those children who received a booster dose of PN23 developed higher serum antibody concentrations for four of the seven serotypes tested and similar opsonic antibody titers to serotype 6B, yet more frequent erythema (p=0.030) and pain or soreness (p=0.024) at the injection site compared to those boosted with PCV. In conclusion, a single dose of PCV at 12-18 months of age primed for responses to booster doses of either PCV or PN23 12 months later. For those children who received a priming dose of PCV, boosting with PN23 resulted in more frequent injection site pain and erythema than boosting with PCV, yet higher antibody concentrations for most of the serotypes tested. PMID- 10738093 TI - Immune responses in mice, cattle and horses to a DNA vaccine for vesicular stomatitis. AB - Vesicular stomatitis (VS) virus causes an important clinical disease of cattle and horses in North America. In order for a vaccine to be useful in the control of VS, it must not only protect against disease, but allow ready differentiation of infected and vaccinated animals. In these studies, we evaluated neutralizing antibody responses in outbred mice, calves, and horses that received a DNA vaccine that expressed the glycoprotein (G) gene of VS New Jersey virus. The vaccine elicited antibody titers in individuals from each species, especially when two doses were administered, but the level of neutralizing antibody needed to confer protection is not known. In mice, co-administration of a plasmid that expressed interleukin-2 resulted in a significant, though modest, increase in antibody titers relative to use of the G gene vaccine alone. The effect of co injecting putative immunostimulatory oligonucleotides was also evaluated. This treatment had no apparent effect in horses and was found to suppress immune responses to the G gene vaccine in mice. If the immune responses obtained in these studies prove to protect cattle and horses from infection with VS virus, DNA vaccination may become a useful tool for control of this disease. PMID- 10738094 TI - Experience with live attenuated varicella vaccine (Oka strain) in healthy Japanese subjects; 10-year survey at pediatric clinic. AB - Live attenuated varicella vaccine (Oka strain, Biken Institute, Osaka, Japan) was administered to 973 healthy individuals over a 10-year period (1987-1997) at the pediatric clinic of Showa Hospital in Japan. We evaluated the relevant serological and clinical data, which were collected by questionnaire. Seroconversion by the immune adherence hemagglutination method was documented in 94% (805/860) of the initially seronegative subjects. Of the initially seropositive subjects, 56% (63/113) showed enhancement of antibody after vaccination. Reactions to the vaccine were generally insignificant, except for a rash at the injection site, seen in the first 3 days post-administration in 17% (41/241) of the recently vaccinated subjects. In March 1998, we conducted a survey of 559 of the initially seronegative subjects who had received the vaccine 0.6-10. 8 (mean 5.4) years earlier. Of these subjects, 21% (119/559) contracted breakthrough varicella. However, their symptoms were milder than those caused by natural varicella seen in unvaccinated children. Seroconversion was demonstrated in 92% (109/119) of these cases. The incidence of breakthrough disease decreased with a rise in postvaccination antibody titer to >==32. Four of the subjects (0.7% of 559) developed herpes zoster following vaccination, two of whom had earlier exhibited breakthrough varicella. Lesions in one case of zoster, without breakthrough varicella, appeared on the cervical dermatome at the injection site. The vaccine was safe and effective. However, there was a relatively high incidence of rash at the injection site with certain lot numbers used in recent years which warrants investigation. PMID- 10738096 TI - Rabies vaccination: comparison of neutralizing antibody responses after priming and boosting with different combinations of DNA, inactivated virus, or recombinant vaccinia virus vaccines. AB - Long-term levels of neutralizing antibody were evaluated in mice after a single immunization with experimental DNA or recombinant vaccinia virus (RVV) vaccines encoding the rabies virus glycoprotein (G), or the commercially available inactivated virus human diploid cell vaccine (HDCV). Anamnestic antibody titers were also evaluated after two booster immunizations with vaccines that were identical to or different from the priming vaccine. Five hundred and forty days (1.5 year) after a single immunization with any of the three vaccines, neutralizing antibody titers remained greater than the minimal acceptable human level of antibody titer (0.5 International Units (IU)/ml). In addition, either an HDCV or DNA booster elicited early and elevated anamnestic antibody responses in mice that had been primed with any of the three vaccines. In contrast, RVV boosters failed to elevate titers in mice that had been previously primed with RVV, and elicited slowly rising titers in mice that had been primed with either DNA or HDCV. Thus, a single vaccination with any of the three different vaccines elicited long-term levels of neutralizing antibody that exceeded 0.5 IU/ml. In contrast, different prime-booster vaccine combinations elicited anamnestic neutralizing antibody responses that increased quickly, increased slowly or failed to increase. PMID- 10738095 TI - Enhancement of common mucosal immunity in aged mice following their supplementation with various antioxidants. AB - Common mucosal immune responses were depressed in aged mice that were orally immunized with Haemophilus influenzae type b oligosaccharide conjugated to Diphtheria CRM197 protein (Hib-DT) vaccine using cholera toxin as the mucosal adjuvant. Both common mucosal and systemic humoral immune responses were also depressed in aged mice that were subcutaneously immunized with vaccine formulations containing Hib-DT plus 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). Dietary supplementation of aged mice with either the antioxidant vitamin E, or with known activators of the alpha isoform of the peroxisome proliferator activated receptor (PPAR-alpha) was capable of restoring their mucosal and systemic humoral immune responses to mature adult levels, by both the oral and subcutaneous routes of immunization. These data support a hypothesis that some aspects of immunosenescence are due to dysregulations in cellular functions, and are not due to any irreversible defects in cellular components of the immune system. PMID- 10738097 TI - Efficacy trial of single-dose live oral cholera vaccine CVD 103-HgR in North Jakarta, Indonesia, a cholera-endemic area. AB - A randomized, double-blind, placebo-controlled efficacy trial of one dose of CVD 103-HgR live oral cholera vaccine was performed in Indonesia from 1993 to 1997. 67,508 persons aged 2-41 years ingested vaccine or placebo and were followed for four years, detecting cholera cases using hospital-based surveillance. A nested reactogenicity study (538 vaccinees, 535 controls) revealed no vaccine attributable side effects. A nested immunogenicity study (N=657) showed vibriocidal seroresponses in 64-70% of vaccinees vs 1-2% of controls. Cholera incidence was lower than expected. 103 cases of Vibrio cholerae O1 El Tor diarrhea were detected, 93 evaluable for vaccine efficacy (43 vaccine, 50 placebo; efficacy=14%). A suggestion of protection was observed among persons with blood group O [P=0.12]. Only seven cases occurred within six months of vaccination, precluding assessment of short-term efficacy. In Jakarta, single dose CVD 103-HgR did not confer long-term protection. Short-term protection from a single-dose and long-term protection from two doses have yet to be studied. PMID- 10738098 TI - Measles vaccine immunogenicity and antibody persistence in 12 vs 15-month old infants. AB - HYPOTHESIS: Maternal measles immunity in the United States today is primarily vaccine induced, with corresponding lower antibody titers in infants, as compared to infants born in an earlier era to mothers with naturally acquired measles immunity. We hypothesized that, due to lower titer of passively transferred maternal measles antibody, administration of measles vaccine at 12 months of age would result in seroconversion and antibody persistence comparable to vaccination at 15 months of age. POPULATION: Children at both an urban hospital and a suburban clinic. METHODS: Informed consent was obtained from mothers for the infants to receive M-M-R(R)II vaccine at either 12 or 15 months and to have serum samples obtained before vaccination and 4 weeks post-vaccination (PV). Between 9 and 39 months PV, a third serum sample was obtained from 28% of seroconverters. A diary of adverse experiences was kept for 3 weeks PV. Sera were assayed by a microneutralization assay (NT) and an enzyme immunoassay (EIA) for measles antibody. RESULTS: Both age groups tolerated vaccination well with minor and transient side effects. Forty-four of 47 (94%) 12-month-old infants seroconverted by NT, compared to 45 of 46 (98%) 15-month-olds (p=NS). There was no statistically significant decline in median NT titers or EIA titers in nineteen 12-month-olds and thirteen 15-month olds followed for 9-39 months PV. CONCLUSION: This study showed comparable serologic responses in 12- vs 15-month-old infants born to measles vaccine-immune mothers; however, the sample size was too small to have adequate power and further study is indicated. Titers of antibody were constant in both the 12-month-old and the 15-month-old infants, over a 9-39 month period, suggesting that waning immunity over this period of time is not a problem in either age group. PMID- 10738099 TI - Monophosphoryl lipid A enhances mucosal and systemic immunity to vaccine antigens following intranasal administration. AB - The induction of protective immunity stemming from vaccines delivered by mucosal routes is dependent on the development of safe and effective mucosal adjuvants. The immunostimulant monophosphoryl lipid A (MPL(R)) was evaluated for its ability to enhance both systemic and mucosal immunity to three distinct antigens. Vaccines formulated with MPL(R) and hepatitis B surface antigen, tetanus toxoid or influenza antigens were administered by intranasal delivery to mice. In each case the vaccines formulated with MPL(R) resulted in enhanced IgA titers from mucosal samples. Enhanced IgA concentrations were detected in samples from both local and distal mucosal sites. In addition, the MPL(R) formulated vaccines induced systemic immunity characteristic of a Th1-type of response. Serum IgG2a antibody titers were elevated and cytotoxic T cell activity was enhanced. PMID- 10738100 TI - Immunogenicity of dengue virus type 1 DNA vaccines expressing truncated and full length envelope protein. AB - Recombinant plasmid DNA constructs expressing truncated or full-length dengue-1 envelope (E) with or without the pre-membrane (prM) were tested for immunogenicity in mice, as candidate dengue DNA vaccines. Two plasmids, one expressing the N-terminal 80% E and the other expressing prM and full length E were immunogenic in intradermally inoculated mice. The vaccinated mice produced dengue-1 specific antibodies that were both neutralizing and long lasting. Data suggested that the plasmid expressing prM and full length E produced virus like particles in transfected cells, and is probably a better immunogen compared to that expressing 80% E. PMID- 10738101 TI - Increased safety level of serotype 3 Sabin oral poliomyelitis vaccine lots by improved seed virus, and tissue culture and virus infection conditions. AB - The content of 472U to 472C revertant virus in serotype 3 oral poliomyelitis monovalent bulk vaccines can be quantified by MAPREC (Mutant Analysis by PCR and Restriction Enzyme Cleavage). Besides other wildtype reversions identified in propagated type 3 Sabin strain populations, the 472U to 472C reversion correlates most prominently with neurovirulence in the monkey neurovirulence test. Therefore, the results can be used for the discrimination of 'good' and 'bad' vaccines on the molecular level. In international collaborative studies it has been well established that vaccine lots containing revertant genomes below a critical threshold pass the in vivo monkey neurovirulence test (MNVT), while vaccine lots containing more revertants fail the MNVT. In this communication we show that the MAPREC test is a sensitive tool for quality control and the demonstration of consistency in large scale production. Furthermore, MAPREC offers a possibility to assess the effect of changed production conditions on the rate of reversion and to find conditions for consistent production with low reversion rates. PMID- 10738102 TI - Immune responses induced by BCG recombinant for human papillomavirus L1 and E7 proteins. AB - Recombinant bacille Calmette-Guerin (BCG) based vaccine delivery systems could potentially share the safety and effectiveness of BCG. We therefore prepared recombinant BCG vaccines which expressed the L1 late protein of the human papillomavirus (HPV) 6b or the E7 early protein of the HPV 16. The two recombinants were evaluated as immunogens in C57BL/6J and BALB/c mice, and compared with a conventional protein/adjuvant system using E7 or L1 mixed with Quil-A adjuvant. rBCG6bL1 and rBCG16E7 primed specific immune responses, represented by DTH, T-proliferation and antibody, and rBCG16E7 induced cytotoxic immune response to E7 protein. The magnitude of the observed responses were less than those elicited by protein/adjuvant vaccine. As recombinant BCG vaccines expressing HPV6bL1 or HPV16E7 persist at low levels in the immunised host, they may be beneficial to prime or retain memory responses to antigens, but are unlikely to be useful as a single component vaccine strategy. PMID- 10738103 TI - Structural differences between foot-and-mouth disease and poliomyelitis viruses influence their inactivation by aziridines. AB - Inactivation of foot-and-mouth disease virus (FMDV) and poliovirus by ethyleneimine (EI) and N-acetylethyleneimine (AEI) has been studied at 25 degrees and at 37 degrees C and in different ionic conditions. FMDV is inactivated rapidly in 100 mM Tris pH 7.6 by each reagent at both temperatures. Poliovirus is also inactivated rapidly in 100 mM Tris by EI at both temperatures and by AEI at 37 degrees C. However, it is inactivated much more slowly by AEI at 25 degrees C; but if the virus is first incubated overnight at 2 degrees C with AEI before transferring to 25 degrees C inactivation then proceeds rapidly. Moreover, the rate of inactivation at 25 degrees C is markedly increased if the virus is suspended in 1 mM Tris. We had interpreted these differences as being due to the greater penetrability of poliovirus (i) in 100 mM Tris at 37 degrees C compared with 25 degrees C and (ii) at lower ionic strength. This interpretation has been confirmed by electron microscopy of FMDV and poliovirus particles stained with phosphotungstic acid. At the elevated temperature, poliovirus had an average diameter of 34+/-0. 21 nm and the stain outlined the nucleic acid core and the individual subunits, whereas at 25 degrees C it averaged 28+/-0.13 nm and the stain did not penetrate the particle. This study also showed that the particle diameter alters with changes in buffer concentration, being 28+/-0.13 nm in 100 mM Tris, 31+/-0.16 nm in 10 mM Tris and 34+/-0.21 nm in 1 mM Tris. The changes in poliovirus are reversible as addition of 1/10 volume of 1 M Tris to the virus in 1 mM Tris resulted in the return of the diameter to 28+/-0.13 nm. FMDV, on the other hand, was less sensitive to osmotic differences as its particle diameter only varied by 7% over the 100-fold change in buffer concentration compared with the 22% change observed for poliovirus. PMID- 10738104 TI - African green monkeys provide a useful nonhuman primate model for the study of human parainfluenza virus types-1, -2, and -3 infection. AB - Human parainfluenza virus (HPIV) types-1, -2, and -3 are significant causes of both upper and lower respiratory tract disease in infants and children. Although there are two live attenuated vaccines for the prevention of HPIV-3 disease in phase 1 clinical trials, vaccines are not currently available for prevention of HPIV-1 or -2 disease. Our laboratory is developing candidate vaccines for the prevention of HPIV-1, -2, and -3 disease, and a suitable nonhuman primate model is needed for evaluation of these vaccine candidates prior to administration to humans. We evaluated the replication of HPIV-1 and -2 in six different species of nonhuman primates and found both viruses to replicate most efficiently in African green monkeys and chimpanzees. We then compared the replication of HPIV-3 in African green monkeys to that in rhesus macaques, which we have used previously, and found that HPIV-3 replicated to higher titer in African green monkeys. In summary, African green monkeys provide a very useful nonhuman primate for the evaluation of HPIV-1, -2, and -3 vaccine candidates, especially for the evaluation of various combinations of these PIV vaccines and for vaccine strategies that employ sequential immunization. PMID- 10738105 TI - Influence of melatonin on proliferation and antioxidant system in Ehrlich ascites carcinoma cells. AB - The effects of oral supplementation of melatonin on growth of Ehrlich ascites carcinoma (EAC) cells implanted intraperitoneally in female mice were studied. Melatonin at 50 mg/kg body wt. reduced the viability and volume of Ehrlich ascites carcinoma cells and increased the survival of the treated mice. No significant change in intracellular reduced glutathione (GSH) content in EAC cells was observed indicating that GSH was not involved in the inhibitory effect of melatonin. The activity of glutathione-S-transferase in EAC cells was significantly increased. Flow cytometirc studies showed that melatonin not only delayed the progression of cells from G(0)/G(1) phase to S-phase of the cell cycle but also reduced DNA synthesis during cell cycle. In addition, the aneuploidy status was depressed in melatonin treated mice. Based on these data and the reduced viability in both in vitro and in vivo, it is suggested that melatonin might induce apoptosis in EAC cells. PMID- 10738106 TI - Absence of evidence for allelic loss or allelic gain for ERCC1 or for XPD in human ovarian cancer cells and tissues. AB - We have previously reported on mRNA expression of ERCC1, XPA and XPD in human ovarian cancer cells and tissues. Several factors can influence mRNA expression for any given gene. Alterations in gene copy number for ERCC1 and/or XPD have been reported to occur in malignant glioma specimens. Human ovarian cancer cell lines and tissues were therefore examined for evidence of altered gene copy number in selected genes within the nucleotide excision repair (NER) pathway. Six ovarian cancer cell lines were studied: A2780, A2780/CP70, SKOV3, MCAS, QvCar3 and Caov4. Cellular sensitivity to cisplatin varies by more than 1 log between some of these cells. In each of these cell lines, the genes examined included ERCC1, XPA, XPB, XPD, XPG, CSB and p53. Genomic DNA was also extracted from ovarian cancer specimens taken from 22 patients and assessed for evidence of allelic loss and/or allelic gain for ERCC1 and XPD. Twelve of the clinical specimens were from patients with platinum-sensitive tumors and ten were from patients with platinum-resistant tumors. In no case could we demonstrate a reproducible variation in gene copy number in any cell line. Among the human tissues studied, there was one case of allelic gain out of 22 specimens. We therefore conclude that alterations in gene copy number is not a common event in human ovarian cancer. Other mechanisms must be invoked to explain differences in mRNA expression for these genes. PMID- 10738107 TI - Melatonin decreases cell proliferation and transformation in a melatonin receptor dependent manner. AB - There are conflicting claims for the role of melatonin in oncogenesis. In addition, the mechanism(s) underlying melatonin's effects in oncogenic processes is (are) unknown. In this study, the effects of melatonin exposure on cell proliferation and transformation were assessed in NIH3T3 cells transfected with either the human mt(1) (NIH-mt1) or MT(2) (NIH-MT2) melatonin receptors. The effects of melatonin exposure on proliferation was assessed by direct cell counts and [(3)H]thymidine uptake assays. The effect of chronic melatonin pretreatment on transformation was assessed by focus assays. In both NIH-mt1 and NIH-MT2 cells, melatonin pretreatment decreased cell proliferation and transformation. Control (NIH-neo) cells did not show this effect. However, as revealed by the [(3)H]thymidine uptake assays, an increase in DNA synthesis occurred in NIH-mt1 cells, whereas no increase occurred in the NIH-MT2 or NIH-neo cells. Upon examination of melatonin receptors, a decrease in the function of both mt(1) and MT(2) receptors occurred. These data suggest that perhaps an attenuation of receptor-mediated processes are involved in the anti-proliferative and anti transformation capabilities of melatonin in NIH3T3 cells. In addition, based on the [(3)H]thymidine assays, receptor mediated signal transduction mechanisms may slow the growth of cells via actions on the cell cycle. The results from this study shed new insight on the putative mechanisms underlying melatonin's effects on cell proliferation and transformation and lends support for a protective role of melatonin in oncogenesis. PMID- 10738108 TI - Dietary fish oil sensitizes A549 lung xenografts to doxorubicin chemotherapy. AB - A549 xenografts were allowed to grow in nude mice to about 5 mm in diameter, then diets were changed to modified AIN-76 diets containing 19% wt./wt. fish oil (FO) or 20% wt./wt. corn oil (CO). Ten days later dietary ferric citrate (0.3% wt./dry wt.) was added and doxoribicin (DOX) treatment (3.6 mg/kg i.v. each of the 5 days for 18 days) commenced. Treatment with DOX halted the growth of tumors in the CO fed mice. However, in those mice, which consumed FO or FO with ferric citrate, treatment with DOX caused significant tumor regression. PMID- 10738109 TI - Ciprofibrate and triiodothyronine do not suppress in vivo induction of placental glutathione S-transferase expression in rat hepatocytes. AB - Studies on hepatocyte primary cultures have suggested that loss of expression of the placental form of glutathione S-transferase in peroxisome proliferator (PP) induced hepatocarcinogenesis is due to inhibition of glutathione S-transferase P (GSTP) transcription by the PPs. In the present study, we have analyzed the effect of a PP, ciprofibrate, and of another ligand of nuclear receptors, 3,3', 5 triiodo-L-thyronine (T3), on GSTP mRNA and protein levels in an in vivo model where GSTP expression was induced in Wistar rats by pre-treatment with a single dose of lead nitrate. Results indicate that administration of ciprofibrate or T3, immediately after lead nitrate treatment, did not exert any inhibitory effect on GSTP mRNA and protein levels, as revealed by both Western and immunohistochemical analysis. The results indicate that PPs do not inhibit hepatocyte GSTP expression induced in vivo by lead nitrate and suggest that inhibition of GSTP expression by PPs may not necessarily be the cause for the rapid disappearance of GSTP-positive preneoplastic lesions observed after a short term exposure to these agents. PMID- 10738110 TI - Methylene blue reverts multidrug resistance: sensitivity of multidrug resistant cells to this dye and its photodynamic action. AB - Photodynamic action has been advocated as an alternative treatment of tumors but the most common used dyes, hematoporphyrin derivatives, are substrate for P glycoprotein. This study investigated the MDR-reverting properties of methylene blue (MB) and compared the sensitivity to its photodynamic action (PDA) in five cell lines that either express or do not express the MDR phenotype. MB was able to revert the MDR phenotype and there was no difference in sensitivity to MB-PDA between MDR and non-MDR cells, suggesting that MB has the advantage of being used simultaneously as a MDR reverser and a photodynamic agent. PMID- 10738111 TI - Inhibition of carcinogen-induced rat mammary tumor growth and other estrogen dependent responses by symmetrical dihalo-substituted analogs of diindolylmethane. AB - 90%) by the haloDIMs at concentrations of 5 or 10 microM, and only 4, 4' dichloroDIM alone increased cell proliferation. With the exception of 5,5' difluoroDIM, the remaining compounds also inhibited E2-induced growth of MCF-7 human breast cancer cells. DihaloDIMs (100 mg/kg/dayx3) were not estrogenic in the immature female B6C3F1 mouse uterus; however, in animals co-treated with E2 (0.02 microg/mouse), 5,5'-dichloro- and 6,6'-dichloroDIM inhibited uterine progesterone receptor (PR) binding and uterine peroxidase activity, whereas 5,5' dichloro- and 5,5'-dichloro-2,2'-dimethylDIM inhibited only the latter response. The antitumorigenic activities of the dihaloDIMs were determined by their inhibition of carcinogen-induced mammary tumor growth in female Sprague-Dawley rats. 4,4'-Dichloro-, 5,5'-dibromo- and 6,6'-dichloroDIM, significantly inhibited mammary tumor growth at doses of 1 mg/kg every second day, and no significant changes in organ weights or liver and kidney histopathology were observed. These three compounds were more active than DIM in the same in vivo assay. PMID- 10738112 TI - Methylenetetrahydrofolate reductase polymorphism and risk of colorectal adenomas. AB - A homozygous mutation at bp 677 in the gene for the methylenetetrahydrofolate reductase (MTHFR) was previously shown to be associated with a decreased risk of colorectal cancer. We examined the relation between the MTHFR genetic polymorphism and risk of colorectal adenoma in Japanese men using 205 cases of colorectal adenomas and 220 controls of normal total colonoscopy. The homozygous mutation was not measurably associated with colorectal adenomas. The findings corroborate the lack of an association between the MTHFR genotype and colorectal adenomas, but do not deny the possibility that the genotype may be involved in the late stage of colorectal carcinogenesis. PMID- 10738113 TI - Psoriasis patients with basal cell carcinoma have more repair-mediated DNA strand breaks after UVC damage in lymphocytes than psoriasis patients without basal cell carcinoma. AB - We have investigated the formation of strand-breaks following UVC irradiation in lymphocytes from psoriasis patients with or without basal cell carcinoma (BCC). Isolated lymphocytes were irradiated with UVC light at a dose of 3.6 J/m(2), and the level of DNA strand-breaks were measured 25 min after the irradiation by the alkaline comet assay. The generation of strand-breaks following UVC irradiation indicates DNA-repair-mediated incisions, as UVC light does not generate strand breaks per se. We found that psoriasis patients with BCC had more DNA-repair incisions than non-cancer patients. The incision level correlated to two polymorphisms of the XPD gene. At present, it is not clear if the association is a primary effect that is related to differences of the XPD protein. Genes encoding for other repair proteins, namely XRCC1, ERCC1, and LIG1 are located close to the XPD gene, and it is possible that the association is due to a cosegregation with a polymorphism in one of these genes. PMID- 10738114 TI - Cyclin E overexpression in transitional cell carcinoma of the bladder. AB - We attempted to clarify the relationship between cyclin E to p27(Kip1), Ki-67 and clinicopathologic features in transitional cell bladder carcinoma. Immunohistochemical staining of archival tissue specimens of transitional cell bladder carcinoma obtained from 94 patients was performed by the labeled streptavidin-biotin-peroxidase method. Overexpression of cyclin E protein was observed in 38 of the 94 (40.4%) specimens, and was positively correlated with histological grade, Ki-67 LI and p27(Kip1) labeling index (LI). These data suggest that cyclin E may be associated with aggressive tumor growth, and may have a relationship with p27(Kip1) for the regulation of cell cycle progression in transitional cell bladder carcinoma. PMID- 10738115 TI - Effect of 9-cis-retinoic acid on oral squamous cell carcinoma cell lines. AB - Retinoic acid (RA) has been shown to be effective in suppressing premalignant lesions and preventing second primary malignancies in patients cured of squamous cell carcinoma of the head and neck. However, the precise mechanisms of these effects are still uncertain. In the present study, we examined the effect of 9 cis-RA on the growth of six oral cancer cell lines (HSC-2, HSC-3, HSC-4, Ca9-22, Ho-1-N-1 and Ho-1-u-1). In addition, the relationship among growth and differentiation of tumor cells, RA responsiveness and the expression of nuclear retinoic acid receptors were also investigated. Among the six cell lines examined, five (HSC-2, HSC-3, HSC-4, Ca9-22 and Ho-1-u-1) displayed growth inhibition after treatment with 1x10(-6) M 9-cis-RA, while Ho-1-N-1 cells were resistant to 9-cis-RA. The expression level of RARbeta in 9-cis-RA resistant Ho-1 N-1 cells was very low in comparison with the sensitive cell lines. On the other hand, all of the six the cell lines expressed RARalpha, RARgamma, and RXRalpha at various levels. 9-cis-RA induced accumulation of cell population in G1 phase in HSC-3 cells on the 6th day of the treatment, followed by a marked reduction in the levels of hyperphosphorylated pRB, whereas p53 level was not altered. Interestingly, 9-cis-RA induced transiently the expression of p21(Waf1/Cip1), p27(Kip1), p300, CBP, BAX, Bak and bcl-2 proteins, respectively. This effect was associated with reduction of cyclin D1, cdk4 and CDK-activating kinase (cyclin H and cdk7) protein in HSC-3 cells. These results suggest that the growth inhibitory effect of 9-cis-RA on oral squamous cell carcinoma may depend on the expression levels of RARs, especially RARbeta proteins and RXRalpha proteins, and that 9-cis-RA may provide a powerful therapeutic agent for head and neck cancers. PMID- 10738116 TI - Increased expression of sialyl Le(x) antigen in non-polypoid growth type of colorectal carcinoma. AB - Colorectal carcinoma can be morphologically divided into two different categories, namely polypoid growth (PG-type) and non-polypoid growth (NPG-type). To ascertain whether the expression of sialyl Le(x) antigen correlates with biologically and clinically important differences, an immunohistochemical assay was performed in 30 PG-type and 119 NPG-type cancers. In contrast to PG-type, the characteristics of the NPG-type include (1) an increased expression of sialyl Le(x); (2) a high rate of lymph node metastasis; (3) a high proportion of moderately differentiated adenocarcinoma cells; (4) young age of onset. It is concluded that differences in sialyl Le(x) expression between the PG-type and NPG type cancers may be at least partly responsible for different tumor progression behavior. PMID- 10738117 TI - Reduced expression of p27(Kip1) correlates with an early stage of cancer invasion in oral squamous cell carcinoma. AB - Down-regulation of p27(Kip1) has been reported to correlate with poor survival of various carcinoma patients including oral squamous cell carcinomas (OSCCs). It is still unclear, however, at what stage of oral carcinogenesis the down-regulation of this protein occurs. In this study, therefore, we evaluated immunoexpression of p27(Kip1) protein in 17 cases of oral epithelial dysplasia and succeeding invasive OSCC in the same patient. We reported here that 88% cases showed high p27(Kip1) expression in dysplastic lesions, whereas 82% cases of succeeding invasive OSCC exhibited reduced expression. The reduction of p27(Kip1) expression was also observed in 16 of 19 (84%) early invasive lesions and well correlated with Ki-67 expression which is good indicator of cell proliferation. We also investigated immunoexpression of p53 protein of which abnormality has been known to occur during the early stage of OSCC development. Overexpression of p53 protein was demonstrated in 29% of dysplastic lesions, 42% of early invasive and 71% of invasive OSCCs. These findings suggest that abnormalities of both p53 and p27(Kip1) are involved in the carcinogenesis of OSCC, but they seem to play their role at different stages of oral cancer development, respectively. Reduced expression of p27(Kip1) may concern the cancer invasion directly or indirectly as well as abnormal proliferation. PMID- 10738119 TI - New directions in the treatment of colorectal cancer: a look to the future. AB - Today, adjuvant 5-fluorouracil based therapy is known to significantly reduce the relapse rates and the risks of dying from resected colon cancer; chemotherapy approximately doubles overall survival of advanced colorectal cancer and second line treatment prolongs the survival of patients compared with best supportive care. At the molecular level a number of key genes are often mutated in cancer of the colon and some of these key regulators of apoptosis are discussed (p53 and bcl-2 family of proteins). Dihydropyrimidine dehydrogenase (DPD) activity may be a potential factor controlling fluorouracil (FU) responsiveness at the tumoral level and its importance is stressed. The rationale of combining FU with DPD inhibitors is fairly strong. Ethynyluracil, UFT and S1 pursue this strategy while capecitabine has another the rationale. Drug resistance should be at least partially overcome by combination chemotherapy (FU plus mitomycin, oxaliplatin, irinotecan) and combined modality (FU+RT) regimens. Improved surgical techniques and radiotherapy have substantially decreased local failure rates for rectal cancers. Finally, innovative treatment modalities such as anti-angiogenetic and antimetastatic agents, farnesyl transferase inhibitors, vaccine and gene therapy are in early clinical trials. PMID- 10738120 TI - Prophylactic mastectomy; evolving perspectives. AB - It is not at all uncommon for surgeons dealing with breast disease to be confronted with the issue of prophylactic mastectomy. Recent advances in understanding the genetic basis of susceptibility to breast cancer and a better identification of the histological factors affecting a woman's lifetime risk of developing breast cancer have contributed to placing prophylactic mastectomy in a proper clinical perspective. Existing data suggest that prophylactic total mastectomy significantly reduces, but does not totally eliminate, the risk of subsequent development of cancer. However, the benefit of prophylactic mastectomy over alternative strategies (surveillance and chemoprevention) remains to be proven. Currently, prophylactic mastectomy may be considered in a few, carefully selected patients. The decision to perform a prophylactic mastectomy should be a multidisciplinary one. Detailed patient' counselling is very important; the patient should understand the limitations of prophylactic mastectomy and the need for postoperative follow-up. Furthermore, she should be well informed about the alternative strategies. PMID- 10738121 TI - Psychosocial problems secondary to cancer: an Italian multicentre survey of consultation-liaison psychiatry in oncology. Italian Consultation-Liaison Group. AB - Data relative to consultation-liaison psychiatry (C-L) in oncology are lacking. In order to examine this area, a multicentre investigation was conducted in 17 general hospitals in Italy. All psychiatric consultation requests for cancer patients referred to C-L during a 1-year period were evaluated. Only 5% (n=217 referred patients: 114 men and 103 women) of all C-L activity were for cancer patients. Most were 'routine' consultations (72%) for current psychiatric symptoms (69%) or coping/compliance problems (12%). Previous psychological or psychiatric disorders were shown in 32% of cases. Approximately 40% of patients were not informed of their referral to C-L. The most frequent ICD-10 psychiatric diagnoses were adjustment disorders (27%) and major affective disorders (23%). Transfer to psychiatric units was low (1%). These findings indicate the need for improvement of referral criteria to C-L and closer attention to continuity of psychosocial care of cancer patients during hospitalisation and post-discharge. PMID- 10738122 TI - ERBB2 overexpression in breast carcinomas: no positive correlation with complete pathological response to preoperative high-dose anthracycline-based chemotherapy. AB - The predictive value of ERBB2 amplification/expression to doxorubicin use is controversial. Preoperative chemotherapy, followed by the pathological assessment of tumour response to treatment provide optimal conditions for the evaluation of the predictive value of biological parameters. We report here data on the predictive value of ERBB2 in a series of 54 cases of breast cancer treated by preoperative high-dose anthracycline-based chemotherapy. Our series consisted of 26 women presenting an inflammatory breast cancer (IBC) and of 28 women with poor prognosis primary cancer (PPPC). Patients received a total of four cycles with doxorubicin (75 mg/m(2) for IBC or 70 mg/m(2) for PPPC) and cyclophosphamide (6 g/m(2) for IBC or 1400 mg/m(2) for PPPC), every 21 days. ERBB2 expression was determined by immunohistochemistry (clone CB11) performed on a tumour biopsy taken before chemotherapy. All patients underwent surgery as a second step of treatment, and the tumour response was assessed on pathological specimens. A complete pathological response was observed in 24 of the 54 cases (44%) (95% confidence interval (CI), 31-57). Pathological complete response was positively correlated with high histological grade (P=0. 02) and with the absence of oestrogen (P=0.003) or progesterone (P=0. 02) receptor expression. ERBB2 overexpression was found in 18 of the 54 cases (33%). A complete pathological response was observed in 33% of these cases (6/18). This figure was not significantly different from the 50% rate of complete response observed for tumours with no detectable ERBB2 expression (18/36). In this small series, ERBB2 overexpression was not a significant predictive marker of the pathological response to high-dose doxorubicin-based chemotherapy. PMID- 10738123 TI - Toxicity of high-dose radiotherapy combined with daily cisplatin in non-small cell lung cancer: results of the EORTC 08912 phase I/II study. European Organization for Research and Treatment of Cancer. AB - The purpose of this work was to study the feasibility of concurrent chemoradiation in patients with inoperable non-small cell lung cancer (NSCLC). 40 patients with inoperable NSCLC were treated with escalating doses of radiotherapy and cisplatin (cDDP). The radiation dose was increased step by step from 60.5 to 66 Gy in daily fractions of 2.75 Gy. Chemotherapy was also increased step by step from 20 to 24 daily doses of cDDP 6 mg/m(2) and given concurrently with radiotherapy. A dose of 40 Gy/2 Gy/20 fractions (fx) was given to the EPTV (elective planning target volume) which included the gross tumour volume with a margin of 2 cm and part of or the entire mediastinum. During each session a boost dose of 0.75 Gy was given simultaneously to the BPTV (boost planning target volume), which encompassed the GTV (gross tumour volume) with a margin of 1 cm, for the first 20 fx, so the total dose to the tumour was 55 Gy. Cisplatin 6 mg/m(2) was given 1 h prior to radiotherapy at each fraction. From then on the dose of radiation to the BPTV and the dose of cDDP were increased step by step. In group I the BPTV was irradiated with two extra fractions of 2.75 Gy to a total dose of 60. 5 Gy without cDDP. In group II the same total dose of 60.5 Gy was given but the last two fractions were combined with cDDP. In group III four extra fractions of 2.75 Gy were given to the BPTV to a total dose of 66 Gy, only two of these fractions combined with cDDP. Finally, in group IV a total dose of 66 Gy was given in 24 fractions, all fractions combined with cDDP. All patients were planned by means of a CT-based conformal treatment planning. The maximal length of the oesophagus receiving >/=60.5 Gy was 11 cm. 40 patients were evaluable for acute and late toxicity and for survival. Acute toxicity grade >/=3 (common toxicity criteria, CTC) was rarely observed; nausea/vomiting in 3 patients (8%), leucopenia in 2 patients (5%), thrombocytopenia in 2 patients (5%), whilst 2 patients (5%) suffered from severe weight loss. Late side-effects (European Organization for Research and Treatment of Cancer/Radiation Therapy Oncology Group, EORTC/RTOG) were: oesophageal toxicity >/=grade 3 in 2 patients (5%) and radiation pneumonitis grades 1 (3%) and 2 (3%) in 1 patient each. Overall actuarial 1- and 2-year survival was 53% and 40%, respectively. The 1- and 2-year local disease-free interval was 65% and 58% respectively. Radiotherapy at a dose of 66 Gy/2.75 Gy/24 fx combined with daily cDDP 6 mg/m(2) given over 5 weeks is feasible and results in a good local disease-free interval and a good survival rate. This treatment schedule is at present being tested as one of the two treatment arms of EORTC phase III study protocol 08972/22973. PMID- 10738124 TI - Vascular endothelial growth factor expression and neovascularisation in non-small cell lung cancer. AB - Many recent studies have demonstrated that tumour angiogenesis is a potent prognostic factor for various malignant tumours, but this has not been clearly shown in non-small cell lung carcinoma (NSCLC). The purpose of this study was to re-evaluate the prognostic value of MVD associated with VEGF in patients with NSCLC by comparing the immunohistochemical results obtained for CD34 with those obtained for vWf. Microvessel density (MVD) and the expression of vascular endothelial growth factor (VEGF) were investigated in 108 cases of NSCLC by immunohistochemistry. The correlation between von Willebrand factor (vWf) and CD34 staining for MVD was not strong, and vWf staining did not correlate with VEGF expression, but CD34 staining did. Staining for CD34 significantly correlated with survival in adenocarcinoma, distant metastasis and postoperative recurrence, but staining for vWf did not. CD34 was more sensitive and specific than vWf for staining endothelial cells associated with VEGF expression. It is suggested that research on neovascularisation should be investigated on every histological subtype or should focus on the early stages of NSCLC which are not under the influence of a variety of complications facilitating tumour neovascularisation. PMID- 10738125 TI - p53 immunoreactivity in biopsy specimens of T1G3 transitional cell carcinoma of the bladder--a helpful parameter in guiding the decision for or against cystectomy? AB - The aim of this study was to determine whether p53 is helpful in making the decision to undergo cystectomy in T1, G3 transitional cell carcinoma (TCC) of the bladder, by prospectively comparing the p53 status of bladder biopsies with the histology and p53 status of the corresponding cystectomy specimens. From January 1996 to August 1997, 38 consecutive patients with T1G3 TCC at 6 different centres were enrolled into the study. Bladder biopsies and cystectomy specimens were examined with three different antibodies against p53. The p53 status of each bladder biopsy was compared with p53 status, tumour stage and grade of the cystectomy specimen. An independent evaluation of the histology and immunohistochemistry was carried out by two pathologists. 15 of 38 patients (39%) were found to have a higher tumour stage in the cystectomy specimen compared with the staging by transurethral resection of the bladder tumour (TUR-B). 3 patients did not show residual tumour in the cystectomy specimen. No differences in p53 positivity were noted between the different antibodies. 14 of 31 evaluable tumours (45%) were p53 positive at the time of the TUR-B. p53 staining of the TUR B specimen did not correctly predict the residual tumour in the cystectomy specimen. We, therefore, concluded that compared with standard histopathology, the p53 status of the TUR-B specimen does not provide additional relevant information with regard to local tumour staging and, thus, is not helpful in making the decision for or against a cystectomy. PMID- 10738126 TI - Clinical impact of dosimetry quality assurance programmes assessed by radiobiological modelling of data from the thermoluminescent dosimetry study of the European Organization for Research and Treatment of Cancer. AB - The European Organization for Research and Treatment of Cancer (EORTC) Radiotherapy Group initiated its mailed thermoluminescence dosimetry (TLD) programme in 1986. The aim of the present study was to evaluate the clinical relevance of variations in beam output detected in the period 1993 to 1996. A total of 140 beam outputs were checked (26 for cobalt-60 units and 114 for linear accelerators) in 35 centres. Clinical dose-response data for tumour control and normal tissue morbidity were used to assess the variation in clinical outcome resulting from variability in beam output. For 75 checked beams with nominal accelerating potentials (n. a.p.) of 6 MV or less the mean ratio, +/- standard deviation (S.D.) of measured to stated output was 1.004+/-0.020. For 65 beams with n. a.p. of 8 MV or more, the ratio was 1.009+/-0.021. Even with this relatively high level of precision, broad distributions of estimated tumour control or normal tissue morbidity were found. In the 10% of the beams with the most pronounced underdosage, the loss in tumour control probability was estimated at 7-8 percentage points. Likewise, in the 10% of the beams with the most pronounced overdosage, the increase in mild/moderate morbidity was 19-22 percentage points. For severe morbidity the same beams raised the estimated incidence of severe complications from 5% to 9-10%. An estimation of the loss of uncomplicated cure probability was about 1% for both high and low energy beams. Sequential mailings considerably improved the uniformity of clinical outcome. We conclude that small deviations in beam output may lead to clinically important variations in outcome. Substantial reductions in the variation between measured and stated output can be achieved by sequential mailings. Mailed TLD checks should be an integral part of a continuously ongoing quality assurance activity in radiotherapy. PMID- 10738127 TI - What influences participation in clinical trials in palliative care in a cancer centre? AB - Like any other specialty, palliative care needs a scientific foundation on which to base its practice. Research in palliative care is particularly difficult because of the characteristics of the patient population under study (e.g. advanced disease, poor performance status and limited prognosis). The aim of this paper was to highlight the challenges of recruitment into clinical trials in palliative care. Information on all patients treated at a specialist cancer centre who were referred for consideration of entry into any one of 23 clinical trials in palliative care was collected prospectively over 4 years to determine factors that influence patients to accept or reject entry into a study. Of the 1206 patients referred, 558 (46%) met the entry criteria. Of these, 362 (30%) agreed to enter and 248 (21% of all those referred) completed the study. Thus, 65% of all eligible patients were entered into trials but only 44% of these completed the study. The relatively high percentage of patients entered probably reflects the site (a cancer centre with a high research profile) and is not typical of other palliative care centres or hospices. The most common reasons given for unwillingness to participate were a wish to defer to a later date, a deterioration in condition, distance from home to hospital, a lack of interest, transfer to another unit, inability to give consent and family objection. In order to maximise patient accrual into trials in palliative care, studies should be designed to suit the patient population under study (e.g. be of short duration with realistic entry criteria) and not necessarily mirror the trial methodology of therapeutic trials in oncology. PMID- 10738128 TI - Cyclopentenyl cytosine inhibits cytidine triphosphate synthetase in paediatric acute non-lymphocytic leukaemia: a promising target for chemotherapy. AB - Cytidine triphosphate (CTP) synthetase is a key enzyme in the anabolic pathways of cytosine and uracil ribonucleotide metabolism. The enzyme catalyses the conversion of uridine triphosphate (UTP) into CTP, and has a high activity in various malignancies, which has led to the development of inhibitors of CTP synthetase for therapeutic purposes. We studied both CTP synthetase activity and ribonucleotide concentrations in leukaemic cells of 12 children suffering from acute non-lymphocytic leukaemia (ANLL), and performed incubation experiments with cyclopentenyl cytosine (CPEC), a nucleoside analogue that is capable of inhibiting CTP synthetase. The CTP synthetase activity in ANLL cells (5.1+/-2.3 nmol CTP/mg/h) was significantly higher compared with granulocytes of healthy controls (0.6+/-0.4 nmol CTP/mg/h, P=0.0002), but was not different from the CTP synthetase activity in non-malignant CD34+ bone marrow cells (5. 6+/-2.4 nmol CTP/mg/h). Major shifts were observed in the various ribonucleotide concentrations in ANLL cells compared with granulocytes: the absolute amount of ribonucleotides was increased with a substantial rise of the CTP (2.4 versus 0.4 pmol/microg protein, P=0.0007) and UTP (8.7 versus 1.6 pmol/microg protein, P=0. 0007) concentrations in ANLL cells compared with granulocytes. Treatment of ANLL cells in vitro with CPEC induced a major depletion (77% with 2.5 microM of CPEC) in the concentration of CTP, whilst the concentrations of the other ribonucleotides remained unchanged. Therefore, the high activity of CTP synthetase in acute non-lymphocytic leukaemic cells can be inhibited by CPEC, which provides a key to a new approach for the treatment of ANLL. PMID- 10738129 TI - Meta-analysis of studies on breast cancer risk and diet: the role of fruit and vegetable consumption and the intake of associated micronutrients. AB - A meta-analysis was carried out, in order to summarise published data on the relationship between breast cancer, fruit and vegetable consumption and/or the intake of beta-carotene and vitamin C. Relative risks were extracted from 26 published studies from 1982 to 1997. Random and fixed effects models were used. Between studies, heterogeneity was found for vegetables, fruit, vitamin C but not for beta-carotene. Summary relative risk (RR) estimates based upon a random effects model, except for beta-carotene, for 'high consumption' compared with 'low consumption', derived from the studies satisfying the inclusion criteria were as follows: vegetable consumption: RR=0.75 (95% CI (confidence interval) 0.66-0.85) from 17 studies; fruit consumption: RR=0.94 (95% CI 0.79-1.11) from 12 studies; vitamin C: RR=0.80 (95% CI 0.68-0.95) from 9 studies; beta-carotene: RR=0.82 (95% CI 0.76-0.91) from 11 studies. This analysis confirms the association between intake of vegetables and, to a lesser extent, fruits and breast cancer risk from published sources. Increasing vegetable consumption might reduce the risk of breast cancer. PMID- 10738130 TI - A cost-effective analysis of the optimum number of stool specimens collected for immunochemical occult blood screening for colorectal cancer. AB - This study was carried out to assess, from the viewpoint of cost-effectiveness, the optimum number of faecal specimens to collect for use in immunochemical occult blood testing as a means of screening for colorectal cancer. 3300 asymptomatic individuals were subjects of this study. They gave samples for an immunochemical faecal occult blood test, monohaem and colonoscopy was carried out during a medical check-up. For evaluation of the optimum number of sampling specimens, the results of the first day of sampling, those of the first and second days, and those of samples taken for 3 consecutive days were considered as the single-day method, the 2-day method and the 3-day method respectively. The average cost to detect 1 patient with colorectal cancer, the detection rate and the false-positive rate of these three faecal sample collection methods were evaluated. The average costs for one cancer case detected were calculated as $3,630.68 for the single-day method, $3,350.65 for the 2-day method and $4,136.36 for the 3-day method, respectively. The detection rate and the false-positive rate were calculated as 47 and 3.5% for the single-day method, 82 and 4.7% for the 2-day method and 88 and 5.3% for the 3-day method, respectively. This detection rate was significantly different between the single- and the 2-day methods, as well as between the single- and the 3-day methods (P<0. 05). No significant differences in the false-positive rate amongst the three testing methods were observed. This analysis suggests that a 2-day faecal collection method is recommended for immunochemical occult blood screening by Monohaem from the aspects of cost-effectiveness and diagnostic accuracy. PMID- 10738131 TI - Genomic imprinting of IGF2 and H19 in human meningiomas. AB - A number of genes, including IGF2 and H19, are normally imprinted with preferential expression of the paternal or maternal allele, respectively. Loss of imprinting (LOI) of IGF2 and H19 is found in a number of tumours, suggesting that LOI of IGF2 and/or H19 may play an important role in tumorigenesis. The IGF2 gene codes for a fetal growth factor and the H19 gene is likely to act as an RNA with an antitumour effect. We investigated the imprinting status of IGF2 and H19 in human meningiomas. The normally imprinted IGF2 gene lacks imprint in the leptomeninges and choroid plexus of the brain. To examine the imprinting status of IGF2 and H19 in human meningiomas we used the ApaI polymorphism in exon 9 for the IGF2 gene and the AluI polymorphism in exon 5 for the H19 gene. In total, 24 meningiomas of WHO grade I, II and III were analysed. 15 meningiomas (63%) were informative for the ApaI polymorphism in the IGF2 gene. Monoallelic expression (MAE) for IGF2 was found in 11 out of 15 tumours (73%) which is in contrast to the lack of imprinting status of IGF2 in leptomeninges. Ten cases (42%) were heterozygous for the H19 gene and biallelic expression was found in 3 out of 10 meningiomas (30%). These results indicate that modulation of the imprinting status of IGF2 and H19 may play an important role for the development of meningiomas. PMID- 10738132 TI - Analysis of Fas and Fas ligand expression and function in lung cancer cell lines. AB - The aim of this study was to investigate the expression of Fas and Fas ligand (FasL) and to determine the significance of these molecules in lung cancer cell lines. Immunoblotting, RT-PCR and flow cytometric analyses were carried out to measure the expression of Fas and FasL and to examine their interactions and effects on cell growth and apoptosis. Fas and FasL were co-expressed in most of the cell lines but to varying degrees. Apoptosis induced by the agonistic anti Fas antibody was significantly correlated with Fas expression (P=0.0075), whereas cisplatin-induced apoptosis was not. Upregulation of Fas and FasL expression by the administration of cisplatin was found in 7 of 11 (64%) and 9 of 11 (82%) cell lines, respectively. However, cisplatin-induced apoptosis was not suppressed by antagonistic anti-FasL antibody. Thus, our data indicated that Fas and FasL were co-expressed in lung cancer cell lines, and that Fas ligation induced by agonistic anti-Fas antibody is functional and induced apoptosis that was dependent on the levels of Fas expression. In contrast, Fas-FasL interactions appeared to be non-functional. Furthermore, our results suggest that cisplatin induced apoptosis in lung cancer cells was independent of the Fas-FasL interaction. PMID- 10738133 TI - The effect of non-steroidal anti-inflammatory drugs on human colorectal cancer cells: evidence of different mechanisms of action. AB - Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit proliferation and induce apoptosis in human colorectal cancer cells in vitro. It remains unclear whether individual NSAIDs act by cyclooxygenase-2 (COX-2) inhibition and how NSAIDs exert their anti-proliferative effects. We investigated the effects of NS-398 (a selective COX-2 inhibitor), indomethacin (a non-selective COX inhibitor) and aspirin on four human colorectal cancer cell lines (HT29.Fu, HCA-7, SW480 and HCT116). NS-398 completely inhibited proliferation, induced G1 arrest and promoted apoptosis in COX-2-expressing cells (HT29.Fu and HCA-7). However, indomethacin had similar effects on all cells, regardless of COX-2 expression. NS 398 also had anti-proliferative activity on COX-2-negative cell lines (SW480 and HCT116). Aspirin inhibited proliferation of all cell lines but did not induce apoptosis. Indomethacin decreased beta-catenin protein expression in all cells (unlike NS-398 or aspirin). NSAIDs act on human colorectal cancer cells via different mechanisms. Decreased beta-catenin protein expression may mediate the anti-proliferative effects of indomethacin. PMID- 10738134 TI - Imaging of tumour neovasculature by targeting the TGF-beta binding receptor endoglin. AB - In vivo imaging of endothelial markers in intact tumour neovasculature would have applications in assessing the efficacy of anti-angiogenic agents in clinical trials. Although a variety of different endothelial markers have been described, few have been evaluated as imaging markers. The transforming growth factor-beta (TGF-beta) binding receptor endoglin is a proliferation-associated endothelial marker. We hypothesised that endoglin would be an ideal target for imaging since it is strongly upregulated in proliferating endothelial cells of the tumour neovasculature. We used a radiolabelled monoclonal anti-endoglin antibody and compared its neovascular binding, accumulation and in vivo behaviour to an isotype-matched control IgG(2a). Our data show that the probe binds specifically and rapidly within minutes in vivo and that correlative autoradiography and immunohistology support the in vivo imaging findings. Imaging of abundantly expressed endothelial targets circumvents delivery barriers normally associated with other tumour targeting strategies, and can potentially be used to quantitate molecular angiogenic markers. PMID- 10738135 TI - Epidemiology of human herpesvirus 6 (HHV-6) infection in pregnant and nonpregnant women. AB - BACKGROUND: Human herpesvirus 6 (HHV-6) is a ubiquitous virus primarily associated with benign conditions such as febrile syndromes and exanthem subitum (roseola infantum). Sexual, horizontal, and vertical transmission have been suggested. Little information is available regarding HHV-6 infection in women of reproductive age. OBJECTIVE: Describe epidemiology of HHV-6 infection in pregnant and nonpregnant women. STUDY DESIGN: The study sample consisted of 569 women, age 18-45, who attended a university family planning clinic (nonpregnant, n=224) and two obstetrics clinics (pregnant [first trimester], n=345) in San Antonio, TX between October 1995 and May 1998. Blood and a vaginal swab, as well as sociodemographic information, were collected from each participant. Plasma was tested for HHV-6 IgG antibodies using a standard immunofluorescence assay (IFA). Lysed material from vaginal swabs was tested for HHV-6 DNA by polymerase chain reaction (PCR). Products were screened by enzyme-linked immunosorbent assay and positive tests were confirmed by repeat PCR followed by Southern analysis. PCR positive samples were subtyped using an established method. RESULTS: All subjects were HHV-6 antibody positive. Geometric mean titers of HHV-6 antibodies were significantly higher among nonpregnant versus pregnant women. Moreover, a higher proportion of nonpregnant versus pregnant women had antibody titers >/=160 and >/=320. This association persisted even after adjusting for a number of sociodemographic and clinical factors. Low rates of HHV-6 shedding in the genital tract were observed for both groups (pregnant, 7/297 [2.0%]; nonpregnant, 8/214 [3.7%]). Of 14 samples subtyped, four (29%) were subtype A. CONCLUSION: The present study showed that 100% of the study sample was infected with HHV-6. Higher HHV-6 antibody titers, however, were noted in nonpregnant women. Both groups shed virus at low rates in the genital tract. HHV-6 subtype A was identified more commonly than previously reported. Further longitudinal studies are required to assess the consequences of maternal HHV-6 infection. PMID- 10738136 TI - The HHV6 paradox: ubiquitous commensal or insidious pathogen? A two-step in situ PCR approach. AB - BACKGROUND: Progressive multifocal leukoencephalopathy (PML) and multiple sclerosis (MS) are demyelinative diseases of the central nervous system (CNS). PML occurs mostly in individuals with AIDS-impaired immunity and is thought to be caused by JC polyoma virus (JCV). In MS a neurotrophic virus trigger is suspected, but the precise etiology remains unknown. Human herpesvirus 6 (HHV6) is a ubiquitous, commensal and usually benign beta-herpesvirus. Some researchers have found evidence for HHV6 infection in MS plaques and sera. We recently demonstrated a high frequency of cells containing HHV6 genome in PML lesions, as well as co-infection of oligodendrocytes by JCV and HHV6. This suggests that HHV6 may be a co-factor in the etiology of PML, and raises questions about its role in other demyelinative diseases. OBJECTIVES: To determine the prevalence and cellular localization of HHV6, JCV and HIV-1 infected cells in PML, MS, AIDS and control CNS tissues, and their potential relationship with disease. STUDY DESIGN: An unconventional, sensitive two-step in situ polymerase chain reaction (ISPCR) procedure was used to amplify and detect HHV6, JCV and HIV-1 genomic DNAs in formalin fixed, paraffin-embedded archival CNS tissues. HHV6, JCV and HIV-1 gene expression was detected by ICC for HHV6 p41 and gp101, JCV large T, and HIV-1 p24 gag and NEF proteins. RESULTS: A high frequency of HHV6 genome was consistently detected in both PML and MS white matter lesional cells; a peri-lesional concentration was notable. HHV6 was found mainly in oligodendrocytes, but neurons were also infected. HHV6 was present in larger amounts than JCV in PML lesions, while more HIV-1 than HHV6 was present in AIDS. Variable amounts of HHV6 genome were detected in normal, AIDS and other control brains; the frequency of infected cells tended to increase with patient age. CONCLUSIONS: High concentrations of HHV6 genome in association with PML and MS lesions, open the possibility that HHV6 activation may play a role in the pathogenesis of these demyelinative diseases. PMID- 10738137 TI - Frequent HHV-6 reactivation in multiple sclerosis (MS) and chronic fatigue syndrome (CFS) patients. AB - BACKGROUND: HHV-6 is a ubiquitous virus and its infection usually occurs in childhood and then becomes a latent infection. HHV-6 reactivation has been shown to play a role in the pathogenesis of AIDS and several other diseases. OBJECTIVES: To determine what role HHV-6 infection or reactivation plays in the pathogenesis of multiple sclerosis (MS) and chronic fatigue syndrome (CFS). RESULTS: Twenty-one MS and 35 CFS patients were studied and followed clinically. In these patients, we measured HHV-6 IgG and IgM antibody levels and also analyzed their peripheral blood mononuclear cells (PBMCs) for the presence of HHV 6, using a short term culture assay. In both MS and CFS patients, we found higher levels of HHV-6 IgM antibody and elevated levels of IgG antibody when compared to healthy controls. Seventy percent of the MS patients studied contained IgM antibodies for HHV-6 late antigens (capsid), while only 15% of the healthy donors (HD) and 20% of the patients with other neurological disorders (OND) had HHV-6 IgM antibodies. Higher frequency of IgM antibody was also detected in CFS patients (57.1%) compared to HD (16%). Moreover, 54% of CFS patients exhibited antibody to HHV-6 early protein (p41/38) compared to only 8.0% of the HD. Elevated IgG antibody titers were detected in both the MS and the CFS patients. PBMCs from MS, CFS and HD were analyzed in a short term culture assay in order to detect HHV-6 antigen expressing cells and to characterize the viral isolates obtained as either Variant A or B. Fifty-four percent of MS patients contained HHV-6 early and late antigen producing cells and 87% of HHV-6 isolates were Variant B. Isolates from CFS, patients were predominately Variant A (70%) and isolates from HD were predominately Variant B (67%). Moreover, one isolate from OND was also Variant B. Persistent HHV-6 infection was found in two CFS patients over a period of 2.5 years and HHV-6 specific cellular immune responses were detected in PBMCs from ten CFS patients. CONCLUSION: In both MS and CFS patients, we found increased levels of HHV-6 antibody and HHV-6 DNA. A decrease in cellular immune responses was also detected in CFS patients. These data suggest that HHV-6 reactivation plays a role in the pathogenesis of these disorders. PMID- 10738138 TI - Increased expression of human herpesvirus 7 in lymphoid organs of AIDS patients. AB - BACKGROUND: Human herpesvirus 7 (HHV-7) interferes reciprocally with the human immunodeficiency virus (HIV) in CD4 T lymphocytes, as infection with HIV results in a down modulation of the CD4 molecule and inhibition of replication of HHV-7, and vice versa. Correlations between HHV-7 and HIV at the organ level have not been studied in detail. OBJECTIVE: To study the presence and cellular distribution of HHV-7 in lymphoid organs, i.e. lymph nodes (LNs) and spleen in AIDS patients and HIV-seronegative individuals. STUDY DESIGN: Cross-sectional study. The detection of HHV-7 specific antigen pp85, the 85 kDa encoded tegument phosphoprotein by U14 gene, was performed by immunohistochemistry (IHC) with a well characterized monoclonal antibody (mAb 5E1) to pp85. Nested polymerase chain reaction (PCR) was applied to detect HHV-7 specific DNA sequences. RESULTS: Cells infected with HHV-7 were detected in five of seven LNs from AIDS patients and in one of five LNs from HIV-seronegative patients. The infected cells were mainly macrophages. In samples from HIV-seropositive patients, a significantly higher number of HHV-7 infected cells could be observed than in specimens from HIV seronegative patients. Neither the antigen nor DNA sequences of HHV-7 could be detected in spleen tissue from HIV-seronegative and AIDS patients. CONCLUSIONS: The data indicate that HHV-7 undergoes a higher extent of reactivation from latency and/or of replication under immunosuppression due to HIV-infection, similar to the other beta-herpesviruses HHV-6 and human cytomegalovirus (HCMV). The data further suggest that LNs, but not the spleen, may be a site of latency and consequently of reactivation of HHV-7 in AIDS patients. PMID- 10738139 TI - Characterization of the human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) oncogene, kaposin (ORF K12). AB - BACKGROUND: Human herpesvirus 8 (HHV-8) has been implicated in the etiology of Kaposi's sarcoma (KS), a highly angiogenic tumor of complex histology, and two lymphoproliferative diseases, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). A number of HHV-8 encoded genes have been proposed to be involved in the pathogenesis of KS and PEL and a few have been shown to be oncogenic in heterologous systems (Reyes GR, LaFemina R, Hayward SD, Hayward GS. Morphological transformation by DNA fragments of human herpesviruses: evidence for two distinct transforming regions in herpes simplex virus types 1 and 2 and lack of correlation with biochemical transfer of the thymidine kinase gene. Cold Spring Harbor Symp Quant Biol 1980;44:629-641; Moore PS, Boshoff C, Weiss RA, Chang Y. Molecular mimicry of human cytokine and cytokine response pathway genes by KSHV. Science 1996;274:1739-1744; Cheng EH, Nicholas J, Bellows DS, Hayward GS, Guo HG, Reitz MS, Hardwick JM. A Bcl-2 homolog encoded by Kaposi sarcoma associated virus, human herpesvirus 8, inhibits apoptosis but does not heterodimerize with Bax or Bak. Proc Natl Acad Sci USA 1997;94:690-694; Li M, Lee H, Yoon DW, Albrecht JC, Fleckenstein B, Neipel F, Jung JU. Kaposi's sarcoma associated herpesvirus encodes a functional cyclin. J Virol 1997;71:1984-1991; Neipel F, Albrecht J-C, Fleckenstein B. Cell-homologous genes In the Kaposi's sarcoma-associated rhadinovirus human herpesvirus 8: determinants of its pathogenicity? J Virol 1997;71:4187-4192; Nicholas J, Ruvolo VR, Burns WH, Sandford G, Wan X, Ciufo D, Hendrickson SB, Guo HG, Hayward GS, Reitz MS. Kaposi's sarcoma-associated human herpesvirus-8 encodes homologues of macrophage inflammatory protein-1 and interleukin-6. Nat Med 1997;3:287-292; Nicholas J, Zong J, Alcendor DJ, Ciufu DM, Poole LJ, Sarisky RT, Chiuo C, Zhang X, Wan X, Guo H, Reitz MS, Hayward GS. Novel organizational features, captured cellular genes, and strain variability within the genome of KSHV/HHV-8. JNCI Monographs 1998;23:79-88; Muralidhar S, Pumfery AM, Hassani M, Sadaie MR, Azumi N, Kishishita M, Brady JN, Doniger J, Medveczky P, Rosenthal LJ. Identification of kaposin (ORF K12) as a human herpesvirus 8 (Kaposi's sarcoma associated herpesvirus) transforming gene. J Virol 1998;72:4980-4988). The kaposin gene (ORF K12) encoded by the abundant latency-associated HHV-8 transcript, T0.7, has been previously shown to induce tumorigenic transformation of Rat-3 cells (Muralidhar S, Pumfery AM, Hassani M, Sadaie MR, Azumi N, Kishishita M, Brady JN, Doniger J, Medveczky P, Rosenthal LJ. Identification of kaposin (ORF K12) as a human herpesvirus 8 (Kaposi's sarcoma associated herpesvirus) transforming gene. J Virol 1998;72:4980-4988). The current study is a further characterization of kaposin protein. OBJECTIVES: Characterization of kaposin expression in transformed and tumor-derived Rat-3 cells as well as PEL cell lines, BCBL-1, BC-3 and KS-1 and analysis of mechanism(s) of transformation. DESIGN: The presence of kaposin DNA in transformed cells was determined by fluorescent in situ hybridization (FISH). Expression of kaposin protein was analyzed by Western blot analysis and indirect immunofluorescence assay (IFA). (ABSTRACT TRUNCATED) PMID- 10738141 TI - Specifics on the refinement and application of two serological assays for the detection of antibodies to HHV-8. AB - BACKGROUND: Serologic assays for the detection of antibodies to human herpesvirus type 8 (HHV-8) are important for epidemiological studies and to further investigate the proposed pathogenesis of the virus in cancer. Although a variety of assays are available, a lack of optimization and standardization makes their usefulness uncertain, and may be responsible for the controversy concerning the prevalence of infection. OBJECTIVES: To refine an indirect immunofluorescent assay (IFA) for the detection of latent antibodies and a recombinant ORF 65 ELISA for the detection of lytic antibodies in order to increase their ability to differentiate individuals at higher and lower risk for HHV-8 infection. STUDY DESIGN: Sera from Kaposi's sarcoma (KS) patients and blood donors (BDs) were used to modify assay parameters in an attempt to better discriminate between the two populations. Modifications included methods of substrate fixation, incubation times, sample dilution, and antigen/conjugate concentrations. RESULTS: Optimal modifications to the latent IFA included acetone fixation of substrate, and dilution of sera to 1:64 which enhanced detection of HHV-8 antibodies from 68 to 92% in the KS population. Similarly, successful refinement of the ORF 65 ELISA to increase the signal-to-noise ratio included the use of 88 ng of ORF 65 antigen per well and serum dilutions of 1:50. Optical density-to-cut-off ratios directly correlated with titers, thereby introducing a strategy to predict antibody concentrations. The ORF 65 ELISA and the latent IFA were both able to discriminate between the two populations but with different efficiencies. CONCLUSIONS: Although neither the latent IFA nor the ORF 65 ELISA produced perfect test indices, improvement in their performances was noted following the optimization strategies. The ELISA produced better detection of antibodies to the virus than the IFA and permitted prediction of sample titers, thus improving cost and time effectiveness. PMID- 10738140 TI - Molecular characterization of HHV-8 positive primary effusion lymphoma reveals pathogenetic and histogenetic features of the disease. AB - BACKGROUND: Primary effusion lymphoma (PEL) associates with HHV-8 infection, preferentially develops in immunodeficient patients and grows in the serous body cavities. PEL derives from post-germinal center, pre-terminally differentiated B cells. The pathogenesis of PEL is unclear and the sole identified genetic lesions are human herpesvirus type-8 (HHV-8) infection in all cases and EBV infection in 70% of cases. Epstein-Barr virus (EBV) infection in PEL displays a latency I phenotype. OBJECTIVES: To clarify the pathogenesis and histogenesis of PEL by investigating (1) the lymphoma karyotype; (2) the expression status of the Met tyrosine kinase receptor and of its ligand hepatocyte growth factor (HGF); (3) the molecular profile of EBV, with particular focus on mutations of EBNA-1 genes, which are thought to affect viral tumorigenicity in EBV-infected neoplasms displaying the latency I phenotype. STUDY DESIGN: Twenty-four PEL (nine cell lines and 15 primary specimens) formed the basis of the study. Karyotypes were investigated by conventional cytogenetics and fluorescent in situ hybridization (FISH) in selected cases. The expression status of Met and HGF was defined by multiple techniques, including RT-PCR, FACS analysis, immunocytochemistry, Western blot studies and ELISA. The molecular profile of EBNA-1 genes of EBV were investigated by DNA direct sequencing. RESULTS: Trisomy 7, trisomy 12 and breaks at 1q21-q25 are recurrently associated with PEL. PEL consistently co-express Met and HGF both at the mRNA and protein level. Among aggressive B-cell lymphomas, Met/HGF co-expression appears to be relatively specific for PEL. The EBNA-1 gene of EBV displays a high degree of genetic heterogeneity in PEL, with no preferential association with one specific variant. CONCLUSIONS: PEL associates with recurrent chromosomal alterations, suggesting that viral infection is not sufficient for tumor development and that lesions of cellular genes may be required. The expression of Met/HGF by PEL cells may bear implications for the lymphoma proliferation and growth pattern, since Met/HGF interactions influence cell mitogenesis and motogenesis. EBV infection in PEL displays a latency I phenotype and fails to associate with specific EBNA-1 variants, suggesting that the role of EBV in PEL is not mediated by the major transforming pathways currently known in EBV positive lymphomas. PMID- 10738142 TI - Human herpesvirus type 8 in salivary gland tumors. AB - BACKGROUND: The new human herpesvirus type 8 (HHV-8) has been detected in all types of Kaposi's sarcomas, as well as in body-cavity lymphomas and Castleman's disease, furthermore molecular biologic studies have identified a number of potential viral oncogenes. There is evidence for sexual transmission of HHV-8 in HIV-seropositive patients, but the route of infection among the HIV-seronegative population is uncertain. Findings of HHV-8 DNA in saliva in some cases are suggestive of nonsexual transmission associated with latent infection of the salivary gland (as it is known for EBV, CMV, HHV-6 and HHV-7). OBJECTIVE: As little is known about the etiological factors of salivary gland tumors and to give more insights into HHV-8 cell tropism normal salivary gland tissue (n=12) and different salivary glands neoplasm (n=58) were tested for HHV-8 sequences and antigens in HIV-seronegative patients. STUDY DESIGN: Biopsies of both normal salivary gland and tumors were investigated for HHV-8 sequences. A nested-PCR method was used for amplification of HHV-8 DNA fragments and the nature of the amplification products was confirmed by Southern blot hybridization. In addition, we used an in situ hybridization technique and immunohistochemical staining for detection of HHV-8 infected cells. The sera of the respective patients were tested for anti-HHV-8 antibodies using commercial IFA and an ELISA-assay. RESULTS: HHV-8 DNA sequences could be detected in one bilateral MALT-lymphoma of the parotid gland of a HHV-8 seropositive female patient suffering from Sjogren's syndrome (SS). The remaining parotid samples did neither show HHV-8 sequences nor HHV-8 antigens. Using above assays only one additional patient was seropositive for HHV-8. CONCLUSION: Our data suggest that HHV-8 does not usually infect the salivary gland in HIV-seronegative patients and does not seem to play a pathogenic role in vascular and epithelial salivary gland neoplasm. Pathogenic role of HHV-8 in Sjogren's syndrome associated MALT-lymphoma remains unclear and should be subject of further studies. PMID- 10738143 TI - Detection of human herpesvirus 8 DNA and antibodies to latent nuclear and lytic phase antigens in serial samples from aids patients with Kaposi's sarcoma. AB - BACKGROUND: human herpesvirus 8 (HHV-8) have recently implicated in the etiology of Kaposi's sarcoma (KS), but the pathophysiologic and immunologic interactions between HHV-8 and the human host are incompletely understood. OBJECTIVE: this paper intends to present partial results of a follow-up study of KS patients, designed to investigate HHV-8 viremia and antibody response. METHODS: ninety-six paired serial samples (PBMCs and sera) were obtained from 12 aids patients with KS who received HAART prior or just after entry in the study. HHV-8 DNA was detected by nested-PCR and antibodies to HHV-8 latent nuclear antigen (LANA) and lytic antigen by immunofluorescence assay (IFA). RESULTS: HHV-8 DNA was detected in 33.3% of the first PBMC samples. Among the eight PCR negative patients, four presented positive samples during the follow-up and four remained negative. Five patients had intermittent viremia. Fifteen of the 96 PBMC samples were PCR positive (15.6%). Four of 39 samples (10.2%) from patients classified as stadio II and 11 of the 53 samples (20.7%) from patients in stadio IV were PCR positive (P=0.2). Six patients (50%) had anti-LANA antibodies at the entry in the study. Among the six seronegative patients, two seroconverted 2 months later and four patients remained seronegative during the 5-8 months of follow-up. All patients had anti-lytic antibodies since the first sample. CONCLUSION: the presence of HHV 8 viremia could be related to the severity of KS and could be intermittent even under HAART. A longer follow-up is needed to confirm these results. PMID- 10738145 TI - [Changes in cadernos de Saude Publica] [In Process Citation] PMID- 10738144 TI - Two distinct lineages of macaque gamma herpesviruses related to the Kaposi's sarcoma associated herpesvirus. AB - BACKGROUND: KSHV, Kaposi's sarcoma-associated herpesvirus, is a necessary cofactor for the development of Kaposi's sarcoma (KS). We have previously reported KSHV-related DNA sequences in retroperitoneal fibromatosis (RF) tissue from two species of macaque. The putative herpesvirus was called RFHV for RF associated herpesvirus. These data suggested that KSHV is a human representative of a larger family of primate herpesviruses. OBJECTIVE: To identify and characterize other members of a putative family of KSHV-related herpesviruses in macaques in order to obtain information on the evolutionary history of KSHV infection in humans. STUDY DESIGN: Lymphoid tissue cells and blood leukocytes from rhesus-, cynomolgus- and pigtailed-macaques were tested for the presence of unknown herpesviruses using degenerate primer-driven PCR amplification. The sequences obtained were compared against known herpesvirus sequences. RESULTS: We have identified new herpesvirus DNA sequences in each of the three macaque species. Sequence comparisons indicate that these new viruses are most related to each other and form a separate phylogenetic lineage within the gamma herpesviruses. Screening of PBMC from Indonesian-origin quarantine animals suggests that these viruses (MGV, macaque gamma virus) are species-specific, and highly prevalent in the wild. They are readily cultured in vivo, and share a common tissue tropism with the previously identified RFHV. CONCLUSIONS: MGV and RFHV represent two independent introductions of an ancestral gamma herpesvirus into macaque precursors. PMID- 10738146 TI - [Present status of traffic accidents in the age of speed: an overview]. AB - This article takes an interdisciplinary perspective to studies on traffic accidents on a national and international scale. It begins by analyzing the great increase in the manufacturing and use of motor vehicles worldwide and the resulting social transformations. Particular attention is given to the degradation of the urban environment and the enormous social costs represented by traffic accidents. The author follows with an epidemiological perspective on the victims of traffic accidents. The relationship between personality and traffic accidents deserved special attention, mainly in that it relates to the consumption of alcohol and other drugs, in addition to other law breaking behavior. The article concludes by emphasizing the need for government to implement specifically consistent public policies to control the problem. PMID- 10738147 TI - [Reflections on the measurement of maternal mortality]. AB - The maternal mortality rate is a highly sensitive indicator for the health level of both women and the general population in a given geographical area. There is extensive variability among different countries, and rates are high in underdeveloped or developing areas, as in Brazil. Health authorities from various countries have launched programs aimed at reducing maternal deaths and have thus needed to estimate the actual rates to allow for a proper assessment and to control the programs' progress. However, there are many obstacles and difficulties in obtaining the real values of these measures, mainly because of incomplete data. The aim of this paper is to present some of the proposed methodologies for estimating maternal mortality rates and to call attention to the limits and biases of these methods. Based on the Brazilian case, the article also recommends an improvement in the quality and coverage of the Civil Registry, the official source of data on births and deaths. PMID- 10738148 TI - [Spatial analysis of Aedes aegypti larval distribution in the Ilha do Governador neighborhood of Rio de Janeiro, Brazil]. AB - This study aims to help expand knowledge on Aedes aegypti in Rio de Janeiro, based on spatial analysis of the mosquito's breeding sites in the neighborhood of Ilha do Governador, Rio de Janeiro, from June 1992 to July 1994. Use of spatial analysis techniques is proposed for vector surveillance and control. Information obtained from the number of dwellings per block that were used for the analysis. A smoothing method, a Gaussian Kernel, was used in the spatial distribution analysis. Breaks in National Health Foundation intervention activities were observed between cycles 4 and 5 and 5 and 6, respectively, followed by increases in vector density and vector-positive areas. Permanently positive areas, like slums and a military compound, display the persistence of favorable environmental conditions for oviposition and larval growth, indicating flaws in the vector control program. Although Kernel is an exploratory analytical method involving subjective interpretation, it provides easy and fast visualization of sites subject to different degrees of risk, unaffected by existing political and administrative territorial apportionment. PMID- 10738149 TI - [Prevalence, risk behavior, and level of information on urinary schistosomiasis in primary school students from the Primeiro de Junho Health District, Maputo, Mozambique]. AB - To assess the prevalence and identify levels of information and risk behavior associated with vesical schistosomiasis, a cross-sectional study was conducted in October-November 1996, involving students from 3 primary schools in the Primeiro de Junho health district in Maputo, Mozambique. Epidemiological interviews and urine samples were taken from 434 male and female students from grades 2 through 5, randomly selected. Estimated prevalence was 11.3% (49/434). Highest prevalence was in the age group ( 15 years old, and the highest intensity of infection among the 10-14-year age group, with 204 eggs/10 ml urine. Approximately 18.9% had heard of schistosomiasis, and of these, only 19.5% (16/82) knew how the disease is manifested. Approximately 50% of the students reported frequent water contacts with potential sources of schistosomiasis. The study shows that vesical schistosomiasis is a public health problem among students in this health district. PMID- 10738150 TI - [Tolerance to administration of massive doses of vitamin A associated to mass immunization of children in Northeast Brazil]. AB - A follow-up study was carried out in two localities in the semi-arid region of the State of Bahia, Northeast Brazil, with the aim of identifying the occurrence and nature of possible acute side effects subsequent to vitamin A megadose supplement given together with mass immunization in children 6-59 months old. The sample consisted of 852 children, 416 from the county of Teofilandia who received vitamin A together with vaccines and 436 from Santa Barbara, who received only vaccine. In the 24 hours before immunization, children from both groups had similar incidences of diarrhea, fever, and vomiting. Anorexia was more prevalent in Teofilandia and remained so throughout the study period. The results suggest that acute side effects like diarrhea, vomiting, fever, or anorexia were not associated with the vitamin A dosage given with mass OPV, DPT, and measles immunization. PMID- 10738151 TI - Toward a public health of situations: the re-contextualization of risk. AB - This paper reviews the role of situational variables in health risk behaviors and the literature on the impact of context and situation in public health. Three postulates for a situational model are presented: that the situation can account for additional variance in explanation of health risk behaviors; that the power of the situation is reciprocal to the degree of individual autonomy; and that the situation is definable and measurable. A situational presentation methodology is presented for measuring situations in public health with preliminary data on its efficacy in the context of sexual behavior and injecting drug use as HIV transmission risks. Interventions which maximize the use of situationally-based information are discussed. It is concluded that situational presentations may offer additional explanatory power in public health and a means for intervening at a situational level. PMID- 10738152 TI - [Understanding the low prevalence of weight-for-height deficit in lower-income Brazilian children: correlations among anthropometric values]. AB - The sample for this study consisted of 386 children from six to 59 months of age. The objective was to study the association between wasting and abdominal circumference. Thirteen anthropometric measurements were taken: weight, height or length, crown-rump length, 4 circumferences, 4 skin fold thicknesses, and 2 breadths. Muscle, fat, and total upper arm areas and leg length were calculated. Indices of body proportionality were obtained by dividing the anthropometric variables by height. Height-for-age, weight-for-age, and weight-for-height deficits were 25.9%, 14.4%, and 3.5%, respectively. The smallest and lightest children were those with the highest abdominal circumferences divided by height. According to this study, abdominal circumference for Brazilian children without height-for-age deficit is, on average, 1.2 cm larger than for US children. Using this as a basis, the study calculated that prevalence of weight-for-height deficit would increase from 3.5% to 7.0% by increasing 2 cm in the abdominal circumference. The low prevalence of wasting and the high prevalence of stunting as indicated by several Brazilian studies could be explained partially by larger mean abdominal circumference values. PMID- 10738153 TI - [Validity of neonatal clinical assessment for estimation of gestational age: comparison of new ++Ballard+ score with date of last menstrual period and ultrasonography]. AB - The objective of this article is to evaluate the accuracy of the New Ballard (NB) score in detecting prematurity. Date of last menstrual period (LMP) and ultrasonography (USG) are used as reference procedures. First, an assessment of inter-observer reliability of the NB score measurements and the USG reports was carried out. Regarding the NB scores, the kappa coefficient and the Intraclass Correlation Coefficient were, respectively, 0.74 (CI 95% 0.49-0.99) and 0.88 (CI 95% 0.78-0.93). For the USG analysis an Intraclass Correlation Coefficient of 0.96 (CI 95% 0.91-0.98) was found. Turning to validity, sensitivity of the NB score was below 0.70 in all but one comparison, irrespective of which procedure was taken as reference. Although specificity was consistently above 0.90, results suggest that the diagnostic ability of the NB score to detect prematurity is far from ideal. The proportion of false negatives may be a warning against its use alone, both in clinical practice and in epidemiological research contexts. Rather, the procedure should be recommended mainly as an accessory diagnostic tool. PMID- 10738154 TI - [Managed care in Latin America: transnationalization of the health sector in the context of reform]. AB - This article presents the results of the comparative research project "Managed Care in Latin America: Its Role in Health Reform". The project was conducted by teams in Argentina, Brazil, Chile, Ecuador, and the United States. The study's objective was to analyze the process by which managed care is exported, especially from the United States, and how managed care is adopted in Latin American countries. Our research methods included qualitative and quantitative techniques. Adoption of managed care reflects transnationalization of the health sector. Our findings demonstrate the entrance of large multinational financial capital into the private insurance and health services sectors and their intention of participating in the administration of government institutions and medical/social security funds. We conclude that this basic change involving the slow adoption of managed care is facilitated by ideological changes with discourses accepting the inexorable nature of public sector reform. PMID- 10738155 TI - [Development of a quantitative questionnaire of food intake in japanese immigrants and their descendants residents in Sao Paulo, Brazil]. AB - The present study describes the development of a questionnaire to assess usual frequency and quantity of food and nutrient intake by people of Japanese descent living in Sao Paulo, Brazil. Both the food list and appropriate serving sizes for food items from the Food Frequency Questionnaire (FFQ) were selected from a cross sectional assessment of diet in a random sample of Japanese immigrants living in the city of Sao Paulo (first- and second-generations, n = 166), aged 45-70 years, using self-administered three-day food records. We used the food records to select food items on the basis of their contribution to total population intake of relevant foods and nutrients. Criteria for grouping separate foods included similarity in nutrient content per usual serving and the importance of a particular food (e.g., typical Japanese foods). Four portion sizes were presented: small, medium, large, and extra-large. We examined the frequency distribution of equivalent-gram weights of each selected food and identified the four serving sizes in the distribution. The quantitative FFQ was designed with 129 food groups and exact frequencies of intake. Instructions for self-reporting and applications of the method for epidemiological use are discussed. PMID- 10738156 TI - [The rural labor process and health in the Southern Brazilian mountains: a descriptive study]. AB - This cross-sectional study among rural workers in the mountainous region of the southernmost Brazilian State of Rio Grande do Sul was designed to identify the characteristics of work performed on family farms. The research focused on the socio-demographic profiles of rural workers, identifying the characteristics of rural labor and describing the prevalence of some disease entities in such populations. Some 1479 rural workers from 495 farms were interviewed. In this sample, 87% of the individuals were members of the farm-owning family, mean age was 41 years, 56% were males, and mean schooling was 5 years. Farms had a mean area of 37 hectares, 50% had at least one type of farm machinery, and fruits constituted the main crop. About 75% of workers handled several types of pesticides, while 12% reported at least one lifetime episode of pesticide poisoning. Prevalence of minor psychiatric disorders was 36%, and annual frequency of occupational injuries was 10%. There was a wide variety of activities and occupational risks. The high prevalence of health problems identified in the study calls attention to the need for measures to promote and protect rural workers' health. PMID- 10738157 TI - [Characterization of the cases of domestic violence against women assisted in 2 public hospitals of Rio de Janeiro]. AB - This paper aims at: (a) to analyze the distribution of the cases of domestic violence against women (adolescent and adult) in relation to emergency care due to external causes; (b) to characterize the victims and the rendered care; (c) to analyze the circumstances in which events happened. In methodological terms, it tries to articulate the quantitative and qualitative approaches. The study was developed in two public hospitals of reference placed in the city of Rio de Janeiro. Of the 72 assisted women, most referred to the husband, the partner or the boyfriend as the aggressor (69.4%) and were beaten (70.4%) especially on the face and on the head. The study comes to the conclusion that the health services should provide a good clinical care and promote prevention's actions. PMID- 10738158 TI - Drug compliance and the elderly: who is publishing, where, and when? AB - Population aging has occurred in a rather heterogeneous way worldwide. The increasing size of the elder age group highlights various health-related problems for the elderly. One such issue is non-compliance with drug prescriptions, a potential source of serious, even life-threatening problems for the patient. The authors have proposed to identify and rank countries that develop research on drug compliance among the elderly, relevant periodicals, and the frequency articles were published between January 1987 and December 1997. A descriptive study was carried out on the basis of a bibliographical review. Three databases were surveyed: MEDLINE, SOCIOFILE and LILACS. The sample included 440 articles, limited to the behavioral sphere. The number of periodicals recorded was 255, varying from one to tem articles. The number of articles published during the study period varied from 23 in 1987 to 49 in 1992, dropping to 29 in 1997. Frequency of the number of articles/year was erratic up to 1992, leveling off from 1993 on at more than double the baseline figure. The authors emphasize the need for developing multi-focused research worldwide. PMID- 10738159 TI - [Adults without caries: a case-control study about knowledge, attitudes and preventive practices]. AB - The purpose of this article was to investigate the effect of personal awareness and attitudes, the dentist's role, oral hygiene habits, and fluoride use on total absence of caries in adults, using a case-control study, matched for sex and age with undergraduate students from two private Brazilian universities. Crude analysis using conditional logistic regression failed to show any effect for the related variables regarding fluoride use and oral hygiene habits (p > 0.20). Use of dental floss was the only exception in this set. It appeared as a risk factor, since individuals who stated not flossing presented a crude odds ratio (OR) of 0.70, or 0.73 when adjusted for regular visits to the dentist. The effect for regular visits to the dentist was that of a risk. Those with a history of caries presented a crude odds ratio of 1.55; after adjusting for dental floss use, the risk was slightly higher (OR = 1.67). Oral hygiene habits, contrary to the formulated hypothesis, did not display the expected protective effect. Likewise, fluoride use was not significantly associated with absence of caries. Individuals with a history of caries were used to visiting more professionals, confirming the formulated hypothesis for this variable. Nevertheless, it is still intriguing that some people fail to develop caries, even they report not practicing measures considered ideal for oral health. PMID- 10738160 TI - [Media, cloning, and bioethics]. AB - This article was based on an analysis of three hundred articles from mainstream Brazilian periodicals over a period of eighteen months, beginning with the announcement of the Dolly case in February 1997. There were two main objectives: to outline the moral constants in the press associated with the possibility of cloning human beings and to identify some of the moral assumptions concerning scientific research with non-human animals that were published carelessly by the media. The authors conclude that there was a haphazard spread of fear concerning the cloning of human beings rather than an ethical debate on the issue, and that there is a serious gap between bioethical reflections and the Brazilian media. PMID- 10738161 TI - [Suicide and its psychopathological relations: a qualitative analysis of 6 cases of rational suicide]. AB - We studied the meaning given to the terms "death" and "suicide" among psychiatric inpatients. Forty-four patients who had been admitted in a psychiatric inpatient unit following a suicide attempt underwent a qualitative interview, using a "general guide interview" approach. The results were analyzed systematically to investigate the relationship between attempted suicide and mental disorder. In 6 cases there was no correlation between the attempted suicide and psychopathological symptoms. One of these patients had a psychiatric diagnosis, but this did not seem to be related to the suicide attempt. All of them made a rational a deliberate suicidal act. CONCLUSION: even in a psychiatric inpatient setting, suicide attempts are not always a behavioural expression of underlying psychopathological disturbances - individual and social factors also play a decisive role. PMID- 10738162 TI - [Pharmaceutical care in primary health care: the experience of 3 Brazilian States]. AB - In the context of recent changes in the Brazilian drug supply system at the national level, mainly the deactivation of CEME (a government agency under the Ministry of Health that dealt with drug supply policy) and the increasing decentralization of activities under the Unified National Health System (SUS), three Brazilian States - Parana, Sao Paulo, and Minas Gerais - have established programs for the distribution of essential drugs for primary health care. A nationwide program for the procurement and supply of 32 drugs for primary health care (the so-called "Basic Pharmacy Program") is currently being implemented by the Ministry of Health, to be decentralized subsequently. This article discusses the establishment of State-level drug policies for primary health care through the analysis of the three pioneering programs encompassing drug selection, procurement, storage, distribution, and use. PMID- 10738163 TI - [Epilepsy, epileptics, the work: conflicting relations]. AB - Participation in the workplace by people with epilepsy is conditioned by various interrelated issues concerning epilepsy itself, the epileptic individual, and society's implicit cultural codes. In order to analyze how such conflictive participation interferes decisively in the quality of life of people with epilepsy, a survey was conducted with 339 patients at the University Hospital of Universidade Federal Fluminense. Patients answered questionnaires including level of schooling, professional qualifications, employment situation, frequency, type, and duration of seizures, associated phenomena, and prejudice towards epilepsy. We observed that satisfactory management of seizures is decisive for their success at work. All patients with daily seizures and 9.3% of those with weekly seizures had never been employed. We conclude that any intervention in this context must include the implementation of multiprofessional and intersectorial therapeutic programs and legislation to protect epileptic patients' rights in a broad effort at demystification of epilepsy. PMID- 10738164 TI - [Gender focus and the relationship between health and work in the context of productive reorganization and underemployment]. AB - This article analyzes the issue of workers' health in the context of productive reorganization, based on the sexual division of labor and gender relations. The author begins with a discussion of cross-cutting issues and moves on to analyze current trends: the increase in female labor, its incorporation by multinational companies in the so-called Third World countries, an increase in differences and greater vulnerability vis-a-vis the process involving underemployment and suspension of social labor clauses. Finally, two examples of female labor (in industry and the school system) foster reflection on the effect of productive reorganization on working women's health, highlighting the issue of excessive workloads for women. PMID- 10738165 TI - Molecular epidemiology of dengue viruses in Brazil. AB - Dengue viruses (DEN) are found as four antigenically distinct serotypes designated DEN-1, 2, 3, and 4. Laboratory evidence that strain-intratypical variation occurs among DEN viruses has been demonstrated since the 1970s, although only with the advances in molecular technologies has it been possible to determine the genetic variability of each serotype. Genotypical identification has proven to be a useful tool for determining the origin and spread of epidemics and to correlate virulence of strains. In this report we present the results of molecular epidemiological studies with the DEN-1 and DEN-2 viruses that caused dengue epidemics in Brazil during the last decade. PMID- 10738166 TI - [Demographic and spatial aspects of scorpion stings in the northwest region of Belo Horizonte City, Minas Gerais, 1993-1996]. AB - This paper analyzes a total of 352 scorpion sting reports in the Northwest quarter of Belo Horizonte, Minas Gerais. Traditionally, this area has shown one of the highest case incidence rates, and the present study pointed to 10.37 stings per 10,000 inhabitants. No gender preference was observed, but most victims were 50 years of age or older. Likewise, no relationship was observed between seasonality and sting rates. According to geographic distribution analyses, the highest incidence rates were in areas with the largest scorpion populations. This preliminary descriptive evaluation is important for guidelines to prevent scorpion stings, which should obviously consider local epidemiological features. PMID- 10738167 TI - [Limits in the applicability of urine delta aminolevulinic acid determination as a screening test in the evaluation of occupational lead poisoning]. AB - The relationship between concentrations of ALA-U and Pb-S for two groups of workers is reported. The first group consisted of workers from a telephone company, and the second, of workers from battery factories with average Pb-S equivalent to 17.3 (g/dl (6.2-39.4) and 61.5 (g/dl (41.1-91.0), respectively. The aim of this study was to evaluate the utilization of ALA-U levels as a screening test for different levels of lead in blood by means of High Performance Liquid Chromatography (HPLC) and of spectrophotometry. A significant correlation was found between measured ALA and levels of blood lead (R = 0.739 - first group; R = 0.902 - second group; p < 0.001). The validity of ALA-U test to evaluate different levels of lead in blood was also studied. By using ALA levels of 3mg/g creatinine as a threshold to detect levels of lead in blood equal to or higher than 20 (g/dl, the test results, for the workers in the first group, showed sensibility of 92% and specificity of 90%. In both groups, the false positives as well the false negatives were lower than 10% which was enough to validate the test. PMID- 10738168 TI - [Operationalizing the categories access and decentralization in health systems analysis]. AB - This paper presents a proposal for operationalization of access and decentralization in a health systems analytical model. It supports the hypothesis that decentralization of health systems fosters better access by users. This proposal is intended to contribute to a closer link between these categories and observed reality. PMID- 10738169 TI - [An epidemiological approach to health promotion: the ideas of Geoffrey Rose]. AB - In this paper, we discuss some of the ideas developed by Geoffrey Rose, which are seldom explicitly expressed in the contemporary debate on health promotion. The following notions are emphasized: many health exposures and outcomes are expressed in a continuum, and do not follow the dichotomy exposed/non-exposed or diseased/ non-diseased; there is a linear relationship between certain exposures and their effects along the range of exposure levels; thus, many individuals exposed to low risk may generate more cases of a disease than few individuals exposed to high risk. In addition, there is a strong relationship between average behaviors and the occurrence of deviance, as a result of the balance between biological and social forces favoring diversity or uniformity. Thus, risk differences between defined populations involve differences in the population distributions as a whole, rather than in the proportion of individuals with high risk. Overall, these concepts favor emphasis on strategies aiming the general population, and not only the individuals considered to be at high risk of disease. PMID- 10738170 TI - [Literature and medicine: the shared territory]. AB - The text De Profundis: Valsa Lenta (De Profundis: Slow Waltz), by Portuguese author Jose Cardoso Pires, describing the aphasia he experienced after a stroke, was used as a starting point to study the different approaches writers and physician have regarding the disease. The differences in form and content of texts describing the disease in literature and medicine fit in the conflict between the two cultures, as described by C.P. Snow, and are typical of the increasing specialization. The addition of literary texts in training programs for doctors and health professionals may help overcome this gap, making it easier to understand the disease in its broader dimension and collaborating to improve the relationship between patients and professional. PMID- 10738171 TI - Physician-patient communication in the prevention of female reproductive tract infections: some limitations. AB - The objective of this study was to describe the information that women with tubal infertility recalled having received from physicians when they consulted for a reproductive tract infection (RTI) and the information physicians reported having provided to women consulting for RTIs. Interviews were conducted with 16 women presenting primary tubal infertility selected on the basis of purposeful sampling criteria. Also, 15 physicians working in the public health network in the city of Campinas, Brazil, were selected at random and interviewed. Analysis of the women's statements concerning previous RTI-related medical consultations showed that they did not recall having received any information on either prevention or the impact of RTI on their reproductive future. Analysis of interviews with physicians showed that the information they provided to women consulting for RTIs was incomplete and unclear. The information women recalled having received and that which physicians remembered having provided at the time of treating a patient with RTI was similar. In conclusion, these women lacked adequate or complete information and that it was probably not possible for them to adopt measures to avoid repetition of RTI and minimize risk of tubal infertility. PMID- 10738173 TI - Community genetics: a new discipline and its application in Brazil. AB - Community genetics is a new discipline which aims to provide genetic services to the community as a whole. As a science, community genetics encompasses all research needed to develop and evaluate its application. There is no question that the development of community genetics is necessary in Brazil. The implementation of such programs in our country, especially for hemoglobinopathies, has been recommended by the World Health Organization and other international organizations. Apart from the need for and appeal of community genetics programs, some aspects require serious review. This article discusses various cultural, social, psychological, and economic factors that can make genetic screening an invasion of individual privacy. PMID- 10738172 TI - [Geographical factors in the epidemiology of intoxication with Karwinskia (tullidora) in Mexico]. AB - Consumption of fruits from the genus Karwinskia may cause a flaccid paralysis that can be confused with the Guillan-Barre syndrome or poliomyelitis. Paralysis resulting from consumption of such fruit has emerged as a public health problem in certain regions of Mexico. The purpose of this study was to investigate geographical factors associated with this intoxication in the 72 cases reported in Mexico from 1990 to 1994. Location of cases coincides with the distribution of the 11 reported species of Karwinskia in the country. The majority of reported cases were related to the consumption of K. humboldtiana, with a smaller number involving K. mollis, K. parvifolia, K. johnstonii, and K. rzedowskii. Most cases were located in regions with dry climates (79.2%), arid vegetation (41. 7%), and altitudes under 1,000 meters above sea level (54.1%). The study establishes three different risk areas: the Balsas river region in the southwestern central part of the country; the arid northern area; and the arid and dry central area. Cases were from rural communities with low levels of schooling and poor socioeconomic conditions. PMID- 10738174 TI - [Sandflies (Diptera, psychodidae) in a secondary forest area in the Paco do Lumiar city, Maranhao, Brazil: a leishmaniasis transmission area]. AB - This paper analyzes the wealth of species, relative abundance, seasonal fluctuation, and nocturnal activity of sandflies. The field survey was conducted in a "capoeira" (secondary forest) area in the county of Paco do Lumiar, Maranhao, where cutaneous and transmission of visceral leishmaniasis frequently occurs. Sandflies were captured by CDC-type light traps from 6:00 PM to 6:00 AM, once a month, from March 1997 to February 1998. A total of 489 specimens were collected (251 males and 238 females), distributed among 10 species: Lutzomyia antunesi (45.19%), Lutzomyia whitmani (29.4%), Lutzomyia longipalpis (7.56%), Lutzomyia sordelli (6.34%), Lutzomyia flaviscutellata (4.5%), Brumptomyia avellari (4.09%), Lutzomyia evandroi (1.85%), Lutzomyia umbratilis (0.61%), Lutzomyia corossoniensis (0.41%), and Lutzomyia trispinosa (0.41%). The sandflies were present year round, with higher abundance during the rainy season. They were present in all intervals studied, with the highest frequency between 12:00 PM and 1:00 AM (31%). PMID- 10738175 TI - [Epidemiological characteristics of snake bites in the state of Roraima, Brazil, 1992-1998]. AB - The author presents a study on snakebites in 8 counties in the State of Roraima, Brazil, from 1992 to 1998. A total of 309 recorded snakebites were described and analyzed by place, fatality, sex, time of day, date, month, and age distribution. Most of the reported cases involved genus Bothrops (81.8%). The most frequently involved counties were Alto Alegre (49.0%), Sao Luiz do Anaua (14.9%), and Normandia (10.3%). Cases occurred most frequently in the months of March and July, and especially during the daytime (74.8%). PMID- 10738176 TI - Short-term culturing influences the number of bcr/abl-fused cells detected by fluorescence in situ hybridisation in bone marrow aspirates of CML patients. AB - Fluorescence in situ hybridisation (FISH) has been proven as a helpful tool in diagnosis and monitoring of bcr/abl fusion in chronic myelogeneous leukaemia (CML). Since long-term cultures are known to decrease the number of bcr/abl-fused cells, it is questionable whether similar effects are detectable in short-term cultures, a technique often preceding FISH analysis. Therefore, we evaluated bone marrow aspirates of 10 CML patients at biopsy and after culturing for between 24 and 144 h by FISH. The percentage of bcr/abl-fused cells in FISH varied between 15 and 70% at biopsy. In samples with 15 and 30% of aberrant cells at biopsy, an increase of about 20% per day was seen within the first 48 h. In longer lasting cultures, the percentage of leukaemic cells then asymptotically approached a value of 60-70%. In patients with 38 and 50% of bcr/abl-fused cells at biopsy, an increase of about 20% could be detected in the first 24 h. Then 65-70% of the cells already bore the bcr/abl fusion, and the percentage of leukaemic cells was almost constant for longer lasting cultures up to 144 h. In patients with a percentage of about 70% before culturing, no increase in positive cells was detected. These results emphasize the impact of short-term culturing on the number of bcr/abl-fused cells. In particular, the importance for monitoring CML patients is obvious. Therefore the effect described should be taken into account in order to avoid misinterpretation and incorrect therapy decisions in CML patients. PMID- 10738177 TI - Reduced expression of cyclin-dependent kinase inhibitor p27(Kip1) in oral malignant tumors. AB - p27(Kip1), a cyclin-dependent kinase (CDK) inhibitor, blocks progression from the G(1) to S phase by binding cyclin E-CDK2 and inhibiting their activities. We studied the expression of p27 in oral tumors by immunohistochemistry to determine whether lack of p27 plays a role in the development and progression of oral cancer. Reduced expression of p27 was detected in 86% of the squamous cell carcinomas and 95% of the mucoepidermoid carcinomas, respectively, while p27 expression was well preserved in the pleomorphic adenomas. The expression of p27 showed an inverse correlation with the expression of cyclin E in the squamous cell carcinomas and mucoepidermoid carcinomas. However, there was no relationship between clinicopathological parameters and p27 expression. These results suggest that the reduction of p27 protein may confer the development of oral squamous cell carcinoma and mucoepidermoid carcinoma partly through the increased expression of cyclin E. PMID- 10738178 TI - Expression of cell-cycle-regulating transcription factor E2F-1 in colorectal carcinomas. AB - The expression of E2F-1 in human colorectal carcinomas was examined immunohistochemically, and the correlation of E2F-1 expression with clinicopathological findings and with the expression of p27(Kip1) was analyzed to elucidate the role of E2F-1 in the development and progression of colorectal carcinomas. In nonneoplastic mucosa, a small number of epithelial cells in the proliferative zone were weakly positive for E2F-1. Weak expression of E2F-1 was detected in many adenoma cells. Most of the colorectal carcinomas expressed E2F-1 at various levels, and strong expression of E2F-1 was detected in 56% (49/88) of the cases. There was no correlation between the expression of E2F-1 and any clinicopathological parameters such as tumor stage, depth of tumor invasion and lymph node metastasis. Reduced expression of p27(Kip1) was confirmed to be significantly correlated with deep tumor invasion and presence of metastasis. No correlation was evident between overexpression of E2F-1 and reduced p27(Kip1) expression. PMID- 10738179 TI - Role of tumor-derived granulocyte-macrophage colony-stimulating factor in mice bearing a highly invasive and metastatic mammary carcinoma. AB - We have examined the role of granulocyte-macrophage colony-stimulating factor (GM CSF) in tumor-bearing BALB/c mice using the syngeneic F3II mammary carcinoma. In the present model, progression of subcutaneous tumors induced massive myelopoiesis in bone marrow and spleen due to GM-CSF secretion by tumor cells. In vitro, the addition of recombinant mouse GM-CSF (5- 25 ng/ml) caused a significant increase in F3II cell growth, either in the presence or absence of serum. Zymographic analysis of conditioned media from F3II monolayers showed that GM-CSF exerted a dose-dependent enhancement in the metalloproteinases MMP-9 (105 kD) and MMP-2 (70 kD), key enzymes in mammary tumor cell invasion. Our data suggest that ectopic GM-CSF production stimulates myelopoiesis and may also play an important role in tumor progression and metastasis formation. PMID- 10738180 TI - Treatment of established tumor is associated with ICAM-1 upregulation and reversed by CD8 depletion in a tumor necrosis factor-alpha gene transfected mouse mammary tumor. AB - INTRODUCTION: We have performed TNF-alpha gene transfection in a mouse mammary cancer line and found significant antitumor effects. We hypothesize that the antitumor effects observed in this model are mediated by ICAM-1 and by the recruitment of CD4+ and CD8+ T cells. In vivo (Balb/c mice) tumor growth inhibition, treatment of established tumor and the effects of ICAM-1 and CD4+ and CD8+ T cells were evaluated. METHODS AND RESULTS: Gene transfection with highly efficient vectors resulted in secretion of large amounts of TNF-alpha (ELISA). In vivo antitumor effects were tested. The number of cells required to generate palpable tumor 7-10 days after subcutaneous injection was determined (1 x 10(6)). The same number of transfected cells were injected subcutaneously and compared to nontransfected controls. Tumors were measured blindly and size was analyzed on day 30 by the Wilcoxon rank sum test. Mean tumor size after injection of transfected cells is compared to that of controls. Control tumors reached the maximum allowable size by day 30 (4 cm(2)). On day 30 EMT6-TNF-alpha tumors were 0.48 cm(2) (p < 0.05). The effect of repeat injection (challenge was also tested. Animals were injected with transfected cells or wild-type control on day -6 and challenged with the same number of wild-type tumor cells on day 0. Significant immune protection against subsequent challenge was seen after first time injection with EMT6-TNF-alpha but not after first time EMT6 wild-type injection (1.62 vs. 4 cm(2)). Treatment of 6-day-old tumor was also evaluated. On day 30, mean tumor size in animals treated with EMT6-TNF-alpha was 0.9 cm(2) compared to 4 cm(2) for controls. In all experiments, CD8+ T cell depletion and CD4+ T cell depletion caused a reversal of TNF-alpha-induced inhibitory effects. In addition, in vivo antibody blocking of ICAM-1 in tumor growth experiments reversed antitumor effects (control 4 cm(2), TNF-alpha 0.2 cm(2) and ICAM-1 blocking 3.14 cm(2)). Using flow cytometry, MHC class I and II and ICAM-1 adhesion molecule expression of transfected tumor was tested. ICAM-I expression was significantly upregulated. MHC class II antigen expression was also increased. TNF-alpha transfected human breast cancer was also evaluated. Three cell lines and fresh tumor were transfected to express TNF-alpha. In vitro analysis revealed ICAM-1 upregulation following transfection. Histologic analysis and immunohistochemical staining revealed TNF-alpha and ICAM-1 in transfected tumors and not in wild-type tumors. CONCLUSION: Highly significant in vivo tumor growth inhibition and immune protection after injection with TNF-alpha-transfected tumors appears to be mediated predominantly by CD8+ T cells and ICAM-1 upregulation. These findings suggest that TNF-alpha increases recruitment and adhesion of effector T cells. PMID- 10738181 TI - Bronchial cartilage atrophy in chronic bronchitis: observations on chondrolytic processes. AB - The status of bronchial cartilage degeneration in chronic bronchitis is unclear, and little is known about the chondrolytic mechanisms involved. The potential contributions of various inflammatory cells, chondrocytes and cartilage-degrading enzymes to cartilage atrophy have been examined. Bronchial cartilage specimens were obtained at autopsy from lobar secondary bronchi from chronic bronchitics and age-matched controls; each was examined by light microscopy and immunohistology for the distributions of mast cells, macrophages, eosinophils, collagenase 1, collagenase 3, and degradation products of cartilage collagen. Most bronchitic specimens showed hypertrophic chondrocytes, some of which were immunostained for collagenase 3, and occasionally for collagenase 1. Evidence for collagen degradation products was demonstrated around the lacunae of a proportion of chondrocytes, and both collagenases were also observed in the soft inflammatory tissues in close association with the cartilage surface, together with variable distributions of mast cells and macrophages. Such observations were generally absent or very much reduced in the control, non-bronchitic specimens. Degenerative changes, atrophy and loss of bronchial cartilage were common features of most chronic bronchitic specimens, this usually being related to intrinsic changes in the chondrocyte phenotype, including proliferative and matrix-degrading properties. Mast cells and macrophages were often observed in close association with the bronchial cartilage, suggesting that inflammatory cells may also contribute to the mechanisms of bronchial cartilage degradation and loss. These observations of bronchial cartilage degeneration were generally lacking in age-matched non-bronchitic control specimens. PMID- 10738183 TI - Magnesium fortification of drinking water suppresses atherogenesis in male LDL receptor-deficient mice. AB - Magnesium, an important cofactor of more than 300 enzymes, has previously been found to modulate blood lipid levels, atherogenesis and atherosclerosis in rabbits, when added to their diet. The aim of this study was to examine whether magnesium fortification of drinking water, without a change in diet content, can affect atherogenesis. The study included six groups of LDL-receptor-deficient mice. The mice received either distilled water or water containing 50 g of magnesium sulfate per liter. In the first (12 weeks) and second (6 weeks) stages of the experiment, the mice received low- and high-cholesterol diets, respectively. At the end of each stage, blood was drawn for the determination of plasma magnesium, calcium and lipid levels. In addition, the extent of atherosclerosis was determined at the aortic sinus. In both males and females, magnesium fortification was associated with higher levels of plasma magnesium (50 and 37% increase, respectively), without any differences in plasma calcium content. The extent of atherosclerosis at the aortic sinus in the male mice that received high levels of magnesium was a third of that of the male mice that received distilled water. However, these differences were not found in the female groups. Surprisingly, the female mice that received water fortified with magnesium had higher levels of cholesterol after stage 2, whereas no differences regarding plasma lipid levels were found among the male mice. These results confirm that magnesium fortification of drinking water is capable of inhibiting atherogenesis in male LDL-receptor-deficient mice. The mechanisms of action are yet to be discovered, and are probably not related to diminished lipid excretion, but possibly to the prevention of calcium influx into vascular smooth muscle cells, elevated antioxidative capacity, or other yet undetermined mechanisms. PMID- 10738182 TI - The p16/Cdkn2a/Ink4a gene is frequently deleted in nitrosourea-induced rat glial tumors. AB - The present study investigates nitrosourea-induced rat (Rattus norvegicus) glioma cell lines for the functional status of the p16/Cdkn2a/Ink4a gene, which encodes the p16 cdk4 inhibitor and the alternative reading frame protein, p19ARF. We detected homozygous deletions of the p16/Cdkn2a/Ink4a gene locus in 4 of 5 glioma cell lines (C6, F98, RG2, and RGL.3), but not in the 9L gliosarcoma cell line or in a rat primary fibroblast cell line. RT-PCR demonstrated expression of the p16 and p19ARF mRNAs only in 9L cells and in rat fibroblasts. Comparative genomic in situ hybridization showed that the copy number of rat chromosome RNO5 was not altered in any of the glioma cell lines investigated, indicating that the deletions result from a discrete loss in the region of the p16/Cdkn2a/Ink4a locus. This is the first report of p16/Cdkn2a/Ink4a deletions present in nitrosourea-induced rat glioma cell lines. Since this genetic alteration is also commonly observed in human malignant glial tumors, our results validate the use of chemically induced rat glioma cell lines as an experimental model in the development of gene therapy strategies. PMID- 10738184 TI - Mitochondrial tRNA(Cys) mutation A5823G in a patient with motor neuron disease and temporal lobe epilepsy. AB - We discovered a new homoplasmic mutation in the mitochondrial cysteine tRNA of a 60-year-old Caucasian male suffering from asymmetrical pure lower motor neuron disease (MND) and temporal lobe epilepsy (TLE). Furthermore, titrations with Amytal, an inhibitor of NADH:CoQ oxidoreductase, revealed mild mitochondrial dysfunction in skeletal muscle tissue, which was described in patients with MND in an earlier report. The mutation was undetectable in 155 Caucasian controls of both sexes, in 40 MND patients and in 13 individuals suffering from TLE. It was, however, detected in a heteroplasmic state in the patient's mother, who did not suffer from a neurological disorder. Since this rare mutation affected a nonconserved base position and was not observed in MND or TLE materials, its relation to disease remains unclear. PMID- 10738185 TI - Mature dendritic cells induce T-helper type-1-dominant immune responses in patients with metastatic renal cell carcinoma. AB - We performed a pilot study on a dendritic cell (DC)-based vaccine in 4 patients with advanced renal cell carcinoma. The vaccine consisted of cultured blood DCs loaded with autologous tumor cell lysate plus keyhole limpet hemocyanin (KLH) and matured with a combination of tumor necrosis factor alpha and prostaglandin E(2). We describe the immune response against KLH induced by DC-based immunization in a patient undergoing an objective partial response and compare it with the responses observed in patients with either stable or progressive disease. The patient with the clinical response developed strong delayed-type hypersensitivity (DTH) against KLH after a single vaccination with antigen-loaded DCs, whereas the other patients failed to develop DTH reactivity even after repeated vaccinations. Antigenic stimulation of mononuclear cells (MNCs) induced proliferation and IFN gamma but not IL-4 production as well as expression of the chemokine receptor CXCR3 consistent with a T-helper (Th) type-1 bias. Exogenous IL-12 enhanced and exogenous IL-4 diminished IFN-gamma production. In the 2 patients with stable disease two or more vaccinations were required to induce maximal MNC responses. In the patient with progressive disease MNC responses were hardly detectable. Anti-KLH antibodies appeared with different kinetics but could be detected in the serum of all patients. Isotype analysis revealed the presence of IgM, IgG(1), IgG(2) and IgG(3) as well as IgA and complete absence of IgE. The patient with progressive disease also developed IgG(4) antibodies indicative of a deviation towards Th2. Cultured blood DCs can be a potent vaccine for the antigen-specific immunization of patients with advanced kidney cancer. KLH serves as a tracer molecule which allows determination of the magnitude, kinetics and Th bias of the cellular and humoral immune response induced by DC-based immunization. The data also suggest that Th type-1-dominant immune responses involving DTH reaction are required for the induction of tumor regression. PMID- 10738186 TI - Multifocality in renal cell carcinoma: A bilateral event? AB - OBJECTIVES: The major disadvantage of nephron-sparing surgery for renal cell carcinoma is the risk of local recurrence. This is most likely a manifestation of undetected small additional tumors in the renal remnant. To define more clearly the incidence and nature of unilateral and bilateral multifocal tumors, an autopsy study was undertaken. MATERIALS AND METHODS: In a series of 14,793 autopsies from 1985 to 1995, 260 renal cell carcinomas were found. In all cases of renal cell carcinoma a search for small renal lesions was performed in the apparently normal-appearing portion of the kidneys. Every kidney was serially and systematically cut (5 mm) to probe for intraparenchymal lesions. RESULTS: Of the 260 renal cell carcinomas 36 cases (13.85%) had multifocal malignant and/or benign nodules. The number of the additional nodules ranged from 2 to 18. 12% of the malignant multifocal carcinomas were limited to the ipsilateral kidney and 88% were bilateral. The average size of the multifocal renal lesions was 8.7 x 9.0 x 9.5 (range 3-23) mm. Renal cell carcinomas with low stage and good grading have a higher incidence of multifocal nodules. No significant difference was found with regard to metastasized and nonmetastasized renal cell carcinomas. In 38.1% of all chromophilic renal cell carcinomas additional nodules were found. CONCLUSIONS: Multifocality in renal cell carcinomas cannot be predicted reliably, although the papillary histological pattern, good grading and low staging seems to be associated with a higher incidence of multifocality. Nearly 90% of the multifocal nodules were bilateral. PMID- 10738187 TI - Cutaneous metastases in renal cell carcinoma. AB - Cutaneous metastasis from renal cell carcinoma is believed to be rare. We present our experience with 10 (3.3%) cases seen in the last 12 years among 306 cases of renal adenocarcinoma treated at our center. There were 9 males and 1 female. Age ranged from 30 to 65 years (average 45 years). 5 patients had skin metastases at the time of presentation (stage IV). In one of them the skin nodule, rather than urologic symptoms, was the presenting complaint. 5 patients presented with skin metastasis during follow-up after nephrectomy. The average time to skin metastasis was 51 months for patients in stage I and 13 months in stage IIIb. The scalp was the most common site of metastasis followed by chest and abdomen. 90% of patients had secondaries in at least one other site, most commonly in lungs (4 cases) and bones (5 cases). 4 patients were treated with interferon-alpha 6 MIU, subcutaneously, three times a week for varying periods from 3 to 4 months but there was no response. In conclusion, cutaneous secondaries from RCC, though uncommon, are not very rare. A few patients may present with a skin mass before detection of the renal tumor. Patients with low-stage disease at presentation may also develop cutaneous secondaries, therefore a prolonged follow-up is required. The commonest site for cutaneous metastasis from RCC is the scalp and face. Most patients had at least one other site of systemic metastasis, hence they were not candidates for curative therapy. Interferon therapy was not helpful. Mean survival after detection of cutaneous metastasis was 7 months. PMID- 10738189 TI - New method of bladder biopsy using combined aspiration and snare. AB - A method of multiple random biopsy combined with bladder wall aspiration and snare in bladder cancer is presented. Our method was used on 23 patients with clinically superficial bladder cancer and was found to have advantages in obtaining an adequate amount of tissue with minimal thermal degeneration. PMID- 10738188 TI - Profile of placental alkaline phosphatase expression in human malignancies: effect of tumour cell activation on alkaline phosphatase expression. AB - Cellular alkaline phosphatases (ALP) are increasingly recognised as important markers for monitoring tumour cell behaviour in human malignancies. Colorimetric, flow-cytometric, and immunocytochemical assays were employed to assess the influence of activation on expression of cellular ALP in human tumour cell lines. The results showed the following: (1) Testis tumour biopsies (16/16) unlike bladder (0/14) and head and neck (0/16) tumours showed positive staining for ALP, particularly the placental type, i.e. PLAP, although this was not always present on all the cells of non-seminoma biopsies. (2) The intensity of ALP expression differed widely in tumour cell lines. Based on biochemical analysis, the profile of ALP fell into two categories: (a) low expressing (MW 70 kD, placental type ALP) like Hep2 and KB lines, and (b) those expressing both low and high molecular (MW 95 kD) bands like testis lines Tera II and Ep2102. In all cases treatment of tumour cell lysates with heat prior to biochemical analysis showed the disappearance of the higher and sharpening of the lower molecular weight ALP band. (3) Exposure of tumour cells to epidermal growth factor (EGF) expressing EGF receptor led to a decreased ALP expression by as much as 54% as assessed by biochemical or flow-cytometric techniques. These data demonstrated that testis tumour tissues and cell lines expressed ALP which were different from others. The data also showed that exposure of tumour cell lines expressing EGFr to EGF resulted in suppression of ALP expression. These observations are consistent with the notion that EGFr and PLAP expression may be taken as a marker of proliferation and differentiation in human malignancies, respectively. PMID- 10738190 TI - Effect of injury to the dorsal funiculus of the thoracic spinal cord on micturition in decerebrate and freely-moving cats. AB - Bladder sensation is transmitted both via the spinothalamic tract in the lateral funiculus and the dorsal system in the dorsal funiculus. We transected the dorsal funiculus in 10 female cats to clarify the functional roles of these two ascending pathways. The dorsal funiculus was transected at T10 in 5 decerebrate and 5 freely-moving cats, and micturition parameters were compared before and after transection. Transection of the dorsal funiculus did not affect any of the parameters of reflex micturition in the 5 decerebrate cats. Within 1 week after transection, 4 of the 5 freely-moving cats used the normal micturition posture, but the remaining one performed micturition in a prone position as if she had lost micturition sensation. All 5 cats urinated with a normal micturition posture by 2 weeks after transection. The mean single voided volume was decreased transiently up to 1 week, but returned to normal by 2 weeks after transection. None of the 5 cats had any residual urine before and after transection. Both the ascending and descending limbs of the micturition reflex pass through the lateral funiculus. Bladder sensation is transmitted both via the spinothalamic tract coursing in the lateral funiculus and the dorsal system in the dorsal funiculus. The dorsal system may play a major role in the transmission of bladder sensation to the cerebral cortex, but may not be essential. PMID- 10738191 TI - Penile prosthesis implantation and couple's satisfaction. AB - OBJECTIVE: Implant surgery is the most acceptable curative choice for patients with erectile dysfunction and their partners, since the results are excellent with regard to the couple's pleasure and the materials used are extremely manageable. MATERIALS AND METHODS: We performed 46 prosthesis implantations in patients with erectile dysfunction: in 22 patients we implanted soft prostheses; in 20 malleable prostheses; in 1 a mono-component prosthesis, and in 3 patients a tri-component model. All patients and partners were evaluated using a questionnaire. RESULTS: We obtained a degree of satisfaction equal to 82% against 18% dissatisfaction due to a sensation of 'unnaturalness' report by the partner, with low perioperative and postoperative complications. CONCLUSION: In our opinion the degree of satisfaction of the patient is currently the only legitimate indicator to establish the effectiveness of therapy. PMID- 10738192 TI - A study of struvite precipitation and urease activity in bacteria isolated from patients with urinary infections and their possible involvement in the formation of renal calculi. AB - Struvite precipitation and urease activity were studied in 72 bacterial strains isolated from patients with an urinary infection. The results revealed struvite precipitation by urease-positive, urease-negative and weakly urealitic bacteria. However, in some strains of each of these groups, no precipitation was observed. Variations in pH within the cultures were also investigated as were the mineralogy and morphology of the crystals precipitated using X-ray diffraction and scanning electron microscopy, respectively. The role of urease activity and alkalinization of the medium in struvite precipitation is discussed, as is the possible involvement of bacteria in nucleation processes. We concluded that urease-negative bacteria or those with weak urease activity may also be involved in the formation of struvite renal calculi although urease-positive bacteria seem to make a greater contribution. PMID- 10738194 TI - A large primary malignant melanoma of the female urethra. AB - We present a case of large, primary malignant melanoma of the female urethra with poor prognosis in this case report. Malignant melanoma is one of the rare tumors of the female urethra and accounts for 0.2% of all malignant melanoma cases. Large (5.6 cm in diameter), primary malignant melanoma of the female urethra is exceedingly rare. PMID- 10738193 TI - Carcinoembryonic antigen and carbohydrate antigen 19-9-producing adenocarcinoma of the prostate: report of an autopsy case. AB - Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) are well known as specific tumor markers of prostate cancer, but carcinoembryonic antigen (CEA)- and carbohydrate antigen 19-9 (CA19-9)-producing adenocarcinoma originating in the prostate is rare. We report here a case of prostatic adenocarcinoma positive for these 4 tumor markers in a 50-year-old man who had initially complained about chest pain due to metastatic bone tumor. In spite of the extensive treatment involving hormone and radiation therapy, the patient died of rapid tumor extension only 4 months after initial diagnosis. Autopsy revealed multiple metastases to the bone, liver, lungs and lymph nodes. Histologically, two types of adenocarcinoma were involved in both primary prostate and metastatic sites: one was a poorly differentiated adenocarcinoma positive for PSA and PAP but not CEA or CA19-9, and the other one was a less differentiated adenocarcinoma partially positive for CEA and CA19-9 but not for PSA or PAP. Based on this case and previous cases by review of the literature, CEA- and CA19-9-producing adenocarcinoma of the prostate was suggested to rapidly progress with multiple metastases and to show poor prognosis with strong resistance to any treatment. PMID- 10738195 TI - Benign ureteral tumors. Four case reports and a review of the literature. AB - The diagnosis of benign ureteral polyps should be considered especially in patients younger than 40 years with filling defects of the proximal ureter on the intravenous pyelogram (IVP). The excellent results of conservative organ-saving surgery (open resection, ureteroscopic resection, laser coagulation) require precise diagnostic management preoperatively in these patients. PMID- 10738196 TI - Multilocular cystic adenomyoma of the pelvic cavity: A rare clinical and histological entity in a young man. AB - We report a large complex cystic pelvic mass encountered in a 16-year-old man complaining of perineal discomfort. A large and elastic hard mass was palpated on the right anterior wall of the rectum with no discernible prostate on digital rectal examination. Prostatic tumor markers were elevated. Excretory urography and urethrocystography showed a filling defect with a smooth edge on the right side of the bladder. Magnetic resonance imaging demonstrated a mass with a mosaic pattern between the right side of the bladder and the rectum. We performed ultrasound-guided transrectal needle core biopsy of the mass. The pathologist suspected hyperplastic glandular epithelium of prostatic origin with focal inflammatory cell infiltration, but there was no sign of malignancy. We thought that the tumor arose from the prostate. Surgery was successfully performed. The tumor was located on the right side of the pelvic cavity and adhered to the right lobe of the seminal vesicle. En bloc excision was performed. This lesion histologically proved to be a multilocular prostatic tissue with a seminal vesicle component, without communication to the surrounding tissue. Such a lesion has not previously been reported in the literature. PMID- 10738197 TI - Accessory scrotum. AB - A 2-year-old male with a perineal swelling, covered with normal skin without any rugosities, was diagnosed as having an accessory scrotum on histopathology. There was no other congenital anomaly. The case is reported due to its rarity and unique clinical picture. PMID- 10738198 TI - The dissociation rate of the EcoRI-DNA-specific complex is linked to water activity. PMID- 10738199 TI - Characterizations of critical processes in liquid-liquid phase separation of the elastomeric protein-water system: microscopic observations and light scattering measurements. AB - Biological self-assembly process of tropoelastin in an extracellular space, viewed as a key step of the elastogenesis, can be mimicked by the temperature dependent coacervation of the elastin-related polypeptide-water system. Early and late stages of the phase separation behavior of the bovine neck ligamental alpha elastin-water system were examined respectively by the laser light scattering photometry and phase contrast microscopy. Changes in the hydrodynamic size of molecular assemblies and visible microcoacervate droplet size were traced as a function of the concentration of alpha-elastin and temperature. Near the critical point, alpha-elastin concentration of 0.11 mg/mL and temperature of 21.5 degrees C, the phase separation was initiated after fast increase of the hydrodynamic size of primary aggregates as scattering particles and followed by the appearance of larger microcoacervate droplets with a broad size distribution. Whereas in the off-critical region, slow decrease of the hydrodynamic size of primary particles induced phase separation with smaller droplets of a narrow size distribution. Observation of the phase separation processes in the alpha-elastin-water system with metal chlorides and hydrophobic synthetic model polypeptide-water system indicated that the fast and slow molecular assembly processes were based on the fundamental hydrophobic interactions and involvements of electrostatic interactions between charged amino acid residues, respectively. PMID- 10738200 TI - Simulations of oligopeptide vibrational CD: effects of isotopic labeling. AB - Simulated ir absorption and vibrational CD (VCD) spectra of four alanine-based octapeptides, each having its main chain constrained to a different secondary structure conformation, were analyzed and compared with experimental results for several different peptides. The octapeptide simulations were based on transfer of property tensors from a series of ab initio calculations for a short L-alanine based segment containing 3 peptide bonds with relative straight phi, psi angles fixed to those appropriate for alpha-helix, 3(10)-helix, ProII-like helix, and beta-sheet-like strand. The tripeptide force field (FF) and atomic polar tensors were obtained with density functional theory techniques at the BPW91/6-31G** level and the atomic axial tensor at the mixed BPW91/6-31G**/HF/6-31G level. Allowing for frequency correction due to the FF limitations, the octapeptide results obtained are qualitatively consistent with experimental observations for ir and VCD spectra of polypeptides and oligopeptides in established conformations. In all cases, the correct VCD sign patterns for the amide I and II bands were predicted, but the intensities did have some variation from the experimental patterns. Predicted VCD changes upon deuteration of either the peptide or side-chains as well as for (13)C isotopic labeling of the amide C=O at specific sites in the peptide chain were computed for analysis of experimental observations. A combination of theoretical modeling with experimental data for labeled compounds leads both to enhanced resolution of component transitions and added conformational applicability of the VCD spectra. PMID- 10738202 TI - The retrogradation of waxy maize starch extrudates: effects of storage temperature and water content. AB - The effects of water content and storage temperature on the kinetics of the retrogradation of nonexpanded waxy maize starch extrudates were studied using (1)H pulsed NMR and wide-angle X-ray diffraction. The increase in crystallinity observed by XRD was accompanied by a decrease in the relaxation times of the solid-like component of the NMR free induction and the spin-echo decays, and an increase in the contribution of the solid-like component to the total signal. The dependence of the rate of starch retrogradation on the storage temperature showed the typical "bell-shaped" behavior, which was successfully modeled using the Lauritzen-Hoffman theory of crystallization of chain-folded polymers. This theory was extended to model the effect of water content on the rate of isothermal crystallization by exploiting the ten-Brinke and Karasz, and the Flory equations to describe the dependence of the glass-transition and the melting temperatures on water content. PMID- 10738201 TI - An NMR study of conformations of substituted dipeptides in dodecylphosphocholine micelles: implications for drug transport. AB - Efficient transport of intact drug (solute) across the intestinal epithelium is typically a requirement for good oral activity. In general, the membrane permeability of a solute is a complex function of its size, lipophilicity, hydrogen bond potential, charge, and conformation. In conjunction with theoretical/computational and in vitro drug transport studies, seven dipeptide (R(1)-D-Xaa-D-Phe-NHMe) homologues were each dissolved in a micellar d(38) dodecylphosphocholine solvent system. In this homologous dipeptide series, factors such as size, lipophilicity, hydrogen-bond potential, and charge were either tightly controlled or well-characterized by other methods in order to investigate by nmr how conformational factors relate to transport. Nuclear Overhauser effect spectroscopy experiments and amide-NH-H(2)O chemical exchange rates showed that the five more lipophilic dipeptides were predominately associated with micelle, whereas the two less lipophilic analogues were not. Rotating frame nuclear Overhauser effect spectroscopy derived interproton distance restraints for each analogue, along with (3)J(HH)-derived dihedral restraints, were used in molecular dynamics/simulated annealing computations. Our results suggest that-other factors being equal-flexible dipeptides having a propensity to fold together nonpolar N- and C-terminal moieties allow greater segregation of polar and nonpolar domains and may possess enhanced transport characteristics. Dipeptides that were less flexible or that retained a less amphiphilic conformation did not have comparably enhanced transport characteristics. We suggest that these conformational/transport correlations may hold true for small, highly functionalized solutes (drugs) in general. PMID- 10738203 TI - Ab initio prediction of the solution structures and populations of a cyclic pentapeptide in DMSO based on an implicit solvation model. AB - Using a recently developed statistical mechanics methodology, the solution structures and populations of the cyclic pentapeptide cyclo(D-Pro(1)-Ala(2) Ala(3)-Ala(4)-Ala(5)) in DMSO are obtained ab initio, i.e., without using experimental restraints. An important ingredient of this methodology is a novel optimization of implicit solvation parameters, which in our previous publication [Baysal, C.; Meirovitch, H. J Am Chem Soc 1998, 120, 800-812] has been applied to a cyclic hexapeptide in DMSO. The molecule has been described by the simplified energy function E(tot) = E(GRO) + summation operator(k) sigma(k)A(k), where E(GRO) is the GROMOS force-field energy, sigma(k) and A(k) are the atomic solvation parameter (ASP) and the solvent accessible surface area of atom k. This methodology, which relies on an extensive conformational search, Monte Carlo simulations, and free energy calculations, is applied here with E(tot) based on the ASPs derived in our previous work, and for comparison also with E(GRO) alone. For both models, entropy effects are found to be significant. For E(tot), the theoretical values of proton-proton distances and (3)J coupling constants agree very well with the NMR results [Mierke, D. F.; Kurz, M.; Kessler, H. J Am Chem Soc 1994, 116, 1042-1049], while the results for E(GRO) are significantly worse. This suggests that our ASPs might be transferrable to other cyclic peptides in DMSO as well, making our methodology a reliable tool for an ab initio structure prediction; obviously, if necessary, parts of this methodology can also be incorporated in a best-fit analysis where experimental restraints are used. PMID- 10738204 TI - Structure of porcine pancreatic elastase complexed with FR901277, a novel macrocyclic inhibitor of elastases, at 1.6 A resolution. AB - Human leukocyte elastase (HLE) is a serine protease that contributes to tissue destruction in various disease states-for example, in emphysema. FR901277 is a natural product isolated from the culture filtrate of Streptomyces resistomicificus and is a potent inhibitor of both HLE and porcine pancreatic elastase (PPE). FR901277 consists of four normal amino acids and three unusual amino acids, and is a unique bicyclic peptide compound. The crystal structure of PPE complexed with FR901277 has been determined at 1.6 A resolution. The Ogamma atom of Ser-195 in PPE did not form a covalent bond with FR901277, but formed a hydrogen bond with the Nvarepsilon atom of His-57. On the other hand, the portion from L-Orn(1) through dehydroxyThr(3) in FR901277 formed an antiparallel beta sheet structure with the backbone of the active site in PPE. The S4 through S2' binding subsites in PPE were all occupied by the hydrophobic side chains of the inhibitor molecule. Especially, the ethylidene moiety of FR901277 occupied the S1 specific pocket, indicating a CH/pi interaction. In addition, the isopropyl side chain of L-Val(7) was located at the enzyme surface between the S2 and S1' pockets with several van der Waals contacts. However, the amino acid (4) residue was not involved in a significant interaction with PPE. Comparison of inhibitor structures in different environments showed that FR901277 has a highly rigid bicyclic framework; however, it can slightly change its conformation according to the circumstances. The binding mode of FR901277 at the active site of PPE was directly applicable to that in HLE, after consideration of induced fit. The structure of the PPE-FR901277 complex provided much information regarding potential sites for modification of the physicochemical properties of FR901277. PMID- 10738205 TI - World Health Organization and International Society of Urological Pathology classification and two-number grading system of bladder tumors. PMID- 10738206 TI - World Health Organization and International Society of Urological Pathology classification and two-number grading system of bladder tumors: reply. PMID- 10738207 TI - Surgery versus radiation therapy for patients with aggressive fibromatosis or desmoid tumors: A comparative review of 22 articles. AB - BACKGROUND: Desmoid tumors (aggressive fibromatoses) are benign neoplasms with high rates of recurrence after surgery. Radiotherapy is sometimes reported to prevent recurrences, but not in all studies. In order to evaluate the effect of radiation, comparative analysis was performed. METHODS: The authors conducted a MEDLINE search and collected all articles in the English language on the treatment of "desmoid tumor" or "aggressive fibromatosis" from the years 1983 1998. They categorized treatment into three groups: surgery alone (S), surgery with radiotherapy (S + RT), or radiotherapy alone (RT). The S and S + RT groups were each subdivided according to whether margins were free (-), positive (+), or unknown. Each subgroup was divided into cases with primary, recurrent, or unknown tumor. RESULTS: The local control rates after treatment for cases in the S group with (-) margins, (+) margins, and overall were 72%, 41%, and 61%, respectively. For the S + RT group the local control results were 94%, 75%, and 75%, respectively, significantly different when compared with the results for the S group. For the RT group, the local control was 78%, significantly superior to that of the S group (61%). Cases with primary and recurrent tumors had significantly superior local control rates with S + RT or RT versus S. Radiotherapy complications noted were fibrosis, paresthesias, edema, and fracture. CONCLUSIONS: RT or S + RT results in significantly better local control than S. Even after dividing the groups into cases with free and positive margins and cases with primary and recurrent tumors, the best local control is achieved with RT or S + RT. PMID- 10738208 TI - Telomerase activity as an indicator of malignant potential in iodine-nonreactive lesions of the esophagus. AB - BACKGROUND: Iodine-nonreactive lesions of esophageal epithelium often are associated with dysplasia and carcinoma. The authors examined the usefulness of telomerase activity as an indicator for esophageal carcinogenesis in such lesions. METHODS: Telomerase activity was measured using the telomeric repeat amplification protocol assay in 18 samples of iodine-nonreactive lesions apart from the primary tumor in surgically resected specimens obtained from patients with esophageal squamous cell carcinoma (ESCC) and 55 endoscopic punch biopsies of iodine-nonreactive lesions obtained from 25 patients with ESCC and 30 patients who had undergone endoscopic examination for other reasons. RESULTS: Ten of 18 iodine-nonreactive samples (56%) obtained from surgically resected specimens showed telomerase activity. In all ten telomerase positive samples, carcinoma in situ (CIS) was observed in iodine-nonreactive mucosa by light microscopy. In eight telomerase negative samples, no tumor tissue was observed in iodine nonreactive lesions. In a parallel study, telomerase activity was detected in 28 of 55 endoscopic punch biopsy specimens (51%). CIS was observed in 25 of 28 iodine-nonreactive lesions with positive telomerase activity (89%), and tumor tissue was not observed in the other 3 samples (11%), which included 2 cases of severe dysplasia and 1 case of moderate dysplasia. No tumor tissue was observed in any of the 27 telomerase negative samples. CONCLUSIONS: Positive and negative telomerase activity was found to be correlated with the presence and absence, respectively, of immortalized tumor cells in iodine-nonreactive lesions. The measurement of telomerase activity in iodine-nonreactive lesions independently contributes to the selection of an appropriate therapeutic strategy. PMID- 10738209 TI - Assessment of gastric carcinoma risk associated with Helicobacter pylori may vary depending on the antigen used: CagA specific enzyme-linked immunoadsorbent assay (ELISA) versus commercially available H. pylori ELISAs. AB - BACKGROUND: Previous epidemiologic studies produced inconsistent results when examining the relation between Helicobacter pylori infection and the risk of gastric carcinoma by measuring various anti-H. pylori antibodies. This study investigated the increased risk of cancer by examining different antibodies, including the specific anti-CagA antibody and antibodies from two commercially available kits. METHODS: An ELISA for the detection of serum anti-CagA was established using a recombinant CagA protein that the authors previously reported. Serum anti-CagA titer was determined for 80 patients with gastric carcinoma and 80 gender- and age-matched controls. Two anti-H. pylori antibodies from the commercially available kits HEL-p (Amrad, Kew Vic, Australia) and HM-CAP (Enteric Product Inc., Westbury, NY) were also evaluated. RESULTS: Anti-CagA seropositivity differed significantly between gastric carcinoma patients and controls (92.5% vs. 55.0%; P = 0. 0001), showing an odds ratio of 10.4 (95% confidence interval [CI]: 4.23-29.74). The difference was less prominent for the seropositivity of HEL-p (77.5% vs. 58.8%; P = 0.0139; odds ratio: 2. 38; 95% CI: 1.20-4.82) and insignificant for that of HM-CAP (65.0% vs. 57.5%; P = 0.4325; odds ratio: 1.30; 95% CI: 0.68-2.49). CONCLUSIONS: The current study revealed that the antibody assay system used could be one important factor in the assessment of gastric carcinoma risk for patients with H. pylori. PMID- 10738211 TI - Prognostic influence of tumor-associated eosinophilic infiltrate in colorectal carcinoma. AB - BACKGROUND: In 1983, Pretlow et al. reported one classical study of the prognostic influence of eosinophil infiltration in human colon carcinoma. Since then, very few reports have analyzed this supposed prognostic influence in this type of tumor, although there have been several reports of other types of tumors with different results. Eosinophils seem to play a central role in the immunologic defense against tumors; their activity can be induced through immunotherapy with interleukin in cases that are unresponsive to conventional therapies. METHODS: To analyze the prognostic influence of eosinophils in colorectal carcinoma, the authors selected 126 patients with this type of tumor treated only with curative surgery and followed for a minimal period of 5 years. They divided these patients into 4 categories according to the number of eosinophils per high-power field (400x): 0 (Grade 1), 1-9 (Grade 2), 10-50 (Grade 3), and more than 50 (Grade 4). RESULTS: The results of this series confirm that high eosinophil counts are associated with a significantly better prognosis. In multivariate analysis, this factor was independent from staging, vascularization, p53 expression, and histologic grade. CONCLUSIONS: Eosinophil count served as a significant independent favorable prognosticator in colorectal carcinoma patients. PMID- 10738210 TI - Thomsen-Friedenreich antigen presents as a prognostic factor in colorectal carcinoma: A clinicopathologic study of 264 patients. AB - BACKGROUND: Up to now, the expression of the tumor-associated Thomsen Friedenreich (TF) antigen in colorectal carcinoma has not been thoroughly investigated with particular emphasis on its correlation with established clinicopathologic characteristics and classifications as well as its prognostic relevance. METHODS: Formalin fixed, paraffin embedded specimens from 264 patients with colorectal carcinoma were stained using an avidin-biotin complex-peroxidase assay. As primary monoclonal antibodies (MAbs), A78-G/A7, which binds to TFalpha and TFbeta antigen irrespective of its carrier, and BW835, which detects TFalpha on MUC1 repeat peptide, were applied. RESULTS: MAbs A78-G/A7 and BW835 labeled 64.8% and 58. 0%, respectively, of carcinomas. None of the binding patterns correlated with gender, tumor localization, or growth type. Only BW835 reactivity exhibited a significant correlation with increasing pTNM staging and histologic grading. Staining of the MAb A78-G/A7 was significantly stronger in carcinomas that contained a mucinous component. In univariate survival analysis, in addition to pTNM staging and histologic grading, reactivity with A78-G/A7 as well as BW835 were significantly correlated with lower survival probability. Multivariate analysis according to the Cox proportional hazards model revealed only pTNM staging, histologic grading, and A78-G/A7 staining to be independent prognostic factors. CONCLUSIONS: According to these results, TF disaccharide represents a cancer-associated antigen in colorectal carcinoma that exhibits qualities of a prognostic marker. As demonstrated by BW835 staining, it is obviously coexpressed with MUC1 peptide core in a great number of cases. These results suggest that TF, in addition to MUC1, might also serve as a useful target antigen in the treatment of patients with colorectal carcinoma. PMID- 10738212 TI - Limited but definite efficacy of prophylactic hepatic arterial infusion chemotherapy after curative resection of colorectal liver metastases: A randomized study. AB - BACKGROUND: Greater than 50% of patients who undergo curative resection of liver metastases from colorectal carcinoma develop recurrent disease in the residual liver. Although several studies have attempted to use hepatic arterial infusion (HAI) chemotherapy to prevent recurrence, to the authors' knowledge the efficacy of the treatment has not yet been determined. METHODS: Nineteen patients who underwent curative hepatectomy for metastatic colorectal carcinoma randomly were assigned into the HAI group (nine patients) or the control group (ten patients). Patients in the HAI group received continuous intraarterial infusion of 5 fluorouracil (5-FU) (500 mg/day) for 4 days followed by a 3-day rest. The treatment was continued for 6 weeks. RESULTS: The median follow-up period was 62.2 months. The recurrence was confirmed in three patients in the HAI group and in eight patients in the control group. Of these, recurrence in the remnant liver was observed in one patient and in six patients, respectively. The median disease free interval after hepatectomy was 62.6 months in the HAI group and 13.8 months in the control group. The 1-year, 2-year, and 3-year disease free survival rates were 77.8%, 77.8%, and 66.7%, respectively, in the HAI group and 50.0%, 30.0%, and 20.0%, respectively, in the control group. Significant prolongation of disease free survival was observed in the HAI group (P = 0.045). No patients in the HAI group reported any adverse effect of >/= Grade 2 (according to the National Cancer Institute Common Toxicity Criteria). Two patients in the HAI group and five patients in the control group were dead of disease at the time of last follow-up. The 1-year, 3-year, and 5-year cumulative survival rates for the HAI group were 88.9%, 77.8%, and 77.8%, respectively, whereas those of the control group were 100.0%, 50.0%, and 50.0%, respectively (P = 0.2686). CONCLUSIONS: This randomized study revealed that short term HAI of 5-FU after curative resection of colorectal hepatic metastases is effective in preventing the recurrence of disease without any serious complications. PMID- 10738213 TI - Unique clinical characteristics of patients with hepatocellular carcinoma who present with high plasma des-gamma-carboxy prothrombin and low serum alpha fetoprotein. AB - BACKGROUND: Although the importance of alpha-fetoprotein (AFP) and des-gamma carboxy prothrombin (DCP) in the clinical treatment of hepatocellular carcinoma (HCC) has been studied extensively, the authors examined the clinical picture of HCC with regard to the state of these two tumor markers. METHODS: The authors categorized 237 HCC cases into 4 groups according to levels of AFP and DCP: high levels of AFP with low DCP levels, high DCP levels with low AFP levels, high levels of both tumor markers, and low levels of both tumor markers. Comparisons of survival rates were made among these groups using the Kaplan-Meier product limit method, and for other comparisons of clinical parameters the Fisher PSLD test was used. Prognostic significance was tested with the Cox proportional hazards model. RESULTS: The cutoff values were set at 100 ng/mL for AFP and 0.0625 AU/mL for DCP. Forty-eight patients (20.7%) had high levels of AFP and low levels of DCP, 22 (9.3%) had high DCP levels and low levels of AFP, 12 (4.6%) had high levels of both AFP and DCP, and 155 (65.4%) had low levels of both DCP and AFP. Patients with high levels of DCP but low levels of AFP were predominantly male and had large lesions but few nodules. Patients with high levels of both tumor markers had the most discouraging outcome observed in this study (death within 3 years). CONCLUSIONS: Patients with high levels of DCP and low levels of AFP exhibited the unique clinical characteristic of large HCC nodules that were few in number. In addition, it was observed that measurement of both AFP and DCP can predict the survival of patients. PMID- 10738214 TI - P53 tumor suppressor gene mutations in hepatocellular carcinoma patients in India. AB - BACKGROUND: Specific mutations of the p53 tumor suppressor gene in hepatocellular carcinoma (HCC) have been reported from several parts of the world, but to the authors' knowledge to date the status of this gene has not been studied in HCC patients in India, where HCC is one of the major cancers and the frequency of chronic hepatitis B virus (HBV) as well as hepatitis C virus (HCV) infection and exposure to dietary aflatoxin B(1) is very high. The most frequent mutation of the p53 gene in HCC is an AGG(Arg) to AGT(Ser) missense mutation at codon 249 of exon 7. METHODS: Liver biopsy specimens from 21 HCC patients and 10 healthy controls were obtained through surgery or by needle biopsy technique. Phenol chloroform-extracted DNA specimens were employed for the detection of HBV infection and p53 gene mutations. Nucleotide mutations of exons 4-9 of the p53 gene were analyzed by polymerase chain reaction (PCR), single strand confirmation polymorphism, and direct sequencing. Third-generation sandwich enzyme-linked immunosorbent assay (ELISA) was used for the serologic detection of HBV and HCV infection. RESULTS: Analysis of exons 4-9 of the p53 gene revealed only 3 mutations (3 of 21 specimens, 14.28%; 95% confidence interval, -0.7-29.3), 2 mutations at codon 249 showing G-->T transversions, and 1 mutation (4.7%) at codon 250 with a C-->T transition. The base substitutions at the third base of codon 249 resulted in a missense mutation leading to a change in amino acid from arginine to serine whereas at codon 250 it caused a change from proline to serine. Dot blot hybridization and PCR for HBV DNA from HCCs revealed 58.8% (10 of 17 specimens) and 90. 47% (19 of 21 specimens), positivity, respectively. ELISA for hepatitis B virus surface antigen in serum showed a positivity of 71. 42% (15 of 21 specimens), but there was only 40% positivity (8 of 20 specimens) for hepatitis B virus envelope antigen whereas 6 of 17 patients (35.29%) showed the presence of antibodies against hepatitis B virus envelope protein. No patient was found to be positive for the HCV antibody. CONCLUSIONS: The very low frequency of p53 mutations and the extremely high frequency of HBV infection (> 90%) in HCC indicate that the mutations in the p53 gene frequently found in HCC reported from different endemic areas of the world may not play a direct role in the development of HCC in India. HBV infection and, possibly, exposure to the dietary aflatoxin B(1) appear to play major roles in the molecular pathogenesis of HCC in India. PMID- 10738215 TI - Transcatheter arterial chemoembolization therapy using iodized oil for patients with unresectable hepatocellular carcinoma: evaluation of three kinds of regimens and analysis of prognostic factors. AB - BACKGROUND: The current study was conducted to evaluate retrospectively the effects of three kinds of regimens used in transcatheter arterial chemoembolization (TACE) in patients with unresectable hepatocellular carcinoma (HCC) and patients' prognosis, and to analyze their prognostic factors. METHODS: The study population was comprised of 152 patients who were treated by TACE alone. Three kinds of regimens were used successively: doxorubicin hydrochloride (ADM) and mitomycin C mixed with iodized oil in 26 patients (ADMOS group), a combination of cisplatin (CDDP) solution and ADMOS in 70 patients (CDDP-ADMOS group), and CDDP powder and pirarubicin hydrochloride mixed with iodized oil in 56 patients (CTLS group). The CTLS group was comprised of patients with significantly worse background factors than the other two groups. RESULTS: The initial tumor response rate with a > 50% reduction was 12%, 23%, and 30%, respectively, in the ADMOS, CDDP-ADMOS, and CTLS groups. CTLS was significantly more effective than ADMOS (P < 0.05), and slightly but not significantly better than CDDP-ADMOS (P <0.1). The cumulative survival rates for the ADMOS, CDDP ADMOS, and CTLS groups were 59.0%, 70.1%, and 72.0%, respectively, at 1 year; 0%, 16. 3%, and 29.8%, respectively, at 3 years; and 0%, 4.1%, and 16.8%, respectively, at 5 years, with median survival times of 448 days, 574 days, and 758 days, respectively. The CTLS group showed a slightly but not significantly better survival than the ADMOS and CDDP-ADMOS groups (P <0.1). Multivariate analysis indicated that the significantly important prognostic factors (in order) were extrahepatic metastasis followed by the TACE regimen, serum alpha fetoprotein levels, and portal vein involvement and that CTLS was the best of the three regimens. CONCLUSIONS: Although TACE, using an effective regimen, improves clinical results, tumor factors appear to be more important when determining prognosis. PMID- 10738216 TI - Hepatoid carcinoma of the pancreas. AB - BACKGROUND: The majority of primary extrahepatic neoplasms exhibiting features of hepatocellular carcinoma in terms of morphology, immunohistochemistry, and behavior have been described in the stomach. To the authors' knowledge only a few cases have involved other organ sites. They frequently are associated with other histologic type tumors such as adenocarcinoma, and portend an aggressive behavior. METHODS: Two examples of hepatoid carcinoma arising from the pancreas are reported in the current study. RESULTS: One case was a malignant islet cell tumor with a full-blown clinical syndrome of glucagon overproduction, histologic evidence of hepatocytic differentiation, bile production, and alpha-fetoprotein (AFP) positivity. The second tumor was a ductal carcinoma showing periodic acid Schiff positive and diastase-resistant hyaline globules, AFP production, and ultrastructural resemblance to hepatocytic cells. CONCLUSIONS: The rare observation of cellular phenotypic transformation that corresponds with the process of hepatocytic transdifferentiation of pancreatic cells demonstrated in animal models and the common embryologic foregut derivation of the pancreas and liver also may explain the phenomenon of pancreatic hepatoid carcinoma. PMID- 10738217 TI - Atypical bronchopulmonary carcinoids show a tendency toward increased apoptotic and proliferative activity. AB - BACKGROUND: Bronchopulmonary carcinoid tumors can be divided into typical and atypical carcinoids according to their histologic and clinical features. Atypical carcinoids tend to have less predictable biologic behavior and are associated with a worse prognosis than typical carcinoids. The authors examined apoptosis and the expression of apoptosis regulating proteins Bcl-2, Bax, Bak, and Mcl-1 in 21 typical and 10 atypical bronchopulmonary carcinoid tumors. To find features distinguishing the growth of these types of tumors, the expression of p53 protein and the proliferation marker Ki-67 were also investigated. Finally, the parameters were compared with clinical follow-up data. METHODS: Thirty-one bronchopulmonary carcinoid tumors were examined by using in situ 3'-end labeling of DNA (TUNEL) for apoptosis and immunohistochemical staining methods for Bcl-2, Bax, Bak, Mcl-1, p53, and Ki-67 in formalin fixed, paraffin embedded tissue specimens. RESULTS: The apoptotic indices (AIs) were low in general (mean 0.18%), but higher AIs (> 0.5%) were observed significantly more often in atypical than in typical carcinoids (P < 0.008) in association with shortened survival (P < 0. 008). No relation between apoptosis and Bcl-2, Bax, Bak, or Mcl-1 expression was found. Expression of p53 protein was detected in only three atypical carcinoids, which also had significantly higher AIs than p53 negative tumors (P < 0.03). Proliferation rates measured by Ki-67 index were unusually low; the highest proliferation rates were observed in atypical carcinoids. There was a positive correlation between AI and Ki-67 indices (P < 0.01). CONCLUSIONS: This study suggests that the extent of apoptosis in bronchopulmonary carcinoid tumors is low. This, together with a low proliferation rate and an apparent absence of p53 mutation, enables a benign growth pattern. Some atypical carcinoids with enhanced apoptosis are associated with shortened survival, clearly deviating from typical carcinoids. PMID- 10738218 TI - Cerebrospinal fluid lactate dehydrogenase isoenzyme analysis for the diagnosis of central nervous system involvement in hematooncologic patients. AB - BACKGROUND: Central nervous system (CNS) involvement is common in hematooncologic diseases. The aim of the current study was to determine the diagnostic value of cerebrospinal fluid (CSF) lactate dehydrogenase (LDH) isoenzyme analysis for the diagnosis of CNS involvement in hematooncologic patients. METHODS: The study was comprised of 63 consecutive hematooncologic patients without previous CNS disease who underwent CSF examination as an integral part of their initial staging procedures (44 patients) or for the evaluation of neurologic symptoms (19 patients). Fifteen of these patients had CNS involvement by leukemia or lymphoma. The LDH isoenzyme pattern was established in the CSF of all patients and analyzed by the classification and regression trees (CART) method to construct a decision tree for the prediction of CNS involvement. An additional group of 30 consecutive patients comprised a validation set that was used for cross-validation of the CART-derived decision tree. RESULTS: A decision tree, with a single split at LDH5 >/= 2.8% for the prediction of CNS involvement, was constructed and validated by data from a validation set of patients. The decision tree had a sensitivity of 93% and a negative predictive value of 98%. One patient (1.6%) and 2 patients (6.6%) were misclassified in the derivation and validation sets, respectively. Overall, in the combined derivation and validation patient population, the decision tree misclassified 3.2% of patients, whereas CSF cytologic examination misclassified 4.3% of patients. CONCLUSIONS: Analysis of the LDH isoenzyme pattern in CSF fluid may be helpful in the evaluation of CNS involvement in patients with hematologic malignancies. The combination of CSF cytology and LDH isoenzyme analysis may improve the sensitivity of CSF cytology significantly. PMID- 10738219 TI - Perineural spread of basal cell carcinomas treated with Mohs micrographic surgery. AB - BACKGROUND: Perineural spread is a well-documented feature of cutaneous tumors and may portend a more aggressive course. The incidence of perineural invasion in basal cell carcinoma (BCC) is reportedly 1%. The authors sought to determine whether perineural spread occurs more commonly than previously thought. METHODS: The authors prospectively evaluated 434 patients with BCC treated with Mohs surgery, assessing the presence or absence of perineural inflammation and invasion in tumors requiring more than one stage of surgery. They also documented the demographic features, clinical characteristics, histologic subtype, and operative data in each case. RESULTS: Seventy-eight BCCs required more than one stage of Mohs surgery. Perineural inflammation, perineural tumor invasion, or both were present in 29 of the 78 tumors (37%), or 6.7% of all 434 prospectively evaluated cases. Twenty-one of the 78 tumors (26.9%) exhibited perineural inflammation, 3 (3.8%) demonstrated perineural invasion, and 5 (6.4%) exhibited both. Tumors with perineural invasion required 5.3 surgical stages on average for clearance, in contrast to tumors without perineural invasion, which required 2.2 stages. Tumors with perineural inflammation, inflammation plus tumor invasion, and invasion alone were, respectively, 138%, 149%, and 194% greater in area preoperatively than tumors without perineural involvement, and their mean defect areas after Mohs surgery were, respectively, 151%, 121%, and 605% larger than those of tumors without perineural involvement. CONCLUSIONS: The incidence of perineural invasion among cases of BCC appears higher than previously recognized. Tumor aggressiveness appears to correlate with the presence of perineural invasion. Surgery with horizontal frozen-section margin control enables easy detection of perineural involvement and should therefore be strongly considered for the treatment of high risk BCC patients. PMID- 10738220 TI - Growth inhibitory effects of paclitaxel on human epithelioid sarcoma in vitro: heterogeneity of response and the multidrug resistance phenotype. AB - BACKGROUND: Epithelioid sarcoma is a highly malignant soft tissue tumor that is largely resistant to conventional chemotherapy and radiotherapy. Because paclitaxel has been proven to be effective in other human malignancies refractory to conventional chemotherapy, the authors analyzed the in vitro growth inhibitory effects of paclitaxel on the human epithelioid-sarcoma cell line GRU-1 and its clonal subpopulations GRU-1A, GRU-1B, and GRU-1C. METHODS: Paclitaxel-induced morphologic alterations were visualized using light microscopy, immunofluorescence microscopy, and transmission electron microscopy. The antiproliferative effects of paclitaxel on the cell lines were determined by 3 [4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium' bromide (MTT) assay. The extent of paclitaxel-induced apoptosis was determined by light microscopy. The expression and function of P-glycoprotein and the multidrug resistance-associated protein (MRP) were defined by reverse transcriptase-polymerase chain reaction and fluorescence-activated cell sorter analysis. RESULTS: Paclitaxel-induced morphologic alterations such as micronucleus formation and microtubule bundles showed no significant differences between the parental cell line and its clonal subpopulations. A significant (P < 0.05) dose-dependent growth inhibition was observed in GRU-1 and its clonal subpopulations, with the IC(50) (concentration that inhibits 50%) values ranging from 0.04-0.49 microM in the different subpopulations. Paclitaxel-induced growth inhibition was accompanied by a slight increase in apoptosis. All cell lines showed an expression of and an effective function of P-glycoprotein and MRP. CONCLUSIONS: The differential response of GRU 1 and its clonal subpopulations to paclitaxel could not be predicted by the expression and function of P-glycoprotein and MRP, suggesting that other drug resistance mechanisms might be relevant in the heterogenous response observed in the epithelioid sarcoma cell lines in the current study. PMID- 10738221 TI - Clinical implication of altered expression of Mad1 protein in human breast carcinoma. AB - BACKGROUND: Mad1 protein is known to repress Myc target genes and antagonize Myc function. The authors undertook this study to investigate the clinical implication of Mad1 expression in human breast carcinoma. METHODS: The authors performed immunohistochemical assays for Mad1 and Myc proteins in human breast carcinoma, along with tissues from normal breast and benign diseases. The data from protein assays were analyzed in terms of the clinical and biologic characteristics of the patients. RESULTS: Of 66 patients with invasive ductal carcinoma, Mad1 expression was detected in 22 (33. 3%). Intensity and area of Mad1 expression significantly decreased in DCIS and invasive cancers, whereas high levels of Mad1 expression were persistent in benign breast lesions. Mad1 expression was significantly reduced in poorly differentiated tumors (P < 0.001). Expression of Mad1 was not associated with tumor size, lymph node status, or stage of disease. The authors did not observe any correlation between S-phase and expression status of Myc or Mad1. Mad1 expression was closely linked to differentiation of the cancer cells and inversely correlated with Myc expression (P = 0.042). In survival analysis, Mad1 was a significant factor in predicting recurrence of the disease, but not overall survival after CMF chemotherapy. CONCLUSIONS: In human breast carcinoma cells, expression of Mad1 seems to be down regulated, whereas expression of Myc is amplified. Altered expression of Mad1 may play a role in the malignant transformation of human mammary epithelial cells and represent an aggressive phenotype in human breast carcinoma. PMID- 10738222 TI - Primary axillary radiotherapy as axillary treatment in breast-conserving therapy for patients with breast carcinoma and clinically negative axillary lymph nodes. AB - BACKGROUND: The objective of the current study was to evaluate the effectiveness and morbidity of primary axillary radiotherapy in breast-conserving therapy for postmenopausal, clinically axillary lymph node negative patients with early stage breast carcinoma. METHODS: Between 1983-1997, 105 patients with clinically negative axillary lymph nodes and breast carcinoma were treated with wide local excision followed by radiotherapy to the breast, and axillary and supraclavicular lymph node areas. Adjuvant treatment with tamoxifen was given to 75 patients. The median follow-up of patients still alive was 41 months (range, 8-137 months). Fifty-five patients with no evidence of disease at last follow-up were examined prospectively with respect to late functional damage. RESULTS: The mean age of the patients was 64 years. Three patients developed a local recurrence. No isolated axillary lymph node recurrence was observed. In two patients, axillary recurrence was accompanied by distant metastases. The 5-year disease free interval and the overall survival were 82% (standard error [SE], 6%) and 83% (SE, 6%), respectively. In five patients, arm edema was reported and impaired shoulder function was reported in seven patients. Prospectively scored, arm edema was reported subjectively by the patient in 4% and objectively measured in 11% of cases. Impaired shoulder function was reported subjectively in 35% and objectively measured in 17% of cases. No brachial plexus neuropathy was noted. CONCLUSIONS: Primary axillary radiotherapy for postmenopausal women with clinically lymph node negative, early stage breast carcinoma was found to result in low axillary lymph node recurrence rates with only limited late complications. Therefore, primary axillary radiotherapy should be considered as axillary treatment in selected patients as an alternative to axillary lymph node dissection. PMID- 10738223 TI - Outcomes and factors impacting local recurrence of ductal carcinoma in situ. AB - BACKGROUND: The optimal management of ductal carcinoma in situ (DCIS) remains controversial. Investigators have focused on identifying patients who are eligible for treatment by excision alone. A retrospective analysis of patients with DCIS treated by various modalities was conducted to compare outcomes and determine factors significant for local recurrence (LR). METHODS: Between 1985 1992, 88 consecutive diagnoses of DCIS were identified in 85 patients. Seventy four percent were detected mammographically. The most common histologic subtypes were comedo (54%) and cribriform (23%). Tumor sizes were < 2.5 cm (49%), > 2.5-5 cm (26%), > 5 cm (23%), and unknown (2%). Final resection margins were tumor free (75%), close/positive (23%), and unknown (2%). Treatment methods included mastectomy (30%), localized surgery and radiation therapy (LSR) (43%), or wide localized surgery alone (LS) (27%). Radiation therapy (RT) was comprised of 50 grays to the breast, and 53% of treated patients received local "boost" irradiation. RESULTS: The median follow up was 8.3 years. The overall recurrence rate was 13. 6%, whereas the median time to LR was 27.8 months. Recurrence rates according to treatment modality were: LS: 25%; LSR: 13%; and mastectomy: 4%. However, if surgical margins were tumor free, LSR had a LR rate of 3.4%. After RT, no LR occurred prior to 15 months, and 4 of 5 tumors were noninvasive. Nine patients treated by excision alone conformed to the criteria of Lagios et al. criteria and LR occurred in three of nine tumors. Of the factors analyzed, margin status was found to be the best predictor for LR (P = 0.05). CONCLUSIONS: If surgical margins are tumor free, the LSR regimen is equivalent to mastectomy for local tumor control. Annual mammograms may be adequate for the follow-up of patients with irradiated breasts, but biannual studies still are recommended for patients treated with excision alone. PMID- 10738224 TI - Human tumor growth is inhibited by a vaccinia virus carrying the E2 gene of bovine papillomavirus. AB - BACKGROUND: Papillomavirus is the etiologic agent associated with cervical carcinoma. The papilloma E2 protein is able to regulate negatively the expression of E6 and E7 papilloma oncoproteins. Therefore, a new, highly attenuated vaccinia virus known as modified vaccinia virus Ankara (MVA), which carries the papillomavirus E2 gene, was used for the treatment of tumors associated with human papillomavirus. METHODS: Analysis of expression of the E2 gene from the recombinant vaccinia virus was performed by reverse transcription-polymerase chain reaction of RNA isolated from infected cells. Detection of the E2 protein was done by immunoprecipitation from proteins labeled with [(35)S]-methionine, isolated from infected cells. The therapeutic effect of the MVA E2 recombinant virus over human tumors was tested in nude mice bearing tumors generated by inoculation of HeLa cells. Series of 10 nude mice with tumors of different sizes were injected with MVA, MVA E2, or phosphate-buffered saline. Tumor size was monitored every week to assess growth. RESULTS: The MVA E2 recombinant virus efficiently expressed the E2 protein in BS-C-1 cells. This protein was able to repress, in vivo, the papillomavirus P105 promoter, which controls the expression of the E6 and E7 oncoproteins. In nude mice the MVA E2 virus reduced tumor growth very efficiently. In contrast, tumors continued to grow in mice treated with MVA or PBS. The life expectancy of MVA E2-treated mice was also increased three- to fourfold compared with that of animals that received MVA or PBS. CONCLUSIONS: The growth of human tumors was efficiently inhibited by the MVA E2 recombinant vaccinia virus. The absence of side effects in treated animals suggested that the MVA E2 virus is a safe biologic agent that could in the future be used in humans for the treatment of cervical carcinoma. PMID- 10738225 TI - Cancer heterogeneity and its biologic implications in the grading of urothelial carcinoma. AB - BACKGROUND: Urothelial carcinoma of the bladder often contains areas with different histologic grades. The influence of cancer heterogeneity on grading and its relation to patient outcome is uncertain. METHODS: The study group consisted of 164 patients with Ta urothelial carcinoma diagnosed at the Mayo Clinic between 1985 and 1986. None had previous or coexistent urothelial carcinoma in situ or invasive carcinoma. The primary (most common) and secondary (second most common if at least 5% of the cancer) patterns of cancer growth were graded by the newly proposed World Health Organization and International Society of Urological Pathology (WHO/ISUP) grading system. Scores of 1, 2, and 3 were assigned to urothelial neoplasms of low malignant potential (LMP), low grade urothelial carcinoma, and high grade urothelial carcinoma, respectively. The mean follow-up was 7.7 years (range, 0-13.3 years; median, 9.2 years). Progression was defined as the development of invasive carcinoma, distant metastasis, or death due to bladder carcinoma. RESULTS: Patient ages ranged from 36 to 96 years (mean, 69 years), and the male-to-female ratio was 4:1. Disease progression developed in 32 patients during a mean follow-up of 7.7 years. The mean interval from diagnosis to progression was 3.1 years (range, 0.01-8.7 years). Progression free survival was 82%, 77%, and 76% at 5, 7, and 10 years, respectively. Primary and secondary grades were different for 52 patients (32%). Based on the worst grade, 19 patients (12%) had urothelial neoplasms of low malignant potential (LMP), 92 (56%) had low grade carcinoma, and 53 (32%) had high grade carcinoma. Histologic grades based on worst, primary, secondary, and combined primary and secondary grades were all significant for predicting progression (P = 0.0009, 0.0004, 0.001, and 0.0001, respectively). Seven-year progression free survival rates for patients with LMP, low grade, and high grade carcinoma (based on worst grade) were 93%, 82%, and 61%, respectively; for patients with combined scores of 2, 3, 4, 5, and 6, survival rates were 93%, 80%, 82%, 68%, and 40%, respectively. The difference between patients with combined scores of 5 or 6 was statistically significant (P = 0.02). CONCLUSIONS: Histologic grade of urothelial carcinoma based on the newly proposed WHO/ISUP grading system stratifies patients into prognostically significant groups. Grading should also take cancer heterogeneity into consideration, and prognostic accuracy appears to be increased when the combined primary and secondary grades are applied. [See editorial counterpoint on pages 1509-12 and reply to counterpoint on pages 1513-6, this issue.] PMID- 10738226 TI - Ifosfamide, paclitaxel, and cisplatin for patients with advanced transitional cell carcinoma of the urothelial tract: final report of a phase II trial evaluating two dosing schedules. AB - BACKGROUND: A combination regimen of ifosfamide, paclitaxel, and cisplatin (ITP), recycled every 4 weeks, was reported in the treatment of previously untreated patients with advanced transitional cell carcinoma (TCC). This study sought to examine ITP at 3-week intervals to assess its feasibility and toxicity, compare the results for different schedules, and assess the impact of prognostic factors and postchemotherapy surgery on outcome. METHODS: ITP (ifosfamide 1.5 g/m(2) daily for 3 days, paclitaxel 200 mg/m(2) over 3 hours, and cisplatin 70 mg/m(2) on Day 1) was administered to patients with metastatic or unresectable TCC and was recycled every 4 weeks (for 30 patients) or 3 weeks (for 15 patients). Granulocyte-colony stimulating factor was given during each cycle. RESULTS: Thirty of 44 assessable patients (68%; 95% confidence interval, 52-81%) demonstrated a major response (10 complete responses [23%], 20 partial [45%]), with durations of response ranging from 4 to 36 months. At a median follow-up of 28 months, the median survival was 20 months. Eleven patients (25%) were disease free at last follow-up. Overall toxicity for the 15 patients whose treatment was recycled at 3 weeks was similar to that for patients treated every 4 weeks. Hematologic toxicity included anemia, thrombocytopenia, and febrile neutropenia. Febrile neutropenia was observed in 7 patients (16%) and in 3.3% of cycles of therapy. No Grade 4 nonhematologic toxicity was observed. Grade 3 nonhematologic toxicity included alopecia, renal insufficiency (11%), and neuropathy (9%). CONCLUSIONS: ITP is an active, well-tolerated regimen for previously untreated patients with TCC of the urothelial tract, resulting in a median survival of 20 months. Treatment can be recycled at 3-week intervals without enhanced toxicity. PMID- 10738227 TI - Squamous papilloma of the urinary tract is unrelated to condyloma acuminata. AB - BACKGROUND: To the authors' knowledge, there is no previous report of squamous papilloma of the urinary tract. It is uncertain whether there is a correlation between squamous papilloma, condyloma acuminatum, and verrucous carcinoma. METHODS: The authors evaluated the clinical and pathologic features of squamous papilloma (5 of the bladder, 2 of the urethra), condyloma acuminatum (3 cases), and verrucous carcinoma (3 cases) of the urinary bladder and performed human papillomavirus (HPV) DNA in situ hybridization studies to determine whether HPV was a common feature shared by these lesions. In addition, DNA ploidy evaluation by image cytometry and p53 immunohistochemical staining were performed. RESULTS: Squamous papilloma of the urinary tract occurred in elderly women and followed a benign clinical course with infrequent recurrence. All squamous papillomas were HPV DNA negative and DNA diploid with no or minimal p53 nuclear accumulation. Condyloma acuminata of the bladder contained HPV DNA, increased p53 protein expression, and aneuploid DNA content. All three cases of condyloma acuminata were associated with coexistent condylomata of the external genitalia, and two required pelvic exenteration for uncontrolled expansile growth. Verrucous carcinoma of the bladder occurred in elderly patients. All three cases of verrucous carcinoma were negative for HPV DNA and DNA aneuploid, and they exhibited consistent p53 expression. CONCLUSIONS: These data indicate that squamous papilloma is a distinct entity not related to condyloma or verrucous carcinoma. These lesions are benign, HPV DNA negative, DNA diploid, and they lack p53 overaccumulation. PMID- 10738228 TI - Grading of late effects in young adult survivors of childhood cancer followed in an ambulatory adult setting. AB - BACKGROUND: The objective of the current study was to describe a multidisciplinary transition program for following young adult survivors of childhood cancer in an adult-based ambulatory medical setting and to report the late effects with grades of toxicity diagnosed in all adult survivors followed in the program. METHODS: The study population was comprised of all young adult survivors (n = 96) of childhood cancer who were seen in the After the Cancer Experience (ACE) Young Adult Program prior to January 31, 1999. The median age of the survivors was 22.8 years (range, 17-34 years) and the median interval from the time of cancer diagnosis was 15.2 years (range, 6-25 years). Primary cancer groups included: leukemia, 33%; sarcoma, 24%; Hodgkin disease, 15%; non-Hodgkin lymphoma, 12%; Wilms' tumor, 9%; and other, 7%. Late effects were graded using the Common Toxicity Criteria, Version 2 (CTCv2), developed by the National Cancer Institute. RESULTS: Approximately 69% of the patients (66 of 96) had at least 1 late effect. Thirty-three percent of patients had a single late effect whereas 36% had >/= 2 late effects. Thirty percent of patients had a CTCv2 Grade 3 or 4 late effect. CONCLUSIONS: The current study represents an example of a successful multidisciplinary transition program in an ambulatory, adult setting for young adult survivors of childhood cancer. Late effects of cancer treatment are common in young adult survivors, with approximately 33% being moderate to severe. Further studies are needed to modify CTCv2 with the aim of developing a reliable and valid tool to assess late effects in long term survivors of childhood cancer. PMID- 10738229 TI - Prognostic factors after non-Hodgkin lymphoma in patients infected with the human immunodeficiency virus: Aquitaine Cohort, France, 1986-1997. Groupe d'Epidemiologie Clinique du SIDA en Aquitaine (GECSA). AB - BACKGROUND: The prognosis for survival of patients infected with the human immunodeficiency virus (HIV) who develop non-Hodgkin lymphoma (NHL) usually is considered to be poor. To the authors' knowledge the impact of highly active antiretroviral therapy, recently introduced in HIV disease case management, has not yet been studied in such circumstances. METHODS: All cases of NHL prospectively diagnosed between January 1986 and December 1997 among patients followed in the Aquitaine Cohort were reviewed. The Kaplan-Meier method and the proportional hazards model were used for statistical analysis. RESULTS: One hundred one NHL diagnoses were validated during the 12-year study period. The median proportional hazards cell count at the time of diagnosis of NHL was 112/mm(3). Histologic findings (Working Formulation classification) were: intermediate grade (N = 23), high grade (N = 61), other (N = 7), and undetermined (N = 10). In 56% of cases, staging classification was Ann Arbor Stage IV. Approximately 73% of patients received a specific NHL chemotherapy. During follow up, 44% were treated with nucleoside reverse transcriptase inhibitors (NRTIs) alone and 18% with triple therapy including a protease inhibitor (PI). The median survival was 6.0 months. In multivariate analysis, after adjusting for age, year of NHL diagnosis, histologic type, medical center, and transmission category, the following factors recorded at the time of diagnosis of NHL were indicative of an increasing risk of death: CD4+ count /= 2 visceral organs (1 with a high baseline serum lactate dehydrogenase level) and a third with soft tissue metastases achieved durable control of disease and were alive a median of 30+ months later. A fourth patient had a palliative response with reversal of melanosis and a survival of 7 months. This regimen was well tolerated and resulted in no serious long term adverse effects. CONCLUSIONS: The response rate for this regimen was no greater than 10% with Type I and II errors each not exceeding 10%. Nevertheless, occasional durable control of disease and the nonoverlapping toxicity profile with prior chemotherapy support consideration of this regimen in these patients who have limited second-line treatment options. PMID- 10738231 TI - Feasibility of oral ciprofloxacin for the outpatient management of febrile neutropenia in selected children with cancer. AB - BACKGROUND: Children with cancer who develop an episode of chemotherapy-induced febrile neutropenia usually are admitted to the hospital for intravenous empiric antibiotic therapy. In the current study, the authors examined the use of ciprofloxacin as outpatient management in selected patients with fever during an episode of neutropenia. METHODS: Febrile neutropenic patients with a diagnosis of cancer were eligible for outpatient management with oral ciprofloxacin if they appeared well and demonstrated the following characteristics: age 1-21 years, malignancy in remission, absolute phagocyte count > 100/mm(3), > 7 days since the initiation of the last course of chemotherapy, and reliable parents. Eligible children received a single dose of ceftazidime and were observed for 2-23 hours. Patients were discharged receiving oral ciprofloxacin (20/mg/kg/day divided in 2 doses) until the patient was afebrile for 24 hours, had sterile blood cultures, and had evidence of bone marrow recovery. Patients were admitted if they appeared toxic, had positive blood cultures, or were febrile for >/= 5 days. RESULTS: Forty-five evaluable episodes occurred in 32 children. Forty of the 45 patients (89%) were treated successfully in the outpatient setting. The 95% lower confidence bound on the proportion of successful outcomes was 70%. Five children required hospitalization: 2 due to noncompliance, 1 to receive intravenous acyclovir for herpes zoster, and 2 (4%) whose blood cultures were positive for Streptococcus viridans and S. pneumoniae. All had uncomplicated hospitalizations. CONCLUSIONS: The current study demonstrates that very carefully selected, low risk patients with febrile neutropenia may be treated successfully without hospitalization using oral ciprofloxacin. Additional research is required to refine further the optimal criteria for the selection of appropriate patients for outpatient management. PMID- 10738232 TI - Measuring quality of life of Chinese cancer patients: A validation of the Chinese version of the Functional Assessment of Cancer Therapy-General (FACT-G) scale. AB - BACKGROUND: Few cancer specific quality-of-life (QoL) measures from the West have been translated for use with Chinese-speaking patients, and no substantial validation of these translations with adequately large cohorts has been published previously, to the authors' knowledge. The Functional Assessment of Cancer Therapy-General (FACT-G) is a well-validated QoL instrument that is specific to cancer patients. The scale was translated into Chinese and the psychometric properties of this translated scale (FACT-G [Ch]) were tested with a Chinese sample in Hong Kong, China. METHODS: A total of 1262 Chinese cancer patients were selected in 3 samples from 5 Hong Kong regional hospitals. Quantitative and qualitative data were used to assess the cultural equivalence, factor structure, reliability, and validity of the FACT-G (Ch). RESULTS: Focus group discussions indicated that the FACT-G was seen as covering QoL domains identified as important and relevant to Chinese cancer patients, though in some respects it was seen as having limited scope in this sample. Psychometrically, the factor structure of the FACT-G deviated from that of the original work. The FACT-G (Ch) had acceptable reliability (Cronbach alpha 0.85). The convergent validity of the FACT-G (Ch) with a generic QoL measure (WHOQOL-BREF[HK]) was 0.72 (P < 0.001), and divergent validity showed low correlations of less than 0.15 (P < 0.05) with non-QoL measures. CONCLUSIONS: Focus group data indicated that the FACT-G translation into Chinese was seen as a conceptually relevant and moderately sufficient QoL measure. Psychometrically, the instrument had acceptable properties, but conceptual differences from the original version were suggested. Although more work is needed to increase its adequacy, the translated scale has reasonable utility for use with Chinese populations in clinical settings. PMID- 10738233 TI - Cancers of the upper aerodigestive tract in Ontario, Canada, and the United States. AB - BACKGROUND: Squamous cancers of the upper aerodigestive tract (UADT) are related to the use of tobacco and/or alcohol, and in North America they are more common among the poor. They are usually locoregionally confined at diagnosis, and local treatment with surgery and/or radiation therapy is often curative. This study compares the incidence and survival of this group of diseases in Canada and the U.S., two North American neighbors with many cultural similarities but significant differences in their health care and social programs. METHODS: To describe and compare the case mix, incidence, and outcome of squamous cancers of the UADT in Ontario, Canada, and the U.S., we used the Ontario Cancer Registry (OCR) and the Surveillance, Epidemiology, and End Results (SEER) registries in the U.S. to identify all cases of cancer with International Classification of Disease (ICD) codes 141, 143-9, 160-1, and a subset of 140, which were diagnosed between 1982 and 1994. ICD-O histology codes were placed into clinically relevant groupings, and ICD-9 site codes were grouped into sites as defined by the International Union Against Cancer and the American Joint Committee on Cancer. Age-adjusted incidence rates were calculated for each site. For the SEER registry, race specific incidence rates were also calculated. Observed and expected survival were plotted by site and registry, and from these, relative survival was calculated. Survival was compared during the first 5 years after diagnosis and during the next 5 years among patients who had survived the first 5 years. RESULTS: Of the 16,577 and 42,990 cases identified in the OCR and SEER registries, respectively, squamous cancer was by far the most common histology (94.1% in OCR, 94.6% in SEER) and will form the main subject of this report. The distribution of squamous cancers by site, subsite, age, and gender were remarkably similar in the two populations. Overall, the incidence was about 17% higher in the U.S. than in Ontario, and this difference was seen for all sites except the nasopharynx, which was more common in Ontario. The higher incidence in the U.S. in part reflects the much higher rate for African Americans than for Americans of other ethnic backgrounds. During the first 5 years after diagnosis, when most deaths from UADT cancer occur, there was a significant relative survival difference in favor of the U.S. for cancer of the supraglottis, and in favor of Ontario for cancer of the oral cavity. There was a nonsignificant trend in favor of Ontario for cancer of the nasopharynx. Within the SEER population, for all sites except the nasopharynx, 5-year relative survival was considerably worse for African Americans than for Americans of other ethnic backgrounds. Examination of survival beyond 5 years after diagnosis for patients who had survived the first 5 years revealed that for all sites, the observed survival continued to diverge markedly from the expected survival. The excess mortality ranged from less than 20% for glottic and nasopharyngeal cancers to about 30-40% for oropharyngeal and supraglottic cancers. CONCLUSIONS: Despite remarkable similarities in case mix between the two countries, UADT cancers were more frequent in the SEER population of the U.S. than in Ontario, and this was partly attributable to the much higher incidence among African Americans. Significant differences between the registries in 5-year survival were seen for several sites. African Americans with UADT cancers had much worse prognoses than did Americans of other ethnic backgrounds. Patients who survive their UADT cancer remain at a higher-than-expected risk of death even after they have been cured. PMID- 10738235 TI - The natural history of breast carcinoma: what have we learned from screening? PMID- 10738234 TI - American Joint Committee on Cancer Prognostic Factors Consensus Conference: Colorectal Working Group. AB - BACKGROUND: The American Joint Committee on Cancer (AJCC), which regularly reviews TNM staging systems, established a working party to develop recommendations for colorectal carcinoma. METHODS: A multidisciplinary consensus conference using published literature developed an arbitrary classification system of prognostic marker value (Category I, IIA, IIB, III, and IV), which forms the framework for this report. RESULTS: The working party concluded that several T categories should be subdivided: pTis into intraepithelial carcinoma (pTie) and intramucosal carcinoma (pTim); pT1 into pT1a and pT1b corresponding to the absence or presence of blood or lymphatic vessel invasion, respectively; and pT4 into pT4a and pT4b according to the absence or presence of tumor involving the surface of the specimen, respectively. The working party also recommended that TNM groups be stratified based on the presence or absence of elevated serum levels of carcinoembryonic antigen (CEA) (>/= 5 ng/mL) on preoperative clinical examination. In addition, the working party also concluded that carcinoma of the appendix should be excluded from the colorectal carcinoma staging system because of fundamental differences in natural history. CONCLUSIONS: The TNM categories and stage groupings for colorectal carcinoma published in the current AJCC manual have clinical and academic value. However, a few categories require subdivision to provide increasing discrimination for individual patients. The serum marker CEA should be added to the staging system, whereas multiple other factors should be recorded as part of good clinical practice. Although many molecular and oncogenic markers show promise to supplement or modify the current staging systems eventually, to the authors' knowledge none have yet been evaluated sufficiently to recommend their inclusion in the TNM system. PMID- 10738236 TI - Author reply PMID- 10738237 TI - Determining the site of the primary cancer in patients with skeletal metastasis of unknown origin: A retrospective study. PMID- 10738238 TI - Author reply PMID- 10738239 TI - Mammalian cell transformation and aneuploidy induced by five bisphenols. AB - Bisphenol-A (BP-A), a monomer of plastics used in numerous consumer products and a xenoestrogen, induces cellular transformation and aneuploidy in Syrian hamster embryo (SHE) cells. In this study, the abilities of 4 other bisphenols to induce cellular transformation and genetic effects in SHE cells were examined and compared to BP-A. Cellular growth was inhibited by all bisphenols in a concentration-related manner. The growth inhibitory effect of the bisphenols ranked: BP-5 > BP-4 > BP-3 > BP-2 or BP-A. Morphological transformation of SHE cells was induced by BP-A, BP-3, BP-4 and BP-5, and the induced-transformation frequencies were highest with BP-4. None of the bisphenols induced gene mutations at the Na(+)/K(+) ATPase locus or the hprt locus, or chromosomal aberrations in SHE cells. By contrast, aneuploidy induction in the near-diploid range was exhibited by BP-A, BP-3, BP-4 or BP-5, corresponding to the transforming activity of each compound. The results indicate that BP-A, BP-3, BP-4 and BP-5 exhibit transforming activity in SHE cells, while BP-2 does not, and that aneuploidy induction may be a causal mechanism of the transforming activity. PMID- 10738240 TI - Loss of heterozygosity and tumor suppressor activity of Bin1 in prostate carcinoma. AB - The genetic events underlying the development of prostate cancer are poorly defined. c-Myc is often activated in tumors that have progressed to metastatic status, so events that promote this process may be important. Bin1 is a nucleocytoplasmic adaptor protein with features of a tumor suppressor that was identified through its ability to interact with and inhibit malignant transformation by c-Myc. We investigated a role for Bin1 loss or inactivation in prostate cancer because the human Bin1 gene is located at chromosome 2q14 within a region that is frequently deleted in metastatic prostate cancer but where no tumor suppressor candidate has been located. A novel polymorphic microsatellite marker located within intron 5 of the human Bin1 gene was used to demonstrate loss of heterozygosity and coding alteration in 40% of informative cases of prostate neoplasia examined. RNA and immunohistochemical analyses indicated that Bin1 was expressed in most primary tumors, even at slightly elevated levels relative to benign tissues, but that it was frequently missing or inactivated by aberrant splicing in metastatic tumors and androgen-independent tumor cell lines. Ectopic expression of Bin1 suppressed the growth of prostate cancer lines in vitro. Our findings support the candidacy of Bin1 as the chromosome 2q prostate tumor suppressor gene. PMID- 10738241 TI - From normal respiratory mucosa to epidermoid carcinoma: expression of human mucin genes. AB - Mucous cells in the respiratory tract contribute to the maintenance of the normal epithelial cell population via mechanisms of cell proliferation and differentiation. Mucous cell hyperplasia often occurs as a basic response to injury in the tracheobronchial epithelium. These cells are also thought to be involved in the histogenesis of epidermoid metaplasia. A typical biochemical feature of these cells is mucus secretion. Aberrant glycosylation or under glycosylation of mucins is well known in cancer; however, the specific role played by mucin genes is at present unclear. To provide information regarding the expression of these genes in squamous metaplasia and squamous cell carcinoma, we analyzed and compared the expression of MUC1-MUC7 genes by in situ hybridization in control respiratory mucosa and lesions associated with neoplasia (hyperplasia, metaplasia and dysplasia) and squamous cell carcinomas. MUC4 was expressed independently of mucus secretion since it was expressed weakly by basal cells and probably by ciliated cells as well as collecting ducts, epidermoid metaplasia with complete squamous cell differentiation, and most of epidermoid carcinomas even well differentiated and keratinized. In squamous metaplasia and dysplasia, MUC4 gene expression was diffuse and less intense than in normal epithelium. MUC5AC was overexpressed in dysplasia as well as in mucous cell and basal cell hyperplasia and undetectable when squamous differentiation was achieved. PMID- 10738242 TI - Microbially produced acetaldehyde from ethanol may increase the risk of colon cancer via folate deficiency. AB - High alcohol and low folate intake are independent risk factors for colorectal cancer. Acetaldehyde has been postulated to be a factor responsible for ethanol associated carcinogenesis. High levels of acetaldehyde accumulate in the large intestine via the microbial oxidation of alcohol. Acetaldehyde degrades folate in vitro. Thus, it is possible that high intracolonic acetaldehyde levels break down folate in the colon. Our aim was to test the effect of high alcohol and acetaldehyde concentrations in the gut on systemic and local intestinal folate levels in rats. Twenty rats received 3 g/kg of ethanol twice a day for 2 weeks with or without concomitant ciprofloxacin administration. Twenty control rats received saline with or without ciprofloxacin. All rats were fed a diet with normal folate content. Alcohol treatment led to very high intracolonic acetaldehyde levels (387 +/- 185 microM), which were markedly decreased by concomitant ciprofloxacin treatment (21 +/- 4 microM). Erythrocyte, serum and small intestinal folate levels were unaffected by alcohol treatment. Alcohol administration decreased significantly colonic mucosal folate levels by 48%, and this effect was prevented by ciprofloxacin. We conclude that alcohol administration for 2 weeks leads to local folate deficiency of colonic mucosa in rats, most probably via the degradation of folate by the high levels of acetaldehyde microbially produced from ethanol. Our findings offer a unique explanation for the increased risk of colonic cancer associated with alcohol intake and folate deficiency. PMID- 10738243 TI - Vegf, Vegf-B, Vegf-C and their receptors KDR, FLT-1 and FLT-4 during the neoplastic progression of human colonic mucosa. AB - Because the crucial role of angiogenesis has been demonstrated in tumor growth and metastasis, the present study was undertaken to characterize the relative expression of vascular endothelial growth factors VEGF (vascular endothelial growth factor), VEGF-B, VEGF-C, and their receptors KDR (kinase insert domain containing receptor), FLT-1 (fms-like tyrosine kinase), and FLT-4 in human colonic cancers, in relation to the Astler-Coller pathological classification, and to prognosis. VEGF and VEGF-B gene expression was quantified by Northern blot in 72 tumor samples matched with control tissues. VEGF gene expression was 1.4 times higher in adenocarcinomas than in control tissues (p = 0.02), but did not increase further between Astler-Coller tumor stages A and D, and did not correlate with disease recurrence for patients at stages B2 or C. In adenomas, VEGF mRNA levels were not significantly different from those in the paired control colonic mucosa. The expression pattern of VEGF isoforms, mainly identified by RT-PCR (reverse-transcriptase-coupled polymerase chain reaction) as VEGF121 and VEGF165 and to a lesser extent VEGF189, was comparable in tumor and control tissues. VEGF-B mRNA levels were unchanged during the neoplastic progression of colonic mucosa. In contrast to KDR and FLT-4, the expression of VEGF-C and FLT-1 genes increased in some pathological tissues. These results provide evidence that the early and sustained increase in VEGF transcripts and the expression of multiple angiogenic factors and receptors contribute to the development of colon cancer, and thus constitute a putative target for anti angiogenic drug therapy. PMID- 10738244 TI - Endothelin receptor blockade potentiates FasL-induced apoptosis in rat colon carcinoma cells. AB - Imbalanced proliferation and apoptosis is important in tumor progression. Endothelin (ET)-1, a 21-amino-acid peptide with vasoconstricting and mitogenic activities, has been shown to be involved in the regulation of apoptosis. Progressive and regressive rat colon (PROb and REGb cells) carcinoma cell lines express the components of the ET-1 system (preproET-1, ET-converting enzyme and ET-receptors) and secrete ET-1. These cells also express the Fas(APO-1, CD95)/FasL system, but are resistant to FasL-induced apoptosis. We thus addressed the role of ET-1 in FasL-dependent cell death. Bosentan, a mixed ET(A)/ET(B) receptor antagonist, potentiated FasL-induced apoptosis in these cells. At low concentrations (10(-13) to 10(-10) M), ET-1 dose-dependently reversed bosentan induced apoptosis. Bosentan sensitization to FasL-induced apoptosis was not mediated by increased expression of Fas receptor and was blocked by the caspase inhibitor zVAD-fmk. The specific inhibition of enzymes involved in ceramide production did not restore survival of cells exposed to FasL and bosentan. Our results suggest that ET-1 is a survival factor able to protect in vitro colon carcinoma cells against FasL-induced apoptosis. PMID- 10738245 TI - bcl-2 over-expression enhances NF-kappaB activity and induces mmp-9 transcription in human MCF7(ADR) breast-cancer cells. AB - bcl-2 expression is often associated with poor prognosis in several types of tumors; however, the role of this molecule in breast cancer is still controversial. We found earlier that over-expression of bcl-2 in a human breast cancer cell line (MCF7(ADR)) enhances its tumorigenicity and metastatic potential by inducing metastasis-associated properties such as increased secretion of the matrix metalloproteinase-9 (mmp-9). In the present study, we investigated the effect of bcl-2 over-expression on the activity of the transcription factor NF kappaB, an important regulator of genes involved in tumor progression and invasion. Transient transfection experiments indicate that over-expression of bcl 2 in the MCF7(ADR) cell line, enhances NF-kappaB-dependent transcriptional activity. Mobility-shift analysis revealed an increase of NF-kappaB DNA-binding in bcl-2-over-expressing clones that correlated with lower levels of the NF kappaB cytoplasmic inhibitor IkappaBalpha. Moreover, point mutations of 2 highly conserved residues within the BH1 and BH2 domains that abrogate the interaction of bcl-2 with bax, or deletion of the N-terminal BH4 domain, completely eliminate the ability of this molecule to up-regulate NF-kappaB-dependent transactivation. Since mmp-9 is a NF-kappaB-regulated gene, we also investigated whether bcl-2 over-expression up-regulated mmp-9 transcription. We found that induction of mmp 9 mRNA correlates with the activation of an mmp-9-promoter-reporter-gene construct in transient transfection assay, and a mutation of the (-600)mmp-9-NF kappaB binding element abolishes this effect. The overall data indicate that bcl 2-mediated regulation of NF-kappaB-transcription-factor activity may represent an important mechanism for the promotion of malignant behavior in MCF-7(ADR) cells. PMID- 10738246 TI - Ectopic p16(ink4) expression enhances CPT-11-induced apoptosis through increased delay in S-phase progression in human non-small-cell-lung-cancer cells. AB - A tumor-suppressor gene, p16(INK4), which is deleted or mutated in tumors, regulates cell-cycle progression through a G(1)-S restriction point by inhibiting CDK4(CDK6)/cyclin-D-mediated phosphorylation of pRb. We have found that ectopic p16(INK4) expression increased cellular sensitivity of human non-small-cell-lung cancer (NSCLC) A549 cells to a selective growth-inhibitory effect induced by the topoisomerase-I inhibitor 11, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy camptothecin (CPT-11) in vitro. In this study, we observed enhanced apoptosis characterized by DNA fragmentation in A549 cells transfected with p16(INK4) cDNA (A549/p16-1) and treated with CPT-11. This apoptosis was suppressed by the inhibitor of interleukin-1beta-converting enzyme (ICE/caspase 1) or ICE-like proteases, Z-Asp-CH2-DCB, as determined by DNA fragmentation and proteolytic cleavage of poly(ADP-ribose) polymerase, a natural substrate for CPP32/caspase-3. In A549/p16-1 cells, cytosolic peptidase activities that cleaved Z-DEVD-7-amino-4-trifluoromethylcoumarin increased during CPT-11-induced apoptosis and were suppressed by a highly specific caspase-3 and caspase-3-like inhibitor, Z-DEVD-fluoromethylketone. These findings indicate that p16(INK) is positively involved in the activation pathway of the caspase-3 induced by CPT-11. The increased delay in S-phase progression and subsequent induction of apoptosis were observed in CPT-11-treated A549/p16-1 cells on the basis of DNA histograms. Specific down-regulation of the cyclin-A protein level in A549/p16-1 cells was observed after CPT-11-treatment, whereas cyclin B, cdk2, and cdc2 protein levels were unaffected. These results suggest that ectopic p16(INK4) expression inappropriately decreases cyclin A and thereby terminates CPT-11-induced G(2)/M accumulation, which is followed by increased apoptosis in p16(INK4)-expressing A549 cells. PMID- 10738247 TI - Increased gelatinase-A and gelatinase-B activities in malignant vs. benign breast tumors. AB - Matrix metalloproteinases (MMP) types 2 and 9 (also known as gelatinase A and B) are thought to be causally involved in cancer invasion and metastasis. In normal as well as in malignant tissue, both these MMPs occur in multiple forms such as inactive precursors, active enzymes and enzyme-inhibitor complexes. Using newly developed quantitative activity assays, the levels of active MMP-2, total (active and activatable) MMP-2 and total MMP-9 were found to be significantly higher in breast carcinomas than in fibroadenomas. In addition, active MMP-2 and MMP-9 were detected more frequently in malignant than in benign breast carcinoma. These new quantitative activity assays are likely to be of use in studying the mechanism of action of both MMP-2 and -9, assessing their potential prognostic value in different cancers and in the design of MMP inhibitors for preventing cancer metastasis. PMID- 10738249 TI - Localisation by in situ hybridisation of S100A4 (p9Ka) mRNA in primary human breast tumour specimens. AB - Rodent S100A4 (p9Ka) induces a metastatic phenotype in benign rat mammary tumour cells and cooperates with the neu oncogene to produce metastatic tumours in a transgenic mouse model system. Human S100A4 possesses similar metastasis-inducing properties. S100A4 mRNA is now sought in human breast tumour-derived cell lines and tumour specimens. S100A4 mRNA is present in some cell lines derived from malignant breast cancers, but is not detectable in cells derived from benign breast tumours. In human tumour specimens, using in situ hybridisation, the mRNA for S100A4 is localised to the epithelial cells of carcinoma specimens, and in some normal breast specimens, to a stromal region surrounding the epithelial ducts. In carcinoma specimens, S100A4 mRNA is also found in the stromal region surrounding islands of cancer cells. For both the epithelial and stromal components, S100A4 mRNA is present at a higher level in carcinomas relative to benign breast tumour specimens. In general, there is a concordance between the S100A4 mRNA signal from the epithelial and stromal elements of the same carcinoma specimens. Using Northern blotting techniques, these results have been extended to a panel of 137 benign and malignant breast tumour specimens. The results show that S100A4 mRNA occurs in the more-malignant, rather than in the more-benign tumour specimens. PMID- 10738248 TI - Involvement of mitochondria and caspase-3 in ET-18-OCH(3)-induced apoptosis of human leukemic cells. AB - The induction of cell death in leukemic HL-60 cells by the ether lipid 1-O octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH(3); edelfosine) followed the typical apoptotic changes in ultrastructural morphology, including blebbing, chromatin condensation, nuclear membrane breakdown and extensive vacuolation. Using a cytofluorimetric approach, we found that ET-18-OCH(3) induced disruption of the mitochondrial transmembrane potential (DeltaPsi(m)) followed by production of reactive oxygen species (ROS) and DNA fragmentation in leukemic cells. ET-18-OCH(3) also induced caspase-3 activation in human leukemic cells, as assessed by cleavage of caspase-3 into the p17 active form and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP). ET-18-OCH(3) analogues unable to induce apoptosis failed to disrupt DeltaPsi(m) and to activate caspase-3. ET-18-OCH(3)-resistant Jurkat cells generated from sensitive Jurkat cells showed no caspase-3 activation and did not undergo DeltaPsi(m) disruption upon ET-18-OCH(3) incubation. Cyclosporin A partially inhibited DeltaPsi(m) dissipation, caspase activation and apoptosis in ET-18-OCH(3)-treated leukemic cells. Overexpression of bcl-2 by gene transfer prevented DeltaPsi(m) collapse, ROS generation, caspase activation and apoptosis in ET-18-OCH(3) treated leukemic T cells. Pretreatment with the caspase inhibitor Z-Asp-2, 6 dichlorobenzoyloxymethylketone prevented ET-18-OCH(3)-induced PARP proteolysis and DNA fragmentation, but not DeltaPsi(m) dissipation. ET-18-OCH(3) did not affect the expression of caspases and bcl-2-related genes. ET-18-OCH(3)-induced apoptosis did not require protein synthesis. Our data indicate that DeltaPsi(m) dissipation and caspase-3 activation are critical events of the apoptotic cascade triggered by the antitumor ether lipid ET-18-OCH(3), and that the sequence of events in the apoptotic action of ET-18-OCH(3) on human leukemic cells is: DeltaPsi(m) disruption, caspase-3 activation and internucleosomal DNA degradation. PMID- 10738250 TI - Radiation-induced expression of functional Fas ligand in EBV-positive human nasopharyngeal carcinoma cells. AB - Ionizing radiation remains a major therapeutic tool against human cancers, especially epithelial tumors, which account for the majority of human malignancies. Although Fas and Fas-L are essential determinants of apoptosis, few data support their role in the cytotoxic effect of ionizing radiation. Epstein Barr-virus (EBV)-positive nasopharyngeal carcinoma (NPC) were chosen to address this question owing to their known sensitivity to ionizing radiation and their constitutive expression of the Fas-receptor. We here report that, in xenografted NPC cells, Fas-L expression, which was very low in basal conditions, was dramatically increased by tumor irradiation. Both the Fas receptor and the Fas ligand were found to be functional in this model, and a high proportion of irradiated NPC cells underwent apoptosis following tumor irradiation. Induction of Fas-L expression and apoptosis were observed for doses as low as 2 Gy. These data show an increase in Fas-L expression upon irradiation exposure, and strongly suggest that, in some epithelial malignancies, Fas-mediated apoptosis can play a major role in the anti-tumor effect of ionizing radiation, in the range of doses used for therapeutic applications. PMID- 10738251 TI - FLT3 ligand gene expression and protein production in human colorectal cancer cell lines and clinical tumor specimens. AB - Dendritic cells (DC) are professional antigen presenting cells (APC) whose proliferation and functional differentiation can be induced by hematopoietic growth factors including GM-CSF and FLT3 ligand (FL). Colorectal cancers are known to be infiltrated by dendritic cells (DC) and neoplastic cells have been shown to produce GM-CSF. In this work we investigated FLT3 ligand (FL) gene expression and protein production in human colorectal cancer cell lines and clinical tumor specimens. Using reverse transcription polymerase chain reaction (RT-PCR), 6 out of 6 established tumor lines were found to express to variable extents FL gene. In 1 of them, SW480, FL immunoreactivity could be observed by taking advantage of specific antibodies. In contrast, soluble FL could not be detected in any culture supernatant. FLT3 receptor (FR) gene was not expressed and exogenous addition to the cultures of recombinant FL (rFL) did not affect the proliferation of the tumor lines. FL gene expression was investigated using a densitometry-assisted, semiquantitative RT-PCR in clinical tumor specimens. Specific FL gene transcripts were amplified from 12 of 12 surgical samples. In these cases, FL gene expression of significantly lower intensity was also detected in healthy mucosa sampled in the vicinity (2 cm) or at a distance (10 cm) from neoplastic outgrowth. Immunohistochemical studies identified FL-positive cancer cells in 5 of 5 cases tested. No positivity was detected in healthy mucosa epithelia at a distance from the tumor or in stromal cells. FL content in preoperative sera from colorectal cancer patients (n = 13) did not exceed the levels detected in healthy donors (10 positive nodes) would have been altered if a level I or level I/II/III dissection were performed. RESULTS: Assuming that all 302 patients had undergone a level I/II dissection, performing only level I dissection would have resulted in a change in nodal category in 15.9% of all patients and 36.1% of patients with positive nodes. The corresponding changes for a level I/II/III dissection would have been 4.3% and 9.5%, respectively. CONCLUSIONS: Variations in the level of axillary node dissection for breast cancer can result in significant changes in the number of positive axillary nodes. This can potentially bias adjuvant chemotherapy recommendations if treatment decisions are based on this prognostic factor. PMID- 10738266 TI - Experience in the treatment of synchronous and metachronous carcinoma of the oesophagus and the head and neck. AB - BACKGROUND AND OBJECTIVES: Treatment of multiple primary squamous cell carcinomas of the head and neck and oesophagus is controversial. The poor prognosis of these 2 types of carcinoma taken individually and their anatomic proximity complicate the therapeutic strategy and limit the treatment choices for each location. METHODS: From 1986 to 1998, 43 patients received curative treatment for multiple synchronous (n = 30) or metachronous (n = 13) primary neoplasms of the oesophagus and head and neck. For synchronous cancers, the therapeutic strategy consisted of first curing the head and neck cancer and then planning oesophagectomy according to the type of head and neck cancer therapy. RESULTS: Ten total oesopharyngolaryngectomies and 33 subtotal oesophagectomies were performed. The postoperative mortality rate was 9.3% (4/43). The rate of anastomotic leakage was 30% (13/43), and all such leaks were cervical. Pulmonary infection occurred in 19% of cases (8/43). A past history of cervical radiation therapy or cervicotomy did not appear to be a significant risk factor for anastomotic leakage or pulmonary complications. Oesophagectomy did not affect the functional results in the 31 patients whose larynx could be preserved. CONCLUSIONS: Oesophagectomy after head and neck cancer treatment is possible with a low mortality rate and acceptable morbidity. PMID- 10738267 TI - Evaluation of p53 alterations in occult lymph node metastases. AB - BACKGROUND AND OBJECTIVES: This study was designed to evaluate p53 alterations in occult lymph node metastases. METHODS: We examined 41 patients with stage I non small-cell lung cancer. We investigated p53 gene mutation by polymerase chain reaction and single-strand conformation polymorphism analysis of exons 5-8, p53 protein accumulation by immunostaining with monoclonal antibody DO-7, and detection of tumor cells in lymph nodes by immunohistochemistry with monoclonal antibodies to cytokeratin (CK). RESULTS: p53 gene mutation was detected in 34% of tumors and nuclear p53 accumulation in 46%. CK-positive cells in the hilar and mediastinal region lymph nodes were detected in 43.9% of patients and 29.3%, respectively. Of the 14 cases with p53 mutation and the 19 cases with p53 accumulation, 12 and 15 had micrometastases in the hilar or mediastinal lymph nodes, respectively. However, p53 alterations were not significantly associated with occult lymph node metastases. In cases with occult lymph node metastases, the 5-year survival was 81. 9% for the p53 wild-type group and 45.8% for the p53 mutation group. CONCLUSIONS: p53 alterations are not correlated with occult lymph node metastases, while p53 gene mutation is considered to be an unfavorable prognostic marker in patients with occult lymph node metastases. J. Surg. Oncol. 2000;73:143-147. PMID- 10738268 TI - Extreme drug resistance assay and response to chemotherapy in patients with primary peritoneal carcinoma. AB - BACKGROUND AND OBJECTIVES: The extreme drug resistance (EDR) assay is an in vitro chemoresistance assay performed on tumor samples grown in culture and is claimed to predict drugs unlikely to produce response. Its clinical value in patients with epithelial ovarian cancer (EOC) has recently been questioned. The aim of this study is to describe EDR assay results and responses to chemotherapy among women with primary peritoneal adenocarcinoma (PPA) and to compare them with those of women with EOC. METHODS: Fresh tumor specimens from 20 consecutive women with PPA were tested for EDR to the following drugs: cisplatin, carboplatin, paclitaxel, doxorubicin, cyclophosphamide, ifosfamide, etoposide, hexamethylmelamine, and topotecan. They were then treated with cisplatin-based combination chemotherapy. The results of the EDR assay and response to chemotherapy were compared with those among women with EOC. RESULTS: There was no significant difference in the incidence of EDR to cisplatin, carboplatin, paclitaxel, doxorubicin, cyclophosphamide, ifosfamide, etoposide, hexamethylmelamine, and topotecan among patients with PPA and those with EOC. The response rate of PPA patients to chemotherapy was 80.0% and unrelated to EDR to the individual drugs used in combination chemotherapy. CONCLUSIONS: The EDR profile and response to cisplatin-based chemotherapy among women with PPA were similar to those among women with EOC. These findings support treating both conditions similarly. EDR to individual drugs does not preclude response to combination chemotherapy. PMID- 10738269 TI - Prognostic significance of immunohistochemical micrometastases in node negative gastric cancer patients. AB - BACKGROUND AND OBJECTIVES: The purpose of this study is to examine the prognostic significance of immunohistochemical (IHC) evidence of lymph node (LN) metastases in histologic node negative gastric cancer patients. METHODS: Retrospective review from 1981 to 1998 revealed 25 patients resected for T1-4N0M0 gastric and gastroesophageal (GE) junction adenocarcinoma. All cases were reviewed and histopathologic parameters were defined for each primary tumor. All LNs underwent IHC analysis with the epithelial marker CAM 5.2. Data are reported as median (range). RESULTS: The median number of LN resected was 7 (range 1-33). The median follow-up time was 25 months (range 4-195) with an overall 5-year survival rate of 55%. For patients with IHC evidence of LN micrometastasis (n = 9), the 5-year survival rate was significantly decreased (35%) compared to a 66% 5-year survival rate for IHC negative patients (n = 16, P = 0.05). CONCLUSIONS: The presence of IHC-detected LN micrometastases correlates with worse prognosis for patients with histologic node negative gastric cancer. IHC may be a useful additional staging modality in this subset of patients. PMID- 10738270 TI - Aberrations of the K-ras, p53, and APC genes in extrahepatic bile duct cancer. AB - BACKGROUND AND OBJECTIVES: The genetic alterations involved in extrahepatic bile duct (EHBD) cancer are poorly understood. Our aim was to identify aberrations of the K-ras, p53, and APC genes in EHBD cancer. METHODS: We investigated aberrations of these genes in 52 EHBD cancers using polymerase chain reaction (PCR) single-strand conformation polymorphism analysis, followed by direct sequence determination and a PCR restriction fragment length polymorphism assay. RESULTS: The K-ras, p53, and APC genes were mutated in 9.6%, 32.7%, and 0% of EHBD cancers, respectively. Loss of heterozygosity at the p53 and APC gene loci was identified in 15.6% and 38.5% of EHBD cancers, respectively. CONCLUSIONS: Our results suggest that an unknown suppressor gene on 5q other than the APC gene may be responsible for EHBD cancer. PMID- 10738271 TI - Tumoricidal effects of onconase on various tumors. AB - BACKGROUND AND OBJECTIVES: The effects of Onconase (Onc) on the tumor growth in vitro and in vivo were examined. Because elevated tumor interstitial fluid pressure (TIFP) is one of the major causes of inadequate drug delivery into solid tumors, we tested if Onc could lower TIFP in solid tumors. METHODS: We used several assays including a clonogenic assay and a growth delay assay for the determination of anti-tumoricidal effects of Onc. We also measured Onc-induced changes in several tumor physiological parameters. RESULTS: Onc demonstrated cytotoxic effects in all eight exponentially growing cell lines in vitro. It effectively inhibited the growth of all four transplanted tumors in vivo, and significantly reduced TIFP in all four tumors. Onc also induced increases in tumor blood flow (TBF) as well as increases in median tumor oxygen partial pressure (pO(2)) in solid tumors. CONCLUSIONS: Onc showed anti-tumoral effects on various tumor cells in vitro as well as in vivo. We also gained some insight regarding the potential physiological benefit of Onc as a new therapeutic agent in cancer treatment. Due to increases in both TBF and tumor pO(2), Onc could be a potential candidate as a novel radiation enhancer; therefore, the study of the radiation response in vivo is warranted. PMID- 10738273 TI - Congenital pleuroperitoneal communication in a patient with pseudomyxoma peritonei. AB - BACKGROUND AND OBJECTIVES: Pseudomyxoma peritonei syndrome is a rare disease arising from a perforated appendiceal adenoma. The syndrome is characterized by progressive accumulation of mucinous ascites and tumor within the peritoneal cavity. Direct extension of pseudomyxoma peritonei to the pleural cavity is uncommon and has been associated with surgical penetration of the diaphragm at the time of cytoreduction. METHODS: We review the case of a patient who presented with mucoid peritoneal and pleural fluid consistent with spontaneous pleural spread of pseudomyxoma peritonei. RESULTS: Surgical exploration confirmed direct pleuroperitoneal communication by macroscopic diaphragmatic fenestration. CONCLUSIONS: This is a rare phenomenon. We outline a therapeutic approach to be applied when pleural involvement is suspected in patients with pseudomyxoma peritonei syndrome. PMID- 10738272 TI - Catheter fracture and cardiac migration: a rare complication of totally implantable venous devices. AB - Totally implantable venous device (TIVD) are widely used for the treatment of patients requiring long-term chemotherapy, total parenteral nutrition and fluid replacement. Until today, many kinds of complications have been reported in the literature. We report an unusual case of catheter fracture as a consequence of pinchoff syndrome, and discuss the potential methods to avoid this complication and its evolution. PMID- 10738274 TI - Hepatic resection of hepatocellular carcinomas based on tumor hemodynamics. PMID- 10738275 TI - Role of surgery in the management of postmastectomy extremity angiosarcoma (Stewart-Treves syndrome). AB - Stewart-Treves syndrome (STS) is the rare occurrence of angiosarcoma in a setting of postmastectomy upper extremity lymphedema. A collective comparison of outcomes following various initial treatment options in STS has not previously been reported. We reviewed 160 cases of STS reported in the literature since 1966. We analyzed the relationship between initial treatment and survival in all 92 of these patients for whom detailed treatment and outcome data had been reported. There was no significant difference in survival comparing those initially treated with wide excision (n = 16) and those treated with amputation (n = 45) (P = 0.40). Even in the setting of initial surgical treatment, overall long-term survival was poor (<40%). There have been even fewer long-term survivors among those treated initially with regional chemotherapy (n = 7) or radiation therapy (n = 24). An update on STS and a discussion of recent advances in the understanding of its molecular pathogenesis that may result in future treatment improvements are presented. PMID- 10738276 TI - Simultaneous quantitative determination of electroporative molecular uptake and subsequent cell survival using gel microdrops and flow cytometry. AB - BACKGROUND: Electroporation is widely used to introduce molecules into cells, but conditions yielding maximal molecular uptake often result in low cell survival. We describe a high throughput method for analyzing populations of culturable cells simultaneously for molecular uptake and cell growth. METHODS: Cells are microencapsulated within agarose gel microdrops (GMDs), exposed to a polar tracer fluorescent molecule, electrically pulsed at various field strengths, and cultured. The GMDs are then analyzed at about 100,000 occupied GMDs per hour by flow cytometry for both uptake and microcolony formation. RESULTS: We demonstrate how the method can be used to optimize a parameter of interest (e.g., the applied field strength) with respect to both uptake and cell survival. Here, the optimal field strength is determined to be 1.7 kV/cm. Below this, there is lower molecular uptake. As the field strength is increased, the cell survival rate goes down. CONCLUSIONS: This method may be applicable to optimization of other electroporation parameters and alternative physical and chemical methods for cell loading. PMID- 10738277 TI - Flow cytometric analysis of immunoprecipitates: high-throughput analysis of protein phosphorylation and protein-protein interactions. AB - BACKGROUND: Activation-induced protein phosphorylation can be studied by Western blotting, but this method is time consuming and depends on the use of radioactive probes for quantitation. We present a novel assay for the assessment of protein phosphorylation based on latex particles and flow cytometry. METHODS: This method employs monoclonal antibodies coupled to latex particles to immobilize protein kinase substrates. Their phosphorylation status is assessed by reactivity with phosphoepitope-specific antibodies. The amount of immobilized protein on the particles was analyzed by direct or indirect immunofluorescence with antibodies to nonphosphorylated epitopes. RESULTS: The assay allowed measurement of phosphorylation of multiple protein kinase substrates in stimulated T cells, including the zeta chain of the T-cell receptor, ZAP-70, CD3, CD5, SHP-1, and ERK 2, using 1-3 microg of total cell protein per sample. The assay provided high resolution of kinetics of phosphorylation and dephosphorylation. Interactions of protein kinase substrates with associated signaling molecules were demonstrated. CONCLUSIONS: The novel assay allows high-throughput quantitative measurement of protein modifications during signal transduction. PMID- 10738278 TI - Selective binding of a monoclonal antibody to Aspergillus niger glucose oxidase by formaldehyde fixed human polymorphonuclear leukocytes. AB - BACKGROUND: Many of the procedures used in handling neutrophils may affect the expression of surface antigens, and hence their quantitation by flow cytometry. METHODS: Because the enzyme glucose oxidase of Aspergillus niger is absent in human tissues, an IgM against it (mAb GO) was used as negative control in a study involving the normal expression of neutrophil specific BH2-Ag in different age groups. RESULTS: When peripheral blood leukocytes (PBL) were freshly prepared, processed and stained with FITC-mAb GO without fixation or when the cells were stained with FITC-mAb GO prior to fixation with 2% formaldehyde, both median fluorescent intensity (MFI) and per cent of positively stained polymorphonuclear leukocytes (PMN) were similar to that obtained with a background sample without any antibody. However, when PBL were fixed after isolation with different concentrations of formaldehyde and for varying durations, MFI and per cent of positively stained PMN but not of monocytes or lymphocytes with FITC-mAb GO increased in a time and concentration dependent manner. Saturation was achieved at a finite concentration of the antibody. In a competition assay unlabelled mAb GO reduced binding of FITC-mAb GO to PMN by 79% and 95% at concentrations 100 and 200 times that of FITC labelled antibody, respectively. CONCLUSIONS: These observations strongly suggest that formaldehyde fixation causes the expression or accessibility of an epitope on PMN that is specifically recognized by the mAb GO. PMID- 10738279 TI - Stochastic modeling of apoptosis tolerance distributions measured by multivariate flow analysis of human leukemia cells. AB - BACKGROUND: Cytofluorometric analysis allows single-cell resolution of all-or none programmed cell death (apoptosis) responses and permits direct measurement of cumulative frequency distributions (CFDs) of apoptosis sensitivity from which the median apoptosis tolerance can be estimated. Robust estimation of susceptibility to apoptosis within neoplastic cell populations provides a means of either accurately determining pharmacologically induced changes in apoptosis sensitivity or comparing cell population responses to different apoptosis inducers. METHODS: Experimentally determined CFDs for VP-16 (etoposide)-induced apoptosis were measured by phosphotidylserine surface expression and mitochondrial membrane potential dissipation (DeltaPsi(m)) in BV173 leukemia cells. CFDs were modelled by a modified Hill equation using a four-parameter nonlinear regression from which median apoptosis tolerance (K) was estimated. RESULTS: Median apoptosis tolerance (K) was estimated from nonlinear regression analysis of CFDs for DeltaPsi(m) collapse and loss of membrane asymmetry. The error distribution of K determined from nonlinear regression analysis of 100 simulated CFDs was shown to exhibit an asymmetrical distribution. The asymmetrical likelihood intervals for K were computed iteratively, thereby providing a measure of experimental error. CONCLUSIONS: A distribution-based approach to apoptosis assay using multivariate flow analysis offers a powerful, quantitative technique for investigating the phenotypical basis of neoplastic cell responsiveness to apoptosis therapy, permitting separation of cell populations on the basis of apoptosis susceptibility. PMID- 10738280 TI - Hue-saturation-density (HSD) model for stain recognition in digital images from transmitted light microscopy. AB - BACKGROUND: Transmitted light microscopy is used in pathology to examine stained tissues. Digital image analysis is gaining importance as a means to quantify alterations in tissues. A prerequisite for accurate and reproducible quantification is the possibility to recognise stains in a standardised manner, independently of variations in the staining density. METHODS: The usefulness of three colour models was studied using data from computer simulations and experimental data from an immuno-doublestained tissue section. Direct use of the three intensities obtained by a colour camera results in the red-green-blue (RGB) model. By decoupling the intensity from the RGB data, the hue-saturation intensity (HSI) model is obtained. However, the major part of the variation in perceived intensities in transmitted light microscopy is caused by variations in staining density. Therefore, the hue-saturation-density (HSD) transform was defined as the RGB to HSI transform, applied to optical density values rather than intensities for the individual RGB channels. RESULTS: In the RGB model, the mixture of chromatic and intensity information hampers standardisation of stain recognition. In the HSI model, mixtures of stains that could be distinguished from other stains in the RGB model could not be separated. The HSD model enabled all possible distinctions in a two-dimensional, standardised data space. CONCLUSIONS: In the RGB model, standardised recognition is only possible by using complex and time-consuming algorithms. The HSI model is not suitable for stain recognition in transmitted light microscopy. The newly derived HSD model was found superior to the existing models for this purpose. PMID- 10738281 TI - Ultra-rare-event detection performance of a custom scanning cytometer on a model preparation of fetal nRBCs. AB - BACKGROUND: The performance of a fully automated scanning cytometer incorporating previously reported high-precision autofocus and accurate image segmentation was evaluated for the detection of "ultra-rare" cells using a model of fetal nucleated red blood cells (fnRBCs) in the maternal circulation. These distinctive scanning cytometry techniques were expected to markedly improve sensitivity and specificity. METHODS: Normal adult blood and fetal red blood cells were stained with fluorescein isothiocyanate-conjugated anti-fetal hemoglobin and 4,6 diamidino-2-phenylindole, a nuclear dye. Adult cells were spiked with fetal cells to create ratios of about 1 fnRBC in 10(7) nucleated cells and deposited in monolayers on slides using centrifugal cytology. Rare-event performance parameters were reviewed, formalized, and applied to test the new instrument using this cell model. RESULTS: Fifteen slides were analyzed to establish performance by comparison with manual detection, and four sets of four slides each were then scanned to explore the limit of detection. Results were an average sensitivity of 91%, an average specificity error of 12.3 false-positives per million cells, and repeatability of 100% at a cell analysis rate of 862 Hz. With addition of a quick interactive step subsequent to scanning, the false-positive rate dropped to a total of only one artifact over the 15 experiments. The instrument succeeded at locating 1 fnRBC in 20 million adult cells, the lowest limit of detection tested. CONCLUSION: This consistently high performance, coupled with the capability of scanning arbitrarily large numbers of cells, validates the considerable potential of precise high-speed autofocus and accurate real-time image segmentation for ultra-rare event detection. PMID- 10738282 TI - Coating of coverslips with glow-discharged carbon promotes cell attachment and spreading probably due to carboxylic groups. AB - BACKGROUND: For high-resolution microscopy, cells have to be analyzed through thin glass coverslips. Therefore, it is necessary to culture cells on coverslips for preservation of cell morphology. We found cell attachment and spreading to be relatively slow processes, even when cells were plated on coated coverslips. This slowness presents a problem, particularly when synchronized cell populations are used. METHODS: In this paper, we present a method that is based on glow discharged carbon coating of coverslips which promotes rapid attachment and spreading of cells, enabling rapid analysis of cells after plating. Results obtained with carbon-coated coverslips were compared with those of other types of coating. Two fibroblast lines, an epithelial cell line, and a carcinoma cell line were tested. RESULTS AND CONCLUSIONS: All cell lines showed a rapid adhesion on carbon-coated coverslips. With fibroblasts we found the carbon coating to be superior to other coatings tested, mainly because the carbon did not influence cell morphology. Using synchronized or irradiated cells produced similar results. The superior performance of carbon coating is probably due to carboxylic groups on the glow-discharged carbon layer. The carbon layer does not interfere with microscopy or immunocytochemical staining procedures. PMID- 10738283 TI - Mean and variance of ratio estimators used in fluorescence ratio imaging. AB - BACKGROUND: The ratio of two measured fluorescence signals (called x and y) is used in different applications in fluorescence microscopy. Multiple instances of both signals can be combined in different ways to construct different ratio estimators. METHODS: The mean and variance of three estimators for the ratio between two random variables, x and y, are discussed. Given n samples of x and y, we can intuitively construct two different estimators: the mean of the ratio of each x and y and the ratio between the mean of x and the mean of y. The former is biased and the latter is only asymptotically unbiased. Using the statistical characteristics of this estimator, a third, unbiased estimator can be constructed. RESULTS: We tested the three estimators on simulated data, real world fluorescence test images, and comparative genome hybridization (CGH) data. The results on the simulated and real-world test images confirm the presented theory. The CGH experiments show that our new estimator performs better than the existing estimators. CONCLUSIONS: We have derived an unbiased ratio estimator that outperforms intuitive ratio estimators. PMID- 10738284 TI - A low-voltage droplet charging circuit with simulative cell-sorting function for flow cytometer-cell sorter. AB - BACKGROUND: Flow cytometer cell sorters have become important tools in many biological laboratories. Commercial electrically-deflected cell sorters that deflect wanted cells in electrically charged droplets need high-voltage amplifiers which are expensive and difficult to obtain. Effort was made to build an alternative droplet charging circuit with low-voltage amplifiers that are much easier to get and have more reasonable price. METHODS: A low-voltage charging circuit was designed. Every time a cell was to be separated, a pair of complementary charging pulses were produced: one was positive and the other was negative with equal amplitude. These were enlarged by two low-voltage charging amplifiers to drive two charging electrodes respectively. RESULTS: Due to the effect of addition, the voltage between the two electrodes was double as high as the output of either amplifier. The result of test experiment proved that the cell sorter with low-voltage amplifiers, which was cheaper and easier to obtain, could separate cells as efficiently as the instrument with high-voltage ones that were more expensive and more difficult to make. In addition, a simulative cell sorting function was provided. CONCLUSIONS: This low-voltage, easily-built and low-price charging circuit for flow cytometer cell sorter is a good alternative to the commonly used high-voltage one, especially to researcher who hopes to build his own personal instrument. PMID- 10738285 TI - Analysis of apoptosis of lymphoid cells in fish exposed to immunotoxic compounds. AB - BACKGROUND: Chemical induction of apoptosis in cells is believed to contribute to toxicity. Techniques for measuring apoptosis have increased in both sensitivity and number and in many cases can be readily extended to nontraditional research species. A comparison of established assays for measuring apoptosis of lymphoid cells has thus far not been performed in the fish and thus would be efficacious in assessing immunotoxicity. METHODS: The present study evaluated chemical induced immune cell apoptosis in fish (tilapia, Oreochromis niloticus) exposed to two known immunotoxic chemicals, azathioprine and T-2 toxin. Cytocentrifugation and light microscopy of leukocyte-enriched cell samples from the pronephros (i.e., the fish primary hematopoietic compartment) demonstrated chemical-related increases in apoptotic bodies. This observation was examined further with the ApoAlert Annexin V Apoptosis kit and two DNA-binding dyes employed for detecting apoptosis, 7-aminoactinomycin D (7-AAD) and propidium iodide (PI). RESULTS: The apoptotic probes confirmed the microscopic observations of increased apoptosis in the chemical-exposed fish. The ApoAlerttrade mark annexin V and 7-AAD assays, which discriminate early and late apoptosis/necrosis, compared well in identifying apoptotic populations. PI staining in Vindelov's solution was unable to detect early apoptosis. CONCLUSIONS: The present data suggest that apoptotic immune cells may be a useful marker for certain immunotoxicant exposures in fish. These findings agree with those of previous reports that fish may respond immunologically in a manner similar to mammals after immunotoxicant challenge. PMID- 10738286 TI - Effect of HeNe and pulsed Nd:YAG laser irradiation on intradental nerve responses to mechanical stimulation of dentine. AB - BACKGROUND AND OBJECTIVE: Our study aimed to determine how lasing affected intradental nerve responses to dentine stimulation. Study Design/Materials and Methods Intradental nerve activity was recorded from canine teeth of anaesthetised ferrets. Dentine exposed at the tip of the tooth was stimulated with a glass probe. After determining baseline responses to mechanical stimulation, dentine was lased using a pulsed Nd:YAG laser at 60-150mJ/pulse and 10-30 pulses/sec (total power = 0.3-3.0 W). RESULTS: The HeNe aiming beam alone and Nd:YAG laser at 0.3 W (+ HeNe) had no effect on intradental nerve responses to dentine stimulation. Lasing at 0.6-1. 5 W could either enhance or suppress intradental nerve responses. Lasing at more than or equal to 2.0 W or repeated lasing at lower intensities depressed intradental nerve responses. Lasing often induced intradental nerve firing. CONCLUSION: HeNe lasing had no effect on intradental nerve excitability. The Nd:YAG laser could depress intradental nerve responses to dentine stimulation. PMID- 10738287 TI - Morphologic changes correlating to different sensitivities of Escherichia coli and enterococcus faecalis to Nd:YAG laser irradiation through dentin. AB - BACKGROUND AND OBJECTIVE: Previous studies demonstrated the disinfecting potential of Nd:YAG laser irradiation on the root canal system from an overall quantitative viewpoint. The aim of this study was to evaluate the specific effect of irradiation through dentin on gram-negative and gram-positive bacteria with regard to their cell structure. STUDY DESIGN/MATERIALS AND METHODS: Sterile dentin samples of standardized size were divided into two sets of four groups with eight samples each. The first set was inoculated with Escherichia coli as the gram-negative test strain, the second set was inoculated with Enterococcus faecalis, which served as the gram-positive test organism. The samples were then irradiated on the bacteria-free side in contact mode under constant scanning movement at an angle of 10 degrees by use of the fiber optic of the Nd:YAG laser. Upon laser treatment they were critical point dried and subjected to SEM investigation. Another two sets of samples were prepared and irradiated in the same manner and evaluated by standard microbiological procedures to verify whether the observed morphologic alterations correlated to cell death. RESULTS: SEM investigations revealed damage pattens that increased with the amount of energy applied. Whereas the gram-negative test organism showed immediate structural injury, the gram-positive test organism required repeated application of irradiation. The microbiological examination showed reduction of both bacterial strains, yet to different extents. CONCLUSION: Our study demonstrates the different morphologic impact of Nd:YAG laser irradiation through dentin on representatives of the two main groups of bacteria. It shows that the construction of the cell wall is crucial for their individual sensitivity to laser treatment. PMID- 10738288 TI - Preliminary investigation of a novel carbon dioxide laser for applications in dentistry. AB - BACKGROUND AND OBJECTIVE: A novel pulsed CO(2) laser was examined for its ability to ablate hard dental tissues. STUDY DESIGN/MATERIALS AND METHODS: Lased human enamel surfaces were viewed using light and scanning electron microscopy for evidence of adverse structural changes. In vitro shear bond strength tests were conducted on composite resin bonded to lased enamel surfaces and compared with conventionally prepared specimens. A thermal camera was used to monitor temperature changes during cavity preparation in tooth slabs to assess likely changes to the dental pulp. RESULTS: No charring or surface cracks were observed on lased enamel surfaces using both microscopic techniques. Bonding of the lased enamel surfaces to composite resin was not significantly different from the acid etched control group. For cavities with a remaining dentine thickness of less than 1 mm, the temperature rise was less than 6 degrees C. CONCLUSION: A novel pulsed CO(2) laser shows promise for cutting cavities in teeth. PMID- 10738289 TI - Nd:YAG laser irradiation of the human dental pulp: implications as a predictor of pulp hemodynamics. AB - BACKGROUND AND OBJECTIVE: The aim of study this was to evaluate in vivo the pulpal blood flow rate, pulpal responsiveness, systemic blood pressure, and pulse rate during Nd:YAG laser irradiation of an isolated tooth. STUDY DESIGN/MATERIALS AND METHODS: Thirteen volunteers from the Tsurumi Dental University faculty participated in this study after giving their consent to the Nd:YAG laser irradiation test protocol. Gingivobuccal areas adjacent to mandibular canines were used for clinical evaluation. Each area was coated with India ink and treated with a Nd:YAG laser for 30 sec at 120-mJ pulses at 10 pulses/sec. Pulpal blood flow was measured by a laser Doppler flowmeter during Nd:YAG laser irradiation of the tooth. All pulp responses were measured by an electric pulp tester before and after Nd:YAG laser irradiation. In addition, the systemic blood pressure and pulse rate were monitored throughout the laser irradiation procedure. RESULTS: For all subjects involved in this study, the pulpal blood flow rate increased during laser irradiation. Threshold values of the electric pulp tests increased in six cases and decreased in six cases. One case showed no change. After 1 month, the threshold values for each subject had returned to previously recorded values. Neither systemic blood pressure nor pulse rate was affected during Nd:YAG laser irradiation. Pulpal blood flow was strongly influenced immediately after Nd:YAG laser irradiation, seen as an increase in the flow rate. CONCLUSION: The results of this study suggest that effects of the Nd:YAG laser irradiation are similar to those of low power laser for the improvement of local blood flow. PMID- 10738290 TI - Evaluation of dentin root canal permeability after instrumentation and Er:YAG laser application. AB - BACKGROUND AND OBJECTIVES: Smear layer removal with EDTA from root canal walls allows greater cleaning and disinfection of root canals. However, because Er:YAG laser acts on the removal of the smear layer, the objective of investigation was to analyze in vitro the effect of Er:YAG laser on dentin root canal wall permeability after endodontic instrumentation and irrigation with water or sodium hypochlorite and Er:YAG laser application. STUDY DESIGN/MATERIALS AND METHODS: A total of 25 extracted human maxillary incisors were divided into five groups: Group I, instrumentation with deionized distilled water as the irrigating solution; Group II, instrumentation with 1% sodium hypochlorite as the irrigating solution; Group III, instrumentation with deionized distilled water as the irrigating solution and Er:YAG laser application; Group IV, instrumentation with 1% sodium hypochlorite solution as the irrigating solution and Er:YAG laser application; Group V, instrumentation only up to #20 file with deionized distilled water as the irrigating solution and Er:YAG laser irradiation. The laser parameters were 15 Hz, 140 mJ, total energy 42 J, 300 pulses (Kavo Key Laser). Copper sulfate (10%) was used to evaluate dentin permeability. The penetration of copper ions into the dentinal tubules was observed using 1% rubeanic acid, which reveals copper ions, forming a stained compound ranging in color from deep blue to black. Transverse sections (500-microm thick) were obtained with a diamond disk from the cervical, middle, and apical thirds. RESULTS: The instrumentation of the root canal that used water as the irrigating solution followed by Er:YAG laser irradiation promoted the greatest increase in dentin permeability. The use of Er:YAG laser, 1% sodium hypochlorite + Er:YAG, and 1% sodium hypochlorite used alone showed an intermediate capacity of increasing dentin permeability. The use of water as the irrigating solution without Er:YAG laser promoted the least dentin permeability. CONCLUSIONS: The use of water as the irrigating solution after instrumentation and Er:YAG laser irradiation was an effective procedure for increasing dentin permeability. PMID- 10738291 TI - Effects of low-energy laser irradiation on bone remodeling during experimental tooth movement in rats. AB - BACKGROUND AND OBJECTIVE: Low-energy laser irradiation has many anabolic effects such as the acceleration of bone formation. However, its effects on tooth movement, performed by bone resorption and formation, have not been well characterized. STUDY DESIGN/MATERIALS AND METHODS: A total of 10 g of orthodontic force was applied to rat molars to cause experimental tooth movement. A Ga-Al-As diode laser was used to irradiate the area around the moved tooth, and after 12 days, the amount of tooth movement was measured. Calcein was injected subcutaneously to label the newly formed alveolar bone for quantitative analysis. Immunohistochemical staining of proliferating cell nuclear antigen was performed to evaluate cellular proliferation. TRAPase staining was also performed to facilitate the identification of osteoclasts. RESULTS: In the laser irradiation group, the amount of tooth movement was significantly greater (1. 3-fold) than that of the nonirradiation group in the end of the experiment. The amount of bone formation and rate of cellular proliferation in the tension side and the number of osteoclasts in the pressure side were all significantly increased in the irradiation group when compared with the nonirradiation group (P < 0. 01). CONCLUSION: These findings suggest that low-energy laser irradiation can accelerate tooth movement accompanied with alveolar bone remodeling. PMID- 10738292 TI - Comparison between isotropic and nonisotropic dosimetry systems during intraperitoneal photodynamic therapy. AB - BACKGROUND AND OBJECTIVE: On-line monitoring of light fluence during intraperitoneal photodynamic therapy (IP PDT) is crucial for safe light delivery. A flat photodiode-based dosimetry system is compared with an isotropic detector based system in patients undergoing IP PDT. STUDY DESIGN/MATERIALS AND METHODS: Flat photodiodes and spherical detectors were placed side by side in the abdomen, for simultaneous light dosimetry in 19 patients. Tissue phantom experiments were performed to provide a preliminary estimate of the tissue optical properties of the peritoneum. RESULTS: The conversion factor between systems for 630-nm light was found to be 1.7 +/- 0.12. The mu(eff) of the tissues in the abdomen is estimated to vary between 0.5 cm(-1) to 1.4 cm(-1) assuming a mu(s)' = 7 cm(-1). CONCLUSIONS: The measured conversion factor should allow for comparison of light fluences with future clinical protocols that use an isotropic-based detector system. Differences in the optical properties of the underlying tissues may contribute to the variability in light measurements. PMID- 10738293 TI - Local 5-aminolevulinic acid application for laser light-induced fluorescence diagnosis of early staged colon cancer in rats. AB - BACKGROUND AND OBJECTIVES: 5-Aminolevulinic acid (ALA) increases the intracellular accumulation of endogenous protoporphyrin IX in colon cancer. Protoporphyrin IX itself is a potential photosensitizer that can be used for laser light-induced fluorescence diagnosis. The aim of this study was to detect cancer in the rat colon before macroscopic visibility. STUDY DESIGN/MATERIALS AND METHODS: Multifocal colon carcinomas were induced by weekly subcutaneous injections of 1,2-dimethylhydrazin-dihydrochloride in male Wistar rats. Local photosensitization was performed with an ALA colon lavage. Red fluorescence (635 nm) was induced by green laser-light irradiation with an Ar-Dye Laser (514 nm) in the colon. Fluorescence was observed by the naked eye with a filter at < 515 nm to eliminate the excitation light. RESULTS: Twenty-five Wistar rats developed 99 macroscopically visible carcinomas and four macroscopically visible dysplasias. The following laser-light-induced fluorescence diagnosis procedure was able to detect 16 additional carcinomas and 41 additional dysplasias. CONCLUSIONS: Local ALA application induces a tumor-specific protoporphyrin IX accumulation in the rat colon and is an efficient method for fluorescence detection of invisible dysplasias and early colon cancer in the rat. PMID- 10738294 TI - Hyperbaric oxygen and photodynamic therapy in the treatment of advanced carcinoma of the cardia and the esophagus. AB - BACKGROUND AND OBJECTIVE: The photochemical reaction of photodynamic therapy (PDT) depends on the presence of molecular oxygen. Because of anoxic regions in tumor tissue and vascular shutdown during PDT, the efficiency is limited. Therefore, the use of hyperbaric oxygen, which increases the oxygen in tumor tissue, as well as the amount of singlet oxygen, may enhance the efficiency of PDT. STUDY DESIGN/MATERIALS AND METHODS: After diagnostic work-up, photosensitization was carried out with a hematoporphyrin-derivate 2 mg/kg body weight 48 hours before PDT. The light dose was calculated as 300 J/cm of fiber tip. Twenty-three patients were treated by PDT alone and 29 patients received PDT under hyperbaric oxygen at a level of two absolute atmospheric pressures. RESULTS: Improvement regarding dysphagia and stenosis-diameter could be obtained in both treatment arms with no significant difference (P = 0.43 and P = 0. 065, respectively). The tumor length also decreased in both groups and showed a significant difference in favour of the PDT/HBO group (P = 0.002). The mean overall survival was 11.3 months. The mean survival time for the PDT group was 8.7 months and for the PDT/HBO group 13.8 months (P = 0.021). CONCLUSION: According to this pilot study, combined PDT/HBO represents a new approach in the treatment of esophageal and cardia cancer, which appears to have enhanced the efficiency of PDT. PMID- 10738295 TI - Preferential localization of varying forms of photoactive 1, 8-naphthalimide compounds within the atheromatous arterial wall. AB - BACKGROUND AND OBJECTIVE: We are currently working with a novel class of photoactivated 4-amino substituted 1,8-naphthalimide compounds for tissue bonding. With promising results in other tissues, we are pursuing potential vascular applications. This study focused on determining the appropriate compound formulation(s), concentration, and exposure times to optimize penetration of the heterogeneous arterial wall. STUDY DESIGN/MATERIALS AND METHODS: Segments of atheromatous rabbit carotid artery were immersed in hydrophilic or lipophilic forms of the compound, then frozen, cryosectioned, and examined by confocal microscopy. RESULTS: The hydrophilic compound exhibited preferential localization within the intima and media and limited presence in the adventitia. Conversely, the lipophilic compound concentrated in the intima and adventitia with virtual exclusion from the media. Exposure to both forms resulted in complete penetration of the arterial wall. CONCLUSION: These results extend our knowledge and permit a more practical approach to potential vascular applications using these photoactivated compounds for tissue bonding. PMID- 10738296 TI - Methylene blue based protein solder for vascular anastomoses: an in vitro burst pressure study. AB - BACKGROUND AND OBJECTIVE: Attempts at sutureless anastomoses have used protein based solders containing chromophores [Oz et al., J Vasc Surg 1990;11:718; Poppas et al., J Urol 1998150:1052] to enhance the strength of laser anastomoses. Reports have described the use of indocyanine green [Oz et al., Surg Forum 1989;316.], fuschin, and fluorescein isothiocyanate as chromophores [Chuck et al. , Lasers Surg Med 1989;9:471; Vance et al., Lasers Med Sci 1988;3:219]. Methylene blue (MB) is a chromophore with absorption peaks in the 600-700 nm region whose use has not been reported in laser-assisted vascular anastomoses. Therefore, we set out to produce and characterise a MB-containing protein solder. The absorption and burst pressure characteristics have been investigated and described as well as a brief review of the chemical and biological properties of MB. STUDY DESIGN/MATERIALS AND METHODS: The MB and porcine serum albumin (PSA) based solder was produced and used to form end-to-end anastomoses in porcine splenic arteries. The solder was activated using a laser diode emitting at 670 nm. The burst pressures of the anastomoses were tested, and the results analysed as a function of MB concentration and absorption. In addition, the relationship between MB concentration and absorption was examined. RESULTS: A dose-response relationship was found between the measured absorption of the solder and the burst pressure of the anastomoses formed. Burst pressures exceeding physiological levels were found. Changes in MB concentration revealed a marked negative deviation from Beer's law at 670 nm, owing to the monomer-dimer-trimer equilibria. CONCLUSION: PSA with MB solder is able to form high-quality end-to end anastomoses, with immediate burst pressure profiles similar to those previously described for sutured [Quigley et al., Microsurgery 1985;6:229], lasered [Quigley et al., Microsurgery 1985;6:229], and soldered anastomoses [Small et al., J Clin Laser Med Surg 1997;15:205]. The relationship between burst pressure strength and chromophore absorption is discussed. PMID- 10738297 TI - Genetics of Beckwith-Wiedemann syndrome-associated tumors: common genetic pathways. AB - A specific subset of solid childhood tumors-Wilms' tumor, adrenocortical carcinoma, rhabdomyosarcoma, and hepatoblastoma-is characterized by its association with Beckwith-Wiedemann syndrome. Genetic abnormalities found in these tumors affect the same chromosome region (11p15), which has been implicated in the etiology of Beckwith-Wiedemann syndrome. This suggests that the development of these tumors occurs along a common genetic pathway involving chromosome 11. To search for additional common genetic pathways, this article reviews the genetic data published for these tumors. It was found that, up until now, the only genetic abnormalities detected in all four tumors affect chromosome band 11p15 and the TP53 gene. In addition, there are several aberrations that occur in two or three of the neoplasms. It is concluded that, of the four tumors, the genetic relationship is most evident between Wilms' tumor and rhabdomyosarcoma. PMID- 10738298 TI - A DNA probe combination for improved detection of MLL/11q23 breakpoints by double color interphase-FISH in acute leukemias. AB - Reciprocal translocations involving the MLL gene on chromosome band 11q23 have been observed in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). In AML, identification of MLL breakpoints is an important prognostic factor. Breakpoints are clustered in an 8 kb DNA fragment (bcr) and can be detected by Southern blotting or fluorescence in situ hybridization (FISH) analysis. Our objective in this study was to design a DNA probe set that enables optimal detection of MLL rearrangements using interphase FISH. Two PAC clones, 217A21 and 167K13, spanning the MLL gene with a minimal overlap in the bcr were isolated and labeled. Twenty-seven AML/ALL patients with cytogenetic 11q23 abnormalities, seven AML/ALL patients without 11q23 abnormalities but MLL rearrangement by Southern blotting, and eight healthy donors were analyzed by FISH. We compared this double-color FISH analysis with FISH using a YAC clone (yB22B2) and with Southern blotting. The PAC probe combination detects an MLL breakpoint in all cases with MLL rearrangement detected by Southern blotting except for cases with a partial tandem duplication detected by reverse transcriptase-polymerase chain reaction (RT-PCR). FISH using the PAC probes also detected MLL breakpoints in four cases with MLL deletions telomeric to the breakpoint that could not be detected by the single probe yB22B2. This new probe set provides a reliable and rapid assay for the diagnosis of AML and ALL patients with MLL/11q23 breakpoints. PMID- 10738299 TI - Presence and expression of the simian virus-40 genome in human giant cell tumors of bone. AB - SV40 DNA sequences have been found in human tumors, such as mesotheliomas, ependymomas, and bone tumors, suggesting that SV40 may be involved in their etiology. The FOS oncogene could play an important role in bone development because SV40 is able to induce FOS in cell culture. In this study, the presence of SV40 sequences, large T antigen (Tag), and FOS protein expression were investigated in 120 giant cell tumors (GCTs), moderately benign bone tumors that in some cases can progress to a malignant phenotype. Polymerase chain reaction (PCR), using primers that amplify the RB1 pocket binding domain and the intron of Tag, was used to analyze GCT for the presence of SV40 DNA. Tag and FOS protein expression was evaluated by immunohistochemistry. SV40 sequences were found in 30/107 GCTs, and of these, 22/30 samples expressed Tag protein (73%) and 15/30 overexpressed the FOS oncogene (50%). FOS was undetectable in 77 SV40-negative GCTs. Sequence analysis of the amplified DNAs confirmed that the amplified sequences corresponded to SV40 DNA. The correlation between FOS overexpression and SV40-positive GCTs was highly statistically significant (P < 0.001). These results show that SV40 DNA sequences and SV40 Tag are present in GCTs and might induce FOS activity. These data suggest that SV40 might play a role in the development and progression of some GCTs. PMID- 10738300 TI - Germline INI1 mutation in a patient with a central nervous system atypical teratoid tumor and renal rhabdoid tumor. AB - We describe a four-month-old child who presented with an atypical teratoid/rhabdoid tumor of the brain and subsequently developed a renal rhabdoid tumor. Distinct histologic features, immunophenotypic profiles, and deletions of chromosome 22 were supportive of two primary tumors. An identical mutation in exon 7 of the INI1 rhabdoid tumor suppressor gene was identified in both tumors, as well as in normal kidney tissue. We propose that this germline INI1 mutation predisposed the child to the development of both malignancies. These findings lend support to the hypothesis that rhabdoid tumors in all sites have a common genetic etiology. PMID- 10738301 TI - Identification of a 1-Mb common region at 16q24.1-24.2 deleted in hepatocellular carcinoma. AB - To identify the location of one or more putative tumor suppressor genes that may be involved in hepatocellular carcinoma (HCC), we examined 96 such tumors for their patterns of allelic loss at 21 microsatellite marker loci distributed along chromosome arm 16q. Allelic loss at one or more loci was observed in 58 (60%) of these tumors. Detailed deletion mapping identified a distinct commonly deleted region located within an interval flanked by D16S534 and D16S3091 at 16q24.1 24.2. By constructing a physical map consisting of a YAC contig across the region, the extent of the deleted region was determined to be less than 1 Mb. Among the tumors for which clinical data were available, allelic loss at 16q24.1 24.2 was more frequent in tumors arising from liver cirrhosis compared to HCCs arising from chronic hepatitis (30/42, 71%, vs. 13/33, 39%; P = 0. 0054). Additionally, allelic loss at 16q24.1-24.2 was frequently observed in small tumors and early-stage tumors as well as in tumors of more advanced phenotype. PMID- 10738302 TI - Mutation screening of the CDKN2A promoter in melanoma families. AB - Germline mutations of CDKN2A, at 9p21, are responsible for predisposition to melanoma in some families. However, evidence of linkage to 9p21 has been demonstrated in a significant proportion of kindreds with no detectable mutations in CDKN2A. It is possible that mutations in noncoding regions may be responsible for predisposition to melanoma in these families. We have analyzed approximately 1 kb of the CDKN2A promoter upstream of the start codon in an attempt to identify causal mutations in 107 melanoma families. Four sequence variants were detected. Two of these (A-191G and A-493T) did not segregate with disease and were present in a control population at a comparable frequency, indicating that they are unlikely to predispose to melanoma. The A-493T variant appeared to be in linkage disequilibrium with the previously described CDKN2A polymorphism Ala148Thr. The variant G-735A was detected in the control population, but segregation of this variant with melanoma within families could not be discounted. The fourth variant (G-34T), located in the 5' UTR, creates an aberrant initiation codon. This variant appeared to segregate with melanoma and was not detected in a control population. G-34T has recently been identified in a subset of Canadian melanoma families and was concluded to be associated with predisposition to melanoma. The creation of an aberrant initiation site in the 5' UTR may have an important role in carcinogenesis in a small percentage of families; however, mutations in the CDKN2A promoter appear to have a limited role in predisposition to melanoma. PMID- 10738303 TI - MEN1 gene mutation analysis of high-grade neuroendocrine lung carcinoma. AB - Neuroendocrine tumors of the lung consist of a spectrum of neoplasms, including typical carcinoids, atypical carcinoids, large-cell neuroendocrine carcinomas (LCNEC), and small-cell lung carcinomas (SCLC). We previously reported frequent inactivation of the gene responsible for multiple endocrine neoplasia type 1 (MEN1) in both typical and atypical carcinoid tumors. In the present study, we extend the analysis of human NE lung tumors to include 9 primary SCLCs, 36 SCLC cell lines, and 13 primary LCNECs for MEN1 gene inactivation. In SCLC, loss of heterozygosity (LOH) at the MEN1 gene on chromosome band 11q13 was detected in one primary tumor and two cell lines. The coding sequence and splice junctions of the MEN1 gene were screened for mutations in all 44 tumors and cell lines, and no mutations were detected. Northern blot analysis of 13 SCLC cell lines showed the MEN1 transcript to be present and of normal size. In LCNECs, a somatic frameshift in the MEN1 gene (1226delC) was found in one of 13 tumors, representing the first mutation observed outside the spectrum of neoplasms associated with MEN1. Interestingly, neither a deletion nor a mutation was detected in the other allele, and wild-type mRNA sequence was expressed in the tumor, suggesting that the MEN1 gene was not inactivated by a conventional two-hit mechanism. The data support the hypothesis that SCLC and lung carcinoids develop via distinct molecular pathways; however, further investigation is necessary to determine the significance of the MEN1 gene mutation observed in a single case of LCNEC. Published 2000 Wiley-Liss, Inc. PMID- 10738304 TI - Nonrandom pattern of cytogenetic abnormalities in squamous cell carcinoma of the larynx. AB - Cytogenetic analysis of short-term cultures from 105 squamous cell carcinomas of the larynx (LSCC) revealed clonal chromosome aberrations in 56 tumors. Simple karyotypic changes (less than four aberrations per clone) were found in 24 cases, and the remaining 32 tumors had complex karyotypes with multiple numerical as well as unbalanced structural rearrangements. Extensive intratumor heterogeneity, in the form of multiple related subclones or unrelated clones, was observed in a large fraction of the tumors. The structural changes most often affected chromosomes 3, 1, 11, 7, 2, 15, 5, 4, 8, and 12, with rearrangements in the centromeric regions, i.e., the centromeric bands p10 and q10 and the juxtacentromeric bands p11 and q11, accounting for 43% of the total breakpoints. The most common imbalances brought about by numerical and unbalanced structural rearrangements were loss of chromosomal region 3p21-pter, chromosome arms 4p, 6q, 8p, 10p, 13p, 14p, 15p, and 17p, and gain of chromosomal regions 3q21-qter, 7q31 pter, and 8q. Among 17 recurrent aberrations identified, the most common were i(8q), hsr(11)(q13), i(3q), i(5p), and del(3)(p11). No statistically significant association was found between major karyotypic features and histological differentiation or TNM stage. The karyotypic features of the LSCC were also compared with previously published oral SCC, a subgroup of SCC that has been more extensively characterized cytogenetically. No clear-cut karyotypic differences were found between LSCC and oral SCC, with the exception that i(8q) was significantly more frequent among the latter. PMID- 10738305 TI - Evidence that haploinsufficiency of Ptch leads to medulloblastoma in mice. AB - The PTCH gene encodes a putative tumor suppressor protein; germline alterations in PTCH have been found in patients with the nevoid basal cell carcinoma syndrome (NBCCS). Medulloblastoma, a brain tumor, develops in about 3% of NBCCS patients, and mutations in PTCH have also been described in a subset of sporadic medulloblastomas. The search for the causes of medulloblastoma has been hindered by the lack of an appropriate model system for this tumor type. Recently, a transgenic mouse hemizygous for the Ptch gene was generated by homologous recombination. Medulloblastomas were found in about 19% of these mice within the first 25 weeks after birth. The status of the wild-type PTCH allele in these tumors has not been investigated. For clearer definition of the role of PTCH as a tumor suppressor in medulloblastoma, 13 cerebellar tumors from transgenic Ptch(+/ ) mice were examined for alterations in the remaining Ptch allele. A single mutation was found in one tumor, a C-to-A substitution changing a tyrosine to a stop codon; all other tumors exhibited a wild-type sequence. Two tumors with normal Ptch cDNA were examined by in situ hybridization. Ptch cDNA was found in tumor cells but not in associated tumor stroma. We also examined the mRNA expression levels for the remaining Ptch allele, as well as for Gli1, a gene known to be transcriptionally activated by Ptch inactivation. Blot analysis of RNA from the 13 tumors shows that Ptch mRNA of appropriate size is expressed in all tumors at varying levels. Expression of Gli1 was increased in tumors compared to normal cerebellum. These results suggest that deletion of one copy of Ptch may be sufficient to promote medulloblastoma development in mice. PMID- 10738306 TI - A functional investigation of tumor suppressor gene activities in a nasopharyngeal carcinoma cell line HONE1 using a monochromosome transfer approach. AB - Monochromosome transfers of selected chromosomes into a nasopharyngeal carcinoma (NPC) cell line were performed to determine if tumor suppressing activity for NPC mapped to chromosomes 9, 11, and 17. Current information from cytogenetic and molecular allelotyping studies indicate that these chromosomes may harbor potential tumor suppressor genes vital to NPC. The present results show the importance of CDKN2A on chromosome 9 in NPC development. There was no functional suppression of tumor development in nude mice with microcell hybrids harboring the newly transferred chromosome 9 containing an interstitial deletion at 9p21, whereas transfection of CDKN2A into the NPC HONE1 cells resulted in obvious growth suppression. Whereas intact chromosome 17 transfers into HONE1 cells showed no functional suppression of tumor formation, chromosome 11 was able to do so. Molecular analysis of chromosome 11 tumor segregants indicated that at least two tumor suppressive regions mapping to 11q13 and 11q22-23 may be critical for the development of NPC. PMID- 10738308 TI - Chromosome-specific telomeric associations in Chinese hamster embryonic cells. AB - Telomeric associations (TAs) represent an important cytogenetic marker of human tumor cells. It has been thought that the primary cause of TAs is telomere shortening. However, we report here a surprising aspect of telomere maintenance in primary Chinese hamster embryonic (CHE) cells: relatively high frequencies of TAs in spite of normal telomere length. These TAs are present in both interphase and metaphase cells, suggesting that metaphase TAs may be relics of interphase chromosome organization. In addition, some TAs observed here are chromosome specific and recurrent in at least three consecutive cell cycles in two different CHE cell strains. In spite of relatively high frequencies of TAs, none of the CHE strains show chromosome instability resulting from breakage-fusion-bridge cycles, as would be expected from tumor cell studies. It appears that TAs in CHE cells may be reversible events. These results are discussed in light of current understanding of telomere biology. PMID- 10738307 TI - Simultaneous molecular karyotyping and mapping of viral DNA integration sites by 25-color COBRA-FISH. AB - Combined binary ratio labeling (COBRA) fluorescence in situ hybridization (FISH) allows 24-color FISH karyotyping of human metaphase chromosomes utilizing only four fluorochromes, instead of the five required for combinatorial labeling procedures. Here we show that by introduction of a fifth fluorochrome, COBRA-FISH permits molecular cytogenetic mapping of viral integration sites in complex karyotypes in the context of a 24-color hybridization. We were able to detect a single copy of the human papillomavirus 16 in the SiHa cell line and to confirm the site of integration at 13q21-31. We also demonstrate the gene mapping possibility of 25-color hybridization by detecting a MYC cosmid on normal metaphase chromosomes. The possibility of mapping single-copy probes in the background of 24-color hybridization expands the tools for cytogenetic mapping of DNA sequences and will contribute to the understanding of the role of viral integration and chromosome rearrangement in virus-mediated carcinogenesis. PMID- 10738309 TI - Chromosome abnormalities in ovarian adenocarcinoma: III. Using breakpoint data to infer and test mathematical models for oncogenesis. AB - Cancer geneticists seek to identify genetic changes in tumor cells and to relate the genetic changes to tumor development. Because single changes can disrupt the cell cycle and promote other genetic changes, it is extremely hard to distinguish cause from effect. In this article we illustrate how 7 techniques from statistics, theoretical computer science, and phylogenetics can be used to infer and test possible models of tumor progression from single genome-wide descriptions of aberrations in a large sample of tumors. Specifically, we propose 4 tree models for tumor progression inferred from the large ovarian cancer data set described in the first 2 articles in this series. The models are derived from 2 different methods to select the non-random genetic aberrations and 2 different methods to infer the trees, given a set of events. Various aspects of the tree models are tested and extended by 5 methods: overall tests of independence, likelihood ratio tests, principal components analysis, directed acyclic graph modeling, and Bayesian survival analysis. All our methods lead to strikingly consistent conclusions about chromosomal breakpoints in ovarian adenocarcinoma, including (1) the non-random breakpoints in ovarian adenocarcinoma do not occur independently; (2) breakpoints in regions 1p3 and 11p1 are important early events and distinguish a class of tumors associated with poor prognosis; and (3) breakpoints in 1p1, 3p1, and 1q2 distinguish a class of ovarian tumors, and the breaks at 1p1 and 3p1 are associated with poor prognosis. PMID- 10738310 TI - Characterization of chromosome 1 abnormalities in malignant melanomas. AB - Chromosome 1 abnormalities are the most commonly detected aberrations in many cancers including malignant melanomas. Specific breakpoints are reported for malignant melanomas throughout the chromosome but especially at 1p36 and at several sites throughout 1p22-q21. In addition, partial deletions and loss of heterozygosity have been found on 1p indicating the possible location of tumor suppressor genes. Here we have characterized the involvement of chromosome 1 in a series of seven malignant melanoma cell lines. Initial chromosome painting studies revealed that six of the cell lines had chromosome 1 rearrangements. Deletions involving 1p10-32, 1q11-44, and 1q25-44 were observed. The other rearrangement breakpoints included three in the 1q10-p11 region with the rest at 1p36, 1p34, 1p32, 1p31, 1p12-13, 1q21, and 1q23. The breaks at 1q10-p11 were investigated further using an alpha-satellite 1 centromere probe and yeast artificial chromosomes (YACs) from the region. Two of the 1q10-p11 breaks mapped in the centromeric region, while the others mapped to variable sites. This suggests that the role of these rearrangements in the pathogenesis of melanomas does not involve the alteration of specific oncogenes in the breakpoint region. During the YAC mapping a previously undetected, small (<1 Mbp) del(1)(p10p11) was identified. This deletion lies within minimal overlapping deleted regions reported in head and neck as well as breast carcinomas and it could therefore facilitate the isolation of a carcinoma-associated tumor suppressor gene. PMID- 10738311 TI - Human E2F5 gene is oncogenic in primary rodent cells and is amplified in human breast tumors. AB - E2F transcription factors (E2F1 to 6) are central players in the control of animal cell proliferation as regulators of genes involved in cell cycle progression and in transformation. In this report, we have investigated the potential involvement of the E2F5 gene in tumorigenesis. We show that E2F5 can promote the formation of morphologically transformed foci in primary baby rat kidney cells (BRK) when it is overexpressed in the presence of its heterodimeric partner DP1 and activated RAS. This suggests that E2F5 behaves like a MYC-type cooperating oncogene in functional assays, prompting us to monitor potential amplifications of the E2F5 gene in primary human tumors. We mapped the human E2F5 gene to 8q21.1-21.3 equidistant from the MOS (8q12) and MYC (8q24) oncogenes. Since the long arm of chromosome 8 is frequently the site of increased gene copy number (ICN) in breast cancer, we screened 442 breast tumor DNAs for gains of E2F5, MOS, and MYC genes. The three genes showed ICN, albeit at variable incidence and levels of amplification, with the ICN of E2F5 occurring concomitantly with those of MOS and/or MYC in almost half of the cases. Moreover, a marked increase of the 2. 5-kb E2F5 transcript was also detected in some tumors and tumor cell lines. In conclusion, the evidence that sustained unregulated expression of E2F5 can cooperate with other oncogenes to promote cell transformation in functional assays, together with the detection of chromosomal amplifications and overexpressions of the E2F5 gene in breast tumors, provides the first indications that E2F5 deregulation may have a role in human tumor development. PMID- 10738312 TI - Role of Helicobacter pylori gastritis in gastric atrophy, intestinal metaplasia, and gastric neoplasia. AB - Helicobacter pylori is the major cause of chronic gastritis worldwide. With an estimated rate of infection of over one half of the world's population, it is responsible for extensive morbidity and mortality. Infection with this organism does not appear to spontaneously resolve. Instead it reaches a chronic stage from which a number of outcomes are possible. This article reviews those outcomes that have been linked to H. pylori and explores the pathogenesis while attempting to resolve the discrepant paths infection can take. The associations include duodenal and gastric ulcers and the majority of gastric lymphomas of B-cell type derived from the mucosa-associated lymphoid tissue (MALT). Chronic gastritis has also been shown to evolve into atrophy with intestinal metaplasia in certain populations. This change in the gastric epithelium has been linked with an increased risk of gastric adenocarcinoma. Microsc. Res. Tech. 48:313-320, 2000. Published 2000 Wiley-Liss, Inc. PMID- 10738313 TI - Role of antigastric autoantibodies in chronic Helicobacter pylori infection. AB - In recent studies a significant association between H. pylori infection and antigastric autoimmunity has been reported. Antigastric autoantibodies can be found in more than 30% of infected patients. Two distinct binding patterns of these autoantibodies have been described, first at the luminal membrane of the foveolar epithelium, and second at the canaliculi membranes of the parietal cells in the body mucosa. The latter type of autoantibodies correlates with histologic and clinical parameters of gastric mucosa atrophy. The gastric H,K-ATPase, which is known to be the autoimmune target in classical autoimmune gastritis, also represents a major autoantigen in atrophic H. pylori gastritis. Molecular mimicry between H. pylori and the host does not seem to be responsible for the generation of this type of autoreactivity. The development of antigastric autoantibodies may be a relevant host factor which contributes to the final clinical outcome of chronic H. pylori gastritis. PMID- 10738314 TI - Histomorphological characteristics of gastric mucosa in patients with Zollinger Ellison syndrome or autoimmune gastric atrophy: role of gastrin and atrophying gastritis. AB - The role of gastrin in the pathophysiology of two diseases affecting the human stomach, the Zollinger Ellison syndrome (ZES) and the pernicious anemia (PA), is reviewed. Both diseases present chronic hypergastrinemia but from different origins. The ZES is characterized by the occurrence of ectopic endocrine gastrin secreting tumors and PA by a fundic atrophic gastritis leading to complete atrophy of fundus and resulting in achlorhydria. In PA, the lack of acid induces continuous gastrin cell activation and is responsible for the subsequent gastrin hypersynthesis and secretion. In ZES, hypergastrinemia causes hypertrophy of the oxyntic mucosa, which, in addition, displays hyperplasia of parietal and mucus cells. In both diseases, hypergastrinemia also induces the hyperproliferation of enterochromaffin-like endocrine cells in the fundic mucosa, which can offer all aspects from hyperplasia, then dysplasia, until true carcinoid tumor. The influence of antisecretory treatments and MEN 1 in the ZES as well as that of several other factors and antrectomy in PA on the behavior of the different gastric cells is evoked. Finally, the role that gastrin and its receptor play in the maintenance of the normal development of gastric mucosa and gastric acid secretion is emphasized by results observed in gene knockout models. PMID- 10738315 TI - Morphological, molecular, and prognostic aspects of gastric endocrine tumors. AB - This paper aims at describing the neuroendocrine cell growths of the gastric mucosa and their pathogenesis. In the corpus-fundus mucosa, gastric neuroendocrine nontumor growths are mostly represented by hyperplastic and, more rarely, dysplastic enterochromaffin-like (ECL) cell changes, while hyperplasia of gastrin-producing (G) cells and, rarely, of somatostatin-producing (D) cells are reported in the antral mucosa. The large majority of gastric neuroendocrine tumors is made by benign, gastrin-dependent, well-differentiated ECL cell growths arising in a background of chronic atrophic gastritis (type I) or, more rarely, associated with type I multiple endocrine neoplasia (MEN I) and Zollinger-Ellison (ZE) syndromes (type II). Rare, aggressive, frequently metastatic, well differentiated gastric neuroendocrine tumors are gastrin-independent and arise as sporadic lesions in the absence of specific gastric pathology (type III). Poorly differentiated neuroendocrine carcinomas (PDEC) are rare, highly aggressive carcinomas. A central role for gastrin is postulated in the pathogenesis of well differentiated type I and II ECL cell tumors with different possible genetic mechanisms. A more complex genetic background, independent of gastrin and possibly implicating altered function or mutation of p53 and other genes is highly suspected for the development of aggressive type III ECL cell carcinomas and PDECs. PMID- 10738316 TI - Gastric MALT lymphoma and its relationship to Helicobacter pylori infection: management and pathogenesis of the disease. AB - Chronic Helicobacter pylori infection is strongly associated with the development of primary gastric B cell lymphoma of MALT-type. Therapeutic decisions in primary gastric MALT lymphomas, e.g., the choice for gastric surgery or stomach conserving treatments in the form of radio-, chemo-, or eradication therapy, should be based on an accurate histopathological diagnosis, grading and clinical staging. Primary gastric low-grade MALT-NHLs in an early clinical stage associated with H. pylori infection were shown to respond with complete remission in approximately 77% of cases upon successful cure of the infection as only treatment modality. The effect of curing H. pylori infection on the course of a high-grade gastric MALT lymphoma is largely uncertain but preliminary results indicate a possible benefit for patients with high-grade MALT lymphoma upon eradication therapy. Concerning the pathogenetic mechanisms of lymphomagenesis, there are many questions to be addressed in the near future. In general, it is still unclear what the exact mechanisms are which lead to the malignant transformation of a reactive infiltrate. Is there a molecular-genetic or immunological point of no return? What is the biological significance of the immunoglobulin rearrangement detected with PCR? The wave of new data each year about the role of H. pylori in gastric MALT lymphoma might help that many of these questions addressed above might be answered within the next years. PMID- 10738317 TI - Morphological changes of the human gastric mucosa under long-term proton pump inhibitor therapy and their clinical relevance. AB - Proton pump inhibitors are potent drugs for the treatment of acid-related diseases. The moderate hypergastrinaemia observed during therapy is a physiological response to low intragastric pH and the increase is limited to the first months of therapy with no further changes thereafter. Reports on endocrine cell changes in the antral mucosa under chronic PPI therapy are controversial and lack clinical relevance. In contrast, in the oxyntic mucosa hyperplastic argyrophil cell changes have been reported, dependent on the degree and duration of hypergastrinaemia, the severity of oxyntic mucosal gastritis, especially atrophy, and the presence of H. pylori infection. Current data do not support a progression from hyperplastic to dysplastic argyrophil cell lesions in humans in the absence of additional genetic factors. Data on the progression of oxyntic gastritis under chronic PPI treatment in comparison to untreated controls could not be confirmed in more recent studies including a well-matched control population. The main factor for gastritis progression is the presence of Helicobacter pylori infection. The bacterium not only causes a chronic inflammation of the gastric mucosa, resulting in atrophy and intestinal metaplasia, but also influences endocrine cell populations involved in the regulation of gastric acid secretion. The clinical benefit of H. pylori eradication in reflux esophagitis patients is still a matter of debate. The complex relations in humans between hypergastrinaemia, (oxyntic) gastritis and atrophy, H. pylori infection, argyrophil cell hyperplasia, and the effects of long-term PPI treatment of acid-related diseases do not allow a quantification of the contribution of each single factor for the observed changes. PMID- 10738318 TI - Three-dimensional graphic reconstruction of the insect exoskeleton through confocal imaging of endogenous fluorescence. AB - The exoskeleton of the cockroach leg was imaged via confocal microscopy to generate digital graphic reconstructions of its three-dimensional structure. The cuticle is autofluorescent and can be visualized without staining, but is maximally imaged in aldehyde-fixed preparations viewed under krypton-argon laser illumination (yellow green (568 nm) excitation, commonly used in confocal microscopes). Images of the entire trochanteral segment of the leg were constructed as montages from optical sections taken as overlapping series that were coincident in the z-axis. Reconstructions of the exoskeleton from these images showed that strain sensing mechanoreceptors are located in association with buttresses and thickenings that form a consistent internal architecture in both juvenile and adult animals. Accuracy of reconstructions was gauged by embedding specimens in Spurr's resin and histologically sectioning them perpendicular to the optical plane of section (z-axis). Comparison of plastic sections with two-dimensional images generated by "resectioning" the software model showed that reconstructed exoskeleton had a high level of accuracy. Imaging of older and larger animals was limited by the sclerotization and increased thickness of the cuticle. Surface extraction algorithms were used to generate vector graphic files in CAD format for export to software used in engineering and design. Among other potential uses, these models have been studied by Finite Element Analysis to examine the distribution of mechanical strains in the exoskeleton that occur during posture and locomotion. The advantages and limitations of the techniques are discussed. These methods may be used in studying the exoskeleton and the anatomy of cuticular mechanoreceptors of other arthropods to similar advantage. PMID- 10738319 TI - Ultrastructure of the echinoderm cuticle after fast-freezing/freeze substitution and conventional chemical fixations. AB - The cuticles of the pedicellaria primordia in the sea urchin Paracentrotus lividus and of the tube foot disk in the sea star Asterias rubens were preserved by different methods, viz., glutaraldehyde fixation followed by osmium tetroxide postfixation, glutaraldehyde-ruthenium red fixation followed by osmium tetroxide ruthenium red postfixation, and two fast freezing / freeze substitution methods (FF/FS). The gross ultrastructure of the cuticle as well as the influence of the preservation method on this ultrastructure were identical for the two tissues studied. The cuticle ultrastructure was poorly preserved after glutaraldehyde fixation / osmium tetroxide postfixation. Its preservation was improved after ruthenium red was added in the fixative and postfixative, but the best preservation was consistently achieved using FF/FS. Both low-pressure freezing (plunge freezing) and high-pressure freezing were tested, the latter giving seemingly better results. With these methods, the cuticle appeared to be composed of a proximal lower cuticle, an intermediate upper cuticle, and a distal fuzzy coat. In particular, cryoimmobilization methods emphasized or revealed the occurrence of a well-developed fibrillar lower cuticle in the pedicellaria, the complexity of the upper cuticle which consisted of several zones, and the importance of the usually poorly preserved fuzzy coat that is actually the thickest layer of the cuticle. These observations bring new insights on the functions of the cuticle, and particularly of the fuzzy coat. According to its preservation characteristics, the fuzzy coat presumably consists mostly of proteoglycans. This composition could give it shock absorption and antifouling properties. Furthermore, its important thickness also implies that molecules detected by the short sensory cilia must diffuse through and could be selected by the fuzzy coat. PMID- 10738320 TI - Informed consent in theory and practice: the new undergraduate curriculum. PMID- 10738321 TI - A 5-year review of 71 consecutive anterior skull base tumours. AB - Techniques used in craniofacial surgery have found extensive application in the management of skull base tumours. The improved exposure gained via osteotomies for facial disassembly has facilitated the en-bloc resection of tumours with clear surgical margins, and the advent of vascularised seals has significantly reduced the risk of meningeal contamination. We present our experience with the extirpation and reconstruction of 71 benign and malignant tumours of the anterior skull base over a 5-year period. Survival and functional outcome data are presented, with an emphasis on the wide range of pathologies and primary treatment strategies seen at presentation. Criteria used in flap selection and the role of prosthetics are discussed. Observations are offered on salvage surgery, demonstrating that excellent palliation can be achieved, even in cases with massive recurrent disease. PMID- 10738322 TI - Does MRI contribute to the investigation of palatal function? AB - The results of a pilot study into the value of MRI scanning in investigation of velopharyngeal function are discussed. MRI offers potential advantage over naso endoscopy in being noninvasive and over video fluoroscopy in avoiding radiation. However, it requires costly equipment and patient cooperation, which limits its use in young patients. Ten normal volunteers and 15 patients with speech problems underwent MRI of the velopharyngeal port at rest and during sustained phonation of word /a/. Optimal planes for scanning were determined. Images were obtained in mid-sagittal, coronal and horizontal planes at the level of the hard palate and in the plane of the levator axis. Computer assisted measurements were possible of the velopharyngeal closure, forward movement of posterior pharyngeal wall, velar lift, velar extensibility and medial movement of the lateral pharyngeal wall. MRI has a potential role in investigation of velopharyngeal incompetence and planning its surgical repair. PMID- 10738323 TI - Custom cranioplasty using stereolithography and acrylic. AB - Numerous methods of cranioplasty have been described. Customization and prefabrication have been reported to reduce operating time and improve cosmesis. An original technique for the manufacture of customized cranioplastic implants has been developed and tested in 30 patients.Thirty patients requiring cranioplasties were selected. Data acquired from computed tomography (CT) were used to manufacture exact plastic replicas (biomodels) of craniotomy defects and master cranioplastic implants using the rapid prototyping technology of stereolithography (SL). The three-dimensional (3D) imaging techniques of mirroring and interpolation were used to extrapolate on existing anatomy to design the master implants. The master implants were hand finished to fit the defect in the corresponding cranial biomodel exactly and were then used to create a cavity mould. The mould was used to cast thermally polymerised custom acrylic implants. The surgeons reported that the customized implants reduced operating time, afforded excellent cosmesis and were cost effective. The patients reported that the opportunity to see the biomodel and implant preoperatively improved their understanding of the procedure. Two complications were noted, one infection and one implant required significant trimming. The simultaneous manufacture of the master implant (male) and biomodel (female) components from SL allowed custom accurate implants to be manufactured. Disadvantages identified were the time required for computer manipulations of the CT data (up to 2 h), difficulty in assessing the accuracy of the computer generated master as a 3D rendering, the potential for SL parts to warp, manufacturing time (minimum 2 days) and the cost of approximately $1300 US per case ($1000 for the SL biomodel and $300 for the acrylic casting). PMID- 10738324 TI - Anatomical features of perforator blood vessels in the deep inferior epigastric perforator flap. AB - The anatomical basis of the blood supply to the deep inferior epigastric perforator (DIEP) flap and the circulation of the cutaneous territory zone IV have been studied in 40 anatomical dissections on fresh cadavers. The pedicle length, diameter and the number and distribution of the major perforator vessels were recorded. The cutaneous territories supplied by the perforator vessels were studied by injecting blue dye in the pedicle of ten specimens. In addition, intravital blue dye studies were performed by injecting the largest perforating vessel of five patients at abdominoplasty. The resulting cutaneous staining was recorded. The deep inferior epigastric artery was present in all cadaver dissections with an average length of 10.3 cm (range 9.0-13 cm) and an average diameter of 3.6 mm (range 2.8-5.0 mm). There were consistently one or two major perforator vessels for each pedicle, located within a radius of 8 cm below the umbilicus. The results of the injection study revealed cutaneous staining of zones I-III. Zone IV was stained weakly or not at all. The same findings were obtained in the clinical cases of abdominoplasty. Zone IV must be critically assessed in clinical cases of the DIEP flap. PMID- 10738325 TI - Long-term outcome of augmentation mammaplasty in male-to-female transsexuals: a questionnaire survey of 107 patients. AB - A retrospective survey of long-term postoperative male-to-female transsexual patients has been performed to evaluate how well augmentation mammaplasty addresses their needs. One hundred and seven (65%) out of 164 anonymous questionnaires sent to the patients were evaluated. Average clinical follow-up of these patients was 4.8 years, whereas the average time lapse between mammaplasty and filling out of the questionnaire was 5.5 years (range, 16 months-17 years). The age of the subjects at the time of this survey ranged from 22 to 76 years (average, 41 years). Seventeen of the 107 patients had undergone further augmentation mammaplasty, on average 57 months after the initial mammaplasty. The average size of implanted prostheses was 258 ml (range, 130- 450 ml). Eighty patients (75%) indicated satisfaction with the final outcome of the mammaplasty. The median postoperative cup size in this group was B (range of postoperative bra size, 30B-40D). The remaining 27 patients (25%) were unhappy with the results of mammaplasty. The median postoperative cup size in the 18 patients who still felt their breasts to be too small was also B (range of bra size, 30B-48E). The average size of current prostheses in these 18 patients was 261 ml. For a male-to female transsexual patient to appreciate the outcome of augmentation mammaplasty, the surgeon should tolerate and address this patient's urge for a distinctly feminine breast configuration. PMID- 10738326 TI - Ultrasonic surgical aspiration with endoscopic confirmation for osmidrosis. AB - Ultrasonic surgical aspiration of axillary apocrine glands with endoscopic confirmation was used for treating osmidrosis in 87 patients. Ultrasound energy liquefies fat and sweat glands via cavitation, but minimally affects blood vessels and nerves at the same energy level. We hypothesised that since the apocrine glands were located within the subcutaneous fat layer, ultrasound liposuction would be effective in its removal and also preserve vasculature of the axillary skin for optimal wound healing. The endoscope was used to visually confirm adequate removal of fat and sweat glands. Our method was effective in 84 patients (96.5%) and recurrence of odour occurred in three patients (3.5%). There were no cases of haematoma, seroma, or skin necrosis. Our method leaves a small inconspicuous scar, maintains normal axillary hair pattern and avoids contracture of the axillary skin after a short and comfortable recovery period. PMID- 10738327 TI - Evaluation of the donor site morbidity after lateral arm flap with skin paddle extending over the elbow joint. AB - The free lateral arm flap may be extended to include the skin over the lateral aspect of the elbow and the forearm. However, extending the flap beyond the lateral condyle has been thought to interfere with the elbow function. The aim of this study was to assess the donor site morbidity after placement of the skin paddle across the lateral aspect of the elbow joint. Seventeen consecutive lateral arm flaps with a skin paddle designed over the elbow joint were performed for different indications. Eleven patients were followed up postoperatively for a minimum of 6 months. The range of motion, torque, power and endurance of the elbow joint were recorded using a Cybex 340 isometric dynamometer. The values of the operated elbow were compared to those of the unoperated elbow and the deficits were expressed in percentages. The touch sensation in the territory of the posterior cutaneous nerve of the forearm was assessed using Semmes-Weinstein monofilaments. Patient satisfaction was evaluated with a self-assessment scale. The donor site scar was stretched in one patient. No patient complained of elbow pain or cold intolerance. Elbow mobility was not significantly affected by flap harvesting (P = 0.06). An extension deficit of 4% was found in four patients. The mean torque, power and endurance deficits were 7.3%, 7.5% and 4.4% respectively as compared to the contralateral side. Complete loss of sensation was not present in any of the donor sites. However, an area of hypoaesthesia with a mean size of 45 cm(2)(range 20-75 cm(2)) was found on the posterolateral surface of the forearm. Patient satisfaction with the donor site was rated high. From this retrospective review it appears that elbow function was not significantly affected by extending or planning the lateral arm flap over the lateral condyle. PMID- 10738328 TI - Quantitative benefits provided by acute tissue expansion: a biomechanical study in human cadavers. AB - The aim of this study was to test the ex vivo biomechanical properties of acutely expanded cutaneous flaps to quantitatively assess the efficacy of intraoperative tissue expansion. A total of 14 fresh male cadavers were used for the study. In each cadaver, a rectangular (15 x 8 cm), proximally based flap was designed on each side of the body, in three different locations: lateral arm, anterior thorax, anterior thigh. In each cadaver, one randomly selected flap per each body region underwent acute-intermittent expansion, whereas the contralateral flap served as control. The biomechanical properties (stress/strain ratio, mean stiffness) of both expanded and control flaps were then assessed by means of a dynamometer and a force-transducer. The obtained data showed that the biomechanical benefits provided by acute tissue expansion were statistically different (P< 0.05) from those obtained by simple subcutaneous undermining. While no changes of length have been observed in the acutely expanded skin flaps as compared to control cutaneous flaps, a statistically significant gain in the compliance of the former has been recorded as compared to the biomechanical behaviour of the latter. PMID- 10738329 TI - Biomechanical properties of thin skin flap after basic fibroblast growth factor (bFGF) administration. AB - This study is designed to examine the effects of basic fibroblast growth factor (bFGF) administration on the biomechanical properties of thin skin flap healing. A total of 42 rats were used in this study, and skin flaps 10 cm long by 3 cm wide were raised in 28 rats. One injection of bFGF was applied at three different times (immediately postoperatively, and 24 h and 48 h postoperatively) between the flap and wound bed of 14 rats (the bFGF treated group), while the other 14 rats with flaps had the same tissue culture medium treatment but without bFGF (the untreated group). The remaining 14 rats without flaps constituted the control group (normal group). The rats were killed 10 days postoperatively, and 1. 0 cm x 6.0 cm sections of the skin flap taken for mechanical and histological testing. The load, deformation and tensile strength at failure were recorded. The average flap survival area in the bFGF treated group was 27 cm(2), significantly higher (P< 0.001) than that seen in the untreated group. The average elastic stiffness of the skin flap in the bFGF treated group was also observed to be higher than in the untreated group although this difference was not significant. The mean tensile strength of the bFGF treated group (61 N) was significantly higher than the untreated group (38 N, P< 0.01) however. Despite this, the tensile strengths at failure of both of these groups were found to be significantly lower than that of normal skin (101 N, P< 0.01). A relaxation in load of about 8% was seen in specimens from the normal group and the bFGF treated group, while the untreated group showed a relaxation of about 15%. PMID- 10738330 TI - A microneurovascular study of the great saphenous vein in man and the possible implications for survival of venous flaps. AB - Cross sections (n = 372) of human long saphenous veins (n = 12) from unsalvageable fresh above knee amputation specimens brought to a trauma centre in a large metropolitan hospital were studied. The veins were harvested longitudinally with an amount of soft tissue equivalent to the diameter of the vein and sectioned at intervals of 5 mm. The sections were stained with haematoxylin and eosin for general study, Van Gieson for better delineation of different coats of the vessel wall and reticulin stain to differentiate between venules and arterioles. The vascular structures within the vein wall and its surrounds were analysed by using analySISpro via a CCD camera attached to a light microscope, with the help of a frame grabber card and image analysis software. Results show that the vein has vasa vasorum within its wall, a capillary network around it, arterioles, venules and the saphenous nerve and its branches with their vascular network a further distance apart. The relevance of the microsurgical anatomy of the saphenous vein and its soft tissue envelope in the survival of venous flaps is discussed. PMID- 10738331 TI - The effects of VEGF on survival of a random flap in the rat: examination of various routes of administration. AB - The purpose of the present study was to determine the effects of vascular endothelial growth factor (VEGF) on survival of a full thickness random pattern, McFarlane musculocutaneous flap in the rat. In addition, this study examined a number of different methods of VEGF delivery in an attempt to determine the most effective route of administration. A 2 x 8 cm full thickness dorsal flap with the pedicle remaining attached at the anterior end was elevated in 72 male Sprague Dawley rats. The rats were randomised into six groups and immediately received the following treatment: Group I (n = 12): systemic VEGF injection into the femoral vein (50 microg/ml); Group II (n = 10): multiple systemic VEGF injections at 0, 24 and 48 h post flap elevation (50 microg/ml); Group III (n = 12): subdermal VEGF injection into the flap (1 microg/ml); Group IV (n = 12): subfascial VEGF injections into the recipient bed (1 microg/ml); Group V (n = 10): topical VEGF onto the recipient bed (1 microg/ml); Group VI (n = 16): control group with no treatment. Following 5 days recovery, the area of flap survival was measured. Mean flap survival ranged from 91% in Group II to 78% in Group V, and was significantly greater in all experimental groups (P< 0.001 for Groups I-IV and P< 0.05 for Group V) as compared to the control group (mean survival of 66%). The only significant difference between the experimental groups was between the mean survival in Group II and Group V (P< 0. 05). Histological analysis demonstrated a qualitatively greater amount of granulation tissue and neovascularisation in the experimental groups. These results support the notion that VEGF rescues tissue at risk of hypoxic damage by inducing angiogenesis, and the use of growth factors such as VEGF holds promise as a method of increasing skin viability. PMID- 10738332 TI - The staggered ellipse. AB - An incision is described in which the two ends of an elliptical excision are displaced. This can be used in two situations; in the reduction in length of one side of a digit and in the excision of two lesions close together. PMID- 10738333 TI - A new model for assessment in plastic surgery: knowledge of relaxed skin tension lines. PMID- 10738335 TI - A combined V-Y advancement-turnover flap for simultaneous perialar and alar reconstruction. AB - A modification to a standard nasolabial flap is described for the simultaneous reconstruction of confluent perialar and full thickness alar defects. The main body of the flap is advanced to cover the external surface of the perialar and alar defects. A side extension to the flap, based only on a dermal blood supply, is turned over, to line the reconstructed alar rim. PMID- 10738334 TI - Johann Friedrich Dieffenbach: successful use of leeches in plastic surgery in the 1820s. AB - Johann Friedrich Dieffenbach (1792-1847) regularly and successfully utilised leeches in sophisticated plastic surgery in Berlin in the 1820s and 1830s, well before anaesthesia, antisepsis and antibiotics. Inexplicably, it took nearly another 150 years before the use of leeches in this context was revived. PMID- 10738336 TI - MRI scan diagnosis of trilucent breast implant rupture. AB - The authors present the case report of a patient who had undergone a latissimus dorsi breast reconstruction plus Trilucent implant. This patient later presented with discomfort in the breast and an MRI scan diagnosed rupture of the implant and also revealed a fluid level within the implant. These findings were confirmed at exploration and were found to be caused by separation of the contents into a serous layer and a lipid layer. PMID- 10738337 TI - Calciphylaxis: a rare limb and life threatening cause of ischaemic skin necrosis and ulceration. AB - Calciphylaxis (uraemic gangrene syndrome) is a rare complication of chronic renal failure and secondary hyperparathyroidism. Patients present with painful purple skin lesions which undergo necrosis and ulceration. The histology is specific. There is medial calcification with intimal hyperplasia and thrombosis of the lumen of small sized arteries in the underlying subcutaneous tissue. Death frequently arises from overwhelming sepsis. Early recognition of this condition and prompt parathyroidectomy can lead to rapid relief of symptoms and ulcer healing and may be life saving. PMID- 10738338 TI - Compartment syndrome complicating metastatic malignant melanoma. AB - Compartment syndrome is well documented in the literature. Neoplasia as a cause is a rare. We report a patient with known metastatic malignant melanoma presenting with a compartment syndrome of the arm caused by a relatively slow growing, non-invasive metastatic deposit. This was excised and the patient made an uneventful recovery. PMID- 10738339 TI - Intramuscular haemangioma of the anterior chest wall. AB - Intramuscular haemangiomas of the chest wall are rare. We present the case of a 33-year-old man with an intramuscular haemangioma of the left side of the anterior chest wall located in the left sixth intercostal space. We resected the tumour and surrounding tissue. Histopathological examination of the tumour demonstrated an intramuscular haemangioma of small-vessel type. The patient has been free of recurrence for 5 years after surgery. PMID- 10738340 TI - Post laser hyperpigmentation and occupational ultraviolet radiation exposure. AB - Hyperpigmentation is an occasional complication of laser therapy. Patients working in an environment with excessive exposure to ultraviolet radiation may be at increased risk. PMID- 10738341 TI - Nasal reconstruction with an expanded dual forehead flap. AB - Nasal reconstruction with an expanded dual forehead flap is described. This method provides for nasal lining and covering with one forehead flap. The relationship between the two flaps is more flexible than a folded flap. Rigid support can be placed between the two flaps. This is an effective method for the reconstruction of a composite nasal defect when it is difficult to find an appropriate local lining flap. PMID- 10738342 TI - Future of plastic surgery in the UK and role of overseas doctors. PMID- 10738343 TI - Application of reduction mammaplasty in treatment of giant breast tumour. PMID- 10738344 TI - A problem of identity following cleft lip repair. PMID- 10738345 TI - Monodigital double cross finger flaps. PMID- 10738346 TI - Facial impaling on a scythe. PMID- 10738348 TI - Introduction PMID- 10738347 TI - Notices PMID- 10738349 TI - RITA. PMID- 10738350 TI - Trials of angioplasty and surgery: CABRI. AB - The medium term (4-year post randomization) results from CABRI indicate that the principal difference between those randomized to coronary angioplasty and those to coronary surgery has been the much greater need for repeat revascularization in the former. A number of factors may play a role in the greater repeat revascularization rate post coronary angioplasty, these include coronary restenosis, residual coronary artery disease, coronary artery disease progression. In the longer term, graft failure in those who have undergone coronary surgery will be important, and it remains to be seen what the effect of this will be. PMID- 10738351 TI - The Emory Angioplasty vs Surgery Trial (EAST). AB - The Emory Angioplasty vs Surgery Trial (EAST) was the outgrowth of the vision of Andreas Gruntzig, expressed as early as 1983, to compare the two revascularization methods in patients with multivessel disease. With sponsorship of the National Heart, Lung and Blood Institute, 392 patients were randomized and followed with 1- and 3-year angiograms and perfusion nuclear scans, and 450 patients were followed in a parallel registry starting in 1987. Now with complete 8-year follow-up, there is no significant survival difference. Revascularization was more complete in the surgery patients, and repeat procedures more common in the PTCA group. Excess procedures in follow-up were concentrated in the first years and the extended 5-year follow-up showed relatively equal use of repeat procedures. The superiority of surgery in diabetic patients seen in the BARI trial was also supported by the EAST findings. PMID- 10738352 TI - The design, patient population and outcomes from the Bypass Angioplasty Revascularization Investigation (BARI) randomized trial and registries. AB - The Bypass Angioplasty Revascularization Investigation (BARI) was designed to compare CAGB and PTCA for patients with symptomatic multivessel coronary artery disease. The randomized trial reported significant differences in 7-year survival favouring CABG. However, for the 353 patients with treated diabetes mellitus, an overwhelming benefit associated with CABG was seen (mortality: 23.6% CABG vs 44. 3% PTCA, p=0.0011), whilst no treatment difference was observed for the 1476 non diabetic patients (13.6% CABG vs 13.2% PTCA, p=0.72). Patients assigned to PTCA experienced fewer in-hospital Q-wave MIs, but these patients received more revascularization procedures and more often had angina during follow-up. PMID- 10738353 TI - Design of the 'Stent or Surgery' trial (SoS): a randomized controlled trial to compare coronary artery bypass grafting with percutaneous transluminal coronary angioplasty and primary stent implantation in patients with multi-vessel coronary artery disease. AB - The 'Stent or Surgery' trial (SoS) is a randomized controlled trial to compare coronary artery bypass grafting with percutaneous transluminal coronary angioplasty and primary stent implantation in patients with multi-vessel coronary artery disease. This is a multicentre, multinational venture involving over 40 centres in 12 countries throughout Europe and Canada. Eligible and consenting patients will be randomly allocated, in equal proportions, to open revascularization by CABG or by PTCA with the primary implantation of intracoronary stents. The trial design is pragmatic and imposes few protocol restrictions in patient selection, surgical and intervention techniques or adjunctive medication schedules. The rationale and design of the trial are discussed, including important sub-studies, examining quality of life, neuropsychological outcome, cost and cost benefit. PMID- 10738354 TI - The ARTS study (Arterial Revascularization Therapies Study). AB - The rising costs of health care have forced policy makers to make choices, and new treatments are increasingly assessed in terms of the balance between additional costs and additional effects. The recent recognition that stenting has a major and long-lasting effect enhancing balloon PTCA procedure has made it imperative to compare in patients with multivessel disease the standard surgical procedure with multiple stenting in a large scale multinational and multicentre approach (19 countries, 68 sites). Selection and inclusion of patients is based on a consensus of the cardiac surgeon and interventional cardiologist on equal 'treatability' of patients by both techniques with analysis of clinical follow-up (event-free survival) on the short (30 day), medium (1 year), and long-term (3 and 5 year) with analysis of cost-effectiveness and quality of life (EuroQol and SF-36). Of the entire trial, the primary null hypothesis which needs to be rejected is that there will be no difference in event-free survival or effectiveness (E), at 1 year and also that the direct and indirect costs (C) per event-free year are not different between surgery or stenting. For this to become significant with a power of 90% one needs 1200 patients. Between April 97 and June 98, 1205 patients have been randomized with a monthly recruitment of 83 patients. Expected costs, effects and cost-effectiveness ratio (CE ratio) are: Stent high costs 2 VDStent high costs 3 VDStent low costs 2 VDStent low costs 3 VDCABG costs (C)$19.297$24.566$16.638$20.456$21.350 effects (E)81%81%81%81%88% CE ratio$23.876$30.397$20.586$25.322$24.348 Clinically, stenting is not expected to be more effective than CABG, but should be cost effective in both the 2- and 3-VD group when using the lower cost estimate and in the 2 VD group when using the higher cost assumptions. PMID- 10738355 TI - Extended use of arterial conduits for myocardial revascularization. AB - Myocardial revascularization by means of surgical coronary artery bypass grafting has proven to provide reproducible and durable relief from the sequellae of coronary atherosclerosis. Despite the proven success of this operation, efforts are ongoing both to reduce the perioperative risks and morbidity, as well as to improve the long-term outcomes. The use of multiple arterial conduits is an example of the latter. This is based on the proven superior long-term patency of arterial grafts as compared with venous conduits. A remarkable wide variety of conduits and configurations are being explored currently. We outline our current thinking with regard to arterial grafting as the field now stands. The early results are encouraging, and suggest a significant improvement in long-term relief from angina pectoris and freedom from reintervention when multiple arterial conduits are employed. PMID- 10738356 TI - Minimally invasive coronary surgery. PMID- 10738357 TI - Analysis of trials of surgery vs angioplasty in myocardial revascularization. AB - AIM: comparison of surgery and angioplasty for the treatment of coronary artery disease. METHOD: comparison using randomized trials for low-risk patients and observational studies for high-risk patients. RESULTS: in low-risk patients, a strategy of initial surgery or angioplasty achieved similar results regarding overall and infarct-free survival rates at 5 years. Residual angina was statistically more prevalent after angioplasty and required more subsequent revascularization procedures. Residual angina negatively impacted on life quality. Angioplasty initially had a cost-effectiveness advantage over surgery, which subsided over time. In high-risk patients, no firm conclusion could be drawn, due to unmatched selection of patients. Angioplasty seems superior in acute myocardial infarction and in very ill patients. Surgery seems superior to treat diseased bypass grafts. CONCLUSIONS: because of similar achievements, the choice of therapy in low-risk patients eventually should depend on patient's preference. During counselling, the deleterious effect of residual angina on life quality and health perception should not be underestimated by practitioners. In high-risk patients, further studies are required to define the best approach to any individual patient. PMID- 10738358 TI - Chlamydia pneumoniae and atherosclerosis. AB - Chlamydia pneumoniae is the third species of the genus Chlamydia and has been known to cause respiratory tract infections. Since the association between the seropositivity of C. pneumoniae and ischemic heart diseases was reported in 1988, the association between C. pneumoniae and atherosclerosis has been noteworthy. Positive findings of the association between C. pneumoniae and atherosclerosis have been reported as the result of seroepidemiological surveys, histological studies to detect C. pneumoniae in human atherosclerotic tissues, and animal infection models. These data supported that C. pneumoniae infection occurs in human vascular walls and may accelerate the foam cell formation of macrophage and smooth muscle cells, and may play a causative role in atherosclerosis. Several large-scale studies of the antimicrobial prevention of secondary cardiac events are in progress. The genome projects for C. pneumoniae have recently been reported. A number of issues remain unclear, however, and further intensive research is necessary. PMID- 10738359 TI - Theiler's murine encephalomyelitis virus (TMEV): the role of a small out-of-frame protein in viral persistence and demyelination. AB - Theiler's murine encephalomyelitis virus (TMEV) belongs to the genus Cardiovirus of the family Picornaviridae and is divided into two subgroups on the basis of different biological activities. GDVII subgroup strains produce acute and fatal polioencephalomyelitis in mice with no virus persistence. In contrast, DA or TO subgroup strains cause an early nonfatal polioencephalomyelitis. TMEV is thought to be an excellent animal model for the human demyelinating disease, multiple sclerosis. Data suggest that macrophages are a major reservoir harboring the virus. A small out-of-frame protein designated L* is synthesized in DA subgroup strains from an alternative, out-of-frame, initiation site. Studies of a DA mutant virus, having an ACG rather than an AUG and therefore does not synthesize L* protein, demonstrate that this protein is important for virus growth in particular cell types and is critical for DA-induced demyelinating disease and virus persistence. In addition, TMEV can be used as a vector for delivering foreign sequences into the central nervous system. PMID- 10738360 TI - Stability of Russell's viper venom toxoid (lyophilized form) on storage. AB - A previously developed Russell's viper venom toxoid in Myanmar is in a liquid form that shows reversion in the form of a reduced number of formaldehyde linkages and toxicity during storage at 37 degrees C and at room temperature. In order to have a safe, potent and stable toxoid, a lyophilized form was prepared in the present study from the liquid toxoid through the use of a freeze dryer. Both the liquid and lyophilized forms were then stored at 4 degrees C and at room temperature, and in addition to safety and immunogenicity tests, biochemical parameters such as the protein content, the activity of venom enzymes (proteinase, phospholipase A, phosphodiesterase, and arginine esterase), and the released free formalin amounts were assessed at 3-month intervals over a period of 1 year. The results indicate that under both conditions, the lyophilized toxoid shows minimum changes in enzyme activity, a reduced tendency toward formaldehyde linkage, no toxicity, and more immunogenicity in comparison with the respective liquid toxoids. It could therefore be hypothesized that Russell's viper venom toxoid in a lyophilized form is more promising in terms of efficacy and stability for prophylactic use in human immunization than the conventional toxoid in a liquid form. PMID- 10738361 TI - Methicillin resistance among Trinidadian isolates of community and hospital strains of Staphylococcus aureus and their patterns of resistance to non-beta lactam antibiotics. AB - The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) strains in Trinidad and the extent of their resistance to other antimicrobial agents in hospital-acquired and community-acquired infections were evaluated over a 2-year period. A total of 450 S. aureus strains were isolated from different patients. The prevalence of methicillin resistance among S. aureus strains was 9.8% (44/450). The proportion of MRSA isolated from hospital sources and community sources was 12.5% (38/305) and 4.1% (6/145), respectively (P < 0.05). The resistant rates of MRSA to the non-beta-lactam antibiotics were as follows: 93.2% resistance to tetracycline, 68.2% to erythromycin, 61.4% to gentamicin, 45.5% to co-trimoxazole, and 20.5% to ciprofloxacin. No MRSA resistant to vancomycin was observed in this study. Study results showed significant increases in MRSA in hospital, 2% in 1995 to 12.5% in 1998 (P < 0.05), and community, 0% in 1995 to 4.1% in 1998 (P < 0.05). It has become apparent that infection control and surveillance initiatives must be focused now on the community in order to monitor and limit the spread of this new and expanding reservoir of MRSA. PMID- 10738362 TI - Exacerbated spongiform lesions in the cerebral cortex in Japanese sheep, in an outbreak of scrapie during 1984-1987. AB - The present study dealt with the pathology of natural scrapie in Japanese Suffolk sheep in a certain selected area. Vacuolations in the cytoplasm of neurons were conspicuous. They were particularly evident in many areas of the medulla and pons, extending into and through pedunculus cerebri and thalamus to the septal area and olfactory tubercle. Proliferation of astrocytes was also easily observed with glial fibrillary acidic protein staining. Neural vacuolations in the cerebral cortex were observed in 73% of the cases. Abnormal prion protein deposits were seen in all cases observed by hydrolytic autoclaving, and subsequent peroxidase and anti-peroxidase immunostaining. Abnormal prion protein staining was the most conspicuous in the polymorphic layers of the hippocampus. PMID- 10738363 TI - Improvement of onychomycosis without antifungal therapy after initiation of highly active anti-retroviral therapy (HAART) in an HIV-infected patient. PMID- 10738364 TI - Emergence and prevalence of a novel Vibrio parahaemolyticus O3:K6 clone in Japan. PMID- 10738365 TI - Mitigation of hepato-cellular injury caused by HAART with glycyrrhizin compound in patients co-infected with HIV and HCV. PMID- 10738366 TI - Genetic analysis of outbreak of hepatitis A virus infection among HIV-1 seropositive men. PMID- 10738367 TI - Integrase of human endogenous retrovirus K-10 supports the replication of replication-incompetent Int- human immunodeficiency virus type 1 mutant. PMID- 10738368 TI - Epidemiological studies of an outbreak of paratyphoid fever in the Shima area of Mie Prefecture. PMID- 10738369 TI - Experiences of microbial contamination of animal colonies maintained in the National Institute of Infectious Diseases, Japan (NIID). PMID- 10738370 TI - The features of imported dengue fever cases from 1996 to 1999. PMID- 10738372 TI - A modest proposal. PMID- 10738371 TI - Nursing science: who cares? PMID- 10738373 TI - Mentoring mistakes. When student knows best. PMID- 10738374 TI - More on hypoglycemia. PMID- 10738375 TI - Women's health alerts. PMID- 10738376 TI - Women's health alerts. PMID- 10738377 TI - Ethics of stem cell storage. PMID- 10738378 TI - Postexposure HIV prophylaxis. PMID- 10738379 TI - Teaching patients to use a numerical pain-rating scale. PMID- 10738380 TI - Responding to 'difficult' patients. PMID- 10738381 TI - Nurses as leaders in health care. Then & now. PMID- 10738382 TI - Primary biliary cirrhosis. PMID- 10738383 TI - Setting behavioral limits. PMID- 10738384 TI - Emergency! Pulmonary edema. PMID- 10738386 TI - Suicide prevention protocol. PMID- 10738385 TI - Getting conscious sedation right. PMID- 10738387 TI - ANA creates new 'house' for all nurses. PMID- 10738388 TI - Computer-based patient records. PMID- 10738389 TI - Protecting nurse whistleblowers. PMID- 10738390 TI - Evolutions of involvement. PMID- 10738391 TI - Nursing's best kept secret. Magnet hospitals can save health care. PMID- 10738392 TI - Planning for the coming shortage. PMID- 10738393 TI - Inherited dystonias. PMID- 10738394 TI - Erythromycin use linked to pyloric stenosis. PMID- 10738395 TI - When a blood pressure isn't routine. PMID- 10738396 TI - Oral patient-controlled analgesia. PMID- 10738397 TI - Hospital caregivers are in a bad mood. PMID- 10738398 TI - The Magnet Nursing Services Recognition Program. PMID- 10738399 TI - The challenge of warfarin therapy. PMID- 10738400 TI - Emergency! Whiplash injury. PMID- 10738401 TI - Hepatitis A: how benign is it? PMID- 10738403 TI - Managing labor: does walking help or hurt? PMID- 10738404 TI - Eliminating racial and ethnic disparities in health care. PMID- 10738405 TI - Books of the Year Awards. The most valuable texts of 1999 as chosen by AJN's panel of judges. PMID- 10738406 TI - Telephone triage. PMID- 10738407 TI - If you're replaced by a younger nurse. PMID- 10738408 TI - Hazards in the workplace. PMID- 10738410 TI - Agenda for women's health research. Improving women's musculoskeletal health. AB - Increased attention and resources focused on women's health during the past decade have resulted in new offices, policies, and programs of the Federal government. The Office of Research on Women's Health, established by the National Institutes of Health in 1990, is a focal point for all National Institutes of Health-supported efforts to improve women's health through biomedical and behavioral research. The Office of Research on Women's Health ensures women's appropriate inclusion in research studies supported by the National Institutes of Health. Through the development of a comprehensive agenda for research on women's health, the Office of Research on Women's Health encourages the consideration of gender issues and gender differences in health and disease in research sponsored by the National Institutes of Health. The importance of research to study and improve women's musculoskeletal health has become increasingly recognized. Clinicians, researchers, and representatives of professional and advocacy organizations concerned with women's musculoskeletal health participated in the development of the initial research agenda on women's health in 1991 and participated in the meetings to update and revise the agenda in 1996 and 1997. As a result of meetings convened to review and revise the agenda on women's health for the twenty-first century, many recommendations for additional research on women's musculoskeletal health have been developed and now are being implemented across the National Institutes of Health. PMID- 10738409 TI - Quantitative computed tomography of vertebral spongiosa: a sensitive method for detecting early bone loss after oophorectomy. 1982. PMID- 10738411 TI - Women and the orthopaedic surgeon. Changing the relationship. AB - Women are disproportionately disabled by musculoskeletal conditions. For biologic and lifestyle reasons, musculoskeletal health is one of the areas of medicine in which the differences between men and women are most marked. Thus, an approach tailored to women, not just an approach based on what has worked for men, often is needed. How orthopaedists treat women will become an even more pressing issue in the years ahead. Demographic trends predict that the majority of senior citizens will be women in whom chronic conditions frequently involve a musculoskeletal problem. Even at earlier ages, women will present more frequently with musculoskeletal conditions because they participate in a broader range of sports and careers. The current study presents a lengthy list of action items that the orthopaedic surgeon should consider as he or she approaches the treatment of women. Many of these items involve anticipating musculoskeletal problems, and convincing patients to take appropriate preventive steps. Because so many patients are treated by physicians who are not orthopaedists for their musculoskeletal problems, a successful preventive approach presents orthopaedists with the opportunity to win the confidence and respect of a new generation of patients. PMID- 10738412 TI - Foot health and shoewear for women. AB - Females are different from males in structure and biomechanics. The foot in the female tends to have a narrower heel in relationship to the forefoot and overall is narrower than a man's foot relative to length. Females tend to pronate their feet more and have smaller Achilles tendons than males, both factors having implications for shoe fit. Although shoes have been worn for thousands of years for the main purpose of protecting feet from the environment, recent studies have implicated shoes as the principal cause of forefoot disorders seen in females. Several authors have reported the harmful effects of shoewear and the greatest factor is a shoe that is improperly fit. With respect to foot disorders in the female, the current study will explore anatomy, biomechanics, common forefoot disorders, and shoewear through the ages, athletic shoewear, and a toe strengthening program. PMID- 10738413 TI - The gender issue: epidemiology of ankle injuries in athletes who participate in basketball. AB - The increased participation of women in organized athletics has resulted in an interest in gender-related injury patterns. Previous reports have indicated an increased incidence in anterior cruciate knee injuries among female intercollegiate basketball players compared with their male counterparts. The current epidemiologic study prospectively evaluated the relative risk of ankle injuries in scholastic and collegiate basketball players during a 2-year period. Eleven thousand seven hundred eighty athletes participated in this study, 4940 females and 6840 males. There were 1052 ankle injuries. Overall, females had a 25% greater risk of sustaining a Grade I ankle sprain compared with their male counterparts. This increased risk was present in the interscholastic and intercollegiate players. There was no significant difference in the risk for Grades II and III ankle sprains, ankle fractures, or syndesmotic sprains. Male and female athletes doubled their risk for sustaining an ankle injury at the intercollegiate level compared with the interscholastic level. PMID- 10738414 TI - Anterior cruciate ligament injuries in the female athlete. Potential risk factors. AB - In the general population, an estimated one in 3000 individuals sustains an anterior cruciate ligament injury per year in the United States, corresponding to an overall injury rate of approximately 100,000 injuries annually. This national estimate is low for women because anterior cruciate ligament injury rates are reported to be two to eight times higher in women than in men participating in the same sports, presenting a sizable health problem. With the growing participation of women in athletics and the debilitating nature of anterior cruciate ligament injuries, a better understanding of mechanisms of injury in women sustaining anterior cruciate ligament injuries is essential. Published studies strongly support noncontact mechanisms for anterior cruciate ligament tears in women, which make these injuries even more perplexing. Speculation on the possible etiology of anterior cruciate ligament injuries in women has centered on anatomic differences, joint laxity, hormones, and training techniques. Investigators have not agreed on causal factors for this injury, but they have started to profile the type of athlete who is at risk. In the current study the most recent scientific studies of intrinsic and extrinsic risk factors thought to be contributing to the high rate of female anterior cruciate ligament injuries will be reviewed, important differences will be highlighted, and recommendations proposed to alleviate or minimize these risk factors among female athletes will be reported where appropriate. PMID- 10738415 TI - The anterior cruciate ligament enigma. Injury mechanisms and prevention. AB - The reasons for the higher frequency of anterior cruciate ligament injuries in women are largely conjecture. These injuries may result from direct contact or, more frequently, from no direct contact to the knee during activities that most athletes consider routine to their sport. This implies that there are intrinsic factors that lead to anterior cruciate ligament rupture. For the anterior cruciate ligament to tear, there must be excess anterior tibial translation or rotation of the femur on the tibia. In the former case, the tibia can move anteriorly during quadriceps activation that is not counterbalanced by hamstring activation. Patients describe their injury as occurring when landing, stopping, or when planting to change directions. The knee typically was near full extension. Mechanically, the angle of the patellar tendon and tibial shaft increases as the knee approaches full extension. This gives a mechanical advantage to the quadriceps. During cutting maneuvers, athletes tend to cut with a knee near extension (0 degree-20 degrees) when the quadriceps are active and the hamstrings are neither very active nor at a knee flexion angle that offers much of a mechanical advantage. In performing cutting and landing maneuvers, women tend to perform the activities more erect; that is, with their knee and hips closer to extension. One possible factor to help reduce the frequency of anterior cruciate ligament injuries in women may be in proper instruction for performing cutting and landing maneuvers which will lower their center of gravity thereby denying the quadriceps the opportunity to shift the tibia anteriorly. PMID- 10738416 TI - Anterior knee pain in females. AB - There are clear differences between men and women regarding anterior knee pain. Anatomic factors including increased pelvic width and resulting excessive lateral thrust on the patella are primary factors that predispose females to anterior knee pain. Effects of estrogen on connective tissue synthesis have been reported, but there is no clear mechanism by which this would affect anterior knee pain. Postural and sociologic factors such as wearing high heels and sitting with legs adducted can influence the incidence and severity of anterior knee pain in women. PMID- 10738417 TI - Idiopathic scoliosis and spondylolysis in the female athlete. Tips for treatment. AB - Idiopathic scoliosis and spondylolysis can be common back problems in female athletes. Diagnosis and treatment can be difficult. With the notable trend toward increasing participation of women and girls in organized sports, it is necessary to know which sports carry additional risks for participants to have these two conditions develop and to determine treatment modalities. In general, idiopathic scoliosis is more prevalent in females and even may be higher in the athletes. Treatment options may include observation, the use of a brace, and surgery. In determining treatment, the type of sport and caliber of athlete must be considered in conjunction with the severity of the curve. Spondylolysis or a stress fracture of the posterior vertebral elements can be a common cause of back pain in an athlete. In many sports that are dominated by females (gymnastics, dancing, figure skating), the athletes carry a high risk of having spondylolysis or a stress fracture. Knowing the risk factors permits precise diagnosis and appropriate treatment. Treatment options include the use of a brace and surgery. In the current study, an extensive review of the literature in conjunction with the extensive experience of a well-established sports medicine clinic at the authors' institution is presented. PMID- 10738418 TI - Upper extremity disorders in women. AB - Carpal tunnel syndrome, Colles' fracture, and osteoarthritis of the basilar joint of the thumb are only three of the many upper extremity conditions that preferentially affect women. With more and more women entering the workplace, these conditions become more disruptive of patients' lifestyles and even may be increasing in incidence. Orthopaedic surgeons traditionally have focused on the surgical treatment of patients with these conditions, but it is becoming increasingly clear that surgeons also must focus on delineating pathophysiology to better identify those individuals at risk and help prevent these potentially disabling disorders. PMID- 10738419 TI - Adhesive capsulitis. A treatment approach. AB - Adhesive capsulitis of the shoulder is a condition of unknown etiology that results in the development of restriction of active and passive glenohumeral motion. The authors will review what currently is known about the etiology of idiopathic adhesive capsulitis, will raise unanswered questions regarding etiology and treatment, and will define a stage-based evaluation and treatment program. Treatment options including benign neglect, home-based and supervised physical therapy, intraarticular corticosteroid injections, closed manipulations, and arthroscopic capsular release will be reviewed critically and the authors' approach to the treatment of patients with idiopathic adhesive capsulitis also will be presented. Additionally, areas of future research will be defined. PMID- 10738420 TI - The lax shoulder in females. Issues, answers, but many more questions. AB - A review of the existing data on shoulder laxity in females reveals there are insufficient data to confirm the commonly held belief that shoulders in females are more lax than shoulders in males. Laxity is not synonymous with instability. Although females may have increased generalized joint laxity relative to males, generalized joint laxity does not correlate with shoulder laxity. There is conflicting data regarding shoulder laxity and gender. A review of patients with multidirectional instability who were treated operatively showed that 55% of the patients were female (N = 94) and 45% were male (N = 77), but the number or gender of patients who were treated nonoperatively was not reported. Multidirectional instability is reviewed in the context of the lax shoulder in the female. Initial treatment should be nonoperative, emphasizing physical therapy and dynamic stabilization of the shoulder. If nonoperative treatment fails, open or arthroscopic inferior capsular shift stabilization is recommended. Additional basic science and clinical trials are needed to determine whether thermal capsulorrhaphy should be considered in the treatment of patients with multidirectional instability of the shoulder. PMID- 10738421 TI - In neck to neck competition are women more fragile? AB - Recent studies indicate numerous differences between female and male athletes regarding injuries of the upper and lower extremities. Studies have been much less clear concerning a gender differential regarding injuries of the cervical spine. For the purposes of the current review, injuries have been divided into three categories. The first category is cervical strain injury. It has been reported that strain injuries are more prevalent in female athletes than male athletes. The second category is cervical disc injury and cervical disc herniation. It has been reported that the male to female incidence is approximately equal in this category. In the third category, which is referred to as major structural injury, studies to date have almost entirely reported on injuries in males showing a significant male preponderance. With increasing participation of women in contact sports that cause major structural injury, a greater incidence of these injuries may be seen in women. PMID- 10738422 TI - Stress fractures and the female athlete. AB - Stress injury to bone is the result of a temporary disturbance in the equilibrium between bone resorption and bone regeneration. Knowledge of the pathophysiology of bone in health and disease currently encompasses an interactive mode that includes biomechanical, hormonal, nutritional, and genetic factors. The current study will review possible etiologic theories for stress injuries to bone with a special emphasis on gender-related factors. Females may be more at risk for a stress injury to bone because of their unique hormonal environment, and other anatomic and gender factors. PMID- 10738423 TI - Osteoporosis. AB - Osteoporosis is a disorder of decreased bone mass, microarchitectural deterioration, and fragility fractures. Osteoporosis is widespread and can affect people of all ethnic backgrounds and many older women and men. An essential element in preventing osteoporosis is the achievement of normal peak bone mass. Adequate nutrition, appropriate calcium and vitamin D intake, regular menstrual cycles and a well balanced exercise program of exercise are essential elements in achieving peak bone mass. At menopause women undergo accelerated bone loss. Thereafter, women and men gradually lose bone mass. A loss of one standard deviation give rise to an enhanced twofold risk of spine fractures or a 2.5 risk of hip fracture. Bone mass is determined by dual energy x-ray absorptiometry, quantitative computed tomography scan, and a peripheral ultrasound. Dual energy x ray absorptiometry has outstanding precision (within 1% to 2%), and has the ability to show the efficacy of drug intervention. Peripheral measurements may identify osteoporosis but only have a 70% correlation with hip and spine bone mass. Dual energy x-ray absorptiometry determines bone mass in a patient but the bone collagen breakdown products (N-telopeptide crosslinks) establish the current rate of bone loss. Major risk factors leading to fragility fracture include low body weight, history of fracture, family history of osteoporosis, and smoking. All individuals should ingest adequate calcium and vitamin D, exercise, and prevent falls. Women with low bone mass, high urinary bone collagen breakdown products, and/or major risk factors should consider hormone replacement therapy or a selective estrogen receptor modulator (Evista), calcitonin and bisphosphonates (alendronate). These agents successfully increase bone mass and limit fracture risk. Men at risk for fragility fractures respond similarly as women to alendronate and calcitonin. Although vertebral compression fractures can occur spontaneously, hip fractures are attributable to low bone mass coupled with a fall. Hence, fall prevention programs in addition to medical treatment are critical in the prevention of fragility fractures. PMID- 10738424 TI - Aging and exercise: are they compatible in women? AB - The benefits of physical exercise and fitness have long been understood. With the aging of society and longevity of women relative to men, interest finally is being given to the female population. There are many benefits to maintaining an active lifestyle as women age. These benefits include maintenance of bone mineral density and prevention of osteoporosis, decreased susceptibility to falls, and decreased susceptibility to breast cancer and other chronic diseases. There are great psychologic benefits to maintaining an active lifestyle, and the connection between cardiovascular fitness and lowering the incidence of cardiac disease is well established. Finally, physical activity and exercise can decrease the symptoms of arthritis. The Centers for Disease Control recommends at least 30 minutes of moderately intensive activity on most days of the week. In older women, the loss of lean body mass (sarcopenia) can be diminished through a strength training program. With these benefits of maintaining an active lifestyle, healthcare providers should become proactive in emphasizing the benefits of physical activity to the older female population. PMID- 10738425 TI - The disproportionate impact of chronic arthralgia and arthritis among women. AB - The heterogeneous group of diseases that causes chronic arthralgia and arthritis is the most common cause of activity limitation and disability among middle age and older women. For reasons that remain poorly understood this group of diseases affects women substantially more frequently than men. In particular, the prevalence rates of the most common causes of arthralgia and arthritis, osteoarthritis and rheumatoid arthritis, and the prevalence rates of less common diseases that cause arthralgia, including systemic lupus erythematosus, systemic sclerosis, and fibromyalgia, are between two and 10 times higher in women. Prevalence rates for most of these conditions increase with age, and may vary among populations. For example, in the United States, systemic lupus erythematosus is approximately three times as common among African-American women as among white women. All of these disorders typically have an insidious onset and variable course that can make diagnosis difficult. Yet, most patients with these diseases benefit from early diagnosis and early nonoperative treatments including patient education, patient participation in disease treatment, activity modification, assistive devices, and medications. Furthermore, early aggressive medical therapy may prevent development of permanent joint and visceral damage in patients with inflammatory diseases including rheumatoid arthritis, systemic lupus erythematosus, and systemic sclerosis. Failure to make the diagnosis of an underlying disease in patients with arthralgia may lead to inappropriate treatment or delay in treatment that can result in irreversible impairment. Because many women with these conditions seek medical care from orthopaedists, orthopaedic residency education and continuing medical education should place emphasis on early diagnosis and nonoperative treatment of patients with arthralgia and arthritis, and, when appropriate, early referral to rheumatologists. PMID- 10738426 TI - The effects of pregnancy on the musculoskeletal system. AB - During pregnancy the female body undergoes many hormonal and anatomic changes that affect the musculoskeletal system. These changes may cause various musculoskeletal complaints, predispose to injury, or alter the course of preexisting conditions. The changes of pregnancy should be taken into account when counseling women who wish to exercise through their pregnancy. Treatment of musculoskeletal complaints during pregnancy must include the potential effects on mother and fetus. PMID- 10738427 TI - Pneumatic compression hemodynamics in total hip arthroplasty. AB - A crossover study was performed to evaluate the effect of several pneumatic compression devices and active dorsoplantar flexion in 10 patients who underwent total hip arthroplasty. Using the Acuson 128XP/10 duplex ultrasound unit with a 5 MHz linear array probe, peak venous velocity and venous volume were assessed above and below the greater saphenous vein and common femoral vein junction. A computer generated randomization table was used to determine the order of the test conditions. The pneumatic compression devices evaluated included two foot pumps, one foot and calf pump, one calf pump, and three calf and thigh pumps. Statistical analyses included analysis of variance and analysis of variance with covariance between devices and patients. The covariates tested were the baseline measurements and the order in which the devices were tested. Differences between devices relate in part to the frequency and rate of inflation and the location and type of compression. Pulsatile calf and foot and calf pneumatic compression with a rapid inflation time produced the greatest increase in peak venous velocity, whereas compression of the calf and thigh showed the greatest increase in venous volume. Because patient and nursing compliance is essential to the success of mechanical prophylaxis for thromboembolic disease, the more simple, yet efficacious, devices that are easier to apply and less cumbersome appear to have a greater likelihood of success. In the active and alert patient, active dorsoplantar flexion should be encouraged. PMID- 10738428 TI - Prevention of loosening in total hip replacements using guided bone regeneration. AB - The aim of this study was to prevent wear debris from reaching the interface of the acetabular cup and femoral component by using a partially occlusive expanded polytetrafluoroethylene membrane. This membrane initially acted as a physical seal, which became incorporated by bone and soft tissue, forming a secondary biologic seal, preventing loosening. An animal model was developed to test the hypothesis. The model replicated the mechanisms of loosening where the effects of wear debris were studied. Using femoral heads with the appropriate roughness, a goat model produced the radiologic and histologic presentation of loosening as observed in total hip replacements in humans. Loosening was assessed by measurement of the radiolucent lines, and was attributed to wear debris by histologic investigation. The expanded polytetrafluoroethylene membrane prevented acetabular implant loosening to a statistical significance of 0.02 in a blinded assessment when compared with the control groups. Loosening of the first 5 mm of the proximomedial aspect of the femur also was prevented. The authors of the current study prevented wear particle-induced osteolysis in the acetabular component by using an expanded polytetrafluoroethylene membrane to seal the bone cement interface. PMID- 10738429 TI - Sensorimotor changes after anterior cruciate ligament reconstruction. AB - The restoration of joint stability is unlikely to be dependent on passive properties of the joint alone, yet the effect of anterior cruciate ligament reconstructive surgery on the sensorimotor system largely remains unexplored. This study evaluated whether surgical reconstruction of the ligament had any effect on one indicator of sensorimotor function, hamstring contraction latency, which previously has been shown to be related to function. Twenty-five patients with unilateral chronic anterior cruciate ligament deficiency had measures of hamstring contraction latency obtained before and after (3 and 6 months) reconstruction. After surgery, the contraction latency difference was found to improve significantly (decrease) in patients who had a preexisting deficit. The mechanism for alteration in response time remains unclear, but an observed relationship between contraction latency and tibial translation supports a mechanical basis for the findings. It was concluded the sensorimotor changes associated with surgical reconstruction of the cruciate ligament may help to restore joint stability. The study highlights the need to appreciate sensorimotor consequences of cruciate ligament surgery. PMID- 10738430 TI - The effect of preoperative gait analysis on orthopaedic decision making. AB - The impact of preoperative gait analysis on the orthopaedic care of 97 patients (101 gait analyses) at the authors' institution was evaluated. For the 70 patients for whom a specific treatment plan had been outlined before the preoperative gait study, the treatment plan was altered in 62 (89%) after the gait analysis study. In 10 of the 70 patients with specific treatment plans before the gait study, the referring physician also served as the physician in the gait laboratory; ultimate treatment was changed in nine of these 10 patients. Of the 273 surgical procedures recommended before the gait study in the 70 patients, 106 (39%) of these procedures were not done when the gait laboratory data were considered. An average of 1.5 procedures per patient that were planned before the gait study ultimately were not deemed necessary by the treating physician after the addition of the gait data. An additional 110 procedures (1.6 per patient) that had not been recommended before the gait study ultimately were performed after addition of the gait laboratory data. This study shows that ultimate surgical intervention frequently is altered by the addition of gait laboratory data. PMID- 10738431 TI - Cementless fixation for primary segmental bone tumor endoprostheses. AB - To combat the high incidence of aseptic loosening for young patients and for patients with failed implants after resection for bone tumors, intramedullary cementless fixation of massive tumor implants was investigated. These implants consist of a hydroxyapatite coated titanium stem. To date, 47 of these prostheses have been inserted for the treatment of primary bone tumors. Radiographs indicate that the stems are osseointegrated. Radiolucent lines have not been seen between the implant and the bone. Bone remodeling changes have been observed. In several cases in which the implant was not seated properly on the transaction site, bone grew to the shoulder of the implant. Bone remodeling was particularly evident in stems that were coated over their entire surface. In these cases, the implant induced local bone resorption so that the bone around the midstream region became thinner, with resorption of cortical bone on the periosteal surface and maintenance of bone on the endosteal surface adjacent to the stem. This effect was attributed to stress shielding, and a three-dimensional finite element model using loading data obtained from a telemetry study indicated that, where the stem was bonded to the bone over the entire surface, stresses in the outer cortex became reduced. In the finite element model, reducing the region of hydroxyapatite coating to approximately 1/3 of the stem length reduced the extent of the low-stress area in the outer cortex. Subsequently, prostheses have been coated with hydroxyapatite over only approximately 1/3 of their stem. This method of fixing the massive endoprosthesis to the bone is thought to be successful in the short-term and offers an alternative to cemented fixation. PMID- 10738432 TI - Sacropelvic resection for recurrent anorectal cancer. A multidisciplinary approach. AB - A multimodal approach including preoperative external beam radiation, surgical resection, and intraoperative electron radiation was used in 23 patients with locally advanced anal or recurrent rectal cancers involving the sacrum. The proximal extent of complete sacral resection was S2 in three patients, S3 in 12 patients, S4 in two patients, and S5 in one patient. The tumor was confined to the anterior sacral cortex in five patients. The resection was marginal in 10, contaminated marginal in 11, and intralesional in two patients. At 19 to 54 months of followup, five patients are alive without evidence of disease and four are alive with disease. Twelve patients died of their disease, and two died of other causes. There was a mean survival of 32.9 months for the patients who were alive at followup. Kaplan-Meier survival for all patients was 82% at 1 year and 73% at 2 years, with death of disease as an endpoint. Thirteen (57%) patients had another local recurrence develop at a mean of 17.2 months. Eight (35%) patients had metastatic disease develop at a mean of 16.3 months. Proper patients selection is important in ensuring a favorable outcome from this aggressive surgery. PMID- 10738433 TI - Population based epidemiologic and morphologic study of femoral shaft fractures. AB - The epidemiologic and morphologic features of all femoral shaft fractures in skeletally mature patients treated during a 10-year period in a semi-urban county were analyzed. Among an average adult population of 202,592 residents, 192 people sustained 201 traumatic femoral shaft fractures during the study period. The incidence was 9.9 fractures per 100,000 person-years. The highest age and gender specific incidences were seen in males from 15 to 24 years of age and in females 75 years of age or older. Seventy-five percent (151) of the fractures were the result of a high-energy trauma, 131 of which occurred in road traffic accidents. Unexpectedly, there were 50 low-energy fractures. Fractures of the middle 1/3 of the diaphysis were 79%. The majority, 155 (77%), of all fractures were transverse, oblique, or oblique transverse. Regarding the degree of comminution, the Winquist and Hansen Grade 0 (noncomminuted) fracture was the most common. Forty-eight percent of fractures were AO Type A, 39% were Type B, and 13% were Type C fractures. Of the 25 open fractures, 14 were Gustilo Type II. All six Type III open injuries were Type IIIA. Based on the data from the current study, most of the femoral fractures in this community might be treated adequately with conventional intramedullary nails, rather than using interlocking nails, provided the stability of fixation and fracture alignment can be maintained. Preventive measures against femoral shaft fractures should focus on protection of automobile drivers, especially young men, and on effective treatment of osteoporosis in elderly women. PMID- 10738434 TI - Salmonella osteomyelitis secondary to iguana exposure. AB - Salmonella osteomyelitis is a rare condition that has been associated with patients having hemoglobinopathies or immunosuppression. Healthy patients with no underlying medical history have been known to have Salmonella osteomyelitis develop. Salmonella infection secondary to reptile exposure is an increasing condition in the United States. Several manifestations of salmonellosis have been described in the literature, but no cases of reptile associated bone or joint infections have been reported. The authors present a case of a 7-month-old girl who contracted Salmonella osteomyelitis of her proximal humerus with septic arthritis of the glenohumeral joint secondary to iguana exposure. PMID- 10738435 TI - Comparison of antibiotic beads and intravenous antibiotics in open fractures. AB - This study compared the efficacy of antibiotic impregnated beads with conventional intravenous antibiotics in the treatment of open fractures. A randomized prospective study was designed and conducted during a 29-month period. Sixty-seven patients with 75 open fractures were treated similarly, with the exception of the method of antibiotic administration, and were followed up for at least 1 year after injury. Infection occurred in two of 24 (8.3%) fractures treated with antibiotic beads alone and in two of 38 (5.3%) fractures treated with conventional intravenous antibiotics. In an unanticipated nonrandomized third cohort group, patients received antibiotic beads and intravenous antibiotics administered for nonorthopaedic reasons or limb threatening injury, or both. Two of 13 (15.4%) fractures in this high risk group became infected. Infection ultimately resolved in all fractures treated with antibiotic beads alone or antibiotic beads in conjunction with conventional intravenous antibiotics. This study was unable to achieve statistical significance; however, the data suggest antibiotic beads may be useful in preventing infection in open fractures. Thus, a larger multicenter randomized prospective study of isolated open fractures, eliminating other variables, is justified. PMID- 10738436 TI - Trapeziometacarpal joint instability affects the moment arms of thumb motor tendons. AB - This study measured the changes in moment arm length of thumb motor tendons after simulated ligamentous instability and subsequent reconstruction of the trapeziometacarpal joint. Excursions of thumb motor tendons were measured simultaneously with the trapeziometacarpal joint angulation during flexion to extension and abduction to adduction motion. Tendon moment arms were calculated based on joint and tendon displacement techniques in the intact joint, after sequential sectionings of the capsuloligamentous restraints, and after the reconstruction procedure of Eaton and Littler. The results showed that moment arms of the abductor pollicis longus and extensor pollicis brevis tendons increased significantly as compared with those for normal joints during flexion to extension motion after sectioning the palmar capsuloligamentous components. After the ulnopalmar structures were cut, the moment arm of the extensor pollicis longus tendon had a statistically significant increase during abduction to adduction motion, and those of the extensor and flexor pollicis longus tendons decreased significantly during flexion to extension motion. Changed moment arms were restored to a normal level after the ligamentous reconstruction. These results indicate that ligamentous disruptions alter the mechanical balance of thumb motor tendons, which may contribute to joint deformities observed in trapeziometacarpal joint arthritis. Restoring joint stability is important to correct mechanical imbalance of the tendons. PMID- 10738437 TI - Effect of microwave oven induced mild hyperthermia on bone viability and strength. AB - Extracorporeal hyperthermia treatment of bone followed by reimplantation may be an option for treating bone tumors. However, intensive heat treatment, such as autoclaving, causes a decline of mechanical and biologic functions of bone tissue. In the current study, a microwave oven was used for minimal hyperthermic treatment, and it was found that complete eradication of all viable cells in rat bone could be achieved with minimal reduction in mechanical function. When the cells were evaluated histologically by special lactate dehydrogenase activity staining, complete bone cell death occurred after 60 seconds of heating in an empty Petri dish and after 30 seconds when heated in a Petri dish containing normal saline. Mechanical stiffness and strength of the bones, tested in three point bending, showed no decrease after this heating. Microwave oven induced hyperthermia eradication of viable cells without significant damage to the mechanical properties may have clinical relevance in limb salvage tumor surgery. PMID- 10738438 TI - Inhibition of inflammatory species by titanium surfaces. AB - Titanium has been used successfully for decades in orthopaedic and dental implants, but the mechanism mediating its biocompatible properties has not been elucidated. The authors investigated the possible role of titanium in modulating reactive oxygen mediators typically produced during the inflammatory response. Peroxynitrite is a highly reactive and unstable inflammatory mediator produced in vivo. This study found a 200% increase in the rate of degradation of peroxynitrite in the presence of titanium. To measure peroxynitrite reactivity, the nitration of 4-hydroxyphenylacetic acid by the peroxynitrite donor 3 morpholinosydnonimine was used. At a pH of 7.4, passivated titanium surfaces led to a 58% decrease of nitration of 4-hydroxyphenylacetic acid by peroxynitrite compared with controls in the absence of proteins. Surface treatments were found to influence the ability of titanium to inhibit peroxynitrite reactivity. Unpassivated titanium surfaces resulted in only a 10% decrease of nitrated 4 hydroxyphenylacetic acid, whereas titanium treated with hydrogen peroxide resulted in a 70% decrease. Decreases in nitration of 4-hydroxyphenylacetic acid also were seen with titanium in the presence of fibrinogen, 10% fetal calf serum, and sodium bicarbonate buffer. These results suggest that titanium is capable of enhancing the breakdown of the inflammatory mediator peroxynitrite and may account for the biocompatible properties of the material. PMID- 10738439 TI - Methodology for long-term wear testing of total knee replacements. AB - This article begins to address the validation requirements of wear testing on total knee replacements in a knee simulator. The knee simulator has four stations. The axial force is variable but reaches a maximum of 2.3 kN. Physiologic anteroposterior shear force and rotational torques are supplied to the knee. The forces and displacements are timed to coincide with those of a typical gait cycle. Kinematics of the simulator are dependent on the type of knee being tested. Tests of designs with well known clinical histories were done to 10 million cycles. The relative amounts and types of wear shown by the designs were similar to that found in their clinical histories. Wear tracks on more conforming designs were larger, and the penetration into the plastic appeared to be less. This did not necessarily mean that wear, as measured by loss of material, was reduced on conforming designs. Delamination of the plastic was achieved only after aging the tibial components. Wear particles isolated from the lubricating fluid were similar in size and shape to those isolated from in vivo specimens. However, the relative amounts of wear particle shapes were different depending on the design. At the start of the tests, all of the flexibly mounted tibial components showed more motion than after 5 million cycles, indicating that the surface of the plastic became more conforming. This study showed that knee wear similar to wear observed in vivo can be reproduced in the laboratory. The parameters and methods elucidated in this introductory study should form the basis for use in preclinical wear tests of total knee replacements. PMID- 10738440 TI - Development of a method to analyze orthopaedic practice expenses. AB - The purpose of the current investigation was to present a standard method by which an orthopaedic practice can analyze its practice expenses. To accomplish this, a five-step process was developed to analyze practice expenses using a modified version of activity-based costing. In this method, general ledger expenses were assigned to 17 activities that encompass all the tasks and processes typically performed in an orthopaedic practice. These 17 activities were identified in a practice expense study conducted for the American Academy of Orthopaedic Surgeons. To calculate the cost of each activity, financial data were used from a group of 19 orthopaedic surgeons in Houston, Texas. The activities that consumed the largest portion of the employee work force (person hours) were service patients in office (25.0% of all person hours), maintain medical records (13.6% of all person hours), and resolve collection disputes and rebill charges (12.3% of all person hours). The activities that comprised the largest portion of the total expenses were maintain facility (21.4%), service patients in office (16.0%), and sustain business by managing and coordinating practice (13.8%). The five-step process of analyzing practice expenses was relatively easy to perform and it may be used reliably by most orthopaedic practices. PMID- 10738441 TI - Shoulder pain in an 8-year-old girl. PMID- 10738442 TI - The fragrance of feminism. PMID- 10738443 TI - Prenotification in cases of death at home. PMID- 10738444 TI - "Sorry, doc, I forgot all about that". PMID- 10738445 TI - Alternative therapies. PMID- 10738446 TI - Overnight dialysis. PMID- 10738447 TI - The journey through the ICU. PMID- 10738448 TI - Career and parenting satisfaction among medical students, residents and physician teachers at a Canadian medical school. AB - BACKGROUND: Studies of career and parenting satisfaction have focused separately on medical students, residents and practising physicians. The objective of this study was to compare satisfaction across a spectrum of stages of medical career. METHODS: A survey of incoming medical students, current medical students, residents and physician teachers at the University of Saskatchewan was conducted in the spring of 1997. Response rates were 77% (43/56), 81% (177/218), 65% (134/206) and 39% (215/554) respectively. Factors assessed in the stepwise regression analysis were the effect of sex, parenting and level of training on the likelihood of recommending parenting to medical students or residents, and on parenting dissatisfaction, job dissatisfaction, career dissatisfaction and the importance of flexibility within the college program to accommodate family obligations. RESULTS: More male than female physician teachers had partners (92% v. 81%, p < 0.01) and were parents (94% v. 72%, p < 0.01). Female physician teachers spent equal hours per week at work compared with their male counterparts (mean 52 and 58 hours respectively) and more than double the weekly time on family and household work (36 v. 14 hours, p < 0.01). Physician teachers were the most likely respondents to recommend parenting to residents and their peers. Residents were the most dissatisfied with their parenting time. At all career stages women were less likely than men to recommend parenting, were more dissatisfied with the amount of time spent as parents and were more likely to regard flexibility within the college program as beneficial. There were no sex related differences in job dissatisfaction and career dissatisfaction. However, married women were more dissatisfied with their jobs than were married men. Job dissatisfaction was greatest among medical students, and career dissatisfaction was greatest among residents. INTERPRETATION: The optimal timing of parenthood appears to be upon completion of medical training. Women were less likely to recommend parenting, less satisfied with the time available for parenting and more likely to value flexibility within the college program to accommodate family needs. These differences did not translate into women experiencing more job or career dissatisfaction. PMID- 10738449 TI - Offering HIV prophylaxis to people who have been sexually assaulted: 16 months' experience in a sexual assault service. AB - The sexual assault service, operated by the Children's & Women's Health Centre of British Columbia in partnership with the Vancouver General Hospital Emergency Department, started offering HIV prophylaxis in November 1996 to patients presenting to the emergency department after a sexual assault. In the first 16 months of the program a total of 258 people were seen by the service, of whom 71 accepted the offer of HIV prophylaxis. Only 29 continued with the drug treatment after receiving the initial 5-day starter pack, and only 8 completed the full 4 week treatment regmen and returned for their final follow-up visit. Patients at highest risk for HIV infection (those who had penetration by an assailant known to be HIV positive or at high risk for HIV infection [men who have sex with men, injection drug users]) were more likely to accept prophylaxis and more likely to complete the treatment than those at lower risk. Compliance and follow-up were the main problems with implementing this service. Service providers found it difficult to give the information about HIV prophylaxis to traumatized patients. After this program evaluation, the service changed its policy to offer HIV prophylaxis only to people at high risk of HIV infection. This targeting of services is expected to make the service providers' jobs easier and to make the program more cost-effective while still protecting sexual assault victims against HIV infection. PMID- 10738451 TI - Would female inmates accept Papanicolaou smear screening if it was offered to them during their incarceration? PMID- 10738450 TI - Neonatal hypernatremic dehydration associated with breast-feeding malnutrition: a retrospective survey. AB - BACKGROUND: Hypernatremic dehydration in neonates is a potentially devastating condition. Recent reports have identified breast-feeding malnutrition as a key factor in its pathophysiology. METHODS: Using a theoretical framework for breast feeding kinetics, a retrospective chart review of all neonates less than 28 days of age who were seen at either British Columbia's Children's Hospital or the Vancouver Breastfeeding Centre between 1991-1994 was conducted to identify and classify possible causes of breast-feeding malnutrition among neonates who developed hypernatremic dehydration. RESULTS: Twenty-one cases hypernatremic dehydration were identified. Infant weight loss ranged from 8% to 30% of birth weight, and serum sodium levels ranged from 146 mmol/L to 207 mmol/L. In each case, maternal or infant factors (e.g., poor breast-feeding technique, lactation failure following postpartum hemorrhage and infant suckling disorders associated with cleft palate or ankyloglossia) that could interfere with either lactation or breast-feeding dynamics and account for insufficient breast milk intake were identified. INTERPRETATION: Prenatal and in-hospital screening for maternal and infant risk factors for breast-feeding malnutrition combined with early postpartum follow-up to detect excessive infant weight loss are important for the prevention of neonatal hypernatremic dehydration. PMID- 10738452 TI - Why don't more women report sexual assault to the police? PMID- 10738453 TI - Parenting, puppies and practice: juggling and gender in medicine. PMID- 10738454 TI - Health care problems in prisons. PMID- 10738455 TI - Is it feasible for women to perform their own Pap smears? A research question in progress. PMID- 10738456 TI - Professionalism: a contract between medicine and society. PMID- 10738458 TI - Africa's "future is frightening" because of HIV. PMID- 10738457 TI - Medicine under threat: professionalism and professional identity. PMID- 10738459 TI - Ovarian cancer in Canada. PMID- 10738460 TI - Quebec says "small is beautiful" when it comes to blood. PMID- 10738461 TI - Organizational depression. PMID- 10738463 TI - True fix. PMID- 10738462 TI - 2000 benchmarking guide. PMID- 10738464 TI - A beacon that would not fade. PMID- 10738470 TI - Lessons from the Westgate Bridge: suicide prevention and publicity. PMID- 10738471 TI - Defibrillation for out-of-hospital cardiac arrest. PMID- 10738472 TI - Preventing hepatitis C virus transmission in Australians who inject drugs. PMID- 10738473 TI - Hepatitis C virus antibody prevalence among injecting drug users at selected needle and syringe programs in Australia, 1995-1997. Collaboration of Australian NSPs. AB - OBJECTIVES: To describe point prevalence of HCV antibody and relevant risk behaviour among people who inject drugs and who attended selected needle and syringe programs throughout Australia in 1995, 1996 and 1997. DESIGN AND SETTING: Repeated cross-sectional surveys of one week's duration were carried out in 21, 20 and 23 needle and syringe program sites throughout Australia in 1995, 1996 and 1997, respectively. PARTICIPANTS: All clients attending participating sites during the designated survey week were asked to complete a self-administered questionnaire and provide a finger-prick blood sample for HCV antibody testing. MAIN OUTCOME MEASURES: Prevalence of HCV antibody. RESULTS: Survey response was 41% (n = 979) in 1995, 51% (n = 1463) in 1996 and 48% (n = 1699) in 1997. HCV prevalence declined significantly from 63% in 1995 to 51% in 1996 and 50% in 1997 (P < 0.001). Among respondents who reported injecting for less than three years, prevalence declined from 22% in 1995 to 13% in 1996 and 1997 (P < 0.001). Reported use of needles and syringes after someone else in the previous month declined from 31% in 1995 and 28% in 1996 to 15% in 1997 (P < 0.001). CONCLUSIONS: Despite an apparent decline in HCV prevalence, carriage rates of HCV antibody remain high. PMID- 10738474 TI - Trends in hospital readmission for asthma: has the Australian National Asthma Campaign had an effect? AB - OBJECTIVES: To describe patterns of hospital readmission for asthma in South Australia from 1989 to 1996, in relation to implementation of the National Asthma Campaign. DESIGN AND SETTING: A comparison of hospital admissions in South Australia of patients aged between one year and 49 years for three conditions: asthma (or respiratory failure with asthma as an underlying condition) and two control conditions--diabetes and epilepsy. Individuals were identified by Medicare number and date of birth. OUTCOME MEASURES: Hospital readmission within 28 days and within one year. RESULTS: Overall, by 1996, there was a statistically significant decline in the risk of readmission for asthma within 28 days of 18% and within one year of 17% compared with 1989 readmission rates. There were no reductions in the risk of readmission for diabetes or epilepsy, suggesting that the decline in risk of readmission for asthma was greater than the underlying effects of general changes in hospital casemix. CONCLUSIONS: The decline in risk of readmission may reflect changes in asthma severity or improved management practices. However, hospital readmission rates still remain high, and to further reduce readmissions for asthma there is a need to identify factors related to presentation for asthma at accident and emergency departments. PMID- 10738475 TI - Jumping from the Westgate Bridge, Melbourne. AB - OBJECTIVE: To describe characteristics of people who jumped from the Westgate Bridge (identifying risk factors for attempted suicide) and to determine why people may survive such a jump. DESIGN AND SETTING: A retrospective case review (coroners' reports and hospital records) of all people known to have jumped from the Westgate Bridge between 1991 and 1998. RESULTS: We identified 62 people who jumped from the Westgate Bridge over the study period. Seven survived. Forty-one (74%) of those who jumped were male. The average age was 33.8 years (range, 15-58 years). Forty-four (71%) had known mental illness (23 schizophrenia, 21 depression). Thirty-nine (63%) landed in water, falling from a height of 58.5 m. Nineteen (31%) fell onto land and in four cases (6%) the landing site was not determined. All survivors landed in water. Six people died from drowning after the fall, and in eight more deaths drowning was a major or contributing factor. All jumps resulted from suicidal intent, and 12 people (19%) had positive toxicology screens for alcohol or other non-prescription drugs at postmortem. CONCLUSIONS: Each year the Westgate Bridge is the scene of about eight suicide attempts by jumping (particularly by men with active psychiatric illness). Some deaths by drowning could be prevented by early detection and rapid emergency service response. The erection of an effective safety barrier would probably prevent more deaths. PMID- 10738476 TI - Phantom gallbladders. AB - The sensitivity of ultrasonography for detecting gallstones is high, but mishaps can occur. The three false positive cases we describe here illustrate problems which may be encountered in interpreting ultrasound images and in communication between the clinician, patient and ultrasonographer. PMID- 10738477 TI - Out-of-hospital cardiac arrest. AB - Out-of-hospital cardiac arrest (OHCA), with its high fatality rate, is a significant public health issue. The aetiology of OHCA is reviewed, and management strategies are discussed, including the "chain of survival", the Utstein method of data collection, and recent developments in advanced cardiac life support emphasising defibrillation. PMID- 10738478 TI - Risk management: how doctors, hospitals and MDOs can limit the costs of malpractice litigation. AB - The concerns of doctors regarding their risk of malpractice litigation and the costs of indemnity premiums are resulting in calls for legal reforms to limit their liability. We do not believe these returns will be successful either practically or politically. Medical defence organisations often attempt to vindicate the doctor rather than settle the dispute--a strategy that might be morally satisfying to doctors but which is also more expensive than the approach taken by commercial insurers. Risk management--the activities required to minimise financial loss for hospitals and the doctors who work in them--is disorganised or absent in most hospitals. Hospital managers lack incentives for risk management because the costs of litigation do not come out of their budgets. The five mainstays of effective risk management are credentialling of medical staff, incident monitoring and tracking, complaints monitoring and tracking, infection control, and documentation in the medical record. The implementation of risk management activities in hospitals is the immediate responsibility of hospital management, not doctors. PMID- 10738479 TI - Effect of ambulance 12-lead ECG recording on times to hospital reperfusion in acute myocardial infarction. AB - OBJECTIVE: To review the evidence that recording a prehospital 12-lead electrocardiogram (ECG) reduces time from hospital arrival to initiation of reperfusion therapy for acute myocardial infarction (AMI). DATA SOURCES: Medline search from 1966 to the present (articles in all languages) and examination of bibliographies. STUDY SELECTION: Published studies of prehospital 12-lead ECG recording that included control groups and reported time intervals from hospital arrival to start of reperfusion therapy. DATA EXTRACTION: Eight articles satisfied selection criteria (two randomised controlled trials, four non randomised interventional studies and two prospective observational studies). DATA SYNTHESIS: Widely varying study methodologies precluded meta-analysis. All studies had methodological problems, but hospital delays were consistently reduced. Such improvements appear to be small in hospitals where delays are already minimal. CONCLUSIONS: Little evidence is available to support routine prehospital 12-lead ECG recording if the median hospital time to reperfusion is already less than 30 minutes. Improvement of in-hospital treatment times may be a better initial strategy than prehospital 12-lead ECG recording, as this will benefit more patients and allow ambulance services to better allocate their available resources. PMID- 10738480 TI - New millennium, new images, new treatments. PMID- 10738481 TI - General practice in the year 2025. PMID- 10738482 TI - New directions for reproduction, obstetrics and gynaecology in Australia beyond 2000. PMID- 10738483 TI - Colorectal cancer: is the surgeon a prognostic factor? PMID- 10738484 TI - Colorectal cancer: is the surgeon a prognostic factor? PMID- 10738485 TI - The effects of bonus payments on emergency service performance in Victoria. PMID- 10738486 TI - Clinically active trachoma versus actual Chlamydial infection. PMID- 10738487 TI - Prevalence of exposure to hepatitis C virus among prison inmates, 1999. PMID- 10738488 TI - Consultation length and chronic illness care in general practice. PMID- 10738489 TI - Organophosphate poisoning versus brainstem stroke. PMID- 10738490 TI - Preventing neonatal HIV infection. PMID- 10738491 TI - Cardiovascular risk factors levels of Pacific people in a New Zealand multicultural workforce. AB - AIMS: To compare cardiovascular risk factors among the major Pacific Island communities participating in a New Zealand multicultural workforce survey. METHOD: There were 650 employed Pacific Island participants (Samoan 357, Cook Islands 177, Tongan 71, Niuean 45), aged 40-65 years, who were interviewed in a work-based, cross-sectional survey. During an oral glucose tolerance test, blood samples were collected for determination of blood glucose and serum lipids. Participants provided information on smoking and leisure time physical activity. Blood pressure, weight and height were measured and body mass index calculated. Ten-year risk of cardiovascular disease was calculated using equations from the Framingham study. RESULTS: Among men, their ten-year risk of a cardiovascular event was similar for the four communities compared (range 11.5% to 13.2%). However, individual risk factors did vary between the ethnic groups with Cook Island men having significantly higher total cholesterol, blood pressure and urinary microalbumin than other Pacific Island ethnic groups, while Tongan men were more likely to smoke and had lower HDL levels than other groups. Among women, Samoan and Cook Island participants had significantly higher ten-year cardiovascular risk scores (5.7%) than Niuean (4.4%) and Tongan (3.7%), due primarily to elevated total cholesterol levels. CONCLUSION: Cardiovascular risk factor levels vary between Pacific Islands communities in New Zealand. Targeted interventions to specific Pacific communities may be more beneficial than the current homogeneous prevention strategy applied to all communities. PMID- 10738492 TI - Cystic fibrosis diagnosed in adult patients. AB - AIM: To review the presentation, diagnosis and long-term, clinical follow-up of cystic fibrosis in adult patients diagnosed in adulthood at Green Lane Hospital. METHODS: A retrospective review of the case notes of patients with cystic fibrosis diagnosed in adulthood at Green Lane Hospital or referred there for management. Information was collected on diagnostic tests, including sweat tests and genotyping. Relevant family history was documented as were spirometry results and microbial colonisation. RESULTS: Six patients conclusively fulfilled the diagnostic criteria for cystic fibrosis. There was a wide range of ages at diagnosis (18-68) and half of the patients had a positive family history. A single mutation was identified in all, but in only one of the cases was the second mutation identified. All patients had evidence of bronchopulmonary suppuration and all had retained pancreatic function. Colonisation with P aeruginosa was associated with marked impairment in lung function. CONCLUSION: The patients at Green Lane Hospital represent part of the broad-spectrum disease in adult patients diagnosed with cystic fibrosis and highlight the differences between this group and those patients diagnosed in childhood with the more classical phenotype. Patients generally have less severe lung disease and retain pancreatic function. Sweat testing is useful diagnostically but gene testing is of limited value in making the diagnosis. PMID- 10738493 TI - Changes to infant sleep practices in Canterbury. AB - AIM: "Reducing the risk" is a public health primary initiative to minimise the incidence of Sudden Infant Death Syndrome (SIDS) in New Zealand. A number of SIDS risks relate to infant sleep practices. We describe current prevalences of these practices. METHODS: A cohort of Canterbury mothers delivering live infants during May 1997 (n = 411) were mailed a questionnaire in July surveying their infant's sleep practices. Survey results were compared to results derived from the Canterbury control infant component of the 1987-90 New Zealand Cot Death Study (NZCDS) (n = 174). Those mothers using either plastic or rubber mattress covers (n = 63) were issued a subsequent questionnaire pertaining to this mattress wrapping practice. RESULTS: Completed questionnaires were returned by 274 (66.7%) mothers. Room sharing with mother was usual for 133 (48.5%) infants, no different from the 94 (54.0%) recorded in the NZCDS (chi 2 = 5.6, df = 2, p = 0.06). However, of those infants sharing a room with their mother, 101 (75.9%) slept in their own bed compared to 46 (48.9%) in the NZCDS (chi 2 = 57.0, df = 2, p < 0.01). Only 8 (2.9%) infants were regularly placed prone to sleep, considerably fewer than the 69 (39.7%) reported in the NZCDS (chi 2 = 100.1, df = 1, p < 0.01). Mattress-wrapping with plastic (14.6% vs. 4.0%; chi 2 = 12.8, df = 1, p < 0.01) and rubber (8.4% vs. 3.4%; chi 2 = 4.4, df = 1, p = 0.04) has significantly increased since the NZCDS. Results from the subsequent questionnaire, completed by 42 (66.7%) respondents, indicated that most, 25 (59.5%), wrapped their infant's mattress to stop soiling. Less than half, 18 (42.9%), wrapped the mattress for the "safety of their baby". CONCLUSION: The "non-prone sleeping" campaign has been successful in Canterbury. Most infants are now routinely placed non-prone for sleep. Of those infants sharing a room with their mothers, an increased proportion is sleeping in separate beds. The use of "drycot" under blankets and sheepskins has diminished. While impermeable mattress-wrapping usage has significantly increased, over three-quarters of Canterbury mothers did not use plastic or rubber mattress-covers on their infant's beds. PMID- 10738494 TI - Methanol poisoning. AB - AIMS: This study examines clinical experience with methanol poisoning during a one-year period. METHODS: All admissions with the diagnosis of suspected methanol toxicity were analysed and the current guidelines for the management of this problem were reviewed. RESULTS: Twenty-four subjects were identified. Most had a history of chronic use of methylated spirits. Four died before admission to hospital and the other 20 patients had 26 admissions to hospital and form the basis for this report. Four patients died in the Intensive Care Unit. In total 11 patients were admitted to the Intensive Care Unit. Seven patients received haemodialysis. There was no correlation between the methanol level and the outcome. The strongest predictor of death or a poor outcome was a blood pH < 7.0. Some patients, in spite of potentially lethal methanol levels of up to 160 mmol/L, did not develop signs of toxicity. CONCLUSIONS: The overall mortality was high and ethanol was given to most of the patients for up to several days. Some patients did not show any toxicity and some of those were not given ethanol. It is recommended that chronic meths drinkers, who are not acidaemic and are generally well, do not require ethanol treatment. Only the complete removal of methanol from methylated spirits will reduce the morbidity of this condition. PMID- 10738495 TI - Variability within general practitioner prescribing over time. AB - AIMS: We have described intra-general practitioner (GP) prescribing variability over time in terms of volume, cost and average item cost of prescription items, within New Zealand general practice. METHODS: Longitudinal data over the financial years 1992-94 were studied for two GP samples. Prescription data for a regional sample of 305 GPs were obtained for the first six months (January to June) from the New Zealand pharmaceutical pricing office, Health Benefits Limited. Prescription data from a second national sample of 74 GPs were obtained from the PreMeC prescription analysis (PAS) database of GPs who had participated in three consecutive September to December prescription analyses. The coefficient of variation was used to measure the intra-GP variability over time in total prescription cost, volume of prescription items and average prescription item cost. RESULTS: The median intra-GP variability over time for the regional GP sample, based on reimbursement data, was 9% in total cost, 9% in total volume and 5% in average item cost. The median intra-GP variability in the national sample was very similar to the regional sample when based on reimbursement data, but when PAS data were used the variability was 16% in total cost, 17% in total volume and 8% average item cost. CONCLUSIONS: The year-on-year, intra-GP variability for cost was 9%, for volume 9-10% and for average item cost 5-6%. Pharmaceutical budget estimates should reflect year-to-year intra-GP prescribing variability of the order of 9%. PMID- 10738497 TI - Dr Bob Tennent and Leptospirosis. PMID- 10738498 TI - Impact of changes in the death registration process upon Maori mortality statistics. PMID- 10738496 TI - ACC and back injuries: the relevance of pre-existing asymptomatic conditions. AB - The purpose of the ARCI (1992) and AI (198) Acts is to cover those who suffered from personal injury by accident. This purpose should not be distorted. This review does not aim to suggest that cover be extended to victims of disease. Equally it is inappropriate that imaging be used to detect asymptomatic, age related change--or reveal asymptomatic abnormalities that do not place the patient at increased risk of symptoms over the general population--so as to unfairly deny coverage to the victims of accidents. This is particularly important to those who suffer personal injury to the lumbar spine where the cause is wholly or substantially an accident, and in whom, without the specified accident having occurred, personal injury (symptoms and disability) would have not been likely to occur. Physicians managing spinal disorders must correlate clinical findings with imaging studies when planning treatment. Those considering entitlement for cover under third party/ACC provisions must pay close attention to the history and clinical evaluation, correlating these with the investigation findings, and not assume that the abnormalities found on sensitive investigations are the cause of the symptoms. PMID- 10738499 TI - Case report: 'necrotic araneism?'. PMID- 10738500 TI - [Treatment of desmoid tumors of the mesenteric root]. AB - OBJECTIVE: To elaborate a therapeutic scheme for desmoid tumors developing within the mesenteric root. These uncommon and often unrecognized tumors are difficult to treat as they lie very close to the superior mesenteric vessels. PATIENTS AND METHODS: Retrospective analysis of 14 cases treated in our center over the last 20 years and a review of the literature. RESULTS: The analysis led to the development of a therapeutic scheme. Patients are placed under regular surveillance to detect any progression of the mesenteric root desmoid tumor. In case of progression, surgery should be proposed as often as possible in spite of the real difficulty of this surgery. When the tumor is unresectable, hormone therapy, sulindac, chemotherapy and radiotherapy can be proposed as appropriate. CONCLUSION: Based on the available literature, the proposed decisional tree is a helpful management tool. This scheme should be validated with the help of a National Observatory focusing on this rare disease. PMID- 10738501 TI - [Coinfection with HIV and HTLV-I infection and survival in AIDS stage. French Guiana Study. GECVIG (Clinical HIV Study Group in Guiana)]. AB - OBJECTIVE: The purpose of this study was to investigate the effect of HTLVI infection on survival in AIDS patients in French Guiana. PATIENTS AND METHODS: A cohort of 151 adult patients with AIDS were followed from January 1992 through June 1996. Kaplan-Meier survival curves were established. Using the Cox model, multivariate analysis was performed to examine different factors affecting survival. RESULTS: The incidence of HTLVI infection in this cohort was 11.9% and 57.6% of the patients died during the study period. Multivariate analysis disclosed that older age at diagnosis of AIDS (over 45 years) and low CD4 count (< 100/mm3) were predictors of poor survival. HIV-HTLVI co-infection was strongly correlated with reduced survival (p = 0.02; RR = 2.2; CI = 1.1-4.5). CONCLUSION: In our region, all patients with HIV infection should be screened for HTLVI infection. In case of co-infection, early care should included adapted antiretroviral regimens. PMID- 10738502 TI - [Agranulocytosis in acute hepatitis B in an HIV seropositive patient]. AB - BACKGROUND: During the course of acute hepatitis B, hematology disorders are common though they are generally mild and occur early. Agranulocytosis is exceptional and occurs late in the disease course. CASE REPORT: We report a case of agranulocytosis which developed 3 weeks after onset of acute hepatitis B in an HIV-positive patient. Peripheral and central hematological disorders led to the diagnosis. Agranulocytosis developed during the cytolytic phase of the primary hepatitis B infection and regressed after administration of hematopoietic growth factors. DISCUSSION: It is sometimes difficult to establish the causal effect of hepatitis B in the development of agranulocytosis in patients with an HIV co infection who are on a multiple drug regimen and subject to multiple bacterial, viral or parasite infections. PMID- 10738504 TI - [Pyoderma gangrenosum simulating erysipelas]. PMID- 10738503 TI - [Malignant melanoma in the child: an uncommon illness]. AB - BACKGROUND: Malignant melanoma are uncommon in children and are sometimes difficult to distinguish from Spitz nevi. Histological diagnosis is particularly difficult. CASE REPORT: Three prepuberty children (aged 14, 10 and 14 years) underwent excision of suspicious nevi found at pathology examination to be malignant melanomas. After complementary treatment the recurrence-free survival has been 18, 14 and 4 years respectively. DISCUSSION: Clinical signs of malignant melanomas are unspecific. As in adults, any modification in a melanic lesion should led to excision and pathology examination. In the literature, there is often a confusion between malignant melanoma developing in children and young adults making it difficult to determine the prognosis of the childhood disease. Prognosis of cutaneous malignant melanomas is generally good (excluding giant nevi and congenital malignant melanoma). PMID- 10738505 TI - [Surgery of the central nervous system: value of preoperative functional brain mapping by direct electric stimulation]. PMID- 10738507 TI - [Can lorazepam prevent recurrent epileptic crises induced by alcohol?]. PMID- 10738506 TI - [Diagnostic and therapeutic value of ambulatory post-voiding bladder ultrasound imaging in evaluating neurogenic bladder dysfunction]. PMID- 10738508 TI - [Diagnosis of hemochromatosis in the era of gentic testing]. PMID- 10738509 TI - [Lipid derivatives of amphotericin B in treatment of systemic candidiasis]. PMID- 10738510 TI - [Apropos of amiodarone: both best and worst]. PMID- 10738511 TI - [Capillary hyperpermeability syndrome. Another case]. PMID- 10738512 TI - [Symptomatic treatment of nephrotic syndrome]. AB - OBJECTIVES OF SYMPTOMATIC TREATMENT: The goal is to maintain quality of life, prevent immediate complications (thromboembolic events, infection, drug reactions), prevent late complications related to atherosclerosis, and limit the progression of the chronic renal failure. THERAPEUTIC ARMAMENTARIUM: Six categories can be described. i) A reduction in proteinuria, essential for controlling the intensity of other manifestations, can be improved with a normal protein content (1 g/kg ideal weight/d) low-salt diet, strict blood pressure control, and most importantly, CEI given alone or in combination with AA2. ii) Restoration of a normal extracellular fluid (edema and high BP) can be achieved by low sodium intake and loop diuretics in fractionated increasing doses (sometimes with combination regimens). It is advisable to keep blood pressure below 125/75 mmHg. iii) Prevention of thromboembolic events (risk level dependent on urine protein output) relies on antivitamin K anticoagulants and low-molecular weight heparins. iv) Adapted prescription of protein-bound drugs. v) Lowering LDL cholesterol, a risk factor for atherosclerosis, with an adapted diet and HMG CoA inhibitors. vi) Prevention of chronic renal failure. The development and course of chronic renal failure depend not only on the histological glomerular lesion and/or the etiology but also on supplementary glomerular and tubulointerstitial damage directly related to the degree of proteinuria. MORE THAN SYMPTOM RELIEF: Symptomatic treatment of nephrotic syndrome must be considered as an integral part of a rigorous goal-oriented therapeutic strategy. PMID- 10738513 TI - [Breast cancer: value of postoperative locoregional irradiation following mastectomy]. AB - A CRUCIAL STEP: Locoregional control is a crucial step in the achievement of cancer cure. After mastectomy, locoregional irradiation (RT) clearly reduces the incidence of chest wall and nodal relapse, especially with initial lesions measuring more than 5 cm or with nodal involvement and/or large lymphatic or vascular emboli. CLINICAL PROOF: Two recent randomized trials have confirmed the benefit of well-adapted locoregional irradiation. In the Danish trial, including premenopausal "high-risk" women treated by mastectomy and chemotherapy (CMF protocol), RT reduced locoregional relapses from 32% to 9% (p < 0.01) and increased the 10-year survival rate from 45% to 54% (p < 0.01). These results are now confirmed in postmenopausal women with an increase in the 10-year survival rate from 36% to 45% (p < 0.001). In the Canadian trial, locoregional relapses decreased from 25% to 13% and the 10-year survival rate increased from 56% to 65%. The meta-analysis published in 1995 by the EBCTCG showed only a modest benefit due to locoregional irradiation in breast cancer. However, when small trials and old trials started before 1970 were excluded because of imperfect methodologies and inadequate irradiation techniques, the benefit of "modern" radiotherapy appeared as significant in 7,840 patients selected in this way. IN CLINICAL PRACTICE: Thus, since locoregional irradiation can avoid some metastatic evolution developed only after "local" or "nodal" relapse, it must be integrated into a multidisciplinary strategy. Nevertheless, this treatment must be safe. This can be achieved with new irradiation techniques including the definition of anatomical volumes and previsional dosimetry. The most important point concerns the treatment of internal mammary nodes, especially when previous chemotherapy including anthracyclines has been performed. The use of a direct field, with at least 40% of the dose delivered by electrons in an alternating scheme is recommended to ensure very good protection of the heart and lungs. PMID- 10738514 TI - [Paraneoplastic neuropathies]. AB - RARE AND INAUGURAL: Paraneoplastic neuropathies (PN) are rare, affecting approximately 4-5% of patients with cancer. Their diagnosis is difficult because the clinical picture is nonspecific and because the neuropathy precedes the discovery of the cancer in a majority of patients. SUBACUTE SENSORY NEUROPATHY (SSN): Initially described by Denny-Brown, subacute sensory neuropathy is characterized by a severe inflammatory loss of the dorsal roots ganglia. The clinical hallmark is a severe, asymmetric sensory neuropathy progressing over a few weeks to months until the patient is bedridden. The underlying cancer is most often a small-cell lung cancer (SCLC) and the presence of anti-Hu antibodies is almost specific of SSN associated with SCLC. MOTOR NEURON DISEASES: Motor neuron diseases are rarely of paraneoplastic origin save for subacute sensory neuropathy associated with Hodgkin's disease. SENSORIMOTOR NEUROPATHIES: Sensorimotor neuropathies are the most frequent PN but constitute a hetereogeneous group. Acute Guillain-Barre syndrome occurs in patients with Hodgkin's syndrome. Paraneoplastic chronic inflammatory demyelinating polyneuropathy probably exists during lymphomas and carcinomas but a fortuitous association has not been formally excluded. Paraneolastic peripheral nerve microvasculitis often presents as mononeuritis multiplex and complicates mainly lymphoma and SCLC. Other PN are generally of the chronic and axonal type. Among them are the so-called "terminal neuropathies" which are linked to weight loss. AUTONOMIC NEUROPATHIES: Autonomic neuropathis occur during SCLC or Hodgkin's disease. Their most frequent clinical presentation is a pseudo-obstruction which is rarely isolated but which generally occurs as part of diffuse encephalomyelitis. PMID- 10738515 TI - [Chief health risks associated with intravenous heroin and cocaine abuse]. AB - THREE CATEGORIES OF COMPLICATIONS: Heroine and cocaine are the main drugs used by injecting drug-users (IDU) in France. There are three categories of complications associated with intravenous drug abuse: effects related to drug toxicity, effects associated with the administration route, and effects associated with social implications of drug-dependence. OTHER HEALTH PROBLEMS: The impact of HIV infection among IDU, particularly in southern Europe, has led to renewed interest in the other health problems raised by this population. A review of recent literature shows that cocaine toxicity is better understood than heroine toxicity. In addition, intravenous drug abuse can lead to a whole series of disease states related to trauma or infection. PMID- 10738516 TI - Detailed haplotype analysis in Ashkenazi Jewish and non-Jewish British dystonic patients carrying the GAG deletion in the DYT1 gene: evidence for a limited number of founder mutations. AB - The DYT1 gene on human chromosome 9q34 appears to be responsible for most cases of early onset primary torsion dystonia (PTD) both in Ashkenazi Jewish (AJ) and in non-Jewish patients. Previous haplotype analysis in a 2 cM region surrounding the DYT1 gene showed that a single founder mutation (DYT1AJ) was responsible for most cases of early onset PTD in the North American AJ population and refined the most likely location of the gene to a 150 kb interval between the marker loci D9S2161 and D9S63. Recently, the majority of cases of early onset PTD in both AJ and non-Jewish patients were found to carry a unique 3-bp (GAG) deletion in the coding region of the DYT1 gene. This deletion appears to have arisen more than once, suggesting independent mutational events. In this study, we analysed the haplotypes surrounding DYT1 in 9 AJ and 15 non-Jewish British patients carrying the GAG deletion in the DYT1 gene. We found that all AJ British patients carried the same haplotype as the North American Jews, sustaining the theory that the current British AJ community descends from the same small group of individuals as the North American Jewry. Furthermore, in the non-Jewish British patients, only a limited number of distinct founder mutations was observed. This supports the hypothesis that the GAG deletion in the DYT1 gene is not a very frequent mutation, and that it has arisen only a limited number of times throughout the centuries. PMID- 10738517 TI - Mucopolysaccharidosis type I: characterization of a common mutation that causes Hurler syndrome in Moroccan subjects. AB - A group of 13 Moroccan patients with MPS I and their families, including three siblings and twin siblings, was screened for mutations of the alpha-L-iduronidase gene using fluorescence-assisted mismatch analysis (FAMA) and cycle sequencing of PCR products. The P533R mutation, which is rare in Europeans, was identified in 92% of mutant alleles (24/26). This is the highest frequency of this mutation detected in patients with Hurler syndrome. None of the patients carried the W402X or Q70X alleles, the most common MPS I mutations in Europeans. These results suggest that the P533R mutation constitutes the genetic lesion which results in MPS I in people of Moroccan descent and provides yet more evidence for the uneven geographical distribution of mutations in MPS I. PMID- 10738518 TI - Combined segregation and linkage analysis of nonsyndromic orofacial cleft in two candidate regions. AB - We applied a complex segregation analysis to 46 pedigrees with a total of 121 nuclear families and 660 individuals, to verify hypotheses regarding the inheritance of OFC and linkage with markers on chromosomes 6 and 2. The POINTER program for segregation analysis strongly rejected the hypothesis of no familial transmission of OFC in these families. When the hypothesis of a two-locus model was tested with COMDS, the analysis showed the presence of at least two loci and the model assuming a dominant major gene and a recessive modifier locus was statistically accepted. Given the fitted two-locus model, we tested for a possible linkage between the major OFC locus and the two markers studied. For D6S259, the estimate of the recombination fraction was theta = 0.098, corresponding to a LOD score around 2.1. On the contrary, the data analysis concerning the D2S378 marker showed an estimate of the recombination fraction not significantly different from the independence hypothesis. PMID- 10738519 TI - Locus and population specific evolution in HLA class II genes. AB - The population genetics of the HLA class II loci was studied with reference to variation in the frequency of (a) alleles at a locus and (b) amino acids at specific sites. Variation was surveyed at 4 loci (DRB1, DQA1, DQB1, and DPB1) in 22 populations from the Twelfth International Histocompatibility Workshop (Saint Malo, 1996). Allele and amino acid variation was measured by computing heterozygosity and the effective number of alleles. Substantial variations in polymorphism were observed among the various populations and loci studied. In the majority of the populations, DRB1 has the highest heterozygosity and effective number of alleles. As previously shown, the Amerindian populations have lower levels of allelic diversity when compared to other populations. At the amino acid level, DRB1 antigen recognition sites (ARS) have the highest heterozygosities and effective number of alleles. For the other loci (DPB1, DQA1, and DQB1) for which there is no crystal structure and for which ARS sites were inferred from DRB1, non-ARS sites were often among the sites with highest levels of variation. It is possible that these putative non-ARS sites do play a role in antigen presentation. The homozygosity test for neutrality was applied to allele and amino acid data. Of the four HLA class II loci studied, only DPB1 failed to show evidence of balancing selection. DQB1 and DQA1 depart significantly from neutrality in the largest number of populations. Genetic distances between populations were computed based on frequency of alleles and amino acids at ARS sites. PMID- 10738520 TI - Evolution of the apolipoprotein B gene and coronary artery disease: a study in low and high risk Asians. AB - This study traces the evolutionary pathways of the apolipoprotein B gene in the low risk Chinese and high risk Asian Indians in relation to coronary artery disease (CAD). Haplotypes were constructed from six apoB polymorphisms sp24/27, Ag(c/g), Ag(a1/d), XbaI, Ag(h/i) and, Ag(t/z). These were genotyped from 474 Chinese (253 healthy, 221 CAD patients) and 248 Asian Indians (164 healthy, 84 CAD patients). The maximum parsimony method was used to construct a cladogram for each ethnic group. Three haplotypes in the Chinese and one in the Indians were found exclusively in CAD patients. These haplotypes were sp27ca1X-ht, sp24ga1X-iz and sp24ca1X + it in Chinese, and sp24cdX-it in Indians. Those in the Chinese all occurred as terminal haplotypes and represented the most recent mutations. Evolutionary pathways for both ethnic groups were similar for majority of the haplotypes except for the presence of an additional third branch in the Indians arising from the ancestral haplotype. However, this third branch does not appear to contribute to the susceptibility of the Indians to CAD. PMID- 10738521 TI - An Asian-Native American paternal lineage identified by RPS4Y resequencing and by microsatellite haplotyping. AB - Human paternal population history was studied in 9 populations [three Native American, three Asian, two Caucasian and one African-derived sample(s)] using sequence and short tandem repeat haplotype diversity within the non pseudoautosegmal region of the Y chromosome. Complete coding and additional flanking sequences (949 base pairs) of the RPS4Y locus were determined in 59 individuals from three of the populations, revealing a nucleotide diversity of 0.0147%, consistent with previous estimates from Y chromosome resequencing studies. One RPS4Y sequence variant, 711C > T, was polymorphic in Asian and Native American populations, but not in African and Caucasian population samples. The RPS4Y 711C > T variant, a second unique sequence variant at DYS287 and nine Y chromosome short tandem repeat (YSTR) loci were used to analyze the evolution of Y chromosome lineages. Three unambiguous lineages were defined in Asian, Native American and Jamaican populations using sequence variants at RPS4Y and DYS287. These lineages were independently supported by the haplotypes defined solely by YSTR alleles, demonstrating the haplotypes constructed from YSTRs can evaluate population diversity, admixture and phylogeny. PMID- 10738522 TI - Relative efficiencies of the chi-square recombination models for gene mapping with human pedigree data. AB - In the paper by Goldstein et al. (Genomics, 1995), the authors carried out a simulation study to investigate the relative efficiencies of a no interference linkage analysis to an analysis with certain models that allow for interference. They showed that, for completely informative and independent recombination data, the analysis with the no interference model was inefficient, in the present of interference. In practice, the assumption of completely informative markers is unrealistic with data from human pedigrees. We report the results of a study investigating whether this conclusion still holds for gametes arising within pedigrees. We consider the same two mapping problems as Goldstein et al.: exclusion mapping and gene ordering. The results obtained were consistent with their findings, although the efficiency gains for analyses using the chi-square model were not as great in some cases. This is not unexpected with less than fully informative data. These results point to the need for research of developing new statistical and computational methods to incorporate interference into multipoint linkage mapping using pedigree data. This would make efficient use of available, but sometimes scarce data, especially in disease gene mapping. PMID- 10738523 TI - Report and abstracts of the Sixth International Workshop on chromosome 9. AB - A meeting on chromosome 9 was held on Tuesday, 27 October 1998 in Denver, with 38 participants (see appendix). Since the last meeting several of the positional cloning efforts on chromosome 9q have come to fruition, and the most detailed discussion was on 9p. Dr Ian Dunham from the Sanger Centre explained the strategy to be used for sequencing chromosome 9, and encouraged collaboration in the preparatory mapping. He indicated that some priority could be given to those regions where people in the field had a strong interest and could identify relevant PAC clones. At this short meeting it was clearly not possible to construct a comprehensive map of chromosome 9, and it was decided that efforts should be made to maintain links to sources of information on the chromosome 9 web page (http:@www.gene.ucl.ac.uk/chr9/). The discussions at the meeting are summarized in four sections: 9p, 9cen-q31, 9q32-9q34 and comparative mapping. Many of the posters presented at the meeting were also presented at the ASHG meeting (28-31 October 1998). They are listed here and are published in The American Journal of Human Genetics, vol. 63 (supplement). Abstracts for posters presented only at this meeting are appended to this report. PMID- 10738524 TI - Identification and characterisation of polymorphisms in human phosphoglucomutase (PGM1). AB - This study is part of our effort to map recombination hotspots in two regions (site A, 18 kb; site B, 40 kb) of the human phosphoglucomutase PGM1 gene. Twenty two PCR amplified fragments comprising six groups, covering about 5.2 kb, were screened for single nucleotide polymorphisms (SNPs) using non-isotopic single stranded conformation polymorphism (SSCP) analysis. Fourteen fragments were variable and seven of these showed common polymorphism. Our strategy for screening for polymorphic sites in the PGM1 gene was based on the results of allelic association analysis between each new marker and the sites of the classical isozyme polymorphism (2/1 in exon 4 and +/- in exon 8). Samples from four populations (Caucasian, Chinese, Vietnamese and New Guinean) were typed for each of the seven polymorphic markers. Between two and four common alleles were found in each case, together with a few rare alleles. Co-dominant inheritance patterns were demonstrated by family studies. The molecular basis of each new marker was determined by direct sequencing of the PCR products: most were SNPs except two that were small insertions/deletions. Direct sequence analysis of a 2.1 kb segment in sixteen individuals revealed no additional nucleotide variation indicating a very high level of efficiency of the SSCP screening method used in this study. The overall nucleotide diversity (theta) for PGM1 was estimated as 0.9 x 10(-3) based on 33 segregating sites in a sequence of 5187 nt and a sample size of 614 individuals. PMID- 10738525 TI - Enzymatic characterization of four new mutations in the glucose-6 phosphatase (G6PC) gene which cause glycogen storage disease type 1a. AB - Glycogen storage disease type 1a (GSD1a) is caused by mutations in the gene of glucose-6 phosphatase (G6PC), encoding the last enzyme of gluconeogenesis and glycogenolysis. To study the effect of mutations previously identified, but not yet enzymatically characterized, in French GSD1a patients, we used an in vitro expression system of the human glucose-6 phosphatase (hGlc6Pase) cDNA. Wild type hGlc6Pase expressed in COS-7 cells exhibited kinetic features comparable to microsomal Glc6Pase from normal human liver and kidney. Four new mutations inducing aminoacid changes in the coding sequence, e.g. W77R, A124T, G184E and L211P, were inserted into the Glc6Pase cDNA by site-directed mutagenesis, and studied after transient expression in COS-7 cells. All four mutations totally abolished Glc6Pase activity. PMID- 10738526 TI - Supportive evidence for contribution of the dopamine D2 receptor gene to heritability of stature: linkage and association studies. AB - Increases in height were reported in children chronically exposed prenatally and postnatally to D2 receptor-blocking drugs. A possible haplotypic association between stature and the DRD2 gene was also reported. In this study, we examined linkage between stature and DRD2 by genotyping a dinucleotide repeat polymorphism in 79 sib-pairs aged 8-17 years. An association between stature and a putative functional polymorphism in the promoter region of the DRD2 gene was examined in the sib-pairs and in 125 unrelated male adults. All the subjects were Japanese. Linkage (p = 0.004, SIBPAL) and an association (p = 0.009, paired t-test, in the sib-pairs; p = 0.006, ANOVA, in the adults) with stature were suggested. These findings indicate that DRD2 is one of the genes that contribute to heritability of stature. PMID- 10738527 TI - Y-chromosome specific YCAII, DYS19 and YAP polymorphisms in human populations: a comparative study. AB - Two hypervariable Y-specific markers, the YCAII and DYS19 STRs, and the more stable Y Alu Polymorphism (YAP) have been analysed in about 1400 individuals of 21 different populations, mainly from Europe but also from the Middle East, Africa and Asia. On the basis of the frequency distributions of these three Y markers we compare, using different statistical analyses, their power in detecting population genetic structure and in distinguishing closely related groups. The pattern of populations' genetic affinities inferred from the three markers considered altogether suggests a strong genetic structure that, with a few exceptions, broadly corresponds to the linguistic relatedness and/or geographic location of the sampled populations. PMID- 10738528 TI - Assessing linkage disequilibrium in a complex genetic system. I. Overall deviation from random association. AB - Linkage disequilibrium is an important tool both at the end stages of positional cloning studies to map genes of particular interest and in reconstruction of population histories. With advances in molecular biology, complex genetic systems involving multiple highly polymorphic markers are becoming more commonly used to study linkage disequilibrium. These systems contain much more genetic information than simple two marker systems. In this article, we introduce a measure to summarize the overall deviation from random association and propose a permutation based estimate for this measure. The performance of the proposed estimation procedure is studied through simulations. The methods proposed in this paper are then applied to population data at the dopamine D2 receptor locus (DRD2) and at the homeobox B (HOXB) gene cluster. PMID- 10738529 TI - The human dendritic cell marker CD83 maps to chromosome 6p23. AB - The chromosomal localization of the human CD83 gene was determined using somatic cell hybrids, a radiation hybrid mapping panel and FISH analysis on human metaphase chromosomes. PCR-based analysis of a single chromosome hybrid panel identified the presence of the CD83 gene on human chromosome 6 and subsequent analysis of the Genebridge4 radiation panel located the gene between AFMa192wg9 and AFMb322wd1 with a lod score of 9.2. Finally, using FISH analysis the CD83 gene was localized to chromosome 6 band p23. PMID- 10738530 TI - MUC1 gene polymorphism does not explain the different incidence of gastric cancer in Portugal and Denmark. AB - MUC1 is a highly polymorphic mucin type glycoprotein expressed on the surface of many epithelia, including gastric mucosa, and is present in several body fluids and mucous secretions. A genetic polymorphism due to variation in length of a 60 bp tandemly repeated sequence domain constitutes more than half of the coding region of the glycoprotein. We demonstrated previously in a Portuguese population sample that the frequency of small MUC1 alleles is increased in patients with gastric carcinoma, suggesting that the possession of small MUC1 alleles confers increased risk for gastric carcinoma development. This finding raised the possibility that the very high prevalence of gastric carcinoma in Portugal could be partly due to a high frequency of small MUC1 alleles in the Portuguese population. In the present study we compared the MUC1 allele distribution in a population of Danish blood donors with the distribution in a population of Portuguese blood donors. The frequency of small MUC1 alleles was significantly higher in the Danish than in the Portuguese sample, thus failing to lend support to the hypothesis that a relatively higher frequency of the small MUC1 alleles might account for the high prevalence of gastric carcinoma in Portugal when compared to Denmark. PMID- 10738531 TI - Rapid and simple determination of hereditary haemochromatosis mutations by multiplex PCR-SSCP: detection of a new polymorphic mutation. AB - Hereditary haemochromatosis is a common inherited disorder leading to excessive accumulation of iron in various organs. Two missense substitutions at the HFE gene have recently been associated with the disease, 187C G and 845G-->A (mutations H63D and C282Y, respectively). We present a simple, rapid PCR-SSCP multiplex screening method allowing the simultaneous detection of both substitutions. Furthermore, testing the method on 420 Danish blood donors revealed the presence of a hitherto undetected third substitution in 13 individuals. The new substitution, a 193A-->T transversion, affects codon 65 changing the code for serine to that of cysteine (S65C). It may thus have functional consequences for the HLA class protein encoded by the HFE-gene. The allele frequencies observed were: H63D 14.8%, C282Y 6.2% and S65C 1.5%, which for the two former alleles are in agreement with frequencies reported for other North European population samples. PMID- 10738532 TI - Mosaic Turner syndrome: cytogenetics versus FISH. AB - Twenty-two cases with Turner syndrome features were subjected to standard cytogenetic techniques using giemsa trypsin (GTG-) banding then fluorescence in situ hybridization (FISH) using a specific whole-X chromosome painting probe, Quint-Essential Y-specific DNA probe (AMELY) for Yp11.2, alpha-satellite (DYZ3) probe and X/Y cocktail-alpha satellite probe (ONCOR) for confirmation of the initial diagnosis and comparison of the two techniques. Eight cases (36%) showed the same karyotype results by both techniques [5 cases: 45,X/46,XX, 2 cases: 45,X/46,X,i(Xq) and one case with a triple cell line 45,X/46,XX/47,XXX]. In the other 14 cases (64%) the FISH technique has identified a third cell line in 7 cases (32%), delineated the origin of the marker in 5 cases (23%) to be derivative X and clarified the deletion of the Yp11.2 region in 2 cases (9%) with the 45,X/46,XY karyotype. The application of FISH has highlighted the differences between the initial diagnosis based on the standard cytogenetic technique and the final diagnosis determined by the application of DNA probes specific for the X and Y chromosomes. FISH proved useful in detection of the low frequency cell lines which need analysis of a large number of metaphase spreads by GTG-banding, helped in identifying the nature and the origin of the unknown markers which has an important implication in the development of gonadal tumours and delineated the deletion of the Yp11.2 region in the 45,X/46,XY Turner patients. PMID- 10738533 TI - Linkage disequilibrium between intra-locus variants in the aminopeptidase n gene and test of their association with coeliac disease. AB - Coeliac disease (CD) is a multigenic and multifactorial enteropathy triggered by gluten-composing proteins. A possible involvement of the intestinal Aminopeptidase N (APN) was investigated by an association analysis. SSCP analysis detected four variants at position 281, 378, 956 and 2957 (referred to no. g178535, GenBank) that were studied in 193 Italian CD families. The haplotypic combinations were determined from family segregation and pairwise linkage disequilibria (D' = D/Dmax) between the polymorphic sites were calculated. Significant D' values ranged between 0.78 and 0.31. Association with CD was tested by TDT (Transmission Disequilibrium Test) utilizing as markers the nucleotide substitutions and their haplotypic combinations. No statistically significant transmission distortion to the probands or to their clinically silent sibs was observed. Our data exclude an involvement in CD of the tested markers and of further undetected variation in strong linkage disequilibrium (D' approximately equal to 1) with them. The power of the test was not adequate to detect an association with an unknown polymorphism which is not in complete linkage disequilibrium with those analysed. PMID- 10738534 TI - Common HLA alleles, rather than rare mutants, confer susceptibility to coeliac disease. AB - Coeliac Disease (CD) is a gluten sensitive enteropathy characterised by villous atrophy and crypt cell hyperplasia. It has a very strong HLA class II association to the DQ locus. The nature of the involvement of the DQ locus in the susceptibility to CD has been examined by tissue culture experiments, association and peptide binding studies. We examined the role of the DQ molecules in the pathogenesis from the perspective of a genetic family study. Using flanking microsatellite markers to the class II region of the MHC to establish the parental origin of the susceptibility DQ alleles, we have evidence suggesting that the HLA association is probably due to the necessity to have these DQ alleles in order to express CD and there is no support for the presence of a rare mutation within the DQ alleles nor any rare HLA-linked gene nearby in linkage disequilibrium with the DQ locus. This approach is applicable to other diseases demonstrating strong association with common alleles, and can be used to predict whether screening the region for rare mutations is likely to be worthwhile. PMID- 10738535 TI - Sequence variants in the 5' flanking region of the leptin gene are associated with obesity in women. AB - Few mutations have been found in the human leptin gene and the relationship between leptin gene sequence variation and human overweight is uncertain. To determine whether sequence variation within the leptin gene and its regulatory elements contribute to extreme obesity, we screened approximately 3 kb of the 5' flanking region and the three exons in 125 unrelated extremely obese (BMI > or = 40 kg/m2) and 86 average weight women (BMI < 27 kg/m2). Within the protein coding regions only one heterozygous silent mutation was found (codon 102; AAC/AAT). Within the 5' flanking region, six frequent sequence variants were detected (q > 0.10), and the allele frequencies of three of these variants differed between obese and average weight Caucasian women (+19, chi 2 = 4.46, p = 0.035; -1823, chi 2 = 4.36, p = 0.037; -2548, chi 2 = 5.73, p = 0.017). Nine infrequent sequence variants were detected (q < 0.05) but they did not occur more often among obese women compared with those of average-weight. For extremely obese women, three polymorphisms (+19, -188, and -633) predicted the degree of obesity. Allelic variants may influence the regulation of the leptin gene and thereby influence body weight, particularly among extremely obese women. However, given the low variability in coding regions and the high variability in the 5' flanking region, discerning the functional significance of each variant is likely to be difficult. PMID- 10738536 TI - New DNA markers with increased informativeness show diminished support for a chromosome 5q11-13 schizophrenia susceptibility locus and exclude linkage in two new cohorts of British and Icelandic families. AB - Genetic linkage of schizophrenia to markers at 5q11.2-13.3 had been reported previously in five Icelandic and two British families, but attempts at replication in independent samples have been unsuccessful. We report here an update on the diagnoses and results of linkage analyses using newer highly polymorphic microsatellite markers at or near the loci D5S76 and D5S39 in the original sample of pedigrees and in two new family samples from Iceland and from Britain. The new results show a reduction in evidence for linkage in the original sample and evidence against linkage in the two new family samples. Although it is possible that a rare locus is present, perhaps in the region 5p14.1-13.1 rather than 5q11.2-13.3, it appears most likely that the original positive lod scores represent an exaggeration of the 'true' lod scores due to random effects and that the small lod scores we now obtain could have arisen by chance. PMID- 10738537 TI - A statistically robust variance-components approach for quantitative trait linkage analysis. AB - Previously we showed (Wang, Guerra & Cohen 1998) that a statistically robust version of the Haseman & Elston (1972) sib-pair method greatly increased power to detect linkage in the presence of outliers. In this paper we report on M estimation to accommodate outliers in the variance-components approach to linkage analysis developed by Amos (1994). Simulations show that in the presence of outliers the robust variance-components approach provides substantially greater power, more precise estimation of heritabilities, and better false-positive rates than the original Gaussian based approach. In the absence of outliers the performance of the robust variance-components approach is similar to that of the Gaussian based approach. For illustration we apply the method to two well characterized lipoprotein systems. PMID- 10738538 TI - Meta-analysis of genome searches. AB - We have developed a method for meta-analysis of genome scans which allows systematic integration of data from published results. The Genome Search Meta analysis method (GSMA) uses a non-parametric ranking method to identify genetic regions that show consistently increased sharing statistics or lod scores. The GSMA ranks genetic regions according to the lod score or p-value achieved in each scan. The summed rank across studies is compared to its probability distribution assuming ranks are randomly assigned. The GSMA can confirm evidence for regions highlighted in the original genome scans, and identify novel regions, which did not reach significance in any scan. In this paper, the GSMA was applied to four genome screens in multiple sclerosis and across 11 screens from autoimmune disorders. The GSMA is appropriate for studies with different family ascertainment, markers, and statistical analysis methods. The method increases the power to detect individual linkages in a clinically homogeneous dataset and has the potential to detect susceptibility loci in clinically distinct diseases which show involvement of common pathogenetic pathways. PMID- 10738539 TI - Allelic diversity of the human plasma alpha(1,3)fucosyltransferase gene (FUT6). AB - The 1080-bp coding region of the human plasma alpha(1,3)fucosyltransferase gene (FUT6) was sequenced in a total of 161 individuals (322 chromosomes) drawn from three populations, involving 56 Africans (Xhosa), 52 European-Africans of South Africa, and 53 Japanese. In addition to six reported base substitutions, eleven new base substitutions and a single base insertion were found in the coding region of the FUT6. Eleven functional and four null alleles were encountered, of which 10 alleles were novel alleles identified in this study. Two null alleles have been identified previously, whereas two novel null alleles, which contained a single base (cytosine) insertion at nucleotide 499, were found in a Xhosa population. The allelic distributions of FUT6 were different among these three populations. The heterozygosity of FUT6 was 0.860, 0.699, and 0.632, in Xhosa, European-African (South Africa), and in Japanese populations, respectively. The extensive DNA sequence diversity of the FUT6 may be suitable for application as a tool in genetic studies for modern human evolution. PMID- 10738540 TI - Search for the PARK3 founder haplotype in a large cohort of patients with Parkinson's disease from northern Germany. AB - A founder haplotype on chromosome 2p for autosomal dominant Parkinson's disease (PD) has been postulated for two families of Northern European descent, and a new mutation in the alpha-synuclein gene (Ala30Pro) has been found in a German PD family. We evaluated 85 German PD patients and 85 ethnically matched controls for shared markers on chromosome 2p and for the new alpha-synuclein mutation. We found no evidence for linkage disequilibrium, suggesting that the putative founder mutation on chromosome 2p is not a common cause of PD in the local population. Furthermore, no patient carried the Ala30Pro change, supporting earlier findings that mutations in the alpha-synuclein gene are extremely rare. PMID- 10738541 TI - Evidence of a major gene effect for angiotensinogen among Nigerians. AB - To dissect the genetic pathway of hypertension, we measured angiotensinogen in 685 members of 186 families recruited from a rural community in southwest Nigeria. Commingling and segregation analyses were carried out. A mixture of two and/or three distributions fits the data significantly better than a single distribution in commingling analysis, suggesting a major gene effect. Segregation analysis confirmed that a recessive major gene model for low values of angiotensinogen provides the best fit to the data and about 13% of the variance was due to the recessive gene segregation. PMID- 10738542 TI - Apolipoprotein E (APOE) allele distribution in the world. Is APOE*4 a 'thrifty' allele? AB - Apolipoprotein E (APOE = gene, apoE = protein) plays a central role in plasma lipoprotein metabolism and in lipid transport within tissues. The APOE shows a genetic polymorphism determined by three common alleles, APOE*2, APOE*3, APOE*4 and the product of the three alleles differs in several functional properties. APOE is involved in the development of certain pathological conditions. In particular, the APOE*4 allele is a risk factor for susceptibility to coronary artery disease (CAD) and Alzheimer's Disease (AD). In the present study we analyzed the APOE allele distribution in the world. The APOE*3 is the most frequent in all the human groups, especially in populations with a long established agricultural economy like those of the Mediterranean basin (0.849 0.898). The frequency of APOE*4, the ancestral allele, remains higher in populations like Pygmies (0.407) and Khoi San (0.370), aborigines of Malaysia (0.240) and Australia (0.260), Papuans (0.368), some Native Americans (0.280), and Lapps (0.310) where an economy of foraging still exists, or food supply is (or was until the recent past) scarce and sporadically available. The APOE*2 frequency fluctuates with no apparent trend (0.145-0.02) and is absent in Native Americans. We suggest that the APOE*4, based on some functional properties it has and on its distribution among human populations, could be identified as a 'thrifty' allele. The exposure of APOE*4 to the contemporary environmental conditions (Western diet, longer lifespans) could have rendered it a susceptibility allele for CAD and AD. The absence of the association of APOE*4 with CAD and AD in Sub-Saharan Africans, and its presence in African Americans, seems to confirm this hypothesis. PMID- 10738543 TI - Influence of apolipoprotein E genotype variation on the means, variances, and correlations of plasma lipids and apolipoproteins in children. AB - The impact of the three most common apolipoprotein E (APOE) genotypes (epsilon 32, epsilson 33, and epsilon 43) on means, variances, and correlations of nine plasma lipid and apolipoprotein traits (total cholesterol, InTriglycerides, HDL cholesterol, and apolipoproteins AI, AII, B, CII, CIII, and InE) was studied in 212 unrelated female and 219 unrelated male children aged 5-21.5 years from 278 pedigrees ascertained without regard to health status from Rochester, Minnesota. There was significant heterogeneity (p < or = 0.05) among genotypes for the mean plasma levels of InApo E, Apo CII, Apo CIII, and InTriglycerides (InTrig) in females, and for the means of InApo E, Apo B, and total cholesterol (Total-C) in males. Significant heterogeneity of intragenotypic variance was observed in males for Apo CII, InTrig, and HDL-C; no significant heterogeneity was observed in females. Pairwise correlations between traits differed significantly among APOE genotypes in both females (6 of 36 pairs) and males (5 of 36 pairs). These results differ from those obtained from studies of the parental generation from the same sample of pedigrees. Our study further demonstrates that, with the exception of mean InApo E levels, the univariate and bivariate distributions of traits that are measures of lipoprotein metabolism are influenced by variation in the APOE gene in a gender- and generation-dependent manner. PMID- 10738544 TI - A logistic regression based extension of the TDT for continuous and categorical traits. AB - The transmission disequilibrium test (TDT), designed as a test of linkage in the presence of association (i.e. linkage disequilibrium), has received considerable attention in the recent statistical genetics literature due to its advantages over other within-family analytic methods. One limitation of the conventional TDT is its application solely to linkage disequilibrium between a genetic marker and a single categorical trait (e.g. presence or absence of a disease). In this paper, we present an extension of the TDT using logistic regression to examine the relation between a candidate gene or genetic marker and one or more continuous or categorical explanatory variables. This logistic regression extension of the TDT possesses all of the desirable features of the conventional TDT, as well as many advantages associated with traditional regression analysis. We describe the model and its properties, as well as a number of its possible applications, and apply it to examine linkage disequilibrium between the dopamine receptor D2 gene (DRD2) and symptoms of childhood attention deficit hyperactivity disorder (ADHD). We also briefly compare the logistic regression TDT to other quantitative TDTs that have been proposed in the literature, and highlight the advantages of a regression-based approach for examining the relation between a candidate gene and one or more continuous or categorical traits. Given its features, we regard the logistic regression extension of the TDT as a flexible new data analytic method with extensive potential applications to problems in medical, psychiatric, and behavioral genetics. PMID- 10738545 TI - Testing the equality of twin correlations with multinomial outcomes. AB - Twin studies are widely used to study genetic and environmental influences on human measurements. Correlations are often used in such studies to compare the levels of similarity between monozygotic and dizygotic twins with respect to a specified trait. In this paper, we compare three procedures for testing the equality of twin correlations when the outcome variable of interest is multinominal. One method is a likelihood ratio test based on an underlying Dirichlet-multinomial distribution. The second method is based on the estimated large sample variance of the estimated correlation, and the third method is based on a chi 2 goodness-of-fit test. The results of a Monte Carlo simulation show that the three methods have similar properties if the number of twin pairs is large (> 100), and the prevalence of the underlying trait is not extreme. Otherwise, the goodness-of-fit approach is to be preferred. We illustrate the methods by analyzing data from a previously published smoking study. PMID- 10738546 TI - Characterization of polymorphisms in the promoter of the human angiotensin II subtype 1 (AT1) receptor gene. AB - In this study eight sequence variants in the functional promoter of the human angiotensin II subtype 1 (AT1 or AGTR1) receptor gene are reported. Six of these variants are in nearly total linkage disequilibrium with each other and occur with a frequency of 15.7%. By haplotype estimation this group of eight sequence variants is characterized by only five haplotypes. There is no linkage disequilibrium between one of these haplotypes and the AT1 + 1166A/C variant. The finding of polymorphic sites in the functional promoter of the human AT1 locus will be beneficial to the study of the role of the AT1 receptor gene in hypertension and other cardiovascular diseases. PMID- 10738547 TI - Modifying the host range properties of retroviral vectors. AB - Gene therapy protocols would be greatly facilitated by the availability of targetable injectable vectors which could deliver genes in vivo to specific target cells or to specific disease sites. Efforts to develop such retroviral vectors are therefore a high priority in gene therapy research. In this review, we describe the current state of our understanding of the structure and function of the retroviral envelope glycoprotein complex. We then discuss the results of the various strategies that have been devised to modify the host range of the retroviral envelope glycoproteins with a view to achieving retroviral vectors capable of delivering their genes in a highly specific manner to selected human target cells. The strengths and limitations of these strategies are examined. PMID- 10738548 TI - Insertion of natural intron 6a-6b into a human cDNA-derived gene therapy vector for cystic fibrosis improves plasmid stability and permits facile RNA/DNA discrimination. AB - BACKGROUND: The gene therapy vector pCMV-CFTR containing human CFTR cDNA shows high segregational instability during growth in Escherichia coli. METHODS: By host strain screening and optimization of fermentation, satisfactory levels of pCMV-CFTR production were achieved. However, the vector was also vulnerable to structural instability manifested by the appearance during fermentation of a more stable mutant form in which the bacterial insertion sequence IS1 had transposed into exon 7 of plasmidborne CFTR. The instability of pCMV-CFTR is attributable to transcription from an upstream cryptic promoter leading to the production of CFTR peptide fragments known to be toxic when expressed in E. coli. To address this, we inserted the 1.1 kb natural human 6a-6b intron into pCMV-CFTR. RESULTS: The new vector pCMV-CFTR-int6ab is more stable in E. coli than either pCMV-CFTR or the IS1 mutant, grows to high cell density giving higher DNA yields and expresses CFTR appropriately in transfected cells. Thus, the intron has a stabilizing effect comparable to the IS1 insertion yet retains full functionality for gene therapy. We describe a PCR assay using primers directed to sequences flanking the intron that allows differentiation between DNA and mature mRNA. The T936C mutation present only in vector DNA has also been exploited to allow transgene CFTR to be distinguished and its dose-dependent expression to be detected in human cellular backgrounds. CONCLUSIONS: Instability of a plasmid vector for gene therapy has been minimized by rational modification. The introduction of an intron for this purpose offers the additional advantage of providing a discriminatory RT-PCR assay. PMID- 10738549 TI - Fluorescence-based selection of retrovirally transduced cells in congenital erythropoietic porphyria: direct selection based on the expression of the therapeutic gene. AB - BACKGROUND: Congenital erythropoietic porphyria (CEP) is an inherited disease caused by a deficiency of uroporphyrinogen III synthase, the fourth enzyme of the haem biosynthesis pathway. It is characterized by accumulation of uroporphyrin I in the bone marrow, peripheral blood and other organs. The prognosis of CEP is poor with death occurring in early adult life and available treatments are only symptomatic and unsatisfactory. In vitro gene transfer experiments have documented the feasibility of gene therapy via haematopoietic stem cells to treat this disease. To facilitate future ex vivo gene therapy in humans, the design of efficient selection procedures to increase the frequency of genetically corrected cells prior to autologous transplantation is a critical step. METHODS: An alternative selection procedure based upon expression of a transferred gene was performed on a lymphoblastoid (LB) cell line from a patient with congenital erythropoietic porphyria to obtain high frequencies of genetically modified cells. The presence of exogeneous delta-aminolevulinic acid (ALA), a haem precursor, induces an increase in porphyrin accumulation in LB deficient cells. Porphyrins exhibit a specific fluorescent emission and can be detected by cytofluorimetry under ultraviolet excitation. RESULTS: In genetically modified cells, the restored metabolic flow from ALA to haem led to a lesser accumulation of porphyrins in the cells, which were easily separated from the deficient cells by flow cytometry cell sorting. CONCLUSION: This selection process represents a rapid and efficient procedure and an excellent alternative to the use of potentially harmful gene markers in retroviral vectors. PMID- 10738550 TI - Interaction of liposomal and polycationic transfection complexes with pulmonary surfactant. AB - BACKGROUND: The delivery of genes to the airways holds promise for the treatment of lung diseases such as cystic fibrosis and asthma. Current non-viral gene delivery systems lack sufficient transfection efficiency. Pulmonary surfactant has been reported to be a barrier to gene transfer into the airways. Here we analyze the interaction of liposomal and polycationic transfection complexes with pulmonary surfactant. METHODS: The efficiency of non-viral transfection of cultured human airway epithelial cells (16HBE14o-), COS7 cells and porcine primary airway epithelial cells was studied in the presence of various surfactant preparations in order to model the conditions prevailing in the airways during transfection. RESULTS: The natural pulmonary surfactant, Alveofact, an extract from bovine lung lavage, was found to inhibit lipofection with lipofectAMINE for all cell lines investigated. Dendrimer meditated polyfection was unaffected for pulmonary cell lines and was weakly affected for COS7 cells. PEI-mediated polyfection was unaffected for all cell lines tested. The synthetic surfactant preparation Exosurf containing L-alpha-phosphatidylcholine-dipalmitoyl (DPPC) as the sole lipid ingredient had no statistically significant effect on polymer- and lipid-mediated transfection. The transfection efficiencies are related to structural changes in the DNA complexes as demonstrated by DNase-accessibility tests and fluorescence spectroscopy. In the presence of the phospholipid POPG, which is a constituent of Alveofact, DNA condensed in lipofectAMINE lipoplexes became accessible to DNaseI, while DNA condensed with PAMAM dendrimer or PEI was less accessible to DNase I as compared to lipoplexes. Consistently, the fluorescence of a DNA-intercalating dye increased after addition of Alveofact only in the case of lipoplexes. CONCLUSIONS: In contrast to lipofection, gene transfer with cationic polymers to airway epithelial cells is not inhibited by pulmonary surfactant in vitro. Depending on the surfactant concentration even an increase in polymermediated transfection can be seen. In conclusion, cationic polymers appear to be the more stable gene delivery systems for topical application into the airways. PMID- 10738551 TI - Activation conditions determine susceptibility of murine primary T-lymphocytes to retroviral infection. AB - BACKGROUND: Genetically modified T-lymphocytes are potential therapeutic agents for the treatment of various disorders. Successful retroviral infection of primary murine T-lymphocytes is a prerequisite to the study of adoptive cell therapies in a small animal model. The definition of factors controlling retroviral infection of T-lymphocytes would also be useful to better understand retroviral diseases. However, retroviral-mediated gene transfer into murine primary T-cells has remained elusive. METHODS: In order to define the requirements for stable and efficient gene transfer in primary murine T lymphocytes, we investigated factors capable of affecting retroviral infection. These include activation conditions (using mitogens or monoclonal antibodies), culture conditions (including media composition and cytokine addition), timing of viral exposure, retroviral receptor selection (ecotropic, amphotropic, or vesicular stomatitis virus G (VSV-G) glycoprotein receptor), and viral titer. RESULTS: We show that efficient gene transfer can be achieved in murine T lymphocytes, provided that a number of favorable conditions are met, in particular optimized T-cell activation conditions, optimal timing of infection, adequate interleukin-2 concentration and T-cell density, and a high viral titer. On a particulate basis, we find that ecotropic particles are more effective than amphotropic or VSV-G-pseudotyped particles, and recommend the use of a specifically selected retroviral packaging cell line. CD4+ T-cells are equally or more infectible than CD8+ lymphocytes, depending on the activation conditions. The Th1 and Th2 subsets are comparably susceptible to retroviral infection, in contrast to what has been reported in some instances of human T-cell infection by human immunodeficiency virus (HIV). CONCLUSIONS: We establish conditions that enable efficient retroviral-mediated gene transfer in murine primary T lymphocytes. T-lymphocytes are not uniformly susceptible to retroviral infection depending on the mode of T-cell activation. These findings have implications for devising approaches to the study of T-cell biology, adoptive cell therapies, and the pathophysiology of retroviral diseases in mouse models. PMID- 10738552 TI - One step screening of retroviral producer clones by real time quantitative PCR. AB - BACKGROUND: Recombinant retroviruses are obtained from either stably or transiently transfected retrovirus producer cells. In the case of stably producing lines, a large number of clones must be screened in order to select the one with the highest titre. The multi-step selection of high titre producing clones is time consuming and expensive. METHODS: We have taken advantage of retroviral endogenous reverse transcription to develop a quantitative PCR assay on crude supernatant from producing clones. We used Taqman PCR technology, which, by using fluorescence measurement at each cycle of amplification, allows PCR product quantification. Fluorescence results from specific degradation of a probe oligonucleotide by the Taq polymerase 3'-5' exonuclease activity. Primers and probe sequences were chosen to anneal to the viral strong stop species, which is the first DNA molecule synthesised during reverse transcription. The protocol consists of a single real time PCR, using as template filtered viral supernatant without any other pre-treatment. RESULTS: We show that the primers and probe described allow quantitation of serially diluted plasmid to as few as 15 plasmid molecules. We then test 200 GFP-expressing retroviral-producing clones either by FACS analysis of infected cells or by using the quantitative PCR. We confirm that the Taqman protocol allows the detection of virus in supernatant and selection of high titre clones. Furthermore, we can determine infectious titre by quantitative PCR on genomic DNA from infected cells, using an additional set of primers and probe to albumin to normalise for the genomic copy number. CONCLUSION: We demonstrate that real time quantitative PCR can be used as a powerful and reliable single step, high throughput screen for high titre retroviral producer clones. PMID- 10738553 TI - Intravenous administration of recombinant adenoviruses causes thrombocytopenia, anemia and erythroblastosis in rabbits. AB - BACKGROUND: Recombinant adenoviruses are highly efficient gene transfer vehicles but their administration to mammals is accompanied by a strong inflammatory response. The present study reports additional side effects observed during adenoviral gene transfer studies in rabbits. METHODS: Hematological and serological parameters, the course of viremia and the organ distribution were analyzed after in vivo administration of E1-deleted adenoviruses in rabbits. RESULTS: The systemic administration of a therapeutic dose of 5 x 10(11) infectious particles/kg (infusion time 20 min) led to an average reduction of 80 90% in the platelet count within 48 h. Full recovery took 10-14 days. Virus administration induced a strong but transient erythroblastosis (peaking 24 h after administration) which settled 48 h later. Normochromic anemia occurred over the next 10 days with hemoglobin levels dropping by about 40% to reach the lowest level 10 days after administration and taking two months for full recovery. Dose dependent thrombocytopenia was also found in mice, but neither erythroblastosis nor anemia was observed (in equivalent doses). The hematological findings did not improve after local injection via the portal vein. Local and systemic administration led to a comparable course of viremia. Only minor differences were found in the biodistribution of viruses between local and systemic administration. Large amounts of viral DNA and transgene expression were found in the lungs, the kidneys and the ovaries, even after local administration via the portal vein. CONCLUSIONS: Local intravenous injection via the portal vein does not prevent systemic spread of viral vectors and the occurrence of vector-related side effects. The hematological changes observed in rabbits suggest the need for careful monitoring of hematological and rheological parameters in clinical trials. PMID- 10738555 TI - All the Virology on the Web. PMID- 10738554 TI - Controlling genes with tetracyclines. PMID- 10738556 TI - A selective amplifier gene for tamoxifen-inducible expansion of hematopoietic cells. AB - BACKGROUND: We have developed a novel system for expansion of gene-modified hematopoietic stem/progenitor cells to overcome the low efficiency of current gene transfer methodology. This system involves 'selective amplifier genes', that encode fusion proteins between the granulocyte colony-stimulating factor receptor (GCR) and the hormone-binding domain of estrogen receptor (ER). Hematopoietic progenitors expressing the chimeras showed estrogen-responsive growth in a controllable manner. However, endogenous estrogen may activate the fusion proteins in vivo, depending on the hormonal status of the subjects. METHODS: We replaced ER with a mutant receptor (TmR) which specifically binds to 4 hydroxytamoxifen (Tm), to overcome limitations with wild-type ER. Interleukin-3 (IL-3)-dependent Ba/F3 cells and hematopoietic progenitor cells transduced with the resultant fusion proteins (GCRTmR and delta GCRTmR) were examined for ligand inducible growth. RESULTS: GCRTmR- and delta GCRTmR-expressing Ba/F3 showed IL-3 independent growth in response to Tm, while the cells were unresponsive to estrogen at concentrations up to 10(-7)-10(-6) M. Furthermore, murine bone marrow cells transduced with GCRTmR and delta GCRTmR formed colonies in methyl-cellulose medium in response to Tm, while virtually no colonies appeared with 10(-7) M estrogen or without cytokines. CONCLUSIONS: These results suggest that influences of the endogenous estrogen can be almost eliminated by using the GCRTmR/Tm or delta GCRTmR/Tm system to expand gene-modified hematopoietic stem/progenitor cells. PMID- 10738557 TI - Erythropoietin secretion and physiological effect in mouse after intramuscular plasmid DNA electrotransfer. AB - BACKGROUND: Direct intramuscular plasmid DNA injection has recently been proposed for erythropoietin therapy, as an alternative to either systemic injection of recombinant erythropoietin or the use of viral vectors for erythropoietin gene transfer. However, direct intramuscular plasmid injection has so far been hampered by low efficiency and high interindividual variability. METHOD: We explored the use of a new method termed 'intramuscular electrotransfer' for erythropoietin gene expression in the mouse. This method is based on intramuscular plasmid injection followed by application of appropriate electric pulses. RESULTS: Intramuscular plasmid electrotransfer in mouse leg led to an increase of approximately 10- to 100-fold in circulating murine erythropoietin level, as compared to naked DNA alone. Using electrotransfer, as little as 1 microgram of an erythropoietin encoding plasmid was sufficient to induce an increase in mouse hematocrit, from 47% up to 80%. This hematocrit increase was stable for at least two months. Moreover, interindividual hematocrit variability was markedly reduced by electrotransfer, as compared with naked DNA injection. CONCLUSION: In vivo electrotransfer appears to be a convenient method for obtaining high erythropoietin expression in mice, and it could also be used for the expression of other secreted therapeutic proteins. PMID- 10738558 TI - Gene transfer by guanidinium-cholesterol: dioleoylphosphatidyl-ethanolamine liposome-DNA complexes in aerosol. AB - BACKGROUND: A major challenge of gene therapy is the efficient transfer of genes to cell sites where effective transfection can occur. The impact of jet nebulization on DNA structural and functional integrity has been problematic for the aerosol delivery of genes to pulmonary sites and remains a serious concern for this otherwise promising and noninvasive approach. METHODS: This study examined effects of cationic liposome-DNA formulation on both transfection efficiency (in vitro and in vivo) and jet nebulizer stability. The effects of nebulization and sonication on liposome-DNA particle size characteristics were examined. Electron microscopy of promising formulations was performed using several fixation methods. RESULTS: The cationic lipid bis-guanidinium-tren cholesterol (BGTC), in combination with the neutral co-lipid dioleoylphosphatidylethanolamine (DOPE), was found to have a degree of stability adequate to permit effective gene delivery by the aerosol route. Optimal ratios of lipids and plasmid DNA were identified. Particle size analysis and ultrastructural studies revealed a remarkably homogeneous population of distinctly liposomal structures correlating with the highest levels of transfection efficiency and nebulizer stability. CONCLUSIONS: Optimizing gene delivery vectors for pulmonary aerosol delivery to respiratory sites must take into account factors other than transfection efficiency in vitro. Effects of liposome-DNA formulation on liposomal morphology (i.e. particle size, multilamellar structure) appear to be relevant to stability during aerosolization. These studies have allowed us to identify formulations that hold promise for successful clinical application of aerosol gene delivery. PMID- 10738559 TI - Quantification of VP22-GFP spread by direct fluorescence in 15 commonly used cell lines. AB - BACKGROUND: The intercellular transport property of VP22 chimeric proteins offers the opportunity for the improvement of gene therapy delivery systems. Since enhanced therapeutic effects of transduced genes already have been exemplified for chimeric proteins VP22-p53 and VP22-tk, we were interested in examining whether spread of VP22 chimeric proteins is a general biological phenomenon not restricted to distinct tissues or species. METHODS: To study intercellular spread of VP22-GFP fusion proteins, 15 different mammalian cell lines were transfected with 200-2000 ng of VP22-GFP or GFP expression plasmids. Expression of VP22-GFP or GFP was monitored by fluorescence microscopy of live GFP fluorescence and direct FACS analysis. For selected cell lines, antibody detection of VP22-GFP spread was analysed by confocal microscopy as a control. RESULTS: Spread of VP22 GFP fusion proteins was detected in all 15 cell lines tested, and quantified by FACS analysis. Experimental conditions were found to be critical in the investigation of VP22-mediated intercellular spread. CONCLUSION: Results of our study indicate that spread of VP22 chimeric proteins is a general biological phenomenon not restricted to distinct tissues or species. Therefore, further evidence is provided that VP22-enhanced gene therapeutic effects may be obtained irrespective of the target organ/tissue to be addressed. PMID- 10738560 TI - Intercellular spread of GFP-VP22. AB - BACKGROUND: The herpes simplex virus type 1 (HSV-1) VP22 polypeptide has been reported to mediate intercellular trafficking of heterologous proteins fused to its C- or N-terminus, a feature making it a useful tool in bystander cell targeted gene therapy. METHODS: Here we show, by detection of Green Fluorescent Protein (GFP) fused to VP22, its subcellular distribution in living producer (transfected) and recipient (non-transfected) cells as well as in cells after fixation. Four cell lines from different species were used. RESULTS: Different fractions of translocated GFP-VP22 fusion protein could be detected in fixed recipient cells by two different methods of fixation. Functional GFP in live recipient cells could not be detected. CONCLUSIONS: Our study indicates that the VP22-chimeric protein transfer in its present form is suboptimal in terms of protein function. However, after fixation, the GFP signal in 100% of cells in a monolayer can be detected even at moderate transfection efficiency. PMID- 10738561 TI - Herpes simplex virus vector-mediated dystrophin gene transfer and expression in MDX mouse skeletal muscle. AB - BACKGROUND: Duchenne muscular dystrophy (DMD) results from mutations that prevent the expression of functional dystrophin in muscle fibers. Herpes simplex virus type-1 (HSV-1) represents a potentially useful vector for treatment of DMD because it has the capacity to accommodate the 14-kb full-length dystrophin cDNA and can efficiently transduce muscle cells. We have tested the ability of first- and second-generation replication-defective HSV vectors to deliver full-length dystrophin to dystrophin-deficient mdx muscle cells in vitro and in vivo. METHODS: First-generation replication-defective HSV vectors harboring full-length or truncated (Becker) dystrophin expression cassettes and lacking a single viral immediate-early (IE) gene were constructed and tested by immunofluorescence and immunoblotting for their ability to direct dystrophin expression in infected mdx cells in culture. To reduce vector cytotoxicity and safety concerns, a second generation dystrophin vector missing additional IE genes was constructed and tested in vitro and in vivo. RESULTS: Dystrophin expression was observed in infected mdx myotubes in vitro in all cases. Confocal microscopy showed exclusive localization of full-length dystrophin to the cell membrane whereas the Becker variant was also found abundantly throughout the cytoplasm. Dystrophin expression in mdx mice was restored in muscle cells near the site of vector injection. CONCLUSION: Highly defective HSV-1 vectors which lack the ability to spread systemically and are greatly reduced in toxicity for infected cells, thus removing an impediment to prolonged transgene expression, can direct the delivery and proper expression of full-length dystrophin whose considerable size is compatible with few other modes of delivery. These vectors may offer a legitimate opportunity toward the development of effective gene therapy treatments for DMD. PMID- 10738562 TI - Prospects for synthetic self-assembling systems in gene delivery. PMID- 10738563 TI - The DNA Vaccine Website. PMID- 10738564 TI - Comparative transfection studies of human ovarian carcinoma cells in vitro, ex vivo and in vivo with poly(2-(dimethylamino)ethyl methacrylate)-based polyplexes. AB - BACKGROUND: Poly(2-(dimethylamino)ethyl methacrylate) (p(DMAEMA)) can be used successfully for in vitro transfection of different cell lines, including the OVCAR-3 human ovarian carcinoma cell line. The aim of this study was to investigate whether it is possible to transfect OVCAR-3 cells in vivo with polyplexes containing p(DMAEMA). METHODS: In order to understand the generally observed gap between in vitro and in vivo transfection, we gradually went from in vitro to in vivo transfection of OVCAR-3 cells, while keeping the exposure conditions the same, as far as possible. To find the reason for the negligible degree of in vivo transfection, in vitro cultured OVCAR-3 cells were transfected in the presence of peritoneal ascites fluid. Next, the influence of hyaluronic acid, one of the ascites components, on the transfection efficiency was studied. RESULTS: P(DMAEMA)-containing polyplexes can transfect OVCAR-3 cells in vitro with an overall transfection efficiency of 10%. Cells grown in vivo can be transfected ex vivo with p(DMAEMA)/plasmid complexes with an overall transfection efficiency of approximately 1-2%. When transfection complexes are injected i.p. into nude mice bearing OVCAR-3 cells in the peritoneal cavity, the degree of in vivo transfection efficiency achieved is negligible. In vitro cultured OVCAR-3 cells were also transfected with polyplexes in the presence of peritoneal ascites fluid. The results indicate that one or more components of ascites had a negative effect on the transfection efficiency of p(DMAEMA)-containing polyplexes. To elucidate which component(s) of ascites may have interfered, the influence of hyaluronic acid, one of the ascites components, on the transfection efficiency was studied. The outcome suggests that hyaluronic acid may have induced a negative effect on the transfection capability of p(DMAEMA)-containing polyplexes. CONCLUSION: P(DMAEMA) is an efficient transfectant in vitro and ex vivo. However, transfected cells were not detected in vivo which may be caused by a negative influence of components of the ascites fluid. PMID- 10738565 TI - Adeno-associated virus-mediated gene delivery. AB - Several gene delivery vehicles are being developed for somatic gene therapy and each of these vectors has unique properties which makes them appropriate for different human disease applications. Recombinant adeno-associated viral (rAAV) vectors are proving themselves to be safe and efficacious for the long-term expression of proteins and correction of genetic diseases following a single administration. The increasing number of tissues and diseases being targeted with rAAV vectors demonstrates their versatility and has resulted in different approaches for enhancing vector performance. Improving the methods for large scale manufacturing, and accumulating safety and efficacy data in animals and humans are areas of intense research. PMID- 10738566 TI - Combined HSV-1 recombinant and amplicon piggyback vectors: replication-competent and defective forms, and therapeutic efficacy for experimental gliomas. AB - BACKGROUND: The versatility of HSV-1 vectors includes large transgene capacity, selective replication of mutants in dividing cells, and availability of recombinant virus (RV) and plasmid-derived (amplicon) vectors, which can be propagated in a co-dependent, 'piggyback', manner. METHODS: A replication defective piggyback vector system was generated in which the amplicon carries either of two genes essential for virus replication, IE2 (ICP27) or IE3 (ICP4), as well as lacZ; the RV is deleted in both these genes, and vector stocks are propagated in cells transfected with one of the complementary genes. In the replication-competent system, the amplicon carries the IE2 and lacZ; the RV had a large deletion in the IE2; and stocks are propagated in untransfected cells. Titers over successive passages, recombination between amplicon and RV, and the structural integrity of vector genomes were evaluated. The replication-competent system was tested for therapeutic efficacy in subcutaneous 9L gliosarcoma tumors in nude mice with activation of ganciclovir via the viral HSV-thymidine kinase gene. RESULTS: Both systems generated high titer amplicon vectors (about 10(7) tu/ml) and amplicon:RV ratios (0.6-3.0). No replication-competent RV was generated in either system. The replication-defective system showed low toxicity and increased packaging efficiency of amplicon vectors, as compared to single mutant RV helper virus. The replication-competent system allowed co-propagation of amplicon and RV; injection into tumors followed by ganciclovir treatment inhibited tumor growth without systemic toxicity. CONCLUSION: New replication defective and replication-competent piggyback HSV, vector systems allow gene delivery via amplicon vectors with reduced toxicity and co-propagation of both RV and amplicon vectors in target cells, with effective tumor therapy via focal virus replication and pro-drug activation. PMID- 10738567 TI - Naked plasmid-mediated gene transfer to skeletal muscle ameliorates diabetes mellitus. AB - BACKGROUND: The ability of tissues to take up naked plasmid DNA in vivo suggests an approach for reconstituting systemic metabolic deficiencies without the disadvantages of viral vectors and lipid-DNA complexes. Plasmid-mediated gene transfer into skeletal muscle was investigated as a means of providing a therapeutic source of insulin. METHODS: Four plasmid constructs, each bearing a mouse furin cDNA transgene and rat proinsulin cDNA (modified for processing by furin) driven by four different promoters were injected into the calf muscles of male Balb/c mice. Insulin and C-peptide concentrations were measured by radio immunoassays having minimal crossreactivity for proinsulin and partially processed proinsulin. RESULTS: Intramuscular insulin concentrations increased by up to 3.6-fold over controls seven days after single injections of CMV, beta actin, hsp70 and myoglobin promoter constructs. The optimal dose for most constructs was 100 micrograms plasmid DNA. Intramuscular plasmid injection into streptozotocin-induced diabetic Balb/c mice raised plasma insulin and C-peptide concentrations, and reduced hyperglycaemia. Two injections (100 micrograms plasmid DNA each) caused higher plasma insulin concentrations and significantly reduced hyperglycemia in diabetic mice than a single injection. Best results were obtained when plasmid injections preceded induction of diabetes by 14 days. CONCLUSIONS: Skeletal muscle is a potentially useful platform for ectopic secretion of insulin using naked plasmid as a gene transfer vector. Injection at two sites 14 days before the onset of severe hyperglycemia is optimal. This approach could protect Type I diabetics from fatal ketoacidosis and enhance the action of agents that sensitize tissues to insulin in type II diabetes. PMID- 10738568 TI - Large-scale manufacturing of safe and efficient retrovirus packaging lines for use in immunotherapy protocols. AB - BACKGROUND: The use of gene modified T lymphocytes for immunotherapy in a cancer or AIDS clinical trial requires an efficient, safe ex vivo method for modification of these cells at manufacturing scale. Since retroviruses have been shown to be a moderately effective means of stably integrating therapeutic genes into T lymphocytes, we wanted to create packaging and producer cell lines that would produce replication competent retrovirus (RCR)-free supernatants, at large scale (> 200 l), and transduce with high efficiency. METHODS: cDNA expression plasmids containing only coding sequences for gagpol or env were built and sequentially transfected into human 293 cells. Packaging and producer clones were characterized for stability, titer and RCR. A producer clone delivering chimeric immune receptors was scaled-up and supernatants used to transduce patient T lymphocytes for clinical studies. PCR and RT-PCR assays were utilized to evaluate the transmission of HERV-H sequences. Relative infectivity of producer clones pseudotyped with different envelopes was determined by transduction and RT assays. RESULTS: RCR-free, human 293 split-genome packaging lines, pseudotyped with amphotropic, xenotropic, or 10A1 envelopes, were created. A CC49 zeta producer clone was scaled-up to 5 x 54 l lots and supernatants used to safely and efficiently transduce patient T lymphocytes with minimal ex vivo manipulation. While 293 cells express HERV-H mRNA, the transmission frequency in our packaging clones was less than 1 HERV-H sequence per 5 x 10(5) proviral integrations. Additionally, 10A1 and xenotropic packaging lines had higher infectivities than the amphotropic clone. CONCLUSION: These packaging lines represent the safest configuration for the large-scale production of retroviral vectors, and are capable of producing high titer, RCR-free retroviral vector for large scale clinical use. While all three clones efficiently transduce human T lymphocytes, the 10A1 clone has the highest infectivity. These packaging cell lines will be valuable for use in human gene therapy protocols. PMID- 10738569 TI - Transfection and physical properties of various saccharide, poly(ethylene glycol), and antibody-derivatized polyethylenimines (PEI). AB - BACKGROUND: The ideal non-viral vector should be cell-type directed and form complexes with DNA that are physically stable, small and electrically neutral. METHODS: We have synthesized several PEI derivatives that coat the PEI/DNA complexes with water-soluble residues able to stabilize the particles, to mask their surface charge and eventually to direct them to a particular tissue. The morphologies and sizes of the complexes were observed by TEM and DLS techniques, and their apparent surface charge was quantitated by zeta potential measurements; in vitro transfection efficacies were determined in serum-containing cell culture medium. RESULTS: When compared to DNA complexes formed with the unmodified PEI, extensive grafting with maltose (15-25% of the amine functions) led to beneficial electrostatic shielding of the particle surface, but was unable to prevent aggregation in physiological salt concentration. More extended hydrophilic residues were therefore explored as a mean of physical repulsion between the particles. Low grafting (2.7%) with a linear dextran non-asaccharide led to small and stable toroids having no apparent surface charge, yet still reaching effective transfection levels. Electron microscopy of complexes with a higher extent of grafting showed worm-like structures unsuited for cell entry. Conjugation of PEI with as little as 0.5% of a terminally galactose-derivatized polyethyleneglycol (PEG)-3400 also gave neutral complexes of another worm-like structure that failed to transfect receptor-expressing hepatocytes. CONCLUSION: These results show that conjugation of large and flexible hydrophilic residues to PEI, while protecting the complexes from parasitic interactions also interfere with DNA condensation. PEG conjugation after PEI/DNA complex formation may avoid this problem, provided intracomplex reorganization is slow. Finally an anti-GD2 antibody (mAb) grafted with PEI was synthesized. The corresponding protein-coated DNA complexes were compact and small (50-60 nm), yet did not enhance transfection of GD2 ganglioside-expressing cells. PMID- 10738570 TI - The use of neural stem cells for gene therapy in the central nervous system. PMID- 10738571 TI - Interpreting cancer vaccine clinical trials. AB - A number of cancer vaccine strategies have entered initial Phase I clinical testing. In this review, Dr. Glenn Dranoff discusses the prospects that appropriate immunologic monitoring of patients in these trials can help delineate important differences among the various immunization strategies and prioritize the most promising approaches for further clinical development. PMID- 10738572 TI - Expression of mucin (MUC-1) from a mini-Epstein-Barr virus in immortalized B cells to generate tumor antigen specific cytotoxic T cells. AB - BACKGROUND: EBV immortalized B-cells can be used as antigen presenting cells (APC) to stimulate specific T-cell responses. Mini-Epstein-Barr virus (mini-EBV) plasmids contain all functional elements of Epstein-Barr virus (EBV) necessary to immortalize B-cells in vitro. These immortalized B-cells are incapable of releasing infectious virus in contrast to cells immortalized by wildtype EBV. In addition, mini-EBVs can be modified in E. coli to alter their genetic composition or adopt new genes. METHODS: We constructed a mini-EBV plasmid carrying an expression cassette for the human tumor antigen mucin encoded by the gene MUC-1. Primary human B-cells were infected with the MUC-1 carrying mini-EBV plasmid packaged into an EBV coat and immortalized B-cell clones were expanded in vitro. These B-cells were analyzed by FACS analyses for the expression of mucin and co stimulatory molecules and were subsequently used as antigen presenting cells (APC) to stimulate peripheral blood mononuclear cells from healthy donors. RESULTS: Several B-cell lines were established that were shown to be free of helper virus or wildtype EBV. These B-cells expressed the relevant tumor-specific epitopes of mucin and the co-stimulatory ligands B7.1 and B7.2 necessary for efficient T-cell activation. Using the mucin expressing B-cells as antigen presenting cells (APC) mucin-epitope specific cytotoxic T-cells were established. CONCLUSIONS: Virus-free B-cell lines expressing tumor-associated epitopes such as mucin or other antigens of interest provide an unlimited and safe source of APC to generate antigen specific T-cells which could be used for clinical trials in adoptive immune therapy or cancer vaccines. PMID- 10738573 TI - Baculovirus vector requires electrostatic interactions including heparan sulfate for efficient gene transfer in mammalian cells. AB - BACKGROUND: Recently, several reports have described the ability of recombinant baculoviruses to transduce a variety of mammalian cells. Yet, mechanisms involved in baculovirus entry in those cells remain largely unexplored, particularly at the primary binding step of the virions to the cell membrane. METHODS: This report focused on the primary virus-cell interactions that lead to in vitro transduction of human 293 cells using a polyhedrin-deleted baculovirus harboring a CMV-driven beta-galactosidase gene (BacLacZ). RESULTS: Infection rate monitored for 8 h and transduction rate with a multiplicity of infection of up to 800 were, both, non-saturable. Temperatures from 37 degrees C to 4 degrees C dramatically impaired BacLacZ but not adenovirus cell attachment. Competitive infections performed with an excess of a non LacZ-expressing baculovirus hardly competed at a 1/1 ratio. Consistent with an adsorptive binding process onto the cell surface, interactions through electrostatic charges between both viral and cell membranes appeared to be critical for BacLacZ transduction. The addition of polybrene to the cells prior to or during the infection prevented both virus binding and LacZ gene transfer, suggesting the involvement of negatively charged epitopes exposed at the cell surface. The simultaneous presence of the highly charged heparin abrogated BacLacZ binding to the cell surface and subsequent gene transfer. Lastly, direct in vitro binding of BacLacZ to heparin but not BSA columns could be demonstrated after elution of infectious BacLacZ virus in high salt molarity. CONCLUSION: Electrostatic charges play a critical role during the first step in mammalian cell transduction mediated by a recombinant baculovirus. PMID- 10738574 TI - Efficient adenoviral gene transfer to kidney cortical vasculature utilizing a fiber modified vector. AB - BACKGROUND: The alpha v integrin is present in vascular endothelium, including that of the kidney. It is also upregulated in the presence of inflammatory cytokines and in some neoplasms. In an effort to transduce vascular endothelial cells, we compare in vitro and in vivo adenoviral gene transfer of a vector with a high affinity peptide ligand to the alpha v integrin incorporated into the fiber coat protein AdZ.F(RGD) to an unmodified vector, AdZ. METHODS: Cell transduction assays were performed on human umbilical vein endothelial cells (HuVEC) and human pulmonary epithelial cells (A549). In vitro competition assays were performed in the presence of either wild type (F5K) or chimeric (F5K(RGD)) soluble recombinant fiber protein. Transduction efficiency was determined by quantitative beta-galactosidase activity. In vivo gene transfer was compared infusing either AdZ or AdZ.F(RGD) into the left renal artery of the rat and assaying beta-galactosidase staining of the kidney. Gene transfer was also evaluated in the presence of a competitive RGD or control RGE peptide. RESULTS: There was a marked increase in transgene expression in HuVEC cells with AdZ.F(RGD) as compared to AdZ. The increased expression with AdZ.F(RGD) was more prominent in the endothelial as opposed to the epithelial cell line. Furthermore pre-incubation of these cells with either F5K or F5K(RGD) soluble fiber protein markedly decreased beta-galactosidase activity of AdZ, whereas only the F5K(RGD) decreased beta-galactosidase activity of AdZ.F(RGD). AdZ.F(RGD) also resulted in significantly enhanced beta-galactosidase expression in the vascular endothelium of the kidney (for comparable amounts of virus injected) as well as significantly higher gene transfer to cortical vasculature. Coinfusion of an RGD peptide with AdZ.F(RGD) blocked gene transfer whereas a control RGD peptide did not. CONCLUSION: We conclude that incorporating a high affinity peptide ligand into the adenoviral fiber protein can preferentially enhance in vitro and in vivo adenoviral transfection efficiency in endothelial cells. Enhancing transfection efficiency will not only broaden the scope of disease processes addressed with adenoviral vectors but also allow the use of lower titer, thereby limiting toxicity. This vector has additional potential to transduce endothelial cells within tumors or ischemic tissue where alpha v integrins are upregulated. PMID- 10738575 TI - Polycation-based DNA complexes for tumor-targeted gene delivery in vivo. AB - BACKGROUND: Efficient and target-specific in vivo gene delivery is a major challenge in gene therapy. Compared to cell culture application, in vivo gene delivery faces a variety of additional obstacles such as anatomical size constraints, interactions with biological fluids and extracellular matrix, and binding to a broad variety of non-target cell types. METHODS: Polycation-based vectors, including adenovirus-enhanced transferrinfection (AVET) and transferrin polyethylenimine (Tf-PEI), were tested for gene delivery into subcutaneously growing tumors after local and systemic application. DNA biodistribution and reporter gene expression was measured in the major organs and in the tumor. RESULTS: Gene transfer after intratumoral application was 10-100 fold more efficient with Tf-PEI/DNA or AVET complexes in comparison to naked DNA. Targeted gene delivery into subcutaneously growing tumors after systemic application was achieved using electroneutral AVET complexes and sterically stabilized PEGylated Tf-PEI/DNA complexes, whereas application of positively charged polycation/DNA complexes resulted in predominant gene expression in the lungs and was associated by considerable toxicity. CONCLUSION: For systemic application, the physical and colloidal parameters of the transfection complexes, such as particle size, stability, and surface charge, determine DNA biodistribution, toxicity, and transfection efficacy. By controlling these parameters, DNA biodistribution and gene expression can be targeted to different organs. PMID- 10738576 TI - Engineered pluripotent mesenchymal cells integrate and differentiate in regenerating bone: a novel cell-mediated gene therapy. AB - BACKGROUND: Among the approximately 6.5 million fractures suffered in the United States every year, about 15% are difficult to heal. As yet, for most of these difficult cases there is no effective therapy. We have developed a mouse radial segmental defect as a model experimental system for testing the capacity of Genetically Engineered Pluripotent Mesenchymal Cells (GEPMC, C3H10T1/2 clone expressing rhBMP-2), for gene delivery, engraftment, and induction of bone growth in regenerating bone. METHODS: Transfected GEPMC expressing rhBMP-2 were further infected with a vector carrying the lacZ gene, that encodes for beta galactosidase (beta-gal). In vitro levels of rhBMP-2 expression and function were confirmed by immunohistochemistry, and bioassay. Differentiation was assayed using alkaline phosphatase staining. GEPMC were transplanted in vivo into a radial segmental defect. The main control groups included lacZ clones of WT C3H10T1/2-LacZ, and CHO-rhBMP-2 cells. New bone formation was measured quantitatively via fluorescent labeling, X-ray analysis and histomorphometry. Engrafted mesenchymal cells were localized in vivo by beta-gal expression, and double immunofluorescence. RESULTS: In vitro, GEPMC expressed rhBMP-2, beta-gal and spontaneously differentiated into osteogenic cells expressing alkaline phosphatase. Detection of transplanted cells revealed engrafted cells that had differentiated into osteoblasts and co-expressed beta-gal and rhBMP-2. Analysis of new bone formation revealed that at four to eight week post-transplantation, GEPMS significantly enhanced segmental defect repair. CONCLUSIONS: Our study shows that cell-mediated gene transfer can be utilized for growth factor delivery to signaling receptors of transplanted cells (autocrine effect) and host mesenchymal cells (paracrine effect) suggesting the ability of GEPMC to engraft, differentiate, and stimulate bone growth. We suggest that our approach should lead to the designing of mesenchymal stem cell based gene therapy strategies for bone lesions as well as other tissues. PMID- 10738577 TI - Glycofection in the presence of anionic fusogenic peptides: a study of the parameters affecting the peptide-mediated enhancement of the transfection efficiency. AB - Gene delivery mediated by polyplexes such as DNA complexed with polylysine conjugates is limited by the low efficiency of escape of DNA from the endosomes. One of the strategies which favors the transmembrane passage of polyplexes consists of adding anionic amphipathic peptides capable of destabilizing membranes in an acidic medium. Although less efficient than replication-defective adenoviruses, fusogenic peptides increase the expression of the reporter gene by a factor between 100 and 1000 depending on the cell line. However, the activity of a given peptide depends on the composition of the lipid bilayer. We were interested in developing a polyplex (glycoplex) formulation comprising a glycosylated polylysine, a fusogenic peptide and a plasmid which would be useful for efficient transfection (glycofection) of a large panel of cells, even in the presence of serum. We synthesized several peptides and tested their efficiency in combination with different glycoplex formulations. We found that glycofection with a quaternary complex (called one pot formulation) made of lactosylated polylysine, polylysine, DNA, and the dimeric peptide (E5-WYGG)2-KA was less cell type dependent than other peptide-based formulations. In addition, its efficiency was not affected by the presence of serum (up to 20%). PMID- 10738578 TI - Optimism regarding the use of RNA/DNA hybrids to repair genes at high efficiency. PMID- 10738579 TI - Gene transfer and therapy clinical trials--update. Part 2: Gene delivery systems used and genes transferred. PMID- 10738580 TI - A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells. AB - BACKGROUND: Tight control of gene activity has been achieved in cells and transgenic organisms using the Tet regulatory systems. Unregulated basal transcription can, however, be observed whenever integration of target genes driven by promoters responsive to tetracycline controlled transcriptional activators (tTA, rtTA) does not occur at suitable chromosomal sites. Moreover, in viral vectors containing both the tTA coding sequence and the regulated target gene, proximity of the enhancer element driving tTA/rtTA expression to the responsive unit will lead to elevated background levels. Similarly when tTA/rtTA responsive transcription units are in a non-integrated state as e.g., during transient expression, intrinsic residual transcription persists in their 'off' state, which can differ in intensity among different cell types. METHODS: To efficiently repress such background activities we generated tetracycline controlled transcriptional silencers (tTS) that bind promoters responsive for rtTA in absence of the effector doxycycline (Dox). Addition of Dox prevents binding of tTS thus relieving repression, promotes binding of rtTA and thereby switches the promoter from an actively repressed to an activated state. RESULTS: Of several tTS--fusions between a modified Tet repressor and transcriptional silencing domains--tTSKid was found to be most effective in reducing the activity of two target promoters. Ten to 200 fold repression is seen in transient expression whereas in stably transfected HeLa cells the regulatory range of the rtTA system was increased by three orders of magnitude. CONCLUSIONS: The new system appears particularly suited for the transfer of toxic genes into appropriate chromosomal sites as well as for tight regulation of genes carried by viral or episomal vectors. PMID- 10738581 TI - Maxillary sinusitis as a surrogate model for CF gene therapy clinical trials in patients with antrostomies. AB - BACKGROUND: Assessing the biological activity and clinical efficacy of gene therapy is critically important in cystic fibrosis (CF). It is widely accepted that clinical testing using surrogate markers including pulmonary function will be useful in assessing clinical efficacy. One problem with pulmonary surrogate markers of CF disease is the large number of patients and length of time required to demonstrate clinical efficacy. An alternative to pulmonary testing of new CF treatments is use of the maxillary sinuses as a surrogate model of CF lung disease. Using CF sinusitis as a surrogate model for testing clinical efficacy of new treatments is attractive because CF upper respiratory disease is similar to the lower respiratory disease with respect to electrophysiology and microbiology. METHODS: Sinusitis recurrence in untreated sinuses was analyzed during a prospective, randomized, unblinded, dose-escalation, within-subjects, phase I clinical trial of the adeno-associated virus mediated cystic fibrosis transmembrane conductance regulator (AAV-CFTR) gene transfer. RESULTS: Clinical symptoms combined with sinus endoscopy proved useful in the diagnosis of unilateral and bilateral sinusitis recurrence. Sinusitis recurred at a rate of 45% during one month of follow-up. IL-8 concentration rose in sinus fluids from affected sinuses. Bacterial cultures and increased sinus leukocytes corroborated recurrent sinusitis. Sinus CT scans were also useful in diagnosing recurrent sinusitis in this surrogate model of CF infectious exacerbations. CONCLUSIONS: CF sinusitis as a surrogate for lung disease is particularly well-suited for phase II clinical trials of gene transfer agents, with the potential for measuring clinical efficacy in relatively small numbers of patients over relatively short periods of time. PMID- 10738582 TI - Keratinocyte growth factor induced epithelial proliferation facilitates retroviral-mediated gene transfer to distal lung epithelia in vivo. AB - BACKGROUND: Cell proliferation, vector titer and accessibility of target cells represent hurdles for efficient gene transfer to lung epithelia in vivo using recombinant murine leukemia (MuLV)-based retroviruses. We tested the hypothesis that the pulmonary epithelium is susceptible to retroviral-mediated gene transfer when stimulated to proliferate by a mitogen, keratinocyte growth factor (KGF). METHODS: Rats received keratinocyte growth factor (KGF, 2.5 micrograms/g x 4 doses, two consecutive days) intratracheally followed by high titer amphotropic retrovirus expressing beta-galactosidase. Gene transfer was assessed five days later. RESULTS: KGF stimulated transient proliferation in the bronchiolar and alveolar epithelia (30-40% PCNA positive cells at peak) which decreased to background levels seven days after administration. Gene transfer to epithelia (X Gal positive cells) occurred more frequently in KGF treated rats, but proliferation exceeded the level of gene transfer. X-gal positive cells were noted in the alveolar epithelium and occasionally in the bronchiolar epithelium. In order to understand the discrepancy between the number of proliferating and transduced cells, primary rat tracheal epithelium cultured at the air-liquid interface was infected from either the apical or basolateral side. Gene transfer was achieved only through basolateral application of vector, suggesting that epithelial polarity represents a barrier to MuLV-based lung gene transfer in vivo. CONCLUSIONS: KGF transiently stimulates epithelial proliferation in vivo, facilitating MuLV-based gene transfer. Retroviral vectors may encounter multiple barriers which have evolved to defend the lung from infections. PMID- 10738583 TI - Selective uptake and sustained expression of AAV vectors following subcutaneous delivery. AB - BACKGROUND: Recombinant adeno-associated viral (rAAV) vectors are capable of long term expression of secreted and intracellular proteins following delivery to muscle, liver, and the central nervous system. In this study, we have evaluated subcutaneous injection of rAAV encoding a variety of transgenes as an alternative route of administration for the systemic delivery of therapeutic proteins. METHODS: rAAV vectors encoding the human factor IX, human interferon-alpha 2a, murine erythropoietin (epo), and Escherichia coli lacZ genes were used for subcutaneous delivery into mature immunocompetent mice. Expression of factor IX and interferon in mouse serum was measured by ELISA. Expression of Epo was monitored by an increase in hemotocrit and by RIA. The tissue tropism of AAV transduction was determined by histochemistry following administration of the lacZ vector. RESULTS: Long-term protein expression (at least one year) is demonstrated in the serum of immunocompetent mice following subcutaneous delivery of AAV vectors encoding the human factor IX and interferon genes. The murine epo gene delivered via this route resulted in levels of Epo that correlate with increased hematocrits of up to 90% for a duration of nine months. rAAV encoding the lacZ gene revealed that the panniculus carnosus, a skeletal muscle layer of the skin, was transduced upon subcutaneous administration. CONCLUSIONS: This study shows that long-term expression of secreted proteins can be achieved using rAAV vectors injected subcutaneously as a single administration. The observation that the panniculus carnosus is the primary tissue transduced by rAAV illustrates the high tropism of rAAV for skeletal muscle. PMID- 10738584 TI - Computerised dystrophic muscle simulator: prospecting potential therapeutic strategies for muscle dystrophies using a virtual experimental model. AB - Inherited muscle diseases are often characterised by widespread muscle damage in the body, limiting the clinical relevance of cell or gene therapy based upon direct injections into muscles. Recent studies have shown, however, that cells originating from the bone marrow are able to target necrosis-regeneration sites as they occur and, in addition, may also participate in the muscle regeneration after undergoing myogenic differentiation. Here, we present a computerised dystrophic muscle simulator that allows the prospecting of different scenarios of both disease evolution and appropriate employment of blood-borne cells as therapeutic shuttles. It provides the option of examining their use either to transfer a healthy gene into the tissue or to impart substances designed to boost its regeneration. One of the major advantages of this tool is that it offers the opportunity of visualising and composing therapeutic strategies in virtual paradigms in which severe clinical situations, not necessarily available in animal models, can be created. The dystrophic muscle simulator is freely accessible via the Genethon web site (www.genethon.fr), and in the online version via http:@www.wiley.co.uk/genmed. PMID- 10738586 TI - Potential of adenovirus as a vector for sustained gene expression. PMID- 10738585 TI - Direct immunologic activities of CpG DNA and implications for gene therapy. AB - Vertebrate immune systems have evolved the ability to detect and be activated by most microbial and viral DNAs by virtue of their content of unmethylated 'CpG motifs', which are selectively suppressed in vertebrate DNA. Because their CpGs are also unmethylated, the DNA in gene therapy vectors routinely induces direct immune stimulation through activating this host defense mechanism. Administration of such 'CpG DNA' by injection or inhalation triggers rapid activation of B cells, monocytes, macrophages, dendritic cells, and natural killer cells, along with the release of pro-inflammatory cytokines. These immune stimulatory effects can be prevented by chloroquine and other drugs that interfere with endosomal maturation or by the presence of certain neutralizing DNA sequences, which block the immune stimulatory CpG motifs. Aside from serving as the genetic code, DNA can have direct immune activities. Vertebrate immune systems have evolved a defense mechanism that is able to broadly detect most microbial and viral DNAs because of differences in the frequency and methylation of CpG dinucleotides in particular base contexts. B cells, monocytes, macrophages, and dendritic cells spontaneously take up DNA of any type. If the DNA contains these immune stimulatory 'CpG-S motifs', the cells become activated within minutes and begin producing pro-inflammatory cytokines such as IL-6 and IL-12 and upregulate expression of co-stimulatory molecules. This results in the activation of both innate and acquired immune responses. The pro-inflammatory effects of CpG-S motifs are opposed by CpG dinucleotides in certain distinct base contexts, termed neutralizing or CpG-N motifs. Increasing the ratio of CpG-S to CpG-N motifs enhances the immune stimulatory effects of DNA, even if the total level of CpGs in the DNA is not altered. While this is useful in generating enhanced genetic vaccines, the opposite strategy is likely to become useful for the generation of gene therapy vectors with reduced inflammatory effects. PMID- 10738587 TI - NIH office of recombinant DNA activities. PMID- 10738588 TI - Gene transfer and therapy clinical trials--update. Part I: Countries, diseases, clinical trial phases, routes of administration. PMID- 10738589 TI - [Diesel particles and allergy: cellular mechanisms]. AB - Urbain air pollutants, particularly diesel exhaust particles are now known to contribute to the increased prevalence of asthma and allergic rhinitis. Diesel exhaust particles act as adjuvants in the immune response and may lead to the enhancement of allergic inflammation. This was first suggested by epidemiological studies and now largely confirmed by numerous experimental studies in animals and humans. We review the different mechanisms involved, including effects on cytokine and chemokine production, as well as activation of different immune cells. We also discuss the metabolic and cellular activation pathways used by polycyclic aromatic hydrocarbons, allergens and their interaction with diesel particles which act in synergy in this immune response toward IgE production and induction of allergic inflammation. PMID- 10738590 TI - [Respiratory allergy to moulds among adults in Eskisehir Anatolia), Turkey]. AB - Allergy to moulds has been known for several decades. But it is rarely isolated in contrast to the other pneumallergens. Prevalence of sensitization to moulds varies from one country to the another. We investigated the allergy to moulds using skin prick tests in adult patients with asthma and/or rhinitis in Eskisehir (Anatolia), Turkey. Cladosporium and Aspergillus were found to be the most common causes in the study population (115 patients). Isolated mould allergy was found as 4.3% with the exclusion of common non-fungal respiratory allergens, among them Cladosporium was the most common cause. PMID- 10738591 TI - Veterinary homeopathy--be openminded! PMID- 10738592 TI - Comment regarding revaccination guidelines. PMID- 10738593 TI - Claims for veterinary chiropractic unjustified. PMID- 10738594 TI - An ethicist's commentary on how to decide whether a given practice is unethical. PMID- 10738595 TI - Agriculture's role in managing antimicrobial resistance: conference report. PMID- 10738596 TI - Standardization of microchip implantation sites PMID- 10738597 TI - The 1999 Reginald Thomson Lecture. Custom-built mice: unique discovery tools in biomedical research. AB - The study of mouse genetics is a science in its own right. Currently, the mouse is the central platform for model building in biomedical research. Recombinant DNA technology and mouse transgenesis have provided an almost unlimited resource of new animals that is constantly expanding. Investigators, through their selection of the most appropriate type of mouse and by imaginative combinations of animals in breeding or culture experiments, have the opportunity for discovery of new information that will benefit the health of both humans and species of veterinary interest. Wider appreciation and use of the mouse as a discovery tool in veterinary research should be encouraged. PMID- 10738598 TI - Esophageal obstruction in horses: a retrospective study of 34 cases. AB - The major purpose of this investigation was to describe the causes, possible complications, and prognoses of horses with esophageal obstruction. Of 34 cases presenting with esophageal obstruction, 28 cases were due to impaction of ingesta. Obstruction due to pre-existing esophageal disease occurred in 4 horses with megaesophagus, in 1 horse with stricture in the upper third of the esophagus, and in 1 horse with esophageal diverticulum. There was no significant difference in the contamination of the trachea between horses that subsequently developed aspiration pneumonia and those that did not. The duration of esophageal obstruction prior to admission was significantly longer in horses that developed aspiration pneumonia (median 18, range 2-48 h) than in those horses that did not (median 4, range 0.5-48 h). Although the obstruction was relieved in all 34 horses, 4 were euthanized because of recurring obstruction due to megaesophagus (n = 2), esophageal diverticulum (n = 1), and esophageal stricture (n = 1). PMID- 10738599 TI - Evaluation of melengestrol acetate and equine chorionic gonadotropin for out-of season breeding in sheep on Prince Edward Island. AB - The purpose of this study was to compare the efficacy of a recommended protocol of oral melengestrol acetate (MGA) to intravaginal medroxyprogesterone acetate (MPA), with or without equine chorionic gonadotropin (eCG), for out-of-season breeding of sheep on Prince Edward Island. One hundred and twenty ewes were assigned to 1 of 4 groups in a factorial design and were treated with either an intravaginal MPA sponge or oral MGA. Ewes received either an intramuscular injection of eCG or a saline placebo at cessation of progestogen treatment. The reproductive performance was best for the MPA-eCG group, followed by the MGA-eCG, MGA-saline, and MPA-saline. Pregnancy rates of 66.7%, 43.3%, 31.0%, and 16.7%, respectively, and ratios of lambs born to ewes exposed to the ram of 1.17, 0.63, 0.52, and 0.23, respectively, were recorded for these groups. We concluded that, under the conditions of this study, the use of oral MGA resulted in fewer lambs than did the use of MPA sponges with eCG. Nonetheless, the use of MGA may be attractive to producers because it is less expensive and more convenient than the use of sponges. PMID- 10738600 TI - Use of recombinant human thyroid-stimulating hormone for thyrotropin stimulation test in euthyroid dogs. AB - The purpose of this study was to evaluate the effects of the recombinant human thyroid-stimulating hormone (rhTSH) on serum total thyroxine (TT4) concentration in euthyroid dogs. Six healthy beagle dogs were used in each of the 3 phases of this study. Phase I: thyroid-stimulating hormone response tests were performed by using a total dose of 25 micrograms, 50 micrograms, and 100 micrograms of rhTSH, administered intravenously. Phases II and III: thyroid-stimulating hormone response tests were performed by using 50 micrograms of rhTSH administered by intramuscular and subcutaneous routes, respectively. In each phase and following all the administered doses of rhTSH, an increase in the serum TT4 concentration was noted, although it was not always significant. For phase I, there was a significant increase in serum TT4 concentrations. Based on this study, 50 micrograms was judged to be the optimal intravenous dose of rhTSH. For phases II and III, there was no significant increase in serum TT4 after the administration of rhTSH. Results of this study suggest that rhTSH could be a good substitute for bovine TSH, when used by the intravenous route, for the TSH stimulation test in dogs. Further studies are required to confirm its clinical usefulness. PMID- 10738601 TI - Comparative cost-effectiveness of ivermectin versus topical organophosphate in feedlot yearlings. AB - A replicated-pen field trial was performed under commercial feedlot conditions in western Canada to determine the cost-effectiveness of administering ivermectin to yearling beef cattle upon entry to the feedlot after the grazing season, and to establish the level of trichostrongylid gastrointestinal parasite infection in this population, as estimated by fecal egg counts. Six thousand eight hundred and eighty-three, mixed breed, yearling steers were randomly allocated upon arrival at the feedlot to one of 2 experimental groups as follows: Ivermectin, which received topical ivermectin (0.5%) at the rate of 1.0 mL/10 kg body weight; or Fenthion, which received topical fenthion (20%) at the rate of 12 mL/295 kg body weight. There were 15 pens in each experimental group. Final weight, weight gain, average daily gain, and dry matter intake to gain ratio were significantly (P < 0.05) improved in the Ivermectin group as compared with the Fenthion group. There were no significant (P > or = 0.05) differences in initial weight, days on feed, or daily dry matter intake between the experimental groups. The geometric mean fecal egg counts at the time of allocation were 14.7 eggs/5 g and 16.6 eggs/5 g for the Ivermectin and Fenthion groups, respectively (P > or = 0.05). There were no significant (P > or = 0.05) differences in morbidity or mortality between the experimental groups. In the economic analysis, the significant improvements in feedlot performance in the Ivermectin group resulted in a net economic advantage of $4.20 CDN per animal. PMID- 10738602 TI - Cervical spinal arachnoid cyst in a dog. AB - An 18-month-old, intact male Akita presented with a 12-month history of progressive ataxia, hypermetria, and loss of conscious proprioception of the thoracic and pelvic limbs. Neurological examination and myelography localized a lesion at cervical vertebrae 1 and 2 consistent with an arachnoid cyst. Hemilaminectomy and cyst fenestration led to virtually full recovery. PMID- 10738603 TI - Anesthesia and analgesia. PMID- 10738605 TI - Clinical pathology: portosystemic shunt in a Labrador retriever. PMID- 10738604 TI - Veterinary nutraceutical medicine. PMID- 10738607 TI - [Hepatic resection for hepatocarcinoma in the cirrhotic patient]. AB - Between January 1993 and June 1999, 54 patients with hepatocellular carcinoma (HCC) complicating cirrhosis underwent hepatic resection. Forty-five (83%) minor hepatic resections were performed. Four (7%) peroperative deaths (within 30 days following surgery) were recorded. During follow-up it was noted that hepatocellular carcinoma recurred in 24 patients (44%) and local recurrence was the most frequent event. Mean disease free interval was 19 months after surgery. By multivariate analysis only vascular invasion proved to be statistically significant to HCC recurrence and survival. The recurrence rate of HCC in patients undergoing liver resection is high and long-term survival is long. An accurate analysis of risk factors for HCC recurrence after liver resection is of the utmost importance and further treatment alternative to and/or in association with surgery requires consideration. PMID- 10738606 TI - Early and long-term prognostic factors after liver resection for HCC. AB - The Authors discuss the principal early and long term predictive factors after liver resection in patients with hepatocellular carcinoma (HCC). The Authors report (131 cases) early mortality as 7.6%, entirely confined in the group, numerically prevalent and affected by cirrhosis. None of the 50 patients with chronic hepatitis (29 cases) or normal liver (21 cases) died after hepatic resection. Mortality is higher in Child B patients (20.7%) and in cases in which a massive haemotransfusion was given (p < 0.05), apart from the width of resection and from the number of hepatic resections. None of 41 cirrhotic Child A patients undergoing a limited hepatic resection (< or = 1 segment) died during the perioperative period. In the group of patients which survived to the resection, global survival at 5 years was 45%. The most important prognostic factor is local recurrence while cirrhosis and the degree of liver failure are not statistically significant. No feature can identify a subgroup of patients with a higher risk of recurrence, which is observed in 52% of patients with a follow up observation after more than 1 year. Among the 29 patients alive after more than 4 years from liver resection, only 11 didn't have local recurrence. The others were treated with iterative hepatic resections or with radiological techniques. In conclusion, the present experience suggests that, in selected cases, hepatic resection could be a low risk therapy (in cirrhotic patients as well). The long term results could improve with an aggressive attitude towards recurrence. PMID- 10738608 TI - [Pneumonectomy for benign disease]. AB - Pneumonectomy for benign disease of the lung is a rather infrequent intervention. A retrospective study based on 1900 pulmonary resections performed in our institute up to 1998, identified a total of 15 patients submitted to pneumonectomy for non-neoplastic disease. Indications were chronic infections in 11 cases, congenital malformations in 2 cases, left primary bronchial stenosis caused by closed thoracic trauma one in case and gunshot wound in one case. Access to the lung was obtained in all cases by a classic postero-lateral thoracotomy: intra-pericardial ligature was required in five cases and in one patient an extrapleural pneumonectomy was performed. The intra-operative mortality was 20% and the average time of hospitalization 27 days. One patient, operated for pulmonary tuberculosis, developed a broncopleural fistula requiring a second operation. In conclusion, the use of pneumonectomy for non-neoplastic diseases presents serious problems and may be associated with major complications. PMID- 10738609 TI - [Quality of life assessment in pancreatic carcinoma: results of an European multicentric study]. AB - Quality of life (Qol) assessment is a mandatory endpoint of cancer clinical trials. Little research has been conducted on pancreatic cancer (Pc) and no disease specific Qol questionnaire exists. We report on the development of the European Organization for Research and Treatment of Cancer (EORTC) Pc Qol module to supplement the core cancer module (QLQ-C30). Literature research and qualitative interviews with 6 specialists and 34 patients in the UK were followed by construction of a questionnaire, a peer review by international specialists and EORTC. Administration of the QLQ-C 30, a provisional Pc module and a qualitative debriefing interview was performed on 78 patients in 8 countries stratified into groups by disease stage and treatment intention. Pretesting identified that 23/26 questions had an adequate internal reliability (Cronbach's alpha > 0.7) and construct validity (Pearson's r 0.4-0.6). The median time of completion was 12 mins. Qualitative analysis indicates that the module is easy to complete and cross culturally applicable. The EORTC QLO-PAN 26 includes 26 items covering symptoms, body image, sexuality and the emotional and social consequences of Pc. It is intended for use in patients undergoing clinical trials for pancreatic cancer including surgery, chemotherapy and radiotherapy and will allow the detection of small but clinically meaningful differences in clinical trials for Pc. PMID- 10738610 TI - [Laparoscopic treatment of complicated colonic diverticulosis]. AB - Despite certain controversies over applying a laparoscopic approach to neoplastic pathologies of the colorectum, it seems to be an ideal form of treatment for benign lesions. The Authors have analysed the results of videolaparoscopic treatment in 23 cases of complicated diverticular disease of the colon using their experience of 205 mininvasive colonic operations. Age, sex, diagnosis, time morbidity and type of intervention such as intra and postoperative, length of hospital stay and the parameters that characterise it (length of nasogastric probe, canalisation and restoration of feeding) were the variables considered in both absolute and comparative methods among the various stages of the disease. The aim was to find the best possible treatment. Out of the 23 patients in the study group, immediate recanalisation was impossible in only one case and all interventions were performed according to the predicted video-assisted technique. A partial ureteral lesion was the only single intraoperatory complication found and was intraoperatively repaired by means of transureteral endoscopy. In three cases non specific complication were encountered. The average length of the intervention was 180 minutes, nasogastric tube was taken 3 days, patients passed and start to feed at the 3rd and 4th postoperative day. These data overlap in the various stages of the disease. An adequate preoperatory study and good experience in major laparoscopic interventions resulted in the common difficulties that surgeons might encounter in this pathology to be overcome. We can therefore conclude that based on our results as well as those found in the literature, diverticular disease can ideally be treated with mininvasive surgery. PMID- 10738612 TI - [Primary hyperparathyroidism: follow-up of the surgically treated patient]. AB - After several remarks on the etiopathogenesis of primary hyperparathyroidism the Authors emphasise the importance of follow-up in operated patients. The evaluation begins right after removal of the lesion in the intraoperative phase, in order to evaluate the validity of the intervention and continues with evaluation of mineral homeostasis, disease itself and function of target structures. In addition to possible hypocalcemic crises, special attention should be placed on possible disease recurrence, and the study of all involved parts such as bones, kidneys, pancreas, digestive apparatus as well as the psychological aspects. These are often involved in a significant manner and condition the quality and duration of the patient's life. PMID- 10738611 TI - [Appendiceal carcinoid. Clinical observations and therapeutic considerations]. AB - The Authors report 3 cases of carcinoid of the appendix discussing the difficult preoperative diagnosis and the different therapeutical options. Between May 1998 and August 1999 36 appendicectomies were performed, in 18 males and 18 females (age 14-89 years; m. a.: 51.5 years). In 3 cases histological response was positive for carcinoid of the appendix. No postoperative mortality or morbility was reported. In one case, the malignancy of histological response made a second operation of typhlectomy necessary, without postoperative complications. The Authors analyze the biological peculiarities and the prognostic factors of appendiceal carcinoid tumors, as size of the tumor and the lymphatic or vascular infiltration of the mesoappendix (and the relative more aggressive surgical treatments) and recommend an appropriate postoperative follow-up since synchronous or metachronous bowel carcinomas are likely to occur. PMID- 10738613 TI - Repair of complex giant ventral hernias with polypropylene and omentoplasty. AB - The Authors in this report describe their experience with complex giant ventral hernias repaired by omentoplasty and polypropylene meshes. They explain their own technique of using the omentum when there is a lack of tissue and the need of an autologous tissue to place between the viscera and the above-lying polypropylene mesh. Among the 5 patients repaired, 4 had a giant sub-umbilical midline ventral hernia and one had a giant border ventral hernia. In all patients, the omentum was placed between the viscera and the mesh, which was fixed by interrupted sutures to the above-lying musculoaponeurotic plane. No complications, signs of bowel obstruction, or recurrences occurred. The Authors stress the omentum as an ideal tissue because of its biocompatibility and prevention of adhesions and fistulas allowing the use of a rigid and efficient prosthesis such as polypropylene. Thus, on the basis of their own short- and long-term good results, they suggest, whenever possible and necessary, the use of omentoplasty to repair complex giant ventral hernias. PMID- 10738614 TI - [Miles operation in rectal carcinoma: history and current status]. AB - The Authors confirm how the advent of mechanical suture has directed the management of rectal carcinoma to an increasingly conservative attitude. In reporting their experience--which consists of about twenty years (222 patients) they specify how not only an increased incidence of recurrence has not appeared, but they have also observed a reduction of theirs (-5.2%). They then attempt to outline basic moments which have characterized the history of rectal carcinoma surgery and they state how today, 80 years since it was conceived, Miles' intervention maintains its validity, even if with more restrictive indications. PMID- 10738615 TI - [Radical trachelectomy with lymphadenectomy]. AB - Radical trachelectomy was described for the first time by Dargent in 1987 as an alternative to the traditional radical hysterectomy; it led to a changed procedure in therapy for early cervical cancer in young women who want to preserve their fertility. With this technique it is possible to remove the uterine cervix and parametrius and at the same time preserve the upper part of the cervical canal and uterus. Only a low-risk subset of young patients with early cancer of the cervix are considered eligible for this treatment; to select them histological and clinical staging of the lesion are the most important criteria. We describe the cases of three patients treated with radical trachelectomy between June 1997 and March 1998 in our Institute, reporting in detail the surgical procedure and the results. PMID- 10738616 TI - Life-threatening digestive haemorrhage from duodenal recurrence of gastric cancer. AB - Invasion of the duodenum by gastric carcinoma is not uncommon. The duodenal invasion by transpiloric infiltration through the submucosal layer or lymphatic spread frequently being microscopic and in minimal number of cases involving the mucosa, is generally asymptomatic and detected only in postmortem examinations. We report a case of life-threatening gastrointestinal bleeding from cancer recurrence at duodenal stump after subtotal gastrectomy for gastric carcinoma. In such cases it can be very hard to find the haemorrhagic source because of the difficulties encountered in endoscopic and radiological evaluation of the duodenal stump. PMID- 10738617 TI - H-type rectovaginal fistula associated with the Currarino triad. AB - We describe a case of H-type rectovaginal fistula associated with the Currarino triad (anorectal stenosis, sacral defect, presacral mass). Presenting symptoms included passage of feces per vaginam, signs of intestinal subocclusion without perianal inflammation, left leg paresis and foul-smelling urine. An anterior sacral meningocele was repaired at the age of three months. At age 18 months the fistula was excised through a perineal approach after creation of a protective colostomy. Diagnostic and therapeutic aspects of this malformation are discussed. PMID- 10738618 TI - Mucinous adenocarcinoma in chronic anorectal fistula. AB - Adenocarcinoma in association with chronic anal fistula is a rare disease which gives rise to difficult problems of diagnosis and treatment. A case of mucinous adenocarcinoma arising on a long standing fistula in ano is described. A patient with a long history of mucinous discharge, pain and perianal induration underwent a biopsy of the external opening of the fistula that showed mucinous infiltrating adenocarcinoma. After a colonoscopy and a preoperative abdominal CT scan, she underwent a successful abdominoperineal resection with adjuvant chemoradiation therapy. Diagnosis of this condition is often difficult; deep and multiple biopsies of the fistulous tracks or perianal mass are necessary to establish the diagnosis. An accurate staging of the neoplasm, using endorectal ultrasound, NMR or CT scans is needed to plan the appropriate treatment. Recent studies have shown that locally advanced anal adenocarcinomas could benefit from pre or postoperative chemoradiation therapy. However, an accurate and complete removal of the tumor, which usually entails abdominoperineal resection, is often necessary to achieve radicality. Despite new therapy protocols, the prognosis of mucinous adenocarcinoma is still poor, mostly due to its advanced nature at the time of diagnosis. This reinforces the importance of biopsy of all perianal abscesses and fistulas for early detection and treatment. PMID- 10738619 TI - Pathologies of travel. Introduction. PMID- 10738620 TI - Letting madness range: travel and mental disorder, c1700-1900. PMID- 10738621 TI - The continental journeys of Andrew Duncan Junior: a physician's education and the international culture of eighteenth-century medicine. PMID- 10738622 TI - Richard Jago's Edge-Hill revisited: a traveller's prospect of the health and disease of a succession of national landscapes. PMID- 10738623 TI - 'The Rime of the Ancient Mariner', a ballad of the scurvy. PMID- 10738624 TI - Lassitude and revival in the warm south: relaxing and exciting travel, 1750-1830. PMID- 10738625 TI - Pathological topographies and cultural itineraries: mapping 'mal'aria' in 18th- and 19th-century Rome. PMID- 10738626 TI - The railway journey and the neuroses of modernity. PMID- 10738627 TI - Mobility, syphilis, and democracy: pathologizing the mobile body. PMID- 10738628 TI - The politics of medical topography: seeking healthiness at the Cape during the nineteenth century. PMID- 10738629 TI - Sleepers wake: Andre Gide and disease in Travels in the Congo. PMID- 10738630 TI - Botanical briefs: daffodils--Narcissus L. PMID- 10738631 TI - Sturge-Weber syndrome. PMID- 10738632 TI - Phaeohyphomycosis in a premature infant. PMID- 10738633 TI - Transgrediens et progrediens palmoplantar keratoderma (Greither's disease) with particular histopathologic findings. AB - We describe a patient with transgrediens et progrediens palmoplantar keratoderma (Greither's disease). Ten of the 25 members of this patient's family in six consecutive generations were affected by the disorder. The pedigree was consistent with an autosomal dominant inheritance pattern, with variable penetrance. The proband's physical examination showed typical signs of Greither's disease. The most striking findings were seen in histopathologic study and consisted of round, focal areas of orthohyperkeratosis located on delled areas of the epidermis. These histopathologic features were present in both the lesions of the palms and dorsum of the hands. We review the clinical manifestations and histopathologic findings of this particular variant of palmoplantar keratoderma and the differential diagnosis. PMID- 10738634 TI - A new family with Papillon-Lefevre syndrome: effectiveness of etretinate treatment. AB - Papillon-Lefevre syndrome is characterized by the association of palmoplantar hyperkeratosis, severe periodontitis, and early loss of deciduous and permanent teeth. We report two patients from the same family, aged 21 and 30 years, who were unaware of their pathology; one was successfully treated with etretinate. PMID- 10738635 TI - Topical imiquimod for recalcitrant facial flat warts. AB - Imiquimod is a unique topical therapeutic agent useful in the treatment of external genital and perianal warts (condyloma acuminata) in adults. The authors report a case of a 21-year-old woman who experienced complete clearance of recalcitrant facial flat warts after 3 weeks of therapy with topical imiquimod 5% cream. PMID- 10738636 TI - Azithromycin eruption in infectious mononucleosis: a proposed mechanism of interaction. AB - The penicillin family of antibiotics may induce drug eruptions when prescribed to patients with infectious mononucleosis. Very similar phenomena have also been cited with other antibiotic families. We report the first case of a cutaneous reaction in a patient with infectious mononucleosis treated with azithromycin. We propose an immune-based hypothesis to explain the transient sensitivity resulting in this secondary cutaneous eruption. PMID- 10738637 TI - Unilateral psoriasis: a case individualized by means of involucrin. AB - Several authors have questioned the existence of unilateral (linear) psoriasis. These authors have suggested that the condition is actually an inflammatory linear verrucous epidermal nevus, or the result of an isomorphic effect on a pre existing epidermal nevus. We report the case of a 25-year-old man, with no relevant personal or family history, who presented with a number of pruritic, punctiform erythematosquamous lesions that were linearly distributed over the left side of the body. Clinical examination and results of histopathologic and histochemical studies indicated unilateral psoriasis. Our findings confirm that involucrin immunohistochemistry can be a useful diagnostic tool in cases of this type. Treatment with keratolytics and topical calcipotriol led to a significant, but only temporary, improvement. PMID- 10738638 TI - The art of learning dermatology. AB - Complete mastery of dermatology is difficult, if not impossible. However, for the open-minded, inquisitive dermatologist, there are many tangential areas that can be studied. This article presents some of the items that have been of interest to me. I hope it inspires others to search out sidelights that will be equally fascinating. PMID- 10738639 TI - Generalized essential telangiectasia in a patient with Graves' disease: should the spectrum of autoimmune diseases associated with generalized telangiectasia be expanded? AB - Generalized essential telangiectasia (GET), as originally described, is not associated with any underlying disease. Although patients with GET lack the typical periungual telangiectases associated with autoimmune collagen vascular diseases, these patients may have an underlying autoimmune process. We present a patient with a history of Graves' disease and low-titer anti-nuclear antibodies, who developed rapidly progressive generalized telangiectases. The gender and age of the majority of patients with GET fit well within the demographics of most autoimmune diseases. The documented occurrence of an autoimmune disease in several of the limited number of patients previously diagnosed with GET provides additional evidence that GET may be associated with an underlying autoimmune disease. PMID- 10738640 TI - Adsorption-desorption of ondansetron on latex particles. AB - Ondansetron is a carbazol with antiemetic properties. It is used primarily to control nausea and vomiting caused by cytotoxic chemotherapy and radiotherapy, as well as for treatment of postoperative vomiting in gynecological surgery. Ondansetron has a shelf life of about 3 hr; hence, it is a matter of great interest to determine the ideal adsorption-desorption conditions for this drug on latex particles for designs of formulations (oral suspensions) containing polymers with the aim of delivering different drugs in a prolonged and controlled fashion. Time, pH, electrolytes, and concentration of the active principal at which maximal adsorption occurred were determined. Desorption of the drug from latex polymer particles was studied in different media. The results obtained suggest that this polymer is suitable as carrier of drug for obtained formulations of controlled release. The findings suggest that pH is the principal factor influencing the release of the ondansetron from Aquateric. The greatest release of drug occurs at acid pH, approximately 70% in the first hour; for the basic medium, the release is about 6%. PMID- 10738641 TI - In vitro-in vivo correlation and comparative bioavailablity of vincamine in prolonged-release preparations. AB - Developing an in vitro dissolution test that gives good correlation with in vivo data for a particular drug product is an important objective. Available dissolution data of vincamine prolonged-release preparations show different in vitro release behavior at different pH using the conventional dissolution techniques. This does not allow development of an in vitro-in vivo correlation (IVIVC). In the present work, the flow-through cell (FTC) dissolution system (USP apparatus 4) was utilized to compare the release rate of three marketed prolonged release oral formulations of vincamine; namely, a brand innovator formulation used as the reference and two formulations from different manufacturers as test products. Both the open and closed systems of FTC were used at variable pH. A comparative bioavailability study was then conducted in 16 healthy volunteers for a test versus the reference product by administering a single dose of 60 mg in a crossover design. Vincamine plasma concentrations were analyzed by a sensitive high-performance liquid chromatography (HPLC) method. This was followed by assessment of IVIVC according to level A as specified by USP 23; the absorbed fraction of vincamine was determined using the Wagner-Nelson method. The results indicated that the pH of the medium affects the release rate pronouncedly. The relative bioavailability based on Cmax and AUC0-12 were found to be 83.15% and 84.15%, respectively. Good correlation was obtained between fraction absorbed in vivo and fraction dissolved in vitro by applying the open system of the FTC. This technique gave the most favorable results with regard to the percentage vincamine released and the IVIVC. PMID- 10738642 TI - Study of omeprazole-gamma-cyclodextrin complexation in the solid state. AB - The possibility of obtaining inclusion complexes between omeprazole (OME) and gamma-cyclodextrin (gamma-CD) by kneading, spray-drying, coprecipitation, and freeze-drying was evaluated. All these methods lead to the isolation of a true inclusion compound, as evidenced by differential scanning calorimetry (DSC), infrared spectroscopy, and X-ray diffractometry on powder (PXRD). Moreover, PXRD and scanning electron microscopy (SEM) afforded data concerning crystallinity and surface characteristics of the solid phases obtained. In all cases, a significant increase of the release rate with respect to the drug alone was found, and it was attributed to the formation of an inclusion compound. Among the solid phases obtained, the comprecipitated product presented the highest dissolution rate. PMID- 10738643 TI - Determination of methotrimeprazine in pharmaceutical preparations by visible spectrophotometry. AB - A spectrophotometric method is described for determination of methotrimeprazine (levomepromazine). The aim of this work was to develop a simple, rapid, precise, and accurate visible spectrophotometric method for determination of methotrimeprazine in tablet, oral solution, and injection. The method is based on methotrimeprazine reaction with bromophenol blue, resulting in a stable, light yellow-green ion-pair complex that, after extraction with chloroform, presented maximum absorption at 409 nm. Beer's law was obeyed in the concentration range from 5.0 to 25.0 micrograms/ml. The proposed standardized method was applied to commercially available and simulated samples. The accuracy of the method was confirmed by recovery tests. PMID- 10738644 TI - Determination of azathioprine and its related substances by capillary zone electrophoresis and its application to pharmaceutical dosage forms assay. AB - The development of a stability-indicating capillary zone electrophoresis (CZE) method for the determination of the drug azathioprine (AZA) and its related substances in bulk and dosage forms is described. Theophylline was used as an internal standard to improve quantitative results. The method was fully validated in terms of repeatability (n = 10, RSD for migration time and peak area ratio were 0.15% and 0.60%, respectively), reproducibility (n = 5, RSD of peak area ratio was 0.84%), linearity at two ranges of the azathioprine concentration, limits of detection (LOD) and quantitation (LOQ), and robustness. The method was applied for determination of the drug in bulk and a commercial tablet dosage form (recovery 98.3-101.3%) and in powder for injection (recovery 98.7-100.6%). The method was fast and reliable for the analysis of AZA and its related substances in bulk and dosage forms. PMID- 10738645 TI - Multivariate methods in developing an evolutionary strategy for tablet formulation. AB - The aim of this study was to develop a new strategy for choosing excipients in tablet formulation. Multivariate techniques such as principal component analysis (PCA) and experimental design were combined in a multivariate design for screening experiments. Of a total 87 investigated excipients, the initial screening experiments contained 5 lubricants, 9 binders, and 5 disintegrants, and 35 experiments were carried out. Considering a reduced factorial design was used, the resulting PCA and partial least squares (PLS) models offered good insight into the possibilities of tablet formulation. It also offered solutions to the problems and clearly gave directions for optimum formulations. Further, it offered several alternatives for achieving quality formulations. Additional experiments conducted to validate and verify the usefulness of the model were successful, resulting in several tablets of good quality. The conclusion is that a multivariate strategy in tablet of formulation is efficient and can be used to reduce the number of experiments drastically. Combining multivariate characterization, physicochemical properties, experimental design, multivariate design, and PLS would lead to an evolutionary strategy for tablet formulation. Since it includes a learning strategy that continuously incorporates data for new compounds and from conducted experiments, this would be an even more powerful tool than expert systems. PMID- 10738646 TI - Lubricant susceptibility of Cellactose and Avicel PH-200: a quantitative relationship. AB - The lubricant susceptibility of two direct compression excipients, Avicel PH-200 and Cellactose, was determined using a model mix with metamizol sodium (dipyrone) and designed experiments to obtain a quantitative relationship by multiple regression. As a result, a new constant called specific lubricant sensibility Ls is proposed for the comparison of lubricant susceptibility in other drug excipient systems. PMID- 10738647 TI - Mucoadhesive and physicochemical characterization of Carbopol-Poloxamer gels containing triamcinolone acetonide. AB - The viscosity and bioadhesive property of Carbopol-Poloxamer gels containing triamcinolone acetonide to mucosa were tested according to various concentrations of Carbopol gels of various pH. The increase in Carbopol concentration caused increased viscosity and bioadhesiveness. The neutralization of pH in various concentrations of Carbopol gels showed the increased viscosity, showing the highest viscosity and highest bioadhesiveness when neutralized to pH 6. A relationship between the viscosity and bioadhesive strength was shown from the neutralized Carbopol gels. The physicochemical interactions between triamcinolone acetonide and polymers were investigated by X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectrophotometry. According to FTIR and XRD studies, the drug did not show any evidence of an interaction with the polymers used and was present in an unchanged state. PMID- 10738648 TI - Preparation and pharmacodynamic evaluation of liposomes of indomethacin. AB - The side effects of indomethacin, such as ulceration of the kidney and central nervous system (CNS) toxicity, limit its use as a drug for rheumatoid arthritis. Encapsulation of this drug in liposomes may reduce the toxic effects. The aim of this study was to determine the factors influencing encapsulation of indomethacin in liposomes and to determine anti-inflammatory potential of liposomal indomethacin. A series of liposomal formulations of indomethacin were prepared using various phospholipids. The effects of method of preparation, lipid composition, charge, and cholesterol (CH) on encapsulation of indomethacin in liposomes were investigated. A significant variation in encapsulation of the drug in liposomes was observed when prepared by different methods. With all the methods of preparation tried, the favorable lipid composition for high encapsulation of this drug was egg phosphatidyl choline:CH: stearlyamine (PC:CH:SA) at a 1:0.5:0.1 molar ratio. Inclusion of cholesterol did not affect the encapsulation efficiency of the drug in liposomes. The drug release profile from the liposomes was biphasic, and the highest percentage drug release was observed with large unilamellar vesicles (LUVs) (100 nm). Inclusion of stearylamine (PC:CH:SA 1:0.5:0.1) and phosphatidyl glycerol (PG) (PC:CH:PG 1:0.5:0.2) in the liposomes reduced the release of the drug in comparison to the neutral liposomes (PC:CH 1:1). The slow release of the drug from stearylamine containing liposomes may be explained by the electrostatic interaction between the acid moiety of the drug and the amine moiety of the lipid. It is assumed that the possible hydrogen bonding between--OH groups of phosphatidyl glycerol and the -COOH group of the drug might be the reason for the slow release of the drug from PC:CH:PG (1:0.5:0.2) containing liposomes. Pharmacodynamic evaluation of the liposomes was performed by carrageenan-induced rat paw edema (acute) and adjuvant arthritis (chronic) models. The anti-inflammatory activity was increased from the first to fifth hour PC:CH:PG (1:0.5:0.2) and PC:CH:SA (1:0.5:0.1) liposomes showed the highest percentage inhibition of edema. In both these models, anti inflammatory activity of liposomal indomethacin was significantly higher than that of free indomethacin (p < .01). The ulcer index of the free drug was about three times more than the encapsulated drug when administered at the same dose intraperitoneally to arthritic rats consecutively for 21 days. PMID- 10738649 TI - The effects of plasticizers on the release of metoprolol tartrate from granules coated with a polymethacrylate film. AB - For coating metoprolol tartrate granules, coating dispersions of Eudragit RS 30 D containing 6%, 12%, or 18% (based on polymer weight) of one of the following plasticizers were used: polyethylene glycol 400 (PEG400), propylene glycol (PG), tributyl citrate (TBC), and triethyl citrate (TEC). The release of metoprolol tartrate from these coated granules was determined at pH 1.2 and 7.4. Slower release resulted from the use of each plasticizer, being slower with increasing concentration of the plasticizer. Release was faster with the more water soluble PEG400 and PG than with TBC and TEC. pH-dependent release was observed with PEG400, PG, and TBC, while TEC gave pH-independent release of drug. PMID- 10738650 TI - The adjuvants aerosil 200 and Gelita-Sol-P influence on the technological characteristics of spray-dried powders from Passiflora edulis var. flavicarpa. AB - Passiflora edulis (passionflower) is a plant widely used in the Brazilian popular medicine and phytopharmaceutical industry for its sedative activity. This work refers to the development of spray-dried powders (SDPs) from the 40% ethanolic extractive solution of P. edulis aerial parts. The SDPs were prepared with a Buchi 190 Mini-spray dryer using as drying adjuvants Aerosil 200 alone (SDP1), an Aerosil 200: Gelita-Sol-P (1:1) mixture (SDP2) and an Aerosil 200:Gelita-Sol-P (1:3) mixture (SDP3). All the powders were obtained using 40 parts adjuvant and 60 parts extract dried residue. The comparison criteria applied were particle size distribution, hygroscopicity at 95%, 60%, and 35% relative humidity (RH), as well as the flavonoid process recovery. The particle size distributions were analyzed by way of (a) normal distribution parameters, (b) the RRSB grid and (c) considering diameter values in terms of an equivalent sphere. All the powders presented nonnormal distribution, and the RRSB analysis appeared to be, therefore, the analysis method of choice. The total flavonoid recovery was around 80%, and it was practically not affected by the SDP1, SDP2, and SDP3 compositions. At the 60% and 90% RH atmospheres, the SDP3 and SDP2 moisture uptakes were higher than that of the SDP1. All the formulations were, on the contrary, stable at 35% RH, showing a slight moisture loss tendency. The results showed in sum that the SDP prepared using Aerosil 200 as the drying adjuvant alone presented the best technological characteristics of all. PMID- 10738651 TI - Validation of tablet dissolution method by high-performance liquid chromatography. AB - Dissolution is a qualitative and quantitative tool that can provide valuable information about biological availability of a drug, as well as batch-to-batch consistency. It is considered one of the most important quality control tests performed on pharmaceutical dosage forms, and validation of dissolution methods is an important part of good manufacturing practices (GMP). Hydroxypropylcellulose (HPC) was formulated with acetaminophen (APAP) and hydrochlorothiazide (HCTZ). Dissolution methods and limits are reported in the USP/NF. Standard operating procedures (SOP) for the HP 8452A spectrophotometer and Vanderkamp 600/6010 Dissolution Tester were followed according to the GMP Manual. A dissolution method was developed for each formulation based on the above. The only discrepancy between high-performance liquid chromatography (HPLC) and standard dissolution testing occurred when comparing the results of the HPC/HCTZ formulation. The ultraviolet (UV) samples were filtered through a 10 micron filter, and the HPLC samples were filtered through a 0.2-micron filter. When the HPC/HCTZ samples were filtered through a 10-micron filter for both UV and liquid chromatography (LC), the results were equal. Filter pore size and area have a large effect on concentration of HPC/HCTZ. The smaller the pore size and the smaller the diameter of the filter, the more HPC/HCTZ is filtered out. HCTZ has a greater tendency to interact with HPC in the filter than other active ingredients tested. HPC and HCTZ levels have little or no effect on the amount of HCTZ lost. PMID- 10738652 TI - Preparation and characterization of spray-dried polymeric nanocapsules. AB - Recently, much interest has been generated by colloidal drug delivery systems such as nanocapsules because of the possibilities for controlled release, increased drug efficacy, and reduced toxicity after parenteral administration. Nanocapsules of poly-epsilon-caprolactone and Eudragit S90 were prepared. However, these systems present physicochemical instability. To dry these nanocapsule suspensions with the view of obtaining a solid form, the spray-drying process was used. Spray-dried powders of nanocapsules of poly-sigma-caprolactone and Eudragit S90 were prepared by atomization in a Buchi 190 Mini-spray dryer using colloidal silicon dioxide as a technological carrier. The morphological analysis of the surface at the powders showed that nanocapsules remain intact, and no change in particle size was detected after the spray-drying process. These results suggest that this method can be an interesting alternative to dry nanocapsule suspensions. PMID- 10738653 TI - Effect of the addition of oxybenzone or octyl-methoxycinnamate on particle size of submicron emulsions. AB - The formulation of sunscreen products requires understanding of the solubilization of these products in different vehicles to obtain aesthetic preparations and to evaluate long-term stability. For this study, two different ultraviolet (UV) filters were selected: oxybenzone (powder) and octyl methoxycinnamate (liquid). First, the solubility of these UV filters was tested using a three-component simplex-centroid design strategy. The mixtures were prepared with three oily phases used in this field of cosmetics: liquid paraffin, isopropyl myristate, and coconut oil. A phase diagram method was used to carry out a systematic study of submicron oil-in-water emulsions. Phase diagrams were produced by diluting fixed binary mixtures with water. The surfactant consisted of polyoxyethylene-20-sorbitan monostearate/sorbitan monostearate (50/50, w/w). The oily phase contained equal quantities of each oil studied. From this water/surfactant/oil ternary system, we selected two reference emulsions with receptively 75/5/20 and 68/7/25 proportions. Photon correlation spectroscopy (PCS) was used to investigate the influence of these two UV filters at several concentrations on droplet size and distribution of the oil droplets in the material. All emulsions were stored and checked every month for 6 months. PMID- 10738654 TI - Statistical comparison of high-shear versus low-shear granulation using a common formulation. AB - Experimental combinations from the ranges assigned to the independent factors were studied using both a low-shear (planetary) mixer and a high-shear mixer for granulation. The independent factors studied were X1 calcium phosphate/mannitol ratio, X2 pregelatinized starch, X3 magnesium stearate, X4 mixer type, and X5 compression pressure. To optimize the tablet properties fully, the experimental range was varied from -2 to +2 experimental units, with the exception of X4, which was assigned -1 for the planetary mixer and +1 for the high-shear mixer. Drug dissolution did not seem to be affected by mixer type, but tablet hardness was affected by mixer type. PMID- 10738655 TI - Biomedical engineering in Israel: a quantum leap. PMID- 10738656 TI - Spatial disorientation: dealing with aeronautical illusions. PMID- 10738657 TI - Classifying spatial disorientation mishaps using different definitions. PMID- 10738658 TI - A spatial disorientation survey of experienced instructor pilots. PMID- 10738659 TI - Naso-occipital vestibulo-ocular reflex responses in normal subjects. PMID- 10738660 TI - The effect of inertial force acceleration on the otolithic membrane. PMID- 10738661 TI - The G-excess effect. PMID- 10738662 TI - Effects of rotation on somatogravic illusions. PMID- 10738663 TI - The good, the bad, and the ugly of head-up displays. PMID- 10738664 TI - An instrumentation solution for reducing spatial disorientation mishaps. PMID- 10738665 TI - Neuropsychological guidelines for aircraft control stations. PMID- 10738666 TI - A fast algorithm for detecting contractions in uterine electromyography. PMID- 10738667 TI - An artificial-intelligence approach to ECG analysis. PMID- 10738668 TI - Modification of a commercially available DNA sequencer to increase sample throughput. PMID- 10738669 TI - When are expert witnesses liable for their malpractice? PMID- 10738670 TI - Bartonellosis. New and old. AB - The number of species that comprise the family of Bartonellaceae, genus Bartonella, has recently increased from one to 11 species, five of which have been associated with different diseases and syndromes in humans. The rapidly growing number of human pathogens has led several investigators to regard bartonellosis and other associated syndromes as important emerging infectious diseases. This article presents the history and epidemiology, clinical features, diagnosis, and treatment of bartonellosis and associated diseases, including Carrion's disease, trench fever, endocarditis and bacteremia, bacillary angiomatosis, and cat-scratch disease. PMID- 10738671 TI - Leptospirosis in Latin America. AB - Leptospirosis is a common disease in Latin America. Transmission to humans occurs by contact with water or soil contaminated with the urine of rodents, dogs, or livestock. Pathogenesis is still poorly understood, and bacterial toxin or virulence factors are probably responsible for many features of the disease. The anicteric form is the most frequent presentation, and its clinical picture resembles influenza or other acute febrile diseases. Icterohemorrhagic leptospirosis, or Weil's syndrome, represents the severe form of the disease. Its clinical picture is similar to bacterial sepsis and multiple organ involvement occurs, mainly in kidneys and lungs, and causes great morbidity and mortality. Death is often related to multiple organ failure and pulmonary hemorrhages. Diagnosis is based on serology or blood, cerebrospinal fluid and urine cultures in specific media. Treatment involves a combination of antibiotics and supportive measures. PMID- 10738672 TI - Shigella and Shiga toxin-producing Escherichia coli causing bloody diarrhea in Latin America. AB - In Latin America, Shigella and shiga toxin-producing Escherichia coli are the two leading agents in the cause of bloody diarrhea. The already high and increasing antimicrobial resistance of Shigella also is a significant problem. Shiga toxin producing E. coli is an emerging disease with life-threatening complications: hemolytic uremic syndrome. Although E. coli O157:H7 remains the most commonly recognized serotype, recently emerging, non-O157 bacteria may be the cause of a similar spectrum of disease in humans. PMID- 10738673 TI - Bacterial resistance to antimicrobial agents in Latin America. The giant is awakening. AB - Resistant bacteria are emerging in Latin America as a real threat to the favorable outcome of infections in community- and hospital-acquired infections. Despite present extensive surveillance, healthcare workers who most need the information may be unaware of this growing problem. Outbreaks of meningococci with diminished susceptibility to penicillin have been reported in the region; a constant increase of resistance to penicillin in pneumococci and poor activity of commonly used oral antibiotics for the treatment of community-acquired urinary tract infections have made the treatment of these infections more difficult. Reports from tertiary hospitals are similar to many other areas of the world, with increasing frequency of Klebsiella pneumoniae-carrying extended-spectrum beta-lactamase, multiresistant strains of Pseudomonas aeruginosa and Acinetobacter baumanni in ICU settings, and reports of methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. A surveillance network readily accessible to those who prescribe antibiotics in Latin America is highly desirable. PMID- 10738674 TI - Plasmodium falciparum infection in the pregnant patient. AB - Malaria should be considered a risk factor in women who are pregnant, principally when the infection is Plasmodium falciparum. Moreover, the risk is greater if the woman is pregnant for the first time; if she has no immunity for malaria; if the diagnosis is made late; or if P. falciparum shows resistance to antimalarial drugs. This article presents the most significant aspects of P. falciparum malaria during pregnancy, including information about treatments and prophylaxis. PMID- 10738675 TI - Taenia solium cysticercosis. AB - Cysticercosis, the infection of the larval tissue stage of the pork tapeworm, Taenia solium, is endemic to most developing countries and increasingly is seen in industrialized countries because of immigration. Human infection is a major cause of acquired epilepsy, and porcine infection causes important economic losses to farm workers. Clinical manifestations vary because of the variable number, size, and location of the cysts and the immune response of the host. Diagnosis is based on imaging techniques (CT and MR imaging) and serology (immunoblot). Current research efforts are centered on control and eradication of the taeniasis cysticercosis complex in endemic areas. PMID- 10738676 TI - Dengue and dengue hemorrhagic fever in Latin America and the Caribbean. AB - Four serotypes of dengue viruses produce dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. They are the most important arbovirus infections of humans, in terms of both morbidity and mortality, constituting one of the most rapidly expanding and re-emerging infectious disease problems in Latin America. In less than 20 years, the region has transformed itself from hypoendemic to hyperendemic, while serotype circulation in most countries has gone from none or single to multiple. Changes in endemicity have coincided with the emergence and increasing incidence of the severer forms of dengue infection. This article reviews the clinical presentations of these diseases. Health care providers who see patients in or returning from areas of Latin America, the Caribbean, and other tropical areas must consider dengue in the differential diagnosis of patients presenting with compatible symptoms, and must be knowledgeable in the current management of this important disease. PMID- 10738677 TI - Influenza. AB - Influenza is an acute respiratory illness of global importance that causes considerable morbidity and mortality every year. At the beginning of the millennium, influenza will still be an emergent or re-emergent infection because of the viral ability to mutate. Global influenza surveillance indicates that influenza viruses may vary within a country and between countries and continents during an influenza season. Virologic surveillance is of critical importance in monitoring antigenic shift and drift. Disease surveillance is important in assessing the impact of the epidemics. Both types of information provide the basis of vaccine composition and the correct use of antivirals. Laboratory diagnosis is of critical importance for the global surveillance of influenza and may allow the timely use of antiviral drugs. Viral isolation remains the gold standard for laboratory diagnosis; however, several new rapid diagnostic tests are available or in development. The clinical spectrum of the disease varies from asymptomatic infection to the classic flu syndrome, and respiratory and nonrespiratory complications are observed particularly in high-risk groups. Current inactivated influenza vaccines have shown efficacy and effectiveness in preventing influenzalike illness, hospitalization for pneumonia, and death and in reducing health care costs. Because of the annual administration of the vaccine and the short period of time where it can be administered, strategies directed at improving vaccine coverage are of critical importance. In this sense, experiences obtained in different countries, such as with the National Immunization Campaigns developed in Argentina, provide one model of massive vaccine administration. In addition to current vaccines, new live attenuated vaccines will permit a most effective prevention of influenza in the community in the near future. A new type of antiviral, neuraminidase inhibitors, offers valuable benefits in the prevention and treatment of influenza A and B. A future pandemic of influenza seems inevitable. There is wide recognition that preparation for the next pandemic requires that infrastructure be in place during interpandemic periods for implementing preventive and therapeutic measures. The WHO has established a pandemic influenza task force, and a number of countries in Latin America have developed formal pandemic plans. These national and international efforts are essential to reduce the mortality and morbidity in the next influenza pandemic. PMID- 10738678 TI - Rodent-borne emerging viral zoonosis. Hemorrhagic fevers and hantavirus infections in South America. AB - Hantaviruses and arenaviruses are naturally occurring viruses of rodents. Four South American hemorrhagic fevers caused by arenaviruses have emerged in the last 5 decades. All have similar clinical manifestations, with a case-fatality rate as high as 15% to 30%. Hantavirus infections have been increasingly recognized in South America since the description in 1993 of Hantavirus pulmonary syndrome. Given the diversity of rodent species in the region, it can be foreseen that many other viruses will be discovered, and some of them will be causing human illnesses of high public health impact. PMID- 10738679 TI - AIDS in Latin America. AB - The epidemic in Latin America has placed an unexpected additional burden on the health care systems and national economies, already weak and affected by severe problems. Specific regional diseases in addition to common opportunistic infections, and particularly the high incidence of TB, produce a different picture compared with the United States and Europe. Access to ARV therapy is far from being universal in Latin America; nevertheless, some countries are providing HAART to all eligible patients, showing that it is not impossible to improve quality of care for people living with HIV infection in the region. Before assuming as definitive and irreversible that at least one or two generations will be sacrificed on the altar of inequity of our uneven world, we as acting scientists should join the struggle of millions of human beings claiming their right to be treated with the best drugs that science can offer today. PMID- 10738680 TI - Human T-cell lymphotropic virus-I in Latin America. AB - HTLV-1 infection is endemic in several Latin American countries. HTLV-1 associated myelophathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia lymphoma (ATLL) are emerging diseases in the region. Documented risk factors for acquiring the virus include breast-feeding, contaminated blood transfusion, and sexual intercourse, all of which are amenable to prevention efforts. Strongyloides stercoralis hyperinfection syndrome and therapeutic failure in apparently healthy patients with nondisseminated strongyloidiasis may be markers of HTLV-1 infection. HTLV-1 co-infection may adversely effect the clinical course of scabies and HIV disease. The new enzyme-linked immunosorbent assays (ELISA) are sensitive and specific, and Western blot technology is reliable for differentiating HTLV-1 from less common HTLV-2. HTLV-1 screening of blood donors and individuals with any disorder that suggests infection has become a necessity in Latin America to prevent the spread of this important emerging pathogen. PMID- 10738681 TI - State of immunization in the Americas. AB - Countries in the Americas have led the world in conquering infectious diseases preventable through vaccination. In 1971, the Western hemisphere achieved smallpox eradication. In 1991, the Americas were free of indigenous transmission of wild poliovirus. In 1998, overall regional vaccination coverage was 86% for diphtheria-pertussis-tetanus, 89% for oral poliovirus vaccine 3, 98% for bacille Calmette-Guerin vaccine, and 85% for measles. These figures confirm that most of the children in the Americas are protected against these diseases. The breakthroughs obtained in immunization have stimulated countries to promote new initiatives aimed at the control and eradication of other vaccine-preventable diseases and to introduce new vaccines into routine schedules. In the 21st century, vaccines will remain the most cost-effective means of preventing diseases and avoiding expensive treatment costs. PMID- 10738682 TI - An electronic manometer for blood-pressure measurement. AB - This article reports the development of an electronic manometer for use in the measurement of blood pressure. It is intended to act as a replacement gauge for the mercury and aneroid manometers used in conventional sphygmomanometers. It measures pressure in the range 0-300 mm Hg (0-40 kPa) with a resolution of 1 mm Hg (0.13 kPa) and an accuracy of +/- 1 mm Hg (+/- 0.13 kPa) which is displayed on a liquid crystal display. It operates from a 9 V PP3 battery over a temperature range of 10-50 degrees C. PMID- 10738683 TI - Influence of anisotropy on the mechanical behaviour of bioprosthetic heart valves. AB - Chemically modified pericardium is commonly used in the fabrication of bioprosthetic heart valves. This material exhibits non-linear elastic behaviour and, as for most other biological soft tissues, it is orthotropic in its extensibility. The influence of the natural orthotropy of pericardium on the mechanical behaviour of pericardial heart valves during the whole cardiac cycle has been studied, using the finite element method. A model of the leaflet of a bicuspid valve has been created, defining the material of the tissue as orthotropic non-linear elastic. Two preferential orthogonal orientations of the tissue have been analysed (axial and circumferential). The results show that even a small amount of orthotropy (an orthotropy index of 1.5 has been used) can significantly affect the mechanical behaviour of the valve, and that an appropriate orientation of the fibres can contribute to optimizing the stress distribution in the leaflets. PMID- 10738684 TI - An effective algorithm for quick fractal analysis of movement biosignals. AB - The problem of numerically classifying patterns, of crucial importance in the biomedical field, is here faced by means of their fractal dimension. A new simple algorithm was developed to characterize biomedical mono-dimensional signals avoiding computationally expensive methods, generally required by the classical approach of the fractal theory. The algorithm produces a number related to the geometric behaviour of the pattern providing information on the studied phenomenon. The results are independent of the signal amplitude and exhibit a fractal measure ranging from 1 to 2 for monotonically going-forwards monodimensional curves, in accordance with theory. Accurate calibration and qualification were accomplished by analysing basic waveforms. Further studies concerned the biomedical field with special reference to gait analysis: so far, well controlled movements such as walking, going up and downstairs and running, have been investigated. Controlled conditions of the test environment guaranteed the necessary repeatability and the accuracy of the practical experiments in setting up the methodology. The algorithm showed good performance in classifying the considered simple movements in the selected sample of normal subjects. The results obtained encourage us to use this technique for an effective on-line movement correlation with other long-term monitored variables such as blood pressure, ECG, etc. PMID- 10738685 TI - Force transducer for measurement of muscle contraction. AB - Single channel force transducers, intended for measurement and evaluation curves of preconditioned fibres in muscles contracting synergistically during direct or indirect electric stimulation of isolated muscle, were designed, developed and experimentally tested. The force transducers were made up of a full Wheatstone bridge composed of four semiconductor strain gauges bonded on a specially designed cantilever. The transducers with a natural frequency 350 Hz and compliance of 0.25 micron g-1 represents a very linear dependence of the output voltage upon the load giving a sensitivity for the transducers of 0.5 mV mN-1 with a bridge excitation voltage of 5 V. The nominal range of each transducer is 0-70 mN. The system is able to record even a contraction of only a few muscle fibres, both single twitches as well as sustained tonic contractions. PMID- 10738686 TI - A portable volumetric infusion pump controller for treatment of thallassemia disease. AB - A design study of a VLSI (very large scale integration) implemented volumetric infusion pump controller with enhanced performance compared to conventional designs is presented. The proposed system is composed of the designed controller and electromechanical equipment consisting of a dc motor, a magnetic drum, and a Hall effect sensor. Medicine dose settings are altered using a keyboard and are processed by the controller to produce the pulses that activate the motor. In advance, the controller supervises the device over all operations performing all the necessary control and fault handling. In this paper, we provide a technical description of the device developed which has a reduced size, power consumption and overall cost, as well as increased reliability and fault tolerance, compared to former implementations. PMID- 10738687 TI - A numerical investigation on the steady and pulsatile flow characteristics in axi symmetric abdominal aortic aneurysm models with some experimental evaluation. AB - Steady and pulsatile flow characteristics in rigid abdominal aortic aneurysm (AAA) models were investigated computationally (using Fluent v. 4.3) over a range of Reynolds number (from 200 to 1600) and Womersley number (from 17 to 22). Some comparisons with measurements obtained by particle image velocimetry under the pulsatile flow conditions are also included. A sinusoidal inlet flow waveform 1 + sin omega t with thin inlet boundary layers was used to produce the required pulsatile flow conditions. The bulk features of the mean flow as well as some detailed features, such as wall shear stress distributions, are the foci of the present investigation. Recirculating vortices appeared at different phases of a flow cycle causing significant spatial and temporal variations in wall shear stresses and static pressure distributions. A high level of shear stresses usually appeared at the upstream and downstream ends of the bulge. Effects of pressure rise caused by the increase in cross-sectional area were transmitted into the downstream tube. Further simulation studies were conducted using simulated physiological waveforms under resting and exercise conditions so as to determine the possible implication of vortex dynamics inside the AAA model. PMID- 10738688 TI - A computer-based system for identification of the static characteristics of medical sensors. AB - This paper presents the design and implementation of a computer-based system for identification of the static characteristics of medical sensors. The static characteristic obtained is then used to correct the input-output non-idealities caused by nonlinearity effects, gain error, hysteresis and environmental effects. The knowledge of this characteristic enables digital signal processing of the sensor response, facilitates correction of input-output non-idealities and forms a compensation formula which is required for the optimal design of the experiment for medical sensor calibration. The identification system consists of a personal computer and a microcontroller-based electronic system. In consideration of practical situations special emphasis is oriented toward its user friendliness and future extendability. PMID- 10738689 TI - Tableting of coated ketoprofen pellets. AB - Ketoprofen pellets were prepared by the method of extrusion-spheronization, and a film coating of guar gum and Eudragit NE was applied to drug cores using pan technology. In an attempt to design a tablet which, on peroral administration, disintegrates rapidly, releasing intact coated pellets which maintain the integrity of both the cores and their release retarding membrane, Avicel PH101, lactose DT and magnesium stearate were used as excipients to prepare tablets comprising ketoprofen pellets or microcapsules. Preliminary experiments were conducted on uncoated pellets to determine the optimum compression force required to prepare tablets of satisfactory mechanical properties and release profiles. Coated pellets containing ketoprofen were used to investigate the influence of excipients levels. In an attempt to minimize problems associated with blending and segregation of microcapsules and excipients, placebo spheres of Avicel PH101 and lactose DT were produced by the method of extrusion-spheronization. The use of placebo spheres produced tablets with improved drug content uniformity and disintegration time. The tensile strength of such compacts was enhanced by excluding magnesium stearate from the mixes without significant problems of sticking or picking. The use of placebo pellets resulted in significant damage to drug microcapsules, which was attributed to the higher hardness and density of the excipients pellets. The role of membrane coating in protecting the drug core during compression was also evaluated. PMID- 10738690 TI - In vitro and in vivo evaluation of diclofenac sodium loaded albumin microspheres. AB - The use of polymeric carriers in formulations of therapeutic drug delivery systems has gained widespread application, due to their advantage of being biodegradable and biocompatible. Among the microparticulate systems, microspheres have a special importance since it is possible to target drugs and provide controlled release. Diclofenac sodium (DS), is a potent drug in the NSAID group having non-steroidal, anti-inflammatory properties, and is widely used in the treatment of rheumatoid arthritis, osteoarthritis and ankylosing spondylitis. In this present study, it was aimed to prepare microsphere formulations of DS using a natural biodegradable polymer as a carrier for intraarticular administration to extend the duration period of the dosage form in the knee joint. Microsphere formulations of DS which were prepared were evaluated in vitro for particle size, yield value, encapsulation efficiency, surface morphology, and in vitro drug release. Two appropriate formulations were selected for in vivo trials. For the in vivo studies, Technetium-99m labelled polyclonal human immunogammaglobulin (99mTc-HIG) was used as the radiopharmaceutical to demonstrate arthritic lesions by gamma scintigraphy. After the induction of arthritis in knee joints of rabbits, the radio-labelled microspheres loaded with DS were injected directly into the articular cavity and at specific time points gamma scintigrams were obtained to find the residence time of the microspheres in knee joints in order to determine the most suitable formulation. PMID- 10738691 TI - Encapsulation of DNA in new multilamellar vesicles prepared by shearing a lyotropic lamellar phase. AB - Encapsulation of DNA in a new non-cationic multilamellar vector (Spherulites), composed of phosphatidylcholine, cholesterol and polyoxyethylene alcohol, is described here for the first time. Spherulites entrapping DNA were prepared by shearing a phospholipid lyotropic lamellar phase, using a recently discovered method. The average diameter of these vesicles ranges around 300 nm, and can be adjusted depending on the conditions of the process. The formulation did not result in cytotoxicity for the human cells and could be used as a DNA delivery system. More emphasis is brought to the role of condensing agents like histones on the encapsulation yield, which has been studied using radiolabelled DNA. It is shown that use of histones (histone to DNA ratio of 0.4) can increase significantly the encapsulation of DNA, thus improving the transfection efficiency. Transfection experiments were done with success using the beta galactoside reporter gene on human primary cells (human skin fibroblasts and human bone marrow stromal cells). The results suggest that the spherulites have to be considered as a new and promising tool for gene transfection. PMID- 10738692 TI - The stability of ascorbic acid microencapsulated in granules of rice starch and in gum arabic. AB - Ascorbic acid (AA) was microencapsulated by spray drying, using gum arabic and rice starch as covering materials. The AA was dissolved in solutions of the wall material prior to processing. For the rice starch, gelatin was used as a binding agent and recovery was effected with calcium pectate. The morphology of the materials was analysed by optical and scanning electron microscopy, it thus being possible to verify the formation and evaluate the structural characteristics of the microcapsules. The capsules produced with gum arabic were smaller (d50% = 8.0 microns) and with a multimode particle size distribution, whilst uncovered starch capsules containing 1-2% gelatin presented a distribution mainly in the range of 5-40 microns. The capsules recovered with calcium pectate had average diameters 10-15 times greater than those obtained only by spray drying. The stability of the encapsulated materials was studied at room temperature (RH 60-65%) and at 45 degrees C (RH 60-65% and 90.7%). AA microencapsulated in gum arabic was shown to be as stable as free crystalline AA under environmental conditions, whereas that encapsulated in rice starch was less stable. Increasing the amount of the binding agent gelatin increased the stability of the uncovered starch encapsulated AA. Recovery with calcium pectate notably increased the stability of the starch encapsulated AA, as compared to the uncovered samples. PMID- 10738693 TI - Microencapsulation by means of step-wise adsorption of polyelectrolytes. AB - Step-wise adsorption of polyelectrolytes is used for the fabrication of micro- and nanocapsules with determined size, capsule wall composition and thickness. The capsule walls made of polyelectrolyte multilayers exclude high molecular weight compounds. Assembling of lipid layers onto these polyelectrolyte capsules prevents the permeation of small dyes. Encapsulation of magnetite nanoparticles is demonstrated and the features of these novel capsules are discussed. PMID- 10738694 TI - Gelatin nanoparticles by two step desolvation--a new preparation method, surface modifications and cell uptake. AB - A new two-step desolvation method for manufacturing gelatin nanoparticles was developed. After the first desolvation step, the low molecular gelatin fractions present in the supernatant were removed by decanting. The high molecular fractions present in the sediment were redesolved and then desolvated again at pH 2.5 in the second step. The resulting particles can then be easily purified by centrifugation and redispersion. The different fractions obtained during the process were analysed by gel permeation chromatography (GPC). Based on these results, it can be concluded that the molecular weight of gelatin has a decisive influence on the stability of the manufactured gelatin nanoparticles. In addition, two fluorescent dyes (Texasred and fluoresceinamine) were coupled to the nanoparticles for cell uptake studies. The fluorescent nanoparticles showed a high uptake into monocytes/macrophages. PMID- 10738695 TI - Poly epsilon-caprolactone nanoparticles containing a poorly soluble pesticide: formulation and stability study. AB - In 1997, a research program was initiated in the laboratories to assess the ability of nanosperes (NS) to improve the biodelivery of new active ingredients (AI) to plants. The goal was to obtain stable poly (epsilon-caprolactone) NS (PeC NS) with the smallest size and the largest amount of encapsulated AI, using a nanoprecipitation method. The smallest particles obtained were in the range of 200-250 nm. The highest encapsulation is obtained with Montanox 80 as surfactant and is between 5-10% (expressed in per cent weight relative to the total weight of polymer), which corresponds to an encapsulation yield of 95%. There is no desorption of the AI with time. In contrast, the dilution of the NS suspension in water is followed by a large removal of the AI in the aqueous phase. This suggests that NS are complex dynamic systems in equilibrium with the external medium and disturbances of this system lead to a loss of AI. PMID- 10738696 TI - Release of bovine serum albumin from preformed porous microspheres of poly(L lactic acid). AB - Preformed porous microspheres of poly(L-lactic acid) (Accurel have been shown to sustain the release of highly water soluble solutes, like dextran and mannitol, for a time period of more than 4 months. The purpose of this investigation was to mechanistically characterize the release of a model protein, bovine serum albumin (BSA), from these highly porous microspheres. The microspheres were loaded with [14C]BSA in three different concentrations of 0.06, 0.26 and 0.59% w/w. The rate of release of [14C]BSA from microspheres was correlated to media ([3H]PBS) uptake. The release of BSA showed a biphasic pattern; an initial rapid release, followed by a sustained release. The initial burst of BSA was found to be inversely proportional to BSA loading and highly correlated to water penetration. The sustained release phase was independent of water penetration kinetics. Washing the microspheres did not remove either the surface bound BSA or the BSA incorporated in the microsphere matrix, indicating the tight binding of BSA to highly porous microspheres. Furthermore, addition of a surfactant induced a dramatic increase in the amount of BSA released, suggesting that the release is controlled by the surface binding of BSA to the polymer. Also, the release rate of BSA beyond the initial burst was found to be much slower than for the lower MW macromolecules like dextran at a similar level. The data from the present work suggests the BSA-polymer interaction to be a major contributing factor in explaining the overall BSA release kinetics. PMID- 10738697 TI - Immune response with biodegradable nanospheres and alum: studies in rabbits using staphylococcal enterotoxin B-toxoid. AB - In this study, the adjuvant effect of the sustained release biodegradable nanospheres (100-150 nm in diameter) has been compared with alum. Nanospheres were formulated using a biodegradable polylactic polyglycolic acid copolymer (PLGA, 50:50) containing Staphylococcal Enterotoxin B (SEB) toxoid as a model vaccine antigen. Systemic immune response of the nanospheres containing toxoid was studied in rabbits by subcutaneous immunization. The data demonstrated that approximately 30% of the toxoid activity was lost following its encapsulation into nanospheres. Under in vitro conditions, nanospheres demonstrated sustained release of the toxoid. However, only 20% of the antigenic toxoid was released over the first 2 weeks of the release study. Immunization of animals with equal doses of toxoid, either using nanospheres or alum induced a comparable systemic immune response (IgG, IgM and IgA). The immune response reached a maximum level at 7 weeks post-immunization, which then gradually declined with time. The booster dose of toxoid at 19 weeks, either using alum or nanospheres induced similar immune response in both the groups, but was greater than the primary immune response. The studies, thus, suggest that biodegradable nanospheres could be used as a vaccine adjuvant. PMID- 10738698 TI - Niosomes as carriers of radiopaque contrast agents for X-ray imaging. AB - Non-ionic surfactant vesicles (niosomes) are considered as carriers of iobitridol, a diagnostic agent used for X-ray imaging. The niosomes, with a diameter between 150 and 175 nm, are prepared using the film-hydration method followed by sonication. These vesicles were obtained with appropriate mixtures of D-alpha tocopheryl polyethylene glycol 1000 succinate, polyoxyethylene glycol 4000 stearate, sorbitan monostearate, cholesterol and dicetylphosphate. Methods allowing the increase of the rate of encapsulation and the stability of the vesicles were carried out. In addition to the study of the formulation of the vesicles, the physico-chemical and morphological properties of the vesicles have been studied. PMID- 10738699 TI - A novel cell encapsulation method using photosensitive poly(allylamine alpha cyanocinnamylideneacetate). AB - A photosensitive alpha-cyanocinnamylideneacetyl group was coupled to poly(allylamine) to obtain a photosensitive polymer. This photosensitive poly(allylamine alpha-cyanocinnamylideneacetate) can cross-link upon light exposure. Microcapsules were fabricated from alginate in contact with Ca+2 ion, followed by coating with the photosensitive poly(allylamine alpha cyanocinnamylideneacetate). The microcapsules, thus formed, can be strengthened significantly by the light-induced cross-linking of poly(allylamine alpha cyanocinnamylideneacetate). Only 16 capsules (out of 50) prepared from the photosensitive poly(allylamine alpha-cyanocinnamylideneacetate) fractured after 48 h of agitation. For microcapsules prepared from the unmodified poly(allylamine), 32 capsules fractured. The photo cross-linked capsular membrane was permeable to cytochrome C, moderately permeable to myoglobin, and least permeable to serum albumin. IW32 (a mouse leukaemia cell line) cells were entrapped and cultured within these microcapsules. The cells proliferated to a density of about 9 x 10(6) cells/ml in the capsules after 7 days of cultivation. PMID- 10738700 TI - Literature alerts. PMID- 10738701 TI - Outcomes comparison of treatment for chronic disabling work-related upper extremity disorders and spinal disorders. PMID- 10738702 TI - ACOEM position statement. Spirometry in the occupational setting. American College of Occupational and Environmental Medicine. AB - This position statement reviews several aspects of spirometric testing in the workplace, where spirometry is employed in the primary, secondary, and tertiary prevention of occupational lung disease. Primary prevention includes pre placement and fitness-for-duty examinations as well as research and monitoring of health status in groups of exposed workers; secondary prevention includes periodic medical screening of individual workers for early effects of exposure to known occupational hazards; and tertiary prevention includes clinical evaluation and impairment/disability assessment. For all of these purposes, valid spirometry measurements are critical, requiring: documented spirometer accuracy and precision, a rigorous and standardized testing technique, standardized measurement of pulmonary function values from the spirogram, adequate initial and refresher training of spirometry technicians, and, ideally, quality assessment of samples of spirograms. Interpretation of spirometric results usually includes comparison with predicted values and should also evaluate changes in lung function over time. Response to inhaled bronchodilators and changes in relation to workplace exposure may also be assessed. Each of these interpretations should begin with an assessment of test quality and, based on the most recent ATS recommendations, should rely on a few reproducible indices of pulmonary function (FEV1, FVC, and FEV1/FVC.) The use of FEF rates (e.g., the FEF25-75%) in interpreting results for individuals is strongly discouraged except when confirming borderline airways obstruction. Finally, the use of serial PEF measurements is emerging as a method for confirming associations between reduced or variable pulmonary function and workplace exposures in the diagnosis of occupational asthma. Throughout this position statement, ACOEM makes detailed recommendations to ensure that each of these areas of test performance and interpretation follow current recommendations/standards in the pulmonary and regulatory fields. Submitted by the Occupational and Environmental Lung Disorder Committee on November 16,1999. Approved by the ACOEM Board of Directors on January 4,2000. PMID- 10738703 TI - Genetic susceptibility research in occupational disease: should subjects have access to interim findings? PMID- 10738704 TI - Effect of ascorbic acid and growth factors on collagen metabolism of flexor retinaculum cells from individuals with and without carpal tunnel syndrome. AB - The effects of ascorbic acid and various growth factors on the proliferation rate and collagen metabolism were studied in cells from the flexor retinaculum of individuals with carpal tunnel syndrome (FR-CTS) and without carpal tunnel syndrome (FR control) and in human dermal fibroblasts. Ascorbic acid and four growth factors, including basic fibroblast growth factor, transforming growth factor, platelet-derived growth factor, and epidermal growth factors, were used. Ascorbic acid stimulates type I collagen production more in FR control than in FR CTS. Growth factor treatment resulted in the following responses by the cells: (1) a higher mitogenic response than in the control cells; (2) a higher stimulation of type III collagen production and a lower stimulation of type I collagen production in CTS cells as compared with control cells; and (3) more alpha 2 (I) than alpha 1 (I) collagen production in CTS cells, unlike in control cells. We concluded that cells of the FR from individuals with CTS are physiologically altered. PMID- 10738705 TI - Dose-response relationships between occupational aerosol exposures and cross shift declines of lung function in poultry workers: recommendations for exposure limits. AB - Numerous articles have been published regarding the adverse respiratory health consequences of working in intensive livestock and poultry housing. Threshold limit exposure guidelines are not currently applied to this environment, but they are essential to implement and monitor effective environmental controls. Previous dose-response research work with swine workers has resulted in exposure limit recommendations of 2.5 mg/m3 total dust, 0.23 mg/m3 respirable dust, 100 EU/m3 endotoxin, and 7 ppm ammonia. No similar recommendations have been reported previously for poultry workers. Therefore, an industry-wide study was conducted to examine dose-response relationships of bioaerosol exposures and worker respiratory health. A total of 257 poultry workers were studied for respiratory symptoms, pulmonary function, and exposure to dust (total and respirable), endotoxin (respirable and total), and ammonia. Details of the sampling plan and environmental assessment are described elsewhere. Relationships between exposures and response were studied by correlation and multiple regressions. Significant dose-response relationships were observed between exposures and pulmonary function decrements over a work shift. Exposure concentrations associated with significant pulmonary function decrements were as follows: 2.4 mg/m3 total dust, 0.16 mg/m3 respirable dust, 614 EU/m3 endotoxin, and 12 ppm ammonia. PMID- 10738706 TI - Effects of short-term exposure to 0.2 ppm ozone on biomarkers of inflammation in sputum, exhaled nitric oxide, and lung function in subjects with mild atopic asthma. AB - To gain further insight into the kinetics of airway inflammatory response and explore the possibility of nitric oxide as a surrogate marker of the lower airway inflammatory response to ozone, nine subjects with mild atopic asthma were exposed to filtered air or 0.2 ppm ozone for 2 hours with intermittent exercise. Lung function was measured at baseline and immediately after exposures. Sputum induction was performed at 6 hours and at 24 hours after exposures, and exhaled nitric oxide levels were measured at baseline, immediately, 6, and 24 hours after both exposures. A significant decline in forced expiratory volume in one second and inspiratory capacity was detectable following exposure to ozone. In addition, a 2-fold increase was observed in the percentage of polymorphonuclear leukocytes 6 hours after exposure to ozone, with no changes in other biomarkers at this time point. By 24 hours after ozone exposure, the neutrophilia had subsided but there was an increase in albumin, total protein, myeloperoxidase, and eosinophil cationic protein. Exhaled nitric oxide levels, histamine, interleukin-8, and growth-related oncogene-alpha in sputum did not change significantly following ozone exposure. It was concluded that short-term ozone exposure induces an acute inflammatory response in asthmatic airways, characterized by early polymorphonuclear leukocyte influx followed by plasma extravasation and activation of eosinophils and neutrophils. Exhaled nitric oxide is not a useful marker for detecting inflammatory response to ozone in persons with mild asthma. PMID- 10738707 TI - Uranium mining and lung cancer among Navajo men in New Mexico and Arizona, 1969 to 1993. AB - Navajo men who were underground miners have excess risk of lung cancer. To further characterize the long-term consequences of uranium mining in this high risk population, we examined lung cancer incidence among Navajo men residing in New Mexico and Arizona from 1969 to 1993 and conducted a population-based case control study to estimate the risk of lung cancer for Navajo uranium miners. Uranium mining contributed substantially to lung cancer among Navajo men over the 25-year period following the end of mining for the Navajo Nation. Sixty-three (67%) of the 94-incident lung cancers among Navajo men occurred in former uranium miners. The relative risk for a history of mining was 28.6 (95% confidence interval, 13.2-61.7). Smoking did not account for the strong relationship between lung cancer and uranium mining. The Navajo experience with uranium mining is a unique example of exposure in a single occupation accounting for the majority of lung cancers in an entire population. PMID- 10738708 TI - Identification of occupational cancer risks in British Columbia. A population based case-control study of 995 incident breast cancer cases by menopausal status, controlling for confounding factors. AB - Lifetime occupational histories as well as information on known and suspected breast cancer risk factors were collected by means of a self-administered questionnaire from 1018 women with incident breast cancer ascertained from the British Columbia Cancer Registry, and from 1020 population controls. A matched case-control study design was used. Conditional logistic regression for matched sets data and the likelihood ratio were used in a two-step procedure and were performed separately for pre-menopausal women, post-menopausal women, and for all cases combined. Excess risk was noted for several white-collar occupations. Significantly increased risk was observed: (1) among pre-menopausal women: in electronic data-processing operators; barbers and hairdressers; in sales and material processing occupations; and in the food, clothing, chemical and transportation industries; (2) among post-menopausal women: in schoolteaching; in medicine, health, and nursing occupations; in laundry and dry-cleaning occupations; and in the aircraft and automotive, including gasoline service station, industries. Several significant associations were also seen in the combined group of pre- and post-menopausal women, particularly in crop farmers and in the fruit and vegetable, publishing and printing, and motor vehicle repair industries. The results of this study suggest excess breast cancer risk in a number of occupations and industries, notably those that entail exposure to solvents and pesticides. PMID- 10738709 TI - Self-reported health in relation to medical health and gender-specific problems in women. AB - Self-reported health was studied in relation to physiological measures, gender specific problems, and clinical evaluation in 61 middle-aged women employed in a male-dominated industry. Using self-reported somatic health as the dependent variable, 50% of the variability was explained by the dimension workplace culture and the ratio low-density lipoprotein/high-density lipoprotein. Some women who reported good health were diagnosed with distress but showed no ill-health factors. However, they reported experiences of gender-specific problems more often than the women who were diagnosed as being healthy. We concluded that self report scales seem not to be enough to identify people who look healthy on standard health scales but who use psychological defenses. Differentiating methods such as gender-specific questions, physiological ratio-measures, and clinical judgment seem to be important for distinguishing genuine from illusory mental or physical health. PMID- 10738710 TI - Is occupation a risk factor for thyroid cancer? Canadian Cancer Registries Epidemiology Research Group. AB - A Canadian case-control study explored the etiology of thyroid cancer, including occupational exposure. Analysis of job history from 1272 thyroid cancer patients and 2666 controls revealed statistically significant risks among the following occupations: Wood Processing, Pulp and Papermaking (odds ratio [OR] = 2.54, 95% confidence interval [CI] = 1.11-5.83); Sales and Service (OR = 1.26, 95% CI = 1.05-1.52); and Clerical (OR = 0.81, 95% CI = 0.67-0.97). ORs were adjusted for age, sex, province, cigarette smoking, education, self-reported exposure to radiation at work, and duration of employment. Exposure to ionizing radiation or electromagnetic fields at work (inferred from job histories) did not affect risk, nor did socioeconomic status, measured by education, income, or occupational prestige. Possible explanations for the results and further investigations are discussed. PMID- 10738711 TI - Physical workplace factors and return to work after compensated low back injury: a disability phase-specific analysis. AB - Little is known about predictors of duration of work disability (DOD). This cohort study of 433 workers' compensation claimants estimated DOD for job, injury, and demographic factors during consecutive disability phases using Cox regression analysis. DOD was calculated from administrative records. Results show that DOD increases with the time spent bending and lifting or pushing or pulling heavy objects at work, but it is unrelated to sitting, standing, or vibration. Younger age, longer pre-injury employment, less severe injuries, and a previous back injury predicted shorter disability, the latter factor only during the subacute/chronic disability phases. The effect of injury severity decayed over time. This study demonstrates the usefulness of a phase-specific analysis and shows that physical job and injury factors have a significant and time-varying impact on DOD. PMID- 10738712 TI - [Universal hearing screening of newborn infants with the BERA-phone]. AB - BACKGROUND: Unidentified and untreated early infant hearing loss leads to speech language deficits as well as to cognitive, intellectual, emotional and psychosocial handicaps. Targeted hearing screening programs may miss approximately 50% of all hearing impaired children. METHODS: In an universal hearing screening program with a two stage protocol, 1349 newborns were examined using the CRESCENDO Newborn Hearing Screener according to Finkenzeller and a clinical ABR system operating with a time course step stimulus algorithm. RESULTS: Five newborns that failed the two stage screening protocol were diagnosed as hearing impaired so that therapy was initiated before the age of six months. The specificity of the method was 98.8%. The CRESCENDO method only required a short examination time and was easy to apply. CONCLUSIONS: Hearing screening programs using ABR offer advantages when compared to evoked otoacoustic emission testing. ABR threshold detection with the time course step stimulus algorithm is a quick and easy to apply method with high specificity that can be recommended for newborn hearing screening. PMID- 10738713 TI - [Bilateral malfunction of peripheral vestibular organs. Observations of 20 cases of Dandy syndrome]. AB - BACKGROUND: Dandy's Syndrome initially provokes dizziness and vertigo. Later on patients suffer from motion unsteadiness especially in dark surroundings and from oscillopsies. Gentamicin is ototoxic mostly for the vestibular part of the inner ear, and it is nephrotoxic. It may cause transitory renal dysfunction. Chronic or acute renal insufficiencies inhibit gentamicin clearance. PATIENTS: Among 20 cases we found 15 who had previously been treated with aminoglycosides (13 with gentamicin and two with streptomycin). Ten of our patients showed symptoms of preexistant chronic nephrosis or of transitory renal insufficiency caused by gentamicin therapy. In all 13 cases, peripheral vestibular function was destroyed or severely damaged by antibiotic. The same patients had no hearing loss. CONCLUSIONS: The different reactions of the cochlear and the vestibular end organs support the theoretical basis for transtympanic gentamicin treatment of Meniere's disease.--Other reasons for Dandy's Syndrome were bilateral Meniere's disease, skull fractures, and bilateral vestibular disorders. PMID- 10738714 TI - [Deletion of chromosome 10q--a marker for metastasis of head-neck carcinomas?]. AB - BACKGROUND: Comparative genomic hybridization (CGH) was applied to squamous cell carcinomas of the head and neck to define genetic alterations that are associated with the metastatic phenotype. METHODS: CGH is a molecular cytogenetic method allowing the comprehensive analysis of a tumor genome for chromosomal imbalances. In total, 23 primary squamous cell carcinomas without evidence of metastasis formation and 20 lymph node metastases were investigated. RESULTS: Prevalent changes observed in more than 50% of the primary tumors included deletions on chromosomes 3p, 4p/q, 5q, 6q, 9p, 11q, 13q, and 18q, and DNA overrepresentations on chromosomes 1p, 3q, 5p, 8q, 9q, 11q13, 16p, 17q, 19p, 20q, and 22q. To evaluate the differences between both groups we used a histogram representation, calculation of a difference histogram, and statistical analysis. The analysis revealed that the lymph node metastases were frequently characterized by deletions on chromosomes 10, 11, and 14. In particular, DNA loss of the chromosomal bands 5p12, 10p11.2-12, 10q21, 10q22-23, 10q24-26, 11p13-14, 11q24 25, and 14q22-24 were significantly associated with metastases formation. The statistical analysis indicated that particularly the deletions on chromosome 10q were highly significant markers for the incidence of lymph node metastases. CONCLUSION: Our data indicate that tumor phenotypes are determined by patterns of chromosomal alterations, and that 10q deletions may predict the metastatic phenotype in head and neck squamous cell carcinomas. PMID- 10738715 TI - [Prognostic relevance of proliferation marker Ki-67 (MIB 1), PCNA and p53 in combined surgically and radiologically treated cancers of the oropharynx and mouth cavity]. AB - STUDY DESIGN: A matched-pair study on the relationship between proliferation associated markers Ki-67, PCNA, and p53 and treatment failure in carcinomas of the oropharynx and oral cavity. MATERIAL AND METHOD: Fifty-six T1-T3 carcinomas of the oropharynx or oral cavity, treated uniformly with primary surgery and postoperative irradiation, were investigated. Twenty-eight patients had recurrent disease and were matched with 28 patients with nonrecurrent disease regarding stage and location of tumor as well as age and therapy. MIB1 staining was used to determine the Ki-67 labeling index. Immunohistochemical studies determined the p53 status and PCNA labeling index. RESULTS: Where treatment failed, the Ki-67 labeling index was significantly (p = 0.032) higher (mean = 59.1%) than in patients without recurrence (mean = 50.5%). Mean time to relapse was 45 months (n = 25) for carcinomas with a Ki-67 (MIB1) labeling index above the median (53.7%) of the general study population, compared to 61.7 months for those cases (n = 31) below this median (p = 0.029). The PCNA labeling index did not correlate significantly with tumor recurrence, nor with time to relapse. In 46% of all tumors, p53 overexpression was present. No significant correlation could be found between p53 overexpression and tumor recurrence or time to relapse. CONCLUSION: Examination of Ki-67 is thought to provide useful prognostic information concerning squamous cell carcinomas of the oropharynx and oral cavity. Overexpression of p53 or PCNA status is not of prognostic value, which is consistent with earlier results. We conclude that the detection of Ki-67 is an unfavorable prognostic factor for squamous cell carcinoma of the oropharynx and oral cavity, at least if treated with a combination of surgery and postoperative irradiation. PMID- 10738716 TI - [Necrotizing lymphadenitis (Kikuchi-Fujimoto disease) as a rare cause of cervical lymphadenopathies. Diagnosis and differential diagnosis]. AB - BACKGROUND: The ENT specialist is often confronted with the inquiry as to the cause of lymph node enlargements. Common causes include inflammations, neoplastic, or autoimmune diseases. We report on three cases of Kikuchi-Fujimoto disease as a benign self-limiting lymphadenopathy of unknown etiology, which is usually found in young women between 20 and 30 years of age. Main symptoms are indolent or light tender, enlarged lymph nodes in the neck area. The correct diagnosis requires the histologic examination of the lymph node. Even for the experienced pathologist the differential diagnosis of malignant systemic disorders can be difficult. PATIENTS: Three female patients suffering from Kikuchi-Fujimoto disease were treated in our department between September 1997 and March 1998. RESULTS: All patients showed indolent and enlarged lymph nodes in the neck. In one case we were able to diagnose an acute cytomegalovirus infection; in another case an acute Epstein-Barr virus infection. After 3-5 months the symptoms disappeared spontaneously in all patients. In two of the three cases, histologic lymph node examination revealed T-cell lymphoma and Hodgkin's disease and in one case led to initial chemotherapeutic treatment. As a result of a second histologic examination, both diagnoses were reviewed and Kikuchi-Fujimoto disease was diagnosed. Chemotherapy was discontinued. CONCLUSIONS: Kikuchi-Fujimoto disease is a benign, self-limiting lymphadenopathy which is usually diagnosed in young women. Distinguishing Kikuchi-Fujimoto disease from malignant systemic disorders may even be difficult for pathologists using histological examination techniques and requires close cooperation between the ENT specialist and the pathologist. In order to minimize the risk of misdiagnosis and incorrect treatment, the ENT specialist should inform the pathologist about the differential diagnosis of Kikuchi-Fujimoto disease in such cases. PMID- 10738717 TI - [Isolated congenital lower lip fistulas]. AB - BACKGROUND: Congenital lip fistulas are very rare and may occur alone or in combination with oral clefts and other dysontogenetic malformations. The etiology of congenital lip fistulas is not yet known. PATIENT AND METHOD: We present a 7 year-old boy with recurrent infections of bilateral congenital fistulas of the lower lip. There were no associated congenital anomalies and a negative family history. An elliptical excision of the fistulas under microscopic control gave satisfactory functional and esthetic results. There was no recurrence two years after surgery. CONCLUSION: Since the possibility of additional severe malformations exists for the patient and the patient's children patients should undergo careful ENT and pediatric assessment, and genetic counselling should be considered. In order to achieve good functional and esthetic results, the corrective operation must be planned carefully taking into consideration important surgical principles. PMID- 10738718 TI - [Environmental health in ENT medicine]. AB - BACKGROUND: Genetic and environmental factors contribute to the development of human diseases. This review article deals with the impact of physical and chemical environmental pollutants on the development of various diseases in otorhinolaryngology. METHODS: Data were obtained from textbooks, review articles, original publications, medical databases, internet searches, and publications of various German and international agencies and organizations concerned with environmental health. RESULTS: Environmental pollutants are important contributing factors in the etiology of head and neck diseases. Health effects include hearing loss due to noisy leisure time activities and cardiovascular diseases due to traffic noise, impairment of hearing and equilibrium induced by various compounds, odor annoyance and olfactory disturbances, irritation and alterations of respiratory mucosa, acute and chronic inflammatory and allergic diseases of the upper airways and the middle ear, disorders of the voice and larynx, and head and neck tumors. CONCLUSION: Environmental medicine may play an increasingly important role in the prevention of head and neck diseases. It requires a considerable toxicological and epidemiological qualification. However, the contribution of specialists well familiar with the complex anatomy, physiology and functional diagnosis of organ systems located in the head and neck are essential for the reliable assessment of environmental diseases in otorhinolaryngology. PMID- 10738719 TI - [Hospital mortality in subarachnoid hemorrhage. Experience of the "Barcelona Registry of Cerebrovascular Diseases"]. AB - OBJECTIVES: To determine clinical predictors of in-hospital mortality in patients with non-traumatic subarachnoid hemorrhage. PATIENTS AND METHODS: Data de 184 patients with subarachnoid hemorrhage were obtained from consecutive stroke included in the prospective "Barcelona Stroke Registry". Demographic, anamnestic, clinical, neuroimaging and outcome variables in the subgroup of patients who died were compared with those in the surviving subgroup. The independent predictive value of each variable on the development of death was assessed with a logistic regression analysis. Three predictive models were constructed. A first model was based on demographic and clinical variables (total 10 variables). A second model was based on demographic, clinical and neuroimaging variables (total 17). A third model was based on demographic, clinical, neuroimaging and outcome variables (total 21). RESULTS: In-hospital death was observed in 44 patients (24%). Transient neurological deficit (OR = 13.92; 95% CI: 1.01-191.95), progressive deficit (OR = 4.21; 95% IC: 1.28-13.86), limb weakness (OR = 3.24; 95% IC: 1.49 7.08) and age (OR = 1.05; 95% CI: 1.02-1.09) appeared to be independent prognostic factors of in-hospital mortality in the first predictive model. In addition to these variables, intraventricular hemorrhage (OR = 5.51; 95% CI: 1.94 16.04) was selected in the second predictive model. Transient neurological deficit (OR = 41.2; 95% CI: 1.61-1056.2), neurological complications (OR = 11.04; CI del 95%: 3.85-31.74), carotid aneurysm (OR = 6.61; 95% CI: 1.23-35.43), intraventricular hemorrhage (OR = 5.51; 95% CI: 1.65-18.4), progressive deficit (OR = 5.35; 95% CI: 1.11-25.90) and hemispheric intracerebral hemorrhage (OR = 4.32; 95% CI: 1.35-13.90), appeared to be independent prognostic factors of in hospital mortality in the third model. CONCLUSIONS: Clinical features easily obtained at the patient's bedside in addition to neuroimaging data easily obtained in routine neuroimaging studies help clinicians to predict in-hospital mortality in patients with subarachnoid hemorrhage. Transient neurological deficit prior to definitive subarachnoid hemorrhage was the main clinical predictor of in-hospital mortality. PMID- 10738720 TI - [Factors associated with the development of atopy in young adults]. AB - BACKGROUND: To determine the risk factors for the development of atopy in Spanish young adults. SUBJECTS AND METHODS: Case-control study over prevalent cases. Carried out in general population between the ages of 20 to 44 years old. Spanish participants of the ECRHS, a random representative sample of Spanish young adults (n = 16,884), and a 20% randomised subsample made by those who answered to a short respiratory questionnaire and had atopy assessed, was studied. Atopy was defined as having serum specific IgE positivity to the following aeroallergnes: cat dander, Cladosporium herbarum, Dermatophagoides pteronyssinus, Parietaria judaica and Phleum pratense. RESULTS: Several factors had a statistically significant effect. In addition to male gender and lower age, maternal allergy (OR = 1.63; 95% CI = 1.11-2.40), having allergic siblings (OR = 1.40; 95% CI = 1.06-1.90) and a higher educational level (OR = 1.69; 95% CI = 1.22-2.34) were associated with the presence of high levels of specific IgE in our sample. Moreover, having had older siblings, especially older brothers appears to be a protective factor to the development of atopy but not in a statistically significant way, while having had pet birds during childhood appears to enhance the risk. CONCLUSIONS: In addition to the familial variables that indicates both environmental and genetic factors, educational level seems to have low degree of association with atopy; this feature shows that variables related with life style are involved in atopy development. PMID- 10738721 TI - [Neutralizing antibodies in patients with multiple sclerosis treated with interferon beta-1b]. AB - BACKGROUND: Neutralising antibodies (NABs) against interferon beta have been described in one third of patients with multiple sclerosis treated with interferon beta. We have analysed the frequency of NABs and their clinical consequences. PATIENTS AND METHODS: We have studied 68 patients. NABs were determined by protein A Myxovirus assay. RESULTS: Positive NABs were detected in 13% of the patients after 2 years of treatment. CONCLUSIONS: It does not seem to exist a relationship between presence of NABs and a poor evolution of the disease in our patients with multiple sclerosis treated with beta interferon. PMID- 10738722 TI - [Costs of a low payment maintenance treatment with methadone]. AB - BACKGROUND: Little is known about the cost of methadone maintenance treatments in opioid-dependent individuals. PATIENTS AND METHODS: Calculation of cost was achieved by applying an activity-based costing and management method from Conselleria de Sanitat of Catalonia, Spain (1997). RESULTS: The cost of each methadone dose was 76 ptas. First medical visits cost 4,517 ptas. Urine tests cost 760 ptas. Transportation amounted to 18,895 ptas. CONCLUSION: Such a costing method is applicable and reveals the low cost of this methadone maintenance treatment in opioid-dependent subjects. PMID- 10738723 TI - [Severity scales at the intensive care units: tools of clinical usefulness or management?]. PMID- 10738724 TI - [Study of 13 cases of anisakiasis in the province of Cordoba]. AB - Anisakiasis, or anisakidosis, is a parasitic zoonosis due to the infestation by nematodes of the Anisakidae family, mainly by Anisakis simplex. Notwithstanding its world wide distribution, in our country its appearance is quite recent (1991) with only 19 cases previously reported. We refer 13 cases diagnosed in different hospitals in the province of Cordoba, Spain, from September 1994 to July 1998 which represents the biggest series described in Spain so far. All the patients had a clinical onset as acute abdomen, so that they required early surgery in which a narrowing and inflammatory intestinal segment was observed and subsequently resected. Pathology revealed in such segments an intense eosinophilic infiltrate in the mucosa. Only in one of the cases parasitic fragments were detected in the intestinal mucosa and in the 12 remaining cases the diagnosis was immunological by IgE specific for Anisakis simplex determination and antigens detection of the nematode with monoclonal antibodies. As interesting epidemiologic antecedent we shall mention the fact that all patients referred a usual raw fish consumption (mainly anchovy with vinegar) which is host of third-stage larval of the parasite. PMID- 10738725 TI - [Thyroid nodules. Diagnosis and treatment]. PMID- 10738726 TI - [Treatment for the eradication of Helicobacter pylori. Recommendations of the Spanish Consensus Conference]. PMID- 10738727 TI - [HIV infection, quality of life and protease inhibitors]. PMID- 10738728 TI - [Aseptic meningitis, erythema nodosum and centrifugal annular erythema as first manifestation of recurrent polychondritis]. PMID- 10738729 TI - [Tetanus at an intensive care unit simulating acute abdomen]. PMID- 10738730 TI - [Meta-analysis, statistical significance and clinical benefit]. PMID- 10738731 TI - [Evaluation of the specialist's training. Should it be quantitative or qualitative?: Reflexions from the point of view of family practice]. PMID- 10738732 TI - [Advances and styles of psychiatry]. PMID- 10738733 TI - [Psychopharmaceuticals and breast feeding]. AB - The frequency of postnatal mental disorders requiring psychopharmacological treatment raises the question whether breast feeding can be recommended in some cases. This paper summarizes recent information regarding benefits and risks of breast feeding for infants of psychopharmacologically treated mothers. If a mother insists upon breast feeding, a careful risk-benefit-consideration has to take place. New results indicate that the advantages of breast feeding can outweight the given risks of maternal pharmacotherapy, if minimal necessary drug doses of an agent with a small amount of passage into milk are prescribed. Monitoring of the serum-levels is advisable especially during the first three months. The nursing infant has to be closely controlled for any adverse effects. PMID- 10738734 TI - [B"ocker FM. Organization of a psychiatric-psychotherapeutic institutional emergency clinic: Needs assessment survey. Psychiat Prax 1999; 26:299-302]. PMID- 10738735 TI - [Neuroleptics and quality of life. A patient survey]. AB - OBJECTIVE: This paper addresses the question as to which positive effects of drug treatment on quality of life are expected by schizophrenic patients, and which negative effects should be avoided by any means. METHODS: A survey, using open ended questions, was carried out among schizophrenic patients from all over Germany (n = 565). RESULTS: Most frequently the patients hoped for a general improvement or stabilisation of their state of health when taking medication. When asked about positive effects, almost every fourth respondent gave a somewhat paradoxical answer by stating that the medication should have less or no side effects. Less frequently, by only 15%, the calming and relaxing effect of the drugs was cited, followed by the antipsychotic effect and relapse prevention. With regard to negative effects, extrapyramidal motor symptoms ranked first with 42% of those questioned regarding them to be a restriction of their quality of life which should definitely be avoided. Second most frequently, sedation was mentioned, followed by weight gain. DISCUSSION: In the light of the introduction of so-called "atypical" neuroleptics, the implications for compliance with the treatment are discussed. PMID- 10738736 TI - ["More is less": a retrospective study of haloperidol dosages in acute schizophrenia]. AB - With the background of a number of meta-analyses on the optimal neuroleptic dosages [1,2] the average daily dosage for the treatment of acute schizophrenic episodes recommended in the internal treatment guidelines of our psychiatric clinic was reduced from 24 mg to 15 mg haloperidol equivalent. In the present retrospective study it was investigated what effect this change in guideline had on the actual dosing behavior and on the efficacy and the side effect rate of the antipsychotic treatment. For this purpose all haloperidol treated patients of a two year interval prior to the change of the guideline (1987/88, n = 103) were compared with all treated of a two year interval (1991/92, n = 87) following it. RESULTS: The evaluation of the treatment data showed that the dosing guideline was adhered to and in 1991/92 on the average actually only 15 mg haloperidol were prescribed daily in acute schizophrenic episodes. The antipsychotic efficacy was just as good under this dosage as under the average daily dose of 24 mg haloperidol given in the preceeding interval; the average time in hospital was even reduced from 76 to 67 days. Under the lower daily doses an additional medication with biperiden was less often required. CONCLUSION: Average daily doses of 15 mg haloperidol appear to be at least equally as effective and more tolerable for the treatment of acute schizophrenic episodes than average daily doses of 24 mg haloperidol. PMID- 10738737 TI - [Relationship between depression and psychosocial stress among Iranian emigrants]. AB - Specific studies on the situation of mentally ill Iranian migrants do not exist in Germany. Studies from the USA analyze the difference between culturally dependent dysphoric affect and clinical depressivity among Iranians. Goal of this study was to discuss specific aspects of migration for depression among Iranians. 94 Iranian migrants were interviewed in different settings. Half of them met criteria for a depressive disorder, which correlated well with the depression scales BDI and HAMD. Depressives showed higher acculturation problems, immigration stress and social isolation than non-depressives. Therapy for depressive Iranian migrants should focus more strongly on barriers of acculturation than on cultural differences in psychopathology. PMID- 10738738 TI - [Mental disorders and the course of opiate dependence]. AB - OBJECTIVE: The present study investigates the hypothesis of a relationship between mental disorders and symptoms on the one hand and the extent of drug consumption and the addiction-related problems of opiate dependents on the other hand. METHODS: In a five-year follow-up study in Hamburg among 350 opiate dependents who, at the time of the initial survey, were in contact with the drug help system, 219 clients (63%) could be interviewed at (so far) three different times of investigation. Standardised questionnaires like the EuropASI, CIDI, SCL 90-R, STAI and BDI were used for the interviews. RESULTS: The general life situation of the surveyed persons has on the whole improved in the course of the last two to three years. Also the average drug consumption clearly decreased. Between mental disorders/disturbances and drug dependence or drug-related problems, a linear correlation could be found: An unfavourable course of mental disorders and symptoms correlates with a problematic current life situation of the client. There is also a global relationship between increased drug consumption and the emergence of mental symptoms like depressiveness and anxiety and the psychosocial functional level. CONCLUSIONS: The expected relationship between mental disturbances and the extent of drug consumption is not very marked. This is in support of the basic assumption that specific constellations of drug consumption and mental disorders do not exist isolated, but that they are related, as elements of a complex pattern, to the development of other areas of life. PMID- 10738739 TI - [Standardized documentation system for the complementary sector of psychiatric care. Development and trial in Saxony]. AB - The necessity for a standardized documentation system for the complementary sector of psychiatric care is pointed out, and the goals and requirements to be met are debated. The test-version of such a documentation system (abbrev. BADO-K) is shown. It consists of modules for the assessment of client-centered data, and for the documentation of the treatment process in various facilities. Furthermore, a module of questionnaires and instruments for assessing outcome variables (e.g. quality of life) is integrated. The principles for using the paper-pencil-version and the EDP-version are outlined and discussed with regard to the legal regulations concerning the protection of personal data. Finally, possibilities and limitations of the presented documentation system are discussed. It is argued that the BADO-K is an useful instrument for evaluating the process and outcome of community psychiatric care. PMID- 10738741 TI - [Ingestion of metals and factitious disorders. Impulse control disorder presenting on a background of situational conflicts]. AB - We describe the case of a 27-year-old woman who in addition to the symptoms of factitious disorder showed deliberate ingestion of foreign objects. The relationship between secret and open self-destructive behavior is discussed. Analysis of situational factors highlights the importance of interactional background for the manifestation of impulsive self-harm. Parallels are pointed out to foreign body ingestion in institutionalized persons. Therapeutic issues are discussed. PMID- 10738740 TI - [Quality assurance and documentation for community mental health services cooperative (GPV). A standardized procedure for regional health care documentation and planning]. AB - This paper describes a standardized assessment-procedure for the so-called "Gemeindepsychiatrische Verbunde" in Germany (GPV-Documentation), which are regional combines of community-based mental health care services. It is the first procedure in Germany which puts data on psychiatric care offers, needs for care and service utilization of all sectors and services in community-based mental health care into standardized measures, thus making offers and care of these services comparable. The procedure can be applied to the daily routine of catchment areas. From a methodical point of view the GPV-Documentation keeps a functional approach to community-based mental health care, but additionally it enables services to describe their specific contribution to regional care separately from others. Annual follow-ups make the GPV-Documentation an ideal tool for planning purposes, care management and quality assurance. Cross-regional comparisons of standards of care are possible. The implementation will also enhance the quality of governmental health reports on the care of chronically mentally ill in Germany remarkably. PMID- 10738742 TI - [Undiagnosed complex focal epilepsy as the cause of a depressive syndrome]. PMID- 10738744 TI - [In Process Citation]. PMID- 10738743 TI - [Cognitive impairment of chronic schizophrenics does not equate to dementia]. PMID- 10738745 TI - [In Process Citation]. PMID- 10738746 TI - [In Process Citation]. PMID- 10738747 TI - [The craniofacial architectural factors predisposing to a skeletal Class II identified by Jean Delaire's architectural analysis]. AB - The current state-of-the-art medical practice involves considering treatment both in terms of symptomatology and etiology. In the case of dento-facial deformities, treatment and prognosis must deal both with constitutional and functional anomalies. Most accurate diagnosis can be achieved using Jean Delaire's architectural cephalometric analysis. We illustrate the usefulness of this system using examples of class II skeletal malocclusions. Class II cranial malocclusion factors appear to be difficult to treat successfully. Maxillary and mandibular functional and constitutional anomalies are described. Functional deformities such as anterior rotation of the maxilla or posterior rotation of the mandibular ramus, can be successfully treated with early orthopedic care. Constitutional anomalies, such as excess length of the premaxilla or maxillary vertical excess, should frequently be treated in a combined orthodontic-surgical sequence as orthopedic care alone is ineffective. This explains the usefulness of Jean Delaire's architectural approach to obtain stable results in the treatment of maxillo-mandibular deformities. PMID- 10738748 TI - Delaire's craniofacial architectural analysis. A reminder of the changes introduced by its designer in 1994. PMID- 10738749 TI - [The importance of early surgery in preventing the esthetic sequelae of cervicofacial mucocutaneous hemangiomas in children]. AB - Infants with cutaneous hemangiomas are classically managed medically, well defined surgery being planned for esthetic correction at the age of 8 to 10 years. We present a series of 65 cases of early surgery in children with head and neck cutaneous or mucosal hemangiomas where irreversible and unesthetic scars were predictable. The surgical procedure was simple and the cosmetic result was better than could be expected after late surgery, limiting psychological consequences. In our opinion, the abstention rule should be changed. A multidisciplinary check-up at 2 years to identify cases with a predictably unesthetic scar after complete resolution of the angioma would help select cases where early surgical correction, taking advantage of the exceptional quality of skin in these young children, would be most beneficial. PMID- 10738750 TI - [Posterior disk displacement of the temporomandibular joint. Apropos 2 cases]. AB - Posterior displacement of the temporo-mandibular joint disk is exceptional. The most typical clinical sign is sudden onset unilateral molar open bite. This lateral open bite is accompanied by a sensation of an intra-articular foreign body and more rarely by painful episodes. The joint sounds are not characteristic. Mouth opening is slightly limited. There is no consensus concerning treatment. Conservative treatment can be prescribed in cases with functional impairment. First line invasive techniques should be avoided. PMID- 10738751 TI - [The use of an alcohol gel of ethyl cellulose in the treatment of venous malformations]. AB - Absolute alcohol is the most effective treatment of venous malformations. We created an alcoholic sclerosing solution less diffusible and we report a preliminary study on 6 patients. The viscosity of the alcohol was enhanced with ethylcellulose and we treated 7 patients with VM. Two benign skin ulcerations were observed and one patient with a large VM of the face could be treated around the eyes in a dangerous area. The results are encouraging and a larger study is being performed. PMID- 10738752 TI - [The nonhealing of the buccal mucosa after tooth extraction. Apropos a case of histiocytosis X]. AB - We report a case of eosinophilic granuloma falsely diagnosed as a radicular cyst in a 28-year-old patient. Between August 1996 and June 1997, successful extraction [correction of avulsion] of teeth 36 and 37 was followed by non healing of the mucosa in the corresponding sockets. Three bone biopsies were needed to establish the diagnosis of histiocytosis X. In case of non-healing of the intraoral mucosa after tooth extraction [correction of avulsion], the first step is to rule out a malignant tumor. A clinical and radiographic work-up as well as biopsy are needed. Less common conditions such as tuberculosis or histiocytosis X requiring specific treatment can be diagnosed by pathology. PMID- 10738753 TI - [Prosthesis fitting after maxillectomy: an indispensable factor in acceptance and rehabilitation]. AB - Surgical resection is often required for maxillary cancer, producing a communication between the oral cavity and the nose or paranasal sinuses. After maxillectomy, patients experience major dysfunction in speech and swallowing which have a very negative psychological effect. These problems can be overcome with an immediate prosthesis but to be fully successful, coordinated work is required between the surgeon and the maxillofacial prosthodontist before, during and after surgery. Ten days after surgery, an interim obturator is made with soft lining materials. This prosthesis will evolve as scar tissue forms, preparing space for the definitive obturator which will restore good quality of life for maxillectomy patients. PMID- 10738754 TI - [Exostosis of the zygoma. Apropos a case]. AB - Exostosis is a benign bony process arising from cortical bone. It is generally localized at the fertile metaphysis of long and some flat bones. Localization to the zygoma and/or the coronoid process is exceptional (5 cases in the world literature). We report an original case of exostosis with isolated zygomatic localization in a 14-year-old girl and review the literature. We emphasize the clinical features and the diagnostic and therapeutic approach in this condition. PMID- 10738755 TI - [The surgical treatment of intracranial aneurysms from the pterion approach using small trephining openings]. AB - In the past decade, there has been a clear trend for the use of low-invasive surgical interventions in many divisions of neurosurgery. Since each operation is bound to be followed by tissue traumatization, a decrease in the sizes of skull trepanation should be regarded as a way of reducing the incidence of intra- and extracranial complications. The proposed variant of a pterion access to intracranial aneurysms by using small trepanation holes may substantially decrease the duration of surgical interventions and to avoid postoperative epidural and subdural hematomas without preventing the visualization of arteries in both anterior and posterior Willis' circle and at the same time the variant exerts no negative effect on the possibilities of hemostasis in intraoperative aneurysm rupture. The operation yields a good cosmetic effect. PMID- 10738756 TI - [The surgical treatment of decubitus ulcers in patients with spinal cord trauma]. AB - The paper summarizes experience in surgically treating decubitus in 429 patients with spinal cord injury. Main criteria for decubitus preparation for surgery, indications for various surgical interventions are defined. The outcomes of treatment are analyzed. The wound healed by first intention in 71% of patients and in 86% of patients within 3 months. Repeated surgery was required in 4.4% of patients. There is evidence that surgical intervention makes it possible to eliminate the existing decubituses in patients with spinal cord injury by score times more rapidly, to greatly improve the quality of their life and to increase survival. PMID- 10738757 TI - [The procedure for the surgical treatment of spinal and spinal cord trauma in the acute period]. AB - Examination and treatment policy is proposed for patients with complicated vertebral injury under the conditions of urgent neurosurgical service. Its practical use at Saint Petersburg city multidisciplinary hospital No. 26 in 207 patients with vertebral and spinal cord injury substantially improved the outcomes of this severe group of patients. A wide range of surgical interventions into the vertebral column was used. Treatment yielded fair and good results in 188 (85.9%) patients. PMID- 10738758 TI - [The characteristics of epidural analgesia during the removal of lumbar intervertebral disk hernias]. AB - Epidural analgesia (EA) was used in 29 patients undergoing surgical removal of lumbar discal hernia. Marcain EA with controlled medicinal sleep and non-assisted breathing allowed to perform the whole operation in 27 patients. EA may be ineffective in combination of sequestrated disk hernia with scarry adhesive process. The technique of the operation demands a single use of the anesthetic drug which is potent enough to make blockade throughout the operation up to the end. PMID- 10738759 TI - [The pathogenesis of elevated intracranial pressure in children and adolescents with craniovertebral pathology]. AB - The paper deals with intracranial hypertension in children and adolescents. Thirty-six patients with the upper cervical dysplasia and identified associated intracranial hypertension were examined. Magnetic resonance imaging and functional tests were used. Some patients were diagnosed as having a thickened ligamentous apparatus at the level of craniovertebral junction, which was a cause of spinal fluid dynamic disorders and led to elevated intracranial pressure. PMID- 10738760 TI - [The morphometric characteristics of the sagittal fissure as the basis for a surgical approach in callosotomy]. AB - Macro-microscopic tomography of the sagittal fissure of the brain has been studied on 10 dead bodies, morphometric studies were performed in 10 healthy subjects (40 NMR-tomograms and 40 cerebral angiograms). The findings made it possible to divide the fissure into three zones and single out basic anatomical structures in each zone. Morphological and morphometrical data obtained provided validation of the approach to the corpus callosum and microsurgical callosotomy. PMID- 10738761 TI - [Lipid-bound sialic acids in the blood of patients with brain diseases]. AB - The results of studies of the blood levels of lipid-bound sialic acids (LBSA) in patients with various brain tumors, subarachnoidal hemorrhage (SAH) caused arterial aneurysmal rupture and in those with brain injury (BI) are analyzed. Significant differences were found in blood LBSA levels in patients with hemispheric tumors and in those with chiasmal sellar ones. The findings suggest that blood LBSA levels cannot be a marker for brain tumors as the blood of patients with benign chiasmal sellar tumors shows high LBSA levels reaching those in patients with malignant hemispheric tumors. The blood levels of LBSA in patients SAH and BI were much higher than those in controls. The blood levels of LBSA are very equal in SAH and BI. PMID- 10738762 TI - [The urological screening of children with myelodysplasia]. AB - All children with myelodysplasia have urological diseases. Nevertheless, errors in urological policy and diagnosis are made just at the first stage of a study, by leading to inadequate treatment, progressive obstructive complications, chronic renal failure. The authors propose a urological screening, simple, accessible, and safe tests, which may evaluate the urinary system, determine the expediency and urgency of further examination during early management of children with myelodysplasia. PMID- 10738763 TI - [Posthemorrhagic cerebral vasospasm in light of the current concepts on the regulation of cerebral blood circulation]. PMID- 10738764 TI - [The history of the Psychiatric Service in the Academician N. N. Burdenko Institute of Neurosurgery of the Russian Academy of Medical Sciences]. PMID- 10738765 TI - Optimum and standard beam widths for numerical modeling of interface scattering problems AB - Gaussian beams provide a useful insonifying field for surface or interface scattering problems such as encountered in electromagnetics, acoustics and seismology. Gaussian beams have these advantages: (i) They give a finite size for the scattering region on the interface. (ii) The incident energy is restricted to a small range of grazing angles. (iii) They do not have side lobes. (iv) They have a convenient mathematical expression. The major disadvantages are: (i) Insonification of an interface is nonuniform. The scattered field will depend on the location of the scatterers within the beam. (ii) The beams spread, so that propagation becomes an integral component of the scattering problem. A standard beam parameterization is proposed which keeps propagation effects uniform among various models so that the effects of scattering only can be compared. In continuous wave problems, for a given angle of incidence and incident amplitude threshold, there will be an optimum Gaussian beam which keeps the insonified area as small as possible. For numerical solutions of pulse beams, these standard parameters provide an estimate of the smallest truncated domain necessary for a physically meaningful result. PMID- 10738766 TI - A boundary that sustains a negligible specular reflection coefficient over a wide frequency band AB - In a previous paper the authors analyzed and discussed the specular reflection coefficient of a plane boundary comprised of a plate, a compliant layer, and a fluid. The analysis showed that a negligible specular reflection coefficient may be derived provided specific resonance conditions are met. The resonance of concern is that between the surface mass of the plate and the surface stiffness of the compliant layer. The conditions of resonance included the value that must be assigned to the loss factor in the compliant layer. In the present paper, an attempt is made to determine the conditions that must be placed on the surface stiffness of the compliant layer in order to increase the frequency range over which a negligible specular reflection coefficient may be maintained. The tolerances in these conditions are also estimated. PMID- 10738767 TI - Inversion of penetrable obstacles from far-field data on narrow angular apertures AB - Reported in this paper are reconstructions of shape and material parameters of two-dimensional, homogeneous, acoustic, penetrable obstacles of arbitrary cross sections which are immersed in an infinite, homogeneous ambience. Reconstructions are based on the far-field scattering patterns of multiple incident plane acoustic waves. For remotely acquired data, practical necessities require not only that the entire data collection region be less than 2 pi, but also that each received "sees" the object over as narrow an angular aperture as possible. The inversions presented here were obtained under such conditions. Two types of data were used for each incidence namely, "near-monostatic" fields of narrow angular apertures (the narrowest aperture reported is 2 degrees), and a "duostatic" geometry consisting of backscatter plus one other receiver angle. These data sets were acquired for a series of incident angles. The theoretical formalism for inversion is algebraic in nature, requires no integral equation, and possesses a number of advantages for the implementation of a Gauss-Newton type of inversion that was used in this study. Moreover, the algorithm is shown to be inheritently parallelizable. PMID- 10738768 TI - Acoustic scattering by baffled flexible surfaces: the discrete optical theorem AB - The optical theorem for acoustic scattering by baffled membranes and plates relates the total cross section of the scattered field, the directivity factor in the specular direction, and the energy dissipated by the structure. It is basically a statement of conservation of power. In this paper it is demonstrated that the discrete formulation of these problems, obtained by a Galerkin approximation, exactly satisfies the optical theorem. This discrete relationship holds regardless of the choice of basis functions and the size of the truncated system N. Thus, the adherence of the approximate numerical results to the power conservation law does not necessarily imply its accuracy. PMID- 10738769 TI - Effects of thermal diffusion on sound attenuation in evaporating and condensing gas-vapor mixtures in tubes AB - An investigation of sound propagation in an air-water vapor mixture contained in a cylindrical tube with wet walls was recently presented [Raspet et al., J. Acoust. Soc. Am. 105, 65-73 (1999)]. The formulation of the problem paralleled the "low reduced frequency method" of Tijdeman [J. Sound Vib. 39, 1-33 (1975)]. It was pointed out that a term of reduced frequency order had been neglected in the radial component of the diffusion equation [G. Swift, personal communication (1999)]. This term represents the additional mass diffusion driven by the temperature gradient, or Soret effect, and is proportional to the thermal diffusion ratio. The solution for the complex wave number of the acoustic mode with this additional term is presented here. Numerically calculated predictions for the air-water vapor mixture show little change in acoustic attenuation due to the coupling. Therefore, a description of the acoustic attenuation where the viscous, thermal, and diffusion processes are decoupled is adequate for the specific case previously discussed by Raspet et al. PMID- 10738770 TI - A transfer-matrix approach for estimating the characteristic impedance and wave numbers of limp and rigid porous materials AB - A method for evaluating the acoustical properties of homogeneous and isotropic porous materials that may be modeled as fluids having complex properties is described here. To implement the procedure, a conventional, two-microphone standing wave tube was modified to include: a new sample holder; a section downstream of the sample holder that accommodated a second pair of microphone holders and an approximately anechoic termination. Sound-pressure measurements at two upstream and two downstream locations were then used to estimate the two-by two transfer matrix of porous material samples. The experimental transfer matrix method has been most widely used in the past to measure the acoustical properties of silencer system components. That procedure was made more efficient here by taking advantage of the reciprocal nature of sound transmission through homogeneous and isotropic porous layers. The transfer matrix of a homogeneous and isotropic, rigid or limp porous layer can easily be used to identify the material's characteristic impedance and wave number, from which other acoustical quantities of interest can be calculated. The procedure has been used to estimate the acoustical properties of a glass fiber material: good agreement was found between the estimated acoustical properties and those predicted by using the formulas of Delany and Bazley. PMID- 10738771 TI - Eigenmode analysis of arbitrarily shaped two-dimensional cavities by the method of point-matching AB - In the proposed method, as in the boundary element method (BEM), a field problem is solved on its boundary along which nodes are distributed. The distinct feature of the proposed method is related to the fact that no interpolation functions between the nodes are required, so that the basic collocation method is employed to satisfy a given boundary condition. This approach reduces a large amount of numerical calculation induced due to the interpolation functions. Unlike the method of auxiliary sources (MAS), in the proposed method only the regular functions that have finite functional values everywhere including the origins are employed as basis functions. As a result, the proposed method does not need the auxiliary surface, the construction of which is a troublesome work because its form, dimension, shifting, etc. strongly influence the accuracy of the MAS. The eigenvalues calculated by the proposed method rapidly converge to the exact values thanks to the simplicity of the method. Moreover, the method gives mode shapes successfully without using the interpolation functions between the nodes. The efficiency and accuracy of the method are verified through several applications. PMID- 10738772 TI - Time-harmonic torsional waves in a composite cylinder with an imperfect interface AB - In this paper, the propagation of time-harmonic torsional waves in composite elastic cylinders is investigated. An imperfect interface is considered where tractions are continuous across the interface and the displacement jump is proportional to the stress acting on the interface. A frequency equation is derived for the rod and dispersion curves of normalized frequency as a function of normalized wave number for elastic bimaterials with varying values for the interface constant F are presented. The analysis is shown to recover the dispersion curves for a bimaterial rod with a perfect (welded) interface (F = 0), and has the correct limiting behavior for large F. It is shown that the modes, at any given frequency, are orthogonal, and it is outlined how the problem of reflection of a torsional mode by a planar defect (such as a circumferential crack) can be treated. PMID- 10738774 TI - Transient acoustic radiation from impulsively accelerated bodies by the finite element method AB - Bodies under impulsive motion, immersed in an infinite acoustic fluid, severely put to test any numerical method for the transient exterior acoustic problem. Such problems, in the context of the finite element method (FEM), are not well studied. FE modeling of such problems requires truncation of the infinite fluid domain at a certain distance from the structure. The volume of computation depends upon the extent of this domain as well as the mesh density. The modeling of the fluid truncation boundary is crucial to the economy and accuracy of solution and various boundary dampers have been proposed in the literature for this purpose. The second order damper leads to unsymmetric boundary matrices and this necessitates the use of an unsymmetric equation solver for large problems. The present paper demonstrates the use of FEM with zeroth, first and second order boundary dampers in conjunction with an unsymmetric, out of core, banded equation solver for impulsive motion problems of rigid bodies in an acoustic fluid. The results compare well with those obtained from analytical methods. PMID- 10738773 TI - Fast analysis of transient acoustic wave scattering from rigid bodies using the multilevel plane wave time domain algorithm AB - The analysis of transient wave scattering from rigid bodies using integral equation-based techniques is computationally intensive: if carried out using classical schemes, the evaluation of the velocity potential on the surface of a three-dimensional scatterer, represented in terms of Ns spatial basis functions for Nt time steps, requires O(NtNs2) operations. The recently developed plane wave time domain (PWTD) algorithm permits the rapid evaluation of transient fields that are generated by bandlimited source distributions. It has been shown that incorporation of the PWTD algorithm into integral equation-based solvers in a two-level setting reduces the computational complexity of a transient analysis to O(NtNs1.5 logNs). In this paper, it is shown that casting the PWTD scheme into a multilevel framework permits the analysis of transient acoustic surface scattering phenomena in O(NtNslog2Ns) operations using O(NtNs) memory. Numerical examples that demonstrate the efficacy of the multilevel implementation are also presented. PMID- 10738775 TI - Sound cancellation by the use of secondary multipoles: experiments AB - Theory related to global, free-field cancellation of a primary monopole field by the use of a displaced, secondary multipole was presented previously: a corresponding experimental investigation is presented here. The construction of multipole source components to octopole order is described, as are procedures for determining their source strengths. Dipoles, longitudinal quadrupoles, and longitudinal octopoles that conformed closely to their theoretical models were constructed using arrays of unbaffled loudspeakers. Two methods of calculating the multipole strengths required to cancel a primary monopole field were implemented in an open-loop manner: a "direct" approach based on a multipole expansion of the primary field, and a least-squares procedure based on fitting the secondary field to the primary field either along a circle enclosing a secondary source, or along a segment of that circle. Cancellation measurements were made on a 1-m-radius circle centered on the secondary source: the primary-to secondary source separation was approximately 0.2 wavelengths. It was found both that a secondary multipole could provide far greater cancellation than a monopole placed at the same distance from the primary source and that the least-squares approach resulted in greater far-field cancellation than did the direct approach. PMID- 10738776 TI - Experimental investigation of transient pressure waves produced in dielectric liquids AB - The experimental results reported in this paper mainly concern the two first transient pressure waves emitted after a fast (approximately ns) and localized (approximately micron) injection of electrical energy Wi in insulating liquids. The influence of various parameters (hydrostatic pressure P infinity, injected energy Wi, etc.) on the relative amplitude of these two transient pressure waves are presented and discussed. The analysis shows clearly a difference of behavior, in particular their dependence as a function of hydrostatic pressure, between these two waves. Basically, the physical processes leading to their emission are quite different. The first pressure transient is a consequence of the plasma relaxation, whereas the second one is due to the bubble collapse. According to these results, the chronology of the events is more improved, as well as the theoretical analysis. PMID- 10738777 TI - Higher-order harmonics of limited diffraction Bessel beams AB - We investigate theoretically the nonlinear propagation of the limited diffraction Bessel beam in nonlinear media, under the successive approximation of the KZK equation. The result shows that the nth-order harmonic of the Bessel beam, like its fundamental component, is radially limited diffracting, and that the main beamwidth of the nth-order harmonic is exactly 1/n times that of the fundamental. PMID- 10738778 TI - Mechanisms for subcritical penetration into a sandy bottom: experimental and modeling results AB - This paper presents preliminary results of a recent study whose overall objectives are to determine the mechanisms contributing significantly to subcritical acoustic penetration into ocean sediments, and to quantify the results for use in sonar performance prediction for the detection of buried objects. In situ acoustic measurements were performed on a sandy bottom whose geoacoustical and geomorphological properties were also measured. A parametric array mounted on a tower moving on a rail was used to insonify hydrophones located above and below the sediment interface. Data covering grazing angles both above and below the nominal critical angle and in the frequency range 2-15 kHz were acquired and processed. The results are compared to two models that account for scattering of sound at the rough water-sediment interface into the sediment. Although all possible mechanisms for subcritical penetration are not modeled, the levels predicted by both models are consistent with the levels observed in the experimental data. For the specific seafloor and experimental conditions examined, the analysis suggests that for frequencies below 5-7 kHz sound penetration into the sediment at subcritical insonification is dominated by the evanescent field, while scattering due to surface roughness is the dominant mechanism at higher frequencies. PMID- 10738779 TI - Impedance-matched absorbers for finite-difference parabolic equation algorithms AB - In this paper, a perfectly matched layer (PML) absorber, recently introduced into the electromagnetic propagation literature by Berenger [J. Comput. Phys. 114, 185 200 (1994)], is adapted for use with both paraxial and wide-angle acoustic parabolic equations (PEs). Our procedure incorporates an imaginary component into the transverse coordinate that mimics the introduction of a fictitious absorber on the edge of the computational grid. Use of such an impedance-matched layer can significantly reduce spurious reflections compared to physical absorbing layer methods and thus allows a smaller number of boundary points to be employed in PE calculations. Numerical results obtained with several higher-order propagator approximations confirm that such impedance-matched absorbers efficiently eliminate reflections. PMID- 10738780 TI - Traction-free vibration of layered elastic and piezoelectric rectangular parallelepipeds AB - A variational method is developed to study the traction-free vibration of layered rectangular elastic and piezoelectric parallelepipeds. The weak form of the equations of motion and the charge equation are formulated in rectangular Cartesian coordinates. Approximate solutions to these equations are sought in a form that combines piecewise linear or quadratic Lagrange basis functions through the layered dimension of the solid with continuous global polynomial or trigonometric functions in the plane. This allows for the necessary discontinuity in the shear strain and normal potential gradient across the interface between layers caused by the mismatch in material properties. Numerical results compare very well with those computed by other techniques for layered elastic and piezoelectric plates with simple support and homogeneous parallelepipeds under stress-free conditions. PMID- 10738781 TI - Acoustic scattering by a three-dimensional elastic object near a rough surface AB - The ensemble-averaged field scattered by a smooth, bounded, elastic object near a penetrable surface with small-scale random roughness is formulated. The formulation consists of combining a perturbative solution for modeling propagation through the rough surface with a transition (T-) matrix solution for scattering by the object near a planar surface. All media bounding the rough surface are assumed to be fluids. By applying the results to a spherical steel shell buried within a rough sediment bottom, it is demonstrated that the ensemble averaged "incoherent" intensity backscattered by buried objects illuminated with shallow-grazing-angle acoustic sources can be well enhanced at high frequencies over field predictions based on scattering models where all environmental surfaces are planar. However, this intensity must compete with the incoherent intensity scattered back from the interface itself, which can defeat detection attempts. The averaged "coherent" component of the field maintains the strong evanescent spectral decay exhibited by flat interface predictions of shallow angle measurements but with small deviations. Nevertheless, bistatic calculations of the coherent field suggest useful strategies for improving long-range detection and identification of buried objects. PMID- 10738782 TI - High-resolution geoacoustic inversion in shallow water: a joint time- and frequency-domain technique AB - High-resolution geoacoustic data are required for accurate predictions of acoustic propagation and scattering in shallow water. Since direct measurement of geoacoustic data is difficult, time-consuming, and expensive, inversion of acoustic data is a promising alternative. However, the main problem encountered in geoacoustic inversion is the problem of uniqueness, i.e., many diverse geoacoustic models can be made to fit the same data set. A key, and perhaps unique, aspect of this approach is the combination of data analysis in both the space-time and the space-frequency domains. This combination attempts to ameliorate the uniqueness problem by exploiting as much independent data as possible. In order to meet the stringent requirements of high spatial resolution and uniqueness, an entire method has been developed including a new measurement technique, processing/analysis technique, and inversion strategy. These techniques are described and then illustrated with a shallow-water data set. Sound-speed gradients in the upper few meters of the sub-bottom appear to be much higher (one order of magnitude) than generally assumed. And, although often ignored, a large density gradient was observed in the top layer that played an acoustically significant role. PMID- 10738783 TI - Target strength of an oily deep-water fish, orange roughy (Hoplostethus atlanticus). II. Modeling. AB - Orange roughy consist of approximately 18% lipids by weight, mostly as wax esters, and the lipids must be taken into account when modeling target strength. A deformed cylinder model incorporating the effect of temperature and pressure on sound speed through wax ester was used to scale experimental measurements of target strength to the temperatures and pressures where orange roughy live (approximately 6 degrees C, depths approximately 800-1300 m). The effect of decreasing temperature and increasing pressure is to increase the sound speed in orange roughy lipids. Modeling shows that the net effect of this is to reduce tilt-averaged target strength, 'TS', by approximately 2 dB. Adjusting experimental results to compensate for temperature and pressure effects gives a predicted 'TS' for a 35-cm orange roughy of -48.3 dB. Adjusting in situ estimates of orange roughy 'TS' for avoidance behavior [McClatchie et al., J. Acoust. Soc. Am. 106, 131-142 (1999)] suggests the correct 'TS' is approximately -47.5 dB, rather than -50 dB as previously reported [Kloser et al., ICES J. Mar. Sci. 54, 60-71 (1997)]. We conclude that experimental and in situ estimates now converge at a 'TS' of approximately -48 dB for a 35-cm fish. PMID- 10738784 TI - Matched-field processing, geoacoustic inversion, and source signature recovery of blue whale vocalizations. AB - Matched-field processing (MFP) and global inversion techniques have been applied to vocalizations from four whales recorded on a 48-element tilted vertical array off the Channel Islands in 1996. Global inversions from selected whale calls using as few as eight elements extracted information about the surrounding ocean bottom composition, array shape, and the animal's position. These inversion results were then used to conduct straightforward MFP on other calls. The sediment sound-speed inversion estimates are consistent with those derived from sediment samples collected in the area. In general, most animals swam from the east to west, but one animal remained within approximately 500 m of its original position over 45 min. All whales vocalized between 10 and 40 m depth. Three acoustic sequences are discussed in detail: the first illustrating a match between an acoustic track and visual sighting, the second tracking two whales to ranges out to 8 km, and the final sequence demonstrating high-resolution dive profiles from an animal that changed its course to avoid the research platform FLIP (floating instrument platform). This last whale displayed an unusual diversity of signals that include three strong frequency-modulated (FM) downsweeps which contain possible signs of an internal resonance. The arrival of this same whale coincided with a sudden change in oceanographic conditions. PMID- 10738785 TI - Matched-field replica model optimization and bottom property inversion in shallow water AB - Matched-field replica models based on an inaccurate knowledge of geoacoustic parameters such as bottom attenuation, shear, and interfacial sound-speed discontinuities, can predict an incorrect number of propagating modes for a shallow-water channel. The resulting degradation in the matched-field ambiguity surface can be substantially reduced by obtaining optimal replica models via modal-sum-limit optimization or bottom-property inversion. The use of these techniques for multi-tone (70, 95, 145, and 195 Hz) source-tow data recorded near San Diego during the first Shallow-Water Evaluation Cell Experiment (SWellEX-1) significantly increased matched-field correlation levels and improved source localization relative to results obtained with a previous nonoptimized model. The predicted number of propagating modes was also reduced substantially. The inversion for bottom properties (attenuation, interfacial sound-speed discontinuities, no shear) provided sediment attenuation estimates which agree well with Hamilton's models and were an order-of-magnitude greater than that used in the nonoptimized model, which accounts for the reduction in the number of modes. A simulated modal decomposition using the inverted optimal replica model verifies the number of modes predicted by the modal-sum-limit optimization. PMID- 10738786 TI - A matched-peak inversion approach for ocean acoustic travel-time tomography AB - A new approach for the inversion of travel-time data is proposed, based on the matching between model arrivals and observed peaks. Using the linearized model relations between sound-speed and arrival-time perturbations about a set of background states, arrival times and associated errors are calculated on a fine grid of model states discretizing the sound-speed parameter space. Each model state can explain (identify) a number of observed peaks in a particular reception lying within the uncertainty intervals of the corresponding predicted arrival times. The model states that explain the maximum number of observed peaks are considered as the more likely parametric descriptions of the reception; these model states can be described in terms of mean values and variances providing a statistical answer (matched-peak solution) to the inversion problem. A basic feature of the matched-peak inversion approach is that each reception can be treated independently, i.e., no constraints are posed from previous-reception identification or inversion results. Accordingly, there is no need for initialization of the inversion procedure and, furthermore, discontinuous travel time data can be treated. The matched-peak inversion method is demonstrated by application to 9-month-long travel-time data from the Thetis-2 tomography experiment in the western Mediterranean sea. PMID- 10738787 TI - The transmission of Lamb waves across adhesively bonded lap joints AB - The transmission of Lamb waves across adhesively bonded lap joints is investigated using finite element analysis. The studies consider three modes for excitation and reception, s0, a0, and a1, applied to lap joints consisting of parallel aluminum sheets bonded with an epoxy adhesive. Transmission coefficient results for a two-dimensional range of bond thicknesses and bond overlap lengths are presented for all three modes. The transmission coefficients are calculated from the spectra of the received and transmitted signals using an approach which is insensitive to the presence of multimode signals and reverberated signals, and which approximates to a power transmission coefficient. Detailed analysis is then performed for one of the modes in order to investigate the nature of the mode conversion in the overlap region of the joint. It is found that the relative amplitudes of the different modes which propagate in the overlap region can be estimated reliably and simply from the properties of the incident wave mode. As well as demonstrating the physics of the mode conversion behavior, the study provides a basis for the selection of modes for nondestructive evaluation (NDE) of the bond region and for measuring the bond dimensions. PMID- 10738788 TI - A two-frequency acoustic technique for bubble resonant oscillation studies AB - A two-frequency acoustic apparatus has been developed to study the dynamics of a single gas or vapor bubble in water. An advantage of the apparatus is its capability of trapping a bubble by an ultrasonic standing wave while independently driving it into oscillations by a second lower frequency acoustic wave. For a preliminary application, the apparatus is used to study resonant oscillations. First, near-resonant coupling between the volume and the n = 3 shape oscillation modes of air bubbles at room temperature is studied, where n is the mode number. The stability boundary, amplitude versus frequency, of the volume oscillation forms a wedge centered at the resonant frequency, which qualitatively agrees with a theoretical prediction based on a phase-space analysis. Next, the resonant volume oscillations of vapor bubbles are studied. The resonant radius of vapor bubbles at 80 degrees C driven at 1682 Hz is determined to be 0.7 mm, in agreement with a prediction obtained by numerical simulation. PMID- 10738789 TI - Line source representation for laser-generated ultrasound in aluminum AB - Modeling the ultrasound generated by a laser source is critical for using noncontact laser-generated ultrasonic systems for the characterization of material properties. In this work, a laser line source was modeled and verified experimentally by measuring the ultrasonic shear wave signal generated in aluminum with a broadband laser generation/electromagnetic acoustic transducer (EMAT)-detection system. Results of calculations and experiments show that the amplitude directivity of a laser line source is identical to that of a point source in the plane perpendicular to the line axis while the temporal dependence differs. PMID- 10738790 TI - A study on ultrasonic solid horns for flexural mode AB - The transfer matrix method is introduced into the analysis of ultrasonic flexural solid horns. The frequency and magnification equations of a horn are derived by using the transfer matrix method. Analyzed are the characteristics of three types of circular cross-section horns: exponent, cone, and catenary. The results are partly verified experimently. PMID- 10738791 TI - Chirp response of an active-controlled thickness-drive tunable transducer AB - The chirp response of a thickness-drive tunable transducer for wide range time bandwidth and sweep rates chirp signals is demonstrated experimentally and computationally. The computational evaluation uses recursive digital-filter model based on the z-transform method. The model is limited to simple lossless structure with no front and backing layers. The model and experimental results show that there is no limit on the maximum sweep rate of the chirp signal but practically the limit is determined by the limitation of the circuit that generates the control voltage that simulates a variable electric load. PMID- 10738792 TI - The measurement of structural mobilities of a circular cylindrical shell AB - Structural mobility is useful for the estimation of structural power flows in coupled systems. Although the methods of measuring structural mobilities are easily found for one-dimensional beam structures, few are available for cylindrical shells. In this paper, a new method is proposed for the measurement of the structural mobilities of a circular cylindrical shell. A point force excitation is used instead of circumferential modal forces which are difficult to implement in practice. This method utilizes the least squares technique to obtain the transfer function components of different circumferential modes from the measured data. Experiments were carried out on a circular cylindrical shell with different end conditions excited by a point force to verify the feasibility of this proposed method. PMID- 10738793 TI - Free localized vibrations of a semi-infinite cylindrical shell AB - Free vibrations of a semi-infinite cylindrical shell, localized near the edge of the shell are investigated. The dynamic equations in the Kirchhoff-Love theory of shells are subjected to asymptotic analysis. Three types of localized vibrations, associated with bending, extensional, and super-low-frequency semi-membrane motions, are determined. A link between localized vibrations and Rayleigh-type bending and extensional waves, propagating along the edge, is established. Different boundary conditions on the edge are considered. It is shown that for bending and super-low-frequency vibrations the natural frequencies are real while for extensional vibrations they have asymptotically small imaginary parts. The latter corresponds to the radiation to infinity caused by coupling between extensional and bending modes. PMID- 10738794 TI - Sound propagation in street canyons: comparison between diffusely and geometrically reflecting boundaries AB - This paper systematically compares the sound fields in street canyons with diffusely and geometrically reflecting boundaries. For diffuse boundaries, a radiosity-based theoretical/computer model has been developed. For geometrical boundaries, the image source method has been used. Computations using the models show that there are considerable differences between the sound fields resulting from the two kinds of boundaries. By replacing diffuse boundaries with geometrical boundaries, the sound attenuation along the length becomes significantly less; the RT30 is considerably longer; and the extra attenuation caused by air or vegetation absorption is reduced. There are also some similarities between the sound fields under the two boundary conditions. For example, in both cases the sound attenuation along the length with a given amount of absorption is the highest if the absorbers are arranged on one boundary and the lowest if they are evenly distributed on all boundaries. Overall, the results suggest that, from the viewpoint of urban noise reduction, it is better to design the street boundaries as diffusely reflective rather than acoustically smooth. PMID- 10738795 TI - The adaptive beam-tracing algorithm AB - The most popular models to predict sound propagation in architectural spaces involve the tracing of rays, images, or beams. Most current beam-tracing methods use conical or triangular beams that may produce overlaps and holes in the predicted sound field. Hence a new method has been developed whereby the shape of reflected beams is governed by the shape of reflecting surfaces so as to produce a geometrically perfect description of the sound propagation for halls with occluding surfaces. The method also facilitates the calculation of diffuse sound propagation by managing the energy transfer from a specular model to a diffuse model. This adaptive beam-tracing method compares well with other methods in terms of speed and accuracy. PMID- 10738796 TI - Prediction of sound transmission loss through multilayered panels by using Gaussian distribution of directional incident energy AB - In this study, a new prediction method is suggested for sound transmission loss (STL) of multilayered panels of infinite extent. Conventional methods such as random or field incidence approach often given significant discrepancies in predicting STL of multilayered panels when compared with the experiments. In this paper, appropriate directional distributions of incident energy to predict the STL of multilayered panels are proposed. In order to find a weighting function to represent the directional distribution of incident energy on the wall in a reverberation chamber, numerical simulations by using a ray-tracing technique are carried out. Simulation results reveal that the directional distribution can be approximately expressed by the Gaussian distribution function in terms of the angle of incidence. The Gaussian function is applied to predict the STL of various multilayered panel configurations as well as single panels. The compared results between the measurement and the prediction show good agreements, which validate the proposed Gaussian function approach. PMID- 10738797 TI - Maximum-likelihood approach to strain imaging using ultrasound AB - A maximum-likelihood (ML) strategy for strain estimation is presented as a framework for designing and evaluating bioelasticity imaging systems. Concepts from continuum mechanics, signal analysis, and acoustic scattering are combined to develop a mathematical model of the ultrasonic waveforms used to form strain images. The model includes three-dimensional (3-D) object motion described by affine transformations, Rayleigh scattering from random media, and 3-D system response functions. The likelihood function for these waveforms is derived to express the Fisher information matrix and variance bounds for displacement and strain estimation. The ML estimator is a generalized cross correlator for pre- and post-compression echo waveforms that is realized by waveform warping and filtering prior to cross correlation and peak detection. Experiments involving soft tissuelike media show the ML estimator approaches the Cramer-Rao error bound for small scaling deformations: at 5 MHz and 1.2% compression, the predicted lower bound for displacement errors is 4.4 microns and the measured standard deviation is 5.7 microns. PMID- 10738798 TI - On the mechanoelectrical coupling in the cochlear outer hair cell. AB - Outer hair cell electromotility, a manifestation of the interconnection between the mechanical and electrical processes occurring in outer hair cells, is believed to be an important contribution to the active cochlea. Two modes of mechanoelectrical coupling in the outer hair cell wall are studied: the potential shift caused by mechanical loading under the wall charge preservation conditions and the current (transferred charge) caused by mechanical loading under the voltage-clamp conditions. By using the previously reported elastic moduli of the wall and components of the active force, the potential shift under the charge preservation conditions is derived. This shift is expressed in terms of the wall strains and the active force derivatives with respect to the wall potential. The magnitudes of the potential shift corresponding to the conditions of cell inflation, axial stretch (compression), and the micropipet aspiration are estimated. In the last case, the distribution of the potential shift along the cell wall is also demonstrated. The potential shift can reach -20(-)-40 mV under the conditions of the micropipet aspiration or cell inflation. Such shift is much smaller under the condition of cell stretch (compression). The current and the charge transfer caused by the cell stretch under the voltage-clamp conditions is analyzed, and shows good agreement of predictions with experimental data. PMID- 10738799 TI - Frequency characteristics of sound transmission in middle ears from Norwegian cattle, and the effect of static pressure differences across the tympanic membrane and the footplate. AB - For 23 cadaver ears from Norwegian cattle, frequency characteristics for the round-window volume displacement relative to the sound pressure at the eardrum have been measured, and are compared to earlier results for human ears [M. Kringlebotn and T. Gundersen, J. Acoust. Soc. Am. 77(1), 159-164 (1985)]. For human as well as for cattle ears, mean amplitude curves have peaks at about 0.7 kHz. At lower frequencies, the mean amplitude for cattle ears is about 5 dB smaller than for human ears. The amplitude curves cross at about 2 kHz, and toward higher frequencies the amplitude for cattle ears becomes increasingly larger. If amplitude curves are roughly approximated by straight lines above 1 kHz, the slope for cattle ears is about -5 dB/octave as compared to about -15 dB/octave for human ears. The phase of the round-window volume displacement lags behind the phase of the sound pressure at the tympanic membrane. The phase lag is close to zero below 0.2 kHz, but increases to about 3.5 pi at 20 kHz for cattle ears, as compared to less than 2 pi for human ears. Further investigations are needed in order to explain the observed differences. Sound transmission in the ear decreases with an increasing static pressure difference across the tympanic membrane, especially at frequencies below 1 kHz, where pressure differences of 10 and 60 cm water cause mean transmission losses of about 10 and 26 dB, respectively, the losses being somewhat larger for overpressures than for underpressures in the ear canal. At higher frequencies, the transmission losses are smaller. For small overpressures, and in a limited frequency range near 3 kHz, even some transmission enhancement may occur. Static pressure variations in the inner ear have only a minor influence on sound transmission. Static pressures relative to the middle ear in the range 0-60 cm water cause mean sound transmission losses less than 5 dB below 1 kHz, and negligible losses at higher frequencies. PMID- 10738800 TI - Individual differences in external-ear transfer functions of cats. AB - Knowledge of the direction-dependent filter characteristics of the external ears is useful for the study of spatial hearing in experimental animals. The present study examined individual differences in the directional components of external ear transfer functions (directional transfer functions, DTFs) among 24 anesthetized cats. Ears were fixed in a frontal position. Inter-cat differences in DTFs were quantified across a mid-frequency range from 8 to 16 kHz and across 30 locations in the horizontal plane and vertical midline. Across cats, DTFs showed similar direction dependence, but tended to differ in regard to the center frequencies of spectral features, such as spectral peaks and notches. Certain mid frequency notches, for instance, varied in frequency across cats by nearly a factor of 2. Scaling of DTFs in frequency could reduce the overall differences between pairs of cats. Scale factors that minimized inter-cat differences ranged as high as 1.57 and correlated moderately with cats' body weights. Nevertheless, appreciable individual differences remained after frequency scaling. Inter-cat differences in DTFs were substantially larger than differences that resulted from variability in positioning the ears. The results suggest some guidelines regarding the conditions under which it is acceptable to apply DTF measurements from one cat to another. PMID- 10738801 TI - Influence of primary frequencies ratio on distortion product otoacoustic emissions amplitude. I. Intersubject variability and consequences on the DPOAE gram. AB - Distortion product otoacoustic emissions (DPOAEs) are used widely in humans to assess cochlear function. The standard procedure consists of recording the 2f1-f2 DPOAE amplitude as a function of the f2 frequency, using a fixed f2/f1 ratio (DPOAE-gram), close to 1.20. DPOAE amplitude, as recorded in the DPOAE-gram, shows a wide range of values in normal-hearing subjects, which can impair the predictive value of the DPOAE-gram for hearing thresholds. This study is aimed at comparing intersubject variability in 2f1-f2 DPOAE amplitude according to three paradigms: a fixed f2/f1 ratio, such as the DPOAE-gram, a variable ratio DPOAE gram (f2/f1 adapted to frequency) and an "optimum" DPOAE-gram, where the f2/f1 is adapted both to subject and frequency. The 2f1-f2 DPOAE amplitude has been investigated on 18 normally hearing subjects at ten different f2 frequencies (from 0.75 to 6 kHz), using an f2 fixed, f1 sweep paradigm, and allowed to define, for each frequency, the f2/f1 ratio giving the greatest 2f1-f2 DPOAE amplitude (or optimum ratio). Results showed a large intersubject variability of the optimum ratio, especially at frequencies below 1.5 kHz, and a significant decrease of the optimum ratio with frequency. The optimum DPOAE-gram was underestimated by up to 5.8 dB on average (up to 14.9 dB for an individual subject) by the fixed ratio DPOAE-gram, and by up to 3 dB on average (up to 10.6 dB for an individual subject) by the variable ratio DPOAE-gram. Intersubject variability was slightly but significantly reduced in the optimum DPOAE-gram versus the fixed-ratio DPOAE-gram. Lastly, correlations between tone-burst evoked otoacoustic emission (TBOAE) amplitudes and maximum DPOAE amplitudes were significantly greater than correlations between TBOAE amplitudes and fixed-ratio DPOAE amplitudes. PMID- 10738802 TI - Influence of primary frequencies ratio on distortion product otoacoustic emissions amplitude. II. Interrelations between multicomponent DPOAEs, tone-burst evoked OAEs, and spontaneous OAEs. AB - Distortion product otoacoustic emissions (DPOAEs) are used widely in humans to assess cochlear function. It is well known that 2f1-f2 DPOAE amplitude increases as the f2/f1 ratio increases from 1.0 to about 1.20, and then decreases as the f2/f1 ratio increases above 1.20, showing an amplitude ratio function, which is thought to be related to cochlear filtering properties. Different lower sideband DPOAEs are believed to show the same amplitude ratio functions as the 2f1-f2 DPOAE, with a magnitude peak situated at a constant DPOAE frequency relative to f2. More recently, several studies have suggested the involvement of a DPOAE component coming from its own distortion product place as well as the DPOAE component coming from the f2 place. To investigate DPOAE generation sites and the importance of the DPOAE frequency place, amplitude ratio functions of 2f1-f2, 3f1 2f2, 4f1-3f2 and 2f2-f1, 3f2-2f1, 4f2-3f1 DPOAE components have been systematically studied in 18 normally hearing subjects, using an f2 fixed, f1 sweep method, and an f1 fixed, f2 sweep method, at ten different f2 frequencies. Results show a dependency of the distortion magnitude peak on f2 frequency for each lower sideband DPOAE, and a small frequency shift of the distortion peak for the high order lower sideband DPOAE components. Strong correlation between the different lower sideband DPOAE amplitude were obtained, whether they were recorded with the same f1 (and a different f2) or with the same f2 (and a different f1), suggesting that lower side-band DPOAE amplitude does not depend on small variations in the f2 frequency. Moreover, correlations between DPOAE amplitude and tone-burst evoked otoacoustic emissions (TBOAEs) are highly significant for TBOAEs centered at the f2 frequency and at 1/2 octave below the f2 frequency, suggesting some degree of importance of the cochlear status at frequencies below f2 in DPOAE amplitude. Subjects presenting spontaneous otoacoustic emissions showed a greater lower sideband DPOAE amplitude recorded for low f2/f1 ratios, and a distortion magnitude peak shifted towards higher frequencies. The best correlation between upper sideband DPOAE amplitude and lower sideband DPOAE amplitude occurred for lower sideband DPOAEs generated by an f2 frequency 1/2 octave to 1 octave below the primaries used to generate upper sideband DPOAEs, suggesting a site of generation basal to f2 for the upper sideband DPOAEs. Correlations between TBOAE amplitude and upper sideband DPOAE amplitude agreed with a site of upper sideband DPOAE generation basal to f2, and which would move with the DPOAE frequency itself. PMID- 10738803 TI - The mechanical waveform of the basilar membrane. II. From data to models--and back. AB - Mechanical responses in the basal turn of the guinea-pig cochlea are measured with low-level broad-band noise as the acoustical stimulus [for details see de Boer and Nuttall, J. Acoust. Soc. Am. 101, 3583-3592 (1997)]. Results are interpreted within the framework of a classical three-dimensional model of the cochlea that belongs to a very wide class of nonlinear models. The use of linear systems analysis for this class of nonlinear models has been justified earlier [de Boer, Audit. Neurosci. 3, 377-388 (1997)]. The data are subjected to inverse analysis with the aim to recover the "effective basilar-membrane impedance." This is a parameter function that, when inserted into the model, produces a model response, the "resynthesized" response, that is similar to the measured response. With present-day solution methods, resynthesis leads back to an almost perfect replica of the original response in the spatial domain. It is demonstrated in this paper that this also applies to the response in the frequency domain and in the time domain. This paper further reports details with regard to geometrical properties of the model employed. Two three-dimensional models are studied; one has its dimensions close to that of the real cochlea, the other is a stylized model which has homogeneous geometry over its length. In spite of the geometric differences the recovered impedance functions are very similar. An impedance function computed for one model can be used in resynthesis of the response in the other one, and this leads to global amplitude deviations between original and resynthesized response functions not exceeding 8 dB. Discrepancies are much larger (particularly in the phase) when a two-dimensional model is compared with a three-dimensional model. It is concluded that a stylized three-dimensional model with homogeneous geometric parameters will give sufficient information in further work on unraveling cochlear function via inverse analysis. In all cases of a sensitive cochlea stimulated by a signal with a stimulus level of 50 dB SPL per octave or less, the resulting basilar-membrane impedance is found to be locally active, that is, the impedance function shows a region where the basilar membrane is able to amplify acoustic power or to reduce dissipation of power by the organ of Corti. Finally, the influence of deliberate errors added to the data is discussed in order to judge the accuracy of the results. PMID- 10738804 TI - The mechanical waveform of the basilar membrane. III. Intensity effects. AB - Mechanical responses in the basal turn of the guinea-pig cochlea were measured with broad-band noise stimuli and expressed as input-output cross-correlation functions. The experiments were performed over the full range of stimulus intensities in order to try to understand the influence of cochlear nonlinearity on frequency selectivity, tuning, signal compression and the impulse response. The results are interpreted within the framework of a nonlinear, locally active, three-dimensional model of the cochlea. The data have been subjected to inverse analysis in order to recover the basilar-membrane (BM) impedance, a parameter function that, when inserted into the (linearized version of that) model, produces a model response that is similar to the measured response. This paper reports details about intensity effects for noise stimulation, in particular, the way the BM impedance varies with stimulus intensity. In terms of the underlying cochlear model, the decrease of the "activity component" in the BM impedance with increasing stimulus level is attributed to saturation of transduction in the outer hair cells. In the present paper this property is brought into a quantitative form. According to the theory [the EQ-NL theorem, de Boer, Audit. Neurosci. 3, 377-388 (1997)], the BM impedance is composed of two components, both intrinsically independent of stimulus level. One is the passive impedance Zpass and the other one is the "extra" impedance Zextra. The latter impedance is to be multiplied by a real factor gamma (0 < or = gamma < or = 1) that depends on stimulus level. This concept about the composition of the BM impedance is termed the "two-component theory of the BM impedance." In this work both impedances are entirely derived from experimental data. The dependence of the factor gamma on stimulus level can be derived by using a unified form of the outer-hair-cell transducer function. From an individual experiment, the two functions Zpass and Zextra are determined, and an approximation (Zpass + gamma Zextra) to the BM impedance constructed. Next, the model response (the "resynthesized" response) corresponding to this "artificial" impedance is computed. The same procedure is executed for several stimulus-level values. For all levels, the results show a close correspondence with the original experimental data; this includes correct prediction of the compression of response amplitudes, the reduction of frequency selectivity, the shift in peak frequency and, most importantly, the preservation of timing in the impulse response. All these findings illustrate the predictive power of the underlying model. PMID- 10738805 TI - The dynamic range of inner hair cell and organ of Corti responses. AB - Inner hair cell (IHC) and organ of Corti (OC) responses are measured from the apical three turns of the guinea pig cochlea, allowing access to regions with best, or most sensitive, frequencies at approximately 250, 1000, and 4000 Hz. In addition to measuring both ac and dc receptor potentials, the average value of the half-wave rectified response (AVEHR) is computed to better reflect the signal that induces transmitter release. This measure facilitates comparisons with single-unit responses in the auditory nerve. Although IHC ac responses exhibit compressive growth, response magnitudes at high levels depend on stimulus frequency. For example, IHCs with moderate and high best frequencies (BF) exhibit more linear responses below the BF of the cell, where higher sound-pressure levels are required to approach saturation. Because a similar frequency dependence is observed in extracellular OC responses, this phenomenon may originate in cochlear mechanics. At the most apical recording location, however, the pattern documented at the base of the cochlea is not seen in IHCs with low BFs around 250 Hz. In fact, more linear behavior is measured above the BF of the cell. These frequency-dependent features require modification of cochlear models that do not provide for longitudinal variations and generally depend on a single stage of saturation located at the synapse. Finally, behavior of dc and AVEHR responses suggests that a single IHC is capable of coding intensity over a large dynamic range [Patuzzi and Sellick, J. Acoust. Soc. Am. 74, 1734-1741 (1983); Smith et al., in Hearing--Physiological Bases and Psychophysics (Springer, Berlin, 1983); Smith, in Auditory Function (Wiley, New York, 1988)] and that information compiled over wide areas along the cochlear partition is not essential for loudness perception, consistent with psychophysical results [Viemeister, Hearing Res. 34, 267-274 (1988)]. PMID- 10738806 TI - Mathematical modeling of vowel perception by users of analog multichannel cochlear implants: temporal and channel-amplitude cues. AB - A "multidimensional phoneme identification" (MPI) model is proposed to account for vowel perception by cochlear implant users. A multidimensional extension of the Durlach-Braida model of intensity perception, this model incorporates an internal noise model and a decision model to account separately for errors due to poor sensitivity and response bias. The MPI model provides a complete quantitative description of how listeners encode and combine acoustic cues, and how they use this information to determine which sound they heard. Thus, it allows for testing specific hypotheses about phoneme identification in a very stringent fashion. As an example of the model's application, vowel identification matrices obtained with synthetic speech stimuli (including "conflicting cue" conditions [Dorman et al., J. Acoust. Soc. Am. 92, 3428-3432 (1992)] were examined. The listeners were users of the "compressed-analog" stimulation strategy, which filters the speech spectrum into four partly overlapping frequency bands and delivers each signal to one of four electrodes in the cochlea. It was found that a simple model incorporating one temporal cue (i.e., an acoustic cue based only on the time waveforms delivered to the most basal channel) and spectral cues (based on the distribution of amplitudes among channels) can be quite successful in explaining listener responses. The new approach represented by the MPI model may be used to obtain useful insights about speech perception by cochlear implant users in particular, and by all kinds of listeners in general. PMID- 10738807 TI - Auditory brainstem responses with optimized chirp signals compensating basilar membrane dispersion. AB - This study examines auditory brainstem responses (ABR) elicited by rising frequency chirps. The time course of frequency change for the chirp theoretically produces simultaneous displacement maxima by compensating for travel-time differences along the cochlear partition. This broadband chirp was derived on the basis of a linear cochlea model [de Boer, "Auditory physics. Physical principles in hearing theory I," Phys. Rep. 62, 87-174 (1980)]. Responses elicited by the broadband chirp show a larger wave-V amplitude than do click-evoked responses for most stimulation levels tested. This result is in contrast to the general hypothesis that the ABR is an electrophysiological event most effectively evoked by the onset or offset of an acoustic stimulus, and unaffected by further stimulation. The use of this rising frequency chirp enables the inclusion of activity from lower frequency regions, whereas with a click, synchrony is decreased in accordance with decreasing traveling velocity in the apical region. The use of a temporally reversed (falling) chirp leads to a further decrease in synchrony as reflected in ABR responses that are smaller than those from a click. These results are compatible with earlier experimental results from recordings of compound action potentials (CAP) [Shore and Nuttall, "High synchrony compound action potentials evoked by rising frequency-swept tonebursts," J. Acoust. Soc. Am. 78, 1286-1295 (1985)] reflecting activity at the level of the auditory nerve. Since the ABR components considered here presumably reflect neural response from the brainstem, the effect of an optimized synchronization at the peripheral level can also be observed at the brainstem level. The rising chirp may therefore be of clinical use in assessing the integrity of the entire peripheral organ and not just its basal end. PMID- 10738808 TI - Studies of interaural attenuation to investigate the validity of a dichotic difference tone response recorded from the inferior colliculus in the chinchilla. AB - In a previous paper (Arnold and Burkard, 1998) a dichotic f2-f1 difference tone (DT) auditory evoked potential from the chinchilla inferior colliculus (IC) was measured while presenting f1 (2000 Hz) to one ear and f2 (2100 Hz) to the other ear. This measurement paradigm could be used as a means to study binaural processing in an unanesthetized animal model. However, it is possible that this response is actually generated peripherally, as a result of acoustic crossover. The purpose of the present set of experiments was to investigate whether the dichotic DT is a true binaural phenomenon. Recordings were made from chronically implanted IC electrodes in unanesthetized, monaural chinchillas (left cochlea destroyed). In experiment 1, interaural attenuation (IA) was measured in two ways. First, IA was measured by comparing IC evoked potential thresholds obtained when stimulating the normal right ear and the dead left ear, using tone bursts (0.5-8 kHz). Mean values of interaural attenuation ranged from 50-65 dB across frequency (55 dB at 2000 Hz). Next, the DT was measured monaurally using f1 = 2000 and f2 = 2100 (L1 = L2). By comparing the mean DT input/output functions for monaural stimulation of the right and left ears, a mean value of IA for the tonal pair was estimated (approximately 69 dB). In experiment 2, the DT was measured with right monaural stimulation, while varying the relative levels of the primaries. A small DT could be seen with primary levels up to 30 dB apart, but not for greater level differences. Differences substantially greater than 30 dB would be expected in the crossover situation based upon IA. In experiment 3, the stimuli were presented dichotically (f1 to right ear, f2 to left ear and vice versa, L1 = L2) to determine whether acoustic crosstalk to the normal right ear would generate a DT. No DT was reliably observed in this condition. Taken together, these results suggest that the dichotic DT is a true binaural phenomenon, and not simply attributable to acoustic crossover. PMID- 10738809 TI - Acoustic mechanisms that determine the ear-canal sound pressures generated by earphones. AB - In clinical measurements of hearing sensitivity, a given earphone is assumed to produce essentially the same sound-pressure level in all ears. However, recent measurements [Voss et al., Ear and Hearing (in press)] show that with some middle ear pathologies, ear-canal sound pressures can deviate by as much as 35 dB from the normal-ear value; the deviations depend on the earphone, the middle-ear pathology, and frequency. These pressure variations cause errors in the results of hearing tests. Models developed here identify acoustic mechanisms that cause pressure variations in certain pathological conditions. The models combine measurement-based Thevenin equivalents for insert and supra-aural earphones with lumped-element models for both the normal ear and ears with pathologies that alter the ear's impedance (mastoid bowl, tympanostomy tube, tympanic-membrane perforation, and a "high-impedance" ear). Comparison of the earphones' Thevenin impedances to the ear's input impedance with these middle-ear conditions shows that neither class of earphone acts as an ideal pressure source; with some middle ear pathologies, the ear's input impedance deviates substantially from normal and thereby causes abnormal ear-canal pressure levels. In general, for the three conditions that make the ear's impedance magnitude lower than normal, the model predicts a reduced ear-canal pressure (as much as 35 dB), with a greater pressure reduction with an insert earphone than with a supra-aural earphone. In contrast, the model predicts that ear-canal pressure levels increase only a few dB when the ear has an increased impedance magnitude; the compliance of the air-space between the tympanic membrane and the earphone determines an upper limit on the effect of the middle-ear's impedance increase. Acoustic leaks at the earphone-to-ear connection can also cause uncontrolled pressure variations during hearing tests. From measurements at the supra-aural earphone-to-ear connection, we conclude that it is unusual for the connection between the earphone cushion and the pinna to seal effectively for frequencies below 250 Hz. The models developed here explain the measured pressure variations with several pathologic ears. Understanding these mechanisms should inform the design of more accurate audiometric systems which might include a microphone that monitors the ear-canal pressure and corrects deviations from normal. PMID- 10738810 TI - Separate mechanisms govern the selection of spectral components for perceptual fusion and for the computation of global pitch. AB - The perceptual fusion of harmonics is often assumed to result from the operation of a template mechanism that is also responsible for computing global pitch. This dual-role hypothesis was tested using frequency-shifted complexes. These sounds are inharmonic, but preserve a regular pattern of equal component spacing. The stimuli had a nominal fundamental (F0) frequency of 200 Hz (+/- 20%), and were frequency shifted either by 25.0% or 37.5% of F0. Three consecutive components (6 8) were removed and replaced with a sinusoidal probe, located at one of a set of positions spanning the gap. On any trial, subjects heard a complex tone followed by an adjustable pure tone in a continuous loop. Subjects were well able to match the pitch of the probe unless it corresponded with a position predicted by the spectral pattern of the complex. Peripheral factors could not account for this finding. In contrast, hit rates were not depressed for probes positioned at integer multiples of the F0(s) corresponding to the global pitch(es) of the complex, predicted from previous data [Patterson, J. Acoust. Soc. Am. 53, 1565 1572 (1973)]. These findings suggest that separate central mechanisms are responsible for computing global pitch and for the perceptual grouping of partials. PMID- 10738811 TI - The perceptual tone/noise ratio of merged iterated rippled noises. AB - Iterated rippled noise (IRN) is constructed by delaying a random noise by d ms, adding it back to the same noise, and repeating the process iteratively. When two IRNs with the same power but slightly different delays are added together, the perceptual tone/noise ratio of the "merged" IRN is markedly reduced with respect to that of either of the component IRNs. In this paper, the reduction in the perceptual tone/noise ratio is measured for IRNs in which one of the delays is always 16 ms and the other is either 16 +/- 0.1 ms or 16 +/- 1.1 ms. The component IRNs have the same number of iterations, and the number varies across conditions from 4 to 256. The perceptual tone/noise ratio is measured using a discrimination matching procedure developed for single IRNs; each merged IRN is compared with a range of "standard" stimuli having varying proportions of a complex tone and a broadband noise [Patterson et al., J. Acoust. Soc. Am. 100, 3286-3294 (1996)]. For single IRNs, the function relating the signal-to-noise ratio of the matching standard to the number of iterations in the IRN was found to be essentially straight. This relationship was explained in terms of the height of the first peak in the autocorrelation of the stimulus wave, or by the first peak in the summary autocorrelogram produced by a time-domain auditory model. For the merged IRNs in the current experiment, the matching-point functions are found to have pronounced downward curvature, in addition to being well below the function for single IRNs. To account for the reduction in the perceptual tone/noise ratio of merged IRNs, the autocorrelation model was extended to include a simple rule for combining adjacent peaks in the autocorrelation function of the wave, and the autocorrelogram model was revised to improve the simulation of the "loss of phase locking" at higher frequencies in the autocorrelogram. PMID- 10738812 TI - Effects of ipsilateral and contralateral precursors on the temporal effect in simultaneous masking with pure tones. AB - In tone-on-tone masking, thresholds often decrease as the onset of the signal is delayed relative to the onset of the masker, especially when the frequency of the masker is higher than the frequency of the signal. This temporal effect was studied here by using a tonal "precursor," whose offset preceded the onset of the tonal masker (and signal). Under the right conditions, the precursor can reduce or eliminate the temporal effect by decreasing the threshold for a signal at masker onset, presumably for the same reason that the threshold decreases as a signal is delayed relative to the onset of a masker. In the present study, the frequency of the signal was 4000 Hz, and the frequency of the masker and precursor was typically 5000 Hz. In experiment 1, the precursor was presented to the ear receiving the masker and signal (ipsilateral precursor); in experiment 2, it was presented to the opposite ear (contralateral precursor). The results from experiment 1 can be summarized as follows: the ipsilateral precursor (a) reaches its maximum effectiveness (in reducing the temporal effect) for precursor durations of 200-400 ms; (b) is ineffective once the delay between its offset and the onset of the masker reaches about 50-100 ms; (c) is generally ineffective when its level is 10 or more dB lower than the level of the masker, but is effective when its level is equal to or greater than the level of the masker; and (d) becomes progressively less effective as its frequency is either increased or decreased relative to the frequency of the masker. The results from experiment 2 can be summarized simply by stating that the contralateral precursor is ineffective in reducing the temporal effect. These results suggest that the effect of the precursor may be mediated peripherally. PMID- 10738813 TI - Detection of quasitrapezoidal frequency and amplitude modulation. AB - It has been proposed that the detection of frequency modulation (FM) of sinusoidal carriers can be mediated by two mechanisms; a place mechanism based on FM-induced amplitude modulation (AM) in the excitation pattern, and a temporal mechanism based on phase locking in the auditory nerve. The temporal mechanism appears to be "sluggish" and does not play a role for FM rates above about 10 Hz. It also does not play a role for high carrier frequencies (above about 5 kHz). This experiment provided a further test of the hypothesis that the effectiveness of the temporal mechanism depends upon the time spent close to frequency extremes during the modulation cycle. Psychometric functions for the detection of AM and FM were measured for two carrier frequencies, 1 and 6 kHz. The modulation waveform was quasitrapezoidal. Within each modulation period, P, a time Tss was spent at each extreme of frequency or amplitude. The transitions between the extremes, with duration Ttrans had the form of a half-cycle of a cosine function. The modulation rate was 2, 5, 10, or 20 Hz, giving values of P of 500, 200, 100, and 50 ms. TSS varied from 0 ms (sinusoidal modulation) up to 160, 80, 40, or 20 ms, for rates of 2, 5, 10, and 20 Hz, respectively. The detectability of AM was not greatly affected by modulation rate or by the value of TSS, except for a slight improvement with increasing TSS for the lowest modulation rates; this was true for both carrier frequencies. For FM of the 6-kHz carrier, the pattern of results was similar to that found for AM, which is consistent with an excitation pattern model of FM detection. For FM of the 1-kHz carrier, performance improved markedly with increasing TSS, especially for the lower FM rates; there was no change in performance with TSS for the 20-Hz modulation rate. The results are consistent with the idea that detection of FM of a 1-kHz carrier is partly mediated by a sluggish temporal mechanism. That mechanism benefits from greater time spent at frequency extremes of the modulation cycle for rates up to 10 Hz. PMID- 10738814 TI - Level discrimination of sinusoids as a function of duration and level for fixed level, roving-level, and across-frequency conditions. AB - The ability of listeners to detect level differences between two sinusoidal stimuli in a two-interval forced-choice procedure was measured as a function of duration and level in three conditions: (1) the pedestal was fixed in level and the stimuli in the two intervals had the same frequency of either 1 or 2 kHz (fixed-level condition); (2) the pedestal was roved in level over a 20-dB range from trial to trial, but the stimuli still had the same frequency of either 1 or 2 kHz (roving-level condition); and (3) the pedestal was roved in level over a 20 dB range and the two stimuli differed in frequency, such that one was around 1 kHz while the other was around 2 kHz (across-frequency condition). In the fixed level conditions, difference limens decreased (improved) with both increasing duration and level, as found in previous studies. In the roving-level conditions, difference limens increased and the dependence on duration and level decreased. Difference limens in the across-frequency conditions were generally highest and showed very little dependence on either stimulus duration or level. The results may be understood in terms of different internal noise components with additive variances: In the fixed-level conditions, sensation noise, which is dependent on stimulus attributes such as duration and level, is dominant. In more difficult conditions, where trace-memory and/or across-channel comparisons are required, a more central, stimulus-independent noise dominates. PMID- 10738816 TI - Tori of confusion: binaural localization cues for sources within reach of a listener. AB - To a first-order approximation, binaural localization cues are ambiguous: many source locations give rise to nearly the same interaural differences. For sources more than a meter away, binaural localization cues are approximately equal for any source on a cone centered on the interaural axis (i.e., the well-known "cone of confusion"). The current paper analyzes simple geometric approximations of a head to gain insight into localization performance for nearby sources. If the head is treated as a rigid, perfect sphere, interaural intensity differences (IIDs) can be broken down into two main components. One component depends on the head shadow and is constant along the cone of confusion (and covaries with the interaural time difference, or ITD). The other component depends only on the relative path lengths from the source to the two ears and is roughly constant for a sphere centered on the interaural axis. This second factor is large enough to be perceptible only when sources are within one or two meters of the listener. Results are not dramatically different if one assumes that the ears are separated by 160 deg along the surface of the sphere (rather than diametrically opposite one another). Thus for nearby sources, binaural information should allow listeners to locate sources within a volume around a circle centered on the interaural axis on a "torus of confusion." The volume of the torus of confusion increases as the source approaches the median plane, degenerating to a volume around the median plane in the limit. PMID- 10738815 TI - Relationships among age-related differences in gap detection and word recognition. AB - The relationships among age-related differences in gap detection and word recognition in subjects with normal hearing or mild sensorineural hearing loss were explored in two studies. In the first study, gap thresholds were obtained for 40 younger and 40 older subjects. The gaps were carried by 150-ms, modulated, low-pass noise bursts with cutoff frequencies of 1 or 6 kHz. The noise bursts were presented at an overall level of 80 dB SPL in three background conditions. Mean gap thresholds ranged between 2.6 and 7.8 ms for the younger age group and between 3.4 and 10.0 ms for the older group. Mean gap thresholds were significantly larger for the older group in all six conditions. Gap thresholds were not significantly correlated with audiometric thresholds in either age group but the 1-kHz gap thresholds increased with age in the younger group. In the second study, the relationships among gap thresholds, spondee-in-babble thresholds, and audiometric thresholds of 66 subjects were examined. Compared with the older subjects, the younger group recognized the spondees at significantly lower (more difficult) spondee-to-babble ratios. In the younger group, spondee-in-babble thresholds were significantly correlated with gap thresholds in conditions of high-frequency masking. In the older group, spondee in-babble thresholds, gap thresholds, and audiometric thresholds were not significantly correlated, but the spondee-in-babble thresholds and two audiometric thresholds increased significantly with age. These results demonstrate that significant age-related changes in auditory processing occur throughout adulthood. Specifically, age-related changes in temporal acuity may begin decades earlier than age-related changes in word recognition. PMID- 10738817 TI - Effects of phase duration and electrode separation on loudness growth in cochlear implant listeners. AB - Loudness estimates were obtained in a group of four adult subjects implanted with the Nucleus-22 multielectrode cochlear implant device, for a range of pulse amplitudes and different fixed phase durations and electrode separations. The stimulus was a 200-ms long train of biphasic pulses presented at 500 pulses/s. Subjects estimated loudness as a number from 0 ("don't hear it") to 100 ("uncomfortably loud"). Loudness was found to grow exponentially with pulse amplitude, at a rate that was dependent upon the phase duration as well as the electrode separation. An equation of the form L = e(lambda + gamma M)(D theta)I, where L is the estimated loudness, M is the separation between electrodes of a stimulating pair, D is the phase duration, I is current amplitude, and lambda, gamma, and theta are constants, appears to describe the observed data adequately. The findings are remarkably consistent across subjects. PMID- 10738818 TI - Place-pitch sensitivity and its relation to consonant recognition by cochlear implant listeners using the MPEAK and SPEAK speech processing strategies. AB - Two related studies investigated the relationship between place-pitch sensitivity and consonant recognition in cochlear implant listeners using the Nucleus MPEAK and SPEAK speech processing strategies. Average place-pitch sensitivity across the electrode array was evaluated as a function of electrode separation, using a psychophysical electrode pitch-ranking task. Consonant recognition was assessed by analyzing error matrices obtained with a standard consonant confusion procedure to obtain relative transmitted information (RTI) measures for three features: stimulus (RTI stim), envelope (RTI env[plc]), and place-of-articulation (RTI plc[env]). The first experiment evaluated consonant recognition performance with MPEAK and SPEAK in the same subjects. Subjects were experienced users of the MPEAK strategy who used the SPEAK strategy on a daily basis for one month and were tested with both processors. It was hypothesized that subjects with good place-pitch sensitivity would demonstrate better consonant place-cue perception with SPEAK than with MPEAK, by virtue of their ability to make use of SPEAK's enhanced representation of spectral speech cues. Surprisingly, all but one subject demonstrated poor consonant place-cue performance with both MPEAK and SPEAK even though most subjects demonstrated good or excellent place-pitch sensitivity. Consistent with this, no systematic relationship between place-pitch sensitivity and consonant place-cue performance was observed. Subjects' poor place-cue perception with SPEAK was subsequently attributed to the relatively short period of experience that they were given with the SPEAK strategy. The second study reexamined the relationship between place-pitch sensitivity and consonant recognition in a group of experienced SPEAK users. For these subjects, a positive relationship was observed between place-pitch sensitivity and consonant place-cue performance, supporting the hypothesis that good place-pitch sensitivity facilitates subjects' use of spectral cues to consonant identity. A strong, linear relationship was also observed between measures of envelope- and place-cue extraction, with place-cue performance increasing as a constant proportion (approximately 0.8) of envelope-cue performance. To the extent that the envelope-cue measure reflects subjects' abilities to resolve amplitude fluctuations in the speech envelope, this finding suggests that both envelope- and place-cue perception depend strongly on subjects' envelope-processing abilities. Related to this, the data suggest that good place-cue perception depends both on envelope-processing abilities and place-pitch sensitivity, and that either factor may limit place-cue perception in a given cochlear implant listener. Data from both experiments indicate that subjects with small electric dynamic ranges (< 8 dB for 125-Hz, 205-microsecond/ph pulse trains) are more likely to demonstrate poor electrode pitch-ranking skills and poor consonant recognition performance than subjects with larger electric dynamic ranges. PMID- 10738819 TI - A self-learning predictive model of articulator movements during speech production. AB - A model is presented which predicts the movements of flesh points on the tongue, lips, and jaw during speech production, from time-aligned phonetic strings. Starting from a database of x-ray articulator trajectories, means and variances of articulator positions and curvatures at the midpoints of phonemes are extracted from the data set. During prediction, the amount of articulatory effort required in a particular phonetic context is estimated from the relative local curvature of the articulator trajectory concerned. Correlations between position and curvature are used to directly predict variations from mean articulator positions due to coarticulatory effects. Use of the explicit coarticulation model yields a significant increase in articulatory modeling accuracy with respect to x ray traces, as compared with the use of mean articulator positions alone. PMID- 10738820 TI - Method for the selection of sentence materials for efficient measurement of the speech reception threshold. AB - A method is described to select sentence materials for efficient measurement of the speech reception threshold (SRT). The first part of the paper addresses the creation of the sentence materials, the recording procedure, and a listening experiment to evaluate the new speech materials. The result is a set of 1272 sentences, where every sentence has been uttered by two male and two female speakers. In the second part of the paper, a method is described to select subsets with properties that are desired for an efficient measurement of the SRT. For two speakers, this method has been applied to obtain two subsets for measurement of the SRT in stationary noise with the long-term average spectrum of speech. Lastly, a listening experiment has been conducted where the two subsets (each comprising 39 lists of 13 sentences each) are directly compared to the existing sets of Plomp and Mimpen [Audiology 18, 43-52 (1979)] and Smoorenburg [J. Acoust. Soc. Am. 91, 421-437 (1992)]. One of the outcomes is that the newly developed sets can be considered as equivalent to these existing sets. PMID- 10738821 TI - Measuring the threshold for speech reception by adaptive variation of the signal bandwidth. II. Hearing-impaired listeners. AB - In a previous study [Noordhoek et al., J. Acoust. Soc. Am. 105, 2895-2902 (1999)], an adaptive test was developed to determine the speech-reception bandwidth threshold (SRBT), i.e., the width of a speech band around 1 kHz required for a 50% intelligibility score. In this test, the band-filtered speech is presented in complementary bandstop-filtered noise. In the present study, the performance of 34 hearing-impaired listeners was measured on this SRBT test and on more common SRT (speech-reception threshold) tests, namely the SRT in quiet, the standard SRT in noise (standard speech spectrum), and the spectrally adapted SRT in noise (fitted to the individual's dynamic range). The aim was to investigate to what extent the performance on these tests could be explained simply from audibility, as estimated with the SII (speech intelligibility index) model, or require the assumption of suprathreshold deficits. For most listeners, an elevated SRT in quiet or an elevated standard SRT in noise could be explained on the basis of audibility. For the spectrally adapted SRT in noise, and especially for the SRBT, the data of most listeners could not be explained from audibility, suggesting that the effects of suprathreshold deficits may be present. Possibly, such a deficit is an increased downward spread of masking. PMID- 10738822 TI - Analysis and synthesis of intonation using the Tilt model. AB - This paper introduces the Tilt intonational model and describes how this model can be used to automatically analyze and synthesize intonation. In the model, intonation is represented as a linear sequence of events, which can be pitch accents or boundary tones. Each event is characterized by continuous parameters representing amplitude, duration, and tilt (a measure of the shape of the event). The paper describes an event detector, in effect an intonational recognition system, which produces a transcription of an utterance's intonation. The features and parameters of the event detector are discussed and performance figures are shown on a variety of read and spontaneous speaker independent conversational speech databases. Given the event locations, algorithms are described which produce an automatic analysis of each event in terms of the Tilt parameters. Synthesis algorithms are also presented which generate F0 contours from Tilt representations. The accuracy of these is shown by comparing synthetic F0 contours to real F0 contours. The paper concludes with an extensive discussion on linguistic representations of intonation and gives evidence that the Tilt model goes a long way to satisfying the desired goals of such a representation in that it has the right number of degrees of freedom to be able to describe and synthesize intonation accurately. PMID- 10738823 TI - Rheo-acoustical study of the shear disruption of reversible aggregates. Ultrasound scattering from concentrated suspensions of red cell aggregates. AB - Shear-induced disruption of reversible aggregates or clusters in a concentrated suspension is investigated by ultrasound backscattering in the low shear regime. Fractal aggregates are considered as non-Brownian scatterers much smaller than the wavelength with acoustic properties close to those of the surrounding liquid, so that the attenuation of the coherent field is weak and multiple scattering can be neglected. The concept of variance in local particle volume fraction is used to deduce a first-order expression of the ultrasound scattering cross section per unit volume for Rayleigh scatterers in a dense suspension. On the basis of a scaling law for the shear-induced disruption of aggregates, the shear stress dependence of the ultrasonic scattered intensity from a dense suspension of clusters is derived. In a second part, the shear breakup of hardened red blood cell aggregates is investigated in plane-plane flow geometry by ultrasound scattering. Rheo-acoustical experiments are analyzed within the framework of the self-consistent field approximation and the scaling laws currently used in microrheological models. Finally, the ability of ultrasonic, light reflectometry and viscometry methods to provide quantitative information about red blood cell aggregation and membrane adhesiveness is discussed. PMID- 10738824 TI - Ultrasound sensitivity in the cricket, Eunemobius carolinus (Gryllidae, Nemobiinae). AB - Extracellular recordings from the cervical connectives in both long- and short winged E. carolinus reveal auditory units that are sensitive to frequencies > 15 kHz with best sensitivity at 35 kHz (79 dB SPL threshold). Stimuli in this frequency range also elicit a startle response in long-winged individuals flying on a tether. For single-pulse stimuli, startle and neck connective thresholds decrease with increasing ultrasound duration, consistent with the operation of an exponential integrator with a approximately 32.5-ms time constant. There is evidence for adaptation to long duration pulses (> 20 ms) in the neck connectives, however, as it is more difficult to elicit responses to the later stimuli of a series. For paired-pulse stimuli consisting of 1-ms pulses of 40 kHz, temporal integration was demonstrated for pulse separations < 5 ms. For longer pulse separations, startle thresholds were elevated by 3 dB and appear to be optimally combined. Startle thresholds to 5 ms frequency modulated (FM) sweeps (60-30 kHz) and pure tone pulses (40 kHz) did not differ. The characteristics and sensitivity of this ultrasound-induced startle response did not differ between males and females. As in some other tympanate insects, ultrasound sensitivity in E. carolinus presumably functions in the context of predation from echolocating bats. PMID- 10738825 TI - Masking by harmonic complexes in budgerigars (Melopsittacus undulatus). AB - In humans, masking by harmonic complexes is dependent not only on the frequency content of the masker, but also its phase spectrum. Complexes that have highly modulated temporal waveforms due to the selection of their component phases usually provide less masking than those with flatter temporal envelopes. Moreover, harmonic complexes that are created with negative Schroeder phases (component phases monotonically decreasing with increasing harmonic frequency) may provide more masking than those created with positive Schroeder phases (monotonically increasing phase), even though both temporal envelopes are equally flat. To date, there has been little comparative work on the masking effectiveness of harmonic complexes. Using operant conditioning and the method of constant stimuli, masking of pure tones by harmonic complexes was examined in budgerigars at several different masker levels for complexes constructed with two different fundamental frequencies. In contrast to humans, thresholds in budgerigars differed very little for the two Schroeder-phase waveforms. Moreover, when there was a difference in masking by these two waveforms, the positive Schroeder was the more effective masker--the reverse of that described for humans. Control experiments showed that phase selection was relevant to the masking ability of harmonic complexes in budgerigars. Release from masking occurred when the components were in coherent phase, compared with a complex with random phases selected for each component. It is suggested that these psychoacoustic differences may emerge from structural and functional differences between the avian and mammalian peripheral auditory systems involving traveling wave mechanics and spectral tuning characteristics. PMID- 10738826 TI - A dual passive cavitation detector for localized detection of lithotripsy-induced cavitation in vitro. AB - A passive cavitation detector (PCD) identifies cavitation events by sensing acoustic emissions generated by the collapse of bubbles. In this work, a dual passive cavitation detector (dual PCD), consisting of a pair of orthogonal confocal receivers, is described for use in shock wave lithotripsy. Cavitation events are detected by both receivers and can be localized to within 5 mm by the nature of the small intersecting volume of the focal areas of the two receivers in association with a coincidence detection algorithm. A calibration technique, based on the impulse response of the transducer, was employed to estimate radiated pressures at collapse near the bubble. Results are presented for the in vitro cavitation fields of both a clinical and a research electrohydraulic lithotripter. The measured lifetime of the primary growth-and-collapse of the cavitation bubbles increased from 180 to 420 microseconds as the power setting was increased from 12 to 24 kV. The measured lifetime compared well with calculations based on the Gilmore-Akulichev formulation for bubble dynamics. The radiated acoustic pressure 10 mm from the collapsing cavitation bubble was measured to vary from 4 to 16 MPa with increasing power setting; although the trends agreed with calculations, the predicted values were four times larger than measured values. The axial length of the cavitation field correlated well with the 6-dB region of the acoustic field. However, the width of the cavitation field (10 mm) was significantly narrower than the acoustic field (25 mm) as bubbles appeared to be drawn to the acoustic axis during the collapse. The dual PCD also detected signals from "rebounds," secondary and tertiary growth-and-collapse cycles. The measured rebound time did not agree with calculations from the single bubble model. The rebounds could be fitted to a Rayleigh collapse model by considering the entire bubble cloud as an effective single bubble. The results from the dual PCD agreed well with images from high-speed photography. The results indicate that single-bubble theory is sufficient to model lithotripsy cavitation dynamics up to time of the main collapse, but that upon collapse bubble cloud dynamics becomes important. PMID- 10738828 TI - Comments on "Ultrasonic flow metering based on transit time differentials which are insensitive to flow profile" PMID- 10738827 TI - Ultrasonic diffraction by a square periodic array of spheres PMID- 10738829 TI - Range estimation and depth discrimination of multiple sources in noisy shallow waters without knowing sound speed profiles PMID- 10738830 TI - Power law absorption in polymers and other systems PMID- 10738832 TI - Surface wave velocity determination by using reflection coefficient method on the liquid-anisotropic-solid interface PMID- 10738831 TI - The radial motion of a sonoluminescence bubble driven with multiple harmonics PMID- 10738833 TI - Flexural edge waves and comments on "A new bending wave solution for the classical plate equation" PMID- 10738834 TI - On the effect of the bending vibration on the acoustic properties of thin poroelastic plates PMID- 10738835 TI - Tinnitus and 2f1-f2 distortion product otoacoustic emissions following salicylate overdose. PMID- 10738836 TI - Limitations in using Golay codes for head-related transfer function measurement. PMID- 10738837 TI - Shape and extinction thresholds in sonoluminescence parameter space PMID- 10738838 TI - Preliminary FoodNet data on the incidence of foodborne illnesses--selected sites, United States, 1999. AB - Each year in the United States, an estimated 76 million persons experience foodborne illnesses. CDC's Emerging Infections Program Foodborne Diseases Active Surveillance Network (FoodNet) collects data on nine foodborne diseases in selected U.S. sites to quantify and monitor foodborne illnesses. This report describes preliminary surveillance data for 1999 and compares them with data from 1996-1998. The data suggest that the incidence of the foodborne illnesses under surveillance declined during 1999 compared with 1996 primarily as a result of decreases in campylobacteriosis and shigellosis and indicate substantial regional variation in the incidence of foodborne diseases. PMID- 10738839 TI - Hantavirus pulmonary syndrome--Panama, 1999-2000. AB - Hantavirus pulmonary syndrome (HPS) is an acute viral rodentborne zoonosis characterized by severe cardiopulmonary illness with a 40%-60% case-fatality rate. Since its identification in the United States in 1993, the recognized clinical spectrum of illnesses associated with human hantavirus infection has expanded to include mild illness, and case-patients have been identified in Canada and South America. This report describes the first confirmed HPS cases from Central America and summarizes preliminary results of clinical, epidemiologic, and ecologic investigations. Investigators identified 12 suspected cases with typical disease and captured four common species of rodents near case households. PMID- 10738840 TI - Outbreaks of Norwalk-like viral gastroenteritis--Alaska and Wisconsin, 1999. AB - Norwalk-like viruses (NLVs) are the most common cause of epidemic gastroenteritis in the United States, resulting in illness in approximately 23 million persons each year . Persons of all ages are affected because previous infection confers only short-term immunity. Most NLV gastroenteritis outbreaks involve foodborne or person-to-person transmission. This report presents investigations of a foodborne NLV outbreak in Alaska and person-to-person transmission in Wisconsin. PMID- 10738841 TI - Assessing the safety of drugs in pregnancy: the role of prospective cohort studies. AB - Since, for obvious reasons, systematic testing of the teratogenic properties of drugs in humans is not possible in the premarketing phase, the epidemiological approaches to postmarketing risk evaluation are of major importance. Cohort studies, with their prospective exposure assessment, their ability to study even exposure to drugs not commonly used in pregnancy, and their ability to monitor both adverse and beneficial fetal outcomes, seem to be the most promising study type from a methodological viewpoint. Although there are numerous cohort studies on the harmful effects of drug use in pregnant women, only a few have been able to demonstrate a risk in terms of an increase in the prevalence of malformations. Most studies with significant findings were those investigating the risk potential of one group of drugs, the anticonvulsants. The lack of cohort studies showing a risk for drug use in pregnancy, however, is not necessarily indicative of some methodological deficiency. Rather, it may suggest that, for the majority of drugs, their use in pregnancy is not associated with an increased risk of congenital malformations. PMID- 10738842 TI - A risk-benefit analysis of methadone maintenance treatment. AB - Methadone maintenance treatment for heroin (diamorphine) addiction has been extensively researched. There is consistent evidence that while in treatment, heroin addicts are at a lower risk of death, are less involved in crime, and feel and function better than while using heroin. Despite the research evidence supporting methadone treatment, there remains widespread public scepticism about this form of treatment. This scepticism is frequently expressed in terms of the perceived risks of methadone treatment. The perceived risk that methadone treatment may maintain people in an addicted lifestyle is not supported by research literature. The risks of treatment include an increased risk of death during induction into treatment, and risks of diversion of drugs to the black market. For some patients, adverse effects of methadone pose a problem and the availability of new pharmacotherapies may provide useful options for these patients. Risks can be reduced and benefits increased by directing greater attention to the quality of treatment. PMID- 10738843 TI - Carbapenems in serious infections: a risk-benefit assessment. AB - The tolerability of the 2 most frequently used carbapenems, imipenem/cilastatin and meropenem, is reviewed. Both of these drugs, but especially imipenem, are potentially neurotoxic and may cause seizures if overdosed relative to renal function and/or bodyweight. The therapeutic margin is considerably narrower with imipenem/cilastatin which cannot be given at doses required for treatment of bacterial meningitis. Meropenem on the other hand, is considerably less prone to cause seizures and its tolerability and efficacy are well documented in 3 relatively large, controlled studies in adults and children with meningitis. They showed that meropenem was as effective and well tolerated as cefotaxime or ceftriaxone. Another potential advantage of meropenem over imipenem/cilastatin is that it can be given intravenously at a high rate without increased risk of nausea or vomiting. An obvious reason for using a carbapenem instead of a cephalosporin for empirical treatment of life-threatening infections is that both imipenem/cilastatin and meropenem have a broader spectrum of activity. They are also more resistant to hydrolysis by the most common beta-lactamases, including the class I cephalosporinase frequently produced by Enterobacter spp. and Pseudomonas spp. and the extended spectrum enzymes, now commonly found in Escherichia coli and Klebsiella spp. PMID- 10738844 TI - Tolerability of atypical antipsychotics. AB - Atypical antipsychotics are expected to be better tolerated than older antipsychotics because of their lower propensity to cause certain adverse effects. All atypical drugs have been shown to cause fewer acute extrapyramidal symptoms (EPS) than a standard typical agent (usually haloperidol) and some (clozapine, sertindole and quetiapine) appear to cause these effects no more often than placebo. In the longer term, clozapine, olanzapine and (less robustly) other atypical antipsychotics are thought to cause less tardive dyskinesia than typical antipsychotics. Problems caused by hyperprolactinaemia occur less often with some atypical antipsychotics than with typical drugs although risperidone and amisulpride appear to have no advantages in this respect. Other adverse effects may occur as frequently with some atypical antipsychotics as with some typical drugs. Clozapine, risperidone and quetiapine are known to cause postural hypotension; clozapine, olanzapine and quetiapine are clearly sedative; and anticholinergic effects are commonly seen with clozapine, and, much less frequently, with olanzapine. Some adverse effects are more frequent with atypical drugs. Idiosyncratic effects seem particularly troublesome with clozapine and, to a lesser extent, sertindole, olanzapine and zotepine. Bodyweight gain is probably more problematic with atypical antipsychotics than with typical drugs. Overall tolerability, as judged by withdrawals from therapy, is not clearly proven to be better with atypical drugs, although some individual trials do indicate an advantage with atypical agents. Differences in tolerability between individual atypical antipsychotics have not been clearly shown. The tolerability profile of atypical drugs certainly benefits from a lower incidence of acute EPS effects, along with less certain or less uniform benefits in symptomatic hyperprolactinaemia or tardive dyskinesia. Other, perhaps more trivial, adverse effects militate against their good tolerability, and effects such as bodyweight gain may severely reduce tolerability. Without clear advantages in tolerability in patient groups used in trials, drug choice in regard to adverse effects should continue to be on a patient to patient basis. PMID- 10738846 TI - Trimethoprim-induced hyperkalaemia: clinical data, mechanism, prevention and management. AB - Cotrimoxazole (trimethoprim-sulfamethoxazole) is a combination antimicrobial that is frequently used to treat a wide variety of infections. Only recently has hyperkalaemia been recognised as a relatively common complication of therapy with trimethoprim. Hyperkalaemia has been demonstrated to occur with the administration of both high and standard dosages of trimethoprim. The recognition of this disorder of potassium homeostasis prompted the investigation and ultimate description of the mechanism by which trimethoprim causes hyperkalaemia. Trimethoprim was found to reduce renal potassium excretion through the competitive inhibition of epithelial sodium channels in the distal nephron, in a manner identical to the potassium-sparing diuretic amiloride. Increased risk for hyperkalaemia with trimethoprim treatment appears to be related to both higher dosages and underlying renal impairment. It is probable that other disturbances in potassium homeostasis, such as hyopoaldosteronism and treatment with medications that impair renal potassium excretion, are also risk factors for hyperkalaemia with trimethoprim therapy. Prevention of this adverse reaction depends upon recognition of patients at risk of developing hyperkalaemia as well as proper dosage selection of trimethoprim for the patient's prevailing glomerular filtration rate. Management of hyperkalaemia often mandates discontinuation of the drug, volume repletion with isotonic fluids, and other therapies specific to hyperkalaemia. In circumstances where continued treatment with trimethoprim is required, induction of high urinary flow rates with intravenous fluids and a loop diuretic, as well as alkalinisation of the urine, have been shown to block the antikaliuretic effect of trimethoprim on distal nephron cells. PMID- 10738845 TI - Drug-induced aseptic meningitis: diagnosis and management. AB - Drug-induced aseptic meningitis (DIAM) has been reported as an uncommon adverse reaction with numerous agents. It is a diagnosis of exclusion, and clinical signs and CSF findings vary greatly. The body of evidence regarding DIAM is largely in the form of anecdotal case reports and must be interpreted carefully bearing this in mind. The major categories of causative agents are nonsteroidal anti inflammatory drugs, antimicrobials, intravenous immunoglobulin, intrathecal agents, vaccines and a number of other less frequently reported agents. There appears to be an association between DIAM and connective tissue disease, particularly systemic lupus erythematosus, and ibuprofen. There are 2 major proposed mechanisms for DIAM. The first involves direct irritation of the meninges by intrathecal administration of the drug, and the second involves immunological hypersensitivity to the drug, most likely type III and type IV hypersensitivity. Recognition and diagnosis of DIAM is important, as it is treatable by withdrawal of the drug and recurrence is prevented. The outcome of DIAM is generally good, usually without long term sequelae. This article describes the case reports of DIAM in the current literature and discusses the diagnosis and management of this rare complication. PMID- 10738849 TI - Geriatric rehabilitation revisited. AB - There is no gold standard for rehabilitation of geriatric patients. Although many studies indicate that a multifaceted team approach, such as seen with specialized stroke units, may enhance outcomes for many patients, how these units achieve this is still unclear. However, the evidence shows that a comprehensive program of both medical and psychosocial support tends to result in patients achieving and maintaining higher levels of function, with less need for readmission, and shorter hospital stays. In addition to interventions aimed at improving function, rehabilitation includes assessment, goal setting, and provision of care to maintain the patient's status. More randomized controlled studies of rehabilitation programs are needed so that we can identify which programs are best for which patients. PMID- 10738848 TI - Are vascular factors involved in Alzheimer's disease? Facts and theories. AB - The hypothesis that vascular factors may contribute to the development of Alzheimer's disease (AD) is supported by epidemiologic and pathologic observations. Arterial hypertension and diabetes have been found to be associated not only with vascular dementia, but also with AD; in addition, the treatment of hypertension with calcium antagonists seems to prevent degenerative dementias. Hypertension and hyperinsulinemia favor the deposition of amyloid substance in the brain. The histopathology of AD is marked not only by neurofibrillary tangles and senile plaques, but also by macro and micro congophilic angiopathy and ischemic white matter rarefaction. The specific AD pathological lesions, if isolated, are not able to lead to an evident clinical picture of dementia, which, on the contrary, becomes evident when vascular, mainly subcortical, lesions are associated. These and other observations suggest that vascular factors may have a role in the development of AD. An aggressive approach to these factors could be of value in the prevention of AD. PMID- 10738847 TI - Drug-induced oesophageal disorders: pathogenesis, incidence, prevention and management. AB - Drug-induced injury of the oesophagus is a common cause of oesophageal complaints. 'Pill-induced' oesophagitis is associated with the ingestion of certain drugs and accounts for many cases of erosive oesophagitis. To date, more than 70 drugs have been reported to induce oesophageal disorders. Antibacterials such as doxycycline, tetracycline and clindamycin are the offending agents in more than 50% of cases. Other commonly prescribed drugs that cause oesophageal injury include aspirin (acetylsalicylic acid), potassium chloride, ferrous sulfate, quinidine, alprenolol and various steroidal and nonsteroidal anti inflammatory agents. However, many physicians and even more patients are not aware of this problem. Capsules or tablets are commonly delayed in their passage through the oesophagus. Highly caustic coatings, direct medication injury and poor oesophageal clearance of pills can lead to acute inflammation. Oesophageal damage occurs when the caustic contents of a drug remain in the oesophagus long enough to produce mucosal lesions. Taking medications at bedtime or without fluids is a common cause of oesophagitis. The possibility of drug-related damage should be suspected in all cases of oesophagitis, chest pain and dysphagia. History and gastrointestinal endoscopy will confirm the diagnosis. Treatment is supportive, although acid reduction is used frequently as an adjunct. This review reflects the current state of knowledge in this field. PMID- 10738850 TI - Circulating thyroid autoantibodies in a sample of Italian octo-nonagenarians: relationship to age, sex, disability, and lipid profile. AB - Circulating thyroid autoantibodies were assessed in a sample of Italian octo nonagenarians living in a restricted area (Val Vibrata, Abruzzo), and selected according to the absence or presence of chronic illness and disability. The study groups included: Group A ("Successful aging"), 98 free-living healthy, fully independent, octo-nonagenarians (57 males and 41 females, average age +/-SD 83.9+/-3 years); Group B ("Unsuccessful aging"), 62 highly disabled octo nonagenarians (24 males and 38 females; average age 86.5+/-3.4 years), and Group C (Controls), 91 randomly selected healthy adult controls (42 males and 49 females; age 53.7+/-16.6 SD years, range 20-70). Serum autoantibodies to thyroglobulin (anti-Tg) and to thyroid peroxidase (anti-TPO) were measured by passive hemagglutination and radioimmunoassays (RIA); serum free thyroxine and thyrotropin by RIA. The prevalence of positive thyroid autoantibody tests was not significantly increased in the elderly groups compared to the controls, with the remarkable exception of anti-Tg detected by RIA which were increased in disabled elderly, compared to free-living elderly and controls. Two/62 disabled elderly had subclinical (1) or mild (1) primary hypothyroidism, and one free-living woman aged 90 years was hyperthyroid (toxic nodular goiter). No correlation was found between serum thyroid autoantibodies, thyroid hormones and TSH and serum lipoprotein fractions. The higher prevalence of thyroid autoantibodies found only in disabled, but not in free-living Italian octo-nonagenarians suggests that thyroid autoimmune phenomena in the elderly may be an expression of age associated disease rather than related to the aging process in itself. PMID- 10738851 TI - The validation of the short form of the Geriatric Depression Scale (GDS) in Greece. AB - The Geriatric Depression Scale-15 (GDS-15) is a short, 15-item instrument specifically designed to assess depression in geriatric populations. Its items require a yes/no response. The Geriatric Depression Scale was first introduced by Yesavage et al. in 1983, and the short form (GDS-15) was developed by Sheikh and Yesavage in 1986. The aim of the current study was the standardization of the GDS 15 for use in Greece. Subjects were divided into Group A: 168 control subjects, and Group B: 103 patients suffering from clinically diagnosed depression. All were over 65 years of age. A score of 6/7 on the GDS-15 was found to be the best cut-off point for diagnosing depression in an elderly Greek population, with Sensitivity = 92.23 and Specificity = 95.24. GDS-15 manifests high internal consistency with Cronbach's alpha = 0.94, and all items seem to be equivalent. Factor Analysis of the GDS-15 revealed 4 factors: a cognitive (thought content), an affective, a functional, and a factor that reflects helplessness and fear for the future. The two diagnostic groups differed on all 4 factors scores at p-value <0.001. PMID- 10738852 TI - Influence of acute and chronic exercise on insulin-like growth factor-I in healthy active elderly men and women. AB - We examined the influence of physical activity (PA) and acute resistance exercise on insulin-like growth factor I (IGF-I) in 39 healthy, independent, community dwelling elderly volunteers (14 men and 25 women) aged from 66 to 84. In a longitudinal non-interventional study, we compared the fluctuations in IGF-I levels and changes in habitual PA over a 6-month period. PA was evaluated by a questionnaire QAPSE (Questionnaire d'Activite Physique Saint-Etienne), and expressed by two activity indices: mean habitual daily energy expenditure (MHDEE), and daily energy expenditure corresponding to leisure time sports activities (sports activity index). In an experimental design we compared IGF-I levels before, at 1 and 15 minutes after short quadriceps muscle exertion (10 consecutive repetitions) carried out with optimal muscle power development. Changes in IGF-I levels over a 6-month period were positively related to changes in MHDEE (r = 0.41; p<0.01), and sports activity (r = 0.40; p<0.02). When analyzed by gender, these correlations were still statistically significant in women (r = 0.39; p = 0.05 and r = 0.41; p<0.05), but not in men (r = 0.36; p = 0.21 and r = 0.20; p = 0.50). No changes (p = 0.17) were observed in serum IGF-I concentrations after the acute exercise protocol. These findings indicate that IGF-I levels are positively related to fluctuations in habitual PA, at least in women. Further research is needed to establish the best intensity and duration of exercise training to induce these changes. PMID- 10738853 TI - A role for leptin in the antiaging action of dietary restriction: a hypothesis. AB - A neuroendocrine signal may play an important role in the antiaging action of dietary restriction (DR). Recent studies have suggested that falling leptin levels by starvation activate the hypothalamic-pituitary-adrenal axis, and suppress gonadal, somatotropic, and thyroid axes as a response for adaptation. Accumulated evidence indicates that similar hormonal changes also occur in DR rodents. In this article, we advance that a reduction in plasma leptin levels in DR rodents might be a critical neuroendocrine modulator in the antiaging action of dietary restriction. PMID- 10738854 TI - Calorie restriction modulates Th-1 and Th-2 cytokine-induced immunoglobulin secretion in young and old C57BL/6 cultured submandibular glands. AB - Immunoglobulin production by the salivary gland plays an important role in oral and upper respiratory tract immunity. Age and/or disease may compromise salivary gland function. In order to gain insight into the role of calorie restriction (CR) on immunoglobulin (Ig) production, we determined the effect of ad libitum (AL) feeding and CR in young (3 months) and old (18-24 months) C57BL/6 mouse submandibular glands (SM). The SM tissues were fragmented and cultured in the absence (control) or presence of either Th-1 cytokines, such as interleukin-2 (IL 2) and interferon-gamma (IFN-gamma), or Th-2 cytokines, e.g. IL-4 and IL-5, for seven days. Culture supernatants were then analyzed for immunoglobulin A (IgA), IgM, and IgG2a levels by ELISA. Aging increased basal (control) IgA and IgM production by 3.1-and 3.7-fold, respectively, in AL mice. CR prevented the age dependent rise of both IgA and IgM, maintaining levels equal to those of young AL mice. Interestingly, age resulted in a decrease of Th-1 cytokine-induced IgA and IgM, and increased IgG2a secretion in AL mice, while Th-2 cytokines did not appear to have an age effect. In general, CR suppressed Ig production induced by both Th-1 and Th-2 cytokines in young mice. In contrast, CR in old mice resulted in enhanced IgA and IgM production to levels similar to those in their young counterparts, while IgG2a was predominantly suppressed by Th-1 and not Th-2 cytokines. The data presented herein show, for the first time, the ability of CR to offset age-induced changes in submandibular gland Ig production, which may play a role in maintaining mucosal immune function, including proper oral health. PMID- 10738855 TI - The effect of different diagnostic criteria on the prevalence of malnutrition in a hospitalized geriatric population. AB - Malnutrition is considered an important clinical problem in geriatric hospitalized patients, but no standard diagnostic criteria are available. The aim of this study was to investigate the effect of 7 different sets of diagnostic criteria (6 in hospitalized elderly and 1 in healthy elderly) from nutritional research studies on the prevalence of malnutrition in 151 elderly patients (49 men, 102 women, mean age 82.8 years) consecutively admitted to an acute geriatric ward. Nutritional status was assessed using anthropometric (body mass index, triceps skinfold thickness, mid arm circumference, arm muscle circumference), and laboratory (serum albumin) parameters, and 1 nutritional questionnaire (Mini Nutritional Assessment). The proportion of patients with malnutrition varied between 6.5 and 85%. It is important to assess the nutritional status in elderly patients because the prevalence of malnutrition in hospitalized elderly patients is substantial whatever criteria are used. However, the criteria for the diagnosis can differ by a factor of more than 10 in the number of patients classified as malnourished. A major goal for further research on malnutrition is to define more standardized and validated criteria in order to make comparisons of prevalence and outcome more meaningful, and ensure that all those who will benefit from a nutritional intervention are identified and treated. PMID- 10738856 TI - Mood and general behavior of patients with severe dementia during one year of supervised, individualized planned care and systematic clinical supervision. Comparison with a similar control group. AB - The aim of this study was to evaluate the effects of one year of systematic clinical supervision, and supervised individually planned care on the mood and general behavior of patients with dementia in relation to their cognitive function and level of confusion. The intervention was carried out in a ward devoted to the care of patients with severe dementia (EW), with a similar ward (CW) where no changes were made serving as a control. Each ward housed 11 patients, of whom 7 patients in each ward survived throughout the study time. Assessment of the patients' mood, general behavior, cognitive functions (MMSE), orientation and confusion (OBS) was made at baseline, and after 6 and 12 months of intervention. Significant deteriorations for the patients in CW were seen during the study period as regards the factor strength, functional performance and orientation in the ward, and speech performance and psychomotor slowing. For the patients in the EW, deterioration was seen only in the factor accessibility, and improvement was seen in the factor sensitivity and euphoria. The differences indicated that the development in the two wards went in opposite directions to the benefit of the patients in the EW. Because of the small sample size, the findings are more the result of a pilot trial, rather than generalizable. The intervention, however, seemed to have a positive effect on the secondary symptoms of the disease. PMID- 10738857 TI - Microembolic signals and clinical outcome in patients with acute stroke--a prospective study. AB - The occurrence of microembolic signals (MES) in patients with transient ischemic attack (TIA) or stroke has already been described; the influence of the time interval between onset of symptoms and transcranial Doppler monitoring (TCD) on the MES rate or MES prevalence and the possible prognostic value of the early detected MES rate on the outcome of TIA or stroke symptoms in a 3 month interval are discussed. In a prospective study we evaluated 61 patients consecutively admitted to our stroke unit after their first ischemic neurological deficit involving the vascular territory of MCA and/or ACA. All of the patients underwent a 30-minute bilateral transcranial Doppler monitoring of their MCAs for the identification of MES. Monitoring was performed within 12.3 + -9.3 (average mean + -SD) hours of stroke onset for the first time, the second time 48 hours after first TCD monitoring. Prognosis for the recovery of neurological deficits was evaluated by using the Barthel index (BI) and Scandinavian Stroke Scale (SSS) at the time of admission of the patient to the stroke unit, and with Barthel indices after one month and after 3 months. As a result, 56% of all patients showed MES in at least one of the two registrations. MES were recorded not only on the symptomatic side. The MES prevalence between both TCD monitorings was significantly different (total MES prevalence: 1st TCD: 26 patients: 2nd TCD: 13 patients; p < 0.04; ipsilateral MES prevalence: 1st TCD: 19 patients; 2nd TCD: 9 patients; p < 0.01). The regression analysis showed a significant influence of the total MES rate on both neurological scores at admission (SSS: 0.03; Barthel index: 0.04), but not for the Barthel scores after one and three months. In conclusion, we found an influence of the time interval between onset of neurological symptoms of TIA or stroke on the MES rate and the prevalence of MES. The prevalence of MES or the MES rate, found after a short time interval to the onset of symptoms, did not have a prognostic value on the outcome of neurological deficits up to a three month follow-up. PMID- 10738858 TI - Prolactin-secreting pituitary adenoma in neuroleptic treated patients with psychotic disorder. AB - Three patients with psychoses and concomitant prolactin-secreting pituitary tumours are described. Patients A and B had bipolar and schizoaffective disorders, respectively. They had both been treated with neuroleptics for 20 years before the prolactinomas were revealed. Patient C developed a paranoid psychosis after two years of continuous bromocriptine treatment for a pituitary tumour. In patient A the prolactin level was successfully normalized and a good antipsychotic effect was maintained by combined therapy with haloperidol and quinagolide but not bromocriptine. In patient B the prolactinoma was removed by surgery, in view of the serious nature of the psychotic disorder, to avoid psychotic relapse by treatment with a dopamine agonist. In patient C a good result was obtained with the combination of clozapine and bromocriptine. These case reports support the view that neuroleptics being dopamine antagonists and dopamine agonistic agents which are the primary treatment of prolactinomas can cancel out each other's effects. The combination of clozapine and quinagolide is recommended as the treatment of choice for most patients. PMID- 10738859 TI - Basal activity of the hypothalamic-pituitary-adrenal axis and cognitive function in anorexia nervosa. AB - Anorexia nervosa is associated with abnormalities in neuroendocrine function including sustained hypercortisolism, which has been shown elsewhere to be associated with impairment of function in learning, memory and attention. Cognitive impairment has also been observed in anorexia nervosa. These effects may be mediated in part through cortisol effects on the hippocampus, which is dense with glucocorticoid receptors. We investigated the association between cortisol levels and cognitive function in anorexia nervosa by measuring both 24 hour urinary cortisol counts and performance on tasks of learning, memory and attention in patients suffering from the disorder. Cortisol secretion was shown to be significantly higher in the patient group than in a matched control group and patients were also shown to be impaired in memory and attention. However, no correlations were found between the cognitive deficits and cortisol measures. It is suggested that more sensitive profiling of cortisol levels throughout the circadian cycle may be useful in future studies of cognitive function in anorexia nervosa. PMID- 10738860 TI - White blood cells and cortisol after sleep deprivation and recovery sleep in humans. AB - Sleep deprivation (SD) has enriched our treatment programme for major depression. SD has been demonstrated to modify different host defence activities. There is some evidence that there are reciprocal relationships between immune function and increased hypothalamic-pituitary-adrenocortical (HPA) axis activity in depression. We therefore investigated the number of leukocytes, granulocytes, monocytes, lymphocytes, B cells, T cells, helper T cells, cytotoxic T cells, NK cells and salivary cortisol in 10 healthy men before and after total SD (TSD) as well as after recovery sleep. Blood samples were drawn on 3 consecutive days at 7 am, 1 pm and 7 pm, respectively. Comparison of the 7 am values by contrast analysis yielded significant differences for granulocytes (p = 0.044) and NK cells (p = 0.001) after SD and recovery sleep. NK cells decreased and granulocytes increased after SD and after recovery sleep. Significant differences between single points in time across the day were found for granulocytes (p = 0.022), monocytes (p = 0.031), T cells (p = 0.005), helper T cells (p = 0.004), cytotoxic T cells (p = 0.005) and NK cells (p = 0.017). No significant difference could be detected for leukocytes, lymphocytes and B cells counts. These results favour the thesis that SD and recovery sleep lead to changes in the distribution of peripheral leukocytes, especially in a reduction of NK cells after SD and recovery sleep. The cortisol rhythm was affected neither by SD nor recovery sleep. PMID- 10738861 TI - Differential sustained attention/vigilance changes over time in schizophrenics and controls during a degraded stimulus Continuous Performance Test. AB - The Continuous Performance Test (CPT) is a widely used procedure for sustained attention/vigilance measurement. However, though the key index of vigilance impairment is the decrement of sensitivity over time during the test period, only few studies have examined whether schizophrenics show a larger drop in CPT performance than do healthy controls. 48 schizophrenic inpatients and 48 controls were investigated with the Munich CPT (480 visual stimuli, 25% target stimuli, one stimulus per second). Stimuli were degraded by randomly inverting 40%, 41%, 42%, or 43% of the pixels. Results were calculated separately for three consecutive trial sections. Additionally, PANSS ratings, medication, and other clinical data were documented. Schizophrenics show a vigilance decrement over time, controls show a vigilance increase. Differential vigilance changes were not related to the level of stimulus degradation. Schizophrenics performed worse than controls only at the lowest degradation level. While overall sensitivity correlated negatively with the dose of atypical neuroleptics and benzodiazepines, vigilance shifts over time correlated negatively with the dose of typical neuroleptics. Furthermore, sensitivity was related to the cognitive PANSS syndrome, number of admissions/duration of illness. Differential sensitivity decrements of schizophrenics and controls can be shown if suited CPT procedures are used. The need for basic research on experimental conditions of the CPT as well as examination of the relationship between sustained attention/vigilance decrements and clinical features of schizophrenia is suggested. PMID- 10738862 TI - Dopamine D3 receptor variant and tardive dyskinesia. AB - In the search for genetic factors contributing to tardive dyskinesia, dopamine receptor genes are considered major candidates. The dopamine D3 receptor is of primary interest as dopamine D3 receptor knock-out mice show locomotor hyperactivation resembling extrapyramidal side-effects of neuroleptic treatment. Furthermore, Steen and colleagues (1997) recently reported an association between tardive dyskinesia and a dopamine D3 receptor gene variant. In the present study we tried to replicate this finding. We investigated 157 patients with schizophrenia or schizoaffective disorder receiving long-term neuroleptic medication who never or persistently displayed tardive dyskinesia. As advanced age is a main risk factor for tardive dyskinesia, we also compared older patients with a long duration of schizophrenia not displaying tardive dyskinesia to younger patients with a shorter duration of the illness displaying tardive dyskinesia. However, we found no evidence that the dopamine D3 receptor gene is likely to confer susceptibility to the development of tardive dyskinesia. PMID- 10738863 TI - Lactate-stress testing in 54 patients with mitochondriopathy. AB - Since there are only few data available about the lactate stress test in a group of patients with mitochondrial myopathy, we investigated the sensitivity of this test in a larger cohort of such patients. Serum lactate was determined before, during and after a 15 minute, constant 30 W workload on a bicycle ergometer in 47 controls, aged 15 to 72 years and 54 patients with mitochondrial myopathy, aged 15 to 74 years. Lactate's upper reference limits at rest, 5, 10, 15 minutes after starting, and 15 minutes after finishing the exercise were 2.0, 2.1, 2.1, 2.1 and 1.8 mmol/l respectively. The sensitivity of the lactate-stress test was 69%. The lactate-stress test complements electrophysiological and bioptical findings and proved to be helpful in diagnosing mitochondrial myopathy. PMID- 10738864 TI - Temperament and character during the course of unipolar depression among inpatients. AB - Cloninger has developed a novel approach concerning relationships between psychopathological syndromes and personality by his biosocial theory. Increased levels of harm avoidance (HA) were consistently found in unipolar disorders. The present study was conducted to cross-validate, in part, previous findings that high harm avoidance (HA) persisted in the course of disorder and to explore the distinct role of character dimensions. One hundred and twenty-six inpatients with an unipolar depressive disorder and 126 healthy controls, strictly matched for age and gender have been included in the study. Our findings underline that higher harm avoidance among unipolar depressives compared to healthy controls persisted after treatment even if a significant reduction could be observed. Recurrent disorders and comorbidity with anxiety disorders seem to be related to a relatively immature character in terms of consistently lower scores for the character dimensions (e.g. self-directedness and cooperativeness) of the patients classified into these groups both at admission and at discharge compared with their healthy counterparts. PMID- 10738865 TI - Comparison between eye movement latency and REM sleep parameters in major depression. AB - Alterations of sleep can be observed polysomnographically in approximately 90 percent of depressed patients. Most of the registered sleep abnormalities in depression also occur in other psychiatric disorders. Only some types of REM sleep alterations-- short REM latency, increase of REM density and shortening of mean latency of eye movements--were reported as more specific for affective disorders. In the present study polysomnograms of 21 medication free patients with major depressive disorder (assessed with a structured interview for DSM-III R and Hamilton Scale) and 21 healthy controls were analysed. REM latency (LREM), REM density (RD), latencies of eye movements (LEM) and mean latency of eye movements (M-LEM) were calculated for both groups. Depressed patients (compared with healthy controls) showed increased RD (38.2% vs. 28.2%, p < 0.0001), shortened M-LEM (35.7 s vs. 48.3 s, p < 0.04) and shortening of LEM in the 1st (28.9 s vs. 48.9 s, p < 0.007) and 4th (27.0 s vs. 59.1 s, p < 0.043) REM sleep periods. LREM was not shortened significantly in depressives (78.5 min vs. 91.3 min, ns). In healthy subjects a negative correlation between M-LEM and RD was found (rho = -0.47, p < 0.03). Since in the current study depressed patients differed from healthy controls, especially concerning phasic activity during REM sleep, presented data support the essential role of REM density for the assessment of sleep in depression. As a quick and easy manner to compute measurement, M-LEM is suggested as additional parameter for the assessment of phasic activity during REM sleep. PMID- 10738866 TI - Depression with DSM-IV atypical features: a marker for bipolar II disorder. AB - The aim of the study was to find the prevalence of atypical features in bipolar II depression versus unipolar depression. Five hundred and fifty seven unipolar and bipolar II depressed outpatients were interviewed with the Structured Clinical Interview for DSM-IV, the Montgomery Asberg Depression Rating Scale, and the Global Assessment of Functioning Scale. DSM-IV atypical features were significantly more common in bipolar II patients than in unipolar patients (45.4% vs 25.4%, odds ratio 2.4). As the diagnosis of bipolar II disorder is often based on diagnosis of past hypomania, which may not be very reliable. depression with atypical features may point to bipolar II disorder diagnosis. PMID- 10738867 TI - Repair of articular cartilage defects with cultured chondrocytes in Atelocollagen gel. Comparison with cultured chondrocytes in suspension. AB - We attempted to repair full-thickness articular cartilage defects in rabbit knee joints with allogeneic cultured chondrocytes embedded in Atelocollagen gel. An articular cartilage defect was created on the patellar groove of the femur. The defect was filled with chondrocytes cultured in the collagen gel and covered with periosteal flap (G group). In three other experimental groups, the same defects were transplanted with chondrocytes in monolayer culture with periosteal flap (M group), periosteal graft only (P group), or left empty (E group). At 4, 12, and 24 weeks after operation, the reparative tissue was analyzed macroscopically and histologically. At 4 weeks after operation, the surfaces of the reparative tissue were smooth, and the defects were filled with reparative tissues that resembled hyaline cartilage in all four groups. However, the reparative tissues degenerated gradually with time in the M, P, and E groups. In contrast, in the G group, the reparative tissue retained its thickness, and there was a steady integration of the grafted tissue into the adjacent normal cartilage at 24 weeks after operation. The results suggest that transplantation of allogeneic chondrocytes cultured in Atelocollagen gel is effective in repairing an articular cartilage defect. PMID- 10738868 TI - Anterior cruciate ligament augmentation under arthroscopy. A minimum 2-year follow-up in 40 patients. AB - We present a new surgical technique of anterior cruciate ligament (ACL) augmentation using autogenous hamstring tendons or allogenic fascia lata under arthroscopy for patients in whom considerable ACL remnants with mechanoreceptors are preserved. We review the clinical results of 40 patients who underwent this ACL augmentation, as assessed by manual instability tests, KT-2000 knee arthrometer, Lysholm and Gillquist score, joint position sense and magnetic resonance imaging (MRI), and compare them with those of ACL reconstruction. The side-to-side differences of anterior displacement measured by the KT-2000 knee arthrometer at 30 lbs, an average of 5.3 +/- 2.6 mm preoperatively, was significantly improved to an average of 0.7 +/- 1.8 mm in the augmentation group, while an average of 6.0 +/- 2.4 mm was improved to 1.8 +/- 2.1 mm in the reconstruction group. There was no statistically significant difference between the preoperative values in the two groups, but the postoperative values of the augmentation group were significantly less than those of the reconstruction group. The ratio of anterior terminal stiffness of the augmentation group was 1.2 +/- 1.8, and that of the ACL reconstruction group was 0.7 +/- 0.4. This was a statistically significant difference (P < 0.05). Thus, as for the KT-2000 knee arthrometer, the ACL augmentation group showed significantly better anteroposterior stability and terminal stiffness than the ACL reconstruction group. The final inaccuracy of joint position sense of the augmentation group was 0.7 degrees +/- 0.7 degrees, while that of the reconstruction group was 1.7 degrees +/- 1.2 degrees indicating a significant difference (P < 0.05). This study demonstrates that ACL augmentation, which can preserve ACL remnants with mechanoreceptors, is superior to ACL reconstruction from the viewpoint of position sense and joint stability. PMID- 10738869 TI - Scaphoid nonunion treated by open reduction, anterior inlay bone grafting, and Kirschner-wire fixation. AB - Forty-three patients with ununited fractures of the scaphoid were treated by open reduction, anterior inlay bone grafting and Kirschner wire fixation. All but one achieved bone union. The one failure was successfully treated with a second anterior inlay bone grafting procedure. The average period of cast immobilization was 7 weeks. The presence of an unstable nonunion did not adversely affect the results. In cases with carpal instability, we used a longer corticocancellous graft to restore a more normal alignment of the scaphoid by opening up the volar cortex. Preoperatively, carpal instability was present in 25 patients. In these cases, carpal alignment demonstrated by the radiolunate and scapholunate angle was improved significantly after surgery: the radiolunate angle from 15 degrees before surgery to 2 degrees after surgery, and the scapholunate angle from 67 degrees to 54 degrees, respectively. Pain, restricted motion of the wrist, and the grip strength were improved after surgery. PMID- 10738870 TI - Hemangioma of skeletal muscle. AB - Intramuscular hemangiomas are rare benign tumors, making up 0.8% of all hemangiomas. They are of interest to the surgeon because their location may present considerable therapeutic challenge since radiographic work-up of the soft tissue mass by magnetic resonance imaging (MRI) may be suspicious for malignancy. The definitive diagnosis is made by histological study of the surgical and/or biopsy specimen. Patients with intramuscular hemangiomas may have soft-tissue complaints, such as pain and swelling, present for years. The gross and microscopic appearance of intramuscular hemangiomas is variable. Grossly, the capillary type is nonvascular and spongy in appearance, whereas the cavernous type is composed of large, thin-walled, dilated vessels lined by flattened endothelial cells. In general, wide excision is the treatment of choice to prevent local recurrence, but every patient with intramuscular hemangioma should be treated individually after evaluating the tumor location, accessibility, and depth of invasion, the patient's age, and cosmetic considerations. From October 1, 1989, to June 30, 1997, 11 patients underwent surgical treatment with the definitive histological diagnosis of intramuscular hemangioma. Pain upon activity but also at rest as well as swelling were the major symptoms. The average duration of symptoms was 13 months (range 1 month to 5 years). After a mean follow- up of 3 years and 4 months (range 12 months to 9 years), one of the patients has developed a recurrence; all remaining patients enjoy pain relief without any recurrence. PMID- 10738871 TI - Subtrochanteric corrective osteotomy for the endoprosthetic treatment of high hip dislocation. Treatment and mid-term results with a cementless straight stem. AB - Total hip arthroplasty is problematical in the case of high dislocation. To reposition the hip, a femoral shortening osteotomy is necessary in order to prevent damage to the neurovascular structures. This paper describes the implantation of a cementless straight stem in 15 patients using a simple technique with a simultaneous, derotating and shortening osteotomy. Femur fractures, pseudoarthroses, stem loosening, paresis and deep infections were not found. One socket loosening was revised. Fourteen patients had good or very good results in the Merle d'Aubigne score after a median of 4.3 (range 2-5.6) years of follow-up. The surgical technique described enables the initial stable fixation of a standard prosthesis without additional osteosynthesis. Additional advantages include a shorter duration of surgery, a lower complication rate and a more rapid consolidation of the osteotomy. PMID- 10738872 TI - Surgical treatment of muscular torticollis for patients above 6 years of age. AB - Eighteen patients with congenital muscular torticollis, aged 6-22 (average 11) years, underwent surgical releases of the contractive bands. There were 8 boys and 10 girls. Preoperative open mouth radiograph of the odontoid process in 16 patients showed asymmetry of articular facets of the axis and tilt of the odontoid process to the side of the torticollis. Sixteen patients received bipolar releases, the others received distal releases. After the operation, a neck collar was applied and an exercise program was started. At an average follow up of 5 years and 7 months, there were excellent or good results in 10 patients. All of the patients except 2 showed improvement of the facial asymmetry and all except 1 had a satisfactory range of motion of the neck. One patient underwent a second operation due to recurrence. The follow-up radiographs showed improvement of the tilt of the odontoid process, but the asymmetry of the articular facets of the axis persisted. We suggest that bipolar release is the treatment of choice for congenital muscular torticollis in patients of more than 6 years of age. To determine the influence of a bipolar release on the functions of the cervical spine, longer follow-up intervals are needed. PMID- 10738874 TI - The incidence of thrombosis in high tibial osteotomies with and without the use of a tourniquet. AB - In a prospective randomised study, 65 high tibial osteotomies were performed in cases of varus osteoarthritis of the knee, and the incidence of thrombosis with and without the use of a tourniquet was studied. With an average incidence of thrombosis of 10.8%, no statistically significant differences between these two groups were seen. PMID- 10738873 TI - Tibial shaft malunion treated with reamed intramedullary nailing: a revised technique. AB - Thirty-seven consecutive adult tibial shaft malunions which had not undergone surgical treatment were prospectively treated with reamed intramedullary nailing. Indications for this treatment modality included a malunion of a tibial shaft which had only been conservatively treated, lesion level fitting for traditional or locked reamed intramedullary nail fixation, less than 2 cm shortening, and without evidence of deep infection at present. The malunions were treated with fibulotomy, closed wedge tibial osteotomy, open reaming of the marrow cavity, stable reamed intramedullary nail stabilization with or without supplementation, and cancellous bone grafting. Thirty-four (92%) patients were followed up for at least 1 year (range 1.0-4.3 years), and all achieved a solid union. The union period was 5.8+/-0.8 months. Complications included 2 (6%) patients with deep infection and 1 (3%) with cortical perforation. However, all 3 patients recovered completely after adequate management. In conclusion, a reamed intramedullary nail is an ideal instrument for tibial shaft malunions in indicated cases. Good exposure of the bony segments to ream the marrow cavity precisely can avoid cortical perforation. Gentle dissection of the soft tissues may lower the infection rate. Concomitant cancellous bone grafting can improve the union rate. PMID- 10738875 TI - Experimental study of bone-implant contact with a parabolic acetabular component (Hofer-Imhof). AB - Incomplete bone contact with threaded acetabular components is usually attributable to the tapered thread design and sclerotic bone, and might be the reason for early loosening. The self-tapping flat threads of the Hofer-Imhof cup allow accurate insertion without incarceration, even in sclerotic bone. To demonstrate the effectiveness of this threaded cup design based on digitized measurements of bone contact area, six Hofer-Imhof cups were implanted into three human cadavers. The bone contact area at the threads was digitally analyzed on cadaver-explanted cups cut into horizontal and coronal sections. In cups fixed with ideal form fit, bone contact was 31.6% (range 8-55%) at the threads and 28.5% (range 0-49%) between the threads on average. In cups with incomplete bone contact secondary to insufficient reaming, the bone contact area averaged 27.4% (range 10-44%) at the threads. In sclerotic bone, the highest bone contact at the threads was 69%. The threads were the first part of the cup to achieve primary bone contact. Factors contributing to poor contact were incomplete reaming, sections which passed through the incisura of the acetabulum, and incomplete contact with the medial aspect of the acetabulum. The aim was to achieve complete bone contact. Because of the higher contact areas observed in sclerotic bone, some sclerotic bone should remain after reaming. PMID- 10738876 TI - Results of posterior cruciate-retaining unconstrained total knee arthroplasty after proximal tibial osteotomy for osteoarthritis. A prospective cohort study. AB - The purpose of the study was to investigate the results of posterior cruciate ligament (PCL)-retaining total knee arthroplasty (TKA) after previous proximal closed wedge tibial osteotomy for degenerative arthritis according to the technique recommended by Coventry and Insall. Thirty-five patients with previous proximal tibial osteotomy were matched to 35 patients without previous osteotomy according to age, sex, and Knee Society patient category. TKA after osteotomy was technically more difficult, often requiring a more extensive exposure. Complications were similar in the two groups, but an extended hematoma was more common in patients with previous osteotomies (2 patients) than in those with primary arthroplasty (1 patient). Knee Society scores were significantly lower in those with previous osteotomy, primarily because of decreased anteroposterior stability and pain. No significant differences were found in function scores, range of motion, or alignment. However, overall results of this PCL-retaining unconstrained TKA did not match those reported after PCL-substituting TKA. PMID- 10738878 TI - Total hip arthroplasty after arthrodesis of the hip joint. AB - The results of 15 conversions of a hip arthrodesis into a total hip arthroplasty performed in the years 1980-1995 are reported. Fifteen patients (8 men, 7 women) underwent total hip arthroplasty 30.9 (range 2-61) years after spontaneous or operative fusion of a hip joint. The primary indications of the conversion were low-back pain (n = 10), knee pain (n = 2) and hip problems (n = 3). At follow-up examination 5.4 (range 2-13.3) years postoperatively, the Harris Hip Score averaged 86.0 (range 70.1-99.0). Six patients were pain-free, 7 had less pain, 2 felt no improvement of pain. All patients confirmed that they would undergo the operation again. The Trendelenburg sign was negative or mild in 8 patients and moderate to severe in 7. Aseptic loosening of 2 stems (1 cemented, 1 cementless) and 2 deep infections required revision surgery. We conclude that this operation can lead to satisfactory results even after a long duration of the arthrodesis. However, full function with no pain and a negative Trendelenburg sign could be obtained in only 20% (3/15) of the cases. PMID- 10738877 TI - Treatment of proximal humerus fracture using multiple intramedullary flexible nails. AB - A total of 61 patients with a proximal humeral fracture was treated between January 1996 and March 1998 by closed reduction and fracture fixation with intramedullary Prevot (or Nancy) nails. Of these, 28 female and 25 male patients with a mean age of 52 years (range 3-91 years) were reviewed clinically and radiologically with a mean follow-up of 17 months (range 4-30 months). The mean Constant score was 63, the mean Neer score 74 and the mean visual analogue scale (VAS) 73. The 14 patients under 24 years old achieved a Constant score of 86, a Neer score of 99 and a VAS of 97, while 13 patients aged between 25 and 60 years had a Constant score of 67, a Neer score of 75 and a VAS of 71. The 26 patients older than 61 years had a Constant score of 48, a Neer score of 61 and a VAS of 61. One patient with total and 6 with partial humeral head necrosis as well as 5 pseudarthroses were noted. Proximal nail perforation of the humeral head due to fracture collapse was seen in 22 cases. Complications were more frequently observed in the elderly. End results were not related to the type of fracture. This minimally invasive technique decreases the rate of occurrence of avascular necrosis of the humeral head. However, fractures are not sufficiently stabilised, mainly because of bone loss induced by impaction and osteoporosis. Bone loss remains an unsolved problem, and alternative methods such as the use of bone substitute combined with minimally invasive techniques should be studied. PMID- 10738879 TI - Total knee arthroplasty in patients with inherited dwarfism--a report of five knee replacements in two patients with Morquio's disease type A and one with spondylo-epiphyseal dysplasia. AB - In Morquio's disease and in hereditary spondylo-epiphyseal dysplasia of longer duration, malalignment, instability and unfavourable anatomy are generally the main surgical problems, but as these patients nowadays do not have a reduced life expectancy, it is worthwhile giving them a functional replacement for their severely deformed and painful knees. We report three patients with inherited dwarfism, in whom a replacement of the knee was performed. PMID- 10738880 TI - Teaching model for intraoperative spinal sonography in spinal fractures. An experimental study. AB - To improve the technique of intraoperative sonography of the spinal canal, a teaching model of the thoracolumbar spine was developed. It allows to simulate the typical spinal stenosis of a vertebral fracture and the sonographic procedure to detect and measure such a lesion. Moreover, partial laminectomy and modification of a fixateur interne set-up, which are preconditions for successful sonography, can be simulated. Independent of the surgical qualification, a high precision in sonographic localisation and measurement of the spinal canal stenosis was achieved by the training. The results could be validated in the cadaveric model. Thus, sonographic expertise acquired with the teaching model proved to be reliable in the clinical situation. PMID- 10738881 TI - The saddle prosthesis in pelvic primary and secondary musculoskeletal tumors: functional results at several postoperative intervals. AB - The first purpose of this study was to evaluate the saddle prosthesis in patients with periacetabular tumors in terms of the functional results obtained after several postoperative intervals. The second purpose was to evaluate the complications and how they might be prevented in the future. Functional results according to the MSTS functional rating system were evaluated at several postoperative intervals in 15 patients treated with internal hemipelvectomy and reconstruction with the saddle prosthesis because of periacetabular primary (n = 9) or secondary (n = 6) malignancies. All complications were evaluated. Three months postoperatively, 7/9 patients with a primary tumor and 2/4 patients with a secondary tumor were able to walk outside without pain. Median functional results 3 and 6 months postoperatively were 40% and 50%, respectively. Deep infection occurred in 4 patients and fracture of the iliac remnant in 2. Heterotopic ossifications along the interpositional component were seen in 5 patients, but they did not negatively influence the functional outcome. Three (relative) contraindications to reconstruction with the saddle prosthesis could be ascertained: osteoporosis, extended involvement of the iliac wing by tumor, and insufficient soft-tissue quality after previous procedures. (Short-term) functional results after reconstruction with the saddle prosthesis are satisfactory if the above-mentioned contraindications are taken into consideration. PMID- 10738882 TI - Evaluation of isolated fractures of the greater trochanter with magnetic resonance imaging. AB - The diagnosis of an isolated fracture of the greater trochanter can be done on routine radiographs, but this may not be the whole story. We evaluated fractures of the greater trochanter of the femur by magnetic resonance imaging (MRI). MR images were obtained within 5 days of the time of clinical presentation. Coronal images were performed on T1- and T2-weighted spin-echo images. Eight elderly patients who were diagnosed as having a greater trochanter fracture on standard radiographs underwent MRI. Three were men aged 62-76 (mean 63.4) years, and five were women aged 80-101 (mean 88.6) years. MRI showed that in seven of the eight cases, the fracture line was observed leading from the greater trochanter towards other trochanter regions. In only one case was the fracture limited to the greater trochanter and corresponded to the line observed on the standard radiographs. We suggest that in cases of greater trochanter fracture with somewhat severe symptoms, MRI should be performed in order to discover the appropriate diagnosis and treatment. PMID- 10738883 TI - Repair of muscle and musculotendinous junction injuries with an autogenous fascial patch. AB - We have used an autogenous fascia lata patch to repair muscle and musculotendinous junction injuries affecting 99 individual muscles in 23 patients with multiple incised wounds to the upper limbs. All patients were followed up for at least 2 years. Two patients with severe dystrophic changes following nerve injuries were excluded from the final analysis. Satisfactory grip strength was restored 6 months following the repair in 19 of the 21 patients we evaluated. Complete active and passive range of wrist and digital motion was seen in 18 of these 21 patients. Tightness of the flexor pollicis longus was seen in 2 patients, one of whom had a deformity of the interphalangeal joint of the thumb that was passively stretched and subsequently reduced. Scar adhesions were noted in 4 other patients, but this did not affect wrist or finger motion, except in one. The autogenous fascial patch repair technique provides strong and good apposition of muscle fibres simply by distributing the force and tension over a large area of the muscle, thereby allowing early mobilization and functional restoration of the extremity. PMID- 10738884 TI - Innervation of nociceptors in the menisci of the knee joint: an immunohistochemical study. AB - Using histology, we studied the innervation of nociceptors in the medial and lateral menisci of the knee joint. Specimens examined were taken from 16 patients during arthroplasty. The patients were 6 men and 10 women, with ages ranging from 14 to 76 years (mean 56 years). Immunohistochemistry with the unlabeled antibody biotin-streptavidin method was employed to detect protein gene product 9.5 (PGP 9.5) or substance P (SP) in the specimen. The antibody for PGP 9.5 detected nerve tissues in the menisci. Most but not all of the nerve fibers were associated with blood vessels. Nerve fibers and sensory receptors were found mainly in the peripheral, vascular zone, representing the outer one-third of the meniscus, and the innervated area was wider in the anterior and posterior horns. Pacinian and Ruffini corpuscles as well as free nerve endings were identified in these areas. Larger fibers coursed circumferentially in the peripheral zone, with smaller branches of nerve fibers running radially into the meniscus. Nerve fibers positive for SP were also detected in the menisci, but were fewer in number. Their branches also were fewer, oriented radially and paralleling blood vessels. This study showed that some of the pain in cases of meniscal tear could originate in the meniscus itself, especially with peripheral tears that may be accompanied by bleeding. PMID- 10738885 TI - Management of the floating elbow injury in children. Simultaneous ipsilateral fractures of the elbow and forearm. AB - Simultaneous ipsilateral fracture of the elbow and forearm--floating elbow--is an uncommon injury. During a 7-year period we prospectively followed 12 children who presented with completely displaced supracondylar fractures of the humerus associated with a forearm fracture of the same limb. All patients underwent emergency operative reduction and percutaneous K-wire stabilisation. At a minimum of 18 months, all patients were assessed clinically and radiologically and the results evaluated according to a conventional scoring system. Ten patients had good or excellent outcomes, and there were two fair results. The incidence of open fractures and nerve injury and the need to perform an open reduction were higher than those recorded for isolated supracondylar fractures. The floating elbow is an indicator of a high energy injury and requires aggressive operative management. PMID- 10738886 TI - Total hip arthroplasty revision using the press-fit CLS Spotorno cementless stem. Twenty-four hips followed between 1987 and 1998. AB - A retrospective study of uncemented femoral revision for loosening after total hip arthroplasty was conducted for the period 1987-1998. The study included 24 hips in 22 patients (7 men, 15 women). The revision procedure consisted of the replacement of the loose hip prosthesis using the CLS (Protek) press-fit stem. Patients were followed for a mean duration of 4.5 years. The mean interval between the primary operation and the revision was 10 years (range 2-16 years). Using the Merle D'Aubigne hip score and radiographic findings, a favorable outcome was noted in 20 hips. We suggest that the femoral revision procedure using the uncemented Spotorno stem is a very useful method of correction of loosening after total hip replacement. PMID- 10738887 TI - Fixation of proximal tibia fractures by a retrograde nail: a biomechanical investigation. AB - Surgical treatment of proximal tibial fractures requires open reduction and internal fixation. The operative exposure causes additional soft-tissue injury and reduces the blood supply to the bone. A cephalograde tibial nail should offer comparable mechanical stability without these disadvantages. We compared the stability of both osteosyntheses in a fracture model with 12 fresh-frozen cadaver bones. While both implants exhibited comparable stiffness under sagittal loading, the plate had a higher rotational and varus stiffness. Despite this higher stiffness, rotational displacements at the fracture gap were nearly twice as large for this implant during loading. We conclude that the retrograde nail provides similar mechanical stability to plate fixation for proximal tibial fractures, while the closed reduction and soft-tissue preservation of this new technique are definite advantages. PMID- 10738888 TI - Limb reconstruction with the callus distraction method after bone tumor resection. AB - The callus distraction method was applied to nine patients who were referred to us because of a bone tumor. Their mean age was 17 years and 3 months (range 7-37 years). Three were female, and 6 were male. All of the tumors were localised on the femur, and the histological diagnosis was two chondrosarcomas, one Ewing's sarcoma, three osteosarcomas, one giant cell bone tumor, and the remainder benign fibrous histiocytoma. The mean length of the defect after resection of the tumor was 11.5 (range 8-20) cm. Preoperative and postoperative chemotherapy were applied to patients with osteosarcoma and Ewing's sarcoma. The patients were followed up for 22 (range 15-30) months on average. The mean period of use of the external fixator was 12.5 (range 8-18) months. One patient suffered a tumour recurrence and died after 20 months. Complications included one deep infection, one skin invagination, and one premature consolidation and bone bridge in the defect area. All of the complications were successfully treated. Functional evaluation gave excellent results in four patients, good in three, and fair in two. This method can be used without any need for massive autogenous bone graft in repairing defects of any length and diameter produced after excision of the lesion and thus can be considered as an alternative to other techniques. PMID- 10738889 TI - Osseous lesions of the distal ulna: atypical location--unusual diagnosis. Report of three cases with similar imaging and different pathologic diagnoses. AB - Three cases with destructive bone lesions of the distal end of the ulna caused by different pathologic entities (Ewing's sarcoma, osteosarcoma, rheumatoid pseudotumoral synovitis) are presented, all with similar clinical and comparable x-ray and magnetic resonance imaging features. Although the distal end of the ulna may be resected without significant functional impairment, careful evaluation of treatment strategies compatible with oncologic standards is warranted. PMID- 10738890 TI - Non-tuberculous cold abscess of the psoas muscle--an unusual manifestation of colocutaneous fistula. AB - We report here a case of colocutaneous fistula drained from the retroperitoneal space mimicking a cold abscess of the psoas muscle. A 60-year-old diabetic woman with a 6-year history of a chronic draining sinus over her right thigh had been treated intermittently with antibiotics. At presentation, she had no systemic toxic signs nor other constitutional symptoms. The patient was inadequately managed by curettage at first under the tentative diagnosis of tuberculous cold abscess. After the correct diagnosis of colocutaneous fistula, right nephrectomy and right hemicolectomy with ileotransverse colostomy were done. The patient was well 5 years later without recurrence. This is an atypical presentation of enterocutaneous fistula in an immunodeficient patient that should be emphasized to facilitate the correct diagnosis and early treatment. PMID- 10738891 TI - Carpal tunnel syndrome caused by an idiopathic calcified mass. AB - This is a case report of carpal tunnel syndrome caused by an idiopathic calcareous lesion within the carpal canal. The median nerve was trapped between the transverse carpal ligament and the calcified mass. The mass was predominantly composed of calcium phosphate. Surgical release of the transverse carpal ligament and removal of the calcareous mass relieved the symptoms. PMID- 10738892 TI - Compression of the ulnar nerve in Guyon's canal by uremic tumoral calcinosis. AB - We describe the case of a 70-year-old woman with chronic renal failure on haemodialysis presenting with an ulnar nerve compression in Guyon's canal secondary to uremic tumoral calcinosis. Excision of calcium deposits and external neurolysis of the ulnar nerve were successfully performed. Simultaneously, the hyperphosphatemia and hypercalcemia were corrected. The pathogenesis of this condition is different from primary tumoral calcinosis. Clinical and radiological features and therapy are discussed. Uremic tumoral calcinosis is an unusual etiology of ulnar nerve compression in Guyon's canal not previously reported in dialysis patients. PMID- 10738893 TI - Giant cell tumor of rib--rare location on the anterior aspect. AB - Giant cell tumor rarely occurs in ribs, where it presents posteriorly. We present a report of a giant cell tumor of bone occurring anteriorly in the rib with a review of the literature. PMID- 10738894 TI - Stress fracture of the cuboid in an 8-year-old boy: a characteristic magnetic resonance imaging diagnosis. AB - A stress fracture of an 8-year-old boy diagnosed on magnetic resonance (MR) images is presented. Stress fractures of the cuboid are probably more common than previously reported. The diagnosis should be considered even when a history of trauma or chronic overload is absent. The MR appearance of the stress fracture was characteristic. PMID- 10738895 TI - Simultaneous anterior and posterior traumatic dislocation of the hip. A case report with review of the literature. AB - The presence of anterior dislocation of the hip along with contralateral posterior dislocation of the hip in the absence of other major trauma is a distinctly rare injury pattern. We report such a case resulting from a motor vehicle striking a pedestrian, along with a review of previous cases. The patient was managed nonoperatively within 6 hours of trauma with an excellent final outcome and no posttraumatic complications over a 3-year follow-up. The possible mechanism of this injury is discussed. PMID- 10738896 TI - Tenosynovial chondromatosis of the third finger. AB - Synovial chondromatosis is an uncommon lesion, characterised by cartilaginous and osseous metaplasia of the joint synovium. It usually involves a large joint and rarely occurs in the hand. A case of tenosynovial chondromatosis of the third finger is reported. PMID- 10738897 TI - MUC1 upregulation by ethanol. AB - MUC1 is a glycoprotein and its expression is altered in breast cancer. Mucin protects epithelia from the external hostile environment. The expression of mucin changes when epithelia come in contact with toxic agents such as ethanol. Previously, we characterized the expression and regulation of tracheo-bronchial mucin (TBM) gene. In the present study, we studied the effect of ethanol on the gene encoding mammary gland mucin MUC1 and observed that ethanol regulates MUC1 expression at the transcription level. Ethanol enhanced the expression of MUC1 mRNA in a dose- and time-dependent manner in MCF-7 cells. At 100 mM concentration (a concentration reported to be present in alcoholics), ethanol induced a three to five-fold increase in mucin transcription as determined by nuclear run on analysis. This concentration of ethanol does not affect the half-life of MUC1 mRNA. PMID- 10738898 TI - Fluorescence spectroscopy of normal, SV40-transformed human keratinocytes, and carcinoma cells. AB - Native fluorescence emission and excitation spectra of SV40 infected human keratinocytes, A431 and SCC324 carcinoma cells, and normal human keratinocytes were measured and compared. A difference in the intracellular metabolic state of NADH was found between the normal cells and the cancer or virus-transformed cells. The observed difference, namely an increased proportion of bound, mitochondrial NADH in the cancer and virus-infected cells, manifests as a blue spectral shift in the emission spectra. PMID- 10738900 TI - Development of intracytoplasmic lumens in a colon cancer cell line cultured on a non-adhesive surface. AB - Cell-matrix interactions have important effects on phenotypic features, such as morphology, differentiation and cell growth. Several papers have suggested that when cell-matrix interactions are interrupted, cells grow as multicellular spheroids and eventually undergo apoptosis. We found that when ET(-), a laminin non-adherent colon cancer cell line, was cultured on poly-2-hydroxyethyl methacrylate (HEMA) coated plastic, the cells floated as cellular aggregates of spheroids or as single cells. Some of the single cells contained a very large intracytoplasmic lumen (ICL) and appeared similar to signet ring cells. These ICL were lined by a layer of short microvilli. The number of the cell did not increased cells when cultured on poly-HEMA. Another type of single cells, usually without ICL, demonstrated the characteristics of apoptotic cells by histologic examination. Acridine orange staining, flow cytometry and electron microscopy confirmed the apoptotic nature of those cells. In immunohistochemical staining for proliferating cell nuclear antigen, spheroids of cells and single cells with ICL were immunoreactive, while most of the single cells without ICL were negative. These results suggest that multicellular aggregation and formation of ICL were induced by the adaptation of ET(-) colon cancer cells in a harmful environment caused by reduced adhesiveness, and these changes might be related to cell survival. PMID- 10738899 TI - Drug metabolizing enzyme system and heme pathway in hepatocarcinogenesis. AB - Chemically induced and spontaneous liver tumors share some metabolic alterations. The decline in hemoprotein levels during hepatocarcinogenesis may result from a diminution of the intracellular heme pool. To elucidate if the onset of the pre initiation stage alters the natural regulation mechanism of heme pathway, animals were fed with p-dimethylaminoazobenzene (DAB) and treated or not with 2 allylisopropylacetamide (AIA). The induction of 6-Aminolevulinic acid synthase (ALA-S) activity and the diminution in microsomal heme oxygenase (MHO) did not change when DAB fed animals were treated with AIA. Cytochrome P-450 (P-450) levels and glutathione S-transferase activity were increased in all the groups tested. Tryptophan pyrrolase, sulphatase and beta-glucuronidase activities were altered in DAB fed animals but AIA treatment did not produce any effect. Changes in drug metabolizing enzymes in livers of DAB fed animals could be the result of a primary deregulation of heme metabolism. These results give additional support to our hypothesis about a mechanism for the onset of hepatocarcinogenesis. PMID- 10738901 TI - Total acid soluble and insoluble carnitine levels in human brain tumors. AB - Carnitine has two main functions, i.e., transporting long-chain fatty acids into the mitochondrial matrix for beta-oxidation to provide cellular energy and modulating the rise in intramitochondrial acyl-CoA/CoA ratio, which relieves the inhibition of many intramitochondrial enzymes involving glucose and amino acid catabolism. The present study examined the acid soluble carnitine (ASCAR) acid insoluble carnitine (AICAR) and total carnitine (TCAR) concentrations of 50 human brain tumor tissues and 11 normal brain tissues. The ASCAR levels significantly higher in gliomas and meningiomas than brain, however similar to brain in metastatic adenocarcinomas. AICAR levels were lower than brain in all tumors with the exception of a medullablastoma. TCAR levels were similar to brain in all tumor types. Decreased AICAR levels may be due to increased utilization of lipids or enhanced phospholipid and cholesterol synthesis which is need for increased membrane synthesis or formation of eicosanoids. Also decreased concentrations may be a reflection of camitine and its acylesters role in preserving the physiologic membrane structure function from oxidative damage. PMID- 10738902 TI - Inhibitory effect of combined administration with CPT-11 and 5-fluorouracil in vitro and in vivo. AB - Combined antitumor activity of CPT-11 and 5-fluorouracil (5-FU) was evaluated in a human cultured cell line derived from lung cancer. After 24 h culture with SN 38 followed by 5-FU 24 h, synergistic effect was observed in the cell line. In addition, the antitumor effect of this combination was studied in in vivo experiments using Donryu rat with Yoshida sarcoma cells. CPT-11 and 5-FU synergistically inhibited tumor growth. There was no significant increase of toxicity as assessed by the body weights. These results might support for the combination with 5-FU and CPT-11 in a chemotherapy for cancer. PMID- 10738904 TI - Modulation of the impaired drug metabolism in sarcoma-180-bearing mice by echitamine chloride. AB - Echitamine chloride (EC), an indole alkaloid, extracted from the bark of Alstonia scholaris has got highly promising anticancer effect. The effect of this drug on the microsomal drug detoxifying system was studied in sarcoma-180 induced mice. When given sub-cutaneously at a dosage of 5 mg/kg body weight, it was able to alter the impaired drug detoxifying system which was observed in the Sarcoma-180 bearing mice. The levels of microsomal protein, Cyt-P450, Cyt-b5, NADH-Cyt-C reductase, NADPH-Cyt-C-reductase, and glu-6 phosphatase were determined. The levels of these drug metabolizing enzymes were decreased in S-180 bearing mice. EC treatment corrected to near normal levels of these enzymes and microsomal hemeproteins. In order to understand the mechanism responsible for the decreased protein level and its normalization after treatment with EC, 3H-Phenylalanine incorporation study was carried out. From the results, it is observed that the synthesis of apoproteins is also altered in tumor-bearing animals. All these changes which were observed in tumor-bearing animals were corrected to near normal levels after treatment with EC. PMID- 10738903 TI - Breast cancer cells express cathepsins B and L but not cathepsins K or H. AB - Lysosomal cysteine proteinases (cathepsins) are considered to play a role in bone degradation mediated by metastatic breast cancers. To evaluate which cathepsin contributes to the osteolysis, we quantitatively determined the expression levels of four cathepsins in two breast cancer cell lines, MCF-7 and MDA-MB-231, by competitive RT-PCR. Cathepsin K, which is the most abundant cathepsin in osteoclasts, was not detected in either cell lines. We also failed to detect cathepsin H mRNA. By contrast, we found significant expression of cathepsins B and L in both cell lines. By Northern blot analysis cathepsin B mRNA was detected in a single form in these cells, whereas osteoclasts contained multiple forms of the mRNA. Cathepsin B protein was also detected by Western blotting as a single immunoreactive band corresponding to its mature enzyme. These findings suggest that osteolysis associated with metastatic breast cancers takes place in a different way from osteoclast-mediated bone resorption. PMID- 10738905 TI - Magnetic field induced inhibition of human osteosarcoma cells treated with adriamycin. AB - Morbidity resulting from the toxicity of chemotherapeutic drugs suggests that novel approaches are worthy of investigation. Development of the use of low intensity magnetic fields as an adjuvant to current treatment regimens to prevent metastatic disease may prove to be efficacious. Using a cell culture model, we have developed a magnetic field (MF) treatment that offers the possibility of lowering the therapeutic dose of these drugs and thereby reducing morbidity. Our studies have found that a low intensity (approximately 2 gauss) MF signal and a relatively low dose (0.1 microg/ml) of Adriamycin (ADR) inhibited proliferation of human osteosarcoma cells by 82%, whereas the MF and ADR acting individually caused only 19% and 44% inhibition, respectively. PMID- 10738906 TI - Modulating effect of new potential antimelanomic agents, spin-labeled triazenes and nitrosoureas on the DOPA-oxidase activity of tyrosinase. AB - The modulating effect of newly synthesized alkylating spin labeled triazene and spin labeled nitrosourea derivatives on the DOPA-oxidase activity of mushroom tyrosinase has been investigated by Bumett's spectrophotometric method (Burnett et al., 1967). All spin labeled triazenes have exhibited activating effect on DOPA-oxidase activity of tyrosinase, whereas clinically used triazene (DTIC), which does not contain nitroxide moiety, have showed inhibiting effect. At the same experimental conditions the spin labeled aminoacid nitrosoureas have showed dual effect - activating, in the beginning of the enzyme reaction and inhibiting later on. It is deduced that the activating effect of the spin labeled compounds is due to the nitroxide moiety and the inhibiting effect of all compounds depends on their half-life time. This study might contribute to make more clear the mechanism of action of the new compounds and on the other hand would come in quite useful as a preliminary prognosis for their antimelanomic activity. PMID- 10738907 TI - Molecular basis of specific inhibition of urokinase plasminogen activator by amiloride. AB - The urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) are very similar serine proteases with the same physiological function, the activation of plasminogen. An increased amount or activity of uPA but not tPA has been detected in human cancers. The PAs are weak proteolytic enzymes, but they activate plasminogen to plasmin, a strong proteolytic enzyme largely responsible for the malignant properties of cancers. It has been shown recently that the administration of uPA inhibitors can reduce tumor size. Inhibitors of uPA could therefore be used as anti-cancer and anti-angiogenesis agents. It has been found that amiloride competitively inhibits the catalytic activity of uPA but not tPA. Modification of this chemical could therefore produce a new class of uPA specific inhibitors and a new class of anti-cancer agents. The X-ray structure of the uPA complex with amiloride is not known. There are structural differences in the specificity pocket of uPA and tPA. However, the potential energy of binding amiloride is lower outside this cavity in the case of tPA. A region responsible for binding amiloride to tPA has been proposed as the loop B93-B101, reached in negatively charged amino acids present in tPA but not uPA. PMID- 10738908 TI - Arginase and ornithine, as markers in human non-small cell lung carcinoma. AB - The arginase activity and ornithine level were determined in tissue obtained from patients with non-small cell lung carcinoma (NSLC). The arginase activity and ornithine level in tumor tissues were 1.89 +/- 1.28 U/mg protein and 42.32 +/- 25.82 nmol/mg protein, respectively versus 0.67 +/- 0.19 U/mg protein and 10.12 +/- 3.69 nmol/mg protein for normal tissues (p < 0.01). PMID- 10738909 TI - Expression of mdm-2 oncoprotein in the primary and metastatic sites of mammary tumor (GI-101) implanted athymic nude mice. AB - The expression of mdm-2 oncoprotein (p90) was determined in a human breast tumor xenograft line (GI-101) that was derived from a 57 year old female cancer patient with recurrent, infiltrating ductal adenocarcinoma (Stage IIIa, T3N2MX). Immunoprecipitation coupled western blot analysis of the primary tumors that have been obtained from xenograft implanted athymic nude mice, using mdm-2 (Ab-1) mouse monoclonal antibody, primarily revealed high level expression of a 90 kD full length mdm-2 protein. In the GI-101 tumor the level of full length mdm-2 (p90) protein expression increased with the increase in the size of the tumor (100 to 2,000 mm(3)) and a maximum expression was detected in 2,000 mm(3) size tumors. In addition to the expression in the primary site, a significantly high level expression of mdm-2 protein (p90) was detected in the lung and liver tissues also, which are the known metastatic sites for GI-101 xenograft tumors. However, the level of mdm-2 protein expression was undetectable in the lung and liver tissues obtained from control mice. A cell line (GI-101A) derived from the GI-101 xenograft tumor also showed a high level expression of mdm-2 protein after several generations of cell passage. When the GI-101A cells were treated with DES (Diethylstilbestrol) the mdm-2 protein expression increased after 10 min treatment and reached a peak level at 40 min. Interestingly, DES (10 and 20 microM) treatment increased the total cell number also after 96 hr treatment compared to the non-treated cells. It appears that mdm-2 (p90) may have a significant role in supporting the tumor cell growth as well as the metastatic process of the GI-101A cells. PMID- 10738910 TI - Investigations on the aminoacid content of tumor associated antigens of rat sarcoma cells induced by virus. AB - In our previous work (Alexandrov et al., 1996) was reported that the rat sarcoma cells induced by SR-RSV express two tumor associated antigens (TAA). The one TAA has a molecular weight of 52 kD and is detected by the help of a monoclonal antibody 2C2 only on the outer side of the plasma membrane of the sarcoma cells. The other antigen, with molecular weight of 28 kD, is expressed on the outher and inner side of the membrane. The antigens were isolated as a pure fraction by polyacrylamide electrophoresis and prepared for aminoacid analysis after that. The consisting 16 bound aminoacids were in different amounts. Both antigens are rich in glycine and poor in aromatic and sulphur-containing aminoacids. The presence of glucosamine and galactosamine in the antigens proves their glycoprotein nature. The received data show that the both TAA-s differ not only in molecular weights, place of expression and functional activity, but also in the amount of the bound aminoacids which constitute their proteins. PMID- 10738911 TI - Lipid peroxidation and antioxidant system in the blood of cancerous patients with metastasis. AB - Free-radical-mediated damages may play an important role during metastasis. To investigate their relevance in the metastatic process MDA levels, glutathione peroxidase (GPX) and superoxide dismutase (SOD) activities, and selenium, zinc and copper contents were determined in plasma and erythrocytes from 20 cancerous patients with metastasis and 30 age-matched controls. Significantly higher concentrations of MDA in plasma as well as in erythrocytes were found comparing to the control group. In both plasma and erythrocytes, GPX activity and selenium and zinc levels were significantly lower in patients than in controls. However, SOD activity in erythrocytes and copper levels in both plasma and erythrocytes were significantly higher in patients. The impaired antioxidant system may favor accumulation of free radicals which may induce the process of metastasis. On the other hand, it is possible that the antioxidant system is impaired as a consequence of abnormality in the antioxidative metabolisms due to the cancer process. PMID- 10738912 TI - Lactoferrin expression in human breast cancer. AB - We analyzed lactoferrin expression in 78 samples from patients with sporadic breast cancer and found 31/78 negative for mRNA expression. Similar results were obtained by immuno-histochemical localization of the lactoferrin protein. We did not find relationship between lactoferrin expression and clinical parameters. We investigated for the absent lactoferrin expression in some cases of breast cancer. In 68 of the samples analyzed, we found an inverse correlation between estrogen receptor expression and lactoferrin expression (P < 0,0001), thus indicating that regulation by the estrogen receptor is not the main element responsible for the expression of lactoferrin in breast cancer. Analysis of methylation of the lactoferrin genomic DNA extracted from the same patients revealed that the degree of methylation does not explain the observed absence of lactoferrin. The 937 bp lactoferrin promoter was investigated for possible mutations. By single-strand conformation polymorphism analysis one polymorphic site was found and characterized. PMID- 10738913 TI - Metabolic myopathies: a clinical approach; part I. AB - Children and adults with metabolic myopathies have underlying deficiencies of energy production, which may result in dysfunction of muscle or other energy dependent tissues, or both. Patients with disorders of glycogen, lipid, or mitochondrial metabolism in muscle may present with dynamic findings (i.e., exercise intolerance, reversible weakness, and myoglobinuria) or progressive muscle weakness, or both. In this first part of the review, we present a brief description of energy metabolism in muscle, a simplified overview of the clinical and laboratory evaluation of the patient with suspected metabolic myopathy, and a diagnostic algorithm aimed at predicting the nature of the underlying biochemical abnormality. The goal is to simplify a complex field of neuromuscular disease and thus lead to early recognition and treatment of these disorders. PMID- 10738914 TI - Clinical and molecular heterogeneity in very-long-chain acyl-coenzyme A dehydrogenase deficiency. AB - Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency is an increasingly recognized defect of mitochondrial fatty acid beta-oxidation manifesting with episodes of metabolic decompensation or isolated recurrent myoglobinuria. In this report the clinical, biochemical, and molecular studies in a series of five patients (four Italian and one Spanish) with this disorder are discussed. Biochemical studies included the determination of fibroblast substrate oxidation rates and enzyme activity and Western blot analysis of VLCAD protein. Molecular analysis was performed by sequencing the VLCAD gene from the genomic DNA. Clinical features were within the spectrum previously reported. Four patients presented in infancy or childhood with episodes of severe metabolic decompensation and dicarboxylic aciduria. Two exhibited cardiomyopathy. The fifth patient presented with isolated recurrent rhabdomyolysis, with no cardiomyopathy or dicarboxylic aciduria. In all patients a significant loss of VLCAD activity associated with a marked reduction of VLCAD protein levels occurred. Molecular analysis disclosed one novel missense mutation (Cys437Tyr) and four previously reported mutations, including two missense substitutions (Phe418Leu and Arg419Trp), a single amino acid deletion (Lys258del), and one splice site mutation (IVS8-C(-2)), which was present in all four Italian patients. All patients exhibited compound heterozygosity. The phenotypic variability and the high genotypic heterogeneity of this hereditary metabolic disorder is reported. PMID- 10738915 TI - Relevance of the cerebellar hemispheres for executive functions. AB - The aim of the present study was to elucidate the role of the cerebellar hemispheres in executive functions. The findings are relevant because of the large number of children who survive cerebellar tumors. Neuropsychologic assessments of four patients (8-21 years of age) who had undergone neurosurgery for removal of tumors in the cerebellar hemispheres were conducted and compared with the assessments of six children who had been diagnosed with temporal lobe tumors or cysts. The executive functions were assessed using the Wisconsin Card Sorting Test. IQs were average in both groups. As expected, patients with cerebellar hemispheric lesions had impaired executive functions. In particular, they appeared to have difficulty generating and testing hypotheses regarding the matching rules on the Wisconsin Card Sorting Test. Patients with temporal lesions had a different pattern of deficits on this test. The findings are consistent with the theories that propose that the cerebellar hemispheres are involved in cognitive processes. The findings also demonstrate that subtle deficits in executive functions can be masked by a normal IQ in survivors of cerebellar tumors and highlight the need to design interventions targeted toward problem solving skills. PMID- 10738916 TI - Dopamine transporter and nitric oxide synthase in hypoxic-ischemic brain. AB - Changes in dopamine transporter and neuronal nitric oxide synthase (nNOS) were investigated by immunohistochemistry in 18 cases of hypoxic-ischemic basal ganglia necrosis. Neuropil dopamine transporter immunostaining in the striatum was increased in seven cases, with relatively mild basal ganglia necrosis, and decreased in four cases, with marked basal ganglia necrosis, compared with age matched control subjects. Correspondingly, some striatal neurons had increased immunoreactivity to dopamine transporter in the cases of increased immunostaining in the neuropil. nNOS-positive neurons did not obviously change in cases of basal ganglia necrosis within 2 days after birth and then decreased or were not detectable in cases of basal ganglia necrosis at more than 3 days after birth. The results suggest that the synthesis of dopamine transporter is up-regulated in relatively mild basal ganglia necrosis to compensate for the uptake of increased dopamine, that this compensative ability is lost in marked basal ganglia necrosis, and that nNOS-containing neurons in the striatum are relatively resistant to hypoxic ischemia. We speculate that glutamate excitotoxicity mediated by glutamate receptors 1, 2/3, and 4 and excessive dopaminergic excitatory activity may play important roles in hypoxic-ischemic basal ganglia necrosis and that nNOS does not contribute to that condition. PMID- 10738917 TI - Mitochondrial biogenesis defects and neuromuscular disorders. AB - A variety of mitochondrial DNA (mtDNA) defects, ranging from point mutations and large-scale deletions to severe reduction in the overall quantity of mtDNA (mtDNA depletion), may be associated with neuromuscular disorders. The nuclear genome, which encodes most of the proteins involved in mitochondrial biogenesis (regulation of maintenance, replication, and transcription of mtDNA), appears to be implicated in many of the mtDNA defects. In this review, we describe some of the mtDNA defects discovered by our laboratory and others in patients with neurologic disorders and analyze their potential relationship with the pathways and mechanisms involved in mitochondrial biogenesis. PMID- 10738918 TI - Brainstem encephalitis resulting from Epstein-Barr virus mimicking an infiltrating tumor in a child. AB - A case of a child with subacute neurologic features and imaging findings consistent with a brainstem encephalitis that was discovered to be related to a primary central nervous system infection caused by Epstein-Barr virus is presented. A brainstem tumor was initially suspected, but a correct diagnosis was formulated on the basis of the favorable clinical course and the detection of positive Epstein-Barr virus serology. In contrast to a prompt recovery of neurologic signs the neuroimaging alterations persisted for a longer time. The present report emphasizes the possible role of Epstein-Barr virus in the pathogenesis of infectious neurologic disorders in childhood, underlining the unusual presentation of a brainstem encephalitis, and considers the discrepancy between the course of neurologic features and the evolution of imaging alterations. PMID- 10738919 TI - Delayed post-traumatic visual loss: a clinical dilemma. AB - A 16-year-old female presented with unilateral blindness in her right eye 2 months after blunt head trauma. Optic nerve edema was demonstrated by funduscopic examination, ultrasonography, and magnetic resonance imaging and failed to respond to medical treatment. Delayed post-traumatic blindness may be a severe complication of head trauma. The late appearance leads to delayed diagnosis and resulting unresponsiveness to treatment. Patients who experience head trauma that could involve the optic nerves should undergo ultrasonography of the optic nerves. An abnormal finding should be followed by an intensive evaluation to determine possible damage. PMID- 10738920 TI - Transcranial magnetic stimulation and other evoked potentials in pediatric multiple sclerosis. AB - In children, multiple sclerosis is rare and has some clinical and paraclinical differences compared with adults. The assessment of corticospinal motor tracts is expected to be relevant because of their frequent early involvement in this disease. Reported are the results of transcranial magnetic stimulation in two children who presented at 12 and 9 years of age with clinically probable and definite multiple sclerosis, respectively. In Patient 1 the excitatory cortical threshold for the upper limbs was abnormally raised. In Patient 2 the latency of the motor-evoked potentials was considerably increased for the right tibialis anterior muscle, with a slowing of the central conduction time. Although these abnormalities may be consistent with central conduction impairment, they may alternatively suggest early axonal damage because irreversible axonal lesions occurring at the onset of the disease have recently been reported. Testing of central motor tracts, in addition to visual, auditory, and somatosensory pathways, therefore appears appropriate in the multimodal assessment of pediatric patients with suspected multiple sclerosis. PMID- 10738921 TI - Basal lamina abnormality in the skeletal muscle of Walker-Warburg syndrome. AB - The basal lamina of skeletal muscle fibers has been reported to be thinned and disrupted in patients with Fukuyama and laminin-alpha-2-deficient congenital muscular dystrophies. The basal lamina is normal in other, later-onset, muscular dystrophies, but the plasma membrane is disrupted. It is unknown whether the dystrophic process in Walker-Warburg syndrome (WWS) is characterized by a basal laminal abnormality, a sarcolemmal abnormality, or both. The present study examined the skeletal muscle of a 3-month-old patient with WWS by immunohistochemistry and electron microscopy and compared the findings with control muscle samples. In control samples the basal lamina of skeletal muscle fibers was a continuous, uniformly dense structure associated with sarcolemma. In WWS the basal lamina appeared deranged, with disruptions in nonnecrotic muscle fibers. Furthermore, in some fibers the basal lamina was thinner, and in others, it was duplicated. Dystrophin, laminin-alpha-2, and adhalin stains revealed normal immunoreactivity. The disruptions in the basal lamina may play a primary role in the degeneration of muscle fibers in WWS. When compared with the dystrophies with a primary sarcolemmal defect, it appears that those with primary basal lamina abnormalities (WWS, laminin-alpha-2-deficient, and Fukuyama congenital muscular dystrophies) present early in life, and the phenotype is more severe. PMID- 10738922 TI - Effects of high-dose intravenous corticosteroid therapy in Landau-Kleffner syndrome. AB - Two children with Landau-Kleffner syndrome were successfully treated with antiepileptic drugs and a high-dose intravenous corticosteroid. A combination of valproate and a benzodiazepine (clonazepam or diazepam) ameliorated epileptic seizures and electroencephalographic spikes and waves, but speech disturbances persisted. Both patients were treated with an intravenous infusion of high-dose methylprednisolone sodium succinate (20 mg/kg daily) for 3 consecutive days. This infusion was repeated three times with a 4-day interval between treatments, which resulted in a rapid improvement in speech ability. After intravenous therapy, prednisolone was given orally (2 mg/kg daily for 1 month, then gradually withdrawn), which maintained the clinical improvement in speech. PMID- 10738923 TI - Muscular carnitine palmitoyltransferase II deficiency in infancy. AB - An 8-month-old female presented with febrile myoglobinuria. The activity of carnitine palmitoyltransferase (CPT) II was decreased to 16% of the control mean, and the oxidation of the long-chain fatty acids was reduced to 25% of the mean in the fibroblasts. Homozygosity for the common mutation, S113L, was identified in the CPT II gene. Residual CPT II activity of more than 10% of the mean and homozygosity for the common mutation S113L are usually associated with a milder reduction of long-chain fatty acid oxidation to about 80% of the control and with a later age of clinical onset. The early clinical presentation in the present patient is unique and was associated with a marked impairment of long-chain fatty acid oxidation, possibly because of other genetic factors. CPT II deficiency should be included in the differential diagnosis of isolated myoglobinuria in infancy. PMID- 10738924 TI - Epidermal nevus syndrome with hypermelanosis and chronic hyponatremia. AB - Epidermal nevus syndrome is seldom encountered, and its association with hypermelanosis and the chronic syndrome of inappropriate antidiuretic hormone secretion (SIADH) has never been reported. A male neonate who developed intractable seizures and hyponatremia soon after birth is reported. He had alopecic patches on the scalp at birth. Large areas of skin hyperpigmentation, and epidermal nevi developed gradually. The clinical picture of hypotonic hyponatremia, high urine osmolality, elevated urine sodium, and euvolemia was compatible with SIADH. The seizures did not correlate with the hyponatremia, and no other cause for the seizures could be identified. The hyponatremia became chronic and was treated with a direct supply of sodium chloride. The development of the patient was markedly delayed at the last visit when he was 1 year of age. It is suggested that hypermelanosis and chronic SIADH may also be a variant presentation of epidermal nevus syndrome. PMID- 10738926 TI - Voiding difficulty in women. PMID- 10738927 TI - Attitudes to vaginal/urethral touching and device placement in women with urinary incontinence. AB - Because of recent development of a number of intravaginal and urethral occlusive devices for the treatment of urinary incontinence, we aimed to develop a linear analog scale that measured women's attitudes to becoming familiar with their genital anatomy and to placing devices into or upon the vagina or urethral orifices. We also investigated whether such attitudes were affected by previous tampon or diaphragm use, or were age dependent. After pilot studies, a 10-item questionnaire was given to 104 consecutive incontinent women, only 30% of whom were quite comfortable about the concept of touching their genitalia: this attitude was age dependent. Only 21% were quite willing to insert a continence device into their vagina: this attitude varied weakly with age but was significantly affected by previous tampon or diaphragm usage. Only 15% felt very comfortable about placing a continence device on to their urethra, but the likelihood of a positive response to this concept was not at all affected by age and was only slightly more common in previous tampon users. Thus older women are less likely to understand the anatomy of their genitalia or to be comfortable about the idea of exploring it, but age is no barrier to willingness to employ urethral or vaginal continence devices per se. We suggest that such a linear analog questionnaire may help gynecologists determine which patients might respond well to the use of vaginal or urethral continence devices. PMID- 10738925 TI - The prenatal pituitary gland--hidden and forgotten. AB - The sella turcica and pituitary gland in a human fetus (18 weeks gestation) with unilateral oro-ocular cleft combined with unilateral cleft lip and palate are described histologically. In this fetus the sella turcica was not a normal sella but a caudally open funnel. The adenopituitary gland tissue was positioned ectopically within the funnel canal and in the pharyngeal submucosa. PMID- 10738928 TI - Urogenital symptoms and resulting discomfort in non-institutionalized Dutch women aged 50-75 years. AB - The aim of this study was to determine the prevalence of urogenital symptoms in non-institutionalized Dutch women aged 50-75 years, and the degree of discomfort suffered as a result. A questionnaire was sent to 2157 women, a sample representative of the female population aged 50-75 years with respect to age, marital status, level of education and menopausal age. The usable response was 81.6% (n = 1761). The overall prevalence of vaginal dryness, soreness and dyspareunia was 27%. The prevalence of micturition symptoms, urinary incontinence and recurrent urinary tract infection was 36%. The prevalence estimates for vaginal dryness and urinary incontinence showed a linear decrease with increasing age. Almost half of the symptomatic women reported moderate to severe discomfort. One-third of those affected received medical care. Previous hysterectomy had no effect on the reported prevalence estimates. Hysterectomized women reported moderate to severe complaints more often than non-hysterectomized ones. There were no significant differences in prevalence estimates between former and current smokers and non-smokers. PMID- 10738929 TI - Reproducibility of the seven-day voiding diary in women with stress urinary incontinence. AB - In this study we aimed to assess the reproducibility of the 7-day voiding diary in women with stress urinary incontinence. We compared two 7-day voiding diaries completed at 4-week intervals by 138 women with stress urinary incontinence enrolled in an interventional trial. The correlation coefficient for the number of weekly incontinence episodes between the two diaries was 0.831. For 280 voiding diaries the results of the first 3 days correlated well with results of the last 4 days (r = 0.887). The number of incontinence episodes, as recorded on a 7-day voiding diary, is a reproducible outcome measure in women with stress urinary incontinence. Results from the first 3 days of a diary correlate well with the last 4 days, suggesting that a 3-day diary is an appropriate outcome measure for clinical trials evaluating treatments for stress incontinence. PMID- 10738930 TI - Perineal assessment of urethrovesical junction mobility in young continent females. AB - This paper describes the application of perineal ultrasound (7 MHz transducer) in the assessment of the urethrovesical junction (UVJ) in 40 continent nulliparous females. Measurements of UVJ vertical and horizontal movements were assessed in relation to the inferior border of the pubic symphysis from resting to the maximum straining position. Participants were examined in the dorsal lithotomy position with no more than 50 ml of urine in the bladder. Volunteers' average age was 16 years (range 10-25). The mean vertical movement was 5.3+/-2.4 mm (maximum 9 mm). In 95% of participants the measurement for horizontal movement did not exceed 11.2 mm. Perineal ultrasound in patients with the bladder practically empty is an easy way to assess urethrovesical junction mobility objectively. It avoids the possible influence of detrusor contraction. A standard method for this assessment can provide accurate essential information for classification, management and follow-up of urinary incontinence. PMID- 10738931 TI - Treatment guidelines for classic and non-ulcer interstitial cystitis. AB - Interstitial cystitis (IC) is a chronic disease of as yet unknown etiology. It commonly affects females, presenting with symptoms of pain on bladder filling, and urinary frequency. Accumulated evidence indicates that IC is a heterogeneous syndrome. Compared to classic IC, the non-ulcer type appears different concerning symptomatic, endoscopic and histological findings, as well as the response to various forms of treatment. This review gives an introduction to the syndrome of IC, concerning epidemiology, clinical characteristics, diagnostic criteria and etiological considerations. A variety of treatment modalities have been suggested and are assessed and reviewed, such as hydrodistension of the bladder, intravesical instillation therapy, oral medication, transcutaneous electrical nerve stimulation, transurethral resection of diseased bladder tissue, and supratrigonal cystectomy followed by enterocystoplasty and urinary diversion. Our algorithm on non-surgical and surgical treatment for classic and non-ulcer IC is presented. PMID- 10738932 TI - Bladder augmentation. AB - This paper reviews bladder augmentation, which has been proved to be an effective way of providing a well functioning urine reservoir that protects the upper urinary tract and allows patients to have a good quality of life. Good results have been achieved with the use of all types of bowel segments. Lifetime follow up and recognition of the complications is mandatory. Very careful patient selection is essential in order to achieve better long-term results. The principle of urothelial preservation, introduced by autoaugmentation, is very promising in the effort to create a compliant urinary reservoir without metabolic disturbance and without the risk of cancer. PMID- 10738933 TI - Urinary stress incontinence among obese women: review of pathophysiology therapy. AB - Obesity is a common condition among women in developed countries and has a major impact on stress urinary incontinence. Women suffering from obesity manifest increased intra-abdominal pressures, which adversely stress the pelvic floor and may contribute to the development of urinary incontinence. In addition, obesity may affect the neuromuscular function of the genitourinary tract, thereby also contributing to incontinence. Accordingly, thorough evaluation of obese women must be performed prior to the institution of treatment. Weight loss may relieve urinary incontinence, but definitive therapy via operative procedures is effective even in obese patients and should be recommended with confidence. PMID- 10738934 TI - Giant calculus in a female urethral diverticulum. AB - Urethral diverticula with calculi are rare. This 5x6 cm calculus presented in a patient with recurrent urinary tract infections. Local excision was effective. PMID- 10738935 TI - Characterizing and reporting pelvic floor defects: the revised New York classification system. AB - The authors have devised a conceptual model and reporting system for characterizing, grading and staging pelvic floor defects. The system is user friendly and simple to learn and apply. It is based on commonly known anatomic landmarks and can be performed without memorizing or referring to a separate characterization and reporting plan. Completing the accompanying forms is self explanatory and provides the information needed for proper comprehension and recording of anatomic defects. The model and reporting format have been used at our institutions for 5 years by medical students, residents, fellows and attendings. It has several advantages over the Pelvic Organ Prolapse Quantitation (POPQ) system: (1) it uses known anatomic landmarks rather than alphabetic labels; (2) it grades lateral wall defects which the POPQ system omits; (3) it recognizes and reports isolated defects or tears which present as bulges in the vaginal walls without downward linear descent (prolapse); (4) it uses a one-page reporting form and a one-page checklist and vaginal profile; (5) it can be done easily in both the supine and the standing positions; (6) it requires simple instruments and a disposable measuring tape available in most office settings; (7) it includes urethral hypermobility in its reporting scheme; (8) it includes cervical length, perineal descent and other measurements in its reporting scheme; and (9) it is similar enough to the POPQ system that easy conversion to, and integration with, the POPQ reporting form and vaginal profile is possible. Prospective trials testing the validity of this system and comparing it with the POPQ system for validity, reliability, reproducibility, test-retest analysis, and interobserver and intraobserver variance are warranted. PMID- 10738937 TI - A histopathological study of lymphoepithelial island formation in labial salivary glands in patients with primary Sjogren's syndrome. AB - The proliferative status of lymphoepithelial islands in the labial salivary glands of primary Sjogren's syndrome (pSS) patients was investigated by counting the number of argyrophilic nucleolar organizer regions (AgNORs) in epithelial cells constituting the islands. The islands were classified into four groups and evaluated in terms of total area and three discrete zones of the islands. In each pSS group, the mean AgNOR number per total island epithelial cell nucleus was significantly higher than in control ductal epithelial cells. The zonal AgNOR number fluctuated during the process of island formation but became more uniform as the islands developed. Furthermore, statistically significant trends among the four pSS groups were observed in the ratio of T lymphocytes, B lymphocytes and plasma cells surrounding the islands. The results indicated that the islands are highly proliferative once island formation begins and that zonal island cell proliferation may be associated with the inflammatory cells. PMID- 10738936 TI - Innervation pattern and Ca2+ signalling in labial salivary glands of healthy individuals and patients with primary Sjogren's syndrome (pSS). AB - We have characterised the innervation pattern and intracellular Ca2+-signalling in labial salivary glands (LSG) of 16 patients with primary Sjogren's syndrome (pSS) and 27 healthy controls. Numerous immunoreactive nerve fibers (IRF) containing vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) were found around acini, ducts and blood vessels. Substance P (SP)-, neuropeptide Y-, tyrosine hydroxylase- and nitric oxide synthase-IRF were mainly surrounding ducts and blood vessels. The majority of pSS patients had inflamed LSG and the presence of focal lymphocytic infiltrates (FI) were more frequent and pronounced as compared with healthy controls. In areas with normal or diffusely inflamed LSG tissue, pSS patients demonstrated the same distribution of IRF as healthy controls with similar histology. However, IRF were absent in central areas of FI both in pSS and age-matched healthy controls. Although all pSS patients had hyposalivation, stimulation with acetylcholine, norepinephrine, phenylephrine, isoproterenol, VIP, PACAP, SP, adenosine 5' triphosphate and uridine 5'-triphosphate induced the same increase in the intracellular free Ca2+ concentration in LSG acini from both pSS patients and healthy controls, indicating the presence of functional receptor systems in vitro. PMID- 10738938 TI - A simple and rapid technique for the detection of Epstein-Barr virus DNA in HIV associated oral hairy leukoplakia biopsies. AB - A method of generating nucleic acid probes by polymerase chain reaction (PCR) for the detection of Epstein-Barr virus (EBV)-DNA by in situ hybridization in oral hairy leukoplakia (OHL) lesions is described. This method has the advantage over older methods of being cheaper, quicker and retaining sensitivity and specificity. Purified PCR products of Epstein-Barr virus DNA of 110 bp and 328 bp were labelled with biotin by nick translation or random primer labelling and were compared in in situ hybridization experiments with probes prepared by incorporation of biotin-labelled nucleotides in the PCR reaction mixture, with EBV viral DNA as a template. These probes were applied to 18 OHL tongue biopsies known to be positive for EBV-DNA, using a commercially available biotin-labelled BamHI "V" fragment EBV-DNA probe. To determine the specificity of the probes, we applied them to 20 normal tongue tissue samples and to 12 biopsies taken from keratotic tongue lesions from patients without risk factors for HIV infection and known to be negative for EBV-DNA. Clear positive signals for EBV-DNA were detected in all 18 cases of OHL biopsies using the amplimer of 328 bp labelled by PCR and random primer labelling. However, nick translation labelling was less efficient and sensitive. All control specimens were negative for EBV-DNA. PMID- 10738939 TI - Oral submucous fibrosis patients have altered levels of cytokine production. AB - Oral submucous fibrosis (OSF) is a pre-malignant fibrotic lesion of the mouth in betel quid chewers and is characterised by dense bands of collagen in the juxta epithelial region preceded by inflammation. We have investigated the spontaneous and stimulated production of cytokines by peripheral blood mononuclear cells (PBMC) from OSF patients and compared them with genetically-related relatives, Indian and Caucasian control subjects. The cytokines studied included: interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). The results show: a) significant differences in the stimulated versus non-stimulated levels of IL-1beta, IL-6, IL-8 and TNF-alpha but not of IFN-gamma production by patients, and in the relatives' stimulated versus non-stimulated levels of IL 1beta, IL-6 and IFN-gamma; b) no difference in the spontaneous cytokine production between any two groups; and c) significant increases in the patients' stimulated cytokines compared to the Caucasian and Indian controls (P< or =0.050). These results demonstrate increased levels of proinflammatory cytokines and reduced anti-fibrotic IFN-gamma in patients with OSF, which may be central to the pathogenesis of OSF. PMID- 10738940 TI - A novel cell line that retains the morphological characteristics of the cells and matrix of odontogenic myxoma. AB - Little is known about the histogenesis of the human odontogenic myxoma or the relation between tumour cells and the matrix. In order to attempt to remedy this situation, we established and investigated a cell line derived from a human odontogenic myxoma. To our knowledge this is the first cell line derived from this tumour. The cell line, named Mix 1, preserved features of the tumour cells. Mix 1 cells expressed vimentin, type I collagen, fibronectin, tenascin and hyaluronic acid. Ultrastructural analysis of cells of the tumour and cell line demonstrated similarities, both containing Golgi apparatus, rough endoplasmic reticulum and mitochondria indicative of secretory cells. Ultrastructural analysis showed the matrix to be represented by bundles of collagen fibrils in the tumour, and by irregular filaments in cultures more than 60 days old. The Mix 1 cell line promises to be an excellent model for investigating the biology of the odontogenic myxoma. PMID- 10738941 TI - Immunohistochemical study of bcl-2 protein, Ki-67 antigen and p53 protein in epithelium of glandular odontogenic cysts and dentigerous cysts. AB - The aim of the present study was the evaluation of the immunohistochemical expression of the apoptosis-inhibiting protein bcl-2, the cell-cycle-related antigen Ki-67 and the p53 protein, which is involved both in cell cycle and apoptosis regulation, in the lining epithelium of glandular odontogenic cysts of the jaws. Formalin-fixed and paraffin-embedded tissue sections of three glandular odontogenic cysts and six dentigerous cysts were immunostained with a standard avidin-biotin peroxidase procedure, after microwave antigen retrieval. The glandular odontogenic cysts showed immunoreactivity for bcl-2 protein in the basal and suprabasal layers, while staining in dentigerous cysts was basal or focal. Most mucous cells and superficial cuboidal cells were negative. The percentage of Ki-67- or p53-positive cells was lower in glandular odontogenic cysts compared with dentigerous cysts. The findings suggest that the biological behavior of glandular odontogenic cysts may be associated with deregulation of cell death in the lining epithelium, while cell proliferation and p53 status do not seem to play a significant role. PMID- 10738942 TI - Opportunities for immune modulation in the spondyloarthropathies with special reference to gut inflammation. AB - The spondyloarthropathies (SpA) are a related group of disorders, characterized primarily by spondylitis, pauci-articular arthritis and enthesitis. The presence of subclinical gut inflammation in patients with SpA ranges from 25 to 75%, depending upon the type of SpA. Several data suggest that the association between gut inflammation and synovitis reflects an etiopathogenetic relationship, and that strategies which interfere with the gut inflammation may also modulate the synovitis. Here we review some standard as well as experimental drugs used in the treatment of patients with inflammatory bowel disease and discuss what is known about their effect on SpA-related locomotor manifestations. For the more experimental drugs, such as cytokines, anticytokines and anti-adhesion compounds, clinical trials in patients with SpA are still very scarce. PMID- 10738943 TI - Increased expression of matrix metalloproteinase-9 in neutrophils in glycogen induced peritoneal inflammation of guinea pigs. AB - OBJECTIVE: Matrix metalloproteinase (MMP)-9 plays an important role in neutrophil extravasation and migration by its ability to degrade the major components of basement membrane. To evaluate the expression of neutrophil MMP-9 under inflammatory conditions, we examined the levels of MMP-9 and its mRNA in neutrophils of glycogen-induced peritoneal inflammation. MATERIALS AND METHODS: Male Hartley guinea pigs weighing 250-300 g were intraperitoneally injected with 0.17% glycogen solution, and 13-15 h after the injection, blood and peritoneal neutrophils were isolated. The levels of MMP-9 and its mRNA were analyzed by gelatin zymography and Northern blotting, respectively. Furthermore, MMP-9 activities in the peritoneal supernatants were measured. RESULTS: MMP-9 level in peritoneal neutrophils was essentially the same as that in blood neutrophils, although peritoneal neutrophils were assumed to have extracellularly released MMP 9 from the granules during infiltration into the peritoneal cavity. Interestingly, MMP-9 mRNA was expressed more abundantly in peritoneal neutrophils than in blood neutrophils (p<0.01). Moreover, MMP-9 levels in blood and peritoneal neutrophils were reduced to 30-45% of non-treated controls by actinomycin D (500 microg/kg) or cycloheximide (10 mg/kg)-treatment (p<0.05). In contrast, MMP-9 activity increased in the peritoneal supernatants of glycogen injected animals was not significantly affected by actinomycin D- or cycloheximide-treatment. In addition, when blood neutrophils of non-injected animals were stimulated with 10(-7) M N-formyl-Met-Leu-Phe, 10 microg/ml lipopolysaccharide, 10 ng/ml phorbol 12-myristate 13-acetate, 10(-8) M IL-8 or 100 U/ml tumor necrosis factor-a, expression of MMP-9 mRNA was markedly increased (p<0.05). CONCLUSIONS: The present observations indicate that MMP-9 gene is transcribed, and MMP-9 protein is synthesized in neutrophils during glycogen injected peritoneal inflammation. Moreover, it is likely that MMP-9 protein in the peritoneal supernatants is mostly derived from preformed MMP-9 which is stored in the neutrophil granules and extracellularly released during infiltration of neutrophils into the peritoneal cavity. Finally, the transcription of MMP-9 gene can be upregulated in neutrophils by stimulation with inflammatory mediators, even after neutrophils have been matured. PMID- 10738944 TI - Molecular mechanisms of lipopolysaccharide induced ICAM-1 expression in A549 cells. AB - OBJECTIVE AND DESIGN: Lung intercellular adhesion molecule-1 (ICAM-1) expression is increased by LPS or hyperoxia on type II cells in vivo. The goals of the present study were to determine the mechanisms of ICAM-1 expression in a lung alveolar epithelial cell line (A549) exposed to lipopolysaccharide (LPS). MATERIALS: A549 cells, a transformed human cell line with characteristics of alveolar epithelial cells, were used. TREATMENT: Cells were exposed to LPS, TNF alpha, IL-1beta, or media alone for up to 12 h. METHODS: Northern blot analyses were done to determine mRNA expression of ICAM-1 after exposures. Protein binding to NF-kappaB sequences were determined by gel mobility shift assays and super shift analysis. RESULTS: ICAM-1 mRNA expression was induced in A549 cells with exposure to LPS for 1 to 4 h, and was diminished to baseline at 8 h, and the inductions were independent of TNF-alpha and IL-1beta expression. Nuclear protein extracts from LPS-exposed cells bound to a NF-kappaB sequence and the timing of increased binding correlated closely with ICAM-1 mRNA induction. Super-shift studies indicated that p65 was involved in the binding to the NF-kappaB sequence and p50 was not. CONCLUSION: LPS inducibility of ICAM-1 mRNA in A549 cells is independent of TNF- and IL-1 in A549 cells, and the similar time course of mRNA induction and NF-kappaB activation suggest the induction of ICAM-1 is mediated, in part, by NF-kappaB. PMID- 10738945 TI - Synergistic actions of antibacterial neutrophil defensins and cathelicidins. AB - OBJECTIVE: Activated neutrophils extracellularly release antibacterial defensins and cathelicidins from the granules. In this study, to elucidate the interactions between defensins and cathelicidins in the extracellular environment, we evaluated the individual and synergistic actions of defensins and cathelicidins in the presence of physiological concentration of NaCl (150 mM). MATERIALS AND METHODS: Antibacterial activities against Escherichia coli and Staphylococcus aureus were assessed using human and guinea pig defensins and cathelicidins. Furthermore, the effect of defensins and cathelicidins on membrane permeabilization was examined using E. coli ML-35p, as a target organism. RESULTS: In the absence of NaCl, human defensin (HNP-1) and guinea pig defensins (GNCPs) exhibited the antibacterial activities in a dose-dependent manner (0.1-10 microg/ml); however, their activities were completely lost in the presence of 150 mM NaCl. In contrast, the antibacterial activities of human cathelicidin (CAP18/LL-37) and guinea pig cathelicidin (CAP11) were resistant to NaCl. Interestingly, HNP-1 and GNCPs synergized with CAP18/LL-37 and CAP11 to enhance the antibacterial activities against E. coli and S. aureus in the presence of 150 mM NaCl (p<0.05). Similarly, HNP-1 and GNCPs were synergistic with CAP18/LL-37 and CAP11 to potentiate the outer and inner membrane permeabilization of E. coli ML-35p (p<0.05). CONCLUSIONS: Together these observations indicate that when extracellularly released from neutrophils, defensins cannot function as antibacterial molecules by themselves, but can synergistically work with cathelicidins to exert the antibacterial activity in the extracellular milieu by augmenting the membrane permeabilization of target cells. PMID- 10738946 TI - The expression of three types of CINCs by lipopolysaccharide-stimulated rat macrophages is inhibited similarly by anti-inflammatory steroids. AB - OBJECTIVE: Expression of CINCs was regulated differentially in lipopolysaccharide stimulated rat macrophages. We examined whether the expression of CINCs in lipopolysaccharide-stimulated rat macrophages is similarly inhibited by anti inflammatory drugs. MATERIALS AND METHODS: Rat peritoneal macrophages were stimulated by lipopolysaccharide in the presence of anti-inflammatory steroids (dexamethasone, prednisolone and hydrocortisone) or non-steroidal anti inflammatory drugs (indomethacin and piroxicam). The production and mRNA expression of three types of CINCs were measured using enzyme-linked immunosorbent assay and northern hybridization. RESULTS: Anti-inflammatory steroids; dexamethasone, prednisolone and hydrocortisone, dose-dependently inhibited the production of CINC-1, -2 and -3, whose inhibitory patterns were similar to each other. Furthermore mRNA expression of each CINC was inhibited by dexamethasone in a dose-dependent manner. In contrast, non-steroidal anti inflammatory drugs, indomethacin and piroxicam were without effect. Expression of each CINC was regulated differently; the production of CINC-1 reached a maximum at 12 h and then slightly decreased after lipopolysaccharide stimulation, whereas that of CINC-2 and CINC-3 increased up to 24 h. Dexamethasone inhibited the CINCs production and mRNA expression at 9 h after lipopolysaccharide stimulation. CONCLUSIONS: These results indicate no difference among CINC-1, -2 and -3 in the inhibition of production and mRNA expression of CINCs by anti-inflammatory steroids, although lipopolysaccharide differentially induces expression of each CINC expression in culture of rat macrophages. PMID- 10738947 TI - The effects of S1319, a novel marine sponge-derived beta2-adrenoceptor agonist, on IgE-mediated activation of human cultured mast cells. AB - OBJECTIVE: This study aimed to evaluate the ability of S1319 (4-hydroxy-7-[1-(1 hydroxy-2-methylamino) ethyl]-1,3-benzothiazol-2(3H)-one acetate), a novel beta2 adrenoceptor selective agonist derived from marine sponge, to inhibit IgE mediated activation of human cultured mast cells (HCMC) in vitro. MATERIALS AND METHODS: We examined the effect of S1319 (racemate) on tryptase release and tumor necrosis factor-alpha (TNF-alpha) production in HCMC generated from human cord blood cells, after cross-linking of high affinity immunoglobulin E receptors (FcepsilonRI), compared with those of the nonselective beta-adrenoceptor agonist, isoproterenol (R-isomer), the selective beta2-adrenoceptor agonist, salbutamol (racemate), and the selective and long-acting beta2-adrenoceptor agonist, formoterol (racemate). We also evaluated the effect of S1319 on the intracellular cAMP level, inositol phosphate production and protein tyrosine phosphorylation in HCMC. RESULTS: S1319 and beta-adrenoceptor agonists inhibited the IgE-mediated release of tryptase. Approximate IC50 values of S1319, formoterol, isoproterenol and albuterol for the inhibition of tryptase release were 0.51+/-0.12, 0.15+/ 0.1, 0.80+/-0.09, and 28+/-32.4 nM, respectively. S1319 and beta-adrenoceptor agonists also inhibited TNF-alpha production by HCMC in a concentration-dependent manner. Approximate IC50 values of S1319, formoterol and isoproterenol for the inhibition of TNF-alpha production were 0.19+/-0.03, 0.28+/-0.02 and 0.32+/-0.03 nM, respectively. S1319 caused a concentration-dependent increase in total cell cyclic AMP levels in HCMC. On the other hand, S1319 inhibited the accumulation of inositol 1,4,5-triphosphate and IgE-mediated protein tyrosine phosphorylation of 42-kDa protein, p42 mitogen activated protein (MAP) kinase (ERK-2). CONCLUSION: These results indicate that S 1319 and beta-adrenoceptor agonists are potent inhibitors of the IgE-mediated release of mediators from HCMC. PMID- 10738948 TI - Response acquisition under direct and indirect contingencies of reinforcement. AB - We compared the effects of direct and indirect reinforcement contingencies on the performance of 6 individuals with profound developmental disabilities. Under both contingencies, completion of identical tasks (opening one of several types of containers) produced access to identical reinforcers. Under the direct contingency, the reinforcer was placed inside the container to be opened; under the indirect contingency, the therapist held the reinforcer and delivered it to the participant upon task completion. One participant immediately performed the task at 100% accuracy under both contingencies. Three participants showed either more immediate or larger improvements in performance under the direct contingency. The remaining 2 participants showed improved performance only under the direct reinforcement contingency. Data taken on the occurrence of "irrelevant" behaviors under the indirect contingency (e.g., reaching for the reinforcer instead of performing the task) provided some evidence that these behaviors may have interfered with task performance and that their occurrence was a function of differential stimulus control. PMID- 10738949 TI - An evaluation of the effects of matched stimuli on behaviors maintained by automatic reinforcement. AB - The purpose of the current investigation was to extend the literature on matched stimuli to three dissimilar forms of aberrant behavior (dangerous climbing and jumping, saliva manipulation, and hand mouthing). The results of functional analyses suggested that each behavior was automatically reinforced. Preference assessments were used to identify two classes of stimuli: items that matched the hypothesized sensory consequences of aberrant behavior (matched stimuli) and items that produced sensory consequences that were not similar to those produced by the aberrant behavior (unmatched stimuli). The effects of providing continuous and noncontingent access to either the most highly preferred matched or the most highly preferred unmatched stimuli were assessed relative to a condition in which no stimuli were available. Overall results suggested that providing access to items that matched the hypothesized sensory consequences of aberrant behavior may be more effective than simply selecting stimuli either arbitrarily or based on the results of preference assessments alone. PMID- 10738950 TI - Effects of increased response effort on self-injury and object manipulation as competing responses. AB - We evaluated the effects of a response-effort intervention on the occurrence of self-injurious hand mouthing and a competing response (object manipulation) with 4 individuals who had profound developmental disabilities. During Phase 1, results of functional analyses showed that all participants engaged in high levels of hand mouthing in the absence of social contingencies, suggesting that the behavior was maintained by automatic reinforcement. In Phase 2, preferred leisure items were identified for participants during assessments in which duration of leisure item manipulation was used as the index of preference. In Phase 3, participants were observed to engage in high levels of hand mouthing and in varying levels of object manipulation when they had free access to their most preferred leisure items during baseline. The effects of increased response effort on hand mouthing and object manipulation were then evaluated in mixed multiple baseline and reversal designs. The response-effort condition was identical to baseline, except that participants wore soft, flexible sleeves that increased resistance for elbow flexion but still enabled participants to engage in hand mouthing. Results showed consistent decreases in SIB and increases in object manipulation during the response-effort condition for all participants. These results suggested that a less preferred reinforcer (produced by object manipulation) may substitute for a more highly preferred reinforcer (produced by hand mouthing) when response effort for hand mouthing was increased. DESCRIPTORS: self-injurious behavior, automatic reinforcement, reinforcer substitutability, response effort PMID- 10738951 TI - Analysis and treatment of finger sucking. AB - We analyzed and treated the finger sucking of 2 developmentally typical children aged 7 and 10 years. The functional analysis revealed that the finger sucking of both children was exhibited primarily during alone conditions, suggesting that the behavior was maintained by automatic reinforcement. An extended analysis provided support for this hypothesis and demonstrated that attenuation of stimulation produced by the finger sucking resulted in behavior reductions for both children. Treatment consisted of having each child wear a glove on the relevant hand during periods when he or she was alone. Use of the glove produced zero levels of finger sucking for 1 participant, whereas only moderate reductions were obtained for the other. Subsequently, an awareness enhancement device was used that produced an immediate reduction in finger sucking. PMID- 10738953 TI - Assessment of a response bias for aggression over functionally equivalent appropriate behavior. AB - We evaluated the effects of a dense (fixed-ratio 1) schedule of reinforcement for an 11-year-old boy's mands for toys while aggression produced the same toys on various schedules chosen on the basis of a progressive-ratio probe. Based on the probe session data, we accurately predicted that aggression would be more probable than mands when the schedules were equal or slightly discrepant, but that mands would be more probable when the schedule discrepancy was large. PMID- 10738952 TI - Evaluating the effects of functional communication training in the presence and absence of establishing operations. AB - We conducted functional analyses of aberrant behavior with 4 children with developmental disabilities. We then implemented functional communication training (FCT) by using different mands across two contexts, one in which the establishing operation (EO) that was relevant to the function of aberrant behavior was present and one in which the EO that was relevant to the function of aberrant behavior was absent. The mand used in the EO-present context served the same function as aberrant behavior, and the mand used in the EO-absent context served a different function than the one identified via the functional analysis. In addition, a free play (control) condition was conducted for all children. Increases in relevant manding were observed in the EO-present context for 3 of the 4 participants. Decreases in aberrant behavior were achieved by the end of the treatment analysis for all 4 participants. Irrelevant mands were rarely observed in the EO-absent context for 3 of the 4 participants. Evaluating the effectiveness of FCT across different contexts allowed a further analysis of manding when the establishing operations were present or absent. The contributions of this study to the understanding of functional equivalence are also discussed. PMID- 10738954 TI - The effects of noncontingent delivery of high- and low-preference stimuli on attention-maintained destructive behavior. AB - An adolescent with severe mental retardation and cerebral palsy who displayed attention-maintained destructive behavior was exposed to noncontingent reinforcer delivery (NCR) with either a high-preference or a low-preference stimulus while reinforcement for destructive behavior with attention remained in effect (i.e., NCR without extinction). NCR without extinction was effective only when the high preference stimulus was available, suggesting that systematic assessment of stimulus quality may enhance the effectiveness of NCR with alternative stimuli. PMID- 10738955 TI - The effects of establishing operations on preference assessment outcomes. AB - Preference assessments were conducted for 4 individuals with developmental disabilities across conditions of (a) control, allowing equal access to all stimuli prior to the preference assessment; (b) deprivation, allowing no access to one stimulus for 48 hr prior to the assessment; and (c) satiation, allowing free access to one stimulus for 10 min immediately prior to the assessment. Deprivation resulted in increased preference, whereas satiation resulted in decreased preference compared to control conditions. PMID- 10738956 TI - The effectiveness of contingency-specific and contingency-nonspecific prompts in controlling bathroom graffiti. AB - This study replicates and extends the work of Watson (1996) in which a sign eliminated graffiti when posted on bathroom walls. The present study investigated the effects of three different signs on walls in six men's bathrooms located on a university campus. Posting the signs was followed by the elimination or sharp reduction of graffiti. Removal of the signs was followed by a resurgence of graffiti. PMID- 10738957 TI - Effects of choice of stimuli as reinforcement for task responding in reinforcement for task responding in preschoolers with and without developmental disabilities. AB - The effects of choice and no choice of stimuli as reinforcement for task responding were investigated across preschoolers with and without disabilities. Five less preferred stimuli were identified for each participant using a stimulus preference assessment. No differences were found for choice and no-choice conditions when the less preferred stimuli were used as reinforcers. PMID- 10738958 TI - Reading, equivalence, and recombination of units: a replication with students with different learning histories. AB - First graders, preschoolers, special education students, and adults received a reading program in which they learned to match printed to dictated words and to construct (copy) printed words. The students not only learned to match the training words but also learned to read them. In addition, most of the students learned to read new words that involved recombinations of the syllables of the training words. The results replicate and extend the generality of a prior analysis of a reading program based on stimulus equivalence and recombination of units. PMID- 10738959 TI - A longitudinal study of informational interventions to save energy in an office building. AB - Informational interventions were employed to promote two behaviors relevant for efficient heating of individual offices in a large office building. In two successive winter seasons, interventions were applied during 4-week periods. Short-term effects were assessed weekly, and long-term effects were assessed 1 year after each of the two intervention periods. Improvements were observed in each intervention period, with partial behavior maintenance 1 year later. The changes observed in the individual offices across conditions are suggestive of the program's capacity to correct relapses in earlier proenvironmental behavior. PMID- 10738960 TI - Further evaluation of low-ranked items in stimulus-choice preference assessments. AB - The generality of the findings reported by DeLeon, Iwata, and Roscoe (1997) was examined by conducting two stimulus-choice preference assessments, the second of which evaluated low-ranked items from the initial assessment. Results for the 2 participants suggested that supplementary assessments of low-ranked items may be useful for identifying a wider variety of reinforcing stimuli. PMID- 10738961 TI - Using brief assessments to evaluate aberrant behavior maintained by attention. AB - We examined the use of brief functional assessments to identify idiosyncratic variables associated with aberrant behavior maintained by positive reinforcement in the form of attention. Two participants with severe developmental disabilities and their parents were involved in the assessment and treatment evaluation process. A modified attention condition was introduced, which involved both parents interacting with a third person. Results of the assessment demonstrated that aberrant behavior occurred only in the modified attention condition for both participants. Treatment consisted of the parents delivering attention on a fixed time schedule during this specific social context. Results indicated that the treatment reduced problem behavior. Follow-up evaluations with 1 participant indicated maintenance of treatment effects for up to 6 months. PMID- 10738962 TI - Functional analysis of aberrant behavior through measurement of separate response topographies. AB - Functional analysis results for multiple topographies of aberrant behavior were graphed in an aggregate fashion and then separately for 48 clients. The results indicated that multiple topographies of behavior may be maintained by different contingencies. These results indicate that graphing functional analysis data in an aggregate fashion and then separately may improve the accuracy of their interpretation. PMID- 10738964 TI - National Institutes of Health stem cell research guidelines. PMID- 10738965 TI - Hematopoiesis and angiogenesis: the same landscape from different points? PMID- 10738963 TI - Integrating basic and applied research and the utility of Lattal and Perone's Handbook of research methods in human operant behavior. AB - Lattal and Perone's Handbook of methods used in human operant research on behavioral processes will be a valuable resource for researchers who want to bridge laboratory developments with applied study. As a supplemental resource, investigators are also encouraged to examine the series of papers in the Journal of Applied Behavior Analysis that discuss basic research and its potential for application. Increased knowledge of behavioral processes in laboratory research could lead to innovative solutions to practical problems addressed by applied behavior analysts in the home, classroom, clinic, and community. PMID- 10738966 TI - The influence of flow cytometric gating strategy and cell preparation procedures on CD34+ cell quantification. PMID- 10738967 TI - Hematopoiesis and angiogenesis: a link between two apparently independent processes. AB - In early ontogeny, hematopoiesis is closely associated with angiogenesis. This article reviews recent studies on the role of angiogenic factors that regulate the proliferation and differentiation of endothelial cells in promoting hematopoietic cell growth and studies on the ability of hematopoietic cytokines to affect several endothelial cell functions. The findings in all these studies support the hypothesis formulated at the beginning of this century that a common ancestral cell, the hemangioblast, gives rise to cells of both the endothelial and the hematopoietic lineages. PMID- 10738968 TI - Unraveling distinct intracellular signals that promote survival and proliferation: study of erythropoietin, stem cell factor, and constitutive signaling in leukemic cells. AB - This review summarizes selected recent studies of the intracellular signals that allow erythroid cells to survive and proliferate under the control of erythropoietin (EPO) and alteration in signals that contribute to EPO-independent survival and proliferation. The hypothesis explored is that the proliferation and survival signals are distinct and can be separately studied with the proper cell lines and growth factor stimulation. The anti- and pro-apoptotic proteins Bcl-XL and BAD are highly implicated in EPO-dependent survival of erythroid cells. Stat5 activity appears to be upstream of Bcl-XL expression such that pathologic, constitutive activation of Stat5 may be a common event in leukemic cells that become resistant to apoptosis by constitutive expression of Bcl-XL. Other signals apparently also control the expression of Bcl-XL, such as the expression of JunB which seem to be required to suppress Bcl-XL expression when EPO is withdrawn. Apoptosis may also be triggered by inactivation of Bcl-XL by BAD. Dephosphorylation of BAD as a result of withdrawal of survival factors converts prosurvival BAD to proapoptotic BAD. Phosphorylation of BAD at the serine 112 residue seems critical to promoting survival. Constitutive activation of a kinase that phosphorylates BAD serine 112 may, therefore, contribute to resistance to apoptosis in leukemic cells. We describe the resistance of erythroleukemic cells to apoptosis induced by EPO withdrawal apparently caused by constitutive BAD phosphorylation. The resistance to apoptosis in these cells is reversed by treatment with the PI3-kinase inhibitor, LY294002, suggesting that resistance to apoptosis in these cells likely results from constitutive P13-kinase that is an upstream activator of an S-112 BAD kinase. The MAP kinase cascade is apparently active in EPO-dependent and stem cell factor (SCF)-dependent proliferation but not survival. In addition, autocrine tumor necrosis factor-a! (TNF-alpha) may also be a proliferation factor not affecting survival. P13-kinase seems to be required for full EPO-dependent proliferation but is not required for EPO dependent survival (but it can promote survival when activated). PMID- 10738970 TI - Modulation of CD4, CXCR-4, and CCR-5 makes human hematopoietic progenitor cell lines infected with human herpesvirus-6 susceptible to human immunodeficiency virus type 1. AB - Two CD34+ human hematopoietic progenitor cell (HPC) lines, KG-1 and TF-1, became susceptible to human immunodeficiency virus type 1 (HIV-1) infection in the presence of a concurrent infection by human herpesvirus-6 (HHV-6). We have analyzed the possible mechanism(s) underlying this phenomenon in light of the recent demonstration that at least two members of the chemokine receptor family, CXCR4 (LESTR/fusin) and CCR5 molecules, are the HIV-1-specific coreceptors necessary, together with the high-affinity receptor CD4, for entry into target cells of T-tropic and M-tropic HIV-1 isolates, respectively. KG-1 cells show CXCR4 and CCR5 surface molecules in a large proportion of the cell population. Therefore, their susceptibility to both T-tropic and M-tropic HIV-1 strains, caused by HHV-6 infection, can be explained by the HHV-6-induced appearance of CD4 molecules in about 40% of the cell population. In TF-1 cells, 10%-15% of which are CD4+ and exhibit a consistent CCR5 presence in a large proportion of the cell population and a hardly detectable amount of CXCR4 in a very limited number of cells, HHV-6 infection does not modify the cell surface availability of HIV-1-specific high-affinity receptor or coreceptors. PMID- 10738969 TI - Application of whole blood and peripheral blood progenitor cells (PBPC) and new strategies for rescue after intensive cyclic chemotherapy in high-risk breast cancer. AB - The efficacy of autologous peripheral stem cells given as mobilized whole blood or leukapheresis product for hematopoietic rescue after intensive chemotherapy was studied in 34 consecutive female patients with high-risk breast cancer. All patients received six cycles of chemotherapy regimen EC (epirubicin 150 mg/m2 and cyclophosphamide 1250 mg/m2) at 14-day intervals. In the first cycle, chemotherapy was given on day 1, and 24 h later mobilization of PBPC was started with G-CSF at a dose of 5 microg/kg/day for 13 days. In all other cycles, G-CSF was given at the same dose from day 7. On days 11, 12, and 13, leukaphereses were performed, and whole blood was collected on day 14 (the peak incidence of colony forming units-granulocyte-macrophage [CFU-GM] burst-forming units-erythrocyte [BFU-E], and colony-forming unit-granulocyte-erythrocyte-macrophage-megakaryocyte [CFU-GEMM]). The second cycle of chemotherapy was started on day 15, and 24 h later, whole blood (collected in the first cycle) was reinfused, and the same was done in the third cycle. In the fourth to sixth chemotherapy cycles, leukapheresis product was used for hematopoietic rescue. The median increment of absolute values in both whole blood and leukapheresis product was as follows: CD34+ cells over baseline was approximately 17.4-fold, CFU-GM was 85.3-fold, BFU E was 95.9-fold, and CFU-GEMM was 44.2-fold. In the cycles with whole blood support, the mean values of applied progenitors per cycle were CD34+ cells 1.52 x 10(6)/kg, CFU-GM, 1.18 x 10(5)/kg, BFU-E 2.54 x 10(5)/kg, CFU-GEMM 0.31 x 10(5)/kg. In the courses with PBPC support, the mean values of progenitors were CD34+ 2.04 x 10(6)/kg, CFU-GM 1.59 x 10(5)/kg, BFU-E 2.87 x 10(5)/kg, and CFU GEMM 0.34 x 10(5)/kg. Leukopenia in patients supported with whole blood versus leukapheresed PBPC was as follows: grade 4, 13/6 (38.2%/17.6%), grade 3, 19/23 (55.9%/70.6%), and grade 2, 1/4 (2.9%/11.8%), respectively. Thrombocytopenia was grade 4, 11/6 (32.4%/17.6%), grade 3, 10/7 (29.4%/20.6%), grade 2, 7/13 (20.6%/38.2%), and grade 1, 6/6 (17.6%/17.6%), respectively. The median follow-up analysis was at 24.6 (7-36) months. High-risk patients previously treated with surgery and adjuvant chemotherapy (n = 5) were not evaluated for response. In 21 patients with locally advanced or inflammatory breast carcinoma the response rate (RR) was 94%, CR was 90%, and PR was 15%. No response to therapy was observed in 1 patient. In 8 patients with metastatic disease, RR was 75%, there was no CR, and PR was 75%. Two patients died during therapy. Relapse-free survival (RFS) in the adjuvant group was 23.7 (range 12-36) months and in the group with locally advanced disease was 18.2 (range 7-27) months. In the group with metastatic disease, time to tumor progression (TTP) was 12.1 (range 1-16) months. Mean duration of hospital stay for whole blood reinfusion in the second and third chemotherapy cycles was 6.7 (range 5-8) days and for PBPC in the fourth to sixth cycles was 6.2 (range 4-8) days, which at p < 0.001 was not statistically significant. PMID- 10738972 TI - Serum levels of stem cell factor in patients during transplantation of bone marrow or peripheral blood stem cells. AB - Stem cell factor (SCF) synergizes with other cytokines in vitro to stimulate the proliferation and differentiation of cells of the myeloid, megakaryocytic, erythroid, and lymphoid lineages. In vivo, it may play a role in engraftment after transplantation of bone marrow (BM) or peripheral blood stem cells (PBSC). Serum levels of SCF were closely monitored in 82 patients before and after allogeneic (n = 38), autologous (n = 6), or syngeneic (n = 1) BM transplantation (BMT) or autologous PBSC transplantation (PBSCT) (n = 37), respectively. SCF serum levels fluctuated around a mean in patients after allogeneic or autologous BMT or after PBSCT. In two patient subgroups (5 patients with acute myeloid leukemia [AML] and 6 patients with chronic myelogenous leukemia [CML]) with identical pretransplant conditioning regimen followed by allogeneic BMT, serum IL 6 levels significantly increased up to day +14 (p < 0.05). Correlation was not found between SCF serum levels and leukocyte or thrombocyte counts or the day of engraftment of these cell types. These data are a basis for further studies and constitute a further mosaic stone in understanding the changes in the complex cytokine network during engraftment. PMID- 10738971 TI - Biological activities of the lectin, abrin-a, against human lymphocytes and cultured leukemic cell lines. AB - The cytoagglutination by abrin-a against human cultured cell lines derived from acute lymphoblastic leukemia (ALL) and human peripheral blood lymphocytes obtained from normal adults and from patients with adult T cell leukemia (ATL) was investigated. Among acute T lymphoblastic leukemia (T-ALL) cell lines, abrin a showed strong cytoagglutination against relatively differentiated cell lines, such as Jurkat and CCRF-HSB-2. Among acute B lymphoblastic leukemia (B-ALL) cell lines, abrin-a strongly agglutinated an immature cell line, NALM6. In comparison with ALL cell lines, cytoagglutination by abrin-a against normal lymphocytes was weak. Abrin-a showed higher cytoagglutination against lymphocytes derived from ATL than lymphocytes derived from normal adults. In connection with the cytoagglutination, abrin-a-induced cytotoxicity against human cultured leukemic cell lines was evaluated. In proportion to the extent of cytoagglutination, abrin a induced cytotoxicity in Jurkat, CCRF-HSB-2, MOLT-4, RPMI8402, and BALL-1 as well. Although CCRF-CEM and BALM-1 were both weakly agglutinated by abrin-a, these cell lines were very sensitive to the abrin-a-induced cytotoxicity. NALM6 was strongly agglutinated by abrin-a, but abrin-a exhibited less strong cytotoxicity against this cell line. These results suggest the feasible application of abrin-a as a tool to distinguish the human leukemic cells and its potential for clinical application. PMID- 10738973 TI - Natural killer (NK) and T cell-associated surface marker expression following allogeneic and autologous bone marrow transplantation (BMT). AB - Natural killer (NK) and T cell development was studied after allogeneic and autologous BMT. We determined the phenotypic expression and lytic ability of these subpopulations after BMT. Following T cell-depleted (TCD) BMT, the number of CD16+ and CD56+ cells peaked at 39 and 46 days, respectively, and constituted the majority of peripheral blood lymphocytes (PBL). Coexpression of CD3 and CD16 was <10% up to 14.5 weeks after transplant. Following allogeneic non-T cell depleted (NTCD) BMT, the number of CD16+ and CD56+ cells peaked at 6 weeks. CD3 expression was normal (70%-80%), % CD8+ cells was high (40%), and % CD4+ cells was low (20%). Following autologous BMT (ABMT), % CD3+ T cells was 80%, of which 70% expressed the CD8 marker. In contrast, CD4 expression was low (20%). CD16+ cells appeared 2.5-3 weeks after ABMT but with low frequency (20%), at which point 20%-30% of the CD3+ cells coexpressed CD16. A positive correlation was found between CD16 expression and cytotoxic capability. In conclusion, a marked difference was observed in NK and T cell-associated markers following TCD BMT, NTCD BMT, and ABMT. Following NTCD or ABMT, but not TCD BMT, a high percentage of cells co-express CD16 and CD3, which may indicate the possibility of a common NK and T cell progenitor. PMID- 10738974 TI - Transduction of human IL-9 receptor cDNA into TF1 cells induces IL-9 dependency and erythroid differentiation. AB - Human growth factor-dependent cell line TF1, which lacks interleukin (IL)-9 receptors (R) and does not grow in IL-9, was transduced with a retroviral vector containing human IL-9R cDNA and a selection marker. An IL-9-dependent TF1 cell line, which could also grow in other cytokines, was established after selection in G418 and could produce mature RBC in response to cytokine stimulation. TF1 cells transduced with the same viral vector without the IL-9R insert cDNA (mock control) and then selected responded the same as nontransduced TF1 cells. They failed to grow in response to IL-9 and did not generate RBC. An increased number and size of burst-forming units-erythroid (BFU-E)-like colonies were detected from IL-9R-transduced TF1 cells, compared with mock-transduced cells, in response to erythropoietin (EPO) and IL-9. To evaluate self-renewal and differentiation capacity, colony-replating assays were performed in the presence of IL-3, GM-CSF, IL-9, and EPO. After four replatings, the cloning efficiency of IL-9R-transduced TF1 cells decreased from 98% to 38%, most likely due to terminal erythroid cell differentiation. In contrast, no change in replating efficiency was detected in mock-transduced cells. TF1 cells stably expressing IL-9R and responding to IL-9 can serve as a cell line model to study the intracellular signals mediating IL-9 induced erythroid cell proliferation and differentiation. PMID- 10738975 TI - Bone marrow processing using the fenwal CS-3000 plus blood cell separator: results of 99 procedures. AB - BMT is used as an established therapy for patients with malignant and nonmalignant diseases. Many techniques for ex vivo treatment have been developed, but these techniques must be preceded by BM processing. We report our experience in processing 99 BM using the Fenwal CS-3000 Plus cell separator using the 1 special program. Ninety-nine procedures were performed in BM harvested from 73 patients and 26 healthy donors. The number of nucleated cells (NC), mononuclear cells (MNC), RBC, platelets, colony-forming units-granulocyte-macrophage (CFU GM), CD34+ cells, relative purity of MNC and PMN, and volume were determined in the unprocessed BM and in the final product. BM processing resulted in NC, MNC, CFU-GM, and CD34+ cell recoveries of 31%, 82.2%, 117.6%, and 97.8%, respectively. RBC, PMN, platelets, and volume removal, respectively, were 96%, 92%, 37.2%, and 85.1%. In pediatric patients, the volume reduction was significantly lower than in adult patients (79.6% versus 88.8%). No other significant differences were found between pediatric and adult results. We conclude that BM processing with the Fenwal CS-3000 Plus cell separator provides a product that can undergo further ex vivo treatments or cryopreservation. PMID- 10738976 TI - The number of CD34+ cells mobilized into the peripheral blood can predict the quality of subsequent collections. AB - PBPC were mobilized using a variety of chemotherapy regimens plus G-CSF in a group of 126 consecutive patients. Data are presented that show a close correlation between the number of CD34+ cells mobilized into the peripheral blood (PB) and the number of CD34+ cells subsequently collected by leukapheresis (R = 0.904). On the basis of this correlation, a regression formula was calculated that could give an estimate of the total number of CD34+ cells likely to be collected by leukapheresis from a given number of CD34+ cells per microliter PB. An easy-to-read table has been compiled to show how this type of analysis can be applied to predict the likely dose of CD34+ cells that will be obtained by leukapheresis over a wide range of patient weights. PMID- 10738977 TI - Optimizing preparation of normal dendritic cells and bcr-abl+ mature dendritic cells derived from immunomagnetically purified CD14+ cells. AB - The goal of this work was to optimize dendritic cell (DC) preparations obtained from patients suffering from chronic myeloid leukemia (CML) and compare them with DC prepared from normal CD14+ mononuclear cells (MNC). We studied normal DC and bcr-abl+ leukemic DC (CML-DC) yields, expression of membrane molecules, differentiation status, and ability to stimulate T cells. We isolated DC precursors from PBMC by CD14-specific immunoadsorption and cultured them for 7 days in GM-CSF and IL-4, followed by a 3-day incubation to fully differentiate the cells. We evaluated cultures of CML-DC using RPMI 1640 medium supplemented with FBS and X-VIVO 15 medium containing human AB serum. In contrast to cells matured in RPMI 1640, virtually all cells incubated in X-VIVO 15 expressed CD83, a marker of mature DC. CML-DC and normal DC were indistinguishable in expression of CD83, resulting in the highest percentage reported so far. The yields of normal DC and CML-DC from CD14+ cells were indistinguishable. The percentage of bcr-abl+ cells in PBMC varied among patients between 65% and 97% and the final CML-DC preparations were >98% bcr-abl+ the highest purity of bcr-abl+ cells to date. Normal DC and CML-DC were equally effective in stimulating proliferation of allogeneic and autologous T cells. These techniques provide highly enriched, mature, functional CML-DC. PMID- 10738978 TI - Factors influencing collection and engraftment of CD34+ cells in patients with breast cancer following high-dose chemotherapy and autologous peripheral blood progenitor cell transplantation. AB - Although autologous PBPC transplantation is being used increasingly for the treatment of breast cancer, there are few data on factors influencing mobilization and engraftment in these patients. We have analyzed these factors in 70 patients with advanced or metastatic breast cancer undergoing autologous PBPC transplantation. All patients were mobilized after stimulation with G-CSF, and a median of 3.16 x 10(6)/kg CD34+ cells (range 0.75-23.33) were infused. All patients received conditioning with a combination of cyclophosphamide, thiotepa, and carboplatin, and postinfusion G-CSF was administered to 60 patients. The median times to reach 0.5 x 10(9)/L and 1 x 10(9)/L neutrophils were 10 and 11 days, respectively. The median times to obtain 20 x 10(9)/L and 50 x 10(9)/L platelets were 12 and 18 days, respectively. An analysis of factors that influence CD34+ cell collection was performed by linear regression. Previous radiation therapy and increasing age were associated with lower numbers of CD34+ cells collected. Those variables that could influence the tempo of engraftment were examined by multivariate analysis using Cox regression models. The number of CD34+ cells infused was found to influence both neutrophil and platelet recovery. The use of G-CSF after transplant, accelerated neutrophil recovery, and having more than six cycles of previous chemotherapy was an unfavorable factor for recovering >50 x 10(9)/L platelets. PMID- 10738979 TI - Comparison of progenitor cell collection on day 4 or day 5 after steady-state stimulation with G-CSF alone in breast cancer patients: influence on CD34+ cell yield, subpopulation, and breast cancer cell contamination. AB - To determine the influence of apheresis timing on CD34+ cell yield, subpopulation, and breast cancer cell contamination, 48 women with breast cancer were stimulated from steady-state hematopoiesis in a prospective but nonrandomized study with 2 x 5 microg/kg G-CSF s.c. alone, and apheresis was started either on day 4 (n = 24) or day 5 (n = 24). Forty-eight women with breast cancer (stage II/III, n = 30; stage IV; n = 12; inflammatory, n = 6) and a median age of 44 years were well balanced between the two groups. In group I, aphersis was started on day 4 and additionally performed on day 5 after G-CSF stimulation, and in group II, apheresis was started on day 5. CD34+ cell count and CD34+ cell subpopulation were determined according to international criteria. Breast cancer cell contamination was detected by immunocytology. The median CD34+ cell harvest on day 4 was 3.3 x 10(6)/kg body weight (range 0.5-12.8) and 6 x 10(6)/kg BW (range 0.3-30) for patients starting on day 5 (p = 0.01). Those patients starting on day 4 achieved a median CD34+ cell count of 4 x 10(6)/kg (range 0.7-13) on day 5 (NS). Twenty-one percent of group I and 71% of group II achieved >5 x 10(6)/kg BW CD34+ cells in the first apheresis, whereas <2.5 x 10(6)/kg BW CD34+ cells in the first apheresis were observed in 38% of group I and 16% of group II. No differences were observed between the CD34+ cell subpopulations, CD34+/CD38+ (10.5% versus 10.5%) and CD34+/Thyl+ (1.5% versus 1.8%). The CD34+ cell harvest from consecutive collecting on days 4 and 5 was nearly identical to the harvest starting on day 5 (6.4 versus 6 x 10(6)/kg). Collecting CD34+ progenitor cells after stimulation with G-CSF alone on day 5 results in a significantly higher cell yield than starting collecting on day 4. No differences in respect to breast cancer cell contamination and CD34+ cell subpopulation were observed. PMID- 10738980 TI - Cellulase hyperproducers constructed from polyploids of Lentinus edodes. AB - A mycelial mat of Lentinus edodes was treated with 0.01% (w/v) colchicine solution for 240 h at 26 degrees C and autopolyploidization occurred. The mycelia were treated subsequently with the haploidizing reagent, benomyl, and fanshaped sectors were produced from colonies. Among such sectors, cellulase hyperproducers could be selected. The cellulase productivity of the hyperproducer, L1, did not decrease through five generations. PMID- 10738981 TI - ATP content of Mycobacterium tuberculosis grown in vivo and in vitro. AB - In order to determine the reason for the slow growth of Mycobacterium leprae either in a host or in vitro, the growth characteristics of Mycobacterium tuberculosis were studied. The ATP content of in vitro-grown M. tuberculosis was about 520 pg/10(6) viable organisms. The ATP levels from in vivo-derived organisms obtained from liver and spleen of mice was about 130 pg (in cases of chronic infection) and about 270 pg (in cases of acute infection). When the in vivo-derived organisms were inoculated into culture medium, the growth rates for both types of organisms, acute as well as chronic infection, were the same and the maximum growth was reached during the fifth subculture. Although the maximum ATP content for both types of organism was the same, it was attained during the 4th subculture for organisms obtained during acute infection and during the 6th subculture for those obtained during chronic infection. The comparison between the ATP content of M. leprae and of M. tuberculosis indicates the reason for the slow growth of M. leprae. PMID- 10738982 TI - Regulation of the thdF gene, which is involved in thiophene oxidation by Escherichia coli K-12. AB - The thdF gene of Escherichia coli encodes a 48 kD protein which is involved in the oxidation of derivatives of the sulphur-containing heterocycle thiophene and which appears to be induced during stationary phase. In this work the upstream regulatory region of the thdF gene was isolated by polymerase chain reaction and inserted in front of the lacZ structural gene. Examination of the resulting thdF lacZ operon fusions showed that expression of the thdF gene increased as E. coli entered the stationary phase. However, the expression of thdF was not dependent on RpoS (KatF), the stationary phase sigma factor. The thdF gene was subject to substantial catabolite repression by glucose and its expression was also greatly decreased in the absence of oxygen. The thdF-lacZ fusions were not significantly affected by elevated temperature or medium of high osmolarity, nor by mutations in thdA, fadR, arcA, arcB, or fnr. Both multicopy, plasmid-borne fusions and single-copy fusions gave similar results in all of the above cases except that the plasmid-borne fusions still showed substantial expression in the absence of oxygen. The heterocyclic compounds thiophene carboxylic acid, furan carboxylic acid and proline increased expression of the thdF gene by 2- to 3-fold, but only during the stationary phase. Tryptophan, indole, and several indole derivatives had no effect. PMID- 10738983 TI - A new factor from Bacillus mesentericus which promotes the growth of Bifidobacterium. AB - It was reported previously that supernatants of cultures of Bacillus mesentericus TO-A promote the growth of Bifidobacterium species. In this study, a new growth promoting factor, BM-1, was purified from the supernatant of such a culture and its chemical structure was determined. BM-1 was identified as 3,3 dihydroxyazetidine, and it promoted the growth of several strains of Bifidobacterium. PMID- 10738984 TI - Adherence of Bacteroides forsythus to host cells. AB - The adherence characteristics of Bacteroides forsythus to host cells, was examined. Four laboratory strains and twelve clinical isolates of B. forsythus were used. All strains demonstrated different haemagglutination activities. The haemagglutination of B. forsythus was inhibited strongly by amino acids such as L arginine, L-histidine, L-lysine and L-alanine. The adherence to polymorphonuclear leucocytes (PMNL) was weak except for B. forsythus ATCC 43037 and OMZ 408. The adherence of these strains was inhibited by L-histidine and L-arginine, and was facilitated by trypsin (0.1 mg/ml) treatment of polymorphonucleocytes. B. forsythus strains showed varied adherence to fibroblasts. It is suggested that the adherence of B. forsythus to host cells is mediated by a factor which is sensitive against some amino acids, and altered by trypsin-like enzymes. PMID- 10738985 TI - Nosocomial outbreak of Salmonella enteritidis in a university hospital. PMID- 10738986 TI - Surgical-site infections and the NNIS SSI Risk Index: room for improvement. PMID- 10738987 TI - Does the Centers for Disease Control's NNIS system risk index stratify patients undergoing cardiothoracic operations by their risk of surgical-site infection? AB - BACKGROUND: In 1991, the Centers for Disease Control and Prevention devised the National Nosocomial Infection Surveillance (NNIS) System risk index to stratify populations of surgical patients by the risk of acquiring surgical-site infections (SSIs). OBJECTIVE: To determine whether the NNIS risk index adequately stratifies a population of cardiothoracic surgery patients by the risk of developing SSI. DESIGN: Case-control study. SETTING: The University of Iowa Hospitals and Clinics, a 900-bed, midwestern, tertiary-care hospital. PATIENTS: 201 patients with SSIs identified by prospective infection control surveillance and 398 controls matched by age, gender, type of procedure, and date of procedure. All patients underwent cardiothoracic operative procedures between November 1990 and January 1994. RESULTS: The SSI rate was 7.8%. Seventy-four percent of cases and 80% of controls had a NNIS risk index score of 1; 24% of cases and 16% of controls had a score of 2 (P=.05). Patients with a NNIS risk score > or =2 were 1.8 times more likely to develop an SSI than those with a NNIS score <2 (odds ratio, 1.83; 95% confidence interval, 1.14-2.94, P=.01). The duration of the procedure was the only component of the index that stratified the population by risk of SSI. CONCLUSIONS: The risk of SSI after cardiothoracic operations increases as the NNIS risk index score increases. However, this index only dichotomized the patient population on the basis of the procedure duration. More research is needed to develop a risk index that adequately stratifies the risk of SSI after cardiothoracic operations. PMID- 10738988 TI - Identification of factors that disrupt negative air pressurization of respiratory isolation rooms. AB - OBJECTIVES: To investigate the airflow characteristics of respiratory isolation rooms (IRs) and to evaluate the use of visible smoke as a monitoring tool. METHODS: Industrial hygienists from the New York State Department of Health evaluated 140 designated IRs in 38 facilities within New York State during 1992 to 1998. The rooms were located in the following settings: hospitals (59%), correctional facilities (40%), and nursing homes (1%). Each room was tested with visible smoke for directional airflow into the patient room (ie, negative air pressure relative to adjacent areas). Information was obtained on each facility's policies and procedures for maintaining and monitoring the operation of the IRs. RESULTS: Inappropriate outward airflow was observed in 38% of the IRs tested. Multiple factors were associated with outward airflow direction, including ventilation systems not balanced (54% of failed rooms), shared anterooms (14%), turbulent airflow patterns (11%), and automated control system inaccuracies (10%). Of the 140 tested rooms, 38 (27%) had either electrical or mechanical devices to monitor air pressurization continuously. The direction of airflow at the door to 50% (19/38) of these rooms was the opposite of that indicated by the continuous monitors at the time of our evaluations. The inability of continuous monitors to indicate the direction of airflow was associated with instrument limitations (74%) and malfunction of the devices (26%). In one facility, daily smoke testing by infection control staff was responsible for identifying the malfunction of a state-of-the-art computerized ventilation monitoring and control system in a room housing a patient infectious with drug-resistant tuberculosis. CONCLUSION: A substantial percentage of IRs did not meet the negative air pressure criterion. These failures were associated with a variety of characteristics in the design and operation of the IRs. Our findings indicate that a balanced ventilation system does not guarantee inward airflow direction. Devices that continuously monitor and, in some cases, control the pressurization of IRs had poor reliability. This study demonstrates the utility of using visible smoke for testing directional airflow of IRs, whether or not continuous monitors are used. Institutional tuberculosis control pro grams should include provisions for appropriate monitoring and maintenance of IR systems on a frequent basis, including the use of visible smoke. PMID- 10738989 TI - Nosocomial Serratia marcescens infections associated with extrinsic contamination of a liquid nonmedicated soap. AB - OBJECTIVE: To determine the role of nonmedicated soap as a source of Serratia marcescens nosocomial infections (NIs) in hospital units with endemic S marcescens NI and to examine the mechanisms of soap colonization. SETTING: University-affiliated tertiary-care hospitals. METHODS: A prospective case control study and an environmental investigation were performed to assess the relationship between S marcescens NIs in hospital units and S marcescens contaminated soap. Soap-bottle use and handwashing practices were reviewed. Cultures of healthcare workers' (HCWs) hands were obtained before and after hand washing with soap. RESULTS: 5 of 7 hospital units with S marcescens NIs had soap bottles contaminated with S marcescens, compared to 1 of 14 other units (P=.006). After hand washing with an S marcescens-contaminated soap pump, HCWs' hands were 54 times more likely to be contaminated with S marcescens (P<.001). CONCLUSIONS: Extrinsic contamination of a non-medicated liquid soap by S marcescens resulted in handborne transmission of S marcescens NIs by HCWs in our setting. This finding led to the application of strict guidelines for nonmedicated soap use and to the reinforcement of alcoholic hand disinfection. PMID- 10738990 TI - Standardized surveillance of hemodialysis vascular access infections: 18-month experience at an outpatient, multifacility hemodialysis center. AB - OBJECTIVE: To develop a standardized surveillance system for monitoring hemodialysis vascular-access infections in order to compare infection rates between outpatient sites and to assess the effectiveness of infection control interventions. DESIGN: Prospective descriptive analysis of incidence infection rates. SETTING: An outpatient hemodialysis center with facilities in Idaho and Oregon. PATIENTS: All outpatients receiving chronic outpatient hemodialysis. RESULTS: There were 38,096 hemodialysis sessions (31,603 via permanent fistulae or grafts, 5,060 via permanent tunneled central catheters, and 1,433 via temporary catheters) during an 18-month study period in 1997 to 1998. We identified 176 total infections, for a rate of 4.62/1,000 dialysis sessions (ds). Of the 176, 80 involved permanent fistulae or grafts (2.53/1,000 ds), 69 involved permanent tunneled central catheter infections (13.64/1,000 ds), and 27 involved temporary catheter infections (18.84/1,000 ds). There were 35 blood-stream infections (0.92/1,000 ds) and 10 episodes of clinical sepsis (0.26 /1,000 ds). One hundred thirty-one vascular-site infections without bacteremia were identified (3.44/1,000 ds), including 65 permanent fistulae or graft infections (2.06/1,000 ds), 42 permanent tunneled central catheter infections (8.3/1,000 ds), and 24 temporary catheter infections (16.75/1,000 ds). CONCLUSIONS: Infection rates were highest among temporary catheters and lowest among permanent native arteriovenous fistulae or synthetic grafts. This represents the first report of extensive incidence data on hemodialysis vascular access infections and represents a standardized surveillance and data-collection system that could be implemented in hemodialysis facilities to allow for reliable data comparison and benchmarking. PMID- 10738991 TI - Outbreak of Pseudomonas aeruginosa ventriculitis among patients in a neurosurgical intensive care unit. AB - OBJECTIVE: To determine the cause of an outbreak of Pseudomonas aeruginosa cerebral ventriculitis among eight patients at a community hospital neurosurgical intensive care unit. All had percutaneous external ventricular catheters (EVCs) to monitor cerebrospinal fluid (CSF) pressure. METHODS: Cohort study of all patients who had EVCs placed during the epidemic period (August 8-October 22, 1997). A case-patient was any patient with P aeruginosa ventriculitis during the epidemic period. Pulsed-field gel electrophoresis (PFGE) was performed on all isolates. RESULTS: P aeruginosa was significantly more likely to be isolated from CSF per EVC placed in the epidemic than pre-epidemic (January 1-August 7, 1997) periods (8/61 [13%] vs 2/131 [1.5%], P=.002). During the epidemic period, ventriculitis was significantly more likely after EVC placement in the operating room than in other units (8/24 vs 0/22, P=.004). EVC placement technique differed for EVCs placed in the operating room (little hair was removed, preventing application of an occlusive dressing) versus other hospital units (more hair was removed, and an occlusive dressing was applied). Among patients who had operating room EVC placement, contact with one healthcare worker was statistically significant (7/13 vs 0/8, P=.02). Hand cultures of this worker were negative. All isolates had closely related PFGE patterns. CONCLUSIONS: These data suggest that a single healthcare worker may have contaminated EVC insertion sites, resulting in an outbreak of P aeruginosa ventriculitis. Affected patients were unlikely to have had an occlusive dressing at the EVC insertion site. Application of a sterile occlusive dressing may decrease the risk of ventriculitis in patients with EVCs. PMID- 10738992 TI - Prevalence of hepatitis C antibodies in a large sample of Belgian healthcare workers. AB - OBJECTIVE: To assess the risk of hepatitis C virus (HCV) infection among Belgian (Flemish) healthcare workers. DESIGN: A seroprevalence survey of HCV IgG antibodies. SETTING AND PARTICIPANTS: A systematic sample of 5,064 employees from 22 general hospitals in Flanders and Brussels, Belgium, was tested at the annual occupational medical examination. Together with demographic and occupational data, information was collected on the frequency of blood contact, needlestick injuries, and medical and surgical history. The blood samples were tested using the third-generation Abbott Screen Kit test, with confirmation by Matrix, LIA, and an in-house polymerase chain reaction and the Quantiplex-HCV b-DNA test. RESULTS: 21 persons were found to be positive for HCV markers. The overall prevalence was 0.41% (95% confidence interval [CI95], 0.24-0.59). A statistically significant association was found with a history of blood transfusion (odds ratio [OR], 4.14; CI95, 1.67-10.31) and with history of a clinically apparent hepatitis (OR, 3.98; CI95, 1.60-9.90). Although the ORs for the frequency of blood contact were slightly elevated (between 1.17 and 2.73), this association was not significant. Moreover, a history of needlestick injuries showed a nonsignificant OR of 1.28 (CI95, 0.53-3.09), and no statistically significant difference was found with a variety of duties and tasks. The ORs for potential occupational risk factors were adjusted according to age, gender, antecedents, and other confounders using a logistic regression analysis. Based on this procedure, the ORs decreased slightly. CONCLUSIONS: Flemish healthcare workers showed a lower HCV seropositivity than is seen in the general population; a history of blood transfusion and of clinically apparent hepatitis was most strongly associated with the presence of HCV markers. We concluded that employees in Flemish regional general hospitals are not at an overall increased risk for HCV infection, although occasional transmission through percutaneous injuries is possible, and prevention therefore remains imperative. PMID- 10738993 TI - Determining the significance of coagulase-negative staphylococci isolated from blood cultures at a community hospital: a role for species and strain identification. AB - OBJECTIVES: To determine the degree to which species identification or strain relatedness assessment of successive blood culture isolates of coagulase-negative staphylococci (CNS) may improve the clinical diagnosis of bloodstream infection (BSI). SETTING: 400-bed community hospital. DESIGN: Prospective laboratory survey during which all CNS blood culture isolates obtained between mid-August 1996 and mid-February 1997 (study period) were saved and later identified to the species level; selected isolates were genotyped using pulsed-field gel electrophoresis at the Centers for Disease Control and Prevention (CDC). Retrospective review of medical records of 37 patients with multiple cultures positive for CNS. RESULTS: During the study period, 171 patients had blood cultures positive for CNS; 130 had single positive cultures and 41 had > or =2 positive cultures. Of these 41, 23 (62%) were from patients with signs and symptoms of BSI according to CDC surveillance definitions. Species identification and strain clonality of CNS isolates from patients with > or =2 positives revealed 3 (13%) of the 23 patients did not have a consistent CNS species, and another 3 (13%) did not have a consistent genotype in the > or =2 positive cultures, suggesting that CNS from these patients probably were contaminants. Thus, species identification and strain clonality assessment reduced by 27% the number of patients with BSI diagnosed based on the presence of symptoms and > or =2 positive blood cultures. CONCLUSIONS: Routine species identification and selected strain genotyping of CNS may reduce the misinterpretation of probable contaminants among patients with > or =2 positive blood cultures. PMID- 10738994 TI - Genotyping by restriction endonuclease analysis compared to phenotyping by antibiogram for typing methicillin-resistant Staphylococcus aureus strains colonizing patients in a nursing home. AB - OBJECTIVES: To assist in defining patterns of methicillin-resistant Staphylococcus aureus (MRSA) colonization in a skilled nursing facility (SNF), we compared genotyping by field-inversion gel electrophoresis (FIGE) restriction endonuclease digestion analysis (REA) with phenotyping by antibiogram for defining strain relatedness among MRSA isolates from SNF patients. DESIGN: Prospective screening culture surveillance for MRSA among patients in a community SNF. METHODS: Nares and stool swab cultures were obtained from newly admitted patients and from all patients quarterly. MRSA were isolated by oxacillin screening agar. Antibiograms were determined by the disk-diffusion method, and genotyping was by FIGE REA. RESULTS: It was shown that, among isolates with the same genotypes, many had different antibiograms; among isolates with the same antibiograms, many had different genotypes; and the discriminatory indices for isolates of MRSA by FIGE REA and by antibiogram were 0.56 and 0.78, respectively. CONCLUSION: Our study demonstrated that, in patients from one SNF, genotyping by FIGE REA identified two prevalent REA DNA types, but with variability of antibiogram patterns within each DNA type; the antibiogram also identified prevalent patterns with variability of REA DNA type within each antibiogram pattern. The discriminatory index of antibiograms alone, or of genotypes alone as determined by FIGE REA, was poor for strains of MRSA isolated from the SNF patients in our study. PMID- 10738995 TI - Nosocomial transmission of tuberculosis (TB) associated with care of an infant with peritoneal TB. AB - Nosocomial transmission of tuberculosis (TB) after exposure to infected peritoneal fluid has not been described. We report the exposure of 111 healthcare workers to infected dialysate from an infant with TB peritonitis. Two (5%) of 39 primary-care nurses, but no doctors or environmental service workers, had apparent tuberculin skin test conversions, raising the concern that patients with peritoneal TB may be a source for nosocomial transmission of TB. PMID- 10738996 TI - Increase in community-acquired methicillin-resistant Staphylococcus aureus at a Naval Medical Center. AB - A retrospective review of methicillin-resistant Staphylococcus aureus isolates from the Naval Medical Center, San Diego, for the years 1994 through 1997, found that the annual number of community-acquired MRSA isolates increased during the period. These outpatient isolates were more likely than inpatient isolates to be sensitive to a greater number of antibiotics. PMID- 10738997 TI - Control of nosocomial Clostridium difficile transmission in bone marrow transplant patients. AB - This is a report of six cases of Clostridium difficile-associated diarrhea (CDAD) that occurred among cancer patients undergoing bone marrow transplantation in a tertiary-care cancer hospital. Specific infection control measures that were taken to minimize the nosocomial spread of CDAD also are discussed. PMID- 10738998 TI - Primer on hepatitis C for hospital epidemiologists. AB - First identified in the late 1980s as the main causative agent of non-A, non-B hepatitis, hepatitis C virus (HCV) infection is now the most common chronic bloodborne infection in the United States. It is likely that the number of deaths attributable to HCV-related chronic liver disease will increase substantially during the next 2 decades, and the potential economic and clinical burden related to HCV is staggering. Accordingly, it is essential to identify risk factors for transmission of HCV and implement appropriate precautions to decrease the prevalence of this emerging infection. The relative importance of the two most common types of exposures associated with transmission of HCV, ie, blood transfusion and injection drug use, has evolved over time. Strict blood-donor selection and the use of reliable serological tests have led to a significant reduction in transfusion-associated risk. Injection drug use is currently the single most important risk factor for HCV infection in the United States. Transmission of HCV poses a serious threat to healthcare workers and patients if infection control techniques or disinfection procedures are inadequate. Potential exposures include contact of the eyes, mucous membranes, broken skin, or needlestick injury. Hospitalized patients may serve as a reservoir for transmission, and the prevalence of anti-HCV seropositivity among such patients can be as high as 20%. The infected healthcare worker is a potential source of transmission. A recent molecular phylogenetic analysis demonstrated that a cardiac surgeon likely transmitted HCV to five of his patients during open heart surgery. In summary, nosocomial transmission has been unequivocally confirmed and underscores the need for strict infection control practices. PMID- 10738999 TI - A phase II study of high dose ARA-C and mitoxantrone for treatment of relapsed or refractory adult acute lymphoblastic leukemia. AB - PURPOSE: The Southwest Oncology Group performed a Phase II study to investigate the effectiveness of an induction regimen of high dose cytosine arabinoside (ara C) with high dose mitoxantrone for treatment of relapsed or refractory adult acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Patients at least 16 years-old with ALL that was in relapse after, or was refractory to, standard induction therapy including at least vincristine and prednisone were eligible, as long as they had no prior treatment with high dose ara-C. The induction regimen included high dose ara-C (3 g/m2 by 3-h i.v. days 1-5) and mitoxantrone (80 mg/m2 by 15-30 min i.v. 12-20 h after the first dose of ara-C). The study design called for a maximum of 55 patients, with early termination if less than nine of the first 30 achieved complete remission. RESULTS: Thirty-three patients entered the study, and 31 were included in the analysis. All 31 completed one course of induction therapy. Four patients died of infection and a fifth of cardiomyopathy with possible sepsis. Seven patients achieved complete remission (23%; 95% confidence interval 10-41%). One of the seven received syngeneic bone marrow transplantation while in remission, and the other six all relapsed within 10 months. All 31 patients died within 25 months after entering the study. CONCLUSIONS: The regimen of high dose ara-C and mitoxantrone was found to be insufficiently effective to warrant further investigation. PMID- 10739000 TI - High-dose cytarabine-based therapy in adults with acute lymphoblastic leukemia. PMID- 10739001 TI - Variability in methotrexate serum and cerebrospinal fluid pharmacokinetics in children with acute lymphocytic leukemia: relation to assay methodology and physiological variables. AB - Methotrexate (MTX) steady state concentrations were evaluated in 42 children who had received high-dose infusions (6-8 g/m2) for acute lymphocytic leukemia. Concentrations in serum and cerebrospinal fluid (CSF) measured by immunoassay were found to be highly variable. Reanalysis by a reference high-pressure liquid chromatography method ruled out analytical factors as a source of this variability. The correlation coefficient between the analytical methods was 0.77 for the serum data and 0.88 for the CSF data. The variability of serum and CSF concentrations was higher in younger patients (serum; P = 0.05 and CSF; P = 0.18), and the CSF concentration decreased with decreasing age and in later courses. Body surface area, body mass index, weight, and gender were not significantly related to MTX variability. We conclude that the pronounced pharmacokinetic variability seen during MTX infusions remains largely unexplained. PMID- 10739002 TI - CD34/CD117 co-expression in childhood acute leukemia. AB - CD117 protein is expressed by the primitive CD34 positive haemopoietic stem cells and also demonstrated on the blasts of 30-100% of AML cases, but rarely on lymphoblasts. Therefore several investigators have used CD117 expression to exclude lymphoblastic origin of blasts. However, conflicting results exist in the literature. We investigated CD34 and CD117 status at initial presentation of 232 children with acute leukemia. CD34 was commonly expressed in all types of acute leukemias, whereas CD117 molecule seemed to be a more specific marker for leukemia of myeloid origin being demonstrated on > 5% of blasts in 60 out of 73 cases of AML patients, but rarely detected in ALL (9/140 patients). Moreover, co expression of CD34/CD117 was extremely rare on lymphoblasts with only 3/140 ALL patients demonstrating > 5% co-expression of CD34 and CD117, and therefore we suggest that it should be used in the exclusion of ALL. PMID- 10739003 TI - Erythroblastic and/or megakaryocytic dysplasia in de novo acute myeloid leukemias M0-M5 show relation to myelodysplastic syndromes and delimit two main categories. AB - Erythroblastic and/or megakaryocytic dysplasia (EMD) was evaluated in diagnostic bone marrow smears of 43 consecutively treated patients under 65 years with de novo acute myeloid leukemia (AML) M0-M5 according to FAB criteria. The evaluation was possible in 39 (91%) patients, i.e. in 32 of 34 patients with non-M3 AML treated in the study UHKT-911 and seven of nine cases with AML M3 treated in other studies. Among non-M3 AML 15 patients were categorized without EMD and 17 cases with EMD. Cytogenetic abnormalities of chromosome 5, 7, 3 or a complex karyotype were found in eight of 17 patients with EMD and in one of 15 cases without EMD (P = 0.018). Seven patients in each category exhibited a normal karyotype. Classical induction therapy with three to four doses of daunorubicin 45 mg/m2 and standard doses of cytosine arabinoside (AraC) for 7 days lead to complete remission in 11 of 14 (78.6%) cases without EMD but only in four of 14 (28.6%) cases with EMD (P = 0.021). High doses (2000 mg/m2 per 12-h x 10) of AraC plus daunorubicin induced complete remission in seven of 10 patients with EMD. Patients with EMD showed significantly worse overall survival (P = 0.03) with a median 13.5 months, while the median survival was estimated to 68.7 months in cases without EMD. The dysplastic features of EMD, karyotypes typical for myelodysplastic syndromes (MDS), poor response to classical therapy and survival show a relation of AML with EMD to MDS. AML without EMD may represent a different biological favorable category. PMID- 10739004 TI - CD14+ peripheral blood mononuclear cells from chronic myeloid leukemia and normal donors are inhibitory to short- and long-term cultured colony-forming cells. AB - Monocyte-induced cell-cytotoxicity has been implicated in the mechanism of suppression of normal haematopoietic progenitors in chronic myeloid leukemia (CML). We examined here the in vitro effect of CML-derived and normal peripheral blood (PB) monocytes on short- and long-term cultured haematopoietic progenitor cells. Short-term coculture (5 days) of CML or normal monocytes with CML or normal peripheral blood mononuclear cells (PBMNC)/CD34+ cells as targets resulted in a significant inhibition of colony-forming cell (CFC) growth. Coculture conditioned medium (CCM) from 5-days cocultures of normal or CML CD14+ monocytes with CD34+ cells were likewise inhibitory to CFC. In 5-week long-term cocultures of monocytes in direct contact with normal bone marrow (BM) progenitors, CML monocytes reduced the proportion of long-term cultured CFC (LTC-CFC) significantly to 52% of the controls, while normal monocytes had a less pronounced inhibitory effect (89% of the controls) on LTC-CFC. Reduction of LTC CFC was great when CML monocytes and target cells were separated by a transwell membrane as compared to control cultures in the absence of CD14+ cells (53.5 vs. 9%). CCM from 5-week cocultures of normal or CML CD14+ monocytes with CD34+ progenitors from bone marrow (BM) cells were also inhibitory to CFC. No difference in cytokine levels for TNF-alpha, IFN-gamma, G-CSF, IL-10, IL-6 was detectable between CML CD14+ CCM and control CCM derived from short- and long term cocultures. Our results suggest that CML monocytes may play a role in the inhibition of normal haematopoiesis through a yet not defined soluble factor supporting the expansion of the malignant clone in CML. PMID- 10739005 TI - The influence of the cell cycle, differentiation and irradiation on the nuclear location of the abl, bcr and c-myc genes in human leukemic cells. AB - abl and bcr genes play an important role in the diagnostics of chronic myelogenous leukemia (CML). The translocation of these genes results in an abnormal chromosome 22 called the Philadelphia chromosome (Ph). The chimeric bcr abl gene is a fundamental phenomenon in the pathogenesis of CML. Malignant transformation of hematopoietic cells is also accompanied by the c-myc gene changes (translocation, amplification). Nuclear topology of the abl, bcr and c myc genes was determined in differentiated as well as in irradiated HL-60 cells using dual-colour fluorescence in situ hybridisation and image analysis by means of a high resolution cytometer. After the induction of the granulocytic differentiation of HL-60 cells with all trans retinoic acid (ATRA) or dimethylsulfoxide (DMSO), the abl and bcr homologous genes were repositioned closer to the nuclear periphery and the average distances between homologous abl abl and bcr-bcr genes as well as between heterologous abl-bcr genes were elongated as compared with untreated human leukemic promyelocytic HL-60 cells. Elongated gene-to-gene and centre-to-gene distances were also found for the c-myc gene during granulocytic differentiation. In the case of the monocytic maturation of HL-60 cells treated with phorbol esters (PMA), the abl and bcr homologous genes were repositioned closer to each other and closer to the nuclear centre. The position of the c-myc gene did not change significantly after the PMA stimulus. The proximity of the abl and bcr genes was also found after gamma irradiation using 60Co (5 Gy). Immediately after the gamma irradiation c-myc was repositioned closer to the nuclear centre, but 24 h after radiation exposure the c-myc position returned back to the pretreatment level. The c-myc gene topology after gamma irradiation (when the cells are blocked in G2 phase) was different from that detected in the G2 sorted control population. We suggest that changes in the abl, bcr and c-myc topology in the case of gamma irradiation are not the effects of the cell cycle. It is possible, that differences in the cell cycle of hematopoietic cells after the gamma irradiation and concurrent proximity of the abl, bcr and c-myc genes could be important from the point of view of contingent gene translocations, that are responsible for malignant transformation of cells. PMID- 10739006 TI - Aclarubicin induces differentiation of leukemic progenitors in myelodysplastic syndrome cooperating with granulocyte colony-stimulating factor. AB - We have reported that low-dose aclarubicin (ACR) therapy is effective in some patients with myelodysplastic syndrome (MDS). Here, we demonstrate that a low concentration of ACR induces the in vitro differentiation of leukemic progenitor cells from patients with MDS. ACR (0.1 ng/ml) significantly increased the number of granulocyte colony-stimulating factor (G-CSF)-dependent colonies from circulating blast cells in vitro in six out of seven MDS patients with refractory anemia with excess of blast in transformation or chronic myelomonocytic leukemia, but not in all four patients with primary acute myelogenous leukemia. In these MDS patients, the effect of ACR gradually disappeared along with the progression of MDS. Interestingly, the majority of G-CSF/ACR-dependent colonies consisted of rather differentiated myeloid cells such as myelocytes and metamyelocytes, whereas colonies formed by G-CSF alone were composed mainly of immature blastic cells. The number of G-CSF-responding progenitors significantly increased during a 24-48 h incubation with ACR alone. The circulating blasts in MDS patients expressed G-CSF receptors at unchanged levels before and after the incubation with ACR. It is suggested that ACR might increase clonogenic progenitor responsiveness to G-CSF in MDS, probably through modulating downstream signaling cascades associated with G-CSF receptors, and induce these progenitors to differentiate in response to G-CSF. PMID- 10739007 TI - Effect of PSC 833 on the cytotoxicity and pharmacodynamics of mitoxantrone in multidrug-resistant K562 cells. AB - We examined the effect of PSC 833, a nonimmunosuppressive cyclosporin analogue, on the cytotoxicity, accumulation and retention of an anthraquinone antileukemia drug mitoxantrone (MIT). This was done in P-glycoprotein (PGP)-overexpressing multidrug-resistant K562/D1-9 cells and compared with the effect of cyclosporin A (CsA). We also compared MIT with the effect of PSC 833 on the cytotoxicity of daunorubicin (DNR) and doxorubicin (DOX). While PSC 833 and CsA had no effect on the cytotoxicity, accumulation and retention of MIT in the parent K562 cells, PSC 833 and CsA restored accumulation and retention of MIT in K562/D1-9 cells dose dependently. Consequently, there was increased sensitivity of K562/D1-9 cells to MIT. The reversing activity of PSC 833 on the cytotoxicity of MIT was stronger than that of CsA, and was almost the same as the reversing activity of PSC 833 on the cytotoxicity of DNR and DOX. The resistance index of MIT decreased from 43.9 fold to 2.8-fold by 0.4 microM PSC 833, which is a clinically achievable plasma concentration. These results suggest that the combination of PSC 833 with MIT could be a promising treatment in reversing PGP-mediated MDR in leukemia patients. PMID- 10739008 TI - Frequent microsatellite instability and BAX mutations in T cell acute lymphoblastic leukemia cell lines. AB - Allelic status of the BAT26 and BAT25 loci was examined in 117 leukemia/lymphoma cell lines consisting of 44 B-lymphoid lineage cell lines, 30 T-lymphoid cell lines and 43 myeloid cell lines to define the lineage specificity of microsatellite instability (MSI) in hematological malignancies. Seventeen (15%) cell lines were defined as having MSI. The incidence of MSI was significantly (P < 0.01) higher in cell lines of lymphoid lineage (15/74; 20%) than in those of myeloid lineage (2/43; 5%). In the cell lines of lymphoid lineage, the incidence of MSI in T cell acute lymphoblastic leukemia (T-ALL) (11/30; 37%) was significantly (P < 0.01) higher than those in B-lineage malignancies (4/44; 9%). The 17 cell lines with MSI were subjected to the mutation analysis of the coding microsatellites in 13 candidate genes. Frameshift mutations were most frequently detected in the BAX gene (14/17, 82%), while the hMSH3, hMSH6, TGFbetaRII, DRP and IGFIIR genes were less frequently mutated (24-47%). The present result indicates that MSI is involved in the development and/or progression of lymphoid malignancies, especially of T-ALL, through the inactivation of BAX and several other genes. PMID- 10739009 TI - Low frequency of expression of dominant-negative Ikaros isoforms in human leukemia and lymphoma cell lines. PMID- 10739010 TI - Important role of thiol compounds to protect oxidative stress on HTLV-I(+) T lymphocytes. PMID- 10739011 TI - Granulocytic sarcoma as the cause of giant abdominal mass: diagnosis by fine needle aspiration and review of the literature. AB - Granulocytic sarcoma (GS) or chloroma is a neoplasia consisting of myeloid precursors in an extramedullary site. It is generally associated with myeloproliferative disorders especially with myeloid neoplasias. A young woman with huge abdominal mass due to GS associated with chronic myelocytic leukemia (CML) has been reported and literature is reviewed. PMID- 10739012 TI - Evaluation by solid vascular casts of arterial geometric optimisation and the influence of ageing. AB - In the theoretical analysis of arterial networks the existence of geometric optimisation has long been suggested, although observational studies have not yet fully corroborated these theories. Since this could be due to experimental flaws, the aim of this study was to establish the validity of arterial geometric optimisation using a new experimental design and to assess the influence of ageing. Solid vascular casts of arterial mesenteric branching systems of 8-wk-old and adult dogs (beagles) were used to examine vascular diameters and branching angles, the latter in a manner that allowed optimisation of the line of view, thus minimising distortion errors due to a line of view not normal to the branching plane. Internal and external vessel diameters were found to be in accordance with the theoretical principle of minimum work (8-wk-old internal: r = 0.994; adult internal: r = 0.971; adult external: r = 0.985). Although branching angles were found to be in agreement with basic qualitative principles of arterial branching geometry, the measurements still showed a large amount of scatter and were generally smaller than expected on theoretical grounds, despite the newly designed measuring technique. These branching angles demonstrated small age-related differences. However, when biological cost was considered per bifurcation, surprisingly, guidance towards minimum lumen volume and pumping power with increase in age could clearly be demonstrated (P < 0.001). It is concluded that our findings support the existence of a degree of arterial geometric optimisation in favour of minimum lumen volume and pumping power, increasing with age. Future investigations should focus on the biofeedback mechanisms involved. PMID- 10739013 TI - Labelling of retinal microglial cells following an intravenous injection of a fluorescent dye into rats of different ages. AB - Retinal microglia were selectively and sequentially labelled in different layers of the retina of postnatal rats following a single intravenous injection of the fluorescent dye, rhodamine isothiocyanate (RhIc). The fluorescent cells were doubly immunostained with OX-42 and ED-1 antibodies that recognise complement type 3 (CR3) receptors and macrophage antigen, respectively. RhIc was first detected in the retinal blood vessels 5 min after injection. At 1 h, a variable number of microglia in the inner layers of the retina, namely, the nerve fibre and ganglion cell layers appeared to emit weak fluorescence. Labelled microglial cells in the inner nuclear and outer plexiform layers were not detected until 1 and 2 d had elapsed following RhIc injection. The number of labelled retinal microglia was progressively increased with time, peaking at 4 d after RhIc injection. The frequency of RhIc labelled cells also increased with age, with the largest number of cells occurring in 7-d-old rats but declined thereafter. In 11 d or older rats, RhIc was confined to the retinal blood vessels. It is concluded that when injected into the circulation, RhIc could readily gain access into the retina tissues due to an inefficient blood-retina barrier in early postnatal stages. It became impeded with maturation of the blood-retina barrier, which was established between 11 and 13 d of age. RhIc that inundated the retinal tissues was thoroughly sequestered by the resident microglial cells. It is therefore suggested that the latter could play a protective role against serum-derived substances that may be deleterious to the developing retina. PMID- 10739014 TI - Bcl-2, tissue transglutaminase and p53 protein expression in the apoptotic cascade in ribs of premature infants. AB - Apoptotic cells of the human growth plate have not previously been demonstrated in situ. We have investigated the distribution of apoptotic cells in costosternal growth plates and bone of premature infants aged 4-11 d with a gestational age of approximately 26 wk. In addition, we investigated the immunolocalisation of apoptosis-related proteins within the growth plates and associated bone. A proportion of late hypertrophic chondrocytes and osteocytes within newly formed primary spongiosa showed evidence of highly fragmented DNA. The incidence of osteocyte apoptosis decreased as the distance from the chondroosseous junction increased. Tissue transglutaminase (tTG) expression was associated with apoptosis of osteocytes and hypertrophic chondrocytes. In contrast the presence of tTG was demonstrated in osteoblasts and bone lining cells but it did not colocalise with evidence of apoptosis. The anti-apoptotic gene product Bcl-2 was absent from the growth plate but was present in osteocytes. Visual assessment indicated a greater occurrence of the protein in cells occupying regions of low apoptosis. P53 was not demonstrated in the growth plate or bone. These findings would indicate that human growth plate chondrocytes appear to show little provision for ensuring cell longevity. In contrast osteocyte apoptosis appears negatively correlated with the skeletal distribution of Bcl-2 protein in the human infant, implying a potential selective vulnerability in individual cells. Lack of Bcl-2 and the high incidence of osteocyte apoptosis in the more rapidly remodelling bone of the human infant suggest a potential role of osteocyte apoptosis in the remodelling process. PMID- 10739015 TI - The arterial supply to the digits of the forelimb in the Bactrian camel (Camelus bactrianus). AB - The arterial supply of the digits of the forelimb of the Bactrian camel is described. The arteries supplying the digits were the palmar metacarpal and common palmar digital arteries III. The palmar metacarpal artery III was the continuation of the deep medial proximal metacarpal branch which was derived from the medial branch of the radial artery. It gave rise to a nutrient branch, medial branch, lateral branch and distal perforating palmar branch at the proximal end of the distal sixth of the cannon bone (fused third and fourth metacarpal bones). The common palmar digital artery III was the continuation of the median artery, which divided into medial and lateral branches. The medial branch of common palmar digital artery III which occasionally arose from the axial palmar proper digital artery III, after giving rise to the axial proximal proximal phalangeal branch, divided into the axial and abaxial palmar proper digital arteries III. The axial palmar proper digital artery III gave off the dorsoaxial distal proximal phalangeal, dorsoaxial proximal middle phalangeal, dorsoaxial distal middle phalangeal, palmoaxial middle phalangeal, palmoaxial distal phalangeal, dorsoaxial distal phalangeal branches, coronal artery and some digital tori branches. The abaxial palmar proper digital artery III gave rise to the abaxial proximal proximal phalangeal, dorsoabaxial distal proximal phalangeal, dorsoabaxial middle phalangeal, palmoabaxial middle phalangeal, palmoabaxial distal phalangeal, dorsoabaxial distal phalangeal branches, coronal artery and some digital tori branches. The lateral branch of the common palmar digital artery III in its origin, course, branching pattern and supply in the fourth digit was similar to the medial branch of common palmar digital artery III in the third digit. PMID- 10739016 TI - Histomorphology of rabbit thigh muscles: establishment of standard control values. AB - The thigh muscles of New Zealand White (NZW) rabbits are frequently used in experimental surgery, particularly for evaluation after reinnervation or ischaemia. Although histomorphometric analyses are regularly performed, morphological data for untreated thigh muscles in previously unoperated animals are not available. Specimens from the rectus femoris (RF), vastus medialis (VM) and adductor magnus (AM) muscles from both thighs were harvested in 7 untreated rabbits and were processed for histomorphometric evaluation. The right RF and VM were harvested in a further 5 rabbit hindlimbs after experimental denervation and reinnervation of the contralateral RF and subsequently processed for histomorphometric analysis. Muscle fibre type distribution, diameter and connective tissue content were evaluated on serial transverse cryosections reacted for ATPase and NADH tetrazolium reductase activity and statistical analysis was performed. In all untreated animals RF revealed the highest proportion of type I muscle fibres (right: 8.4+/-4%, left: 11.4+/-4.9%), whereas VM showed the highest percentage of IIa fibres (right: 31.9+/-5.5%, left: 28.3+/ 7.8%) and AM the highest proportion of IIb/d fibres (right: 80.5+/-8.6%, left: 84.4+/-6.3%). Fibre type distribution and diameter in rabbits after contralateral experimental operations revealed a statistically significant difference from the data obtained in bilaterally untreated animals. Knowledge of the morphology of untreated muscles is fundamental to the understanding of changes induced by intervention to the ipsi and/or contralateral thigh muscles. PMID- 10739017 TI - Accumulation of PDGF+ cells and internalisation of the PDGF receptor at myotendinous junction following modified hindlimb muscle use in the rat. AB - Morphological observations have shown previously that myotendinous junctions (MTJs) are sites where the associations between the cytoskeleton and the cell membrane are extensively remodelled during muscle growth and modified mechanical loading. The platelet derived growth factor (PDGF) molecule has been shown to induce cytoskeletal remodelling at focal contact sites of myoblasts in culture, the analogous structures of MTJs. The goals of the study were to determine whether PDGF is synthesised by mononuclear cells and whether PDGF receptors are internalised at the MTJs of the soleus muscle experiencing reloading. We also examined whether ED2+ macrophages that are nonphagocytic and activated inflammatory cells at MTJs during reloading secrete PDGF. Results obtained by immunohistochemistry showed that there was an increase in the number of cells expressing PDGF at remodelling MTJs and that the ED2+ macrophage population does not express PDGF at MTJs. According to morphological criteria, fibroblasts would be the logical candidates to secrete PDGF molecules near MTJs. Furthermore, the modification in muscle loading resulted in internalisation of PDGF receptors concentrated at the MTJ which accumulated predominantly around muscle nuclei. The enrichment of PDGF receptors and PDGF+ cells at MTJs and the internalisation of PDGF receptors during remodelling of MTJs suggest that PDGF may influence remodelling of MTJs following modified muscle use. PMID- 10739018 TI - Spermiogenesis and spermiation in a monotreme mammal, the platypus, Ornithorhynchus anatinus. AB - Spermatogenesis in the platypus (Ornithorhynchus anatinus) is of considerable biological interest as the structure of its gametes more closely resemble that of reptiles and birds than marsupial or eutherian mammals. The ultrastructure of 16 steps of spermatid development is described and provides a basis for determining the kinetics of spermatogenesis. Steps 1-3 correspond to the Golgi phase of spermatid development, steps 4-8 correspond to the cap phase, steps 9-12 are the acrosomal phase, and steps 13-16 are the maturation phase. Acrosomal development follows the reptilian model and no acrosomal granule is formed. Most other features of spermiogenesis are similar to processes in reptiles and birds. However, some are unique to mammals. For example, a thin, lateral margin of the acrosome of platypus sperm expands over the nucleus as in other mammals, and more than in reptiles and birds. Also, a tubulobulbar complex develops around the spermatid head, a feature which appears to be unique to mammals. Further, during spermiation the residual body is released from the caudal end of the nucleus of platypus sperm leaving a cytoplasmic droplet located at the proximal end of the middle piece as in marsupial and eutherian mammals. Other features of spermiogenesis in platypus appear to be unique to monotremes. For example, nuclear condensation involves the formation of a layer of chromatin granules under the nucleolemma, and development of the fibrous sheath of the principal piece starts much later in the platypus than in birds or eutherian mammals. PMID- 10739019 TI - Peptidergic hormones and neuropeptides, and aminergic neurotransmitters of the pancreatic islets of the Houbara bustard (Chlamydotis undulata). AB - Immunoreactivity to insulin (Ins), somatostatin (Som), glucagon (Glu) and pancreatic polypeptide (PP) was found in 70%, 22%, 15% and 11% respectively of Houbara pancreatic endocrine islet cells. Whilst Ins occurred centrally and SOM was observed both in peripherally and centrally located islets, the other hormones were localised in peripheral islet cells; Som was also observed in neuronal cell bodies and nerve fibres. In addition, the islet cells contained substance P (SP) (65%) in the centre and vasoactive intestinal polypeptide (VIP) (2%) at the periphery. Immunoreactivity to choline acetyltransferase (ChAT), VIP and galanin (Gal) occurred in the walls of blood vessels located mainly at the periphery of islets. Occasionally, VIP and Gal immunoreactive varicose nerve terminals and ChAT immunoreactive cell bodies were also observed in the centre of islets. SP neuronal cell bodies were not observed but prominent SP immunoreactive varicose terminals were discernible in capillary walls within the islets. Neuropeptide Y (NPY) immunoreactive neurons were detected in neuronal cell bodies located mainly peripherally. Neuronal nitric oxide synthase (nNOS) immunoreactivity occurred in neuronal cell bodies and nerve fibres mainly at the periphery and also in centrally located islet endocrine cells. Immunoreactivity to tyrosine hydroxylase (TH) was similar in distribution to that of ChAT. In comparison with other avian species, the islets of the dorsal pancreatic lobe of the bustard contain all the peptidergic hormones normally present in the islets of other avian species, but are not segregated into dark A and light B cells. Many of the insulin containing cells also contained SP. The islets also contained several neuropeptides which are probably involved in their regulation. PMID- 10739020 TI - Rearrangement of extracellular matrix during cluster formation by human luteinising granulosa cells in culture. AB - Human ovarian granulosa cells were cultured on a basement membrane preparation (Matrigel) to investigate the role of extracellular matrix components in granulosa cell cluster formation. Time-lapse videomicroscopy of these cultures revealed a rapid aggregation of cells which was initiated during the first 2-4 h of culture so that by 8 h most of the granulosa cells were incorporated into clusters. Further amalgamation then occurred with the transfer of cells along 'bridges' between combining clusters. The clustering process, which was complete by about 24 h, was accompanied by reorganisation of matrix which was visualised by immunolabelling of laminin. Clustering cells appeared to gather matrix which became distributed around the clusters. Confocal microscopy showed matrix to be present over the surface of each cluster as well as around the base apparently anchoring the aggregate to the culture surface. Results suggest the potential for active rearrangement of matrix by granulosa-derived cells during corpus luteum development. PMID- 10739022 TI - Location of the spinal nucleus of the accessory nerve in the human spinal cord. AB - The segmental extent and topography of the spinal nucleus of the accessory nerve (SNAN) was investigated in the adult human spinal cord. Transverse sections of segments between the lower medulla and C6 were stained with cresyl violet and the motor cell columns identified according to the numerical locations defined by Elliott (1942). The segmental extent and topography of the cervical part of column 2 resembled that previously described for the SNAN of primates. PMID- 10739021 TI - Characterisation of Kupffer cells in some Amphibia. AB - A study on the Kupffer cells (KCs) of Amphibia was undertaken in order to compare these cells with those of endothermic animals. Liver tissue and isolated and cultured KCs were studied by light microscopy and by transmission and scanning electron microscopy. We have shown that amphibian KCs can be divided into 2 principal types: 'small' and 'large'. Both cell types possess the distinctive KC morphology. They show nonspecific esterase activity, weak endogenous peroxidase activity in the nuclear envelope and in the rough endoplasmic reticulum, and the ability to engulf naturally present cell debris or experimentally administered zymosan or latex particles. The principal difference between the small and the large cells consists in the substantial quantity of inclusion bodies that exist only in the latter. We conclude that amphibian KCs, apart from their ability to build melanosomes and synthesise melanins, are very similar to mammalian KCs. PMID- 10739023 TI - Ultrastructural characterisation of the olfactory mucosa of the armadillo Dasypus hybridus (Dasypodidae, Xenarthra). AB - The ultrastructure of the olfactory mucosa of the armadillo Dasypus hybridus was studied. A comparison with the olfactory mucosa of another armadillo (Chaetophractus villosus) was made. The olfactory mucosa of D. hybridus shows many features which are similar to those of other mammals. Interestingly, it differs from the olfactory mucosa of the armadillo C. villosus. A suggestion is made that these differences may be due to differences in the digging habits of these species. In Dasypus, the supporting cells (SCs) showed dense vacuoles, multivesicular bodies and lysosome-like bodies probably related with the endocytotic system. The SCs show a dense network of SER presumably associated with xenobiotic mechanisms. The olfactory receptor neurons exhibit lysosome-like bodies and multivesicular bodies in their perikarya. These organelles suggest the presence of an endocytotic system. Duct cells of Bowman's glands exhibit secretory activities. Bowman's glands are compound-branched tubulo-acinar mixed glands with merocrine secretory mechanisms. PMID- 10739024 TI - Neurotrophins and other growth factors in the regenerative milieu of proximal nerve stump tips. AB - Classic ideas on mechanisms for axon sprouting and nerve regeneration from peripheral nerves suggest that there is a prominent role for neurotrophin support. There has been comparatively less attention towards features of the regenerative process that develop from the proximal nerve trunk without the support of target tissues or the denervated trunk of a peripheral nerve. We studied early (2-14 d) expression of local growth factors in proximal nerve stump tips of transected sciatic nerves in rats. Immunohistochemical labelling was used to address specific deposition of BDNF, NGF, NT-3, bFGF, CNTF and IGF-1. We observed a unique localisation of BDNF, and to a much lesser extent, NGF in mast cells of injured nerve trunks but they were also observed in intact uninjured nerves. Macrophages did not express either BDNF or NGF. CNTF and IGF-1 were expressed in Schwann cells of intact nerves and stumps. We did not observe bFGF or NT-3 expression in any of the samples we studied. Mast cells may represent an important reservoir of BDNF in peripheral nerves. PMID- 10739025 TI - Galanin-like immunoreactive endocrine cells in bovine pancreas. AB - Pancreata of fetal, neonatal and adult cattle were studied immunohistochemically for galanin. The results revealed galanin-like immunoreactivity both in the endocrine cells and in the neural elements. The galanin-like immunoreactive endocrine cells (Gal-LIEC) were confined to the large islets, and were not observed in the islets of Langerhans and exocrine pancreas. They were first detected at the third prenatal month. Their developmental profile showed an increase from fetal to early neonatal stage with a subsequent decrease towards adulthood. The considerable number of Gal-LIEC from late prepartum to early postpartum stage may imply functional significance of galanin during the perinatal development of cattle. Coexistence of galanin and insulin was also observed which may suggest autocrine interaction between the 2 hormones. PMID- 10739026 TI - Chromatographic enantiomer separation of chiral xenobiotics and their metabolites -a versatile tool for process studies in marine and terrestrial ecosystems. AB - A review discussing methodical aspects of enantioselective chromatographic separation of chiral environmental xenobiotics as well as examples for process studies reported in literature. The process studies include microbial transformation of chiral pollutants in aquatic ecosystems, their enzymatic transformation in biota, their photochemical degradation, air/sea exchange processes and atmospheric long range transport, and enantioselective toxic effects. PMID- 10739027 TI - Separation of non-ortho polychlorinated biphenyl congeners on pre-packed carbon tubes. Application to analysis in sewage sludge and soil samples. AB - The analysis of planar (non-ortho) polychlorinated biphenyls (PCB) by HRGC-ECD or HRGC-HRMS requires a fractionation step to avoid the interferences of the bulk of PCB, usually in much higher concentration than the planar ones. In this paper, a new method, based on the fractionation of PCB on SPE commercial tubes pre-packed with Carbopack B, has been developed. After the extract has been applied on the stationary phase, the bulk of PCD are eluted with 15 ml of hexane (fraction I), mono-ortho PCB with 20 ml of hexane/toluene 99:1 (fraction II) and planar PCB with 20 ml of toluene (fraction III) in a station under vacuum. The method has been validated: accuracy (expressed as recovery in %) is >70% and precision (expressed as % RSD) is <20% considering changes of day, analyst and batch of tubes. The method is linear in the range studied. Other advantages are that the method is simple, rapid and it can be easily automated. The application of this separation to the determination of planar PCB in fly-ash extracts from an intercalibration exercise and to sewage sludge, sediment and soil samples has been successful. In addition, this method removes hydrocarbons from the planar PCB fraction and allows its concentration to very small volumes. PMID- 10739028 TI - Development and validation of a GC/MS method for determination of phenolic xenoestrogens in aquatic samples. AB - A simple and sensitive GC/MS method for the quantitative determination of the estrogenic phenolic compounds 4-nonylphenol, 4-t-octylphenol, bisphenol A, 3-t butyl-4-hydroxyanisole, 2-t-butyl-4-methylphenol, 4-hydroxybiphenyl, 2 hydroxybiphenyl, 4-chloro-3-methylphenol, and 4-chloro-2-methylphenol in aquatic samples was developed. The method for assessing their occurrence in sewage, surface and drinking waters consists of solid phase extraction (SPE) using a polystyrene copolymer phase. After methylation of the extract HRGC/LRMS analysis was possible without any clean up, even in raw sewage samples. Limits of detection and determination were between <0.01 and 0.05 ng/l and 0.01 and 0.05 ng/l, respectively. Recoveries were above 70% with exception of 3-t-butyl-4 hydroxyanisole. PMID- 10739030 TI - Degradation half-life times of PCDDs, PCDFs and PCBs for environmental fate modeling. AB - Literature search of the knowledge on the degradation of persistent organic pollutants (POPs) in environmental compartments air, water, soil and sediment was done in purpose to find properties of POPs of interest for modeling. One degradation process, hydrolysis (chemical degradation), was omitted as negligibly slow for POPs studied. The other two, photolysis and biodegradation processes, were considered separately in purpose to develop estimation procedures. The estimates can be given as pseudo first-order rate constants kP for photolysis and kB for biodegradation. For each compartment, an overall degradation rate is k(tot) = kP + kB and lifetime t(1/2) = ln 2/k(tot). The latter values, lifetimes in each compartment, will be used as input parameters to the Baltic Sea model. PMID- 10739029 TI - Analysis of flame retarded polymers and recycling materials. AB - Recycling activities on polymeric materials are increasing and becoming more and more important in recent years. For polymers containing no flame retardants, suitable recycling strategies already exist. In order to investigate the recyclability of flame retarded polymers that contain brominated flame retardants, a number of samples were analysed as received from a recycling company. Following the identification and sorting of the samples according to type of polymers and flame retardants, material recycling was tested for the flame retarded polymers identified to occur most frequently. The reactivity of the flame retardants during the recycling procedure was studied by analysing for brominated dioxins and furans. The results demonstrate that flame retarded polymers can be recycled under certain experimental conditions. PMID- 10739031 TI - Further study on the photochemistry of non-ortho substituted PCBs by UV irradiation in alkaline 2-propanol. AB - The photochemical behaviors of six non-ortho substituted PCB congeners, i.e., 3,4 DiCB, 3,5-DiCB, 3,3',5-TriCB, 3,4,5-TriCB, 3,3',4,5-TetraCB, and 3,4,4',5 TetraCB, irradiated at 254 nm in alkaline 2-propanol were investigated. Besides the determination of the photodechlorination pathways of these compounds, the presence of photorearrangement was observed in the case of 3, 4-DiCB with its products being identified. The results indicate that dechlorination is much more important than rearrangement during the process of PCB photolysis. PMID- 10739032 TI - An investigation of the in vivo formation of octachlorodibenzo-p-dioxin. AB - The in vivo formation of dioxins from chemical precursors was investigated in rats. Sprague-Dawley rats were fed pentachlorophenol or a predioxin in peanut oil for 14 days. Mass balance calculations indicated that pentachlorophenol was not converted to dioxins; however, the predioxin, nonachloro-2-phenoxyphenol, was converted to OCDD. Conversion of the predioxin ranged from 0.5% to 153% and depended on the amount of predioxin and OCDD present in the diet. The analytical procedures used for sample preparation did not appear to cause conversion of the predioxin to OCDD. The mechanism for biological conversion may be enzymatic or spontaneous. PMID- 10739033 TI - Myeloperoxidase-catalyzed formation of PCDD/F from chlorophenols. AB - Chlorophenols (CP) are transformed in vitro to polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F) by a biochemical-catalyzed oxidation. This is shown for 2,4,5-tri-, 2,3,4,6-tetra-and pentachlorophenol with myeloperoxidase recovered from human leucocytes in the presence of hydrogen peroxide. The yield, the reaction, and the PCDD/F-pattern found depend on the CP. The formation rates are in the micromol-per-mol range for all substrates. The experiments confirm the suspicion that a biochemical formation of PCDD/F from precursors such as CPs can take place in the human body and that this metabolic pathway may lead to a higher inner exposure with PCDD/F than is now assumed. PMID- 10739034 TI - Simulation of long-term environmental dynamics of polychlorinated dibenzo-p dioxins and polychlorinated dibenzofurans using the dynamic multimedia environmental fate model and its implication to the time trend analysis of dioxins. AB - Long term environmental fate of polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were studied using the fugacity-based dynamic multimedia environmental fate model. New formulation about soil wind erosion into air was included into the model. Effect of process assumption, sensitivity analysis for parameters, and tentative validation against the measured sediment core analysis was performed. Mass fluxes between compartments were estimated by the dynamic modeling. From the model estimates, major mass fluxes coming from emission source were shown to go to the soil and water through wet/dry deposition, then go to degradation mainly in the soil and sediment. Major mass fluxes of TCDD and OCDD come from the impurities in CNP (Chlornitrofen) and PCP (Pentachlorophenol) directly into the soil. Consideration about multimedia environmental dynamics using the modeled mass fluxes was shown in the discussion. PMID- 10739035 TI - Tissue distribution of 2,2',4,4'-tetrabromo[14C]diphenyl ether ([14C]-PBDE 47) in pike (Esox lucius) after dietary exposure--a time series study using whole body autoradiography. AB - In the present study, the tissue distribution of [14C]-labelled 2,2',4,4' tetrabrominated diphenyl ether (PBDE 47) and its possible metabolites was investigated after dietary exposure in pike (Esox lucius) using whole-body autoradiography. The study is a time series with pike examined 9, 18, 36 and 65 days after exposure. PBDE 47 was efficiently absorbed from the food (>90%) and radioactivity remained in the body in considerable amounts even after the longest period examined. The results indicate that PBDE 47 is not rapidly metabolised to hydrophilic, but possible to hydrophobic metabolites and that PBDE 47 and possible hydrophobic metabolites are accumulated in the lipid rich tissues of the pike. Melanin binding of PBDE 47 and possible metabolite(s) is suggested. The levels of PBDE 47 and/or metabolite(s) declines with time in most tissues except for the most lipid rich, where no decline in radioactivity is observed even after the longest period studied. Signs of irreversible incorporation of PBDE 47 derived radioactivity were detected but considered as too uncertain to conclude that covalent binding of PBDE 47-metabolites to macromolecules occurs. PMID- 10739036 TI - Influence of combustion conditions on the PCDD/F-, PCB-, PCBz- and PAH concentrations in the post-combustion chamber of a waste incineration pilot plant. AB - Experiments at a pilot scale waste incinerator (0.5 MW thermal power) showed that the conditions in the post-combustion chamber (650-900 degrees C) are strongly influencing the formation of chlorinated and non-chlorinated aromatics. Non optimal combustion conditions resulted in increased concentrations of mono- to trichlorinated dibenzo-p-dioxins (PCDD), dibenzofurans (PCDF) and polycyclic aromatic hydrocarbons (PAH), while chlorinated benzenes (PCBz), polychlorinated biphenyls (PCB) and the higher chlorinated PCDD/F are only weakly affected or even decrease. The changes in concentration of the compounds investigated over a time span of hours gave hints on 'memory effects' in this combustion zone. For mono- and dichlorinated benzenes, a high correlation (r2 = 0.80) with the international toxicity equivalent (I-TEQ) value of PCDD/F was observed. As recently has been demonstrated, this correlation can be utilized for an indirect on-line measurement of the I-TEQ by a novel laser mass spectrometric technique (REMPI-TO-FMS). PMID- 10739037 TI - Comparison of 2,4,6-trichlorophenol conversion to PCDD/ PCDF on a MSWI-fly ash and a model fly ash. AB - We performed experiments on two different matrices with 2,4,6-trichlorophenol as precursor to Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD)/F. A municipal solid waste incinerators (MSWI) and a model fly ash were spiked in two different ways. The experiments demonstrated a three times higher formation potential of the trichlorophenol to PCDD on MSWI fly ash compared with the model fly ash used. For both fly ashes the PCDD yield was higher when gaseous trichlorophenol was fed continuously compared to mixing the fly ashes prior to the experiments with the total amount of the precursor. Despite dilution of the fly ashes tenfold with an inactive matrix the conversion of the chlorophenol was very high. PMID- 10739038 TI - Formation of dioxins (PCDDs/PCDFs) by dioxin-free fly ash as a catalyst and relation with several chlorine-sources. AB - Simplified thermal formation experiments have been conducted using dioxin-free fly ash as a catalyst with many kinds of combustible samples such as newspaper, kerosene, paraffin, PE (polyethylene), PP (polypropylene) and PVC. Chlorine sources were PVC, NaCl and HCl. The combustion of samples containing chlorine in the absence of dioxin-free fly ash produced dioxins at a low level although HCl was present in the gas stream. On the other hand, the combustion of samples without chlorine with dioxin-free fly ash increased dioxins formation to a level around 10 times higher than that upon heating dioxin-free fly ash alone. This result is considered to be due to the presence of metal chloride in the fly ash and hydrocarbons in the gas stream. The combustion of samples containing either an organic or inorganic chlorine source or using a HCl stream with dioxin-free fly ash increased dioxin level dramatically. PMID- 10739039 TI - The formation of polychlorinated dibenzo-p-dioxins/dibenzofurans from carbon model mixtures containing ferrous chloride. AB - The potential to form polychlorinated dibenzo-p-dioxins/furans (PCDD/F) was investigated in carbon model systems containing ferrous chloride tetrahydrate and a matrix representative of that found in particle emission from the catalytic extraction process (CEP) for wastes. Various types of carbons were used resulting in different PCDD/F yields but, with one exception, similar homologue distributions. Due to the similarity between the turbostratic structure of the carbon in the representative CEP dusts and the carbon blacks used in the model system, experiments were performed using two carbon blacks (termed CBA and CBB). On a mass basis, CBB was more reactive over the temperature range of 275-325 degrees C and reaction times of 20-60 min in the formation of PCDD/F; as well as more adsorptive in terms of the desorption of PCDD/F. On a volume basis, the reactivities and adsorptivities were similar. A maximum in PCDD/F formation occurred at an oxygen concentration of 2% in nitrogen. PMID- 10739040 TI - Mono- to tri-chlorinated dibenzodioxin (CDD) and dibenzofuran (CDF) congeners/homologues as indicators of CDD and CDF emissions from municipal waste and waste/coal combustion. AB - Total homologue concentrations and select congener concentrations from amongst the mono- to tri-chlorinated dibenzodioxins (CDDs) and dibenzofurans (CDFs) are used to model both Total (mono- to octa-) CDD + CDF emissions and the toxicity equivalent (TEQ) of the 2,3,7,8-chlorine-substituted emissions. Analysis of emission data from two facilities indicates that use of total homologue concentrations shows limited, facility-specific correlations with Total CDDs/CDFs and TEQ. Concentrations of select mono- to tri-CDD/CDF congeners show promising correlation with CDD/CDF TEQ across facilities, suggesting that these compounds can act as TEQ indicators. PMID- 10739041 TI - Fingerprints of dioxin from thermal industrial processes. AB - PCDD/F homologue profiles and 2,3,7,8 chlorine-substituted congener patterns are studied using principal component analysis and hierarchical cluster analysis. Measurement data from different industrial metallurgical processes such as iron and steel manufacturing, copper smelters and aluminium plants are compared. It appears that the PCDD/F homologue profiles from industrial metallurgical processes are similar to those from municipal solid waste incinerators. The 2,3,7,8 chlorine-substituted congener pattern from copper smelters is also very similar to that of municipal solid waste incinerators. However, the 2,3,7,8 chlorine-substituted congener pattern from an aluminum plant is very different, 2,3,7,8-TCDF being by far the most important TCDF congener. PMID- 10739042 TI - Certification of a long-term sampling system for PCDFs and PCDDs in the flue gas from industrial facilities. AB - AMESA (adsorption method for sampling of dioxins) is a fully automatic system for long term monitoring of dioxin emissions from industrial processes based on the adsorption method. The system has been tested and undergoing a certification procedure according to the German guidelines for certification of systems for continuous monitoring of special substances. The certification covered parameters such as disposability of the system, reproducibility of the results and comparability of the sampling method with German and European standard methods. Furthermore break through, blanks and sample storability were investigated. The results prove that AMESA is a state of the art sampling system for continuous monitoring of dioxin/furan emissions. PMID- 10739043 TI - Polypropylene as regenerable absorber for PCDD/F emission control. AB - A new method for the PCDD/F reduction in exhaust gases of incineration plants is based on the property of polypropylene (PP) of interacting with PCDD/F to a variable degree depending on the temperature. After having been loaded with PCDD/F at approx. 80 degrees C, the PP absorber material can be regenerated by a temperature increase of about 50 degrees C. Hence, PP can be applied in a closed cycle as a thermally regenerable PCDD/F separator without an additional waste flow being generated. The setup of the pilot plant and the first results achieved shall be described. PMID- 10739044 TI - Results of an "emergency quality control study" as confirmation of a PCDD/PCDF contamination of milk and butter samples. AB - Elevated PCDD/F levels in four butter samples and one milk sample had to be confirmed under considerable time pressure. These samples should demonstrate a raising dioxin contamination in different regions of Germany caused by the use of Brazilian citrus pulp as feedstuff. Above all, the increase of 2,3,7,8-TCDD and 1,2,3,7,8-PeCDD had to be confirmed. Thus, an "emergency quality control study" was performed including five laboratories from the official food control in Germany. The results had to be reported about two to three weeks after the first announcement of the samples (including time for shipment). The five laboratories applied different extraction and clean-up methods, used different GC-columns and GC/MS-equipment and different standard solutions. The results are in a good agreement. Slightly different results for most individual congeners were of minor importance. A difference was observed only in the 2,3,7,8-TCDF content of one laboratory which was not relevant for the evaluation of the increase of the dioxin contamination in milk and dairy products. The laboratory's individual methods proved to give correct results even under a very tight schedule. PMID- 10739045 TI - Increase of the PCDD/F-contamination of milk, butter and meat samples by use of contaminated citrus pulp. AB - In Germany from 1993 to 1997, the polychlorinated dibenzo-p-dioxins and -furans (PCDD/F) contamination of food decreased slowly but constantly. However, for milk and butter, this trend was gradually reversed beginning in September 1997: From summer 1997 to February 1998 the average PCDD/F contamination of dairy products increased from a low level of about 0.6 pg I-TEQ/g fat in summer 1997. The dioxin content, of a limited number of randomly collected samples, rose on average to 1.41 pg I-TEQ/g fat (median 1.06 pg I-TEQ/g fat) in different regions of Germany in February 1998. A butter sample from the Netherlands with 1.96 pg I-TEQ/g fat hinted at the same source. The congener pattern in all contaminated milk and butter samples had elevated amounts of 2,3,7,8-TCDD and 1,2,3,7,8-PeCDD. Also, meat samples (beef, cow's meat and veal) with the same dioxin pattern were found to contain between 1.72 and 4.26 pg I-TEQ/g fat (background contamination of 0.53 pg I-TEQ/g fat). Large number of samples were analyzed to find a key for the cause. With a farmer producing milk with about 4.9 pg I-TEQ/g fat, a new source of PCDD/F for food contamination was discovered: the use of PCDD/F contaminated citrus pulp from Brazil as feed material for ruminants on a very large scale. Containing about 5-10 ng I-TEQ/kg, this component was about 20-100 times more highly contaminated than average feed with background contamination. Very complex pieces of circumstantial evidence were gathered to prove the correlation between this component of feed and the increase of dioxin contamination in milk. With respect to the huge trade of this feed ingredient on the global market, many other countries were involved. As an immediate response on these findings, the European Community fixed a preliminary maximum permitted level of 500 pg I-TEQ/kg citrus pulp, valid since August 1998. PMID- 10739046 TI - PCB-blood levels in teachers, working in PCB-contaminated schools. AB - In order to get more information about potential health hazards due to indoor air PCBs the present study investigated the PCB indoor concentration in schools as well as the blood levels of 6 PCB-indicator congeners in teachers from these schools. 151 teachers (78 male and 73 female; mean age 48 years) from 3 contaminated and 2 control schools participated in the study. Maximal indoor air values for total PCBs (6 PCB-indicator congeners times 5) in schools ranged from 1587 to 10655 ng/m3. Blood analyses indicated an increase in mean PCB 28 level from 0.036 (control group) to 0.098 microg/l in teachers from a school with heavy contamination of low chlorinated PCB. But there was no significant increase of PCB 138, 153 and 180 in blood above the normal background concentrations in any of the contaminated schools (mean values of all groups: PCB 138 = 0.66, 153 = 0.95, 180 = 0.70 microg/l blood). The results of blood analyses and additional toxicokinetic calculations suggested that inhalative PCB-uptake in the most contaminated schools caused a minor increase above mean background-PCB concentrations in blood. In conclusion, despite high PCB indoor air levels in schools, there was only a moderate increase in blood concentrations of teachers, mainly due to congeners with low chlorination (PCB 28 to PCB 101). PMID- 10739047 TI - Dioxin incinerator emissions exposure study Times Beach, Missouri. AB - PURPOSE: To determine whether living in the vicinity of a hazardous waste incinerator that was burning material contaminated with 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) increased TCDD and toxicity equivalencies (TEQ) in individuals living near the incinerator. METHODS: Participants were randomly chosen from an area close to the incinerator and compared to participants outside of the exposure area. TCDD and related compounds were measured in blood serum before incineration, four months after incineration started, and at the end of incineration. RESULTS: Lipid adjusted serum levels of TCDD and TEQ decreased from pre-incineration to four months after incineration, and decreased further by the end of incineration. CONCLUSION: Incineration of TCDD did not result in any measurable exposure to the population surrounding the incinerator. PMID- 10739048 TI - Potential pathways and exposure to explain the human body burden of organochlorine compounds: a multivariate statistical analysis of human monitoring in Wurzburg, Germany. AB - By means of multivariate statistical analysis of human monitoring in the vicinity of Wurzburg it is examined whether the organochlorine levels in blood data of 309 persons can in part be explained by pollution from outdoor and/or indoor air. Although diet is the main source for intake this study concentrated on the remaining sources. Using 12 personal and geographical variables, which describe some aspects or components of indoor or outdoor exposure, the quantities of the different pathways for the intake are calculated and shown. Besides the impact of the indoor air, which can be pointed out for intake at the workplace for some PCB congeners, also the hypothesis of the existence of an intake from outdoor air for some organochlorine compounds cannot be rejected. Here, the origin is the spatial variation of the pollution, which can be directly traced back to micro-climatic processes, local relief conditions, and the functional set-up of the town. PMID- 10739049 TI - Multivariate data evaluation of PCB and dioxin profiles in the general population in Sweden and Spain. AB - Thirty-three polychlorinated biphenyls and 20 dioxin-like congeners were determined by GC-MS in blood and adipose tissue samples from two general population groups, one in Spain (n = 35) and one in Sweden (n = 28). The results were evaluated by multivariate projection (Simca), after normalisation to the most abundant and stable congener in each data set, in order to reveal differences in the "national" congener profiles. The mean values of each population group did not differ significantly. The sum of PCBs in the Spanish was 1450 ng/g and in the Swedish 1310 ng/g. The sum of the dioxin-like compounds in Spain was 1180 pg/g and in Sweden 804 pg/g. The congener profiles differed between the countries. Mainly the higher chlorinated congeners for both the dioxin-like compounds and the PCBs are more dominant in the Spanish group compared to the Swedish. PMID- 10739050 TI - The reliability of 2,3,7,8-tetrachlorodibenzo-p-dioxin and toxicity equivalence measurements in Missouri residents. AB - This study evaluates the reliability of serum measurements of 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) and toxicity equivalence (TEQ) in individuals with no prior exposure to TCDD or dioxin-like compounds above background levels. Reliability was measured over three time-periods in the same individuals. TCDD and TEQ were measured in 127 and 128 participants. The values declined during the testing periods. Reliability coefficients for TCDD was 0.69 and for TEQ 0.91 but increased to 0.79 and 0.92, respectively, after removal of three outlying cases. Study results show good reliability over 22 months suggesting a consistency in the uptake and excretion of TCDD and other dioxin-like compounds. PMID- 10739051 TI - Dioxins: WHO's tolerable daily intake (TDI) revisited. AB - In December 1990, the World Health Organization (WHO) established a tolerable daily intake (TDI) of 10 pg/kg b.w. (body weight) for TCDD, based on liver toxicity, reproductive effects and immunotoxicity in experimental animals, and making use of kinetic data in humans and experimental animals. Since then new epidemiological and toxicological data have emerged, in particular with respect to neurodevelopmental and endocrine effects of dioxin. Therefore, the European Centre for Environment and Health of the World Health Organization (WHO-ECEH) and the International Programme on Chemical Safety (IPCS) jointly organized a consultation on the "Assessment of the health risk of dioxins: re-evaluation of the TDI", May 1998, Geneva, Switzerland. The participants discussed the health risks for infants, cancer and non-cancer endpoints in humans and animals, mechanistic aspects, kinetic behaviour, modelling, exposure, and the applicability of the toxic equivalency (TEQ) concept. For the health risk assessment of dioxin-like compounds, the WHO Consultation focused on the most sensitive effects that are considered adverse (hormonal, reproductive and developmental effects) seen at low doses in animal studies (rats and monkeys). Human daily intakes corresponding with body burdens similar to those associated with adverse effects in animals could be estimated to be in the range of 14-37 pg/kg b.w./day. To arrive at a TDI expressed as TEQ, a composite uncertainty factor of 10 was recommended. By applying this uncertainty factor a TDI range of 1-4 pg TEQs/kg body weight was established. An extensive executive summary of the results of this WHO Consultation with all the underlying background documents will be published in Food Additives and Contaminants (in press). PMID- 10739052 TI - Decrease of PCDD/F levels in human blood from Germany over the past ten years (1989-1998). AB - 744 whole blood samples of normal subjects from Germany collected in 1989-1998 have been analyzed for polychlorinated dibenzo-p-dioxins (PCDD) and dibenzofurans (PCDF) by capillary gas chromatography/high resolution mass spectrometry. Over the examined time period a continuous decrease of the PCDD/F concentrations in human blood was observed. The mean levels found were 43.7 pg I-TEq/g (lipid basis) in 1989 and 20.7 pg I-TEq/g (lipid basis) in 1996/98 [median: 42.2 and 19.4]. The reduction to about the half was found for most congeners. Each one year subset of the collective and the entire collective shows a positive correlation of the PCDD/F blood levels with age for most of the congeners, the sum values and the calculated toxicity equivalents. For statistical evaluation a multiplicative model was used: Concentration = A x Age(B). The correlation is mostly pronounced for lower chlorinated PCDD and for 2,3,4,7,8-PentaCDF. The PCDD/F concentrations in human blood in relation to the year of examination and the age of the subjects can be described by a linear model: I-TEq [pg/g (lipid basis)] = 6176 - 3.097 x Year + 0.6482 x Age or by a multiplicative model: I-TEq [pg/g(lipid basis)] = 10(89.08-0.04415 x Year + 0.008468 x Age). PMID- 10739053 TI - Certain organochlorine and organobromine contaminants in Swedish human milk in perspective of past 20-30 years. AB - The investigations of organochlorine compounds in breast milk from women living in the Stockholm region started in 1967. The present study summarises the investigations of polychlorinated biphenyls (PCBs), naphthalenes (PCNs), dibenzo p-dioxins (PCDDs), dibenzofurans (PCDFs), polybrominated diphenyl ethers (PBDEs) and pesticides (DDT, DDE, hexachlorobenzene, dieldrin) as well as methylsulfonyl metabolites of PCBs and DDE in human milk sampled during different periods up to 1997. During the course of 20-30 yr the levels of organochlorine compounds in human milk have decreased to various extent. A decrease to the half of the original concentration was attained in the range of 4-17 yr periods. On the contrary to the organochlorine compounds, the concentrations of PBDEs have increased during the period 1972-1997, indicating a doubling of the levels by 5 yr. The levels reflect the environmental contamination and background levels in the population. The accumulation and ongoing increase in the levels of PBDEs calls for immediate measures to stop the environmental pollution and human exposure to PBDEs. PMID- 10739054 TI - Steps towards a European dioxin emission inventory. AB - The results of a project aiming at collection and evaluation of information regarding the industrial and non-industrial emission sources for dioxins and furans (PCDD/PCDF) in 17 European Countries (EU 15, CH, N) are presented. An overview about national documents covering dioxin emission inventories for the period 1990-1995 is given. Some data on emissions associated with residual materials, waste and waste water are presented additionally. Based on the air emission data contained in these documents the most important emission sources were determined which are assumed to cover about 90% of the overall emissions. For the selected sources and for all 17 countries new estimates of the atmospheric PCDD/PCDF emissions were derived from average emission factors and statistical activity rates for the year 1994. As a result, on the European scale the largest annual PCDD/PCDF emission is assessed to be released from municipal waste incineration, quite closely followed by emissions from iron ore sintering. Considerable releases of dioxins and furans--based, however, on highly uncertain data--are further assessed for domestic burning, accidental fires and (former) use of contaminated wood preservatives (pentachlorophenol). A lower but still significant emission is further assigned to the sector of non-ferrous metal production; particular processes used in this branch proved to generate very high PCDD/PCDF flue gas concentrations. PMID- 10739055 TI - Input/output balance of estrogenic active compounds in a major municipal sewage plant in Germany. AB - 24 h samples of untreated and treated wastewater were taken in parallel from a modern municipal sewage plant in southern Germany in March and June 1998. After solid phase extraction, total estrogenic activity was quantitatively measured with a miniaturized E-screen assay and the levels of nine estrogenic phenolic chemicals analyzed by HRGC/LRMS. 17Beta-estradiol equivalent concentrations (EEQ) were 58 and 70 ng/l in the influent and 6 ng/l in the effluent, indicating that the load of estrogenic activity of the wastewater was reduced by about 90% in the sewage plant. Less than 3% of the estrogenic activity was found in the sludge. 4 t-octylphenol, 4-nonylphenol, bisphenol A, 2-hydroxybiphenyl, and 4-chloro-3 methylphenol were detected in the untreated wastewater at levels from 0.13 to 3.6 microg/l. 4-t-octylphenol, 4-nonylphenol, and bisphenol A were present in the effluent at concentrations from 0.16 to 0.36 microg/l, 2-hydroxybiphenyl and 4 chloro-3-methylphenol were not detectable. The contribution of the quantified levels of phenolic xenoestrogens to total estrogenic activity in the sewage was 0.7-4.3%. PMID- 10739056 TI - Dioxin mass balance in a municipal waste incinerator. AB - A dioxin mass balance in an Spanish municipal waste incinerator (MWI) is presented. Input and output inventories from two sampling collection episodes including the analysis of PCDD/PCDF in urban solid waste (USW), stack gas emissions, fly ash and slag are reported. In one collection the levels of USW were around 8 pg I-TEQ/g and non-thermal destruction was observed overall. In the other collection the levels of USW were higher (around 64 pg I-TEQ/g) and the dioxin balance revealed a thermal destruction. Analysis of the different waste materials (textile, organic, plastic, wood and paper) was performed separately and the textile samples presented the highest levels. PMID- 10739057 TI - Investigation on organic pollutants from a domestic heating system using various solid biofuels. AB - Various herbaceous biofuels (straw, whole plant cereals and set aside hay) and spruce wood were tested for their potential to form PCDD/F, PCPh, PCBz and PAH during combustion. The trials were conducted in an automatically charged multi fuel furnace for domestic applications (50 kWth). Both, flue gas and the different ash fractions were analysed. CO-emission results show, that combustion conditions were relatively uniform (mean CO-level: 200 mg/m3 at 13% O2 in flue gas). Likewise, the TOC- and PAH-emissions in the fuel gas remained constantly on a relatively low level. However, for the PCDD/F, PCPh and PCBz increased emissions were detected when herbacious fuels were applied. This may be attributed to their higher chlorine concentration and the high ash content, which is responsible for increased dust emissions during combustion. Similar observations were also found for the PCDD/F-concentrations in the ashes. Combustion chamber ashes usually showed a drastically reduced contamination with highly toxical compounds, compared to the ash fraction from the heat exchanger ash or to chimney soot. PMID- 10739058 TI - Regional aspects and statistical characterisation of the load with semivolatile organic compounds at remote Austrian forest sites. AB - Spruce needles and humus layer of 25 remote forest sites spread all over Austria were investigated for their concentrations of PCDD/F, PCB, HCH, HCB, PCP, DDX and PAH. Influences of the sites' location on the detected concentrations have been identified: The north of Austria was characterised by a comparably higher overall pollutant load. In addition, altitudinal aspects were addressed. Between several compounds significant positive correlations have been identified, which were more pronounced for compounds with a stronger causal relationship. Pattern analyses allowed to identify--even for the remote sites--differences between the relative PCDD/F, HCH, DDX and PAH patterns of the sites. Partly, these different patterns were related to significantly higher or lower corresponding absolute concentrations of the sites. PMID- 10739059 TI - Study of evolution of PCDD/F in sewage sludge-amended soils for land restoration purposes. AB - The evolution of polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) in sewage sludge-amended soils used in the restoration of degraded lands, like quarries, has been studied. Two experiments were performed: one in the lab, under controlled conditions, and another in a quarry. Two different doses of sewage sludge were applied in both experiments (with two types of application in the quarry experiment) and the evolution of the amended soils were compared with that of the respective control soils (without addition of sewage sludge). The samples were analyzed with a previously validated method by HRGC HRMS after the extraction and the necessary clean-up steps. The results reveal that polluted sewage sludge increases PCDD/F concentration in soils and that these compounds are persistent in the matrix after long periods of time. PMID- 10739060 TI - Screening-level ecological risk assessment of polychlorinated dibenzo-p-dioxins and dibenzofurans in sediments and aquatic biota from the Venice Lagoon, Italy. AB - Recent monitoring data indicate that portions of Italy's Venice Lagoon ecosystem have been degraded due to biological and chemical pollution from a variety of potential sources. Using polychlorinated dibenzo-p-dioxin (PCDD) and dibenzofuran (PCDF) data collected from sediment, fish and shellfish in the Lagoon, a screening-level ecological risk assessment (ERA) was performed to evaluate the risks to representative aquatic biota and wildlife receptors. Risks to aquatic invertebrates posed by PCDD/Fs in sediment were evaluated by comparing measured tissue concentrations in fish and shellfish to appropriate ecotoxicological reference values. For mammalian and avian receptors, risks posed by theoretical exposures to PCDD/Fs through the food chain were calculated using conservative wildlife exposure models. Results of the screening-level approach indicate that the potential for adverse effects to fish and aquatic invertebrate receptors from PCDD/Fs in surficial sediments are unlikely. Adverse effects to wildlife are possible but highly uncertain, and warrants further investigation in a more comprehensive ERA. PMID- 10739061 TI - Uptake of dioxin-like compounds from sewage sludge into various plant species- assessment of levels using a sensitive bioassay. AB - A bioassay for the detection of dioxin-like compounds was used to estimate uptake of dioxin-like compounds in carrots, oil seed rape seeds, zucchinis and cucumbers grown in soil amended with sewage sludge from Swedish sewage treatment plants (STP). This sensitive bioassay is based on 7-ethoxyresorufin O-deethylase (EROD) induction in cultured chicken embryo livers and reflects the combined biological effect of all dioxin-like compounds in a sample, including ones that seldom are analyzed. The bioassay detected low concentrations of dioxin-like compounds in all carrot, zucchini and cucumber samples, but did not detect any dioxin-like compounds in the rape seeds. In carrots the concentrations were increased up to seven times when grown in soil amended with high applications of some of the sludge samples, while others did not increase the concentrations compared to control. More realistic sludge applications only increased the concentrations slightly. The sludge-fertilized carrots contained the highest concentrations of the investigated plants (up to 14 pg bioassay-derived TCDD equivalents (bio TEQs)/g d.w.). In the carrots, differences in uptake of dioxin-like compounds depended on the sludge origin, which may be due to more easily bioaccumulated dioxin-like compounds in some sludge samples, or other components that facilitated uptake into the carrots. In the cucumbers, a more than two-fold increase (from 0.2 to 0.5 pg bio-TEQs/g d.w.) was observed in specimens grown in sludge-amended soil when compared to controls, suggesting a small uptake from the roots to the shoots. No sludge-dependent increase in uptake was seen in the zucchini fruits. The bio-TEQ levels were generally low in the consumable above ground plant parts of the investigated species. However, the question if repeated sludge application results in a soil accumulation of dioxin-like compounds, thereby increasing the risk of plant uptake, remains to be investigated. PMID- 10739062 TI - Search for chlorobornanes in river sediments and in influents and effluents. AB - In 1997, we collected nine river sediment samples, including one duplicate, from above and below three pulp and paper mills on the Wisconsin River in Wisconsin, USA, and one mill on Beaver Dam Creek in Ontario, Canada. We also obtained twenty split sediment samples from the Wisconsin River that were collected by the US EPA in collaboration with Indiana University. Finally, we collected influent and effluent samples from eight pulp and paper related industries in the US and Canada. We analyzed all sediment and water samples for hexa- through decachlorobornanes. We did not detect any chlorobornanes in most of the sediments. When chlorobornanes were detected, the concentration in sediments below pulp mills were similar to or less than in those from above the same mills. Chlorobornanes could not be detected in any of the influent or effluent samples. Thus, pulp and paper mills and relate facilities are not a source of chlorobornanes. PMID- 10739063 TI - Organochlorine compounds in the Gulf of Bothnia: sediment and benthic species. AB - Surface sediment, amphipods (Monoporeia affinis), isopods (Saduria entomon) and fourhorn sculpins (Oncocottus quadricornis) were collected at two coastal stations in the Gulf of Bothnia, one in the Bothnian Bay and the other in the Bothnian Sea. The objective was to study the concentrations, composition profiles, bioaccumulation features and spatial differences of organochlorine compounds such as hexachlorocyclohexanes (HCHs), DDTs, hexachlorobenzene (HCBz), chlordanes (CHLs), dieldrin, Mirex and polychlorinated biphenyls (PCBs). All groups of compounds were found in every sample investigated, with the exception of Mirex that was not detected in the sediment samples. The concentrations for e.g. PCBs and CHLs ranged from 700 to 2400 and 70 to 400 ng/g lipid in the specimens. For the corresponding sediments the results were 9.0-9.3 ng/g dw for PCBs and 0.54-0.57 ng/g dw for CHLs, respectively. Bioaccumulation differences between the species with regard to both degree of and type of compound were observed. The highest accumulation potential was found for the cyclodiene compounds including CHLs and Mirex in isopod. Finally, there were only small concentration and bioaccumulation differences between the two stations. PMID- 10739064 TI - Rapid anaerobic degradation of toxaphene in sewage sludge. AB - We studied the degradation of technical toxaphene in anaerobic sewage sludge from a municipal waste water treatment plant. Chlorobornanes, chlorocamphenes and related compounds were rapidly degraded, with degradation rates in the order of decachloro>nonachloro>octochloro>heptachloro approximately = hexachloro compounds. The half-lives of individual congeners ranged from <1 day to several days. We also studied the degradation of technical toxaphene in previously sterilized sludge (control), and found it was slower than in the anaerobic sludge. The chlorobornanes that degraded most rapidly in the non-sterilized anaerobic sludge were those with gem chloro substitution on the 6-member carbon ring, including the toxic congeners, Toxicant A and B. Non-gem chloro substituted congeners, like the biologically persistent P26 and P50, also degraded, but less rapidly. Toxaphene degradation in sewage sludge proceeded primarily via reductive dechlorination, leading to HxSed, HpSed, TC2 and other persistent metabolites. Enantioselective determinations indicated little, if any, enantioselectivity in the formation and/or degradation of these compounds. The isomer and enantiomer profiles of the hexa-, hepta-, and octachlorobornanes are similar to those observed in sediment from the Baltic Sea, suggesting that technical toxaphene is the source of these compounds and that its composition was changed via similar anaerobic degradation pathways. PMID- 10739065 TI - Toxaphene congeners in the Canadian Great Lakes basin: temporal and spatial food web dynamics. AB - Samples of a top predator fish species, lake trout (Salvelinus namaycush) and predominant forage species including smelt (Osmerus mordax), alewife (Alosa pseudoharengus), slimy sculpin (Cottus cognatus), deepwater sculpin (Myoxocephalus quadricornis) and lake herring (Coregonus artedii) were, collected from throughout 4 of the 5 Great Lakes (Superior, Huron, Erie and Ontario) (Fig. 1). Lake trout were also collected from three isolated lake systems (Lakes Nipigon, Simcoe and Opeongo), all located within the basin. All the samples were analysed for body burdens of total toxaphene and 22 toxaphene congeners. In addition, from each of the Great Lakes sites samples of major invertebrate dietary items, which included Mysis relicta, Diporeia hoyi and plankton, were similarly analysed. Whole lake trout samples, archived at -80 degrees C, were reanalysed to determine historical trends of toxaphene congeners plus carbon and nitrogen stable isotope signatures. The Lake Superior food web consistently had the highest levels of total toxaphene of all the Great Lakes monitored. The primary source of toxaphene to Lake Superior has been identified as atmospheric transport and deposition from sites in the southern US, Mexico and Central America (Hoff, R.M., Strachan, W.M.J., Sweet, C.W., Chan, C.H., Shackelton, M., Bidleman, T.F., Brice, K.A., Burnison, D.A., Cussion, S., Gatz, D.F., Harlin, K., Schroeder, W.H., 1996. Atmospheric deposition of toxic chemicals to the Great Lakes: A review of data through 1994. Atmospheric Environ. 30, 3505-3527). Of the offsystem lakes surveyed. Lake Nipigon, situated due north of Lake Superior and with a Lake Basin area of about 6% of Lake Superior (Hendendorf, C.E., 1982. J. Great Lakes Res. 8(3), 379-412) had total toxaphene levels in lake trout equivalent to about 50% of those found in lake trout from Lake Superior. Temporal trend toxaphene congener analysis was conducted on archived whole fish samples collected over the period 1980 through to the 1990's. Initially a nonachlorobornane congener (Parlar #50) was predominant, with congeners #40, #62 and #21 being the next most prominent in the 1980 samples. Samples from the 1990's showed a significant decline in the presence of lower chlorinated congeners #40 and #21. Analysis of total toxaphene in food webs, indicated elevated levels in lower trophic level species such as Diporeia and Cottus sp. which have a benthic association. The stable isotope temporal trend 13C signature identified a significant shift in the lake trout diet over the period 1993 to 1996. PMID- 10739066 TI - Levels of toxaphene congeners in fish from Danish waters. AB - The levels of toxaphene congeners, in addition to PCB congeners and organochlorine pesticides, were determined in various fish samples from different Danish waters. While PCB-153 and p,p'-DDE show different levels depending on the fishing area, with highest levels in fish from the Western Baltic Sea, toxaphene was detected in all the samples investigated at a more constant level. The distribution of the three toxaphene congeners Parlar #26, #50 and #62 depends on the fishing area, with the Western Baltic Sea being different from the other waters by having almost equal levels of toxaphene congeners #26 and #50. PMID- 10739067 TI - Reduction of serum thyroxine concentrations by methylsulfonyl metabolites of tetra-, penta- and hexachlorinated biphenyls in male Sprague-Dawley rats. AB - Male Sprague-Dawley rats received four consecutive intraperitoneal doses of nine methylsulfonyl (MeSO2) metabolites of tetra-, penta- and hexachlorinated biphenyls (tetra-, penta- and hexaCBs) to determine their effects on thyroid hormone levels. Nine MeSO2 metabolites, major MeSO2-PCBs detected in human milk, liver and adipose tissue, were 3-MeSO2-2,2',4',5-tetraCB (3-MeSO2-CB49), 3-MeSO2 2,3',4',5-tetraCB (3-MeSO2-CB70), 3-MeSO2-2,2',3',4',5-pentaCB (3-MeSO2-CB87), 3 MeSO2-2,2',4',5,5'-pentaCB (3-MeSO2-CB101), 4-MeSO2-2,2',4',5,5'-pentaCB (4-MeSO2 CB101), 3-MeSO2-2,2',3',4',5,6-hexaCB (3-MeSO2-CB132), 3-MeSO2-2,2',3',4',5,5' hexaCB (3-MeSO2-CB141), 3-MeSO2-2,2',4',5,5',6-hexaCB (3-MeSO2-CB149) and 4-MeSO2 2,2',4',5,5',6-hexaCB (4-MeSO2-CB149). All nine MeSO2 metabolites (20 micromol/kg once daily for four days) reduced serum total thyroxine levels (16-44%) at a much lower dose than phenobarbital (431 micromol/kg once daily for four days) on days 2, 3, 4 and 7 after the last dosage. Total triiodothyronine level was reduced 37% by treatments with 3-MeSO2-CB49 and 3-MeSO2-CB149 at day 7, but increased 35% and 38% by 3-MeSO2-CB70 and 4-MeSO2-CB101 at days 3 and 4, respectively. The reductions in thyroxine levels led to an increase in thyroid-stimulating hormone levels by 3-MeSO2-CB49, 3-MeSO2-CB87, 3-MeSO2-CB101, 3-MeSO2-CB132, 3-MeSO2 CB141, 3-MeSO2-CB149 and 4-MeSO2-CB149. A 30% increase in thyroid weight was produced by 3-MeSO2-CB101 and 3-MeSO2-CB141 treatments. Total cytochrome P450 content and the activity of 7-pentoxyrosorufin O-dealkylase were increased by all seven 3-MeSO2-PCBs. 3-MeSO2-CB49, 3-MeSO2-CB87, 3-MeSO2-CB101 and 3-MeSO2-CB132 also increased the activity of 7-ethoxyresorufin O-dealkylase. Thus, it is likely that all nine tested MeSO2 metabolites could influence thyroid hormone metabolism. The results show that tested 3- and 4-MeSO2 metabolites of tetra-, penta- and hexaCBs reduce thyroid hormone levels in rats, suggesting that the metabolites may act as endocrine-disrupters. PMID- 10739068 TI - Analysis of difference in vivo effects of TCDD between c-src +/+ mice, c-src deficient, -/+ and -/- B6, 129-Src(tm 1 sor) mice and their wild-type littermates. AB - Toxicities of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were tested on two strains of c-src deficient B6, 129-Src(tm 1 sor) mice, c-src -/+ and c-src -/- and their matched wild-type littermates c-src +/+ mice along with another c-src +/+ mice, from the same genetic lineage, B6, 129-Fos(tm 1 Pa) mice. The most conspicuous effect of c-src deficiency on the toxicity of TCDD appears to be the reduced hepatotoxicity. TCDD-treated c-src deficient mice show only modest degrees of hepatomegaly and triglycerides accumulation as compared to treated wild-type mice. PMID- 10739070 TI - Lowered birth weight among infants born to women with a high intake of fish contaminated with persistent organochlorine compounds. AB - In Sweden fatty fish from the Baltic Sea (at the Swedish east coast) constitute the main exposure route for persistent organochlorine compounds (POC). The present study assessed reproductive outcomes among fishermen's sisters from the Swedish east coast, who were shown to had had a relatively high consumption of contaminated fish in early life. For relevant comparison, we used a similar cohort from the Swedish west coast, where the contamination of fish is considerable less. The east coast cohort women had an increased risk of having an infant with low birth weight (LBW; <2500 g) as compared with the west coast cohort women (odds ratio (OR) 1.6. 95% confidence interval 1.1-2.3). The results strengthen the hypothesized association between exposure to POC during childhood and adolescence and an increased risk for LBW. PMID- 10739069 TI - Seveso Women's Health Study: a study of the effects of 2,3,7,8-tetrachlorodibenzo p-dioxin on reproductive health. AB - Although reproductive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure have been reported in numerous investigations of animals, studies of this association in humans are limited. In 1976, an explosion in Seveso, Italy exposed the surrounding population to among the highest levels of TCDD recorded in humans. The relatively pure exposure to TCDD and the ability to quantify individual level TCDD exposure from sera collected in 1976 for the Seveso cohort affords a unique opportunity to evaluate the potential dose-response relationship between TCDD exposure and a spectrum of reproductive endpoints. The Seveso Women's Health Study (SWHS) is the first comprehensive study of the reproductive health of a human population exposed to TCDD. The primary objectives of the study are to investigate the relationship of TCDD and the following endpoints: (1) endometriosis; (2) menstrual cycle characteristics; (3) age at menarche; (4) birth outcomes of pregnancies conceived after 1976; (5) time to conception and clinical infertility; and (6) age at menopause. Included in the SWHS cohort are women who were 0-40 yr old in 1976, who have adequate stored sera collected between 1976 and 1980, and who resided in Zones A or B at the time of the accident. All women were interviewed extensively about their reproductive and pregnancy history and had a blood draw. For an eligible subset of women, a pelvic exam and transvaginal ultrasound were conducted and a menstrual diary was completed. More than 95% of the women were located 20 yr after the accident and roughly 80% of the cohort agreed to participate. Data collection was completed in July 1998, serum TCDD analysis of samples for analysis of endometriosis as a nested case-control study was completed in October 1998, and statistical analysis of these data should be completed in early 1999. Serum samples are now being analyzed in order to relate TCDD levels with the remaining reproductive outcomes. PMID- 10739071 TI - Open chemical combustions resulting in a local increased incidence of orofacial clefts. AB - HYPOTHESIS: The open chemical combustions in Zeeburg, Amsterdam, The Netherlands, during the years 1961 up to and including 1969, resulted in a local increased incidence of orofacial clefts during this period. STUDY: A retrospective observational epidemiological study was performed, comparing the trend of the incidence of non-syndromal orofacial clefts during the sixties, for the Zeeburg maternity with that of the Wilhelmina Gasthuis. Both clinics were situated in Amsterdam, but varying in distance and compass direction from the incineration works. Thereafter, the addresses of the mothers giving birth to infants with orofacial clefts were plotted on a map of Amsterdam. RESULTS: Of the 8803 children born in the Zeeburg clinic during this period, 21 had a non-syndromal orofacial cleft, producing an average incidence of 2.4 per 1000 births. For the years 1963 through 1965 the incidence rose dramatically to peak at 7.1 per 1000, before plateauing at an average incidence of 1.68 per 1000 births, still 155% higher than in the Wilhelmina clinic (average incidence of 0.66 per 1000 during the years 1966 through 1969). During the 10 year period the Wilhelmina clinic exhibited no such rise. The incidence of non-syndromal orofacial clefts at the Wilhelmina clinic at no time exceeded 2.3 per 1000 births during the 10 year period. The addresses of the mothers of the Zeeburg clefts were grouped primarily to the northwest (and a smaller group to the west) of the incineration works. CONCLUSION: A relation between the open incineration of the chemicals and a local increased incidence of orofacial clefts seems very likely. PMID- 10739072 TI - Psychological effects upon exposure to polyhalogenated dibenzodioxins and dibenzofurans. AB - Thirty workers who had been exposed to combustion products for several years due to testing of flame retarding qualities of building materials and 30 controls from the same facility were investigated. Concentrations found in samples taken from different places of the facility were up to 14,660 microg/kg for polybrominated dibenzofurans and up to 67.1 microg/kg for polychlorinated dibenzodioxins (PCDDs) and dibenzofurans (PCDFs). Physical examination, routine laboratory parameters, and blood fat concentrations of PCDDs and PCDFs revealed normal findings. Neurotoxic symptoms showed a weak tendency of overrepresentation among the exposed workers. The frequency of neurobehavioural symptoms increased significantly with trait anxiety independent of exposure to combustion products. PMID- 10739073 TI - Testicular cancer and occupational exposures with a focus on xenoestrogens in polyvinyl chloride plastics. AB - In a case-control study of 148 cases of testicular cancer and 314 healthy controls information was collected on lifetime working histories and specific exposures. The main finding was a six-fold increase in the risk for seminoma, one type of testicular cancer, among plastic workers exposed to polyvinyl chloride (PVC). No overrisks were observed for other plastics than PVC. There may be an aetiological link to xenoestrogens, i.e. chemicals in the environment with oestrogenic properties. Phthalates are used in PVC as plasticizer and have oestrogenic properties that could promote the growth of endocrine sensitive tumour cells. However, this "endocrine disruptors" hypothesis must be further evaluated in experimental models with regard to the causative mechanisms of the oestrogenic tumour promotion. PMID- 10739074 TI - Biotransformation rates of Ugilec 141 (tetrachlorobenzyltoluenes) in rat and trout microsomes. AB - In vitro biotransformation rates of tetrachlorobenzyltoluene (TCBT) isomers 3,3',4,4'-Cl4-2-Me (TCBT 87), 3,3',4,4'-Cl4-5-Me (TCBT 88), and 3,3',4',5-Cl4-4 Me (TCBT 94) were determined using trout and rat hepatic microsomes. The disappearance of the TCBTs from the in vitro system followed first-order kinetics. The estimated biotransformation constants (k) for the rat ranged from 0.96 to 4.14 h(-1). Biotransformation rates for trout microsomes were much lower and ranged from 0.009 to 0.017 h(-1). PMID- 10739075 TI - Psychological distress in grandmother kinship care providers: the role of resources, social support, and physical health. AB - OBJECTIVE: The purpose of the present study was to investigate predictors of psychological distress in grandmother kinship care providers. More specifically, it was hypothesized that social support, family resources, and physical health would predict psychological distress in grandmothers raising grandchildren. METHOD: One hundred and two grandmothers raising grandchildren in parent-absent homes completed the Brief Symptom Inventory, Short Health Form-36, Family Resource Scale, Family Support Scale, and a questionnaire requesting background and demographic data. RESULTS: Results indicated that psychological distress was predicted by family resources, participants' physical health, and to a lesser extent, social support. CONCLUSION: The findings indicate that family resources, social support, and physical health affected psychological distress in grandmothers raising grandchildren. Grandmothers who reported fewer resources, less social support, and poorer physical health tended to experience higher levels of psychological distress. This study suggests that greater attention be given to interventions aimed to decrease psychological distress and improve the financial resources and physical health of grandmothers raising grandchildren. PMID- 10739077 TI - Psychological consequences of child maltreatment in homeless adolescents: untangling the unique effects of maltreatment and family environment. AB - OBJECTIVE: This study examined the differential effects of various forms of abuse, as well as their combined effects. The study also sought to separate the factors uniquely associated with abuse from those associated with the more general problems present in an abusive family environment. METHODOLOGY: Data were collected from 329 homeless adolescents. Preliminary analyses indicated some degree of matching for family background variables. Any differences in demographic or family characteristics were controlled for statistically. RESULTS: Chi-square analyses revealed significant differences across groups for rates of assault, rape, depression/dysthymia, and attempted suicide. Multivariate analyses (MANOVAs) indicated significant differences in severity of internalizing problems and cognitive problems. Without exception, the group with histories of both physical and sexual abuse exhibited the most severe symptomatology and was at greatest risk for revictimization. Multiple regression analyses suggested that abuse histories were predictive of internalizing problems while family characteristics were more predictive of externalizing problems. CONCLUSIONS: The findings indicate that both abuse type and family characteristics contribute to the development of symptomatology. Future prospective and longitudinal studies are needed to clarify the sequelae of abuse, as well as the possible cause and effect relations between abuse, family characteristics, and psychological outcome. PMID- 10739076 TI - Risk factors and context of men who physically abuse in Bogota, Colombia. AB - OBJECTIVES: The aim of this study was to identify risk factors for physical abuse caused by male perpetrators, as well as to describe the context of abuse and the role of the female partner in these cases in Bogota, Colombia. METHODS: Information from in-depth interviews of males reported to authorities for physical child abuse and their female partners (n = 45) was quantitatively and qualitatively analyzed and compared to to males and their female partners from the same neighborhood living with a child of the same gender and age (+/-3 years) as the abused child (n = 44). RESULTS: Situations of abuse occurred more often on a weekday, in the afternoon or early evening hours, with the mother present, exceptionally involved substance abuse, and tended to be repetitive. Male subjects' lower level of education, stepfather status, perceived stress, substance abuse and mental illness, lack of social support, history of childhood physical abuse, negative perceptions, attributions and unrealistic expectations of the child's behavior were associated with abuse. Cases' female partners were more likely to have a lower occupational level, a higher frequency of dependent personality, a history of childhood physical and sexual abuse and be herself physically and emotionally abused by her spouse. At least three scenarios for abuse emerged from the analyses: "explosive" men, "abusive disciplinarians," and "children out of parental control." CONCLUSIONS: Most of the findings are consistent with existing research despite the different social and cultural context. The different scenarios suggest the need to tailor preventive and rehabilitative interventions for abusers. PMID- 10739078 TI - Jewish immigrant parents from the former Soviet Union: a method for studying their views of how to respond to children's misbehavior. AB - OBJECTIVES: First to acquire knowledge about measures which Jewish immigrant parents from the Former Soviet Union may apply when facing misbehavior of children, and second to develop a methodology which would overcome barriers to talking about their child rearing practices. METHOD: Interviews with 273 immigrant parents were conducted in Israel. An adaptation and modifications were made to an instrument developed by Gardner, Scarr, and Schwartz to assess parents' approach to disciplinary methods titled "The Parental Discipline Techniques Instrument." Parents were presented with situations which describe misbehavior of children and they were asked in each situation to assess the child's behavior and for three alternative measures. RESULTS: Talking to the child (in a unidirectional manner) was the most common method suggested by the respondents as a first option. When they were asked for alternatives, the most common disciplinary method suggested was setting "restrictions" for the child. "Isolating" the child and "ignoring" the child were also suggested to a certain extent. There were methods, mostly indirect methods (e.g., the use of formal and informal networks), which were suggested in response to one type of misbehavior and did not appear in responses to other misbehavior. CONCLUSIONS: The methodology utilized in the study was found to be adequate in overcoming impediments which may hinder learning about the participants' views of how to respond to children's misbehavior. Such information could contribute to assessment and intervention in situations of maltreatment by enabling professionals to place the parent's behavior within the range of responses common within the specific cultural context of the immigrants. PMID- 10739079 TI - Differences and similarities between violent offenders and sex offenders. AB - OBJECTIVES: To investigate differences and similarities between violent offenders and two types of sex offenders, rapists and child molesters, in terms of their personality, the nature of the victim, the role of alcohol, and their confession to their crime. METHOD: Thirty-six adult sex offenders, 23 child molesters, and 32 violent offenders were compared on personality measures, their relationship with the victim, the presence of alcohol intoxication, their confession rate and retraction at trial, and the reasons they gave for having confessed to the police. RESULTS: Child molesters and adult sex offenders (rapists) were significantly more introverted than the violent offenders. The child molesters had higher social desirability scores than the other groups, they tended to assault relatives and friends, they were rarely intoxicated while committing the offense, and they had strong internal need to confess to the police. Rapists and violent offenders were more commonly intoxicated during the commission of the offense; the former tended to assault acquaintances, where violent offenders most commonly assaulted strangers. Exactly half of the rapists retracted their confession when the case went to trial; in contrast none of the other offenders retracted their confession. CONCLUSIONS: The results show that there are significant differences as well as similarities between the three groups of offenders, which have implications for assessment and treatment. PMID- 10739080 TI - Re-focusing child protective services. PMID- 10739081 TI - Alternative responses to child protective services: emerging issues and concerns. AB - PROBLEM: Increased calls to "do something" about child protective services (CPS) have resulted in proposals or new "paradigms" for services to at-risk or abusive families. These new paradigms call for the reform or revamping of CPS through the development of a community-based alternative response to some reports of child abuse and/or neglect. METHOD: This article reports on outcomes for 1,263 "low" risk CPS referrals diverted to a community-based alternative response system. Data on child, family, and case characteristics and services provided are presented as well as outcomes associated with re-referral and placement post service provision. RESULTS: The risk level and severity of some of the referrals to alternative response systems seems inappropriately high. The rates of re referral were similar for families who did or did not engage in assessment services, and were highest for families where domestic violence was present. CONCLUSIONS: Criteria for diversion to community alternatives to CPS must be clearly articulated and applied. Both CPS and alternative response system workers must have the skills required to address a family's recognition of the problem and degree of motivation to engage in problem resolution, and to understand their relationship to continued risk of CA/N. PMID- 10739082 TI - Recalling childhood experiences. PMID- 10739083 TI - Mental health professionals' attitudes and practices towards male childhood sexual abuse. AB - OBJECTIVE: This study aims to test the hypothesis of Holmes, Offen, and Waller (1997) that mental health professionals are not asking male patients about histories of sexual abuse. It also aims to investigate general attitudes and practices of mental health professionals to the issue of male sexual abuse. METHOD: One hundred and seventy-nine questionnaires were given to nurses, psychologists, and psychiatrists asking 10 questions about their attitudes and practice towards male sexual abuse. RESULTS: The majority of staff questioned rarely inquire about sexual abuse in male patients; staff are generally using ineffective and unsystematic methods of enquiry when they do ask; knowledge of prevalence rates of male sexual abuse are extremely variable; and 2/3 of staff report having had no specific training in assessment/treatment of sexual abuse and a similar number do not feel sufficiently trained to be able to inquire about sexual abuse in male patients. CONCLUSIONS: The study provides evidence for Holmes and colleagues' (1997) hypothesis that men are not being asked about sexual abuse histories. It also highlights a need for training professionals about male sexual abuse. PMID- 10739084 TI - The veridicality of punitive childhood experiences reported by adolescents and young adults. AB - OBJECTIVE: The primary goal of the present research was to determine whether retrospective reports of childhood disciplinary experiences and perceptions of that discipline correspond to actual childhood events and whether the accuracy of that report was influenced by the affective state of the respondent. METHOD: Eighty-three adolescent and young adult males completed a retrospective measure of physical child maltreatment, Assessing Environments (AEIII), and the Brief Symptom Inventory (BSI). As children the participants had been observed naturalistically in their homes interacting with their parents an average of 10 years earlier. RESULTS: Analyses were consistent with the hypothesis that both current mood and actual observations of parent-child interactions during childhood predict self-reported recollections of childhood maltreatment by one's parents. Further the veridicality of such recollections appears to depend upon the objective specificity versus the perceptive nature of the questions used to elicit the recollections. CONCLUSIONS: The findings suggest that assessment instruments suitable for obtaining information regarding earlier childhood victimization must utilize behaviorally specific items. Thus, items that are either global or intimate a normative comparison should be avoided. PMID- 10739085 TI - Reported anxieties concerning intimate parenting in women sexually abused as children. AB - OBJECTIVE: The study had two aims: (1) To investigate whether mothers with a history of contact child sexual abuse were anxious about the intimate aspects of parenting compared with a clinical comparison group. (2) To determine if there was any relationship between the mother's anxieties and the kind of parenting they recalled receiving themselves. METHOD: Two groups of mothers in mental health out-patient care were interviewed; 34 women with a history of contact child sexual abuse and 29 women with no history of sexual abuse. They completed the Intimate Aspects of Parenting Questionnaire, The Parenting Stress Index (Short form) The Parental Bonding Instrument and The General Health Questionnaire 28. The index group also completed a sexual abuse history questionnaire. RESULTS: Mothers with a history of child sexual abuse were significantly more anxious about intimate aspects of parenting than the comparison group. They also reported significantly more overall stress as parents. The index group recalled that their own parents were significantly less caring and that their fathers more controlling than the comparison group. A low score on Father Care was significantly associated with concerns about intimate parenting, but not with total parenting stress. By contrast, a low score on Mother Care was significantly associated with higher stress experienced as a parent, but not as strongly with anxieties about intimate parenting. CONCLUSIONS: Mothers with a history of contact child sexual abuse who attend mental health services are often worried that their normal parenting behaviors may be inappropriate or seen as such by other people. These anxieties seem associated with their history of childhood sexual abuse. PMID- 10739086 TI - Antidepressant-induced lipidosis with special reference to tricyclic compounds. AB - Cationic amphiphilic drugs, in general, induce phospholipid disturbances. Tricyclic, as well as other antidepressants belong to this group. In experimental animals, antidepressants induce lipid storage disorders in cells of most organs, a so-called generalized phospholipidosis. This disorder is conveniently detected by electron microscopic examination revealing myelin figures. Myelin figures or myeloid bodies are subcellular organelles containing unicentric lamellar layers. The lipidotic induction potency during in vivo is related to the apolarity of the compound. Metabolism of phospholipids takes place within the cell continuously. Several underlying mechanisms may be responsible for the induction of the phospholipid disturbance. For instance, it has been suggested that the compounds bind to phospholipids and such binding may alter the phospholipid's suitability as a substrate for phospholipases. Free TCA or metabolites thereof may also inhibit phospholipases directly, as has been demonstrated for sphingomyelinase in glioma and neuroblastoma cells. Both these mechanisms might result in phospholipidosis. Interaction between drug and phospholipid bilayer has been investigated by nuclear magnetic resonance technique. There seems to be large differences in the sensitivities amongst different organs. Steroid-producing cells of the adrenal cortex, testis and ovaries are in particular susceptible to drug-induced lipidosis. The so-called foam cells are lung macrophages located in the interstitium which become densely packed with myelin figures during TCA exposure. It requires about 3-6 weeks of treatment to develop this converted cell. In cell cultures however, phospholipidosis is demonstrated already after 24 h only. It appears that the cells that undergo TCA-induced lipidosis may recover after withdrawal of the drug. The time required to achieve complete recovery ranges from 3-4 weeks to several months, depending on the organ affected. Little is known about the functional significance of lipidosis. Even if TCA and other antidepressants show other effects, it has not been possible to exclusively relate it to phospholipidosis. However, few attempts have been made to correlate the physiological effects of TCAs in experimental animals to the morphological changes associated with phospholipidosis. There is an increasing evidence however, that cationic amphiphilic drugs may have effects on immune function, signal transduction and receptor-mediated events, effects that to some extent might be related to disturbances in phospholipid metabolism. PMID- 10739087 TI - Basal nitric oxide limits immune, nervous and cardiovascular excitation: human endothelia express a mu opiate receptor. AB - Nitric oxide (NO) is a major signaling molecule in the immune, cardiovascular and nervous systems. The synthesizing enzyme, nitric oxide synthase (NOS) occurs in three forms: endothelial (e), neuronal (n) and inducible (i) NOS. The first two are constitutively expressed. We surmise that in many tissues there is a basal level of NO and that the actions of several signaling molecules initiate increases in cNOS-derived NO to enhance momentary basal levels that exerts inhibitory cellular actions, via cellular conformational changes. It is our contention that much of the literature concerning the actions of NO really deal with i-NOS-derived NO. We make the case that cNOS is responsible for a basal or 'tonal' level of NO; that this NO keeps particular types of cells in a state of inhibition and that activation of these cells occurs through disinhibition. Furthermore, naturally occurring signaling molecules such as morphine, anandamide, interleukin-10 and 17-beta-estradiol appear to exert, in part, their beneficial physiological actions, i.e., immune and endothelial down regulation by the stimulation of cNOS. In regard to opiates, we demonstrate the presence of a human endothelial mu opiate receptor by RT-PCR and sequence determination, further substantiating the role of opiates in vascular coupling to NO release. Taken together, cNOS derived NO enhances basal NO actions, i.e., cellular activation state, and these actions are further enhanced by iNOS derived NO. PMID- 10739088 TI - MAL, a proteolipid in glycosphingolipid enriched domains: functional implications in myelin and beyond. AB - The myelin and lymphocyte protein MAL (VIP17/MVP17) is a proteolipid of 17 kD with a hydrophobicity pattern that indicates a four transmembrane domain structure. The MAL cDNA has been cloned from human T-cells, rat oligodendrocytes and the Madin-Darby canine kidney (MDCK) cell line. In the nervous system both myelinating cells, oligodendrocytes and Schwann cells, express MAL protein. MAL expression parallels myelin formation, and MAL is predominantly localized in compact myelin. Prior to myelin formation MAL is also found in immature Schwann cells. Outside the nervous system MAL expression is found in T-cells and in distinct epithelial cells, e.g. in kidney, stomach and thyroid gland, where MAL is localised in the apical plasma membrane. Specific glycosphingolipids, e.g. galactosylceramide and sulfatide, are enriched in such apical kidney and stomach membranes as well as in myelin. MAL copurifies with these glycosphingolipids in detergent insoluble domains, indicating a close association and possible functional interactions of MAL with glycosphingolipids in these tissues. Moreover, recent reports point to additional functions of MAL-glycosphingolipid complexes in signalling, cell differentiation and apical sorting. The role of MAL in the formation, stabilisation and maintenance of glycosphingolipid-enriched membrane microdomains and its contribution to specific membrane properties in myelin and epithelial cells are discussed. PMID- 10739089 TI - Statistics of transmitter release at nerve terminals. AB - This review presents an historical account of the developments of the statistical analysis of quantal transmission over the past half century and of the progress made in using this approach to reveal new properties of nerve terminals. In the early 1950s, Katz and his colleagues showed that evoked transmitter release occurred in quanta at the neuromuscular junction, opening up the study of transmitter release at nerve terminals to statistical analysis. In the subsequent two decades attempts were made to see if evoked quantal release could be described by binomial or compound binomial statistics, as originally suggested by Katz, and to relate the parameters of the statistic to various structures of the nerve terminal. During this period two hypotheses were enunciated, namely the 'vesicle hypothesis', which states that quanta arise as a consequence of the packaging of transmitter in vesicles; and the 'active zone hypothesis', which states that vesicles undergo exocytosis at discrete sites on the nerve terminal. Unsuccessful attempts were made to relate the binomial parameter n to the elements in these hypotheses, that is to the number of active zones possessed by the terminal or the number of vesicles available for release at these zones. This difficulty was part resolved in the late 1970s with the application of non uniform binomial statistics to transmitter release from nerve terminals, in which n is the number of active zones each with their individual probabilities, p(j). Autocorrelation functions were subsequently introduced to detect if transmitter release is quantised at a particular nerve terminal. Statistical methods which would allow discrimination between different models of transmitter release over the active zones of a terminal were then developed. The introduction of maximum likelihood estimation procedures then allowed estimates to be made of the parameters in the statistical models of quantal release. The application of these procedures to experimental data from a variety of nerve terminals provided evidence for the concept that each synapse, taken as possessing a single active zone, possesses its own individual probability of secretion of a quantum by the exocytosis of a vesicle. In the late 1960s Stevens introduced the first stochastic approach to the analysis of the kinetics of the release of a quantum of transmitter at the neuromuscular junction following an impulse. In the subsequent decades this was developed into an explicit theory for the interaction of proteins involved in regulated exocytosis of a vesicle at an active zone. The parameters were the number of transition steps in the release process (k), each occurring at the same rate (alpha), with the possibility of each of these steps becoming blocked at the same rate (gamma). Maximum likelihood estimation procedures could then be used to obtain these parameter values. The discovery was made in the 1990s of the core proteins of the SNARE complex that govern regulated exocytosis. This offers the possibility in the near future of identifying the kinetic interaction of these proteins with the parameters of the stochastic process of exocytosis which confer a particular probability on individual synapses. PMID- 10739090 TI - The key role of butyrylcholinesterase during neurogenesis and neural disorders: an antisense-5'butyrylcholinesterase-DNA study. AB - The wide tissue distribution of butyrylcholinesterase (BChE) in organisms makes specific roles possible, although no clear physiologic function has yet been assigned to this enzyme. In vertebrates, it appears e.g. in serum, hemopoietic cells, liver, lung, heart, at cholinergic synapses, in the central nervous system. in tumors and not at least (besides acetylcholinesterase, AChE) in developing embryonic tissues. Here, a functional role of BChE can be found in regulation of cell proliferation and the onset of differentiation during early neuronal development--independent of its enzymatic activity. For studies concerning this point, we have established a strategy for a specific and efficient inhibition of BChE to investigate how the expected decrease of enzyme and, therefore, the manipulation of cellular cholinesterase-equilibrium influences embryonic neurogenesis--among others to gain information about the significance of noncholinergic, activity-independent and cell growth functions of BChE. The antisense-5'BChE-DNA strategy is based on inhibition of BChE mRNA transcription and protein synthesis. For this, the BChE gene is cloned into a suitable vector system; this is done in antisense-orientation, so that a transfected cell will produce their own antisense mRNA to inhibit gene expression. For such investigations in neurogenesis, the developing retina is a good model and we are able to create organotypic, three-dimensional retinal aggregates in vitro (retinospheroids) using isolated retinal cells of 6-day-old chicken embryos. Using this in vitro retina and "knock out" of BChE gene expression, we could show a key role of BChE during neurogenesis. The results are of great interest because in tumorigenesis and some neuronal disorders, the BChE gene is amplified or abnormally expressed. It has to be discussed how the antisense-5'BChE strategy can play a role in the development of new and efficient therapy forms. PMID- 10739091 TI - A new oxyprenyl coumarin and highly methylated flavones from the exudate of Ozothamnus lycopodioides (Asteraceae). AB - A new oxyprenyl coumarin was isolated from the lipophilic exudate of Ozothamnus lycopodioides. Its structure was established as 7-(3,3'-dimethylallyloxy)-5 hydroxy-6-methoxycoumarin from its UV, MS and NMR spectral data, especially two dimensional experiments. In addition to six earlier reported flavonols, we found four highly substituted flavones, including two rare methylenedioxyflavones. PMID- 10739092 TI - Epicuticular flavonoids of some Scrophulariaceae. AB - Twenty-two species of Scophulariaceae have been found to accumulate flavonoid aglycones externally on their leaves and stems. They belong to the genera Anarrhinum, Antirrhinum, Asarina, Calceolaria, Mimulus, and Odontites. Most of the flavonoids are methylated flavones and flavonols, some with 6-O and/or 8-O substitution. One of them is the natural isobutyryl ester of a rare flavone. PMID- 10739093 TI - Downstream processing for xylitol recovery from fermented sugar cane bagasse hydrolysate using aluminium polychloride. AB - Xylitol, a sweetener comparable to sucrose, is anticariogenic and can be consumed by diabetics. This sugar has been employed successfully in many foods and pharmaceutical products. The discovery of microorganisms capable of converting xylose present in lignocellulosic biomass into xylitol offers the opportunity of producing this poliol in a simple way. Xylitol production by biotechnological means using sugar cane bagasse is under study in our laboratories, and fermentation parameters have already been established. However, the downstream processing for xylitol recovery is still a bottleneck on which there is only a few data available in the literature. The present study deals with xylitol recovery from fermented sugar cane bagasse hydrolysate using 5.2 g/l of aluminium polychloride associated with activated charcoal. The experiments were performed at pH 9, 50 degrees C for 50 min. The results showed that aluminium polychloride and activated charcoal promoted a 93.5% reduction in phenolic compounds and a 9.7% loss of xylitol from the fermented medium, which became more discoloured, facilitating the xylitol separation. PMID- 10739094 TI - Changes of the photosynthetic apparatus in Spirulina cyanobacterium by sodium stress. AB - Spirulina platensis trichomes grown in Zarrouks medium having total Na+ concentration as 0.14 M when transferred to fresh Zarrouks medium containing enhanced level of Na+ ions equal to 0.86 M showed 30% more accumulation of Na+ intracellularly as compared to the control. An inhibition of photosystem II activity to almost 66% was observed. Also due to this exposure to high Na+, the room temperature absorption characteristics of Spirulina trichomes and the thylakoid membrane preparations were altered indicating changes in the chromophore protein interactions and alterations in the phycocyanin/allophycocyanin ratio; there by affecting the energy harvest and energy transfer processes. An increase in the carotenoid absorption was two fold over the control in the treated sample. Similarly, room temperature and low temperature (77 K) fluorescence emission spectra collectively suggested alterations in the chlorophyll a emissions, F 726 of photosystem I reflecting changes in the lipid protein environment of the thylakoid. Our results indicate that in Spirulina the enhanced Na+ level alters the energy harvest and transfer processes. It also affected the emission characteristics of chlorophyll a of photosystem I. PMID- 10739095 TI - Participation of the tightly-bound (putative cytoskeleton-bound) polysomes in translation during germination of dormant and non-dormant cereal caryopses. AB - Research was done on dormant and non-dormant barley cv. Ars caryopses and triticale cv. Grado caryopses treated and non-treated with abscisic acid (ABA). During germination higher participation of populations of so-called tightly-bound polysomes (TBP) in embryos of dormant barley caryopses was observed, as well as their high metabolic activity. In embryos of triticale caryopses of which dormancy was imposed in an artificial way by ABA (100 microM), the strongest incorporation of 14C-amino acids into nascent polypeptide chains in vivo was found in population of TBP, as well as the highest participation among three of the studied fractions (free polysomes, membrane-bound polysomes and tightly-bound polysomes). These results may indicate the significant role of TBP (putative cytoskeleton-bound polysomes--CBP) in maintaining dormancy during imbibition of cereal caryopses. PMID- 10739096 TI - Occurrence of avenanthramides and hydroxycinnamoyl-CoA:hydroxyanthranilate N hydroxycinnamoyltransferase activity in oat seeds. AB - Oat phytoalexins, avenanthramides, occur as constitutive components in seeds. The amounts of each avenanthramide were analyzed. The composition of avenanthramides in dry seeds was different from that in elicitor-treated leaves. In seeds, avenanthramide C was most abundant with an amount two times larger than that of avenanthramide A or B. On the other hand, avenanthramide A was the major component in elicitor-treated leaves. The total amount of avenanthramides in seeds increased 2.5 times during imbibition for 48 h although the composition did not change. The hydroxycinnamoyl-CoA:hydroxyanthranilate N hydroxycinnamoyltransferase (HHT, EC 2.3.1.-) activity, which is responsible for the final condensation step in the avenanthramide biosynthesis, was detected in dry seeds. The activity was localized in endosperm and scutellum, and slightly increased during 48-h imbibition. The enzyme was partially purified by anion exchange chromatography from both dry seeds and elicitor-treated leaves The activity was separated into two peaks by chromatography, indicating that HHT consists of at least two isoforms. The substrate specificities of HHT isoforms from seeds were different from each other. PMID- 10739097 TI - Selective induction of glutathione S-transferase subunits in wheat plants exposed to the herbicide acifluorfen. AB - Exposure to the herbicide acifluorfen resulted in marked increase of glutathione S-transferase (GST) enzyme activity in wheat seedlings, primarily in shoot tissues. From the six major, constitutively expressed GST subunits found in untreated wheat shoots subunits 2 and 3 were selectively induced by acifluorfen. No new subunit could be detected. The induced subunits belong to those GST isoenzymes, which metabolize diphenyl ether herbicides. PMID- 10739098 TI - Cytotoxic activity of flavonoids and extracts from Retama sphaerocarpa Boissier. AB - Seven flavonoids isolated from chloroform, ethyl acetate and butanol extracts, obtained from the aerial parts of Retama sphaerocarpa, have been assessed for cytotoxic activity against three human cancer cell lines: TK-10 (renal adenocarcinoma), MCF-7 (breast adenocarcinoma) and UACC-62 (melanoma), using the SRB assay. All of them, extracts and flavonoids, were actives in, at least, one of the three cell lines at the recommended National Cancer Institute doses. They produce a dose-dependent inhibition of cell growth at concentrations in the 10( 6)-10(-4) M and 25-250 microg/ml range for the flavonoids and extracts respectively, being the flavonol rhamnazin the most cytotoxic. PMID- 10739099 TI - Genes expressed in Ascochyta rabiei-inoculated chickpea plants and elicited cell cultures as detected by differential cDNA-hybridization. AB - In response to the exogenous application of elicitors and attempted invasion by pathogens, plants exhibit a wide range of defense reactions. To understand the defense mechanisms at the level of gene activation and deactivation, differential screenings were performed to isolate cDNA clones which are differentially expressed in pathogen-inoculated resistant chickpea plants and elicitor-treated cell cultures. A plenty of genes were isolated and arranged in 5 groups, namely defense-related pathways, signal transduction pathways, regulation of gene expression, catabolic pathways and primary metabolism. Most of these genes were activated although several genes were also found to be suppressed. We discuss the plausible functions of cDNA products in plant defense responses. The cDNAs provide a variety of tools to investigate molecular mechanisms of defense responses and clearly reflect the massive genomic and metabolic changes which occur during manifestation of antimicrobial defense. PMID- 10739100 TI - Catalytic decomposition of 3-chloroperoxybenzoic acid by immobilized catalase in a non-aqueous medium. AB - Catalytic activities of catalase (CAT) immobilized on graphite--GMZ, soot- "NORIT" and "PM-100" to mediate decomposition of 3-Cl-C6H4COOOH (3-CPBA) in acetonitrile have been investigated. Under these conditions, the kinetic parameters Km, k, Ea, Vmax, and Z0 were calculated. Conclusions on a probable mechanism of the catalytic process observed were drawn from the calculated values of deltaG*, deltaH*, and deltaS*. A quantitative UV-spectrophotometrical approach was used as the basic analytical tool. The electrochemical reduction of oxygen generated in enzyme catalysed 3-CPBA decomposition was examined with polarization curves method. PMID- 10739101 TI - Study of xanthine oxidase immobilized electrode based on modified graphite. AB - Xanthine oxidase (E. C. 1.2.3.2) was immobilized by adsorption on electrochemically modified graphite plate to obtain an enzyme electrode. The current of the enzyme electrode in substrate (xanthine) solutions was found to be a result of the electrooxidation of H2O2 generated in the enzyme layer. The linearity of the amperometric signal was up to a substrate concentration of 65 microM at 0.6 V (vs. Ag/AgCl). The response time was 2 minutes. The enzyme electrode preserves 80% of its initial activity after a three-week storage in air at room temperature. PMID- 10739102 TI - Extracellular xylanase production by two thermophilic alkali-tolerant Bacillus strains in batch and continuous cultures. AB - Xylanase production of newly isolated thermophilic alkali-tolerant Bacillus sp. strain SP and strain BC was investigated in batch and continuous cultures. Enzyme synthesis was inducible with both strains and was observed only in xylan containing media. Xylan from oat spelt is a better inducer than xylan from birch for strain Bacillus sp. BC while such difference was not observed for strain SP. Compared with batch cultures xylanase production of both strains increased about two times and its rate became more than four times faster in continuous cultures at a dilution rate of 0.2 h(-1). PMID- 10739103 TI - Chemical composition and antimicrobial activity of European propolis. AB - Three propolis samples from Austria, Germany and France were investigated by GC/MS, where eleven compounds were being new for propolis. The samples showed some similarities in their qualitative composition. Phenylethyl-trans-caffeate, benzyl ferulate and galangin were predominant in German propolis. Benzyl caffeate was predominant in French sample. Pinocembrin was predominant in French and Austrian propolis and trans-p-coumaric acid was predominant in all samples. The antimicrobial activity against Staphylococcus aureus; Escherichia coli, and Candida albicans was evaluated. German propolis showed the highest antimicrobial activity against Staphylococcus aureus and Escherichia coli. While Austrian propolis has the highest activity against Candida albicans. French propolis was effective against all pathogens but less than German and Austrian propolis. PMID- 10739104 TI - Evaluation of phenolic compounds in Brazilian propolis from different geographic regions. AB - Chemometrics has been shown quite efficient to uncover relationships between chemical composition of a sample and its geographical origin. Forty propolis samples originated from the the South and South East of Brazil were analyzed by HPLC and 18 compounds of interest were studied which included: caffeic, p coumaric and ferulic acids, and some of their derivatives, pinobanksin, a derivative of kaempferol and five phenolic compounds (assigned as 3-prenyl4 hydroxycinnamic acid (PHCA); 2,2-dimethyl-6-carboxyethnyl-2H-1-benzopyran (DCBE); 3,5-diprenyl-4-hydroxycinnamic acid (DHCA); compound E (still unknown) and 6 propenoic-2,2-dimethyl-8-prenyl-2H-1-benzopyran acid (DPB). Principal Component Analysis (PCA) indicated three different groups of propolis samples, having the same typical chromatogram, evaluated by HPLC. Samples from the South East group were rich in derivatives of kaempferol. Samples from the South group I had a high content of DPB compound, but a low concentration of kaempferol derivatives and of DCBEN compound. Samples from the South group II were characterized by a high concentration of DCBEN, DHCA, p-coumaric and DPB compounds. Therefore, the identification of new compounds in Brazilian propolis can give, useful information about the plant sources of a given geographic region. PMID- 10739105 TI - Bioactive terpenes from the soft coral Heteroxenia sp. from Mindoro, Philippines. AB - A marine soft coral species of the genus Heteroxenia collected from Mindoro Island, Philippines yielded two cadinene sesquiterpenes, (+)-alpha-muurolene (1) and a novel derivative (+)-6-hydroxy-alpha-muurolene (2), as well as the biologically active polyhydroxysterol, sarcoaldosterol A (3). The structure of the novel compound was unambiguously established on the basis of NMR spectroscopic (1H, 13C, COSY, 1H-detected direct and long range 13C-1H correlations) and mass spectrometric (EIMS) data. All compounds were active against the phytopathogenic fungus Cladosporium cucumerinum. The isolated terpenes were also active in the brine shrimp lethality test. PMID- 10739106 TI - Antiinfluenza virus effect of extracts from marine algae and invertebrates. AB - Sixty products, derived from marine organisms, typical of the Bulgarian Black Sea coast, were examined for inhibitory activity on the reproduction of influenza viruses in tissue cultures. The antiviral effect was investigated by the reduction of virus infectivity. Using representative strains of influenza virus it was shown that apparently the inhibitory effect was strain-specific. The most effective products were further studied in fertile hen's eggs and in experimental influenza infection in white mice. PMID- 10739107 TI - Lethal attribute of serine protease secreted by Vibrio alginolyticus strains in kuruma prawn Penaeus japonicus. AB - Toxicity of the extracellular products (ECP) and the lethal attribute of serine protease secreted by five pathogenic Vibrio alginolyticus strains from various sources in kuruma prawn Penaeus japonicus were studied. The ECPs of organisms originally isolated from diseased kuruma prawn or small abalone Haliotis diversicolor supertexta were more lethal (LD50 value of 0.48 or 0.41 microg protein/g prawn) than those from diseased tiger prawn P. monodon, yellowfin porgy Acanthopagrus latus or horse mackerel (LD50 value of 0.98-1.17 microg protein/g prawn). All the ECPs manifested strong, weak and no activities against gelatin, sheep erythrocytes and chitin, respectively. In immunodiffusion tests using rabbit antiserum to a purified 33 kDa serine protease of strain Swy against ECP of each tested strain produced one single precipitation band in each treatment. Furthermore, the serine protease was suggested to be the dominant protease secreted by V. alginolyticus strains tested since the majority of enzymatic activity of the respective ECP was inhibited by phenylmethanesulfonyl fluoride (PMSF). A higher inhibition of serine protease activity by PMSF resulted in lower mortality rate of the ECPs injected into the prawns suggesting that the protease is one of the major lethal factor(s) secreted by V. alginolyticus. PMID- 10739108 TI - Tissue cholinesterases. A comparative study of their kinetic properties. AB - The substrate saturation and temperature-dependent kinetic properties of soluble and membrane-bound forms of acetylcholinestarase (AChE) from brain and butyrylcholinesterase (BChE) from heart and liver were examined. In simultaneous studies these parameters were also measured for AChE in erythrocyte membranes and for BChE in the serum from rat and humans. For both soluble and membrane-bound forms of the enzyme from the three tissues, two components were discernible. In the brain, Km of component I (high affinity) and component II (low affinity) was somewhat higher in membrane-bound form than that of the soluble form components, while the Vmax values were significantly higher by about five fold. In the heart, Km of component II was lower in membrane-bound form than in the soluble form, while Vmax for both the components was about four to six fold higher in the membrane-bound form. In the liver, Vmax was marginally higher for the two components of the membrane-bound enzyme; the Km only of component I was higher by a factor of 2. In the rat erythrocyte membranes three components of AChE were present showing increasing values of Km and Vmax. In contrast, in the human erythrocyte membranes only two components could be detected; the one corresponding to component II of rat erythrocyte membranes was absent. In the rat serum two components of BChE were present while the human serum was found to possess three components. Component I of the human serum was missing in the rat serum. Temperature kinetics studies revealed that the Arrhenius plots were biphasic for most of the systems except for human serum. Membrane binding of the enzyme resulted in decreased energy of activation with shift in phase transition temperature (Tt) to near physiological temperature. PMID- 10739109 TI - Flexibility of amino acid residues at position four of nonapeptides enhances their binding to human leucocyte antigen (HLA) molecules. AB - The binding affinity of synthetic nonapeptides to human leucocyte antigens (HLA) molecules of the A0201 allotype, the most common in Caucasian, is enhanced or reduced by suitable amino acid substitutions at position 4, as a result of increased or decreased chain flexibility. A higher flexibility of the bond at this position correlates with an easier accommodation of the fragment into the HLA groove, while rigidity of the peptide chain appears to interfere. These data are based on two lines of evidence: a) most natural high affinity ligands for HLA A0201 possess, at position 4, flexible residues b) substitutions of such residues by rigid amino acids results in a decrease of binding affinity. PMID- 10739110 TI - Oxidation of plasmalogens produces highly effective modulators of macrophage function. AB - Model derivatives of plasmalogens and chemically synthesized oxidative degradation products as found e.g. during oxidation of low density lipoproteins show strong effects on phagocytosis induced secretion of reactive oxygen species of macrophages which was measured by luminol-enhanced chemiluminescence. Whereas a plasmalogen epoxide showed enhancing effects in submicromolar range, inhibition was found with higher concentrations as well as with alpha-hydroxyaldehydes. The substances showed only little effects on the non-cellular ROS-dependent chemiluminescence of the reaction between hydrogen peroxide and opsonized zymosan and no cytotoxic effects under the assay conditions used. These results show that oxidative modification and degradation of plasmalogens occuring also under pathophysiological situations in vivo produces effective modulators of macrophage function which could be important; e.g. during inflammation or atherogenesis. PMID- 10739111 TI - Endothelium modulates contractile response to simvastatin in rat aorta. AB - Simvastatin is an inhibitor of HMG-CoA reductase used in the treatment of hypercholesterolemia. In the present study simvastatin-induced contraction was observed in rat aortic thoracic rings, this effect increased when the endothelium was removed and when NO synthase was blocked by L-NOARG (3 x 10(-5) M). The contractile effect of simvastatin on intact aortic rings diminished when cyclo oxygenase was inhibited with indomethacin (10(-5) M). Also in the presence of endothelium, pretreatment with mevalonate (1 mM), the product of HMG-CoA reductase activity, significantly inhibited the contraction. In other experiments carried out on endothelium-removed preparations and in medium containing the calcium antagonist, diltiazem (10(-5) and 10(-6) M), the contraction dose response curves were significantly reduced and the same happened in the presence of the inhibitor of sarcoplasmic reticulum Ca-2+-ATPase, cyclopiazonic acid (CPA) (3 x 10(-6) M). The results suggest that simvastatin might increase intracellular calcium concentration. This effect could lead to an activation of NO synthase and cyclooxygenase pathways in endothelial cells and to contraction in vascular smooth muscle cells. This rise in Ca2+ concentration could be due to an inhibition of isoprenoid synthesis prevented by mevalonate. PMID- 10739112 TI - Molecular dynamics study on an adipokinetic hormone peptide in aqueous solution. AB - Lom-AKH-I is a member of the adipokinetic hormone/red pigment concentrating hormone (AKH/RPCH) family of peptides found in flying insects. A molecular dynamics simulation at room temperature (293 K) in water has been performed to survey the folding path of the Lom-AKH-I peptide in water and to establish the secondary structure of Lom-AKH-I. The obtained results indicate the presence of an undefined extended conformation. PMID- 10739113 TI - The prognostic significance of membrane transport-associated multidrug resistance (MDR) proteins in leukemia. AB - A major problem in the treatment of leukemia is the development of resistance to chemotherapeutic agents. There are several ways for cancer cells to develop resistance or defense mechanisms against cytotoxic drugs. This review paper will focus on membrane transport-associated multidrug resistance (MDR). The proteins involved, P-glycoprotein (P-gp), MRP1 and LRP/MVP, share the ability to act as drug transport proteins. Following upregulation of the mdr-1 gene, the energy dependent transmembrane P-gp overexpression results in diminished intracellular concentrations of anthracyclins, vinca-alkaloids and epipodophyllotoxins. The other transmembrane protein, MRP1, also has intracellular epitopes which are involved in intracellular redistribution and sequestration of drugs. The last named mechanism has also been ascribed to LRP, a protein which only occurs intracellularly. In leukemia patients, cellular drug resistance profiles determined in vitro at the time of presentation show a strong correlation with outcome. In AML, mdr-1 overexpression at diagnosis is a strong independent predictor for CR and long-term survival. In ALL, mdr-1 expression is of minor importance for prediction of outcome. In AML, MRP1 expression at diagnosis is not correlated with clinical response and survival in most studies. In ALL, MRP1 expression at diagnosis is not associated with response and long-term survival in the few studies on this aspect which have been published. The studies on LRP in AML emphasize the importance of the correlation between LRP-expression and anthracycline accumulation and suggest that LRP-expression has prognostic value at diagnosis. However, there is an equal number of studies where a predictive value in the case of LRP-expression in de novo AML cannot be shown. The highest levels of LRP have been reported in multiple relapses of ALL. Furthermore, new membrane-associated drug transport proteins have been reported including the transporter associated with antigen processing (TAP), the anthracyclin resistance associated protein (ARA), five new homologues of MRP (MRP2, or MOAT, MRP3, MRP4, MRP5, and MRP6), the sister of P-glycoprotein (sP-gp) and breast cancer resistance protein (BCRP). Studies on the (clinical) significance of these proteins have not yet been reported. PMID- 10739114 TI - Substrate recognition by P-glycoprotein and the multidrug resistance-associated protein MRP1: a comparison. AB - OBJECTIVES: It has recently been suggested that substrate recognition patterns for human P-glycoprotein encoded by mdr1 consist of two electron donor groups with a spatial separation of 2.5 +/- 0.3 A (type I units) or three electron donor groups with a spatial separation of the two outer groups of 4.6 +/- 0.6 A (type II units) [Seelig 1998]. Since P-gp and the multidrug resistance-associated protein (MRP1) have overlapping substrate specificity, we screened the chemical structures of 21 compounds, previously tested as MRP1 substrates, for electron donor units. In addition, we searched the putative transmembrane domains (TMD 1 12) of P-gp and (TMD 6-17) of MRP1 for amino acid side chains having the potential to interact with the respective substrates. METHODS: The three dimensional structures of potential MRP1 substrates were modeled with a force field approach and were then screened for electron donor units. Helical wheel projections of the 12 putative transmembrane domains of P-gp (1-12) and MRP (6 17) were analyzed for their content of amino acid residues with hydrogen bonding side chains, charged amino acid residues, and amino acid residues with pi electron systems. RESULTS: MRP1 recognizes compounds with type I and type II units. At least one electrically neutral together with either one negatively charged type I unit or two electrically neutral type I units are required for the compound to be bound and transported. Transport increases with increasing number of electron donor units. Compounds which carry exclusively electrically neutral type I units (P-gp substrates) are transported only weakly by MRP1, and compounds with cationic type I units (P-gp substrates) are not transported at all. An analysis of the putative transmembrane alpha-helices of MRP1 and P-gp reveals that the amino acid residues with hydrogen-bond donor side chains are arranged preferentially on one side of the helix and amino acid residues with inert (non hydrogen-bonding) side chains on the other side. In the case of MRP1, the hydrogen-bonding face also contains several cationic residues whereas, in the case of P-gp, it contains clusters of amino acid residues with beta-electron systems. CONCLUSIONS: We propose that P-gp and MRP1 recognize type I or type II units in chemical compounds having diverse structures, and that these transporters bind their substrates via hydrogen bond formation. Furthermore, we propose that transport of anionic substrates by MRP1 is facilitated by cationic amino acid residues present in the transmembrane helices of MRP1, whereas the transport of cationic substrates by P-gp is facilitated by a beta-electron slide guide. PMID- 10739115 TI - Use of membrane vesicles to investigate drug interactions with transporter proteins, P-glycoprotein and multidrug resistance-associated protein. AB - BACKGROUND: The ATP-dependent drug transporter proteins, P-glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP) are known to be involved in drug efflux that reduces drug accumulation and so renders tumor cells resistant to the cytotoxic effects of a number of anticancer agents. The ways in which these transporters bring about drug expulsion are not fully explained and may involve intracellular factors as well. Thus detailed evidence may be difficult to obtain from studies on intact cells. MATERIAL AND METHODS: Inside-out plasma membrane vesicles prepared from multidrug-resistant cells expressing high amounts of Pgp or of MRP provide a simpler system for investigating the interactions of putative substrates and resistance modifiers with the transport process. We consider here some aspects of the accumulation of radiolabelled vincristine and of dinitrophenol glutathione conjugate by these vesicles and demonstrate the usefulness of this approach for determining whether potential inhibitors have their effects on transport at the cell membrane or by more indirect means. CONCLUSIONS: We show that information gained from analysis of the ATP-dependence, time course and osmotic sensitivity of accumulation is helpful in distinguishing between transport and changes in binding. We have also used the technique to demonstrate the effects of the resistance modifier, XR-9051 on Pgp-mediated transport and to explore interactions of MK571, indomethacin and ethacrynic acid with MRP. PMID- 10739116 TI - Localization of the 1,4-dihydropyridine drug acceptor of P-glycoprotein to a cytoplasmic domain using a permanently charged derivative N-methyl dexniguldipine. AB - INTRODUCTION: P-glycoprotein (P-gp) is a 170 kDa ATPase which can transport a wide range of natural product cytotoxic drugs out of cells, thus conferring the multidrug resistance (MDR) phenotype. METHODS: In this paper we used the 1,4 dihydropyridine (1,4-DHP) MDR-reversing agent dexniguldipine (DN), and a derivative with a quaternary nitrogen which is permanently charged, N-methyl-DN, to explore the sidedness of block of [3H]-vinblastine transport by P-gp. RESULTS: In cytotoxicity assays, 1 microM DN sensitized MCF7 ADR cells, causing a 13-fold decrease in the EC50 of vinblastine from 400 +/- 80 nM to 30 +/- 25 nM. In marked contrast, N-methyl-DN was without effect. In intact MCF7 ADR cells, DN reversed the [3H]vinblastine uptake deficit with an EC50 of 445 +/- 100 nM, again, N methyl-DN was inactive. In photoaffinity labelling studies using the arylazide [3H]-B9209-005 in whole cells, DN potently inhibited incorporation of the photoaffinity label into P-gp whilst N-methyl-DN was without effect. However, in photoaffinity labelling studies in membrane fragments, both DN and N-methyl-DN potently inhibited [3H]-B9209-005 photoaffinity labelling of P-gp. Furthermore, in membrane fragments [3H]-vinblastine binding to P-glycoprotein was potently inhibited by both N-methyl-DN (Ki 10.7 +/- 4.9 nM) and DN (Ki 11.2 +/- 3.8 nM), and both N-methyl-DN and DN blocked ATP-dependent [3H]-vinblastine transport into inside-out vesicles. Thus, in intact cells the permanently charged 1,4 dihydropyridine, N-methyl-DN is unable to reverse the MDR phenotype or photoaffinity labelling of P-gp. However, in cell fragments and inside-out vesicles, N-methyl-DN binds avidly to P-gp and this binding blocks [3H] vinblastine transport. CONCLUSION: These data are consistent with the hypothesis that 1,4-DHPs block [3H]-vinblastine binding, and thereby transport by P-gp, by acting at a domain accessible only from the cytoplasm. PMID- 10739117 TI - Genetic linkage groups in the Japanese brown frog (Rana japonica). AB - To elucidate the genetic linkage groups of the Japanese brown frog (Rana japonica) and compare them with those of other amphibians, we analyzed the inheritance of alleles at 15 enzyme and blood protein loci and one pigment locus (Blk) in 3,298 offspring derived from 37 crosses using 28 males heterozygous at these loci. Of 63 pairs of loci tested for linkage, 55 pairs showed independent assortment in all crosses examined. In another six pairs, that is, between FUM/ME 1, alpha-GDH/MDH-2, MDH-1/ME-1, MDH-2/ME-2, MDH-2/PEP-C, and ME-2/PEP-C, all the offspring analyzed were parental, and none were recombinant. In the other two pairs, that is, between GPI/PEP-D and AAT-1/Blk, most of the offspring were parental, and some were recombinants with recombination rates ranging from 4.8% to 8.0%. Thus the following four linkage groups comprising 11 loci were established in R. japonica. The first group included the loci for alpha-GDH, MDH 2, ME-2, and PEP-C; the second group included the loci for MDH-1, ME-1, and FUM; the third group included the loci for GPI and PEP-D; and the fourth group included the loci for AAT-1 and Blk. No linkage between the other five loci--ADA, MPI, PEP-A, PGM, and Alb--was observed in the present study. PMID- 10739118 TI - Genetic differentiation among Oregon lake populations of the Daphnia pulex species complex. AB - Gene flow among invertebrate populations inhabiting bodies of nonflowing freshwater such as ponds or lakes must at some stage involve transport across habitat unsuitable for adult stages. Consequently the potential for interpopulational differentiation is high in these species, yet empirical studies of lake populations of Cladocerans such as Daphnia have failed to reveal high levels of genetic distinctiveness among populations and have led to much speculation about how these populations exchange genes and remain cohesive evolutionary units. In this study we surveyed 42 Oregon lake populations of Daphnia from the D. pulex species complex for genetic variation within the mitochondrial DNA control region. We have used this data to test the relative abilities of various ecological factors to explain the observed patterns in genetic differentiation among lakes. Despite limited genetic variation detected among our samples--11 very similar RFLP-defined mtDNA genotypes from 388 individuals--analyses of nucleotide variance using analogs to Wright's F statistics indicate that when multilake populations are defined in terms of the river drainage basin to which they belong, strong and significant amounts of among-population genetic variation can be detected at this locus (F(ST) estimates between 0.5 and 0.6). In contrast, we fail to detect consistent significant among population variation when populations are defined on the basis of regional physical geography, bird migratory flyways, or lake trophic status. The manner in which the data are compiled, that is, whether RFLPs or nucleotide sequences are used, has little effect on the overall conclusions, yet it is clear that nucleotide sequence data would lower the standard errors of F(ST) estimates. We propose that periodic widescale flooding during the late Pleistocene may be an important mechanism to homogenize genetic differences among lake Daphnia continent-wide south of the southern-most extent of Pleistocene glaciation. PMID- 10739119 TI - NOR regions of polychaete worms of the genus Ophryotrocha studied by chromosome banding techniques and FISH. AB - This article reports the results of cytogenetic analyses carried out on 10 species of polychaete worms belonging to the genus Ophryotrocha (Dorvilleidae). Nucleolar organizer regions (NORs) were characterized by Ag staining, C-banding, CMA3 staining, and ribosomal fluorescent in situ hybridization (rDNA FISH). Extensive intraspecific variation in NOR number and distribution were observed in O. costlowi, O. sp. macrovifera, O. notoglandulata, O.l. labronica, O. l. pacifica (2n = 6), O. p. puerilis, O. diadema (2n = 8), O. hartmanni, O. gracilis (2n = 10). In O. sp. robusta (2n = 10), Ag-NORs were always located on a single chromosome pair. CMA3 staining suggests a possible trend toward a GC-rich rDNA compartmentalization. In O.l. labronica, O. p. puerilis, O. diadema, and O. sp. robusta rDNA FISH shows that Ag and FISH signals coincide. Results from C-banding seem to indicate that the increased genome size (GS) observed in O. sp. macrovifera (0.8 pg) and O. hartmanni (1.16 pg) compared to the base GS value of the genus (0.4 pg) cannot be attributed to variation in the heterochromatin content. PMID- 10739120 TI - Detection of RFLP markers associated with antibody response in meat-type chickens: haplotype/genotype, single-band, and multiband analyses of RFLP in the major histocompatibility complex. AB - Improving disease resistance in poultry by direct selection or by selecting for immune response is hardly feasible due to the quantitative nature of these traits, their low heritability, and the difficulties associated with reliable measurements. In this situation, marker-assisted selection (MAS) is expected to be a more effective breeding approach. The major histocompatibility complex (MHC), known to affect immune response and disease resistance, was examined as a set of candidate genes for association between DNA markers and antibody response. Backcross (BC1) and F2 families were generated from a cross between lines divergently selected for high or low antibody response to Escherichia coli vaccination. Restriction fragment length polymorphism (RFLP) analysis of the highly polymorphic MHC class IV (B-G) region suggested an association with antibody response to several antigens (E. coli, SRBC, NDV). The multiband data generated with the class IV probe were used to compare the efficacies of three alternative analyses: "single-band" (carriers versus noncarriers of each RFLP band separately), "multiband" (multiple regression on all RFLP bands), and "genotype" (determined from family analysis of RFLP patterns/haplotypes). Groups of birds identified by the "multiband" analysis were identical to the haplotype based genotypes, suggesting that the laborious step of haplotype determination can be omitted without unduly sacrificing power of analysis. PMID- 10739121 TI - Mitochondrial DNA variation in the European otter (Lutra lutra) and the use of spatial autocorrelation analysis in conservation. AB - To add genetic information to the international conservation efforts on European otters Lutra lutra, we investigated the genetic population structure in and around a known "source" population of the otter, the Oberlausitz (OL) in eastern Germany. This was complemented by a first survey of genetic variation levels in the Central European otter population. Sequence analysis of 300bp of the mitochondrial control region in 76 specimens from the eastern German study region and 53 individuals from several other European populations revealed a low level of genetic variation, with only 5 haplotypes present and nucleotide diversities within populations ranging from 0.00% to 0.17%. Apart from eastern Germany, one haplotype was by far the most abundant one, from which other, only locally occurring types, could be derived by a single point mutation. This suggests a single Pleistocene refugium from which the analyzed European regions have been reinvaded after the glaciations. Within eastern Germany, two abundant haplotypes were found. Their occurrence differed significantly among subregions of eastern Germany. The uneven distribution of a locally restricted but abundant haplotype could be explained by isolation-by-distance and might reflect emigration from the OL source population to surrounding regions. This suggests that vital local populations can indeed serve as "sources" for the invasion of surrounding areas. Given a suitable genetic marker, we suggest a spatial autocorrelation analysis to monitor the genetic effect of such an emigration from a source population. PMID- 10739122 TI - Establishing a captive broodstock for the endangered bonytail chub (Gila elegans). AB - It is crucial for endangered species to retain as much genetic variation as possible to enhance recovery. Bonytail chub (Gila elegans) is one the most imperiled freshwater fish species, persisting as a declining population of large and old individuals primarily in Lake Mohave on the lower Colorado River. Establishment of a new captive broodstock from the 1981 F1 progeny of at most 10 wild fish plus any newly captured wild fish is evaluated and reviewed. The effective number of founders contributing to the 1981 F1 progeny appears quite small, varying from approximately 3.5, based on F1 allozyme data and supported by mtDNA data, to approximately 8.5, based on the original production records. Using a sample of these progeny to initiate a new broodstock further reduces the effective number of founders. With even the most optimistic evaluation of the amount of genetic variation in F1 progeny, it is obvious that including wild fish in the broodstock is essential to increase the amount of genetic variation. The approach given here could be applied to retain genetic variation in other endangered species in a captive broodstock until they have stable natural populations of adequate size. PMID- 10739123 TI - Mitochondrial DNA haplotyping of Testudo graeca on both continental sides of the Straits of Gibraltar. AB - Testudo graeca is an endangered species of tortoise that inhabits Mediterranean areas of Africa, Asia, and Europe. Western populations are found on both sides of the Straits of Gibraltar. The effects of geographical isolation on genetic divergence were assessed by the sequence analysis of two mitochondrial DNA regions of the 12S rRNA and cytochrome b genes. Four different haplotypes were identified. A single haplotype was shared by all Spanish and some east Moroccan specimens. Two haplotypes were unique to the west Moroccan T. graeca populations and allowed the clear discrimination between individual specimens found west of the Moulouya River. Phylogenetic analysis based on the estimation of nucleotide sequence distances of the haplotypes suggests an African origin for the Spanish populations and a subspecies status for the west Moroccan pool. PMID- 10739124 TI - Turkish honeybees: genetic variation and evidence for a fourth lineage of Apis mellifera mtDNA. AB - The mtDNA of bees from 84 colonies of Turkish honeybees (Apis mellifera) was surveyed for variation at four diagnostic restriction sites and the sequence of a noncoding intergenic region. These colonies came from 16 locations, ranging from European Turkey and the western Mediterranean coast to the Caucasus Mountains along the Georgian border, the eastern Lake Van region, and the extreme south. Combined restriction site and sequence data revealed four haplotypes. Three haplotypes belonged to the eastern Mediterranean mtDNA lineage. The fourth haplotype, which had a novel restriction site pattern and noncoding sequence, was found in samples from the extreme south, near the Syrian border. We found two different noncoding sequences among the eastern Mediterranean haplotypes. The "Caucasian" sequence matches that described from A. m. caucasica, and the "Anatolian" sequence matches that of A. m. carnica. The frequency of the "Caucasian" sequence was highest (98-100%) in sites near the Georgian border and decreased steeply to the south and west. Elsewhere the Anatolian sequence was found. In European Turkey (Thrace) a restriction site polymorphism previously reported from A. m. carnica in Austria and the Balkans was present at high frequency. A novel mtDNA haplotype with a unique restriction site pattern and noncoding sequence was found among bees from Hatay, in the extreme south near the Syrian border. This haplotype differed from the three previously known lineages of honeybee mtDNA--African, western European, and eastern Mediterranean-and may represent a fourth mitochondrial lineage. PMID- 10739125 TI - Classifying genealogical origins in hybrid populations using dominant markers. AB - In hybrid studies, potential for error is high when classifying genealogical origins of individuals (e.g., parental, F1, F2) based on their genotypic arrays. For codominant markers, previous researchers have considered the probability of misclassification by genotypic inspection and proposed alternative maximum likelihood approaches to estimating genealogical class frequencies. Recently developed dominant marker systems may significantly increase the number of diagnostic loci available for hybrid studies. I examine probabilities of classification error based on the number of dominant loci. As in earlier studies, I assume that only parental and first- and second-generation hybrid crosses between two taxa potentially exist. Thirteen loci with dominant expression from each parental taxon (i.e., 26 total loci) are needed to reduce classification error below 5% for F2 individuals, compared to 13 codominant loci for the same error rate. Use of loci in similar numbers from both taxa most efficiently increases power to characterize all genealogical classes. In contrast, classification of backcrosses to one parental taxon is wholly dependent on loci from the other taxon. Use of dominant diagnostic markers may increase the power and expand the use of maximum-likelihood methods for evaluating hybrid mixtures. PMID- 10739126 TI - Is there really natural selection affecting the l frequencies (long hair) in the Brazilian cat populations? AB - The scientific literature on cat genetics contains a presumed typical example of natural selection affecting l frequencies (long hair) in 16 Brazilian cat populations. It has been observed that the hotter and more tropical the climate in Brazil, the lower the values of l frequencies in the cat populations. Nevertheless, this study of some new cat populations in Latin America showed that all of them, independent of the climate, had high or very high l frequencies. l postulate that an alternative migrational-historical hypothesis exists that explains the correlation between the l frequencies and climate characteristics (which are correlated with the latitude) without using natural selection explanations concerning the appearance of the l allele in Brazil. PMID- 10739127 TI - Improved estimation of the proportion of triploids in populations with diploid and triploid individuals. AB - We consider the estimation of the proportion of triploids in populations of plants or animals in which diploid and triploid individuals coexist, using data from electrophoretic analysis of isozyme or microsatellite markers. Individuals that have three distinct alleles at a locus are unambiguously triploid. However, other individuals cannot be classified with certainty as diploid or triploid, unless allelic dosage can be determined reliably. This is impossible for microsatellite markers, and for many isozyme markers. We therefore present a maximum likelihood method of estimating the proportion of triploids based only on the presence or absence of different alleles. PMID- 10739128 TI - Inheritance of unique fruit and foliage color mutation in NuMex pinata. AB - The inheritance of mature fruit color in peppers (Capsicum spp.) is controlled by several genes. However, the inheritance of the transition of colors the fruit undergo during ripening has not been described extensively. The authors describe the inheritance of a unique gene which affects foliage color and fruit color transition occurring in the jalapeno cultivar NuMex Pinata. The gene responsible is designated the tra gene. PMID- 10739129 TI - Population genetics of geographically restricted and widespread species of Myrica (Myricaceae). AB - Allozyme variation of 11 putative loci in five populations of the rare Myrica adenophora Hance, and four populations of its widespread congeneric species, M. rubra (Lour.) Sieb. & Zucc. was studied. Among the 21 alleles studied, no unique allele was detected for M. adenophora, whereas M. rubra had 3 alleles not found in the former species. In terms of genetic diversity, populations of the rare species contained fewer alleles per locus (1.5 versus 1.7), fewer effective number of alleles per locus (1.12 versus 1.20), fewer number of alleles per polymorphic locus (2.14 versus 2.46), lower percentage of polymorphic loci (30.9 versus 40.9), and lower expected heterozygosity (0.106 versus 0.163) than populations of the widespread species. Genetic distances within species average 0.043 for M. adenophora and 0.045 for M. rubra, and between species ranged from 0.052 to 0.177, with a mean of 0.103, which agrees with the very similar gross morphologies of these two species. Intrapopulation differentiation was similar in both species: G(ST) = 0.152 for M. adenophora, and 0.146 for M. rubra, whereas estimated gene flow based on G(ST) values were moderate in these two species (Nm = 1.39 versus 1.46). We inferred that M. rubra and M. adenophora are a progenitor derivative species pair that emerged before migrating into Taiwan during the last glacial period. We consider the Hengchun population (Chiupeng, Hsuhai, and Chufengpi) and Taitung population (Tienkuan and Lanshan) of M. adenophora which probably arose from two subsets of the genome of M. rubra. Genetic drift was inferred to be one of the forces shaping the observed genetic structure in M. adenophora and M. rubra. PMID- 10739130 TI - A major gene for time of flowering in chickpea. AB - A major gene for the number of days from sowing to appearance of the first flower (time of flowering) was identified in a cross between an extrashort duration chickpea (Cicer arietinum L.) variety, ICCV 2, and a medium duration variety, JG 62. The F2 population was advanced through the single-seed-descent method to develop random recombinant inbred lines (RILs). Time of flowering was recorded for the parents and 66 F(6) RILs from this cross that were grown in a Vertisol field in the post-rainy season of 1996-1997. Similarly the parents, F(1) and F(10) RILs were evaluated in 1997-1998. The F(1) flowered along with JG 62. The time of flowering for the two sets of RILs showed bimodal distributions with nearly equal peaks. One peak corresponded with ICCV 2 and the other with JG 62. This suggests that a single gene controls the difference for the time of flowering between ICCV 2 and JG 62 and the allele carried by the latter parent is dominant. To our knowledge no gene has been identified for the time of flowering in chickpea. Therefore the allele carried by JG 62 is designated as Efl-1 and that by ICCV 2 as efl-1. The proposed genotype for ICCV 2 is efl-1 efl-1 and for JG 62 is Efl-1 Efl-1. The genotype efl-1 efl-1 reduces the time of flowering at ICRISAT by nearly 3 weeks. The significance of this gene for breeding for early maturity and genome mapping has been discussed. PMID- 10739131 TI - Heritability estimates for octyl acetate and octyl butyrate in the mature fruit of the wild parsnip. AB - The aliphatic esters octyl acetate and octyl butyrate occur as major components of essential oils in the vittae, or oil tubes, of the wild parsnip (Pastinaca sativa). We determined phenotypic variation and narrow-sense heritabilities of these octyl esters in wild parsnip fruits from 30 maternal families. The mean octyl acetate content was 1.56 microg/mg dry fruit (0.08-5.51 microg/mg dry fruit) and the mean octyl butyrate content was 4.28 microg/mg dry fruit (1.28 14.22 microg/ mg dry fruit). Narrow-sense heritabilities for each ester's content were calculated by analysis of half-sib families (HS) and parent-offspring regression (OP). Heritabilities were 0.389 (HS) and 0.654 (OP) for octyl acetate and 0.670 (HS) and 0.626 (OP) for octyl butyrate. The amounts of the esters were phenotypically correlated with each other and with the linear furanocoumarins bergapten and xanthotoxin, phototoxic compounds that co-occur in the vittae with the esters. Ester amounts were not genetically correlated, indicating that these compounds could respond independently to selection pressures. These octyl esters may serve as carrier solvents that enhance penetration of these furanocoumarins into herbivore integuments and gut walls. PMID- 10739132 TI - Outcrossing rates and relatedness estimates in pecan (Carya illinoinensis) populations. AB - Estimates of single and multilocus outcrossing rates as well as relatedness among progeny of individual seed trees were obtained for 14 populations of pecan [Carya illinoinensis (Wangenh.) K. Koch]. Mean outcrossing estimates were not significantly different from 1.0 and relatedness values indicate that most progeny within families are half sibs. Biparental inbreeding was insignificant in all study sites, and inbreeding coefficients indicated that populations were close to inbreeding equilibrium. PMID- 10739133 TI - Chloroplast inheritance and DNA variation in sweet, sour, and ground cherry. AB - Sour cherry (Prunus cerasus L.) is an allotetraploid and both sweet cherry (P avium L.) and ground cherry (P. fruticosa Pall.) are the proposed progenitor species. The study investigated the maternal species origin(s) of sour cherry using chloroplast DNA (cpDNA) markers and a diverse set of 22 sweet, 25 sour, and 7 ground cherry selections. Two cpDNA restriction fragment length polymorphisms (RFLPs) and one polymerase chain reaction (PCR) fragment length polymorphism were identified among the 54 selections. The three polymorphisms considered together resolved four haplotypes. Analysis of sour cherry progeny indicated that the chloroplast genome is maternally inherited and therefore appropriate to use in determining maternal phylogenetic relationships. Ground cherry was found more likely than sweet cherry to be the maternal progenitor species of sour cherry since 23 of 25 of the sour cherry selections had the most prevalent ground cherry haplotype. However, the other two sour cherry selections tested had the most prevalent sweet cherry haplotype and a wild French sweet cherry selection had the most prevalent ground cherry haplotype. The results underscore the importance of using diverse Prunus germplasm to investigate phylogenetic relationships. PMID- 10739134 TI - A gene for leaf necrosis in chickpea (Cicer arietinum L.). AB - Necrosis of leaves was observed in the glabrous mutant (ICC 15566) of desi chickpea (Cicer arietinum L.). It was characterized by drying of leaflet margins to drying of complete leaflets of older leaves. The oldest leaves were the most affected and the intensity of necrosis decreased toward the apical meristem. A single recessive gene, designated nec, was found to govern the necrotic characteristic. The nec locus was linked to gl (glabrous shoots) with a map distance of 16 +/- 3 cM. The loci slv (simple leaves), mlv (multipinnate leaves), nlv (narrow leaflets), hg (prostrate growth habit), P (pink corolla), and shp (round seed shape) segregated independently of nec. PMID- 10739135 TI - Single-locus inheritance in the allotetraploid Coffea arabica L. and interspecific hybrid C. arabica x C. canephora. AB - Molecular cytogenetic analysis has indicated that Coffea arabica is an amphidiploid formed from the hybridization between two closely related diploid progenitor species, C. canephora and C. eugenioides. Our aim was to determine the mode of inheritance in C. arabica and in a tetraploid interspecific hybrid (called arabusta) between C. arabica and C. canephora as revealed by segregation analyses of restriction fragment length polymorphism (RFLP) loci markers. The observed RFLP allele segregations in an F(2) progeny of C. arabica conform to disomic inheritance as expected, with regular bivalent pairing of homologous chromosomes in the F1 hybrid. In contrast, RFLP loci followed tetrasomic inheritance in the arabusta interspecific hybrid, although bivalents have been reported to predominate greatly at meiosis in its hybrid. These results suggest that homologous chromosomes do not pair in C. arabica, not as a consequence of structural differentiation, but because of the functioning of pairing regulating factors. Moreover, the arabusta hybrid seems to offer the possibility of gene exchange between the homologous genomes. PMID- 10739136 TI - PLABSIM: software for simulation of marker-assisted backcrossing. PMID- 10739137 TI - WHICHRUN (version 3.2): a computer program for population assignment of individuals based on multilocus genotype data. PMID- 10739138 TI - Submicrosecond real-time fluorescence sampling: application to protein folding. AB - Time-resolved fluorescence detection has become a central tool in the study of protein folding. This article briefly reviews modern fluorescence techniques and then focuses on recent improvements made possible by array photomultipliers, computer-controlled data gating, and long-memory multi-channel digitizers. It is now possible to detect fluorescence wavelength profiles and/or fluorescence decay transients very cost effectively with sub-microsecond kinetic time resolution out to long times. Folding kinetics can be analyzed by singular value decomposition (SVD) or chi-analysis. The latter provides an objective method for detecting nonexponential kinetics in two-state systems. PMID- 10739139 TI - Study of the chemical structures of the photo-cross-linking products between Tyr and the 5-azido-2-nitrobenzoyl residue. AB - Irradiation of N-(tyrosyl)-N'-(5-azido-2-nitrobenzoyl)-1,2-diaminoethane (I) initiates chemical reactions that lead to different products depending on the experimental conditions. All of these products are attributed to the reactions of triplet 4-nitrobenzoyl nitrene (4NBN). The reactions of triplet 4NBN with the tyrosyl residue result in the formation of two distinct products: compound II, which is unstable in aqueous solution, and the stable compound cyclo-[1-(4'-nitro 3'-benzoyl)-2-(aminotyrosyl)-N,N'-ethylenediami ne] (III). The formation of II is detected only in aerobic conditions. The unstable photoproduct II converts almost completely into compound III when its solution is concentrated. The photoproducts II and III have absorption spectra that are close to those of the photolabelled peptides. This finding is important for speculating about the chemical nature of the photomodification products of protein tyrosyl residues by the arylazide group. PMID- 10739140 TI - A time-resolved near-infrared fluorescence assay for glucose: opportunities for trans-dermal sensing. AB - We report a time-resolved near-infrared fluorescence assay for glucose detection that incorporates pulsed diode laser excitation. Reduction in fluorescence resonance energy transfer to a malachite green-Dextran complex from allophycocyanin bound to concanavalin A (ConA) due to displacement of the complex by glucose from ConA provides the basis of the assay. The fluorescence quenching kinetics are analysed and discussed in detail. The change in fluorescence decay kinetics in the presence of glucose is found from dimensionality studies to be brought about by a change in the distribution of malachite green-Dextran acceptors. Glucose concentrations are measured in solution to within +/- 10% over the range 0-30 mM. PMID- 10739141 TI - Changes in the endoplasmic reticulum structure of Paramecium primaurelia in relation to different cellular physiological states. AB - The fluorochrome 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)], a vital dye utilized to stain the endoplasmic reticulum (ER) of animal and plant cells, has been used to visualize the ER-type structures of Paramecium primaurelia under confocal laser scanning microscopy (CLSM). The morphology of the ER has been studied in paramecia in different physiological conditions. Cells are analysed in early and late logarithmic growth phases, in stationary and in death phases, during shift-up by refeeding after starvation and shift-down by using a starvation medium. In log-phase growing paramecia, the ER constitutes an anastomosing membrane system consisting of short tubules and flattened sacs forming a peripheral network, which is abundant in the cortical region around the trichocysts and the ciliary basal bodies. The tubular network and cytoplasmic membranes are reduced in stationary-phase cells; the original conditions are restored in starved cells after refeeding. The analysis of serial optical sections collected by CLSM at 0.5 microm intervals and three-dimensional reconstruction from these sections allow us to visualize differences between differently growing cells. PMID- 10739142 TI - Contrasting effects of excess ferritin expression on the iron-mediated oxidative stress induced by tert-butyl hydroperoxide or ultraviolet-A in human fibroblasts and keratinocytes. AB - Iron and/or ferritin accumulation are known to occur under pathological conditions in many inflammatory skin diseases or in human skin chronically exposed to UV light. Under such conditions, ferritin is believed to play an effective protective role in accommodating and 'deactivating' excess 'free' iron produced by the inflammatory process or the UV illumination. The present study compares the relationship between ferritin over-expression and effects of an oxidative stress induced chemically by tert-butyl hydroperoxide or photochemically by UV-A radiation. As shown by immunoassay, cultured MRC 5 and HS 68 fibroblasts treated for at least one day with transferrin or overnight with non-toxic concentrations of the ferric nitrilotriacetate complex express up to 10 times more ferritin than untreated cells, whereas a five-fold increase is obtained with NCTC 2544 keratinocytes. In all cases a parallel increase in soluble cellular iron is measured by inductive plasma emission spectroscopy. The superoxide dismutase and catalase activities and total glutathione levels are not modified by the iron treatment, whereas a transient increase in the Se-dependent glutathione peroxidase activity of keratinocytes is observed after a short incubation with the iron complex. In keratinocytes and fibroblasts, ferritin over expression after iron treatment markedly inhibits lipid peroxidation but, paradoxically, not the mortality induced by tert-butyl hydroperoxide. In contrast, this excess ferritin does not protect cells from both the peroxidation and mortality induced by moderate doses (30 J/cm2) of UV-A radiation. As a consequence, protection against oxidative damage by excess ferritin synthesis clearly depends on the nature of the oxidative stress on cell targets and it seems to be of lesser importance in the case of photochemically induced oxidation. PMID- 10739143 TI - Effects of 780 nm diode laser irradiation on blood microcirculation: preliminary findings on time-dependent T1-weighted contrast-enhanced magnetic resonance imaging (MRI). AB - Laser therapy by low light doses shows promising results in the modulation of some cell functions. Various clinical studies indicate that laser therapy is a valuable method for pain treatment and the acceleration of wound healing. However, the mechanism behind it is still not completely understood. To explore the effect of a low-power diode laser (lambda = 780 nm) on normal skin tissue, time-dependent contrast enhancement has been determined by magnetic resonance imaging (MRI). In the examinations, six healthy volunteers (four male and two female) have been irradiated on their right planta pedis (sole of foot) with 5 J/cm2 at a fluence rate of 100 mW/cm2. T1-weighted magnetic resonance imaging is used to quantify the time-dependent local accumulation of Gadolinium-DPTA, its actual content in the local current blood volume as well as its distribution to the extracellular space. Images are obtained before and after the application of laser light. When laser light is applied the signal to noise ratio increases by more than 0.35 +/- 0.15 (range 0.23-0.63) after irradiation according to contrast enhanced MRI. It can be observed that, after biomodulation with light of low energy and low power, wound healing improves and pain is reduced. This effect might be explained by an increased blood flow in this area. Therefore, the use of this kind of laser treatment might improve the outcome of other therapeutic modalities such as tumour ionizing radiation therapy and local chemotherapy. PMID- 10739144 TI - Effect of ultraviolet-B radiation on intact cells of the cyanobacterium Spirulina platensis: characterization of the alterations in the thylakoid membranes. AB - Intact trichomes of Spirulina platensis are exposed to ultraviolet- B (UV-B) radiation (270-320 nm; 1.9 mW m(-2)) for 9 h. This UV-B exposure results in alterations in the pigment-protein complexes and in the fluorescence emission profile of the chlorophyll-protein complexes of the thylakoids as compared with thylakoids isolated from control dark-adapted Spirulina cells. The UV-B exposure causes a significant decrease in photosystem II activity, but no loss in photosystem I activity. Although there is no change in the photosystem I activity in thylakoids from UV-B-exposed cells, the chlorophyll a emission at room temperature and at 77 K indicates alterations associated with photosystem I. Additionally, the results clearly demonstrate that the photosystem II core antennae of chlorophyll proteins CP47 and CP43 are affected by UV-B exposure, as revealed by Western blot analysis. Furthermore, a prominent 94 kDa protein band appears in the sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS PAGE) profile of UV-B-exposed cell thylakoids, which is absent from the control thylakoids. This 94 kDa protein appears not to be newly induced by UV-B exposure, but could possibly have originated from the UV-B-induced cross-linking of the thylakoid proteins. The exposure of isolated Spirulina thylakoids to the same intensity of UV-B radiation for 1-3 h induces losses in the CP47 and CP43 levels, but does not induce the appearance of the 94 kDa protein band in SDS-PAGE. These results clearly demonstrate that prolonged exposure of Spirulina cells to moderate levels of UV-B affects the chlorophyll a-protein complexes and alters the fluorescence emission spectral profile of the pigment-protein complexes of the thylakoid membranes. Thus, it is clear that chlorophyll a antennae of Spirulina platensis are significantly altered by UV-B radiation. PMID- 10739145 TI - H2O2-induced cross-protection against UV-C killing in Escherichia coli is blocked in a lexA (Def) background. AB - Pretreatment with 2.5 mM H2O2 protects E. coli cells against UV-C killing, a phenomenon independent of LexA cleavage. In this paper, we observe that this cross-protection response is neither dependent on the dinY gene product nor on the system that controls dinY, since H2O2 is able to induce cross-protection but not to induce the dinY gene in a lexA-noninducible strain [lexA (Ind-)]. Moreover, this response is not induced in a lexA (Def) background, suggesting that the expression of the SOS regulon may inhibit this cross-protection response. PMID- 10739146 TI - 5-Aminolevulinic acid and its derivatives: physical chemical properties and protoporphyrin IX formation in cultured cells. AB - Protoporphyrin IX (PpIX) is used as a fluorescence marker and photosensitizing agent in photodynamic therapy (PDT). A temporary increase of PpIX in tissues can be obtained by administration of 5-aminolevulinic acid (ALA). Lipophilicity is one of the key parameters defining the bioavailability of a topically applied drug. In the present work, octanol-water partition coefficients of ALA and several of its esters have been determined to obtain a parameter related to their lipophilicity. The influence of parameters such as lipophilicity, concentration, time, and pH value on PpIX formation induced by ALA and its esters is then investigated in human cell lines originating from the lung and bladder. ALA esters are found to be more lipophilic than the free acid. The optimal concentration (c(opt), precursor concentration at which maximal PpIX accumulation is observed) is then measured for each precursor. Long-chained ALA esters are found to decrease the c(opt) value by up to two orders of magnitude as compared to ALA. The reduction of PpIX formation observed at higher concentrations than c(opt) is correlated to reduced cell viability as determined by measuring the mitochondrial activity. Under optimal conditions, the PpIX formation rate induced by the longer-chained esters is higher than that of ALA or the shorter-chained esters. A biphasic pH dependence on PpIX generation is observed for ALA and its derivatives. Maximal PpIX formation is measured under physiological conditions (pH 7.0-7.6), indicating that further enhancement of intracellular PpIX content may be achieved by adjusting the pharmaceutical formulation of ALA or its derivatives to these pH levels. PMID- 10739147 TI - Charlotte's chocolate ice cream soda. PMID- 10739148 TI - The results of a primary and staged pantalar arthrodesis and tibiotalocalcaneal arthrodesis in adult patients. AB - Twenty-three patients (twenty-seven feet) with either a primary or staged pantalar arthrodesis or a tibiotalocalcaneal arthrodesis were evaluated to determine their clinical status. The main indication for the operation was the presence of severe pain unresponsive to non-operative treatment. Fourteen feet (twelve patients) had a pantalar arthrodesis; a fusion of the ankle, subtalar, talonavicular and calcaneocuboid joints. Half the feet in this group had either a triple arthrodesis or an ankle fusion performed at an earlier time. The remaining seven feet had all joints fused during the same operation. Thirteen feet (eleven patients) had a tibiotalocalcaneal arthrodesis. Two of these feet had an ankle arthrodesis performed four and six years previously. The other eleven had the ankle and subtalar joints fused during the same operation. All patients were followed for a mean of fifty-five months (14 to 159 months) from the time of their final arthrodesis procedure. Overall, twenty-three of the twenty-seven feet achieved a solid arthrodesis of all joints operated upon. Four feet had a failure of fusion of only a single joint and all were in the pantalar group. The mean time to radiographic fusion was twenty-three weeks and resulted in a plantigrade foot with an average tibia-floor angle of 87 degrees. Complications occurred in ten feet (37%); of which there were three deep infections; two ankles and one subtalar joint. These arthrodeses procedures resulted in marked relief of the patients' preoperative pain, the main indication for performing the surgery. Postoperatively there was no pain in eleven feet, mild occasional pain in thirteen feet, and moderate pain in only three feet. However, when all parameters of our clinical rating scale were evaluated, only five patients had an excellent clinical result, nine were rated good, three were rated fair and six patients had a poor result. These operations must be considered to be salvage procedures. They are technically difficult to perform and major complications may occur. Pain relief appears to be the main indication for performing these operations, and may account for whatever improvement occurs in the patient's function. PMID- 10739149 TI - Displaced intra-articular calcaneal fracture treated surgically with limited posterior incision. AB - The incidence of calcaneal fracture has been slowly increasing; however, the ideal treatment for displaced intra-articular fracture is not available yet, even though the fracture brings frequent complication and disability. Between April 1991 and March 1998, we treated 103 displaced intra-articular calcaneal fractures of 92 patients surgically with limited posterior incision, modified Gallie approach. There were thirty-seven tongue-type fractures, fifteen tongue-type fractures with moderate comminution, nineteen joint-depression fractures, twenty nine joint-depression fractures with moderate comminution, and three extensively comminuted fractures. The fracture fragments were fixed mainly with partly threaded small cancellous screws or Steinmann pins without any bone graft. Ankle and subtalar motion was permitted immediately if fixation were stable enough. Otherwise, a short period of cast immobilization was utilized. With a mean follow up of 28 months (range, 12 to 66 months), eighty six percent of feet had no pain or only occasional pain not requiring medication. Using American Orthopedic Foot and Ankle Society hindfoot score system for assessment, ninety percent of feet rated as good to excellent. We used "Circle draw test" for evaluation of subtalar motion during follow-up visitation and found eight-seven percent of feet showed good to excellent correlation with the functional recovery. We recommend a limited posterior incision for reduction and internal fixation of displaced intra articular calcaneal fractures. For displaced intra-articular fractures with three or four large fragments without further comminution and without a displaced fracture of the calcaneal cuboid joint, this method is particularly useful. We also recommend a Circle draw test for evaluation of subtalar joint motion as well as an indicator of functional recovery after displaced calcaneal fractures. PMID- 10739150 TI - Interdigital neuroma: intermuscular neuroma transposition compared with resection. AB - This prospective, randomized study compares the treatment of an interdigital neuroma (IDN) by the standard resection operation with a technique in which the IDN is transposed into the inter-muscular space between the adductor hallucis and the interossei muscles after division of the digital nerves distal to the IDN. The resection group contained 22 patients and 22 neuromas and the transposition group contained 22 patients and 23 neuromas. An interviewer, blinded as to the operative technique used, telephoned each patient preoperatively, and at 1 month, 3 months, 6 months, 12 months, and 36-48 months postoperatively. The interviewer recorded the patient's reported pain level on a numerical rating scale of 0 to 100. In the resection group the average pain level was slightly lower through the first 6 month period, but at the 12 month review the resection group had a slightly higher average pain level . At the 36-48 month survey the resection group again reported a greater average pain level and fewer asymptomatic patients. It was concluded that it is unnecessary to excise the IDN to obtain excellent relief of pain. It was also concluded that transposition of the IDN into an intermuscular position between the adductor hallucis and the interossei muscles produced significantly better long term results than did the standard resection operation. PMID- 10739151 TI - Anatomical and radiological considerations of the fifth metatarsal bone. AB - Twenty cadaver fifth metatarsals were harvested from cadaver feet. They were then sectioned coronally in three locations. The cortical thickness (medial, lateral, dorsal, and plantar) and the intra-medullary canal diameter (dorsoplantar and mediolateral) were measured at the three sectional sites. The intra-medullary canal of six specimens was outlined with radiopaque solder wire. The canal was then examined radiographically with the lateral and dorsoplantar views. A lateral bow on the dorsoplantar view was observed in some specimens, which could contribute to surgical complications. On lateral view the intramedullary canal appeared straight in all specimens. The canal projects at least partially into the fifth metatarsal cuboid joint. When considering intra-medullary fixation a surgeon must take into account quality of bone stock and bowing of the canal. A bowed intra-medullary canal lends to vulnerability of the medial cortex at roughly mid-shaft of the fifth metatarsal. The canal has a narrower diameter in the dorsoplantar dimension than the mediolateral dimension. The cortical thickness was found to be less in the dorsal and plantar areas of the fifth metatarsal when compared to medial and lateral cortex. All of these findings lead to causes for complication in intra-medullary fixation of the fifth metatarsal. PMID- 10739152 TI - Subtalar pronation--relationship to the medial longitudinal arch loading in the normal foot. AB - A three-dimensional biomechanical model was used to calculate the mechanical response of the foot to a load of 683 Newtons with the subtalar joint in the neutral position, at five degrees of pronation, and at five degrees of supination. Pronation causes the forefoot to evert, increasing the load borne by the first metatarsal. This results in a 47% increase in the moment about the talonavicular joint and a 58% increase in the moment about the navicular-medial cuneiform joint. Subtalar joint supination causes the forefoot to invert and results in a 55% increase in the moment about the calcaneal-cuboid joint. PMID- 10739153 TI - An insole pressure measurement system: repeatability of postural data. AB - This study analyzed the ability of an in-shoe plantar pressure measurement system to provide repeatable measurements of postural sway data for both healthy and clinical patients. Each participant's in-shoe pressure data were recorded for three trials during each test session during quiet stance. Healthy individuals (n = 9) participated on three consecutive days while clinical participants (n = 5) were tested on one day. Nine response variables were measured to assess their postural stability. Intrasubject measures were evaluated using the Kerlinger reliability procedure. Values provided directly by the Parotec System for a single day of testing yielded the following average coefficients: r = 0.95 (left), r = 0.97 (right) with mean coefficient values from the three day tests of: r = 0.98 (left), r = 0.98 (right). Variables calculated from raw data on a single day produced mean coefficients of: r = 0.77 (left), r = 0.76 (right) and over three days of: r = 0.65 (left), r = 0.66 (right). The ability to record highly reproducible data of postural sway parameters should assist clinicians to treat patients more confidently for balance deficiencies. PMID- 10739154 TI - The plantar loading variations to uphill and downhill gradients during treadmill walking. AB - The purpose of the study was to examine the plantar loading changes during 5 gradient conditions on a treadmill (-15%, -8.5%, Level, 8.5%, 15%) for 20 participants using the Pedar in-shoe pressure measurement system. The measurement system uses EMED insoles, each consisting of 99 capacitive sensors, sampled at 50 Hz. Data was collected from the last 20 seconds at each gradient condition while participants walked. As the treadmill gradient increased, loading (peak pressure [PP] and peak force [PF]) increased in the hallux and 1st metatarsal regions and decreased in the heel region. With negative gradients, loading (PP and PF) increased in the heel region and decreased in the 4th and 5th metatarsal regions. PMID- 10739155 TI - Movement coupling at the ankle during the stance phase of running. AB - The purpose of this study was to quantify movement coupling at the ankle during the stance phase of running using bone-mounted markers. Intracortical bone pins with reflective marker triads were inserted under standard local anaesthesia into the calcaneus and the tibia of five healthy male subjects. The three-dimensional rotations were determined using a joint coordinate system approach. Movement coupling was observed in all test subjects and occurred in phases with considerable individual differences. Between the shoe and the calcaneus coupling increased after midstance which suggested that the test shoes provided more coupling for inversion than for eversion. Movement coupling between calcaneus and tibia was higher in the first phase (from heel strike to midstance) compared with the second phase (from midstance to take-off). This finding is in contrast to previous in-vitro studies but may be explained by the higher vertical loads of the present in-vivo study. Thus, movement coupling measured at the bone level changed throughout the stance phase of running and was found to be far more complex than a simple mitered joint or universal joint model. PMID- 10739156 TI - The reliability and validity of a first ray measurement device. AB - The need for measuring the mobility of the first ray has been identified. The purpose of this study was to test the reliability and validity of a device built to measure the relative vertical displacement of the first ray. Twenty fresh frozen cadaver feet were sampled (mean age of donor was 70+/-13 years). Dorsal mobility of the first ray was measured by device over three trials of repeated loading using 20 N, 35 N, 55 N and 85 N of force. Radiographs served as the criterion standard for validation of the device. Vertical displacement was measured from x-ray by a digitizing procedure that recorded movement of the first ray during loading. Reliability of the device assessed by intraclass correlation coefficients (ICC) was .98 with standard errors of the measurement calculated to be 0.35 mm. A correlation of agreement value of .97 was determined for the two methods of measurement. Analysis of variance testing found a significant interaction between force and method of measurement. Tukey post-hoc analysis found no difference (F<1.70) between the two methods of measuring first ray displacement in cadaver specimens when the force applied did not exceed 55 N. At 85 N of force (F = 10.05), unwanted movement of the second metatarsal caused the device to overestimate the amount of displacement that occurred specific to the first ray. This measure of first ray mobility should help clinicians and researchers to better investigate foot pathology resulting from faulty mechanics of the first ray. PMID- 10739157 TI - Avascular necrosis of the talus following subtalar arthrorisis with a polyethylene endoprosthesis: a case report. PMID- 10739158 TI - Specialty day of the European Foot and Ankle Society (EFAS), June 6, 1999 at the 4th Congress of the European Federation of National Associations of Orthopaedics and Traumatology (EFORT). PMID- 10739159 TI - Analysis of the retinal edema of full-thickness macular holes by scanning laser ophthalmoscopy and optical coherence tomography. AB - The purpose of this study was to investigate the relationship between the dark area illuminated by scanning laser ophthalmoscopy (SLO) and cystic spaces around macular holes as shown by optical coherence tomography (OCT). SLO allows for two dimensional retinal examination, using short wave length (514 nm, argon) which is useful for the vitreoretinal surface and inner retina; red helium-neon laser (633 nm), which is capable of imaging deeper tissues; and infrared diode laser (780 nm), for choroidal examination. OCT is analogous to ultrasound except that optical rather than acoustic reflectivity is measured. OCT can produce the cross sectional view of retina. Using SLO (helium-neon laser) and OCT, we examined 8 eyes with full-thickness macular holes. Eight normal eyes served as controls. Cystic spaces were in proportion to dark areas with statistical significance as shown by correlation analysis. Evaluation of the full-thickness macular holes by using both SLO and OCT is highly useful. PMID- 10739160 TI - Strong immunoreactivity of platelet-derived growth factor and its receptor at human and mouse neuromuscular junctions. AB - Platelet-derived growth factor (PDGF) and its alpha-receptor were localized at human and mouse neuromuscular junctions (NMJs) using specific polyclonal antibodies against each, anti-PDGF-A and anti-PDGF alpha-receptor, respectively. By applying double fluorescence labeling, immunoreactivity for PDGF and its receptor was closely co-localized with acetylcholine receptors, which were identified with alpha-bungarotoxin. PDGF might be involved in the interaction between the presynaptic and postsynaptic components. This is the first demonstration of PDGF and its receptor concentrated at human and mouse NMJs. PMID- 10739161 TI - Risk factors and triggers of sudden death in the working generation: an autopsy proven case-control study. AB - In Japan, studies on the risk factors of sudden death in the working generation have been rarely carried out, especially among extremely rare cases of causative disease. Thus, the present study aimed to identify the risk factors and triggers of sudden death in cases whose causes of death were definitely proven by autopsy. We investigated the legal medical records for four years from May 1994 to February 1998. Out of 271 cases, 176 patients 20 to 59 years were enrolled as cases of sudden death in the working generation. Among these, 91 cases, 52%, could be analyzed by telephone interviews from close family members. Only one examiner undertook all phone questions to the case subjects. As control subjects, 1167 persons who consulted us for a health check were employed. Of the sudden death cases, the final diagnosis in 29 cases was coronary artery disease (31.9%), 18, acute cardiac dysfunction (19.8%), 6, other cardiac diseases (6.6%), 4, acute aortic dissection (4.4%), 4, cerebrovascular disease (4.4%) and 30, other diseases (32.9%). Through conditional logistic analysis, the following risk factors emerged as candidates: Long-term stress, history of heart disease, hypertension, chest symptoms, autonomic disturbance, short-term stress and a smoking habit. Short-term stress, autonomic disturbance and a smoking habit increased the risk of sudden death due to coronary artery disease. Long-term stress was associated with an increased risk of sudden death due to acute cardiac dysfunction. It was also demonstrated that autonomic disturbance and stress were closely related to the occurrence of sudden death. Therefore, to prevent sudden death, it would be helpful to identify subjective symptoms to relieve such stress in some way. PMID- 10739162 TI - Elevated plasma level of plasminogen activator inhibitor-1 (PAI-1) in patients with relapsing-remitting multiple sclerosis. AB - Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system and one of the earliest changes in inflammatory focus involves the activation of vascular endothelial cells. We determined the plasma level of plasminogen activator inhibitor-1 (PAI-1), a key regulator of fibrinolysis and cell migration, in patients with MS. The level of plasma PAI-1 was significantly higher in active MS cases when compared to stable MS and controls. Plasma concentrations of tissue plasminogen activator, transforming growth factor beta 1, and lipoprotein-a remained normal in spite of disease activity. These results suggested that PAI-1 plasma levels are associated with MS disease activity and is a good marker for MS relapse. PMID- 10739163 TI - In vitro toxicity of gallium arsenide in alveolar macrophages evaluated by magnetometry, cytochemistry and morphology. AB - Gallium arsenide (GaAs), a chemical compound of gallium and arsenic, causes various toxic effects including pulmonary diseases in animals. Since the toxicity is not completely investigated, GaAs has been used in workplaces as the material of various semiconductor products. The present study was conducted to clarify the toxicity of GaAs particles in the alveolar macrophages of hamsters using magnetometry, enzyme release assays and morphological examinations. Alveolar macrophages obtained from hamsters by tracheobronchial lavage and adhered to the disks in the bottom of wells were exposed to ferrosoferric oxide and GaAs particles. Ferrosoferric oxide particles were magnetized externally and the remanent magnetic field was measured. Relaxation, a fast decline of the remanent magnetic fields radiated from the alveolar macrophages, was delayed and decay constants were decreased dose-dependently due to exposure to GaAs. Because the relaxation is thought to be associated with cytoskeleton, the exposure of GaAs may have impaired the motor function of them. Enzyme release assay and morphological findings indicated the damage to the macrophages. Thus the cytotoxicity causes cytostructural changes and cell death. According to DNA electrophoresis and the TUNEL method, necrotic changes occur more frequently than apoptotic changes. PMID- 10739164 TI - Right ventricular diastolic filling assessed by conventional doppler and tissue Doppler imaging in normal children. AB - To examine age-related changes in right ventricular filling dynamics, we performed conventional pulsed Doppler (n=99) and tissue Doppler (n=30) echocardiographic studies in normal subjects aged 7 days to 273 months. The tricuspid flow velocity during early diastole (peak E) wave correlated significantly but weakly with the logarithm of age. The peak E wave in the early neonatal period was almost 80% of the older children's values and increased to 100% by 36 months of age. In the right ventricular tissue Doppler imaging, the peak myocardial velocity during early diastole also increased significantly with the logarithm of age. However, the tissue Doppler peak A did not change with age. There was a significant correlation between the tissue Doppler peak E wave and the tricuspid peak E wave and between the tissue Doppler peak E/A wave and the tricuspid peak E/A wave. The age-related changes in the tricuspid inflow velocity patterns were similar to the age-related alterations in the right ventricular myocardial velocity patterns. Age-related changes in the tricuspid inflow velocity and myocardial velocity patterns may be related to age-related maturation in the right ventricular diastolic performance. PMID- 10739165 TI - Functional analysis after auto iris pigment epithelial cell transplantation in patients with age-related macular degeneration. AB - Recent transplantation studies indicate that subretinal space is not always an immunologically privileged site and non-autologous cells may be rejected in patients with exudative age-related macular degeneration (AMD). We performed autologous iris pigment epithelial (IPE) cell transplantation by cell suspension after autologous IPE cell culture in 8 patients with AMD. These patients were followed without immunosuppression between 1.5 and 8 months and the retinal function was analyzed. No cystoid macular edema or fluorescein leakage was observed. Six of the 8 patients improved visual acuity of more than two lines and the other two patients retained preoperative visual acuity. Five patients had increased visual field sensitivity, one patient retained pretransplantation sensitivity, and one patient showed a gradual decrease in sensitivity (one patient was not examined). Although 2 of the 8 patients showed decreased amplitude of flicker electroretinography (ERG) (about 60 to 70% as that of preoperative level), the average improvement of each amplitude of a single white flash (a wave), photopic, or flicker ERG was 123, 102, and 107%, respectively. No proliferative change in the submacular lesion or vitreous cavity was observed after transplantation. From this functional analysis, transplanted autologous IPE may have, in part, an alternative function in regard to the retinal pigment epithelium in the subretinal space. PMID- 10739166 TI - Human pulmonary dirofilariasis: report of six cases. AB - We report six cases of pulmonary dirofilariasis diagnosed at our laboratory with clinical and pathological features. The nodules of dirofilariasis were round in three cases as previously reported, however dumbbell-shaped in two cases. The nodule did not attach to the pleura in four cases. Microscopically, the nodules were granulomas composed of central coagulation necrosis and peripheral fibrosis with round cell infiltration, histiocytes, and multinucleated giant cells. Necrotic pulmonary artery with single or multiple sections of degenerated nematode was observed in the center of the nodule. Dilated bronchioles with inflammation were observed in the nodule in four cases. Collapse of the alveoli, organizing pneumonia, hemosiderin-laden macrophages were observed around the nodule. We suppose that the nodule is not an infarction but a granuloma caused by antigen released from the nematode. Because the pulmonary dirofilariasis is difficult to be differentiated from primary or metastatic lung carcinoma, and the inflammation exists around the nodule, the nodule should be removed surgically. PMID- 10739167 TI - CYP2D6 ultrarapid metabolizer genotype as a potential modifier of smoking behaviour. AB - Some 3-10% of Caucasians are deficient in CYP2D6 metabolism (poor metabolizers), due to inheritance of two defective alleles, whereas amplification of the CYP2D6 gene results in ultrarapid metabolism in 1-2% of Caucasian populations. To examine the possible association between CYP2D6 polymorphism and individual smoking behaviour, we analysed the prevalence of CYP2D6 genotypes among 292 long term heavy smokers, 382 individuals with more variable smoking histories, and 302 never-smokers. The prevalence of ultrarapid metabolizers in heavy smokers (7.9%) was twofold compared to individuals with variable smoking habits (3.7%; odds ratio 2.3, 95% confidence interval 1.2-4.4), and fourfold compared with never smokers (2.0%) (odds ratio 4.2, 95% confidence interval 1.8-9.8). The frequency of poor metabolizer genotype was approximately 2%, in each smoker group. However, when men and women were studied separately, the prevalence of poor metabolizer genotype was higher in male never-smokers (3.6%) than in variable smokers (2.7%) and heavy smokers (2.2%). Moreover, a trend test, adjusted by age, gender and cancer status, revealed a significant trend for the increased tobacco usage with increased metabolic capacity. Our results are in agreement with the assumption that increased CYP2D6 activity may contribute to the probability of being addicted to smoking. PMID- 10739168 TI - Variation in induced CYP1A1 levels: relationship to CYP1A1, Ah receptor and GSTM1 polymorphisms. AB - The genotypic basis of interindividual variation in levels of induced CYP1A1 activity has been investigated by screening both the CYP1A1 gene and the Ah receptor gene (AhR) for both previously described and novel polymorphisms. A 103 fold level of interindividual variation in induced CYPlA1 activity [ethoxyresorufin O-deethylase (EROD)] was observed in lymphocytes from a group of 30 Caucasian volunteers. High levels of induced EROD activity did not correlate with the presence of CYP1A1*2 or CYP1A1*4 alleles or with the GSTM1 null genotype. Novel CYP1A1 alleles with the base substitutions C4151T, G-469A and C 459T respectively, were detected by screening the coding exons and approximately 1 kb of upstream sequence in 20 individuals by single-strand conformational polymorphism (SSCP) analysis but none of the three novel alleles appeared to be associated with high induced CYP1A1 activity in the study group. Screening of the 11 exons of the AhR gene by SSCP analysis confirmed the existence of the previously described G1721A polymorphism in a Caucasian population and a novel allele (G1768A which results in the amino acid substitution V5701) was also detected. The novel allele was very rare in Caucasians though more common in African-Americans. Individuals with at least one copy of the G1721A AhR variant allele showed a significantly higher level of induced CYP1A1 activity compared with individuals negative for the polymorphism (P = 0.0001). A similar finding was obtained for induced CYP1A1 protein levels determined by immunoblotting. Levels of induced CYP1A1 activity were also found to show a sex difference with women showing a significantly lower induced activity compared with men. We conclude that genotypes for the G1721A AhR polymorphism and gender appear to be determinants of levels of induced CYP1A1 activity and that interindividual variation in levels of induced CYP1A1 activity appears to be associated more with regulatory factors than polymorphism in the CYP1A1 gene. PMID- 10739169 TI - Association of CYP1B1 genetic polymorphism with incidence to breast and lung cancer. AB - Cytochrome P450 1B1 (CYP1B1) participates in the metabolic activation of a number of procarcinogens including benzo[a]pyrene and the hydroxylation of 17beta estradiol at the C-4 position. In this study, we investigated the association between CYP1B1 genetic polymorphism and breast or lung cancer incidence. The Ala Ser polymorphism at codon 119 in presumed substrate recognition site 1 was significantly associated with the incidence of breast or squamous cell carcinoma of the lung. On the other hand, Leu-Val polymorphism at codon 432 did not show any association to the cancers. An allele containing both Ala and Leu simultaneously, comprised 75% of alleles among 315 Japanese healthy controls, was significantly inversely associated with breast cancer incidence. When expressed in a recombinant system, this CYP1B1 cDNA showed the lowest 17beta-estradiol 4 hydroxylase activity among four different variant forms of CYP1B1. Thus, inter individual differences in activation of procarcinogens or metabolism of oestrogen originating from genetic polymorphisms of the human CYP1B1 gene may contribute to the susceptibility of human cancers. PMID- 10739170 TI - Polymorphisms of NAT2 in relation to sulphasalazine-induced agranulocytosis. AB - Agranulocytosis is a rare, but serious adverse reaction to sulphasalazine. The polymorphic enzyme N-acetyltransferase 2 (NAT2) plays an important role in the metabolism of sulphasalazine. This study was conducted to analyse whether the risk of sulphasalazine-induced agranulocytosis is increased in slow acetylators. Patients were treated for inflammatory disease, mostly joint disease, with a mean dose of 2 g sulphasalazine daily. Thirty-nine patients reacted with agranulocytosis, while 75 patients had been treated for a minimum of 3 months without haematological side-effects. A population-based control panel of 448 individuals was used for comparison. All subjects were genotyped for NAT2 by polymerase chain reaction followed by restriction enzyme digestion. The six most common allelic variants were analysed: NAT2*4, NAT2*5A, NAT2*5B, NAT2*5C, NAT2*6 and NAT2*7. The proportion of slow acetylators was significantly higher in patients with sulphasalazine-induced agranulocytosis (69%) and population-based controls (64%) compared to patients who tolerated sulphasalazine (45%); odds ratio 2.71 [95% confidence interval (CI) 1.20; 6.15], P = 0.015, and odds ratio 2.17 (95% CI 1.32; 3.56), P = 0.002, respectively. Patients who developed agranulocytosis did not differ from population-based control subjects in the frequency of slow acetylators; odds ratio 1.25 (95% CI 0.62; 2.53), P = 0.535. The risk of agranulocytosis did not appear to be increased in slow acetylators, provided that the difference compared with sulphasalazine-treated control subjects was not due to a predominance of fast acetylators among patients with inflammatory joint disease. Instead, selection bias was suspected since more slow acetylators may have discontinued sulphasalazine therapy because of drug intolerance. PMID- 10739171 TI - Association study of dopamine receptor gene polymorphisms with drug-induced hallucinations in patients with idiopathic Parkinson's disease. AB - Some patients with idiopathic Parkinson's disease experience hallucinations as a result of treatment with levodopa and dopamine agonists. There is evidence for some heterogeneity in these hallucinating patients based on duration of Parkinson's disease at onset of hallucinations. We compared the frequency of polymorphisms in the dopamine D2 and D3 receptor genes between patients with drug induced hallucinations and non-hallucinating patients. Two polymorphisms close to DRD2 and one in DRD3 were studied. No association was found with the whole group of hallucinating patients and their controls. However, an association was found with late-onset hallucinations and the C allele of the TaqIA polymorphism, 10.5 kb 3' to DRD2. This polymorphism may be in linkage disequilibrium with a mutation in DRD2 or a nearby gene that predisposes to drug-induced hallucinations which occur later in the course of idiopathic Parkinson's disease. PMID- 10739172 TI - Discovery of a functional polymorphism in human glutathione transferase zeta by expressed sequence tag database analysis. AB - Analysis of the expressed sequence tag (EST) database by sequence alignment allows a rapid screen for polymorphisms in proteins of physiological interest. The human zeta class glutathione transferase GSTZ1 has recently been characterized and analysis of expressed sequence tag clones suggested that this gene may be polymorphic. This report identifies three GSTZ1 alleles resulting from A to G transitions at nucleotides 94 and 124 of the coding region, GSTZ1*A A94A124; GSTZ1*B-A94G124; GSTZ1*C-G94G124. Polymerase chain reaction/restriction fragment length polymorphism analysis of a control Caucasian population (n = 141) showed that all three alleles were present, with frequencies of 0.09, 0.28 and 0.63 for Z1*A, Z1*B and Z1*C, respectively. These nucleotide substitutions are non-synonymous, with A to G at positions 94 and 124 encoding Lys32 to Glu and Arg42 to Gly substitutions, respectively. The variant proteins were expressed in Escherichia coli as 6X His-tagged proteins and purified by Ni-agarose column chromatography. Examination of the activities of recombinant proteins revealed that GSTZ1a-1a displayed differences in activity towards several substrates compared with GSTZ1b-1b and GSTZ1c-1c, including 3.6-fold higher activity towards dichloroacetate. This report demonstrates the discovery of a functional polymorphism by analysis of the EST database. PMID- 10739173 TI - Characterization of human polymorphic DNA repair methyltransferase. AB - The O6-methylguanine-DNA methyltransferase (MGMT) is a critical defence against alkylation-induced mutagenesis and carcinogenesis. More than a 20-fold interindividual difference in the MGMT activity is known to exist among human cultured fibroblasts. We previously reported three allelic variants of the human MGMT gene, namely V1, V2, and V3. Both V1 and V2 carry amino acid substitutions, Leu84Phe and Trp65Cys, respectively, while V3 has a silent mutation. In order to reveal the pharmacogenetic and ecogenetic significance of polymorphism in the human MGMT gene, we investigated the in-vivo characteristics of V1 and V2 methyltransferase enzyme. Escherichia coli strain KT233 (ogt-, ada-) and mer- HeLa MR cells carrying a V1 sequence exhibited almost the same level of sensitivity against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as did those with a wild-type sequence. The level of methyltransferase protein in those cells was essentially the same as for the wild-type and V1 samples. On the other hand, E. coli and human cells expressing V2 cDNA showed a significantly reduced level of survival. In these cells, V2 protein was hardly detected, even though mRNA was produced normally. An in-vitro translation experiment revealed that the V2 sequence had the potential to produce methyltransferase protein, as did the wild type and V1 sequences. There was also evidence for a small amount of V2 protein being produced but rapidly degraded, thus implying that the V2 molecule is unstable in vivo. Using purified recombinant proteins, we estimated the kinetic values of wild-type and variant form of enzymes, which would support these views. From these results, we concluded that the wild-type and V1 protein have similar enzymatic and physicochemical properties, while V2 protein is considered to be unstable and rare. PMID- 10739174 TI - Phenotypes of flavin-containing monooxygenase activity determined by ranitidine N oxidation are positively correlated with genotypes of linked FM03 gene mutations in a Korean population. AB - A non-invasive urine analysis method to determine the in-vivo flavin-containing mono-oxygenase (FMO) activity catalysing N-oxidation of ranitidine (RA) was developed and used to phenotype a Korean population. FMO activity was assessed by the molar concentration ratio of RA and RANO in the bulked 8 h urine. This method was used to determine the FMO phenotypes of 210 Korean volunteers (173 men and 37 women, 110 nonsmokers and 100 smokers). Urinary RA/RANO ratio, representing the metabolic ratio and the reciprocal index of FMO activity, ranged from 5.67-27.20 (4.8-fold difference) and was not different between men and women (P = 0.76) or between smokers and nonsmokers (P = 0.50). The frequencies of RA/RANO ratios were distributed in a trimodal fashion. Among the 210 Korean subjects, 93 (44.3%) were fast metabolizers, 104 (49.5%) were intermediate metabolizers and 13 (6.2%) were slow metabolizers. Subsequently, the relationship between the ranitidine N oxidation phenotypes and FMO3 genotypes, determined by the presence of two previously identified mutant alleles (Glu158Lys: FMO3/Lys158 and Glu308Gly: FMO3/Gly308 alleles) commonly found in our Korean population was examined. The results showed that subjects who were homozygous and heterozygous for either one or both of the FMO3/Lys158 and FMO3/Gly308 mutant alleles had significantly lower in-vivo FMO activities than those with homozygous wild-type alleles (FMO3/Glu158 and FMO3/Glu308) (P < 0.001, Mann-Whitney U-test). Furthermore, the FMO activities of subjects with either FMO3/Lys158 or FMO3/Gly308 mutant alleles were almost identical to those having both FMO3 mutant alleles (FMO3/Lys158 and FMO3/Gly308). These two mutant alleles located, respectively, at exons 4 and 7 in the FMO3 gene appeared to be strongly linked by cis-configuration in Koreans. Therefore, we concluded that presence of FMO3/Lys158 and FMO3/Gly308 mutant alleles in FMO3 gene is responsible for the low ranitidine N-oxidation (FMO3 activity) in our Korean population. PMID- 10739175 TI - Arylamine N-acetyltransferase 1 (NAT1) genotypes in a Lebanese population. AB - The frequency distributions of human N-acetyltransferase 1 (NAT1*) alleles in various ethnic groups are largely unknown. This lack of information is in contrast to the many studies of ethnic differences in NAT2* alleles and phenotypes. Increasing interest in NAT1 due to its potential roles in carcinogen metabolism and cancer risk makes it desirable to know the distribution of NAT1* alleles in various populations. Using a polymerase chain reaction-restriction fragment length polymorphism genotyping assay, the frequency of NAT1* alleles in a Lebanese population was determined. Of 84 NAT1* alleles assayed, 56% were NAT1*4. Alleles NAT1*3, *10, and *14 were found at frequencies of 0.036, 0.107, and 0.238, respectively. Five additional alleles (6%) differed from previously reported alleles. Nearly 50% of the population were heterozygous for a NAT1*14 allele. The unusually high frequency of NAT1*14 alleles in Lebanese may be useful for epidemiological studies of the effects of the NAT1 polymorphism in this population. PMID- 10739176 TI - Polymorphism of the cytochrome P450 (CYP) 2C9 gene in Japanese epileptic patients: genetic analysis of the CYP2C9 locus. PMID- 10739177 TI - Human cytochrome P450 (CYP) genes: recommendations for the nomenclature of alleles. PMID- 10739179 TI - Atmospheric pressure MALDI PMID- 10739178 TI - Challenges for the analytical chemistry curriculum PMID- 10739181 TI - QC lasers changing spectroscopy PMID- 10739180 TI - Instant film: microdevices in 2 h PMID- 10739182 TI - News from the 4th symposium on the analysis of well-characterized biotechnology pharmaceuticals. PMID- 10739183 TI - Pittcon faces new challenges PMID- 10739184 TI - Voltammetry--spanning the kinetic timescale PMID- 10739185 TI - SFC of drug enantiomers. PMID- 10739186 TI - Flow injection analysis: from beaker to microfluidics. PMID- 10739187 TI - Fluorometers dissected. Fluorometry offers lower detection limits for various applications PMID- 10739188 TI - MS/MS: multimedia shopping for a mass spec. How to use the internet to research a mass spectrometer purchase PMID- 10739189 TI - Molecular orientation and angular distribution probed by angle-resolved absorbance and second harmonic generation AB - Second harmonic generation (SHG) and angle-resolved absorbance with photoacoustic detection were combined to evaluate both the mean orientation angle and the width of the angular distribution for two surface-bound molecular systems. Assuming a Gaussian distribution function, a physisorbed stilbene dye on fused silica exhibited a narrow distribution centered at 73 degrees with a root-mean-square (rms) width of less than approximately 8 degrees. In contrast, a covalently bound azo dye resulted in a broad orientation distribution (rms width of approximately 30 degrees) centered at 60 degrees. It was also demonstrated that the combination of nonlinear (SHG) and linear (absorbance) spectroscopic techniques provides valuable insight into molecular orientation that a combination of two linear techniques (such as fluorescence and absorbance) is unable to provide. PMID- 10739190 TI - Ion trap collisional activation of the (M + 2H)2+ - (M + 17H)17+ ions of human hemoglobin beta-chain. AB - The parent ions of human hemoglobin beta-chain ranging in charge from 2+ to 17+ have been subjected to ion trap collisional activation. The highest charge-state ions (17+ to 13+) yielded series of products arising from dissociation of adjacent residues. The intermediate charge-state ions (12+ to 5+) tended to fragment preferentially at the N-terminal sides of proline residues and the C terminal sides of acidic residues. Many, but not all, of the possible cleavages at proline, aspartic acid, and glutamic acid residues were represented in the spectra. The lowest charge-state ions were difficult to dissociate with high efficiency and yielded spectra with poorly defined product ion signals. This observation is attributed to sequential fragmentations arising from losses of small molecules such as water and/or ammonia. The poor fragmentation efficiency observed for the low charge states is due at least in part to the low trapping wells used to store the ions. Higher ion stabilities due to lower Coulombic repulsion and charges being sequestered at highly basic sites may also play an important role. Ion/ion proton-transfer reactions involving protein parent ions allows for the formation of a wide range of parent ion charge states. In addition, the ion/ion proton-transfer reactions involving protein dissociation products simplify interpretation of the product ion spectra. PMID- 10739191 TI - Adsorption-based electrochemical detection of nonelectrochemically active analytes for capillary electrophoresis AB - A sensitive electrochemical detection method (ECD) for capillary electrophoresis has been developed that is applicable to a much wider range of analytes than more conventional ECD methods. Using a modified Osteryoung square-wave voltammetry method, the adsorption of what are normally considered nonelectrochemically active analytes onto a platinum electrode was found to produce a concentration proportional response. Although the mechanisms that cause this response may be complex, it appears that it is due to changes in the electrode/solution interface that accompany adsorption of the analyte onto the electrode rather than a simple redox process. Analytes that possess pi-electron density appeared to chemisorb rather than only physically adsorb onto the electrode and gave the best response with detection limits of < 10(-8) M while maintaining good linearity. Because this detection method requires only that the analyte adsorb onto the electrode, it has the advantage of much wider applicability than previously reported electrochemical detection methods. The applicability of this detection method was investigated for a variety of analytes and background electrolyte conditions (varied pH, ionic strength, buffer additives). Comparisons of the sensitivity of this method to UV detection showed that, even for analytes that have good UV chromophores, sensitivities greater than 1 order of magnitude were obtained using adsorption-based electrochemical detection. PMID- 10739193 TI - Multilayered assembly of dendrimers with enzymes on gold: thickness-controlled biosensing interface AB - A new approach to construct a multilayered enzyme film on the Au surface for use as a biosensing interface is described. The film was prepared by alternate layer by-layer depositions of G4 poly(amidoamine) dendrimers and periodate-oxidized glucose oxidase (GOx). The cyclic voltammograms obtained from the Au electrodes modified with the GOx/dendrimer multilayers revealed that bioelectrocatalytic response is directly correlated to the number of deposited bilayers, that is, to the amount of active enzyme immobilized on the Au electrode surface. From the analysis of voltammetric signals, the coverage of active enzyme per GOx/dendrimer bilayer during the multilayer-forming steps was estimated, which demonstrates that the multilayer is constructed in a spatially ordered manner. Also, with the ellipsometric measurements, a linear increment of the film thickness was registered, supporting the formation of the proposed multilayered structure. The E5D5 electrode showed the sensitivity of 14.7 microA x mM(-1) glucose x cm(-2) and remained stable over 20 days under day-by-day calibrations. The proposed method is simple and would be applicable to the constructions of thickness- and sensitivity-controllable biosensing interfaces composed of multienzymes as well as a single enzyme. PMID- 10739192 TI - Development of a creatinine ELISA and an amperometric antibody-based creatine sensor with a detection limit in the nanomolar range. AB - Creatinine-specific antibodies have been generated and used for highly sensitive and specific immunochemical creatinine determinations. Creatinine was derivatized at N3 and coupled to KLH carrier protein. On the basis of this immunogen, monoclonal antibodies were developed by hybridoma technology. Antibodies from various clones have been characterized with BIAcore 2000 with respect to the dissociation constant and specificity. Antibodies of clone B90-AH5 exhibited the lowest dissociation constant (0.74 microM) and the highest specificity for creatinine and were chosen for the development of a competitive ELISA and an amperometric creatinine sensor. The creatinine sensor was constructed by fixing a creatinine-modified membrane on the top of a platinum working electrode which was then incorporated into a stirred electrochemical measuring cell. For creatinine determination the creatinine-containing sample was incubated with B90-AH5 and anti-IgG(mouse)-glucose oxidase conjugate and applied to the measuring cell. After a washing step glucose was added and the produced hydrogen peroxide was registered at Eappl = +600 mV vs Ag/AgCl. The measuring range was 0.01-10 microg/mL. The highest sensitivity for creatinine was achieved at 330 ng/mL (3 microM) and the lower detection limit at 4.5 ng/mL (40 nM). This is far below the relevant clinical range, which is 5-17 microg/mL (44-150 microM) and allows a reliable determination of very low creatinine concentrations in serum, where standard methods cannot be applied. After each measurement the sensor was regenerated with 10 mM HCl without any loss in binding activity. PMID- 10739194 TI - Sensing of acetylcholine by a tricomponent-enzyme layered electrode using faradaic impedance spectroscopy, cyclic voltammetry, and microgravimetric quartz crystal microbalance transduction methods. AB - A three-enzyme layered assembly on Au electrodes or Au-quartz crystals, consisting of horseradish peroxidase, HRP, choline oxidase, ChO, and acetylcholine esterase, AChE, is used to sense acetylcholine by the HRP-mediated oxidation of 3,3',5,5'-tetramethylbenzidine, TMB (1), by H2O2, and the formation of the insoluble product (2) on the respective transducers. The analyte substrate, acetylcholine, is hydrolyzed by AChE to choline that is oxidized by ChO and O2 to yield the respective betaine and H2O2. The amounts of generated H2O2 and the resulting insoluble product on the transducers correlate with the concentration of acetylcholine in the samples. The formation of the insoluble product (2) on electrode supports is followed by faradaic impedance spectroscopy that probes the increased interfacial electron-transfer resistance upon the formation of 2, and by cyclic voltammetry that reflects electron-transfer barriers upon the formation of the precipitate. The frequency of the Au-quartz crystal decreases as a result of the accumulation of the insoluble precipitate. The amount of insoluble product formed on the transducers is controlled by the concentration of acetylcholine and by the time interval of biocatalyzed precipitation. The generation of the insoluble product provides a means to amplify the sensing processes. Acetylcholine concentrations corresponding to 1 x 10(-5) M are easily sensed by the different transducers. PMID- 10739195 TI - Voltammetric and reference microelectrodes with integrated microchannels for flow through microvoltammetry. 1. The microcell AB - This paper describes the construction of 2 microsensor units for on-line voltammetric detection inside a cylindrical microcell (a working microsensor unit and a reference and auxiliary microsensor unit), for application to heavy-metal analysis in complex media such as natural waters. Both microsensor units include a channel for the solution renewal in the microcell after analysis. The working microsensor, a Hg-plated Ir microelectrode, is protected against fouling with an agarose gel including a hydrophobic chromatographic phase (C18). The fabrication steps and the quality tests related to long-term use and reliability, as well as to precision, are described. The application of the protective gel layer against fouling by hydrophobic or surface active small molecules is of general application, reliable, and very efficient. The reference and auxiliary unit is composed by an iridium oxide based mini reference electrode, an auxiliary Pt electrode, and a circulation channel. It is built to enable its use inside a 700 microm-diameter tubing connected to a hollow fiber supported liquid membrane lumen (volume, 5-10 microL) for heavy-metal analysis. However, it can be used in any other microanalytical system. The reference electrode is sufficiently stable for voltammetric applications (1-2 mV drift/day), and its lifetime is more than one year. The Ti/IrO2 core is immersed in a pH-buffered agarose gel, to guarantee potential stability even when the electrode is immersed in variable pH solutions. PMID- 10739196 TI - Voltammetric and reference microelectrodes with integrated microchannels for flow through microvoltammetry. 2. Coupling the microcell to a supported liquid membrane preconcentration technique AB - The paper describes the assembly and functioning of the microvoltammetric cell described in Part 1, with a hollow Fiber Supported Liquid Membrane (HFSLM), for trace metal analysis. Membrane stability, working-electrode behavior, mercury film lifetime inside the HFSLM, hydrodynamic conditions, as well as transport kinetics of the metal through the SLM have been studied in detail. System calibrations have been performed in the range 5-120 nM Pb(II). The reproducibility and sensitivity of the whole microsystem is discussed as well as limitations and possible improvements. PMID- 10739197 TI - Subnanoliter volume wall-jet cells combined with interdigitated microarray electrode and enzyme modified planar microelectrode AB - Miniaturized wall-jet type flow cells with an active volume of 0.042-15 nL were fabricated for use as highly sensitive electrochemical detectors for capillary electrophoresis/electrochemical detection and small on-line enzyme sensors. The cells consisted of three glass plates and a fused-silica capillary. Two of the plates had microfabricated flow channels and guide trenches for the capillary and working, reference, and counter electrodes. The other plate had a film electrode. When an interdigitated microarray electrode (total area, 66 microm x 64 microm; bandwidth and gap, 2 microm) was installed in the flow cell, the redox cycling enhanced the current at flow rates of less than 100 nL/min even though there were only eight pairs of microbands. A sharp dopamine peak enhanced by the redox cycling was observed when the cell was used for capillary electrophoresis. A square film electrode modified with glutamate oxidase and Os-poly(vinylpyridine) containing HRP was also installed in the flow cell and used to measure neurotransmitter release from cultured nerve cells. When the flow rate was relatively high, the response time of the modified electrode was comparable to that of a cylindrical carbon fiber electrode (33 microm o.d.) modified with the same enzyme and mediator. We observed a transient cathodic current response assigned to the glutamate release with the electrode in the flow cell in a suction mode measurement when we stimulated cultured nerve cells electrically with a dual microelectrode. PMID- 10739198 TI - Salicylate-selective electrodes based on AI(III) and Sn(IV) salophens AB - New salicylate-selective electrodes based on aluminum-(III) and tin(IV) salophens are described. The electrodes were prepared by incorporating the ionophores into plasticized poly(vinyl chloride) (PVC) membranes, which were directly coated on the surface of graphite electrodes. These novel electrodes display high selectivity for salicylate with respect to many common inorganic and organic anions. The influence of membrane composition and pH and the effect of lipophilic cationic and anionic additives on the response properties of the electrodes were investigated. The electrode based on aluminum salophen, with 32% PVC, 65.8% plasticizer, and 2.2% ionophore, shows the best potentiometric response characteristics and displays a linear log [Sal-] vs EMF response over the concentration range 1 x 10(-6) - 1 x 10(-1) M in phosphate buffer solutions of pH 7.0, with a Nernstian slope of -59.2 mV/decade of salicylate concentration. Highest selectivity was observed for the membrane incorporating 38.8% PVC, 57.3% plasticizer, 2.6% Sn(salophen), and 1.3% sodium tetraphenylborate. The electrodes exhibit fast response times and micromolar detection limits (approximately 1 x 10(-6) M salicylate) and could be used over a wide pH range of 3-8. Applications of the electrodes for determination of salicylate in pharmaceutical preparations and biological samples are reported. PMID- 10739199 TI - Probing solvent fractionation in electrospray droplets with laser-induced fluorescence of a solvatochromic dye AB - Laser-induced fluorescence spectroscopy is used to profile solvent fractionation in an electrospray plume containing Nile Red, a solvatochromic dye (i.e., a dye for which spectral features are sensitive to solvent polarity). The results confirm gradual enrichment of the less volatile component in spray droplets as a result of solvent evaporation. Changes in solvent composition are evident in both axial and lateral profiles. The influences of capillary voltage, nebulizing gas, an electrolyte modifier, and flow rate on the extent of solvent fractionation are assessed. The results suggest that, in addition to volatility of the solvents, major factors affecting solvent fractionation include initial droplet size, droplet velocity, and the cooling effect of the nebulizing gas. Isolating contributions from these factors will require complementary data from other experiments. PMID- 10739200 TI - Charge capacity limitations of radio frequency ion guides in their use for improved ion accumulation and trapping in mass spectrometry. AB - The use of radio frequency (rf) ion guides as "linear" two-dimensional ion traps and ion guides for ion storage and accumulation, respectively, is becoming increasingly important for realizing improved sensitivity in mass spectrometry. Analytical relationships describing the ion accumulation operation mode of rf ion guides are reported. Comparisons are made between the rf quadrupole ion guide, higher-order rf multipoles and rf stacked ring ion guides, in terms of the charge capacity limitations due to the instability of ions, rf focusing efficiency limits, and effects due to rf ion heating (i.e., collisional activation due to rf oscillations of ions). Analytical relations for the stored charge quantity are derived in the low ion energy approximation, which is shown to be reasonable for the systems considered. The ion density spatial distribution is derived, an exponential form of which proved to provide a good approximation for high-order rf multipoles and stacked ring rf ion guides. The limit on the stored charge dependence upon rf is shown to be directly related to the thermal dissociation thresholds for the ions being studied; the limitation is weaker for higher-order multipoles and stacked ring ion guides. These results suggest that rf quadrupoles provide an optimum configuration when accumulation of a moderate ion density is sufficient (below 10(9) elementary charges/m). Alternatively, accumulation of an appreciable density for more fragile species, such as noncovalent complexes, may be realized using higher-order multipoles and stacked ring ion guides. PMID- 10739201 TI - Higher throughput bioanalysis by automation of a protein precipitation assay using a 96-well format with detection by LC-MS/MS. AB - Generic methodology for the automated preparation and analysis of drug levels in plasma samples within a drug discovery environment was achieved through the redesign of a protein precipitation assay to a microtiter (96-well) plate format and the application of robotic liquid handling for performance of all transfer and pipetting steps. Validation studies revealed that the application of robotics to sample preparation, in general, maintained the analytical accuracy and precision compared with preparing samples manually. The use of rapid gradient LC MS/MS for analysis coupled with flow diversion of the solvent front allowed the introduction of protein-precipitated samples into the mass spectrometer without the necessity for source cleaning. The problem inherent in automatically pipetting plasma, caused by fibrinogen clots, was overcome by storing samples at 80 degrees C and thus precluding clot formation. The resulting methodology allowed sample preparation for a 96-well plate designed to accommodate 54 unknowns, duplicate 12-point calibration curves, and 6 sets of quality controls at three levels in approximately 2 h. This approach allowed an increase in throughput of sample preparation and analysis to >400 samples per day per LC MS/MS instrument with minimal manual intervention. Overall, substantial time savings were realized, demonstrating that automation is an increasingly essential tool in a drug discovery bioanalytical environment. PMID- 10739202 TI - Identification of metal cations, metal complexes, and anions by electrospray mass spectrometry in the negative ion mode AB - Pneumatically assisted electrospray mass spectrometry (ES-MS) is used in the negative ion mode for aqueous metal (M) solutions in an excess of hydrochloric or nitric acid, where the major anion X = Cl- or NO3-. A collision energy of approximately 20 eV removes anion-solvent clusters for most elements and leaves negative complex ions of the general form (Mn+Xn+1)-. Complexation with anions prevents charge reduction reactions at least to n = 3, even in cases where the third ionization energy of M greatly exceeds the first ionization energy of the solvent. These negative ions thus preserve the charge state of the metal cation from the solution and allow identification of both cations and anions in a single set of electrospray conditions. Cations such as Fe3+ or Cu2+ that have a lower oxidation state in solution produce a distribution of negative ions, each with a single negative charge overall; e.g., an Fe3+ solution produces both Fe(III)X4- and Fe(III)X3-. This distribution of FeIII and FeII species is attributed to electrochemical reduction of Fe3+ at the negatively charged ES needle. "Native" anions such as perrhenate or molybdate produce singly charged analogues such as ReO4- or HMoO4-. Metal-EDTA complexes are seen as M(III)Y- or M(II)HY-. The sensitivity for these "native" anions is suppressed by competition with the excess chloride or nitrate used to produce the metal-containing complex ions. PMID- 10739203 TI - Characterization of dyes and other pollutants in the effluent of a textile company by LC/NMR and LC/MS AB - LC/NMR and LC/MS (the latter technique in the MSn mode) were used to characterize the organic constituents of industrial wastewater with emphasis on polar, nonvolatile compounds. In the effluent of a textile company, various compounds such as anthraquinone-type dyes and their byproducts, a fluorescent brightener, a byproduct from polyester production, and auxiliaries such as anionic and nonionic surfactants and their degradation products were identified. It is shown that the combined use of both hyphenated techniques provides complementary structural information. If applied under comparable chromatographic conditions, they are well-suited for the nontarget analysis. PMID- 10739204 TI - A statistical basis for testing the significance of mass spectrometric protein identification results. AB - A method for testing the significance of mass spectrometric (MS) protein identification results is presented. MS proteolytic peptide mapping and genome database searching provide a rapid, sensitive, and potentially accurate means for identifying proteins. Database search algorithms detect the matching between proteolytic peptide masses from an MS peptide map and theoretical proteolytic peptide masses of the proteins in a genome database. The number of masses that matches is used to compute a score, S, for each protein, and the protein that yields the best score is assumed as the identification result. There is a risk of obtaining a false result, because masses determined by MS are not unique; i.e., each mass in a peptide map can match randomly one or several proteins in a genome database. A false result is obtained when the score, S, due to random matching cannot be discerned from the score due to matching with a real protein in the sample. We therefore introduce the frequency function, f(S), for false (random) identification results as a basis for testing at what significance level, alpha, one can reject a null hypothesis, H0: "the result is false". The significance is tested by comparing an experimental score, S(E), with a critical score, S(C), required for a significant result at the level alpha. If S(E) > or = S(C), H0 is rejected. f(S) and S(C) were obtained by simulations utilizing random tryptic peptide maps generated from a genome database. The critical score, S(C), was studied as a function of the number of masses in the peptide map, the mass accuracy, the degree of incomplete enzymatic cleavage, the protein mass range, and the size of the genome. With S(C) known for a variety of experimental constraints, significance testing can be fully automated and integrated with database searching software used for protein identification. PMID- 10739205 TI - A method for the chemical generation of N-terminal peptide sequence tags for rapid protein identification. AB - We describe a method for generating multiple small sequences from the N terminal of peptides in unseparated protein digests by stepwise thioacetylation and acid cleavage. The mass differences between a series of N-terminally degraded peptides give short sequences of defined length. Such short "sequence tags" together with the mass of the parent peptide can be used to identify the protein in a database. The sequence ladders are generated without the use of chain terminators or sample aliquoting and the degradation reagents are water soluble so that the chemistry can be carried out on peptides immobilized on C-18 reversed-phase supports without any peptide loss due to washing with organic solvents as occurs in Edman type sequencing. The entire procedure can be automated, and we describe a prototype device for the parallel analysis of multiple samples. We demonstrate the effectiveness of this chemical tagging method in a comparison with Edman sequencing, peptide mass fingerprinting, and MS/MS analysis of crude protein fractions obtained from an HPLC separation of the Escherichia coli ribosome complex which consists of 57 proteins. We show that chemical tagging is a viable first-pass high-throughput identification method to be used prior to an in depth MS/MS analysis. PMID- 10739206 TI - A microdevice with integrated liquid junction for facile peptide and protein analysis by capillary electrophoresis/electrospray mass spectrometry. AB - A novel microfabricated device was implemented for facile coupling of capillary electrophoresis with mass spectrometry (CE/MS). The device was constructed from glass wafers using standard photolithographic/wet chemical etching methods. The design integrated (a) sample inlet ports, (b) the separation channel, (c) a liquid junction, and (d) a guiding channel for the insertion of the electrospray capillary, which was enclosed in a miniaturized subatmospheric electrospray chamber of an ion trap MS. The replaceable electrospray capillary was precisely aligned with the exit of the separation channel by a microfabricated guiding channel. No glue was necessary to seal the electrospray capillary. This design allowed simple and fast replacement of either the microdevice or the electrospray capillary. The performance of the device was tested for CE/MS of peptides, proteins, and protein tryptic digests. On-line tandem mass spectrometry was used for the structure identification of the protein digest products. High efficiency/high-resolution separations could be obtained on a longer channel (11 cm on-chip) microdevice, and fast separations (under 50 s) were achieved with a short (4.5 cm on-chip) separation channel. In the experiments, both electrokinetic and pressure injections were used. The separation efficiency was comparable to that obtained from conventional capillary electrophoresis. PMID- 10739207 TI - Approaching a million-fold sensitivity increase in capillary electrophoresis with direct ultraviolet detection: cation-selective exhaustive injection and sweeping AB - A novel method that combines two on-line concentration techniques in capillary electrophoresis (CE), namely, sample stacking with electrokinetic injection (field-enhanced sample injection, FESI) and sweeping, afforded the detection of positively chargeable analytes in parts per trillion (ppt) levels. The main idea is to selectively introduce by FESI as many molecules of cationic analytes as possible from a very dilute sample solution and focus the resulting zone by sweeping. Limit of detection values (signal-to-noise ratio 3) of 4.1 and 8.0 ppt the lowest concentration reported by direct UV detection in CE-with average plate numbers of 3.6 x 10(5) and 4.4 x 10(5) are obtained for laudanosine and naphthylamine (standard solutions), respectively. This translates to improvements in peak heights compared with usual injection approaching a million-fold. Optimization schemes and application to quantitative and qualitative analyses are also investigated. PMID- 10739208 TI - Stationary-phase contribution of 1-propanol organic modifier to changes in sorption of 1-hexanol on an ODS-bonded phase AB - Using the reversed-phase bonded-phase HPLC packing Partisil-10 ODS-3, sorption isotherms have been measured for the alcohols 1-propanol (PrOH) and 1-hexanol (HexOH), and as well, a simultaneous sorption curve for the two alcohols has been measured from solutions containing a low and constant concentration of HexOH as sample with increasing concentrations of PrOH as organic modifier. The mobile phase effect of PrOH is quantified by solution-phase activity coefficients obtained either from vapor/solution equilibrium measurements or from cloud point measurements. Since sorbed alcohols are located at the ODS/solution interface, the stationary-phase effect of PrOH is modeled in terms of three processes: (i) competition for space; (ii) decrease of space required per mole with increasing concentration of sorbed PrOH; and (iii) change of free energy of sorption with increasing concentration of sorbed PrOH. The model yields excellent fits to the isotherms and to the simultaneous sorption curve. Comparison of the model-fitting parameters for the simultaneous sorption curve with those for the PrOH isotherm confirms that the stationary-phase effect of PrOH on HexOH is due exclusively to processes i and ii. Sorbed PrOH causes rearrangement of the C18 chains of the ODS phase. For volume percent PrOH less than 15% in the mobile phase, the effect of PrOH on sample sorption is nearly exclusively in the stationary phase. Between 15 and 30%, both mobile- and stationary-phase effects are important. PMID- 10739209 TI - Quantitative separation of aliphatic and aromatic hydrocarbons using silver ion- silica solid-phase extraction AB - A solid-phase extraction (SPE) method employing silver nitrate impregnated silica has been developed and evaluated for the separation of defined aliphatic and aromatic hydrocarbons from crude oils. The versatility of the SPE method is demonstrated using a light crude oil from the North Sea and a heavy crude oil from Orcutt field (Monterey, California, U.S.A.). The coefficients of variation for a number of geochemical parameters measured on both aliphatic and aromatic hydrocarbons were excellent. The separation efficiency of SPE is demonstrated using quantification of monoaromatic steroid hydrocarbons which are notoriously difficult to efficiently sequester into the aromatic hydrocarbon fraction using traditional liquid chromatographic procedure. The selectivity and efficiency of the SPE technique is comparable with that of silica gel TLC. However, losses of volatile compounds such as naphthalene are limited during SPE since the sample remains in solvent. We conclude that solid-phase extraction affords rapid sample turnover suitable for processing large sample numbers with high reproducibility. PMID- 10739210 TI - DNA sequencing up to 1300 bases in two hours by capillary electrophoresis with mixed replaceable linear polyacrylamide solutions. AB - This paper presents results on ultralong read DNA sequencing with relatively short separation times using capillary electrophoresis with replaceable polymer matrixes. In previous work, the effectiveness of mixed replaceable solutions of linear polyacrylamide (LPA) was demonstrated, and 1000 bases were routinely obtained in less than 1 h. Substantially longer read lengths have now been achieved by a combination of improved formulation of LPA mixtures, optimization of temperature and electric field, adjustment of the sequencing reaction, and refinement of the base-caller. The average molar masses of LPA used as DNA separation matrixes were measured by gel permeation chromatography and multiangle laser light scattering. Newly formulated matrixes comprising 0.5% (w/w) 270 kDa and 2% (w/w) 10 or 17 MDa LPA raised the optimum column temperature from 60 to 70 degrees C, increasing the selectivity for large DNA fragments, while maintaining high selectivity for small fragments as well. This improved resolution was further enhanced by reducing the electric field strength from 200 to 125 V/cm. In addition, because sequencing accuracy beyond 1000 bases was diminished by the low signal from G-terminated fragments when the standard reaction protocol for a commercial dye primer kit was used, the amount of these fragments was doubled. Augmenting the base-calling expert system with rules specific for low peak resolution also had a significant effect, contributing slightly less than half of the total increase in read length. With full optimization, this read length reached up to 1300 bases (average 1250) with 98.5% accuracy in 2 h for a single stranded M13 template. PMID- 10739211 TI - Electroosmotic capillary flow with nonuniform zeta potential AB - The present work is an analytical and experimental study of electroosmotic flow (EOF) in cylindrical capillaries with nonuniform wall surface charge (zeta potential) distributions. In particular, this study investigates perturbations of electroosmotic flow in open capillaries that are due to induced pressure gradients resulting from axial variations in the wall zeta-potential. The experimental inquiry focuses on electroosmotic flow under a uniform applied field in capillaries with an EOF-suppressing polymer adsorbed onto various fractions of the total capillary length. This fractional EOF suppression was achieved by coupling capillaries with substantially different zeta-potentials. The resulting flow fields were imaged with a nonintrusive, caged-fluorescence imaging technique. Simple analytical models for the velocity field and rate of sample dispersion in capillaries with axial zeta-potential variations are presented. The resulting induced pressure gradients and the associated band-broadening effects are of particular importance to the performance of chemical and biochemical analysis systems such as capillary electrokinetic chromatography and capillary zone electrophoresis. PMID- 10739212 TI - Membrane extraction with a sorbent interface for headspace monitoring of aqueous samples using a cap sampling device AB - A cap-shaped device was employed for headspace sampling. This sampling device coupled to membrane extraction with a sorbent interface (MESI) is intended to perform on-site and on-line aqueous sample monitoring. A laboratory sampling testwas performed both at the water surface and under water, and it showed some advantages in underwater monitoring. A group of volatile organic compounds (VOCs), varying in PDMS/gas and gas/water distribution constants, benzene, toluene, ethylbenzene, o-xylene, and trichloroethylene (TCE), was used for the sampling study. Magnetic stirring of the sample and circulation of the headspace air with a microfan were used for the enhancement of mass transfer between sample matrix and membrane to obtain higher extraction rate and efficiency. The agitation approaches were investigated individually and compared. The results showed that simultaneous agitation in water and air could greatly improve the extraction efficiency. Good linearity and precision and low detection limits were obtained for water-surface monitoring. The study demonstrated that Cap-MESI is a useful tool for field headspace monitoring of volatile organic compounds. PMID- 10739213 TI - Calibration of membrane extraction for air analysis AB - Calibration methods based on the recently developed mathematical model are proposed for air monitoring by membrane extraction. In membrane extraction, analytes permeate through the membrane at a constant rate controlled by the distribution constant and the diffusion coefficient. The principle of the proposed calibration approach is based on either theoretical or experimental determination of both constants at the extraction conditions. A group of selected compounds was employed for the experimental testing, and the results indicated practical feasibility of the approach. On-line determination of partition coefficients and distribution constants was proposed and investigated, producing very promising results. Both approaches to calibration facilitate quantitative monitoring. PMID- 10739214 TI - Selective fluorescence quenching of polycyclic aromatic hydrocarbons by aliphatic amines AB - The ability of primary, secondary, and tertiary mono- and diamines to serve as fluorescence quenchers for polycyclic aromatic hydrocarbons (PAHs) has been evaluated in acetonitrile and methanol. In general, the efficiency of quenching increases with the electron-donating ability of the amine and with the number of amine groups. The selectivity for nonalternant PAHs relative to the alternant isomers appears to decrease as the quenching efficiency increases. Nevertheless, all of the amines exhibit greater selectivity than 1,2,4-trimethoxybenzene, a previously characterized selective quencher for nonalternant PAHs. The experimentally measured rate constants for dynamic quenching show good agreement to those predicted by Rehm-Weller electron-transfer theory in the solvent acetonitrile. In contrast, the rate constants in methanol are anomalously low and do not conform well to Rehm-Weller theory. Presumably this is due to explicit hydrogen bonding between the solvent and quencher, which decreases the concentration of the free amine that is available for fluorescence quenching. PMID- 10739215 TI - Optical sensors for the determination of concentrated hydroxide AB - An optical sensor system has been developed for the determination of concentrated strong bases ([OH-] = 1-10 M). The base sensors consist of SiO2/ZrO2-organic polymer composites and doped high-pKa indicators. Films were obtained by spin casting these composite materials on glass substrates and were used as sensor elements for the spectrometric determination of hydroxide. The hydrophilic nature of the mixed oxide SiO2/ZrO2 and its chemical stability in concentrated alkali made it attractive as support in the composites. The organic polymers in the composites either provided better mechanical stability and dye immobilization or enhanced OH- diffusion and sensor response. The composite sensors showed a relative standard deviation of less than 2%. The response time of a SiO2/ZrO2 Nafion composite (sensor 2) was short (5 s), and a small hysteresis was observed during reproducibility measurements with 1-4 M NaOH solutions. The sensors were found to be stable in 4 M NaOH during a 30-day durability test, showing a standard deviation of 3.0-4.7%. The diffusion kinetics and hysteresis performance of the sensors were also evaluated. PMID- 10739216 TI - Using N-aminoperylene-3,4:9,10-tetracarboxylbisimide as a fluorogenic reactand in the optical sensing of aqueous propionaldehyde AB - Aldehydes are usually determined via chemical derivatization using a chromogenic and fluorogenic reagent followed by chromatographic separation and UV-visible detection. As a consequence, continuous on-line monitoring is impossible. Following our concept of reversible chemical reactions as the basis of optical sensors, we have investigated N-amino-N'-(1-hexylheptyl)perylene-3,4:9,10 tetracarboxylbisimide for aldehyde sensing. The fluorogenic reactand has been embedded in plasticized PVC, and the resulting thin layers have been exposed to aqueous samples of aliphatic aldehydes and ketones. The reactand exhibits a pronounced increase in fluorescence upon interaction with aldehydes since the chemical reaction causes a dequenching of perylene fluorescence. Upon interaction with aqueous propionaldehyde, sensor layers typically exhibit a dynamic range from 5 to 100 mM propionaldehyde, and the limit of detection amounts to 0.08 mM. The forward and reverse response time (t95) for a decade change in activity is in the range of 2-7 min, when measured at pH 2.5. The selectivity of sensor layers toward aldehydes correlates with their lipophilicity in that aldehydes with higher lipophilicity are more efficiently extracted into the polymer layer. PMID- 10739217 TI - Electroosmotic flow control of fluids on a capillary electrophoresis microdevice using an applied external voltage AB - Independent control of electroosmosis is important for separation science techniques such as capillary zone electrophoresis and for the movement of fluids on microdevices. A capillary electrophoresis microdevice is demonstrated which provides more efficient control of electroosmosis with an applied external voltage field. The device is fabricated in a glass substrate where a 5.0 cm separation channel (30 microm wide) is paralleled with two embedded electrodes positioned 50 microm away in the substrate. With this structure, greatly reduced applied external potentials (< or = 120 V compared to tens of kilovolts) still effectively altered electroosmosis. The efficiency for the control of electroosmosis by the applied external field is improved by approximately 40 times over published values. PMID- 10739218 TI - Flame hydrolysis deposition of glass on silicon for the integration of optical and microfluidic devices AB - Flame hydrolysis deposition (FHD) of glasses has previously found applications in the telecommunications industry. This paper shows how the technology can be used to deposit silica with different refractive indices and thereby produce low-loss planar waveguides for use in analytical applications. We also show that the glasses can be patterned using a new reactive ion etch and sealed using a modification of anodic bonding, such that the resulting microstructures can be readily incorporated within a lithographically defined "chip", integrating both optical and fluidic circuitry on the same device. In the example described in this paper, waveguides, analytical microtiter chambers and fluidic capillary channels, with the necessary high aspect ratio features (and with depths up to 40 microm) were all produced in glass, using the appropriate deposition and etching technologies. The performance of the chip was assessed in the framework of a low volume fluorescence assay, using waveguides to address miniaturized microtiter chambers with volumes of 230 and 570 pL. Devices featuring different optical detection configurations, including both in-line and orthogonal waveguide geometries, were fabricated. In the optimal configuration, the experimental detection limit was determined as ca. 20 pM (equivalent to 10 zmol) of a cyanine fluorophore, Cy5. The applicability of the device as a biochip platform was further illustrated by analytical measurements on fluorescently labeled oligodeoxynucleotides. PMID- 10739219 TI - Homicidal asphyxia. AB - Homicides due to asphyxia are relatively uncommon. To better understand the presentation of such cases, the files of the Bexar County Medical Examiner's Office were reviewed from January 1, 1985, through December 31, 1998, for all such homicides. A total of 133 cases were found. The largest category was ligature strangulation with 48 deaths (21 male, 27 female). Petechiae were present in the conjunctivae and/or sclerae in 86% of the cases; fractures of the hyoid and/or thyroid cartilage were present in 12.5%. There were a total of 41 deaths from manual strangulation (27 female, 14 male). Petechiae were present in 89% of the cases. In cases of manual strangulation, fractures of the hyoid, thyroid, or cricoid cartilage were found in all the male victims and slightly more than one half of the female victims. Twenty-six cases of suffocation were found; 20 of the victims were < or =2 years of age. Only 1 of these children had petechiae and/or scleral hemorrhage. Five deaths were due to choking. Three of the deaths involved adults who were gagged; 2 deaths involved infants with foreign material pushed into the mouth. Other categories of asphyxia were as follows: 9 deaths due to more than one form of asphyxia; 1 death due to hanging, and 3 deaths due to drowning. Rape was the motive in 66% of the female victims of ligature strangulation and 52% of those due to manual strangulation. PMID- 10739220 TI - Characteristics of child abuse by anteroposterior manual compression versus cardiopulmonary resuscitation: case reports. AB - Lethal and sublethal injuries to infants caused by anteroposterior manual compression can be accompanied by lateral rib fractures, hemorrhage into the viscera, and when severe, rupture of large vessels and solid organs. Abusers may claim that these injuries were sustained in an attempt to perform cardiopulmonary resuscitation (CPR). The difference between injuries caused by CPR and injuries caused by inflicted anteroposterior compression is examined in one sublethal and two lethal cases. PMID- 10739221 TI - Level of agreement between opinions of medical examiner investigators and forensic pathologist medical examiners regarding the manner of death. AB - To evaluate the level of agreement between medical examiner investigators' opinion of the manner of death and what the manner of death was as certified by forensic pathologist medical examiners (MEs), we reviewed the case records stored in a database of all deaths reported to the office of the medical examiner in Fulton County, Georgia. Of 15,771 deaths reported to the office during a 10-year period, a difference exists in 1908 cases. In 900 natural deaths, the investigators recorded 135 accident, 10 homicide, 10 suicide, and 745 undetermined manners of death. In 755 accidental deaths, the investigators recorded 16 natural, 8 homicide, 13 suicide, and 718 undetermined manners of death. In 107 homicides, the investigators recorded 12 natural, 8 accident, 0 suicide, and 87 undetermined manners of death. In 70 suicides, the investigators recorded 9 natural, 9 accident, 3 homicide, and 49 undetermined manners of death. In 61 deaths classified as undetermined, the investigators recorded 25 natural, 13 accident, 17 homicide, and 6 suicide manners of death. In 15 deaths, the discrepancy exists due to an apparent error in the database information. This study confirms a high concordance between investigator and ME opinion regarding manner of death but also documents the need for case review and autopsies by forensic pathologists to confirm the investigators' opinion of the manner of death, determine the manner of death when the investigator selects undetermined, and on occasion, refute the investigators' opinion regarding the manner of death. PMID- 10739222 TI - Advances in the diagnosis of wound vitality: a review. AB - The diagnosis of the vital origin of wounds in many cases remains an unsolved problem for the forensic pathologist. Practical experience enables the expert to diagnose the vital or postmortem origin of wounds on the basis of macroscopic examination. In some cases, optic microscopy is used to confirm the diagnosis. In many other cases, additional more sensitive and specific markers of vitality are required. In the past 50 years, comprehensive research on this topic has resulted in a better understanding of the acute inflammatory reaction. The development and application of sensitive and specific markers through research in the areas of histochemistry, enzymology, and biochemistry has provided a partial solution to the problems involved in wound vitality diagnosis. A review of this challenging area of forensic pathology, including an explanation of these methods and markers, is presented in this paper. PMID- 10739223 TI - Multiple self-inflicted gunshot wounds to the head: report of a case and review of the literature. AB - Multiple self-inflicted gunshot wounds to the head are rare and usually present a challenge to the pathologist and to the police in determining the manner of death. We report a case of two suicidal gunshot wounds to the head. The literature is reviewed, and the pertinent findings, including location of the wounds, location of the brain injuries, types of weapons used, and criteria important to determine the manner of death, are discussed. PMID- 10739224 TI - Frequency of pulmonary hemosiderosis in Eastern North Carolina. AB - Pulmonary hemosiderosis has been attributed to airborne fungi in water-damaged homes in studies of a cluster of cases in infants and children in Cleveland, Ohio, in 1997. We have searched for such emerging infectious agents in the Coastal Plain of North Carolina, which is subject to intermittent flooding. Pulmonary tissue from 206 infants and young children whose deaths had been investigated from October 1978 to September 1996 was retained at East Carolina University School of Medicine. Ages ranged from premature newborns to 49-months. One hundred and ten deaths were attributed to sudden infant death syndrome (SIDS). New sections were cut and analyzed using hematoxylin and eosin, Prussian blue for iron, and Gomori methenamine silver for fungal organisms. Twenty-three infants and children had iron-containing macrophages. Sixteen of these had underlying illnesses, but 4 were originally diagnosed as SIDS. Only one of these had sufficient hemosiderosis to be considered as having pulmonary hemosiderosis and no fungi were present. One case without iron had fungi and was not originally diagnosed as SIDS. The single case of pulmonary hemosiderosis found in this rural area is not similar to the cluster in Cleveland. The study identifies no fungal organisms as emerging infectious diseases in this area. PMID- 10739225 TI - Asphyxial death during prone restraint revisited: a report of 21 cases. AB - Determining the cause of death when a restrained person suddenly dies is a problem for death investigators. Twenty-one cases of death during prone restraint are reported as examples of the common elements and range of variation in these apparently asphyxial events. A reasonable diagnosis of restraint asphyxia can usually be made after ruling out other causes and collecting supportive participant and witness statements in a timely fashion. Common elements in this syndrome include prone restraint with pressure on the upper torso; handcuffing, leg restraint, or hogtying; acute psychosis and agitation, often stimulant drug induced; physical exertion and struggle; and obesity. Establishing a temporal association between the restraint and the sudden loss of consciousness/death is critical to making a correct determination of cause of death. PMID- 10739226 TI - An unusual foreign body in the distal small intestine: case report. AB - A case of a deceased 77-year-old woman presenting to the coroner for postmortem examination scribed. A plastic tie used to seal loaves of sliced bread and other plastic-wrapped food stuffs was found clamped by its "teeth" to a length of small bowel proximal to the cecum, resulting in localized mucosal ulceration, thickening, and edema of the bowel wall. There was also infarcted small bowel due to aortic atherosclerosis, which was submitted as the cause of death. The presence of the bread tie probably did not significantly contribute to death but was as an unusual finding at postmortem examination and has not been previously described. PMID- 10739227 TI - An unusual death by propranolol ingestion. AB - A case of acute intoxication with propranolol is reported. A 60-year-old man was found dead in his car with no evidence of trauma or signs of asphyxia. Near the car, a pharmaceutical box of Inderal, a pharmaceutical formulation containing propranolol, was found. The man was not under therapy with this medication, but circumstantial data suggested the use of this medicine by his sister. High blood levels of propranolol confirmed the suspected drug use and induced us to discuss the probable lethal mechanism of this substance. The analysis of this compound was performed by liquid-liquid extraction followed by gas chromatography/mass spectrometry determination. PMID- 10739228 TI - An unusual drug death involving maggots. AB - Toxicologic analysis of decomposed specimens provides greater analytical challenges than those encountered with fresh postmortem specimens. Despite the difficulties involved, in cases in which the cause of death is not determined at autopsy or when there is a strong indication of drug intoxication, all reasonable steps must be undertaken to perform as comprehensive a drug screen as possible. An unidentified white male was found in a field near a river. The body was decomposed and skeletonized, and 3- to 4-mm maggots were present on the body. Near the body was an empty bottle of secobarbital that had been prescribed to a female. There was no evidence of injury. Calf muscle and maggots were sent for toxicologic analysis. No volatile substances or drugs were detected in the calf muscle. Because intoxication due to secobarbital was strongly suggested from the scene investigation, the only other specimen available, the maggots, were tested for acid-neutral drugs. Secobarbital was identified by retention time and was confirmed by full-scan electron ionization gas chromatography/mass spectrometry. Based on the available information, the medical examiner ruled that the cause of death was secobarbital intoxication and the manner of death was suicide. PMID- 10739229 TI - Sudden infant death, large intestinal volvulus, and a duplication cyst of the terminal ileum. AB - A previously well 7-week-old boy was found unresponsive and cyanotic in his cot in the morning. Resuscitative attempts achieved only partial response with subsequent terminal cardiac arrest occurring later in the day. At autopsy a large intestinal volvulus was found associated with disseminated Clostridium perfringens sepsis. In addition, a duplication cyst of the terminal ileum was present. Although rare, right-sided colonic volvulus may be a cause of unexpected death in infancy. While predisposing factors to volvulus may include intestinal duplication cysts, the anatomic relation of the duplication to the volvulus must be carefully determined before a causal relation can be accepted; the cyst in this case was most likely incidental to the terminal event. PMID- 10739230 TI - Unusual fatal mechanisms in nonasphyxial autoerotic death. AB - The diagnosis of autoerotic death is most often made when there has been accidental asphyxia from ropes or ligatures used by the deceased as a part of his or her autoerotic ritual. Three cases of probable autoerotic death are reported in which the mechanisms of death involved hyperthermia, sepsis, and hemorrhage, respectively. Case 1: A 46-year-old man was found dead in bushland clothed in a dress, female undergarments, and seven pairs of stockings/pantyhose. The underwear had been cut to enable exposure of the genitals. The recorded daily maximum temperature was 39 degrees C, and the deceased had been taking the drug benztropine. Death was attributed to hyperthermia due to a combination of excessive clothing, high ambient temperature, and prescription drug side effect. Case 2: A 40-year-old man was found dead in his boarding house. At autopsy, a pencil was found within his abdominal cavity with perforation of the bladder and peritonitis. Death was attributed to peritonitis/sepsis following intraurethral introduction of a pencil. Case 3: A 56-year-old man was found dead lying on his bed following massive rectal hemorrhage. A blood stained shoe horn was found nearby. Death was attributed to hemorrhage following laceration of the anal canal with a shoe horn. The diagnosis of autoerotic death may be difficult when typical features are absent, however, any unusual injury associated with genitourinary manipulation must raise this possibility. PMID- 10739231 TI - Pulmonary arterial fibromuscular dysplasia: a rare cause of fulminant lung hemorrhage. AB - Arterial fibromuscular dysplasia (FMD) represents a collection of noninflammatory and nonatherosclerotic vascular diseases with a poorly understood etiology. Classically occurring in renal and cerebral arteries, this entity has also been reported in coronary, carotid, and other medium and small arteries. One case occurring in the pulmonary vasculature has been reported. Fatal hemothorax and lung hemorrhage have multiple causes, including other vascular malformations and connective tissue disorders; however, cases of pulmonary FMD are exceedingly rare. We report what appears to be the second such association, occurring in a 69 year-old man. The patient presented with a 3-week history of increasing dyspnea, fatigue, and productive cough; 3 days of increasing back and chest pain; and syncope. Chest radiograph showed a "white-out" of the left lung. The patient died shortly after admission from a fulminant respiratory disease of undetermined etiology. At autopsy he was found to have a massive left hemothorax resulting from an unsuspected pulmonary arterial fibromuscular dysplasia. PMID- 10739233 TI - A deadly anti-SIDS device. AB - The diagnosis of sudden infant death syndrome (SIDS) has been an enigma to medical examiners and coroners for decades. The recent drastic decrease in the number of SIDS cases has been associated with infants sleeping supine instead of prone. The apparent relation between sleeping position and SIDS has led to the marketing of several positioning sleep aids. We report a case of the improper use of one such device which resulted in the death of an infant. PMID- 10739232 TI - Arterial-esophageal fistulae in patients requiring nasogastric esophageal intubation. AB - A rare and potentially fatal cause of hematemesis is fistula formation between the esophagus and the vascular system. A case report of a 39-year-old woman with congenital aortic arch anomalies hospitalized for treatment of head injuries demonstrates the potential for iatrogenic esophageal trauma to initiate fistula formation between the esophagus and an anomalous arterial system. A literature review revealed 6 other cases of vascular-esophageal fistulae caused by nasogastric esophageal intubation. It is concluded that aortic arch anomalies increase the risk of esophageal injury and subsequent fistula formation from nasogastric esophageal intubation. In addition, the clinical features and pathologic findings of vascular-esophageal fistulae are reviewed. PMID- 10739234 TI - Cerebellar agenesis in a suicide. AB - A case of a suicidal death associated with autopsy findings of right cerebellar and vermian agenesis is described. A review of the literature points out the various recurrence of agenesis, symptoms, and the likelihood of motor deficits. The singular coexistence of only behavioral and intellectual deficiency with suicidal inclination in an adult man with partial cerebellar agenesis is discussed. PMID- 10739235 TI - Myotonic dystrophy CTG repeats in an Italian population sample: evaluation of the polymorphism for forensic applications. AB - The myotonic dystrophy (DM) CTG repeat polymorphism has been studied in an Italian population sample. Polymerase chain reaction (PCR) amplification, manual polyacrylamide gel electrophoresis (PAGE), and silver staining were employed. Alleles were typed by comparison with a sequenced allelic ladder. A total of 25 different alleles, spanning the range from 5 to 31 CTG triplets, was observed. The heterozygosity was 79%, and no significant deviation from Hardy-Weinberg equilibrium was found. Eighty-one meioses from parentage testing were also analyzed, and a Mendelian pattern of inheritance was observed in all cases. In addition, we could successfully type the DM locus in 20 laboratory-prepared bloodstains, with 1 ng of DNA allowing clear definition of alleles. We conclude that the CTG repeats at the DM locus may be useful for forensic applications. PMID- 10739236 TI - Unexpected childhood death due to hemolytic uremic syndrome. AB - Two cases of unexpected childhood death due to hemolytic uremic syndrome are reported. A 21-month-old girl who was discovered dead in bed following a short illness was found at autopsy to have overwhelming sepsis resulting from transmural colitis. Escherichia coli serotype 0157A was isolated from the intestine, and renal changes of hemolytic uremic syndrome were found. A 4-year old girl died suddenly in hospital from intracranial hemorrhage while being treated for hemolytic uremic syndrome-related renal failure. Culture of urine and feces grew verocytotoxin producing E. coli. These cases demonstrate that hemolytic uremic syndrome may be a rare cause of unexpected childhood death and that the diagnosis may not be established prior to autopsy. Postmortem culture of tissues and fluids in cases of suspected sepsis in children may be essential in establishing this diagnosis, because histologic evaluation may be compromised by profound sepsis and tissue putrefaction. Accuracy in diagnosis may have significant public health and medicolegal consequences. PMID- 10739237 TI - Restraint position and positional asphyxia. PMID- 10739238 TI - Forensic interpretation of blood in the anus. PMID- 10739239 TI - Death due to concussion and alcohol. PMID- 10739240 TI - Effect on the incidence of SIDS due to amendment of the death certificate form in Japan. PMID- 10739241 TI - Substrate-assisted catalysis: molecular basis and biological significance. AB - Substrate-assisted catalysis (SAC) is the process by which a functional group in a substrate contributes to catalysis by an enzyme. SAC has been demonstrated for representatives of three major enzyme classes: serine proteases, GTPases, and type II restriction endonucleases, as well as lysozyme and hexose-1-phosphate uridylyltransferase. Moreover, structure-based predictions of SAC have been made for many additional enzymes. Examples of SAC include both naturally occurring enzymes such as type II restriction endonucleases as well as engineered enzymes including serine proteases. In the latter case, a functional group from a substrate can substitute for a catalytic residue replaced by site-directed mutagenesis. From a protein engineering perspective, SAC provides a strategy for drastically changing enzyme substrate specificity or even the reaction catalyzed. From a biological viewpoint, SAC contributes significantly to the activity of some enzymes and may represent a functional intermediate in the evolution of catalysis. This review focuses on advances in engineering enzyme specificity and activity by SAC, together with the biological significance of this phenomenon. PMID- 10739242 TI - Folding and binding cascades: dynamic landscapes and population shifts. AB - Whereas previously we have successfully utilized the folding funnels concept to rationalize binding mechanisms (Ma B, Kumar S, Tsai CJ, Nussinov R, 1999, Protein Eng 12:713-720) and to describe binding (Tsai CJ, Kumar S, Ma B, Nussinov R, 1999, Protein Sci 8:1181-1190), here we further extend the concept of folding funnels, illustrating its utility in explaining enzyme pathways, multimolecular associations, and allostery. This extension is based on the recognition that funnels are not stationary; rather, they are dynamic, depending on the physical or binding conditions (Tsai CJ, Ma B, Nussinov R, 1999, Proc Natl Acad Sci USA 96:9970-9972). Different binding states change the surrounding environment of proteins. The changed environment is in turn expressed in shifted energy landscapes, with different shapes and distributions of populations of conformers. Hence, the function of a protein and its properties are not only decided by the static folded three-dimensional structure; they are determined by the distribution of its conformational substates, and in particular, by the redistributions of the populations under different environments. That is, protein function derives from its dynamic energy landscape, caused by changes in its surroundings. PMID- 10739243 TI - NMR analysis of cleaved Escherichia coli thioredoxin (1-73/74-108) and its P76A variant: cis/trans peptide isomerization. AB - Inspection of high resolution three-dimensional (3D) structures from the protein database reveals an increasing number of cis-Xaa-Pro and cis-Xaa-Yaa peptide bonds. However, we are still far from being able to predict whether these bonds will remain cis upon single-site substitution of Pro or Yaa and/or cleavage of a peptide bond close to it in the sequence. We have chosen oxidized Escherichia coli thioredoxin (Trx), a member of the Trx superfamily with a single alpha/beta domain and cis P76 to determine the effect of single-site substitution and/or cleavage on this isomer. Standard two-dimensional (2D) NMR analysis were performed on cleaved Trx (1-73/74-108) and its P76A variant. Analysis of the NOE connectivities indicates remarkable similarity between the secondary and supersecondary structure of the noncovalent complexes and Trx. Analysis of the 2D version of the HCCH-TOCSY and HMQC-NOESY-HMQC and 13C-filtered HMQC-NOESY spectra of cleaved Trx with uniformly 13C-labeled 175 and P76 shows surprising conservation of both cis P76 and packing of 175 against W31. A similar NMR analysis of its P76A variant provides no evidence for cis A76 and shows only subtle local changes in both the packing of 175 and the interstrand connectivities between its most protected hydrophobic strands (beta2 and beta4). Indeed, a molecular simulation model for the trans P76A variant of Trx shows only subtle local changes around the substitution site. In conclusion, cleavage of R73 is insufficient to provoke cis/trans isomerization of P76, but cleavage and single-site substitution (P76A) favors the trans isomer. PMID- 10739244 TI - The crystal structures of human alpha-thrombin complexed with active site directed diamino benzo[b]thiophene derivatives: a binding mode for a structurally novel class of inhibitors. AB - The crystal structures of four active site-directed thrombin inhibitors, 1-4, in a complex with human alpha-thrombin have been determined and refined at up to 2.0 A resolution using X-ray crystallography. These compounds belong to a structurally novel family of inhibitors based on a 2,3-disubstituted benzo[b]thiophene structure. Compared to traditional active-site directed inhibitors, the X-ray crystal structures of these complexes reveal a novel binding mode. Unexpectedly, the lipophilic benzo[b]thiophene nucleus of the inhibitor appears to bind in the S1 specificity pocket. At the same time, the basic amine of the C-3 side chain of the inhibitor interacts with the mostly hydrophobic proximal, S2, and distal, S3, binding sites. The second, basic amine side chain at C-2 was found to point away from the active site, occupying a location between the S1 and S1' sites. Together, the aromatic rings of the C-2 and C-3 side chains sandwich the indole ring of Trp60D contained in the thrombin S2 insertion loop defined by the sequence "Tyr-Pro-Pro-Trp." [The thrombin residue numbering used in this study is equivalent to that reported for chymotrypsinogen (Hartley BS, Shotton DM, 1971, The enzymes, vol. 3. New York: Academic Press. pp 323-373).] In contrast to the binding mode of more classical thrombin inhibitors (D-Phe-Pro-Arg-H, NAPAP, Argatroban), this novel class of benzo[b]thiophene derivatives does not engage in hydrogen bond formation with Gly216 of the thrombin active site. A detailed analysis of the three-dimensional structures not only provides a clearer understanding of the interaction of these agents with thrombin, but forms a foundation for rational structure-based drug design. The use of the data from this study has led to the design of derivatives that are up to 2,900-fold more potent than the screening hit 1. PMID- 10739245 TI - Expression, purification, and structural analysis of the trimeric form of the catalytic domain of the Escherichia coli dihydrolipoamide succinyltransferase. AB - The dihydrolipoamide succinyltransferase (E2o) component of the alpha ketoglutarate dehydrogenase complex catalyzes the transfer of a succinyl group from the S-succinyldihydrolipoyl moiety to coenzyme A. E2o is normally a 24-mer, but is found as a trimer when E2o is expressed with a C-terminal [His]6 tag. The crystal structure of the trimeric form of the catalytic domain (CD) of the Escherichia coli E2o has been solved to 3.0 A resolution using the Molecular Replacement method. The refined model contains an intact trimer in the asymmetric unit and has an R-factor of 0.257 (Rfree = 0.286) for 18,699 reflections between 10.0 and 3.0 A resolution. The core of tE2oCD (residues 187-396) superimposes onto that of the cubic E2oCD with an RMS difference of 0.4 A for all main-chain atoms. The C-terminal end of tE2oCD (residues 397-404) rotates by an average of 37 degrees compared to cubic E2oCD, disrupting the normal twofold interface. Despite the alteration of quaternary structure, the active site of tE2oCD shows no significant differences from that of the cubic E2oCD, although several side chains in the active site are more ordered in the trimeric form of E2oCD. Analysis of the available sequence data suggests that the majority of E2 components have active sites that resemble that of E. coli E2oCD. The remaining E2 components can be divided into three groups based on active-site sequence similarity. Analysis of the surface properties of both crystal forms of E. coli E2oCD suggests key residues that may be involved in the protein-protein contacts that occur between the catalytic and lipoyl domains of E2o. PMID- 10739246 TI - Crystal structures of two mutants (K206Q, H207E) of the N-lobe of human transferrin with increased affinity for iron. AB - The X-ray crystallographic structures of two mutants (K206Q and H207E) of the N lobe of human transferrin (hTF/2N) have been determined to high resolution (1.8 and 2.0 A, respectively). Both mutant proteins bind iron with greater affinity than native hTF/2N. The structures of the K206Q and H207E mutants show interactions (both H-bonding and electrostatic) that stabilize the interaction of Lys296 in the closed conformation, thereby stabilizing the iron bound forms. PMID- 10739247 TI - The use of nucleotide analogs to evaluate the mechanism of the heterotropic response of Escherichia coli aspartate transcarbamoylase. AB - As an alternative method to study the heterotropic mechanism of Escherichia coli aspartate transcarbamoylase, a series of nucleotide analogs were used. These nucleotide analogs have the advantage over site-specific mutagenesis experiments in that interactions between the backbone of the protein and the nucleotide could be evaluated in terms of their importance for function. The ATP analogs purine 5' triphosphate (PTP), 6-chloropurine 5'-triphosphate (Cl-PTP), 6-mercaptopurine 5' triphosphate (SH-PTP), 6-methylpurine 5'-triphosphate (Me-PTP), and 1 methyladenosine 5'-triphosphate (Me-ATP) were partially synthesized from their corresponding nucleosides. Kinetic analysis was performed on the wild-type enzyme in the presence of these ATP analogs along with GTP, ITP, and XTP. PTP, Cl-PTP, and SH-PTP each activate the enzyme at subsaturating concentrations of L aspartate and saturating concentrations of carbamoyl phosphate, but not to the same extent as does ATP. These experiments suggest that the interaction between N6-amino group of ATP and the backbone of the regulatory chain is important for orienting the nucleotide and inducing the displacements of the regulatory chain backbone necessary for initiation of the regulatory response. Me-PTP and Me-ATP also activate the enzyme, but in a more complex fashion, which suggests differential binding at the two sites within each regulatory dimer. The purine nucleotides GTP, ITP, and XTP each inhibit the enzyme but to a lesser extent than CTP. The influence of deoxy and dideoxynucleotides on the activity of the enzyme was also investigated. These experiments suggest that the 2' and 3' ribose hydroxyl groups are not of significant importance for binding and orientation of the nucleotide in the regulatory binding site. 2'-dCTP inhibits the enzyme to the same extent as CTP, indicating that the interactions of the enzyme to the O2 carbonyl of CTP are critical for CTP binding, inhibition, and the ability of the enzyme to discriminate between ATP and CTP. Examination of the electrostatic surface potential of the nucleotides and the regulatory chain suggest that the complimentary electrostatic interactions between the nucleotides and the regulatory chain are important for binding and orientation of the nucleotide necessary to induce the local conformational changes that propagate the heterotropic effect. PMID- 10739248 TI - Conformational substates in different crystal forms of the photoactive yellow protein--correlation with theoretical and experimental flexibility. AB - The conformational changes during the photocycle of the photoactive yellow protein have been the subject of many recent studies. Spectroscopic measurements have shown that the photocycle also occurs in a crystalline environment, and this has been the basis for subsequent Laue diffraction and cryocrystallographic studies. These studies have shown that conformational changes during the photocycle are limited to the chromophore and its immediate environment. However, spectroscopic studies suggest the presence of large conformational changes in the protein. Here, we address this apparent discrepancy in two ways. First, we obtain a description of large concerted motions in the ground state of the yellow protein from NMR data and theoretical calculations. Second, we describe the high resolution structure of the yellow protein crystallized in a different space group. The structure of the yellow protein differs significantly between the two crystal forms. We show that these differences can be used to obtain a description of the flexibility of the protein that is consistent with the motions observed in solution. PMID- 10739249 TI - X-Ray crystal structure and molecular dynamics simulations of silver hake parvalbumin (Isoform B). AB - Parvalbumins constitute a class of calcium-binding proteins characterized by the presence of several helix-loop-helix (EF-hand) motifs. In a previous study (Revett SP, King G, Shabanowitz J, Hunt DF, Hartman KL, Laue TM, Nelson DJ, 1997, Protein Sci 7:2397-2408), we presented the sequence of the major parvalbumin isoform from the silver hake (Merluccius bilinearis) and presented spectroscopic and structural information on the excised "EF-hand" portion of the protein. In this study, the X-ray crystal structure of the silver hake major parvalbumin has been determined to high resolution, in the frozen state, using the molecular replacement method with the carp parvalbumin structure as a starting model. The crystals are orthorhombic, space group C2221, with a = 75.7 A, b = 80.7 A, and c = 42.1 A. Data were collected from a single crystal grown in 15% glycerol, which served as a cryoprotectant for flash freezing at -188 degrees C. The structure refined to a conventional R-value of 21% (free R 25%) for observed reflections in the range 8 to 1.65 A [1 > 2sigma(I)]. The refined model includes an acetylated amino terminus, 108 residues (characteristic of a beta parvalbumin lineage), 2 calcium ions, and 114 water molecules per protein molecule. The resulting structure was used in molecular dynamics (MD) simulations focused primarily on the dynamics of the ligands coordinating the Ca2+ ions in the CD and EF sites. MD simulations were performed on both the fully Ca2+ loaded protein and on a Ca2+ deficient variant, with Ca2+ only in the CD site. There was substantial agreement between the MD and X-ray results in addressing the issue of mobility of key residues in the calcium-binding sites, especially with regard to the side chain of Ser55 in the CD site and Asp92 in the EF site. PMID- 10739250 TI - Deleterious effects of beta-branched residues in the S1 specificity pocket of Streptomyces griseus proteinase B (SGPB): crystal structures of the turkey ovomucoid third domain variants Ile18I, Val18I, Thr18I, and Ser18I in complex with SGPB. AB - Turkey ovomucoid third domain (OMTKY3) is a canonical inhibitor of serine proteinases. Upon complex formation, the inhibitors fully exposed P1 residue becomes fully buried in the preformed cavity of the enzyme. All 20 P1 variants of OMTKY3 have been obtained by recombinant DNA technology and their equilibrium association constants have been measured with six serine proteinases. To rationalize the trends observed in this data set, high resolution crystal structures have been determined for OMTKY3 P1 variants in complex with the bacterial serine proteinase, Streptomyces griseus proteinase B (SGPB). Four high resolution complex structures are being reported in this paper; the three beta branched variants, Ile18I, Val18I, and Thr18I, determined to 2.1, 1.6, and 1.7 A resolution, respectively, and the structure of the Ser18I variant complex, determined to 1.9 A resolution. Models of the Cys18I, Hse18I, and Ape18I variant complexes are also discussed. The beta-branched side chains are not complementary to the shape of the S1 binding pocket in SGPB, in contrast to that of the wild type gamma-branched P1 residue for OMTKY3, Leu18I. Chi1 angles of approximately 40 degrees are imposed on the side chains of Ile18I, Val18I, and Thr18I within the S1 pocket. Dihedral angles of +60 degrees, -60 degrees, or 180 degrees are more commonly observed but 40 degrees is not unfavorable for the beta-branched side chains. Thr18I Ogamma1 also forms a hydrogen bond with Ser195 Ogamma in this orientation. The Ser18I side chain adopts two alternate conformations within the S1 pocket of SGPB, suggesting that the side chain is not stable in either conformation. PMID- 10739251 TI - Dynamics of the Hck-SH3 domain: comparison of experiment with multiple molecular dynamics simulations. AB - Molecular dynamics calculations provide a method by which the dynamic properties of molecules can be explored over timescales and at a level of detail that cannot be obtained experimentally from NMR or X-ray analyses. Recent work (Philippopoulos M, Mandel AM, Palmer AG III, Lim C, 1997, Proteins 28:481-493) has indicated that the accuracy of these simulations is high, as measured by the correspondence of parameters extracted from these calculations to those determined through experimental means. Here, we investigate the dynamic behavior of the Src homology 3 (SH3) domain of hematopoietic cell kinase (Hck) via 5N backbone relaxation NMR studies and a set of four independent 4 ns solvated molecular dynamics calculations. We also find that molecular dynamics simulations accurately reproduce fast motion dynamics as estimated from generalized order parameter (S2) analysis for regions of the protein that have experimentally well defined coordinates (i.e., stable secondary structural elements). However, for regions where the coordinates are not well defined, as indicated by high local root-mean-square deviations among NMR-determined structural family members or high B-factors/low electron density in X-ray crystallography determined structures, the parameters calculated from a short to moderate length (less than 5-10 ns) molecular dynamics trajectory are dependent on the particular coordinates chosen as a starting point for the simulation. PMID- 10739252 TI - Affinity cleavage at the divalent metal site of porcine NAD-specific isocitrate dehydrogenase. AB - A divalent metal ion, such as Mn2+, is required for the catalytic reaction and allosteric regulation of pig heart NAD-dependent isocitrate dehydrogenase. The enzyme is irreversibly inactivated and cleaved by Fe2+ in the presence of O2 and ascorbate at pH 7.0. Mn2+ prevents both inactivation and cleavage. Nucleotide ligands, such as NAD, NADPH, and ADP, neither prevent nor promote inactivation or cleavage of the enzyme by Fe2+. The NAD-specific isocitrate dehydrogenase is composed of three distinct subunits in the ratio 2alpha:1beta:1gamma. The results indicate that the oxidative inactivation and cleavage are specific and involve the 40 kDa alpha subunit of the enzyme. A pair of major peptides is generated during Fe2+ inactivation: 29.5 + 10.5 kDa, as determined by SDS-PAGE. Amino terminal sequencing reveals that these peptides arise by cleavage of the Val262 His263 bond of the alpha subunit. No fragments are produced when enzyme is incubated with Fe2+ and ascorbate under denaturing conditions in the presence of 6 M urea, indicating that the native structure is required for the specific cleavage. These results suggest that His263 of the alpha subunit may be a ligand of the divalent metal ion needed for the reaction catalyzed by isocitrate dehydrogenase. Isocitrate enhances the inactivation of enzyme caused by Fe2+ in the presence of oxygen, but prevents the cleavage, suggesting that inactivation occurs by a different mechanism when metal ion is bound to the enzyme in the presence of isocitrate: oxidation of cysteine may be responsible for the rapid inactivation in this case. Affinity cleavage caused by Fe2+ implicates alpha as the catalytic subunit of the multisubunit porcine NAD-dependent isocitrate dehydrogenase. PMID- 10739253 TI - Folding of beta-sandwich proteins: three-state transition of a fibronectin type III module. AB - An analysis of the folding of the 94 residue tenth fibronectin type III (fnIII) domain of human fibronectin (FNfn10) is presented. Use of guanidine isothiocyanate as a denaturant allows us to obtain equilibrium and kinetic data across a broad range of denaturant concentrations that are unavailable in guanidine hydrochloride. Equilibrium unfolding experiments show that FNfn10 is significantly more stable than has been reported previously. Comparison of equilibrium and kinetic parameters reveals the presence of an intermediate that accumulates at low denaturant concentrations. This is the first demonstration of three-state folding kinetics for a fnIII domain. We have previously shown that a homologous domain from human tenascin (TNfn3) folds by a two-state mechanism, but this does not necessarily indicate that the two proteins fold by different folding pathways. PMID- 10739254 TI - Interaction with magnesium and ADP stabilizes both components of nitrogenase from Klebsiella pneumoniae against urea denaturation. AB - The nitrogenase enzyme of Klebsiella pneumoniae consists of two separable proteins, each with multiple subunits and one or more oxygen sensitive metallocenters. The wild-type nitrogenase proteins are stable to electrophoresis in high concentrations of urea under anaerobic conditions. Addition of Mg+2 and ADP greatly increases the stability of the smaller Fe protein (from <4 to >6 M for full unfolding), an effect directly analogous to stabilization in p21ras induced by Mg+2 and GDP. Stabilization by Mg+2 is slight for the holo MoFe protein (from approximately 1.5 to approximately 2.4 M) but more dramatic for the apo protein form of the MoFe protein accumulated by certain Fe protein (nifH gene) mutants. The potent product inhibitor of nitrogenase function, MgADP, increases stability of the MoFe protein more than Mg+2 alone, to approximately 3.6 M, showing that nucleotides interact with the MoFe protein. Mutations of the nifM gene result in slower accumulation of less stable Fe protein, indicating that NifM is involved in correct folding of the Fe protein. Mutationally altered proteins are often difficult to purify for study because of their inherent instability, low expression level, or oxygen lability. Crude extracts of 11 different mutants of Fe protein (nifH gene) were examined by transverse urea gradient gels to rapidly screen for stabilizing interactions in the presence or absence of substrate or inhibitor analogs. Amino acid alterations D44N and R188C, at the interface of the dimer, in the vicinity of the nucleotide binding site(s), have significantly lower stability than the wild-type enzyme in the absence of Mg+2 but comparable stability in its presence, showing the importance of Mg+2 in the subunit interactions. Mutations N163S and E266K, in which residues normally involved in hydrogen bonding far from the active site were altered, are more labile than the wild-type even with Mg+2 added. Seven other mutants, though nonfunctional, did not appear altered in stability compared to the wild-type. PMID- 10739255 TI - Antibody-detected folding: kinetics of surface epitope formation are distinct from other folding phases. AB - The rate of macromolecular surface formation in yeast iso-2 cytochrome c and its site-specific mutant, N52I iso-2, has been studied using a monoclonal antibody that recognizes a tertiary epitope including K58 and H39. The results indicate that epitope refolding occurs after fast folding but prior to slow folding, in contrast to horse cytochrome c where surface formation occurs early. The antibody detected (ad) kinetic phase accompanying epitope formation has k(ad) = 0.2 s(-1) and is approximately 40-fold slower than the fastest detectable event in the folding of yeast iso-2 cytochrome c (k2f approximately 8 s(-1)), but occurs prior to the absorbance- and fluorescence-detected slow folding steps (k1a approximately 0.06 s(-1); k1b approximately 0.09 s(-1)). N5I iso-2 cytochrome c exhibits similar kinetic behavior with respect to epitope formation. A detailed dissection of the mechanistic differences between the folding pathways of horse and yeast cytochromes c identifies possible reasons for the slow surface formation in the latter. Our results suggest that non-native ligation involving H33 or H39 during refolding may slow down the formation of the tertiary epitope in iso-2 cytochrome c. This study illustrates that surface formation can be coupled to early events in protein folding. Thus, the rate of macromolecular surface formation is fine tuned by the residues that make up the surface and the interactions they entertain during refolding. PMID- 10739256 TI - Anti-HIV and anti-tumor protein MAP30, a 30 kDa single-strand type-I RIP, shares similar secondary structure and beta-sheet topology with the A chain of ricin, a type-II RIP. AB - MAP30 is a 30 kDa single-stranded, type-I ribosome inactivating protein (RIP) possessing anti-tumor and anti-HIV activities. It binds both ribosomal RNA and the HIV-1 long-terminal repeat DNA. To understand the structural basis for MAP30 activities, we undertook the study of MAP30 by solution NMR spectroscopy. We report nearly complete 1H, 13C, and 15N chemical shift assignments of its 263 amino acids. Based upon an analysis of secondary 13C chemical shifts, 3J(HNHA) coupling constants, hydrogen exchange data, and nuclear Overhauser effect patterns, we find that the secondary structure and beta-sheet topology of MAP30 are very similar to those of the ricin A chain, a subunit of the well-known type II RIP, even though two proteins display distinct activities. We therefore suggest that MAP30 and ricin A chain share a similar three-dimensional fold, and that the reported functional differences between two proteins arise primarily from differences in local three-dimensional structure and other structural properties such as surface electrostatic potentials. PMID- 10739257 TI - Apoflavodoxin (un)folding followed at the residue level by NMR. AB - The denaturant-induced (un)folding of apoflavodoxin from Azotobacter vinelandii has been followed at the residue level by NMR spectroscopy. NH groups of 21 residues of the protein could be followed in a series of 1H-15N heteronuclear single-quantum coherence spectra recorded at increasing concentrations of guanidinium hydrochloride despite the formation of protein aggregate. These NH groups are distributed throughout the whole apoflavodoxin structure. The midpoints of unfolding determined by NMR coincide with the one obtained by fluorescence emission spectroscopy. Both techniques give rise to unfolding curves with transition zones at significantly lower denaturant concentrations than the one obtained by circular dichroism spectroscopy. The NMR (un)folding data support a mechanism for apoflavodoxin folding in which a relatively stable intermediate is involved. Native apoflavodoxin is shown to cooperatively unfold to a molten globule-like state with extremely broadened NMR resonances. This initial unfolding step is slow on the NMR chemical shift timescale. The subsequent unfolding of the molten globule is faster on the NMR chemical shift timescale and the limited appearance of 1H-15N HSQC cross peaks of unfolded apoflavodoxin in the denaturant range studied indicates that it is noncooperative. PMID- 10739258 TI - A step toward the prediction of the fluorescence lifetimes of tryptophan residues in proteins based on structural and spectral data. AB - A method is presented that allows the calculation of the lifetimes of tryptophan residues on the basis of spectral and structural data. It is applied to two different proteins. The calcium binding protein from the sarcoplasm of the muscles of the sand worm Nereis diversicolor (NSCP) changes its conformation upon binding of Ca2+ or Mg2+. NSCP contains three tryptophan residues at position 4, 57, and 170, respectively. The fluorescence lifetimes of W57 are investigated in a mutant in which W4 and W170 have been replaced. The time resolved fluorescence properties of W57 are linked to its different microconformations, which were determined by a molecular dynamics simulation map. Together with the determination of the radiative rate constant from the wavelength of maximum intensity of the decay associated spectra, it was possible to determine an exponential relation between the nonradiative rate constant and the distance between the indole CE3 atom and the carbonyl carbon of the peptide bond reflecting a mechanism of electron transfer as the main determinant of the value for the nonradiative rate constant. This result allows the calculation of the fluorescence lifetimes from the protein structure and the spectra. This method was further tested for the tryptophan of Ha-ras p21 (W32) and for W43 of the Tet repressor, which resulted in acceptable values for the predicted lifetimes. PMID- 10739259 TI - Phosphorylation and subcellular redistribution of high mobility group proteins 14 and 17, analyzed by mass spectrometry. AB - High mobility group (HMG) proteins 14 and 17 are nonhistone nuclear proteins that have been implicated in control of transcription and chromatin structure. To examine the posttranslational modifications of HMG-14 and -17 in vivo, HMG proteins were prepared from nuclear vs. cytosolic fractions of human K562 cells treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) or okadaic acid (OA) and examined by electrospray mass spectrometry. Analysis of full-length masses demonstrated mono-, di-, and triphosphorylation of HMG-14 and mono- and diphosphorylation of HMG-17 from OA treated cells, whereas HMG-14 and -17 from TPA treated cells were monophosphorylated. Peptide mass and sequence analysis showed major and minor phosphorylation sites, respectively, at Ser24 and Ser28 in HMG-17, and Ser20 and Ser24 in HMG-14. These sites were found in the consensus sequence RRSARLSAK, within the nucleosomal binding domain of each protein. A third phosphorylation site in HMG-14 was located at either Ser6 or Ser7. Interestingly, the proportion of HMG-14 and -17 found in cytosolic pools increased significantly after 1 h of treatment compared to control cells and showed preferential phosphorylation compared with proteins from nuclear fractions. These results suggest that phosphorylation of HMG-14 and -7 interferes with nuclear localization mechanisms in a manner favoring release from nuclei. PMID- 10739260 TI - Common EF-hand motifs in cholinesterases and neuroligins suggest a role for Ca2+ binding in cell surface associations. AB - Comparisons of protein sequence via cyclic training of Hidden Markov Models (HMMs) in conjunction with alignments of three-dimensional structure, using the Combinatorial Extension (CE) algorithm, reveal two putative EF-hand metal binding domains in acetylcholinesterase. Based on sequence similarity, putative EF-hands are also predicted for the neuroligin family of cell surface proteins. These predictions are supported by experimental evidence. In the acetylcholinesterase crystal structure from Torpedo californica, the first putative EF-hand region binds the Zn2+ found in the heavy metal replacement structure. Further, the interaction of neuroligin 1 with its cognate receptor neurexin depends on Ca2+. Thus, members of the alpha,beta hydrolase fold family of proteins contain potential Ca2+ binding sites, which in some family members may be critical for heterologous cell associations. PMID- 10739262 TI - Kinetic evidence of an on-pathway intermediate in the folding of lysozyme. AB - By means of a kinetic test, it was demonstrated that one of the folding intermediates (Ialpha) of hen lysozyme with alpha-domain folded and beta-domain unfolded is on the folding pathway under the classical definition. Ialpha folds to the native (N) state directly (unfolded (U) <==> Ialpha <==> N) without having to unfold to U and then refold to N through alternative folding pathways as in Ialpha <==> U <==> N. PMID- 10739261 TI - Amide proton hydrogen exchange rates for sperm whale myoglobin obtained from 15N 1H NMR spectra. AB - The hydrogen exchange behavior of exchangeable protons in proteins can provide important information for understanding the principles of protein structure and function. The positions and exchange rates of the slowly-exchanging amide protons in sperm whale myoglobin have been mapped using 15N-1H NMR spectroscopy. The slowest-exchanging amide protons are those that are hydrogen bonded in the longest helices, including members of the B, E, and H helices. Significant protection factors were observed also in the A, C, and G helices, and for a few residues in the D and F helices. Knowledge of the identity of slowly-exchanging amide protons forms the basis for the extensive quench-flow kinetic folding experiments that have been performed for myoglobin, and gives insights into the tertiary interactions and dynamics in the protein. PMID- 10739263 TI - Expectations from structural genomics. AB - Structural genomics projects aim to provide an experimental structure or a good model for every protein in all completed genomes. Most of the experimental work for these projects will be directed toward proteins whose fold cannot be readily recognized by simple sequence comparison with proteins of known structure. Based on the history of proteins classified in the SCOP structure database, we expect that only about a quarter of the early structural genomics targets will have a new fold. Among the remaining ones, about half are likely to be evolutionarily related to proteins of known structure, even though the homology could not be readily detected by sequence analysis. PMID- 10739264 TI - The pi-helix translates structure into function. AB - A search for the occurrence of the rare pi-helix was performed with Iditis from the Oxford Molecular Group upon the Protein Data Bank. In 8 of the 10 confirmed crystal structures that harbor the pi-helix, its unique conformation has been linked directly to the formation or stabilization of a specific binding site within the protein. In the discussion to follow, the role for each of these eight pi-helices will be addressed in regard to protein function. It is clear upon closer examination that the conformation of the pi-helix has evolved to provide unique structural features within a variety of proteins. PMID- 10739265 TI - Physical activity and the prevention of type 2 diabetes mellitus: how much for how long? AB - From observational studies to clinical trials in a variety of populations and age groups, evidence is mounting in support of the hypothesis that physical activity plays a significant role in the prevention of type 2 diabetes mellitus. Based on the current findings, it is likely that physical activity can reduce the risk of diabetes mellitus development. What is less clear is how much physical activity is necessary, and for how long. Obviously, we are more likely to see the anticipated physiological changes if we maximise the dose. Yet, although maximal is better from a physiological point of view, we 'in the trenches' have also recognised the fact that a sedentary individual will most likely not continue to undergo a high intensity activity exercise regimen. In contrast, evidence is mounting regarding long term compliance to moderate levels of activity, which appear to be easier to adopt in one's lifestyle and are less likely to result in injury. More importantly, there appear to be beneficial changes in insulin sensitivity and glucose tolerance in the sedentary individual who incorporates moderate levels of activity such as walking into the their lifestyle, although the onset of beneficial metabolic changes appear to occur much more slowly and less dramatically than what occurs with a high intensity regimen. Even if activity is shown to be beneficial, we are faced with the challenge of reaching the sedentary individuals who would most likely benefit from an increase in physical activity in the first place. This task is a difficult one because of the difficulty in quantifying the sum total of an entire day's worth of movement, rather than that of a few relatively higher intensity leisure activities. What is needed is a measure or combination of measures of physical activity that are simple to use, relatively inexpensive, and adequately capture the subtle changes in physical activity through the day that we are encouraging. It is unlikely that the protective nature of a physically active lifestyle in preventing diabetes mellitus will have a lasting impact once a switch to a sedentary way of life is made. Therefore, from a public health viewpoint, long term commitments to increased activity are required. This is the ultimate challenge. PMID- 10739266 TI - Whole body fatigue and critical power: a physiological interpretation. AB - Critical power (CP) is a fundamental concept describing fatigue and exhaustion. The main physiological determinant of CP is the ability to utilise oxygen. This in turn is dependent primarily on diffusion distance. During exercise, many different tissue systems must increase their metabolic demand. It is argued that each tissue system, such as cardiac, respiratory and leg muscles, has their own CP. Cardiac muscle has the greatest CP relative to its maximum power because it has the shortest diffusion distances. Respiratory muscle also has a substantially higher relative CP than leg muscle. The higher relative CPs of cardiac and respiratory muscle are due in part to the homeostatic functions these tissues provide. This built in protective design can be disrupted in certain conditions such as hypoxia. During high intensity exercise, fatigue and ensuing exhaustion will occur if any contributing physiological system functions above its CP. PMID- 10739267 TI - Physical activity and mental health: current concepts. AB - Physical activity may play an important role in the management of mild-to moderate mental health diseases, especially depression and anxiety. Although people with depression tend to be less physically active than non-depressed individuals, increased aerobic exercise or strength training has been shown to reduce depressive symptoms significantly. However, habitual physical activity has not been shown to prevent the onset of depression. Anxiety symptoms and panic disorder also improve with regular exercise, and beneficial effects appear to equal meditation or relaxation. In general, acute anxiety responds better to exercise than chronic anxiety. Studies of older adults and adolescents with depression or anxiety have been limited, but physical activity appears beneficial to these populations as well. Excessive physical activity may lead to overtraining and generate psychological symptoms that mimic depression. Several differing psychological and physiological mechanisms have been proposed to explain the effect of physical activity on mental health disorders. Well controlled studies are needed to clarify the mental health benefits of exercise among various populations and to address directly processes underlying the benefits of exercise on mental health. PMID- 10739269 TI - Infectious diseases in rugby players: incidence, treatment and prevention. AB - Participation in rugby football can expose individuals to a variety of infectious diseases both on and off the field of play. The close physical contact and trauma inherent in playing rugby facilitates the transmission of viral, bacterial and fungal pathogens between players and may also lead to the acquisition of potentially lethal infections from the environment, such as tetanus. In the past few years there have been a number of reported outbreaks of infection amongst rugby players in the medical literature. The appearance of HIV infection has focused attention on the potential for transmission of this and other blood-borne viruses such as hepatitis B and C viruses from bleeding wounds sustained on the rugby field. As a result, various expert bodies have produced guidelines on the management of players with bleeding wounds. Opportunities are now available to rugby players to play outside their own countries, including the third world. This can bring them into contact with a wide range of travel-associated infections, some of which may be life threatening. In view of the above it is clear that rugby players and those who coach and manage rugby teams require information and education on the subject of infection and its prevention, as well as access to appropriate medical care and expertise. Many of the infections seen in rugby players are preventable, e.g. by promoting hygienic facilities and conduct in changing rooms and on the field of play, by exclusion of infected players from contact with others and, in some cases, by immunisation or chemoprophylaxis. Players who present with infections should be assessed, correctly diagnosed (using laboratory investigations where appropriate) and treated, and measures should be taken to prevent spread to team-mates and other contacts while respecting the confidentiality of the individual. Any outbreaks of infection should be reported to the appropriate authorities. There is evidence to suggest that strenuous physical exercise such as playing rugby can make individuals susceptible to certain types of infection and prolong time to recovery. More information is required on the true frequency and effects of infection in rugby players. PMID- 10739270 TI - A new order of things: the future of academics in pediatric orthopaedics in the 21st century. PMID- 10739268 TI - The effectiveness of commercially available sports drinks. AB - The purpose of this review is to evaluate the effectiveness of commercially available sports drinks by answering the questions: (i) will consuming a sports drink be beneficial to performance? and (ii) do different sports drinks vary in their effectiveness? To answer these questions we have considered the composition of commercially available sports drinks, examined the rationale for using them, and critically reviewed the vast number of studies that have investigated the effectiveness of sports drinks on performance. The focus is on the drinks that contain low carbohydrate concentrations (<10%) and are marketed for general consumption before and during exercise rather than those with carbohydrate concentrations >10%, which are intended for carbohydrate loading. Our conclusions are 3-fold. First, because of variations in drink composition and research design, much of the sports drinks research from the past cannot be applied directly to the effectiveness of currently available sports drinks. Secondly, in studies where a practical protocol has been used along with a currently available sports beverage, there is evidence to suggest that consuming a sports drinks will improve performance compared with consuming a placebo beverage. Finally, there is little evidence that any one sports drink is superior to any of the other beverages on the market. PMID- 10739271 TI - Growth patterns after lengthening of congenitally short lower limbs in young children. AB - The purpose of this study was to assess growth patterns after lengthening of the congenitally short femur or tibia in children younger than 6 years. Twenty such children underwent 28 bone segment lengthenings (13 femora and 15 tibiae) by distraction osteogenesis. Our results show that femoral lengthening in children younger than 6 years does not lead to growth inhibition, whereas isolated femoral lengthening may be associated with growth stimulation. Isolated tibial lengthening in children younger than 6 years does not lead to growth inhibition, whereas simultaneous femoral and tibial lengthening or two tibial lengthenings in close succession can lead to tibial growth inhibition. PMID- 10739272 TI - The effect of increased distraction frequency on soft tissues during limb lengthening in an animal model. AB - The purpose of this study was to compare the effect of distraction frequency on soft tissues. Ten rabbits underwent high-frequency, automated distraction at 1,440 increments per day, whereas five rabbits were manually distracted at a low frequency of three increments per day. The tibias were lengthened approximately 20% at a rate of 1.05 mm/day. The results indicate that all animals had decreased range of motion of the ankle; however, the high-frequency group fared better compared with the low-frequency group (p<0.01). In addition, the P1 latency of the somatosensory evoked potentials did not change in the high-frequency group, but was statistically higher for the low-frequency group (p<0.05). There was no significant difference found in the histologic studies of muscle of either group. Thus increasing the distraction frequency resulted in better range of motion of the ankle joint and maintained normal somatosensory evoked potentials. PMID- 10739273 TI - Attempted limb lengthenings beyond twenty percent of the initial bone length: results and complications. AB - In response to historic guidelines suggesting limits to the amount of limb lengthening, we report the results and complications of those patients in whom the initial goal of lengthening exceeded 20% of the initial segment length. Thirty-one patients underwent a total of 35 attempted lengthenings with a mean follow-up of 38.6 months. Limb-length discrepancy was the primary indication in all but one case. With an average goal of 35% of the original bone length, we achieved a mean gain of 33%. Lengthening to within 2.5 cm was achieved in 31 (89%) of 35 cases, and significantly more successful with goals extending < or =55% of the initial bone length (p<0.05). Treatment times extended a mean of 8.7 months with a healing index of 37 days/cm. By Paley's classification scheme, all had problems, in addition to an average of 1.3 obstacles and 0.9 complications per segment lengthened. In 23 patients with extended follow-up, good to excellent results were achieved in 78%, and were significantly more successful with goals < or =55% (< or =0.05). PMID- 10739274 TI - Ilizarov lengthening in centralized fibula. AB - Tibial hemimelia often produces major limb length problems (1,6,9,15) as well as foot deformity. The decision to perform reconstructive surgery depends on the expected leg-length discrepancy, the anomalies of the foot, and the status of the knee (4,6,8,15). Congenital bone deficiencies usually have a constant rate of growth inhibition (8), and leg lengthening is often associated with more complications (5,13). The complication rate is also increased with the increased leg-length discrepancy (5). In tibial hemimelia with functioning quadriceps (types I and II) and a functional foot, centralization of the fibula onto the talus and synostosis with the proximal tibia is an accepted reconstructive procedure (1,4,6,7,9,15). However, when the transplanted fibula produces a functional limb for the patient, the correction of leg-length inequality would be a challenge. This is a report of such a case. PMID- 10739275 TI - Pin site care during external fixation in children: results of a nihilistic approach. AB - We prospectively followed 27 consecutive children with tibial circular external fixators applied between July 1, 1995, and June 30, 1997. A simple pin care system with no physical pin cleansing except that provided by daily showers was used. Children with inflamed or infected pin sites were placed on an oral antibiotic (cephalexin) for 10 days. Pin sites were graded according to the system of Dahl et al. on a 0 to 5 scale. A total of 4,473 observations was made. Patients developed 178 pin tract infections (4.0% per observation), with 151 (85%) grade 1 and 27 (15%) grade 2 infections. No pin was removed because of infection. Diaphyseal half pin sites were less commonly infected (1.6%) than periarticular wire or half pin sites (4.5%). We recommend only showering without other physical pin cleaning procedures in children undergoing external fixation procedures. PMID- 10739276 TI - Postaxial hypoplasia of the lower extremity. AB - Congenital deficiency of the fibula may present a variety of patterns. In a series of 20 patients with this diagnosis, we have observed limb-length inequality and a spectrum of musculoskeletal anomalies involving the ipsilateral hip, femur, knee, tibia/fibula, ankle, and foot. Considering the frequently associated abnormalities of the lower extremity, the term postaxial hypoplasia may be more descriptive than the traditional terms fibular hemimelia or fibular a/hypoplasia. By raising the awareness of associated deformities, the clinician is better prepared to advise patients and to intercede accordingly. Based on our experience, we advocate a modular treatment approach combining limb lengthening with hemiepiphysiodesis of the distal femur and/or ankle to correct valgus alignment and establish a neutral mechanical axis. Contralateral epiphysiodesis as an adjunct may be preferable to double or repeated lengthening. The goal is to achieve symmetry and stable joints at skeletal maturity with a minimal number of well-timed surgical interventions. By using this strategy and with minimal morbidity, 10 of our patients who have reached skeletal maturity have achieved a satisfactory outcome. PMID- 10739277 TI - Fracture separation of the distal humeral epiphysis in children younger than three years old. AB - This study evaluated the change of carrying angle and the causes of cubitus varus after the fracture separation of the distal humeral epiphysis in young children. Twelve cases of fracture separation of distal humeral epiphysis were treated from January 1995 to July 1997. The age of all patients was younger than 3 years old. A metaphyseal fragment was seen in all cases (Salter-Harris type II), but the size of the metaphyseal fragment was either a very small flake or a large Thurston-Holland fragment. Posteromedial displacement was seen in all cases. There were three treatment groups: closed reduction with percutaneous pinning, closed reduction with cast, and cast without reduction. Follow-up period averaged 23.5 months (range, 12-40). Cubitus varus deformity was seen in seven cases, and six of them had a partial defect of the medial condyle that was avascular necrosis. The methods of treatment, age of injury, and the type of epiphyseal injury had no influence on the development of cubitus varus, but avascular necrosis of the medial condyle was related to the cubitus varus deformity (p<0.05). The results of this study suggest that fracture separation of distal humeral epiphysis in young children is likely to produce cubitus varus deformity with the development of avascular necrosis of the medial humeral condyle. PMID- 10739278 TI - The Ilizarov fixator for pediatric and adolescent supracondylar fracture variants. AB - Most displaced pediatric and adolescent supracondylar elbow fractures can be treated with closed reduction and percutaneous pinning (CRPP). The Ilizarov method provides a minimally invasive technique for obtaining and maintaining a reduction for supracondylar variants when CRPP fails. This study is a retrospective review of 13 patients who underwent treatment of acute atypical supracondylar fractures with the Ilizarov fixator. Each fracture exhibited one or more of the following indications for the Ilizarov method: inability to obtain reduction due to anterior displacement of the distal humerus, inability to obtain reduction due to the interval since the injury, comminution, and intercondylar extension of the fracture. Ages ranged from 4 to 16 years. Time in the fixator ranged from 29 to 61 days. There were no permanent nerve injuries, deep infections, broken implants, or heterotopic ossification. All healed with a satisfactory carrying angle and range of motion. PMID- 10739279 TI - Open femur fractures in children: treatment, complications, and results. AB - We evaluated retrospectively the treatment of 44 open femur fractures occurring between the lesser trochanter and the distal femoral physis in 43 children aged 16 years and younger. Fractures that involved the physis or that were a consequence of gunshot wounds were excluded. There were 25 grade I, 9 grade II, and 9 grade III fractures. The mean age at injury was 9.5 years. Ninety percent of the fractures were automobile related. More than 70% of the children had associated injuries. The average time to healing for all fractures in this study was 17 weeks. Our data indicate that there is a statistically significant increased time to heal with increasing age of the child (p = 0.04). Additionally, grade III fractures healed more slowly than grade I or II fractures (p = 0.0006). Fractures treated with external fixation took longer to unite than those treated with other methods (p = 0.05). The presence of complications increased the time to fracture union (p = 0.00001). Grade III injuries were the most difficult to manage; 50% of the fractures in this group developed osteomyelitis and 20% malunited. In contrast, none of the fractures in either the grade I or II groups developed deep infection. After aggressive debridement, grade I and grade II fractures may be stabilized with age-appropriate fixation methods. Grade III injuries should be managed with vigorous debridement and vigilance, as these injuries are prone to deep infection and malunion. The optimal method of skeletal stabilization in grade III fractures remains unresolved. PMID- 10739280 TI - Early spica treatment of pediatric femoral shaft fractures. AB - The purpose of this prospective study was to document results of early spica casting in treating all children aged 10 years or younger presenting during a 30 month period with a femoral shaft fracture. The outcome of 101 femoral shaft fractures thus treated demonstrated excellent results with few complications. Four spicas had to be removed at 7 to 10 days for unacceptable shortening and an additional four probably should have been. Control of alignment was not a problem, with only one patient's femur on cast removal being in a position different from that accepted at 7 to 10 days. An age older than 7 to 8 years is the only variable that might be used to predict the need for a change in treatment at 7 to 10 days. PMID- 10739281 TI - Premature physeal closure after tibial diaphyseal fractures in adolescents. AB - Seven cases of premature physeal closure secondary to diaphyseal fractures of the tibia in adolescents between 12 and 15 years of age are presented. At the time of the accidents, there was no evidence of physeal lesion in any of the patients. After 4 to 13 months of follow-up (mean, 9 months), early closure was observed in the radiologic controls of one or more physes of the affected leg: distal femoral and proximal and distal tibial in three cases, isolated distal femoral physis in three cases, and both tibial physes without femoral damage in one case. Physeal closure was always central, and there was no case of angular deformity. After 15 to 42 months of follow-up (mean, 27 months), all patients had a leg-length discrepancy in the 8- to 30-mm range (mean, 18 mm). Only one patient required surgical correction (proximal epiphysiodesis of the contralateral tibia followed by tibial lengthening). Adolescents with diaphyseal fractures of the long bones should be monitored until they have stopped growing because of the risk of developing leg-length discrepancy as a consequence of premature closure of one or more leg physis. PMID- 10739282 TI - Fracture patterns in children and young adults who fall from significant heights. AB - Trauma is a leading cause of morbidity and mortality for children and young adults. When all causes of trauma are considered, falls are the most common mechanism of injury. To address specifically age-related fracture patterns in children who fall, we identified 125 consecutive patients, 21 years old or younger, who fell from a height of 10 feet or greater. The medical records and radiographs for 110 of these patients were available for review. Patients were divided into three groups based on age: there were 25 infant/toddlers (0-2 years), 55 children (3-10 years), and 30 adolescent/young adults (11-21 years). We found statistically significant differences in fracture distribution between the groups. The adolescent/young adult group sustained a greater number of vertebral fractures (p<0.003) and total fractures per fall (p<0.015). The children, in contrast, had a greater number of long bone fractures (p<0.05). Knowledge of age-related fracture patterns could result in improved diagnosis and treatment of these injuries. PMID- 10739283 TI - Scoring of pediatric orthopaedic polytrauma: correlations of different injury scoring systems and prognosis for hospital course. AB - Trauma scoring systems were compared among themselves and to recovery variables in a cohort of 91 pediatric polytrauma patients with orthopaedic injuries. They included the Trauma Score (TS), Revised Trauma Score (RTS), Injury Severity Score (ISS), Modified Abbreviated Injury Severity Scale (MISS), Pediatric Trauma Score (PTS), and TRISS-b survival statistic. Significant correlations between scoring systems and hospital course parameters existed. TRISS-b had the strongest correlation for days in the intensive care unit and total complications (r2 = 0.59, 0.58). PTS correlated poorly with recovery variables when compared to other scoring systems. The TS correlated most strongly with ventilatory days and complications of immobilization (r2 = 0.77, 0.58). The TS should be used early in the assessment of the pediatric polytrauma patient, along with the TRISS-b statistic. If they predict prolonged intensive care unit and ventilatory days and hospital complications, operative fracture management should be strongly considered. PMID- 10739284 TI - The efficacy of tone-reducing features in orthotics on the gait of children with spastic diplegic cerebral palsy. AB - This study analyzed the effects of tone-reducing features in ankle-foot orthotics (AFOs) on the gait of eight children (ages 4-11 years) with spastic diplegic cerebral palsy. A standard gait analysis was performed on each subject in each of three trial orthotics and in a baseline shoes-only condition. A 4-week accommodation period was allotted for each of the three devices: a standard hinged AFO, an AFO with tone-reducing features, and a supramalleolar orthotic with tone-reducing features. Most significant differences were at the ankle, between free-ankle and plantar flexion-limiting conditions. No significant functional changes in gait were evident with the addition of tone-reducing properties to a standard articulating AFO. PMID- 10739285 TI - Evaluation of rotational gait abnormality in the patients cerebral palsy. AB - Internal rotation of hip is commonly seen in children with cerebral palsy. Existing muscle imbalance causes persistence of femoral deformity, which may contribute to rotational asymmetry. In cerebral palsy, gait deviations are the result of dynamic and static components, both caused by muscle imbalance. In this study we investigated the predictability of hip rotation in gait from the measurement of anatomic deformity. Computed tomography (CT) measurements of femoral anteversion and physical examination data failed to predict the hip rotation in gait. However, tibial (CT) measurements and physical examination data highly correlated with tibial rotation in gait. We conclude the dynamic component of hip rotation during gait is significant, as anatomic deformity did not predict gait deviations. PMID- 10739286 TI - Serial casting in idiopathic toe-walkers and children with spastic cerebral palsy. AB - Serial casting to stretch the plantar-flexors has been advocated for idiopathic toe-walkers (ITW) and children with spastic cerebral palsy (CP), although outcomes have not been well studied. Neuromuscular function and gait were examined in eight children with CP (mean age, 7.1 years) and eight ITW (mean age, 7.5 years) casted for 3 to 6 weeks. Baseline comparisons indicated that children with CP produced lower isometric plantar-flexor torques (p<0.02) concomitant with marked co-contraction (p<0.001). greater ankle mobility (p<0.02), and higher reflex excitability (p<0.001) than ITW. After casting, both groups increased dorsiflexion range (p<0.001), decreased resistance to passive stretch (p<0.005), and produced maximal plantar-flexor torques in dorsiflexed positions (p<0.001). Reflex excitability was reduced in CP (p<0.05). Immediately postcasting, no children toe-walked, but two with CP resumed a digitigrade pattern 6 weeks later. Gait velocity and stride length did not change (p>0.05). Serial casting yielded positive outcomes that may be longer lasting in ITW. PMID- 10739287 TI - Surgical treatment of severe hindfoot valgus by medial displacement osteotomy of the os calcis in children with myelomeningocele. AB - This is a retrospective review of 27 patients with severe hindfoot valgus who were treated with a medial sliding osteotomy of the os calcis. The group consisted of 38 feet in 27 patients with myelomeningocele, ranging in age from 7 to 17 years. Follow-up averaged >5 years. The amount of hindfoot deformity, problems with brace and shoe wear, and overall patient satisfaction were analyzed. Eighty-two percent of the patients were completely satisfied. Most had complete correction of the hindfoot deformity. There was one superficial pin site infection and no nonunions. PMID- 10739288 TI - Accurate determination of cast weight for neonates with clubfoot. AB - The purpose of this study was to determine changes in cast weight during the first 48 hours after application so that the true weight of a neonate can be estimated without the need for removing the cast. Five types of cast materials were compared. Cast weight measurements were obtained before and after application and at intervals during 48 hours. Final cast weight averaged 107.5% of dry weight for plaster and 99% of dry weight for synthetic cast materials. For very low birth weight infants, the difference between the initial wet weight of plaster of Paris and its final dry weight may be significant for calculating drug and fluid dosages. The weight of an infant can now be calculated without the necessity of cast removal. PMID- 10739289 TI - Coxa pedis dysplasia in congenital convex pes valgus. AB - A dissection was carried out on bilateral congenital convex pes valgus in a newborn who died of congenital visceral malformations. The main finding was a modification in the neck-body relations of the talus. Nearly all muscles showed morphologic alterations. No insertional or histologic anomalies were shown. The plantar calcaneonavicular ligament was stretched and attenuated. The alterations observed in our case affected all the anatomic structures forming and stabilizing the coxa pedis. Our case suggests that the dysplastic evolution of the talar body neck angle halts, and the neck assumes an adducted, medially inclined and plantar tilted angle with respect to the talar body. We propose the term "congenital dysplasia of the coxa pedis" to describe this condition, because it emphasizes the primary alteration in the morphogenesis of the talocalcaneonavicular joint. PMID- 10739290 TI - Resistant talipes equinovarus associated with congenital constriction band syndrome. AB - Thirty-seven clubfeet in 28 patients with concomitant congenital constriction band syndrome were reviewed. The bands were considered to be of significance if located in the calf region (zone 2). The severity of the bands was classified. Grade 1 bands involved subcutaneous tissue, grade 2 bands extended to the fascia, grade 3 bands extended to the fascia and required release, and grade 4 bands were congenital amputations. The patients were divided based on neurologic deficit. Group A consisted of 26 clubfeet without neurologic deficit and had 1.4 surgeries per clubfoot. Group B consisted of 11 clubfeet with neurologic deficit and had 3.7 surgeries per clubfoot. Children with grade 3 bands in zone 2 were most likely to have a neurologic deficit. Twenty-three clubfeet had a good clinical result, seven clubfeet fair, and seven clubfeet poor. Group B had poorer results than Group A. These feet ultimately required numerous and more extensive surgeries to obtain correction, and ultimately had a poorer result. PMID- 10739291 TI - Clinical and radiographic analysis of osteochondromas and growth disturbance in hereditary multiple exostoses. AB - Hereditary multiple exostoses (HME) is traditionally described as a skeletal dysplasia. However, the discovery that the EXT family of tumour suppressor genes are responsible for HME suggests that it is more appropriate to classify HME as a familial neoplastic trait. In a clinical and radiographic analysis of paired bone length and exostoses number and dimensions in a HME cohort, the local presence of osteochondromas was consistently associated with growth disturbance. In particular, an inverse correlation between osteochondroma size and relative bone length (p<0.01) was found. These data suggest that the growth retardation in HME may result from the local effects of enlarging osteochondromas rather than a skeletal dysplasia effect. This study provides the first clinical rationale for ablation of rapidly enlarging exostoses to reduce growth disturbance. PMID- 10739292 TI - The risk of carcinogenesis from radiographs to pediatric orthopaedic patients. AB - This study set out to determine whether cumulative radiograph exposure of children significantly increases their risk of radiation-induced carcinogenesis or hereditary defects. Records of children treated for idiopathic scoliosis, hip dysplasia, or leg-length discrepancy between 1980 and 1993 at the Shriners Hospital in Spokane, WA, were retrospectively reviewed. Total radiation and organ dose exposures were calculated using information from individual radiology reports. Surgically treated idiopathic scoliosis patients had the largest total radiation skin entrance and organ dose exposures. This group's risks for developing leukemia, breast cancer, or a heritable defect, respectively, were 0.8%, 2.1%, and 3.0% higher than baseline risks. The other treatment groups had increased carcinogenic risks of <1%. The use of serial radiographs during the treatment of idiopathic scoliosis, hip dysplasia, and leg-length discrepancy appears relatively safe. The increased risk of carcinogenesis or hereditary defects in these patients is minimal. PMID- 10739293 TI - Free tissue transfer in the treatment of linear scleroderma. AB - Linear scleroderma is a subtype of localized scleroderma that most commonly affects children. The disease is rare, and the course is unpredictable. Most cases spontaneously resolve in 3-5 years. A small number, however, slowly progress to involve deeper subcutaneous tissues, muscles, and periosteum. Subsequent joint contractures and gross disturbances are frequent. Little on treatment is recorded in the literature. We report on four children who, in addition to bony procedures and release of joint and ligamentous contractures, had excision of the diseased skin and free soft-tissue transfer for coverage. PMID- 10739294 TI - Objective evaluation of knee laxity in children. AB - This study was designed to measure objectively knee laxity in children. Physical examination and the KT 1000 arthrometer were used to test the knee laxity of 150 healthy, uninjured children between 6 and 18 years of age. Data from the knee examinations and the KT 1000 measurements were compared and statistically analyzed to determine the change in knee laxity with age, laxity differences between boys and girls, and the correlation between the KT1000 measurements and subjective tests for laxity described by Carter and Wilkinson. There was no statistical difference in knee laxity between boys and girls of similar ages. We found that knee laxity, determined by measuring the millimeters of tibial translation using the KT 1000 arthrometer, was significantly greater in younger children. PMID- 10739295 TI - The normal width of anterior hip synovial recess in children. AB - To establish the diagnostic criteria for hip joint effusion, the normal width of the anterior hip synovial recess in children and the difference between both hips of the same child were examined in this study. Eight kindergarten schools were randomly selected for study in Kaohsiung City. The width of anterior hip synovial recess of 1,568 hips in 784 children (421 boys, 363 girls) was measured by ultrasound scanner with 7.5-MHz linear probe. The age of children ranged from 2.5 to 6.6 years old. During measurement, the children were put in supine position with hips and knees in extension and patella facing up. The results revealed that the mean width of the anterior synovial recess was 7.29+/-1.15 mm. Linear correlation of the width of synovial recess to age and height was disclosed and expressed as follows: width (millimeters) equal to 6.52+0.013x age (months), or width (millimeters) equal to 3.97+0.030x height (centimeters). The mean difference between both hips of the same child was 0.611+/-0.523 mm, and the difference was independent from the factors of age, gender, height, or weight (p>0.05). Ninety-five percent of the differences should be < or =1.46 mm, so that if the difference between hips is >1.46 mm, hip joint effusion should be suspected. PMID- 10739296 TI - Intramedullary rodding in osteogenesis imperfecta. AB - The results of intramedullary rodding of long bones of 16 children with osteogenesis imperfecta, over a 10-year period, were analyzed. Sheffield elongating rods or non-elongating rods were used. The frequency of fractures was dramatically reduced after implantation of either type of rod, and the ambulatory status improved in all instances. The results were significantly better after Sheffield rodding with regard to the frequency of complications requiring reoperations and the longevity of the rods. Migration of the rods, encountered frequently, appears to be related to improper placement of the rods in the bone. It seems likely that if care is taken to ensure precise placement of a rod of appropriate size, several of these complications may be avoided. PMID- 10739297 TI - Protective influence of ossific nucleus. PMID- 10739298 TI - A neuroendocrine releasing effect of melatonin in the brain of an insect, Periplaneta americana (L.). AB - Melatonin exists in nearly all organisms, but little is known of its function in non-vertebrates. Long-term perifusions as well as short-term batch incubations of brains and molting glands of the cockroach Periplaneta americana were used to test the influence of melatonin on the prothoracicotropic hormone, a glandotropic neuropeptide in the brain, which stimulates the production of the molting hormone ecdysone in the molting gland. Changes of ecdysteroid production in molting glands were determined by radioimmunoassay as ecdysone equivalents. Melatonin (10 nmol/L) was without effect on the prothoracic gland but stimulated the prothoracicotropic effect of brains in both in vitro investigations, long-term perifusions and short-term batch incubations. The effect was dose-dependent. The melatonin effect on the release of prothoracicotropic hormone in the brain was suppressed by luzindole (10 nmol/L), a pre-synaptic receptor antagonist of melatonin. The retro-cerebral complex (corpora cardiaca-corpora allata) did not seem to be involved in the effect of melatonin on the brain. Serotonin (10 nmol/L) suppressed the release of prothoracicotropic hormone. This is the first experimental evidence of a neurohormonal releasing effect of melatonin in the insect nervous system. PMID- 10739299 TI - Melatonin inhibits oxidative modification of low-density lipoprotein particles in normolipidemic post-menopausal women. AB - In this study, we investigated the short-term effect of melatonin on the susceptibility of low-density lipoprotein (LDL) to oxidation in normolipidemic post-menopausal women. Fifteen post-menopausal women received 6.0 mg melatonin daily for 2 wk. Blood samples were obtained before and after the treatment and the plasma levels of total cholesterol, total triglyceride, high-density lipoprotein (HDL)-cholesterol, LDL-cholesterol, LDL-triglyceride, and LDL apolipoprotein B were determined. LDL oxidation was performed by incubation with copper ions and was analyzed by monitoring the kinetics of conjugated diene formation and measuring the concentration of thiobarbituric-acid-reactive substances (TBARS). LDL-apolipoprotein B derivatization was analyzed by measuring trinitrobenzene sulfonic acid (TNBS) reactivity. Melatonin treatment significantly increased the plasma triglyceride levels (P<0.05), but did not significantly alter the plasma levels of total cholesterol, HDL-cholesterol, or LDL-lipids. The kinetics analysis of conjugated diene production revealed that melatonin treatment significantly prolonged the lag time of conjugated diene formation (from 64.71+/-11.89 to 70.15+/-10.52 min, P<0.05). The oxidation rate and the amount of conjugated diene, however, did not change significantly. The TBARS concentration was significantly reduced by melatonin treatment (from 49.31+/-7.57 to 38.69+/-23.90 nM/mg LDL, P<0.05). Furthermore, melatonin treatment significantly reduced the copper-induced decrease of TNBS reactivity (from 79.43+/-6.19 to 86.50+/-9.07% at 1 hr and from 71.03+/-6.74 to 76.31+/ 4.99% at 2 hr, P<0.05). These results indicate that melatonin treatment may reduce LDL susceptibility to oxidative modification in normolipidemic post menopausal women. PMID- 10739301 TI - Serum cholesterol and lipid peroxidation are decreased by melatonin in diet induced hypercholesterolemic rats. AB - The purpose of this study was to investigate the effect of melatonin, at pharmacological doses, on serum lipids of rats fed with a hypercholesterolemic diet. Therefore, different groups of animals were fed with either the regular Sanders Chow diet or a diet enriched in cholesterol. Moreover, animals were treated with or without melatonin in the drinking water for 3 months. We show that melatonin treatment did not affect the levels of cholesterol or triglycerides in rats fed with a regular diet. However, the increase in total cholesterol and low-density lipoprotein (LDL)-cholesterol induced by a cholesterol-enriched diet was reduced significantly by melatonin administration. On the other hand, melatonin administration prevented the decrease in high density lipoprotein (HDL)-cholesterol induced by the same diet. No differences in the levels of very low-density lipoprotein (VLDL)-cholesterol and triglycerides were found. We also found that melatonin administration slightly decreased serum uric, bilirubin and increased serum glucose levels. Other biochemical parameters, including total proteins, creatinine, urea, phosphorus, calcium, glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), gamma glutamyltranspeptidase (gamma-GT), acetyl cholinesterase (AcCho), and alkaline phosphatase (ALP) were not modified by melatonin treatment. Finally, lipid peroxidation (LPO) was studied in membranes of liver, brain, spleen, and heart as an index of membrane oxidative damage. Results show that hypercholesterolemic diet did not modify the LPO status in any of the tissues studied. However, chronic melatonin administration significantly decreased LPO. Results confirm that melatonin participates in the regulation of cholesterol metabolism and in the prevention of oxidative damage to membranes. PMID- 10739300 TI - Protective effect of melatonin against oxidative stress induced by ligature of extra-hepatic biliary duct in rats: comparison with the effect of S-adenosyl-L methionine. AB - In the present research, we studied the effect of the administration of melatonin or S-adenosyl-L-methionine (S-AMe) on oxidative stress and hepatic cholestasis produced by double ligature of the extra-hepatic biliary duct (LBD) in adult male Wistar rats. Hepatic oxidative stress was evaluated by the changes in the amount of lipid peroxides and by the reduced glutathione content (GSH) in lysates of erythrocytes and homogenates of hepatic tissue. The severity of the cholestasis and hepatic injury were determined by the changes in the plasma enzyme activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), g-glutamyl-transpeptidase (GGT), and levels of albumin, total bilirubin (TB) and direct bilirubin (DB). Either melatonin or S-AMe were administered daily 3 days before LBD, and for 10 days after biliary obstruction. LDB caused highly significant increases in plasma enzyme activities and in bilirubin and lipid peroxides levels in erythrocytes and hepatic tissue. At the same time, this procedure produced a notable decrease in the GSH pools in these biological media. Both melatonin and S-AMe administration were effective as antioxidants and hepatoprotective substances, although the protective effects of melatonin were superior; it prevented the GSH decrease and reduced significantly the increases in enzyme activities and lipid peroxidation products produced by biliary ligature. S-AMe did not modify the increased GGT activity nor did it decrease greatly the TB levels (43% melatonin vs. 14% S-AMe). However, S-AMe was effective in preventing the loss of GSH in erythrocytes and hepatic tissue, as was melatonin. The obtained data permit the following conclusions. First, the LDB models cause marked hepatic oxidative stress. Second, the participation of free radicals of oxygen in the pathogenecity and severity of cholestasis produced by the acute obstruction of the extra-hepatic biliary duct is likely. Third, the results confirm the function of S-AMe as an antioxidant and hepatoprotector. Finally, melatonin is far more potent and provides superior protection as compared to S-AMe. Considering the decrease in oxidative stress and the intensity of cholestasis, these findings have interesting clinical implications for melatonin as a possible therapeutic agent in biliary cholestasis and parenchymatous liver injury. PMID- 10739302 TI - Evidence for a melatonin receptor within pancreatic islets of neonate rats: functional, autoradiographic, and molecular investigations. AB - In a recent perifusion investigation, we showed that the pineal secretory product melatonin reduces insulin secretion from isolated pancreatic islets of neonate rats stimulated with potassium chloride (KCl), glucose, and forskolin. This effect of melatonin was reproduced with doses ranging from 200 pmol/L to 5 micromol/L. Because it is generally accepted that melatonin exerts some of its biological effects through specific, high-affinity pertussis-toxin-sensitive G protein-coupled receptors, we blocked the putative melatonin receptor of pancreatic islets using both the non-hydrolyzable guanosine triphosphate analog guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS, 30 micromol/L) and the melatonin antagonist luzindole (10 micromol/L). Both GTPgammaS and luzindole caused a near normalization of the melatonin-induced inhibition of the forskolin-stimulated insulin secretion. To localize putative melatonin receptors within the pancreatic islets autoradiographic studies were additionally carried out. These investigations showed specific binding of 2-[125I]iodomelatonin, which were in exact correspondence with the localization of the islets. In addition, gray-level analysis showed that unlabeled melatonin was able to reduce the binding of 2 [125I]iodomelatonin in a dose-dependent manner. Concentrations of unlabeled melatonin of 10(-9) mol/L produced a 50% reduction in specific binding, whereas concentrations of 10(-6) mol/L displaced the binding completely. Likewise, the results of molecular investigations showed that the rat pancreas contains a melatonin receptor, since reverse transcription polymerase chain reaction (RT PCR) experiments, using specific primers for the rat melatonin receptor Mel1a, showed that mRNA for this melatonin receptor type is expressed in pancreatic tissue of newborn rats. In summary, it may be said that our functional. autoradiographic, and molecular results indicate that the Mel1a receptor is located on the pancreatic islets, possibly in the beta cells. PMID- 10739303 TI - Melatonin receptors in human uveal melanocytes and melanoma cells. AB - Previous work has demonstrated that melatonin inhibits growth of cultured human uveal melanoma cells. The goal of this study was to determine the expression of mRNA encoding the melatonin receptor subtypes and the effect of specific melatonin receptor agonists on cell growth of uveal melanoma cells and melanocytes. RNA expression of the human melatonin Mel1a and Mel1b receptor subtypes was determined by reverse transcription-polymerase chain reaction (RT PCR) amplification of RNA isolated from two melanoma cell lines and from one cell line of normal melanocytes. PCR-amplified cDNA encoding the Mel1b melatonin receptor subtype, but not the Mel1a subtype, was detected in reverse-transcribed RNA obtained from both normal uveal melanocytes and melanoma cell lines. Uveal melanoma cells and melanocytes were cultured for 24 hr, then melatonin or one of its membrane receptor agonists, 6-chloromelatonin (Mel1a-1b) or S-20098 (Mel1b) or its putative nuclear agonist, CGP-52608 (Mel2), was added to the medium. After 5 days, the cells were detached, counted, and compared to untreated controls. Melatonin and its membrane receptor agonists (Mel1a-1b and Mel1b), but not its putative nuclear receptor agonist (Mel2), inhibited the growth of uveal melanoma cells, but not normal melanocytes, at very low concentrations. In uveal melanoma cells, the expression of RNA encoding the Mel1b receptor suggests that the growth inhibiting effect of melatonin on uveal melanoma cells is related to activation of the melatonin Mel1b membrane receptor. Furthermore, the expression of RNA encoding melatonin receptors in normal uveal melanocytes suggests that melatonin may play a role in the function of these cells. PMID- 10739304 TI - Melatonin protects against the free radical-induced impairment of nitric oxide production in the human umbilical artery. AB - We evaluated melatonin's antioxidative effect on the free radical-induced impairment of nitric oxide production in the human umbilical artery, which may play an important role in fetal hypoxia and ischemia during preeclampsia. Umbilical artery sections with intact endothelium were obtained from healthy pregnant women who were delivered between 37 and 40 wk of gestation. The production of nitric oxide in the umbilical arteries was stimulated by adding L arginine followed by incubation for 60 min. Nitric oxide concentrations were estimated by measuring nitrite ions (NO2), using high-performance liquid chromatography. Prior to the addition of L-arginine, the segments were treated with hydrogen peroxide (H2O2) alone (1, 10, 100 microM), or were pretreated with either 50 mM mannitol or melatonin (20, 100, 500 microM) before adding H2O2. Changes in L-arginine-induced NO2 production were expressed as a percentage of NO2 production at the end of preincubation. NO2 production was significantly increased by incubating the umbilical artery sections with L-arginine (P<0.01). Treatment with H2O2 significantly reduced L-arginine-induced NO2-production in a concentration-dependent manner (P<0.01). Pretreatment with melatonin significantly increased NO2 production that had been decreased by H2O2 in a concentration-dependent manner (P<0.01). Similarly, pretreatment with mannitol reversed the H2O2-induced reduction in NO2- production (P<0.001). These results indicate that H2O2 may impair nitric oxide synthesis in the endothelium of human umbilical arteries. Melatonin significantly suppresses the H2O2-induced inhibition effect of nitric oxide production, most likely through its ability to scavenge hydroxyl radicals. PMID- 10739305 TI - Immunohistochemical study of the glutamate transporter proteins GLT-1 and GLAST in rat and gerbil pineal gland. AB - Several investigations performed during this decade have led to the hypothesis that small secretory vesicles of pinealocytes (generally referred to as synaptic like microvesicles, SLMVs) are components of a system for intercellular paracrine communication between pineal cells, which shares many features with the process of synaptic neurotransmission. According to a recent study, one parallel that can be drawn to synaptic signal transduction seems to be the presence of pineal re uptake systems for messenger molecules released from SLMVs, i.e. for neuroactive amino acids such as L-glutamate. In order to further characterize these uptake mechanisms, we have carried out an immunohistochemical study to explore the presence and cellular localization of the glutamate transporters GLT-1 and GLAST in rat and gerbil pineal glands. GLT-1 and GLAST were always detected in a subpopulation of pineal parenchymal cells in both species. Using immunostaining of serial semithin sections with antibodies against marker proteins of pineal cell types, GLT-1- and GLAST-positive cells were identified as interstitial glial cells. In addition, some pinealocytes also displayed immunoreactivity for GLT-1. In contrast to current thinking, our findings show that GLT-1 is not the only glutamate transporter subtype expressed in the pineal gland. Moreover, our observations point to a significant participation of interstitial cells in the process of pineal glutamatergic communication, reminiscent of the role of glial cells during glutamatergic neurotransmission in the central nervous system. PMID- 10739306 TI - Circadian rhythm of mt1 melatonin receptor expression in the suprachiasmatic nucleus of the C3H/HeN mouse. AB - This report studied the diurnal and circadian rhythms of mt1 melatonin receptor expression in the SCN of C3H/HeN mice maintained in either a light:dark (LD) cycle or in constant dark for a minimum of 6 wk. Diurnal times (ZT) were assessed with reference to the onset of the light period (ZT0) and circadian times (CT) were established by determining the phase of wheel running activity of each mouse before sacrifice. 2-[125I]-Iodomelatonin binding in the SCN revealed low amplitude diurnal and circadian rhythms with highest levels of binding 2 hr after lights on (41.3+/-1.7 fmol/mg protein, n = 5, at ZT2) or at the beginning of the subjective day (48.6+/-2.1 fmol/mg protein, n = 6, CT2), respectively. The expression of mt1 mRNA, determined by in situ hybridization with a 35S-labeled mouse mt1 riboprobe, showed robust diurnal and circadian rhythms. In animals housed under a LD cycle, low levels of expression were observed during the day, with a rapid rise in mt1 melatonin receptor expression at the beginning of the dark period (ZT14), coincident with an abrupt increase in levels of circulating melatonin measured by radioimmunoassay. In animals housed under constant dark conditions, a robust peak of mt1 mRNA expression occurred in the middle of the subjective night (CT18), 8 hr before the peak of protein expression, while the lowest levels of mt1 mRNA expression were observed during the day (CTI10). Results suggest that mt1 melatonin receptor rhythm in the C3H/HeN mouse SCN is regulated both by light and by the biological clock as distinct rhythms of both mRNA and protein are differentially expressed under a LD cycle and constant dark conditions. PMID- 10739307 TI - Recognition of heterospecific alarm vocalizations by bonnet macaques (Macaca radiata). AB - Recognition of heterospecific alarm vocalizations is an essential component of antipredator behavior in several prey species. The authors examined the role of learning in the discrimination of heterospecific vocalizations by wild bonnet macaques (Macaca radiata) in southern India The bonnet macaques' flight and scanning responses to playbacks of their own alarm vocalizations were compared with their responses to playbacks of vocalizations of Nilgiri langurs (Trachypithecus johnii), Hanuman langurs (Semnopithecus entellus), and sambar deer (Cervus unicolor). The study was conducted in 3 regions that differed in the frequency with which bonnet macaques encountered these species and included an urban setting. Call recognition was highest in adults and in regions where individuals were frequently exposed to the calling species; calls were not recognized by urban monkeys. Thus, age and experience are important factors in heterospecific call recognition by bonnet macaques. PMID- 10739308 TI - Hand preferences in unimanual and bimanual feeding by wild vervet monkeys (Cercopithecus aethiops). AB - Lateral preference was examined in spontaneous feeding actions in 2 troops of wild vervet monkeys (Cercopithecus aethiops). Processing of 4 foods (termites, leaf shoots, sugarcane, and fruit) was studied. Actions included unimanual reaching to moving objects, operating from an unstable posture, and coordinated bimanual processing. Between 19 and 31 subjects were available, according to the task. In 2 tasks, laterality of 2 independent stages was measured separately, giving 6 measures in all. On 4 of these measures, most monkeys were ambipreferent, and only a few showed significant hand preferences. Only for termite feeding and detaching material from fruits did the majority show significant lateralization; no tasks elicited exclusive use of 1 hand. Preference appeared labile, because in 2 tasks, population trends reversed with increasing age. No population trends to left or right were found; instead, these monkeys showed ambilaterality, with lateralization associated with task complexity. PMID- 10739309 TI - Low-frequency acoustic modulations generated by the high-frequency portion of the cochlea, noninvasively recorded from the scalp of mice (Mus musculus). AB - Vocalizations often contain low-frequency modulations of the envelope of a high frequency sound. The high-frequency portion of the cochlear nerve of mice (Mus musculus) generates a robust phase-locked response to these low-frequency modulations, and it can be easily recorded from the surface of the scalp. The cochlea is most sensitive to envelope modulation frequencies of approximately 500 to 2000 Hz. These responses have detection thresholds that are approximately 10 dB more sensitive than auditory brainstem responses, and they are very sharply tuned. These measurements may provide a nontraumatic means of repeatedly assessing cochlear functions involved in sound localization and perception of vocalizations. PMID- 10739310 TI - Proximate factors mediating "contact" calls in adult female baboons (Papio cynocephalus ursinus) and their infants. AB - "Contact" calls are widespread in social mammals and birds, but the proximate factors that motivate call production and mediate their contact function remain poorly specified. Field study of chacma baboons (Papio cynocephalus ursinus) revealed that contact barks in adult females were motivated by separation both from the group at large and from their dependent infants. A variety of social and ecological factors affect the probability of separation from either one or both. Results of simultaneous observations and a playback experiment indicate that the contact function of calling between mothers and infants was mediated by occasional maternal retrieval rather than coordinated call exchange. Mothers recognized the contact barks of their own infants and often were strongly motivated to locate them. However, mothers did not produce contact barks in reply unless they themselves were at risk of becoming separated from the group. PMID- 10739311 TI - Recognizing facial cues: individual discrimination by chimpanzees (Pan troglodytes) and rhesus monkeys (Macaca mulatta). AB - Faces are one of the most salient classes of stimuli involved in social communication. Three experiments compared face-recognition abilities in chimpanzees (Pan troglodytes) and rhesus monkeys (Macaca mulatta). In the face matching task, the chimpanzees matched identical photographs of conspecifics' faces on Trial 1, and the rhesus monkeys did the same after 4 generalization trials. In the individual-recognition task, the chimpanzees matched 2 different photographs of the same individual after 2 trials, and the rhesus monkeys generalized in fewer than 6 trials. The feature-masking task showed that the eyes were the most important cue for individual recognition. Thus, chimpanzees and rhesus monkeys are able to use facial cues to discriminate unfamiliar conspecifics. Although the rhesus monkeys required many trials to learn the tasks, this is not evidence that faces are not as important social stimuli for them as for the chimpanzees. PMID- 10739312 TI - Multiple levels of representation of song by European starlings (Sturnus vulgaris): open-ended categorization of starling song types and differential forgetting of song categories and exemplars. AB - Four European starlings (Sturnus vulgaris) were trained to discriminate among conspecific and heterospecific song segments in a go/no-go operant task. In Experiment 1, the starlings discriminated among novel starling and heterospecific songs, indicating an open-ended category of conspecific song types. The starlings also showed excellent memory for reinforced conspecific songs and discriminated among subordinate categories of conspecific song. In Experiment 2, the starlings were presented with the song segments from Experiment 1 after an 8-month delay period. The starlings retained the discrimination between conspecific and heterospecific songs but not among conspecific songs. The starlings also retained memory for individual singers over the 8-month delay. Starlings categorize song at the level of species, and at subordinate categories of song types, and may have superior long-term retention of song categories relative to song exemplars. PMID- 10739313 TI - Numerical competence in rats (Rattus norvegicus): Davis and Bradford (1986) extended. AB - The task of rats (Rattus norvegicus) was to enter 1 box of a defined ordinal number among an array of boxes. In Experiments 1 and 2, the rats correctly chose the 4th box from arrays of 6 and 12 boxes, respectively. In Experiments 3 and 4, in which the ordinal number of the correct box was increased in a graduated fashion, they were able to select the correct box even when its position was higher than 10th among 12 and 18 boxes, respectively. In Experiment 5, the possibility that the rats had used cues to the openability of the box doors was ruled out. In Experiments 6 and 7, the rats succeeded in the task even when the total number of boxes was varied from trial to trial. In Experiment 8, both small and large-sized boxes were used to control for the possibility of using the cumulative length of the boxes as a cue. Overall, the results suggest that performance was based on numerical cues. Intentional acts were occasionally observed but appeared not to be essential for solving the task. PMID- 10739314 TI - Heritable variation for latent inhibition and its correlation with reversal learning in honeybees (Apis mellifera). AB - Latent inhibition (LI) in honeybees (Apis mellifera) was studied by using a proboscis extension response conditioning procedure. Individual queens, drones, and workers differed in the degree to which they revealed LI. The authors hypothesized that individual differences would have a substantial genetic basis. Two sets of progeny were established by crossing virgin queens and individual drones, both of which had been selected for differential expression of inhibition. LI was stronger in the progeny from the queens and drones that had shown greater inhibition. The expression of LI was also dependent on environmental factors that are most likely associated with age, foraging experience outside of the colony, or both. Furthermore, there was a correlated response in the speed at which progeny reversed a learned discrimination of 2 odors. These genetic analyses may reveal underlying mechanisms that these 2 learning paradigms have in common. PMID- 10739315 TI - Bubble ring play of bottlenose dolphins (Tursiops truncatus): implications for cognition. AB - Research on the cognitive capacities of dolphins and other cetaceans (whales and porpoises) has importance for the study of comparative cognition, particularly with other large-brained social mammals, such as primates. One of the areas in which cetaceans can be compared with primates is that of object manipulation and physical causality, for which there is an abundant body of literature in primates. The authors supplemented qualitative observations with statistical methods to examine playful bouts of underwater bubble ring production and manipulation in 4 juvenile male captive bottlenose dolphins (Tursiops truncatus). The results are consistent with the hypothesis that dolphins monitor the quality of their bubble rings and anticipate their actions during bubble ring play. PMID- 10739316 TI - Automatic segmentation of the colon for virtual colonoscopy. AB - Virtual colonoscopy is a minimally invasive technique that enables early detection of colorectal polyps and cancer. Normally, a patient's bowel is prepared with colonic lavage and gas insufflation prior to computed tomography scanning. An important step for 3D analysis of the image volume is segmentation of the colon. The high-contrast gas/tissue interface that exists in the colon lumen makes segmentation of the majority of the colon relatively easy; however, two factors inhibit automatic segmentation of the entire colon. First, the colon is not the only gas-filled organ in the data volume: lungs, small bowel, and stomach also meet this criterion. User-defined seed points placed in the colon lumen have previously been required to spatially isolate the colon. Second, portions of the colon lumen may be obstructed by peristalsis, large masses, and/or residual feces. These complicating factors require increased user interaction during the segmentation process to isolate additional colonic segments. To automate the segmentation of the colon, we have developed a method to locate seed points and segment the gas-filled lumen sections without user supervision. We have also developed an automated approach to improve lumen segmentation by digitally removing residual contrast-enhanced fluid. Experimental results with 20 patient volumes show that our method is accurate and reliable. PMID- 10739317 TI - Quantitative analysis of the neonatal brain by ultrasound. AB - Clinical research has shown a clear correlation between white matter disorders of the neonatal brain and neuromotoric handicap at a later age. Ultrasound imaging is a proven method to detect the white matter damage at an early stage. However, since subjective visual examination of the images by neonatologists not always leads to an unambiguous diagnosis, a need for quantitative characterization is felt. Reproducibility is the first requirement in order to be able to perform objective quantitative analysis. This paper proposes a software-based method to compensate for variable acquisition factors that negatively affect the reproducibility of the measurements. The results of some basic experiments will illustrate the usefulness of the developed compensation algorithm. PMID- 10739318 TI - Infantile tuberous sclerosis changes in the brain: proton MR spectroscopy findings. AB - A parietal hamartoma of a three-month-old boy with tuberous sclerosis was studied with magnetic resonance (MR) imaging, and proton MR spectroscopy. MR spectra were obtained with the single-voxel PRESS (point resolved spectroscopy; TR = 1500 ms, TE = 135 ms) sequence, in a 8 cc region of interest. Apparently low NAA/Cho (0.28), and NAA/Cr (0.37) ratios were noted in the hamartoma, that could suggest a neoplasm. The lesion and the surrounding brain tissue were studied again after seven months with spectroscopic imaging using the chemical shift sequence (TR = 1500 ms. TE = 40 ms). This study revealed apparently improved NAA/Cho (2.63), NAA/Cr (2.13) ratios in the hamartoma compared to the initial examination at three months of age, excluding the possibility of a neoplasm. PMID- 10739319 TI - Shortcomings of low-cost imaging systems for viewing computed radiographs. AB - OBJECTIVE: To assess potential advantages of a new PC-based viewing tool featuring image post-processing for viewing computed radiographs on low-cost hardware (PC) with a common display card and color monitor, and to evaluate the effect of using color versus monochrome monitors. MATERIALS AND METHODS: Computed radiographs of a statistical phantom were viewed on a PC, with and without post processing (spatial frequency and contrast processing), employing a monochrome or a color monitor. Findings were compared with the viewing on a radiological Workstation and evaluated with ROC analysis. RESULTS: Image post-processing improved the perception of low-contrast details significantly irrespective of the monitor used. No significant difference in perception was observed between monochrome and color monitors. The review at the radiological Workstation was superior to the review done using the PC with image processing. CONCLUSION: Lower quality hardware (graphic card and monitor) used in low cost PCs negatively affects perception of low-contrast details in computed radiographs. In this situation, it is highly recommended to use spatial frequency and contrast processing. No significant quality gain has been observed for the high-end monochrome monitor compared to the color display. However, the color monitor was affected stronger by high ambient illumination. PMID- 10739320 TI - Catheter and MR angiography of persistent trigeminal artery associated with occipital arteriovenous malformation. AB - We present a patient with an occipital arteriovenous malformation fed by the posterior cerebral artery coexisting with an ipsilateral persistent trigeminal artery. These anomalies were well demonstrated by MR angiography and confirmed by catheter angiography. PMID- 10739321 TI - Single-shot turbo spin-echo MR myelography: comparison with 3D-turbo spin-echo MR myelography and T2-turbo spin-echo at 1 T. AB - In order to reduce the acquisition time, we compared the single-shot-TSE-MR myelography (MRm) and 3D-TSE-MRm. The T2-TSE sequence was the standard of reference. Fifty patients with low back pain, sciatica or cervical radiculopathy were examined at 1.0 T. The shortest AP diameter of the spinal canal, signal-to noise ratio (SNR) for CSF and cord, contrast-to-noise ratio (CNR) and relative contrast (ReCon) between CSF and cord were calculated. No statistically significant difference was found between the three sequences with regard to the AP diameter of the spinal canal. A significant difference was found in: (i) SNRcord; (ii) SNRCSF; (iii) SS-TSE-MRm (showed the highest CNR) and (iv) SS-TSE MRm (showed higher ReCon compared to 3D-TSE-MRm). In conclusion, SS-TSE-MRm can be used alternatively to 3D-TSE-MRm reducing the acquisition time down to only 8 s per image. PMID- 10739322 TI - Spin-echo echo-planar MR imaging of hepatocellular carcinoma arising from chronic liver damage: comparison with turbo spin-echo imaging. AB - The aim of this work was to evaluate the value of echo-planar imaging (EPI) in the detection of hepatocellular carcinomas arising in a chronic liver damage to respiratory triggered turbo spin-echo (TSE) imaging. With spin-echo EPI sequences, better lesion-liver contrast was obtained than TSE imaging. Although severe artifact is seen, this imaging produce good contrast, and may be useful as an adjunct to TSE imaging in evaluating patients with chronic liver damage. PMID- 10739323 TI - Ultrafast fetal MR images of sacrococcygeal teratoma: a case report. AB - A case of sacrococcygeal teratoma diagnosed at 31 weeks' gestation by fetal MR imaging is presented. For fetal MR imaging, an ultrafast imaging sequence, Half Fourier acquisition single shot turbo spin echo (HASTE) was employed. The HASTE sequence enabled us to obtain high resolution images in a short time and was particularly useful in enabling better contrast between the cystic and solid components of the tumor. PMID- 10739324 TI - Tardive dyskinesia: pathophysiology and animal models. AB - Tardive dyskinesia stimulated extensive research into the mechanisms of antipsychotic drug action. A wide range of homologous, analogous, and correlational animal models have been developed to explore how typical neuroleptic drugs do and atypical antipsychotic agents do not seem to cause tardive dyskinesia. The leading hypotheses of the underlying pathophysiology of tardive dyskinesia include dopamine receptor hypersensitivity, GABA insufficiency, and/or structural abnormalities. All these hypotheses have data both for and against them. The roles of psychosis and aging must also be considered in any explanation of tardive dyskinesia. The challenge still remains of how to accurately attribute the relative contributions of each of these factors to the pathogenesis and pathophysiology of tardive dyskinesia. Fortunately, the atypical antipsychotic agents appear to greatly decrease the liability of developing tardive dyskinesia, but how this occurs remains an open and fascinating line of inquiry. PMID- 10739325 TI - Prevalence of spontaneous dyskinesia in schizophrenia. AB - Spontaneous abnormal involuntary movements phenomenologically identical to neuroleptic-induced tardive dyskinesia have been described in schizophrenia for over a century. Because at present nearly all patients with schizophrenia are exposed to neuroleptic medications, information about the prevalence of spontaneous dyskinesia is obtained from accounts from the preneuroleptic era, evaluations of first-episode patients before neuroleptic treatment, and the identification and assessment of drug-naive patients in developing countries. In this report, data from 14 studies of neuroleptic-naive patients with schizophrenia are used to generate age-adjusted estimates of the prevalence of spontaneous dyskinesia. While the precision of this estimate is limited by the difficulty of obtaining large, untreated samples, available data suggest a spontaneous dyskinesia rate of approximately 4% in first-episode schizophrenic patients, 12% for patients ill several years but below age 30 years, 25% for those aged between 30 and 50 years, and 40% for those aged 60 years or older. Relative to the incidence and accrued prevalence of spontaneous dyskinesia expected during the natural history of untreated schizophrenia, the cumulative impact of treatment with new neuroleptic agents has yet to be determined. PMID- 10739326 TI - Review of incidence studies of tardive dyskinesia associated with typical antipsychotics. AB - Unless researchers make an organized effort, patients with tardive dyskinesia are difficult to study and easily lost to follow up. Because there is no treatment for tardive dyskinesia, investigators are obliged to study the natural history of the disorder and identify risk and prognostic factors to further understand pathophysiologic mechanisms and to guide prevention and treatment efforts. Abundant variability exists among incidence studies of tardive dyskinesia, depending to some extent on design issues. In this article, the design concepts of incidence and prevalence studies are described, along with results, methodological problems, and identified risk factors in various tardive dyskinesia incidence studies involving the use of typical antipsychotic medications. PMID- 10739327 TI - Expected incidence of tardive dyskinesia associated with atypical antipsychotics. AB - Given the problematic nature of tardive dyskinesia in persons taking conventional antipsychotics, evaluation of newer atypical antipsychotic agents should include a systematic assessment of tardive dyskinesia liability. Results of a prospective double-blind, randomized study of schizophrenic patients who participated in 3 preclinical olanzapine studies and were treated with 5 to 20 mg/day of olanzapine (N = 1192) or haloperidol (N = 522) recently indicated a significantly lower risk of development of tardive dyskinesia with olanzapine treatment than haloperidol treatment. This article discusses the known effects of atypical antipsychotic medications on tardive dyskinesia movements (both withdrawal and persistent) and the incidence rate of tardive dyskinesia among schizophrenic patients undergoing long-term treatment with olanzapine or haloperidol. PMID- 10739328 TI - Tardive dyskinesia in older patients. AB - Neuroleptic-induced tardive dyskinesia, which often appears in middle-aged and older adults early in the course of treatment with low doses of conventional antipsychotics, is 5 to 6 times more prevalent in elderly than in younger patients. In addition to age, other risk factors for tardive dyskinesia include early extrapyramidal symptoms (EPS), cumulative amounts of neuroleptics, duration of neuroleptic treatment, and history of alcohol abuse and/or dependence. The atypical antipsychotics, which have a low liability for EPS, are likely to also have low potential for tardive dyskinesia, despite the paucity of controlled studies. Starting and maintenance doses of the atypical antipsychotics should generally be lower in older than in younger adults. PMID- 10739329 TI - Antipsychotics in the treatment of mood disorders and risk of tardive dyskinesia. AB - Psychosis occurs commonly in patients with mood disorders and has traditionally been treated with typical antipsychotics. Exposure to typical antipsychotics poses a risk for the emergence of tardive dyskinesia. Atypical antipsychotics may have advantages over typical agents in the treatment of patients with mood disorders complicated by psychotic features. The studies of typical and atypical antipsychotics in the treatment of mood disorders were reviewed. Similarly, studies regarding the risk of tardive dyskinesia from typical and atypical agents in patients with mood disorders were surveyed. Typical and atypical antipsychotics appear to be comparably effective in the treatment of acute mania. Limited data regarding these medications in psychotic depression are available. Advantages of atypical antipsychotics include, for most agents, minimal extrapyramidal and prolactin effects, inherent thymoleptic activity, and lower rates of tardive dyskinesia. Atypical antipsychotics appear to have a number of advantages over typical agents in the treatment of patients with psychotic mood disorders. PMID- 10739331 TI - Update on legal issues associated with tardive dyskinesia. AB - Of the various drug therapies, antipsychotic medication presents some novel twists to old issues in law and psychiatry. From what is known, its benefits are high but so are its risks, notably the risk of tardive dyskinesia in the case of the neuroleptics. Presented here for consideration are legal issues of standard of care, informed consent, the right of institutionalized patients to refuse treatment, statute of limitations, and causal nexus. PMID- 10739330 TI - The treatment of tardive dyskinesia and tardive dystonia. AB - The enthusiasm produced by the introduction of antipsychotic medication in the 1950s gave way to a certain frustration in the 1970s and 1980s. Despite the development of a large number of new drugs, little progress was made in treatment because these new agents were, in essence, therapeutically equivalent. This lack of progress was perhaps also related to an emphasis on tardive dyskinesia in the 1970s, i.e., the preoccupation with a negative effect of treatment. The reverse is taking place today. Clozapine and the other atypical antipsychotics are associated in people's minds with fewer or absent extrapyramidal symptoms and less tardive dyskinesia than the older typical agents. As a result, a certain amount of complacency exists. Tardive dyskinesia not only may be painful and disfiguring, but it also predicts poor outcome in patients with schizophrenia. Although many treatments have been tried, none have proven completely efficacious. The best treatment for tardive dyskinesia and dystonia is prevention, which is a function of medication choice. Pharmacologic interventions for tardive dyskinesia include clozapine and the other atypical antipsychotics. If typical antipsychotics must be used, they should be started at the lowest possible levels. Studies of risperidone suggest that it, too, should be used at very low doses to minimize the risk of tardive dyskinesia. It is also possible that schizophrenic patients taking atypical antipsychotics may experience fewer spontaneous dyskinesias, although further study is warranted. PMID- 10739332 TI - Characterization of murine autoimmune myocarditis induced by self and foreign cardiac myosin. AB - Previously we showed that autoimmune myocarditis could be induced in mice by immunization with purified murine cardiac myosin (MCM). In this study, we found that identical disease could also be induced in genetically susceptible mice by immunization with porcine cardiac myosin (PCM). The cardiac lesions induced by both antigens were characterized by extensive infiltration of the myocardium accompanied by myocyte necrosis. A novel finding was the presence of multinucleated giant cells and eosinophils in the cardiac infiltrates, in addition to a mixture of mononuclear cells and polymorphonuclear cells described previously. Immunohistochemical staining demonstrated that the mononuclear cells consisted predominantly of macrophages, CD4+ T cells and, to a lesser extent, CD8+ T cells and B cells. In addition, increased cardiac expression of adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) were demonstrated in mice that developed myocarditis as compared with those that did not develop disease upon immunization with either PCM or MCM. The levels of TNFalpha detected in spleen cell culture supernatant were found to be higher in mice that developed myocarditis than in those that did not develop the disease. Mice immunized with PCM generated T cells and B cells reactive not only with PCM but also with MCM, and vice versa. In addition, the serum levels of IgG1 anti-MCM antibodies produced in mice immunized with PCM as well as MCM were found to correlate positively with the development of myocarditis. Such a detailed characterization of the murine model of autoimmune myocarditis induced by PCM or MCM allowed us to compare the disease process induced by homologous self and foreign antigens. PMID- 10739333 TI - Peripheral blood and intrathyroidal T cell clones from patients with thyroid autoimmune diseases. AB - For a better understanding of the pathogenesis of thyroid autoimmune diseases, we have studied morphological and functional properties of T clones from peripheral blood lymphocytes (PBL) and from intrathyroidal lymphocytes (ITL) obtained from 3 patients with Graves' disease or 1 Hashimoto's thyroiditis. Investigations were carried out on clones cultured alone or cocultured with autologous thyrocytes. Clonage efficiency ranged from 30% to 33% for PBL and 10% to 36% for ITL. A predominance of CD4-positive clones was observed whatever the origin of the lymphocytes or the autoimmune pathology. Gamma interferon (IFN-gamma) was detected in the majority (17/19) of the clones tested. Intracytoplasmic interleukin (IL-4) was secreted in 7/19 clones and both cytokines were produced in 5/19 clones. In coculture a proliferative response and tumour necrosis factor (TNF-alpha) production were observed with 6 clones (4 from Graves thyrocytes and 2 from thyroiditis). No cytotoxic clone was derived from Graves or thyroiditis tissues. These data demonstrate that the large majority of T clones are principally CD4-T cells; all the clones secreted TNF-alpha and a large majority produced IFN-gamma. Only a few clones produced IL-4 alone or associated with IFN gamma. Six T clones induced proliferative response and of TNF-alpha secretion in coculture. Further investigations must be performed on these antigen-reactive T clones to analyse their role in the pathogenesis of the human thyroid autoimmune diseases. PMID- 10739334 TI - Prevalence of beta-cell and thyroid autoantibody positivity in schoolchildren during three-year follow-up. AB - The prevalence of autoantibodies against the 65 kD isoform of glutamic acid decarboxylase (GAD65Ab), insulin (IAA), islet cells (ICA), thyroid peroxidase (TPOAb) and thyroglobulin (TgAb), in relation to HLA-DR types, was assessed in 310 (HLA in 280) twelve-year-old children during three-year follow-up. Altogether, 26.8% (83/310) of the children were found to carry at least one autoantibody. The HLA-DR3/DR4 genotype was significantly more prevalent in the subgroup of children GAD65Ab-positive on at least one occasion than among GAD65Ab negative children [33% (2/6) vs. 5% (12/274); p = 0.031, as was the HLA-DR4/x genotype among children seropositive for at least one thyroid autoantibody, compared to the corresponding seronegative subgroup 152% (34/65) vs. 34% (74/215); p=0.01]. The proportion of children seropositive in at least one of the three tests was 1.9% (6/310) for GAD65Ab, 2.6% (8/310) for IAA, 5.2% (16/310) for ICA, 11.3% (35/310) for TPOAb and 19.4% (60/310) for TgAb. All autoantibodies except GAD65Ab tended to disappear during follow-up, and at the three-year follow up IAA had disappeared in 50% (2/4) of cases, ICA in 67% (6/9), TPOAb in 30% (6/20) and TgAb in 38% (18/47) of cases. The turnover of seropositive subjects and the large proportion of children seropositive for at least one islet or thyroid autoantibody during a three-year follow-up suggest transient autoantibodies to be more common than is discernible in cross-sectional investigations. PMID- 10739335 TI - Avidity of islet cell autoantibodies in non-diabetic children and children with insulin-dependent diabetes. AB - Low-avidity immunoglobulin G antibodies, which commonly occur after microbial infections and form unstable complexes with the antigen, are within weeks replaced by high-avidity antibodies, which bind the antigen tightly and characterise mature immune response. We hypothesised that avidity of islet cell autoantibodies (ICA), found in the sera of patients with insulin-dependent diabetes mellitus (IDDM) usually months or years before disease onset, might reflect the stage of progression towards overt IDDM. ICA were quantitated before and after treatment of antigen-antibody complexes with 5 M urea in a new assay, in which the fluorescence intensity of europium chelate-labelled detecting antibody is measured from a digital time-resolved fluorescence image. The proportion of urea-resistant ICA of all ICA was defined as the avidity index. The median avidity indices (range) of 119 children with recent-onset IDDM and 64 non diabetic ICA-positive children were 74% (23-100%) and 11% (0-100%), respectively (p = 0.0001). The avidity indices of only 3 children with IDDM (2.5%), but of 55 non-diabetic ICA-positive children (86%) were < 40%. In conclusion, our data show that ICA avidity indices of ICA-positive non-diabetic children and children with IDDM differ, suggesting that measurement of ICA avidity may improve prediction of the time of onset of clinical IDDM. PMID- 10739336 TI - Autoimmunity and vascular involvement in systemic sclerosis (SSc). AB - Endothelial injury, obliterative microvascular lesions, and increased vascular wall thickness are present in all involved organs in scleroderma. The vascular pathology is associated with altered vascular function with increased vasospasm, reduced vasodilatory capacity and increased adhesiveness of the blood vessels to platelets and lymphocytes. The extent of injury and dysfunction is reflected by changes in the circulating levels of vascular markers. The initial triggers for the vascular pathology are not known. Possible viral triggers are visited here, including cytomegalovirus in view of increased levels of anti-CMV antibodies in scleroderma, and the remarkable similarities between CMV vasculopathies and scleroderma vascular disease. Endothelial apoptosis in scleroderma may be related to viral infection, immune reactions to viral or environmental factors, reperfusion injury or to anti-endothelial antibodies. The impact of the vascular pathology on the evolution of tissue fibrosis is not known; still, cytokines (TGFbeta, IL4), vascular factors (endothelin), and growth factors (PDGF) are possibly crucial signals that link the vascular disease to tissue fibrosis. Knowledge of the regulation of these and other factors will provide the opportunity to develop more rational therapeutic approaches to the disease. PMID- 10739337 TI - Mycolic acids from Rhodococcus, Gordonia, and Dietzia. AB - The mycolic acids from 11 species of Rhodococcus, seven species of Gordonia, and one species of Dietzia were analyzed using capillary gas chromatography and mass spectrometry (GLC/MS). All strains tested in this study were divided into three groups according to the degree of double bonds and the average carbon number (Av.Nc.) of their mycolic acids. The genus Gordonia belongs to the first group possessing an Av.Nc. in the upper 50s and 60s with 0 to 5 double bonds. Some Rhodococcus species possessed Av.Nc. in the 40s with a variety of distributions of polyunsaturated fatty acids from 0 to 4. The rest of the Rhodococcus species and the genus Dietzia possessed Av.Nc. in the 30s with saturated fatty acids. We previously reported on Nocardia strains whose Av.Nc. were in the 50s. Considering the identification of mycolic acid-containing Actinomycetales at the generic level, the Av.Nc. proved to be useful as a means of differentiating the genera Rhodococcus, Gordonia and Nocardia. The genus Dietzia was found to have its own characteristic constitution of mycolic acid molecular species. The mycolic acids from D. maris 58001T were characterized by an almost equal amount of constituents of even- and odd-numbered carbon chains, whereas the major components of mycolic acids in all other strains had even-numbered carbon chains. Another characteristic of Dietzia was some even-numbered mycolic acids which contained odd-numbered straight chains with odd-numbered alpha-branches. These characteristics indicated that Dietzia might possess a novel fatty acid biosynthesis system. PMID- 10739338 TI - Immunomagnetic separation of Cryptosporidium parvum oocysts using MACS MicroBeads and high gradient separation columns. AB - We evaluated the MACS immunomagnetic separation (IMS) system for concentrating Cryptosporidium parvum. Oocysts were first labeled with fluorescein isothiocyanate (FITC) or rabbit anti-C. parvum antibodies, then linked to MicroBeads coated with anti-FITC or anti-rabbit IgG, and separated through a high gradient separation column. Results indicated that over 95% of oocysts were recovered and their fluorescence and infectivity were retained. The presence of MicroBeads showed no effect on genomic DNA extraction and subsequent polymerase chain reaction (PCR)-based analyses, as sensitivity of PCR (10 oocysts) and the band pattern of randomly amplified polymorphic DNA (RAPD) were identical to those using DNAs extracted from normally purified oocysts. IMS-PCR consistently detected as few as 10 oocysts from 100 ml of apple juice or homogenized milk and IMS-IFA could detect 100 oocysts from 1 g of deer manure, demonstrating the efficiency of IMS in recovering oocysts from environmental and food samples. Our results suggest that the MACS IMS system could be used for multiple applications in Cryptosporidium research. PMID- 10739340 TI - Differentiation of Salmonella enterica serotype gallinarum biotype pullorum from biotype gallinarum by analysis of phase 1 flagellin C gene (fliC). AB - Salmonella enterica serotype gallinarum biotype gallinarum and biotype pullorum are non-motile and pathogenic avian strains. Biotype gallinarum causes fowl typhoid and biotype pullorum is the cause of pullorum disease in chickens. The two biotypes could be differentiated based on biochemical characteristics. However, conventional culture and biochemical assays are time-consuming, laborious and need sterile laboratory practices. Although the two biotypes, gallinarum and pullorum are non-motile, they possess the phase 1 flagellin C gene. The variable regions of the flagellin C gene from 41 biotype pullorum and 52 biotype gallinarum were amplified by colony-PCR and analyzed by single strand conformational polymorphism (SSCP) method. Differences in SSCP electrophoretic patterns were confirmed by nucleotide sequencing. In addition, PCR-RFLP with Hinp1I was also successfully applied to differentiate the two biotypes. These results suggested that the variable regions of fliC could be used as a genetic marker to differentiate biotype gallinarum from biotype pullorum. PMID- 10739339 TI - Phylogenetic analysis of aerobic freshwater and marine enrichment cultures efficient in hydrocarbon degradation: effect of profiling method. AB - Aerobically grown enrichment cultures derived from hydrocarbon-contaminated seawater and freshwater sediments were generated by growth on crude oil as sole carbon source. Both cultures displayed a high rate of degradation for a wide range of hydrocarbon compounds. The bacterial species composition of these cultures was investigated by PCR of the 16S rDNA gene using multiple primer combinations. Near full-length 16S rDNA clone libraries were generated and screened by restriction analysis prior to sequence analysis. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was carried out using two other PCR primer sets targeting either the V3 or V6-V8 regions, and sequences derived from prominent DGGE bands were compared to sequences obtained via cloning. All data sets suggested that the seawater culture was dominated by alpha subgroup proteobacteria, whereas the freshwater culture was dominated by members of the beta- and gamma-proteobacteria. However, the V6-V8 primer pair was deficient in the recovery of Sphingomonas-like 16S rDNA due to a 3' terminal mismatch with the reverse primer. Most 16S rDNA sequences recovered from the marine enrichment were not closely related to genera containing known oil degrading organisms, although some were detected. All methods suggested that the freshwater enrichment was dominated by genera containing known hydrocarbon degrading species. PMID- 10739341 TI - Quantitative analysis of phagocytosis of Cryptococcus neoformans by adherent phagocytic cells by fluorescence multi-well plate reader. AB - Macrophages and monocytes are adherent phagocytic cells which play an important role in host defence against the yeast-like fungus Cryptococcus neoformans. Before, phagocytosis by adherent phagocytes could only be measured by means of microscopy or by a radioactive assay, which both have obvious disadvantages. We have developed a new, rapid and objective method to measure phagocytosis of C. neoformans by adherent phagocytes (e.g. alveolar macrophages) using a fluorescence multi-well plate reader. This method allows us to discriminate accurately between adherence and internalisation of C. neoformans by macrophages during long term incubation. In addition, the method was used to study the role of the mannose receptor in phagocytosis of the acapsular yeast in the absence of serum by human monocyte-derived macrophages (MDM). PMID- 10739342 TI - Extraction of violacein from Chromobacterium violaceum provides a new quantitative bioassay for N-acyl homoserine lactone autoinducers. AB - Fatty acyl homoserine lactones (AHLs) are used as extracellular quorum sensing signals by a variety of gram-negative bacteria. By activating proteins belonging to the LuxR family of transcriptional regulators, these signal metabolites allow population density-dependent gene regulation within a species, as well as interspecies communication among different bacteria. The experimental detection of AHLs is important in the identification of quorum sensing capabilities in bacteria. Chromobacterium violaceum is a gram-negative bacterium that produces the purple pigment violacein in response to the presence of the AHL N-hexanoyl homoserine lactone (C6HSL). The mini-Tn5 mutant strain C. violaceum CV0blu is deficient in the production of this signal molecule but retains the ability to synthesize violacein in response to the presence of C6HSL and a variety of other short-chain AHLs. We have developed a quantitative bioassay that measures the amount of violacein produced by this strain in response to the presence of different concentrations of various AHL molecules. This new assay provides a means of quantifying the amount of a given AHL present in a bacterial culture and can be used to measure differences in AHL production among different strains or different batch cultures of a given species. PMID- 10739343 TI - Second-order functions are the simplest correlations between flow cytometric light scatter and bacterial diameter. AB - Second-order mathematical relationships between bacterial cell diameter determined by electric particle analyser and flow cytometric forward light scatter in axenic cultures are obtained and discussed. Since it is technically impossible today to obtain both measurements for each individual cell, standard regression techniques cannot be applied. To overcome this limitation, we assume that these two parameters are related by a monotone increasing function that enables their mathematical relationships to be studied. Our conclusion is that forward light scatter data cannot be linearly transformed into bacterial size values by an accurate and universal function. However, second-order relationships seem to be the simplest satisfactory relationships between cell diameter and forward light scatter in eubacteria. PMID- 10739344 TI - Usefulness of the BACTEC MYCO/F lytic system for detection of mycobacteremia in a clinical microbiology laboratory. AB - 278 BACTEC MYCO/F lytic system blood cultures for mycobacteria were evaluated between 1997 and 1999. Sixty of them were read as positive by the system, being considered 15 of them as false positives. Twenty-seven yielded mycobacterial growth (13 Mycobacterium avium from 3 patients and 14 Mycobacterium tuberculosis from 8 patients). Other bacteria isolated were coagulase-negative Staphylococcus (13 samples), Corynebacterium sp. (5 samples), Salmonella enteritidis (2 samples) and Klebsiella pneumoniae (1 sample). Five of these isolates were considered as true episodes of bacteremia. The average time for detection of mycobacteria was 12.6 days for M. avium and 26.4 days for M. tuberculosis. BACTEC MYCO/F lytic system is useful for detection of mycobacteremia in clinical microbiology laboratories. PMID- 10739345 TI - Flow cytometric techniques to characterise physiological states of Acinetobacter calcoaceticus. AB - Monitoring biotechnological processes involves acquiring information about key metabolic events and, ideally, single cell states should be determined to obtain comprehensive data on the physiological status of the surveyed population. In this paper, growth stages of the strain Acinetobacter calcoaceticus 69-V were characterised at the single cell level using flow cytometry. Four methods for analysing bacterial cellular characteristics by fluorescence were compared with respect to their sensitivity to changes in the physiological states induced by changing micro-environmental conditions. DNA analysis was confirmed to be highly informative with regard to the multiplication activity of the population. Measuring the membrane potential related fluorescence intensity (MPRFI) and the rRNA content were found to be useful for describing high-active cell states. A method for the measurement of the fluidity related fluorescence intensity (FRFI) was developed, since it allowed changes in the fluidity of the bacterial membrane to be detected, and thereby provided a valuable means of tracking adaptation of the population to micro-environmental deviations from optimal growth conditions. PMID- 10739346 TI - Development and evaluation of a broad-range PCR-ELISA assay with Borrelia burgdorferi and Streptococcus pneumoniae as model organisms for reactive arthritis and bacterial meningitis. AB - We have developed an assay based on a 16S rDNA broad-range amplification system followed by species-specific detection with a commercially available PCR-ELISA kit. B. burgdorferi and S. pneumoniae were used as model systems for arthritis and meningitis, respectively. The sensitivity of the B. burgdorferi assay was comparable to that of a species-specific PCR, whereas for S. pneumoniae the detection limit was one to three organisms as determined by plate counts. To specifically differentiate two species, two discontinuously located nucleotide differences in the region complementary to the capture probe are required during the detection step with the PCR-ELISA kit. A preliminary clinical evaluation was performed with eight specimens (joint and cerebrospinal fluids) previously shown to contain B. burgdorferi DNA. Except for one sample which was positive by the broad-range PCR-ELISA system only, the results were in agreement with those obtained by B. burgdorferi species-specific PCR. None of the 23 control samples were positive by either method. Thus, broad-range amplification in combination with the PCR-ELISA kit promises to be a sensitive and specific format for the detection of agents causing reactive arthritis, meningitis or other diseases associated with a limited number of different bacteria. PMID- 10739347 TI - Quantification of bacterial adhesion forces using atomic force microscopy (AFM). AB - This study demonstrated that atomic force microscopy (AFM) can be used to obtain high-resolution topographical images of bacteria, and to quantify the tip-cell interaction force and the surface elasticity. Results show that the adhesion force between the Si3N4 tip and the bacteria surface was in the range from -3.9 to -4.3 nN. On the other hand, the adhesion forces at the periphery of the cell substratum contact surface ranged from -5.1 to -5.9 nN and those at the cell-cell interface ranged from -6.5 to -6.8 nN. The two latter forces were considerably greater than the former one, most likely due to the accumulation of extracellular polymer substance (EPS). Results also show that the elasticity varied on the cell surface. PMID- 10739348 TI - A rapid technique for screening of lovastatin-producing strains of Aspergillus terreus by agar plug and Neurospora crassa bioassay. AB - The success of strain improvement programme depends on the number of isolates that can be screened after mutagenic treatment. A technique to rapidly screen large number of high-yielding isolates was developed. The 'agar plug' method that utilizes the anti-fungal property of lovastatin to produce a zone of inhibition against Neurospora crassa was not only economical but also less labour-intensive. We were able to isolate a high-yielding strain, the productivity of which increased by 138% as compared to the parent strain in the submerged fermentation process. PMID- 10739349 TI - Use of gel electrophoresis for the study of enzymatic activities of cold-adapted bacteria. AB - Glutamate dehydrogenase (GDH) and lactate dehydrogenase (LDH) activity of 13 cold adapted strains, isolated from cold soils and showing GDH and/or LDH activity in spectrophotometric assays, were revealed by the use of electrophoresis on a nondenaturing acrylamide gel (zymogram). Psychrophilic strains were grown at 4 degrees C and 10 degrees C and the psychrotolerant strains at 4 degrees, 20 degrees and 28 degrees C. Incubation with the specific substrate and staining were done at 4, 28 or 37 degrees C. In the most cold-adapted strains, LDH and GDH production was high at 4 degrees C. In psychrotrophic strains, enzyme production and activity were greater at 20 or 28 degrees C than at lower temperatures. LDH remained active up to 37 degrees C while GDH activity was more thermolabile. GDH activity was NAD-dependent in some psychrophilic strains. In other strains, it was dependent on NAD(P) only or on both NAD and NAD(P). Two bands were seen for GDH or LDH activity in some strains. This method, which does not require a dialysis step, can be used to study the influence of temperature on enzyme production and activity, and the co-factor dependence. It detects phenotypic differences between isozymes, providing data for systematics. PMID- 10739350 TI - The value of spiritual support in cancer patients. PMID- 10739351 TI - Au revoir, from Derek Doyle: farewell address held in Geneva. PMID- 10739352 TI - Integrated palliative care within a general hospital. AB - The goal of the project was to integrate essential elements of palliative care into the regular duties of an internal ward located in a general hospital serving a rural area. To achieve this goal, the medical and nursing staff was moderately expanded, which was made possible by a grant from the Deutsche Krebshilfe, and training and supervision were instituted. Patients with malignant diseases in advanced and terminal phases were enrolled in the project and evaluated using a specifically designed documentation system. On average, 8.5 (18%) of the 46 beds on the ward were occupied by patients being cared for as part of the project at any one time. Effective relief of pain, nausea and respiratory distress were documented. For those dying in the hospital, a single room and the presence of family members were possible in the majority of cases. A high impact on the team became apparent through an anonymous questionnaire given during supervision. The project shows that it is possible to integrate palliative care into the work of a regular internal medical ward, with positive consequences for the patients and the team. PMID- 10739353 TI - Health-related quality-of-life assessment in clinical trials of supportive care in oncology. AB - A primary objective of symptom control and supportive care in clinical trials is to improve health-related quality of life. However, in the past, most such clinical trials have concentrated on limited outcomes, such as control of anorexia or pain, and have not taken into account the broader outcome of health related quality of life. The multidimensional tools needed to carry out these trials are now available, and several studies have yielded results that are informative and useful. These include studies on ameliorating anorexia and weight loss, fatigue and anemia, postchemotherapy nausea and vomiting, and pain from bone metastases. Examples of such studies are given. However, there is still much to learn, and investigators are urged to continue to measure health-related quality of life in clinical trials of symptom control and supportive care. PMID- 10739354 TI - Psychopharmacology in supportive care of cancer: a review for the clinician: II. Neuroleptics. AB - Neuroleptics are frequently used in patients with advanced cancer. Most relevant and practical aspects of their use in supportive cancer care are reviewed, to assist the clinical oncologist and palliative care specialist when prescribing these drugs. This article reviews pharmacological properties, indications, such as delirium, nausea and vomiting, pain, anxiety and other symptoms, adverse effects, and drug interactions of neuroleptics and compares the profiles of different compounds. Special emphasis is put on the role of neuroleptics in the management of delirium. PMID- 10739355 TI - Future trends in oncology. AB - The incidence of cancer will continue to increase more rapidly than the population in both industrialized and developing countries. The number of older patients will rise. Cancer will be a major health challenge in the future. Patients will become more and more involved in decisions relating to their care and their quality of life, behaving in a more adult way than hitherto. Specific treatments will continue to develop, leading to more cures and to longer survival for patients who are not cured. The fight against cancer could be organized in such a way as to make it possible to provide global care of patients through comprehensive cancer networks. Supportive care could increasingly take the form of continuous care covering every step in the fight against cancer, starting with prevention and screening. PMID- 10739356 TI - A multidimensional measure of religious involvement for cancer patients: the Duke Religious Index. AB - Despite increasing interest in the relationship between religious involvement and health outcomes for cancer patients, research has been limited by the lack of appropriate measures. Few of the many instruments available are well suited to cancer patients. The current study examined the psychometric properties of one recently developed measure, the Duke Religious Index (DRI), which assesses several aspects of religious involvement. The DRI was evaluated in two distinct samples: 104 cancer patients receiving treatment at a bone marrow transplantation program and 175 gynecology clinic patients. The instrument demonstrated good internal consistency (coefficient alphas 0.87-0.94). Moderate to high correlations with other measures of religiosity provided support for convergent validity. Modest relationships with other measures commonly used in psychosocial oncology (e.g., optimism, social support, purpose in life) indicated that the instrument provides unique information (all rs's < 0. 42). Small relationships with social desirability response bias, negative affect, and relationship cohesion further supported the divergent validity of the instrument (all rs's < 0.22). The DRI was significantly associated with demographic characteristics but not with medical variables. Findings support the value of the DRI for use in oncology settings. PMID- 10739357 TI - Faith among patients with advanced cancer. A pilot study on patients offered "no more than" palliation. AB - Spiritual wellbeing is an important topic in cancer care. Being religious is reported by patients facing dilemmas concerning the quality and meaning of life to be potentially helpful. However, the fear of death may be close to the surface and easily stimulated. The aim of this study was to clarify patients' attitudes to faith. Between February 1998 and February 1999, 20 patients aged 37-74 years and suffering from ten different incurable cancers were enrolled in the study. An interview technique focusing on the topic by way of an open question about faith was employed. The topic was only continued if the patient signalled a clear wish for this. Half the patients had a close relative present during the conversation, and an oncology nurse was present in all cases. Most patients (18, or 90%) intimated that the topic was of interest: 85% responded by saying they believed in God, and 75% reported that they prayed. A quarter (25%) mentioned that they had visited their local Lutheran pastor before their admission to hospital. One patient reported being a Jehovah's Witness and one, a member of the Norwegian Humanistically Ethical Association (HEA). Following the conversation, 4 patients requested a visit from the hospital chaplain, 1 asked for contact with the Salvation Army to be arranged, and 1 wanted to talk to the local leader of HEA. Following the conversation all patients were observed by a nurse, and no raised level of anxiety was reported. Sixteen of the patients died within a median of 18 (1-180) days after the conversation. In conclusion, most patients responded positively to a question about faith. The topic should be addressed in the treatment of patients with advanced disease. However, care must be taken to avoid frightening the patients. Patients' attitudes with regard to what death brings deserve respect. PMID- 10739358 TI - Economic analysis of terminal care for patients with malignant osteolytic bone disease and pain treated with pamidronate. AB - The goals of this study were the assessment (1) of all costs of terminal care of patients with osteolytic bone disease and pain and (2) of the economic consequences of the pamidronate treatment as observed in a prospective clinical trial on the effectiveness of pamidronate. A total of 70 patients were recruited, who were all suffering from advanced tumour diseases (60% breast cancer, 21% multiple myeloma, and 19% other tumours). In a single-institution study the patients were randomly assigned to receive, in a double-blinded setting, pamidronate 60 mg i.v. or 90 mg i.v. every 3 weeks for a maximum of six cycles. Perception of pain intensity was recorded by self-assessment, using a linear analogue scale. Follow-up lasted 6 months after treatment. All elements of direct costs of in-patient and out-patient care were recorded in cooperation with the hospital administration and the health insurance companies [Krankenkassen]. Average monthly direct costs amounted to ECU 1,290 (+/-410) and 1,050 (+/- 430) during the treatment phase and follow-up, respectively. Average in-patient costs were about three times the out-patient costs. Significantly higher costs (by a factor of 2) were observed for terminal care in hospital (last 3 months before death) than for continued care (of patients surviving the study period). The treatment with pamidronate reduced pain significantly but did not add noticeably to the costs. The study showed that it is practicable and quite efficient to combine a pharmaco-economic evaluation with a clinical trial, although it may be difficult (depending on the setting and availability of information) to assess true costs, i.e. total resource usage. PMID- 10739359 TI - Factors influencing the opioid response in advanced cancer patients with pain followed at home: the effects of age and gender. AB - The aim of this study was to evaluate the influence of age and gender on pain characteristics and opioid response in advanced cancer patients followed at home. A perspective study was carried out in a sample of 181 consecutive advanced cancer patients who required opioids in the last 4 weeks before death. Pain intensity and symptoms associated with opioid therapy at weekly intervals for 4 weeks were recorded, as were the previous oncological treatments. Opioid doses increased over time, but remained stable in the last 2 weeks of life, while pain intensity decreased over time despite unchanged use of NSAIDs. A considerable increase in symptom intensity was observed in the last weeks of life, except for nausea and vomiting. Visceral pain was more often reported in women. Male patients more often presented somatic pain mechanisms. Neuropathic pain was associated with higher mean VAS intensity and was equally reported in male and female patients and in the different age groups. Very old patients, who received less chemotherapy, required less opioid doses and reported a lower intensity of some symptoms, while reporting similar pain relief. Dry mouth was more frequent in adults than in very old patients. The identification of specific factors and pain characteristics may be useful in suggesting the likelihood of response in terms of analgesia and opioid-related adverse effects. Age and gender analysis should be included in all cancer pain and symptom control studies, as they may have an influence on cancer pain prognosis. PMID- 10739360 TI - Granisetron, tropisetron, and ondansetron in the prevention of acute emesis induced by a combination of cisplatin-Adriamycin and by high-dose ifosfamide delivered in multiple-day continuous infusions. AB - The antiemetic efficacy of granisetron, ondansetron and tropisetron was evaluated in patients treated with cisplatin-Adriamycin (CDP/ADM) and ifosfamide (IFO) by continuous infusion (CI). In all, 90 patients with osteosarcoma were randomly assigned to receive granisetron (2 mg/m2), or ondansetron (5.3 mg/m2), or tropisetron (3.3 mg/m2) plus dexamethasone 8 mg/m2. Chemotherapy consisted of CDP (120 mg/m2, 48-h CI) followed by ADM (75 mg/m2, 24-h CI) and then, in the second cycle, delivered 3 weeks later, IFO 15 g/m2 (120-h CI). Complete protection (CP) from emesis was obtained on 59% of the 717 days of treatment, without significant differences among the three study drugs. A significantly higher rate of CP was obtained during chemotherapy with IFO than with CDP/ ADM (69% vs 44%; P<0.0001). The rate of CP declined from the first to the last day of treatment for both CDP/ADM (61% to 27%, P<0.0001) and IFO (95% to 43%) cycles (P<0.0001). When CDP/ ADM and IFO are delivered on multiple days by CI, granisetron, ondansetron and tropisetron have the same antiemetic efficacy, which declines from the first day onward through successive days. PMID- 10739361 TI - Successful palliation of hypoactive delirium due to multi-organ failure by oral methylphenidate. AB - Delirium is frequently observed in terminally ill cancer patients, but complete remission is often difficult to achieve. Therefore, symptom palliation is of great importance to improve patients' quality of life. Although it has been suggested that psychostimulants would be beneficial to patients with hypoactive delirium, there have been very few empirical reports so far. We report on a terminally ill cancer patient with hypoactive delirium caused by multi-organ failure, in whom methylphenidate was effective in improving the ability to maintain communication. This case highlights the efficacy of methylphenidate for hypoactive delirium in terminally ill cancer patients. PMID- 10739362 TI - Methadone as a rescue for failed high-dose opiate therapy for catastrophic pain. AB - Methadone is a unique opioid with several special properties besides being a mu agonist, which makes for difficulty in the calculation of equianalgesic doses. A series of three cases is presented to illustrate this point. PMID- 10739363 TI - Exploring the role of the cerebellum in sensory anticipation and timing: commentary on Tesche and Karhu. AB - The past decade has witnessed a paradigm shift concerning the study of the cerebellum. Results from various studies employing a variety of methodologies suggest that the functional role of this structure is not limited to motor control. The article by Tesche and Karhu appearing in this issue, provides strong evidence that the cerebellum in humans is activated in anticipation of somatosensory events, even when these events do not require overt responses. In their study, the sensory response is observed when the stimuli fail to occur at expected points in time, consistent with the hypothesis that the cerebellum is specialized for representing the temporal relationships between events, motoric or otherwise. Timing and sensory expectancy likely reflect nested hypotheses, and it remains to be seen if one provides a more encompassing yet specific view of cerebellar function. PMID- 10739364 TI - Anticipatory cerebellar responses during somatosensory omission in man. AB - The traditional view of cerebellum is a structure that modifies and synchronizes elements of motor performance. Recent evidence indicates that human cerebellum is involved in a wide range of nonmotor sensory and cognitive functions. A common feature in these diverse motor and nonmotor tasks may be the capacity of cerebellar neuronal circuits to process and anticipate sensory input with high temporal acuity. We present evidence supporting this hypothesis from measurements of the magnetic field at the scalp evoked by neuronal population activity in human cerebellum. Intermittent electrical stimulation of the finger and the median nerve elicited stimulus-locked cerebellar responses with oscillatory components at 6-12 Hz and 25-35 Hz. Sustained oscillatory activity followed random stimulus omissions, with initiation of cerebellar responses prior to the next overt stimulus. These responses indexed processing of temporal features of somatosensory input independent of motor performance or response. The refractory behavior of the responses suggested that a neuronal trace of the temporal pattern of somatosensory stimulation remained in cerebellar circuits for 2-4 s. The cerebellar activity elicited by violation of an established temporal pattern was enhanced when attention was directed to somatosensory stimuli, in concordance with recent imaging studies suggesting participation of cerebellum in attentional networks. The attentional enhancement of the cerebellar responses supports the salience of cerebellar activity in the processing of purely somatosensory input. The short-term maintenance of cerebellar templates for predictable sensory input may reflect a physiological substrate for fine-grained temporal tuning and optimization of performance in large-scale sensory and integrative systems. PMID- 10739365 TI - Robust estimation of the probabilities of 3-D clusters in functional brain images: application to PET data. AB - Recently, we presented a method (the CS method) for estimating the probability distributions of the sizes of supra threshold clusters in functional brain images [Ledberg A, Akerman S, Roland PE. 1998. Estimating the significance of 3D clusters in functional brain images. NeuroImage 8:113-128]. In that method, the significance of the observed test statistic (cluster size) is assessed by comparing it with a sample of the test statistic obtained from simulated statistical images (SSIs). These images are generated to have the same spatial autocorrelation as the observed statistical image (t-image) would have under the null hypothesis. The CS method relies on the assumptions that the t-images are stationary and that they can be transformed to have a normal distribution. These assumptions are not always valid, and thus limit the applicability of the method. The purpose of this paper is to present a modification of the previous method, that does not depend on these assumptions. This modified CS method (MCS) uses the residuals in the linear model as a model of a dataset obtained under the null hypothesis. Subsequently, datasets with the same distribution as the residuals are generated, and from these datasets the SSIs are derived. These SSIs are t distributed. Thus, a conversion to normal distribution is no longer needed. Furthermore, no assumptions concerning the stationarity of the statistical images are needed. The MCS method is validated on both synthetical images and PET images and is shown to give accurate estimates of the probability distribution of the cluster size statistic. PMID- 10739366 TI - FMRI of visual encoding: reproducibility of activation. AB - fMRI, a noninvasive technique to measure brain activation, is gaining clinical interest, because its sensitivity enables individual assessments. However, more insight in the reproducibility of these measurements during higher cognitive tasks is necessary. We performed an fMRI study involving within- and between subject reproducibility during encoding of complex visual pictures. Ten healthy subjects were studied on three occasions: twice in the same scanning session (study 1 and 2), and a third time, 3-24 days later (study 3). On all 30 occasions but one, activation was found in areas expected on the basis of previous studies, including the fusiform and lingual gyri, occipital and parietal areas, the (para)hippocampal area, and the frontal inferior sulcus. The reproducibility of the number of activated voxels in the whole brain was 72% and 63% (respectively, studies 1 and 2, and 1 and 3). The reproducibility of anatomical identical pixels that supplement these results was 49% and 36%. These reproducibility measures increase about 5-15% when only areas of expected activation are included. The quantitative measurements indicate that there is substantial variation in the volume of activation. The recognition of pictures as tested afterward explains part of this variation between subjects. Our findings indicate that whereas consistent patterns of activation exist, more insight is needed into what determines the volume of activation, especially to assess cognitive alterations in patients over time. PMID- 10739367 TI - On the relation between brain images and brain neural networks. AB - The relationship between brain images observed by PET and fMRI and the underlying neural activity is analysed using recent results on the detailed nature of averaged and synchronised activity of coupled neural networks and on a simplifying model of the level of blood flow caused by neural activity. The conditions on the coupled neural systems are specified that lead to structural equation models, giving support to analysis of the covariance structural equation modelling of brain imaging data. The relation between the resulting models and possible neural codes are analysed. Furthermore, a new form of structural equation model is derived, in which all neuronal activity arises as hidden variables. We discuss how the results of such analyses can be transported back to the domain of coupled temporally dynamic neural systems in the brain appropriate to EEG and MEG observations. PMID- 10739368 TI - D-dimer assays for the exclusion of venous thromboembolism: which test for which diagnostic strategy? PMID- 10739369 TI - Comparison of a clinical probability estimate and two clinical models in patients with suspected pulmonary embolism. PMID- 10739370 TI - Blood coagulation factor X deficiency causes partial embryonic lethality and fatal neonatal bleeding in mice. AB - Mice with a total deficiency in blood coagulation Factor X (FX) were generated by targeted replacement of an 18-kb fragment of the FX gene, comprising all exons encoding the mature FX protein, with a neo(r) cassette. The genotype distribution among the offspring from heterozygous breeding pairs suggested that FX deficiency resulted in partial embryonic lethality, with approximately one-third of the FX-/ embryos dying around embryonic day (E) 11.5-12.5. Two of 44 non-resorbed FX-/- embryos analyzed at these stages showed signs of massive bleeding, one of which into the brain ventricles, but no histological defects in the vasculature of these embryos or their yolk sac were observed. The remainder of the FX-/- embryos appeared normal and survived to term, but the majority of neonates (90%) died within 5 days, most frequently from intraabdominal bleeding. The remaining FX-/- animals succumbed between postnatal day (P)5 and P20 with intraabdominal, subcutaneous, or intracranial bleeding or a combination thereof. The lethal phenotype of the FX-/- mice illustrates the importance of FX function in embryonic and postnatal survival and demonstrates that these mice serve as effective models of the bleeding disorders observed in severe FX deficiency in humans. PMID- 10739371 TI - Exclusion of deep venous thrombosis with D-dimer testing--comparison of 13 D dimer methods in 99 outpatients suspected of deep venous thrombosis using venography as reference standard. AB - In a direct assay comparison we evaluated the diagnostic performance of 10 novel D-Dimer assays for the exclusion of deep venous thrombosis (DVT). In addition, 3 conventional ELISA D-Dimer assays were included as reference tests. The study was performed in 99 consecutive outpatients referred to the emergency department for clinical suspicion of DVT. Venography was used as reference standard and demonstrated the presence of DVT in 50 patients (6 patients with isolated distal DVT and 44 patients with proximal DVT). The qualitative D-Dimer assays Minutex and SimpliRED and the quantitative BC DD showed overall sensitivities (for proximal and distal DVT) of only 80-83% with specificities that ranged from 87 to 94%. Overall sensitivity was 94% for the qualitative INSTANT I.A. and 98% for the quantitative Turbiquant at a cut-off level equal to the detection limit. Using different cut-off levels a sensitivity of 100% for proximal DVT and for proximal as well as distal DVT could be obtained for NycoCard, IL DD, Liatest, Tinaquant and VIDAS D-Dimer assays with specificities that ranged from 31% (NycoCard) to 71% (VIDAS) for proximal DVT and from 12% (NycoCard) to 47% (IL DD) for overall DVT. At a cut-off level equal to the upper limit of the reference range only Tinaquant and VIDAS showed a sensitivity of 100% for proximal as well as for distal DVT with a specificity of 39% and 41% respectively. The results of this study suggest that the VIDAS and Tinaquant D-Dimer assays have the highest sensitivity for the exclusion of DVT in outpatients. In outpatients that have a low or moderate pretest probability for DVT, these tests may be used in management studies where anticoagulation is withheld on the basis of D-Dimer testing alone. PMID- 10739372 TI - Comparison of a clinical probability estimate and two clinical models in patients with suspected pulmonary embolism. ANTELOPE-Study Group. AB - Recent studies have suggested that both the subjective judgement of a physician and standardized clinical models can be helpful in the estimation of the probability of the disease in patients with suspected pulmonary embolism (PE). We performed a multi-center study in consecutive in- and outpatients with suspected PE to compare the potential diagnostic utility of these methods. Of the 517 study patients, 160 (31%) were classified as having PE. Of these patients, 14% had a low probability as estimated by the treating physician, while 25 to 36% were categorized as having a low clinical probability with the use of two previously described clinical models. The objectively confirmed prevalence of PE in these three low probability categories was 19%, 28% and 28%, respectively. The three methods yielded comparable predictive values for PE in the other probability categories. We conclude that a physician's clinical judgement alone and two standardized clinical models, although comparable, perform disappointingly in categorizing the pre-test probability in patients with suspected PE. PMID- 10739373 TI - The factor V R2 allele: risk of venous thromboembolism, factor V levels and resistance to activated protein C. AB - Case-control studies have yielded conflicting results regarding the relative risk of venous thromboembolism associated with the factor V R2 allele. We calculated odds ratios in 581 patients and 469 age-matched controls. The odds ratio for the R2 allele in patients relative to controls was 1.21 (95% CI 0.84 to 1.74). These results do not support the hypothesis that the R2 allele is a risk factor for venous thromboembolism. There was no relationship between factor V levels and R2 carrier status. Normalised APC sensitivity ratios were not lower in carriers of the R2 allele. In an in vitro model progressive APC resistance was observed with factor V levels of 60% and less but ratios less than 2.4 (equivalent to a normalised ratio of 0.73) did not occur until factor V levels were less than 20%. The relationship between APC resistance and factor V level was not observed in a factor VIII-independent model. PMID- 10739374 TI - Outpatient treatment of pulmonary embolism with dalteparin. AB - BACKGROUND: Pulmonary embolism is a common complication of deep vein thrombosis. It has been established that low molecular weight heparin may be used to treat deep vein thrombosis or pulmonary embolism and randomized studies have established that outpatient management of deep vein thrombosis with low molecular weight heparin is at least as effective as in-hospital management with unfractionated heparin. METHODS: This was a prospective cohort study of eligible patients with pulmonary embolism managed as outpatients using dalteparin (200 U/kg s/c daily) for a minimum of five days and warfarin for 3 months. Outpatients included those managed exclusively out of hospital and those managed initially for 1-3 days as inpatients who then completed therapy out of hospital. Reasons for admission included hemodynamic instability; hypoxia requiring oxygen therapy; admission for another medical reason; severe pain requiring parenteral analgesia or high risk of major bleeding. Patients were followed for three months for clinically apparent recurrent venous thromboembolism and bleeding. RESULTS: Between three teaching hospitals, a total of 158 patients with pulmonary embolism were identified. Fifty patients were managed as inpatients and 108 as outpatients. Of the outpatients, 27 were managed for an average of 2.5 days as inpatients and then completed dalteparin therapy as outpatients. The remaining 81 patients were managed exclusively as outpatients with dalteparin. For all outpatients the overall symptomatic recurrence rate of venous thromboembolism was 5.6% (6/108) with only 1.9% (2/108) major bleeds. There were a total of four deaths with none due to pulmonary embolism or major bleed. CONCLUSIONS: This prospective study suggests that outpatient management of pulmonary embolism is feasible and safe for the majority of patients. PMID- 10739375 TI - Genetic polymorphisms and coronary artery disease in the south of France. AB - Vascular disease is a multifactorial disease that involves atherosclerotic and thrombotic factors. Genetic polymorphisms have been associated with myocardial infarction and angina pectoris. The aim of the present study was to assess the relationship between some genetic polymorphisms and myocardial infarction (MI) or vasospastic angina pectoris in a population from southern France. Genetic polymorphisms of the renin angiotensin system (the D/I polymorphism of the ACE gene and the A1166C polymorphism of the angiotensin II type 1 receptor [AT1R]) and of haemostatic factors (the -675 4G/5G polymorphism of the plasminogen activator inhibitor 1[PAI-1] gene, and the G to T common point mutation in exon 2, codon 34 of the Factor XIII A-subunit gene) were examined. We assessed the genotype distribution in consecutive coronary artery disease (CAD) patients with MI (n = 201) and vasospastic angina pectoris (n = 43) and in 244 healthy controls comparable in age, sex, body mass index and total cholesterol level. The genotype distribution of AT1R polymorphism was significantly different between controls and patients, the prevalence of the C allele carriers being higher in patients with MI after the age of 45 than in control individuals (61 vs 45%, p <0.01), leading to an odds ratio (OR) of 2 (CI: 1.2-3.4). When looking at the group of patients with vasospastic angina the difference was even higher (76 vs 45%, p <0.01) yielding an OR of 4.3 (CI: 1.4-17.4). Genotype distributions of ACE, PAI-1 and Factor XIII polymorphisms were similar in patients and in controls. This study is in favor of a role of ATIR gene polymorphism in myocardial infarction and vasospastic angina. PMID- 10739376 TI - c7E3 Fab inhibits low shear flow modulated platelet adhesion to endothelium and surface-absorbed fibrinogen by blocking platelet GP IIb/IIIa as well as endothelial vitronectin receptor--results from patients with acute myocardial infarction and healthy controls. AB - The c7E3 Fab reduces ischemic complications in patients undergoing high-risk coronary angioplasty or atherectomy. The present study investigated how c7E3 Fab inhibition of the platelet receptor glycoprotein IIb/IIIa and the endothelial vitronectin receptor affected platelet adhesion to endothelium and surface adsorbed fibrinogen under flow conditions. Platelet adhesion was examined using a stagnation point flow device with shear stress and shear rates up to 2.2 dynes/cm2 and 170 s(-1), respectively. Ex vivo adhesion was compared between two groups of patients with acute myocardial infarction (AMI) treated with angioplasty and stent implantation and a group of healthy controls. Only one AMI group received c7E3 Fab therapy. Patients in both groups were administered acetyl salicylic acid (ASA) and heparin. In AMI patients c7E3 Fab reduced platelet adhesion to adsorbed fibrinogen by 79% compared to AMI patients without c7E3 Fab treatment and by 74% compared to healthy controls. Thirty hours after termination of c7E3 Fab infusion adhesion had slightly recovered with an inhibition of 61% and 52% still present, respectively. Additionally, in vitro platelet adhesion to intact endothelium and to adsorbed fibrinogen was measured during superfusion with ADP stimulated platelet rich plasma of healthy controls to which c7E3 Fab was added at a final concentration fc of 20 microg/ml. In spite of ADP stimulation c7E3 Fab completely blocked platelet adhesion to adsorbed fibrinogen and, moreover, to intact endothelium. Preincubation of endothelial cells with c7E3 (fc = 20 [microg/ml) blocked adhesion of ADP-stimulated platelets by approximately 50%. Apart from the inhibition of platelet aggregation, c7E3 Fab added in vitro and given therapeutically in patients effectively blocks platelet adhesion to components of the injured as well as intact vessel wall under stagnation point flow conditions. PMID- 10739377 TI - Relationship of cardiac troponin T and procoagulant activity in unstable angina. AB - OBJECTIVE: The present study sought to determine the incidence of increased procoagulant activity in patients with unstable angina (UAP), and to evaluate the relationship between cardiac troponin T (cTnT) and molecular markers of hemostatic activation. Method. We studied 44 patients with UAP further classified by plasma cTnT levels. All patients received an antithrombotic therapy consisting of therapeutic doses of unfractionated heparin and acetylsalicylic acid. Quantitative levels of cTnT and plasma concentrations of fibrin monomers (FM), prothrombin fragments F1+2, thrombin antithrombin III complexes (TAT), plasminogen and alpha2-antiplasmin were sampled serially within the first 48 h. RESULTS: Increased plasma concentrations of FM were detected in 45.5% of patients and were more frequently present among those with cTnT concentrations > or =0.1 ng/ml (13 of 18 vs 7 of 26 patients, p = 0.003). In these patients, mean plasma concentrations of FM were significantly higher than in patients with cTnT <0.1 ng/ml (7.93 +/- 2.3 vs 3.12 +/- 0.6 microg/ml, p = 0.02). There was a close relationship between plasma levels of cTnT and FM (r = 0.74, p <0.004), prothrombin fragments F1+2 (r = 0.71, p = 0.046) and a trend to significance was noted for TAT (r = 0.42, p = 0.055). No significant correlation was observed with markers of the fibrinolytic system (plasminogen and alpha2-anti-plasmin). Plasma levels of cTnT > or =0.1 ng/ml identified a concomitant increase of hemostatic markers with a sensitivity, specificity and positive predictive value of 65, 79, and 72% for FM, 63, 76, and 67% for prothrombin fragments F1+2, and 58, 66, and 39% for TAT, respectively. CONCLUSIONS: In patients with UAP, cTnT identifies patients with increased procoagulant activity and is closely related to plasma levels of molecular markers of hemostatic activation. Therefore, cTnT alone or in combination with one of these markers may be helpful to identify patients requiring more potent antithrombin or antiplatelet therapy. PMID- 10739379 TI - The impact of Glu102Lys on the factor X function in a patient with a doubly homozygous factor X deficiency (Gla14Lys and Glu102Lys). AB - Two homozygous point mutations were found in a patient with factor X (FX) deficiency; One results in substitution of Lys for Gla+ 14 and the second causes a Lys substitution for Glu102. The proposita has a severely reduced FX coagulant activity in the extrinsic (<1% of normal) and in the intrinsic (30% of normal) system of coagulation and after activation with Russel's viper venom (18% of normal). The FX antigen is reduced in this patient to 20% of normal. The substitution of Lys for Glu102 in FX deficiency has been reported previously in a heterozygous state in conjunction with a Lys for Gla+14 substitution and with a Pro for Ser334 substitution. The contribution of the Lys for Glu102 substitution in the observed combined FX defect in these patients was unclear. The mutation causing the Glu102Lys substitution was introduced by site directed mutagenesis into a wild-type FX cDNA, and recombinant protein was expressed in HEK 293 cells. Compared to the wild-type FX cDNA, the mutant construct had a 67% activity upon activation in the extrinsic system, 93% activity upon activation in the intrinsic system and 72% after activation with RVV. The data presented show that the substitution of Lys for Glu102 results in a minor functional defect of the FX molecule. PMID- 10739378 TI - Inherited thrombophilia as a risk factor for the development of ischemic stroke in young adults. AB - INTRODUCTION: Several recent studies have analyzed a possible effect of thrombophilia risk factors such as factor V Leiden, the prothrombin variant (allele 20210 A), and homozygosity for thermolabile methylenetetrahydrofolate reductase (MTHFR-T) on the development of ischemic stroke (IS). In the present study, we determined the role of these prothrombotic polymorphisms in the early onset of arterial IS or cerebral venous thrombosis (CVT) in a group of young Brazilian adults of Caucasian and African descent. MATERIALS AND METHODS: We conducted a cross-sectional study of 167 survivors of IS (153 patients with arterial IS and 14 cases of CVT; 66 men: 101 women; 124 of Caucasian and 43 of African origin; median age: 32.6 years; range: 15 to 45 years) and compared the prevalence of inherited thrombophilia risk factors with a control group of 225 sex and age matched individuals of the same ethnic background. To determine the interaction with atherogenic risk factors, the following diagnoses were considered: hypertension, hyperlipoproteinemia, diabetes mellitus, smoking status and use of oral contraceptives. RESULTS: In the arterial IS group, no significant variation was found between patients and controls of Caucasian origin regarding the prevalence of factor V Leiden (P = 0.92), the prothrombin variant (P = 0.13) or homozygosity for MTHFR-T (P = 0.61). Among Brazilians of African descent, 10.3% were homozygous for MTHFR-T, which was significantly elevated, odds ratio of 5.9 (95% CI: 0.88 to 49.15). In the CVT group, two Caucasian patients (20%) were heterozygous for the prothrombin variant, odds ratio of 9.7 (95% CI: 0.95 to 89.71) and one patient was carrier of factor V Leiden (P = 0.49). No prothrombotic polymorphism was identified in patients with CVT of African descent. All women in the CVT group were in use of oral contraceptives or in the post-partum state. DISCUSSION: Inherited thrombophilia risk factors were not found to increase the risk of arterial IS among young patients of Caucasian descent. However, a potential role of homozygosity for MTHFR-T was observed in a small group of patients of African origin. The analysis of patients with CVT revealed an increased risk due to the prothrombin gene variant or oral contraceptive use. Further studies including all incoming patients with IS are necessary to evaluate the impact of inherited thrombophilia risk factors on early mortality. PMID- 10739380 TI - Severe factor VII deficiency caused by a novel mutation His348 to Gln in the catalytic domain. AB - Factor VII is a vitamin K-dependent zymogen that plays a key role in the initiation of the extrinsic pathway. A severe factor VII deficiency was identified in a 45-year old male whose plasma factor VII antigen was less than 60 ng/ml and expressed 5.2% of normal factor VII activity. DNA sequence analysis of the patient's factor VII gene showed a thymidine to guanine transversion at nucleotide 10968 in exon VIII that results in a novel amino acid substitution of His348 to Gln. The patient was homozygous for this mutation, whereas some of his family members were heterozygous. Both wild type and mutant factor VII were transiently expressed in COS-1 cells. The level of secreted mutant factor VII antigen was only 11.0% of the level of wild type factor VII. In CHO cells stably transfected with the mutant factor VII, only 37.3% of the total labeled FVII was secreted into the conditioned media and the remainder was retained inside the cells. These data suggest this mutation leads to factor VII deficiency due to the impaired secretion of the molecule. PMID- 10739381 TI - Fast and efficient mutation detection method using multiplex PCR and cycle sequencing--application to haemophilia B. AB - A method using multiplex PCR followed by cycle-sequencing has been developed to detect mutations in the FIX gene. The procedure was evaluated in 45 severe or mild haemophilia B patients from 45 unrelated families. At least one deleterious mutation was identified in every haemophiliac demonstrating the efficiency of the method. Furthermore the described procedure offers many advantages compared to other screening detection methods: it is fast (less than 48 h), simple (partly automated) and of relatively low cost (it requires only one PCR). PMID- 10739382 TI - An automated heteroduplex assay for the Pi(A) polymorphism of glycoprotein IIb/IIIa, multiplexed with two prothrombotic genetic markers. AB - Screening for the PIA polymorphism has been made faster and simpler with the advent of heteroduplex technology. Simultaneous screening for three common prothrombotic polymorphisms pl(A), factor V Leiden, and MTHFR(C677T) has been achieved with multiplex heteroduplex analysis. We describe a quick and simple method for PlA heteroduplex probe production. The probe was multiplexed with heteroduplex probes for MTHFR(C677T) and factor V Leiden polymorphisms in a one tube assay, allowing rapid automated genotyping of all three. This automated multiplex assay was applied to a cohort of 165 patients and showed excellent correlation with gel-based assays, both PAGE and RFLP. This approach will facilitate the analysis of multiple polymorphisms in complex disease in large populations. PMID- 10739384 TI - Bovine protein C inhibitor has a unique reactive site and can transiently inhibit plasmin. AB - Protein C inhibitor (PCI) regulates the anticoagulant protein C pathway by neutralizing activated protein C and thrombin-thrombomodulin complex in the human hemostatic system. In this study, we cloned a full-length bovine PCI cDNA encoding a putative 19-residue signal peptide and a 385-residue mature protein; this showed 70.6%, 70.6%, 57.5% and 59.6% amino acid sequence homology with the human, rhesus monkey, rat and mouse PCIs, respectively. Bovine PCI mRNA (2.1 kb in size) was expressed strongly in the liver, and moderately in the kidney and testis, but not in other tissues tested. Bovine PCI has a putative reactive site peptide bond, Lys-Ser, that is different from the reactive site sequence (Arg Ser) of other species' PCI. We found that bovine PCI transiently inhibits bovine plasmin, but not human plasmin. Western blot analysis showed that the reactive site of bovine PCI is cleaved during the course of complex formation with bovine plasmin; degraded PCI is released from the complex gradually concomitant with the recovery of plasmin activity. These findings suggest that bovine PCI plays a role not only in the protein C pathway but also in the fibrinolytic activity of bovine hemostatic system. PMID- 10739383 TI - Oxidation of human alpha-thrombin by the myeloperoxidase-H2O2-chloride system: structural and functional effects. AB - The myeloperoxidase-H2O2-chloride system (MPOS) is exploited by white blood cells to generate reactive oxygen species in many processes involved in the pathogenesis of inflammation and atherothrombosis. This, study investigated the biochemical and functional effects of alpha-thrombin oxidation by MPOS. This system, in the presence of 100 microM L-tyrosine, caused in the thrombin molecule loss of tryptophan and lysine residues and formation of dityrosine, chloramine and carbonyl groups. The same changes could be directly induced by thrombin incubation with reagent HOCI, but not with H2O2 alone. Exposure to either MPOS or HOCl caused major functional abnormalities in human alpha-thrombin. The interaction of oxidized (ox-)thrombin with Protein C and antithrombin III-heparin complex were most sensitive to oxidation, being the kcat/Km value for Protein C hydrolysis roughly reduced 13-fold and the affinity for the antithrombin III heparin complex decreased approximately 15-fold. Ox-thrombin interaction with small synthetic peptides showed several changes, arising from a perturbation of the S2-S3 specificity of the enzyme. Ox-thrombin was also characterized by a 5 fold decrease of the kcat/Km value for both fibrinopeptide A and B release from fibrinogen, a 5.8-fold increase of the EC50 value for platelet activation and a 2 fold decrease of binding affinity for thrombomodulin. The above results indicate a high sensitivity of thrombin to oxidative modifications by myeloperoxidase. Perturbed interactions with Protein C and the heparin-ATIII complex were the most relevant functional abnormalities of ox-thrombin. PMID- 10739385 TI - A simple, quick one-step ELISA assay for the determination of complex plasma factor XIII (A2B2). AB - A new highly sensitive sandwich ELISA assay was developed for the determination of plasma factor XIII (FXIII). Plasma FXIII is a tetrameric complex of two types of subunits (A2B2). A biotinylated monoclonal capture-antibody against the B subunit and a peroxidase-labelled monoclonal tag-antibody against the A-subunit were added to the plasma dilution and the amount of the complex attached to streptavidin-coated microplate was quantitated by measuring peroxidase activity. Only the tetrameric plasma FXIII reacted in the assay, non-complexed A or B subunits showed no reaction. The assay is linear up-to 40 microg/L of FXIII in the assay mixture. It is a quick one-step assay which can be performed within two hours. At normal and low FXIII concentration within batch reproducibility was 2.0% and 3.3%, day to day variation was 5.5% and 8.7%, respectively. Its high sensitivity allows reliable measurement at FXIII concentrations below 1% of normal average. Plasma samples can be stored for the assay at -20 degrees C for at least one month. Plasma levels of healthy individuals were normally distributed and no gender difference was observed. A reference interval of 14-28 mg/L (67-133%) was established. PMID- 10739386 TI - Mapping and functional studies of two alloantibodies developed in patients with type 3 von Willebrand disease. AB - Inhibitors against von Willebrand factor (vWF) developed in two unrelated multitransfused patients (patients 1 and 2) with severe (type 3) von Willebrand disease (vWD) were analyzed. Both inhibitors were identified as antibodies of the IgG class by ELISA using immobilized purified vWF and either serum or purified Ig from the patients. Typing, mapping and functional studies of both antibodies revealed significantly distinct properties. Patient 1 antibody contained all subclasses of IgG (1, 2, 3 and 4) whereas antibody from patient 2 was a mixture of only IgG1 and 4. By ELISA using a series of immobilized purified proteolytic fragments of vWF, patient 1 antibody mainly bound to fragment SpIII and, to a lower extent, to fragments SpII and SpI; it poorly bound to P34 and the 39/34 kDa fragment. In contrast, patient 2 antibody only bound to fragments corresponding to the N-terminal portion of vWF but failed to bind to SpII. Functional studies were performed by testing the capacity of each antibody to inhibit vWF binding to its various ligands. Both antibodies blocked vWF binding to Factor VIII (FVIII), fibrillar type III collagen, bitiscetin and the subsequent induced binding to GPIb. Patient 1 antibody also blocked vWF binding to platelet GPIb when induced by ristocetin. However it failed to block vWF binding to GPIb when induced by botrocetin as well as the binding of botrocetin itself to vWF. Our data thus suggest that this inhibitor does not recognize the GPIb-binding site on vWF but the sites of vWF involved in its interaction with ristocetin. In contrast, we observed that patient 2 antibody blocked vWF binding to platelet GPIb induced by either agonist as well as vWF binding to botrocetin. Finally, the effect of the antibodies was tested on vWF binding to GPIIb/IIIa. As expected from the mapping experiments, only IgG from patient 1 blocked the interaction while IgG from patient 2 had no effect. In conclusion, we have shown that two multitransfused patients with type 3 vWD have developed alloantibodies with similar properties to those of polyclonal antibodies but with distinct effects on the functions of vWF. PMID- 10739387 TI - The influence exerted by a restricted phospholipid microenvironment on the expression of tissue factor activity at the cell plasma membrane surface. AB - Phosphatidylserine (PhtdSer) is an anionic aminophospholipid necessary for the development of optimal tissue factor (TF) activity at the cell surface. This study investigates the implication of a restricted lipid environment with respect to PhtdSer availability on TF expression and activity. K562 cells, showing a reduced ability to externalize PhtdSer, were transfected with human TF cDNA. PhtdSer exposure and TF activity were examined in transfected cells and compared to monocytic THP-1 cells expressing constitutive and inducible TF or megakaryocytic HEL cells showing a high PhtdSer externalization potency. TF expression was evidenced by flow cytometry and its activity measured using functional assays. PhtdSer exposure was monitored by enzymatic prothrombinase assay. One clone (DC9) expressed a stable amount of TF antigen without global modification of its membrane status. Despite a noticeable TF expression level, clone DC9 presented only a weak TF activity even after ionophore stimulation. The apparent Km, relative to factor X (FX) activation by TF-factor VIIa (FVIIa) complex, was 335 nM versus 70 nM for THP-1 cells. The velocity of the reaction was found 3-fold slower in DC9 than THP-1 cells. Ionophore treatment resulting in slightly enhanced amounts of available PhtdSer abolished this difference. The DC9 clone appears suitable for further investigations on the biology of TF expressed at the surface of cells where the contribution of PhtdSer is significantly attenuated. Such cells should enable further assessment of the role of TF as a receptor coupled to intracellular signaling pathways and its fate during apoptotic cell death. PMID- 10739388 TI - Cellular degradation of free and inhibitor-bound tissue-type plasminogen activator--requirement for a co-receptor? AB - The low density lipoprotein receptor-related protein (LRP) is a multiligand clearance receptor that removes free tissue-type plasminogen activator (t-PA) or complexes of t-PA with plasminogen activator inhibitor type 1 (PAI-1) from the blood circulation or the pericellular space. Co-receptors are essential for LRP mediated clearance of several ligands (e.g. glycosaminoglycans for thrombin/protease nexin and lipoprotein lipase, and the urokinase receptor for urokinase/PAI-1 complexes). The present study was undertaken to investigate whether LRP-mediated t-PA clearance requires a co-receptor as well. In five cell lines from different organs and species degradation of t-PA and t-PA/PAI-1 was mediated by LRP (or LRP-like receptors). No degradation of t-PA and t-PA/PAI-1 occurred in THP-1 or U-937 human monocyte-like cells, despite the presence of functional LRP. As glycosaminoglycans can bind t-PA and PAI-1 we investigated whether they are involved in t-PA/PAI-1 degradation. Pre-treatment of COS cells or HT1080 cells with chlorate, an inhibitor of glycosaminoglycan sulfation, did not decrease t-PA/PAI-1 degradation. Furthermore, CHO cells genetically deficient in glycosaminoglycans efficiently degraded t-PA/PAI-1. Thus it is unlikely that glycosaminoglycans are co-receptors for degradation of t-PA or t-PA/PAI-1. This study indicates that THP-1 and U-937 cells lack a critical component (co receptor?) for the LRP-mediated degradation of t-PA. PMID- 10739389 TI - Characterization of mouse thrombin-activatable fibrinolysis inhibitor. AB - Based on in vitro studies, thrombin-activatable fibrinolysis inhibitor (TAFI) has been hypothesized as a link between coagulation and fibrinolysis, but the physiological role of TAFI in vivo has not yet been established. To anticipate on the availability of genetically modified mouse models, we studied the endogenous expression of TAFI in mice. Functional TAFI was found in mouse plasma. TAFI mRNA was only detectable in the liver, showing a hepatocyte-specific expression with a pericentral lobular distribution pattern. The murine TAFI cDNA was cloned and sequenced. The deduced amino acid sequence revealed that murine TAFI is highly identical to human TAFI. The murine cDNA was stably expressed and the activated recombinant protein was functionally active; it converted the substrate hippuryl arginine, and prolonged the clot lysis time of TAFI depleted plasma. We conclude that mice have functional TAFI in plasma, which is highly similar to human TAFI. Therefore, genetically modified mice may provide useful models to study the role of TAFI in vivo. PMID- 10739390 TI - Role of gender and genetic variance in plasminogen activator inhibitor-1 secretion from human adipose tissue. AB - Gender and the 4G/5G polymorphism in the plasminogen activator inhibitor 1 (PAI 1) gene are believed to play a role in the regulation of plasma PAI-1 activity. Adipose tissue has been found to be an important source of PAI-1. The possible influence of gender and the 4G/5G polymorphism in the PAI-1 gene on PAI-1 secretion from abdominal subcutaneous adipose tissue was investigated in 59 women and 32 men. The subjects were apparently healthy, although they differed markedly inter-individually in body mass index (21-53 kg/m2). The 4G/5G polymorphism did not influence the adipose secretion rate of PAI-1 or plasma PAI-1 activity. There was no gender difference in the adipose secretion of PAI-1. In multiple regression, including body mass index (BMI), waist-to-hip ratio (WHR), plasma insulin and plasma triglycerides as the independent and adipose PAI-1 secretion as the dependent variable, only BMI and plasma triglycerides correlated independently with adipose PAI-1 secretion (r = 0.54, p <0.05; r = 0.51, p <0.05, respectively). Men had a two times higher plasma PAI-1 activity than women (p <0.05). This gender difference was mainly due to gender differences in WHR. In multiple regression analysis, BMI and WHR were identified to be independently correlated with plasma PAI-1 activity (r = 0.60, p <0.05; r = 0.52, p = 0.01, respectively). In conclusion, neither gender nor the 4G/5G polymorphism in the PAI-1 gene are associated with secretion of PAI-1 from abdominal subcutaneous adipose tissue. PMID- 10739391 TI - Randomized, placebo-controlled, blinded and cross-matched study on the antiplatelet effect of inhaled nitric oxide in healthy volunteers. AB - The platelet inhibitory effect of 0-40 ppm inhaled nitric oxide (NO) was investigated in healthy men and women. In both groups, ADP-and collagen-induced platelet aggregation was significantly inhibited 20 (T20) and 40 min (T40) after the beginning of inhalation of 5, 10, and 40 ppm. Moreover, in both men and women, the in vitro bleeding time was significantly prolonged at T20 and T40 during inhalation of 40 ppm. Inhalation of NO also inhibited P-selectin expression at 5, 10, and 40 ppm and fibrinogen binding to the GPIIb/IIIa-receptor at 40 ppm. In conclusion, in healthy volunteers, the platelet inhibitory effect of inhaled NO was not dose-related, since it was significant at 5 and 10 ppm but did not increase during the administration of higher NO concentrations. In addition, gender-related differences were only observed in ADP-induced platelet aggregation at 10 ppm and in bleeding time prolongation at 40 ppm. PMID- 10739392 TI - Monitoring of aspirin (ASA) pharmacodynamics with the platelet function analyzer PFA-100. AB - BACKGROUND: Anti-platelet drug therapy is currently performed without monitoring, because the established method of platelet aggregometry is cumbersome. The recently developed platelet function analyzer PFA-100 measures shear stress dependent, collagen epinephrine (CEPI) and collagen adenosine diphosphate (CADP) induced platelet plug formation. As the PFA-100 provides a valuable tool to detect patients with platelet dysfunction more efficiently and cost-effectively than aggregometry, we investigated its potential to monitor the efficacy of aspirin treatment. METHODS: All healthy volunteers (n = 10) received a fractionated infusion of L-aspirin to establish individual dose-response curves. Further, in a randomized, double-blind, placebo controlled two-way cross over study the same volunteers received either 50 or 100 mg aspirin/day p.o. for a period of 11 days to determine the day-to-day variability CEPI induced closure time (CT) under constant intake of low dose aspirin, and to compare the efficacy of those two doses. RESULTS: Intra- and intersubject variability of CEPI-CT averaged 9% and 22%, respectively. Seven volunteers exceeded the maximum of CEPI CT (>300 s) already after infusion of 100 mg L-aspirin. Intake of 100 mg of aspirin elicited a more rapid onset of effect than 50 mg, which was only significant on days 3 and 4 of aspirin intake. The aspirin induced CEPI-CT prolongation correlated positively with basal CEPI-CT values (r = 0.86; p = 0.001) and were strongly dependent on von Willebrand Factor levels (r = -0.9; p = 0.001). CONCLUSION: Thus, the PFA-100 system appears suitable to demonstrate an aspirin-induced platelet effect in a longitudinal study, and may be adequate to monitor a patient's compliance. However, prospective trials have to be conducted to demonstrate whether the EPI-CT achieved under ASA-intake has predictive value for cardiovascular outcome. PMID- 10739393 TI - An in-vitro model to study device-induced thrombosis and embolism: evaluation of the efficacy of tirofiban, aspirin, and dipyridamole. AB - A bovine in-vitro model was developed to investigate device-induced thromboembolism (TE) and its pharmacological intervention, using a stent as a prototype device. Emboli were assessed continuously using a light-scattering microemboli detector (LSMD). Thrombus on the stent was assessed gravimetrically at the end of the experiment. The contribution of the stent as the predominant source of detectable thromboemboli in this model was verified by placing LSMD probes upstream and downstream of the stent. The effectiveness of ethylenedinitrilo-tetraacetic-acid (EDTA) and three anti-thrombogenic agents (aspirin, dipyridamole, and tirofiban) for mitigating device-induced TE was also assessed. The results show that 1) the model has potential to study device induced TE and the efficacy of possible interventional strategies, 2) the LSMD is capable of continuous, non-invasive, real-time assessment of embolism, 3) the assessment of embolization may constitute an important part of evaluating hemocompatibility, 4) tirofiban is effective in reducing both stent-induced thrombosis and embolism above certain concentrations. PMID- 10739394 TI - GPV is a marker of in vivo platelet activation--study in a rat thrombosis model. AB - Thrombin plays a central role in the genesis of thrombotic events and is the most potent known platelet agonist. This enzyme activates platelets by cleaving G protein coupled protease activated receptors (PARs) and by binding to glycoprotein (GP) Ib. Thrombin also cleaves platelet GPV to liberate a soluble 69 kDa fragment (GPVf1), leaving a 20 kDa fragment (GPVf2) attached to the membrane. The aim of this study was to assess the value of GPV as an in vivo marker of the activation of platelets by thrombin. Newly developed monoclonal and polyclonal antibodies recognizing rat GPVf1 and GPVf2 respectively were used to detect soluble GPV by ELISA and the new NH2-terminus exposed by thrombin using flow cytometry. These assays were employed in a rat thrombosis model designed to trigger thrombin formation in vivo. When thromboplastin (4.8 ml/kg/h) was infused for 30 min, thrombin generation was reflected by a rapid increase in thrombin antithrombin (TAT) complexes in plasma and by the appearance of GPVf2 at the surface of circulating platelets. Simultaneously, GPVf1 disappeared from the surface of platelets and accumulated as a soluble fragment in plasma, where it was detected by GPV ELISA. These effects were inhibited by pretreatment of the rats with hirudin. Levels of plasma PF4 also increased in this model, but unlike GPV levels which returned slowly (> 2 hours) to baseline, PF4 had a very short half-life. In conclusion, GPV is cleaved by thrombin in vivo, circulates and is a reliable in vivo marker of the activation of platelets by thrombin. Monitoring of GPV levels in rats should be useful to evaluate the effects of antithrombotic and antiplatelet drugs, while further studies will be required to confirm the potential interest of GPV as a marker of thrombotic states in humans. PMID- 10739395 TI - Protamine sulphate inhibits platelet membrane glycoprotein Ib-von Willebrand factor activity. AB - Platelet adhesion to the injured vessel wall is essential in haemostasis and thrombosis. This process involves the interaction of the platelet glycoprotein Ib (GPIb) with surface bound von Willebrand factor (vWF). Since synthetic polycationic peptides of the general formula (Arg)n, (Lys)n or (Arg-Lys)n inhibit GPIb-vWF interaction, they were suggested as potential antithrombotics. Protamine sulphate is a highly cationic polypeptide, arginine accounting for approximately 60% of the primary sequence, utilized to neutralize the anticoagulant effect of heparin after cardiac surgery. We have investigated potential effects of protamine sulphate on the function of GPIb-vWF. Addition of protamine sulphate to platelet-rich plasma (PRP), reduced significantly the GPIb-vWF activity as assessed by ristocetin-induced platelet agglutination. When protamine sulphate was added to PRP containing heparin, even at clinically relevant neutralizing doses the GPIb-vWF activity was reduced by 20-30% (p < 0.001). Protamine sulphate in excess of heparin nearly abolished the activity. Furthermore, the direct effect of protamine sulphate on collagen-induced platelet thrombus formation in non-anticoagulated human blood was investigated by employing an ex-vivo parallel plate perfusion chamber device. Protamine sulphate (200 microg/mL) reduced platelet-collagen adhesion at shear rates of 650 and 2600 sec(-1) by 40% (p< 0.004) and 45% (p < 0.0001), respectively. The corresponding platelet thrombus volumes were concomitantly reduced by 90% (p < 0.006) and 84% (p < 0.05). Our data are questioning the rationale for empirical repetitive protamine sulphate administration when so-called "heparin rebound" after cardiac surgery is suspected, since protamine sulphate in excess of heparin may impair the platelet GPIb-vWF interaction necessary for normal haemostasis. PMID- 10739396 TI - Homocysteine and oxidized low density lipoprotein enhanced platelet adhesion to endothelial cells under flow conditions: distinct mechanisms of thrombogenic modulation. AB - We investigated the effects of two well established risk factors for cardiovascular disease, homocysteine and oxidized low density lipoprotein (ox LDL), on endothelial cell thrombogenicity. For this purpose we studied platelet adhesion to human endothelial cells (EC) under flow conditions at a shear rate of 350 s(-1) following EC treatment with either homocysteine or ox-LDL. Treatment of EC with either homocysteine (1 or 10 mmol/L for 16 h) or ox-LDL (100 microg/ml for 16 h) resulted in a 2-3 fold enhancement in platelet adhesion. The enhancement in platelet adhesion induced by 1 mmol/L homocysteine, but not that induced by 10 mmol/L homocysteine, was absolutely dependent on fibrin formation. Homocysteine treatment has significantly increased the cell surface tissue factor (TF) activity and slightly reduced the expression of the intercellular adhesion molecule I (ICAM-1). In contrast, ox-LDL treatment upregulated ICAM-1 expression and had no significant effect on endothelial TF activity. Neither homocysteine nor Ox-LDL affected surface expression of the alpha(v)beta3 integrin. The homocysteine-induced enhancement in platelet adhesion was almost completely abolished by blockade of the EC TF activity by a polyclonal antibody. The enhancing effect of homocysteine was also greatly reduced by inhibition of the EC alpha(v)beta3 integrin, but was not affected by blockade of EC ICAM-1. On the other hand, ox-LDL-induced enhancement in platelet - EC adhesion was greatly inhibited by blocking ICAM-1 or alpha(v)beta3, but remained unaffected by inhibition of TF activity. Preincubation of platelets with the glycoprotein IIb IIIa (GPIIb-IIIa) antagonist Reo-Pro has virtually abolished the enhancing effect of both homocysteine and ox-LDL. Our results suggest that homocysteine and ox-LDL might increase endothelial thrombogenicity by distinct mechanisms: homocysteine - by inducing TF activity, and ox-LDL - by upregulating ICAM-1, both of which enhance GPIIb-IIIa/fibrinogen dependent platelet adhesion to EC. The alpha(v)beta3 integrin, although not affected by EC stimulation, seems to play a crucial role in platelet-EC interaction regardless of the mechanism of EC perturbation. PMID- 10739397 TI - Effect of instruments on lupus anticoagulant testing. PMID- 10739398 TI - A case report of a paroxysmal nocturnal hemoglobinuria with increased coagulation activation markers while on treatment with LMWH with anti-Xa levels above the therapeutic range. PMID- 10739399 TI - The contribution of plasma (R)- & (S)-warfarin and vitamin K concentrations to intra-individual variability in anticoagulation. PMID- 10739401 TI - Thrombophilic mutations in Iran. PMID- 10739400 TI - Evaluation of lipoprotein(a) and genetic prothrombotic risk factors in patients with recurrent foetal loss. PMID- 10739402 TI - Comparison of the risk of pulmonary embolism and deep vein thrombosis in the presence of factor V Leiden or prothrombin G20210A. PMID- 10739403 TI - Automated, simultaneous detection of the factor V Leiden and prothrombin (G20210A) variants using multiplex PCR and a line probe assay. PMID- 10739404 TI - Monitoring platelet inhibition during chronic oral platelet glycoprotein IIb/IIIa blockade: are we missing something? PMID- 10739405 TI - Genes, environment, and mental health wellness. PMID- 10739406 TI - Cognition: long-term potentiation. PMID- 10739407 TI - Neuroanatomical hypothesis of panic disorder, revised. AB - OBJECTIVE: In a 1989 article, the authors provided a hypothesis for the neuroanatomical basis of panic disorder that attempted to explain why both medication and cognitive behavioral psychotherapy are effective treatments. Here they revise that hypothesis to consider developments in the preclinical understanding of the neurobiology of fear and avoidance. METHOD: The authors review recent literature on the phenomenology, neurobiology, and treatment of panic disorder and impressive developments in documenting the neuroanatomy of conditioned fear in animals. RESULTS: There appears to be a remarkable similarity between the physiological and behavioral consequences of response to a conditioned fear stimulus and a panic attack. In animals, these responses are mediated by a "fear network" in the brain that is centered in the amygdala and involves its interaction with the hippocampus and medial prefrontal cortex. Projections from the amygdala to hypothalamic and brainstem sites explain many of the observed signs of conditioned fear responses. It is speculated that a similar network is involved in panic disorder. A convergence of evidence suggests that both heritable factors and stressful life events, particularly in early childhood, are responsible for the onset of panic disorder. CONCLUSIONS: Medications, particularly those that influence the serotonin system, are hypothesized to desensitize the fear network from the level of the amygdala through its projects to the hypothalamus and the brainstem. Effective psychosocial treatments may also reduce contextual fear and cognitive misattributions at the level of the prefrontal cortex and hippocampus. Neuroimaging studies should help clarify whether these hypotheses are correct. PMID- 10739408 TI - A multidimensional twin study of mental health in women. AB - OBJECTIVE: While researchers have increasing insight into the role of genetic and environmental factors in the etiology of psychiatric and substance use disorders, they know much less about how such factors influence the dimensions of healthy psychological functioning. METHOD: In a population-based sample of 794 female female twin pairs, the authors examined, by using multivariate structural equation modeling, six dimensions of mental health: perceived physical health, nonconflictual interpersonal relationships, anxious-depressive symptoms, substance use, social support, and self-esteem. RESULTS: The best-fit model was complex and constituted five common factors (two genetic, one family environmental, and two unique environmental); variable-specific genetic effects for physical health, substance use, and social support; and variable-specific family environmental effects for interpersonal relationships and substance use. Genetic effects were seen for all six dimensions; total heritabilities ranged from 16% to 49%. Family environment was an important influence on interpersonal relationships, substance use, and social support. CONCLUSIONS: Mental health is a complex phenotype that is influenced by a diverse array of genetic and environmental factors. While genetic factors appear to be of moderate etiologic importance in all major dimensions of mental health, the family environment is an important influence on only interpersonal relations, social support, and substance use. PMID- 10739409 TI - Relationship between dopamine D(2) occupancy, clinical response, and side effects: a double-blind PET study of first-episode schizophrenia. AB - OBJECTIVE: Since all antipsychotics block dopamine D(2) receptors, the authors investigated how well D(2) receptor occupancy in vivo predicts clinical response, extrapyramidal side effects, and hyperprolactinemia. METHOD: In a double-blind study, 22 patients with first-episode schizophrenia were randomly assigned to 1.0 or 2. 5 mg/day of haloperidol. After 2 weeks of treatment, D(2) receptor occupancy was determined with [(11)C]raclopride and positron emission tomography, and clinical response, extrapyramidal side effects, and prolactin levels were measured. Patients who showed adequate responses continued taking their initial doses, those who did not respond had their doses increased to 5.0 mg/day, and evaluations were repeated at 4 weeks for all patients. RESULTS: The patients showed a wide range of D(2) occupancy (38%-87%). The degree of receptor occupancy predicted clinical improvement, hyperprolactinemia, and extrapyramidal side effects. The likelihood of clinical response, hyperprolactinemia, and extrapyramidal side effects increased significantly as D(2) occupancy exceeded 65%, 72%, and 78%, respectively. CONCLUSIONS: The study confirms that D(2) occupancy is an important mediator of response and side effects in antipsychotic treatment. The data are consistent with a "target and trigger" hypothesis of antipsychotic action, i.e., that the D(2) receptor specificity of antipsychotics permits them to target discrete neurons and that their antagonist properties trigger within those neurons intracellular changes that ultimately beget antipsychotic response. While limited to haloperidol, the relationship between D(2) occupancy and side effects in this study helps explain many of the observed clinical differences between typical and atypical antipsychotics. PMID- 10739410 TI - Barriers to the treatment of social anxiety. AB - OBJECTIVE: This article evaluates barriers to treatment reported by adults with social anxiety who participated in the 1996 National Anxiety Disorders Screening Day. METHOD: The background characteristics of screening day participants with symptoms of social anxiety (N=6,130) were compared with those of participants without social anxiety (N=4,507). Barriers to previous mental health treatment reported by participants with and without symptoms of social anxiety were compared. RESULTS: Social anxiety was strongly associated with functional impairment, feelings of social isolation, and suicidal ideation. Compared to participants without social anxiety, those with social anxiety were significantly more likely to report that financial barriers, uncertainty over where to go for help, and fear of what others might think or say prevented them from seeking treatment. However, they were significantly less likely to report they avoided treatment because they did not believe they had an anxiety disorder. Roughly one third (N=1,400 of 3,682, 38.0%) of the participants with symptoms of social anxiety who were referred for further evaluation were specifically referred for an evaluation for social phobia. CONCLUSIONS: Social anxiety is associated with a distinct pattern of treatment barriers. Treatment access may be improved by building public awareness of locally available services, easing the psychological and financial burden of entering treatment, and increasing health care professionals' awareness of its clinical significance. PMID- 10739411 TI - Identity disturbance in borderline personality disorder: an empirical investigation. AB - OBJECTIVE: Identity disturbance is one of the nine criteria for borderline personality disorder in DSM-IV, yet the precise nature of this disturbance has received little empirical attention. This study examines 1) the extent to which identity disturbance is a single construct, 2) the extent to which it distinguishes patients with borderline personality disorder, and 3) the role of sexual abuse in identity disturbance in patients with borderline personality disorder. METHOD: The authors constructed an instrument that consisted of 35 indicators of identity disturbance culled from relevant clinical and theoretical literature and asked clinicians to rate a patient on each of the items. The patient group consisted of 95 subjects diagnosed with borderline personality disorder (N=34), another personality disorder (N=20), or no personality disorder (N=41). Relevant diagnostic, demographic, and developmental history data were also collected. The authors used factor analysis to ascertain whether identity disturbance is a unitary construct and then examined the relation between dimensions of identity disturbance and borderline diagnosis after controlling for sexual abuse history. RESULTS: Four identity disturbance factors were identified: role absorption (in which patients tend to define themselves in terms of a single role or cause), painful incoherence (a subjective sense of lack of coherence), inconsistency (an objective incoherence in thought, feeling, and behavior), and lack of commitment (e.g., to jobs or values). All four factors, but particularly painful incoherence, distinguished patients with borderline personality disorder. Although sexual abuse was associated with some of the identity factors, particularly painful incoherence, borderline pathology contributed unique variance beyond abuse history to all four identity disturbance factors. The data also provided further evidence for an emerging empirical distinction between two borderline personality disorder types: one defined by emotional dysregulation and dysphoria, the other by histrionic characteristics. CONCLUSIONS: Identity disturbance is a multifaceted construct that distinguishes patients with borderline personality disorder from other patients. Some of its components are related to a history of sexual abuse, whereas others are not. Identity disturbance appears to be characteristic of borderline patients whether or not they have an abuse history. PMID- 10739412 TI - Regional neural dysfunctions in chronic schizophrenia studied with positron emission tomography. AB - OBJECTIVE: Whether chronicity of illness produces progressive neural abnormality is an important question in current schizophrenia research. Positron emission tomography (PET) offers an opportunity to visualize and measure blood flow in vivo to address this issue. The authors previously compared healthy volunteers with neuroleptic-naive patients experiencing their first episode of schizophrenia and reported that abnormalities in blood flow, including lower flow in prefrontal regions and higher flow in the thalamus and cerebellum, are present at the early stage of schizophrenic illness. The goal of the present study was to measure blood flow with PET in patients with chronic schizophrenia. METHOD: PET was used to examine regional cerebral blood flow (rCBF) in 30 patients with chronic schizophrenia and 30 normal comparison subjects. To determine if the patterns of flow abnormality in the patients with chronic schizophrenia were similar to those of patients experiencing their first episode of schizophrenia, the same cognitive condition was examined as in the earlier study. The patients with chronic schizophrenia in the current study had been neuroleptic-free for at least 3 weeks. RESULTS: As in the authors' previous study, the chronically ill patients showed lower flow in prefrontal areas and higher flow in thalamic and cerebellar regions than normal comparison subjects, suggesting that a similar neural dysfunction occurs in both first-episode and chronic schizophrenia. CONCLUSIONS: rCBF abnormalities in patients with chronic schizophrenia are not due to chronicity of illness or the effects of medication. These results provide evidence that the primary neural abnormalities in schizophrenia may occur in cortical, cerebellar, and thalamic regions and that the dysfunction in these regions may explain the "loosening of associations" that Bleuler considered to be the fundamental cognitive phenotype of schizophrenia. These abnormalities can be reconceptualized as "cognitive dysmetria." PMID- 10739413 TI - Neuropsychology of first-episode schizophrenia: initial characterization and clinical correlates. AB - OBJECTIVE: Neuropsychological impairments are well documented in schizophrenia and are important targets of treatment. Information about the severity and pattern of deficits after treatment for the first psychotic episode and about relationships between these deficits and syndromal characteristics remains limited. METHOD: Comprehensive neuropsychological assessments including 41 individual tests were given to 94 patients with first-episode schizophrenia after initial stabilization of psychosis and to a comparison group of 36 healthy volunteers. Profiles of neuropsychological deficits and the relationship of deficits to sex and handedness were examined. Correlations of neuropsychological deficit with a broad range of historical and clinical characteristics, including outcome, were explored. RESULTS: Patients had a large generalized neuropsychological deficit (1.5 standard deviations compared to healthy volunteers). Patients also had, superimposed on the generalized deficit, subtle relative deficits (less than 0.5 standard deviation compared to their own average profile) in memory and executive functions. Learning/memory dysfunction best distinguished patients from healthy individuals; after accounting for this difference, only motor deficits further distinguished the groups. Patients with higher neuropsychological ability had only memory deficits, and patients with lower ability had both memory and executive deficits. No sex differences were observed beyond the normal advantage for men in motor speed. Dextral patients had less severe generalized deficit. Severity of residual symptoms was associated with greater generalized deficit. Executive and attentional deficits were most linked to global functional impairment and poor outcome. CONCLUSIONS: The results document a large generalized deficit, and more subtle differential deficits, in clinically stabilized first-episode patients. Learning/memory deficits were observed even in patients with less severe generalized deficit, but the pattern was unlike the amnestic syndrome and probably reflects different mechanisms. Executive and attentional deficits marked the more severely disabled patients, and may portend relatively poor outcome. Failure to develop typical patterns of cerebral dominance may increase the risk for greater generalized deficit. PMID- 10739414 TI - Neurological signs and the heterogeneity of schizophrenia. AB - OBJECTIVE: More than 20 studies of schizophrenia have found a three-factor model of symptom complexes or syndromes consisting of hallucinations/delusions, disorganization of thought and behavior, and negative symptoms. Several lines of evidence suggest that these syndromes relate to neurobiological differences. We examined the relationship of these three syndromes to neurological signs. METHOD: The relationships among the subscales of the Neurological Evaluation Scale and hallucinations/delusions, disorganization, and the deficit syndrome were examined in 83 clinically stable outpatients with schizophrenia. Patients with the deficit syndrome have enduring, idiopathic (or primary) negative symptoms. RESULTS: Each of the three syndromes had a distinctive pattern of relationships to neurological signs. Disorganization was significantly related to the total score on the Neurological Evaluation Scale, to sensory integration, and to the sequencing of complex motor acts. The deficit syndrome was significantly related to sensory integration only. Neither hallucinations/delusions nor a continuous measure of negative symptoms derived from the Brief Psychiatric Rating Scale (that measured both primary and secondary negative symptoms, as well as enduring and transient symptoms) was related to any of the Neurological Evaluation Scale subscales or total score. Drug treatment was not related to neurological impairment. CONCLUSIONS: The results further support the neurobiological significance of the three clinical syndromes of schizophrenia. Ratings on a scale measuring negative symptoms appear to be less sensitive to neurobiological correlates than is the categorization of the presence or absence of the deficit syndrome. PMID- 10739415 TI - Violence and delusions: data from the MacArthur Violence Risk Assessment Study. AB - OBJECTIVE: Previous work has suggested that delusions are associated with a higher risk of violence, particularly delusions in which patients believe that people are seeking to harm them or that outside forces are controlling their minds (denoted as "threat/control override" delusions). This study explores the relationship between delusions and violence among patients recently discharged from acute psychiatric hospitalization. METHOD: Data were drawn from the MacArthur Violence Risk Assessment Study, a study of violence in the community that followed 1,136 recently discharged psychiatric patients for 1 year. Interviews at discharge and at five 10-week intervals gathered clinical, historical, situational, and dispositional information, including the presence and nature of delusional thoughts. Violence was ascertained from reports of subjects, collateral informants, and official records. RESULTS: Neither delusions in general nor threat/control override delusions in particular were associated with a higher risk of violent behavior. Comparisons with prior studies suggest that reliance on subject self-reports of delusional symptoms may result in mislabeling as delusions other phenomena that can contribute to violence. CONCLUSIONS: Although delusions can precipitate violence in individual cases, these data suggest that they do not increase the overall risk of violence in persons with mental illness in the year after discharge from hospitalization. PMID- 10739416 TI - Empirical examination of current depression categories in a population-based study: symptoms, course, and risk factors. AB - OBJECTIVE: Research studies on the validity of current diagnostic and subthreshold categories of depression that use a population-based follow-up design are rare. The authors examined the validity and utility of four current depression categories by examining subject transition between categories and the symptoms, course, and risk factors of each. METHOD: A general population sample of 1,920 adults from the Baltimore Epidemiologic Catchment Area 13-year follow-up study were examined. Data on diagnoses, symptoms, course, and risk factors were collected by using the National Institute of Mental Health Diagnostic Interview Schedule, the Life Chart Interview, and an office visit. Polychotomous regression was used to examine the heterogeneity of four diagnostic categories: major depressive disorder, depressive syndrome, dysthymia, and a comorbid depression condition (major depressive disorder and dysthymia). RESULTS: Transitions between the four depression categories occurred over the 13 years. Symptom profiles for the four categories were parallel but differed in severity. Course characteristics among the four categories slightly differed. Risk factor profiles showed significant differences. Family history was associated with both depressive syndrome and major depressive disorder. Stressful life events were most strongly associated with depressive syndrome. Female gender was most strongly associated with the comorbid depression category. CONCLUSIONS: The evidence suggests that except for dysthymia, the depression categories are genetically homogeneous and environmentally heterogeneous. Stress is associated with mild depression, and gender is associated with severe depression. The apparent familial transmission of the subthreshold entity, depressive syndrome, needs further investigation. PMID- 10739418 TI - Use of exclusion criteria in selecting research subjects and its effect on the generalizability of alcohol treatment outcome studies. AB - OBJECTIVE: Researchers have not systematically examined how exclusion criteria used in selection of research subjects affect the generalizability of treatment outcome research. This study evaluated the use of exclusion criteria in alcohol treatment outcome research and its effects on the comparability of research subjects with real-world individuals seeking alcohol treatment. METHOD: Eight of the most common exclusion criteria described in the alcohol treatment research literature were operationalized and applied to large, representative clinical patient samples from the public and private sectors to determine whether the hypothetical research samples differed substantially from real-world samples. Five hundred ninety-three consecutive individuals seeking alcohol treatment at one of eight treatment programs participated. A trained research technician gathered information from participants on demographic variables and on alcohol, drug, and psychiatric problems as measured by the Addiction Severity Index. RESULTS: Large proportions of potential research subjects were excluded under most of the criteria tested. The overall pattern of results showed that African Americans, low-income individuals, and individuals who had more severe alcohol, drug, and psychiatric problems were disproportionately excluded under most criteria. CONCLUSIONS: Exclusion criteria can result in alcohol treatment outcome research samples that are more heavily composed of white, economically stable, and higher-functioning individuals than are real-world samples of substance abuse patients seen in clinical practice, potentially compromising the generalizability of results. For both scientific and ethical reasons, in addition to studies that use exclusion criteria, outcome research that uses no or minimal exclusion criteria should be conducted so that alcohol treatment outcome research can be better generalized to vulnerable populations. PMID- 10739417 TI - Diagnosis of nonpsychotic patients in community clinics. AB - OBJECTIVE: Diagnosis-specific, proven efficacious treatments are a major recent advance in psychiatry. Appropriate use of such treatments presupposes patients who meet the diagnostic criteria and clinicians who have accurately diagnosed the target disorder and comorbid conditions. Since little is known about whether these prerequisites are commonly met, the authors conducted a study at two community treatment sites to determine the frequency of various axis I diagnoses and the concordance between the diagnoses recorded in patient charts and those obtained by a structured interview. Given that a DSM diagnosis may not be sufficient to understand a patient's problems, the authors also obtained ratings of interpersonal functioning. METHOD: The subjects were 164 nonpsychotic patients who were seen at a rural (N=114) or urban (N=50) community treatment facility. Raters trained to reliably use the Structured Clinical Interview for DSM-IV (SCID) conducted diagnostic interviews. Clinical charts were reviewed to obtain clinical diagnoses. Patients completed questionnaires regarding interpersonal functioning. RESULTS: Most (N=145, 88%) of the patients met the SCID criteria for a current axis I diagnosis, and 53% (N=87) met the criteria for two or more disorders. Clinical and SCID diagnoses had poor agreement. Evidence was found for interpersonal dysfunction. CONCLUSIONS: Most patients met the diagnostic criteria for conditions for which there are proven treatments; however, inaccurate diagnosis proved common. This barrier to optimal treatment could be ameliorated with the use of structured interviews for common diagnoses. Scores on social/interpersonal measures support the premise that DSM symptoms provide only part of the relevant information about patients' conditions. PMID- 10739419 TI - Evaluation of competence to consent to assisted suicide: views of forensic psychiatrists. AB - OBJECTIVE: Mental health evaluation of competence to consent has been proposed as an important safeguard for patients requesting assisted suicide, yet mental health professionals have not developed guidelines or standards to aid in such evaluations. The authors surveyed a national sample of forensic psychiatrists in the United States regarding the process, thresholds, and standards that should be used to determine competence to consent to assisted suicide. METHOD: An anonymous questionnaire was sent to board-certified forensic psychiatrists between August and October 1997. RESULTS: Of the 456 forensic psychiatrists who were sent the questionnaire, 290 (64%) responded. Sixty-six percent believed that assisted suicide was ethical in at least some circumstances, and 63% thought that it should be legalized for some competent persons. Twenty-four percent indicated that it was unethical for psychiatrists to determine competence; however, 61% thought such an evaluation should be required in some or all cases. Seventy-eight percent recommended a very stringent standard of competence. Seventy-three percent believed that at least two independent examiners were needed to determine competence, and 44% favored requiring judicial review of a decision. Fifty-eight percent believed that the presence of major depressive disorder should result in an automatic finding of incompetence. Psychiatrists with ethical objections to assisted suicide advocated a higher threshold for competence and more extensive review of a decision. CONCLUSIONS: The ethical views of psychiatrists may influence their clinical opinions regarding patient competence to consent to assisted suicide. The extensive evaluation recommended by forensic psychiatrists would likely both minimize this bias and assure that only competent patients have access to assisted suicide, but the process might burden terminally ill patients. PMID- 10739420 TI - Characteristics of suicide attempts of patients with major depressive episode and borderline personality disorder: a comparative study. AB - OBJECTIVE: Suicidal behavior is highly prevalent in borderline personality disorder and major depressive episode, although the characteristics of suicide attempts in the two disorders are believed to differ. Comorbidity of borderline personality disorder and major depressive episode may obscure characteristics of suicide attempts that are uniquely related to the psychopathology of each disorder. We compared suicidal behavior in patients with borderline personality disorder, major depressive episode, and borderline personality disorder plus major depressive episode to determine whether characteristics of suicide attempts differed between groups and if aspects of core psychopathology predicted specific attempt characteristics. METHOD: Eighty-one inpatients with borderline personality disorder, including 49 patients with borderline personality disorder plus major depressive episode, were compared to 77 inpatients with major depressive episode alone on measures of depressed mood, hopelessness, impulsive aggression, and suicidal behavior, including lifetime number of attempts, degree of lethal intent, objective planning, medical damage, and degree of violence of suicide methods. RESULTS: No significant differences were found in the characteristics of suicide attempts between patients with borderline personality disorder and those with major depressive episode. However, patients with both disorders had the greatest number of suicide attempts and the highest level of objective planning. An increase in either impulsive aggression or hopelessness or a diagnosis of borderline personality disorder predicted a greater number of attempts. Hopelessness predicted lethal intent in all three groups and predicted objective planning in the group with both disorders. Medical damage resulting from the most serious lifetime suicide attempt was predicted by number of attempts. CONCLUSIONS: Comorbidity of borderline personality disorder with major depressive episode increases the number and seriousness of suicide attempts. Hopelessness and impulsive aggression independently increase the risk of suicidal behavior in patients with borderline personality disorder and in patients with major depressive episode. PMID- 10739421 TI - Association of aggressive behavior with altered serotonergic function in patients who are not suicidal. AB - OBJECTIVE: The purpose of this study was to determine whether aggression and serotonergic dysfunction are related in the absence of a history of suicidal behavior. Although serotonergic dysfunction has been implicated in aggressive and impulsive behavior, most studies of such behavior have included individuals with a history of suicide attempts. Low concentrations of CSF 5-hydroxyindoleacetic acid (5-HIAA) have been consistently associated with suicidal behavior, presenting a potential confound in the link between aggression and serotonergic dysfunction. METHOD: The authors examined the association between aggression and CSF 5-HIAA concentrations in a group of 64 patients who had different DSM-III-R axis I diagnoses and no past suicidal behavior. Aggressive (N=35) and nonaggressive (N=29) groups were defined by a median split on a six-item history of adulthood aggressive behavior. RESULTS: The aggressive group had significantly lower CSF 5-HIAA concentrations than the nonaggressive group. Aggressive individuals also scored significantly higher on self-report measures of hostility, impulsiveness, and sensation seeking. CSF 5-HIAA concentrations, however, did not correlate with self-reported hostility and impulsivity. CONCLUSIONS: There is an association between aggressive behavior and serotonergic dysfunction independent of suicidal behavior in patients with axis I disorders who exhibit relatively milder forms of aggressive behavior. Analogous to findings with suicidal behavior, a low concentration of CSF 5-HIAA is related to aggressive behavior but does not show the same relationship to the continuum of aggressive feelings and thoughts. PMID- 10739422 TI - Posttraumatic stress disorder among Hispanic Vietnam veterans. AB - OBJECTIVE: The purpose of this study was to examine posttraumatic stress disorder (PTSD) among Hispanics who served in the Vietnam War. METHOD: The authors conducted secondary data analyses of the National Vietnam Veterans Readjustment Study, a national epidemiologic study completed in 1988 of a representative sample of veterans who served during the Vietnam era (N=1,195). RESULTS: After adjustment for premilitary and military experiences, the authors found that Hispanic, particularly Puerto Rican, Vietnam veterans had significantly more severe PTSD symptoms and a higher probability of experiencing PTSD than nonminority veterans. However, they had no greater risk for other mental disorders, and their greater risk for PTSD was not explained by acculturation. Despite their more severe symptoms, Hispanic veterans, especially Puerto Rican veterans, showed no greater functional impairment than non-Hispanic white veterans. CONCLUSIONS: Hispanic Vietnam veterans, especially Puerto Rican Vietnam veterans, have a higher risk for PTSD and experience more severe PTSD symptoms than non-Hispanic white Vietnam veterans, and these differences are not explained by exposure to stressors or acculturation. This high level of symptoms was not accompanied by substantial reduction in functioning, suggesting that the observed differences in symptom reporting may reflect features of expressive style rather than different levels of illness. PMID- 10739423 TI - Treating an Orthodox Jewish woman with obsessive-compulsive disorder: maintaining reproductive and psychologic stability in the context of normative religious rituals. PMID- 10739425 TI - Two-year prospective evaluation of the relationship between acute stress disorder and posttraumatic stress disorder following mild traumatic brain injury. AB - OBJECTIVE: To assess the ability of acute stress disorder to predict posttraumatic stress disorder (PTSD), the relationship between acute stress disorder and PTSD over the 2 years following mild traumatic brain injury was determined. METHOD: Survivors of motor vehicle accidents who sustained mild traumatic brain injuries were assessed for acute stress disorder within 1 month of the trauma (N=79) and for PTSD at 6 months (N=63) and 2 years (N=50) posttrauma. RESULTS: Acute stress disorder was diagnosed in 14% of the patients. Among the patients who participated in all three assessments, 80% of the subjects who met the criteria for acute stress disorder were diagnosed with PTSD at 2 years. Of the total initial group, 73% of those diagnosed with acute stress disorder had PTSD at 2 years. CONCLUSIONS: This study provides further support for the utility of the acute stress disorder diagnosis as a predictor of PTSD but indicates that the predictive power of the diagnostic criteria can be increased by placing greater emphasis on reexperiencing, avoidance, and arousal symptoms. PMID- 10739426 TI - Posttraumatic stress disorder after severe traumatic brain injury. AB - OBJECTIVE: This study indexed the profile of posttraumatic stress disorder (PTSD) after severe traumatic injury to the brain. METHOD: Patients who sustained a severe traumatic brain injury (N=96) were assessed for PTSD 6 months after the injury with the PTSD Interview, a structured clinical interview based on DSM-III R criteria. RESULTS: PTSD was diagnosed in 26 (27.1%) of the patients. While only 19.2% (N=5) of the patients with PTSD reported intrusive memories of the trauma, 96.2% (N=25) reported emotional reactivity. Intrusive memories, nightmares, and emotional reactivity had very strong positive predictive values for the presence of PTSD. CONCLUSIONS: These findings indicate that PTSD can develop after severe traumatic brain injury. The predominance of emotional reactivity and the relative absence of traumatic memories in patients with PTSD who suffered impaired consciousness during trauma suggest that traumatic experiences can mediate PTSD at an implicit level. PMID- 10739427 TI - High levels of dopamine activity in the basal ganglia of cigarette smokers. AB - OBJECTIVE: The authors' goal was to study presynaptic dopamine activity in smoking and nonsmoking human subjects in vivo. METHOD: [(18)F]Fluorodopa ([(18)F]DOPA) uptake K(i) values in the basal ganglia of nine smoking and 10 nonsmoking healthy men were measured with positron emission tomography. RESULTS: Significantly higher [(18)F]DOPA uptake was observed in both the putamen (average 17.3% higher) and the caudate (average 30.4% higher) of smokers than in those of nonsmokers. CONCLUSIONS: Smoking is related to greater dopamine activity in the human basal ganglia. Nicotine-induced dopamine activity may be a relevant mechanism in dependence on cigarette smoking. PMID- 10739428 TI - Age-related cognitive deficits mediated by changes in the striatal dopamine system. AB - OBJECTIVE: The study examined the influence of losses in dopaminergic function on age-related cognitive deficits. METHOD: Eleven healthy subjects (21-68 years of age) completed a set of cognitive tasks used to assess perceptual speed and episodic memory. D(2) receptor binding was measured in the caudate and the putamen by using positron emission tomography. RESULTS: A gradual age-related deterioration was found for all cognitive tasks and for D(2) binding in both striatal structures. Statistical control of D(2) binding eliminated the age related cognitive variation, whereas residual effects of D(2) binding were seen after the analysis controlled for age. CONCLUSIONS: D(2) receptor binding is a more important factor than chronological age in accounting for variation in cognitive performance across the adult lifespan. Changes in dopaminergic neurotransmission play an important role in aging-related cognitive decline. PMID- 10739430 TI - Proton magnetic resonance spectroscopy of the medial prefrontal cortex in patients with deficit schizophrenia: preliminary report. AB - OBJECTIVE: Proton magnetic resonance spectroscopy (1H-MRS) was used to study medial prefrontal metabolic impairments in schizophrenic patients with the deficit syndrome. METHOD: The subjects were 22 schizophrenic patients categorized as deficit (N=5) or nondeficit (N=17) and 21 healthy subjects. (1)H-MRS was performed for the right and the left medial prefrontal cortex. RESULTS: The patients with the deficit syndrome had significantly lower ratios of N acetylaspartate to creatine plus phosphocreatine than did the healthy subjects or nondeficit patients. CONCLUSIONS: As N-acetylaspartate levels could reflect neuronal density and/or viability, this finding suggests a neuronal loss in the medial prefrontal cortex of deficit patients. PMID- 10739429 TI - Cognitive deficit induced by acute tryptophan depletion in patients with Alzheimer's disease. AB - OBJECTIVE: The study assessed the effects on global cognitive function and mood of a reduction of brain serotonin by means of acute tryptophan depletion in 16 patients with dementia of the Alzheimer type and in 16 cognitively intact comparison subjects. METHOD: In a double-blind, crossover design, subjects received a tryptophan-free amino acid drink to induce acute tryptophan depletion and, on a separate occasion, a placebo drink containing a balanced mixture of amino acids. On each occasion, ratings of depressed mood were made at baseline and 4 and 7 hours later, and the Modified Mini-Mental State was administered at baseline and 4 hours later. RESULTS: Patients with dementia of the Alzheimer type had a significantly lower mean score on the Modified Mini-Mental State after acute tryptophan depletion than after receiving placebo. The comparison group showed no difference in mean score on the Modified Mini-Mental State after acute tryptophan depletion and after receiving placebo. No significant changes in mood were found in either group. CONCLUSIONS: Acute tryptophan depletion significantly impaired cognitive function in patients with dementia of the Alzheimer type. Compromised serotonergic function, in combination with cholinergic deficit, may make an important contribution to cognitive decline in dementia of the Alzheimer type. PMID- 10739431 TI - Reduced concentrations of thalamic N-acetylaspartate in male patients with schizophrenia. AB - OBJECTIVE: The authors measured N-acetylaspartate (a putative neuronal marker) in the right and left thalamus of 17 male patients with schizophrenia using in vivo proton magnetic resonance spectroscopic imaging ((1)H MRSI). METHOD: (1)H MRSI was performed on 17 medicated male patients with schizophrenia and 10 male comparison subjects. Concentrations of N-acetylaspartate, creatine, and choline were determined in the thalamic regions bilaterally. RESULTS: The patients with schizophrenia demonstrated significantly lower concentrations of N acetylaspartate than the comparison subjects in both the right and left thalamic regions. Right thalamic N-acetylaspartate and left thalamic N-acetylaspartate were significantly correlated in the patients but not in the comparison subjects. There was no association between N-acetylaspartate and duration of illness or medication dose. No group differences or lateralized asymmetries in choline or creatine were noted. CONCLUSIONS: The finding of reduced concentrations of N acetylaspartate bilaterally suggests neuronal dysfunction and/or loss in both the right and left thalamic regions in male patients with schizophrenia. PMID- 10739432 TI - High serum cholesterol and risk of suicide. AB - OBJECTIVE: The study was conducted to estimate the association between serum total cholesterol concentration and mortality from suicide. METHOD: The baseline serum total cholesterol concentration of 37,635 adults was determined in five independent population surveys conducted during 1972-1992 in Finland. Mortality from different causes of death was monitored for a mean of 14.6 years after the survey dates. The means for violent suicides (N=130) included hanging, firearms, cutting, jumping, and unspecified means. The means for nonviolent suicides (N=46) included drug overdose, poisoning with gases, and drowning. RESULTS: Serum total cholesterol concentration was positively related to the risk of violent suicide. Among subjects whose serum total cholesterol concentration was in the highest category, the adjusted relative risk was more than twofold compared with the lowest category. The violent/nonviolent suicide ratio increased linearly with increasing cholesterol category. No association between serum total cholesterol concentration and the risk of nonviolent suicide was found. CONCLUSIONS: This is the first study to demonstrate the positive relationship of high serum total cholesterol concentration with increased risk of violent suicide. PMID- 10739446 TI - Olanzapine and panic attacks. PMID- 10739448 TI - Vitamin B(12) deficiency manifested as psychosis without anemia. PMID- 10739450 TI - Older patients and health care utilization. PMID- 10739451 TI - "Cognitive dysmetria" in schizophrenia. PMID- 10739453 TI - Sympathoadrenal hyperactivity and neuroleptic malignant syndrome. PMID- 10739454 TI - Periconception care. AB - If we are to provide periconception care to all American men and women of reproductive age, we must consider it all "routine visits." For prevention of birth defects, all reproductive-age women should be counseled about folic acid supplementation and rubella immunity. Many clinicians consider risk reduction and anticipatory activities important in periconception care. This includes counseling families about avoiding tobacco and substance abuse, using passenger restraints, avoiding teratogens, seeking appropriate genetic counseling and early prenatal care, and a variety of other periconceptional health issues. Reproductive-age women with underlying medical problems should be identified and counseled about pregnancy risks. PMID- 10739455 TI - Maternal infections: diagnosis and management. AB - An understanding of the common infections that occur during pregnancy is crucial for prenatal care providers. This article discusses specific screening and diagnostic tests, along with appropriate treatment regimens for common bacterial, viral, protozoal, and fungal infections that occur during pregnancy. Additionally, methods to reduce the likelihood of infection, such as preconception immunization, are reviewed. PMID- 10739456 TI - Drug use during pregnancy and lactation. AB - This article reviews the basic principles of pharmacology and teratogenicity of drugs for the pregnant and lactating woman and summarizes the Food and Drug Administration, American College of Obstetrics and Gynecology, and American Academy of Pediatrics classifications. These categories were devised for caregivers of both women and their babies. The authors propose a two/three letter category system to assist those who, like family physicians, must care for women while pregnant and while lactating. Common problems from everyday practice are reviewed, with an emphasis on the important drugs to use and avoid in a wide variety of everyday clinical settings to help the busy primary care physician in making decisions about medications in pregnancy and lactation. PMID- 10739457 TI - Prenatal screening. AB - The prenatal period is an ideal time to look at screening issues in an effort to maximize the health of the woman and effect a good outcome for her baby. As primary care providers for family, it is important to look beyond a traditional medical model for screening to examine a woman in the context of her family and her experiences. This article takes a broader view of screening to include some social and family issues. It also seeks to examine the evidence around some of the controversial issues in screening to help the provider be a thorough advisor to his or her patients. PMID- 10739458 TI - Risk assessment. AB - Risk assessment is the process by which clinicians screen for conditions that, if unmanaged, result in complications of pregnancy or adverse birth outcomes and for which an intervention would improve the well-being of the mother, child, and family. One of the major US health care goals is that by the year 2000, at least 90% of pregnant women will receive risk appropriate care. This article discusses the major risks to the mother and child during pregnancy, and presents tools to assess pregnancy well-being. PMID- 10739459 TI - Nutrition, exercise, work, and sex in pregnancy. AB - This article focuses on the effect of daily living on pregnancy outcome. Although most patients can continue their everyday activities without any concern about effect on pregnancy outcome, all pregnant patients want and need their physician's advice about all of these areas. PMID- 10739460 TI - Alternative medicine in maternity care. AB - Primary care physicians are confronted daily with questions from their patients about alternative medicine. When maternity care patients seek information about such therapies, careful attention must be paid to issues of safety and efficacy for both the mother and her unborn child. This article clarifies the role of alternative medicine in maternity care by looking at the definitions and history of common alternative therapies, documenting the evidence for alternative therapies in prenatal, intrapartum, and postpartum care, and suggesting ways to incorporate alternative medicine into primary care practice. PMID- 10739461 TI - Vaginal bleeding associated with pregnancy. AB - Vaginal bleeding during pregnancy provokes physical and emotional stress to patients and physicians. Physicians must be prepared to assess the medical implications of acute blood loss to these patients and their unborn children quickly. When mother and fetus are stable, the recognition and treatment of the underlying cause is essential to decreasing additional maternal and fetal morbidity and mortality associated with the bleeding episode. PMID- 10739462 TI - Spontaneous pregnancy loss: evaluation, management, and follow-up counseling. AB - Spontaneous pregnancy loss is a common problem requiring a logical and systematic approach to evaluation and management. This article outlines a practical method for primary care physicians to use throughout the diagnosis, management, and follow-up periods. It integrates collaborative decision making and attention to the emotional and informational needs of the patient experiencing spontaneous pregnancy loss. PMID- 10739463 TI - Ectopic pregnancy. AB - Ectopic pregnancy occurs in approximately 2% of all pregnancies in the United States, and is the nation's leading cause of first trimester maternal death. Its incidence has increased sixfold in the past 25 years, despite significant improvements in techniques for early diagnosis and management. This article reviews the epidemiology, risk factors, and common clinical presentations of ectopic pregnancy. Both traditional and newly developed strategies for diagnosis and management are described. The primary care physician is in an excellent position to screen for and diagnose ectopic pregnancy, and to counsel patients regarding treatment options and future risks. With the increasing trend toward outpatient nonsurgical management of ectopics, it is expected that the roll of the primary care physician in managing patients with ectopic pregnancy will continue to increase. PMID- 10739464 TI - Hypertensive disorders, diabetes mellitus, and anemia: three common medical complications of pregnancy. AB - Hypertensive disorders, diabetes mellitus, and anemia are three common medical complications of pregnancy. In many cases, primary care physicians manage them. The understanding of these conditions and recommendations for their treatment are in constant evolution. Care for patients with these problems requires a working knowledge of current information. This article reviews the current knowledge about and treatment of these three disorders. PMID- 10739465 TI - Obstetric analgesia. AB - Several analgesic options are available for patients during labor. Selection of the appropriate technique must be individualized. Education and preparation begins during prenatal care. If medications are to be used, the risks and benefits to the mother and infant must be considered. Continued patient-doctor communication throughout labor is essential. Patient preferences, tempered by sound medical judgement, should guide the selection of the optimal modality for pain control during labor. PMID- 10739466 TI - Keeping normal labor normal. AB - Labor and delivery have been viewed by physicians as processes that can and must be managed by physicians for their pregnant patients. This article asserts that most births do not need to be interventionally managed and that a birth attendant's highest order skill is knowing when and how not to intervene. Further, the article looks at what birth interventions are likely to keep normal labor normal. The authors propose a new paradigm: The 10 "P's" of keeping normal labor normal. PMID- 10739467 TI - Breastfeeding and postpartum maternal care. AB - The early postpartum period is a time of transition for families. Breastfeeding, the optimal form of feeding for newborns, doesn't always come naturally. Medical interventions at birth can affect early breastfeeding, and close follow up of breastfeeding couplets is essential. New mothers often deal with challenges such a postpartum depression, thyroid disease, perineal pain, and other sexual concerns. Physicians can support families in this transition period to help optimize it for the mother and her baby. PMID- 10739468 TI - Issues in newborn care. AB - During the provision of newborn care, the primary care provider must identify any apparent or potential health concerns, while at the same time dealing with the concerns of new mothers and other members of the now expanded family. Efficiently managing outpatient problems such as colic, dysfunctional sleep, skin disorders, neonatal jaundice, feeding problems, and newborn circumcision is the purpose of this article. PMID- 10739469 TI - The development of A. N. Belozersky's ideas in polyphosphate biochemistry. AB - This review covers some trends and approaches to the study of inorganic polyphosphates that originated from the fruitful ideas and pioneering works of A. N. Belozersky. This is, first of all, the elucidation of a close relationship between these biopolymers and nucleic acids in organisms at different evolutionary stages; second, the study of "fossil" reactions in polyphosphate metabolism that permit an understanding of their role in the evolution of phosphorus turnover and cell bioenergetics; third, the possible use of the conservative enzymes of polyphosphate metabolism, e.g., exopolyphosphatases, as molecular chronometers for obtaining additional data concerning the theory of the endosymbiotic origin of eukaryotic cells from prokaryotes. PMID- 10739470 TI - Transmembrane ion transport by polyphosphate/poly-(R)-3-hydroxybutyrate complexes. AB - Transmembrane ion transport, a critical process in providing energy for cell functions, is carried out by pore-forming macromolecules capable of discriminating among very similar ions and responding to changes in membrane potential. It is widely regarded that ion channels are exclusively proteins, relatively late arrivals in cell evolution. Here we discuss the formation of ion selective, voltage-activated channels by complexes of two simple homopolymers, namely, inorganic polyphosphates (polyPs) and poly-(R)-3-hydroxybutyrates (PHBs), derived from phosphate and acetate, respectively. Each has unique molecular characteristics that facilitate ion selection, solvation, and transport. Complexes of the two polymers, isolated from bacterial plasma membranes or prepared from the synthetic polymers, form voltage-dependent, Ca2+-selective channels in planar lipid bilayers that are selective for divalent over monovalent cations, permeant to Ca2+, Sr2+, and Ba2+, and blocked by transition metal cations in a concentration-dependent manner. Recently, both polyP and PHB have been found to be components of ion-conducting proteins: namely, the human erythrocyte Ca2+-ATPase pump and the Streptomyces lividans potassium channel. The contribution of polyP and PHB to ion selection and/or transport in these proteins is yet unknown, but their presence gives rise to the hypothesis that these and other ion transporters are supramolecular structures in which proteins, polyP, and PHB cooperate in forming well-regulated and specific cation transfer systems. PMID- 10739471 TI - Polyphosphate in bone. AB - Human bone-forming osteoblasts are an excellent model to investigate the multiple functions of inorganic polyphosphates (polyP) for the following reasons: 1) they contain relatively high amounts of polyP and polyP-dependent enzymes; 2) they allow the study of both general and specific functions of these polymers, and 3) medically relevant results can be expected from these studies. PMID- 10739472 TI - Molecular analysis of polyphosphate accumulation in bacteria. AB - The dynamic behavior of inorganic polyphosphate (polyP), its accumulation and disappearance, is the most striking aspect of polyP metabolism in bacteria. Imbalance between polyP synthesis and degradation results in fluctuations of polyP by 100- to 1000-fold. We here review recent results with respect to this polyP metabolism in bacteria. PolyP accumulation in response to amino acid starvation, accompanied by increased levels of stringent factors, has been observed in Escherichia coli. Inhibition by stringent factors of polyphosphatase interrupts the dynamic balance between the synthesis and degradation of polyP, accounting for polyP accumulation. Polyphosphate kinase is required for activation of intracellular protein degradation, which is required for adaptation at the onset of amino acid starvation. The adaptation to amino acid starvation is mediated by the network of stringent response and polyP metabolism. PolyP accumulation independent of stringent response has also been observed. Novobiocin, an inhibitor for DNA gyrase, stimulated accumulation of polyP but not that of stringent factors. However, a temperature-sensitive DNA gyrase mutant did not exhibit polyP accumulation at the non-permissive temperature. Antagonistic relationship of polyP to nucleic acid synthesis, explored by Harold, appears to be more complicated. We discuss relationship of Pi regulation to polyP accumulation in E. coli and Klebsiella aerogenes. A function of polyP as an in vivo phosphagen affecting polyP accumulation is also discussed. PMID- 10739473 TI - Polyphosphates and enzymes of polyphosphate metabolism in Escherichia coli. AB - This review summarizes the results of our study of polyphosphate and enzymes of polyphosphate metabolism in E. coli and their regulation by exogenous orthophosphate and other physiological and genetic factors. PMID- 10739474 TI - Inorganic polyphosphate and polyphosphate kinase: their novel biological functions and applications. AB - In this review, we discuss the following two subjects: 1) the physiological function of polyphosphate (poly(P)) as a regulatory factor for gene expression in Escherichia coli, and 2) novel functions of E. coli polyphosphate kinase (PPK) and their applications. With regard to the first subject, it has been shown that E. coli cells in which yeast exopolyphosphatase (poly(P)ase), PPX1, was overproduced reduced resistance to H2O2 and heat shock as did a mutant whose polyphosphate kinase gene is disrupted. Sensitivity to H2O2 and heat shock evinced by cells that overproduce PPX1 is attributed to depressed levels of rpoS expression. Since rpoS is a central element in a regulatory network that governs the expression of stationary-phase-induced genes, poly(P) affects the expression of many genes through controlling rpoS expression. Furthermore, poly(P) is also involved in expression of other stress-inducible genes that are not directly regulated by rpoS. The second subject includes the application of novel functions of PPK for nucleoside triphosphate (NTP) regeneration. Recently E. coli PPK has been found to catalyze the kination of not only ADP but also other nucleoside diphosphates using poly(P) as a phospho-donor, yielding NTPs. This nucleoside diphosphate kinase-like activity of PPK was confirmed to be available for NTP regeneration essential for enzymatic oligosaccharide synthesis using the sugar nucleotide cycling method. PPK has also been found to express a poly(P):AMP phosphotransferase activity by coupling with adenylate kinase (ADK) in E. coli. The ATP-regeneration system consisting of ADK, PPK, and poly(P) was shown to be promising for practical utilization of poly(P) as ATP substitute. PMID- 10739475 TI - Application of polyphosphate metabolism to environmental and biotechnological problems. AB - The synthesis and degradation of polyphosphate (polyP) are influenced by the energy state of the cell and extracellular phosphate levels. The import of excess phosphate and its incorporation into polyP under phosphate- and energy-rich growth conditions allows organisms to survive when phosphate or energy are depleted. Under phosphate-starvation conditions, phosphate can be recovered from polyP by hydrolysis. When the organism is energy starved, energy can be recovered either by regenerating the high-energy phosphoanhydride bond donor (ATP in most cases) or by hydrolysis of polyP and subsequent secretion of orthophosphate to recharge the transmembrane proton gradient. Understanding how the energy state of the cell and environmental phosphate levels affect polyP metabolism is essential to improving such environmental processes as enhanced biological phosphorus removal, a treatment process that is widely used to remove excess phosphate from wastewater. Manipulation of the genes responsible for polyP metabolism can also be used to improve gene expression from phosphate-starvation promoters and to remove heavy metals from contaminated environments. PMID- 10739476 TI - Recent developments in the biochemistry and ecology of enhanced biological phosphorus removal. AB - Most of the genes encoding the enzymes involved in polyP synthesis and degradation and in phosphate transport have been studied in various Gram-negative bacteria. Progress has also been made in studying the biochemical mechanisms underlying the process of enhanced biological phosphorus removal (EBPR), in particular in lab-scale systems fed with acetate or acetate plus glucose as the sole carbon and energy sources. By applying 13C-NMR, previous models concerning anaerobic carbon metabolism have been advanced and the role of glycogen in providing reducing equivalents in EBPR is definitely demonstrated. The role of the citric acid cycle in supplying reducing equivalents for the conversion of acetyl-CoA into poly-beta-hydroxybutyrate and poly-beta-hydroxyvalerate has been discussed. An incomplete citric acid cycle has been proposed to provide a small part of the reducing equivalents. Polyphosphate:AMP phosphotransferase and polyphosphatase were readily detectable in EBPR sludge fed with acetate plus glucose, but polyphosphate kinase remained undetected. In a lab-scale EBPR system, fed for several months with only acetate as carbon source, a Rhodocyclus like bacterium (R6) was highly enriched and is therefore probably responsible for EBPR in systems fed with acetate only. This R6-type bacterium was however also present in other EBPR sludges (but to a lesser extent), and may therefore play an important role in EBPR in general. This organism accumulates polyhydroxyalkanoates anaerobically and polyP under aerobic conditions. Unlike members of the genus Rhodocyclus, bacterium R6 cannot grow phototrophically. Therefore a provisional new genus Candidatus and species Accumulibacter phosphatis was proposed. PMID- 10739477 TI - Microbial selection of polyphosphate-accumulating bacteria in activated sludge wastewater treatment processes for enhanced biological phosphate removal. AB - Activated sludge processes with alternating anaerobic and aerobic conditions (the anaerobic-aerobic process) have been successfully used for enhanced biological phosphate removal (EBPR) from wastewater. It is known that polyphosphate accumulating bacteria (PAB) play an essential role for EBPR in the anaerobic aerobic process. The present paper reviews limited information available on the metabolism and the microbial community structure of EBPR, highlighting the microbial ecological selection of PAB in EBPR processes. Exposure of microorganisms to alternate carbon-rich anaerobic environments and carbon-poor aerobic environments in the anaerobic-aerobic process induces the key metabolic characteristics of PAB, which include organic substrate uptake followed by its conversion to stored polyhydroxyalkanoate (PHA) and hydrolysis of intracellular polyphosphate accompanied by subsequent Pi release under anaerobic conditions. Intracellular glycogen is assumed to function as a regulator of the redox balance in the cell. Storage of glycogen is a key strategy for PAB to maintain the redox balance in the anaerobic uptake of various organic substrates, and hence to win in the microbial selection. Acinetobacter spp., Microlunatus phosphovorus, Lampropedia spp., and the Rhodocyclus group have been reported as candidates of PAB. PAB may not be composed of a few limited genospecies, but involve phylogenetically and taxonomically diverse groups of bacteria. To define microbial community structure of EBPR processes, it is needed to look more closely into the occurrence and behavior of each species of PAB in various EBPR processes mainly by molecular methods because many of PAB seem to be impossible to culture. PMID- 10739478 TI - Dependence of inorganic polyphosphate chain length on the orthophosphate content in the culture medium of the yeast Saccharomyces cerevisiae. AB - The content of inorganic linear polyphosphate (polyP) and the polymeric degree (n) of these compounds were determined in the process of growth of the yeast Saccharomyces cerevisiae VKM Y-1173 in a medium, which contained varying Pi amount with the constant level of all the necessary components. For this purpose, a combination of chemical methods of polyP extraction and 31P-NMR spectroscopy studies of their chain length were used. After 7 h of phosphate starvation, the yeast was shown to use almost completely the phosphate reserve in the form of polyP localized in various cell compartments to support their vitality. The polyP drop was followed by a considerable shortening of the polymer chain length of acid-soluble (polyP1) and two alkali-soluble (polyP3 and polyP4) fractions. Under the same conditions, the content of a salt-soluble fraction (polyP2) decreased almost 20-fold followed by a simultaneous increase of the chain length nearly 2 fold. As a result, fraction chain length ranged up to n = 40-45. Replacement of the yeast cells after phosphate starvation to a complete phosphate- and glucose containing medium resulted in super-accumulation ("overcompensation") of polyP within 2 h mainly in polyP3 and, to a lesser degree, in polyP1, polyP2, and polyP5 fractions. In polyP4 fraction localized as polyP3 at the cell surface, the polyP super-accumulation was not detected. The increase of polyP amount in the fractions mentioned turned out not to be accompanied by simultaneous elongation of their chain length and occurred at the lowest level that is characteristic of a polymer level for each fraction. Further cultivation of the yeast on the complete medium during 2 h had little or no effect on polyP content in the cells but led to elongation of polyP chain length especially in the polyP3 and polyP4 fractions. A phenomenon of considerable elongation of polyP chain length against the background of their fixed content revealed in the yeast growing on the complete medium suggests that these organisms possess a previously unknown discrete way of polyP biosynthesis, which results first in the formation of comparatively low-molecular-mass chains followed by that of high-molecular-mass polymers. PMID- 10739479 TI - Purification and characterization of a soluble polyphosphatase from mitochondria of Saccharomyces cerevisiae. AB - A polyphosphatase with the specific activity 2.2 U/mg was purified to apparent homogeneity from a soluble preparation of mitochondria of Saccharomyces cerevisiae. The polyphosphatase is a monomeric protein of approximately 41 kD. The purified enzyme hydrolyzes polyphosphates with an average chain length of 9 to 208 phosphate residues to the same extent, but its activity is approximately 2 fold higher with tripolyphosphate. ATP, PPi, and p-nitrophenyl phosphate are not substrates of this enzyme. The apparent Km values are 300, 18, and 0.25 microM obtained at hydrolysis of polyphosphates with a chain length of 3, 15, and 188 phosphate residues, respectively. Several divalent cations stimulated the enzyme activity 1.2-27-fold (Mg2+ = Co2+ = Mn2+ > Zn2+). Determination of the protein N terminal sequence and its comparison with the EMBL data library indicates that the soluble polyphosphatase of mitochondria of S. cerevisiae is not encoded by the gene of the major yeast polyphosphatase PPX1. PMID- 10739480 TI - Active site interactions in oligomeric structures of inorganic pyrophosphatases. AB - Recent progress in studies of the mode of action of cytoplasmic inorganic pyrophosphatases is mainly due to the analysis of a dozen and a half structures of the apoenzyme, its complexes, and mutants. However, despite considerable research on the mechanism of action of these enzymes, many important problems remain unclear. Among them is the problem of active site interactions in oligomeric structures and their role in catalysis; this review focuses on this problem. The abundant experimental data requires generalization and comprehensive analysis. A characteristic feature of the spatial structure of inorganic pyrophosphatases is a flexible system of noncovalent interactions between protein groups penetrating the whole molecule of the oligomeric enzyme. Binding of metal ions, sulfate (an analog of the product of the enzymatic reaction), and affinity phosphorus-containing inhibitors at the active site or site-directed mutagenesis induce rearrangements in the set of hydrogen and ionic interactions, which change active site properties and in some instances, cause molecule asymmetry. In the trimeric form of Escherichia coli pyrophosphatase obtained by dissociation of a hexamer, active sites also interact with each other, which is manifested by negative cooperativity upon substrate binding. The association of trimers into the hexamer leads to perfect organization of active sites and to their coordinated functioning, probably due to the restoration of communication channels between the trimers. PMID- 10739481 TI - Mechanism of Ca2+-induced inhibition of Escherichia coli inorganic pyrophosphatase. AB - The causes of inhibition of Escherichia coli inorganic pyrophosphatase (PPase) by Ca2+ were investigated. The interactions of several mutant pyrophosphatases with Ca2+ in the absence of substrate were analyzed by equilibrium dialysis. The kinetics of Ca2+ inhibition of hydrolysis of the substrates MgPPi and LaPPi by the native PPase and three mutant enzymes (Asp-42-Asn, Ala, and Glu) were studied. X-Ray data on E. coli PPase complexed with Ca2+ or CaPPi solved at atomic resolution were analyzed. It was shown that, in the course of the catalytic reaction, Ca2+ replaces Mg2+ at the M2 site, which shows higher affinity for Ca2+ than for Mg2+. Different properties of these cations account for active site deformation. Our findings indicate that the filling of the M2 site with Ca2+ is sufficient for PPase inhibition. This fact proves that Ca2+ is incapable of properly activating the H2O molecule for nucleophilic attack on PPi. It was also demonstrated that Ca2+, as a constituent of the non-hydrolyzable substrate analog CaPPi, competes with MgPPi at the M3 binding site. As a result, Ca2+ is a powerful inhibitor of all known PPases. Other possible reasons for the inhibitory effect of Ca2+ on the enzyme activity are also considered. PMID- 10739482 TI - Engineering a new magnesium binding site in the subunit contact region of Escherichia coli inorganic pyrophosphatase. AB - Three Gln-80 residues belonging to different subunits of homohexameric Escherichia coli pyrophosphatase are separated by only one water molecule to which they are hydrogen bonded. Substitution of Glu for Gln-80 stabilizes quaternary structure of the enzyme but has only a small effect on enzyme activity. The substitution stimulates Mg2+ binding and changes the appearance of the Mg2+ concentration dependence of the rate constant for the trimer --> hexamer transition. These data suggest that a new Mg2+ binding site is formed in the intersubunit contact region as a result of the substitution. Three-dimensional modeling of the mutated protein showed that a chelate complex might form involving two of the three Glu-80 residues. PMID- 10739483 TI - Domains of nursing intervention after sudden cardiac arrest and automatic internal cardioverter defibrillator implantation. AB - PURPOSE: The purpose of the study was to explore individual and family experiences after sudden cardiac arrest and automatic internal cardioverter defibrillator implantation during the first year of recovery. This report specifically addresses the domains of concern expressed and helpful strategies used by participants that are relevant to the development of future intervention programs. DESIGN: A grounded theory approach was used to gain an understanding of areas of concern of sudden cardiac arrest survivors and families that could be used when designing future nursing interventions. Semistructured interviews were conducted with both sudden cardiac arrest survivors and 1 family member each at 5 points during the first year of recovery (hospitalization; 1, 3, 6, and 12 months after hospitalization). Participants were asked to identify those specific areas that most concerned them and that they would like assistance with during the first year. A total of 150 interviews were conducted with 176 hours of data generated. SETTING: The study focused on 10 northwest urban community medical centers and participants' homes within a 50-mile driving distance from the medical centers. SAMPLE: The sample included 15 first-time sudden cardiac arrest survivors (13 men and 2 women) and 1 family member each between the ages of 31 and 72 years. RESULTS: Domains of concern identified by participants that can be used to design future nursing intervention programs included preventive care, dealing with automatic internal cardioverter defibrillator shocks, emotional challenges, physical changes, activities of daily living, partner relationships, and dealing with health care providers. Suggestions of helpful strategies used by participants during the first year are outlined. IMPLICATIONS: Domains of concern and helpful strategies identified by participants provide a framework for the development and testing of nursing intervention programs to enhance recovery following sudden cardiac arrest for survivors and their families. PMID- 10739484 TI - Help seeking in a support group for recipients of implantable cardioverter defibrillators and their support persons. AB - PURPOSE: To understand shared meanings of help-seeking experiences in support groups of people with implantable cardioverter defibrillator (ICD) and their support persons. SETTING: ICD support group at an urban medical center. SAMPLE: Fifteen individuals with ICD and 9 support persons. RESULTS: Six related themes and 1 constitutive pattern emerged. Themes included hearing and telling stories, help seeking encouraged by triggers, seeking meaningful information, forming a therapeutic friendship through group camaraderie, gaining assistance from the facilitator, and the sharing of a similar view by support persons. The constitutive pattern is coping with the possibility of death. IMPLICATIONS: Health care providers may recommend storytelling as the central mechanism of interactions in support groups that assist in coping with daily anxieties of living with an ICD. Nurses would be appropriate facilitators to guide discussion, to provide technical information, and to promote anticipatory guidance in coping with potential firing events. PMID- 10739485 TI - Psychologic distress among spouses of patients undergoing cardiac rehabilitation. AB - OBJECTIVE: The goal of this study was to identify common sources of distress in spouses of patients undergoing cardiac rehabilitation and to compare spouses classified as being in distress and those classified as nondistressed on demographic variables, their experiences of stress, coping strategies employed, marital intimacy, and level of family functioning. The study aimed to describe the needs of spouses with respect to possible interventions. DESIGN: A cross sectional analysis was used. SETTING: The study was located at the University of Ottawa Heart Institute Prevention and Rehabilitation Centre's cardiac rehabilitation program. OUTCOME MEASURES: Scores on the Brief Symptom Inventory, Heart Disease Hassles Scale, Coping Strategies Inventory, Miller Intimacy Scale, and McMaster Family Assessment Device were used as outcome measures. RESULTS: Two hundred thirteen female spouses of patients undergoing cardiac rehabilitation participated in this study. Sixty-six percent of the spouses met the criteria for distress. This was more common in younger spouses (51.99 +/- 9.94 years) than in older spouses (55. 74 +/- 10.54 years) (t = -2.45; P =.013). Distressed spouses used disengagement coping strategies significantly more than the nondistressed spouses (t = 6.91; P =.0001). Distressed spouses also reported significantly less intimacy in their marriages (t = -3.99; P =.0001) and poorer family functioning (t = 5.86; P =.0001). The most prevalent symptoms of psychologic distress included feeling tense, having trouble falling asleep and feeling easily hurt. The most prevalent stressors were as follows: (1) worries about treatment, recovery, and prognosis (75.5%); (2) moodiness of the patient (66.7%); (3) worries about the patient returning to work and about money (38.8%); (4) sexual concerns (36.7%); and (5) helplessness or apathy on the part of the patient and increased spousal responsibility (36.1%). CONCLUSIONS: Spouses of patients undergoing cardiac rehabilitation should be screened for psychologic distress, and those in distress should be offered interventions focused on assisting them to deal with specific stressors related to their experience with a spouse with heart disease. Interventions indicated include stress-management techniques and encouraging the use of engagement coping strategies. In addition, marital and family concerns need to be directly addressed in support interventions. PMID- 10739487 TI - Spectrum of upper-extremity deep venous thrombosis in a community teaching hospital. AB - OBJECTIVE: The goal of this study was to characterize the spectrum of upper extremity deep venous thrombosis in a community teaching hospital. DESIGN AND SETTING: A retrospective analysis was used at a large urban teaching hospital. MATERIAL AND METHODS: We reviewed the records of 90 patients with ultrasound documented thrombosis of the internal jugular, subclavian, axillary, or brachial veins to determine clinical characteristics, risk factors, and outcome. RESULTS: The most common underlying conditions associated with upper-extremity deep venous thrombosis were the presence of a central venous catheter in 65 patients (72%), infection in 25 (28%), extrathoracic malignancy in 20 (22%), thoracic malignancy in 19 (21%), renal failure in 19 (21%), and a prior lower-extremity deep venous thrombosis in 16 (18%). Pain was noted in 31 (34%) patients, and 76 patients (84%) had edema of the involved extremity. The left subclavian vein was involved in 44 patients (49%), and 35 patients (39%) had a central venous catheter in the left subclavian vein. When a central venous catheter was present, the deep venous thrombosis was usually ipsilateral (P <.001). Heparin and warfarin were administered to 65 (72%) and 53 (59%) of the patients, respectively. Eleven patients (12%) died. Of these patients, 8 (73%) had an underlying infection, whereas only 22% of survivors had an infection (P =.0012). CONCLUSION: Upper extremity deep venous thrombosis typically occurs in patients with a systemic illness in the presence of a central venous catheter. The left subclavian vein is frequently involved because this is a common site for placement of a central venous catheter. Pain is uncommon, but edema of the involved extremity is noted in the majority of patients. The mortality rate of patients in this study with an upper-extremity deep venous thrombosis was 12%; most patients who died had a central venous catheter and an underlying infection. PMID- 10739486 TI - Meaning of illness for women with coronary heart disease. AB - OBJECTIVE: The goal of this study was to describe the meaning of an acute cardiac event and cardiac illness for women with coronary heart disease. DESIGN: A longitudinal, descriptive design was used. SETTING: The study was undertaken at a tertiary care hospital in the Pacific Northwest. PATIENTS: The study considered a purposive sample of 6 women ranging in age from 47 to 67 years, who were hospitalized for coronary heart disease (3 for coronary bypass surgery and 3 for coronary angioplasty). METHODS: We completed a series of 2 focused semistructured interviews, 1 while women were hospitalized for coronary heart disease and the second 3 months later. The qualitative method of grounded theory guided the collection and analysis of data. RESULTS: The meaning of illness for women with coronary heart disease evolved over time in a process we termed seeking understanding. After the onset of symptoms, women described 3 responses: denial, acknowledging, and being scared. These did not occur in sequence, but as iterative processes. They described 4 additional themes after obtaining medical attention: naming, seriousness of illness, comparing self with others, and causality. CONCLUSIONS: A substantive theory of seeking understanding was described in this study and contributes to our understanding of meaning and behavior of women with coronary heart disease. Future research with use of similar methods may lead to an understanding of women's decision-making process regarding their illness. PMID- 10739488 TI - Evaluation of advanced cardiac life support written examinations. AB - OBJECTIVE: To examine the quality of 2 advanced cardiac life support written examinations and the relationship between demographic variables and test performance. DESIGN: Descriptive. SETTING: Advanced cardiac life support courses held at 4 midwestern hospitals, one outpatient surgery center, and one community college. SUBJECTS: Nonprobability sample of 367 participants. OUTCOME MEASURE: Two versions of the American Heart Association advanced cardiac life support written examinations. RESULTS: Reliability coefficients of test A and test B were 0.45 and 0.54, respectively. Discrimination and difficulty indices calculated on both tests revealed that test B questions were more difficult, with better discriminators. Prior experience, sex of participant, and profession were identified as factors influencing test performance. CONCLUSION: Although test B surpassed the performance of test A, both examinations were problematic. Additional investigation and refinement of the advanced cardiac life support tests are warranted. PMID- 10739489 TI - Bronchopulmonary hygiene physical therapy in bronchiectasis and chronic obstructive pulmonary disease: a systematic review. AB - OBJECTIVES: This study had two objectives: (1) to examine the effects of bronchopulmonary hygiene physical therapy on patients with chronic obstructive pulmonary disease and bronchiectasis; (2) to determine any differences between manual and mechanical techniques for bronchopulmonary hygiene physical therapy. DESIGN: The study design was a systematic review of the literature that used an exhaustive search for trials and review methods prescribed by the Cochrane Collaboration. INCLUSION CRITERIA: Randomized controlled trials examined patient groups, interventions, and dependent variables. PATIENTS: Patients included those with chronic obstructive pulmonary diseases (emphysema or chronic bronchitis) or bronchiectasis. INTERVENTIONS: Any of the following interventions or combinations thereof were included: manual interventions, such as postural drainage, chest percussion, vibration, chest shaking, directed coughing, or forced exhalation technique. CONTROLS: Controls of the study were as follows: no intervention; placebo; coughing; and mechanical interventions, such as mechanical vibration. RESULTS: The search identified 99 potential trials; inclusion or exclusion analysis left 7, which examined a total of 126 patients. Mean score on trial quality was 1.4 (5 = greatest). Three separate trials (N = 51) found statistically significant effects for bronchopulmonary hygiene physical therapy on sputum production and radioaerosol clearance. No trials (N = 126) found statistically significant effects on pulmonary function variables or differences between manual and mechanical techniques. CONCLUSIONS: Considering the small sizes, low quality, and mixed results from the trials, the research on bronchopulmonary hygiene physical therapy is inconclusive. There is a need for adequately sized, high-quality, randomized controlled trials with uniform patient populations to examine the effects of bronchopulmonary hygiene physical therapy. PMID- 10739491 TI - Communication patterns and decision making among parents and health care providers in the neonatal intensive care unit: a case study. AB - This case study illustrates salient issues in the communication and decision making of parents in a neonatal intensive care unit. The case provides descriptions that may be typical features of interaction between parents and professionals in a neonatal intensive care unit. Conclusions are drawn from this case with recommendations for practice. PMID- 10739490 TI - Assessment of alternative hand hygiene regimens to improve skin health among neonatal intensive care unit nurses. AB - OBJECTIVE: The frequent handwashing and gloving required in high-risk, high volume patient care areas such as critical care units damages skin of the hands. The purpose of this exploratory study was to compare 2 hand care regimens (traditional antiseptic wash with chlorhexidine-containing detergent versus mild soap wash with subsequent alcohol-based rinse for degerming as necessary) in a neonatal intensive care unit (NICU). DESIGN: Prospective, quasi-experimental, random assignment. SETTING: One NICU (47 beds) in a New York City children's hospital. SUBJECTS: Sixteen full-time NICU nurses. OUTCOME MEASURES: Microbial flora and skin condition of hands. INTERVENTION: Nurses were randomly assigned to one of the 2 hand care regimens. RESULTS: No significant differences in microbial counts or types of organisms from hands of staff were found, but after 2 weeks nurses in the mild soap and alcohol group had significant improvements in their skin condition (P =.005). CONCLUSIONS: Use of a mild soap for cleaning and an alcohol-based product for degerming may offer an acceptable alternative to the traditional antiseptic handwash and may reduce skin damage to health care professionals' hands. PMID- 10739493 TI - Get to know the Respiratory Nursing Society. PMID- 10739492 TI - Broad-complex tachycardia in a young man. PMID- 10739495 TI - The Filshie clip for female sterilization: a review of world experience. AB - Laparoscopic tubal electrocoagulation continues to be widely practiced; however, mechanical devices such as the Yoon band, the Hulka clip, and, most recently, the Filshie clip are becoming more popular because of the avoidance of accidental electrical burns, the diminished likelihood of subsequent ectopic pregnancy, and, in the case of the clips, the minimal degree of tubal destruction, thus allowing for maximum reversibility. This survey of worldwide reports from 1981 to the present reveals a high level of acceptance of the Filshie clip because of its effective design and ease of application. PMID- 10739496 TI - First-trimester screening for aneuploidy: research or standard of care? AB - First-trimester screening for Down syndrome has been proposed as a significant improvement with respect to second-trimester serum screening programs, the current standard of care, because of apparently higher detection rates and an earlier gestational age at diagnosis. First-trimester nuchal translucency on ultrasonography forms the basis of this new form of screening, although studies of its efficacy have yielded widely conflicting results, with detection rates ranging from 29% to 91%. Studies of first-trimester serum screening with measurements of pregnancy-associated plasma protein A and free beta-human chorionic gonadotropin serum concentrations have been much more consistent, with Down syndrome detection rates of 55% to 63% at a 5% false-positive rate. The combination of first-trimester ultrasonographic and serum screening has the potential to yield a Down syndrome detection rate of 80% at a 5% false-positive rate, although this approach has not been adequately studied. There have been no studies performed to date to directly compare the performance of first-trimester and second-trimester methods of screening. Two major trials are underway that will address this issue, one in the United Kingdom and one in the United States. Until the results of these trials are available, the current standard of care with respect to Down syndrome screening should not be changed, and first trimester screening should remain investigational. PMID- 10739497 TI - The effects of L-arginine on the release of prolactin from decidual explants in vitro. AB - OBJECTIVE: Our aim was to elucidate a role for the L -arginine-nitric oxide cyclic guanosine monophosphate pathway in the control of decidual prolactin release in vitro. STUDY DESIGN: In decidua obtained from normal vaginal deliveries, we examined the effects of L -arginine; a stable analog of cyclic guanosine monophosphate, 8-bromo-cyclic guanosine monophosphate; and two nitric oxide synthase inhibitors, L -nitro arginine methyl ester (L -NAME) and L -nitro monomethyl arginine (L -NMMA) on the release of prolactin from decidual tissue in vitro. RESULTS: L -arginine, at 100 micromol/L, produced an increase in medium prolactin concentration after a 2-hour exposure; however, its inactive isomer, D arginine, at the same concentration, did not. The increase in prolactin release initiated by L -arginine was sustained after a 24-hour incubation. In addition, 8 bromo-cyclic guanosine monophosphate (10 micromol/L) stimulated prolactin release. At 2 hours, the effects of L -arginine were reversed by L -NAME (100 micromol/L), which by itself was without effect. Incubation of tissue with L NAME for 24 hours inhibited prolactin secretion (24 hours; P <.05, vs control); however, L -NMMA was without effect. CONCLUSIONS: These results suggest that the L -arginine-nitric oxide-cyclic guanosine monophosphate pathway is involved in the regulation of prolactin secretion by decidual tissue. PMID- 10739498 TI - Conservative management of ovarian cystic teratoma during pregnancy and labor. AB - OBJECTIVE: Our goal was to evaluate the adequacy of conservative management during pregnancy and labor in women with an ultrasonographically diagnosed ovarian cystic teratoma. STUDY DESIGN: Forty-nine women with ultrasonographically diagnosed ovarian cystic teratoma <6 cm were followed for detection of possible complications through pregnancy and labor. Serial ultrasonographic examinations before pregnancy, during pregnancy, and after delivery were performed to detect changes in the size of the cystic teratoma. RESULTS: In a group of 49 women with dermoid cysts (mean age, 30 years), 68 pregnancies resulted. Of the 68 pregnancies, 4 ended in miscarriages, 1 was electively terminated, and in the remaining 63 pregnancies, a total of 64 healthy infants were delivered. Five patients needed treatment with assisted reproductive techniques. Fifty-five pregnancies ended in normal vaginal deliveries and 8 were delivered by cesarean (cesarean delivery rate of 16%). None of the classical complications attributed to dermoid cysts such as torsion, dystocia, or rupture occurred in the study group. In a follow-up of 56 dermoid cysts throughout pregnancy, cyst size remained unchanged. CONCLUSIONS: Ovarian dermoid cysts <6 cm are not expected to grow during pregnancy or to cause complications in pregnancy and labor. PMID- 10739499 TI - Risk factors for hyperplasia-associated versus atrophy-associated endometrial carcinoma. AB - OBJECTIVE: Endometrial cancer can be divided into atrophy-associated and hyperplasia-associated subtypes. It has been suggested that these subtypes have different pathologic features and prognoses. This preliminary study explores whether these subtypes are associated with different risk factors. STUDY DESIGN: Hysterectomies performed in cases of endometrial carcinoma with evaluable benign endometrium on routine processing were reviewed, and clinical data were abstracted from medical records. Forty-eight subjects with atrophy-associated and 28 subjects with hyperplasia-associated cancers were studied. RESULTS: We found younger age, higher weight, absence of cigarette smoking, and earlier menarche in subjects with hyperplasia-related cancers. CONCLUSIONS: Our findings support the idea that hyperplasia-associated endometrial cancer is estrogen-related but also suggest that atrophy-associated cases may result from a different causal pathway. Epidemiologic studies may yield more precise and accurate measures of association if atrophy-associated and hyperplasia-associated endometrial cancers are considered separately. PMID- 10739500 TI - Transvaginal ultrasonography of the endometrium in women with postmenopausal bleeding: is it always necessary to perform an endometrial biopsy? AB - OBJECTIVE: This study was undertaken to evaluate whether it was possible to abstain from performing an endometrial biopsy when endometrial thickness according to transvaginal ultrasonography was /=50 years who were referred because of postmenopausal bleeding or irregular bleeding during hormone replacement therapy. If endometrial thickness was /=5 mm underwent either curettage or endometrial biopsy. RESULTS: One hundred sixty-three women had an endometrial thickness /=5 mm. The corresponding figure when atypical hyperplasia and endometrial metastases were included was 20. 2%. CONCLUSION: If the false-negative rate of endometrial biopsy techniques is taken into account, then the combination of transvaginal ultrasonography and cervical cytologic examination is an adequate form of management for women with postmenopausal bleeding or irregular bleeding during hormone replacement therapy as long as endometrial thickness is /=3 months after the loop excision of the transformation zone. Simple regression analysis and the Student paired t test was performed to determine whether the length of the cervix had changed significantly between the measurements. RESULTS: The mean cervical lengths as measured by transvaginal ultrasonography before and after loop excision of the transformation zone were 3.1 +/- 0.8 cm and 3.1 +/- 0.7 cm, respectively. The correlation between ultrasonographic measurements before and after loop excision of the transformation zone was r = 0.88 (P <.0001). A paired t test resulted in a P value of 1.0000, which indicates that the ultrasonographic measurement after loop excision of the transformation zone was not different from the ultrasonographic measurement before loop excision of the transformation zone. The mean difference between measurements was 0.0 +/- 0.4 cm. CONCLUSION: After adequate healing time after loop excision of the transformation zone, the length of the cervix, as measured by transvaginal ultrasonography, does not appear to remain shortened. PMID- 10739502 TI - Coronary and uterine vascular responses to raloxifene in the sheep. AB - OBJECTIVE: We sought to determine whether raloxifene increases coronary and uterine blood flow in ovariectomized ewes. STUDY DESIGN: Twelve ewes were chronically instrumented for measurement of mean arterial pressure, heart rate, cardiac output, coronary blood flow, and uterine blood flow. Sheep received 17beta-estradiol, Estrace, raloxifene, or KY Jelly vehicle on separate days. RESULTS: 17beta-Estradiol increased uterine blood flow from 21 +/- 3 to 254 +/- 36 mL/min and coronary blood flow by 21% +/- 2% within 2 hours. Estrace increased uterine blood flow from 30 +/- 7 to 260 +/- 62 mL/min and coronary blood flow by 8% +/- 4% within 3 hours. Raloxifene increased uterine blood flow from 20 +/- 3 mL/min to 220 +/- 53 mL/min by 6 hours and coronary blood flow by 22% +/- 5% within 24 hours. To determine whether hemodynamic responses were mediated by nitric oxide, L -nitroarginine methyl ester was administered and produced an approximate 50% decrease in uterine blood flow for all 3 compounds. L Nitroarginine methyl ester attenuated increases in coronary blood flow induced by 17beta-estradiol, Estrace, and raloxifene. CONCLUSION: Raloxifene has significant coronary and uterine vascular effects in the ovariectomized ewe. The coronary and uterine responses are partially mediated by nitric oxide. PMID- 10739503 TI - Assessment by quantitative ultrasonometry of the effects of hormone replacement therapy on bone mass. AB - OBJECTIVE: This study was undertaken to evaluate the impact of hormone replacement therapy on results of quantitative ultrasonometry of the heel. STUDY DESIGN: A total of 2006 healthy perimenopausal women (mean age, 52.2 (10.3 years) were recruited in 5 German centers: 611 women (30%) had received hormone replacement therapy and 1395 (70%) had not. About 90% of the hormone replacement therapy users were current users, and the rest had stopped <6 months before the study. Speed of sound, broadband ultrasonographic attenuation, and the stiffness index were compared among the following groups: all users and nonusers of hormone replacement therapy, hormone replacement therapy users and nonuser control subjects matched for age and body mass index, and hormone replacement therapy users grouped in relation to the duration of hormone replacement therapy use and age and control subjects matched for body mass index. RESULTS: Women who were using hormone replacement therapy had significantly higher values (P <.001) than did nonusers for all ultrasonographic variables, even after we controlled for age and body mass index. Women who had used hormone replacement therapy for >3 years had significantly higher values (P <.001) than did matched control subjects for all variables. Differences increased with the duration of hormone replacement therapy use. CONCLUSION: Quantitative ultrasonometric measurement at the heel differentiates hormone replacement therapy users from nonusers, reflects duration of hormone replacement therapy use, and could be useful in both clinical trials and patient management. PMID- 10739504 TI - Prevalence of enlarged ovarian follicles among users of levonorgestrel subdermal contraceptive implants (Norplant). AB - OBJECTIVE: The aim of this study was to determine the prevalence of enlarged follicles, as detected by a single clinical or ultrasonographic examination, among users of levonorgestrel subdermal contraceptive implants (Norplant implants). STUDY DESIGN: This was a cross-sectional study of 103 users of Norplant implants and 50 users of the TCu380A intrauterine contraceptive device, all of whom received reproductive health services from PROFAMILIA, Santo Domingo, Dominican Republic. Bimanual pelvic examination and vaginal ultrasonography were performed. Enlarged follicles (>25 mm) were followed up weekly. The chi(2) test was applied to these data. RESULTS: Enlarged follicles were detected by ultrasonography in 17. 5% of Norplant implants users and 4% of TCu380A intrauterine contraceptive device users, respectively (P <.04). There was no difference according to duration of use. The longest time to involution of the follicles was 4 weeks. Forty percent of the enlarged follicles detected by ultrasonography were also detected by bimanual pelvic examination. CONCLUSION: Enlarged follicles are a frequent finding among women who use Norplant implants, but they are less frequent than described in previous studies, which were based on serial ultrasonographic scans in selected groups of users. Physicians and users should be aware of the transient nature of these enlarged follicles, which do not require intervention. PMID- 10739505 TI - The depth of the pouch of Douglas in nulliparous and parous women without genital prolapse and in patients with genital prolapse. AB - OBJECTIVES: This study was undertaken to assess the depth of the pouch of Douglas among nulliparous women and to compare it with those among parous women and patients with genital prolapse. STUDY DESIGN: The depth of the pouch of Douglas was measured in 22 nulliparous women and 28 parous women without genital prolapse and in 18 patients with prolapse of the posterior vaginal wall. RESULTS: The pouch of Douglas extended from 11% to 89% of the total vaginal length among nulliparous and parous women, without significant difference between these groups (mean, 49% and 45%, respectively). It was significantly deeper in patients with prolapse, ranging from 25% to 96% (mean, 72%). CONCLUSION: There is an extensive variation in the depth of the pouch of Douglas. A deep pouch of Douglas is frequently present in young nulliparous women and may increase the risk for development of a posterior vaginal wall prolapse. PMID- 10739506 TI - Clinical equivalence of intranasal and oral 17beta-estradiol for postmenopausal symptoms. AB - OBJECTIVE: The aim of this study was to demonstrate clinical equivalence between a novel intranasal estradiol formulation and a reference oral drug. STUDY DESIGN: In this multinational, double-blind, parallel-group study 659 postmenopausal women with moderate to severe postmenopausal symptoms were randomly assigned to receive either 300 microg/d intranasal 17beta-estradiol (S21400) or 2 mg/d oral micronized estradiol, plus the appropriate placebo, for 24 weeks. All patients also received 10 mg/d dydrogesterone for 14 days per 28-day cycle. Adjustment of intranasal dosage was permitted from week 14 on. The primary efficacy criterion was the Kupperman index at week 14, with a predefined limit of equivalence of 4. RESULTS: Kupperman index scores improved similarly in the 2 groups, from 28.4 +/- 6.2 to 10.0 +/- 8.6 (mean +/- SD) for S21400 and from 28.1 +/- 6.0 to 8.9 +/- 8.0 for oral therapy, with a difference between groups at week 14 of 1.1 +/- 0.6 (90% confidence interval, 0. 0 to 2.2). This was below the predefined equivalence limit of +4 for statistical noninferiority (P <.001). The daily number and intensity of hot flushes decreased similarly in the two treatment groups. Withdrawal bleeding was 20% less frequent with intranasal therapy (90% confidence interval, 12.5 to 27.6). Severe mastalgia was less frequent in the S21400 group (1.0%) than in the group with oral therapy (5.2%; P <.01). Triglyceride and angiotensinogen levels increased significantly with oral therapy but not with S21400. The same number of patients required dose adaptation in the 2 groups (approximately 20%). CONCLUSION: Intranasal administration of 300 microg/d estradiol was at least as effective as oral administration of 2 mg/d estradiol in alleviating postmenopausal symptoms, with less frequent mastalgia and uterine bleeding and without the metabolic consequences of the first-pass effect. PMID- 10739507 TI - Ovarian cancer gene therapy: repeated treatment with thymidine kinase in an adenovirus vector and ganciclovir improves survival in a novel immunocompetent murine model. AB - OBJECTIVE: Our purpose was to assess the effect of multiple injections of the system of herpes simplex virus thymidine kinase in an adenovirus vector and ganciclovir on survival in a murine model of human epithelial ovarian cancer. STUDY DESIGN: In this work we tested the ability of the system of thymidine kinase delivered by an adenovirus vector and ganciclovir to treat ovarian cancer in a novel murine model for epithelial ovarian cancer, SaskMouse. SaskMouse was developed by injecting LM-1 cells, a murine epithelial ovarian cancer cell line, intraperitoneally into a syngeneic C57BL/6N x C3H/He mouse strain. The cells developed into multiple cancer implants on different abdominal organs, leading to ascites and rapid death. The model has an intact immune system, as evidenced by the inability of different human cancer cells to develop into cancers when injected into the mice intraperitoneally. RESULTS: The system of thymidine kinase delivered by an adenovirus vector and ganciclovir was applied to SaskMouse. Mice were either untreated (group 1), treated with one intraperitoneal injection of adenovirus- thymidine kinase at 250 plaque-forming units/cell (group 2), or treated with two intraperitoneal injections of adenovirus-thymidine kinase at 250 plaque-forming units/cell on days 0 and 23 (group 3). Survivals were 23 +/- 2, 27 +/- 2, and 35 +/- 4 days, respectively (P <.05). Antiadenoviral antibodies were assayed both in the serum and in the peritoneal fluid of treated mice. Despite high antibody titers in serum, there were no detectable antibodies in the peritoneal fluid. CONCLUSION: Our data suggest that multiple intraperitoneal injections of the combination of thymidine kinase delivered by an adenovirus vector and ganciclovir are effective in prolonging survival in the presence of ovarian cancer. There are potential implications for other abdominal malignancies. PMID- 10739508 TI - Optimizing continuous-combined hormone replacement therapy for postmenopausal women: a comparison of six different treatment regimens. AB - OBJECTIVE: We sought to determine the optimum estradiol valerate medroxyprogesterone acetate regimens for efficacy and safety. STUDY DESIGN: We performed a 24-month, randomized, double-blind phase II study. Four hundred nineteen women who were postmenopausal for at least 3 years were placed in six parallel treatment groups and received 1 or 2 mg estradiol valerate with either 2.5 or 5 mg medroxyprogesterone acetate. In two groups the dose of estradiol valerate was increased from 1 to 2 mg estradiol valerate after 6 months. RESULTS: A marked improvement of climacteric symptoms was observed, and most women had no bleeding even during the first 3 months of treatment. The best bleeding pattern was achieved with 1 mg estradiol valerate and 2.5 or 5 mg medroxyprogesterone acetate, and in most groups the bleeding pattern improved over time. No cases of hyperplasia were observed. CONCLUSION: All regimens alleviated climacteric symptoms and provided excellent bleeding control, even during the early weeks of treatment. A choice of various dose combinations offers flexibility of dosing, thus enabling therapy to be tailored to the needs of individual women. PMID- 10739509 TI - Uterine effects of raloxifene in comparison with continuous-combined hormone replacement therapy in postmenopausal women. AB - OBJECTIVE: We sought to compare the uterine effects of raloxifene with those of continuous-combined hormone replacement therapy. STUDY DESIGN: This randomized, double-blind 24-month study involved 136 postmenopausal women who received raloxifene 150 mg/d or conjugated equine estrogens 0.625 mg/d with medroxyprogesterone acetate 2.5 mg/d. After baseline evaluations, endometrial biopsy specimens were obtained, and endometrial thickness was measured annually by means of transvaginal ultrasonography. Statistical analyses were performed with an intention-to-treat approach. RESULTS: In the raloxifene group at the end point of the study 94.4% of biopsy specimens showed normal benign postmenopausal endometrium and 5.6% were classified as benign stimulatory endometrium. In the continuous-combined hormone replacement therapy group at the end point of the study 78.7% of biopsy specimens showed normal benign postmenopausal endometrium, 19. 1% were classified as benign stimulatory endometrium, and 2.1% showed benign abnormal postmenopausal endometrium. Mean endometrial thickness was unchanged from baseline with raloxifene and was increased significantly by 0.5 mm at 12 months with continuous-combined hormone replacement therapy. CONCLUSION: Raloxifene 150 mg/d did not increase endometrial thickness or cause endometrial proliferation in healthy postmenopausal women. PMID- 10739510 TI - The effect of low-dose 3-keto-desogestrel added to a copper-releasing intrauterine contraceptive device on menstrual blood loss: a double-blind, dose finding, placebo-controlled study. AB - OBJECTIVE: We sought to establish the lowest dose of 3-keto-desogestrel, when added to a copper-releasing intrauterine contraceptive device, that is needed to reduce menstrual blood loss to a very low level and to treat (essential) menorrhagia successfully. STUDY DESIGN: A double-blind group comparative study was designed in which 203 healthy women with or without menorrhagia were enrolled and randomized to four different Multiload Cu250 intrauterine contraceptive devices, releasing 0 (control), 1.5, 3, or 6 microg of 3-keto-desogestrel daily. Menstrual blood loss, hemoglobin, and serum ferritin levels were determined before and during treatment. RESULTS: All 3-keto-desogestrel-copper intrauterine contraceptive devices reduced menstrual blood loss significantly, causing a reduction of up to 30 to 40 mL after 12 months of use. All women with essential menorrhagia were considered to have been successfully treated after 6 months of use. Serum ferritin levels rose with all three 3-keto-desogestrel-loaded devices. CONCLUSION: A Multiload Cu250 intrauterine contraceptive device releasing 1.5 microg of 3-keto-desogestrel daily is able to reduce menstrual blood loss to a very low level and to replete body iron stores in women with or without menorrhagia. Higher doses have no superior effect. PMID- 10739511 TI - Prostaglandin F2alpha-induced Ca++ oscillations in human myometrial cells and the role of RU 486. AB - OBJECTIVE: We sought to examine the change of cytosolic calcium concentration caused by prostaglandin F(2)(alpha) and RU 486 in cultured human myometrial cells. STUDY DESIGN: Human myometrial cells obtained from 16 nonpregnant women were loaded with fura 2, and the intracellular cytosolic calcium concentrations were measured by the use of wavelength spectrophotofluorometry. RESULTS: Application of prostaglandin F(2)(alpha) (2.8 micromol/L) caused an initial rapid rise in cytosolic calcium concentration followed by sustained cytosolic calcium oscillations at an average frequency of 0.43 +/- 0.04 min(-1) and an amplitude in the range of 296.82 +/- 27. 16 nmol/L. The oscillatory activity was not affected by increasing the concentration of prostaglandin F(2)(alpha) but varied by changing the concentration of extracellular cytosolic calcium concentration. The cytosolic calcium oscillations were suppressed by caffeine, 2,5-di-tert butylhydroquinone, and lanthanum but not affected by ryanodine. Verapamil decreased the amplitude but not the frequency of oscillations. The progesterone antagonist RU 486 at a concentration of 10(-8) to 10(-5) mol/L had no significant effect on the basal intracellular cytosolic calcium. However, RU 486 (10(-5) mol/L) significantly increased the frequency but not the amplitude of intracellular cytosolic calcium oscillations induced by prostaglandin F(2)(alpha). CONCLUSION: The results indicate that prostaglandin F(2)(alpha) stimulated cytosolic calcium oscillations are mediated by an increase in both cytosolic calcium release from inositol 1,4,5-trisphosphate-sensitive cytosolic calcium stores and a cytosolic calcium influx from the extracellular space. Moreover, RU 486 seems to directly regulate prostaglandin F(2)(alpha)-induced intracellular cytosolic calcium in human myometrial cells. PMID- 10739512 TI - Can antenatal clinical and biochemical markers predict the development of severe preeclampsia? AB - OBJECTIVE: This study was undertaken to develop a multivariable clinical predictive rule for severe preeclampsia using second-trimester clinical factors and biochemical markers. STUDY DESIGN: We performed a retrospective cohort study of all pregnant patients with single gestations from 1995 through 1997 for whom we had complete follow-up data. Through medical record review we determined whether patients had severe preeclampsia develop according to American College of Obstetricians and Gynecologists criteria. Case patients with severe preeclampsia were compared with control subjects with respect to clinical data and multiple marker screening test results. With potential predictive factors identified in the bivariate and stratified analyses both an explanatory logistic regression model and a clinical prediction rule were created. Patients were assigned a predictive score according to the presence or absence of predictive factors, and receiver operating characteristic analysis was used to determine the optimal score cutoff point for prediction of severe preeclampsia with maximal sensitivity. RESULTS: Among the 1998 patients we found 49 patients with severe preeclampsia (prevalence, 2.5%). After we controlled for confounding variables, case patients and control subjects had similar human chorionic gonadotropin and alpha-fetoprotein levels, and the only variables that remained significantly associated with severe preeclampsia were nulliparity (relative risk, 3.8; 95% confidence interval, 1.7-8.3), history of preeclampsia (relative risk, 5.0; 95% confidence interval, 1.7-17.2), elevated screening mean arterial pressure (relative risk, 3.5; 95% confidence interval, 1.7-7.2), and low unconjugated estriol concentration (relative risk, 1.7; 95% confidence interval, 0.9-3.4). Our predictive model for severe preeclampsia, which included only these 4 variables, had a sensitivity of 76% and a specificity of 46%. CONCLUSION: Even after incorporation of the strongest risk factors, our predictive model had only modest sensitivity and specificity for discrimination of patients at risk for development of severe preeclampsia. The addition of the human chorionic gonadotropin and alpha-fetoprotein biochemical markers did not enhance the model's predictive value for severe preeclampsia. PMID- 10739513 TI - Failure of serum beta2-microglobulin levels as an early marker of preeclampsia. AB - OBJECTIVE: Our purpose was to determine whether second-trimester maternal serum beta(2)-microglobulin levels could be used to predict subsequent development of preeclampsia. STUDY DESIGN: We first did a cross-sectional study to compare serum concentrations of beta(2)-microglobulin between women with preeclampsia and normotensive women. Serum beta(2)-microglobulin concentrations of 11 consecutive patients hospitalized for preeclampsia were compared with those of 11 normotensive women hospitalized for threatened premature labor. The second part of the study consisted of a nested case-control study in which each woman in whom preeclampsia ultimately developed was matched with 2 women who remained normotensive throughout gestation. For that purpose a total of 450 consecutive healthy nulliparous women were studied prospectively. Blood samples were collected between 20 and 24.9 weeks' gestation and frozen at -20 degrees C until assay after groups had been selected. RESULTS: In the cross-sectional study serum beta(2)-microglobulin levels were significantly higher in women with preeclampsia than in control women (1.87 +/- 0.36 mg/L vs 1.01 +/- 0. 12 mg/L; t = 7.61; P <.0001). Among the 450 women who were followed up prospectively, preeclampsia developed in 7 (1.5 %). Fourteen of the women who remained normotensive were matched with the 7 women in whom preeclampsia ultimately developed. No difference was found in early serum beta(2)-microglobulin concentrations between women in whom preeclampsia subsequently developed and those who remained normotensive throughout gestation (1.02 +/- 0.12 vs 0.95 +/- 0.12 mg/L). CONCLUSIONS: Serum beta(2)-microglobulin levels do not predict subsequent preeclampsia. PMID- 10739514 TI - Is vaginal birth after cesarean less expensive than repeat cesarean delivery? AB - OBJECTIVE: This study was undertaken to compare total medical costs of trial of labor after cesarean with those of elective repeat cesarean without labor, with both short- and long-term neonatal costs associated with such procedures taken into account. STUDY DESIGN: Costs associated with All Patient Refined diagnosis related groups and Current Procedural Terminology for a large not-for-profit health care system were applied to an algorithm describing maternal and neonatal outcomes of trial of labor. Perinatal morbidity rates and cost estimates for long term neurologic damage associated with uterine rupture were derived from published literature. RESULTS: If a 70% vaginal birth rate for women undergoing a trial of labor and delivery in a tertiary center with a mean uterine rupture to delivery time of 13 minutes is assumed, the net cost differential ranged from a saving of $149 to a loss of $217, depending on morbidity assumptions. For vaginal birth after cesarean success rates <70%, trial of labor in the presence of two previous scars, and institutional factors increasing the perinatal morbidity rate by just 4% with respect to that seen in tertiary centers, trial of labor resulted in a net financial loss to the health care system regardless of all other assumptions made. CONCLUSIONS: When costs as opposed to charges are considered and the cost of long-term care for neurologically injured infants is taken into account, trial of labor after previous cesarean is unlikely to be associated with a significant cost saving for the health care system. Recent government-mandated length-of-stay requirements are likely to make the economic benefit of vaginal birth after cesarean even less favorable. Factors other than cost must govern decisions regarding trial of labor or repeat cesarean. PMID- 10739515 TI - The fetal electrocardiogram: relationship with acidemia at delivery. AB - OBJECTIVE: This study was undertaken to analyze the relationship between the fetal electrocardiogram and arterial pH and base excess at delivery. STUDY DESIGN: In the labor wards of three teaching hospitals a retrospective observational study was conducted on fetal monitor recordings of 679 women for whom there was an indication for continuous fetal monitoring during labor. These women had been recruited as part of either observational studies or a prospective randomized trial related to the Nottingham fetal electrocardiographic project. Fetal heart and uterine contraction data were obtained with the Nottingham fetal electrocardiographic analyzer. Morphologic and time interval analyses of the fetal electrocardiogram were performed. Evaluation was carried out for the last half hour before delivery. Main outcome measures were umbilical arterial pH and base excess at delivery. RESULTS: The study demonstrated a relationship between time interval analysis of the fetal electrocardiogram and a low umbilical arterial pH and base excess at delivery. Analysis of the morphologic characteristics of the fetal electrocardiogram (ST segment and T-wave height) showed no significant relationship. CONCLUSIONS: Time interval analysis of the fetal electrocardiogram during labor is related to relative acidemia at delivery. PMID- 10739516 TI - Predictors of neonatal outcome in women with severe preeclampsia or eclampsia between 24 and 33 weeks' gestation. AB - OBJECTIVE: We sought to characterize predictors of neonatal outcome in women with severe preeclampsia or eclampsia who were delivered of their infants preterm. STUDY DESIGN: We performed a retrospective analysis of 195 pregnancies delivered between 24 and 33 weeks' gestation because of severe preeclampsia or eclampsia. Multiple logistic regression and univariate chi(2) analysis were performed for the dependent outcome variables of survival and respiratory distress syndrome by use of independent fetal and maternal variables. A P value of <.05 was considered significant. RESULTS: In the multivariate analysis, respiratory distress syndrome was inversely related to gestational age at delivery (P =.0018) and directly related to cesarean delivery (P =.02), whereas survival was directly related to birth weight (P =.00025). There was no correlation in the multivariate analysis between respiratory distress syndrome or survival and corticosteroid use, composite neonatal morbidity, mean arterial pressure, eclampsia, or abruptio placentae. In the univariate analysis respiratory distress syndrome was associated with cesarean delivery (odds ratio, 7.19; 95% confidence interval, 2. 91-18.32). The incidence of intrauterine growth restriction increased as gestational age advanced. Furthermore, intrauterine growth restriction decreased survival in both the multivariate (P =. 038; odds ratio, 13.2; 95% confidence interval, 1.16-151.8) and univariate (P =.001; odds ratio, 5.88; 95% confidence interval, 1. 81-19.26) analyses. CONCLUSION: The presence of intrauterine growth restriction adversely affected survival independently of other variables. Presumed intrauterine stress, as reflected by the severity of maternal disease, did not improve neonatal outcome. PMID- 10739517 TI - The role of cyclic nucleotides in the spontaneous contractility and responsiveness to nitric oxide of the rat uterus at midgestation and term. AB - OBJECTIVES: The aim of this investigation was to study the effects of pharmacologic manipulation of cyclic nucleotide levels on the uterine spontaneous contractile activity and responsiveness to nitric oxide in pregnant rats at midgestation and term. STUDY DESIGN: Uterine rings from pregnant rats at midgestation and term were placed in organ chambers for isometric tension recording. Concentration-response curves were obtained for phosphodiesterase and guanylate cyclase inhibitors, membrane-permeable cyclic nucleotide analogs, and forskolin. In addition, the effects of minimal inhibitory concentrations of these agents on the concentration-response relationships for diethylamine nitric oxide (a nitric oxide donor) were studied. RESULTS: Nonselective phosphodiesterase inhibitors induced more inhibition of contractions of uterine rings from pregnant rats at term than at midgestation and zaprinast induced less. Inhibitors of guanylate cyclase and membrane-permeable analogs of cyclic guanosine monophosphate were equally effective in tissues from pregnant rats at midgestation and term. All agents attenuated the inhibitory effect of diethylamine nitric oxide at midgestation; however, dibutyryl cyclic adenosine monophosphate and papaverine increased the inhibitory effect of diethylamine nitric oxide in tissues from pregnant animals at term. CONCLUSIONS: Cyclic nucleotides modulate both spontaneous and nitric oxide-induced changes in uterine contraction during pregnancy. Application of nonselective inhibitors of phosphodiesterase, as well as membrane-permeable analogs of cyclic adenosine monophosphate, may counteract refractoriness to nitric oxide at term. PMID- 10739518 TI - Adenylate cyclase and potassium channels are involved in forskolin- and 1,9 dideoxyforskolin-induced inhibition of pregnant rat uterus contractility. AB - OBJECTIVE: We sought to study the contribution of potassium channels in the effect of forskolin and 1,9-dideoxyforskolin on uterine contractility in the pregnant rat. STUDY DESIGN: Rings taken from the middle portions of uterine horns from rats at 16 days of gestation were positioned in organ chambers containing physiologic salt solution bubbled with 5% carbon dioxide in air (37 degrees C, pH approximately 7.4) for isometric tension recording under 2 g passive tension. The effects of cumulative concentrations of forskolin and 1,9-dideoxyforskolin in the absence or presence of an adenylate cyclase inhibitor (MDL-12,330A, 10(-5) mol/L), a nonselective potassium channel blocker (tetrabutylammonium, 10(-4) mol/L), or an adenosine triphosphate-dependent potassium channel blocker (glibenclamide 10(-5) mol/L) were studied. RESULTS: Both forskolin and, to a lesser extent, 1,9-dideoxyforskolin inhibit uterine contractions. Tetrabutylammonium, glibenclamide, and MDL-12, 330A attenuated the effects of forskolin, whereas glibenclamide was less effective against 1,9-dideoxyforskolin. CONCLUSION: Activation of adenylate cyclases, as well as adenosine triphosphate dependent potassium channels and, to a greater extent, calcium-dependent potassium channels, is involved in the inhibitory effect of forskolin in uterine rings from rats at 16 days of gestation. Inhibition of uterine contractions by 1,9-dideoxyforskolin is less than that by forskolin and involves activation of adenylate cyclase and calcium-dependent potassium channels. Whether activation of guanylate cyclase is involved in the effect of the agents on calcium-dependent potassium channels needs further investigation. 1, 9-Dideoxyforskolin is not an inactive isomer of forskolin in rat uterine rings. PMID- 10739519 TI - The preterm prediction study: granulocyte colony-stimulating factor and spontaneous preterm birth. National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network. AB - OBJECTIVE: Granulocyte colony-stimulating factor is elevated in the amniotic fluid and plasma of women with chorioamnionitis and active preterm labor. We investigated the relationship between plasma granulocyte colony-stimulating factor and subsequent spontaneous preterm birth in pregnant women without symptoms. STUDY DESIGN: We performed a nested case-control study involving 194 women who had a singleton spontaneous preterm birth and 194 matched term control subjects from the patient pool (n = 2929) enrolled in the Preterm Prediction Study. Plasma collected at 24 and 28 weeks' gestation was analyzed for granulocyte colony-stimulating factor, and the results were compared with subsequent spontaneous preterm birth. RESULTS: Compared with term control subjects, women who were delivered of their infants spontaneously at <28 weeks' gestation had increased mean granulocyte colony-stimulating factor values at 24 weeks' gestation (84.7 +/- 38.4 vs 67.7 +/- 28.6 pg/mL; P =.049), and women who were delivered of their infants at <32 weeks' gestation had increased mean plasma granulocyte colony-stimulating factor values at 28 weeks' gestation (80.4 +/- 24.1 vs 55.9 +/- 16.5 pg/mL; P =. 001). At 24 weeks' gestation a granulocyte colony-stimulating factor value >75th percentile in control subjects (approximately 80 pg/mL) was found in 48.9% (23/47) of all women delivered of their infants at <32 weeks' gestation versus 14.9% (7/47) of the term control subjects (adjusted odds ratio, 6.2; 95% confidence interval, 1.8-20. 8). At 28 weeks' gestation a granulocyte colony-stimulating factor value >75th percentile was found in 36.8% (7/19) of women delivered of their infants at <32 weeks' gestation versus 5.3% (1/19) of term control subjects (adjusted odds ratio, 25.7; 95% confidence interval, 1.5-470.4). When measured at 24 or 28 weeks' gestation, granulocyte colony-stimulating factor did not predict spontaneous preterm birth at 32 to 34 weeks' gestation or at 35 to 36 weeks' gestation. CONCLUSION: In pregnant women without symptoms at 24 and 28 weeks' gestation, elevated plasma granulocyte colony-stimulating factor levels are associated with subsequent early (<32 weeks' gestation) spontaneous preterm birth, especially within the next 4 weeks, but not with late spontaneous preterm birth. These data provide further evidence that early spontaneous preterm birth is associated with an inflammatory process that is identifiable by the presence of a cytokine in maternal plasma several weeks before the early spontaneous preterm birth; however, later spontaneous preterm birth is not associated with this process. PMID- 10739520 TI - The preterm prediction study: cervical lactoferrin concentration, other markers of lower genital tract infection, and preterm birth. National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network. AB - OBJECTIVE: This study was undertaken to determine the relationship among cervical lactoferrin concentration, other cervical markers potentially related to infection, and spontaneous preterm birth. STUDY DESIGN: Cervical lactoferrin concentrations obtained at 22 to 24 weeks' gestation among 121 women who had a spontaneous preterm birth <35 weeks' gestation were compared with cervical lactoferrin concentrations among 121 women matched for race, parity, and center who were delivered at >/=37 weeks' gestation. Results were compared against levels of cervical interleukin 6, fetal fibronectin, and sialidase, against cervical length according to ultrasonography, and according to the bacterial vaginosis Gram stain score. RESULTS: Cervical lactoferrin concentrations ranged from not measurable (19% of the concentrations were below the threshold for this assay) to a titer of >/=1:64. There was no significant difference in the overall distributions of lactoferrin concentrations between the case patients and control subjects (P =.18). Only when the highest titers of lactoferrin were considered were there more women in the spontaneous preterm birth group (6/121 vs 0/121; P =.03). According to Spearman correlation analyses the cervical lactoferrin concentrations were strongly related to interleukin 6 concentration (r =.51; P =.0001), sialidase activity (r =.38; P =.0001), and bacterial vaginosis (r =.38; P =.0001), were weakly related to fetal fibronectin (r =. 16; P =.01), and were not related to cervical length. With the 90th percentile (a dilution of 1:32) used as a cutoff to establish a dichotomous variable, lactoferrin concentration had the following odds ratios and 95% confidence intervals for associations with other potential markers of infection: bacterial vaginosis odds ratio, 4.8 (95% confidence interval, 2.2-10.3); interleukin 6 concentration odds ratio, 2.8 (95% confidence interval, 1.2-6.5); sialidase activity odds ratio, 5. 5 (95% confidence interval, 2.2-13.7); fetal fibronectin concentration odds ratio, 0.6 (95% confidence interval, 0.2-2.0); chlamydiosis odds ratio, 2.3 (95% confidence interval, 0.8-6.9); and short cervix odds ratio, 0.5 (95% confidence interval, 0.2-1.4). CONCLUSIONS: Lactoferrin found in the cervix correlated well with other markers of lower genital tract infection. High lactoferrin levels were associated with spontaneous preterm birth but had a very low predictive sensitivity. PMID- 10739521 TI - The Preterm Prediction Study: sequential cervical length and fetal fibronectin testing for the prediction of spontaneous preterm birth. National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network. AB - OBJECTIVES: This study was undertaken to further elucidate the pathogenesis of preterm birth by means of traditional risk factors and new markers for preterm birth derived from the Preterm Prediction Study. STUDY DESIGN: A total of 3076 women (2929 with singleton gestations and 147 with twin pregnancies) were categorized according to the presence of risk factors including black race, low body mass index, the presence of bacterial vaginosis, and previous preterm birth. At 24 and 28 weeks' gestation cervical length was measured and categorized as short (25 mm). Vaginal and cervical fetal fibronectin concentrations were measured at 24, 26, 28, and 30 weeks' gestation and results were categorized as positive (>/=50 ng/mL) or negative (<50 ng/mL). RESULTS: At 24 to 26 weeks' gestation women with each of the risk factors were more likely to have positive fibronectin test results or to have a short cervix. Among women with negative fetal fibronectin results at 24 to 26 weeks' gestation those with a short cervix were more likely to have positive fetal fibronectin results at 28 to 30 weeks' gestation, and among those with normal cervical length those women who had positive fetal fibronectin results were more likely to have a short cervix at later evaluation. Most women who had positive fetal fibronectin results at 24 to 26 weeks' gestation had negative results at 28 to 30 weeks' gestation, whereas most but not all women who had a short cervix at 24 to 26 weeks' gestation still had a short cervix at 28 to 30 weeks' gestation. In each period women with both a positive fetal fibronectin result and a short cervix were at substantially increased risk of spontaneous preterm birth; women with either marker alone had intermediate and approximately equal risks of spontaneous preterm birth, and women without either marker had a low risk of spontaneous preterm birth. CONCLUSION: Regardless of other risk factors, a short cervix predicts a subsequent positive fetal fibronectin result, and a positive fetal fibronectin result predicts subsequent cervical shortening. These data do not support a single sequence of events leading to spontaneous preterm birth. PMID- 10739522 TI - Elevation of total nitrite and nitrate concentration in vaginal secretions as a predictor of premature delivery. AB - We measured the total concentration of nitrite and nitrate, metabolites of nitric oxide, in vaginal secretions from pregnant women at 22 to 32 weeks' gestation. Total nitrite and nitrate concentrations in patients with preterm premature rupture of membranes and in those with preterm labor and subsequent premature delivery were significantly higher than concentrations in patients who were delivered at term. Elevated total nitrite and nitrate concentration may predict premature delivery. PMID- 10739523 TI - Is the angle of needle insertion influencing the created defect in human fetal membranes? Evaluation of the agreement between specialists' opinions and ex vivo observations. AB - OBJECTIVE: We sought to evaluate the agreement between the opinions of specialists in fetal medicine with ex vivo observations on the potential influence of the angle of needle insertion on the fluid leak through the created defect in human fetal membranes. STUDY DESIGN: Membranes from placentas of women who were delivered by elective cesarean were harvested, cut in pieces, and secured to the bottom of plastic tubes filled with Hartmann solution. They were punctured with 18-, 20-, or 22-gauge needles, with an angle of insertion of 90 degrees (group 1) or 45 degrees (group 2), and the flow rate (in milliliters per minute) through the created defect at a constant pressure of 150 mm H(2)O was measured. Fifty physicians performing amniocentesis at fetal medicine reference centers were interviewed about their impression and clinical attitude with respect to the angle of needle insertion at the time of amniocentesis. RESULTS: In the ex vivo study, puncture with a 45 degrees angle was associated with a significantly lower flow of fluid through the membrane defect for all needle sizes tested. Regarding survey answers, 82% of physicians try to perform amniocentesis with a given angle (ie, 90 degrees in the vast majority of cases). Among the reasons for doing so, minimizing membrane damage was mentioned in more than half of cases. CONCLUSION: These results provide evidence that the angle of needle insertion influences the type of defect and therefore the flow rate through human membranes. Membrane damage is a concern of a proportion of specialists while performing amniocentesis, but ex vivo observations do not agree with clinical assumptions on the potential influence of the angle of insertion. PMID- 10739524 TI - Adrenomedullin is increased in the fetoplacental circulation in intrauterine growth restriction with abnormal umbilical artery waveforms. AB - OBJECTIVE: To examine whether adrenomedullin, a novel vasoactive peptide produced by the placenta, participates in the uteroplacental hemodynamic alterations in intrauterine growth restriction, we studied the correlation between adrenomedullin levels and fetoplacental blood flow. STUDY DESIGN: Maternal and umbilical blood samples were collected in pregnancies complicated by intrauterine growth restriction with abnormal umbilical artery Doppler findings and in control pregnancies. Adrenomedullin levels were measured by means of a specific radioimmunoassay, and flow velocimetry waveforms were recorded from uterine, umbilical, and fetal middle cerebral arteries. RESULTS: Mean adrenomedullin values in umbilical plasma were higher (P <.05) in patients with intrauterine growth restriction (63.7 +/- 34.2 pg/mL; n = 16) than in control subjects (38.1 +/- 14.8 pg/mL; n = 16). A significant correlation was found between maternal adrenomedullin levels and umbilical artery pulsatility index. Moreover, fetal adrenomedullin concentrations correlated negatively with middle cerebral artery pulsatility index and positively with umbilical artery pulsatility index/middle cerebral artery pulsatility index ratio. CONCLUSION: This study provides evidence that adrenomedullin is increased in fetuses with intrauterine growth restriction in response to reduced uteroplacental blood flow and suggests that it may participate in the fetal hemodynamic modifications. PMID- 10739525 TI - Routine second-trimester ultrasonography in the United States: a cost-benefit analysis. AB - OBJECTIVE: The objective of this study was to perform a cost-benefit analysis of routine second-trimester screening ultrasonography in the United States as compared with performing ultrasonography only in the presence of indications. STUDY DESIGN: It was assumed that 1 million pregnant women are available annually who otherwise would not have an indication for an ultrasonographic examination. Cost savings from early detection and therapeutic abortion were considered only for fetal conditions for which lifetime cost estimates are available, including spina bifida, major cardiac disease, cleft lip or palate, renal agenesis or dysgenesis, urinary obstruction, lower or upper limb reduction, omphalocele, gastroschisis, and diaphragmatic hernia. Two separate cost-benefit analyses were considered with the range of fetal anomaly detection rates before 24 weeks' gestation as reported by tertiary and non-tertiary centers in the Routine Antenatal Diagnostic Imaging with Ultrasound (RADIUS) trial. Potential cost savings from averting treatment for preterm labor and postdate gestations were also considered. RESULTS: The ratio of savings to cost was between 1.35 and 1.70 (savings of $1.35-$1.70 per $1 spent) if the ultrasonographic examinations were performed in tertiary care centers. The ratio of savings to cost was between 0.40 and 0.74 (loss of $0.26-$0.60 per $1 spent) if the examinations were performed in nontertiary centers. If the screening ultrasonography was performed in tertiary centers, the expected annual net benefits were estimated at $97 to 189 million. If ultrasonographic screening was performed in nontertiary centers, the expected annual net losses were estimated at $69 to 161 million. CONCLUSION: Routine second-trimester ultrasonographic screening appears to be associated with net benefits only if the ultrasonography is performed in tertiary care centers. PMID- 10739526 TI - Amniotic gas values and acid-base status during acute maternal hyperoxemia and hypoxemia in the early fetal sheep. AB - OBJECTIVE: This study was undertaken to determine amniotic fluid gas values and acid-base balance during maternal hyperoxemia and hypoxemia in early pregnancy. STUDY DESIGN: Anesthetized sheep (n = 12) in early and mid pregnancy (0.3 to 0.5 gestation) were subjected to hyperoxemia followed by hypoxemia. Amniotic fluid PO (2), pH, PCO (2), bicarbonate concentration, and base excess were monitored continuously with a multiparameter Paratrend (Diametrics Medical Inc, St Paul, Minn) sensor and compared between 0.3 and 0.5 gestation. RESULTS: During maternal normoxemia all parameters were constant. At all gestational ages maternal hyperoxemia caused no changes apart from a rapid increase in amniotic fluid PO (2) (P <.001). Maternal hypoxemia led to a reduced amniotic fluid PO (2) (P <.001), whereas the PCO (2) and the bicarbonate concentration increased (P <.001). Changes in amniotic fluid gas values and acid-base balance were more pronounced at 0.3 gestation than at 0.5 gestation. Amniotic fluid Po(2) responded earlier to maternal hyperoxemia than to hypoxemia (9. 5 vs 14.3 minutes; P <.001). During hypoxemia maternal PaCO (2) changed faster than did amniotic fluid PCO (2) (P <.001). CONCLUSIONS: Acute maternal hypoxemia during early pregnancy was quickly reflected in amniotic fluid gas values and acid-base balance, whereas hyperoxygenation induced the quickest changes in amniotic fluid PO (2). The metabolic effects of maternal hyperoxemia and hypoxemia were also more pronounced during early pregnancy than at mid pregnancy. PMID- 10739527 TI - Effect of fetal magnetic resonance imaging on fetal heart rate patterns. AB - OBJECTIVE: Our aim was to record the fetal heart rate before and during magnetic resonance imaging to observe the effects of the magnetic resonance imaging process on fetal heart rate parameters during imaging. STUDY DESIGN: Fetal heart rate recordings were obtained in 10 pregnant volunteers at the time of magnetic resonance imaging. All the pregnant women were at term (37-41 weeks) with singleton fetuses in the cephalic presentation. The scanning was performed on a 0.5-T purpose-built superconductive magnet by use of echo-planar imaging. The fetal heart recordings were obtained with a modified Sonicaid Meridian 800 (Oxford) Doppler ultrasound monitor. Recordings of the fetal heart were made for a period of at least 15 minutes outside the magnet and then for at least 15 minutes inside the magnet. RESULTS: There were no significant changes in any fetal heart rate parameters before and during the magnetic resonance imaging, as determined by the Wilcoxon matched-pairs signed-ranks test (P >.3). CONCLUSION: This is the first report of fetal heart rate recording during magnetic resonance imaging of the fetus. Magnetic resonance imaging does not produce demonstrable effects on fetal heart rate patterns. PMID- 10739528 TI - Localization of p43 placental isoferritin in human maternal-fetal tissue interface. AB - OBJECTIVE: Human placental isoferritin, which is composed of a 43-kd protein subunit, is exclusively reactive with the CM-H9 monoclonal antibody. The p43 exerts immunosuppressive activity during pregnancy. The aim of this study was to localize the expression of p43 in the maternal-fetal tissue interface during normal gestation. STUDY DESIGN: Villous tissues samples were collected between 5 and 20 weeks' gestation and at term from uncomplicated pregnancies. Immunohistochemical localization of p43 was performed with CM-H9 monoclonal antibody. RESULTS: During the first trimester p43 was highly expressed in syncytiotrophoblast, Hofbauer cells, and decidual macrophages. From 15 weeks' gestation onward expression in the syncytiotrophoblast was below the level of detection; however, p43 was demonstrated in villous Hofbauer cells and decidual macrophages throughout gestation. CONCLUSIONS: Expression of p43 was demonstrated on both sides of the maternal-fetal tissue interface, with localization dependent on gestational age. This may suggest its immunologic function throughout pregnancy. First-trimester syncytiotrophoblast displayed high p43 levels, which disappeared later on, whereas maternal serum p43 level continued to increase, which suggests an extraplacental source for p43. PMID- 10739529 TI - Fetal exposure to an intra-amniotic inflammation and the development of cerebral palsy at the age of three years. AB - OBJECTIVE: The aim of this study was to determine whether fetal exposure to intra amniotic inflammation and a systemic fetal inflammatory response (funisitis) are associated with the development of cerebral palsy at the age of 3 years. STUDY DESIGN: This cohort study included 123 preterm singleton newborns (gestational age at birth, /=3 years. The presence of intra-amniotic inflammation was determined by elevated amniotic fluid concentrations of proinflammatory cytokines such as interleukins 6 and 8 and by amniotic fluid white blood cell count. Cytokine concentrations were measured with sensitive and specific immunoassays. Funisitis was diagnosed in the presence of neutrophil infiltration into the umbilical vessel walls or Wharton jelly. Cerebral palsy was diagnosed by neurologic examination at the age of 3 years. RESULTS: Newborns with subsequent development of cerebral palsy had a higher rate of funisitis and were born to mothers with higher median concentrations of interleukins 6 and 8 and higher white blood cell counts in the amniotic fluid compared with newborns without subsequent development of cerebral palsy (funisitis: 75% [9/12] vs 23% [24/105]; interleukin 6: median, 18.9 ng/mL; range, 0. 02-92.5 ng/mL; vs median, 1.0 ng/mL; range, 0.01-115.2 ng/mL; interleukin 8: median, 13.0 ng/mL; range, 0.1-294.5 ng/mL; vs median, 1.2 ng/mL; range, 0.05-285.0 ng/mL; white blood cell count: median, 198 cells/mm(3); range, 0->1000 cells/mm(3); vs median, 3 cells/mm(3); range, 0-19,764 cells/mm(3); P <.01 for each). After adjustment for the gestational age at birth, the presence of funisitis and elevated concentrations of interleukins 6 and 8 in amniotic fluid significantly increased the odds of development of cerebral palsy (funisitis: odds ratio, 5.5; 95% confidence interval, 1.2-24.5; interleukin 6: odds ratio, 6.4; 95% confidence interval, 1. 3 33.0; interleukin 8: odds ratio, 5.9; 95% confidence interval, 1. 1-30.7; P <.05 for each). CONCLUSION: Antenatal exposure to intra-amniotic inflammation and evidence of a systemic fetal inflammatory response (funisitis) are strong and independent risk factors for the subsequent development of cerebral palsy at the age of 3 years. PMID- 10739530 TI - Ethanol exposure induces oxidative stress and impairs nitric oxide availability in the human placental villi: a possible mechanism of toxicity. AB - OBJECTIVE: We undertook this investigation to explore the effects of ethanol exposure on nitric oxide synthase levels and nitric oxide release. Our hypothesis was that ethanol exposure modifies nitric oxide activity within the placenta as a result of oxidative stress. STUDY DESIGN: Four 10-g samples of term normal human placental villous tissue were perifused with nonrecirculating Dulbecco's modified Eagle's medium and 25-mmol/L N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] with 0-, 50-, 100-, or 200-mmol/L ethanol. After 2 hours of exposure, tissue was removed, fixed, and frozen for analysis. Immunohistochemical analysis was performed for subtype I or neuronal nitric oxide synthase (nNOS), subtype II or inducible nitric oxide synthase (iNOS), and subtype III or endothelial nitric oxide synthase (eNOS) localization. Western blot analysis was performed for eNOS quantitation. Cyclic guanosine monophosphate and copper-zinc superoxide dismutase levels were measured by electroimmunoassay and kinetic assay, respectively. Nitric oxide release was analyzed by a Sievers nitric oxide analyzer. RESULTS: Immunohistochemical examination confirmed that only eNOS was localized to the syncytiotrophoblasts. After ethanol exposure, eNOS protein expression increased 2.5- to 3.0-fold over that of the control. Tissue cyclic guanosine monophosphate content and nitric oxide release into the effluent were decreased, whereas superoxide dismutase levels were increased at higher ethanol levels (P <.05). CONCLUSION: Ethanol exposure appears to induce oxidative stress, which may account for the decreased nitric oxide release, because nitric oxide may be shunted toward scavenging free radicals. Increased eNOS protein expression may be a response to the increased demand for nitric oxide. Decreased nitric oxide availability could adversely affect placental blood flow regulation, which could, in turn, account for the growth restriction seen in ethanol-exposed fetuses. PMID- 10739531 TI - Mathematic modeling of forces associated with shoulder dystocia: a comparison of endogenous and exogenous sources. AB - OBJECTIVE: A mathematic model was developed to estimate the compressive pressure on the fetal neck overlying the roots of the brachial plexus by the symphysis pubis during a shoulder dystocia event. The induced pressure was calculated for both exogenous (clinician applied) and endogenous (maternal and uterine) forces during the second stage of labor. STUDY DESIGN: Intrauterine pressure and clinician-applied force data were taken from the existing literature. A free-body diagram was generated and equilibrium equations were used to calculate the contact pressure between the base of the fetal neck and the symphysis pubis during a shoulder dystocia event. RESULTS: Clinician-applied traction to the fetal head (exogenous force) led to an estimated contact pressure of 22.9 kPa between the fetal neck and the symphysis pubis. In contrast, uterine and maternal expulsive efforts (endogenous forces) resulted in contact pressures that ranged from 91.1 to 202.5 kPa. The estimated pressures resulting from endogenous forces are 4 to 9 times greater than the value calculated for clinician-applied forces. CONCLUSION: Neonatal brachial plexus injury is not a priori explained by iatrogenically induced excessive traction. Spontaneous endogenous forces may contribute substantially to this type of neonatal trauma. PMID- 10739532 TI - Keratinocyte growth factor in the promotion of human chorionic gonadotropin production in human choriocarcinoma cells. AB - OBJECTIVE: Several growth factors and cytokines appear to participate in the proliferation or differentiation of trophoblast cells. The purpose of this study was to investigate the extent to which keratinocyte growth factor participates in the development of human embryonic and trophoblast cells at the maternal-fetal interface. STUDY DESIGN: The reverse transcriptase-polymerase chain reaction method was used to determine the gene expression of keratinocyte growth factor and keratinocyte growth factor receptor in human choriocarcinoma cells (BeWo), human teratocarcinoma cells (PA-1), and human endometrial stromal cells. We also examined the effects of keratinocyte growth factor on cell proliferation and production of human chorionic gonadotropin in BeWo and PA-1 cells. RESULTS: Keratinocyte growth factor gene was expressed in all cell types. The expression was pronounced in stromal cells of the endometrium collected during the secretory phase and early pregnancy. The keratinocyte growth factor expression was also enhanced in the differentiated BeWo cells. The expression of keratinocyte growth factor receptor gene was observed only in the BeWo cells. The addition of keratinocyte growth factor to the medium did not affect cell proliferation of the BeWo and PA-1 cells. On the other hand, keratinocyte growth factor (100 ng/mL) significantly enhanced human chorionic gonadotropin production in the BeWo cells. Stimulatory action of keratinocyte growth factor on human chorionic gonadotropin production in the BeWo cells was markedly enhanced after forskolin-induced differentiation. CONCLUSIONS: We conclude that keratinocyte growth factor may play an important role in promotion of human chorionic gonadotropin production in the trophoblast cells. PMID- 10739533 TI - Effect of esophageal ligation on amniotic fluid volume and urinary flow rate in fetal sheep. AB - OBJECTIVE: Although the fetus normally swallows large volumes of amniotic fluid each day, it is unclear whether amniotic fluid volume increases after fetal esophageal obstruction or whether fetal urine production changes. Our objective was to determine the effects of fetal esophageal ligation on amniotic fluid volume and urinary flow rate over time. STUDY DESIGN: Seven late-gestation fetal sheep underwent esophageal ligation, and 7 served as time control animals. The urachus was ligated to eliminate urine flow to the allantoic cavity. On days 1, 3, 5, 7, and 9 after surgery, we measured the composition of amniotic fluid, fetal urine, and fetal and maternal blood, as well as amniotic fluid volume and fetal urinary flow rate. A 3-factor analysis of variance was used for statistical analysis. RESULTS: Amniotic fluid volume did not change with time in the control group, averaging 876 +/- 142 mL (mean +/- SEM), and it decreased in the esophageal ligation group (P =.020), averaging 309 +/- 75 mL on day 9. Fetal urinary flow rate was lower (P =.0063) in the esophageal ligation group (431 +/- 27 mL/d) than in the control group (631 +/- 54 mL/d). There were no differences in fetal or maternal blood compositions between the two groups. Amniotic fluid sodium and chloride increased in the ligated animals. CONCLUSION: Polyhydramnios did not occur after esophageal ligation, even though the fetuses excreted approximately 4000 mL of urine over the 9-day study period. This suggests that intramembranous absorption is substantially increased. With only small changes in amniotic solute concentrations, intramembranous solute absorption must occur simultaneously with water, suggesting a near-zero reflection coefficient for solutes. We speculate that fetal urine, lung secretions, or both contain a factor that increases intramembranous permeability. PMID- 10739534 TI - Obstetric and perinatal outcomes from the australian and new zealand twin-twin transfusion syndrome registry. AB - OBJECTIVE: Our purpose was to investigate the antepartum characteristics and perinatal outcomes of twin-twin transfusion syndrome cases from a multicenter national registry. STUDY DESIGN: Perinatal centers in Australia and New Zealand voluntarily notified a central evaluation registry with information on identified pregnancies with twin-twin transfusion syndrome during 1995 through 1998. RESULTS: One hundred twelve cases of twin-twin transfusion syndrome were registered. The median gestation at diagnosis was 21.5 weeks (range, 14.4-34.6 weeks). Oligohydramnios-polyhydramnios sequence was the most common presentation, with 84% of cases involving "stuck" twinning. Therapeutic amnioreduction was used in 92 cases (82.1%), with the median number of procedures per case being 2 (range, 1-23). The median gestation at delivery was 29 weeks (range, 18-38 weeks). The overall perinatal survival rate was 62.5%. Abnormal findings on cranial ultrasonography were present in 27.3% of live neonates, and periventricular leukomalacia was reported in 10.8%. Increased gestational age at delivery, the presence of umbilical artery diastolic flow, and a prolonged interval from final amnioreduction to delivery were positively associated with the delivery of live fetuses without complications. CONCLUSION: The majority of antenatally identified cases of twin-twin transfusion syndrome are managed with serial amnioreduction. Despite contemporary obstetric and neonatal management strategies, perinatal mortality and morbidity rates are high. PMID- 10739535 TI - Nitric oxide modulates angiotensin II-induced drinking behavior in the near-term ovine fetus. AB - OBJECTIVE: Human and ovine fetuses demonstrate an enhanced rate of spontaneous and angiotensin II-stimulated swallowing. Angiotensin II and nitric oxide synthase have been localized to thirst centers in the brain. This study was performed to determine whether central nitric oxide contributes to the regulation of angiotensin II-induced fetal swallowing. STUDY DESIGN: Six pregnant ewes with near-term singleton fetuses were chronically prepared with fetal vascular and lateral ventricle catheters and electrocorticogram and esophageal electromyogram electrodes. After a 2-hour control period, fetuses were administered serial lateral ventricle injections (1 mL) of angiotensin II (3.2 microg; time, 2 hours) and N omega-nitro-L -arginine methyl ester (3 mg; time, 3 hours) and a repeat angiotensin II injection (3.2 microg; time, 5 hours). All fetuses received an additional control study of lateral ventricle injections of artificial cerebrospinal fluid on a previous day. RESULTS: Angiotensin II injection significantly increased mean +/- SEM fetal swallowing (0.9 +/- 0.1 to 2.7 +/- 0.4 swallows/min). N omega-nitro-L -arginine methyl ester significantly decreased fetal swallowing to below the basal rate (0.4 +/- 0.1 swallows/min), and swallowing did not increase with the second angiotensin II dose (in the presence of nitric oxide blockade). CONCLUSIONS: These results demonstrate that inhibition of central nitric oxide suppresses fetal swallowing behavior in response to central angiotensin II. We speculate that tonic nitric oxide facilitates angiotensin II swallowing stimulation by maintenance of glutamate activation of hypothalamic N -methyl-D -aspartate receptors. PMID- 10739536 TI - Perineal application of talc and cornstarch powders: evaluation of ovarian cancer risk. AB - Some epidemiologic studies have reported associations between perineal talc exposure and epithelial ovarian cancer, which raises parallel questions about the consequences of perineal exposure to cornstarch. Cornstarch powder is an alternative to talc powder that by its nature is a completely different substance. In this review of the literature the epidemiologic data on ovarian cancer risk and perineal application of both powders are reviewed, and the chemical natures of the two powders are compared. All available data indicate that whereas associations between talc exposure and ovarian cancer have suggested but not proved a casual relationship, the application of perineal powder containing cornstarch exclusively is not predicted to be a risk factor for ovarian cancer. PMID- 10739537 TI - Recurrent leiomyomatosis peritonealis disseminata exacerbated by in vitro fertilization. AB - Leiomyomatosis peritonealis disseminata is a rare disease that is most often discovered during pregnancy or in patients with a history of oral contraceptive use. We report the first case of a patient with the disease who conceived by in vitro fertilization and the complications that occurred. PMID- 10739538 TI - Absence of flow velocity waveform changes in uterine arteries after bilateral internal iliac artery ligation. AB - A case is reported of bilateral internal iliac artery ligation during cesarean delivery for intractable hemorrhage. Uterine artery Doppler flow velocity waveforms were documented before and after the procedure. After the ligation the uterine arteries could still be visualized in the appropriate anatomic location, and no changes in Doppler flow velocity waveforms were documented. PMID- 10739539 TI - Complications of elective cesarean delivery necessitating postpartum hysterectomy. AB - A 34-year-old primiparous woman underwent an elective primary cesarean delivery. Five days later a hysterectomy had to be performed to save the patient's life because of generalized peritonitis. Although risks have decreased dramatically with modern obstetric care, awareness of the potential for severe complications is mandatory even in cases of elective cesarean delivery. PMID- 10739540 TI - Treatment of vaginal dysplasia: just a simple loop electrosurgical excision procedure? AB - We report a case of sigmoid perforation, peritonitis, and adult respiratory distress syndrome after a loop excision procedure for carcinoma in situ of the cervix and dysplasia of the vagina. Treatment of vaginal dysplasia with loop excision is potentially dangerous. Safer methods of management include carbon dioxide laser, topical 5-fluorouracil, and fulguration. PMID- 10739541 TI - Fistula in ano as a rare complication of mediolateral episiotomy: report of three cases. AB - Fistula in ano has been reported as a rare complication of midline episiotomy. We report here 3 cases of fistula in ano complicating mediolateral episiotomy. One was suprasphincteric and 2 were transsphincteric. All patients had perineal pain and chronic suppuration. Before referral, 2 patients underwent incision and drainage alone. As a definitive procedure fistulotomy was performed, and long term functional results have been satisfactory. PMID- 10739542 TI - Laparoscopic port site implantation with ovarian cancer. AB - We report the cases of 3 patients in whom tumor implantation developed at the port site at which ovarian cancer was removed laparoscopically. The 3 patients, who were aged 30, 32, and 40 years, all had an ovary that did not appear cancerous removed by laparoscopy through a port site. All 3 patients underwent re exploration within 3 weeks and were found to have tumoral spread and port site implantation of tumor. When ovarian cancer is removed laparoscopically, the potential exists for intra-abdominal tumoral spread. When surgical staging is undertaken after laparoscopic removal of ovarian cancer, the port site should be excised in a full-thickness fashion. PMID- 10739543 TI - Highly abnormal maternal inhibin and beta-human chorionic gonadotropin levels along with severe HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome at 17 weeks' gestation with triploidy. AB - A 17-week pregnancy complicated by severe hypertension is reported. The fetus had multiple anomalies and was found to have triploidy. Assay of maternal serum markers for trisomy 21 revealed elevated levels of inhibin (137.51 multiples of the median) and human chorionic gonadotropin (41.51 multiples of the median). PMID- 10739544 TI - Tuberculous peritonitis: part of the differential diagnosis in ovarian cancer. AB - We report a case of tuberculous peritonitis in a young woman who was initially thought to have ovarian cancer. We emphasize the misleading raised CA 125 levels and radiologic pictures and the importance of frozen-section analysis for definitive diagnosis to avoid unnecessary surgery. PMID- 10739545 TI - Ovarian vein thrombosis associated with Crohn's disease: a case report. AB - A nonpuerperal patient with Crohn's disease and pelvic pain was incidently diagnosed with ovarian vein thrombosis. The patient's laboratory data were negative for an inherited hypercoagulopathy state. Ovarian vein thrombosis is a possible cause of pelvic pain in patients with Crohn's disease. Modern imaging technology has made it easier to diagnose the once-elusive ovarian vein thrombosis. PMID- 10739546 TI - Primary aldosteronism as a cause of severe postpartum hypertension in two women. AB - Two women who first had the clinical features of primary aldosteronism in the postpartum period are described. Their gestations were virtually uneventful. After delivery, however, progressively severe hypertension (Joint National Committee VI, stage 3) with hypokalemia developed. Pregnancy may conceal the clinical symptoms of primary aldosteronism that causes unexpected severe hypertension in the postpartum period. PMID- 10739547 TI - Ovarian hyperstimulation caused by a gonadotropin agonist. PMID- 10739548 TI - Accuracy of clinical assessment of paravaginal defects in women with anterior vaginal wall prolapse. PMID- 10739550 TI - Perinatal morbidity associated with misoprostol-induced labor. PMID- 10739552 TI - Perinatal risks associated with borderline amniotic fluid index. PMID- 10739554 TI - Breast cancer in dense breast: detection with contrast-enhanced dynamic MR imaging. AB - The purpose of this study was to identify contrast enhancement patterns of dense breast parenchyma and to investigate the ability of dynamic magnetic resonance imaging (MRI) to detect cancer in the dense breast. Thirty-two patients with breast cancer in dense breast underwent gadolinium-enhanced dynamic MRI. The detectability of cancer by dynamic MRI, mammography, and physical examination was compared. Two parenchymal enhancement patterns could be identified. One was increasingly multiple patchy enhancement found predominantly in the periphery (type A), and the other was faint enhancement without any nodular opacification (type B). Type A was thought to reflect severe proliferative fibrocystic change (PFC). For both patterns, the detection rate of primary cancers by dynamic MRI was found to be superior to that by other modalities. Dynamic MRI also could detect multifocal cancers, which could not be found with other modalities, although the detectability of these small cancers might be reduced in patients with severe PFC. PMID- 10739555 TI - Gadolinium-enhanced arterial-phase MR imaging of hypervascular liver tumors: comparison between tailored and fixed scanning delays in the same patients. AB - The purpose of this study was to compare in the same patients tailored and fixed scanning delays during gadolinium-enhanced arterial-phase magnetic resonance imaging of hypervascular liver tumors. Tailored scanning delays were obtained with automated region of interest threshold triggering. A delay of 23 seconds between the start of contrast material injection and imaging was used for fixed delay examinations. Quantitative and qualitative evaluation was performed in 21 patients with normal cardiac function referred for MR assessment of hypervascular liver tumors. In the tailored examinations, the median time delay between the start of contrast material injection and the start of magnetic resonance imaging was 21 seconds (range, 18-34 seconds). The median tumor-to-liver contrast during tailored examinations was 19.1 versus 14.7 during fixed delay examinations. This difference, however, was not significant. Similarly, the enhancement in the aorta, the portal vein, the liver, and the tumor did not differ significantly between examinations performed with tailored and fixed delays. It is concluded that in our group of patients with hypervascular liver tumors and normal cardiac function, no significant improvement in tumor-to-liver contrast and enhancement during the arterial phase was found when gadolinium-enhanced magnetic resonance imaging was performed with a tailored scanning delay rather than with a fixed delay. PMID- 10739556 TI - Ultra-fast three-dimensional MR perfusion imaging of the entire brain in acute stroke assessment. AB - We sought to evaluate a three-dimensional (3D) whole-brain perfusion technique based on echo-shifting (PRESTO) for its performance in evaluation of acute stroke. Twenty-six patients were scanned within 6 hours after onset of hemispheric symptoms, and the results were compared with results of diffusion weighted imaging (DWI) and digital subtraction angiography (DSA). The signal-to noise ratio of the images was 61 +/- 3 pre-contrast and 47 +/- 3 at the bolus peak. Brain coverage on perfusion parameter maps was 95% +/- 2% compared with that displayed on T2-weighted images, with only minor artifacts related to susceptibility at the skull base. Measured regional cerebral blood volume (rCBV) reduction closely correlated to lesion size on initial DWI and to final clinical outcome (P = 0.006), consistent with results previously reported for 2D perfusion methods. Mismatches between DWI and perfusion imaging characterized the total extent of tissue at risk, and the contrast timing correlated with the amount of collateral circulation as shown on DSA. In conclusion, 3D imaging using the PRESTO technique permits high-quality perfusion imaging of the entire brain. PMID- 10739557 TI - Measurement of global brain atrophy in Alzheimer's disease with unsupervised segmentation of spin-echo MRI studies. AB - In 16 patients with probable Alzheimer's disease (AD; NINDS criteria, age range 56-78 years), gray matter (GM), white matter (WM), and cerebrospinal fluid (CSF) absolute and fractional volumes were measured with an unsupervised multiparametric post-processing segmentation method based on estimates of relaxation rates R1, R2 (R1 = 1/T1; R2 = 1/T2) and proton density [N(H)] from conventional spin-echo studies (Alfano et al. Magn. Reson. Med. 1997;37:84-93). Global brain atrophy, and GM and WM fractions significantly correlated with Mini Mental Status Examination and Blessed Dementia Scale scores. Compared with normals, brain compartments in AD patients showed decreased GM (-6.84 +/- 1.58%) and WM fractions (-9.79 +/- 2.47%) and increased CSF fractions (+58.80 +/- 10.37%). Changes were more evident in early-onset AD patients. In AD, measurement of global brain atrophy obtained by a computerized procedure based on routine magnetic resonance studies could complement the information provided by neuropsychological tests for the assessment of disease severity. PMID- 10739558 TI - Tissue segmentation on MR images of the brain by possibilistic clustering on a 3D wavelet representation. AB - An algorithm for the segmentation of a single sequence of three-dimensional magnetic resonance (MR) images into cerebrospinal fluid, gray matter, and white matter classes is proposed. This new method is a possibilistic clustering algorithm using the fuzzy theory as frame and the wavelet coefficients of the voxels as features to be clustered. Fuzzy logic models the uncertainty and imprecision inherent in MR images of the brain, while the wavelet representation allows for both spatial and textural information. The procedure is fast, unsupervised, and totally independent of any statistical assumptions. The method is tested on a phantom image, then applied to normal and Alzheimer's brains, and finally compared with another classic brain tissue segmentation method, affording a relevant classification of voxels into the different tissue classes. PMID- 10739559 TI - Cerebral metabolic changes in patients with a symptomatic occlusion of the internal carotid artery: a longitudinal 1H magnetic resonance spectroscopy study. AB - The objective of this 1H magnetic resonance spectroscopy study was to investigate the time course of the brain metabolites N-acetyl-aspartate (NAA), choline, and lactate in patients with transient or minor disabling neurological deficits associated with an occlusion of the internal carotid artery (ICA). Fifty patients had had symptoms of hemispheric ischemia, and 16 had suffered symptoms of retinal ischemia. Single-voxel proton spectra were obtained from uninfarcted cerebral regions on three occasions: 0-6, 6-12, and 12-18 months after symptoms. Reference values were obtained from 29 control subjects. In the 0-6 month period, patients with hemispheric ischemia showed a significantly lower NAA/creatine ratio in the hemisphere ipsilateral to the ICA occlusion, compared with control subjects and patients with retinal ischemia, and a significantly higher choline/creatine ratio, compared with control subjects. The prevalence of lactate did not differ significantly between patient groups. In the following time periods, the NAA/creatine ratio in patients with hemispheric ischemia tended to return to control values and no longer differed from that in patients with retinal ischemia; the choline/creatine ratio decreased significantly and returned to control values. These results demonstrate that cerebral metabolism is altered in patients with an ICA occlusion who have had a hemispheric ischemic event, but returns (choline) or tends to return (NAA) to control values over time. The metabolic changes occur primarily in the hemisphere ipsilateral to the symptomatic ICA occlusion and are related to the occurrence of the hemispheric ischemic event. PMID- 10739560 TI - 2D CSI proton MR spectroscopy of human spinal vertebra: feasibility studies. AB - This report focuses on proton magnetic resonance spectroscopy (1H MRS) of spine vertebra acquired with two-dimensional chemical shift imaging (2D CSI), utilizing the stimulated echo acquisition mode (STEAM) sequence. Both validity and reproducibility studies were performed. To validate the 2D CSI method, its spectra were compared with those obtained with the single-voxel (SV) method. Five normal volunteers were scanned. The reproducibility of 2D CSI was examined by performing spectroscopy on two different occasions, on three normal volunteers. Data show that the STEAM 2D CSI technique results in MRI spectra comparable to those obtained with the STEAM SV method. 2D CSI offers significant time savings and convenient multi-voxel spectral analysis at a substantially higher signal-to noise ratio. The 2D CSI method was then applied to a patient with a small vertebral hemangioma. The results demonstrated that the voxels containing the hemangioma exhibit different spectra than the neighboring voxels of the same vertebra. Additionally, a case of vertebral osteoporosis was investigated. Results showed a significant increase in the lipid-to-water ratio (LWR). It is suggested that 2D CSI may be powerful in identifying physiological as well as pathological changes of the bone marrow. Furthermore, covering a more extensive area of the vertebral body will maximize the chances of depicting a small focus of pathologic tissue. A more detailed bone marrow pattern was noticed in on one subject whose spectra show more lipid peaks. PMID- 10739561 TI - Cervical spondylosis: contrast-enhanced magnetization transfer prepulsed 3D turbo field echo MR imaging. AB - Magnetic resonance (MR) imaging of the cervical spine with axial, low flip angle three-dimensional (3D) gradient-echo sequences is limited by long acquisition times and also by increased sensitivity to extrinsic and intrinsic magnetic field inhomogeneity, magnetic susceptibility differences, chemical shifts, and cerebrospinal fluid pulsatility. We attempted to assess the performance of gadolinium-enhanced, magnetization transfer (MT) prepulsed 3D fast gradient-echo sequences in demonstrating spondylotic changes of the cervical spine. Twenty patients with known cervical spine spondylosis were prospectively imaged in the axial plane using two gradient-echo-based MR techniques: 3D fast field echo (FFE) and gadolinium-enhanced, MT prepulsed, segmented turbo field echo (TFE). An average of 58 neural foramina on the 3D FFE images and 47 neural foramina on the contrast-enhanced TFE images were judged to be narrowed. The degree of neural foraminal narrowing was significantly less on the contrast-enhanced TFE images compared with the FFE images (P <0.001). Contrast-enhanced, MT prepulsed, segmented 3D TFE MR imaging has potential for ameliorating some of the limitations encountered in the more widely used gradient-echo techniques. PMID- 10739563 TI - Benefits and pitfalls of keyhole imaging, especially in first-pass perfusion studies. AB - A comparison of dynamic results of a multi-echo contrast-enhanced perfusion study obtained from a keyhole imaging experiment and the results from low-resolution updates is presented. If, for each dynamic state, a separate reference image exists, high spatial resolution in the dynamic results can be preserved through keyhole imaging. If only one reference image can be used, the dynamic key-hole results still offer high spatial frequency content due to spatial phase discontinuities in the images. These often exist at the outline of organs and result from the fat in connective tissues. If the basic assumption of keyhole imaging, namely, that the relevant information is centered in k-space, is violated, as in T2*-weighted gradient-echo images, keyhole imaging can lead to erroneous results even though the update images themselves seem to be free of any artifacts. PMID- 10739562 TI - Reconstruction of blood flow patterns in a human carotid bifurcation: a combined CFD and MRI study. AB - The carotid bifurcation is a common site for clinically significant atherosclerosis, and the development of this disease may be influenced by the local hemodynamic environment. It has been shown that vessel geometry and pulsatile flow conditions are the predominant factors that determine the detailed blood flow patterns at the carotid bifurcation. This study was initiated to quantify the velocity profiles and wall shear stress (WSS) distributions in an anatomically true model of the human carotid bifurcation using data acquired from magnetic resonance (MR) imaging scans of an individual subject. A numerical simulation approach combining the image processing and computational fluid dynamics (CFD) techniques was developed. Individual vascular anatomy and pulsatile flow conditions were all incorporated into the computer model. It was found that the geometry of the carotid bifurcation was highly complex, involving helical curvature and out-of-plane branching. These geometrical features resulted in patterns of flow and wall shear stress significantly different from those found in simplified planar carotid bifurcation models. Comparisons between the predicted flow patterns and MR measurement demonstrated good quantitative agreement. PMID- 10739564 TI - Preoperative MRA assessment of the coronary arteries in an ascending aortic aneurysm. AB - We present a patient with an aneurysm that included both the aortic root and the ascending aorta. Visualization of the coronary arteries by x-ray angiography was not technically feasible. Magnetic resonance angiography (MRA) was thus performed and allowed an accurate evaluation of the involvement of the coronary arteries in the aneurysm and the patency of the proximal coronaries, as well as visualization of the aneurysm itself. PMID- 10739565 TI - Anomalous inferior vena cava demonstrated by contrast-enhanced magnetic resonance venography. AB - We discuss the technique of contrast-enhanced magnetic resonance venography and its advantages over other methods of imaging the veins of the chest. An example is presented of the results of this technique in a patient with agenesis of the hepatic segment of the inferior vena cava and azygos continuation. PMID- 10739566 TI - Measurement of relative fat content by proton magnetic resonance spectroscopy using a clinical imager. AB - The aim of this study was to determine the applicability of a proton magnetic resonance (MR) spectroscopy-based technique using a clinical 1.5-T MR imager for assessment of relative fat content. Proton MR spectra were obtained from a trunk phantom and 23 volunteers using a single free induction decay measurement. The ratios of fat methyl and methylene proton resonance to the water proton resonance were compared with the ratio of oil weight to water weight for the phantom, and with the ratio of body fat to lean body mass estimated by bioelectrical impedance analysis for the human subjects. Good linear relationships were found between the MR metabolite ratio and the ratio of oil weight to water weight (r = 0.9989), and the ratio of body fat to lean body mass (r = 0.9169). This MR spectroscopy-based technique is sufficiently accurate and may be applicable to assessment of human body composition. PMID- 10739567 TI - Slice-selective proton double quantum filtered MRI of joint connective tissues. AB - 1H double quantum filtered (DQF) imaging has been shown to highlight tendons. In this work the DQF magnetic resonance imaging pulse sequence is extended to include slice selection. The short transverse relaxation time of the 1H nuclear magnetic resonance in connective tissues, presents a stringent demand on the application of gradients and soft radiofrequency pulse lengths needed for slice selection. In the present work a slice selection pulse sequence is implemented by postponing the application of the slice refocusing gradient to the period after the last pulse just before the acquisition. Slice-selective DQF images of rat lower leg and knee are given to demonstrate the efficacy of the technique. PMID- 10739568 TI - A brief history of myelinated nerve fibers: one hundred and fifty years of controversy. AB - The early controversies over myelinated nerve fibers focused on whether nerves are hollow or not, whether the fatty "marrow" (myelin) is inside the nerve fiber or around it, whether myelin is secreted by the axon or formed by another cell, whether nerve fibers are discrete or part of a syncytial network, whether nodes of Ranvier are present in central myelin or only in peripheral myelin. Since Geren's seminal discovery that peripheral myelin is formed by the Schwann cell plasma membrane wrapped around the axon, the focus has shifted. Myelin is clearly a living cell appendage, and the myelin sheath is dependent upon intercellular interactions not only during its formation, but throughout its lifetime and during pathological processes affecting either the axon or the myelin-forming cell. The myelinated fiber is a functional unit, an exquisite symbiosis, whose ability to perform optimally, in some cases whose very survival, depends on the effects the respective cells exert on one another. How are these interactions mediated? Which structures and functions depend on such interaction and which are independent of it? How do cells of the size and shape of myelin-forming cells cope with their metabolic demands and support their most distal components? What are the mechanisms and mutual consequences of demyelination or axonopathy? Relevant studies have burgeoned with the development of molecular biological and genetic engineering methods, and with improvements in microscopy, in vitro culture and specific immunostaining methods. This introductory essay provides an overview of the structural background and continuing controversies relevant to the articles that follow, which represent a sampling of current work and present new information on the molecular structure, function and pathology of myelin and axoglial interactions. PMID- 10739569 TI - RNA on the road to myelin. AB - In oligodendrocytes some mRNAs are transported from the perikaryon to the distal processes and localized in the myelin compartment where they are translated. This review describes the cis-acting signals and trans-acting factors that mediate intracellular trafficking of myelin basic protein (MBP) RNA, the prototype for such mRNAs in myelinating glia. PMID- 10739570 TI - Genetic dissection of myelin galactolipid function. AB - The roles that the myelin galactolipids galactocerebroside (GalC) and sulfatide play in cellular differentiation, myelin formation and maintenance have been investigated for nearly 3 decades. During that time the primary approach has been to perturb lipid activity using antibodies and chemical agents in artificial systems. Recently, the isolation of the gene that encodes UDP-galactose:ceramide galactosyltransferase (CGT), the enzyme that catalyzes an essential step in the synthetic pathway of GalC and sulfatide, has enabled the generation of mice that lack myelin galactolipids. These mice display a severe tremor, hindlimb paralysis and electrophysiological defects. In addition, the CGT null mutants exhibit: 1) impaired oligodendrocyte differentiation, 2) myelin sheaths that are thin, incompletely compacted and unstable, and 3) structural abnormalities in the nodal and paranodal regions including disrupted axo-glial junctions. Collectively, these findings suggest that GalC and sulfatide are essential in myelin formation and maintenance, possibly by mediating intra- and intercellular interactions. PMID- 10739571 TI - Myelin glycosphingolipid/cholesterol-enriched microdomains selectively sequester the non-compact myelin proteins CNP and MOG. AB - Plasma membranes are complex arrays of protein and lipid subdomains. Detergent insoluble, glycosphingolipid/cholesterol-enriched micro-domains (DIGCEMs) have been implicated in protein sorting and/or as sites for signaling cascades in the plasma membrane. We previously identified the presence of DIGCEMs in oligodendrocytes in culture and purified myelin and characterized a novel DIGCEM associated tetraspan protein, MVP17/rMAL (Kim et al. (1995) Journal of Neuroscience Research 42, 413-422). We have now analyzed the association of known myelin proteins with DIGCEMs in order to provide a better understanding of their roles during myelin biogenesis. We used four well-established criteria to identify myelin DIGCEM-associated proteins: insolubility in a non-ionic detergent Triton X-100 at low temperature (4 degrees C), flotation of the insoluble complexes to low density fractions in sucrose gradients, and TX-100 solubilization at 37 degrees C, or at 4 degrees C following treatment with the cholesterol-binding detergent saponin. We demonstrate that these proteins fall into four distinct groups. Although all tested proteins could be floated to a low density fraction, proteolipid protein (PLP), myelin basic protein (MBP) and myelin associated glycoprotein (MAG) were solubilized by the detergent extraction, and connexin32 (Cx32) and oligodendrocyte-specific protein (OSP) met only some of the criteria for DIGCEMs. Only the non-compact myelin proteins 2',3' cyclic nucleotide 3'-phosphodiesterase (CNP) and myelin/oligodendrocyte glycoprotein (MOG) satisfied all four criteria for DIGCEM-associated proteins. Significantly, only approximately 40% of CNP and MOG were selectively associated with DIGCEMs. This suggests that they may have both non-active "soluble", and functionally active DIGCEM-associated, forms in the membrane, consistent with current views that DIGCEMs provide platforms for bringing together and activating components of the signal transduction apparatus. We therefore propose that CNP and MOG may have unique roles among the major myelin proteins in signaling pathways mediated by lipid-protein microdomains formed in myelin. PMID- 10739572 TI - Clustering of neuronal sodium channels requires contact with myelinating Schwann cells. AB - Efficient and rapid conduction of action potentials by saltatory conduction requires the clustering of voltage-gated sodium channels at nodes of Ranvier. This clustering results from interactions between neurons and myelinating glia, although it has not been established whether this glial signal is contact dependent or soluble. To investigate the nature of this signal, we examined sodium channel clustering in co-cultures of embryonic rat dorsal root ganglion neurons and Schwann cells. Cultures maintained under conditions promoting or preventing myelination were immunostained with antibodies against the alpha subunit of the sodium channel and against ankyrin(G), a cytoskeletal protein associated with these channels. Consistent with previous in vivo studies (Vabnick et al., 1996), sodium channels and ankyrin G cluster at the onset of myelination. These clusters form adjacent to the ends of the myelinating Schwann cells and appear to fuse to form mature nodes. In contrast, sodium channels and ankyrin G do not cluster in neurons grown alone or in co-cultures where myelination is precluded by growing cells in defined media. Conditioned media from myelinating co-cultures also failed to induce sodium channel or ankyrin G clusters in cultures of neurons alone. Finally, no clusters develop in the amyelinated portions of suspended fascicles of dorsal root ganglia explants despite being in close proximity to myelinated segments in other areas of the dish. These results indicate that clustering of sodium channels requires contact with myelinating Schwann cells. PMID- 10739573 TI - Physiological roles of axonal ankyrins in survival of premyelinated axons and localization of voltage-gated sodium channels. AB - 440 kD ankyrin-B and 480/270 kD ankyrin-G are membrane skeletal proteins with closely related biochemical properties yet distinctive physiological roles in axons. These proteins associate with spectrin-actin networks and also bind to integral membrane proteins including the L1 CAM family of cell adhesion molecules and voltage-gated sodium channels. 440 kD ankyrin-B is expressed with L1 in premyelinated axon tracts, and is essential for survival of these axons, at least in the case of the optic nerve. 440 ankyrin-B may collaborate with L1 in transcellular structures that mediate axon fasciculation and mechanically stabilize axon bundles, although these proteins may also be involved in axon pathfinding. Ankyrin-B (-/-) mice exhibit loss of L1 from premyelinated axon tracts and a similar, although much more severe, phenotype to L1 (-/-) mice and humans with L1 mutations. Ankyrin-B and L1 thus are candidates to collaborate in the same structural pathway and defects in this pathway can lead to nervous system malformations and mental retardation. 480/270 kD ankyrin-G are highly concentrated along with the L1CAM family members neurofascin and NrCAM at nodes of Ranvier and axon initial segments. Voltage-gated sodium channels bind directly to ankyrins, and are likely to associate in a ternary complex containing neurofascin/NrCAM, and ankyrin-G. Mice with ankyrin-G expression abolished in the cerebellum exhibit loss of ability of Purkinje neurons to fire action potentials, as well as loss of restriction of neurofascin/NrCAM to axon initial segments. Ankyrin-G thus is a key component in assembly of functional components of the axon initial segment and possibly the node of Ranvier. PMID- 10739574 TI - K+ channel distribution and clustering in developing and hypomyelinated axons of the optic nerve. AB - The localization of Shaker-type K(+) channels in specialized domains of myelinated central nervous system axons was studied during development of the optic nerve. In adult rats Kv1.1, Kv1.2, Kv1.6, and the cytoplasmic beta-subunit Kvbeta2 were colocalized in juxtaparanodal zones. During development, clustering of K(+) channels lagged behind that for nodal Na(+) channels by about 5 days. In contrast to the PNS, K(+) channels were initially expressed fully segregated from nodes and paranodes, the latter identified by immunofluorescence of Caspr, a component of axoglial junctions. Clusters of K(+) channels were first detected at postnatal day 14 (P14) at a limited number of sites. Expression increased until all juxtaparanodes had immunoreactivity by P40. Developmental studies in hypomyelinating Shiverer mice revealed dramatically disrupted axoglial junctions, aberrant Na(+) channel clusters, and little or no detectable clustering of K(+) channels at all ages. These results suggest that in the optic nerve, compact myelin and normal axoglial junctions are essential for proper K(+) channel clustering and localization. PMID- 10739575 TI - Myelinating Schwann cells determine the internodal localization of Kv1.1, Kv1.2, Kvbeta2, and Caspr. AB - We examined the localization of Caspr and the K(+) channels Kv1.1 and Kv1.2, all of which are intrinsic membrane proteins of myelinated axons in the PNS. Caspr is localized to the paranode; Kv1. 1, Kv1.2 and their beta2 subunit are localized to the juxtaparanode. Throughout the internodal region, a strand of Caspr staining is flanked by a double strand of Kv1.1/Kv1.2/Kvbeta2 staining. This tripartite strand apposes the inner mesaxon of the myelin sheath, and forms a circumferential ring that apposes the innermost aspect of Schmidt-Lanterman incisures. The localization of Caspr and Kv1.2 are not disrupted in mice with null mutations of the myelin associated glycoprotein, connexin32, or Kv1.1 genes. At all of these locations, Caspr and Kv1.1/Kv1.2/Kvbeta2 define distinct but interrelated domains of the axonal membrane that appear to be organized by the myelin sheath. PMID- 10739576 TI - Analysis of potassium channel functions in mammalian axons by gene knockouts. AB - Mammalian axons express a rich repertoire of various K channel subtypes whose distribution is profoundly affected by myelination. In the past two decades, functional analysis of axonal K channels has been approached primarily through pharmacology. Recently, gene knockout techniques have been used to specifically delete a particular K channel subtype from axons. This is significant since the bulk of K channels in a myelinated nerve are covered by the myelin, making functional analysis of specific K channel subtypes by traditional means difficult. This review summarizes the first mutational analysis of this sort performed on an axonal fast K channel termed Kv1.1. This K channel is concealed by the myelin loops in the paranodes of all major myelinated fiber tracts, and exhibits highly heterogeneous distribution even in certain non-myelinated CNS axons. Physiological analysis of Kv1.1 null mutants suggest novel functions for this axonal K channel subtype, including modulation of conduction failures at branch points and stabilization of transition zones in myelinated nerves. PMID- 10739577 TI - Generation of oligodendroglial progenitors in acute inflammatory demyelinating lesions of the rat brain stem is associated with demyelination rather than inflammation. AB - Remyelination is an extremely efficient process in the adult rodent central nervous system yet the source of new oligodendroglia that appear following primary demyelination is still subject to much debate. Using a reliable marker for oligodendroglial progenitor cells in vivo, the NG2 chondroitin sulphate proteoglycan, we have evaluated the response of endogenous NG2(+) cells in the adult rat brain stem and cerebellum to inflammatory demyelinating lesions in an experimentally induced animal model of multiple sclerosis (MS), antibody augmented experimental allergic encephalomyelitis (ADEAE). We have manipulated T cell mediated EAE in Lewis rats by injecting in addition, either anti myelin/oligodendroglial glycoprotein (MOG) antibodies to induce inflammatory demyelination, or non-specific mouse immunoglobulins to induce an inflammatory response without demyelination. We have examined the relationship of NG2(+) progenitor cells to microglia (OX-42(+)), astrocytes (GFAP(+)) and mature oligodendroglia (CNP(+)), in the normal and demyelinated CNS. In the normal CNS NG2-expressing cells are closely intermingled with other glia but represent a distinct cell population. A prominent inflammatory response, identified by the presence of large perivascular and periventricular accumulations of reactive OX42(+) macrophages/microglia, occurred in animals with ADEAE at 7-9 days post injection (DPI), coinciding with severe clinical symptoms. In animals injected with anti-MOG antibodies inflammation was followed by the appearance of large areas of demyelination at 11-14 DPI, at which point the animals had recovered clinically. The response of NG2(+) cells was different depending on whether the inflammation was accompanied by demyelination. In the presence of inflammation, NG2(+) cells responded by an increase in immunoreactivity and an alteration in their morphology, exhibiting enlarged cell bodies and an increased number of intensely stained processes. In areas of demyelination NG2(+) cells had fewer intensely stained processes reminiscent of progenitor cells seen during development. Quantitative analysis revealed a 3-fold increase in the number of NG2(+) cells in demyelinated lesions at 11 DPI, whereas no change was observed in areas of inflammation in the absence of demyelination. Mitotic figures were only seen in NG2(+) cells in areas of demyelination. NG2(+) cell numbers appeared to return to control levels following remyelination. These results suggest that endogenous oligodendroglial progenitors divide and/or migrate, in response to signals triggered by demyelinating rather than inflammatory events, to generate a large progenitor population sufficient to promote the rapid and successful remyelination observed in this model. PMID- 10739578 TI - Axonal pathology in myelin disorders. AB - Myelination provides extrinsic trophic signals that influence normal maturation and long-term survival of axons. The extent of axonal involvement in diseases affecting myelin or myelin forming cells has traditionally been underestimated. There are, however, many examples of axon damage as a consequence of dysmyelinating or demyelinating disorders. More than a century ago, Charcot described the pathology of multiple sclerosis (MS) in terms of demyelination and relative sparing of axons. Recent reports demonstrate a strong correlation between inflammatory demyelination in MS lesions and axonal transection, indicating axonal loss at disease onset. Disruption of axons is also observed in experimental allergic encephalomyelitis and in Theiler's murine encephalomyelitis virus disease, two animal models of inflammatory demyelinating CNS disease. A number of dysmyelinating mouse mutants with axonal pathology have provided insights regarding cellular and molecular mechanisms of axon degeneration. For example, the myelin-associated glycoprotein and proteolipid protein have been shown to be essential for mediating myelin-derived trophic signals to axons. Patients with the inherited peripheral neuropathy Charcot-Marie Tooth disease type 1 develop symptomatic progressive axonal loss due to abnormal Schwann cell expression of peripheral myelin protein 22. The data summarized in this review indicate that axonal damage is an integral part of myelin disease, and that loss of axons contributes to the irreversible functional impairment observed in affected individuals. Early neuroprotection should be considered as an additional therapeutic option for these patients. PMID- 10739579 TI - Antibody-mediated CNS demyelination: focal spinal cord lesions induced by implantation of an IgM anti-galactocerebroside-secreting hybridoma. AB - O1 hybridoma cells, which secrete an IgM antigalactocerebroside, were implanted into the spinal cord of cyclosporine-treated juvenile or adult rats, and the animals were sacrificed approximately 2-3 wk later. About half the recipient animals developed myelin lesions. In some, sharply circumscribed foci of demyelination formed within the dorsal columns. Cellular reaction consisted of macrophages containing refractile globules in the parenchyma and within enlarged perivascular spaces as well as thickened endothelial cells. "Shadow plaques" also developed, i.e. regions in which axons were surrounded by thin myelin sheaths, compatible with remyelination. In addition, we found damaged axons, some of which were swollen with organelles, comparable to the enlarged axon profiles seen at sites of constriction or interruption. Compromise of the blood-brain barrier at sites of hybridoma growth was demonstrated by extravasation of Evans blue dye. Discontinuation of cyclosporine was followed by an anti-hybridoma, complement fixing antibody response within 2-3 d. This model of focal CNS demyelination and remyelination, with evidence of some axon damage, is mediated by a defined IgM antiglycolipid monoclonal antibody secreted within the spinal cord parenchyma. The lesions, which are similar to those of multiple sclerosis, probably result from the interaction between the intrathecally secreted IgM antibody and complement entering from the circulation at foci of compromised blood-brain barrier plus activation of endogenous or hematogenous macrophages via their complement receptors. PMID- 10739581 TI - Neuropsychology in the new millennium: prognostications, dreams, and warnings PMID- 10739582 TI - Brain, mind, and the evolution of connectivity. PMID- 10739580 TI - Death of oligodendrocytes and microglial phagocytosis of myelin precede immigration of Schwann cells into the spinal cord. AB - Small, circumscribed electrolytic lesions were made in the upper cervical corticospinal tract in adult rats. In the centre of the lesion, the axons and all other tissue elements were totally destroyed. Surrounding this region of destruction is an area of tissue which is only partially damaged. In this area TUNEL positive staining of contiguous rows of tract glial cells indicates massive oligodendrocytic apoptosis at 1-3 days after operation, but axons, astrocytes and blood vessels survive. From around 4 days, the corticospinal axons in this area are demyelinated, and the microglia contain ingested myelin, identified in electron micrographs as characteristic MBP immunoreactive laminar cytoplasmic bodies. After around 3 weeks, large numbers of Schwann cells, continuous with those on the pial surface of the spinal cord, accumulate along the lesion track and selectively infiltrate the perilesional reactive area, where they mingle intimately with the phagocytic microglia. Electron micrographs show that at this time basal lamina-enclosed Schwann cell processes establish non-myelinated ensheathment of axons. From around 4 weeks after operation, prominent Schwann cell myelination is indicated by P0 immunoreactivity, and peripheral type, one-to one myelination in electron micrographs. Thus the effect of the selective loss of oligodendrocytes is to first activate microglia, and then to induce a replacement of myelin by Schwann cells. PMID- 10739583 TI - All the king's horses and all the king's men: putting the brain back together again. PMID- 10739584 TI - Picking two scientific roses for the next century. PMID- 10739585 TI - New models for old: taking the neural network seriously. PMID- 10739586 TI - Call for abstraction. PMID- 10739587 TI - Memory, consciousness, and the brain. PMID- 10739588 TI - The "lumping" and "splitting" of function and brain. PMID- 10739589 TI - The issue of representation in brain research. PMID- 10739590 TI - One of twenty questions for the twenty-first century: how do brain regions interact and integrate information? PMID- 10739591 TI - Chaos, brain, and cognition: toward a nonlinear order? PMID- 10739592 TI - Searching for the neural correlates of consciousness: clues from face recognition research. PMID- 10739593 TI - Darwin's contribution to neuropsychology in the twenty-first century. PMID- 10739594 TI - What matters in the third millenium. PMID- 10739595 TI - Biology and culture in the literate mind. PMID- 10739596 TI - Culture and brain organization. PMID- 10739597 TI - Convergence of functional magnetic resonance imaging and event-related potential methodologies. PMID- 10739598 TI - Optical monitoring of neuronal activity: brain-mapping on a shoestring. PMID- 10739599 TI - Imaging aphasia: the coming paradigm shift. PMID- 10739600 TI - Exploiting cognitive brain maps. PMID- 10739601 TI - The additive factor method in brain imaging. PMID- 10739603 TI - From chronograph to functional image: what's next? PMID- 10739602 TI - Functional neuroimaging of mental chronometry. PMID- 10739604 TI - The when and where of reading in the brain. PMID- 10739605 TI - Excitatory and inhibitory influences on the remembering brain. PMID- 10739606 TI - Achieving convergent evidence through divergent approaches. PMID- 10739608 TI - The importance of autonomic nervous system function for theories of cognitive brain function. PMID- 10739607 TI - Cognitive neuroscience needs affective neuroscience (and vice versa). PMID- 10739609 TI - Toward a computational affective neuroscience. PMID- 10739610 TI - Applying cognitive research in the twenty-first century: event-related potentials in assessment. PMID- 10739611 TI - New survivors for the new millennium: cognitive risk and reserve in adults with childhood brain insults. PMID- 10739612 TI - Capitalizing on neuroplasticity. PMID- 10739613 TI - Neurorehabilitation in the third millenium: new roles for our environment, behaviors, and mind in brain damage and recovery? PMID- 10739614 TI - The study of the regenesis of mind in the twenty-first century. PMID- 10739615 TI - Clinical neuropsychology, functional neurosurgery, and restorative neurology in the next millennium: beyond secondary outcome measures. PMID- 10739616 TI - Cognitive rehabilitation in clinical neuropsychology. PMID- 10739617 TI - Cortical specialization for higher cognitive functions: beyond the maturational model. PMID- 10739618 TI - Mapping cognitive processes onto the brain: mind the gap. PMID- 10739619 TI - Toward a biological account of music experience. PMID- 10739620 TI - A new millennium in cognitive neuropsychology research: the era of individual differences? PMID- 10739621 TI - Laughing matters: toward a structural and neural account. PMID- 10739622 TI - A new paradigm for investigating category-specific agnosia in the new millennium. PMID- 10739623 TI - Organization of semantic knowledge in the human brain: toward a resolution in the next millennium. PMID- 10739624 TI - Representational gestures as actions in space: propositions for a research program. PMID- 10739625 TI - Moving into the new millennium: some perspectives on the brain in action. PMID- 10739626 TI - Reaching in the new millenium: defining coordinate systems. PMID- 10739627 TI - How laterality will survive the millennium bug. PMID- 10739628 TI - Dynamics and variability of brain activation: searching for neural correlates of skill acquisition. PMID- 10739629 TI - Toward the study of knowledge representation in the brain. PMID- 10739630 TI - Exposure to prenatal carbon monoxide and postnatal hyperthermia: short and long term effects on neurochemicals and neuroglia in the developing brain. AB - The effects of prenatal exposure to carbon monoxide (CO), a major component of cigarette smoke, was studied alone or in combination with postnatal hyperthermia, on the structural and neurochemical development of the postnatal brain at 1 and 8 weeks. Pregnant guinea pigs (n = 11) were exposed to 200 p.p.m CO for 10 h/day from midgestation until term (68 days), whereas control mothers (n = 10) breathed room air. On postnatal day 4, neonates from the control and CO-exposed pregnancies were exposed to hyperthermia (35 degrees C) for 75 min or remained at ambient (23 degrees C) temperature. Using semiquantitative immunohistochemical techniques the following neurotransmitter alterations were found in the medulla at 1 week: a decrease in met-enkephalin-immunoreactivity (IR) following postnatal hyperthermia and an increase in 5-hydroxytryptamine-IR following a combination of CO and hyperthermia. No alterations were observed in substance P- or tyrosine hydroxylase-IR in any paradigm. At 8 weeks of age the combination of prenatal CO exposure followed by a brief hyperthermic stress postnatally resulted in lesions throughout the brain and an increase in glial fibrillary acidic protein-IR in the medulla. Such effects on brain development could be of relevance in cardiorespiratory control in the neonate and could have implications for the etiology of Sudden Infant Death Syndrome, where smoking and hyperthermia are major risk factors. PMID- 10739631 TI - Glucose hypometabolism and neuropathological correlates in brains of dementia with Lewy bodies. AB - Cerebral glucose metabolism using positron emission tomography (PET) with (18)F fluorodeoxyglucose was examined in 11 patients with probable Alzheimer's disease (AD), 6 patients with probable, and 1 patient with autopsy-confirmed dementia with Lewy bodies (DLB) as well as in 10 age-matched normal control subjects. Among widespread cortical regions showing glucose hypometabolism in the DLB group, the metabolic reduction was most pronounced in the visual association cortex compared to that in the AD group. Using a metabolic ratio of 0.92 in the visual association cortex as a cutoff (mean-2 SD of normal control subjects), DLB could be distinguished from AD with a sensitivity of 86% and a specificity of 91%. In contrast, apolipoprotein E4 allele frequency and cerebrospinal fluid tau levels did not differ significantly between the two groups. In order to further dissect out neuropathological correlates of the dysfunctional occipital lobe, postmortem brains from 19 patients with AD and 17 with DLB as well as 11 brains from normal controls were examined. A distinct and extensive spongiform change with coexisting gliosis was variably noted throughout cerebral white matter with relative sparing of gray matter in DLB. Notably, the white matter spongiform change and gliosis was most prominently and consistently found in the occipital region of DLB, and the severity of the spongiform change in each brain region generally paralleled to the regional difference in reduced glucose metabolism between the living AD and DLB patients. These findings suggest that (1) among several potential antemortem biomarkers in the diagnosis of DLB, measures of the glucose metabolism in the occipital cortex may be an informative diagnostic aid to distinguish DLB from AD; and (2) a pathological process that generates widespread spongiform change and gliosis in long projection fibers may contribute, at least in part, to the characteristic imaging features of DLB. PMID- 10739632 TI - Dexamethasone induces TrkA and p75NTR immunoreactivity in the cerebral cortex and hippocampus. AB - Nerve growth factor (NGF) plays a crucial role in synaptic plasticity during brain development and adulthood by activating a dual receptor system composed of TrkA and p75 (p75NTR) receptors. Exogenous NGF modulates the expression of both receptors. Little is known about the ability of endogenous NGF to regulate the expression of these receptors in basal forebrain cholinergic terminals. The ability of glucocorticoids to increase NGF expression in the hippocampus prompted us to investigate whether the synthetic glucocorticoid dexamethasone (DEX) increases TrkA and p75NTR expression in NGF-target cholinergic neurons in developing rats. We first examined the effect of DEX on NGF mRNA by in situ hybridization. DEX given systemically (0.5 mg/kg, sc) for 1 week to 7-day-old rats elicited an increase in NGF mRNA levels in the dentate gyrus of the hippocampus and superficial layers II and III of the cerebral cortex. Immunohistochemical analysis of p75NTR and TrkA levels revealed a dramatic increase in p75NTR immunoreactivity (IR) in both basal forebrain and hippocampus and TrkA IR in the hippocampus. Interestingly, in DEX-treated rats more axonal terminals were immunopositive for p75NTR in the hippocampus and cortex, suggesting an increase in p75NTR IR in cell bodies as well as in terminals. Our data indicate that the endogenously produced NGF elicits biological changes similar to those of the exogenously delivered NGF. We suggest that glucocorticoids might regulate and coordinate cholinergic neuronal maturation by increasing the biosynthesis of NGF. PMID- 10739634 TI - Tolerance to the anticonvulsant effects of lamotrigine on amygdala kindled seizures: cross-tolerance to carbamazepine but not valproate or diazepam. AB - Using an amygdala-kindled seizure paradigm, we evaluated the acute and chronic anticonvulsant effects of lamotrigine (LTG). Lamotrigine produced dose-dependent inhibitory effects on seizure stage, afterdischarge (AD), and seizure duration. Lamotrigine (15 mg/kg) also increased the afterdischarge and seizure thresholds. Following repeated LTG administration and stimulation at 48-h intervals, tolerance developed to LTG's (15 mg/kg) anticonvulsant effects, and cross tolerance was observed to the anticonvulsant effects of carbamazepine (CBZ, 15 mg/kg). In a separate group of kindled rats, CBZ (15 mg/kg) was repeatedly administered to induce tolerance. This led to a partial cross-tolerance to LTG, manifesting as an increased rate of tolerance development to LTG, and seizures following the first injection in some animals, which were not observed in CBZ nontolerant controls. When these rats were made fully tolerant to LTG and then exposed to higher doses of LTG (30 and 50 mg/kg), no anticonvulsant effects were observed. In contrast, higher doses of CBZ (30 mg/kg) did restore efficacy in CBZ tolerant animals. Cross-tolerance from LTG to valproate and diazepam was not observed, although cross-tolerance from CBZ to valproate has been reported previously. These data suggest that LTG has both shared and distinct anticonvulsant mechanisms from those of CBZ on amygdala-kindled seizures. The implications of these results for clinical therapeutics remain to be evaluated. PMID- 10739633 TI - Protein synthesis inhibition in neocortical grafts evaluated by systemic amino acid uptake autoradiography. AB - The temporal pattern of protein synthesis inhibition was examined in grafted neocortical neurons using [(3)H]valine in vivo autoradiography. Neuronal uptake levels of systemically administered (3)H-labeled amino acids which cross the blood-brain barrier (BBB) via endothelial cell neutral carriers have long been a hallmark in studies of experimental ischemic pathology; there is likely a strong correlation between persistent protein synthesis inhibition and the progression of cell damage. Because the grafting procedure involves the loss of blood flow and the subsequent reperfusion of the donor tissue there are, mechanistically, important similarities to reversible ischemia models. The effects of ischemic injury on grafted CNS neurons are not fully understood. Quantitative analysis of grain distribution in individual graft or control (adjacent host cortex) neurons indicated an initial breakdown of the amino acid barrier system, subsequent recovery, and progressive reduction of amino acid uptake by 1 year. Up to 3 weeks after surgery grafts were flooded with the [(3)H]valine tracer but individual neurons contained relatively few silver grains. After this time, the tracer was normally distributed within graft neurons but at significantly lower levels than in controls. Grain density gradually decreased over time such that 12-month grafted neurons had approximately half that compared to control and only 58% of that in 2-month grafts; the 12-month levels were comparable to those observed at early (10 days) postoperative times. Autoradiography of immunostained sections for MAP-2, SMI 311 (neurofilament marker), and neuron-specific enolase showed reduced expression of these proteins in neurons coupled with weak amino acid tracer uptake. The results further suggest that grafted neurons bear intriguing similarities to neurons placed at ischemic risk, particularly "penumbral" neurons, which are affected by reduced blood flow and are metabolically weakened. The loss of BBB properties in early grafts may also extend to the endothelial cell amino acid carrier system, and the delayed revascularization process could affect neuronal uptake mechanisms. PMID- 10739635 TI - Interferon-gamma induces apoptosis and augments the expression of Fas and Fas ligand by microglia in vitro. AB - Activation of microglia by interferon-gamma (IFN-gamma) has been implicated in a number of central nervous system (CNS) inflammatory disease processes. Because IFN-gamma has also been shown to play a role in programmed cell death, we investigated its cytotoxicity and its effect on the Fas apoptotic pathway in microglia. Flow cytometry was used to quantify the IFN-gamma-mediated apoptotic response and Fas and Fas ligand (FasL) expression in two well-characterized murine microglia cell lines (BV-2 and N9). Nuclear fragmentation, suggestive of apoptosis, was noted within 24 h of incubation of microglia with IFN-gamma (10 U/ml). After a 72-h incubation, almost every BV-2 and N9 microglia, but not GL261 glioma cells, underwent cell death and detached from the culture plates. This cytotoxicity occurred even at low IFN-gamma concentrations (1 U/ml) and was inhibited by BAF, a pan-caspase inhibitor. Incubation of BV-2 and N9 microglia, but not GL261 glioma cells, with IFN-gamma also potentiated the expression of Fas and FasL in a similar dose-response and time-course manner, as seen for the apoptotic response. Whereas Fas expression increased by 100% in both microglia cells, FasL upregulation was more pronounced and increased by as much as 200% in the N9 cells. These findings suggest that in addition to its role as a microglia activator, IFN-gamma may also induce apoptosis of microglia, possibly through simultaneous upregulation of Fas and FasL. Interferon-gamma modulation of the Fas pathway and apoptosis in microglia may be important in the pathogenesis of inflammatory CNS disease processes. PMID- 10739636 TI - BDNF is needed for postnatal maturation of basal forebrain and neostriatum cholinergic neurons in vivo. AB - Neurotrophins regulate survival, neurite outgrowth, and phenotypic maturation of developing neurons. Brain-derived neurotrophic factor (BDNF) can promote the survival of developing cholinergic forebrain neurons in vitro and reduce their degeneration following injury in adult rats. We investigated the role of endogenous BDNF during postnatal development of these cholinergic neurons by analyzing homozygous BDNF-deficient (-/-) mice and their littermates (+/+, +/-). At P6, the number of choline acetyltransferase- (ChAT) positive neurons in the medial septum was approximately 23% lower in BDNF-/- mice, although their brain and body weight was normal. At P15, control (+/+) littermates had approximately 45% more and approximately 45% larger ChAT-positive neurons and a much denser cholinergic hippocampal innervation than at P6, indicative of maturation of the septohippocampal system. In BDNF-/- mice, the number, size, and ChAT immunostaining intensity of the cholinergic neurons remained the same between P6 and P15 (few mice survive longer). BDNF-/- mice had about three times more TUNEL labeled (a marker of apoptosis) cells in the medial septum at P6, consistent with (but not proof of) the possibility that the cholinergic neurons were dying. The cholinergic hippocampal innervation in BDNF-/- mice expanded to a lesser extent than in controls and had reduced levels of acetylcholinesterase staining at P15. The developmental deficits were largely similar in the neostriatum of BDNF-/- mice. These findings suggest that BDNF is critical for postnatal development and maturation of cholinergic forebrain neurons. PMID- 10739637 TI - Alterations in contractile properties and expression of myofibrillar proteins in wobbler mouse muscles. AB - The purpose of this study was to characterize the alterations in muscle contractile (tension-pCa relationship) and biochemical (myosin heavy and light chains, troponin C content) properties in a hereditary motoneuron disease. The study was performed on wobbler mouse mutants which presented a neuronal degeneration. The time course of the disease was followed at 5 and 7 weeks in sternocleidomastoid (SCM) and soleus muscles. The wobbler disease was found to induce a shift from fast to slow myosin heavy-chain isoform expression in SCM and soleus muscles. The analysis of the myosin light-chain (MLC) composition revealed, for the SCM muscles, the appearance of the slow isoforms at 5 weeks and an increase in the regulatory MLC2 content at 7 weeks. A significant increase in the slow troponin C isoform content was found in both types of wobbler muscles at 7 weeks. The wobbler soleus and SCM muscles presented an age- and fiber-type related atrophy, characterized by a decline in absolute maximal tension and fiber diameter. A decrease in calcium sensitivity was observed at 7 weeks for the soleus fibers and at both 5 and 7 weeks for the SCM. The results indicated fast to-slow changes in contractile and biochemical properties of the wobbler soleus and SCM muscles, which occurred during the motoneuron degeneration process previously described in the wobbler pathology. PMID- 10739638 TI - Combined use of the adenosine A(2A) antagonist KW-6002 with L-DOPA or with selective D1 or D2 dopamine agonists increases antiparkinsonian activity but not dyskinesia in MPTP-treated monkeys. AB - The novel selective adenosine A(2A) receptor antagonist KW-6002 improves motor disability in MPTP-treated parkinsonian marmosets without provoking dyskinesia. In this study we have investigated whether KW-6002 in combination with l-DOPA or selective D1 or D2 dopamine receptor agonists enhances antiparkinsonian activity in MPTP-treated common marmosets. Combination of KW-6002 with the selective dopamine D2 receptor agonist quinpirole or the D1 receptor agonist SKF80723 produced an additive improvement in motor disability. Coadministration of KW-6002 with a low dose of L-DOPA also produced an additive improvement in motor disability, and increased locomotor activity. The ability of KW-6002 to enhance antiparkinsonian activity was more marked with L-DOPA and quinpirole than with the D1 agonist. However, despite producing an enhanced antiparkinsonian response KW-6002 did not exacerbate L-DOPA-induced dyskinesia in MPTP-treated common marmosets previously primed to exhibit dyskinesia by prior exposure to L-DOPA. Selective adenosine A(2A) receptor antagonists, such as KW-6002, may be one means of reducing the dosage of L-DOPA used in treating Parkinson's disease and are potentially a novel approach to treating the illness both as monotherapy and in combination with dopaminergic drugs. PMID- 10739639 TI - Decrease in striatal enkephalin mRNA in mouse models of Huntington's disease. AB - Huntington's disease is a devastating progressive neurodegenerative illness characterized by massive neuronal loss in the striatum. It is caused by the presence of an expanded CAG repeat in the gene encoding huntingtin, a protein of unknown function. We have examined the expression of neurotransmitters and other antigens present in striatal neurons with immunohistochemistry, and the level of expression of mRNAs encoding enkephalin, substance P, and glutamic acid decarboxylases with quantitative in situ hybridization histochemistry, in the striatum of two mouse models of Huntington's disease: transgenic animals expressing exon 1 of the human huntingtin gene with 144 CAG repeats and "knock in" mice containing a chimeric mouse/human exon 1 with 71 or 94 CAG repeats inserted by homologous targeting. Although the transgenic (but not the knock-in) mice were previously shown to display prominent huntingtin- and ubiquitin containing nuclear inclusions in striatal neurons, in situ nick translation followed by emulsion autoradiography did not reveal any DNA damage in striatum or cortex in these mice. Immunolabeling for calbindin D 28K, enkephalin, substance P, glutamic acid decarboxylases (M(r) 65,000 or 67,000, GAD65 and GAD67), somatostatin, choline acetyltransferase, parvalbumin, and glial fibrillary acidic protein were remarkably similar in transgenic, knock-in, and wild-type mice. Both transgenic and knock-in mice, however, showed a marked decrease in the level of expression of enkephalin mRNA in striatal neurons without significant decreases in mRNAs encoding substance P, GAD65, or GAD67. The data indicate that decreased expression of enkephalin mRNA may be an early sign of neuronal dysfunction due to the Huntington's disease mutation. PMID- 10739640 TI - Nerve injury-induced mechanical but not thermal hyperalgesia is attenuated in neurokinin-1 receptor knockout mice. AB - Mice lacking the gene encoding for substance P and neurokinin A, or the NK-1 receptor, exhibit alterations in behavior to various acute nociceptive stimuli. However, behavioral responses of NK-1 mutant animals have not been well characterized in models of chronic pain. We studied the behavioral responses of NK-1 knockout and wild-type control mice to thermal and mechanical stimuli before and after inducing chronic neuropathic pain by unilateral ligation of the L5 spinal nerve. Mechanical hyperalgesia was evaluated by determining the frequency of withdrawal to von Frey monofilaments applied to the hind paws. Nerve injury induced hyperalgesia to thermal stimuli was examined by determining responses to radiant heat and cooling stimuli. The contribution of the sympathetic nervous system to mechanical hyperalgesia was evaluated by administering 3 mg/kg phentolamine, an alpha-adrenergic antagonist, subcutaneously. Following spinal nerve injury, withdrawal frequencies to mechanical stimulation increased in wild type mice within 1 day and persisted during the 9-week observation period, whereas in the knockout mice, withdrawal frequencies did not increase significantly. In contrast, withdrawal latencies to radiant heat decreased up to 2 weeks after nerve injury in both the NK-1 and the wild-type mice. Similarly, the increase in withdrawal frequency to the cooling stimuli following the nerve injury was not different in the NK-1 knockout and wild-type mice. Mechanical hyperalgesia in the wild-type mice was not reversed by systemic administration of phentolamine, suggesting that the pain is not sympathetically maintained. The results indicate that NK-1 receptors contribute to the development of mechanical, but not thermal, hyperalgesia in neuropathic pain. PMID- 10739641 TI - Intrastriatal and intranigral grafting of hNT neurons in the 6-OHDA rat model of Parkinson's disease. AB - The clinical findings on neural transplantation for Parkinson's disease (PD) reported thus far are promising but many issues must be addressed before neural transplantation can be considered a routine therapeutic option for PD. The future of neural transplantation for the treatment of neurological disorders may rest in the discovery of a suitable alternative cell type for fetal tissue. One such alternative may be neurons derived from a human teratocarcinoma (hNT). hNT neurons have been shown to survive and integrate within the host brain following transplantation and provide functional recovery in animal models of stroke and Huntington's disease. In this study, we describe the transplantation of hNT neurons in the substantia nigra (SN) and striatum of the rat model for PD. Twenty seven rats were grafted with one of three hNT neuronal products; hNT neurons, hNT DA neurons, or lithium chloride (LiCl) pretreated hNT-DA neurons. Robust hNT grafts could be seen with anti-neural cell adhesion molecule and anti-neuron specific enolase immunostaining. Immunostaining for tyrosine hydroxylase (TH) expression revealed no TH-immunoreactive (THir) neurons in any animals with hNT neuronal grafts. THir cells were observed in 43% of animals with hNT-DA neuronal grafts and all animals with LiCl pretreated hNT-DA neuronal grafts (100%). The number of THir neurons in these animals was low and not sufficient to produce significant functional recovery. In summary, this study has demonstrated that hNT neurons survive transplantation and express TH in the striatum and SN. Although hNT neurons are promising as an alternative to fetal tissue and may have potential clinical applications in the future, further improvements in enhancing TH expression are needed. PMID- 10739642 TI - Effect of axotomy on expression of NPY, galanin, and NPY Y1 and Y2 receptors in dorsal root ganglia and the superior cervical ganglion studied with double labeling in situ hybridization and immunohistochemistry. AB - Using double-labeling techniques for both in situ hybridization and immunohistochemistry some peptides and peptide receptors were studied quantitatively in a sensory and a sympathetic ganglion after axotomy. In the lumbar 5 dorsal root ganglion (DRG) normally no neuropeptide Y- and only a few galanin-positive cell bodies are seen. Following complete transection of the sciatic nerve around 60% of all neuropeptide Y (NPY) neuron profiles (NPs) were galanin positive (+) and 33-44% of all galanin NPs were NPY(+). A good agreement between immunohistochemistry and in situ hybridization was observed for NPY and galanin. NPY Y1- and Y2-receptor (R) mRNAs were found in around 40% of all NPY mRNA(+) NPs, and more than half of the Y1-R mRNA(+) NPs and two-thirds of the Y2 R mRNA(+) NPs were NPY(+). In addition, more than one-third of the galanin mRNA containing NPs showed colocalization with NPY receptor mRNAs and up to 70% of the Y2-R mRNA(+) NPs also expressed galanin mRNA. In the control superior cervical ganglion (SCG) 10% of the NPY(+) NPs were Y2-R mRNA(+), and 85% of the Y2-R(+) NPs were NPY mRNA(+), and the corresponding percentages after axotomy were around 35 and 45%, respectively. Following axotomy of the carotid nerves around half of all NPY(+) NPs were galanin(+), and conversely around 50% of all galanin NPs were NPY(+) at the mRNA level, whereas much lower percentages (15 and 9%, respectively) were observed with immunohistochemistry. These results demonstrate that double-labeling procedures are valid tools to quantitatively evaluate coexistence situations in sensory and sympathetic ganglia, showing a high degree of coexistence for NPY and galanin in axotomized neurons both in the lumbar 5 DRG and in the SCG. However, the immunohistochemical analysis in the SCG demonstrated much lower numbers of peptide-positive neurons than seen with in situ hybridization, suggesting that the latter technique is more sensitive. The fact that a considerable number of neurons express NPY together with Y1- and/or Y2-Rs indicates that both receptors may act as autoreceptors, the Y1-R presumably at the level of the cell body and the Y2-R on nerve terminals in the dorsal horn and/or the periphery. The present results also show that in both sensory and sympathetic neurons there is a strong upregulation of the Y2-R after nerve injury, suggesting a possible role in trophic and regenerative events. PMID- 10739643 TI - Cyclosporin ameliorates traumatic brain-injury-induced alterations of hippocampal synaptic plasticity. AB - Although traumatic brain injury (TBI) often results in impaired learning and memory functions, the underlying mechanisms are unknown and there are currently no treatments that can preserve such functions. We studied plasticity at CA3-CA1 synapses in hippocampal slices from rats subjected to controlled cortical impact TBI. Long-term potentiation (LTP) of synaptic transmission was markedly impaired, whereas long-term depression (LTD) was enhanced, 48 h following TBI when compared to unoperated and sham control rats. Post-TBI administration of cyclosporin A, a compound that stabilizes mitochondrial function, resulted in a highly significant amelioration of the impairment of LTP and completely prevented the enhancement of LTD. Our data suggest that alterations in hippocampal synaptic plasticity may be responsible for learning and memory deficits resulting from TBI and that agents such as cyclosporin A that stabilize mitochondrial function may be effective treatments for TBI. PMID- 10739644 TI - Analysis of BDNF production in the aging gerbil cochlea. AB - Degeneration of cochlear neurons is the most commonly observed cellular change in the aging human and gerbil cochlea. Although it is unclear what leads to this neuronal loss, changes in the production of target-derived trophic factors may be the ultimate cause of cochlear neuron degeneration. The present study used an enzyme-linked immunosorbent assay to investigate whether BDNF is produced by the organ of Corti or cochlear ganglia of young, middle aged, or aged gerbils. The results revealed an age-related increase in BDNF in the organ of Corti, but not in the cochlear ganglia. PMID- 10739645 TI - Therapeutic applications of apoptosis research. PMID- 10739646 TI - Apoptotic DNA fragmentation. AB - Degradation of nuclear DNA into nucleosomal units is one of the hallmarks of apoptotic cell death. It occurs in response to various apoptotic stimuli in a wide variety of cell types. Molecular characterization of this process identified a specific DNase (CAD, caspase-activated DNase) that cleaves chromosomal DNA in a caspase-dependent manner. CAD is synthesized with the help of ICAD (inhibitor of CAD), which works as a specific chaperone for CAD and is found complexed with ICAD in proliferating cells. When cells are induced to undergo apoptosis, caspases-in particular caspase 3-cleave ICAD to dissociate the CAD:ICAD complex, allowing CAD to cleave chromosomal DNA. Cells that lack ICAD or that express caspase-resistant mutant ICAD thus do not show DNA fragmentation during apoptosis, although they do exhibit some other features of apoptosis and die. In this review, the molecular mechanism of and the physiological roles played by apoptotic DNA fragmentation will be discussed. PMID- 10739647 TI - The mitochondrion in cell death control: certainties and incognita. AB - Apoptosis research has recently experienced a change from a paradigm in which the nucleus determined the apoptotic process to a paradigm in which caspases and, more recently, mitochondria constitute the center of death control. Mitochondria undergo major changes in membrane integrity before classical signs of cell death become manifest. These changes concern both the inner and the outer mitochondrial membranes, leading to the dissipation of the inner transmembrane potential (DeltaPsi(m)) and/or the release of intermembrane proteins through the outer membrane. An ever-increasing number of endogenous, viral, or xenogeneic effectors directly act on mitochondria to trigger permeabilization. At least in some cases, this is achieved by a direct action on the permeability transition pore complex (PTPC), a multiprotein ensemble containing proteins from both mitochondrial membranes, which interact with pro- and antiapoptotic members of the Bcl-2 family. At present, it is elusive whether opening of the PTPC is the only physiological mechanism leading to mitochondrial membrane permeabilization. Proteins released from mitochondria during apoptosis include caspases (mainly caspases 2, 3, and 9), caspase activators (cytochrome c, hsp 10), as well as a caspase-independent death effector, AIF (apoptosis inducing factor). The functional hierarchy among these proteins and their actual impact on the decision between death and life is elusive. PMID- 10739648 TI - Protein complexes activate distinct caspase cascades in death receptor and stress induced apoptosis. AB - Caspases play a central role in the execution phase of apoptosis and are responsible for many of the morphological features normally associated with this form of cell death. Caspases can activate one another and consequently can initiate specific caspase cascades. Caspases-8 and -9 appear to be the apical caspases activated in death receptor- and mitochondrial stress-induced apoptosis, respectively. The role of large protein complexes in mediating these pathways is discussed. PMID- 10739649 TI - Serine/threonine protein kinases and apoptosis. AB - Over the past decade, our understanding of apoptosis, or programmed cell death, has increased greatly, with the identification of some of the major components of the apoptotic programme and the processes regulating their activation. Although apoptosis is an intrinsic process present in all cells, it can be regulated by extrinsic factors, including hormones, growth factors, cell surface receptors, and cellular stress. The actions of both pro- and antiapoptotic factors are often affected by modulation of the phosphorylation status of key elements of the apoptotic process. This minireview will focus on the role of protein kinases in apoptosis. Apoptosis is a multistep process and protein kinases have been implicated both in the upstream induction phase of apoptosis and in the downstream execution stage, as the direct targets for caspases. Due to the space constraints of this review it is not possible to discuss all of the kinases involved in the apoptotic process and we have focused here on the role of the serine/threonine protein kinases. The kinases of this family that have been suggested to play a role in apoptosis are the mitogen-activated protein kinase (MAPK) family, specifically p42/44 ERK, p38 MAPK and c-Jun N-terminal kinase (JNK), cyclic AMP-dependent protein kinase (PKA), protein kinase B (PKB), or Akt and protein kinase C (PKC). We have also considered briefly the potential for the regulation of these kinases by tyrosine protein kinases, such as c-abl. PMID- 10739650 TI - Induction of apoptosis by cancer chemotherapy. AB - Studies performed over the past five years have demonstrated that there are two major cell-intrinsic pathways for inducing apoptosis, one that begins with ligation of cell surface death receptors and another that involves mitochondrial release of cytochrome c. Several reports have suggested that anticancer drugs kill susceptible cells by inducing expression of death receptor ligands, especially Fas ligand (FasL). Other reports have indicated that chemotherapeutic agents trigger apoptosis by inducing release of cytochrome c from mitochondria. In this review, we describe the two prototypic death pathways, indicate experimental approaches for distinguishing whether chemotherapeutic agents trigger one pathway or the other, summarize current understanding of the role of the two pathways in chemotherapy-induced apoptosis, and discuss the implications of these studies for mechanisms of resistance to chemotherapeutic agents. PMID- 10739651 TI - The Bcl-2 protein family. PMID- 10739652 TI - The CD95 (APO-1/Fas) and the TRAIL (APO-2L) apoptosis systems. PMID- 10739653 TI - Divinations and surprises: genetic analysis of caspase function in mice. AB - Caspases are critical mediators of apoptosis, the principle mechanism by which extra and harmful cells are eliminated to ensure proper development and maintain cellular homeostasis in all multicellular organisms. While compelling evidence suggests that the activation of these otherwise latent intracellular proteases is required for the execution of most, if not all apoptosis in mammals, the presence of more than a dozen caspases presents a major challenge to our understanding of the precise function of individual caspases in vivo. Using a genetic approach, several groups have generated transgenic mice deficient in various caspases so as to investigate their physiological functions. In this review, we will discuss what these studies have revealed about the role of individual caspase in development, apoptosis, and inflammation, with a particular focus on the predictable phenotypes versus the surprises based on in vitro results, as well as the implications of these findings. PMID- 10739654 TI - Modulation of EGF receptor activity by changes in the GM3 content in a human epidermoid carcinoma cell line, A431. AB - Gangliosides have been described as modulators of growth factor receptors. For example, GM3 addition in cell culture medium inhibits epidermal growth factor (EGF)-stimulated receptor autophosphorylation. Furthermore, depletion of ganglioside by sialidase gene transfection appeared to increase EGF receptor (EGFR) autophosphorylation. These data suggested that changes in GM3 content may result in different responses to EGF. In this study, the ceramide analog d-threo 1-phenyl-2-decannoylamino-3-morpholino-1-propanol ([D]-PDMP), which inhibits UDP glucose-ceramide glucosyltransferase, and addition of GM3 to the culture medium were used to study the effects of GM3 on the EGFR. Addition of 10 microM [D]-PDMP to A431 cells resulted in significant GM3 depletion. Additionally, EGFR autophosphorylation was increased after EGF stimulation. When exogenous GM3 was added in combination with [D]-PDMP, the enhanced EGFR autophosphorylation was returned to control levels. [D]-PDMP also increased EGF-induced cell proliferation, consistent with its effect on autophosphorylation. Once again, the addition of GM3 in combination with [D]-PDMP reversed these effects. These results indicate that growth factor receptor functions can be modulated by the level of ganglioside expression in cell lines. Addition of GM3 inhibits EGFR activity and decrease of GM3 levels using [D]-PDMP treatment enhances EGFR activity. Modulation of growth factor receptor function may provide an explanation for how transformation-dependent ganglioside changes contribute to the transformed phenotype. PMID- 10739655 TI - Age-related alterations in the activation of heat shock transcription factor 1 in rat hepatocytes. AB - The induction of hsp70 transcription by heat shock is significantly reduced in hepatocytes isolated from old rats compared to hepatocytes isolated from young/adult rats, and the decline in hsp70 transcription is correlated with a decrease in the induction of heat shock transcription factor 1 (HSF1) binding to the heat shock element. However, the decreased HSF1 binding activity to DNA is not due to reduced levels of HSF1 that are available for activation by heat shock. In fact, the levels of HSF1 are two- to threefold higher in hepatocytes from old rats, and the age-related increase in the levels of HSF1 protein in hepatocytes appears to arise from a decrease in the degradation of the HSF1 because HSF1 mRNA levels do not change and the synthesis of HSF1 decreases approximately 50% with age. No evidence was found for an impairment in HSF1 oligomerization in hepatocytes from old rats, e.g., the level of HSF1 trimers, the nuclear translocation of HSF1, and the phosphorylation of HSF1 after heat shock are similar in hepatocytes isolated from young/adult and old rats. However, the thermostability of the DNA binding activity of HSF1 was significantly reduced with age in a cell-free system as well as in isolated hepatocytes. PMID- 10739656 TI - Vesicle-mediated phosphatidylcholine reapposition to the plasma membrane following hormone-induced phospholipase D activation. AB - Phospholipase D (PLD) activation involved in signal transduction may lead to the hydrolysis of conspicuous amounts of phosphatidylcholine (PC). This study shows that PLD activation significantly alters the plasma membrane (PM) environment and the membrane exchange dynamics. PC-PLD activation in vasopressin (AVP)-stimulated L6 myogenic cells was accompanied by increased exocytosis and decreased membrane fluidity, as shown by transmission EM and fluorescence spectroscopy of trimethylammonium-diphenyl-hexatriene. AVP-induced exocytosis appeared to be brefeldin A-insensitive. PLD inhibition by Zn(2+) and PC de novo synthesis inhibition by hexadecylphosphocholine abolished AVP-induced vesicle traffic. Upon AVP stimulation, metabolically labeled PC decreased in PM, then transiently increased in microsomes, and returned to the prestimulus level in the PM within 5 min, a phenomenon requiring PC neosynthesis and microtubule functionality. Vesicle traffic with similar features was also observed after endothelin-1 induced PC-PLD activation in rat peritubular myoid cells. These results indicate that, in nonsecretory cells, exocytosis coupled to PC de novo synthesis restores PM-PC, conspicuously consumed during PLD-mediated signal transduction. PMID- 10739657 TI - Heart, brain, and body wall defects in mice lacking calreticulin. AB - Calreticulin is a ubiquitously expressed protein, which has been implicated in a large number of cellular functions, including calcium storage and signaling, protein folding, and cell attachment. To examine the role of calreticulin during in vivo development, mice deficient in calreticulin were generated by targeted inactivation of the calreticulin gene. Calreticulin-deficient mutants die in utero, mostly in late gestation. Half of these embryos had decreased cardiac cell mass, associated with increased apoptosis of cardiac myocytes. In vitro differentiation cultures of calreticulin-deficient embryonic stem cells resulted in fewer embryoid bodies with contractile activity than cultures derived from calreticulin +/- stem cells (P < 0.001). Sixteen percent of the mutants exhibited exencephaly secondary to a defect in neural tube closure. Embryos surviving until Embryonic Day 16.5 had omphalocele. Lack of calreticulin did not influence survival of embryonic fibroblasts under various endoplasmic reticulum stress conditions. However, calreticulin did influence cell migration in a calcium- and substrate-dependent manner. We conclude that calreticulin is not essential during the early stages of embryonic development, but is important for the development of heart and brain and for ventral body wall closure. The observed abnormalities are compatible with a role of calreticulin in the modulation of cellular calcium signaling. PMID- 10739658 TI - Effects of cross-linked profilin:beta/gamma-actin on the dynamics of the microfilament system in cultured cells. AB - There is evidence that the profilin:actin complex is the immediate precursor in the formation of actin filaments in cells. This paper describes the cell morphology and microfilament distribution after microinjection of covalently cross-linked profilin:beta/gamma-actin (PxA) in two different cell lines. Injected cells were either kept unstimulated or stimulated with platelet-derived growth factor (PDGF) before fixation and visualization of filamentous actin. After injection of low doses of PxA, the cells displayed an actin organization characterized by a clearance of diffuse fluorescence from a region immediately interior of ruffling edges and the appearance of small dots of fluorescence in the same region. At higher concentrations, PxA effectively inhibited outgrowth of lamellae and microspikes, and there was a drastic reduction of actin staining in the zone behind the advancing edge. This effect is reminiscent of the effect of cytochalasin B on fibroblasts and the growth cone of neuronal cells. As in these cases, there remained a rim of actin-dependent fluorescence on the very edge of the membrane lamella, particularly in the PxA-treated fibroblasts. The interference of PxA with the formation of surface structures was pronounced after PDGF stimulation. Here, PxA effectively eliminated the enhancement of the ruffling activity in the cell edges and on the dorsal surface of the cells. In contrast to PxA, injection of non-cross-linked profilin:beta/gamma-actin had no apparent effect on cell morphology and microfilament distribution except for an increased concentration of filamentous actin in one of the cell lines. PMID- 10739659 TI - p53 localizes to the centrosomes and spindles of mitotic cells in the embryonic chick epiblast, human cell lines, and a human primary culture: An immunofluorescence study. AB - Immunofluorescent staining of mitotic centrosomes and spindles by anti-p53 antibodies was observed in the embryonic chick epiblast by epifluorescence microscopy and in three human cancer cell lines, an SV40-immortalized cell line, and a normal human fibroblast culture by confocal microscopy. In the chick epiblast, the centrosomes stained from early prophase through to the formation of the G1 nuclei and the spindle fibers stained from prophase through to telophase. In the human cells, the staining was observed from late prophase to telophase. The epiblast was stained by the anti-p53 antibodies DO-1, Ab-6, and Bp53-12. The human cells were also stained by these antibodies as well as by other anti-p53 antibodies. Preabsorption of DO-1 and Bp53-12 with purified tubulin did not diminish the immunostaining, showing that the antibodies were not reacting with tubulin in the mitotic centrosomes and spindles. The immunostaining in the chick epiblast was very clearly localized to the mitotic centrosomes and spindles, revealing a cytoplasmic location for p53 during mitosis and accounting for earlier reports of an association between p53, tubulin, and centrosomes. The localization of p53 to the spindle supports an involvement of p53 in spindle function. PMID- 10739660 TI - Nuclear topology of murine, cerebellar Purkinje neurons: changes as a function of development. AB - The interphase nucleus is a structurally ordered, three-dimensional structure, in which specific chromatin domains occupy distinct spatial positions that can, in turn, be modified with changes in cell function. A fundamental goal in developmental neurobiology is the identification of mechanisms that dictate the orderly expression of genes in a cell-specific manner. Given that different neuronal populations feature a characteristic spatial topology of centromeric sequences, the positioning of specific DNA sequences may constitute such a mechanism. We tested the hypothesis that the cell-specific nuclear topology in fully differentiated neurons is acquired before or during that stage at which neuron-specific sequences are first expressed. For this, we assessed the number and spatial distribution of centromeric domains in the murine, cerebellar Purkinje neuron as a function of postnatal development. Centromeric domains were localized by immunofluorescence of centromere-associated kinetochore proteins and visualized by confocal microscopy. Kinetochores are known to cluster in Purkinje neurons. Thus, the number of signals discerned is always less than the chromosome complement of the species. The number of signals observed in adults (10.8 +/- 0.46) (mean +/- SEM) is established by postnatal day 15 (P15), after a transient decrease from 11.44 +/- 0.44 at P0 to 8.78 +/- 0.24 at P3. The distribution of signals characteristic of the adult, with the majority located at the nucleolus, is established by P5 and is associated with a decrease in the fraction of signals at the nuclear periphery. These changes are temporally associated with the onset of processes such as dendritic differentiation and synaptic maturation and might serve the process of differentiation by placing specific sequences into transcriptionally competent, nuclear sites. PMID- 10739661 TI - Preferential expression of NuMA in the nuclei of proliferating cells. AB - Nuclear mitotic apparatus protein (NuMA) has an indispensable function in normal mitosis as an organizer of the mitotic spindle. NuMA is a prominent component of interphase cell nuclear matrix but its role during interphase is largely unknown. We examined the presence of NuMA in several human tissues. The majority of cells were positive for NuMA but a few negative cell types were found, including spermatozoa, superficial keratinocytes, neutrophil granulocytes, syncytiotrophoblasts, and some neurons, fibroblasts, and smooth and skeletal muscle cells. We further investigated the presence of NuMA in a cultured estrogen dependent human breast cancer cell line and observed the disappearance of nuclear NuMA in the quiescent cells. The percentage of NuMA-positive cells diminished from an initial approximately 100 to 60% during 6 days of culture. The presence of NuMA correlated positively with the presence of proliferation marker Ki-67 antigen and negatively with the culture time, confluence, and size of the cell islets. These results show that some nonproliferating, highly differentiated cell types lack NuMA and that cells may lose their NuMA without dramatic effects on the nuclear shape. This suggests that NuMA may be a nonessential component of the interphase nucleus. PMID- 10739662 TI - Dispersive initiation of replication in the Chinese hamster rhodopsin locus. AB - Several higher eukaryotic replication origins appear to be composed of broad zones of potential nascent strand start sites, while others are more circumscribed, resembling those of yeast, bacteria, and viruses. The most delocalized origin identified so far is approximately 55 kb in length and lies between the convergently transcribed dihydrofolate reductase (DHFR) and the 2BE2121 genes on chromosome 2 in the Chinese hamster genome. In some of our studies, we have utilized the rhodopsin origin as an early replicating internal standard for assessing the effects of deleting various parts of the DHFR locus on DHFR origin activity. However, it had not been previously established that the rhodopsin locus was located at a site far enough away to be immune to such deletions, nor had the mechanism of initiation at this origin been characterized. In the present study, we have localized the rhodopsin domain to a pair of small metacentric chromosomes and have used neutral/neutral 2-D gel replicon mapping to show that initiation in this origin is also highly delocalized, encompassing a region more than 50 kb in length that includes the nontranscribed rhodopsin gene itself. The initiation zone is flanked at least on one end by an actively transcribed gene that does not support initiation. Thus, the DHFR and rhodopsin origins belong to a class of complex, polydisperse origins that appears to be unique to higher eukaryotic cells. PMID- 10739663 TI - The Ser252Trp fibroblast growth factor receptor-2 (FGFR-2) mutation induces PKC independent downregulation of FGFR-2 associated with premature calvaria osteoblast differentiation. AB - We recently showed that the Apert Ser252Trp fibroblast growth factor receptor-2 (FGFR-2) mutation causes premature osteoblast differentiation and increased subperiosteal calvaria bone matrix formation. To gain further insight into the cellular mechanisms involved in these effects, we examined the effects of the mutation on the expression of FGFRs in relation to cell proliferation and differentiation markers in vivo and in vitro, and we analyzed the underlying signaling pathways in mutant cells. Immunohistochemical analysis of the Apert calvaria suture showed that the Ser252Trp FGFR-2 mutation increased type 1 collagen, osteocalcin, and osteopontin expression in preosteoblasts compared to normal, whereas cell growth was not affected. The premature osteoblast differentiation induced by the mutation was associated with lower than normal FGFR-2 immunolabeling, whereas FGFR-1 and FGFR-3 levels were not decreased. Immunocytochemical analysis in osteoblasts isolated from Apert coronal suture showed that the Ser252Trp mutation induced constitutive downregulation of FGFR-2 in mutant cells. Western blot analysis of FGFRs in immortalized mutant osteoblastic cells confirmed that the mutation induced FGFR-2 downregulation. FGFR-2 mRNA levels were not altered in mutant cells, indicating that FGFR-2 downregulation resulted from receptor internalization rather than from changes in receptor mRNA. The signaling pathway involved in FGFR-2 downregulation was studied using specific inhibitors of FGF signaling molecules. The selective PKC inhibitor calphostin C markedly reduced FGFR-2 protein levels in mutant cells, in contrast to the p38 MAP kinase inhibitor SB 203580 or the Erk 1,2 MAP kinase inhibitor PD-98059, showing that PKC is involved in FGFR-2 regulation, but not in FGFR-2 downregulation in mutant cells. The results indicate that the premature osteoblast differentiation induced by the FGFR-2 Ser252Trp mutation is associated with a PKC-independent downregulation of FGFR-2 in human calvaria cells. PMID- 10739664 TI - Crk-associated substrate p130(Cas) interacts with nephrocystin and both proteins localize to cell-cell contacts of polarized epithelial cells. AB - Crk-associated substrate (p130(Cas), Cas) is a docking protein first recognized as having elevated phosphotyrosine content in mammalian cells transformed by v Src and v-Crk oncoproteins. Subsequent studies have implicated Cas in the control of normal cell behavior through its roles in integrin-mediated signal transduction and organization of the actin cytoskeleton at sites of cell adhesion. In this study, we sought to gain new insight into normal Cas function by identifying previously unrecognized interacting proteins. A yeast two-hybrid screen using the C-terminal region of Cas as a bait identified the Src homology 3 (SH3) domain of the mouse "nephrocystin" protein-orthologous to a human protein whose loss of function leads to the cystic kidney disease familial juvenile nephronophthisis. The putative full-length mouse and partial canine nephrocystin sequences were deduced from cDNA clones. Additional studies using epitope-tagged mouse nephrocystin indicated that nephrocystin and Cas can interact in mammalian cells and revealed that both proteins prominently localize at or near sites of cell-cell contact in polarized Madin-Darby canine kidney epithelial cells. Our findings provide novel insight into the normal cellular activities regulated by both Cas and nephrocystin, and raise the possibility that these proteins have a related function in polarized epithelial cells. PMID- 10739665 TI - Stress-induced activation of c-Jun N-terminal kinase in sensory ganglion neurons: accumulation in nuclear domains enriched in splicing factors and distribution in perichromatin fibrils. AB - In response to cellular stress, the activation of the JNK cascade mediates phosphorylation of c-Jun that promotes its transactivation, which in turn activates the transcription of specific genes. In an experimental model of neuronal stress in vivo, by means of immunofluorescence and kinase assays we have found a reversible activation of JNK induced by the administration of the anti cancer drug Adriamycin. In control neurons, a considerable basal level of the active, phosphorylated JNK was detected in neuronal nuclei, with a speckled distribution in addition to a diffuse nucleoplasmic signal. Adriamycin-induced neuronal stress was associated with a notable increase of this nuclear immunostaining, indicating activation of the JNK pathway which was confirmed by the increase of JNK enzymatic activity, while no changes in the total JNK were detected by Western blots. The JNK neuronal response to stress was also accompanied by an increase in the nuclear immunoreactivity for c-Jun and also by the de novo appearance of a strong nuclear phospho-c-Jun signal. These effects tend to revert to the control situation after 24 h of Adriamycin treatment. The nuclear compartmentalization of phospho-JNK and its substrate c-Jun was analyzed by confocal laser microscopy. Phospho-JNK strongly colocalizes with snRNPs in nuclear speckles, while the former was not concentrated in the coiled bodies. Upon stress induction, both c-Jun and phospho-c-Jun show a nucleoplasmic distribution in euchromatin domains, with the nucleoli free of immunolabeling. Furthermore, the nuclear speckles enriched in phospho-JNK exhibit a very low or undetectable signal with both c-Jun antibodies. Immunogold electron microscopy confirms the accumulation of phospho-JNK in interchromatin granule clusters (nuclear speckles), while in the nucleoplasm this kinase is mainly localized in perichromatin fibrils. Both c-Jun and phospho-c-Jun were also detected in perichromatin fibrils. Double labeling experiments show the colocalization of phospho-JNK and phospho-c-Jun in certain perichromatin fibrils. These results indicate that the neuronal response to the Adriamycin-induced stress is mediated by the activation of the JNK pathway. The accumulation of phospho-JNK in nuclear speckles raises the possibility that this kinase may be involved in the phosphorylation of an unknown splicing factor. Moreover, the colocalization of phospho-JNK and c-Jun in perichromatin fibrils, which are associated with sites of active transcription, suggests that these nuclear structures may be putative sites for the phosphorylation of JNK substrates. PMID- 10739666 TI - Delineation of the protein domains responsible for SYT, SSX, and SYT-SSX nuclear localization. AB - In the vast majority of synovial sarcomas the N-terminal part of the SYT protein is fused to the C-terminal part of an SSX protein, either SSX1 or SSX2. The wild type proteins, as well as the resultant SYT-SSX1 and SYT-SSX2 fusion proteins, are localized in the nucleus. Recent studies in experimental systems indicated that the SYT protein may function as a transcriptional activator whereas the SSX proteins may act as transcriptional repressors. In the present work we created a series of deletion mutants and found that SYT and SSX depend on N-terminal and highly conserved C-terminal domains for nuclear localization, respectively. Our results also show that the SYT-SSX proteins colocalize with SSX2, a feature that depends on the presence of the C-terminal SSX sequences in the chimeric proteins. Absence of these sequences led to an altered subcellular localization, coinciding with that of SYT. Besides, we found that endogenously expressed SSX proteins colocalize with polycomb-group proteins and condensed chromosomes during mitosis, features that are also conferred by the C-terminus of SSX. Taken together, these results led us to conclude that the SSX moiety, especially the most C-terminal 34 amino acids, of the SYT-SSX fusion proteins is crucial for aberrant spatial targeting and transcriptional control within the nucleus. PMID- 10739667 TI - Secretory leukocyte protease inhibitor is a novel inhibitor of fibroblast mediated collagen gel contraction. AB - Cultured epithelial cells, including those from the oral epithelium, have been successfully applied in the promotion of scarless wound healing. Factors released from the epithelial cells are thought to contribute significantly to the beneficial effects. In the conditioned medium of human oral epithelial cells, we found a factor that inhibited fibroblast-mediated collagen gel contraction, an in vitro model of wound healing and scar formation. Biochemical analysis identified the factor to be human secretory leukocyte protease inhibitor (SLPI). Fibroblasts transfected with SLPI cDNA showed reduced gel-contracting activity. SLPI purified from the conditioned medium inhibited gel contraction in a dose-dependent manner, and anti-SLPI antibody counteracted this activity. Upon SLPI treatment, human skin fibroblasts in collagen gel became shorter in length and were inhibited in pseudopodia extension. Furthermore, after SLPI treatment, alpha(1)-integrin immunoreactivity decreased, and cyclic AMP levels increased. Excessive gel contraction was observed when fibroblasts treated with TGF-beta1 and fibroblasts from hypertrophic and from keloid scar tissue were cultured in collagen gel. SLPI was also effective in inhibiting gel contraction in the above three models of scar formation. These results suggest that SLPI may be useful in promoting scarless wound healing. PMID- 10739668 TI - A comparison of the activity, sequence specificity, and CRM1-dependence of different nuclear export signals. AB - Nuclear export sequences (NESs) have been identified in many cellular proteins, but it remains unclear how different NESs compare in activity. We describe a sensitive new in vivo export assay which we have used to assess the relative export activity of different types of NES. The most common type of export sequence resembles that first identified in the HIV-1 Rev protein and typically comprises a core of large hydrophobic amino acids that specify recognition by the CRM1 export receptor. We compared 10 previously identified Rev-type NESs in our assay, and whereas all were functional, the relative export activities of these signals varied considerably. We further identified 3 new Rev-type NESs from a computer database search, and each export signal was assigned a score of 1 to 9 and ranked in order of activity (e.g., PKI > c-ABL > Ran-BP1 > FMRP > PML > IkappaB-alpha > hdm2). The weakest NESs were found in the p53 tumor suppressor and the p53-regulated proteins p21 and hdm2, which are all normally localized to the nucleus. All of the Rev-type NESs were inactivated by mutation of key hydrophobic residues and by treatment with the CRM1-specific export inhibitor, leptomycin B. In contrast, a different type of export signal, the KNS shuttling element derived from hnRNP K, exhibited a modest export activity that was insensitive to leptomycin B treatment. KNS thus appears to mediate export via a CRM1-independent pathway. Mutagenesis of the KNS sequence identified, for the first time, specific serines and acidic residues necessary for its export activity, thereby distinguishing KNS from other types of nuclear transport signal. We have shown that different nuclear export signals can vary profoundly in activity and therefore conclude that the nuclear export rate of a specific shuttling protein largely depends on both the strength and the accessibility of its NES. PMID- 10739669 TI - Differential expression of human topoisomerase IIIalpha during the cell cycle progression in HL-60 leukemia cells and human peripheral blood lymphocytes. AB - Human topoisomerase IIIalpha (huTop IIIalpha) has been demonstrated to belong to type IA subfamily. In this study, we found that huTop IIIalpha expressed constitutively and remained at high levels throughout the cell cycle in HL-60 cells when compared to the cell-cycle-dependent expression of huTop IIIalpha in phytohemagglutinin-activated peripheral blood lymphocytes. During the cell cycle progression, this protein remained accentuated in the nucleolus without significant translocation from the nucleolus to the nucleoplasm. In addition, during the course of granulocytic maturation in DMSO-treated HL-60 cells, huTop IIIalpha levels decreased when cells stopped proliferation and nucleoli diminished in size. However, its level remained unchanged during the course of monocytic maturation of vitamin D(3)-treated HL-60 cells which still retained its proliferative capacity and did not change the size of the nucleolus. The data suggested that huTop IIIalpha is involved in rDNA metabolism, such as rDNA transcription. Its cellular level appeared to be under control during the cell cycle progression of normal lymphocytes, but was found to be deregulated in HL-60 cells which may be associated with the tumor transformed cell phenotypes. PMID- 10739670 TI - N-cadherin function is required for differentiation-dependent cytoskeletal reorganization in lens cells in vitro. AB - Members of the cadherin family of cell adhesion molecules participate in calcium dependent cell-cell adhesions that are necessary for the cell sorting events that regulate early developmental processes. Although individual cadherin molecules have been shown to participate in tissue histogenesis, the regulation of function of these receptors in cell differentiation has been more difficult to identify. We have determined that N-cadherin linkage to the cytoskeleton is correlated with lens cell differentiation in vivo. Through the use of a chick embryo lens culture system that mimics differentiation in vivo, we have determined that N-cadherin linkage to the cytoskeleton is altered and lens differentiation is blocked by function-blocking antibodies to N-cadherin. In the presence of the N-cadherin function-blocking antibody, NCD-2, both N-cadherin and filamentous actin are prevented from organizing at the cortical membranes. This correlates with an inhibition of lens morphogenesis and differentiation. These results are paralleled by changes in the expression of the molecular components of the cadherin-catenin complex and their linkage to the actin cytoskeleton. In the presence of NCD-2, expression of N-cadherin, alpha-catenin, and beta-catenin is inhibited and their association with the cytoskeleton blocked. Overall cadherin expression, however, remains unchanged as demonstrated by studies with a pan cadherin antibody. This is accompanied by an increase in expression of the cadherin cytoskeletal protein plakoglobin. Although the cells have tried to compensate for the loss of N-cadherin by up-regulation of another cadherin(s) and plakoglobin, this is unable to compensate for N-cadherin function. The data strongly suggest that N-cadherin and its associated cytoskeleton play an important role in the differentiation process that leads to the formation of the crystalline lens. PMID- 10739671 TI - The interferon regulatory factors 1 and 2 bind to a segment of the human c-myb first intron: possible role in the regulation of c-myb expression. AB - The preferential expression of the protooncogene c-myb in hematopoietic cells is in part regulated by a mechanism of transcriptional block in the first intron. By electrophoresis mobility shift assays using probes corresponding to different segments of the putative human c-myb intron 1 transcription pause region and nuclear extracts from myeloid leukemia HL 60 and fibroblast WI 38 cells, we detected a HL-60-specific DNA-protein complex with a 123-bp fragment containing binding sites for the interferon regulatory factors (IRFs) nuclear proteins. Formation of the DNA-protein complex was abrogated by competition with an oligomer containing the wild-type, but not the mutated, IRF binding site and the complex was specifically supershifted by the anti-IRF-1 or the anti-IRF-2 antibody. Moreover, in vitro translated IRF-1 or IRF-2 protein did interact with the 123-bp c-myb intron 1 fragment. Upon TPA-induced differentiation, c-myb expression was readily down-modulated in parental HL 60 cells, but not in cells transfected with an antisense IRF-1 plasmid. Moreover, chloramphenicol acetyltransferase activity driven by a c-myb promoter containing the entire intron 1 was suppressed upon IRF-1, but not IRF-2 expression. Together, these results are consistent with the existence of a functional relationship between IRF-1 and c-myb in which IRF-1 negatively regulates c-myb expression at the transcriptional level by a mechanism that may depend on the interaction of IRF-1 with a segment of the c-myb gene implicated in transcription pausing. PMID- 10739672 TI - Clustering of beta(2)-integrins on human neutrophils activates dual signaling pathways to PtdIns 3-kinase. AB - The beta(2)-integrins on leukocytes can serve as a signaling unit during cell adhesion and locomotion, and to further clarify this important property we investigated the possible mechanisms of beta(2)-integrin-induced activation of PtdIns 3-kinase. It has previously been demonstrated that clustering of beta(2) integrins activates p21(ras) by a tyrosine kinase-dependent pathway, and here we show that active p21(ras) interacts with its downstream target, PtdIns 3-kinase. Engagement of beta(2)-integrins also activates the tyrosine kinases p58(c-fgr) and p59/61(hck) and causes them to associate with the p85 subunit of PtdIns 3 kinase. These findings suggest a mechanism whereby p58(c-fgr) and p59/61(hck) are directly involved in the activation of PtdIns 3-kinase. No coupling between p58(c fgr) and p59/61(hck) could be detected; hence these kinases probably trigger independent but parallel signals to PtdIns 3-kinase. The effect of beta(2) integrin clustering on PtdIns 3-kinase activity was monitored as the activation of protein kinase B (PKB). Stimulation of PKB by beta(2)-integrins was abolished by genistein and wortmannin but not by using methyl transferase inhibitors to abrogate the influence of p21(ras)-related proteins. Thus, even if PtdIns 3 kinase is not activated by p21(ras), it can maintain full enzyme activity due to the mentioned interaction with p58(c-fgr) or p59/61(hck). These tyrosine kinases apparently activate similar pathways that operate in parallel and therefore have the potential to substitute for each other in mediating adhesion and regulating cell locomotion. PMID- 10739674 TI - Hepatocyte growth factor signal coupling to various transcription factors depends on triggering of Met receptor and protein kinase transducers in human hepatoma cells HepG2. AB - Hepatocyte growth factor (HGF) regulates a wide variety of biological activities by binding to the tyrosine kinase receptor Met. In HGF-treated hepatocarcinoma cells, we observed a biphasic activation of AP-1 and AP-2 transcription factors. For NF-kappaB complex the p50-p50 homodimer was activated before the p50-p65 heterodimer, and c-Myc/Max DNA-binding activity increased thereafter. Since these transcription factors are responders to mitogenic stimulation through protein kinase transducers, we tested the effects of inhibitors of these enzymes on the DNA binding after HGF treatment. Inhibition of protein kinase C (PKC) with H7 strikingly activated NF-kappaB above the values observed after HGF alone. Under this inhibitory condition, Met tyrosine phosphorylation was elevated as though the phosphorylation-dependent activity of the receptor was partially blocked by activation of PKC due to HGF. NF-kappaB DNA binding seems to be related to Met triggering by HGF since it was largely prevented by genistein treatment, which blocks receptor activity. Phosphatidylinositol 3-kinase seems to be involved in AP-1 binding activity stimulated by HGF. It is noteworthy that Met is responsive to HGF stimulating postreceptor signaling, which converges on the activation of transcription factors acting coordinately to regulate target gene expression. PMID- 10739673 TI - A role for serine proteases in mediating phorbol ester-induced differentiation of HL-60 cells. AB - Treatment of human HL-60 promyelocytic leukemia cells with the phorbol ester 12-O tetradecanoylphorbol-13-acetate (TPA) results in increases in proteolytic activity and maturation toward the monocytic lineage. To investigate the potential roles that different classes of proteases play in the monocytic differentiation of HL-60 cells, cells were treated with phorbol ester in the presence of various serine and cysteine protease inhibitors. The serine protease inhibitors 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), N-alpha-tosyl phenylalanine chloromethyl ketone (TPCK), and N-alpha-tosyl-lysine chloromethyl ketone (TLCK) repressed a number of phenotypic markers of monocytic differentiation including surface expression of the CD11b integrin, cell aggregate formation, cell cycle exit, and cell death. CD11b was not detected at the cell surface by FACS analysis up to 24 h after induction of differentiation; however, both CD11b mRNA and protein were present. Downregulation of c-myc mRNA and upregulation of c-fos and egr-1 mRNA and protein, which normally occur during TPA-induced differentiation, were not affected by inclusion of the protease inhibitors. These data indicate that serine proteases specifically mediate many of the phenotypic aspects of TPA-induced monocytic differentiation but are not involved with the induction or repression of differentiation-sensitive transcription factors and suggest that serine protease activity is required for intracellular processing of CD11b. PMID- 10739675 TI - Heat-inducible expression of a reporter gene detected by transient assay in zebrafish. AB - Heat-inducibility of two reporter constructs expressing lacZ gene under the control of mouse and Xenopus hsp70 promoters was tested in zebrafish (Danio rerio) embryos using a transient expression system. Cells expressing beta galactosidase were stained blue by histochemical staining and their average number per embryo was used as an indicator of the expression level of the reporter gene. Both constructs were heat-inducible in the embryonic tissues and showed similar heat dependence (increasing expression levels from 35-36 degrees C up to 39 degrees C with an apparent decrease at 40 degrees C), resembling that of the zebrafish hsp70 genes. However, their induction kinetics were different, which might be due to differences in their 5' UTRs. Spatial expression patterns of the two hsp/lacZ constructs and an endogenous hsp70 gene were mostly similar on the RNA level. These results indicate that our approach is applicable for in vivo analysis of the heat-shock response and that exogenous heat-shock promoters may be useful for inducible expression of transgenes in fish. PMID- 10739676 TI - Accumulation of short telomeres in human fibroblasts prior to replicative senescence. AB - The loss of telomere repeats has been causally linked to in vitro replicative senescence of human diploid fibroblasts (HDFs). In order to study the mechanism(s) by which telomere shortening signals cell senescence, we analyzed the telomere length at specific chromosome ends at cumulative population doublings in polyclonal and clonal HDFs by quantitative fluorescence in situ hybridization. The rate of telomere shortening at individual telomeres varied between 50 and 150 bp per population doubling and short telomeres with an estimated 1-2 kb of telomere repeats accumulated prior to senescence. The average telomere length in specific chromosome ends was remarkably similar between clones. However, some exceptions with individual telomeres measuring 0.5-1 kb were observed. In the fibroblast clones, the onset of replicative senescence was significantly correlated with the mean telomere fluorescence but, strikingly, not with chromosomes with the shortest telomere length. The accumulation of short telomeres in late passages of cultured HDFs is compatible with selection of cells on the basis of telomere length and limited recombination between telomeres prior to senescence. PMID- 10739677 TI - Downregulation of JNK/SAPK activity is associated with the cross-resistance to P glycoprotein-unrelated drugs in multidrug-resistant FM3A/M cells overexpressing P glycoprotein. AB - In the present study, cross-drug resistance in multidrug-resistant (MDR) cells, which overexpress P-glycoprotein (Pgp), a mdr1 gene product, against Pgp unrelated drugs, and its relevance to c-Jun N-terminal kinase (JNK)/stress activated protein kinase (SAPK) activity were examined. The multidrug-resistant FM3A/M cells overexpressing Pgp were resistant to apoptotic cell death induced either by Pgp-related drugs including vincristine and vinblastine, which are pumped out by Pgp, or by the Pgp-unrelated drugs including 5'-fluorouracil (5-FU) and bleomycin, which are not targets for Pgp, compared with the parental FM3A cells. Verapamil reversed the resistance of FM3A/M cells to apoptosis induced by the Pgp-related drugs but not that induced by the Pgp-unrelated drugs. Interestingly, FM3A/M cells have shown significantly lower basal and drug stimulated JNK/SAPK activities than FM3A cells. After transfection with pEBG-SEK or pEBG-SAPK constructs, FM3A/M cells recovered the basal and Pgp-unrelated drug stimulated activities of JNK/SAPK and the susceptibility to Pgp-unrelated drug induced apoptotic cell death comparable to those of FM3A cells. Furthermore, FM3A cells became resistant to apoptotic cell death induced by vincristine and 5-FU after transfection with pEBG-SEK(K --> R), a dominant negative inhibitory mutant of SEK. These results suggest that downregulation of JNK/SAPK activity appears to confer on Pgp-associated FM3A/M cells a cross-resistance to Pgp-unrelated drugs. PMID- 10739678 TI - Gene-specific DNA repair of pyrimidine dimers does not decline during cellular aging in vitro. AB - A large number of studies have demonstrated that various kinds of DNA damage accumulate during aging and one of the causes for this could be a decrease in DNA repair capacity. However, the level of total genomic repair has not been strongly correlated with aging. DNA repair of certain kinds of damage is known to be closely connected to the transcription process; thus, we chose to investigate the level of gene-specific repair of UV-induced damage using in vitro aging of human diploid skin fibroblasts and trabecular osteoblasts as model systems for aging. We find that the total genomic repair is not significantly affected during cellular aging of cultures of both human skin fibroblasts and trabecular osteoblasts. Gene-specific repair was analyzed during cellular aging in the dihydrofolate reductase housekeeping gene, the p53 tumor suppressor gene, and the inactive region X(754). There was no clear difference in the capacity of young and old cells to repair UV-induced pyrimidine dimers in any of the analyzed genes. Thus, in vitro senescent cells can sustain the ability to repair externally induced damage. PMID- 10739679 TI - An established light ear mutant (C57BL/6J-Pdeb(rd1) le) mouse cell line exhibits a block to secretion of lysosomal enzymes. AB - The hypopigment mutant mice, light ear, pallid, and beige, possess defects in melanosomes, lysosomes, and platelet dense granules, suggesting that these organelles share a common biogenesis and processing. Light ear and pallid mutants are animal models for Hermansky Pudlak syndrome, whereas the beige mouse is an animal model for Chediak Higashi syndrome. An established skin cell line from the light ear mouse was tested along with pallid and beige cell lines for mutant effects on secretion of lysosomal hydrolase activities of six different lysosomal glycosidases and the trafficking of N-[5-(5,7-dimethyl BODIPY)-1-pentanoyl]-D erythrosphingosine (C(5)-DMB-ceramide). There were no consistently significant differences between the pallid and the beige mutant cell lines or between these two mutant lines and the control cell line in the percentage secretion of lysosomal hydrolase activities. The light ear mutant cell line, however, displayed a significantly lower percentage secretion of lysosomal hydrolase activities than all other cell lines tested. The light ear mutant cells processed C(5)-DMB-ceramide completely, as seen in the control cell line, whereas pallid and beige cell lines retained fluorescent material and exhibited a block in the complete processing of C(5)-DMB-ceramide 20 h after labeling. The block to secretion of lyososomal hydrolase activities in the light ear mutant cell line will be useful for further studies on this mutant's lysosomal defect. PMID- 10739680 TI - The cytoplasmic domain of the alpha1 integrin subunit influences stress fiber formation via the conserved GFFKR motif. AB - Integrins are heterodimeric transmembrane proteins that mediate substrate adhesion and migration but also the bidirectional transfer of information across the plasma membrane via their cytoplasmic domains. We addressed the question of whether the very short cytoplasmic tail of the alpha1 integrin subunit of alpha1beta1 integrin is required for alpha1beta1-specific adhesion, spreading, and migration. For this purpose we transfected the alpha1 integrin subunit and two cytoplasmically truncated alpha1 subunits into Chinese hamster ovary (CHO) cells. Elimination of the entire cytoplasmic domain of the alpha1 subunit does not affect adhesion but leads to inhibition of spreading and stress fiber formation. The defect in spreading could not be rescued by lysophosphatidic acid, which has been reported to stimulate actin stress fiber formation via Rho. Additionally, deletion of the entire cytoplasmic domain of the alpha1 subunit abolishes migration toward alpha1beta1-specific substrates. Migration and stress fiber formation are similar in CHO-alpha1 cells and CHO cells carrying an alpha1 subunit still containing the conserved GFFKR motif. So, the GFFKR motif of the alpha1 subunit is essential and sufficient for these processes. PMID- 10739681 TI - A simple method for accurate estimation of apoptotic cells. AB - A simple, sensitive, and reliable "DNA diffusion" assay for the quantification of apoptosis is described. Human lymphocytes and human lymphoblastoid cells, MOLT-4, were exposed to 0, 12.5, 25, 50, or 100 rad of X-rays. After 24 h of incubation, cells were mixed with agarose, microgels were made, and cells were lysed in high salt and detergents. DNA was precipitated in microgels by ethanol. Staining of DNA was done with an intense fluorescent dye, YOYO-1. Apoptotic cells show a halo of granular DNA with a hazy outer boundary. Necrotic cells, resulting from hyperthermia treatment, on the other hand, show an unusually large homogeneous nucleus with a clearly defined boundary. The number of cells with apoptotic and necrotic appearance can be scored and quantified by using a fluorescent microscope. Results were compared with other methods of apoptosis measurement: morphological estimations of apoptosis and DNA ladder pattern formation in regular agarose gel electrophoresis. Validation of the technique was done using some known inducers of apoptosis and necrosis (hyperthermia, hydrogen peroxide, mitoxantrone, novobiocin, and sodium ascorbate). PMID- 10739682 TI - Gene structure, expression analysis, and membrane topology of RDH4. AB - The murine retinol dehydrogenase RDH4 oxidizes several cis-isomers of retinol into their corresponding aldehydes. We have determined the structure of the murine gene, investigated the temporal and spatial expression of the enzyme, and analyzed the membrane topology of the enzyme. The gene has four translated exons, and several alternatively spliced exons in the 5'-untranslated region were identified. Immunohistochemical analysis showed expression of RDH4 in developing and adult mouse eye, particularly in the retinal pigment epithelium. In nonocular adult tissues, including liver, kidney, lung, and skin, RDH4 expression was widespread. The results suggest that RDH4 may have a dual and tissue-specific role in oxidation of 9-cis- and 11-cis-isomers of retinol into 9-cis-retinal and 11-cis-retinal, respectively. Furthermore, the lumenal orientation of the enzyme domain in the ER suggests that oxidation of both cis-isomers of retinol occurs in the ER. PMID- 10739684 TI - Molecular characteristics of cancers: the way of the future? PMID- 10739683 TI - Molecular and cytokinetic pretreatment risk assessment in endometrial carcinoma. AB - OBJECTIVE: Our objective was to determine whether cytokinetic and molecular analyses of curettage specimens can provide a mechanism for triage of patients with endometrial cancer before initiating definitive surgical treatment. METHODS: Pretreatment analysis consisted of flow cytometric determination of ploidy, S phase fraction (SPF), and proliferative index (PI) and immunohistochemical determination of expression of proliferating cell nuclear antigen, HER-2/neu, and p53 in curettage specimens from 134 patients with endometrial carcinoma who subsequently had surgical staging and definitive surgical treatment. Fisher's exact test or chi(2) was used to examine the association between pretreatment variables and traditional surgical-pathologic indices. The log-rank test was used for univariate survival analysis. Cox proportional hazards identified the most important molecular factors. RESULTS: Nondiploid status, SPF >/=9%, and PI >/=14% were associated with the traditional posttreatment prognostic indices, stage, grade, and histologic subtype. Univariate survival analysis demonstrated a correlation between nondiploid status, SPF >/=9%, PI >/=14%, and p53 overexpression and decreased progression-free survival (PFS) and disease-related survival (DRS). Stepwise Cox regression analysis identified p53 overexpression and SPF >/=9% as the most significant pretreatment molecular risk factors. A model stratifying patients according to whether none, one, or both of these two pretreatment factors were present showed that when both factors are present the risk for recurrence was higher (RR = 7.07; 95% confidence interval [CI], 3.06 16.38; P < 0.01) and death due to disease was higher (RR = 9.93; 95% CI, 3.92 25.19; P < 0.01) than when no factors are present. In the group with both factors, 5-year PFS and DRS estimates were 41 and 44%, respectively, compared with 86 and 86% and 90 and 92% for the "none" and "one" groups, respectively. CONCLUSION: When observed simultaneously, increased SPF and p53 overexpression defined a group of patients at high risk for rapid recurrence and death due to disease. Pretreatment molecular analysis of curettage specimens could provide a mechanism of triage that could be applied before definitive surgical treatment. PMID- 10739685 TI - Apoptosis, bcl-2 expression, and proliferation in benign and malignant endometrial epithelium: An approach using multiparameter flow cytometry. AB - OBJECTIVE: Disturbances in the regulation of cell proliferation and differentiation play an important role in the formation of neoplastic lesions. Consequently, abnormalities in apoptosis regulation may contribute to this process. Expression of a neoepitope on cytokeratin 18, unmasked by an early caspase cleavage event and recognized by the novel monoclonal antibody M30, is an indicator of early epithelial cell apoptosis. The purpose of this study was to evaluate the quantitative relation among apoptosis (M30), cell persistence (bcl 2), and proliferation (S-phase fraction; SPF) in malignant and benign endometrium. METHODS: Using multiparameter DNA flow cytometry on 54 formalin fixed paraffin-embedded samples from benign (proliferative, secretory, inactive, and hyperplastic endometrium) and malignant (grades 1-3 endometrial adenocarcinoma) endometrial tissue, bcl-2 expression and M30 reactivity were assessed together with the SPF in the cytokeratin-positive epithelial cells. RESULTS: Benign cyclic endometrium showed a relatively high bcl-2 expression and low M30 reactivity in the proliferative phase whereas in the secretory phase this relation was inverse. In endometrial hyperplasia the expression of bcl-2 was increased compared to that in secretory and postmenopausal endometrium, but still below the level of proliferative samples. The expression of M30 also increased compared to normal proliferative endometrium but did not reach the level of endometrium in the secretory phase of the menstrual cycle. In cancer the expression of bcl-2 decreased with the progression of differentiation grade. For M30 expression this relation was inverse. Overall there was a significant increase of M30 reactivity in cancerous compared to hyperplasia and normal cyclic endometrium. CONCLUSION: Transition of endometrial epithelium from hyperplasia to cancer seems to involve both increased apoptosis and decreased bcl-2 expression. Flow cytometric evaluation of M30 and bcl-2 expression levels, with the SPF, in currettage specimens from postmenopausal patients complaining of bleeding provides a quantitative assessment of endometrial apoptosis, anti-apoptosis, and proliferation. Further studies are needed to determine the relationship among these three processes as indicators of the biological behavior of gynecological tumors. PMID- 10739686 TI - Atypical clustering of gynecologic malignancies: A family study including molecular analysis of candidate genes. AB - OBJECTIVE: We set out to determine whether hereditary nonpolyposis colorectal cancer (HNPCC) was responsible for cancer susceptibility in a family with gynecologic malignancies in three consecutive generations. METHODS: A detailed family history study, including review of medical records, was undertaken. Tumor DNAs from affected family members were evaluated for microsatellite instability (MSI). Linkage between cancer susceptibility and the candidate DNA mismatch repair genes MLH1, MSH2, MSH3, and MSH6 (GTBP) was investigated. MLH1 and MSH2 protein expression was evaluated by immunohistochemistry and MSH2 was investigated for mutation. RESULTS: Four gynecologic malignancies in the core family were confirmed. MSI was seen in six of seven cancers studied. The only MSI negative tumor was an ovarian cancer from the proband's maternal grandmother, which arose at the age of 92. Haplotype analysis using chromosome 2p markers implicated the MSH2 gene in this family's cancer susceptibility. MSH2 protein expression was absent in an MSI-positive colon cancer from an affected family member. CONCLUSIONS: The inability to exclude linkage of MSH2 with the disease susceptibility, the presence of the MSI phenotype in cancers from family members sharing the same region of chromosome 2p, and the lack of immunodetectable MSH2 point to MSH2-associated HNPCC as a cause for this family's cancer susceptibility. Continued efforts to increase awareness of the heritability of endometrial cancer should improve our understanding of the disease, with resultant improved surveillance strategies, recommendations for surgical and chemoprophylaxis, and identification of patients at risk for malignancy as a result of HNPCC. PMID- 10739687 TI - Expression of thymidine phosphorylase in epithelial ovarian cancer: correlation with angiogenesis, apoptosis, and ultrasound-derived peak systolic velocity. AB - OBJECTIVE: The objective of this study was to determine whether the expression of thymidine phosphorylase (TP) by epithelial ovarian cancer cells correlates with the density of microvessels within the tumor, apoptotic index (AI) within the tumor cells, and ultrasound-derived blood flow. METHODS: Color Doppler imaging and pulsed Doppler spectral analysis ultrasonography were used to scan 44 patients with an overt ovarian mass immediately before laparotomy. Sections of malignant tumors were analyzed for the cellular expression of TP and the intratumoral density of microvessels by immunohistochemistry using antibodies to TP and factor-VIII-related antigen, respectively. Moreover, AI was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling methods. RESULTS: Forty-four epithelial ovarian cancers were studied (6 low malignant potential, 15 serous cystadenocarcinoma, 9 mucinous cystadenocarcinoma, 8 endometrioid adenocarcinoma, 4 clear cell carcinoma, and 2 malignant Brenner tumors); 19 were Stage I, 6 Stage II, 15 Stage III, and 4 Stage IV. Fourteen tumors (32%) were classified as being TP positive. The proportion of Stage I tumors that was TP positive (16%) was significantly lower (P = 0.022) that the corresponding value for Stages II-IV (44%), although the values for microvessel count, AI, and peak systolic velocity (PSV) were similar. AI was significantly lower in TP-positive tumors than in TP-negative tumors (P = 0.015). The PSV was significantly higher in TP-positive tumors (P = 0.02). There was a significant correlation between the microvessel count and the PSV (r = 0.34, P = 0.024). Moreover, AI was significantly inversely related to the PSV (r = 0. 35, P = 0.023). The PSV in a subgroup with a high microvessel count and low AI was significantly higher than that in a subgroup with a low microvessel count and high AI (P = 0.0006). These findings significantly associated with TP expression (P = 0.024). CONCLUSIONS: The intratumoral PSV, as determined by color Doppler imaging and spectral analysis, might reflect the coordination of angiogenesis and apoptosis associated with TP expression in epithelial ovarian cancer. PMID- 10739688 TI - Expression of carbohydrate antigens in advanced-stage ovarian carcinomas and their metastases-A clinicopathologic study. AB - OBJECTIVE: Up-regulated expression or loss of expression of various carbohydrate antigens on the surface of cancer cells has been associated with a metastatic phenotype and poor survival in epithelial malignancies of different origins. The object of this study was to investigate the expression of carbohydrate antigens in two groups of patients diagnosed with advanced-stage ovarian carcinoma-one with an extremely favorable outcome and the other with a uniformly poor survival. METHODS: Sections from 76 paraffin-embedded blocks (primary ovarian carcinomas and metastatic lesions) from 45 patients diagnosed with advanced-stage ovarian carcinomas (FIGO stages III-IV) were immunohistochemically stained using five monoclonal antibodies for Lewis(y) (Le(y))(two antibodies), Sialyl Lewis(x) (Slex), Tn, and Sialyl Tn (STn) antigens. Patients were divided in two groups based on outcome. Long-term survivors (21 patients) and short-term survivors (24 patients) were defined using a double cut-off of 36 months for disease-free survival (DFS) and 60 months for overall survival (OS). Staining results for primary tumors and metastases were analyzed separately. RESULTS: Mean follow-up period was 70 months. The mean values for DFS and OS were 109 and 125 months for long-term survivors and 3 and 25 months for short-term survivors. Staining for all four antigens was seen in the majority of cases (range = 72-96%) and tended to be comparable in primary tumors and their metastases. However, absence of immunoreactivity for STn was seen in 9/38 (24%) metastatic lesions and only 1/38 (3%) primary tumors. This finding did not reach statistical significance (P > 0.05). A combined pattern of membranous and cytoplasmic staining was predominant in the majority of cases. Enhanced staining for Le(y) and STn was detected in the invasive front of some tumors, while Slex and Tn immunoreactivity did not relate to cell location. Primary tumors and metastatic lesions of long-term survivors displayed immunoreactivity patterns that were comparable to those of short-term survivors. In the evaluation of survival curves, more diffuse staining for Slex showed marginal correlation with poor survival (P = 0.05), while a trend toward poorer survival was seen in tumors that were more extensively stained for Le(y) and Tn (P > 0.05). CONCLUSIONS: Le(y), Slex, STn, and Tn antigens are widely expressed in primary ovarian carcinomas and their metastases. Altered expression of Sialyl Tn is observed with tumor progression in a fraction of ovarian carcinomas. Expression of membrane carbohydrate residues is prevalent in tumors of both long-term and short-term survivors and does not appear to be a strong predictor of disease outcome. However, larger studies are needed to further elucidate the role of these molecules in ovarian carcinogenesis. PMID- 10739689 TI - Whole-body PET with (fluorine-18)-2-deoxyglucose for detecting recurrent primary serous peritoneal carcinoma: An initial report. AB - OBJECTIVE: Because of the limited sensitivity and specificity of conventional tools such as computerized tomography (CT) or magnetic resonance imaging (MRI) for detecting persistent or recurrent primary serous peritoneal carcinoma (PSPC), a reliable means of diagnosis remains elusive. Positron emission tomography (PET) scanning may offer another approach to this problem. METHODS: A prospective study of three patients requiring surgical exploration for suspected recurrence of PSPC received a whole-body PET (fluorine-18)-2-deoxyglucose (FDG) scanning in a teaching hospital from July 1995 to December 1998. The suspected recurrence was based upon clinical findings including a detailed physical examination, serum CA 125 marker ultrasound, CT, and MRI. Three patients were enrolled in this study. RESULTS: In all three patients, PET images demonstrated increased FDG uptake in a distribution that correlated with surgical-pathologic findings (100%); on the contrary, CT can detect 33.3% of these patients with malignant diseases and MRI can detect two-thirds of cases. Serum CA-125 was also elevated in all three patients, although one patient showed an equivocal elevation of 25.7 IU/ml. CONCLUSIONS: Conventional imaging studies are neither sensitive nor specific for detecting recurrent PSPC. In contrast, besides CA-125, PET might offer a relatively effective tool for detecting recurrent primary serous peritoneal carcinoma. Due to the very small number of patients available in this study, considerable research must be performed to clarify the impact of PET on detecting recurrence of PSPC. PMID- 10739690 TI - Efficacy and hematologic toxicity of salvage chemotherapy following stem cell supported high-dose chemotherapy in women with recurrent ovarian cancer. AB - OBJECTIVE: The objective of this study was to determine the efficacy and hematologic toxicity of salvage chemotherapy in patients with recurrent ovarian cancer following high-dose chemotherapy and peripheral blood stem cell transplantation (PBSCT). METHODS: A retrospective analysis of 19 Massachusetts General Hospital case records of women with relapsed ovarian cancer following PBSCT was conducted. RESULTS: Between February 1996 and September 1998, 24 women with ovarian cancer were treated with PBSCT. Nine patients were treated with an upfront PBSCT regimen to consolidate first-line chemotherapy and 15 patients were treated with PBSCT after a median of two lines (range: 1-3) of prior chemotherapy. Sixteen patients presented with relapsed disease at a median of 230 days post-PBSCT and 3 patients had persistent disease through high-dose chemotherapy. Each of these 19 patients has been treated with salvage chemotherapy following PBSCT. Patients received one of six different first-line salvage chemotherapy regimens. Sixteen of nineteen patients are alive a median of 383 days (range: 156-868) after relapse following PBSCT. Three patients died of progressive disease at a median of 284 days (range: 224-648) after post-PBSCT relapse. Six patients achieved a complete response, four patients had a partial response, three patients had stable disease, and six patients had progressive disease in response to first-line salvage chemotherapy. Seven patients experienced grade III/IV neutropenia, and three patients experienced grade III/IV thrombocytopenia. CONCLUSIONS: We conclude that in a patient population selected for chemotherapy sensitive and low-volume disease prior to PBSCT, patients with recurrent tumor appear to respond to salvage chemotherapy, and associated hematologic toxicity is acceptable and manageable. PMID- 10739691 TI - The outcome of stage I-II clinically and surgically staged papillary serous and clear cell endometrial cancers when compared with endometrioid carcinoma. AB - PURPOSE: The aim of this study was to compare survival and recurrence in clinical and surgical stage I-II papillary serous (PS), clear cell (CC), and endometrioid (EM) cancers of the endometrium and examine the prognostic utility of myometrial invasion. METHODS: Clinical, surgicopathologic, and survival data were retrospectively collected on 574 clinical stage I-II endometrial cancer patients, including 53 PS and 18 CC (based on postoperative histology), undergoing hysterectomy at Duke University Medical Center between 1967 and 1990. All staging material was available and reexamined prior to this analysis, and FIGO surgical staging was retrospectively assigned. Prognostic variables examined included age, stage, grade, myometrial invasion, lymph-vascular space invasion (LVSI), and histology. PS and CC histologic subtypes were compared as both common category and discrete categories versus EM, EM grade 1 (EM1), EM grade 2 (EM2), and EM grade 3 (EM3). Statistical analyses were performed using chi(2), Fisher's exact, and Wilcoxon rank sum tests, Cox regression analysis, and Kaplan-Meier survival analysis. RESULTS: PS tumors accounted for 9%, CC for 3%, and EM for 88% of cases. Recurrences were more frequent among PS (38%) and CC (22%) compared with EM (9%) (P < 0.001 and 0.08, respectively), and PS recurred more frequently than EM3 alone (20%) (P = 0.06). Among PS, CC, and EM3 patients with recurrences there were no statistical differences in the proportion that received preoperative or postoperative radiotherapy or chemotherapy. Prognostic factors for shorter survival included age >=60, surgical stage III+IV, presence of LVSI, histology (PS, CC, or EM3), and >=50% myometrial invasion. The estimated 5-year survival of PS+CC patients with <2 mm myometrial invasion is 0.56 compared to 0.93 for EM patients (P < 0. 001). PS + CC tumors confined to the endometrium had a 5-year survival of 0.60 compared to 0.98 and 1.00 for EM and EM3, respectively. The 5 year survival for surgically staged IA patients (0.57) was not different from stages IB and IC combined (0.53) (P = 0.72). The 5-year survival for surgical stage I + II PS + CC patients (0.56) was comparable to that for clinical stage I + II PS + CC patients (0.46) and remained significantly smaller than that for EM patients (0.86) (P < 0.001). CONCLUSION: Recurrences are more frequent among PS and CC tumors compared with EM and among PS compared with EM3. When controlled for surgical stage I-II tumors, 5-year survival for PS + CC patients remains comparable to that of clinical stage I-II patients and below that of EM. Prognostic factors for survival in PS and CC patients include age, stage, and LVSI. PS, CC, and EM3 subtypes together are predictors of poor survival. Thorough extended surgical staging is indicated in PS and CC tumors, and prospective trials of aggressive adjuvant therapies for surgical stage I-II tumors are needed to improve outcome in PS and CC patients. PMID- 10739693 TI - Sexual dysfunction following vulvectomy. AB - OBJECTIVE: This is a pilot study to evaluate sexual dysfunction in women after vulvectomy. METHODS: An 88-question survey was used to assess body image and the DSM IV criteria for sexual dysfunction on women who had undergone vulvectomy. RESULTS: Forty-seven women agreed to participate in the study and 41 women (87%) returned the survey. There was a significant alteration of body image in these women after vulvectomy (P = 0.004). Sexual frequency significantly decreased after surgery (P = 0.001) and there was significant sexual dysfunction in the categories of sexual aversion disorder (P = 0.01), arousal disorder (P = 0.02), and hypoactive sexual disorder (P = 0. 001). The extent of surgery did not correlate with degree of sexual dysfunction in any category. Women who were depressed at the time of survey (as determined by the PRIME-MD scale) were more likely to suffer sexual aversion disorder (P = 0.05) and tended to have more body image disturbance (P = 0.1) and global sexual dysfunction (P = 0.06). CONCLUSIONS: Women experience significant sexual dysfunction after vulvectomy and the extent of surgery or type of vulvectomy did not correlate with degree of sexual dysfunction. There is a significant need to address sexual problems with all women after any vulvectomy. Age, depression, worsening GOG performance status, and preoperative hypoactive sexual dysfunction were risk factors for sexual dysfunction after vulvar surgery. Appropriate counseling and treatment of depression may be of benefit to this patient population. PMID- 10739692 TI - Results of radical radiotherapy for recurrent endometrial cancer. AB - OBJECTIVES: The aims of this study were to determine the overall survival (OS) and local control (LC) achieved in patients developing a locoregional recurrence of endometrial carcinoma and to define those prognostic factors that predict for improved LC and OS. METHODS: Between 1984 and 1988, 958 women were referred to Princess Margaret Hospital (PMH) with a diagnosis of endometrial carcinoma. Of these, 58 were treated for recurrent disease with radical radiotherapy (RT). Forty-two were referred with recurrence and 16 relapsed during follow-up at PMH for their primary tumor. None had received prior RT. The majority (n = 49) were treated with combined external beam RT followed by an intracavitary cesium insertion. RESULTS: The median time to relapse from original diagnosis was 1.3 years (range 0.2-13.4 years). The actuarial 5- and 10-year OS was 53 and 41%, respectively. The respective results for LC were 65 and 62%. All end-points were measured from the time of relapse. The median total dose received was 81.5 Gy. Univariate analysis showed that favorable histological features at original diagnosis (<50% myometrial involvement, grade 1-2, P = 0.007) and Perez modified staging (P = 0.02) were significant predictors for OS. The Perez staging (P = 0.02) and size of recurrence (<2 cm versus >/=2 cm, P = 0.04) were predictors for LC. CONCLUSION: Patients with localized relapse of endometrial carcinoma in whom radical radiotherapy can be administered should be treated aggressively and may be cured in over half the cases treated. Pathological findings in the original surgical specimen, size of recurrent disease, and a modified vaginal carcinoma staging system are significant predictors of local pelvic control and survival. PMID- 10739694 TI - Combined transvaginal B-mode and color Doppler sonography for differential diagnosis of ovarian tumors: results of a multivariate logistic regression analysis. AB - OBJECTIVE: Transvaginal sonography is limited in its ability to assess early stage cancers of the ovary as well as in distinguishing benign processes. As a method for characterization of tumor vascularization, color-coded Doppler sonography may be able to improve the diagnostic accuracy of B-mode sonography. METHODS: Preoperative transvaginal B-mode and Doppler sonography was performed in 63 patients with unclear adnexal lesions prior to operation. Using multiple logistic regression, the independent variables of each procedure were selected and combined to yield a diagnostic flow chart. The diagnostic accuracy of this decision matrix was tested on 257 patients with unclear adnexal tumors. RESULTS: In the 63 adnexal tumors investigated, the diagnostic impact of isolated sonomorphological assessment with evidence of a "solid area" was 78%. Using Doppler sonography, the best discrimination was achieved by displaying the vascular distribution ("central vascularization"). Combining these independent significant variables of the two procedures raised the diagnostic accuracy to 90% (sensitivity 86%, specificity 93%). The validity achieved by this combination was confirmed by the independent application of this method to the 257 adnexal tumors with unclear malignancy status (diagnostic accuracy 93%, sensitivity 92%, specificity 94%). CONCLUSIONS: The combination of sonography and Doppler sonography achieves high and reproducible diagnostic accuracy in preoperative malignancy status assessment of adnexal tumors. The additional use of Doppler sonography can thus provide significant aid both for differential diagnostics of adnexal lesions and for the choice of surgical route in the case of an existing indication for operative therapy. PMID- 10739695 TI - Technical development and results of left extraperitoneal laparoscopic paraaortic lymphadenectomy for cervical cancer. AB - OBJECTIVE: The aim of this study was to describe the development of our technique for laparoscopic paraaortic lymphadenectomy for cervical cancer and to evaluate the accuracy of the left extraperitoneal route to perform complete paraaortic lymphadenectomy. METHODS: A retrospective study of a consecutive series of 44 patients with cervical cancer undergoing laparoscopic paraaortic lymphadenectomy between July 1992 and November 1998 was performed, as well as a comparison of the three routes successively used to perform paraaortic lymphadenectomy: transperitoneal, bilateral extraperitoneal, and left extraperitoneal. RESULTS: The initial choice of surgical access was transperitoneal (n = 9) in 20%, bilateral extraperitoneal (n = 14) in 32%, and left extraperitoneal (n = 21) in 48% of cases. Success rates of laparoscopic paraaortic lymphadenectomy were 78% for the transperitoneal approach, 93% for the bilateral extraperitoneal approach, and 95% for the left extraperitoneal approach. Conversion from extraperitoneal to transperitoneal laparoscopic paraaortic lymphadenectomy, because of a peritoneal tear, was necessary in 3 cases (21.4%) for the bilateral extraperitoneal route and in 3 cases (14.3%) for the left extraperitoneal route (P = 0.43). The extent of dissection varied with experience. Systematic paraaortic lymphadenectomy (up to the left renal vein) was performed via the transperitoneal route in 1 case with 19 aortic nodes removed (common iliac nodes excluded) in 160 min, via the bilateral extraperitoneal route in 6 cases with a mean of 16 +/- 2 (range: 14-19) aortic nodes removed in 153 +/- 22 min (range: 130-180), and via the left extraperitoneal route in 12 cases with a mean of 15 +/- 3 (range: 10-19) aortic nodes removed in 119 +/- 14 min (range: 100-150). There were no statistically significant differences in the total number of nodes removed between the two extraperitoneal routes, although the bilateral extraperitoneal route yielded more right-sided aortic nodes (P < 0. 01). The operating time was significantly shortened using the left extraperitoneal route (P < 0.05). CONCLUSION: Systematic paraaortic lymphadenectomy by a left extraperitoneal route is feasible. Information on right-sided aortic nodes can be obtained although the sampling is reduced compared to that of bilateral extraperitoneal route. It provides the advantages related to the use of the extraperitoneal route while reducing manipulations and thus the risk of peritoneal tearing compared to those of the bilateral extraperitoneal route. PMID- 10739696 TI - Inguinal node status by ultrasound in vulva cancer. AB - OBJECTIVE: The objective of this study was to determine the value of ultrasound in preoperative assessment of groin node status in vulva cancer. MATERIALS AND METHODS: Women with clinically uninvolved groins who were undergoing groin node dissection for vulva cancer in our department over an 18-month period were recruited into the study. A preoperative scan of each groin to be dissected was performed to identify any suspicious lymph nodes containing metastases. Suspicious nodes were defined by two sonographic criteria: short axis diameter (>8 mm) and a long axis/short axis ratio (L/S 0.10). In the single PSCP case with a K-ras mutation, the mutation was found in only one of five tumor sites tested. All four mutations involved a single nucleotide alteration in codon 12 (GGT to GTT, Gly to Val). To evaluate the ras pathway in PSCP, we used the known activated ras binding domain on Raf-1 to perform an assay to test for activated ras. We identified ras activation in 4 of 5 PSCP cases tested and, to confirm that the activation was functional, we tested and found similar activation of MAP kinase, a downstream mediator for K-ras expression. CONCLUSIONS: K-ras mutations occur at low rates in both PSCP and high-grade, late stage SEOC, and therefore K-ras mutations are not involved in the development of these two diseases. Finding the mutation in only one of multiple tumor sites in the PSCP case supports growing evidence for a multifocal origin of PSCP. Our findings of ras activation in four of five cases of PSCP suggest that ras activation by mechanisms other than genetic mutation is important for PSCP tumorigenesis. PMID- 10739699 TI - Phase II evaluation of 24-h continuous infusion topotecan in recurrent, potentially platinum-sensitive ovarian cancer: A Gynecologic Oncology Group study. AB - OBJECTIVES: The aim of this study was to develop an alternative effective and more convenient administration schedule for intravenous topotecan when used as palliative treatment in ovarian cancer. METHODS: The Gynecologic Oncology Group conducted a Phase II trial of 24-h infusional topotecan (8.5 mg/m(2)) with treatment repeated every 3 weeks in 29 patients with platinum-sensitive recurrent ovarian cancer (prior response to platinum-based chemotherapy with a minimum treatment-free interval >/=6 months). RESULTS: The major toxicities of therapy were grade 4 neutropenia and thrombocytopenia which developed in 86 and 14% of patients, respectively. Other severe side effects were uncommon. Only 2 partial responses (7%) were observed in the 28 patients evaluable for response. CONCLUSIONS: Despite the relatively favorable ovarian cancer patient population treated in this trial (platinum-sensitive recurrent disease), the response rate was disappointingly low. Considering the three- to fivefold higher objective response rates observed in other trials employing topotecan in individuals with platinum-sensitive ovarian cancer utilizing a 5-day treatment program (delivered every 3 weeks), the results of the current study provide strong support for the conclusion that clinically relevant antineoplastic activity of this agent is highly schedule dependent. PMID- 10739700 TI - Radiation therapy for elderly patient with squamous cell carcinoma of the uterine cervix. AB - OBJECTIVE: The purpose of this study was to profile cervical squamous cell carcinoma in elderly patients undergoing radiation therapy and to study the treatment outcome and side effects of therapy. MATERIALS AND METHODS: A retrospective analysis was carried out from the records of 380 patients with squamous cell carcinoma of the uterine cervix who had been given radiation therapy between 1970 and 1994. The patients were divided into three age groups: under 70 years (youngest group; n = 215), 70 to 79 years (intermediate group; n = 124), and 80 years or older (oldest group; n = 41). Radiation therapy was performed by a combination of external beam therapy and three brachytherapy fractions using low-dose-rate sources. RESULTS: The 5-year overall survival rates in the youngest, intermediate, and oldest groups were 58, 50, and 33%, respectively, while cause-specific survival rates were 68, 70, and 65%, respectively. For the patients with stage III, the 5-year overall survival rates in the youngest, intermediate, and oldest groups were 59, 48, and 36%, respectively, while cause-specific survival rates were 72, 70, and 70%, respectively. There was no statistical significance in the 5-year intrapelvic recurrence rates among the three groups. Grade 3 or 4 complications occurred in 6.5% of the youngest, 11.3% of the intermediate, and 7.3% of the oldest groups. CONCLUSION: Radiation therapy with external beam combined with three fractions of low-dose-rate brachytherapy proved both highly effective and safe for senior patients with cervical squamous cell carcinoma. PMID- 10739701 TI - Establishment and characterization of a breast cell strain containing a BRCA1 185delAG mutation. AB - OBJECTIVE: The aim of this study was to examine whether cells containing the heterozygous form of a BRCA1 185delAG mutation would exhibit abnormal growth or an altered response to DNA damage. METHODS: A primary culture of human mammary epithelial cells (90P) was obtained from the nontumor breast tissue of a 35-year old patient who had undergone a mastectomy for removal of a breast tumor. These cells were immortalized (90PE6E7) following retroviral infection with HPV-16 viral E6/E7. genes. Both the 90P cell strain and the cell line were characterized for their ability to grow in culture, form colonies in soft agar, and produce tumors in athymic nude mice compared to normal breast epithelial cells containing wild-type BRCA1. 90P cells were also analyzed for cellular response to gamma radiation and H(2)O(2). RESULTS: These cells were confirmed to contain a frameshift mutation, 185delAG, of the BRCA1 gene. Despite being heterozygous for wild-type BRCA1, the 220-kDa full-size BRCA1 protein was abundantly expressed. 90P and 90PE6E7 cells grew at a similar rate and were anchorage dependent. 90PE6E7 also failed to form tumors in athymic nude mice. Finally, 90P cells exhibited a survival response similar to that of normal mammary epithelial cells to radiation damage and exposure to oxidative stress. CONCLUSION: To our knowledge the 90P cells and the 90PE6E7 cells are the first characterized, non tumor-derived breast epithelial cells that are heterozygous for the BRCA1 germline mutation 185delAG. Our conclusion is that these BRCA1 mutant cells appear to have growth and stress response characteristics similar to those of normal human breast cells, which is consistent with the hypothesis that loss of heterozygosity must occur to impair putative BRCA1 function. PMID- 10739702 TI - The possible role of p53 and bcl-2 expression in cervical carcinomas and their premalignant lesions. AB - OBJECTIVE: The purpose of this study was to assess the expression and clinical significance of bcl-2 and p53 in the progression of cervical neoplasias. METHODS: One hundred seventy-one cervical specimens, consisting of normal cervical epithelium (n = 13), lesions with histological features of HPV infection (n = 14), CIN (cervical intraepithelial neoplasia) lesions (n = 63), and cervical carcinomas (n = 81) were examined immunohistochemically in paraffin sections. RESULTS: Twenty-three specimens showed p53 expression [3/20 (15%) CIN III, 18/63 (29%) ISCC (invasive squamous cervical carcinoma), and 2/18 (11%) adenocarcinomas] while 63 cases expressed the bcl-2 gene [10/13 (77%) normal, 0/14(0%) condylomas, 6/23 (26%) CIN I, 9/20 (45%) CIN II, 15/20 (75%) CIN III, 18/63 (29%) ISCC, and 5/18 (28%) adenocarcinomas]. The expression of bcl-2 was found to increase in direct relation to the grade of CIN (P = 0.02) whereas such a trend was not observed for p53. p53 was not detected in normal or premalignant lesions (except 3 out of 20 cases of CIN III). There was no significant correlation between the expression of p53 and the histological type of cervical carcinoma, even though expression of p53 was higher in ISCC than in adenocarcinomas (29% vs 11%, respectively). In cervical cancer patients, expression of bcl-2 was correlated to a greater than 5-year survival (P < 0.01) while no prognostic significance of p53 expression was found. CONCLUSION: Evaluation of bcl-2 expression may provide additional and independent prognostic information for the clinical course of the disease and therefore to be developed as a prognostic indicator for cervical cancer. PMID- 10739703 TI - Development and assessment of a general theory of cervical carcinogenesis utilizing a severe combined immunodeficiency murine-human xenograft model. AB - OBJECTIVE: Currently, we lack a theoretical explanation for why squamous cell cervical cancer develops predominantly in specific sites (i.e., along the squamocolumnar junction). We therefore implanted human cervical tissues containing the transformation zone in severe combined immunodeficiency (SCID) mice and studied morphology, steroid effects, gene expression, and human papillomavirus (HPV) factors. METHODS: Normal and dysplastic human cervical tissues (3 x 2 mm) were placed subcutaneously in SCID-beige mice and later assessed by in situ hybridization for HPV 16/18 DNA and by immunohistochemistry for expression of CD31, keratin, proliferating-cell nuclear antigen, HPV 16 E6, p53, and Notch-1 (a binary cell fate determination protein). Some normal tissues were implanted with either a 90-day release 1.7-mg 17beta-estradiol pellet or a 5 mg tamoxifen pellet; others were infected prior to implantation with human recombinant adenovirus 5 vector containing a human cytomegalovirus promoter driven beta-galactosidase gene and later assessed by X-gal staining. RESULTS: Murine and human vessels formed anastomoses by 3 weeks. For at least 11 weeks, normal tissue retained the transformation zone and normal cell-type-specific keratin expression and exhibited normal proliferation; Notch-1 was present only in the basal cell layer. Dysplastic tissues exhibited koilocytosis, increased levels of cellular proliferation, and aberrant keratin, p53, and Notch-1 expression; HPV 16/18 DNA and HPV 16 E6 protein were detected for at least 6 weeks. Squamous metaplasia of normal cervical epithelium resulted from estrogen exposure, and a predominant columnar differentiation pattern was associated with tamoxifen administration. Through stable adenovirus infection, beta-galactosidase was expressed for at least 6 weeks. CONCLUSIONS: This small manipulatable xenograft model maintains normal and dysplastic human cervical epithelium through neovascularization. Neoplastic tissue retains HPV 16/18 DNA and a premalignant phenotype, including elevated levels of cellular proliferation and aberrant keratin, p53, and Notch-1 expression. These attributes constitute essential features of a biologic model through which one may study HPV-mediated human disease and may be superior to cell culture and transgenic murine systems. Furthermore, this may serve as a model for gene therapy. Finally, we suggest that the normal cervical epithelium is maintained through putative interactions between the Notch locus and cell cycle growth regulators such as p53 and pRb. Neoplastic cervical epithelium may arise through disruption of this pathway. This theory may be testable in our animal model. PMID- 10739704 TI - Use of hormone replacement therapy and adenocarcinomas and squamous cell carcinomas of the uterine cervix. AB - INTRODUCTION: Exogenous hormones may influence the development of cervical adenocarcinomas. Incidence rates of adenocarcinomas and use of noncontraceptive hormones have increased since the 1970s, but few studies have investigated this potential relationship. METHODS: We conducted a multicenter case-control study of 124 women with adenocarcinomas, 139 women with squamous cell carcinomas matched on age, diagnosis date, clinic, and stage of disease (in situ or invasive) to adenocarcinoma cases, and 307 healthy community controls who were also matched on age, ethnicity, and residence to adenocarcinoma cases. Participants completed in person interviews regarding exogenous hormone use before diagnosis and other risk factors and volunteered cervical samples for human papillomavirus (HPV) testing via a PCR-based method. Odds ratios (ORs) with 95% confidence intervals (CIs) estimated relative risks. RESULTS: Only 13 adenocarcinoma cases (10.5%), 7 squamous carcinoma cases (5%), and 20 controls (6.5%) had used noncontraceptive hormones for menopausal symptoms, irregular periods, or disease prevention; most use was short-term, former use. Ever-use was associated with adenocarcinomas (OR = 2.1, 95% CI 0.95-4.6) but not squamous carcinomas (OR = 0.85, 95% CI 0.34-2.1). No trends were seen with duration of use or ages at first use, but unopposed estrogens were positively associated with adenocarcinomas (OR = 2.7). Unopposed estrogens remained associated with adenocarcinomas (OR = 2.0) when analyses were restricted to the HPV-positive controls. Menopausal status was not associated with adenocarcinomas or squamous carcinomas and did not modify the other associations. CONCLUSION: Although small numbers warrant tentative conclusions, exogenous estrogens, especially unopposed estrogens, were positively associated with adenocarcinomas. Noncontraceptive hormones were negatively but weakly associated with squamous carcinomas. PMID- 10739705 TI - Radical hysterectomy: An anatomic evaluation of parametrial dissection. AB - OBJECTIVES: This study was designed to demonstrate a reduction in the amount of blood loss for vesicouterine ligament dissection and to investigate the intrapelvic autonomic nerve pathway and its preservation by means of anatomic analysis. METHODS: The anchoring mechanism of the pelvic viscera to the pelvic wall was divided into a supporting system facing laterally and a suspensory system facing dorsoventrally. An operative procedure was designed in which both systems were separated and dissected independently. RESULTS: Between the two systems, an artificial space was developed, which required a new dissection method for the parametrium and revealed a new anatomic pathway for the ureter and autonomic nerve. The amount of blood loss (mean +/- SD) during dissection of the vesicouterine ligament was ultimately 260.1 +/- 114.8 ml. Postoperatively, the maximum capacity of the bladder was 393.9 +/- 40.4 ml, maximum detrusor pressure 6.3 +/- 4.1 cm H(2)O, mean compliance >10 ml/cm H(2)O, residual urine 23.8 +/- 9.4 ml, and maximum flow rate 25. +/- 8 2.2 ml/s, respectively. CONCLUSION: A new classification for the parametrium and its dissection method have been established. Development of this new operative procedure has also contributed to a decrease in blood loss and preservation of bladder function. PMID- 10739706 TI - Can initial serum cyfra 21-1, SCC antigen, and TPA levels in squamous cell cervical cancer predict lymph node metastases or prognosis? AB - OBJECTIVE: The aim of this study was to determine whether lymph node metastases or prognosis can be predicted by initial serum Cyfra 21-1, tissue polypeptide antigen (TPA), and squamous cell carcinoma antigen (SCC-Ag) levels in squamous cell cervical cancer. METHODS: Pretreatment serum levels of 92 patients were correlated with clinicopathologic parameters and prognostic data. The clinical performance of the tests was evaluated by their receiver operating characteristic curves. The prognostic power of the variables was assessed using Cox regression analysis. RESULTS: Serum levels of each marker were significantly related to tumor stage, size, and depth of infiltration. The clinical performance of each marker in predicting lymph node metastases or parametrial involvement was poor. In the stepwise Cox regression analysis, regarding patients with early stage cervical cancer (stage Ib/IIa, n = 63), tumor size (P = 0.0005) was the only independent prognostic factor for disease-free interval. Lymph node status (P = 0.0014), tumor size (P = 0.004), and parametrial involvement (P = 0.025) were independent risk factors for survival. Considering all patients with stages Ia through IVb disease, tumor size (P = 0.0001) and TPA level (P = 0. 026) were independent risk factors for disease-free interval, whereas tumor size (P = 0.0001) and parametrial involvement (P = 0. 0002) were risk factors for survival. CONCLUSIONS: Pretreatment Cyfra 21-1, TPA, and SCC-Ag levels were strongly related to tumor burden, but insufficiently reliable for identifying patients at risk of the presence of lymph node metastases or parametrial involvement. Serum levels of each marker showed no independent prognostic value in early stage cervical cancer. PMID- 10739707 TI - Allelic imbalance in lichen sclerosus, hyperplasia, and intraepithelial neoplasia of the vulva. AB - BACKGROUND: Few studies have addressed in detail the genetic alterations that occur in vulvar squamous carcinomas (VSCC) and their precursor lesions. In a previous study, we determined the most common chromosomal loci for allelic imbalance (AI) in HPV-positive and -negative VSCCs. The present study was designed to determine whether AI and the microsatellite instability phenotype (MIN) were present in epithelial lesions known to be associated with VSCC. DESIGN: Fifty-seven epithelial loci were analyzed, including HPV-positive (classic) and -negative (differentiated) vulvar intraepithelial neoplasms (VINs), lichen sclerosus (LS), and nonatypical hyperplasias. Thirty-one epithelial loci (55%) were obtained from patients with associated invasive vulvar carcinoma. HPV status was determined by polymerase chain reaction analysis. AI and MIN were determined by comparisons of microdissected target tissues with stromal controls, targeting 11 chromosomal loci. RESULTS: AI was identified in all epithelial categories, involving at least one chromosomal locus in 67, 53, 50, and 43% of classic VIN, differentiated VIN, hyperplasia, and LS. MIN was infrequent (10 13%), but confined to HPV-negative epithelial changes. HPV-positive lesions generally scored for AI more frequently, but certain loci scored nearly equally in both HPV-positive and -negative lesions, including 8p, 11q, and 17p. There were no differences in frequency of AI between epithelia with and without associated invasive carcinoma. CONCLUSIONS: The presence of allelic imbalance in vulvar hyperplasia and LS supports the hypothesis that these alterations are at greater risk for neoplasia despite the absence of conspicuous cellular atypia. A model is proposed in which these changes represent monoclonal expansion and are at empirically greater risk for subsequent "critical events" leading to morphologic atypia (VIN). The possibility that these early genetic changes influence both HPV-positive and -negative pathways merits further study. PMID- 10739709 TI - Paget's disease of the vulva: pathology, pattern of involvement, and prognosis. AB - OBJECTIVE: The aim of this study was to determine prognostic factors and risk factors for recurrence in patients with Paget's disease of the vulva. METHODS: The medical records of 76 patients with a diagnosis of Paget's disease of the vulva were retrospectively reviewed. The diagnosis in each case was confirmed by reviewing the pathology. Patients were then divided into four groups by diagnosis: intraepithelial Paget's disease (IEP) (n = 46), invasive Paget's disease (IP) (n = 9), intraepithelial Paget's disease with underlying adenocarcinoma (IEPUA) (n = 13), and intraepithelial Paget's disease with a coexisting cancer (CCA) (n = 8). Comorbid conditions, location of disease, pathologic diagnosis, method of treatment, margin status, and current status of the patient were evaluated. Descriptive statistical data and univariate analysis were generated using the Statview statistical package. RESULTS: A diagnosis of IEPUA, IP, or CCA predicted a poor survival (P = 0. 0017). Patients who had received chemotherapy or radiation as treatment had a poor survival (P < 0.0001 and 0.0002). Patients with clitoral Paget's disease had a higher incidence of death from disease (P = 0.026). When death from all causes was considered, patients treated with wide local excision (WLE) had a significantly longer survival than patients treated with other more radical treatments (P = 0.02). Risk factors for recurrence included treatment with WLE (P = 0.004). CONCLUSIONS: Patients with IP, IEPUA, or CCA have a poorer prognosis than patients with IEP. Location of Paget's disease is important for prognosis; and patients with clitoral Paget's disease may require more aggressive treatment. WLE is associated with a higher risk of recurrence, but overall patients with WLE tend to survive longer than patients treated more radically. PMID- 10739708 TI - RU486 increases radiosensitivity and restores apoptosis through modulation of HPV E6/E7 in dexamethasone-treated cervical carcinoma cells. AB - OBJECTIVE: Cervical carcinoma tumors containing radioresistant cells are associated with decreased local control and survival. Therefore, strategies to increase cell kill during radiotherapy have a clear rationale. It was previously determined that treatment with the corticosteroid dexamethasone increased radioresistance and decreased apoptosis in C4-1 cervical carcinoma cells. The goal of this study was to determine whether hormone antagonists, specifically Mifepristone (RU486), could reverse the effects of dexamethasone on clonogenic survival and apoptosis following gamma-irradiation. METHODS: Cervical carcinoma cell line C4-1 cells were exposed to 1 microM dexamethasone in the presence or absence of 1 microM Mifepristone (RU486), a hormone antagonist, and irradiation. Cells were analyzed for steroid-dependent HPV E6/E7 mRNA expression (by Northern blot analysis), clonogenic survival, and apoptosis (by Annexin V staining and the DNA fragmentation assay). In addition, p53 protein levels were determined by Western blot analysis. RESULTS: The hormone antagonist RU486 reversed dexamethasone-dependent upregulation of E6/E7 mRNA and restored radiation-induced p53 expression, apoptosis, and clonogenic survival to levels similar to those observed following irradiation alone. CONCLUSION: RU486 reverses glucocorticoid dependent upregulation of HPV E6/E7, which corresponds to restoration of p53 expression, and restores radiosensitivity and apoptosis following gamma irradiation. Therefore, it appears that along with radiation, RU486 may be a beneficial agent in the treatment of hormone-reactive cervical tumors. PMID- 10739710 TI - Alternative medicine in gynecologic oncology: A case report. AB - A patient with Stage IIB squamous cell cancer of the cervix presented 10 days into her primary radiation therapy with neutropenic fever and diarrhea requiring hospitalization and delay in treatment. The patient's history revealed extensive use of alternative therapies including potential toxic botanicals. This case raises the possibility that a patient's use of alternative therapies may have caused toxicity and delayed primary therapy. Gynecologic oncology patients may be using alternative therapies justifying the need for incorporating this classification into the historical exam. PMID- 10739711 TI - High-dose-rate interstitial brachytherapy of Bartholin's gland: A case report. AB - OBJECTIVE: To preserve the organs and function of the anus and ureter, radiotherapy was chosen for an advanced vulvar tumor originating from Bartholin's gland. METHODS: The patient was a 74-year-old female with stage III (FIGO) vulvar cancer derived from the left Bartholin's gland. She received 63 Gy to the vulvar lesion with external beam irradiation followed by 30 Gy of high-dose-rate interstitial brachytherapy. RESULTS: One year after radiotherapy, simple vulvectomy and reconstructive surgery were performed. The anal and ureteral functions were preserved. Histological examination revealed no evident malignant cells. CONCLUSION: Owing to the technological development of radiotherapy and improved reconstructive surgery, radiotherapy in treatment of the vulvar area has become effective and safe and could be indicated more as a treatment option for the sake of organ preservation. PMID- 10739712 TI - Complete hydatidiform mole and a coexistent viable fetus: report of two cases and review of the literature. AB - OBJECTIVE: The aim of this study was to report the clinical features, management, and outcome of two cases of complete hydatidiform mole with a coexisting viable fetus and to review the literature. PATIENTS: In this article, we report on the well-documented follow-up of 2 cases of twin pregnancies with complete hydatidiform mole and a viable fetus, both of which ended with the delivery of a normal infant at 41 and 26 weeks of gestation. It is of interest that both pregnancies were achieved following induction of ovulation with hMG/hCG. Since 1977, the year in which complete and partial moles were characterized as distinct pathologic entities, 15 cases (including our 2) have been reported. RESULTS: Persistent GTT developed in eight patients (53.3%) and four patients (27.7%) developed metastatic disease. Seventy-five percent patients with persistent GTT were treated with single-agent chemotherapy. The median gestational age of the patients with subsequent persistent GTT was 34.5 weeks compared to 38 weeks in the patients without persistent GTT. CONCLUSION: Complete hydatidiform mole and coexistent fetus is a rare occurrence and is associated with an increased risk of persistent gestational trophoblastic tumor. Based on currently available information, it seems that in the presence of a stable pregnancy, normal karyotype, and a normal sonogram it is reasonable to allow the pregnancy to continue. PMID- 10739713 TI - Alpha-fetoprotein production by a malignant mixed Mullerian tumor of the ovary. AB - Elevated levels of alpha-fetoprotein (AFP), a fetal serum protein, usually signal the development of hepatoma or germ cell tumors, including endodermal sinus tumors. We report the case of a 52-year-old woman with an alpha-fetoprotein producing malignant mixed Mullerian tumor (MMMT) of the ovary. Serum AFP was 5348 ng/ml at diagnosis. Immunohistochemistry confirmed that the carcinomatous component of this biphasic tumor was the seat of AFP production. After three cycles of combination chemotherapy, the patient achieved a complete remission. Serum AFP was strongly correlated with response to treatment. This is the first report of AFP production by a MMMT of the ovary without a yolk sac component. PMID- 10739714 TI - Metachronous carcinoma of the vulva and fallopian tube. AB - BACKGROUND: Metachronous carcinoma of the vulva and fallopian tube is an unusual co-occurrence of gynecological malignancies. A report of such a case that developed and recurred over a 7-year period is presented. CASE: A 53-year-old G3P3 female presented with a verrucous carcinoma of the vulva and a serous papillary adenocarcinoma of the left fallopian tube metachronously. To investigate a possible association between the co-occurrence of the rare neoplasms and factors associated with multiple gynecological malignancies, we analyzed the status of human papillomavirus infection and DNA mismatch repair deficiency as indicated by microsatellite instability. All samples analyzed were negative for these factors. CONCLUSION: The present results support the possibility that metachronous carcinomas of the vulva and fallopian tube involve unknown etiological factors or arise independently. PMID- 10739715 TI - Recurrent rectoneovaginal fistula caused by an incidental squamous cell carcinoma of the neovagina in Mayer-Rokitansky-Kuster-Hauser syndrome. AB - OBJECTIVE: Mayer-Rokitansky-Kuster-Hauser syndrome is a congenital malformation characterized by an absence of the vagina associated with a variable abnormality of the uterus and the urinary tract but functional ovaries. Surgical correction requires the creation of a neovaginal canal by the performance of a neovaginoplasty and an accurate long-term application of an artificial phallus phantom to avoid secondary shrinkage of the canal. Due to the chronic alteration of the posterior neovaginal wall, ulcers and consecutive fistulae may occur. We report the clinical course of a female who required surgical intervention for a rectoneovaginal fistula and developed a recurrence of the fistula due to one of the extremely rare squamous cell carcinomas of the neovaginal epithelium in order to show potential diagnostic and therapeutic features. METHOD: The systematic report of a case is presented. RESULT: Almost 13 years following the initial construction of a neovagina the patient developed a single-tract rectoneovaginal fistula. After surgical repair she represented with a recurrence due to a vast squamous cell carcinoma of the former operation site. Tumor en bloc resection was performed and currently (follow-up: 4 months) she has no signs of new tumor progression. CONCLUSION: Creation of a neovagina is the standard procedure for treating vaginal atresia or aplasia. Because of the long clinical course postoperatively, complications may occur. This report of a case of a malignant transformation in neovaginal epithelium shows the potential risk of malignancy and underlines the necessity of a close follow-up. PMID- 10739716 TI - Successful preservation of fertility subsequent to a complete pathologic response of a squamous cell carcinoma of the uterine cervix treated with primary systemic chemotherapy. AB - This is the first report about a successful pregnancy following treatment for advanced cervical cancer. PMID- 10739717 TI - Uterine metastasis from a heterologous metaplastic breast carcinoma simulating a primary uterine malignancy. AB - OBJECTIVE: To describe the first distant metastasis of a heterologous metaplastic breast carcinoma in the uterus and discuss its differential diagnosis. METHODS: Light microscopy, immunohistochemistry, and flow cytometry were used to evaluate the tumor. RESULTS: A 58-year-old woman underwent mastectomy for metaplastic breast carcinoma confined to the breast. She presented 4 years later with vaginal bleeding. The endometrial curettage showed a poorly differentiated carcinoma. She underwent hysterectomy and bilateral salpingo-oophorectomy as well as pelvic and periaortic lymphadenectomy. Clinical and intraoperative findings favored a primary uterine malignancy. The uterus was markedly distorted with multiple gray white, solid subserosal, and intramural tumor nodules. The tumor diffusely infiltrated the endometrium sparing benign endometrial glands. The tumor nodules were distributed full thickness of the myometrium. These nodules were composed of high-grade malignant epithelial cells with areas of chondroid metaplasia. Extrauterine microscopic tumor was present in left ovary, pelvic, and periaortic lymph nodes. The histologic features and estrogen/progesterone receptors (ER/PR) as well as DNA ploidy analysis of the uterine tumor showed striking similarity with those of the primary metaplastic breast carcinoma. A diagnosis of metastatic metaplastic breast carcinoma in the uterus was rendered. CONCLUSION: A metastatic heterologous metaplastic breast carcinoma with cartilaginous metaplasia should be considered in the differential diagnosis of heterologous uterine malignant mixed mesodermal tumor (MMMT) and high-grade endometrioid carcinoma with rare foci of cartilage. PMID- 10739718 TI - Choroidal metastasis presented as the initial symptom of the recurrence from ovarian endometrioid adenocarcinoma: A case report. AB - Choroidal metastasis from recurrent ovarian cancer is extremely rare. To our knowledge, this report is the first such case of endometrioid adenocarcinoma. A 30-year-old pregnant woman presented to an ophthalmologist in January 1999 because of a rapid visual deterioration. Ophthalmologic evaluation revealed a metastatic choroidal tumor from ovarian cancer which had been diagnosed 10 years ago. After cesarean section, multiple metastases were found including the choroid, scalp, bone, and lung, and she received four courses of single agent carboplatin chemotherapy. She died of disease in July 1999. This case highlights the need to investigate the etiology of visual complaints in patients with a history of ovarian cancer even in the early stage. PMID- 10739719 TI - The fatal attraction of saphenous venous bypass grafts. PMID- 10739720 TI - Heart failure: we need more trials in typical patients. PMID- 10739721 TI - Intensive insulin treatment of diabetic patients with myocardial infarction is highly cost-effective. PMID- 10739722 TI - What have we learned from the recent large trials in acute coronary syndromes without ST-segment elevation? PMID- 10739723 TI - Percutaneous transluminal septal myocardial ablation: a new treatment for hypertrophic obstructive cardiomyopathy. PMID- 10739724 TI - Coronary angiography and outcome following acute myocardial infarction. PMID- 10739725 TI - The bigger, the better: true also for in-stent restenosis? PMID- 10739726 TI - Cost effectiveness of the implantable cardioverter defibrillator. PMID- 10739727 TI - Electron-beam computed tomography for detection of early signs of coronary arteriosclerosis. PMID- 10739728 TI - Cost-effectiveness of intense insulin treatment after acute myocardial infarction in patients with diabetes mellitus; results from the DIGAMI study. AB - AIMS: The aim of the present analysis was to estimate the cost-effectiveness of intense insulin treatment after acute myocardial infarction in patients with diabetes mellitus based on the results of the Diabetes Mellitus Insulin Glucose Infusion in Acute Myocardial Infarction (DIGAMI) study. In this study 620 patients with diabetes mellitus and acute myocardial infarction were randomized to intense insulin treatment (insulin group) or to serve as controls given standard antidiabetic therapy. Mortality was significantly reduced in the insulin group. METHODS AND RESULTS: The cost-effectiveness ratio was estimated as the incremental cost per life-year and quality-adjusted life-year gained of intense insulin treatment. The incremental costs were estimated as the difference in health care costs and indirect costs (labour production) during the first year of follow-up plus the future costs of increased survival. The life-years gained were based on the 5-year long-term follow-up experience and an assumed annual 20% mortality risk for all patients thereafter. The health care costs were Euro 975 higher in the insulin group during the first year of follow-up, mainly due to a longer period of initial hospitalization related to the institution of multidose insulin. The estimated discounted gain in life-years of the insulin treatment was 0.94 years without and 0.66 with quality of life adjustment, respectively. The cost per life-year gained by intense insulin treatment was Euro 16 900 and the cost per quality-adjusted life-year gained was Euro 24 100. Thus the estimated cost-effectiveness ratios were relatively low. CONCLUSION: The results of the DIGAMI study indicate that intense insulin treatment after an acute myocardial infarction in patients with diabetes mellitus has an acceptable level of cost effectiveness. PMID- 10739729 TI - Differences in use of coronary angiography and outcome of myocardial infarction in Toulouse (France) and Gerona (Spain). The MONICA-Toulouse and REGICOR investigators. AB - AIMS: Differences in the management and organization of health services may account for some of the variability in myocardial infarction case fa tality in different geographic areas. The 28-day outcome was compared i n two regions with similar myocardial infarction incidence and mortality rates that had opposing patients in the use of coronary angiography an d coronary revascularization. METHODS AND RESULTS: The 28-day case fatality of patients aged 35 to 64 years with myocardial infarction, surviving the first hour post-admission to hospitals with coronary care units, was compared in the population of myocardial infarction registries of Toulouse, France and Gerona, Spain. Patient characteristics were similar. In Toulouse, 93% of the 819 registered patients underwent coronary angiography compared with only 6% of the 454 in Gerona. Among hospitalized patients 28-day case fatality was 4.3% and 9.3% in Toulouse and Gerona, respectively (P=0.0003). Rates of thrombolysis and beta-blocker use were higher in Toulouse, although severity indicators were similar to those of Gerona. A model adjusted for these variables showed that the risk of death was 1.90 (95% confidence interval: 1.17-3.07) in Gerona patients compared with those of Toulouse. CONCLUSIONS: Routine angiography use is associated with better 28-day myocardial infarction prognosis than restrictive use. However, the optimum proportion of myocardial infarction patients who have to receive angiography procedures remains unclear. PMID- 10739730 TI - Mortality and repeat interventions up until 20 years after aorto-coronary bypass surgery with saphenous vein grafts. A follow-up study of 1041 patients. AB - AIMS: To determine very long-term survival and incidence of recurrent interventions following aorto-coronary bypass surgery using venous grafts. METHODS AND RESULTS: A group of 1041 consecutive patients operated upon between 1971 and 1980 were followed for a median of 19 years (range 13-26). Peri operative mortality was 1.2%. Survival probability at 5, 10, 15, and 20 years was 92%, 77%, 57%, and 40%, respectively. After 5 or more years following operation the mortality was higher than in the matched Dutch population. Age, extent of coronary artery disease, and ejection fraction are independent predictors of mortality. Of the 593 deceased patients at least 63% died of a probable cardiac cause, while cardiovascular mortality is 40% in the general Dutch population. Repeat revascularization procedures (aorto-coronary bypass surgery or percutaneous transluminal coronary angioplasty) were performed in 343 patients (33%), with an increasing incidence after 7 years. CONCLUSION: Aorto-coronary bypass surgery using vein grafts is safe and has a reasonable long-term prognosis for survival, although less than a matched population. After approximately 7 years both mortality and the need for repeated revascularizations increased. Since a majority of patients died of a cardiac cause and a substantial number of patients required repeated revascularization, aorto-coronary bypass surgery is a palliative treatment of a progressive disease. PMID- 10739731 TI - Predictors of event-free survival after repeat intracoronary procedure for in stent restenosis; study with angiographic and intravascular ultrasound imaging. AB - AIMS: Lumen enlargement during repeat percutaneous coronary intervention for in stent restenosis has been shown to be the result of both stent over-expansion and decrease in neointimal tissue. How these two different mechanisms of action may influence outcome and target lesion revascularization after repeat intervention for in-stent restenosis is unclear. METHODS: Intravascular ultrasound guided repeat intervention for in-stent restenosis was carried out either with balloon angioplasty, or with a combination of rotational atherectomy plus balloon angioplasty. Clinical follow-up at 1 year, including death, myocardial infarction, or need for revascularization, was obtained. RESULTS: Seventy patients were included in this study; 40 were treated by balloon alone, and 30 by combination of rotational atherectomy plus balloon. Event-free survival probability was 76+/-5%. The mechanism of lumen enlargement, be it stent over expansion or tissue removal, had no influence on long-term clinical evolution. The only independent predictor was the minimal lumen cross-sectional area at the end of the procedure, the larger the lumen cross-sectional area, the higher the event-free probability. The cut-off point of the lumen cross-sectional area was set at 4.7 mm(2)by discriminant analysis. Event-free survival was 69+/-15% in patients with <4.7 mm(2)lumen cross-sectional area and 91+/-8% in patients with >4.7 mm(2)lumen cross-sectional area (P=0. 008). CONCLUSIONS: This study showed that the only independent predictor of late clinical outcome after percutaneous re-intervention for in-stent restenosis was final lumen size, no matter which means were used to achieve it. PMID- 10739732 TI - A national survey of heart failure in French hospitals. The Myocardiopathy and Heart Failure Working Group of the French Society of Cardiology, the National College of General Hospital Cardiologists and the French Geriatrics Society. AB - Rationale The epidemiology of heart failure and patient management procedures may vary from one country to another. This study was designed to analyse the spectrum of patients hospitalized in France for heart failure. Patients and Methods A registry involving 120 departments (cardiology, general medicine and geriatrics) and 1058 patients.Results The patients' median age was 76 years, and the male/female ratio was 55/45. Echocardiography was performed in 77% of the cases: the left ventricular ejection fraction was <30% in 22%, between 30 and 40% in 25% and greater than 40% in 53%. Angiotensin-converting enzyme (ACE) inhibitors were prescribed to, respectively, 78% and 63% of patients with ejection fractions above and below 40%. Conclusion In France, patients hospitalized for heart failure tend to be old and are often women; about one in two have relatively preserved left ventricular systolic function. ACE inhibitor prescription seems to be more frequent than currently reported. This survey confirms the clear gap between the populations in clinical trials and those in routine clinical practice. PMID- 10739733 TI - Left ventricular outflow tract gradient decrease with non-surgical myocardial reduction improves exercise capacity in patients with hypertrophic obstructive cardiomyopathy. AB - OBJECTIVES: This study was undertaken to evaluate mid-term clinical results of non-surgical myocardial reduction in patients with hypertrophic obstructive cardiomyopathy. METHODS: Twenty-five patients with left ventricular outflow tract obstruction (mean gradient of 84. 54+/-31.38 mmHg) and symptoms of dyspnoea, angina and/or syncope were treated with non-surgical myocardial reduction. The patients were followed-up for a mean period of 10.44+/-1.8 months. In all patients clinical examination with echocardiography was repeated after every 3 months of follow-up, and a symptom-limited treadmill test was repeated at the 6 month follow-up. Eighteen patients underwent simultaneous respiratory gas analysis. RESULTS: Clinical follow-up examinations were achieved in all 25 patients. Persistent left ventricular outflow tract gradient reduction was seen in 23 patients. Seventeen patients had a reduction of left ventricular outflow tract gradient >50% of baseline value. Twenty patients showed a clinical improvement from 2.8+/-0.5 up to 1.2+/-0.5 NYHA class (P<0.001). The clinical improvement was matched by an improvement in objective measures of exercise capacity in patients with significant left ventricular outflow tract gradient reduction. Exercise time increased from 571.9+/-192.2 to 703.5+/-175.4 s, P<0. 001, and peak VO(2)increased from 14.6+/-5.2 to 20.5+/-8.6 ml. kg(-1)min(-1), P<0.05. CONCLUSION: Significant left ventricular outflow tract gradient reduction with exercise capacity improvement was achieved in the majority of patients treated with non-surgical myocardial reduction. We recommend this method as an alternative to surgery for symptomatic patients with hypertrophic obstructive cardiomyopathy. PMID- 10739735 TI - TNF-alpha system in CHF. A reply. PMID- 10739734 TI - Medical history of hypercholesterolaemia adversely affects the outcome of out-of hospital cardiopulmonary resuscitation; the 'Shahal' experience in Israel. AB - AIMS: To evaluate the impact selected risk factors for cardiac death may have on the success rate in a large cohort of subscribers to 'SHAHAL' who were resuscitated from out-of-hospital cardiac arrest. METHODS AND RESULTS: In this medical facility currently serving 50 000 subscribers, data were prospectively gathered from between 1987-1998. The information retrieved from the patients' medical records included a medical history of hypertension, diabetes, hypercholesterolaemia (>220.mg. dl(-1)) smoking, angina, previous myocardial infarction, and congestive heart failure. A total of 998 patients aged 74+/-12 years (mean+/-1 SD) were included. Death was announced at the scene for 659 (66%) victims, while 339 (34%) patients were taken to hospital. Of these 140 (14% of the total cohort) survived and were discharged from the hospital. A comparison of various selected parameters between survivors and non-survivors of resuscitation revealed that survivors were younger, had a higher rate of pulseless ventricular tachycardia/ventricular fibrillation, more were among the arrests witnessed by the 'SHAHAL' team, and that more had a shorter time lag to initiation of cardiopulmonary resuscitation than non-survivors. None of the studied risk factors predicted the outcome of cardiopulmonary resuscitation, with the exception of hypercholesterolaemia, which carried a significantly worse prognosis for cardiopulmonary resuscitation (P=0.009). CONCLUSIONS: A medical history of hypercholesterolaemia appears to be an important risk factor which adversely affects the outcome of cardiopulmonary resuscitation. PMID- 10739737 TI - Myocardial perfusion imaging and pilot certification. PMID- 10739736 TI - TNF-alpha system in CHF. PMID- 10739738 TI - ESC News and Appointments. PMID- 10739739 TI - Testing methods for developmental neurotoxicity of environmental chemicals. AB - Human brain development is slow and delicate, involving many unique, though interrelated, cellular events. The fetus and child are often more susceptible to chemical toxins that alter the structure and/or function of the brain, although susceptibility varies for individual neurotoxicants. Early exposure to neurotoxins has been implicated in neurological diseases and mental retardation. Pesticide exposures pose a particular concern since many are designed to be neurotoxic to pests and can also affect humans. Acknowledging the potential for vulnerability of the developing brain, EPA recently began to "call in" data on developmental neurotoxicity (DNT) from manufacturers of pesticides already registered and considered to be neurotoxic-around 140 pesticides. Chemicals are to be tested following the DNT testing guideline (OPPTS 870.6300). This paper assesses whether tests performed according to this guideline can effectively identify developmental neurotoxicants. We found the testing guideline deficient in several respects, including: It is not always triggered appropriately within the current tiered system for testing; It does not expose developing animals during all critical periods of vulnerability; It does not assess effects that may become evident later in life; It does not include methodology for consideration of pharmacokinetic variables; Methodology for assessment of neurobehavioral, neuropathological, and morphometry is highly variable; Testing of neurochemical changes is limited and not always required. We propose modifications to the EPA testing guideline that would improve its adequacy for assessing and predicting risks to infants and children. This paper emphasizes that deficiencies in the testing methodology for developmental neurotoxicants represent a significant gap and increase the uncertainty in the establishment of safe levels of exposure to developing individuals. PMID- 10739740 TI - Evidence for basolateral uptake of cadmium in the kidneys of rats. AB - In three separate sets of studies, the effects of ureteral ligation and coadministration of cadmium with cysteine or glutathione (GSH) (in either a 4:1 or 2:1 ratio of thiol to cadmium) on the renal disposition of cadmium were assessed in rats 1 h after the administration of cadmium. In all experiments, co administration of cadmium with either cysteine or GSH caused the renal accumulation of cadmium to increase significantly (by approximately 60-70%) 1 h after injection. Moreover, in all experiments in which both ureters had been ligated in a rat prior to the administration of cadmium, the net total renal accumulation of cadmium was only about 20% less than that in control animals that had not undergone bilateral ureteral ligation when cadmium was administered as cadmium chloride. Furthermore, in animals in which only one ureter had been ligated, the net accumulation of cadmium in the kidney whose ureter had been ligated was between 25 and 30% less than that in the contralateral kidney. Coadministration of cadmium with cysteine or GSH also caused the net accumulation of cadmium to be increased in rats whose ureter(s) had been ligated. Overall, the present findings indicate that there is a significant basolateral component in the acute, in vivo, renal tubular uptake of cadmium. Moreover, the findings indicate that the basolateral uptake of cadmium is enhanced when cadmium is coadministered with cysteine or GSH. PMID- 10739741 TI - TCDD induces CYP1A4 and CYP1A5 in chick liver and kidney and only CYP1A4, an enzyme lacking arachidonic acid epoxygenase activity, in myocardium and vascular endothelium. AB - The toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other Ah receptor ligands, species differences in sensitivity and the relationship of CYP1A induction to the toxicity, are poorly understood. Ah receptor ligands induce formation of CYP1A1 and 1A2 in mammals and of a different set of enzymes, CYP1A4 and 1A5, in chicks. We examined induction by TCDD of CYP1A4 and 1A5 mRNA and protein in chick embryo liver, heart, kidney, lung, intestine, bursa, spleen, thymus, brain, and muscle by in situ hybridization and immunohistochemistry and verified the histochemical findings by CYP-specific assays, 7-ethoxyresorufin deethylase for CYP1A4 and arachidonic acid epoxygenation for CYP1A5. CYP1A4 alone was extensively induced in the cardiovascular system, in cardiac myocytes, in perivascular cells having the same location as impulse-conducting Purkinje cells, and like CYP1A1, in vascular endothelium in every organ examined. Unlike mammalian CYP1A, CYP1A4 and 1A5 were both substantially induced in kidney proximal tubules as well as liver, and neither enzyme was induced in kidney glomeruli or lung or brain parenchymal cells. The findings demonstrate (a) a route for CYP1A4 to affect cardiac function, (b) that vascular endothelium is a major site of CYP1A induction across species, and (c) that CYP1A induced in heart or endothelial cells cannot affect cardiac or vascular function via generation of arachidonic acid epoxides because the CYP1A enzymes induced in those organs are not arachidonic acid epoxygenases. Further, the specificity of CYP1A induction sites and of the catalytically active enzymes induced at each site support a significant role for CYP1A induction in Ah receptor ligand toxicity and species differences in sensitivity. PMID- 10739742 TI - Use of human skin equivalent Apligraf for in vitro assessment of cumulative skin irritation potential of topical products. AB - The main goal of the present study was to investigate the response of the human skin equivalent Apligraf in vitro to the application of irritant substances and its predictivity as a screening tool for cumulative skin irritant potential in humans. Vaseline, calcipotriol, trans-retinoic acid, and sodium lauryl sulfate were applied to Apligraf in vitro for 24 h. Cell viability (lactate dehydrogenase leakage), release and mRNA expression of the proinflammatory cytokines IL-1alpha and IL-8, and morphological changes were assessed. The same products were applied to 30 healthy volunteers in a double-blind, randomized, vehicle-controlled within subject study. The skin reactions after repeated 24-h applications over 3 weeks under Finn chamber patches were monitored by visual scoring and biophysical methods (trans-epidermal water loss, chromametry, and blood flow). Sodium lauryl sulfate was cytotoxic to Apligraf, and increased the release and expression of cytokines at low (0.2%, 0. 4%), but not at high (0.8%, 1%) concentrations. It induced severe irritancy in vivo. Trans-retinoic acid increased the expression and release of cytokines with no detectable cytotoxicity and showed moderate irritancy in humans. Although calcipotriol did neither affect cell viability nor the production of cytokines, it induced morphological signs of irritation and was mildly irritant for healthy volunteers. Vaseline was innocuous in vivo and induced no changes in Apligraf. In conclusion, the cumulative skin irritation potential of the tested products could be predicted with Apligraf in a sensitive and specific manner, by monitoring cytotoxicity, proinflammatory cytokines, and morphological changes. PMID- 10739743 TI - Contribution of direct solvent injury to the dose-dependent kinetics of trichloroethylene: portal vein administration to rats. AB - Presystemic elimination of trichloroethylene (TCE), a common contaminant of drinking water, has been shown by Lee et al. (Toxicol. Appl. Pharmacol. 139, 262 271, 1996) to be inversely related to dose. When relatively high doses were administered to rats via the portal vein (PV), first-pass hepatic extraction became negligible. This phenomenon could result not only from metabolic saturation, but from suicidal destruction of cytochromes P450 and hepatocellular injury as well. The objectives of the current investigation were to: (a) clarify the relative roles of P450 depletion and hepatocellular toxicity in the apparent cessation of hepatic elimination of TCE in animals given relatively high doses of TCE via the PV; and (b) investigate mechanism(s) of hepatocellular injury under such exposure conditions. TCE (16 and 64 mg/kg body weight (bw) was incorporated into a 5% aqueous Alkamuls emulsion and injected via an indwelling jugular vein (JV) or PV cannula into male Sprague-Dawley rats. Some animals received 73.5 micromol/kg of p-nitrophenol (PNP), a competitive metabolic inhibitor of TCE, through the PV cannula 3 min before TCE. Administration of TCE via the PV resulted in deposition of relatively high levels of TCE in the liver. PV dosing resulted in lower total hepatic P450 levels than did JV dosing. PV dosing produced marked elevations of cytoplasmic enzymes in serum, but JV dosing did not. Decreases in hepatic P450 were not selective for cytochrome P4502E1. Histological examination of the liver of PV-dosed rats revealed periportal rather than centrilobular necrosis. PNP pretreatment failed to prevent the increase in serum enzymes, decrease in hepatic P450 content, and hepatic necrosis following PV TCE. It is concluded that PV injection of bolus doses of TCE >/= 16 mg/kg causes liver injury within minutes in rats, primarily through direct solvent action on hepatocellular membranes rather than by P450-mediated effects. This liver damage likely plays a modest role in reducing the liver's capacity to metabolize high PV doses of TCE. PMID- 10739744 TI - Mechanisms of the dose-dependent kinetics of trichloroethylene: oral bolus dosing of rats. AB - Trichloroethylene (TCE), a common contaminant of drinking water, is oxidized by high-affinity, low-capacity cytochrome P450 isozymes and subsequently converted to metabolites, some of which are carcinogenic in mice and rats. Although the initial oxidation step is known to be rate-limiting and saturable, the oral dosage-range over which saturation materializes is unclear. One objective of this study was to characterize the dose-dependency of gastrointestinal (GI) absorption of TCE and its kinetics over a wide range of oral bolus doses. A related objective was to investigate cause(s) of the apparent saturation kinetics observed. Cannulas were surgically implanted into a carotid artery and the stomach of male Sprague-Dawley rats. TCE was incorporated into a 5% aqueous Alkamuls emulsion and given in doses of 2 to 1200 mg/kg bw via the stomach tube. Serial blood samples were taken from the arterial cannula for up to 14 h postdosing and analyzed for TCE content by headspace gas chromatography. The rate of GI absorption of TCE diminished as the dosage increased. Pharmacokinetic analysis indicated that TCE was eliminated by capacity-limited hepatic metabolism, with incursion into nonlinear kinetics with bolus doses >/=8 to 16 mg/kg. Effects of p-nitrophenol, a competitive metabolic inhibitor, were manifest at a high, but not at a low TCE dose. Gavage bolus doses as high as 1200 mg/kg did not cause rapid elevation of serum enzyme levels, typical of the solvation of hepatocellular membranes observed after portal vein administration of TCE (Lee et al., Toxicol. Appl. Pharmacol. 163, 000-000, 2000). No evidence of cytochrome P4502E1 (CYP2E1) destruction was seen with oral doses up to 1000 mg/kg. Instead, CYP2E1 activity was induced as early as 1 h postdosing. Induction was maximal at 12 h, then returned toward controls during the next 12 h. Pretreatment with cycloheximide did not reduce CYP2E1 activity in rats given 432 or 1000 mg TCE/kg, suggesting that binding of TCE to CYP2E1 may stabilize the isozyme. Metabolic saturation, in concert with relatively slow GI absorption, are responsible for the prolonged elevation of blood TCE levels in rats given high TCE doses, while suicidal inactivation of CYP2E1 and hepatocellular injury apparently play little role. PMID- 10739745 TI - Acute and chronic nicotine exposures modulate the immune system through different pathways. AB - We have previously shown that T cells from rats exposed chronically to cigarette smoke or nicotine (NT) exhibit T cell anergy and decreased proliferation to T cell mitogens. Effects of chronic NT on T cell function persist for at least 2 weeks after the termination of NT treatment. Moreover, these effects of NT are causally related to the decreased Ca(2+) response to T cell receptor (TCR) ligation and constitutive activation of protein tyrosine kinase (PTK) and phospholipase C (PLC)-gamma1 activities. Acute NT treatment also suppresses the Con A-induced T cell proliferation; however, it is not known whether the mechanism(s) by which acute and chronic NT treatments inhibit T cell proliferation are identical. To evaluate this question, LEW rats were acutely treated with NT (1 mg/kg body wt) for 1, 2, or 24 h by an ip injection or implanted with constant-release miniosmotic pumps containing saline or NT (1 mg/kg body wt/day) for a 3-week chronic exposure. Inhibition of Con A-induced proliferation of peripheral blood cells (PBC) by both acute and chronic treatments was reversed by the inhibitor of nicotinic acetylcholine receptors, mecamylamine (MEC), indicating that these receptors are required for T cell proliferation. However, the effect of acute NT on the Con A response was short lived (i.e., observed at 1 and 2 h but not at 24 h after NT administration) and was seen in PBC but not in spleen cells. Unlike the chronic treatment, acute NT administration neither suppressed significantly the TCR-mediated [Ca(2+)](i) response nor did it cause the constitutive activation of PTK and PLC-gamma1 activities in blood lymphocytes. Acute, but not chronic, NT administration increased the plasma corticosterone concentration, and this increase was also inhibited by MEC. Moreover, adrenalectomy abrogated the acute but not chronic NT effects on the Con A response. Thus, the acute and chronic effects of NT on T lymphocytes are mechanistically distinct phenomena. Whereas chronic administration of NT causes T cell anergy, acute effects are primarily mediated via the activation of the hypothalamus-pituitary-adrenal axis. PMID- 10739746 TI - Pathological and biochemical characterization of microcystin-induced hepatopancreas and kidney damage in carp (Cyprinus carpio). AB - Mass occurrences of cyanobacteria, due to their inherent capacity for toxin production, specifically of microcystins (MC), have been associated with fish kills worldwide. The uptake of MC-LR and the sequence of pathological and associated biochemical changes was investigated in carp (Cyprinus carpio) in vivo over 72 h. Carp were gavaged with a single sublethal bolus dose of toxic Microcystis aeruginosa (PCC 7806) amounting to an equivalent of 400 microg MC LR/kg body wt. Damage of renal proximal tubular cells and hepatocytes was observed as early as 1 h, followed by pathological changes in the intestinal mucosa at approximately 12 h postdosing. These alterations were characterized in hepatopancreas by a dissociation of hepatocytes, an early onset of apoptotic cell death, and delayed cell lysis. In the renal proximal tubules (P2) observations included increased vacuolation of individual tubular epithelial cells, apoptosis, cell shedding, and finally proteinaceous casts at the cortico-medullary junction. Concurrently with the pathological alterations, MC-immunopositive staining was observed in hepatocytes and the proximal tubular cells; the staining increasing in the hepatopancreas in intensity with increasing time postdosing. The presence of apoptotic cell death was determined using in situ fragment end labeling (ISEL) of the respective tissue sections and agarose gel electrophoresis for detection of DNA-laddering. The analysis of carp tissue extracts (hepatopancreas, kidney, GI tract, skeletal muscle, brain, heart, spleen, and gills) demonstrated MC-LR adducts having molecular weights of 38 kDa (putatively catalytic subunit of protein phosphatases-1 and -2A) and 28 kDa, respectively. An additional band was found to be present at 23 kDa in both hepatopancreas and kidney. The present data demonstrate that, in comparison to the pathological events in salmonids exposed to MC, where a slower development of pathology and primarily necrotic cell death prevails, the pathology in carp develops rapidly and at lower toxin concentrations. This is most likely due to a more efficient uptake of toxin, while the mechanism of cell death is primarily apoptosis. PMID- 10739747 TI - Growth inhibition, cell-cycle dysregulation, and induction of apoptosis by green tea constituent (-)-epigallocatechin-3-gallate in androgen-sensitive and androgen insensitive human prostate carcinoma cells. AB - Prostate cancer (PCA) is the most prevalent cancer diagnosed and the second leading cause of cancer-related deaths among men in the United States. Descriptive epidemiological data suggest that androgens and environmental exposures play a key role in prostatic carcinogenesis. Since androgen action is intimately associated with proliferation and differentiation, at the time of clinical diagnosis in humans most PCA represent themselves as a mixture of androgen-sensitive and androgen-insensitive cells. Androgen-sensitive cells undergo rapid apoptosis upon androgen withdrawal. On the other hand, the androgen insensitive cells do not undergo apoptosis upon androgen blocking, but maintain the molecular machinery of apoptosis. Thus, agents capable of inhibiting growth and/or inducing apoptosis in both androgen-sensitive and androgen-insensitive cells will be useful for the management of PCA. In the present study, we show that (-)-epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent present in green tea, imparts antiproliferative effects against both androgen sensitive and androgen-insensitive human PCA cells, and this effect is mediated by deregulation in cell cycle and induction of apoptosis. EGCG treatment was found to result in a dose-dependent inhibition of cell growth in both androgen insensitive DU145 and androgen-sensitive LNCaP cells. In both the cell types, EGCG treatment also resulted in a dose-dependent G(0)/G(1)-phase arrest of the cell cycle as observed by DNA cell-cycle analysis. As evident by DNA ladder assay, confocal microscopy, and flow cytometry, the treatment of both DU145 and LNCaP cells with EGCG resulted in a dose-dependent apoptosis. Western blot analysis revealed that EGCG treatment resulted in (i) a dose-dependent increase of p53 in LNCaP cells (carrying wild-type p53), but not in DU145 cells (carrying mutant p53), and (ii) induction of cyclin kinase inhibitor WAF1/p21 in both cell types. These results suggest that EGCG negatively modulates PCA cell growth, by affecting mitogenesis as well as inducing apoptosis, in cell-type-specific manner which may be mediated by WAF1/p21-caused G(0)/G(1)-phase cell-cycle arrest, irrespective of the androgen association or p53 status of the cells. PMID- 10739748 TI - Epigenetic properties of fumonisin B(1): cell cycle arrest and DNA base modification in C6 glioma cells. AB - Fumonisin B(1) produced by the fungus Fusarium moniliforme is a member of a new class of sphinganine analogue mycotoxins that occur widely in the food chain. Epidemiological studies associate FB(1) with human oesophageal cancer in China and South Africa. FB(1) also causes acute pulmonary edema in pigs and equine leucoencephalomalacia. This disease is thought to be a consequence of inhibition by FB(1) of cellular ceramide synthesis in cells. To investigate further on this pathogenesis, the effect of FB(1) was studied on cell viability (3 to 54 microM of FB(1)), protein (2.5 to 20 microM of FB(1)) and DNA syntheses (2.5 to 50 microM of FB(1)), and cellular cycle (3 to 18 microM of FB(1)) of rat C6 glioma cells after 24 h incubation. The results of the viability test show that FB(1) induces 10 +/- 2% and 47 +/- 4% cell death with, respectively, 3 and 54 microM, in C6 cells. This cytotoxicity induced by FB(1) was efficiently prevented when the cells were preincubated 24 h with vitamin E (25 microM). FB(1) displays epigenetic properties since it induced hypermethylation of the DNA (9-18 microM). Inhibition of protein synthesis was observed with FB(1) with an IC(50) of 6 microM showing that C6 glioma cells are very sensitive to FB(1); however, the synthesis of DNA was only slightly inhibited, up to 20 microM of FB1. The flow cytometry showed that the number of cells in phase S decreased significantly as compared to the control p = 0.01 from 18. 7 +/- 2.5% to 8.1 +/- 1.1% for 9 microM FB(1). The number of cells in phase G(2)/M increased significantly as compared to the control (p /=125 microM of the hydrophobic bile acid, glycochenodeoxycholic acid (GCDC), underwent a time- and dose-dependent decrease of intracellular GSH levels by 4-h incubation. This loss of intracellular GSH was not associated with an increase of intracellular GSH disulfide (GSSG). Rather, GCDC stimulated the dose dependent accumulation of extracellular GSSG. The mechanism for extracellular GSSG accumulation by GCDC was through increased efflux of reduced GSH from hepatocytes into the media, where it subsequently oxidized to GSSG. Treatment of hepatocytes with GCDC (0-750 microM) did not directly alter GSH-dependent enzyme activities. The reduction of intracellular GSH with 125 microM GCDC correlated with extensive apoptosis at this concentration as determined by fluorescence microscopy of DAPI (4, 6-diamindino-2-phenylindole hydrochloride)-stained nuclei. Higher concentrations of GCDC (>/=500 microM) favored cellular necrosis and lipid peroxidation. Depleting GSH by treating hepatocytes with 1-bromoheptane increased their sensitivity toward GCDC-induced cellular necrosis, but not apoptosis. However, enhancing the hepatocyte GSH content by supplementation with GSH ethylester (GSH-EE) failed to protect hepatocytes against either mode of cellular death. In conclusion, while GCDC-induced cytotoxicities were associated with an increased efflux of GSH from rat hepatocytes, GSH status modulated GCDC-induced necrosis, but not apoptosis. PMID- 10739751 TI - Mutational and haplotype analyses of families with familial partial lipodystrophy (Dunnigan variety) reveal recurrent missense mutations in the globular C-terminal domain of lamin A/C. AB - Familial partial lipodystrophy (FPLD), Dunnigan variety, is an autosomal dominant disorder characterized by marked loss of subcutaneous adipose tissue from the extremities and trunk but by excess fat deposition in the head and neck. The disease is frequently associated with profound insulin resistance, dyslipidemia, and diabetes. We have localized a gene for FPLD to chromosome 1q21-q23, and it has recently been proposed that nuclear lamin A/C is altered in FPLD, on the basis of a novel missense mutation (R482Q) in five Canadian probands. This gene had previously been shown to be altered in autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD-AD) and in dilated cardiomyopathy and conduction-system disease. We examined 15 families with FPLD for mutations in lamin A/C. Five families harbored the R482Q alteration that segregated with the disease phenotype. Seven families harbored an R482W alteration, and one family harbored a G465D alteration. All these mutations lie within exon 8 of the lamin A/C gene-an exon that has also been shown to harbor different missense mutations that are responsible for EDMD-AD. Mutations could not be detected in lamin A/C in one FPLD family in which there was linkage to chromosome 1q21-q23. One family with atypical FPLD harbored an R582H alteration in exon 11 of lamin A. This exon does not comprise part of the lamin C coding region. All mutations in FPLD affect the globular C-terminal domain of the lamin A/C protein. In contrast, mutations responsible for dilated cardiomyopathy and conduction-system disease are observed in the rod domain of the protein. The FPLD mutations R482Q and R482W occurred on different haplotypes, indicating that they are likely to have arisen more than once. PMID- 10739752 TI - Similar splice-site mutations of the ATP7A gene lead to different phenotypes: classical Menkes disease or occipital horn syndrome. AB - More than 150 point mutations have now been identified in the ATP7A gene. Most of these mutations lead to the classic form of Menkes disease (MD), and a few lead to the milder occipital horn syndrome (OHS). To get a better understanding of molecular changes leading to classic MD and OHS, we took advantage of the unique finding of three patients with similar mutations but different phenotypes. Although all three patients had mutations located in the splice-donor site of intron 6, only two of the patients had the MD phenotype; the third had the OHS phenotype. Fibroblast cultures from the three patients were analyzed by reverse transcriptase (RT)-PCR to try to find an explanation of the different phenotypes. In all three patients, exon 6 was deleted in the majority of the ATP7A transcripts. However, by RT-PCR amplification with an exon 6-specific primer, we were able to amplify exon 6-containing mRNA products from all three patients, even though they were in low abundance. Sequencing of these products indicated that only the patient with OHS had correctly spliced exon 6-containing transcripts. We used two different methods of quantitative RT-PCR analysis and found that the level of correctly spliced mRNA in this patient was 2%-5% of the level found in unaffected individuals. These findings indicate that the presence of barely detectable amounts of correctly spliced ATP7A transcript is sufficient to permit the development of the milder OHS phenotype, as opposed to classic MD. PMID- 10739753 TI - Manitoba aboriginal kindred with original cerebro-oculo- facio-skeletal syndrome has a mutation in the Cockayne syndrome group B (CSB) gene. AB - Cerebro-oculo-facio-skeletal (COFS) syndrome is a rapidly progressive neurological disorder leading to brain atrophy with calcification, cataracts, microcornea, optic atrophy, progressive joint contractures, and growth failure. Cockayne syndrome (CS) is a recessively inherited neurodegenerative disorder characterized by low-to-normal birth weight; growth failure; brain dysmyelination with calcium deposits; cutaneous photosensitivity; pigmentary retinopathy, cataracts, or both; and sensorineural hearing loss. CS cells are hypersensitive to UV radiation because of impaired nucleotide excision repair of UV radiation induced damage in actively transcribed DNA. The abnormalities in CS are associated with mutations in the CSA or CSB genes. In this report, we present evidence that two probands related to the Manitoba Aboriginal population group within which COFS syndrome was originally reported have cellular phenotypes indistinguishable from those in CS cells. The identical mutation was detected in the CSB gene from both children with COFS syndrome and in both parents of one of the patients. This mutation was also detected in three other patients with COFS syndrome from the Manitoba Aboriginal population group. These results suggest that CS and COFS syndrome share a common pathogenesis. PMID- 10739754 TI - Homozygosity mapping identifies an additional locus for Wolfram syndrome on chromosome 4q. AB - Wolfram syndrome, which is sometimes referred to as "DIDMOAD" (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness), is an autosomal recessive neurodegenerative disorder for which only insulin-dependent diabetes mellitus and optic atrophy are necessary to make the diagnosis. Researchers have mapped Wolfram syndrome to chromosome 4p16.1, and, recently, a gene encoding a putative transmembrane protein has been cloned and mutations have been identified in patients. To pursue the possibility of locus heterogeneity, 16 patients from four different families were recruited. These patients, who have the Wolfram syndrome phenotype, also have additional features that have not previously been reported. There is an absence of diabetes insipidus in all affected family members. In addition, several patients have profound upper gastrointestinal ulceration and bleeding. With the use of three microsatellite markers (D4S432, D4S3023, and D4S2366) reported to be linked to the chromosome 4p16.1 locus, we significantly excluded linkage in three of the four families. The two affected individuals in one family showed homozygosity for all three markers from the region of linkage on chromosome 4p16.1. For the other three families, genetic heterogeneity for Wolfram syndrome was verified by demonstration of linkage to chromosome 4q22-24. In conclusion, we report the unique clinical findings and linkage-analysis results of 16 patients with Wolfram syndrome and provide further evidence for the genetic heterogeneity of this disorder. We also provide data on a new locus that plays a role in the etiology of insulin-dependent diabetes mellitus. PMID- 10739755 TI - Effects of HFE C282Y and H63D polymorphisms and polygenic background on iron stores in a large community sample of twins. AB - The aim of this study was to assess and to compare the role of HFE polymorphisms and other genetic factors in variation in iron stores. Blood samples were obtained from 3,375 adult male and female twins (age range 29-82 years) recruited from the Australian Twin Registry. There were 1,233 complete pairs (562 monozygotic and 571 dizygotic twins). Serum iron, transferrin, transferrin saturation with iron, and ferritin were measured, and the HFE C282Y and H63D genotypes were determined. The frequency of the C282Y allele was.072, and that of the H63D allele was.141. Significant sources of variation in the indices of iron status included age, sex, age-sex interaction, body-mass index, and both the C282Y and H63D genotypes. The iron, transferrin, and saturation values of CC and CY subjects differed significantly, but the ferritin values did not. After correction for age and body-mass index, 23% and 31% of the variance in iron, 66% and 49% of the variance in transferrin, 33% and 47% of the variance in transferrin saturation, and 47% and 47% of the variance in ferritin could be explained by additive genetic factors, for men and women, respectively. HFE C282Y and H63D variation accounted for <5% of the corrected phenotypic variance, except for saturation (12% in women and 5% in men). We conclude that HFE CY and HD heterozygotes differ in iron status from the CC and HH homozygotes and that serum transferrin saturation is more affected than is serum ferritin. There are highly significant effects of other as-yet-unidentified genes on iron stores, in addition to HFE genotype. PMID- 10739756 TI - BRCA1 and BRCA2 mutation analysis of 208 Ashkenazi Jewish women with ovarian cancer. AB - Ovarian cancer is a component of the autosomal-dominant hereditary breast-ovarian cancer syndrome and may be due to a mutation in either the BRCA1 or BRCA2 genes. Two mutations in BRCA1 (185delAG and 5382insC) and one mutation in BRCA2 (6174delT) are common in the Ashkenazi Jewish population. One of these three mutations is present in approximately 2% of the Jewish population. Each mutation is associated with an increased risk of ovarian cancer, and it is expected that a significant proportion of Jewish women with ovarian cancer will carry one of these mutations. To estimate the proportion of ovarian cancers attributable to founding mutations in BRCA1 and BRCA2 in the Jewish population and the familial cancer risks associated with each, we interviewed 213 Jewish women with ovarian cancer at 11 medical centers in North America and Israel and offered these women genetic testing for the three founder mutations. To establish the presence of nonfounder mutations in this population, we also completed the protein-truncation test on exon 11 of BRCA1 and exons 10 and 11 of BRCA2. We obtained a detailed family history on all women we studied who had cancer and on a control population of 386 Ashkenazi Jewish women without ovarian or breast cancer. A founder mutation was present in 41.3% of the women we studied. The cumulative incidence of ovarian cancer to age 75 years was found to be 6.3% for female first-degree relatives of the patients with ovarian cancer, compared with 2.0% for the female relatives of healthy controls (relative risk 3.2; 95% CI 1.5-6.8; P=.002). The relative risk to age 75 years for breast cancer among the female first-degree relatives was 2.0 (95% CI 1.4-3.0; P=.0001). Only one nonfounder mutation was identified (in this instance, in a woman of mixed ancestry), and the three founding mutations accounted for most of the observed excess risk of ovarian and breast cancer in relatives. PMID- 10739757 TI - A multipoint method for detecting genotyping errors and mutations in sibling-pair linkage data. AB - The identification of genes contributing to complex diseases and quantitative traits requires genetic data of high fidelity, because undetected errors and mutations can profoundly affect linkage information. The recent emphasis on the use of the sibling-pair design eliminates or decreases the likelihood of detection of genotyping errors and marker mutations through apparent Mendelian incompatibilities or close double recombinants. In this article, we describe a hidden Markov method for detecting genotyping errors and mutations in multilocus linkage data. Specifically, we calculate the posterior probability of genotyping error or mutation for each sibling-pair-marker combination, conditional on all marker data and an assumed genotype-error rate. The method is designed for use with sibling-pair data when parental genotypes are unavailable. Through Monte Carlo simulation, we explore the effects of map density, marker-allele frequencies, marker position, and genotype-error rate on the accuracy of our error-detection method. In addition, we examine the impact of genotyping errors and error detection and correction on multipoint linkage information. We illustrate that even moderate error rates can result in substantial loss of linkage information, given efforts to fine-map a putative disease locus. Although simulations suggest that our method detects cysteine substitution in type I collagen, localized at position 134 of the alpha1(I) collagen chain. The arginine residue is highly conserved and localized in the X position of the Gly-X-Y triplet. As a consequence, intermolecular disulfide bridges are formed, resulting in type I collagen aggregates, which are retained in the cells. Whereas substitutions of glycine residues in type I collagen invariably result in osteogenesis imperfecta, substitutions of nonglycine residues in type I collagen have not yet been associated with a human disease. In contrast, arginine-->cysteine substitutions in type II collagen have been identified in a variety of chondrodysplasias. Our findings show that mutations in other fibrillar collagens can be causally involved in classical EDS and point to genetic heterogeneity of this disorder. PMID- 10739763 TI - Familial posterior fossa brain tumors of infancy secondary to germline mutation of the hSNF5 gene. AB - We have identified a family afflicted over multiple generations with posterior fossa tumors of infancy, including central nervous system (CNS) malignant rhabdoid tumor (a subset of primitive neuroectodermal tumors, or PNET) and choroid plexus carcinoma. Various hereditary tumor syndromes, including Li Fraumeni syndrome, Gorlin syndrome, and Turcot syndrome, have been linked to increased risk of developing CNS PNETs and choroid plexus tumors. Malignant rhabdoid tumors of the CNS and kidney show loss of heterozygosity at chromosome 22q11. The hSNF5 gene on chromosome 22q11 has recently been identified as a candidate tumor-suppressor gene in sporadic CNS and renal malignant rhabdoid tumors. We describe a family in which both affected and some unaffected family members were found to have a germline splice-site mutation of the hSNF5 gene, leading to exclusion of exon 7 from the mature cDNA and a subsequent frameshift. Tumor tissue shows loss of the wild-type hSNF5 allele, in keeping with a tumor suppressor gene. These findings suggest that germline mutations in hSNF5 are associated with a novel autosomal dominant syndrome with incomplete penetrance that predisposes to malignant posterior fossa brain tumors in infancy. PMID- 10739766 TI - Surnames and the Y chromosome. AB - A randomly ascertained sample of males with the surname "Sykes" was typed with four Y-chromosome microsatellites. Almost half the sample shared the same Y chromosome haplotype, which has not been observed in control samples either from the same geographic region or from the United Kingdom as a whole. This points to a single surname founder for extant Sykes males, even though written sources had predicted multiple origins. The distribution of other Sykes Y-chromosome haplotypes were not significantly different from those in controls and may be accounted for by the historical accumulation of nonpaternity during the past 700 years, in which case the average rate estimate is 1.3%/generation. If this pattern is reproduced with other surnames, it may have important forensic and genealogical applications. PMID- 10739764 TI - Different mutations in the LMNA gene cause autosomal dominant and autosomal recessive Emery-Dreifuss muscular dystrophy. AB - Emery-Dreifuss muscular dystrophy (EMD) is a condition characterized by the clinical triad of early-onset contractures, progressive weakness in humeroperoneal muscles, and cardiomyopathy with conduction block. The disease was described for the first time as an X-linked muscular dystrophy, but autosomal dominant and autosomal recessive forms were reported. The genes for X-linked EMD and autosomal dominant EMD (AD-EMD) were identified. We report here that heterozygote mutations in LMNA, the gene for AD-EMD, may cause diverse phenotypes ranging from typical EMD to no phenotypic effect. Our results show that LMNA mutations are also responsible for the recessive form of the disease. Our results give further support to the notion that different genetic forms of EMD have a common pathophysiological background. The distribution of the mutations in AD-EMD patients (in the tail and in the 2A rod domain) suggests that unique interactions between lamin A/C and other nuclear components exist that have an important role in cardiac and skeletal muscle function. PMID- 10739765 TI - Segregation analysis in Shwachman-Diamond syndrome: evidence for recessive inheritance. AB - Shwachman-Diamond syndrome is a rare disorder of unknown cause. Reports have indicated the occurrence of affected siblings, but formal segregation analysis has not been performed. In families collected for genetic studies, the mean paternal age and mean difference in parental ages were found to be consistent with the general population. We determined estimates of segregation proportion in a cohort of 84 patients with complete sibship data under the assumption of complete ascertainment, using the Li and Mantel estimator, and of single ascertainment with the Davie modification. A third estimate was also computed with the expectation-maximization (EM) algorithm. All three estimates supported an autosomal recessive mode of inheritance, but complete ascertainment was found to be unlikely. Although there are no overt signs of disease in adult carriers (parents), the use of serum trypsinogen levels to indicate exocrine pancreatic dysfunction was evaluated as a potential measure for heterozygote expression. No consistent differences were found in levels between parents and a normal control population. Although genetic heterogeneity cannot be excluded, our results indicate that simulation and genetic analyses of Shwachman-Diamond syndrome should consider a recessive model of inheritance. PMID- 10739767 TI - Primary, nonsyndromic vesicoureteric reflux and its nephropathy is genetically heterogeneous, with a locus on chromosome 1. AB - Primary vesicoureteric reflux (VUR) affects 1%-2% of whites, and reflux nephropathy (RN) causes up to 15% of end-stage renal failure in children and adults. There is a 30-50-fold increased incidence of VUR in first-degree relatives of probands, compared with the general population. We report the results of the first genomewide search of VUR and RN; we studied seven European families whose members exhibit apparently dominant inheritance. We initially typed 387 polymorphic markers spaced, on average, at 10 cM throughout the genome; we used the GENEHUNTER program to provide parametric and nonparametric linkage analyses of affected individuals. The most positive locus spanned 20 cM on 1p13 between GATA176C01 and D1S1653 and had a nonparametric LOD score (NPL) of 5.76 (P=.0002) and a parametric LOD score of 3.16. Saturation with markers at 1-cM intervals increased the NPL to 5.94 (P=.00009). Hence, VUR maps to a locus on chromosome 1. There was evidence of genetic heterogeneity at the chromosome 1 locus, and 12 additional loci were identified genomewide, with P<.05. No significant linkage was found to 6p, where a renal and ureteric malformation locus has been reported, or to PAX2, mutations of which cause VUR in renal coloboma syndrome. Our results support the hypothesis that VUR is a genetic disorder. PMID- 10739768 TI - Autosomal-dominant congenital cataract associated with a deletion mutation in the human beaded filament protein gene BFSP2. AB - Congenital cataracts are a common major abnormality of the eye that frequently cause blindness in infants. At least one-third of all cases are familial; autosomal-dominant congenital cataract appears to be the most-common familial form in the Western world. Elsewhere, in family ADCC-3, we mapped an autosomal dominant cataract gene to chromosome 3q21-q22, near the gene that encodes a lens specific beaded filament protein gene, BFSP2. By sequencing the coding regions of BFSP2, we found that a deletion mutation, DeltaE233, is associated with cataracts in this family. This is the first report of an inherited cataract that is caused by a mutation in a cytoskeletal protein. PMID- 10739769 TI - A novel locus (DFNA24) for prelingual nonprogressive autosomal dominant nonsyndromic hearing loss maps to 4q35-qter in a large Swiss German kindred. AB - Nonsyndromic hearing loss is one of the most genetically heterogeneous traits known. A total of 30 autosomal dominant nonsyndromic hearing-loss loci have been mapped, and 11 genes have been isolated. In the majority of cases, autosomal dominant nonsyndromic hearing loss is postlingual and progressive, with the exception of hearing impairment in families in which the impairment is linked to DFNA3, DFNA8/12, and DFNA24, the novel locus described in this report. DFNA24 was identified in a large Swiss German kindred with a history of autosomal dominant hearing loss that dates back to the middle of the 19th century. The hearing impaired individuals in this kindred have prelingual, nonprogressive, bilateral sensorineural hearing loss affecting mainly mid and high frequencies. The DFNA24 locus maps to 4q35-qter. A maximum multipoint LOD score of 11.6 was obtained at 208.1 cM at marker D4S1652. The 3.0-unit support interval for the map position of this locus ranges from 205.8 cM to 211.7 cM (5.9 cM). PMID- 10739770 TI - The gene for May-Hegglin anomaly localizes to a <1-Mb region on chromosome 22q12.3-13.1. AB - The May-Hegglin anomaly (MHA) is an autosomal dominant platelet disorder of unknown etiology. It is characterized by thrombocytopenia, giant platelets, and leukocyte inclusion bodies, and affected heterozygotes are predisposed to bleeding episodes. The MHA gene has recently been localized, by means of linkage analysis, to a 13.6-cM region on chromosome 22, and the complete chromosome 22 sequence has been reported. We recently performed a genome scan for the MHA gene in 29 members of a large, multigenerational Italian family, and we now confirm that the MHA locus is on chromosome 22q12. 3-13.1. The maximal two-point LOD score of 4.50 was achieved with the use of marker D22S283, at a recombination fraction of.05. Haplotype analysis narrowed the MHA critical region to 6.6 cM between markers D22S683 and D22S1177. It is of note that the chromosome 22 sequence allowed all markers to be ordered correctly, identified all the candidate genes and predicted genes, and specifically determined the physical size of the MHA region to be 0. 7 Mb. These results significantly narrow the region in which the MHA gene is located, and they represent the first use of chromosome 22 data to positionally clone a disease gene. PMID- 10739771 TI - A novel X-linked dominant condition: X-linked congenital isolated ptosis. AB - We present a large family with a previously undescribed condition: X-linked dominant congenital bilateral isolated ptosis. Linkage analysis defined a critical region between Xq24 and Xq27.1, with a maximum single-point LOD score of 2.88 at DXS1047 and DXS984. Male and female family members are equally affected, providing an example of an X-linked, truly dominant condition. PMID- 10739772 TI - Terminal osseous dysplasia with pigmentary defects maps to human chromosome Xq27.3-xqter. AB - We have identified a four-generation family with 10 affected females manifesting one or more of the following features: osseous dysplasia involving the metacarpals, metatarsals, and phalanges leading to brachydactyly, camptodactyly, and other digital deformities; pigmentary defects on the face and scalp; and multiple frenula. There were no affected males. We performed X-inactivation studies on seven affected females, using a methylation assay at the androgen receptor locus; all seven demonstrated preferential inactivation of their maternal chromosomes carrying the mutation, and two unaffected females showed a random pattern. These findings indicate that this disorder is linked to the X chromosome. To map the gene for this disorder, we analyzed DNA from nine affected females and five unaffected individuals, using 40 polymorphic markers evenly distributed throughout the X chromosome. Two-point and multipoint linkage analyses using informative markers excluded most of the X chromosome and demonstrated linkage to a region on the long arm between DXS548 and Xqter. A maximum LOD score of 3.16 at recombination fraction 0 was obtained for five markers mapping to Xq27.3-Xq28. The mapping data should facilitate the identification of the molecular basis of this disorder. PMID- 10739773 TI - Mutations in the mitochondrial tRNA Ser(UCN) and in the GJB2 (connexin 26) gene are not modifiers of the age at onset or severity of hearing loss in Spanish patients with the 12S rRNA A1555G mutation. PMID- 10739775 TI - The HLA component of type I diabetes. PMID- 10739776 TI - New insights into the epidemiologic and clinical manifestations of atherosclerotic renovascular disease. AB - Atherosclerotic renovascular disease (ARVD) continues to challenge the clinician as we enter the third millenium. ARVD frequently complicates patients with other vascular pathological states, and it is an increasingly common cause of end-stage renal failure. Although renovascular interventional procedures are now widely available and are of benefit to some patients with ARVD, a large proportion still progress to dialysis. Recent epidemiological investigations have emphasized the relationship between ARVD and other vascular diseases, and these are notable in patients with coronary artery disease and/or cardiac failure. Increased awareness of the possible coexistence of ARVD in patients with these latter conditions may allow earlier diagnosis and a minimization of complications (eg, angiotensin converting enzyme inhibitor-related uremia or flash pulmonary edema). Contemporary studies also highlight the importance of intrarenal vascular and parenchymal injury in the cause of chronic renal failure in many patients with ARVD. Severe renal structural damage often coexists with proximal renal arterial narrowing, and this can explain the variability of renal functional outcomes known to accompany revascularization procedures. More appropriate selection of those patients likely to benefit from renovascular revascularization is now required. Large-scale trials that will identify the optimal approach to improving renal functional and survival outcomes in this high-risk group of patients are now long overdue. PMID- 10739777 TI - Calcified subcutaneous arterioles with infarcts of the subcutis and skin ("calciphylaxis") in chronic renal failure. AB - Patients with chronic renal failure (CRF) are at increased risk for pathological calcifications because of increased serum calcium-phosphorus products. A minority, including those undergoing dialysis, develop a syndrome of deep skin ulcerations in association with calcification of subcutaneous arterioles. The body distribution of the skin lesions may be proximal (central), distal (peripheral), or both. Since 1968, this syndrome has been called "calciphylaxis" in the belief that it is the human analogue of Selye's experimental models of tissue calcification. Our review emphasizes that this syndrome comprises two separate processes not found in calciphylaxis: calcification of subcutaneous arterioles and infarctions of subcutaneous adipose tissue (panniculus adiposus) and skin. The infarctions are acute and clinically dramatic, whereas the calcific arteriolopathy is preexistent, having developed slowly, sometimes over years, and silently. Separating these two processes facilitates analyses of pathogenetic factors, such as those that target subcutaneous arterioles for calcification and those that interfere with blood flow through the calcified arterioles, sufficient in some patients to cause the infarctions, and of why obesity in CRF is a syndrome risk factor. This approach further helps to provide a much needed standardized definition of the syndrome, thereby facilitating comparisons of the results of such treatments as parathyroidectomy, anticoagulants, and phosphate binders. Finally, the separation shows why the application of such terms as calciphylaxis and calcifying panniculitis to this syndrome is inappropriate. PMID- 10739778 TI - Comparing the urea reduction ratio and the urea product as outcome-based measures of hemodialysis dose. AB - The urea reduction ratio (URR) and normalized treatment ratio (Kt/V) are related quantities that have become accepted measures of hemodialysis dose. Recent studies, however, have suggested that they combine two elements, both favorably associated with clinical outcome, as a single ratio. These elements, Kt and V, may offset each other, producing a complex quantity that does not reflect a true relationship between dialysis exposure and clinical outcome. This project explored and compared the associations of the URR and the ?urea clearance x time? product (Kt) with mortality in a large sample of hemodialysis patients (37,108 patients) during 1998. Survival analyses using conventional techniques were the primary analytic tools. The relationship between URR and survival was U-shaped or J-shaped, with greater relative mortality at both extremes of the URR distribution than at its middle. Thus, identifying a threshold for adequate dialysis was not possible unless one considers also a threshold for overdialysis. Conversely, the association between Kt and outcome was much simpler, reflecting progressive improvement over the range of Kt evaluated here. These analyses suggest that such measures as URR and Kt/V are compound and complex, and that a simpler, more direct, measure, such as the Kt, should be considered to describe hemodialysis dose. PMID- 10739779 TI - Effect of biocompatibility of hemodialysis membranes on serum albumin levels. AB - Hypoalbuminemia in end-stage renal disease is a marker of high morbidity and mortality. In some patients, the cause of low serum albumin levels is easily identified and therefore treatable, but in many patients, the cause is not clear. We studied the effect of changing the dialysis membrane from a bioincompatible to a biocompatible membrane on serum albumin level. Stable hemodialysis patients dialyzed with cuprammonium membranes who had serum albumin levels less than 3.5 g/dL were switched to the more biocompatible membrane, polysulfone. Serum albumin levels increased from 3.22 +/- 0.037 to 3.35 +/- 0.038 g/dL (mean +/- SE; P < 0.002). The increase was seen in patients both with and without diabetes. Thus, dialyzer membrane may affect serum albumin levels and should be considered in the differential diagnosis of hypoalbuminemia in patients undergoing hemodialysis with bioincompatible membranes. Membrane choice may have an important effect on the outcome of morbidity and mortality of hemodialysis patients. PMID- 10739780 TI - Initiation of hemodialysis treatment leads to improvement of T-cell activation in patients with end-stage renal disease. AB - Patients with chronic renal failure show an immunodeficiency characterized by frequent infectious complications and a low response to vaccinations. This is paralleled in vitro by a low T-cell proliferation on mitogenic stimuli because of an impaired costimulation by accessory cells. Furthermore, alterations of the cytokine profile are correlated with impaired immune function. The immune system is influenced by both uremia and renal replacement therapy. To evaluate the influence of hemodialysis on immune parameters, we studied patients before and after the initiation of chronic hemodialysis therapy. Fourteen patients with end stage renal failure were tested before dialysis initiation and during the first 6 weeks of hemodialysis treatment. We determined the in vitro T-cell proliferation, as well as plasma levels of interleukin-6 (IL-6) and the release of IL-6 and IL 10 into culture supernatant poststimulation with lipopolysaccharide. After 6 weeks of intermittent hemodialysis, in vitro T-cell proliferation on stimulation improved significantly (stimulation index, 21.6 +/- 18.5 versus 58.1 +/- 45.5; P < 0.01). This improvement occurred regardless of whether synthetic dialyzers or cellulosic membranes were used for the initiation of dialysis. Plasma IL-6 levels, as well as IL-6 and IL-10 secretion, did not change during the study period. In patients with end-stage renal disease, the initiation of hemodialysis led to a significant improvement of in vitro T-cell proliferation. This effect may have a role for an improvement of immune function in vivo. The expected normalization of IL-6 and IL-10 production may be masked by cytokine induction through hemodialysis membranes. PMID- 10739781 TI - Reduced blood pressure diurnal variability as a risk factor for progressive left ventricular dilatation in hemodialysis patients. AB - Cardiovascular mortality places a considerable burden on chronic renal replacement therapy programs. Left ventricular hypertrophy (LVH) increases the risk for cardiovascular mortality. Risk factors for LVH in the dialysis population are numerous and include arterial distensibility, hypertension, anemia, arteriovenous fistula, and hyperparathyroidism. An important factor to consider in the diagnosis and evaluation of hypertension in this clinical setting is blood pressure (BP) level variation, only accessible using ambulatory BP monitoring (ABPM). In uremic patients, a relative elevation of BP during sleep periods leading to an increased 24-hour BP load is frequently described. Whether this additional BP burden is pathophysiologically significant has not been resolved. This study is designed to examine the effect on echocardiographically derived measurements of the left ventricle in 60 stable chronic hemodialysis patients of abnormal (reduced) diurnal BP variability, measuring ambulatory BP on three occasions and performing echocardiography twice over a 12-month period. First, we found that most dialysis patients (76%) had consistent diurnal BP rhythms over a 12-month period, and second, those patients with persistently reduced diurnal BP rhythm tended to develop a dilated left ventricle and left atrium in the absence of other known and/or relevant risk factors (persistently increased sleep BP group; n = 36; LV end-diastolic diameter, 38.2 +/- 2.5 mm/m(2) versus persistently normal sleep BP group; n = 10; LV end-diastolic diameter, 30.6 +/- 3. 3 mm/m(2); P < 0.05). These results suggest that persistent abnormal BP variability is a risk factor for a dilated heart on dialysis, independent of the BP level. PMID- 10739783 TI - Differences in quality of life across renal replacement therapies: a meta analytic comparison. AB - A meta-analysis compared emotional distress and psychological well-being across renal replacement therapies (RRTs) and examined whether differences could be explained by: (1) treatment modalities, (2) case mix, or (3) methodologic rigor. Standard meta-analytic procedures were used to evaluate published comparative studies. Successful renal transplantation was associated with: (1) lower distress (effect size, d = -0.43 SD) and greater well-being (d = 0. 62 SD) than in-center hemodialysis (CHD) and (2) lower distress (d = -0.29 SD) and greater well-being (d = 0.53 SD) than continuous ambulatory peritoneal dialysis (CAPD). CAPD was characterized by greater well-being (d = 0.18 SD) than CHD and CHD was associated with greater distress (d = 0.16 SD) than home hemodialysis. Although methodologic rigor and case-mix differences did not correlate with the magnitude of psychosocial differences across RRTs, 10 of the 12 comparisons (83%) were threatened by publication bias (ie, that nonsignificant comparisons may have been underrepresented in the published literature). Thus, although significant quality of-life differences were evident across treatment groups, the types of patients representative of the various RRTs also differed significantly in terms of case mix variables relevant to psychosocial well-being and emotional distress. Published findings indicating differential quality of life across RRTs may thus be attributable to: (1) valid differences in effective renal replacement, reduced medical complications, and lifestyles afforded by these treatment modalities; (2) case-mix differences in the patient samples selected to represent them in research comparisons; or (3) both of these alternative explanations. PMID- 10739782 TI - Novel dialysis cannulas: are they worth the higher price tag? AB - Accessory hemodialysis equipment, including dialysis cannulas, usually lack controlled and independent testing before being introduced onto the market. The aim of this study is a prospective comparison of a newly designed curved-tip dialysis cannula with a standard dialysis cannula of the same size from the same manufacturer. Fifteen chronic dialysis patients were enrolled onto a prospective 4-month crossover study. All patients had arteriovenous fistulas, except for two patients with polytetrafluoroethylene grafts. The routinely used standard cannulas were replaced by either a curved-tip 15G cannula or a new standard 15G cannula from the same manufacturer. The two cannulas were compared with respect to puncture-related pain and/or problems and bleeding complications, as well as blood-flow dynamics. Venous and arterial access pressures were recorded at blood flow rates of 100 to 400 mL/min. Linear regression analyses of arterial and venous pressure profiles showed the same regression lines for the standard and curved-tip cannulas. Plasma haptoglobulin levels and occlusion times necessary to stop bleeding after removal of the cannulas did not differ between the two cannulas. Both patients and nurses independently reported equal puncture-related pain and/or problems for both cannulas on visual analogue scales. No correlation was found between puncture problems reported by nurses and puncture pain reported by patients. The curved-tip cannula does not offer an advantage compared with the less expensive standard cannula. Controlled testing of advertised advantages by manufacturers of accessory equipment should be a prerequisite before introduction into routine clinical treatment. PMID- 10739784 TI - Patient assessment of quality of care in a chronic peritoneal dialysis facility. AB - The percentage of patients with end-stage renal disease (ESRD) maintained on chronic peritoneal dialysis (CPD) in the United States remains well less than the percentage in several other countries. Furthermore, there has recently been a decline in the percentage of patients with ESRD in the United States undergoing CPD. The reasons for this decline are uncertain, and investigators have implicated problems with the kinetics of peritoneal dialysis, peritonitis and exit-site infections, and psychosocial stresses imposed by the therapy. Few studies, however, have considered the role of the dialysis facility itself and patient perceptions of the facility as contributing to problems with the long term acceptance of CPD. This study is designed to examine patients' perceptions of the organization and structure of the peritoneal dialysis facility and their interactions with the facility, focusing attention on areas of patient satisfaction and dissatisfaction with the facility. The study was conducted in a large, freestanding peritoneal dialysis program in an urban area that currently treats 140 patients undergoing CPD. Thirty patients were randomly selected to participate in the present study. A structured interview that included open-ended questions was administered and tape-recorded by a trained interviewer not affiliated with the dialysis unit. Patient responses were then reviewed by two investigators, and a taxonomy of patient satisfaction and dissatisfaction was developed, using a modification of the classification proposed by Concato and Feinstein. Patient responses were then categorized according to the taxonomy. The most frequently cited areas of patient satisfaction included the amount of information and instruction provided by the staff (n = 30), personal atmosphere of the facility (n = 30), efficiency of delivery of the dialysis supplies (n = 23), and availability of the primary nurse (n = 18). The importance of the nurse patient interaction was emphasized by all 30 patients, whereas the physician patient interaction was cited by only 14 patients. The most frequently cited area of dissatisfaction noted by all 30 patients concerned the dialysis regimen itself. The present study focuses attention on patient perceptions of their CPD facility, identifying areas of satisfaction and dissatisfaction. The analysis is important not only in providing a framework for CPD facilities with which to review their own interactions with CPD patients, but also for identifying those areas that require attention to maintain the long-term viability of CPD therapy. PMID- 10739785 TI - Changes of cytokine profiles during peritonitis in patients on continuous ambulatory peritoneal dialysis. AB - Continuous ambulatory peritoneal dialysis (CAPD) has emerged as an important dialysis treatment modality worldwide. One of the major complications is bacterial peritonitis, which may result in subsequent technique failure because of loss of peritoneal clearance or peritoneal fibrosis. Bacterial peritonitis leads to the release of proinflammatory cytokines from resident and infiltrating cells in the peritoneal cavity. We studied 35 patients undergoing CAPD with acute bacterial peritonitis. All patients treated with antibiotics for 2 weeks after the clinical diagnosis of peritonitis had a good recovery. Peritoneal dialysate effluent (PDE) was collected on days 1, 3, 5, 10, 21, and 42 after the start of treatment. Cell populations were monitored by flow cytometry. PDE levels of interleukin-1beta (IL-1), IL-6, transforming growth factor-beta (TGF-beta), and basic fibroblast growth factor (FGF) were measured by enzyme-linked immunosorbent assay. Gene transcription of TGF-beta in macrophages from PDE was measured by quantitative polymerase chain reaction. Bacterial peritonitis was associated with a sharp increase in total cell and neutrophil counts (400-fold) in PDE up to 3 weeks after peritonitis despite clinical remission (P < 0.0001). There was an increased absolute number of macrophages during the first 3 weeks despite the reduced percentage of macrophages among total cells in PDE compared with noninfective PDE. There was a progressive increase in the percentage of mesothelial cells or dead cells in the total cell population in PDE over the entire 6-week period. PDE levels of IL-1, IL-6, TGF-beta, and FGF increased markedly on day 1 before their levels decreased gradually. PDE levels of these cytokines or growth factors were significantly greater than those in noninfective PDE (n = 76) throughout the study period (P < 0.01). Similarly, TGF-beta complementary DNA (cDNA) molecules per macrophage were significantly greater than those of macrophages in noninfective PDE throughout this period (P < 0.01). There was no significant correlation between PDE levels of TGF-beta and TGF-beta cDNA molecules per macrophage, suggesting that peritoneal macrophages are not the only source of TGF-beta in PDE. We conclude there is an active release of proinflammatory cytokines and sclerogenic growth factors through at least 6 weeks despite apparent clinical remission of peritonitis. The peritoneal cytokine networks after peritonitis may potentially affect the physiological properties of the peritoneal membrane. PMID- 10739786 TI - Using renal transplantation to evaluate a simple approach for predicting the impact of end-stage renal disease therapies on patient survival: observed/expected life span. AB - The effectiveness of therapy for a chronic disease can be assessed by evaluating the length of time that a patient survives after receiving treatment. We used a novel means for measuring the effectiveness of renal replacement therapy for patients with end-stage renal disease (ESRD): the ratio of observed life span divided by expected life span. This ratio incorporated observed life span for patients from the time of ESRD and expected life span based on state-specific life-table analyses. A total of 3,782 individuals with ESRD were analyzed (average follow-up, 14.2 +/- 4.9 years); 3, 192 patients in that group received a kidney transplant at some point during their course of ESRD. For each patient, we determined a curve of observed/expected life span. Separate patient groups were analyzed to determine the median population observed/expected life span or the percentage of patients who reached 0.5 observed/expected life span. Younger transplant recipients (<21 years) had a median observed/expected life span of 67%, significantly greater than the median observed/expected life span for those aged 21 to 40 years (49%; P = 0.01) and 41 to 60 years (47%; P = 0.01). Surprisingly, 57% of the patients aged older than 60 years reached their median observed/expected life span (P = 0.02 versus <21 years; P = not significant against all others). A Cox proportional hazards model identified era of immunosuppression (hazards ratio, 0.32) and atherosclerotic vascular disease related ESRD (hazards ratio, 2.07) as significant variables influencing patient survival and observed/expected life span. This simple ratio is easy to use and may be a helpful tool for assessing the survival benefits of risk-factor modifications and therapeutic advances in transplantation and ESRD care. PMID- 10739787 TI - Abbreviated tacrolimus area-under-the-curve monitoring for renal transplant recipients. AB - The area under the concentration time curve (AUC) for oral tacrolimus (FK) may provide a more precise model for FK monitoring after renal transplantation. The purpose of this study is to identify a simple, cost-effective method for predicting FK AUC. FK concentrations were measured at 0, 1, 2, 4, 6, 8, and 12 hours after the morning dose. The predicted AUCs (AUC(p)s) derived from regression equations were used to estimate the actual 12-hour AUCs (AUC(12)s). The relationship between AUC(p) and AUC(12) was validated by determining the coefficient of multiple determination (R(2)), percentage of prediction error (PE%), and percentage of absolute prediction error (APE%). Eighteen stable Oriental renal transplant recipients (9 men, 9 women) with a mean age of 42.6 +/- 6 years and mean body weight of 62.7 +/- 10 kg were recruited for the study. The FK AUC(12), trough, 2-hour, and 4-hour concentrations were 125 +/- 24 h. ng/mL (range, 87.7 to 181.9 h. ng/mL), 6 +/- 1.3 ng/mL, 18.1 +/- 4.7 ng/mL, and 11 +/- 2.4 ng/mL, respectively. Trough FK concentration did not have a significant correlation with AUC(12) (r = 0.34; P = 0.17). AUC(p) obtained by a two-time point regression equation using 2-hour (C2) and 4-hour (C4) FK concentrations: (AUC(P) = 16.2 + 2.4*C2 + 5.9*C4) obtained an R(2), PE%, and APE% of 0.93, -0.2% +/- 5.2% (range, -13% to 9.3%), and 3. 6% +/- 3.7% (range, 0.02% to 13%), respectively. We conclude that a two-point sampling method using C2 and C4 may be a more cost-effective FK monitoring strategy than morning FK trough levels in transplant recipients. PMID- 10739789 TI - Transplant glomerulopathy as a cause of late graft loss. AB - Several pathophysiological processes contribute to chronic kidney transplant rejection. Among the most distinctive is transplant glomerulopathy, characterized by widening of the subendothelial space with accumulation of flocculent material and duplication of the basement membrane. The current study assessed the course of graft loss in patients with and without this form of injury. Twenty-five patients with prominent transplant glomerulopathy were identified from biopsies performed at a single center during 4 years. These patients were compared with control patients with a similar degree of renal dysfunction in whom biopsies showed chronic rejection without transplant glomerulopathy. Patients with transplant glomerulopathy showed an increased rate of graft loss after biopsy. Biopsies were performed longer after transplantation in these patients, however, than in control patients with an equal degree of graft dysfunction. Graft survival from the time of transplantation was therefore not different between the two groups. Morphological studies showed that transplant glomerulopathy was not associated with increased severity of chronic vascular injury characterized by arterial and arteriolar intimal thickening or hyalinosis. These findings show that transplant glomerulopathy may develop late after transplantation and separately from chronic vascular rejection. The appearance of transplant glomerulopathy on a biopsy specimen is followed by accelerated graft loss. PMID- 10739788 TI - Role of humoral immune reactions as target for antirejection therapy in recipients of a spousal-donor kidney graft. AB - Excellent graft outcome has been reported for spousal-donor kidney transplantation. In husband-to-wife transplantation, however, a tendency toward inferior graft survival has been described for recipients who were previously pregnant. In our series of spousal-kidney transplantations (nine transplantations; three female recipients), actual graft survival is 100% (median observation time, 339 days). Five patients experienced early allograft rejection. In four transplant recipients, rejection was easily reversible by conventional antirejection therapy. In a multiparous recipient, however, mild interstitial allograft rejection associated with early graft dysfunction was resistant to anticellular treatment (antilymphocyte antibody, tacrolimus rescue therapy). The particular finding of polymorphonuclear neutrophils in peritubular capillaries and the finding of diffuse capillary deposits of the complement split product, C4d, in a posttransplantation biopsy specimen suggested a role of antibody mediated graft injury. Retrospective flow cytometry cross-matching showed the presence of preformed immunoglobulin G (IgG) antibodies to HLA class I antigens that were not detectable by pretransplantation lymphocytotoxic cross-match testing or screening for panel reactive antibodies. After transplantation, however, complement-fixing antibodies, also presumably triggered by reexposure to spousal-donor HLA antigens, could be detected in the patient's serum. These findings suggested antibody-mediated allograft rejection and led to the initiation of immunoadsorption therapy (14 sessions) with staphylococcal protein A. Selective removal of recipient IgG resulted in complete reversal of graft dysfunction. Our findings suggest that in husband-to-wife transplantation, donor specific antibodies, presumably triggered by previous pregnancies, might occasionally induce sustained allograft dysfunction. Thus, in this particular setting, a detailed immunologic and histopathologic work-up regarding antibody mediated allograft dysfunction is warranted because immunoadsorption may be a highly effective treatment modality. PMID- 10739790 TI - Abnormal 24-hour blood pressure patterns in children after renal transplantation. AB - Hypertension after renal transplantation occurs commonly and is associated with decreased allograft survival. Hypertension is usually diagnosed by casual blood pressure (BP) measurements in the outpatient clinic that may not reflect the overall 24-hour BP pattern. To better describe the pattern of BP in children after renal transplantation, 24-hour ambulatory BP monitoring (APBM) was performed in 42 patients with stable renal function. BP was measured every 20 minutes during the daytime and every 30 minutes at night. Mean patient age was 12.8 +/- 5.2 years, and mean time after transplantation was 34 +/- 36 months. Seventy-six percent of the patients were administered antihypertensive medications. Twenty-four-hour mean systolic BP (SBP) was 127 +/- 11 mm Hg, and diastolic BP (DBP) was 80 +/- 11 mm Hg. Mean 24-hour BP load values (percentage of BP readings > 95th percentile based on Task Force criteria) were 59% for SBP and 50% for DBP, which were significantly elevated compared with healthy children (P < 0.001). An attenuated decline in sleep BP (nondipping) was found in 78% of the patients for SBP and 50% for DBP. Sleep BP exceeded awake BP in 24% of the patients for SBP and 17% for DBP. Boys had a greater SBP load (66% versus 45%; P = 0.03) and DBP load (57% versus 38%; P = 0.04) than girls. These results confirm in children the high prevalence of hypertension by ABPM criteria after renal transplantation and show attenuation of normal sleep BP decreases. These BP disturbances may shorten renal allograft survival and predispose children to long term hypertensive end-organ damage. PMID- 10739791 TI - Cigarette smoking is associated with augmented progression of renal insufficiency in severe essential hypertension. AB - Hypertension-associated renal disease is a major cause of end-stage renal disease (ESRD) in the United States, but its risk factors remain incompletely defined. Identification and correction of amendable ESRD risk factors among patients with essential hypertension could reduce ESRD prevalence. Patients referred by their primary care physician for hypertension management to an academic nephrology clinic during calendar year 1995 were followed up prospectively. Studied patients had no evidence of secondary hypertension, diabetes, or primary renal disease. All were treated pharmacologically toward a target mean blood pressure (MBP) of 100 mm Hg or less. The course of renal function during follow-up was assessed as the slope of the reciprocal of plasma creatinine concentration (1/P(cr)) plotted against months of follow-up and as the change in calculated glomerular filtration rate (GFR) in milliliters per minute per month. The following patient characteristics were prospectively examined as possible predictive factors for altered renal function: age, sex, ethnicity, initial MBP, initial P(cr) level, initial level of urine protein excretion, and smoking status. Fifty-three patients were enrolled, and follow-up data were available for 51 patients after a mean follow-up of 35.5 months. Despite MBP reduction from 126.8 +/- 1.3 to 96.5 +/- 1.1 mm Hg (P < 0.0001), P(cr) level increased from 1.5 +/- 0.1 to 1.9 +/- 0. 2 mg/dL (P < 0.01). Multivariate regression analysis showed that smoking, greater initial P(cr) level, and black ethnicity were the only examined parameters that independently predicted both a decrease in the 1/P(cr) slope and calculated GFR with at least 95% confidence. Smoking was by far the most powerful of the examined factors, with initial P(cr) and ethnicity being much less predictive. These studies show for the first time that smoking is an independent risk factor for renal function decline in patients with severe essential hypertension. PMID- 10739792 TI - Effect of ACE inhibitors in diabetic and nondiabetic chronic renal disease: a systematic overview of randomized placebo-controlled trials. AB - Clinical trials have shown the beneficial effects of angiotensin-converting enzyme (ACE) inhibitors in delaying the progression of diabetic renal disease. There is less evidence from primary clinical trials of nondiabetic renal disease. We performed an updated meta-analysis to determine the efficacy of ACE inhibitors in slowing the progression of renal disease over a broad range of functional renal impairment. We included published and unpublished randomized, placebo controlled, parallel trials with at least 1 year of follow-up available from January 1970 to June 1999. In nine trials of subjects with diabetic nephropathy and microalbuminuria, the relative risk for developing macroalbuminuria was 0.35 (95% confidence interval [CI], 0.24 to 0.53) for individuals treated with an ACE inhibitor compared with placebo. In seven trials of subjects with overt proteinuria and renal insufficiency from a variety of causes (30% diabetes, 70% nondiabetes), the relative risk for doubling of serum creatinine concentration or developing end-stage renal disease was 0.60 (95% CI, 0.49 to 0.73) for individuals treated with an ACE inhibitor compared with placebo. Treatment of individuals with chronic renal insufficiency with ACE inhibitors delays the progression of disease compared with placebo across a spectrum of disease causes and renal dysfunction. PMID- 10739793 TI - Increased prevalence of proteinuria in diabetic sibs of proteinuric type 2 diabetic subjects. AB - There is strong evidence for clustering of renal disease in type 1 diabetes, but few data exist with respect to type 2 diabetes. The objective of this case control study is to determine whether there is a familial predisposition to the development of proteinuria in patients with type 2 diabetes. Fifty patients with type 2 diabetes with macroproteinuria (protein > or = 500 mg/24 h) with no evidence of causes other than diabetic nephropathy on investigation were identified through routine screening. These patients had 25 living sibs with diabetes, of whom 24 sibs agreed to participate on the study. For each of these sibs, two controls with non-insulin-dependent diabetes were randomly selected, individually matched for age, sex, and duration of diabetes. Twelve of 24 sibs (50%) and 9 of 48 controls (18.8%) had proteinuria (P < 0.01). Systolic and diastolic blood pressure and the proportion on antihypertensive treatment were similar in the two groups. Our data suggest there is increased prevalence of macroproteinuria in diabetic sibs of macroproteinuric patients with type 2 diabetes in a population of white, Caucasian, European descent. PMID- 10739794 TI - Differential diagnosis of prerenal azotemia from acute tubular necrosis and prediction of recovery by Doppler ultrasound. AB - Acute renal failure (ARF) is a life-threatening disease that often causes multiple organ dysfunction. The accurate and rapid diagnosis of the cause of ARF is particularly important for selecting the appropriate therapy. Ultrasound Doppler is a noninvasive diagnostic method that has recently been introduced to clinical nephrology. We report the diagnostic value of Doppler ultrasound in differentiating acute tubular necrosis (ATN) from prerenal azotemia by comparing this study with the fractional excretion of sodium (FENa), renal failure index (RFI), and urinary/serum creatinine (Cr) ratio. Doppler ultrasound was able to differentiate prerenal azotemia from ATN, equivalent to FENa, RFI, and the urinary/serum Cr ratio. Doppler ultrasound does not require blood or urine samples and can be performed at the bedside. Of note, Doppler is unaffected by changes in Na or Cr in urine or serum after diuretics or hemodialysis. Furthermore, one can predict recovery from ATN by Doppler findings. Thus, we consider Doppler ultrasound an effective diagnostic tool in ARF. PMID- 10739795 TI - Laparoscopic nephrectomy in patients with end-stage renal disease and autosomal dominant polycystic kidney disease. AB - Autosomal dominant polycystic kidney disease (ADPKD) is often characterized by end-stage renal disease (ESRD) and problems including pain, hematuria, and infection. Open nephrectomy is curative; however, the morbidity of the procedure is considerable. Between 1995 and 1998, 11 laparoscopic nephrectomies were performed on nine symptomatic patients (five men and four women) with ESRD and ADPKD. Two patients underwent a staged bilateral laparoscopic nephrectomy. All patients presented with abdominal or flank pain and an abdominal mass. Other clinical problems included hypertension in eight patients, urinary tract infections in two patients, and gross hematuria in one patient. Seven patients were receiving long-term dialysis treatment, and two patients had undergone prior renal transplantation. Patients were evaluated for preoperative and postoperative pain, analgesic use, hospital course, and convalescence. The overall average operative time was 6.3 hours, with an average estimated blood loss of 153 mL. Eight nephrectomy specimens were removed by morcellation, and three specimens were removed intact through a 7- to 12-cm incision. The average hospital stay was 3 days, and the average time to normal activity was 5 weeks. With a mean follow up of 31 months, all nine patients reported elimination of their preoperative pain based on a pain analogue score. Six major and two minor complications occurred, including blood transfusion, a vena cavotomy, splenic cyanosis, pulmonary embolism, clotted arteriovenous fistula, and brachial plexus injury. Incisional hernias occurred in two of the three patients who underwent open removal. One patient noted improvement, and two patients noted resolution of their hypertension postoperatively. Laparoscopic nephrectomy in patients with ADPKD and ESRD offers an effective alternative to open nephrectomy to manage renal-related pain. This procedure provides the benefits of minimal intraoperative blood loss, minimal postoperative pain, brief hospital stay, and rapid convalescence. PMID- 10739796 TI - Tissue factor, plasminogen activator inhibitor-1, and thrombin receptor expression in human crescentic glomerulonephritis. AB - Glomerular fibrin deposition is a common histological feature of crescentic glomerulonephritis (CGN). Tissue factor (TF) is the most powerful activator of the coagulation system, whereas plasminogen activator inhibitor (PAI)-1 is a key modulator of the fibrinolytic pathway. Thrombin, released locally as the final step of the coagulation cascade and trapped within the fibrin clots, can induce the activation of glomerular cells, through the interaction with a specific receptor. To investigate the mechanisms underlying coagulation cascade activation and fibrin deposition and the role of this phenomenon in the pathogenesis of human CGN, TF, PAI-1, and thrombin receptor expression were studied in CGN biopsy specimens. Glomerular TF gene and protein expression were strikingly increased in CGN, in particular within the crescents and in the mesangial area, with the same distribution of fibrin deposits. Interestingly, very few infiltrating mononuclear cells were stained in TF immunohistochemistry. To better evaluate the involvement of monocytes in TF expression, TF mRNA and CD68 protein were studied by an in situ hybridization/immunohistochemistry combined technique. Only 16% of the cells expressing TF mRNA were CD68 positive. However, most of the TF signal was localized in the proximity of monocytes, suggesting that soluble mediator(s) released by these cells could induce TF expression. Indeed, interleukin-1 (IL-1), one of the main monocyte-derived cytokines, upregulated TF mRNA levels in cultured human mesangial cells in a time-dependent manner. Moreover, a striking increase in IL-1 expression was present within the cellular crescents in CGN biopsy specimens. Finally, we observed a marked upregulation of both PAI-1 and thrombin receptor mRNA levels in CGN with a pattern resembling TF and fibrin distribution. Surprisingly, thrombin receptor protein expression was strikingly downregulated in CGN, suggesting its continuous activation and degradation. In conclusion, we can hypothesize that TF and PAI-1, mainly expressed by resident cells, may play a pivotal role in the development and preservation of fibrin deposits in CGN. In addition, thrombin, released locally and accumulated within the fibrin clots, may represent a pathogenetic mediator of crescentic lesions. PMID- 10739797 TI - Evaluation of a new fluorescent dye method to measure urinary albumin in lieu of urinary total protein. AB - Urinary total protein (UTP) determinations are notoriously inaccurate, poorly reproducible, and difficult to interpret in early renal disease, causing many investigators to measure urinary albumin instead. In this study, we compare a new nonimmunologic fluorescent dye (AB-dye) for measuring albumin with the more expensive and cumbersome radioimmunoassay. We tested 207 urine specimens from patients with variable protein concentrations and divided the results into five arbitrary ranges (0 to 20, 21 to 50, 51 to 100, 101 to 200, and 201 to 400) for chi-square analysis. There was a high degree of correlation between the two methods (chi-square = 260. 8 with 16 degrees of freedom; P < 0.001). The correlation was also high when analyzed by linear regression (R = 0.86; F < 0.01). Based on our comparison of total protein and albumin concentration in the same urine samples, we hypothesized that patients with mild proteinuria may not necessarily have microalbuminuria. Urine samples with UTP between 150 and 400 microg/mL were tested for albumin by the AB-dye. Of 41 samples in this range, 18 (44%) had normal albumin levels. We conclude that measuring urinary albumin with the AB-dye is comparable in performance to radioimmunoassay and could replace UTP determinations, especially for patients with borderline elevations of UTP, many of whom do not have microalbuminuria. PMID- 10739798 TI - MR imaging features of acute bilateral renal cortical necrosis. AB - Bilateral renal cortical necrosis (BRCN) is an uncommon cause of acute renal failure. Kidney biopsy, arteriography, and contrast-enhanced computed tomography (CT) are usually used to diagnose BRCN. However, these methods can have potentially serious side effects. We report two cases in which magnetic resonance imaging (MRI) evidenced characteristic features of BRCN, which were confirmed by histological findings and arteriography and correlated with clinical evolution. In the first case report, the diagnosis of a massive and complete cortical necrosis variety was suggested on MRI that showed a thin rim of low signal intensity along border of kidneys. It was confirmed on kidney biopsy, and the renal function did not recover. The second case is an incomplete form with cortical patchy areas of low signal intensity. In these two patients, MRI helped to establish an early diagnosis of BRCN with characteristic representative findings, without the potential nephrotoxic effects of iodinated contrast that has to be used in CT and arteriography. Kidney biopsy, besides the risks of complications, provides only a parceled analysis of the renal tissue and therefore does not allow any conclusion as to the extension of cortical necrosis. MRI may be of great help for the diagnosis and follow-up of acute renal cortical necrosis. PMID- 10739799 TI - Recurrent type I membranoproliferative glomerulonephritis in a renal allograft: successful treatment with plasmapheresis. AB - Recurrent disease is increasingly recognized as a cause of renal allograft dysfunction and failure. We describe a patient with type I membranoproliferative glomerulonephritis not associated with hepatitis C. The glomerular disease recurred in the renal allograft within 1 month of transplantation, leading to acute allograft dysfunction and nephrotic syndrome. Aggressive treatment with prednisone and plasmapheresis resulted in improvement in kidney function, improvement of the light microscopic picture, and removal of immune complexes from the glomerular subendothelial space. PMID- 10739800 TI - Co-occurrence of autosomal dominant polycystic kidney disease and Marfan syndrome in a kindred. AB - Several reports exist of the co-occurrence of autosomal dominant polycystic kidney disease (ADPKD) and Marfan syndrome, including a report of ADPKD and "overlap" connective tissue disorder in a family with linkage to the PKD1 locus. We report the results of clinical and linkage investigations of an ADPKD family in whom several affected subjects also had aortic vascular complications as well as features of Marfan syndrome. Detailed clinical assessment and linkage analysis were performed with polymorphic microsatellite markers closely linked to the PKD1 and FBN1 loci. Survival data were compared with 10 geographically matched PKD1 families. Although several subjects had features of both ADPKD and Marfan syndrome, detailed clinical examination of the extended family indicated that the two conditions had converged within the kindred. For those with ADPKD, linkage was established to the PKD1 locus (lod score, 6.04). Among those with features of Marfan syndrome, linkage was confirmed to the FBN1 locus (lod score, 1.87). Five of six subjects with both ADPKD and the high-risk FBN1 haplotype had associated vascular complications. In contrast, among the remaining nine individuals with PKD1 alone, seven had aortic assessments, and none were found to have aortic complications. Our experience suggests that when prominent features of connective tissue disease or vascular complications are found in ADPKD patients, alternative additional diagnoses should be considered, including the possibility of a coinherited FBN1 mutation responsible for Marfan syndrome or, alternatively, an associated milder FBN1 phenotype in the absence of sufficient other clinical features to allow Marfan syndrome to be diagnosed. PMID- 10739801 TI - Severe ectopic calcification of the intestinal wall in a patient on long-term continuous ambulatory peritoneal dialysis therapy. AB - We report autopsy findings of a 69-year-old man on long-term CAPD therapy for 13 years who showed linear peritoneal calcification. Continuous ambulatory peritoneal dialysis (CAPD) was started in 1982. He has been administered excessive amounts of vitamin D(3) derivatives (VitD) (2.0 to 2.5 microg daily) and calcium carbonate (4 g daily) for secondary hyperparathyroidism since initiation of CAPD. In May 1995, his intact parathyroid hormone (PTH) level increased over 1,000 pg/mL. Immediately after VitD was changed from pill to liquid, the dose was increased to 5 microg daily. Although the serum calcium level remained between 4.5 and 4.9 mEq/L, and serum phosphate level was 5.0 to 7.2 mg/dL, plain abdominal radiography and computed tomography showed continuous calcification along the intestinal wall in October 1995. In spite of the continuation of CAPD therapy, he remained asymptomatic until he died of congestive heart failure in January 1997. He experienced eight episodes of peritonitis during his clinical course. Autopsy showed that numerous calcified plaques were present on the submucosal portion between the thickened serosa and the longitudinal layer of the muscularis externa. The remainder of the subserosa was fibrotic, and the small arteries had markedly thickened intima and severely narrowed lumina. PMID- 10739802 TI - Adverse effect of smoking on the renal outcome of patients with primary hypertension. PMID- 10739803 TI - Pain management in polycystic kidney disease. PMID- 10739804 TI - Effect of maximal reduction of angiotensin in renal fibrosis: bad news-good news from a pediatric mouse. PMID- 10739805 TI - Fanconi syndrome with free kappa light chains in the urine. PMID- 10739807 TI - Continuing medical education exercise, april 2000 PMID- 10739806 TI - Acute rejection, type I (Interstitial) PMID- 10739808 TI - Thin basement membrane disease with heavy proteinuria or nephrotic syndrome at presentation. AB - Thin basement membrane disease (TBMD) is a condition originally defined as diffuse thinning of the glomerular basement membrane (GBM) associated with hematuria in all patients. Although proteinuria has been described in up to 60% of patients with TBMD, it is almost always mild, with a 24-hour excretion mostly of less than 500 mg. We describe eight patients (four men and four women between 32 and 66 years of age) with TBMD who presented with heavy proteinuria or nephrotic syndrome. Among the seven cases with family history, hematuria was noted in five. All patients had a long history of microscopic hematuria, with episodic gross hematuria in two. Renal biopsies showed diffuse thinning of the GBM in each patient (mean between 185.3 x 29.8 nm and 232.6 x 34.5 nm versus control between 325 x 35 nm and 350 x 15 nm). Three cases showed thinning of GBM only (group I); the remaining five cases showed thinning of GBM associated with focal segmental glomerulosclerosis. All three patients of group I presented with nephrotic syndrome and normal renal function. Treatment with steroids resulted in remission of nephrotic syndrome in two, whereas nephrotic syndrome persisted in the untreated patient. Among the five patients in group II, nephrotic syndrome and normal renal function at presentation were noted in two, whereas the other three had heavy proteinuria (2.2, 2. 5, and 2.6 g/d, respectively) associated with mildly decreased renal function (serum creatinine 1.8, 1.3, and 1.5 mg/dL, respectively). At last follow-up, although the renal function was stable in all five, only the three who received steroid treatment had remission or marked improvement of proteinuria. Hematuria, however, persisted in all eight patients of both groups. Whether specific gene mutations are translated into structural changes responsible for both excessive GBM thinning and increased transcapillary permeability remains to be elucidated. Alternatively, the heavy proteinuria/nephrotic syndrome may not be related to TBMD, but rather is the manifestation of associated glomerular diseases. Follow-up, including a response to steroids, supports the latter hypothesis. PMID- 10739809 TI - Acute tubulointerstitial nephritis attributable to indinavir therapy. AB - Indinavir sulfate has been reported to cause asymptomatic crystalluria and nephrolithiasis in patients with human immunodeficiency virus (HIV) infection. Patients taking indinavir may present with asymptomatic crystalluria, nephrolithiasis with frank renal colic and obstruction, flank pain in the absence of nephrolithiasis, and dysuria or urgency. Asymptomatic crystalluria has been described as benign. Discontinuation of the drug has not been recommended in the absence of nephrolithiasis. We report two HIV-positive patients receiving indinavir who developed acute interstitial nephritis with foreign body giant cell reaction on renal biopsies. Both patients had asymptomatic crystalluria, although crystals were associated with clumps of white blood cells (WBCs) on urinalysis in one patient. Both cases show that the inflammatory response was significant enough to lead to tubular injury and acute renal impairment. Our findings suggest that asymptomatic crystalluria attributable to indinavir may illicit an inflammatory response with acute renal insufficiency, warranting monitoring of renal function, especially in patients with crystalluria. PMID- 10739810 TI - Antihypertensive therapy for nondiabetic nephropathy PMID- 10739811 TI - Gender reassignment and assisted reproduction: An ethical analysis. Bristol Centre for Reproductive Medicine Ethics Advisory Committee. PMID- 10739812 TI - Ethics of postmortem sperm retrieval: ethics of sperm retrieval after death or persistent vegetative state. PMID- 10739813 TI - Luteal phase start of low-dose FSH priming of follicles results in an efficient recovery, maturation and fertilization of immature human oocytes. AB - In this prospective study we investigated whether the maturation and fertilization of immature oocytes can be improved by administration of recombinant follicle stimulating hormone (rFSH) starting in the late luteal phase in two groups of women: group 1 (n = 6) women with regular menstrual cycles; and group 2 (n = 6) women with irregular cycles and polycystic ovaries (PCO) on ultrasound examination. Low-dose (37.5 IU) rFSH was commenced 11 days after LH surge during a spontaneous menstrual cycle and on the ninth day of progesterone administration in an irregular cycle. Recombinant FSH was continued until the leading follicle was approximately 10 mm in diameter. The oocytes were retrieved after withdrawing rFSH for 2-5 days. In total, 136 oocytes were recovered (group 1, 67 oocytes; group 2, 69 oocytes). Nine of the oocytes from PCO women were atretic at retrieval. Oocytes complete with cumulus cells were cultured for 44 h in complex tissue culture medium supplemented with gonadotrophins and fetal calf serum. After maturation, the cumulus cells were removed and metaphase II oocytes were injected with spermatozoa. Respectively, the oocyte maturation and fertilization rates were 64 and 72% in group 1, and 78 and 57% in group 2 (not significant). After fertilization, the zygotes (group 1, n = 22; group 2, n = 11) and cleavage stage embryos (group 1, n = 9; group 2, n = 15) were frozen in propanediol. All women except one (11/12) had approximately five zygotes or cleaved embryos frozen. The viability of in-vitro matured frozen-thawed embryos was generally poorer than that (81%) seen after conventional intracytoplasmic sperm injection, with 61% survival in group 1 and 23% in group 2. Fifteen embryo transfers resulted in one miscarriage at 6 weeks gestation. The late luteal start of low-dose rFSH yielded a good number of immature oocytes in women with both regular and irregular cycles. Two out of three of these oocytes matured and fertilized. However, cryosurvival of the zygotes and cleaved embryos was unsatisfactory and thus cryopreservation of in-vitro matured embryos may not be an optimal procedure. PMID- 10739814 TI - Side of ovulation and cycle characteristics in normally fertile women. AB - This study was undertaken to establish whether ovulation in humans alternates consistently from right to left ovary in successive cycles and whether the site of ovulation affects the next cycle length or the hormonal profiles. A total of 199 cycles in 80 normally fertile women were studied. The volunteers were monitored with ultrasonography to determine the day and side of ovulation and to calculate follicular and luteal phase lengths. Urinary hormone concentrations were also assayed. Right-sided ovulations occurred in 104 of the 199 cycles (52.3%; not significantly different from 50%). Alternate ovulations occurred in 61 of the 119 pairs of succeeding cycles (51.3%, not significant). The follicular phase length in contralateral ovulation (14.59 +/- 0.33 days; mean +/- SEM) did not differ significantly from that of ipsilateral ovulation (14.59 +/- 0. 37 days). There were also no significant differences in urinary concentrations of oestrone-3-glucuronide, pregnanediol-3alpha glucuronide, follicle stimulating hormone, and luteinizing hormone between ipsilateral and contralateral ovulation in either early follicular, peri-ovulatory or luteal phase of the cycle. It is concluded that in normally fertile women, the cycle length and the hormonal profile are independent of the, most probably random, site of ovulation. PMID- 10739815 TI - Secretory characteristics and viability of human term placental tissue after overnight cold preservation. AB - Collection of human term placentae for research purposes is generally limited during working hours. Preserving placental tissue overnight might help to postpone experiments and, by extent, to increase material availability. In this study, fragments from normal placentae were incubated at 37 degrees C either immediately after delivery or after preservation at 4 degrees C in a HEPES buffered solution or in a Roswell Park Memorial Institute (RPMI) 1640 culture medium. Protein, human chorionic gonadotrophin (HCG), human placental lactogen (HPL) and lactate dehydrogenase (LDH) contents within preserved explants were similar to those within freshly delivered ones. In contrast, HCG and HPL amounts released during incubation of preserved tissue were lower than with freshly delivered tissue. Differences were significant only during the first 3 h of incubation. Hormone releases were similarly Ca(2+)-stimulated, and Co(2+)- and low temperature-inhibited in preserved and freshly delivered tissues. After preservation, LDH leakage was also reduced. Furthermore, before and after 37 degrees C incubation during 6 h, preserved tissue was morphologically indistinguishable from freshly delivered tissue and showed neither higher incidence of DNA fragmentation, nor elevated caspase-3 activity, both of which are markers of apoptosis. This study validates an original, useful and rapid method to preserve placental tissue. Consequently, this preservation model may facilitate the study of physiological processes regulating placental hormone secretion in normal and pathological conditions. PMID- 10739816 TI - Testicular and sperm DNA damage after treatment with fludarabine for chronic lymphocytic leukaemia. AB - This study investigated whether chemotherapy using fludarabine (FLU) caused testicular damage and if cytotoxicity could be detected as sperm DNA damage in the single cell Comet assay. A patient with chronic lymphocytic leukaemia requesting preservation of fertility was treated with seven monthly cycles of fludarabine (45.8 mg total dose per cycle). Testicular assessments, serum follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone measurements, semen analysis and sperm Comet assays were carried out at presentation (pre-FLU therapy), after 1 and 7 months of FLU treatment, and finally at 11 months after completion of chemotherapy. We found that testicular damage occurred within a month, as indicated by reduced testicular volume, oligozoospermia, elevated FSH and LH, and lower testosterone concentrations. Spermatozoa with a large range of DNA damage were detected in the samples from both the control and treated men. DNA damage in the spermatozoa was marked by 7 months of FLU treatment. The high levels of sperm DNA damage seen during and possibly persisting after treatment suggests that caution should be exercised if the ejaculates from these men are used for in-vitro fertility treatment. Further experiments are needed to assess the biological significance of these DNA changes; it may, however, be prudent at present to be cautious when counselling these patients. PMID- 10739817 TI - Smooth muscles are frequent components of endometriotic lesions. AB - Deep infiltrating endometriosis (deeper than 5 mm under the peritoneum) often takes the form of a nodular lesion (or 'adenomyotic nodule') consisting of smooth muscles and fibrosis with active glands and scanty stroma. Thus, among endometriotic lesions, a certain distinction is drawn between musculo-glandular lesions and glandular lesions composed of endometrial-like epithelium surrounded by a cell-producing (cytogenous) stroma. The aim of this study was to detect by immunohistochemistry, with a monoclonal antibody against muscle-specific actin, the presence of smooth muscles in 54 endometriotic lesions originating from four different pelvic locations (peritoneum, ovary, rectovaginal septum and uterosacral ligaments) and to quantify the smooth muscle content. Smooth muscles were frequent components of endometriotic lesions in pelvic locations. In addition, smooth muscles were significantly (P < 0. 001) more abundant in endometriotic lesions than in their respective unaffected sites. This finding supports, at least partially, the occurrence of a metaplastic phenomenon in the pathogenesis of endometriotic lesions. The definition of distinct endometriotic entities based on the difference in the tissue composition of the lesions (endometriotic nodules versus adenomyotic nodules) is inconsistent with the very frequent presence of smooth muscle cells in endometriosis irrespective of its localization. PMID- 10739818 TI - Adverse effects of hydrosalpinx fluid on sperm motility and survival. AB - The negative impact of hydrosalpinx on IVF outcome is well recognized but some reports indicate that tubal infertility with hydrosalpinx is a heterogeneous entity and may have different effects on the outcome. The embryotoxic effects of hydrosalpinx fluid (HF) have been documented in mouse but not human embryos. This study examined the effects of HF on sperm motility and survival after various periods of incubation. Fifteen infertile patients with hydrosalpinx shown on ultrasound monitoring during ovarian stimulation underwent aspiration of HF after egg collection. Electrolytes, glucose and pyruvate concentrations were within the physiological ranges found in normal human tubal fluid. Sperm motility and velocities remained unchanged after 5 h of incubation with various concentrations of HF but the percentage of motile spermatozoa was significantly reduced after 24 h of incubation. Both 50 and 100% HF were potentially cytotoxic (survival indices <85%). The detrimental effect seemed to be dependent on the concentrations of HF. Low osmolarity, low lactate concentrations or the protein content may be responsible for the loss of sperm motility. A human sperm survival test using HF may be useful in selecting appropriate treatment options for patients with hydrosalpinx undergoing IVF treatment or tubal surgery. PMID- 10739819 TI - Antimicrobial activity of human cervical mucus. AB - The antibacterial activity of human cervical mucus (CM) was examined on standardized microbial colonized agar plates (agar diffusion test). In parallel, the lysozyme content of CM was determined by means of a turbidimetric test system in aliquots of the same CM specimens. Suspensions of living lyophilized Micrococcus lysedeikticus were used as bacterial substrate. Testing was performed in a total of 133 CM samples, obtained at mid-cycle from sexually active women from unselected infertile couples with a median age of 30 (range 21-42) years. All mucus specimens showed considerable antibacterial activity with clearly visible circular inhibition zones around the CM-filled holes in the colonized agar plates. Related to the effect of hen's egg white (HEW)-lysozyme on the same plates, the median activity of the CM specimens in the agar diffusion test was equivalent to 33.0 (range 6.4-391.4) microg/ml HEW-lysozyme. However, there was a wide inter-individual range of antibacterial effects of cervical secretions. The cervical index did not significantly influence the outcome of either test. The pH of the endocervical CM also was not correlated with the antibacterial effect. Sexual activity leading to the presence of spermatozoa in CM considerably increased its antibacterial effect. The activity was markedly higher in samples obtained within hours after intercourse compared with those taken after sexual abstinence of >/=5 days (P < 0.05). In microbially colonized CM specimens compared to sterile CM, all obtained under hormonally standardized conditions, the antibacterial activity in the agar plate test was significantly lower (P < 0.05). The results of this pilot study demonstrate the considerable antibacterial activity of human CM. PMID- 10739820 TI - Increased risk of non-insulin dependent diabetes mellitus, arterial hypertension and coronary artery disease in perimenopausal women with a history of the polycystic ovary syndrome. AB - The aim of the study was to determine the prevalence of non-insulin dependent diabetes mellitus (NIDDM), arterial hypertension, coronary artery disease and the risk factors for these diseases in perimenopausal women with a history of polycystic ovary syndrome (PCOS) treatment. A group of 28 women was selected from a large group of patients who had undergone wedge ovarian resection. A total of 752 controls was selected by age (45-59 years) from a random female population sample. There was no difference between the two groups in body mass index, waist circumference or waist-hip ratio. Both groups were found to have identical family histories of NIDDM, hypertension, and coronary artery disease and identical smoking habits. We did not find a difference between the mean concentrations of lipids and fasting glucose. The two groups did not differ in the proportions of women with elevated lipid concentrations. The prevalence of NIDDM and coronary artery disease was significantly higher in PCOS women. In conclusion, women in the general population have the same level of risk factors at perimenopausal age as PCOS women. Patients with markedly expressed clinical symptoms of PCOS made up a subgroup in the general population at high risk for developing NIDDM and coronary artery disease. PMID- 10739822 TI - Response to activated protein C during normal menstrual cycle and ovarian stimulation. AB - Oestrogen has been pointed out as a pre-thrombotic factor. Protein C is a key enzyme in the down-regulation of blood coagulation. Recent data suggest that activated protein C (APC) resistance which is not due to the factor V:Q 506 Leiden mutation and appears to be acquired, is also a risk factor for thrombosis. In this study, we evaluated the endogenous oestradiol production and its possible influence on APC. Eighteen normally menstruating women were studied during one ovulatory cycle. Furthermore, 20 women undergoing controlled ovarian stimulation, and achieving extremely high oestradiol concentrations, were investigated. Normalized APC (nAPC) ratio (clotting time of tested sample/clotting time of pooled control plasma) was measured. Samples collected on menstrual cycle days 1 3, 6-8, 13-14, 20-24 corresponded to nAPC ratios 1.02 +/- 0.19 (mean +/- SD), 1.05 +/- 0. 15, 1.02 +/- 0.16 and 1.03 +/- 0.21 respectively. During ovarian stimulation, the nAPC ratios were 0.99 +/- 0.12, 1.03 +/- 0.18, 1.01 +/- 0.16 and 0.97 +/- 0.13 at oestradiol minimum, days 5-8 pre-oocyte retrieval, oestradiol maximum and at oocyte retrieval respectively. In spite of the great difference in the concentrations of oestradiol between women in normal menstrual cycle and women undergoing ovarian stimulation, no difference in nAPC ratios was observed. PMID- 10739821 TI - Sigmoid endometriosis and ovarian stimulation. AB - In-vitro fertilization (IVF) and ovarian stimulation are frequently performed in patients with endometriosis. Although endometriosis is a hormone-dependent disease, the rate of IVF complications related to endometriosis is low. We report four cases of severe digestive complications due to the rapid growth of sigmoid endometriosis under ovarian stimulation. In three patients, sigmoid endometriosis was diagnosed at laparoscopy for sterility. Because of the absence of digestive symptoms or repercussion on the bowel, no bowel resection was performed before ovarian stimulation. All patients experienced severe digestive symptoms during ovarian stimulation, and a segmental sigmoid resection had to be performed. Analysis of endoscopic and radiological data demonstrated that bowel lesions of small size may rapidly enlarge and become highly symptomatic under ovarian stimulation. At immunohistochemistry, these infiltrating lesions displayed high populations of steroid receptors and a high proliferative index (Ki-67 activity), suggesting a strong dependence on circulating ovarian hormones and a potential for rapid growth under supraphysiological oestrogen concentrations. Clinicians should be aware of this rare but severe digestive complication of ovarian stimulation. The early diagnosis of such lesions may help the patients to avoid months of morbidity falsely attributed to ovarian stimulation side effects. Further experience is necessary to determine the optimal attitude when diagnosing a small and asymptomatic endometriotic bowel lesion before ovarian stimulation. PMID- 10739823 TI - A qualitative follow-up study of women's experiences with oocyte donation. AB - Oocyte donation is growing at an exponential rate. Currently, thousands of women donate each year. The health services that donors receive deserve evaluation. Thirty-three former donors were recruited from IVF clinics, a matching agency, the Internet, advertisements, and word of mouth. In-depth interviews were conducted to learn what motivated the donation, to determine how satisfied donors were with the experience and what issues played a role in donor satisfaction, and to identify recommendations to improve the process. None of the participants regretted their decision to donate, but they were not always completely satisfied with the donation experience. The physical process, compensation, quality of medical care, and level of involvement in the process were the primary factors that affected satisfaction. Matching agencies and IVF clinics may improve donor satisfaction by: minimizing trips to the clinic; using protocols that limit the number of intramuscular injections; reducing the risk of hyperstimulation syndrome; providing follow-up care; reimbursing for expenses such as lost work, travel, and child care; separating direct reimbursements from 'income' to decrease the amount of taxes donors must pay on compensation; treating donors with respect and appreciation; and informing them about the outcome. Improved donor satisfaction is likely to improve donor recruitment and retention. PMID- 10739824 TI - Vaginal sildenafil (Viagra): a preliminary report of a novel method to improve uterine artery blood flow and endometrial development in patients undergoing IVF. AB - Endometrial growth is thought to depend on uterine artery blood flow and the importance of endometrial development on in-vitro fertilization (IVF) outcome has been previously reported. Nitric oxide (NO) relaxes vascular smooth muscle through a cGMP-mediated pathway and NO synthase isoforms have been identified in the uterus. Sildenafil citrate (Viagra), a type 5-specific phosphodiesterase inhibitor, augments the vasodilatory effects of NO by preventing the degradation of cGMP. In this preliminary report we describe the use of vaginal sildenafil to improve uterine artery blood flow and sonographic endometrial appearance in four patients with prior failed assisted reproductive cycles due to poor endometrial response. The uterine artery pulsatility index (PI) was measured in a mock cycle after pituitary down-regulation with Lupron. The PI was decreased after 7 days of sildenafil (indicating increased blood flow) and returned to baseline following treatment with placebo. The combination of sildenafil and oestradiol valerate improved blood flow and endometrial thickness in all patients. These findings were reproduced in an ensuing gonadotrophin-stimulated cycle. Three of the four patients conceived. Although greater numbers of patients and randomized evaluation are needed to validate this treatment, vaginal sildenafil may be effective for improving uterine artery blood flow and endometrial development in IVF patients with prior poor endometrial response. PMID- 10739825 TI - Transmission risk of hepatitis C virus in assisted reproductive techniques. AB - Medical assistance for procreation in a couple where one or both parents has hepatitis C viral infection (HCV) raises the issue of the transmission of the infection to the baby and/or of possible contamination of both the technicians and the gametes or embryos from virus-free parents in the laboratory. It becomes essential to assess transmission risk in assisted reproductive techniques in order to define clearly the management of couples according to their viral status. To define the HCV transmissibility risk in assisted reproduction related to the presence of virus in semen from infected infertile men, HCV RNA detection was performed in sera, and semen and sperm fractions obtained after Percoll gradient centrifugation. HCV RNA was detected in 5% (2/39) of the semen samples tested: in the raw semen, in the seminal fluid and in the cell pellet but never after Percoll selection. According to these results, we suggest a strategy for HCV-infected infertile men who need assistance for procreation. PMID- 10739826 TI - Recurrent use of newer oral contraceptives and the risk of venous thromboembolism. AB - The epidemiological studies that assessed the risk of venous thromboembolism (VTE) associated with newer oral contraceptives (OC) did not distinguish between patterns of OC use, namely first-time users, repeaters and switchers. Data from a Transnational case-control study were used to assess the risk of VTE for the latter patterns of use, while accounting for duration of use. Over the period 1993-1996, 551 cases of VTE were identified in Germany and the UK along with 2066 controls. Totals of 128 cases and 650 controls were analysed for repeat use and 135 cases and 622 controls for switching patterns. The adjusted rate ratio of VTE for repeat users of third generation OC was 0.6 (95% CI:0.3-1.2) relative to repeat users of second generation pills, whereas it was 1.3 (95% CI:0.7-2.4) for switchers from second to third generation pills relative to switchers from third to second generation pills. We conclude that second and third generation agents are associated with equivalent risks of VTE when the same agent is used repeatedly after interruption periods or when users are switched between the two generations of pills. These analyses suggest that the higher risk observed for the newer OC in other studies may be the result of inadequate comparisons of pill users with different patterns of pill use. PMID- 10739827 TI - CDB-2914: anti-progestational/anti-glucocorticoid profile and post-coital anti fertility activity in rats and rabbits. AB - Our goal was to determine the endocrine and post-coital anti-fertility activity of CDB-2914. Concurrent administration of progesterone to rats on day 4 post mating blocked the anti-fertility activity of a single oral 2 mg dose of CDB 2914. CDB-2914 did not exhibit progestational activity in the oestradiol-primed immature female rabbit at doses that exhibited anti-progestational activity. CDB 2914 antagonized exogenous and endogenous progesterone-stimulated uterine haptoglobin synthesis and secretion in immature and adult mated rabbits respectively. Neither CDB-2914 nor mifepristone exhibited glucocorticoid activity as determined by thymus involution in rats; mifepristone was twice as potent as CDB-2914 in antagonizing glucocorticoid action. Post-coital CDB-2914 treatment resulted in a dose-dependent reduction in implantation sites and pregnancy rates in rabbits. CDB-2914-induced inhibition of uterine weight increase, endometrial glandular arborization and uterine haptoglobin synthesis/secretion correlated with inhibition of pregnancy in mated rabbits. A single oral dose of 64 mg CDB 2914/rabbit was effective at blocking pregnancy when administered on day 4, 5, or 6 post-mating, whereas 32 mg/rabbit was only partially effective in this regard. These data demonstrate that CDB-2914 is a potent, orally active anti-progestin with weak anti-glucocorticoid activity. CDB-2914 inhibited implantation in adult rats and rabbits demonstrating its potential as a post-coital contraceptive drug. PMID- 10739828 TI - Study of apoptotic DNA fragmentation in human spermatozoa. AB - The aim of our work was to define and better understand apoptosis in the spermatozoa of normal subjects, infertile patients and patients affected by specific tumoral diseases employing the method of the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling and confirming the results by electron microscopy. We studied 23 healthy, normozoospermic subjects (group A), 29 oligoasthenoteratozoospermic patients, affected by various andrological pathologies (group B), 28 patients with Hodgkin's disease (C1) and 30 patients with testicular cancer (C2). Our data demonstrate that the percentage of apoptosis in normozoospermic subjects (group A) is significantly lower than in all the other groups (B, C1, C2) (P < 0.001). This confirms that high DNA fragmentation is one of the characteristics of spermatogenetic failure. The induction of apoptosis, which can also be a basic response to neoplastic disease, can even act right up to the mature male gamete. Our results suggest that apoptosis could be the final result of various pathologies and of a deregulation of spermatogenesis control systems. PMID- 10739830 TI - Laser-induced immobilization and plasma membrane permeabilization in human spermatozoa. AB - We evaluated the potential use of a non-contact, 1.48 microm wavelength diode laser for immobilization of human spermatozoa and permeabilization of the sperm membrane in different culture media. When we applied a single laser shot near to the middle region of the sperm tail, spermatozoa could be immobilized either temporarily or permanently, depending on the energy used. Above an energy of 2 mJ in polyvinylpyrrolidone and 2-3 mJ in culture medium, a reliable permanent immobilization was achieved by permeabilization of the sperm tail membrane. We then explored the use of a double laser shot technique. Spermatozoa were temporarily immobilized by a first laser shot applied near to the sperm tail followed by permeabilization with a second laser shot aimed directly at the sperm tail. This sequential approach yielded permanent immobilization at much lower energy values compared with the single shot technique. Following the injection of laser-treated spermatozoa, mouse oocytes underwent normal activation and pronuclear formation. We conclude that a non-contact 1.48 microm diode laser system can be used for immobilization of spermatozoa and for permeabilization of the sperm tail membrane. This laser procedure may offer an alternative to currently used sperm pretreatment prior to intracytoplasmic sperm injection. PMID- 10739829 TI - Relationship between semen quality and the seminal plasma components carnitine, alpha-glucosidase, fructose, citrate and granulocyte elastase in infertile men compared with a normal population. AB - The seminal plasma components neutral alpha-glucosidase, carnitine, fructose, citrate, and polymorphonuclear granulocyte (PMN) elastase in 253 men were determined. The seminal plasma of 221 infertile men, a control group with proved fertility and 13 patients after vasectomy were investigated. The concentrations of free carnitine (212 versus 521 micromol/l, n = 219, P < 0.001), total carnitine (437 versus 743 micromol/l, n = 219, P < 0.001), and the activity of neutral alpha-glucosidase (15.1 versus 23.4 IU/l, n = 236, P < 0.05) were significantly reduced in the infertile patient group as compared to the fertile control group, the concentration of PMN elastase (102 versus 48 microg/l, n = 234, P < 0.05) being significantly increased in the infertile patients. In the patients after vasectomy the activity of neutral alpha-glucosidase was the only epididymal marker that was significantly reduced (4.3 versus 9. 8 IU/l, n = 35, P = 0.002) in comparison with the patients with testicular azoospermia. At a limit of 6 IU/l the sensitivity of the method was 92% and the specificity was 72%. Altogether, the epididymal markers were reduced in subfertile patients compared with the control group. For the differential diagnosis of azoospermia only the determination of the neutral alpha-glucosidase activity is useful. PMID- 10739831 TI - Detection of chromosomal abnormalities by fluorescent in-situ hybridization in immotile viable spermatozoa determined by hypo-osmotic sperm swelling test. AB - If randomly selected immotile spermatozoa are used for intracytoplasmic sperm injection (ICSI), pregnancy rates are significantly decreased. The hypo-osmotic swelling test (HOST) is the only method available to detect the viable, but immotile spermatozoa for ICSI. However, evidence is still lacking for the chromosomal abnormalities for the normal-looking, but immotile spermatozoa positive for HOST. Sperm samples from 20 infertile men with normal chromosomal constitution were obtained. After Percoll separation, morphologically normal but immotile spermatozoa were transported individually into HOST solution for 1 min using micropipettes. Cells that showed tail curling with swelling in HOST were then transferred back into human tubal fluid solution to allow reversal of swelling. These sperm cells were fixed and processed for the multi-colour fluorescence in-situ hybridization (FISH) for chromosomes X, Y and 18. The same FISH procedure was applied for the motile spermatozoa from the same cohort, which formed the control group. The average aneuploidy rates were 1.70 and 1.54% in 1000 HOST positive immotile and motile spermatozoa respectively detected by FISH for each patient. Our results indicate that morphologically normal, immotile but viable spermatozoa have an aneuploidy rate similar to that of normal motile spermatozoa. PMID- 10739832 TI - The use of silane-coated silica particles for density gradient centrifugation in in-vitro fertilization. AB - Silane-coated silica particles (PureSperm) were evaluated as an alternative to Percoll for gradient separation of spermatozoa, for use in assisted reproduction. Recovery of motile and morphologically normal spermatozoa after using a four layer Percoll and a two- and four-layer PureSperm gradient respectively was recorded. In-vitro fertilization (IVF) results after using PureSperm for the sperm preparation were also evaluated. No difference in sperm recovery or sperm motility was found when comparing the use of Percoll and the four-layer gradient of PureSperm. When using a two-layer PureSperm gradient, motility was significantly decreased (P < 0.05) compared to Percoll. Normal sperm morphology increased from 8-17.2% after using Percoll and to 12.7% and 11.4% after using a four-layer and a two-layer PureSperm gradient respectively. All gradient preparations showed a significant decrease in the teratozoospermia index compared to the ejaculate (P < 0.01). No significant differences in IVF results regarding fertilization and pregnancy rates were found when PureSperm or the swim-up technique were used for the sperm preparation. PureSperm seems to be an acceptable alternative to Percoll but although the percentage of sperm recovery was higher after PureSperm we still recommend the swim-up technique to be the first choice, as a higher percentage of progressive motile spermatozoa is obtained without using other chemicals than IVF culture medium. PMID- 10739833 TI - Birth after treatment of a male with seminoma and azoospermia with cryopreserved thawed testicular tissue. AB - The case of an infertile couple in which a testicular seminoma and azoospermia were discovered in the husband during infertility treatment is described. A small piece of testicular tissue, obtained by biopsy from the healthy testis [testicular sperm extraction (TESE)], was deep-frozen before oncology therapy was initiated. The patient's lymphocyte karyotype was normal and no Y microdeletions were found. After conclusion of oncology treatment, the tissue was thawed and successfully used in the intracytoplasmic sperm injection (ICSI) procedure. A healthy girl was born. Testicular tumours are known to impair fertility in the majority of patients, and fertility deteriorates further after cytotoxic and surgical oncology treatment. Until recently in Slovenia, for young oncology patients cryopreservation was applied only to high quality ejaculate fulfilling the criteria for intrauterine insemination or in-vitro fertilization after thawing. Failing that, the only remaining options were fertilization by donor spermatozoa or child adoption. New assisted reproductive technologies, of which the ICSI procedure is the most successful, are suitable for the treatment of only the most severe cases of male infertility. It is reasonable to cryopreserve even poor quality ejaculate prior to the oncology therapy, as well as testicular tissue in cases of azoospermia. PMID- 10739834 TI - Bipolar coagulation with small diameter forceps in animal models for in-utero cord obliteration. AB - The objective of this study was to evaluate the feasibility, efficacy and safety of bipolar coagulation using endoscopic forceps of diameters of 2.7 mm and less in animal models mimicking in-utero conditions. Forceps of 2.2, 2.3, 2.5 and 2.7 mm were tested in rabbits (n = 36). Vessel pairs were dissected and coagulated in a fluid environment under endoscopic vision at 15 and 25 W. The main outcome measure was the perforation rate. In fetal lambs (n = 25), umbilical cords were coagulated under sono-endoscopic control with power settings from 10 to 35 W. Main outcome measures were the duration of coagulation, perforation rate, change in the temperature of the amniotic fluid and efficacy of vessel occlusion rate. At 20-25 W, all cords were coagulated successfully without any perforation using 2.3, 2.5 or 2.7 mm forceps. Coagulation with the 2. 2 mm forceps was associated with a high perforation rate, although the design rather than the diameter of the forceps may have influenced this outcome. Bipolar coagulation with forceps between 2. 3 and 2.7 mm and appropriate power settings achieves efficacious and safe coagulation in animal models for umbilical cord occlusion. PMID- 10739835 TI - Pregnancy outcome and deliveries following laparoscopic myomectomy. AB - Uterine rupture after myomectomy by laparotomy is not a common occurrence. Some case reports of uterine rupture after laparoscopic myomectomy (LM) raise the question of the quality of the uterine scar produced when this technique is performed. In order to assess the outcome of pregnancies and deliveries after LM and to assess the risk of uterine rupture, we performed an observational study. Questionnaires were mailed to all women who had had LM for at least one intramural or subserosal myoma of more than 20 mm diameter and who were aged <45 years. Ninety-eight patients became pregnant at least once after LM, giving a total of 145 pregnancies. Among the 100 patients who had delivery, there were three cases of spontaneous uterine rupture. Because only one of these uterine ruptures occurred on the LM scar, the risk of uterine rupture was 1.0% (95% CI 0.0-5. 5%). Seventy-two patients (72.0%) had trials of labour. Of these, 58 (80.6%) were delivered vaginally. There was no uterine rupture during the trials of labour. Spontaneous uterine rupture seems to be rare after LM. This risk should not deter the use of LM if needed. When performing LM, particular care must be given to the uterine closure. PMID- 10739836 TI - Post-implantation development of mouse androgenetic embryos produced by in-vitro fertilization of enucleated oocytes. AB - We report here on the precise ability of mouse androgenetic embryos produced by in-vitro fertilization of enucleated oocytes to develop to day 9.5 of gestation when cultured with M16 and CZB media. Androgenetic embryos cultured with CZB rather than M16 medium developed to the blastocyst stage in a more significant proportion (56.6% versus 45.0%, P < 0.001). However, after cavitation, the rate of cell proliferation of androgenetic embryos cultured with CZB medium was significantly decreased (P < 0.05). Embryo transfer experiments showed that blastocysts cultured with M16 medium were superior to those cultured with CZB medium in their ability to develop to 9.5-day-old fetuses (28.1% versus 11.1%, P < 0.001). These results showed that the present procedure for producing androgenetic mouse embryos is reliable and that M16 medium is superior for culturing the embryos. Fetal sexing by polymerase chain reaction (PCR) also demonstrated that both XX and XY embryos develop to 9.5-day fetuses at theoretical rates (1:2). This is the first finding that mouse XX androgenones survive after implantation. PMID- 10739837 TI - Cellular characterization of blastocysts derived from rabbit 4-, 8- and 16-cell embryos and isolated blastomeres cultured in vitro. AB - The purpose of this study was to investigate the developmental potential of isolated rabbit blastomeres under various culture conditions to gain insight into their ability to form the two cell lineages of a viable blastocyst. Intact embryos at the 4-cell, 8-cell, 16-cell stages and blastomeres isolated from 4-, 8 and 16-cell rabbit embryos (1/4, 1/8 or 1/16 blastomeres respectively) were cultured in drops of one of three different media, each supplemented with either fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA). The effects of the extracellular matrix fibronectin (FN) on the development of isolated rabbit blastomeres were also investigated. Supplementation of the medium with FCS yielded a higher (P < 0.05) proportion of blastocysts than BSA or PVA, predominantly from 1/4 blastomeres. No major differences were found between the three basic culture media. In 1/4, 1/8 or 1/16 blastomeres, blastocyst formation rates were greater (P < 0.05) in groups cultured in matrix-free (54.5, 59.6 and 54.6% respectively) than in FN-coated groups (35.4, 46.0 and 26.1% respectively). Only in blastocysts derived from 1/4 blastomeres, were the numbers of inner cell mass (ICM) and total cells of blastocysts higher (P < 0.05) in FN-coated groups than in matrix-free groups (12.7 +/- 1.1 versus 8.5 +/- 0.7 ICM, 73.8 +/- 3. 7 versus 57.8 +/- 3.3 total cells). The percentage of blastocysts derived from single blastomeres with ICM cells decreased with increasing cell stage of the parent embryos in FN-coated (93.6, 78.3 and 44.0%, respectively) as well as matrix-free groups (96.2, 69.3 and 55.2%). In FN-coated groups, after 96 h (1/4) or 72 h (1/8 and 1/16) of culture, approximately 20-30% of blastomeres did not develop into normal blastocysts but formed sheets with 30-50 cells attached to the bottom of the dishes. These results indicate that the development of rabbit blastomeres shares important characteristics with those from mouse and domestic species and may thus aid in developing an efficient culture system for blastomeres, derived from human embryos. PMID- 10739838 TI - Human zona pellucida micromanipulation and monozygotic twinning frequency after IVF. AB - To assess the association of zona pellucida micromanipulation and subsequent development of monozygotic twins, cases of assisted embryo hatching (AH) and intracytoplasmic sperm injection (ICSI) were identified and related to treatment type, implantation and zygosity data. Embryology records from all patients undergoing in-vitro fertilization (IVF) at this centre from January 1995 to March 1998 were reviewed. In this study, 3546 transfer cycles were completed, with clinical pregnancy established in 1911 (54% per transfer) patients undergoing a single IVF cycle. These pregnancies occurred in 1674 (88%) IVF cycles, 120 (6%) donor oocyte cycles (DER), and 117 (6%) frozen embryo transfer (FET) cycles. During the study period, 23 cases of monozygotic (MZ) twins were identified, representing an overall frequency of 1.2%. Chorionicity was determined by transvaginal ultrasound at 7 weeks when the number of embryos transferred was less than the number of fetal heart-beats, or when >1 fetal heartbeat per gestational sac was seen. Zygosity was confirmed by placental evaluation at delivery, and corroborated the antenatal diagnosis in all cases. Among IVF study patients the frequency of MZ twinning was not statistically different between zona manipulated and zona intact subgroups. While this investigation is the largest to date describing the relationship between MZ twins and zona procedures, studies with even greater statistical power are needed to clarify it more precisely, particularly in DER and FET settings. A greater overall frequency of MZ twinning for IVF patients may be a function of the higher number of embryos transferred in IVF, rather than discrete zona manipulations. PMID- 10739839 TI - Intracellular pH regulation in human preimplantation embryos. AB - We report here that intracellular pH (pH(i)) in cleavage-stage human embryos (2-8 cell) is regulated by at least two mechanisms: the HCO(3)(-)/Cl(-) exchanger (relieves alkalosis) and the Na(+)/H(+) antiporter (relieves acidosis). The mean pH(i) of cleavage-stage embryos was 7.12 +/- 0.008 (n = 199) with little variation between different stages. Embryos demonstrated robust recovery from alkalosis that was appropriately Cl(-)-dependent, indicating the presence of the HCO(3)(-)/Cl(-) exchanger. This was further confirmed by measuring the rate of intracellular alkalinization upon Cl(-) removal, which was markedly inhibited by the anion transport inhibitor, 4,4'-diisocyanatostilbene-2,2'-disulphonic acid, disodium salt. The set-point of the HCO(3)(-)/Cl(-) exchanger was between pH(i) 7.2 and 7.3. Embryos also exhibited Na(+)-dependent recovery from intracellular acidosis. Na(+)/H(+) antiporter activity appeared to regulate recovery up to about pH(i) 6.8; this recovery was HCO(3)(-)-independent and amiloride-sensitive, with a pH(i) set-point of approximately 6.8-6.9. A second system that was both Na(+)- and HCO(3)(-)-dependent appeared to mediate further recovery from acidosis up to about pH(i) 7.1. Thus, pH(i) of early human preimplantation embryos appears to be regulated by opposing mechanisms (HCO(3)(-)/Cl(-) exchanger, Na(+)/H(+) antiporter, and possibly a third acid-alleviating transporter that was both Na(+) and HCO(3)(-)-dependent) resulting in the maintenance of pH(i) within a narrow range. PMID- 10739840 TI - Comparison of ethylene glycol, 1,2-propanediol and glycerol for cryopreservation of slow-cooled mouse zygotes, 4-cell embryos and blastocysts. AB - The aim of the study was to analyse the toxicity, the osmolar and cryoprotective activity of ethylene glycol (ETG) in terms of survival rate (SR), cleavage rate (CR) and expanded blastocysts percentage (EBP) of mouse embryos. Early mouse embryos and blastocysts were slowly cooled with ETG, 1,2-propanediol (PROH) or glycerol, and thawed. The Van t'Hoff curve for 1.5 mol/l ETG showed recovery of initial volume within 4 min. No differences were observed in CR and EBP of ETG exposed compared with non-exposed mouse zygotes. The SR of zygotes frozen with PROH was significantly better than with ETG (92% and 60% respectively; P < 0.01), and a significantly better EBP was achieved for blastocysts frozen with glycerol compared with ETG (75% and 50% respectively; P < 0.05). For 4-cell stage embryos, no differences were observed in SR and EBP between ETG and PROH. Higher EBP was observed for 4-cell stage embryos (53%) frozen with ETG compared with pronucleate stage (19%) and blastocysts (48%). Low toxicity, good SR and EBP were observed for mouse embryos frozen with ETG, the best results being obtained at the 4-cell stage. At other embryonic stages, PROH and glycerol respectively seemed to provide better results. PMID- 10739841 TI - A microsurgical technique for enucleation of multipronuclear human zygotes. AB - Tripronuclear human zygotes were used to determine the feasibility of an enucleation procedure without the use of cytoskeleton inhibitors to obtain intact cytoplasts for nuclear transfer research. Of 61 tripronuclear human zygotes manipulated, 100% of the zygotes survived after removal of one pronucleus and 90.1% after complete enucleation, proving the reliability of the proposed microsurgical technique. Morphological changes were observed in the resulting cytoplasts during extended culture. At 36 h post-insemination, 87.5% of cultured cytoplasts showed morphological changes, which included cleavage into two 'blastomeres', three 'blastomeres' with various degrees of fragmentation, one cell with fragments or gross fragmentation. Comparison with the diploid human zygotes from the same harvests, which had undergone the first cleavage division by this time, showed similar timing in cleavage suggesting autonomous cytoplasmic activity in human zygotes. PMID- 10739843 TI - Association of molecular variants of luteinizing hormone with male infertility. AB - Luteinizing hormone (LH) stimulates the interstitial Leydig cells to produce testosterone, which is essential for spermatogenesis. Abnormalities in the function of LH may affect the process of spermatogenesis and thus result in infertility. The aim of this study was to determine the association of three known variants of LH (Gln54Arg [Trp8Arg; Ile15Thr] and Gly102Ser) with male infertility. A total of 145 infertile men and 200 healthy fertile men were recruited and screened for the presence of these three LH variants. The Gln54Arg variant could not be detected in either of the groups studied. Twelve infertile (8.2%) and 15 fertile (7.5%) men were found to carry the [Trp8Ile; I15Thr] variant, but its occurrence did not show any significant difference between the patient and control groups. The Gly102Ser variant was detected in five patients with infertility (3.4%), but not in the control subjects (P = 0.013). This study showed that the Gln54Arg and [Trp8Ile; I15Thr] variants in the LHbeta gene were not associated with male infertility, whereas the Gly102Ser variant might be implicated in infertility in some Singapore Chinese men. PMID- 10739842 TI - Inhibition by human embryos of mouse granulosa cell progesterone production: development of a sensitive bioassay. AB - Reproduction technologies could be improved by the development of methods to evaluate oocyte or embryo quality in a non-invasive, quantitative manner. Since human embryos secrete a factor that inhibits granulosa cell progesterone production, an interspecies bioassay was established to investigate whether the presence of this progesterone-inhibitory factor (PIF) in human embryo-conditioned (HEC) media is related to the health and developmental capacity of the embryos. Oocytes were microsurgically removed from oocyte-cumulus complexes isolated from superovulated mouse ovaries, and the oocytectomized complexes were cultured in HEC media in the presence of follicle stimulating hormone and testosterone. Progesterone accumulation in the media was determined by radioimmunoassay. Despite the potential limitations of very small volumes of HEC media to evaluate, and the need to freeze these media at the source, the bioassay was able to detect PIF activity in HEC media. Most embryos produced PIF activity, but the degree of inhibition was not correlated with the ability of oocytes to be fertilized, nor with embryo morphology or ability to cleave and develop after transfer. These results demonstrate that secretion of PIF by human embryos can be measured by this bioassay and that human PIF can inhibit murine granulosa cell steroidogenesis; however, PIF activity is not correlated with human embryo quality or developmental competence. PMID- 10739844 TI - Rapid determination of zygosity and common aneuploidies from amniotic fluid cells using quantitative fluorescent polymerase chain reaction following genetic amniocentesis in multiple pregnancies. AB - Following second-trimester twin amniocentesis, we used quantitative fluorescent polymerase chain reaction (QF-PCR) assays and polymorphic small tandem repeats (STR) for rapid determination of zygosity and common aneuploidies from amniotic fluid (AF) cells in four pregnancies with like-sex twins, fused placentae and inconclusive chorionicity. The first and the second cases were suspected to have inadvertent sampling of the same amniotic cavity twice. The first case showed a dizygotic (DZ) pattern and repeat amniocentesis was thus avoided. The second case was monozygotic (MZ) and was complicated by discordant fetal growth and twin-twin transfusion syndrome. The third case was associated with a co-twin malformation, occipital encephalocele. DNA studies revealed MZ twinning with a discordant structural defect. The fourth case was associated with co-twin abnormalities of cystic hygroma and hydrops fetalis. DNA studies showed DZ twinning with discordant structural and chromosomal defects. The QF-PCR assay with STR has the advantages of rapid determination of zygosity and common aneuploidies in AF cells. This simple test appears to be useful in the instances of possible inadvertent puncture of the same amniotic cavity twice during amniocentesis and of discordant fetal structural and/or chromosomal abnormalities following genetic amniocentesis in multiple pregnancies with uncertain chorionicity. PMID- 10739845 TI - Obstetric outcome of twin pregnancies after in-vitro fertilization: a matched control study in four Dutch university hospitals. AB - In singleton pregnancies after in-vitro fertilization (IVF), increased rates of obstetric and perinatal complications have been reported. Studies that compared IVF twin pregnancies with spontaneously conceived twins have yielded conflicting results. We compared 96 IVF twin pregnancies to 96 controls after elaborate matching. The design of our study precluded matching by zygosity. The monozygosity rate was higher in the control group and this implies that beforehand the risk for a less favourable outcome in the control group was higher than in the IVF group. However, the average birthweight of the IVF children was less than that of children in the control group (P = 0.04). This was not due to more intrauterine growth retardation in the IVF group. The mean gestational age at birth was 5 days shorter in IVF than control pregnancies, and although this difference was not significant it might explain the lower birthweight in the IVF group. The discordance rate in the IVF group was significantly increased. We found no difference in perinatal mortality and morbidity. We conclude that this study provides further evidence for a different outcome of IVF twin pregnancies in comparison with spontaneously conceived twin pregnancies. PMID- 10739846 TI - Mannan-binding lectin concentration during normal human pregnancy. AB - Relative deficiency of mannan-binding lectin (MBL) is associated with recurrent miscarriage. To investigate whether MBL concentration alters as a result of pregnancy, serial measurements were made in 14 patients before and during normal early pregnancy. Additionally, a longitudinal study was made of one individual over several years and including two normal pregnancies. Three (20%) subjects experienced a significant increase in MBL concentration during pregnancy. It was concluded that a modest but significant increase in MBL concentration can result from pregnancy, but it is a rare occurrence in the first trimester. PMID- 10739847 TI - Incidence of congenital malformations in children born after ICSI. AB - The aim of this study was to determine the incidence of congenital malformations in a complete cohort of children born after intracytoplasmic sperm injection (ICSI). The medical records were retrieved for 1139 infants, 736 singletons, 200 sets of twins and one set of triplets. The total number of infants with an identified anomaly was 87 (7.6%), 40 of which were minor. The incidence of malformations in children born after ICSI was also compared with all births in Sweden using data from the Swedish Medical Birth Registry and the Registry of Congenital Malformations. For ICSI children, the odds ratio (OR) for having any major or minor malformation was 1.75 [95% confidence interval (CI) 1.19-2.58] after stratification for delivery hospital, year of birth and maternal age. If stratification for singletons/twins was also done, the OR was reduced to 1.19 (95% CI 0.79-1.81). The increased rate of congenital malformations is thus mainly a result of a high rate of multiple births. The only specific malformation which was found to occur in excess in children born after ICSI was hypospadias (relative risk 3.0, exact 95% CI 1. 09-6.50) which may be related to paternal subfertility. PMID- 10739848 TI - Pregnancy and sex steroid hormones enhance circulating calcitonin gene-related peptide concentrations in rats. AB - Calcitonin-gene-related peptide (CGRP) is a 37 amino acid neuropeptide synthesized primarily in dorsal root ganglia (DRG) and distributed widely in the perivascular nerves, suggesting that this peptide may play a role in the regulation of peripheral vascular tone. Since female sex steroid hormones have been implicated in the regulation of peripheral vascular tone during pregnancy, we postulated that they may alter the concentration of CGRP in the circulation and thus modulate the increased blood flow observed during pregnancy. In the present study, we measured changes in plasma concentrations of CGRP in non pregnant, pregnant, and post-partum rats. Groups of ovariectomized rats were treated s.c. for 3 days either with 17beta-oestradiol (2.5 microg per injection twice daily), progesterone (2 mg per injection twice daily), or vehicle. Another group of adult, non-pregnant rats at dioestrus stage of the oestrous cycle was also used in this study. Plasma concentrations of CGRP were higher (P < 0.05) in rats on day 19 of pregnancy (22.0 +/- 3.0 pmol/l) compared to that during delivery (5. 0 +/- 2.0), post-partum day 2 (2.0 +/- 0.7) or in non-pregnant (4.9 +/- 1.6) state. Furthermore, in adult ovariectomized (6.0 +/- 0.6) rats, plasma CGRP concentrations were increased significantly (P < 0. 05) by oestradiol (10.0 +/- 1.0), progesterone (9.5 +/- 1.0) and oestradiol + progesterone (14.0 +/- 1.0). Thus, circulating concentrations of CGRP are elevated during pregnancy and by oestrogen and progesterone, suggesting that the elevated concentrations of CGRP may play an important role in vascular adaptations that occur during pregnancy. PMID- 10739849 TI - Maternal homocysteine and chorionic vascularization in recurrent early pregnancy loss. AB - Defective chorionic villous vascularization has been suggested to be associated with embryonic death. There are no reports, however, describing chorionic vascular profiles in spontaneous miscarriage tissue. Therefore, we investigated chorionic villous vascularization by both histopathology and an image analysis system combined with CD34 immunohistochemistry in spontaneous miscarriage tissue of 19 women with recurrent early pregnancy loss (REPL). Subsequently, we studied the vascular profile parameters (median vascular area, perimeter, number of vascular elements per measured chorionic area, and the median area, perimeter and diameter per vascular element) in relation to the maternal plasma total homocysteine concentrations (an independent risk factor for REPL). The histopathological scores and the measured number of vascular elements per mm(2) chorionic tissue were not significantly different between women with elevated and those with normal total homocysteine concentrations. However, women with elevated total homocysteine concentrations (fasting >18.3 micromol/l and/or 6 h after methionine load >61.5 micromol/l) showed (per measured chorionic area) significant smaller median vascular areas and perimeters. The single chorionic vascular elements in these women had significant smaller median areas, perimeters and diameters. Furthermore, the fasting total homocysteine was negatively correlated with the perimeter of the vascular element (r = -0.54: P <0.05). In conclusion, in REPL, elevated maternal total homocysteine concentrations are associated with defective chorionic villous vascularization. PMID- 10739850 TI - Influence of oestrous cycle and pregnancy on the reactivity of the rat mesenteric vascular bed. AB - In isolated, perfused mesenteric vascular beds from female rats, it was assessed whether the constrictor response to cirazoline, an alpha(1)-adrenergic agonist, or acetylcholine (ACh)-induced relaxation was altered by oestrous cycle or pregnancy and the ability of nitric oxide (NO), prostanoids and endothelium derived hyperpolarizing factor (EDHF) to modulate these responses. Mesenteries, removed from female rats on each oestrous cycle day and gestation day 16, were perfused with physiological salt solution. Tone was induced with cirazoline (1 micromol/l), and concentration-response curves to ACh generated. Responsiveness to ACh was tested in the presence of N(omega)-nitro-L-arginine (L-NA), ibuprofen (IBU) and tetrabutylammonium (TBA), to inhibit nitric oxide synthase (NOS), cyclo oxygenase and K(+) channels respectively. Cirazoline-induced tone was smaller in pro-oestrous and pregnant groups, but the increase in tone to L-NA was larger in pregnant compared with oestrous and dioestrous groups. Control responses to ACh were not different, but L-NA attenuated the response in virgin groups only. IBU did not affect the ACh response, but TBA attenuated it in all groups. When TBA was introduced first, ACh-induced dilatation was significantly reduced and not altered by L-NA addition. These results suggest that in the mesenteric vascular bed from cycling and pregnant rats, EDHF is the major mediator of ACh-induced dilatation and NOS may be up-regulated in pregnant and pro-oestrous rats. PMID- 10739851 TI - Congenital malformations after intracytoplasmic injection of spermatids. AB - Spermatid microinjection into oocytes was applied in cases of intracytoplasmic sperm injection (ICSI)/testicular sperm extraction (TESE) where no spermatozoa could be found in numerous testicular samples. Although several pregnancies were obtained with this procedure, serious concerns remain regarding its safety. Although the relevance of the injection of spermatids is by no means certain, we wish to report that from four pregnancies obtained after injection of elongated spermatids, two cases of major malformation resulted. PMID- 10739852 TI - The causes of preterm birth. PMID- 10739853 TI - Endogenous LH surge detection versus administration of HCG to correctly time intrauterine insemination: which provides a better pregnancy rate? PMID- 10739854 TI - Human reproductive failure is not a clinical feature associated with beta(2) glycoprotein-I antibodies in anticardiolipin and lupus anticoagulant seronegative patients. PMID- 10739855 TI - Changes in whole saliva in patients with coeliac disease. AB - Many systemic diseases impair salivary flow rate and composition and therefore incite oral pathological processes. This study analyses the composition of whole saliva in patients with diagnosed coeliac disease (CD) and in healthy controls, and monitors possible changes in saliva composition after a short oral gluten challenge. Paraffin-stimulated whole saliva was collected from 128 CD patients and 55 healthy controls. In a separate study, paraffin-stimulated whole saliva samples were collected from 33 CD patients and 10 controls both before and 24 h after an oral mucosal and submucosal gluten challenge. No difference in saliva flow rate was observed, but total protein (P 450 showed an average measurement of less than 15 microns, and 10 of 14 cases with a cardiac score < or = 450 showed an average measurement of > 15 microns = 0.0498). Histologic and ultrastructural evidence of collagen damage was identified in the pericardium from the 900-g heart with the 1,000-ml effusion. CONCLUSIONS: We propose that collagen stretching and slippage of obliquely oriented collagen layers contribute to the increased surface area needed to accommodate larger volumes. When these limits are exceeded, collagen damage ensues. PMID- 10739901 TI - An ultrastructural study of cardiac myocytes in postmyocardial infarction ventricular aneurysm representative of chronic ischemic myocardium using semiquantitative and quantitative assessment. AB - To elucidate the characteristic myocytic changes in chronic ischemic myocardium, an electronmicroscopic (EM) study was carried out in surgically excised postmyocardial infarction left ventricular aneurysm (LVAN) (n = 15) using semiquantitative (in all 15 cases) and quantitative assessment methods (in 10 cases). The control group, representing normal ultrastructure, included left ventricular endomyocardial biopsy specimens of sick sinus syndrome (SSS) (n = 3), Wolff-Parkinson-White (WPW) Syndrome (n = 1), and intraoperative left ventricular endomyocardial biopsy in mitral stenosis (MS) (n = 3). Myocardial condition was assessed at the ultrastructural level according to the severity of morphologic changes, first semiquantitatively, thereafter with the use of the Image Processor Analyzer LUZEX III for morphometric analysis. The most marked EM findings were mitochondrial regressive changes, glycogen accumulation, nuclear deformities, increased rough-surfaced endoplasmic reticulum at the perinuclear portion, lysis of myofibrils, and myofibrillar degeneration. The quantitative analysis revealed significant (p < 0.05) increase of glycogen deposition, only at the perinuclear portion in the LVAN group. The myofibril to mitochondria ratio at the intercalated disc (ID) portion of the cardiac myocytes significantly increased (p < 0.01) in the LVAN group as compared with the normal control group. Light microscopically evaluated quantitative analysis, using toluidine-blue stained semithin sections which underwent EM observation, showed that the fractional area of interstitial fibrous tissue was significantly increased in the LVAN group compared to the normal controls (p < 0.01). These results signify that in chronic ischemic myocardium, decreased consumption of glycogen in oxidative phosphorylation occurs in the surviving myocytes, and that hypertrophy of the myocytes appears. The results of this study may lead to the proper ultrastructural interpretation of biopsied human myocardium, regardless of etiology. PMID- 10739903 TI - Structural changes of atrial myocardium during chronic atrial fibrillation. AB - Of all known arrhythmia's, atrial fibrillation (AF) is the most often met in the clinical setting and it is associated with an increase in mortality risk. Several risk factors for AF have been described and several mechanisms of induction and maintenance have been proposed. Studies in patients with AF have shown that structural changes occur in the atria, but the relationship between the structural remodelling and the chronicity of the arrhythmia are not well understood. The changes mainly concern adaptive (dedifferentiation of cardiomyocytes) and maladaptive (degeneration of cells with replacement fibrosis) features. In order to characterise the time course of the structural remodelling the need for animal models which adequately mimic chronic atrial fibrillation in humans is felt essential. In this review, the structural changes that are observed during prolonged sustained AF in patients and animal models, are described. Furthermore, the time course and potential mechanisms of structural remodelling are discussed and methods for elucidation of the underlying molecular mechanisms are presented. PMID- 10739904 TI - Symptomatic solitary right renal vein aneurysm: a case report. AB - Renal vein aneurysms are very uncommon. Those located in the right side are exceptional. A symptomatic, solitary, voluminous aneurysm of the right renal vein in a 33-year-old man is reported. The lesion was diagnosed preoperatively as a renal tumor. The pathologic study of the nephrectomy specimen disclosed a thrombosed saccular aneurysm measuring 5.5 cm with marked medial atrophy of its wall. Recognition that a mass like this reported is of vascular origin is very important to avoid percutaneous biopsy which could be potentially hazardous; and for undertaking conservative prophylactic surgery. Renal venography is indicated for the diagnosis of these lesions. PMID- 10739905 TI - In situ interleukin-6 transcription in embryonic nonmuscle myosin heavy chain expressing immature mesenchyme cells of cardiac myxoma. AB - Cardiac myxomas are benign tumors which sometimes secrete interleukin-6 (IL-6), however, the pathogenesis and the IL-6 secreting cells are not clear. There are vascular myosin heavy chain isoforms; SM2 expression is specific to mature smooth muscle cells, while SMemb is a nonmuscle-type isoform which is expressed in immature mesenchyme cells. We hypothesized that immature mesenchyme cells play pivotal roles in the secretion of IL-6; we studied these expression in resected samples of myxoma. SMemb expression was increased but SM2 expression was not in the channels of myxoma. Increased IL-6 transcription was observed in the SMemb expressing cells in the channel. Therefore, mesenchyme cells with immature phenotype in the channel play pivotal roles of inflammation and pathogenesis of cardiac myxoma. PMID- 10739906 TI - In utero development of hypertensive necrotizing pulmonary arterial lesions: report of a case associated with premature closure of the ductus arteriosus and pulmonary hypoplasia. AB - Premature closure of the ductus arteriosus (PCDA) is an uncommon defect in which pulmonary hypertension (PH) has been documented by echocardiography in patients and by direct measurement after experimental PCDA in animals. The pulmonary vascular histology in human cases has received little attention but in the few recorded observations the vessels were either normal or showed increased muscularity. We report the case of a 31 week hydropic female stillborn monozygotic twin in whom postmortem examination disclosed PCDA and hypoplasia of the lungs. Atypical plexiform lesions with necrotizing pulmonary arteritis were present. These lesions represent vascular consequences of severe pulmonary hypertension produced by greatly enhanced blood flow through a restricted vascular bed resulting from the combined effects of these two abnormalities. The findings in this case of PCDA with presumed severe PH indicate that severe pulmonary vascular changes can develop in utero and that the interval of time needed for development of such chances in secondary PH is relatively short. PMID- 10739907 TI - Anaplastic large cell lymphoma (CD 30+), T-phenotype, in the heart of an HIV positive man. AB - Non-Hodgkin's lymphomas described in patients with HIV-infection are most often high-grade B-cell lymphomas. Anaplastic large cell lymphoma (CD 30+) has been described in a minority of immunocompromised patients. Although sporadic reports of T-cell lymphomas associated with HIV infection are found in the literature, they have not been described to occur in the myocardium. We present a case of anaplastic large cell lymphoma (CD 30+), T-phenotype involving the heart in a 42 year-old HIV-positive patient. PMID- 10739908 TI - Severe heart valve calcification in a young patient with Werner syndrome. AB - Werner syndrome is a rare autosomal recessive disorder characterized by the appearance of premature aging. We report on severe aortic and mitral valve calcification in an 18-year-old girl, necessitating double valve replacement. These special cardiovascular findings are discussed with regard to diagnosis and treatment. PMID- 10739909 TI - Polypoid (pedunculated) subepicardial lipoma: a cardiac lesion resembling the epiploic appendage. AB - This report describes a 75-year-old woman with an asymptomatic pedunculated lipoma involving the epicardium of the right atrium. The lipoma was an incidental finding at autopsy. The twisted lesion showed many similarities with the infarcted epiploic appendages observed in the visceral peritoneum. PMID- 10739910 TI - An alternate binding site for the P1-P3 group of a class of potent HIV-1 protease inhibitors as a result of concerted structural change in the 80s loop of the protease. AB - Structures of the complexes of HIV protease inhibitor L--756,423 with the HIV-1 wild-type protease and of the inhibitors Indinavir, L-739,622 and Saquinavir with the mutant protease (9X) containing nine point mutations (Leu10Val, Lys20Met, Leu24Ile, Ser37Asp, Met46Ile, Ile54Val, Leu63Pro, Ala71Val, Val82Thr) have been determined. Comparative analysis of these structures reveals an alternate binding pocket for the P1-P3 group of Indinavir and L--756, 423. The alternate binding pocket is a result of concerted structural change in the 80s loop (residues 79 82) of the protease. The 80s loop is pulled away from the active site in order to accommodate the P1-P3 group, which is sandwiched between the flap and the 80s loop. This structural change is observed for the complexes of the wild type as well as the 9X mutant protease. The study reveals that the 80s loop is an intrinsically flexible loop in the wild-type HIV-1 protease and that mutations in this loop are not necessary to result in conformational changes. Conformation of this loop in the complex depends primarily upon the nature of the bound inhibitor and may be influenced by mutations in the protease. The results underscore the need to understand the intrinsic structural plasticity of the protease for the design of effective inhibitors against the wild-type and drug-resistant enzyme forms. In addition, the alternate binding pocket for the P1-P3 group of Indinavir and L--756,423 may be exploited for the design of potent inhibitors. PMID- 10739911 TI - Structure of spinach acetohydroxyacid isomeroreductase complexed with its reaction product dihydroxymethylvalerate, manganese and (phospho)-ADP-ribose. AB - Acetohydroxyacid isomeroreductase catalyses a two-step reaction composed of an alkyl migration followed by an NADPH-dependent reduction. Both steps require a divalent cation and the first step has a strong preference for magnesium. Manganese ions are highly unfavourable to the reaction: only 3% residual activity is observed in the presence of this cation. Acetohydroxyacid isomeroreductase has been crystallized with its substrate, 2-aceto-2-hydroxybutyrate (AHB), Mn(2+) and NADPH. The 1.6 A resolution electron-density map showed the reaction product (2,3 dihydroxy-3-methylvalerate, DHMV) and a density corresponding to (phospho)-ADP ribose instead of the whole NADP(+). This is one of the few structures of an enzyme complexed with its reaction product. The structure of this complex was refined to an R factor of 19.3% and an R(free) of 22.5%. The overall structure of the enzyme is very similar to that of the complex with the reaction-intermediate analogue IpOHA [N-hydroxy-N-isopropyloxamate; Biou et al. (1997), EMBO J. 16, 3405-3415]. However, the active site shows some differences: the nicotinamide is cleaved and the surrounding amino acids have rearranged accordingly. Comparison between the structures corresponding to the reaction intermediate and to the end of the reaction allowed the proposal of a reaction scheme. Taking this result into account, the enzyme was crystallized with Ni(2+) and Zn(2+), for which only 0.02% residual activity were measured; however, the crystals of AHB/Zn/NADPH and of AHB/Ni/NADPH also contain the reaction product. Moreover, mass-spectrometry measurements confirmed the -cleavage of nicotinamide. PMID- 10739912 TI - Structure determination of bacteriophage PP7 from Pseudomonas aeruginosa: from poor data to a good map. AB - The structure of bacteriophage PP7 from Pseudomomas aeruginosa was determined to 3.7 A resolution. Triclinic crystals of three forms were obtained, diffracting to between 4.5 and 3.4 A resolution. The quality of the crystals was exceptionally poor, leading to problems in the evaluation of the recorded images and to a final data set which would appear to be useless with standard criteria for protein crystals. In all crystal forms, the unit cell contains two icosahedral particles, providing 120-fold non-crystallographic symmetry. For two of the crystal forms, the particle orientations were calculated using the self-rotation function. The two particles in the asymmetric unit had very similar but distinct orientations. The position of the second particle was found using the Patterson function. Initial phases to 15 A resolution were calculated using the related phage MS2 as a model. Real-space averaging was performed and phases were extended from 15 A resolution to the limit of the data. The map was improved significantly by using only the 'high' resolution data in the resolution range 7-3.7 A, allowing the positions of most side chains to be determined. The better quality of the 7-3.7 A resolution map is presumably a consequence of the presence of satellite crystals. The position of the second particle was improved using the correlation coefficient in the averaging process to monitor the refinement by moving the particle around in small steps. PMID- 10739914 TI - The refined structure of canavalin from jack bean in two crystal forms at 2.1 and 2.0 A resolution. AB - The structure of canavalin was refined to 2.1 and 2.0 A resolution in cubic and hexagonal crystals of space group P2(1)3 and P6(3), respectively. The threefold molecular symmetry is expressed in the symmetry of both crystals, where each identical subunit is an asymmetric unit. The canavalin subunit consists of two very similar domains, each comprised of a core subdomain having Swiss-roll topology with a loop subdomain that contains helices. The refined canavalin models resolved the discrepancy in amino-acid registers of the secondary structural elements compared with phaseolin. The presence of strand Z in both domains of canavalin was confirmed and a new helix in the loop between strands A and B of each domain was observed. The models were analyzed in terms of the duplicated vicilin domains. Three strictly conserved residues, two glycines and a proline, were identified. The similarity between entire vicilin molecules is greater than that between separate domains of canavalin and phaseolin. Homology modeling of the sucrose-binding protein (SBP) from soybean showed a plausible trimeric assembly of subunits similar to that of vicilins. PMID- 10739913 TI - Structure of the Ser195Ala mutant of human alpha--thrombin complexed with fibrinopeptide A(7--16): evidence for residual catalytic activity. AB - The Ser195Ala mutant of human alpha-thrombin was complexed with fibrinopeptide A(7-22) (FPA) in an effort to describe the (P1'-P6') post-cleavage binding subsites of the fibrinogen-recognition exosite and define more clearly the nature of the Michaelis complex and the scissile peptide bond bound at the catalytic site. The thrombin mutant, however, has residual catalytic activity and proteolysis occurred at the Arg16-Gly17 bond. Thus, the structure of the thrombin complex determined was that of FPA(7-16) bound at the active site, which is very similar to the ternary FPA(7-16)cmk-human thrombin-hirugen complex (r.m.s.d. approximately 0.4 A; Stubbs et al. , 1992). It is further shown by subsidiary experiments that the cleavage is the result of residual catalytic activity of the altered catalytic machinery. PMID- 10739915 TI - MAD analyses of yeast 5-aminolaevulinate dehydratase: their use in structure determination and in defining the metal-binding sites. AB - MAD experiments attempting to solve the structure of 5--aminolaevulinic acid dehydratase using Zn and Pb edges are described. The data obtained proved insufficient for a complete structure solution but were invaluable in subsequent identification of metal-binding sites using anomalous difference Fourier analyses once the structure of the enzyme had been solved. These sites include the highly inhibitory substitution of an enzymic cofactor Zn(2+) ion by Pb(2+) ions, which represents a major contribution towards understanding the molecular basis of lead poisoning. The MAD data collected at the Pb edge were also used with isomorphous replacement data from the same Pb co-crystal and a Hg co-crystal to provide the first delineation of the enzyme's quaternary structure. In this MADIR analysis, the Hg co-crystal data were treated as native data. Anomalous difference Fouriers were again used, revealing that Hg(2+) had substituted for the same Zn(2+) cofactor ion as had Pb(2+), a finding of fundamental importance for the understanding of mercury poisoning. In addition, Pt(2+) ions were found to bind at the same place in the structure. The refined structures of the Pb- and the Hg complexed enzymes are presented at 2.5 and 3.0 A resolution, respectively. PMID- 10739916 TI - Applications of single-wavelength anomalous dispersion at high and atomic resolution. AB - Two examples of the application of single-wavelength anomalous dispersion (SAD) in macromolecular structure determination are described, both using the statistical phasing program SHARP. For the holmium-substituted calcium-binding protein psoriasin (22.7 kDa), a set of accurate phases has been obtained to a resolution of 1.05 A without recourse to an atomic model of the molecule. The accuracy of the phases resulted in an electron-density map of a quality comparable to sigma(A)-weighted 2mF(o) - DF(c) maps derived from the final model refined with SHELX97. Comparison of the refined and SAD electron-density maps showed significant discrepancies resulting from the iterative refinement in reciprocal space. Additionally, it is shown that the structure of psoriasin can be determined from native data extending to 2.0 A alone by exploiting the minute anomalous signal from a bound zinc ion. PMID- 10739917 TI - Rfree and the rfree ratio. II. Calculation Of the expected values and variances of cross-validation statistics in macromolecular least-squares refinement. AB - The free R factor is used routinely as a cross-validation tool in macromolecular crystallography. However, without any means of deriving quantitative estimates of its expected value and variance, its application has been rather subjective and its usefulness therefore somewhat limited. In the first part of this series, estimates of the expected value of the ratio of the free R factor to the standard R factor at the convergence of the structure refinement were given. Here, estimates of the variance of this ratio are given and are compared with the observed deviations from the expected values for a selection of refined structures. It is discussed how errors in the functional form of the structure factor model as well as other types of errors might influence this ratio. PMID- 10739918 TI - Bulk-solvent correction in direct-methods phasing. AB - It is shown that for crystals of large proteins at low diffraction resolution, with N approximately 10 000 independent non-H protein atoms and d(min) approximately 8 A, a simple bulk-solvent correction yields the Sayre equation in its classical form, F(h) = q summation operator(k)F(k)F(h - k). In the low resolution protein case, the proportionality factor becomes q = 1/[( - rho(S))V], where V is the unit-cell volume, rho(S) is the assumed constant electron density in the solvent regions of the crystal and is the average electron density in the protein regions. The classical form of the tangent formula follows from the bulk-solvent-corrected Sayre equation and its validity at low resolution is verified in empirical calculations. PMID- 10739919 TI - Crystallization and preliminary X-ray analysis of cephalosporin C acylase from Pseudomonas sp. strain N176. AB - Cephalosporin C acylase from Pseudomonas sp. strain N176, a heterodimer of 25 and 58 kDa, has been crystallized using polyethylene glycol 6000 as precipitant. The crystals are orthorhombic and have unit-cell parameters a = 141.41, b = 192.10, c = 80.75 A. They belong to space group P2(1)2(1)2(1) and diffract to at least 2.7 A resolution. Calculations indicate that there are two heterodimers in the asymmetric unit. The structure is being solved by molecular replacement using penicillin G acylase from Escherichia coli as a search model and by multiple isomorphous replacement. PMID- 10739920 TI - Structural studies of streptococcus agalactiae hyaluronate lyase. AB - The bacteria Streptococcus agalactiae, part of normal human flora, produce an enzyme, hyaluronate lyase, which appears to contribute to the invasive capacity of this pathogen by degrading hyaluronan and chondroitin sulfates of the extracellular matrix of host tissues. The native enzyme, the product of the hylB(3502) allele, has a molecular mass of 111 kDa but undergoes an autocatalytic conversion to a smaller enzymatically active 92 kDa form. To initiate the determination of the catalytic mechanism of action of these enzymes, the 111 and 92 kDa enzymes were crystallized by a vapor-diffusion method using polyethylene glycol monomethyl ether 5000 and potassium thiocyanate as precipitating agents. The 111 kDa enzyme crystals are of poor quality and diffract X-rays to a very low resolution. However, the crystals of the truncated 92 kDa enzyme diffract X-rays to 2.1 A resolution. The crystal symmetry is C222(1) and the unit-cell parameters are a = 51.69, b = 157.03, c = 239.20 A (alpha = beta = gamma = 90 degrees ). The V(m) of 2.64 A(3) Da(-1) is consistent with the presence of one molecule of hyaluronate lyase in the asymmetric unit and a crystal solvent content of 53%. An isomorphous ethylmercuricthiosalicylic acid heavy-atom derivative diffraction data set has been obtained in order to solve the structure. PMID- 10739921 TI - Crystallization and initial X-ray analysis of alkaline xylanase. AB - Crystals of alkaline xylanase (xylanase J) from alkaliphilic Bacillus sp. 41M-1 were grown by decreasing the temperature of the protein solution. Initial X-ray analysis of the crystal showed a tetragonal system with space group P4(1) or P4(3) and unit-cell parameters a = b = 115.7, c = 46.0 A. Assuming two molecules per asymmetric unit, V(m) = 2.31 A(3) Da(-1). The crystals diffract X-rays to 2.6 A resolution at 100 K. PMID- 10739922 TI - Crystallization and preliminary X-ray diffraction studies of phospholipase D from Streptomyces sp. AB - Crystals of purified phospholipase D (E.C. 3.1.4.4) from Streptomyces sp. strain PMF have been grown under two different crystallization conditions using vapour diffusion. Both conditions gave monoclinic crystals in space group P2(1). The unit-cell parameters were a = 57.28, b = 57.42, c = 68.70 A, beta = 93.17 degrees. The crystals diffract at 110 K to a resolution beyond 1.4 A using synchrotron radiation. PMID- 10739923 TI - Crystallization and X-ray diffraction studies of the fatty-acid responsive transcription factor FadR from Escherichia coli. AB - FadR, an acylCoA-dependent Escherichia coli transcription factor controlling the expression of genes involved in fatty-acid degradation and synthesis, has been crystallized. Crystals of two binary complexes were obtained. The FadR-CoA complex crystallized in space group C222(1), with unit-cell parameters a = 61.3, b = 102.0, c = 91.3 A. The FadR-octanoyl-CoA complex crystallized in space group P6(5)22, with unit-cell parameters a = b = 59.7, c = 296.2 A. Both crystal forms diffracted to 3.5 A on a rotating-anode generator. In both crystal forms, the asymmetric unit contains one subunit. The protein is known to be a homodimer; each subunit consists of two domains of unknown fold. For the acyl-CoA-binding domain, a previously undetected sequence homology to PAS domains, in particular the photoactive yellow protein, is reported. PMID- 10739924 TI - Crystallization of native and selenomethionyl yeast orotidine 5'-phosphate decarboxylase. AB - Crystals of the Saccharomyces cerevisiae pyrimidine biosynthetic enzyme orotidine 5'-phosphate decarboxylase (ODCase) were grown by the hanging-drop vapor diffusion technique at 277 K using polyethylene glycol 4000 as the precipitant. Crystals of native and selenomethionyl ODCase diffract to less than 2.2 A and belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 90.1, b = 116.2, c = 117.0 A. Crystals of ODCase grown in the presence of the postulated transition-state analog inhibitor 6-hydroxyuridine 5'--phosphate (BMP) diffract to less than 2.5 A and belong to space group P2(1), with unit-cell parameters a = 79.9, b = 80.0, c = 98.2 A, beta = 108.6 degrees. PMID- 10739925 TI - Cloning, expression and crystallization of VMA13p, an essential subunit of the vacuolar H+-ATPase of Saccharomyces cerevisiae. AB - The expression and crystallization of the VMA13p subunit of the vacuolar proton translocating ATPase (V-ATPase) of Saccharomyces cerevisiae is described. This 478 amino-acid subunit is essential for activity but not for the assembly of this multisubunit complex. The protein has been recombinantly overexpressed in Escherichia coli and purified. Diffraction-quality crystals have been obtained using the hanging-drop vapor-diffusion method with ammonium sulfate as precipitant. Several different crystal forms were obtained. The most suitable crystal form for crystallographic characterization belongs to space group P3(1)21 or its enantiomorph, with unit-cell parameters a = b = 118.8, c = 119.3 A. Using an in-house X-ray source, the crystals diffract to about 3.5 A resolution under rapidly frozen conditions. PMID- 10739926 TI - Crystallization and preliminary X-ray analysis of native and recombinant human bile-salt dependent lipase: strategies for improvement of diffraction quality. AB - Human bile-salt dependent lipase (BSDL), secreted into both the digestive tract and human milk, is integral to the effective absorption of dietary lipids. In attempts to obtain crystals suitable for high-resolution X-ray crystallographic studies, various forms of the enzyme have been crystallized, including native and desialidated human milk BSDL and both intact recombinant BSDL and a truncated form lacking the heavily glycosylated C-terminal repeat region. Trigonal crystals of native BSDL, with unit-cell parameters a = b = 90.0, c = 156.1 A, were obtained using 15-20%(w/v) PEG 8000 as precipitant. These crystals diffract to 3.5 A along the unique axis, but to only 5-7 A in orthogonal directions. Crystals of recombinant truncated BSDL grown from 15-20%(w/v) PEG 6000 are orthorhombic, space group P2(1)2(1)2(1), with unit-cell parameters a = 59.2, b = 90.0, c = 107.7 A, and diffract to 2.6 A resolution. These are suitable for structural analysis by X-ray crystallography. PMID- 10739927 TI - Crystallization and preliminary X-ray diffraction studies of a monooxygenase from Streptomyces coelicolor A3(2) involved in the biosynthesis of the polyketide actinorhodin. AB - The aromatic monooxygenase ActVA-Orf6 from Streptomyces coelicolor A3(2) that catalyses an unusual oxidation on the actinorhodin biosynthetic pathway has been crystallized. The crystals diffract to 1.73 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 46.95, b = 59.29, c = 71.67 A. Solvent-content (44%) and self-rotation function calculations predict the presence of two molecules in the asymmetric unit. Structure determination should provide further insight into the enzyme mechanism and aid in the design of biosynthetic pathways to produce new polyketide natural products with novel functionality. PMID- 10739928 TI - Screening orthologs as an important variable in crystallization: preliminary X ray diffraction studies of the tRNA-modifying enzyme S-adenosyl-methionine:tRNA ribosyl transferase/isomerase. AB - The genes encoding the tRNA-modifying enzyme S-adenosylmethionine:tRNA ribosyl transferase/isomerase (QueA) from 12 eubacterial sources were overexpressed in Escherichia coli and the resulting products were purified to homogeneity and subjected to crystallization trials. Using the hanging-drop vapour-diffusion method, crystals suitable for X-ray diffraction experiments were only obtained for the queA gene product from Bacillus subtilis. The crystals belong to the space group P422, with unit-cell parameters a = b = 100.7, c = 150.9 A. Using highly focused synchrotron radiation from the EMBL/ESRF beamline ID13 (Grenoble, France), diffraction to at least 3.2 A could be achieved. A selenomethionyl derivative of the protein was prepared and crystallized for future multiwavelength anomalous diffraction (MAD) experiments. PMID- 10739929 TI - The NADP(H)-binding component (dIII) of human heart transhydrogenase: crystallization and preliminary crystallographic analysis. AB - Transhydrogenase is a membrane protein which uses the energy of the proton motive force to drive the reduction of NADP(+) by NADH. The enzyme has three domains: dII spans the membrane, while dI and dIII protrude from the membrane and contain the binding sites for NAD(H) and NADP(H), respectively. DIII from human heart transhydrogenase has been expressed in Escherichia coli. The purified protein has been crystallized with bound NADP(+) using the hanging-drop vapour-diffusion method with ammonium sulfate as a precipitant. The crystals belong to the tetragonal space group P4(1)22 or P4(3)22, with unit-cell parameters a = b = 58.1, c = 251.0 A. A 2.1 A resolution native data set has been collected with an R(merge) of 6. 8%. PMID- 10739930 TI - Crystallization and preliminary X-ray crystallographic analysis of yeast arginyl tRNA synthetase-yeast tRNAArg complexes. AB - Three different crystal forms of complexes between arginyl-tRNA synthetase from the yeast Saccharomyces cerevisae (yArgRS) and the yeast second major tRNA(Arg) (tRNA(Arg)(ICG)) isoacceptor have been crystallized by the hanging-drop vapour diffusion method in the presence of ammonium sulfate. Crystal form II, which diffracts beyond 2.2 A resolution at the European Synchrotron Radiation Facility ID14-4 beamline, belongs to the orthorhombic space group P2(1)2(1)2, with unit cell parameters a = 129.64, b = 107.47, c = 71. 38 A. This crystal form presents the highest resolution obtained for an active form of an aminoacyl-tRNA synthetase-tRNA complex. The estimated V(m) of 2.6 A(3) Da(-1) indicates one molecule of complex in the asymmetric unit. The three crystal forms were solved by the molecular-replacement method using the coordinates of the free yArgRS. PMID- 10739931 TI - Purification, crystallization and preliminary X-ray analysis of haemoglobin I from the armoured catfish Liposarcus anisitsi. AB - Liposarcus anisitsi is an armoured catfish that presents accessorial air oxygenation through a modified stomach, which allows this species to survive in waters with very low oxygen content. Analysis of its haemolysate has shown the presence of four haemoglobins; this work focuses on the main component, haemoglobin I. It has been crystallized in two different forms and X-ray diffraction data have been collected to 2.77 and 2.86 A resolution using synchrotron radiation. Crystals were determined to belong to the space groups C2 and P2(1) and preliminary structural analysis revealed the presence of one tetramer in the asymmetric unit in both crystal forms. The structure was determined using a standard molecular-replacement technique. PMID- 10739932 TI - Crystallization of engineered Thermus flavus 5S rRNA under earth and microgravity conditions. AB - Thermus flavus 5S rRNA with a molecular weight of about 40 kDa was modified at the 5' and 3' ends. Crystals were obtained under earth and microgravity conditions. The best crystals were obtained during NASA space mission STS 94. For the first time, it was possible to collect a complete data set from 5S rRNA crystals to 7.8 A resolution and to assign the space group as R32, with unit-cell parameters a = b = 110.3, c = 387.6 A, alpha = beta = 90, gamma = 120 degrees. PMID- 10739933 TI - Crystallization and preliminary X-ray analysis of squid neuronal Sec1. AB - Sec1 protein family members are involved in the regulation of all intracellular SNARE-mediated (SNARE = soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) vesicle-fusion processes in a step preceding membrane fusion and have been shown to interact with t-SNAREs. To better understand the structural basis and the role of Sec1 in the regulation of the SNARE-complex formation, neuronal Sec1 from the squid Loligo pealei has been expressed and crystallized; this invertebrate protein shows a high sequence homology to the human neuronal Sec1, Munc18a. Here, the production of diffraction quality native crystals, which belong to space group P3(1)21 and diffract to 3.3 A resolution, is described. In addition, selenomethionyl n-Sec1 crystals in space groups P3(1)21 and P2(1) have been generated. Preliminary analysis of the monoclinic space group indicates that these crystals diffract to a resolution higher than 2.5 A. PMID- 10739934 TI - Crystallization and preliminary X-ray crystallographic analysis of human nucleoside diphosphate kinase A. AB - Human nucleoside diphosphate kinase A catalyzes phosphoryl transfer and acts as a suppressor of metastasis. It has been crystallized using 2-methyl-2,4-pentanediol as a precipitant at 288 K. The crystal is monoclinic, belonging to the space group P2(1), with unit-cell parameters a = 74.21, b = 78.11, c = 82.29 A, beta = 101. 33 degrees. The asymmetric unit contains a homohexamer, with a corresponding crystal volume per protein mass (V(m)) of 2.27 A(3) Da(-1) and a solvent content of 46%. Native X-ray data to 2.15 A resolution have been collected using synchrotron X-rays. PMID- 10739935 TI - Crystallization and preliminary X-ray diffraction studies of mutants of B1 IgG binding domain of protein L from Peptostreptococcus magnus. AB - The small 62-residue IgG-binding domain B1 of protein L from Peptostreptococcus magnus (Ppl-B1) has proven to be a simple system for the study of the thermodynamics and kinetics of protein folding. X-ray diffraction studies have been initiated in order to determine how the thermostability, folding and unfolding rates of a series of point mutations spanning Ppl-B1 correlate with the high-resolution structures. To this end, a tryptophan-containing variant of Ppl B1 (herein known as wild type) and two mutants, Lys61Ala and Val49Ala, have been crystallized. Full data sets have been collected for the wild type and the Lys61Ala and Val49Ala mutants to resolutions of 1. 7, 2.3 and 1.8 A, respectively. Interestingly, all three crystallize using different precipitants and in different space groups. This may be a consequence of the relatively large effects of single-site mutations on surface-charge distribution or structural conformation, which might affect crystal contact sites. PMID- 10739936 TI - Crystallization and preliminary crystallographic studies of a new crystal form of Escherichia coli L--asparaginase II (Ser58Ala mutant). AB - Periplasmic Escherichia coli L-asparaginase II with an Ser58Ala mutation in the active-site cavity has been crystallized in a new orthorhombic form (space group P2(1)2(1)2). Crystals of this polymorph suitable for X-ray diffraction have been obtained by vapour diffusion using two sets of conditions: (i) 1% agarose gel using MPD as precipitant (pH 4.8) and (ii) liquid droplets using PEG-MME 550 (pH 9.0). The crystals grown in agarose gel are characterized by unit-cell parameters a = 226.9, b = 128.4, c = 61.9 A and diffract to 2.3 A resolution. The asymmetric unit contains six protein molecules arranged into one pseudo-222-symmetric homotetramer and an active-site competent dimer from which another homotetramer is generated by crystallographic symmetry. PMID- 10739937 TI - Crystallization and preliminary X-ray analysis of shikimate dehydrogenase from Escherichia coli. AB - Shikimate dehydrogenase from Escherichia coli has been crystallized by the vapour diffusion method using ammonium sulfate as a precipitant. Mass spectrometry confirmed the purity of the enzyme and dynamic light scattering was used to find the appropriate additives to yield a monodisperse enzyme solution. The crystals are monoclinic, space group C2, with unit-cell parameters a = 110.0, b = 139.8, c = 102.6 A, beta = 122.2 degrees (at 100 K). Native crystals diffract to 2.3 A in house on a rotating-anode X-ray source. The asymmetric unit is likely to contain four molecules, related by 222 symmetry, corresponding to a packing density of 2.86 A(3) Da(-1). PMID- 10739938 TI - Preliminary X-ray analysis of a new crystal form of the Escherichia coli KDO8P synthase. AB - 3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the biosynthesis of an essential component of the lipopolysaccharide of all Gram negative bacteria. The structure and mechanism of KDO8P synthase are being actively studied as this enzyme represents an important target for antibiotic therapy. The structure of the Escherichia coli KDO8P synthase in cubic crystals (space group I23) has recently been determined and the enzyme shown to be a tetramer of identical subunits. However, this information is challenged by biochemical studies, which suggest that the enzyme behaves in solution as a homotrimer. Here, the preparation and preliminary X-ray analysis of monoclinic crystals of KDO8P synthase are reported. The crystals belong to space group P2(1), with unit-cell parameters a approximately 50, b approximately 140, c approximately 74 A, beta approximately 105 degrees. The structure of KDO8P synthase in the monoclinic crystal form was determined by molecular replacement, using as a search model one of the subunits of the enzyme in the cubic crystals. A tetramer of KDO8P synthase with 222 local symmetry is also present in the asymmetric unit of the P2(1) crystals, with a solvent content of 43%. The observation that the same quaternary structure of KDO8P synthase is observed in two different crystal forms belonging to distinct crystal systems (monoclinic and cubic) suggests that a tetramer is the native form of the enzyme. PMID- 10739939 TI - Atomic resolution structure of native porcine pancreatic elastase at 1.1 A. AB - A data set from the serine protease porcine pancreatic elastase was collected at atomic resolution (1.1 A) with synchrotron radiation. The improved resolution allows the determination of atom positions with high accuracy, as well as the localization of H atoms. Three residues could be modelled in alternative positions. The catalytic triad of elastase consists of His57, Asp102 and Ser195. The His57 N(delta1) H atom was located at a distance of 0.82 A from the N(delta1) atom. The distance between His57 N(delta1) and Asp102 O(delta2) is 2.70 +/- 0.04 A, thus indicating normal hydrogen-bonding geometry. Additional H atoms at His57 N(varepsilon2) and Ser195 O(gamma) could not be identified in the F(o) - F(c) density maps. PMID- 10739940 TI - alpha-L-iduronidase forms semi-crystalline spherulites with amyloid-like properties. AB - While seeking conditions for single crystals of human alpha-L-iduronidase, solutions were discovered (pH 3.0-8.5 containing calcium or zinc salts) that transform soluble alpha-L-iduronidase to a solid aggregate. This aggregate is a spherulite of semi-crystalline protein. The X-ray diffraction pattern and ability to bind Congo red characterize the alpha-L-iduronidase spherulite as 'amyloid like', in that it displays two of the characteristics of amyloidogenic proteins. In addition, alpha-L-iduronidase also interacts with heparin, as do some amyloid forming proteins. PMID- 10739941 TI - Expression of cytokines in bacterial and viral infections and their biochemical aspects. AB - Cytokines are very important in the host defense system, and play a critical role in protection against bacterial and viral infections. Cytokines are also involved in the pathogenesis and development of symptoms in infections. In this article, Helicobacter pylori (H. pylori) infection as bacterial infection, and influenza virus infection, encephalomyocarditis virus (EMCV) infection, and herpes simplex virus (HSV) infection as viral infection are mentioned. In H. pylori infection, various chemokines, especially interleukin (IL)-8, induce inflammatory responses in the gastroduodenal mucosa. Furthermore, IL-6, IL-7, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma are involved in both protection and pathogenesis. In influenza virus infection, IFN-alpha/beta, IFN-gamma, and IL-6 play protective roles. In EMCV infection, IL-6 and TNF-alpha play important roles as a protective and exacerbative factor in acute myocarditis, respectively. Furthermore, in HSV infection, the production of inflammatory cytokines is closely correlated with the pathogenesis of herpetic keratitis, and IFN-gamma plays an important role in enhancing viral clearance from the cornea and trigeminal ganglions. PMID- 10739942 TI - Functional domain-assembly in hairpin ribozymes. AB - Functional structures of hairpin ribozymes have been investigated by constructing various chemically modified molecules. Domain-exchange and linker insertion experiments were performed to find active conformations of the RNA enzyme showing cleavage activity. Our experiments and other evidence suggest that the active structure has a bent conformation, and that domain-interactions are essential for the cleavage activity. PMID- 10739943 TI - The iteron regions necessary for the RepE-iteron interaction in vivo in mini-F plasmids of Escherichia coli. AB - We have determined the nucleotide positions of an incC iteron essential for RepE binding by analyzing mutated incC iterons defective in exerting incompatibility towards mini-F plasmids. The mutations affecting this incompatibility occurred mostly at two positions within the incC iteron, i.e. an iteron conserved position and a mini-F specific position. Most of the iterons with a base-change at either of these two positions had lost the binding affinity for RepE. This agrees with the crystallographic structure of the RepE-iteron complex which showed that the N and C terminal domains of RepE interact with the two major grooves on one face of the iteron DNA. These grooves contain the iteron conserved and mini-F specific positions necessary for RepE binding. Thus the binding mode may be common to in the case of mini-F like plasmids. PMID- 10739944 TI - Membrane structure of the hepatitis B virus surface antigen particle. AB - Expression of S protein, an envelope protein of hepatitis B virus, in the absence of other viral proteins, leads to the secretion of hepatitis B virus surface antigen (HBsAg) particles that are formed by budding from the endoplasmic reticulum membranes. The HBsAg particles produced by mouse fibroblast cells show a unique lipid composition, with 1,2-diacyl glycerophosphocholine being the dominant component. The lipid organization of the HBsAg particles was studied by measuring electron spin resonance (ESR) using various spin-labeled fatty acids, and the results were compared with a parallel study on HVJ (Sendai virus) and vesicles reconstituted with total lipids of the HBsAg particles (HBs-lipid vesicles). HVJ and the HBs-lipid vesicles showed typical ESR spectra of lipids arranged in a lipid bilayer structure. In contrast, the ESR spectra obtained with the HBsAg particles showed that the movement of lipids in the particle is severely restricted and a typical immobilized signal characteristic of tight lipid-protein interactions was also evident. Phosphatidylcholine (PC) in the HBsAg particles was not exchangeable by a PC-specific exchange protein purified from bovine liver, while phospholipase A(2) from Naja naja vemon was able to hydrolyze all the PC in the particles. These analyses suggest that the lipids in the HBsAg particles are not organized in a typical lipid bilayer structure, but are located at the surface of the particles and are in a highly immobilized state. Based on these observations we propose a unique lipid assembly and membrane structure model for HBsAg particles. PMID- 10739945 TI - Energy-yielding properties of SoxB-type cytochrome bo(3) terminal oxidase: analyses involving Bacillus stearothermophilus K1041 and its mutant strains. AB - We isolated a K17q8 mutant from K17 mutant cells of Bacillus stearothermophilus which contain SoxB-type cytochrome bo(3) as well as cytochrome bd but not SoxM type cytochrome caa(3), which is the main terminal oxidase in B. stearothermophilus K1041. The respiration of K17q8 was highly sensitive to as little as 10 microM cyanide, indicating that the main terminal oxidase is cytochrome bo(3). The aerobic growth yield of K17q8 was lower than that of wild type K1041, but higher than that of parental K17. The H(+)/O ratio of K17q8 was about 5, i.e. a little lower than the 6.1-6.5 of K1041, but higher than the 2.9 3.1 of K17 [Sone et al. (1999) J. Biosci. Bioeng. 87, 495-499]. Analyses of membrane fragments indicated that K17q8 contains about 0.2 nmol cytochrome bo(3) per mg membrane protein, and scarcely any subunits of cytochromes caa(3) and bd. From the membrane fraction of K17q8, cytochrome bo(3) was purified and shown to be composed of two subunits with apparent molecular masses of 56 and 19 kDa. The enzyme contained protoheme IX and heme O, as the main low-spin heme and high-spin heme. Analysis of the substrate specificity indicated that the high-affinity site is very specific to cytochrome c-551, a cytochrome c which is a membrane-bound lipoprotein of thermophilic Bacillus. The I(50) of purified cytochrome bo(3) was determined to be 4 microM, indicating that cytochrome bo(3) among the three terminal oxidases in B. stearothermophilus was most susceptible to cyanide. The respiration of K17q8 was mostly inhibited by the addition of cyanide at this concentration. PMID- 10739946 TI - Kinetic and mutational studies of three NifS homologs from Escherichia coli: mechanistic difference between L-cysteine desulfurase and L-selenocysteine lyase reactions. AB - We have purified three NifS homologs from Escherichia coli, CSD, CsdB, and IscS, that appear to be involved in iron-sulfur cluster formation and/or the biosynthesis of selenophosphate. All three homologs catalyze the elimination of Se and S from L-selenocysteine and L-cysteine, respectively, to form L-alanine. These pyridoxal 5'-phosphate enzymes were inactivated by abortive transamination, yielding pyruvate and a pyridoxamine 5'-phosphate form of the enzyme. The enzymes showed non-Michaelis-Menten behavior for L-selenocysteine and L-cysteine. When pyruvate was added, they showed Michaelis-Menten behavior for L-selenocysteine but not for L-cysteine. Pyruvate significantly enhanced the activity of CSD toward L-selenocysteine. Surprisingly, the enzyme activity toward L-cysteine was not increased as much by pyruvate, suggesting the presence of different rate limiting steps or reaction mechanisms for L-cysteine desulfurization and the degradation of L-selenocysteine. We substituted Ala for each of Cys358 in CSD, Cys364 in CsdB, and Cys328 in IscS, residues that correspond to the catalytically essential Cys325 of Azotobacter vinelandii NifS. The enzyme activity toward L cysteine was almost completely abolished by the mutations, whereas the activity toward L-selenocysteine was much less affected. This indicates that the reaction mechanism of L-cysteine desulfurization is different from that of L selenocysteine decomposition, and that the conserved cysteine residues play a critical role only in L-cysteine desulfurization. PMID- 10739947 TI - Purification and characterization of a membrane-bound sialidase from pig liver. AB - A membrane-bound sialidase in pig liver microsomes was solubilized with a nonionic detergent, IGEPAL CA630, and purified to homogeneity by sequential chromatographies on SP-Toyopearl, Butyl-Toyopearl (1st), SuperQ-Toyopearl, Hydroxyapatite, Butyl-Toyopearl (2nd), GM1-Cellulofine affinity, and sialic acid Cellulofine affinity columns. The molecular weight of the purified enzyme was estimated to be 57 kDa on SDS-PAGE. The pH optimum was 4.8 for the activity measured using 4-methylumbelliferyl-alpha-N-acetylneuraminic acid (4MU-Neu5Ac) as the substrate. The enzyme activity was inhibited by 2-deoxy-2,3-dehydro-N acetylneuraminic acid, iodoacetamide and p-chloromercuribenzoic acid. While the enzyme could effectively hydrolyze 4MU-Neu5Ac, it failed to significantly cleave a sialic acid residue(s) from sialyllactose, glycoproteins or gangliosides at pH 4.8. These results suggest that the purified enzyme is a novel sialidase with a substrate specificity distinct from those of known membrane-bound sialidases in mammalian tissues. PMID- 10739948 TI - Characterization of the binding of spike H protein of bacteriophage phiX174 with receptor lipopolysaccharides. AB - The spike H protein of bacteriophage phiX174 was prepared as a hexa histidine tagged fusion (HisH). On enzyme-linked plate assaying, HisH was found to bind specifically to the lipopolysaccharides (LPSs) of phiX174-sensitive strains, Escherichia coli C and Salmonella typhimurium Ra chemotype, having the complete oligosaccharide sequence of the R-core on the LPSs. In sharp contrast, HisH bound weakly to the LPSs of phiX174-insensitive strains, i.e. E. coli F583 (Rd(2)) lacking some terminal saccharides and E. coli O111: B4 (smooth strain) having additional O-repeats on the R-core. The fluorescence spectra of HisH changed dose dependently in the case of the LPS of E. coli C, the intensity increasing and the emission peak shifting to the shorter wavelength side, which was attributable to the hydrophobic interaction of HisH with the LPS. The binding equilibrium was analyzed by fluorometric titration to determine the dissociation constant K(d), 7.02 +/- 0.37 microM, and the Gibbs free energy change DeltaG(0), -29.1 kJ mol( 1) (at 22 degrees C, pH 7.4). Based on the temperature dependence of (K)d in a van't Hoff plot, the standard enthalpy change DeltaH(0) and the entropy change DeltaS(0) were calculated to be +23.7 kJ mol(-1) and 179 J mol(-1) K(-1) at 22 degrees C, respectively, and this binding was thereby concluded to be an entropy driven reaction. PMID- 10739949 TI - Mutational analysis of tyrosine-191 in the catalysis of Cephalosporium acremonium isopenicillin N synthase. AB - Isopenicillin N synthase (IPNS) is a key enzyme responsible for the catalytic conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N in the beta-lactam antibiotic biosynthetic pathway. The Aspergillus nidulans IPNS crystal structure implicated amino acid residues tyrosine-189, arginine-279, and serine-281 in the substrate-binding of the valine carboxylate portion of ACV via hydrogen bonds. In previous reports, we provided mutational evidence for the critical involvement of the corresponding arginine 281 and serine-283, which constitute a conserved R-X-S motif, for the catalysis of Cephalosporium acremonium IPNS (cIPNS). In this study, we report the site directed mutagenesis of the corresponding tyrosine-191 in cIPNS to four amino acids from different amino acid groups, namely, phenylalanine, serine, histidine, and aspartate. The mutants Y191F, Y191H, and Y191R respectively yielded specific activities at levels of 3, 8.6, and 18.8% relative to the wild-type when enzyme bioassays were performed using purified protein fractions. These results were surprising, as previous mutational analyses involving arginine-281 and serine-283 resulted in non-measurable specific activities, thus suggesting that tyrosine-191 is important but not critical for the activity of cIPNS due to its involvement in ACV binding. Hence, it is likely that tyrosine-191 is the least critical of the three residues involved in binding the ACV valine carboxylate moiety. PMID- 10739950 TI - Effect of human granulocyte-macrophage colony stimulating factor (hGM-CSF) on lymphoid and myeloid differentiation of sorted hematopoietic stem cells from hGM CSF receptor gene transgenic mice. AB - Bone marrow lineage-negative (Lin(-)) c-Kit(+) Sca-1(+) hematopoietic cells from human GM-CSF receptor gene transgenic mice were cultured on established bone marrow stromal cell (TBR59) layers and on semisolid medium. In the semisolid assay, an increasing number of larger colonies were observed in the presence of hGM-CSF. By coculture with the stromal cells, cobblestones containing myeloid and lymphoid lineages of cells were formed from the stem cell enriched fraction, and addition of hGM-CSF strongly stimulated formation of the cobblestones containing both lineages. Repeating passages of the cobblestones on TBR59 stromal cells in the presence of hGM-CSF gradually decreased cobblestone formation and inversely increased macrophages and granulocytes, while mast cells were generated when the cells derived from the semisolid assay were cultured in a liquid medium containing hGM-CSF. These results consistently suggest that cytokines such as GM CSF may costimulate the immature hematopoietic cells at their stroma-dependent phase before lineage commitment, and after commitment that occurs by an intrinsic program of the cells, they may stimulate maintenance and maturation of progenitor cells. PMID- 10739952 TI - A chemometric approach to the estimation of the absorption spectra of dye probe merocyanine 540 in aqueous and phospholipid environments. AB - Merocyanine 540 (MC540) is a widely used dye probe for membranous environments. However, fundamental knowledge of the spectral features of this dye in aqueous and hydrophobic environments is still lacking. Such knowledge is important because biomembranes involve a hydrophobic environment surrounded by a hydrophilic environment. Because many investigations so far have been performed based on indistinct spectral estimations, the interpretation of the data obtained using this dye as a fluorescent transmembrane probe remains controversial. In order to determine the exact spectra in both aqueous and hydrophobic environments, we adopted principal factor analysis (PFA), a method of multivariate analysis. The PFA method can also determine the number of molecular species present in the reaction mixture, which is three in pure water and two in phospholipid suspension. Two of the species in both water and phospholipid suspension were the monomer and dimer. The third species in water was the trimer, but its amount was so small at 10 microM MC540 solution that the spectral data in water can be approximated neglecting this molecular species. The monomer spectrum changed its form markedly with a bathochromic shift when transferred from the water to phospholipid environment, whereas the dimer remained similar in its shape except for a remarkable red shift. In water, the dissociation constants, K(1) and K(2), for the assumed stacking-model reactions, M+M <--> M(2) and M+M(2) <--> M(3), were 3.1 x 10(-4) M and 5.7 x 10(-4) M, respectively. In the phospholipid environment, the dissociation constant K* for the assumed stacking model reaction, M(*)+M(*) <--> *M(2), was 1.9x10(-5)M. The fluorescent intensities of MC540 were also measured in both water and phospholipid environments. A comparison based on the absorption and fluorescence spectra suggested that the temporal increase in the amount of the monomer on the excitable membrane contributes to the fluorescent intensity change observed in the transmembrane potential change. PMID- 10739951 TI - Glutathione as an essential factor for chaperon-mediated activation of lactonizing lipase (LipL) from Pseudomonas sp. 109. AB - Pseudomonas sp. 109 produces a unique lipase (LipL) which efficiently catalyzes intramolecular transesterification of omega-hydroxyesters to form macrocyclic lactones. The production of the enzymatically active LipL requires a specific molecular chaperon (LimL protein) together with a low-M(r) lipase-activation factor (LAF) of unknown structure. From 50 g of Pseudomonas cells, 2.15 mg of LAF was purified as a sulfobenzofurazanyl derivative after methanol extraction, derivatization, and C(18) reverse-phase HPLC. One-dimensional and two-dimensional 600 MHz (1)H-NMR and fast atom bombardment mass spectrometry (FAB-MS) revealed that LAF is glutathione. Because several SH compounds (L-cysteine and mercaptoethanol) were similarly effective to native LAF in the activation of LipL, and because only LipL contains two cysteinyl residues forming an intramolecular disulfide bond, it is concluded that the reduction of and reformation of the intramolecular disulfide bond of LipL is essential to liberate free and fully active LipL. PMID- 10739953 TI - Expression of plant group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed functional divergence among LEA proteins. AB - To study functions of late embryogenesis abundant (LEA) proteins, which accumulate in plant cells under water deficit conditions, in vivo functional analyses were carried out using a yeast (Saccharomyces cerevisiae) heterologous expression system. Two lea genes, tomato le4 (group 2) and barley HVA1 (group 3), were expressed under the GAL1 promoter, and the gene products were detected using specific antisera. The growth of the transformants was scored and compared with a control strain to analyze the effect of these proteins on yeast cells under stress conditions. The yeast cells expressing HVA1 showed shorter lag period when transferred to a medium containing 1.2 M NaCl as compared to a control strain, while the cells expressing le4 did not show improved growth. Attenuated growth inhibition in a medium containing 1.2 M KCl was observed in the yeast cells expressing le4 and HVA1. No obvious growth improvement was observed in a high sorbitol medium in the cells expressing either le4 or HVA1. Increased freezing tolerance was observed in both lea-expressing cells, while no effect on heat tolerance was observed. These results support the hypothesis that different LEA proteins play a distinctive role in the protection against cellular dehydration. PMID- 10739954 TI - Engineering subtilisin E for enhanced stability and activity in polar organic solvents. AB - We examined the effect of a novel disulfide bond engineered in subtilisin E from Bacillus subtilis based on the structure of a thermophilic subtilisin-type serine protease aqualysin I. Four sites (Ser163/Ser194, Lys170/Ser194, Lys170/Glu195, and Pro172/Glu195) in subtilisin E were chosen as candidates for Cys substitutions by site-directed mutagenesis. The Cys170/Cys195 mutant subtilisin formed a disulfide bond in B. subtilis, and showed a 5-10-fold increase in specific activity for an authentic peptide substrate for subtilisin, N-succinyl-L Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide, compared with the single-Cys mutants. However, the disulfide mutant had a 50% decrease in catalytic efficiency due to a smaller k(cat) and was thermolabile relative to the wild-type enzyme, whereas it was greatly stabilized relative to its reduced form. These results suggest that an electrostatic interaction between Lys170 and Glu195 is important for catalysis and stability in subtilisin E. Interestingly, the disulfide mutant was found to be more stable in polar organic solvents, such as dimethylformamide and ethanol, than the wild-type enzyme, even under reducing conditions; this is probably due to the substitution of uncharged Cys by charged surface residues (Lys170 and Glu195). Further, the amino-terminal engineered disulfide bond (Gly61Cys/Ser98Cys) and the mutation Ile31Leu were introduced to enhance the stability and catalytic activity. A prominent 3-4-fold increase in the catalytic efficiency occurred in the quintet mutant enzyme over the range of dimethylformamide concentration (up to 40%). PMID- 10739955 TI - A new approach to gene mutation analysis using "GFP-Display". AB - The unique behavior of green fluorescent protein (GFP) on SDS-PAGE was applied to the detection of a single amino acid substitution in GFP-tagged polypeptides. This simple detection method using SDS/urea gels was designated GFP-display. The N-terminal 18 or 37 amino acids of K-Ras was used as a model GFP-tagged polypeptide. K-ras exon 1 was fused to a gfp cDNA at each end and expressed in Escherichia coli. Amino acid number 12 of K-Ras (wild type; Gly) was changed to Ser, Arg, Cys, Asp, Ala, or Val, and the mobility shift of the greenish fluorescent bands in the SDS/urea gel was analyzed. These mutants were easily detected by GFP-display; however, detection depended strongly on the urea concentration and electrophoresis temperature. Subsequently, GFP-display was applied to the 36 amino acids encoding human p53 exon 7. Amino acid number 248 (wild type; Arg) was changed to Gly, Trp, Gln, Pro, or Leu, and similar mobility shifts were observed. GFP-display could be coupled with an in vitro translation system. Fluorescent active GFP and GFP-Ras fusion proteins were synthesized within a few hours. GFP-display shows potential as a modern approach to gene mutation analysis at the protein level, and is a useful method for protein engineering studies. PMID- 10739956 TI - Substrate specificity of bovine cathepsin B and its inhibition by CA074, based on crystal structure refinement of the complex. AB - The crystal structure of the bovine spleen cathepsin B (BSCB)-CA074 complex was refined to R = 0.152 using X-ray diffraction data up to 2.18 A resolution. BSCB is characterized by an extra Cys148-Cys252 disulfide bridge, as compared with rat and human CBs. Although the crystal structures of these enzymes showed similar overall folding, a difference was observed in the occluding loop, a structural element specific only to CB. Comparison of the torsion angles indicated the different flexibilities of their loop structures. The oxirane C6 atom of CA074 was covalently bonded to the Cys29 S(gamma) atom (C3-S(gamma)=1.81 A), where the S-configuration was transformed to the R-form. Concerning the oxirane carbon atom that participates in the covalent bonding with the Cys residue, an acceptable rule has been proposed. The substrate specificities at the Sn (n = 1-3) and Sn' (n=1 and 2) subsites of CB, together with the interaction features as to CA074, have been discussed in comparison with the crystal structure of the papain-CA028 (a CA074-related inhibitor) complex. PMID- 10739957 TI - Cell surface activities of the human type 2b phosphatidic acid phosphatase. AB - Several isozymes of mammalian type 2, Mg(2+)-independent phosphatidic acid phosphatase (PAP-2) have recently been cloned, and they are predicted to have their catalytic sites exposed at the cell surface membranes. We investigated the mode of utilization of extracellular lipid substrates by the human PAP-2b expressed in HEK293 cells as a green fluorescent fusion protein. We first confirmed the plasma membrane localization of the expressed PAP-2b. PAP-2b actively hydrolyzed exogenously added lysophosphatidic acid and short-chain phosphatidic acid. In the case of dephosphorylation of lysophosphatidic acid, the reaction products, including inorganic phosphate and monoacylglycerol, were recovered exclusively in the extracellular medium. Interestingly, PAP-2b exhibited negligible activities toward long-chain phosphatidic acid either exogenously when added or generated within the membranes by treating the cells with bacterial phospholipase D. These findings indicate that PAP-2b acts at the outer leaflet of cell surface bilayers and can account for the ecto-PAP activities previously described for various types of cells. PMID- 10739959 TI - Functional expression of two Arabidopsis aldehyde oxidases in the yeast Pichia pastoris. AB - To investigate the biochemical and enzymatic properties of two aldehyde oxidase (AO) isoforms of Arabidopsis thaliana, we expressed AAO1 and AAO2 cDNAs in a heterologous yeast (Pichia pastoris) system and successfully obtained the proteins in active forms. The expressed AAO1 and AAO2 proteins gave activity bands with the same mobilities on native gel electrophoresis and exhibited the same substrate preferences on zymograms with 8 aldehydes as those of AOalpha and AOgamma in Arabidopsis seedlings, respectively. Furthermore, anti-AAO1 and anti AAO2 antibodies, which specifically recognize the seedling AOalpha and AOgamma, respectively, reacted with the AAO1 and AAO2 proteins produced in P. pastoris, respectively. These results indicate that these AO proteins are accurately produced in the yeast system, as in Arabidopsis seedlings. Using AO preparations from P. pastoris, the enzymatic properties of Arabidopsis AOalpha and AOgamma were investigated. AOalpha showed a relatively wide substrate specificity for 7 aldehydes tested, with high affinity to benzaldehyde and indole-3-aldehyde, while AOgamma could most efficiently oxidize naphthaldehyde. AOalpha was strongly inhibited by iodoacetate and KCN, while AOgamma was inhibited not only by iodoacetate and KCN but also by 2-mercaptethanol, dithiothreitol, menadion, and estradiol. AOalpha and AOgamma showed the highest activity at around 65 and 50 degrees C, respectively, and exhibited pH dependence around pH 8.0. These results indicate that the two AO isoforms in Arabidopsis seedlings have different enzymatic properties and may have different physiological roles in vivo. PMID- 10739958 TI - Inhibitory effects of flavonoids on rabbit heart carbonyl reductase. AB - The inhibitory effects of flavonoids (galangin, kaempferol, quercetin, myricetin, morin, and taxifolin) on rabbit heart carbonyl reductase (RHCR) were investigated using 4-benzoylpyridine (4BP) as the substrate. The stereochemical characteristics of the flavonoids were found to be a factor determining their inhibitory potencies toward RHCR. Furthermore, the lipophilicity, and the scavenging or antioxidative effects of the flavonoids were likely to complicate the structure-activity relationship of their inhibitory effects on RHCR. Quercetin inhibited RHCR uncompetitively with respect to NADPH and competitively with respect to 4BP, suggesting that it competes with 4BP at the substrate binding site of RHCR. RHCR efficiently reduced benzoquinones (1,4-benzoquinone and 2-methyl-1, 4-benzoquinone) and naphthoquinones (1,4-naphthoquinone and menadione). Galangin was a potent inhibitor of RHCR when menadione was used as the substrate, and prevented the formation of the superoxide anion radical in the presence of RHCR, NADPH, and menadione. Flavonoids may be useful compounds for suppressing the cardiotoxicity of quinones by inhibiting RHCR. PMID- 10739960 TI - Mitochondrial targeting signal-induced conformational change and repression of the peroxisomal targeting signal of the precursor for rat liver serine:pyruvate/alanine:glyoxylate aminotransferase. AB - In the rat liver, two mRNAs for serine:pyruvate (or alanine:glyoxylate) aminotransferase are generated from a single gene by alternative transcription initiation. The longer mRNA encodes a precursor of a mitochondrial enzyme that has a mitochondrial targeting signal at the N-terminus and is translocated into mitochondria. The shorter mRNA encodes a peroxisomal enzyme of mature size that is imported into peroxisomes. We have been interested in the mechanism of selective targeting to mitochondria of the precursor protein that also contains a peroxisomal targeting signal in the molecule. In this study, we examined the effect of the mitochondrial targeting signal on the conformation of the protein and on the function of the peroxisomal targeting signal in the precursor molecule. The results suggest that the mitochondrial targeting signal causes the conformation of the protein to become unfolded and that this conformational change in turn causes repression of the putative peroxisomal targeting signal contained in the precursor protein. PMID- 10739961 TI - Functional construction of the anti-mucin core protein (MUC1) antibody MUSE11 variable regions in a bacterial expression system. AB - A bacterial expression system for the variable region fragments (Fvs) of the anti MUC1 tumor antigen antibody MUSE11 has been constructed. The Fv fragment showed binding specificity toward TFK-1 cells, with slightly reduced affinity compared to its parent IgG. The single-chain Fv fragment was arranged in two orders, VH linker-VL and VL-linker-VH. However, linking the regions with a flexible peptide linker (GGGGS)(3) or with a shorter linker (GGGGS) led to a dramatic decrease in the biological activity toward the target antigen in both arrangements, suggesting that the MUSE11 antibody loses its activity when the domains are linked with polypeptide linkers. These results indicate that the variable region domains of the anti-MUC1 antibody MUSE11 have specificity only in the Fv form, and that linking the domains strongly reduces the association with its target antigen. Gel filtration analysis indicates that the scFv has a dimeric structure, suggesting that the inactivation of MUSE11 scFv is due to unfavorable intermolecular associations of the scFv chains. To our knowledge, this is the first report of a significant reduction in affinity caused by linking the variable domains in both arrangements, i.e., VH-VL and VL-VH. PMID- 10739962 TI - NMR analysis of intra- and inter-molecular stems in the dimerization initiation site of the HIV-1 genome. AB - Two positive-strand HIV-1 genomic RNAs form a dimer in virion particles through interaction of the dimerization initiation sites (DIS). The DIS RNA fragment spontaneously formed a "loose-dimer" and was converted into a "tight-dimer" by supplementation with nucleocapsid protein NCp7. This two-step dimerization reaction requires the whole DIS sequence [Takahashi et al. (2000) RNA 6, 96-102]. In the present study, we measured imino proton resonances to investigate the secondary structures of the two types of dimers in a 39-mer RNA covering the entire DIS (DIS39), including discrimination between intra- and inter-molecular base pairing. Both the presence and absence of inter-molecular NOE between (15)N labeled and unlabeled DIS39 were unambiguously detected in an equimolar mixture of (15)N-labeled and unlabeled DIS39. The stem-bulge-stem structures in both dimers were confirmed and found to be very close to each other from clear superimposition of the NMR spectra in the two dimeric states. Nevertheless, the modes of base pairing in the stems of the loose- and tight-dimers were intra- and inter-molecular, respectively. Our results suggest a large structural alteration of genomic RNA occurs during virion maturation. PMID- 10739963 TI - Structures of ovine corticotropin-releasing factor and its Ala32 mutant as studied by CD and NMR techniques. AB - The corticotropin-releasing factor (CRF) is a 41-amino acid peptide-amide hormone, which mediates a general stress-response. It has been reported that the substitution of His-32 in the ovine CRF (oCRF) with Ala brings about a 4.5-fold increase in activity [Kornreich et al. (1992) J. Med. Chem. 35, 1870-76]. Here, we have determined the secondary structure of this Ala-substituted ovine CRF ([Ala32]oCRF) and compare it with that of oCRF using circular dichroism (CD) and NMR techniques in trifluoroethanol (TFE) solution, which is known to stabilize the alpha-helix formation. In contrast to an earlier report, it was observed the alpha-helical structure extends to the C-terminus of oCRF. By analyzing the CalphaH and NH chemical shifts, the properties of local structures of oCRF were elucidated. The oCRF and [Ala32]oCRF have stable alpha-helical structures in the middle region, regardless of pH and temperature, and the alpha-helix initiation regions of these peptides are stabilized as the pH is decreased. However, the [Ala32]oCRF has a more stable alpha-helical structure than oCRF in the vicinity of the substitution region, and it is thought that this is the cause of the increased activity of [Ala32]oCRF. PMID- 10739964 TI - Chimeric glycosaminoglycan oligosaccharides synthesized by enzymatic reconstruction and their use in substrate specificity determination of Streptococcus hyaluronidase. AB - A method was developed for the reconstruction of glycosaminoglycan (GAG) oligosaccharides using the transglycosylation reaction of an endo-beta-N acetylhexosaminidase, testicular hyaluronidase, under optimal conditions. Repetition of the transglycosylation using suitable combinations of various GAGs as acceptors and donors made it possible to custom-synthesize GAG oligosaccharides. Thus we prepared a library of chimeric GAG oligosaccharides with hybrid structures composed of disaccharide units such as GlcA-GlcNAc (from hyaluronic acid), GlcA-GalNAc (from chondroitin), GlcA-GalNAc4S (from chondroitin 4-sulfate), GlcA-GalNAc6S (from chondroitin 6-sulfate), IdoA-GalNAc (from desulfated dermatan sulfate), and GlcA-GalNAc4,6-diS (from chondroitin sulfate E). The specificity of the hyaluronidase from Streptococcus dysgalactiae (hyaluronidase SD) was then investigated using these chimeric GAG oligosaccharides as model substrates. The results indicate that the specificity of hyaluronidase SD is determined by the following restrictions at the nonreducing terminal side of the cleavage site: (i) at least one disaccharide unit (GlcA-GlcNAc) is necessary for the enzymatic action of hyaluronidase SD; (ii) cleavage is inhibited by sulfation of the N-acetylgalactosamine; (iii) hyaluronidase SD releases GlcA-GalNAc and IdoA-GalNAc units as well as GlcA GlcNAc. At the reducing terminal side of the cleavage site, the sulfated residues on the N-acetylgalactosamines in the disaccharide units were found to have no influence on the cleavage. Additionally, we found that hyaluronidase SD can specifically and endolytically cleave the internal unsulfated regions of chondroitin sulfate chains. This demonstration indicates that custom-synthesized GAG oligosaccharides will open a new avenue in GAG glycotechnology. PMID- 10739965 TI - Upstream regions directing heart-specific expression of the GATA6 gene during mouse early development. AB - The expression of murine transcription factor GATA6 is restricted to tissues including the heart and gastrointestinal systems during embryogenesis, and is maintained throughout postnatal life. We have characterized the 5' upstream region (6.4 kb) of the mouse GATA6 gene, and identified two closely spaced transcription initiation sites. The flanking sequence lacks a typical TATA-box, and is rich in guanine and cytosine. The role of the 5' upstream region was examined using the lacZ reporter gene in transgenic mice. A construct containing the 5' flanking sequence (4.9 kb), untranslated exon 1 and 1.3 kb intron 1 could drive the gene expression in the embryonic and adult heart regions. Weak expression was also observed in the stomach, liver, and bronchial arch in addition to the cardiac region. Deletion of the 5' upstream region ( approximately 1.2 kb) or intron 1 abolished all this expression, indicating that at least two cis-acting control elements are necessary for heart-specific expression of GATA6 in vivo. PMID- 10739966 TI - A review of outcome after moderate and severe closed head injury with an introduction to life care planning. AB - Forensic consultation regarding moderate and severe closed head injury (CHI) generally focuses on determination of severity of residual deficits and the implications of these deficits for future health care needs, personal independence, and employment. This information can be used to develop a life care plan that describes the patient's needs for continued medical care, rehabilitation, and daily assistance or supervision and estimates the long-term costs for these services. This article provides brief reviews of CHI classification, epidemiology, residual deficits, expected outcomes, and factors predictive of outcome. An introduction to the process of developing a life care plan is presented. PMID- 10739967 TI - Diagnosis of mild head injury and the postconcussion syndrome. AB - Mild head injuries can cause acute transient cognitive inefficiency that typically resolves within 3 months. Postconcussion syndrome may initially be related to acute cerebral dysfunction but can also arise as a psychological consequence of head trauma. The syndrome persists beyond 3 months in a significant number of patients with mild head trauma as a psychological disorder. International Classification of Diseases diagnostic criteria for postconcussion syndrome are currently recommended for clinical purposes. These criteria are contrasted with research diagnostic criteria used in the Diagnostic and Statistical Manual of Mental Disorders. The differential diagnosis of persistent cognitive and postconcussive symptoms in forensic practice is reviewed. PMID- 10739968 TI - Neurobehavioral recovery after pediatric head trauma: injury, pre-injury, and post-injury issues. AB - This article reviews the most significant demographic, neurological, and psychosocial factors affecting the occurrence of and recovery from traumatic head injury (THI) in children and adolescents. Review of the available literature suggests that, as with adults, there is no compelling evidence for persistent neurobehavioral deficits after mild THI in children. In contrast, neurobehavioral deficits are common in children who have sustained moderate to severe THI. This article emphasizes that a long-term developmental perspective that considers in concert injury, pre-injury, and post-injury variables is needed for a proper appreciation of possible sequelae of pediatric THI. Specific pitfalls in forensic assessments of these children are reviewed. Empirical findings are presented to support the position that neuropsychological evaluations of children with THI that do not consider pre-injury status are likely to lead to misattribution errors. Clinical implications are illustrated with a case example. PMID- 10739969 TI - Clinical neuropsychology in the criminal forensic setting. AB - This article reviews the application of clinical neuropsychology to criminal court proceedings, a complex, underserved, yet growing area of neuropsychological practice. The authors write from the perspective that the audience is primarily neurorehabilitation clinicians with limited experience in criminal matters. Discussions on the theoretical differences between clinical and forensic work, the forensic evaluation process with conceptual model, historical and current perspectives on criminal competencies and responsibility, prediction of dangerousness, and professional and ethical issues often encountered in criminal neuropsychology are provided. PMID- 10739970 TI - Detecting exaggeration and malingering in neuropsychological assessment. AB - Magnification of symptoms or nonoptimal effort on neuropsychological tests, within the context of head injury litigation, can have several independent or related underlying causes. Therefore, detecting exaggeration does not automatically indicate that the individual is malingering. This article reviews the evaluative and differential diagnostic process and provides the clinician with suggestions regarding assessment methods. A forensic evaluation that does not include careful consideration of possible negative response bias should be considered incomplete. PMID- 10739971 TI - Functional neuroimaging and quantitative electroencephalography in adult traumatic head injury: clinical applications and interpretive cautions. AB - Functional neuroimaging and quantitative electroencephalographic procedures are being used increasingly in brain injury research and clinical care. These procedures are also seeing increased use in the context of forensic evaluations, particularly in cases of mild head trauma. This article provides an overview of the use of procedures such as positron emission tomography, single photon emission computed tomography, and quantitative electroencephalogram in adults. Also discussed are the clinical limitations of each procedure within the context of myriad interpretive confounds that can interfere with accurate differential diagnosis of mild head trauma. PMID- 10739972 TI - [Analysis of the literature about drug-induced aphthous ulcers]. AB - OBJECTIVES: Analysis of the literature on drug-induced aphthous ulcers and mucosal ulcerations and evidence-based grading. MATERIAL AND METHODS: Four literature sources were analyzed. Three groups of key words were used: 1) oral, buccal, genital, mucosal; 2) ulcer, ulceration, aphthous, aphthosis; 3) induced, drug, adverse-effects, with cross-overs. Four grading patterns were used: presence of aphthous term or synonym, typical clinical description of aphthous ulcer, presentation suggesting diagnosis of aphthous ulcer, criteria defining likelihood of drug causality. RESULTS: We examined 66 of the 220 publications responding to our selection criteria. Typical clinical description of aphthous ulcer and/or clinical presentation suggesting the diagnosis of aphthous ulcer were noted for 8 compounds with likely or palausible patterns of causality. For 21 compounds, we found only aphthous term or synonym without a clinical description or presentation. DISCUSSION: Our review of the literature individualized a group of 8 compounds where the diagnosis of aphthous ulcers was plausible and another group of 21 compounds where the diagnosis of aphthous ulcers requires confirmation. The clinical relevance and limitations of this analysis are discussed. CONCLUSION: Drug-induced aphthosis is probably a real phenomenon. Causality of the 8 compounds in the first group is simply more fully documented than for the 21 compounds in the second group. A low evidence level may not confirm these hypotheses. Some drugs may have been incorrectly ruled out due to lack of information. PMID- 10739973 TI - [Melanoma in organ transplant patients]. AB - OBJECTIVE: The incidence of cutaneous melanoma has rapidly increased in the white population over the last decades. It has been estimated that the incidence doubles world-wide every 10 years. Different risk factors have been identified, including immunosuppression. The aim of our study-was to determine the relative risk of developing melanoma in the organ transplant population and the clinical and histological features of their melanomas. PATIENTS AND METHODS: This retrospective study was conducted with the collaboration of 9 University Hospital Centers: Besancon, Brest, Caen, Dijon, Lille, Lyon, Nantes, Paris (Pitie Salpetriere) and Rennes. A questionnaire was sent to the different departments of dermatology of these hospitals to obtain information on patients who had presented a melanoma after a transplantation between 1971 and 1997. During this period, there were 12,477 organ transplant recipients in the transplantation units of these 9 hospitals. Average follow-up for these patients was about 5 years and the average duration of immunosuppressive therapy was about 4.5 years. RESULTS: Among 12,477 organ transplant recipients, we found 17 cases of melanoma but no data could be obtain on one case: 14 occurred in renal transplant recipients and 3 in cardiac transplant recipients. Clinical and histological data were only available in 16 patients. The average time between transplantation and diagnosis of melanoma was 63 months, but it was 5 times shorter for 2 patients who had a past history of melanoma before transplantation. Two patients had a mucosal melanoma; for the cutaneous melanomas, 2 appeared on Dubreuilh melanosis, 2 were in situ melanomas, 7 were superficial spreading melanomas and 3 were nodular melanomas. The histological review of 11 cutaneous melanomas revealed a precursor nevus in 6 cases and a weak or no stroma reaction in 7/7 cases. Complete excision of the melanoma was performed in all patients except one with anorectal melanoma. Four patients died of visceral metastasis within a mean 15 months. The other 12 patients are still alive with a mean 3 year course since tumor treatment. We tried to determine the relative risk of developing melanoma in the renal transplant population (14 cases). The number of expected cases of melanoma was 5.54, giving a relative risk of 2.5. DISCUSSION: Only 4 studies have shown an increase in the incidence of melanoma in the renal transplant population: approximately 2 to 5-fold. In our study, the 2.5-fold increase in melanoma was estimated with an average 5 year follow-up and an average 5 year immunosuppressive therapy. This is probably an underestimation of risk because we were unable to make an exhaustive collection of cases of melanomas even though transplant recipients undergo more physical examinations than a reference population. The mean latency period from transplantation to melanoma diagnosis was 63 months, as in other studies. Histological examination showed that a precursor nevus is frequent with weak host cellular response to the tumor. The prognosis of these melanomas remains difficult to predict, but in our study, it would not appear to be as poor as expected. Discontinuation of immunosuppressive therapy would not appear to be necessary except in the presence of metastasis. Finally, our study demonstrates the importance of good patient follow-up, even after graft rejection due to the persistent risk of melanoma. PMID- 10739974 TI - [Extracorporeal photopheresis as an alternative therapy for drug-resistant graft versus host disease: three cases]. AB - BACKGROUND: Graft versus host reaction is a life-threatening complication of allogenic bone marrow transplantation. Extracorporeal photopheresis has been used for some years in the treatment of graft versus host reaction. We report on three children treated with extracorporeal photopheresis for a graft versus host reaction resistant to immunosuppresive drugs. MATERIAL AND METHODS: Three children with a graft versus host reaction were submitted to 18, 30 and 46 extracorporeal photopheresis courses respectively. In the same time, the other immunosuppressive treatments were tapered or definitively stopped (ciclosporin). RESULTS: A dramatic improvement of cutaneous status and biological data was observed after the first courses. However, the extracorporeal photopheresis treatment did not improve the mucous lesions. No serious adverse effect was encountered. COMMENTS: As published elsewhere, extracorporeal photopheresis was effective on the graft versus host reaction lichenoid cutaneous lesions and in case of visceral involvement. In all of our cases, the immunosuppressive drug could have been tapered. No adverse event was observed. Thus, extracorporeal photopheresis should be indicated in case of resistance to immunosuppressive drugs. PMID- 10739975 TI - [Thalidomide and thrombosis]. AB - BACKGROUND: Teratogenicity and neuropathy are the well known serious side effects induced by thalidomide. We describe 5 cases of thrombotic events occurring within a brief delay after the onset of thalidomide in a manner that suggests that thalidomide could have acted as a precipiting or as a starting factor in these events. OBSERVATIONS: Five patients including 4 patients with lupus erythematosus (1 discoid lupus, 1 subacute lupus and 2 systemic lupus erythematosus) and one patient with a severe atopic dermatitis, all without previous history of vascular events, developed an arterial thrombosis (2 cases) or a venous thrombosis (3 cases), severe in 4 cases, few days or weeks after the onset of thalidomide treatment (50 to 100 mg daily). DISCUSSION: All the patients had risk factors of thrombosis: the presence of antiphospholipids and/or anticardiolipin antibodies in lupus erythematosus patients and a trauma in the atopic case. However the absence of a previous story of thrombosis, its rapid occurrence after the onset of thalidomide and its severity are intriguing. In addition, recent studies demonstrate that thalidomide has various effects that would act, among other things, on angiogenesis. Thus, we think that a doubt exists on a negative effect of thalidomide in thrombosis risk factors patients and that this hypothesis has to be confirmed. PMID- 10739976 TI - [Cryosurgery of basal cell carcinoma: a study of 358 patients]. AB - INTRODUCTION: Cryosurgery is a well-established therapeutic modality for basal cell carcinoma. We report herein an important series of basal cell carcinoma treated by cryosurgery, with a five year cure rate evaluation. PATIENTS AND METHOD: Retrospective study with 395 basal cell carcinomas (over 358 patients - sex ratio H/F: 0.85) treated by cryosurgery between 1981 and 1992. For each patient the data were: age, sex, size of the lesion, location, clinical sub-type, tissue-temperature monitoring, recurrence date and esthetic outcome. RESULT: The lesions were located on the face (93 p. 100) and back (7 p. 100). Mean size was 17 mm. Clinical sub-types were known for 178 lesions; 11 p. 100 of BCC were morpheaform. Tissue-temperature monitoring was performed for 55 p. 100 of cases. 111 tumors were observed for more than 5 years. The 5-year actuarial failure rate was 9 p. 100. The 5-year cure rate was not significantly altered by sex, size of lesions, location and clinical sub-type. No frequent recurrences were observed when tissue-temperature monitoring had not been performed. Complications were rare and esthetic outcome was good. DISCUSSION: The 5-year actuarial recurrence rate with cryosurgery is similar to conventional surgery. It is not essential to control tissue-temperature for preventing recurrences. Cryosurgery is a reliable treatment and outcome depends on surgeon experience. Quickness and low cost of this procedure argue for choosing cryosurgery when treating elderly patients. PMID- 10739977 TI - [Renal function during long-term cyclosporin treatment of psoriasis]. AB - INTRODUCTION: The goal of this retrospective study was to evaluate the effects of cyclosporin A on renal function in patients treated for five years or more. PATIENTS AND METHODS: Two hundred twenty-four psoriasis patients were treated with cyclosporin at St. Louis Hospital, in Paris, from 1988 to 1997. Eleven patients (5 p. 100 of the total) took cyclosporin for at least five years. Nine case histories were available for the analysis. RESULTS: The group studied consisted of eight men and one woman, ranging in age from 28 to 48 with an average age of 38 years. The average cyclosporin dosage was 3.6 mg/kg/day (2.0 5.0 mg/kg/day). The changes in renal function were not significant as compared to the baseline level. The patients did not show persistent increases of serum creatinine more than 30 p. 100 of the pre-treatment value. Two patients had to discontinue treatment after 6 years because of a 20 p. 100 decrease in glomerular filtration rate. DISCUSSION: Previous studies have shown that cyclosporin A is not a suitable long-term continuous monotherapy for psoriasis. However, with precise and regular monitoring of kidney function, a minority of patients can benefit from prolonged treatment without experiencing side effects. PMID- 10739978 TI - [Pretibial epidermolysis bullosa and hypothyroidism]. AB - BACKGROUND: We report a case of primary non-autoimmune hypothyroidism causing pretibial epidermolysis bullosa. CASE REPORT: A 70-year-old man with primary non autoimmune hypothyroidism developed blisters of different ages on the lateral aspect of both legs. Pathology reported blisters with subepidermal cleavage. Direct immunofluorescence was negative. Electron microscope examination showed a variable cleavage level and diffuse infiltration of a granulous and amorphous microfibrillar substance. After hormone replacement therapy, euthyroidism was associated with a reduction in the number of bullae and finally complete remission. After 12 months follow-up, the patient has not experienced recurrence. DISCUSSION: Recurrence-free clinical improvement after hormone replacement therapy suggests the diagnosis of hypothyroidism pretibial epidermolysis bullosae. Mochizuki et al. described a similar case which rapidly regressed after hormone therapy but where the electron microscope showed a different cleavage level. These bullae appear to result from a mechanical mechanism due to their localization in areas exposed to friction and also to the presence of bullae of different ages. This hypothesis is confirmed by the presence of a variable level of cleavage and a substance dense to electrons at electron microscopy as well as by the skin weakness. Our case confirms the reality of hypothyroidism pretibial epidermolysis bullosa. Thyroid hormones should be assayed in patients presenting pretibial bullae. PMID- 10739979 TI - [Large crusted ulceration of the scalp: first manifestation of cryptococcosis in an AIDS patient]. AB - BACKGROUND: Cryptococcosis is an infection caused by the yeast-like fungus Cryptococcus neoformans. It primarily occurs in immunocompromised hosts. Crytptococcosis is usually a systemic disease and may be serious if the lungs or the central nervous system are involved. CASE REPORT: We describe a case of cutaneous cryptococcosis without systemic involvement occurring as the first manifestation of AIDS. The skin lesion in our patient consisted of a large crusted ulceration on the scalp. Fluconazole was successful. DISCUSSION: This case is a new observation of primary cutaneous cryptococcosis in a patient with AIDS. Clinical appearance may be misleading; skin lesions may be the primary sign in immunocompromised patients. Rapid treatment and secondary prophylaxis are required. PMID- 10739980 TI - [Contact dermatitis to topical antiviral drugs]. AB - BACKGROUND: Topical antiviral drugs are frequently used. Repeated applications on a skin injured by the viral infection can lead to irritating dermatitis or contact eczema. This secondary effect is often unrecognized because imputed to an increase of the initial dermatosis. CASES REPORTS: Case 1 - A 58 year-old woman had an eczema around the mouth after the use of Zovirax(R) cream (acyclovir). The patch test with this cream was positive (++ at 96 h) but the detail of all the constituents was negative. Case 2 - A 39 year-old woman had an eczema surrounding the mouth after each use of Zovirax(R) cream and Cuterpes(R) (ibacitabin). The patch tests were doubtful for Zovirax(R) cream, positive for Cuterpes(R) (++ at 96 h) and ibacitabin 1 p. 100 and 10 p. 100 in petrolatum (++ at 96 h). Case 3 - A 58 year-old man had an acute eczema of the face after the use of Zovirax(R) cream and Vira-MP(R) (vidarabin phosphate). The patch tests were positive for the both antiviral topical drugs but negative for each one of their constituents. DISCUSSION: Contact dermatitis to antiviral topical drugs are rare, due to active molecule or its excipients. The propyleneglycol, component found in the three antiviral drugs, can induce irritation or allergy. In the most of cases, like in our case number 1 and 3, the patch tests with each constituents of the topical drugs were negative, we conclude to a compound allergy. PMID- 10739981 TI - [Acquired peeling skin syndrome in an adult]. AB - BACKGROUND: Peeling skin syndrome is a rare form of congenital ichthyosis. The term was coined in 1982 by Levy and Goldsmith and the syndrome is clinically characterized by generalized scaling. Histologically, there is an epidermal separation in the stratum corneum. CASE REPORT: We report the case of a 73-year old woman who had ichthyosis without cicatricial progressive alopecia since her first pregnancy. An ultrastructural study was performed confirming the clinical diagnosis of peeling skin syndrome. DISCUSSION: The peeling skin syndrome designates several different clinical entities classed by Traupe in type A and type B. Mevorah and al. expanded this classification with a type C. This classification has remained valid after additional information provided by ultrastructural studies and may suggest different pathogenic mechanisms underlying the dermatosis. A critical review of the literature shows that the case reported here is exceptional and had a late clinical onset. PMID- 10739982 TI - [Linear radiodermatitis following total body electron beam therapy]. AB - BACKGROUND: We describe herein a peculiar clinical presentation of a linear overlapping radiodermatitis, localized on the internal side of the limbs, following a total body electron beam therapy. CASE REPORT: A 68-year-old-man was treated in April 1998 by a total body electron beam therapy, for a stage I mycosis fungoides. Few days after the last irradiation, the patient suffered from a linear eruption localized on the internal side of the limbs and the external side of the abdominal wall. DISCUSSION: Total body electron beam therapy can be proposed in early stage mycosis fungoides with localized or generalized cutaneous lesions. Because penetration depth of electron is well controlled and is limited to the skin, usual side effects of total body electron beam therapy do not concern internal organs (bone marrow,.). We report here a peculiar clinical presentation of a linear radiodermatitis in frontal plane which has not been reported to date, to our knowledge. This radiodermatitis corresponds to overlapping of posterior and anterior fields of irradiation. PMID- 10739983 TI - [Extensive primary cutaneous mucinosis associated with refractory anemia with excess of blast]. AB - BACKGROUND: We describe an original case of a primary cutaneous mucinosis, associated with a refractory anemia with excess of blast. CASE REPORT: A 65 year old female consulted for erythematous-purple blush reticulated and large plaques that progressively developed on the trunk and limbs. Biopsies showed abundant dermis mucin deposits with a perivascular lymphocytic infiltrate. The epidermis was normal. Laboratory evaluation showed refractory anemia with excess of blast, associated with cytogenetic abnormalities. After two years course, the progressive aggravation of the myelodysplastic syndrome was associated with a progressive extension of the cutaneous mucinosis. DISCUSSION: This is the first report of an atypical primary diffuse and extensive cutaneous mucinosis. Primary cutaneous mucinosis may be associated with systemic diseases. To our knowledge, the association with refractory anemia with excess of blast has not been described previously. Mucine deposits may be considered as a consequence of neutrophilic dysfunction that are observed in myelodysplastic syndromes. PMID- 10739984 TI - [Pili multigemini: a pilar dysplasia with linear disposition]. AB - BACKGROUND: Pili multigemini is an uncommon pilar dysplasia with linear disposition which could be explained by the pattern of Blaschko's lines. CASE REPORT: A 37-year-old man with no medical history developed pili multigemini over a heavily bearded chin. DISCUSSION: Mili multigemini is an uncommon developmental defect of hair follicles resulting from hairs with multiple matrices and papillae emerging through a single pilosebaceous canal. This defect has a linear distribution on the chin we found to follow Blaschko's lines. Pili multigemini has been observed in association with a few rare malformations. Treatment is difficult. PMID- 10739985 TI - ["Milia en plaque" with multiple and successive localizations]. PMID- 10739986 TI - [Immersion oils for microscopy: a new source of occupational eczema]. PMID- 10739987 TI - [Multiple unilateral angiofibromas of the face]. PMID- 10739988 TI - [Cutaneous metastasis of a cholangiocarcinoma at the site of a percutaneous biliary catheter]. PMID- 10739989 TI - [Angio-eccrine hamartoma]. PMID- 10739990 TI - [Lichen planus-like keratosis or (solitary) benign lichenoid keratosis]. PMID- 10739991 TI - [Hirsutism]. PMID- 10739992 TI - [Use of growth factors for treatment of chronic wounds. Results of controlled studies]. PMID- 10739993 TI - [Subacute cutaneous lupus erythematosus and interferon beta-1 a]. PMID- 10739994 TI - [Strasbourg dermatology meeting - special meeting on erythema nodosum, may 8th, 1938] [In Process Citation] PMID- 10739996 TI - [Recurrent laryngeal nerve paralysis: current knowledge and treatment]. AB - The aim of this report is to summarize current concepts in unilateral recurrent laryngeal nerve paralysis (URLNP). Important aspects of laryngeal phylogenesis, physiology and anatomy are reviewed. Recent advances in the neurophysiology of URNLP are discussed. Revised and updated principles of diagnosis and treatment are provided. Glottic configuration and prognosis vary according to the type of neural lesion (neurapraxia, axonotmesis or neurotmesis). Therapeutic indications depend on glottic configuration and prognosis. Treatment options include voice therapy, vocal fold augmentation by intrafold injection, medialization thyroplasty, arytenoid adduction, and laryngeal reinnervation. Each treatment option is summarized, and the results reported in the medical literature are reviewed. PMID- 10739997 TI - [Prognostic influence of cutaneous involvement in malignant tumors of the oral cavity]. AB - The aim of this study was to assess the prognostic influence of cutaneous involvement in T4 squamous cell carcinoma of the oral cavity. The population was a homogeneous group of 137 patients. Cutaneous tumor localizations were observed in 20 of them. Surgery and radiotherapy were given in 103 cases and salvage surgery was performed in 34. Local control at 5 years was achieved in 68.5 % of the patients. Carcinologic failure rate was 55 % and 27.3 % in patients with and without cutaneous involvement respectively (p =0. 013). 5-year survivor rate was also correlated with cutaneous involvement: 10 % versus 32.2 % (p <0.0001). PMID- 10739999 TI - [A numerical simulation of the aerodynamics of the nasal cavity]. AB - We present the results of the numerical simulation of the flow in the nasal cavity, going from the tip of the nostril to the nasopharyngeal region. The volume of the nasal cavity, obtained from axial and coronal scans, takes into account the geometries of the nasal valve and turbinates. The simulation is carried out with the FLUENT code which solves the equations of fluid mechanics. The obtained results for the inspiratory phase are analyzed from the velocities and pressures, paying special attention to the separation of the streamlines in the region located between the middle meatus and the olfactory area. The presented results show the potential of simulation when used in parallel with the clinical approach. PMID- 10739998 TI - [Nasal soluble levels of ICAM-1 in allergic rhinitis]. AB - Adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), play an important role in the development of the inflammatory allergic response in the nose. ICAM-1 expression on nasal epithelial cells during allergic reaction is regarded as a major hallmark of allergic inflammation. OBJECTIVE: The aim of the study was to evaluate the levels of soluble ICAM-1 (sICAM-1) in nasal epithelial lining fluid (ELF) in patients with allergic rhinitis. PATIENTS: Seventeen patients with perennial allergic rhinitis (age: 34,6 +/- 14,6) were screened and the results were compared with those from 11 seasonal allergic rhinitis patients (age: 25.9 +/- 7.4) and 10 non-allergic patients. METHODS: The study was performed outside the pollen season. The scores of subjective symptoms were estimated by two differents methods. First, on the basis of a visual analog scale for the symptoms including nasal obstruction, rhinorrhea, sneezing and pruritus. This score was called "Autoscore" (fullmark =40 points). Second, on the basis of a four mark scale for each symptom defined (0 =no symptom, 1 =mild, 2 =moderate, 3 =severe). This score was called "Heteroscore" (fullmark =12 points). Specimens of nasal mucosa were collected by brushing the surface of nasal cavity. Levels of sICAM-1 and sECP (soluble Eosinophilic cationic protein) were measured by specific enzyme-linked immunosorbent assay. RESULTS: The levels of sICAM-1 in ELF were significantly higher (p <0.01) in patients with perennial rhinitis compared to patients with seasonal rhinitis outside the pollen season and to non allergic patients. Levels of sICAM-1 in patients with perennial allergic rhinitis were correlated with levels of sECP (p <0.003) and with the four mark scale scores (p <0. 03) but did not correlate with the visual analog scale scores. CONCLUSIONS: sICAM-1 increased in nasal secretions during natural perennial rhinitis and could be considered as a representative hallmark for clinical severity and follow-up evaluation in perennial allergic rhinitis. PMID- 10740000 TI - [Mastoid cortical bone grafts in ossiculoplasty]. AB - Ossicular homografts have been left for safety reasons with regard to viral transmission diseases. Several means are usable to reconstruct ossicular chain: synthetic prosthesis and autologous bone. On grounds of disponibility, biocompatibility, cost and use easiness we have been using mastoid cortical bone since 1995. We have studied hearing results and tolerance of 45 ossiculoplasties performed with cortical bone. Two years after, we have been obtaining as good or even better functional results with cortical bone graft than with auto or homologous ossicular bones (air bone gap inferior or equal to 20 DB in 89 % of the cases) and no extrusion. Thus, cortical bone seems to be, the better material when autologous ossicular bones are not available. PMID- 10740001 TI - [Our current results with acoustic neurinoma surgery]. AB - Out of 130 CPA tumors operated on between 1993 and 1997, 91 were vestibular schwannomas less than 25 mm with preoperative hearing and normal facial function. The pre and postoperative facial and hearing functions were analyzed prospectively. The surgical technique applied was the complete tumor removal via retrosigmoid approach under facial nerve monitoring and CPA endoscopy. Clinical features were analyzed to determine postoperative facial grading (House Brackmannn) and tonal and vocal audiometrical datas (PTA-SDS) at 8 days, 90 days and 1 year. We achieved 96 % of good facial results (Grade I and II/HB) and 46,5 % of postoperative conserved hearing. For tumors less than 25 mm in the CPA, good preoperative hearing level, preoperative AOE and BER seem to be predictive factors for hearing preservation. Early diagnosis associated with the advances in minimal invasive otoneurological techniques leads to perform complete removal of this type of tumors with respect of facial and hearing functions. PMID- 10740003 TI - [VHL, angiogenesis and renal carcinoma: the puzzle is complete]. PMID- 10740002 TI - [Head and neck desmoid tumor in children: a case report and review of the literature]. AB - We report a case of desmoid tumor in the submandibular region in an 18-month-old girl. Head and neck desmoid tumors are uncommon in children and diagnosis is a difficult task because the tumors are often classified as different types of fibromatosis. This histologically benign affection is characterized by local expansion or destruction and tends to recur. Pathology gives the positive diagnosis, showing fibroblastic monoclonal proliferation between the cellular center and the collagen periphery. Electron microscopy evidences an abundant collagen network enclosing a polymorphous cellular proliferation. Immunohistochemistry defines vimentin and actin positive desmoid tumors. Surgery is usually the therapeutic choice. Chemotherapy and radiotherapy may be used in conjunction with surgery in situations of recurrence or unsatisfactory surgical margin. There is a risk of recurrence which can be detected with regular follow up examinations. PMID- 10740004 TI - [Carcinoma arising within mammary fibroadenomas. A study of six patients]. AB - We report six cases of carcinomas arising within fibroadenomas. Fibroadenoma is a benign neoplasm occurring in young women. Its association with carcinomas is unfrequent and particularly reported in older women. Few data are available on the histologic features of fibroadenomas harboring malignant lesions. In this study, most cases of fibroadenomas showed cysts, sclerosing adenosis, epithelial calcifications or papillary apocrine changes. These fibroadenomas are classified as complex and are a long-term risk factor for breast cancer. The complex fibroadenoma may be specific of fibroadenoma associated with carcinoma. PMID- 10740005 TI - [Fibrillin network in normal bone tissue]. AB - The molecular basis for Marfan's syndrome is known to reside in mutations in FBN1, the gene for fibrillin 1. The skeletal manifestations of Marfan syndrome include morphologic abnormalities and osteopenia. Presence and distribution of fibrillin 1 in adult bone (healthy or with Marfan syndrome) has not been studied extensively. We evaluated distribution of fibrillin and type III collagen in bone and cartilage of children and adults without bone disease, using monoclonal antibodies. Fibrillin is mostly present in attachment sites for tendons. In cartilage and bone tissue, fibrillin is identified at the junction between cartilage and bone in children, and in the areas with intense osteoblastic activity. These data suggest participation of fibrillin in bone formation and growth during youth and in bone mineralisation in adult. PMID- 10740007 TI - [Pancreatic lesions in von Hippel-Lindau disease]. PMID- 10740006 TI - [Diagnostic value of percutaneous biopsy of the renal masses. 73 cases]. AB - The aim of this study is to evaluate the diagnostic value of percutaneous biopsies of renal masses with comparison of pathological data between the biopsies and the surgical specimens. Thus, we retrospectively studied 73 biopsies of patients, who were referred to our institution between 1995 and 1997. The mean age of the patients was 60 +/- 14 years with a male predominance (67%). Twenty per cent of the biopsies were negative. Eleven per cent of the biopsies were benign lesions (oncocytoma, angiomyolipoma, infectious lesions). For renal cell carcinomas, the correlation coefficient between biopsy histology and final pathology was 0.87. Conversely, it was only 0.36 for the Fuhrman grading. Nevertheless, low (1-2) and high (3-4) grades were accurately separated (0.92). We conclude that the percutaneous biopsy is a useful tool for diagnosis of renal masses. PMID- 10740008 TI - [Pancreatic neuroendocrine tumors and von Hippel-Lindau disease]. AB - Pancreatic neuroendocrine tumors are rare in von Hippel-Lindau disease, most often asymptomatic, nonfunctioning, non secreting, and benign. We report a case of low grade malignant pancreatic, secreting and asymptomatic neuroendocrine tumors, occurring in a 27 year old woman in the setting von Hippel-Lindau disease with recurrent pheochromocytoma, retinal and medullary hemangioblastomas, paraganglioma of the carotid body and ovarian cystadenoma. Neuroendocrine pancreatic tumors of von Hippel-Lindau disease are often constituted by clear cells, in the contrary of other neuroendocrine tumors of the pancreas. Occurrence of a pancreatic neuroendocrine tumor, especially in association with pheochromocytoma, may be misdiagnosed with a type 2 multiple endocrine neoplasia syndrom instead of von Hippel-Lindau disease. PMID- 10740009 TI - [Malignant mesenchymoma of mesentery. A controversial entity]. AB - We report a case of malignant mesenchymoma developed in the mesentery in a 62 year-old man with no past history. It was an huge mass in the mesentery of the ileum. The tumor was composed of areas of chondrosarcoma and rhabdomyosarcoma in addition to an undifferentiated fusiform component. Malignant mesenchymoma is a rare sarcoma occurring preferentially in the retroperitoneum or the thigh. The location in the mesentery is exceptional. Malignant mesenchymoma is discussed more particularly with dedifferentiated liposarcoma with heterologous elements. The patient is alive without recurrence three years after surgery. PMID- 10740010 TI - [Hepatic MALT lymphoma disclosing a nodal extension]. AB - We report a case of a 50-year-old man with chronic viral hepatitis B presenting with a primary hepatic lymphoma of mucosa-associated lymphoid tissue, revealed clinically by a pedicular nodal mass. The liver biopsy showed an active chronic hepatitis and a dense portal lymphoid infiltrate with centrocyte-like cells inducing typical biliary lympho-epithelial lesions. The lymph-node biopsy revealed a marginal zone lymphoma pattern. A monoclonal rearrangement of the immunoglobulin heavy chain gene was detected in the lymph node by polymerase chain reaction. This case of primary hepatic mucosa-associated lymphoid tissue lymphoma confirms that the liver also contains mucosa-associated lymphoid tissue, in which low grade lymphoma can arise. PMID- 10740011 TI - [Infiltrating syringoadenoma of the nipple: a new case]. AB - Infiltrating syringomatous adenoma of the nipple is composed of small sweat ducts and solid strands, surrounded by desmoplastic stroma and preferentially develops in the superficial breast tissue and specially in the nipple. This particular and exceptional lesion deserves to be acknowlegded by pathologists concerned with breast pathology since this tumor mimics grade 1 invasive breast ductal carcinoma. PMID- 10740012 TI - [How fine needle aspiration biopsy make it possible to avoid extensive surgery. A case of abdominopelvic actinomycosis]. AB - A case of abdominopelvic actinomycosis diagnosed by fine needle aspiration biopsy is reported. The patient, using an intrauterine device for five years, had a pelvic mass infiltrating the left ovary and five hepatic masses. Cytological smears prepared from an ultrasound-guided fine needle aspiration biopsy of one of the hepatic masses, showed actinomycotic granule. In the literature, few cases of abdominopelvic actinomycosis diagnosed by fine needle aspiration biopsy are reported. This affection, able to simulate a neoplasia on clinical and radiological grounds, is usually diagnosed on the histology of the surgical specimen. Ultrasound and computerized tomography allows to guide fine needle into necrotic areas where the probability to meet actinomycotic granules is maximum and so to avoid an extensive surgery. PMID- 10740013 TI - [Renal angiomyolipoma with predominant muscular epithelioid components. 2 cases]. AB - We report two cases of pararenal angiomyolipomas, in two middle-aged women without tuberous sclerosis. Both tumors were made of muscular epithelioid cells without atypia, some of which were stained by HMB-45 antibody. The fibrous stroma contained numerous thick-walled blood vessels but no adipose component. The ultrastructural examination revealed that muscular epithelioid cells contained myofilaments and melanosomas. Because of their immunohistochemical and ultrastructural profiles, both tumors may be included in the family of lesions composed of perivascular epithelioid cells (PEC). This tumor type is considered as a particular variant of angiomyolipoma with predominant muscular epithelioid component. The immunohistochemical expression of progesterone and estrogen receptors by both tumors may suggest their hormonodependent character. PMID- 10740014 TI - [Sclerosing epithelioid fibrosarcoma: a fibrosarcoma with a very long course]. AB - Sclerosing epithelioid fibrosarcoma is a rare and slowly growing variant of fibrosarcoma occurring in adults. Its carcinomatous and/or hemangiopericytomatous patterns may mislead to many different tumors. These differential diagnoses are generally ruled out by the clinical data, the morphology and the immunohistochemical study. We report two typical cases of this fibrosarcoma and we highlight the different criterias which are indispensable to make the diagnosis. The very slow course of this disease goes along with a very low proliferative rate, but an accumulation of p53 by immunohistochemistry is paradoxically always founds, even though this accumulation is more often found in highly aggressive sarcomas. PMID- 10740015 TI - [Infiltrating granular cell tumor of the esophagus: a description of two cases]. AB - Granular cell tumor of the esophagus is an unusual tumor. It presents usually as a small and well limited lesion, localized in the mucosa or the submucosa. We report two cases of granular cell tumor of the esophagus, remarkable for their infiltrative growth. The tumor invaded the esophageal muscularis propria in one case and went through the adventitia in the other. There was no recurrence 1 year and 7 years after surgery, despite an incomplete resection in the second case. Thirteen cases of infiltrative granular cell tumors of the esophagus have been published. They are usually responsible for dysphagia. They can invade the muscularis propria and the adventitia as well as the periesophageal organs. There is no recurrence, even after an incomplete resection. The infiltrative feature of the granular cell tumors of the esophagus, by itself, cannot be considered as a malignant feature. The diagnosis of malignant granular cell tumor of the esophagus lies on the discover of metastases. PMID- 10740016 TI - [An unusual subcutaneous nodule of parasitic origin]. PMID- 10740017 TI - [A misleading spinal metastasis]. PMID- 10740018 TI - [A cutaneous neck flow]. PMID- 10740019 TI - [A skin nodule]. PMID- 10740020 TI - [One heat-induced antigen retrieval step allows the sequential detection of two antigens on the same slide of tissue fixed and enclosed in paraffin]. AB - The heat-induced antigen retrieval (HIAR) procedure allows the immunohistochemical detection of various antigens on paraffin-embedded sections. The re-use of slides negative for the detection of a first set of antigens may be an interesting alternative in case of a limited number of slides. After HIAR, a series of Bouin's liquid-fixed tonsil sections was stained for Epithelial Membrane Antigen (EMA) which labelled epithelial cells and plasma cells. A second immunostaining for CD20 (L26) was performed on the same slides divided in two sets. The HIAR was repeated in the first set but not in the second one. A similar staining of follicular B-cells was observed in the two sets. However background staining was enhanced by repetition of HIAR. Analogous results were obtained using anti-cytokeratin (KL1) instead of anti-EMA. This was confirmed on slides for which three or four cycles of HIAR were performed prior immunostaining. Our data suggest that the renewal of the HIAR procedure must be avoided since it was found stable for at least 1 year. PMID- 10740021 TI - [Colorectal cancer: an new role for the anatomic pathologist]. PMID- 10740022 TI - [Isolated necrotizing arteritis of the uterine cervix]. PMID- 10740045 TI - [Familial amyloid polyneuropathy type I complicated by chronic glaucoma: 3 cases]. AB - PURPOSE: We report 3 cases of familial amyloid polyneuropathy type I (FAP) with amyloid infiltration of the vitreum and glaucoma. PATIENTS AND METHODS: We reviewed the records of three patients, 2 females and one male, aged 41, 47 and 83 years respectively. The 3 patients had familial amyloid polyneuropathy type I with vitreous infiltration and open angle glaucoma. The two women underwent a liver graft four years earlier. Vitrectomy allowed confirmation of the diagnosis in the 83-year-old patient. Two patients underwent trabeculectomy, histological analysis of the iris and the trabeculum was obtained for one patient. RESULTS: The tree patients presented a polyneuropathy, vitreous infiltration and open angle glaucoma. Results of light microscopy of the iris and the trabeculum showed amyloid deposits in the iris and the trabecular meshworks. CONCLUSION: Familial amyloid polyneuropathy is a hereditary disease which may have a wide range of ocular manifestations. Glaucoma is among the most serious complications of familial amyloid polyneuropathy. PMID- 10740046 TI - [Final visual acuity in patients undergoing posterior vitrectomy for Terson's syndrome: a series of 11 cases]. AB - PURPOSE: The purpose of this study was to assess visual acuity after vitrectomy for Terson's syndrome. METHODS: This is a retrospective study of 11 eyes (7 patients), who underwent pars plana vitrectomy between 1996 and 1998. Visual acuity was evaluated at 1, 5 and 12 months. We also report immediate and delayed complications. RESULTS: Initial visual acuity was below 20/200 for 9 patients (81%). Final visual acuity was 20/25. An epiretinal membrane was noticed in two patients during the surgical procedure. Final visual acuity for those two patients was only 20/40 and 20/100. Two patients developed cataract following vitrectomy, one developed an endophthalmitis with good vision recovery. CONCLUSION: Vitrectomy for vitreous hemorrhage in Terson's syndrome after three months follow up is a solution without major risks and has a good visual prognosis. Final visual acuity of patients after vitrectomy for Terson's syndrome is good. However, Terson's complications such as epiretinal membrane and cataract are limiting factors for good visual recovery. PMID- 10740047 TI - [Acquired oculomotor paralysis: a new therapeutic approach. Apropos of 10 cases]. AB - PURPOSE: The aim of this work is to report our rehabilitation scheme and outcome in patients with acquired oculomotor palsy. PATIENTS AND METHODS: We cared for 10 patients with oculomotor palsy between January 1996 and March 1998 at the Casablanca University Hospital. Our orthoptic rehabilitation scheme was based on reinforcing the patient's sensorial potential using a prism dioper and motor capacities by soliciting vergency and version movements. RESULTS: The 10 patients (7 males, 3 females, mean age 39.5 years) had unilateral IV palsy (n =3), bilateral IV palsy (n= 1), unilateral VI palsy (n= 2), bilateral VI palsy (n= 1), partial unilateral III palsy (n= 1) and dissociated bilateral III palsy (n= 2). Mean delay to initiation of rehabilitation was 49 days and mean duration for treatment was 3 months. We achieved total recovery in 50% of the cases and partial recovery in 40%. Intermittent diplopia persisted in 10 per thousand of the cases. These results differ slightly from those in the literature where total regression is reported in about 50% of the patients after therapeutic abstention. CONCLUSION: This small series is insufficient to validate our method. The results obtained do however show that rehabilitation is safe and should be applied more widely in patients with oculomotor palsy. PMID- 10740048 TI - [Lyell's syndrome and its ophthalmologic manifestations in Cameroon]. AB - BACKGROUND: To study basic epidemiological datas and ocular involvment during toxic epidermal necrolysis (T.E.N.) in Cameroonian patients. METHODS: Ten consecutive patients in the Central Hospital of Yaounde were included during a 4 years period. Anamnesis, clinical features, dermatological, ophthalmological and pathological examinations were done. Minimal follow up of 3 months. RESULTS: Mean age: 20.1 +/- 11.4 yrs (SD); culprit drugs: sulfadoxin (5/10), sulfamethoxazol (3/10); phenytoin (1/10). Mean of skin detachment: 41 +/- 17%. 2 patients died with sulfadoxin as culprit drug. Early ocular findings: conjunctivitis (all patients), symblepharon (1/8), ankyloblepharon (3 patients over 8 with sulfadoxin as culprit drug). All cases with ankyloblepharon (3) evoluated toward formation of a fibrovascular precorneal membrane with blindness. Two of the patients received a kerothoprosthesis on one eye. The 3 patients had a severe dry eye syndrome. CONCLUSION: TEN appears to be uncommon in Cameroon. Use of sulfonamides for treatment and prevention of malaria and AID'S opportunistic infections may increase the incidence of disease in tropical african areas. Ophthalmologists should be aware of early management of ocular manifestations in Lyell syndrome to avoid severe sequelae. That sequelae may be due to usual intensive healing in African patients. Severe corneal damages can be corrected only by keratoprosthesis. PMID- 10740049 TI - [Evaluation of free radical production by conjunctival impression cytology of patients treated with long-term antiglaucoma drugs or of contact lens wearers]. AB - PURPOSE: To investigate free radical production in impression cytology specimens using microplate cold light cytofluorimetry. METHODS: 60 impression cytology specimens (IC) were harvested in 30 patients aged from 20 to 90 years. IC were taken in 10 healthy subjects, 10 glaucoma patients receiving longterm treatment for glaucoma and 10 contact lens wearers for more than five years. A complete ophthalmologic examination (BUT, corneal staining) was performed. Evaluation of free radical production was done using a dichlorofluoresceine diacetate (DCFHDA) test for H(2)O(2) evaluation and a hydroethidine test for O(2)(.)- measurement. RESULTS: The BUT values in the two pathological groups were significantly lower than those of controls (p<0.001). A free radical production was found in both contact lens wearers (p<0.001 compared to control values) and glaucoma patients (p<0.001 compared to control values). Both hydrogen peroxyde and superoxide anion productions were significantly correlated to the decrease of BUT values and the increase of superficial punctuate keratitis. CONCLUSION: The results of this preliminary study confirm that conjunctival epithelial cells may abnormally produce oxygen free radicals in chronic ocular surface disorders. This may participate to the cellular alterations observed in inflammatory diseases of ocular surface. PMID- 10740050 TI - [Congenital corneal anesthesia in children: diagnostic and therapeutic problems]. AB - Congenital corneal anesthesia (CCA) is an uncommon condition difficult to diagnose. We report the case of a 9-month-old girl who presented bilateral congenital corneal anesthesia. The child had a corneal ulcer which had been unresponsive to adapted local treatment. Self-inflicted corneal injuries were present. Local treatment and arm splints led to quick healing. CCA occurs either alone or in association with neurological diseases (familial dysautonomia) or systemic congenital abnormalities (Goldenhar Gorlin syndrome). It is important to search for corneal anesthesia in children with chronic ulcerations of the cornea and self-inflicted injuries. Early diagnosis is important due to the risk for the visual prognosis. Short and longterm prevention of self-inflicted corneal injuries should be associated with a local treatment to assure rapid healing and a relapse free outcome. PMID- 10740051 TI - [Complications of hydroxyapatite orbital implants in children: a series of 105 cases]. AB - PURPOSE: To describe the complications encountered using hydroxyapatite as an orbital implant in the pediatric population after enucleation, and try to define the risk factors for their occurrence. MATERIAL: and methods: Retrospective review of charts of children who underwent enucleation with placement of an hydroxyapatite implant between January 1991 and 1998. Complications and their type (conjunctival, extrusion, eyelids complications) have been specifically looked for. A statistical analysis trying to isolate the risks factors was performed. RESULTS: 105 implants have been used during this period with 26.7% of complications appearing during a median followup of 22 months. Extrusion of the implant was observed in 1.9% of the cases, a chronic conjunctival erosion in 15%, a conjunctival lesion without erosion in 4.75% and no eyelid lesion could be seen. Treatment (medical or surgical) achieved 75% good results. No risk factor could be found. CONCLUSION: Hydroxyapatite orbital implants were initially thought to have no or few complications. More recently, chronic erosions in particular were described. Little data exists on the pediatric population. Our series confirms that complications occur and that they are comparable to the adult population. Nevertheless the hydroxyapatite orbital implant seems better tolerated than other porous or non porous implants and warrants its use. PMID- 10740052 TI - [Definitive internal tamponade with silicone oil in certain cases of retinal detachment]. AB - PURPOSE: The aim of this study was to analyze the effect of definitively maintaining silicone oil in the treatment of certain cases of retinal detachment. MATERIAL AND METHODS: Sixteen cases of retinal detachment seen from July 1995 to April 1999 were retrospectively studied. For these eyes with residual visual acuity, we voluntarily maintained silicone oil. There were 11 men and 5 women, mean age 56 (range 24 - 82). Followup ranged from 18 to 45 months. RESULTS: We analyzed visual acuity outcome, complications, and main reasons for maintaining longterm silicone oil CONCLUSION: This study provided interesting insight concerning certain clinical situations where silicone oil has to be maintained definitively. In some cases, low, but useful, visual acuity can be achieved for long periods of time. This therapeutic approach would be particularly helpful for mono-ophthalmic patients. PMID- 10740053 TI - [Melkersson-Rosenthal syndrome: anatomo-clinical and evolutive concepts]. AB - PURPOSE: To inform ophthalmologists about the Melkersson-Rosenthal Syndrome. A new clinical classification is suggested. METHODS: Commented observation and review of the litterature. RESULTS: The presented case is characterized by an exclusive involvement of orbital sphere, due to inflammation of palpebral and orbital fat. CONCLUSION: Melkersson-Rosenthal Syndrome can be divided into type 1 characterized by perioral lesions and type 2 characterized by periocular lesions, with preferential involvement of fat and evolution towards fibrosis. PMID- 10740055 TI - [ [In Process Citation] PMID- 10740054 TI - [Phacoemulsification following radial keratotomy. Topographic and refractive analysis concerning an 18-month period (apropos of a case)]. AB - We followed a 45-year-old man who underwent clear cornea phacoemulsification cataract extraction 3 years after radial keratotomy during an 18 month period. We report early and late refractive analysis and corneal topography during the followup period. The patient experienced an hyperopic shift which was different from the aim of 5 diopters due to an early corneal flattening of 3 diopters and an underestimation of the IOL power of 2 diopters using SRKII calculation formula. Corneal flattening regressed after 3 weeks. We recommend that these patients should be informed of an eventual early hyperopic shift and late refractive error, that Binkhorst II or Hoffer-Q intraocular lens calculation formulas be used and keratometry measurements be obtained from computerized videokeratography. PMID- 10740057 TI - [ [In Process Citation] PMID- 10740056 TI - [ [In Process Citation] PMID- 10740058 TI - [ [In Process Citation] PMID- 10740059 TI - [Consensus on neovascular glaucoma]. AB - Neovascular glaucoma is a dreadful pathology with a rapid spontaneous evolution responsible for painful and blind eye. The main cause is an anterior neovascular proliferation following a broad retinal ischemia. Early diagnosis and treatment are required in order to maintain a good visual status and a satisfactory IOP control with medical, surgical or cylodestructive procedures. In any case, the treatment of the retinal ischemia has to be performed. One must keep in mind that the most efficient way to avoid the incidence of neovascular glaucoma is a strict control of clinical situations potentially responsible for retinal ischemia, namely VRO in elderly patients and diabetic retinopathy in younger patients. PMID- 10740060 TI - [Good practices in traumatic glaucoma]. AB - We review different aspects of trauma induced glaucoma and hypertonia, discussing pathophysiology, patient management, and the main clinical presentations. One can distinguish schematically first acute hypertonia related to changes in the position of the crystalline lens or to a hyphema which requires emergency medical or surgical care and second, chronic situations generally related to definitive angle lesions. We also discuss particular presentations such as phantom cell glaucoma and glaucoma by chemical burn or siderosis. PMID- 10740061 TI - [ [In Process Citation] PMID- 10740062 TI - [ [In Process Citation] PMID- 10740063 TI - [ [In Process Citation] PMID- 10740064 TI - [ [In Process Citation] PMID- 10740065 TI - [ [In Process Citation] PMID- 10740066 TI - [The urgency of emergencies?]. PMID- 10740067 TI - [ [In Process Citation] PMID- 10740068 TI - [Spiral (helical) CT of tracheobronchial diseases]. AB - The acquisition techniques, including some important dosimetric considerations, and the reconstruction methods reviewing the tomographic, bronchographic and endoscopic rendering techniques are first taken into consideration. They are applied to the most usual problems and diseases encountered in the daily activityThe main purpose of this review, which merges the modern technology of spiral (helical) CT with the multislice acquisition in the very near future, consists in delivering, whenever possible, an integrated diagnostic and therapeutic approach of imaging, endoscopy and surgery. PMID- 10740069 TI - [Ulnar dimelia: imaging modalities and surgical implications]. AB - PURPOSE: To report 2patients with ulnar dimelia. METHODS: Plain radiographs (2patients), angiography (1patient), MR (2patients), CT (2patients) with 3Dreconstruction (1patient) were performed. RESULTS: Unilateral polydactyly with absence of the thumb, ulnar duplication with absence of radius were observed in both patients. Shoulder dislocation was present in one patient. Vascular anomalities were observed in one patient who underwent angiography but did not interfere with polydactyly surgical treatment and pollicisation. CT with 3D helped in delineating the elbow and shoulder bone abnormalities and thus in planning elbow osteotomies. MR was helpful in imaging muscle anatomy. CONCLUSIONS: Ulnar dimelia or "mirror hand" is a rare congenital disorder of the upper limb. Approximately 70cases have been reported, including 3 with shoulder dislocation. A multimodality imaging approach, exploring the various aspects of the malformation is mandatory to help the surgeon in order to obtain a functional and aesthetic upper limb after complex surgical procedures taking into account the various aspects of the malformation. PMID- 10740070 TI - [Corticosteroid injections of the sacroiliac joint during magnetic resonance: preliminary results]. AB - PURPOSE: The aim of this study was to evaluate the advantages of MRguided injections of corticosteroids into the sacroiliac joints (SIJ) in patients suffering from refractory sacroiliitis despite appropriate oral therapy. MATERIALS AND METHODS: At a lowfield open MR, we performed twentyfour infiltrations in 12 patients suffering from persistent buttock pain. Clinical followup and laboratory findings were evaluated every 4 up to 6 weeks. A subjective rating using a dolorimetry scale (0= no pain, up to=10 for most severe pain) was assessed prior to intervention and at three months followup. Morphological changes of the SIJ were examined on a 1.5 Tesla imager before and three months after steroid injections. RESULTS: Adequate positioning of the needle was obtained in all but one case due to a software defect. Clinical improvement was observed in 10 patients with a mean painfree period of 9.6 months. Dolorimetry index decreased from 7.6 +/- 1.5 to 2.9 +/- 1.5. Nonsteroidal antiinflammatory drugs could be discontinued in 4 patients and reduced in 4 others. CONCLUSION: We therefore conclude that MRguided injection of the SIJ is feasible and could be preferred to more conventional methods when technical equipment is available. PMID- 10740071 TI - [CT imaging of fungal and nonfungal caseous sinusitis. A report of 50 cases]. AB - PURPOSE: Evaluate the contribution of CT for the diagnosis of caseous fungal and not fungal sinusitis, mostly of dental origin and often improperly called aspergillus sinusitis. MATERIAL: and methods. About 50 cases of caseous maxillary sinusitis, including 34 fungal sinusitis, the authors analyse the most important CT criteria. All patients underwent thereafter surgery, and the removal material was submitted to mycological investigations. RESULTS: Constructive and destructive modifications of the bone walls, fillings of sinus cavities and middle meatus are aspecific signs of chronicity. Dental origin foreign bodies and microcalcifications are more evocative but also non specific. Only the mycological investigation leads to the diagnosis of fungal and identifies by round fifty percent cases to the exact nature of fungal. CONCLUSION: There are no valid predictive criteria for fungal etiology by CT investigation. PMID- 10740072 TI - [A case of progressive multifocal leukoencephalopathy in AIDS: neuroimaging with clinical and pathologic correlation]. AB - We present the neuroimaging features with clinical and pathologic correlation in a case of Progressive Multifocal Leukoencephalopathy (PML) in AIDS. CT and MRI showed typical lesions of PML. At pathology examination, characteristic lesions of PML were found in association with HIV encephalitis due to CNS infection by the virus. PMID- 10740073 TI - [Primitive neuroectodermal tumor of the kidney manifested as a spontaneous hematoma]. AB - Ultrasound and CT in a young man, admitted for violent left flank pain, revelead a large heterogeneous left renal mass, with hypodense collection along the psoas. The diagnosis was spontaneous renal hematoma. A check CT 3 weeks later found a large tumor of the inferior pole. After radical nephrectomy, histopathology diagnosed a primitive neuroectodermal tumor. The patient died one year later despite neoadjuvant chemotherapy. This article reports the clinical, radiological and histopathological aspect of a primitive neuroectodermal tumor of the kidney, compared with the other cases in literature. PMID- 10740074 TI - [Legionella pneumophila: unusual lung and hepatic manifestations]. AB - We report an uncommon presentation of Legionella pneumophila infection following a placentalblood transplantation in an immunodeficient child. The patient presented with lung nodules and liver abscesses. We discuss how a digestive contamination can be suspected. PMID- 10740075 TI - [Osteosarcoma manifesting as Paget's bone disease]. AB - Osteosarcoma is a malignant bone tumour of mesenchymal osteoid tissue origin. Osteosarcoma can be primitive, but it may also complicated a Paget's disease of bone. We described a 75yearold man with a right thigh mass discovered after a motoring accident. Histopathologic examination revealed the diagnosis of osteosarcoma. Plain film Xrays showed Paget's lesion of the right pelvis bone. CT and MRI allowed complete evaluation of this lesion. Final diagnosis was those of malignant transformation of Paget disease. The patient was refused for surgery in view of the size of the tumour and chimiotherapy followed. PMID- 10740076 TI - [Quid? Wilson's disease]. PMID- 10740077 TI - [Preoperative devascularization of a vertebral metastasis with a spinal artery at the same level]. AB - An hypervascular and lytic thoracic (Th10) vertebral metastasis from kidney neoplasm causing cord compression was successfully resected after embolization. Because the anterior spinal artery arose at the same level, arterial embolization was only partial and completed by percutaneous vertebroplasty using Nbutyl cyanoacrylate. PMID- 10740078 TI - [Concerning the article "the need for examination as seen by the radiologist, the report as seen by the clinician"]. PMID- 10740079 TI - [Breast cancer. Text of recommendations. National Association of Accreditation and Health Evaluation]. PMID- 10740080 TI - [Protection of personal data: mandatory in epidemiology research]. PMID- 10740081 TI - [Factors associated with medical management of HIV infected persons in two French districts, 1993-1995]. AB - BACKGROUND: The regularity of medical followup of HIVinfected patients greatly influences the effectiveness of antiretroviral treatments and of prophylaxis of opportunistic infections. METHODS: To identify potential barriers to a regular followup, a retrospective study was conducted among the adult AIDS patients diagnosed between July 1993 and May 1995 in two French districts (Gironde and HautsdeSeine). Medical followup was described based on the frequency of CD4 counts in the medical file and on a confidential interview among patients whose HIV infection had been diagnosed at least 6 months before AIDS. Irregular followup (less than one CD4 count per year when CD4> 500/ mm(3), and per 6months when CD4< 500/ mm(3)) within the two years preceding AIDS diagnosis was analysed according to socioeconomic status and to social and behavioral factors. RESULTS: Among 290 patients, followup was irregular in 51% of the patients (injecting drug users: 66%, homo/bisexual men: 41%, patients infected through heterosexual contact: 49%, p<0.01). Factors independently associated with irregular followup were low income level (adjusted odds ratio (aOR)= 2.4; 95% confidence interval (CI), 1.44.1); absence of regular practitioner at HIV diagnosis (aOR= 1.9; 95% CI 1.13.3); imprisonment between HIV diagnosis and AIDS (aOR= 3.8; 95% CI 1.310. 9), and being non homo/bisexual male (aOR= 2.4; 95% CI 1.15.1) versus homo/bisexual male (aOR= 1.3; 95% CI 0.72.7) and female (reference). CONCLUSIONS: Results of this study show that several socioeconomic, psychological and cultural barriers to a regular preAIDS medical followup still exist. Impact of new antiretroviral therapy may only be optimal if these barriers are overcome. PMID- 10740082 TI - [Effect of dapsone on survival in HIV infected patients: a meta- analysis of finished trials]. AB - BACKGROUND: The aim of the study was to estimate the effect of dapsone on survival in HIVinfected patients. METHOD: The method was a metaanalysis. Data searches used MEDLINE, AIDS TRIALS, and AIDS DRUGS databases from 1983 to January 1996, clinical trials registries of appropriate collaborative research groups, abstract books of International Conferences on AIDS and infectious diseases between 1988 and 1996, references listed within selected articles and active experts in HIV infection. Were considered as eligible: randomized clinical trials, conducted in adults, with one arm evaluating dapsone as prophylactic agent for Pneumocystis Carinii Pneumonia (PCP). Each primary investigator was asked to provide the most recent aggregated study data by completing a standardized questionnaire and to provide files of individual patient data whenever possible. RESULTS: Overall, 17 trials (4343 patients) were eligible for the metaanalysis. The analysis of all available aggregated data included 16 trials (4267 patients) and showed no deleterious effect of dapsone on survival: OR=1.11, 95% Confidence Interval (CI)=0.961.29. There was no evidence of heterogeneity among studies (p=0.50). The analysis of individual data included 10 trials (3115 patients) (OR for aggregated data from those trials=1.10, CI=0. 931.29) and confirmed the absence of deleterious effect of dapsone on survival: stratified Hazard Ratio=1.12, CI=0.991.27 (logrank test: p=0.08). In this subsample, there was evidence of a deleterious effect of dapsone used as secondary prophylaxis. However, this result did not remain when the trial reporting the greatest negative effect of dapsone on survival was omitted. CONCLUSION: Dapsone may be used safely as a primary prophylactic regimen for PCP or toxoplasmosis. However, no definitive recommendation can be made for the use of dapsone as secondary PCP prophylaxis. PMID- 10740083 TI - Predictors of stroke - associated mortality in Africans. AB - BACKGROUND: The objective of the study was to examine the association of the stroke-related mortality with gender, age, ethnicity, social class, blood pressure, fibrinogen, selected clinical data and meteorologic parameters in hospitalized Africans. METHODS: A series of 1032 consecutively hospitalized incident cases of acute stroke between 1987 and 1991 was studied. Univariate and multivariate analyses were used to estimate the risk (odds ratio=OR) of stroke mortality for meteorologic parameters on the month before the accident onset and selected sociodemophysiological variables on the day of admission. RESULTS: The variables significantly associated with stroke mortality in multivariate analysis were male sex (OR= 2.3 [1.3 - 4.1]), low social class (OR= 2.0 [1.2 - 4.0]), migrant tribes (OR= 1.7 [1.5 - 1.8]), ischemic stroke (OR= 1.4 [1.2 - 1.6]), heart rate >=100 bpm (OR= 1.1 [1.0 - 1.2]), age > or =60 years (OR= 1.03 [1.01 - 1.06]), systolic blood pressure> 160 mmHg (OR= 1. 02 [1.01 - 1.03]), and fibrinogen > or =400 mg/dl (OR= 1.01 [1.002 - 1. 02]). However, diastolic blood pressure > 90 mmHg and global radiation< 340 Cal/cm(2)/day were significantly (p< 0.05) and inversely associated with stroke mortality. CONCLUSION: Our results indicate that male sex, older age, low social class, migration, ischemic stroke and higher baseline levels of heart rate, systolic blood pressure and fibrinogen are significant predictors of stroke mortality, but lower global radiation and higher diastolic blood pressure are inversely linked. PMID- 10740084 TI - [How to calculate the completeness of ascertainment of a morbidity register? Example of the Isere childhood handicap register in Grenoble, France]. AB - BACKGROUND: It should no longer be necessary to demonstrate the importance of knowing the completeness of ascertainment for a morbidity register, particularly with respect to the interpretation of prevalence rates and their trends, but also when using register data for etiological studies. METHOD: The study covered 9 generations of children born between 1980 and 1988. All of these children lived in the Isere department in SouthEast France, and each of them had at least one major deficiency, according to the inclusion criteria laid down by the RHEOP ("Registre des Handicaps de l'Enfant et Observatoire Perinatal", in French, or Childhood Handicap Register and Perinatal Observatory). These children were recruited from four different data sources. The completeness of ascertainment of the register was estimated first by means of the capturerecapture method, based on two sources that were shown to be independent by the Wittes method. Following this, loglinear models were used. The advantage of this was the absence of restrictions involved in adhering to the necessary validity conditions before applying the capture-recapture method, and the possibility of introducing heterogeneity variables, such as the number of deficiencies per child, for example. RESULTS: The applied capturerecapture method, with two main sources that have been found to be independent, gives an overall completeness of ascertainment of 86% CI(95%)[8291], with a variation of between 76% CI(95%)[6787] and 97% CI(95%)[93100] when the number of deficiencies per child is taken into account. After application of the loglinear models, the results obtained are very close to those obtained with the capture-recapture method, both in the case of estimation of the overall completeness of ascertainment and in the case of the completeness of ascertainment that is estimated according to the number of deficiencies variable. The similarity of the results obtained by the two methods appears to support our empirical study, but is only possible because of the validity of certain conditions (the interactions of the order of three were not significant) which can only be verified using statistical tests in the linear log models. CONCLUSION: If the application conditions of the capture-recapture method are carefully adhered to, it becomes possible, without the help of software, to produce a correct estimate of the number of missing cases. Nevertheless, it would be unreasonable to continue using this method alone since log linear models have been found to be independent of these validity conditions. PMID- 10740085 TI - [A study based on national DRG data to evaluate work load and practice relating to cancer patients in not-for-profit hospitals]. AB - BACKGROUND: In France there is no reliable information describing the organisation of hospital care for patients with cancer. The present study attempts to clarify this issue taking advantage of an information source that has up to now been unused, namely the national PMSI (Information System Medical program) data base. METHODS: A quantitative study has been carried out regarding cancer management in France using information filed with the PMSI which compiles data related to hospital admissions in all institutions with more than 100 beds and subject to a defined global budget. The "cancer" component of hospital activity was extracted using a specific algorithm which utilized the diagnostic and intervention codes included in the admission summaries. By using the unit of activity as defined by the ISA (Activity Synthetic Index) and the scale of relative cost according to the GHM (Homogeneous Group of patients) it was possible to analyse the information in terms of a balance sheet. RESULTS: The study provided information regarding the costs and methods of management, including therapeutic strategies, for each type of hospital establishment. It is shown that with one death out of six, cancer covers a quarter of all hospital stays, and one sixth of annual hospital expenses. This accounts for 16.2% of ISA ie approximately 29 billion francs (4.6 billion dollars) for the public and semipublic sectors. Surgery, which accounted for 32% of expenditures, appeared to be the most expensive intervention, ahead of chemotherapy (16.3%) and radiotherapy (9.1%). Each type of hospital organisation (university, cancer centre, district hospital) had their own relative figures. CONCLUSION: Through this study the current situation regarding cancer care in hospital has been documented. It has also demonstrated the value of the PMSI data base as a source of information for large scale quantitative studies of health care economics. However, the PMSI does not yet provide details regarding infrastructure or succession of hospital stay. Ultimately, this analysis does not provide any information on the quality or efficacy of care but does define a typological system for health care organisations which could provide information on distribution of resources. PMID- 10740086 TI - [A socio-anthropological study of birth in Morocco]. AB - BACKGROUND: To understand the role of beliefs, notions of risk and local practices in explaining the low utilization of maternal health services in Morocco. METHODS: Ethnographic research in three sites in Morocco, including interviews with 126 women and observation of medical encounters at health facilities. RESULTS: Local practices are not incompatible a priori with the use of health services, and women manifest a relatively pragmatic and flexible attitude towards the decisions they make regarding birth. The different factors that influence women's choices are analyzed, in particular those related to obstacles of accessibility and quality of services. CONCLUSION: The pluralism and eclecticism that characterize beliefs and behaviors related to birth in Morocco suggest that an improvement in the quality of care would encourage more women to seek care from health facilities. PMID- 10740087 TI - [Security services: an overview of the French legislation on cryptography]. AB - This paper deals with the main security services of data protection: integrity, authentication, nonrepudiation and confidentiality. It describes the different tools which may be used to achieve these security objectives: encryption, digital signature, access controls, integrity control, audit and certification. The French legislation concerning encryption has been recently updated in order to simplify the use of encryption techniques for personal data security. This legislation describes different authorization or reporting procedures according to the type of the request (application, equipment, import or export of encryption techniques) and the security level required. PMID- 10740088 TI - [Security of healthcare data networks used for epidemiological studies]. AB - Record linkage, for compiling sameperson records from various source files, can improve the feasibility of epidemiological research using populationbased studies. The question is comply with the European legislation on data privacy and data security. For example, a computerized record hash coding and linkage procedure is described to link medical information within the framework of epidemiological followup. Before their extraction, files are rendered anonymous using a oneway hash coding based on the standard hash algorithm (SHA) function. Once rendered anonymous using the software ANONYMAT, the linkage of patient information can be accomplished by means of a mixture model, taking into account several identification variables. An application of this anonymous record linkage procedure was carried out in order to link medical files on cancer, from 3 hospitals of the French RhoneAlpes region. This application stresses how the use of the ANONYMAT software allows compliance with the legislation on data confidentiality without entailing problems on data availability. PMID- 10740089 TI - The natural history in slow onset Crohn's disease and lack of early markers influences the delay to diagnosis. PMID- 10740090 TI - Autor's reply PMID- 10740091 TI - [Is it possible to improve the health behaviour of the elderly?]. PMID- 10740092 TI - [Oligodendrogliomas revisited]. PMID- 10740093 TI - [Development of dyskinesias induced by treatment for Parkinson's disease: potential role of first exposure to L-DOPA (or phenomenon of priming)]. AB - L-DOPA-induced dyskinesias are one of the main problems encountered in treating patients with Parkinson's disease (PD). They are induced by the antiparkinsonian medications and primarily related to the degree of dopaminergic depletion, as shown by the fact that they tend to appear several years after the onset of the disease. Do the initial therapeutic decisions taken in treating a PD patient influence the point at which dyskinesias first occur? This question is raised in view of the apparent priming phenomenon that occurs in first exposure to L-DOPA. L-DOPA administrated to an MPTP intoxicated monkey rapidly corrects the animals' motor symptoms but generate dyskinesias. In contrast, the administration of dopaminergic agonists with a long half-life has a similar therapeutic effect but without inducing dyskinesias. However, a parkinsonian monkey that had received L DOPA and developed dyskinesias, which were subsequently abolished when the treatment was withdrawn for several months, proceeded to develop dyskinesias when treatment with dopaminergic agonists with long half-life was introduced. The monkeys' previous exposure to L-DOPA (i.e. priming) thus increased its susceptibility to develop dyskinesias after exposure to drugs which would not otherwise have had this effect. Pulsatile activation of type D2 dopamine receptors is reported to be the principal factor in the triggering of dyskinesias and may well be involved in the priming phenomenon. While the pathophysiological basis of priming is not yet known, the phenomenon would not appear to be related to a hyperexpression of dopamine receptors (types D1 and D2) in the sensorimotor striatum. The results of recent experiments have given rise to several different hypothesis for the mechanisms involved in priming: the role of internalization of dopamine receptors after administration of dopaminergic drugs; change in the distribution of D3 dopamine receptor; changes in the expression of peptides (substance P, enkephalin) in efferent neurons of the striatum; and reorganization of connections at the level of the dopaminergic neurons and their target tissue. While many questions remain unanswered, it may well be that the initial therapeutic decisions taken when treating de novo patient are crucial in trying to delay the onset of dyskinesias. PMID- 10740094 TI - [Neuronal expression of foreign genes with recombinant rabies virus variants]. AB - Rabies virus variants obtained by recombinant DNA techniques enabled us to use the high neurotropism of rabies virus to express foreign genes (e.g: Chloramphenicol Acetyl Transferase gene) in neuronal cell cultures as well as in rodent brain. The foreign gene was inserted in the viral pseudogene region; this insertion did not affect the neurotropism of rabies virus, as shown by infection of neuronal cell cultures without any major cytopathic effects for several days. Stereotaxic inoculation of these rabies virus variants into rat striatum indicated that insertion of the foreign gene did not alter the viral axonal transport and the subsequent widespread brain infection. These data allow to consider rabies virus as a vector for the selective expression of foreign genes in neurons. PMID- 10740095 TI - [The first episode of central nervous system demyelinization and hepatitis B virus vaccination]. AB - BACKGROUND: Central nervous system (CNS) demyelinating episodes have been described following numerous vaccines but there is no definite conclusion about a causal relationship. Recently, in France, in the context of an Expanded Program on Immunization, several cases of CNS demyelination have been observed following injection of recombinant hepatitis B (HB) vaccine, leading to great concern. METHODS: We performed a hospital-based case-control study of 121 patients with a first episode of CNS demyelination occuring between July 1993 and December 1995 and 121 age and sex matched controls seen in the same period. Data on vaccinations history of cases and controls were collected by a postal questionnaire and confirmed by a phone interview. RESULTS: Adjusted odds ratio (OR) obtained from conditional logistic regression between a first episode of CNS demyelination and any vaccination were equal to 1.4 (95 p. 100 CI 0. 5-4.3) for an exposure within the 60 previous days and 2.1 (95 p. 100 CI 0.7-6.0) for an exposure within the 61-180 previous days. Similar results were found for HB vaccine exposure within the 60 previous days (adjusted OR=1.7, 95 p. 100 CI 0.5 6.3) or within the 61 to 180 previous days (adjusted OR= 1.5, 95 p. 100 CI 0.5 5.3). CONCLUSION: These findings did not permit to exclude confidently an association between HB vaccine and the occurrence of a first CNS demyelinating episode. PMID- 10740096 TI - [Quality of life and multiple sclerosis: validation of the french version of the self-questionnaire (SEP-59)]. AB - We conducted a prospective study among 166 multiple sclerosis (MS) patients (103 from an university hospital, 63 from a MS rehabilitation center) to assess the properties of the French version of the Multiple Sclerosis Quality Of Life - 54 items (MS QOL-54) which combines the MOS SF36 together with MS specific items. The SF-36 had been translated into French through the IQOLA project. We translated and adapted the MS specific items with the help of three different teams. The translation into French has an addition of five items, because we kept the MS specific items of an earlier unpublished form. Acceptability is excellent with a response rate over 90p.100. Test-retest reliability is good except for the "role limitation-emotional" scale of the SF-36. Construct validity, based on factor analysis, shows no change in the SF-36 internal consistency and the specific items provided their own information. External validity, tested against both medical (Expanded Disability Status Scale, Kurtzke scale, Mini-Mental-State and disease stage) and rehabilitation (Functional Independence Measure) parameters is excellent. The French MS QOL questionnaire contains 59 items including both the SF-36 and the MS QOL-54 items. This will permit international comparisons of MS patients' care and therapy. PMID- 10740097 TI - [Late cerebrovascular complications of cardiac transplantation]. AB - The occurrence of stroke after the post-operative period of cardiac transplantation is a rare event, and the role of the cardiac transplant in these patients, who often have various vascular risk factors, is unclear. We reviewed the clinical records of 303 consecutive patients with orthotopic cardiac transplantation performed from March 1985 to December 1996 and selected those who developed a stroke over the first 2 months postoperatively, in order to evaluate the frequency and the mechanisms of late cerebrovascular complications. Four patients had presented cerebral infarct and two intracranial hemorrhage. The overall risk of late cerebrovascular complications was 2.6p.100 at 5 years. Ischemic stroke was related to cardiac embolus in one patient, lacunar infarction in another, and was of undetermined etiology in two cases. In addition to vascular risk factors prior to transplantation and the development of intracardiac thrombus, immunosuppressive therapy, the surgical procedure and cardiac rejection may play a role in the occurrence of stroke in such patients. PMID- 10740098 TI - [Cysticercosis and epilepsy in the city of Tone, north of Togo]. AB - An epidemiology study was conducted in Tone, a city in the very north of Togo from November 1 to 25, 1995. The prevalence of epilepsy was studied in 9,155 subjects and seroprevalence of cysticercosis in 1,343. This was a representative sample of this region with 194,000 inhabitants. The prevalence of epilepsy was 18.6 per 1 000 inhabitants. A causal relationship was found betwen cysticercosis and epilepsy. The prevalenced of cysticercosis was 38 per 1 000 persons in the general population and 135.29 per 1 000 epileptic patients. The difference was highly statistically significant (X( 2)=74.17, p<10(-6)). PMID- 10740099 TI - [Left unilateral melokinetic apraxia and left dynamic apraxia following partial callosal infarction]. AB - A 69 year-old right-handed man suffered from an infarct in the left anterior cerebral territory's artery, involving the anterior and middle parts of the corpus callosum, and the cingulum. He had a right crural hemiparesis with a grasp reflex of the right hand, and ipsilaterally a melokinetic and a dynamic apraxia. Agility of the left fingers was lost: fast and nimble movements of theses fingers, and repetitive gestures of the left hand were defective. Execution of motor sequences with the left hand was disrupted by a lack of litheness in gesture series. By analogy with unilateral left ideomotor apraxia, we suggest melokinetic and dynamic apraxia could be symptomatic of an interhemispheric disconnection. Premotor cortex could have a dominance for both digital movements and programmation of gestual sequences. As a general rule, the left cortex could be preferentially activated by the nature of some tasks, particularly the execution of complex gestual series, which implie an internal speech. In theses conditions, an interhemispheric disconnection could lead some difficulties to make theses gestures with the left hand. PMID- 10740100 TI - [Cervicobrachial neuralgia without deficits associated with 2 cases of extracranial vertebral artery dissection]. PMID- 10740101 TI - [Pathological laughter after the brainstem infarction]. AB - We report a case of 'fou rire prodromique' heralding a brainstem infarct with favorable prognosis after thrombolysis. Different clinical forms of pathological laughter, the pathophysiological mechanisms and clinico-anatomical correlations of this rare phenomenon are discussed. PMID- 10740103 TI - [Idiopathic]. PMID- 10740102 TI - [Pseudomigraine with transient neurological signs and lymphocytic pleiocytosis]. AB - We report the case of a 32-year-old man without previous medical history of migraine, who presented with severe headache and temporary focal neurological deficits. Lumbar puncture revealed aseptic lymphocytic pleiocytosis. The patient completly recovered within 2 months. This condition was suggestive of a transient syndrome of headache with neurologic deficits and CSF pleiocytosis. The main characteristics and the physiopathology of this uncommon disorder are discussed. PMID- 10740104 TI - [Motor and vocal tic after severe head trauma]. PMID- 10740105 TI - [Partial motor epilepsy, intracranial hematoma and protein S defiency in a 49 year-old woman]. PMID- 10740106 TI - [MRI aspects of intracranial hypotension]. PMID- 10740107 TI - [Recent advances in understanding the mechanism of sleep]. PMID- 10740108 TI - [ [In Process Citation] PMID- 10740109 TI - [Thoracic hydatidosis. A review of 1619 cases]. AB - We analyzed the cases of 1619 patients who underwent surgery for hydatidosis in a chest localization to present a review of this parasitosis, endemic in Southern Mediterranean countries. A lung localization was observed in 1527 patients (94.6%). Extrapulmonary localizations were less common (5.62%) and included 42 heart cysts, 21 primary pleural cysts, 13 diaphragm cysts, 8 mediastinum cysts and 8 ribs cysts. For pulmonary cysts the sex ratio was 1: 1 (51.46% men, 48.85% women). Mean age was 24 years. A context of hydatid exposure was present in 81.8% of cases. The diagnosis was established on the basis of associated functional signs and an opacity with regular borders on the chest x-ray. Surgical treatment was simple cystecomy in 85.9% of the cases and lung resection in 14. 1%. Operative mortality was 0.785%. Pleuropulmonary recurrence was observed in 21 patients. Thoracic and pulmonary hydatidosis is easily diagnosed and treatment is well defined. It remains however a major challenge for health care with an important social and economic impact. Prevention can only be achieved by carefully designed actions against parasite transmission. PMID- 10740110 TI - [Evaluation and symptomatic treatment of surinfectious exacerbations of COPD: preliminary study of antibiotic treatment combined with fenspiride (Pneumorel 80mg) versus placebo]. AB - Exacerbations of chronic obstructive pulmonary disease (COPD) have an inflammatory component in addition to the possible infectious component. The antiinflammatory properties of fenspiride (Pneumorel(R) 80 mg) should be evaluated in this frequent clinical situation. OBJECTIVES: Assess the supplementary therapeutic benefit provided by fenspiride administered in combination with antibiotics in COPD patients presenting an episode of bronchial infection. PATIENTS AND METHODS: A preliminary randomized placebo-controlled double-blind study was conduced in 7 centers. Patients under 80 years of age of both sexes were included. All patients had COPD and presented a bronchial infection defined as the presence of at least 2 of the 3 criteria defined by Anthonisen. Patients were randomly assigned to group F or group P. Group F received an antibiotic therapy from day 1 to day 11 plus fenspiride (3 x 80mg/d from day 0 to day 30). Group P received the same antibiotic therapy plus placebo. Amoxicillin 500mg plus clavulanic acid 125, 3 tablets/day, was administered in both groups. RESULTS: Thirty-nine patients were included (group F 19 patients, group P 20 patients; 6 women and 33 men; mean age 61.1 +/- 9.8 years). The 3 Anthonisen criteria were present in 79% and 75% of the patients in group F and P respectively (NS). On day 11, expectoration resolved in 39% and 32% (NS) and cough in 44% and 16% (NS) of the patients in groups F and P respectively. Lung auscultation returned to normal in 83% of the patients in group F compared with 47% in group P (p=0.05). A composite clinical score including expectoration cough and auscultation findings showed that 28% of the patients in group F were symptom free on day 11 compared with 0% in group P (p=0.04). On day 30, the two groups were comparable. CONCLUSION: In this preliminary study of patients with COPD presenting a bronchial superinfection, there was a significant improvement in lung auscultation and in the composite clinical score in patients given fenspiride. Fenspiride was thus found to provide an early clinical benefit. PMID- 10740111 TI - [Yellow nail syndrome. Three cases]. AB - Yellow nail syndrome is an uncommon diagnosis established on the basis of clinical presentation with slow-growing yellow discolored nails, lymphedema, and pulmonary manifestations. We report 3 new cases with their pulmonary component. PMID- 10740112 TI - [Active tuberculosis in a patient with Hodgkin's disease. A case report]. AB - We report a case of active tuberculosis in a patient with Hodgkin's disease. The two diagnoses were established simultaneously at the Respiratory Disease Unit of the Yalgado National Hospital, Burkina Faso. The clinical presentation was misleading as the signs and symptoms of the two disease are similar. Certain diagnosis was established after isolation of tuberculosis bacilli and histocytology findings for Hodgkin's disease. Both diseases are potentially curable. The clinical course depends on early diagnosis and treatment. PMID- 10740113 TI - [Candida albicans fungemia with pulmonary localization treated with fluconazole. A case report]. AB - We report a case of Candida albicans fungemia complicated by a pulmonary localization in a non-immunocompromised patient. Complete recovery was obtained after a long course of high-dose fluconazole in spite of in vitro resistance of the Candida to fluconazole. The usefulness of fluconazole therapy, the best dosage regimen and the in vitro and in vivo correlations are discussed. PMID- 10740114 TI - [Hydatid pulmonary embolism. Two case reports]. AB - Hydatid pulmonary embolism is an uncommon condition resulting from the rupture of a hydatid heart cyst or the opening of a visceral hydatid cyst (often in the liver) into the venous circulation. We report two cases of hydatid pulmonary embolism following rupture of a hydatic cyst in the right ventricle. One case progressed to chronic cor pulmonale. We examine the pathophysiological mechanisms as well as the clinical, therapeutic and evolutive aspects. PMID- 10740115 TI - [Caseous pneumonia in a 14-month-old infant]. AB - We report a case of caseous pneumonia in a 14-month-old HIV-negative infant. This clinical picture is usually observed in healthy young adults, rarely in infants. The clinical and radiological signs of caseous pneumonia often simulate acute non tuberculous respiratory disease. Bacteriological confirmation is required, but the diagnosis can be suggested in case of a pulmonary syndrome with no bacterial isolate and unresponsive to well-conducted antibiotic therapy. The clinical course is rapid with caseous involution of the lung. Early diagnosis is required for specific curative treatment and to limit sequelae. PMID- 10740117 TI - [Did you read the last issue?] PMID- 10740116 TI - [Clinical and economical assessment of surgical treatment for obstructive sleep apnea. National Agency for Accreditation and Evaluation in Health]. PMID- 10740118 TI - Proceedings of a symposium. How should phase II trials in cardiovascular medicine be conducted? Hilton Head, South Carolina, USA. October 3-4, 1997. PMID- 10740119 TI - Current definitions of phases of investigation and the role of the FDA in the conduct of clinical trials. PMID- 10740120 TI - Challenges in the conduct and interpretation of phase II (pilot) randomized trials. PMID- 10740121 TI - The role of clinical nonfatal end points in cardiovascular phase II/III clinical trials. PMID- 10740122 TI - Economic end points in phase II trials. PMID- 10740123 TI - Select-drop designs in clinical trials. PMID- 10740124 TI - Sample size and interim analysis issues for dose selection. PMID- 10740125 TI - Equivalence and noninferiority trials. PMID- 10740126 TI - Design and interpretation of equivalence trials. PMID- 10740127 TI - Estimating the distribution of quality-adjusted life with censored data. PMID- 10740128 TI - Chronic ischemic heart disease. PMID- 10740129 TI - Acute ischemic heart disease. PMID- 10740130 TI - Surrogate end points in cardiovascular disease trials. PMID- 10740131 TI - An FDA perspective on antiarrhythmic drugs in phase II trials. PMID- 10740132 TI - The development of antiarrhythmic drugs. PMID- 10740133 TI - Current perspectives on the design of phase II trials of new drugs for the treatment of heart failure. PMID- 10740134 TI - Design of phase II trials in congestive heart failure. PMID- 10740135 TI - Patient-specific dosing of IIb/IIIa antagonists during acute coronary syndromes: rationale and design of the PARAGON B study. The PARAGON B International Steering Committee. AB - BACKGROUND: Acute coronary syndromes, the leading cause of hospitalizations among adults, are frequently the sequelae of atherothrombotic events associated with coronary arterial plaque rupture. Beyond the usual antithrombotic therapies (aspirin and heparin), potent antiplatelet agents, glycoprotein IIb/IIIa receptor antagonists, have been shown to improve patient outcome. Lamifiban is a short acting, renally excreted IIb/IIIa antagonist that was found in post hoc analyses of the Platelet IIb/IIIa Antagonism for the Reduction of Acute Coronary Syndrome Events in a Global Organization Network (PARAGON A) study to reduce the 30-day incidence of death or myocardial infarction by 40% when plasma concentrations of 18 to 42 ng/mL were achieved. METHODS AND RESULTS: To determine if lamifiban, dosed according to creatinine clearance, could decrease the rates of death, myocardial infarction, or refractory ischemia, a randomized, double-blind, placebo-controlled trial was undertaken. In 26 countries, 5228 patients seen within 12 hours of symptom onset of a non-ST-elevation acute coronary syndrome were randomly assigned to placebo or lamifiban bolus and infusion. CONCLUSION: The plasma concentration of small-molecule IIb/IIIa inhibitors is strongly influenced by renal function, and renal-specific dosing of these agents may improve outcome among patients with acute coronary syndromes. The PARAGON B trial is testing this hypothesis. PMID- 10740136 TI - Medicaid, managed care, and the care of patients hospitalized for acute myocardial infarction. AB - BACKGROUND: TennCare, beginning in January 1994, channeled all Medicaid-eligible patients into managed care while expanding Medicaid coverage to large numbers of previously uninsured patients. We assessed the impact of TennCare on (1) coronary revascularization of patients who had had an acute myocardial infarction (AMI), (2) the likelihood of the patient having a usual provider of care after discharge from the hospital, and (3) health and functional status 1 to 3 years after the index AMI. METHODS AND RESULTS: With the use of 1996 to 1997 survey data from 438 patients hospitalized for AMI in 1993 and 1995 who were under age 65 years at the index admission, multivariate analysis was used to calculate effects of TennCare on utilization and outcomes. TennCare patients were as likely as privately insured patients to have received coronary revascularization within 30 days of the index AMI (odds ratio 0.87, P =.69). Persons enrolled in TennCare and in traditional Medicaid who received a revascularization procedure were much less likely to have received coronary angioplasty than coronary bypass surgery than were the privately insured (TennCare: odds ratio 0.37, P =.05; Medicaid: odds ratio 0.28, P =.08). Virtually all TennCare enrollees (94%) reported having a usual provider of care in the year before the survey versus 85% for privately insured patients with AMI in 1995 (P =.05). On health and functional status, TennCare enrollees overall fared as well as those with private insurance. CONCLUSIONS: Our results suggest that TennCare brought patients who otherwise would have been uninsured or enrolled in Medicaid into the medical mainstream, measured both in terms of utilization of services and health and functional status. PMID- 10740137 TI - Statins: effective antiatherosclerotic therapy. AB - BACKGROUND: Statins are the most effective agents currently available for lowering plasma levels of low-density lipoprotein cholesterol (LDL-C) and are the mainstay of therapy for hyperlipidemia. The statins are highly liver-selective, inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, a key enzyme in the synthesis of cholesterol. Several large, controlled clinical trials have confirmed significant reductions in rates of coronary heart disease morbidity and death with long-term statin therapy in patients with mild to severe hypercholesterolemia. METHODS AND RESULTS: This review article is based on a literature search of more than 60 relevant articles from peer-reviewed journals. Search engines included Medline and Embase. In surveying clinical and angiographic evidence, we found that statins appear to reduce the incidence of coronary events by slowing the progression of atherosclerosis and preventing atheromatous lesion formation. We found that the 6 statins currently marketed atorvastatin, cerivastatin, fluvastatin, lovastatin, pravastatin, and simvastatin differ in their inhibitory action on the HMG-CoA reductase enzyme. CONCLUSIONS: The use of more potent statins such as atorvastatin and simvastatin affords greater lowering of LDL-C and triglyceride levels, allowing more patients to achieve target goals. The question of how low LDL-C levels should be lowered will be answered by ongoing clinical trials. PMID- 10740138 TI - beta-blocker improvement in diastolic performance: the yin and yang of ventricular function changes. PMID- 10740139 TI - Neurohormonal activation in severe heart failure: relations to patient death and the effect of treatment with flosequinan. AB - BACKGROUND: Flosequinan is a direct-acting vasodilator that exerts beneficial hemodynamic effects and improves the exercise tolerance of patients with heart failure. However, a multicenter trial has demonstrated that long-term administration of flosequinan is associated with increased mortality rate. To explore a possible role of neurohormonal activation on this adverse outcome, we conducted a substudy to examine the plasma levels of 3 neurohormonal systems known to have prognostic implications in heart failure. METHODS: At 20 participating Canadian centers, paired plasma samples at baseline and 1 month after randomization for the measurement of N-terminal atrial natriuretic peptide (N-ANP), angiotensin II, and norepinephrine were obtained in 234 patients (114 receiving flosequinan and 120 receiving placebo). RESULTS: Treatment with flosequinan was associated with a decline in median plasma N-ANP levels (2139 pmol/L at baseline to 1625 pmol/L at 1 month [P =. 0001]), unchanged plasma angiotensin II levels (40 to 50 pmol/L [P =. 2700]), and a modest increase in plasma norepinephrine levels (391 to 439 pg/mL [P =.002]). These changes were not observed in the placebo group. Multivariate analysis of baseline variables revealed that plasma norepinephrine level predicted patients' death whereas analysis incorporating both baseline and 1-month variables indicated that plasma N-ANP level predicted patients' death. Furthermore, in the flosequinan group, a significant decline in plasma N-ANP level was observed in the survivors only. On multivariate analysis of baseline and 1-month data, the increase in plasma norepinephrine level did not predict the increase in heart rate associated with the use of flosequinan, suggesting that the 2 effects might be mediated by separate mechanisms. CONCLUSIONS: Results of our study demonstrate that in patients with severe heart failure, baseline norepinephrine level predicts death. Flosequinan increases plasma norepinephrine level and heart rate in these patients through mechanisms that override its beneficial hemodynamic effects. Our study reinforces the concept that the direct actions of a pharmacologic agent may have a more profound impact on the prognosis of these patients than the hemodynamic effects. PMID- 10740140 TI - Beta-blockade therapy in chronic heart failure: diastolic function and mitral regurgitation improvement by carvedilol. AB - BACKGROUND: In patients with chronic heart failure, the use of carvedilol therapy induces clinical and hemodynamic improvement. However, although the benefits of this beta-blocker have been established in patients with chronic heart failure, the mechanisms underlying them and the changes in left ventricular systolic function, diastolic function, and mitral regurgitation during long-term therapy remain unclear. OBJECTIVE: To identify the clinical and functional effects of carvedilol, focusing on diastolic function and mitral regurgitation variations. METHODS: Forty-five consecutive patients with chronic heart failure (ejection fraction 24% +/- 7%), 17 with dilated ischemic and 28 with nonischemic cardiomyopathy, were treated with carvedilol (mean dose 44 +/- 30 mg) and matched for clinical (New York Heart Association functional class and heart failure duration) and hemodynamic (cardiac index and pulmonary wedge pressure) characteristics to a control group. Clinical and echocardiographic variables were measured in the 2 groups at baseline and after 6 months and the results compared. RESULTS: After 6 months of treatment with carvedilol, left ventricular ejection fraction had increased from 24% +/- 7% to 29% +/- 9% (P <.0001); this change was caused by a reduction in end-systolic volume index (106 +/- 41 vs 93 +/- 37 mL/m(2); P <. 0001). Deceleration time of early diastolic filling increased (134 +/- 74 vs 196 +/- 63 ms; P <.0001). Seventeen of the 27 patients with demonstrated improvement of left ventricular diastolic filling moved from having a restrictive filling pattern to having a normal or pseudonormal left ventricular filling pattern. In the control group, no significant changes in deceleration time of early diastolic filling were found (139 +/- 74 vs 132 +/- 45 ms; P = not significant). The effective regurgitant orifice area decreased significantly in the carvedilol group but not in the control group. These changes were associated with a significant reduction of the mitral regurgitant stroke volume in the carvedilol group (50 +/- 25 vs 16 +/- 13 mL; P <.0001) but not in the control group (57 +/- 29 vs 47 +/- 24 mL; P = not significant). These changes of mitral regurgitation were closely associated with significant improvement of forward aortic stroke volume (r = -.57, P <.0001). These findings were not observed in patients in the control group. CONCLUSIONS: The results of this study show that long-term carvedilol therapy in patients with chronic heart failure was able to prevent or partially reverse progressive left ventricular dilatation. The effects on left ventricular remodeling were associated with a concomitant recovery of diastolic reserve and a decrease of mitral regurgitation, which have been demonstrated to be powerful prognostic predictors in such patients. Overall these findings provide important insights into the pathophysiologic mechanisms by which carvedilol improves the clinical course of patients with chronic heart failure. PMID- 10740141 TI - Randomized trial of candesartan cilexetil in the treatment of patients with congestive heart failure and a history of intolerance to angiotensin-converting enzyme inhibitors. AB - BACKGROUND: Many patients with congestive heart failure do not receive the benefits of angiotensin-converting enzyme (ACE) inhibitors because of intolerance. We sought to determine the tolerability of an angiotensin II receptor blocker, candesartan cilexetil, among patients considered intolerant of ACE inhibitors. METHODS: Patients with CHF, left ventricular ejection fraction less than 35%, and history of discontinuing an ACE inhibitor because of intolerance underwent double-blind randomization in a 2:1 ratio to receive candesartan (n = 179) or a placebo (n = 91). The initial dosage of candesartan was 4 mg/d; the dosage was increased to 16 mg/d if the drug was tolerated. A history of intolerance of ACE inhibitor was attributed to cough (67% of patients), hypotension (15%), or renal dysfunction (11%). RESULTS: The study drug was continued for 12 weeks by 82.7% of patients who received candesartan versus 86.8% of patients who received the placebo. This 4.1% greater discontinuation rate with active therapy was not significant; the 95% confidence interval ranged from 4.8% more discontinuation with placebo to 13% more with candesartan. Titration to the 16-mg target dose was possible for 69% of patients who received candesartan versus 84% of those who received the placebo. Frequencies of death and morbidity were not significantly different between the candesartan and placebo groups (death 3.4% and 3.3%, worsening heart failure 8.4% and 13.2%, myocardial infarction 2.8% and 5.5%, all-cause hospitalization 12.8% and 18.7%, and death or hospitalization for heart failure 11.7% and 14.3%). CONCLUSIONS: Candesartan was well tolerated by this population. The effect of candesartan on major clinical end points, including death, remains to be determined. PMID- 10740142 TI - Endothelial cell dysfunction correlates differentially with survival in primary and secondary pulmonary hypertension. AB - BACKGROUND: Plasma von Willebrand factor antigen (vWF:Ag) has been used as a marker of endothelial perturbation in a number of vascular disorders. In this study, vWF:Ag was determined as an attempt to evaluate the severity of endothelial cell dysfunction in primary pulmonary hypertension (PPH) and congenital heart disease-associated pulmonary hypertension (CHD-PH) comparatively and to determine its impact on short-term survival. METHODS AND RESULTS: Clinical, hemodynamic, and biochemical data were obtained from 11 patients with PPH and 24 with CHD-PH. Patient groups were similar in terms of age and pulmonary artery pressure. vWF:Ag was measured by electroimmunodiffusion. Patients were followed up for 1 year and at that time, data collected at the beginning of the study were subjected to univariate and multivariate analyses. vWF:Ag was increased in patients (normal reference value 87% +/- 23% activity, mean +/- SD), with higher levels in the PPH group (231% +/- 89%) in comparison with the CHD-PH group (127% +/- 68%) (P <.001). Multivariate analysis showed that survival was influenced by the underlying cause of pulmonary hypertension and vWF:Ag levels but not by patient age, sex, or pulmonary artery pressure. Seven of 10 nonsurvivors but only 4 of 25 survivors had PPH (P =.007). vWF:Ag was 255% +/- 90% in the nonsurvivor group and 121% +/- 54% in the survivors (P <.001). CONCLUSIONS: Our findings suggest that short-term survival is related to the severity of endothelial cell dysfunction in pulmonary hypertension. In addition, exceedingly high vWF:Ag levels in PPH might reflect a particular pattern of endothelial cell dysfunction that could be associated with decreased short-term life expectancy in this disorder compared with secondary forms of pulmonary hypertension. PMID- 10740143 TI - Determinants of angiotensin-converting enzyme inhibitor prescription in severe heart failure with left ventricular systolic dysfunction: the EPICAL study. AB - BACKGROUND: Angiotensin-converting enzyme (ACE) inhibitors have been demonstrated to reduce morbidity and mortality rates in patients with heart failure with left ventricular systolic dysfunction. Nevertheless, these drugs are underutilized in current practice and prescribed at doses below those usually recommended. The aim of this work was to identify the social, demographic, laboratory, clinical, and therapeutic factors associated with nonprescription of ACE inhibitors and/or their prescription at doses below those recommended in the treatment of severe long-term congestive heart failure (CHF). METHODS AND RESULTS: An epidemiologic observational study, EPICAL (EPidemiologie de l'Insuffisance Cardiaque Avancee en Lorraine), studied 417 patients with severe CHF surviving after the index hospitalization. Multivariate logistic regression determined the factors associated with ACE inhibitor nonprescription and with their prescription at lower-than-recommended doses. ACE inhibitors were taken by 75% of the patients but 38% took lower-than-recommended doses. Factors shown to be associated with nonprescription included patients >65 years of age with renal impairment (odds ratio 19.5, confidence interval [CI] 7.9-48.0), nonsinus cardiac rhythm (odds ratio 2.0, CI 1.2-3.2), and prescription of potassium-sparing diuretics (odds ratio 2.4, CI 1. 2-4.7). Renal impairment was the single most important factor associated with prescription of lower-than-recommended doses, particularly in elderly patients. CONCLUSIONS: Our results underline the need for optimal and better use of ACE inhibitor therapy. CHF treatment guidelines must be more uniformly applied by all physicians caring for patients with heart failure. PMID- 10740144 TI - Reproducibility of neointima quantification with motorized intravascular ultrasound pullback in stented coronary arteries. AB - BACKGROUND: Intravascular ultrasound (IVUS) imaging has shown excellent reproducibility immediately after coronary stent implantation. However, the variability of measurements in lesions late after stent implantation, when neointima formation is present, has not been studied. Neointimal tissue is generally low echogenic and thus difficult to quantify. We therefore sought to analyze the reproducibility of morphometric measurements late after stent implantation. METHODS AND RESULTS: Fifty consecutive patients were investigated 6 months after Palmaz-Schatz stent implantation (motorized catheter pullback 0.5 mm/s). Two experienced investigators independently identified the stent area, lumen area, and neointimal area at different sites within the stent. Planimetric measurements were performed with commercially available software. Correlation coefficient and mean difference for corresponding measurements were calculated for the intraobserver and interobserver comparisons. Variability for the intraobserver and interobserver comparisons was similar. Observer agreement regarding the presence of neointimal hyperplasia was as high as 71% (interobserver comparison 62%). The mean difference for neointima area was 0.06 +/- 1.5 mm(2) (-0.6 +/- 1.5 mm(2)); mean differences for lumen area were 0.02 +/- 0.19 mm(2) (0.03 +/- 0.17 mm(2)) and for stent area 0.01 +/- 0.09 mm(2) (-0.02 +/ 0.12 mm(2)) (values for interobserver comparison are given in parentheses). Correlation between measurements was high for all structures: correlation coefficients were 0.66 (0.69) for neointima, 0.94 (0.95) for lumen, and 0.95 (0. 91) for stent area. CONCLUSIONS: Morphometric measurements of IVUS investigations with motorized IVUS pullback late after stent placement show good reproducibility. Intraobserver variability and interobserver variability are low. Differences for corresponding measurements were more pronounced for neointima area. Motorized catheter pullback guarantees high reliability of IVUS measurements and should be used routinely for clinical IVUS studies. PMID- 10740145 TI - Multivessel coronary stenting versus bypass surgery in patients with multivessel coronary artery disease and normal left ventricular function: immediate and 2 year long-term follow-up. AB - BACKGROUND: Compared with coronary artery bypass surgery (CABG), the clinical benefits of intracoronary stenting have not been established in patients with multivessel coronary lesions. METHODS AND RESULTS: To compare the clinical outcomes of intracoronary stenting with that of CABG, we reviewed the outcomes of patients with multivessel coronary artery disease from an observational database. Two hundred consecutive patients with multivessel coronary artery disease and normal left ventricular function were evaluated. In 200 patients, multivessel stenting was performed in 100 and CABG was performed in 100. Complete revascularization was achieved in 95% in the CABG group and in 69% in the stent group (P <.05). The duration of total hospital stay and coronary care unit admission was significantly shorter in the stent group (P <.05). The long-term survival was similar between the 2 groups. There were no significant differences of cardiac events between the 2 groups except for the recurrence of angina (19% in stenting vs 8% in CABG, P =.03) and target lesion revascularization (19% vs 2%, P <.01) in the patients with stents. CONCLUSIONS: In selected patients with multivessel coronary artery disease and normal left ventricular function, intracoronary stenting may offer an effective alternative to coronary bypass surgery. PMID- 10740146 TI - Role of intracoronary ultrasound after high-pressure stent implantation. AB - BACKGROUND: Poststent high-pressure balloon inflation has been shown to improve clinical outcomes. However, it is unknown whether intracoronary ultrasound (ICUS) provides additional clinical guidance after initial high-pressure balloon inflation is used during stent placement. Thus the purpose of this study was to determine if stent deployment techniques are improved with ICUS imaging despite an optimal angiographic result achieved with high-pressure balloon inflation. METHODS AND RESULTS: Prospective data were collected on 96 consecutive patients in whom 151 stents were deployed. Stents and high-pressure balloons were angiographically sized 1:1 by visual estimation. High-pressure (> or =12 atm in all cases) balloon inflations were continued until angiographic completion (<10% residual stenosis), after which index ICUS imaging was performed. Stent apposition, symmetry, and lumen dimensions were evaluated. An optimal ICUS result was defined as full apposition of the stent, symmetry ratio > or =0.80, and acute gain > or =0.80 of the reference lumen area. If inadequate ICUS results were found, further dilations with higher pressures or larger balloons and subsequent stent reevaluation with ICUS were performed. Sixty-nine (46%) stents required additional balloon inflations. Of these stents, 35 (23%) had initial acute gains that were <80% of the reference lumen area. Forty-six (30%) stents were found to have unapposed struts and 24 (16%) had a symmetry ratio <0.80. In patients requiring additional inflations, minimum stent area increased from 7.6 +/- 2.2 mm(2) to 9.2 +/- 2.4 mm(2) (P <.0001). Similarly, complete stent apposition improved from 33% to 68% of total stents (P <.0001). After initial ICUS, higher pressure dilations were performed in 40 patients, whereas larger balloons greater than or equal to ICUS reference vessel diameter were used in 33 patients. Follow up was obtained in 95 (99%) patients. The overall major adverse cardiac event rate at 6 months was 9.3%, which consisted of 8 target vessel revascularizations and 1 abrupt closure requiring repeat intervention. CONCLUSIONS: Even when poststent high-pressure balloon inflation achieves an optimal angiographic result, ICUS assists in optimizing acute gain, symmetry, and apposition of intracoronary stents in approximately 50% of patients. Moreover, ICUS guidance is associated with low rates for target vessel revascularization and major adverse cardiac events at 6-month follow-up. PMID- 10740147 TI - Sex differences in coronary artery size assessed by intravascular ultrasound. AB - BACKGROUND: Women have worse outcomes after myocardial infarction and coronary revascularization. The explanations are likely multifactorial but may include smaller coronary artery size. Smaller luminal diameter has been confirmed angiographically; however, because of possible confounding effects of coronary remodeling, angiographically silent atherosclerosis, and body size, it is unclear if there is a true sex influence on arterial size. METHODS: We performed intravascular ultrasound on left main (LM) and proximal left anterior descending (LAD) coronary artery segments that were free of significant atherosclerosis in 50 men and 25 women. Arterial and luminal areas were measured by planimetry and corrected for body surface area. We evaluated associations between sex and coronary dimensions with univariate and then multiple linear regression analyses. RESULTS: Mean uncorrected LM and LAD arterial areas were smaller in women than in men (21.53 vs 26.95 mm(2), P <.001, and 14. 68 vs 19.94 mm(2), P =.002, respectively), as were mean LM and LAD luminal areas (15.94 vs 18.79 mm(2), P =.020, and 10.13 vs 12.71 mm(2), P =.036, respectively). In multivariate models accounting for body surface area and controlling for other factors, sex independently predicted corrected LM and LAD arterial area. In analyses that additionally controlled for plaque area, sex independently predicted corrected LAD luminal area. CONCLUSIONS: LM and LAD arteries are smaller in women, independent of body size. This suggests an intrinsic sex effect on coronary dimensions. Future studies should investigate underlying mechanisms because they may lead to novel therapeutic strategies and improved outcomes for women with coronary artery disease. PMID- 10740148 TI - Three-dimensional helical computed tomographic angiography in neonates and infants with complex congenital heart disease. AB - BACKGROUND: For the clinical management of patients with complex congenital heart disease (CHD), accurate evaluation of their morphologic conditions is critical. Three-dimensional (3D) helical computed tomography (CT) angiography has been used to assess the vascular system in adult patients; the indication for complex CHD, especially in the neonatal period, has not yet been defined. Therefore the purposes of our study were to determine the quality and limitations of current 3D helical CT angiography for neonates and infants with complex CHD and to assess the clinical utility of this technique. METHODS AND RESULTS: 3D helical CT angiography was performed in 17 patients with various types of complex CHD. Their median age was 41 days (range 3 days to 9 months), and mean body weight was 3.6 kg (range 2.2 to 8.5 kg). All 3D images were produced with the 3D reconstruction algorithm of shaded-surface display. Oral sedation was required in only 4 infants during the procedure. 3D helical CT angiography clearly demonstrated the shape and spatial relation of great arteries, proximal branch pulmonary arteries, anomalous pulmonary venous connections, the patent ductus arteriosus, and a shunt. The 3D information of extracardiac morphologic characteristics and 3D anatomic relation of each extracardiac structure were easily recognized by this imaging process. However, intracardiac structure could not be visualized because of blurred and/or unclear edges of the ventricular wall caused by respiratory movement. CONCLUSIONS: 3D helical CT angiography represents an important additional diagnostic tool and may become an alternative method to angiography or other noninvasive techniques used in the evaluation of extracardiac anomalies in neonates and infants with complex CHD. PMID- 10740149 TI - Peripheral resistance vessel dysfunction in Marfan syndrome. AB - BACKGROUND: Patients with Marfan syndrome show a hereditary abnormality of elastin metabolism that may cause aortic enlargement and dissection. We have hypothesized that abnormal elastin may alter peripheral vascular structure and function. METHODS: Forearm blood flow (FBF) (in milliliters per minute per 100 mL) response to the endothelium-dependent dilator acetylcholine (0.75 to 4.5 microg/min per 100 mL), the endothelium-independent dilator sodium nitroprusside (0.05 to 0.3 microg/min per 100 mL), and structure-related maximum dilator response (10-minute occlusion-induced reactive hyperemia) were measured by plethysmograph in 10 patients with Marfan syndrome (mean age 44 years) and 10 healthy age- and sex-matched controls. Patients with the complications of hypercholesterolemia, diabetes mellitus, or heart failure were excluded from the study. RESULTS: Basal FBF (mean +/- SE) did not differ between the 2 groups (2.7 +/- 0.3 vs 2.3 +/- 0.4). Maximum FBF response to acetylcholine in patients with Marfan syndrome was significantly lower than that of healthy controls (8.5 +/- 2.1 vs 15.4 +/- 1.7 mL/min per 100 mL; P <.05). Reactive hyperemia was also lower in patients with Marfan syndrome (at peak 23.0 +/- 2.5 vs 29.5 +/- 2.3 mL/min per 100 mL; P <.05), but sodium nitroprusside-induced FBF changes did not differ between the 2 groups (10.3 +/- 1.1 vs 10.2 +/- 1.5 mL/min per 100 mL; P = not significant). CONCLUSION: These observations suggest that endothelium-dependent dilation and maximum dilator reserve capacity are both abnormal in peripheral resistance vessels of patients with Marfan syndrome. These peripheral vasomotion abnormalities may have a detrimental impact on the cardiovascular system in this disorder. PMID- 10740150 TI - Vasodilatation with felodipine in chronic asymptomatic aortic regurgitation. AB - BACKGROUND: Afterload reduction decreases volume overload on the left ventricle and may thereby delay the need for valve replacement in chronic asymptomatic aortic regurgitation. The aims of this randomized double-blind, placebo controlled trial were to examine short- and long-term hemodynamic effects of felodipine in chronic asymptomatic aortic regurgitation. METHODS: Sixteen patients were randomly assigned to an intravenous infusion of either felodipine 0. 3 mg or placebo followed by 3 months' treatment with felodipine 10 mg or placebo orally once daily. Magnetic resonance imaging was performed at baseline, immediately after intravenous treatment, and after 3 months of oral treatment. RESULTS: Intravenous felodipine caused a statistically significant reduction in the systemic vascular resistance from (mean +/- SD) 1160 +/- 400 to 970 +/- 320 dynes. s. cm(-5) (P <.05), in the regurgitant volume index from 1.5 +/- 0.8 to 1.3 +/- 0.8 L. min(-1). m(-2) (P <.05), and in the regurgitant fraction from 0.31 +/- 0.15 to 0.26 +/- 0.14 (P <.05). The forward cardiac output index increased significantly from 3.2 +/- 0.9 to 3.5 +/- 0.7 L. min(-1). m(-2) (P <.05). Three months of oral treatment with felodipine caused a corresponding but more pronounced decrease in systemic vascular resistance of 880 +/- 330 dynes. s. cm( 5) (P <.05), regurgitant volume index of 1.2 +/- 0.7 L. min(-1). m(-2) (P <.05), and regurgitant fraction 0.25 +/- 0.11 (P <.05), whereas the forward cardiac output index increased to 3.6 +/- 0.7 L. min(-1). m(-2) (P <.05). No significant changes were found in the placebo group. Left ventricular volumes and ejection fraction remained unaffected by treatment, but compared with the placebo group left ventricular myocardial mass decreased significantly from 137 +/- 24 to 132 +/- 21 g. m(-2) (P <.01). CONCLUSION: In chronic asymptomatic aortic regurgitation, felodipine causes beneficial hemodynamic effects that may postpone the need for valve replacement. PMID- 10740151 TI - Detection of angiographically significant coronary artery disease with accelerated intermittent imaging after intravenous administration of ultrasound contrast material. AB - BACKGROUND: Accelerated intermittent harmonic imaging (AII) is used to detect myocardial perfusion abnormalities after intravenous injection of ultrasound contrast medium. A low mechanical index and frame rates of 10 to 20 Hz are used to allow simultaneous wall motion analysis. The purpose of this study was to determine whether the myocardial contrast enhancement achieved with AII can be used to detect angiographically significant coronary artery disease during stress echocardiography. METHODS: We gave intravenous perfluorocarbon containing microbubbles to 45 patients (total of 270 regions) during dobutamine (n = 27) or exercise (n = 18) stress testing with AII. Quantitative angiography was performed on all patients after the stress echocardiograms were interpreted. RESULTS: Quantitative angiography showed >50% diameter stenosis of at least 1 vessel in 32 patients (total of 118 regions). There were visually evident contrast defects in 100 (85%) of these regions, and wall motion was abnormal in 64 (54%). Overall, there was agreement between regional perfusion and quantitative angiographic findings in 217 of the 270 regions (kappa = 0.61; 80% agreement). Agreement with findings at quantitative angiography was good for both dobutamine stress (kappa = 0.66; 83% agreement) and exercise (kappa = 0.53; 77% agreement). The greatest incremental benefit of AII versus wall motion was gained during dobutamine stress. The contrast studies depicted 90% of the regions supplied by a vessel with >50% stenosis, whereas wall motion depicted only 32% (P =.001). CONCLUSIONS: The results of this study indicated that accelerated intermittent perfusion imaging during stress echocardiography can improve the sensitivity of the study in detecting angiographically significant coronary artery disease, especially during dobutamine stress. PMID- 10740152 TI - Signal-averaged P-wave abnormalities and atrial size in patients with and without idiopathic paroxysmal atrial fibrillation. AB - BACKGROUND: The relation between abnormalities in the signal-averaged P wave and atrial size has not been determined in patients with paroxysmal atrial fibrillation (PAF) without structural heart disease. METHODS: Signal-averaged electrocardiograms of P waves were recorded in 38 patients with idiopathic PAF and 34 control subjects. Filtered P-wave duration (FPD) and root-mean-square voltages for the last 20 ms of the vector magnitude were measured. Atrial volume was calculated by cine magnetic resonance imaging. RESULTS: FPD was longer (131.7 +/- 10.9 ms vs 120.8 +/- 8.6 ms, P <.0001) and root-mean-square voltage was lower (2.89 +/- 1.29 microV vs 3.62 +/- 1.48 microV, P <.05) in the PAF group than in control subjects. However, the various atrial volumes were similar in the 2 groups. In controls, FPD was significantly correlated with left (r = 0.593, P <.0001) and total (r = 0.492, P <.005) atrial volume but not with right atrial volume. In patients with PAF, no significant correlations were found between FPD and any of the atrial volumes. Elderly patients with PAF (age > or =60 years) showed longer FPD than younger patients with PAF (139.2 +/- 9.4 ms vs 125.6 +/- 8.0 ms, P <.0001). CONCLUSIONS: FPD is influenced by the left and total atrial volumes in the normal heart without PAF. Prolonged FPD seems to be a useful predictor of idiopathic PAF among patients without atrial enlargement, especially in the elderly. PMID- 10740153 TI - Long-term follow-up of patients requiring intravenous amiodarone to suppress hemodynamically destabilizing ventricular arrhythmias. AB - BACKGROUND: Intravenous amiodarone is effective for the acute suppression of recurrent hemodynamically destabilizing ventricular arrhythmias. There are no follow-up data on patients undergoing long-term therapy with intravenous amiodarone. The objective of this investigation was to evaluate long-term outcome. METHODS AND RESULTS: We reviewed the clinical courses of 245 patients given intravenous amiodarone for sustained ventricular tachyarrhythmias. Of the 107 survivors (84% men; mean age 64 years) released from the hospital taking oral amiodarone, 41 were discharged with an empiric prescription for oral amiodarone. For 64 patients a decision regarding further therapy was based on results of an electrophysiologic study. Two patients were treated empirically with oral amiodarone and an implantable cardioverter defibrillator. Clinical variables and survival curves were the same for the empirically treated group and the group whose treatment was based on electrophysiologic findings (P =.89). Survival at 6, 12, and 18 months was 88%, 81% and 71%, respectively, for empirically treated patients, and 83%, 80% and 73%, respectively, for patients whose therapy was directed with an electrophysiologic study. Of the 64 patients who underwent electrophysiologic studies, 33 received an implantable cardioverter defibrillator. The Kaplan-Meier survival curves for patients with and patients without an implantable cardioverter defibrillator were similar (P =.46). CONCLUSIONS: Patients for whom recurrent ventricular tachycardia and fibrillation are suppressed with intravenous amiodarone and who are discharged receiving oral amiodarone have an 80% 1-year survival rate. Although not randomized, our data suggested that among such patients, electrophysiologic testing, implantation of a cardioverter defibrillator, or both may not be necessary. Ascertaining the best management strategy for these patients will require a prospective randomized trial. PMID- 10740154 TI - Therapeutic heparin concentrations augment platelet reactivity: implications for the pharmacologic assessment of the glycoprotein IIb/IIIa antagonist abciximab. AB - BACKGROUND: This study evaluated the effect of heparin on the platelet reactivity and the pharmacodynamic profile of abciximab. METHODS AND RESULTS: Ex vivo studies were performed on patients undergoing elective percutaneous coronary intervention (n = 26) who were at moderate to high risk of ischemic complications. Patients received a 12,000-U bolus of heparin followed by a 0.25 mg/kg bolus of abciximab. Before abciximab treatment, platelet aggregation responses to a variety of stimuli were assessed immediately before and 10 minutes after the heparin bolus. Heparin increased platelet aggregation to 2 and 5 micromol/L adenosine diphosphate (ADP) and 5 microg/mL collagen by 36%, 25%, and 46%, respectively (P < or =.001), but did not influence platelet reactivity to thrombin receptor-activating peptide or 20 micromol/L ADP and had no appreciable effect on platelet surface glycoprotein (GP) IIb/IIIa receptor numbers. To assess the impact of heparin on the pharmacodynamic profile of abciximab, GP IIb/IIIa receptor blockade and platelet aggregation inhibition estimates obtained after abciximab administration were calculated relative to the basal levels observed both before and after the heparin bolus. At 2 and 24 hours after the abciximab bolus, GP IIb/IIIa receptor blockade measurements normalized to either the preheparin or postheparin baseline determinations were equivalent. For all ADP concentrations tested, the 2-hour post-abciximab bolus platelet aggregation inhibition estimates based on the preheparin and postheparin baseline values were comparable. However, for 2 and 5 micromol/L ADP, the 24-hour post-abciximab platelet aggregation inhibition measurements based on preheparin baseline values were significantly lower than postheparin baseline determinations (both P < or =.003). In vitro studies revealed that therapeutic heparin doses induced a concentration-dependent reduction in the extent of platelet inhibition produced by amounts of abciximab that elicit partial inhibition of platelet aggregation. However, at abciximab concentrations that achieved platelet aggregation blockade of >80%, the levels of inhibition of platelet aggregation in the presence and absence of heparin were equivalent. CONCLUSIONS: The cumulative ex vivo and in vitro data indicate that for certain stimuli, heparin alters the platelet inhibitory profile of abciximab at concentrations of the agent that yield partial suppression of platelet function. PMID- 10740155 TI - Alcohol consumption and plasminogen activator inhibitor type 1: the National Heart, Lung, and Blood Institute Family Heart Study. AB - BACKGROUND: Plasminogen activator inhibitor type 1 (PAI-1) plays a key role in fibrinolytic activity, which is important for thrombotic cardiovascular events. It has been suggested that moderate alcohol consumption may protect against coronary heart disease and ischemic stroke. However, little is known about the effects of moderate doses of alcohol on PAI-1. METHODS AND RESULTS: We assessed the association between different levels of alcohol consumption and PAI-1 among 1862 participants of the National Heart, Lung, and Blood Institute Family Heart Study. We fitted a regression model, adjusting for anthropometric, metabolic, and lifestyle factors. Individuals in the highest alcohol intake category were leaner, had higher high-density lipoprotein cholesterol levels, smoked more cigarettes, and consumed less dietary fiber compared with never-drinkers. For drinking categories of never-drinkers, ex-drinkers, and current drinkers of 0.1 to 1.4, 1.5 to 4.9, 5.0 to 14.9, and > or =15 g/d of alcohol, multivariate adjusted geometric mean PAI-1 levels among women were 10.77, 9.41, 9.99, 11.21, 11.28, and 16.40 ng/mL, respectively. With similar categories except the top category divided into 15.0 to 29.9 and > or =30 g/d, PAI-1 levels among men were 18.43, 15.77, 15.19, 16.20, 17.27, 24.02, and 29.46 ng/mL, respectively. CONCLUSIONS: These results show that alcohol consumption up to 14.9 g/d is not associated with increased PAI-1, whereas the findings suggest increased PAI-1 with greater alcohol intake. PMID- 10740156 TI - Right ventricular function in patients with first inferior myocardial infarction: assessment by tricuspid annular motion and tricuspid annular velocity. AB - BACKGROUND: Unlike left ventricular function, right ventricular (RV) function has not been widely studied after a myocardial infarction (MI). The current study describes RV function determined by tricuspid annular motion and tricuspid annular velocity after MI. METHODS AND RESULTS: Thirty-eight patients with a first acute inferior MI were prospectively compared with 33 patients with a first anterior MI and 24 age-matched healthy individuals. Association of RV infarction in inferior MI was defined as the presence of >/=1-mm ST-segment elevation at the right precordial lead, V(4)R, of the electrocardiograms. From the echocardiographic apical 4-chamber views, the systolic motion of the tricuspid annulus was recorded at the RV free wall with the use of 2-dimensional guided M mode recordings. Peak systolic and peak early and late diastolic velocities of the tricuspid annulus at the RV free wall also were recorded with the use of pulsed-wave Doppler tissue imaging. The tricuspid annular motion was reduced in inferior MI compared with that in healthy individuals (20.5 and 25 mm, P <.001). The peak systolic velocity of the tricuspid annulus was significantly reduced in inferior MI compared with that in healthy individuals (12 vs 14.5 cm/s, P <.001) and patients with anterior MI (12 and 14.5 cm/s, P <.001). Patients with inferior MI were divided into 2 subgroups: those with and those without electrocardiographic signs of RV infarction. The tricuspid annular motion was significantly lower in patients with RV infarction than in patients without RV infarction (17 and 22.7 mm, P <.001). In addition, compared with patients without electrocardiographic signs of RV infarction, patients with RV infarction also had a significantly decreased peak systolic tricuspid annular velocity (13.3 and 10.3 cm/s, P <.001) and peak early diastolic velocity (13 and 8.2 cm/s, P <.001). CONCLUSIONS: These results suggest that tricuspid annular motion and tricuspid annular velocity can be used to assess RV function in association with inferior MI. PMID- 10740157 TI - Impact of Doppler-derived left ventricular diastolic performance on exercise capacity in normal individuals. AB - BACKGROUND: Doppler-derived left ventricular (LV) diastolic indexes have been shown to correlate with exercise capacity in patients with heart diseases as well as healthy individuals. However, it is uncertain whether they predict exercise capacity independent of noncardiac factors. METHODS: To clarify the impact of the LV diastolic index on exercise capacity, 160 healthy individuals were investigated. All underwent Bruce protocol treadmill stress testing and 2 dimensional and Doppler echocardiography. Estimated metabolic equivalent was calculated from exercise time (metabolic equivalent = 1.11 + 0.016 x exercise time). Diastolic performance was assessed by Doppler transmitral flow velocity pattern. Pulmonary function tests and complete blood cell count were also performed. RESULTS: LV diastolic indexes correlated well with metabolic equivalent (peak transmitral filling velocity (A): r = -0.51, P <.0001; ratio of early and late transmitral filling velocities (E/A): r = 0.58, P <. 0001). However, there was no significant correlation between LV systolic indexes and metabolic equivalent. Independent predictors for a higher metabolic equivalent by multivariate analysis were higher E/A (P <.0001), higher vital capacity (P =.001), smaller body mass index (P =.0003), younger age (P =.0050), and higher hemoglobin concentration (P =.0026). CONCLUSION: Doppler-derived LV diastolic index may help in predicting exercise capacity in normal individuals independent of other cardiac and extracardiac factors. PMID- 10740158 TI - Aortic root dilatation among young competitive athletes: echocardiographic screening of 1929 athletes between 15 and 34 years of age. AB - BACKGROUND: Aortic dilatation can be lethal for young competitive athletes. The prevalence among athletes is not known, however, and thus a reasonable approach to early recognition remains uncertain. METHODS AND RESULTS: Echocardiograms of 1929 normotensive athletes 15 to 34 years of age were analyzed. Five (0.26%) athletes had aortic dilatation; 4 of the 5 played basketball. This made the prevalence of aortic dilatation 0.96% (4 of 415) among basketball and volleyball players, who represented a population of especially tall athletes. Tallness aside, only 2 of the 5 athletes had features of Marfan syndrome. Among the athletes without aortic dilatation, the relation between body surface area and aortic root dimension was nonlinear and best described with a quadratic regression model. Athletes with aortic dilatation fell well outside the 95% confidence interval. CONCLUSION: Because a higher incidence of aortic dilatation is to be anticipated among very tall athletes, inclusion of echocardiography in screening before participation in certain sports should be considered. PMID- 10740159 TI - Changes in lipoprotein(a) concentration after orthotopic heart transplantation. AB - BACKGROUND: Elevated concentrations of lipoprotein(a) have been considered an important risk factor in the development of premature cardiovascular disease and have been proposed as a risk factor in the development of accelerated cardiac allograft vasculopathy after orthotopic heart transplantation. METHODS: We prospectively measured lipoprotein(a), fasting cholesterol, and triglyceride concentrations before (n = 38), 6 months (n = 38), and 1 year (n = 21) after orthotopic heart transplantation. The mean age of the patients was 52 +/- 2 years. Eighty-seven percent of the patients were men, 82% were white, and 61% had ischemic cardiomyopathy. RESULTS: Mean lipoprotein(a) concentration was lower 6 months after transplantation than it was before the operation (23 +/- 3 mg/dL vs 17 +/- 3 mg/dL; P =.014) and remained low 1 year after transplantation (23 +/- 3 mg/dL vs 18 +/- 4 mg/dL; P = not significant). In contrast, mean cholesterol concentration was higher 6 months after transplantation (171 +/- 8 mg/dL vs 221 +/- 8 mg/dL; P <.001) and 1 year (171 +/- 8 mg/dL vs 205 +/- 10 mg/dL; P <.01) than it was before transplantation. Triglyceride concentration was higher 1 year after transplantation than it was before the operation (146 +/- 13 mg/dL vs 184 +/- 20 mg/dL; P =.017). CONCLUSIONS: Lipoprotein(a) concentrations decrease during the 6 months after transplantation and stay low for at least 1 year after the operation. Additional studies are needed to ascertain the effect these changes in lipoprotein(a) concentration on the development of cardiac allograft vasculopathy. PMID- 10740160 TI - Benefits of lipid lowering on vascular reactivity in patients with coronary artery disease and average cholesterol levels: a mechanism for reducing clinical events? AB - BACKGROUND: The favorable effects of lowering low-density lipoprotein (LDL) cholesterol on reducing clinical events in patients with coronary disease have been well established. The mechanisms responsible for this benefit, however, have not been fully understood. This study examined the impact of lipid-lowering therapy on endothelium-dependent vasoreactivity in a subgroup of patients after myocardial infarction with average cholesterol levels who participated in the Cholesterol Recurrent Events (CARE) study to determine whether an effect on endothelial function is a viable mechanism for the observed reduction in clinical events. METHODS AND RESULTS: Participants were recruited from among volunteers in the CARE trial at 2 university-based outpatient cardiology clinics. Patients were randomly assigned to pravastatin or placebo. Plasma lipids were measured at baseline and semiannually thereafter. During the final 6 months of the trial, vasoreactivity was assessed by change in ultrasound-determined brachial artery diameter in response to blood pressure cuff-induced ischemia (endothelium dependent) and to nitroglycerin, a direct vasodilator. Differences in response were examined between the 2 randomized groups. The relation between change in LDL cholesterol from baseline to year 5 and the magnitude of endothelium-dependent vasodilation also was examined. There was significantly greater endothelium dependent vasodilation observed in the pravastatin group compared with the placebo group (13% vs 8%, P =.0002), with no difference between the groups in their response to the endothelium-independent vasodilator nitroglycerin. The magnitude of the endothelium-dependent vasodilation was significantly correlated with the percent change in LDL-cholesterol from baseline to final visit (r = 0.49, P =.015). CONCLUSIONS: These findings indicate that the use of pravastatin in patients after myocardial infarction with average cholesterol levels is associated with greater endothelium-dependent vasodilation compared with those who received placebo. The magnitude of this vasodilatory response is correlated to the reduction in LDL-cholesterol. This improvement in endothelium-dependent vasoreactivity may be a likely mechanism, at least in part, for the reduction in recurrent clinical events observed and reported in the CARE study. PMID- 10740162 TI - Benefits of magnesium in acute myocardial infarction: timing is crucial. AB - OBJECTIVE: To evaluate magnesium deficiency during and after acute myocardial infarction (AMI) and the role of intravenous magnesium therapy given in the early postinfarction period. METHODS: One hundred patients with AMI were randomly assigned to 2 equal groups and monitored over a 4-week period. The placebo group received intravenous dextrose solution and the trial group was given 15 g intravenous magnesium (62 mmol) over a 48-hour period. Serum magnesium levels were measured on days 1, 2, 4, and 6 after admission by calorimetry with methyl thymol blue. RESULTS: The groups were comparable in prevalence of risk factors for coronary artery disease and other acute parameters of AMI. The serum magnesium levels of a group of 50 controls were higher (1.61 +/- 0. 21 vs 1.23 +/ 0.27mEq/L) than in patients with AMI (P <.001). There was an increase in serum magnesium levels in the trial group on day 2 (1.73 +/- 0.27 vs 1.29 +/- 0.28 mEq/L; P <.001) as well as on day 4 (1.62 +/- 0.25 vs 1.38 +/- 0.36 mEq/L; P <.001). The trial group also showed significantly lower incidence of arrhythmias (8% vs 34%) and death (4% vs 20%). The mortality rate from pump failure was reduced in the trial group (4% vs 14%). CONCLUSIONS: The serum magnesium levels in patients with AMI were lower compared with controls. The administration of intravenous magnesium to patients in the immediate postinfarction period is cardioprotective and decreases the incidence of arrhythmia, pump dysfunction, and death. Thus intravenous magnesium is a safe, effective, and inexpensive modality of treatment in AMI. PMID- 10740161 TI - Long-term estrogen replacement therapy is associated with improved exercise capacity in postmenopausal women without known coronary artery disease. AB - BACKGROUND: Short-term estrogen administration improves vasodilation and has been shown to improve exercise capacity. However, it is unknown whether long-term estrogen replacement therapy is associated with improved exercise capacity in postmenopausal women without known coronary artery disease. METHODS AND RESULTS: We studied 248 postmenopausal women without known coronary artery disease (mean age 63.5 years); 158 (64%) were current or past hormone replacement therapy (HRT) users and 108 (44%) were current users of HRT. Attributes potentially affecting exercise capacity and cardiac risk factors were carefully measured. These included duration of estrogen replacement therapy, all variables in the Framingham risk index, physical activity level, body mass index, waist-to-hip ratio, presence of osteoporosis, and family history of heart disease. We measured maximal oxygen uptake (MVO (2)) and anaerobic threshold as objective markers of exercise capacity. The relation between exercise capacity and use of HRT was analyzed with the use of logistic regression, controlling for confounding variables. We found that fitness, as measured by MVO (2) and anaerobic threshold, was significantly greater in women who had used HRT currently or in the past compared with women who had never used HRT. This difference in fitness was not confounded by age or physical activity level. CONCLUSIONS: Estrogen replacement therapy is associated with increased exercise capacity as measured by MVO (2) and anaerobic threshold in postmenopausal women without coronary artery disease. This finding is consistent with the beneficial effect of short-term estrogen administration on improved endothelium-dependent and endothelium-independent vasodilation. PMID- 10740163 TI - Efficacy of serologic testing in asymmetric sensorineural hearing loss. AB - PURPOSE: The goal of this study was to determine the efficacy of a detailed questionnaire, auditory brain stem response testing (ABR), MRI, and an extensive battery of serologic tests in diagnosing asymmetric sensorineural hearing loss (ASNHL). METHODS AND MATERIAL: Patients with audiograms demonstrating ASNHL of 10 dB or greater in 2 consecutive frequencies or 15 dB in any 1 frequency between 250 and 6000 Hz were asked to participate. Patients underwent MRI scanning of the cerebellopontine angle, internal auditory canals, and posterior fossa with gadolinium contrast, ABR, and an extensive battery of tests. The causative diagnosis was made by the individual clinician based on each patient's history, physical examination, and test results. RESULTS: Forty-five patients completed the study. A review of the data confirmed the utility of a detailed history and physical examination, MRI, and fluorescent treponemal antibody test in all cases. Erythrocyte sedimentation rate, glycosylated hemoglobin, Lyme antibody titers, and total hemolytic component (CH50) were helpful in selected cases. Thyroid function testing, complete blood count, Sequential Multiple Analysis-7, prothrombin time/partial thromboplastin time, lipid profile, and ABR were of no value in these patients. CONCLUSION: A careful history and physical examination, MRI, and fluorescent treponemal antibody test should be performed for the evaluation of all patients with ASNHL; however, more extensive serologic testing, including sedimentation rate, glycosylated hemoglobin, Lyme antibody titers, and CH50, should be selectively performed, based on a suggestive history or suspicious physical findings. PMID- 10740164 TI - External and middle ear effects on infant hearing screening test results. AB - This study investigated the relationship between external and middle ear factors and hearing screening results by automated auditory brain stem response (ABR) and transient-evoked otoacoustic emissions (EOAEs). The ears of 200 healthy newborns aged 5 to 48 hours underwent screening by ABR and EOAE, followed by otoscopic examination. The pass rates for ABR and EOAE were 91% and 58.5%, respectively. On otoscopic examination, 28% (112/400) ears had occluding vernix obscuring the view of the tympanic membrane. Cleaning of vernix was successfully performed in all but 2 ears that had occluding vernix. Cleaning of vernix significantly increased the pass rates of all 400 ears for ABR and EOAE to 96% and 69%. Decreased tympanic membrane mobility was found in 22.7% (90/396) of ears that were evaluated otoscopically. Decreased tympanic membrane mobility had a significant effect on EOAE screening; only 33.4% of ears passed EOAE testing. Decreased tympanic membrane mobility did not significantly affect pass rates for ABR screening; 95% of these ears passed the automated ABR screen. Implications for newborn hearing screening are discussed. PMID- 10740165 TI - Vagal paraganglioma: the Jefferson experience. AB - Vagal paraganglioma is a rare tumor of neural crest origin. Although the literature is in agreement with regard to epidemiology, diagnosis, and tumor biology, there is some controversy over treatment modalities for these patients. We performed a nonrandomized retrospective study in a large single-institution series of patients (n = 19) in whom vagal paraganglioma was diagnosed. General statistics included age, male/female ratio, tumor size, and duration of follow up. Other variables such as signs and symptoms at presentation, family history, multicentricity, metastatic disease, and secretion of catecholamines were included. CT scan, MRI, and angiography were used in combination for diagnostic purposes as well as for treatment planning. Preoperative embolization was performed in 5 of the more recently treated patients. Current issues regarding the use of preoperative embolization and choice of surgical approach were analyzed. In this article the possibility and sequela of vagus nerve-sparing procedures will be presented. Operative complications and postoperative morbidity related to cranial neuropathies will be discussed. The rationale for performing adjunct procedures, including cricopharyngeal myotomy and vocal fold medialization, to facilitate the rehabilitation of patients with postoperative cranial nerve deficits will be given. Our findings and recommendations will be compared with currently accepted treatment protocols in conjunction with a review of the literature. PMID- 10740166 TI - Effect of stenting after laryngotracheal reconstruction in a subglottic stenosis model. AB - OBJECTIVES: This study addresses the impact of stenting on early wound healing after laryngotracheal reconstruction (LTR) in a rabbit model with established subglottic stenosis. METHODS: Subglottic stenosis was created in 42 New Zealand white rabbits through a transoral, endoscopic technique. Three weeks later, endoscopy and axial CT were performed to document and grade the degree of stenosis. Subsequently, LTR was performed in all animals, with half of the animals receiving an intraluminal stent. Four rabbits from each group were euthanized on postoperative days 6, 9, 14, 21, and 28. Measurements of graft vascularization were obtained with a computerized image measurement program, and a comparison was made regarding the rate of vascularization. RESULTS: There was a statistically significant increase in the rate of vascularization in the stented group (mean 75% +/- 5% vs 56% +/- 3% at day 14; P < 0. 05). However, clinical and radiographic comparisons of the stented and nonstented specimens revealed a trend toward increased mucosal edema and granulation tissue in the stented group at later time intervals (days 21 and 28). CONCLUSION: This analysis suggests that stenting does not inhibit graft vascularization in the early postoperative period after LTR; however, complications were seen in the stented group at longer time intervals. PMID- 10740167 TI - Factors affecting survival for floor-of-mouth carcinoma. AB - OBJECTIVES: The treatment of extensive floor-of-mouth carcinoma has remained a challenging problem for head and neck surgeons. We have reviewed our experience in the surgical management of floor-of-mouth cancer in an attempt to identify factors influencing survival. METHODS: A total of 144 patients with cancer involving the floor of the mouth were treated between March 1988 and November 1995. A retrospective chart review was conducted that captured information including clinical staging, therapeutic modalities, pathologic findings, and patient follow-up. Factors affecting survival were assessed by nonparametric analysis and analysis of variance. RESULTS: There was no statistical significance for the effects of vascular invasion (P = 0.4019), lymphatic invasion (P = 0.3430), bone invasion (P = 0.1548), or positive margins (P = 0.1113) on survival. Extranodal extension and recurrent disease were strongly suggestive of influencing survival but were not statistically significant (P = 0.0650 and P = 0.0504, respectively). Nodal disease significantly affected survival (P = 0.0138) but did not affect recurrence (P = 0.451). CONCLUSION: Mean survival for this cohort was 30.6 months. Positive node status significantly affected mean overall survival in this series, whereas extracapsular disease did not. These data suggest that aggressive surgical management of neck disease is mandated to maximize survival. PMID- 10740168 TI - Malignant neoplasms of the nasal cavity and paranasal sinuses: a series of 256 patients in Mexico City and Monterrey. Is air pollution the missing link? AB - Air pollution is a serious health problem in major cities in Mexico. The concentrations of monitored criteria pollutants have been above the US National Ambient Air Quality Standards for the last decade. To determine whether the number of primary malignant nasal and paranasal neoplasms has increased, we surveyed 256 such cases admitted to a major adult oncology hospital located in metropolitan Mexico City (MMC) for the period from 1976-1997 and to a tertiary hospital in Monterrey, an industrial city, for the period from 1993-1998. The clinical histories and histopathologic material were reviewed, and a brief clinical summary was written for each case. In the MMC hospital the number of newly diagnosed nasal and paranasal neoplasms per year for the period from 1976 1986 averaged 5.1, whereas for the next 11 years it increased to 12.5. The maximal increase was observed in 1995-1997, with an average of 20.3 new cases per year (P = 0.0006). The predominant neoplasms in these series were non-Hodgkin's lymphoma, squamous cell carcinoma, melanoma, adenocarcinoma, Schneiderian carcinoma, and nasopharyngeal carcinoma. In the Monterrey hospital a 2-fold increase in the numbers of newly diagnosed nasal and paranasal neoplasms was recorded between 1993 and 1998. The predominant MMC neoplasm in this series, namely nasal T-cell/natural killer cell non-Hodgkin's lymphoma, is potentially Epstein-Barr virus related. Nasal and paranasal malignant neoplasms are generally rare. Environmental causative factors include exposure in industries such as nickel refining, leather, and wood furniture manufacturing. Although epidemiologic studies have not addressed the relationship between outdoor air pollution and sinonasal malignant neoplasms, there is strong evidence for the nasal and paranasal carcinogenic effect of occupational aerosol complex chemical mixtures. General practitioners and ear, nose, and throat physicians working in highly polluted cities should be aware of the clinical presentations of these patients. Identification of this apparent increase in sinonasal malignant neoplasms in two urban Mexican polluted cities warrants further mechanistic and epidemiologic studies. PMID- 10740169 TI - Sensory recovery in melolabial flaps used for oral cavity reconstruction. AB - Current literature advocates the use of complex reinnervated free flaps to re establish oral sensation after resection of oral cavity cancers. It has been demonstrated that noninnervated flaps can also re-establish sensation. We assessed the return of sensation in local melolabial flaps used in oral reconstruction. Seven patients underwent sensory testing at intervals from 12 to 18 months after surgery. The ability to distinguish differences in temperature was present in all patients. Spontaneous return of sensitivity to touch was documented by clinical testing in 71% of the patients. Less return of sensation was seen in flaps used for defects of the buccal mucosa relative to the floor of mouth. We conclude that spontaneous return of flap sensation does occur with local melolabial flaps. Given the simplicity of these procedures, melolabial flaps represent a reasonable alternative in floor-of-mouth reconstruction for selected patients. PMID- 10740170 TI - Bacteria-mucin interaction in the upper aerodigestive tract shows striking heterogeneity: implications in otitis media, rhinosinusitis, and pneumonia. AB - The mucociliary system of the upper and lower respiratory tracts is a critical nonspecific pathway for the elimination of bacteria and other particulate matter. The interaction between bacteria and purified mucin of the upper and lower respiratory tracts has been a major focus of our laboratory for the past decade. We have previously demonstrated that nontypable Haemophilus influenzae and Moraxella catarrhalis adhere to human purified nasopharyngeal mucin and human middle ear mucin by a very limited number of specific outer membrane proteins. There have been no previous studies on the interaction of Streptococcus pneumoniae and purified mucin. Such information would be of extreme importance in identifying specific mechanisms of preventing colonization of this important pathogen to nasopharyngeal mucin. Using an overlay technique of purified radiolabeled mucins of the upper and lower respiratory tracts in a solid phase assay with 4 predominant pathogens of the upper and lower respiratory tracts, we found a striking heterogeneity of bacteria-mucin interaction. The implications of these interactions in the development of otitis media, rhinosinusitis, and lower respiratory infections are briefly discussed. PMID- 10740171 TI - Dexamethasone pharmacokinetics in the inner ear: comparison of route of administration and use of facilitating agents. AB - There is growing otologic interest in treating inner ear disorders, such as sudden sensorineural hearing loss and acute or unremitting Meniere's disease, with intratympanic dexamethasone (IT-DEX). Although anecdotally reported, there are no scientific clinical papers and few prior laboratory research publications on the subject. This study compares perilymph dexamethasone concentrations after systemic and intratympanic administration and assesses the role of 3 potential transport facilitators of IT-DEX into perilymph. Forty guinea pigs (79 ears) were randomly separated into 5 groups. Dexamethasone levels were measured by radioimmunoassay. IT-DEX resulted in higher perilymph steroid levels than intravenous dexamethasone (P < 0.05). Histamine facilitator resulted in significantly higher perilymph steroid levels than IT-DEX alone (P < 0.05). Neither hyaluronic acid nor dimethylsulfoxide was a potent facilitator. This study demonstrates that IT-DEX administration results in superior perilymph levels within 1 hour of administration and does not result in systemic absorption. Histamine is a potent facilitating agent. The clinical implications are considerable. PMID- 10740172 TI - Auricular composite grafting to repair nasal vestibular stenosis. AB - Nasal vestibular stenosis is defined as a narrowing of the nasal inlet resulting in airway obstruction. Causes include nasal trauma, infection, and iatrogenic insults. The objectives of this article are to illustrate nasal vestibular stenosis and to analyze common causes and surgical treatments. The operative technique of the senior author (M.S.K.), will be presented. Donor site morbidity and patient outcomes will be discussed. Sixteen patients were seen during a 5 year period. Eleven women and 5 men were evaluated and found to have nasal vestibular stenosis. Three patients had stenosis as a result of nasal fracture or laceration. Stenosis developed in the remaining 13 as a result of nasal surgery. All patients underwent auricular composite grafting, and grafts took in 100%. Partial skin slough occurred in up to 50%; however, re-epithelialization was seen within 3 weeks. Complications were seen at the donor site. Immediate patient satisfaction was seen and continues during long-term follow-up. PMID- 10740173 TI - Thyroplasty type I: short- versus long-term results. AB - OBJECTIVE: After thyroplasty type I, significant improvement has been reported in objective measures of vocal function. The purpose of this investigation was to compare the short- and long-term results in patients undergoing thyroplasty type I. METHODS: Data on 26 patients who had undergone thyroplasty type I for management of unilateral vocal fold paralysis were compared from before surgery to the short-term (1-month) and long-term (>1-year) postoperative assessment points. Statistical analysis included paired tests to assess the significance of between-group differences. RESULTS: Significant differences were found between the preoperative and both postoperative evaluations for the measures of mean glottal flow rate, maximum phonation time, jitter, shimmer, and harmonic-to-noise ratio. However, no significant differences were found between the 1-month and >1 year assessment points. CONCLUSIONS: The results for the parameters studied appeared to reach maximum improvement within 1 month after surgery. It is possible that the effects of time, including the normal aging process, hormonal changes, or other alterations in general health, may require longer follow-up to better address these issues. PMID- 10740174 TI - Vocal fold paresis. AB - Vocal fold paresis (VFP) is a relatively common and often overlooked condition that can be difficult to diagnose based on the laryngeal examination alone. A retrospective review of the records of 50 consecutive adult patients with VFP was performed. In each case, the diagnosis of VFP was confirmed by laryngeal electromyography. The presenting symptoms were dysphonia (100%), vocal fatigue (76%), diplophonia (40%), and odynophonia (12%), and the findings were unilateral vocal fold hypomobility (50%), unilateral bowing (36%), and bilateral bowing (22%). Laryngoplasty and/or lipoinjection was performed in 54% of the subjects, and significant vocal improvement was achieved in 85%. VFP appears to be underdiagnosed because many VFP patients have compensatory hyperkinetic disorders at presentation. Although the diagnosis of VFP may be suspected based on the patient's symptoms and findings, the diagnostic sine qua non is laryngeal electromyography. In addition, surgical treatment for VFP appears to be safe and effective. PMID- 10740175 TI - Health status in patients with disturbed sleep and obstructive sleep apnea. AB - The health status of 435 consecutive patients with sleep disturbances necessitating polysomnography was investigated. Patients underwent overnight polysomnography and health status assessment, including the Medical Outcomes Study SF-36 Health Survey and the Pittsburgh Sleep Quality Index. Based on a respiratory distress index (RDI) greater than 10 to define apnea, patients with apnea were significantly (P < 0.05) more likely to be male, be older, and have higher body mass index and lower oxygen saturation levels than patients without apnea. Multiple domains of the SF-36 Health Survey and the Pittsburgh Sleep Quality Index were significantly worse (P < 0.05) for this population when normative data were compared. Although few differences were observed between the apneic and nonapneic patients when a cutoff point for apnea was defined as an RDI greater than 10 or 20, increasing RDI was significantly associated with worsening physical functioning scores. Overall, decrements in health status measures were more strongly correlated with the number of oxygen desaturations below 85% than with increasing RDI. We conclude that patients with sleep disturbances demonstrate significant decrements in general and sleep-specific health status, but these decrements are more closely associated with oxygen desaturation than RDI. PMID- 10740176 TI - Cautery-assisted palatal stiffening operation. AB - Outpatient surgical therapy of habitual snoring and mild obstructive sleep apnea has evolved significantly in recent years. We introduce the cautery-assisted palatal stiffening operation (CAPSO) and detail its important advantages over uvulopalatopharyngoplasty, laser-assisted uvulopalatoplasty, and palatal radiofrequency ablation. CAPSO is critically analyzed with regard to extent of surgery, need for repetition of procedure, results, complications, predictors of success, and cost analysis. CAPSO is a mucosal surgery that induces a midline palatal scar that stiffens the floppy palate. Two hundred six consecutive patients underwent CAPSO over an 18-month period, followed by office examination and telephone evaluation. The success rate was initially 92% and dipped to 77% after 1 year. CAPSO eliminates excessive snoring caused by palatal flutter and has success rates that were comparable with those of traditional palatal surgery. CAPSO is a simple and safe office procedure that avoids the need for multiple stage operations and does not rely on expensive laser systems or radiofrequency generators and hand pieces. PMID- 10740177 TI - Magnetic resonance imaging of the facial nerve in children with idiopathic facial paralysis. AB - The role of gadolinium-enhanced MRI (Gd-MRI) in the diagnosis of idiopathic facial paralysis (IFP) in children is not well defined. Fourteen children with IFP were evaluated to assess the use of Gd-MRI for the presence and pattern of enhancement and its usefulness in predicting the recovery of facial function. Six of 14 children had enhancement of the facial nerve on Gd-MRI, whereas 8 had none. Enhancement was noted in the tympanic, mastoid, and most commonly in the distal intracanalicular and labyrinthine segments. The average time from onset of paresis to recovery in patients with enhancement was 19.3 weeks, whereas in those with no enhancement, mean recovery time was 9.5 weeks (P = 0.003, t test). All 14 patients eventually had recovery to House-Brackmann grade I or II. Gd-MRI is not required for all children with IFP but may yield information about the time course of recovery of facial function. PMID- 10740178 TI - Operative times, postanesthesia recovery times, and complications during sinonasal surgery using general anesthesia and local anesthesia with sedation. AB - OBJECTIVE: The goal was to compare complication rates and recovery times in patients undergoing elective septoplasty or endoscopic sinus surgery using local anesthesia with sedation (LAS) versus general anesthesia (GA). METHODS AND PATIENTS: A retrospective chart review of a consecutive sample of 177 patients undergoing elective septoplasty or endoscopic sinus surgery between July 1, 1994, and June 30, 1996, was carried out at our university-based outpatient surgery unit. Outcome measures included total operative time, surgical time, recovery time, and perioperative complications. RESULTS: Total operative and recovery times were shorter in patients undergoing LAS. The frequency of emesis, epistaxis, and nausea were less in the LAS population than in the GA population. Three patients who underwent GA required unplanned admissions. CONCLUSION: This study suggests that in selected patients undergoing sinonasal surgery, LAS may result in shorter total operative times, shorter recovery times, and less frequent nausea, emesis, and epistaxis than GA. PMID- 10740179 TI - Ossification of eustachian tube cartilage and Ostmann's fatty tissue in chronic renal failure. AB - Ossification of the eustachian tube (ET) cartilage in 2 cases of chronic renal failure is reported for the first time. In both cases, the ossification was observed in the medial lamina of the ET cartilage. In addition, ossification of Ostmann's fatty tissue was observed in case 1, and ossification of the lateral lamina of the ET cartilage was seen in case 2. Correlation between ossification in chronic renal failure and dysfunction of the ET caused by ossified ET cartilage and Ostmann's fatty tissue is discussed. PMID- 10740180 TI - Brain stem stroke associated with bupivacaine injection for adenotonsillectomy. PMID- 10740181 TI - Cherubism and upper airway obstruction. PMID- 10740182 TI - Jugular diverticulum: clinical significance. PMID- 10740183 TI - Possible role of anticoagulation in the treatment of rhinocerebral mucormycosis. PMID- 10740184 TI - Surgery on the larynx and pharynx in Byzantium (AD 324-1453): early scientific descriptions of these operations. AB - We present the techniques of various operations on the larynx and pharynx (incision of abscesses of the tonsils, tonsillectomy, tracheotomy, uvulectomy, and removal of foreign bodies) found in the Greek texts of Byzantine physicians. The techniques of these operations were the first to be so meticulously described and were compiled from the texts, now lost, of the ancient Greek physicians. These medical texts, which followed and enriched the Hippocratic, Hellenistic, Roman, and Galenic medical traditions, later influenced medieval European surgery, either directly through Latin translations or indirectly through works of Arab physicians. PMID- 10740185 TI - Deterioration of intraoral recognition of shapes after treatment of oral and pharyngeal cancer. AB - Thirty patients with diagnosed malignant tumors of the oral cavity or pharynx were tested in regards to intraoral shape recognition at 4 test occasions: before all treatment, after radiotherapy, 6 months after surgery, and 1 year after surgery. They were compared within groups as well as with a group of healthy reference individuals of the same age who underwent the same test procedure at a 2-month interval. The tumor itself did not influence the capability of shape recognition. The reference individuals demonstrated significantly better results on the second test occasion, which is known as a learning effect. Learning improvement was not seen in the patients whose second test occasions were after radiotherapy, implying an impediment amounting to the magnitude of the learning effect. At 6 months after surgery the patients' capabilities of shape recognition had deteriorated significantly with no difference between the oral cancer group and the pharyngeal cancer group. No spontaneous rehabilitation had taken place 1 year after surgery. The presence or absence of surgical lingual nerve damage did not influence the results. The nonoperated side does not compensate for the operated one. It is plausible that decreased oral sensory acuity in recognizing the shape of the bolus contributes to postoperative swallowing problems. PMID- 10740186 TI - Variations of the great arteries in the carotid triangle. AB - The variations of the common carotid artery, as well as of the external and internal carotid arteries, are described. During anatomic dissection on adult cadavers, we investigated the variability of appearance of 40 carotid arterial systems. Special consideration was given to the topographic relations such as the level of the bifurcation of the common carotid artery, the relationship between the external and internal carotid arteries, and the origin of the great collateral branches. Special attention was paid to the origin of the superior thyroid artery. In this article the practical importance of these variations is stressed. PMID- 10740187 TI - Diagnosis and documentation of central nervous system dysfunctions with craniocorpography after surgical removal of acoustic neurinomas. AB - Among other tests, craniocorpography (CCG) was performed in 21 patients after acoustic neurinoma surgery. After surgery, 17 patients (81%) had a developing vestibular compensation or an already normal CCG pattern; 3 patients (14%) had signs of persisting central nervous system dysfunction, either localized to the brain stem or in combination with a cerebellar dysfunction, and 1 patient showed a delayed but sufficient compensation after removal of a neurinoma that compressed central nervous system structures. Brain stem and cerebellar dysfunctions caused by tumor compression demonstrated a better vestibular compensation than dysfunctions caused by surgical manipulation, despite no evidence of cerebellar alteration. As an adjunct to complete neuro-otologic and neurologic examinations CCG could become a useful tool in the topodiagnosis of central nervous system dysfunctions after acoustic neurinoma surgery and therefore in the documentation and follow-up process of these patients. PMID- 10740188 TI - Prognostic assessment of sonography and tumor volumetry in advanced cancer of the head and neck by use of doppler ultrasonography. AB - OBJECTIVE: The objective of this trial was to examine the degree of tumor vascularity in lymph node metastases as depicted by computer-assisted color Doppler sonography and the tumor volumes associated with prognosis in carcinomas of the oropharynx and hypopharynx after primary radiochemotherapy. PATIENTS AND METHODS: In a prospective trial, 25 patients with advanced squamous cell carcinomas of the oropharynx and hypopharynx (stage IV UICC 1997) were treated with radiochemotherapy. The color Doppler findings were quantified with a computer-assisted protocol that quantitatively describes color Doppler images by the relative color pixel density (CPD). As important prognostic cofactor, total tumor volume (TTV) was calculated from CT sections and related to the degree of vascularity. RESULTS: Low CPD in neck metastases showed a correlation with better overall survival. A high-CPD group and a high-TTV group (median survival 10.1 months) were determined and were compared with all other CPD/TTV combinations (median survival 28.4 months); the difference in survival was significant (P = 0.002). CONCLUSION: The results indicate that high tumor vascularity in combination with high TTV indicates a particularly bad prognosis in patients treated with primary radiochemotherapy for head and neck cancer. PMID- 10740190 TI - Effect of gastroesophageal reflux on hypertrophy of the base of the tongue. AB - The objective of this study was to determine a possible association between pharyngolaryngeal signs of gastroesophageal reflux (GER) and hypertrophy of the lymphoid follicles at the base of the tongue (HBT). For this purpose, 306 patients submitted to videolaryngoscopy were studied retrospectively and classified according to the presence and size of follicles on the base of the tongue. HBT was considered to be present when the follicles prevented the view of the vallecula. The patients were grouped according to the presence of videolaryngoscopic signs of GER and further subdivided according to the presence of esophageal, pharyngolaryngeal, and esophagopharyngolaryngeal symptoms, with an attempt made to relate these symptoms to the presence of HBT. HBT was detected in 62.4% (63/101) of the patients with signs of GER and in 29.3% (60/205) of patients with no signs of GER. When HBT incidence was studied according to the symptoms reported by the patient, the condition was found to be present in 57.2% (8/14) of patients with exclusively esophageal symptoms, in 63.6% (21/33) with esophagopharyngolaryngeal symptoms, and in 75% (15/20) with exclusively pharyngolaryngeal symptoms. We conclude therefore that HBT is associated with GER. PMID- 10740191 TI - Extensive salivary gland choristoma of the pterygopalatine fossa. PMID- 10740189 TI - Benefits of cochlear implantation in elderly patients. AB - Cochlear implantation in elderly patients is a questionable subject. The purpose of this study was to evaluate the procedure and its outcome, the postoperative course, and the audiologic and social benefits of cochlear implantation in this population. Twenty-seven patients older than 60 years were compared with a control group of 15 adult patients. This retrospective study analyzed data concerning the outcome of the procedure, postoperative course, postoperative orthophonic test results, and answers of a questionnaire assessing the changes in communication, perception, and social outcomes. The procedure was uneventful in both groups. Minor complications were not more frequent in elderly patients. Orthophonic test results were comparable in both groups. At 12 months, 83% of the elderly patients had an open-set speech discrimination score above 60%. The benefits of cochlear implantation in terms of the quality of life are not statistically different with younger patients. Cost-utility analysis might support these findings. PMID- 10740192 TI - Histopathology of multiple temporal bone metastasis from pancreatic adenocarcinoma: a case showing bilateral hearing loss and Bechterew's phenomenon. PMID- 10740193 TI - Facial paralysis: An unusual presentation of congenital cholesteatoma. PMID- 10740194 TI - Chondrosarcoma of the larynx. PMID- 10740195 TI - External ear involvement in epidermolysis bullosa. PMID- 10740196 TI - Does it take a village to write a case report? PMID- 10740197 TI - Does it take a village to write a case report? PMID- 10740198 TI - An assessment of the use of interphase FISH with chromosome specific probes as an alternative to cytogenetics in prenatal diagnosis. AB - We have assessed the effects that would have been observed if we had changed from standard prenatal diagnosis to interphase fluorescence in situ hybridization (FISH) on our amniocentesis samples. We aimed to estimate the number of cases with aberrations other than chromosomes 13, 18, 21, X and Y, which would not have been detectable by FISH and to assess the potential clinical implications for these cases. In 1687 prenatal diagnoses, 111 cases had abnormal cytogenetic reports (6.5% aneuploidy rate). Out of those 111 cases, 14 had chromosomal abnormalities not detectable by FISH but four of these had major structural abnormalities diagnosed on ultrasound, which would have lead to counselling of a very poor prognosis anyway. In 10 cases without abnormal ultrasound findings, if FISH had been used rather than cytogenetics, it appears that there may have had no detrimental effects on the clinical outcomes of the cases studied. Out of those 10 cases, two pregnancies were terminated because of abnormal cytogenetic results (one was due to maternal age and the second one was due to abnormal biochemical screening) (mosaic 46,XY, /47,XY,+mar and 46,X,del(8)(p21) respectively) and their post-mortem results also did not show any abnormalities. One pregnancy was continued in spite of a de novo chromosomal rearrangement and resulted in an apparently normal live birth. Five cases (including a set of twins) with inherited balanced translocations resulted in four normal live births and one unexplained intrauterine death at 32 weeks' gestation and post-mortem was declined. One case with a paternally derived abnormal chromosome 21, decided to continue the pregnancy and resulted in a normal live birth. The last case in this group resulted in a rhesus related intrauterine death in the second trimester, and although an abnormal chromosome 13 insertion (paternally derived known aberration) there was no abnormality found at post-mortem. Therefore, we suggest that it is reasonable to use FISH as an alternative prenatal diagnosis for indications such as advanced maternal age and abnormal maternal serum biochemical screening when high quality ultrasound scanning is performed, but FISH should only be used as an additional test to conventional cytogenetics for the other indications, especially when abnormalities are found on ultrasound scan. PMID- 10740199 TI - Comparison of fetal cell recovery from maternal blood using a high density gradient for the initial separation step: 1.090 versus 1.119 g/ml. AB - The purpose of this study was to improve recovery of fetal nucleated erythrocytes (NRBCs) from maternal blood for non-invasive prenatal diagnosis. Peripheral blood samples were obtained from 27 women who had just undergone pregnancy termination at 6 to 23 weeks. Samples were split and mononuclear cells were isolated using Histopaque gradient at densities of 1.090 g/ml and 1.119 g/ml. CD45 depletion using magnetic activated cell-sorting, followed by flow-sorting with antibody to gamma-globin and fluorescence in situ hybridization (FISH) analysis, were used to evaluate the number of fetal NRBCs recovered. In samples separated with the 1.119 g/ml density gradient, the yield of true anti-gamma haemoglobin positive cells (median, 14. 9; range, 0-717.5) was significantly higher than that with the 1.090 g/ml density gradient (median, 4.9; range, 0-532.5). After FISH analysis, in the 14 samples in which the fetal karyotype differed from the mother, the median number of fetal NRBCs separated by the 1. 119 g/ml density gradient was 22.9 (2 717.5), which was significantly higher than that by the 1.090 g/ml gradient (median, 11.5; range, 0-532.5, p=0.022). Increased density of the gradient used for the initial enrichment of fetal cells results in improved fetal cell recovery in fresh post-termination blood samples, which may permit better non-invasive detection of fetal cells in maternal blood. PMID- 10740200 TI - Pathologic correlation of sonographic echogenic foci in the fetal heart. AB - The goal of the current paper is to present, on the basis of six investigated fetal hearts, the pathological substrate of prenatally, sonographically diagnosed echogenic intramyocardial foci. The right ventricle, left ventricle, interventricular septum and papillary muscles of both ventricles of six hearts of the fetuses of high-risk pregnant women showing sonographically diagnosed intramyocardial echogenic foci were investigated. At termination of pregnancy all the women were between the 20th and the 24th week of gestation. Of the six cases, four were induced abortions and two were spontaneous abortions. For the control group we investigated the hearts of two fetuses without heart defects at the 22nd and 23rd week of gestation which had proven extracardial anomalies and a normal karyotype. Both cases were induced abortions which prenatally showed no echogenic cardiac foci. In all eight cases a prenatal karyotyping was performed. In the six cases with echogenic foci the location and number of the foci were determined both prenatally and by means of histologic sections; the size of the foci was measured as well. Histological staining was with routine haematoxylin-eosin; Masson-Goldner was used for connective tissue; Kossa for calcification; and Berlin blue for haemosiderin. Histologically, in all six cases, intramyocardial calcifications surrounded by fibrotic tissue were in: papillary muscle of the left ventricle in six cases; papillary muscle of the right ventricle in one case; and the subendocardial myocardium of the right ventricle in one case. Four cases had one, one case had two and one case had three calcified foci. Three cases had cardiac and two cases had extracardiac anomalies. Four cases had chromosome anomalies: two were trisomy 21, one trisomy 13 and one triploidy. The remaining two cases had a normal karyotype. No case showed pathological changes of the chordae tendinae, myocardial cell necrosis or inflammation. There were no patho morphological differences between the intramyocardial calcifications of the papillary muscles in cases with chromosome anomalies and with normal karyotype. As the patho-morphological correlate of the sonographically diagnosed echogenic foci, histological investigation on all six fetal hearts showed coarse intramyocardial calcifications surrounded by myocardial fibrosis. Conventional histological methods gave no indication of the cause of the intramyocardial calcifications. PMID- 10740201 TI - Prenatal ultrasound findings in affected and unaffected pregnancies that are screen-positive for trisomy 18: the California experience. AB - This study investigated whether significant differences in ultrasound findings exist between trisomy 18 affected and unaffected pregnancies positive by serum screening. Ultrasound reports were reviewed for 335 screen-positive women. This represented 65% of all trisomy 18 screen-positive patients who had follow-up services at any of 117 Californian state-approved Prenatal Diagnosis Centers during a six-month period from November 1, 1995 to April 30, 1996. Ultrasound reports were available for 100% of trisomy 18 fetuses diagnosed during the six month period (n=23). Ultrasound findings were reported as normal in 35% of the fetuses affected with trisomy 18. The number and type of abnormalities observed in the affected and unaffected groups are described. When compared to unaffected cases, the trisomy 18 affected fetuses had a greater re-dating discrepancy on follow-up ultrasound evaluation and significantly lower femur length to biparietal diameter (FL/BPD) ratio measurements. We recommend that all women who are screen positive for trisomy 18 be encouraged to have amniocentesis, regardless of ultrasound findings, since affected fetuses may not be detected otherwise. PMID- 10740202 TI - DNA methylation analysis with respect to prenatal diagnosis of the Angelman and Prader-Willi syndromes and imprinting. AB - The Angelman (AS) and Prader-Willi syndromes (PWS) are clinically distinct neurobehavioural syndromes resulting from loss of maternal (AS) or paternal contributions (PWS) of imprinted genes within the chromosomal 15q11-q13 region. The molecular diagnosis of both syndromes can be made by a variety of techniques, including DNA methylation, DNA polymorphism and molecular cytogenetic analyses. DNA methylation analysis at three major loci (ZNF127, PW71 and 5' SNRPN) has been successfully used for the postnatal diagnosis of AS and PWS. Methylation analysis, in contrast to other techniques, can reliably be used to diagnose all three major molecular classes (deletion, uniparental disomy and imprinting mutation) of PWS, and three of the four major classes of AS. In this study we demonstrate that methylation analysis can also be successfully used in prenatal diagnosis, by examining specimens obtained from amniocentesis and chorionic villus sampling. Correct prenatal diagnoses were obtained in 24 out of 24 samples using the 5' SNRPN locus; 4 out of 15 using the ZNF127 locus; and 10 out of 18 using the PW71 locus. Therefore, our data indicate that although the DNA methylation imprints of ZNF127 and 5' SNRPN arise in the germline and are present in brain, only 5' SNRPN maintains the imprint in tissues suitable for the prenatal diagnosis of AS and PWS. PMID- 10740203 TI - A tobacco-specific carcinogen in the fetus. AB - A tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and its metabolite, 4-[(methylnitrosamino)-1-(3-pyridyl)but-1-yl]beta-O-D glucosiduronic+ ++ acid (NNAL-Gluc), have been found in the urine of newborns whose mothers smoked during pregnancy. We set out to determine whether this carcinogen is present in the fetus in early pregnancy. Cell-free amniotic fluid (AF) was obtained through routine amniocentesis for prenatal genetic studies from groups of smokers and non-smokers. NNAL and NNAL-Gluc were quantified by previously published methods. A history of smoking was confirmed by assays for cotinine plus N-beta-D-glucosiduronosyl-(S)-(-) cotinine inner salt (cotinine Gluc) in AF. NNAL was detected in the AF of 11/21 (52.4%) of smokers and in 2/30 (6.7%) of non-smokers, a statistically significant difference (p=0.0006). There was not convincing evidence of NNAL-Gluc in the AF. This study documents for the first time that the tobacco-specific carcinogen NNAL is present in the fetus in early pregnancy. Further rigorous epidemiological studies are needed to determine whether the offspring of smoking mothers have an increased lifetime risk of cancer. PMID- 10740204 TI - Is maternal serum total hCG a marker of trisomy 21 in the first trimester of pregnancy? AB - In a study of 130 first trimester cases of trisomy 21 and 959 controls we have shown that the median MoM for alpha-fetoprotein (AFP) is lower (0.82) and that for total human chorionic gonadotrophin (hCG) is higher (1.31) than in the control group. For AFP 15.3% of cases were below the 5th centile and for total hCG 19. 8% were above the 95th centile. The median shift observed for AFP and total hCG is poorer than that for pregnancy associated plasma protein-A (PAPP-A) or free beta-hCG and together with maternal age, AFP and total hCG could only be expected to detect 40% of cases. In combination with PAPP-A, total hCG would identify 52% of cases, somewhat less than the 67% observed with free beta-hCG and PAPP-A. However, we have demonstrated for total hCG a significant temporal change in median MoM with gestational age. Before 70 days the median MoM was less than 0.5, between 70 and 83 days this increased to 1.13, and between 84 and 97 days this increased to 1.52. This median shift has significant implications for interpreting previous studies and even more significant implications for detection rates. When population parameters specific to the gestational age in question are used, detection rates with total hCG and PAPP-A increase from 47% at 70-83 days to 60% at 84-97 days. This observation explains much of the confusion around total hCG in the first trimester and shows the importance of selecting analyte pairs and population parameters appropriate to the time in gestation when screening is performed. PMID- 10740205 TI - Dandy-walker malformation: prenatal diagnosis and outcome. AB - Prenatal ultrasound identified Dandy-Walker malformation (DWM) in ten singleton pregnancies with concurrent central nervous system (CNS) anomalies and extra-CNS anomalies in eight cases. DWM was confirmed by postnatal magnetic resonance imaging (MRI) or pathological examination in nine cases. Karyotypes were normal in the seven infants tested. Postnatal neurological and developmental testing in the five survivors showed a spectrum of clinical outcome from minor defects to severe handicap. Postnatal investigation also disclosed additional CNS and extra CNS findings not detected on ultrasound, as did autopsy in the other five infants. However, ultrasound diagnosis of DWM is accurate and is an indication for exhaustive screening for concurrent anomalies both within and outside the CNS and in chromosome structure and number, as the prognosis is heavily dependent on associated malformations and karyotype. PMID- 10740206 TI - The sonographic diagnosis of Dandy-Walker and Dandy-Walker variant: associated findings and outcomes. AB - Outcomes of pregnancies with sonographically diagnosed Dandy-Walker (DW) or Dandy Walker variant (DWV) syndromes vary widely. We examined our own experience with these diagnoses in an effort to identify those sonographic features that best predicted neonatal outcome. We identified 50 fetuses with DW and 49 with DWV diagnosed sonographically. Eighty-six per cent of fetuses with DW and 85% of fetuses with DWV had other sonographically identifiable anomalies, the most common being ventriculomegaly (DW: 32%; DWV: 27%) and cardiac defects (DW:38%; DWV: 41%). Forty-six per cent and 36% of available karyotypes in cases of DW and DWV, respectively, were abnormal. 50 out of 99 women in our series elected pregnancy termination. Only three pregnancies with DW resulted in a living infant, and only one of these had a normal paediatric examination at six-week follow-up. Thirteen out of 49 infants with DWV survived the neonatal period and 7 of 13 were reported initially as normal infants, including six with an isolated finding of DWV. We conclude that overall, the prognosis for these posterior fossa defects is grim but not uniformly fatal. The presence of other anomalies is associated with the worst prognosis. Isolated Dandy-Walker variant has the highest chance of leading to a normal neonate. PMID- 10740207 TI - Severe fetal cytomegalic inclusion disease after documented maternal reactivation of cytomegalovirus infection during pregnancy. AB - Recurrent cytomegalovirus infection during pregnancy is considered less dangerous for the fetus than primary infection. We present a case of severe fetal cytomegalic inclusion disease after maternal reactivation of cytomegalovirus during the first trimester of pregnancy. The possibility of such fetal injury is an argument for prenatal diagnosis in seropositive pregnant women when ultrasonographic findings suggest cytomegalovirus infection. PMID- 10740208 TI - Exclusion of late infantile neuronal ceroid lipofuscinosis (LINCL) in a fetus by assay of tripeptidyl peptidase I in chorionic villi. AB - We report the exclusion of late infantile neuronal ceroid lipofuscinosis in a fetus by assay of tripeptidyl peptidase I activity and by mutational analysis in chorionic villi. This is the first pregnancy at risk for LINCL to be monitored by enzyme assay. No morphological abnormalities were detected. PMID- 10740209 TI - Prenatal diagnosis of Gaucher's disease type 2. Ultrasonographic, biochemical and histological aspects. AB - We report on the early prenatal diagnosis of fetal Gaucher disease type 2 by ultrasound examination and beta-glucosidase activity assay on amniocytes from a fetus of 15 weeks' gestation whose first sibling fetus had previously been affected with hydrops fetalis. These cases emphasize the importance of the pathological examination of all fetuses presenting with hydrops fetalis and also stress that minimal and precocious echographic signs can be suggestive of such a lysosomal storage disease. PMID- 10740210 TI - Aicardi syndrome: prenatal sonographic findings. A report of two cases. AB - The prenatal sonographic findings in two children with Aicardi syndrome are reported. PMID- 10740211 TI - Acute recurrent polyhydramnios and amniotic prolactin. AB - A 25 year-old patient in her third pregnancy presented with acute polyhydramnios at 24 weeks' gestation, which was the same time as in the two previous pregnancies. In both she had preterm premature rupture of membranes, preterm delivery and neonatal deaths. In the third pregnancy, amnioreductions combined with medical treatment resulted in the birth by Caesarean section of a normally formed live male at 31 weeks of pregnancy. Acute recurrent polyhydramnios is an extremely rare condition of unknown aetiology. We hypothesized that amniotic prolactin plays a role in this pathology. It was measured serially in amniotic fluid and high levels were found. PMID- 10740212 TI - Prenatal diagnosis of partial trisomy 4q26-qter and monosomy for the Wolf Hirschhorn critical region in a fetus with split hand malformation. AB - We describe the results of prenatal analyses and postnatal findings in a male fetus with a partial trisomy for the long arm and a small terminal monosomy for the short arm of chromosome 4 with the following karyotype: 46,XY,add(4)(p16.3).ish dup(4)(q26qter)(wcp4+, D4S2336x3,AFMb280xa5x2,4ptel-,WHCR ). G-banding did not identify the origin of the additional chromosomal segment, but this was achieved prenatally by application of RxFISH and whole chromosome painting probes. Subsequent FISH analysis with region-specific YAC clones was used to relate the phenotypic findings such as bilateral split hand formation, specific cardiac and kidney anomalies, microtia, and hypoplastic thorax more exactly to the partial trisomy of the segment 4q26-qter. PMID- 10740213 TI - Nuchal translucency and trisomy 18. PMID- 10740214 TI - Maternal blood superoxide dismutase in Down syndrome. PMID- 10740215 TI - Fetal DNA in maternal plasma: the prenatal detection of a paternally inherited fetal aneuploidy. PMID- 10740216 TI - Current awareness in prenatal diagnosis. Bibliography. PMID- 10740217 TI - Batten's disease: clues to neuronal protein catabolism in lysosomes. AB - Neuronal ceroid lipofuscinosis (Batten disease) encompasses a group of 8 or more inherited lysosomal storage diseases, with an overall frequency of 1 in 12,500 births. All are characterized by progressive blindness and dementia and were initially classified on the basis of age of onset, clinical phenotype and ultrastructural characterization of the storage material as granular osmiophilic deposits, curvilinear bodies or fingerprint bodies. Recent research has shown that the various forms of Batten disease result from mutations in at least 8 genes which code for proteins involved in different aspects of lysosomal protein catabolism. These include palmitoyl:protein thioesterase 1 (CLN1), tripeptidylpeptidase 1 (CLN2), cathepsin D (CLN8), and two membrane proteins of unknown function (CLN3 and CLN5). Biochemically, Batten disease is characterized by the accumulation in neurons and other cells of an autofluorescent pigment which has resisted many attempts at analysis. In this review we attempt to relate our current understanding of the nature of the storage material in Batten disease with this genetic information. We conclude that the 8 genes probably code for proteins which facilitate the degradation of post-translationally modified proteins in lysosomes, suggesting that the turnover of these proteins is highest in cortical neurons. PMID- 10740218 TI - Does ceramide play a role in neural cell apoptosis? AB - Ceramide is a lipid second messenger, that is generated in response to stimulation of the cell death pathways by a number of ligands binding to surface receptors, growth factor withdrawal, treatment with chemotherapeutic agents, or high doses of ionizing radiation or oxidizing agents. Depending on the target cell, ceramide induces diverse biological responses including apoptosis, cell cycle arrest, differentiation, and also proliferation. In this review we consider the evidence for its role in apoptosis in cells of the nervous system. PMID- 10740219 TI - Glyceraldehyde-3-phosphate dehydrogenase and apoptosis. AB - Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has long been recognized as playing an integral role in glycolysis. During the past 20 years, however, a number of novel, additional functions for GAPDH have been described. These include acting as an uracil DNA glycosylase, activator of transcription, binding to RNA, and an involvement in tubulin assembly. One of the most intriguing functions which has recently been recognized is an involvement in the initiation of apoptosis. Further, GAPDH associates with proteins implicated in human neurodegenerative disorders. This review summarizes the evidence implicating GAPDH in the initiation of one or more apoptotic cascades. The possible functions of GAPDH in the nucleus which could result in the initiation of apoptosis are also discussed. PMID- 10740220 TI - Overexpression of rapsyn modifies the intracellular trafficking of acetylcholine receptors. AB - Rapsyn is a protein that interacts with the cytoplasmic face of the nicotinic acetylcholine receptors (AChR) to cluster them within postsynaptic membrane of muscle. Here we show that intracellular AChRs are also affected by rapsyn. When rapsyn was co-transfected with AChR into QT-6 fibroblasts, (125)I-alpha bungarotoxin binding indicated a reduction in the fraction of AChRs expressed on the cell surface, compared to cells expressing AChRs alone. Double fluorescent labeling showed that intracellular AChRs accumulated in patches at the cell periphery, beneath rapsyn-associated cell surface AChR clusters. These patches were observed even when cells were grown in medium containing excess unlabelled alpha-bungarotoxin to mask internalized AChRs, suggesting that they arose from hindered trafficking of newly formed AChRs to the cell surface. Similarly, in the muscle cell line, C2, overexpression of rapsyn resulted in the co-localization of aggregates of intracellular alpha-bungarotoxin binding sites with rapsyn beneath cell surface AChR microaggregates. The results indicate that rapsyn can modify the trafficking of AChRs within the cell and suggest a role in selectively targeting newly synthesized intracellular AChRs to the postsynaptic membrane. PMID- 10740221 TI - Involvement of GIRK2 in postnatal development of the weaver cerebellum. AB - We demonstrate that the homozygous weaver granule neurons cultured on a laminin substratum fail to express inwardly rectifying potassium currents, including a functional G-protein coupled inwardly rectifying potassium (GIRK)2 potassium channel. By contrast, both normal and weaver Purkinje cells express inwardly rectifying potassium currents, and normal granule cells exhibit inwardly rectifying potassium currents inducible with GTP-gamma-S. In protein extracts of the vermal postnatal day (P)5-9 weaver cerebellum, the GIRK2 protein could not be detected by Western analysis, although the GIRK2 protein was detectable in extracts of the normal vermis. Northern analysis indicated that during early postnatal cerebellar development, the GIRK2 mRNA is expressed at extremely low levels being detectable at P18-23 in the normal but not yet in the homozygous weaver cerebellum. Using reverse transcriptase-polymerase chain reaction (RT PCR), the GIRK2 mRNA was detected in both normal and weaver cerebella, but quantitative PCR confirmed that the weaver cerebellum expressed the GIRK2 gene at significantly lower levels as compared to the normal cerebellum (P = 0.01, paired t-test). Sequencing indicated that the weaver GIRK2 channel gene had the point mutation proposed to be responsible for the weaver phenotype. Rescue of both survival and neurite outgrowth of the cultured vermal weaver granule neurons by verapamil (Liesi and Wright, 1996; Liesi et al., 1999) induced expression of immunocytochemically detectable levels of the GIRK2 protein. Sequencing revealed that the GIRK2 mRNA of the rescued weaver granule neurons remained the mutated variant of the GIRK2 channel gene. Our results indicate that expression of the mutated GIRK2 protein and/or mRNA in the weaver granule neurons may be an indicator of rescue rather than death of the weaver granule neurons. That the weaver granule neurons expressed no functional GIRK2 receptors during a time period of neuronal death and migration failure suggests that the point mutation in the H5 membrane spanning region of the GIRK2 gene may associate with, but not be responsible for the weaver phenotype. PMID- 10740222 TI - Isolation and heterologous expression of two genomic clones encoding Shaker related potassium channels of trout CNS. AB - Two Shaker-related potassium channel genes (termed tsha1 and tsha2) expressed in the CNS of trout were cloned and sequenced. The coding regions of both genes were not interrupted by introns and exhibited a high overall sequence similarity to other members of the Shaker subfamily. By computer-assisted sequence alignments, tsha1 was identified as a fish homologue to the mammalian Kv1.2 subtype of potassium channels, whereas tsha2 did not show a preferential sequence homology but shared a uniform similarity to Kv1.1, Kv1.2, and Kv1.3. Upon heterologous expression in a mammalian glial cell line, both channels exhibited delayed rectifier current properties that differed from each other by their threshold potentials of activation and their pharamcological features: wheras the tsha1 mediated current was efficiently blocked by submicromolar concentrations of alpha DTX but not by TEA, tsha2 was highly TEA-sensitive, correlating well with differences in the amino acid structure of the pore outer mouth region. As revealed by RT-PCR, Shaker-related potassium channels were sequentially expressed during trout brain development: tsha2 was found initially at stage 34, followed by tsha1 at stage 36, whereas two other members of the Shaker family (tsha3 and tsha4) were detectable much earlier (stage 30, hatching). In the mature brain tissue, no regional specialization of Shaker channel subtype expression was noted. PMID- 10740223 TI - Dynamics of cell aggregation during in vitro neurogenesis by immortalized neuroectodermal progenitors. AB - Early events of in vitro neuronal development were studied by inducing neuron formation in a neuroectodermal cell line, NE-4C/A3, derived from the embryonic forebrain vesicles of p53-deficient mice. Neuronal differentiation was initiated by treating the cells with all-trans retinoic acid (RA). By the second day of RA treatment compact cell aggregates were formed. The first signs of neuronal cell fate decision were revealed inside the aggregates. To elucidate the process of aggregate formation, the dynamics of cell clustering and the migration of individual cells were investigated by a novel computer-controlled videomicroscopic system. Besides real-time observation of cell motility, the system allowed statistical analysis of large sets of data providing quantitative evaluation of cell locomotion during an early, critical phase of RA induced neuron formation. The results showed that chemoattractants did not play a principal role in cell aggregation. Retinoic acid, on the other hand, was found to cause a rapid decrease in the average migratory velocity without changing the randomness of migratory routes. The data indicated that aggregation was facilitated by increased cohesion upon incident collision of randomly encountering cells. The resulting compact cell clusters provided the structural conditions for contact communication apparently needed for the neuronal differentiation of NE-4C/A3 cells. PMID- 10740224 TI - Recoverin negative photoreceptor cells. AB - Recoverin, a calcium-binding protein, is unique with respect to its cellular regulation. It is present in retinal rods, cones, cone bipolar cells, and in a rare population of cells in the ganglion cell layer. Inappropriate turn-on or turn-off of recoverin expression has been reported both in small cell lung carcinoma cells from patients with cancer-associated retinopathy (Matsusara et al. [1996] Br. J. Cancer 74:1419-1422; Adamus et al. [1998] J. Autoimmun. 11: 523 533; Ohguro et al. [1999] Invest. Ophthalmol. Vis. Sci. 40:82-89) and in cultured retinal neurons (McGinnis et al. [1999] J. Neursci. Res. 55:252-260). In a recent report using double labeling immunofluorescence microscopy methods with antibodies against either rhodopsin and recoverin or arrestin and recoverin, two unique cell phenotypes, rhodopsin-positive and recoverin-negative, and arrestin positive, and recoverin-negative were observed in vitro. These two unique cell types could be nonphotoreceptor cells in which rhodopsin and arrestin are inappropriately turned on or they are photoreceptor cells in which the recoverin gene is inappropriately turned off. In this study, multiple antibodies were used to study, on a single-cell basis, whether the photoreceptor cell-specific marker, rhodopsin, is inappropriately expressed in nonphotoreceptor cells in our retinal neuronal culture system. We also examined the hypothesis that the two unique cell phenotypes represent the same population of cells. A triple labeling method has been established to visualize recoverin, rhodopsin, and arrestin protein expression simultaneously in cultured retinal neurons. Our data clearly and directly demonstrate that the previously described unique cell phenotypes are the same population of cells, rod photoreceptors. The existence of recoverin-negative photoreceptors demonstrates that the recoverin gene can be regulated independently of other photoreceptor cell-specific proteins and suggests that this primary cell culture may be useful as a model system for investigating the illicit expression of the recoverin gene in cancer associated retinopathy. PMID- 10740225 TI - N-methyl-D-aspartate (NMDA) receptors in the ventral tegmental area: subcellular distribution and colocalization with 5-hydroxytryptamine(2A) receptors. AB - Glutamate receptors of the N-methyl-D-aspartate (NMDA) subtype have been implicated in behavioral sensitization to psychostimulants and in psychotic behaviors involving excitation of ventral tegmental area (VTA) dopaminergic neurons. Antagonists of serotonin (5-hydroxytryptamine, 5-HT) receptors of the 5 HT(2A) subtype are potent antipsychotics that attenuate these NMDA-evoked responses. We examined the electron microscopic immunocytochemical localization of antisera against the NMDA R1 subunit (NMDAR1) and 5-HT(2A) receptors to determine potential sites for their dual activation in the rat paranigral and parabrachial VTA subdivisions that are distinguished, in part, by their respective striatolimbic and cortical projections. In both regions, NMDAR1 immunoreactivity was localized mainly to the cytoplasm of somata and dendrites, and was only occasionally seen near or within excitatory-type asymmetric synapses. Many of the NMDAR1-labeled somata and dendrites also expressed 5-HT(2A) receptors, having a similar, but largely non-overlapping, neuronal distribution. The mean area density of NMDAR1 and dually labeled dendritic profiles was significantly greater in the paranigral than in the parabrachial VTA. NMDAR1 was also present in small axons showing a similar regional difference in area density. No regional difference in area density was seen in dendrites or small axons containing only 5-HT(2A) receptors. Our results indicate that NMDA and 5 HT(2A) receptors in the VTA are transiently expressed on synaptic plasma membranes of single neurons showing widespread cytoplasmic distributions of each of the receptors. They also suggest a major role for NMDA receptors in modulating the output of paranigral neurons and the release of transmitters from axons passing through this region. PMID- 10740226 TI - Involvement of glial fibrillary acidic protein (GFAP) expressed in astroglial cells in circadian rhythm under constant lighting conditions in mice. AB - To clarify the role of glial fibrillary acidic protein (GFAP)-expressed glial cells in the circadian clock, we examined GFAP expression in the suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL) under various lighting conditions in mice. We demonstrated that GFAP expression did not show daily change in the SCN under a light-dark cycle; however, long-term housing under constant lighting conditions led to dramatic changes in GFAP expression, i.e., a decrease in the SCN and an increase in the IGL. Furthermore, mice that had a targeted deletion in the GFAP gene (GFAP mutant mice) showed longer and more arrhythmic circadian activity rhythms in constant lighting conditions than wild type mice, while GFAP mutant mice exhibited stable circadian rhythms both in a light-dark cycle and constant darkness, and showed normal entrainment to environmental light stimuli. These results suggest that the GFAP-expressed astroglial cells in the SCN and the IGL may have some role in circadian oscillation under constant lighting conditions. PMID- 10740227 TI - Increased expression of glutamate decarboxylase (GAD(67)) in feline lumbar spinal cord after complete thoracic spinal cord transection. AB - To determine changes in gamma-aminobutyric acid (GABA) in the spinal cord in response to a complete transection, we examined the cellular and tissue changes of the two forms of GABA synthetic enzyme glutamate decarboxylase (GAD(65) and GAD(67)). In situ hybridization, immunohistochemistry, and Western blot analyses show that spinal cord transection between thoracic segments 12 and 13 results in an increase of GAD(67), but not GAD(65), protein and mRNA in the lumbar spinal cord. This increase occurs mainly in the dorsal horn and persists for at least 12 months. In addition, there was relatively high GAD(67)-immunoreactivity around the central canal, with dorsolateral GAD(67)-immunoreactive fibers extending toward the ependyma and into the central canal in the transected animals. We suggest that an increase in GAD(67) leads to increased GABA production in spinal neurons below the injury site, resulting in altered inhibition and trophic support during posttrauma recovery and adaptation. Increased GABA synthesis around the central canal, in the vicinity of ependymal cells, may represent part of a regenerative process in the mammalian spinal cord, reminiscent of that observed in lower vertebrates. PMID- 10740228 TI - L-cycloserine slows the clinical and pathological course in mice with globoid cell leukodystrophy (twitcher mice). AB - Globoid cell leukodystrophy (Krabbe's disease) is an autosomal recessive disease that affects the lysosomal enzyme galactosylceramidase. Galactosylceramidase removes galactose from galactosylceramide and psychosine, which are derived from sphingosine. In the present study, L-cycloserine (an inhibitor of 3 ketodyhydrosphingosine synthase) was administered to the twitcher mouse, an authentic model of globoid cell leukodystrophy. Twitcher mice treated with L cycloserine had a significantly longer life span and a delayed onset of weight loss than vehicle-injected twitcher mice. Pathological features such as macrophage infiltration and astrocyte gliosis also were less in treated twitcher mice. These results indicate that substrate reduction therapy may have therapeutic value for individuals with residual enzymatic activity, e.g., individuals with late onset disease or individuals with partial enzyme replacement via bone marrow transplantation. In these cases, a reduction in galactosylceramide and psychosine synthesis would enable residual enzymatic activity to keep up with the accumulation of these substrates that would otherwise lead to pathology. PMID- 10740229 TI - rhGGF2 protects against cisplatin-induced neuropathy in the rat. AB - In many patients treated with cisplatin a peripheral sensory neuropathy develops. This side-effect is considered dose-limiting, and therefore restricts the total dose of cisplatin that can be administered. Recent in vitro and in vivo studies suggest that recombinant human Glial Growth Factor 2 (rhGGF2) has neuroprotective effects. This prompted us to investigate in a rat model whether rhGGF2 ameliorates cisplatin neuropathy. A total of 48 rats were randomly divided into four groups of 12 rats each. Three groups received cisplatin and were treated with either 0.1 mg/kg rhGGF2, 0.3 mg/kg rhGGF2 or placebo. The fourth group (saline/placebo) served as age-matched controls. In the cisplatin/placebo treated rats a neuropathy developed, as determined by measurements of the nerve conduction velocity (NCV). Treatment with rhGGF2 dose-dependently protected against the neuropathy. Histological examination and morphometric analysis revealed that rhGGF2 also protects against cisplatin-induced changes in the morphology and size of DRG satellite cell nuclei. In a control study rhGGF2 did not affect normal NCV development. We conclude that rhGGF2 treatment is of benefit in the treatment of cisplatin neuropathy in the rat. PMID- 10740230 TI - Effects of inducible glial fibrillary acidic protein on glioma cell motility and proliferation. AB - We have studied the effect of induced glial fibrillary acidic protein (GFAP) on motility, cell morphology, and proliferation of two originally GFAP-negative human glioma cell lines. Glioma cell lines U-1242 MG and U-251 MG sp subclone 3A were transfected with a vector system that allows for an inducible GFAP expression. This experimental system creates an "on/off" situation in which GFAP expression is suppressed by tetracycline. Inducible expression of GFAP in the absence of tetracycline was confirmed by immunofluorescence staining and Northern and Western blotting. The study showed that forced GFAP expression resulted in an inhibition of cell motility measured as the phagokinetic track area of individual cells seeded sparsely on a surface covered with gold particles. It also resulted in a change in cell morphology, with extended cell processes, and it was associated with a low fraction of cells in S-phase. We conclude that the down regulation of GFAP expression that is often seen in gliomas in vivo may be an important parameter of tumor progression related mainly to the motile and thereby invasive properties of malignant glioma cells. PMID- 10740231 TI - Age-dependent changes in adenosine A1 receptor and uptake site binding in the mouse brain: an autoradiographic study. AB - Ageing is a multifactorial, inevitable event of life span, which affects neurotransmission in the CNS. Since adenosine is a major neuromodulator of the synaptic activity, it was of interest to investigate the possible modification of the adenosinergic system in the brain during ageing. Using "in vitro" quantitative autoradiography and the radioactive ligands [(3)H]Cyclohexyladenosine and [(3)H]Nitrobenzylthioinosine, we have studied the distribution of A1 adenosine receptors and adenosine uptake sites in the aged mice (26 months) compared to the young ones (3 months). Our results showed a widespread reduction in A1 receptor binding in the aged animals, which was brain area-specific, occurring in areas where adenosine plays a significant neuromodulatory role such as the hippocampus, cortex, basal ganglia, and thalamus. Interestingly, the significant reduction in NBI-sensitive adenosine uptake sites was restricted to few areas of the aged brain, mainly in thalamic nuclei. Since the alterations in the density of A1 receptors and adenosine uptake sites showed no regional correlation and since no significant changes in either neuronal or glial cell number are observed, at least in hippocampus and cortex in this mouse strain during ageing, our findings could be explained by a selective age-dependent reduction of these adenosinergic components rather than by a general neuronal cell degeneration. As adenosine depresses electrical activity in hippocampus, a downregulation of adenosinergic function could probably be related to enhanced excitability seen in hippocampal neurons of the CA1 subregion and dentate gyrus of aged animals. PMID- 10740232 TI - Increased expression of mRNA encoding interleukin-1beta and caspase-1, and the secreted isoform of interleukin-1 receptor antagonist in the rat brain following systemic kainic acid administration. AB - Kainic acid, an analogue of glutamate, injected systemically to rats evokes seizures that are accompanied by nerve cell damage primarily in the limbic system. In the present study, we have analyzed the temporal profile of the expression of the cytokines interleukin-1beta (IL-1beta) and IL-1 receptor antagonist (IL-1ra), and the related IL-1beta-converting enzyme (ICE/caspase-1), in different regions of the rat brain in response to peripheral kainic acid administration (10 mg/kg, i.p.). In situ hybridization histochemistry experiments revealed that IL-1beta mRNA-expressing cells, morphologically identified as microglial cells, were mainly localized to regions showing pronounced neuronal degeneration; hippocampus, thalamus, amygdala, and certain cortical regions. The strongest expression of IL-1beta mRNA was observed after 12 hr in these regions. A weak induction of the IL-1beta mRNA expression was observed already at 2 hr. Similar results were obtained by RT-PCR analysis, showing a significantly increased expression of IL-1beta mRNA in the hippocampus and amygdala after 12 hr. In addition, RT-PCR analysis revealed that IL-1ra mRNA, and specifically mRNA encoding the secreted isoform of IL-1ra (sIL-1ra), was strongly induced in the hippocampus and amygdala at 12 and 24 hr post-injection. RT-PCR analysis of mRNA encoding caspase-1 showed a significantly increased expression in the amygdala after 12 hr. In conclusion, in response to systemic kainic acid injection IL 1beta mRNA is rapidly induced and followed by induction of IL-1ra mRNA and caspase-1 mRNA, supporting a role of the IL-1 system in the inflammatory response during excitotoxic damage. PMID- 10740235 TI - Abundant perforin expression at the maternal-fetal interface: guarding the semiallogeneic transplant? PMID- 10740236 TI - The prime-boost strategy: exciting prospects for improved vaccination. PMID- 10740237 TI - Induction of specific immune tolerance with hybrid antibodies. PMID- 10740238 TI - The structure and origin of rheumatoid factors. PMID- 10740239 TI - Localization of Th-cell subsets in inflammation: differential thresholds for extravasation of Th1 and Th2 cells. PMID- 10740240 TI - Inhibitory MHC class I receptors on gammadelta T cells in tumour immunity and autoimmunity. PMID- 10740241 TI - Authors' corrections PMID- 10740242 TI - Authors' corrections PMID- 10740243 TI - Bioenergetics of immune functions: fundamental and therapeutic aspects. PMID- 10740245 TI - Reply to cohen PMID- 10740244 TI - Cytokine profile data. PMID- 10740246 TI - Reply to cohen PMID- 10740248 TI - Closing in on the elusive liver stem cell? PMID- 10740247 TI - Innate immunity in pregnancy. PMID- 10740250 TI - X marks the spot in lung cancer. PMID- 10740249 TI - Wielding more power over angiogenesis. PMID- 10740251 TI - How HIV hijacks host cells. PMID- 10740252 TI - Cervical cancer risk: is there a genetic component? AB - Human papilloma virus (HPV) is a major risk factor for the development of cervical cancer. As only some infected women develop cancer, other factors must be important for disease development. Genetic epidemiological studies show that genetic factors contribute significantly to disease risk. Genetic susceptibility to HPV exposure and/or infection appears to be important in determining the individual risk to develop this virally induced cancer. PMID- 10740253 TI - Matrix metalloproteinases: multifunctional contributors to tumor progression. AB - Matrix metalloproteinases (MMPs) are a family of extracellular matrix degrading proteinases. Owing to their matrix-degrading abilities and high expression in advanced tumors, MMPs were originally implicated in invasion and metastasis during cancer progression. However, recent work extends a role for MMPs during multiple stages of tumor progression to include other functions such as growth, angiogenesis and migration. Based on studies in animal models implicating MMP activity in cancer, synthetic MMP inhibitors are currently being tested in a clinical setting. PMID- 10740254 TI - Exploiting the hypoxic cancer cell: mechanisms and therapeutic strategies. AB - Human solid tumours are considerably less well oxygenated than normal tissues. This leads to resistance to radiotherapy and anticancer chemotherapy, as well as predisposing to increased tumour metastases. However, tumour hypoxia can be exploited in cancer treatment. One such strategy is to use drugs that are toxic only under hypoxic conditions, and the first drug of this class to enter clinical testing, tirapazamine, is showing considerable promise. The second way to exploit hypoxia is to take advantage of the selective induction of the transcription factor hypoxia-inducible factor 1 (HIF-1) under hypoxic conditions; gene therapy strategies based on this are in development. PMID- 10740255 TI - Dynamics of intercellular communication during melanoma development. AB - Melanoma development involves processes determined collectively by various microenvironmental factors, among which intercellular communication has drawn increased attention. Cell-cell crosstalk mediated by cadherins and connexins results in coordinated regulation of cell growth, differentiation, apoptosis and migration. Abnormal expression of adhesion receptors and dysregulated intercellular communication appears to drive tumor development and progression. PMID- 10740256 TI - Genes influencing HDL metabolism: new perspectives and implications for atherosclerosis prevention. AB - Atherosclerotic cardiovascular disease (ASCVD) is the most common cause of morbidity and mortality in Western societies. Current therapies, such as reduction of plasma cholesterol, significantly reduce, but do not come close to eliminating, the complications of ASCVD. Therefore, novel therapeutic approaches to the prevention of acute coronary events and progression of atherosclerosis are still needed. The complex metabolism of high density lipoproteins represents an attractive potential target for therapeutic intervention. Here, we will discuss those components of the high density lipoprotein metabolism and lipid transport pathways that are potential preventative or therapeutic targets for ASCVD. PMID- 10740257 TI - Animal models for immune defects caused by hepatitis C virus. PMID- 10740258 TI - Issues in the development of an industrial bioprocess advisory system. AB - The background and motivation for the construction of a fault detection and advisory system for an industrial fermentation process plant are described. Here, the knowledge extracted from the operators (implemented in the form of production rules) is integrated with multivariate data-based methods for fault detection. The industrial benefits arising from this integrated system include: (1) reduced variability, (2) increased mean performance levels, (3) reduced operator-training time and (4) knowledge management in the broader organization. PMID- 10740259 TI - Safe biotechnology 10: DNA content of biotechnological process waste. The Safety in Biotechnology Working Party of the European Federation of Biotechnology. AB - The adequacy of the existing treatment, disposal and recycling processes of waste streams from biotechnological laboratories and industrial processes, especially those using genetically modified microorganisms, have been repeatedly discussed. Here, we focus on the discussions linked to the DNA content of these wastes, the properties of extracellular (or 'naked') DNA and the ability to transfer genetic information between bacteria (e.g. antibiotic resistances) or into higher organisms. PMID- 10740260 TI - Nanopores and nucleic acids: prospects for ultrarapid sequencing. AB - DNA and RNA molecules can be detected as they are driven through a nanopore by an applied electric field at rates ranging from several hundred microseconds to a few milliseconds per molecule. The nanopore can rapidly discriminate between pyrimidine and purine segments along a single-stranded nucleic acid molecule. Nanopore detection and characterization of single molecules represents a new method for directly reading information encoded in linear polymers. If single nucleotide resolution can be achieved, it is possible that nucleic acid sequences can be determined at rates exceeding a thousand bases per second. PMID- 10740261 TI - Single-cell MALDI: a new tool for direct peptide profiling. AB - Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry (MS) is a rapid and sensitive analytical approach that is well suited for obtaining molecular weights of peptides and proteins from complex samples. MALDI-MS can profile the peptides and proteins from single-cell and small tissue samples without the need for extensive sample preparation, except for the cell isolation and matrix application. Strategies for peptide identification and characterization of post-translational modifications are presented. Furthermore, several recent enhancements in MALDI-MS technology, including in situ peptide sequencing as well as the direct spatial mapping of peptides in cells and tissues are discussed. PMID- 10740262 TI - Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. AB - As a result of high production costs, commercial products from microalgae must command high prices. Astaxanthin produced by Haematococcus is a product that has become a commercial reality through novel and advanced technology. Cultivation methods have been developed to produce Haematococcus containing 1.5-3.0% astaxanthin by dry weight, with potential applications as a pigment source in aquaculture, poultry feeds and in the worldwide nutraceutical market. PMID- 10740263 TI - Genetic modulation of tumor antigen presentation. AB - An effective cancer-cell vaccine is created by expressing major histocompatibility-complex (MHC) class II molecules without the invariant chain protein (Ii) that normally blocks the antigenic-peptide-binding site of MHC class II molecules at their synthesis in the endoplasmic reticulum. Such tumor-cell constructs are created either by the transfer of genes for MHC class IIalpha and beta chains, or by the induction of MHC class II molecules and Ii protein with a transacting factor, followed by Ii suppression using antisense methods. Preclinical validation of this approach is reviewed with the goal of using this immunotherapy for metastatic human cancers. PMID- 10740264 TI - Developments and improvements in the manufacturing of human therapeutics with mammalian cell cultures. AB - During recent years, biopharmaceutical products manufactured by processes that use mammalian cell cultures have gained increasing importance. At the same time, a strong awareness of the importance of the safety and quality of such products has also emerged. This has led to improvements in cultivation and production technology, validation procedures and process organization. PMID- 10740265 TI - The nuclear envelope, muscular dystrophy and gene expression. AB - Lamins and other nuclear envelope proteins organize nuclear architecture through structural attachments that vary dynamically during the cell cycle and cell differentiation. Genetic studies have now shown that people with mutations in either lamins A/C or emerin, a nuclear membrane protein, develop Emery-Dreifuss muscular dystrophy. A mouse model for this rare disease has been created by knocking out the gene that encodes lamin A/C. This article discusses these and other recent results in the wider context of nuclear envelope function, as a framework for thinking about the possible ways in which defects in nuclear envelope proteins can lead to disease. PMID- 10740266 TI - The IKK complex: an integrator of all signals that activate NF-kappaB? AB - The NF-kappaB family of transcription factors plays a crucial role in the immune, inflammatory and apoptotic responses. These proteins are normally found in the cytoplasm, retained by interaction with an inhibitory molecule called IkappaB. Activation of the NF-kappaB signalling cascade results in phosphorylation and degradation of IkappaB, allowing nuclear translocation of the NF-kappaB complexes. The recent identification of a high-molecular-weight complex containing two kinases and a regulatory subunit has led to a flurry of new results that shed light on some of the most complex mechanisms contributing to the exquisite regulation of NF-kappaB activity. PMID- 10740267 TI - Much ado about M cells. AB - The M cell is a remarkable cell type found in the epithelium that covers mucosa associated lymphoid tissue in the digestive tract and the airways. M cells internalize macromolecules and microorganisms efficiently and deliver them to the underlying lymphoid tissue. In the gut, M cells, unlike the neighbouring absorptive enterocytes, lack a highly organized apical brush border and glycocalyx, and are poorly equipped with digestive enzymes. An insight into the role of immune cells in the differentiation of this unique cell type has been gained recently by using immunodeficient mice and an in vitro model of M cells. These and other recent findings suggest that M cells have a highly plastic phenotype and raise interesting questions about how cell differentiation is controlled in the gut. PMID- 10740268 TI - Plant GTPases: the Rhos in bloom. AB - In animal cells and in fungi, small GTP-binding proteins of the Rho family have well-established roles in morphogenesis, cell-cycle progression, gene transcription and the generation of superoxide anions. The presence of these proteins in plant cells, however, has been established only recently, and the role of Rho GTPases in plants is now coming into view. Already, it is apparent that there are both striking similarities and fascinating differences in how Rho GTPases are regulated and used in plant versus animal and fungal cells. These new findings define certain core properties that might be common to members of this protein family in all eukaryotes. PMID- 10740269 TI - Understanding Ras: 'it ain't over 'til it's over'. AB - Since 1982, Ras has been the subject of intense research scrutiny, focused on determining the role of aberrant Ras function in human cancers and defining the mechanism by which Ras mediates its actions in normal and neoplastic cells. The long-term goal has been to develop antagonists of Ras as novel approaches for cancer treatment. Although impressive strides have been made in these endeavours, and our knowledge of Ras is quite extensive, it appears that we are at the beginning, rather than at the end, of fully understanding Ras function. This review highlights new issues that have further complicated our efforts to understand Ras. PMID- 10740270 TI - The spindle checkpoint: two transitions, two pathways. AB - The spindle checkpoint is an evolutionarily conserved mitotic regulatory mechanism that ensures that anaphase is not attempted until chromosomes are properly aligned on the spindle. Two different cell-cycle transitions must be inhibited by the spindle checkpoint to arrest cells at metaphase and prevent mitotic exit. The checkpoint proteins interact in ways that are more complex than was originally envisioned. This review summarizes the evidence for two pathways of spindle-checkpoint regulation in budding yeast. We describe how the proteins are involved in these pathways and discuss the ways in which the spindle checkpoint inhibits the cell-cycle machinery. PMID- 10740271 TI - Protein traps: using intracellular localization for cloning. AB - A much-sought goal - the rapid cloning of genes whose protein products have specific intracellular localizations - has now been made possible. A visual screen of cells expressing fusions between coding DNA sequences and a reporter, such as green-fluorescent protein (GFP) or beta-galactosidase, identifies cells with the pattern of interest. The DNA sequences encoding these targeted fusions can then be cloned either directly from these cells or by repeated rounds of screening and subdivision of library pools. It is expected that systematic screenings based on these methods will identify additional components for every compartment and define new domains, thus facilitating a more comprehensive understanding of the cellular architecture. PMID- 10740272 TI - Careers-perspective interview. PMID- 10740273 TI - Affective blindsight? PMID- 10740274 TI - Affective blindsight: are we blindly led by emotions?Response to Heywood and Kentridge (2000). PMID- 10740276 TI - A conventional approach to chimpanzee cognitionResponse to M.D. Hauser (2000). PMID- 10740275 TI - Homologies for numerical memory span? PMID- 10740277 TI - Towards a functional neuroanatomy of speech perception. AB - The functional neuroanatomy of speech perception has been difficult to characterize. Part of the difficulty, we suggest, stems from the fact that the neural systems supporting 'speech perception' vary as a function of the task. Specifically, the set of cognitive and neural systems involved in performing traditional laboratory speech perception tasks, such as syllable discrimination or identification, only partially overlap those involved in speech perception as it occurs during natural language comprehension. In this review, we argue that cortical fields in the posterior-superior temporal lobe, bilaterally, constitute the primary substrate for constructing sound-based representations of speech, and that these sound-based representations interface with different supramodal systems in a task-dependent manner. Tasks that require access to the mental lexicon (i.e. accessing meaning-based representations) rely on auditory-to meaning interface systems in the cortex in the vicinity of the left temporal parietal-occipital junction. Tasks that require explicit access to speech segments rely on auditory-motor interface systems in the left frontal and parietal lobes. This auditory-motor interface system also appears to be recruited in phonological working memory. PMID- 10740278 TI - Inhibition of return. AB - Immediately following an event at a peripheral location there is facilitation for the processing of other stimuli near that location. This is said to reflect a reflexive shift of attention towards the source of stimulation. After attention is removed from such a peripheral location, there is then delayed responding to stimuli subsequently displayed there. This inhibitory aftereffect, first described in 1984 and later labeled 'inhibition of return (IOR)', encourages orienting towards novel locations and hence might facilitate foraging and other search behaviors. Since its relatively recent discovery, IOR has been the subject of intensive investigation, from many angles and with a wide variety of approaches. After describing the seminal contribution of Posner and Cohen ('Who'), this review will discuss what causes IOR and, once initiated, what effects IOR has on subsequent processing ('What'). The time course ('When') and spatial distribution ('Where') of IOR, and what is known about IOR's neural implementation ('How') and functional significance ('Why') are also discussed. PMID- 10740279 TI - Attentional capture and inattentional blindness. AB - Although we intuitively believe that salient or distinctive objects will capture our attention, surprisingly often they do not. For example, drivers may fail to notice another car when trying to turn or a person may fail to see a friend in a cinema when looking for an empty seat, even if the friend is waving. The study of attentional capture has focused primarily on measuring the effect of an irrelevant stimulus on task performance. In essence, these studies explore how well observers can ignore something they expect but know to be irrelevant. By contrast, the real-world examples above raise a different question: how likely are subjects to notice something salient and potentially relevant that they do not expect? Recently, several new paradigms exploring this question have found that, quite often, unexpected objects fail to capture attention, a phenomenon known as 'inattentional blindness'. This review considers evidence for the effects of irrelevant features both on performance ('implicit attentional capture') and on awareness ('explicit attentional capture'). Taken together, traditional studies of implicit attentional capture and recent studies of inattentional blindness provide a more complete understanding of the varieties of attentional capture, both in the laboratory and in the real world. PMID- 10740280 TI - The item-based nature of children's early syntactic development. AB - Recent research using both naturalistic and experimental methods has found that the vast majority of young children's early language is organized around concrete, item-based linguistic schemas. From this beginning, children then construct more abstract and adult-like linguistic constructions, but only gradually and in piecemeal fashion. These new data present significant problems for nativist accounts of children's language development that use adult-like linguistic categories, structures and formal grammars as analytical tools. Instead, the best account of these data is provided by a usage-based model in which children imitatively learn concrete linguistic expressions from the language they hear around them, and then - using their general cognitive and social-cognitive skills - categorize, schematize and creatively combine these individually learned expressions and structures. PMID- 10740281 TI - A radical approach to treating inflammation. PMID- 10740282 TI - Is EDHF an epoxyeicosatrienoic acid? PMID- 10740284 TI - Reply: cytochrome P450-derived eicosanoids and the vascular wall. PMID- 10740283 TI - New role for epoxyeicosatrienoic acids as anti-inflammatory mediators. PMID- 10740285 TI - The PTH2 receptor and TIP39: a new peptide-receptor system. PMID- 10740286 TI - Pro-inflammatory effects of substance P: new perspectives for the treatment of airway diseases? PMID- 10740287 TI - Anandamide: an endogenous activator of the vanilloid receptor. PMID- 10740288 TI - Receptors for diadenosine polyphosphates P2D, P2YApnA, P4 and dinucleotide receptors: are there too many? PMID- 10740290 TI - Elucidating cell signaling mechanisms using antisense technology. AB - Many diseases result from defects in cell signaling. Achieving an in-depth understanding of the complex mechanisms by which cells transduce extracellular signals into cellular responses in both normal and diseased systems is a crucial step in the discovery of more effective drugs to treat human diseases. Traditional approaches for studying cell signaling have some limitations. Antisense oligonucleotides represent a novel approach for studying signal transduction processes that offers significant advantages in terms of specificity and versatility. This article reviews the opportunities that antisense oligonucleotides offer for the study of signal transduction pathways and identification of inhibitors of these pathways for drug development. PMID- 10740289 TI - G-quadruplex DNA: a potential target for anti-cancer drug design. AB - In addition to the familiar duplex DNA, certain DNA sequences can fold into secondary structures that are four-stranded; because they are made up of guanine (G) bases, such structures are called G-quadruplexes. Considerable circumstantial evidence suggests that these structures can exist in vivo in specific regions of the genome including the telomeric ends of chromosomes and oncogene regulatory regions. Recent studies have demonstrated that small molecules can facilitate the formation of, and stabilize, G-quadruplexes. The possible role of G-quadruplex interactive compounds as pharmacologically important molecules is explored in this article. PMID- 10740291 TI - Development and therapeutic potential of kynurenic acid and kynurenine derivatives for neuroprotection. AB - Manipulation of the kynurenine pathway of tryptophan metabolism has yielded a plethora of agents that are now being developed as neuroprotectants and anticonvulsants. This pathway is involved in the production of the excitotoxin quinolinic acid and the neuroprotectant kynurenic acid. Approaches used in the development of therapeutic agents include production of analogues or pro-drugs of kynurenic acid and inhibitors of the enzyme responsible for the synthesis of quinolinic acid. Indeed, analogues of the amino acid receptor antagonist kynurenic acid are now in, or are about to enter, clinical trials for stroke and related disorders. This review summarizes the mechanism of action of these various agents, the development of glutamate receptor antagonists from kynurenic acid and the range of their potential uses in neurology and psychiatry. PMID- 10740292 TI - Transport in and out of plastids: does the outer envelope membrane control the flow? PMID- 10740293 TI - . . . response: the chloroplast outer envelope: a molecular sieve? PMID- 10740294 TI - ReplyellipsisWater ascent in plants PMID- 10740295 TI - The nitrogen physiology of the marine N2-fixing cyanobacteria Trichodesmium spp. AB - Trichodesmium spp. have proved to be enigmatic organisms, and their ecology and physiology are unusual among diazotrophs. Recent research shows that they can simultaneously fix N2 and take up combined nitrogen. The co-occurrence of these two processes is thought to be incompatible, but they could be obligatory in Trichodesmium spp. if only a small fraction of cells within a colony or along a filament are capable of N2 fixation. Combined nitrogen is released from cells during periods of active growth and N2 fixation, and concomitantly taken up by Trichodesmium spp. or cells living in association with colonies. Although the nitrogenase of Trichodesmium spp. is affected by high concentrations of combined nitrogen, it might be relatively less sensitive to low concentrations of combined nitrogen typical of the oligotrophic ocean and culture conditions. Nitrogenase activity and synthesis exhibits an endogenous rhythm in Trichodesmium spp. cultures, which is affected by the addition of nitrogen. PMID- 10740296 TI - CDPKs - a kinase for every Ca2+ signal? AB - Numerous stimuli can alter the Ca2+concentration in the cytoplasm, a factor common to many physiological responses in plant and animal cells. Calcium-binding proteins decode information contained in the temporal and spatial patterns of these Ca2+ signals and bring about changes in metabolism and gene expression. In addition to calmodulin, a calcium-binding protein found in all eukaryotes, plants contain a large family of calcium-binding regulatory protein kinases. Evidence is accumulating that these protein kinases participate in numerous aspects of plant growth and development. PMID- 10740297 TI - Pre-mRNA splicing in higher plants. AB - Most plant mRNAs are synthesized as precursors containing one or more intervening sequences (introns) that are removed during the process of splicing. The basic mechanism of spliceosome assembly and intron excision is similar in all eukaryotes. However, the recognition of introns in plants has some unique features, which distinguishes it from the reactions in vertebrates and yeast. Recent progress has occurred in characterizing the splicing signals in plant pre mRNAs, in identifying the mutants affected in splicing and in discovering new examples of alternatively spliced mRNAs. In combination with information provided by the Arabidopsis genome-sequencing project, these studies are contributing to a better understanding of the splicing process and its role in the regulation of gene expression in plants. PMID- 10740298 TI - The cell and developmental biology of alkaloid biosynthesis. AB - Plants produce unique natural products as a result of gene mutation and subsequent adaptation of metabolic pathways to create new secondary metabolites. However, their biosynthesis and accumulation remains remarkably under the control of the biotic and abiotic environments. Alkaloid biosynthesis, which requires the adaptation of cellular activities to perform specialized metabolism without compromising general homeostasis, is accomplished by restricting product biosynthesis and accumulation to particular cells and to defined times of plant development. The cell and developmental biology of alkaloid biosynthesis, which is remarkably complex, evolved in part by recruiting pre-existing enzymes to perform new functions. PMID- 10740299 TI - Membrane heredity and early chloroplast evolution. AB - Membrane heredity was central to the unique symbiogenetic origin from cyanobacteria of chloroplasts in the ancestor of Plantae (green plants, red algae, glaucophytes) and to subsequent lateral transfers of plastids to form even more complex photosynthetic chimeras. Each symbiogenesis integrated disparate genomes and several radically different genetic membranes into a more complex cell. The common ancestor of Plantae evolved transit machinery for plastid protein import. In later secondary symbiogeneses, signal sequences were added to target proteins across host perialgal membranes: independently into green algal plastids (euglenoids, chlorarachneans) and red algal plastids (alveolates, chromists). Conservatism and innovation during early plastid diversification are discussed. PMID- 10740300 TI - Neural tube defect: a preventable congenital malformation. PMID- 10740301 TI - Role of ethamsylate in preventing periventricular-intraventricular hemorrhage in premature infants below 34 weeks of gestation. AB - OBJECTIVE: To determine the role of ethamsylate in prevention of PVH-IVH in premature infants <34 weeks gestational age. DESIGN: Prospective, randomized, controlled study. METHODS: Infants less than 34 weeks gestational age were included in the trial. Neonates with congenital malformations, family history of bleeding disorders and with Apgar scores <5 at 5 minutes were excluded. Subjects were randomized into two groups--Group A infants received intravenous ethamsylate (12.5 mg/kg) six hourly for four days and Group B infants served as a control group. Regular cranial ultrasounds to detect the presence of PVH-IVH were done between days 3-5, 10-14 and 28-30 of post natal age, and before hospital discharge in all infants and weekly in infants detected to have PVH-IVH on earlier scans. Various antenatal and postnatal factors known to affect the incidence of PVH-IVH were recorded. RESULTS: A total of 192 infants underwent the trial, 93 in Group A and 99 in Group B. Antenatal corticosteroids (1 or 2 doses) were administered to 32 ( 34.4%) and 36 (36.3%) women in Group A and Group B, respectively. None of the mothers received phenobarbitone, vitamin K or indomethacin antenatally and none of the infants received phenobarbitone, vitamin E or indomethacin postnatally during the study period. PVH-IVH was seen in 26 infants in Group A, of which Grade I IVH occurred in 9, Grade II in 14, Grade III in 2 and Grade IV in one infant. Twenty-nine infants had PVH-IVH in Group B of which 11 had Grade I, 15 Grade II and 3 Grade III. None of the differences were statistically significant. CONCLUSION: Postnatal administration of ethamsylate did not decrease the incidence of PVH-IVH in the study infants. PMID- 10740302 TI - Autoimmune thyroid disease in childhood: a study of children and their families. AB - OBJECTIVE: To study the clinical and laboratory profile of children with autoimmune thyroid disease (AITD) and its familial prevalence. DESIGN: Clinical and investigative evaluation of 96 children and adolescents 5 to 16 years old suspected of having AITD based on clinical and family data and similar assessment of parents and siblings of 30 confirmed cases of AITD. SETTING AND SUBJECTS: Of these 96 cases, 66 were from a private clinic and 30 were institution based thyroid antibody positive with confirmed AITD. On initial testing 36 (55%) of 66 clinic cases were thyroid antibodies (ab) positive and 30 were (ab) negative. In 12 of 30 ab -ve cases retesting for antibodies by newer technique or FNAC confirmed AITD. Clinical and laboratory evaluation of 90 of a total of 106 parents and siblings of the 30 institution based cases. METHODS: Clinical evaluation with goiter grading by WHO criteria was done in all. Family history of thyroid disease was inquired for in all. Clinical examination and thyroid antibody status was assessed in 90 family members as stated above. Thyroid antimicrosomal (AMA) and antithyroglobulin (ATG) antibodies were tested by standard hemogglutination kits. Titers of > 1:100 considered +ve for children and >1:400 for adults. Thyroid (ab) could be tested in ten of the ab-ve cases by ECI technique on follow up. Bone age was assessed. Ultrasonographic or TCM 99 scanning of thyroid gland and FNAC were done as indicated. RESULTS: Of the 96 children suspected to have AITD, thyroid antibodies were positive in high titers in 66 (36+30) cases (69%) on initial testing but with more sensitive ECI technique significant antibody titres were detected in 10 more cases (79%) and FNAC confirmed AITD in 2 more subjects (total 78 - initial 66 + 12). F:M ratio was 2.9:1. Sixty one per cent of children were between 6 to 12 years of age; mean age 10.12+/-2.9 years. Seventy seven per cent had hypothyroidism, 10% had thyrotoxicosis and only 13% were euthyroid. Family history of thyroid disease was elicited in 33% of the series. Survey of 90 parents and siblings of the institution based group revealed, euthyroid goiters in 17%, subclinical hypothyroidism in 10% and significant AMA titers in 43% (65% of mothers, 30% siblings and 43% fathers). CONCLUSION: Juvenile AITD is a common cause of acquired thyroid disease in children above 5 years of age with a 3-fold higher prevalence in girls. The manifestations are heterogeneous. Hypothyroidism was most common (77%), euthyroid goiters (13%) and thyrotoxicosis (10%) were less frequent. Familial aggregation was noted in adult family members (33%) with positive thyroid antibodies in 65% of mothers. Sibling affection was less frequent. The familial and genetic implications of AITD are important; diagnosis of AITD in children may also help detect subclinical disease in adult family members. PMID- 10740303 TI - Pune low birth weight study--a six year follow up. AB - OBJECTIVE: To evaluate the intelligence, visuo-motor perception, emotional problems and preschool skills in low birth weight (LBW) infants and the impact of social and environmental factors on their development. DESIGN: A prospective cohort study. SETTING: Infants discharged from a Neonatal Special Care Unit of a referral hospital with birth weight less than 2000 g followed up in the High Risk Clinic. METHODS: Low birth weight infants were assessed by Stanford Binet Scales of intelligence, Bender Gestalt Test for visuo-motor perception, Human Figure Drawing for emotional indicators and occupational therapy assessment. A detailed evaluation of their environment and socio-economic status was done. Hearing and ophthalmic assessment was also done and the school progress report was scrutinized. RESULTS: Two hundred and one LBW and seventy one control children were assessed. The mean IQ of LBW children was within normal limits (94.3), though significantly lower than controls (101.3). Preterm SGA children had the lowest mean IQ. Visuo-motor perception and preschool skills and language development was poorer in LBW children. There was no difference in the emotional indicators. Thirteen per cent of LBW children had borderline IQ, as compared to 5.6% in controls (p<0.05). Mother's education and spaciousness of the house had a positive impact and chronic medical problems had a negative impact on the IQ. CONCLUSIONS: The mean IQ of LBW children was within normal limits. The incidence of children with borderline intelligence (IQ 70-85) was significantly higher than controls. Mother's education had a positive impact on the intelligence of the children. A longer follow up is necessary to identify "slow learners". PMID- 10740304 TI - IAP guidelines on optional vaccines and related matters. PMID- 10740305 TI - A neonate with bilateral renal masses. PMID- 10740306 TI - Designs of medical studies. PMID- 10740307 TI - Serology in congenital infections: experience in selected symptomatic infants. PMID- 10740308 TI - Relactation-manipal experience. PMID- 10740309 TI - Revised look at micro-erythrocyte sedimentation rate in neonates. PMID- 10740310 TI - Successful bone marrow transplantation in an infant with Wiskott-Aldrich syndrome. PMID- 10740311 TI - Herpes simplex meningoencephalitis and HIV infection in a newborn. PMID- 10740312 TI - True tail in a neonate. PMID- 10740313 TI - Familial hypoparathyroidism. PMID- 10740314 TI - Fanconi's constitutional aplastic anemia. PMID- 10740316 TI - Typhoid vaccination after enteric fever - reply PMID- 10740315 TI - Typhoid vaccination after enteric fever. PMID- 10740317 TI - Use of a eutectic mixture of local anesthesia in newborns. PMID- 10740318 TI - Child and maternal health in rural areas of Chandigarh. PMID- 10740319 TI - The brachioradialis reflex. PMID- 10740320 TI - National Neonatology Forum National Database on Neonatal Morbidity and Mortality. PMID- 10740321 TI - National neonatology forum national database on neonatal morbidity and mortality reply PMID- 10740322 TI - Well baby shows--some more suggestions. PMID- 10740323 TI - Nifedipine in pregnancy. PMID- 10740324 TI - The surgical management of cervical carcinoma within the South West of England: progress through an audit loop. Gynaecology Tumour Panel. AB - OBJECTIVE: To define and use a minimum clinical dataset for prospective data collection in order to audit the surgical management of cervical cancer in the South West of England. To compare this data set with a retrospective audit allowing assessment of the quality of care offered to patients. DESIGN: Prospective collection of a defined dataset on paper forms which were put into a computerised database for analysis. Registrations validated against histopathology databases and hospital coding. SETTING: All 13 hospitals in the South West of England which participated in the retrospective audit. PARTICIPANTS: One hundred and sixty-five women with cervical cancer diagnosed in 1997. MAIN OUTCOME MEASURES: Distribution of cases by hospital and surgeon; workload of individual surgeons; adequacy and accuracy of FIGO staging; adequacy of histological information; and adequacy of surgery. RESULTS: There is a trend to centralisation of cancer care and radical surgery in the region. Prospective collection of data has dramatically improved FIGO staging with 92% of all cases staged. For cases greater than Stage Ia, 98% were staged suggesting that a target of 100% staging is feasible. The histological dimensions of tumours were not measured in a high proportion of cases (20% of tumour diameters and 28% of tumour thicknesses). Apparent inadequacies in surgical management are explored. In 10/165 cases (6%) inappropriate conservative surgery may have been unavoidable, suggesting that a quality standard of 95% for appropriate radical surgical management of cervical cancer can be achieved. An anatomically complete removal of pelvic node-bearing tissue, yielding greater than 10 nodes in more than 95% cases, should be achievable with each surgeon/pathologist achieving a mean of more than 20 nodes. CONCLUSION: Regional audit of cervical cancer management is feasible. It can be used to improve the quality of information on management and guide improved service provision. PMID- 10740325 TI - Ambulatory urodynamics: do they help clinical management? AB - OBJECTIVE: To assess the contribution of ambulatory urodynamics to the treatment of women with urinary incontinence. DESIGN: A retrospective casenote review of all women referred from the urogynaecology clinic for ambulatory urodynamic monitoring from 1 April 1994 to 31 December 1997. SETTING: A teaching hospital tertiary referral centre urodynamic laboratory. PARTICIPANTS: Notes were retrieved of 71 women, 80% of whom had had the investigation because the conventional cystometrogram had been normal. RESULTS: Technical difficulties occurred in 30 traces, two of which were not interpretable. Detrusor instability was diagnosed in 32 women, including three women who also had stress incontinence (42% of interpretable traces). Of these, 20 women were treated with anticholinergics compared with nine of 37 women where detrusor instability was not diagnosed. None of the women with detrusor instability were offered anti incontinence surgery, compared with five of those where the bladder remained stable. Less than half the women who were treated with anticholinergic medication improved, but none were considered suitable for more aggressive treatment. CONCLUSIONS: Although the diagnosis of detrusor instability may be increased by ambulatory urodynamics, this does not always translate into more effective treatment. Ambulatory urodynamic testing does not yet result in clinical improvements in diagnosis and treatment. PMID- 10740326 TI - Assessment of menstrual blood loss using a pictorial chart: a validation study. AB - OBJECTIVE: To assess the accuracy of a pictorial blood loss assessment chart (PBAC) as a method for estimating menstrual blood loss in women complaining of heavy periods. DESIGN: A prospective analysis of 103 consecutive women complaining of excessive menstrual blood loss. POPULATION: Women recruited into an on-going study of menstrual blood loss in a large district general hospital. MAIN OUTCOME MEASURES: Correlation between pictorial assessment of blood loss and actual measured menstrual blood loss. RESULTS: There was poor correlation between observed PBAC score and menstrual blood loss. CONCLUSION: We have not been able to validate previously published work. The pictorial assessment chart offers no significant improvement in the quality of objective diagnosis in women complaining of menorrhagia. PMID- 10740327 TI - A double-blind randomised trial of leuprorelin acetate prior to hysterectomy for dysfunctional uterine bleeding. AB - OBJECTIVE: To evaluate the use of pre-operative leuprorelin acetate for reducing the morbidity from hysterectomy for nonfibroid menorrhagia. DESIGN: A double blind, randomised, placebo-controlled trial. SETTING: Gynaecology department in a large university teaching hospital. SAMPLE: Fifty-one women without uterine fibroids awaiting abdominal or vaginal hysterectomy for dysfunctional uterine bleeding. METHODS: Participants received leuprorelin acetate or placebo for eight weeks prior to hysterectomy. MAIN OUTCOME MEASURES: Operative blood loss, operative difficulty, first day morphine use, speed of return to 'normal health'. RESULTS: The study and control groups were similar as regards prognostic factors. Two women in the study group withdrew because of side-effects. Although a 34% reduction in uterine volume was seen in those treated with leuprorelin, there were no significant differences in operative blood loss (183 mL in the study group vs 285 mL in controls, P = 0.27), operation time (39 vs 49 min, P = 0.64) or operative difficulty (visual analogue scale 3.0 vs 4.0, P = 0.09). Furthermore, there was no difference between the groups in post-operative morbidity or rate of recovery. CONCLUSIONS: Treating women with leuprorelin acetate for 8 weeks prior to surgery for nonfibroid menorrhagia has no significant operative or post-operative benefits. PMID- 10740328 TI - The effect of submucous fibroids on the dose-dependent modulation of uterine bleeding by trimegestone in postmenopausal women treated with hormone replacement therapy. AB - OBJECTIVE: To assess the value of identifying endometrial structural abnormalities at baseline hysteroscopy in predicting the pattern of bleeding in postmenopausal women treated with hormone replacement therapy. DESIGN: A randomised, double-blind, dose-ranging study. SETTING: A teaching hospital in the UK. POPULATION: One hundred and seventy-six healthy postmenopausal women. METHODS: Women were randomised to receive one of four doses of oral trimegestone (0.05, 0.1, 0.25 and 0.5 mg per day), from day 15-28, and a daily dose of 2 mg oral micronised oestradiol for six treatment cycles. Women completed diaries in which the bleeding episodes were recorded. Hysteroscopy under local anaesthesia and endometrial biopsy were performed at baseline and on day 24 of the last treatment cycle. RESULTS: Women with submucous fibroids had more prolonged (P = 0.026) and heavier (P = 0.002) progestogen-associated bleeding (odds ratio 4.54). The incidence of intermenstrual bleeding, but not its duration or severity, was higher in women with submucous fibroids (P = 0.017). There was a clear dose dependent effect of trimegestone, with a consistently later onset of progestogen associated bleeding occurring with increasing doses of trimegestone (P < 0.001), and such episodes became progressively lighter and of shorter duration over time (P < 0.001). CONCLUSION: Hysteroscopic evaluation of the endometrial cavity in women treated with hormone replacement therapy, predicts the occurrence of heavy and unscheduled bleeding. PMID- 10740329 TI - Length of use and symptoms associated with premature removal of the levonorgestrel intrauterine system: a nation-wide study of 17,360 users. AB - OBJECTIVE: To establish the continuation rates of the levonorgestrel intrauterine system (LNG IUS) and symptoms associated with its premature removal. SAMPLE AND SETTING: All women in Finland who had a LNG IUS inserted between April 1990 and December 1993 and whose doctor had filled in and returned a form at the insertion visit. This study population consists of 46% of all the LNG IUSs sold in Finland between 1990 and 1993. DESIGN: A questionnaire on reproductive and contraceptive history, gynaecological problems and symptoms experienced during the use of the LNG IUS was sent to 23,885 LNG IUS users. A total of 17,914 questionnaires were returned (response rate 75%). The results cover experience from 58,600 woman years. A log-rank-test was used to test differences in continuation rates. Multivariate analyse were performed using Cox's proportional hazard model. RESULTS: The LNG IUS was prematurely removed from 5175 women. The one, two, three, four and five year continuation rates were 93%, 87%, 81%, 75% and 65%, respectively. The symptoms during the use of the LNG IUS most strongly associated with its premature removal were excessive bleeding and spotting, and infections and pain. The risk of premature removal was markedly lower among women who had occasional or total absence of menstruation. Premature removal was less likely in the oldest age group. CONCLUSIONS: The continuation rate of the LNG IUS compares favourably with other long-acting contraceptive systems. Totally or occasionally absent menstruation was strongly associated with prolonged continuation. PMID- 10740330 TI - A cost effectiveness analysis of goserelin compared with danazol as endometrial thinning agents. AB - OBJECTIVE: To analyse the cost, effectiveness and cost effectiveness of two endometrial thinning agents prior to laser ablation for dysfunctional uterine bleeding: danazol and goserelin. SETTING: A district general hospital. DESIGN: A retrospective cost effectiveness analysis, from the perspective of the health service, based on data from an open, randomised, parallel group comparative study of 160 pre-menopausal women with dysfunctional uterine bleeding. METHODS: Within the trial, length of operation and duration of hospital stay was recorded for each woman. Resource use due to complications of surgery and adverse drug events was evaluated by one of the authors (R.G.). Additional surgery after completion of the study was collected using a postal questionnaire which was distributed to every woman who had undergone surgery. Resource use was costed using detailed unit costs from a specific NHS trust and from published sources. A cost effectiveness analysis was undertaken relating differential cost to differential rates of amenorrhoea at women's last point of follow up. RESULTS: Information on amenorrhoea was available from 138 women, of whom 111 had completed the questionnaire to indicate longer term follow up. Women who did not complete the clinical trial were not included in this economic evaluation. On average, women randomised to goserelin spent less time in theatre and on the ward. Based on longer term follow up, rates of retreatment were similar in the two groups. The mean (SD) health service cost of women in the goserelin group was pound sterling 323.84 (pound sterling 309.94), compared with pound sterling 243.45 (pound sterling 265.23) in the danazol group; median (range) costs were pound sterling 220.29 (pound sterling 191-pound sterling 2127) and pound sterling 159.76 (pound sterling 140-pound sterling 1426) in the two groups, respectively. These costs were significantly higher for goserelin (P = 0.0001). The goserelin group also had a higher rate of amenorrhoea (38.8% vs 28.6%, P = 0.23). Based on mean differences in cost, the incremental cost of goserelin per additional woman with amenorrhoea was pound sterling 788; based on median differences in cost the ratio was pound sterling 590. CONCLUSIONS: The shorter duration in theatre and stay in hospital provided a modest offset of the higher acquisition cost of goserelin, but the overall cost of management remained significantly higher than managing women with danazol. The rates of amenorrhoea indicated that goserelin was more effective at 24 weeks and approximately two years after surgery, although statistical significance was only achieved at 24 weeks. The economic impact of women withdrawn from treatment was not considered, but sensitivity analysis indicates that these women may have had a large effect on the overall result of this study. Purchasers will need to decide whether the additional cost of management with goserelin is justified by the increased rates of amenorrhoea and the reduced withdrawals from treatment. PMID- 10740331 TI - Piperazine oestrone sulphate and interrupted norethisterone: effects on the postmenopausal endometrium. AB - OBJECTIVE: To assess the effects on the postmenopausal endometrium of two doses of oral piperazine oestrone sulphate and interrupted norethisterone in comparison with a continuously combined regimen and placebo. DESIGN: A prospective randomised trial. PARTICIPANTS: Two hundred healthy postmenopausal women. METHODS: Random assignment to two years of treatment with alternating three-day cycles of 1.5 mg piperazine oestrone sulphate and 1.5 mg piperazine oestrone sulphate + 0.7 mg norethisterone (highEP), or alternating three-day cycles of 0.75 mg piperazine oestrone sulphate and 0.75 mg piperazine oestrone sulphate + 0.35 mg norethisterone (lowEP), or 2 mg 17 beta-oestradiol continuously combined with 1 mg norethisterone acetate (E2+NETA), or placebo. MAIN OUTCOME MEASURES: Effect of treatment on endometrial histology, endometrial thickness, occurrence of uterine bleeding, endometrial oestrogen and progesterone receptor content, endometrial isocitrate dehydrogenase activity, and serum placental protein 14. RESULTS: The incidence of bleeding declined with time. In the second treatment year, the women receiving lowEP reported on average 7.3 days of bleeding, highEP 16.7 days, and E2+NETA 11.2 days. Histological assessment of endometrial biopsies revealed an atrophic or slightly secretory endometrium. Serum placental protein 14 increased slightly, but was statistically highly significant, during treatment, but no cyclical variation was observed. Endometrial isocitrate dehydrogenase was low in all three hormone groups and the same low level of endometrial oestrogen receptor and progesterone receptor was found comparable to the level in the placebo group. CONCLUSIONS: Histological and biochemical assessment of the endometrium showed that interrupted hormone replacement therapy induced the same pattern in endometrial parameters as continuous combined hormone replacement therapy. PMID- 10740332 TI - Piperazine oestrone sulphate and interrupted norethisterone in postmenopausal women: effects on bone mass, lipoprotein metabolism, climacteric symptoms, and adverse effects. AB - OBJECTIVE: To compare the effects of two doses of piperazine oestrone sulphate combined with interrupted norethisterone, with that of oestradiol continuously combined with norethisterone acetate, and with placebo, in postmenopausal women. DESIGN: A prospective randomised trial. PARTICIPANTS: Two hundred postmenopausal women. SETTING: Monocentre study with expertise in osteoporosis. METHODS: The participants were randomly assigned to two years of treatment with alternating three-day cycles of 1.5 mg of piperazine oestrone sulphate plus 0.7 mg of norethisterone (highEP), or alternating three-day cycles of 0.75 mg of piperaine oestrone sulphate plus 0.35 mg of norethisterone (lowEP), or 2 mg of 17beta oestradiol continuously combined with 1 mg of norethisterone acetate (E2+NETA), or placebo. MAIN OUTCOME MEASURES: Change in bone mineral density, lipoprotein metabolism, climacteric symptoms, and adverse effects. RESULTS: One hundred and twenty-one women completed the study. Spinal bone mineral density was increased about 9% over two years by E2+NETA, about 6% by highEP, 4% by lowEP, but remained unchanged in the placebo group. The same pattern was seen in the hip and forearm. All hormone regimens decreased markers of bone turnover and alleviated climacteric symptoms. Serum lipoproteins decreased by about 10% in all hormone groups. CONCLUSIONS: All hormone regimens studied prevented bone loss completely and lowered serum lipids. PMID- 10740333 TI - The effect of a group programme on women with the Mayer-Rokitansky-Kuster-Hauser syndrome. AB - OBJECTIVE: To evaluate the effect of a group programme on psychological distress in women with the Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome. SETTING: Gynaecologic outpatient clinic. SAMPLE: Seventeen women with MRKH syndrome participated in this study. METHOD: A semi-structured programme of seven sessions was offered dealing with themes of the MRKH syndrome. Psychological distress was measured at the first visit 3-6 month before the group programme was started (pre test-0), at the first (pre-test) and then at the last group session (post-test). MAIN OUTCOME MEASURE: The Symptom Check List-90 was used to assess feelings of psychological distress. RESULTS: The post-test subscale scores for anxiety, depression, interpersonal sensitivity and the total score for psychological distress were significantly lower than the pre-test-0 and pre-test scores (P < 0.05). No significant differences were found between the pre-test-0 and pre-test subscale scores and total score. CONCLUSION: A semi-structured group programme seems valuable in helping women with the MRKH syndrome to deal with their psychological stress. PMID- 10740334 TI - Trends in the incidence of ectopic pregnancy in England and Wales from 1966 to 1996. AB - OBJECTIVE: To examine the incidence of ectopic pregnancy over the period 1966 to 1996. SETTING: England and Wales. DESIGN: Use of official statistics on hospital discharges, maternities, legal abortions and estimated populations of women aged 15-44 years. MAIN OUTCOME MEASURES: Incidence rates of ectopic pregnancies. RESULTS: Between 1966 to 1970 and 1994 to 1996 the recorded incidence increased 4.5-fold from 3.45 to 15.5 per 1000 maternities, 3.8-fold from 3.25 to 12.4 per 1000 pregnancies and 3.1-fold from 30.2 to 94.8 per 100,000 women aged 15-44. The rate of increase was not uniform. Incidence approximately doubled between 1966 and 1985, when the official data collection system changed. By 1989, when data from the new system became available, there had been a further almost doubling of recorded incidence. Subsequently, the upward trend appears to have continued until 1991 to 1992 and has remained stable in the last four years of the study. The trends were similar in each of three 10-year age groups. CONCLUSIONS: The recorded incidence of ectopic pregnancy has increased markedly over the last three decades. This may be partly due to artefacts of data recording and more sensitive diagnostic tests, but it is likely that the actual incidence has increased, probably due to a sexually transmitted agent. PMID- 10740335 TI - Genetic influence on birthweight and gestational length determined by studies in offspring of twins. AB - OBJECTIVE: To determine the relative importance of genetic effects on birthweight, gestational length and small for gestational age. DESIGN: A cohort study, using individual record linkage between the population-based Swedish Twin and Birth Registers to estimate twin similarities in twins with known zygosity. POPULATION: Included were 868 monozygotic and 1141 dizygotic female twin pairs, born in Sweden before 1959, who both delivered single births from 1973-1993. METHODS: Quantitative genetic methods, offspring birthweight, gestational length and small for gestational age birth in twin sisters. MAIN OUTCOME MEASURES: Twin similarities measured as probandwise concordance rates and intra-class correlations for birthweight, gestational length and small for gestational age births. RESULTS: Concordance rates and intra-class correlations for birthweight, gestational length and small for gestational age were consistently higher in monozygotic compared with dizygotic twins. Model fitting suggested heritability estimates in the range from 25% to 40%. CONCLUSIONS: This study suggests genetic effects not only for birthweight and fetal growth, but also for gestational length. The mediation of these genetic effects may partly be due to similarities in maternal antropometric measures, lifestyle and medical complications during pregnancy. The study does not distinguish between fetal and maternal genetic effects. PMID- 10740336 TI - Randomised clinical trials of fish oil supplementation in high risk pregnancies. Fish Oil Trials In Pregnancy (FOTIP) Team. AB - OBJECTIVE: To test the postulated preventive effects of dietary n-3 fatty acids on pre-term delivery, intrauterine growth retardation, and pregnancy induced hypertension. DESIGN: In six multicentre trials, women with high risk pregnancies were randomly assigned to receive fish oil (Pikasol) or olive oil in identically looking capsules from around 20 weeks (prophylactic trials) or 33 weeks (therapeutic trials) until delivery. SETTING: Nineteen hospitals in Europe. SAMPLES: Four prophylactic trials enrolled 232, 280, and 386 women who had experienced previous pre-term delivery, intrauterine growth retardation, or pregnancy induced hypertension respectively, and 579 with twin pregnancies. Two therapeutic trials enrolled 79 women with threatening pre-eclampsia and 63 with suspected intrauterine growth retardation. INTERVENTIONS: The fish oil provided 2.7 g and 6.1 g n-3 fatty acids/day in the prophylactic and therapeutic trials, respectively. MAIN OUTCOME MEASURES: Preterm delivery, intrauterine growth retardation, pregnancy induced hypertension. RESULTS: Fish oil reduced recurrence risk of pre-term delivery from 33% to 21% (odds ratio 0.54 (95% CI 0.30 to 0.98)) but did not affect recurrence risks for the other outcomes (OR 1.26; 0.74 to 2.12 and 0.98; 0.63 to 1.53, respectively). In twin pregnancies, the risks for all three outcomes were similar in the two intervention arms (95% CI for the three odds ratios were 0.73 to 1.40, 0.90 to 1.52, and 0.83 to 2.32, respectively). The therapeutic trials detected no significant effects on pre-defined outcomes. In the combined trials, fish oil delayed spontaneous delivery (proportional hazards ratio 1.22; 1.07 to 1.39, P = 0.002). CONCLUSIONS: Fish oil supplementation reduced the recurrence risk of pre-term delivery, but had no effect on pre-term delivery in twin pregnancies. Fish oil had no effect on intrauterine growth retardation and pregnancy induced hypertension, affecting neither recurrence risk nor risk in twin pregnancies. PMID- 10740337 TI - A randomised evaluation of two techniques of management of the third stage of labour in women at low risk of postpartum haemorrhage. AB - OBJECTIVE: To determine whether early placental drainage plus cord traction reduces the incidences of manual removal and blood loss, and to determine the risk factors associated with blood loss after delivery. DESIGN: Prospective randomised study. SETTING: University teaching hospital, Montpellier, France. METHODS: A randomised study compared 239 women who had placental cord drainage plus cord traction with 238 women with expectant delivery. The need for manual removal of the placenta and the drop in haemoglobin after delivery were assessed. The duration of the third stage of labour and the time between birth and the beginning of perineal suturing were measured. Statistical analysis used the paired t test for continuous variables, the Kruskal-Wallis test for nonparametric data and chi2 test for categoric variables. Stepwise logistic regression analyses were performed with a drop in haemoglobin as the outcome variable. RESULTS: No significant difference was found in the two groups with regard to the incidence of manual removal of retained complete or incomplete placenta or postpartum haemorrhage. The median values of the duration of the third stage of labour, birth-to-perineal suture time and drop in haemoglobin were significantly lower in the cord drainage group than in the control group. After controlling for confounding variables, parity proved to be the only significant predictor of drop in haemoglobin. CONCLUSION: Cord drainage decreases the duration of the third stage of labour and reduces blood loss but not the incidence of manual removal of the placenta. PMID- 10740338 TI - Outcome of labour after successful external cephalic version at term complicated by isolated transient fetal bradycardia. AB - OBJECTIVES: To investigate factors associated with the occurrence of transient fetal bradycardia after external cephalic version, and labour outcome after isolated transient fetal bradycardia. DESIGN: Cohort study. SETTING: Teaching hospital with a policy of offering external cephalic version for breech presentation at or beyond 36 weeks of gestation. POPULATION: Four hundred and twenty-nine external cephalic versions performed over a 5-year period. METHODS: Between group differences were compared with the unpaired t test or the chi2 test. Logistic regression analysis was performed to exclude confounding effects. MAIN OUTCOME MEASURES: Incidence of caesarean section for fetal distress. RESULTS: Transient fetal bradycardia occurred in 8.4% of external cephalic versions, and was associated with a successful version (OR 16.45, P < 0.001), a difficult procedure (OR 3.70, P = 0.001), and nulliparity (OR 2.83, P = 0.007). The incidence of intrapartum caesarean section for fetal distress was 16.7% in pregnancies with transient fetal bradycardia, compared with 7.9% in those without (OR 2.34, 95% CI 0.81, 6.71). CONCLUSIONS: Transient fetal bradycardia after external cephalic version may be associated with a higher risk of intrapartum caesarean section for fetal distress. PMID- 10740339 TI - External cephalic version with epidural anaesthesia after failure of a first trial with beta-mimetics. AB - OBJECTIVE: To assess the efficacy, tolerance, and cost of external version under epidural anaesthesia and beta-mimetic tocolysis after the failure of an initial attempt with tocolysis alone. DESIGN: Prospective open study. PARTICIPANTS: Sixty eight women with breech presentation at around 36 weeks of gestation and an attempted external cephalic version under salbutamol that failed, who consented to try a second attempt under epidural anaesthesia. RESULTS: The overall success rate under epidural anaesthesia was 39.7% (27/68), and complications occurred in two cases. The total cost of attempting external version was higher than the cost of expectant management. CONCLUSIONS: The efficacy of external cephalic version under epidural reduces the rate of caesarean sections associated with breech presentation, but its relative safety remains in question. Moreover, our economic analysis discourages the hope of lowered costs suggested by earlier reports that this technique is more expensive than expectant management, except in institutions with a policy of systematic caesarean sections when version fails. PMID- 10740340 TI - Reactive thrombocytosis after caesarean section and vaginal delivery: implications for maternal thromboembolism and its prevention. AB - OBJECTIVE: To assess the duration and severity of reactive thrombocytosis after caesarean section and vaginal delivery. DESIGN: A prospective cohort study. SETTING: A large teaching hospital. METHODS: Women admitted for delivery at the Leicester Royal Infirmary were recruited into the study. The platelet count was measured before delivery and postnatally on days 3, 8, 12, 16, 20 and 24. Women who had antepartum haemorrhage, postpartum haemorrhage and those delivered by instrumental delivery were excluded from the study. Sixty-five were recruited, and 45 completed the study, 20 of whom were delivered by a normal vaginal delivery and 25 by caesarean section. A random effects model was used to compare platelet counts within and between the two groups to assess the severity and the timing of reactive thrombocytosis. RESULTS: There were no statistically significant differences in booking and pre-delivery platelet counts between the two groups (mean values 248.4 x 10(9)/L and 245 x 10(9)/L in the normal vaginal group and 269.4 x 10(9)/L and 251.6 x 10(9)/L in the caesarean section group, respectively). Postnatally, a rise in the platelet count was noted in the normal vaginal delivery group, reaching statistically significant peak values, compared with booking and pre-delivery at days 8 and 12 of the postnatal period (mean value 365.8 x 10(9)/L; P < 0.001 and 369.4 x 10(9)/L; P < 0.001 respectively). In the caesarean section group, the platelet count was raised to a statistically significant high value, compared with booking and pre-delivery at day 8 of the postnatal period. The platelet count peaked at days 12 and 16 of the postnatal period (mean value 522.5 x 10(9)/L; P < 0.0001 and 526.5 x 10(9)/L; P < 0.0001, respectively) and remained significantly higher than booking and predelivery values for 24 days after the caesarean section. There was a greater rise in the platelet count in the caesarean section group compared with the vaginal delivery group. The platelet counts in the caesarean section group were significantly higher than these in the normal vaginal delivery group from day 12 to day 24 of the postnatal period. CONCLUSION: A significant rise in platelet count occurred eight to twelve days after normal vaginal delivery and caesarean section. The increase in platelet count continued to rise for 16 days after caesarean section, and it stayed significantly higher for more than 24 days after the delivery. PMID- 10740341 TI - The effect of thrombophylaxis on pregnancy outcome in patients with recurrent pregnancy loss associated with factor V Leiden mutation. AB - OBJECTIVE: To observe the effect of thrombophylaxis on pregnancy in women with a history of unexplained recurrent pregnancy loss also carrying the factor V Leiden mutation. METHODS: Between 1 January and 31 December 1996, activated protein C (APC) resistance and factor V Leiden mutation were prospectively measured in 56 nonpregnant women, with a history of two or more unexplained recurrent pregnancy losses. During the same study period, seven women carrying the factor V Leiden mutation conceived, and were subsequently followed throughout their pregnancy. Subcutaneous low molecular weight heparin (LMWH, enoxaparin, 40 mg/day) and oral low dose aspirin (100 mg/day) were administered throughout the pregnancies, starting at early first trimester. Ultrasound and Doppler umbilical and fetal middle cerebral arterial flow studies were performed in the second and third trimesters, and the course and outcome of the pregnancies were documented. RESULTS: Activated protein C resistance and factor V Leiden were found in 20 (36%) and 12 (21%) women of the study, respectively. Five of the seven pregnancies occuring progressed uneventfully to term with normal fetal growth, normal Doppler flow studies and uneventful neonatal outcome. Two of the seven women had early missed abortions. CONCLUSIONS: Thrombophylaxis, beginning in early pregnancy, in women with unexplained recurrent pregnancy loss associated with factor V Leiden mutation, seems to be safe and allow normal fetal development and good neonatal outcome. To prove the efficacy of thrombophylaxis by LMWH and low dose aspirin in this setting prospective controlled studies seem to be justified. PMID- 10740342 TI - Fetal distress increases interleukin-6 and interleukin-8 and decreases tumour necrosis factor-alpha cord blood levels in noninfected full-term neonates. AB - OBJECTIVE: To assess the influence of fetal distress on interleukin-1beta, interleukin-6, interleukin-8 and on tumour necrosis factor-alpha blood levels in noninfected full-term neonates. STUDY DESIGN: In a multicentre prospective study, cord blood samples were obtained at time of delivery from 234 noninfected full term neonates for the purposes of measuring serum levels of interleukin-1beta, interleukin-6, interleukin-8 and tumour necrosis factor-alpha using immunoassays. Women were classified into four groups according to the mode of delivery (vaginal delivery or caesarean section) and the presence or absence of fetal distress. The role of labour was also investigated. RESULTS: No significant relationship was found between cytokine cord blood levels and the mode of delivery. Fetal distress was associated with an increase in interleukin-6 (P = 0.01) and interleukin-8 (P < 0.001) levels, and a decrease in tumour necrosis factor-alpha (P < 0.001). Labour was also associated with a significant increase in interleukin-6 and interleukin-8 cord blood levels (P = 0.01 and P < 0.001, respectively). CONCLUSION: Fetal distress and labour were each associated with elevated interleukin-6 and interleukin-8 cord blood levels in noninfected full term neonates while only fetal distress was associated with decreased tumour necrosis factor-alpha levels. PMID- 10740343 TI - Hyperechogenic fetal bowel: a prospective analysis of sixty consecutive cases. AB - A two year prospective analysis of all second trimester fetuses (16-22 weeks of gestation) with hyperechogenic bowel was undertaken. Hyperechogenic fetal bowel (sonographic echogenicity similar to or greater than that of surrounding fetal bone) was diagnosed using strict criteria. Outcome of affected fetuses was ascertained from hospital records, health care workers and autopsy reports, up to six months of age. Sixty consecutive fetuses were identified, of which 48 (80%) were liveborn. Six pregnancies were terminated, four ended with an intrauterine fetal death and two died at birth. The incidence of cystic fibrosis and aneuploidy were each 5%, and there were no cases of congenital infection. Intrauterine growth retardation was recorded in six fetuses (10%), four of whom died perinatally. Eighty-two percent of fetuses (28/34) with isolated hyperechogenic bowel had a normal outcome. PMID- 10740344 TI - Compartment syndrome following postpartum haemorrhage. PMID- 10740345 TI - Sub-aponeurotic haemorrhage: a rare but life-threatening neonatal complication associated with ventouse delivery. PMID- 10740346 TI - The effect of oestrogen supplementation on postmenopausal urinary stress incontinence: a double-blind placebo-controlled trial. PMID- 10740347 TI - Antecedents of neonatal encephalopathy with fetal acidaemia at term. PMID- 10740348 TI - Are we prescribing multiple courses of antenatal corticosteroids? A survey of practice in the UK. PMID- 10740349 TI - Randomised trial comparing expectant with medical management for first trimester miscarriages. PMID- 10740350 TI - A feasibility study of adolescent sex education: medical students as peer educators in Edinburgh schools. PMID- 10740351 TI - [Alternative medicine between adaptation and demise: exemplified by homeopathy]. AB - For the past several years, conventional health care systems--often called "conventional medicine"--have been criticized widely. Reasons mentioned are increasing costs and its ill equipment to handle multifaceted chronic illnesses. Such a decline of legitimacy is paralleled by an increase of alternative and complementary medicine. As the example of homeopathy shall demonstrate, successful strategies of professionalization in orthodox medicine can not simply be applied to its "holistic challengers". Important reasons to be outlined encompass the economic impacts of health care which have reached both orthodox and alternative medicine as well. PMID- 10740352 TI - [Are children properly classified within the scope of expert assessment to determine need for services?]. AB - This project deals with assessment directives issued by the leading statutory care insurance companies to define the need for care of children at certain ages in coping with the activities of daily life specified by the German Social Legislation. The ages in question relate to healthy, normally developed children. They are used as a measure of comparison for the assessment of diseased children. Besides, they have a decisive significance in defining the need for care. Only if a child is not able to perform a certain activity, although it is meant to be able to do so at that age, can we define the need for help and assistance, i.e. care. The project is divided into two sections. 1. Comparison of age mentioned in the assessment directives for each of the daily life activities of healthy and normally developed children aged 1 to 12 years with the age mentioned in technical literature. 2. Comparison of age mentioned in the assessment directives for each ot the daily life activities of healthy and normally developed children at the age of 1 to 12 years with those mentioned in retrospective interviews with the same group of children. In the first part of the project, an analysis of the literature dealing with the subject was done. The literature included the stock of technical literature in the German language. Studying the literature, ages for children could be isolated, that could be classed with the activities of daily life mentioned in the German Social Code. These ages were compared with those in the assessment directives. In a second part of the project, 81 mothers and fathers of healthy, normally developed children aged 1 to 12 years were asked via a questionnaire. They were asked to estimate retrospectively from what age on their child had been able to perform certain activities of daily life without any help. The comparison of ages mentioned in the assessment directives with those of the analysis of literature conformed only regarding daily life activity. Regarding the other activities of daily life the age for children was set higher in the assessment directives than in the literature. Comparing the age of children in the assessment directives with the results of the questionnaires the age was also set higher in the directives than shown by the results of the questionnaires. The results of the literature analysis and the inquiry by questionnaires show that the ages mentioned in the assessment directives for an independent performance of daily life activities do not correspond to the development according to the age of healthy normally developed children aged 1 to 12 years. As a result of this project it seems to be justified to be doubtful about an adequate assessment of children who are in the need of care. Due to this reason it would be desirable to reexamine the data of the assessment directives by means of a new representative prospective study. PMID- 10740353 TI - [Risk of endangering patients by hepatitis B infected surgeons: monitoring the health of medical personnel in hospitals must be evaluated]. AB - In connection with the HBV infections by a cardiothoracic surgeon at a universitary hospital in Germany it must be clarified whether the detected omissions (no HBV vaccination of the surgeon, parts of the medical staff lacking health checks) indicate generally relevant problems in occupational and patient health protection. This is very necessary because in international literature more than 40 cases of HBV transmission from doctors to patients are documented and a high rate of undetected cases must be supposed. The case under reference shows up weak points with regard to the established legal mechanisms of control and refers to a problem of insufficient coordination between hospital hygiene and occupational health protection. Local health departments and occupational cooperative societies should urgently draw the necessary conclusions: A quantified report (with specified numbers) is required stating the health checks and non-performance of vaccinations in hospitals; defaults in this regard must be remedied without delay. The work which infectious employees can do must be defined by guide lines at every hospital. Expert committees considering the circumstances of the individual cases should determine the necessary safety measures. PMID- 10740354 TI - [Serologic study of the prevalence and course of Hantavirus infections in Mecklenburg-Vorpommern]. AB - 694 sera obtained from 1994 till 1998 in the general population were examined with a recombinant enzyme immunoassay for hantavirus antibodies against the serotypes Hantaan and Puumala. Positive samples were retested for confirmation with an indirect immunofluorescence assay and a recombinant immunoblot. The observed antibody prevalence of 0.9% signities that Mecklenburg-Vorpommern is not an endemic area. 12 out of 196 patients with a suspected hantavirus infection were confirmed. All proved antibodies were anti-Hantaan. Due to strong cross reactivity in the Hantaan group, definite determination was not possible with the methods employed. The clinical symptoms corresponded to epidemic nephropathy. One patient developed the hantavirus pulmonary syndrome. Titre controls were carried out. In all professional groups with a high risk of infection (forestry employees, zoo keepers, workers in a stud farm, pest controllers) no increased antibody prevalence was seen. Dialysis patients had the same antibody prevalence as the general population (0.9%). PMID- 10740355 TI - [Promoting non-smoking behavior in 13-year-old students in primary schools and high schools. A prospective, randomized intervention study with 1,956 students]. AB - 1956 7th grade students of high schools and secondary modern schools in three cities with 110,000 to 180,000 inhabitants were asked about cigarette, alcohol and drug consumption using questionnaires. Subsequently 1080 of the students took part in a newly invented non-smoking curriculum which was held during 8 lessons within 4 weeks (intervention group (I)). The lessons were performed by non smoking physicians who had passed a training programme before. 876 students served as control (C) group. 2 years later the questioning was repeated. The number of students who started smoking within the two years was significantly lower in the intervention group. In the high schools a significant effect was seen in boys (I: 13.0%, K: 22.4%; p < 0.01) and girls (I: 21.4%, K: 28.4%; p < 0.05) whereas in secondary modern schools a significant effect was seen only in boys (I: 17.4%, K: 25.2%; p < 0.05) not in girls (I: 18.0%, K: 22.0%; n.s.). First-time consumers of drugs like cannabis, marihuana and organic solvents were found less often in the intervention group among boys in high schools than in the control group (I: 14.9%; K: 23.6%; p < 0.05). No effects of the intervention were seen in girls of high schools and in students of secondary modern schools. No effects of the intervention were seen on alcohol consumption. Students who drank beer, wine or liquor several times a week were found at the same rate in the intervention and control groups. We conclude that a non-smoking intervention of 8 lessons in the 7th grade of high schools and secondary modern schools may lower the rate of first-time consumers of cigarettes but not of alcohol and drugs. PMID- 10740356 TI - [Decontamination of highly polychlorinated biphenyl contaminated indoor areas by complete removal of primary and secondary sources]. AB - A new sanitization concept in a school highly contaminated with polychlorinated biphenyls (PCBs) is described. The maximum indoor air concentration was 13,100 ng/m3 PCB. Initially the average indoor PCB concentrations attained 6,000-7,000 ng/m3. A reduction to 226 +/- 89.1 ng/m3 (n = 24) was achieved. Ten months later the PCB concentration was 171 +/- 57.6 ng/m3 (n = 3). Primary sources were PCB contaminated elastic sealants and the paint of radiators or heating elements. Also PCB-contaminated materials were walls, floors and ceilings. These secondary contaminations were also relevant for PCB indoor air concentrations. The primary sources were removed dust-free by manual procedures. Contrary to other sanitization concepts all secondary-contaminated PCB materials were also removed with a high-pressure water method specially developed for such cases. Because of the removal of all relevant primary and secondary PCB deposits, a lasting effect can be expected. By this sanitization concept, the use of new and less intensively explored products suppressing the diffusion of PCBs into the indoor air and whose toxicity is hardly known, may be prevented, thus avoiding the need for prolonged control measurements. Before using the above-mentioned new sanitization concept, another concept had been rejected for lack of efficiency, thus avoiding unnecessary costs. The companies performing sanitization should be compelled to accept a defined lowest PCB concentration. PMID- 10740357 TI - [Organ shortage in transplant centers--approaches to a solution from the economic viewpoint]. AB - In the article the regulations of the German transplantation law, which came into force on 1 December 1997, are considered from an economic perspective. First, the essential regulations of the law are described and then analyzed with respect to economic aspects. The law regulates in particular the administration of the demand surplus for organs. This could be reduced by rationing the demand or by increasing the supply. Apart from the medical features economic attributes such as cost effectiveness are also applicable as criteria for rationing. In addition, the method of the quality-adjusted life-years (QALYs) is introduced and described on the basis of the results of a study about costs and benefits of liver transplantation. To increase the organ supply the authorities in Germany have so far established information campaigns and made appeals to the public. In contrast, an organ donation club the allocation of donors is made considering if and when the recipient declared his readiness to organ donation. In this way the willingness to donate his own organs could be sanctioned positively and an additional incentive for supply could be set. PMID- 10740358 TI - ["Use of patient-related information for research in medicine and public health"]. PMID- 10740359 TI - [5 years nursing care insurance legislation. Comprehension of medical aspects by the expert physician]. PMID- 10740360 TI - [Certification according to DIN EN ISO 9001 in MDK in Hessia--a report of experiences]. AB - Quality assurance is a routine and indispensable part of the work of the Medizinische Dienst der Krankenversicherung (MDK) (Medical Services of statutory health insurance bodies in Germany). To optimize the internal organisational processes, a quality assurance system (QAS), certified by EQ Zert, Ulm (an accredited certifier of the TGA), was implemented for the first time in one of the departments of the MDK, namely, the hospital department. Employees and management consider the advantages to be as follows: the aims of the department are clearly described. the processes are now self-evident and more strictly observed, overlaps to other fields of the company are defined. Both an Error Management System with follow up measures, and a Project Management System to control projects have been implemented. The documentation system was standardized, and consequently relocating documents has become more streamlined. An MDK self-evaluation was performed as part of an internal audit. The audit showed that the QAS of the hospital department of the MDK in Hesse is indeed highly developed. It is anticipated that gradually other fields of the MDK will also be certified, to comply with the changing requirements. PMID- 10740361 TI - Cocaine. PMID- 10740363 TI - User centred health care. PMID- 10740362 TI - The challenge of evaluating and treating HIV related cutaneous disease. PMID- 10740364 TI - Literature and psychiatry. PMID- 10740365 TI - Hormone replacement therapy: a survey of Irish general practitioners. AB - We report findings of a cross-sectional postal survey of current prescribing practices of hormone replacement therapy (HRT) by Irish General Practitioners from a random sample of 600 Irish College of General Practitioners members. Median estimated prescribing rate of HRT was 17.5% (interquartile range 10 to 30%). The majority of General Practitioners would prescribe for the prevention of osteoporosis but there was some reluctance to prescribe solely for the prevention of CVD. Common cardiovascular conditions were regarded as contraindications to HRT by nearly one-third of GPs. Female GPs were more likely than males to request mammography (p < 0.002), to consider a first degree relative with breast cancer a contraindication (p < 0.01) and less likely to prescribe HRT for longer than 10 years (p < 0.0001), because of breast cancer risk. 78% of GPs would offer HRT to all eligible women. We conclude the estimated prescribing rate is comparable to rates in other developed countries. PMID- 10740366 TI - Hospital services for patients with acute stroke in Ireland: the Volunteer Stroke Scheme survey of consultant opinion. AB - A national postal survey of hospital based consultants with responsibility for acute care of stroke admissions was performed in November, December 1998. Of 162 survey forms, 140 (86.4%) were returned representing consultants working in all 38 acute general hospitals (total 10,067 hospital beds) of whom 135 indicated that stroke patients were admitted under their care. Patients were admitted under 11 different subspeciality groups to various medical and surgical wards. Only 18.5% of consultants worked in hospitals where there was a physician/neurologist with specific responsibility for stroke, whilst only 19.5% were aware of a policy in their hospital for implementation of minimum standards of care for stroke patients or a recent audit of stroke care (9%). A substantial number of hospitals in certain health board areas have no access to a consultant led rehabilitation unit within their own health board area whilst 18/38 hospitals have no on-site CT brain scanning. Despite the proven value of organised hospital stroke care, this survey documents major deficiencies in this country. We suggest that each health board would review its services to include in each hospital a consultant physician with special responsibility for co-ordination and development of appropriately staffed and funded stroke services. PMID- 10740367 TI - The fetal and neonatal outcomes of Rhesus D antibody affected pregnancies in Northern Ireland. AB - It has been suggested that routine antenatal prophylactic anti-D should be introduced for prevention of Rhesus D (RhD) haemolytic disease. Before making changes to the current prevention program it is important, therefore, to have up to-date data on affected infants. Pregnant women with anti-D antibodies between September 1994 and February 1997 were identified by the Northern Ireland Blood Transfusion Service. The records of 124 women and 130 babies were examined. 26% of planned deliveries occurred in hospitals without paediatric cover. Rhesus isoimmunisation affected 78 babies. Fifty-nine infants were admitted to one of seven neonatal units. There were 2 stillbirths and 1 neonatal death. Two infants have severe neurodevelopmental delay. There is still significant morbidity associated with RhD haemolytic disease. Care for RhD affected pregnancies should be centralised to guarantee training opportunities and maintenance of expertise. The current management of these pregnancies should be re-examined before changing the Rhesus prevention program. PMID- 10740368 TI - Open carpal tunnel release under local anaesthesia: a patient satisfaction survey. AB - The patient acceptability of open carpal tunnel release under local anaesthesia (LA) and tourniquet control was assessed by a postal survey. 58 releases on 44 patients over a 3-year period were surveyed, with a questionnaire response rate of 91%. Use of LA for this procedure was effective and acceptable to most patients, but the incidence of severe pain due to infiltration of LA and the tourniquet intolerance rate were unacceptably high and have been addressed by use of LA with adrenaline and bipolar diathermy to dispense with tourniquet and use of dental syringe and injecting LA from proximal to distal direction to make infiltration more comfortable. PMID- 10740369 TI - Caroli's disease--a case presentation. PMID- 10740370 TI - Idiopathic haemorrhagic cerebellar infarction in an 11 year old child. PMID- 10740372 TI - A case study of non-binding mediation in practice: Rush-Presbyterian-St. Luke's Medical Centre, Chicago, Medical Malpractice Mediation Programme: continued.... PMID- 10740371 TI - Acute myocarditis precipitated by Salmonella Montevideo infection: a case report. PMID- 10740373 TI - [The connection between magnetic fields and cancer is still of current interest]. PMID- 10740374 TI - [Great risks of undertreatment. Two of three hospitalized children are not receiving approved drug treatment]. PMID- 10740375 TI - [Help to children and adolescents with malnutrition or eating disorders. Percutaneous endoscopic gastrostomy with button: simple, safe and cost effective]. AB - Percutaneous endoscopic gastrostomy (PEG) has gained great popularity for children with malnutrition and eating disorders secondary to chronic illness. However, the procedure is not without risks. We report on 62 infants and children, median age 4 years (1 month-20 years), who underwent PEG placement. Cerebral palsy with or without mental retardation was the most common diagnosis (50%). No complications related to the PEG procedure itself occurred, but postoperative pneumonia was seen in 10%. Late complications were few: intraperitoneal migration of the button in one child and prolapse of the stoma in another. At the time of button placement, after median 14 weeks, mean weight had increased from a standard deviation score of -2.7 to -2.2 (P < 0.001). We consider PEG to be a safe procedure for children with malnutrition requiring enteral feeding. Due to potential risks and complications related to this method, a multidisciplinary approach, as found in a "nutritional support team", is recommended. PMID- 10740376 TI - [Current ambulatory treatment of renal calculi]. PMID- 10740377 TI - [Four new instrument for blood gas analysis are tested: handy cassettes for easier use]. AB - An entirely new type of blood gas analyser has made its way into the marketplace, to be used, for example, in emergency rooms, intensive care units, ambulances, and bedside with quarantined patients in infectious diseases units. The instruments reviewed here employ new miniaturised analysis circuitry, integrated into the cassette on which the blood sample is applied. These instruments are designed for use by care-givers without specific laboratory training. Four point of-care blood gas analysers are tested: OPTI 1 (AVL), I-STAT (HP), IRMA (Infiniti) och ABL 70 (Radiometer). PMID- 10740378 TI - [Adulteration of urine drug testing--an exaggerated cause of concern]. AB - In a study performed at a Stockholm clinic for young people with drug abuse problems, where urine adulteration was suspected to be fairly frequent, a total of 594 patient specimens were subjected to Adultacheck test strip screening for nitrite, glutaraldehyde, pH, and creatinine. Creatinine measurement was also performed at the laboratory, together with drug screening using EMIT reagents, and a subsample was spiked with phencyclidine to verify EMIT test function. The frequency of dilute urine (creatinine < 4 mmol/L) was 11%. Otherwise no evidence of urine adulteration was observed. Thus the Adultacheck strip might be useful in detecting dilute urine specimens already at the clinic, though the test strip levels did not agree well with the respective laboratory results. It was concluded that adulteration of urine specimens was not common at the clinic, and that specimen collection for urine drug testing could be performed in a reliable manner. PMID- 10740379 TI - [A retrospective study among diabetics in the county of Kronoberg. The St. Vincent declaration has not affected the frequency of amputations]. PMID- 10740380 TI - [More demands on internal medicine from patients with multiple diseases. The specialties of internal medicine should have an overall leadership]. PMID- 10740381 TI - [I'm trying to be as nice as the radiologist I met 23 years ago]. PMID- 10740382 TI - [Frogs, fish and reflection of the sun gave a clue to understanding how the visual impressions reach the brain]. PMID- 10740383 TI - [The National Board of Health and Welfare: stronger evidence basis for the benefits of mammography]. PMID- 10740384 TI - [No evidence that routine mammography screening is effective]. PMID- 10740385 TI - [The debate on MedAnalys goes on: no basis so far for the suspicions]. PMID- 10740386 TI - [Apropos a book on euthanasia in the Netherlands: we read, we see and interpret differently]. PMID- 10740387 TI - [A lot of changes: internal medicine, quo vadis?]. PMID- 10740388 TI - [What the SvD really wrote about the research on mammography]. PMID- 10740389 TI - [Chronic fatigue is a problem not yet solved--new viewpoints are required]. PMID- 10740390 TI - [Do we know how well science transmits knowledge?]. PMID- 10740392 TI - [What is power and why specifically 80%?]. PMID- 10740391 TI - [What is p and why specifically 0.05?]. PMID- 10740393 TI - [Multivariate analysis--do we understand what we are doing?]. PMID- 10740395 TI - [Clinical trials--what is the question?]. PMID- 10740394 TI - [Surviving is not equal to survival?]. PMID- 10740396 TI - [Meta-analysis of clinical studies: value for the wise or risk for harm?]. PMID- 10740398 TI - [Statistically significant--also relevant for the patient?]. PMID- 10740397 TI - ["Statistically significant"--an overestimation of data value?]. PMID- 10740399 TI - [Facing the Third Millennium]. PMID- 10740400 TI - [Nutrition and colorectal cancer]. AB - Colorectal cancer is the most common tumor behind lung cancer in men and breast cancer in women. Its prevalence shows a wide geographic variability, which, along with epidemiological and experimental evidence, suggests a considerable participation of environmental factors, mainly dietary in its cause. It is not known which dietary components have an undisputed influence on the risk of colorectal cancer, nor how they condition the different genetic susceptibilities of the individuals. However, we do know of the protective action that consuming vegetables has, and the increased risk associated with saturated fats and red meats, especially when associated with other factors like a hypocaloric diet, obesity, sedentary life, or the use of alcohol. Fiber, however, has not proven the risk reducing properties that were attributed to it by epidemiological studies, possibly because this term encompassed several very different substances. Moreover, it is very difficult to know the effect of an isolated nutrient as these are intimately entwined in the foods. It should be kept in mind that many substances, like plants, as well as the usual nutrients, contain a large number of components with unknown effects and that the manner in which the foods are prepared or eaten, no doubt are of influence. A healthy lifestyle with regard to the risk of colorectal cancer, should no doubt include the consumption of large amounts of vegetables and whole cereals, a limit of the caloric intake with fats not exceeding 30%, to eat fish and chicken rather than red meat, to avoid alcohol, and to regularly partake of physical exercise. PMID- 10740401 TI - [The effect of fatty emulsions with distinct triglyceride compositions on the lipid metabolism of the septic patient]. AB - OBJECTIVE: To assess the effects on the intravascular lipid mechanism of fatty emulsions with an identical lipid concentration and a different triglyceride composition administered as part of the total parenteral nutrition (TPN) in septicemic patients hospitalized in the intensive care unit (ICU). One emulsion will be made up of long chain triglycerides, LCT (20% Intralipid) (group I) and the other will be made up by a mixture of medium- and long-chain triglycerides, MCT/LCT (1:1) (20% Lipofundina) (group II). AREA: Vall d'Hebron General University Hospital. Intensive care unit, Biochemistry laboratory, nutritional support unit, and Department of Pharmacy. PATIENTS: 12 septicemic patients who required TPN were studied, and these patients were randomly given one of the two lipid emulsions for a five day period. Prior to initiating the TPN and before ending it, blood samples were drawn for the analysis of the lipoprotein components VLDL, LDL, and HDL isolated by ultracentrifugation, and the basic lipid and nutritional parameters. RESULTS: The baseline statistical analysis shows that even though both groups are not comparable, the composition of the VLDL, LDL, and HDL lipoproteins differs from the reference values. After five days of TPN, the metabolic behavior of the groups is different, in group I the concentrations of reactive C protein (RCP) decreased as did the HDL phospholipids, while group II presented an increase in the plasma triglyceride levels, the VLDL cholesterol, the LDL triglycerides, and the HDL proteins. CONCLUSIONS: Septicemic patients present in altered lipoprotein pattern that tends to normalize after 5 days of lipid emulsions administration. PMID- 10740402 TI - [Drug administration in patients on enteral nutrition: the nasoenteral route versus the parenteral route]. AB - The administration of medications through a nasoenteral tube or enterostomy is an alternative to the parenteral route, as this is more comfortable, less aggressive for the patient, and cheaper. The objective of this study was to potentiate the nasoenteral route versus the parenteral route and the quantify the economic repercussions. For this we carried out a prospective study of the prescriptions in 100% of the patients with enteral nutrition through a tube over a six month period. Informative notes were sent to the clinicians for those patients who met the established inclusion criteria and who were candidates for changing the route of administration. Of the 542 medications susceptible to the change, 217 were changed to the nasoenteral route. The economic savings during this period were 1,401,095 pesetas, and we consider the main advantage of the change in method is not economical, but rather the greater degree of satisfaction that the patient derives from this, as well as making a release from hospital with at home enteral nutrition easier. PMID- 10740403 TI - [The evaluation of gastrointestinal tolerance for a new infant feeding formula (Similac) in healthy infants]. AB - There is a pediatric consensus that maternal milk is the best food for the new born, but when this is impossible, one turns to artificial nutrition. The main objective of infant formulae is to achieve a gastrointestinal tolerance that is similar to maternal milk. With this in mind, we carried out an observational, prospective, and multicentric study with the objective of evaluating the gastrointestinal tolerance of Similac, taking maternal milk and other formulae on the market as reference. The study included 2 pediatric visits, a diary in which the parents recorded the gastrointestinal parameters, and a final questionnaire to evaluate the degree of satisfaction. Information was obtained from 6,617 evaluable cases, 82.2% fed with Similac (S), 8.8% with maternal milk (MM) and 9% with other formulae (OF). The analysis of the data showed that the percentage of children with signs of gastrointestinal intolerance, was reduced to more than half when using S compared to OF (5.6% S, 5% LM, and 14.1% OF). The color and consistency of the feces was significantly associated with the formula group, and in both cases S showed a greater similarity to the pattern obtained with MM compared to the OF group. 18.6% of the children with OF presented hard feces, and this percentage was reduced to at least half with S (6.2%). The S group showed a greater percentage of lack of regurgitations and no aerophagia and the lowest percentage for three or more regurgitations and severe aerophagia. Those questioned considered that S was easier to reconstitute up, is liked better, and is better tolerated than the OF used. Similac achieved a greater similarity in gastrointestinal tolerance to that of maternal milk than other formulae did. PMID- 10740404 TI - [The zinc status in a selected Spanish population. A multivariate analysis]. AB - There are no previous studies on the zinc status in the healthy, adult, Spanish population. The objective of this study was to obtain the levels of zinc in the serum and hair of this population to determine the normal nutritional status of zinc, and evaluating the influence of certain factors like age, sex, type of residence, body mass index (BMI) hair color, or recent food intake (zinc and fiber contents of the diet) on this status. Using a simple randomized sampling, 186 blood donors of both sexes with ages comprised between 18 and 65 years, were selected. The results were analyzed using uni- and multi-variate statistical techniques. The average concentration of zinc in serum was 97.22 micrograms/dl (95% CI: 95.1-99.4); the average zinc level in hair was 163.86 micrograms/g (95% CI: 157.8-169.9). The serum zinc was individually associated with the type of residence and the zinc and fiber contents of the diet. The zinc in hair was associated with sex, age, body mass index, and hair color. Using a multivariate analysis, predictive models of multiple regression were obtained for the serum zinc: y = 109.69 - 1.39 (dietary zinc) - 4.63 (sex) - 0.65 (dietary fiber), for the zinc in the serum, and y = 199.34 + 26.62 (sex) - 2.08 (BMI) - 0.47 (age) for the zinc in the hair. The serum concentration decreases as the dietary zinc and fiber decreases and women have lower levels. The zinc in hair is increased in women and is reduced with increasing age and BMI. PMID- 10740405 TI - [The kidneys--are they the culprit or/and the victim of elevated blood pressure?]. PMID- 10740406 TI - [Evaluation of the relationship between possible control of anemia in hemodialysis patients and the concentration of leptin]. AB - There are many growth factors (GFs), which stimulate the proliferation and maturation of erythroid progenitors. The main one is erythropoietin (Epo). Epo acts in concert with other GFs. Recently it was suggested that leptin (Lep) could be involved in a very early stage of erythropoiesis (E). The aim of this study was to analyse the relations between ability of idiopathic compensation of anemia by hemodialysis patients (HD pts) and concentration of Lep in HD men and women separately. The study was performed in 25/13M, 12F/HD pts, who idiopathically compensate anemia (group 1) and 29/16M, 13F/HD pts who required rHuEpo therapy (group 2). The mean Lep level in all women together was significantly higher than in all HD men together (26.9 +/- 6.3 ng/mL vs 6.8 +/- 0.9 ng/mL) but BMI was similar in men and women. We did not find significant differences in level of Lep in both studied groups of HD pts. Perhaps the lower influence of Epo and testosterone on E in HD women is compensated by significantly higher Lep concentration. PMID- 10740407 TI - [Tissue factor and inhibitor of the blood coagulation pathway in nephrotic syndrome]. AB - Some parameters of extrinsic coagulation pathway, vitronectin and thrombomodulin were studied in 21 patients with nephrotic syndrome. Concentrations of tissue factor and tissue factor pathway inhibitor were found to be significantly elevated in patients with nephrotic syndrome when compared to control group. Activities of tissue factor and tissue factor pathway inhibitor as well as vitronectin did not differ significantly from the healthy volunteers. Activities of factor VII and X were significantly higher in nephrotic patients when compared to the control group. Thrombomodulin--a marker of endothelial cell injury was significantly higher in patients with nephrotic syndrome relative to controls. PMID- 10740408 TI - [Expression of CD5 antigen in B and T cells from umbilical cord blood, from blood of healthy adults and patients with chronic lymphocytic B-cell leukemia (PBL-B)]. AB - Antigen CD5 is the glycoprotein which belong to the scavenger receptor cysteine rich family. Mainly there is on the T cells subpopulation. During fetal life B CD5+ cells are major subpopulation of B cells in the spleen, lymph nodes and there are also in the cord blood. In adult CD5+ cells are minor subpopulation (27%) of B cells from the peripheral blood. CD5 there are on chronic lymphocyte leukaemia B cells (B-CLL) also. Usually expression CD5 on B-CLL cells associated with weak or lack expression of the surface immunoglobulins and CD79 beta, CD20, CD22, CD21 (CR-2), CD35 (CR-1) antigens. It appeared interesting to compare the expression of CD5 antigen (the mean fluorescence intensity--MFI of CD5) on B cells from the cord blood, adults peripheral blood and B-CLL patients. MFI of CD5 on B and T cells were also compared in each groups. MFI of CD 19 was studied too. Lymphocytes from the cord blood (11 assays), adult peripheral blood of healthy volunteers (18 assays) and the peripheral blood of no treated patients with B-CLL (56 assays) were studied. The immunological phenotype of lymphocytes was evaluated with the monoclonal antibodies anti-CD5 and anti-CD19 by the flow cytometry method. We have demonstrated that MFI of CD5 on B cells from patients with B-CLL was strongest and weakest from normal individuals. MFI of CD5 on T cells from patients with B-CLL is stronger in comparison to healthy volunteers. MFI of CD19 is weakest on cells from patients with B-CLL and strongest in normal individuals. On the basis of the our results and other medical papers we suggest on the one hand that biology of B-CLL depend on deficit antigens specific for B cells lines on the other hand depend on overexpression of CD5 antigens on leukaemic B and T cells also. PMID- 10740409 TI - [Effects of anti-inflammatory drugs on adhesion and migration of lymphocytes]. AB - The first crucial step of cell recruitment to the sites of inflammation is adhesion and motility, which are interdependent phenomena. The purpose of our study was investigate the influence used anti-inflammatory drugs (Aspirin, Cyclosporin A, Methotrexate) and endogenous substances of anti-inflammatory activity (Cortisol, TGF-beta) on these phenomena. Lymphocytes from healthy subjects were preincubated with various concentrations of these substances and adhesion to plastic bound CD11a/CD18, CD11c/CD18, CD44 and CD62L mAbs was determined. Simultaneously, cell motility was investigated after Boyden method. We found significant reduction of cell adhesion to all the ligands used. Furthermore we observed significant dose dependent increase in motility of these cells. These data indicate that simultaneous enhancement of motility and reduction of adhesion might be a common pathway for the mechanisms of action of anti-inflammatory agents. PMID- 10740410 TI - [Evaluation of the importance of antibodies against islet cells (ICA) and Glutamate Decarboxylase(anti-GAD) for determining prognosis of diabetes type 1 in the Polish population--5 year observation]. AB - It was recently shown that there are significant differences between the frequencies of antibodies against pancreatic islet cell antigens (ICA, GADA) in the first degree relatives of IDDM patients in different regions of Poland. There are however no published studies concerning their predictive value in the development of IDDM in the Polish population. The aim of the present study was to evaluate the PPV (positive predictive value) of ICA and GADA antibodies and to analyse diabetes-free survival in association with titre, antibodies co-existence and relatives age in the Polish population. The study was performed in 225 first degree relatives of IDDM patients with ICA and/or GADA and 100 relatives without antibodies, in whom ICA and/or GADA were performed in 1993-1994. We have observed significantly lower percentages of diabetes-free survival in subjects with ICA > 20 JDF in comparison to relatives without ICA or with ICA < 20 JDF. The highest predictive value for diabetes type 1 development was associated with the ICA > 80 JDF and with the co-existence of ICA--20-79 JDF and GADA (+). There was also a statistically lower diabetes-free survival in first degree relatives (with ICA > 20 JDF) older than 20 years of age in comparison to the younger subjects. Detection of 2 antibodies: ICA and GADA made it possible to identify 80% of first degree relatives who have developed diabetes type 1 in the following 5-6 years, this suggests that the combined measurement of ICA and GADA could be a useful marker in screening for diabetes type 1 in first degree relatives of IDDM subjects in the Polish population. For the diabetes type 1 risk assessment in relatives with ICA > 20 JDF the age of the studied subjects should be taken into consideration. PMID- 10740411 TI - [Clinical aspects, diagnosis and treatment of hypercalcemia in a patient after removal of an adrenal tumor producing cortisol. Case report]. PMID- 10740412 TI - [Extramedullary hemopoiesis presenting as a tumor of the posterior mediastinum in a patient with hemolytic anemia]. AB - Extramedullary hemopoietic focus is a rare cause of tuberous enlargement of the posterior mediastinum. A 62-year-old man who had been diagnosed as having congenital spherocytic anemia 20 years ago, was admitted to hospital with initial diagnosis of pneumonia. Chest X-ray disclosed inflammatory infiltration in the lower lobe of the right lung and a tumor in the posterior mediastinum. Pneumonia subsided following treatment with antibiotics. Spiral computer tomography with concomitant transthoracic biopsy of the lesion, confirmed the presence of tumor located on the right side of the spinal column, as well as the presence of two minor focci on its opposite side. Cytological examination revealed extramedullary hemopoiesis. Differential diagnosis of tumor in posterior mediastinum, should take into consideration the possibility of extramedullary hemopoietic focci. PMID- 10740413 TI - [Clinical role of the Fas/Fas ligand system]. PMID- 10740414 TI - [Lipocytes--their importance in physiology and pathology of the liver]. PMID- 10740415 TI - [Right-sided infective endocarditis]. PMID- 10740416 TI - [N-acetyl-beta-D-glucosaminidase and its clinical significance]. PMID- 10740417 TI - [Do leptin and neuropeptide Y influence blood pressure regulation in healthy pregnant women and women with preeclampsia?]. AB - Leptin (LP) and neuropeptide Y (NPY) are involved in the regulation of appetite and energy expenditure. As was shown in our previous studies healthy non pregnant and pregnant women are characterized by a significant positive correlation between maternal body mass index (BMI) and plasma leptin concentration. On the other side participation of both leptin and obesity in the pathogenesis of essential hypertension is presumed. The present study aimed to answer the following question: to what extend LP and NPY are involved in the pathogenesis of arterial hypertension in pregnant women with EPH gestosis. One to 2 days before delivery plasma LP and NPY concentration were estimated in 43 healthy pregnant women, in 18 pregnant women with EPH gestosis and in 26 healthy non pregnant women. In pregnant women with EPH gestosis, mean arterial blood pressure (MAP) (114.6 +/- 1.3 mm Hg) and mean leptinaemia (21.9 +/- 8.5 ng/ml) were significantly higher than in healthy pregnant women (89.1 +/- 0.9 mm Hg and 15.0 +/- 1.3 ng/ml respectively) and in non pregnant women (MAP--91.56 +/- 1.4 mm HG i LP--10.9 +/- 1.7 ng/ml). In healthy pregnant women, in women with EPH gestosis and in healthy nonpregnant women plasma NPY concentrations were of similar magnitude (42.3 +/- 4.1 vs 43.7 +/- 8.5 vs 50.7 +/- 6.1 pg/ml respectively). In pregnant women with EPH gestosis a significant positive correlation was found between diastolic blood pressure or MAP and plasma NPY concentration. Leptinaemia was significantly correlated with systolic, diastolic and MAP respectively only when results obtained in both groups of pregnant women were analyzed together. CONCLUSIONS: 1) leptin seems to be involved in the regulation of blood pressure both in healthy and preeclamptic pregnant women, 2) participation of NPY in the pathogenesis of hypertension in preeclamptic women is likely. PMID- 10740418 TI - [QT interval dispersion and the type of obesity in women]. AB - The risk of sudden cardiac death is higher in patients with obesity, particularly in the upper body obesity. The most common cause of cardiac events are serious ventricular arrhythmias. Delayed cardiac repolarization leading to the prolongation of the QT interval is a well characterised precursor of arrhythmias. The QT interval dispersion reflects inhomogeneity of repolarization. The aim of this study was to assess QT interval dispersion (QTd) in obese women and to establish the relationship between obesity and QTd. 62 patients with obesity (group 1) and 15 apparently healthy women (group 2) were included in our study. Obese subjects were distinguished in accordance to the waist to hip ratio (WHR) into three subgroups: 1a--obese women with upper body obesity; 1b--obese women with WHR: 0.75-0.85; 1c--obese women with lower body obesity. A standard 12-lead ECG was performed in each subjects and QTd, QTdc (QT correlated interval dispersion), QTdR (QTd ratio) were calculated. The left ventricular mass (LVM) and left ventricular mass index (LVMI) were obtained from echocardiograms. We found QTd, QTdc, QTdR and LVM, LVMI to be significantly higher in obese women as well as positive correlation between BMI and both LVM, LVMI. QTdR was significantly higher in subgroup 1a compared with subgroup 1c. CONCLUSIONS: 1) QT interval dispersion was increased in obese women compared with healthy subjects, 2) supposedly increased QTd was associated with the type of obesity. PMID- 10740419 TI - [Electrical instability of the heart in patients with left ventricular hypertrophy in the course of hypertension]. AB - Left ventricular hypertrophy (LVH) in course of the systemic hypertension (HT) is found as independent risk factor for ventricular arrhythmia and sudden cardiac death. OBJECTS: The aim of the study was to evaluate the relationship between occurrence of LVH and electrical instability. Study group included 64 patients (pts) with mild to moderate HT, without clinical signs of coronary artery disease. Pts were divided into two group: with and without LVH (LVH was diagnosed by echocardiography (ECHO) when left ventricular mass index (LVMI) was > 106 g/m2 for women, and > 111 g/m2 for men). By 12-lead ECG QTc, dispersion QT (QTd) were calculated. From SAECG the QRS duration (tQRS), the root mean square of the last 40 ms of the filtered QRS complex (RMS40) and the duration of the terminal low amplitude QRS signal < 40 microV (LAS 40) were calculated. Ventricular arrhythmias were recorded during 24 hour Holter monitoring and classified into the Lown classification. RESULTS: No difference in QTd between two groups was observed. Nobody has nonsustained ventricular tachycardia in 24-hour Holter monitoring. In group with LVH tQRS was longer and RMS higher in SAECG than in group without LVH. CONCLUSION: LVH in hypertensive pts influences the increase of electrical instability that is evaluated by SAECG. PMID- 10740420 TI - [The diagnostic value of resting electrocardiography in stable coronary artery disease]. AB - The relation of resting electrocardiographic (ECG) patterns to angiographic features was assessed in 566 patients with chest pain regarded as definite or probable stable angina pectoris. The indications for catheterization in each patient were determined at the discretion of the attending physician. All patients underwent diagnostic coronary angiography (clinically important coronary artery disease was defined as > or = 70 per cent narrowing of the diameter of at least one major vessel or > or = 50 per cent of the left main coronary artery) and standard 12 lead electrocardiography which was interpreted by 2 cardiologists independently in coordinating centre. The signs of impaired coronary blood flow were assessed by abnormalities of repolarization (among others S-T segment, the T wave), depolarization and presence of disturbances of cardiac rythm. The resting routine electrocardiogram was assigned to one of three categories: normal, nonspecific abnormalities or typical for coronary insufficiency. The typical pattern for ischemia was present in 104 patients (18%), nonspecific abnormalities were present in 185 patients (33%) and electrocardiogram was normal in 277 patients (49%). Sensitivity and specificity of the typical for coronary insufficiency resting ECG was calculated: 23% and 87% respectively for the entire group, 33% and 81% in women, 20% and 93% in men. In the group with normal resting electrocardiographic pattern 55% of patients have significant stenosis in at least one major coronary artery. PMID- 10740421 TI - [Should sinus rhythm be restored in patients with chronic atrial fibrillation? Preliminary results from the Polish "Hot Cafe" study]. AB - Atrial fibrillation (AF) is the most common arrhythmia occurring in clinical practice. It is the most frequent cause of hospitalization in cardiac rhythm disturbance. Despite comprehensive progress in the research into electrophysiological mechanisms leading to this loss of normal rhythm and new procedures dealing with it, the main problem being the conversion to and maintaining the normal sinus rhythm (SR) has not been solved. The polish study "Hot Cafe" is trying to evaluate the advantages and risks of the two procedures widely used in clinical practice in patients (pts) with chronic AF, i.e. conversion and maintaining SR vs leaving pts with the arrhythmia. Pts with non valvular chronic AF fulfilling the criteria for including them into the sample are randomly assigned to two procedures: conversion to SR by means of direct current cardioversion and maintaining it or leaving pts with AF. Pts left with AF are treated by rate control and antithrombotic treatment. The project is of prospective kind and it will be carried out by many medical institutions. It is planned to include 200 pts. The observation period will last at least 12 months. Preliminary results after inclusion of the first 121 pts are shown. PMID- 10740422 TI - [Fabry's disease--late diagnosis in men with chronic nephropathy and skin changes]. AB - We report a case of Fabry's disease, diagnosed in a 39-year-old patient treated for 4 years because of glomerulonephritis. The disease manifested itself by the presence of typical petechiae-like skin lesions in the bathing trunk area (angiokeratoma), eye changes, paresthesia, and--in additional investigations- mild proteinuria, lowered creatinine clearance, along with changes in the central nervous system. A biopsy of the kidney revealed the presence of foamy cells in all glomeruli, and in electron microscopy multilamellar bodies (zebra bodies). The diagnose of the disease was confirmed by a marked decrease in leucocyte alpha galactosidase activity. An early diagnosis of non-inflammatory character of Fabry's disease allows to avoid an unnecessary immunosuppressive treatment. PMID- 10740423 TI - [Hyponatremia in congestive heart failure]. PMID- 10740424 TI - [Periodontal state and cardiovascular diseases]. PMID- 10740425 TI - [Spiral computed tomography in diagnosis of pulmonary embolism]. PMID- 10740426 TI - [The role of adhesion molecule ICAM-1 in pathogenesis of Lesniowski's and Crohn's disease and ulcerative colitis]. PMID- 10740427 TI - [Genetic alterations in gastric cancer]. PMID- 10740428 TI - [Results of experimental and clinical investigations on icodextrin --review of studies presented in Charlotte (02/28-03/02/1999)]. PMID- 10740429 TI - [Urinary incontinence, a guideline--and so what?]. PMID- 10740430 TI - [Venous ulcers. An age-related disease, too]. PMID- 10740431 TI - [Meckel's diverticulum. From embryology to therapy]. AB - Meckel's diverticulum (MD), an remnant of the omphalomesenteric duct, is the most common congenital abnormality of the small bowel. The aim of this paper is to give a review of MD, from embryology to diagnosis, complications and treatment. PMID- 10740432 TI - [Reduction mammaplasty]. AB - The aim of this retrospective study was to determine the pre- and postoperative symptoms among 170 women, who had modified Robbins reduction mammaplasties. The major part of the breast tissue was removed superiorly and laterally. The operations were carried out between January 1994 and April 1996. The journals were reviewed, and a questionnaire was sent to the patients. Ninety-one percent were returned. Many of the preoperative symptoms were significantly reduced in frequency post-operatively. Prior to operation, some of the patients had had symptoms that influenced their ability to manage their jobs and/or education, and 22% of the women took analgesics daily or several times weekly before surgery. After the operations the need for medicine was significantly reduced. A modified Robbins reduction mammaplasty significantly relieved many preoperative symptoms. The need for analgetics medicine was reduced, and the cosmetic results were good. PMID- 10740433 TI - [Organochlorine compounds and breast cancer--is there a connection between environmental pollution and breast cancer?]. AB - Some organochlorine compounds may have weak oestrogenic effects and are therefore suspected of increasing the risk of breast cancer. The risk of breast cancer was assessed prospectively in relation to the serum concentration of several organochlorines. In all 240 women, who developed breast cancer between 1976 and 1993 were together with 477 breast cancer-free controls enrolled in a cohort nested case-control study. The serum dieldrin concentration was associated with a significantly increased dose-related risk of breast cancer (Odds Ratio 2.05; 95% confidence interval 1.17-3.57; p for trend 0.01). There was no overall association between risk of breast cancer and DDT or polychlorinated biphenyls. The results support the hypothesis that exposure to oestrogenic organochlorines may increase the risk of breast cancer. PMID- 10740434 TI - [Cochlear implantation in adults. Results and significance for communication and quality of life]. AB - The aim of this study was to assess the benefits and complications from cochlear implantation and to evaluate the average implant usage per day. Ten profoundly deaf adults were implanted during the period April 1994 to September 1997. The patients answered questionnaires one year or more after receiving their cochlear implants. The average implant usage per day was 16 hours. The patients would again opt for a cochlear implant and would recommend a cochlear implant for a deaf friend. A paired comparison showed that the implants led to statistically significant improvement in a number of factors; self perceived communication skills, frequency of conversation with others, telephone usage, self-confidence, and the hearing impairment's impact on family life. No serious surgical complications were found. PMID- 10740435 TI - [Prognostic factors in soft tissue sarcomas. Experiences from the Sarcoma Center in Aarhus]. AB - In the present study, the outcome, patterns of local recurrence and survival, as well as prognostic factors, were evaluated in patients surgically treated for soft tissue sarcomas. Between January 1979 and July 1993, 316 consecutive patients were referred to the Sarcoma Centre in Aarhus with localised malignant soft tissue sarcoma of the extremities or trunk. There were 161 men (51%) and 155 women (49%), the median age was 56 years (1-94). Histologically 52 patients (16%) had a grade I, 60 patients (19%) a grade 2 and 204 patients (65%) a grade 3A or 3B tumour. The five-year local recurrence rate was 18% and the five-year survival rate was 75%. Multivariate analysis indicated the following variables as independent unfavourable factors for local recurrence: extracompartmental location, histological high-grade (i.e. histologically highly malignant) local excision, no adjuvant radiotherapy and intralesional/marginal excision. Independent unfavourable factors for survival were advanced age, extracompartmental location, histological high-grade, lower extremity location and large tumour size. Based on these variables, a prognostic model was made. PMID- 10740436 TI - [Ciprofloxacin and pivampicillin in acute epididymitis in men above the age of 40 years]. AB - Ciprofloxacin 500 mg orally twice daily was compared to pivampicillin 700 mg orally twice daily for 10 days in 172 men (41-85 years) with acute epididymitis. Failure of treatment occurred in 48 patients, in 15/76 (19.7%) receiving ciprofloxacin versus in 33/82 (40.2%) receiving pivampicillin (p = 0.006). Ciprofloxacin 500 mg orally twice daily is more effective than pivampicillin 700 mg orally twice daily and has fewer adverse events. PMID- 10740437 TI - [Suture foreign body reaction as a mammographic pitfall]. AB - A mammographic pitfall is presented. After lumpectomy owing to cancer in the breast a woman developed an immediate allergic reaction presumably caused by the subcutaneous sutures (vicryl). After adjuvant irradiation the breast became swollen and mammography gave suspicion of a recurrent breast cancer. Surgical excision was performed and examination of the biopsy specimen showed a foreign body reaction to the suture material with fibrosis and eosinophilia. PMID- 10740438 TI - [Picture of the month. Intracranial abscess]. PMID- 10740439 TI - [New international guidelines for treatment of uncomplicated, acute bacterial cystitis and pyelonephritis in women]. PMID- 10740440 TI - Trends in pregnancies and pregnancy rates by outcome: estimates for the United States, 1976-96. AB - OBJECTIVES: This report presents national estimates of pregnancies and pregnancy rates according to women's age, race, and Hispanic origin, and by marital status, race, and Hispanic origin. Data are presented for 1976-96. Data from the National Survey of Family Growth (NSFG) are used to show information on sexual activity, contraceptive practices, and infertility, as well as women's reports of pregnancy intentions. METHODS: Tables of pregnancy rates and the factors affecting pregnancy rates are presented and interpreted. Birth data are from the birth registration system for all births registered in the United States and reported by State health departments to NCHS; abortion data are from The Alan Guttmacher Institute (AGI) and the National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention (CDC); and fetal loss data are from pregnancy history information collected in the NSFG. RESULTS: In 1996 an estimated 6.24 million pregnancies resulted in 3.89 million live births, 1.37 million induced abortions, and 0.98 million fetal losses. The pregnancy rate in 1996 was 104.7 pregnancies per 1,000 women aged 15-44 years, 9 percent lower than in 1990 (115.6), and the lowest recorded since 1976 (102.7). Since 1990 rates have dropped 8 percent for live births, 16 percent for induced abortions, and 4 percent for fetal losses. The teenage pregnancy rate has declined considerably in the 1990's, falling 15 percent from its 1991 high of 116.5 per 1,000 women aged 15-19 years to 98.7 in 1996. Among the factors accounting for this decline are decreased sexual activity, increases in condom use, and the adoption of the injectable and implant contraceptives. PMID- 10740441 TI - Should you take an ACE inhibitor? PMID- 10740442 TI - Peripheral vascular disease: walking toward a cure. PMID- 10740443 TI - The right prescription for easing back pain. PMID- 10740444 TI - Dyspepsia: soothing the sour stomach. PMID- 10740445 TI - New insomnia drug. Are there any medications to treat insomnia that won't leave me feeling groggy the next day? PMID- 10740446 TI - Vitamin E recommendations. Is there a difference between natural and synthetic vitamin E? PMID- 10740447 TI - Osteoporosis screening. What is the best way to measure bone density? PMID- 10740448 TI - Creating an environmentally friendly dental practice. PMID- 10740449 TI - Disclosure and the consent form. PMID- 10740450 TI - Dental management service organizations. PMID- 10740451 TI - Placing esthetic bonded direct posts and cores. PMID- 10740452 TI - Lack of integration of smooth titanium surfaces: a working hypothesis based on strains generated in the surrounding bone. AB - It has been observed that the polished neck of dental implants does not osseointegrate as do textured surfaces. Similar findings were reported in the orthopedic literature on artificial hip endoprostheses. In Dentistry, lack of osseointegration was attributed to increased pressure on the osseous bed during implant placement, establishment of a physiological "biologic width", stress shielding and lack of adequate biomechanical coupling between the load-bearing implant surface and the surrounding bone. Among the many variables that may affect osseointegration, this analysis proposes to include stress transfer as a significant one. Therefore the present report discusses the relationship between the stresses applied and bone homeostasis. Any viable osseous structure (including the tissue that surrounds the polished implant neck) is subjected to periodic phases of resorption and formation. Clinical and experimental data have shown the detrimental effects of lack of function in that bone mass decreases with time. Due to inadequate mechanical stimuli, bone that is resorbed during normal turnover is redeposited in lesser amounts than previously, a process observed clinically as resorption. The stress ranges which cause bone to resorb, maintain or increase its mass and the level that eventually causes bone to fracture have been delimited in the literature. Applying these values to the situation to dental implants, it follows that if it is to be stable, crestal bone must be subjected to suitable levels of mechanical stimulation. We suggest that smooth surfaces will not provide adequate biomechanical coupling with the bone surrounding the implant neck in that the stress range induced by a polished surface is limited. We propose that the surface texture of threaded, plasma coated or sandblasted implants generates a heterogeneous stress field around an implant in function. By design, such a stress field includes force levels which are conducive to bone formation. Hence, during the formation phase of bone turnover, osteoblast lineages are much more likely to be stimulated by biomechanical signals of appropriate magnitude. PMID- 10740453 TI - Adherence of periodontopathic bacteria to bioabsorbable and non-absorbable barrier membranes in vitro. AB - Guided tissue regeneration (GTR) techniques are increasingly used for the treatment of periodontal defects, or in conjunction with dental implant procedures. As adhesion of bacteria to barrier membranes used in these techniques may lead to failure, a prerequisite for treatment success is an infection-free healing process. The present study examined the adhesion of 3 periodontal pathogenic bacteria: Actinobacillus actinomycetemcomitans, Treponema denticola and Porphyromonas gingivalis, to 3 barrier membranes: Collagen, (Biomend) PTFE, (TefGen-FD) and e-PTFE, (Gore-Tex). The membranes were incubated with 3[H] thymidine labeled bacteria, and the number of adherent bacteria was calculated using a scintillation counter. The effect of albumin coating on bacterial adherence to the membranes was also studied. Bacterial adherence to the membranes was further examined by scanning electron microscopy (SEM). The results show that the adherence of all bacterial strains to collagen membranes was significantly higher than to the other membranes tested. Precoating of the membranes with albumin did not change the bacterial adherence significantly. These findings are of importance in evaluating the ability of periodontal bacteria to colonize and infect different types of barrier membranes. PMID- 10740454 TI - A prospective clinical evaluation of different single-tooth restoration designs on osseointegrated implants. A 3-year follow-up of Branemark implants. AB - Since single-tooth implant restorations were introduced 12 years ago (Jemt 1986), there has been continuous development both in the technical design and the aesthetic outcome of the treatment. In order to ensure high quality in single tooth implant treatments a clinical follow-up study was carried out on patients treated with modifications to the original regimen. In this study 69 consecutive patients were provided with 80 single-tooth implant restorations. The patients were followed for 3 years. There was continuous development of the prosthetic design during the time of the study, allowing us to analyse possible prognostic differences for the different prosthetic treatments. This study confirms earlier reports which describe the single-tooth implant treatment as a safe method with few surgical complications and minimal marginal bone loss. Only 1 implant was lost during the follow-up period and the average marginal bone loss was 0.48 mm over the 3-year follow-up period. Crowns veneered with acrylic and with gold casted directly to the abutments, screwed onto the implants, led to recurring prosthetic complications and gave an appearance of rapid ageing. The first generation of crowns made following the Cera-One design, sometimes produced a gap between the crown and the abutment associated with significant marginal bone loss during the first year. Few surgical or prosthetic complications were noted with cemented all-ceramic constructions, although the number of these crowns in this study was limited. PMID- 10740455 TI - Characteristic features of trabecular bone in edentulous maxillae. AB - Following tooth loss, the maxillary alveolar ridge is affected by extensive resorption and its cancellous bone substance undergoes intense remodeling processes. This is particularly important for endosseous implant surgery as the primary stability and thus the prognosis of endosseous implants depends on the cancellous bone density and structure of the alveolar ridge. To analyze the structure of alveolar trabecular bone, 156 sections were obtained from 52 edentulous maxillae (29 female, 23 male; mean age: 72.5 years) from the lateral incisor, first premolar, and first molar regions. The structural histomorphometric analysis was performed on cancellous bone of the section surfaces using semiautomatic image analysis. The following parameters were measured: trabecular bone volume, trabecular number, trabecular thickness, trabecular plate separation and trabecular interconnection. All examined parameters showed an extreme range of variation. A difference of more than 45% between the highest (= 51.93%) and the lowest (= 6.73%) trabecular bone volumes was found. Furthermore, the measurements showed that trabecular bone volume, thickness and number were distinctly lower in the molar region than in the incisal and premolar regions. Significant sex-specific differences were found in all investigated regions, female maxillae showing a smaller amount and a lower connectivity of cancellous bone than male maxillae. PMID- 10740456 TI - Augmentation of calvarial tissue using non-permeable silicone domes and bovine bone mineral. An experimental study in the rat. AB - The aim of the present study was to evaluate the effect of bovine bone mineral placement as an adjunct to a guided bone regeneration (GBR)-barrier in experimental bone augmentation. Some 18 Sprague-Dawley rats underwent flap surgery in which non-permeable silicone domes were placed on the calvaria. A test group of 9 animals received domes filled with cancellous deproteinized bovine bone mineral (Bio-Oss). The domes of 9 control animals were left empty. The experiment was terminated after 8 weeks of healing. Inspection after histologic preparation found no signs of any foreign body reaction to the implanted bovine bone mineral, which seemed to be well integrated with the augmented tissue. The control specimens healed uniformly with new bone inside the domes at the base of the calvaria. The tissue inside the domes of the test specimens had a completely different architecture with new bone both at the base of the calvaria and at a distance from the base, surrounded by and in close contact with non-mineralized tissue and remaining bovine bone mineral. Some ingrowth of soft connective tissue, most likely from the sagittal skull suture could be seen in most test and control specimens. Histomorphometric analysis revealed that the total amount of augmented tissue was significantly greater in the test group (x = 1.93 +/- 0.21 mm2) than in the control group (x = 0.99 +/- 0.22 mm2), P < 0.001). The control domes contained 0.73 +/- 0.28 mm2 augmented bone, while the corresponding value for the test specimens was 0.35 +/- 0.13 mm2, significantly less than the controls (P < 0.01). The test domes contained 0.83 +/- 0.15 mm2 newly-formed non mineralized calvarial tissue, significantly more than the controls (x = 0.26 +/- 0.16 mm2), P < 0.001). Remaining particles of bovine bone mineral constituted 0.76 +/- 0.14 mm2, or 39%, of the total amount of augmented tissue in the test group. A considerable shrinkage to about 69% of the height of the initial fill of bovine bone mineral at surgery appeared in the test domes after 8 weeks of healing. The reason for this was probably more due to a denser packing of the bovine bone mineral during healing than to resorptive activity. It was concluded that implantation of bovine bone mineral combined with GBR technique significantly enhances augmentation of calvarial tissue. Early mineralization, however, seems to be retarded compared to non-filled control specimens. Further studies should be made to determine the long-term maturation of augmented tissue including resorption pattern of the bovine bone mineral. PMID- 10740457 TI - Long-term stability of jaw bone tuberosities formed by "guided tissue regeneration". AB - The aim of the present study was to evaluate whether bone tuberosities produced by GTR on the lateral surface of the mandibular ramus in rats are stable on a long-term basis. Thirty male 6-month-old albino rats of the Wistar strain were used in the study. Tissue flaps were elevated on the lateral aspect of the mandibular ramus. The periosteum was preserved (P+) on one side of the jaw while the bone was denuded (P-) on the other. A rigid, non-porous oval-shaped teflon capsule was placed on both sides with its opening facing the ramus. Six months following surgery, 10 rats were sacrificed and prepared for histology while the remaining 20 rats were subjected to a second operation during which the capsules were removed. Standardized radiographs, taken immediately before and after removal of the capsule and after 3, 6, 9 and 12 months, were subjected to planimetric measurements and subtraction radiography. Ten animals were sacrificed and prepared for histological analysis after 6 months following removal of the capsules and the remaining 10 animals after 12 months. Histology revealed that at 6 months after the placement of the capsules, 17 were completely filled with new bone. The remaining 3 capsules which were displaced exhibited only partial bone fill. The radiographic analysis revealed that after 6 months 98.6 +/- 7.6% (mean +/- SD) in average of the cross-sectional area created by the capsules was filled with new bone. Within 3 months after removal of the capsules a slight resorption of the new bone had occurred, thereby reducing the area of the bone tuberosities by 4 to 8%. No further resorption of the bone tuberosities took place from 3 to 12 months. These observations indicating that new bone produced by GTR is stable on a long-term basis, may question the general belief that non-functional bone will resorb over time. PMID- 10740458 TI - The combined use of bioresorbable membranes and xenografts or autografts in the treatment of bone defects around implants. A study in beagle dogs. AB - The aim of the present investigation was to test the effect of a bioresorbable membrane supported by xenografts or autografts in regenerating bone into peri implant defects. In 3 dogs, the mandibular premolars P2, P3, P4 and M1 were extracted bilaterally. After 4 months of healing, 3 standardized bone defects were prepared on each side of the mandible and 1 implant per defect was placed. The 6 sites in each dog were distributed into 4 different treatment groups: 2 sites received a Bio-Gide membrane alone (BG); 2 sites received a Bio-Gide membrane supported by Bio-Oss (BG + BO); 1 site received the Bio-Gide membrane supported by autogenic bone harvested from the prepared defects (BG + Aut); 1 site received neither membrane nor bone graft and served as control (C). The soft tissue flaps were adapted and sutured for primary healing. No adverse events occurred during the experimental period. After 16 weeks, the dogs were sacrificed and histomorphometric examinations on non-decalcified ground sections were carried out. The vertical bone growth amounted to 45% (SD +/- 13%) of the defect height in the BG group, to 78% (SD +/- 29%) in the BG + BO group, to 69% (SD +/- 9%) in the BG + Aut group, and to 22% (SD +/- 10%) in C group. The horizontal bone growth measured 78% (SD +/- 16%) in the BG group, 81% (SD +/- 21%) in the BG + BO group, 82% (SD +/- 12%) in the BG + Aut group, and 46% (SD +/- 21%) in the C group. The vertical height of bone growth in contact with the implant measured 17% (SD +/- 12%) in the BG group, 20% (SD +/- 12%) in the BG + BO group, 17% (SD +/- 7%) in the BG + Aut group, and 12% (SD +/- 8%) in the C group. The surface fraction of the graft in direct bone contact measured 89% (SD +/- 9%) in the BG + BO group and 93% (SD +/- 3%) in the BG + Aut group. It is concluded that the bioresorbable membrane tested enhances bone regeneration, in particular in conjunction with the use of a supporting graft material. In addition, deproteinized bovine bone mineral and autogenic bone grafts appeared to be equally well integrated into regenerating bone. Finally, no additional effects in the bone growth was observed with the autogenous bone in comparison with the hydroxyapatite. PMID- 10740459 TI - Histomorphometrical analysis of bone formed in human maxillary sinus floor elevations grafted with OP-1 device, demineralized bone matrix or autogenous bone. Comparison with non-grafted sites in a series of case reports. AB - Bone morphogenetic proteins have proven to be effective bone inductors in animals and are therefore promising as inductors of bone formation in humans. In the present study we investigated the tissue formed after grafting osteogenic protein 1 on a collagen carrier (OP-1-device) in the human sinus floor elevation procedure. Three patients were grafted with OP-1 device. For comparison 3 groups of 3 patients were included in the study receiving respectively, autogenous bone, human freeze-dried demineralized bone matrix (DBM) or no graft. This last group had a sufficient alveolar bone height for dental implantation. Six months after grafting, at the time of implantation, biopsies were taken from the grafted area and/or the future dental positions. Undecalcified sections were used for histological and histomorphometrical analysis. All grafted sinuses showed an increased osteoid percentage when compared to non-grafted sinuses. Autogenous bone grafts all showed lamellar bone formation. In the DBM grafts mostly woven bone had been formed, predominantly by what appeared to be osteo-conduction. The OP-1 device gave rise to bone formation in 2 of the 3 patients. After 6 months implants could only be placed in 1 out of the 3 patients treated with OP-1 device. This patient showed mature lamellar bone formation, comparable to autogenous bone grafts. In the second patient all bone found was woven and the presence of a high osteoid percentage and large osteocyte lacunae indicated that this was recently-formed bone. Remnants of the collagen carrier were rare and new bone was never found against them, suggesting that this bone was formed by osteo induction. In the third patient no new bone had been formed. The device had been encapsulated with fibrous tissue and inflammatory reaction was present. We conclude that in the human sinus floor elevation OP-1 has potential bone inductive capacity, but that results in the 3 patients tested with the current OP 1 device are inconsistent. PMID- 10740460 TI - Sinus floor augmentation with simultaneous placement of dental implants using a combination of deproteinized bone xenografts and recombinant human osteogenic protein-1. A histometric study in miniature pigs. AB - Maxillary sinus floor augmentation with autogenous bone has become a widely accepted procedure in implant dentistry. The use of osteoconductive bone substitutes in this indication is controversial, since their use can lead to a prolonged healing time, inhomogenous ossification, foreign body reaction, migration of particles and low bone-implant contact (BIC). The purpose of this study was to examine whether the combination of an osteoinductive protein (recombinant human osteogenic protein-1 (rhOP-1 = bone morphogenetic protein-7) with natural bovine bone mineral (BioOss) would improve ossification and the bone implant contact (BIC) in a sinus floor augmentation with simultaneous placement of implants. In this study, the maxillary sinus floors in 5 miniature pigs were augmented with 3 ml BioOss containing 420 micrograms rhOP-1 on the test side and 3 ml BioOss alone on the control side. At the time of augmentation a titanium implant (ITI) was inserted from a laterocaudal direction. After 6 months of healing the sites of augmentation were removed and examined in non-decalcified sections by microradiography, fluorescence microscopy of sequentially labelled specimens and by histometry. On both sides, significant amounts of newly-formed bone were observed. However, on the test sites, the percentage of BIC in the augmented area was 80.0% versus 38.6% on control sites. It can be concluded that the application of bone morphogenetic proteins caused a more rapid and enhanced osseointegration of simultaneously placed implants when compared to the bone substitute alone. Therefore recombinant human osteogenic protein-1 delivered by natural bone mineral has the potential to become a clinical alternative for autogenous bone grafts in sinus floor augmentation. PMID- 10740461 TI - Four-handed dentistry revisited. PMID- 10740462 TI - Toxic waste and the dental assistant. PMID- 10740463 TI - Education, leadership, recognition: the ADAA at 75. PMID- 10740464 TI - Memoirs of a "lady in attendance". PMID- 10740465 TI - The choice of gold standard for evaluating tests for caries diagnosis. PMID- 10740466 TI - Accurate linear measurements in the anterior maxilla using orthoradially reformatted spiral computed tomography. AB - OBJECTIVES: To validate measurement accuracy in proximity to the incisive canal from two-dimensional (2D) reformatted spiral computed tomography (CT). MATERIALS AND METHODS: Eight cadaver heads were examined with spiral CT with 1 mm thick axial slices and 1 mm.s-1 table feed. The data was transferred to a networked computer workstation to generate 2D orthoradially reformatted images. The length of the incisive canal and width of the alveolar crest were measured independently by two oral and maxillofacial radiologists. The soft tissues in the region of the incisive foramen were removed and physical measurements made using an electromagnetic digitizer. RESULTS: There was no statistically significant difference between measurements on orthoradial 2DCT images and physical measurements (P > 0.05). CONCLUSIONS: Accurate measurements for dental implant placement in proximity to the incisive canal can be made from reformatted spiral CT images. PMID- 10740467 TI - The reproducibility of the mandibular cortical index. AB - OBJECTIVES: To assess the reproducibility and diagnostic validity of Mandibular Cortical Index (MCI) when used by minimally trained observers. METHODS: Four 'experts' and 45 final-year dental students classified the appearance of the lower border of the mandibular cortex in 30 panoramic radiographs using the MCI. Experts viewed the original radiographs. The students received instruction in the MCI and viewed slides of the radiographs in a lecture theatre. RESULTS: For intra observer agreement, the experts had significantly higher overall values of weighted kappa, indicating substantial agreement in MCI assessment, whereas the students showed moderate agreement. For inter-observer agreement, there was fair agreement between the experts and poor agreement between the students. Using the experts' MCI assessment as the 'gold standard', the mean sensitivity of the students in diagnosis of C3 was 0.71 (maximum 0.95, minimum 0.25) and mean specificity was 0.56 (maximum 0.9, minimum 0.20). CONCLUSIONS: The MCI has important limitations in terms of intra- and inter-observer agreement. Minimal training in its use, such as might be given in a lecture format to dentists, was ineffective and associated with poor inter-observer agreement and limited diagnostic validity in identifying signs of osteoporosis. More lengthy training and experience in using the MCI would be needed for it to be effective as a diagnostic tool in general dental practice. PMID- 10740468 TI - Evaluation of maxillofacial fibrosarcoma using computer graphics and spiral computed tomography. AB - This paper describes the appearance of three cases of maxillofacial fibrosarcoma in multiplanar (MPR) and three-dimensional reconstructed computed tomography (3DCT). The images were analysed by two oral and maxillofacial radiologists and one neuroradiologist. All of the cases clearly demonstrated the usefulness of the interactive MPR and 3DCT in the diagnosis and evaluation of the extent of these maxillofacial tumors. PMID- 10740469 TI - Two- and three-dimensional imaging modalities for the detection of caries. A comparison between film, digital radiography and tuned aperture computed tomography (TACT). AB - OBJECTIVES: To compare the diagnostic performance provided by two- (film and digital radiography) and three-dimensional imaging modalities (TACT slices and TACT pseudoholograms) in the detection of primary caries. METHODS: Forty-two extracted human posterior teeth were mounted and imaged with conventional film and direct digital radiography. Free-hand positioning of a dental X-ray source was used for all exposures. From the digital images, iteratively restored TACT slices and TACT pseudoholograms were generated. Film images were viewed on a viewbox. Digital format images were viewed on a high-resolution monitor. Eight observers used a five-point scale to score the presence or absence of occlusal and proximal caries using the four image modalities. Observers' assessments were compared with the histological examination of tooth sections. Possible differences in ROC curve areas among image modalities, observers, and surfaces were assessed by ANOVA. Intra- and interobserver reliability as indicated by intraclass correlation was also calculated. RESULTS: There were no statistically significant differences between the diagnostic performances of film, digital radiography, TACT slices and TACT pseudoholograms in the detection of caries (P = 0.310). Intraclass correlation indicated the highest concordance both within and between observers when film was used for the evaluation. CONCLUSIONS: Under the experimental conditions of this study, three-dimensional TACT images did not improve caries detection over film or digital radiography. Further research should investigate the effects of imaging variables on TACT's diagnostic efficacy. PMID- 10740470 TI - An audit of occipitomental radiographs. AB - OBJECTIVES: To audit the justification for and image quality of occipitomental (OM) radiographs obtained at a dental teaching hospital. METHODS: One hundred OM radiographs taken at Glasgow Dental Hospital and their associated request forms were analysed by two examiners retrospectively. Information recorded from the request form included the age and gender of the patient, requesting department, grade of requesting clinician, reason for the request and exposure factors. The radiographs were assessed for radiographic density, patient positioning, presence of abnormality and whether the maxillary sinuses were normal or not. It was then decided by reference to the clinical records if there had been an indication for taking the radiograph. RESULTS: The majority of the requests were from junior staff, most frequently for pain or sinusitis. Almost all were from the Departments of Oral Surgery, Oral Medicine or Dental Accident and Emergency (A&E). The technical quality of the radiographs was variable, especially with respect to superimposition of the petrous temporal bone. Over half of the cases had normal sinuses, no abnormality and no indication for the radiograph. CONCLUSIONS: Many requests were inappropriate and the resultant radiographs had a low diagnostic yield. The technical quality of the radiographs was variable. Guidelines for clinicians for the prescription of OM radiographs were produced and the protocol for radiographic technique changed. PMID- 10740471 TI - Frequency of portrayal of foramen transversarium of the second cervical vertebra on rotational panoramic radiographs. AB - OBJECTIVES: To determine the prevalence of portrayal of the foramen transversarium of the second cervical vertebra on rotational panoramic radiographs. METHODS: A stratified random sample of 1600 diagnostic-quality rotational panoramic radiographs were retrospectively reviewed. Approximately 100 radiographs each for males and females were reviewed for each of the decades of life up to age 69 and for subjects 70 or older. RESULTS: The foramen was portrayed 29.3% of all radiographs, 35% of females and 22.6% of males. However, when considering only those radiographs in which cervical vertebrae were visible, the foramen was more frequently portrayed in males than in females. Portrayal of the foramen did not differ substantially among age groups. CONCLUSIONS: The foramen transversarium is found sufficiently often on rotational panoramic radiographs that this structure should be identifiable by clinicians who use these images. PMID- 10740472 TI - Radiological and clinical features of aneurysmal bone cyst of the jaws. AB - PURPOSE: To evaluate the clinical and radiological features of aneurysmal bone cyst (ABC) of the jaws. MATERIALS AND METHODS: A total of 64 cases (60 from the English-language literature and four new cases) were studied and critically evaluated with emphasis on the radiological features. RESULTS: Age at time of initial diagnosis ranged from 4-78 years, with a mean age of 21.5 years and median age of 17 years. There were no differences in gender distribution. The ratio between the mandible and maxilla was 2.4:1. Ninety-two per cent of the lesions were located in the posterior region of the jaws. Lesions were radiolucent in 87% of cases, radiopaque in 2% and mixed in 11%. Fifty-three per cent were multilocular, 43% unilocular and 3% not loculated. The border of the lesions was defined but not corticated in 39%, well defined in 33% and diffuse in 28%. CONCLUSIONS: ABC has a variable radiological appearance and should be considered in the differential diagnosis of any unilocular or multilocular radiolucent lesion of the jaws as well as any mixed radiolucent-radiopaque lesion. PMID- 10740473 TI - Radiomorphometric indices of the mandible in a British female population. AB - OBJECTIVES: To examine the radiomorphometric indices of the mandible on panoramic radiographs in a population of British female patients, to identify their normal ranges and to investigate their relationships with age, detentition and social class. METHODS: Five indices, cortical width at the gonion (GI) and below the mental foramen (MI), the panoramic mandibular index (PMI), the mandibular cortical index (MCI) and one new index (measurement of cortical width at the antegonion; AI), were measured bilaterally on 500 panoramic radiographs of females by one trained observer. The measurements were analysed for ease of application, repeatability, relationships with age, dentition and social class and interrelationships between the variables. RESULTS: All quantitative indices (GI, MI, PMI, AI), showed a significant, negative correlation with age. MCI showed an age-related distribution. Mandibular dentition exerted a significant influence on some indices, but social class had no influence. Intra-observer repeatability of MI, PMI and AI was fair (precision < 20%) but that for GI was poor. Intra-observer agreement in MCI assessments was excellent. CONCLUSIONS: The age-related changes in mandibular radiomorphometric indices and their variation within each age band lend support to their potential use in identification of skeletal osteopenia. However, problems with repeatability and measurement precision identified in the pilot study, notably with GI, may be a considerable obstacle to their use in general practice. PMID- 10740474 TI - Comparison of microscopy and radiography as gold standards in radiographic caries diagnosis. AB - OBJECTIVES: To compare the effect of the choice of gold standard on the diagnostic outcome of approximal caries detection in original and compressed digital radiographs. METHODS: 116 extracted teeth radiographed with a storage phosphor system constituted the original images. These images were compressed at 1:20 and 1:33 with the JPEG irreversible compression standard. Five radiologists scored the three sets of images for the presence of approximal caries on a five rank confidence scale. The radiographic scores were validated by stereomicroscopy (the true gold standard). The individual ROC areas for the five observers were used to select the worst (obsworst) and the best (obsbest) performer: Their scores in the original images were used as the second and third 'gold standards' for the remaining observers. Mean ROC areas for the three observers with the three types of images were calculated using these two new 'gold standards'. Differences between the ROC areas when using microscopy, obsworst, and obsbest as the 'gold standard' were compared. RESULTS: The mean ROC areas in the original images were 0.66, 0.74 and 0.91 using the true gold standard and obsbest and obsworst as the 'gold standards' respectively. The difference between the true gold standard and obsworst was statistically significant (P < 0.001). The mean ROC areas using the true gold standard decreased with increasing compression whereas they were constant or increased using obsworst and obsbest as 'gold standards', respectively. CONCLUSIONS: Accuracy in approximal caries diagnosis was significantly higher when an observer was the 'gold standard' than when the true gold standard was obtained by microscopy. Paradoxically, the compressed, degraded images were more accurate than the originals when an observer was the 'gold standard' while they were less accurate with the true gold standard. Thus, results obtained using observers' scores from the radiographs which are being evaluated, as validation for the presence of caries may mislead the clinician. PMID- 10740475 TI - Metallic residues as a source of artifacts in magnetic resonance imaging of the temporomandibular joint. AB - OBJECTIVES: To establish the source of metallic artifacts on postoperative MRI of the temporomandibular joint after arthroplasty. METHODS: An experiment was carried out to evaluate MR artifacts which were thought to have been caused by minute metallic particles deposited at surgery. Four types of cutting instruments were used to cut an extracted tooth: a diamond bur attached to a dental air turbine handpiece, a steel bur and bone bur attached to a dental micromotor handpiece and a bone file. The tooth fragments were collected in plastic centrifuge tubes in water. These samples were evaluated with six MR sequences. RESULTS: MR artifacts were clearly demonstrated in samples using the bone bur and the bone file but not the diamond and steel burs. CONCLUSIONS: The artifacts seen on postoperative MRI were derived from minute metal particles from the bone bur or file used for condylar arthroplasty. Metal artifacts should be considered when interpreting postoperative MR images. PMID- 10740476 TI - Impedance type electronic apex locators. PMID- 10740477 TI - Managing the endodontic emergency. PMID- 10740478 TI - The three wave concept of Hero 642. PMID- 10740479 TI - The 10 commandments: optimum quality endodontics. PMID- 10740480 TI - The C-shaped root canal system and its endodontic implications: a clinical review. PMID- 10740481 TI - Sealer selection: a considered approach. PMID- 10740482 TI - Irrigating the pulp space. PMID- 10740483 TI - The Quantec rotary nickel titanium instrumentation system. PMID- 10740484 TI - Making your own retrograde carrier. AB - One of the problems faced by manufacturers is the difficulty in constructing a robust and reliable, angled applicator tip. This can be overcome by handmaking your own retrograde carrier. The applicator tip may be bent to about 50 degrees and, if a kink occurs while bending the tip, it can be replaced easily by a new modified needle. Because the wire used is flexible, it can adapt to the bend without a problem. Narrower carriers can also be made using a 20-G needle, perhaps more suitable for retrograde fillings of molar apices. Because the carrier is designed to be used once only, the problems of it being difficult to load and liable to blockages should not arise. PMID- 10740485 TI - Conservative management of a crown-root fracture. PMID- 10740486 TI - Ethics: more than living between right and wrong. PMID- 10740487 TI - Options for posterior ceramic restorations. PMID- 10740488 TI - Ankylosed central incisor--a multidisciplinary approach to implant replacement. PMID- 10740489 TI - Periodontal treatment for elderly patients. AB - The largest growing segment of our society is that of older adults. The population explosion of older adults challenges both general practitioners and periodontists to provide the highest level of care available. More of our patients will have concurrent medical conditions that alter or modify the delivery and provision of periodontal care. This paper reviews some of the common conditions occurring in the older patient and suggests some modifications in periodontal therapy which may be beneficial. The therapist must be knowledgeable about the medications commonly used for treating the chronic diseases of the older population. Finally, the therapist must become adept in performing functional assessments of patients so that the types of therapy administered contribute to the quality of life desired by patients. PMID- 10740490 TI - Diagnosing molar furcation invasions. AB - Molar teeth with deep, horizontal furcation invasions (FI) are among the most difficult to treat periodontally. Therefore, it is important that these defects be detected early when the chances for successful, long-term treatment results are good. Even advanced FI are often treatable if their extent is properly diagnosed. The role of radiographs in the diagnosis of FI is discussed with emphasis on their limitations. The importance of accurate horizontal and vertical probing of FI is stressed. Proper probing techniques are presented along with anatomical factors which may adversely affect accuracy. PMID- 10740491 TI - Diagnostic tests for periodontal disease. AB - This paper reviews the principles and the types of diagnostic tests for periodontal disease. The relevance of the tests, such as sensitivity and specificity, and the consequences after the tests are discussed. Several clinical and laboratory tests such as DNA probes; test for the presence of aspartate aminotransferase or collagenase in gingival crevicular fluid (GCF); and the genetic test for the patients having higher risk to the disease are presented. These tests can serve as the adjunctive tools for traditional periodontal examinations. Understanding and properly utilizing these diagnostic tests would enhance treatment and provide better prognosis for patients. PMID- 10740492 TI - Metal reinforced provisional restoration. PMID- 10740493 TI - Soft and hard tissue resection in periodontal therapy. AB - The stabilization of inflammatory periodontal diseases (gingivitis and periodontitis) and result periodontal maintenance is achieved most effectively by first establishing health within the dento-gingival complex (connective tissue, junctional epithelium, and sulcus). Two forms of surgical periodontal therapy commonly employed to reach this endpoint are resective in nature:, the gingivectomy/gingivoplasty technique for soft tissue resection and the ostectomy (osteoectomy)/ osteoplasty approach for hard tissue resection. These two forms of therapy are presented here from a historical perspective through to today's accepted methodology. The are agumented by literature references and presentations of clinical cases. PMID- 10740494 TI - Adjuncts used to enhance the results of guided tissue regeneration. AB - Guided Tissue Regeneration involves procedures designed to regenerate lost periodontal structures. There are several adjunctive procedures used in conjunction with barrier membranes. This review article discusses the most commonly used adjuncts and the benefits and limitations of each. A brief synopsis of possible future directions is also discussed. PMID- 10740495 TI - Evaluation of an illuminated mirror. AB - The viewing of oral structures with an adequate level of illumination is necessary to make accurate diagnoses of normalities and abnormalities in the oral cavity. The purpose of this study is to determine if a device with an illuminated mirror is heat sterilizable, easy to use, and compares in illumination potential to a traditional overhead dental operating light. We sterilized the Denlite in both chemical vapor and autoclave units (including a group that was cleaned ultrasonically prior to autoclave), testing the illuminance level every 10 cycles, for 100 cycles. We installed devices in the Graduate Operative Dentistry Clinic and requested those operators assigned to the units to rate the devices. Their acceptance rate was high in visibility, ease of use, acceptable temperature of handle, and lack of problems with the device. The device had an illuminance level of 14,200 lux, which is just below the maximum level of illuminance required by the American Dental Association's Specification on Dental Operating Lights. PMID- 10740496 TI - Using two surgical protocols to restore the edentulous patient with implant supported overdentures. AB - Long-term success rates are reported for dental implant systems using both the two-stage and one-stage surgical protocols. Although the one-stage offers several advantages, prudent diagnosis is a key factor for selecting the appropriate surgical protocol. This article will review the relevant literature for both protocols and will illustrate their use in patient treatment. PMID- 10740497 TI - An alternative technique for an accurate implant-retained prosthesis impression. AB - A technique is described to help ensure the accurate reproduction of rigid implant components and the edentulous soft tissue. Splinted implant impression copings are luted together with resin to a specially designed custom tray. PMID- 10740499 TI - Distortion of three-unit implant frameworks during casting, soldering, and simulated porcelain firings. AB - PURPOSE: The aim of this study was to assess distortion inherent in casting, soldering, and simulated porcelain firings of screw-retained, implant-supported three-unit fixed partial dentures (FPDs). MATERIALS AND METHODS: Ten wax patterns were fabricated on a die-stone cast containing two implants, 20 mm apart from center to center. Five specimens were cast in a high-palladium alloy, exposed to simulated porcelain firings, sectioned, and then soldered with low-fusing solder. Five specimens were cast, sectioned, soldered with high-fusing solder, and then exposed to simulated porcelain firings. For each specimen, two horizontal and six vertical distances between appropriately scribed reference points were measured with a traveling microscope. Comparisons were made among the various measurements taken after wax-pattern fabrication, casting, high- and low-fusing soldering, and each porcelain firing. Data were analyzed using a repeated-measures factorial ANOVA (alpha = 0.05). RESULTS: Significant difference was detected in the amount of horizontal distortion during casting (53 +/- 24 microns) and high-fusing soldering (-49 +/- 50 microns), as well as in the amount of horizontal distortion during high-fusing soldering (-49 +/- 50 microns) and low-fusing soldering (17 +/ 26 microns). However, no clinically significant difference was found in the amount of horizontal distortion during casting, low-fusing, and high-fusing soldering. The greatest amount of distortion during the simulated porcelain firings took place during the oxidizing cycle. CONCLUSIONS: Soldering did not improve the casting misfit of a three-unit implant-retained FPD model. Metal ceramic implant frameworks should be oxidized before intraoral fit evaluation. PMID- 10740498 TI - Current methods of finish-line exposure by practicing prosthodontists. AB - PURPOSE: The purpose of this study was to survey members of The American College of Prosthodontists to evaluate current methods of finish-line exposure. In addition, frequency of use of epinephrine compounds and observed side effects were assessed. MATERIALS AND METHODS: A questionnaire was mailed to the 2,436 members of The American College of Prosthodontists. Group differences were evaluated using chi 2 analysis. RESULTS: Completed questionnaires were returned by 1,246 prosthodontists, which is a return rate of 51%. Ninety-eight percent of respondent prosthodontists use retraction cord, and 48% use a double-cord technique. Plain cord is the most commonly used cord (44%), followed by aluminum chloride-impregnated cord (18%), and epinephrine-impregnated cord (14%). Nine hundred one respondents (81%) soak the cord before placing it in the gingival sulcus. The most common medicament for soaking the cords is buffered aluminum chloride (55%). Side effects to epinephrine were reported by 387 respondents (33%), with the most common side effect reported being increased pulse rate, followed by anxiety. Approximately one quarter (24%) of the prosthodontists surveyed had observed side effects to chemical agents other than epinephrine. CONCLUSIONS: Prosthodontists throughout the country use many different techniques and agents to expose finish lines. No statistically significant differences (p > .05) were found between year of specialty training completion groups with respect to retraction cord use. Copper bands are used significantly more frequently (p < .05) in the northwest region of the United States than elsewhere. PMID- 10740500 TI - Infrared gold alloy brazing on titanium and Ti-6Al-4V alloy surfaces and its application to removable prosthodontics. AB - PURPOSE: This study investigated the area size of the flow of a gold braze alloy on commercially pure titanium and Ti-6Al-4V alloy plates, and elemental composition at the interface was determined. In the second part of this study, the tensile strengths of titanium plates brazed using a gold alloy were investigated. MATERIALS AND METHODS: Chips of Type IV gold alloy and silver braze alloys were melted onto commercially pure titanium and Ti-6Al-4V surfaces in a dental infrared radiation unit. Flow area of each braze alloy was measured using a digital image analyzer. Tensile specimens (n = 5) were also prepared by infrared brazing using the braze alloys. Five specimens for each combination of the two titanium plates and the two braze alloys were subjected to tensile loading using a Universal testing machine. Electronprobe microanalysis of x-rays at cross-section of the brazed joints to determine elemental composition across the interface, as well as scanning electron microscopic observation at the fracture surfaces, were also conducted. RESULTS: The braze alloys flowed well and spread over the Ti and Ti-6Al-4V plates. Braze alloy type significantly influenced flow, and the gold alloy flowed less on the titanium materials. The mean tensile strengths of Ti and Ti-6Al-4V plates brazed using the gold braze alloy were 219 MPa and 417 MPa, respectively. The fracture surfaces of Ti-6Al-4V specimens with the gold braze alloy exhibited typical ductile behavior. Ti with the same braze alloy showed brittle surfaces. A greater concentration of Cu was found at the Ti with gold braze interface. CONCLUSION: The flow and the tensile strength of the gold alloy coating on titanium surface by means of an infrared brazing is adequate for dental use. PMID- 10740501 TI - Flexural properties and surface finishing of acetal resin denture clasps. AB - PURPOSE: This study examined flexural properties of acetal resin to determine the appropriate design for an acetal resin removable partial denture clasp. The effectiveness of various acetal resin polishing procedures was also evaluated using scanning electron microscopy. MATERIALS AND METHODS: Loads of up to 1,500 g were applied to cylindrical acetal resin specimen lengths of 5, 10, and 15 mm, and the degree of deflection of the specimen tip was measured. Specimens were 2 mm and 1.5 mm in diameter with a round cross-section (R2 and R1.5, respectively), as well as 2 mm in diameter and half-round in cross-section. Half-round specimens were loaded with their flat surface vertical and then horizontal (HRV and HRH, respectively). Contour plots showing load and deflection for varying lengths of resin were developed for each cross-sectional type based on specimen length, and the stiffness was calculated. Additional acetal resin specimens underwent polishing techniques including tungsten carbide burs, aluminium oxide stones, abrasive impregnated rubber points, and "Polish-D." Polished specimens were examined with scanning electron microscopy to compare surface roughness. RESULTS: For the 5-mm-length specimens, the median stiffness values decreased in the following order: R2 > HRV > HRH > R1.5. The median stiffness values for the 10-mm and 15-mm lengths followed a similar trend. At each length, the R2 specimen was generally at least twice as stiff as other designs. Regarding polishing, the smoothest surface was seen on the specimen that was polished with the rubber point followed by "Polish-D." CONCLUSIONS: To have stiffness similar to a cast cobalt-chromium clasp 15 mm long and 1 mm in diameter, a suitable acetal resin clasp must be shorter (approximately 5 mm) with greater cross-sectional diameter (approximately 1.4 mm). To obtain a smooth surface, the acetal resin clasp should be polished with a rubber point followed by a material such as "Polish-D." PMID- 10740502 TI - Effective taper criterion for the full veneer crown preparation in preclinical prosthodontics. AB - PURPOSE: Effective taper criteria must define a realistic, measurable goal that the student can visualize and achieve. Six degrees is widely accepted as the taper criterion for the full veneer crown preparation. However, studies show the actual taper of most preparations to be greater than 12 degrees. The purpose of this study was to determine whether 12 degrees is an effective taper criterion for the full veneer crown preparation in preclinical prosthodontics instruction. MATERIALS AND METHODS: A group of 191 full veneer crown preparations with 6 degrees as the taper criterion, and a group of 130 full veneer crown preparations with 12 degrees as the taper criterion were evaluated. All preparations were accomplished by preclinical dental students working on typodonts under examination conditions. RESULTS: The overall mean taper for each group exceeded the targeted criterion. The overall mean taper for the 6 degrees group was 14,490. When 12 degrees was the criterion, the overall mean taper was 15,580. The t test results indicate significant differences (p < .0001) between the targeted criterion and the actual preparation mean taper in all samples except one: the faciolingual measurements in the 12 degrees group. The faciolingual measurement of 12,920 was not statistically significantly different (.0542) from the targeted criterion of 12 degrees. CONCLUSION: The use of a 12 degrees taper criterion did not result in preclinical students achieving the goal of a 12 degrees taper. However, a 12 degrees criterion is more realistic than a 6 degrees criterion for full veneer crown preparations. PMID- 10740503 TI - Oral rehabilitation of a child with primary failure of tooth eruption. AB - This article describes the prosthetic evaluation and treatment of a 10-year-old female patient with primary failure of tooth eruption. This case is unique in that the failure of eruption affected both the deciduous and permanent dentition in all quadrants, in the absence of any other systemic abnormalities. After diagnostic articulation and a thorough discussion of the treatment with the patient and her parents, complete maxillary and mandibular overdentures were fabricated. The process followed with this preadolescent patient to ensure maximum compliance, success, and encouragement toward a positive attitude in wearing complete dentures is reported. PMID- 10740504 TI - The history of articulators: a perspective on the early years, Part I. PMID- 10740505 TI - Comparison of perceptions of computer-animated left- and right-facing profiles. AB - PURPOSE: Although scientific publications differ in displaying left- or right facing profiles, there are few systematic studies of the possible effects of directional biases and laterality on patients' and clinicians' perceptions of treatment need, outcomes, and satisfaction. As part of a research program to quantitate the physical bases of the perceived zone of acceptability and most pleasing facial profiles, responses to computer-animated left- and right-facing soft-tissue profile images were compared and related to eye and hand preference. MATERIALS AND METHODS: Standardized left-facing, soft-tissue profile images were captured in color and digitized. The right-facing profile was created by reversing the left profile image to display a mirror image. Using imaging and customized morphing software, retrusive and protrusive extremes were created, from which transitional frames were developed to display five features of the soft-tissue profile: upper lip, chin, bimaxillary position, lower face height, and mandible. One left- and one right-facing profile for a Class II division 1 female, and Class III male were randomly presented to 24 subjects who were asked to indicate: 1) acceptability by pressing a mouse button and releasing the button when the images were no longer acceptable; and 2) most-pleasing image by pressing the mouse button once. RESULTS: No differences were found between left- and right profile images for zone of acceptability, midpoint of the zone of acceptability, or most pleasing; nor was there any relation to laterality measures. However, consistently higher intercorrelations among the features for the zone of acceptability were found for the left- than for the right-facing profiles for retrusive to protrusive and protrusive to retrusive excursions measured in millimeters (p < .001). The bimaxillary position accounted for most of the variance in judgments of acceptability with greater influence on the left than on the right profile. CONCLUSION: With the increasing use of computer-imaging in dental practices, the influence of psychophysical and other environmental variables on perception must be considered. PMID- 10740506 TI - Compressive and tensile zones in the cement interface of full crowns: a technical note on the concept of resistance. AB - PURPOSE: The objectives of the study were: 1) to map the stresses acting on the cement interface of crown and abutment analogs during loading; and 2) to provide a theoretical basis for the hypothesis that resistance to lateral dislodgment is a function of the distribution of compressive force vectors acting on the cement lute. MATERIALS AND METHODS: Three-dimensional finite element (FE) meshes of crown and abutment analogs were constructed and loaded in a direction perpendicular to the axes of symmetry of the abutments. Three parameters were investigated: taper (10 degrees and 20 degrees of convergence), abutment substrate (Ni-Cr alloy and dentin), and type of cement (zinc oxide eugenol, zinc phosphate, glass ionomer, and composite resin). The tensile and compressive components of the resulting force systems were plotted along two axes (z: parallel to the axis of symmetry of the crown/abutment complex; and y: perpendicular to z, i.e., parallel to the direction of loading). Von Mises stresses were also generated. RESULTS: First, it was shown that the restoration's axis of rotation was located inside the abutment cone and was perpendicular to and intersected the axis of symmetry of the crown/abutment complex. Second, stress distribution was dependent on the three parameters investigated. Varying taper led to shifts due mainly to alterations in specimen geometry, whereas the abutment substrate and the cement type had a bearing on the level of the axis of rotation. The smaller the modulus of elasticity of the abutment substrate or the cement lute, the farther apical the location of the axis of rotation. CONCLUSIONS: Conventional schemes for explaining crown dislodgment in which the restoration rotates around an axis located at the preparation margin should be reassessed. The results of the FE analysis are compatible with the hypothesis that resistance to lateral dislodgment is a function of the distribution of compressive force vectors acting on the cement interface. PMID- 10740507 TI - Physical-property comparison of a chairside- or laboratory-polymerized permanent soft-liner during 1 year. AB - PURPOSE: This investigation examined the influence of polymerization mode and water storage duration on the Shore A hardness, water sorption, resin solubility, and glass transition temperature of Permasoft, a commercial soft denture liner that is polymerized either chairside or in the laboratory. MATERIALS AND METHODS: Specimen disks (31-mm diameter x 10 mm thick) and bars (44 x 8.5 x 1.2 mm) were polymerized following manufacturer's recommendations. The chairside polymerization process was simulated by pouring the mixed components into a mold and processing at 70 degrees C for 15 minutes under 2-psi pressure. Laboratory polymerized specimens of the same dimensions were fabricated by processing under 500-psi pressure at 100 degrees C for 45 minutes. Specimens were stored in distilled water at 37 degrees C for 1, 7, and 30 days, and 6 and 12 months. Specimens were tested for Shore A hardness, water sorption, resin solubility, and glass transition temperature after the prescribed interval. To determine the effects of polymerization mode and storage time on material properties, a repeated-measures ANOVA (hardness data) and a two-way ANOVA (sorption and solubility data) with appropriate post-hoc tests were used. RESULTS: Shore A hardness values increased from a low of 9.4 (+/- 0.5) units immediately after fabrication to a maximum of 15.9 (+/- 1.1) units after 1 year. Mode of polymerization did not influence hardness (p = .9851). Water-sorption values ranged from 4.2 (+/- 0.2%) of dry weight to 14.7 (+/- 2.5%) after 1 year. Resin solubility varied from 10.3 (+/- 0.6%) of preimmersion weight to 15.4 (+/- 1.1%), and immersion duration had no effect on solubility. In addition, after 1 year of storage, no difference in resin solubility or water sorption was found with respect to cure mode. The glass transition temperature for chairside-polymerized samples approximated -10 degrees C, while that for labpolymerized samples approximated -15 degrees C. CONCLUSIONS: With regard to the material properties evaluated in this study, clinically processed Permasoft liner was equivalent to the laboratory-processed material. PMID- 10740508 TI - Evaluation of a computer-aided learning program in removable partial denture framework designing. AB - Assessment of a computer-assisted learning (CAL) program dealing with designing removable partial dentures was performed as both formative and summative evaluation. Formative evaluation was obtained through open-ended questions in a written questionnaire administered to 60 junior students from two Australian Dental Schools and through observation of four groups of these students as they worked through the program. Formative evaluation, fundamental to completion of the program, led to several changes and additions. Observation of the students using the program during its final stages of development was one of the most informative and helpful parts of the evaluation. It allowed nuances of expression and body language to be noted that would not have been apparent in written or numerical data. Summative evaluation was obtained through structured questions in the questionnaire and through a post-CAL test under examination conditions. Summative assessment showed that students could attain a desired standard of knowledge if a lecture series was replaced by the CAL program; that the program was user-friendly, even for students with little or no computer experience; and that most students considered it to be a useful learning resource. More than half the students were concerned about using CAL to replace human instruction. It is significant that students were particularly impressed by aspects of the program that could not be duplicated by a book; the interactive nature of the learning process and the ability to rotate the diagnostic casts. This highlights the need for CAL programs to use the full potential of computer capability, rather than merely replicate a textbook on a screen. PMID- 10740509 TI - The esthetic width in fixed prosthodontics. AB - With the evolution of adhesive dentistry and the increasing use of porcelain veneers, single-unit crowns generally are restricted to the replacement of pre existing full-coverage crowns and the restoration of nonvital and/or severely damaged teeth. Porcelain-fused-to-metal restorations are still widely used to generate single-unit crowns and fixed partial dentures. Collarless metal-ceramic restorations represent the most successful evolution among efforts to meet maximum esthetic requirements using porcelain-fused-to-metal restorations. Extended metal frameworks and opaque aluminous ceramic cores are associated with unpleasant optical effects in the soft tissues surrounding such restorations. This problem is particularly evident in the presence of the upper lip, which can generate an "umbrella effect" characterized by gray marginal gingivae and dark interdental papillae. Based on the concept of the biologic width, a systematic approach is proposed for the elaboration of an "esthetic width," including: 1) positioning of preparation margins; 2) reduction of the metal framework; and (c) appropriate marginal design of porcelain-fused-to-metal restorations. Strategic features of pontics and a specific interdental design are suggested to compensate for deficient anatomical features of the soft tissue and the edentulous ridge. PMID- 10740510 TI - A survey of private prosthodontic practice. AB - The data from a survey of private prosthodontic practitioners are reported in this article. The author provides a brief look at employment status, number of employees, time spent in the office, number of patient visits per week, gross billings, net income, and practice expenses of prosthodontic practices. In addition, the author has developed a profile for a median prosthodontic practice and practitioner. PMID- 10740511 TI - A soldering index for cement-retained implant restorations. AB - Recent advances in implant abutment interface design purport to offer more repeatable, accurate abutment connection. Improved circumferential indexing of the abutments along with a decrease in micromovement may result in a more predictable fit of cement-retained implant prostheses through a simple variation of soldering technique. A technique for developing a more accurate soldering relationship is described. PMID- 10740512 TI - A technique for border molding edentulous impressions using vinyl polysiloxane material. AB - A technique for making selective tissue placement final impressions for complete dentures using vinyl polysiloxane and custom trays with peripheral relief is presented. This technique is an alternative to the conventional method of border molding using modeling plastic impression compound. PMID- 10740513 TI - Using a facsimile (fax) as an aid for communication in maxillectomy planning between the prosthodontist and surgeon. AB - Preoperative communication between the prosthodontist and surgeon is required for properly planning the surgical prostheses that are often indicated for maxillectomy patients. When geography or long distances preclude face-to-face consultation, electronic means of correspondence are necessary to minimize delays in the fabrication of surgical prostheses. This article presents a time-efficient procedure for facilitation of the necessary communication between the prosthodontist and surgeon before maxillectomy surgical procedures. PMID- 10740514 TI - Silicone thermoplastic sheeting for treatment of facial scars: an improved technique. AB - Silicone thermoplastic sheeting has been used successfully in the management of hypertrophic and keloid scars resulting from thermal burn injuries. A technique is described that incorporates silicone thermoplastic sheeting for fabrication of a compression face mask. This technique combines the moldability of thermoplastic splinting materials with the therapeutic surface of silicone, yielding the advantages of both in a one-step process. PMID- 10740515 TI - The future of dental care for the elderly population. AB - The U.S. population is aging, and the elderly are maintaining many of their natural teeth. Studies have shown that if older people have teeth, they tend to utilize dental services to a similar extent as younger cohorts. Geriatric dental care is the diagnosis, treatment, and prevention of dental and oral diseases for all older adults. A functional categorization of the aging population is more useful in dentistry than a chronological one, and 70 percent of this population, or 23.2 million people, is able to visit a dentist in his or her office. The oral health care of older adults has become more challenging because they will no longer accept extraction and dentures as a solution to complex restorative needs. This paper discusses these issues and looks at the future of geriatric dental care. PMID- 10740516 TI - Doing the right thing: resolving ethical issues in geriatric dental care. AB - This article reviews key ethical concerns facing dental professionals caring for older adults and offers guidelines for resolving them. Approaches to securing informed consent for treatment and assessing decision making capacity are discussed. Next, the question of deciding among several treatment options when patient preferences are unknown is considered. Finally, guidelines are offered on restraint use for older adults with behavioral problems. PMID- 10740517 TI - Oral health care for homebound and institutional elderly. AB - The provision of oral health care to homebound and institutionalized patients presents enormous challenges as well as several advantages for the dental professional. This article discusses the rapid growth of this segment of the population, the barriers to their receiving dental care, and the objectives for provision of that care. PMID- 10740518 TI - Alzheimer's disease and cognitively impaired elderly: providing dental care. AB - With the number of adults reaching older ages, the number of Americans who develop Alzheimer's disease and other neurological impairments will also increase. The dental management of these patients requires a great deal of understanding and patience coupled with background knowledge of the disease and proficiency in providing behavior modification techniques. This paper discusses five major areas that dental practitioners should consider prior to caring for patients with Alzheimer's disease or other neurological impairments. PMID- 10740519 TI - Providing oral cancer examinations for older adults. AB - Although cancer is not a part of the aging process, malignant neoplasms occur primarily in older adults. As the size of the elderly population increases, there will be many more older adults at risk for oral cancer. Many older adults do not seek dental care because they do not think they need it; and, therefore, they do not receive routine oral examinations. Dental practitioners need to encourage older patients to seek dental care so they can receive oral cancer examinations. PMID- 10740520 TI - Customers for life: marketing oral health care to older adults. AB - Respect for and awareness of the needs of older patients from dental office staff will help such patients feel welcome in a practice. Marketing to older patients is built upon this foundation. In addition, there are other strategies for internal and external marketing aimed at older people. This article addresses the concept of turning aging patients into "customers for life." PMID- 10740521 TI - Rapid healing of gingival incisions by the helium-neon diode laser. AB - Fifty-eight extraction patients had one of two gingival flap incisions lased with a 1.4 mw helium-neon (670 nm) diode laser for 30 seconds (fluence = 0.34 J/cm2). Healing rates were evaluated clinically and photographically. Sixty-nine percent of the irradiated incisions healed faster than the control incisions. No significant difference in healing was noted when patients were compared by age, gender, race, and anatomic location of the incision. This study concludes that helium-neon diode lasers, at the previously mentioned energy level, increase the rate of gingival wound healing in 69 percent of patients, without any side effects. For the last 30 years, low-power lasers in dentistry have appeared to stimulate healing rates and increase the rate of repair of injured tissue. Helium neon and similar lasers emit light in the red (600-700 nm) spectrums and produce energy densities (fluences) below 20 Joules/cm2. They have been studied in a variety of animal tissue culture and human evaluations to determine their ability to increase the rates of wound healing by biostimulation. Over the last three decades, researchers have found that ruby and gas helium-neon (low-power laser radiation) have a biostimulatory effect on living tissue. Studies show that under specific conditions, red spectrum laser light speeds the healing of wounds. Photons from the red light lasers, which include ruby lasers (694 nm), helium neon gas lasers (632 nm), and helium-neon diode lasers (650-670 nm), appear to stimulate rapid epithelialization and fibroblast (collagen) proliferation in animal and human tissue cultures. Low-power lasers have been reported to reduce post-extraction pain and swelling and to increase rates of wound healing (including scar formation, phagocytosis) in cell culture, animal, and human clinical studies. The new, compact, and inexpensive (under $50) helium-neon diode lasers have produced similar effects. These FDA Class IIIa lasers have no hazards associated with them, although one should avoid direct exposure to the eye for a prolonged period of time. In the past, many biostimulation studies using red spectrum lasers produced confusing data and conflicting results. Some studies reported that the biostimulation effect did not occur in all cases of laser irradiation, while other research reported that it did. Results seem to depend on the delivery of appropriate energy fluence levels (between 1 and 20 J/cm2) and the type of laser (wavelength) used. Several of these studies never described the levels of laser energy used to promote the described biostimulatory results. This caused controversy when determining whether or not helium-neon lasers influence wound healing. Studies suggest that low-power laser exposure can significantly increase the healing rate during the first few days of the healing process; however, studies do not show appreciable net benefit as compared to controls toward the end of a two-week wound repair cycle. The increased healing effect appears to be centered around the early, most sensitive stages of the healing process. Several studies showed optimum tissue healing rates at helium-neon laser exposure levels between 1 J/cm2 and 20 J/cm2. Laser-enhanced biostimulation has been reported to produce metabolic changes within the cells. This results in faster cell division, rapid matrix production (increased collagen, myofibroblasts, etc.), and cell movement. There have been few controlled studies using adequate numbers of human subjects in identifying the beneficial effects of helium-neon laser biostimulation. Ethical concerns, bulky equipment, and problems with biased study designs have frustrated a practical evaluation of laser biostimulation for general dental practice. A recently published "preliminary" study involving 52 patients was designed to reduce these issues. The purpose of this study is to complement the above research and to evaluate whether helium neon diode laser radiation at average fluences of 0.34 J/c PMID- 10740522 TI - Current concepts in nonsurgical endodontic retreatment. PMID- 10740523 TI - PST: the role of genetic testing for periodontal susceptibility in clinical practice. PMID- 10740524 TI - Bases and liners: current concepts. PMID- 10740525 TI - Neck exercises triggering dental pain. PMID- 10740526 TI - Managing dental care. PMID- 10740527 TI - A revolution in implant dentistry. PMID- 10740528 TI - Tongue cleaning: a necessary part of the oral hygiene regimen. PMID- 10740529 TI - Dentistry's role in identification of missing and unknown children: update on the dental bite impression technique. PMID- 10740530 TI - The radiographic image of sublingual tissues. PMID- 10740531 TI - Surgical exposure and orthodontic repositioning of an impacted mandibular premolar. AB - This article is intended to give an overview of mandibular second premolar impactions and a detailed description of treatment. Practitioners who possess a basic knowledge of oral surgery technique and comprehensive corrective orthodontic capabilities could successfully treat such an entity. PMID- 10740532 TI - Alveolar distraction osteogenesis: a new alternative to bone grafts. Report of three cases. AB - Case reports of distraction osteogenesis of the alveolar bone, case selection, methods and follow-up of patients seen in the Oral Surgery Group office, New Brunswick, NJ. PMID- 10740533 TI - Enhancement of cross-arch stabilization of implants with a semi-precision lingual bar. PMID- 10740534 TI - Dental images in Anglo-Saxon literature. PMID- 10740535 TI - The treatment of a localized osseous sequestrum with porous bone mineral in combination with a collagen membrane and resorbable bone pins. AB - The ultimate goal of periodontal therapy should not be limited to the establishment and maintenance of periodontal health. The potential regeneration of the hard and soft periodontal tissues lost due to disease also should be considered. This case presentation evaluated a new surgical technique for the treatment of a variety of localized bone defects, utilizing porous bone mineral in combination with collagen membrane and resorbable bone pins. PMID- 10740536 TI - Dental treatment guidelines for use of restraints within the nine Louisiana developmental centers. Louisiana State University Dental Health Resources Program. AB - There are approximatively 2000 mentally retarded or developmentally disabled (MR/DD) individuals in Louisiana's nine developmental centers (LDC). Two-thirds resist the delivery of preventive and clinical dental services. Therefore, for these patients to receive quality, comprehensive dental care, some form of restraint is necessary. Physical holds, mechanical and/or chemical restraints allow caregivers and dental professionals to provide medical/dental services. However, prior to the use of restraint, a clear understanding and acquisition of informed consent are needed. The dentist, developmental center and its administration are responsible for developing a plan to obtain informed consent and to express the importance of restraint to each resident's guardian. This article defines the types of restraint based upon resistance encountered and defines informed consent according to the Louisiana Medical Consent Law. PMID- 10740537 TI - A shame about the patient? PMID- 10740538 TI - Aprotinin and renal function in patients undergoing cardiac surgery. PMID- 10740539 TI - Patient satisfaction after anaesthesia and surgery: results of a prospective survey of 10,811 patients. AB - Patient satisfaction after anaesthesia is an important outcome of hospital care. We analysed our anaesthetic database to identify potentially modifiable factors associated with dissatisfaction. At the time of analysis, our database contained information on 10,811 in-patients interviewed on the first day after operation. The major subjective outcome measure was patient satisfaction. We also measured other predetermined outcomes, such as nausea, vomiting, pain and complications. The overall level of satisfaction was high (96.8%); 246 (2.3%) patients were 'somewhat dissatisfied' and 97 (0.9%) were 'dissatisfied' with their anaesthetic care. After adjustment for patient and surgical factors, there was a strong relation between patient dissatisfaction and: (i) intraoperative awareness (odds ratio (OR) 54.9, 95% confidence intervals (CI) 15.7-191); (ii) moderate or severe postoperative pain (OR 3.94, 95% CI 3.16-4.91); (iii) severe nausea and vomiting (OR 4.09, 95% CI 3.18-5.25); and (iv) any other postoperative complications (OR 2.04, 95% CI 1.61-2.56). Several factors associated with dissatisfaction may be preventable or better treated. PMID- 10740540 TI - Validity and reliability of a postoperative quality of recovery score: the QoR 40. AB - Quality of recovery after anaesthesia is an important measure of the early postoperative health status of patients. We attempted to develop a valid, reliable and responsive measure of quality of recovery after anaesthesia and surgery. We studied 160 patients and asked them to rate postoperative recovery using three methods: a 100-mm visual analogue scale (VAS), a nine-item questionnaire and a 50-item questionnaire; the questionnaires were repeated later on the same day. From these results, we developed a 40-item questionnaire as a measure of quality of recovery (QoR-40; maximum score 200). We found good convergent validity between QoR-40 and VAS (r = 0.68, P < 0.001). Construct validity was supported by a negative correlation with duration of hospital stay (rho = -0.24, P < 0.001) and a lower mean QoR-40 score in women (162 (SD 26)) compared with men (173 (17)) (P = 0.002). There was also good test-retest reliability (intra-class ri = 0.92, P < 0.001), internal consistency (Cronbach's alpha = 0.93, P < 0.001) and split-half coefficient (alpha = 0.83, P < 0.001). The standardized response mean, a measure of responsiveness, was 0.65. The QoR-40 was completed in less than 6.3 (4.9) min. We believe that the QoR-40 is a good objective measure of quality of recovery after anaesthesia and surgery. It would be a useful end-point in perioperative clinical studies. PMID- 10740541 TI - Aprotinin does not impair renal haemodynamics and function after cardiac surgery. AB - Patients undergoing cardiac surgery with moderate hypothermic cardiopulmonary bypass (CPB) were allocated randomly to receive either saline (control group, n = 29) or a high-dose regimen of aprotinin (aprotinin group, n = 28). In both groups, CPB was associated with similar and transient increases in effective renal plasma flow (+54% in controls and +48% in aprotinin-treated patients) and in fractional excretion of sodium and potassium, but glomerular filtration rate remained unchanged. Plasma and urinary ratios of 6-keto-PGF1 alpha to thromboxane B2 (TxB2) increased significantly, indicating systemic and renal release of vasodilatory prostaglandins. Osmolar clearance correlated with urinary excretion of cyclic GMP (r = 0.79 and 0.86 in the control and aprotinin groups, respectively) and 6-keto-PGF1 alpha (r = 0.63 and 0.69 in the control and aprotinin groups, respectively). Compared with preoperative values, plasma atrial natriuretic peptide increased after weaning from CPB (+71% and +93% in the control and aprotinin groups, respectively). Aprotinin had no apparent adverse effect on renal function and it did not alter mechanisms involving prostanoids and atrial natriuretic peptide during cardiac surgery. PMID- 10740542 TI - A placebo-controlled study of the effects of dopexamine on gastric mucosal perfusion in infants undergoing hypothermic cardiopulmonary bypass. AB - We used a laser Doppler probe to measure gastric mucosal blood flow (flux) and a paediatric tonometer to intermittently calculate gastric-arterial PCO2 difference (PrCO2-PaCO2 gap) in 50 infants aged 0.3-52 weeks who required hypothermic cardiopulmonary bypass (CPB). During CPB, patients in group 2 (n = 25) were given dopexamine 1.0 mg kg-1 over 5 min, followed by an infusion of 2 micrograms kg-1 min-1. Patients in group 1 (n = 25) received an equal volume of saline. Drug allocation was random and blinded. Measurements of flux, PrCO2-PaCO2 gap and mean femoral arterial pressure (MAP) were made over 5 min during steady state before and after cooling on CPB to 18-24 degrees C. MAP and blood lactate concentrations were similar in both groups throughout CPB. Mean flux decreased from 182 (SD 60) at the beginning of CPB to 158 (51) after rewarming on CPB in group 1, whereas it increased from 180 (56) to 196 (49) in group 2. This post-rewarm flux was significantly greater in group 2 than in group 1 (P = 0.01). Similarly, mean PrCO2-PaCO2 gap increased significantly from 3.6 (6.3) to 8.2 (6.7) in group 1 (P = 0.01) compared with a significant decrease from 5.8 (5.5) to 2.1 (5.5) in group 2 (P = 0.02). Mean PrCO2-PaCO2 gap after rewarming in group 2 was significantly higher than that in group 1 (P = 0.001). These data indicate that dopexamine may be useful in maintaining normal gut perfusion in infants requiring hypothermic CPB. PMID- 10740543 TI - Effects of changes in packed cell volume on the specific heat capacity of blood: implications for studies measuring heat exchange in extracorporeal circuits. AB - Extracorporeal circuits such as cardiopulmonary bypass (CPB) and renal dialysis machines cause active and/or passive loss of body heat. Attempts to quantify this heat loss are generally based on the Fick principle which requires knowledge of the specific heat capacity (SHC) of blood. As changes in packed cell volume are common, we investigated the effect of these changes on the SHC of blood over a range of packed cell volumes (PCV) from whole blood at 43.1% (3594 J kg-1 degrees C-1) to pure Hartmann's solution (4153 J kg-1 degrees C-1). The SHC of other fluids used during CPB was also measured and found to be 4139 J kg-1 degrees C-1 and 4082 J kg-1 degrees C-1 for normal saline and Gelofusine, respectively. The maximum variability in SHC over the range of PCV values encountered during CPB was calculated to be small (5.5%). We conclude that use of a constant value of SHC for calculation of thermal energy transfer is currently justified. PMID- 10740544 TI - Increasing isoflurane concentration may cause paradoxical increases in the EEG bispectral index in surgical patients. AB - We have studied the effects of increases in isoflurane concentration on the EEG bispectral index (BIS) in 70 patients anaesthetized with isoflurane-nitrous oxide sufentanil for major abdominal surgery. During surgery, baseline BIS was recorded at 0.8% end-tidal isoflurane with nitrous oxide in oxygen (FIO2 0.35). After this, end-tidal isoflurane was increased to 1.6% for 15 min and decreased subsequently to 0.8% for 20 min to assess recovery. In 20 patients, BIS decreased from a mean value of 40 (SD 9) during baseline to 25 (10) at 1.6% isoflurane. In contrast, BIS did not change in 23 patients and increased in 27 patients from 35 (6) to 46 (8) as isoflurane was increased to 1.6%. In all patients, BIS recovered to baseline values at 0.8% isoflurane. The changes in BIS with increasing isoflurane concentration were not related to drugs or differences in physiological variables, which did not differ between groups. Patients with a decrease in BIS were significantly younger (38 (range 18-68) yr) than those with unchanged (55 (26-70) yr) or increased (60 (40-70) yr) BIS values (P < 0.001). It is possible that the paradoxical increase in BIS is related to continuous pre burst EEG patterns consisting of high-frequency activity. This suggests that the use of BIS as a guide for isoflurane administration may be misleading in some patients undergoing surgical procedures. PMID- 10740545 TI - Behaviour of near-infrared light in the adult human head: implications for clinical near-infrared spectroscopy. AB - To test theoretical assumptions supporting the use of near-infrared spectroscopy (NIRS) in clinical practice, we examined the behaviour of NIR light transmission and attenuation in the human head. Sterile probes for emitting and detecting NIR light at a fixed separation of 40 mm were placed in turn on intact skin, skull, dura and cerebral cortex of 10 patients undergoing elective neurosurgery. In the first five patients, the detecting probe was moved through successive extracerebral layers with the emitter on the skin surface. In the second five patients, the process was reversed, with the emitting probe moved and the detector in the same place on the scalp. NIR intensity was measured at each tissue interface and compared with the intensity measured at the skin surface with all layers intact. Removal of bone and dura from the light path caused a significant reduction in detected intensity. The largest mean reduction in light intensity was a 14-fold decrease with removal of bone (unadjusted P < 0.0001; paired t test). The assumptions that extracerebral tissues contribute little to attenuation of NIR light in the adult head and that most of this attenuation occurs superficially in the scalp are drawn into question by this study. We postulate that the skull and/or its interface with other layers may act as an optical 'channel', distorting the behaviour of NIR light in the human head. PMID- 10740546 TI - Rocuronium potency and recovery characteristics during steady-state desflurane, sevoflurane, isoflurane or propofol anaesthesia. AB - We have studied the potency and recovery characteristics of rocuronium during 1.25 MAC of isoflurane, desflurane, sevoflurane or propofol anaesthesia in 84 patients using electromyography. Potency was determined by a cumulative bolus technique. The mean ED50 of rocuronium was 169 (SD 41), 126 (32), 121 (28) and 136 (25) micrograms kg-1 during propofol, isoflurane, sevoflurane and desflurane anaesthesia, respectively (ns), and ED90 values were 358 (62), 288 (29), 289 (28) and 250 (28) micrograms kg-1, respectively. The reduction in ED90 was statistically significant for all three inhalation anaesthetics (P < 0.05) compared with propofol. After 120 min, the cumulative infusion rate of rocuronium to obtain twitch depression of 90-95% was 9.0 (1.9), 6.3 (1.6), 6.1 (2.0) and 6.1 (1.1) micrograms kg-1 min-1 during propofol, isoflurane, sevoflurane and desflurane anaesthesia, respectively (P < 0.01). Recovery index was 22 (13), 27 (10), 28 (13) and 26 (14) min under propofol, isoflurane, sevoflurane and desflurane anaesthesia, respectively (ns). There were no significant differences between the three potent inhalation anaesthetics in relation to potency, infusion requirements or recovery characteristics of rocuronium. PMID- 10740547 TI - Single-dose ketorolac and pethidine in acute postoperative pain: systematic review with meta-analysis. AB - For a systematic review of postoperative analgesic efficacy and adverse effects of single doses, injected or oral, of pethidine and ketorolac compared with placebo, we sought published randomized studies in moderate to severe postoperative pain. Information on summed pain intensity or pain relief outcomes over 4-6 h was extracted and converted to dichotomous information to produce the number of patients with at least 50% pain relief. This was used to calculate the relative benefit and number-needed-to-treat (NNT) for one patient to achieve at least 50% pain relief. Minor and major adverse effect data were extracted and summarized. For pethidine 100 mg i.m., eight randomized, controlled studies met the inclusion criteria, with 203 patients given pethidine and 161 placebo. The NNT to produce at least 50% pain relief was 2.9 (95% confidence interval 2.3 3.9). At this dose, pethidine produced significantly more drowsiness and dizziness than placebo, with numbers-needed-to-harm (NNH) of 2.9 (2.2-4.4) and 7.2 (4.8-14), respectively. For ketorolac, 14 reports met the inclusion criteria (six i.m. and eight oral). Most i.m. information (176 patients) was available for the 30 mg dose, which had an NNT of 3.4 (2.5-4.9). Most oral information was available for the 10 mg dose, which had an NNT of 2.6 (2.3-3.1). Oral ketorolac 10 mg was consistently at least as effective as ketorolac 30 mg i.m. Only with oral ketorolac 10 mg were there significantly more adverse effects than with placebo, with an NNH for any adverse effect of 7.3 (4.7-17). PMID- 10740548 TI - Hyperbaric bupivacaine for spinal anaesthesia in 7-18 yr old children: comparison of bupivacaine 5 mg ml-1 in 0.9% and 8% glucose solutions. AB - We have compared two hyperbaric bupivacaine solutions for spinal anaesthesia in 7 18-yr-old school-aged children in a double-blind, randomized, parallel group, prospective study. Children were premedicated with diazepam orally. Half of the patients were sedated with either midazolam or thiopental. After lumbar puncture with a 27-gauge spinal needle, bupivacaine 5 mg ml-1 in either 0.9% or 8% glucose was injected in a dose of 0.3 mg kg-1. Maximum cephalad spread and regression of block were tested by transcutaneous electrical stimulation. Success rate, spread and duration of sensory block were similar in both groups. The highest median level of sensory block was T4 (10-90th percentiles T1-T7) in the 0.9% glucose group and T4 (T1-T5) in the 8% glucose group. Time to two segment regression of block was 83 (50-143) min in the 0.9% glucose and 85 (53-150) min in the 8% glucose group. The incidence of adverse effects was similar. Six children were given etilefrin to treat hypotension and six atropine for bradycardia. Nausea was associated with a high level of block. Shivering was detected in 16 children. PMID- 10740549 TI - Fentanyl augments block of sympathetic responses to skin incision during sevoflurane anaesthesia in children. AB - We studied 61 healthy ASA 1 patients (aged 2-6 yr) to determine if fentanyl affects the minimum alveolar concentration which blocks adrenergic responses to skin incision (MAC-BAR) in 50% of children in the presence of 60% nitrous oxide. Patients were allocated randomly to one of three fentanyl groups to receive 0, 2 or 4 micrograms kg-1. Patients also received sevoflurane at a preselected end tidal concentration according to an 'up-and-down' design. After a steady-state sevoflurane concentration had been maintained for at least 15 min, fentanyl was given i.v. Skin incision was performed 5 min after administration of fentanyl. The response was considered positive if heart rate (HR) or mean arterial pressure (MAP) increased by 15% or more. The MAC-BAR of sevoflurane was 1.45 MAC (95% confidence intervals 1.25-1.65 MAC), and this was reduced markedly to 0.63 MAC and 0.38 MAC by addition of fentanyl 2 and 4 micrograms kg-1, respectively. A ceiling effect was not observed and there was a significant difference between the 2 and 4 micrograms kg-1 groups. PMID- 10740550 TI - Microdialysis study of the blood-brain equilibration of thiopental enantiomers. AB - Thiopental is a racemate of equimolar R- and S-thiopental enantiomers that have different potencies in laboratory experiments. We measured concentrations of R- and S-thiopental in plasma, tissues and brain microdialysate of rats after computer-controlled infusion of thiopental i.v. to a plasma concentration of 40 micrograms ml-1 for 20 min in two pharmacokinetic studies. In study 1, animals were found to maintain their target plasma concentrations, which then decayed biphasically after infusion. Brain microdialysate concentrations of both enantiomers increased from about 3% of corresponding plasma concentrations at 1 min to 9% at 20 min. In study 2, thiopental concentrations were found to be highest at 20 min in CNS tissue, at 30 min in muscle and at 60 min in fat. Tissue:plasma distribution coefficients of R-thiopental were greater than those of S-thiopental when calculated from total or unbound plasma concentrations. We found no pharmacokinetic evidence to support differences between the thiopental enantiomers in rates of equilibration across the blood-brain barrier after infusion of rac-thiopental. PMID- 10740551 TI - Gastrointestinal luminal PCO2 tonometry: an update on physiology, methodology and clinical applications. PMID- 10740552 TI - Wound infiltration with bupivacaine after surgery to the cervical spine using a posterior approach. AB - We have compared pain scores, morphine consumption and duration of stay for 50 adults who underwent elective cervical spine surgery via a posterior incision in a prospective, double-blind, placebo-controlled, randomized study. During wound closure, the paravertebral muscles and subcutaneous tissues were infiltrated with 40 ml of saline (control) or 0.25% bupivacaine. There were no significant differences in pain scores, morphine consumption or duration of stay between groups. In view of the potential risks of wound infiltration in the cervical region, we consider that this practice should be abandoned. PMID- 10740553 TI - Comparison of 1% ropivacaine with 0.75% bupivacaine and 2% lidocaine for peribulbar anaesthesia. AB - We have compared the efficacy of 1% ropivacaine with a mixture of 0.75% bupivacaine and 2% lidocaine for peribulbar anaesthesia in cataract surgery. We used the time to adequate block for surgery, and ocular and eyelid movement scores at 8 min after block as clinical end-points. Ninety patients were allocated randomly to receive 7-10 ml of a mixture of equal parts of 0.75% bupivacaine and 2% lidocaine or an equal volume of 1% ropivacaine alone. Hyaluronidase 15 iu ml-1 was added to both solutions. There were no differences between groups in clinical end-points. Median time at which the block was adequate to start surgery was 8 min (interquartile range 4-10 min) in each group. Median eyelid movement scores were similar in both groups, but the bupivacaine and lidocaine mixture produced a significantly decreased ocular movement score at 2, 4 and 6 min (P < 0.05). There was no difference between groups in the incidence of minor complications. Based on clinical end-points, time to adequate block for surgery and median ocular and eyelid movement scores at 8 min, 1% ropivacaine as the sole agent for peribulbar anaesthesia was comparable with a mixture of 0.75% bupivacaine and 2% lidocaine. PMID- 10740554 TI - Effects of respiratory and metabolic pH changes and hypoxia on ropivacaine induced cardiotoxicity in dogs. AB - We have studied the effects of acute changes in acid-base status and hypoxia on the cardiotoxic effects of intracoronary injection of ropivacaine in anaesthetized dogs. The effects of intracoronary ropivacaine were compared when ropivacaine was administered during eucapnia and during each of another nine states in random order: hypocapnia, hypercapnia, hypoxia, metabolic alkalosis, metabolic acidosis, combined metabolic acidosis and hypocapnia, combined metabolic alkalosis and hypercapnia, combined hypoxia and hypercapnia, and combined metabolic acidosis and hypoxia. Hypocapnic alkalosis consistently reduced the cardiotoxic effects of intracoronary ropivacaine (P < 0.01). Our findings indicate that induction of hypocapnic alkalosis may provide a useful adjunct to standard resuscitative measure after inadvertent administration of amide local anaesthetic agents. PMID- 10740555 TI - I.v. regional diamorphine for analgesia after foot surgery. AB - Opioids administered to peripheral tissues can have significant analgesic effects in doses which would not be effective centrally. We have assessed the effects of regional diamorphine 2.5 mg i.v. in 14 patients undergoing surgical correction of bilateral arthritic foot deformities in a prospective, randomized, double-blind study. Patients acted as their own controls as only one foot received the active drug. Visual analogue scale (VAS) pain scores and wound tenderness were measured over 72 h. Diamorphine did not improve median VAS area under the curve pain scores during the first 6 h after surgery (33 (95% confidence intervals (CI) 25 46) vs 24 (17-35)). It also did not effect wound hypersensitivity when tested at 72 h after surgery (95 (47-125) vs 90 (50-125) g). There were no significant adverse effects. PMID- 10740556 TI - Analgesic action of i.v. morphine-6-glucuronide in healthy volunteers. AB - The pharmacodynamics of morphine-6-glucuronide (M-6-G) i.v. were assessed in 12 healthy male volunteers in an open study. After a single bolus dose of M-6-G 5 mg i.v., we measured antinociceptive effects, using electrical and cold pain tests, and plasma concentrations of M-6-G, morphine-3-glucuronide (M-3-G) and morphine. Pain intensities during electrical stimulation (at 30, 60 and 90 min after injection) and ice water immersion (at 60 min) decreased significantly (P < 0.005) compared with baseline. Mean plasma peak concentrations of M-6-G were 139.3 (SD 38.9) ng ml-1, measured at 15 min. Our data demonstrate that M-6-G has significant analgesic activity. PMID- 10740557 TI - Comparison of different doses of remifentanil on the cardiovascular response to laryngoscopy and tracheal intubation. AB - We have compared three bolus and infusion regimens of remifentanil on the cardiovascular response to laryngoscopy and orotracheal intubation in three groups of 20 ASA I-II female patients, in a randomized, double-blind study. Patients in group 1 received glycopyrolate 200 micrograms i.v. followed by a bolus dose of remifentanil 1 microgram kg-1 over 30 s and an infusion of remifentanil at a rate of 0.5 microgram kg-1 min-1. The other patients received remifentanil 0.5 microgram kg-1 over 30 s and an infusion of 0.25 microgram kg-1 min-1 with (group 2) or without (group 3) pretreatment with glycopyrrolate 200 micrograms. All patients then received a sleep dose of propofol, rocuronium 0.6 mg kg-1 and 1% isoflurane with 67% nitrous oxide in oxygen. Laryngoscopy and tracheal intubation were performed 3 min later. Heart rate and arterial pressure were recorded at 1-min intervals from before induction of anaesthesia until 5 min after intubation. Baseline heart rate was similar in all groups, but decreased in group 3 (no glycopyrrolate) after induction and remained significantly lower after intubation compared with the other groups (P < 0.05). Heart rate and arterial pressure increased slightly after intubation in each group but there were no significant differences in mean arterial pressure between groups at any time. The incidence of bradycardia (one patient in group 2) and hypotension (two patients in groups 1 and 2 and three patients in group 3) was low. PMID- 10740558 TI - Comparison of laryngeal mask and intubating laryngeal mask insertion by the naive intubator. AB - Seventy-five inexperienced participants were timed inserting the laryngeal mask airway (LMA) and the intubating laryngeal mask (ILM) in one of five cadavers. Adequacy of ventilation was assessed on a three-point scale depending on chest expansion and air leak. Participants were also asked to intubate the trachea via the ILM. The ILM was inserted faster than the LMA (P < 0.05) with a greater proportion achieving adequate ventilation after their first attempt (P < 0.05). Tracheal intubation via the ILM was completed successfully by 67% (52 of 75) of participants. In a questionnaire, participants stated that the ILM was easier to use and the preferred device in an emergency. The results suggest that inexperienced practitioners should use the ILM rather than the LMA for emergency ventilation. PMID- 10740559 TI - Pain on injection of rocuronium: influence of two doses of lidocaine pretreatment. AB - We have assessed the incidence of pain on injection of rocuronium and evaluated if pretreatment with lidocaine i.v. reduced it, in a randomized, controlled study in 90 patients. We found that 37% of patients who received lidocaine 10 mg pretreatment had pain on injection of rocuronium compared with 77% of patients who received saline pretreatment and 7% of patients who were pretreated with lidocaine 30 mg (P < 0.05 in each instance compared with control). In addition, patients pretreated with lidocaine were less likely to suffer moderate or severe pain. Both lidocaine 10 mg and 30 mg i.v. given before administration of rocuronium significantly reduced the incidence and severity of pain on injection of rocuronium, and the higher dose was more effective. PMID- 10740560 TI - Histaminoid reactions associated with rocuronium. AB - We describe three histaminoid reactions occurring on induction of anaesthesia. The patients were all resuscitated successfully and subsequent skin testing suggested sensitivity to rocuronium. In this hospital, the incidence of such reactions is of the order of 1 in 3000. This may be coincidental but suggests that there should be close monitoring of the incidence of reactions to rocuronium. Review of the cases suggests that current guidelines on management are not always followed. PMID- 10740561 TI - Fatal paradoxical air embolism during liver transplantation. AB - We describe a case of fatal paradoxical coronary air embolism during liver transplantation. The literature on the diagnosis and prophylaxis of paradoxical air embolism during liver transplantation is reviewed and discussed. PMID- 10740562 TI - Tension pneumocephalus after neurosurgery in the supine position. AB - Tension pneumocephalus has been reported most frequently after posterior fossa surgery performed in the sitting position. We present a paediatric patient who developed tension pneumocephalus in the postoperative period after decompression of a craniopharyngioma performed with the patient in the supine position. PMID- 10740563 TI - Severe vasovagal attack during regional anaesthesia for caesarean section. AB - A patient experienced a severe vasovagal attack during regional anaesthesia for elective Caesarean section. The combination of vagal over-activity and sympathetic block produced profound hypotension that threatened the life of the mother and infant. The vasovagal syndrome is described, and its prevention and management discussed. PMID- 10740564 TI - The Woolley and Roe case. AB - Albert Woolley and Cecil Roe were healthy, middle-aged men who became paraplegic after spinal anaesthesia for minor surgery at the Chesterfield Royal Hospital in 1947. The spinal anaesthetics were given by the same anaesthetist, Dr Malcolm Graham, using the same drug on the same day at the same hospital. The outcome for the patients and their families was devastating, as it was for the use of spinal anaesthesia in the UK. At the trial 6 yr later, and against the opinion of leading neurologists, the judge accepted Professor Macintosh's suggestion that phenol, in which the ampoules of local anaesthetic had been immersed, had contaminated the local anaesthetic through invisible cracks. In an interview 30 yr after the verdict, Dr Graham believed tha the tragedy was caused by contamination of the spinal needles or syringes during the sterilization process. The subsequent explanation that, on the day in question, descaling liquid in the sterilizing pan had not been replaced by water, supported his belief and finally offered a credible explanation. We review the Woolley and Roe case, the status of spinal anaesthesia before and after 1947, and the relevant medico-legal judgments in claims for negligence in the early days of the National Health Service. PMID- 10740565 TI - Putting the sterile syringe packaging to good use. PMID- 10740566 TI - Identifying tracheomalacia. PMID- 10740567 TI - Haemodynamic and/or tonometric monitoring in cardiac surgery. PMID- 10740568 TI - Implications of the Relenza ruling. PMID- 10740569 TI - Accountability, clinical governance and the acceptance of imperfection. PMID- 10740570 TI - Medical relief in earthquakes. PMID- 10740571 TI - Genetics of diabetic nephropathy and microalbuminuria. PMID- 10740572 TI - Elder abuse: do general practitioners know or care? AB - A pilot survey in Tower Hamlets, London, indicated that many general practitioners (GPs) might not be recognizing abuse of elderly patients through lack of training. The survey was replicated on a large scale in Birmingham, to allow further analysis. 561 Birmingham GPs were mailed questionnaires and responses from 291 were analysed, providing data from 95% of the practices. The findings were similar to those in Tower Hamlets: just under half had diagnosed elder abuse in the previous year. Regression analysis of the combined data-sets (n = 363) indicated that the strongest factor predicting GP diagnosis of abuse was knowledge of 5 or more risk situations (odds ratio 6.77, 95% confidence interval 4.19, 10.93). The findings of these surveys suggest that research-based education and training would help GPs to become better at identifying and managing elder abuse. PMID- 10740573 TI - Larva therapy in wound management. AB - The use of maggots for wound debridement has a long history and has lately gained ground in several countries. We collected prospective data to examine the current use of larva therapy (LT) in the UK. Quantitative information was collected on 70 patients treated in nine hospitals. LT is used primarily to treat leg ulcers and generally involves three applications of larvae at two to three day intervals. This method is judged effective in wound debridement and promotes the growth of granulation tissue. Wound exudate, odour, infection and pain are all reduced by the treatment. Adverse reactions are infrequent but include pain, bleeding, pyrexia and influenza-like symptoms. Prevention of hospital admission and surgery, reduced need for antibiotics and reduced hospital stay are all identified as outcomes of LT. The nurse practitioners who used LT believed it to have an important role in wound management. A randomized clinical trial, comparing LT with other debriding agents, is required for evaluation of cost effectiveness. PMID- 10740574 TI - Anaphylactic reaction to radioimmunotherapy despite plasmapheresis to remove anti mouse antibodies. PMID- 10740575 TI - Dipyridamole in the treatment of a neonate with persistent pulmonary hypertension. PMID- 10740576 TI - Two aorto-iliac bypass graft infections associated with appendicitis. PMID- 10740577 TI - Bilateral sixth and fourth cranial nerve palsies in idiopathic intracranial hypertension. PMID- 10740578 TI - Snoring with sleep apnoea: deceptive appearances. PMID- 10740579 TI - Sclerosing mediastinitis with aortic valve regurgitation and aortic root dilatation. PMID- 10740580 TI - Chicken bone injury of the common bile duct. PMID- 10740581 TI - Extrarectal mass: a tailgut cyst. PMID- 10740582 TI - Sleep disorders in the Macbeths. PMID- 10740583 TI - Beauty is only skin deep. PMID- 10740584 TI - Sir William Osler--Abroad with Jane. PMID- 10740585 TI - Medicine at the Battle of Stalingrad. PMID- 10740586 TI - A national database of medical error. PMID- 10740587 TI - Fibrolipoma of the median nerve. PMID- 10740588 TI - The struck-off mystery. PMID- 10740589 TI - The limits of pressure sore prevention. PMID- 10740590 TI - Non-senile squalor. PMID- 10740591 TI - Ethics committees. PMID- 10740592 TI - Imprint cytology of parathyroid tissue in relation to other tissues of the neck and mediastinum. AB - OBJECTIVE: To retest the hypothesis that imprint cytology may be used to reliably diagnose parathyroid tissue and, if so, to ascertain whether accuracy in this technique may be easily attained. STUDY DESIGN: Imprint preparations from 15 parathyroid, 10 thyroid, 8 lymphoreticular and 2 adipose tissue specimens were assessed blindly by two pathologists, one of whom (pathologist B) had only limited experience with endocrine tissue imprint cytology. RESULTS: Both assessors consistently distinguished parathyroid and thyroid preparations from lymphoreticular and adipose tissue preparations. While there was occasional difficulty in distinguishing between parathyroid and thyroid preparations, this was usually attributable to the scanty nature of the preparations. No single cytologic feature allowed a distinction between parathyroid and thyroid tissue. However, by considering several relatively diagnostic features collectively, pathologist B showed an increase in specificity and sensitivity rates for distinguishing parathyroid from thyroid imprints from 82% to 100% and 57% to 83%, respectively. CONCLUSION: The high accuracy rates and rapid [table: see text] learning curve shown by imprint cytology in distinguishing between different neck or mediastinal tissue types, together with its time- and cost-cutting potential, support a role for the technique in the intraoperative diagnosis of parathyroid tissue. PMID- 10740593 TI - Detection of Chlamydia trachomatis in pregnant women by the Papanicolaou technique, enzyme immunoassay and polymerase chain reaction. AB - OBJECTIVE: To compare Papanicolaou staining, enzyme immunoassay (EIA) and the polymerase chain reaction (PCR) techniques for detecting Chlamydia trachomatis in pregnant women. STUDY DESIGN: Endocervical specimens were taken randomly from 125 pregnant women with or without symptoms. These women attended their first medical consultation at the Regional General Ignacio Zaragoza Hospital. Samples were analyzed for detection of C trachomatis. When results differed between tests, specimens were evaluated by direct immunofluorescence staining. RESULTS: The prevalence of chlamydial infection was 2.4%. The characteristics of patients positive for Chlamydia were: average age, 24 years; first sexual encounter at age 21 years, one partner and six to nine months of gestation. The sensitivity, specificity, accuracy, positive predictive values and negative predictive values were 100%, 99.18%, 99.20%, 75% and 100%, respectively, for Papanicolaou staining; 100%, 92.62%, 92%, 25% and 100% for EIA; and 100%, 100%, 100% and 100% for PCR. CONCLUSION: Both Papanicolaou staining and PCR were adequate for diagnosis of C trachomatis infection. EIA was not reliable and therefore is not recommended for use as a diagnostic technique in a pregnant population with low risk and low prevalence. PMID- 10740594 TI - Imprint cytology in the diagnosis of Helicobacter pylori. Does imprinting damage the biopsy specimen? AB - OBJECTIVE: To investigate the efficacy of imprint cytology in the diagnosis of Helicobacter pylori infection and whether it damages the biopsy specimen for subsequent histologic examination. STUDY DESIGN: Two antral biopsies were taken from 76 patients with dyspeptic symptoms undergoing upper gastrointestinal endoscopy. Imprint cytology was made from the first specimen. This specimen was fixed in 10% formalin and sent for histopathologic examination. The second specimen was directly fixed in 10% formalin for routine histopathologic examination without being used for an imprint. The imprint smears were examined by cytopathologists. The biopsy specimens were examined by pathologists who did not know which specimens were used for the imprints. RESULTS: H pylori was seen in smears from 55 (72%) patients and in both biopsy specimens from the same patients. The pathologists could not recognize the biopsy specimens from which the imprints were made. Concordance between imprint cytology and histopathology was 100%. CONCLUSION: Imprint cytology is a suitable test for H pylori diagnosis, and imprints do not adversely affect the quality of the biopsy specimen. PMID- 10740595 TI - Evaluation of the AutoCyte SCREEN system in a clinical cytopathology laboratory. AB - OBJECTIVE: To compare the AutoCyte SCREEN (AutoCyte, Burlington, North Carolina, U.S.A.) system with manual screening by experienced cytotechnologists using thin layer preparations that had been previously extensively studied and their cytologic abnormalities well defined. STUDY DESIGN: AutoCyte PREP (AutoCyte) samples prepared for a previous split-sample study comparing thin-layer preparations to conventional smears were used. These 1,992 AutoCyte PREP samples were in a cohort the abnormal findings of which had been confirmed via independent review by two sets of pathologists. For the current study, these samples were remasked and evaluated by the AutoCyte SCREEN system in a clinical laboratory. The instrument scanned each slide and selected six overview fields and 120 single objects for storage and display. The computer classified each slide in one of the following categories: abnormal, uncertain, normal or unsatisfactory. Independently for each case, a cytotechnologist evaluated the six fields and 120 objects selected by the instrument as abnormal, normal or unsatisfactory. For those cases classified as uncertain by AutoCyte, the technologist then reexamined the cellular displays and entered a consensus classification. These results were then compared to those of an independent review by cytotechnologists of the identical set of slides using routine manual screening. RESULTS: The AutoCyte SCREEN selected 35% of slides for manual review. Technologist and computer rendered equivalent classifications in 79%. Of the total slides screened by the AutoCyte SCREEN, 57% were classified as "uncertain," and 88% of these were subsequently classified as normal by consensus. Using the well-defined abnormal values of the cellular sample as a basis for calculation, the AutoCyte SCREEN-assisted practice had a diagnostic sensitivity of 85% and diagnostic specificity of 97.6%. Comparable values for manual screening of the identical cellular sample were a diagnostic sensitivity of 80% and specificity of 97.4%. CONCLUSION: The AutoCyte SCREEN achieves comparable or greater sensitivity in detecting cervical abnormalities in comparison with manual screening. When combined with the substantial advantage of thin-layer preparations over conventional smears, the AutoCyte SCREEN provides a screening system of superior sensitivity over conventionally prepared and examined cervical smears. PMID- 10740596 TI - Vaginal intraepithelial neoplasia and the Pap smear. AB - OBJECTIVE: To assess the sensitivity of the vaginal smear cytologic examination in detecting vaginal intraepithelial neoplasia (VAIN) and to evaluate the cytologic findings of cases of VAIN. STUDY DESIGN: Cases with a histologic diagnosis of VAIN were identified from the Barnes-Jewish Hospital South and North Campus over a period of five and nine years, respectively. Only posthysterectomy patients with a tissue biopsy diagnosis of VAIN and with a vaginal smear obtained within three months of the biopsy were included in the study. Pertinent clinical information was obtained by reviewing the medical records. Two pathologists reviewed the pathologic samples. RESULTS: Thirty-five vaginal smears from 31 posthysterectomy patients were included in the study. The mean age was 57 years (range, 29-84). The cytologic diagnoses of smears from patients with VAIN included: high grade squamous intraepithelial lesion (19 cases), low grade squamous intraepithelial lesion (10 cases), atypical squamous cells of uncertain significance (5 cases) and negative for malignancy (1 case). CONCLUSION: The sensitivity of the vaginal smear cytologic examination in detecting VAIN is 83%. Obscuring inflammation contributed to false negative diagnoses in two cases. PMID- 10740597 TI - Effects of tamoxifen on cervicovaginal smears from patients with breast cancer. AB - OBJECTIVE: To evaluate the effect of tamoxifen on cervicovaginal epithelium and determine the value of cervicovaginal smears in identifying patients at risk for endometrial carcinoma. STUDY DESIGN: A group of 48 women with prior breast cancer were divided into three groups: A, tamoxifen-treated patients who developed endometrial carcinoma (n = 20); B, patients with endometrial cancer not treated with tamoxifen (n = 22); and C, tamoxifen-treated patients with no endometrial carcinoma (n = 16). A total of 114 cervicovaginal smears from these patients were evaluated for maturation index, histiocytes, benign and malignant endometrial cells, reactive cellular changes and microorganisms. All patients treated with tamoxifen had received doses of 10 mg twice daily. RESULTS: The maturation index was increased in tamoxifen-treated patients (A and C) versus nontreated patients (B) P < or = .001). The number of cases with endometrial cells was significantly higher in smears of treated patients who developed endometrial cancer (A) as compared to groups B and C (P = .01 and .02, respectively). Histiocytes were also significantly increased in the two groups that subsequently developed endometrial carcinoma (A and B) as compared to the group that did not (group C) (P = .02). There was no significant difference in the presence of reactive cellular changes between the three groups. CONCLUSION: Patients treated with tamoxifen exhibited a partial estrogenic effect in their smears regardless of whether they developed endometrial cancer. However, the presence of endometrial cells in the smears indicated a higher risk of endometrial adenocarcinoma. PMID- 10740598 TI - Fine needle aspiration cytology of vaginal cuff lesions. AB - OBJECTIVE: To assess the usefulness of fine needle aspiration (FNA) of the vaginal cuff in various lesions in the female genital tract. STUDY DESIGN: Twenty six FNAs of vaginal cuff lesions were performed at our institution from 1994 to 1998. All cases were reviewed. Data on clinical histories and follow-up, if available, were collected. Histologic material was reviewed. Fourteen gynecologic (cervicovaginal) smears performed on these patients within six months prior to the FNA were also reviewed. RESULTS: Two of twenty-six (7.7%) FNAs were non diagnostic due to poor cellular yield. Four lesions were classified as benign (16.7%). The remaining 20 FNAs were classified as malignant (83%) and were consistent with the clinical history in all cases. CONCLUSION: The results show that FNA of the vaginal cuff appears to be a simple and accurate procedure for detecting benign and malignant lesions of the female genitourinary tract. Clinical indications, cytologic techniques and potential pitfalls are identical to those of FNAs at other sites. PMID- 10740599 TI - Quality control of cervical cytology in high-risk women. PAPNET system compared with manual rescreening. AB - OBJECTIVE: To compare the effectiveness of the PAPNET System with conventional rescreening of negative cervical smears in a high-risk population. STUDY DESIGN: Three thousand ninety-seven negative cervical smears from women with past history of cervical abnormalities were rescreened manually and with the PAPNET System. There were two reviews of PAPNET images: the first by two cytotechnologists with limited exposure to the instrument, and the second, limited to smears with discrepant diagnoses, by an expert in the use of the system. The remaining discrepant smears were submitted to a blinded microscopic review by a third party. The a priori consensus diagnosis was arbitrarily established when the result of two of the three reviews--manual, PAPNET and the independent third review--were concordant. The results of rescreening were compared with available biopsies. RESULTS: On manual rescreening of the 3,097 smears, 2,901 (93.66%) were reported as negative and 170 (5.49%) as abnormal. On the first PAPNET review, 2,938 (94.87%) were reported as negative and 150 (4.84%) as abnormal. There were 144 smears with discrepant diagnoses. After the second PAPNET review of these discrepant smears, the agreement between manual and PAPNET rescreening rose from 94.27% to 95.58%. A final, blinded review of 89 residual discrepant smears was used to establish consensus diagnoses. The diagnoses made by PAPNET-assisted rescreening agreed much better with the consensus diagnoses than did manual rescreening (Kappa = .61 vs. Kappa = -.32, P < .001). When compared with the results of 50 available biopsies, PAPNET-assisted rescreening also had a somewhat lower false negative rate (sensitivity 58.82% vs. 41.18%, P = .17) and a statistically significant lower false positive rate (specificity 63.64% vs. 36.36%, P = .01). CONCLUSION: PAPNET-assisted rescreening, when carried out by an experienced person, is more efficient than manual rescreening. PMID- 10740600 TI - Transcutaneous fine needle aspiration biopsy of the preepiglottic space. AB - OBJECTIVE: To evaluate transcutaneous fine needle aspiration biopsy of the preepiglottic space for staging supraglottic squamous cell carcinoma. STUDY DESIGN: We studied 28 patients who underwent total or horizontal supraglottic laryngectomy as their main therapy modality due to supraglottic squamous cell carcinoma, followed in some cases by adjuvant radiation therapy. All the patients underwent transcutaneous fine needle aspiration. RESULTS: The cytopathologic examination of the material obtained by aspiration was compared to the histopathologic analysis of the laryngectomy specimens; the efficiency was 96.4%. The method did not cause any morbidity. CONCLUSION: Transcutaneous fine needle aspiration biopsy of the preepiglottic space is useful for preepiglottic space evaluation, with a high correlation with histopathologic results and no morbidity. PMID- 10740601 TI - Cystic degeneration in phyllodes tumor. A source of error in cytologic interpretation. AB - OBJECTIVE: To examine problems encountered in the cytologic interpretation of phyllodes tumor (PT) with cystic degeneration and solutions thereof. STUDY DESIGN: Cystic degeneration was found in seven PTs (five benign, one low grade and one high grade). Aspirates from these yielded fluid and were usually labelled fibrocystic change on the original cytology. Smears were retrospectively analyzed, with special attention to the background, presence and nature of the epithelial and stromal fragments, foam cells and naked nuclei in the background. RESULTS: PTs with cystic degeneration on cytology showed thick fluid in the background, foamy macrophages (100%), apocrine cells (28%) and epithelial fragments, which showed nuclear atypia in two cases. On reviewing the smears, five of seven PTs had stromal fragments, albeit in small numbers. Most important, even in the absence of stromal fragments, all cases showed 5-50% naked nuclei of the fibroblastic type dispersed within the fluid background. CONCLUSION: In cases of fluid aspirates from well-defined lumps, one must search for fibroblastlike naked nuclei or stromal fragments within the fluid to clinch the diagnosis of phyllodes tumor. PMID- 10740602 TI - Cytologic appearances in invasive lobular carcinoma of the breast. A study of 21 cases. AB - OBJECTIVE: To examine the fine needle aspiration cytologic features of invasive lobular carcinoma of breast and to discuss problems that may occur in cytodiagnosis. STUDY DESIGN: Fine needle aspiration cytologic smears from 21 cases of invasive lobular carcinoma (ILC) of breast were subjected to detailed cytomorphologic analysis. Features studied included pattern of cells, size of cells, nuclear placement, pleomorphism, presence of intracytoplasmic lumina (ICL) and signet ring cells. RESULTS: Cellularity was generally moderate or high, and the pattern was predominantly or partly dissociated in 86% of cases. Rosettelike pattern was discerned in alveolar-type ILC. Cell size was usually small or intermediate, with nuclei placed eccentrically in most cases. ICLs with or without signet ring cells were present in 12 cases (57%). CONCLUSION: A cytologic picture consisting of predominantly dissociated small or intermediate-sized tumor cells with eccentric nuclei, with some of the cells showing ICLs, is highly suggestive of ILC. Indian file pattern, another characteristic feature of ILC, is, however, focal and inconsistent. Variant patterns of ILC may show other cytologic features, such as rosettelike pattern (alveolar variant of ILC) or large cell pattern (pleomorphic variant of ILC) and may consequently be difficult to categorize on cytologic smears. PMID- 10740603 TI - Telecytologic diagnosis of breast fine needle aspiration biopsies. Intraobserver concordance. AB - OBJECTIVE: To determine the intraobserver concordance between telecytologic and glass slide diagnosis of breast fine needle aspirates. STUDY DESIGN: Twenty-five cases, originally received in consultation, were each examined by three cytopathologists. An average of seven compressed digital images per case were presented, together with a brief clinical history, using the http protocol and an internet browser. RESULTS: Agreement between the telecytologic and glass slide diagnosis ranged from 80% to 96%. Nevertheless, two cases that had been unequivocally diagnosed as malignant based upon video images were considered to be benign by the same pathologist when reviewing the glass slides. Both diagnostic confidence and self-concordance were higher for one pathologist having significant previous video microscopy experience. CONCLUSION: Although intraobserver concordance between telecytologic and glass slide diagnoses of breast fine needle aspirates is high, refinement of existing criteria for diagnosis of malignancy, taking account of the particular limitations associated with telecytologic diagnosis, may be prudent prior to widespread use of telecytology for fine needle aspiration evaluation. PMID- 10740604 TI - Medullary thyroid carcinoma. Rare cytologic findings. AB - OBJECTIVE: To describe some rare cytologic findings in medullary thyroid carcinoma. STUDY DESIGN: Review of the fine needle aspiration smears from 15 cases of medullary thyroid carcinoma that were confirmed on histologic sections. The ages ranged between 31 and 67 years; 10 were female and 5 male. Thirteen were sporadic forms, and two were familial forms. RESULTS: Eight cases were classified as pleomorphic cell type and seven as monomorphic cell type. The smears revealed round, oval, triangular, polygonal and spindle-shaped cells, intracytoplasmic red granules, occasional intranuclear inclusions, amyloid, binucleated and multinucleated cells, and thick, granular chromatin. CONCLUSION: The rare cytologic findings in this study were grape cells, cytoplasmic nippling, elongated cytoplasmic processes, carrot-shaped nuclei, nuclear buddings, mast cell-like cells and a Burkitt's lymphoma-like appearance. These findings were rarely reported before. PMID- 10740605 TI - Cytomorphologic features of Merkel cell carcinoma in fine needle aspiration biopsies. A study of two atypical cases. AB - OBJECTIVE: To report atypical cytomorphologic features in fine needle aspiration biopsies (FNABs) from two cases of Merkel cell carcinoma (MCC), a primary neuroendocrine neoplasm of skin. STUDY DESIGN: Retrospective review of FNABs with histologic correlation from six patients with MCC and a report of findings from two whose smears showed atypical features. RESULTS: Typically the aspirates produce highly cellular smears of loosely clustered and individual, relatively monomorphic, small tumor cells with round to oval, regularly contoured nuclei. In two of our cases, the tumor cell nuclei exhibited a spectrum of pleomorphism ranging from moderately complex nuclear membranes with cleaves, indentations and protrusions in one case to large, markedly bizarre, convoluted nuclei and multinucleate tumor cells in the extreme case. Both cases were primary neoplasms, and the diagnosis was based on clinical, histologic and immunohistochemical data. Additionally, electron microscopy was performed on the tumor with bizarre nuclei and demonstrated rare, dense core neurosecretory granules and paranuclear bundles of intermediate filaments. PMID- 10740606 TI - Apoptotic index from fine needle aspiration cytology as a criterion to predict histologic grade of non-Hodgkin's lymphoma. AB - OBJECTIVE: To investigate whether the assessment of apoptotic index (AI) from fine needle aspiration (FNA) smears of non-Hodgkin's lymphomas (NHL) is reliable and has potential utility as a criterion to predict histologic grade. STUDY DESIGN: AI was independently determined by four cytopathologists as a percentage from routine FNA smears in 96 NHLs and 15 lymphoid hyperplasias. Working formulation (WF) grades from corresponding surgical biopsies were modified to include mantle zone-derived NHLs as intermediate grade and to make diffuse large cell NHL a separate category called "high" grade, whereas WF high grade NHLs were called "very high" grade. Histologic grades were also derived from the Revised European American Lymphoma (REAL) classification. AI was compared with histologic grade using the unpaired, two-tailed Student t test. These data were used to determine potential thresholds for AI that separate lower from higher grade NHLs. RESULTS: Measurements of AI strongly correlated between cytopathologists (median r = .93). Low and intermediate grade NHLs had indistinguishable AIs, whereas higher grade NHLs had significantly higher AIs. Appropriate potential AI thresholds between low or intermediate grade and higher grade NHLs were in the range of 1.5-2.5% (modified WF) and 1-2% (REAL). CONCLUSION: There is excellent interobserver reliability in the measurement of AI from FNAs of NHLs. Higher AIs distinguish higher from lower grade NHLs. Diffuse large cell NHLs had AIs that were similar to WF high grade NHLs. PMID- 10740607 TI - Cytologic and biomolecular diagnosis of polyomavirus infection in urine specimens of HIV-positive patients. AB - OBJECTIVE: To evaluate the frequency of human polyomavirus reactivation in urine specimens from HIV-positive patients; compare the sensitivity of cytology, immunohistochemistry and molecular biology; differentiate viral genotypes; and correlate the results with urinary cytologic abnormalities. STUDY DESIGN: Urine specimens from 78 unselected HIV-positive patients were evaluated by means of cytology, immunohistochemistry and nested polymerase chain reaction (n-PCR) to evaluate the presence of polyomaviruses. Restriction fragment length polymorphism (RFLP) was carried out in positive cases in order to differentiate BK virus (BKV) from JC virus (JCV). CD4 cells and serum creatinine levels were evaluated as indices of immune status and renal function, respectively, whereas the presence of red blood cells was used as an index of urogenital damage. RESULTS: Cytologic evidence of polyomavirus infection was found in 17 samples and immunohistochemically confirmed in 9; another 6 cytologically negative cases were detected by means of immunohistochemistry. In all cases, only one or two cells showed typical viral inclusions or positive staining. n-PCR identified 44 positive samples, thus confirming all of the cytologically and immunohistochemically positive cases and detecting polyomavirus genome in a further 21. RFLP detected 39 JCV, 1 BKV and 4 JCV-BKV infections. No correlation was found between the presence or type of polyomavirus and immune status, but red blood cells were found more frequently in the positive than in the negative samples. Serum creatinine levels fell within the normal range in all cases. CONCLUSION: Molecular biology is the most sensitive tool for detecting polyomavirus urinary infection in HIV-positive patients and the only reliable method of differentiating JCV and BKV viral genotypes. PMID- 10740608 TI - A low-power, "architectural," clue to the follicular variant of papillary thyroid adenocarcinoma in aspiration biopsy. AB - OBJECTIVE: To search for low-power, or "architectural," clues to the diagnosis of the follicular variant of papillary thyroid adenocarcinoma (FVP) in Diff-Quik stained aspiration biopsy smears in order to aid in the rapid diagnosis of FVP, especially as a complement to frozen sections. STUDY DESIGN: The smears of 23 cases, each with tissue-proven FVP, were compared to those of 23 cases of classic papillary thyroid adenocarcinoma (PTA), 23 cases of tissue-proven follicular neoplasms (FN) (i.e., adenoma or well-differentiated adenocarcinoma) and 23 samples of colloid nodules (CN). The low-power (10x) features of the four groups were studied and compared. RESULTS: Our study showed that FVP exhibited monolayered cellular sheets with branched, irregular contours, which can be distinguished from the uniform microfollicles with smooth contours formed in FN and the large, round or oval monolayered sheets of follicular cells found in CN. FVP shared all of the features of classic PTA except for the larger, complex sheets of epithelial cells and psammoma bodies found in the latter. CONCLUSION: The branched sheets of epithelial cells evident during low-power examination of Diff-Quick-stained smears of thyroid aspirates are a ready first clue to the rapid diagnosis of FVP. The diagnosis can be confirmed by subsequent careful evaluation of nuclei in Papanicolaou-stained smears and sections of tissue. PMID- 10740610 TI - Adult rhabdomyoma in fine needle aspirates. A report of two cases. AB - BACKGROUND: Adult rhabdomyoma (ARh) is a rare, benign tumor arising most frequently in the head and neck region and sometimes mimicking malignant tumors clinically. Correct preoperative evaluation of this tumor is of crucial importance as its treatment is complete excision only and not radical surgery. CASES: Two patients with ARh, one tumor presenting near the submandibular gland and the other in the thyroid area, are reported. The first tumor was correctly diagnosed by fine needle aspiration cytology. The second, clinically suspected to be a colloid goiter, was preoperatively diagnosed as such cytologically as well. After the tumor was excised, reexamination of the cytologic specimen disclosed follicle cells admixed with single cells from ARh; these had been interpreted as colloid fragments at the time of primary evaluation. CONCLUSION: Fine needle aspiration evaluation of ARh may be problematic due to the rarity of the tumor and to the similarity of the tumor cells to normal striated muscle and to other tumors in which cells with abundant granular cytoplasm are characteristic. With an awareness of the cytologic features of this uncommon tumor, cytopathologists can render a correct diagnosis. PMID- 10740609 TI - Utility of CD34 reactivity in evaluating focal nodular hepatocellular lesions sampled by fine needle aspiration biopsy. AB - OBJECTIVE: To determine the patterns of CD34 reactivity in hepatocellular adenoma and focal nodular hyperplasia and to evaluate the utility of CD34 reactivity in the diagnosis of hepatocellular carcinoma. STUDY DESIGN: Seventeen cases of well differentiated hepatocellular carcinoma, 14 cases of cirrhosis, 9 cases of focal nodular hyperplasia and 7 cases of hepatocellular adenoma were stained with immunoperoxidase antibodies to CD34. The slides were scored according to the degree of lesional reactivity. RESULTS: Fourteen of 17 cell blocks with hepatocellular carcinoma showed unequivocal sinusoidal or peripheral reactivity for CD34. Five of seven cases of hepatocellular adenoma and four of nine cases of focal nodular hyperplasia showed > 50% sinusoidal reactivity for CD34. All 14 cases of cirrhosis showed peripheral to no sinusoidal reactivity. CONCLUSION: CD34 reactivity in a diffuse sinusoidal pattern can be helpful in the diagnosis of hepatocellular carcinoma. However, consideration should be given to the possibility of hepatocellular adenoma and focal nodular hyperplasia, which can also exhibit significant diffuse CD34 reactivity. In these cases, a reticulin stain may be helpful with the differential diagnosis. PMID- 10740612 TI - Cytologic diagnosis of metastatic basal cell carcinoma. Report of a case with immunocytochemical and molecular pathologic considerations. AB - BACKGROUND: Metastases of basal cell carcinoma (BCC) are extraordinarily rare events, with only about 200 published reports. The usefulness of immunohistochemical markers in the diagnosis of metastatic BCC was previously established on cytologic material. Furthermore, in recent years, numerous molecular markers have been studied to explain its pathogenesis and relatively indolent behavior. CASE REPORT: A 62-year-old, white male presented with lymphadenopathy in the right side of the neck. The patient had a long-standing history of multiple excisions of BCCs during the previous 30 years. Fine needle aspiration biopsy revealed tight clusters and sheets of small, round tumor cells with hyperchromatic nuclei, small nucleoli and minimal cytoplasm. In addition, in some of the clusters the tumor cells showed peripheral palisading. Based on the cytomorphology and diffuse immunohistochemical positivity for a low-molecular weight cytokeratin marker, MNF 116, and negativity for AE1/AE3, Cam5.2, synaptophysin and chromogranin, a diagnosis of metastatic BCC was rendered. Subsequent histopathologic examination of metastatically involved lymph nodes removed in a radical neck dissection confirmed this diagnosis. In addition, on histologic sections the metastatic tumor cells were found to express bcl-2 and CD44, markers that have been recently studied in cutaneous tumors. CONCLUSION: In acquiring metastatic potential, this lesion did not lose the molecular characteristics of bcl-2 and CD44 expression, the two features deemed to be important in the indolent behavior of BCC. PMID- 10740611 TI - Bronchial brushing cytology features of primary malignant fibrous histiocytoma of the lung. A case report. AB - BACKGROUND: Malignant fibrous histiocytoma (MFH) of the lung is rare. Early diagnosis is very important because of its poor prognosis. Long-term survivors of pulmonary MFH are patients who had surgical resection. When the patient can undergo surgery after a prompt diagnosis, the prognosis improves more than with other therapy. However, it is not easy to establish the diagnosis of thoracic MFH. In general, the small fragments from bronchial or percutaneous transthoracic fine needle aspiration (FNA) biopsies are inadequate for cytologic or pathologic analysis. Bronchial brushing cytology is greatly superior to FNA cytology because one can obtain a large amount of cells. Therefore, bronchial brushing cytology may play a useful role in diagnosis when endobronchial involvement is found. CASE: A 65-year-old female was admitted with a cough, yellow sputum and exertional dyspnea. A chest roentgenogram showed a 12 x 12-cm mass in the left lung field. Bronchial brushing cytology revealed many fibroblastlike, histiocytelike, bizarre and multinucleated giant cells in a background of necrosis. Atypical mitotic figures were also found. The cytologic findings strongly suggested MFH. Although the pathologic findings from FNA biopsy showed storiform clusters structured by pleomorphic, fibroblastlike cells with bizarre nuclei and mitotic figures, the material was too small to diagnose it definitively. Six months later the patient died. An autopsy confirmed the diagnosis of MFH: the typical storiform clusters were composed of many fibroblastlike and histiocytelike cells that were positive for CD68 (PGM1) antibody. CONCLUSION: Bronchial brushing cytology may be a useful method for early, definitive diagnosis of MFH. The presence of pleomorphic, spindle-shaped fibroblastlike and histiocytelike cells with the clusters showing a storiform pattern may permit the diagnosis of MFH. PMID- 10740613 TI - Circulating cancer cells in peripheral blood. A case report. AB - BACKGROUND: Carcinocythemia, the presence of circulating cancer cells in peripheral blood, is a rare complication of solid neoplasms. When the number of such cells is very high, they can be detected during routine laboratory tests. They are associated with a dismal prognosis. CASE REPORT: Carcinocythemia occurred in a patient with disseminated breast cancer. Eighteen cases were identified from a review of the literature. The most common neoplasms associated with circulating cancer cells in peripheral blood were breast adenocarcinoma, small cell lung carcinoma and rhabdomyosarcoma. All the patients had stage IV disease at the time of diagnosis, and all had involvement of the reticuloendothelial system. Patients survived for an average of a few days or weeks. CONCLUSION: Circulating cancer cells in peripheral blood are an unusual manifestation of disseminated neoplasms that occurs as a terminal event. PMID- 10740614 TI - High cellular atypia in a pulmonary tumorlet. Report of a case with cytologic findings. AB - BACKGROUND: Pulmonary tumorlets are localized lesions of neuroendocrine cell proliferation, usually found in association with chronic pulmonary inflammation. Since they are mostly incidental histologic or radiologic discoveries, they have received little attention, and there have been no reports on their detailed cytology. We describe for the first time the cytologic features of a pulmonary tumorlet and discuss its differential diagnosis. CASE: An abnormal nodule in the right lung field was discovered on a regular checkup by chest roentgenogram in a 70-year-old, nonsmoking female. Intraoperative aspiration cytology demonstrated cohesive, spindle-shaped cells arranged in fascicles or singly. Since these cells showed nuclear atypia, such as hyperchromasia, a coarsely granular chromatin pattern and nuclear grooving, a nonepithelial malignant lesion was suspected and upper lobectomy performed. The final diagnosis was a pulmonary tumorlet on the basis of histologic examination of the resected material. CONCLUSION: This is the first cytologic report of a pulmonary tumorlet. In this case, differential diagnosis was made of a tumor consisting predominantly of spindle-shaped cells. Although cytologic findings included nuclear atypia, the lesion was not malignant. PMID- 10740615 TI - Cytopathology of oncocytic carcinoid tumor of the lung mimicking granular cell tumor. A case report. AB - BACKGROUND: The cytopathologic features of oncocytic carcinoid tumor of the lung, a rare variant of carcinoid tumor that is composed exclusively of oncocytes, have not been described before in detail. CASE: The bronchial brush smears from an 80 year-old female with an endobronchial obstructive tumor showed single and loose clusters of tumor cells with abundant granular, eosinophilic cytoplasm. The differential diagnoses included oncocytic carcinoid tumor, granular cell tumor, other oncocytic tumors of bronchial origin and metastatic oncocytic tumors. Immunocytochemistry and electron microscopy confirmed the diagnosis of oncocytic carcinoid tumor. CONCLUSION: Oncocytic carcinoid tumor of the lung has cytopathologic features similar to those of granular cell tumor and pulmonary oncocytoma. Immunocytochemistry, electron microscope or both are necessary to distinguish these neoplasms. PMID- 10740616 TI - Basement membrane material and tigroid background in a fine needle aspirate of clear cell adenocarcinoma of the cervix. A case report. AB - BACKGROUND: Although cytologic findings of clear cell adenocarcinoma of the female genital tract have been reported sporadically, the background on the smear has received little attention. CASE: A 16-year-old female had a large, necrotic mass in the cervix. As cervical brushing cytology and a punch biopsy of the mass could not make a definitive diagnosis, fine needle aspiration cytology was performed from the mass. The smears revealed loose, three-dimensional clusters and sheet arrangements of atypical cells. Dispersed atypical cells were also seen. The atypical cells were large and had abundant, weakly stained cytoplasm and round or oval nuclei with large nucleoli. There were a few tumor cells with clear cytoplasm and distinct cell borders in Papanicolaou-stained smears. The background in Diff-Quik-stained smears revealed a tigroid background and basement membrane material. CONCLUSION: The malignant tumor, revealing both a tigroid background and basement membrane material, seems to have been clear cell adenocarcinoma. Both features are diagnostic clues to clear cell adenocarcinoma in the female genital tract. Our case indicates that aspiration cytology is also an effective method of diagnosing a cervical tumor when the tumor is polypoid and the surface is extensively necrotic. PMID- 10740617 TI - Aspiration biopsy of pleomorphic lipoma of the breast. A case report. AB - BACKGROUND: It is well known that subcutaneous lumps in the breast may create diagnostic confusion. We report, for the first time in the cytologic literature, the features of a pleomorphic lipoma in the breast. CASE: Pleomorphic lipoma of the breast was aspirated, showing typical floret cells and mononuclear, hyperchromatic cells with scalloped nuclei, masquerading as malignancy. CONCLUSION: Pleomorphic lipoma has rather typical cytologic features that may allow its recognition when they are present in the proper clinical setting. This report illustrates another diagnostic pitfall in fine needle aspiration biopsy of the breast. PMID- 10740618 TI - Fine needle aspiration cytology of high grade mucoepidermoid carcinoma of the thyroid. A case report. AB - BACKGROUND: Although mucoepidermoid carcinoma is considered a very rare, low grade thyroid neoplasm, in two patients a very rapid and aggressive outcome occurred. We describe the cytologic, histologic and immunohistochemical findings of a high grade mucoepidermoid carcinoma that evolved into an anaplastic carcinoma. CASE: A 57-year-old man was admitted with dysphagia, dysphonia and odynophagia. The patient had begun to develop symptoms over the previous two months. Ultrasound and computed tomography revealed diffuse enlargement of the thyroid gland with multiple, bilateral, palpable lymph nodes in the cervical, supraclavicular, paratracheal and retrocaval chains. The patient died four weeks after receiving the first cycle of treatment with adriamycin and cisplatin. The smears were highly cellular, with a background rich in neutrophilic, inflammatory infiltrate and necrotic debris. Two main types of tumor cell were identified: squamoid and mucus secreting. Squamoid cells were polygonal, with well-defined borders and dense cytoplasm. Nuclei varied greatly in shape and size and displayed clumped chromatin and prominent nucleoli. Mucussecreting cells were ring shaped and dispersed among the squamoid cells; they contained a large vacuole, with condensed acid and neutral mucins, that peripherally displaced the nucleus. Small and large clusters of large, polygonal cells with single or multiple bizarre nuclei and less-dense cytoplasm were also present. Histology revealed tumor cells distributed in irregular nests, with necrosis surrounded by a fibrous stroma. The predominant cells were squamoid, but dispersed mucus secreting cells were frequently seen in the better-differentiated areas. Sparse anaplastic spindle cells were observed adjacent to the squamoid focus. Immunohistochemistry revealed a reaction positive for cytokeratin (AE3/AE1) in tumor nests and negative staining for thyroglobulin and neuroendocrine markers. CONCLUSION: Although mucoepidermoid carcinoma of the thyroid is a very rare neoplasm, its peculiar cytomorphologic features in fine needle aspiration cytology may contribute to its correct diagnosis. PMID- 10740619 TI - Fine needle aspiration cytology of pilomatrixoma of the breast. PMID- 10740620 TI - Amyloid spherules in prolactinoma. PMID- 10740621 TI - Microfilariae in fine needle aspiration smears from lesions of nodular leprosy. PMID- 10740622 TI - Metastatic breast adenocarcinoma diagnosed by aspiration of a pulmonary artery embolism. PMID- 10740623 TI - Fine needle aspiration of cryptococcal lymphadenitis: further observations using autofluorescence. PMID- 10740625 TI - [Genome analyses for precancerous lesions in the gastrointestinal tract]. AB - We herein summarize the reports on genetic changes in precancerous lesions in the gastrointestinal tract. It has been reported that with esophageal lesions such as dysplasia and Barrett's esophagus there is a high frequency of p53 mutations. Among gastric lesions, some cases of chronic atrophic gastritis have been shown to harbor K-ras mutations. p53 and APC mutations in intestinal metaplasia have also been demonstrated, as have APC mutations in flat adenomas. With colorectal lesions, it has been reported that K-ras, DCC, p53 mutations commonly occur while APC mutations are also seen in cases of adenoma-carcinoma. p53 and K-ras mutations have been demonstrated with serrated adenoma, and K-ras mutations with hyperplastic polyps APC mutations in familial polyposis coli, LKB1 mutations in Peutz-Jeghers syndrome, and SMAD4/DPC4 mutations in juvenile polyposis syndrome have been found. Besides these genes, other genetic changes likely occur in carcinogenesis among those with hereditary diseases. K-ras mutations in aberrant crypt foci and hMSH2 mutations in ulcerative colitis have been found. Research into the genetic changes associated with cancerous lesions should lead to the development of early diagnosis and treatment methods for gastrointestinal cancer as well as the improved comprehension of carcinogenesis. PMID- 10740624 TI - Diagnosis of microfilariae in testicular fine needle aspiration biopsy. PMID- 10740626 TI - [Recent regimen of chemotherapy for advanced head and neck carcinomas]. AB - Standard treatment for squamous cell carcinomas in the head and neck consists of surgery and/or radiotherapy. However, this standard treatment alone has not improved the poor prognosis of advanced head and neck carcinomas. A new curative treatment modality for advanced head and neck carcinomas including chemotherapy with an impact is needed. The clinical results of chemotherapy over the past twenty years have shown chemotherapy including cisplatin (CDDP) and 5 fluorouracil (5-FU) to be the most efficacious for advanced cases. In this paper, the problems with this chemotherapy are discussed in terms of predictive factors of the response to this treatment, and the recent clinical results with chemotherapy including CDDP, 5-FU, and other drugs are reviewed. Furthermore, new chemotherapeutic regimens and the clinical results including commonly applied taxanes, e.g., paclitaxel and docetaxel, are reviewed. PMID- 10740627 TI - [New combination therapies for gastrointestinal cancer]. AB - Recently, cisplation (CDDP) and CPT-11 have joined the other drugs used for gastrointestinal chemotherapy, and the combination of I-leucovorin (I-LV)/5-FU has become available for use in medical treatment in Japan. This enables doctors to make a variety of regimens for gastrointestinal cancers. In this paper, we explain the new combinations, especially LV/5-FU/platinum, CPT-11/CDDP, and LV/5 FU/CPT-11. The combination of 5-FU/LV/CPT-11 has shown a higher antitumor activity than 5-FU/LV alone, with increased progression free survival or time-to treatment failure. This combination will be considered the new standard regimen for colorectal cancer. It is worthy of note that the combination of UFT/LV provided on equally effective but safe and more convenient oral alternative to the standard i.v. 5-FU/LV regimen for colorectal cancer. PMID- 10740628 TI - [Treatment of unresectable non-small-cell lung cancer]. AB - Current progress in the treatment of unresectable non-small-cell lung cancer (NSCLC) is reviewed. Several new agents including vinorelbine, paclitaxel, docetaxel, gemcitabine, and irinotecan have been shown to have distinct activity for NSCLC. Combinations of a new agent with cisplatin or carboplatin were highly active for advanced NSCLC, and randomized trials are in progress to establish the standard chemotherapy regimen for advanced NSCLC. The effectiveness of concurrent chemoradiotherapy has been established in Japan, while that of induction chemotherapy has yet to be confirmed. Induction chemoradiotherapy may be useful and randomized trials comparing chemotherapy alone with chemoradiotherapy as an induction therapy are needed. PMID- 10740629 TI - [New combination chemotherapies for breast cancer]. AB - Doxorubicin has been a pivotal role in combination chemotherapy for breast cancer (BC) since 1970's. Over the past decade, a number of new and effective cytotoxic agents have become available for the treatment of breast cancer. The most active agents may be the taxanes, paclitaxel and docetaxel, because their clinical efficacy exceeds that of the anthracyclines, previously the most effective agents against breast cancer. To obtain better quality of life and longer survival for our patients, we need to improve our therapeutic strategy and tactics by developing new combination chemotherapies using taxanes, anthracyclines, and other new promising agents such as vinorelbine, capecitabine, S-1, gemcitabine, liposomal doxorubicin, MTA and so on. Recombinant humanized anti-HER 2 monoclonal antibody is also very active for patients with BC, when used together with taxanes, showing survival advantage compared with taxanes alone. Extensive clinical investigations have been performing with such active agents and biotherapeutics. PMID- 10740630 TI - [New regimens for the treatment of gynecologic cancers]. AB - Until the late-1980s, combination CDDP (or CBDCA)/cyclophosphamide (CPA) and CDDP (or CBDCA)/CPA/adriamycin (ADM) were the two most common choices for treating patients with ovarian cancer. In the last several years, the identification of paclitaxel (TXL) activity in previously treated ovarian cancer patients has led to its incorporation into primary chemotherapy regimens for newly diagnosed patients. Based on prospective trials by the Gynecologic Oncology Group (GOG) and European-Canadian investigators, CDDP/TXL became the new standard regimen in 1998. Now, three randomized trials by the GOG (158), AGO, and a Dutch group comparing CDDP/TXL and carboplatin (CBDCA)/TXL are also in progress. These study groups recommended that CBDCA/TXL be used as the standard regimen in 1999 because CBDCA/TXL is less toxic, allows a better quality of life during treatment, and results in an equivalent response rate and progression-free survival. In patients with high-risk corpus cancer, clinical trials comparing platinum/TXL or platinum/ADM/TXL versus platinum/ADM are in progress. In Japan, CPT-11/CDDP has been shown to be an effective and safe regimen in patients with advanced ovarian cancer, especially clear cell carcinoma and cervical cancer. PMID- 10740631 TI - [New combination chemotherapy in urological cancers]. AB - Although several effective therapeutic modalities are currently available for each urological cancer, there are still many patients for whom cure is not possible. Interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) are both standard agents for patients with metastatic renal cell carcinoma (RCC). However, only up to 20% of patients can attain a complete response with these agents. It has been reported that for bladder transitional cell carcinoma (TCC), a cisplatin-based chemotherapy such as M-VAC chemotherapy can give patients a prognostic benefit in an adjuvant setting. There has been no therapy to improve upon M-VAC for more than a decade. In this review, several trials with new combination chemotherapies that were developed to overcome the limitations of the current therapy are discussed. These include the combination of IL-2, IFN-alpha and 5-fluorouracil for RCC, paclitaxel/gemcitabine and cisplatin/carboplatin for TCC, and paclitaxel/docetaxel and estramustine for hormone-refractory prostate cancer. The results of initial trials with these new combination therapies are promising. Large scale clinical trials, however, have yet to be done. PMID- 10740632 TI - [New combination therapies in hematological malignancies]. AB - New combination therapies against hematological malignancies have recently been reported. Anti-CD20 chimeric antibody adds a therapeutic benefit to standard-dose CHOP therapy without causing significant additional toxicity in the treatment of indolent B cell lymphoma. Multidrug resistant modifiers such as PSC833 and MS209 in combination with chemotherapy are useful for treating poor risk AML patients, whose leukemia/lymphoma cells express P-glycoprotein. Fludarabine containing FL and FLAG therapy were effective in patients with AML in relapse. The early addition of chemotherapy to ATRA, and maintenance therapy with chemotherapy and intermittent ATRA, can reduce the incidence of relapse in cases of acute promyelocytic leukemia. PMID- 10740633 TI - [Interim report of JFMTC study no. 20 on the effectiveness of high dose CDDP plus 5-FU regimen as an adjuvant therapy for far-advanced cancer of the stomach]. AB - This interim analysis of the JFMTC study as of May, 1998 covers 321 gastrectomized patients with far-advanced stomach cancer from 135 institutions between November, 1993 and March, 1996. The intensive therapy group (I-group) received CDDP i.p. administration on resective surgery with 70 mg/m2 followed by CDDP i.v. of 80 mg/m2 (day 1, i.v.), accompanying 5-FU of 350 mg/m2/day (day 1-5, c.v.i.) in the 4th, 8th and 12th weeks. The I-group was randomly compared with the standard therapy group (S-group) of MMC of 6 mg/m2 i.v. in the 4th, 8th and 12th weeks and UFT of 3-4 capsules daily for postoperative one year. The results obtained were that 1. adverse reactions were found more in the I-group than in the S-group, particularly notable in the decrease in blood cells, loss of appetite and nausea/vomiting, and incidence of grade 3 or more being 13% (neutrophile leukocytes), 26% and 21%, respectively; 2. there was no significant difference between I- and S-groups in terms of 3-year survival or disease-free survival rates. (JFMTC: Japanese Foundation of Multidisciplinary Treatment for Cancer). PMID- 10740634 TI - [Anti-emetic treatment for patients with lung cancer after chemotherapy, and usefulness of prochlorperazine for psychiatric emesis]. AB - Clinical study of cisplatin 80 mg/m2 and other agents was undertaken to determine whether they could prevent psychiatric emesis during chemotherapy in 50 patients with lung cancer. YG character tests revealed that 9 patients had a coupled rightward shift in N (nervous) and I (inferior complex) components, and were classified as emotionally unstable. In these 9 cases, addition of 15 mg prochlorperazine to the preventive combined administration of 3 mg granisetoron, 500 mg methylpredonisolon, and 40 mg metoclopramide decreased the post chemotherapy psychiatric emesis from 100% in 3 control cases to 16.7% in 6 added cases. In the remaining 41 cases, the preventive combined administration alone was effective. The good control of acute emesis is the first priority in emotionally unstable patients. The addition of prochlorperazine might be a useful therapy for psychiatric emesis induced by anticancer agents in emotionally unstable patients. PMID- 10740635 TI - [A phase II study of irinotecan combined with cisplatin in non-small cell lung cancer. CPT-11 Lung Cancer Study Group]. AB - Based upon the results of phase I study of irinotecan (CPT-11) combined with cisplatin (CDDP) on non-small cell lung cancer (NSCLC), a combination phase II study on NSCLC was carried out from Feb., 1992 to Sep., 1992. CPT-11 (60 mg/m2) and CDDP (80 mg/m2) were administered by i.v. drip infusion, with administration schedules of Days 1, 8, 15 and only Day 1, respectively. This therapy course was repeated every 4 weeks. Subjects were NSCLC patients of stage III B or IV disease. Those without prior chemotherapy (Group A) and those with prior therapy (Group B) were enrolled separately. Seventy patients were entered into Group A and 32 patients into Group B. One of the patients of Group A was ineligible. The characteristics of the eligible cases of Group A were: male/female, 51/18; median age, 61 years old; PS 0/1/2, 18/39/12; stage IIIB/IV, 26/43; and adeno/squamous/large, 51/15/3. Those of group B were: male/female, 20/12; median age, 62 years old; PS 0/1/2, 5/18/9; stage I/IIIB/IV, 1/7/24, adeno/squamous/large/ad-sq, 28/2/1/1. Thirty-three patients (47.8%) responded in Group A and B patients (25.0%) responded in Group B. Major adverse reactions (grade 3 or higher) of Group A/Group B were neutropenia (80.3%/73.3%), anemia (35.3%/34.4%), diarrhea (18.8%/28.1%) and nausea/vomiting (34.8%/34.4%). Median survival times for Group A and Group B were 308 and 295 days, respectively. CPT 11 in combination with CDDP is effective against NSCLC, suggesting that further studies are needed to determine the usefulness of this therapy. PMID- 10740636 TI - [Efficacy of docetaxel for recurrent breast cancer: evaluation based on chemosensitivity test and clinical response]. AB - We investigated the chemosensitivity of anticancer agents against primary (230 patients, 268 tumors) and recurrent breast cancer (40 patients, 51 tumors) using histoculture drug response assays (HDRA) of surgical specimens. Of the 40 recurrent breast cancer patients, 26 were pretreated with anthracycline. The efficacy of the agents was assessed according to an inhibition index of optical density detected by an ELISA reader. The inhibition rate of docetaxel against recurrent tumors was similar that against primary ones, although the rates of adriamycin, 5 fluorouracil, mitomycin and cisplatin against recurrent tumors were significantly lower than those against primary ones. The clinical response of docetaxel was also evaluated in ten patients with recurrent breast cancer. Of the ten patients with recurrent breast cancer, eight were pretreated with anthracycline, and seven showed a partial response. These results indicate that docetaxel is effective against recurrent breast cancer, even anthracycline resistant breast cancer. PMID- 10740637 TI - [Neoadjuvant chemotherapy with cisplatin and CPT-11 for advanced cervical cancer]. AB - Neoadjuvant chemotherapy (NAC) is currently used for the treatment of advanced cervical cancer by many institutions. We investigated the value of NAC followed by radical surgery and/or radiotherapy for patients with locally advanced cervical cancer. Sixteen patients with stage Ib2-IIIb cervical cancer were enrolled in this study. CPT-11 (60 mg/m2) in 500 m/5% glucose was given intravenously on Days 1, 8, and 15, before cisplatin (60 mg/m2) in 500 ml normal saline. The treatment was repeated every 4 weeks for 2 or 3 cycles. All patients were evaluable for response and toxicity. Two achieved a clinical complete response (CR), 11 had a partial response (PR), 2 had no change (NC) and 1 had progressive disease (PD), for an overall response of 81.3%. Neutropenia was observed in 100% (> grade 3: 70.6%) and diarrhea was recorded in 55.9% (> grade 3: 5.9%). The combination of CPT-11 and cisplatin in locally advanced cervical cancer is thus an active regimen with a manageable toxicity as a neoadjuvant chemotherapy. PMID- 10740638 TI - [G-CSF administration following autologous peripheral blood stem cell transplantation--the effect of G-CSF level on neutrophil recovery]. AB - We studied the usefulness of rhG-CSF (filgrastim) administration in patients who received autologous peripheral blood stem cell transplantation (PBSCT) combined with super-high dose chemotherapy. Twenty patients received 0-8.3 micrograms/kg/day filgrastim after PBSCT. There was a significant relationship between G-CSF dose and the neutrophil recovery rate, and the highest levels of serum G-CSF tended to correlate with neutrophil recovery rate. The highest G-CSF level after 75 micrograms injection in normal volunteers is reported to be 1,500 pg/ml. On the other hand, as one patient in our series exhibited extremely high endogenous G-CSF of 11,500 pg/ml, measurements of G-CSF might reduce the over administration of rhG-CSF. PMID- 10740639 TI - [Chronological observation of nausea and vomiting in outpatients given oral antimetabolites as chemotherapy--two patients receiving ondansetron hydrochloride tablets]. AB - The incidence of nausea and vomiting or anorexia was investigated in 16 outpatients receiving oral antimetabolites such as 5-FU (fluorouracil) as chemotherapy, during a maximum observation period of 28 days. In those patients who experienced the above symptoms which meet the standard defined in the study protocol, ondansetron hydrochloride tablets in a 4 mg/day dose were given based on the decision of the physician in charge, and its efficacy in those patients was examined. Nausea and emesis or anorexia was observed in six cases (37.5%) during the period of observation. Anorexia appeared in a majority of the above cases, with an incidence rate was 31.3% (5/16 cases). In two of the cases, anorexia improved after ondansetron tablets were administered. No adverse drug reaction was reported with ondansetron tablets. We conclude that although antimetabolites have low emetogenicity, as anorexia appeared in approximately 30% of the patients, the use of ondansetron tablets or other antiemetics should be considered in order to maintain patients' QOL and drug compliance. PMID- 10740640 TI - [Antitumor effects of TZT-1027, a novel dolastatin 10 derivative, on human tumor xenografts in nude mice]. AB - TZT-1027 was evaluated for its antitumor effects in sixteen human tumors xenografted in nude mice from gastric (H-81, H-106, H-30, H-154), breast (H-31, H 62), colon (H-110, H-143), lung (LC-376, H-74, Mqnu-1, LC-351), liver (H-181), renal cell (H-12) and ovarian (H-OC-3, SOC-4) cancer lines. In the latter three and lung (Mqnu-1, LC-351) cancers the results were compared with those obtained with CPT-11, VCR, CDDP, ADM. TZT-1027 showed effective antitumor activity (IR > or = 58%) against fifteen of the tumor lines, all but LC-351, and showed markedly effective activity (IR > or = 80%) against twelve tumor lines, including drug resistant colon (H-110), lung (H-74) and ovarian (SOC-4) cancer lines. The complete regression was shown in five H-OC-3 tumor-bearing mice out of seven. Moreover, TZT-1027 was shown to be more potent in three cancer models (Mqnu-1, H 81, SOC-4) than CPT-11, and to have markedly effective antitumor activity in two cancers (H-12, H-OC-3) in which VCR was ineffective and in ovarian cancer (SOC-4) in which CPT-11, CDDP and ADM were ineffective. The administration of TZT-1027 induced fewer side effects; transient reduction of body weight was observed in four lines out of sixteen tested. These results suggest that TZT-1027 is an excellent candidate for clinical trials for the treatment of cancer. PMID- 10740642 TI - [Staging laparoscopy for a patient with advanced gastric cancer whose serum CA 19 9 level decreased remarkably after 2 courses of F/P therapy]. AB - The prognosis of patients with Stage IVb gastric cancer remains poor, and therapy for these patients is not easy. Recently, neoadjuvant chemotherapy is thought to be one of the better strategies for such patients. In this paper, we report a 48 year-old female patient with advanced gastric cancer whose serum CA 19-9 level decreased remarkably after 2 courses of F/P therapy. We also operated on this patient with staging laparoscopy, a new and more useful technique for the patients such as the present one. The advantages of staging laparoscopy are as follows: 1. The entire intraabdominal space can be observed from a small wound, and the clinical stage of the patient can be diagnosed more correctly. 2. It is easy to obtain cells from small amounts of ascites or tissues of small nodules in the abdominal wall and lymph node involvements. 3. Patients suffer less pain and movement of the body is not as limited after operation. 4. Administering drugs into the intra abdominal space is easy using this technique. Staging laparoscopy might therefore be one of the better surgical strategies for the patients with advanced patients when selecting a suitable therapy. PMID- 10740641 TI - [Seven patients with stage IVb advanced gastric cancer who were treated with preoperative chemotherapy]. AB - We examined the usefulness of preoperative chemotherapy using 5-FU and low-dose CDDP in patients with stage IVb gastric cancer. Between 1996 and 1998, seven patients with stage IVb gastric cancer who received preoperative chemotherapy achieved complete or partial response. One course of the chemotherapy was as follows: arterial or venous infusion of 5-FU (500 or 250 mg/day on day 1-5) and low-dose CDDP (5-10 mg/day on day 1-5) for three weeks. In addition to preoperative chemotherapy, biological response modifiers such as OK-432 and lentinan were used. We evaluated the response with abdominal or chest CT and tumor markers. Although preoperative chemotherapy did not improve the survival rate significantly, the prognosis of these patients seemed to be relatively good. Only slight side effects were found. These results suggest that preoperative chemotherapy using 5-FU and low-dose CDDP may be useful for patients with stage IVb gastric cancer. PMID- 10740643 TI - [A case of advanced hepatocellular carcinoma with lung and bone metastases effectively treated by orally administered UFT]. AB - A 52-year-old male underwent hepatic subsegmentectomy for hepatocellular carcinoma (HCC). Five months later, a recurrent tumor was found in the liver and transcatheter arterial embolization (TAE) was performed. However, recurrent tumors were growing rapidly with multiple lung and bone metastases. The titer of serum AFP was elevated to 896,095 ng/ml and the titer of serum PIVKA-II was elevated to 1294.5 AU/ml. The patient was treated by oral administration of UFT (600 mg/day). Two weeks later, his general condition was improved, and several months later, the liver tumor, multiple lung metastases and multiple bone metastases had almost disappeared. The titers of serum AFP and PIVKA-II were reduced to the normal range. He has maintained a good state of health for about four years now. This case suggests the clinical usefulness of UFT for advanced HCC. PMID- 10740644 TI - [Toxicosis of high-dose methotrexate (HD-MTX) for osteosarcoma, cured with treatment by leucovorin (LV) rescue and hemoperfusion--a case report]. AB - A 17-year-old boy suffered from osteosarcoma in his left distal femur. He was treated with 4 courses of HD-MTX preoperatively, then a wide resection and replacement with endprosthesis was performed. After surgery, 4 more courses of HD MTX were administered. In the last course of HD-MTX, the serum level of MTX had not decreased to a safe level after 48 hours following MTX administration. Liver and renal dysfunction then occurred, so massive leucovorin rescue and hemoperfusion were done. Fortunately, all complications disappeared. The patient is alive and well, and has been disease free for six years since surgery. PMID- 10740645 TI - [Irinotecan (topoisomerase-I inhibitor) for the treatment of recurrent primary intracranial malignant lymphoma]. AB - Primary intracranial malignant lymphoma is a fetal disease with poor prognosis, and there is no effective treatment against recurrent primary intracranial malignant lymphomas. We report 3 cases of malignant lymphoma treated with irinotecan (topoisomerase-I inhibitor, camptothecin derivatives), an aromatic drug extracted from camptotheca acuminata. After the initial diagnosis, surgical resection followed by radiation therapy was performed for one cerebral, and two cerebellar malignant lymphomas. The tumors recurred 1 month, 18 months, and 18 months after the initial treatment, respectively. The former two cases were treated with additional radiation therapy and/or radiosurgery for the recurrent tumors; however, the tumors recurred again. All cases were treated finally with a combination therapy of irinotecan and cis-platinum followed by a maintenance therapy with irinotecan only. All cases showed a sharp roentgenographical response to the chemotherapy even after cumulative recurrences. One patient died of systemic infection, and another died of intracranial tumor recurrence 11 and 29 months after the initial diagnosis, respectively. Autopsies revealed multiple tumor recurrences in both these cases. The other patient died 31 months after the initial diagnosis, also due to intracranial tumor recurrences. These results indicate the usefulness of irinotecan for the treatment of recurrent primary intracranial malignant lymphoma; however, further investigation is necessary to establish a better protocol for irinotecan treatment against primary intracranial malignant lymphoma. PMID- 10740646 TI - [Two cases of acute myelogenous leukemia complicated with fatal gastrointestinal tract bleeding after treatment with idarubicin and cytarabine]. AB - We describe herein two newly diagnosed patients with acute myelogenous leukemia (AML), who were treated twice with an idarubicin hydrochloride (IDR)-containing regimen as a response-orientated induction therapy. Both patients had severe gastrointestinal tract hemorrhage complications at their nadir. The two patients were as follows: a 35-year-old male, FAB-M4, and a 47-year-old female, FAB-M0. They received the same induction chemotherapy (IDR 12 mg/m2 for four days and cytarabine 100 mg/m2 for ten days). No response (NR) was obtained in either, so they underwent the same regimen again. During the period of myelosuppression, they developed severe gastrointestinal hemorrhage. One died of sepsis, and the other of acute respiratory distress syndrome without a recovery in bone marrow. The fetal gastrointestinal tract complications may have been due to severe myelosuppression and mucosal damage in these patients. Careful observation will be needed to prevent such severe complications after the treatment with IDR. PMID- 10740647 TI - Pretargeted radioimmunotherapy of non-Hodgkin's lymphoma: best of both worlds? PMID- 10740648 TI - Radiobiology of low-dose-rate radiation relevant to radioimmunotherapy. AB - Systemically delivered targeted radioisotopes are being used ever-increasingly for cancer therapy. Compared with conventional external beam radiation therapy, the radiation dose-rate associated with these novel systemic therapies is approximately two orders of magnitude lower in intensity (dose-rate). Hence it is referred to as low dose-rate (LDR) radiotherapy. An awareness of the unique radiobiology associated with LDR-based therapy provides a basis for cautious enthusiasm as these new therapies are explored. PMID- 10740649 TI - Pretargeted radioimmunotherapy (PRIT) for treatment of non-Hodgkin's lymphoma (NHL): initial phase I/II study results. AB - Pretargeted radioimmunotherapy (PRIT) was investigated in patients with non Hodgkin's lymphoma (NHL). The PRIT approach used in this study is a multi-step delivery system in which an antibody is used to target streptavidin to a tumor associated antigen receptor, and subsequently biotin is then used to target 90Y radioisotope to the tumor localized streptavidin. A chimeric, IgG1, anti-CD20 antibody, designated C2B8 or Rituximab, was conjugated to streptavidin (SA) and administered to patients with NHL. Thirty-four hours later, a clearing agent, synthetic biotin-N-acetyl-galactosamine, was administered to remove non-localized conjugate from the circulation. Finally, a DOTA-biotin ligand, labeled with 111In for imaging and/or 90Y for therapy was administered. Ten patients with relapsed or refractory NHL were studied. In three patients, the C2B8/SA conjugate was radiolabeled with a trace amount of 186Re in order to assess pharmacokinetics and biodistribution using gamma camera imaging. Seven patients received 30 or 50 mCi/m2 90Y DOTA-biotin. Re-186 C2B8/SA images confirmed that the conjugate localized to known tumor sites and that the clearing agent removed > 95% of the conjugate from the circulation. Radiolabeled biotin localized well to tumor. Unbound radiobiotin was rapidly excreted from the whole body and normal organs. The mean tumor dose calculated was 29 +/- 23 cGy/mCi 90Y and the average whole body dose was 0.76 +/- 0.3 cGy/mCi 90Y, resulting in a mean tumor to whole body dose ratio of 38:1. Only grade I/II non-hematologic toxicity was observed. Hematologic toxicity was also not severe; i.e., five of the seven patients who received 30 or 50 mCi/m2 of 90Y-DOTA-biotin experienced only transient grade III (but no grade IV) hematologic toxicity. Although six of ten patients developed humoral immune responses to the streptavidin, these were delayed and transient and hence may not preclude retreatment. Six of seven patients who received 30 or 50mCi/m2 90Y achieved objective tumor regression, including three complete and one partial response. The estimate of tumor to whole body dose ratio (38:1) achieved with PRIT in these NHL patients is higher than has been achieved in other studies using conventional RIT. Toxicity was mild and tumor response encouraging. PRIT clearly deserves additional study in patients with NHL. PMID- 10740650 TI - Experimental radiation pneumonitis studied with indium-111-pentetreotide. AB - OBJECTIVES: To understand the evolution of lung uptake of 111-In-Pentetreotide in a rat model of pulmonary radiation pneumonitis. METHODS: A 15 Gy 60-Co thoracic irradiation (1.4 Gy/min) was delivered to Wistar rats. Irradiated and control animals were studied during 8 weeks after irradiation. 24 hours after an injection of 111-In-pentetreotide (12-18 MBq), the uptake in the lung tissue (ULT), in the alveolar cells (UpC) and in different organs, was determined. Histological examinations were performed. RESULTS: ULT and UpC after irradiation increased significantly peaking at 4 weeks (ULT: 32.8 +/- 13.0 in 10(-5) of the injected dose versus 10.8 +/- 2.0 for control; and, UpC was 19.3 +/- 7.2 versus 7.3 +/- 4.1) and decreased afterwards. Pre-injection of cold octreotide decreased the lung uptake. This evolution parallels the histological changes: alveolitis with granulomas in the interstitium at 4 weeks followed by development of sites of interstitial fibrosis. These observations suggest that the uptake is due to activated cells, mainly macrophages within the granulomas and in the alveoli, expressing somatostatin receptors. CONCLUSION: 1) The uptake of 111-In pentetreotide in injured lungs after irradiation, already described in man, was confirmed in a rat model; 2) our results suggest that it is possible to follow the evolution of radiation lung injury by using In-111-pentetreotide. PMID- 10740651 TI - The antitumor effect of intraperitoneal treatment with rhTNF-alpha muteins on Ehrlich ascites tumor growth. AB - The intraperitoneal (i.p.) treatment with recombinant human tumor necrosis factor alpha (rhTNF-alpha) is one of the possible therapies for tumors that are confined to the abdominal cavity. Clinical trials aiming at the exploitation of the antitumor effects of rhTNF-alpha have been largely disappointing. In this model the activity of some rhTNF-alpha derivatives was studied. Ehrlich's ascites tumor (EAT) bearing Swiss albino male mice were treated i.p. three times a week with 10 micrograms/mice of rhTNF-alpha, mutein V or mutein VI for two weeks, starting on the 4th day after tumor inoculation. Control mice received PBS. The effect of the rhTNF-alpha derivatives on the course of EAT was evaluated basing on: total ascites volume (TAV); packed cell volume (PCV); total packed cell volume (TPCV); inhibitory growth rate (IGR); cellular population of EAT fluid; morphological EAT cell changes and mean survival time (MST). In the study mutein VI had only a slight effect on MST but significant on TAV- and TPCV-IGR (p < 0.001). In mice treated with rhTNF-alpha and mutein V the enhancement of MST (p < 0.01) was accompanied by TAV- and TPCV-IGR (p < 0.001). The number of EAT cells in ascites decreased after rhTNF-alpha and mutein V administration (p < 0.001). We conclude that treatment with high-dose of this modified molecule lacking the possibility of binding with p75R and not producing so intensified side effects is likely to find wider application in therapy and prevent the ascites growth just as rhTNF alpha dosage. PMID- 10740652 TI - Effect of hemostatics used during operations for digestive organ on cancer cells present in the peritoneal cavity. AB - We investigated effects of hemostatics used during operations for digestive organ on cancer cells present in the peritoneal cavity using BALB/c mice inoculated with Meth A tumor cells (fibrosarcoma) intraperitoneally (i.p.) and C3H/He mice inoculated with MH134 tumor cell (hepatic cell carcinoma) i.p. Microfibrillar collagen hemostat (Avitene) or fibrinogen preparation (Beriplast P) did not affect survivals of those tumor-bearing mice. Gelatin sponge (Spongel)prolonged survivals of MH134 tumor-bearing mice. Liquid form gelatin used instead of Spongel displayed in vitro antitumor effect on MH134 tumor cells at the concentration of 15 mg/ml. Radioactive sodium chromate-labeled MH134 and Meth A tumor cells were not lysed when they were incubated with 15 mg/ml of liquid form gelatin for 24 hours. On the other hand, the tritium thymidine (3H-TdR) uptake by MH134 or RL male 1 tumor cells was suppressed when they were incubated with 15 mg/ml of liquid form gelatin for 24 hours. Proliferation of Meth A tumor cells were not affected by the treatment. Effect of liquid form gelatin on phytohemagglutinin (PHA)-stimulated spleen cells as a benign counter-part of RL male 1 tumor cells (T cell lymphoma) was examined. Liquid form gelatin (15 mg/ml) did not suppress 3H-TdR uptake by PHA-stimulated spleen cells. PMID- 10740653 TI - Intraperitoneal radioimmunotherapy with human monoclonal IGM in nude mice with peritoneal carcinomatosis. AB - In an effort to improve loco-regional control of ovarian cancer, intraperitoneal (i.p.) administration of an yttrium-90 (90Y) labeled human IgM was studied in a nude mouse model of the disease. METHODS: Athymic nude mice bearing i.p. nodules of SKOV3 NMP2, a human ovarian carcinoma cell line, received single (50-400 microCi) or fractionated (150-510 microCi) administrations of 90Y-labeled 2B12. Untreated mice and mice treated with unlabeled immunoconjugate served as controls. Mice were monitored for weight loss, blood counts and survival. RESULTS: Mice that received at least 300 microCi of 90Y-labeled 2B12 in a single administration lost more than 10% of their body weight with some early deaths, both of which were prevented with fractionated administration. Granulocytes and lymphocytes declined with treatment while red blood cell counts were relatively stable. Untreated mice and mice treated with unlabeled immunoconjugate had a median survival time of 20 and 17 days respectively. Treatment with 90Y-labeled 2B12 increased median survival by 11-12 days per 100 microCi for single (50-300 microCi) and fractionated administrations (150-510 microCi). The highest fractionated activity produced over three logs of tumor cell kill without significant toxicity. CONCLUSION: Intraperitoneal RIT with 90Y-labeled 2B12 appears to be an attractive modality to treat peritoneal carcinomatosis and warrants further development. PMID- 10740654 TI - Intraarterial HCC therapy with I-131-Lipiodol. AB - We report on our 2-year experience with intraarterial liver therapy with I-131 Lipiodol in patients with hepatocellular carcinoma in Germany. 30 therapies with intraarterial delivery of I-131-labeled Lipiodol were performed in 14 patients with hepatocellular carcinoma (HCC) with or without portal vein thrombosis during hepatic angiography. The patients were monitored for 1) distribution of Lipiodol by CT, 2) distribution of applied activity by planar scintigraphy and SPECT, and 3) tumor response by CT, MRT and 18-FDG-PET. In 5 patients the tumor size was reduced after the first treatment (responder). Eight patients, primarily with big tumors (> 7 cm), had stable (4) or progressive disease (4), and 1 patient died because of renal failure. CT and SPECT showed pronounced I-131-lipiodol accumulation in the tumor tissue in all patients with variable distribution patterns. One patient had an acute pancreatitis like syndrome together with elevation of liver enzymes, probably due to arterial spasm. 9 patients had transient and mild symptoms in the upper abdomen, fever to 40 degrees C, and a leukocytosis. Two patients had a transient mild elevation of pancreatic enzymes. All patients had a transient rise in liver enzymes. In conclusion, therapeutic efficacy was dependent on the tumor mass. Side effects due to the radiopharmaceutical were tolerable, and other side effects may result from the angiography procedure related manipulations. These results are encouraging for tumors up to a moderate mass. PMID- 10740655 TI - Neovascular targeting with cyclic RGD peptide (cRGDf-ACHA) to enhance delivery of radioimmunotherapy. AB - Radioimmunotherapy (RIT) has been hampered by delivery of only a small fraction of the administered dose of radiolabeled MAb to tumor. A strategy for creating and controlling tumor vascular permeability would enable more effective RIT. The alpha v beta 3 integrin receptor is an appealing target for strategies designed to enhance permeability of tumor vessels because it is highly and preferentially expressed in most tumors. In human tumor mouse models, apoptosis of neovascular endothelial cells has been demonstrated after treatment with alpha v beta 3 antagonists. Since this apoptotic effect could transiently increase permeability of tumor blood vessels, radiolabeled antibodies (MAb) circulating during this period would have increased access to extravascular tumor. To determine if this hypothesis was correct, a pharmacokinetic study of an immunospecific MAb given after an alpha v beta 3 antagonist was performed in nude mice bearing human breast cancer xenografts. The alpha v beta 3 antagonist, cyclic RGD pentapeptide (c-RGDf-ACHA; cyclo arginine glycine aspartic acid D-phenylalanine -1 amino cyclohexane carboxylic acid), inhibits alpha v beta 3 binding to its vitronectin ligand at nanomolar levels. Cyclic RGD peptide (250 micrograms i.p.) given 1 hour before 111In-ChL6 MAb resulted in a 40-50% increase in tumor uptake (concentration), when compared to the control tumor uptake, of MAb 24 hours after administration. When cyclic RGD peptide was given as a continuous infusion (17.5 micrograms/hr) for 1 or 24 hours before 111In-ChL6, tumor uptake of 111In-ChL6 was increased less, and, these data were not statistically different from the control data. There were no differences for any of the groups in the groups in the concentrations of 111In-ChL6 in normal organs or blood when compared to the control group. The results suggest that cyclic RGD peptide provided a temporary, selective increase in tumor vascular permeability, that allowed a larger fraction of the 111In-ChL6 to accumulate in the tumor. PMID- 10740656 TI - The role of 99mTc-tetrofosmin Pinhole-SPECT in breast cancer axillary lymph node staging. AB - The number of metastatic axillary nodes represents one of the most important prognostic factors in preoperative breast cancer patients. 99mTc-Tetrofosmin high resolution Pinhole (P)-SPECT was employed in 112 patients, 100 with breast cancer and 12 with benign mammary lesions, to ascertain axillary lymph node involvement. Axillary P-SPECT images were acquired utilizing specific software connected to a circular high resolution, single-head gamma camera equipped with a pinhole collimator with aperture size of 4.45 mm, rotating 180 degrees around the involved axilla. At the same time, patients also underwent conventional SPECT and planar acquisitions. Per-patient sensitivity and specificity were 100% and 93.6% for P-SPECT, 96.2% and 93.6% for SPECT and 56.6% and 100% for planar imaging, respectively. Moreover, P-SPECT detected more than 51% of lesions ascertained by histology, whereas SPECT and planar detected 32.6% and 20.3%, respectively. Only P-SPECT succeeded in identifying the exact number of metastatic axillary lesions in patients with multiple nodes; this procedure was able to correctly differentiate 88.67% of patients with 3 or less nodes from those with more than 3, thus giving important prognostic information. These data suggest 99mTc Tetrofosmin P-SPECT is a reliable imaging method both for staging and prognostic purposes in breast cancer, and its routine use is recommended. PMID- 10740657 TI - Advances in sputum analysis for screening and early detection of lung cancer. AB - BACKGROUND: Screening for lung cancer using currently available techniques is not effective in reducing mortality from the disease. METHODS: Archived sputum specimens and clinical data linking specimens to lung cancer outcomes from prior screening programs have been reexamined to evaluate altered gene expressing, including specific oncogene activation and tumor suppressor gene deletion, as well as genomic instability and abnormal methylation. RESULTS: Several of these tests allow determination of a molecular diagnosis of cancer years before clinical presentation. CONCLUSIONS: These sputum tests provide an impetus to reconsider screening for lung cancer. Prospective trials are required to confirm test performance characteristics, and management and intervention strategies must be developed that are appropriate to the stage at which lung cancer is diagnosed. PMID- 10740658 TI - Innovative treatment strategies in locally advanced and/or unresectable non-small cell lung cancer. AB - BACKGROUND: While small improvements in outcome have occurred for patients with locally advanced non-small cell lung cancer (NSCLC), 5-year survival results remain low, ranging from 5% to 20%. Distant metastases and local-regional progression remain significant patterns of failure. METHODS: Trials investigating innovative treatment strategies for patients with locally advanced and/or unresectable NSCLC are reviewed, including altered radiation fractionation schema, conformal 3-dimensional radiotherapy, and combined chemoradiotherapy regimens. RESULTS: Whereas hyperfractionated radiation therapy (HFRT) alone does not appear to be beneficial, combined HFRT and chemotherapy appears promising in several trials. Patients treated with accelerated RT compared with standard RT have an improved survival. As higher radiation doses appear to enhance local tumor control, strategies involving 3-dimensional conformal radiotherapy merit further investigation. RT plus chemotherapy is superior to RT alone, albeit with greater toxicity. Amifostine is currently being investigated as a radioprotector. The optimal chemotherapy agents and their integration with radiotherapy are the subject of randomized trials. CONCLUSIONS: Ongoing investigations are warranted to combat both local-regional and systemic failures for patients with locally advanced NSCLC. Treatment strategies need to consider not only the traditional endpoints of survival and local control, but also quality of life. PMID- 10740659 TI - Endoscopic treatment of early-stage lung cancer. AB - BACKGROUND: Disease-free survival after surgical resection of lung carcinoma in situ has been reported as over 90%. After resection of stage IA non-small cell lung cancer, survival at 5 years is approximately 60% to 70%. If endoscopic or bronchoscopic treatments of early-stage lung cancer can provide similar disease free survival with less perioperative mortality, morbidity, and cost, then they may be alternative front-line therapies. METHODS: The authors review early-stage lung cancer detection by fluorescence bronchoscopy and the potential treatment of this disease by endoscopic techniques (photodynamic therapy, brachytherapy, Nd:YAG laser, electrocautery, and cryotherapy). RESULTS: Several reports have noted improved outcomes using endoscopic therapies for early-stage lung cancer, but insufficient data preclude firm conclusions regarding the role of fluorescence bronchoscopy, endobronchial brachytherapy, or electrocautery in early-stage lung cancer. Other than resection, photodynamic therapy may represent the best approach at this time. The principal indication for laser bronchoscopy is palliation of central airway obstruction. CONCLUSIONS: The identification of early-stage lung cancer provides no advantage if we have little to offer the patient short of traditional therapy. The value of newer treatment techniques and methods requires verification. PMID- 10740660 TI - The role of induction therapy in the management of resectable non-small cell lung cancer. AB - BACKGROUND: Combined-modality therapy has become standard for many patients with non-small cell lung cancer. Although surgical resection offers the best chance for long-term survival, the limited number of resectable patients and the presence of occult micrometastatic disease has limited the effectiveness of this modality alone. METHODS: The authors reviewed several trials involving the use of induction chemotherapy in managing resectable non-small cell lung cancer. RESULTS: Extensive phase II experience in patients with stage III disease has confirmed the feasibility of this approach. Unfortunately, heterogeneous patient populations and treatment regimens limit the ability to draw firm conclusions from these trials alone. While the phase III experience has been limited, long term follow-up is now available suggesting that induction therapy may have a beneficial impact on survival, especially for those patients who can be sufficiently downstaged. Recent phase II trials have included stage III patients who have traditionally been considered inoperable. Although encouraging, the role of surgery after chemoradiotherapy for this population of patients remains undefined. CONCLUSIONS: Results from ongoing randomized trials studying the impact of induction therapy on well-defined patient populations will be necessary before the optimal regimen and patient population can be identified. PMID- 10740661 TI - Contemporary smoking cessation. AB - BACKGROUND: Tobacco smoking is the single most preventable cause of death in the United States today. Oncologists are in a unique position to affect the health and economic burdens of smoking by encouraging cessation among their patients who smoke. METHODS: The authors describe and review the effectiveness of current smoking cessation interventions as well as strategies that can be used to encourage cessation among patients. Three types of smoking cessation interventions are described: minimal (or self-help) interventions, behavioral interventions, and pharmacotherapy. The effectiveness of combinations of these types of treatments is also discussed. RESULTS: Oncology professionals can improve their patients' chances for success by implementing various cost effective and easily executed smoking cessation interventions. Advice from a physician to quit smoking has resulted in long-term quit rates of up to 10.2%, and nicotine transdermal patches, nicotine gum, inhalers, and nasal sprays are also effective. CONCLUSIONS: Oncologists are encouraged to adopt the "4As" treatment protocol recommended by the National Cancer Institute: ask patients about their smoking status, advise them to quit, assist by recommending pharmacotherapy, counseling, and psychosocial self-help materials, and arrange follow-up. PMID- 10740662 TI - A progressively enlarging cystic renal mass in a previously healthy 48-year-old woman. PMID- 10740663 TI - Clinical approach to neuroendocrine carcinoma of the skin (Merkel cell carcinoma). PMID- 10740664 TI - Clotted arteriovenous grafts: a silent source of infection. PMID- 10740665 TI - Impact of end-stage renal disease and dialysis on glycemic control. PMID- 10740666 TI - The generation of non-dextran intravenous iron: is iron dextran obsolete? PMID- 10740667 TI - How can survival of the well-dialyzed patient be increased? PMID- 10740668 TI - Staphylococcus aureus bacteremia in patients undergoing hemodialysis. PMID- 10740669 TI - Complications of autosomal dominant polycystic kidney disease in hemodialysis patients. PMID- 10740670 TI - Use of androgens in patients with renal failure. AB - Anabolic steroids have been used for the treatment of the anemia of chronic renal failure for more than 25 years. Due to concerns over adverse effects, their use historically has been limited to nandrolone decanoate given to men, usually over age 50, who have intact kidneys. The introduction of epoetin alfa in 1989 has led to reduced androgen use for the treatment of anemia. Nevertheless, there continues to be scientific investigation into the possible role that androgens may play in combination with or as an alternative to erythropoietin. Whether combination therapy will prove to be useful remains to be determined in a large, prospective, randomized trial. There is little likelihood, based on present literature, that androgen therapy alone will replace epoetin alfa in U.S. dialysis units. This topic was addressed recently by the Anemia Work Group of the National Kidney Foundation Dialysis Outcomes Quality Initiative (27). While acknowledging androgen treatment may be less expensive than epoetin alfa, the group stated that the potential risks of primary androgen therapy alone make this form of treatment "unacceptable." The work group did not offer any recommendations on the combined use of erythropoietin and androgens, stating that published data are inconclusive. If future reimbursement policies are changed to include epoetin alfa within a capitated rate, economic incentives may lead to increased use of androgens to achieve targeted hematocrit values. The potential value of anabolic steroids for treating malnutrition in dialysis patients is an intriguing idea. Very little has been done to explore this issue, and this clinical practice has not become widespread nor universally recommended (28). Prospective clinical trials in this area may be warranted as well. PMID- 10740671 TI - Antithrombotic agents and the prevention of access thrombosis. PMID- 10740672 TI - Peritoneal defenses against infection: winning the battle but losing the war? PMID- 10740673 TI - Hemorrhage versus cancer in acquired cystic disease. PMID- 10740674 TI - Clinical utility of C-reactive protein measurements. PMID- 10740675 TI - Consequences of pica in dialysis patients. PMID- 10740676 TI - Use of serum cardiac troponin-I levels in dialysis patients. PMID- 10740677 TI - Risks of vascular port access devices. PMID- 10740678 TI - The decade of behavior: an opportunity for family psychology. PMID- 10740679 TI - Parental factors and the young child's postdivorce adjustment: a meta-analysis with implications for parenting arrangements. AB - A model-based meta-analytic review highlighted relationships critical for understanding the young child's postdivorce adjustment when planning parenting arrangements. This review confirmed the utility of an interactional model that includes preseparation information and information about father, mother, and the parental alliance. Interactions among the quality and frequency of father's involvement in relation to postdivorce child adjustment that were not clear in studies looking only at direct effects of father-access variables were identified. Results indicated that maternal variables should not be viewed in isolation from the parental alliance or from the father-child relationship. The review discusses variables promoting developmental competence as well as risk factors that clearly interfere with both parental and child functioning. PMID- 10740680 TI - Stressful life events, psychological distress, coping, and parenting of divorced mothers: a longitudinal study. AB - This was a prospective longitudinal study of the relationships among life stress, psychological distress, coping, and parenting behaviors in a sample of divorced custodial mothers. First, the differential effects of major events and daily stressors on psychological distress and parenting were explored. Second, the mediational links among stress, distress, and 3 dimensions of parenting behaviors were studied. Third, 3 coping strategies were studied as moderators of the relationship between distress and parenting. The results showed that both major and small events had significant effects on parental distress, with the effects of daily negative events being greater than those of major events. Parental distress mediated the relationships between stressful life events and parental acceptance of their children's behaviors. Parental coping strategies moderated the relationship between mothers' psychological distress and mothers' discipline practice. PMID- 10740681 TI - Predicting marital stability and divorce in newlywed couples. AB - A longitudinal study with 95 newlywed couples examined the power of the Oral History Interview to predict stable marital relationships and divorce. A principal-components analysis of the interview with the couples (Time 1) identified a latent variable, perceived marital bond, that was significant in predicting which couples would remain married or divorce within the first 5 years of their marriage. A discriminant function analysis of the newlywed oral history data predicted, with 87.4% accuracy, those couples whose marriages remained intact or broke up at the Time 2 data collection point. The oral history data predicted with 81% accuracy those couples who remained married or divorced at the Time 3 data collection point. This study offers support for causal linkages between perceptual biases and selective attention on the path of marriage. PMID- 10740682 TI - The baby and the marriage: identifying factors that buffer against decline in marital satisfaction after the first baby arrives. AB - This longitudinal study identified factors of couples' marital friendship in the beginning months of marriage that predicted stability versus decline in marital satisfaction over the transition to parenthood. Newlywed couples (N = 130) were followed longitudinally for 6 years. Forty-three couples because parents, and 39 childless couples served as a control group. Couples were interviewed about the history and philosophy of their relationship as newlyweds. What predicted the stable or increasing marital satisfaction of mothers were the husband's expression of fondness toward her, the husband's high awareness for her and their relationship, and her awareness for her husband and their relationship. In contrast, what predicted the decline in marital satisfaction of mothers were the husband's negativity toward his wife, the husband's disappointment in the marriage, or the husband or wife having described their lives as chaotic. PMID- 10740683 TI - Living with a depressed spouse. AB - The depressed mood and specific burdens experienced by spouses of patients in treatment for depression were examined. Forty-nine wife-depressed couples and 30 husband-depressed couples were administered the Structured Clinical Interview for DSM-IV (M. B. First, R. L. Spitzer, M. Gibbon, & J. B. W. Williams, 1995), and spouses completed measures of depressed mood and burden. Overall, spouses living with a depressed patient reported significantly more depressed mood than general population norms and numerous specific burdens. Regression analyses showed that these burdens as well as gender of the spouse accounted for the spouses' depressed mood that would otherwise be attributed to mood contagion. It is suggested that as an alternative to an exclusive therapeutic focus on patient outcomes, attention might profitably be directed to the distress and burden experienced by spouses. PMID- 10740684 TI - Behavioral observations of parenting in battered women. AB - The current study assessed observational data of parenting in 95 battered women and their school-aged children's behavioral adjustment and analyzed them within an ecological framework. Results indicated that maternal experience of psychological and physical abuse predicts maternal warmth, but not maternal authority-control, over and above ontogenic and exosystemic factors. In addition, maternal experience of psychological abuse and maternal authority-control predicted children's observed behavior, over and above child characteristics. An ecological framework for understanding the effects of domestic violence on parenting was supported. PMID- 10740685 TI - Affective, organizational, and relational characteristics of maltreating families: a systems perspective. AB - In-home structured interactions of 42 maltreating families (neglect: n = 12; physical abuse: n = 19; sexual abuse: n = 11) and 23 low-income comparison families with preschool-aged children were examined to determine whether maltreating and nonmaltreating families could be distinguished by system-level processes. Coding from videotapes of family interactions yielded ratings for affective, organizational, and relational features of each family unit. Results from family coding demonstrated that sexually abusive families had significantly more difficulties regulating anger, evidenced more chaos and less role clarity, and relied less on adaptive-flexible relationship strategies than nonmaltreating families. The importance of family climate and structure, above and beyond individual maltreatment acts, are high-lighted. Treatment and social policy implications and directions for future research in the family study of child maltreatment are discussed. PMID- 10740686 TI - Emotion regulation as a mediator of associations between mother-child attachment and peer relationships in middle childhood. AB - Although a link between attachment and peer relationships has been established, the mechanisms that account for this link have not been identified. The 1st goal of this study was to test emotion regulation as a mediator of this link in middle childhood. The 2nd goal was to examine how different aspects of emotion regulation relate to peer competence. Fifth graders completed self-report and semiprojective measures to index mother-child attachment, mothers reported on children's emotionality and coping strategies, and teachers reported on children's peer competence. Constructive coping was related to both attachment and peer competence, and mediated the association between attachment and peer competence, suggesting that emotion regulation is one of the mechanisms accounting for attachment-peer links. Constructive coping was more strongly associated with peer competence for children high on negative emotionality than for children low on negative emotionality. PMID- 10740687 TI - Attachment and control in family and mentoring contexts as determinants of adolescent adjustment to college. AB - This study examined whether adolescents' perceptions of attachment security and behavioral and psychological control as experienced in family and mentoring contexts are predictive of their adjustment to college. One hundred fifty-eight academically at-risk adolescents (63 men and 95 women, 16-20 years old) completed questionnaires twice during their first semester: before and after they participated in a mentoring program. Analyses yielded 4 findings: (a) Paternal control was predictive of adolescent adjustment to college; (b) above and beyond perceptions of parental attachment and control, perception of a secure relationship with a mentor was predictive of adolescent adjustment; (c) this relationship was found to be stronger for adolescents who reported having high levels of security in the relationship with their mother; and (d) psychological control by both parents appeared to be a significant determinant of academic achievement. PMID- 10740688 TI - Marital behavior and the security of preschooler-parent attachment relationships. AB - Longitudinal and concurrent relations among positive and negative marital behaviors in 2 contexts and preschoolers' security of attachment were examined for 53 families. At 6 months postpartum, couples were observed in their homes during couple discussion and family play. At 3 years, parents completed the Attachment Q-Set (E. Waters, 1987); marital and parenting behavior was also observed. Interparental hostility during family play at 6 months predicted less secure preschooler-mother attachment. Greater marital conflict at 3 years was associated with less security with mother and father, whereas positive marital engagement at 3 years was associated with more secure child-father attachment. Mothers' parenting partially explained the linkages between marital behavior and child-mother attachment. These results highlight the impact of positive and negative marital behaviors on children's abilities to use their parents as a secure base. PMID- 10740690 TI - Outbreak of kala-azar in Bombay. AB - A chance diagnosis of kala-azar in a patient referred from Acworth Leprosy Home in Bombay was followed up, resulting in an investigation of a total of 25 patients (inpatients and residents) for the presence of the disease. 30.3% of the patients investigated were found to be suffering from the disease. This confirms the earlier suspicion that Bombay and especially the Acworth Leprosy Home is an endemic area for kala-azar. PMID- 10740689 TI - Non-specific granulomatous inflammatory lesions of small bowel. AB - The entity of nonspecific granulomatous inflammatory lesions(NSGIL) of the small bowel is a diagnostic and therapeutic dilemma. Data of 52 histopathologically proven cases of NSGIL seen by us between 1986 and 1991 were analysed. All these patients presented with either intestinal obstruction or perforation. They were thoroughly evaluated and investigated for tuberculosis. Of the 52 patients, 6 patients received antitubercular therapy (ATT) before and after surgery and 32 patients only after surgery. Fourteen patients did not receive ATT. Surgical procedures undertaken included stricturoplasty, resection/anastomosis and simple suturing of perforation. No complications were seen in patients who received ATT; however, six of 14 patients who did not receive ATT developed wound sepsis and 2 developed partial wound dehiscence. Many of these NSGIL lesions could be tuberculous in etiology though typical caseating granulomas were not seen. PMID- 10740691 TI - Conceptual and methodologic challenges of assessing the short-term efficacy of Guggulu in obesity: data emergent from a naturalistic clinical trial. AB - An open comparative trial was conducted in 58 adult obese patients (Body Mass Index > or = 25 kg/square metre). Group I (n = 27), non-drug, was advised diet (1200-1600 cals) and a brisk walk for 30 minutes. Group II, in addition, received Guggulu (Medohar) 1.5-3 gms/day for 30 days. Mean difference in weight loss between Guggulu and non-drug group was 0.32 kg (ns) on day 15 and 0.58 kg on day 30 (ns). The mean weight reduction in patients (> 90 kgs) was 1.92 kg (ns) and 2.25 kg (ns) higher in Guggulu group. All patients weighing > 90 kg lost weight in Guggulu group whilst 3 in non-drug group did not lose weight. Guggulu was tolerated well. The data from this pilot study suggest a synergistic diet-Guggulu interaction over 30 days in patients weighing > 90 kgs which needs to be confirmed in a large placebo controlled study. PMID- 10740692 TI - Simultaneous pterygium and cataract surgery. AB - In our country both pterygium and cataract have a high incidence. Hence in this study, thirty patients with pterygium and cataract were treated with a simultaneous pterygium excision and cataract extraction procedure. These patients after pterygium excision were treated intra-operatively with 500 rads of beta radiation over the pterygium site. Then, the cataract was extracted and the patients were treated post-operatively with topical betamethasone 0.1% for a duration of three months. They were followed up for a duration of 6 months postoperatively. Nineteen patients (63%) had visual recovery to 6/12. Twelve of 30 patients (40%) had recurrence of pterygium. The combined procedure did not result in any surgical complications following cataract removal. Post operatively, after 6 months 13 patients had with the rule astigmatism (WRA) for a mean WRA of 1.3 D, and 17 had against the rule astigmatism (ARA) for a mean ARA of 1.2 D. PMID- 10740693 TI - Detection of medullary carcinoma of thyroid, with liver metastasis, using 99mTc DMSA(V) scintigraphy. AB - A sixty year old female referred for thyroid and liver scintigraphy had a clinical history of progressive swelling in the neck with hepatomegaly. A large cold area was detected in the right thyroid lobe using 99mTc pertechnetate and in the right lobe of liver using 99mTc phytate. Subsequent whole body scan with 99mTC DMSA(V) showed avid tracer uptake in right lobe of thyroid and liver. Aspiration cytology of thyroid and liver showed medullary carcinoma of thyroid with its metastasis in liver. Histopathology following thyroidectomy confirmed the diagnosis. Thus 99mTc pentavalent DMSA contributes specificity to diagnose medullary carcinoma of thyroid and metastatic lesions. PMID- 10740694 TI - Multimodality management of a case of primary osteogenic sarcoma of the zygoma. AB - Craniofacial osteogenic sarcomas are rare primary malignant bone tumors and very few cases involving zygomatic bone were reported in literature. We present our experience of multimodality management of a case of primary osteogenic sarcoma of zygoma. Wide radical excision of the tumor including the parotid gland was done followed by three cycles of adjuvant chemotherapy and fifty Gy of external radiotherapy. The patient is disease-free at two years follow-up. Till 1970s, craniofacial osteogenic sarcomas were managed mainly by radical surgery with a high local failure rate. With the advances made in the field of radiotherapy and chemotherapy, multimodality therapy is playing a major role in the treatment of these aggressive tumors with better overall and disease-free survival. PMID- 10740695 TI - Scintigraphic detection of bile leak and follow-up in a post-cholecystectomy patient with recognition of tail sign. AB - Early detection of site and extent of biliary tract disruption can significantly reduce mortality and morbidity in a postoperative biliary leak. We report a case in whom extent and location of post surgical biliary leak was detected with the help of 99mTc BULIDA cholescintigraphy and showed a good correlation with "T" tube cholangiography. Cholescintigraphy was also useful in assessing the follow up of this patient. We conclude that 99mTc BULIDA cholescintigraphy is a non invasive, safe, simple and sensitive procedure in the detection of the site, extent of the leak and in follow up of the postoperative biliary leak. PMID- 10740696 TI - Renal calculi in primary hyperaldosteronism. AB - Increased urinary calcium (Ca++) excretion and the presence of negative Ca++ balance is well documented in primary hyperaldosteronism. However, renal calculi as a major manifestation of this disorder is not previously described. This report describes probably the first patient who presented with renal calculi in association with primary hyperaldosteronism. We believe that primary hyperaldosteronism was a major pathogenetic factor in formation of renal calculi since increased urinary excretion of Ca++ and uric acid noted at the onset declined following short-term spironolactone administration and remission from renal calculi has persisted following initial nephrolithotomy and continued spironolactone therapy which also corrected hypertension and hyperkalemia, a hallmark of this disorder. PMID- 10740697 TI - Carcinoma in a fibroadenoma. AB - A carcinoma arising within a fibroadenoma is an unusual occurrence, with only a little over 100 reported cases. The purpose of this report is to increase the awareness of this entity and to discourage the practice of rendering a diagnosis on gross examination of the tumor. We are reporting a case with two distinct primary tumors within the same breast, one of which was arising within the fibroadenoma. Only two such cases have been previously reported. PMID- 10740698 TI - Neonatal mydriasis due to effects of atropine used for maternal Tik-20 poisoning. AB - A neonate was born to a mother who had consumed an organophosphorus(OPC) compound with suicidal intent. The mother was administered atropine and this caused mydriasis in the neonate without any other pharmacological effects. There was no evidence of placental dysfunction. There are no case reports of OPC consumed in pregnancy and its effect on neonates or of effects of massive doses of atropine in the mother and its effects on the fetus or the newborn. PMID- 10740699 TI - Hallerman-Streiff syndrome. AB - A 3 day old female neonate with Hallerman Streiff Syndrome presented with white spots in both the eyes. Both eyebrows and eyelashes were found to be sparse. Anterior chamber was found to be shallow. Total cataract was detected with posterior synechiae. Fundus could not be viewed. General examination revealed other features of Hallerman-Streiff Syndrome--short stature, bird like face, atrophy of skin and natal teeth. Lensectomy was carried out for left eye at the age of 10 weeks. However, the child had repeated respiratory tract infections and died at the age of 22 weeks. PMID- 10740700 TI - Salmeterol: a long acting bronchodilator. PMID- 10740701 TI - Preventing distortion of quick couplers of hoses of central pipe lines--a cheap and simple method. AB - A cheap and practical approach of steel chains attached to the station outlet quick couplers helps in maintaining the shape of the quick couplers and ensures their effective functioning over a long period of time and avoids mishap of connection due to damage of these couplers. PMID- 10740702 TI - Ethical dilemmas. PMID- 10740703 TI - Tricortical cervical inter-body screw fixation. AB - A new tricortical method of screw implantation for anterior cervical interbody plate fixation is described. The screws are placed obliquely such that they engage the anterior cortex of the body and traverse through the cortices adjoining the disc space. By this method the screws not only hold the plate firmly with a tricortical purchase, but by virtue of their course stabilize the two adjoining vertebral bodies by themselves. Sixteen patients were treated by this method. In three of these cases only tricortical screws without the metal plate were used for fixation. The advantages of the technique are discussed. PMID- 10740704 TI - Ebstein's anomaly--an autopsy study of 28 cases. AB - Twenty eight autopsy specimens of Ebstein's anomaly were studied in order to evaluate the morphologic features of the abnormal tricuspid valve. All cases showed marked dilatation of the original tricuspid annulus, a normally positioned anterior leaflet and variable downward displacement of the posterior and septal leaflets. Sixteen cases showed a very large anterior leaflet. All three leaflets showed dysplastic features and a wide range of anatomic abnormalities in the valve and valve apparatus. A thin walled atrialised right ventricle was present in nine cases. Associated cardiac anomalies were seen in 21 cases, the commonest being an atrial septal defect (17 cases). PMID- 10740705 TI - Effect of oral administration of Terminalia chebula on gastric emptying: an experimental study. AB - Terminalia chebula is a commonly advocated agent in Ayurveda for improving gastrointestinal motility. Charles Foster rats (150-200 gms of either sex) were divided into four groups as follows--Group 1 (n = 15) normal animals; Group II (n = 6) rats administered metoclopramide (1.35 mg/kg); Group III (n = 8) rats given atropine (0.45 mg/kg). These agents were injected intramuscularly, 30 mins before the experiment. Rats from Group IV (n = 8) were administered Terminalia chebula (100 mg/kg/day for 15 days orally). Metoclopramide and atropine have established prokinetic and antikinetic activities respectively and are therefore included for comparison. All rats were then given a test meal of methyl cellulose (1.5%) mixed with phenol red (50 mg/100 ml) orally and gastric emptying was measured 20 mins later. Gastric emptying of normal rats (Group I) was found to be 51.6 +/- 7.79%. Metoclopramide significantly increased the gastric emptying (76.33 +/- 12.37%; p < 0.01) and atropine inhibited the motility (% gastric emptying being 7.26 +/- 19.76%; p < 0.01). Terminalia chebula was found to increase the percent gastric emptying (86.57 +/- 6.65%; p < 0.01). Thus from this study it appears that Terminalia chebula can serve as an useful alternative to prokinetic drugs available today. PMID- 10740706 TI - Intraparotid facial nerve schwannoma. AB - Intraparotid facial nerve schwannoma are uncommon. Preoperative diagnosis of parotid tumour as schwannoma is difficult when facial nerve function is normal. A rare case of solitary schwannoma involving the upper branch of the facial nerve is described and the literature on the subject is reviewed. PMID- 10740707 TI - Echinococcosis as a cause of infertility. PMID- 10740708 TI - Migration of craniotomy flap: an unusual complication. AB - An unusual complication following a craniotomy is reported. The free bone flap migrated over the adjacent bone four weeks following surgery and needed operative readjustment. The probable causes for such a complication are analysed and discussed. PMID- 10740709 TI - Irreducible lateral dislocation of the elbow. AB - A rare case of an irreducible post-traumatic lateral dislocation of elbow is presented. The mechanism of injury was fall on a flexed elbow with trauma on its medial aspect resulting in pronation of the forearm. At open reduction, the brachialis muscle was in the form of a tight band which prevented reduction. The ulnar nerve was entrapped in the joint. PMID- 10740710 TI - Pseudomyxoma peritoneii. AB - A middle aged male patient presented with gradual distension of the abdomen. Imaging modalities showed classical features of pseudomyxoma peritoneii which was confirmed by aspiration cytology. Details of the case are described and relevant literature is reviewed. PMID- 10740711 TI - Pulmonary involvement in idiopathic hypereosinophilic syndrome. AB - A case of idiopathic hypereosinophilic syndrome (HES) is presented. The patient had been symptomatic and had documented peripheral blood eosinophilia for 9 years. The patients having only pulmonary involvement, seem to have a good prognosis and hence must be considered as a separate subgroup of HES. PMID- 10740712 TI - Nasolacrimal duct foreign body--endoscopic removal. AB - Foreign body in the nasolacrimal duct is extremely rare. We present a case of foreign body of nasolacrimal duct responsible for recurrent dacryocystitis which was removed with the help of an endoscope. PMID- 10740713 TI - Paroxysmal nocturnal haemoglobinuria: the current scenario. PMID- 10740714 TI - TRAS principles blight arterial bypass and plasty. AB - A new concept--Tissue Requisitions (Principle I)/Relinquishes (Principle II) Arterial Supply--of TRAS principles is introduced to help appreciate the failures/successes of modern medicine's attempts at restoring arterial flow in luminally compromised coronary/carotid fields, an invasive branch rightly called vascular ReRheology, which comprises diagnosing/treating arterial blocks. The technical wizardry of arterial reconstruction (bypass) or lumen--restoration (plasty) has to reckon with the TRAS principles all the time. PMID- 10740715 TI - Seminal profiles of lysosomal enzymes in normal and infertile men. AB - Human seminal plasma is known to possess considerable proteolytic activity, much of which is associated with lysosomes. The activities of lysosomal hydrolases like alkaline proteinase, cathepsin-D, aryl-sulfatase and N-acetyl-beta-D glucosaminidase in seminal plasma from randomly chosen infertile and vasectomised men have been compared. These enzymes have been implicated in the coagulation and liquefaction processes. The role of fructose and proteins in these processes has also been studied. The results indicate that cathepsin-D and aryl-sulfatase activity in infertile men were significantly lower than normo-spermic subjects. N acetyl-beta-D-glucosaminidase was lowest in azoospermia suggesting that it could be used as a biochemical marker for azoospermia. Conversely, alkaline proteinase showed increased levels in all the infertile cases. PMID- 10740716 TI - Effect of closed mitral valvotomy on spirometric pulmonary function tests in mitral stenosis. AB - The effect of closed mitral valvotomy on the spirometric pulmonary functions was studied in 25 patients with mitral stenosis. The tests were performed before and after operation, the latter at varying intervals (4 to 6 weeks and 8 to 12 months). The preoperative values were considerably low. After 4 to 6 weeks following surgery, further significant reduction in Forced Vital Capacity (FVC) and Forced Expiratory Volume in one second (FEV1) was observed. This was ascribed to the residual healing process and thoracotomy pain. However, Forced expiratory flow rate during mid segment of FVC (FEF25-75%), which reflects obstruction in small airways, did not show any variation. There was improvement in all the above parameters, 8-12 months after surgery. This suggests definite reversibility in the pulmonary functions following valvotomy. PMID- 10740717 TI - Multiple dermoid cysts of omentum. AB - Dermoid cyst of the omentum is an extremely rare condition. We report a case of multiple dermoid cysts of omentum in a 50 years old woman. The aetiopathogenesis, clinical presentation and relevant literature is briefly reviewed. PMID- 10740718 TI - Duplication cyst of the pylorus--a rare cause of gastric outlet obstruction in the newborn. AB - Duplication cysts of the pylorus are the rarest of alimentary tract duplications with very few reported cases. We present such a cyst in a neonate presenting with gastric outlet obstruction. We have also reviewed the literature and outlined the theories of origin, modes of presentation, diagnosis and the surgical procedures. Even though several modes of surgery have been described, it is best to individualize the surgical option in each case. The results depend on the nature and site of duplication, complications and the associated anomalies. PMID- 10740719 TI - Gastric volvulus in childhood. AB - Gastric volvulus is an uncommon condition more so in the paediatric age group. The cause of gastric volvulus may be idiopathic or secondary to various congenital or acquired conditions. In this short series of three patients, one had volvulus which was due to ligamentous laxity and mobile spleen, second had congenital postero-lateral diaphragmatic defect and the third had hiatus hernia. PMID- 10740720 TI - Sphenoid wing meningioma--an unusual cause of duro-optic calcification. AB - Sphenoid ridge is the third commonest site of intracranial meningiomas. Although sphenoid ridge meningiomas often involve the optic canal, calcification along the optic nerve has not been reported with these tumors. We describe CT features of a calcified optic nerve in a patient with a calcified sphenoid ridge meningioma. PMID- 10740721 TI - A giant vascular hamartoma of the breast in a child. AB - Vascular tumors of the breast are uncommon. Perilobular hemangiomas, the commonest histologic subtype of breast hemangiomas, constitute majority of microscopic, vascular mammary tumors. Macroscopic hemangiomas and vascular hamartomas of the breast are distinctly rare. We describe a giant, vascular hamartoma of the right breast in a five year old girl and discuss pertinent literature. PMID- 10740722 TI - Surgical physiology of wound healing: a review. AB - The healing of wounds caused by accident, assault, welfare and surgical operations has always been a central consideration in surgical practice because any breach in continuity of skin or mucous membrane exposes the deeper tissues to the danger of infections. The understanding of the mechanism of wound healing has increased dramatically during last few years. Today wound healing abnormalities are among the greatest causes of disability and deformity. "I dressed the wound, God healed it" (Ambroise Pare) wound healing involves multiple complicated events. It is the amount and quality of scar tissue and ultimately its remodelling that is of greater importance. The understanding of this process of wound healing and factors affecting it forms the basis of any surgical procedure. PMID- 10740723 TI - An epitaph for the gene. An obituary for genetics. An adieu for heredity. AB - Modern medicine has been researching on cancer cell, cancer, hypertension, heart attack and so on without once defining any of these clearly. It swears by these terms much like mankind swears by sunset and sunrise, which are just not there. It is possible that the pet hobbyhorses of modern times, namely, gene, genetics, and heredity may belong to the above mythical group-entities that are logically absent, but whose illogic is strong enough to sustain research and publication world over. Gene, genetics and heredity have outlived their utility and must be replaced in near future by new concepts and terms. PMID- 10740724 TI - Polygraphic recordings of respiration in neonates: a pointer to SIDS? AB - Polygraphic respiratory recordings of 60 neonates were obtained from the infants in paediatric intensive care unit of a large public hospital. Thirty infants were pre-term and thirty infants were in the term (control) group. The recordings were analysed for periodic breathing and apnoeic episodes. Results were also determined for apnoeic density in pre-term and term Infants. Apnoeic episodes were found to be statistically significant in the pre-term infants over term Infants. PMID- 10740725 TI - Effect of Y virilin on male infertility. AB - A prospective placebo controlled double blind study was conducted in patients attending male infertility clinics of our hospital to evaluate effects of a herbal formulation for male infertility--'Y-virilin'. In phase 1 forty patients with oligospermia with or without asthenospermia were randomly allocated to 2 treatment groups--Treatment Group A i.e. formulation under test and treatment Group B (Placebo). Therapy with these agents was given twice a day for 6 months. In phase 2, 12 patients with azospermia were administered either 'Y virilin' or the placebo (n = 6/Gp). In all patients along with semen analysis (sperm count, percentage of motile sperms and grade of motility) was done monthly for 6 months. Serum FSH levels were estimated before and at the end of therapy. A significant increase in sperm count was observed from 2-3 months in oligospermics receiving Y virilin as compared to basal values (p < 0.05). In Group B the follow-up sperm counts were either comparable to basal values or were lesser. However, the percentage and grade of motility did not differ in two groups at the end of respective treatment. No change was found in mean FSH value. During the therapy period incidence of conception was 20% in treatment Group A and 5% in Group B. Of the azospermic receiving 'Y-virilin' 50% showed a count of 10-20 millions/cmm while none from the placebo group. This study highlights the therapeutic potential of the tested formulation in the patients with infertility. PMID- 10740726 TI - Comparative pathogenicity of trichomonas vaginalis isolated from symptomatic & asymptomatic cases. AB - Pathogenicity of 19 isolates of T.vaginalis obtained from vaginal specimens were studied in the murine model by intraperitoneal route. Sixteen isolates were recovered from the females with various clinical conditions and 3 isolates were from normal healthy females. Pathogenicity level of these isolates were studied by inoculating 5 mice per isolates through intraperitoneal route and the animals were sacrificed on tenth day post-inoculation. In general, all the isolates recovered produced infection in mice. On comparison with the reference strain obtained from Hoechst India Ltd., seven isolates recovered from symptomatic cases and one strain from healthy females produced severe infection in mice. Though variation in pathogenicity level was observed among the isolates, a definite correlation between clinical picture in natural host and pathogenicity in mice was not observed. PMID- 10740727 TI - Bone imaging in sports medicine. AB - Increased participation in sports by the general public leads to increase in sports induced injuries including stress fractures, shin splints, arthritis and host of musculotendenous maladies. We have studied twenty patients referred from sports clinic for bone scanning to evaluate clinically difficult problems. It showed stress fracture in twelve patients, bilateral shin splint in five patients and normal bone scan in three patients. Present study highlights the utility of bone imaging for the diagnosis of various sports injuries in sports medicine. PMID- 10740728 TI - Methaemoglobinemia in nitrobenzene poisoning. AB - A young girl with nitrobenzene induced methaemoglobinaemia was saved by the timely use of mechanical ventilator, administration of oral methylene blue and parenteral ascorbic acid. Though parenteral methylene blue is the antidote of choice, due to its non-availability, the laboratory preparation of methylene blue have been utilized orally. The rare occurrence of such cases, and the efficacy of oral methylene blue and other supportive measures in evading death due to Nitrobenzene poisoning have been highlighted. PMID- 10740729 TI - Atlantoaxial dislocation associated with stenosis of canal at atlas. AB - Three rare cases of stenosis of spinal canal at the level of atlas associated with atlantoaxial dislocation are presented. An atlantoaxial lateral mass fixation with plate and screws after posterior midline bony decompression was successfully performed in these cases. PMID- 10740730 TI - Benign osteoblastoma mimicking malignancy of the spine. AB - A case of osteoblastoma of the spine in a sixty year old female is presented. These lesions usually get misdiagnosed as tuberculosis or malignancy of the spine. Salient diagnostic features have been discussed. PMID- 10740731 TI - Lipoprotein lipase deficiency. AB - A rare case of a 3 month old child with lipoprotein lipase deficiency who presented with bronchopneumonia is reported. After noticing lipaemic serum and lipaemia retinalis, a diagnosis of hyperlipoproteinaemia was considered. Lipoprotein lipase deficiency was confirmed with post heparin lipoprotein lipase enzyme activity estimation. PMID- 10740732 TI - Two stage reconstruction for the Shepherd's crook deformity in a case of polyostotic fibrous dysplasia. AB - Polyostotic fibrous dysplasia leads to progressive and disabling deformity involving the proximal femur. Conventional methods of treatment have been ineffective in controlling this problem. Two stage reconstruction was carried out in a case of polyostotic fibrous dysplasia with bilateral shepherd's crook deformity. Bilateral subtrochanteric osteotomies with intramedullary fixation in the first stage and intertrochanteric osteotomies in the second stage with nail plate fixation was done to provide definitive control of the deformity. Bone graft was not used. PMID- 10740733 TI - Qualtum cosmics-and-chaotics--the ultimate tortoise in physics and modern medicine. AB - Qualtum cosmics is the qualitative opposite of quantum mechanics. The flip-side of qualtum cosmics is qualtum chaotics, the two governing much of what is seen as inscrutable in medicine. The Ultimate (Last) Tortoise is close to Einsteinean idea of a Unified Theory, a single concept that can explain whatsoever there is in physics, (and in medicine, or what have you). PMID- 10740734 TI - A prospective study of seroprevalence of Toxoplasmosis in general population, and in HIV/AIDS patients in Bombay, India. AB - Two hundred and seventy nine sera (age group 13-50 years) were tested for antitoxoplasma IgG/IgM antibodies by ELISA techniques; the diagnostic titer for positive test is 10 iu/ml or > 1:100. Sera were obtained from (i) 165 (100 men/65 women) healthy adult voluntary blood donors (HIV, HBsAg, VDRL negative); (ii) 89 consecutive HIV/AIDS patients (82 men/7 women); and (iii) 25 patients (HIV negative: 12 men/13 women) treated for cerebral Tuberculoma or Neurocysticercosis during this study from January 1996-June 1997. The overall seroprevalence was 30.9% (51/165) in the immunocompetent adult (group i) 34% (34/100) men and 26.2% (17/65) in women [range: 10-899 iu/ml; (mean: 376.8)]. In HIV infected hosts the seroprevalence [range: 21-340 iu/ml; (mean; 180)] was 67.8% (56/82 men, 04/07 women). The seroprevalence was 20.5% (8/39), 32.8% (22/67), 34.8% (16/46) and 38.4% (5/13) in the 2nd, 3rd, 4th and 5th decades respectively in healthy adults. In HIV/AIDS patients, 69% (29/42) in the 3rd and 70.6% (24/34) in 4th decade were seropositive. The risk of cerebral Toxoplasmosis (encephalitis-02, granuloma-24) was 43.3% (26/60, mean 250 iu/ml). The seroprevalence was 28% in group iii (range 12-80 iu/ml, mean 21 iu/ml). Anti-toxo IgM was negative in all. Primary Toxoplasma infection appears to be subclinical and prevalent throughout life. T. gondii has emerged as an important opportunistic infection in HIV/AIDS patients in Bombay. Recrudescence of cerebral toxoplasmosis (CTOX) is observed with low IgG response during mid-late stage of the disease, as seen in our patients (mean IgG 250 iu/ml, CD4+ = 283/cmm (range 43-504 in 5 patients). Primary prophylaxis for CTOX seems rationale and can be targeted to asymptomatic HIV/AIDS population at risk who are seropositive for T. gondii (mean IgG 111.5 iu/ml in our study). The very high predictive value of a negative test for TOX remains the best serological parameter for excluding acute episode of TOX. PMID- 10740735 TI - Klebocin typing of Klebsiella species isolated from nosocomial infection in intensive care unit. AB - Klebocin typing and antibiotic resistance have been studied for 518 strains of Klebsiella pneumoniae, [106 from intensive care unit (ICU) sites, 182 from ICU staff flora, 192 from patient flora and 38 from clinical specimens]. The overall typability was 71.62%. The most common mnemonic types among various sources were 111, 211, and 112. Of the total strains tested, 28.37% strains were found to be untypable. These strains are labelled as "444". When klebocin typing was used in association with antibiogram, in 86.84% cases of clinical infection probable source of infection could be detected. Thus a combination of two typing methods poses a significant contribution in epidemiological studies. PMID- 10740736 TI - Persistent frontal fistula. AB - The frontal sinus is prone to various complications--usually secondary to blockage of the fronto-nasal duct and stagnation of frontal sinus secretions. These pent-up secretions may result in pressure necrosis of the inferior or posterior sinus wall. Involvement of anterior wall is uncommon. We present a case of an anterior wall frontal sinus fistula and discuss its management. PMID- 10740737 TI - Single unilateral ectopic bifid ureter with contralateral orthotopic quadrufid ureter--a rare combination. AB - This is a report a case with a unilateral single ectopic ureter associated with ipsilateral incomplete duplication of upper third of the ureter, contralateral quadrufid ureter and L3 hemivertebra. These findings provide further evidence of a generalised mesenchymal or epithelial defect, which would explain a defective ureteral bud and metanephric development, besides associated anomalies. PMID- 10740739 TI - Fundamental interpretation of the peak profiles in linear reversed-phase liquid chromatography PMID- 10740738 TI - Sacral chordoma--a case report. AB - Chordoma, a rare malignant tumour of early adulthood, rarely presents in children. We report such a case of rare malignant tumour which was diagnosed in the first decade of life. PMID- 10740740 TI - Dispersion in micellar electrokinetic chromatography. PMID- 10740741 TI - In search of a chromatographic model for biopartitioning. PMID- 10740742 TI - Advances in physico-chemical measurements using inverse gas chromatography PMID- 10740743 TI - Fundamental aspects of aerosol-based light-scattering detectors for separations. PMID- 10740744 TI - New developments in liquid-chromatographic stationary phases PMID- 10740745 TI - Non-silica-based supports in liquid chromatography of bioactive compounds PMID- 10740746 TI - Overview of the surface modification techniques for the capillary electrophoresis of proteins. PMID- 10740748 TI - Countercurrent chromatography: fundamentally a preparative tool PMID- 10740747 TI - Continuous bed for conventional column and capillary column chromatography. PMID- 10740749 TI - Analysis of oligonucleotides by ESI-MS. PMID- 10740751 TI - Effect of adsorption phenomena on retention values in capillary gas-liquid chromatography PMID- 10740750 TI - Determination of herbicides in water using HPLC-MS techniques. PMID- 10740752 TI - Modern history of women in sports. Twenty-five years of Title IX. AB - The impact of federal anti-discrimination laws such as Title IX and the Amateur Sports Act have opened the doors of sport participation opportunity to girls and women in sports over the last 25 years. Such participation has created new and more lucrative consumer markets for sporting goods manufactures, as well as college and professional sports teams. The prospect of economic gain has created significant social changes: Women are now being encouraged to participate in sports and embrace the resulting benefits, and the general public is showing increased support for gender equity in sports. PMID- 10740753 TI - The benefits of exercise for women. AB - This article describes the benefits of exercise, including why exercise improves blood pressure control, how it may reduce the risk for cancer, and increase mental health, and how it may improve bone mineral density. The article also explores the gender-independent and gender-specific benefits of exercise for women. PMID- 10740754 TI - Harassment, sexual abuse, and safety of the female athlete. AB - Sexual harassment and abuse by authority figures in sports are discussed in relation to how female athletes might improve their personal safety to guard against such practices. The origins of sport research on this theme are traced, and the processes of sexual harassment and abuse are identified. Risk factors for the coach, the athlete, and the sport are presented, and, finally, sources of prevention measures for coaches, athletes, parents, and clubs are provided. PMID- 10740755 TI - Disordered eating and the female athlete triad. AB - Female athletes are under intense pressure to have a low percentage of body fat for performance, which may result in a vulnerable athlete resorting to disordered eating, developing amenorrhea, and suffering the consequences of osteoporosis. Prevention of disordered eating practices among female athletes requires a de emphasis of a low percentage of body fat and a good nutrition education program. The female athlete triad is a serious syndrome that requires a multidisciplinary approach to diagnosis and treatment. PMID- 10740756 TI - A lifetime of fitness. Exercise in the perimenopausal and postmenopausal woman. AB - The peri- and postmenopausal woman experiences physiologic changes of aging that include alterations in hormone levels. Research has shown that the perimenopausal and postmenopausal woman can benefit significantly from exercise, whether endurance or strength training. Exercise can improve the quality of life and attenuate some of the physiologic changes associated with aging. Additionally, exercise can ameliorate the decline in fitness and bone, prevent chronic disease, and promote functional independence. Women who exercise regularly throughout life are physiologically 20 to 30 years younger than their sedentary counterparts. Fitness is a lifetime endeavor that has many positive benefits. Weightbearing activities are especially important as bone loss increases in the perimenopausal phase of life. Women should perform aerobic exercise 3 to 7 days per week for 15 to 60 minutes at 65% to 70% HRreserve. Strengthening exercises should be done 2 to 3 days per week at 40% to 80% 1RM with appropriately selected exercises. An exercise program should be functional and enjoyable. There is no better motivation to exercise than having a partner to work out with and keep the motivation alive. Most important, age itself is not a deterrent to exercise. PMID- 10740757 TI - Bone health and osteoporosis. AB - Good bone health is important to everyone, regardless of age. Osteoporosis is a preventable disease, and steps for increasing bone mass should begin at an early age. To increase bone mass, much of the recent evidence suggests that the best time for intervention with calcium or exercise may be during the prepubertal years. In the older female athlete, BMD is typically well above normal, but even young amenorrheic athletes can have bone density values equal to that of an elderly osteoporotic woman. Myriad treatment options are available for the person with low bone mass, but prevention is a much more preferable alternative. PMID- 10740758 TI - Menstrual cycle, contraception, and performance. AB - Although understanding of the unique physiology of the female athlete has increased, there are still many questions to be answered. Endogenous and exogenous female sex steroids have been shown to influence various cardiovascular, respiratory, and metabolic parameters, but these changes probably have minimal impact on the ability of most recreational athletes to participate in and enjoy their sport. Statistically significant data may or may not have clinical or performance relevance. By the same token, a statistically nonsignificant change may mean the difference between first and second place to an elite athlete. For an athlete concerned about maximizing performance, individual variability in menstrual cycle changes to various performance parameters must be considered. It is difficult to predict how accurately controlled laboratory findings from a study population apply to an individual competitor on the playing field. Athletes taking OCs for contraception or for menstrual cycle control may be able to minimize any potential side effects and performance influences by taking the lower dose triphasic pills and the newer progestins. For women with menstrual dysfunction, OCs may provide a predictable hormonal milieu for training and competition. Further scientific study is needed using large-scale, prospective, randomized clinical trials on trained athletes and accurate hormonal measurements to determine the phase of the menstrual cycle to determine short- and long-term effects of cycle phase and OCs in exercising women. As more questions continue to be answered, physicians and sport scientists will be better able to guide women not only to maximize their performance but to ensure lifelong good health. PMID- 10740759 TI - Exercise during pregnancy. A clinical update. AB - Research dealing with exercise during pregnancy continues to demonstrate marked benefits for mother and fetus. The type, intensity, frequency, and duration of the exercise seem to be important determinants of its beneficial effects. Maternal benefits include improved cardiovascular function, limited weight gain and fat retention, improved attitude and mental state, easier and less complicated labor, quick recovery, and improved fitness. Fetal benefits may include decreased growth of the fat organ, improved stress tolerance, and advanced neurobehavioral maturation. Currently, the offspring are leaner at 5 years of age and have a slightly better neurodevelopmental outcome. Postpubertal effects are still unknown. In the absence of medical contraindications, women should be encouraged to maintain their prepregnancy activity level. PMID- 10740760 TI - Gender differences in noncontact anterior cruciate ligament injuries. AB - Female athletes have an increased incidence of ACL rupture. The cause of this increased injury rate is unclear, but it is most likely from a complex interplay between multiple variables. The relative risk of incurring an ACL injury is still low. The increased risk of ACL injury in women compared with men should not discourage female participation in sports. Instead, the focus should be on strategies to prevent injuries. Intrinsic factors are difficult or impossible to change; modifiable risk factors need to identified and prevention strategies should be employed. PMID- 10740761 TI - Stress fractures in women. AB - Stress fractures are common overuse injuries, reported more frequently in female athletes than in male athletes. The clinician should have a high index of suspicion for this injury whether an athlete presents with complaints of acute pain and disability or insidious onset of pain that is aggravated by activity and relieved by rest. Radiographs, bone scans, CT scans, and MR imaging may all be useful in confirming the diagnosis. Conservative treatment is usually successful, although one must be especially vigilant in treating a stress fracture prone to complications. Identifying risk factors for susceptibility to stress fracture development may help to avoid recurrence of the injury. More research is needed to determine cause versus association of these various risk factors in relationship to stress injury to bone. PMID- 10740762 TI - Patellofemoral pain syndrome in the female athlete. AB - Although PFS will continue to be a therapeutic challenge, the prognosis for most female athletes is good, especially if they are motivated and compliant with their treatment program. Particularly in women, care should be taken to avoid placing too much emphasis on biomechanical variants that may not be clinically significant or correctable because such findings can reinforce a feeling that "nothing can be done." In many cases, muscle dysfunction and repetitive loading of the patellofemoral joint rather than fixed biomechanical factors contribute to the development of PFS. Nonetheless, the importance of a detailed biomechanical assessment on physical examination must not be neglected, particularly in athletes who are not improving with conservative treatment and who may become surgical candidates. A practical initial treatment program for most athletes with nontraumatic PFS begins with relative rest, quadriceps strengthening, and stretching of tight myotendinous units. The introduction of NSAIDs, orthoses, taping, knee sleeves, and more specific rehabilitative exercises should be an individualized decision based on physical findings, past treatment results, and athletic expectations. Surgical referral should be considered in cases of PFS or patellar instability refractory to prolonged maximal nonoperative treatment. PMID- 10740763 TI - Multidirectional instability of the shoulder in the female athlete. AB - Multidirectional instability (MDI) of the shoulder is a complex entity, characterized by symptomatic global laxity of the glenohumeral joint. Because symptoms vary widely, making the diagnosis of MDI is not always straightforward. In addition, relatively few series of patients with this disorder have been reported. Thus, precise definition, causes, and treatment remain elusive. This article reviews the current literature regarding anatomy, biomechanics, clinical diagnosis, and treatment of MDI that are relevant to care of the female athlete. PMID- 10740764 TI - Foot and ankle injuries in dance. AB - Acute traumatic injuries are common in ballet dancers. A careful history, thorough examination, and appropriate imaging should allow for the diagnosis of most problems. The clinician must have a high index of suspicion for occult bony injuries, especially if the patient fails to recover as expected. Aggressive treatment of the sprained ankle is essential to maintain foot and ankle mobility and prevent prolonged disability and subsequent overuse injuries. Kinetic chain dysfunctions are common in ballet dancers with overuse injuries and commonly follow ankle sprains. They may represent a secondary phenomenon that developed in response to the compensatory movement changes caused by the initial injury. It is important to remember, however, that these dysfunctions may have been long standing and a causative factor in the injury. Regardless of the time of onset of the dysfunction, residual kinetic chain dysfunction associated with incomplete rehabilitation of an injury may predispose the dancer to further injuries. Untreated dysfunctions at one site in the kinetic chain may predispose to compensatory dysfunction at other sites in the chain. Accordingly, it is essential to thoroughly examine the entire chain for functional movements when dealing with an injury, because identification and treatment of the kinetic chain dysfunction is important in the rehabilitation of the dancing athlete. Kinetic chain dysfunctions are common in injured ballet dancers and may be a cause of repeated injury. Why then are these dysfunctions left untreated? Medical personnel caring for dancers are sometimes guilty of tunnel vision, and focus solely on the injured site without considering what is happening at other sites in the kinetic chain. This oversight is compounded when the physicians or therapists are satisfied with discovering simply what injury has occurred rather than asking why the injury has occurred. The significance of kinetic chain dysfunctions is only just beginning to be recognized, and many examiners are not aware of the relationship between abnormal motion and injury. Generally, people see only what they look for, and they look only for what they know. Kinetic chain dysfunctions can easily be detected with simple tests of functional movement if the examiners include these tests in their assessment of the injured dancer. As long as clinicians are either unaware of or unwilling to perform these tests, these dysfunctions will remain untreated and may put the dancer at risk of failed rehabilitation or predispose them to further injury. PMID- 10740765 TI - Injuries and special concerns of female figure skaters. AB - Figure skaters are often young athletes involved in an extremely stressful, structured environment that is focused solely on skating. These athletes are at risk for a multitude of psychologic concerns, including poor communication skills, performance anxiety, stress, and family issues. Helping athletes develop self-awareness, techniques to control stress and anxiety, motivational strategies, ability to maintain concentration, and emotional balance is best done with the guidance of a sports-trained counseling professional. Communication among the athlete, parents, and coaches is extremely important. Often, coaches tell the athlete how and what to feel, and the athlete falls into a routine of trying to please coaches and parents instead of developing her own persona. The communication between pairs skaters and ice dancers is integral to their success. Excellent interpersonal skills among members of pair teams are advantageous in creating a lasting relationship. Honesty, respect, clarity, consistency, and sincerity are essential components of outstanding teammates and should be cultivated. On the other hand, sarcasm, disrespect, castigation, and ridicule should be addressed immediately and should be discouraged strongly. PMID- 10740766 TI - Principles of digital imaging. AB - Electronic sensors in diagnostic radiology are gradually replacing radiographic film. The advantages of this new technology include a lower radiation dose to the patient, an almost instantaneous availability of images without the need for chemical film processing, and the possibility of image enhancement and computer aided feature extraction. Digital radiography is a promising technology, opening the door to new diagnostic procedures not available with traditional film-based imaging. PMID- 10740767 TI - Intraoral detectors. CCD, CMOS, TFT, and other devices. AB - The first real-time intraoral detectors were introduced in 1997. Since then, charge-coupled device sensors have dramatically improved. The spatial and gray scale resolution has increased and the dimensions of the sensitive areas are now comparable to those of conventional film. More recently, complementary metal oxide semiconductors were introduced. This technology offers the potential of integration, manufacturability, and low costs. Digital radiographic technology has matured and clinicians should adopt the systems and invest the time and effort to master them. PMID- 10740768 TI - Extraoral and panoramic systems. AB - Digital solutions for extraoral and panoramic radiology have been available for more than 15 years. The costs, however, have been too high for adoption in private dental practice. The past 2 years have seen tremendous growth in the number of relatively cost-effective digital options available to the dentist and dental specialist. Furthermore, computer speed and data storage capacities have progressed. This article presents an overview of systems available at present, and their enabling technologies and enhancement capabilities. PMID- 10740769 TI - Dental photostimulable phosphor radiography. AB - Photostimulable phosphor radiography (PPR), a mature industry in medical imaging, became available in dentistry in 1994. At least three dental PPR systems have been made available commercially since that time. Although dental PPR offers a number of advantages over film-based methods, some aspects of dental PPR need improvement. Further research is needed to more fully compare and contrast this modality with film-based methods. PMID- 10740770 TI - Image processing tools for dental applications. AB - Digital image processing has seen a wide variety of applications in dental radiography. Examples of operations from any of the fundamental classes can be found in the literature, and the increasing role of digital technology in dental imaging will promote new developments and clinical applications. Further progress is not limited by a lack of available image processing tools; rather, improvement of our understanding of the various components of diagnostic imaging will facilitate the meaningful use of new imaging technologies in dentistry. Image processing cannot be separated from other aspects of the diagnostic imaging chain. Its diagnostic value fundamentally depends on image acquisition as well as on aspects of vision and cognition. If used properly, image processing can help to improve diagnostic outcomes and provide insight into other aspects of the imaging process that may be limiting the diagnostic yield. Although novelty is still a powerful force driving some scientific endeavors, advances in dental imaging will be based on the purposeful, goal-oriented development and application of new technology. Specifications for useful image processing operations need to be defined with the diagnostic problem in mind as well as the final outcome for the patient. PMID- 10740771 TI - Digital imaging for dental caries. AB - Laboratory studies show that digital intraoral radiography systems are as accurate as dental film for the detection of caries when a good-quality image is obtained, although more re-takes might be necessary because of positioning errors with the digital systems, particularly the charge-coupled device sensors. The phosphor plate is more comfortable for the patient than nondigital systems, and the dose can be further reduced with the storage phosphors. Cross-contamination does not pose a problem with digital systems if simple hygiene procedures are observed. PMID- 10740772 TI - Digital radiographic image archival, retrieval, and management. AB - Data in the health care environment can encompass a wide range of contents, each representing one aspect of health care delivery. Examples of such a diverse and yet related content are diagnostic images, laboratory results, patient history, hospital administration, and insurance and payer coverage. The focus of this article is the handling of diagnostic imaging in general and radiographic imaging in particular, therefore fundamental concepts and issues are presented and discussed. PMID- 10740773 TI - Teleradiology. AB - The technology of digital imaging and image transmission is here. Notwithstanding all the advancements that have been made in teleradiology, this technology remains in its adolescence with respect to medicine--and infancy with respect to dentistry. As with any new technology, acceptance is a function of time. Individual practitioners' comfort levels with computerization, digital imaging, and long-distance communication will invariably affect the scenario. The advantages of savings in terms of cost and time, coupled with an increased demand for tertiary health care in underserved urban and rural areas, will facilitate the acceptance of teleradiology. Rapid advances of the last decades have created an electronic society only dreamed about in 1970. These changes can be expected to continue at an ever-increasing rate, and dentists must prepare themselves to be in step with these innovations. PMID- 10740774 TI - Computed tomography applications for dentistry. AB - The technologic advances in CT made over the last 25 years have made it possible for CT to be an integral component of the dentist's diagnostic armamentarium. CT can be used to image the extent of pathologic conditions as well as help to unravel complex facial fractures. CT can also be used to assess the temporomandibular joints and the paranasal sinuses and for presurgical implant treatment planning. As the speed of image acquisition and reconstruction increases, more dental applications for CT will be realized and requested. PMID- 10740775 TI - Three-dimensional computed tomography imaging in dentistry. AB - The authors have presented several 3-D CT imaging techniques that may be useful in dentistry. MPR and dental MPR provides cross-sectional images of the jaws and the dental arches, and its utility in implant treatment planning is widely recognized. SSD allows the clinician to visualize the surface of an anatomic structure that is selected by a threshold value. Volume rendering is a powerful and flexible three-dimensional imaging technique that can create various unique images. MIP may be useful in contrast-enhanced CT examinations, such as sialography. Model production and virtual reality three-dimensional imaging may become major techniques in the near future. Although the increased radiation dose to the patients has to be considered, future imaging demands will bring more access to 3-D CT imaging by dental patients and, with adequate three-dimensional rendering techniques, more accurate diagnosis and treatment planning. PMID- 10740776 TI - Magnetic resonance imaging in dentistry. AB - MR imaging has found a place in the imaging sciences. These images are unique in that no ionizing radiation is used, the same tissues may or may not have different appearances with different machine settings, a unique set of terms is used to describe the findings in the images, and these findings can be analyzed with the view of obtaining more diagnostic information not readily available with any other imaging modality. Cases have been presented to help demonstrate differences in T1-weighted and T2-weighted sequences as well as the value of contrast material. Additionally, plain film and CT views were included so that they may be contrasted with the MR images. The role and application of CT versus MR imaging has been further discussed and illustrated by Nortje and van Rensburg. A typical temporomandibular joint case has also been presented. In the future, it is hoped that clinicians will refer more patients for MR imaging so that diagnoses can be made more accurately with analytic software. PMID- 10740777 TI - The future of digital imaging in dentistry. AB - Digital radiographic imaging in dentistry has matured. All the reasons dentists have cited for keeping conventional, film-based systems have been refuted: cost, sensors, storage, and training are not issues. There are many reasons to switch to digital. This article outlines external factors that dentists may not have considered that will influence the way they conduct business in their offices related to radiographic imaging. The evidence points to the inevitable adoption of digital imaging systems. PMID- 10740778 TI - [Effects of blood oxygenation on the shape of a NMR signal]. AB - The relationship of the width of a NMR proton signal from blood to its oxygenation degree was examined. Its width is shown to consist of a symmetrical spread caused by a dipole-dipole interaction, and increasing (with reduced oxygenation) asymmetrical spreading induced by the fact that erythrocytes are distorted from the spherical shape. The shape of red blood cells can be controlled by the ratio of asymmetrical spreading to the shift of signal resonance frequency. PMID- 10740779 TI - [An experimental study of acoustic noise of heart valve prostheses]. AB - The paper deals with the acoustic noise that accompanies the work of artificial cardiac valves (ACV). The generation of a noise signal is a substantial disadvantage of ACV work after implantation. A highly sensitive solid-state probe was used to measure the noise signal. The disc ACVs Bjork-Shiley (USA), Omniscience" (Italy), MIKS, a basic (Russia), MIKS with an acoustic gap (Russia), and MIKS-3 were tested. The valves worked under model physiological flow. The disc valves of the MIKS series with an acoustic gap manufactured by ROSINVEST (Russia) were found to have the least noise. PMID- 10740780 TI - [Mathematical models of dose fractionation based on LQ function. (population tissue models)]. AB - A mathematical model was developed to calculate the probability of tumor tissue sterilization. It is assumed that tumor tissue contains normal and radio resistant tumor cells and the survival of both types of tumor cells can be described by LQ functions. A package of programmes was created to solve the extreme problems in the determination of the parameters of LQ functions and the relative count of radio-resistant cells in the volume of tumor tissue. A programme complex was devised to solve practical tasks in radiological care. The series of tasks, which illustrate various aspects of determination of the parameters of the mathematical model by using clinical data and calculating the probability of tumor tissue radiation sterilization. PMID- 10740781 TI - [Formation of diagnostic rules by using neural networks]. AB - Neural networks were trained on unrepresentative patient data sets proposed by skilled clinicians. In all cases, the trained neural networks correctly classified all presented cases. There were 2 intervals introduced for encoding estimates. The number of both signs and neurons in the trained neural networks was minimized. The trained neural networks adequately represent a set of logical formulas that are easy to perceive. These formulas are similar to syndrome complexes, they can be tabulated and presented as diagnostic tables the physicians usually use. The proposed decisions are accompanied by the estimates of their affinity, which allows their validity to be controlled. The clinical studies performed showed that the proposed decisions were in good agreement with medical conclusions in most cases (over 88%). PMID- 10740782 TI - [Determination of limb loading parameters on standard external fixators]. AB - For the compression-traction osteosynthesis system CTS, the grounds for evaluating the existing parasitic deformity of loaded apparatus elements, such as pins and rings are shown. The measuring procedure for testing the axial and angular rigidity of a regenerate zone is presented. The angular rigidity is estimated by the ellipse of rigidities, i.e. by three parameters: a vector of the maximum rigidity (CU), a vector of the minimum rigidity (CV) and an azimuth of CU position in the ring plane. Some more useful functions of CTS, including the posthospital treatment of patients are described. PMID- 10740783 TI - [Mineral density of the femoral diaphysis after hip prosthesis implantation]. AB - The aseptic instability of endoprostheses, which occurs after their good operation, is a severe complication. All developed countries study this problem from different points of view. This paper presents the data of up to 5-year follow-ups of the mineral saturation of the microstructures of the hip bone diaphysis during endoprosthesis. The paper has been the only to be performed by latest techniques in the former Soviet Union. It is of great interest for researchers who are engaged in searching materials for endoprostheses, for their designers. It is also useful for surgeons in solving the problems how to raise patients after surgery and in giving further recommendations. PMID- 10740784 TI - [Methods of digital processing of pulse and motor signals]. AB - The paper outlines the authors' developed and practically applied procedure for digital processing of pulsomotorograms obtained by monitoring hemomotorodynamic parameters during abdominal surgical interventions whose purposes are to diminish the influence of interference, to eliminate systematic distortions, to standardize and identify motor and pulse waves from the baseline interferential wave. PMID- 10740785 TI - [Computer-assisted method of representation of sperm movement in the evaluation of the quality of ejaculate]. AB - The paper describes computer-aided processing of video images of mobile biological objects in view of forming tracks and of evaluating their individual kinetic characteristics. The proposed processing procedure allows one avoid lower information quality associated with resultant image contract reduction when combining video frames are summarized and the signal/ratio decreases. PMID- 10740786 TI - [Equipment for ultrasound densitometry: problems and prospects]. AB - Summary, The paper analyzes the Russian market of ultrasound densitometric devices. Based on the sales statistics, the development of this market is forecast. A comparative review of various types of equipment is given and the optimal outline of an ultrasound densitometer is proposed. PMID- 10740787 TI - [A "neuromyo-test-response 01" device for neuromuscular function control during anesthesia]. AB - The paper outlines the technical data and procedure of using a new "neuromyotest response-01" device designed by the Novye Pribory (New Devices) Medical Engineering Center (Samara) for estimation of the level of neuromuscular block. PMID- 10740788 TI - [Method of evaluation of the efficiency of laboratory systems]. AB - The paper proposes a model criterion for evaluating the sociomedical efficiency of the operation of laboratory data measuring systems. The information theory was used to compare the goal of diagnostic use of laboratory systems, which shown up through the prevalence of diseases, the sensitivity and specificity of diagnostic tests with the amount of information got at the outlet of the system. Thus, one criteria combines medical and engineering approaches to evaluating the efficiency of complex systems. PMID- 10740790 TI - [Russian-American Seminar on Management of Medical Technologies]. PMID- 10740789 TI - [Improvement in cryopreservation of blood platelets]. AB - The first Russian solution Krimolit designed for cryopreservation of platelet concentrates down -196 degrees C was clinically tested. The therapeutical efficacy of the cryopreserved platelets was evaluated on the basis of clinical and laboratory monitoring of transfusions in 20 cancer hematological patients. They were found to have the same therapeutical action as fresh cells. The introduction of cryopreserved platelets into clinical practice allows one to form stores and to transfuse autological cells. Krimolit is recommended for clinical application. PMID- 10740791 TI - [The 6h International Conference on Medical Physics. "Medical Physics--Patras 99"]. PMID- 10740792 TI - Removal of ocular artifact from the EEG: a review. AB - Eye movements cause changes to the electric fields around the eyes, and consequently over the scalp. As a result, EEG recordings are often significantly distorted, and their interpretation problematic. A number of methods have been proposed to overcome this problem, ranging from the rejection of data corresponding temporally to large eye movements, to the removal of the estimated effect of ocular activity from the EEG (EOG correction). This paper reviews a number of such methods of dealing with ocular artifact in the EEG, focusing on the relative merits of a variety of EOG correction procedures. Issues discussed include the distinction between frequency and time domain approaches, the number of EOG channels required for adequate correction, estimating correction coefficients from raw versus averaged data, differential correction of different types of eye movement, the most suitable statistical procedure for estimating correction coefficients, the use of calibration trials for the estimation of correction coefficients, and the distinction between 'coefficient estimation' and 'correction phase' error. A suggested EOG correction algorithm is also described. PMID- 10740793 TI - [Scapular pain and supra-scapular neuropathy in sports medicine]. AB - Eight patients with shoulder pain are reported with a history of athletic activities. On examination, performed with a delay of several months, all patients had painful paresis and atrophy of spinati fossa. Electroneuromyography was carried out in all cases and showed a suprascapular nerve axonal loss from the spinati muscles or infraspinatus muscle, signs of denervation-reinnervation in spinati or infraspinatus muscles, normal examination of other scapular girdle muscles, and a coordinate spinati contraction with shoulder displacement excluding rotator cuff tears. All patients had conservative treatment and only two improved. Six patients underwent surgical decompression of the suprascapular nerve; in three, motor function clearly improved, and in three others pain improved. The factors leading to entrapment include stretch mechanisms associated with shoulder movements, leading to suprascapular nerve liability to mechanical lesions. In patients with shoulder pain, the authors recommend an early electrophysiological work-up to recognize an isolated suprascapular neuropathy. The surgical decompression of the nerve should be based on persistent shoulder pain after conservative treatment. PMID- 10740794 TI - Neurophysiological studies of thin myelinated (A delta) and unmyelinated (C) fibers: application to peripheral neuropathies. AB - Dysfunction of small fibers may appear in isolation or associated with large fiber lesions. In some acute neuropathies, such as pandysautonomia, small-fiber impairment is relatively pure but it may also appear in disorders with prominent somatic damage, such as Guillain-Barre syndrome, in which autonomic failure worsens the prognosis. At the present time, chronic idiopathic distal small-fiber neuropathy is diagnosed more frequently, and in some prevalent disorders, such as diabetic or amyloidotic polyneuropathies, small-fiber dysfunction is very noticeable. In pure autonomic failure, a peripheral autonomic failure exists, distinguishing it from multiple-system atrophy. Complex regional pain syndrome is a severe condition in which small fibers are responsible for disabling signs and symptoms, and only instrumental recordings lead to the proper treatment. Standard neurophysiological techniques evaluate large myelinated fibers exclusively. Small fiber polyneuropathy has been considered as a type of somatic neuropathy, but thin myelinated and unmyelinated fibers are responsible not only for temperature and pain perception but also autonomic function. For instance, full autonomic evaluation is needed in some clinical situations such as autonomic failure in the elderly or orthostatic intolerance syndrome. To evaluate small-fiber impairment we need a battery of sensitive, reproducible, specific and noninvasive tests covering somatic and autonomic systems. In this review, we describe and analyze a number of neurophysiological techniques used to diagnose and characterize small fiber dysfunction in humans. These include cardiovascular monitoring, sudomotor testing, pupillary responses and quantitative sensory tests, and also to some extent thermography and laser evoked potentials. The use of such techniques has proven useful not only for diagnosis, but also to guide adequate therapy and optimize follow-up. PMID- 10740795 TI - Intrathecal baclofen normalizes motor strategy for squatting in familial spastic paraplegia: a case study. AB - We aimed to assess whether intrathecal baclofen could alter the motor strategy for squatting of a patient with pure familial spastic paraplegia. Before baclofen injection and two, four and six hours after it, the patient was evaluated as follows: self-report of walking stiffness and movement initiation; muscle tone with the Ashworth scale; and kinematic and electromyographic analysis of the squatting movement using the opto-electronic ELITE system. The patient's subjective improvement and decrease in muscle tone were dramatic after baclofen injection. Kinematic analysis of squatting showed gradual improvement. Before the injection, the movement was performed with loss of trunk verticality, backward shift of the hip, multiphasic ascending phase of the knee angular velocity and dynamic ankle stiffening. After baclofen injection, the movement was made with vertical translation of body segments and monophasic ascending phase of the knee angular velocity. The effect was maximal six hours after the injection. Electromyographic activities showed a non-specific co-contraction pattern before the injection, and a reciprocal pattern two hours after it. Moreover, a physiological anticipatory deactivation of the hamstring muscles appeared two hours after the injection. In this study of a single patient with familial spastic paraplegia, intrathecal baclofen has facilitated the emergence of normal, supraspinally determined movement patterns. PMID- 10740796 TI - [Design of a force sensor to evaluate the grasping movement]. AB - Grasping phenomena are abnormal motor behaviours described after cerebral lesions. They are defined as a stereotyped response, consisting of a progressive forced closure of the patient's hand on the examiner's fingers, when they are slowly moved exerting a strong pressure across the palm in an upward direction. Several types are described. The aim of the study was to develop a force sensor to evaluate the force of grasping in accordance with the force of dragging. The characteristics of this force sensor are presented and the clinical application in two selected patients demonstrated. PMID- 10740797 TI - Prognostic significance of fallopian tube cytology: a study of 99 endometrial malignancies. AB - The purpose of the study was to relate fallopian tube lavage-cytology (tubal washings) from patients treated for endometrial malignancies with other recognized prognostic factors and also with patient survival. In 99 patients the following prognostic variables were analyzed: patient age, peritoneal washing cytology (PW), endometrial tumor grade, depth of myometrial invasion, myometrial vascular involvement, cervical stromal infiltration and peritoneal metastases. The association between tubal washings and preoperative hysteroscopy was also examined. Of the 99 patients with endometrial malignancy, 18 experienced their first tumor recurrence or died from tumor during the follow-up period (median 53 months, range 5-137 months). The remaining 81 patients were disease-free on their last visit or died from unrelated causes. Detailed statistical analysis revealed a complex inter-relationship between the variables but no independent prognostic significance for tubal washings. Furthermore, the absence of any statistical association between hysteroscopy and survival suggests that preoperative hysteroscopy has no deleterious effect. Although the small number of patients in this study limits any definitive conclusion, the analyzed data suggest that tubal washing cytology plays no useful role in the current management of patients with endometrial malignancies. PMID- 10740798 TI - Rosai-Dorfman disease of the breast: a mimic of breast malignancy. AB - Rosai-Dorfman disease (RDD) or sinus histiocytosis with massive lymphadenopathy (SHML) is primarily a nodal-based, idiopathic, benign proliferative disorder of histiocytes with 43% of these cases also involving extranodal sites. The breast is an unusual site of occurrence of RDD. We report two cases of this exceptional event. The first represents an intramammary nodal Rosai-Dorfman disease, while the second is an extranodal disease with sole involvement of the breast. In both, the possibility of malignancy was raised. Histological examination of the two breast lesions revealed sheets of characteristic large histiocytes displaying emperipolesis, a microscopic hallmark of this disease. Immunohistochemical and ultrastructural studies were also performed; the former showed cytoplasmic staining of histiocytes for S-100 protein, while histiocytes that engulfed lymphocytes and plasma cells were identified on electron microscopy. PMID- 10740799 TI - Fine needle aspiration diagnosis of neuroendocrine tumors in the liver. AB - Ten neuroendocrine tumors (NET) in the liver are presented, in which the diagnosis was made on fine needle aspiration cytology and cell blocks from the aspirate. In seven of the patients with liver metastasis, a biopsy-proven extrahepatic primary NET had been previously diagnosed, while in three patients no extrahepatic neoplasm could be identified, suggesting that the NET may have been a primary in the liver. Based on cytomorphological findings the cases were typed as either round cell type, spindle cell type or polygonal cell type. In all cases, immunopositivity for neuroendocrine markers provided reliable evidence of the cell of origin and distinguished them from well-differentiated hepatocellular carcinoma, adenocarcinomas and other neoplasms, which sometimes may present a diagnostic dilemma. PMID- 10740800 TI - Hemorrhagic lymphadenopathy as a presenting feature of primary al amyloidosis. AB - Lymphadenopathy associated with hemorrhage as a presenting feature of primary (AL) amyloidosis has not previously been described. We report two such cases one of whom had an acquired factor X and IX deficiency. The clinical presentations were characterized by sudden spontaneous enlargement of lymph nodes followed by partial regression. In both cases significant delay in diagnosis, and hence treatment, occurred due to the mode of presentation. One patient died with rapidly progressive disease but the other has had an excellent response to therapy with high-dose melphalan (HDM, 200 mg/m2) and peripheral blood stem cell rescue. AL amyloid should be considered in all patients presenting with hemorrhagic lymphadenopathy. PMID- 10740801 TI - Broadsheet number 54. Hereditary hemochromatosis. PMID- 10740802 TI - Test and teach. Number Ninety Seven. Granulomatous inflammation and CNS germinoma. PMID- 10740803 TI - Test and teach. Number Ninety Eight. Solid pseudopapillary tumor of pancreas. PMID- 10740804 TI - Test and teach. Number Ninety Nine. Carcinosarcoma of the gallbladder. PMID- 10740805 TI - Detection and serogroup determination of Neisseria meningitidis in CSF by polymerase chain reaction (PCR). AB - A PCR protocol for the detection and serogroup determination of Neisseria meningitidis in CSF from 85 cases of suspected meningitis was evaluated. Screening assays for both IS1106 and the ctrA gene were used to detect meningococcal DNA, and a further two assays using the siaD gene were performed to determine the serogroup. PCR results were compared with results of bacteriological culture and discrepant results resolved by analysis of clinical data and further laboratory test results. The resolved sensitivity and specificity of the PCR screening assay were 89 and 100%, and those of bacteriological culture were 37 and 100%, respectively. The siaD B/C PCR assay was able to determine a serogroup in 85% of cases positive by the PCR screening assay compared with 50% of cases where a serogroup was determined by traditional methods. PCR is a useful tool for diagnosis of meningococcal meningitis when Gram stain and culture tests are negative, a situation that may arise when antibiotic treatment has commenced prior to lumbar puncture. PMID- 10740806 TI - Group B streptococcus screening in pregnant women: a comparison of three media. AB - This study compared the relative isolation rate of Group B Streptococcus (GBS) from 663 low vaginal swabs, collected from antenatal patients, on three media: horse blood agar plus neomycin (75 mg/l) (Neo), Islam agar (Islam) and Islam agar plus nalidixic acid (15 mg/l) and colistin sulphate (10 mg/l) (Islam Plus). One hundred and forty-seven (22%) GBS were isolated. At 24 hours the isolation rate was highest using Neo, but within 72 hours there was little difference. The isolation rates for Neo, Islam and Islam Plus at 24 hours were 124 (18.7%), 103 (15.6%), 109 (16.4%) (P < 0.05); at 48 hours 125 (18.9%), 116 (17.5%), 121 (18.1%) (P > 0.1); and at 72 hours 125 (18.9%), 121 (18.3%) and 127 (19.1%) (P > 0.1), respectively. Twenty-two isolates were missed on Neo, 26 on Islam and 20 on Islam Plus. Of those missed on Islam agars, 12 failed to produce pigment and were only detected on Neo. The disadvantage of Neo is the need to perform additional tests to confirm the identity as GBS. In the present study 100 suspicious colonies were identified as Group D. PMID- 10740807 TI - Early diagnosis of Murray Valley encephalitis by reverse transcriptase-polymerase chain reaction. AB - A 4-year-old aboriginal boy developed encephalitis due to Murray Valley encephalitis virus (MVE) following an earlier infection with Kunjin virus (KUN). The illness was severe, resulting in cerebral atrophy and profound physical and intellectual disability. The earlier KUN infection complicated his serological profile and delayed antibody responses to MVE. By contrast, the reverse transcriptase-polymerase chain reaction (RT-PCR) assay detected MVE in serum 3 days after the onset of illness and 4 days before the appearance of MVE-specific IgM. We suggest that MVE-specific RT-PCR provides rapid and specific diagnosis of MVE and should be used more widely for the diagnosis of acute viral encephalitis in cases originating from flavivirus endemic areas. PMID- 10740808 TI - Acute basophilic leukemia presenting with abnormal liver function tests and the absence of blast cells in the peripheral blood. AB - Acute basophilic leukemia is an uncommon form of acute leukemia, rarely occurring as a de novo disease. We describe a case of de novo acute basophilic leukemia occurring in a 47-year-old man who presented with abnormal liver function tests in the absence of leukemic infiltration of the liver. We postulate that this presentation occurred as a consequence of pathophysiological features of the malignant basophilic blast cells. PMID- 10740809 TI - Nephrotic syndrome in a patient with IgA deficiency-associated mesangioproliferative glomerulonephritis. AB - A case of mesangioproliferative glomerulonephritis in a 55-year-old woman with selective IgA deficiency and serum antinuclear antibodies who presented with nephrotic syndrome is described. The patient did not have clinical or laboratory features of systemic lupus erythematosus (SLE) other than antinuclear antibodies. Histology of the patient's renal biopsy revealed a mesangioproliferative glomerulonephritis and direct immunofluorescence showed that paramesangial deposits contained predominant IgM with lesser IgG, C3 and C1q. These findings are identical to those previously described in a form of glomerulonephritis associated with IgA deficiency and would be atypical for lupus nephritis. Glomerulonephritis is not a well recognized complication of IgA deficiency, though it has been rarely reported in the literature. This case provides further evidence that IgA deficiency is associated with a unique immune complex-mediated glomerulopathy with characteristic immunopathological and ultrastructural features. It is the first reported case to present with nephrotic syndrome. PMID- 10740810 TI - Relationship between Ras pathways and cell cycle control. AB - The ordered execution of the two main events of cellular reproduction, duplication of the genome and cell division, characterize progression through the cell cycle. Cultured cells can be switched between cycling and non-cycling states by alteration of extracellular conditions and the notion that a critical cellular control mechanism presides on this decision, whose temporal location is known as the restriction point, has become the focus for the study of how extracellular mitogenic signalling impinges upon the cell cycle to influence proliferation. This review attempts to cover the disparate pathways of Ras-mediated mitogenic signal transduction that impact upon restriction point control. PMID- 10740811 TI - Cell-anchorage, cell cytoskeleton, and Rho-GTPase family in regulation of cell cycle progression. AB - It has been well known that cell-anchorage and the cell cytoskeleton are deeply involved in the regulation of cell proliferation and cell cycle. However, the precise molecular mechanism involved in cell-anchorage and the cell cytoskeleton have remained be to elucidated. The recent great volume of information regarding cell cycle regulators such as cyclin, cyclin-dependent kinases (CDKs) and CDK inhibitors (CKI) has facilitated the understanding of the cell cycle in mammalian cells. In this review, we will focus on these cell cycle regulators to discuss the regulation of cell proliferation controlled by cell-anchorage and the cytoskeleton, and especially the roles of Rho family GTPases. PMID- 10740812 TI - The continuum model and G1-control of the mammalian cell cycle. AB - The continuum model of the mammalian division cycle proposes that there are no G1 phase specific controls or events. The G1 phase is simply the time when processes begun in the previous cell cycle are completed. In this review, the continuum model is applied the variability of the G1-phase, the existence of G1-less cells, the ubiquitous G1-phase arrest phenomenon, the effect of over-expressed cyclins on G1-phase length, the statistical variation of the cell cycle, the reports of G1-phase syntheses, the proposed variation in retinoblastoma protein phosphorylation in G1-phase, and the myriad findings put forward to support the G1-control model of the mammalian division cycle. The continuum model is a valid description of the mammalian division cycle. PMID- 10740813 TI - Histone acetylation and the control of the cell cycle. AB - The critical steps of the cell cycle are generally controlled through the transcriptional regulation of specific subsets of genes. Transcriptional regulation has been recently linked to acetylation or deacetylation of core histone tails: acetylated histone tails are generally associated with active chromatin, whereas deacetylated histone tails are associated with silent parts of the genome. A number of transcriptional co-regulators are histone acetyl transferases or histone deacetylases. Here, we discuss some of the critical cell cycle steps in which these enzymes are involved. PMID- 10740814 TI - 14-3-3 proteins and growth control. AB - The 14-3-3 proteins constitute a family that is highly conserved in a wide range of organisms, including higher eukaryotes, invertebrates and plants. Variants of 14-3-3 proteins assembled in homo- and heterodimers were found to interact with diverse cellular proteins. Until recently, the biological role of 14-3-3 members was still poorly understood. However, the results of an increasing number of studies on their structure and function are converging to define 14-3-3 proteins as a novel type of adaptor that modulates interactions between components involved in signal transduction pathway and in cell cycle control. PMID- 10740815 TI - A Cdc7p-Dbf4p protein kinase activity is conserved from yeast to humans. AB - DBF4 and CDC7 were identified as budding yeast cell cycle mutants that arrest immediately before S phase. The Dbf4p and Cdc7p proteins interact to form a protein kinase, Cdc7p being the catalytic subunit and Dbf4p is a cyclin-like molecule that activates the kinase in late G1. Dbf4p also targets Cdc7p to origins of replication where likely substrates include the Mcm proteins. Dbf4p and Cdc7p related proteins occur in the fission yeast and in metazoans. These also phosphorylate Mcm proteins and preliminary evidence indicates a similar function to Dbf4p/Cdc7p in budding yeast. The Dbf4p/Cdc7p activity will therefore very likely be conserved in all eukaryotes. PMID- 10740816 TI - Alternative product of the p16/CKDN2A locus connects the Rb and p53 tumor suppressors. AB - Two distinct products are specified by the CDKN2A locus, the p16INK4a cyclin dependent kinase inhibitor and a protein termed ARF. ARF has been shown to bind to the Mdm2-p53 complex, resulting in stabilisation of both proteins, and a feedback loop exists through which ARF levels are negatively regulated by p53. Significantly, ARF expression is positively regulated by members of the E2F family of transcription factors. This provides a link between the Rb and p53 pathways and a mechanism whereby inactivation of Rb and release of E2F will lead to the stabilisation and functional activation of p53. The alternative exon encoding the functional amino terminal portion of ARF presumably represents an independent gene that has become co-localized with p16INK4a in order to exploit a common regulatory mechanism or purpose. PMID- 10740817 TI - Phosphorylation-dependent prolyl isomerization: a novel cell cycle regulatory mechanism. AB - Protein phosphorylation by proline-directed protein kinases plays an essential role in triggering a programmed set of cell cycle events. We have recently isolated an essential and conserved mitotic regulator, Pin1. Pin1 is a phosphorylation-dependent prolyl isomerase that specifically isomerizes the phosphorylated serine/threonine-proline bond. Pin1 also binds and regulates the function of a conserved set of mitosis-specific phosphoproteins. These results suggest phosphorylation-dependent prolyl isomerization to be a novel cell cycle regulatory mechanism. This new post-translational regulation may allow the general increase in protein phosphorylation to be converted into the organised and programmed set of structural modifications that occur during mitosis. In addition, since inhibition of Pin1 induces mitotic arrest and apoptosis, Pin1 may be a potential new drug target. PMID- 10740818 TI - Functions of Pho85 cyclin-dependent kinases in budding yeast. AB - Pho85 is a multifunctional cyclin-dependent kinase (Cdk) in Saccharomyces cerevisiae that has emerged as an important model for the role of Cdks in both cell cycle control and other processes. Pho85 was originally discovered as a regulator of phosphate metabolism but roles for Pho85 in glycogen biosynthesis, actin regulation and cell cycle progression have since been discovered. Ten genes encoding known or putative Pho85 cyclins (Pcls) have been identified and the Pcls appear to target Pho85 to specific cellular functions and substrates. In this chapter, we review the functions of the various Pcl-Pho85 complexes in budding yeast. We focus on the known biological roles of Pho85 with an emphasis on Pho85 substrates and cyclin-Cdk specificity. PMID- 10740819 TI - Cell cycle regulation by the Cdc25 phosphatase family. AB - Activation of cyclin-dependent kinases in higher eukaryotic cells can be achieved through dephosphorylation by members of the Cdc25 phosphatase family, Cdc25A, Cdc25B and Cdc25C. Cdc25A plays an important role at the G1/S-phase transition. Cdc25B undergoes activation during S-phase and plays a role in activating the mitotic kinase Cdk1/cyclin B in the cytoplasm. Active Cdk1/cyclin B then phosphorylates and activates Cdc25C leading to a positive feedback mechanism and to entry into mitosis. Cdc25A and B are potential human oncogenes. In addition, Cdc25 is a main player of the G2 arrest caused by DNA damage or in the presence of unreplicated DNA. PMID- 10740820 TI - Molecular mechanisms of the initiation of oocyte maturation: general and species specific aspects. AB - Stimulated by maturation-inducing hormone secreted from follicle cells surrounding the oocytes, fully-grown oocytes mature and become fertilisable. During maturation, immature oocytes resume meiosis arrested at the first prophase and proceed to the first or second metaphase at which they are naturally inseminated. Paying special attention to general and species-specific aspects, we summarise the mechanisms regulating the initial phase of oocyte maturation, from the reception of hormonal signals on the oocyte surface to activation of the maturation-promoting factor in the cytoplasm, in amphibians, fishes, mammals and marine invertebrates. PMID- 10740821 TI - The activation of MAP kinase and p34cdc2/cyclin B during the meiotic maturation of Xenopus oocytes. AB - G2-arrested Xenopus oocytes are induced to enter M-phase of meiosis by progesterone stimulation. This process, known as meiotic maturation, requires the activation of p34cdc2/cyclin B complexes (pre-MPF) which is brought about by the prior translation of specific maternal mRNAs stored in the oocyte. One of these mRNAs encodes for the protein kinase Mos which has an essential role in oocyte maturation, most likely due to its ability to activate MAP kinase (MAPK). Here we review our current knowledge on the Mos/MAPK signalling pathway and a recently found connection between MAPK-activated p90rsk and the p34cdc2 inhibitory kinase Myt1. We also discuss a pathway that involves the protein kinase Plx1 and leads to the activation of the phosphatase Cdc25, as well as other regulators of p34cdc2/cyclin B activity which may have a role in oocyte maturation. PMID- 10740822 TI - Premature chromatin condensation caused by loss of RCC1. AB - Hamster rcc1 mutant, tsBN2, prematurely enter mitosis during S phase. RCC1 is a guanine nucleotide exchanging factor for a small G protein Ran and localised on the chromatin, whereas RanGTPase activating protein is in the cytoplasm. Consistently, Ran shuttles between the nucleus and the cytoplasm, carrying out nucleus-cytosol exchange of macromolecules, which regulates the cell cycle. The finding that loss of RCC1 which disturbs nuclear protein export due to loss of RanGTP, abrogates the check point control suggests that RCC1 senses the status of the chromatin, such as replication, and couples it to the cell cycle progression through Ran. PMID- 10740823 TI - HTLV-I Tax and cell cycle progression. AB - Human T-cell leukemia virus type I (HTLV-I) is the etiological agent for adult T cell leukemia (ATL) and various human myopathies/neuropathies. HTLV-I encodes a 40 kDa phosphoprotein, Tax, which has been implicated in cellular transformation. In similarity with several other oncoproteins such as Myc, Jun, and Fos, Tax is a transcriptional activator. How Tax mechanistically dysregulates the cell cycle remains unclear. Recent findings from us and others have shown that Tax targets key regulators of G1/S and M progression such as p16INK4a, cyclin D1, cyclin D3 cdk, and the mitotic spindle checkpoint apparatus. Thus, Tax influences the progression of cells in various phases of the cell cycle. In this regard, we will discuss three distinct mechanisms through which Tax affects cell-cycling: a) through direct association Tax can abrogate the inhibitory function of p16INK4a on the G1-cdks, b) Tax can also directly influence cyclin D-cdk activities by a protein-protein interaction, and c) Tax targets the HsMAD1 mitotic spindle assembly checkpoint protein. Through these varied routes, the HTLV-I oncoprotein dysregulates cellular growth controls and engenders a proclivity of cells toward a loss of DNA-damage surveillance. PMID- 10740824 TI - The cell cycle in protozoan parasites. AB - Research into cell cycle control in protozoan parasites, which are responsible for major public health problems in the developing world, has been hampered by the difficulties in performing classical genetic analysis with these organisms. Nevertheless, in a large part thanks to the data gathered in other eukaryotic systems and to the acquisition of the sequences of parasite genes homologous to cell cycle regulators, many molecular tools required for an in-depth study of the cell cycle in protozoan parasites have been collected over the past few years. Despite the considerable phylogenetic divergence between these organisms and other eukaryotes, and notwithstanding important specificities such as the apparent lack of checkpoints during cell cycle progression, available data indicate that the major families of cell cycle regulators appear to operate in protozoan parasites. Functional studies are now needed to define the precise role of these regulators in the life cycle of the parasites, and to possibly validate cell cycle control elements as potential targets for chemotherapy. PMID- 10740825 TI - Circadian control of cell division in unicellular organisms. AB - Cell division cycles and circadian rhythms are major periodic phenomena in organisms. Circadian oscillators control biochemical, physiological, and behavioral events in a wide range of living systems including almost all eukaryotes that have been tested and some prokaryotes-in particular, the cyanobacteria. Gating of cell division is one of the key processes that has been reported to be regulated by circadian clocks in many organisms. We survey studies of the circadian control of cell division in eukaryotic microorganisms and introduce recent progress on understanding the interaction between circadian rhythms and cell division cycles in cyanobacteria. PMID- 10740826 TI - Circadian variation of cell proliferation and cell cycle protein expression in man: clinical implications. AB - Most physiological, biochemical and behavioural processes have been shown to vary in a regular and predictable periodic manner with respect to time. This review focuses on the circadian rhythm in cell proliferation in bone marrow and gut and how this is associated with a circadian expression of cell cycle proteins in human oral mucosa. The control of circadian rhythms by the suprachiasmatic nuclei and the evolving understanding of the genetic and molecular biology of the circadian clock is outlined. Finally, the potential clinical impact of chronobiology in cancer medicine is discussed. PMID- 10740827 TI - Molecular switches that govern the balance between proliferation and apoptosis. AB - Tissue modelling during embryogenesis and tissue homeostasis during adult life is governed by a dynamic equilibrium between growth and programmed cell death (apoptosis). Growth control and apoptosis are intimately associated, and a disturbance of the balance between these two processes often leads to pathological situations, such as for example cell accumulations in cancer. To date many of the molecular mechanisms controlling growth control on the one hand, and apoptosis on the other hand are known, whereas the switch that controls the decision between both pathways remains elusive. A cell is continuously exposed to multiple opposing "death" and "survival" triggers. A challenging question is how a cell senses these signals and decides to live or die. A decision in favour of survival should automatically result in a shut down of the death pathways. Alternatively, a decision for death should result in inhibition of futile attempts to survive. The molecular events controlling this balance of signals will be discussed with special emphasis on the role of cyclin-dependent kinases and the ubiquitin-dependent and proteasome-mediated protein degradation pathway. PMID- 10740828 TI - Molecular events that regulate cell proliferation: an approach for the development of new anticancer drugs. AB - Cancer chemotherapy is the object of many fundamental and clinical researches. The development in molecular techniques and structural studies at the molecular level have led to the discovery of key proteins involved in the regulation of cell proliferation. This opened perspectives to characterize new anticancer drugs in order to reduce the limitations found with conventional drugs such as the lack of selectivity for cancer cells and resistance phenomena. This review presents the anticancer drugs in clinical investigations that target molecules involved in the signal transduction impairment, the cell cycle deregulation and the differentiation with comments on their mechanisms of action. PMID- 10740829 TI - Abortive oncogeny and cell cycle-mediated events in Alzheimer disease. AB - Alzheimer disease, the leading cause of senile dementia, is characterised by the degeneration of select neuronal populations. While the mechanism(s) underlying such cell loss are largely unknown, recent findings indicate inappropriate re entry into the cell cycle resembling an abortive oncogeny. In postmitotic neurons, such mitotic re-entrance is deleterious and one that involves virtually the entire spectrum of the described pathological events in Alzheimer disease including, ultimately, cell death. PMID- 10740830 TI - [Reactivity of blood vessels in vitro: the role of perfusate pH and the initial tone of myocytes]. AB - In the rat mesenteric artery ring, acidic solution (pH 7.0) inhibited the electrical field stimulation (EFS)-evoked response in a wide range of stretch. Acidosis potentiated the EFS-evoked constriction of the ring precontracted with 0.5 mu noradrenaline. The EFS dilated rings precontracted with 0.7 mu noradrenaline in control solution but contracted it in the acidic solution. Tone dependent mechanisms of the acidosis effects on the EFS-evoked responses are discussed. PMID- 10740831 TI - [Aromatase (estrogen synthetase) gene expression in human leukocytes]. AB - Extragonadal aromatization plays an important role in many physiological and pathological conditions. It has been found that incubation of the peripheral blood mononuclears in the RPMI-1640 medium with 10% fetal calf serum during 48 hrs is able to induce the aromatase gene expression. The latter did not occur in 11 samples of mononuclears of the intra-tissue leukocyte infiltration which finding needs further investigation. The data obtained suggest that the possibility of the aromatase gene induction in leukocytes is associated with the cellular fraction capable of the substrate adhesion. PMID- 10740832 TI - [Expression of urokinase and its receptor correlate with proliferation of smooth muscle cell in injured arteries]. AB - Using the immunohistochemistry and a standard reverse transcriptase-polymerase chain reaction assay optimized to estimate the mRNA levels, we observed a 2-fold increased uPA expression by the SMCs in injured vessels as compared with uninjured vessels. The uPA elevation occurred within 6 hours from the injury and persisted for 96 hours after the injury. The uPAR expression was also elevated after an injury although it occurred slower and more gradually. The data obtained suggest that the uPA is capable of contributing to both the SMC migration, replication and accumulation in the neointima early after balloon catheter injury of the carotid artery in rats. PMID- 10740833 TI - [Cerebrovascular circulation during blockade of angiotensin receptors]. AB - In experiments on anaesthetized rats, Losartan was found to cause an obvious decrease in the ABP in normotensive rats. The cerebral blood flow differed independence on cerebral vascular resistance and the ABP level. The autoregulation of the cerebral blood flow remained unaltered. In hypertensive rats Losartan caused a significant decrease in the ABP as compared with normotensive rats. A shift of lower limits of the cerebral blood flow autoregulation towards a lower ABP level, was observed. PMID- 10740834 TI - [Physiological manifestations of action of a gene regulating predisposition to a pendulum-like movements in rodents]. AB - Physiological effects of the gene determining predisposition to a stereotypical hyperkinesis in the form of pedulum movements (PM) are manifested in some behavioral peculiarities (total motor activity, emotionality, intensity of the startle reflex, sensitivity of the 5-HT2 receptors, predisposition to catalepsy). High frequency of the PM occurring in Wistar rat stock suggests that the gene determining the PM expression is not a pathological one but controls some adaptive properties of the nervous system. Relationships between the PM expression and the degree of predisposition to catalepsy are biphasic and may be described by an inverted U-shaped curve. PMID- 10740835 TI - [Characteristics of the acute phase reaction in humans with various types of autonomic nervous system regulation during simulated hyperthermia]. AB - Depending on the type of autonomous regulation, differences in basic levels of interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha) were revealed under conditions of hyperthermia in healthy subjects aged 19-21. A parasympathetic type of autonomous regulation corresponded to higher initial levels of proinflammatory cytokinesis, whereas a dominating sympathetic type corresponded to lower levels of the IL-1 beta and TNF alpha. The subjects with the latter type of regulation revealed an increase in the IL-1 beta TNF alpha combined with a higher heat tolerance. The subjects with the former type of regulation revealed a lower heat tolerance. The increase in the alpha2 macroglobulin appeared to be a most typical acute phase response of the human body to hyperthermia. PMID- 10740836 TI - [The role of thyroid status in the cardiac protection during heat adaptation in rats]. AB - Thyroid status is important for realization of protective cardiac effects on heat adaptation as evaluated by the changes induced by the heat stress (40-42 degree C during 3 hrs) in parameters of the heart main contractile function and in contraction of lipid peroxidation products. Adaptation to heat against the background of administration of small doses of thyroid in potentiated these effects, whereas heat training against the background of thyrostatic agent Mercasolil prevented them. PMID- 10740837 TI - [The role of serum 'adhesive factor' and leukocyte sensitization in pathogenesis of multiple sclerosis]. AB - The reactivity to the myelin basic protein, brain gangliosides purified derivative and also the influence of the serum on the cells adherence were determined by micromodification of the leukocyte adherence inhibition (LAI) test. 46 multiple sclerosis patients, 21 patients with hereditary disease and 41 donors were examined. The cellular sensitization to the myelin basic protein and brain gangliosides was revealed in 57% of the multiple sclerosis patients. The leukocyte adherence was increase in 53.5% patients when autologous serum was added. The adherence factor was most often found (90% case) in patients with severe neurological deficit. It is suggested that this factor is a connected t the increased levels of circulating adhesion molecules in multiple sclerosis patients. PMID- 10740838 TI - [The role of local stress-limiting myocardial systems in the protective cardiac effect of low doses of thyroid hormones during immobilization stress in rats]. AB - Thyroid hormones seem to activate some fundamental cellular protective mechanisms and thus to be an important regulatory factor for local myocardial systems in their stress-limiting activity. PMID- 10740839 TI - [The role of sodium-proton exchange in the regulation of the electric and contractile activity of smooth muscles]. AB - The Na+/H+-exchange inhibitor aethylisopropylamyloride was found to enhance the membrane resistance, the AP duration, and the duration of the guinea pig ureter's smooth muscle contraction. Tetraethylammonium and incubation in the Ca-free EG-TA containing solution eliminated the activating effect of this inhibitor. Gramycidin A shortens the AP plateau. The Na+/H+-exchange mechanism seems to act under normal conditions. Alterations occurring in electrical and contractile activities of smooth muscle under the effect of the Na+/H+-exchange seem to result from the changes in the membrane Ca-dependent potassium conductivity. PMID- 10740840 TI - [Functional role of V1- and V2-receptors of the apical and basolateral membranes of the frog bladder epithelial cells]. AB - Arginine vasotocin, 0.02--1 nM, increases osmotic water permeability of frog urinary bladder, arginine vasotocin after a simultaneous addition to the mucosal and serosal Ringer solutions rises the water permeability to a lesser degree than on the hormone addition only to the serosal solution. 1 nM remestyp, an agonist of V1-receptors, from the apical membrane decreases the hydroosmotic effect of arginine vasotocin added to the serosal Ringer solution. When added to the mucosal solution, combination of the same concentrations of arginine vasotocin and SR 49059, an antagonist of V--receptors, or desmopressin, agonist of V2 receptor alone, increases the effect of the same concentration of arginine vasotocin added to the serosal solution. 1 nM arginine vasotocin at the luminal membrane increases secretion into the Ringer solution of prostaglandin E, and prostaglandin E1 but not of prostaglandin F2 alpha. The data obtained indicate the presence of the arginine vasotocin receptors responsible for the hydroosmotic effect only in the basolateral membranes, while arginine prostaglandin E, participation is shown in modulation of the arginine vasotocin effect. PMID- 10740842 TI - [Forming of the Pavlov pouch changes functional condition of the adherent mucosal layer in upper regions of the digestive tract]. AB - In result of forming the Pavlov small gaster, secretion of pepsine in isolated part and, probable, in other parts of the gaster, was increased. This entailed a decrease in the pepsin sorption level on the luminal surface of adherent mucous layer of the gaster parts and intestine under study. This led to a drop of the level of structure glycoproteins and to increase in the per cent contents of polymeric glycoproteins in the whole pool of the mucous layer glycoproteins. The data obtained suggest that forming of the Pavlov small gaster indices significant changes of the adherent mucous layer structure-functional condition both in the gaster and in lower parts of the intestine. PMID- 10740841 TI - [Age characteristics of K(+) transport in the rat distal colon]. AB - In vitro and in vivo experiments with perfusion in 20- and 60-day old rats revealed that K+ absorption in the gut as well as 86Rb uptake in the distal colon was significantly higher in younger rats due probable, to the higher activity of the apex-located K+-dependent ATPase and lower activity of the basolateral K+ carriers. The luminal blockade of K+ absorbing pumps with ouabain or omeprazole resulted in a decrease of the K+ absorption and K2 accumulation in skeletal muscles. The higher K+-absorbing/K_ secreting mechanisms ratio in younger rats contributes to the positive potassium balance. PMID- 10740843 TI - [Effect of the long-term exposure of the isolated myometrium from nonpregnant rats to serum from pregnant women on its contractile activity and the beta adrenergic reactivity]. AB - Pregnant women's blood serum (the whole as well as 50- and 100-diluted) did not alter parameters of spontaneous motility, i.e. it did not affect the uterus myocytes' spontaneous motility, whereas the whole or 50-diluted serum raised considerable the rat myometrium's beta-adrenoreaction. The data obtained suggest that the beta-adrenoreaction of the rat uterus' myocytes increases after a prolonged contact with pregnant women's blood serum which may be due to the beta adrenoreceptor synthesis activation and to an increase in their concentration because of diffusion from the blood serum. This suggests existence of humoral mechanisms of the myometrium beta-adrenoreaction regulation, the mechanisms creating the optimum uterus' contractile activity. PMID- 10740844 TI - [24-Hour monitoring of the serum lipid profile in normal and obese women]. AB - In fast and in fat-load elevated the free cholesterol level and decreased the total phospholipids contents in the blood serum of both normal and obese women. Subsequently, normal women revealed a drop in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, whereas in obese women the phosphatidylcholine level remained stable during the test. Contents of phosphatidylcholine, phosphatic acid and glycerophosphate was still lowering in 24 hours after the fat load. PMID- 10740845 TI - [Model study of the heat exchange between arterioles and venules]. PMID- 10740846 TI - Isoelectric focusing nonporous RP HPLC: a two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI-TOF mass spectrometry. AB - A novel two-dimensional liquid-phase separation method was developed that is capable of resolving large numbers of cellular proteins. The proteins are separated by pI using isoelectric focusing in the first dimension and by hydrophobicity using nonporous reversed-phase HPLC in the second dimension (IEF NP RP HPLC). Proteins were mapped using original software in order to create a protein pattern analogous to that of the 2-D PAGE image. RP HPLC peaks are represented by bands of different intensity in the 2-D image, according to the intensity of the peaks eluting from the HPLC. Each peak was collected as the eluent of the HPLC separation in the liquid phase. The proteins collected were identified using proteolytic enzymes, MALDI-TOF MS and MSFit database searching. Using IEF-NP RP HPLC, approximately 700 bands were resolved in a pI range from 3.2 to 9.5 and 38 different proteins with molecular weights ranging from 12,000 to 75,000 were identified. In comparison to a 2-D gel separation of the same human erythroleukemia cell line lysate, the IEF-NP RP HPLC produced improved resolution of low mass and basic proteins. In addition, the proteins remained in the liquid phase throughout the separation, thus making the entire procedure highly amenable to automation and high throughput. It is demonstrated that IEF-NP RP HPLC provides a viable alternative to the 2-D gel separation method for the screening of protein profiles. PMID- 10740847 TI - Protein identification with a single accurate mass of a cysteine-containing peptide and constrained database searching. AB - A method for rapid and unambiguous identification of proteins by sequence database searching using the accurate mass of a single peptide and specific sequence constraints is described. Peptide masses were measured using electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry to an accuracy of 1 ppm. The presence of a cysteine residue within a peptide sequence was used as a database searching constraint to reduce the number of potential database hits. Cysteine-containing peptides were detected within a mixture of peptides by incorporating chlorine into a general alkylating reagent specific for cysteine residues. Secondary search constraints included the specificity of the protease used for protein digestion and the molecular mass of the protein estimated by gel electrophoresis. The natural isotopic distribution of chlorine encoded the cysteine-containing peptide with a distinctive isotopic pattern that allowed automatic screening of mass spectra. The method is demonstrated for a peptide standard and unknown proteins from a yeast lysate using all 6118 possible yeast open reading frames as a database. As judged by calculation of codon bias, low-abundance proteins were identified from the yeast lysate using this new method but not by traditional methods such as tandem mass spectrometry via data-dependent acquisition or mass mapping. PMID- 10740848 TI - Improving the thromboresistivity of chemical sensors via nitric oxide release: fabrication and in vivo evaluation of NO-releasing oxygen-sensing catheters. AB - The development and in vivo analytical performance of a nitric oxide (NO) releasing amperometric oxygen sensor with greatly enhanced thromboresistivity are reported. Gas permeable coatings formulated with cross-linked silicone rubber (SR) containing NO-generating compounds (diazeniumdiolates) are shown to release NO for extended periods of time (> 20 h) while reducing platelet adhesion and activation. Oxygen-sensing catheters prepared by dip-coating the NO-releasing films over the outer SR tubes of the implantable devices display similar analytical response properties in vitro (sensitivity, selectivity, response times) when compared to analogous sensors prepared without the NO release coatings. Superior analytical accuracy (relative to blood PO2 values measured in vitro) and greatly reduced thrombus formation on the outer surface of the sensors are observed in vivo (in canine model) with the NO release PO2 sensors compared to control sensors (without NO release) implanted simultaneously within the same animals. Based on these preliminary studies, the use of NO release polymers to fabricate catheter-style chemical sensors may be a potential solution to lingering biocompatibility and concomitant performance problems encountered when attempting to employ such devices for continuous intravascular measurements of blood gases and electrolytes. PMID- 10740849 TI - Selectivity of potentiometric ion sensors. AB - Selectivities of solvent polymeric membrane ion-selective electrodes (ISEs) are quantitatively related to equilibria at the interface between the sample and the electrode membrane. However, only correctly determined selectivity coefficients allow accurate predictions of ISE responses to real-world samples. Moreover, they are also required for the optimization of ionophore structures and membrane compositions. Best suited for such purposes are potentiometric selectivity coefficients as defined already in the 1960s. This paper briefly reviews the basic relationships and focuses on possible biases in the determination of selectivity coefficients. The traditional methods to determine selectivity coefficients (separate solution method, fixed interference method) are still the same as those originally proposed by IUPAC in 1976. However, several precautions are needed to obtain meaningful data. For example, errors arise when the response to a weakly interfering ion is also influenced by the primary ion leaching from the membrane. Wrong selectivity coefficients may be also obtained when the interfering agent is highly preferred and the electrode shows counterion interference. Recent advances show how such pitfalls can be avoided. A detailed recipe to determine correct potentiometric selectivity coefficients unaffected by such biases is presented. PMID- 10740850 TI - Site-specific mass tagging with stable isotopes in proteins for accurate and efficient protein identification. AB - Proteolytic peptide mass mapping as measured by mass spectrometry provides a major approach for the identification of proteins. A protein is usually identified by the best match between the measured and calculated m/z values of the proteolytic peptides. A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization. Without ultrahigh instrumental accuracy, it is possible to increase the specificity of the assignments of particular proteolytic peptides by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence. Here we report this novel method of generating residue-specific mass-tagged proteolytic peptides for accurate and efficient protein identification. Selected amino acids are labeled with 13C/15N/2H and incorporated into proteins in a sequence-specific manner during cell culturing. Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern. Through their characteristic patterns, the peptides with mass tags can then be readily distinguished from other peptides in mass spectra. This method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency, and accuracy for protein identifications. PMID- 10740851 TI - Integration of an immunosorbent assay system: analysis of secretory human immunoglobulin A on polystyrene beads in a microchip. AB - An immunosorbent assay system was integrated into a glass microchip. Polystyrene beads were introduced into a microchannel, and then human secretory immunoglobulin A (s-IgA) adsorbed on the bead surface was reacted with colloidal gold conjugated anti-s-IgA antibody and detected by a thermal lens microscope. The scale merits of liquid microspace on the molecular behavior remarkably contributed to reduced assay time. The integration cut the time necessary for the antigen-antibody reaction by 1/90, thus shortening the overall analysis time from 24 h to less than 1 h. Moreover, troublesome operations required for conventional immunosorbent assays could be replaced by simple operations. PMID- 10740852 TI - Chemiluminescent reaction of fluorescent organic compounds with KHSO5 using cobalt(II) as catalyst and its first application to molecular imprinting AB - The decomposition of peroxomonosulfate (HSO5-) has been investigated by chemiluminescence (CL). A weak CL was observed during mixing the HSO5- solution with the Co2+ solution in unbuffered conditions. An appropriate amount of fluorescent organic compounds (FOCs), such as dansyl amino acids and pyrene, was added to the KHSO5/Co2+ solution, a strong CL was recorded. A possible CL mechanism, based on studies of the fluorescence, CL, and UV-visible spectra and comparison of Co3+ oxidation ability with the SO4.- radical ion, was discussed. The CL from HSO5-/Co2+ is the emission of singlet oxygen produced from the catalytic decomposition of HSO5-. It was suggested that the decomposition of HSO5 in aqueous solution with Co2+ proceeds via one-electron transfer to yield SO4.- radical ion. The FOC was attacked by SO4.- radical ion and oxidized to decompose into small molecules. During this proceeding, CL emission was given out. The present CL system has been developed as a flow injection analysis for FOCs. The detection limits (S/N = 3) were in the concentration range 10(-9)-10(-7) M for FOCs. Oxidation decomposition and CL emission of the analytes have been used in the molecular imprinting recognition. As an initial attempt, dansyl-L phenylalanine was used as a template molecule and methacrylic acid and 2 vinylpyridine were used as functional monomers. The network copolymer imprinted with dansyl-L-phenylalanine exhibits an affinity for the template molecule. When the flowing streams of HSO5- and Co2+ solutions mixing through the molecularly imprinted polymer particles filled the flow cell, the template molecule, dansyl-L phenylalanine reacted with the HSO5-/Co2+ solution and CL was emitted. The dansyl L-phenylalanine was decomposed during the CL process, and the cavities of a defined shape and an arrangement of functional groups complementary to the template in the polymer were left for the next sample analysis. PMID- 10740853 TI - Electrostatic axially harmonic orbital trapping: a high-performance technique of mass analysis AB - This work describes a new type of mass analyzer which employs trapping in an electrostatic field. The potential distribution of the field can be represented as a combination of quadrupole and logarithmic potentials. In the absence of any magnetic or rf fields, ion stability is achieved only due to ions orbiting around an axial electrode. Orbiting ions also perform harmonic oscillations along the electrode with frequency proportional to (m/z)-1/2. These oscillations are detected using image current detection and are transformed into mass spectra using fast FT, similarly to FTICR. Practical aspects of the trap design are presented. High-mass resolution up to 150,000 for ions produced by laser ablation has been demonstrated, along with high-energy acceptance and wide mass range. PMID- 10740855 TI - Surfactant-aided, matrix-assisted laser desorption/ionization mass spectrometry of hydrophobic and hydrophilic peptides. AB - The analysis of hydrophobic and hydrophilic peptides in an aqueous medium using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is reported. The key development allowing for simultaneous analysis of both hydrophobic and hydrophilic components of the sample mixture is the use of surfactants to solubilize the hydrophobic components in the MALDI matrix solution. A wide variety of anionic, cationic, zwitterionic, and nonionic surfactants were evaluated for their ability to assist in the generation of an abundant pseudomolecular ion from a model hydrophobic peptide ([tert butoxycarbonyl]Glu[gamma-O-benzyl]-Ala-Leu-Ala[O-phenacyl ester]). The results indicate that the most successful surfactant among those studied for analyzing the model hydrophobic peptide is sodium dodecyl sulfate (SDS). SDS exhibited no interfering surfactant background ions, little to no loss of the acid-labile protecting groups from the model hydrophobic peptide, and an abundant pseudomolecular ion of the analyte. In addition, the use of surfactants is shown to be compatible with hydrophilic peptides as well. Mixtures of hydrophobic and hydrophilic peptides were characterized using surfactant-aided (SA) MALDI-MS, and it is demonstrated that all components are detectable once the surfactant is included in the sample solution. We conclude that the key benefit of using SA MALDI-MS is its ability to simultaneously analyze hydrophobic and hydrophilic peptides from a single sample mixture, including synthetic peptides containing acid- and base-labile protecting groups. PMID- 10740854 TI - Identification of in-gel digested proteins by complementary peptide mass fingerprinting and tandem mass spectrometry data obtained on an electrospray ionization quadrupole time-of-flight mass spectrometer. AB - The present study reports a procedure developed for the identification of SDS polyacrylamide gel electrophoretically separated proteins using an electrospray ionization quadrupole time-of-flight mass spectrometer (Q-TOF MS) equipped with pressurized sample introduction. It is based on in-gel digestion of the proteins without previous reduction/alkylation and on the capability of the Q-TOF MS to provide data suitable for peptide mass fingerprinting database searches and for tandem mass spectrometry (MS/MS) database searches (sequence tags). Omitting the reduction/alkylation step reduces sample contamination and sample loss, resulting in increased sensitivity. Omitting this step can leave disulfide-connected peptides in the analyte that can lead to misleading or ambiguous results from the peptide mass fingerprinting database search. This uncertainty, however, is overcome by MS/MS analysis of the peptides. Furthermore, the two complementary MS approaches increase the accuracy of the assignment of the unknown protein. This procedure is thus, highly sensitive, accurate, and rapid. In combination with pressurized nanospray sample introduction, it is suitable for automated sample handling. Here, we apply this approach to identify protein contaminants observed during the purification of the yeast DNA mismatch repair protein Mlh 1. PMID- 10740856 TI - A multiple channel electrospray source used to detect highly reactive ketenes from a flow pyrolyzer AB - This work detects protonated molecular ions of highly reactive pyrolytic products -cyclopentadienylideneketene, cyclohexadienylideneketenimine, and acetylketene- using a flow pyrolyzer connected to a multiple-channel electrospray mass spectrometry. The ketene generated in the flow pyrolyzer is directly conducted into the central channel of a seven-channel electrospray ionization source by a stream of nitrogen gas. Concurrently, a methanol solution containing 0.1% trifluoroacetic acid is electrosprayed through the outside six channels. The protonated methanol ions and the charged droplets generated from the outside six electrospray channels facilitate the ionization of the neutral ketenes through ion-molecule reactions or absorption followed by protonation. PMID- 10740857 TI - Identification and C-terminal characterization of proteins from two-dimensional polyacrylamide gels by a combination of isotopic labeling and nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry. AB - We propose a novel method for the identification and C-terminal characterization of proteins separated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE). Proteins were digested in a gel in a buffer solution containing 50% 18O labeled water, and mixtures of 18O/16O-labeled peptides were analyzed by nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry (FT ICR MS). This method was evaluated using horse skeletal muscle myoglobin as the model protein in SDS gel. The high resolution of FT-ICR MS minimized the overlapping of peptide peaks and facilitated identification of the C-terminal peptide, which was done by observing the undisrupted isotope peak pattern. As well, with its low ppm-level high mass accuracy, it can rapidly and reliably identify the in-gel-separated protein and determine its C-terminal by peptide mass fingerprinting alone. Therefore, this method should be applicable to routine and high-throughput proteome studies. Here, the method was applied to the analysis of rat liver proteins separated by 2D-PAGE. The C-termini of eight proteins were successfully identified out of 10 randomly picked Coomassie brilliant blue-stained spots. The feasibility and limitations of this approach are reported in this paper. PMID- 10740858 TI - Direct determination of dibutyl and monobutyl phosphate in a tributyl phosphate/nitric aqueous-phase system by electrospray mass spectrometry AB - Electrospray ionization mass spectrometry was tested for its potential use in the quantification of monobutyl phosphate (H2MBP) and dibutyl phosphate (HDBP), two degradation products of tributyl phosphate (TBP), the extractant used in the nuclear fuel reprocessing known as the PUREX process. Detection and quantification of these phosphate esters by electrospray in positive and negative ionization mode are reported in this study. This fast and reliable method, which does not require any preliminary sample extraction, appears to be very attractive for process control. Negative ionization mode gave abundant [M - H]- ions for both HDBP and H2MBP products. Thus, the concentration of H2MBP between 0.1 and 10 g/L in concentrated aqueous nitrate solutions can be precisely determined. Moreover, the concentration of HDBP up to 1 g/L in a TBP matrix was evaluated in this mode. For HDBP concentrations below 1 g/L, detection in the positive ionization mode appeared to be attractive. TBP and HDBP cluster detection allowed quantitative HDBP determination. Indeed, small amounts of HDBP in commercial TBP (60 mg/L) could be directly quantified using the specific [2TBP, HDBP + H]+ cluster at m/z 743. PMID- 10740860 TI - Determination of hydrolysis products of sulfur mustards by reversed-phase microcolumn liquid chromatography coupled on-line with sulfur flame photometric detection and electrospray ionization mass spectrometry using large-volume injections and peak compression AB - On-line coupling of reversed-phase microcolumn liquid chromatography (micro-RPLC) and sulfur-selective flame photometric detection (S-FPD) was studied for the selective and direct determination of thiodiglycol, bis(2 hydroxyethylthio)methane, 1,2-bis(2-hydroxyethylthio)ethane, 1,3-bis(2 hydroxyethylthio)propane, and 1,4-bis(2-hydroxyethylthio)butane, which are breakdown products of the chemical warfare agents called sulfur mustards. Both isocratic and gradient elution were used. To improve sensitivity, large-volume injections were applied together with peak compression by displacement for late eluting analytes. With S-FPD, detection limits of 1 microgram/mL were obtained for all compounds. Using the same approach, the target analytes as well as various oxidation products could be identified by micro-RPLC with electrospray ionization mass spectrometry (ESI-MS) and ESI-MS/MS. The optimized micro-RPLC-S FPD system was successfully used for the analysis of a spiked soil sample. PMID- 10740859 TI - Neural network recognition of chemical class information in mobility spectra obtained at high temperatures. AB - A minimal neural network was applied to a large library of high-temperature mobility spectra drawn from 16 chemical classes including 154 substances with 2000 spectra at various concentrations. A genetic algorithm was used to create a representative subset of points from the mobility spectrum as input to a cascade type back-propagation network. This network demonstrated that significant information specific to chemical class was located in the spectral region near the reactant ions. This network failed to generalize the solution to unfamiliar compounds necessitating the use of complete spectra in network processing. An extended back-propagation network classified unfamiliar chemicals by functional group with a mean for average values of 0.83 without sulfides and 0.79 with sulfides. Further experiments confirmed that chemical class information was resident in the spectral region near the reactant ions. Deconvolution of spectra demonstrated the presence of ions, merged with the reactant ion peaks that originated from introduced samples. The ability of the neural network to generalize the solution to unfamiliar compounds suggests that these ions are distinct and class specific. PMID- 10740861 TI - Microsequencing of glycans using 2-aminobenzamide and MALDI-TOF mass spectrometry: occurrence of unique linkage-dependent fragmentation. AB - 2-Aminobenzamide-derivatized oligosaccharides were separated by three lectin column chromatographies and then subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for structural characterization of the carbohydrates. The combination of sequential exoglycosidase digestion and MALDI-TOF MS greatly facilitates the monosaccharide sequencing and is more feasible than size-exclusion column chromatography in terms of the time consumed and the laboriousness of the procedure. By this strategy, microsequencing of 2-3 pmol of oligosaccharide derivatives could be achieved. Furthermore, spectra obtained by the post source decay (PSD) mode provide excellent sequence information. The relative intensities of metastable ions due to fragmentation at glycosidic linkages were different among linkage isomers of particular oligosaccharides. These results demonstrate that PSD analysis possesses significant potential for the estimation of glycosidic linkage in carbohydrate structures. PMID- 10740863 TI - Macroporous photopolymer frits for capillary electrochromatography AB - Macroporous polymer frits have been fabricated in fused-silica capillaries by the UV photopolymerization of a solution of glycidyl methacrylate and trimethylopropane trimethacrylate. This in situ preparation is a simple, rapid, and reproducible process. The frit can be placed at any desired position along the column. Photopolymer frits can withstand the short exposure to a high pressure (over 6000 psi). Bubble formation is no observed to occur with these frits under our experimental conditions. By choice of porogens, it is possible to control the porous properties. The use of such frits in capillaries to retain particles of chromatographic packing has been demonstrated to be stable and robust with continuous operation over 3 days. PMID- 10740862 TI - An algorithm for automated bacterial identification using matrix-assisted laser desorption/ionization mass spectrometry. AB - An algorithm for bacterial identification using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is being developed. This mass spectral fingerprint comparison algorithm is fully automated and statistically based, providing objective analysis of samples to be identified. Based on extraction of reference fingerprint ions from test spectra, this approach should lend itself well to real-world applications where samples are likely to be impure. This algorithm is illustrated using a blind study. In the study, MALDI-MS fingerprints for Bacillus atrophaeus ATCC 49337, Bacillus cereus ATCC 14579T, Escherichia coli ATCC 33694, Pantoea agglomerans ATCC 33243, and Pseudomonas putida F1 are collected and form a reference library. The identification of test samples containing one or more reference bacteria, potentially mixed with one species not in the library (Shewanella alga BrY), is performed by comparison to the reference library with a calculated degree of association. Out of 60 samples, no false positives are present, and the correct identification rate is 75%. Missed identifications are largely due to a weak B. cereus signal in the bacterial mixtures. Potential modifications to the algorithm are presented and result in a higher than 90% correct identification rate for the blind study data, suggesting that this approach has the potential for reliable and accurate automated data analysis of MALDI-MS. PMID- 10740864 TI - Congeneric behavior in estimations of octanol-water partition coefficients by micellar electrokinetic chromatography. AB - Linear Solvation Energy Relationships (LSERs) are used to explain the congeneric behavior observed when using Micellar Electrokinetic Chromatography (MEKC) to estimate the octanol-water partition coefficient scale of solute hydrophobicity. Such studies provide useful insights about the nature of solute interactions that are responsible for the sources of congeneric relationships between MEKC retention and log Po/w. It was determined that solute dipolarity/polarizability and hydrogen-bonding character play the most important roles in the congeneric behavior observed for many surfactant systems. The individual dipolarity/polarizability and hydrogen-bonding contributions to the free energy of transfer were also investigated. PMID- 10740865 TI - Amino acid analysis by capillary electrophoresis electrospray ionization mass spectrometry. AB - A method for the determination of underivatized amino acids based on capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) is described. To analyze free amino acids simultaneously a low acidic pH condition was used to confer positive charge on whole amino acids. The choice of the electrolyte and its concentration influenced resolution and peak shape of the amino acids, and 1 M formic acid was selected as the optimal electrolyte. Meanwhile, the sheath liquid composition had a significant effect on sensitivity and the highest sensitivity was obtained when 5 mM ammonium acetate in 50% (v/v) methanol-water was used. Protonated amino acids were roughly separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath flow electrospray ionization interface. Under the optimized conditions, 19 free amino acids normally found in proteins and several physiological amino acids were well determined in less than 17 min. The detection limits for basic amino acids were between 0.3 and 1.1 mumol/L and for acidic and low molecular weight amino acids were less than 6.0 mumol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. This method is simple, rapid, and selective compared with conventional techniques and could be readily applied to the analysis of free amino acids in soy sauce. PMID- 10740866 TI - Selective focusing of catecholamines and weakly acidic compounds by capillary electrophoresis using a dynamic pH junction. AB - A systematic study of selective analyte focusing in a multisection electrolyte system by capillary electrophoresis (CE) is presented. It was found that a dynamic pH junction between sample and background electrolyte zones can be used to focus zwitterionic catecholamines and weakly acidic compounds without the use of special ampholytes. Differences in pH and concentration of complexing agents, such as borate, in the sample and background electrolyte zones were determined to cause focusing through changes in the local velocity of the analyte in two different segments of the capillary. Velocity-difference induced focusing (V-DIF) of analytes using a dynamic pH junction allowed the injection of large sample volumes and significantly improved the concentration sensitivity of CE. Under optimized conditions, the limit of detection for epinephrine was determined to be about 4 x 10(-8) M (the original sample) with conventional UV absorbance detection. Moreover, separation efficiencies greater than a million theoretical plates can be achieved by focusing such large sample volumes into narrow zones. Multisection electrolyte systems, which lead to the formation of a dynamic pH junction, can be tuned toward improving the concentration sensitivity of specific analytes if their chemical properties are known. PMID- 10740867 TI - High-temperature ultrafast liquid chromatography. AB - A novel liquid chromatographic system which enables high temperature ultrafast liquid chromatography (HTU-FLC) has been designed through the careful consideration of heat transfer, band broadening, and pressure drop. Studies of the effect of linear velocity on the HETP show that column efficiency at high velocity, especially of well-retained solutes, dramatically improves at higher temperatures. At 150 degrees C, at a flow rate of 15 mL/min with a 5 cm by 4.6 mm (i.d.) column packed with 3 microns polystyrene-coated zirconia porous particles, long chain alkylphenones were completely resolved, and the analysis time could be decreased by a factor of 50 compared to that at room temperature (25 degrees C) at a conventional flow rate (4 mL/min). In addition, using pure water as the mobile phase, five phenols were separated in less than 30 s. PMID- 10740868 TI - Degree of solute inclusion in native beta-cyclodextrin: chromatographic approach AB - The reversed-phase liquid chromatography retention and separation of a series of D,L dansyl amino acids were investigated over a wide range of salting-out agent (sucrose) concentrations using native beta-cyclodextrin as a chiral stationary phase. An original treatment was developed to determine the number of sucrose molecules (n) excluded from the solute-beta-cyclodextrin cavity interface when the analyte transfer occurred. Using the n values, the relative degrees of compound inclusion were calculated and correlated to the steric bulkiness of the solute. Thermodynamic parameter variations are discussed in relation to the inclusion degree of the dansyl amino acids. This numerical approach is a valuable tool to explore the steric effects implied in the host-guest complex formation. PMID- 10740869 TI - Improved solvent trapping of volatiles in supercritical fluid extraction by pressurizing the collection vial AB - The high flow rates that result after decompression make the trapping of analytes one of the more difficult aspects of a supercritical extraction. By elevation of the pressure on the collection vial, the flow may be reduced and trapping efficiency improved, considerably. The effects of different collection vial pressures were evaluated. The best results were obtained at 25 atm. With the collection vial at 25 atm, the trapping efficiencies of different solvents were then investigated. Also considered were the effects of solvent volume, extraction flow rate, collection solvent geometry, and restrictor temperature. For a 30 min extraction--with the restrictor line at 200 degrees C--quantitative recoveries were obtained with analytes as volatile as chlorobenzene (approximately 90%) in 1 mL of solvent. Mild precooling of the collection vessel allowed quantitative recoveries to be obtained down to benzene (94.2%). PMID- 10740870 TI - Performance of a monolithic silica column in a capillary under pressure-driven and electrodriven conditions AB - A continuous macroporous silica gel network was prepared in a fused-silica capillary and evaluated in reversed-phase liquid chromatography. Under pressure driven conditions, the monolithic silica column derivatized to C18 phase (100 microns in diameter, 25 cm in length, silica skeleton size of approximately 2.2 microns) produced plate heights of about 23 and 81 microns at 0.5 mm/s with a pressure drop of 0.4 kg/cm2, and at 4.0 mm/s with 3.6 kg/cm2, respectively, in 90% acetonitrile for hexylbenzene with a k value of 0.7. The separation impedance, E, calculated for the present monolithic silica column was much smaller at a low flow rate than those for particle-packed columns, although higher E values were obtained at a higher flow rate. Considerable dependence of column efficiency on the linear velocity of the mobile phase was observed despite the small size of the silica skeletons. A major source of band broadening in the HPLC mode was found in the A term of the van Deemter equation. The performance of the continuous silica capillary column in the electrodriven mode was much better than that in the pressure-driven mode. Plate heights of 7-8 microns were obtained for alkylbenzenes at 0.7-1.3 mm/s, although the electroosmotic flow was slow. In HPLC and CEC mode, the dependency of plate height on k values of the solutes was observed as seen in open tube chromatography presumably due to the contribution of the large through-pores. Since monolithic silica capillary columns can provide high permeability, the pressure-driven operation at a very low pressure can afford a separation speed similar to CEC at a high electric field. PMID- 10740871 TI - Differentiation and authentication of Panax ginseng, Panax quinquefolius, and ginseng products by using HPLC/MS. AB - An LC/MS-based method is established for the differentiation and authentication of specimens and commercial samples of Panax ginseng (Oriental ginseng) and Panax quinquefolius (American ginseng). This method is based on the separation of ginsenosides present in the ginseng methanolic extracts using high-performance liquid chromatography (HPLC), followed by detection with electrospray mass spectrometry. Differentiation of ginsenosides is achieved through simultaneous detection of intact ginsenoside molecular ions and the ions of their characteristic thermal degradation products. An important parameter used for differentiating P. ginseng and P. quinquefolius is the presence of ginsenoside Rf and 24-(R)-pseudoginsenoside F11 in the RICs of Oriental and American ginsengs, respectively. It is important to stress that ginsenoside Rf and 24(R) pseudoginsenoside F11, which possess the same molecular weight and were found to have similar retention times under most LC conditions, can be unambiguously distinguished in the present HPLC/MS method. The method developed is robust, reliable, reproducible, and highly sensitive down to the nanogram level. PMID- 10740872 TI - Single-molecular AFM probing of specific DNA sequencing using RecA-promoted homologous pairing and strand exchange. AB - The specific sequence in a linearlized double-stranded DNA target has been identified at a single-molecular level by atomic force microscopy (AFM). This was accomplished using RecA-coated, single-stranded DNA probes which were paired with a specific complementary DNA sequence in a linear double-stranded DNA target by strand-exchange reaction at a homologous sequence site with target DNA. The sites of interaction between the nucleoprotein filaments and the double-stranded DNA targets were directly visualized by AFM in solution containing 4 mM magnesium acetate. Measurements of the position of RecA-coated probes paired to individual target DNA showed that DNA probes specifically paired at their corresponding homologous target sequences. Strand exchange promoted by RecA and the visualization by AFM provided a rapid and efficient way to identify homologous sequence on a single-molecule target DNA. PMID- 10740873 TI - High extraction efficiency for POPs in real contaminated soil samples using accelerated solvent extraction AB - Systematic investigations were performed to study the dependence of the extraction efficiency of persistent organic pollutants (POPs), including chlorobenzenes, HCH isomers, DDX, PCB congeners, and PAHs, on the accelerated solvent extraction (ASE) operating variables solvent and temperature. Mixed soil samples from two locations with considerable differences in soil properties and contamination in the Leipzig-Halle region (Germany) were used. The objective was to optimize ASE for the extraction of POPs from real soil samples and to improve on the results achieved with Soxhlet extraction (SOX). Solvents with differing polarities were tested. Quadruple and triple determinations were performed on the two soils, respectively, between 20 and 180 degrees C in 20 degrees C steps. All the results were compared with those obtained by SOX, as well as, in some cases during preliminary studies, by ultrasonic extraction (USE). In ASE, the optimum conditions proved to be two extraction steps at 80 and 140 degrees C (average RSD 10.7%) with three static cycles (extraction time 35 min) using toluene as solvent and at a pressure of 15 MPa. Owing to the superior analyte/matrix separation by ASE, in many cases for real soil samples analytical values better by up to 1 order of magnitude or even more were obtained compared to SOX results. PMID- 10740874 TI - C60 and C70 HPLC retention reversal study using organic modifiers AB - A novel equation (Guillaume Y. C. et al. Anal. Chem. 1998, 70, 608) modeling the weak polar solute retention in reversed-phase liquid chromatography (RPLC) was applied to fullerene molecules C60 and C70. In RPLC, with an organic modifier (OM)/water mobile phase, the fullerene cluster solvation energies were calculated for OM = methanol, ethanol, propanol, butanol, and pentanol. An enthalpy-entropy compensation revealed that the type of interactions between fullerenes and the stationary phase was independent of both the fullerene and organic modifier structures. The energetics of OM and OM-water cluster exchange processes in the mobile phase were investigated in relation to the carbon atom number of the hydrophobic chain of the OM. Two linear correlations were found between the Gibbs free energy changes in the solvent exchange processes which confirmed that (i) a reversal elution order existed for C60 and C70 when methanol was changed into ethanol, propanol, butanol, pentanol and that (ii) the mobile phase was dominant in governing selectivity changes in nonpolar solutes. PMID- 10740875 TI - Retention of ions on nonporous charged stationary phases AB - The interactions between ion-exchange resins and counterions consist of several mechanisms, such as ion-pair formation between active sites and counterions, specific adsorption, solvation changes, and double-layer accumulation. The double layer accumulation of ions, which is a typical nonstoichiometric mechanism, is an important factor governing overall ion-exchange chromatographic retention when a major part of the stationary-phase surface is in contact with eluent flows. Nonporous stationary phases, where solutes are accessible to the surfaces by convection as well as by diffusion, possibly highlight this nonstoichiometric contribution through the coupling of a flow profile with an electrostatic potential function. The retention of ions on nonporous stationary phases has been interpreted by a model derived on the basis of the Poisson-Boltzmann equation including solvation change terms. Unusual retention behaviors have been confirmed only for anions, and can be explained by the model including the assumption that anions undergo solvation changes in a thin layer (approximately 5 nm thickness) at the vicinity of the stationary phase; the thickness should be a function of eluent flow rates. This strongly suggests that there is a difference in solvation nature between cations and anions. It can be inferred that water molecules interacting with polymer domains of the stationary phase behave like single molecules and cannot form a stable hydration shell around an anion as usually seen in bulk solution. PMID- 10740876 TI - Carbon film-based interdigitated array microelectrode used in capillary electrophoresis with electrochemical detection AB - A carbon film based interdigitated ring-shaped array (IDRA) microelectrode was applied to capillary electrophoresis with electrochemical detection to enhance the detection sensitivity on the basis of the redox cycling of electrochemical reversible species at the IDRA microelectrode. We propose a simple capillary electrode connection device that consists of an X-Y-Z fiber aligner, an electrochemical cell, and a Nafion tubing joint that will enable the detection capillary to be aligned easily on the IDRA microelectrode and isolate the separation voltage from the electrochemical detection system. We used the off column amperometric detection of aqueous ferrocene and catecholamines by capillary electrophoresis with an IDRA microelectrode to investigate the effects of the capillary-to-electrode distance and the separation voltage on the response currents in single and dual modes and the collection efficiencies (CE) and redox cycles (Rc) at the IDRA microelectrode. The results show that CE and Rc increase when we increase the distance and lower the separation voltage. The limiting currents also increase as the separation voltage decreases in the dual mode. Under optimum conditions, the CE and Rc of catechol, with good reversibility, reach 83.9% and 3.67, respectively. Our results showed that dual-mode detection with the IDRA microelectrode was capable of achieving lower detection limits than single-mode detection. PMID- 10740877 TI - Anomalous conductance in electrolyte solutions: a potentiometric and conductometric study of the dissociation of moderately strong acids AB - Properties of the associated forms of five moderately strong acids in aqueous solution (trichloro-, trifluoro-, chlorodifluoro-, and dichloroacetic acids) and sulfamic acid are studied by potentiometric and conductometric methods. Conductometric estimates of the acidity constants for the haloacetic acids are substantially different from the pH potentiometric values. However, the conductometric and pH potentiometric estimates of the acidity constants for sulfamic acid are in agreement. We interpret these results in terms of a neutral associated acid species of each of the haloacids which contributes to the electrolytic conductance. PMID- 10740878 TI - Permeation liquid membrane metal transport: studies of complex stoichiometries and reactions in Cu(II) extraction with the mixture 22DD-laurate in toluene/phenylhexane AB - The role of lauric acid (LAH) in the transport of copper(II) through a permeation liquid membrane (PLM) comprising 1,10-didecyldiaza-18-crown-6 (22DD) and lauric acid (ratio 1:1) in 1:1 v/v toluene/phenylhexane has been investigated by determining the stoichiometry of metal extraction and of the metal complex formed in the organic phase by performing 1H NMR and liquid/liquid and liquid/membrane extraction measurements. In the absence of copper(II), the 1H NMR data suggest that there is a strong interaction between the proton of LAH and the nitrogen of the 22DD macrocycle but no interaction between the aliphatic long chains of LAH and 22DD. Thus, in the organic solution, the two compounds are associated as (22DD-H)(+)-LA-, the laurate being away from (22DD-H)+. The signal intensity of the acidic proton was found to decrease when the metal Pb(II) was incorporated by the carrier after its extraction from the aqueous phase. Additionally, liquid/liquid as well as liquid/membrane extraction results reveal that Cu(II) extraction proceeds via the loss of two protons from the organic phase. The Cu(II) is found to be located in the 22DD cavity and the stoichiometry of the complex in the organic phase is (22DD-Cu)(2+)-2LA-. Metal extraction is governed by 22DD and laurate acts only as counteranion. An unexpected feature was observed in the liquid/liquid extraction which was that, at low 22DD and LAH concentrations, the slope for log(Kp) = f(pH) was 2 whereas it was much lower at high carrier concentration. This unexpected result seems to stem from impurities present in 22DD: only 0.1 mol% of impurity can indeed influence the exchange ratio of Cu(II) and H+. This type of anomaly, however, is not found in the normal procedure of liquid/membrane extraction possibly due to the lower carrier/metal molar ratio which is used in the classical PLM conditions. PMID- 10740879 TI - DNA sensing on a DNA probe-modified electrode using ferrocenylnaphthalene diimide as the electrochemically active ligand. AB - Naphthalene diimide derivative 1 carrying ferrocenyl moieties at the termini of imide substituents binds intact calf thymus DNA 4 times more strongly than the denatured DNA, and its complex with the intact DNA dissociates 80 times more slowly than that with the denatured DNA. On the basis of these observations, ligand 1 was applied to a probe of electrochemical DNA sensing. A thiol-linked single-stranded DNA probe was immobilized through the S-Au bonding to 20-30 pmol/mm2 on a gold electrode. Following hybridization with the complementary DNA, the electrode was soaked in a solution containing 1 (intercalation step) and then washed with buffer for 5 s. The cyclic voltammogram and differential pulse voltammogram for this electrode gave an electrochemical signal due to the redox reaction of 1 that was bound to the double-stranded DNA on the electrode. Thus, dA20 and the yeast choline transport gene were quantitated at the subpicomole level. The sensitivity of DNA detection was improved to 10 zmol by reducing the amount of immobilized DNA probe and protecting the uncovered surface of the electrode with 2-mercaptoethanol. PMID- 10740881 TI - Solubilization of hydrophilic compounds in 1,1,1,2-tetrafluoroethane with a cationic surfactant AB - Solubilization of hydrophilic compounds was examined in liquid 1,1,1,2 tetrafluoroethane (R134a) in the presence of the cationic surfactant trioctylmethylammonium chloride (TOMAC). The absorption spectra of methyl orange in the TOMAC-containing R134a solutions were obtained. Significant blue shifts were observed in comparison with the spectrum of methyl orange in aqueous solution. The shifts decreased as the water-to-surfactant ratio, W0, increased. In addition, spectral measurements confirmed the dissolution of cytochrome c in R134a in the presence of TOMAC. R134a remains as a liquid under mild applied pressure and becomes gas under ambient conditions; it therefore separates from analytes of interest directly without further concentration when used as an extraction solvent. Accordingly, it may be applied to recover valuable hydrophilic substances of low concentration from aqueous solutions. PMID- 10740880 TI - Characterization of cellular optoporation with distance. AB - We have developed and characterized cellular optoporation with visible wavelengths of light using standard uncoated glass cover slips as the absorptive media. A frequency-doubled Nd:YAG laser pulse was focused at the interface of the glass surface and aqueous buffer, creating a stress wave and transiently permeabilizing nearby cells. Following optoporation of adherent cells, three spatial zones were present which were distinguished by the viability of the cells and the loading efficiency (or number of extracellular molecules loaded). The loading efficiency also depended on the concentration of the extracellular molecules and the molecular weight of the molecules. In the zone farthest from the laser beam (> 60 microns under these conditions), nearly all cells were both successfully loaded and viable. To illustrate the wider applicability of this optoporation method, cells were loaded with a substrate for protein kinase C and the cellular contents then analyzed by capillary electrophoresis. In contrast to peptides loaded by microinjection, optoporated peptide showed little proteolytic degradation, suggesting that the cells were minimally perturbed. Also demonstrating the potential for future work, cells were optoporated and loaded with a fluorophore in the enclosed channels of microfluidic devices. PMID- 10740882 TI - Liquid chromatography/mass spectrometry methods for distinguishing N-oxides from hydroxylated compounds AB - This study describes the application of liquid chromatography/mass spectrometry (LC/MS) methods for distinguishing between aliphatic and aromatic hydroxylations and between hydroxylations and N-oxidations. Hydroxylations and N-oxidations are common biotransformation reactions of drugs. Electrospray (ESI) and atmospheric pressure chemical ionization (APCI) were used to generate ions from liquid chromatographic effluents. ESI-MS, ESI-MS/MS, APCI-MS, and APCI-MS/MS experiments were performed on several metabolites and derivatives of loratadine (a long acting and nonsedating tricyclic antihistamine) using an ion trap mass spectrometer (LCQ) and a triple-quadrupole mass spectrometer (TSQ). The observations are as follows: (1) LC/ESI-MS produced predominantly [M + H]+ ions with minor fragmentation. (2) LC/ESI-MS/MS data, however, showed a predominant loss of water from metabolites with aliphatic hydroxylation while the loss of water was not favored when hydroxylation was phenolic. N-Oxides (aromatic and aliphatic) showed only a small amount of water loss in the MS/MS spectra. (3) Under LC/APCI-MS conditions, aliphatic hydroxylation could be readily distinguished from aromatic hydroxylation based on the extent of water loss. In addition, N-oxides produced distinct [M + H - O]+ ions. These [M + H - O]+ ions were not produced in the APCI-MS spectra of hydroxylated metabolites. (4) Similar to the ESI-MS/MS spectra, the APCI-MS/MS spectra from the (M + H)+ ions of N oxides yielded a small amount of water loss but no [M + H - O]+ ions. These results indicate that LC/APCI-MS can be used to distinguish between hydroxylated metabolites and N-oxides. PMID- 10740883 TI - Determination of biological toxins using capillary electrokinetic chromatography with multiphoton-excited fluorescence. AB - We report a highly sensitive and rapid strategy for characterizing biological toxins based on capillary electrokinetic chromatography with multiphoton-excited fluorescence. In this approach, aflatoxins B1, B2, and G1 and the cholera toxin A subunit are fractionated in approximately 80 s in a narrow-bore electrophoretic channel using the negatively charged pseudostationary phase, carboxymethyl-beta cyclodextrin. The aflatoxins--highly mutagenic multiple-ringed heterocycles produced by Aspergillus fungi--are excited at the capillary outlet through the simultaneous absorption of two to three 750-nm photons to yield characteristic blue fluorescence; cholera toxin A-subunit, the catalytic domain of the bacterial protein toxin from Vibrio cholera, is excited through an unidentified multiphoton pathway that apparently includes photochemical transformation of an aromatic residue in the polypeptide. The anionic carboxymethyl-beta-cyclodextrin, used to chromatographically resolve the uncharged aflatoxins, enhances emission from these compounds without contributing substantially to the background. Detection limits for these toxins separated in 2.1-micron-i.d. capillaries range from 4.4 zmol (approximately 2700 molecules) for aflatoxin B2 to 3.4 amol for the cholera toxin A-subunit. Larger (16-micron-i.d.) separation capillaries provide concentration detection limits for aflatoxins in the 0.2-0.4 nM range, severalfold lower than achieved in 2.1-micron capillaries. These results represent an improvement of > 10(4) in mass detectability compared to previously published capillary separations of aflatoxins and demonstrate new possibilities for the analysis of proteins and peptides. PMID- 10740885 TI - Simulation studies of optimum energies for DXA: dependence on tissue type, patient size and dose model. AB - Dual-energy x-ray absorptiometry (DXA) is a well established technique for measuring bone mineral density (BMD). However, in recent years DXA is increasingly being used to measure body composition in terms of fat and fat-free mass. DXA scanners must also determine the soft tissue baseline value from soft tissue-only regions adjacent to bone. The aim of this work is to determine, using computer simulations, the optimum x-ray energies for: a number of dose models, different tissues, i.e. bone mineral, average soft tissue, lean soft tissue and fat; and a range of anatomical sites and patient sizes. Three models for patient dose were evaluated: total beam energy, entrance exposure and absorbed dose calculated by Monte Carlo modelling. A range of tissue compositions and thicknesses were chosen to cover typical patient variations for the three sites: femoral neck, PA spine and lateral spine. In this work, the optimisation of the energies is based on: (1) the uncertainty that arises from the quantum statistical nature of the number of x-rays recorded by the detector, and (2) the radiation dose received by the patient. This study has deliberately not considered other parameters such as detector response, electronic noise, x-ray tube heat load etc, because these are technology dependent parameters, not ones that are inherent to the measuring technique. Optimisation of the energies is achieved by minimisation of the product of variance of density measurement and dose which is independent of the absolute intensities of the x-ray beams. The results obtained indicate that if solving for bone density, then Elow in the range 34 to 42 keV, Ehigh in the range 100 to 200 keV and incident intensity ratio (low energy/high energy) in the range 3 to 10 is a reasonable compromise for the normal range of patient sizes. The choice of energies is complicated by the fact that the DXA unit must also solve for fat and lean soft tissue in soft tissue-only regions adjacent to the bone. In this case the ranges of energies suggested above are acceptable only for the average phantom. In extreme cases the variance-dose product can be up to 50 times higher than for the ideal energies. PMID- 10740884 TI - Contrast enhancement in microplanar beam radiography. AB - In x-ray radiography, the target produces a useful shadow from absorption of the primary beam, while the scattered radiation into the off-target region decreases the contrast of the target image. A bundle of closely spaced microplanar beams can reduce the scattered radiation and give superior image contrast compared with a single macrobeam of the same dimensions. To further reduce the scattered radiation and increase the image contrast, we place an air gap between the tissue phantom and the detector. The primary and scattered photon flux of a single microplanar beam is measured as a function of thickness inside the phantom and in the air gap. Results show that a bundle of closely spaced, microplanar beams increase the image contrast by 22% and a 2 cm air gap decreases the scattered photon flux by about half, improving the contrast by an additional 16%. Thus an overall improvement of 41% in contrast can be achieved with microplanar beam radiography. PMID- 10740887 TI - Implementation of a stereotactic radiotherapy system. AB - The tests carried out on a recently-installed stereotactic radiotherapy system capable of fully frameless stereotaxy are outlined. The two principle investigations carried out on the planning system were a dosimetry test, and a test to verify the accuracy of localisation from CT examination through to patient treatment. The method employed in frameless relocation for fractionated stereotactic radiotherapy--position detection using stereoscopic infra-red cameras and a set of fiducials attached to a bite-block--was tested for reproducibility. The results of these tests suggest a mean accuracy in treatment localisation of 1.23 mm (maximum 2.72 mm) for single fraction techniques, and 1.77 mm (maximum 4.53 mm) for treatments involving repeated (frameless) localisation. The implications of these accuracies for treatment with single, multiple-fraction and multi-isocentre techniques are discussed. PMID- 10740886 TI - Positron emission tomography: radioisotope and radiopharmaceutical production. AB - A Centre for Positron Emission Tomography (PET) has been operational within the Department of Nuclear Medicine at the Austin & Repatriation Medical Centre (A&RMC) in Melbourne for seven years. PET is a non-invasive imaging technique based on the use of biologically relevant compounds labelled with short-lived positron-emitting radionuclides such as carbon-11, nitrogen-13, oxygen-15 and fluorine-18. The basic facility consists of a medical cyclotron (10 MeV proton & 5 MeV deuteron), six lead-shielded hotcells with associated radiochemistry facilities, radiopharmacy and a whole body PET scanner. A strong radiolabelling development program, including the production of 15O-oxygen, 15O-carbon monoxide, 15O-carbon dioxide, 15O-water, 13N-ammonia, 18F-FDG, 18F-FMISO, 11C-SCH23390 and 11C-flumazenil has been pursued to support an ambitious clinical and research program in neurology, oncology, cardiology and psychiatry. PMID- 10740888 TI - Patient doses from neuroradiological procedures. AB - Patient doses have been determined for a sample of 20 patients in a hospital department undergoing various interventional neuroradiological procedures. Skin entrance dose, dose-area product and effective dose have been estimated. Effective dose was estimated from published conversion factors. The highest dose on the patients body was recorded as the maximum skin entrance dose. The maximum skin entrance dose varied from 0.2 to 2.3 Gy. The largest maximum skin entrance dose is similar to the threshold for skin injuries (erythema). The effective dose varied from 2 to 34 mSv. The results of this study are compared with the results of other studies on radiation dose to patients from neuroradiological procedures. PMID- 10740889 TI - Monitor unit calculations using a 3D computerised treatment planning system: verification in an anthropomorphic phantom. AB - The aim of this work was to assess the monitor unit calculation accuracy of a new 3D computer planning system in a volume containing significant lung heterogeneity. An anthropomorphic phantom drilled to accommodate a cylindrical ionisation chamber was used for measurements. Results were also compared to an older 2D system and a manual calculation method. Only a slight improvement was achieved with the new system and an energy dependence was evident. This type of test is recommended before implementing a new 3D planning system. PMID- 10740890 TI - The use of round ended needles for high dose rate brachytherapy implants. AB - The use of Speiser needles for interstitial implants has resulted in malfunctioning of HDR afterloading equipment due to seepage of blood. We have successfully used rounded ended needles in the patient after removal of the trocar ended needles, preventing the seepage of blood. This technique also helps to prevent any possible cross contamination between patients. PMID- 10740891 TI - Adaptive explanation in socio-ecology: lessons from the Equidae. AB - Socio-ecological explanations for intra- and interspecific variation in the social and spatial organization of animals predominate in the scientific literature. The socio-ecological model, developed first for the Bovidae and Cervidae, is commonly applied more widely to other groups including the Equidae. Intraspecific comparisons are particularly valuable because they allow the role of environment and demography on social and spatial organization to be understood while controlling for phylogeny or morphology which confound interspecific comparisons. Feral horse (Equus caballus Linnaeus 1758) populations with different demography inhabit a range of environments throughout the world. I use 56 reports to obtain 23 measures or characteristics of the behaviour and the social and spatial organization of 19 feral horse populations in which the environment, demography, management, research effort and sample size are also described. Comparison shows that different populations had remarkably similar social and spatial organization and that group sizes and composition, and home range sizes varied as much within as between populations. I assess the few exceptions to uniformity and conclude that they are due to the attributes of the studies themselves, particularly to poor definition of terms and inadequate empiricism, rather than to the environment or demography per se. Interspecific comparisons show that equid species adhere to their different social and spatial organizations despite similarities in their environments and even when species are sympatric. Furthermore, equid male territoriality has been ill-defined in previous studies, observations presented as evidence of territoriality are also found in non-territorial equids, and populations of supposedly territorial species demonstrate female defence polygyny. Thus, territoriality may not be a useful categorization in the Equidae. Moreover, although equid socio-ecologists have relied on the socio-ecological model derived from the extremely diverse Bovidae and Cervidae for explanations of variation in equine society, the homomorphic, but large and polygynous, and monogeneric Equidae do not support previous socio-ecological explanations for relationships between body size, mating system and sexual dimorphism in ungulates. Consequently, in spite of the efforts of numerous authors during the past two decades, functional explanations of apparent differences in feral horse and equid social and spatial organization and behaviour based on assumptions of their current utility in the environmental or demographic context remain unconvincing. Nevertheless, differences in social cohesion between species that are insensitive to intra- and interspecific variation in habitat and predation pressure warrant explanation. Thus, I propose alternative avenues of inquiry including testing for species-specific differences in inter-individual aggression and investigating the role of phylogenetic constraints in equine society. The Equidae are evidence of the relative importance of phylogeny and biological structure, and unimportance of the present day environment, in animal behaviour and social and spatial organization. PMID- 10740892 TI - Why do females mate multiply? A review of the genetic benefits. AB - The aim of this review is to consider the potential benefits that females may gain from mating more than once in a single reproductive cycle. The relationship between non-genetic and genetic benefits is briefly explored. We suggest that multiple mating for purely non-genetic benefits is unlikely as it invariably leads to the possibility of genetic benefits as well. We begin by briefly reviewing the main models for genetic benefits to mate choice, and the supporting evidence that choice can increase offspring performance and the sexual attractiveness of sons. We then explain how multiple mating can elevate offspring fitness by increasing the number of potential sires that compete, when this occurs in conjunction with mechanisms of paternity biasing that function in copula or post-copulation. We begin by identifying cases where females use pre copulatory cues to identify mates prior to remating. In the simplest case, females remate because they identify a superior mate and 'trade up' genetically. The main evidence for this process comes from extra-pair copulation in birds. Second, we note other cases where pre-copulatory cues may be less reliable and females mate with several males to promote post-copulatory mechanisms that bias paternity. Although a distinction is drawn between sperm competition and cryptic female choice, we point out that the genetic benefits to polyandry in terms of producing more viable or sexually attractive offspring do not depend on the exact mechanism that leads to biased paternity. Post-copulatory mechanisms of paternity biasing may: (1) reduce genetic incompatibility between male and female genetic contributions to offspring; (2) increase offspring viability if there is a positive correlation between traits favoured post-copulation and those that improve performance under natural selection; (3) increase the ability of sons to gain paternity when they mate with polyandrous females. A third possibility is that genetic diversity among offspring is directly favoured. This can be due to bet-hedging (due to mate assessment errors or temporal fluctuations in the environment), beneficial interactions between less related siblings or the opportunity to preferentially fertilise eggs with sperm of a specific genotype drawn from a range of stored sperm depending on prevailing environmental conditions. We use case studies from the social insects to provide some concrete examples of the role of genetic diversity among progeny in elevating fitness. We conclude that post-copulatory mechanisms provide a more reliable way of selecting a genetically compatible mate than pre-copulatory mate choice. Some of the best evidence for cryptic female choice by sperm selection is due to selection of more compatible sperm. Two future areas of research seem likely to be profitable. First, more experimental evidence is needed demonstrating that multiple mating increases offspring fitness via genetic gains. Second, the role of multiple mating in promoting assortative fertilization and increasing reproductive isolation between populations may help us to understand sympatric speciation. PMID- 10740893 TI - Plant invasions--the role of mutualisms. AB - Many introduced plant species rely on mutualisms in their new habitats to overcome barriers to establishment and to become naturalized and, in some cases, invasive. Mutualisms involving animal-mediated pollination and seed dispersal, and symbioses between plant roots and microbiota often facilitate invasions. The spread of many alien plants, particularly woody ones, depends on pollinator mutualisms. Most alien plants are well served by generalist pollinators (insects and birds), and pollinator limitation does not appear to be a major barrier for the spread of introduced plants (special conditions relating to Ficus and orchids are described). Seeds of many of the most notorious plant invaders are dispersed by animals, mainly birds and mammals. Our review supports the view that tightly coevolved, plant-vertebrate seed dispersal systems are extremely rare. Vertebrate dispersed plants are generally not limited reproductively by the lack of dispersers. Most mycorrhizal plants form associations with arbuscular mycorrhizal fungi which, because of their low specificity, do not seem to play a major role in facilitating or hindering plant invasions (except possibly on remote islands such as the Galapagos which are poor in arbuscular mycorrhizal fungi). The lack of symbionts has, however, been a major barrier for many ectomycorrhizal plants, notably for Pinus spp. in parts of the southern hemisphere. The roles of nitrogen fixing associations between legumes and rhizobia and between actinorhizal plants and Frankia spp. in promoting or hindering invasions have been virtually ignored in the invasions literature. Symbionts required to induce nitrogen fixation in many plants are extremely widespread, but intentional introductions of symbionts have altered the invasibility of many, if not most, systems. Some of the world's worst invasive alien species only invaded after the introduction of symbionts. Mutualisms in the new environment sometimes re-unite the same species that form partnerships in the native range of the plant. Very often, however, different species are involved, emphasizing the diffuse nature of many (most) mutualisms. Mutualisms in new habitats usually duplicate functions or strategies that exist in the natural range of the plant. Occasionally, mutualisms forge totally novel combinations, with profound implications for the behaviour of the introduced plant in the new environment (examples are seed dispersal mutualisms involving wind-dispersed pines and cockatoos in Australia; and mycorrhizal associations involving plant roots and fungi). Many ecosystems are becoming more susceptible to invasion by introduced plants because: (a) they contain an increasing array of potential mutualistic partners (e.g. generalist frugivores and pollinators, mycorrhizal fungi with wide host ranges, rhizobia strains with infectivity across genera); and (b) conditions conductive for the establishment of various alien/alien synergisms are becoming more abundant. Incorporating perspectives on mutualisms in screening protocols will improve (but not perfect) our ability to predict whether a given plant species could invade a particular habitat. PMID- 10740894 TI - Toxic action/toxicity. AB - Some six or so physiological systems, essential to normal mammalian life, are involved in poisoning; an intoxication that causes severe injury to any one of them could be life threatening. Reversible chemical reactions showing Scatchard type binding are exemplified by CO, CN- and cyclodiene neurotoxin insecticide intoxications, and by antigen-antibody complex formation. Haemoglobin (Hb) molecular biology accounts for the allosteric co-operativity and other characteristics of CO poisoning, CN- acts as a powerful cytochrome oxidase inhibitor, and antigen binding in a deep antibody cleft between two domains equipped with epitopes for antigen-binding groups explains hapten-specific immune reactions. Covalent chemical reactions with second-order (SN2) kinetics characterize Hg and Cd poisonings, the reactions of organophosphates and phosphonates with acetylcholinesterase and neurotoxic esterase and the reaction sequence whereby Paraquat accepts electrons and generates superoxide under aerobic conditions. Indirect carcinogens require cytochrome P450 activation to form DNA adducts in target-organ DNA and cause cancer, but a battery of detoxifying enzymes clustered with the P450 system must be overcome. Thus, S metabolism competes ineffectively with target DNA for reactive vinyl chloride (VC) metabolites, epoxide hydrolase is important to the metabolism and carcinogenicity of alfatoxins and polycyclic aromatic hydrocarbons (benzo[a]pyrene, etc.), and the non-toxic 2-naphthylhydroxylamine N-glucuronide acts as a transport form in 2-naphthylamine bladder cancer. VC liver-cancer pathogenesis is explicable in terms of the presence of the glutathione S transferase detoxifying system in hepatocytes and its absence from the fibroblastic elements, and of the VC concentrations reaching the liver by different administrative routes. In VC carcinogenicity, chemical reactions give imidazo-cyclization products with nucleoside residues of target DNA, and in benzene leukaemia, Z,Z-muconaldehyde forms cyclic products containing a pyrrole residue linked to purine. Increased HbCO concentrations reduce the O2-carrying capacity of the blood, and the changed shape of the O2-Hb dissociation curve parallels disturbance in O2 unloading. CN- acts on electron transport and paralyses respiration. In telodrin poisoning, preconvulsive glutamine formation abstracts tricarboxylic acid intermediates incommensurately with normal cerebral respiration. Antigen-antibody complexing depletes the antibody titre, available against infection. At high doses of Cd, Cd-thionein filtered through the kidneys is reabsorbed and tubular lesions produced. Some organophosphate insecticides promote irreversible acetylcholinesterase phosphorylation and blockade nerve function, and others react with neurotoxic esterase to cause delayed neuropathy. The evidence for Paraquat pulmonary poisoning suggests a radical mechanism involving three interrelated cyclic reaction stages. The action of N- and O8 (O substituent in 6-position of the purine) demethylases explains deletion mechanisms for DNA-alkyl adducts. DNA-directed synthesis in the presence of ultimate carcinogens provides for an estimation of misincorporations, which implicate the same transversions as those found by direct mutagenicity testing. Chemical carcinogens recognize tissue-sensitive cells and modify their heritable genetic complement. Oncoproteins encoded by activated oncogenes signal the transformation of normal cells into cancer cells. The importance of the H-ras oncogene and p53 tumour-suppressor gene is stressed. Antidotal action is analysed; for example, parenteral glutamine administration to telodrin intoxicated rats restores the depleted cerebral glutamate level and prevents seizures. Glutamate acts as anticonvulsant in petit mal epilepsy. In general, therefore, the reaction of the toxicant-related substance with the relevant target-tissue macromolecule accounts for the biochemical/biological events at a cellular level a PMID- 10740895 TI - Flight adaptations in Palaeozoic Palaeoptera (Insecta). AB - The use of available morphological characters in the interpretation of the flight of insects known only as fossils is reviewed, and the principles are then applied to elucidating the flight performance and techniques of Palaeozoic palaeopterous insects. Wing-loadings and pterothorax mass/total mass ratios are estimated and aspect ratios and shape-descriptors are derived for a selection of species, and the functional significance of wing characters discussed. Carboniferous and Permian ephemeropteroids ('mayflies') show major differences from modern forms in morphology and presumed flight ability, whereas Palaeozoic odonatoids ('dragonflies') show early adaptation to aerial predation on a wide size-range of prey, closely paralleling modern dragonflies and damselflies in shape and wing design but lacking some performance-related structural refinements. The extensive adaptive radiation in form and flight technique in the haustellate orders Palaeodictyoptera, Megasecoptera, Diaphanopterodea and Permothemistida is examined and discussed in the context of Palaeozoic ecology. PMID- 10740896 TI - Now you see it ... PMID- 10740897 TI - Orthodontics should shoulder responsibility. PMID- 10740898 TI - Still making its mark. PMID- 10740899 TI - Carcinogen exposure in oral cancer sufferers. PMID- 10740900 TI - Treat phobic children and adults alike. PMID- 10740901 TI - Do we really want a quick fix? AB - Dental practitioners have been encouraged recently to take a more structured long term view of their continuing professional development, in particular being wary of any 'weekend' courses apparently offering 'quick fix' solutions to their clinical problems. PMID- 10740902 TI - The 1999 BDA Heathrow Timings inquiry. AB - In the summer of 1999 the BDA conducted a Timings Inquiry in order to provide information on treatment timings within the General Dental Services (GDS) and to develop a model of an hourly rate for general practice. A panel of dentists measured the absolute time taken to carry out 21 key treatments. The relative times of related treatments were then estimated. The panel also came to consensus about the variables to construct the hourly rate model. The absolute timings exercise was applied to the hourly rate model in order to draw some conclusions about the average earnings of a full-time dentist committed to the NHS. This information formed a central part of the BDA's 1999 evidence to the Doctors and Dentists Review Body. PMID- 10740903 TI - Effectiveness on oral health of a long-term health education programme for mothers with young children. AB - AIM: To determine the effect of dental health education (DHE) on caries incidence in infants, through regular home visits by trained DH Educators over a period of 3 years. METHOD: A randomly selected cohort of 228 children born between 1st January and 30th September 1995, in a low socioeconomic/high caries suburb of Leeds (UK) were divided into the following groups: A) DHE focused on diet; B) DHE focused on oral hygiene instruction (OHI) using fluoride toothpaste; and C) DHE by a combined diet and OHI message. DHE was given using an interview and counselling for at least 15 minutes at home every 3 months for the first 2 years and twice a year in the third year of the study. A fourth group D was given diet and OHI, at home, once a year only. All children and mothers were examined for caries and oral hygiene. A fifth group E (control) received no DHE and were never visited but examined at 3 years of age only. RESULTS: In the groups of children visited regularly only two developed caries and three had gingivitis (all in group A). In group E, however, 33% of children had caries and nine (16%) had gingivitis. The differences in caries levels and caries risk factors between study and control groups were statistically significant (P < 0.001). Mothers of the study groups also showed an improvement in their own levels of gingivitis, debris and calculus scores by the second and third examinations (P < 0.001). CONCLUSION: Regular home visits to mothers with infants, commencing at or soon after the time of the eruption of the first deciduous teeth, was shown to be effective in preventing the occurrence of nursing caries. PMID- 10740904 TI - An evaluation of an oral health training programme for carers of the elderly in residential homes. AB - OBJECTIVE: The objectives of this study were: to evaluate carers' knowledge of oral health; to provide a high quality, consistent, oral health training programme for carers in residential homes; to evaluate the quality of this programme by examining both carers' changes in knowledge and any changes in carers' behaviour as reported by residents and to assess any changes in the oral health of the elderly residents after one year. DESIGN: A cross-sectional, multi centre study using a carer training programme, evaluated by both a questionnaire conducted with carers and residents and oral examination of residents. SETTING: In August 1996, 20 (20%) of the residential/nursing homes, in West Hertfordshire were chosen at random and all managers contacted and offered an oral examination for all their residents. Ten (10%) of the homes were also offered an oral health training programme for their carers. Eighteen homes accepted the oral examination for all consenting residents and 7 of the 10 homes offered accepted the carer training. SUBJECTS: Thirty-nine carers from 7 of the residential homes attended an oral health training course and 213 elderly residents in the 18 homes were examined both at baseline and after 12 months. RESULTS: Carers' baseline knowledge about oral health was poor; the oral health training programme was enjoyed and their knowledge gain after one week was high. However, the elderly residents perceived no change in the oral care given by carers either after one week or after one year and there was no measurable improvement in the oral health of residents after carer training, except for an increase in filled coronal surfaces. Few of the carers originally trained were still working in the same residential homes after one year. CONCLUSION: Although the carer training programme was well received, no changes in oral health practice resulted. Barriers to practice of oral care by carers remained and training, even when including practical skills, evaluation by peers and a high knowledge gain, failed to reduce these barriers. PMID- 10740905 TI - A survey of the teaching of conscious sedation in dental schools of the United Kingdom and Ireland. AB - AIM: To assess and compare, for the first time, the quantity and quality of dental undergraduate teaching in conscious sedation in the dental schools of the UK and Ireland. This was achieved using a prospective, questionnaire-based survey. METHODS: Questionnaires were designed to collect information about undergraduate sedation education from teaching staff and final year dental undergraduates at the 16 dental schools in the UK and Ireland. Staff questionnaires were distributed to a nominated sedation teacher at each dental school and sought details of didactic and clinical sedation teaching methods, plus the quantity and perceived quality of sedation teaching. Student questionnaires were distributed to 5th year dental students and enquired about the quantity and quality of clinical sedation teaching received. The survey was undertaken during May-June 1998. RESULTS: Thirteen dental schools returned staff questionnaires (81%). Seven also provided a student response (44%). The proportion of final year students within the 7 schools who returned completed questionnaires was 38%. Sedation teaching was undertaken primarily by oral surgery and paediatric dental departments. Three schools also utilised anaesthetic departments and 2 schools had dedicated dental sedation departments. All but 2 schools provided didactic teaching on sedation (mean: 4.2 lectures, 1.8 seminars). Of the 7 schools which returned staff and student questionnaires, all provided some clinical training using inhalational and intravenous demonstration cases (mean 5.1 and 4.4 cases, per student, respectively). All but one school provided hands-on inhalational sedation experience (mean 2.6 cases per student) but only two schools provided any hands-on intravenous sedation experience. The quantity of hands-on experience was greater at the two dental schools with dedicated dental sedation departments. Across the schools students rated the overall quality of sedation teaching at average or above, but most staff graded the overall quality of teaching at below average. CONCLUSION: Dental undergraduate sedation teaching shows considerable variation across the dental schools surveyed. At most schools students gained little or no hands-on experience in sedation, especially in intravenous techniques. The undergraduate foundation for sedation education must improve if conscious sedation is to become the principal alternative to general anaesthesia in dental practice. PMID- 10740906 TI - Branding: the patient's perspective. PMID- 10740907 TI - The retreat of plague from Central Europe, 1640-1720: a geomedical approach. PMID- 10740909 TI - Economically practical and critically necessary? The development of intensive care at Chestnut Hill Hospital. PMID- 10740908 TI - Producing development: The anatomy of human embryos and the norms of Wilhelm His. PMID- 10740910 TI - Long waits, small spaces, and compassionate care: memories of race and medicine in a mid-twentieth-century southern community. PMID- 10740911 TI - Implementation of a new e-mail application in a health sciences center. Case study. PMID- 10740912 TI - Computer use among elderly people. AB - This study investigates the literature on the state of knowledge on computer use among elderly people. Results of this review suggest that benefits may ensue if computer use is advocated. Functional limitations secondary to visual, hearing, and mobility changes; financial inadequacy; and cognitive limitation related to memory use are identified as barriers that may inhibit this population from learning how to use the computer. Methods and techniques dealing with these barriers are presented. Finally, teaching strategies that may be implemented to facilitate the learning process among elderly population on computer use are discussed and elucidated. PMID- 10740914 TI - Development and evaluation of a pathophysiology CD-ROM for nurse practitioner distance education. AB - Increasing interest in computer-mediated learning technologies has prompted educators to incorporate them into many learning environments; however, there is still little evaluative evidence to support their effectiveness. This report describes the development and evaluation of an instructional CD-ROM on pathophysiology for Nurse Practitioner students. Researcher-designed questionnaires were used to assess the characteristics of our students, and to solicit their ratings of the CD-ROM on ease of use, clarity of instruction, interest, and convenience of the program, using 5-point Likert scales. The respondents indicated that the package was easy and convenient to use, with high technical quality, and of a level challenging to some but not all of the students. On-line quizzes were most highly rated. There were also some indications of higher levels of achievement among users than among nonusers. These data suggest use of CD-ROM as a distance education strategy can be effective for Nurse Practitioner students. PMID- 10740913 TI - Computerized test development software. A comparative review updated. AB - This article provides a comparative review of nine commercially available software programs: A+ Test Manager and Test Taker, CATSoftware, Diploma 97, Examaker for Windows, FastTEST, Microtest Pro, Pedagogue, Question Mark, and Test Construction Set. Issues for using test development software are covered including security, pedagogical uses, and test design and administration. Nurse educators are encouraged to consider the unique needs of each setting when selecting a software package to support test development and administration. PMID- 10740915 TI - User-friendliness of computerized information systems. AB - Despite the plethora of research on nurses and the use of computers and information systems, there have been few attempts to examine the everyday experiences of nurses who use such systems in practice. This qualitative study builds on our limited understanding of practitioners' experiences regarding use of Computerized Patient Information Systems (CPIS). Focus group interviews wer held across Australia with practitioners from a wide range of clinical settings and specialties. The study findings suggest that participants were predominantly critical of systems in almost every area related to "user-friendliness." The perspectives and views of practitioners are important to understand if future generations of CPIS hardware and software are to be developed with a greater appreciation of the needs of the system's front-line users. PMID- 10740916 TI - Tetrad analysis shows that gene conversion is the major mechanism involved in mutation at the human minisatellite MS1 integrated in Saccharomyces cerevisiae. AB - Minisatellites are arrays of tandemly repeated DNA sequences which occur at thousands of locations in the human genome. They are frequently hypervariable with respect to allele length as a result of high rates of complex and incompletely understood recombination-based germline mutation events that alter the repeat copy number. MS1 is one of the most variable minisatellites so far isolated from the human genome. We have integrated MS1, flanked by synthetic markers, in the vicinity of a hot spot for meiotic double-strand breaks upstream of the LEU2 locus in chromosome III of Saccharomyces cerevisiae. Here we present the first tetrad analysis of mutations at a human minisatellite locus. The data showed that mutant alleles occur as single mutants in one of the spores in a tetrad, also when the mutant structure was the result of a combination of intra- and inter-allelic rearrangements. The conversional transfer of repeat units from one allele to the other was associated with flanking marker conversion which always involved the same flank of the minisatellite. The results demonstrate that conversion is the predominant mechanism by which minisatellite alleles mutate to new lengths, and also support the assumption that cis-acting elements are involved in the regulation of the mutational process in humans. PMID- 10740917 TI - A scenario for the hobo transposable element invasion, deduced from the structure of natural populations of Drosophila melanogaster using tandem TPE repeats. AB - Temporal surveys of hobo transposable elements in natural populations reveal a historical pattern suggesting a recent world-wide invasion of D. melanogaster by these transposons, perhaps following a recent horizontal transfer. To clarify the dynamics of hobo elements in natural populations, and thus to provide further data for our understanding of the hobo invasion, TPE tandem repeats, observed in the polymorphic S region of the element, were used as molecular markers. The number of TPE repeats was studied in 101 current populations from around the world, and in 63 strains collected in the past. This revealed a geographical distribution which seems to have been stable since the beginning of the 1960s. This distribution is compatible with a number of hypotheses for the dynamics of hobo elements. We propose a scenario based on an invasion in two stages: first, a complete invasion by elements with three TPE repeats, followed by the beginning of a new invasion involving hobo elements with five or seven repeats. PMID- 10740919 TI - Localization of genes affecting species differences in male courtship song between Drosophila virilis and D. littoralis. AB - The males of six species of the Drosophila virilis group (including D. virilis) keep their wings extended while producing a train of sound pulses, where the pulses follow each other without any pause. The males of the remaining five species of the group produce only one sound pulse during each wing extension/vibration, which results in species-specific songs with long pauses (in D. littoralis about 300 ms) between successive sound pulses. Genetic analyses of the differences between the songs of D. virilis and D. littoralis showed that species-specific song traits are affected by genes on the X chromosome, and for the length of pause, also by genes on chromosomes 3 and 4. The X chromosomal genes having a major impact on pulse and pause length were tightly linked with white, apricot and notched marker genes located at the proximal third of the chromosome. A large inversion in D. littoralis, marked by notched, prevents more precise localization of these genes by classical crossing methods. PMID- 10740918 TI - Microsatellite variation in populations of Drosophila pseudoobscura and Drosophila persimilis. AB - We have isolated, characterized and mapped 33 dinucleotide, three trinucleotide and one tetranucleotide repeat loci from the four major chromosomes of Drosophila pseudoobscura. Average inferred repeat unit length of the dinucleotide repeats is 12 repeat units, similar to D. melanogaster. Assays of D. pseudoobscura and populations of its sibling species, D. persimilis, using 10 of these loci show extremely high levels of variation compared with similar studies of dinucleotide repeat variation in D. melanogaster populations. The high levels of variation are consistent with an average mutation rate of approximately 10(-6) per locus per generation and an effective population size of D. pseudoobscura approximately four times larger than that of D. melanogaster. Consistent with allozymes and nucleotide sequence polymorphism, the dinucleotide repeat loci reveal minimal structure across four populations of D. pseudoobscura. Finally, our preliminary recombinational mapping of 24 of these microsatellites suggests that the total recombinational genome size may be larger than previously inferred using morphological mutant markers. PMID- 10740920 TI - Temporal uniformity of the spontaneous mutational variance of quantitative traits in Drosophila melanogaster. AB - Spontaneous mutations were allowed to accumulate over 209 generations in more than 100 lines, all of them independently derived from a completely homozygous population of Drosophila melanogaster and subsequently maintained under strong inbreeding (equivalent to full-sib mating). Traits scored were: abdominal (AB) and sternopleural (ST) bristle number, wing length (WL) and egg-to-adult viability (V). On two occasions--early (generations 93-122) and late (generations 169-209)--ANOVA estimates of the mutational variance and the mutational line x generation interaction variance were obtained. Mutational heritabilities of morphological traits ranged from 2 x 10(-4) to 2 x 10(-3) and the mutational coefficient of variation of viability was 0.01. For AB, WL and V, temporal uniformity of the mutational variance was observed. However, a fluctuation of the mutational heritability of ST was detected and could be ascribed to random genotype x environment interaction. PMID- 10740921 TI - Sex ratio distortion in bovine sperm correlates to recombination in the pseudoautosomal region. AB - A total of 2122 single sperm from 35 bulls belonging to six different paternal half-sib groups were analysed with respect to two markers in the bovine pseudoautosomal region (PAR) and sex-specific loci on the X and Y chromosomes, respectively. A segregation ratio significantly different from 1:1 was observed in a test over all families, with a higher proportion of X-bearing gametes (53.5%). The analysis of recombination conducted separately for X- and Y-bearing sperm showed that X-bearing sperm cells possess highly significant individual and between-family variability in recombination rate, whereas Y-bearing sperm show linkage homogeneity. To test whether the two phenomena are related, different logistic regression models were fitted to the data. The results show that sex ratio significantly correlates with changes in recombination rate among X-bearing but not among Y-bearing sperm. Different hypotheses to explain these observations are discussed. PMID- 10740922 TI - Distinguishing recombination and intragenic gene conversion by linkage disequilibrium patterns. AB - Deterministic theory suggests that reciprocal recombination and intragenic, interallelic conversion have different effects on the linkage disequilibrium between a pair of genetic markers. Under a model of reciprocal recombination, the decay rate of linkage disequilibrium depends on the distance between the two markers, while under conversion the decay rate is independent of this distance, provided that conversion tracts are short. A population genetic three-locus model provides a function Q of two-locus linkage disequilibria. Viewed as a random variable, Q is the basis for a test of the relative impact of conversion and recombination. This test requires haplotype frequency data of a sufficiently variable three-locus system. One of the few examples currently available is data from the Human Leukocyte Antigen (HLA) class I genes of three Amerindian populations. We find that conversion may have played a dominant role in shaping haplotype patterns over short stretches of DNA, whereas reciprocal recombination may have played a greater role over longer stretches of DNA. However, in order to draw firm conclusions more independent data are necessary. PMID- 10740923 TI - Inbreeding depression due to mildly deleterious mutations in finite populations: size does matter. AB - We studied the effects of population size on the inbreeding depression and genetic load caused by deleterious mutations at a single locus. Analysis shows how the inbreeding depression decreases as population size becomes smaller and/or the rate of inbreeding increases. This pattern contrasts with that for the load, which increases as population size becomes smaller but decreases as inbreeding rate goes up. The depression and load both approach asymptotic limits when the population size becomes very large or very small. Numerical results show that the transition between the small and the large population regimes is quite rapid, and occurs largely over a range of population sizes that vary by a factor of 10. The effects of drift on inbreeding depression may bias some estimates of the genomic rate of deleterious mutation. These effects could also be important in the evolution of breeding systems in hermaphroditic organisms and in the conservation of endangered populations. PMID- 10740924 TI - Predicting long-term response to selection. AB - Lande's equation for predicting the response of trait means to a shift in optimal trait values is tested using a stochastic simulation model. The simulated population is finite, and each individual has a finite number of loci. Therefore, selection may cause allele frequencies and distributions to change over time. Since the equation assumes constant genetic parameters, the degree to which such allelic changes affect predictions can be examined. Predictions are based only on information available at generation zero of directional selection. The quality of the predictions depends on the nature of allelic distributions in the original population. If allelic effects are approximately normally distributed, as assumed in Lande's Gaussian approximation to the continuum-of-alleles model, the predictions are very accurate, despite small changes in the G matrix. If allelic effects have a leptokurtic distribution, as is likely in Turelli's 'house-of cards' approximation, the equation underestimates the rate of response and correlated response, and overestimates the time required for the trait means to reach their equilibrium values. Models with biallelic loci have limits as to the amount of trait divergence possible, since only two allelic values are available at each of a finite set of loci. If the new optimal trait values lie within these limits, predictions are good, if not, singularity in the G matrix results in suboptimal equilibria, despite the presence of genetic variance for each individual trait. PMID- 10740925 TI - On the number of reproductives contributing to a half-sib progeny array. AB - We address various statistical aspects of biological parentage in multi-offspring broods that arise via multiple paternity or multiple maternity and, hence, consist of mixtures of full- and half-sibs. Conditioned on population genetic parameters, computer simulations described herein permit estimation of: (1) the mean number of offspring needed to detect all parental gametes in a brood and (2) the relationship between the number of distinct parental gametes found in a brood and the number of parents. Results are relevant to the design of empirical studies employing molecular markers to assess genetic parentage in polygynous or polyandrous species with large broods, such as are found in many fishes, amphibians, insects, plants and other groups. The utility of this approach is illustrated using two empirical data sets. PMID- 10740926 TI - Models of human genetic disease: how biased are the standard formulae? AB - Standard theory provides a simple prediction for the frequency of a recessive lethal allele conferring heterozygous protection against an infectious disease (the best-known example being sickle cell protection against malaria). This relationship allows historic disease mortality rates to be estimated. There are, however, hidden biases in this approach. Reproductively active human females in archaic societies normally produce children at intervals of around 4 years. If death of the fetus or young infant (less than around 3 years of age) occurs, then the mother re-enters oestrus and produces another child. This 'reproductive compensation' reduces selection against the agent causing early mortality (the recessive allele or infective agent) and biases our estimates of historic mortality rates. The magnitude of these biases is investigated. Re-conception also constitutes a demographic selective pressure acting alongside natural selection: lethal genetic diseases (or tightly linked loci) will be selected to become ever more virulent, killing at ever decreasing ages, to allow the mother to re-enter oestrus and re-conceive a (hopefully unaffected) sibling; this effect also invalidates statistical tests using the number of alleles to distinguish overdominance from drift as explanations for high allele frequency. The same bias affects calculations of mutation/selection balance: for any given mutation rate, syndromes which kill early in life will reach much higher frequencies than those killing at later ages. An intriguing consequence is that lethal recessive disorders in humans will increase in frequency by up to 45% as a consequence of the recent demographic transition to planned family size. PMID- 10740927 TI - A transmission/disequilibrium test approach to screen for quantitative trait loci in two selected lines of large white pigs. AB - Pedigree and marker data from a multiple-generation pig selection experiment have been analysed to screen for loci affecting quantitative traits (QTL). Pigs from a base population were selected either for low backfat thickness at fixed live weight (L-line) or high live weight at fixed age (F-line). Selection was based on single-trait own performance and DNA was available on selected individuals only. Genotypes for three marker loci with known positions on chromosome 4 were available. The transmission/disequilibrium test (TDT) was originally described in human genetics to test for linkage between a genetic marker and a disease susceptibility locus, in the presence of association. Here, we adapt the TDT to test for linkage between a marker and QTL favoured by selection, and for linkage disequilibrium between them in the base population. The a priori unknown distribution of the test statistic under the null hypothesis, no linkage, was obtained via Monte Carlo simulation. Significant TDT statistics were found for markers AFABP and SW818 in the F-line, indicating the presence of a closely linked QTL affecting growth performance. In the L-line, none of the markers studied showed significance. This study emphasizes the potential of the TDT as a quick and simple approach to screen for QTL in situations where marker genotypes are available on selected individuals. The results suggest that previously identified QTL in crosses of genetically diverse breeds may also segregate in commercial selection lines. PMID- 10740928 TI - A review of workers' compensation claims: the frequency of claim denial and medical treatment delay. AB - A retrospective chart review of 257 patients who presented between January to March 1998, was conducted to identify the frequency of workers' compensation claim denial among patients who reported to Straub Clinic and Hospital. Results showed that 12.8% of claims were either "denied" or temporarily "denied pending investigation". Analysis of all the claims found that only 2% resulted in delayed medical care. Further analysis of the denied claims revealed the vast majority of claims (78.8%) were denied because the medical provider and the patient had incorrect workers' compensation carrier information. Other independent variables studied (size of the employer, category of employer, injury type, status of case, length of case and number of visits) did not predict either denial of the claim or treatment delay. This study suggests that the efficiency of our State's Workers' Compensation system would be greatly improved by developing a system to provide medical care providers with accurate insurance information. PMID- 10740929 TI - Learning to implement the medical home in communities. AB - Practicing the Medical Home within communities is prefaced by university training through pediatric residency programs. Through collaboration experiences, future and practicing pediatricians can learn skills to form effective interprofessional relationships, thereby supporting families and children. Seeing parents as enabled partners and creating interdependent relationships with community members and professionals, enhances the medical home's broad base of support for families. PMID- 10740930 TI - Neuropsychological differentiation of depression and anxiety. AB - The high comorbidity of depression and anxiety is well established empirically but not well understood conceptually, in terms of either psychological or biological mechanisms. A neuropsychological model of regional brain activity in emotion provides contrasting hypotheses for depression and anxiety, with depression associated with a relative decrease and anxiety with a relative increase in right-posterior activity. These hypotheses received support in a comparison of individuals diagnosed with depression and community controls, and also in a separate study of nonpatients administered a measure of perceptual asymmetry. Hierarchical regressions revealed that depression and anxiety were uniquely and jointly associated with perceptual asymmetry. In light of consistent empirical support for the model, implications for conceptualizations of the comorbidity of depression and anxiety are discussed. PMID- 10740931 TI - Clinical depression and day-to-day social interaction in a community sample. AB - Adult participants recruited from the community, one half of whom met criteria for clinical depression, described their day-to-day social interactions using a variant of the Rochester Interaction Record. Compared with the nondepressed participants, depressed participants found their interactions to be less enjoyable and less intimate, and they felt less influence over their interactions. Differences between the two groups in intimacy occurred only in interactions with close relations and not in interactions with nonintimates, and differences in influence were more pronounced for those who were cohabiting than for those who were not. There were no differences in how socially active depressed and nondepressed people were or in the amount of contact they had with different relational partners. PMID- 10740933 TI - Emotional processing in posttraumatic stress disorder. AB - The emotional deficits associated with posttraumatic stress disorder (PTSD) are the least understood and the most understudied aspect of the syndrome. In this study, the connection was evaluated between trauma-context reactivity and subsequent emotional deficits in PTSD. Combat veterans with PTSD and well adjusted veteran control participants were exposed to reminders of combat, after which their emotional behavior was assessed in response to a series of emotionally evocative images. Under the neutral condition, both groups exhibited emotional behavior modulated by stimulus valence. Partially consistent with the conceptual model described by B. Litz (1992), the PTSD group exhibited suppressed expressive-motor responses to positively valenced images, in comparison with the control group, only after being exposed to a trauma-related prime. Contrary to expectations, the PTSD group showed no augmentation of emotional response to negatively valenced cues after being exposed to trauma reminders. However, the PTSD group responded to all images, in both prime conditions, with higher heart rate reactivity, suggesting an automatic preparation for demand or threat in any uncertain emotional context. Possible causes and consequences of these results are discussed. PMID- 10740932 TI - Prior interpersonal trauma: the contribution to current PTSD symptoms in female rape victims. AB - The purpose of the current study was to disentangle the relationship of childhood sexual abuse and childhood physical abuse from prior adult sexual and physical victimization in predicting current posttraumatic stress disorder (PTSD) symptoms in recent rape victims. The participants were a community sample of 117 adult rape victims assessed within 1 month of a recent index rape for a history of child sexual abuse, child physical abuse, other adult sexual and physical victimization, and current PTSD symptoms. Results from path analyses showed that a history of child sexual abuse seems to increase vulnerability for adult sexual and physical victimization and appears to contribute to current PTSD symptoms within the cumulative context of other adult trauma. PMID- 10740934 TI - Personality and expressed emotion. AB - Expressed emotion (EE) is an established psychosocial predictor of relapse in patients with schizophrenia and mood disorders; however, nothing is currently known about the personality characteristics of high- and low-EE relatives. A total of 45 relatives of schizophrenia patients completed the California Psychological Inventory, a widely used measure of common personality traits. Compared with low-EE relatives, high-EE relatives were more conventional in their behavior (higher norm-favoring) and less satisfied with themselves and their lives (lower self-realization). High-EE relatives were also less flexible, tolerant, and lower in empathy and achievement via independence than were low-EE relatives. Even with statistical control of potential demographic confounds, flexibility remained a significant predictor of EE status. PMID- 10740935 TI - Posttraumatic stress disorder following political imprisonment: the role of mental defeat, alienation, and perceived permanent change. AB - An interview study of 81 former political prisoners investigated whether posttraumatic stress disorder (PTSD) is related to the way individuals process the prison experience. In contrast to participants without PTSD, those with chronic or remitted PTSD were more likely to perceive mental defeat and an overall feeling of alienation from other people. Chronic PTSD was also related to perceived negative and permanent change in their personalities or life aspirations. The groups did not differ in their attempts to gain control during imprisonment. Evidence for a relationship between political commitment and PTSD was mixed. The results suggest that mental defeat, alienation, and permanent change are related to PTSD after interpersonal trauma and may need to be addressed in treatment. PMID- 10740936 TI - Continuity and discontinuity models of bulimia nervosa: a taxometric investigation. AB - Past research evaluating the continuity and discontinuity models of bulimia has produced inconclusive results. In the current study, we performed a taxometric analysis of bulimia nervosa using means above minus below a sliding cut and maximum covariance analysis with a sample of women diagnosed with bulimia nervosa (n = 201) or women college students (n = 412). Indicators were derived from the Bulimia Test--Revised and the Eating Attitudes Test--26, and both a mixed sample and the nonclinical sample were analyzed. With both taxometric methods and both mixed and nonclinical samples, results were consistently suggestive of a latent taxon for bulimia. These results challenge a dimensional model of bulimia nervosa. PMID- 10740937 TI - Predicting PTSD symptoms in victims of violent crime: the role of shame, anger, and childhood abuse. AB - To examine the role of cognitive-affective appraisals and childhood abuse as predictors of crime-related posttraumatic stress disorder (PTSD) symptoms, 157 victims of violent crime were interviewed within 1 month post-crime and 6 months later. Measures within 1 month post-crime included previous physical and sexual abuse in childhood and responses to the current crime, including shame and anger with self and others. When all variables were considered together, shame and anger with others were the only independent predictors of PTSD symptoms at 1 month, and shame was the only independent predictor of PTSD symptoms at 6 months when 1-month symptoms were controlled. The results suggest that both shame and anger play an important role in the phenomenology of crime-related PTSD and that shame makes a contribution to the subsequent course of symptoms. The findings are also consistent with previous evidence for the role of shame as a mediator between childhood abuse and adult psychopathology. PMID- 10740938 TI - Smoking withdrawal dynamics in unaided quitters. AB - Considerable research shows that withdrawal severity is inconsistently related to smoking cessation outcomes. This may result from measurement problems or failure to scrutinize important dimensions of the withdrawal experience. Two recent studies demonstrated that withdrawal elevation and variations in the time course of withdrawal were related to relapse in smokers treated with the nicotine patch (T. M. Piasecki, M. C. Fiore, & T. B. Baker, 1998). This article reports a conceptual replication and extension of those findings in unaided quitters. Evidence for temporal heterogeneity was found across different types of withdrawal symptoms. Patterns or slopes of affect and urge reports over time predicted smoking status at follow-up, as did mean elevation in withdrawal symptoms. These results suggest that affect and urge withdrawal symptoms make independent contributions to relapse and that relapse is related to both symptom severity and trajectory. PMID- 10740939 TI - Hedonic capacity and schizotypy revisited: a taxometric analysis of social anhedonia. AB - P. E. Meehl (1962) originally conjectured that hedonic capacity was an indicator of the latent class or taxon of schizotypy. However, P. E. Meehl (1989, 1990) subsequently diminished the role of hedonic capacity in his theory, indicating that hypohedonia is one of a dozen normal-range (nontaxonic) individual differences factors that may potentiate the expression of schizophrenia. This dimensional-only view of hedonic capacity was tested by applying taxometric procedures to the Revised Social Anhedonia Scale (RSAS; M. L. Eckblad, L. J. Chapman, J. P. Chapman, & M. Mishlove, 1982) in a sample of college students (N = 1,526). Analyses indicated that the construct measured by the RSAS is taxonic in nature with a base rate approximating .10. These data are interpreted in the context of other findings suggesting that social anhedonia is an indicator of schizotypy. PMID- 10740940 TI - Effects of nicotine deprivation on alcohol-related information processing and drinking behavior. AB - This study examined the influence of smoking cues and nicotine deprivation on responses to alcohol among hazardous drinkers. Fifty-six daily smoking, hazardous drinkers were exposed to either smoking cues or control cues after either 6 hr of nicotine deprivation or no deprivation. Urges to drink alcohol, alcohol-related cognitive processing, and alcohol consumption were assessed after cue exposure. Results indicated that nicotine deprivation increased urges to drink, the accessibility of alcohol outcome expectancies, and the volume of alcohol consumed. There was little influence of the smoking cue manipulation on these processes. Implications for understanding the mechanisms underlying alcohol tobacco interactions are discussed. PMID- 10740941 TI - The influence of alcohol expectancy priming and mood manipulation on subsequent alcohol consumption. AB - Studies showing that verbal priming can implicitly affect alcohol consumption have been used to support cognitive models of expectancies. However, because expectancy words reflect affective states as well as drinking outcomes, mediation through an affective pathway remains theoretically plausible (i.e., such words inadvertently may affect mood, which in turn influences drinking). The primary pathway was identified (and expectancy theory was tested) by comparing memory priming (using alcohol expectancy or neutral words) with mood induction (using positive or neutral music); an unrelated experiment paradigm allowed the priming manipulation to implicitly affect drinking. Men in the alcohol priming group drank significantly more than men in each of the other conditions, and, consistent with theory, men with histories of heavier drinking drank the most when primed with alcohol expectancies, indicating that expectancies can function as automatic memory processes. PMID- 10740942 TI - Right-posterior face processing anomaly in depression. AB - Evidence of a right-posterior brain anomaly was found in a study of 19 individuals with major depression and 15 controls. Participants performed a recognition-memory task involving positive, neutral, and negative face and word stimuli. Scalp brain wave topography suggested a region-specific anomaly in the depressed group. Individuals with major depression demonstrated a reduction in the N200 component of the event-related brain potential to faces and not words. Furthermore, results indicate that the regional anomaly is specific to positive facial stimuli. Findings are interpreted in light of a model of regional brain specialization in emotion and psychopathology. PMID- 10740944 TI - Demographic, clinical, and neurocognitive correlates of everyday functional impairment in severe mental illness. AB - Although cognitive deficits often accompany severe mental illness, their implications for everyday functioning remain poorly understood. In this study, an occupational therapist (OT) rated the everyday functioning of 105 adult psychiatric patients. Using demographic, clinical, and cognitive variables, the authors tested alternative models to account for the observed variability in OT ratings. Although age, education, and the presence of schizophrenia each contributed to a model that accounted for 27% of the variation in functional independence, adding terms for auditory divided attention and verbal learning increased the proportion of explained variance to 45% and decreased the beta weights for age and education--but not schizophrenia--to nonsignificant levels. These findings demonstrate the relevance of cognitive performance to everyday functioning in severe mental illness. They are discussed with respect to hypothesized determinants of psychiatric disability. PMID- 10740943 TI - The effects of familial risk, personality, and expectancies on alcohol use and abuse. AB - This study tested a structural model of the association between familial risk, personality risk, alcohol expectancies, and alcohol abuse in a sample of 224 young adult offspring of alcoholics and 209 offspring of nonalcoholics. The results provided support for 2 personality-risk pathways, a social deviance proneness and an excitement/pleasure seeking path, that accounted for a significant portion of the association between a familial alcoholism and alcohol abuse. The path from familial alcoholism to social deviance proneness lead directly to alcohol problems. The path from familial alcoholism to excitement/pleasure seeking was associated with increased drinking, which, in turn, was associated with alcohol problems. Positive alcohol expectancies accounted for part of the association between excitement seeking and alcohol use. The results suggest 2 different biopsychosocial mechanisms that elevate risk for abuse in the offspring of alcoholics. PMID- 10740945 TI - Life events can trigger depressive exacerbation in the early course of schizophrenia. AB - Previous studies have linked life events with depression in chronic schizophrenia, but those studies had methodological limitations. Using a prospective research design and examining events that were clearly independent of the patients' illnesses, the authors sought to determine whether stressful life events could trigger depressive symptoms in the early course of schizophrenia. Schizophrenia patients (n = 99) were followed for 1 year from a point of outpatient stabilization. Life event interviews were conducted every 4 weeks and symptom assessments every 2 weeks. Survival analyses showed a significantly increased risk for an exacerbation of significant depressive symptoms following an independent life event. Of interest is that an analysis of competing risk showed that the odds of psychotic exacerbation following a major independent life event were not significantly greater than the odds of depressive exacerbation. The risk of depression and of psychosis after experiencing a stressful life event is significantly increased for the first month, but the risk period can extend to 3 months. PMID- 10740946 TI - Predicting anxiety and depression from personality: is there a synergistic effect of neuroticism and extraversion? AB - In a longitudinal study of college students, B. S. Gershuny and K. J. Sher (1998) found that high neuroticism and low extraversion had a synergistic effect in predicting anxiety and depression 3 years later. This article attempted to replicate their finding using data from 2 community samples: (a) a cross sectional survey of 2,677 persons aged 18-79 years, and (b) a longitudinal study in which 441 persons aged 70 or older were followed over 3-4 years. Both studies found that neuroticism predicted anxiety and depression, but there were no Neuroticism x Extraversion interaction effects. These results cast doubt on the generalizability of the original findings. PMID- 10740947 TI - Mindfulness-based cognitive therapy reduces overgeneral autobiographical memory in formerly depressed patients. AB - Previous research on depressed and suicidal patients and those with posttraumatic stress disorder has shown that patients' memory for the past is overgeneral (i.e., patients retrieve generic summaries of past events rather than specific events). This study investigated whether autobiographical memory could be affected by psychological treatment. Recovered depressed patients were randomly allocated to receive either treatment as usual or treatment designed to reduce risk of relapse. Whereas control patients showed no change in specificity of memories recalled in response to cue words, the treatment group showed a significantly reduced number of generic memories. Although such a memory deficit may arise from long-standing tendencies to encode and retrieve events generically, such a style is open to modification. PMID- 10740949 TI - Smoking, anxiety, and attention: support for the role of nicotine in attentionally mediated anxiolysis. AB - Based on J.D. Kassel and S. Shiffman's (1997) study, the hypothesis was tested that cigarette smoking's anxiolytic effects are attentionally mediated and depend on the presence of benign distraction. This study explored whether the attentionally mediated anxiolytic effects of smoking observed in the J. D. Kassel and S. Shiffman (1997) study could be replicated and are attributable to nicotine. The study examined the effect on anxiety of smoking a high- or low yield nicotine cigarette with or without a current distraction in 67 smokers. As predicted, only those who smoked a high-yield cigarette paired with participation in a distracting activity experienced a significant reduction in anxiety. Those who smoked a high-yield cigarette in the absence of distraction experienced a slight exacerbation of anxiety. These findings suggest that nicotine--not the behavioral or sensory aspects of smoking--interacts with distraction and leads to alleviation of anxiety. PMID- 10740948 TI - Effects of disclosure of traumatic events on illness behavior among psychiatric prison inmates. AB - To assess the health effects of writing about traumatic events in a clinical population, 98 psychiatric prison inmates were randomly assigned to 1 of 3 conditions in which they were asked to write about their deepest thoughts and feelings surrounding upsetting experiences (trauma writing condition), write about trivial topics (trivial writing control), or go about their daily routine without writing (no-writing control). Both writing groups wrote for 20 min per day for 3 consecutive days. Participants in the trauma condition reported experiencing more physical symptoms subsequent to the intervention relative to those in the other conditions. Despite this, controlling for prewriting infirmary visits, sex offenders in the trauma writing condition decreased their postwriting infirmary visits. These results are congruent with predictions based on stigmatization and inhibition. PMID- 10740950 TI - Controllability perceptions and reactions to symptoms of schizophrenia: a within family comparison of relatives with high and low expressed emotion. AB - In a sample of 35 family members of patients with recent-onset schizophrenia, attributions of control and the content of critical comments were compared for 2 relatives of the same household who held discrepant expressed emotion (EE) attitudes (1 high and 1 low) toward their mentally ill family member. Attributions and the content of critical comments were also compared for low-EE relatives from low-EE homes versus low-EE relatives from high-EE homes. Our results indicate that high-EE relatives tend to attribute more control over behavior to patients than do low-EE relatives of the same patient. In addition, low-EE relatives from high-EE homes attribute more behavioral control to patients than do low-EE relatives from low-EE homes. These findings suggest that EE status is linked to attributions of control over behaviors, but additional patient factors or influences among family members may also affect EE attitudes. PMID- 10740951 TI - An examination of the nature and correlates of ethnic harassment experiences in multiple contexts. AB - The authors examined the nature and correlates of ethnic harassment experiences in 4 predominantly Hispanic samples of working men and women (N = 575) using a newly developed scale. Confirmatory factor analyses supported a 2-factor structure for the Ethnic Harassment Experiences scale (i.e., verbal ethnic harassment and exclusion due to ethnicity). Most experiences of ethnic harassment in the workplace during the previous 24 months included verbal ethnic harassment (i.e., ethnic slurs, derogatory ethnic comments, or ethnic jokes). It was also found that participants who experienced verbal ethnic harassment and exclusion reported negative correlates that could not be attributed to affective disposition. For some correlates, those who experienced low levels of verbal harassment and high levels of exclusion were more negatively affected than others, perhaps as a result of the attributional ambiguity of the situation. PMID- 10740952 TI - Identifying international assignees at risk for premature departure: the interactive effect of outcome favorability and procedural fairness. AB - Two studies examined factors that predict expatriate managers' tendencies to think seriously about departing prematurely from their international assignments. Previous research (conducted outside of the expatriate context) has shown that individuals' willingness to stay with or leave their positions is an interactive function of outcome favorability and procedural fairness. A conceptually analogous interaction effect was found in the present studies. Whereas expatriates more seriously thought of departing prematurely when they perceived the non-work-related outcomes of their overseas assignments to be less favorable, this tendency was much less pronounced when procedural fairness was relatively high. Theoretical and practical implications are discussed, as are limitations of the studies and suggestions for future research. PMID- 10740953 TI - Score comparability of a state mathematics assessment across students with and without reading accommodations. AB - This study investigated the factorial invariance of a fourth-grade state mathematics assessment across groups of general education students and students with learning disabilities with and without reading accommodations. Confirmatory factor analysis was used to assess the fit of a 2-factor model to each of the 3 groups. In addition to the overall fit of this model, several levels of constraint were investigated. Invariance across the 3 groups was supported for factor loadings and intercepts. However, invariance of the factor covariances across the general education group and the groups of students with learning disabilities was not supported. Because of the implications for aggregating reported scores, further research is needed into the relationship between the factors in the different groups. PMID- 10740954 TI - Using response time measures to assess "guilty knowledge". AB - How can a suspect's guilt or innocence be reliably tested? The validity of the polygraph, which measures changes in physiological arousal during a "guilty knowledge" test, is controversial (e.g., T. R. Bashore & P. E. Rapp, 1993; T. P. Cross & L. Saxe, 1992; D. T. Lykken, 1998; J. P. Rosenfeld, 1995; R. Steinbrook, 1992). One alternative to the polygraph examines event-related potentials recorded during a memory interference task (L. A. Farwell & E. Donchin, 1991). The present study extended this paradigm to determine whether response times (RTs) can accurately identify participants possessing specific guilty knowledge. Results from Experiment 1 showed that RT alone can reliably discriminate "guilty" from "innocent" participants. Experiments 2a and 2b indicated that an RT-based paradigm is more resistant to strategic manipulation than previously suggested (Farwell & Donchin, 1991). This RT-based paradigm may be a viable alternative to the polygraph for detecting guilty knowledge. PMID- 10740955 TI - Allegations of wrongdoing: the effects of reinforcement on children's mundane and fantastic claims. AB - S. Garven, J. M. Wood, R. S. Malpass, and J. S. Shaw (1998) found that the interviewing techniques used in the McMartin Preschool case can induce preschool children to make false allegations of wrong doing against a classroom visitor. In this study, 2 specific components of the McMartin interviews, reinforcement and cowitness information, were examined more closely in interviews of 120 children, ages 5 to 7 years. Children who received reinforcement made 35% false allegations against a classroom visitor, compared with 12% made by controls. When questioned about "fantastic" events (e.g., being taken from school in a helicopter), children receiving reinforcement made 52% false allegations, compared with 5% made by controls. In a second interview, children repeated the allegations even when reinforcement had been discontinued. The findings indicate that reinforcement can swiftly induce children to make persistent false allegations of wrong doing. PMID- 10740956 TI - The influence of job familiarity and impression management on self-report measure scale scores and response latencies. AB - Two studies were conducted to examine the effects of job familiarity and impression management on response latencies and scale scores for measures of personality and situational judgment. In a laboratory study using university students and a field study using U.S. Border Patrol Agent applicants, impression management was generally associated with faster personality item responses when job familiarity was high and with slower responses when job familiarity was low. Both impression management and job familiarity were associated with personality item responses that were more likely to lead to a job offer. The field study revealed a similar pattern of results for situational judgment scale response latencies, although only impression management was associated with item responses that were more likely to lead to a job offer. The implications for using response latencies to detect impression management on self-report measures are discussed. PMID- 10740957 TI - An empirical examination of self-reported work stress among U.S. managers. AB - This study proposes that self-reported work stress among U.S. managers is differentially related (positively and negatively) to work outcomes depending on the stressors that are being evaluated. Specific hypotheses were derived from this general proposition and tested using a sample of 1,886 U.S. managers and longitudinal data. Regression results indicate that challenge-related self reported stress is positively related to job satisfaction and negatively related to job search. In contrast, hindrance-related self-reported stress is negatively related to job satisfaction and positively related to job search and turnover. Future research directions are discussed. PMID- 10740958 TI - How do people evaluate social sexual conduct at work? A psycholegal model. AB - The authors tested a psycholegal model of how people evaluate social sexual conduct at work with videotaped reenactments of interviews with alleged complainants, perpetrators, and other workers. Participants (200 full-time male and female workers) were randomly assigned to evaluate the complaints with either the reasonable person or reasonable woman legal standard. Participants answered questions about sexual harassment law and completed the Ambivalent Sexism Inventory. Participants who took the reasonable woman perspective, as compared with those who took the reasonable person perspective, were more likely to find the conduct harassing; this was especially the case among participants high in hostile sexism. Medium-sized gender effects were found in the severe case but were absent in the weaker, more ambiguous case. The implications of these findings for hostile work environment law are discussed. PMID- 10740959 TI - Climbing the corporate ladder: do female and male executives follow the same route? AB - This study compares the careers of matched samples of 69 female executives and 69 male executives by examining perceived barriers and facilitators of advancement, self-reported developmental experiences, and career histories. Consistent with tokenism theory, women reported greater barriers, such as lack of culture fit and being excluded from informal networks, and greater importance of having a good track record and developing relationships to facilitate advancement than did men. Career success, measured by organizational level and compensation, was positively related to breadth of experience and developmental assignments for both genders, but successful women were less likely than successful men to report that mentoring facilitated their advancement. Developmental experiences and career histories were similar for female and male executives, but men had more overseas assignments and women had more assignments with nonauthority relationships. PMID- 10740960 TI - Task conflict and relationship conflict in top management teams: the pivotal role of intragroup trust. AB - Task conflict is usually associated with effective decisions, and relationship conflict is associated with poor decisions. The 2 conflict types are typically correlated in ongoing groups, however, which creates a prescriptive dilemma. Three explanations might account for this relationship--misattribution of task conflict as relationship conflict, harsh task conflict tactics triggering relationship conflict, and misattribution of relationship conflict as task conflict. The authors found that intragroup trust moderates the relationship between task conflict and relationship conflict in 70 top management teams. This result supports the "misattribution of task conflict" explanation. The authors also found a weak effect that is consistent with the argument that tactical choices drive the association between the 2 conflict types. We infer that trust is a key to gaining the benefits of task conflict without suffering the costs of relationship conflict. PMID- 10740961 TI - Correction for range restriction: an expanded typology. AB - A common research problem is the estimation of the population correlation between x and y from an observed correlation rxy obtained from a sample that has been restricted because of some sample selection process. Methods of correcting sample correlations for range restriction in a limited set of conditions are well-known. An expanded classification scheme for range-restriction scenarios is developed that conceptualizes range-restriction scenarios from various combinations of the following facets: (a) the variable(s) on which selection occurs (x, y and/or a 3rd variable z), (b) whether unrestricted variances for the relevant variables are known, and (c) whether a 3rd variable, if involved, is measured or unmeasured. On the basis of these facets, the authors describe potential solutions for 11 different range-restriction scenarios and summarize research to date on these techniques. PMID- 10740962 TI - Facial composite production: a comparison of mechanical and computer-driven systems. AB - Computer-driven systems for constructing composite faces of suspects (E-fit; Mac a-Mug) have largely replaced mechanical systems (Photofit; the Identikit) in police use, yet little is known of their comparative effectiveness in rendering an accurate likeness. Participants (N = 24) constructed 2 of 4 familiar or unfamiliar faces, for one of which they used Photofit and for the other, E-fit. A likeness of each face was made first under target-absent conditions and then with photographs of the target present. The accuracy of the resulting composites was assessed by familiarity ratings, names elicited, and matching accuracy. The computer-driven system showed consistent superiority only when a familiar face was constructed in the presence of photographs; when participants worked from memory, E-fit was no better than Photofit. The implications of these findings for theories of face retrieval and the operational use of composites are discussed. PMID- 10740963 TI - On measures of explained variance in nonrecursive structural equation models. AB - Whereas measures of explained variance in a regression and an equation of a recursive structural equation model can be simply summarized by a standard R2 measure, this is not possible in nonrecursive models in which there are reciprocal interdependencies among variables. This article provides a general approach to defining variance explained in latent dependent variables of nonrecursive linear structural equation models. A new method of its estimation, easily implemented in EQS or LISREL and available in EQS 6, is described and illustrated. PMID- 10740964 TI - Predictors and outcomes of openness to changes in a reorganizing workplace. AB - It is becoming increasingly important for employees to be able to cope with change in the workplace. This longitudinal study examined a set of individual differences and context-specific predictors of employee openness (i.e., change acceptance and positive view of changes) toward a set of workplace changes. Personal resilience (a composite of self-esteem, optimism, and perceived control) was related to higher levels of change acceptance. Three context-specific variables (information received about the changes, self-efficacy for coping with the changes, and participation in the change decision process) were predictive of higher levels of employee openness to the changes. Lower levels of change acceptance were associated with less job satisfaction, more work irritation, and stronger intentions to quit. PMID- 10740965 TI - Item selection counts: a comparison of empirical key and rational scale validities in theory-based and non-theory-based item pools. AB - Little explicit attention has been given to the impact of item pools on the validities and cross-validities of different background data scoring approaches. This study tests the idea that pools of items theoretically related to the performance of interest will outperform pools of items with no hypothesized relationship with the criterion. Validities and cross-validities of rational scales and empirical keys created from theory- and non-theory-based item pools were compared for 3 criteria. When size of the item pools was held constant, theory-based empirical keys (correlational and vertical percent) and rational scales showed larger validities and cross-validities than non-theory-based empirical keys (correlational and vertical percent) and showed minimal shrinkage in cross-validities. Even when item pool for the non-theory-based keys was expanded to include all items in the instrument, the theory-based keys showed comparable or slightly better validities and cross-validities for 2 of the 3 criteria, including college GPA, which was separated from the predictors by 4 years. PMID- 10740966 TI - The phonological acquisition of Putonghua (Modern Standard Chinese). AB - The phonological acquisition of 129 monolingual Putonghua-speaking children, aged 1;6 to 4;6, is described. Putonghua (Modern Standard Chinese) syllables have four possible elements: tone, syllable-initial consonant, vowel, and syllable-final consonant. The children's errors suggested that Putonghua-speaking children mastered these elements in the following order: tones were acquired first; then syllable-final consonants and vowels; and syllable-initial consonants were acquired last. Phonetic acquisition of the 21 syllable-initial consonants was complete by 3;6 for 75% of children. By 4;6 the children were using the syllable initial consonants correctly on two thirds of occasions (with the exception of four affricates). Simple vowels emerged early in development. However, triphthongs and diphthongs were prone to systematic errors. Tone errors were rare, perhaps because of their role in distinguishing lexical meaning. In contrast, acquisition of 'weak stress' and 'rhotacized feature' was incomplete in the oldest children assessed. Phonological processes used by the children were identified. Two of these processes, syllable-initial consonant deletion and backing, would be considered atypical error patterns in English. Existing theories of phonological acquisition (e.g. concepts of markedness, functional load, feature hierarchies) cannot account for some of the patterns revealed. A satisfactory explanation of the findings requires more attention to the specific characteristics of the linguistic system the children are learning. It is proposed that the saliency of the components in the language system determines the order of acquisition. PMID- 10740967 TI - The Hebrew Communicative Development Inventory: language specific properties and cross-linguistic generalizations. AB - Cultural, linguistic, and developmental evidence was taken into consideration in constructing the HCDI, a Hebrew adaptation of the MCDI. The HCDI was then administered to a stratified sample of Israeli mothers of 253 toddlers aged 1;6 to 2;0 (M = 1;8.18). Hebrew results are presented and compared with scores from the original MCDI sample (Fenson, Dale, Reznick, Bates, Thal & Pethick, 1994). The HCDI is a reliable and sensitive measure of lexical development and emergent grammar, capturing wide variability among Israeli toddlers. In comparison with English, the relation between vocabulary size and age, as well as the shape of the growth curves for nouns, predicate terms, and closed class words relative to size of lexicon, were strikingly similar. These results indicate that conclusions concerning cross-linguistic similarities can be best documented by using parallel methods of measurement. The HCDI results support the claim that early lexical development in Hebrew and in English follow remarkably similar development patterns, despite the typological differences between the two target languages. PMID- 10740968 TI - Caregiver input in English and Korean: use of nouns and verbs in book-reading and toy-play contexts. AB - This study investigates structural and pragmatic aspects of caregiver input in English and Korean that relate to the early development of nouns and verbs. Twenty mothers in each language were asked to interact with their one-and-a-half year-old children in two contexts: book-reading and toy-play. Overall, English speaking mothers use more nouns than verbs, and focus more on objects than on actions. In contrast, Korean-speaking mothers provide a balanced treatment of nouns and verbs, and focus on objects and actions to a similar degree. A significant context effect indicates that whereas English-speaking mothers emphasize nouns in both contexts, Korean-speaking mothers do so only in the Books context. In the Toys context, they provide more verbs and focus more on actions. These data suggest that systematic comparisons of caregiver input within and across different contexts provide a richer and more accurate account of the variability that can occur across languages and cultures. PMID- 10740969 TI - Animacy effects on the production of object-dislocated descriptions by Catalan speaking children. AB - This paper presents an experiment that examined two related questions. First, it examined the effects of animacy on the production of different syntactic structures and word orders by Catalan-speaking children. Secondly, it explored the relationship between age and the production of different syntactic structures by these children. The results of a picture description experiment run with eighty Catalan-speaking children aged 4;11 to 11;11 show that participants tended to produce more object-dislocated descriptions when the patient was animate than when the patient was inanimate. The results also show that while the dislocation of object clauses is a construction already consolidated at 5;0, the passive structure is a construction still not fully acquired at 11;0. A comparison between the results obtained in the present experiment with existing results from similar experiments with English-speaking children shows that there is a cross language difference in the age at which children start producing passive clauses. We argue that frequency of exposure to a particular syntactic structure is an important factor that contributes to the acquisition of that syntactic structure. We also suggest that the effects of animacy on the production of object dislocated descriptions can be explained by means of conceptual and lexical accessibility. PMID- 10740970 TI - Case, inflection and subject licensing in child Catalan and Spanish. AB - The development of specified tense and number morphology in child Catalan and Spanish is found to correlate with the onset of overt subject use. The data come from four monolingual child Catalan-speakers (from the Serra & Sole corpus) and one monolingual child Spanish-speaker (from the Linaza corpus) who were studied longitudinally from 1:0 to 3;6, approximately. The simultaneous emergence of tense and number morphology on one hand and overt subjects on the other in the children's speech is taken as evidence that a particular aspect of Universal Grammar, Case Theory, determines the possible co-occurrences of verbal inflections and subject types in developing grammatical systems. Parallels in verbal inflectional development are found in other child languages, while such parallels are not found in regard to subject use. Possible modifications to Case Theory, which would allow a unified account of the cross-linguistic developmental patterns of subject use, are considered. The possibility of explaining the early absence of overt subjects in these null subject languages as the result of an early sentence processing deficit is explored and rejected. The children's knowledge of whether their language is a null subject or overt subject language even before acquiring adult-like verbal inflection is taken as further evidence for what has been called 'early convergence' on parameter settings. PMID- 10740971 TI - Subject-auxiliary inversion errors and wh-question acquisition: 'what children do know'? AB - The present paper reports an analysis of correct wh-question production and subject-auxiliary inversion errors in one child's early wh-question data (age 2;3.4 to 4;10.23). It is argued that two current movement rule accounts (DeVilliers, 1991; Valian, Lasser & Mandelbaum, 1992) cannot explain the patterning of early wh-questions. However, the data can be explained in terms of the child's knowledge of particular lexically-specific wh-word + auxiliary combinations, and the pattern of inversion and uninversion predicted from the relative frequencies of these combinations in the mother's speech. The results support the claim that correctly inverted wh-questions can be produced without access to a subject-auxiliary inversion rule and are consistent with the constructivist claim that a distributional learning mechanism that learns and reproduces lexically-specific formulae heard in the input can explain much of the early multi-word speech data. The implications of these results for movement rule based and constructivist theories of grammatical development are discussed. PMID- 10740972 TI - More overregularizations after all: new data and discussion on Marcus, Pinker, Ullman, Hollander, Rosen & Xu. AB - Marcus, Pinker, Ullman, Hollander, Rosen & Xu (1992) claim that when the irregular past form of a verb is known, it is immediately known to be the correct form, such that over-regularizations only occur as speech errors, not as a genuine grammatical alternative; as a result, they argue, over-regularization rates are, when carefully inspected, very low. In the present paper: (1) it is shown that even if over-regularizations are a genuine grammatical alternative, overall rates in samples would still be low for most children; (2) careful analysis shows evidence for substantial over-regularization periods in three longitudinal subjects ages 2;5-5;2 (Abe), 2;3-5;2 (Adam) and 2;3-5;0 (Sarah); (3) Abe's much higher rates follow from general developments in his past tense acquisition, in ways not consonant with Marcus et al.'s formulations. PMID- 10740973 TI - Fertilization failure and oocyte activation. AB - Recent reports have suggested that when unfertilized oocytes with a spermatozoon after intracytoplasmic sperm injection (ICSI) are properly activated, the activated oocytes develop normally similar to fertilized oocytes. However, human oocytes do not easily respond to universal activators of mammalian oocytes like ethanol or calcium ionophore A23187, which induce a calcium transient in ooplasm. Puromycin activates human oocytes at a rate of 90%, but more than two thirds of the parthenogenesis possess 2 haploid pronuclei or 1 diploid pronucleus without extrusion of the second polar body. Therefore, the activation method which produces one pronucleus with extrusion of the second polar body in oocytes without a spermatozoon is necessary for producing embryos with normal karyotypes. Recently, we found the oocyte activation method which produced parthenogenesis displaying one pronucleus with extrusion of the second polar body. Using our method (a combination of calcium ionophore A23187 and puromycin), the activation rate was approximately 90% and the proportion of parthenogenesis displaying one pronucleus with extrusion of the second polar body was approximately 80% in human aged and mouse young oocytes. When human unfertilized oocytes following ICSI were activated by this method, two pronuclei were formed with extrusion of the second polar body in 30% of the oocytes. Four cleaved parthenogenesis (or embryos) showed normal karyotypes. However, the cytotoxic, teratogenetic and mutagenetic activity of Ca ionophore and puromycin should be approved prior to the clinical adaptation of the method. PMID- 10740974 TI - Synergistic effect of combining theophylline and drugs that potentially elevate serum creatine kinase. AB - An increase in the serum creatine kinase (CK) level is one of the side effects of theophylline; on rare occasions, the increase may be followed by rhabdomyolysis. Theophylline is often administered with drugs that potentially elevate the serum CK level (CK-elevating drugs) such as beta-agonists and steroids. However, the effects of the combined treatment of theophylline and CK-elevating drugs have not been reported. We, therefore, retrospectively investigated the effects of combined treatment on the serum CK level, in 391 asthmatic outpatients. In this study, the number and type of the CK-elevating drugs administered, and the serum levels of CK and theophylline, were investigated. The patients were divided into four groups: the theophylline-treated and CK-elevating drug-treated group, the theophylline-treated and non-CK-elevating drug-treated group, the non theophylline-treated and CK-elevating drug-treated group, and the non theophylline-treated and non-CK-elevating drug-treated group. The theophylline treated and CK-elevating drug-treated group showed about 100% higher serum CK levels (225 IU/L) than any other group (102-124 IU/L), and no increase in the serum theophylline level. This result indicates that there is a synergistic effect of theophylline and CK-elevating drugs on the serum CK level. The combined treatment of theophylline and CK-elevating drugs induces a synergistic increase in the serum CK level, indicating not pharmacokinetic but pharmacodynamic interactions with these drugs. PMID- 10740975 TI - The relation between anxiety and depressive symptoms in normal subjects and patients with anxiety and/or mood disorders. AB - OBJECTIVE: We investigated the associations between anxiety and depressive symptoms in normal subjects and patients with mood and/or anxiety disorders, using the Japaneses version of Spielberger's STAI and the Zung SDS. METHODS: The subjects for the present study were 60 normal subjects, 15 patients with anxiety disorders and, 12 patients with mood disorders meeting the DSM-IV diagnostic criteria. Informed consent was obtained from all subjects. RESULTS: 1) Both the mean total state-anxiety (S-anxiety) and trait-anxiety (T-anxiety) scores were significantly higher in patients with anxiety disorders and mood disorders than in normal subjects. 2) The mean total SDS score was significantly higher in patients with anxiety disorders and mood disorders than in normal subjects. 3) In normal subjects, there were significant positive correlations between the total T and S-anxiety scores and total SDS scores. 4) In patients with anxiety disorders, there were significant positive correlations between the total T- and S-anxiety scores and total SDS scores. 5) In patients with mood disorders, there were nonsignificant positive correlations between the total T- and S-anxiety scores and total SDS scores. CONCLUSION: Our results might provide support for the existence of common underlying mechanisms to anxiety and depression. PMID- 10740976 TI - Activation of G-protein coupled fMLP or PAF receptor directly triggers glucose transporter type 1 (GLUT1) translocation in Chinese hamster ovary (CHO) cells stably expressing fMLP or PAF receptor. AB - The chemoattractants, fMLP and PAF, stimulate glucose uptake in phagocytes to obtain an energy source for host defense. Glucose uptake in phagocytes is mainly regulated via glucose transporter type 1 (GLUT1). To examine molecular mechanisms of facilitated glucose uptake in response to fMLP or PAF, we established CHO cells stably expressing fMLP or PAF receptor with c-myc epitope tagged GLUT1 which could immunologically detect GLUT1 on the cell surface. In the CHO cells, both fMLP and PAF directly triggered GLUT1 translocation from the intracellular pool to the cell surface, and stimulated glucose uptake. Therefore, in phagocytes, we propose that fMLP and PAF also trigger GLUT1 translocation to stimulate glucose uptake as an energy source for host defense. PMID- 10740977 TI - Spectral analysis of erector spinae muscle surface electromyography as an index of exercise performance in maximal treadmill running. AB - Thirteen male athletes (mean 20.7 years) participated in the present study which investigated the relationship between mean power frequency (MPF) and exercise intensity determined from gas analysis during maximal treadmill running. All subjects performed two consecutive ramp exercise tests on the treadmill. Myoelectric signals from surface electrodes on the erector spinae muscles were digitized and MPF was calculated every ten seconds. Gas exchange data was collected using an automated breath-by-breath system, from which the anaerobic threshold (AT), respiratory gas exchange ratio (R = VCO2/VO2), and %VO2 = VO2/VO2max were obtained. During loading, MPF showed a steady decrease, followed by a sudden fall to a base level in both tests. After loading, MPF recovered within 30 seconds in all subjects. The test-retest reliability coefficient of MPF and R at the point of sudden fall in MPF were 0.757 (p = 0.0018), and 0.808 (p = 0.0004). These findings suggest that a sudden fall and a base level of MPF indicate local muscle fatigue, and the spectral analysis of trunk muscle surface EMG provides a reliable index of exercise performance in maximal treadmill running. PMID- 10740978 TI - Mucosal change of the stomach with low-grade mucosa-associated lymphoid tissue lymphoma after eradication of Helicobacter pylori: follow-up study of 48 cases. AB - Low-grade mucosa-associated lymphoid tissue (MALT) lymphoma of the stomach has been demonstrated to be closely linked to Helicobacter pylori (H. pylori) and to be frequently remissioned after the cure of H. pylori infection. Several previous studies have focused on proliferating lymphocytes but little is known about gastric epithelial change and the duration of the remission after the cure of H. pylori infection. We performed a long-term follow-up investigation on the effects of anti-H. pylori treatment on MALT lymphoma and chronic gastritis at the histologic and molecular levels. Forty-eight patients with low-grade gastric MALT lymphoma and 28 chronic gastritis patients in whom H. pylori infection was eradicated were studied. After eradication, 43 MALT lymphoma patients showed complete histologic remission and continuous remission was observed during follow up for up to 43 months (mean, 17.8 months). As for epithelial changes after eradication, "emptiness of lamina propria" was more pronounced in the mucosa with MALT lymphoma than that with chronic gastritis, and its severity in MALT lymphoma cases significantly decreased during the observation period whereas the glandular area increased. Cystic change of the fundic gland also occurred more frequently in MALT lymphoma cases than chronic gastritis cases. B-cell clonality before eradication analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) was detected in almost all MALT lymphoma cases (43 cases), but rare in chronic gastritis cases (6 cases). After eradication, in spite of histologic regression, 21 MALT lymphoma patients had a persistent monoclonal population during the follow-up period. B-cell monoclonality preceding the malignant transformation was noted in 4 cases. These observations indicate that 1) complete histologic remission of low-grade gastric MALT lymphomas seems stable even if a monoclonal B cell population is detectable in some cases, 2) there may be a stage of disease where monoclonal B cells are present but there is no histologic evidence of MALT lymphoma, and 3) regenerative change of the damaged glands may occur in histologic regressed MALT lymphoma cases. PMID- 10740979 TI - Overexpression of wild-type Akt1 promoted insulin-stimulated p70S6 kinase (p70S6K) activity and affected GSK3 beta regulation, but did not promote insulin stimulated GLUT4 translocation or glucose transport in L6 myotubes. AB - We have developed a simple, direct and sensitive method to detect GLUT4 on the cell surface. Using this system, we found that PI3-kinase plays a key role in the signaling pathway of insulin-stimulated GLUT4 translocation. One of the down stream effectors of PI3-kinase is serine-threonine kinase Akt (protein kinase B, RAK-PK), but the involvement of Akt in insulin-stimulated GLUT4 translocation is controversial. To investigate whether Akt1 regulates insulin-stimulated GLUT4 translocation and glucose uptake in L6 myotubes, we established L6 myotubes stably expressing c-myc epitope-tagged GLUT4 (GLUT4myc) and mouse wild type (WT) Akt1. We found that overexpression of WT Akt1 promoted insulin-stimulated p70S6 kinase (p70S6K) activity and increased the basal activity of GSK3 beta, but did not promote insulin-stimulated GLUT4 translocation or glucose uptake. These data supported the result that Akt is not a main signaling molecule to transmit the signal of insulin-stimulated GLUT4 translocation or glucose uptake from insulin activated PI3-kinase. PMID- 10740980 TI - Histological reaction of auditory bulla bone to synthetic auditory ossicle (Apaceram) in rats. AB - To investigate the biocompatibility of a synthetic auditory ossicle to host bone, small thin Apaceram disks composed of dense hydroxyapatite were implanted under the periosteum of the left auditory bulla in 32 rats for periods ranging from 1 day to 270 days. A sham operation performed on 10 rats served as a control. Decalcified histological sections stained with hematoxylin and eosin were observed using light microscopy. The experiment showed: 1) a time-dependent mature fibrous connective tissue surrounding the Apaceram disk, 2) no evidence of inflammatory reaction caused by the implant from 90 days after implantation until the end of the experiment, 3) no evidence of osteolysis by osteoclasts caused by the implant, and 4) direct contact of bone to the implant on the bone-disk interface at 180 and 270 days after implantation. The findings suggest that Apaceram has a high degree of implant biocompatibility, making it a satisfactory substitute biomaterial for otological reconstructive surgeries. PMID- 10740981 TI - Cathepsins B and L in synovial fluids from patients with rheumatoid arthritis and the effect of cathepsin B on the activation of pro-urokinase. AB - To clarify the pathophysiological role of cathepsins in rheumatoid arthritis (RA), we investigated whether cathepsin B or cathepsin L was increased in synovial fluid (SF) of RA joints, and whether the cathepsin isolated from SF of RA patients activated pro-urokinase or not. Thus, we estimated the content of cathepsins in SF of RA patients by measuring their activities by fluorospectrometry, using Z-Phe-Arg-MCA as the substrate. Cathepsin activity was approximately 4-fold higher in the SF of RA patients than in those of patients with osteoarthritis. Cathepsin B and cathepsin L were separated by cation exchange column chromatography. As a result, a large peak corresponding to cathepsin B and a very small peak corresponding to cathepsin L were detected. Biochemical sequential fractionation of the cathepsin purified from the SF showed that the large peak was mainly composed of cathepsin B. This purified enzyme induced conversion of pro-urokinase to urokinase, and the Km for pro-urokinase was approximately 8.27 microM. These findings indicated that an imbalance between cathepsin B and its inhibitors occurred due to increased concentrations of active cathepsin B in RA articular lesions, and that cathepsin B might be related to the degradation of cartilage in RA by activating the fibrinolytic cascade. PMID- 10740982 TI - HSP70 and c-Fos expression of brain stem hypoglossal nucleus in drowning. AB - The brain stem hypoglossal nucleus (HN) is the center of nerves innervating the upper respiratory tract and is related to control of mastication, deglutition, speech and respiration. To elucidate the relationship between asphyxia and the HN, we investigated the change of hypoglossal neurons in cases of hanging, strangulation, smothering, choking, drowning and respiratory failure. Using immunohistochemical techniques, we observed the brain stem HN with antibodies against microtubule-associated protein 2 (MAP2), muscarinic acetylcholine receptor (mAChR), c-fos gene product (c-Fos) and 72 kD heat-shock protein (HSP70). MAP2, a cytoskeletal protein of the neuron, is a marker of neuronal damage. Muscarinic AChR was used as a marker of neuronal membrane and ACh signaling. We employed both HSP70 and c-Fos as markers of stress- or damage related events. We measured the percentage of immunopositive neurons in total neurons of HN. Drowning produced higher expression of HSP70 and c-Fos than other causes of asphyxia, suggesting that drowning induces more severe damage in HN neurons. Furthermore, it was suspected that neuronal changes in drowning might relate to functions of the HN. These observations indicate that immunohistochemical examination of the brain stem HN could provide useful information for determining the cause of asphyxia. PMID- 10740983 TI - Novel antipsychotics and the neuropsychological deficiencies of schizophrenia. PMID- 10740984 TI - Measuring neuropsychological change in schizophrenia with novel antipsychotic medications. PMID- 10740985 TI - Treating cognitive dysfunction in patients with schizophrenia. AB - Cognitive dysfunction is a common, chronically disabling component of schizophrenia. It has been proposed that many of the symptoms of schizophrenia can be understood as a result of disruption of fundamental cognitive processes. This paper reviews treatment strategies aimed at improving cognitive function in patients with schizophrenia. Nonpharmacologic interventions include instruction in the performance of tasks such as the Wisconsin Card Sorting Test. Mixed results have been achieved, but it appears that instruction methods involving reinforcement of information held in working memory are more successful. Computer aided remediation has also been used with variable success. Novel antipsychotic drugs appear to have an advantage over conventional antipsychotic drugs in terms of their effect on cognitive function. The development of more precisely tailored methods of remedial teaching, along with optimal pharmacologic treatment, may lead to more effective treatment of cognitive dysfunction in patients with schizophrenia. PMID- 10740986 TI - Pathways to relapse: the neurobiology of drug- and stress-induced relapse to drug taking. AB - Relapse is a major characteristic of drug addiction, and remains the primary problem in treating drug abuse. Without an understanding of the factors that determine renewed drug-seeking, the urge to use drugs, and the persistent craving for them, it is unlikely that health care professionals can provide effective treatment. Using an animal model of relapse, the author and her team are studying factors that induce reinstatement of drug-taking behaviour after short and long periods of abstinence, and they are exploring the neurobiological basis of these effects. In their experiments, rats are trained to self-administer drugs intravenously by pressing 1 of 2 levers. During a subsequent period, the drug is no longer available, but the rats are free to try to obtain the drug (a period of "extinction training"). After extinction of responding, the investigators test for the ability of various events to reinitiate drug-seeking. On this background of renewed drug-seeking or relapse, the investigators search for pharmacological and neurochemical manipulations that might block or attenuate such behaviour. They have found that the 2 most effective events for reinstating responding after both short and long drug-free periods are re-exposure to the drug itself and exposure to a brief period of stress. The critical neurochemical pathways mediating drug-induced relapse are not identical to those mediating stress induced relapse. Relapse induced by "priming" injections of heroin or cocaine involves activation of the mesolimbic dopaminergic pathways, whereas relapse induced by stress involves actions of corticotropin-releasing factor (CRF) in the brain, and of brain noradrenergic (NE) systems. In addition, evidence shows that CRF and NE may interact at the level of the bed nucleus of the stria terminalis in stress-induced relapse. By contrast, relapse induced by "priming" injections of drugs is relatively unaffected by manipulation of CRF and NE systems of the brain. PMID- 10740988 TI - Antipsychotic agents and QT changes. AB - Recently, antipsychotic medications of the novel or atypical classes have received increased attention because of concerns with respect to potential lengthening of the QT interval, yet the currently available and commonly prescribed conventional antipsychotics are significantly more cardiotoxic, particularly agents in the butyrophenone and phenothiazine classes. Lengthening of the QT interval can be associated with a fatal paroxysmal ventricular arrhythmia known as torsades de pointes. The specific duration of the QT interval at which the risk of an adverse cardiac event is greatest, is not established. There is not only significant variation in the applied definition of an abnormal interval, but the maximal QT interval in healthy volunteers is greater than the currently accepted standards. The QT interval is influenced by normal physiological and pathologic factors, but the mechanisms remain unclear. Using recombinant technology, haloperidol and sertindole have been demonstrated to be high-affinity antagonists of a human cardiac potassium channel encoded by the human ether-a-go-go-related gene. Pimozide, however, has been shown to act principally through calcium channel antagonism, and chlorpromazine may affect sodium channels. Nevertheless, it is possible that these effects are significant only in the presence of predisposing factors, either genetic or acquired. Despite proven efficacy in clinical trials and subsequent supervised use in Europe, a number of recently developed antipsychotic medications are not available to patients in North America. Yet, conventional antipsychotic medications that would not be approved by current safety standards continue to be widely used. PMID- 10740987 TI - Novel antipsychotics: issues and controversies. Typicality of atypical antipsychotics. AB - The typicality of atypical antipsychotic drugs remains debatable. Preclinical studies and findings from randomized, controlled and open trials of clozapine, olanzapine, risperidone, quetiapine, sertindole, ziprasidone and a substituted benzamide were examined. A MEDLINE search was conducted using key words, including "extrapyramidal side effects," "cognition," "schizophrenia" and the generic drug names. Over 140 articles from peer-reviewed journals were reviewed, some of which were based on a meta-analysis. New-generation neuroleptic agents were found to have greater efficacy on the negative symptoms of schizophrenia and to cause fewer unwanted extrapyramidal side effects (EPS) than the traditional antipsychotic drugs. On one hand, atypical neuroleptic agents could be strictly defined as any neuroleptic agent with antipsychotic effects at a dosage that does not cause extrapyramidal side effects. Thus, clozapine is regarded as the "standard" atypical antipsychotic drug. On the other hand, typicality is about dimension rather than category, and we suggest the use of the term "spectrum of atypicality." For example, an emphasis is placed on quetiapine to illustrate where a new compound fits in this spectrum. Although dose-related, atypicality may be more a question of prescription attitude than of a specific characteristic of a compound. The degree to which a new compound is clinically superior to another atypical antipsychotic drug, in terms of improving positive, negative or affective symptoms, cognitive function and long-term outcome, will require further a priori hypotheses based on conceptual frameworks that are clinically meaningful. In addition, the results from industry-sponsored trials should be more comparable to those obtained from investigator-leading trials. Finally, the patient characteristics that define a patient's response to a specific antipsychotic drug are unknown. PMID- 10740989 TI - Antipsychotic agents differ in how fast they come off the dopamine D2 receptors. Implications for atypical antipsychotic action. AB - RATIONALE AND OBJECTIVE: While the blockade of dopamine D2 receptors are necessary for antipsychotic action, antipsychotic agents differ nearly a thousand fold in their affinity for the D2 receptor. This affinity is determined by the rate at which the antipsychotic agent binds to (kon) and the rate at which it dissociates from (koff) the D2 receptors. The objective of this study was to determine the relationship between kon, koff and the affinity (Ki) of antipsychotic agents for the D2 receptors, with particular reference to typical and atypical antipsychotic agents. DESIGN: The koff of several typical as well as atypical antipsychotic agents (nemonapride, spiperone, haloperidol, chlorpromazine, raclopride, olanzapine, sertindole, clozapine and quetiapine) was measured in vitro using the 3H-radiolabelled analogues of these drugs. The affinity of these drugs for the D2 receptor was determined by competition with 3H raclopride in vitro. The kon was derived from values of affinity and ++koff. MAIN OUTCOME MEASURES: kon, koff, and the Ki of antipsychotic drugs. RESULTS: The range of affinity values was similar to that conventionally accepted (0.025-155 nmol/L). The koff values varied a thousand-fold from 0.002 to 3.013 min-1, with relatively little variation in kon. The rate at which antipsychotic agents come off the receptor (koff) accounted for 99% of the variation in their affinity for the D2 receptor; differences in kon did not account for differences in affinity. CONCLUSIONS: The differences in the affinity of antipsychotic agents are entirely determined by how fast they come off the D2 receptor. These differences in koff may lead to functionally different kinds of dopamine blockade. Drugs with a higher koff will be faster in blocking receptors, and once blocked, will provide more access to surges in dopamine transmission. Since atypical drugs show a lower affinity and a faster dissociation, a higher koff for the D2 receptor is proposed as a mechanism for "atypical" antipsychotic effect. PMID- 10740990 TI - Influence of a chronic ultramild stress procedure on decision-making in mice. AB - OBJECTIVE: To test the influence of a chronic ultra mild stress (CUMS) procedure, based solely on socio-environmental stressors, on cognitive-behavioural function in mice. DESIGN: Behavioural study. PARTICIPANTS: B6D2F1 mice. INTERVENTIONS: Mice were exposed to various stressors and then tested using a decision-making task. RESULTS: We observed that stress facilitated "choice" behaviour, with an absence of "no choice" behaviour. Stress also facilitated a more rapid capacity to process information, a decrease in the level of evaluation of the choice situation and less hesitation. These stress-related consequences on decision making may be attributed to a higher level of distractability in the stressed mice. CONCLUSIONS: The CUMS model may be useful for the study of stress-related disorders by proposing a new method for assessing gene-environment interactions in cognitive-affective behaviours. PMID- 10740991 TI - An examination of the sensitivity of the six-item Hamilton Rating Scale for Depression in a sample of patients suffering from major depressive disorder. AB - OBJECTIVES: To compare the sensitivity of the 6-item Hamilton Rating Scale for Depression (HRSD6) with the more widely used 17-item Hamilton Rating Scale for Depression (HRSD17) in patients suffering from major depressive disorder, with or without melancholia and/or dysthymic disorder. A secondary objective was to compare the sensitivity of the HRSD6 to the Montgomery-Asberg Depression Rating Scale (MADRS). DESIGN: Retrospective analysis of 4 clinical trials that tested antidepressant therapies. SETTING: Outpatient treatment in a major psychiatric hospital. PARTICIPANTS: One hundred and forty-three male and female outpatients meeting the criteria of the DSM-III-R or DSM-IV for major depressive disorder. OUTCOME MEASURES: HRSD17, HRSD6 and MADRS. RESULTS: The HRSD6 correlated strongly with the HRSD17, both at baseline and termination of treatment, and for the subgroups of double depression and melancholia. The HRSD6 was also correlated significantly with the MADRS at both measurement times, and for the subgroups. Paired t-tests with the HRSD6, HRSD17 and MADRS demonstrated equal sensitivity to change over the course of treatment, both in the full sample and in the dysthymic and melancholic subgroups. CONCLUSIONS: The HRSD6 appears to be as sensitive to change over treatment as the HRSD17 and the MADRS. A shorter, less time-consuming measure of depression may have utility in clinical practice and research. PMID- 10740992 TI - Sulpiride addition in a case of fluvoxamine-refractory obsessive-compulsive disorder without comorbid psychopathology. PMID- 10740993 TI - How does grapefruit juice affect psychotropic medications? PMID- 10740994 TI - Tarsal functions, movement, and stabilization mechanisms in foot, ankle, and leg performance. AB - The authors investigated the function of the tarsus and the skeletal segments of the foot in terms of the biomechanical significance of the individual links of the foot-ankle-leg complex. A performance model based on pressure-washed trabecular systems and the interface of the different segments making up the articular foot skeleton is presented and discussed. The biomechanical influence of these segments on each other and on their appendicular companion bones is assessed, and an explanation of their function is provided. PMID- 10740995 TI - Effect of physical therapy on limited joint mobility in the diabetic foot. A pilot study. AB - Eleven patients with limited joint mobility and neuropathy were enrolled in a physical therapy program of passive joint mobilization at a rate of two sessions per week. Treatment resulted in a significant improvement in joint mobility after 10 sessions. Further improvement after 20 sessions did not reach the level of statistical significance, although near-normal joint mobility was attained. After completion of therapy, there was a progressive deterioration in joint mobility. No serious adverse effects were noted during treatment. This study provides some evidence that use of physical therapy may result in significant, although temporary, improvement in the mobility of the ankle and foot joints in diabetic patients with limited joint mobility and neuropathy. As limited joint mobility has been associated with the development of abnormally high pressures under the feet, which in turn may contribute to plantar ulceration in the susceptible neuropathic foot, the results indicate that physical therapy may be useful in the prevention of plantar ulceration in diabetic patients with limited joint mobility and neuropathy, although this must be verified by additional research. PMID- 10740996 TI - The evolution of the study of the mechanics of running. Relationship to injury. AB - The mechanics of running have been studied for hundreds of years. With the increase in interest in running as a recreational activity in recent years, running-related injuries have become common. Hundreds of studies on the mechanics of running have been conducted over the past few decades. However, the relationship between the mechanics of running and injury is still not well understood. This article describes the evolution of the study of the mechanics of running, with a focus on their relationship to injury. A brief historical review is presented, followed by a summary of the era of descriptive research and, finally, an overview of the more recent generation of hypothesis-driven research. The article concludes with suggestions for future research that may lead to greater insight into the relationship between lower-extremity biomechanics and injury. PMID- 10740997 TI - Efficacy of foot orthoses. What does the literature tell us? AB - This paper reviews the literature relating to foot orthoses, in particular foot orthoses that attempt to alter biomechanical function. Whilst few well-controlled studies have been performed, the findings from the available literature are generally positive. The authors provide an overview of this literature and then summarise the findings. The literature is categorised into six research outcome areas: (i) patient satisfaction, (ii) pain and deformity, (iii) plantar pressures, (iv) position and motion, (v) muscle activity, and (vi) oxygen consumption. In addition, the difficulties associated with researching foot orthoses are discussed. PMID- 10740998 TI - [Is MBSL-level ward needed for the treatment of viral hemorrhagic diseases and pest?]. AB - The recently revised Japanese Law on Infectious Diseases designates pest, Lassa, Marburg, Ebola and Crimean-Congo hemorrhagic diseases should be treated in an MBSL-level ward and that it should be constructed in each prefecture. However, pest can be treated with several antibiotics easily in an ordinary infectious disease ward. Lassa, Marburg and Ebola virus diseases are endemic in tropical Africa and only Lassa fever was imported into Japan in 1987. The probability of its importation to each prefecture is calculated on an assumption that a Lassa fever patient may be imported into Japan once in 10 years. Its incidence was calculated in comparison with the incidence of imported malaria from the African continent. Its probability P is calculated as follows. Corrected number of imported malaria patients from the African continent per year for each prefecture CN is divided by 445. 445 is the number of imported malaria patients from the African continent in ten years. Finally 445/CN is the number of years needed for each prefecture to import one case of Lassa fever. The results indicate that it takes 37 years for Metropolitan Tokyo where the largest number of malaria patients are imported annually. Other prefectures need more than 100 to 10,000 years, with an average of 1,017 years, for importation of one patient of Lassa fever. It is concluded that construction of an MBSL-level ward in each prefecture is unnecessary. The reports that the above mentioned viral hemorrhagic diseases can be treated safely in the ordinary infectious disease ward should be carefully reviewed. PMID- 10740999 TI - [Epidemiological study on Staphylococcus epidermidis isolated from bloodstream and blood vessel catheter]. AB - An epidemiological study on 35 strains of Staphylococcus epidermidis was conducted in Juntendo University Hospital between 1994 and 1996. The strains were isolated from blood and blood vessel catheters. Three epidemiological markers; PFGE type (pulsed-field gel electrophoresis using SmaI), biotype by STAPHYOGRAM and antibiotype (antibiotic resistant pattern) were used. There were 12 types in PFGE type, 6 types in biotype and 7 types in antibiotype. 1. The predominant types were PFGE type A (57.1%), biotype 1 (62.9%), and antibiotype I (resistant for oxacillin, ampicillin and gentamicin; 34.3%) in Juntendo University Hospital. 2. The strains with antibiotic V-VII (resistant for over 6 antibiotics) showed only PFGE type A and B. All strains with PFGE type B showed biotype 4-6 (negative nitrate reduction strain). 3. The strains having PFGE type A and B were isolated from various patient wards. The strains showing PFGE type A and antibiotype I were isolated from the pediatric ward. 4. There was no strain with PFGE type C or D in 1996. 5. Three patients in whom S. epidermidis was frequently isolated for a few months had the same types of PFGE type, biotype as well as antibiotype. PMID- 10741000 TI - [Epidemiological analysis of Shiga toxin-producing Escherichia coli O157 isolates from familial infection]. AB - A total of 201 Shiga toxin-producing Escherichia coli (STEC) O157:H7 isolates from 22 epidemiologically unrelated familial infections in Osaka were analyzed by various epidemiological markers, such as Shiga toxin (STx) typing, antimicrobial resistant patterns, colicine typing, plasmid profiles and pulsed-field gel electrophoresis (PFGE) typing. There were two cases where different type strains were detected in a family (family No. 21 and 22). In the family No. 21, three different strains were isolated from a 5-year-old male infant; one identical with that from his mother, and the others different in 4 markers except STx type. In the family No. 22, two kinds of strain were detected in a 48-year-old father; one identical with those from other members of the family, and the other different in STx, plasmid profile and PFGE types. These facts showed the possibility of a simultaneous double infection from the common sources of infectious factors or a successive reinfection with different types of the agents. Identical marker strains were detected from 8 out of 12 familial infection cases from July to September. Although infectious sources of these cases are not yet clearly identified, these results of epidemiological markers analysis indicate a probable circulation of the common contaminated foodstuffs. A combined use of phenotypic and genotypic tests were shown to be useful for the epidemiological analysis. Further, it seemed necessary to examine epidemiological markers of more than one strain in familial infection or identical facilities generation cases. And also a collective analysis of the relating factors such as biological markers of the causative agents, the list of eaten foodstuffs, and successive outbreaks of the patients was thought most important. PMID- 10741001 TI - [A SHV-derived extended-spectrum beta-lactamase (SHV-12) produced by an Escherichia coli recovered from wound abscess in post operative case with rectal carcinoma]. AB - A 62-year-old woman admitted for rectal carcinoma suffered from a post-operative bacterial infection. Oxy-imino-beta-lactams including cefotiam (CTM) and cefozopran (CZOP) were prescribed for this case, but the patient developed a wound abscess followed by peritonitis. She recovered from the bacterial infection after drainage and recurrent washing of the abscess. An ephemeral aggravation of infectious signs was observed just after creation of an artificial anus, and CZOP was again administered, and no evident bacterial infection occurred. The patient recovered, then was followed as an outpatient to date. A CAZ-resistant (MIC, > 16 micrograms/ml) E. coli was recovered from pus of her wound abscess. Since the CAZ resistance decreased (MIC, 64 micrograms/ml-->0.13 microgram/ml) by the presence of clavulanate (CVA) in this isolate, this strain was speculated to be an extended spectrum beta-lactamase (ESBL) producer at an early stage of infection. A similar strain was also isolated from the feces. Therefore, we immediately took measures to block the nosocomial spread of this microorganism, and we succeeded in preventing a nosocomial outbreak of this strain. It was later confirmed by PCR analysis and DNA sequencing analysis that this CAZ-resistant E. coli strain produces an ESBL (SHV-5-2a = SHV-12). This is the first report of a case of infection with SHV-derived ESBL producing E. coli strain in Japan. We are concerned that further dissemination of this kind of microorganism might occur in the near future also in Japan, as it has been widely observed in European countries and the US. We believe that it will be very important to distinguish the type of beta-lactamases for rigorous bacterial infection control with the prudent use of antibiotics. In other words, we in Japan must recall that various gram-negative bacterial species that produce TEM-, SHV-derived ESBLs, Toho-1, AmpC, or IMP-1 are already widespread. Thus, we should take this fact into consideration when we do antibiotic susceptibility tentings and interpretation of the results for promotion of accurate chemotherapy. PMID- 10741002 TI - [The bacteriocidal effect of the electrolysed functioning water against Helicobacter pylori]. AB - The bacteriocidal effect of electrolysed functioning water against Helicobacter pylori was examined by both the culture method (viable count on agar plate) and the bacterial ATP content analysis (biolumminescence assay). The culture method revealed the water's reduced bacteriocidal effect on organic substances such as horse serum and bovine serum albumin. The bacterial ATP content determined by the treatment with lysis solution correlated with the bacterial cell count. The ATP of bacteria treated with electrolysed functioning water, benzalkonium chloride, and chlorhexidine gluconate was still present after contact with these solutions for 30 minutes, while the bacterial growth was completely inhibited by the culture method. Therefore, it was suggested that the bacterial ATP was derived from the coccoid form which was observed morphologically due to the action of these solutions. These results demonstrate that these solutions are not effective bacteriocidally against the coccoid form. Consequently, when we utilize functioning water in the field, we should keep in mind its nonbacteriocidal effect against the coccoid form, as well as its reduced effect under the condition of the contamination by organic substances. PMID- 10741003 TI - [A two-year follow-up of zidovudine plus lamivudine combined with indinavir in antivival naive HIV-infected patients]. AB - Efficacy of a 3-drug combination (Zidovudine (AZT) + Lamivudine (3TC) + Indinavir (IDV)) has been evaluated in 17 anti-viral naive patients with HIV infection for 24 months. Our genotypic resistance assay was able to analyze more than 80% of the patients whose viral load (VL) was over 3,000 copies/ml. This therapy was continued in 11 patients (65%) for 24 months. Among them, VL was undetectable (VL < 400 copies/ml) in patients at 24 month (47% by intent-to-treat, 72% by on treatment). Of the 11 patients, a 3TC resistance-related mutantion was detected in only one case. The therapy was discontinued in 6 cases. Main reasons of the discontinuation were side effects. However, if the therapy was switched to other combinations when VL was undetectable, VL remained undetectable in 5 cases at 24 month. PMID- 10741004 TI - [The prevalence of virulence-related genes, eaeA, aggR and astA, of localized and aggregative-adherent Escherichia coli (EPEC and EAggEC) in healthy children and age-matched patients with diarrhea]. AB - The prevalence of virulence-related genes of localized- and aggregated-adherent Escherichia coli (EPEC and EAggEC), such as eaeA, aggR and astA was compared between E. coli isolated from 0 to 5 year old children with and without diarrhea in Saga Prefecture. In the case of eaeA, 233 cases in Aichi Prefecture were included. The subjects were 74 diarrheal patients from which no diarrheagenic bacteria were detected besides E. coli. The control subjects were 304 nursery school children without diarrhea, and E. coli was isolated from 278 children in which 105 strains were of 0-serotype. EaeA-positive E. coli was isolated from nine (12.2%) Saga cases, 19 (8.2%) Aichi cases and 6 (5.7%) control subjects; aggR-positive E. coli was isolated from 10 (13.5%) cases and 6 (5.7%) control subjects and astA-positive E. coli from 10 (13.5%) cases and 14 (13.3%) control subjects. No significant difference (p > 0.05) was observed in the prevalence of eaeA, aggR and astA between healthy and diarrheal children, even in age-matched and 0-serotypable E. coli limited comparisons. The pathogenicity of EPEC and EAggEC should be investigated, considering other known or unidentified factors. PMID- 10741005 TI - Salmonella paratyphi A is more genetically homogeneous than Salmonella typhi, as indicated by pulsed-field gel electrophoresis. AB - We analyzed 18 Salmonella Paratyphi A and 12 Salmonella Typhi isolates from domestic and imported cases in Aichi, Japan, using pulsed-field gel electrophoresis. Paratyphoid fever cases have increased and outbreaks of Salmonella Paratyphi A occasionally occur in Japan, but S. Paratyphi A has not been extensively analyzed. Our study suggests significant genetic homogeneity among Salmonella Paratyphi A belonging to different phage types, which is in contrast to the genetic heterogeneity of Salmonella Typhi. These results suggest that a limited number of clones are responsible for paratyphoid fever. PMID- 10741006 TI - [Antibody levels for diphtheria, tetanus and pertussis in young adult females immunized with whole cell pertussis-diphtheria-tetanus toxoid vaccine in infancy]. AB - Antibody levels for diphtheria, tetanus and pertussis in 84 young adult females were measured. They had been immunized with whole cell pertussis-diphtheria tetanus toxoid (DTwP) vaccine as a routine immunization in their infancy. Their history of DTwP vaccination were confirmed in their Maternal and Child Health Handbook, which includes their immunization record. Among the 84 cases, 4 cases (4.7%) had been immunized with the first dose of DTwP, 5 cases (6.0%) with the second dose, 23 cases (27.4%) with the third dose and 52 cases (61.9%) with the fourth dose. Of the 84 cases, 89.3% had received DTwP vaccine more than the third dose. In the 15-19 years after the last DTwP vaccination, the antibody positive rate for diphtheria and tetanus (> or = 0.01 IU/ml) were 86.9% and 94.0%, respectively. On the other hand, antibody positive rate for anti-pertussis toxin (anti-PT) and anti-filamentous hemaggulutinin (anti-FHA) (> or = 10 EU/ml) were 35.7% and 55.9%, respectively. The positive rate for pertussis compared with those for diphtheria and tetanus were lower. These findings suggested that DTwP vaccination in infancy does not provide sufficient immunity for young adults against pertussis, but DTwP vaccination provides adequate immunity against diphtheria and tetanus. PMID- 10741007 TI - [Isolation of Aeromonas species from patients with sporadic diarrhea and characterization of Aeromonas hydrophila isolates]. AB - A total of 16 strains of Aeromonas species were isolated from feces of 348 patients with sporadic diarrhea in western Kanagawa, Japan from 1996 to 1998. Of the 16 isolates, 7 were Aeromonas hydrophila, 1 was A. sobria and 8 were A. caviae. The strains of A. hydrophila were examined for hemolytic activities, hemolysin gene types and O-serogroups. Although all 7 strains of A. hydrophila showed hemolytic activities on sheep blood agar, in the test for hemolytic activities in culture supernatant, only 1 of the these strains showed no hemolytic activity against sheep erythrocytes. From the results of PCR assay, the tested strains of A. hydrophila were grouped into 2 hemolysin gene types of [ahh1 + ahh3 + aerA] (n = 6) and [ahh1 + aerA] (n = 1) both of which are recognized to be enteropathogenic. Five of the 7 strains of A. hydrophila belonged to serogroup O11. These results suggest that 7 strains of A. hydrophila isolates are recognized to be enteropathogenic strains and serogroup O11 is the major O serogroup of enteropathogenic A. hydrophila in humans. PMID- 10741008 TI - [Japanese spotted fever complicated by acute respiratory failure]. AB - A 66-year-old male was admitted to our hospital, presenting a high fever and generalized erythema on June 9, 1999. Physical examination revealed many eschars on his legs. Laboratory examinations were as follows: platelet counts, 5.5 x 10(4)/microliter: FDP, 25 micrograms/ml: TAT, 70.9 ng/ml: GOT, 177 IU/l, GPT, 174 IU/l: CRP, 32.3 mg/dl. Based on these findings, he was diagnosed as having rickettsiosis with DIC, and minocycline (200 mg/day) and heparin were started immediately, but had no clinical effect for 3 days. Blood gas analysis showed severe hypoxia and the chest CT scan revealed increased CT value in all lung fields with reticular shadows in the lower fields and pleural effusion, suggested interstitial pneumonia. Methyl-prednisolone pulse therapy was started on June 12, after which he completely recovered. Anti-Rikettia japonica IgM antibody was found to be x8,192 by immunofluorescent test, establishing the diagnosis of Japanese spotted fever. Acute respiratory failure with interstitial pneumonia shadows should be emphasized as a complication of severe rickettsiosis. PMID- 10741009 TI - Detection of circulating tumor cells in venous blood of nasopharyngeal carcinoma patients by nested reverse transcriptase-polymerase chain reaction. AB - Nasopharyngeal carcinoma (NPC) is a radiosensitive tumor. Because of recent advances in radiation oncology, distant metastasis has become the predominant failure site after adequate radiotherapy. The purpose of this study is to establish a nested reverse transcriptase-polymerase chain reaction (RT-PCR) system and to evaluate the potential of cytokeratin 19 (CK-19) mRNA as a target for detecting micrometastasis in the blood of NPC patients. Venous blood samples from 40 patients with biopsy-proven NPC (25 previously untreated and 15 after radiotherapy) and 20 healthy volunteers were tested. We divided the 40 patients into 4 groups: cured, early stage, advanced stage, and metastasized, according to results of clinical staging work-up. Under our nested RT-PCR experimental conditions, 2 of 8 early stage patients (25.0%), 6 of 15 advanced stage patients (40%), and 6 of 8 patients with distant metastasis (75%) had CK-19 positive cells in peripheral blood (P = 0.11). No CK-19 positive cells were detected in 9 "cured" patients and 20 healthy volunteers. Our data indicated that the positive detection rate for CK-19 mRNA in peripheral blood increased as the clinical stage of disease increased, but the difference did not reach statistical significance. Longer follow-up is needed to assess the significance of CK-19 mRNA in blood, as well as its relation to subsequent metastasis and prognosis of NPC. PMID- 10741010 TI - Mutation analysis of PTEN/MMAC 1 in sporadic thyroid tumors. AB - Recently, a putative tumor suppressor gene, PTEN/MMAC1, has been identified at chromosome 10q23.3. This gene encodes a 403 amino acid dual specificity phosphatase containing a region of homology to tensin and auxillin. Somatic mutations of the PTEN/MMAC1 gene have been found in a number of cancer cell lines and primary cancers. Cowden disease, an autosomal dominant harmartoma syndrome associated with thyroid and breast tumors, has been found to be associated with mutations of PTEN/MMAC1 gene. To evaluate the role of the PTEN/MMAC1 gene in sporadic thyroid tumors, we studied 17 sporadic thyroid tumors, of which 12 were papillary thyroid carcinomas, 1 was follicular thyroid carcinoma, 1 was medullary thyroid carcinoma and 3 were thyroid adenomas. Direct sequencing of PCR-amplified products was performed for all nine exons of PTEN/MMAC1. No mutations of PTEN/MMAC1 gene were observed in any of the sporadic thyroid tumors. Our results indicate that the PTEN/MMAC1 gene may not play a major role in sporadic thyroid tumors. PMID- 10741011 TI - Increase of leptin levels following exogenous administration of estrogen in women with normal menstruation. AB - To investigate the acute effect of exogenous estrogen on the changes of leptin production, the time sequence of changes in plasma leptin concentration were determined following i.v. administration of premarin, a conjugated estrogen, during the follicular phase (days 5-9) in women (n = 12) with normal menstruation. The mean circulating estradiol level abruptly (P < 0.0001) increased from 4 h to a level of 72-fold from baseline and was still significantly (P < 0.05) elevated at 72 h after injection of conjugated estrogen. The mean leptin level showed a significant (P < 0.05) gradual increase from 24 h (10.8 +/- 1.0 ng/mL) continuing on up to a peak at 32 h (11.6 +/- 0.9 ng/mL); significantly (P < 0.05) sustained to 48 h (10.7 +/- 0.7 ng/mL) and then slowly decreased at 56 h after injection of premarin, as compared with the preinjection level (7.8 +/- 0.7 ng/mL). However, there were no changes of leptin levels in women (n = 10) who received normal saline injection. These data clearly suggest that a higher level of estrogen could stimulate the increase of plasma leptin concentration during the follicular phase of normal cyclic women. This result provides evidence that supraphysiological level of estrogen may act on the adipocyte to modulate leptin production through the endocrine mechanism. PMID- 10741012 TI - Prolonged morphine treatment relieves thermal hyperalgesia in rats with sciatic nerve constriction injury. AB - We examined the effects of chronic morphine treatment with regular intermittent administration in a modified chronic constriction injury (CCI) model of the rat sciatic nerve originally introduced by Bennett and Xie. A painful neuropathy was induced over the left hind limb with sciatic nerve ligation, and sham surgery was done on the opposite side in male Sprague-Dawley rats. Paw-with-drawal latency (PWL) was obtained one day before surgery (pre-op baseline) and on the fourth day after surgery (post-op) to assure the development of thermal hyperalgesia. Morphine hydrochloride (5, 10, 15, and 20 mg/kg per day) was subcutaneously administered for 7 days to four experimental groups. The control group received normal saline rather than morphine under the same injection protocol. PWLs were evaluated on days 5, 7, 9, and 11 of the treatment. PWL decreased to 50-60% of the pre-op baseline or sham limb on the fourth day after surgery. Morphine's ability to reverse PWL appeared dose-related, and no tolerance developed during treatment with chronic intermittent administration. This may indicate that prolonged use of intermittently-administered morphine can be a feasible regimen for relief of neuropathic pain. PMID- 10741013 TI - Imaging of invasiveness of pituitary adenomas. AB - The purpose of our study was to examine the tumor size, imaging invasiveness of the pituitary macroadenomas (PMA) and to evaluate the directions of PMA spread. One hundred and thirty-five patients with PMA were examined with MRI and/or CT for pre-operative evaluation. We retrospectively reviewed the CT and MRI to identify tumor size, extension and to evaluate the directions of tumor spread. One hundred and seventeen patients (87%) had suprasellar extension with compression of optic apparatuses, twelve patients (9%) had extension of tumor upward to hypothalamus and third ventricle. Infrasellar extension via the floor of the sella and sphenoid sinus was found in thirty-eight patients (28%), and further downward extension to ethmoid sinus, nasopharynx and/or skull base was depicted in five patients (4%). Twenty-two patients (16%) had lateral invasion to the cavernous sinus and associated cranial nerves. Temporal and frontal extensions were depicted in seven patients (5%) and six patients (4%), respectively. Five patients (4%) had posterior subtentorial extension to posterior fossa. Histologically, only two patients showed microscopic invasive features. There was no correlation between histologic features and imaging invasiveness. The PMA had the potential of multi-directional extension. This experience indicated any type of pituitary adenoma could invade surrounding structures. Suprasellar invasion was the most common direction of pituitary adenoma spread, followed by infrasellar, lateral, anterior and posterior routes. PMID- 10741014 TI - The prevalence of betel chewing among the students of the different senior high schools in southern Taiwan. AB - A cross-sectional study was designed to determine the prevalence of betel chewing and the high-risk groups among the students of the first and second grades in five types of senior high schools in southern Taiwan. The results reveal that more male than female students were betel chewers: the prevalence rate of current chewers is from 0.96% to 16.15% among male students compared with that of 0% to 5.58% among female students. The students in the vocational schools was found have the higher prevalence rate of betel chewing. The students in the agricultural schools are the highest-risk group for betel chewing. The highest odds were found in the agricultural school students when they were compared with the general school students. It is 31.74 for the current chewers and 5.93 for the ex-chewers. PMID- 10741015 TI - A study of the professional commitment changes from nursing students to registered nurses. AB - The purposes of this longitudinal study were to understand the professional commitment changes from nursing students to registered nurses. A total of 890 subjects completed the questionnaires of the nursing professional commitment and personality test before their graduation, and nursing professional commitment one year after their graduation, respectively. The data were analyzed by mean, correlation, t-test and repeated measures ANOVA. The results showed that: (1) the nurses exhibited a medium-high degree of professional commitment--the average score was 2.99 before graduation and 2.85 after graduation; (2) there was a highly significant correlation between professional commitments for nursing students and registered nurses; (3) The scores of overall commitment and four factors of professional commitment decreased from nursing students to registered nurses significantly; (4) personality traits were positively correlated with professional commitment for nursing students and registered nurses; and (5) there was no significant relationship between nurses' work backgrounds and the changes of professional commitment from nursing students to registered nurses. Implications for nursing education and for hospital management are suggested. PMID- 10741016 TI - A low temperature plastic anterior encased ankle foot orthosis: construction and preliminary clinical results. AB - There are two basic plastic AFO designs, either posterior or anterior design, used to resolve spastic ankle and foot problems. The alternative designs of plastic AFOs, of either anterior or posterior design, may include modification with different shapes and materials. This paper describes a new low temperature plastic anterior AFO design, named as anterior encased ankle foot orthosis (AEAFO), which is useful to correct typical hemiplegic gait problems. It can be easily and quickly fabricated, as well as readjusted in OT clinics. The AEAFO has demonstrated an excellent effect on controlling foot inversion and ankle plantar flexion that are commonly encountered by stroke and head injury patients during ambulation. The preliminary effects, strength and durability of AEAFO in clinical applications are most satisfactory. PMID- 10741017 TI - Glandular odontogenic cyst: a case report. AB - Glandular odontogenic cyst (GOC) is an extremely rare lesion occurring in the jawbones. Sialo-odontogenic cyst was first described as a multicystic lesion resembling a botryoid odontogenic cyst (BOC) or a central mucoepidermoid carcinoma by Padayachee in 1987. In 1988, Gardner used the term "glandular odontogenic cyst" and considered it as a histologic variant of BOC. Most authors agreed that GOC was odontogenic because of the concurrent ball-like epithelial structure, ameloblastoma, squamous odontogenic tumor-like proliferation in its wall, or hyaline bodies in the epithelium lining. Recently, immunohistochemical studies of the cytokeratin profiles have also supported this concept. Its aggressive behavior and the recurrent tendency make it important. A new case of GOC in a 59-year-old male presented as a multilocular radiolucency in the anterior region of the mandible, invading the marrow space by epithelial islands is described with other clinicopathologic features and the literature is briefly discussed. PMID- 10741018 TI - [Family is the most important resource within family therapy. Fault-finding and putting a burden on the relatives should be counteracted]. PMID- 10741019 TI - [Reduced endothelial function is hardly a cause of primary Raynaud's phenomenon. Alternative mechanisms should be studied to clarify the causes]. PMID- 10741020 TI - [Endometriosis--an inflammatory disease in women. Pain and reduced fertility are serious symptoms]. AB - Endometriosis is a common disease with serious consequences for the patient and for society. Until now there has been no reliable method for diagnosing endometriosis besides direct visualization, e.g. utilizing laparoscopy. IVF offers the most successful treatment of endometriosis-related infertility, and a patient should be offered that treatment after one year of infertility. Waiting lists are often rather long, and other treatment modalities might be attempted while waiting for IVF. Pharmacological treatment does not increase the chances of pregnancy, while surgical extirpation of mild to moderate endometriosis might well result in a pregnancy earlier than anticipated. PMID- 10741021 TI - [Current aspects on the treatment of endometriosis]. AB - The aim of pharmacological treatment of endometriosis is to reduce estrogen levels, which may be achieved using gestagens or GnRH-agonists. The effects of the different hormones are mainly the same, while side effects differ. If the GnRH-agonist dose is modified, or if a low dose of estrogen and/or gestagen is given in addition, the hypo-estrogenic side effects of GnRH-agonists can be reduced. Surgical treatment can often be performed in conjunction with diagnostic laparoscopy. Supplementary hormonal treatment may postpone recurrence. Because endometriosis is in many women a chronically recurring disorder, continuity in the doctor-patient relationship is essential. PMID- 10741022 TI - [Unified methods for vaginal cytological diagnosis are required. Accuracy of the ASCUS respective CIN I compared]. AB - A study is focused on the evaluation and comparison of the Bethesda ASCUS diagnosis (Atypical Squamous Cells of Undetermined Significance) with the CIN I classification (cervical intraepithelial neoplasia). From 1996 to 1999 2070 cases were reported as being ASCUS, which represented 2.5 per cent of 81,518 gynecological cases diagnosed during the same period. In 479 cases (23 per cent) a subsequent biopsy and/or endocervical curettage was performed. The control group was 740 (24 procent) of 3113 cases with CIN I diagnosis. Out of these two groups only those cases were selected that had undergone biopsy after either ASCUS diagnosis (135 cases) or CIN I diagnosis (740 cases). Out of the 135 ASCUS cases 25 (19 per cent) showed condylomata, 39 (29 per cent) cervical intraepithelial neoplasia (CIN I) and 20 (14 per cent) CIN II-III. No case of carcinoma was detected after histological examination. The control group of 740 cases included 102 (14 per cent) with condylomata, 208 (28 per cent) with CIN I and 118 (16 per cent) with CIN II-III. Among patients with an ASCUS diagnosis after cytology and subsequent cervical biopsy 43 per cent had cervical intraepithelial neoplasia, either CIN I or CIN II-III, as against 44 per cent in the control group. These findings indicate that ASCUS cytologically defines a group of patients with or at risk for developing of a squamous intraepithelial lesion. PMID- 10741023 TI - [Gynecology in the new millenium. Increased responsibility of gynecologists for women's health]. PMID- 10741024 TI - [Breast reduction surgery are medically justified. Follow-up of 49 cases shows permanent pain reduction and increased quality of life]. PMID- 10741025 TI - [Adjuvant therapy of breast cancer in the USA]. PMID- 10741026 TI - [The clinical impact of gestational diabetes mellitus]. AB - In Denmark, gestational diabetes mellitus (GDM) develops in about 2% of all pregnant women. The discussion of GDM is complicated by lack of consensus regarding screening methods, diagnosis and treatment. Observational studies indicate that untreated GDM is associated with an increased risk of maternal and perinatal morbidity, and that the offspring of GDM mothers tend to be at increased risk of developing diabetes and adiposity as a result of an abnormal intrauterine environment. Several follow-up studies have shown that women with previous GDM run a considerable risk of developing diabetes (especially type 2 diabetes) later in life. Intervention strategies for this high risk group are suggested. PMID- 10741027 TI - [Foreign body can be extracted by radiologic intervention technique. A case report with a successful extraction of a pivot tooth from peripheral bronchi]. PMID- 10741028 TI - [The debate on mammography--the National Board of Health and Welfare is answering Sjonell and Stahle]. PMID- 10741029 TI - [The debate on MedAnalys: the laboratory provided analyses for other than clinical chemistry]. PMID- 10741030 TI - [Length of treatment in deep venous thrombosis--some comments]. PMID- 10741031 TI - [Reply: effects and side-effects of the length of therapy]. PMID- 10741032 TI - [Arm edema in cancer therapy: the number of examined lymph nodes a significant factor]. PMID- 10741033 TI - [A reply: the aim was to survey an underestimated problem among a high-risk group]. PMID- 10741034 TI - [Prescription of Viagra: the Medical Products Agency does recommend]. PMID- 10741035 TI - [Future challenge--both passive and active patients]. PMID- 10741036 TI - [Paternalistic views on the physician-patient relation and sick-leave certificates]. PMID- 10741037 TI - [Dr Knock--a satire which has become a reality]. PMID- 10741038 TI - [Question from an anonymous general practitioner: When a patient takes his life- who is to share the responsibility with me?]. PMID- 10741039 TI - [The homeless have right to health and social care. Special outpatient clinics are badly needed]. PMID- 10741040 TI - [Reduced risk of cardiovascular disease with moderate alcohol drinking]. PMID- 10741041 TI - [Moderate alcohol drinking protects against heart disease]. PMID- 10741042 TI - [Multimodal imaging in pre-surgical evaluation of epilepsy patients]. AB - In the pre-surgical evaluation of epilepsy patients, different functional investigations can be combined and co-registered to computerised image processing methods to enhance their diagnostic value. A method for subtraction of interictal from ictal SPECT is used, and the result is co-registered to MR, demonstrating the change in blood flow during seizure in the patient's MR-image. The level of significance is calculated and a statistical map is produced. Recent methods for EEG source localization use the MR-image to create a model of the head, producing more accurate solutions. Cases are demonstrated illustrating results concordant with these independent methods. PMID- 10741043 TI - [Fertility and contraception in women older than 40 years]. AB - Women in their later reproductive years constitute a large proportion of contraceptive users. Although increasing age is accompanied by a general decline in fecundity, each individual needs to know her own risk or possibility of becoming pregnant and when it is safe to stop using contraceptives. Women over 40 need special consideration since pregnancy and childbirth is sometimes associated with higher health risks both for mother and infant. Women over 40 are more likely than younger women to have medical problems, menstrual disorders and previous experience that may affect their choice and use of contraceptive methods. Recommendations for counselling, health screening and choice of contraceptive method in women in their later reproductive years are given. PMID- 10741044 TI - [Risk of early therapeutic failure after prostatectomy is relatively great. Tumor grade and preoperative serum level of PSA are significant prognostic variables]. AB - Prostate cancer is the most common malignancy among males in the West. Any reduction in mortality would require early detection of cases in which curative treatment is possible. From 1994 through 1998, 105 patients with clinically localised T1-T2 tumours were operated via radical prostatectomy at Karolinska sjukhuset in Stockholm. Three patients were lost to follow-up. We obtained clinical data from patients' records and used pathologist reviews to characterise tumours regarding grade and histopathological stage. We used PSA in serum as a surrogate endpoint, with a value above 0.6 ng/ml designated as treatment failure. Outcome was examined with respect to tumour grade, histopathological stage and pre-operative PSA level. Altogether 22% of patients showed PSA failure during follow-up. No mortality due to prostate cancer was recorded. We found that both tumour grade and pre-operative serum PSA level correlated well with treatment failure (P < 0.01). Our findings illustrate the diagnostic difficulties involved, since a relatively large percentage of patients were registered for treatment failure shortly after radical prostatectomy. PMID- 10741045 TI - [Obesity doesn't increase the risks of coronary surgery. Results from a study of 7,248 patients who underwent the surgery]. AB - Out of 7,248 patients who underwent coronary artery bypass surgery from 1970 1995, 17% of females and 13% of males were severely obese, defined as body mass index (BMI) 30 kg/m2. Risk of death within 30 days of operation was 2.1% in patients with BMI < 25 kg/m2, 2.1% if BMI was 25-30 kg/m2 and 1.3% in patients with BMI 30 kg/m2 (P = 0.20). The low risk in obese patients persisted after multivariate correction for year of surgery, age, sex, hypertension, diabetes, unstable angina and number of diseased vessels. The risk of reoperation for bleeding decreased (P < 0.001) whereas the risk of wound infection and sternal dehiscence increased (P = 0.09 and P = 0.05, respectively) with increasing BMI. We conclude that coronary artery bypass surgery can be performed safely in obese patients. PMID- 10741046 TI - [Prognosis is often poor in chest pain not interpreted as angina pectoris. Simultaneous occurrence of cardiovascular risk factors increases the risk of premature death]. AB - BACKGROUND: Typical angina pectoris is easy to recognize, but coronary insufficiency may present with nonspecific chest discomfort. AIMS OF STUDY: We wanted to investigate long-term prognosis in men with different types of chest pain. METHODS: A random population sample comprising 5,773 men aged 51-57 years at baseline were followed for 16 years. RESULTS: Mortality due to coronary heart disease was 8.0% among men without chest pain, 19.5% (total mortality 44%) among those with non-specific chest pain, 24.8% (total mortality 45%) among those with typical angina and 48.5% among those with a history of myocardial infarction at baseline. CONCLUSION: Non-specific chest pain is associated with poor prognosis, and coronary risk factors have strong predictive value. PMID- 10741047 TI - [A referral for exercise? No shortage of life style programs in the databases, but the information can't be efficiently used in practice]. PMID- 10741048 TI - [Nils Forsberg, soldier and medical orderly in the French-German war, painted the suffering and death]. PMID- 10741049 TI - [Identity crisis of Swedish physicians? Use possibilities of the information society]. PMID- 10741050 TI - [Randomized trials should be interpreted carefully]. PMID- 10741051 TI - [Rh-negative women should receive prenatal prophylaxis]. PMID- 10741052 TI - [Time to put the Ps in health care! Patients, Professionalism, Personnel in focus]. PMID- 10741053 TI - [The Medical Society should abandon the HSAN!]. PMID- 10741054 TI - [The SBU is planning a prospective study on concussion]. PMID- 10741055 TI - [The plan for development was in reality a plan for cost savings]. PMID- 10741056 TI - [Strengthen the role of family and the child with severe epilepsy]. PMID- 10741057 TI - [Same requirements for bedside testing?]. PMID- 10741058 TI - [Prognosis of the thermal status of man in view of the impact of a variety of factors]. AB - Mathematic and statistic analysis helped to determine classes of human functional state characterized by variable degree of thermoregulatory and cardiovascular strain. The authors represented technology forecasting a class of human functional state under influence of combined factors that change heat status and overcharge cardiovascular system and technology forecasting reliable time tolerance interval for the complex of factors influencing human. PMID- 10741059 TI - [The immune system status among those working under the influence of biological, chemical and physical factors]. AB - The authors compared main immune parameters in workers contacting various occupational hazards. The studies revealed that actual hazards induced variable associations of disorders in cellular, humoral immunity, nonspecific protective factors, local immunity of saliva and allergic reactivity. PMID- 10741060 TI - [The influence of the optical irradiation of natural and artificial sources on humans]. AB - The authors studied parameters of human natural insolation in relationships with season and functioning of main body systems. The functioning rhythms appeared to depend on natural saturation with optic energy. Regulation of light energy emitted by artificial sources requires consideration of natural background insolation in various seasons. PMID- 10741061 TI - [The influence of transportation noise and vibration on population]. AB - Transport noise affects dwellers, changing functional state of central nervous and cardiovascular systems. Combination of noise and vibration induces changes that are more severe. PMID- 10741062 TI - [Physiological mechanisms fo the formation of the adaptation- compensatory process with the impact of local vibration accompanied by noise and dust]. AB - Based on studies and mathematic analysis of 72 parameters of body systems, the authors arranged correlation matrix and multiple regression models for 109 tunnelers and drillers. The parameters characterizing mobility of retardation and balance of nervous processes were proved to play a leading role among adaptation and compensation mechanisms of human response to local vibration, noise and dust. The authors justified criteria to reliably characterize individual sensitivity to local vibration. PMID- 10741063 TI - [Occupational aging (biological age modeling)]. AB - Based on workers' biologic age, the authors created a systemic model of general and occupational ablebodiedness and tested the model on selective determination of biologic age among workers of various Ukrainian regions. The results proved increase in occupational aging of the population. General and occupational ablebodiedness in males of the select was lower than that in the females. Occupational ablebodiedness of population, especially of males, is considerably decreased. The latter evidence is a function of economic circumstances: heavy, hazardous and dangerous work conditions hardly compensated by payments. PMID- 10741064 TI - [The effectiveness of antioxidants and adaptogenes in the increase of defense reaction of the organism in the course of influence from factors of occupational and ecological environment (review of literature)]. PMID- 10741065 TI - [The new hand ergometer in the evaluation of fatigue from physical labor]. AB - Experimental laboratory studies compared informativeness of new hand ergometer and that of traditional one in diagnosis of muscular fatigue. Ergometric method using the device suggested was proved to be more informative than dinanometry. PMID- 10741066 TI - [Psycho-emotional profile of personality among teenage girls living in the Volgograd industrial region]. AB - Social, economic and other factors appeared to form psychoemotional personal profile of teenager girls living in significant industrial region subjected to pollution with chemicals varying in jeopardy. The studies proved that ecologic factors may play an important role in formation of psychoemotional personal profile. All this could be a pathogenetic basis for endocrine disorders among teenagers and subsequent reproductive disturbances and birth of diseased children. PMID- 10741067 TI - [The formation of knowledge of electromagnetic ecology among high-school students]. AB - The article tackle the problem of ecologic education, training and development in electromagnetic safety. Electromagnetic ecology is associated with effects of electromagnetic fields, so these topics are expedient in comprehensive courses of physics, ecology, natural science, biology, geography. The authors necessitate specification of regulatory document, program, methods and means of giving such knowledge. PMID- 10741069 TI - [Unlicensed therapy in children]. PMID- 10741068 TI - [The aspects of intellectual work capacity among thermal electric power stations operators]. PMID- 10741070 TI - [Monoclonal antibodies in hematologic neoplasms]. PMID- 10741071 TI - [Motility disorders in the digestive tract. Physiology--physiopathology- therapy]. PMID- 10741072 TI - [Bad breath. Etiology, diagnosis and therapeutic problems]. PMID- 10741073 TI - [Which lipid class for which lipid disorder?]. PMID- 10741074 TI - Pyridostigmine bromide intake during the Persian Gulf War is not associated with postwar handgrip strength. AB - Many Persian Gulf War veterans took pyridostigmine bromide (PB) during the Persian Gulf War. Previous research suggests that PB intake and insecticide exposure may reduce muscular strength. During 1994 and 1995, we examined the relationships between self-reported PB intake, self-reported exposures, and handgrip strength among 527 Gulf War veterans (GWVs) and 969 nondeployed veterans of that era (NDVs). We found that 25.4% and 6.7% of the GWVs and NDVs, respectively, reported generalized musucle weakness (for 1 month or longer) since the Gulf War (July 1990). Many veterans also reported exposure to insecticide during the war. Dominant handgrip strength was measured three times with a hand held dynamometer in subjects standing with the elbow bent at a right angle. Multiple linear regression revealed that handgrip strength was negatively associated with age (p = 0.001) and female gender (p < 0.001). Handgrip strength was also found to be positively associated with height (p < 0.001), but it was not associated with PB intake (p = 0.558). Exposure to insecticides had no major effect on handgrip strength. These data suggest no association between PB intake and postwar handgrip strength. PMID- 10741075 TI - First psychotic episodes among Israeli youth during military service. AB - The military setting offers unique opportunities for the study of the epidemiology of first psychotic episodes among Israeli youth. The aim of the present study was to describe characteristics of first psychotic episodes among a cohort of Israeli soldiers. Ninety soldiers, 67 males and 23 females, who were hospitalized for a first psychotic episode were studied and compared with 90 soldiers hospitalized in psychiatric units during the same year who were not psychotic. The results include a high rate of schizophreniform psychoses and schizophrenia; no gender difference in the occurrence of psychoses; within average mean ratings on the preinduction psychometric intelligence test; no history of substance abuse; and a remarkable occurrence of psychiatric hospitalizations, including first psychotic episodes, during the stressful beginning of military service, supporting the hypothesis that psychotic symptoms are likely to occur in a stressful situation among vulnerable individuals. Follow up studies will indicate whether early detection and treatment may improve the outcome of the psychoses. PMID- 10741076 TI - The problems and characteristics of hepatic war trauma management in central Dalmatia during the 1991-1995 war in Croatia. AB - OBJECTIVE: War injuries to the liver treated during the 1991-1995 war in central Dalmatia, Croatia, were analyzed retrospectively. METHODS: There were 713 patients with abdominal injuries, 125 (17%) of whom were operated on for liver injuries. Mobile surgical teams operated on 91 patients (73%) in rear war hospitals (echelons II and III); only 34 patients (27%) were immediately transferred to and operated on at the Split University Hospital. RESULTS: After wounds to the small and large intestine, liver injuries were the third most common abdominal injuries. Morbidity was 63%, and reoperations were required in 20% of cases. Fourteen patients (11%) died. The highest rate of complications was observed in patients with postoperative inflammatory intra-abdominal collection, disseminated intravascular coagulation, adult respiratory distress syndrome, sepsis, or ileus. The highest mortality was found in those patients requiring a right hemihepatectomy. CONCLUSIONS: The best results were achieved in patients on whom, after hepatotomy, selective hemostasis with liver debridement was carried out. PMID- 10741077 TI - Chronic liver disease among U.S. military patients: the role of hepatitis C and G virus infection. AB - Thirty-nine patients with chronic liver disease who were being evaluated in a U.S. military treatment facility were tested for antibody to hepatitis C virus (anti-HCV) and for hepatitis G virus (HGV) RNA by reverse transcriptase polymerase chain reaction. Serum samples from 20 patients (51%) were positive for anti-HCV by immunoblot assay. HGV RNA was found in the sera of only two patients, both of whom were also positive for anti-HCV. HGV appears to have a limited role in causing chronic liver disease in this population of military patients, many of whom had traveled outside the United States. However, HCV infection was commonly associated with chronic hepatitis and cirrhosis, as in civilian patients. PMID- 10741078 TI - The vision for the Department of Defense's computer-based patient record. AB - To meet the Department of Defense's clinical information management mission for the next century, a vision for the computer-based patient record (CPR) was needed. This article describes the generation of that vision as well as the resulting definition, characteristics, and essential functions of the Department's CPR. PMID- 10741079 TI - Colorectal war injuries. AB - During the war in Croatia (1991-1995), 851 war casualties were treated by the surgical teams of Rijeka Clinical Hospital. Among them, 66 patients (7.8%) had colon and/or rectum injuries, usually combined with trauma of other abdominal organs, most often jejunum and ileum (24.2%). Regarding the site of injury, right and sigmoid colon were the most frequently traumatized areas (30.3% and 31.8%, respectively). All patients received surgical treatment within 3 hours from wounding. In 89% of patients, relieving colostomy was formed after the resection of the damaged part of the intestine; 11% of patients were treated with primary resection or sutures without colostomy. The mortality rate of 3% (2 patients) was caused by mutilating multiple organ abdominal trauma combined with massive brain injury. PMID- 10741080 TI - The Walter Reed Army Medical Center Nephrology Service website: a tool for military-wide provider and patient education. AB - OBJECTIVES: Military medical subspecialists consult with referring physicians, direct patient care and education, support isolated subspecialists, and maintain cohesive readiness plans. The World Wide Web appears promising in fulfilling these roles. METHODS: A website providing directed resources, the Walter Reed Army Medical Center Nephrology Service website (http:@www.wramc.amedd.army. mil/departments/medicine/nephro/NEPHROLOGY/index.htm!), is described in the context of its role in military medicine and online publishing. RESULTS: The Walter Reed Army Medical Center Nephrology Service website provides educational resources for both patients and physicians worldwide. It remains the only Army internal medicine website to offer online category I continuing medical education credits free to military physicians, and it also allows online graduate medical education. CONCLUSIONS: Military medical subspecialty programs can use the World Wide Web to provide consultation and education to distant and isolated patients and providers in a practical, feasible manner. This process can be expanded to education in operational medicine and other military-specific medical topics. PMID- 10741081 TI - Skin disease in military personnel. AB - Skin disease, disease of the musculoskeletal system, and respiratory infections are the most frequent reasons for military personnel to seek medical care. The Oslo Military Clinic serves all of the military personnel in Oslo and the surrounding region, including officers and civilian employees. From September 1996 to May 1997, 1,360 patients were diagnosed and treated by the author, and the data are included in the following study. Upper respiratory disease was the primary reason for seeking medical attention in 26% of the patients, 21% visited the clinic because of disease or pain in the musculoskeletal system, and 16% suffered from a skin disease. Apart from the low number of female patients, the patient population and the disease spectrum observed in the military clinic are very similar to those in a general medical practice. Among the 222 patients suffering from a cutaneous disease, eczema (42 patients), allergy (excluding dermatitis) (34 patients), acne vulgaris (23 patients), and sexually transmitted diseases (28 patients) were the most prevalent processes. Other less prevalent skin diseases were fungal infections, herpes simplex infection, nevi, common warts, and superficial bacterial skin infections. Skin diseases seen in one patient only included erysipelas, herpes zoster, dermatitis herpetiformis, and Ehlers-Danlos syndrome. Good clinical skills in dermatology are of paramount importance in military medicine, and if possible, the military should appoint a dermatologist to its medical team to rapidly diagnose and treat the large number of patients with skin disorders. PMID- 10741082 TI - Simplified use of mixed propofol and alfentanil for anesthesia in remote locations. AB - Total intravenous anesthesia (TIVA) is a useful technique in precarious situations in which anesthesia ventilators and medical gas can be difficult to obtain. The aim of the study is to compare TIVA technique using a simplified infusion scheme for propofol and alfentanil mixed together (45 ml of propofol 1% and 2,500 micrograms of alfentanil in a 50-ml syringe) with an inhalational anesthetic technique (isoflurane/N2O, sufentanil). Thirty-two American Society of Anesthesiologists physical status I patients undergoing orthopedic surgery were studied. Intubation conditions and hemodynamic responses to intubation were comparable in the two groups. Only patients receiving TIVA had responses to surgery. In the TIVA group, time to extubation was shorter (16 +/- 5 vs. 25 +/- 7 minutes) and postoperative requirement for morphine was lower (6.2% vs. 25%) than in the inhalation group (p < 0.05). TIVA using a mixture of propofol and alfentanil is a reliable technique of anesthesia in patients without multiple injuries. PMID- 10741083 TI - Behavioral treatment of insomnia: the Wilford Hall Insomnia Program. AB - Persistent insomnia is one of the most common patient complaints reported in primary care settings. This paper reviews the behavioral assessment and treatment approaches for persistent insomnia and reports the results of 42 consecutively treated patients in the Wilford Hall Insomnia Program. Patients were treated in a six-session, multiple-component behavioral group treatment program consisting of sleep restriction, stimulus control, sleep hygiene, relaxation training, and cognitive therapy. In addition, patients were seen for two individual treatment sessions in conjunction with the program and for two individual post-group treatment sessions to assess treatment progress. The results indicated that sleep onset latency was improved by 53%, wake after sleep onset was decreased by 40%, and sleep efficiency was improved by 22%. A detailed description of the behavioral treatment program for insomnia is provided as well as recommendations for practitioners working in primary care settings. PMID- 10741084 TI - Self-reported needlestick injuries in dental health care workers at Armed Forces Hospital Riyadh, Saudi Arabia. AB - The objective of the present study was to assess the incidence of sharps/needlestick injuries among dental health care workers (DHCWs) at Armed Forces Hospital Riyadh. A questionnaire was distributed among all dental staff, including dentists, hygienists, and dental surgery assistants. Results show that 65 individuals (58%) had sharps/needlestick injuries, and of these, more than half did not report these injuries to the appropriate department. At the time of injury, the majority of the DHCWs were vaccinated or immune, but a few of them were not vaccinated against hepatitis B virus. This study concludes that every DHCW should be immunized against hepatitis B virus to avoid cross-infection from sharps/needlestick injuries, which are quite common in a dental practice. The high frequency of these injuries could be reduced by simple interventions. PMID- 10741085 TI - The Army American College of Physicians Annual Scientific Meeting: attendee motivation and educational value. PMID- 10741086 TI - Understanding women who are violent in intimate relationships: implications for Army family advocacy. AB - Women who are violent in intimate relationships is a controversial and neglected subject in the area of spouse abuse in the civilian and military communities. Researchers report that women initiate more acts of violence than their male partners. This article provides a review of the literature, which identifies the high rates of violence by women against their male partners. In addition, this article discusses the context in which women offend and the motivations of women offenders. The implication for the Army Family Advocacy Program (FAP) is to enhance providers' clinical knowledge and increase community members' awareness so that FAP personnel can appropriately intervene with abusive couples. The goal of this author is to argue for broadening the scope of spouse abuse to include violence perpetrated by women. PMID- 10741087 TI - Military service and mental health in later life. AB - This study sought to determine if the self-assessed mental health of older, community-dwelling veterans differs from that of older nonveterans controlling for known demographic, socioeconomic, and clinical covariates of health and well being. Participants are the older male respondents (65+ years) to the Medical Expenditure Panel Survey (Round 1), conducted in 1996 (N = 1,068). In bivariable analyses using population weights, veterans were significantly less likely than nonveterans to report their mental health as fair to poor. However, when demographic, socioeconomic, and health-related characteristics were controlled, no statistically significant difference in self-assessed mental health was detected between veterans and nonveterans. Military service per se is not a risk factor for poorer self-appraised mental health in old age. Nor does military service confer protection from late-life deficits in mental health. Rather, mental health in later life is largely a function of an individual's health and socioeconomic status. PMID- 10741088 TI - Polish and American collaboration on zoonotic parasitic studies, 1960 to 1997. AB - The Marshall Plan of 1947 kindled interest in research in Europe. The U.S. Public Health Service encouraged the use of blocked national currencies to research disease problems. The parasitic diseases were epidemic/epizootic problems in Poland. The initial project was trichinellosis. The 10-year study emphasized the natural history, epidemiology, diagnosis, and therapy of trichinellosis in Poland. The wildlife source of trichinellosis was widespread. Clinical studies discounted the effectiveness of mebendazole but found steroids useful. Taenia saginata is common in Eastern Europe. T. saginata cystericosis in cattle is not easily diagnosed. Physical examination by meat inspectors missed 50% of the infected carcasses. Taenia solium is uncommon. Giardiasis is declining in Poland. The epidemiology of giardiasis in Poland is different from that in the United States, where water-borne infections are common. A study of toxoplasmosis revealed a low prevalence in women but a high prevalence in cats. No cases were identified in 4,311 newborns. PMID- 10741089 TI - Shipboard medical admissions during peacetime and combat support deployments. AB - Hospitalizations aboard aircraft carriers were examined to ascertain differences in illness type attributable to theater of operations and combat deployment status. Percentage distributions and lengths-of-stay statistics of major diagnostic categories are provided and compared between Vietnam combat support and peacetime modes of operations and between Western Pacific and Mediterranean theaters of operations. Respiratory disease proportions were found to be significantly higher for Vietnam combat support deployments than during subsequent peacetime deployments. For peacetime deployments, the Western Pacific deployments had higher percentages of infective, respiratory, and skin disorders, whereas the Mediterranean theater had a larger proportion of total admissions accounted for by accidents. PMID- 10741090 TI - Gluteal gunshot wounds. AB - Experience with advanced surgery for the treatment of civilian gunshot injuries supports the changes in approach and indications for the treatment of war gunshot injuries. Eight patients with gluteal gunshot wounds are presented with typical war injuries. They were wounded during 1992 and 1993 in the war in Croatia and Bosnia and Herzegovina. Surgical complications were experienced in two cases. Complications developed in cases of colonic and rectal injuries for which advanced surgery was performed in the treatment of the war wound. Poor conditions for advanced surgery during war can change the final result, making the outcome of the war wound worse. PMID- 10741091 TI - Bilateral peroneal nerve palsy induced by prolonged squatting. AB - External or internal pressures on peripheral nerves may result in compression neuropathies. Although compressive common peroneal nerve palsy is well known, to date very few cases with bilateral palsies have been reported. The clinical and electrophysiological manifestations of three patients with bilateral peroneal nerve palsies are reported, and their clinical outcomes are discussed. The first patient's transient bilateral palsy was corrected by conservative means. The second patient, with a more severe axonal lesion, did not improve within 3 months, and nearly complete recovery occurred after operative decompression. For the third patient, who had been suffering for a long time, no improvement could be hoped for. Prolonged squatting was the etiological factor in all three cases. Bilateral compression neuropathies of the peroneal nerve, like unilateral lesions, may recover spontaneously. Surgical intervention is recommended for patients with predominantly axonal lesions and for those who do not improve within 3 months. PMID- 10741092 TI - Respiratory intermediate intensive care units in Europe. PMID- 10741093 TI - Asthma in adolescence: a problem. PMID- 10741094 TI - Lymphocyte expression of human leukocyte antigen class II molecules in patients with chronic obstructive pulmonary disease. AB - Chronic obstructive pulmonary disease (COPD) is a multifactorial disorder, deriving from a combination of environmental and genetic factors. Polymorphisms of genes of the human major histocompatibility complex in COPD have been poorly studied in the past. In a preliminary approach, it was difficult to type human leukocyte antigen (HLA) at the protein level and it was hypothesized that there was a reduced surface density of HLA class II molecules. The aims of this study were to analyse, by cytofluorimetry, HLA class I and II molecule densities on peripheral mononuclear cells of COPD patients and to investigate whether there was a correlation with the polymorphisms of DQA and DQB promoter regions which are supposed to be important factors involved in surface expression of HLA-DQ molecules. The study investigated 27 male COPD patients admitted because of disease exacerbation and 49 healthy male controls. Quantitative analysis of fluorescence intensity of HLA class I (A, B, C) and class II (DR, DP, DQ) molecules was performed on blood mononuclear cells by cytoron cytofluorimetry. Polymorphisms of DQA and DQB promoters (QAP and QBP) were determined from the DNA (PCR-SSO). The surface densities of HLA class I and HLA-DQ molecules did not differ between the COPD patients and controls. HLA-DP molecule density seemed to be slightly, but not significantly lower in COPD, whereas surface HLA-DR molecules were significantly reduced (p < 0.005 vs controls). Frequencies of QAP alleles were not different between the COPD patients and controls, but the QBP 5.12 allele was significantly more frequent in COPD than in controls (chi 2 = 10.83, p = 0.0182, RR 5.5). In conclusion, individuals with exacerbated chronic obstructive pulmonary disease have reduced surface DR molecule expression and an increased frequency of the QBP 5.12 allele. The possible relationship between these two features and the possible role of cytokines in reducing human leukocyte antigen-DR expression in exacerbated chronic obstructive pulmonary disease is explored. PMID- 10741096 TI - Fibreoptic bronchoscopy in the diagnosis of lung cancer. AB - A total of 562 patients with lung cancer was evaluated by fibreoptic bronchoscopy (FOB) by three bronchoscopic diagnostic procedures: biopsy, bronchial brushing and bronchial washing. Endoscopically visible tumours (EV) were detected in 264, while 257 had endoscopically nonvisible tumours > or = 2 cm in diameter and FOB was done without fluoroscopy because of limited availability (ENV). Forty-one had small (< 2 cm), endoscopically nonvisible tumours with FOB performed under uniplanar fluoroscopy (ENV + F). The overall diagnostic yield rates of FOB were 98.1%, 61.5% and 58.5% for the EV, ENV and ENV + F cases, respectively. Reviewing the differential yield rates of the three diagnostic techniques and comparing them with the results of previous studies led to the following conclusions. (1) Combinations of biopsy with brushing and biopsy with washing can diagnose more than 95% of all fibreoptic bronchoscopy positive cases with endoscopically visible tumours. Performing either of these combinations may be more cost effective than doing all three techniques routinely. (2) For cases with endoscopically nonvisible tumours, performance of all three diagnostic techniques is recommended, especially when fibreoptic bronchoscopy is performed without fluoroscopic guidance, as washing and brushing seem to compensate for a lower yield of the biopsy. (3) For tumours < 2 cm in diameter, knowledge on the diagnostic efficacy of fibreoptic bronchoscopy was limited owing to the small size of previous studies. The yield of 58.5% for fibreoptic bronchoscopy in these patients with performance of all three diagnostic procedures was comparatively high. It could be further increased to 75.6% if supplemented by percutaneous needle biopsy when fibreoptic bronchoscopy turned out to be nondiagnostic. If available, the use of transbronchial needle aspiration may also increase the overall diagnostic yield of fibreoptic bronchoscopy in these cases. PMID- 10741095 TI - Comparison of different long-term asthma treatments in subjects with mild-to moderate asthma. AB - In order to compare the efficacy of different asthma treatment in subjects with mild-to-moderate asthma, three groups of 11 patients were treated with nedocromil sodium (NS), beclomethasone dipropionate (BDP) and beclomethasone dipropionate plus salmeterol (BDP + S) in an open, randomized study. Symptom score, peak expiratory flow (PEF) maximal amplitude, forced expiratory volume in one second (FEV1), and methacholine reactivity were measured at the baseline and at intervals of 3 months up to 12 months. After 3 months, symptoms reduced significantly in all treatment groups, while PEF variability improved in BDP and BDP + S groups; FEV1 and bronchial responsiveness to methacholine were significantly improved in comparison with baseline value in the BDP + S group only. No significant difference was observed after 6 and 12 months of treatment in PEF variability, FEV1 or bronchial hyperreactivity in the NS group compared with baseline values, while a significant difference was observed in symptom score. BDP group showed a significant improvement in FEV1 and bronchial reactivity to methacholine after 6 and 12 months of treatment. In the BDP + S group, the improvement in symptoms and pulmonary function persisted until the end of the study. In conclusion, the combination of beclomethasone dipropionate and salmeterol improved pulmonary function and bronchial reactivity earlier than beclomethasone dipropionate alone, while nedocromil sodium improved symptoms but not pulmonary function. Assuming that bronchial reactivity could be an indirect measurement of airway inflammation, overtreatment of asthma in relationship with the classification of asthma severity of the International Guidelines could improve both airway inflammation and the prognosis of airway obstruction. PMID- 10741097 TI - Varix of the inferior pulmonary vein: computed tomography and magnetic resonance angiography findings. AB - A 37-yr-old male presented with a 2-week history of intermittent episodes of mild haemoptysis. Computed tomography and magnetic resonance imaging revealed a varix of the inferior pulmonary vein. The diagnosis was confirmed using magnetic resonance angiography and conventional angiography. This condition is relatively rare, and few cases have previously been demonstrated by means of magnetic resonance imaging and angiography. PMID- 10741098 TI - Posterior mediastinal goiter. AB - A patient with chronic cough and recent dysphagia was found to have a retrotracheal mass extending into the mediastinum on chest radiography. A computed tomographic scan confirmed a retrotracheal posterosuperior mediastinal lesion which was believed to have a neurogenic origin. A thyroid 131I scan revealed no uptake of tracer in the chest and results of thyroid function tests were normal. A large retrotracheal colloidal nodular goiter was excised through a right thoracotomy. The diagnostic approach and the safety of surgical access by thoracotomy for thyroid lesions in this unusual site are discussed. PMID- 10741099 TI - Endobronchial granular cell tumour. AB - Granular cell tumour of the lung is a rare benign tumour. We report a case of endobronchial granular cell tumour which occurred in a 70-yr-old male with haemoptysis. Because of the size of the tumour (greater than 8 mm) and the associated pseudoepitheliomatous hyperplasia with marked atypia, surgical resection was performed. No neoplastic relapse has been observed in nearly 4 yrs of follow-up. PMID- 10741100 TI - The Italian AIPO study on tuberculosis treatment results, report 1997. National AIPO "Tuberculosis" Study Group. AB - In Italy no national data have been available on tuberculosis (TB) treatment results. In 1995 the AIPO (Italian Association of Hospital Pneumologists) TB Study Group, in collaboration with the Istituto Superiore di Sanita (technical branch of the Ministry of Health) started a prospective monitoring activity based on the recommendations of the World Health Organization and the International Union Against Tuberculosis and Lung Disease. The aim of this study was to analyse the case findings and treatment results during 1997. Data were collected from a representative network of TB units nation-wide, managing a significant proportion of all TB cases notified in Italy each year. A total of 715 TB cases were reported (56% males; 24% immigrants), of which 635 (89%) were new cases. Of these cases 493 (69%) were pulmonary, 187 (26%) extra-pulmonary cases and 35 (5%) both pulmonary and extrapulmonary. The main risk factors for TB were history of recent contact and diabetes among native Italians, human immunodeficiency virus seropositive status and a history of recent contact among immigrants. The majority of immigrants came from Africa and Central and South America, and stayed in Italy for more than 24 months before the diagnosis of TB was made. Thirty-six per cent of patients had a positive direct sputum smear examination for alcohol acid-fast bacilli and 27% were resistant to any drug (monoresistance to isoniazid: 2.4%; multi-drug resistance: 7.5%). In 97% of cases the duration of treatment was < 12 months. The overall success rate (cured plus treatment completed) was 78.1%. A significantly higher percentage of deaths, which was age related, was found in native Italians, while immigrants had a higher default rate. In conclusion, case finding and treatment results in Italy in 1997 are similar to those described in 1995 and 1996. PMID- 10741101 TI - Physiological changes during severe airflow obstruction in chronic obstructive pulmonary disease. AB - Chronic expiratory flow limitation and hyperinflation are the mechanical hallmarks of chronic obstructive pulmonary disease (COPD). Although carbon dioxide retention is dependent on the severity of airflow limitation, there is considerable variability in the relationships between arterial carbon dioxide tension (Pa,CO2) and forced expiratory volume in one second (FEV1) or total lung resistance (RL). In stable COPD patients with severe airflow obstruction, shallow breathing and inspiratory muscle weakness are the main factors associated with CO2 retention. In stable COPD patients, the diaphragm is less effective than in normal subjects and, with increasing airflow obstruction and hyperinflation, the contribution to the generation of ventilatory pressure of the ribcage inspiratory muscles increased. Abdominal muscles are recruited during expiration in severe COPD patients and the expiratory rise in gastric pressure is directly related to intrinsic positive end-expiratory (alveolar) pressure (PEEPi). During acute bronchoconstriction, COPD patients with severe airflow obstruction recruited the rib cage inspiratory muscles proportionally more than the diaphragm. The associated recruitment of abdominal muscles results in a reduction in abdominal volume at end-expiration and contributes to a significant extent to PEEPi. Dynamic hyperinflation can be overestimated during chronic and acute airway obstruction if abdominal muscle function is not evaluated. PMID- 10741102 TI - Requirements for appropriate evaluation of diagnostic tests in suspected pulmonary embolism. AB - In contrast to the development of new drugs, strict guidelines for the development of new diagnostic methods do not exist. A diagnostic test can be made available without proper evaluation of its clinical utility, which can lead to its premature introduction and inappropriate use. In this review suggestions are made regarding the criteria that should be met during the various phases of development of new diagnostic techniques. It is suggested that a new diagnostic test should only be implemented in routine clinical use after all phases of development have been properly performed with good results. Several diagnostic tests for pulmonary embolism (pulmonary angiography, ventilation-perfusion scintigraphy, D-dimer assays, and spiral computed tomography), and the studies evaluating them, are thereafter reviewed. It is concluded that present pulmonary angiography and ventilation-perfusion scintigraphy are the only properly evaluated diagnostic tests for pulmonary embolism. Although new developments, such as D-dimer assays and the spiral computed tomographic scan are certainly promising, further studies are needed to determine their real value and safety in the diagnostic work-up of patients suspected of pulmonary embolism. PMID- 10741103 TI - Systemic corticosteroids for chronic obstructive pulmonary disease: benefits and risks. AB - Systemic corticosteroids have become an increasingly common therapy for patients with stable and unstable chronic obstructive pulmonary disease (COPD), despite uncertain clinical efficacy and known adverse effects. A recently completed, large clinical trial has confirmed that systemic corticosteroids hasten recovery from exacerbations of COPD. Maximum benefits occur with only 2 weeks of therapy. The appropriate role of systemic corticosteroids in stable COPD has not been adequately defined. Prolonged therapy with systemic corticosteroids confers a large risk for developing osteoporotic fractures. Measures for reducing this risk are described. PMID- 10741104 TI - Treatment of small cell lung cancer. AB - Small cell lung cancer represents approximately 25% of all lung cancers. During the 1980s enormous efforts have been made to try to create a breakthrough in this peculiar disease but, despite the excellent results obtained in the early 1980s with multidrug chemotherapy, no significant improvements have been achieved in the 1990s. Overall response rates are in the range of 60-90% for patients with limited disease and 40-70% for those with extended disease, with pathological remissions in the range of 20-50%. Nevertheless, the majority of patients with small cell lung cancer continues to relapse and ultimately dies of the disease. New strategies under clinical evaluation are the multimodality approach, such as concurrent chemoradiation, and the use of dose-intensive regimes or even high dose chemotherapy supported by autologous haemopoietic rescue. In the near future other strategies will be possible, owing to the tremendous increase in the knowledge of biological properties of this disease. This article aims to summarize the research so far, the current strategies and the probable future of the treatment of small cell lung cancer. PMID- 10741105 TI - Respiratory high-dependency care units in Italy. PMID- 10741106 TI - Intensive- and high-dependency care medicine in Greece. PMID- 10741107 TI - Respiratory intensive care in Spain. PMID- 10741108 TI - Respiratory high dependency unit care in the UK. PMID- 10741109 TI - Respiratory high-dependency units in Germany. AB - In Germany, there is a well-established scene being engaged in non-invasive mechanical ventilation (NIMV), but only a minority of centres for home mechanical ventilation simultaneously run a respiratory high-dependency unit (RHDU). There are no standardized concepts, and RHDU do not have official government recognition. However, due to the increased usage of NIMV in both intensive care units and RHDU, and the cost pressure in the health system, the current atmosphere is open for a co-operative strategy and gives respiratory medicine a unique opportunity to acquire a new profile. Therefore, both nursing and medical staff must receive training in NIMV-associated strategies. PMID- 10741110 TI - Respiratory high dependency care units (RHDCU) in France. AB - The concept of intermediary intensive care units appeared in France with the progress of respiratory intensive care in the 1970s and an increment of the percentage of patients who were surviving an acute respiratory episode. It then became necessary to provide them with an optimal transition between the medical intensive care unit (ICU) and the home, thus explaining the emerging need for units of post-intensive care. At the present time, French health regulations recognize medical and surgical ICUs with a specific budget for equipment and nurses, and the specialized ICU which exists through a means share with the medical units (pulmonology, cardiology, etc.) inside which they are located. New regulations should make official the separation between medical and surgical intensive care units, intermediate (specialized) intensive care units and wards of reinforced follow-up, in a general perspective of improvement of the health network between the patient, home, and the different sectors of hospitalization (acute/chronic, public and private). PMID- 10741111 TI - [Endovascular neurointervention for cerebral aneurysm]. AB - Embolization would have an advantage in the treatment of cerebral aneurysms by eliminating the need for craniotomy. The recent protocol of endovascular treatment for cerebral aneurysm is classified into two main categories; is parent artery occlusion and intra-aneurysmal embolization. For aneurysms with a broad neck and large sac, it is recommended to consider proximal arterial occlusion if tolerance is confirmed by cerebral blood flow study at the time of balloon Matas' test. Parent artery occlusion is a classical technique, but still mandatory and effective clinically. In patients who have aneurysms with a has well-defined neck and who are not good candidates for craniotomy, intra-aneurysmal embolization would be recommended as an alternative means of treatment. Care should be taken to prevent problems and complications, including careful catheter and guidewire manipulation. Careful selection of cases and appropriate pre-, intra-, and post procedure patient management is essential. Since the clinical application of Gugliemi's detachable coil (GDC) started, the potential of endovascular treatment for cerebral aneurysm has changed considerably. Preliminary results of embolization using GDC suggest that endovascular treatment would offer marked improvement in the management of patients harboring cerebral aneurysms. PMID- 10741112 TI - [Endovascular neurointervention for intracranial dural arteriovenous fistula]. AB - Dural arteriovenous fistula(DAVF) is an abnormal shunt within the dura matter or near the venous sinuses. Various congenital and idiopathic causes have been suggested, including such as venous sinus occlusion, trauma, surgery, and changes in hormone levels, but the exact etiology of the disease is unknown. The pattern of venous drainage seen on angiography was used as the basis for a classification of DAVF by Djindjian. Recent classification suggests that lesions that drain into the venous sinus or meningeal vein will behave only in a benign manner, whereas those that have subarachnoid venous drainage or alone or in addition will behave aggressively. Selection of treatment can be made from observation, carotid manual compression, transarterial or transvenous embolization, radiotherapy, or surgical intervention. The goal of treatment and consequently the techniques used, depends on the intensity of symptoms or the neurologic and, in particular, hemorrhagic risk posed by the type of venous drainage. The therapeutic decision depends also on the patient's general clinical status. Embolization can create a reduction of flow, which results in disappearance in symptoms and sometimes complete cure as seen at angiography. Complex DAVF must be treated with combined endovascular techniques plus neurosurgery or radiotherapy. PMID- 10741113 TI - [Detectability of metastatic brain tumors using gadoteridol-enhanced MRI: usefulness of standard-dose T1-weighted spin-echo image with magnetization transfer and enhanced FLAIR]. AB - PURPOSE: The aim of this study was to compare the detectability and image contrast of metastatic brain tumors depicted by T1-weighted MR imaging (T1WI) with the magnetization transfer (MT) technique after the administration of a standard dose(MT-SD-T1WI) or T1WI without MT after the administration of a double dose of gadoteridol(DD-T1WI). We also assessed the usefulness of enhanced fluid attenuated inversion recovery (FLAIR) for depicting very small metastatic tumors. METHODS: Forty-six MRI procedures were performed in 31 patients with metastatic brain tumors that had been diagnosed clinically and radiologically. An incremental dose technique was used with intravenous injections of 0.1 and 0.1 mmol/kg of gadoteridol. In 28 MRI procedures, enhanced FLAIR was carried out after an MT-SD-T1WI study. RESULTS: Detectability was significantly greater with both MT-SD-T1WI and DD-T1WI than with SD-T1WI. However, there was no significant difference between MT-SD- and DD-T1WI. Although an MT pulse increased the contrast between the enhanced tumor and white matter, the contrast between edema and white matter was decreased. Both MT-SD- and DD-T1WI showed small but conspicuous enhanced foci, but we could not determine whether these were vessels or small metastases on the brain surface. However, enhanced FLAIR only demonstrated foci that were thought to be small metastases. CONCLUSIONS: MT-SD- and DD-T1WI had equal ability to detect metastatic brain tumors. Enhanced FLAIR was useful for assessing very small metastases on the brain surface. PMID- 10741114 TI - [Optimal dosage of contrast material in screening upper abdominal enhanced CT]. AB - PURPOSE: To examine the possibility of decreasing the amount of intravenous contrast materia for screening upper abdominal CT scanning. MATERIALS AND METHODS: Eighty-seven patients(53 men and 34 women; 56.0 +/- 15.7 y.o.; range 20 88 y.o.; body weight 57.6 +/- 11.1 kg, range 29-85 kg) were randomly divided into two groups according to the amount of i.v. contrast material: 100 ml independent of patient weight(group 1) and 2 ml/kg (group 2). In both groups, contrast material(Iopamiron 300 mgI/ml) was intravenously administrated at an infusion speed of 1.5 ml/sec. Spiral scanning was started 90 sec after the start of contrast material injection. CT numbers of the portal vein, hepatic parenchyma of the right lobe, and background were measured on pre- and postenhanced images, and the contrast-to-noise ratio(CNR) and increases in CT the number of the right hepatic lobe(CNIliver) and portal vein(CNIportal) were calculated. RESULTS: In group 1, patient's weight showed negative linear correlations with CNR(r = 0.442, p = 0.0027), CNIliver(r = 0.422, p = 0.0043), and CNIportal(r = 0.509, p = 0.00042). In group 2, such correlations were not observed. CONCLUSION: Contrast enhancement in screening upper abdominal CT was negatively correlated with patients' weight in the group of contrast material injected with 100 ml. PMID- 10741115 TI - Role of early phase helical CT images in the evaluation of wall invasion of colorectal cancer: pathological correlation. PMID- 10741116 TI - [Evaluation of hepatic toxicity following high-dose 5-FU arterial infusion chemotherapy: analysis of 42 cases of colorectal liver metastases]. AB - The regional toxicity of an anticancer agent for normal liver tissue following hepatic arterial infusion chemotherapy (HAI) was evaluated in terms of morphology, function, and histopathology. Forty-two patients(M:F = 30:12; mean age, 59.9 years) with liver metastases from colorectal cancer were treated with HAI using a totally implantable vascular access port system from July 1994 to March 1999. The regimen used here was so-called weekly high-dose 5-fluorouracil(5 FU) infusion(5-FU, 1,000 mg/m2/week). Volume measurement of the liver demonstrated not only whole liver atrophy including the tumor but also volume reduction of the non-tumorous lobe. Atrophic change of the liver was seen in patients who were administered over 20 g/m2 of 5-FU(p < 0.01). The CT attenuation values of the liver were examined, and fatty infiltration was seen in six patients. Histologic examination of liver biopsies from the non-tumorous part revealed steatosis and infiltration of inflammatory cells in the portal triad, which were not seen in specimens prior to HAI. On clinical laboratory findings, enzymes representing bile duct, including alkaline phosphatase, leucine amino peptidase, and gamma-glutamyltranspeptidase, were increased in 22 patients. In terms of regional toxicity for long-term HAI, 20 g/m2 of 5-FU, is the key dose at which to consider temporary cessation or dose reduction. PMID- 10741117 TI - [Simultaneous query and retrieve from multiple DICOM image archives using a proxy server]. AB - PURPOSE: DICOM clients could not access more than one archive simultaneously in the previous PACS systems. We developed a proxy server which makes it possible for clients to query and retrieve images from multivendor DICOM archives simultaneously. METHODS: We used a Web server that supports the DICOM query and retrieve(Q/R) service class as a proxy server. This system consisted of two DICOM servers and six DICOM clients of different vendors in the Radiology Department. Through Web browsers on the clients, requests for Q/R were transmitted to the proxy server by hypertext transfer protocol. The proxy server communicated with DICOM servers by DICOM Q/R Service Class(C-FIND, C-MOVE), and then DICOM servers directly transmitted images to DICOM clients by DICOM Storage Service Class(C STORE). RESULTS: DICOM clients could transmit Q/R requests to two DICOM servers simultaneously via the proxy server and receive requested images promptly. Confirmation of conformance among the proxy server, DICOM archives, and DICOM clients became much easier than previously. CONCLUSION: A Web server working as a proxy server of the DICOM Q/R Service Class is an effective solution for allowing DICOM clients to communicate with multiple DICOM archives. PMID- 10741118 TI - [In Process Citation] PMID- 10741119 TI - [In Process Citation] PMID- 10741120 TI - [Development of lymphocytes]. AB - Many subtypes in malignant lymphoma, especially those of B-cell lineage, have been established by linking morphologically each of them to one of the normal lymphoid development stages. Recent researches justified some of those entities in terms of clinical aspects and biological natures. Moreover, new techniques and findings in immunology settled many problems in the disease which had been unsolved by conventional ones. Single cell analysis on somatic hypermutation, for instance, shed light on the nature of Hodgkin's disease. A good knowledge of lymphocyte development makes it easier to understand malignant lymphoma, and is very important to hematologists and hematopathologists dealing with the disease. Development of lymphoid tissue and recent advances in this field will be briefly reviewed. PMID- 10741121 TI - [Intracellular signal transduction pathway and its relation to lymphomagenesis]. AB - An increasing number of reports have suggested that aberration in signal transduction and apoptosis is involved in the development of malignant lymphoma. This review summarizes the signal transduction pathway and the apoptotic cascade that are tightly regulated in relation to each other. Increased cell growth and proliferation is as important in lymphomagenesis as decreased programmed cell death. NPM/ALK chimeric protein and Bcl-2 overexpression are such good examples respectively. TNFR family including its viral analogue, LMP1 display diverse effects on cell proliferation and apoptosis through TRAF mediated NF-kappa B signaling pathway, and are key molecules especially in virus-associated lymphoma, such as EBV and HTLV-I. PMID- 10741122 TI - [Epidemiology and geo-pathology of malignant lymphomas with special emphasis on Japanese lymphomas]. AB - The descriptive epidemiology and geo-pathology of malignant lymphomas have been hampered by the absence of a unified classification. Recent proposal of the REAL classification which will soon become the WHO classification is a significant step toward better understandings of the epidemiology of this interesting neoplasia. Worldwide epidemic of non-Hodgkin's lymphoma was discussed with possible relationships to herbicides. General characteristics of Japanese lymphomas as compared to Western lymphomas were described with special emphasis on the correlation between the low incidence rates of follicular lymphomas and Hodgkin's lymphomas. PMID- 10741123 TI - [Oncogenes and tumor suppressor genes involved in the development of malignant lymphomas]. AB - In comparison with the molecular mechanism of myeloid malignancies, the characteristic point in that of lymphoid tumors is that they develop from errors in the recombination of either immunoglobulin genes or T cell receptor genes. In myeloid malignancies, chimeric fusion genes are frequently detected as a results of chromosomal translocation, whereas unregulated expression of oncogenes or loss of expression of tumor suppressor genes are often found in lymphoid tumors. Recent advances in understanding the molecular development of lymphomas have provided a new insight into the roles of the cell cycle machinery in human lymphomagenesis. In this review, representative mechanisms are discussed to understand a conceptual framework for the growing literatures. PMID- 10741124 TI - [Epstein-Barr virus and malignant lymphoma]. AB - Association between Epstein-Barr virus and malignant lymphomas such as Burkitt's lymphoma, Hodgkin's disease(HD), nasal T/NK cell lymphoma(NTL), immunodefficiency associated lymphoma, and pyothorax associated lymphoma (PAL) has been suggested. Among these, HD, SNL, PAL are relatively common in Japan. For HD, EBV association was found in the disease peak incidences; those are peak in older adults in Japan, older peak of the bimodal peaks in Western countries, and unimodal peak in the childhood in the developing countries. For NTL, EBV was associated with almost all cases with CD56+ phenotype and cases with polymorphic reticulosis morphology with CD56- phenotype. For PAL, most cases are EBV-associated, about 40% of these show Type B subtype, the ratio of type A EBV to type B EBV in PAL was similar to those in immunodeficiency-associated lymphomas. PMID- 10741125 TI - [Hepatitis virus and malignant lymphoma]. AB - Hepatitis virus, especially hepatitis C virus(HCV), is suggested to be associated with lymphomagenesis. A high prevalence(33%) of HCV among non-Hodgkin's lymphoma(NHL) patients has been reported mainly in Italy, but the prevalence is low in other countries. HCV-related NHL is varied histopathologically, including diffuse large B cell lymphoma, immunocytoma or follicular lymphoma(REAL). The HCV genotypes involved are 1b, 2a or 2c(Simmonds). Although HCV RNA + strand has been detected in lymphoma tissue in various studies, there are not many studies in which HCV RNA-strand has been detected. Recent studies have shown that BCL-2 plays an important role in lymphoproliferation by suppressing apoptosis, that HCV core protein regulates c-myc transcription and that BCL-2 and c-myc work together in lymphomagenesis. It seems difficult to provide reasonable explanations regarding these puzzling epidemiological findings and lymphomagenesis. PMID- 10741126 TI - [Human herpesvirus 6(HHV-6) and HHV-8 in malignant lymphoma]. AB - Over the past 14 years three new human herpesviruses(HHV-6, -7 and -8) have been recognized and implicated as etiological agents of several human diseases. HHV-6 has been cited as a possible cause or as a modulating element of certain human lymphoproliferative disorders, particularly malignant lymphomas including Hodgkin's disease and Burkitt's lymphoma. Despite the genomic homology and serological cross-reactivity between HHV-6 and HHV-7, evidence for the association of HHV-7 with lymphoproliferative diseases has been scarce. HHV-8 was originally found in patients with Kaposi's sarcoma, and subsequently shown to be implicated in primary effusion lymphoma(PEL), a novel lymphoma entity. We found that HHV-6 genome is integrated into host DNA of lymphoma cells. Further studies are warranted to determine whether chromosomally integrated HHV-6 has any role in the pathogenesis of viral genome-positive lymphoproliferative diseases. PMID- 10741127 TI - [Malignant lymphoma and Helicobacter pylori infection]. AB - Lymphoid tissue is acquired in the stomach in response to antigenic stimulation, so called mucosa-associated lymphoid tissue(MALT). In 1983, Isaacson had named a type of malignant B-cell lymphoma "MALT lymphoma" which grow in the marginal zone of lymphoid foliclies in the gastric mucosa. MALT lymphoma has lately attracted attention because of the relation of Helicobacter pylori(H. pylori). There are several studies that H. pylori can be detected in more than 90% of patients with MALT lymphoma and cure of H. pylori infection is followed by a complete regression of these tumors in most patient. This paper reviews the current knowledge about MALT lymphoma, and immunological and molecular aspects in the pathogenesis of the disease. PMID- 10741128 TI - [Malignant lymphoma: REAL classification to new WHO classification]. AB - The Revised European-American Classification of Lymphoid Neoplasms(REAL) proposed in 1994 represented a new paradigm for the classification of lymphomas. This classification emphasized that each disease was a distinct entity, defined by a constellation of clinical and laboratory features, i.e., morphology and genetic features, immunophenotype, clinical presentation, and course. And it also noted that the site(s) of presentation were a signpost for important underlying biologic distinctions. A new WHO classification is planed to be proposed, re categorizing entities of the REAL classification. Now, WHO members planed to publish the new classification as the bluebook of WHO at first in 1998 and now in 2000. This paper reports mainly different points in the new WHO classification of malignant lymphoma from the REAL classification. PMID- 10741129 TI - [Phenotypic classification of malignant lymphoma]. AB - The phenotypic characters of the lymphoma cell are important in the diagnosis of this disease. We recently tested whether the flow cytometry with fresh biopsy sample might be useful in the diagnosis of the lymphoma. In our date, 1. a rise and fall of the surface immunoglobulin kappa/lambda ratio indicated the monoclonal proliferation of the B-cell, 2. the increased proportion of the CD5/CD23 double positive cells indicated B-cell chronic lymphocytic leukemia or small lymphocytic lymphoma, 3. the decreased proportion of the CD2 positive cells and the increased proportion of the CD19 positive cells indicated B-cell lymphoma. These findings suggest that the flow cytometry is of adjunctive importance in making a diagnosis of the lymphoma. PMID- 10741130 TI - [Immunophenotypic analysis in the diagnosis of malignant lymphoma]. AB - Immunophenotypic analysis is capable of distinguishing benign and malignant or determining the lineage of neoplastic cells in most of lymphoproliferative disorders. The methodological approaches can be divided into two major categories; immunohistochemistry and flow cytometry. Both of these techniques can be important adjuncts to histological diagnosis although flow cytometry is not yet common in Japan. The basic features to be checked by immunophenotyping are as follows. IPT-I: Light chain restriction of immunoglobulin. IPT-II: Loss or stronger expression of one or more antigens normally expressed in reactive lymphocytes. IPT-III: Significant increase of a particular cell population that is quite rare or absent from normal tissue at the biopsy site. While IPT-I could be taken as direct evidence of a clonal proliferation of B-lineage cell, IPT-II and -III are the only strong suggestions of a malignant lymphoid proliferation. PMID- 10741131 TI - [Clinical staging classification of non-Hodgkin's lymphoma]. AB - Ann Arbor staging classification (with Cotswolds modifications) is widely used to evaluate the cases of non-Hodgkin's lymphoma(NHL). Contrary to Hodgkin's disease, most patients with NHL have advanced and extranodal disease, and therefore Ann Arbor staging system is not satisfactory in all the cases of NHL. However, Ann Arbor clinical stage is a powerful prognostic factor when it is used with other prognostic factors, such as serum lactate dehydrogenase and performance status(International Prognostic Index). Clinical features of NHL are closely related to histologic subtypes, so newer staging system will be based on pathological diagnosis and clinical parameters. PMID- 10741132 TI - [Prognostic factors and predictive model of non-Hodgkin's lymphoma: pathological prognostic groupings and international prognostic index]. AB - The non-Hodgkin's lymphomas are a diverse group of neoplasms, pathological prognostic groupings, based on survival, are essential to facilitate clinical comparisons of therapeutic trials. In addition, a model, based on important prognostic factors, is also necessary for predicting therapeutic outcome in patients with certain pathological prognostic group. In this paper, four models of pathological prognostic groupings, such as Working Formulation classification, National Cancer Institute classification, lymphoma clinico-pathologic(LCP) classification, and LCP schema, are reviewed for clinical usage. Comments on international prognostic index as a predictive model for aggressive lymphoma are described with its perspective for predictive capacity of therapeutic outcome. PMID- 10741133 TI - [Immunoglobulin and T-cell receptor gene rearrangements as markers of lineage and clonality in malignant lymphoma]. AB - In the process of lymphocyte differentiation, genes encoding variable regions of immunoglobulin and T-cell receptor undergo rearrangement. Such gene rearrangements represent markers of lineage and clonality of lymphocytes, allowing molecular diagnosis of human lymphoid neoplasms. Gene rearrangements are analyzed by Southern hybridization, using DNA probes specific for immunoglobulin heavy chain (IgH) and T-cell receptor beta (TCR beta) chain. This approach can detect monoclonal lymphoid populations that constitute 1-5% of total cells in tissues, and can be used successfully to distinguish lymphoid neoplasms from polyclonal lymphoid proliferations and to determine the lymphocytic lineage of neoplasms; i.e., rearrangement of IgH genes without TCR beta gene rearrangement strongly supports B-lineage, while rearrangement of TCR beta without IgH gene rearrangement implies T-lineage. PMID- 10741134 TI - [Methodology and clinical implication of the identification of clonality in malignant lymphoma]. AB - A variety of methods is now available to identify clonality of lymphoid neoplasia by recent progress in immunology and molecular biology. The target molecules for these include physiologically or pathologically altered genes/their products and virus-derived DNAs. Identification of clonality is important for more accurate diagnosis of lymphoma and, especially, related borderline lesions. It is also of considerable value in staging and follow-up study of the patients because higher sensitivity of polymerase-chain reaction made it possible to detect submicroscopic lesions including minimal residual disease after treatment. However, many lesions and phenomena which are clonal, but do not appear to be malignant, have been characterized. These may prompt an update of the conventional concept of "malignant lymphoma". PMID- 10741135 TI - [CT-guided needle biopsy in malignant lymphoma: current techniques and its usefulness]. AB - Histological examination of adequate biopsy specimens is fundamental to the management of patients with malignant lymphoma. Core-needle biopsy with computed tomography potentially provides a better basis for this assessment. Image-guided needle biopsy should be the first procedure performed in the diagnosis of lymphoma, except in easily accessible superficial neck, inguinal, and axillary nodal sites. Recent studies have shown a diagnostic accuracy of 83% to 87%. We describe the biopsy technique that have been successful in our personal experience. From our experience, CT-guided core-needle biopsies prove sufficient information for the diagnosis of and subsequent therapeutic decision to treat most cases of lymphoma. PMID- 10741136 TI - [Diagnostic imaging of malignant lymphoma: current usefulness and limitation]. AB - The recent developments of imaging modalities allow us to make a diagnosis of malignant lymphoma, to determine its staging, and to evaluate its response to treatments noninvasively. Especially, CT and MR imagings can visualize almost all organs involved by lymphoma with high spatial and contrast resolutions. However, it is difficult to detect the small diseased lymph nodes and splenic lesions. The tumor viability after treatments can not be accurately evaluated with imaging studies. The coverage of the whole lymph nodes during one examination is not easily achieved, either. The multidetector CT system, fast MR imaging techniques, new MR contrast media, and PET are expected to overcome these difficulties. PMID- 10741137 TI - [Clinicopathological characteristics of Burkitt lymphoma]. AB - In the new WHO classification, the category of Burkitt lymphoma includes classic Burkitt lymphoma and a variant-Burkitt-like-lymphoma. In addition, three subcategories--endemic, non-endemic, and immunodeficiency-associated--were proposed to reflect the major clinical and genetic subtypes of this disease. Endemic Burkitt lymphoma is well known to carry EB virus(EBV). However, not more than 20% of the sporadic Burkitt lymphoma carry EBV. One of the three alternative forms of the Ig/myc translocation are regularly present in all Burkitt lymphomas, whether EBV positive or negative. Thus, translocation, rather than EBV, must be considered as the main rate-limiting event in the development of Burkitt lymphoma. EBV may increase the probability of this event by expanding the target cell population at risk. PMID- 10741138 TI - [MALT lymphoma]. AB - MALT lymphoma was first described by Isaacson et al., in 1983, as an extranodal lymphoma arising in the marginal zone of the mucosa-associated lymphoid tissue, and has recently aroused much interest among investigators. This disease was incorporated in the REAL classification in 1994, and has since been accepted as an established independent disease entity involving the lymphoid tissues. The purpose of this paper is to comment on the disease concept, pathology, diagnosis and therapy of MALT lymphoma as well as the outlook for its study and management, and to survey the relevant literature. PMID- 10741139 TI - [NK-cell lymphomas and NK-like T-cell lymphomas]. AB - Two types of NK(natural killer)-cell lymphomas have been described. One is "nasal and nasal-type NK-cell lymphoma", and the other is "blastic NK-cell lymphoma". Nasal and nasal-type NK-cell lymphoma is characterized by an extranodal presentation, angiocentric and angiodestructive proliferation, LGL(large granular lymphocyte) morphology, CD2+CD3-CD16-/+CD56+CD57- phenotype, and an aggressive clinical course. Blastic NK-cell lymphoma, a recently described rare disease, is characterized by a frequent manifestation into the skin, lymphoblastic morphology, CD2-/+CD3-CD4+/-CD16-CD56+CD57- phenotype, and a poor prognosis. Recently described "NK-like T-cell lymphomas" are defined as lymphomas of NK-like T-cell(MHC-unrestricted CTL) origin, and are characterized by an extranodal manifestation, LGL morphology, CD3+CD4-CD8+CD16+/-CD56+CD57- phenotype, and an aggressive clinical course. PMID- 10741140 TI - [Ki-1 lymphoma]. AB - ALCL is widely recognized with its broad morphologic and phenotypic spectrum causing controversy in the diagnosis of this peculiar neoplasm. It is now beyond doubt that a significant proportion(64 to 84%) of the cases diagnosed as ALCL is closely associated with the expression of chimeric NPM-ALK protein activated by the (2;5) (p23;q35) chromosomal translocation, which can be detected by anti p80NPM/ALK or ALK1 antibodies. Recently, some investigators including us asserted that these p80NPM/ALK or ALK1-positive(p80/ALK+) ALCLs represent a distinct genetic entity with occurrence in young patients and a favorable prognosis, and should be differentiated from the p80/ALK- tumors with the relatively aggressive clinical course. The p80/ALK+ lymphomas also revealed the characteristic morphology such as horseshoe-like, kidney-like or doughnut-like nuclei and frequent expression of EMA and cytotoxic molecules. However, these features are shared, though to a lesser degree, by other p80/ALK-negative lymphoid neoplasms. Indeed, it is indicated that cytotoxic ALCL cases may be either p80/ALK positive or negative, suggesting that the cytotoxicity and expression of p80/ALK are independent phenomena among the cases of ALCL of T- and null-cell type. Thus, several areas of disagreement and controversy that surround the diagnosis and categorization of ALCL remain. PMID- 10741141 TI - [Clinico-pathological aspects of adult T-cell leukemia/lymphoma(ATL)]. AB - Adult T-cell leukemia/lymphoma(ATL) was first discovered and reported in Japan, where it has a high incidence in the southwestern region of the country. The retrovirus human T-cell leukemia virus type I(HTLV-I) is considered to be related to its etiology. ATL shows divers clinical features. It can be divided into four types of smoldering, chronic, acute, and lymphoma. ATL cells originate from the CD4-positive subset of peripheral T cells showing a characteristic notch in the nucleus and a tendency for lobulation. A definit diagnosis of ATL is made by documenting the presence of HTLV-I proviral DNA in the DNA of leukemic or lymphoma cells. Crinico-Pathological aspects of ATL are more complexed than other types of lymphoma because of the verity of the disease type, state of immunodeficiency, hypercalcemia, cytokine activation, and so on. PMID- 10741143 TI - [Lymphoma-associated hemophagocytic syndrome(LAHS)]. AB - Hemophagocytic syndrome(HPS) is characterized by a systemic proliferation of histiocytes showing hemophagocytosis in bone marrow, liver, or spleen. Clinical features of HPS are idiopathic fever, cytopenia, liver dysfunction, and coagulopathy. Adult HPS includes lymphoma-associated hemophagocytic syndrome(LAHS) and the incidence of LAHS was about 40% of the patients with HPS according to our study. About 70% of patients with LAHS had hepatosplenomegly without significant peripheral lymphoadenopathy or nodal lesions and showed HPS at the time of presentation. These clinical features were previously diagnosed as malignant histiocytosis (MH). The prognosis of LAHS depends mostly on the immunophenotype of lymphomas, T/NK-LAHS or B-LAHS. The median survival is 69 days in T/NK-LAHS and 242 days in B-LAHS. PMID- 10741142 TI - [Clinicopathological differential diagnosis of mycosis fungoides/Sezary syndrome from the cutaneous type of adult T-cell leukemia/lymphoma]. AB - Mycosis fungoides/Sezary syndrome(MF/SS) is considered as a distinct clinical entity in the latest classifications, including the revised European-American classification of lymphoid neoplasm(REAL), and the ones by the WHO and the European Organization for Research and Treatment of Cancer(EORTC). In Japan, where is an endemic area for human T-cell lymphotropic virus type 1(HTLV-1), we have been facing some problems concerning the differential diagnosis of MF/SS. We have observed many cutaneous types of adult T-cell leukemia/lymphoma(cATL/L) that can initiate as erythematous infiltrating plaques similar to MF or as an erythrodermic lesion similar to SS and also as tumoral and nodular lesions, solitary or multiple that resemble d'emblee type MF. The importance of the differential diagnosis between MF/SS and cATL/L lies on the significant difference on the prognosis. It is worth to differentiate MF/SS from cATL/L, especially in MF patients positive for HTLV-1 antibody. We emphasize on the characteristics that differentiate MF/SS from cATL/L as well as on the new findings regarding clinical, histopathological, phenotypical and genotypical aspects of MF/SS. PMID- 10741144 TI - [AIDS-related malignant lymphoma]. AB - Patients with HIV-1 infection have high risk for malignant lymphoma(AIDS lymphoma). In Japanese patients, more than 90% of AIDS lymphoma are related to EBV. All 22 cases which we have experienced are non-Hodgkin's lymphoma and their histological classification is either diffuse large or diffuse immunoblastic. Their mean CD4 count is 13.6/mm3. The reports from United States and Europe show that a half of AIDS lymphoma cases are small noncleaved type and the mean CD4 count is approximately 200/mm3. This difference between Japan and other countries may be caused by difference of EBV prevalence, but precise reason is not clear. PMID- 10741145 TI - [Primary extranodal malignant lymphoma]. AB - Primary extranodal lymphoma with high frequency and/or unique feature in Japan is discussed. Waldeyer ring lymphoma is most frequent(30%). Most tumors consist of diffuse, large cell type of B cell. CHOP followed by radiation achieved 94.1% of 5 years survival. Gastrointestinal lymphoma is another frequent lymphoma. Surgical rejection followed by chemotherapy and radiation therapy achieved high response. Pyothorax-associated lymphoma is a unique entity reported in Japan. EB virus and artificial pneumothorax are related to its occurrence. In intravascular lymphomas, lymphoma cells regionally exist in small vessel and are diffuse, large cell type of B cells. Brain is frequently involved. Bone lymphoma is found in 4 8%. The prognosis depend on the site and 5 year relapse-free survival is 70%. PMID- 10741146 TI - [Treatment for Hodgkin's disease]. AB - The mainstay of treatment for patients with localized stage HD was historically radiation therapy. However, chemotherapy has been substituting radiation therapy because of remarkable effects of chemotherapy and the late adverse effects of radiation therapy. In the treatment of advanced stage HD, ABVD therapy is currently identified as the best chemotherapy on the basis of equivalent efficacy and reduced toxicity comparing with MOPP therapy. The newer, intensified regimens such as escalated BEACOPP or Stanford V have demonstrated excellent disease control in preliminary results. For patients with primary refractory or relapsed HD after chemotherapy, current data support the use of high-dose chemotherapy with autologous stem cell rescue. PMID- 10741147 TI - [Therapeutic advances for indolent lymphomas]. AB - New therapeutic strategies for indolent lymphomas are described. Relapsed or advanced staged follicular lymphoma is assumed to have an indolent course when compared with intermediate- and high-grade NHL. Although treatment of low-grade follicular lymphomas with standard chemotherapeutic regimens is characteristically associated with a high initial response rate, the clinical course consists of a pattern of repeated relapse. Subsequent remissions occur, but at a progressively lower rate and with a shorter duration. Patients eventually succumb to the disease or its complications with a median survival of approximately 6.2 years. For these reasons, novel therapeutic agents and strategies need to be evaluated in this group of patients. Recently, high-dose chemoradiotherapy with autologous hematopoietic stem cell transplantation with ex vivo purging method was reported to be very effective therapeutic strategy. Very promising reports of chimeric anti-CD20 monoclonal antibody(rituximab), radioimmunotherapy by radioisotope-conjugated monoclonal antibodies, interferon, and allogeneic mini-transplantation followed by non-myeloablative preconditioning regimens are reviewed. Recent therapeutic approaches for other subtypes of indolent lymphomas including MALT and mantle cell lymphoma are also reviewed. PMID- 10741148 TI - [Treatment of intermediate and high grade non-Hodgkin's lymphoma]. AB - Since the study by SWOG in 1993, CHOP regimen has been the standard therapy for patients with advanced stage intermediate-grade or high-grade non-Hodgkin's lymphoma. Three-year estimated survival for all patients was about 50%. However, five-year disease free survival of the patients with high or high-intermediate risk by age-adjusted international prognostic index(IPI) is less than 30%. For the patients with low and low-intermediate IPI, high cure rate can be achieved by CHOP, or modified CHOP therapy. For the patients in high or high-intermediate risk groups, the CHOP regimen is not satisfactory. High-dose chemotherapy with haematopoietic stem cell rescue has been tried for those patients. We are conducting pilot study of high-dose chemotherapy with peripheral blood stem cell transplant(PBSCT) following induction with double CHOP regimen. Although the results are encouraging, confirmation with randomized prospective trials are necessary. PMID- 10741149 TI - [Salvage therapy in relapsed or refractory malignant lymphoma]. AB - High dose therapy(HDT) followed by autologous stem cell transplantation(ASCT) is a therapeutic options in chemotherapy-sensitive aggressive non-Hodgkin's lymphoma(NHL) and Hodgkin's disease at relapse. Of the patients with NHL, primary refractory disease should also be treated with such therapy. In patients with indolent lymphoma at relapse, disease free survival after treatment with purged ASCT has been shown to reach a plateau, although the therapy is a matter for debate at present. Anti-CD-20 monoclonal antibody in combination with standard- or high-dose chemotherapy is also quite effective for indolent NHL at relapse. In aggressive NHL with high- or high-intermediate international prognostic index, Burkitt's lymphoma, and mantle cell lymphoma. ASCT as front-line therapy might improve the clinical outcome. PMID- 10741150 TI - [Hematopoietic stem cell transplantation for malignant lymphoma]. AB - The clinical results and indications for hematopoietic stem cell transplantation (HSCT) including malignant lymphoma(ML) will be reviewed in this paper. In aggressive non-Hodgkin's lymphoma, patients with chemosensitive relapses and induction failures are appropriate candidates for HSCT. Purged HSCT may improve outcome of patients with relapses in follicular lymphoma and mantle cell lymphoma. High dose therapy with HSCT is proposed as a potentially curative treatment for ML that is not curable with conventional chemotherapy such as induction failure, relapse, poor-risk and indolent lymphoma. PMID- 10741151 TI - [Radiation therapy for malignant lymphoma]. AB - For the treatment of malignant lymphoma(ML), it is important to recognize its clinical features. Recently, the role of radiation therapy(RT) has been reconsidered in the treatment of ML because of the effectiveness of combined modality treatment (CMT) for early-stage aggressive non-Hodgkin's lymphoma(NHL). Recent data suggest that adjuvant radiotherapy to the bulky site in advanced stage aggressive NHL may significantly improve the relapse-free survival and overall survival. On the other hand, in the treatment of early-stage Hodgkin's disease(HD), meta-analysis data showed no apparent survival benefit from CMT or extended RT compared with RT alone. So in the initial treatment of early-stage HD, RT alone with adequate field should be recommended. Low-grade mucosa associated lymphoid tissue(MALT) lymphoma is considered to be an additional indication for the use of RT because it is very sensitive to radiation and potentially curable with RT. PMID- 10741152 TI - [The current status and future applications of gene therapy and immunogene therapy for malignant lymphoma]. AB - The present therapy for malignant lymphoma including stem cell transplantation, has been greatly developed. However, treatments still remain ineffective for many patients. Gene therapy is providing new strategies for the treatments of malignant lymphoma. There are three major approaches; 1) killing the tumor cell itself by introducing anti-sense genes against oncogene, tumor suppressor genes or drug-sensitive genes. 2) modifying the immune response by introducing genes that will trigger anti-tumor response or tumor specific genes to antigen presenting cell. 3) decreasing the sensitivity of hemopoietic cells by introducing drug resistance genes. We describe here the current and future applications of gene therapy for malignant lymphoma. PMID- 10741154 TI - [Search for an endogenous ligand of the orphan G protein-coupled receptor- discovery of apelin, a novel biologically active peptide]. AB - In the search for an endogenous ligand of the human orphan receptor APJ, we have isolated from bovine stomach extracts a novel 36-amino acid peptide, designated apelin. The APJ receptor is one of the G protein-coupled orphan receptors, many of which have been considered to be specific receptors for unidentified hormones and neuropeptides. We recently found the presence of apelin in the adipocytes and vascular walls as well as in the stomach. We examined biological activities of apelin and found that apelin lowered blood pressure in rats and also released cholecystokinin(CCK) from dispersed intestinal endocrine cells. Since apelin is an endogenous ligand for the HIV entry coreceptor APJ, we tested the effect of apelin on the entry of HIV in association with CD4, and found that apelin blocked the entry of HIV-1 and HIV-2. PMID- 10741153 TI - [Hepatology of the 20th century--what then?]. PMID- 10741155 TI - [Cholecystokinin (CCK) type-A receptor gene abnormality and life style related diseases]. PMID- 10741156 TI - [Capsules containing contrast medium for the assessment of gastric emptying in functional dyspepsia patients]. AB - Usefulness of No. 00 capsules containing contrast medium tests for functional dyspepsia was evaluated in 13 healthy volunteers and 22 patients with dysmotility like symptoms. Each subject swallowed 15 No. 00 capsules with Okunosu A (200 Kcal; Horika Canned Food Co., Niigata, Japan), and at 0, 15, 30, 45, 60, 90, 120 min, the capsule movement and size were monitored fluoroscopically and radiograms were obtained. And more, we tried to examine 5 healthy volunteers using 15 No. 3 capsules or 20 sitzmarks with Okunos A. In addition, they were examined by using 15 No. 00 capsules and sitzmarks meal. As a result, visualized capsule movement from the proximal to distal stomach up to 90 min was faster in the patients. The number of capsules remaining in the distal stomach from 30 min up to 120 min was significantly larger in the patients (p < 0.05). This test method was evaluated as being useful in monitoring solid emptying and we recognized that the way of the examination using No. 00 capsules and Okunos A could be an aptitude for evaluating functional dyspepsia up to date. PMID- 10741157 TI - [Factor V inhibitor developed after the surgery of esophageal cancer]. PMID- 10741158 TI - [A case of gastrointestinal stromal tumor (GIST) in the stomach]. PMID- 10741159 TI - [A case of primary malignant fibrous histiocytoma of the stomach]. PMID- 10741160 TI - [Infected hepatic cyst: report of two cases]. PMID- 10741161 TI - [An autopsy case of cholangiocellular carcinoma producing granulocyte colony stimulating factor]. PMID- 10741162 TI - [A case of autoimmune pancreatitis (AIP) diagnosed by pancreatic biopsy]. PMID- 10741163 TI - [A case of nonfunctioning minute malignant pancreatic endocrine tumor, showing regional stenosis of the main pancreatic duct]. PMID- 10741164 TI - [A case of pseudoaneurysm of the dorsal pancreatic artery presenting with gastrointestinal bleeding--successful management by transcatheter arterial embolization]. PMID- 10741165 TI - [A case of amebic colitis with liver abscess]. PMID- 10741166 TI - [Molecular, cellular and clinical aspects of myocardial ischemia]. AB - The authors summarize the fundamentals and actualities of the molecular and cellular background of myocardial ischaemia-reperfusion for the clinician. Metabolic changes following acute and chronic ischaemia and the role of free radicals, white blood cells, the endothelium, the heat shock proteins are reviewed. "New ischaemic syndromes" are important intrinsic adaptation mechanisms. Stunning is a transient contractile dysfunction following short periods of coronary occlusion in spite of restored perfusion. Hibernating myocardium is resulted after progressive chronic ischaemia including reversible contractile dysfunction, reduced metabolism and cellular dedifferentiation. Preconditioning means concomitant development of a protected state against lethal ischaemia after mild noxious stimuli. Several strategies are considered for myocardial cytoprotection based on the complex physiopathology of ischaemia induced injury. PMID- 10741167 TI - [Menopause and hormone replacement therapy]. AB - Due to the improving life expectancy of women spend third of their active life after the menopause. Estrogen deficiency can be caused by both natural and artificial menopause. The lack of estrogen can directly worsen the quality of life and epidemiological evidence suggests association with development of certain diseased states. Hormone replacement with natural estrogens has been proven to be successful for various indications: it reduces the menopausal vasomotor and psychological symptoms thus improving quality of life. It can also be used to prevent harmful effects of estrogen deficiency in various organs. Literature review supports the role of estrogen in atherosclerosis and osteoporosis prevention. Further evidence required establishing the role of estrogens in secondary prevention of coronary artery disease. Also needs to be explained why the beneficial effects of estrogen therapy in osteoporosis seem to disappear soon after cessation of therapy. Currently the relative risk increase of breast cancer during long-term hormone replacement therapy cannot be exactly measured. Nevertheless, substantial reduction of mortality in estrogen receptor positive breast cancer can also be seen with women on hormone replacement as compared to controls. Some data support the negative correlation of residual but still detectable, endogen estrogen and atherosclerosis and similarly to osteoporosis. The same residual estrogen levels seem to correlate positively with breast cancer. The recognition (and further acceptance) of the role of the residual estrogens might have influence on the indication, choice and dosage of preparation and duration of hormone replacement therapy. Overall evidence is in favor of the need medical attention for menopause: which ranges from preventive screening to long term hormone replacement therapy. The decision to treat requires the risks and benefits taken into consideration. This highly specialized care is provided in menopause clinics in Hungary. New oestrogen like agents are being developed like the selective estrogen receptor modulators, the tibolone and the phyto-estrogens. They provide tissue-specific effect acting as estrogen agonistics, sustaining the beneficial preventive and therapeutic effects of the estrogens, but in the breast and endometrial tissue they behave like estrogen antagonists avoiding the side effects of the current used oestrogens. They might play a significant role in the treatment of menopause in the future. PMID- 10741168 TI - [Surgical treatment of cholelithiasis in the era of minimal invasiveness]. AB - Three hundred and eighty-four patients with cholelithiasis have been operated on at the author's department in two years. There were 186 laparoscopic cholecystectomies, 162 micro-minilaparotomies and 36 traditional operations. All but one patients recovered. The author's opinion is, that laparoscopic and micro minilaparotomic cholecystectomies are nearly identical operations in the treatment of cholelithiasis, but the indication of the two minimal invasive methods is not the same. PMID- 10741169 TI - [Life perspectives for patients treated with chronic hemodialysis]. AB - The authors studied life perspectives of patients in a nephrology department using up Kaplan and Meier's analysis. Data of 51 of the patients included in the chronic hemodialysis programme between 1991 and 1997 were studied. The patients were under treatment for 19 months on average, 18% received renal allograft. The three-year life expectation for dialysed patients was 55%, without the transplant patients it was only 40%. The causes of deaths were infection and cardiovascular diseases in 40-40%. The patients life perspectives were better not only in primary renal diseases, but in vascular renal failure also, than in diabetic nephropathy. The authors made comparison between theirs and the national data, and discuss the necessary works to improve their results. PMID- 10741170 TI - [Chiari I. malformation, a new childhood disease?]. AB - From July 1, 1994 to July 1, 1997 a total of 17 children with Chiari I malformation diagnosed by MRI was seen in our hospital. Six of them underwent craniocervical decompression and tonsilar resection. The major complaints of all these children were gait ataxia and persisting (longer than 6 month) headache, and one child had acutely presenting vertigo and problem of swallowing. The MRI showed that in all cases the tonsils were displaced below the level of CI. Hydrocephalus or syrinx was not seen. At surgery the tonsils were resected. All the patients can be considered cured and symptom free at an average follow up of 13 month. PMID- 10741171 TI - Emerging viral diseases: some comments from a regional perspective. PMID- 10741172 TI - Diarrhoea morbidity in children in the Asaro Valley, Eastern Highlands Province, Papua New Guinea. AB - A morbidity study of diarrhoea covering 1926 children aged less than 5 years was carried out in Goroka town and the Lowa and Asaro Census Divisions, Eastern Highlands Province between 1986 and 1989. The study involved weekly demographic surveillance of the total population and morbidity surveillance of children by lay reporters who enquired about the presence or absence on any of the preceding 8 days of a range of symptoms associated with diarrhoeal and respiratory diseases. A three-day symptom-free period was used to define distinct episodes of diarrhoea. The average number of episodes/child-year for all children in the study population was 3.0. Boys suffered a significantly higher incidence of diarrhoea under 48 months of age than girls (4.4 episodes compared to 3.6/child year). Incidence was highest among those aged 6-17 months (5.5/child-year) with a rapid decline after age 35 months. Incidence of diarrhoea was highest in the more remote Asaro Census Division and was higher in periurban areas than in Goroka town. Incidence also varied significantly between villages, some villages experiencing up to 10 times the incidence of diarrhoea found in Goroka town. The incidence of diarrhoea was significantly higher in January than at other times of year. Duration of diarrhoea varied with age, the longest duration being an average of 4.7 days in the 12-17 months age group. In order to reduce diarrhoea morbidity, it is necessary to improve access to water, encourage improved hygiene practices and breastfeeding and warn people about the risks of sleeping with pigs. PMID- 10741173 TI - Serological cross-reactivities between the retroviruses HIV and HTLV-1 and the malaria parasite Plasmodium falciparum. AB - Serum samples from three populations of Papua New Guinea, where Plasmodium falciparum malaria and human T-lymphotropic virus type 1 (HTLV-1) are coendemic at high prevalence rates, showed statistically significant ELISA co seropositivity and co-seronegativity. Cross-reactivity was further indicated by the presence of 10 bands ranging from 134 kDa to 18 kDa on immunoblots of electrophoresed whole lysate P. falciparum antigen against serum of HTLV-1 seropositive patients from an area where malaria is not present. Similarly, sera from patients positive for human immunodeficiency virus (HIV) from a non malarious region produced immunoblot bands ranging from 134 kDa to 33 kDa to the P. falciparum antigen. The HTLV-1 and HIV serum samples yielded a number of immunoblot bands when reacted to an electrophoresed human O type red cell membrane antigen, but those bands had no identity to the cross-reactive bands on the P. falciparum antigen immunoblots. Malaria-positive sera from Papua New Guinean subjects presumed to be uninfected with HIV produced a variety of bands, some of intense prominence, to HIV antigen on diagnostic Western blots. PMID- 10741174 TI - Halofantrine in the treatment of uncomplicated falciparum malaria with a three dose regimen in Papua New Guinea: a preliminary report. AB - We evaluated the efficacy and safety of halofantrine in 19 patients with acute uncomplicated falciparum malaria. Each patient received oral halofantrine hydrochloride 500 mg every 6 hours for 3 doses (total 1.5 g). In almost all the patients clinical symptoms of malaria and parasitaemia disappeared within 2 and 3 days, respectively, of starting treatment. We observed no recurrence of parasitaemia during 14 days of follow-up. Tolerance to halofantrine was good except for minor and self-limiting gastrointestinal side-effects. Haematological and biochemical indices were not seriously affected. Halofantrine-induced prolongation of Q-T/Q-Tc intervals was the consistent cardiac manifestation in 84% of patients. The Q-T/Q-Tc interval prolongation increased with each dose; it reached a maximum between 18 and 24 hours and thereafter returned to baseline. These preliminary data indicate that, apart from the cardiac side-effects, halofantrine is an effective and safe drug, well tolerated by most of the patients in the study. PMID- 10741175 TI - In vitro susceptibility of Plasmodium falciparum isolates to halofantrine in the Central Province of Papua New Guinea. AB - Halofantrine is a newer antimalarial drug which has not been approved for clinical use in Papua New Guinea. We assessed 21 Central Province isolates of Plasmodium falciparum for their in vitro susceptibility to halofantrine. The concentration required to inhibit 50% of parasite growth (IC50) ranged from 0.05 to 7.0 nM with a mean of 1.90 nM and a median of 1.50 nM. The minimum inhibitory concentration (MIC) values ranged from 2.5 to 50 nM with a median of 5.0 nM. All but one isolate had an MIC of 10 nM or less. These results indicate that halofantrine would be a suitable alternative for the treatment of P. falciparum malaria in the region in the future, if and when the need arises, provided that its use was carefully monitored. PMID- 10741176 TI - Intraoperative use of mitomycin C in the treatment of recurrent pterygium. AB - The prevalence rate of pterygium in Papua New Guinea (PNG) is as high as 15%. Recurrence rates up to 50% are encountered after primary excision. In a country such as PNG where resources in terms of funds and manpower are limited, a simple procedure had to be identified to reduce this alarmingly high rate of pterygium recurrence. This article compares the results of a randomized masked study involving the single intraoperative application of 0.02% mitomycin C solution in 65 eyes undergoing surgery for recurrent pterygium using the bare sclera technique with a similar group of 65 patients in which the drug was not used. The results indicate that a single intraoperative application of mitomycin C solution was enough to reduce the recurrence rate of pterygium to 3% in the treated group as compared to 48% in the untreated group at the end of a 12-month follow-up. In the study it was also seen that, in PNG, pterygia were more common in females and that recurrences tended to occur early and were obvious in the first few weeks following surgery. PMID- 10741177 TI - [A. B. Fokht--1 of the founders of experimental pathology and cardiology in Russia (on the 150th anniversary of his birth)]. PMID- 10741178 TI - [A comprehensive study of the neurochemical and immune mechanisms of morphine tolerance: the effects of naloxone]. AB - To test the authors' hypothesis about the role of endopeptidase (enkephalinase A, in particular) in mechanisms of morphine tolerance and blocking action of small doses of naloxone, they studied nociception reactions, morphine antibodies titres and enkephalinase A activity after morphine, d-phenylalanine and naloxone injection in brain structures. It is shown that activity of enkephalinase A in structures of endogenous antinociceptive system increased simultaneously with morphine antibodies titres in tolerance condition. Injection of small dose naloxone inhibited enkephalinase activity in brain structures and decreased morphine antibodies titres to these in control morphine-sensitive rats and therefore suppressed morphine tolerance. Prolonged naloxone injection decreased morphine antibodies titres to the levels of intact animals and highly increased titers of antiidiotypic morphine antibodies. Thus, these results confirm the role of enkephalinase as a neuromodulator. A strong relationship exists between enkephalinase and immune mechanisms of development of morphine tolerance which can be blocked by small naloxone doses. It is concluded that naloxone in small doses can be used in patients to suppress morphine tolerance. PMID- 10741179 TI - [The actualization of heteronomous monosynaptic connections of IA afferents in the muscle motor centers of the human hand]. AB - Reflex excitability of spinal centers of hand muscles was examined in normal subjects and patients with traumatic lesion of forearm nerves. Central and peripheral muscle responses were evoked by stimulation of homonymous and heteronymous nerves. Patients with median and/or ulnar nerve lesion showed the heteronymous abductor pollicis brevis and abductor digit minimi H-responses. The cause of appearance of heteronymous responses is supposed to be actualization of latent heteronymous monosynaptic connections following traumatic lesion and afferent deprivation. It seems obvious that activation of heteronymous pathways corresponds to the process in afferent system known as "revitalization" of median to-ulnar anastomoses after damage. PMID- 10741180 TI - [The correlation of the different mechanisms of adaptation of the circulatory system to hypovolemia in ontogeny]. AB - It was established in experiments on narcotized adult cats and pigs of different age that postnatal mammal development is characterized by the presence of a period of the smallest resistance to massive blood loss (33% of circulating blood volume) when the ability for fast spontaneous restitution of plasma volume is already lost but the mechanism of increasing resistance of peripheral vessels to hypovolemia for support of perfusion pressure of adult mammal is still weak. PMID- 10741181 TI - [The vasoactive properties of different classes of blood lipoproteins in hypercholesterolemia and atherosclerosis]. AB - Studies were made of the effects of various classes of lipoproteins (LP) from the blood of rabbits with experimental hypercholesterolemia and human subjects with pronounced atherosclerosis on the vascular tone of rat thoracic aorta specimens. In hypercholesterolemia, there was a considerable weakening of a vasodilatory action of atherogenic LP fractions--LDL and VLDL. A vasodilatory effect of HDLP was not changed. In subjects suffering from atherosclerosis vasodilatory effects of both atherogenic and antiatherogenic HDLP decreased significantly. The revealed changes were related to shifts in lipid and protein components of LP complexes manifesting in the development of dyslipoproteinemia and dysapoproteinemia. In atherosclerosis accompanied with more profound changes suppression of vasodilatory effects of LP is greater than in transient experimental hypercholesterolemia. Consequently, changes of lipid and apo composition of LP and weakening of their vasodilatory action play a considerable role in the vessel tone changes in atherogenic situation. PMID- 10741182 TI - [The clinical and pathophysiological characteristics of the combined use of diaphragmatic electrostimulation and ultrasonic inhalation in patients with obstructive bronchitis]. AB - Efficiency of diaphragm electrostimulation (DES) in combination with ultrasound inhalations (USI) was tried in 172 patients with chronic obstructive bronchitis (COB) in the exacerbation phase. They were divided into 3 groups by the degree of respiratory distress (RD) and circulatory insufficiency (CI). Group 1 consisted of 61 patients with RD of the first degree, group 2--of 57 patients with RD of the second degree and CI stage I, group 3--of 54 patients with RD of the second degree and CI stage IIA. Each group was subdivided into 3 subgroups exposed to DES + USI, USI, DES (subgroups 1, 2 and 3, respectively). Before and after the treatment, the patients underwent spirography, ECG, stimulation electromyography of the diaphragm, x-ray of the chest. Positive effects of DES + USI were reported on respiration mechanics, diaphragm contractility, pulmonary ventilation, function of the bronchopulmonary system and diaphragm. This combination promoted also deeper inhalation of the aerosol, faster relief of clinical symptoms. DES in COB patients is contraindicated unless bronchospasm is properly reduced. PMID- 10741183 TI - [The dependence of the temperature response to pyrogens on the ambient temperature and the initial body temperature]. AB - The dependence of the temperature response to injection of Freund's complete adjuvant (FCA) containing killed and dried Mycobacterium butyricum on ambient temperature within 8-10, 14-16, 20-22, 26-28 degrees C ranges and initial body temperature was studied in experiments on rats, guinea pigs, and rabbits. The duration of the hyperthermic response was maximal at an ambient temperature 14-16 degrees C, and its dynamics can be divided in 2 phases, initial febrile and subsequent subfebrile. An increase in rectal temperature after application of the agent was the higher the lower was its initial value and vice versa. The hyperthermic response to FCA did not occur at 8-10 degrees C. PMID- 10741184 TI - [The problem of censored samples in experimental medicine]. AB - The paper analyses the problem of censored samples in experimental medicine. Some cases in such samples have indefinite digital values. Censoring may result from death of some animals in the test group of the disease studied. When the animals that survived in the test group are compared to the control group in which all the animals survived, the difference between the groups is conventionally interpreted as the result of the disease pathogenesis, but as well it can be a result of censoring. The author gives the examples of censored samples and how to manage the problem. PMID- 10741185 TI - [An experimental model of suppurative osteomyelitis]. AB - A new experimental rabbit model of acute purulent osteomyelitis is proposed. The model is reproduced by percutaneous introduction of 4 million St. aureus microorganisms into the medullary channel of the femoral bone after aseptic destruction of the bone marrow by ethyl alcohol and hot saline. The model satisfies the demands of good reproduction, reduces probability of contamination of periosteal tissues during intraosteal inoculation and provides reproduction of local inflammation without its generalization which results in reduced number of fatal outcomes among experimental animals. PMID- 10741186 TI - Tonsillectomy--current practice and audit. PMID- 10741187 TI - Benzodiazepine prescriptions on the rise. PMID- 10741188 TI - The incidence and management of post-tonsillectomy haemorrhage: a Singaporean experience. AB - AIM OF STUDY: To audit the incidence and management of post-tonsillectomy haemorrhage in the Singaporean context. METHODS: Three hundred and sixty-six consecutive tonsillectomies done over a 2-year period were retrospectively studied. Demographic details and indication for operation were evaluated in conjunction with the incidence and management of post-operative haemorrhage. RESULTS: 84.4% of the patients were adults while 58.7% were males. Chronic tonsillitis was the most common indication for operation (84.5%). The incidence of primary and secondary haemorrhage was 0.6% and 7.1% respectively. 3.8% of the patients had to return to the operation theatre to secure haemostasis. The use of post-operative antibiotics did not significantly affect the incidence of haemorrhage. There was no mortality from this complication. The haemorrhage rate was significantly higher among the adults (p = 0.048) and males (p = 0.019) in this study. CONCLUSION: Our overall post-tonsillectomy haemorrhage rate was higher than that reported in most of the Western publications because adults rather than children predominated in our series. We report a low incidence of primary haemorrhage which is in keeping with the results of studies on electrodissection tonsillectomies. Our findings indicate that tonsillectomy is a safe procedure which can be performed on a day surgery basis if the patients are carefully selected. PMID- 10741189 TI - Stress and the undergraduates. AB - OBJECTIVES: To assess the psychological health of undergraduates during their initial entry to the University, and to identify common sources of stress in their lives. METHOD: All first year law and medical undergraduates were administered a series of questionnaires. RESULTS: Of the 135 medical and 128 law first year undergraduates surveyed using the General Health Questionnaire (GHQ) to measure psycho-emotional distress, 77 medical students (57%) and 69 law students (47.3%) scored above the traditional cut-off of 4/5 points. Law students had significantly lower (p < 0.05) Life-Events Scores (mean 40.62, SD 29.30) than medical students (mean 55.62, SD 31.70). There was no statistical significant difference between medical (mean 89.32, SD 18.36) and law (mean 93.39, SD +/- 19.76) students (p = 0.88) in Type A Behavioural Pattern (TAPB). Hostelites (n = 67) reported significantly higher (p < 0.05) LES (mean 56.22, SD +/- 28.17) than non-hostelites (n = 196, mean 45.40, SD +/- 32.04). The commonest life events experienced in the past twelve months by the students were: (1) Difficulty keeping up with reading (84.4%), (2) Increased amount of academic work (82.5%), (3) Difficulty in tutorials (66.5%), (4) Little time for personal activities (61.6%) and (5) Difficulties in lectures as well as (6) peer competition (each 46.8%). Law students cited more relationship problems, while medical students were more troubled by academic work. When faced with a problem, more than seven out of ten students would turn to friends and classmates for help. The other common avenues of support were the family, and religion. A substantial proportion of law (37.5%) and medical (31.1%) students preferred to keep their problems to themselves; significantly (p < 0.00001) more of them (72.2%) were high GHQ scorers than other students (high GHQ scorers 47.4%). Interestingly 27.8% of these students turn to religion for emotional support. CONCLUSION: Health education programmes, mentorship and a reduction in information overload in the curriculum can be important strategies to enable undergraduates cope better with the demands of tertiary education. PMID- 10741190 TI - Autologous transfusion in obstetrics. AB - AIM OF STUDY: To assess the safety of blood collection for autologous transfusion in pregnant women and the effects of the procedure on the fetus. METHOD: Prospective study involving obstetric patients undergoing elective lower segment Caesarean section (LSCS) between 1993 and 1994 were recruited for autologous transfusion. Continuous cardiotocograph monitoring was carried out throughout the duration of the procedure and the fetal haemodynamics was studied before and after by doppler blood flow ultrasound. RESULTS: The results show that there was no fetal deceleration or increased uterine activity as monitored by cardiotocography. The mean pulsatility index (PI) for the umbilical artery before the procedure was 0.9560 and after was 0.0820 which is a significant difference (p value = 0.035). CONCLUSION: Autologous blood collection seems to be a safe procedure for both the mother and the fetus. However, autologous transfusion remains an unpopular choice amongst pregnant women. PMID- 10741191 TI - Functional status of the elderly in Singapore. AB - BACKGROUND: This study was done to look at prevalence and factors affecting functional dependence. The data in this paper were part of the 'Community Study on State of Health, Function, Cognitive and Social State of Elderly People in Singapore' conducted in Singapore between September 1992 and December 1993. PATIENTS: Four hundred and one elderly, aged 60 years and above were studied. Sixty-eight subjects (17%) were dependent in at least one function of Activities of Daily Living (ADL) as measured by the Barthel's Index (BI). RESULTS: The most common problem identified using this index was urinary incontinence. Forty subjects (10.4%) were dependent in at least one Instrumental Activities of Daily Living (IADL) function. The most common mode of transportation was by public transport, of which travelling by bus was the most common (59.9%) and mass rapid transit (MRT) was the least common (4%). Female gender and age > or = 75 were significant factors associated with dependence measured on Barthel's index but not on IADL. CONCLUSION: Results of this study were also compared with two previous surveys done in Singapore. Discrepancies in results noted could be attributed to different definitions in which the functions were assessed. We should aim to standardise the definition and measures of function in the local context in order to measure changes in the elderly population. PMID- 10741192 TI - Combined spinal epidural for labour analgesia--duration, efficacy and side effects of adding sufentanil or fentanyl to bupivacaine intrathecally vs plain bupivacaine. AB - AIM OF STUDY: The aim of the study was to evaluate the efficacy and side effects of adding sufentanil 10 micrograms, fentanyl 10 micrograms or a control of 1 mL saline to 2.5 mg bupivacaine given intrathecally via combined spinal epidural (CSE) for labour pain relief in the first stage. METHOD: Sixty ASA I or II patients who requested for epidural analgesia were randomised to three groups. CSE was performed with a 16G Touhy needle and 27G Whitacre needle. RESULTS: Patients in the sufentanil/bupivacaine group had a significantly longer duration of analgesia (162.9 min +/- 63.4) compared with fentanyl/bupivacaine (110.0 min +/- 44.6) compared with plain bupivacaine (70.0 min +/- 32.1). Pruritus was significant in patients with sufentanil (80%) and fentanyl (47.4%) but did not occur with plain bupivacaine. There was no significant difference in the incidence of nausea or vomiting, hypotension and motor blockade although blood pressures in the sufentanil group were consistently lower than the other two groups. Pain scores were lowest in the sufentanil group. Fetal heart rate changes and Apgar scores were not significantly different between the groups. CONCLUSION: In combined spinal epidural for labour analgesia, adding sufentanil 10 micrograms to intrathecal bupivacaine 2.5 mg provided fast onset and good analgesia for a longer duration compared with adding fentanyl 10 micrograms and with plain bupivacaine. The main side effect was pruritus. Neonatal outcome was similar. PMID- 10741193 TI - Screening for glaucoma in the Chinese elderly population in Singapore. AB - AIM: To determine the performance of different tests in screening for glaucoma. METHOD: The results from a glaucoma prevalence study of 479 Chinese elderly Singaporeans were analysed. Glaucoma screening tests evaluated in this study included Goldmann applanation tonometry (GAT), non-contact tonometry (NCT), optic disc assessment, screening Humphrey visual field and gonioscopy. The sensitivity, specificity and positive predictive values of the various screening tests in detecting glaucoma were calculated. RESULTS: The sensitivity of GAT and NCT at a specificity of at least 90% was 26% and 30% respectively with a low positive predictive value (PPV) of 0.13. Optic disc assessment with a vertical cup-disc ratio cut-off of more than 0.7 had a sensitivity of 100% and a specificity of 94% and a PPV of 0.46. The sensitivity of visual fields was 100% with a specificity of 76% and a PPV of 0.17. Gonioscopy had a sensitivity of 100% and specificity of 93% and a PPV of 0.14 in detecting primary angle closure glaucoma. CONCLUSION: Optic nerve assessment performed better than other glaucoma screening tests. There are, however, many other considerations that need to be taken into account in determining the ideal test for population screening. PMID- 10741194 TI - A 20-year-old man with eosinophilia and easy bruisability. AB - Acquired platelet dysfunction with eosinophilia is a rare, benign self-limiting disorder characterised by platelet dysfunction and an association with parasitic infections. A 20-year-old national serviceman who presented with easy bruising and spontaneous epistaxis is reported. Investigations revealed eosinophilia and an abnormal platelet function test similar to that seen in patients with Glanzmann's thrombasthenia infections. He also had evidence of hookworm infestation. Following a course of mebendazole, his symptoms and platelet dysfunction resolved. Hence, this disorder of acquired platelet dysfunction with eosinophilia should be considered in patients with bleeding disorders secondary to platelet dysfunction. PMID- 10741195 TI - Transpelvic rectus abdominis flap for perineal reconstruction following abdominal perineal resection with en bloc partial cystectomy and prostatectomy for locally advanced rectal cancer. AB - We report a case of locally advanced carcinoma of the rectum invading the bladder and prostate in a young man treated initially with neoadjuvant chemoradiotherapy. This was followed by an en bloc resection of the tumour, partial cystectomy and prostatectomy and an abdominal perineal resection. The urinary bladder was reconstructed and a new bladder neck reimplanted into the proximal urethra where the sphincter had been preserved. There was extensive radiation changes to the perineal skin where a wide excision had been performed. The pelvic defect was reconstructed with a pedicled transpelvic rectus abdominis myocutaneous flap. The patient recovered uneventfully and remains well with no clinical evidence of recurrence 18 months post-operatively. In very selected cases there is a definite role for neoadjuvant therapy and enbloc resection of the tumour followed by reconstruction of the perineum. PMID- 10741196 TI - Psychiatric and surgical management of male genital self-mutilation. AB - Cases of genital self-mutilation are usually seen in the general hospital setting and can be difficult to manage especially in those patients who have psychiatric illness. A joint effort between the psychiatric and the surgical services will be required right from the beginning of hospital admission to diagnosis and later, to follow-up. Psychiatric consultation strategies at the different phases of intervention will be needed to cater for the special needs of the surgical team, patient and family. We describe three cases of genital self- mutilators and the general management of these patients. PMID- 10741197 TI - The glossopharyngeal nerve, glossopharyngeal neuralgia and the Eagle's syndrome- current concepts and management. AB - Glossopharyngeal neuralgia is not just a painful condition. At times, it may be life-threatening as a result of associated cardiovascular consequences. Even in the absence of life-threatening consequences, it can be a severe debilitating disease with depression, suicidal tendencies, fear of swallowing, loss of weight and under-nutrition. The treatment for glossopharyngeal neuralgia and Eagle's syndrome has evolved over time. This review summarises the scientific evidence and philosophy about current management and therapy. Emphasis is placed on the importance of excluding secondary causes of glossopharyngeal neuralgia before embarking on nerve section through the posterior cranial fossa approach. The Eagle's syndrome due to an elongated styloid process is the most important cause of secondary glossopharyngeal neuralgia. Stylectomy is effective and should be considered before embarking on any neurosurgical procedure. Peripheral cervical and trans-tonsillar approaches to the glossopharyngeal nerve are also discussed. PMID- 10741198 TI - Clinics in diagnostic imaging (42). Shepherd's fracture. AB - A 21-year-old woman presented with severe ankle pain during a soccer match. Radiographs showed a Shepherd's fracture of the talus. She responded well to conservative treatment. The imaging anatomy of the posterior talus and os trigonum is reviewed, together with radiological features of osteochondritis dissecans of the talar dome. PMID- 10741200 TI - Annual report of the Section on Medical History PMID- 10741199 TI - Report of the President of the College of Physicians of Philadelphia July 1998 June 1999 PMID- 10741201 TI - Annual report of the Section on Public Health and Preventive Medicine PMID- 10741203 TI - Annual report of the Section on Medical Ethics PMID- 10741202 TI - Annual report of the Section on Arts-Medicine PMID- 10741205 TI - Annual report on public relations PMID- 10741204 TI - Annual report of the Executive Director July 1998-June 1999 PMID- 10741206 TI - Financial report of the College of Physicians of Philadelphia 1997-June 1998 PMID- 10741207 TI - Annual report of the Katherine A. Shaw Division of Library and Public Services July 1998-June 1999 PMID- 10741208 TI - Annual report of the library July 1998-June 1999 PMID- 10741209 TI - Annual report of the Francis C. Wood Institute for the History of Medicine July 1998-June 1999 PMID- 10741210 TI - Making mothers: medical discourse and the birth of the middle-class mother, 1750 1850. PMID- 10741211 TI - Global psychoactive resources PMID- 10741213 TI - Narcotic addiction: modernity and professional medical authority in the United States, 1870-1920 PMID- 10741212 TI - The origins of orthopsychiatry? Child psychopharmacology at the Philadelphia Orthopedic Hospital and Infirmary for Nervous Diseases, 1881-1889. PMID- 10741214 TI - Albert S. Ashmead and the First International Leprosy Conference, Berlin, 1897. PMID- 10741215 TI - Yellow fever: politics and class relations in Philadelphia, 1793-1805. PMID- 10741216 TI - Medicine's problem children: tobacco, alcohol, and amphetamines. PMID- 10741217 TI - The "agrarian myth" and student health at the University of Pennsylvania. PMID- 10741218 TI - The campaign for reform: medical education and professional improvement in antebellum America. PMID- 10741219 TI - The compulsory registration of patients with tuberculosis: an 1894 debate at the College of Physicians. PMID- 10741220 TI - Magic bullets: relations between science, magic, and religion. PMID- 10741221 TI - Anatomical drawings submitted for the Hugh Lenox Hodge Prize, 1878. PMID- 10741222 TI - [Quality of psychiatric treatment and organization]. PMID- 10741223 TI - [Assertive community treatment of psychoses]. AB - Long-term institutionalization is no longer the preferred treatment for the severely mentally ill. Several models for outpatient treatment of the severely mentally ill have been developed, among them Assertive Community Treatment (ACT). The literature on this model is reviewed in a Cochrane review and in randomized trials comparing ACT with hospital admission. ACT is a clinically effective approach to managing the care of severely ill people in the community. ACT, if correctly targeted on high users of in-patient-care, can substantially reduce costs of hospital care whilst improving outcome and patient and relatives satisfaction. Setting up ACT teams should be supported by politicians, professionals and consumers. PMID- 10741224 TI - [Homeless persons residing in shelters in the county of Funen I. Psychosocial characteristics and need of treatment]. AB - In the County of Funen (population 460,000) 120 homeless people resided in shelters for homeless people during week 49, 1996. Of these homeless persons, 94 (or 78%) were examined using ICD-10 and the Addiction Severity Index. Ninety percent were men, 90% were aged 20-59 years, 98% were unmarried or separated/divorced. Seventy-one percent had lived alone during the past three years, only 2% were employed, and sources of income for 83% were public assistance or disability pensions. The population of homeless people was divided into three groups: 1) Psychoses were found in 13 cases or 14%--schizophrenia (4%), paranoid psychosis (4%) and affective disorder (5%). About 2/3 had double disorder, psychosis plus substance abuse. 2) Sixty-four or 68% were non-psychotic homeless people, who had alcohol/substance abuse (53 or 83%) and/or personality disorder (57 or 89%). 3) Seventeen or 18% had no mental illness. About 60% had been sentenced to prison at least once. About 20% needed psychiatric treatment. PMID- 10741225 TI - [Homeless persons residing in shelters in the county of Funen II. Comparison between 1990 and 1996 and occurrence of mental disease]. AB - The question has been raised whether the change from hospital to community psychiatry is accompanied by an increasing number of the mentally ill in institutions for homeless people. During week 49, 1996, the residents in institutions for homeless people in the County of Funen were interviewed and diagnosed according to ICD-8, and the results were compared with a similar study from week 47, 1990. The prevalence of psychoses was 11% in 1990 and 14% in 1996. This difference was not significant. Significantly fewer psychotic homeless people were treated for their psychotic disorders in 1996 compared with 1990. The prevalence of alcoholics had fallen by 35%, while the prevalence of narcotics and cannabis addicts had risen by 78% and 110%, respectively. These changes were statistically significant. There was still a high prevalence of personality disorders, 70% in 1990 and 60% in 1996. Even though the deinstitutionalization of the psychiatric service has not been accompanied by more psychotic patients in institutions for the homeless, the clientele is still heavily strained with psychosis, personality disorder and substance abuse. PMID- 10741226 TI - [From psychiatric hospital to residential home for younger psychiatric patients. A cross-over study of residents in Sundbygaard in Copenhagen]]. AB - Along with the implementation of community psychiatry, an increasing number of younger psychiatric patients live in social institutions, with psychiatric treatment offered on a consultation basis. On the basis of hospital records and interviews with staff members, the study describes two groups of patients in a former psychiatric nursing hospital, group 1 offered time-limited stay with the aim of social rehabilitation, and group 2 offered permanent stay. Patients of group 1 were younger and more frequently treated with atypical antipsychotics. Apart from this, no differences were found. The vast majority of both groups suffered from schizophrenia. The intensity of symptoms was high, despite extensive medication. The two groups might represent the same category of the most severely disabled patients, seen at different ages. More intensive psychiatric consultant service is found necessary, and the process of rehabilitation should be evaluated prospectively. PMID- 10741227 TI - [Readmissions for schizophrenia in Denmark 1979-1998]. AB - The aim of the present study was to produce a nation-wide quantitative description of re-admissions for schizophrenia in Denmark in the period 1979 1998. The study is based on anonymous data from the Psychiatric Central Register. All persons with a main diagnosis of schizophrenia are included. The number of re admissions of schizophrenics has constantly increased since 1979 (approx. 65%), and the number of persons who account for these re-admissions has increased by approximately 55%. Patients who for the first time are diagnosed with schizophrenia have in the first year after the first admission an average risk of re-admission of 50%, a risk that has not decreased during the 20 years studied. The median age of re-admitted patients has increased by five years for males and is almost unchanged for females indicating a continuous need for re-admission of schizophrenic patients, also the older patients. Danish psychiatry has each year contact with approximately 50% of the persons who at least once have been diagnosed with schizophrenia since 1969 and who are still alive. Certain precautions must be taken in the interpretations of general aggregated statistics such as these. PMID- 10741228 TI - [Quality assurance of treatment and care of schizophrenic patients]. AB - The object of the study was to investigate the significance of quality assurance of treatment and care for schizophrenic patients in relation to psychosocial functioning and prognosis. In a period of one year prior to the quality assurance, quality measurements were carried out on 53 schizophrenic patients. Then quality standards with structure, process- and result criteria were implemented, and quality measurements for 57 patients admitted after the implementation were carried out. At admission there was no significant differences between the two groups. Before the implementation of quality standards, the patients did not regain their habitual level of psychosocial functioning (measured by the GAF-scale) at discharge. After the implementation the patients regained their habitual level. After implementation there was a significant lower rate of readmissions. Due to the fact that readmission is identical with relapse, the quality improvement therefore improved the prognosis for the schizophrenic patients. PMID- 10741229 TI - [Causes of death of German refugee children in 1945]. AB - In the last months of the second World War, 250,000 German refugees landed in Denmark. A third of them were children under the age of 15. Seven thousand German refugee children under the age of five died in Denmark in 1945. Using birth certificates and death certificates from the Danish national archives and burial lists from the German refugee cemetaries I have collected data to reveal causes of death, age distributions and time of the deaths of the 7000 fatal cases among children under the age of five. Three thousand children under the age of one, 2000 children one year old and 2000 children 2-4 years old died. Most of them died just before and after the German surrender, but many died in the months following the German surrender. The infant mortality was extremely high all during 1945. The infants died from diseases due to malnutrition, but the older the children the more likely the causes of death were due to infectious diseases such as pneumonia, measles, diphtheria and gastroenteritis. PMID- 10741230 TI - [Information, satisfaction and anxiety in connection with anesthesia. A questionnaire study]. AB - The purpose of the study was to assess the level of information, anxiety and satisfaction with treatment and care among patients undergoing general anaesthesia. A questionnaire was answered by 406 patients. Seventeen criteria comprising various aspects of the total course of anaesthesia were defined to characterise a "satisfactory anaesthetic experience". After the preoperative visit 65% of the nervous patients were less nervous. Only 47% were informed of possible postanaesthetic side effects. Although 86% were "satisfied" with the treatment and care, only 36% met the criteria of satisfaction. The main problems were anxiety (especially fear of not waking up again), nausea and pain after waking up. We conclude that patients are not told enough about anaesthesia and side effects. Many patients, particularly the younger patients and the female patients, are afraid of anaesthesia. PMID- 10741231 TI - [Occurrence of Enterobius vermicularis in children hospitalized at a central hospital]. AB - The aim of the study was to determine the prevalence of EV among hospitalized children and to estimate if abdominal pain is related to EV-infestation. A perianal tape-test for identification of eggs of EV was done on 186 children, aged 1-17, hospitalized in the period 8.3.1996 to 31.12.1997. The children were separated into two groups: 123 children with abdominal pain, and 63 children with symptoms thought not to be related to EV (trauma, testicular retention and hernia). Clinical data were collected from case records. Altogether 40 children (22%) were infested with EV. Among 111 children aged 5-12 the prevalence was 32 (29%). No association was found between abdominal pain and infestation with EV. In conclusion, EV is a common disease among hospitalized children. According to this study there is no indication for routine perianal tape-test of children hospitalized for abdominal pain. PMID- 10741232 TI - [Endoscopic diagnosis of enterobiasis]. AB - This article describes two cases of worm-infestation with symptomatology, diagnosis and treatment. The cases are discussed in respect to the other cases in the literature emphasizing symptomatology and diagnosis by endoscopy. PMID- 10741233 TI - [Increased risk of cancer among younger patients on dialysis]. PMID- 10741234 TI - [Echinococcus multiocularis--a new zoonotic infection in Denmark and a new differential diagnosis in hepatic tumors]. PMID- 10741235 TI - [A comment. Lymphocytosis and headache]. PMID- 10741236 TI - [Hearing tests performed by specialists prior the use of hearing aids]. PMID- 10741237 TI - [Parasitologic diagnosis. Enterobius vermicularis]. PMID- 10741238 TI - [Anticoagulant treatment]. AB - In 1998, the sale of vitamin K antagonists (VKA) in Denmark corresponded to the amount used for treatment of more than 20,000 patients for one year. This is more than three times more than ten years earlier. The reasons for the increasing use of VKA are new indications for permanent anticoagulant treatment, especially chronic atrial fibrillation and venous thromboembolism associated with permanent thromboembolic risk factors. The risk of bleeding is higher in the introductory phase of anticoagulant treatment than later on. It is now recommended to commence anticoagulant therapy without a loading dose. This seems to hasten a good estimate of the maintenance dose. The metabolism of VKA depends on a number of genetic and acquired factors. Knowledge of these factors is crucial for optimal regulation of the treatment, and it is important that patients at start of treatment are thoroughly informed about these factors in order to minimize the risk of complications. PMID- 10741239 TI - [Interactions with peroral anticoagulants (AC)]. PMID- 10741240 TI - [Continuous medical education--an evaluation]. PMID- 10741241 TI - [Percutaneous endoscopic gastrostomy]. PMID- 10741242 TI - [Psychological consequences of breast cancer screening among healthy women]. AB - The number of healthy women undergoing screening for cancer is very large. It is therefore important to know about the psychological consequences of screening. The literature for this review was found by searching in national and international databases and reading lists of references. This review finds that both women with negative test results and women with false positive test results report on psychological consequences such as fear and raised anxiety, depression, impaired working capacity, reduced self-esteem and self-confidence, as well as effects on their relationship to other people. They also report on physical symptoms such as impaired sleep or appetite. This review suggests that methods other than psychometric questionnaires would offer more knowledge on the subject and that future research should concentrate on those women experiencing serious psychological effects of screening. PMID- 10741243 TI - [Thromboembolic diseases in children]. AB - The incidence of thromboembolic diseases in children is increasing, probably as a consequence of medical progress and modern intensive care. Due to lack of scientific evidence, clinical recommendations for treatment of children are mainly based on opinions and extrapolations from adult medicine. This article presents current concepts of thromboembolic diseases in children, based on a review of the literature. Characteristic features are presented and differences between the disease in adults and children are outlined. Guidelines for antithrombotic treatment are provided. We suggest that screening for thrombophilic disorders should be performed on wide indications, and that modern principles of treatment in adult medicine should also be applied for children with thromboembolic diseases. PMID- 10741244 TI - [Problem-based learning in medical education]. AB - Problem Based Learning (PBL) has not yet been instituted systematically at medical schools in Denmark. We therefore introduced the method in a 10th term course in internal medicine and surgery, eighteen months before graduation, and evaluated the experience after two terms with a total of 93 students and 15 tutors. Compared with traditional education such as bed-side clinics and lectures etc., PBL was the preferred method by 67% of the students, while 28% found the methods equally good and only 2% discredited PBL. The main advantage of PBL was ascribed to motivation and activation, the students finding themselves as being part of the problem-solving situation. The tutors estimated PBL highly when teaching clinical coping strategies, stressing the need for a realistic and appropriate setting. This experience supports the decision to introduce PBL throughout the new medical curriculum in Copenhagen. PMID- 10741245 TI - [Logbook. A possible method]. AB - The European board of psychiatry recommends the use of a logbook and checklist. There has been discussion about the extent of registration--and the resources involved. We present a simple method of registering supervision. Trainees make a daily report of the amount of supervision received. The trial period was divided into two sections: March, April and May 1997; (training intensive) and June, July and August 1997; (holiday season). The trainees responded well to the logbook, the answering rate was 94.2% in the first period, 60.9% in the holiday season. Calculations based on the first period show that trainees receive supervision during 9.8% of their working hours (range 4.8-16.0). Twenty-nine percent of supervision is of the direct variety, where both trainer and trainee are present. This variety suffers during holiday season. This type of logbook provides an opportunity to be directed in training activities, with a minimum of resources involved. PMID- 10741246 TI - [Medical continuing education in the county of Vestsjaelland 1995-1997. Young physicians are generally satisfied with their medical training]. AB - During the last few years there has been some criticism concerning the medical training of young doctors. The present study analyses the satisfaction with the medical training (hospitals and general practices) in 401 courses of medical training in the county of West Zealand, Denmark, during the years 1995-1997. The 401 courses resulted in 3136 statements with respect to satisfaction. Only 4.9% of the statements was "discontent", 16.5% "somewhat content", 36.4% "content" and 42.3% "very content". The 4.9% with the statement "discontent" was especially related to the introduction to place of education, focus on medical training and the theoretical lessons. In conclusion the present study revealed general satisfaction in young doctors with the medical training in the county of West Zealand. PMID- 10741247 TI - [Quality development of continuing medical clinical education in the county of Roskilde]. PMID- 10741248 TI - [Congenital tracheobiliary fistula]. AB - A case of congenital bronchobiliary fistula in a three-year old girl is reported. The patient was born after 40 weeks of gestation and referred due to a long history of respiratory tract problems. Bronchoscopy disclosed a third opening at the level of the carina. The opening was cannulated with an X-ray positive catheter, and a fistula crossing the diaphragm and communicating with the left hepatic duct was found. Cholescintigraphic examination (HIDA-scanning) confirmed the presence of the fistula, but found no evidence of abnormalities in the rest of the biliary system. The thoracic portion of the fistula was excised through a right side thoracotomy. The postoperative course was uncomplicated. HIDA-scanning one month after the operation showed normal uptake, and the girl's symptoms had disappeared. PMID- 10741249 TI - [Traumatic abdominal hernia]. AB - Traumatic abdominal hernia remains a rare clinical entity despite an overall increase in blunt abdominal traumas. A case is presented, in which the left colonic flexure herniated due to direct trauma to the left side of the abdomen. The diagnosis of traumatic hernia may be established by CT scan and due to a high incidence of associated intraabdominal injuries, early exploration and repair is advocated. PMID- 10741250 TI - [A reference program on hip fractures. June 1999]. PMID- 10741251 TI - [Cell-to-cell interactions between iris and trabecular meshwork in primary open angle glaucoma]. AB - Pathomorphological study of the ocular draining system in the presence of an ischemic iris was carried out in patients with primary open-angle glaucoma (POAG). A total of 173 biopsy specimens of the sclera and iris were collected during anti-glaucoma operations in 115 patients aged 32-90 years. The specimens were stained for amyloid with Congo red, thioflavin, and toluidine blue at pH 4.6 and 5.8 for detecting glycosaminoglycanes. The specimens were examined by histochemical and electron microscopic methods. Morphological changes in the trabecular system in POAG are due to abnormalities in the iridal microcirculatory bed. The status of the trabecular system during the initial and advanced stages of glaucoma depends on the severity of involvement of iridal arterioles, capillaries, and venules, modulating the biochemical processes in the anterior segment of the eye. Endotheliocyte and collagen matrix pathology and subsequent degradation of the trabecular system are closely related to dystrophic processes in the iris (release of plasma protein from the vessels and melanin granules into anterior chamber humor, cytotoxic effects of degradation products on adjacent tissues). The detected changes imply the use of drugs improving blood supply to the eye at the microcirculatory level and antihypoxants and antioxidants in local and total-systems therapy of patients with POAG. PMID- 10741252 TI - [Experimental study of cytokines complex inhibitory effect on wound healing after glaucoma filtering operations: histopathological and immunochemical findings]. AB - Experiments on a model of glaucoma filtering operation in 25 rabbits showed that the natural cytokine complex inhibited the operation wound healing in comparison with the control. The complex was prepared by an original method (culturing peripheral blood leukocytes) at Immunology Department of the Russian State Medical University. Histopathological study revealed delayed formation of cicatricial tissue under the effect of cytokines. Decreased proliferation of fibroblasts and their decreased functional activity (decreased synthetic activity of glycosaminoglycanes and collagen) was detected in cicatricial tissue. Immunohistochemical studies showed that in remote periods (after 3-8 months) type IV membrane collagen formed in new cicatricial tissue, while in the control, types III and I collagens predominated in cicatricial tissue. Immunohistochemical structure of the cicatrix in experiment correlated with fine fibrous structure of cicatricial tissue, characterized by high permeability. Possible mechanisms of the inhibitory effect of the natural cytokine complex on the course of operation wound healing after glaucoma-filtering surgery are discussed. PMID- 10741253 TI - [Submuscular decompression multisclerectomy - new operation for neovascular glaucoma]. AB - A new intervention for neovascular glaucoma is suggested: submuscular decompression multisclerectomy with collagen implant impregnated with 5 fluorouracyl. Immediate and remote (up to 2.5 years) results in 29 patients were analyzed. Intraoperative complications were observed in 24.1% cases: vitreous prolapse in 13.8% and hyphema in 10.3%. Ophthalmic tone compensation was attained in 96% cases in the early postoperative period and in 63% in remote period. Painful syndrome was arrested in 96 and 65% cases, respectively. The hypotensive effect of the operation is due to an increase in the volume of the eyeball and enhancement of uveoscleral discharge. PMID- 10741254 TI - [New modified method of phacoemulsification in lenticular subluxation]. AB - A new method for cataract removal by phacoemulsification in impaired lenticular joints consists in fixation during surgery of the anterior lenticular chamber along the edge of the capsulorrhexis by 4 retractor hooks and subsequent implantation of a soft intraocular lens. The operation was performed in 6 patients with cataracts of different types complicated by subluxation of the lens. The operation did not involve such complications as vitreous prolapse or displacement of the incompletely dislocated lens. All operations were finished with complete evacuation of lenticular mass and implantation of soft silicone intraocular lenses. The proposed method widens the category of patients in whom modern phacoemulsification can be used. PMID- 10741255 TI - [Clinical manifestations and possible causes of dystrophic changes in corneal flap after laser specialized keratomileusis]. AB - Main types of dystrophic complications on the corneal flap associated with laser specialized keratomileusis (LASIK) are discussed. A total of 6507 down-up LASIK operations were performed. The most grave form of corneal flap disadaptation syndrome (CFDS) (central dystrophy) occurred in 1.59% cases only in patients with myopic keratoablation. Slight manifestations of this syndrome were observed in patients with myopia and hypermetropia. The authors discuss a concept of CFDS development in which the leading role is attributed to the initial disagreement between the size of corneal flap before and after keratoablation. The incidence of such complications depends on the method of corneal flap positioning during the final phase of the operation. Clinical picture, outcome, and treatment of CFDS are discussed. PMID- 10741256 TI - [Evaluation of therapeutic effect of new Russian drug semax in optic nerve disease]. AB - Semax is a synthetic analog of adrenocorticotropic hormone 4-10 with a pronounced nootropic effect. It was used in the treatment of vascular, toxic allergic, and inflammatory diseases of the optic nerve and partial atrophy of the optic nerve in parallel with basic neurotrophic and antiinflammatory therapy. For evaluating the drug efficiency, the patients were divided into 3 groups, administered the drug via different routes. In group 1 semax was administered intransally as nasal drops, in group 2 by endonasal electrophoresis, and group 3 was control. Addition of semax to therapeutic complex in patients with diseases of the optic nerve had a favorable impact on the intensity and rate of recovery and improved the visual functions. Semax effectively protected nervous tissue from aftereffects of injury, particularly during the acute stage of optic nerve disease: it stimulated positive changes in the clinical picture, which were evaluated by improvement of visual acuity, extension of the total visual field, increase in the electric sensitivity and conductivity of the optic nerve, and improvement of color vision. PMID- 10741257 TI - [Ampli-pulse phoresis in patients with partial optic nerve atrophy]. AB - A new complex method for treating partial atrophies of the optic nerve unites surgical, physiotherapeutic, and drug effects. One electrode is attached directly to the anterior segment of the optic nerve by a collagen infusion system and the other is fixed at the back of the neck. The involved optic nerve is exposed to sinusoidal modulated currents in the rectified mode, permitting direct drug electrophoresis in parallel with electric stimulation of nerve fibers. The efficiency of the method is two times higher than of standard treatment. PMID- 10741258 TI - [Early changes in the bioelectrical activity of retina after eyeball contusion]. AB - Characteristic signs of changes in the bioelectrical activity of the retina soon after contusion of the eyeball supplement the functional criteria used for accurate diagnosis of the severity of contusion (classification of the injury). The most sensitive indicator of electroretinography is the amplitude of the electroretinogram (ERG) a-wave, which is sharply suppressed during the first three days in injury of any severity. Recovery of the bioelectrical activity of photoreceptors is delayed in comparison with the time course of the function of the second-order neurons (more rapid recovery of the b-wave in comparison with the a-wave). The reactivity of Muller cells increases in severe contusion, which is shown by a more pronounced decrease in the ERG b-wave amplitude and less expressed changes in low-frequency ERG. Analysis of the time course of biopotentials during repeated examinations of the patients helps control the efficacy of treatment. The results indicate that electroretinographic examinations are the most desirable 5-7 days after the contusion, because at this period the differences in the bioelectrical activity of the retina are the most expressed. The most informative parameters of electroretinography are the time course of total ERG a- and b-waves and the low-frequency rhythmic ERG. PMID- 10741259 TI - [Activities of trypsin-like serine proteases and their inhibitors in blood and subretinal fluid of patients with different stages of proliferative vitreoretinopathy in rhegmatogenous retinal detachment]. AB - Concentrations of protein, trypsin-like and antitryptic activities, and the level of alpha 2-macroglobulin were measured in the subretinal fluid (SRF) and blood of patients with different stages of proliferative vitreoretinopathy (PVR). The content of protein in SRF of patients with retinal detachment with PVR was by an order of magnitude higher than in the vitreous body and increased from stage B to stage D1, which indicates increased permeability of the blood-eye barrier. Trypsin-like proteolytic enzymes and their main inhibitors are present in the SRF, and changes in their activity indicate augmentation of the inflammatory component of PVR from stage B to stage C3 and transition to a process similar to tissue cicatrization during stage D1. Changes in the proteinase-inhibitor balance in the peripheral blood in PVR are characteristic of a sluggish inflammatory process which activates from stage B to stage C and eventuates in stage D1. Increase in the blood level of alpha 2-macroglobulin during stages C3 and D1 seems to indicate the development of total autoimmune reaction. Antiinflammatory steroid therapy before surgery had a favorable impact on the course of the postoperative period. PMID- 10741260 TI - [Gamma-interferon in patients with uveal melanoma]. PMID- 10741261 TI - [Epidemiology of refraction abnormalities in Northern Navy young soldiers]. AB - Screening ophthalmological studies were carried out in 2851 soldiers arriving to Northern Navy, in order to detect ocular diseases, refraction abnormalities, and functional disorders. Visual disorders were detected in 529 subjects (19%), refraction abnormalities in 87% of these. Myopia predominated (344 subjects, 66%) in the structure of ophthalmological disorders. Hypermetropia was detected in 114 patients (21%), anisometropia in 85 (16%). Twelve percent of all examinees were in need of optic correction. Epidemiological analysis showed an obvious increase in the prevalence of myopia from South to North: 204/1000 in Northern regions, 99/1000 in the central areas, and 56/1000 in Southern regions. Service in the Extreme North can be regarded as a factor of risk of myopia progress, requiring active preventive measures. PMID- 10741262 TI - [Therapy of primary open-angle glaucoma by oftan-dipivefrin]. AB - Choice of optimal hypotensive therapy with the minimal side effects remains a serious problem in therapy of glaucoma. The efficacy and safety of oftan dipivefrin (Sante, Finland) and glaucon (Alcon, USA) are compared in a controlled balanced randomized open parallel study preceded by a period for discharge of the previous drugs. The period of discharge was 7 days, active treatment was administered for 8 weeks. Sixty patients aged 61-69 years were treated. Thirty one patients were treated by oftan-dipivefrin (group 1) and 29 by glaucon (group 2). In parallel with this, a 1% pilocarpin solution was administered in both groups. The examinations carried out during all consultations included measurements of intraocular pressure before instillations every day at the same time, evaluation of visual acuity, tonography, and study of the central and peripheral visual fields. The time course of hypotensive effects of both drugs showed a sufficient decrease in intraocular pressure in both groups; the effect of oftan-dipivefrin was more pronounced, while glaucon caused the least number of side effects. PMID- 10741263 TI - [Comparison of clinical efficacy of trental various routes administration in hypoxic states of eye posterior segment]. AB - Ninety-four patients with acute and chronic diseases of the posterior segment of the eye (optic nerve atrophy, central chorioretinal dystrophy, ischemic neuropathy, circulatory disorders in the central retinal artery and its branches) were examined. Clinical efficacy of various routes of administration of trental (intravenous, parabulbar, and by subtenon implantation of a collagen infusion system--SICIS) was evaluated in patients with ischemic states of the posterior segment of the eye. The most pronounced improvement of visual acuity was observed after administration through collagen infusion system. In acute and chronic hypoxic diseases of the posterior segment of the eye the time course of the rheographic coefficient was virtually the same after intravenous and parabulbar administration, while after SICIS the rheographic coefficient was significantly higher. Therefore, SICIS is the most effective method for trental administration in patients with acute and chronic diseases of the posterior segment of the eye; parabulbar administration ranks second in efficiency. This recommends SICIS as the most effective method for trental administration. PMID- 10741264 TI - [Mechanism of optic nerve congestive disk development during acute period of closed craniocerebral injury]. AB - The mechanism of development of congestion on the fundus oculi in acute posttraumatic intraocular hypertension was studied. A total of 410 patients were examined during the acute period of closed craniocerebral injury of different severity. Pressure in the central retinal vein was tentatively measured by transpalpebral compression of the eyeball with monitoring venous pulsation by direct ophthalmoscopy. The presence of spontaneous pulsation of retinal veins was regarded as an indicator of normal intracranial pressure. Analysis of the severity and location of the intracranial focus of injury together with evaluation of pressure in the retinal vein suggest that congestion on the fundus oculi in closed craniocerebral injury is triggered by the reaction of cavernous sinus intramural baroreceptors to a sudden rise of intracranial pressure. PMID- 10741265 TI - [Typical difficulties of neonatologists and ophthalmologists in the treatment of infants with retinopathy of prematurity]. AB - A perinatally, somatically, and neurologically aggravated child born at 26 weeks weighing 860 g with preterm neuropathy is described. Typical difficulties of neonatologists and pediatric ophthalmologists in the treatment of preterm babies with this grave ocular disease are described: the diagnosis cannot be confirmed by ophthalmoscopy because the optic media are not transparent, cryo- or surgical treatment has to be delayed because of severe somatic and neurological status, and intra- and postoperative complications develop despite thorough preoperative preparation. PMID- 10741266 TI - [Formula for estimating the optic force of elastic intraocular lenses]. AB - Formula for estimating the optic force of intraocular lenses made from elastic materials has been tried in 106 patients. The formula takes account of forward deflection of the elastic intraocular lens in the posterior chamber of the eye. The proposed method for estimation more than threefold decreases the mean error in estimation of optic force of elastic intraocular lenses in comparison with calculations with the use of known formulas. The mean error is 0.78 +/- 0.13 diopters, which corresponds to modern requirements to the accuracy of formulas for calculating the optic force of intraocular lenses. PMID- 10741267 TI - [Case of bilateral electric cataract]. AB - A girl aged 14 years had a severe electric injury after contact with an earth electrode of a high voltage power transformer. Two months after the injury, bilateral electric cataract developed in the presence of posttraumatic cerebral angiodystonia and polyneuropathy. Both eyes were operated on and intraocular lenses were implanted with good results. PMID- 10741268 TI - [Surgical activation of anterior and posterior routes of humor discharge in glaucoma]. PMID- 10741269 TI - [Anatomic diagnostic parallels in ocular vascular and orbital status as shown by color doppler mapping]. PMID- 10741270 TI - Smallpox eradication. WHO Advisory Committee on variola virus research. PMID- 10741271 TI - Influenza in the world. 1 October 1998-30 September 1999. PMID- 10741272 TI - Tamoxifen for the prevention of breast cancer. PMID- 10741273 TI - The relationship between angiogenesis and the immune response in carcinogenesis and the progression of malignant disease. AB - Recent studies have demonstrated that angiogenesis and suppressed cell-mediated immunity (CMI) play a central role in the pathogenesis of malignant disease facilitating tumour growth, invasion and metastasis. In the majority of tumours, the malignant process is preceded by a pathological condition or exposure to an irritant which itself is associated with the induction of angiogenesis and/or suppressed CMI. These include: cigarette smoking, chronic bronchitis and lung cancer; chronic oesophagitis and oesophageal cancer; chronic viral infections such as human papilloma virus and ano-genital cancers, chronic hepatitis B and C and hepatocellular carcinoma, and Epstein-Barr virus (EBV) and lymphomas; chronic inflammatory conditions such as Crohn's disease and ulcerative colitis and colorectal cancer; asbestos exposure and mesothelioma and excessive sunlight exposure/sunburn and malignant melanoma. Chronic exposure to growth factors (insulin-like growth factor-I in acromegaly), mutations in tumour suppressor genes (TP53 in Li Fraumeni syndrome) and long-term exposure to immunosuppressive agents (cyclosporin A) may also give rise to similar environments and are associated with the development of a range of solid tumours. The increased blood supply would facilitate the development and proliferation of an abnormal clone or clones of cells arising as the result of: (a) an inherited genetic abnormality; and/or (b) acquired somatic mutations, the latter due to local production and/or enhanced delivery of carcinogens and mutagenic growth factors. With progressive detrimental mutations and growth-induced tumour hypoxia, the transformed cell, to a lesser or greater extent, may amplify the angiogenic process and CMI suppression, thereby facilitating further tumour growth and metastasis. There is accumulating evidence that long-term treatment with cyclo-oxygenase inhibitors (aspirin and indomethacin), cytokines such as interferon-alpha, anti-oestrogens (tamoxifen and raloxifene) and captopril significantly reduces the incidence of solid tumours such as breast and colorectal cancer. These agents are anti angiogenic and, in the case of aspirin, indomethacin and interferon-alpha have proven immunomodulatory effects. Collectively these observations indicate that angiogenesis and suppressed CMI play a central role in the development and progression of malignant disease. PMID- 10741274 TI - Assessment of HER2 status in breast cancer: why, when and how? AB - Human epidermal growth factor receptor 2 (HER2) is overexpressed, usually as a result of HER2 proto-oncogene amplification, in 20-30% of breast cancers. A HER2 positive status is generally associated with more aggressive disease and a worse prognosis. Furthermore, a positive HER2 status may predict the likelihood of resistance to some conventional therapies, as well as probably being predictive of sensitivity to anthracycline dose intensification. In addition to this prognostic/predictive value, HER2 is a target for specific therapy, with anti HER2 monoclonal antibody therapy available in the USA. This article reviews the different assays used to determine HER2 status, discussing their relative advantages/disadvantages and the need for their standardisation before integration alongside other pathological indices into the clinical management of breast cancer. PMID- 10741275 TI - Two weekly vinorelbine: administration in patients who have received at least two prior chemotherapy regimes for advanced breast cancer. AB - This study examined the response to and toxicity of two weekly vinorelbine administration in patients with at least two prior chemotherapeutic treatments for advanced breast cancer. This single centre study enrolled 20 patients, 19 of whom had received prior taxane treatment for advanced breast cancer. Taxane treatment was in the form of docetaxel for all but 1 patient who had received paclitaxel. All patients had received two or more prior chemotherapeutic regimes for advanced breast carcinoma, including anthracyclines (epirubicin) in 19 patients. Vinorelbine 25 mg/m2 two weekly was given for 6 months, until disease progression or toxicity precluded further treatment. 5 earlier studied patients started vinorelbine at 25 mg/m2/week; all changed to the two weekly schedule, limiting the incidence and severity of neutropenia. 7 partial responses (PRs) out of 20 assessable patients (35% overall response rate, 95% confidence interval 15 59%) were noted, all PRs occurring in taxane pretreated patients. The median duration of response was 4 months whilst the median time to progression was 2.75 months. Overall, there were 7 neutropenic events (35%) of 2 week median duration, spanning common toxicity criteria (CTC) grades 1-3 in severity. 5 neutropenia cases (25%) occurred in patients whilst on two weekly vinorelbine. 2 cases (10%) required granulocyte colony stimulating factor support, 1 having had febrile neutropenia (52%). One case of thrombocytopenia, neurotoxicity and nausea (each CTC grade 1) were recorded. Although this study involves a small number of cases, these preliminary results suggest that two weekly vinorelbine is effective in heavily pretreated (including taxane pretreated) advanced breast carcinoma. Response is comparable with that of traditionally used weekly regimes, with markedly less toxicity. PMID- 10741276 TI - Paclitaxel by 1-h infusion in combination with carboplatin in advanced non-small cell lung carcinoma (NSCLC). AB - In our previous study, FCCC 93-024, paclitaxel by 24-h infusion combined with carboplatin yielded a response rate of 62% and median survival of 54 weeks in advanced non-small cell lung cancer (NSCLC). Myelosuppression proved dose limiting, requiring the routine use of granulocyte-colony stimulating factor (G CSF). Based on the reported activity of 1-h paclitaxel infusion in NSCLC and minimal myelosuppression at doses of 135 and 200 mg/m2 every 3 weeks and the suggestion of a dose-response relationship, we launched an intrapatient dose escalation trial of combination carboplatin and 1-h paclitaxel. Chemotherapy naive patients with advanced NSCLC received paclitaxel 175 mg/m2 1-h and carboplatin dosed to a fixed targeted area under the concentration-time curve (AUC) of 7.5 at three weekly intervals for six cycles. In the absence of grade 4 myelosuppression, paclitaxel was escalated by 35 mg/m2/cycle on an intrapatient basis to a maximum dose of 280 mg/m2 by cycle 4. G-CSF was not routinely used. 57 patients (pts) were accrued from November 1994 through to April 1996. 44 pts (77%) had Eastern Cooperative Oncology Group (ECOG) performance status 1. Median age was 64 (range: 34-80) years. Cumulative peripheral sensory neuropathy proved dose-limiting and prohibitive in the first 20 evaluable patients (cohort A): grade > or = 1 in 15 patients (75%), grade 3 in 6 (30%), generally occurring at paclitaxel doses > or = 215 mg/m2 and obligating 3 patients to have treatment halted in the absence of disease progression. The protocol, therefore, was revised and the initial paclitaxel dose reduced to 135 mg/m2 with intrapatient dose escalation of 40 mg/m2/cycle to a maximum dose of 215 mg/m2, recapitulating the original dosing schema used in FCCC 93-024. 35 patients were enrolled in this second cohort (B); 33 proved evaluable. Whilst 17 (52%) experienced peripheral sensory neuropathy, grade 3 neurotoxicity developed in only 3 (9%). Myelosuppression also was less pronounced, with 42% exhibiting grade 4 granulocytopenia and 30% grade > or = 3 thrombocytopenia in cohort B compared with 70% and 50%, respectively in cohort A. Of the first 22 patients accrued to cohort A, 12 (55%) had major objective responses. Median survival was 48.5 weeks, 1-year survival rate 45% and 2-year survival rate 18%. Of 33 evaluable patients in cohort B, 9 (27%) had major objective responses. Median survival was 46 weeks, 1-year survival rate 47% and 2-year survival rate 12%. Combination paclitaxel by 1-h infusion and carboplatin at a fixed targeted AUC of 7.5 is active in advanced NSCLC. Neurotoxicity, not myelosuppression, proved dose-limiting at paclitaxel doses exceeding 215 mg/m2. Lower doses may be associated with lower response rates, but do not appear to compromise survival. PMID- 10741277 TI - Phase II trial of vinorelbine in patients with advanced and/or recurrent cervical carcinoma: an EORTC Gynaecological Cancer Cooperative Group Study. AB - The objective of this phase II study was to assess the efficacy and toxicity of vinorelbine administered as a single agent in the treatment of chemonaive cervical cancer patients. 46 patients (41 eligible) with cervical cancer (epidermoid or adenocarcinoma) and measurable metastatic and/or recurrent disease localised outside irradiated areas were treated with weekly intravenous (i.v.) vinorelbine 30 mg/m2 infused over 20 min. No prior chemotherapy was allowed. Median age was 53 years (range: 33-73), and performance status 1 (0-2). 31 patients (76%) had prior radiation therapy. There were 7 partial responders (17, 95% confidence interval (CI) 7-32) and 8 stable diseases (20%). Median duration of response was 5 months (4-11). Granulocytopenia was the major toxicity, with 47% of patients exhibiting grade 3 or 4 toxicity. Dose reduction and/or treatment delay was necessary in 28 patients (78%). Peripheral neuropathy reported in 10 patients was mild (grade 1 in 9 patients and grade 2 in 1 patient). In conclusion, single agent vinorelbine has moderate activity in recurrent or metastatic cervical cancer, but its reduced neurotoxicity warrants further study in combination with cisplatin. PMID- 10741278 TI - Are 18fluorodeoxyglucose positron emission tomography and magnetic resonance imaging useful in the prediction of relapse in lymphoma residual masses? AB - Treatment of both Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL) frequently results in a residual mass visible radiologically. Such patients may receive radiotherapy unnecessarily because the residual mass may represent benign fibrotic tissue rather than residual active lymphoma. Radiotherapy has been shown to have significant short and more worrying long-term toxicity. Refining the criteria for its use would be a major advance. A number of clinical investigations have been evaluated to more accurately determine the nature of such lesions, including erythrocyte sedimentation rate (ESR), magnetic resonance imaging (MRI) and high-dose gallium-67 scanning (HDGS) but none has proven utility. 18[F]-fluorodeoxyglucose positron emission tomography (FDG-PET) is an imaging technique that has been shown to be useful in distinguishing fibrosis from residual active disease in solid tumours. The aim of this study was to compare FDG PET and MRI in the assessment of residual masses following treatment for lymphoma. Patients with NHL/HD who had a residual mass following chemotherapy were eligible for this study. Patients had a combination of MRI and/or PET. All scans were completed within 5 months of the end of treatment. Patients were followed-up for relapse. 56 patients had an MRI scan, 24 had a PET scan and 22 patients had both investigations. Overall sensitivity and specificity, respectively, were for MRI 45% and 74%, PET 50% and 69%, and PET/MRI concurring 50% and 67%. There was a trend for improved relapse-free survival (RFS) with a negative result of both MRI and PET, but this was not statistically significant. The predictive value for both tests failed to reach statistical significance. Subgroup analysis suggests that PET may be better at predicting relapse in patients with NHL, especially those with masses above the diaphragm. There is no convincing evidence that either MRI or PET or the combination can reliably predict relapse within residual masses after treatment for lymphoma. A negative PET scan however appears to be more informative than a positive result and may well aid clinical decision making. There are a number of factors that may produce false-positive results, including post-treatment inflammatory changes, the sensitivity of the test in the setting of minimal residual disease and the heterogeneity of the histological subtypes studied. A negative PET (or MRI) result in lymphoma residual masses following therapy may negate the necessity for further therapy such as chemotherapy or radiotherapy and their concomitant toxicities. PMID- 10741279 TI - Non-Hodgkin's lymphoma presenting with spinal cord compression; a clinicopathological review of 25 cases. AB - The aim of this study was to retrospectively examine 25 patients with newly diagnosed non-Hodgkin's lymphoma (NHL) presenting with spinal cord or cauda equina compression as the first symptom that were referred to our department between 1985 and 1996. At presentation 17 patients were non-ambulatory; dual sphincter impairment was found in 9 patients with a further 8 patients having bladder dysfunction only. All patients had a tissue diagnosis. Five low-grade and 20 intermediate or high-grade tumours were identified. In this latter group 4 patients were treated palliatively and the remaining 16 patients received combination chemotherapy and/or radical radiation therapy. The overall survival at 5 years is 59%. The majority of patients became ambulatory, even if paretic at presentation. This is in marked contrast to reports of patients presenting in this fashion due to metastatic carcinoma. We urge this diagnosis be considered in all patients presenting with spinal cord compression attributed to malignancy. PMID- 10741280 TI - Thyroid cancer in children: the Royal Marsden Hospital experience. AB - The first child with well-differentiated thyroid cancer treated at the Royal Marsden Hospital presented in 1917. Since that time 30 children under the age of 16 years have been treated over a period during which many new treatments have been introduced. We have reviewed their management and outcome. The median follow up is 22.5 years (range: 1-66). The median time to recurrence was 7 years (range: 2-44). There were events up to 44 years after presentation. The risk of recurrence was higher in children aged 10 years or younger [HR 3.45, 95% CI (1.04 11.11) P = 0.03]. Thyroid stimulating hormone (TSH) suppression was the only intervention to be shown to reduce the recurrence rate [HR 11, 95% CI (2.27-50) P = 0.0003]. The median overall survival is 53 years. The only presenting feature predictive of poorer survival was the presence of metastases (HR 28.96, 95% CI 2.51-334, P < 0.001). Patients who developed recurrence had a higher risk of death (HR 9.90, 95% CI 0.98-100, P = 0.02) and a shorter median survival of 30 years. No therapeutic intervention could be shown statistically to impact on survival. Our recommendation for treatment is total or near-total thyroidectomy for all patients and radioiodine ablation for all except those with early T stage node-negative disease aged over 10 years. Modified neck dissection is recommended for children with clinically positive neck nodes and TSH suppression for all. Follow-up with serial thyroglobulin measurement should be lifelong. PMID- 10741281 TI - Engraftment and growth of patient-derived retinoblastoma tumour in severe combined immunodeficiency mice. AB - The development of an in vivo model of retinoblastoma could be important for studying its biological behaviour and developing novel therapeutic strategies. We examined the ability of patient-derived retinoblastoma cells to grow and disseminate in severe combined immunodeficiency CB-17-SCID mice after subcutaneous (s.c.) inoculation without conditioning treatment. 24/30 (80%) of patient-derived tumours engrafted and grew as s.c. nodules in SCID mice. Whilst most xenografted tumours appeared to be localised, by PCR assay a positive DNA band of human minisatellite region (YNZ.22) was determined in the bone marrow of 19/25 (76%), in the spleen of 14/25 (56%) and in the liver of 16/25 (64%) mice, respectively, indicating dissemination to distant organs. Cytogenetic analysis demonstrated i(6p) in 5/12 (42%) and trisomy 1 or 1q abnormalities in 8/12 (67%) of the xenografted tumour samples studied, respectively, suggesting that retinoblastoma tumour cells maintain their cytogenetic abnormalities following adoptive growth in SCID mice. In this report we demonstrate the ability to propagate human primary retinoblastoma cells in SCID mice after s.c. inoculation and suggest the possibility of using the SCID mouse model to study the intrinsic biological behaviour of human retinoblastoma and to develop novel therapeutic strategies in the treatment of this disease. PMID- 10741282 TI - Familial prostate cancer from the family-cancer database. AB - The aim of this study was to calculate the familial risk for prostate cancer (PC) for different family relationships. PC was studied in the Swedish Family-Cancer Database, updated in 1999 to cover individuals born after 1934 with their biological parents, totalling 9.6 million persons. Cancer data were obtained from the Swedish Cancer Registry from 1958 to 1996 and included 1035 PC cases amongst offspring. 188 families were identified where a father and a son had PC, giving a familial standardised incidence ratio (SIR) of 2.44 (2.10-2.80). The proportion of familial cancers was 18.2% amongst all PC amongst all PC amongst sons. There were only 5 pairs of affected brothers, of which 3 had an affected father. Age of onset modified familial risks modestly; the highest SIR of 4.43 (1.40-9.17) was for sons diagnosed before 50 years of age when the father was diagnosed before 65 years of age. When analysed across sites, an association of PC in one generation and stomach, liver and skin cancer and myeloma in another generation was observed. The link was most consistent for skin cancer. No maternal site was associated with a son's PC, although the SIR of breast cancer was 1.22 (0.95 1.53). No increased risk of malignancy was observed in wives of affected men excluding any shared environmental effect for PC and female cancers. PMID- 10741283 TI - Risk perception, screening practice and interest in genetic testing among unaffected men in families with hereditary prostate cancer. AB - Approximately 5-10% of prostate cancer cases are caused by dominantly inherited susceptibility to the disease. Although advances have been made in research concerning the genetic mechanisms of hereditary prostate cancer, little is known about the psychological consequences for men at high risk of developing the disease. The aims of the present study were to examine risk perception, interest in genetic investigations, cancer-specific worry, and screening practice among unaffected men, aged 40-72 years old, with a pedigree consistent with hereditary prostate cancer and an estimated lifetime risk of prostate cancer of 35-45%. A questionnaire was sent by mail to 120 subjects, of whom 110 responded. Most of the men (n = 90, 82%) worried about having an inherited susceptibility to prostate cancer, and 34 (31%) claimed that worry about prostate cancer affected their daily life (3 (3%) fairly much, 31 (28%) slightly). As many as 40% of the study subjects perceived their lifetime risk of prostate cancer as 67% or more. Perceived high risk was associated with symptoms of depression and with cancer worry affecting daily living. Two-thirds of the men aged 50 years old or more were regularly screened for prostate cancer. Subjects with high levels of cancer specific stress, as measured by the avoidance subscale of the Impact of Event Scale, were less likely to opt for screening. Almost all of the men (94%) were interested in presymptomatic genetic testing (84 (76%) "definitely yes" and 20 (18%) "probably yes"). We conclude that hereditary susceptibility to prostate cancer has significant psychological consequences although it rarely causes psychiatric morbidity. The present study underlines the importance of giving thorough, repeated information to men at high risk of prostate cancer. PMID- 10741284 TI - Involvement of adenomatous polyposis coli (APC)/beta-catenin signalling in human breast cancer. AB - We studied the relevance of adenomatous polyposis coli (APC)/beta-catenin signalling in the development of breast cancer by analysing the expression of beta-catenin in 54 primary breast tumours (34 ductal and 20 lobular). We showed that 13% of the tumours exhibited upregulated levels of beta-catenin in the cytosol suggesting that defects in APC/beta-catenin signalling components had lowered the rate of beta-catenin degradation. No mutations were observed in the amino-terminal region of beta-catenin, which comprises conserved serine residues important for phosphorylation-dependent degradation of the protein, but the APC protein was altered in 6% of the tumours. Tyrosine phosphorylation of beta catenin was detected in only one tumour and could, therefore, not have been responsible for the observed increased levels of this protein. Although 9% of the tumours displayed upregulation of c-MYC protein, there was no correlation with beta-catenin overexpression, suggesting that increased beta-catenin expression is not the major cause of c-myc gene activation in breast cancer. It is imperative that elements that selectively drive the oncogenic activity of beta-catenin in breast cancer be identified. PMID- 10741285 TI - Changes in p21WAF1, pRb, Mdm-2, Bax and Bcl-2 expression in cervical cancer cell lines transfected with a p53 expressing adenovirus. AB - The aim of this study was to provide some insights into the molecular mechanisms involved in p53-dependent apoptosis and growth arrest. Changes in the levels of p53 protein and proteins regulated by p53 were studied in relation to events of the cell cycle and apoptosis in cervical cancer cell lines upon transfection with a p53 expressing adenovirus (Ad5-p53). The post-transfection level of p53 protein in SiHa cells was found to be unchanged during the 24-48 h period. In contrast, the level of p21WAF1 protein was shown to increase to its highest level at 24 h, and decreased gradually up to 48 h after the Ad5-p53 transfection. We further noted that the increase of p21WAF1 was accompanied by G1 arrest at 24 h and the decrease of p21WAF1 was associated with apoptosis at 36-48 h after transfection. An anti-p21WAF1 antibody cross-reactive protein band of approximately 14 kDa was observed in HeLa and C-33A cells when these cells were committed to apoptosis upon Ad5-p53 transfection. In SiHa cells, phosphorylation of pRb was inhibited during the early stage of Ad5-p53 transfection. This was followed by the cleavage of pRb. However, Ad5-p53 transfection did not change the levels of Bax and Bcl-2 proteins. Our results suggested that, Bax and Bcl-2 may not be important for the apoptosis of these cells, whereas cleavage of Rb, and the decrease of p21WAF1 could play important roles in p53-dependent apoptosis. PMID- 10741286 TI - Expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase in human gallbladder lesions. AB - The aim of this study was to investigate the expression of platelet-derived endothelial growth factor (PD-ECGF) in human gallbladder carcinomas to elucidate its role in angiogenesis and tumour progression. To this end, 56 archival surgical specimens of gallbladder lesions were examined for PD-ECGF/thymidine phosphorylase (TP) expression by immunohistochemistry and the PD-ECGF/TP protein level was assessed in five fresh specimens of gallbladder carcinoma by enzyme linked immunosorbent assay (ELISA). Hyperplastic epithelial cells and adenoma cells showed no or faint staining with PD-ECGF/TP. Out of 43 gallbladder carcinomas, 27 (63%) showed moderate to strong immunoreactivity in the cytoplasm and nuclei of the tumour cells. PD-ECGF/TP immunoreactivity in stromal infiltrating cells was detected in 43% (3/7) hyperplasias, 17% (1/6) adenomas and 86% (37/43) carcinomas. PD-ECGF/TP protein levels in carcinoma tissues were higher than those in corresponding normal mucosa. PD-ECGF/TP expression did not correlate with angiogenesis, but significantly correlated with depth of invasion, lymph node metastasis, and tumour stage. These results overall suggest that PD ECGF/TP produced by both cancer cells and infiltrating cells is associated with tumour progression in human gallbladder carcinoma. PMID- 10741287 TI - A limited sampling strategy for determining carboplatin AUC and monitoring drug dosage. AB - There are several convincing reports showing relationships between the area under the curve of ultrafilterable concentration versus time (AUC) and pharmacodynamics of carboplatin. It is advisable, in treated patients, to check the AUC that is effectively delivered as compared with the prescribed AUC. To this end, limited sampling strategy seems to be an adequate approach since it limits the constraints of repeated blood sampling for both patients and nursing staff. A flexible limited sampling method for assessing ultrafilterable carboplatin AUC was developed. This method was based on a Bayesian estimation of carboplatin clearance using the NON linear Mixed Effect Model (NONMEN) program and a large pharmacokinetic and covariates database (103 patients). The optimal sampling design was a two-sample schedule (1 and 4 h after the end of infusion). During a prospective evaluation, it allowed an adequate estimation of carboplatin clearance with a non-significant bias (-4.5%) and a good precision (9%). In a second stage, this method was clinically applied to monitor carboplatin AUC in a group of 5 patients with metastatic germ cell tumours treated with intensified high dose carboplatin-based chemotherapy for 4 days. Dosage adjustments were performed according to daily controls of their AUC in order to obtain a total AUC of 20 mg/ml x min. By using this strategy all patients effectively received a total AUC very close to this targeted AUC, thus proving the clinical usefulness of this limited sampling method. PMID- 10741288 TI - Comments on: Multidrug resistance-associated protein (MRP) expression is correlated with expression of aberrant p53 protein in colorectal cancer, Fukushima Y, Oshika Y, Tokunaga T, et al., Eur J Cancer 1999, 35, 935-938. Mutant p53 and high expression of MRP are associated in acute myeloblastic leukaemia. PMID- 10741289 TI - The treatment of ovarian cancer has reached new standards of care. PMID- 10741290 TI - A new standard of care for treatment of ovarian cancer. PMID- 10741291 TI - Federation of European Cancer Societies. Full report. Economic evaluation in cancer care: questions and answers on how to alleviate conflicts between rising needs and expectations and tightening budgets. AB - All Western countries have experienced a fast growth in their healthcare expenses over recent decades. It is expected that pressure for such growth will continue in the future. But spending an ever larger share of our nation's resources on healthcare cannot be afforded. As a consequence, making choices will become more and more inevitable, even in cancer care. Economic evaluation is a very supportive tool for such decisions. This position statement concludes with recommendations for providers and healthcare policy-makers, to safeguard and further improve good clinical decision making and healthcare policy in cancer care under tightening budgets. PMID- 10741292 TI - Can dihydropyrimidine dehydrogenase impact 5-fluorouracil-based treatment? AB - More than 80% of an administered 5-fluorouracil (5-FU) dose is degraded by dihydropyrimidine dehydrogenase (DPD), making it an important regulator of this commonly used anticancer agent. The high variation in population DPD activity, association with 5-FU activity, and development of DPD inhibitors have all contributed to the current focus on this enzyme. This review details the impact of DPD on 5-FU pharmacology, catalogues recent information on DPD mutations, evaluates the case for tumour DPD as a source of 5-FU resistance and introduces the clinical case for DPD inhibitors as a mechanism for the use of oral fluoropyrimidine therapies. PMID- 10741293 TI - Prognostic effect of amenorrhoea and elevated serum gonadotropin levels induced by adjuvant chemotherapy in premenopausal node-positive breast cancer patients. AB - The purpose of the study was to determine the correlation between prognosis and chemotherapy induced amenorrhoea or elevated gonadotropin levels in node-positive breast cancer patients. Since we have previously found a better prognosis in patients with more profound leucopenia induced by adjuvant chemotherapy, we examined whether this effect was mediated through more efficient induction of amenorrhoea. The study population consisted of 126 premenopausal, primarily operable, node-positive breast cancer patients treated with cyclophosphamide, methotrexate and 5-fluorouracil (CMF) adjuvant chemotherapy at the Department of Oncology, Helsinki University Central Hospital between 1990 and 1993. 12 months after the beginning of adjuvant chemotherapy, the patients were divided into groups with respect to their menstrual function (regular menstruation, irregular menstruation or amenorrhoea). Information about menstruation status and serum concentration of follicle stimulating hormone (FSH) and oestradiol were recorded at 12 and 24 months from the beginning of adjuvant chemotherapy. Median follow-up time was 72 months. Women who experienced amenorrhoea or had irregular menstruation after chemotherapy had a significantly better 5-year disease-free survival (DFS) in univariate analysis than women who continued to menstruate (P = 0.02). Amenorrhoea and irregular menstruation were associated with a better DFS among patients with oestrogen receptor (ER) positive primary tumours (P = 0.007), whereas no such association was found in ER negative cases (P = 0.86). 5-year overall survival (OS) in univariate analysis was also better in patients who experienced amenorrhoea (81%) or who had irregular menstruation (90%) after chemotherapy as compared with patients with regular menstruation (68%; 81 versus 68%, P = 0.05). The serum FSH level did not correlate significantly with outcome irrespective of the cut-off point chosen. Nodal status, tumour size and menstruation status after chemotherapy were also significantly associated with DFS in a multivariate analysis. The menstruation status after chemotherapy lost its significance for OS in a multivariate analysis whilst the number of affected lymph nodes, tumour size and oestrogen/progesterone receptor status retained their impact. There was no association between the degree of leucopenia and induction of amenorrhoea by CMF. Chemotherapy-induced ovarian function suppression (amenorrhoea/irregular menstruation) after chemotherapy had a favourable effect on DFS in premenopausal breast cancer patients. The post chemotherapy menstruation status is a clinically usable marker for sufficient endocrine effect of chemotherapy in ER/PR-positive patients in all premenopausal age groups. FSH level seemed to be a less reliable indicator of the castration effect of adjuvant chemotherapy in this study. PMID- 10741294 TI - Value of pedigree/clinical data, immunohistochemistry and microsatellite instability analyses in reducing the cost of determining hMLH1 and hMSH2 gene mutations in patients with colorectal cancer. AB - The aim of this study was to evaluate the significance of pedigree/clinical data, immunohistochemistry (IHC) and microsatellite instability (MI) analyses in the reduction of costs of constitutional hMLH1 and hMSH2 gene mutation diagnosis in patients with colorectal cancers (CRC). Pedigree/clinical data were evaluated on a series of 168 patients with CRC, including 43 consecutive sporadic late-onset and 25 consecutive, definitive or suspected hereditary non-polyposis colorectal cancer (HNPCC) cases, examined by IHC and MI analyses. In the latter group, 6/25 (24%) constitutional mutations were found. We detected no germline mutations in the sporadic late-onset patients. The lowest costs (880 Euro/mutation detected) were achieved by performing pedigree/clinical data (for exclusion of late-onset sporadic CRC) in conjuction with IHC only. In this model 1/6 (17%) mutations was missed. Additional preselection by IHC and MI analyses before sequencing was required to detect all mutations. In this approach, which seems to be the most effective in the search for hMLH1 and hMSH2 gene mutation, the cost was 1767 euro/mutation detected. PMID- 10741295 TI - Vascular enumeration as a prognosticator for colorectal carcinoma. AB - Vascular enumeration is thought to be an independent prognosticator for several human tumours, including breast, bladder and colorectal carcinomas. There have been 12 reports on the prognostic influence of vascular enumeration in colorectal carcinoma with different results. To test the prognostic influence of this factor in our patients, we have selected 126 patients with colorectal carcinoma Dukes' stages A to C treated only with curative surgery with no further adjuvant therapy. The minimal follow-up time was 5 years (60 months). After immunostaining with CD34, we performed a manual count of the vessels following Gasparini's criteria. In our series vascular enumeration showed significant association with the histological grade (P = 0.03) with a cut-off point at 77 vessels/200x, but not with tumour staging and vascular and neural invasion (P > 0.05). Vascular enumeration was a prognosticator for RFS (relapse-free survival) (P = 0.009) and OS (overall survival) (P = 0.01) in all Dukes' stages in the univariate analysis, but this prognostic influence was lost in the multivariate analysis, in which only stage, histological differentiation, location and vascular and neural invasion behaved as significant independent prognosticators. PMID- 10741297 TI - Vascular endothelial growth factor and platelet-derived endothelial cell growth factor expression are implicated in the angiogenesis of endometrial cancer. AB - Although many angiogenic factors have been described, it is not well defined which factors are expressed in endometrial cancer. The object of this study was to examine mRNA levels of the two angiogenic factors, vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) in endometrial cancer tissues and their association with clinicopathological features including microvessel density. The level of VEGF and PD-ECGF mRNAs was assessed by semi-quantitative reverse transcription-polymerase chain reaction using beta-actin as an internal standard in 38 patients with endometrial cancer. Microvessel counts were also assessed by immunostaining for factor VIII-related antigen in the most vascularised area of the specimen. VEGF/beta-actin ratios of non-endometrioid tumours were significantly higher than those of endometrioid tumours (P = 0.013). VEGF/beta-actin ratios of cases with lymph-vascular space involvement were significantly higher than those of cases without lymph-vascular space involvement (P = 0.021). Although it was not statistically significant, PD ECGF/beta-actin ratios in grade 3 tumours were higher than those in grade 1 and 2 tumours (P = 0.066). The microvessel density was significantly correlated with the level of VEGF and PD-ECGF mRNA expression (P = 0.041 and P < 0.0001, respectively). Our findings provide evidence that the expression of both VEGF and PD-ECGF is involved in the promotion of angiogenesis in endometrial cancer. In addition, VEGF and PD-ECGF might contribute to the aggressive potential of high grade tumours or certain histological subtypes with unfavourable prognosis through the induction of angiogenesis. PMID- 10741296 TI - Effect of high-dose ifosfamide in advanced soft tissue sarcomas. A multicentre phase II study of the EORTC Soft Tissue and Bone Sarcoma Group. AB - In this phase II study the effect of high-dose ifosfamide (HDI) given as a 3-day continuous infusion at a dose of 12 g/m2 repeated every 4 weeks with adequate mesna protection and hydration was evaluated in patients with advanced soft tissue sarcomas. A total of 124 patients entered the trial of which 10 were ineligible. HDI was given both as first-line and second-line chemotherapy. Median age was 46 years (19-66 years). Median World Health Organization (WHO) performance status was 1 (0-1). Fifty two per cent of the patients were males. The predominant histology was leiomyosarcoma (33%). A maximum of six cycles was given. At the time of analysis 55 patients have died. The partial response (PR) rate was 16%. The median time to progression was 15 weeks. 8 of the 18 responding patients (44%) had synovial sarcomas, whereas only 5% of the patients having leiomyosarcomas responded. The grade 3 + 4 haematological toxicity encountered was neutrophils in 78% and platelets in 12%. The major grade 3 + 4 non haematological toxicities encountered were febrile neutropenia in 39%, infection in 20%, and acute renal failure in 4%. In conclusion, it is possible to administer HDI on a multicentre basis, but the toxicity is substantial. HDI given as a continuous infusion at this dose cannot be recommended as the standard treatment of advanced soft tissue sarcomas, even in selected patients. PMID- 10741298 TI - Chemotherapy with cisplatin, epirubicin and docetaxel in transitional cell urothelial cancer. Phase II trial. AB - Cisplatin (CDDP), epirubicin (EPI) and docetaxel have single agent activity against urothelial transitional cell carcinoma (TCC). We evaluated the efficacy and toxicity of this combination in locally advanced or metastatic urothelial TCC. Patients with urothelial TCC who had no prior chemotherapy (prior adjuvant chemotherapy > 6 months allowed) were eligible for entry the study. Eligibility criteria were performance status 0-3, granulocyte count (AGC) > or = 1.5 (10(9)/l), platelet count > or = 100 (10(9)/l), clearance creatine > or = 60 ml/min and total bilirubin level < or = 1.5 mg/dl. Treatment consisted of EPI 40 mg/m2 intravenous push, docetaxel 75 mg/m2 in 1 h infusion with premedication and CDDP 75 mg/m2 with pre- and posthydration. Treatment was repeated every 21 days. Antiemetics with dexamethasone and 5-HT3 antagonists were used routinely. Prophylactic haematopoietic growth factors were not used. Patients were evaluated for toxicity weekly and assessed for response every two cycles of treatment. 32 patients were entered into the study and 30 patients (7 with locally advanced and 23 with metastatic disease) were assessable for response. There were 9 (30.0%) complete responses (2, 28.6% in locally advanced and 7, 30.4% in metastatic disease) and 11 (36.7%) partial responses (3, 42.9% in locally advanced and 8, 34.8% in metastatic disease) with an overall response rate (RR) of 66.7% (71.5% in locally advanced, 65.2% in metastatic disease). Overall median survival was 14.5 months (15 months for locally advanced, 12.5 months for metastatic disease). The median duration of response in patients with metastatic disease was 8.5 months. 16 (53.3%) patients required one dose reduction and 5 (16.7%) patients required two dose reductions for a nadir AGC < or = 500/mm3. Four episodes of febrile neutropenia and sepsis occurred. No patient had a dose reduction or treatment delay for any other grade 3/4 toxicity. There were no treatment delays due to myelotoxicity. Alopecia was universal. Non-haematological toxicity including mucositis, fluid retention, allergy, cutaneous toxicity, diarrhoea and neurotoxicity were mild and infrequent. The combination of EPI, docetaxel and CDDP is an active regimen for urothelial TCC. The response rate and toxicity were comparable with the M-VAC (methotrexate, vinblastine, doxorubicin, cisplastin) regimen. Phase III trials comparing this regimen with M-VAC are warranted. PMID- 10741299 TI - De novo cancers in paediatric renal transplant recipients: a multicentre analysis within the North Italy Transplant programme (NITp), Italy. AB - The purpose of this study was to determine the frequency and the outcome of de novo malignancies in a cohort of renal transplant paediatric patients. The records of 493 kidney transplants, carried out in 454 paediatric recipients at the three paediatric transplant centres of the North Italy Transplant programme (NITp, Italy) were reviewed. 10 cases of malignancies (2.2%) comprising both PTLD (post-transplant lymphoproliferative disorders) (6 cases, 1.3%) and non-PTLD malignancies (4 cases, 0.88%) were reported. Non-PTLD included one urothelial carcinoma and one Wilms' tumour of the recipient's left native kidney, one abdominal dysgerminoma and one optic nerve glioma of the left eye. The PTLD consisted of localised or disseminated Epstein-Barr virus (EBV)--associated B lymphocyte monoclonal (5 cases) and polyclonal (1 case) proliferations. All patients suffering from PTLD had been EBV-negative at the time of transplantation, but developed EBV primary infection after transplantation. All PTLD patient donors were EBV-positive. In addition, all but 1 patient received, before and/or after transplantation, a range of immunosuppressive drugs in addition to the baseline prophylactic immunosuppressive regimen. Moreover, 3 patients suffered from syndromes associated with a genetic predisposition to cancer. Finally, the malignancies reported here were associated with 20% graft failure and 20% mortality rates. PMID- 10741300 TI - Granulocyte colony stimulating factor permits dose intensification by interval compression in the treatment of Ewing's sarcomas and soft tissue sarcomas in children. AB - 71 children with sarcomas were treated in a prospective pilot study to determine whether granulocyte colony stimulating factor (G-CSF) permits compression of the interval between chemotherapy cycles. Patients had Ewing's sarcoma/primitive neuroectodermal tumour (PNET), rhabdomyosarcoma, non-rhabdo soft tissue sarcomas or other advanced soft tissue tumours. The chemotherapy alternated vincristine doxorubicin-cyclophosphamide and ifosfamide-etoposide, with G-CSF between courses. Therapy had two phases: induction (six cycles) and continuation (six cycles), which included primary tumour treatment with surgery and/or radiation. Chemotherapy cycles began every 14 days, or upon absolute neutrophil count (ANC) and platelet count recovery. The median chemotherapy cycle interval was 16 (11 48) days in the induction phase, with a median average relative dose intensification (ARDI) of 1.27 compared with every-21-day therapy. In the continuation phase, the median cycle interval was 21 days, with a median ARDI of 1.10. Radiation therapy prolonged chemotherapy intervals, whilst erythropoietin shortened them. Toxicity was modest for such chemotherapy. Event-free survival is comparable with or superior to that in recent large studies. G-CSF permits intensification of this regimen through interval compression. The impact of this approach on efficacy remains to be determined in a randomised trial. PMID- 10741301 TI - Comorbidity and colorectal cancer according to subsite and stage: a population based study. AB - In developed countries the growing proportion of elderly colorectal cancer patients with comorbidity will probably complicate clinical management. The aim of this study was to investigate the prevalence of prognostically relevant comorbidity in unselected colorectal cancer patients diagnosed in the Eindhoven Cancer Registry, according to age, gender and subsite and the association with stage of disease, treatment and short-term survival. Comorbid conditions were recorded, according to Charlson's index. The most common concomitant illnesses were cardiovascular diseases, previous cancers and hypertension. The prevalence of comorbidity, especially of cardiovascular disease, previous cancer and diabetes, was highest in the ascending colon. It was slightly higher in patients with Dukes' stage A, probably due to early detection because of regular monitoring for the comorbid condition. Comorbidity was not associated with the resection rate, but was negatively associated with short-term survival. Elder male colorectal cancer patients particularly suffer from substantial comorbidity, influencing the prognosis. PMID- 10741302 TI - Overestimated risk of second primary malignancies in ovarian cancer patients. AB - Registry-based cohort studies have established an increased risk of developing second primary malignancies (SPM) in patients with a primary ovarian cancer. In order to examine the accuracy of cancer registration with emphasis on registration of SPM, 344 women with ovarian cancer and 379 subsequent SPM, registered between 1958 and 1992 in the Stockholm-Gotland Cancer Registry (SGCR), a division of the Swedish Cancer Registry (SCR), were investigated. Complete records including pathology reports were examined and an additional histopathological evaluation was conducted for a sample of the group. The results revealed that 28 diagnoses of SPM were incorrectly registered (14 cases were misdiagnosed SPM of the gastrointestinal tract, mainly colon and rectum) and 34 women (with 38 SPM) were incorrectly registered with ovarian cancer. Recalculations of the risk of a subsequent cancer were performed on the basis of these findings and the results suggest an overestimation of the risk of developing SPM. Inferences of these findings to other primary sites of multiple malignancies should be made with caution and further studies are needed. PMID- 10741303 TI - Lack of p53 protein expression in preneoplastic rat hepatocytes in vitro after exposure to N-acetoxy-acetylaminofluorene, X-rays or a proteasome inhibitor. AB - Clonal expansion of initiated cells is an important process in carcinogenesis. Loss of functional p53 protein in initiated, preneoplastic cells might be involved in this process because such a loss would favour cell growth at the expense of normal cells upon exposure to genotoxic compounds. We have tested the hypothesis that p53 is not expressed in preneoplastic cells in the rat liver. Hepatocytes were isolated from livers of 10-week-old female rats that contained foci of preneoplastic hepatocytes, generated by 6-7 weekly injections of diethylnitrosamine (0.15 mmol/kg body wt intraperitoneally (i.p.)), starting 24 h after birth. The mixture of phenotypically normal and preneoplastic hepatocytes was exposed to X-rays or N-acetoxy-acetylaminofluorene (NAAAF), both causing DNA damage directly. At 24 and 48 h after exposure the cells were fixed and double stained for glutathione-S-transferase 7-7 (GST7-7), to identify preneoplastic cells, and p53. The percentage of p53-positive cells was much lower in GST7-7 positive (GST7-7+) than in GST7-7 negative (GST7-7-) hepatocytes. Exposure of cells to X-rays or NAAAF induced p53 in GST7-7- cells after 24 h, but GST7-7+ hepatocytes failed to do so. These results suggest that preneoplastic cells do not express p53 or have an attenuated p53 response to genotoxic treatments. This was confirmed when the cells were exposed to a proteasome inhibitor, PSI, which inhibits p53 degradation: a 12-fold increase in p53-positive cells was found after 48 h in GST7-7- hepatocytes, but in GST7-7+ hepatocytes no increase was observed. The percentage of GST7-7+ hepatocytes among surviving cells was increased after exposure to NAAAF, suggesting that these are more resistant to NAAAF than GST7-7- cells. This was not observed with PSI. These results indicate that preneoplastic hepatocytes have a lower p53 protein content and are not able to increase p53 upon inhibition of p53 breakdown or upon induction of DNA damage. Therefore, loss of p53 may favour clonal expansion of preneoplastic hepatocytes in the rat after administration of hepatocarcinogens or X-rays. PMID- 10741304 TI - Forskolin and phorbol ester have opposite effects on the expression of mucin associated sialyl-Lewis(a) in pancreatic cancer cells. AB - The carbohydrate antigen sialyl-Lewis(a) is important to pancreatic tumour biology because the circulating antigen is used in serological tests for malignancy and because cell surface antigen is involved in tumour cell binding to the endothelial adhesion molecule, E-selectin, in extravasation. In this study, we examined the effects of the adenylyl cyclase activator, forskolin, and the diacylglycerol analogue, phorbol 12-myristate 13-acetate (PMA), on the expression and release of sialyl-Lewis(a) in human pancreatic cancer cells. Increases in the release of sialyl-Lewis(a) from SW1990 cells produced by forskolin and PMA were associated with increases in the activities of protein kinases A and C, respectively, and could be blocked by inhibitors specific for these enzymes. Immunoprecipitation experiments showed that sialyl-Lewis(a) was associated with MUC1 mucin. Forskolin also increased the cellular content of antigen and MUC1 mRNA. Actinomycin D and a protein kinase A inhibitor, H8, blocked these effects. In contrast, PMA reduced cellular antigen and MUC1 mRNA levels, although it produced a temporary increase in release of the antigen. The effects of PMA were blocked by the protein kinase C inhibitor, H7. PMA also reduced cell binding to the adhesion molecule E-selectin. In summary, PKA and PKC alter cell MUC1 associated sialyl-Lewis(a) in opposite directions. These changes may have clinical utility in the diagnosis of pancreatic cancer and the prevention of metastases. PMID- 10741305 TI - E-selectin can mediate the arrest type of adhesion of colon cancer cells under physiological shear flow. AB - The aim of this study was to determine whether colon cancer cells flowing in blood exhibit the same adhesion pattern to the vascular bed as leucocytes using a flow adhesion system. In shear flow conditions, five colon cancer cell lines showed less tethering to E-selectin substrates than polymorphonuclear cells (PMN). However, some of the Colo201 cells formed complete arrest on E-selectin in continuous shear flow which was never observed in PMN cells. Colo201 cells expressed both sialyl Le-x and sialyl Le-a at similar levels in flow cytometry. However, the staining pattern showed marked contrast under the fluorescein microscope. The cell membrane of Colo201 cells was uniformly stained with anti sialyl Le-a MAb, whereas anti-sialyl Le-x MAb only stained in the patchy areas. Pretreatment of Colo201 cells with anti-sLe-a decreased tethering, while anti-sLe x significantly inhibited the arrest formation. Our data suggest that E-selectin alone can mediate colon cancer cell lodgement and subsequent metastasis without the contribution of integrin molecules and that the different distribution of E selectin ligands may affect the adhesion behaviour of colon cancer cells in flow conditions. PMID- 10741306 TI - Antagonists of growth hormone-releasing hormone (GH-RH) inhibit in vivo proliferation of experimental pancreatic cancers and decrease IGF-II levels in tumours. AB - Insulin-like growth factors (IGF-I and IGF-II) are implicated in the pathogenesis of pancreatic carcinoma. Antagonists of growth hormone-releasing hormone (GH-RH) suppress the GH-RH-GH-IGF-I axis and also act directly on tumours to reduce production of IGF-I or II. The aim of this study was to investigate the effects of two potent GH-RH antagonists in two experimental models of pancreatic cancer. Syrian golden hamsters with nitrosamine-induced pancreatic tumours were treated with 10 micrograms/day of GH-RH antagonist MZ-4-71 for 60 days. The therapy reduced the number of tumorous animals, decreased the weight of tumorous pancreata by 55%, and lowered AgNOR numbers in tumour cells. In two other experiments, GH-RH antagonists MZ-4-71 and MZ-5-156 significantly inhibited growth of SW-1990 human pancreatic cancers xenografted into nude mice, as shown by a reduction in tumour volume and tumour weights, and a decrease in AgNORs in cancer cells. IGF-I levels in serum and in pancreatic cancer tissue remained unchanged after therapy, suggesting that an effect on IGF-I is not involved in tumour inhibition. In contrast, IGF-II concentrations in tumours were significantly reduced by 50-60% after treatment with the GH-RH antagonists as compared with controls. In vitro studies showed that the concentration of IGF-II in the culture medium was increased after seeding of SW-1990 cells, indicating that this pancreatic cancer cell line produced and released IGF-II. This finding was also supported by the expression of IGF-II mRNA in the SW-1990 cells. Addition of 3 x 10(-6) M of GH-RH antagonist MZ-5-156 to the reduced-serum medium decreased cell proliferation, IGF-II mRNA expression in the cells and IGF-II concentration in the medium. Our findings indicate that inhibitory effects of GH RH antagonists on the growth of experimental pancreatic cancers, may result from a decrease in the production and concentration of IGF-II in the tumours. PMID- 10741307 TI - Seattle, the World Trade Organization and the NHS. PMID- 10741308 TI - Health improvement and the RSM. Interview by Robin Fox. PMID- 10741309 TI - Too ill to learn: double jeopardy in education for sick children. PMID- 10741310 TI - Surgical correction of refractive errors. PMID- 10741311 TI - Imported fever: a survival guide. PMID- 10741312 TI - The course of severe chronic fatigue syndrome in childhood. AB - Little has been reported on prognostic indicators in children with chronic fatigue syndrome (CFS). We used interviews with children and parents, a mean of 45.5 months after illness onset, to follow up 25 cases of CFS referred to tertiary paediatric psychiatric clinics. At its worst, the illness had been markedly handicapping (prolonged bed-rest and school absence in two-thirds); mean time out of school was one academic year. Two-thirds, however, had recovered and resumed normal activities--mean duration of illness to recovery/assessment 38 months--and none had developed other medical conditions. Recovery was associated with specific physical triggers to the illness, with start of illness in the autumn school term and with higher socioeconomic status. Severe fatigue states in children can cause serious and longlasting handicap but most children recover. PMID- 10741313 TI - Hospital referrals for low back pain: more coherence needed. AB - Low back pain is a common reason for hospital referral but little is known of the resulting workload in different specialties. All new outpatient attendances for conditions with low back pain were recorded over one month in a teaching hospital and a district general hospital. The patients were seen in at least ten specialties and two-fifths of them had been seen previously with the same symptom in another department. In the two hospitals, low back pain accounted for 15% and 12% of all new outpatient attendances. A more coherent referral policy is needed. PMID- 10741314 TI - Underutilization of antithrombotic therapy in atrial fibrillation. AB - Most patients with atrial fibrillation should be considered for antithrombotic therapy. In a retrospective survey we investigated practice in two hospitals. For patients at high risk, established guidelines recommend warfarin, or aspirin if anticoagulants are contraindicated; for those at medium risk, either may be used. Of 156 with atrial fibrillation (acute, chronic or paroxysmal), 119 were at high risk, mean age 79 years. According to the guidelines, 89 of these were suitable for anticoagulation but only 49 (55%) received warfarin; 27 received aspirin and 13 neither. Of 27 patients at medium risk (mean age 70 years), 6 were not prescribed any antithrombotic therapy. This survey indicates that guidelines on antithrombotic therapy are commonly disregarded and that, in particular, warfarin is underutilized in the group for whom it is most indicated. PMID- 10741315 TI - Unexplained loss of consciousness: systemic mastocytosis. PMID- 10741316 TI - Bilateral scapular fractures after electrocution. PMID- 10741317 TI - Combined Galeazzi and Monteggia forearm fracture. PMID- 10741318 TI - Stromal tumour of the small bowel. PMID- 10741319 TI - Insulin coma therapy in schizophrenia. PMID- 10741320 TI - So you want to write a textbook? PMID- 10741321 TI - The placebo effect and endogenous opioids. PMID- 10741322 TI - Clinical examination of the respiratory system. PMID- 10741323 TI - Causes of impaired fetal growth. PMID- 10741324 TI - The health of the Cornish tin miner. PMID- 10741325 TI - The sin of Onan. PMID- 10741326 TI - How do GPs respond to reports of abnormal chest X-rays? PMID- 10741327 TI - Systemic lupus with aneurysms. PMID- 10741328 TI - Tyger tyger. PMID- 10741329 TI - Multiple chemical sensitivity. PMID- 10741332 TI - Astrophysical Turbulence and Convection. Proceeding from the Florida Workshops in Nonlinear Astronomy and Physics. Gainesville, Florida, USA. February 18-20, 1999. PMID- 10741331 TI - American Society of Anesthesiologists 1999 annual meeting. Dallas, Texas, USA. October 9-13, 1999. Abstracts. PMID- 10741330 TI - Attitudes to organ donation among South Asians in the UK. PMID- 10741333 TI - Experimental Biology 2000. San Diego, California, USA. April 15-18, 2000. Abstracts. PMID- 10741334 TI - British Society of Gastroenterology annual meeting. Glasgow, 23-25 March 1999. Abstracts. PMID- 10741336 TI - 19th Joint meeting of the British Endocrine Societies, with the European Federation of Endocrine Societies. Birmingham, 13-16 March 2000. Abstracts. PMID- 10741337 TI - American Urological Association 95th annual meeting. Atlanta, Georgia, USA. April 29-May 4, 2000. Abstracts. PMID- 10741339 TI - 26th Annual meeting of the European Group for Blood and Marrow Transplantation and 16th meeting of the Nurses Group. Innsbruck, Austria, March 5-8, 2000. Abstracts. PMID- 10741338 TI - American Society for Artificial Internal Organs 46th annual conference. New York, New York, USA. June 28-July 1, 2000. Abstracts. PMID- 10741335 TI - An evidence-based appraisal of reflux disease management--the Genval Workshop Report. PMID- 10741340 TI - 31st Annual meeting of the British Medical Ultrasound Society. Glasgow, 8-10 December 1999. Abstracts. PMID- 10741341 TI - Society of Toxicology 39th annual meeting. Philadelphia, Pennsylvania, USA. March 19-23, 2000. Abstracts. PMID- 10741342 TI - Can involving diabetes specialists in developing a new insulin pen result in patient benefits? PMID- 10741343 TI - Acceptability and safety of a new 3.0 ml re-usable insulin pen (HumaPen) in clinical use. AB - A new re-usable insulin pen (the HumaPen) was tested in a multinational study in patients with Type 1 (45.1%) or Type 2 (54.9%) diabetes. Prior to study entry, patients used either syringes or a comparison pen. Patients changed to the HumaPen for all insulin injections during the 5-to 7-week study and a 12- to 13 week extension period. Acceptability of the HumaPen was assessed from a questionnaire completed at the end of the study by 72 previous syringe users and 209 previous pen users. HumaPen features considered easy/very easy by pre-study syringe users or pre-study pen users were correcting a mis-dialled dose (97.2%; 95.1%, respectively), reading the dose numbers (95.8%; 97.1%) and ease of learning (95.8%; 92.3%). Convenience, comfort and ease of use were stated to be better by a majority of pre-study syringe users and similar by pre-study pen users. At the end of the initial study, 73.6% of syringe users and 55.5% of previous pen users said they would continue to use the HumaPen, and 171 patients continued into the extension. As expected, the most common adverse event was hyperglycaemia but the majority were mild and none caused discontinuation from HumaPen use. The main reason for not continuing into the extension period, and for return of pens, was the injection force, which was subsequently reduced by modification of design and production prior to launch. The HumaPen therefore appeared well accepted and safe in this global study and a majority said they would recommend it to other patients. PMID- 10741344 TI - Apolipoprotein B metabolism in dyslipidaemia and the effect of drugs. PMID- 10741345 TI - Muscle amino acid metabolism at rest and during exercise. PMID- 10741346 TI - Amino acid availability controls muscle protein metabolism. PMID- 10741347 TI - Physiological effects and potential applications of GH and IGF-I. PMID- 10741348 TI - Treatment of growth hormone deficiency syndrome in adults. PMID- 10741349 TI - Physiologic effects and potential use of androgens. PMID- 10741350 TI - Guidelines for diabetes care. A desktop guide to type 1 (insulin-dependent) diabetes mellitus. European Diabetes Policy Group 1998. PMID- 10741351 TI - Aging and auditory distraction. PMID- 10741352 TI - Frequency-following response: effects of interaural time and intensity differences. AB - This research investigated whether brainstem neural mechanisms that mediate lateralization of sounds can be extracted from the frequency-following response (FFR). Monaural and binaural FFRs were obtained from normal-hearing subjects to low-frequency (500 Hz) linearly gated tone bursts (4-4-4 msec) at 40, 50, and 60 dB SL and four interaural time differences (ITDs) (0, 333, 500, and 667 microsec). FFRs were also recorded to ITDs and intensity presented in concert and in opposition (lateralization stimuli). The results show that overall intensity and interaural time differentially affect the FFR. The FFRs evoked by ITDs and intensity (in concert and in opposition) are strikingly different. The normalized amplitudes of the binaural interaction component (BIC) are minimally altered by ITDs and intensity. The study presents strong evidence that ITDs of 0, 333, 500, and 667 microsec and lateralization stimuli, easily discriminated perceptually, evoke clearly distinguishable FFR waveforms. These ITDs provide the cues that mammals use to localize sound in a freefield. The BIC is essentially unaffected by overall intensity, ITDs, and lateralization stimuli. Based on the findings of this study, the FFR has the potential to become a tool for identification of normal and abnormal binaural processing at lower brainstem levels. PMID- 10741353 TI - Identification of the mismatch negativity in the responses of individual listeners. AB - The mismatch negativity (MMN) was recorded from normal adults in three stimulus conditions: two contrast conditions and a control condition in which standard and deviant stimuli were identical. Averaged waveforms were analyzed by examiners blind to the evoking stimulus condition. Hit rates, a false alarm rate, and d' values were determined based on the number of MMNs identified in each condition. Hit rates were low and the false alarm rate was relatively high, resulting in unacceptably small d' values. The relationship between MMN findings and corresponding behavioral discrimination data for individual listeners was not systematic. Factors that may contribute to ambiguity and error in MMN data analysis are discussed. PMID- 10741354 TI - Speech recognition with in-the-ear and behind-the-ear dual-microphone hearing instruments. AB - The primary purpose of this study was to compare the overall listening benefit in diffuse noise provided by dual-microphone technology in an in-the-ear (ITE) hearing instrument to that provided by dual-microphone technology in a behind-the ear (BTE) hearing instrument. Further, the study was designed to determine whether the use of the dual-microphone + the manufacturer's party response algorithm in the ITE and BTE hearing instruments provided listening benefit in diffuse noise over their respective omnidirectional microphone modes. Twenty-four adults with mild to moderately severe sensorineural hearing loss were evaluated while wearing binaural BTE and ITE hearing instruments. The results indicated that the dual-microphone + party response mode did provide significant benefit in diffuse noise for both the ITE (3.27 dB signal-to-noise ratio [SNR] improvement) and BTE (5.77 dB SNR improvement) hearing instruments relative to their respective conventional omnidirectional microphones. No significant difference in performance was found between the ITE and BTE hearing instruments when each device was in the dual-microphone + party response mode. It is concluded that the use of dual-microphone technology in both ITE and BTE hearing instruments can improve speech recognition in diffuse noise. PMID- 10741355 TI - Electrophysiologic signs of auditory distraction in elderly listeners. AB - Brain mapping was used to investigate the ability of young and elderly female listeners to attend to /ga/ syllabic events at one ear in the presence of speech babble competition at the opposite ear. An oddball stimulus presentation paradigm was used to record the N1 and P2 components of the late auditory evoked potential (LAEP) from 19 scalp locations. With speech competition, elderly listeners exhibited significantly larger reductions in P2 amplitude than did young listeners. The competition produced no changes in N1 amplitude in either group. These findings contrast with those of an earlier study in which age-related reductions in N1 but not P2 amplitude were found when listeners attended to tones rather than speech stimuli in the presence of speech competition. These studies suggest that amplitude reductions in different LAEP components may provide electrophysiologic indices of age-related breakdowns in processing sounds in the presence of competition. Which LAEP components are affected may depend on experimental variables such as task difficulty or the nature of the stimuli (e.g., speech vs nonspeech). PMID- 10741356 TI - The CON-SOT-LOT test for nonorganic hearing loss. AB - The efficacy of a screening procedure for detecting nonorganic hearing loss using standard audiometric instrumentation was examined. Thirty normal-hearing subjects' responses to continuous and standard and lengthened off-time pulsed tones, using an ascending-descending method, were compared. Subjects were asked to respond to test stimuli in normal-hearing and simulated hearing loss conditions. Data show that there are clinical and statistical differences between subjects' response patterns in the normal-hearing condition as compared to the feigned hearing loss condition. The procedure is offered as a simple method of screening for nonorganic hearing loss. PMID- 10741357 TI - Anticipatory strategy training: implications for the postlingually hearing impaired adult. AB - The present study was designed to determine if speech recognition performance will improve after subjects prepare for an unfamiliar communication situation as opposed to a familiar one. Forty-five normal-hearing subjects were divided into three groups: one trained using a well-known fairytale, one trained using an obscure fable, and one without training. Post-training, all groups performed similarly when tested on the familiar tale. When test material involved the unfamiliar fable, only the group trained on that material obtained significantly better scores than the other groups. Results support teaching clients that increasing their knowledge of upcoming unfamiliar events can improve subsequent speech recognition. PMID- 10741358 TI - Mechanism of injury in foot fractures. PMID- 10741359 TI - Piriformis syndrome. PMID- 10741360 TI - "Bungee cord" effect in hamstring tendon ACL reconstruction. PMID- 10741361 TI - What is going on in medicine? PMID- 10741362 TI - Screw augmentation: the gold standard for cementless cup fixation. PMID- 10741363 TI - Noncemented fixation without screws is better. PMID- 10741364 TI - Iliac crest bone-muscle transfer in periacetabular osteotomy. AB - Ganz et al reported acetabular osteotomy for extensive acetabular reorientation without changing the acetabular diameter. This article reports our modification of this surgical technique, which preserves the integrity of the tensor fascia latea muscle as well as providing viable bone for bone graft at the anterior iliac osteotomy site. The interval between the sartorius and tensor fascia lata is developed distal to the anterosuperior iliac spine. After iliac osteotomy through the anterosuperior iliac spine, the distal portion of the anterosuperior iliac spine (acetabular segment), along with its slip of tensor fascia lata (1-2 cm), is separated and shaped to fit the anterior bone gap. Two small holes are drilled into that segment and also into the distal cut surface in the anterior portion of the remaining crest so that when the sutures are tightened, the tensor muscle is preserved anatomically and bone grafting is achieved. PMID- 10741365 TI - Contact stresses with an unresurfaced patella in total knee arthroplasty: the effect of femoral component design. AB - Compressive contact stresses between the patella and the anterior femur were measured with a digital electronic sensor before and after total knee arthroplasty (TKA) in 10 cadaver knee specimens. Contact stresses were measured first in normal knees, then after TKA with the Insall-Burstein Total Condylar, Miller Galante II, Ortholoc II, Porous Coated Anatomic, and Profix knee prostheses implanted without resurfacing the patella. The Insall-Burstein, Miller Galante II, and Ortholoc II prostheses had significantly higher contact stresses than the normal knee throughout the flexion arc. The Porous Coated Anatomic, which has a smooth patellar groove, maintained contact area as in the normal knee and did not have significantly higher contact stresses at flexion angles <90 degrees. At flexion angles > or =105 degrees, patellofemoral contact occurred in two small areas as the patella encountered the intercondylar notch in all components except the Profix. The Profix maintained full contact and low compressive stresses throughout the full flexion arc because of its posteriorly extended patellar groove. Design features of the patellofemoral portion of TKA components are important factors that affect contact stresses in the patellofemoral joint. These features likely will affect the clinical results of TKA with an unresurfaced patella. PMID- 10741366 TI - Long-term results of periosteal transplantation in osteochondritis dissecans of the knee. AB - Between 1986 and 1991, a total of 18 patients (11 men and 7 women) with osteochondritis dissecans of the knee were treated with periosteal transplantation. Median patient age was 19 years (range: 16-45 years). Eight patients were reoperated up to 8 years postoperatively, due to reduced range of motion, synovitis, or formation of an exostosis in the transplanted area. Of 14 patients who were available for follow-up after 8 years (range: 5-10), 2 were completely pain free. Six patients had reduced range of motion, knee instability, or quadriceps muscle atrophy. The number of reoperations and the presence of continued knee pain in most patients does not justify the extensive procedure of periosteal transplantation. PMID- 10741367 TI - Factors related to hip muscle weakness following fixation of acetabular fractures. AB - Two hundred twenty-five acetabular fractures treated surgically by one surgeon using a single surgical approach were reviewed to identify the incidence of clinically significant hip muscle weakness. Clinical grade of fair or poor and presence of radiographic signs of osteoarthritis were most strongly correlated with hip muscle weakness (P<.0001). Other factors related to muscle weakness were >21 days to repair, abdominal trauma, infection, avascular necrosis, heterotopic ossification resection, chest trauma, and head trauma. The presence of arthritis at follow-up increased the incidence of hip muscle weakness (46% [41/89]; P<.0001) independent of surgical approach. PMID- 10741368 TI - The impact of simulated distal interphalangeal joint fusion on grip strength. AB - This study assessed the impact of distal interphalangeal (DIP) joint fusion on grip strength and how this may be related to a profundus quadriga. Nineteen adults (12 men and 7 women) with an average age of 32.6 years (range: 24-61 years) underwent a series of grip strength measurements with simulation of DIP arthrodesis to the index, long, and index and long (index-long) fingers. The nondominant hand served as a control for the testing. All participants were tested in the same manner: baseline dominant and nondominant hands without DIP block followed by blocking of DIP flexion on the dominant hand only for index, index-long, and long finger alone. Nondominant hands also were tested each time, but without blocking to serve as a control for normal changes in grip strength with repeated testing. Grip strength values were compared to baseline for each trial using the Student's t test. Significant decreases in grip strength were seen for all DIP blocking compared with baseline, but no significant differences were noted in the equivalent trial in the nondominant, nonblocked hand. These findings may have clinical relevance when performing DIP arthrodesis. PMID- 10741369 TI - The lumbosacral nerves in relation to dorsal S1 screw placement and their locations on plain radiographs. AB - Seven adult cadaver lumbopelvises were harvested to study the anatomic relationship of the L4 and L5 nerves to S1 dorsal screw placement and the location of the L4, L5, and S1 nerves on plain radiographs. The mean lateral angle of S1 screw trajectory toward the L4 nerve was 31+/-8 degrees, and the mean screw trajectory length was 53+/-8 mm. The mean lateral angle of the screw trajectory toward the L5 nerve was 21+/-8 degrees, and the mean screw trajectory length was 38+/-4 mm. On both inlet and outlet radiographs, the lateral angle of the nerves increased from L4 to S1. The L4 nerve coursed over the middle third of the superior ala in the inlet view and the middle third of the lateral mass in the outlet view. The L5 nerve coursed over the inner third of the superior ala and inner third of the lateral mass. On the lateral view, the mean distances from the sacral promontory to the L4, L5, and S1 nerves along the anterior border of the sacrum were 4+/-7 mm, 12+/-5 mm, and 28+/-8 mm, respectively. This study suggests that S1 sacral screws be directed between 30 degrees and 40 degrees lateral to avoid compromising the lumbosacral trunk and sacroiliac joint. PMID- 10741370 TI - Indications for instrumentation in degenerative lumbar spinal disorders. PMID- 10741371 TI - Osteosarcoma of the coracoid process. PMID- 10741372 TI - Migration of a Knowle's pin from the neck of the femur to the knee joint. PMID- 10741373 TI - Brodie's abscess of the hip simulating osteoid osteoma. PMID- 10741374 TI - Radiologic case study. Popliteal cyst (Baker's cyst). PMID- 10741375 TI - A prostaglandin E1 analog, OP-1206, alleviates 5-fluorouracil-induced injury of rat small intestine. AB - 5-Fluorouracil (5-FU) often causes the gastrointestinal toxicity, including enterocolitis. We investigated effects of OP-1206 (17S, 20-dimethyl-trans-delta2 prostaglandin E1) on 5-FU-induced leukocyte infiltration and epithelial barrier dysfunction of rat small intestine. Myeloperoxidase (MPO) activity of the small intestine was assayed as an index of leukocyte infiltration. Intestinal epithelial permeability was determined by the small intestinal absorption of a paracellular permeation marker, fluorescein isothiocyanate-labeled dextran (molecular weight; 4,400) (FD-4) using the in situ closed intestinal loop technique. The MPO activity and FD-4 permeation were significantly increased by the administration of 5-FU to rats for 4 days, while on the coadministration of 5 FU and OP-1206, they were similar to those of control rats treated with saline solution alone, respectively. These observations indicate that OP-1206 reduced the leukocyte infiltration and the change in epithelial permeability of rat small intestine induced by 5-FU. PMID- 10741376 TI - Changes of hepatic vitronectin levels in patients with chronic hepatitis C treated with interferon alpha. AB - Hepatic vitronectin expression was assessed in 27 patients with chronic hepatitis C before and after interferon alpha treatment and in 7 control patients. Before interferon therapy, vitronectin was localized in the hepatocytes and in the portal and central venous regions. A high correlation was found for the vitronectin expression level with the histological grading and staging scores in the hepatocytes as well as in the portal region. After interferon therapy, the hepatic vitronectin was significantly decreased in the sustained and transient responders, but it was not as markedly decreased in the nonresponders and the non treated group. A good correlation was found for the vitronectin expression with the staging scores but not with the grading scores in the portal region. These findings suggest that hepatic vitronectin is influenced by interferon therapy and that it may play an important role as a hepatic adhesion molecule through the improvement of inflammation, necrosis and fibrogenesis. PMID- 10741377 TI - Age- and sex-related changes in toluene metabolism by rat hepatic microsomes in vitro. AB - Age- and sex-related changes in toluene metabolism by hepatic microsomes of male and female Sprague-Dawley (SD) rats (1 to 20 weeks) were investigated. A major metabolite of toluene, benzyl alcohol (BA), was measured by high-performance liquid chromatography (HPLC). At low substrate toluene concentrations (0.4 mM), in male rats, BA increased dramatically with development, reaching a peak at 5 weeks of age, rapidly decreasing thereafter. In female rats, BA increased dramatically with development at 3 to 5 weeks of age, and then declined gradually to a low level. Gender differences were obtained at 5 and 20 weeks of age, with BA products being higher in males than in females. At high substrate toluene concentrations (5.0 mM), in male rats, the BA formation pattern was similar to that at the low substrate concentration, although the rate of increase with age was slower. In female rats, a peak was obtained at 3 weeks of age, and then declined gradually to a low level. Gender differences were obtained at 5, 15 and 20 weeks of age, with BA products being higher in males than in females. These results indicate that toluene metabolism exhibits age and gender differences. PMID- 10741378 TI - Interaction of hyaluronic acid with mucin, evaluated by gel permeation chromatography. AB - Hyaluronic acid (HA) is known to increase the ocular bioavailability of ophthalmic drugs not only for its viscous properties but also for its specific affinity for ocular mucins. This phenomenon, called bio- or mucoadhesion, can be evaluated in vitro by mechanical tests which, however, require considerable amounts of mucin (M) that are difficult to obtain from ocular surfaces. Thus, we developed an alternative method, based on gel permeation liquid chromatography, to examine the interaction of HA with microgram quantities of mucin. HA (from human umbilical cord or rooster comb) were fractionated using a Sepharose CL-4B column, before and after incubation with porcine gastric mucin (PGM), and the fractions were analyzed by a specific assay based on the histological dye Stains all. PGM interacted with high molecular weight (M.W). HA, causing the displacement of low M.W., non-covalently bound, HA fragments, which were eluted under a distinct chromatographic peak. By quantitating the relative area of this peak, an evaluation of the mucoadhesion of HA could be obtained. This method could be useful to study the interaction between HA and microgram quantities of ocular M (mucin), obtained from individual patients or normal subjects. PMID- 10741379 TI - Mild hyperthermia-induced apoptosis is dependent on p53 in human lymphoid cells. AB - Mild hyperthermia is known to enhance apoptosis. The p53 tumor-suppressor gene product has been shown to function in apoptosis in response to genotoxic stress. However, there is little information regarding the mechanism of p53-dependent apoptosis induced by heat stress. In present study, a p53 contribution in mild hyperthermia-induced apoptosis was investigated in human lymphoid system. After 30-minute exposure at 44 degrees C, the accumulation of p53 protein was clearly observed in TK6 and ML-1 cells. Using comet assay, the more significant sensitivity to hyperthermic apoptosis was found in TK6 (wild-type p53) than in WI L2-NS (mutated in p53). Furthermore, the significantly rapid shifting from early apoptotic phase to late apoptotic was observed in heat-induced p53 TK6 cells. These findings suggest that p53-dependent apoptosis is efficaciously induced by mild hyperthermia as non-genotoxic stress in human lymphoid system. PMID- 10741380 TI - Nephrotoxic effects of allopurinol in dinitrofluorobenzene-sensitized mice: comparative studies on TEI-6720. AB - Allopurinol is widely used and generally well-tolerated. However, when used in patients with renal insufficiency it may have life-threatening toxic effects known as allopurinol hypersensitivity syndrome (AHS). We previously found that allopurinol increased ear swelling and mortality in a DNFB-induced contact hypersensitivity mouse model. In the present study, we investigated the toxic effect of allopurinol on DNFB-sensitized mice in order to clarify the mechanism responsible for the lethal effect of allopurinol. Allopurinol increased plasma GPT and GOT in DNFB-sensitized mice and markedly increased plasma creatinine and BUN. The increase in plasma GPT and GOT was moderate and declined time dependently. In contrast, the increase in plasma creatinine and BUN was striking and continued until 18 hr after administration of allopurinol at 100 mg/kg/day. Although allopurinol increased GOT and GPT in DNFB-sensitized mice, no effect was observed in non-sensitized mice even at 100 mg/kg/day, indicating that allopurinol essentially has no toxic effect on the liver. A high dose of allopurinol induced renal impairment even in non-sensitized mice. These observations indicate that there is some biological interaction between allopurinol and DNFB, and suggest that allopurinol may modulate or enhance the inflammatory reactions induced by DNFB, and/or that DNFB may cause metabolic changes via inflammation, leading to the enhanced toxicity of allopurinol. In contrast, TEI-6720, a newly synthesized XOD/XDH inhibitor, had almost no effect on DNFB-sensitized mice. TEI-6720 at 1 mg/kg, in terms of hypouricemic effect, appeared to be more potent than allopurinol at 3 mg/kg. Therefore, the nephrotoxic effect of allopurinol observed in the present study may not be related to XOD/XDH inhibitory activity. PMID- 10741381 TI - A comparative study on the hypouricemic activity and potency in renal xanthine calculus formation of two xanthine oxidase/xanthine dehydrogenase inhibitors: TEI 6720 and allopurinol in rats. AB - In this study, the hypouricemic efficacy of a novel xanthine oxidase/xanthine dehydrogenase inhibitor, TEI-6720, was compared with that of allopurinol in a hyperuricemic rat model established by feeding the animals oxonate, a uricase inhibitor. In addition, using normal rats, the changes in xanthine concentration in plasma and the concentrations and absolute quantities of uric acid, allantoin and xanthine in urine were analyzed during a 28-day period of repeated administration of TEI-6720 to determine the changes occurring during this period and the conditions required for the formation of xanthine crystals and calculi in comparison with allopurinol. TEI-6720 and allopurinol caused a significant dose dependent decrease in plasma uric acid levels in the hyperuricemic rat model and the ED50 of TEI-6720 was lower than that of allopurinol, indicating that in terms of hypouricemic efficacy TEI-6720 is more potent than allopurinol. TEI-6720 also showed more potent activity than allopurinol in decreasing urinary uric acid and allantoin levels in normal rats. In addition, TEI-6720 and allopurinol showed similar dose-response curves for the decrease in uric acid or allantoin concentration, and the associated increase in xanthine concentration, indicating that TEI-6720 and allopurinol have similar pharmacological characteristics although the dosage required differs. The efficacy of TEI-6720 in increasing plasma and urinary xanthine levels in normal rats was approximately 10- to 30 fold greater than that of allopurinol. However, with respect to renal xanthine calculus formation, there was only about a 3-fold difference in dosage comparing TEI-6720 and allopurinol. This difference suggests that there may be another factor independent of xanthine, and dependent on the drug itself, involved in renal calculus formation caused by allopurinol. The daily excretion of purine metabolites per body weight was about 20-fold higher in rats than in humans. From these results, it is concluded that TEI-6720 has potent hypouricemic activity and that, compared to allopurinol, administration of TEI-6720 is not likely to result in a higher incidence of calculus formation. PMID- 10741382 TI - Effects of mibefradil, a selective T-type Ca2+ channel antagonist, on sino-atrial node and ventricular myocardia. AB - The effects of mibefradil, a non-dihydropyridine Ca2+ channel antagonist, on the action potential configuration of isolated rabbit sino-atrial node preparations, membrane currents of guinea-pig ventricular myocytes and the contractile force of isolated ventricular papillary muscles were examined. In sino-atrial node preparations, 10 microM mibefradil decreased the slope of the pacemaker depolarization (phase 4 depolarization) and maximum rate of rise, and shifted the threshold potential to the positive direction with no effect on action potential duration. In ventricular myocytes, 1 microM mibefradil inhibited the T-type Ca2+ current by about 40% while it had no effect on the L-type Ca2+ current. At 10 microM, mibefradil inhibited the L-type and T-type Ca2+ currents by about 40% and 90%, respectively. Mibefradil had no effect on contractile force at concentrations up to 1 microM. Thus, mibefradil was shown to produce potent prolongation of the pacemaker depolarization, mainly through inhibition of the T type Ca2+ current. It is suggested that the T-type Ca2+ current may not be involved in ventricular contraction. PMID- 10741383 TI - D2 autoreceptors are not involved in the down-regulation of the striatal dopamine transporter caused by alpha-methyl-p-tyrosine. AB - The mechanisms by which the brain dopamine neuronal transporter is regulated by chronic alteration of dopamine transmission are not well understood. It has been shown previously that chronic inhibition of dopamine synthesis decreases dopamine transporter (DAT) density and function. The purpose of the present study was to determine whether these effects involve dopamine D2 receptors. Chronic treatment with alpha-methyl-p-tyrosine decreased binding of [3H]mazindol and dopamine release by d-amphetamine. The down-regulation of the DAT by alpha-methyl-p tyrosine was not altered by co-treatment with a D2 receptor agonist or antagonist. However, chronic treatment with a D2 agonist, quinpirole, also decreased mazindol binding and amphetamine-induced release of dopamine. The results indicate that chronic inhibition of dopamine synthesis and stimulation of D2 receptors have similar, but independent, effects on DAT binding and function. PMID- 10741384 TI - Determination of a new asiatic acid derivative, AS 2-006A in rat plasma and urine, and human plasma by high-performance liquid chromatography. AB - A high-performance liquid chromatographic method was developed for the determination of a new wound healing agent, AS 2-006A (ethoxymethyl 2-oxo-3, 23-O isopropylideneasiatate), in rat plasma and urine, and human plasma. The sample preparation was simple: 2 volumes of acetonitrile were added to the biological samples to deproteinize it. A 50-microl aliquot of the supernatant was injected onto the reversed-phase column. The mobile phase employed was acetonitrile : H2O (9:1, v/v) and run at a flow rate of 1.1 ml/min. The column effluent was monitored by a UV detector set at 205 nm. The retention time for AS 2-006A was approximately 29.5 min. The detection limits for AS 2-006A in rat and human plasma were both 1 microg/ml, and in rat urine was 2 microg/ml. The coefficients of variation of the assay (within-day and between-day) were generally low (below 10.8%) for rat plasma and urine, and human plasma. No interferences from endogenous substances were found. PMID- 10741385 TI - Inhibition of calcium ionophore-stimulated leukotriene generation from intact human neutrophils by captopril. AB - Effects of captopril on the formation of leukotrienes (LTs) from stimulated intact human neutrophils were investigated. Neutrophils were stimulated with 1 microM calcium ionophore A23187 for the generation of LTs. A reverse phase high performance liquid chromatography technique and UV spectroscopy were used to detect and quantitate the released LTs namely, LTB4. LTC4. delta6-trans-LTB4 and delta6-trans-12-epi-LTB4. Preincubation of neutrophils with captopril significantly reduced LTB4 formation in a concentration-dependent manner, as compared to diluent-treated control cells. Since LTA4 is the substrate for both LTB4 and LTC4, thus in presence of captopril, shunting of LTA4 from synthesis of LTB4 was not directed to LTC4 formation. This finding was evidenced by the significant decrease of LTC4 production under the influence of high concentration of the drug. Formation of LTB4 stereoisomers, delta6-trans-LTB4 and delta6-trans 12-epi-LTB4 was not markedly altered by captopril. In subsequent experiments, when neutrophils were stimulated with A23187 after preincubation with exogenous arachidonic acid (75 microM), and treatment with captopril, similar findings were obtained for LTB4 and LTC4. Meanwhile, formation of the nonenzymatic hydrolysis products of LTA4 tended to rise reaching significant level in case of delta6 trans-LTB4, at the high concentration of captopril. These results demonstrate that captopril is an inhibitor of enzymatically generated LTs produced by intact human neutrophils, being more potent against LTB4. These effects of captopril on LTs are not mediated via an inhibition of arachidonic acid formation from membrane phospholipids. It could be suggested that captopril, at doses used clinically. could inhibit the generation of LTB4 without affecting LTC4. Consequently, these findings might account for possible antiinflammatory activity for captopril, and further suggest that some of the observed side effects of captopril might not be related to an overproduction of LTC4. PMID- 10741386 TI - Tumor counterattack--concept and reality. PMID- 10741387 TI - Intrinsic defects of B cell function in X-linked severe combined immunodeficiency. AB - The cytokine receptor common gamma chain mutation in X-linked SCID results in a failure of T and NK cell development and an as yet undefined defect of B cells. Using immunoglobulin isotype-specific reverse transcription-PCR we show that although hematopoietic stem cell transplantation restores a diverse repertoire of class-switched B cell clones, on further analysis these are almost all of donor origin. This suggests that host B cells, which predominate after unconditioned transplantation, are still defective even in the presence of normal T cells. These studies imply that effective humoral reconstitution can only be achieved by the engraftment of normal donor B cells. PMID- 10741388 TI - Regulation and kinetics of premigrant thymocyte expansion. AB - Normal mature thymocytes proliferate before emigrating to the periphery, and continuous bromodeoxyuridine labeling showed that more than 30 % of fully mature thymic emigrants have replicated DNA in the 24 h before exit. The percentage of DNA-synthetizing single-positive (SP) thymocytes is transiently augmented during the postnatal period, with peaks on days 2 and 4 for CD4 and CD8 cells, respectively. Similar kinetics were observed in mouse chimeras made by transfer of normal bone marrow cells into RAG-2-deficient mice. These data show that proliferation of mature thymocytes is developmentally regulated. The proliferation peaks (on days 16 and 18 post transfer) observed in simple bone marrow chimeras were abolished when lymph node T cells were mixed with the bone marrow cell inoculum, suggesting that the peripheral pool controls the late thymic expansion. The phenotype of cycling SP thymocytes is atypical: they do not regulate activation and adhesion surface molecules like peripheral activated T cells. PMID- 10741389 TI - Induction of resistance to active experimental allergic encephalomyelitis by myelin basic protein-specific Th2 cell lines generated in the presence of glucocorticoids and IL-4. AB - We have produced T cell lines with a Th2 phenotype in the presence of IL-4 and the glucocorticoid dexamethasone (DEX). IL-4 and DEX together were more effective in inducing a Th2 response than IL-4 alone. Myelin basic protein (MBP)-specific Th2 lines were obtained and their ability to induce experimental allergic encephalomyelitis (EAE) was studied. Lines treated with IL-4 and DEX did not transfer passive EAE and did not induce cellular infiltration into the central nervous system as opposed to the encephalitogenic Th1 lines. Lines treated with IL-4 and DEX did not protect animals from the effect of encephalitogenic Th1 lines when the two were injected together. However, a high proportion of animals injected with IL-4 + DEX-treated lines became refractory to the development of EAE after immunization with MBP; that is, it was possible to induce resistance to active EAE without prior episodes of disease. Interestingly, animals injected with T cell lines had accelerated antibody responses against MBP and the predominant isotype was dependent on the cytokines synthesized by the T cell line injected. There was not evidence that the resistance to active EAE was due to anergy of MBP-reactive cells or the action of CD8+ cells. Our data suggest that MBP-specific T cell lines prevent the induction of disease by deviating the reactivity to MBP from a cell-mediated to a humoral one and not merely from a Th1 to a Th2 response. PMID- 10741390 TI - The B cell receptor, but not the pre-B cell receptor, mediates arrest of B cell differentiation. AB - B cell development in organ cultures of fetal liver from mice at day 14 of gestation resembles in kinetics and cell numbers generated the one observed in vivo. This development in vitro can be blocked by an IL-7 receptor-specific monoclonal antibody. Monoclonal antibodies specific for the pre-B cell receptor, i. e. for VpreB, lambda5, or muH chains, do not perturb B cell development in these organ cultures up to and including the CD25+ small pre-BII cell stage. However, muH chain-specific antibodies inhibit the appearance of the subsequent surface IgM+ immature B cells. In organ cultures of muH chain allotype heterozygous (muHa x muHb)F1 fetal livers a dose-dependent inhibition by allotype specific monoclonal antibodies of sIgM+ immature B cells expressing the corresponding, but not the other, allotype was observed. By combining cell sorting with limiting dilution analysis of lipopolysaccharide-reactive cells, the probable target cell of this muH chain-specific inhibition was identified as an IgM+, CD23-immature B cell. Hence, engagement of the pre-B cell receptor by specific antibodies does not influence B cell development, while engagement of the B cell receptor on immature B cells does. PMID- 10741391 TI - Modulation of host immune responses stimulated by Salmonella vaccine carrier strains by using different promoters to drive the expression of the recombinant antigen. AB - We evaluated whether immune responses stimulated by Salmonella vaccine carriers can be modulated by using different promoters to drive antigen expression. Mice were orally immunized with strains transfected with plasmids carrying beta galactosidase (beta-gal) under the control of either a constitutive or an in vivo activated promoter. While alpha-gal-reactive IgG1, IgG2a, IgG2b and IgG3 were detected in sera of mice immunized with Salmonella expressing constitutively beta gal, higher titers dominated by IgG2a and IgG2b were detected in sera when the in vivo-activated promoter was used. beta-gal-specific proliferative responses of spleen-derived CD4+ T lymphocytes were similar in both groups. However, CD4+ T lymphocytes from mice immunized with the constitutive promoter secreted IL-4, IL 5, IL-6, IL-10 and IFN-gamma (Th1/Th2 pattern), whereas CD4+ cells mainly secreted IFN-gamma (Th1 pattern) when the second construct was used. The spleens of all immunized mice contained beta-gal-reactive CD8+ CTL precursors. The vaccine prototypes were tested for their capacity to control seeding and/or development within the lung of an intravenously delivered aggressive fibrosarcoma transfected with beta-gal. Reduced metastasis and significantly increased mean survival times were observed in all vaccinated mice. However, protection was improved when the carrier expressed beta-gal upon infection (80 % versus 50% survival, p < 0.05). PMID- 10741392 TI - Thymosin-alpha1 regulates MHC class I expression in FRTL-5 cells at transcriptional level. AB - In this study we examined the effect of the synthetic peptide thymosin-alpha1 (T(alpha)1) on MHC class I expression in FRTL-5 cells. Treatment with T(alpha)1 increased expression of MHC class I surface molecules and mRNA, which reached its peak (153 +/- 8 % of the control value) after 12 h. Chloramphenicol acetyltransferase (CAT) analysis, following transfection with a plasmid containing the regulatory sequence of MHC class I (or its deletion derivatives) with the CAT reporter gene, and electrophoretic mobility shift assay experiments demonstrated that the action of T(alpha)1 was at the transcriptional level, and its mechanism of action is likely due to increased binding between the complex p50/fra-2 and the enhancer A sequence of the 5' flanking region of a swine class I gene (PD1). An increase in the expression of MHC class I surface molecules was also observed by flow cytometry in murine and human tumor cell lines and in primary cultures of human macrophages. This study shows for the first time an effect of Talpha1 on the regulation of gene expression at the molecular level, and may further contribute to explaining the results obtained using Talpha1 in the control of infectious diseases and tumor growth. PMID- 10741393 TI - 2B4 functions as a co-receptor in human NK cell activation. AB - Natural cytotoxicity receptors (NKp46, NKp44 and NKp300) play a predominant role in human NK cell triggering during natural cytotoxicity. Human 2B4 also induced NK cell activation in redirected killing assays using anti-2B4 monoclonal antibodies (mAb) and murine targets. Since this effect was confined to a fraction of NK cells, this suggested a functional heterogeneity of 2B4 molecules. Here we show that activation via 2B4 in redirected killing against murine targets is strictly dependent upon the engagement of NKp46 by murine ligand (s) on target cells. Thus, NK cell clones expressing high surface density of NKp46 (NKp46bright) were triggered by anti-2B4 mAb, whereas NKp46dull clones were not although they expressed a comparable surface density of 2B4. mAb-mediated modulation of NKp46 molecules in NKp46bright clones had no effect on the expression of 2B4 while it rendered cells unresponsive to anti-2B4 mAb. Finally, anti-2B4 mAb could induce NK cell triggering in NKp46dull clones provided that suboptimal doses of anti-NKp44 or anti-CD16 mAb were added to the redirected killing assay. These results indicate that differences in responses do not reflect a functional heterogeneity of 2B4 but rather depend on the co-engagement of triggering receptors. PMID- 10741394 TI - Dendritic cells and differential usage of the MHC class II transactivator promoters in the central nervous system in experimental autoimmune encephalitis. AB - In the normal central nervous system (CNS) expression of MHC class II is minimal, but has been found to be highly up-regulated on microglia cells in experimental autoimmune encephalitis (EAE). Here we used the EAE model to examine the regulation of expression of the class II transactivator (CIITA), which is required for activation of MHC class II genes. EAE was induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein peptide 35-55. CIITA mRNA form I (specific for dendritic cells) and form IV (IFN-gamma inducible) but not form III (B cell specific) were detected in brain and spinal cord of mice with acute EAE. In unimmunized or mock-immunized mice, none of the three CIITA forms was found to be induced. Dendritic cells (DC) were identified by immunostainings for CD11c in perivascular and meningeal cell infiltrates in EAE spinal cord and brain. Time-course analysis showed (1) the appearance of DC in the CNS shortly before onset of disease, (2) the recruitment of CD11b cells occuring much earlier and (3) the absence of CIITA and MHC class II expression in these CD11b+ cells at preclinical stages. PMID- 10741395 TI - The cancer germ-line genes MAGE-1, MAGE-3 and PRAME are commonly expressed by human myeloma cells. AB - In this study, we have investigated the mRNA expression of the cancer germ-line genes MAGE, BAGE, GAGE, RAGE and the tumor-overexpressed gene PRAME by human myeloma cell lines and malignant plasma cells from patients with multiple myeloma (MM). By reverse transcription-PCR, we show that all myeloma cell lines (n = 16) express at least one of these genes, except RAGE-1 that was never expressed. We show that malignant plasma cells from the majority of MM patients (n = 21) expressed MAGE-1, MAGE-3 and PRAME. On the contrary, polyclonal reactive plasma cells did not express any of these genes. By flow cytometry, we show that mage-1 protein is expressed within myeloma cells and cell lines and that anti-mage-1.HLA A1 cytotoxic T lymphocytes efficiently killed MAGE-1+HLA-A1+ MDN myeloma cells. Taken together, our data show that mage-1 and mage-3 could constitute specific targets for tumor immunotherapy of MM patients. PMID- 10741396 TI - Binding of T lymphocytes to hippocampal neurons through ICAM-5 (telencephalin) and characterization of its interaction with the leukocyte integrin CD11a/CD18. AB - Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a member of the immunoglobulin superfamily expressed on telencephalic neurons, and serves as a ligand for the leukocyte integrin CD11 a/CD18. We studied here the binding site in ICAM-5 for CD11a/CD18. Protein constructs containing the first immunoglobulin domain of ICAM-5 were able to support CD11a/CD18 interaction, while deletion of the first domain abolished binding. Monoclonal antibodies reacting with the first domain of ICAM-5 also completely blocked the interaction. The soluble first domain of ICAM-5 inhibited the binding of T cells to immobilized ICAM-5 at concentrations of 50 nM and higher. Interestingly, the sixth domain of ICAM-5 was also able to support leukocyte binding, but this binding activity may not involve leukocyte integrins. To test the involvement of ICAM-5 in leukocyte-neuron interactions, an assay using human T cells binding to rat hippocampal neurons was established. This binding was blocked by monoclonal antibodies against CD11a/CD18 and ICAM-5. Thus ICAM-5 may act as a major adhesion molecule for leukocyte binding to neurons in the central nervous system. PMID- 10741397 TI - Human intestinal lamina propria and intraepithelial lymphocytes express receptors specific for chemokines induced by inflammation. AB - To determine which chemokine receptors might be involved in T lymphocyte localization to the intestinal mucosa, we examined receptor expression on human intestinal lamina propria lymphocytes (LPL), intraepithelial lymphocytes (IEL) and CD45RO+beta7hi gut homing peripheral blood lymphocytes (PBL). Virtually all LPL and IEL expressed CXCR3 and CCR5, receptors that have been associated with Th1(Tc1)/Th0 lymphocytes, while CCR3 and CCR4, receptors associated with Th2 (Tc2)lymphocytes, CCR7, CXCR1 and CXCR2 were not expressed. CXCR3 and CCR5 receptors were functional, as LPL and IEL migrated to their respective ligands I TAC and RANTES. In addition, most alphaEbeta7- LPL and IEL expressed high levels of CCR2. While the majority of CD45RO(-)beta7hi PBL also expressed CXCR3 and CCR5, a proportion of these cells were CXCR3- and/or CCR5- and some expressed CCR4 and/or CCR7, indicating that lymphocytes recruited to the intestinal mucosa represent a subset of these cells. In summary, our results show that LPL and IEL within the normal intestine express a specific and similar array of chemokine receptors whose ligands are constitutively expressed in the intestinal mucosa and whose expression is up-regulated during intestinal inflammation. These results support the view that CXCR3, CCR5 and CCR2 may play an important role in lymphocyte localization within the intestinal mucosa. PMID- 10741398 TI - Engagement of CD33 surface molecules prevents the generation of dendritic cells from both monocytes and CD34+ myeloid precursors. AB - Although CD33 represents an important marker of myeloid cell differentiation, its function remains poorly defined. In view of its homology with p75/AIRM1, a recently identified surface molecule which exerts a potent inhibition on NK cell function, we re-evaluated the effect of CD33 engagement in defined myeloid cell functions. Addition of anti-CD33 mAb to cultures of CD14+ monocytes supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4 and TNF alpha, prevented the generation of dendritic cells. In these cultured cells, engagement of CD33 resulted in an increased surface binding of annexin-V, followed by cell death. Mature dendritic cells were resistant to the CD33 mediated effect. Also in CD34+ precursors, cultured in the presence of flt3 ligand, c-Kit-ligand, GM-CSF, IL-4 and TNF-alpha, addition of anti-CD33 mAb prevented the recovery of mature dendritic cells. These data suggest a regulatory role of CD33 in the myeloid cell maturation and may offer a tool to interfere with the monocyte/macrophage cell function as well as with the development of dendritic cells. PMID- 10741399 TI - IL-12 gene-deficient C57BL/6 mice are susceptible to Leishmania donovani but have diminished hepatic immunopathology. AB - To determine the in vivo role of IL-12 in the development of protective immunity in visceral leishmaniasis caused by Leishmania donovani, we examined the course of L. donovani infection in IL-12-deficient C57BL/6 (IL-12-/-) mice. IL-12-/- mice displayed significantly higher parasite burdens in their livers and spleens than wild-type C57BL/6 mice throughout the course of infection. Despite high parasite burdens, the onset of hepatosplenomegaly was significantly delayed in L. donovani-infected IL-12-/-. Moreover, livers and spleens from IL-12-/- mice displayed significantly less inflammation and poorly formed granulomatous lesions than those from IL-12+/+ mice throughout the course of infection. Antigen stimulated splenocytes from IL-12-/- mice produced significantly less IFN-gamma but more IL-4 than IL-12+/+ mice. These findings indicate that although endogenous IL-12 is critical for the development of protective immunity to L. donovani, it is also responsible for inducing the significant immunopathology associated with visceral leishmaniasis. PMID- 10741401 TI - Class I-restricted presentation of exogenous antigen acquired by Fcgamma receptor mediated endocytosis is regulated in dendritic cells. AB - We evaluated MHC class I- and II-restricted presentation of exogenous antigen by mouse bone marrow-derived dendritic cells (DC) and splenic B cells. DC presented to class I-restricted transgenic T cells femtomolar concentrations of antigens from liposomes targeted to the IgG Fc receptor. Targeting these liposomes to surface immunoglobulin did not permit B cells to stimulate class I-restricted responses. Nevertheless, both DC and B cells presented antigen from liposomes targeted to these same receptors with equivalent efficiency to class II restricted T cells. Acquisition of the capacity to present class II-restricted antigens required shorter periods of differentiation of DC than presentation of exogenous class I-restricted antigens. The latent period for class I-restricted presentation of exogenous antigen by DC could not be shortened by exposing them to lipopolysaccharide, double-stranded RNA or antibody to CD40. Class I presentation depended on expression of the TAP1 transporter. Our data are consistent with the existence of a regulated transport process present in DC which can convey exogenous antigen from endocytic vesicles to the cytosol. PMID- 10741400 TI - Site of antigen delivery can influence T cell priming: pulmonary environment promotes preferential Th2-type differentiation. AB - Delivery of foreign antigens to mucosal surfaces, such as the pulmonary airways, has been shown to preferentially induce Th2-mediated responses in humans and in mice. What is not clear from these studies is whether this preferential skewing in responses is the result of the limited types of antigen being administered and/or a bias towards using particular genetic strains of mice, or whether the lung environment in itself provides a favored site for the priming of Th2-type cells. We have addressed this issue using an antigen/mouse strain combination that, under typical conditions of immunization, is strongly biased towards priming for Th1 CD4+ T cells. We show that Leishmania major parasites delivered to C57BL/6 mice via an intranasal route fail to induce the expected Th1-dominated responses and instead preferentially prime for Th2 responses. These included an influx in lymphocytes and eosinophils into alveoli, as well as the induction of Th2-type foci of inflammation around pulmonary blood vessels and airways. Moreover, high levels of Th2-associated cytokines (IL-4 and IL-5) were generated when lung-draining lymph node and tissue cells were restimulated with L. major lysate. These data suggest that the lung environment per se favors Th differentiation towards the Th2 phenotype. PMID- 10741402 TI - Molecular cloning and protein analysis of divergent forms of the complement component C3 from a bony fish, the common carp (Cyprinus carpio): presence of variants lacking the catalytic histidine. AB - Unlike mammals, some bony fish species have been reported to possess multiple forms of the complement component C3. To explore the structural and functional diversity of bony fish C3, we have isolated eight distinct cDNA clones encoding C3 from a single carp (Cyprinus carpio). The eight sequences were grouped into five C3 types, designated C3-H1, C3-H2, C3-S, C3-Q1 and C3-Q2, each sharing 80-86 % amino acid sequence identity with the others. A striking amino acid substitution was noted at the position corresponding to the catalytic histidine, which is conserved in C3 from all the animals analyzed to date and provides the thioester with the ability to bind covalently to hydroxy groups on the target cells or to be hydrolyzed quickly; C3-S, C3-Q1 and C3-Q2 have serine, glutamine and glutamine residues, respectively, in place of the histidine which is conserved in C3-H1 and C3-H2. On the other hand, five distinct C3 forms, named C3 1 to C3-5, were purified from the serum of a single carp. N-terminal sequencing and covalent binding to [3H]glycine identified C3-1 as the translated product of C3-S, while C3-2 was that of C3-H1, and C3-5 that of C3-H2. C3-1 showed a hemolytic activity threefold higher than that of C3-2, whereas C3-5 was inactive, suggesting that the thioester catalytic mechanism is not a necessary determinant for C3 activity and that C3 lacking the catalytic histidine plays a significant role in the complement system of carp and probably other bony fish. PMID- 10741403 TI - The type II decoy receptor of IL-1 inhibits murine collagen-induced arthritis. AB - IL-1 is a key cytokine involved in the inflammatory response. The type II receptor of IL-1 (IL-1RII) acts as a decoy receptor, binding and inhibiting the effect of IL-1. This study was undertaken to establish whether IL-1RII can ameliorate collagen-induced arthritis, a model of inflammatory arthritis in mice. We used human keratinocytes transfected with the human (h)IL-1 RII gene as a source of hIL-1 RII protein. We showed that these cells expressed both the membrane and soluble form of receptor. In vitro, IL-1-stimulated murine macrophage cells showed a decreased expression of TNF-alpha in the presence of hIL-1 RII. We engrafted the hIL-1RII-transfected cells in the back of mice developing collagen-induced arthritis. We found that clinical and histological parameters of arthritis were significantly decreased in mice treated with cells producing hIL-1RII. In addition, hIL-1RII administration was able to reduce the expression of mRNA for IL-6 and myeloperoxidase in the joints of treated animals. These data show that hIL-1 RII anti-inflammatory properties in the model of collagen-induced arthritis in mice and could have a regulatory role in rheumatoid arthritis. PMID- 10741404 TI - Autonomous induction of proliferation, JNK and NF-alphaB activation in primary resting T cells by mobilized CD28. AB - Induction of proliferation in primary resting T cells requires engagement of both the antigen-specific TCR and the co-stimulatory receptor CD28. Here we report that CD28 functions as an autonomous mitogenic receptor which is mobilized by TCR signaling through cytoskeletal rearrangement. Shortcutting of TCR-dependent CD28 recruitment by stimulation with monoclonal antibodies specific for mobilized CD28 results in maximum proliferation and IL-2 secretion in primary resting T cells without activation of ZAP-70, a central component of the TCR's signal transduction machinery. Engagement of mobilized CD28 fully activates the c-Jun N terminal kinase cascade and translocation of NF-alphaB, two key targets of signal integration in co-stimulation. We propose a two-step activation model for co stimulation in primary resting T cells in which antigen recognition recruits co stimulatory receptors which then autonomously transduce signals promoting T cell proliferation. PMID- 10741405 TI - Donor cell persistence and activation-induced unresponsiveness of peripheral CD8+ T cells. AB - We studied the impact of the duration of donor cell persistence on CD8+ T cell responsiveness after adoptive transfer of antigen-expressing lymphoid cells. Naive or immunized female mice were treated by adoptive transfer of spleen cells from mice ubiquitously expressing a lymphocytic choriomeningitis virus-derived cytotoxic T lymphocyte (CTL) epitope (gp33-41) either alone or in combination with the male H-Y antigen providing additional antigenic CTL and T helper cell determinants. Low doses of male spleen cells (or sorted B cells) primed CTL, while high doses of the same cells rendered them unresponsive. CTL unresponsiveness induced by high numbers of male spleen cells was dependent upon prolonged persistence of antigen-expressing donor cells. Unresponsive CTL reverted to a state of activation when the duration of donor cell chimerism was limited. Memory CTL could be rendered unresponsive if antigen-expressing donor cells were allowed to persist. These results suggest that, irrespective of the type of antigen-presenting cell and the functional state of the responding T cell, activation and unresponsiveness can represent two different outcomes critically determined by quantitative and kinetic differences of antigen persistence. PMID- 10741406 TI - Cofilin: a missing link between T cell co-stimulation and rearrangement of the actin cytoskeleton. AB - The actin cytoskeletal network plays a regulatory role in receptor-mediated signal-transducing events. Recently, we have shown that the small actin depolymerizing protein cofilin represents a component of a co-stimulatory signaling pathway in human T cells. Cofilin is dephosphorylated on phosphoserine residues following co-stimulation via accessory receptors such as CD2, CD4, CD8 or CD28, but not in response to TCR engagement alone. Here we demonstrate that accessory receptor triggering induces the transient association of cofilin with the actin cytoskeleton. Only the dephosphorylated form of cofilin binds to cytoskeletal actin in vivo. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 block dephosphorylation of cofilin and its association with the actin cytoskeleton. These results suggest that cofilin provides an as yet missing link between functionally crucial T cell surface receptors and rearrangements of the actin cytoskeleton. PMID- 10741407 TI - CD46 (membrane cofactor protein) associates with multiple beta1 integrins and tetraspans. AB - The tetraspans associate with a large number of surface molecules, including a subset of beta1 integrins and, indirectly through CD19, with the complement receptor CD21. To further characterize the tetraspan complexes we have raised and selected monoclonal antibodies (mAb) for their ability to immunoprecipitate a molecule associated with CD9. A unique mAb was identified which recognizes the complement regulator CD46 (membrane cofactor protein). CD46 associated in part with several tetranspans and with all beta1 integrins that were tested (CD29/CD49a, CD29/CD49b, CD29/CD49c, CD29/CD49e, CD29/CD49f) but not with beta4 integrins. These data, together with cross-linking experiments showing the existence in living cells of CD46/integrin complexes, suggest that CD46 associates directly with beta1 integrins and indirectly with tetraspans. CD46 also acts as a receptor for measles virus; however, mAb to various integrins and tetraspans did not modify the virus fusion entry step. PMID- 10741408 TI - Th2 immune regulation induced by T cell vaccination in patients with multiple sclerosis. AB - T cell responses to myelin basic protein (MBP) are potentially involved in the pathogenesis of multiple sclerosis (MS). In this study, we demonstrated that subcutaneous inoculations with irradiated autologous MBP-reactive T cell clones (T cell vaccination) elicited CD8+ anti-idiotypic T cell responses and CD4+ Th2 cell responses in patients with MS. Both regulatory cell types induced by T cell vaccination contributed to the inhibition of MBP-reactive T cells while they differed in the recognition pattern and functional properties. We describe for the first time that the Th2 regulatory cells reacted with activated but not resting T cells in the context of MHC class II molecules and inhibited the proliferation of MBP-reactive T cells through the secretion of IL-4 and IL-10. The T-T cell interaction mediated by Th2 regulatory cells was independent of the antigen specificity of activated T cells. The findings have important implications for our understanding of the regulatory mechanism induced by T cell vaccination. PMID- 10741409 TI - Chemokines and CD40 expression in human fibroblasts. AB - Chemokines are cytokines specialized for recruiting leukocytes in inflammatory responses. Recent data indicate that besides macrophages and leukocytes fibroblasts may also be a source of these important immune molecules. We assayed chemokine expression (mRNA/ protein) in cultured fibroblasts isolated from a variety of human tissues and different pathologic states: normal bone marrow vs. myelometaplastic spleen, normal lung vs. metastasis stroma, and normal breast vs. radiation fibrosis and tumor stroma. In all fibroblasts, transcripts for chemokines IL-8, stromal cell-derived factor-1, monocyte chemotactic protein (MCP)-1 and eotaxin were detected. Although the production of IL-8 was abundant in most of the fibroblasts studied, fibroblasts from lung and pathologic breast tissue produced significantly less. Conversely, eotaxin production was low in most fibroblasts except in those isolated from myelometaplastic tissue where it was highly produced. Moreover, chemokines MCP-4, RANTES and macrophage inflammatory protein-1alpha were found to be expressed only in fibroblasts from select tissues. When the expression of CD40, an activating surface molecule for immune cells, was investigated, we found that most of the fibroblasts expressed this antigen. Overall these results indicate that cultured human fibroblasts from various tissues and pathologic settings produce a distinct panel of chemokines and express CD 40, suggesting a possible fundamental role of fibroblasts in immune responses and disease processes. PMID- 10741410 TI - NK cell expression of the killer cell lectin-like receptor G1 (KLRG1), the mouse homolog of MAFA, is modulated by MHC class I molecules. AB - Using a new mAb, 2F1, we characterize a mouse natural killer (NK) cell antigen termed 'killer cell lectin-like receptor G1' (KLRG1; formerly mouse MAFA or 2F1 Ag). KLRG1 is expressed on 30-60% of murine NK cells, and a small fraction of T cells, and is composed of a homodimer of glycosylated 30-38-kDa subunits. Strikingly, cell surface expression of KLRG1 by NK cells was substantially down regulated in mice deficient for expression of class I molecules, in contrast to the Ly49 lectin-like NK receptors, which are up-regulated in class I-deficient mice. We could not demonstrate binding of KLRG1 to class I molecules in a cell cell adhesion assay. Transgenic expression of KLRG1 under heterologous transcription elements was unaffected by class I deficiency, indicating that class I molecules do not affect the KLRG1 protein directly, and suggesting that regulation is at the level of expression of the endogenous KLRG1 gene. Evidence is presented that class I molecules regulate KLRG1 via interactions with class I specific inhibitory Ly49 molecules and SHP-1 signaling. Thus, although KLRG1 and Ly49 molecules are both lectin-like inhibitory receptors that are regulated by class I expression, the effects of class I on the cell surface expression of the molecules are opposing, and the underlying regulatory mechanisms are distinct. PMID- 10741411 TI - Protection of T cells from activation-induced cell death by Fas+ B cells. AB - Naive CD4+ T cells proliferate strongly in response to stimulation by superantigens such as staphylococcal enterotoxin B (SEB). However, when these same cells revert to a resting phenotype and are subjected to restimulation with either SEB or anti-CD3, the majority of these SEB-responsive cells undergo Fas ligand (FasL)-mediated activation-induced cell death (AICD). We investigated the impact of Fas expression on T cell AICD by utilizing B cell stimulators that lacked functional FasL and either expressed or did not express the Fas receptor. Our results indicate that B cells play an important role in modulating the level of T cell AICD via the Fas/FasL pathway. Activated B cells expressing high levels of Fas receptor can redirect the FasL expressed by T cells primed to undergo AICD away from the T cells and prevent the induction of AICD in these cells. Furthermore, B cells stimulated through both the CD40 receptor and membrane IgM appear to mediate a stronger protective effect on T cells by virtue of their resistance to FasL-mediated cytolysis. These observations suggest a mechanism by which normal B cell and T cell responses to foreign antigen are maintained, while responses to self antigen are not. PMID- 10741412 TI - The human neural cell adhesion molecule L1 functions as a costimulatory molecule in T cell activation. AB - L1 is a neural cell adhesion molecule (CAM) known to be important for normal neurological development. Despite being described as a neural CAM, we have documented L1 expression by antigen-presenting cells of myelomonocytic origin. Here we demonstrate that L1 can function as a costimulatory molecule in T cell activation. A monoclonal antibody that abrogates L1-L1 homophilic binding significantly reduced mixed leukocyte responses initiated by allogeneic L1+ dendritic cells. Autologous T cell activation in response to phytohemagglutinin was also inhibited by blockade of L1. In accordance with these results, transfection of human L1 into a murine myeloma cell line significantly increased the capacity of these cells to stimulate xenogeneic T cell responses. As a costimulatory ligand L1 could represent a novel target for immunotherapeutic intervention and may act as an important intermediary in neuroimmunological processes and disease. PMID- 10741413 TI - Dual role for macrophages in vivo in pathogenesis and control of murine Salmonella enterica var. Typhimurium infections. AB - Salmonella spp. are regarded as facultative intracellular bacterial pathogens which are found inside macrophages (Mphi) after i. v. infection. It is generally assumed that Mphi restrict the replication of the bacteria during infection. In this study we examined the in vivo activities of Mphi during experimental S. typhimurium infections, using a selective liposome-based Mphi elimination technique. Unexpectedly, elimination of Mphi prior to infection with virulent S. typhimurium decreased morbidity and mortality, suggesting that Mphi mediate the pathology caused by S. typhimurium. Removal of Mphi) during vaccination with attenuated S. typhimurium did not affect protection against challenge with virulent S. typhimurium, suggesting that Mphi are not required for the induction of protective immunity and that other cells must function as antigen-presenting cell to elicit T cell-mediated protection. However, Mphi appeared to be important effectors of protection against challenge infection since elimination of Mphi from vaccinated mice prior to challenge infection with virulent S. typhimurium significantly decreased protection. These results enhance our understanding of the control of S. typhimurium growth in vivo, and moreover suggest that Mphi play a major role in the pathology of virulent S. typhimurium infections. As such, these cells may present a novel target for therapeutic intervention. PMID- 10741414 TI - Cholesterol depletion disrupts lipid rafts and modulates the activity of multiple signaling pathways in T lymphocytes. AB - Lipid rafts are specialized plasma membrane microdomains, in which glycosphingolipids and cholesterol are major structural components. In T lymphocytes, several signaling proteins are associated with lipid rafts including the protein tyrosine kinase LCK and the adapter protein LAT. To investigate their importance in T cell signaling, lipid rafts were disrupted by depleting cholesterol with methyl-beta-cyclodextrin (MbetaCD). This transiently induced tyrosine phosphorylation of multiple proteins, including the ZAP-70 tyrosine kinase, its associated T cell antigen receptor zeta chain, LAT and phospholipase Cgamma1. Tyrosine phosphorylation was dependent on expression of LCK in lipid rafts. Depletion of cholesterol also resulted in activation of the Ras-ERK pathway. This was largely dependent on phorbol ester-sensitive protein kinase C (PKC) and the PKC-theta isoform translocated to the plasma membrane following MbetaCD treatment. MbetaCD did not stimulate intracellular Ca2+ fluxes; however, consistent with its ability to stimulate Ras, MbetaCD synergized with a Ca2+ ionophore to induce formation of the transcription factor NF-AT. These data indicate a crucial role for cholesterol in the regulation of signaling pathways in T cells, which is likely to reflect its importance in the formation of plasma membrane lipid rafts. PMID- 10741415 TI - A flavonoid sulfate antigen activates human alphabeta CD8+ Th2 lymphocytes in pollen allergy. AB - Cellular immune responses are initiated when T lymphocytes expressing alphabeta TCR recognize peptide antigens bound to MHC molecules or, less frequently, double stranded glycolipid antigens bound to CD1 molecules. In the allergy to Parietaria judaica, human alphabeta CD8+ Th2 lymphocytes react to a non-peptide pollinic antigen presented by B cells. The environmental allergen was purified and identified as a new flavonoid pigment: 2'-O-sulfate, 6-O-betaD glucuronopyranosyl, 2',5,6-trihydroxy-isoflavone. Its specific recognition by alphabeta CD8+ Th2 T cells (1) depends upon an MHC- and CD1-independent presentation mediated by B cells, (2) is determined by the flavonoid carbohydrate and sulfate groups and (3) leads to positive skin prick test in allergic patients. Hence, an unusual mode of aromatic sulfated antigen recognition by alphabeta CD8+ Th2 T lymphocytes might underlie the cellular mediation of human allergy to plant allergens. PMID- 10741416 TI - Enhanced and sustained activation of human B cells by anti-immunoglobulin conjugated to the EBV glycoprotein gp350. AB - We coupled a monoclonal anti-human IgD to the gp350 gylcoprotein of Epstein-Barr virus, which has been shown to bind to the complement receptor 2 (CR2), and compared its B cell stimulatory ability to that of anti-Ig and to a multivalent anti-Ig-dextran conjugate. The anti-Ig-gp350 conjugate stimulated higher levels of human B cell proliferation in vitro than did anti-Ig or anti-Ig conjugated to control viral protein, comparable to the proliferation stimulated by the multivalent anti-Ig-dextran. This enhanced proliferation was dependent on binding of the conjugate to CR2, inasmuch as an anti-CD2 antibody blocked the enhanced proliferative response. This enhanced proliferative response was associated with prolonged elevations of intracellular ionized calcium, which was comparable to the response stimulated by anti-Ig-dextran. These findings suggest the use of gp350 as a carrier molecule for weakly immunogenic peptides or antigens which, when bound to gp350, would enhance B cell clonal expansion and activation of antigen-specific B cells. PMID- 10741417 TI - Cloning and expression throughout mouse development of mfat1, a homologue of the Drosophila tumour suppressor gene fat. AB - We present the entire sequence of the mouse Fat orthologue (mFat1), a protein of 4,588 amino acids with 34 cadherin repeats, 27 potential N-glycosylation sites, five EGF repeats and a laminin A G-motif in its extracellular domain. A single transmembrane region is followed by a cytoplasmic domain containing putative catenin-binding sequences. mFat1 shows high homology to human FAT and lesser homology to Drosophila Fat. The sequence of this giant cadherin suggests that it is unlikely to have a homophilic adhesive function, but may mediate heterophilic adhesion or play a signalling role. Expression analysis shows that the mfat1 gene is expressed early in pre-implantation mouse development, at the compact eight cell stage. Whole-mount and section in situ analyses show that transcripts are widely expressed throughout post-implantation development, most notably in the limb buds, branchial arches, forming somites, and in particular in the proliferating ventricular zones in the brain, being down-regulated as cells cease dividing. RT-PCR detects widespread expression in the adult suggesting a role in proliferation and differentiation of many tissues and cell types. PMID- 10741418 TI - TGFbeta2 acts as an "activator" molecule in reaction-diffusion model and is involved in cell sorting phenomenon in mouse limb micromass culture. AB - It was previously speculated that TGFbeta acts as an "activator"-molecule in chondrogenic pattern formation in the limb micromass culture system, but its precise role and relationship with the cell sorting phenomenon have not been properly studied. In the present study, we examined whether the TGFbeta2 molecule satisfies the necessary conditions for an "activator"-molecule in the reaction diffusion model. Firstly, we showed that TGFbeta2 became localized at chondrogenic sites during the establishment of a chondrogenic pattern, and exogenous TGFbeta2 promoted chondrogenesis when added in the culture medium. Secondly, TGFbeta2 protein was shown to promote the production of its own mRNA after 3 hr, indicating that a positive feedback mechanism exists which may be responsible for the emergence of the chondrogenic pattern. We then found that when locally applied with beads, TGFbeta2 suppressed chondrogenesis around the beads, indicating it induces the lateral inhibitory mechanism, which is a key element for the formation of the periodic pattern. We also examined the possible effects of TGFbeta2 on the cell sorting phenomenon and found that TGFbeta2 exerts differential chemotactic activity on proximal and distal mesenchyme cells of the limb bud, and at very early phases of differentiation TGFbeta2 promotes the expression of N-cadherin protein which is known to be involved in pattern formation in this culture system. These findings suggest that TGFbeta2 acts as an "activator"-like molecule in chondrogenic pattern formation in vitro, and is possibly responsible for the cell sorting phenomenon. PMID- 10741419 TI - Development of skeletal muscles in transforming growth factor-beta 1 (TGF-beta1) null-mutant mice. AB - Fetal transforming growth factor-beta 1 (TGF-beta1) has been postulated to regulate the onset of myotube formation and/or pattern formation in developing skeletal muscles. In apparent contradiction of these hypotheses, the development of the extensor digitorum longus and soleus in TGF-beta1 null-mutant muscle was normal. The onset of secondary myotube formation, the numbers of myotubes formed, the proportion of fast and slow fibers, and the patterns of fiber types and connective tissues were essentially identical in TGF-beta1(+/+) and TGF-beta1(-/ ) mice. A portion of the TGFbeta1 in skeletal muscles is derived from the mother, via the placenta. This maternal-derived TGF-beta1 was also not essential for the development of skeletal muscles, as the characteristics of pups born to a TGF beta1(-/-) mother were normal TGF-beta1(-/-) mice die at weaning due to a generalized autoimmune attack. This postnatal death was circumvented by breeding the TGF-beta1 null mutation into nude mice (Whn(-/-)). Like many other strains of TGF-beta1(-/-) mice, extensive loss of Whn(-/-), TGF-beta1(-/-) embryos occurred in utero. However, a portion of the Whn(-/-), TGF-beta1(-/-) mice survived past weaning, remained healthy, and were fertile. The TGF-beta1(-/-) x Whn(-/-) mouse thus represents a valuable tool for the study of the function of TGF-beta1 in the adult, including its putative role as a pregnancy-related hormone. PMID- 10741420 TI - Stabilization and remodeling of the membrane skeleton during lens fiber cell differentiation and maturation. AB - Actin filaments are integral components of the plasma membrane-associated cytoskeleton (membrane skeleton) and are believed to play important roles in the determination of cell polarity, shape, and membrane mechanical properties, however the roles of actin regulatory proteins in controlling the assembly, stability, and organization of actin filaments in the membrane skeleton are not well understood. Tropomodulin is a tropomyosin and actin-binding protein that stabilizes tropomyosin-actin filaments by capping their pointed ends and is associated with the spectrin-actin membrane skeleton in erythrocytes, skeletal muscle cells, and lens fiber cells, a specialized epithelial cell type. In this study, we have investigated the role of tropomodulin and other membrane skeleton components in lens fiber cell differentiation and maturation. Our results demonstrate that tropomodulin is expressed concomitantly with lens fiber cell differentiation and assembles onto the plasma membrane only after fiber cells have begun to elongate and form apical-apical contacts with the undifferentiated epithelium. In contrast, other membrane skeleton components, spectrin, actin, and tropomyosin, are constitutively expressed and assembled on the plasma membranes of both undifferentiated and differentiated fiber cells. Tropomodulin, but not other membrane skeleton components, is also enriched at a novel structure at the apical and basal ends of newly elongated fiber cells at the fiber cell-epithelium and fiber cell-capsule interface, respectively. Once assembled, tropomodulin and its binding partners, tropomyosin and actin, remain membrane-associated and are not proteolyzed during fiber cell maturation and aging, despite proteolysis of alpha-spectrin and other cytoskeletal filament systems such as microtubules and intermediate filaments. We propose that actin filament stabilization by tropomodulin, coupled with partial proteolysis of other cytoskeletal components, represents a programmed remodeling of the lens membrane skeleton that may be essential to maintain plasma membrane integrity and transparency of the extremely elongated, long-lived cells of the lens. The unique localization of tropomodulin at fiber cell tips further suggests a new role for tropomodulin at cell-cell and cell-substratum contacts; this may be important for cell migration and/or adhesion during differentiation and morphogenesis. PMID- 10741421 TI - The lymphatic endothelium of the avian wing is of somitic origin. AB - It has recently been shown that there are lymphangioblasts in the early avian wing bud, but fate map studies on the origin of these cells have not yet been performed. The lymphatics in the wings of 10-day-old chick and quail embryos are characterized by both the position along with all major blood vascular routes and by the Vascular Endothelial Growth Factor Receptor-3 (VEGFR-3) expression. In the quail, the endothelium of both blood vessels and lymphatics can be marked with the QH1 antibody. We have grafted the dorsal halves of epithelial somites of 2 day-old quail embryos homotopically into chick embryos. The grafting was performed at the wing level and the host embryos were reincubated until day 10. The chimeric wings were studied with the QH1 antibody alone and with double staining consisting of VEGFR-3 in situ hybridization and QH1 immunofluorescence. Our results show that in the wing the endothelium of both the blood vessels and the lymphatics is derived from the somites. QH1-positive endothelial cells form the vasculature of the chimeric wings. Chimeric lymphatics of the wing can be identified because of their typical position and their VEGFR-3 and QH1 double positivity. This shows that not only the blood vascular cells but also the lymphatic endothelial cells of the avian wing are born in the paraxial/somitic mesoderm. PMID- 10741422 TI - Expression of sox11 gene duplicates in zebrafish suggests the reciprocal loss of ancestral gene expression patterns in development. AB - To investigate the role of sox genes in vertebrate development, we have isolated sox11 from zebrafish (Danio rerio). Two distinct classes of sox11-related cDNAs were identified, sox11a and sox11b. The predicted protein sequences shared 75% identity. In a gene phylogeny, both sox11a and sox11b cluster with human, mouse, chick, and Xenopus Sox11, indicating that zebrafish, like Xenopus, has two orthologues of tetrapod Sox11. The work reported here investigates the evolutionary origin of these two gene duplicates and the consequences of their duplication for development. The sox11a and sox11b genes map to linkage groups 17 and 20, respectively, together with other loci whose orthologues are syntenic with human SOX11, suggesting that during the fish lineage, a large chromosome region sharing conserved syntenies with mammals has become duplicated. Studies in mouse and chick have shown that Sox11 is expressed in the central nervous system during development. Expression patterns of zebrafish sox11a and sox11b confirm that they are expressed in the developing nervous system, including the forebrain, midbrain, hindbrain, eyes, and ears from an early stage. Other sites of expression include the fin buds and somites. The two sox genes, sox11a and sox11b, are expressed in both overlapping and distinct sites. Their expression patterns suggest that sox11a and sox11b may share the developmental domains of the single Sox11 gene present in mouse and chick. For example, zebrafish sox11a is expressed in the anterior somites, and zebrafish sox11b is expressed in the posterior somites, but the single Sox11 gene of mouse is expressed in all the somites. Thus, the zebrafish duplicate genes appear to have reciprocally lost expression domains present in the sox11 gene of the last common ancestor of tetrapods and zebrafish. This splitting of the roles of Sox11 between two paralogues suggests that regulatory elements governing the expression of the sox11 gene in the common ancestor of zebrafish and tetrapods may have been reciprocally mutated in the zebrafish gene duplicates. This is consistent with duplicate gene evolution via a duplication-degeneration-complementation process. PMID- 10741423 TI - Expression and subcellular localization of SF-1, SOX9, WT1, and AMH proteins during early human testicular development. AB - Many transcription factors have been identified and implicated in male sex determination pathway. Specifically involved in Sertoli cell differentiation and subsequent anti-Mullerian hormone (AMH) secretion in eutherian mammals, they include steroidogenic factor-1 (SF-1), SOX9 (SRY HMG box related gene 9), WT1 (Wilms' tumor 1), and GATA-4 (a zinc finger transcription factor). These factors have been described to execute their function in the male sex determination pathway by controlling AMH transcriptional expression. To understand the hierarchies of these factors and their involvement in the developing testis, for the first time we show the expression and subcellular localization of these factors by immunohistochemistry in the early human testis during embryogenesis compared with AMH expression. If these studies do not refute their possible synergistic interaction to control AMH expression in human embryo, they also reveal a new sexual dimorphism in SOX9 expression during the sex determination process. We show that SOX9 sex specifically shifts from the cytoplasmic to the nuclear compartment at the time of testis differentiation and AMH expression. Putative models for this subcellular distribution are discussed. PMID- 10741424 TI - Neurotrophins enhance electric field-directed growth cone guidance and directed nerve branching. AB - Neurotrophins play major roles in the developing nervous system in controlling neuronal differentiation, neurite outgrowth, guidance and branching, synapse formation and maturation, and neuronal survival or death. There is increasing evidence that nervous system construction takes place in the presence of dc electric fields, which fluctuate dynamically in space and time during embryonic development. These have their origins in the neural tube itself, as well as in surrounding skin and gut. Early disruption of these endogenous electric fields causes failure of the nervous system to form, or else it forms aberrantly. Nerve growth, guidance, and branching are controlled tightly during pathway construction and in vitro dc electric fields have profound effects on each of these behaviours. We have used cultured neurones to ask whether neurotrophins and dc electric fields might interact in shaping neuronal growth, given their coexistence in vivo. Electric field-directed nerve growth generally was enhanced by the simultaneous presentation of several neurotrophins to the growth cone. Under certain circumstances, more nerves turned cathodally, they turned faster, further, and in lower field strengths. Intriguingly, other combinations of dc electric field and neurotrophins (low field strength and neurotrophin 3 (NT-3) switched the direction of growth cone turning. Additionally, cathodally directed nerve growth was faster and directed branching was much more common when electric fields and neurotrophins interacted with neuronal growth cones. Given such profound changes in growth cone behaviour in vitro, neurotrophins and endogenous electric fields are likely to interact in vivo. PMID- 10741425 TI - Characterization of gill-specific genes of the acorn worm Ptychodera flava. AB - Acorn worms are hemichordate deuterostomes that have remarkable gills thought to be homologous to pharyngeal gills in urochordates and cephalochordates, and pharyngeal pouches in vertebrates. In search of molecular keys to analyzing the origin and evolution of the anterior gut and neck region of the chordate body, the present study isolated cDNA clones for six gill-specific genes, designated PfG1 to PfG6, from Ptychodera flava using differential screening of a cDNA library of RNA from gills. Northern blotting confirmed that these genes were all expressed only in the gills. In situ hybridization showed that the expression of these genes is limited to the endodermally derived columnar epithelium of the pharynx. PfG1 encodes a 42-kDa polypeptide containing sequence similar to D domains, protein domains characteristic of extracellular proteins. Expression of PfG1 is localized in a delimited pattern along the columnar epithelium of the inner gill apparatus. Expression in the epibranchial ridge appears as two stripes running longitudinally in the epithelium just lateral of the midline. A stripe of expression also appears in a slightly posterior portion on the curve of each band of columnar epithelium on the pharyngeal surface of the secondary gill bars. The five other gill-specific genes, PfG2 to PfG6, encode a family of C-type lectin polypeptides that appear to be secreted proteins. PfG2 to PfG6 are also expressed in the columnar epithelium of the epibranchial ridge as two parallel stripes, but at the lateral margin of the ridge. One of the genes, PfG6, is additionally expressed in the innermost curve of the epithelium on the pharyngeal surface of each secondary gill bar. The localization of expression of PfPax1/9, a gill specific transcription factor gene, was examined and shown to also be primarily in the endodermal columnar epithelium on the pharyngeal faces of the gill bars. On the secondary gill bars, where PfG1 and PfG6 are also expressed in the columnar epithelium, PfPax1/9 is expressed in the anterior and posterior portions but signal is not evident in the epithelium on the central, innermost curve of the gill bar. The anterior domain of PfPax1/9 expression is more extensive but overlaps the anterior domain of PfG1 expression, whereas its posterior domain of expression is more posterior and complementary to that of PfG6. PMID- 10741426 TI - Specific expression of the LIM/homeodomain protein Lim-1 in horizontal cells during retinogenesis. AB - The LIM/homeodomain transcription factor Lim-1 has been shown to play an essential role in early embryonic patterning during vertebrate development. Here we report the spatial and temporal expression patterns of Lim-1 during retinal development as detected by immunohistochemistry using a specific anti-Lim-1 antibody. By double-immunostaining, we have demonstrated for the first time that Lim-1 is exclusively expressed within the horizontal cell type in the adult retina. In the developing mouse retina, Lim-1 commences its expression in migratory horizontal cell precursors streaming toward the future horizontal cell layer in the ventricular zone. Moreover, its expression during retinogenesis is spatially and temporally coincident with that of the calcium-binding protein calbindin D-28k in horizontal cells. These data together suggest a possible role for Lim-1 in terminal differentiation and maintenance of horizontal cells, and that Lim-1 can serve as a specific molecular marker for the study of horizontal cell specification. PMID- 10741427 TI - Ion channels as molecular coulter counters to probe metabolite transport. PMID- 10741428 TI - Transient and permanent fusion of vesicles in Zea mays coleoptile protoplasts measured in the cell-attached configuration. AB - Exocytosis in protoplasts from Zea mays L. coleoptiles was studied using patch clamp techniques. Fusion of individual vesicles with the plasma membrane was monitored as a step increase of the membrane capacitance (Cm). Vesicle fusion was observed as (i) An irreversible step increase in Cm. (ii) Occasionally, irreversible Cm steps were preceded by transient changes in Cm, suggesting that the electrical connection between the vesicle with the plasma membrane opens and closes reversibly before full connection is achieved. (iii) Most frequently, however, stepwise transient changes in Cm did not lead to an irreversible Cm step. Within one patch of membrane capacitance steps due to transient and irreversible fusions were of similar amplitude. This suggests that the exocytosis events do not result from the fusion of vesicles with different sizes but are due to kinetically different states in a fusion process of the same vesicle type. The dwell time histogram of the transient fusion events peaked at about 100 msec. Fusion can be described with a circular three-state model for the fusion process of two fused states and one nonfused state. It predicts that energy input is required to drive the system into a prevailing direction. PMID- 10741429 TI - Spontaneously opening GABA(A) channels in CA1 pyramidal neurones of rat hippocampus. AB - Spontaneous, single channel, chloride currents were recorded in 48% of cell attached patches on neurones in the CA1 region of rat hippocampal slices. In some patches, there was more than 1 channel active. They showed outward rectification: both channel conductance and open probability were greater at depolarized than at hyperpolarized potentials. Channels activated by gamma-aminobutyric acid (GABA) in silent patches on the same neurones had similar conductance and outward rectification. The spontaneous currents were inhibited by bicuculline and potentiated by diazepam. It was concluded that the spontaneously opening channels were constitutively active, nonsynaptic GABA(A) channels. Such spontaneously opening GABA(A) channels may provide a tonic inhibitory mechanism in these cells and perhaps in other cells that have GABA(A) receptors although not having a GABA(A) synaptic input. They may also be a target for clinically useful drugs such as the benzodiazepines. PMID- 10741430 TI - Roles of charged residues in the conserved motif, G-X-X-X-D/E-R/K-X-G-[X]-R/K R/K, of the lactose permease of Escherichia coli. AB - The lactose permease is a polytopic membrane protein that has a duplicated conserved motif, GXXX(D/E)(R/K)XG[X](R/K)(R/K), located in cytoplasmic loops 2/3 and 8/9. In the current study, the roles of the basic residues and the acidic residue were investigated in greater detail. Neutral substitutions of two positive charges in loop 2/3 were tolerated, while a triple mutant resulted in a complete loss of expression. Neutral substitutions of a basic residue in loop 8/9 (i.e., K289I) also diminished protein stability. By comparison, neutral substitutions affecting the negative charge in loop 2/3 had normal levels of expression, but were defective in transport. A double mutant (D68T/N284D), in which the aspartate of loop 2/3 was moved to loop 8/9, did not have appreciable activity, indicating that the negative charge in the conserved motif could not be placed in loop 8/9 to recover lactose transport activity. An analysis of site directed mutants in loop 7/8 and loop 8/9 indicated that an alteration in the charge distribution across transmembrane segment 8 was not sufficient to alleviate a defect caused by the loss of a negative charge in loop 2/3. To further explore this phenomenon, the double mutant, D68T/N284D, was used as a parental strain to isolate suppressor mutations which restored function. One mutant was obtained in which an acidic residue in loop 11/12 was changed to a basic residue (i.e., Glu374 --> Lys). Overall, the results of this study suggest that the basic residues in the conserved motif play a role in protein insertion and/or stability, and that the negative charge plays a role in conformational changes. PMID- 10741431 TI - H+-and K+-dependence of Ca2+ uptake in lung lamellar bodies. AB - Lung lamellar bodies maintain an acidic interior by an energy-dependent process. The acidic pH may affect the packaging of surfactant phospholipids, processing of surfactant proteins, or surfactant protein A-dependent lipid aggregation. The electron-probe microanalysis of lamellar body elemental composition has previously suggested that lamellar bodies contain high levels of calcium some of which may be in ionic form. In this study, we investigated the Ca2+ uptake characteristics in isolated lung lamellar bodies. The uptake of Ca2+ was measured by monitoring changes in the fluorescence of Fluo-3, a Ca2+ indicator dye. The uptake of Ca2+ in lamellar bodies was ATP-dependent and increased with increasing concentrations of Ca2+. At 100 nM Ca2+, the uptake was almost completely inhibited by bafilomycin A1, a selective inhibitor of vacuolar type H+-ATPase, or by NH4Cl, which raises the lamellar body pH, suggesting that the pH gradient regulates the uptake. The uptake of Ca2+ increased as the Ca2+ concentration was increased, but the relative contribution of bafilomycin A1-sensitive uptake decreased. At 700 nM, it comprised only 20% of the total uptake. These results suggest the presence of additional mechanism(s) for uptake at higher Ca2+ concentrations. At 700 nm Ca2+, the rate and extent of uptake were lower in the absence of K+ than in the presence of K+. The inhibitors of Ca2+-activated K+ channels, tetraethylammonium, Penitrem A, and 4-aminopyridine, also inhibited the K+-dependent Ca2+ uptake at 700 nM Ca2+. Thus the uptake of Ca2+ in isolated lung lamellar bodies appears to be regulated by two mechanisms, (i) the H+-gradient and (ii) the K+ transport across the lamellar body membrane. We speculate that lamellar bodies accumulate Ca2+ and contribute to regulation of cytosolic Ca2+ in type II cells under resting and stimulated conditions. PMID- 10741432 TI - Maturational changes in rabbit renal basolateral membrane vesicle osmotic water permeability. AB - We have recently demonstrated that while the osmotic water permeability (Pf) of neonatal proximal tubules is higher than that of adult tubules, the Pf of brushborder membrane vesicles from neonatal rabbits is lower than that of adults. The present study examined developmental changes in the water transport characteristics of proximal tubule basolateral membranes by determining aquaporin 1 (AQP1) protein abundance and the Pf in neonatal (10-14 days old) and adult rabbit renal basolateral membrane vesicles (BLMV). At 25 degrees C the Pf of neonatal BLMV was significantly lower than the adult BLMV at osmotic gradients ranging from 40 to 160 mOsm/kg water. The activation energies for osmotic water movement were identical in the neonatal and adult BLMV (8.65 +/- 0.47 vs. 8.86 +/ 1.35 kcal x deg(-1) x mol(-1). Reflection coefficients for sodium chloride and sodium bicarbonate were identical in both the neonatal and adult BLMV and were not different from one. Mercury chloride (0.5 mM) reduced osmotic water movement by 31.3 +/- 5.5% in the adult BLMV, but by only 4.0 +/- 4.0% in neonatal vesicles (P < 0.01). Adult BLMV AQP1 abundance was higher than that in the neonate. These data demonstrate that neonatal BLMV have a lower Pf and AQP1 protein abundance than adults and that a significantly greater fraction of water traverses the basolateral membrane lipid bilayer and not water channels in neonates compared to adults. The lower Pf of the neonatal BLMV indicates that the basolateral membrane is not responsible for the higher transepithelial Pf in the neonatal proximal tubule. PMID- 10741433 TI - Single-channel characterization of a nonselective cation channel from human placental microvillus membranes. Large conductance, multiplicity of conductance states, and inhibition by lanthanides. AB - The rate-limiting step for the maternofetal exchange of low molecular-weight solutes in humans is constituted by transport across a single epithelial layer (syncytiotrophoblast) of the placenta. Other than the well-established presence of a large-conductance, multisubstate Cl- channel, the ionic channels occurring in this syncytial tissue are, for the most part, unknown. We have found that fusion of apical plasma membrane-enriched vesicle fractions with planar lipid bilayers leads, mainly (96% of 353 reconstitutions), to the reconstitution of nonselective cation channels. Here we describe the properties of this novel placental conductance at the single-channel level. The channel has a large (>200 pS) and variable conductance, is cation selective (P(Cl)/P(K) approximately or approximately equal 0.024), is reversibly inhibited (presumably blocked) by submillimolar La3+, has very unstable kinetics, and displays a large number (>10) of current sublevels with a "promiscuous" connectivity pattern. The occurrence of both "staircaselike" and "all-or-nothing" transitions between the minimum and maximum current levels was intriguing, particularly considering the large number of conductance levels spanned at a time during the concerted current steps. Single-channel data simulated according to a multistate linear reaction scheme, with rate constants that can vary spontaneously in time, reproduce many aspects of the recorded subconductance behavior. The channel's sensitivity to lanthanides is reminiscent of stretch-sensitive channels which, in turn, suggests a physiological role for this ion channel as a mechanotransducer during syncytiotrophoblast-volume regulation. PMID- 10741434 TI - Cation permeability and selectivity of a root plasma membrane calcium channel. AB - Calcium channels in the plasma membrane of root cells fulfill both nutritional and signaling roles. The permeability of these channels to different cations determines the magnitude of their cation conductances, their effects on cell membrane potential and their contribution to cation toxicities. The selectivity of the rca channel, a Ca2+-permeable channel from the plasma membrane of wheat (Triticum aestivum L.) roots, was studied following its incorporation into planar lipid bilayers. The permeation of K+, Na+, Ca2+ and Mg2+ through the pore of the rca channel was modeled. It was assumed that cations permeated in single file through a pore with three energy barriers and two ion-binding sites. Differences in permeation between divalent and monovalent cations were attributed largely to the affinity of the ion binding sites. The model suggested that significant negative surface charge was present in the vestibules to the pore and that the pore could accommodate two cations simultaneously, which repelled each other strongly. The pore structure of the rca channel appeared to differ from that of L type calcium channels from animal cell membranes since its ion binding sites had a lower affinity for divalent cations. The model adequately accounted for the diverse permeation phenomena observed for the rca channel. It described the apparent submillimolar Km for the relationship between unitary conductance and Ca2+ activity, the differences in selectivity sequences obtained from measurements of conductance and permeability ratios, the changes in relative cation permeabilities with solution ionic composition, and the complex effects of Ca2+ on K+ and Na+ currents through the channel. Having established the adequacy of the model, it was used to predict the unitary currents that would be observed under the ionic conditions employed in patch-clamp experiments and to demonstrate the high selectivity of the rca channel for Ca2+ influx under physiological conditions. PMID- 10741435 TI - The impact of laparoscopy on cancer management. PMID- 10741436 TI - Endoscopic retrograde cholangiopancreatography in the management of choledocholithiasis. AB - Surgeons today have a wide range of therapeutic options for the management of patients with choledocholithiasis. Endoscopists, interventional radiologists, and surgeons employ a variety of techniques to access and remove common bile duct stones (CBDS) successfully. Although earlier studies have been done to assess the relative merits of laparoscopic and endoscopic management of CBDS, few of them have employed a randomized prospective trial for the comparison. Without recognized parameters for comparison, no definitive conclusions can be drawn. Herein, we examine the role of therapeutic endoscopic retrograde cholangiopancreatography (ERCP) as an important adjunct to laparoscopic cholecystectomy (LC) in the management of CBDS. The three main scenarios in which this modality is employed for CBDS removal are selective preoperative ERCP, intraoperative ERCP, and postoperative ERCP. We conclude that an appropriate balance must be struck to maintain a high yield of positive or therapeutic ERCP, avoid unnecessary ERCP, and not miss CBDS, while ensuring acceptably low rates of morbidity and mortality and controlling costs. As we await the publication of prospective data, we may look for direction from decision analysis in order to develop optimal management strategies and define the "best practice" results that should be expected of operators before new procedures and innovative technology are accepted on a widespread basis. PMID- 10741437 TI - Endoscopic treatment of distal bile duct stricture from chronic pancreatitis. AB - BACKGROUND: Endoscopic placement of biliary stents is an effective initial treatment for jaundice and cholangitis caused by common bile duct (CBD) strictures secondary to chronic pancreatitis; however, the role of endoscopic treatment for long-term management of these strictures is less clear. In 1992, we designed a protocol of balloon dilatation and stenting for > or =12 months. This study evaluates endoscopic therapy as a definitive long-term treatment for these strictures. We have treated 25 patients with this protocol. METHODS: All patients had an endoscopic sphincterotomy, balloon dilatation of the stricture, and then placement of a polyethylene stent (7-11.5 F). Stents were exchanged at 3-4-month intervals to avoid the complications of clogging and cholangitis. We were particularly interested in how many patients would achieve resolution of the stricture and tolerate removal of the stent. RESULTS: The length of the CBD strictures ranged from 8 to 40 mm. Within days of stenting, all patients achieved relief of jaundice and cholestasis. Complications consisted of six episodes of cholangitis and nine episodes of pancreatitis. There were no deaths. Twenty of the 25 patients are now stent-free after an average stenting period of 13 months (range, 3-28). To date, there has been no recurrence of stricture, for a mean of 32 months. Three patients still have stents in place, and two patients required operation--one for persistent stricture and recurrent cholangitis after 8 months of stenting, and one for a mass in the head of the pancreas that was thought to be cancer. CONCLUSIONS: Our results indicate that these strictures will respond and dilate after a course of stenting in 80% of patients, with an acceptable morbidity. Although these are medium-term results at 32 months, we would expect most recurrences within the 1st year following stent removal. In some cases, stenting is necessary for >12 months. Thus, the data suggest that endoscopic stenting provides definitive treatment in most patients with CBD stricture due to chronic pancreatitis and may be considered a viable alternative to standard surgical bypass. PMID- 10741438 TI - Combined intraoperative laparoscopic cholecystectomy and endoscopic retrograde cholangiopancreatography: lessons from 29 cases. AB - BACKGROUND: The role and timing of endoscopic retrograde cholangiopancreatography (ERCP) in patients with suspected choledocholethiasis remains a controversial subject. There have been few studies exploring the role of intraoperative ERCP. Therefore, we set out to perform a retrospective review of 29 patients who underwent combined laparoscopic cholecystectomy (LC) and intraoperative ERCP (LC/ERCP). Our objective was to assess the feasibility of a one-stage approach using intraoperative ERCP. METHODS: We identified 29 patients in whom LC/ERCP was attempted between January 1996 and November 1998 at a university-affiliated hospital with a large private faculty. Parameters reviewed included preoperative diagnosis, liver function tests (LFT), finding on transcystic cholangiogram (TCC), ERCP, stone retrieval, failure of ERCP, length of stay, morbidity, and mortality. RESULTS: Twenty-eight of 29 patients (97%) underwent successful combined LC/ERCP. Successful TCC followed by ERCP was performed in 21 of 26 patients (81%). Five TCC were technically unsuccessful; in these patients, ERCP was performed on the basis of preoperative criteria. In three patients, TCC was not attempted. Stones were successfully retrieved from 20 of 21 patients (95%) with abnormal finding on TCC, one of five patients (20%) with failed TCC, and two of three patients (67%) with ERCP but without TCC. Overall morbidity was 14%, comprising two patients with postoperative hyperamylasemia and two with cystic duct leaks. There were no deaths in the group. The mean time for the combined procedure was 173 min (range, 50-290). Mean length of hospitalization was 3.4 days, and mean postoperative stay was 2.2 days. CONCLUSIONS: LC/ERCP can be performed safely. The advantages of the combined procedures include one-stage treatment of cholelithiasis and choledocholithiasis, avoidance of unnecessary preoperative ERCP and their concomitant complications, and elimination of potential return to the operating room when postoperative ERCP is technically impossible. PMID- 10741439 TI - Lymphocyte proliferation in mice after a full laparotomy is the same whether performed in a sealed carbon dioxide chamber or in room air. AB - PURPOSE: Our laboratory has demonstrated that significantly more cell-mediated immunosuppression occurs after full laparotomy than after either anesthesia control or carbon dioxide (CO2) pneumoperitoneum. We further demonstrated that the postoperative immunosuppression is related to the length of the incision. Other investigators believe that the immunosuppression observed after laparotomy is caused by peritoneal exposure to small amounts of lipopolysaccharide found in circulating air. They believe that the better-preserved immune function associated with laparoscopic surgery results from the avoidance of air contamination of the peritoneal cavity. To investigate this hypothesis, we determined and compared postoperative lymphocyte proliferation rates after (a) laparotomy in room air, (b) laparotomy in a CO2 chamber, (c) CO2 insufflation in a murine model, and (d) anesthesia alone. METHODS: Female C3H/He mice (n = 21) were divided randomly into four groups: (a) anesthesia control, (b) air laparotomy, (c) CO2 laparotomy, and (d) CO2 insufflation. The control mice underwent no procedure. The group 2 animals underwent a full midline incision (xiphoid to pubis) and exposure to room air for 20 min and then were clipped closed. The group 3 mice underwent a full midline incision in a sealed CO2 chamber for 20 min, and the group 4 mice insufflation with CO2 gas at 4 to 6 mm Hg for 20 min. Splenocytes were harvested from all the animals on day 2 after the interventions. Lymphocyte proliferation then was assessed using the nonradioactive colorimetric MTS/PMS system 72 h after concanavalin-A stimulation. RESULTS: There was no significant difference in lymphocyte proliferation between the air and CO2 laparotomy groups. Lymphocyte proliferation in the anesthesia control and CO2 insufflation groups was significantly higher than in both the air laparotomy (p<0.05) and CO2 laparotomy (p<0.05) groups (p values by Tukey-Kramer test). There was no significant difference between the anesthesia control and CO2 pneumoperitoneum groups. CONCLUSIONS: Our results suggest that full laparotomy performed in a sealed CO2 chamber compared to room air laparotomy resulted in similar suppression of lymphocyte proliferation. Furthermore, no significant suppression of lymphocyte proliferation was observed in the CO2 pneumoperitoneum group. These results, with regard to lymphocyte proliferation rates, refute the hypothesis that postoperative immunosuppression is related to air exposure and support the alternative hypothesis that immunosuppression is related to incision length. PMID- 10741440 TI - Pneumoperitoneum with carbon dioxide enhances liver metastases of cancer cells implanted into the portal vein in rabbits. AB - BACKGROUND: Little is known about the role of the CO2 pneumoperitoneum on tumor cells that spread from the portal system into the liver during laparoscopic surgery for gastrointestinal malignancies. Therefore, we designed a study to investigate the effect of CO2 pneumoperitoneum on cancer cells implanted in the portal vein in a rabbit model. METHODS: Immediately after intraportal inoculation of 2.5x10(5) cells of VX2 cancer, the rabbits received either CO2 pneumoperitoneum at a pressure of 10 mm Hg for 30 min (pneumoperitoneum group, n = 14) or laparotomy alone for 30 min (laparotomy group, n = 14). RESULTS: The number (p<0.01) and area of cancer nodules (p = 0.045) on the liver surface on day 17 were greater in the pneumoperitoneum group than in the laparotomy group. The frequency of cancer nodules >3.0 mm in diameter was higher in the pneumoperitoneum group than in the laparotomy group (p<0.001). CONCLUSIONS: Compared with laparotomy, CO2 pneumoperitoneum enhanced the development of liver metastases in this experimental model. PMID- 10741441 TI - Laparoscopic treatment of hydatid cysts of the liver and spleen. AB - BACKGROUND: The short-term results from laparoscopic treatment of hydatid cysts of the liver and spleen were reported previously. The procedure was shown to be feasible and safe, offering the advantages of laparoscopic surgery. This is the first report on the long-term follow-up of this operation in a large group of patients. METHODS: In this study, 108 hydatid cysts of the liver and spleen in 83 consecutive patients (43 males [52%] and 40 females [48%]) were approached laparoscopically. The mean age of the patients was 40 years (range, 13-85 years). There were 104 liver cysts and 4 spleen cysts. The liver cysts were located in the right lobe in 42 patients (53%), in the left lobe in 21 patients (26%) and in both lobes in 16 patients (21%). Of the 104 cysts, 44 (42%) were uniloculated and 60 (58%) were multiloculated. RESULTS: All cysts were approached laparoscopically. The mean operative time was 80 min (range, 40-180 min). The conversion rate was 3%. The mean hospital stay was 3 days (range, 2-7 days). There were no mortalities, and complications occurred in nine patients (11%). All were managed conservatively except one patient in whom a laparotomy was needed. All patients were followed up for a mean period of 30 months (range, 4-54 months) with serological testing and ultrasonography if needed. In three patients (3.6%) recurrence of the disease developed. CONCLUSION: The laparoscopic approach to uncomplicated hydatid cysts of the liver and spleen is a safe and effective option with favorable long-term results. PMID- 10741442 TI - Vascular injuries within the hepatoduodenal ligament: recognition by laparoscopic color Doppler ultrasound. AB - BACKGROUND: Lesions of vascular structures are rare but serious complications of laparoscopic cholecystectomy. The purpose of this blind randomized animal study was to investigate the possibility of detecting different vascular lesions within the hepatoduodenal ligament using laparoscopic color Doppler ultrasound (LCDU). METHODS: Twenty-four lesions of the hepatic artery and portal vein were created laparoscopically in six farmer pigs using titanium clips. The following injuries were studied: (a) partial occlusion of the hepatic artery (eight cases), (b) complete occlusion of the hepatic artery (eight cases), (c) partial occlusion of the portal vein (eight cases). There were also eight cases without lesions of the vascular vessels. The order in which the injuries were created was randomly assigned. The study was performed in a blind fashion. Recognition of the injuries was attempted with LCDU. RESULTS: All injuries were recognized correctly by LCDU. There were no false positive results. The clips were reliably located. Using color Doppler imaging, partial occlusions of the hepatic artery and portal vein were visualized by changes of the blood flow from laminar to turbulent behind the clip. Complete occlusion of the hepatic artery was recognized as a complete cessation of the colored blood flow. CONCLUSION: LCDU is a very efficient tool for visualizing vascular structures and evaluating the bloodstream. Partial or complete vascular occlusion by clips that may occur as a result of difficult dissection during laparoscopic cholecystectomy can be visualized reliably using this technique. PMID- 10741443 TI - Minimally invasive surgery and clinical decision-making for pediatric malignancy. AB - BACKGROUND: Minimally invasive surgery (MIS) is an ideal way to obtain biopsy specimens in children with cancer. We examined the safety, reliability and outcome of decisions made based on tissue obtained using MIS. METHODS: Fifty-nine oncology patients underwent 62 MIS procedures between January 1994 and July 1998. Complications, biopsy results, and outcomes were reviewed. RESULTS: The study population comprised 32 boys and 27 girls, with an average age of 8.8 years. There were 47 thoracoscopic and 15 laparoscopic operations. Laparoscopic procedures included initial biopsy, determination of resectability, and second look exam. Thoracoscopic procedures included 40 lung biopsies and seven biopsies/resections of mediastinal masses. Diagnostic accuracy was 100% in all cases. No patient was found retrospectively to have been inadequately treated based on decisions made from tissue obtained by MIS. CONCLUSION: MIS is a safe and accurate means of obtaining tissue in pediatric oncology patients. Treatment decisions can be made accurately and with confidence using these techniques. PMID- 10741444 TI - Preoperative abdominal ultrasound may be misleading in risk stratification for presence of common bile duct abnormalities. AB - BACKGROUND: Following the advent of laparoscopic cholecystectomy (LC), the preoperative predictors of common bile duct (CBD) abnormalities became more important in perioperative decision making. Preoperative transabdominal ultrasound (US) is used to assess the preoperative risks associated with CBD abnormalities. This study attempts to determine the sensitivity and specificity of US in determining CBD abnormalities in patients prior to LC. METHODS: US measurements of the CBD diameter and presence of stones were ascertained from radiology reports in 100 patients who had LC with a routine intraoperative cholangiogram (IOC). The same information was obtained from the patients' IOC. A supraduodenal CBD diameter of >8 mm was considered dilated. RESULTS: US demonstrated a sensitivity of 25% and a specificity of 70% for the detection of CBD dilatation compared to IOC. The sensitivity of US for predicting CBD dilatation was 55% when the IOC-derived diameter was >10 mm and 100% when it was >15 mm. The overall sensitivity of US for detection of stones was 10%; it improved to 17% in patients with a dilated CBD on US. CONCLUSIONS: Preoperative ultrasound is neither sensitive nor specific for detecting CBD dilatation or presence of stones. A negative preoperative US report may be misleading in risk stratification for the presence of these CBD abnormalities. In order to avoid missing any CBD pathology, we recommend the routine use of intraoperative cholangiography. PMID- 10741445 TI - Determinants of outcomes in laparoscopic colorectal surgery: a multiple regression analysis of 416 resections. AB - BACKGROUND: To date, most large series of laparoscopic colorectal procedures have been descriptive reports that do not account for the potentially complex interaction of outcome predictors. The purpose of this study was to identify the preoperative factors that predict operative time, conversion to open surgery, and intraoperative and postoperative complications in laparoscopic colorectal surgery. METHODS: Multiple regression techniques were used to analyze 416 laparoscopic resections from a prospective database of laparoscopic colorectal procedures performed between April 1991 and April 1998. The preoperative factors considered were patient-specific (age, gender, weight) or disease-specific (diagnosis of cancer, Crohn's disease, diverticulitis, fistula). Surgical experience of < or =50 cases was also considered. Finally, all resections were represented by a combination of the following five procedure components: resections of the (a) hepatic flexure, (b) splenic flexure, (c) sigmoid, and (d) rectum, or (e) a perineal dissection. RESULTS: Patient weight, Crohn's disease, and each of the five individual procedure components incrementally lengthened operative time. Conversion to open surgery was influenced by the patient's weight, malignancy, and early experience of the surgeon. The risk of a postoperative complication was increased by the patient's age, resection of the perineum, and the presence of a fistula. No factors significantly influenced the risk of an intraoperative complication. CONCLUSIONS: Several preoperative factors that significantly affect outcomes in laparoscopic colorectal resections have been identified. Consideration of these factors may help in case selection and estimation of operating time; they should also be valuable when patients are informed of their risk of conversion and complications. PMID- 10741446 TI - Intraoperative cholangiography and postoperative pancreatitis. AB - BACKGROUND: It has been suggested that intraoperative cholangiography (IOC) can cause postoperative pancreatitis. METHODS: We studied the relationship between IOC and pancreatitis by reviewing the case histories of 500 patients (1992-97) who underwent cholecystectomy at our institution. In 82% of cases, the cholecystectomies were done laparoscopically, whereas, 7% were converted to an open procedure. An IOC was performed in 435/500, or 87%. During these studies, common bile duct (CBD) stones (or the possibility of a stone) were noted in 14% of the cases. An intraoperative CBD investigation was required in nine of 435 cases or 8.5%, while 28/435 (9.2%) underwent postoperative endoscopic retrograde cholangiopancreatography (ERCP). RESULTS: Follow-up was available in 90% of our patients (452/500). We found six cases of postoperative pancreatitis; only three of 452 (0.6%) occurred <1 year after cholecystectomy. None of these patients had a preoperative history of pancreatitis. In all six cases, there appeared to be an etiology for the pancreatitis unrelated to IOC. CONCLUSION: We could find no statistical association between IOC and postoperative pancreatitis. Postoperative pancreatitis is uncommon at our institution, where routine IOC is employed. Therefore, we conclude that IOC does not cause pancreatitis. PMID- 10741447 TI - Laparoscopic cholecystectomy and interventional endoscopy for gallstone complications during pregnancy. AB - BACKGROUND: Symptomatic or complicated gallstone disease is the most common reason for nongynecological operations during pregnancy. Gallstones are present in 12% of all pregnancies, and more than one-third of patients fail medical treatment and therefore require surgical endoscopy or laparoscopy. Gallstone pancreatitis and jaundice during pregnancy is associated with a high recurrence rate, exposing both fetus and mother to an increased risk of morbidity and mortality. METHODS: During a 4-year period, all pregnant patients (n = 37) with symptomatic or complicated gallstone disease were studied prospectively at the Landeskrankenhaus in Salzburg, Austria. Five patients had an endoscopic retrograde cholangiopancreatogram (ERCP) for biliary pancreatitis or jaundice; two of these underwent subsequent laparoscopic cholecystectomy. Another seven patients required laparoscopic cholecystectomy for severe pain or cholecystitis; all were in their 13th-32nd gestational week. Access was established by Veress needle in all cases. Insufflation pressure was 8-10 mm Hg, and mean operative time was 62 min. RESULTS: All patients delivered full-term, healthy babies. There were no postendoscopic or postoperative complications. All patients enjoyed full relief from their symptoms; there were no recurrences of pancreatitis or jaundice. CONCLUSIONS: The combination of ERCP and laparoscopic cholecystectomy offers a safe and effective option for the definitive treatment of complicated gallstone disease and intractable pain during pregnancy, and there is sufficient access for the combined treatment to be employed. PMID- 10741448 TI - The adverse hemodynamic effects of anesthesia, head-up tilt, and carbon dioxide pneumoperitoneum during laparoscopic cholecystectomy. AB - BACKGROUND: The increased intra-abdominal pressure during pneumoperitoneum, together with the head-up tilt used in upper abdominal laparoscopies, would be expected to decrease venous return to the heart. The goal of our study was to determine whether laparoscopy impairs cardiac performance when preventive measures to improve venous return are taken, and to analyze the effects of positioning, anesthesia, and increased intra-abdominal pressure. METHODS: Using invasive monitoring, hemodynamic changes were investigated in 15 ASA class I or II patients under isoflurane-fentanyl anesthesia during laparoscopic cholecystectomy. Before laparoscopy, the patients received an intravenous (IV) infusion of colloid solution if cardiac filling pressures were low, and their legs were wrapped from toes to groin with elastic bandages. Measurements were taken while the patients were awake in the supine (baseline) and head-up tilt (15 20 degrees) positions, and after the induction of anesthesia in the same positions. Measurements were repeated at regular intervals during laparoscopy (intra-abdominal pressure at 13-16 mm Hg), after deflation of the gas, and in the recovery room. RESULTS: With the passive head-up tilt in awake and anesthetized patients, the cardiac index (CI), stroke index (SI), central venous pressure (CVP), and pulmonary capillary wedge pressure (PCWP) decreased, and systemic vascular resistance increased. With the patient under anesthesia, SI decreased, but CI did not change significantly as a result of the compensatory increase in heart rate. Carbon dioxide (CO2) insufflation at the start of laparoscopy produced increases in CVP and PCWP as well as mean systemic and mean pulmonary arterial pressures without changes in CI or SI. Toward the end of the laparoscopy, CI decreased by 15%. The hemodynamic values returned to nearly prelaparoscopic levels after deflation of the gas, and CI was elevated during the recovery period, whereas systemic vascular resistance was decreased in comparison with the baseline. CONCLUSIONS: By correcting relative dehydration and preventing the pooling of blood, CI decreased less than 20% during pneumoperitoneum as compared with the baseline awake level. The head-up positioning accounts for many of the adverse effects in hemodynamics during laparoscopic cholecystectomy. PMID- 10741449 TI - Cost-effectiveness of different diagnostic strategies in patients with nonresectable upper gastrointestinal tract malignancies. AB - BACKGROUND AND METHODS: Using a simple model, this retrospective study evaluated the cost-effectiveness of different diagnostic strategies used for pretherapeutic detection of patients with disseminated or locally nonresectable upper gastrointestinal tract malignancies (UGIM). Of 162 consecutive UGIM patients referred for treatment, 73 (45%) had disseminated or locally nonresectable disease, and these patients were eligible for evaluation. RESULTS: The noninvasive diagnostic strategies (computed tomography [CT] with ultrasonography [US] and endoscopic ultrasonography [EUS]) had a low procedure cost, but a diagnostic strategy based on CT with US or CT with US and laparoscopy was not cost-effective. The inclusion of endoscopic or laparoscopic ultrasonography seemed necessary to the provision of a cost-effective strategy because both techniques had a high diagnostic accuracy combined with a low cost. A change in diagnostic strategy from CT with US to CT with US and EUS resulted in a net saving regarding the cost of each additional nonresectable patient detected, but this strategy still required up to 20% futile explorative laparotomies. CONCLUSIONS: The combination of endoscopic and laparoscopic ultrasonography was cost-effective and had no complications in this study. We use this strategy as our standard in the pretherapeutic evaluation of UGIM patients. PMID- 10741450 TI - A prospective multicenter study on laparoscopic treatment of gastroesophageal reflux disease in Italy: type of surgery, conversions, complications, and early results. Study Group for the Laparoscopic Treatment of Gastroesophageal Reflux Disease of the Italian Society of Endoscopic Surgery (SICE). AB - BACKGROUND: A national survey was undertaken by the Italian Society for Laparoscopic Surgery to investigate the prevalence, indications, conversion rate, mortality, morbidity, and early results of laparoscopic antireflux surgery. METHODS: Beginning on January 1, 1996, all of the centers taking part in this study were asked to complete a questionnaire on each patient. The questionnaire was divided into four parts and covered such areas as indications for surgery and preoperative workup, type of operation performed and certain aspects of the surgical technique, conversions and their causes, intraoperative and postoperative complications (within 4 weeks), and details of the postoperative course. The last part of the questionnaire focused on the follow-up period and was designed to gather data on recurrence of preoperative symptoms, postoperative symptoms (dysphagia, gas bloat), and postoperative test findings. RESULTS: As of June 30, 1998, 21 centers were taking part in the study and 621 patients were enrolled, with a median of 27 patients per center (less than one patient/month). The most popular technique was the Nissen-Rossetti (52%), followed by the Nissen (33%) and Toupet procedures (13%). Other techniques, such as the Dor and Lortat Jacob, were used in the remainder of cases. Patients who received a Toupet procedure had a higher incidence of defective peristalsis (p<0.05). The conversion rate to open surgery was 2.9%. The most common causes of conversion were inability to reduce the hiatus hernia or distal esophagus in the abdomen and adhesions from previous surgery. Perforation of the stomach and esophagus occurred in <1% of patients. Mortality was nil. Postoperative complications were observed in 7.3% of cases. The most common complication was acute dysphagia (19 patients), which required reoperation in 10 patients. No differences in the incidence of acute dysphagia were found for the different surgical techniques employed. Follow-up data were obtained for 319 patients (53%): 91.5% of the patients remained GERD symptom-free; severe esophagitis (grade 2-3) healed in 95% of the patients; lower esophageal sphincter (LES) manometric characteristics (pressure, abdominal length, and overall length) improved significantly after surgery (p<0.005); and acid exposure of the distal esophagus decreased. CONCLUSIONS: Laparoscopic antireflux surgery has no mortality and a low morbidity. Symptoms and esophagitis are resolved in >90% of patients. Despite these favorable results, however, this type of surgery is not yet as widely employed in Italy as in other countries. PMID- 10741451 TI - Scrotal hernias: a contraindication for an endoscopic procedure? Results of a single-institution experience in transabdominal preperitoneal repair. AB - INTRODUCTION: Endoscopic repair was introduced for use with inguinal hernia therapy more than 10 years ago. The technique as well as the indications for this method are debated, however. As a borderline inguinal hernia situation, the scrotal hernia in particular evokes vehement objections to an endoscopic procedure because of the anticipated problems and complications in dissecting the extended hernia sac. The efficiency of the laparoscopic transabdominal preperitoneal (TAPP) technique in the treatment of scrotal hernia therefore is discussed in this article. METHODS: Laparoscopic hernia repair (TAPP) has been performed in our department since 1993. Data are collected by a prospective documentation of operative and follow-up results. For evaluation, a comparison of scrotal and normal hernia repair was performed. RESULTS: Between April 1993 and June 1998 the TAPP technique was used to treat 191 scrotal hernias, 42 (22%) of which were recurrent hernias. The median operating time for a normal inguinal hernia repair was 45 min, whereas scrotal hernias required a median of 65 min and irreducible scrotral hernias a median of 68.5 min. Major complications were observed in 1.6% of scrotal and 0.6% of normal inguinal hernia repairs. The most frequent scrotal hernia repair problem was the formation of a seroma, 10.5% of which had to be evacuated. During a follow-up period of 30 months, we found a total of two recurrences (1.05%). CONCLUSION: In scrotal hernia repair, TAPP is not associated with higher complication rates and can be performed with efficiency comparable with that in normal inguinal hernia repair. PMID- 10741452 TI - Laparoscopic treatment of symptomatic lymphoceles after kidney transplantation. AB - BACKGROUND: The incidence of lymphocele after kidney transplantation ranges from 0.6% to 18%. This study examines the use of laparoscopic ultrasound for the location of lymphoceles during laparoscopic drainage. METHODS: Between July 1993 and October 1998, we performed 147 kidney transplants. A symptomatic lymphocele was observed in 19 patients (12.9%). All of these patients underwent peritoneal laparoscopic fenestration of the lymphocele. The graft, kidney hilum, ureter, iliac vessels, and lymphoceles were identified by laparoscopic ultrasound. RESULTS: All but one patient were discharged within 24 h. One recurrence (5.2%), which was successfully treated by laparoscopy, was observed at a mean follow-up of 15.5 months. We had one complication (5.2 %)-a left hydrocele that occurred 2 days after drainage of a lymphocele located in the left iliac fossa. CONCLUSIONS: Laparoscopic peritoneal drainage of posttransplant lymphoceles shares the well known advantages of laparoscopy. Furthermore, laparoscopic ultrasound is a useful tool that allows the recognition of anatomical structures and decreases the risk of iatrogenic lesions. PMID- 10741453 TI - Laparoscopic Nissen fundoplication: a technique for the easy and precise manufacture of a true fundoplication. AB - Passing the stomach behind the esophagus during laparoscopic Nissen fundoplication is a common source of frustration for the laparoscopic surgeon. It often leads to an incorrect formation of the fundoplication, resulting in a wrapping or twisting of the fundus around the distal esophagus. The correct technique should result in the distal esophagus being enveloped inside the fundus without distorting the orientation of the greater curve. We have developed an easy, precise, and reproducible technique to perform this maneuver. The steps for performance of this maneuver are described. PMID- 10741454 TI - Comparison of insufflation vs. retractional technique for laparoscopic-assisted intervertebral fusion of the lumbar spine. AB - Laparoscopic transperitoneal fusion of the L5-S1 spinal interspace has become a common procedure. Retroperitoneal retraction and laparoscopic instrumentation without insufflation also allows visualization of the upper lumbar spaces, but this procedure is much more difficult to accomplish. We review and compare our results using each of these techniques for the treatment of mechanical instability and chronic back pain. A total of 35 selected patients underwent intervertebral fusion between February 1996 and August 1998. Their mean age was 48 years. There were 22 female and 13 male patients. Standard CO2 insufflation was used in 10 patients with L5-S1 fusions. Retractional gasless technique was used in nine patients with fusions at L5-S1, 16 patients at L4-L5, one patient at L3-L4, three patients at L2-3, and one patient at L1-L2. Thus, we performed a total of 40 lumbar fusions in 35 patients. In the 19 patients with the gasless technique, a balloon dissector and retractor facilitated the retroperitoneal exposure. Seven of these 19 patients were converted to open procedures, most commonly due to lacerations of the peritoneal lining that prohibited visualization. None of the L5-S1 patients with insufflation were converted to open. Mean operative time in the insufflated patients was 152 min vs. 181 min for the retractional technique. There were seven complications in the transperitoneal group: one fusion device migration, one postoperative UTI, one intracerebral hemorrhage, one severe postoperative pancreatitis, and three iliac vein lacerations. There were 16 complications in the retroperitoneal group: one deep vein thromboses, one serosal bowel injury, one small tear in the spleen, one cage migration, one postoperative pulmonary atelectasis, one postoperative hydrocele, four postoperative ileus, and six peritoneal tears. The mean postoperative stay was three days for both groups. There were no deaths. The L5-S1 interspace is best approached transperitoneally for anterior fusion. Although the retroperitoneal retractional technique is much more difficult and has a longer and steeper learning curve, it does allow laparoscopic anterior fusion of the upper lumbar spine. PMID- 10741455 TI - Polymer sheet for covering tumors during endoscopic surgery. AB - It is recommended that tumor surface should be covered before resection in endoscopic surgery, but this is difficult to do, and no satisfactory method for this purpose has been reported. Therefore, we developed a polymer sheet to cover the wet surfaces of tumors. The sheet we developed is composed mainly of a cellulose derivative, ethyl citrate, and polyacrylic acid. Experimental and clinical study was performed to investigate the usefulness of the sheet. The sheet became attached to the organ surface by absorbing fluid secreted by tissues, and remained fixed for a period of 2 to 3 hours. No foreign body or allergic reaction was observed, and no postoperative infection occurred. The polymer sheet can be used safely for the purpose of covering tumors during endoscopic surgery. PMID- 10741456 TI - Trocar for laparoscopic placement of peritoneal dialysis catheter. PMID- 10741457 TI - Liver retraction techniques for laparoscopic cholecystectomy. PMID- 10741458 TI - Evidence for a noncholinergic function of acetylcholinesterase during development of chicken retina as shown by fasciculin. AB - Fasciculin 2 (FAS), an acetylcholinesterase (AChE) peripheral site ligand that inhibits mammalian AChE in the picomolar range and chicken AChE only at micromolar concentrations, was used in chick retinal cell cultures to evaluate the influence of AChE on neuronal development. The effects of other AChE inhibitors that bind the active and/or the peripheral site of the enzyme [paraoxon, eserine, or 1,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51)] were also studied. Morphological changes of cultured neurons were observed with the drugs used and in the different cell culture systems studied. Cell aggregates size decreased by more than 35% in diameter after 9 days of FAS treatment, mainly due to reduction in the presumptive plexiform area of the aggregates. Eserine showed no effect on the morphology of the aggregates, although it fully inhibited the activity of AChE. In dense stationary cell culture, cluster formation increased after 3 days and 6 days of FAS treatment. However, FAS, at concentrations in which changes of morphological parameters were observed, did not inhibit the AChE activity as measured histochemically. In contrast, paraoxon treatment produced a slight morphological alteration of the cultures, while a strong inhibition of enzyme activity caused by this agent was observed. BW284c51 showed a harmful, probably toxic effect, also causing a slight AChE inhibition. It is suggested that the effect of an anticholinesterase agent on the morphological modifications of cultured neurons is not necessarily associated with the intensity of the AChE inhibition, especially in the case of FAS. Moreover, most of the effects of AChE on culture morphology appear to be independent of the cholinolytic activity of the enzyme. The results obtained demonstrate that FAS is not toxic for the cells and suggest that regions of the AChE molecule related to the enzyme peripheral site are likely to be involved with the nonclassical role of AChE. PMID- 10741459 TI - Horizontal basal cell proliferation in the olfactory epithelium of transforming growth factor-alpha transgenic mice. AB - Transgenic mice in which overexpression of the transforming growth factor alpha (TGF-alpha) gene was directed by the keratin-14 promoter were used to study the regulation of cell cycle progression and proliferation in vivo in the olfactory epithelium. The level of TGF-alpha protein was 73% greater in the nasal-olfactory epithelium of the transgenic mice than in that of nontransgenic littermate controls. Increased levels of TGF-alpha protein were accompanied by a 5.8-fold selective increase in the proliferation of phenotypically characterized horizontal basal cells in the transgenics compared with nontransgenics; in contrast, globose basal cells exhibited a similar low level of proliferation in both transgenics and nontransgenics. The level of expression of epidermal growth factor receptor protein, the receptor for TGF-alpha, was also upregulated in the transgenics, indicating a role for the ErbB tyrosine kinase receptor family in the response to TGF-alpha in the olfactory epithelium. TGF-alpha overexpression was also associated with increased expression of several early cell-cycle associated proteins, including the growth factor sensor cyclin D1, retinoblastoma, E2F-1 transcription factor, and cyclin E, indicating the progression of relatively quiescent progenitor cells in the G1 phase of the cell cycle toward the G1/S restriction point, after which the cells become refractive to mitogens. These results demonstrate a role for the growth factor TGF-alpha in the in vivo regulation of cell cycle progression and proliferation in the mitotically active olfactory epithelium in these transgenic mice. PMID- 10741460 TI - Retrograde tracing and neuropeptide immunohistochemistry of sensory neurones projecting to the cartilaginous distal femoral epiphysis of young rats. AB - Although cartilage is considered to be devoid of innervation, axons occur in the perichondrium and during development in cartilage canals, thereby having a relatively close spatial relationship to chondroblasts and chondrocytes. The present study locates the source of the sensory innervation of the femoral cartilaginous epiphyses of young rats and investigates whether the neuropeptide calcitonin gene-related peptide (CGRP) can influence chondrocytes. Retrograde tracing from the distal femoral epiphysis of young rats with Fast Blue (FB) showed labelled neuronal profiles in the L2-L5 dorsal root ganglia. Sample countings indicated that 50% of the FB-labelled neuronal profiles were located at the L3 level and 25% at the L4 level. The labelled neurones had diameters of 15 40 microm, with a peak at 25-30 microm. Immunohistochemistry showed that about 50% of the FB-labelled profiles contained CGRP. Together with the finding that CGRP influences bone cells to generate the second messenger cAMP, this result suggested the hypothesis that chondrocytes might be similarly influenced by CGRP. However, stimulation of cartilage slices with CGRP in vitro followed by an assay of the cAMP content did not provide support for this hypothesis. We conclude that primary sensory neurones containing CGRP project to the perichondrium and to cartilage canals of growing cartilage, and that exogenous CGRP does not elevate the cAMP content of cartilage slices in vitro. PMID- 10741461 TI - Calcium homeostasis and nerve growth factor secretion from vascular and bladder smooth muscle cells. AB - Bladder and vascular smooth muscle cells cultured from four rat strains (WKY, SHR, WKHA, WKHT) differing in rates of nerve growth factor (NGF) production were used to determine whether a relationship exists between intracellular calcium and NGF secretion. Basal cytosolic calcium was related to basal NGF secretion rates in bladder and vascular smooth muscle cells from all four strains with the exception of WKHT bladder muscle cells. Thrombin is a calcium-mobilizing agent and increases NGF production from vascular but not bladder smooth muscle cells. Strain differences were found in the magnitude of the calcium peak induced by thrombin in vascular smooth muscle cells, but these differences did not correlate with NGF secretion. Thrombin caused a calcium response in bladder smooth muscle cells without influencing NGF production. Quenching the calcium transient with a calcium chelator had no effect on thrombin-inducted NGF secretion rates in vascular smooth muscle cells. Thus, basal intracellular calcium may establish a set point for NGF secretion from smooth muscle. In addition, transient elevations in cytosolic calcium were unrelated to the induction of NGF output. PMID- 10741462 TI - Taxol impairs anterograde axonal transport of microinjected horseradish peroxidase in dorsal root ganglia neurons in vitro. AB - We have investigated the effects of taxol on the axonal transport of horseradish peroxidase (HRP) in dorsal root ganglia (DRG) cells and their neuronal cytoskeleton. The former were analysed by microinjection of HRP into single DRG cells and the latter was studied by means of immunohistochemistry and cryo electron microscopy. In cultured and untreated DRG cells, microinjected HRP was typically transported anterogradely several hundred micrometres along their neurites. Different exposure periods (1, 2 and 3 days) to taxol were analysed. The axonal transport of HRP in DRG cells was time-dependently impeded by taxol. After the drug had been washed out, a recovery of the axonal transport of HRP was observed and confirmed by quantitative analysis. Cryo-electron microscopy revealed an abnormal aggregation of axonal and cytoplasmic microtubules, associated with a decreased amount of cross-linking structures, in taxol-treated DRG cell cultures. After 3 days of taxol exposure, microtubule-associated proteins and Tau-protein were restricted to the cellular somata but the neurofilament network and tubulin-proteins seemed to be unaffected. Our results demonstrate, for the first time, an inhibition of anterograde axonal transport of HRP in single neurons by taxol. This effect is reversible and seems not to be caused by cellular damage, but is rather a consequence of an altered organisation of microtubules and/or microtubule-associated proteins. PMID- 10741463 TI - Neurotrophin receptors and nerve growth factor are differentially expressed in adjacent nonneuronal cells of normal and injured tooth pulp. AB - High-affinity tyrosine kinase A (trkA) neurotrophin receptors on neurons and nonneuronal cells elicit differentiation or survival functions in response to nerve growth factor (NGF), whereas the low-affinity neurotrophin (p75) receptor modulates trkA activity or can independently cause apoptosis or NFkappaB-mediated survival functions. We examined dental tissues for the presence of trkA-like immunoreactivity (trkA-IR), to determine which nonneuronal cell types express it in normal compared with inflamed teeth and how the trkA-positive cells relate to those expressing the p75 receptor and/or NGF. Normal and injured rat molars (dentin cavity for 4 h, 16-24 h, 3 days, 16 days, or 5 weeks) were immunoreacted using the ABC detection system for two anti-trkA antibodies (sTA, Santa Cruz Biotechnology; rTA, L. Reichardt) and antibodies against p75 and NGF, all of which also stained pulpal nerve fibers. We report that, when using the sTA antibody (recognizing the intracellular carboxy terminal), nonneuronal trkA-IR was found in odontoblasts of normal teeth and also in invading polymorphonuclear leukocytes (PMNs) and reparative odontoblasts after injury. When using rTA (recognizing the extracellular domain of the receptor), nonneuronal trkA-IR was only found in odontoblasts. Odontoblasts also had NGF-IR but did not label for NGF mRNA. The lack of odontoblast NGF mRNA suggests that NGF is passed from fibroblasts to the adjacent odontoblasts, where it is picked up by receptor mediated mechanisms for regulation of odontoblast function. Tooth injury disrupts this system such that trkA-IR decreases in injured odontoblasts, p75 decreases in fibroblasts, and NGF is upregulated by fibroblasts and accumulates in the injured pulp and surviving odontoblasts. Pulpal NGF may contribute to chemoattraction for the invading leukocytes or their sTA-IR may have been induced in response to pulpal NGF. Thus, tooth pulp has a different distribution of nonneuronal NGF and its paracrine receptors during inflammation compared with normal conditions. PMID- 10741464 TI - Epidermal growth factor, insulin, and estrogen stimulate development of prolactin secreting cells in cultures of GH3 cells. AB - Pituitary tumor GH3 cells synthesize and secrete both growth hormone (GH) and prolactin (PRL). Morphological and functional changes of GH3 cells induced by epidermal growth factor (EGF, 10 nM), insulin (300 nM), and estradiol-17beta (E2, 1 nM) were studied. Treatment of cultures of GH3 cells for 6 days with EGF, insulin, or E2 alone, and with EGF plus E2 did not affect the total number of GH3 cells, but a combination of EGF, insulin, and E2 decreased the total number of GH3 cells compared with control treatment. DNA-synthesizing cells were detected by monitoring 5-bromo-2'-deoxyuridine (BrdU) uptake. EGF, E2, or a combination of EGF, insulin, and E2 significantly decreased the proportion of BrdU-labeled cells (21.1+/-1.7%, 21.0+/-1.4%, 18.2+/-1.3%; P<0.05, P<0.05, P<0.01, respectively) compared with control treatment (28.6+/-1.5%), but insulin did not (31.4+/-2.4%). Immunocytochemical analysis of GH3 cells cultured in 5% fetal calf serum supplemented medium (control) showed that about 70% of all GH3 cells were GH immunoreactive cells (GH-ir cells), apparently containing only GH, and 14% were mammosomatotrophs (MS cells), containing both GH and PRL, while PRL immunoreactive cells (PRL-ir cells), containing only PRL, were not detected. No GH or PRL immunoreactivity could be detected in the remaining cells (15%). EGF decreased the proportion of GH-ir cells. The effects of EGF were enhanced by simultaneous exposure to insulin and E2; this decreased the proportion of GH-ir cells to about 20% of the total GH3 cells and significantly increased the proportion of MS cells to 300% of controls. Treatment with EGF plus insulin, EGF plus E2, or a combination of EGF, insulin, and E2 all stimulated the appearance of PRL-ir cells. Exposure to EGF caused a significant decrease in GH mRNA (P<0.01) and a significant increase in PRL mRNA (P<0.05). These observations suggest that EGF is closely involved in differentiation of PRL-ir cells from GH ir cells via MS cells in GH3 cell cultures. Cytosine arabinoside (10(-7) M), an inhibitor of cell division, did not affect the changes in proportion of the three cell types induced by treatment with a combination of EGF, insulin, and E2. It is therefore probable that the transdifferentiation does not require mitosis of the GH3 cells. PMID- 10741465 TI - Colocalization of pinopsin with two types of G-protein alpha-subunits in the chicken pineal gland. AB - Pinopsin is a photoreceptive molecule present in the outer segments of chicken pinealocytes. In this paper, the localization of alpha-subunits of G-proteins, rod transducin (Gt1) and Gq/11, was examined by immunoelectron microscopy to investigate whether these G-proteins colocalize with pinopsin in the outer segments. Ultrathin sections of the chicken pineal gland were double immunolabeled with antibodies to pinopsin and either Gt1alpha or Gq/11alpha. As shown previously, the outer segments around the follicular lumen exhibited divergent morphology with ciliary, bulbous, or lamellate shapes, and most of them displayed pinopsin immunoreactivity. The majority (>90%) of pinopsin immunopositive outer segments were labeled by anti-Gt1alpha and/or anti Gq/11alpha antibodies. Application of double-immunolabeling to serial sections demonstrated that a large number of the pinopsin-immunopositive outer segments contained both Gt1alpha and Gq/11alpha immunoreactivities. These results suggest that Gt1alpha and Gq/11alpha are functionally coupled with light-activated pinopsin within a single outer segment. PMID- 10741466 TI - The subcommissural organ of the frog Rana perezi is innervated by nerve fibres containing GABA. AB - The innervation of the frog subcommissural organ was studied by light-microscopic and ultrastructural immunocytochemistry using antisera against serotonin, noradrenaline, dopamine, gamma-aminobutyric acid (GABA), glutamic acid decarboxylase, different GABA receptor subunits and bovine Reissner's fibre material (AFRU). In the proximity of the organ, serotonin- and noradrenaline containing fibres were rare whereas dopamine-immunoreactive fibres were more numerous. Many GABA- and glutamic acid decarboxylase-containing nerve fibres were found at the basal portion of the ependymal cells of the subcommissural organ. Under the electron microscope, these GABA-immunolabelled nerve endings appeared to establish axoglandular synapses with secretory ependymal cells of the subcommissural organ. In addition, the secretory ependymal cells expressed high amounts of the beta2-subunit of the GABA(A) receptor. Since GABA-immunoreactive neurons were present in the frog pineal organ proper and apparently contributed axons to the pineal tract, we suggest that at least part of the GABAergic fibres innervating the frog subcommissural organ could originate from the pineal organ. PMID- 10741467 TI - Immunohistochemical studies on regulation of alternative splicing of fast skeletal muscle troponin T: non-uniform distribution of the exon x3 epitope in a single muscle fiber. AB - Troponin T (TnT) isoforms of chicken fast skeletal muscle are classified into two types, breast-muscle-type (B-type) and leg-muscle-type (L-type) isoforms. These isoforms are produced from a single gene by differential alternative splicing of pre-mRNA. We investigated immunohistochemically the distribution of B-type TnT isoforms in chicken leg muscle (musculus biceps femoris), using anti-exon x3 that was raised against a synthetic peptide corresponding to exon x3 and recognized B type, but not the L-type, TnT isoforms. Mosaic patterns of immunostaining showing locally different expression of B-type TnT isoforms in a single fiber were observed among fibers, and the non-uniform distribution of the isoforms was also detected in sectioned fibers and myofibrils from the muscle. The results indicated that regulation of pre-mRNA splicing of fast skeletal muscle TnT was different not only among the muscle fibers but also within a single fiber, suggesting that heterogeneous myonuclei in regulation of alternative splicings occur in a single muscle fiber. PMID- 10741468 TI - Sialic acid inhibits agrin signaling in C2 myotubes. AB - Acetylcholine receptor (AChR) clustering is an early event in neuromuscular synapse formation that is commonly studied using muscle cell culture. Motor neuron-derived agrin induces the postsynaptic tyrosine phosphorylation of both a muscle-specific kinase (MuSK) and the AChR beta-subunit. These phosphorylation events are required for AChR clustering, suggesting an agrin-driven signaling pathway. Both the phosphorylation events and AChR clustering can also be induced by neuraminidase, an enzyme that cleaves sialic acid from glycoconjugates, suggesting that neuraminidase is able to activate the agrin signaling pathway. A postulated signal for postsynaptic differentiation at sites of nerve-muscle contact during vertebrate development is the enzymatic removal of basal lamina components. We show here that bath-applied sialic acid has an effect directly opposite that of agrin or neuraminidase. Sialic acid not only decreases AChR clustering but also diminishes the tyrosine phosphorylation of MuSK and the AChR beta-subunit signal-transduction events normally driven by agrin. However, sialic acid does not prevent agrin-binding molecules from colocalizing with the decreased number of AChR clusters that do form, suggesting that sialic acid is acting to inhibit the agrin signaling pathway downstream of agrin binding to the muscle cell membrane. We propose a regulatory role for sialic acid in the signal transduction events of neuromuscular synapse formation, in which agrin or neuraminidase can overcome this sialic acid repression, resulting in the clustering of AChRs and other postsynaptic molecules. PMID- 10741469 TI - In situ localization of anion exchanger-2 in the human kidney. AB - Na+-independent anion exchangers (AE) are a family of membrane carriers that mediate the electroneutral exchange of Cl- for HCO3- ions across plasma membranes. They are involved in intracellular pH and cell volume regulation as well as in transepithelial acid-base transport. While anion exchanger-1 (AE1) has been localized previously in the human kidney, thus far there has been no definite report on anion exchanger-2 (AE2) in this human tissue. Accordingly, immunohistochemistry was carried out on surgical specimens of the human kidney (fixed in formalin and embedded in paraffin), using a specific AE2 monoclonal antibody. Strong immunostaining was observed at the basolateral membrane of cells of thick ascending limbs and distal convoluted tubules, colocalizing with the basal membranous labyrinth of cellular interdigitations, typical of these segments. In fact, AE2 staining was attenuated at the macula densa, where basal infoldings are scarce. Additionally, in situ hybridization experiments on formalin-fixed tissue demonstrated the presence of AE2 mRNA in the same segments of the distal nephron. On the other hand, control immunohistochemistry with a monoclonal antibody against AE1 gave the expected immunoreactivity at the basal pole of the type A intercalated cells of connecting tubules and cortical collecting ducts, and in erythrocytes. Our results indicate that, depending on the nephron segment and corresponding cell types, AE1 and AE2 proteins are differentially involved in the Na+-independent exchange of Cl- for HCO3- at the basolateral membrane of polarized kidney epithelial cells. PMID- 10741470 TI - Expression and cell-specific localization of the cholecystokinin B/gastrin receptor in the human stomach. AB - Gastrin stimulates gastric acid secretion by acting on the cholecystokinin B/gastrin receptor (CCK-BR). The localization of this receptor at the cellular level showed conflicting results in animal studies and has not been described in man by immunohistochemistry. The aim of the present study is to characterize the precise cellular location of the CCK-BR in the human stomach. Polyclonal antisera were raised against different epitopes of the CCK-BR molecule and used for immunohistochemical investigations. CCK-BR mRNA was detected in paraffin tissue sections by the highly sensitive method of in situ reverse transcriptase polymerase chain reaction (RT-PCR). Using immunohistochemistry, CCK-BR could successfully be localized in gastric parietal cells. In the majority of parietal cells, CCK-BR immunoreactivity was present a he basolateral cell membrane domain. In some parietal cells, a granular pattern of immunoreactivity was exclusively confined to the cytoplasm of the cells. CCK-BR mRNA was found in parietal cells and in enterochromaffin-like (ECL) cells by means of in situ RT-PCR. No expression of CCK-BR was found in the gastric antral mucosa. Our data support the concept that gastrin stimulates gastric acid secretion directly via CCK-B receptors on parietal cells and indirectly by inducing histamine release from histamine-containing ECL cells, which contributes to acid secretion by parietal cells. PMID- 10741471 TI - Fluoride-induced ultrastructural changes in exocrine pancreas cells of rats: fluoride disrupts the export of zymogens from the rough endoplasmic reticulum (rER). AB - Influence of fluoride on exocrine pancreas cells was examined morphologically with traditional and prolonged osmium fixation techniques for electron microscopy in the enamel fluorosis model rats injected subcutaneously twice a day with 20 mg/kg body weight of sodium fluoride. Although the rough endoplasmic reticulum (rER) of exocrine pancreas cells in control rats was laminated and oriented parallel to the circumference of the nucleus, the rER of the cells in NaF-treated rats was dilated, disrupted the laminated arrangement, and changed to the globular-shape rER. Many intracisternal granules were formed in these globular shape rER of the cells exposed to fluoride. Lots of autophagosomes were also seen in the exocrine cells with NaF treatment. The autophagosomes were limited with a double or multiple membranes, and contained cytoplasmic organelles and/or the intracisternal granules. The outer and inner leaflets of double membranes of the autophagosomes were usually separated by a distinct electron-lucent area. In prolonged osmium fixation, the area between the double membranes of the autophagosome was filled with osmiun reaction deposits. Many autophagosomes were encircled with the single or multiple osmiophilic layers. In some cases, the osmium positive saccules also surrounded the free surface of the globular-shape rER containing intracisternal granules. These findings indicate that fluoride disrupts the export of zymogens from the rER, resulting in formation of intracisternal granules and autophagosomes, and that the osmiophilic saccules participate in sequestration of cytoplasmic organelles in forming autophagosomes. PMID- 10741472 TI - CYP2E1 expression in bone marrow and its intra- and interspecies variability: approaches for a more reliable extrapolation from one species to another in the risk assessment of chemicals. AB - When characterizing the health risks for man by exposure to chemicals, species specific differences have to be taken into consideration, otherwise extrapolation from animal data to the human situation would be inadequate. The site-specific toxicity of chemicals may be explained by the following alternatives: (1) reactive metabolites are generated in the liver and subsequently transported to the target tissue(s); (2) metabolism of the parent compound occurs in the target tissue, a pathway by which the enzymes necessary for activation must be expressed in the target tissue. Cytochrome P450 2E1 (CYP2E1) is an important phase-I enzyme activating several chemicals. In the study described in this paper, myeloid intra and interspecies variability in the expression of CYP2E1 has been investigated in rats, rabbits and man, because the bone marrow represents an important target organ for toxic effects of several chemicals, e.g. benzene. CYP2E1 at the protein level was detected by Western blotting and enzyme activities were determined by CYP2E1-dependent hydroxylation of chlorzoxazone (CLX). In the bone marrow of Wistar rats, the CLX hydroxylase activities were within the same order of magnitude (range: 0.1-0.4 pmol/mg protein per min) as previously described for mice (range 0.2-0.8 pmol/mg protein per min), whereas the CYP2E1 activities in two strains of rabbits were significantly higher (range: 1.7-4.7 pmol/mg protein per min) than in the rodents (P < 0.05). In human CD34+ bone marrow stem cells, CYP2E1 could also be detected on the protein level by Western blotting. The data demonstrate a presence of CYP2E1 in the bone marrow of all species investigated, thus supporting the hypothesis of CYP2E1-dependent local metabolism of several chemicals as a factor possibly contributing to their myelotoxicity and haematotoxicity. The data show that intraspecies/intrastrain variability of CYP2E1 activity in rodents is small. However, CYP2E1 activity between rodents and a non-rodent species was quite different indicating considerable interspecies variability. PMID- 10741473 TI - Effect of 4-tert-octylphenol on cytochrome P450 enzymes in rat liver. AB - The effects were studied of 4-tert-octylphenol (OP) on hepatic cytochrome P450 enzymes in rats. Rats were treated intraperitoneally with OP twice, at doses of 5, 10, and 20 mg/kg. Among the cytochrome P450-dependent monooxygenase activities, testosterone 2alpha-hydroxylase activity, which is associated with CYP2C11, was significantly decreased by OP at all doses. The level relative to control activity was 67-22%. CYP3A2-dependent monooxygenase, testosterone 6beta hydroxylase activity was also decreased by 51% by OP at 20 mg/kg. Furthermore, immunoblotting showed that OP (10 or 20 mg/kg) significantly decreased CYP2C11/6 and CYP3A2/1 protein levels. However, the reduction ratio of CYP2C11/6 and CYP3A2/1 protein levels by OP treatment was lower than that of testosterone 2alpha-hydroxylase and testosterone 6beta-hydroxylase activities. The Cl(int) (Vmax/Km) value for testosterone 2alpha-hydroxylase was significantly decreased by OP at all doses, whereas the Cl(int) value for testosterone 6beta-hydroxylase was only decreased by OP at 20 mg/kg. In addition, 7-ethoxycoumarin O-deethylase activity was significantly decreased by 32% by the highest dose of OP. By contrast, CYP1A1-, CYP1A2-, CYP2A1-, CYP2B1/2-, CYP2D1-, CYP2E1- and CYP4A1/2/3 dependent monooxygenase activities were not affected by OP at any dose. These results suggest that OP changes the male-specific cytochrome P450 isoforms in rat liver, and that these changes closely relate to the toxicity of OP. PMID- 10741474 TI - Biological monitoring of standardized exposure to ethylbenzene: evaluation of a biological tolerance (BAT) value. AB - The results of standardized 8 h lasting exposures of n = 18 volunteers to ethylbenzene (EthBz) at levels of 25 and 100% of the maximum allowable concentrations at the workplace (MAK) value of 100 ppm as well as the results of field studies are considered to evaluate a biological tolerance (BAT) value for EthBz. On the basis of the relationship between the external and internal exposure a BAT value of 1.5 mg/l has been set for the EthBz concentration in blood as the most sensitive and specific parameter of exposure to this aromatic hydrocarbon. The interpretation of EthBz blood values has to take into account the short half-life of t1/2 = 0.5 +/- 0.08 h in the first hour after the end of exposure in which this aromatic hydrocarbon is eliminated from the blood. The additional determination of the EthBz metabolites mandelic acid (MA) and phenylglyoxylic acid (PGA), respectively, excreted in post shift urine as well as in urine samples at the beginning of the next shift shows good correlations with the external exposure. The biological half-life of MA was calculated to t1/2 = 5.3 +/- 1.1 h. Because the time of sampling can vary the relationship between the levels of MA to PGA the total concentration of the excreted metabolites depends less on this influence and is therefore better suited for monitoring exposed persons. On the basis of the standardized experiments a BAT value has been proposed of 2 g MA plus PGA corrected per gram creatinine. Both BAT values are adjusted to data which result from earlier standardized exposures during 30 min to EthBz under physical activity of 50 watt on a bicycle ergometer. PMID- 10741475 TI - Experimental exposure of rat skin to methyl bromide: a toxicokinetic and histopathological study. AB - Methyl bromide was experimentally exposed to a 12 cm2 area of the back skin of Wistar rats for 30 s, and for 1, 3, and 5 min, and time courses of both changes in plasma bromide concentration and of histopathological changes were examined. To measure bromide ion, a head space gas chromatography was used. The concentration of plasma bromide ion showed a sharp increase immediately after the exposure in all exposed groups, reaching a peak level after 1 h, then decreased rapidly. The ion level gradually decreased after 72 h to 1 week, and returned to a normal level after 4 to 8 weeks. Calculating from a regressive curve, the biological half lives of plasma bromide ion were 5.0 days to 6.5 days. Histopathologically, the impairments to the epidermal cells, fibroblasts and blood vessels were observed in the early phase. These cellular changes could be due to the direct cytotoxicity of the compound. In the next phase, newly infiltrating cells showed degeneration and necrosis. Subsequently, an impairment of the collagen bundles was observed. Our experiments suggested an immediate permeation and rapid metabolization of methyl bromide in the skin and a multistep formation of the skin damage induced by the compound. These processes of methyl bromide-induced skin damage are quite different from chemical skin injuries caused by the representative causative agents such as alkaline and acid. PMID- 10741476 TI - Measurement of furancarboxylic acid, a candidate for uremic toxin, in human serum, hair, and sweat, and analysis of pharmacological actions in vitro. AB - 3-Carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF), a candidate for uremic toxin, was measured in human hair for examining a possible utility as indicator of renal dysfunction. The serum concentration of CMPF was much higher (32.3 +/- 2.7 microg/ml, n = 17; mean +/- SEM) in uremic patients aged 40-55 years receiving hemodialysis treatment than in healthy younger subjects (3.61 +/- 0.19 microg/ml, n = 22), aged 18-23 years. However, the hair concentration of CMPF tended to be lower in the patients (6.8 +/- 1.7 ng/10 mg hair) than in the healthy younger subjects (15.8 +/- 4.5 ng/10 mg) and was significantly lower than that in the healthy age-matched subjects (22.4 +/- 5.3 ng/10 mg, n = 12), aged 40 47 years. Since CMPF was measurable in the sweat (4.4 +/- 3.7 ng/mg) collected from six out of seven healthy subjects examined, it was suggested that the contribution of sweat to the measurement of CMPF in hair was considerable. The fact that the uremic patients undergoing hemodialysis therapy had less sweat than healthy subjects may explain the lower concentration of CMPF in the patients' hair. The pathophysiological roles of CMPF in the body were attempted to be explored by using excised guinea pig organs, and human platelets and neutrophils. CMPF showed no remarkable effects in the concentration range of < or =10(-4) M except for only slight suppression of spontaneous contracture of guinea pig tenia coli at 10(-4) M. As far as the organs and tissues examined in the present study are concerned, the biological activity of CMPF itself, if any, may be very weak. Precaution should be taken against the delivery of a substance through sweat to hair when a small amount of substance is attempted to be measured in hair by employing a sensitive analytical method. PMID- 10741477 TI - The relevance of inhibitor-substrate interactions when measuring neuropathy target esterase inhibition. AB - Neuropathy target esterase (NTE), thought to be the target for organophosphate polyneuropathy, is operationally defined as that neural phenyl valerate esterase resistant to paraoxon (40 microM) and sensitive to mipafox (50 microM; 20 min, pH 8.0, 37 degrees C). The time course of inhibition of particulate paraoxon pretreated esterases by mipafox showed that the lines indicating the rate of inhibition did not pass through the log 100% activity when extrapolated at zero time. Slopes of inhibition of NTE were not linearly related to the concentration of mipafox. Kinetic parameters derived from Wilkinson type plots were: Ka = 49 199 microM, k(+2) = 0.24-0.64 min(-1) and k(a) = 3.1-5.0 mM(-1) m(-1). When mipafox was removed (either by dilution or centrifugation) before the addition of phenyl valerate intercepts below 100% disappeared. We confirm that the formation of Michaelis complex between NTE and mipafox is not prevented by phenyl valerate and that inhibition proceeds after addition of phenyl valerate. We compared inhibitions obtained with experiments by using the traditional method (sequential incubation with inhibitors and phenyl valerate) to those obtained with a method where mipafox is removed before the addition of substrate. When calculating fixed time 50% inhibitory concentrations (IC50s) of some inhibitors for NTE, the longer the hydrolysis time, the lower were the IC50s. Therefore, the inhibitory potency of certain NTE inhibitors, is accurately assessed only when calculating second order rate constants (k(a)). PMID- 10741478 TI - Post-mortem orthopantomography--an aid in screening for identification purposes. AB - Ante-mortem orthopantomograms may be of great value in the identification of human remains. This x-ray technique provides an overall view of the teeth and jaws and thus of numerous individual structural characteristics within a short time. Standardised post-mortem orthopantomography has previously not been feasible in the forensic practice. The present study shows how orthopantomography can be applied to identification procedures. The reproduction of ante-mortem x ray conditions is implemented here in the production of post-mortem x-rays, using a purpose-designed radiographic tripod. For the first time, account is taken not only of the size and structure but also of the nature of the soft tissue covering of exhibits. For post-mortem preparation of these radiographs, appropriate positioning aids, a spinal column substitute and a soft tissue filter were constructed. Individual macerated jaws as well as the complete cranium can now be positioned correctly in the upright orthopantomograph (OPG). The method presented expands the spectrum of forensic radiology for the individual case and in our opinion also offers a reliable aid for victim identification in the wake of mass disasters, aircraft crashes and terrorist attacks, where a large number of bodies have to be identified under great pressure. PMID- 10741479 TI - Immunohistochemical investigations on the course of astroglial GFAP expression following human brain injury. AB - The course of GFAP expression by astrocytes has been immunohistochemically investigated during the first 30 weeks after human brain injury. In order to provide reliable data for a forensic wound age estimation, a quantitative morphometric analysis was performed considering the different topographic regions of the cortex as well as of the white matter. Compared to the GFAP immunoreactivity in unaltered control tissue, significantly increased numbers of GFAP positive astroglial cells could be detected adjacent to the cortical contusion from 1 day up to 4 weeks after brain injury. PMID- 10741480 TI - Further sequence and length variation at the STR loci HumFES/FPS, HumVWA, HumFGA and D12S391. AB - This paper reports population data and statistics for the HumFES/FPS, HumVWA, HumFGA and D12S391 loci in Austria. The sequences of some rare and new variant alleles which have been identified in the course of the present population study and other investigations are described. Sequence variation occurred in a HumFES/FPS allele revealing an (ATTT)9 structure and an A to C transversion in the 5' flanking region. At the HumVWA locus the structural type of the common allele 14 has been found in one allele 13 and in three examples of allele 15. Additionally the TCTA (TCTG)3(TCTA)n structure has been observed in three examples of allele 13 and one allele 14, which is very uncommon. Another allele 14 showed a C to T transition in the third of nine TCTA repeats. The sequences of three length variations at the HumFGA locus, namely the alleles 16, 19.2 and 21.2 are reported. At the D12S391 locus a novel 19.1 allele was found in this study. An extended nomenclature is proposed for the HumVWA locus to denominate sequence variants in a precise but simple way. PMID- 10741481 TI - Colour measurements of pallor mortis. AB - Little interest has yet been focused on the development of postmortem paleness (pallor mortis). Using an opto-electronical colour measurement device, we examined pallor mortis in 126 bodies and compared these findings to the average skin colour of 72 living Caucasian volunteers. It was shown that (a) hairy skin influences the results and any hair must be removed by shaving before colour determination, (b) among the living, there is a skin colour difference between the sexes which disappears after death, (c) postmortem paleness is caused by lack of capillary circulation after death and (d) paleness develops so rapidly after death that it has no or little use in determining time of death. PMID- 10741482 TI - Relationship between impact velocity and injuries in fatal pedestrian-car collisions. AB - The most common type of fatal pedestrian-car collision, i.e. a passenger car with a wedge or pontoon shaped front striking an erect adult with this front, was investigated. A reliable calculation of the impact velocity range by a technical expert and a comprehensive autopsy suitable for traffic accidents were performed in every case. A total of 47 fatalities form the material of this study and the impact velocities varied between 18 and 142 km/h. Primary and secondary injuries did not show a relationship to impact velocity. The occurrence of four types of indirect injuries revealed a clear relationship to impact velocity, i.e. spinal fractures, ruptures of the thoracic aorta, inguinal skin ruptures and dismemberment of the body. Important parameters such as the type of car, impact velocity range and indirect injuries are listed for each individual case. Because of the limited number of cases, the impact velocity ranges (3-30 km/h) instead of mean values were considered. A cautious interpretation of the data can be summarised in the following conclusions: If there is no spinal fracture, the velocity was below 70 km/h and probably below 50 km/h. Aortic and inguinal skin ruptures are always present if the velocity was above 100 km/h but never occurred below 50-60 km/h. If dismemberment occurs, the velocity was above 90 km/h. Consequently, an estimation of the impact velocity from the presence or absence of indirect injuries is possible in pedestrian-car collisions of the type examined. However, the selection criteria applied in this study and additional parameters influencing the collision dynamics have to be considered carefully. PMID- 10741483 TI - Forensic evaluation of mtDNA in a population from south west Switzerland. AB - The polymorphism of the two hypervariable segments (HVI and HVII) of the control region of mtDNA was analyzed in a population of 154 unrelated individuals from south west Switzerland using a fluorescent based capillary electrophoresis sequencing method. In our population data of 154 random individuals, 137 mtDNA types were observed. Of these, 124 sequences were observed only in one individual whereas 10 sequences were observed in 2 individuals, 2 sequences in 3 individuals and 1 sequence in 4 individuals. The probability of two unrelated individuals having the same sequence was 0.84%. The results were compared with four other Caucasian populations. Furthermore, the usefulness of the mtDNA sequencing was tested, for exclusion and inclusion, in 18 forensic cases including 69 evidence samples and 44 reference samples. Despite the fact that 55% of the evidence samples yielded a negative result for the nuclear DNA with the human dot quantitation system, the success rate of the mtDNA sequencing was 71.0%. This validation study proves the great usefulness and sensitivity of the mtDNA sequencing technique using nested PCR and fluorescent capillary electrophoresis. PMID- 10741484 TI - Forensic identification of skeletal remains from members of Ernesto Che Guevara's guerrillas in Bolivia based on DNA typing. AB - We report the positive identification of several members of the guerrillas led by Ernesto "Che" Guevara on the 1960 s in Bolivia by means of DNA fingerprinting. Successful DNA typing of both short tandem repeat loci and the hypervariable region of the human mitochondrial DNA was achieved after extracting total DNA from bones obtained from two burial sites. Given the size of the Cuban database for the STR allele frequencies, a conservative approach was followed to estimate the statistical significance of the genetic evidence. The estimated probabilities of paternity for the two cases in which the paternity logic was applied were higher than 99%. One case was analyzed using mitochondrial DNA and could not be excluded from the identity proposed by the forensic anthropology team. A fourth case was identified by exclusion, on the basis of the positive identification of the other remains, the historical and other anthropological evidence. PMID- 10741485 TI - Effect of the shampoo Ultra Clean on drug concentrations in human hair. AB - The influence of the special shampoo Ultra Clean (Zydot Unlimited, Tulsa, Oklahoma) on the results of hair analyses was investigated. Hair samples from persons (n = 14) with a known history of drug abuse were collected at autopsy. The hair samples were divided into separate strands which were analyzed both after washing with Ultra Clean and without treatment. Hair analyses were performed by methanol extraction under sonication, purification by solid phase extraction and GC/MS in SIM mode according to routine procedures for tetrahydrocannabinol (THC), cocaine, amphetamine, methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), methylenedioxyethylamphetamine (MDE), heroin, 6-monoacetylmorphine (6-MAM), morphine, codeine, dihydrocodeine and methadone. All drugs originally present in the hair fibers were still detected after a single application of Ultra Clean. However, a slight decrease in drug concentrations could mostly be observed e.g. cocaine (n = 10) -5%, 6-MAM (n = 12) -9%, morphine (n = 12) -26%, THC (n = 4) -36%. The findings clearly demonstrated that drug substances had not been sufficiently removed from human hair by a single Ultra Clean treatment to drop their concentrations below the limit of detection of the analytical method applied. PMID- 10741486 TI - A rare case of evisceration. AB - A case of homicide is described where the body of a young woman was found in the countryside in a state of advanced decomposition. The autopsy revealed that almost all the internal organs were missing, but not due to putrefaction. The thoracic cavity was opened in the professional manner normally used during an autopsy. The remains of the small intestine were tied with strands of hair braided into a thin cord. PMID- 10741487 TI - Sudden death due to cerebrotendinous xanthomatosis confirmed by mutation analysis. AB - A case of sudden death of a 52-year-old mentally retarded Caucasian male is described where the rectal temperature was 43.4 degrees C 3 h postmortem. The autopsy revealed cerebrotendinous xanthomatosis (CTX), a rare hereditary metabolic disorder, as the primary disease. The diagnosis was confirmed by postmortem identification of two mutations (compound heterozygosity for R237X and IVS6+1G-->A) in the sterol 27-hydroxylase (CYP27) gene. Both mutations have already been described in patients with CTX and can be considered the most likely cause of the disease. The pathomechanism of the excessive hyperthermia could not be completely elucidated. PMID- 10741488 TI - Influence of different staining techniques on the DNA analysis of histological sections. AB - The use of stained histological sections as a source for DNA may be necessary in forensic case work if confusion of tissue is suspected, for identification or paternity cases. To elucidate the influence of different staining techniques on the PCR amplification of DNA, histological sections of liver tissue were prepared using eight different staining techniques and two histochemical methods. The DNA of the sections was extracted by a modified Chelex extraction and amplified using a commercial triplex kit. Staining with hematoxylin-eosin, hemalum-eosin, azan, periodic acid-schiff and prussian blue showed no adverse effect on the amplification of DNA while the extracts of tissue stained by Masson-Goldner, Ladewig and elastica-van Gieson methods had to be purified before amplification was possible. Staining with phosphoric tungsten acid hematoxylin and Gomori led to a degradation of DNA probably due to the use of potassium permanganate solution. PMID- 10741489 TI - Demonstration of morphine in ganglion cells of the hippocampus from victims of heroin overdose by means of anti-morphine antiserum. AB - To investigate the topography of morphine distribution in the human brain, a method has been developed to detect morphine immunohistochemically. In this study hippocampus tissue from victims of heroin overdose (blood morphine concentrations 220 ng/g-1500 ng/g; 6-MAM positive urine sample), known for its high concentration of mu-opiate receptors was used. The immunohistochemical staining was performed with an anti-morphine antiserum originally developed for radio immunoassays. In comparison with control specimens from cases of sudden death without morphine exposition or a history of heroin abuse, the brains from victims of heroin overdose showed selectively stained ganglion cells, axons and dendrites, suggesting a massive concentration of morphine in the neuronal structures. PMID- 10741490 TI - Stability of carbohydrate deficient transferrin (CDT) in stored blood samples. AB - The alcoholism marker CDT was determined on 257 blood samples 1-2 days after the blood samples were taken and again after storage for 7 months at +4 degrees C. The differences between the pairs of CDT values were so large that the determination of CDT after long term storage of the blood sample has no evidential value. PMID- 10741491 TI - Frequency data on the loci vWA, FES/FPS, F13A01, TH01, TPOX and CSF1P0 in a population from South Poland. AB - Allele and genotype frequencies for six short tandem repeat (STR) loci were determined in a sample of 124 inhabitants from South Poland with commercial PCR based typing kits. No deviations from Hardy-Weinberg expectations were found. The combined power of discrimination for the six loci was 0.9999982. There was no genotypic disequilibrium between the loci except for vWA and F13A01. The set of PCR loci was validated as useful for paternity testing and individual identification in the Polish population. PMID- 10741492 TI - Population study of Aymara Amerindians for the PCR-DNA polymorphisms HUMTH01, HUMVWA31A, D3S1358, D8S1179, D18S51, D19S253, YNZ22 and HLA-DQalpha. AB - Allele and genotype frequencies for eight DNA polymorphisms (HUMTH01, HUMVWA31A, D3S1358, D8S1179, D18S51, D19S253, YNZ22 and HLA-DQalpha) were determined in a population sample of Aymara Indians from Bolivia using PCR. No deviations of the observed allelic frequencies from Hardy-Weinberg equilibrium were found for all the systems studied. Significant differences in the allele frequencies were found between the Aymara and Quechua populations only for HUMVWA31A, which suggests a certain degree of genetic differentiation between the two populations. PMID- 10741493 TI - Benign vascular lesions of bone: radiologic and pathologic features. AB - The benign vascular tumors of bone represent a diverse group of tumors that can present with a broad spectrum of clinical signs and symptoms. They can also present a significant diagnostic challenge due to their widely variable radiographic imaging and histologic features. Some of the tumors manifest as clearly benign lesions with tissue-specific diagnostic imaging features, while others have non-specific imaging features that may simulate malignant neoplasm. This article will provide a review of the nomenclature and the characteristic radiographic and pathologic features of the benign vascular lesions of bone. The information will aid in improving our diagnostic accuracy and enhance our understanding of the biologic potential of this diverse group of osseous lesions. PMID- 10741494 TI - "Spinolaminar breach": an important sign in cervical spinous process fractures. AB - OBJECTIVE: To report the sign of "spinolaminar breach" and its likely importance in fractures of the cervical spinous processes. DESIGN: Six cases of spinous process fractures demonstrating disruption of the spinolaminar line or "spinolaminar breach" were analyzed. Lateral and anteroposterior radiographs (n=6), CT scans (n=3) and MRI scans (n=1) were reviewed together by the authors, with consensus being reached as to the radiographic findings. Clinical records were also reviewed. RESULTS: The levels of injury were C6 (n=5) and C5 (n=2). Injuries were associated with delayed anterior subluxation (n=4) and neurological deficit (n=2). Five patients were male and one was female with a mean age of 31 years (range 8-59 years). Injuries resulted from motor vehicle accidents (n=4), a motor cycle accident (n=1) and a fall (n=1). CONCLUSION: "Spinolaminar breach", or disruption of the spinolaminar line, indicates a complex spinous process fracture with extension into the lamina and spinal canal. Spinous process fractures with spinolaminar breach may have associated posterior ligamentous injury with potential for delayed instability and neurological deficit. It is important that radiologists and physicians caring for the trauma patient be aware of this sign in order to avoid misdiagnosis as a "clay shoveler's fracture", which can lead to adverse outcome. PMID- 10741495 TI - MR arthrography of the shoulder and hip after fluoroscopic landmarking. AB - PURPOSE: To describe a technique for intra-articular injection in the MR suite after conventional fluoroscopic landmarking in order to streamline MR arthrography. DESIGN AND PATIENTS: This technique was performed on 33 consecutive patients referred for MR arthrography of the shoulder to evaluate the glenoid labrum and on 15 consecutive patients referred for MR arthrography of the hip to evaluate the acetabular labrum. The patients were landmarked in the fluoroscopy suite, followed by a conventional MR examination. The intra-articular injection was then performed on the MR table and the MR arthrographic sequences obtained. RESULTS: One of the 48 injections was extra-articular, requiring a second injection. The other injections were performed without incident, and the average total procedure time for all injections was 10 min. CONCLUSIONS: This technique is a reliable method of streamlining intra-articular injections when performing conventional MR imaging prior to the MR arthrographic portion of the examination. It shortens the total MR examination time by eliminating a visit to the fluoroscopy suite in the middle of the MR study, and its use of a straight anterior approach for both the shoulder and hip joints should be familiar to most people who perform conventional arthrography. PMID- 10741496 TI - Are magnetic resonance flexion views useful in evaluating the cervical spine of patients with rheumatoid arthritis? AB - OBJECTIVE: To determine whether MR imaging in flexion adds value relative to imaging in the neutral position with respect to displaying involvement of the subarachnoid space, brainstem and spinal cord. DESIGN AND PATIENTS: T1-weighted MR images of the cervical spine in 42 rheumatoid arthritis patients with cervical spine involvement were obtained and analyzed prospectively. We assessed changes between images obtained in the neutral position and following active flexion, especially horizontal atlantoaxial and subaxial motion, presence or absence of brainstem compression, subarachnoid space involvement at the atlantoaxial and subaxial level and the cervicomedullary angle. Vertical atlantoaxial subluxation and the amount of pannus were correlated with motion and change in subarachnoid space. RESULTS: The flexion images showed horizontal atlantoaxial motion in 21 patients and subaxial motion in one patient. The flexion view displayed brainstem compression in only one patient. Involvement of the subarachnoid space increased at the atlantoaxial level in eight (19%) patients (P=0.004) and at the level below C2 in five (12%) patients (P=0.03). There were no patients with a normal subarachnoid space in neutral position and compression in the flexed position. The cervicomedullary angle changed significantly with flexion. Vertical atlantoaxial subluxation and the amount of pannus did not show a significant correlation with motion or subarachnoid space involvement. CONCLUSION: MR imaging in the flexed position shows a statistically significant narrowing of the subarachnoid space at the atlantoaxial level and below C2. Cord compression is only observed on flexion views if the subarachnoid space in neutral position is already decreased. MR imaging in the flexed position might be useful, since subarachnoid space involvement may be an indicator for the development of neurologic dysfunction. PMID- 10741497 TI - Lumbar and radial bone mineral density in children and adolescents with X-linked hypophosphatemia: evaluation with dual X-ray absorptiometry. AB - OBJECTIVE: To evaluate the bone mineral status of children being treated for X linked hypophosphatemia, including potential differences between cortical bone in the radial diaphysis and combined cortical and trabecular bone in the lumbar spine. DESIGN AND PATIENTS: Forty-four bone mineral evaluations were performed in 11 children and adolescents with X-linked hypophosphatemia. Bone mineral density (BMD) of the lumbar spine and the radial diaphysis were measured by dual X-ray absorptiometry (DXA), second metacarpal cortical thickness was measured on hand radiographs, and these results were expressed as Z-scores (standard deviations from the mean). RESULTS: For the 11 initial examinations, Z-scores (mean+/-SD) were: radial BMD, -2.73+/-1.15, lumbar BMD, +1.28+/-1.53; and cortical thickness, -2.21+/-0.95. Lumbar BMD Z-scores were significantly greater than those for radial BMD and cortical thickness. On follow-up examinations there was a mild increase in radial BMD and decrease in lumbar BMD. Although these changes were statistically significant, they were quite small and the discordance between radial and lumbar BMD was not corrected. CONCLUSIONS: Children and adolescents who are being treated for X-linked hypophosphatemia manifest a bone mineral disorder characterized by decreased BMD in the appendicular skeleton and increased BMD in the lumbar spine. Although current therapy is successful in its anti-rachitic effects, it does not correct this bone mineral disorder and additional therapeutic trials should be considered. PMID- 10741498 TI - Premature epiphyseal fusion and degenerative arthritis in chronic recurrent multifocal osteomyelitis. AB - A 9-year-old boy was diagnosed with chronic recurrent multifocal osteomyelitis affecting multiple sites. During an 8-year follow-up he developed premature closure of a distal radial epiphysis and degenerative changes in the adjacent radiocarpal joint. PMID- 10741499 TI - Intra-articular regional migratory osteoporosis of the knee. AB - We report a case of intra-articular regional migratory osteoporosis of the knee in a 53-year-old man. The case demonstrates an unusual pattern of migration of the marrow edema within the knee joint. This phenomenon has received scant attention in the radiological literature. PMID- 10741500 TI - Proteus syndrome: MRI characteristics of plantar cerebriform hyperplasia. AB - Proteus syndrome is a rare congenital hamartomatous syndrome with a variety of abnormalities. It shares many features with other congenital hamartomatous disorders, but cerebriform hyperplasia of the soles and the palms is known as a quite distinctive characteristic in the dermatologic literature. The purpose of this case report is to demonstrate the MRI features of plantar cerebriform hyperplasia in a 9-year-old boy with known Proteus syndrome. PMID- 10741501 TI - Dumb-bell-type teratoma in the lumbar spine. AB - We report a case of a lumbar teratoma in a 50-year-old woman. The teratoma showed a dumb-bell-type expansion at the level of the left L3/4 foramen with massive erosion of the L3 vertebral body. MRI revealed inhomogeneous signal changes in the tumor, which were histologically compatible with a mixture of bone, muscle, fat, and cyst containing sebaceous material. Complete resection of the tumor and spinal arthrodesis with pedicle screw fixation was necessary to obtain stability of the affected spinal segment. PMID- 10741502 TI - Elastofibroma of the neck. AB - Elastofibromas are benign lesions of the chest wall. We describe the first reported case of elastofibroma in the neck. Imaging features as well as location of the lesion were atypical. On computed tomography and magnetic resonance imaging the lesion contained a marked preponderance of fat, because the lesion arose within fat. PMID- 10741503 TI - Hyperfunctioning parathyroid carcinoma. AB - Primary hyperparathyroidism is rarely caused by carcinoma. We report a patient who manifested many of the clinical and radiographic features of the disease. When encountering symptomatic hypercalcemia with or without a palpable neck mass, carcinoma should be considered in the differential diagnosis. Patient survival depends on an aggressive surgical approach to the primary lesion and recurrent disease. PMID- 10741504 TI - Metabolic genotype in relation to individual susceptibility to environmental carcinogens. AB - Earlier research indicates that within the human population there are considerable differences in the response to the carcinogenic activity of environmental carcinogens. Genetic polymorphism associated with several variants of the gene products participating in the biotransformation of various xenobiotics (including carcinogens) found in human populations constitutes a major cause of those differences. Enzymes coded by different variants of the same gene can differ in their catalytic activities. Up to the present time, most information on the effect of genetic polymorphism on the individual's ability to activate or deactivate environmental carcinogenic xenobiotics, and the associated risk of cancer, has been collected from studies of cytochromes P-450 belonging to gene families CYP1, CYP2 and CYP3, and of glutathione S-transferases and N acetyltransferases. As carcinogen metabolism comprises a chain of chemical reactions involving numerous enzymes and enzyme-coding genes, research performed hitherto is able to offer only a very limited explanation of the associations between genetic polymorphism and the individual's susceptibility to cancer. PMID- 10741505 TI - Normal liver function in women in the general Japanese population subjected to environmental exposure to cadmium at various levels. AB - OBJECTIVES: Whereas it is well established that environmental exposure to cadmium (Cd) may induce kidney dysfunction, less attention has been paid to the possible disturbance of liver function by Cd exposure. The possibility that liver function is adversely affected by current levels of environmental exposure to Cd as investigated in women in the general population in Japan, where the background level of exposure to Cd is known to be high. METHODS: From 1991 to 1997, 24-h food duplicate, peripheral blood and morning spot urine samples were collected from 607 non-smoking and non-habitually drinking women (age range 19-78 years) at 30 survey sites (with no known environmental pollution from heavy metals) throughout Japan. Liver function parameters in serum were examined by conventional methods. After wet-ashing, the food duplicate, blood and urine samples were analyzed for Cd intake via food (Cd-F), Cd in blood (Cd-B), and Cd in urine (Cd-U) by inductively-coupled plasma mass spectrometry. RESULTS: The geometric mean values for Cd-F, Cd-B, and Cd-U were 24.7 (27.1) microg/day, 1.76 (2.07) microg/l, and 3.94 (4.61) microg/g creatinine (values in parentheses for 41- to 60-year-old women), respectively. It as found that the three parameters of ALP, ALT, and AST activity were positively and significantly related to the age of the subjects (whereas no association as detected in cases of gamma-GTP, LAP, and albumin). Accordingly, a further analysis as made with 367 women selected by age (41-60 years; about 60% of the total population). Essentially, no Cd dose dependent changes in liver function parameters were observed in the selected population of this narrower age range. CONCLUSIONS: Overall, it seemed prudent to conclude that liver function as not disturbed by the current environmental exposure to Cd in Japan. PMID- 10741506 TI - Further reduction in lead exposure in women in general populations in Japan in the 1990s, and comparison with levels in east and south-east Asia. AB - OBJECTIVE: The objective of the study was to elucidate the current level of environmental lead (Pb) exposure of women in general population in Japan, where the use of organic Pb in automobile gasoline was phased out from 1973 to reach a zero level early in the 1980s. METHODS: A survey was conducted in 27 sites throughout Japan from 1991 to 1997. Five hundred and eighty-eight non-smoking women from the sites offered 24-h food duplicate, peripheral blood, and spot urine samples. Pb in food duplicates (Pb-F), blood (Pb-B), and urine (Pb-U) were analyzed by inductively-coupled plasma mass spectrometry. The results of Pb-F and Pb-B were compared with observations from a study conducted from 1977 to 1981 on 339 women at the same sites. Log-normal distribution was assumed for the evaluation of the results. RESULTS: Geometric means (GMs) of Pb-F, Pb-B, and Pb-U in the 1991-1997 study were 9.0 microg/day, 20.2 microg/l, and 2.18 microg/g creatinine, respectively. The values for Pb-F and Pb-B were substantially lower than the values (32.8 microg/day for Pb-F and 31.7 microg/l for Pb-B) obtained in the 1977-1981 study, which were already low when compared internationally. Cd-U values in the period from 1991 to 1997 also appeared to be among the lowest in the world. Analysis for time-dependent changes in Pb-U was, however, not possible at the time of this study because no values were available for the period from 1977 to 1981. CONCLUSIONS: Substantial reductions from 1977-1981 levels in environmental Pb exposure were observed among the study populations in Japan. Current exposure levels appear to be lower than those in other parts of Asia, the USA, and Europe. PMID- 10741507 TI - Field evaluation of a passive sampler for the exposure assessment of 2 methoxyethanol. AB - OBJECTIVE: This paper presents a field evaluation of 3 M 3500 passive badges for measuring 2-methoxyethanol (ME) in a humid working environment. METHODS: A total of 93 pairs of side-by-side active/passive samples, 48 pairs of duplicate active samples, 52 pairs of duplicate passive samples, and three groups of six replicate active/passive samples were compared. Three groups of six replicate active charcoal/active charcoal tube with drying tube samples were also compared to evaluate the humidity effect. RESULTS: No statistical difference was found between the passive badges and active samplers. Linear regression showed the correlation to be high (r = 0.992, slope = 0.973, n = 93) over the range of 0.17 163 ppm. The mean concentration difference was -0.34 ppm and the mean relative error was 3.50%. The intraclass correlation coefficients of 48 duplicate active samples and 52 duplicate passive samples were 0.994 and 0.989, respectively. The precision of replicate passive, active, and active/drying tube samples (n = 6) were 3.84%, 7.14%, and 5.12%, respectively. CONCLUSIONS: The humidity effect for active samples was insignificant at the low sampling rate (36.3 ml/min). It is therefore concluded that the use of the passive samplers to assess ME exposure produces comparable findings to that of active sampling. PMID- 10741508 TI - Neurobehavioural effects of experimental isopropanol exposure. AB - OBJECTIVES: The study investigated the acute effects of isopropanol exposure at the maximum allowable concentration (MAC) level on the performance of neurobehavioural functions. METHODS: In an exposure chamber 20 healthy subjects aged between 21 and 30 years were exposed to isopropanol at a concentration of 400 ppm (Swiss MAC value) for 8 h. A control group of another 20 subjects of the same age range, gender and education as used in a previous study with methylformate were subjected to the same test procedures without exposure. Mood was measured with the profile of mood states (POMS) in the morning, at noon and in the evening. Neurobehavioural performance tests were administered using the Wiener Test System (Wiener reaction test, Stroop, nonverbal learning, Wiener determination test, "Konturtracking" test), and the digit span test of the Neurobehavioural evaluation system (NES2). A test for postural sway was also conducted. During the POMS and the Wiener determination test the electromyography of the forehead and the left neck muscle as well as the pulse were registered. In the morning and evening spirometry (FVC, FEV, MEF and PEF) and odour threshold were measured. RESULTS: Only postural sway in bipedal standing at noon showed stronger deterioration in the exposed than in the control group when compared with the morning values. The evening values of monopedal standing were also more impaired in the exposed than the control group. In the other tests, no solvent effect could be established. CONCLUSIONS: It can be assumed, that, similar to other alcohols, isopropanol affects postural balance. Our results point to such a disturbance, but because it is the only study so far using the MAC value, any conclusions about safety risks would be premature. PMID- 10741509 TI - Biological monitoring of workers exposed to N,N-dimethylformamide in the synthetic fibre industry. AB - OBJECTIVES: Monitoring of workplace air and biological monitoring of 23 workers exposed to N,N-dimethylformamide (DMF) in the polyacrylic fibre industry was carried out on 4 consecutive days. The main focus of the investigation was to study the relationship between external and internal exposure, the suitability of the metabolites of DMF for biological monitoring and their toxicokinetic behaviour in humans. METHODS: Air samples were collected using personal air samplers. The limit of detection (LOD) for DMF using an analytical method recommended by the Deutsche Forschungsgemeinschaft (DFG) was 0.1 ppm. The urinary metabolites, N-hydroxymethyl-N-methylformamide (HMMF), N-methylformamide (NMF), and N-acetyl-S-(N-methylcarbamoyl)-cysteine (AMCC), were determined in one analytical run by gas chromatography with thermionic sensitive detection (GC/TSD). The total sum of HMMF and NMF was determined in the form of NMF. The LOD was 1.0 mg/l for NMF and 0.5 mg/l for AMCC. RESULTS AND CONCLUSIONS: The external exposure to DMF vapour varied greatly depending on the workplace (median 1.74 ppm, range < 0.1-159.77 ppm). Urinary NMF concentrations were highest in post-shift samples. They also covered a wide range (< 1.0-108.7 mg/l). This variation was probably the result of different concentrations of DMF in the air at different workplaces, dermal absorption and differences in the protective measures implemented by each individual (gloves, gas masks etc.). The urinary NMF concentrations had decreased almost to zero by the beginning of the next shift. The median half-time for NMF was determined to be 5.1 h. The concentrations of AMCC in urine were determined to be in the range from < 0.5 to 204.9 mg/l. Unlike the concentrations of NMF, the AMCC concentrations did not decrease during the intervals between the shifts. For the exposure situation investigated in our study, a steady state was found between the external exposure to DMF and the levels of AMCC excreted in urine about 2 days after the beginning of exposure. AMCC is therefore excreted more slowly than NMF. The half-time for AMCC is more than 16 h. Linear regression analysis for external exposure and urinary excretion of metabolites was carried out for a sub-group of 12 workers. External exposure to 10 ppm DMF in air (the current German MAK value) corresponds to an average NMF concentration of about 27.9 mg/l in post-shift urine from the same day and an average AMCC concentration of 69.2 mg/l in pre-shift urine from the following day. NMF in urine samples therefore represents an index of daily exposure to DMF, while AMCC represents an index of the average exposure over the preceding working days. AMCC is considered to be better suited for biomonitoring purposes because (1) it has a longer half-time than NMF and (2) its formation in humans is more closely related to DMF toxicity. PMID- 10741510 TI - Dermal absorption of N,N-dimethylacetamide in human volunteers. AB - OBJECTIVES: We investigated the potential for the dermal absorption of N,N dimethylacetamide (DMAC: CAS No. 127-19-5) vapor, the biological half-life of N methylacetamide (NMAC) in urine as the biological exposure item of DMAC, and the adjustment method for urinary concentrations. METHODS: Twelve healthy male volunteers (mean age 25.2 years, range 21-43 years) were exposed to DMAC for 4 h on two occasions at intervals of 96 h or above. Each volunteer sat inside a whole body-type exposure chamber for the dermal exposure experiment or outside the chamber for the inhalation exposure experiment. The temperature and relative humidity in the chamber were controlled at approximately 26 degrees C and 40% in order to keep the skin (90% naked) of the volunteers dry. DMAC concentrations were 6.1 +/- 1.3 ppm for dermal exposure and 6.1 +/- 1.3 ppm for inhalation exposure. Urine samples were collected from 0 h through 36 h and at 48 h and 72 h after the exposure. Extrapolations from exposure concentrations for 4 h to 10 ppm for 8 h were performed. RESULTS: Mean dermal absorption was estimated to be 40.4% of the total DMAC uptake. The biological half-lives of urinary NMAC were 9.0 +/- 1.4 h and 5.6 +/- 1.3 h via skin and lung, respectively. Mean NMAC in urine just after 5 consecutive workdays (8 h/day) at 10 ppm DMAC exposure was assumed to be 33.7 mg/g x Cr (18.6-70.0 mg/g x Cr). Creatinine-adjusted NMAC concentration in urine for each volunteer within 12 h after the exposure was more closely correlated with the total excretion amount of NMAC up to 36 h than with urinary volume-adjusted or specific-gravity-adjusted NMAC concentration in both the dermal and inhalation exposure experiments. CONCLUSIONS: DMAC vapor was significantly absorbed through the skin. Estimated NMAC values indicate that 20 mg/g x Cr NMAC seems to be appropriate as the biological exposure index. PMID- 10741511 TI - Aromatic DNA adducts in coke-oven workers, in relation to exposure, lifestyle and genetic polymorphism of metabolic enzymes. AB - OBJECTIVE: This study investigates the effect of multiple factors, including exposure to polycyclic aromatic hydrocarbons (PAHs), lifestyle, genetic polymorphism of cytochrome P450 (CYP)1A1, glutathione transferase (GST)M1, GSTP1, N-acetyltransferase (NAT)2 and gene p53, as well as any family history of cancer, on DNA adduct levels in coke-oven workers. METHODS: Sixty-five coke-oven workers employed at the largest iron-steel factory in China were recruited for the study. Personal data were collected at the interview. DNA adduct levels in total white blood cells (WBCs) were detected using 32P-postlabeling techniques. Genetic polymorphisms were analyzed by polymerase chain reaction (PCR) methods. RESULTS: The subjects were divided into low and high exposure groups, according to personal exposure to PAHs. The mean adduct value was 1.57 (range 0.54 to 4.35) per 10(8) nucleotides. A tendency for increased levels of DNA adducts in the high exposure group was observed, compared with the low exposure group (P = 0.07). In the low exposure group, DNA adducts were found to be positively associated with urinary cotinine (r = 0.44, P = 0.01). The rare allele homozygotes of CYP1A1 showed significantly higher DNA adduct levels than those of other CYP1A1 genotypes. Individuals with the NAT2 wild type had significantly increased DNA adduct levels than those with other NAT2 genotypes in the high exposure group. The p53 genetic polymorphism revealed a significantly positive effect on DNA adducts formation. There was a significantly higher adduct level in the subjects with a family history of cancer than those without, in the high exposure category. CONCLUSIONS: Effects of several variables, such as smoking, genetic polymorphism of 2 CYP1A1, NAT2, and gene p53, and a family history of cancer on DNA adduct levels were found, suggesting that these variables should be considered when evaluating the genotoxic effect of occupational exposure to PAHs using WBCs DNA adducts. PMID- 10741512 TI - Respiratory findings in mail carriers. AB - The results of the recording of respiratory symptoms and the measurement of lung function in 136 male postal workers employed as mail carriers were studied. In addition, the prevalence of chronic respiratory symptoms in 87 male nonexposed control workers was also examined. There was a significantly higher prevalence of chronic bronchitis (25.0%) and sinusitis (38.9%) in mail carriers than in control workers (13.8%; P < 0.05 and 2.3%; P < 0.01). A logistic regression analysis performed on the results of the study of chronic respiratory symptoms of mail carriers indicated a significant (P < 0.001) effect of smoking in this cohort, with the exception of occupational asthma. Mail carriers who smoked had a significantly higher prevalence of chronic cough (45.3%), chronic phlegm (39.1%), chronic bronchitis (39.1%) and sinusitis (53.1%) than mail carriers who were nonsmokers (18.1%; 12.5%; 12.5% and 26.4% respectively.) (P < 0.01). A high prevalence of acute symptoms developing during the work-shift was recorded, in both smokers and nonsmokers, being highest for upper airway symptoms, headache (50.0%), nasal catarrh (42.6%), and eye irritation (57.4%). The results of tests for average measured ventilatory capacity (as a percentage of predicted capacity) were significantly lower than expected, particularly for maximum flow rates at the last 25% of the vital capacity (FEF25), in both smokers (68.5%) and in nonsmokers (74.2%). A multivariate analysis of lung function parameters indicated a significant effect of employment conditions. The only major identifiable occupational exposure of mail carriers was to ambient air pollution for an average of 6 h per day as well as to adverse meteorological conditions. The measured ambient concentrations of major outdoor pollutants, primarily total suspended particulates, sulfur dioxide (SO2) and black smoke exceeded considerably the recommended Croatian maximum air quality standards over the past 10 years. Our study of mail carriers demonstrated that these workers were subject to respiratory symptoms associated with their smoking habits. Lung function findings suggested that occupational exposures, possibly to atmospheric pollution in combination with adverse meteorological conditions, may have led to lung function impairment in these workers. PMID- 10741513 TI - The prognostic significance of DNA cytophotometry and proliferation index (Ki-67) in giant cell tumors of bone. AB - We studied DNA ploidy by smear cytophotometry and proliferation activity by Ki-67 MIB immunohistochemistry in 69 primary and recurrent giant cell tumors (GCT) from 50 randomly selected patients. The obtained results were evaluated with comparisons made to the available clinical data. From the 46 primary tumors 63% showed diploidy and 37% aneuploidy. A significantly (P=0.026) higher recurrence rate (64%) was observed in aneuploid than in diploid tumors (31%). In the course of the recurrences, both the ratio of aneuploid tumors as well as the proliferation index of the tumors increased, though the degree of the latter was non-significant. Aneuploidy did not mean an unambiguous tendency towards malignant transformation; however, a close follow-up of recurrent aneuploid tumors, and wide excision of the recurrence instead of intralesional curettage are the recommended procedures. The DNA cytophotometry and proliferation index of GCTs--as compared to other histologic examinations--are of prognostic value in the evaluation of the recurrence potential of the GCTs. PMID- 10741514 TI - Complications of Marchetti locked nailing for humeral shaft fractures. AB - In this retrospective study 50 humeral fractures (36 acute, 6 pathological fractures and 8 non-unions) were treated by retrograde locked bundled Marchetti nailing. No intraoperative complications occurred. Postoperative complications included 7 non-unions (4/36 acute fractures and 3/8 delayed union), and 2 intraarticular penetrations of the secondary nails. However, at the subsequent removal of the implant 5 supracondylar fractures occurred. PMID- 10741515 TI - Bone densitometry: influence of prosthetic design and hydroxyapatite coating on regional adaptive bone remodelling. AB - The objective of this prospective study was to determine if bone densitometry can detect disparities in regional adaptive bone remodelling surrounding the cementless porous-coated femoral component of a hip prosthesis in two titanium alloy implants of different design . These prostheses were the S-ROM (n=69) and the Multilock (n=65). The Multilock implants consisted of two groups; 25 had a 50 micron layer of hydroxyapatite (HA) sprayed over the porous surface of the femoral component and the remaining 40 femoral components were not coated with HA. Densitometry was performed with dual energy X-ray absorptiometry (DXA) utilizing the LUNAR ORTHO software to analyse the seven Gruen zones. Bone mineral density measurements were obtained within a week of surgery as a baseline reference and at 6, 12, 24, 36 and 48 months thereafter. At 6 months there was significant mineral loss in all Gruen zones in the three prostheses. By 48 months there were differences in mineral loss between the three prostheses. In the zones adjacent to the porous surface, predominantly zones 1 and 7, the S-ROM exhibited 60% less mineral loss than the Multilock in zone 1, and there was no significant difference in zone 7. Compared to the Multilock-HA, the S-ROM lost 35% less mineral in zone 1, but the Multilock lost 70% less mineral than the S-ROM in zone 7. The Multilock-HA lost 37% and 75% less mineral than the Multilock in zones 1 and 7, respectively, i.e., hydroxyapatite coating tended to preserve bone stalk. Using the Gruen zone area measurements provided by the software, the S-ROM had significantly greater bone resorption in zone 7 at 24 months than either of the Multilocks, which did not differ from each other. In conclusion, DXA has shown differences in periprosthetic adaptive bone remodelling between implants of different design and composition as a function of time. PMID- 10741516 TI - Sociodemographic factors and failure of hip arthroplasty. AB - To identify the sociodemographic and lifestyle factors related to development of aseptic loosening after a cemented total hip arthroplasty (THA), 151 consecutive primary THAs performed at a single hospital in a rural district of Japan, at a mean follow-up of 7.5 years, were reviewed. Based on the medical records collected during hospital admission for THA, in which sociodemographic and lifestyle backgrounds had been recorded by the nurse, answers regarding residence, education, employment, engagement in agricultural work, marital status, family members living together, religion, recreational activity, smoking, and alcohol intake were analyzed in relation to the development of loosening, using a multivariate Cox proportional hazards model. With adjustment for cementing technique, diagnosis, age, and sex, agricultural work had a significant relation with prosthetic loosening (relative risk=2.85, P=0.03). Restriction of physical work in agriculture for patients undergoing THA should be considered. PMID- 10741517 TI - Uncemented total hip arthroplasty in the elderly. AB - Between January 1991 and December 1994, 132 uncemented total hip arthroplasties (THA) were performed on 125 patients over 65 years of age; of which 102 arthroplasties, performed in 90 patients, were followed for at least 4 years. One revision was necessary following fracture of an acetabular component secondary to trauma. The post-operative Harris hip score (HHS) ranged from 87 to 99. Radiologically there were no signs of subsidence of more than 3 mm, nor of osteolysis. Five patients experienced thigh pain. Based on the clinical and radiological results, uncemented total hip arthroplasties can give satisfactory function in elderly patients. PMID- 10741518 TI - Intertrochanteric osteotomy with a short intramedullary locking nail. AB - In this study the Gamma nail was used for fixation after intertrochanteric osteotomy in osteoarthritis. In 31 patients varus, valgus and complex intertrochanteric osteotomies were stabilized. The patients were allowed full weight bearing 2 weeks postoperatively. After a mean follow-up of 25.6 (13-42) months 29 osteotomies were healed. Two patients were lost to follow-up. No femoral shaft fractures or thigh pain occurred. PMID- 10741519 TI - Anterior knee pain syndrome. AB - The aim of this study was to correlate the distribution of substance-P nerve fibres within the knee joint in 20 patients complaining of anterior knee pain syndrome, with the clinical picture, patellar motion, power of the quadriceps mechanism, and the psychological profile. A comparison was made with 2 similar groups of patients with knee problems (patients with osteoarthrosis and patients with anterior cruciate ligament rupture or meniscal lesions), and with a further group of 20 healthy volunteers. The results suggest that anterior knee pain might be a psychosomatic syndrome associated with sub-clinical patellar instability and with little if any relation to levels of physical activity. PMID- 10741520 TI - Patterns of single segment non-physeal extremity fractures in children. AB - Five hundred and fifteen children with single segment non-physeal extremity fractures were retrospectively reviewed. The male to female ratio was 2.7:1. The average age of study group was 9.9+/-4.7 years; 273 fractures (53%) were on the left, 242 (47%) were on the right. A fall was the main cause of the injury in most of the cases (80%). The forearm was the most commonly seen fracture site. Summer was the season when the fracture incidence was the highest. Highest surgical treatment rate (50%) was seen in supracondylar humerus fractures. It has been stated in the literature that non-physeal fractures are far more common than physeal fractures. We assume that this study not only will help understanding of the injury patterns of single segment non-physeal fractures in children but also, be a base for future studies in prevention and treatment of this type of fracture. PMID- 10741521 TI - Total hip arthroplasty for tuberculous coxitis. AB - We report a case of tuberculous arthritis of the hip in a 22 year old male patient, treated with arthrotomy and antituberculous antibiotic therapy for 9 months; the joint deteriorated and 2 years later he underwent uncemented total hip arthroplasty. He received antibiotic therapy for 3 months preoperatively and for 6 months postoperatively. At 5 year follow-up there was no evidence of recurrent infection. PMID- 10741522 TI - Ceramic femoral head fractures in total hip replacement. AB - Two cases of fracture of the ceramic head of a total hip replacement are presented. In both cases the damaged components were replaced with satisfactory results. PMID- 10741523 TI - Hip dislocations associated with ipsilateral femoral neck fracture. AB - Two cases of subcapital fracture associated with hip dislocation, treated with primary uncemented Total Hip Replacement are presented. PMID- 10741524 TI - Complete fracture-dislocation of the lumbar spine without paraplegia. AB - A case of fracture-dislocation of the lumbar spine with complete anterior dislocation of L4 without any neurological deficit. PMID- 10741525 TI - Synovial chondromatosis of the subcoracoid bursa. AB - Synovial chondromatosis, is the chondroid metaplasia of the synovial membrane. Large joints such as the knee and hip are most commonly involved. Extraarticular involvement is rarely described. Synovial chondromatosis may be associated with impingement syndrome of the shoulder. We report a case of synovial chondromatosis of the subcoracoid bursa, which resulted in impingement symptoms. PMID- 10741526 TI - Spontaneous pneumothorax without any detectable pulmonary metastases in a patient with osteosarcoma. AB - An 11-year-old girl with osteosarcoma in the left distal femur, developed unilateral spontaneous pneumothorax. Pneumothorax was found at the initial presentation, but chest CT failed to reveal pulmonary metastases, bullae or blebs. PMID- 10741527 TI - Unusual proximal tibiofibular synostosis. AB - Proximal tibiofibular synostosis without multiple hereditary exostosis is extremely rare and only 7 cases have been reported in the literature. All of the previously reported cases accompanied deformities such as distal positioning of the proximal tibiofibular joint, leg length discrepancy, bowing of the fibula, and valgus deformity of the knee. The present case of a 24-year-old man had neither a history of trauma nor deformity around the knee. Therefore, it was suggested that this type of synostosis occurred after epiphyseal plate closure. PMID- 10741528 TI - Medial subtalar dislocation. AB - We report a medial subtalar dislocation without fracture in an eighteen year old male injured during basketball game. He was successfully treated with closed reduction and cast immobilization. At one year follow-up he was symptomless. PMID- 10741529 TI - Assessing candidate genes as risk factors for mental disorders: the value of population-based epidemiological studies. AB - BACKGROUND: It is now possible to routinely collect DNA for the investigation of genetic risk factors in psychiatric epidemiology. However, concerns have been raised about the investigation of genetic risk factors in population surveys and in case-control studies with population controls, because of spurious associations arising through population stratification. To overcome this problem, some geneticists advocate the use of family controls and studies on isolated populations. However, such approaches may not be the best for psychiatric epidemiology. METHOD: A critical analysis is made of arguments against the investigation of genetic risk factors in studies with population controls. RESULTS: It is argued that concerns about population stratification have been overblown. It is simply a form of confounding and can occur in studies of environmental as well as genetic risk factors. Epidemiologists have traditionally dealt with this and other types of confounding by matching on potential confounders in the design of the study, or stratification by confounders in the analysis. Family controls overcome population stratification, but not other forms of confounding. They are a good choice when the sole interest is genetic risk factors, but psychiatric epidemiologists are typically interested in both genetic and environmental factors. Family controls are poor for examining environmental risk factors because they can give rise to over-matching. Isolated populations may produce genetic associations that do not generalize to other populations, and they may have a restricted range of environments. CONCLUSION: We conclude that population surveys and case-control studies using population controls are suitable for the investigation of both genetic and environmental risk factors and have greater public health relevance than alternative designs. PMID- 10741530 TI - The persistence of psychiatric deviance from the age of 8 to the age of 15 years. AB - BACKGROUND: Earlier research has shown that psychiatric problems in children tend to persist over years. This investigation assessed the persistence of psychiatric deviance among children over a 7-year period from the age of 8 to the age of 15 years. We also explored the relationship between problems leading to special attention at the well-baby clinics before school age and future psychiatric deviance. METHODS: The study material consisted of three questionnaires filled out by the parents, teachers and children themselves (N = 1268) at three time points, together with data concerning the children gathered from the records of well-baby clinics. RESULTS: At the age of 15 years, girls scored higher than boys on the parental scale and on the Beck's Depression Inventory (BDI), while boys scored higher than girls on the teachers' scale. Parental ratings had a high correlation over 3 years and a moderate correlation over 7 years. Teachers' and children's ratings correlated moderately over 3 years, but did not correlate significantly over 7 years. The probability of being deviant at the age of 15 years was elevated if the child scored high on the parents' or teachers' scale at the age of 8 or 12 years, or on the Children's Depression Inventory (CDI) at the age of 12 years. Problems noted by health professionals (problems in growth, somatic diseases, emotional/behavioural problems of the child, psychosocial problems of the family) before school age were related to future deviance on the parental scale. Emotional/behavioural problems before school age elevated the probability of scoring high on the teachers' scale at the age of 15, and problems in psychomotor development elevated the probability of scoring high on the BDI. CONCLUSIONS: Psychiatric deviance is persistent over several years in children. Primary health care professionals can identify children who are at risk for future psychiatric problems. PMID- 10741531 TI - Correlations between socioeconomic status, IQ and aetiology in mental retardation: a population-based study of Norwegian children. AB - BACKGROUND: Mental retardation (MR) (IQ< or = 70) is one of the most frequent and debilitating neurological handicaps in children. The aetiologies of MR are multiple and to a certain extent related to social class and the degree of MR. However, recent epidemiological data assessing these relationships are scarce. The objective of the present study was therefore to estimate the associations between socioeconomic status (SES), level of IQ, and causative factors in children with MR. METHODS: The investigation was designed as a cross-sectional population-based study of Akershus County, outside Norway's capital Oslo. The source population comprised 30 037 children born between 1980 and 1985. Of 178 children aged 8-13 years, 79 had severe MR (SMR) (IQ<50) and 99 had mild MR (MMR) (IQ 50-70). SES was grouped from I to V according to parental education. Diagnostic work-up regarding aetiology and medical diagnoses was extensive. The aetiology of MR was divided into two main groups: biopathological MR (n = 143), and unspecified MR, meaning MR of unknown aetiology (n = 35). Biopathological MR was further subdivided into four sub-groups: prenatal, perinatal, postnatal and undetermined timing of the damaging event. RESULTS: SES III, IV and V (the lower grades) increased the risk of MMR. In SES IV and V there was an increased risk of unspecified MR, odds ratio 7.0 and 5.6 (95% CI 1.3, 39.0 and 1.1, 30.0) respectively, compared to SES I. IQ in unspecified MR was significantly higher than in the biopathological aetiology sub-groups. CONCLUSIONS: The study showed that parents of children with SMR had a higher socioeconomic level than parents of children with MMR. The findings were also consistent with a partial overlap between unspecified MR and the lower end of normal IQ distribution in the general population. PMID- 10741532 TI - Postnatal depression-myth and reality: maternal depression before and after the birth of a child. AB - BACKGROUND: Much has been written about postnatal depression as a clinical condition. There is some evidence to suggest that a substantial proportion of women who give birth experience a depression in the postnatal period. This paper reports the results of a longitudinal study of the mental health of a large sample of women who were in the early stages of pregnancy at entry to the study. METHODS: Each participant was assessed for symptoms of depression at the first clinic visit (entry to the study), and reassessed at various intervals--at 3-5 days, at 6 months, and again at 5 years after the birth of the child--using the DSSI-D (Delusions-Symptoms-States Inventory). RESULTS: Retrospective recall questions indicate that shortly after the birth the majority of women experienced some depressed mood. Of those who experienced depressed mood, the data suggest that the symptoms were not severe, nor did these symptoms generally continue beyond a few weeks. The longitudinal data indicate that levels of depression in our sample are highest either at the first clinic visit or at the 5-year follow up. Rates of depression at the 6-month follow-up are relatively low by comparison. CONCLUSION: While most mothers experience periods of depressed mood after the birth of their baby, these periods are generally of short duration and of lesser intensity than a major depression. Mothers appear to experience increasing levels of symptoms of depression as their child grows up. Many of the "cases" of depression experienced at the 5-year follow-up represent a recurrence of a previous experience of depression. PMID- 10741533 TI - Common mood and anxiety states: gender differences in the protective effect of physical activity. AB - BACKGROUND: We wished to examine the impact of the duration and intensity of physical activity on common anxiety and depressive states. METHOD: A nested case control design was applied to data from the Health and Lifestyle Survey. Anxiety and depressive states were measured by caseness on the General Health Questionnaire. Physical activity variables were defined from a detailed activity schedule. RESULTS: After adjustment for potential confounders, the findings suggest that compared to men who reported 0-44 min of daily physical activity, there is benefit to men who exercise for at least 92 min a day (92-161 min a day: OR = 0.57, 95% CI = 0.37-0.87, P<0.01; 162-554 min a day: OR = 0.65, 95% CI = 0.43-0.97, P<0.05), but not to women. The protective effect does not appear to vary according to the intensity of activity in men or women. CONCLUSIONS: Physical activity of long duration amongst men confers protection against common mood and anxiety states. This study found no such protection for women. PMID- 10741534 TI - Trauma exposure, resilience, social support, and PTSD construct validity among former prisoners of war. AB - BACKGROUND: The aim of this study was to investigate predictors of persistent symptoms of posttraumatic stress disorder (PTSD) and to examine the construct validity of PTSD in a national sample of 270 World War II and Korean Conflict prisoners of war (POWs). METHOD: POWs were interviewed at two points in time (1965 and 1990). Predictors included PTSD symptomatology measured in 1965 by items from the Cornell Medical Index (CMI), severity of captivity trauma, resilience factors, and post-trauma social support. The criterion, symptomatology in the early 1990s, was evaluated with the PTSD module of the Structured Clinical Interview for DSM (SCID). RESULTS: The CMI provided only partial coverage of PTSD criteria and appeared to provide only a general index of distress. Clustering of SCID items in two-dimensional space via multidimensional scaling analysis offers some construct validation for the DSM's differentiation of PTSD symptoms into criterion groups, although there was not a perfect match. Trauma severity is best related to PTSD symptomatology experienced in 1990, mitigated in part by greater education level and age at the time of trauma exposure. Surprisingly, 1965 distress added only a modest amount to the prediction of current distress, while post-trauma social support added none. CONCLUSIONS: These findings support previous work showing the severe psychological sequelae of POW status 40-50 years after captivity, and indicate that trauma severity during captivity is the best predictor of current PTSD symptomatology. Results also add to our understanding of the conceptual differentiation of PTSD symptoms into separate and distinct symptom clusters. PMID- 10741535 TI - What do case managers do? An investigation of case manager interventions and their relationship to client outcome. AB - BACKGROUND: The aim of the present study, which forms part of a wider case management multicenter study, was to explore the content of case managers' work, service patterns, and their relationship to client outcome. METHODS: Client background characteristics were registered at admission, and needs of care and psychosocial functioning were assessed at admission to the service and after 18 months. In seven of the services an extended follow-up was performed, which included assessments regarding quality of life, level of symptoms and social network. The study comprised 176 severely mentally ill clients, of whom 153 participated in a general 18-month follow-up (87%), with 113 clients out of 134 participating in an extended follow-up sample (84%). RESULTS: The investigation of service patterns showed that clients received services in a great variety of life areas using a number of different types of interventions. The results support the assumption that severely mentally ill clients are in need of case management services that offer more than brokerage services and coordination. A more active rehabilitation-oriented approach was found towards younger clients and clients who had a job, which may reflect a higher level of ambition in assisting younger clients to gain access to and stay in education and the labor market. Several types of intervention were related to client outcome. Brokerage, intervention planning and more interventions in the area of skills relating to activities of daily living were related to a more pronounced decrease in needs of care. More time spent on indirect work on behalf of the clients was related to a better outcome with regard to psychiatric symptoms and social network. CONCLUSIONS: The results indicate that specific service components have a more obvious distinct impact than others on outcome, and that this increase in effectiveness varies with the outcome targeted. PMID- 10741536 TI - Smoking cessation treatments in Spain. A thirty-year perspective: 1966-1996. AB - This paper examines the evolution of the scientific literature over the past thirty years (1966-1996) about smoking studies conducted in Spain and determines where the trend is leading. We utilized local (Indice Medico Espanol) and international databases (MedLine, PsycLit) to find papers related to the treatment of smoking in Spain from 1966 to 1996, including such approaches as medical advice, pharmacological, psychological, community, and combined approaches. The total number of articles about studies related to tobacco contained in the three databases in the 1966-1996 period was 1,026. The largest number of articles, a total of 455 (44.3%), was published between 1991 and 1996. Specifically, of the 125 articles about smoking cessation treatments, empirical studies constitute 48.8% (n = 61) of all the articles published. The results of the studies and the increasing number of publications indicate that in the present decade there has been intense interest from both health professionals and the Health Administration in promoting formal smoking treatment programs. Nevertheless, too few empirical studies about smoking cessation methods have been undertaken to date. It is still necessary to perform more studies with nicotine replacement therapy, behavior therapy, and combined therapy, as well as controlled studies of treating specific smoking populations, and community approaches. PMID- 10741537 TI - Understanding condom use among heroin addicts in methadone maintenance using the information-motivation-behavioral skills model. AB - The current study represents the application of a health behavior model to account for unsafe sexual behavior (as opposed to unsafe needle use) among heroin addicts in methadone treatment. The Information-Motivation-Behavioral Skills (IMB) model of HIV preventive behavior asserts that HIV prevention information, motivation, and behavioral skills are fundamental determinants of HIV preventive behavior. Participants (N = 156 heroin-addicted individuals in methadone treatment) completed assessments of their levels of HIV prevention information, motivation, behavioral skills, and safer sexual behavior. Overall measures of fit generated via structural equation modeling indicate that the IMB model adequately fits the data obtained. The constructs of the model accounted for a substantial proportion of the variance in safer sexual behavior, and tests of parameter estimates indicate that while information and motivation had direct and reliable associations with safer sexual behavior in this population, behavioral skills did not. Discussion focuses on the primary roles of HIV prevention information and motivation as determinants of safer sexual behavior in this population, on the lack of a significant contribution of HIV prevention behavioral skills, and on the implications for intervention of this pattern of findings. PMID- 10741538 TI - The family and alcohol: effects of excessive drinking and conceptualizations of spouses over recent decades. AB - There is much debate in the addiction literature about the extent to which excessive drinking affects nondrinking family members. The issue is considered in this review by examining and evaluating research relating to the effects of drinking on children, family systems, and partners of drinkers. The latter group have, historically, been blamed and apologized for their partner's drinking, although more recent theories have adopted a stress and coping paradigm, thus normalizing individuals and their behaviors. Conceptualizations of spouses over the last five decades are described and evaluated in the second part of the review. Finally, the review considers the impact of the recent stress and coping paradigm on clinical interventions for excessive drinkers and their families, and suggestions are made for future research. PMID- 10741539 TI - Nurses' attitudes toward substance misusers. II. Experiments and studies comparing nurses to other groups. AB - Experimental investigations of nurses consistently indicate that a patent labeled as a substance misuser is perceived far more negatively across a range of personal attributes than an identical patient who is not so labeled. Comparative evaluations suggest that nurses are less tolerant of social drinking and drug use and are more morally condemnatory of the chemically dependent than are other health-care professionals. PMID- 10741540 TI - "Alcohol dependence" and death: survival analysis of the Baltimore ECA sample from 1981 to 1995. AB - OBJECTIVE: Evidence is provided about the association between "alcohol-use disorders" and the 14-year risk of death in a community sample. Most prior descriptions of this association come from treatment samples. METHOD: 3,481 adult household residents were recruited into the NIMB Baltimore Epidemiologic Catchment Area survey and interviewed in 1981. The Diagnostic Interview Schedule (DIS) was employed to assess alcohol drinking and other drug-taking behaviors, and to determine fulfillment of DSM-III criteria for "alcohol abuse" and/or "dependence" diagnoses. Participants were followed-up in 1993-1996, by which time 24% of the sample had died. Median age of death was estimated for persons with and without alcohol disorders, and for "heavy" and "nonheavy" drinkers. Cox proportional hazards models adjusted for the influence of age, sex, race, "drug use disorders," and tobacco smoking. RESULTS: "Alcohol abuse" and/or "dependence" was associated with a higher risk of death and a younger median age of death (adjusted relative risk = 1.3, p = .016). "Heavy" alcohol consumption was also associated with a significantly elevated risk of death. The DIS diagnosis of "alcohol use disorder" helped predict mortality over and above a prediction based solely upon "heavy drinking" (p < .01). CONCLUSIONS: These findings indicate that the observed increased risk of death associated with "alcohol dependence" is not limited to cases severe enough to have been treated but is also present among cases in the household population. PMID- 10741541 TI - Substance misuse and related infectious diseases in a soup kitchen population. AB - Representative samples of female (N = 119) and male (N = 100) guests were selected at two inner city soup kitchens. In the preceding month, 75% used cocaine/crack and 25% used heroin/opiates as determined by hair analysis. Relatively few guests (25%) were in substance dependency treatment. Infectious disease rates were: HIV (16%), hepatitis B exposure (21%), hepatitis B carrier (6%), syphilis exposure (15%). Years of injecting drug use and homelessness/marginal housing were associated with HIV infection and hepatitis B exposure. Soup kitchens should be prime locations for outreach to cocaine/crack and heroin users in need of treatment, medical care, and interventions to prevent infectious disease transmission. PMID- 10741542 TI - Predicting treatment-seeking behavior: psychometric properties of a brief self report scale. AB - Development of a brief instrument to assess attitudes toward treatment and predict treatment-seeking behavior among out-of-treatment substance misusers is described. Exploratory factor analysis of an initial pool of 41 items identified four subscales: Perceived need for treatment: perceived drug problem severity; motivation to quit; and negative attitudes toward treatment. Psychometric analyses of data from 535 substance misusers participating in an outreach intervention project provided substantial support for the reliability and construct validity of the first three subscales, and marginal support for the fourth. Evidence of predictive validity was provided by further analyses indicating significant relationships between the three primary scales and both treatment admissions and treatment-seeking during a 3-month follow-up period. The final instrument, the Treatment Attitude Profile (TAP), contains 25 items in a self-report format suitable for use with limited literacy populations in field or office settings. PMID- 10741543 TI - Utilization of common analgesic and anxiolytic medications by registered First Nations residents of western Canada. AB - The study examined utilization of acetaminophen with codeine and benzodiazepine drugs among Non-Insured Health Benefits (NIHB) program registered First Nations residents in the four western provinces of Canada and sought preliminary indicators of factors influencing utilization. A small percentage of NIHB clients in the four western provinces were excessive users of acetaminophen with codeine and/or benzodiazepines, but overall utilization of these central nervous system drugs was moderate and within the bounds of non-First Nations population use examined in this study. The study also demonstrated that utilization of acetaminophen with codeine among NIHB claimants was inversely related to the stringency of provincial regulation. PMID- 10741544 TI - Perceived social support from parents and peers: which is the stronger predictor of adolescent substance use? AB - A number of factors influence adolescent substance use. The main goal of this study has been to investigate how sociodemographics, psychosocial health, and perceived support from parents and friends might predict smoking, drinking, and drug use in adolescence. Data were collected using self-administered questionnaires among secondary school students (n = 1,039) in Szeged, Hungary. Sociodemographics (gender, age, and the type of school) proved to be the key predictors of substance use. While neither friend nor mother support appeared to be strong predictors, a low level of perceived father support increased the chance of all types of substance use. PMID- 10741545 TI - Design, synthesis and biological evaluation of 3-amino-3-phenylpropionamide derivatives as novel mu opioid receptor ligands. AB - 3-Amino-3-phenylpropionamide derivatives were produced as small molecule mimics of the cyclic octapeptide octreotide from readily available imine 1. The compounds exhibit high affinity for the mu opioid receptor. PMID- 10741546 TI - Parallel synthesis of a series of subtype-selective NMDA receptor antagonists. AB - A series of 1-(heteroarylthioalkyl)-4-benzylpiperidines was rapidly synthesized through the use of parallel synthesis to investigate the binding affinity for the NR1A/2B receptor subtype. PMID- 10741547 TI - Novel peptidomimetic hematoregulatory compounds. AB - The activity of a novel series of peptidomimetic hematoregulatory compounds, designed based on a pharmacophore model inferred from the structure activity relationships of a peptide SK&F 107647 (1), is reported. These compounds induce a hematopoietic synergistic factor (HSF) which in turn modulates host defense. The compounds may represent novel therapeutic agents in the area of hematoregulation. PMID- 10741548 TI - Preparation and use of a photoactivatable glucose-6-phosphate analogue. AB - A benzophenone-containing derivative of glucose-6-phosphate, 6-[(3-([2,3-3H2]-p benzoyl dihydrocinnamidoylpropyl-1-oxy)phosphoryl]-D-glucopyranose ([3H]BZDC-Glc 6-P) was synthesized and employed to photoaffinity label proteins on intact rat liver microsomes. The use of a non-photoactivatable, UV-transparent desoxy analogue of BZDC, named p-benzyldihydrocinnamoyl (BnDC), is introduced as a general method to achieve competition when hydrophilic ligands are modified with hydrophobic photophores. PMID- 10741549 TI - Structure-based design of a novel synthetic spiroketal pyran as a pharmacophore for the marine natural product spongistatin 1. AB - SPIKET-P, a novel synthetic spiroketal pyran, was rationally designed as a pharmocophore for the tubulin depolymerizing marine natural product Spongistatin 1. SPIKET-P was prepared from the commercially available benzyl (R)-(-)-glycidyl ether using a versatile 11-step synthetic scheme in a stereocontrolled fashion. At nanomolar concentrations, SPIKET-P caused tubulin depolymerization in cell free turbidity assays and exhibited potent cytotoxic activity against cancer cells as evidenced by destruction of microtubule organization, and prevention of mitotic spindle formation in human breast cancer cells. PMID- 10741550 TI - Cysteine chloromethyl and diazomethyl ketone derivatives with potent anti leukemic activity. AB - A series of cysteine diazomethyl- and chloromethyl ketone derivatives has been synthesized and evaluated against human B-lineage (Nalm-6) and T-lineage (Molt-3) acute lymphoblastic leukemia cell lines. The chloromethyl ketone compounds showed potent cytotoxicity against these cell lines, with IC50 values in the low micromolar range. The best compounds were N-acetyl-S-dodecyl-Cys chloromethyl ketone (IC50 = 2.0 microM against Nalm-6, 2.3 microM against Molt-3) and N-acetyl S-trans,trans-farnesyl-Cys chloromethyl ketone (IC50 = 3.0 microM against Nalm-6 and 1.4 microM against Molt-3). PMID- 10741551 TI - The S-alkyl chain length as a determinant of the anti-leukemic activity of cysteine chloromethyl ketone compounds. AB - A series of cysteine chloromethyl ketone compounds with a systematic variation of the S-alkyl chain length have been synthesized in order to gauge the effect of the alkyl chain length on the cytotoxicity of these compounds against human acute lymphoblastic leukemia cells. Comparable activities were observed for compounds with S-alkyl chains ranging from pentyl to dodecyl, with the best being undecyl (IC50= 1.7 microM) and dodecyl (IC50=2.0 microM) against B-lineage leukemia cells and hexyl (IC50 = 0.7 microM) against T-lineage leukemia cells. PMID- 10741552 TI - Synthesis, DNA binding, topoisomerases inhibition and cytotoxic properties of 4 arylcarboxamidopyrrolo-2-carboxyanilides. AB - Three 4-arylcarboxamidopyrrolo-2-carboxyanilides bearing different substituents on the pyrrole nitrogen were synthesized and evaluated for their capacities to bind to specific sequences within the minor groove of DNA and to inhibit human topoisomerases I and II in vitro. The cytotoxicity of the drugs correlates with their DNA binding affinities. The two drugs bearing a N-methyl or N-benzyl pyrrole stabilize topoisomerase I-DNA complexes. PMID- 10741553 TI - Fluoresceinated FKBP12 ligands for a high-throughput fluorescence polarization assay. AB - Several fluoresceinated FKBP12 ligands have been prepared for a high-throughput fluorescence polarization assay. K(i)s for FKBP12 rotamase inhibition by these ligands range from 1.3 microM to 32 nM, and their design is based on X-ray crystal structures of FKBP12 complexed with known immunophilin ligands. PMID- 10741554 TI - Synthesis and dopaminergic activity of heterocyclic analogues of 5,6-dihydroxy-2 aminotetralins. AB - The heterocyclic analogues of 5,6-dihydroxy-2-aminotetralins (6) were synthesized and their in vitro dopaminergic activity was compared to that of (-)-DP-5,6-ADTN and the novel potent agonist Z12571. The results show that changing the cathecol ring for a heterocycle decreases the D1-like activity of the target molecules 6. However, the D2-like activity of tetrahydroquinoline (6j) was comparable to that of (-)-DP-5,6-ADTN. PMID- 10741555 TI - 2-Substituted paullones: CDK1/cyclin B-inhibiting property and in vitro antiproliferative activity. AB - 9-Trifluoromethyl-paullones with a carbon chain in the 2-position were synthesized by palladium-catalyzed coupling reactions of a 2-iodoprecursor with terminal alkenes or alkynes, respectively. The introduction of a 2-cyanoethyl substituent led to a significant enhancement of CDK1/cyclin B inhibiting property and in vitro antiproliferative activity. PMID- 10741556 TI - Membrane-permeant, bioactivatable analogues of cGMP as inducers of cell death in IPC-81 leukemia cells. AB - We report an improved single-step synthesis to generate the membrane-permeant acetoxymethyl esters (AM-esters) of cGMP and three cGMP-analogues. These bioactivatable compounds were found to induce cell death in rat IPC-81 cells, a model system for acute myelocytic leukemia, in micromolar doses, while the corresponding non-modified cGMP-analogues were inactive. PMID- 10741557 TI - Naphthyl ketones: a new class of Janus kinase 3 inhibitors. AB - Potent inhibition of Janus kinase 3 was found for a series of naphthyl(beta aminoethyl)ketones (e.g. 7, pIC50 = 7.1+/-0.3). Further studies indicated that these compounds fragment in less than 1 h by retro-Michael reaction in the Jak3 in vitro ELISA assay procedure. The breakdown product of 7, 2-naphthylvinyl ketone (22, pIC50 = 6.8+/-0.3) showed very similar inhibitory activity to 7. Compounds 7 (in neutral buffer) and 22 will be useful pharmacological tools for the investigation of the Janus tyrosine kinase Jak3. PMID- 10741558 TI - Pyrrolo[3,2-c]pyridine derivatives as inhibitors of platelet aggregation. AB - A series of pyrrolo[3,2-c]pyridines, isosteres of the antithrombotic drug ticlopidine, has been synthesized and evaluated in vitro for the ability to inhibit aggregation of human platelet-rich plasma induced by adenosin 5' diphosphate (ADP). Structure-activity relationships showed their antiplatelet effects to be related to the lipophilicity. PMID- 10741559 TI - Resistance-modifying agents. Part 7: 2,6-disubstituted-4,8 dibenzylaminopyrimido[5,4-d]pyrimidines that inhibit nucleoside transport in the presence of alpha1-acid glycoprotein (AGP). AB - The synthesis and biological evaluation of potent 4,8 dibenzylaminopyrimidopyrimidine nucleoside transport inhibitors, with reduced binding to alpha1-acid glycoprotein, is reported. PMID- 10741560 TI - 8-Methylureido-4,5-dihydro-4-oxo-10H-imidazo[1,2-a]indeno[1,2-e]pyrazines: highly potent in vivo AMPA antagonists. AB - A novel series of readily water soluble 8-methylureido-4,5-dihydro-4-oxo-10H imidazo[1,2-a]indeno[1,2-e]++ +pyrazines were synthesized. The -10-yl acetic acid ((+)-3) and -10-carboxylidene (4) derivatives exhibit potent affinities (IC50=4 and 19 nM, respectively) and antagonist properties (IC50 = 2 and 3 nM, respectively) at the ionotropic AMPA receptor. These compounds also display anticonvulsant properties against both electrically and sound-induced convulsions in mice after ip, sc and iv administration with ED50 values between 0.9 and 11 mg/kg, thus suggesting adequate brain penetration. PMID- 10741561 TI - Inhibition of inducible nitric oxide synthase by acetamidine derivatives of hetero-substituted lysine and homolysine. AB - The synthesis and in vitro evaluation of the acetamidine derivatives of hetero substituted lysine and homolysine analogues have identified potent inhibitors of human nitric oxide synthase enzymes, including examples with marked selectivity for the inducible isoform. PMID- 10741562 TI - 1,3-Diarylcycloalkanopyrazoles and diphenyl hydrazides as selective inhibitors of cyclooxygenase-2. AB - Novel 1,3-diarylcycloalkanopyrazoles 1, and diphenyl hydrazides 2 were identified as selective inhibitors of cyclooxygenase-2. The 1,3-diaryl substitution pattern of the pyrazole ring in 1 differentiates these compounds from most of the known selective COX-2 inhibitors that contain two aryl rings at the adjacent positions on a heterocyclic or a phenyl ring. Similarly, the two phenyl rings in 2 are also separated by three atoms. SAR of both phenyl rings in 1 and 2, and the aliphatic ring in 1 will be discussed. PMID- 10741563 TI - Probes for imidazoline binding sites: synthesis and evaluation of a selective, irreversible I2 ligand. AB - An irreversible ligand (7) has been prepared based on the selective I2 ligand 2 BFI. Compound 7 displayed high affinity and selectivity for I2-sites and has been shown to irreversibly bind to these sites in rat brain. Compound 7 should, therefore, prove an invaluable tool for the further elucidation of I2-site function. PMID- 10741564 TI - An islet-specific CD8+ T cell hybridoma generated from non-obese diabetic mice recognizes insulin as an autoantigen. AB - Although CD8+ T cells play a major role in beta cell destruction in insulin dependent diabetes in the non-obese diabetic mouse, the T cell autoantigen(s) recognized by such cells remains to be identified. Therefore, an islet-reactive, CD8+ T cell line was generated from islet-infiltrating cells and hybridized by fusion with a CD8+ alphabeta TCR- BW5147 thymoma. In the presence of islets, none of the 12 CD3+ CD8+ T cell hybridomas isolated secreted IL-2/IL-4 or IFNgamma but three were islet specific, as shown by activation induced cell death. Subclone 4A7.7.15 recognized only islets expressing H-2Kd, demonstrated islet-specific inhibition of proliferation and concomitant partial arrest in the G2/M phase of the cell cycle. Further analysis using a panel of cell lines, expressing H-2Kd, and transfected with the cDNA for various putative autoantigens in type 1 diabetes showed that 4A7.7.15 recognizes insulin as an antigen. PMID- 10741565 TI - (AC)23 [Z-2] polymorphism of the aldose reductase gene and fast progression of retinopathy in Chilean type 2 diabetics. AB - A recent case-control study suggests that the allele (AC)23 of a variable number tandem repeat (VNTR) associated to the aldose reductase (ALR2) gene could be related to early retinopathy in Type 2 diabetics. By means of a longitudinal retrospective study, we aimed to seek for a relationship between the rate of progression of retinopathy and the (AC)23 allele of the VNTR associated to the ALR2 gene. A random sample was obtained of 27 Type 2 diabetics (aged 68.1 +/- 10.6 years, diabetes duration = 20.7 +/- 4.8 years, mean HbA1 = 10.6 +/- 1.6%). The mean HbA1 was the arithmetic average of 2.2 measurements per patient per year of total glycosilated hemoglobin (Gabbay method, normal range: 4.2-7.5%). Retinopathy was graded by an Ophthalmologist in a scale from zero to four score points. The genotype of the (AC), VNTR was determined by 32P-PCR plus sequenciation in a Perkin-Elmer laser device. The Mann-Whitney test and either chi2 or Fisher's exact test were used. A P < 0.05 was considered as statistically significant. The retinopathy progression rate (RPR, points x year(-1)) was calculated by dividing the increment of retinopathy score (delta Retinopathy Score, [points]), by the duration of the follow up [years]. The 12 diabetics having the (AC)23 allele had a mean RPR 8.9 times higher (0.40 +/- 0.61 points x year(-1)) than the 15 patients who had alleles other than (AC)23 (0.045 +/- 0.099 points x year(-1), P = 0.037). Both groups were similar with respect to: mean HbA1 (10.5 +/- 1.4 and 10.7 +/- 1.7%, P = 0.95), age at diagnosis (48.5 +/- 6.3 and 46.3 +/- 14.0 years, P = 0.81), diabetes' duration (21.3 +/- 4.7 and 20.2 +/- 4.9 years, P = 0.41) and serum creatinine (0.89 +/- 0.2 and 1.13 +/- 0.5 mg dl( 1), P = 0.35). We concluded that, in Type-2 diabetics having similar glycemic control, the (AC)23 allele of the VNTR associated to the ALR2 gene, is associated to a 8.9 times faster progression of retinopathy than in patients who have other alleles. PMID- 10741566 TI - Validation of the insulin sensitivity index (ISI(0,120)): comparison with other measures. AB - The purpose of this study was to explore possible calculations using oral glucose tolerance test (OGTT) values in order to develop a simple measure of insulin sensitivity. We devised a formula for an insulin sensitivity index, ISI(0,120), that uses the fasting (0 min) and 120 min post-oral glucose (OGTT) insulin and glucose concentrations. It appears to be generalizable across a spectrum of glucose tolerance and obesity. Most importantly, our data show that ISI(0,120) correlates well, when applied prospectively in comparative studies, with the insulin sensitivity index obtained from the euglycemic hyperinsulinemic clamp (r = 0.63, P < 0.001). This correlation was demonstrably superior to other indices of insulin sensitivity such as the HOMA formula presented by Matthews, and performed comparably to the computerized HOMA index. Measurement of insulin sensitivity has traditionally been possible only in research settings because of the invasiveness and expense of the methods used. Clinical investigators have therefore sought more practical methods to obtain an index of insulin sensitivity. Such an index should approximate insulin sensitivity as measured by the euglycemic hyperinsulinemic clamp (M). We present ISI(0,120), a simple yet sensitive measure of insulin sensitivity which is adaptable for use in clinical settings as well as large epidemiologic studies. PMID- 10741567 TI - Impairment of insulin-induced vasodilation is associated with muscle insulin resistance in type 2 diabetes. AB - To clarify the association between the actions of insulin on the vascular wall and on the muscles in diabetes, we evaluated insulin-mediated vasodilation and muscle glucose uptake simultaneously using the euglycemic hyperinsulinemic glucose clamp technique and the calculation of total peripheral vascular resistance (TPR) from arterial pulse wave analysis in 19 Japanese patients with type 2 diabetes who had no signs of atherosclerosis. During the clamp study, the plasma norepinephrine (NE) level and plasma renin activity (PRA) increased without showing any significant correlation to the glucose infusion rate (GIR); a marker of muscle insulin sensitivity, and no changes of other plasma vasoactive hormone levels were observed. TPR decreased over time during the clamp study. The decrease of TPR from baseline was 0.88 +/- 0.02 at 1 h (mean +/- S.E.M., P < 0.01) and 0.79 +/- 0.03 at 2 h (P < 0.01), and the relative change in TPR from baseline was negatively correlated with GIR (r = -0.48 at 1 and 2 h; both P < 0.05). Our results suggest that there is also insulin resistance in the vascular wall, and this phenomenon may be associated with muscle insulin resistance in type 2 diabetes. PMID- 10741568 TI - Insulin and high glucose modulation of phosphatase and reductase enzymes in the human erythrocytes: a comparative analysis in normal and diabetic states. AB - The ability of insulin to influence activities of various protein kinases and protein phosphatases, that are thought to mediate insulin action, are limited in patients with insulin resistance. Because numerous responses to insulin are affected, we undertook studies to determine whether protein tyrosine phosphatases (PTPs) activities are altered in patients with diabetes syndrome. In order to evaluate abnormal PTP activities, we done a comparative study using erythrocytes from normal and diabetic patients. We determined the activity of the cytosolic acid PTP in basal and insulin-dependent states. Mean basal PTP activities, were found to be significantly higher in diabetics than in normal subjects (type 1 diabetics: 0.36 +/- 0.01 vs 0.28 +/- 0.01 mmol p-nitrophenolate/h per g hemoglobin (Hb), P < 0.001; type 2 diabetics: 0.35 +/- 0.01 vs 0.28 +/- 0.01 mmol p-nitrophenolate/h per g Hb, P < 0.001). Insulin, at concentrations above physiological levels (1 mIU/ml), inhibited the PTP activities in erythrocytes from normal subjects (-15 +/- 4.1%, P < 0.01). Insulin could also modulate glycolysis, probably as a consequence of receptor tyrosine kinase activation, inducing phosphorylation of protein band 3 and hence the release of glycolytic enzymes. We have previously reported that a reductase enzyme in human erythrocytes is dependent on glycolysis being significantly activated (+28 +/- 3.1%, P < 0.001) by high insulin levels (1 mIU/ml). Mean basal reductase activities were found to be significantly lower in diabetics than in normal subjects (type 1 diabetics: 0.77 +/- 0.03 vs 0.97 +/- 0.02 mmol ferrocyanide/20 min per l cells, P < 0.001; type 2 diabetics: 0.77 +/- 0.04 vs 0.97 +/- 0.02 mmol ferrocyanide/20 min per l cells, P < 0.001), indicating altered erythrocyte metabolism in the diabetic patients. High glucose levels were used to mimic hyperglycemia condition, using erythrocytes from normal subjects. At 30 mM glucose, erythrocytic phosphatase activity was stimulated (+32 +/- 4.2%, P < 0.0001), although no effect was observed on the reductase enzyme at the same glucose levels. Results indicated that diabetic disorders appear to be associated with quantitative alterations of erythrocyte acid phosphatase activity and other enzymes that depend on the glycolytic rate (reductase). The overall data suggest that erythrocyte acid phosphatase may have a role in the modulation of glycolytic rates through the control of insulin receptor phosphorylation. PMID- 10741569 TI - The ABBOS-peptide from bovine serum albumin causes an IFN-gamma and IL-4 mRNA response in lymphocytes from children with recent onset of type 1 diabetes. AB - The ABBOS-peptide from bovine serum albumin (BSA) in cow's milk has been suggested to initiate the autoimmune process against the beta-cells leading to type 1 diabetes. The aim of this study was to elucidate if the ABBOS-peptide is a possible trigger of type I diabetes. The cytokines IL-4 and IFN-gamma were determined at the level of transcription as mRNA in lymphocytes, stimulated with the ABBOS-peptide. Sixteen children with newly diagnosed type 1 diabetes were compared with 10 healthy controls matched for the diabetes associated HLA-type DR3/4. Antibodies to bovine serum albumin (BSA), insulin antibodies (IA), and antibodies against islet cells (ICA) were determined, as well as serum C-peptide. Increased mRNA expression for IFN-gamma and/or IL-4 could be observed in lymphocytes from 13/16 children with recent onset of diabetes after in vitro stimulation with the ABBOS-peptide. Low expression of IFN-gamma mRNA was associated with high secretion of C-peptide, whereas a positive relationship could be observed between expression of IL-4 mRNA and insulin antibodies. Expression of IFN-gamma and/or IL-4 mRNA was also detected in lymphocytes from 6/10 healthy controls. ABBOS may have a role as a reactive epitope in the upregulation of the autoimmune process against the beta-cells but ABBOS does not seem to cause any specific Th1 response. An increased mRNA expression could also be seen in lymphocytes from healthy controls. Thus, the ABBOS-peptide might just cause or reflect an unspecific immune activity. PMID- 10741570 TI - Genetic markers of immunoglobulins and diabetes mellitus in the multiracial population of New Caledonia. The CALDIA Study Group. AB - GM and KM immunoglobulin allotypes, which are the markers, respectively, of the constant parts of the heavy and the light chains of the IgG1, IgG2 and IgG3 subclasses, have been analysed in diabetic mellitus patients and controls living in New Caledonia. We tested 40 Europeans, 256 Melanesians and 44 Polynesians, as well as their 340 matched controls, in order to search for a genetic susceptibility at those polymorphic loci. All the subjects were tested for G1M (1, 2, 3, 17), G2M (23), G3M (5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 28) and KM (1) by the classical hemagglutination method. The frequencies of GM haplotypes and KM alleles have been estimated by a maximum likelihood method. The results are in favour of no influence of the GM and KM loci. The prevalence of diabetes mellitus varies in the populations of New Caledonia: Polynesians are at much higher risk than Melanesians or Europeans. The GM haplotype distribution differs among ethnic groups; so they provide a useful marker to measure genetic admixture. The higher prevalence of diabetes observed among New Caledonians of European origin compared to the prevalence in Europe may be explained by genetic admixture with neighbouring Pacific populations, notably Polynesians (Asian haplotypes are present at a frequency of 9.4%). So, the genetic admixture should be measured in any genetic epidemiological study. PMID- 10741571 TI - Apolipoprotein E polymorphism in non-insulin-dependent diabetics of Mumbai, India and its effect on plasma lipids and lipoproteins. AB - The role of apolipoproteinE (apoE) phenotypes in modulating the plasma lipid and lipoprotein levels was studied in a group of NIDDM patients and healthy individuals residing in Mumbai. The apoE phenotype frequencies were similar in the diabetic and healthy persons. The elevations in the lipid/lipoprotein levels were higher in diabetic subjects (53.3%) than in the controls (8%), showing the frequency of increase to be highest in the apoE4/4 group of diabetics, followed by apoE4/3 and apoE3/2 groups. In the controls as well, a similar trend was observed in different groups, indicating that the susceptibility to changes in lipid concentrations differs among apoE phenotypes. The apoE3/3 bearing individuals in both the categories showed close to normal lipid levels, suggesting it to be the wild type. The occurrence of apoE4 allele was higher than the apoE2 allele in diabetic individuals. Diabetic subjects with the apoE4 allele showed hypercholesterolemia, while those with apoE2 showed the presence of hypertriglyceridemia. One of the striking features of our work is a significant relationship between apoE4/3 phenotype in NIDDM persons and elevated levels of plasma triglyceride, thus suggesting a delayed catabolism of VLDL relative to production. In conclusion, the work suggests that the apoE2 and apoE4 alleles are associated with elevations in lipid levels and these changes are more pronounced in the diabetic individuals in whom most of the lipid levels were higher, indicating a gene environment/disease interaction. PMID- 10741572 TI - Fertility, mortality and gender bias among tribal population: an Indian perspective. AB - The present paper critically reviews the existing literature on fertility, mortality and its gender bias among India's tribal population in the post Independence period. Despite difficulties and limitations of available literature on tribal demography--most of which has been produced by anthropologists--our review extracts several interesting and important points. First, although fertility and mortality levels for some tribes and for some regions are either lower or higher or even the same as those for nontribal groups, India's aggregate tribal population evinces both lower fertility and mortality than the levels for their closest comparable nontribal group, namely low caste people. Several sociocultural and lifestyle features of tribals are historically favourable to maintaining a relatively low fertility and mortality. Despite baseline aggregative patterns of demographic differential being favourable to tribes, there is rather strong indication that of late and in the near future Indian tribals might be lagging behind the nontribal population in demographic transition (e.g. in terms of slower pace of tribal fertility and mortality declines). Also, while gender relations among Indian tribes have historically been more balanced and egalitarian, an unfortunate trend of tribal gender bias conforming to the mainstream anti-female pattern (along with acculturation, assimilation and similar 'modernizing' processes) is increasingly discernable under current circumstances. PMID- 10741573 TI - Gender and the stigma of onchocercal skin disease in Africa. AB - This paper reports results from a multicenter study of gender differences in the stigma associated with onchocercal skin disease (OSD) in five African sites: Cameroon, Ghana, Nigeria (Awka and Ibadan) and Uganda. The studies used a common protocol to compare affected and unaffected respondents, that is, men and women with onchodermatitis in highly endemic areas and respondents from communities with low endemicity or no onchocerciasis. The methods were both quantitative and qualitative, allowing for the comparison of stigma scores and people's verbal descriptions of their experiences and attitudes. Questions to the unaffected were asked after providing them with photographs and short descriptions (vignettes) depicting typical cases. We found that stigma was expressed more openly by the unaffected, who perceived OSD as something foreign or removed from themselves, whereas the affected tended to deny that they experienced stigma as a result of the condition. Gender differences in stigma scores were not significantly different for men and women, but qualitative data revealed that stigma was experienced differently by men and women, and that men and women were affected by it in distinctive ways. Men were more concerned about the impact of the disease on sexual performance and economic prospects, whereas women expressed more concern about physical appearance and life chances, especially marriage. Similar trends were found in the different sites in the responses of affected and unaffected respondents, and differences between them, despite geographical and cultural variations. PMID- 10741574 TI - Sexually transmitted diseases in Morocco: gender influences on prevention and health care seeking behavior. AB - Increased awareness of the medical and social costs of sexually transmitted diseases (STD) has resulted in greater attention to the control of these illnesses. STDs are responsible for a significant amount of morbidity in Morocco and have become a key target of the HIV control program. In 1996, the Ministry of Health conducted a qualitative study in order to enhance information, education and communication strategies in the national STD/HIV program. Data on the conceptualization and knowledge of STD, information sources and health-care seeking behavior were gathered through 70 semidirected, in-depth interviews conducted with men and women in the general population and health care providers (HCPs). Two commonly applied health behavior theories in STD/HIV prevention, the Health Belief Model (HBM) and the Theory of Reasoned Action (TRA) served as a framework for data analysis. The most common name for STD is berd, which means "the cold" in Moroccan Arabic. Berd is caused either by cold striking the genital area or sexual intercourse and most often designates a syndrome of genital discharge. However, the term was also often used to indicate STD in general. The dual causality of berd maintains social stability by providing an honorable excuse for individuals who become infected, while warning against unsanctioned sexual behavior. Clear gender differences in understanding STDs and health-care seeking behavior emerged through these interviews. STDs in Morocco are viewed as women's illnesses and men with STD often reported feeling victimized by women. Men appear to have more extensive informal information sources for STD than women. Consequences of STD, both physical and psychosocial, were viewed as more severe for women than men, and men had greater access to treatment, for both social and economic reasons. PMID- 10741575 TI - Constructions of masculinity and their influence on men's well-being: a theory of gender and health. AB - Men in the United States suffer more severe chronic conditions, have higher death rates for all 15 leading causes of death, and die nearly 7 yr younger than women. Health-related beliefs and behaviours are important contributors to these differences. Men in the United States are more likely than women to adopt beliefs and behaviours that increase their risks, and are less likely to engage in behaviours that are linked with health and longevity. In an attempt to explain these differences, this paper proposes a relational theory of men's health from a social constructionist and feminist perspective. It suggests that health-related beliefs and behaviours, like other social practices that women and men engage in, are a means for demonstrating femininities and masculinities. In examining constructions of masculinity and health within a relational context, this theory proposes that health behaviours are used in daily interactions in the social structuring of gender and power. It further proposes that the social practices that undermine men's health are often signifiers of masculinity and instruments that men use in the negotiation of social power and status. This paper explores how factors such as ethnicity, economic status, educational level, sexual orientation and social context influence the kind of masculinity that men construct and contribute to differential health risks among men in the United States. It also examines how masculinity and health are constructed in relation to femininities and to institutional structures, such as the health care system. Finally, it explores how social and institutional structures help to sustain and reproduce men's health risks and the social construction of men as the stronger sex. PMID- 10741576 TI - Workforce diversity: implications for the effectiveness of health care delivery teams. AB - This paper examines the implications of racial diversity for the self-perceived communication effectiveness of nursing care teams. An RN leads the nursing care team (NCT) and delivers care in collaboration with two or more nonlicensed caregivers. Overlap is intentionally designed into the roles of NCT members and the range of duties the team performs is generally expanded to include functions previously performed by personnel from centralized departments. NCTs are highly reliant on mutual respect and effective communication among team members. Team conflict and miscommunication can be exacerbated by the strong correlation between role on the nursing care team (NCT) and race. Verbatim transcripts of fourteen focus groups from two study hospitals were used to develop a grounded theory of the role that race plays in the self-perceived communication effectiveness of nursing care teams. Two themes that emerged from the focus group discussions constitute the overarching framework within which racially diverse team members evaluate team communication effectiveness: different perspectives and alternative realities. Three additional themes, social isolation, selective perception and stereotypes, that serve as reinforcing factors were also identified, i.e., these factors deepen the conflict and dissatisfaction with team communication that occurs as a natural consequence of the overarching framework of different perspectives and alternative realities. Leadership emerged as a powerful mitigating factor in the model of how race influences the self-perceived communication effectiveness of nursing care teams. Leaders who can transcend racial identity as evidenced by the ability to validate alternative realities and appreciate different perspectives appear to moderate the potential negative effects of racial diversity on team communication processes and strengthen the positive aspects of diversity. PMID- 10741577 TI - Work stress among six professional groups: the Singapore experience. AB - Recent developments in stress research have called for attention to how social structures influence the stress and coping processes. This paper examines the experience of work stress among professionals in Singapore and argues that workers' experiences in the workplace are influenced not only by individual personality and job nature, but also by structural forces shaping the profession, the social organization of work institutions and the development of the economy. Data were collected from a survey of professionals in Singapore conducted in 1989 1990. The sample consisted of 2570 men and women from six different professions and para-professions, namely general practitioners, lawyers, engineers, teachers, nurses and life insurance personnel. Results showed that performance pressure and work-family conflicts were perceived to be the most stressful aspects of work. These two stressors also significantly contributed to the experience of overall work stress. Further, stress arising from work-family conflicts, performance pressure and poor job prospects was negatively associated with the level of work satisfaction. These findings were discussed in the contexts of increasing professionalization and de-professionalization and the growing emphases on productivity and efficiency in a quickly developing economy. PMID- 10741578 TI - Professional commitment among US physician executives in managed care. AB - This paper examines professional commitment among physician executives working in managed care settings in the United States. The rise of an 'administrative elite' in medicine is central to the notion that physicians preserve their professional dominance despite changes in their prestige, work and employment status. Implicit in the notion of Freidson's restructuring perspective, physician executives presumably remain dedicated to professional interests in their management roles. The findings of a national survey support this assumption. Physician executives maintain meaningful, stable levels of professional commitment over time in management and the organization. This commitment is positively related to work related characteristics involving favorable perceptions of the management job and physical and mental 'connection' to the practice of medicine. Belief in one's ability to successfully deliver appropriate clinical care, however, moderates the positive association between involvement in the management job and professional commitment. The findings provide a rationale for the maintenance of professional loyalty among physicians in management rooted in the work-related perceptions and activities of the individual physician executive. PMID- 10741579 TI - Family self-medication and antibiotics abuse for children and juveniles in a Chinese city. AB - To identify the determinants of self-medication and antibiotics abuse by parents treating their children aged between 2 and 18 over the previous year, an investigation was conducted in Hefei City, China in April, 1995. A total of 1596 students from a kindergarten, a primary school and a high school were included in the study, and 1459 completed questionnaires were collected (the response rate: 91.4%). The results showed the rate of parental self-medication for their children in the sample was 59.4%. It increased with children's age; about 51% of children had received parental self-medication on six or more occasions during the 1-year period and 32.8% on four to five occasions; there were associations between parental self-prescribers and sources of medicine and severity of disease. The rate of antibiotics abuse was 35.7%. Logistic regression analysis showed that there were significant associations between self-medication and payment of the mother's medical fees by employers, severity of diseases as well as the mother's educational level. PMID- 10741580 TI - Community-perceived benefits of ivermectin treatment in northeastern Nigeria. AB - A 3-step approach involving focus group discussion, structured interviews and informal conversations with key individuals was used to investigate community usage and perceived benefits of ivermectin in nine Nigerian villages participating in a WHO-sponsored investigation of community-directed treatment with ivermectin (CDTI). Only 27% of 284 persons interviewed had received treatment. An under-estimation of the district's ivermectin needs led to inadequate supply of ivermectin to the communities, which was cited as the main reason (65%) for non-treatment. All those treated (N=76) were further interviewed using questionnaires. Worm expulsion (80%) and blindness prevention (68%) were the most frequently stated benefits. Other perceived benefits were an increase in vitality (68%), sexual drive and performance (29%). The sudden relief from a heavy burden of worms, which had built up over a long period, may have indirect effect on all aspects of an individual's health and account for the diverse experiences. The feeling of vitality, good appetite and general health following ivermectin treatment is an animating experience to many communities. Health planners face the challenge of preparing communities for fewer 'sensational' experiences and preventing a possible feeling of disappointment that may result from frequent usage. PMID- 10741581 TI - Low-income mothers' views on breastfeeding. AB - Nourishing infants presents women today with choices, desires, obligations and constraints. Despite mounting evidence about the health, psychosocial and societal benefits of breastfeeding both for women and infants, current breastfeeding rates worldwide are far from optimal, particularly among low-income women. Many mothers choose to use infant formula. Drawing from structured interviews with 154 mothers from an urban low-income multiethnic population in the United States, a typology of mothers' feelings about their infant feeding method is developed. Findings indicate that regardless of their feeding method, mothers tended to attribute higher health benefits to breastfeeding and perceived community norms as probreastfeeding. They differed in their rating and perceptions of logistics and the extent to which benefits mattered in their infant-feeding decision. Contradictions associated with the practice of breastfeeding even among mothers who breastfed, were reflected in their perceptions of social disapproval of breastfeeding in public, reports of ridicule by friends, lack of support from some health providers, and difficulties associated with working. A typology of mothers' emotional states resulting from such contradictions summarizes the findings and underscores how some mothers who did not, but would have liked to breastfeed, may be subjected to feelings of guilt and deprivation. Implications for educational interventions are to amplify prenatal infant feeding consultations and address ways to overcome logistical and apprehension barriers. PMID- 10741582 TI - The experience of chronic illness and post-traumatic stress disorder: the consequences of cumulative adversity. AB - In this paper the experiences of the chronically ill are examined to explore the impact of post-traumatic stress disorder (PTSD), accumulated burden of adversity and trauma spectrum disorder on subsequent illness and coping behaviors. Individuals experiencing chronic diseases have been studied with regard to depression, anxiety and a variety of coping maladaptions, but negligible attention has been given to the PTSD potential of chronic disease over the life course. Yet, growing evidence suggests that the traumatogenic potential of chronic diseases, some sudden and unexpected onsets, and the traumatogenic changes in life circumstance, may produce maladaptive illness coping over the life course. More importantly, attention needs to focus on the additive effect of co-morbid life events and the traumatic potential of invasive medical therapies. Consideration of PTSD and a continuum of cumulative adversity provide a more complex and fully drawn understanding of the circumstances surrounding chronic illness coping and reasons for maladaptive coping following invasive therapies and changes in the disease trajectory. The pathophysiology that produces a chronic diseases does not begin at symptom onset, and the psychosocial strategies to cope with a chronic illness, whether efficacious or maladaptive, also do not begin at symptom onset, but develops over the life course. PMID- 10741583 TI - The many meanings of deinsuring a health service: the case of in vitro fertilization in Ontario. AB - There is currently much international interest in principles and processes for determining which services should qualify for health insurance packages. However, there has yet been little analysis of the implications of actual deinsurance decisions made by such priority-setting exercises. This paper reports experience from the case of in vitro fertilization (IVF) deinsurance in Ontario, Canada. The analysis addresses some of the more social aspects of financial incentives by characterizing funding structures as means of communicating complex policy objectives, rather than mechanistic reward-penalty systems. Deinsuring IVF was intended to support several policy goals, including: controlling public expenditures, restricting public coverage to 'medically necessary' services, applying evidence of effectiveness as a criterion for medical necessity and implementing part of a policy program to control new reproductive technologies. It may seem a modest step to remove one dubious service from public insurance coverage. Nevertheless, as interpreted by stakeholders, deinsurance of IVF may inadvertently promote the opposite of what policy makers intended. This case suggests that priority-setting decisions based on incomplete information, inconsistently applied principles and too little attention to health system dynamics (perhaps the norm in 'real-world' priority-setting) can have the perverse effect of undermining progress toward health reform goals of improved health, reduced expenditures and more rigorously evaluated services. The case of IVF in Ontario offers several lessons for other jurisdictions engaged in priority setting and service deinsurance: (1) individual services interact with the rest of the system and cannot be removed without systemic effects, (2) inconsistent use of coverage principles can undermine legitimacy of both priority-setting principles and processes and (3) 'evidence-based' decisions can founder on differing stakeholder ideas about appropriate evidence and on the inconsistent message given by commercializing ineffective or unproven care. PMID- 10741584 TI - Addicts' narratives of recovery from drug use: constructing a non-addict identity. AB - Sociological accounts of the process of recovery from dependent drug use have emphasised the importance of the individual constructing a non-addict identity for themselves. Following Giddens we identify the process of providing a narrative of their recovery as one of the mechanisms by which addicts may seek to achieve this. The narratives of recovery which are the subject of this paper were elicited in the course of semi-structured interviews with a sample of 70 recovering addicts. There were three key areas in which the addicts' narratives of recovery could be seen to be constructing a non-addict identity for the individual; firstly, in relation to the reinterpretation of aspects of their drug using lifestyle; secondly, in relation to the reconstruction of their sense of self and thirdly, in relation to the provision of convincing explanations for their recovery. In certain respects, the addicts' narratives of recovery are similar to the accounts of recovery provided by drug workers and addictions researchers. The paper argues that the correspondence between addicts' own accounts of their recovery and those of professional drug workers may be not so much the result of the intrinsic nature of the recovery process as a product of the socially constructed nature of the narratives and the fact that the latter may have been developed in conjunction with those working in the drug treatment industry. PMID- 10741585 TI - A review on the nonoperative removal of necrotic tissue from burn wounds. AB - The study of nonoperative debridement of burns got underway during the Second World War. A large number of substances such as enzymes of plant origin, acids and proteolytic enzymes of bacterial origin were examined since. The proteolytic enzymes derived from filtrates of C. histolyticum and B. subtilis have attracted the greatest interest. Although enzymatic debridement would seem at first sight to be an attractive form of treatment, unfortunately the results are highly variable. PMID- 10741586 TI - Generation of peroxynitrite in localised, moderate temperature burns. AB - Simultaneous generation of nitric oxide (NO*) and superoxide (O2-) can lead to the formation of peroxynitrite (ONOO-), a potent oxidant that has been implicated in the pathogenesis of a number of disease states. This study was designed to investigate the possible generation of ONOO- in local cutaneous tissues following thermal injury. Male Wistar rats were anaesthetised in a nonrecovery procedure and subjected to a small (1 cm diameter), abdominal burn of moderate temperature (50 degrees C, 5-15 min). At either the 60 or 180 min time point postburn the animals were killed, and skin sites were removed and homogenised. An ELISA was used to quantify protein bound 3-nitrotyrosine (3NT), a biomarker for ONOO- in the rat skin. In separate experiments the accumulation of [125I]-albumin in thermally injured skin was used to calculate plasma extravasation. Thermal injury (50 degrees C, 10 min) to rat abdominal skin caused a significant increase in both 3NT (p < 0.05) and oedema formation (p < 0.001) when compared to unheated control sites at the 180 min time point postburn. This data is the first to show protein nitration in thermally injured, oedematous skin and strongly suggests that ONOO- is generated in thermally damaged cutaneous tissue. PMID- 10741587 TI - Comparison of lymphocyte populations in cutaneous and electrical burn patients: a clinical study. AB - Immunosuppression following thermal injury has been noted in recent years. Both cellular and humoral immune systems have been reported to be affected. The present study aimed to compare the quantitative differences between cutaneous and electrical burn patients in respect to the partition and levels of lymphocyte populations. From March 1997 through February 1998, 15 patients with major thermal injury or high voltage electrical injury were included in this clinical prospective study. Blood samples were collected at three and seven days postburn. The evaluation of lymphocyte populations of patients was performed by SimulTest IMK plus. T cell and B cell populations, activated T cells, natural killer and helper T cell levels were all suppressed in both groups. Suppressor T cell levels were elevated in electrical burn group at both three and seven days. Therefore, CD 4/CD 8 ratios were more suppressed in electrical burn group. In conclusion, lymphocyte populations in electrical burn patients and also contributing factors which play important roles in the development of sepsis in both group need to be investigated further. PMID- 10741588 TI - Heat shock response reduces mortality after severe experimental burns. AB - The heat shock response has imparted protective effects in animal models of septic shock and endotoxemia. This study has tested the hypothesis that it could be protective in experimental burns. One hundred and fifteen adult male Fischer rats were randomly divided into four groups. Rats in the first group (n = 12) were anesthetized and shaved. In the second group (n = 15) rats were anesthetized and heated in a 45 degrees C water bath. In the third group (n = 44), rats were anesthetized, shaved and submitted to 26-30% body surface third-degree burns using a brass bar. In the fourth group (n = 44), rats were anesthetized, heated and, 1 day after, they were burnt. Mortality rates were measured at 3, 7, 15 and 25 days. Liver and lung samples were collected from all groups for heat-shock protein 70 detection. Heat-shock protein 70 was positive in heated animals. No animals died in the first or second group. Heated and burnt animals showed significantly decreased mortality at days 3 (p < 0.05, Fischer's exact test) and at days 7, 15 and 25 (p < 0.01) after burns, when compared to unheated burnt animals. In conclusion, eliciting the heat-shock response significantly reduced mortality rates in this model of experimental burns. PMID- 10741589 TI - Tumor necrosis factor-alpha and tumor necrosis factor receptor I, II levels in patients with severe burns. AB - Tumor necrosis factor alpha (TNF-alpha) and tumor necrosis factor receptor I and II (TNFRI and TNFRII) were studied in 24 burn patients who had a total burn surface area (TBSA) of 50.2 +/- 20.4%. Immediately after the injury, both the TNFRI and TNFRII levels correlated significantly with TBSA (r = 0.7344, P < 0.0001; r = 0.6074, P = 0.0012). The TNFRI and TNFRII levels immediately after the injury were significantly higher in the 11 patients who later died of their burns than in the 13 patients who survived (0.8 +/- 0.4 ng/ml vs. 1.8 +/- 0.7 ng/ml, P = 0.0002; 2.3 +/- 1.1 ng/ml vs. 4.5 +/- 1.6 ng/ml, P = 0.0009). The TNF alpha levels immediately after the injury did not differ significantly between the group that survived and the group that died. The TNFRI and TNFRII values for the entire follow-up period also correlated significantly with TBSA. Peak TNFRI and TNFRII levels were significantly higher in the group that died than in the group that survived (6.0 +/- 4.7 ng/ml vs. 14.1 +/- 7.8 ng/ml, P = 0.0009; 7.0 +/ 5.1 ng/ml vs. 16.7 +/- 5.2 ng/ml, P = 0.0003). The TNF-alpha levels correlated significantly with both the TNFRI and the TNFRII levels. The TNFRI and TNFRII levels thus closely reflected the severity of the burns in both the acute postburn period and the subsequent follow-up period. In other words, these parameters well reflected the severity and outcome of the burns, irrespective of the presence or absence of accompanying infection. PMID- 10741590 TI - Non-invasive quantification of skin injury resulting from exposure to sulphur mustard and Lewisite vapours. AB - The severity and progression of skin lesions resulting from exposure to the chemical warfare agents Lewisite (L) and sulphur mustard (SM) have been investigated using the non-invasive biophysical methods of evaporimetry and reflectance spectroscopy in large white pigs in vivo. Erythema (redness) expressed immediately after exposure to L or SM vapours appeared to be related to the lesion severity as demonstrated by histopathological analysis. Skin brightness correlated well with scab formation whereas blueness (cyanosis) did not appreciably alter throughout the study. Rates of transepidermal water loss (TEWL) changed both with occlusion (during vapour exposure) and also mirrored the progression of macroscopic skin injury after 12 h. Whilst no single parameter could be used in isolation to ascertain the severity and subsequent progression of the skin lesions, measurement of erythema, skin brightness and TEWL could provide quantitative, non-invasive methods for determining the efficacy of antidotes or therapies to prevent the toxic effects of chemical warfare agents. However, neither colourimetry or TEWL provided a clinical evaluation of such lesions that were comparable with the prognostic capabilities of laser Doppler imaging. PMID- 10741591 TI - Burns sustained by hot bath and shower water. AB - An 8-year retrospective review of patients admitted to Stoke Mandeville Hospital (Aylesbury, UK) because of burns sustained by hot bath and shower water was undertaken. Fifty-seven patients of all ages were identified and stratified into paediatric (< 16 years) and adult groups. Nine patients died. The main characteristics of the burns, causes and outcomes of treatment were analysed for each group. Children were predominantly under three years of age (83%), sustaining most frequently only superficial burns (41%) with areas of less than 10% total body surface area (72%). Parents' supervision was inadequate in 85% of cases. Eighty-three percent of the adults were over the age of 60. Two thirds had some form of psycho-motor disorder that predisposed to an accident which should have been anticipated. In comparison to children, adults suffered more extensive and deeper burns that resulted in a mortality of 44% (8/18). In both groups, the lower parts of the body were most frequently involved. The observed decline in the number of admissions for the period of investigation is encouraging. It supports an ultimate need for further development and actual implementation of preventative measures for hot water burns in the homes of people who are at greatest risk. PMID- 10741592 TI - Ten years epidemiological study of paediatric burns in Manipal, India. AB - Three hundred and nine children of burns injuries treated over last 10 years (1989-1998) in Kasturba Hospital, Manipal (India) were studied retrospectively and were analysed for incidence, severity, extent, causes, risk factors and overall mortality. Children of age < 5 years were affected more than children of age > 5 years (76.1 vs. 23.9%). Females were affected more than males (74.1 vs. 25.9%). Most of the children received burn injuries in the range of 0 to 20% BSA (63.1%). Scald (72.5%) followed by flame (22.7%) and electrical burn (3.2%) were most common cause of burn injuries. Overall paediatric burn mortality was 7.4%. PMID- 10741593 TI - Cooling the burn wound: evaluation of different modalites. AB - A study was undertaken to investigate the cooling and healing effect of different modalities: Melaleuca Alternifolia Hydrogel (Levtrade International (Pty) Ltd.) was compared with tap water as a coolant following application onto a fresh deep partial thickness hot water burn in a porcine model. Four identical circular scalds were created on the backs of 10 pigs. One wound was not treated and served as a control. The other 3 wounds were either cooled with tap water (15 degrees C) or had Melaleuca Hydrogel dressing applied immediately, or after a 30 min delay. Intradermal temperatures were monitored in all wounds: preburn, during the burn and at regular intervals for 1 h. The wounds were biopsied for histological assessment. These samples were repeated at 24 h and 3 weeks. The mean decrease in final temperature at 1 h was in comparison to the preburn temperature; control +0.44 degrees C (i.e. a temperature increase); water -7.82 degrees C; Melaleuca Hydrogel -3.87 degrees C; Melaleuca Hydrogel after 30 min delay -2.67 degrees C. Clinical and histological assessment at 21 days indicated more rapid healing in both the Melaleuca Hydrogel and water-cooled burns compared with the untreated controls. Effective cooling of the burn wound and an increased rate of wound healing was achieved by both repeated tap water compresses and by immediate or delayed application of Melaleuca Hydrogel. Cooling is an effective means to reduce tissue damage and increase wound healing. PMID- 10741594 TI - Pediatric burn patients from Vietnamese camps in Hong Kong from 1989 to 1997. AB - Over the 9 years from 1989 to 1997, many children who suffered from scald burns in the Vietnamese camps in the New Territories of Hong Kong were treated. The profile of these children was examined and analyzed. PMID- 10741595 TI - Rapid induction analgesia for the alleviation of procedural pain during burn care. AB - Burn patients must often endure intense pain during their regular dressing changes. The aim of the present study was to investigate the therapeutic effect of rapid induction analgesia (RIA) on resting and procedural pain, anticipatory anxiety, relaxation levels and medication consumption in 30 hospitalized burn patients. Patients rated levels of pain and relaxation for four burn care sessions. RIA was conducted twice on 15 patients, whereas dressing changes proceeded as usual in 15 control patients. When asked to recall pain during the dressing changes, patients remembered an experience which was worse in its entirety than the average of spot ratings taken during the burn care procedure. However, self-reported ratings of the sensory and affective components of pain decreased significantly during and after RIA, particularly in patients who became readily absorbed, and relaxation increased during burn care. Anticipatory anxiety decreased before dressing changes in the RIA group, and analgesic intake decreased between treatment sessions. The promising outcome of this study confirms RIA as a viable adjunct to narcotic treatment for pain control during burn care. PMID- 10741596 TI - Resurfacing deep wound of upper extremities with pedicled groin flaps. AB - A total of 29 axial pedicled groin skin flaps were applied clinically with satisfactory result excepting for one flap which tailed on transplantation. These cases included severe scar contracture of the dorsum of hands in 20 patients, deeply burned wounds with infection and exposure of deep structure in upper extremities in nine patients, such as electrical burns and hot-crushing injuries. The flap is supplied by two groups of nutrient vessels with abundant vascularization and located in a hidden area. Therefore, the pedicled groin skin flap is still valuable due to its advantages as safe, easy operation and strong antiinfective ability although the free groin flap is more widely used today. PMID- 10741597 TI - Analysis of cost of dressings in the care of burn patients. AB - This study aimed at analyzing the costs of material used in the occlusive dressings of burn patients. The subjects of the study were patients over the age of 18 years who were admitted to the Burns Unit of the Ribeirao Preto Medical School Clinical Hospital, University of Sao Paulo-Brazil, from 15 January to 11 September 1998. Data were collected concerning the costs and duration of bathing and dressing of these patients. We concluded that there was a positive relation between the real costs of the materials used and the percentage of the body surface burned. In addition, there was a positive correlation between the number of staff involved in care and the size of the burns. There was no significant difference between the cost of materials used in dressings for burns of different depths. PMID- 10741598 TI - Tissue expansion for frontal hairline restoration in severe alopecia in a child. AB - Burn alopecia has serious sequelae, both aesthetic and psychological, particularly in children. A case of 70% scalp alopecia due to a flame burn, and the modality of treatment is described. This consisted of an expanded temporo parietal flap, which was transposed to create a frontal hairline. A relatively simple surgical procedure had both substantial aesthetic, and psychological benefits. PMID- 10741599 TI - The use of temporary laparoscopic loop ileostomy in lumbosacral burns. AB - The technique of laparoscopic formation of loop ileostomies has been previously described for use in a variety of conditions. We present this as an option for faecal diversion in severe burns involving the back and buttock region and describe its use in two cases. Faecal diversion allows for easier wound care and nursing. Intra-abdominal assessment of these very sick patients can also be performed simultaneously. The technique is relatively simple, readily available and associated with minimal morbidity. Furthermore, this technique minimises interference with the abdominal wall as a donor site for skin grafts. PMID- 10741600 TI - Cardiac compromise in a patient with Down's Syndrome: a lesson learnt. AB - A 49 year old burn victim with Down's Syndrome (Trisomy 21) was admitted with 15% body surface area (BSA) superficial burns. This was complicated by a large atrioseptal defect. Her course was stormy with difficulties encountered in managing her fluid status. Adequate fluid resuscitation was difficult to maintain with a fragile compromise between pulmonary insufficiency and renal impairment. She expired 12 days post-injury. Cardiac anomalies are not uncommon in the subgroup of patients with major burns who respond poorly to fluid resuscitation. PMID- 10741601 TI - An unusual long-term complication of burn injury: malignant fibrous histiocytoma developed in chronic burn scar. AB - Development of malignant tumors in chronic burn wounds is a well-known complication. These tumors are almost always squamous cell carcinomas, although other types of malignancies such as basal cell carcinoma, malignant melanoma and sarcomas can be seen rarely. There are only three previously reported cases of malignant fibrous histiocytoma developed in chronic burn scar in the literature. Two cases with malignant fibrous histiocytoma developed in chronic, badly treated burn wounds are presented. One of the tumors was multifocal and overexpression of the p53 gene was present. Both tumors were excised widely and skin grafted. Regional lymph node dissection was performed in one case. One of the patients died due to tumor recurrence and lymphatic metastases. These cases represent a very uncommon complication of burn injury and indicate the importance of the appropriate primary treatment of the burn wound. PMID- 10741602 TI - Recent references. PMID- 10741603 TI - Energy-loss special issue based on the presentations of the 14th Congress on Electron Microscopy, Cancun, Mexico, September 1998. Session K: electron energy loss spectroscopy (EELS), energy-loss near-edge structure (ELNES) and extended energy-loss fine structure (EXELFS). PMID- 10741604 TI - The physical significance of the mixed dynamic form factor AB - We show that the mixed dynamic form factor for inelastic scattering of fast electrons in crystals is closely related to the density matrix of the probe electron and to that of the scatterer. With this insight it is possible to calculate both energy filtered diffraction patterns and energy spectroscopic high resolution images. As an example we discuss the Si-K and -L edges. PMID- 10741605 TI - Methods for ELNES-quantification: characterization of the degree of inversion of Mg-Al-spinels AB - The energy loss near-edge structures in electron energy-loss spectra contain information about bonding characteristics, the electronic structure and coordinations of the excited atoms. We have calculated sets of reference spectra for the normal and for the inverse Mg-Al-spinel using a full multiple scattering approach. By a quantitative comparison of these reference spectra with experimental data ELNES-quantification becomes possible. We characterized the degree of inversion lambda by the analysis of the relative peak-intensities and the relative peak-positions within 35 eV beyond the edge onset. The results demonstrate that by using the provided methods ELNES-quantification will become possible when uncertainties in the experiment are reduced and a better fit of the simulations to the experiment is achieved. PMID- 10741606 TI - Valence state mapping of cobalt and manganese using near-edge fine structures AB - The properties of transition metal oxides are related to the presence of elements with mixed valences. The spectroscopy analysis of the valence states is feasible experimentally, but a spatial mapping of valence states of transition metal elements is a challenge to existing microscopy techniques. In this paper, with the use of valence state information provided by the white lines and near-edge fine structures observed using the electron energy-loss spectroscopy (EELS) in a transmission electron microscope (TEM), a novel experimental approach is demonstrated to map the valence state distributions of Mn and Co using the ratio of white lines in the energy-filtered TEM. The valence state map is almost independent of specimen thickness in the thickness range adequate for quantitative EELS microanalysis. An optimum spatial resolution of approximately 2 nm has been achieved for a two-phase Co oxides. PMID- 10741607 TI - Understanding STM images and EELS spectra of oxides with strongly correlated electrons: a comparison of nickel and uranium oxides AB - Using a theoretical approach combining the local spin density approximation (LSDA) of density functional theory and the Hubbard U term (LSDA + U), we analyse the connection between the experimentally observed electron energy loss spectra and elevated temperature scanning tunnelling images of surfaces of semiconducting nickel monoxide NiO and uranium dioxide UO2. We show that a combination of electron energy loss spectroscopy, atomic-resolution tunnelling imaging and first principles ab initio calculations provides a powerful tool for studying electronic and structural properties of surfaces of transition metal and actinide oxides. PMID- 10741608 TI - Branching ratio and L2 + L3 intensities of 3d-transition metals in phthalocyanines and the amine complexes AB - L(2,3) inner-shell excitation spectra were obtained by electron energy-loss spectroscopy (EELS) for the divalent first transition series metals in phthalocyanine complexes (MPc) such as titanium oxide phthalocyanine (TiOPc), fluoro-chromium phthalocyanine (CrFPc), manganese phthalocyanine (MnPc), iron phthalocyanine (FePc), cobalt phthalocyanine (CoPc), nickel phthalocyanine (NiPc) and copper phthalocyanine (CuPc). It was found that the value of normalized total intensity of I(L2 + L3) was nearly proportional to the formal electron vacancies of each 3d-state, and the values of the branching ratio, I(L3)/I((L2 + L3), represented a high-spin-state rather than low-spin-state for MnPc, FePc and NiPc. EELS was also applied to charge-transfer complexes of FePc with an amine such as pyridine or gamma-picoline. It was concluded that their I(L2 + L3) intensity of Fe showed the decrease in vacancies of 3d-states on the formation of the charge transfer complex with these amines, which suggests some electron transfer from the amine to Fe in phthalocyanine. The EELS study provides beneficial information for investigating the electronic states of the specific metal sites in organic materials. PMID- 10741609 TI - Simulating the oxygen K-edge spectrum from grain boundaries in ceramic oxides using the multiple scattering methodology AB - In this paper we demonstrate the use of the multiple scattering methodology to interpret oxygen K-edge spectra from both the bulk and grain boundaries in a variety of ceramic oxides. The experimental electron energy loss spectra (EELS) used in this study, were obtained from a dedicated scanning transmission electron microscope (STEM). Using the STEM to obtain the spectra has the advantage that each spectrum can be acquired with atomic spatial resolution. While the energy resolution is limited to approximately 0.8 eV, and the angular integration in the microscope apertures precludes momentum resolved spectroscopy, this unprecedented spatial resolution allows the electronic structure at individual defect sites to be determined. Additionally, as the microscope can also provide an atomic resolution image of the defect, the relationship between the atomic structure of the defect and its local electronic structure can be determined. In practice, this is achieved by using the structure observed in the image to build the real space atomic cluster for multiple scattering simulations. Detailed interpretation of the simulations of oxygen K-edge spectra from bulk MgO, CaO, SrTiO3, TiO2, MnO2, Mn3O4, Mn2O3 and MnO are presented. In addition, the simulations from grain boundaries in TiO2 (undoped) and SrTiO3 (undoped and Mn doped) are discussed in relation to quantifying the changes in the local electronic structure that are a direct consequence of the defect structure. The simulations are used to make interpretations of the structure-property relationships at these grain boundaries. PMID- 10741610 TI - Identification by TEM and EELS of the products formed at the surface of a carbon electrode during its reduction in MClO4-EC and MBF4-EC electrolytes (M = Li, Na) AB - The electrochemical intercalation of lithium and sodium into graphite was carried out using a liquid electrolyte containing ethylene carbonate (EC) as solvent and MClO4 or MBF4 (M = Li, Na) as salts. The first intercalation of alkali metals into graphite is accompanied with irreversible reactions attributed to the reduction of the electrolyte. These reactions contribute to the development of a passivating layer, formed on graphite surface prior to intercalation. This layer is impervious to solvent molecules but allows alkali ions to diffuse through its bulk. The surface chemistry of the electrodes was characterized using transmission electron microscopy (image, selected area electron diffraction) as well as EELS. The effect of the nature of the alkali salts on the properties of the passivating layer is studied. PMID- 10741611 TI - Theoretical and experimental limits of the analysis of III/V semiconductors using EELS AB - The optimisation of acquisition conditions for EELS spectroscopy of Al(x)Ga(1 x)As heterostructures permits one to find the absolute concentration with a precision of better than delta = +/-0.02 and to detect changes in concentration of +/-0.01 for x = 0-0.5. In order to achieve this concentration precision, we investigated ways to reduce the influence of three major sources on the inaccuracies of the measurement: the effect of electron channelling which biases the ionisation probabilities on the different atomic sites, the contribution of the sample surface layers and the accuracy in the spectral analysis. An optimal specimen orientation that maximises the stability of the electron densities on Al and As sites without introducing an unacceptable loss of spatial resolution due to sample tilt is found by computing the electron channelling intensity as a function of the specimen tilt angle. The influence of a Ga enriched surface layer on the analysis is demonstrated. Two methods for the extraction of the edge intensity from the spectra are compared. These methods are shown to give the upper and the lower limit of the Ga concentration. PMID- 10741612 TI - Local chemical states and microstructure of photoluminescent porous silicon studied by means of EELS and TEM AB - Photoluminescent porous Si (PS) layers were formed on low resistivity p-type Si(100) wafers by anodization at a current density (Ia) of 100-350 mA cm(-2) at room temperature. The local chemical states and the microstructures of the PS layers were studied by means of electron energy loss spectroscopy (EELS) and transmission electron microscopy (TEM), and were correlated to the red photoluminescence (PL). The PS layer consists of sponge-like and tree-like structures. The tree-like structure becomes finer with increase of Ia. The fine tree-like structure is shown to be favorable for intensive PL. The EELS analysis reveals that the Si-4O structure, namely the basic structure of SiO2, and Si crystals coexist in the PS layers. The relative content of SiO2 to Si crystallites (R) increases with increase in Ia and decreases with the depth from the top surface of the PS layer. High R corresponds to intensive PL. These results suggest that PL may be connected with some kind of defects in SiO2 and/or defects in the boundary between Si crystallites and SiO2 structure in anodized PS layers, and that PL may be mainly emitted from the layer near the top surface of the PS layers. PMID- 10741613 TI - Nanoscale EELS analysis of dielectric function and bandgap properties in gaN and related materials AB - The low loss region of an EEL spectrum (<50 eV) contains information about excitations of outer shell electrons and thus the electronic structure of a specimen which determines its optical properties. In this work, dedicated electron energy loss spectroscopy (EELS) methods for the experimental acquisition and analysis of spectra are described, which give improved information about the electronic structure near the bandgap region at a spatial resolution in the range of nanometers. For this purpose, we made use of a cold field emission scanning transmission electron microscope (STEM) equipped with a dedicated EELS system. This device provides a subnanometer electron probe and offers an energy resolution of 0.35 eV. Application of suitable deconvolution routines for removal of the zero loss peak extracts information on the bandgap region while the Kramers-Kronig transformation deduces the dielectric properties from the measured energy loss function. These methods have been applied to characterize the optical properties of wide-bandgap materials for the case of III-nitride compounds, which are currently the most promising material for applications on optoelectronic devices working in the blue and ultraviolet spectral range. The obtained results are in excellent agreement with experimental measurements by synchrotron ellipsometry and theoretical studies. The potential of the superior spatial resolution of EELS in a STEM is demonstrated by the analysis of dielectric properties of individual layers of heterostructures and individual defects within wurtzite GaN. PMID- 10741614 TI - High-detective-quantum-efficiency fast-electron detector for electron energy loss spectroscopy AB - In order to increase the sensitivity of the parallel electron detector used in electron energy loss spectroscopy (EELS), we have developed a direct electron exposed detector, based on a photodiode array (PDA). This work investigates the performance of this detector at 100 keV incident electrons in terms of the detective quantum efficiency (DQE), the modulation transfer function (MTF) and radiation damage. PMID- 10741615 TI - Visibility spectroscopy with electron waves using a Wien filter: higher order corrections AB - Fourier spectroscopy, one of the most precise spectrometry methods in light optics, has been realized in the past years with electron waves by including a crossed-field analyzer (Wien filter) as a wave packet shifting device into a biprism interferometer. To fully exploit the high precision of this method of determining the energy spectrum, higher order corrections stemming from the Wien filter's focusing action are necessary in the evaluation of the measurement. We show that these can be taken into account by using a transformation variable being proportional to the quotient of electric field strength of the Wien capacitor over fringe spacing. Finally, we make some remarks about the possibility of interference of inelastically scattered electrons. PMID- 10741616 TI - Clinical trials of hematopoietic cell transplantations: current needs and future strategies. PMID- 10741617 TI - Relevance and potential of natural killer cells in stem cell transplantation. PMID- 10741618 TI - Humoral immune response to proteins of human cytomegalovirus latency-associated transcripts. AB - Latent human cytomegalovirus (CMV) infection of hematopoietic progenitor cells is associated with the presence of latency-associated transcripts that may express 6 proteins larger than 44 amino acids in size (open reading frame [ORF] 55, ORF45, ORF94, ORF59, ORF154, ORF152/UL124). The serologic response to these proteins was evaluated in healthy seropositive individuals as well as in individuals undergoing active CMV infection. Individual recombinant GST-fusion proteins, prepared from bacteria, were found by enzyme-linked immunosorbent assay to be recognized by between 8% and 44% long-term healthy seropositive individuals, with ORF94 and ORF55 being the most broadly and significantly recognized. Although nearly all of serum samples (85%) recognized at least 1 of these proteins, none reacted with all 6. Patterns of antibody prevalence to these proteins in long term seropositive individuals were similar to many antigens expressed during productive replication (IE1, ppUL57, ppUL83/pp65), but none were broadly detected by a majority of individuals, a characteristic of only a few productive-phase antigens, including ppUL44/ICP36 and ppUL32/pp150. Consistent with prevalence in long-term seropositive individuals, commercial preparations of pooled human gamma globulin were also found to recognize latency-associated proteins. Serologic reactivity to latency-associated proteins was slow to develop following primary infection, in a pattern distinct from any of the characterized replication-phase proteins tested here, and was boosted late after secondary infection or reactivation in solid-organ transplant recipients without showing a correlation with viremia or disease. These results provide evidence that proteins expressed from the latent region during natural infection exhibit immunogenicity comparable with most other characterized viral antigens, although the narrow response to individual latency-associated proteins likely precludes their use in serologic assays to investigate clinical correlates or outcome in transplant recipients. PMID- 10741619 TI - Early outcomes after allogeneic stem cell transplantation for leukemia and myelodysplasia without protective isolation: a 10-year experience. AB - Although it is common practice to use some form of isolation to protect allogeneic stem cell transplant patients from infection, the necessity for these practices in all environments has not been demonstrated. The current study evaluated patterns of infection and 100-day transplant-related mortality in 288 patients with myelodysplasia and leukemia transplanted without isolation. Patients were allowed out of hospital at any time within constraints of the medication schedule. Fever, foci of infection, and positive cultures within 28 days and death within 100 days because of the transplant procedure were recorded. Fever occurred in 57% of patients, and 10% had a clinical or radiographic focus of infection. Most infections were apparently endogenous; blood cultures from 24% of recipients grew organisms, 87% of which were gram-positive bacteria. Four patients (1%) died with aspergillus infection in circumstances indicating that isolation would not have been helpful. Twenty percent of patients remained without evidence of infection throughout. Transplant-related mortality at 100 days was 1% for 108 patients with early leukemia receiving transplants from matched siblings. For patients at higher risk, by virtue of donor and/or disease status, mortality was 21%. These figures compare favorably with those reported to the International Bone Marrow Transplant Registry, the majority of patients having been subjected to some form of isolation. We conclude that allogeneic stem cell transplantation can be safely performed in some environments without confining patients continuously to the hospital. PMID- 10741620 TI - Are we treating health or physical appearance when we prescribe drugs for obesity? PMID- 10741621 TI - Assessment and reporting of clinical pharmacology information in drug labeling. PMID- 10741622 TI - Relationship between time after intake of grapefruit juice and the effect on pharmacokinetics and pharmacodynamics of nisoldipine in healthy subjects. AB - A clinical study was performed in eight healthy volunteers to investigate the effect of various timing of grapefruit juice intake on nisoldipine pharmacokinetics and pharmacodynamics, and to validate our pharmacokinetic model. The subjects were given 10 mg oral nisoldipine with water (control), or 5 mg oral nisoldipine with 200 mL grapefruit juice (G0) or with water at 14 (G14), 38 (G38), 72 (G72) or 96 hours (G96) after a 7-day period of thrice-daily intake of grapefruit juice. Grapefruit juice ingestion did not affect heart rate or the effect area during the first 8 hours of heart rate after nisoldipine administration, although significant decreases of systolic and diastolic blood pressure were caused in G0 by coadministration of grapefruit juice with nisoldipine. Headaches were reported by 3, 2, and 1 persons in G0, G14, and G38, respectively, but no subjects in G72 and G96 reported headaches. Compared with the control group, the maximum plasma concentration of nisoldipine was significantly increased after grapefruit juice intake in G0 and G14, and the plasma concentration was significantly increased at each time in G0 to G72. Therefore the effect of grapefruit juice decreased time dependently and lasted for at least 3 days after intake. Furthermore, our model gave predicted values in good agreement with the observed values. It is therefore necessary to withhold grapefruit juice for at least 3 days before administration of the drug to prevent grapefruit juice-nisoldipine interaction. PMID- 10741623 TI - Diltiazem and mibefradil increase the plasma concentrations and greatly enhance the adrenal-suppressant effect of oral methylprednisolone. AB - OBJECTIVE: To examine the possible interaction of the calcium channel blockers diltiazem and mibefradil with orally administered methylprednisolone. METHODS: In this randomized, double-blind, placebo-controlled, three-phase crossover study, nine healthy SUBJECTS received 60 mg diltiazem three times a day, 50 mg mibefradil once a day, or placebo orally for 3 days. On day 3, each subject received a 16-mg oral dose of methylprednisolone. Plasma concentrations of methylprednisolone and cortisol were determined by HPLC up to 47 hours. RESULTS: Compared with placebo, diltiazem and mibefradil increased the total area under the plasma concentration-time curve of methylprednisolone [AUC(0-infinity)] 2.6 fold (P < .001) and 3.8-fold (P < .001), the peak plasma concentration 1.6-fold (P < .001) and 1.8-fold (P < .001), and the elimination half-life 1.9-fold (P < .001) and 2.7-fold (P < .001), respectively. The nighttime exposure to methylprednisolone [AUC(12-23)] was increased 28.2-fold (P < .01) and 72.1-fold (P < .001) by diltiazem and mibefradil, respectively, and correlated negatively (r = -0.81, P < .001) with the morning plasma cortisol concentration (measured at 8 AM, 23 hours after the administration of methylprednisolone). During the diltiazem phase, the morning plasma cortisol concentration was 12% of that during the placebo phase (P < .001); during the mibefradil phase, the morning plasma cortisol concentration was 2% of that during the placebo phase (P < .001). CONCLUSIONS: Coadministration of diltiazem or mibefradil with methylprednisolone resulted in increased plasma concentrations and a greatly enhanced adrenal suppressant effect of oral methylprednisolone. Care should be taken if methylprednisolone is coadministered with a potent CYP3A4 inhibitor for a long period. PMID- 10741624 TI - Effect of cimetidine and probenecid on pilsicainide renal clearance in humans. AB - OBJECTIVE: To investigate the effect of cimetidine and probenecid on the renal clearance of pilsicainide in healthy subjects. METHODS: Nine healthy men (age range, 21 to 38 years) were given oral doses of 50 mg pilsicainide hydrochloride alone, with coadministration of 800 mg oral cimetidine, or with coadministration of 1,500 mg oral probenecid on three occasions in a Latin-square order. Urine and venous blood samples were collected on a timely basis. The concentration of pilsicainide in plasma and urine were determined by an HPLC method. RESULTS: Concomitant administration of cimetidine significantly increased the area under the plasma concentration-time curve of pilsicainide by a mean of 33%, prolonged elimination half-life by a mean of 24% (from 5 to 6.2 hours), reduced apparent oral clearance by a mean of 26% (from 14.7 +/- 0.1 to 10.8 +/- 0.8 L/h) and reduced renal clearance by a mean of 28% (from 196.8 +/- 53.9 to 141.8 +/- 25.9 mL/min). The net renal clearance by tubular secretion was significantly reduced by a mean value of 38%, from 151.4 +/- 62.9 to 93.0 +/- 31.1 mL/min. Coadministration of probenecid did not show any changes in plasma concentrations of pilsicainide, pharmacokinetics, or the net renal clearance by tubular secretion of pilsicainide. CONCLUSIONS: Pilsicainide appeared to be secreted by the active transport system for organic bases in the proximal tubule, and the excretion of pilsicainide was inhibited by cimetidine. PMID- 10741625 TI - Target site concentrations after continuous infusion and bolus injection of cefpirome to healthy volunteers. AB - BACKGROUND: Recent data indicate a higher level of effectivity of beta-lactam antibiotics if serum concentrations are kept above the minimal inhibitory concentration (MIC) of the pathogen. This concept would favor continuous infusion over bolus dosing. However, it is usually not the serum concentration but the free interstitial concentration in the target tissue that determines antibiotic activity. We therefore set out to measure effective drug concentrations in the interstitial space of muscle and subcutaneous adipose tissue and to compare trough levels and times above the MIC after bolus versus continuous infusion of cefpirome. METHODS: Twelve healthy volunteers received a single dose of 2 g cefpirome as an intravenous bolus or as a continuous infusion over 8 hours in a crossover design, and the resulting free interstitial tissue concentrations were measured with use of microdialysis. RESULTS: After bolus injection, mean interstitial trough concentrations were 3.0 +/- 1.9 microg/mL and 2.1 +/-1.0 microg/mL for muscle and subcutaneous tissue, respectively; continuous infusion resulted in trough levels of 10.1 +/- 6.8 microg/mL and 10.1 +/- 4.6 microg/mL for muscle and subcutaneous tissue, respectively. This resulted in significantly longer times above the MIC with continuous infusion for Staphylococcus epidermidis and Enterobacter cloacae. Bacteria with an MIC < or =1 would be covered by either method, whereas higher doses seem to be necessary for Pseudomonas aeruginosa. CONCLUSION: Although susceptible organisms will usually be covered sufficiently with standard dosing regimens, soft tissue infections with bacteria that have MIC values of 2 to 8 may profit from continuous application. Coverage of P aeruginosa, however, would be inadequate with conventional daily doses of 4 g cefpirome regardless of the method of application. PMID- 10741626 TI - The disposition of chloroquine and its main metabolite desethylchloroquine in volunteers with and without chloroquine-induced pruritus: evidence for decreased chloroquine metabolism in volunteers with pruritus. AB - The pharmacokinetics of chloroquine and its main metabolite desethylchloroquine have been carried out in volunteers with and without chloroquine-induced pruritus. It was shown that the volunteers with pruritus tended to metabolize chloroquine slower than the volunteers without pruritus because the metabolic ratio was lower in the volunteers with pruritus than that in the volunteers without pruritus. However, the overall pharmacokinetic patterns were comparable between the two groups and agreed with published data. The 24-hour urinary collections in the two groups of volunteers indicated that the volunteers with pruritus excreted more chloroquine (although not statistically significant) than the volunteers without pruritus. This also indicates that they metabolized less chloroquine. There were no side effects of note in any of the volunteers. The volunteers who gave positive histories of chloroquine-induced pruritus had mild episodes of itching after intake of the drug; the pruritus subsided within 48 hours in all instances. PMID- 10741627 TI - Prediction of cyclosporine clearance in liver transplant recipients by the use of midazolam as a cytochrome P450 3A probe. AB - BACKGROUND: Interindividual differences in the kinetics of cyclosporine (INN, ciclosporin) result in part from variations in the activity of cytochrome P450 3A (CYP3A). The biotransformation of midazolam to 1'-hydroxymidazolam is also catalyzed by CYP3A. The objective of this study was to examine the usefulness of midazolam as a CYP3A probe to predict cyclosporine clearance. METHODS: Twenty-six stable liver transplant recipients receiving immunosuppressive therapy with oral cyclosporine (Neoral) were studied. Midazolam (0.015 mg/kg) was administered intravenously and a blood sample was obtained 1 hour later. The plasma concentration of midazolam and 1'-hydroxymidazolam was measured by gas chromatography-mass spectrometry. Blood concentration of cyclosporine was measured by a fluorescence polarization assay. Cyclosporine clearance was calculated as daily dose divided by trough level. RESULTS: There were large interindividual variations in cyclosporine clearance and in midazolam metabolism. Cyclosporine blood levels correlated poorly with dose (r = -0.016). However, there was a significant correlation between cyclosporine clearance and the plasma concentration of 1'-hydroxymidazolam (r = 0.559; P < .001) or the midazolam/1' hydroxymidazolam plasma concentration ratio (r = 0.668; P < .001). CONCLUSION: Heterogeneity in CYP3A activity contributes to interpatient differences in cyclosporine dosage requirements after liver transplantation. Midazolam metabolism correlated with cyclosporine clearance, but it accounted for only about 40% of the variability in the apparent oral clearance of cyclosporine and this relationship is not tight enough to be useful in the prediction of cyclosporine dosage requirements in the clinical setting. PMID- 10741628 TI - Nonlinear kinetics and pharmacologic response to mibefradil. AB - BACKGROUND: Increasing oral doses of mibefradil (10 to 320 mg) decrease its apparent oral clearance; however, intravenous doses up to 80 mg do not reduce its systemic clearance. This study aimed to understand the mechanisms underlying the zero-order kinetics of mibefradil. METHODS: A group of 10 normotensive volunteers received 50 mg/day oral mibefradil for 8 days and, on days 1 and 8, 5 mg deuterated mibefradil by infusion. Ten additional volunteers observed the same protocol with a daily oral dose of 100 mg mibefradil. Serial blood samples were withdrawn, and mibefradil plasma concentrations were assayed by liquid chromatography-mass spectrometry. Blood pressure and heart rate were measured for 4 hours, and an ECG was performed 2 hours after drug administration. RESULTS: Repeated oral administration of 50 mg mibefradil generated zero-order kinetics secondary to a decrease in mibefradil systemic clearance. Compared with the 50-mg dose, single and repeated oral doses of 100 mg further decreased mibefradil clearance. Mibefradil bioavailability was not affected by increasing mibefradil doses. Mean diastolic blood pressure was decreased by single and repeated doses of 100 mg to the same extent. Repeated doses of 100 mg reduced heart rate and prolonged the PR and QTc, changes that were associated with mibefradil plasma concentrations. CONCLUSIONS: Repeated doses of 50 mg or doses of 100 mg mibefradil generated zero-order kinetics secondary to a decrease in hepatic extraction of the drug. Zero-order kinetics did not affect the response concentration relationship of mibefradil. PMID- 10741629 TI - Effect of age and smoking on in vivo CYP1A2, flavin-containing monooxygenase, and xanthine oxidase activities in Koreans: determination by caffeine metabolism. AB - OBJECTIVES: To assess the effect of gender, age, and smoking habits on the in vivo activities of CYP1A2, flavin-containing monooxygenase (FMO), and xanthine oxidase in Korean subjects. METHODS: One hundred thirty-three age- and gender matched healthy Korean volunteers (age range, 21 to 78 years; mean age, 35.3 +/- 16.6 years) with and without smoking habits participated. After drinking a cup of coffee (200 mL) that contained 110 mg caffeine, a 1-hour urine sample (between 4 and 5 hours) was collected and caffeine metabolites were analyzed by HPLC. RESULTS: There were marked individual variations in CYP1A2 [(1,7-dimethylurate + paraxanthine)/caffeine], FMO (theobromine/caffeine), and xanthine oxidase (1 methylurate/1-methylxanthine) activities (14-, 42-, and 9-fold, respectively). However, the mean values of these enzyme activities in the nonsmokers were not different between men and women. In the nonsmoking subjects in their 20s, the mean values of CYP1A2 and FMO activities (13.5 +/- 5.9 and 2.1 +/- 1.9, respectively) were higher than those (7.9 +/-1.8 and 0.95 +/- 0.22) of older decennial age groups. Xanthine oxidase activities were the same for all age groups (subjects in their 20s through their 70s). CYP1A2 activity of the smokers (20.0 +/- 9.6) was higher than that of the nonsmokers (10.8 +/- 5.8; P < .001). Similarly, the FMO activity in smokers (3.4 +/- 2.7) was higher than that of the nonsmokers (1.8 +/- 1.7; P < .001). The xanthine oxidase activity (1.3 +/- 0.5) was not increased in smokers (1.4 +/- 0.5; P = .46). CONCLUSIONS: Results of this caffeine metabolism study conducted with age- and gender-matched healthy Korean volunteers with and without smoking habits provided the baseline and the widely varying interindividual activities of CYP1A2, FMO, and xanthine oxidase in a Korean population. The results also suggested that drugs metabolized by CYP1A2 and FMO may require individualized dose adjustment according to the age and smoking habits of the subjects. PMID- 10741630 TI - The interaction of diltiazem with simvastatin. AB - BACKGROUND: Simvastatin is an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase that is used as a cholesterol-lowering agent and is metabolized by cytochrome P450 3A (CYP3A) enzymes. Diltiazem is a substrate and an inhibitor of CYP3A enzymes and is commonly coadministered with cholesterol lowering agents such as simvastatin. The objective of this study was to quantify the effect of diltiazem on the pharmacokinetics of simvastatin. METHOD: A fixed order study was conducted in 10 healthy volunteers with a 2-week washout period between the phases. In one arm of the study, a single 20-mg dose of simvastatin was administered orally; the second arm entailed administration of a single 20-mg dose of simvastatin orally after 2 weeks of treatment with 120 mg diltiazem twice a day. RESULTS: Diltiazem significantly increased the mean peak serum concentration of simvastatin by 3.6-fold (P < .05) and simvastatin acid by 3.7 fold (P < .05). Diltiazem also significantly increased the area under the serum concentration-time curve of simvastatin 5-fold (P < .05) and the elimination half life 2.3-fold (P < .05). There was no change in the time to peak concentration for simvastatin and simvastatin acid. CONCLUSION: Diltiazem coadministration resulted in a significant interaction with simvastatin, probably by inhibiting CYP3A-mediated metabolism. Concomitant use of diltiazem or other potent inhibitors of CYP3A with simvastatin should be avoided, or close clinical monitoring should be used. PMID- 10741631 TI - Contribution of CYP2E1 and CYP3A to acetaminophen reactive metabolite formation. AB - BACKGROUND: CYP2E1, 1A2, and 3A4 have all been implicated in the formation of N acetyl-p-benzoquinone imine (NAPQI), the reactive intermediate of acetaminophen (INN, paracetamol), in studies in human liver microsomes and complementary deoxyribonucleic acid-expressed enzymes. However, recent pharmacokinetic evidence in humans has shown that the involvement of CYP1A2 is negligible in vivo. The purpose of this study was to evaluate the respective roles of CYP2E1 and 3A4 in vivo. METHODS: The involvement of CYP2E1 was assessed through pretreatment of adult human volunteers with disulfiram to inhibit the enzyme and the role of CYP3A4 through its induction in a second cohort of adults with rifampin (INN, rifampicin). Each of the respective studies was an open-label, balanced randomized crossover design. Blood samples were obtained serially for 12 hours and urine was collected for 24 hours after acetaminophen administration. Acetaminophen was assayed in plasma, and acetaminophen and metabolites were assayed in urine. RESULTS: The recovery of the thiol metabolites formed by conjugation of NAPQI with glutathione was decreased by 69%, and the formation clearance of NAPQI was decreased by 74% (both P < .01) by pretreatment with disulfiram. Rifampin pretreatment had no effect on the formation of NAPQI or the recovery of thiol metabolites formed by conjugation of NAPQI with glutathione. CONCLUSIONS: CYP2E1 accounts for the formation of NAPQI in intact humans; the contribution of other isozymes of cytochrome P450 appears to be negligible. Under some conditions, disulfiram may be useful in diminishing the formation of NAPQI after acetaminophen overdose. PMID- 10741632 TI - Paroxetine affects metoprolol pharmacokinetics and pharmacodynamics in healthy volunteers. AB - OBJECTIVE: To investigate the effect of multiple-dose paroxetine intake on the stereoselective pharmacokinetics and the pharmacodynamics of metoprolol. METHODS: We conducted an open trial with two sessions in eight healthy male volunteers. Racemic metoprolol (100 mg single oral dose) was administered before and after paroxetine treatment (20 mg/day for 6 days). The (R)- and (S)-metoprolol pharmacokinetics, metoprolol metabolic ratio (MR), exercise heart rate and blood pressure were assessed for 12 (pharmacodynamic data) to 24 (pharmacokinetic data) hours after each metoprolol intake. RESULTS: Paroxetine treatment increased the mean area under the plasma concentration-time curve extrapolated to infinity (AUC) of (R)- and (S)-metoprolol significantly (169 to 1,340 ng x h/mL [P < .001] and 279 to 1,418 ng x h/mL [P < .001], respectively), with an approximately twofold increase in both maximum plasma concentration and terminal elimination half-life. Furthermore, the (S)/(R) AUC ratio was significantly decreased, from 1.72 to 1.07 (P < .001). The mean metoprolol MR was significantly increased, from 0.17 to 5.69 (P < .05). The AUC of the metoprolol-induced decrease in exercise heart rate versus time curve was increased, with 46% (P < .01) after multiple dose paroxetine intake, reaching significance from 6 hours after metoprolol intake, illustrating a more sustained beta-blockade. Similar results were obtained for the effect on exercise systolic blood pressure. Multiple-dose metoprolol administration combined with paroxetine can lead to an accumulation of the beta-blocking (S)-enantiomer of metoprolol, possibly resulting in unacceptable bradycardia, loss of cardioselectivity, or both. CONCLUSION: Multiple-dose paroxetine intake affects both metoprolol pharmacokinetics and pharmacodynamics and suggests that when paroxetine is added to an ongoing metoprolol therapy, caution is warranted and a reduction of the metoprolol dose may be required to prevent undesired adverse effects. PMID- 10741633 TI - Venous neuropeptide Y receptor responsiveness in patients with chronic heart failure. AB - BACKGROUND: Chronic heart failure is associated with increased sympathetic nerve activity and elevated plasma neuropeptide Y levels. The aim of this study was to investigate whether increased neuropeptide Y release altered vascular neuropeptide Y responses in the dorsal hand veins in patients with chronic heart failure. METHODS AND RESULTS: Neuropeptide Y responsiveness was studied in vivo with use of a hand vein tonometry technique in 14 patients with chronic heart failure and left ventricular ejection fraction (LVEF) values <20%, 16 patients with LVEF values from 20% to 35%, and 16 age-similar healthy control subjects. Plasma norepinephrine and neuropeptide Y levels were significantly elevated in patients with chronic heart failure and LVEF values <20% compared with control subjects (P < .01). Plasma neuropeptide Y but not norepinephrine levels were significantly elevated in patients with chronic heart failure and LVEF values from 20% to 35% compared with control subjects (P < .01). Increasing doses of neuropeptide Y (25 to 2,000 pmol/min) were infused into a dorsal hand vein of each subject. Dose-dependent venoconstriction to neuropeptide Y was observed in all subjects studied. The neuropeptide Y dose-response curve in patients with LVEF values from 20% to 35% was significantly shifted to the left compared with patients with LVEF values <20% and control subjects (P < .01), whereas no significant difference was observed between the control subjects and the patients with LVEF values <20%. No significant difference in neuropeptide Y dose responses was observed between patients with chronic heart failure with plasma neuropeptide Y levels above the median and patients with chronic heart failure with plasma neuropeptide Y levels below the median. CONCLUSIONS: In vivo venous neuropeptide Y receptor responsiveness is increased in patients with chronic heart failure and LVEF values from 20% to 35%. This increased neuropeptide Y responsiveness may contribute to venoconstriction at this stage of heart failure. PMID- 10741634 TI - Venous responsiveness to norepinephrine in healthy subjects: effects of single doses of 325 mg aspirin. AB - BACKGROUND: Antithrombotic doses of aspirin, which are widely used in patients with cardiovascular disease, may inhibit prostaglandin synthesis but the physiologic significance with regard to vascular tone is not well defined. We hypothesized that inhibition of vasodilator prostaglandin synthesis by aspirin would significantly increase sympathetic-mediated venoconstriction. METHODS: Twelve healthy volunteers (mean age, 24.5 +/- 0.8 years; age range, 19 to 30 years) were studied on two mornings approximately 7 days apart and not less than 90 minutes after a randomized single dose of 325 mg aspirin or matching placebo. Distension of dorsal hand veins was measured with use of the linear variable differential transformer technique during local infusions of exogenous norepinephrine (0.125 to 1,024 ng/min) and during release of endogenous norepinephrine from sympathetic activation by a forehead cold pressor test. Hemodynamic parameters and venous plasma catecholamine levels were measured. Antiplatelet activity of the dose of aspirin was confirmed in three subjects. RESULTS: Aspirin increased venoconstriction to norepinephrine, causing a significant shift to the left (P < .03) of the norepinephrine dose-response curve and a significant decrease in logED50 (P < .03), representing a decrease in the dose of norepinephrine required to reduce vein distension by 50% from 50 to 25 ng/min. Venoconstriction to the cold pressor test was significantly increased by aspirin (10% +/- 3% versus 3% +/- 1% for placebo; P < .02). Cold pressor-induced increases in mean arterial pressure were significantly larger with aspirin compared with placebo (18 +/- 1 versus 14 +/- 1 mm Hg, respectively; P < .03). Baseline levels and stress-induced increases in plasma norepinephrine were not different between days. CONCLUSIONS: The results suggest that aspirin inhibits the role of vasodilator prostaglandins in modulating peripheral venoconstriction and increases vascular resistance during physiologic stress in young healthy subjects. PMID- 10741636 TI - Use of calcium antagonists and hemoglobin loss in hospitalized elderly patients: a cohort study. Gruppo Italiano di Farmacoepidemiologia nell'Anziano (GIFA) investigators. AB - OBJECTIVE: To assess the association between in-hospital use of calcium antagonists and incident reduction in hemoglobin levels, as well as the impact of individual baseline risk for gastrointestinal bleeding on such an association. METHODS: The association between calcium antagonists and hemoglobin decrease > 1.2 g/dL was examined in 6,721 patients enrolled in a collaborative pharmacoepidemiology study who did not take calcium antagonists before admission and with baseline hemoglobin > or =12 g/dL. Among these participants, 1,076 patients started taking calcium antagonists during their hospital stays. Demographic variables, comorbid conditions, medications, and objective tests that were associated with incident hemoglobin loss in separate age- and sex-adjusted logistic regression models were examined as potential confounders in a summary model. Higher risk for gastrointestinal bleeding was defined by diagnosis, treatment for peptic disease, or both. RESULTS: Hemoglobin decrease was detected in 24% of participants who started treatment with calcium antagonists and in 19% of other patients (P < .0001). In addition, use of calcium antagonists was independently associated with increased probability of hemoglobin loss (odds ratio [OR], 1.22; 95% confidence interval [CI], 1.03 to 1.45; P = .018) after adjusting for potential confounders. Treatment with calcium antagonists was associated with hemoglobin loss in patients with higher baseline risk for gastrointestinal bleeding (OR, 1.67; 95% CI, 1.26 to 2.22; P < .0001) but not among other participants (OR, 1.02; 95% CI, 0.82 to 1.25). CONCLUSION: Starting treatment with calcium antagonists is associated with a reduction in hemoglobin levels during a hospital stay. However, the increased risk of hemoglobin loss seems to be limited to patients with diagnosis or symptoms of peptic disease. PMID- 10741635 TI - Ex vivo--in vitro interaction between aspirin, clopidogrel, and the glycoprotein IIb/IIIa inhibitors abciximab and SR121566A. AB - OBJECTIVES: To assess the interaction between aspirin and clopidogrel in healthy male volunteers and the interaction of the glycoprotein IIb/IIIa (GPIIb/IIIa) inhibitors abciximab and SR121566A with blood from those pretreated subjects (ex vivo-in vitro). METHODS: Aspirin (300 mg/day), clopidogrel (75 mg/day), or the combination of both drugs were administered orally for 8 days. Group 1 (n = 5) started with aspirin and group 2 (n = 5) with clopidogrel. From day 4 to day 8, subjects of both groups received the combined treatment. Blood from these subjects was spiked with abciximab (0.5 and 1.5 microg x mL(-1)) and SR121566A (31 and 62 ng x mL(-1)). RESULTS: In vivo, average bleeding times were 6.8 minutes at baseline, 20.3 minutes for clopidogrel alone (P < .01), 10.9 minutes for aspirin alone (difference not significant), and 24.0 minutes (P < .01) for the combined treatment. Fibrinogen binding to the platelet GPIIb/IIIa receptor was reduced for aspirin to 69% (difference not significant), to 63% for clopidogrel (difference not significant), and to 63% for the clopidogrel plus aspirin combination (P < .01). CD62 expression as a marker of platelet granular secretion was reduced to 66% by clopidogrel (P < .01) and to 41% by the combination of clopidogrel and aspirin; aspirin alone had no effect. In vitro, with pretreatment with aspirin and clopidogrel, inhibitory effects of the GPIIb/IIIa inhibitors on fibrinogen binding were additive to changes observed with aspirin or clopidogrel alone. No effect on CD62 expression was observed with either GPIIb/IIIa inhibitor. Aspirin and clopidogrel reinforced effects of the GPIIb/IIIa inhibitors on adenosine diphosphate (5 micromol/L)-induced aggregation in an additive manner, a supra-additive effect was observed with collagen (2 microg x mL(-1))-induced aggregation. CONCLUSION: The augmentation of the antiaggregatory effects of GPIIb/IIIa inhibitors by aspirin and clopidogrel and the lack of antisecretory effects of GPIIb/IIIa inhibitors may favor their combination with clopidogrel. PMID- 10741637 TI - Life-threatening 5-fluorouracil-like cardiac toxicity after treatment with 5 fluorocytosine. PMID- 10741638 TI - Eletriptan and coronary artery. PMID- 10741639 TI - The forgotten author. PMID- 10741640 TI - Aberrant crypt foci in colorectal carcinogenesis. Cell and crypt dynamics. AB - Aberrant crypt foci (ACF) have been identified on the colonic mucosal surface of rodents treated with colon carcinogens and of humans after methylene-blue staining and observation under a light microscope. Several lines of evidence strongly suggest that ACF with certain morphological, histological, cell kinetics, and genetic features are precursor lesions of colon cancer both in rodents and in humans. Thus, ACF represent the earliest step in colorectal carcinogenesis. This paper has the main purpose of reviewing the evidence supporting this view, with particular emphasis on cell and crypt dynamics in ACF. ACF have been used as intermediate biomarkers of cancer development in animal studies aimed at the identification of colon carcinogens and chemopreventive agents. Recently, evidence has also shown that ACF can be effectively employed in chemopreventive studies also in humans. PMID- 10741641 TI - Dilazep, a nucleoside transporter inhibitor, modulates cell cycle progression and DNA synthesis in rat mesangial cells in vitro. AB - The direct effects of the nucleoside transporter inhibitor dilazep on the cell cycle of mesangial cells have not before been investigated. The purpose of this study was to elucidate whether dilazep can inhibit the proliferation of mesangial cells and how it interferes with the cell cycle of these cells. DNA histograms were used and BrdUrd uptake rate was measured by flow cytometry. There was no significant difference in the cell numbers among the untreated group and the 10( 5) M, 10(-6) M or 10(-7) M dilazep-treated groups at 24 h of incubation. However, at 48 and 72 h, the cell numbers in the dilazep-treated groups were significantly lower compared with that of the untreated group (P < 0.005). The DNA histograms of cultured rat mesangial cells at 12, 24, and 48 h of incubation with 10(-5) M dilazep showed that the ratio of the S phase population in the dilazep-treated group decreased by 2.2% at 12 h, by 9.6% at 24 h, and by 18.9% at 48 h compared with the untreated group. The ratio of the G0/G1 phase population in the dilazep treated group significantly increased: 6.8% at 12h (P < 0.05), 13.9% at 24 h (P < 0.001), and 76.5% at 48 h (P < 0.001) compared with the untreated group. A flow cytometric measurement of bivariate DNA/BrdUrd distribution demonstrated that the DNA synthesis rate in the S phase decreased after 6 h (P < 0.005) and 12 h (P < 0.05) of incubation compared with the untreated group. These results suggest that dilazep inhibits the proliferation of cultured rat mesangial cells by suppressing the G1/S transition by prolonging G2/M and through decreasing the DNA synthesis rate. PMID- 10741642 TI - Cell membrane changes of structure and function in protein kinase inhibitor induced polyploid cells. AB - Exogenous cyclic AMP has been thought to be a chemical without marked pharmacological effect until now, as it is not capable of penetrating the cell membrane in most eucaryotic cells. The present study obtained results consistent with those of most previous studies, showing that exogenous cyclic AMP itself did not interfere with the cell cycle even at the high dose of 100 microM. However, it was found that K252a, a potent inhibitor of protein kinases including protein kinase C, induced DNA re-replication, i.e. DNA synthesis at a elevated DNA ploidy in cells that had not undergone cytokinesis (leading to polyploidization), and that exogenous cyclic AMP markedly potentiated the K252a-induced polyploidization at a very low dose similar to the effective dose of membrane-permeable cyclic AMP analogue dibutyryl cyclic AMP. These findings suggested that the cell membrane changed during the formation of polyploid cells. This supposition was confirmed by scanning electron microscopy to observe structural changes and by determination of cellular attachment to investigate functional changes. PMID- 10741643 TI - Influence of the G2 cell cycle block abrogator pentoxifylline on the expression and subcellular location of cyclin B1 and p34cdc2 in HeLa cervical carcinoma cells. AB - The progression of cells from G2 into mitosis is mainly controlled by formation of the cyclin B1/p34cdc2 complex. The behaviour of this complex in the irradiation-induced G2 cell cycle delay is still unclear. A prior study demonstrated that the expression of the cyclin B1 protein is reduced by irradiation, and restored to control levels by the methylxanthine drug pentoxifylline, which is a potent G2 block abrogator. The present study shows that irradiation, and 2 mM pentoxifylline affect the expression of the cyclin dependent kinase p34cdc2 in HeLa cells. Irradiation induces p34cdc2 levels to increase and cyclin B1 levels to decrease. Addition of pentoxifylline at the G2 maximum reverses these trends. This is also evident from the cyclin B1/p34cdc2 ratios which decline after irradiation and are rapidly restored to control levels upon addition of pentoxifylline. It is concluded that cyclin BI and p34cdc2 protein expression are important events and act in concert to control the irradiation induced G2 block. Analysis of cyclin B1 expression in whole cells and in isolated nuclei furthermore show that cyclin B1 is translocated from the nucleus into the cytoplasm when the G2 block is abrogated by pentoxifylline. PMID- 10741644 TI - Nuclear chromatin texture and sensitivity to DNase I in human leukaemic CEM cells incubated with nanomolar okadaic acid. AB - It is now known that the analysis of chromatin texture can be used in oncology as a sensitive detection method, either to define diagnostic classifications or to locate a lesion along a defined trend curve. However, the functional significance of these variations in textural features remains sometimes unclear. Several drugs have been shown to be able to modulate chromatin structure. Among them, the phosphatase inhibitor okadaic acid at low concentration can increase accessibility to DNA in chromatin of carcinoma cells. This paper demonstrates that short exposures (0-3 h) to a 10-nM dose of okadaic acid induced an increased sensitivity to DNase I digestion in human CEM leukaemic cell nuclei and that this sensitization was associated to variations of nuclear texture characteristics, as evaluated by image cytometry. CEM cells treated with okadaic acid for 0-3h displayed changes in chromatin supraorganization with a more homogeneous and fine chromatin texture, as compared to control cells. This suggests that the appearance of an open configuration of chromatin structure as evaluated by biochemical methods corresponds to a more decondensed texture of nuclei measured by image cytometry. Longer exposures (6-24h) of CEM cells to 10 nM okadaic acid lead to apoptosis. As reported previously for camptothecin-treated HL60 cells, okadaic acid-treated CEM cells display biphasic nuclear chromatin texture changes, i.e. a decondensation phase followed by the appearance of typical apoptotic cells with a smaller nuclear area and a highly condensed chromatin. Finally, using the multidrug-resistant CEM-VLB cell line, it was confirmed that these multidrug-resistant cells also display cross-resistance to okadaic acid, as this compound was unable to induce either increased DNase I sensitivity, apoptosis, or altered nuclear texture in this particular cell line. PMID- 10741645 TI - A cantilever array-based artificial nose AB - We present quantitative and qualitative detection of analyte vapors using a microfabricated silicon cantilever array. To observe transduction of physical and chemical processes into nanomechanical motion of the cantilever, swelling of a polymer layer on the cantilever is monitored during exposure to the analyte. This motion is tracked by a beam-deflection technique using a time multiplexing scheme. The response pattern of eight cantilevers is analyzed via principal component analysis (PCA) and artificial neural network (ANN) techniques, which facilitates the application of the device as an artificial chemical nose. Analytes tested comprise chemical solvents, a homologous series of primary alcohols, and natural flavors. First differential measurements of surface stress change due to protein adsorption on a cantilever array are shown using a liquid cell. PMID- 10741646 TI - Environmental sensors based on micromachined cantilevers with integrated read-out AB - An AFM probe with integrated piezoresistive read-out has been developed and applied as a cantilever-based environmental sensor. The probe has a built-in reference cantilever, which makes it possible to subtract background drift directly in the measurement. Moreover, the integrated read-out facilitates measurements in liquid. The probe has been successfully implemented in gaseous as well as in liquid experiments. For example, the probe has been used as an accurate and minute thermal sensor and as a humidity sensor. In liquid, the probe has been used to detect the presence of alcohol in water. PMID- 10741647 TI - Multiple-input microcantilever sensors AB - A surface-micromachined micro-electro-mechanical-system (MEMS) process has been used to demonstrate multiple-input chemical sensing using selectively coated cantilever arrays. Cantilever motion due to absorption-induced stress was readout using a custom-designed, eight-channel integrated circuit. Combined hydrogen and mercury vapor detection was achieved with a palm-sized, self-powered module with spread-spectrum telemetry reporting. PMID- 10741648 TI - Microcantilever-based biosensors AB - The use of surface stress-based sensors as bio-chemical sensors was investigated. In principle, adsorption of biochemical species on a functionalised surface of a microfabricated cantilever will cause surface stress and consequently the cantilever bends. Two applications are presented: first lipoproteins and their oxidised form which are responsible for cholesterol accumulation in arteries were differentiated by measuring the surface stress involved in their adsorption on a sugar (heparin); secondly, the surface stress resulting from surface induced conformational changes in protein was monitored. That provided experimental evidence of long time-scale surface processes. PMID- 10741649 TI - Microfabrication of a combined AFM-SNOM sensor AB - The objective of this work is to fabricate a scanning probe sensor that combines the well-established method for atomic force microscopy, employing a micro machined Si cantilever and integrated tip, with a probe for the optical near field. A photosensitive pn-junction is integrated into the tip for that purpose and an Al coating is applied to the tip. It comprises an aperture of 50-70 nm in diameter at the apex of the tip in order to spatially limit the interaction of the tip to the optical near field of the sample. Characterization of the tip and first results of simultaneously recorded force and photon images are presented. PMID- 10741650 TI - Piezoresistive sensors for scanning probe microscopy AB - In this article we summarize the efforts devoted to the realization of our ideas of the development of piezoresistive sensor family used in scanning probe microscopy. All the sensors described here are fabricated based on advanced silicon micromachining and standard CMOS processing. The fabrication scenario presented in this article allows for the production of different sensors with the same tip deflection piezoresistive detection scheme. In this way we designed and fabricated, as a basic sensor, piezoresistive cantilever for atomic force microscopy, which enables surface topography measurements with a resolution of 0.1 nm. Next, by introducing a conductive tip isolated from the beam we obtained a microprobe for scanning capacitance microscopy and scanning tunneling microscopy. With this microprobe we measured capacitance between the microtip and the surface in the range of 10(-22) F. Furthermore, a modification of the piezoresistors placement, based on the finite element method (FEM) simulation permits fabrication of the multipurpose sensor for lateral force microscopy, which enables measurements of friction forces with a resolution of 1 nN. Finally, using the same basic device idea and only slightly modified process sequence we manufactured femtocalorimeter for the detection of heat energy in the range of 50 pJ. PMID- 10741651 TI - Detection of infrared photons using the electronic stress in metal-semiconductor cantilever interfaces AB - We report on a new method for detecting photons using the stress caused by photoelectrons emitted from a metal film surface in contact with a semiconductor microstructure which forms a Schottky barrier. The detection of photons results from measuring the photo-induced bending of the Schottky barrier microstructure due to electronic stress produced by photoelectrons diffusing into the microstructure. Internal photoemission has been used in the past to detect photons, however, in those cases the detection was accomplished by measuring the current due to photoelectrons and not due to electronic stress. In this work we studied the photon response of 500 nm thick Si microcantilevers coated with a 30 nm layer of Pt. Photons with sufficient energies produce electrons from the platinum-silicon interface which diffuse into the Si and produce an electronic stress. Since the excess charge carriers cause the Si microcantilever to contract in length but not the Pt layer, the bimaterial microcantilever bends. The charge carriers responsible for the photo-induced stress in Si, were produced via internal photoemission using a diode laser with wavelength lambda = 1550 nm. PMID- 10741652 TI - Whisker probes AB - A new technology for preparation of AFM probes based on whiskers is proposed. The technology posseses significant advantages to the whisker probes over standard ones as related to the shapes of the probes, as well as to the properties of the cantilevers on which the whiskers are grown. PMID- 10741653 TI - Phase detection of electrostatic force by AFM with a conductive tip AB - Electrostatic force measurements were performed by the Kelvin probe force microscopy, which consists of the atomic force microscopy and a conductive tip. Sample surface potential was evaluated through the electrostatic force, which works between the sample and the tip when we apply an AC bias at a frequency f(s) with a DC offset. If the DC offset voltage is equal to the surface potential difference between the sample and the tip, the amplitude of the f(s) component in the electrostatic force becomes zero, and the phase of the f(s) component jumps 180 degrees there. We found that the complementary use of the amplitude and phase signals improved the accuracy of the determination of the surface potential. We measured both the work functions of some metals and the surface Fermi levels of an InAs layers on (1 0 0) and (1 1 0) GaAs substrates based on the phase detection method of the electrostatic force. PMID- 10741654 TI - Finite element calculations and fabrication of cantilever sensors for nanoscale detection AB - Finite element analysis (FEA) is used to study the effect of geometric variations on the properties of rectangular cantilevers and U-shaped Joule-heated cantilevers. Simulations of locally thinned cantilevers as well as of cantilevers modified by the implementing of a hole or a side cut are compared with fabricated cantilevers, which are tuned by focused ion beam (FIB) milling. By locally thinning the cantilevers, the resonance frequency and the spring constant are reduced. For a hole, the internal stress is increased while for a side cut, the lateral spring constant is decreased. Good agreement between the measured and the simulated resonance frequencies is observed. Simulations of the current density and the temperature distributions attained during the passage of current through a doped silicon layer are performed to optimize the design of Joule-heated cantilevers (U-shaped) for thermal gravimetric applications. A very uniform temperature distribution over a region near the apex can be realized by slitting the U-shaped cantilever. In such a way, the heating power can be minimized by effecting only a small variation in the geometry of a U-shaped cantilever. A simple fabrication process for the fabrication of Joule-heated cantilevers is presented, which consists mainly of a uniform conductive p-doped layer. PMID- 10741655 TI - Amplitude curves and operating regimes in dynamic atomic force microscopy AB - The experimental dependence of the amplitude on the average tip-sample distance has been studied to understand the operation of an atomic force microscope with an amplitude modulation feedback. The amplitude curves can be classified in three major groups according to the existence or not of a local maximum and how the maximum is reached (steplike discontinuities vs. smooth transitions). A model describing the cantilever motion as a forced nonlinear oscillator allows to associate the features observed in the amplitude curves with the tip-sample interaction force. The model also allows to define two elemental tip-sample interaction regimes, attractive and repulsive. The presence of a local maximum in the amplitude curves is related to a transition between the attractive and the repulsive regime. PMID- 10741656 TI - Data analysis of interaction forces measured with the atomic force microscope AB - The force-distance cycle mode of the atomic force microscope (AFM) allows for detection of interaction forces between the AFM-tip and a substrate (probe). This can either be a direct tip-sample interaction or an interaction between molecules coupled to the tip and probe, respectively. The interaction forces are typically in the range of a few pN to some hundred pN. In this article we describe algorithms for the analysis of force-distance cycles, to quantify interaction forces between tip and probe. Both, the direct tip-probe interaction as well as the interaction between specifically bound molecules are analyzed. The molecules bound to tip and probe have to be either long and flexible or have to be bound via a flexible cross linker. The algorithms are exemplified on direct tip-probe interactions and on unbinding events of cadherins which are bound via PEG-spacers to the AFM-tip and to the probe. PMID- 10741657 TI - Scanning tunneling microscopy nanofabrication of electronic industry compatible thermal Si oxide AB - We report here a nanofabrication result on a 2.7 nm thermal oxide layer using the low-energy e-beam/scanning tunneling microscope (STM) technique in conjunction with thermal annealing, in which line windows with average width of 50 nm can be formed. Comparing to the low-energy e-beam processing on thin layers of native Si oxide, this nanofabrication shows a uniform etching of the electronic industry compatible Si oxide, with which nanoscale trenches can be formed. In addition to demonstrating further the nanofabrication capability of this technique on the thermal oxide, the results present critical evidence to our previous discussions on the mechanism of the low-energy e-beam/STM nanofabrication. PMID- 10741658 TI - Observations of sudden structural-changes of the faulted-halves of the DAS structure on quenched Si(1 1 1) by STM AB - We have carried out scanning tunneling microscopy (STM) observations of unreconstructed regions on quenched Si(1 1 1) surfaces at 380 degrees C at a scanning speed of 1.7 s per frame. In the regions, it is found that single faulted-halves of the dimer-adatom-stacking-fault (DAS) structure are formed isolatedly or at the edges of the surrounding DAS domains sharing one corner hole. In such "living" regions, we have succeeded to observe sudden structural changes of the faulted-halves during line scans in single frames of STM images. PMID- 10741659 TI - STM analyses of surface phenomena in Si(1 0 0) under proton irradiation AB - Detailed investigation of surface phenomena (sputtering, blistering, flaking) at silicon irradiated with 700 keV protons to fluences in the range of 10(16)-10(18) p/cm2 was carried out with the help of an open-air scanning tunneling microscope of original design. Multiple STM images of irradiated sample surfaces containing both intact and broken blisters were analyzed, and their distributions by sizes and evolution under thermal annealing estimated. PMID- 10741660 TI - Lift-off patterning of thin Au films on Si surfaces with atomic force microscopy AB - We studied a new lift-off process of thin Au film on silicon surfaces in nanometer-scale, combining anodic oxidation patterning with AFM, deposition of Au thin film on the patterned substrate and chemical etching processes of the Si oxide underneath the Au film. For Au films of thickness of 2-5 nm, the Au films on the Si oxide patterns were left unbroken and bent down to stick to Si surface after the removal of the oxide by the chemical etching. For an Au film of 1 nm in thickness, it was possible to lift-off the Au film on oxide patterns of the lines and dots in nanometer-scale using Si oxide as a sacrificial mask. PMID- 10741661 TI - Analysis of failure mechanisms in electrically stressed gold nanowires AB - An analysis of polycrystalline Au thin-film interconnects of widths ranging from 850 to 25 nm, and lengths ranging from 1 microm to 20 nm which have been electrically stressed to the point of failure is presented. A new method for testing failure of interconnects is proposed, based on a quantity we call the failure current density. The mean time to failure for fixed current density and also the failure current density are seen to decrease with decreasing wire width contrary to expectations. The failure current density for a given wire width increases as the length decreases. An analysis of the temperature and stress profiles based on calculations of a simple model is presented which shows that the length dependence is due to thermal stresses rather than electromigration, and the width dependence is due to enhanced electromigration due to surface diffusion. PMID- 10741662 TI - Manipulation of gold nanoparticles in liquid environments using scanning force microscopy AB - Precise and controlled manipulation of individual gold nanoparticles (deposited on a Si/SiO2 surface) in liquid environments using the tip of a scanning force microscope is reported for the first time. Experiments were performed in deionized water and in ethanol as a prototype for an organic solvent. Analysis of the amplitude signal of the cantilever before and during manipulation reveals that the particles are pushed across the surface, similar to the manipulation of nanoparticles in air. PMID- 10741663 TI - Investigations into local ferroelectric properties by atomic force microscopy AB - In this article, we describe nanometer scale characterization of piezoelectric thin films of Lead-Zirconate-Titanate (PZT). Using the electric field from a biased conducting atomic-force microscopy (AFM) tip, we show that it is possible to form and subsequently image ferroelectric domains. Using a sphere-plane model for the tip-sample system we calculate the distribution of electric potential, field and polarization charge, and find good agreement with the experimental values. We also discuss the effects of surface contaminants on domain formation. PMID- 10741664 TI - AFM characterization of domain structure of ferroelectric TGS crystals on a nanoscale AB - Atomic force microscopy has been used for the study of the surface topography and domain structures of triglycin sulfate (TGS) crystals. The images of various types of domains at the polar (0 1 0) surfaces of as-cleaved TGS crystals have been obtained (in contact mode and resonant mode). The crystals grown old, after annealing, gamma-irradiated by various doses, were studied. It has been found that the surface relief of crystals after annealing differs from that of crystals grown old and irradiated: in the first case the peak-to-peak value is nearly constant whereas in the second case it varies in wide limits. The parameters of domain structure were determined. These parameters have been shown to be different for crystals grown old, crystals after annealing and irradiated crystals. It has been shown that, after cantilever scanning after a long period in different AFM modes the TGS surface experiences a partial polarization reversal. PMID- 10741665 TI - Fabrication of a quantum point contact by the dynamic plowing technique and wet chemical etching AB - We have fabricated extremely confined ballistic constrictions using a nanolithography technique based on an atomic force microscope. Vector-scan controlled dynamic plowing with the vibrating tip enables to plastically indent a thin resist layer along a prearranged path. Transfer of the resist pattern into the semiconductor substrate is achieved by a strongly diluted aqueous etchant. In this way approximately 30 nm deep gooves were etched in the channel area of a modulation-doped GaAs/GaA1As field-effect transistor. The quantum point contacts were defined by a broken line whose 60 nm width represents the length and the sub 100 nm gap determines the width of the constriction. At liquid-helium temperature the conductance as a function of gate voltage shows a stepwise increase in units of 2e2/h. Signatures of the conductance quantization persist up to 50 K, which indicates a large subband spacing. PMID- 10741666 TI - Nanoscale devices fabricated by direct machining of GaAs with an atomic force microscope AB - We demonstrate a lithography wherein the tapping mode of an atomic force microscope the Si tip is used as a chiseling tool for direct machining of a GaAs surface. Single-groove drawing movements in a vector-scan mode result in approximately 3-4 nm deep and 30 nm wide furrows, which can be combined to arbitrary noncontiguous polygon patterns. Beneath such a groove a barrier arises in the electron channel of a GaAs/A1GaAs modulation-doped field effect transistor (MODFET). Using appropriate sub-100 nm line patterns we prepared quantum point contacts and single electron devices. At T = 4.2 K the transconductance characteristics of these nanoscale MODFETs exhibit structures, which represent signatures of either the quantized conductance or Coulomb-blockade effects. PMID- 10741667 TI - Formation of nano-pyramids of layered materials with AFM AB - We have been able to raise squares of around 2 A in height of the layered materials Bi1.6Sb0.4Se3, Bi1.9Sb0.1Se3 and Bi2Se3 by means of contact atomic force microscopy in air. By raising squares on/beneath previously produced squares, Mayan-like pyramids have been constructed, each step of the pyramid being around 2 A in height. We neither remove matter from the surface nor produce a localized oxidation of the sample; we elevate a portion of it. When the surface is oxidized, the AFM cantilever pulls the oxide off producing holes. The raising of the squares is also possible in a nitrogen atmosphere. PMID- 10741668 TI - Comprehensive surface analysis of hydrophobically functionalized SFM tips AB - Tip-sample interactions have been of interest since the early development of the scanning force microscope. Investigations of interfacial interactions at the molecular level are of importance for fundamental studies of bi-molecular interactions and for possible applications in biomedical research and industrial settings. By engineering the surface chemical properties of the SFM probes, specific force interactions may be measured. However, as these modification schemes become more widely applied, detailed chemical analysis of the modified cantilever surfaces becomes crucial. In this paper, we describe two approaches to coat SFM cantilevers with hydrophobic coatings: a silanization protocol and ratio frequency plasma enhanced chemical vapor deposition. PMID- 10741669 TI - A novel cleaning method of gold-coated atomic force microscope tips for their chemical modification AB - For chemical modification of gold-coated AFM tips with thiol or sulfide compounds, a new two-step precleaning procedure was studied. The two-step cleaning procedure involves (i) oxidation of organic contaminants on the AFM tips with ozone treatment and (ii) reduction of the oxidized gold surface by immersing the oxidized tip into pure hot ethanol at ca. 65 degrees C. The chemically modified tips prepared from gold-coated AFM tips precleaned by the two-step procedure gave almost the same tip characteristics as those chemically modified immediately after gold vapor deposition in a factory. The present two-step cleaning procedure can be used widely for chemical modification of commercially available gold-coated AFM tips with thiol or disulfide compounds for chemical force microscopy. PMID- 10741671 TI - Chemical force microscopy of microcontact-printed self-assembled monolayers by pulsed-force-mode atomic force microscopy AB - A novel chemically sensitive imaging mode based on adhesive force detection by previously developed pulsed-force-mode atomic force microscopy (PFM-AFM) is presented. PFM-AFM enables simultaneous imaging of surface topography and adhesive force between tip and sample surfaces. Since the adhesive forces are directly related to interaction between chemical functional groups on tip and sample surfaces, we combined the adhesive force mapping by PFM-AFM with chemically modified tips to accomplish imaging of a sample surface with chemical sensitivity. The adhesive force mapping by PFM-AFM both in air and pure water with CH3- and COOH-modified tips clearly discriminated the chemical functional groups on the patterned self-assembled monolayers (SAMs) consisting of COOH- and CH3-terminated regions prepared by microcontact printing (microCP). These results indicate that the adhesive force mapping by PFM-AFM can be used to image distribution of different chemical functional groups on a sample surface. The discrimination mechanism based upon adhesive forces measured by PFM-AFM was compared with that based upon friction forces measured by friction force microscopy. The former is related to observed difference in interactions between tip and sample surfaces when the different interfaces are detached, while the latter depends on difference in periodic corrugated interfacial potentials due to Pauli repulsive forces between the outermost functional groups facing each other and also difference in shear moduli of elasticities between different SAMs. PMID- 10741670 TI - Quantitative interrogation of micropatterned biomolecules by surface force microscopy. AB - Synthetic biomaterials are widely used in medical implants with success in improving and extending quality of life. However, these materials were not originally designed to interact with cells through specific signaling pathways. As a result, the interaction with the body is mediated through passive adsorption of a disorganized protein monolayer. Next generation biomaterials have been proposed to be active in modifying the biological response of the host through the incorporation of specific biorecognition moieties. An important tool in the development of these novel active biomaterials is the scanning force microscope (SFM). The SFM allows for interrogation of bioactive biomaterials in mapping or spectroscopic modes. In this work, micropatterned protein surfaces were prepared using biomolecules implicated in wound healing. The surfaces were imaged via SFM and the specific binding forces between surface associated biomolecules and antibody functionalized tips were quantified. PMID- 10741672 TI - The mechanism of cellulase action on cotton fibers: evidence from atomic force microscopy. AB - Two cellulases from Trichoderma reesei--an exoglucanase, CBH I, and an endoglucanase, EG II--alone and in combination were incubated with cotton fibers. The effects of the cellulases on the surfaces of the cotton fibers were examined by atomic force microscopy. At high magnification, the physical effects on the fibers caused by the two types of enzymes were considerably different. Treatment with CBH I resulted in the appearance of distinct pathways or tracks along the length of the macrofibril. Treatment with EG II appeared to cause peeling and smoothing of the fiber surface. In combination, their effect was observed to be greatest when both enzymes were present simultaneously. When fibers smoothed by treatment with EG II were treated subsequently with CBH I, further evidence of path way formation caused by the action of CBH I along the fibers was observed. Incubation with a cellulase from Thermotoga maritima that lacks a cellulose binding domain had no effect on the surface of cotton fibers. These images provide the first physical evidence of differences in the effect of cellulase components action on the surface of cotton fibers and provide evidence for the movement or tracking of CBH I along the fibers. The first AFM image of CBH I molecules are presented. PMID- 10741673 TI - Comparing microscopic with macroscopic elastic properties of polymer gel AB - Measurements of the local elastic modulus of agar gels obtained with atomic force microscope (AFM) force mapping were compared with values obtained by the tensile creep method. The observed spatial distributions of the local elastic modulus over the gel surface in AFM elastic images clearly corresponded to the network structure of agar fibers observed both in AFM topographic and scanning electron microscope (SEM) images. Both peak and average values of distribution functions in the histograms of local elastic modulus increase monotonically with the agar concentration. Values obtained by AFM force mapping were found to be proportional to values obtained from creep experiments. PMID- 10741674 TI - Optimal sensitivity for molecular recognition MAC-mode AFM AB - Molecular recognition force microscopy (MRFM) using the magnetic AC mode (MAC mode) atomic force microscope (AFM) was recently investigated to locate and probe recognition sites. A flexible crosslinker carrying a ligand is bound to the tip for the molecular recognition of receptors on the surface of a sample. In this report, the driving frequency is calculated which optimizes the sensitivity (S). The sensitivity of MRFM is defined as the relative change of the magnetically excited cantilever deflection amplitude arising from a crosslinker/antibody/antigen connection that is characterized by a very small force constant. The sensitivity is calculated in a damped oscillator model with a certain value of quality factor Q, which, together with load, defines the frequency response (unloaded oscillator shows resonance at Q > 0.707). If Q < 1, the greatest value of S corresponds to zero driving frequency omega (measured in units of eigenfrequency). Therefore, for Q < 1, MAC-mode has no advantage in comparison with DC-mode. Two additional extremes are found at omegaL = (1 - 1/Q)(1/2) and omegaR = (1 + 1/Q)(1/2), with corresponding sensitivities S(L) = Q2/(2Q - 1), S(R) = Q2/(2Q + 1). The L-extreme exists only for Q > 1, and then S(L) > S(R), i.e. the L-extreme is the main one. For Q > 1, S(L) > 1, and for Q > 2.41, S(R) > 1. These are the critical Q-values, above which selecting driving frequency equal to sigmaL or sigmaR brings advantage to MAC mode vs. DC mode. Satisfactory quality of the oscillator model is demonstrated by comparison of some results with those calculated within the classical description of cantilevers. PMID- 10741675 TI - Identifying sequence similarities between DNA molecules. AB - An atomic force microscope (AFM) imaging technique is described to compare sequences between two different DNA molecules and precisely locate nonhomologies in DNA strands. Sequence comparisons are made by forming heteroduplexes between the two molecules and, by AFM imaging the intact molecules formed, identifying both homologous and nonhomologous regions. By forming heteroduplexes between linearized wildtype pSV-beta-galactosidase plasmid (6821 bp) and a series of deletion mutants we have identified nonhomologies (deletions) as small as 22 bp and as large as 418 bp. Furthermore, by incorporating our technique for AFM mediated restriction mapping of DNA these mutations can be positioned relative to EcoRI restriction sites. These results suggest AFM can be useful in identifying molecular level similarities and differences in DNA. PMID- 10741676 TI - Human chromosome structure studied by scanning force microscopy after an enzymatic digestion of the covering cell material. AB - In standard preparations, metaphase human chromosomes are covered by a cell material film composed mainly of proteins and RNA. This film (approximately 30 nm thickness) hides the chromosome structure to the tip of a scanning force microscope. In this work, a mild enzymatic treatment is applied to remove the cell material film. After treatment, the individual chromatin fibers at the surface were resolved. Furthermore, the chromosome shows a thickness modulation, in which thicker/thinner regions could be associated with G/R bands. Finally, the topography of the chromosomes in solution is presented. The chromosome volume swelled about five-fold and chromatin packaging in bands and coils was observed. PMID- 10741677 TI - Elasticity mapping of living fibroblasts by AFM and immunofluorescence observation of the cytoskeleton. AB - Using the force mapping mode of atomic force microscopy (AFM), we measured spatial distribution of elastic moduli of living mouse fibroblasts (NIH3T3) in a physiological condition. The nuclear portion of the cellular surface is about 10 times softer than the surroundings. Stiffer fibers are confirmed in the elastic images. In order to investigate origin of the softer nuclear portion and the stiffer fibers, we fixed the identical cells imaged by the AFM, and carried out immunofluorescence observation for three types of cytoskeletal filaments--actin filaments, microtubules, and intermediate filaments, using confocal laser scanning microscopy (CLSM). A comparison between the AFM and the CLSM images revealed that the elasticity of the cells was concerned not only with the distribution of actin network, but also with intermediate filaments, whereas microtubules had no large effect on the measured elasticity. PMID- 10741678 TI - Effect of streptolysin O on the microelasticity of human platelets analyzed by atomic force microscopy. AB - Atomic force microscopy (AFM) has been shown to be a suitable tool to probe biophysical properties of cells and cell fragments. We analysed biophysical alterations of human platelets by AFM using streptolysin O (SLO) as a model for pore forming proteins. Permeabilization of platelet membrane by SLO was confirmed by transmission electron and confocal microscopy. Using force volume imaging combined with FIEL analysis we were able to show dynamically the increase in the elasticity of platelets during the pore formation by SLO and could correlate the viscoelasticity to the morphology of platelets. Stabilizing the actin cytoskeleton by phalloidin resulted in partial restoration of the elasticity indicating that loss of stability in platelets by SLO is mediated by alterations of both plasma membrane and cytoskeleton. PMID- 10741679 TI - Mechanical stimulation of individual stereocilia of living cochlear hair cells by atomic force microscopy. AB - This paper describes the investigation of elastical properties and imaging of living cochlear hair bundles of inner (IHC) and outer hair cells (OHC) on the level of individual stereocilia. A custom-made AFM-setup was used, allowing to scan the mechano-sensitive structures of the inner ear under direct control of an upright differential interference contrast (DIC) microscope with a water immersion objective. Scanning electron microscopy (SEM) images of the identical hair bundles obtained after AFM investigation demonstrated that forces up to 1.5 nanonewton (nN) did not cause obvious damage of the surface morphology of the stereocilia. These are the first images of hair bundles of living sensory cells of the organ of Corti by AFM. They display the tips of individual stereocilia and the typical V-shape of ciliary bundles. Since line scans clearly show that slope and force interaction depend on the elastical properties of stereocilia, quantitative stiffness measurements and stimulation of single transduction channels are suggested. PMID- 10741680 TI - Tapping-mode atomic force microscopy on intact cells: optimal adjustment of tapping conditions by using the deflection signal. AB - Difficulties in the proper adjustment of the scanning parameters are often encountered when using tapping-mode atomic force microscopy (TMAFM) for imaging thick and soft material, and particularly living cells, in aqueous buffer. A simple procedure that drastically enhances the successful imaging of the surface of intact cells by TMAFM is described. It is based on the observation, in liquid, of a deflection signal, concomitant with the damping of the amplitude that can be followed by amplitude-distance curves. For intact cells, the evolution of the deflection signal, steeper than the amplitude damping allows a precise adjustment of the feedback value. Besides its use in finding the appropriate tapping conditions, the deflection signal provides images of living cells that essentially reveal the organization of the membrane cytoskeleton. This allows to show that changes in the membrane surface topography are associated with a reorganization of the membrane skeleton. Studies on the relationships between the cell surface topography and membrane skeleton organization in living cells open a new field of applications for the atomic force microscope. PMID- 10741681 TI - Adapting atomic force microscopy for cell biology. AB - We present details of our AFM modifications to produce an adaptable imaging system for the cell biologist. We have designed and validated a new inverted microscope interface and a scan head with increased Z-range, based upon the TopoMetrix Explorer AFM. We have utilised these changes, together with home-made glass ball cantilevers, to obtain topographical information over cells with large Z-dimension (over 15 microm high), and mapped calcitonin-calcitonin receptor binding forces in living bone cells. We conclude that modified AFM can be used to evaluate intermolecular events in living cells and that this approach will ensure general application to the study of receptor-ligand interactions under truly physiological conditions. PMID- 10741682 TI - Atomic force microscopy imaging of living cells: a preliminary study of the disruptive effect of the cantilever tip on cell morphology. AB - Recent studies have demonstrated that atomic force microscopy (AFM) is a potential tool for studying important dynamic cellular processes in real time. However, the interactions between the cantilever tip and the cell surface are not well understood, and the disruptive effect of the cantilever tip on cell morphology has not been well characterized. In this study, the disruptive effect of the scanning cantilever tip on cell morphology, in the AFM contact mode, has been investigated. The aims of this study are to identify what kinds of cell morphological changes generally occurred under normal AFM imaging conditions and to find out how long cells remain viable during scanning. Two cell lines, SK-N-SH (human neuroblastoma cells) and AV12 (Syrian hamster cells) were studied in the experiment because these are widely used in biomedical research as an expression system for studying cellular functions of neuronal receptors. The experimental results suggest that the sensitivity of cells to the cantilever disruptive effect is dependent on cell type and that there are patterns observed in the changes of cell morphology induced by the cantilever force in these two cell lines. PMID- 10741683 TI - An adipocyte-derived plasma protein, adiponectin, adheres to injured vascular walls. AB - Adipose tissue secretes a variety of proteins into the bloodstream. We have previously reported a novel cDNA, apM1 (adipose most abundant gene transcript 1), which is specifically and abundantly expressed in adipose tissue [1]. Primary structure analysis predicted that the apM1 gene product possesses significant homology to collagens VIII, X and complement factor C1q, and we named it adiponectin. In the current study, we analyzed characteristics of adiponectin in vitro and in vivo. Adiponectin protein was proved to be secreted into the medium when the cDNA was transfected to COS cells. Anti-adiponectin cross-reactivities were abundantly detected in the human plasma. In solid-phase binding assays, adiponectin specifically bound to collagen types I, III and V, which are present in vascular intima. Immunohistochemical analysis revealed that adiponectin was detected in the walls of the catheter-injured vessels but not in the intact vascular walls. These data suggest that adiponectin is a plasma protein produced by adipose tissue and accumulates in vascular walls when the endothelial barrier is injured. PMID- 10741684 TI - Endomorphin-1, an endogenous mu-opioid receptor-selective agonist, stimulates oxygen consumption in mice. AB - This study was undertaken to investigate the effect of endogenous mu-receptor selective peptide endomorphin 1, administered intracerebroventricularly, on oxygen consumption in mice. The intracerebroventricular injection of endomorphin 1 (3-30 nmol) significantly increased oxygen consumption in unrestrained mice. The effect of endomorphin 1 (30 nmol) was significantly antagonized by the simultaneous intraperitoneal administration of naloxone (100 nmol). These results suggest that endomorphin 1 stimulates oxygen consumption, and that the mu-opioid receptor influences energy balance in mice. PMID- 10741685 TI - Glucose-induced insulin mRNA accumulation is impaired in islets from neonatal streptozotocin-treated rats. AB - According to the "glucose toxicity" hypothesis, hyperglycemia contributes to defective beta-cell function in type 2, non-insulin-dependent diabetes mellitus. This concept is supported by substantial data in rodent models of diabetes. However, the ability of glucose to stimulate the accumulation of insulin mRNA, a critical feature of normal beta-cell physiology, has not been investigated in in vivo models with chronic hyperglycemia. The aim of this study was to determine whether glucose-induced insulin mRNA accumulation is impaired in the neonatal streptozotocin-treated rat (n0-STZ rat), a model of non-obese, non-insulin dependent diabetes mellitus. Islets of Langerhans isolated from n0-STZ and control rats were cultured for 24 h in the presence of 2.8 or 16.7 mmol/l glucose, and insulin mRNA levels were measured by Northern analysis. Insulin mRNA levels were increased more than twofold by glucose in control islets. In contrast, no significant effect of glucose was found on insulin mRNA levels in n0 STZ islets. We conclude that insulin gene regulation by glucose is impaired in n0 STZ rat islets. PMID- 10741686 TI - Influence of insulin status on extra-mitochondrial oxygen metabolism in the rat. AB - The effects of alloxan diabetes and subsequent treatment with insulin on extra mitochondrial oxygen metabolism in terms of D-amino acid oxidase (DAAO), xanthine oxidase and catalase were examined. The DAAO activity in the liver with D-alanine and D-serine decreased by 33-62% in the diabetic group while the decrease in the kidneys was 61-74%. Insulin treatment resulted in overstimulation of DAAO activity in the liver but not in the kidneys. Tissue glycogen content was lowered in the diabetic animals but was restored by insulin treatment. Tissue glycogen content and DAAO activity showed an inverse relationship. The xanthine oxidase activity in the two tissues decreased from 40-55%; the catalase activity decreased from 34-54%. Insulin treatment was unable to restore the xanthine oxidase and catalase activities in both the tissues. PMID- 10741687 TI - Amylin and insulin interact to reduce food intake in rats. AB - We investigated the hypothesis that amylin and insulin, hormones co-secreted by pancreatic B-cells in response to a nutrient stimulus, interact to reduce food intake. A paradigm was employed that assessed food intake in adult male rats after bolus intravenous (i.v.) infusion at dark onset. In one experiment, rats received saline or amylin (0.1, 0.5 or 1.0 nmol). All amylin doses significantly suppressed 1 h intake, and although significant decreases in cumulative intake persisted for 2 h after 0.5 and 1.0 nmol, a significant increase of food intake actually occurred relative to saline during the interval from 1 to 2 h post infusion. In another experiment, rats received saline, 0.25 nmol amylin, 10 mU insulin, or the combination of amylin plus insulin. Neither amylin nor insulin alone significantly changed cumulative food intake at any time point as compared to saline. However, the combination significantly reduced intake relative not only to saline but also to amylin and insulin alone after 1, 2, and 4 hours. These data are consistent with the hypothesis that endogenous amylin and insulin interact to reduce food intake and, ultimately, body weight. PMID- 10741688 TI - Effects of long-term treatment with GH in the bone mineral density of adults with hypopituitarism and GH deficiency and after discontinuation of GH replacement. AB - BACKGROUND: Only few previous studies have assessed the effects of long-term growth hormone (GH) replacement therapy on bone mineral density (BMD) in adult patients with GH deficiency. The aim of this study was to investigate the effects of long-term GH therapy on bone metabolism and BMD. MATERIAL AND METHODS: At the start of the study, 20 adults with GH deficiency were randomized to receive either GH, 0.25 IU x kg per week, or placebo. After 6 months, patients in the placebo group were switched to GH therapy, and they received GH for a further 18 months. Of the 20 patients, 14 were male and 6 female with GH deficiency of adult onset. The mean age of the patients at the start of the study was 40.3+/-10.9 years and the duration of GH deficiency was 10.6+/-6.4 years. Patients deficient in pituitary hormones other than GH had been receiving stable replacement doses of appropriate hormones for at least 6 months before the start of the study. Rates of bone metabolism were assessed by measuring calcium, phosphate, alkaline phosphatase, calciuria, phosphaturia and osteocalcin. BMD was measured by dual X ray absorptiometry. Body composition was calculated from measurements of bioelectrical impedance. RESULTS: Before GH treatment, BMD in the femoral neck was lower in patients than in controls. The rate of bone resorption markers increased significantly after 6 months and remained stable during the whole treatment period. BMD significantly increased in L2-L4 after 12 months of treatment with an increase of Z-score. The total BMD increase was 4.5+/-6.5%. BMD in the femoral neck increased after 12 months with an increase of Z-score after 18 months. The total increase was 10.4+/-18%. The total BMD increase was not different among patients with or without basal osteopenia. In both groups BMD in L2-L4 and in the femoral neck remained stable after 12 months without GH treatment. Sex, age, BMI and the time in which GH deficiency started, before or after the end of the peak of BMD, did not correlate with BMD. The BMD values and their response to GH treatment did not correlate with other associated deficiencies, and we did not find differences among BMD increase and GH dose, levels of insulin-growth factor-I, insulin growth factor binding protein-3, and parameters of body composition. CONCLUSIONS: The results of the study support previous ones that BMD is subnormal in adults with GH deficiency; that GH replacement therapy stimulates bone turnover with initial biochemical changes; and that in the long term, this stimulation results in a significant augmentation in BMD that continues to increase after 2 years and remains stable after 12 months of GH withdrawal. PMID- 10741690 TI - Sequence analysis of the MEN I gene in two patients with multiple cutaneous lipomas and endocrine tumors. AB - The discovery of mutations of the menin gene in a few multiple endocrine neoplasma type 1 (MEN I)-associated lipomas and loss of heterozygosity (LOH) on chromosome 11q13 in some sporadic lipomas has stimulated the hypothesis that lipomas may belong to the group of sporadic tumors caused by defects of the gene responsible for MEN I. Since it is unclear if the above hypothesis applies to all patients with lipoma or just to specific subsets, we searched to enlarge the database on this topic. For this purpose, we identified two patients with multiple cutaneous lipomas. One had an additional pituitary adenoma and familial presentation of multiple lipomas, the other had recurrent goiter in the setting of a family history of adenomatous goiter. Deoxyribonucleic acid (DNA) was analyzed by complete direct DNA sequencing of all coding exons and splice junctions of the MEN I gene. No mutation was identified in the coding exons of the menin gene. In contrast to former data on sporadic lipomas, these data are the first to render evidence that mutations of the MEN I gene may not be responsible for the formation of multiple lipomas, even if they appear in the context of other endocrine tumors. PMID- 10741689 TI - Depot-specific effects of early growth retardation on adipocyte insulin action. AB - Male offspring of rats protein restricted during pregnancy and lactation are growth restricted and have changes in insulin action on epididymal adipocytes. Adipocytes from different anatomical depots are thought to have distinct metabolic functions. The aim of the present study was to determine whether the alterations in metabolism of adipocytes from early growth restricted rats is depot-specific or more generalised. Epididymal, intra-abdominal and subcutaneous adipocytes were studied from three-month-old male offspring of control and protein restricted dams. Basal glucose uptakes were higher in low protein adipocytes (p<0.01) compared to controls. However, insulin stimulation was less in the low protein group in all depots compared to controls (p<0.05). Isoproterenol-stimulated lipolysis was greater in low protein adipocytes (p<0.0001), but the magnitude was greater in epididymal (p<0.0001) and intra abdominal (p<0.0001) adipocytes than in subcutaneous adipocytes. Epididymal and intra-abdominal adipocytes from low protein offspring were also resistant to the anti-lipolytic action of insulin. These results suggest that certain changes associated with early growth retardation are depot-specific, being enhanced in the more metabolically active intra-abdominal and epididymal tissues. PMID- 10741691 TI - Lack of association between an ecNOS gene polymorphism and diabetic nephropathy in type 2 diabetic patients with proliferative diabetic retinopathy. AB - The development of diabetic nephropathy shows remarkable variation among individuals. Therefore, not only hyperglycemia but also genetic factors may contribute to the development of diabetic nephropathy. The aim of the present study was to examine the contribution of the 27-bp repeat polymorphism in intron 4 of the endothelial constitutive nitric oxide synthase gene (ecNOS4) to the development of diabetic nephropathy. For this purpose, we analyzed this polymorphism in 167 Japanese type 2 diabetic patients with proliferative diabetic retinopathy consisting of 102 patients with diabetic nephropathy (with macroalbuminuria) and 65 patients without diabetic nephropathy (with normoalbuminuria). The genotype and allele frequencies were not significantly different between patients with diabetic nephropathy and those without diabetic nephropathy (ecNOS4 "b/b" 79.4% vs. 84.6%, ecNOS4 "b/a" 20.6% vs. 15.4%, "b" allele 89.7% vs. 92.3%, "a" allele 10.3% vs. 7.7%). We conclude that the ecNOS4 polymorphism does not contribute to the development of diabetic nephropathy. PMID- 10741692 TI - Melatonin inhibits NADPH-dependent lipid peroxidation in human placental mitochondria. PMID- 10741693 TI - Blockade of receptors for growth factors: an anticancer therapy--the fourth annual Joseph H Burchenal American Association of Cancer Research Clinical Research Award Lecture. PMID- 10741694 TI - The RB2/p130 gene: the latest weapon in the war against lung cancer? AB - Lung cancer is the second cause of death after cardiovascular diseases and is the major cause of cancer deaths in the Western world. Large scale screening trials conducted 15-20 years ago using chest X-rays and sputum cytology were able to detect stage I cancers but failed to impact on survival. This is because of the early metastatic potential of small primary tumors. It is important then to detect lung cancer at an earlier stage, studying and identifying genetic lesions that could indicate a new target(s) for gene therapy. The retinoblastoma-related gene pRb2/p130, a new tumor suppressor gene cloned in 1993, is emerging as one of the candidate markers and targets for gene therapeutic approach. Effective genetic therapy requires both a genetic material to be used therapeutically and a means to deliver it. A scope for this review is to examine some of the gene delivery systems mostly used, discussing their weaknesses and strengths, and to discuss the role of pRb2/p130 in lung cancer. PMID- 10741695 TI - Antibody-directed enzyme prodrug therapy: efficacy and mechanism of action in colorectal carcinoma. AB - In antibody-directed enzyme prodrug therapy, an enzyme conjugated to an antitumor antibody is given i.v. and localizes in the tumor. A prodrug is then given, which is converted to a cytotoxic drug selectively in the tumor. Ten patients with colorectal carcinoma expressing carcinoembryonic antigen received antibody directed enzyme prodrug therapy with A5B7 F(ab')2 antibody to carcinoembryonic antigen conjugated to carboxypeptidase G2 (CPG2). A galactosylated antibody directed against the active site of CPG2 (SB43-gal) was given to clear and inactivate circulating enzyme. A benzoic acid mustard-glutamate prodrug was given when plasma enzyme levels had fallen to a predetermined safe level, and this was converted by CPG2 in the tumor into a cytotoxic form. Enzyme levels derived from quantitative gamma camera imaging and from direct measurements in plasma and tumor biopsies showed that the median tumor:plasma ratio of enzyme exceeded 10000:1 at the time of prodrug administration. Enzyme concentrations in the tumor (median, 0.47 units g(-1)) were sufficient to generate cytotoxic levels of active drug. The concentration of prodrug needed for optimal conversion (Km) of 3 microM was achieved. Prodrug conversion to drug was shown by finding detectable levels of drug in plasma. There was evidence of tumor response; one patient had a partial response, and six patients had stable disease for a median of 4 months after previous tumor progression (one of these six had a tumor marker response). Manageable neutropenia and thrombocytopenia occurred. Conditions for effective antitumor therapy were met, and there was evidence of tumor response in colorectal cancer. PMID- 10741696 TI - Expression of ERCC1 antisense RNA abrogates gemicitabine-mediated cytotoxic synergism with cisplatin in human colon tumor cells defective in mismatch repair but proficient in nucleotide excision repair. AB - Gemcitabine, or 2',2'-difluorodeoxycytidine (dFdC) is a new anticancer agent with significant activity against a broad spectrum of tumors either as a single agent or in combination with other active anticancer drugs. Studies in vitro and in vivo have demonstrated that dFdC produces cytotoxic synergism with cisplatin, or cis-diamminedi-choloroplatinum(II) (CDDP); however, the mechanism by which the synergism occurs has not been elucidated. We proposed that the nucleotide excision repair (NER) process, which is responsible for the cellular removal of CDDP-DNA adducts, may be a target for the mechanism of the cytotoxic synergism of dFdC and CDDP. Because the mismatch repair (MMR) pathway is involved in mediating CDDP cytotoxicity, making determination of the role of the NER in the cytotoxic synergism more complicated, and because tumors are often defective in MMR, we selected an NER-proficient, MMR-deficient, CP2.0 human colon carcinoma cell line as a model for this study. By an in vitro repair synthesis assay, we found that dFdC triphosphate (dFdCTP), the active metabolite of dFdC, inhibited the incorporation of [alpha-32P]dATP as well as the incorporation of [alpha-32P]dCTP, suggesting that the repair inhibition by dFdCTP does not result simply from competition for the incorporation site but rather is also due to prevention of chain elongation during the DNA resynthesis process. To determine whether the repair inhibition contributes to the cytotoxic synergism, we examined the effect of the constitutive expression of ERCC1 antisense RNA on the interaction of dFdC and CDDP. CP2.0 cells were transfected with pERCC1/AS, an ERCC1 antisense expression vector; eight hygromycine-resistant clones expressing various levels of the antisense RNA were selected for quantification of and correlation between the repair activity and cytotoxic synergism. The results show that stable expression of ERCC1 antisense RNA down-regulated the level of mRNA and repair activity; the down-regulation of the repair activity significantly correlated with the reduction of the cytotoxic synergism of the two agents. These data provide direct evidence to support the hypothesis that inhibition of the repair of CDDP-induced DNA lesions plays a critical role in dFdC-mediated cytotoxic synergism with CDDP in MMR-deficient tumor cells. PMID- 10741697 TI - The pathology of familial breast cancer: histological features of cancers in families not attributable to mutations in BRCA1 or BRCA2. AB - Breast cancers arising in carriers of mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, differ histologically from each other and from breast cancers unselected for a family history. However, a substantial proportion of families with multiple cases of breast cancer is not attributable to these two genes (non-BRCA1/2 families). We have now characterized the pathology of 82 breast cancers from non-BRCA1/2 families. Breast cancers in non BRCA1/2 families were of lower grade (P = 0.0018), showed fewer mitoses (P < 0.0001), less nuclear pleomorphism (P = 0.0014), less lymphocytic infiltrate (P < 0.0001), a lesser extent of the tumor with a continuous pushing margin (P = 0.004), a lesser extent of the tumor composed of solid sheets of cells (P = 0.0047), less necrosis (P = 0.002), and wereparison with BRCA2 tumors, non BRCA1/2 tumors were lower grade (P = 0.017) and exhibited less pleomorphism (P = 0.01) and more tubule formation (P = 0.05). In comparison with control breast cancers unselected for a family history of the disease, non-BRCA1/2 tumors were of significantly lower grade (P = 0.001), showed less pleomorphism (P = 0.0002), and had a lower mitotic count (P = 0.003). The results indicate that non-BRCA1/2 breast cancers differ histologically from both BRCA1 and BRCA2 breast cancers and are overall of lower grade. They also suggest that non-BRCA1/2 breast cancers differ from nonfamilial breast cancers, but these differences may be attributable to various types of bias. PMID- 10741698 TI - Differential expression of E-cadherin and type IV collagenase genes predicts outcome in patients with stage I non-small cell lung carcinoma. AB - Because routine histopathological examination of primary non-small cell lung cancer does not predict disease outcome, we correlated disease outcome with the expression level of multiple genes that regulate distinct steps of the metastatic process in 60 formalin-fixed, paraffin-embedded, archival specimens of stage I lung carcinoma from patients undergoing curative surgery at the M. D. Anderson Cancer Center. The expression of E-cadherin (related to cell cohesion), type IV collagenase [matrix metalloproteinase (MMP)-2 and MMP-9, related to invasion], and three angiogenic molecules, basic fibroblast growth factor, vascular endothelial growth factor/vascular permeability factor, and interleukin 8, were examined by a colorimetric in situ mRNA hybridization technique. The expression levels of the individual genes analyzed by a Cox univariate analysis were not prognostic. In contrast, the ratio between expression of type IV collagenases (mean of the expression of MMP-2 and MMP-9) and E-cadherin, the MMP:E-cadherin ratio (measured at the periphery of each tumor), was significantly higher in patients with recurrent disease than in patients who remained disease free (P = 0.00003). Longer overall survival and reduced disease recurrence rates were significantly associated with a lower MMP:E-cadherin ratio (<2) by a Kaplan-Meier survival analysis (P = 0.0002 and P = 0.0001, respectively). Multiple covariate analyses of overall and disease-free survival also concluded that the MMP:E cadherin ratio was a significant prognostic factor when corrected for age (P = 0.0001). Determination of this gene expression ratio in individual human lung cancers might therefore be used to direct tailored treatment for individual patients with resectable lung cancer. PMID- 10741699 TI - A phase I study of Onyx-015, an E1B attenuated adenovirus, administered intratumorally to patients with recurrent head and neck cancer. AB - An E1B 55 kDa gene-deleted adenovirus, Onyx-015, which reportedly selectively replicates in and lyses p53-deficient cells, was administered by a single intratumoral injection to a total of 22 patients with recurrent head and neck cancer. The objectives of this Phase I study were to determine the safety, feasibility, and efficacy of this therapy and determine any correlation to p53 status. Six cohorts were investigated with a dose escalation from 10(7)-10(11) plaque-forming units. Toxicity was assessed using NCIC criteria. Tumor response was assessed by clinical and radiological measurement. Blood samples were taken to detect adenovirus DNA and neutralizing antibody to adenovirus. Tumor biopsies were taken to detect adenovirus by in situ hybridization. Treatment was well tolerated, with the main toxicity being grade 1/2 flu-like symptoms. Dose limiting toxicity was not reached at the highest dose of 10(11) plaque-forming units. Twenty-one of the 22 patients treated showed an increase in neutralizing antibody to adenovirus. In situ hybridization showed viral replication in 4 of 22 patients treated, all of whom had mutant p53 tumors. Using conventional response criteria, no objective responses were observed. However, magnetic resonance imaging scans were suggestive of tumor necrosis at the site of viral injection in five patients, three of whom were classified using nonconventional criteria as partial responders, and two of whom were classified using nonconventional criteria as minor responders. Of these five cases, four had mutant p53 tumors. The response duration for the three partial responders was 4, 8, and 12 weeks. An additional eight patients had stable disease in the injected tumors lasting from 4-8 weeks. These preliminary results show that intratumoral administration of Onyx-015 is feasible, well tolerated, and associated with biological activity. Further investigation of Onyx-015, particularly with a more frequent injection protocol and in combination with systemic chemotherapy, is warranted. PMID- 10741701 TI - Pharmacokinetics of irinotecan and its metabolites SN-38 and APC in children with recurrent solid tumors after protracted low-dose irinotecan. AB - Irinotecan (IRN), a topoisomerase I interactive agent, has significant antitumor activity in early Phase I studies in children with recurrent solid tumors. However, the disposition of IRN and its metabolites, SN-38 and APC, in children has not been reported. Children with solid tumors refractory to conventional therapy received IRN by a 1-h i.v. infusion at either 20, 24, or 29 mg/m2 daily for 5 consecutive days for 2 weeks. Serial blood samples were collected after doses 1 and 10 of the first course. IRN, SN-38, and APC lactone concentrations were determined by an isocratic high-performance liquid chromatography assay. A linear four-compartment model was fit simultaneously to the IRN, SN-38, and APC plasma concentration versus time data. Systemic clearance rate for IRN was 58.7 +/- 18.8 liters/h/m2 (mean +/- SD). The mean +/- SD ng/ml x h single-day lactone SN-38 area under the concentration-time curve (AUC(0-->6) was 90.9 +/- 96.4, 103.7 +/- 62.4, and 95.3 +/- 63.9 at IRN doses of 20, 24, and 29 mg/m2, respectively. The relative extent of IRN conversion to SN-38 and metabolism to APC measured after dose 1 were 0.49 +/- 0.33 and 0.29 +/- 0.17 (mean +/- SD). No statistically significant intrapatient difference was noted for SN-38 area under the concentration-time curve. Large interpatient variability in IRN and metabolite disposition was observed. The relative extent of conversion and the SN 38 systemic exposure achieved with this protracted schedule of administration were much greater than reported in adults or children receiving larger intermittent doses. PMID- 10741700 TI - Suppression of telomerase activity and cytokine messenger RNA levels in acute myelogenous leukemia cells in vivo in patients by amifostine and interleukin 4. AB - High levels of telomerase activity and high rates of cell proliferation are associated with a poor prognosis in acute myelogenous leukemia. Furthermore, cytokine production by leukemia cells is believed to play an important role in determining the proliferative characteristics of leukemia. The in vivo effects of two noncytotoxic agents on these parameters were determined in 33 acute myelogenous leukemia patients. Three daily doses of interleukin (IL) 4 or a single dose of amifostine reduced telomerase activity in the leukemia marrow cells in 7 of 9 and 11 of 13 patients, respectively. The administration of a single dose of amifostine resulted in a reduction in tumor necrosis factor alpha and IL-6 transcript levels in the marrow cells of 10 of 13 and 12 of 13 patients in which these transcripts were present. The administration of only three doses of IL-4 or a single dose of amifostine has a significant effect on leukemia cell parameters, which are believed to have a significant impact on the in vivo biology of the disease and on its response to remission induction therapy. PMID- 10741702 TI - Quickly predicting chemotherapy response to paclitaxel-based therapy in non-small cell lung cancer by early technetium-99m methoxyisobutylisonitrile chest single photon-emission computed tomography. AB - The purpose of this study was to retrospectively predict the chemotherapy response to paclitaxel in non-small cell lung cancer (NSCLC) using technetium-99m methoxyisobutylisonitrile (Tc-99m MIBI) chest single-photon-emission computed tomography (SPECT) to detect the expression of multidrug-resistance-mediated Mr 170,000 P-glycoprotein. Before chemotherapy with Paclitaxel (Taxol), 30 patients with stage IIIb or IV NSCLC were enrolled in this study. Early chest SPECT 10 min after i.v. injection of Tc-99m MIBI was performed to qualitatively interpret Tc 99m MIBI chest SPECT visually and quantitatively calculate early tumor:normal lung ratios (T:NL) for quick assessment of multidrug-resistant P-glycoprotein expression in NSCLC. On the basis of qualitatively visual interpretation of early Tc-99m MIBI chest SPECT, all of 15 (100%) cases with good response to chemotherapy with Taxol could be detected but 10 (67%) of 15 cases with poor response could not be detected. Early Tc-99m MIBI chest SPECT could correctly predict chemotherapy response in 25 (83%) of 30 of cases. The early T:NL were 3.30 +/- 0.82 for 15 patients with good response and 2.02 +/- 0.19 for 5 patients with poor response. The differences were significant (P < 0.05) by independent Student t tests. However, no significant differences were found for other prognostic factors (age, sex, tumor size, tumor location, stage, and cell type) between good-response and poor-response patients. Early Tc-99m MIBI chest SPECT has the potential to predict chemotherapy response to Paclitaxel. PMID- 10741703 TI - Phase II trial of bryostatin 1 in patients with relapsed low-grade non-Hodgkin's lymphoma and chronic lymphocytic leukemia. AB - Bryostatin 1 is a natural product isolated from the marine bryozoan Bugula neritina in 1982 and is currently undergoing evaluation in a number of malignancies. Twenty-five patients with relapsed, low-grade non-Hodgkin's lymphoma or chronic lyphocytic leukemia (CLL) received bryostatin 1 by 72-h continuous infusion every 2 weeks at a dose of 120 microg/m2 per course. Patients who progressed while receiving bryostatin 1 alone could participate in a feasibility study by receiving vincristine administered by bolus i.v. injection immediately after the completion of the bryostatin 1 infusion. The dose of vincristine was escalated in groups of three patients as follows: level 1, 0.5 mg/m2; level 2, 1.0 mg/m2; and level 3, 1.4 mg/m2 with vincristine doses capped at 2.0 mg for all patients. Bryostatin 1 alone resulted in one complete remission and two partial remissions. Nine patients received sequential treatment with bryostatin 1 and vincristine. The addition of vincristine at a dose of 2 mg was feasible and caused the expected dose-related sensory neuropathy. Phenotypic analysis by flow cytometric analysis on pre- and post-bryostatin 1-treated peripheral blood lymphocytes revealed up-regulation in the coexpression of CD11c/ CD22 on CD20+ B cells in two of four CLL patients studied, which is consistent with in vitro findings of differentiation of CLL cells to a hairy cell phenotype. PMID- 10741704 TI - Flow cytometric measurement of intracellular cytokines detects immune responses in MUC1 immunotherapy. AB - The detection of tumor-specific T cells in immunized cancer patients usually relies on lengthy and difficult CTL assays; we now report on flow cytometry to detect the intracellular cytokines interleukin 2 (IL-2), IL-4, IFN-gamma, and tumor necrosis factor alpha (TNF-alpha) produced by CD4+CD69+ and CD8+CD69+ activated T cells after MUC1 antigen stimulation. Peripheral blood mononuclear cells were obtained from 12 patients with adenocarcinoma injected with mannan MUC1; cells were exposed in vitro for 18 h to MUCI peptide in the presence of CD28 monoclonal antibody and Brefeldin; permeabilized cells were used for the expression of cytokines. After stimulation in vitro with MUC1-variable number of tandem repeats peptides, CD8+CD69+ T cells from all immunized patients generated 3-9 times higher levels of TNF-alpha(P < 0.038) and IFN-gamma (P <0.010) than did cells from 12 normal subjects; minor increases in IL-4 occurred. By contrast, CD4+CD69+ cells showed no overall alteration in TNF-alpha and IFN-gamma cytokine production, although in some patients, their measurement was informative; the measurement of IL-2 was not useful in either CD4+CD69+ or CD8+CD69+ cells. We conclude that in MUC1-immunized patients, the measurement of TNF-alpha and IFN gamma in activated CD69+CD8+ T cells may be indicative of their immune status. PMID- 10741705 TI - The development of biologic end points in patients treated with differentiation agents: an experience of retinoids in prostate cancer. AB - The evaluation of new therapies in prostate cancer requires unique end points for agents with diverse mechanisms of action. Because retinoic acid may have a confounding effect on prostate-specific antigen, we incorporated a pathological end point into the outcome assessment of two sequential clinical trials using all trans-retinoic acid (ATRA) and the combination of 13-cis-retinoic acid and IFN-2a (cRA?IFN). Pre- and posttherapy tumor biopsy specimens were studied for histological changes, apoptosis (terminal deoxynucleotidyl transferase-mediated nick end labeling assay), and proliferation index (Ki67). Prostate-specific membrane antigen (PSMA) expression was also evaluated using two different monoclonal antibodies to its intracellular domain (Cytogen 7E11 and Hybritech PM2). Fourteen patients with androgen-independent disease were treated with ATRA (50 mg/m2 p.o. every 8 h daily) and 16 androgen-independent and 4 androgen dependent patients were treated with cRA?IFN (10 mg/kg/day cRA plus 3, 6, or 9 million units daily IFN). Both therapies were well tolerated, with fatigue and cheilitis being the most common adverse events. Clinical activity, assessed by radiographs and serum prostate-specific antigen, was minimal, and the majority of patients progressed within 3 months. One patient with androgen-dependent disease had prolonged stabilization for >1 year. The majority of cases (95%) showed no gross histological changes and no difference in apoptotic or proliferative indices. Increased PSMA immunoreactivity was seen in seven of nine (78%) cases using PM2 antibody and in two of nine (22%) cases using the 7E11 antibody. Although antitumor effects were modest, the results suggest a role for retinoids in modulating the expression of PSMA on prostate cancer cells. PMID- 10741706 TI - Phase I trial of all-trans retinoic acid in patients with treated head and neck squamous carcinoma. AB - Although retinoids show promise for prevention of second primary upper aerodigestive tract tumors, the optimum retinoid, dose, and schedule are unknown. All-trans retinoic acid (ATRA) has greater affinity for retinoic acid receptors and may be more active than other retinoids but has a shorter plasma half life and may up-regulate its own metabolism. We defined the maximum long-term tolerable dose, dosing frequency, pharmacokinetics, and toxicity of ATRA in patients with treated squamous cell carcinoma of the head and neck (SCCHN). Twenty-one patients were randomized to 45, 90, or 150 mg/m2 ATRA either once daily, or as divided doses every 8 h, for 1 year. Pharmacokinetics were assessed periodically. Fourteen men and seven women with previous SCCHN of initial stage I IV were treated. Grade > or =3 toxicities (reversible) included headache and hypertriglyceridemia in 5 and 6 patients each, mucositis in 2 patients, and hyperbilirubinemia, elevated alkaline phosphatase, colitis, lipasemia, xerostomia, eczema, and arthritis in 1 patient each. The 150-mg/m2 dose was not tolerable. Doses were reduced for grade > or =3 toxicity in seven of eight patients at 90 mg/m2 daily. Three of nine patients at 45 mg/m2/day required dose reduction, two at the once-daily dose. Day 1 ATRA area under the plasma concentration versus time curve (AUC) increased with dose, and after 1-2 months of continued dosing, the AUC declined in 7 of 13 patients (54%) studied. ATRA AUC did not correlate with toxicity severity or frequency. Fifteen mg/m2/day every 8 h is a tolerable dose for 1 year in patients with treated SCCHN. ATRA pharmacokinetics did not correlate with toxicity. PMID- 10741707 TI - A clinical study of hypoxia and metallothionein protein expression in squamous cell carcinomas. AB - The objective was to discover whether the oxygen-regulated protein, metallothionein, is expressed in the hypoxic cells of squamous cell carcinomas. Twenty patients with squamous cell carcinoma of the uterine cervix or head and neck were infused with a solution of the hypoxia marker, pimonidazole hydrochloride, at a dose of 0.5 g/m2. The following day, biopsies were collected, formalin fixed, paraffin embedded, and sectioned at 4 microm. Sections from each biopsy were immunostained for pimonidazole binding, metallothioneins I and II, involucrin, and proliferating cell nuclear antigen. A total of 84 biopsies were analyzed. Sixty-four of 84 biopsy sections contained hypoxia. Of the hypoxia containing sections, 43 of 64 or 67% showed no microregional overlap between hypoxia and metallothionein; 7 of 64 showed overlap; and 14 of 64 showed a combination of overlap and no overlap. On a tumor-by-tumor basis, 5 of 7 head and neck and 7 of 13 cervix tumors showed no overlap between metallothionein and hypoxia at the microregional level. Ranges for the percentage of the area of hypoxia in head and neck (<0.9 to 17%) and cervix (<0.1 to 14%) tumors were similar. In the hypoxia-containing sections, immunostaining for involucrin, a molecular marker for differentiation, overlapped with that for hypoxia in 82% of the cases. The majority of hypoxic cells in squamous cell carcinomas do not express metallothionein protein, although metallothionein is induced by hypoxia in human tumor cells in vitro. Hypoxic cells in human tumors tend to be in regions immunostaining for involucrin, and it seems possible that differentiation of hypoxic cells in squamous cell carcinomas might affect metallothionein I and II expression. PMID- 10741708 TI - Electrochemotherapy with cisplatin: clinical experience in malignant melanoma patients. AB - Electrochemotherapy consists of chemotherapy followed by local application of electric pulses to the tumor to increase drug delivery into the cells. The aim of this Phase II clinical study was to evaluate the antitumor effectiveness of electrochemotherapy using intratumoral cisplatin administration on cutaneous tumor nodules in malignant melanoma patients. In 10 patients, 133 tumor nodules of different sizes were treated: (a) 82 tumor nodules were treated with electrochemotherapy; (b) 27 tumor nodules were treated with cisplatin; (c) 2 tumor nodules were treated with electric pulses; and (d) 22 tumor nodules were untreated. Four weeks after therapy, 78% objective responses were obtained in the electrochemotherapy group, and 38% objective responses were obtained in the cisplatin group. Exposure of tumor nodules to electric pulses without cisplatin treatment had no effect on tumor growth. Electrochemotherapy was well tolerated by all patients, and a good cosmetic effect was obtained, with only minimal scarring and a slight depigmentation of the skin. At 124 weeks of follow-up, a 77% control rate of the tumor nodules treated by electrochemotherapy was observed, compared to 19% for those that were treated with cisplatin only (P < 0.0001). Our results clearly demonstrate that electrochemotherapy with cisplatin is a highly effective approach for treatment of cutaneous malignant melanoma nodules. The advantages of this therapy include its simplicity, the short duration of treatment sessions, low cisplatin doses, and insignificant side effects, as well as the fact that it can be done on an outpatient basis. PMID- 10741709 TI - Phase II study of oral topotecan in advanced non-small cell lung cancer. AB - This study was designed to assess the activity of oral topotecan (TPT) in patients with advanced non-small cell lung cancer previously untreated with chemotherapy. Eligible patients had inoperable stage III or stage IV non-small cell lung cancer and were chemotherapy-naive. Other inclusion criteria were Eastern Cooperative Oncology Group performance status 0, 1, or 2, adequate bone marrow, and renal and hepatic function. Of 30 patients, 29 were assessable for response. Oral TPT was administered for 5 days every 21 days for up to six cycles unless disease progression or unacceptable toxicity occurred. Patients received a dose of 2.3 mg/m2/day for the first cycle. Dose modification for subsequent cycles was based on tolerability. Patients completed symptom questionnaires every 3 weeks. Pharmacokinetics were evaluated in all patients during cycle 1. Three patients had radiological responses with a reduction in tumor size of 30-40%. No patients achieved complete or partial responses to treatment. Thirteen patients had a stable disease (43.3%), and the median survival was 39.9 weeks with a 1 year survival of 33.3%. At the time of analysis, 27 patients had died. Median time to progression was 12.3 weeks. Treatment was well tolerated. A total of 125 cycles of treatment were completed. Twelve patients (40%) experienced grade III/IV neutropenia. Five patients (16.6%) had grade III/IV anemia. There were two episodes of grade III/IV thrombocytopenia. The main nonhematological toxicities consisted of grade III nausea (13%) and grade III vomiting (13%). The most frequently reported disease-related symptoms at baseline were dyspnea, cough, and fatigue. There was a subsequent improvement in patient scores of dyspnea in 17% of patients, 31% showed improvement in cough, and 32% showed improvement in fatigue. The mean area under the curve of TPT following 2.3 mg/m2 p.o. was 51.6 ng.h/ml (%SD, 25%). The area under the curve of TPT on day 1 of the first cycle was correlated with the percentage fall in leukocytes. Although oral TPT at the applied dose and schedule showed modest activity as a single agent, almost one half of the patients had a stable disease, and median time to progression was 12.3 weeks. The overall median survival was a promising 39.9 weeks, and useful palliation of symptoms was seen. PMID- 10741710 TI - Induction of antibodies against GM2 ganglioside by immunizing melanoma patients using GM2-keyhole limpet hemocyanin + QS21 vaccine: a dose-response study. AB - In a previous randomized Phase III trial (P. O. Livingston et al, J. Clin. Oncol., 12: 1036-1044, 1994), we demonstrated that immunization with GM2 and bacille Calmette-Guerin reduced the risk of relapse in stage III melanoma patients who were free of disease after surgical resection and who had no preexisting anti-GM2 antibodies. That vaccine formulation induced IgM anti-GM2 antibodies in 74% but induced IgG anti-GM2 antibodies in only 10% of the patients. To optimize the immune response against GM2, a reformulated vaccine was produced conjugating GM2 to keyhole limpet hemocyanin (KLH) and using the adjuvant QS21 (GM2-KLH/QS21). In pilot studies, 70 microg of vaccine induced IgG anti-GM2 antibodies in 76% of the patients. We wished to define the lowest vaccine dose that induced consistent, high-titer IgM and IgG antibodies against GM2. Fifty-two melanoma patients who were free of disease after resection but at high risk for relapse were immunized with GM2-KLH/QS21 vaccine at GM2 doses of 1, 3, 10, 30, or 70 ILg on weeks 1, 2, 3, 4, 12, 24, and 36. Serum collected at frequent and defined intervals was tested for anti-GM2 antibodies. Overall, 88% of the patients developed IgM anti-GM2 antibodies; 71% also developed IgG anti GM2 antibodies. GM2-KLH doses of 3-70 microg seemed to be equivalent in terms of peak titers and induction of anti-GM2 antibodies. At the 30-microg dose level, 50% of the patients developed complement fixing anti-GM2 antibodies detectable at a serum dilution of 1:10. We conclude that the GM2-KLH/QS21 formulation is more immunogenic than our previous formulation and that 3 microg is the lowest dose that induces consistent, high-titer IgM and IgG antibodies against GM2. PMID- 10741711 TI - In vivo and in vitro ovarian carcinoma growth inhibition by a phosphatidylinositol 3-kinase inhibitor (LY294002). AB - Phosphatidylinositol 3-kinase (PI3-K) induces mitogenesis, cell growth, and cell transformation. Amplification of the gene encoding the P110alpha subunit likely is an important event in ovarian cancer progression, and PI3-K inhibitors are possible therapeutic agents for this disease. We evaluated effects of LY294002, a potent inhibitor of PI3-K, on growth of ovarian carcinoma in vivo and in vitro, and on ascites formation in vivo. Athymic mice were inoculated i.p. with the ovarian cancer cell line OVCAR-3. Seven days after inoculation, mice were treated with or without LY294002 (100 mg/kg of body weight) for 3 weeks. Body weight and abdominal circumference were measured twice weekly. At the end of the experiment, mice were sacrificed, ascites volume was measured, and tumors were excised. Mean tumor burden in the LY294002-treated group was reduced by approximately 65% versus controls. Virtually no ascites developed in the treatment group; mean volume of ascites in controls was 3.3 +/- 0.38 ml. OVCAR-3 cells also were cultured in vitro without and with LY294002 (1, 5, and 10 microM) for 24 h. The number of cells in 1, 5, and 10 microM LY294002-treated wells was reduced by 27, 56, and 75%, respectively, versus controls. In vivo and in vitro morphological studies demonstrated that LY294002 induced marked nuclear pyknosis and diminished cytoplasmic volume in the tumor cells, confirmed as apoptosis. Thus, LY294002 significantly inhibits growth and ascites formation of ovarian carcinoma in vivo and markedly inhibits ovarian cancer cell proliferation in vitro, suggesting an important role of PI3-K inhibitors as a potentially useful treatment for women with ovarian carcinoma. PMID- 10741712 TI - Up-regulation of the proapoptotic mediators Bax and Bak after adenovirus-mediated p53 gene transfer in lung cancer cells. AB - Overexpression of wild-type p53 in cancer cells by adenovirus-mediated p53 gene transfer can result in the induction of apoptosis. To identify the potential mediators of this p53-induced apoptosis, we examined apoptotic protein levels in human lung cancer cells after Adp53 gene transfer. We observed up-regulation of Bax and Bak protein levels 18-36 h after transduction with Adp53 in H1299, H358, and H322 lung cancer cells. Contrary to expected observations, no changes in Bcl 2 and Bcl-X(L) protein levels were observed. Morphological cell changes and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining showed evidence of apoptosis in all cell lines 48 h after transduction with Adp53. These results indicate that the induction of apoptosis by adenovirus mediated p53 transfer may be mediated by the induction of proapoptotic mechanisms rather than suppression of antiapoptotic mechanisms. PMID- 10741713 TI - Ciprofloxacin mediated cell growth inhibition, S/G2-M cell cycle arrest, and apoptosis in a human transitional cell carcinoma of the bladder cell line. AB - The second most prevalent urological malignancy in middle aged and elderly men is bladder cancer, with 90% of the cases being transitional cell carcinomas. The success of current systemic and intravesical therapeutic agents, such as cisplatin, thiotepa, Adriamycin, mitomycin C, and bacillus Calmette-Guerin, is limited with recurrence rates reduced to 17-44%. In addition, most of these agents require instrumentation of the urinary tract and are delivered at a significant cost and potential morbidity to the patient. Fluroquinolone antibiotics such as ciprofloxacin, which can be administered p.o., may have a profound effect in bladder cancer management. This is primarily based on limited in vitro studies on tumor cells derived from transitional cell carcinoma of the bladder that revealed a dose- and time-dependent inhibition of cell growth by ciprofloxacin at concentrations that are easily attainable in the urine of patients. However, the mechanism(s) by which ciprofloxacin elicits its biological effects on bladder cancer cells is not well documented. Our experimental data confirm previous studies showing the in vitro cell growth inhibition of the transitional cell carcinoma of the bladder cell line HTB9 and further showed the induction of cell cycle arrest at the S/G2-M checkpoints. In addition, we found down-regulation of cyclin B, cyclin E, and dephosphorylation of cdk2 in ciprofloxacin-treated bladder tumor cells. There was also an up-regulation of Bax, which altered the Bax:Bcl-2 ratio, which may be responsible for mitochondrial depolarization reported to be involved prior to the induction of apoptosis. The cyclin-dependent kinase inhibitor p21WAF1 level was found to be decreased within 12 h of ciprofloxacin treatment and disappeared completely when HTB9 cells were treated with 200 microg/ml ciprofloxacin for 24 h. The down regulation of p21WAF1 closely correlated with poly(ADP-ribose) polymerase cleavage and CPP32 activation. Recent studies revealed that p21WAF1 protects cells from apoptosis by arresting them in G1 and further binds to pro-caspase-3, preventing its activation and thus, inhibiting the apoptotic cascade. Hence, the down-regulation of p21WAF1, together with the alterations in Bax and cdk2 as observed in our studies, may define a novel mechanism by which ciprofloxacin inhibits tumor cell growth and induces apoptotic cell death. The results of our current studies provide strong experimental evidence for the use of ciprofloxacin as a potential preventive and/or therapeutic agent for the management of transitional cell carcinoma of the bladder. PMID- 10741714 TI - The in vitro evaluation of 25-hydroxyvitamin D3 and 19-nor-1alpha,25 dihydroxyvitamin D2 as therapeutic agents for prostate cancer. AB - Prostate cancer cells contain specific receptors [vitamin D receptors (VDRs)] for 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), which is known to inhibit the proliferation and invasiveness of these cells. These findings support the use of 1alpha,25(OH)2D3 for prostate cancer therapy. However, because 1alpha,25(OH)2D3 can cause hypercalcemia, analogues of 1alpha,25(OH)2D3 that are less calcemic but that exhibit potent antiproliferative activity would be attractive as therapeutic agents. We investigated the effects of two different types of less calcemic vitamin D compounds, 25-hydroxyvitamin D3 [25(OH)D3] and 19-nor-1alpha,25 dihydroxyvitamin D2 [19-nor-1,25(OH)2D2], and compared their activity to 1alpha,25(OH)2D3 on (a) the proliferation of primary cultures and cell lines of human prostate cancer cells; and (b) the transactivation of the VDRs in the androgen-insensitive PC-3 cancer cell line stably transfected with VDR (PC-3/ VDR). 19-nor-1alpha,25(OH)2D2, an analogue of 1alpha,25(OH)2D3 that was originally developed for the treatment of parathyroid disease, has been shown to be less calcemic than 1alpha,25(OH)2D3 in clinical trials. Additionally, we recently showed that human prostate cells in primary culture possess 25(OH)D3 1alpha-hydroxylase, an enzyme that hydroxylates the inactive prohormone, 25(OH)D3, to the active hormone, 1alpha,25(OH)2D3, intracellularly. We reasoned that the hormone that is formed intracellularly would inhibit prostate cell proliferation in an autocrine fashion. We found that 1alpha,25(OH)2D3 and 19-nor 1alpha,25(OH)2D2 caused similar dose-dependent inhibition in the cell lines and primary cultures in the [3H]thymidine incorporation assay and that both compounds were significantly more active in the primary cultures than in LNCaP cells. Likewise, 25(OH)D3 had inhibitory effects comparable to those of 1alpha,25(OH)2D3 in the primary cultures. In the chloramphenicol acetyltransferase (CAT) reporter gene transactivation assay in PC-3/ VDR cells, 1alpha,25(OH)2D3 and 19-nor 1alpha,25(OH)2D2 caused similar increases in CAT activity between 10(-11)and 10( 9) M. Incubation of PC-3/VDR cells with 5 x 10(-8) M 25(OH)D3 induced a 29-fold increase in CAT activity, similar to that induced by 10(-8) M 1alpha,25(OH)2D3. In conclusion, our data indicate that 25(OH)D3 and 19-nor-1alpha,25(OH)2D2 represent two different solutions to the problem of hypercalcemia associated with vitamin D-based therapies: 25(OH)D3 requires the presence of 1alpha-hydroxylase, whereas 19-nor-1alpha,25(OH)2D2 does not. Both drugs are approved for human use and may be good candidates for human clinical trials in prostate cancer. PMID- 10741715 TI - Immunoprotective activities of multiple chaperone proteins isolated from murine B cell leukemia/lymphoma. AB - Although the use of tumor-derived heat shock/chaperone proteins (HSPs) as anticancer vaccines is gaining wider study and acceptance, there have thus far been no reports concerning chaperone antitumor activities against disseminated hematological malignancies. We have devised an efficient and effective method for purification of the chaperone proteins grp94/gp96, HSP90, HSP70, and calreticulin from harvested A20 murine leukemia/lymphoma tumor material. We have demonstrated that these purified proteins, when used as vaccines, can induce potent and specific immunity against a lethal tumor challenge. Individual chaperone proteins were differentially effective in their abilities to provide immune protection. The increase in survival generated by the most effective chaperone vaccine, HSP70, resulted from at least a 2-log reduction in tumor burden. Syngeneic granulocyte macrophage colony-stimulating factor producing fibroblasts were injected at the site of vaccination in an attempt to augment the immune response. Surprisingly, localized granulocyte macrophage colony-stimulating factor production inhibited the protective effects of chaperone vaccination. These studies provide evidence that chaperone proteins can be isolated from B-cell tumors and used effectively to immunize against disseminated lymphoid malignancies. PMID- 10741716 TI - p27Kip1 accumulation by inhibition of proteasome function induces apoptosis in oral squamous cell carcinoma cells. AB - Ubiquitin-mediated proteolysis controls intracellular levels of various cell cycle regulatory proteins, and its inhibition has been shown to induce apoptosis in proliferating cells. In the present study, we examined induction of apoptosis in oral squamous cell carcinoma (OSCC) cells by treatment with specific proteasome inhibitors, carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal and lactacystin. In all three OSCC cell lines examined, apoptotic changes such as apoptotic body formation and DNA fragmentation were observed at various degrees after 24 h of the carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal or lactacystin treatment. HSC2 cells showed the most prominent apoptotic changes among the cell lines examined and demonstrated the highest level of accumulation of p27Kip1 protein after the treatment with proteasome inhibitor. Reduced expressions of cyclin D1 and phospho pRb were also observed after the treatment with proteasome inhibitor. Moreover, 12 h of treatment with the proteasome inhibitor inhibited cdk2/cyclin E kinase activity and increased the ratio of the cell cycle population at the G1 phase. The proteasome inhibitor led to inhibition of cell cycle progression. In addition, activation of CPP32 and reduced expression of Bcl 2 were observed. Because apoptosis induced by the proteasome inhibitor was inhibited by treatment with antisense p27Kip1 oligonucleotide, accumulation of the p27Kip1 protein might play an important role in the apoptosis induced by proteasome inhibitor. The present results suggest that inhibition of proteasome function may be used as a possible target of novel therapy for OSCC. PMID- 10741717 TI - Flavopiridol, a cyclin-dependent kinase inhibitor, prevents spindle inhibitor induced endoreduplication in human cancer cells. AB - Defects in cell cycle checkpoints can lead to chromosome abnormality, aneuploidy, and genomic instability, all of which can contribute to tumorigenesis. Recent studies and data presented in this study indicate that cells with compromised G1 checkpoint endoreduplicate and become polyploid in response to microtubule inhibitors. Previous studies have shown that polyploid cells are unstable and lose chromosomes randomly to give aneuploidy. In this study, we show that endoreduplication and polyploidation can be prevented by inhibiting the cyclin dependent kinases (Cdks) by flavopiridol, a synthetic flavone presently undergoing phase II clinical trials. In our initial studies, we treated MCF-7 cells with paclitaxel, which results in the arrest of cells in G1 with 4n DNA content (pseudo G1). This was coincident with increased p53 and p21 protein expression and decreased cyclin E/Cdk2 kinase activity. In contrast, G1 checkpoint-compromised MDA-MB-468 (p53-/- and pRb-/-) and p21-/- HCT116 do not arrest in the pseudo G1 state after exposure to microtubule inhibitors and enter in the S phase with 4n DNA content. More than 60% of MDA-MB-468 cells accumulate with >4n DNA content after 72 h of nocodazole treatment. The MPM-2 labeling showed that 8n cells also undergo mitosis. These cells display deregulated and persistent activation of cyclin E/Cdk2 and cyclin B1/cdc2 kinase activity. Administration of flavopiridol after mitotic block results in the arrest of cells in the pseudo G1 state and the dramatic decrease in cells containing >4n DNA content in MDA-MB-468 cells. The cyclin E/Cdk2 and cyclin B1/cdc2 kinase activities remained low after exit from mitosis. Furthermore, pRb was hypophosphorylated after the addition of flavopiridol in p21-deficient HCT116 cells, indicating the arrest of cells at the pseudo G1 state. Based on these studies, we propose that flavopiridol preserves the genomic stability by preventing endoreduplication and polyploidy and thus has the potential to be used as a chemopreventive agent to prevent the occurrence of neoplasia. PMID- 10741718 TI - Peroxisome proliferator-activated receptor gamma as a novel target in cancer therapy: binding and activation by an aromatic fatty acid with clinical antitumor activity. AB - Aromatic fatty acids, of which phenylacetate is a prototype, constitute a class of low toxicity drugs with demonstrated antitumor activity in experimental models and in humans. Using in vitro models, we show here a tight correlation between tumor growth arrest by phenylacetate and activation of peroxisome proliferator activated receptor gamma (PPARgamma), a member of the nuclear receptor superfamily. In support are the following observations: (a) the efficacy of phenylacetate as a cytostatic agent was correlated with pre-treatment levels of PPARgamma, as documented using established tumor lines and forced expression models; (b) in responsive tumor cells, PPARgamma expression was up-regulated within 2-9 h of treatment preceding increases in p21waf1, a marker of cell cycle arrest; (c) inhibition of mitogen-activated protein kinase, a negative regulator of PPARgamma, enhanced drug activity; and (d) phenylacetate interacted directly with the ligand-binding site of PPARgamma and activated its transcriptional function. The ability to bind and activate PPARgamma was common to biologically active analogues of phenylacetate and corresponded to their potency as antitumor agents (phenylacetate < phenylbutyrate < p-chloro-phenylacetate < p-iodo phenylbutyrate), whereas an inactive derivative, phenylacetylglutamine, had no effect on PPARgamma. These findings point to PPARgamma as a novel target in cancer therapy and provide the first identification of ligands that have selective antitumor activity in patients. PMID- 10741719 TI - 1-phenyl-2-decanoylamino-3-morpholino-1-propanol chemosensitizes neuroblastoma cells for taxol and vincristine. AB - In this study, we show that an inhibitor of glycosphin-golipid biosynthesis, D,L threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), increases the chemosensitivity of neuroblastoma tumor cells for Taxol and vincristine. At noneffective low doses of Taxol or vincristine, the addition of a noneffective dose of PDMP resulted in 70% cytotoxicity, indicating synergy. Such an effect was not observed for etoposide (VP16). PDMP caused an early (6 h) increase in ceramide (Cer) levels, but the excess Cer was metabolically removed in the long term (96 h). However, upon incubation with PDMP in combination with Taxol, but not with etoposide, Cer levels remained elevated at 96 h. These results suggest that neuroblastoma cells are normally able to metabolically remove excess Cer, but lose this capacity upon exposure to microtubule modulating anticancer agents (Taxol or vincristine). In addition, PDMP treatment resulted in a decreased efflux of [14C]Taxol and [3H]vincristine from neuroblastoma cells, similar to treatment with PSC833 or MK571, suggesting an effect of PDMP on the transporter proteins P-glycoprotein and/or multidrug resistance protein. PDMP did not further reduce [14C]Taxol or [3H]vincristine efflux in PSC833-treated cells, although it did further diminish cell survival under these conditions. We conclude that a combined administration of nontoxic concentrations of PDMP and either Taxol or vincristine results in highly sensitized neuroblastoma cells. This appears to involve a sustained elevation of Cer levels, possibly in concert with increased drug accumulation. PMID- 10741720 TI - Colon cancer chemopreventive drugs modulate integrin-mediated signaling pathways. AB - Epidemiological studies of colorectal cancer incidence suggest that the development of this disease can be modulated by dietary factors. Among the micronutrients showing significant efficacy in tumor prevention are polyphenolic antioxidants found in fruits and vegetables. Epidemiological studies also indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) decrease the incidence of colorectal cancer. Integrin-mediated cell-matrix contact provides critical signaling that regulates cellular proliferation, migration, and apoptosis. A signaling mediator for this system is focal adhesion kinase (FAK). Thus far, FAK has not been identified as a target for the inhibitory action of any chemopreventive drug in vivo or in vitro. However, the loss of integrin mediated cell-matrix contact can induce apoptosis (anoikis), and effective chemopreventive agents typically increase the rate of enterocyte apoptosis. Therefore, we asked whether the NSAID, sulindac sulfide, and the phenolic antioxidant, caffeic acid phenethyl ester (CAPE), affected FAK expression or tyrosine phosphorylation in human colon carcinoma cells. We show that subapoptotic doses of both sulindac sulfide and CAPE caused a rearrangement of the actin cytoskeleton and consequently the loss of focal adhesion plaques. These drugs also reduced the tyrosine phosphorylation of FAK and an associated factor, p130Cas. Steady-state levels of these proteins, together with other relevant signaling molecules, remained unchanged after treatments. Finally, we show that both CAPE and sulindac reduced cell invasion, a functional assay for the inhibition of signaling downstream of FAK. These data strongly suggest that chemopreventive drugs can regulate FAK activity. In conclusion, these novel studies add modulation of integrin-mediated signaling to the spectrum of activity of NSAIDs and plant phenolics. PMID- 10741721 TI - Treatment for malignant pleural effusion of human lung adenocarcinoma by inhibition of vascular endothelial growth factor receptor tyrosine kinase phosphorylation. AB - Malignant pleural effusion (PE) is associated with advanced human lung cancer. We found recently, using a nude mouse model, that vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is responsible for PE induced by non-small cell human lung carcinoma cells. The purpose of this study was to determine the therapeutic potential of a VEGF/VPF receptor tyrosine kinase phosphorylation inhibitor, PTK 787, against PE formed by human lung adenocarcinoma (PC14PE6) cells. PTK 787 did not affect the in vitro proliferation of PC14PE6 cells, whereas it specifically inhibited proliferation of human dermal microvascular endothelial cells stimulated by VEGF/VPF. A specific platelet derived growth factor receptor tyrosine kinase inhibitor, CGP57148 (used as a control because PTK 787 also inhibits platelet-derived growth factor receptor tyrosine kinases), had no effect on proliferation of PC14PE6 or human dermal microvascular endothelial cells. i.v. injection of PC14PE6 cells into nude mice produced lung lesions and a large volume of PE containing a high level of VEGF/VPF. Oral treatment with CGP57148 had no effect on PE or lung metastasis. In contrast, oral treatment with PTK 787 significantly reduced the formation of PE but not the number of lung lesions. Furthermore, treatment with PTK 787 significantly suppressed vascular hyperpermeability of PE-bearing mice but did not affect the VEGF/VPF level in PE or expression of VEGF/VPF protein and mRNA in the lung tumors of PC14PE6 cells in vivo. These findings indicate that PTK 787 reduced PE formation mainly by inhibiting vascular permeability, suggesting that this VEGF/VPF receptor tyrosine kinase inhibitor could be useful for the control of malignant PE. PMID- 10741722 TI - Macrophage inflammatory protein 1alpha attenuates the toxic effects of temozolomide in human bone marrow granulocyte-macrophage colony-forming cells. AB - Macrophage inflammatory protein 1alpha (MIP-1alpha) is a chemokine that may act principally by preventing hemopoietic cells from entering G1, thereby attenuating the cytotoxic effects of cell cycle-specific chemotherapeutic agents. Here we examine the effect of MIP-1alpha on the sensitivity of human granulocyte macrophage hemopoietic progenitor cells (granulocyte-macrophage colony-forming cells; GM-CFCs) with the cytotoxic effects of antitumor agents that act mainly via alkylation at the O6 position of guanine in DNA. Mononuclear cell preparations from human bone marrow were used in an in vitro GM-CFC colony forming assay. The GM-CFC survival from individual patients displayed a range of sensitivities to the methylating agent temozolomide [(Tz) 20-55% survival at 10 microg/ml Tz]. However, in all 16 cases, MIP-1alpha (50 ng/ml) protected against GM-CFC killing: survival in the presence of MIP-1alpha ranged from 65-97% at 10 microg/ml Tz, with GM-CFCs being 1.5-4.5-fold more resistant than control cells from the same patient. The highest levels of protection were seen in the GM-CFCs with the highest sensitivity in the absence of MIP-1alpha. Similar degrees of protection were seen for the methylating agent streptozotocin, but no protection was detected for the chloroethylating agents carmustine or mitozolomide in the samples for which there was protection against the toxic effects of Tz. Whereas the mechanism of this effect remains to be established, the results may have potential immediate clinical application in the attenuation of hematological toxicity after administration of methylating antitumor agents. PMID- 10741723 TI - Therapeutic synergy of TNP-470 and ionizing radiation: effects on tumor growth, vessel morphology, and angiogenesis in human glioblastoma multiforme xenografts. AB - We examined the effect on tumor growth, vessel morphology, and expression of angiogenic factors of combining radiotherapy and antiangiogenesis in the human glioblastoma line U87 grown in the flank or intracranially in the nude mouse. The antiangiogenic agent TNP-470 was given 6.7 mg/kg s.c. daily on day 1-7 starting 1 week after transplantation. Irradiation (IR), 10 Gy x 1, was administered on day 7. A series of tumors were excised 8 and 48 h after the end of treatment. The vascular morphology was evaluated in CD31 immunostained cryosections and by electron microscopy, and the pattern of expression of angiogenic factors (mRNA and protein) was quantitatively analyzed by phosphorimaging of Northern blots and Western blots. Significant inhibition of s.c. flank tumor growth relative to untreated controls was achieved by monotherapy with both TNP-470 (P < 0.001) and IR (P < 0.001). A significant enhancement of this effect was obtained by combining TNP-470 and IR (P < 0.05). We saw no effect of TNP-470 either alone or in addition to the effect of IR on the survival of mice with intracranial tumors. CD31 immunostaining of s.c. tumors showed acute endothelial swelling and luminal protrusion in irradiated tumor vessels but never in tumors pretreated with TNP 470, and not in the untreated controls. The vessel density (Chalkley point counts) was unchanged by TNP-470 therapy. In the TNP-470-treated tumors, we observed a distinct broadening of the endothelial basement membrane by an approximately 400-700-nm-thick electron-dense yet uncharacterized fibrillar material. TNP-470 treated tumors +/- IR also had a significantly increased mRNA expression of angiopoietin-1, whereas angiopoietin-2, vascular endothelial growth factor and basic fibroblast growth factor mRNA were unchanged by the treatments. In conclusion, TNP-470 significantly enhanced the tumor effect of ionizing IR, and our findings strongly indicate that acute microvascular damage after IR is effectively prevented by concurrent TNP-470 treatment. A significant up regulation of angiopoietin-1 seems to play a role in this protective mechanism, which as yet is not fully elucidated. PMID- 10741724 TI - A wild-type sequence p53 peptide presented by HLA-A24 induces cytotoxic T lymphocytes that recognize squamous cell carcinomas of the head and neck. AB - Evidence has accumulated indicating that HLA-A2-restricted CTLs specific for human wild-type sequence p53 epitopes lyse tumor cells expressing mutant p53. To explore the possibility that wild-type sequence p53 peptides could also be used in vaccines for patients expressing HLA-A24 antigen, another frequent HLA class I allele, we investigated the induction of HLA-A24-restricted p53-specific CTLs from the peripheral blood lymphocytes of normal donors. Of six p53-derived peptides possessing an HLA-A24 binding motif, the p53 peptide 125-134 (p53(125 134)) was found to have a high binding capacity and induced peptide-specific CTLs from peripheral blood mononuclear cells, using peptide-pulsed autologous dendritic cells and subsequent cultivation with cytokines interleukin 2 and interleukin 7. Bulk CTL populations lysed peptide-pulsed HLA-A24+ targets as well as HLA-A24+ squamous cell carcinoma of the head and neck (SCCHN) cell lines. However, IFN-gamma pretreatment of HLA-A24+ SCCHN cell lines was necessary for lysis, suggesting that a ligand density higher than that normally expressed by tumor cells is required for these CTLs to mediate lysis. Moreover, a cloned CTL, designated TH#99, isolated from the bulk population by limiting dilution, lysed HLA-A24+ SCCHN targets more efficiently than the bulk CTL population. Lysis was inhibited by anti-HLA class I monoclonal antibody but not by anti-HLA-DR monoclonal antibody. These results indicate that HLA-A24-restricted CTLs recognizing the wild-type sequence p53(125-134) can be generated using autologous dendritic cells from precursors present in peripheral blood lymphocytes obtained from normal HLA-A24+ donors. This finding suggests that vaccine strategies targeting wild-type sequence p53 epitopes can be extended to a wider range of cancer patients. PMID- 10741725 TI - Effect of telomere and telomerase interactive agents on human tumor and normal cell lines. AB - Shortening of telomeres along with an up-regulation of telomerase is implicated in the immortality of tumor cells. Targeting either telomeres or telomerase with specific compounds has been proposed as an anticancer strategy. Because telomerase activity and telomeres are found in normal cells, telomere or telomerase targeting agents could induce side effects in normal tissues. We evaluated the effects of telomere and telomerase interactive agents in human tumor and normal cell lines to try to determine the potential side effects those agents might induce in patients. Toxicity of the G-quadruplex interactive porphyrins (TMPyP4, TMPyP2) and azidothymidine (AZT) were tested using a cell counting technique against normal human cell lines (CRL-2115 and CRL-2120, fibroblasts; NHEK-Ad, adult keratinocytes; CCL-241, small intestinal cells; NCM 460, colonic mucosal epithelial cells) and human tumor cell lines (MDA-MB 231 and Hs 578T, breast cancer; SK-N-FI, neuroblastoma; HeLa, cervix cancer; MIA PaCa-2, pancreatic cancer; HT-29 and HCT-116, colon cancer; DU 145, prostatic cancer cell line). Telomerase activity of these cell lines was measured by a non-PCR-based conventional assay. The effects of TMPgammaP2, TMPyP4, and AZT were also evaluated against normal human bone marrow specimens, using a granulocyte macrophage colony-forming assay (CFU-GM). AZT showed very low cytotoxic effects against normal and tumor cell lines, with the IC50 values above 200 microM. The IC50 values for TMPyP2 and TMPyP4 in normal human cell lines were in the range of 2.9-48.3 microM and 1.7-15.5 microM, respectively, whereas in tumor cell lines the IC50 values were 11.4-53 microM and 9.0-28.2 microM, respectively. Within the tissue types, keratinocytes were more sensitive to TMPyP4 than fibroblasts, and small intestinal cells were more sensitive than colonic mucosal epithelial cells. The IC50 for TMPyP2 and TMPyP4 in the normal marrow colony-forming assays were 19.3 +/- 5.1 microM and 47.9 +/-1.0 microM, respectively. In conclusion, the in vitro cytotoxicity of the telomere interactive agent TMPyP4 is comparable in human tumor and normal cell lines, which indicates that TMPyP4 could have effects on normal tissues. PMID- 10741726 TI - Pharmacokinetics and cerebrospinal fluid penetration of CI-994 (N-acetyldinaline) in the nonhuman primate. AB - CI-994 is a substituted benzamide derivative that has demonstrated significant antitumor activity in vitro and in vivo against a broad spectrum of murine and human tumor models. Its mechanism of action is still unknown but seems to be novel compared with existing anticancer drugs. We studied the plasma and cerebrospinal fluid (CSF) pharmacokinetics of CI-994 in nonhuman primates. Three animals (total 4 doses) received an 80 mg/m2 dose of CI-994 administered over 20 min, and one animal received a dose of 100 mg/m2. Serial plasma and fourth ventricular CSF samples were obtained from 0 to 4320 min after administration of the 80-mg/m2 dose, and only plasma samples were obtained after the 100-mg/m2 dose. CI-994 was measured using a previously validated reverse-phase high performance liquid chromatography assay. Elimination of CI-994 from plasma was triexponential (4 of 5 cases) or biexponential (1 of 5 cases), with a terminal half life (t1/2) of 7.4 +/- 2.5 h, volume of distribution of 15.5 +/- 1.8 L/m2, and clearance of 40 +/- 6 ml/min/m2. The area under the concentration-time curve (AUC) for the 80-mg/m2 dose was 125 +/- 17 microM x hr. CI-994 was first detected in CSF at the completion of the i.v. infusion. Peak concentrations of CI-994 in CSF were 3.4 +/- 0.3 microM. Elimination from CSF was monoexponential (2 of 4 cases) or biexponential (2 of 4 cases) with a terminal t1/2 in CSF of 12.9 +/- 2.5 h and AUC of 55 +/- 18 microM x hr. The AUC(CSF):AUCplasma ratio was 43 +/- 10%. This study demonstrates that there is excellent CSF penetration of CI-994 after i.v. administration. Additional studies are needed to evaluate the potential role of CI-994 in the treatment of central nervous system neoplasms. PMID- 10741727 TI - Biochemical correlates of temozolomide sensitivity in pediatric solid tumor xenograft models. AB - The antitumor activity of the methylating agent temozolomide has been evaluated against a panel of 17 xenografts derived from pediatric solid tumors. Temozolomide was administered p.o. daily for five consecutive days at a dose level of 66 mg/kg. Courses of treatment were repeated every 21 days for three cycles. Tumor lines were classified as having high, intermediate, or low sensitivity, determined by complete responses, partial responses, or stable disease, respectively. Overall, temozolomide induced complete responses in five lines and partial responses in three additional tumor lines, giving objective regressions in 47% of xenograft lines. Analysis of temozolomide plasma systemic exposure indicated that this dose level was relevant to exposure achieved in patients. Tumors were analyzed by immunoblotting for levels of O6-methylguanine DNA methyltransferase (MGMT) and two mismatch repair proteins, MLH-1 and MSH-2. Tumors classified as having high or intermediate sensitivity had low or undetectable MGMT and expressed detectable MLH-1 and MSH-2 proteins. Tumors classified as having low sensitivity had either (a) high MGMT or (b) low or undetectable MGMT but were deficient in MLH-1. The relationship between p53 and response to temozolomide was also examined. In vitro temozolomide did not induce p21cip1 in p53-competent NB-1643 neuroblastoma cells. Suppression of p53 function in NB1643 clones through stable expression of a trans dominant negative p53 (NB1643p53TDN) did not confer temozolomide resistance. Similarly, tumor sensitivity to temozolomide did not segregate with p53 genotype or p53 functional status. These results indicate that MGMT is the primary mechanism for temozolomide resistance, but in the absence of MGMT, proficient mismatch repair determines sensitivity to this agent. PMID- 10741728 TI - Antitumor activity of N-(2-hydroxypropyl) methacrylamide copolymer-Mesochlorine e6 and adriamycin conjugates in combination treatments. AB - This study demonstrates the selective tumor targeting and the antitumor efficacy of the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound mesochlorin e6 monoethylenediamine (Mce6) and HPMA copolymer-bound Adriamycin (ADR) in combination photodynamic therapy (PDT) and chemotherapy against human ovarian OVCAR-3 carcinoma xenografted in female athynmic mice. The concentrations of Mce6 and ADR in blood and tissues, in free or HPMA copolymer-bound form, were determined by fluorescence and high-performance liquid chromatography fluorescence assays, respectively. Xenograft responses to single and combination therapies were recorded. The peak concentration of HPMA copolymer-Mce6 conjugate in tumor was achieved 18 h after administration. For HPMA copolymer-bound drugs, the concentration ratios of liver and spleen versus muscle were significantly higher than those of free drugs. The HPMA copolymer-bound drugs demonstrated selective targeting and accumulation in the tumor, probably attributed to the enhanced permeability and retention effect. In vivo studies revealed that all tumors in the treatment groups showed significant responses after receiving any of the various types of therapy as compared with controls (P < 0.001). PDT with HPMA copolymer-Mce6 conjugate (PDTMC) at a dose of 13.4 mg/kg (1.5 mg/kg of Mce6 equivalent) and light doses of 110 J/cm2 at 12 and 18 h, respectively, resulted in significant suppression of the growth of OVCAR-3 tumors. Three courses of chemotherapy using 35 mg/kg (2.2 mg/kg of ADR equivalent) of HPMA copolymer-ADR conjugate (CHEMO) were effective in suppressing the growth of tumors. Single PDTMC plus multiple CHEMO exhibited significantly greater therapeutic efficacy than multiple CHEMO. In the group of mice receiving multiple PDTMC, tumor recurrence became obvious after day 20. However, 10 of 12 tumors exhibited complete responses in the group of mice receiving multiple PDTMC plus multiple CHEMO. The least to most effective treatments were ranked as follows: multiple CHEMO < single PDTMC plus multiple CHEMO < multiple PDTMC < multiple PDTMC plus multiple CHEMO. The results clearly demonstrate that: (a) HPMA copolymer-bound drugs exhibited selective tumor accumulation contrary to free drugs; (b) PDT using HPMA copolymer-Mce6 conjugate with multiple light irradiations was a better therapy than that with single light irradiation; and (c) combination chemotherapy and photodynamic therapy with HPMA copolymer-ADR and HPMA copolymer-Mce6 conjugates was the most effective regimen. PMID- 10741729 TI - Treatment regimens including the multitargeted antifolate LY231514 in human tumor xenografts. AB - The scheduling of antifolate antitumor agents, including the new multitargeted autofolate LY231514 (MTA), with 5-fluorouracil was explored in the human MX-1 breast carcinoma and human H460 and Calu-6 non-small cell lung carcinoma xenografts to assess antitumor activity and toxicity (body weight loss). Administration of the antifolate (methotrexate, MTA, or LY309887) 6 h prior to administration of 5-fluorouracil resulted in additive growth delay of the MX-1 tumor when the antifolate was methotrexate or LY309887 and greater-than-additive tumor growth delay (TGD) when the antifolate was MTA. In the H460 tumor, the most effective regimens were a 14-day course of MTA or LY309887 along with 5 fluorouracil administered on the final 5 days. In addition, the simultaneous combination of MTA administered daily for 5 days for 2 weeks with administration of gemcitabine resulted in greater-than-additive H460 TGD. MTA was additive with fractionated radiation therapy in the H460 tumor when the drug was administered prior to each radiation fraction. MTA administered along with paclitaxel produced greater-than-additive H460 TGD and additive responses along with vinorelbine and carboplatin. In the Calu-6 non-small cell lung carcinoma xenograft, MTA administered in combination with cisplatin or oxaliplatin was highly effective, whereas MTA administered in combination with cyclophosphamide, gemcitabine, or doxorubicin produced additive responses. Administration of MTA along with paclitaxel or doxorubicin resulted in additive MX-1 TGD. Thus, MTA appears to be especially effective in combination therapies including 5-fluorouracil or an antitumor platinum complex. PMID- 10741730 TI - Antisense suppression of proline-directed protein kinase FA enhances chemosensitivity in human prostate cancer cells. AB - Initial clinic studies revealed that proline-directed protein kinase FA (PDPK FA) is overexpressed manyfold in various human cancerous tissues relative to the normal control. However, the role of overexpressed PDPK FA in cancers remains unknown and needs to be established. To determine whether PDPK FA is associated with drug sensitivity, we investigated the effects of partial inhibition of this kinase on the human prostate carcinoma cell line (PC-3). PDPK FA antisense expression vector and its specific antibody were successfully developed. Two stable transfected antisense clones (PA7 and PA3) of human prostate carcinoma cell were subcloned, and they expressed approximately 75% and approximately 35% of the total PDPK FA existing in the control-transfected clone as determined by both immunoprecipitate activity assay and immunoblot analysis. In sharp contrast, the PDPK FA antisense clones expressed no significant suppression of any other related proline-directed protein kinase member expression, demonstrating the specificity of these two antisense clones. When compared with parental or control transfected cells, the low-PDPK FA-expressing antisense clones displayed an enhanced sensitivity to carboplatin, 5-fluorouracil, paclitaxel, and hydroxyurea. Estimation of the IC50 index further revealed that the antisense clones displayed up to > 100-fold drug sensitivity, and there was a correlation between suppressed levels of PDPK FA and drug sensitivity. Taken together, the results demonstrate that specific antisense suppression of overexpressed PDPK FA in human prostate cancer cells is sufficient to enhance various drug sensitivity, indicating that PDPK FA is an important regulator in controlling multiple drug resistance of human prostate cancer cells. PMID- 10741731 TI - p53 mutations in defined structural and functional domains are related to poor clinical outcome in non-small cell lung cancer patients. AB - The prognostic value of p53 status in non-small cell lung cancer has been investigated in 148 patients with clinical stage I-IIIB disease. Tumor tissues were examined for mutations in exons 4-9, with emphasis on defined structural and functional domains. Eighty-four mutations were detected in 83 (54%) of the patients. Eighty-eight percent of the mutations were within exons 5-8, and 12% of the mutations were within exons 4 and 9. Missense mutations occurred in 67% of the tumors, and 30% were null mutations (10% stop mutations, 15% frameshift mutations, and 5% splice site mutations). Patients with mutations in p53 had a significantly higher risk for lung cancer-related death and for death from all causes than those with wild-type p53 [hazard ratio (HR) = 2.09 and 95% confidence interval (CI) = 1.20-3.64 and HR = 1.69 and 95% CI = 1.06-2.70, respectively]. Mutations in p53 related to even still poorer lung cancer-related prognosis were found at the following locations: (a) exon 8 (HR = 3.5; 95% CI, 1.59-7.71)]; (b) the structural domains L2 + L3 (HR = 2.36; 95% CI, 1.18-4.74), and (c) codons involved in zinc binding (HR = 11.7; 95% CI, 3.56-38.69). Together, the biologically functional group of severe flexible mutants (codons 172, 173, 175, 176, 179, 181, 238, 245, and 267) and severe contact mutants (248, 282) were significantly related to shorter lung cancer-related survival (HR = 4.16; 95% CI, 1.93-8.97). Squamous cell carcinoma was the dominant histological type in tumors involved in poor prognosis in exon 8 (HR = 3.19; 95% CI, 1.07-9.45). These results indicate that mutations in defined structural and functional domains of p53 may be useful molecular biological markers for prognosis and treatment strategy in non-small cell lung cancer patients. PMID- 10741732 TI - Rapid ("warm") autopsy study for procurement of metastatic prostate cancer. AB - In this report, we describe the distribution of metastases from 14 patients who had hormone-refractory adenocarcinoma of the prostate and agreed while alive to undergo directed autopsies after their deaths. These autopsies were undertaken specifically to document the distribution of metastases, characterize tumors phenotypically and immunohistochemically, harvest fresh and snap frozen tumor and normal control tissues suitable for molecular examination, and establish cell lines via passages through generations of severe combined immunodeficient and athymic mice. Achievement of these goals was obtained through the development of a multidisciplinary team approach. Team members included a medical oncologist, pathologists, urologists, and researchers. The autopsy and tissue procurement teams were available on a round-the-clock basis. The tissues harvested from these autopsies yielded high-quality tumor samples, as evidenced by excellent preservation seen by light microscopy, strong prostate-specific antigen immunostaining, and the successful development of xenografts. The development and expansion of this program represent a valuable resource for molecular and clinical researchers. PMID- 10741733 TI - Epstein-Barr virus DNA in serum/plasma as a tumor marker for nasopharyngeal cancer. AB - Nasopharyngeal cancer (NPC) constitutes a type of carcinoma encountered frequently in Southern China, among Eskimos of the Arctic region, and to a lesser extent in Southeast Asia. Because EBV DNA present in plasma or serum of NPC patients has proven to represent a promising noninvasive tumor marker, the present study was designed to determine the incidence of serum/plasma EBV DNA by nested PCR during various disease management stages. By this method, we could detect EBV DNA in plasma/serum of 98 of 167 NPC patients prior to treatment, compared with 10 of 77 samples derived from healthy blood donors serving as controls, with a similar prevalence observed in plasma versus serum. Investigation of 13 patients subjected to radiotherapy revealed plasma EBV DNA to persist in the plasma of one case, whereas among the remaining patients, it had vanished during the early phase of treatment. Finally, with 52 samples derived from 37 NPC patients during follow-up, we established 100% specificity and 0% false-positive rate for plasma DNA detection by nested PCR. Moreover, we subjected 24 known EBV DNA-positive serum samples to DNase digestion prior to DNA extraction and amplification to differentiate between free and encapsulated viral DNA, which demonstrated complete absence of the human beta-globin genomic DNA in contrast to EBV DNA detectable in 14 samples. In conclusion, applying this noninvasive method, serum/plasma EBV DNA constitutes a reliable tumor marker prior to, during, and after treatment of NPC. PMID- 10741734 TI - Prognostic significance of cysteine proteinases cathepsins B and L and their endogenous inhibitors stefins A and B in patients with squamous cell carcinoma of the head and neck. AB - Cysteine proteinases cathepsins (Cats) B and L and their endogenous inhibitors stefins (Stefs) A and B are implicated in the processes of local and metastatic tumor spread. They were identified as potential prognosticators in various malignant diseases, particularly in breast cancer. The aim of the present study was to determine the concentrations of Cats B and L and Stefs A and B in the tumor and adjacent normal tissue samples collected from 49 patients (the present group) with squamous cell carcinoma of the head and neck (SCCHN), using quantitative immunosorbent assays (ELISA; KRKA d.d., Novo mesto, Slovenia). Their clinical significance was compared with that from a previous study (the reference group, 45 patients; Budihna et al., Biol. Chem. Hoppe-Seyler, 377: 385-390, 1996). The follow-up of patients from the latter report was updated for this purpose. In the present group, significantly higher concentrations of Cat B (P < 0.0001), Cat L (P < 0.0001) and Stef A (P = 0.006) were found in tumors compared with concentrations in their normal tissue counterparts. Cat concentrations in normal laryngeal tissue were significantly/marginally elevated compared with nonlaryngeal tissue (Cat B, P = 0.02; Cat L, P = 0.06). The tumor concentration of Cat L was found to correlate with pT classification (P = 0.005) and tumor-node metastasis stage (P = 0.05), whereas the concentrations of Stefs A and B correlated with pN classification (P = 0.007 and P = 0.03, respectively) and tumor-node-metastasis stage of the disease (P = 0.02 and P = 0.03, respectively). There was no statistically significant difference between low and high Cat B or Cat L groups, regarding either disease-free survival or disease-specific survival, using a minimum P approach to determine cutoff concentrations. The risk of disease recurrence and SCCHN-related death was significantly higher in patients with low Stef A (P = 0.0006 and P = 0.0005, respectively) and Stef B (P = 0.0009 and P = 0.0007, respectively) tumors, compared with those with high-Stef A and Stef B tumors. These results remained significant even after Ps were adjusted for a possible bias in the estimated effect on survival. The survival analysis in the reference group also confirmed these findings (Stef A: P = 0.0009 and P = 0.002, respectively; Stef B: P = 0.03 and P = 0.009, respectively). To avoid any possible bias arising from the differences between the laboratories that performed the biochemical analysis, the concentrations of both Stefs in the present group and in the reference group were standardized and coupled together to form a uniform group. In univariate survival analysis, standardized values of Stef A and Stef B correlated inversely with the rate of relapse (P = 0.0000) and mortality rate (P = 0.0000). Multivariate regression analysis showed that the standardized value of Stef A is the strongest independent prognostic factor for both disease-free survival and disease-specific survival. These findings show the specific role of Cats B and L and Stefs A and B in the invasive behavior of SCCHN. Furthermore, Stef A proved to be a reliable prognosticator of the risk of relapse and death in patients with this type of cancer. PMID- 10741735 TI - Prognostic role of thymidylate synthase, thymidine phosphorylase/platelet-derived endothelial cell growth factor, and proliferation markers in colorectal cancer. AB - 5-Fluorouracil (5FU)-based therapy is given to patients with advanced colorectal cancer and as adjuvant treatment. Thymidylate synthase (TS) is the target for 5FU, and may have a prognostic role for the outcome of 5FU-based therapy together with proliferation markers such as p53 and Ki67. Thymidine phosphorylase (TP, also known as platelet-derived endothelial cell growth factor) may be of importance both in the 5FU drug activation pathway and in tumor angiogenesis, similar to vascular endothelial growth factor (VEGF). TS and TP levels were determined biochemically in fresh-frozen tumor specimens of 32 untreated patients with colorectal cancer, whereas in paraffin-embedded tissue samples, immunohistochemistry was performed for TS, TP, and additional prognostic markers such as p53, Ki67, and VEGF as well as microvessel density. All factors were correlated with patient characteristics such as age, gender, Dukes' stage, angio invasion, and differentiation grade. TS and TP as measured by various assays were correlated with overall and disease-free survival in this patient group. TP enzyme activity and protein expression correlated with each other. A significant correlation was found between TP enzyme activity and 5-fluoro-2'-deoxyuridine-5' monophosphate binding activity. VEGF expression correlated significantly with TP immunostaining and Ki67 index. Survival analysis revealed a significant relation of TS levels to the overall survival in this small patient group and a significant correlation between TP activity and disease-free survival. TS and TP both were of prognostic significance in these patients with colorectal cancer. The interesting relationship of TS and TP with angiogenesis and proliferation needs further investigation. PMID- 10741736 TI - DNA-dependent protein kinase activity correlates with Ku70 expression and radiation sensitivity in esophageal cancer cell lines. AB - We investigated the relationship between DNA-dependent protein kinase (DNA-PK) activity and radiation sensitivity using 14 esophageal cancer cell lines, TE 1 14. DNA-PK activities differed significantly among the cell lines. The highest DNA-PK activity observed in TE-8 was more than two times higher than the lowest DNA-PK activity observed in TE-5. Significant correlation was observed between DNA-PK activity and D0 (r = 0.766; P = 0.0008). Western blots analysis revealed a significant correlation between DNA-PK activity and Ku70 expression, suggesting that the regulation in DNA-PK activity was associated with Ku70 expression. The data suggest that the measurement of DNA-PK activity and/or Ku70 expression may provide a useful way to predict radiation sensitivity. PMID- 10741737 TI - Loss of heterozygosity on chromosome 6p21.2 as a potential marker for recurrence after radiotherapy of human cervical cancer. AB - Cervical carcinomas develop as a result of multiple genetic alterations, and specific alterations lead to specific clinical behavior. However, the effect of such alterations on the recurrence of cervical cancer after radiotherapy remains unknown. Chromosome arm 6p is one of those most frequently involved in a loss of heterozygosity (LOH) in patients with cervical carcinoma. The aim of this study was to identify the correlation between the LOH on chromosome 6p21.2 and the recurrence of cervical cancer after radiotherapy. A total of 62 patients with cervical cancer (stage I, 4 patients; stage II, 9 patients; stage III, 37 patients; and stage IV, 12 patients) were included in this study. All patients were treated with definitive radiotherapy. We analyzed specimens from the tumors and venous blood of all patients. Tumors and normal DNA were analyzed by PCR for genetic losses at three polymorphic microsatellite loci (D6S276, D6S1624, and D6S1583). Chromosome 6p21.2 is involved in the LOH in 46.8% (29 of 62) of the informative carcinomas. Ten patients had a local recurrence, 4 had distant metastases, and 13 had both local recurrence and distant metastases after radiotherapy. To evaluate the relationship between the recurrence after radiotherapy and LOH on chromosome 6p21.2, we divided the patients into those with cancer recurrence (n = 27) and those without recurrence (n = 35). LOH on chromosome 6p21.2 was correlated with recurrence after radiotherapy (P = 0.006). The tumors in patients with recurrence were significantly larger than those in patients without recurrence (P = 0.003). However, there was no correlation between the sizes and stages of tumors and the LOH on chromosome 6p21.2. In addition, both overall survival and relapse-free survival were significantly worse for the patients with LOH as compared with those without LOH (P = 0.02 and P = 0.002, respectively). The results of this study suggest that LOH on 6p21.2 is correlated with recurrence of cervical carcinoma after radiotherapy. PMID- 10741738 TI - Overexpression level of stromelysin 3 is related to the lymph node involvement in non-small cell lung cancer. AB - Proteases contribute to tumor invasion and metastasis via their potential to degrade basement membranes and extracellular matrix. Our aim was to compare the level of several proteases: urokinase-type plasminogen activator (u-PA), matrix metalloproteinase 2 (MMP-2; 72-kDa type IV collagenase, also known as gelatinase A), MMP-11 [also known as stromelysin 3 (STR3)], and cathepsins B and L in resected non-small cell lung cancer. Between June 1996 and March 1998, samples of lung tumor tissues were taken from 119 surgically treated patients. Thirty out of the 119 tumor samples were matched with corresponding adjacent normal tissue. u PA was measured by a commercially available immunoluminometric assay. Metalloproteinases and cathepsins have been evaluated at the RNA level by Northern blot and quantified with a PhosphorImager. Expression of these proteases was compared to the following clinicopathological parameters: pathological diagnosis, tumor size, exposure to asbestos, radiotherapy, neo-adjuvant chemotherapy, tumor-node-metastasis stage, lymph node involvement, presence of metastasis. u-PA, MMP-2, MMP-11/STR3, and cathepsin B were significantly increased in tumor (the tumor:normal ratio was on average increased by 5.4-, 2.2 , 83.5-, and 2.2-fold, respectively). The tumor:normal ratio of MMP-11/ STR3 was found to be significantly linked to the lymph node involvement (P < 0.05). Our results suggest that several proteases are involved in the invasive potential of non-small cell lung cancer and that the quantification of MMP-11/ STR3 could represent an useful prognostic marker. PMID- 10741739 TI - Pattern of radiation-induced RET and NTRK1 rearrangements in 191 post-chernobyl papillary thyroid carcinomas: biological, phenotypic, and clinical implications. AB - Molecular genetic aberrations and the related phenotypes were investigated in 191 papillary thyroid carcinomas (PTCs) from patients exposed at young age to radioiodine released from the Chernobyl reactor. A high prevalence of RET gene rearrangements (62.3%) with a significant predominance of ELE1/RET (PTC3) over H4/RET (PTC1) rearrangements was found in PTCs of the first post-Chernobyl decade. NTRK1 rearrangements were rare (3.3%). In 3.3%, we observed novel types of RET rearrangements: GOLGA5/ RET (PTC5), HTIF/RET (PTC6), RFG7/RET (PTC7), and an as yet undefined RFGX/RET.RET rearrangements, preferentially ELE1/RET, are related to rapid tumor development. At longer intervals after exposure to ionizing radiation, the prevalence of RET rearrangements declines with a shift from ELE1/RET to H4/RET, most significantly in female patients. The prevalence of specific types of rearrangements is independent of age at irradiation. A significantly higher prevalence of ELE1/RET was observed in the most heavily contaminated Oblasts, Gomel and Brest, suggesting a preferential formation of this type of rearrangement after high thyroid doses. RET rearrangement is related to aggressive growth: Rearrangement-positive PTCs were in a more advanced pT category and more frequently in the pN1 category at presentation than rearrangement-negative PTCs. ELE1/RET is related to the solid variant of PTC, H4/RET more frequently to typical papillary structures. The genotype/phenotype evaluation of post-Chernobyl PTCs reveals a characteristic spectrum of gene rearrangements that lead to typical phenotypes with important biological and clinical implications. PMID- 10741740 TI - Disease control of uterine cervical cancer: relationships to tumor oxygen tension, vascular density, cell density, and frequency of mitosis and apoptosis measured before treatment and during radiotherapy. AB - Identification of biological parameters of major importance for the control of malignant diseases can be useful for the design of optimal treatment regimes for individual patients. Tumor oxygen tension (pO2), vascular density, cell density, and frequency of mitosis and apoptosis were measured before treatment (40 patients) and after 2 weeks of radiotherapy (22 patients) in patients with uterine cervical cancer. The aim was to investigate whether one of the parameters was more important for disease control than the others. Three sets of data were considered; the pretreatment parameters, the parameters measured after 2 weeks of radiation, and the changes in the parameters during this time. The pO2 was measured polarographically; the other parameters were determined by histological analyses of tumor biopsies. Hypoxic subvolume (HSV5), ie., the fraction of pO2 readings <5 mm Hg multiplied with tumor volume, showed the strongest correlation to control. Patients with a small HSVs before treatment had a higher control probability after a median follow-up time of 50 months than patients with a large HSV5 (P < 0.001). All other parameters or changes in parameters showed impaired correlation to control compared with pretreatment HSV5. The present results suggest that pretreatment oxygenation is more important for disease control of cervical cancer than the oxygenation after 2 weeks of radiotherapy or the changes in oxygenation during this time. Moreover, vascular density, cell density, and frequency of mitosis and apoptosis before treatment or after 2 weeks of therapy are probably not as important as pretreatment oxygenation as well. Although significant correlations between disease control and some of the parameters other than pretreatment oxygenation can occur in studies based on a large number of patients, the specificity of these parameters in the prediction of control is probably not as high as for oxygenation. PMID- 10741741 TI - Expression of cell cycle control proteins in primary colorectal tumors does not always predict expression in lymph node metastases. AB - Analysis of tumor markers focuses on expression in primary tumors with the assumption that this is representative of metastatic tumor, against which treatment is targeted. Few studies have compared the expression of such markers in primary and secondary tumors. In this study, several key genes involved in cell cycle regulation were investigated in colorectal tumors and corresponding lymph node metastases. The cell cycle regulators p53, cyclin D1, p21, p27, retinoblastoma protein (Rb), and proliferating cell nuclear antigen (PCNA) were examined in a series of 42 paired samples of primary colorectal and secondary lymph node tumors by immunohistochemistry. Expression of p53, p27, and Rb was similar in virtually all paired samples (p53, 38 of 42; p27, 39 of 42; Rb, 40 of 42), indicating that the pattern of these proteins in colorectal tumors may be used to predict that in lymph node tumors. It also suggests a lack of direct involvement in the metastatic process. A lower concordance for p21 and cyclin D1 staining was observed between primary and secondary tumors (p21, 19 of 42; cyclin D1, 22 of 42). p21 expression was more often observed in primary colorectal cancers, whereas cyclin D1 expression was more frequently seen in lymph node metastases, in keeping with the contrasting roles of these proteins as a cell cycle inhibitor (p21) and activator (cyclin D1). The PCNA-labeling index was found to vary considerably in a number of cases, thus limiting the ability to predict expression of this protein in lymph node metastases from the primary tumor. In addition, PCNA-labeling indices between paired samples were neither consistently higher nor lower, suggesting that the proliferative capacity of tumor cells is not directly related to their ability to metastasize. PMID- 10741742 TI - Microsatellite alterations plasma DNA of primary breast cancer patients. AB - The aim of this study was to analyze plasma DNA from primary and metastatic breast cancer cases for tumor-specific alterations and to compare these findings with immunocytochemistry and estimation of cytokeratin 19 (CK19) mRNA for detection of micrometastases. DNA was extracted from plasma, lymphocytes, and microdissected tumor tissue sections obtained from 71 patients with breast cancer and 9 controls. DNA samples were analyzed for loss of heterozygosity (LOH) and/or microsatellite instability (MI) by PCR with two polymorphic markers (DM-1 and D16S400). Reverse transcription-quantitative PCR (QPCR) and immunocytochemistry were used for detection of CK19 mRNA and protein. Breast cancer plasma DNA displayed frequent LOH (31.3%) and MI (11.6%) supported by the same alteration in microdissected tumor DNA. Most notably, 10 of the 39 patients with primary breast cancer showed LOH (n = 6) or MI (n = 4). We compared plasma tumor DNA, plasma and bone marrow QPCR, and blood and bone marrow immunocytochemistry in 32 of the patients with primary cancer. Of these, only one patient had immunocytochemically detectable carcinoma cells in the blood, and three showed abnormally high levels of plasma CK19 mRNA. All four of these patients had plasma DNA alterations. We then compared bone marrow findings: of the 10 primary breast cancers that showed LOH or MI, 6 had elevated CK19 mRNA and 5 had immunocytochemically positive cells. Tumor DNA is readily detectable in plasma of primary and metastatic breast cancer patients, and plasma DNA alterations (LOH and MI) reflect those seen in the tumor. The application of microsatellite analyses to plasma DNA may be useful in assessing tumor burden in breast cancer patients, particularly when combined with QPCR, and is preferable for patients with breast cancer, for whom sequential bone marrow aspiration is undesirable. PMID- 10741743 TI - Predictive survival markers in patients with surgically resected non-small cell lung carcinoma. AB - Among patients with resected non-small cell lung carcinoma, about 50% will present a tumor recurrence. Thus, it would be of major importance to be able to predict and try to prevent these relapses by an active chemotherapy and/or radiotherapy. In an attempt to answer this question, the tumors of 227 patients with a surgically resected non-small cell lung carcinoma were evaluated as follows: tumors were classified as squamous cell carcinoma (n = 132) or adenocarcinoma (n = 95), and tumor differentiation was evaluated for each type. Then, all tumors were classified in respect to their pathological TNM staging (WHO) and screened by immunohistochemistry for the detection of the expression of the following antigens: Bcl-2, A+B+H blood group antigens, c-erb-b2, p53, and Pan Ras antigens. Furthermore, adenocarcinomas were screened for the presence of point mutations in Ki-Ras codons 1-31. Finally, the patient blood group was defined, and patient survival was analyzed using nonparametric tests and proportional hazard Cox models. Using Kaplan-Meier survival curves, disease pathological TNM staging was shown to be a strong predictive factor of survival for both squamous cell carcinoma and adenocarcinoma. Patients with squamous cell carcinoma experienced fewer relapses than those with adenocarcinoma (42% versus 63%; P = 0.0002) and had a significantly better survival. All evaluated antigens were more often present in squamous cell carcinoma than in adenocarcinoma except for Pan-Ras (three times more frequent in adenocarcinoma). In patients with squamous cell carcinoma, only tumor staging had a significant prognosis value (P = 0.01). In patients with lung adenocarcinoma, a well-differentiated tumor (P = 0.009) as well as a positive Bcl-2 staining (P = 0.009) and an A+B+H antigen tumor staining (P = 0.024) were associated with a better survival. In contrast, patients with a stage I or II disease and a p53-positive tumor staining and patients with the O blood group (P = 0.01) had a shorter survival. Interestingly, no relation with patient survival was related to c-erb-b2 and Pan-Ras staining. Finally, 12 point mutations were found out of 81 tumors (15%) evaluated for Ki Ras codons 1-31; they involved codon 12 but also 8, 14, and 15 without any relationship to survival. In respect to lung adenocarcinoma, using Cox proportional hazard models stratified on tumor staging, the following markers were shown to be related to survival: (a) Independent markers of longer survival (ie., high histological degree of tumor differentiation and positive Bcl-2 and A+B+H blood group antigen expression by tumor cells); and (b) Independent markers of shorter survival (i.e., O blood group for all patients and p53 tumor staining in patients with stage I and II diseases). This study suggests that, in patients who undergo surgery for lung adenocarcinoma, the presence or absence of these criteria could be used to define a subset of patients who may benefit from a more specific follow-up. PMID- 10741744 TI - Elevated levels of p66 Shc are found in breast cancer cell lines and primary tumors with high metastatic potential. AB - The adapter molecule Shc has been implicated in specific steps of metastasis. In the current study, we show that the expression and activation of the p66 Shc isoform increased in a highly metastatic variant (F-11) of the human breast cancer cell line MDA-MB-231 compared to the parent cell line, whereas the p46 and p52 Shc isoforms were unchanged. Despite reports that p66 Shc can negatively regulate epidermal growth factor signaling to the mitogen-activated protein kinase pathway, we found no change in epidermal growth factor-stimulated activation of mitogen-activated protein kinase in the F-11 cell line. We determined the level of Shc expression by immunoblot in primary breast cancer specimens obtained from patients with or without axillary node involvement. p66 Shc expression increased in tumors obtained from node-positive patients (Spearman correlation coefficient = 0.43377; P = 0.0058) compared to the node-negative specimens. Furthermore, increasing levels of p66 Shc correlated with an increasing number of positive nodes (P = 0.032). This study shows that p66 Shc expression increased in cultured breast cancer cells selected for metastasis and in primary human breast cancer specimens obtained from patients with lymph node involvement, suggesting a possible role for Shc in human breast cancer metastasis. PMID- 10741745 TI - Differential expression of osteonectin/SPARC during human prostate cancer progression. AB - The precise mechanism(s) involved in invasion and metastasis of prostate cancer (CaP) is poorly understood. Osteonectin [ON (also known as SPARC or BM-40)] is an antiadhesive protein known to be involved in cell-matrix interactions, migration, and angiogenesis. In this report, we studied the expression of ON in human prostate cell lines, primary tumors, and metastatic foci of CaP. Reverse transcription-PCR and nonradioactive in situ hybridization (ISH) techniques were used to determine ON gene expression. Immunohistochemistry was carried out using the polyclonal antibody LF37 and/or the monoclonal antibody ON-mAb. Low to moderate levels of ON mRNA and protein were observed in glandular epithelial cells of normal tissue as well as a few primary CaPs. However, high levels of ON mRNA and protein were observed in most of the CaP metastatic foci, both osseous and nonosseous. This correlated well with our findings that multiple different CaP cell lines including four CaP cell lines derived from metastases show high levels of ON gene expression. Furthermore, ISH analyses and cell-specific reverse transcription-PCR evaluation showed that both the luminal and basal cells express the ON gene. We conclude that the differential pattern of ON expression suggests that it may play an important role in the progression of CaP. PMID- 10741746 TI - Prognostic factors in Hodgkin's disease: multivariate analysis of 327 patients from a single institution. AB - On the basis of a retrospective study of 327 patients with Hodgkin's disease (HD), the prognostic significance of several factors, accepted previously and recently proposed, has been analyzed with regard to response to treatment and the survival time. Multivariate regression analysis strongly decreased the number of potentially prognostic parameters. The only independent, pretreatment factors negatively influenced by either time of survival or response to treatment were the following: age at diagnosis of more than 45 years, advanced (IIIB/IV) clinical stage, poor clinical status according to Karnofsky's scale (score less than 70), presence of systemic symptoms, mixed cellularity/lymphocyte depletion histological type, multisite peripheral nodal localization of the disease, abdominal lymphadenopathy, and large primary tumor mass (bulky disease). Short time to achieve complete remission (during the first four courses of chemotherapy) has proven to be significantly positive predictive factor. Cumulative dose of cytostatics lower than programmed was a significantly negative prognostic factor that correlated with a shorter time of survival. Lack of or a too-low dose of radiotherapy had the same predictive value. High activity of serum lactate dehydrogenase correlated moderately with poor response to the first line treatment but did not influence the survival time. Other clinical, morphological, and biochemical parameters influenced neither the prognosis nor the response to treatment. Additionally, immunohistochemical examinations for proliferating cell nuclear antigen and the protein products of the p53 and bcl-2 genes were performed on the lymph nodes obtained from the patients with HD. High expression of proliferating cell nuclear antigen, p53, and BCL-2 correlated with poor response to the treatment and/or short time of survival. Statistical analysis has led us to the conclusion that the pretreatment expression of these oncoproteins can be taken into consideration as a new prognostic factor in HD. PMID- 10741747 TI - Vascular endothelial growth factor expression predicts outcome and lymph node metastasis in squamous cell carcinoma of the esophagus. AB - Vascular endothelial growth factor (VEGF) expression and tumor microvessel density (MVD) were examined by immunohistochemical staining in 117 cases of thoracic esophageal squamous cell carcinoma. Thirty-six (31%) of the 117 cases were evaluated as VEGF-positive. The average number of metastatic lymph nodes at surgery was 5.6 in the VEGF-positive cases and 3.0 in the VEGF-negative cases and was significantly higher in those with VEGF-positive cases (P = 0.04). The incidence of pathological tumor (PT)2-4 cases among the high-MVD cases was significantly higher than among the low-MVD cases (P = 0.01). MVD was 59.4 +/- 4.7 (mean +/- SE)/mm2 in the VEGF-positive cases and 47.9 +/- 3.8/mm2 in the VEGF negative cases. The MVD of the VEGF-positive tumors was higher than that of VEGF negative tumors, but the difference was not significant (P = 0.08). The survival rate of the patients with high-MVD tumors was significantly poorer than those with low-MVD tumors, and the survival rate of those patients with VEGF-positive tumors was significantly poorer than in those with VEGF-negative tumors (P = 0.009 and P = 0.04, respectively). The cumulative survival rates in the VEGF positive groups were found to be significantly poorer in the pT3 and pathological node (pN)1 groups when stratified according to pT factor (pathological T category) and pN factor (pathological N category) in the tumor-node-metastasis (TNM) classification. VEGF expression had the second highest hazard ratio in the multivariate analysis, after pN factor. These results indicate that VEGF is a useful marker for predicting the outcome in patients with more advanced esophageal squamous cell carcinoma. It seems that TNM factors and VEGF expression are important factors in the selection of appropriate treatments. PMID- 10741748 TI - Clinical significance of matrix metalloproteinase-7 expression in esophageal carcinoma. AB - Matrix metalloproteinase-7 (MMP-7) is a member of MMP family and has a wide variety of substrate spectra. It is reported to play an important role in carcinoma invasion and metastasis. There is, however, little information on the clinical significance of MMP-7 in human esophageal carcinoma. We thus studied 48 tumor/normal pair samples of human esophagus by Northern blot analysis. The results demonstrated that the tumor tissue (T) of esophageal carcinoma showed a higher expression of MMP-7 mRNA than the corresponding normal tissue (N) in 31 cases (65%). We also statistically evaluated tumor MMP-7 value (T value) corrected for MMP-7-positive control (KYSE150 transfected with the MMP-7 gene). Fourteen cases with T value > or = 0.3 showed a higher frequency of lymph node metastasis than 34 cases with T value < 0.3 (P < 0.05). The cases with T value > or = 0.3 showed a significantly poorer prognosis than those with T value < 0.3 (P < 0.01). Multivariate analysis demonstrated that the MMP-7 expression status was the independent factor relating to the prognosis (P = 0.0005). The findings indicated that MMP-7 might be a novel prognostic factor for patients with esophageal carcinoma. PMID- 10741749 TI - Clinical significance of serum soluble intercellular adhesion molecule 1 in gastric cancer. AB - We studied the correlation between serum soluble intercellular molecule 1 (sICAM 1) and clinicopathological features in patients with gastric cancer. The impact of sICAM-1 on prognosis was also evaluated. The sera from 224 patients with gastric cancer, 44 healthy individuals, and 35 patients with benign gastrointestinal diseases (4 patients with submucosal stomach tumors, 6 patients with gastric ulcers, 1 patient with Crohn disease, 2 patients with ulcerative colitis, 7 patients with gall stones, 5 patients with chronic pancreatitis, and 10 patients with liver cirrhosis) were measured for sICAM-1 titer using a sandwich enzyme immunoassay method. There was no correlation between the serum titer of sICAM-1 and the age or gender of healthy controls. Among patients with benign gastrointestinal diseases, the patients with liver cirrhosis had a significantly higher mean serum sICAM-1 titer than that of healthy controls (P < 0.0001). The mean serum sICAM-1 titer of all patients with gastric cancer was not significantly different from that of healthy controls. However, among the patients with stage IV and recurrent disease, the serum sICAM-1 titer of those with hematogenous metastasis was significantly higher than that of patients without hematogenous metastasis (P = 0.001). The patients with a high serum sICAM 1 titer of more than 304 ng/ml (mean of healthy controls plus SD) showed a significantly worse prognosis than patients with a low serum sICAM-1 titer (P = 0.010). Nevertheless, serum sICAM-1 titer was not an independent predictor of prognosis by multivariate analysis. In conclusion, serum sICAM-1 cannot be used as a tumor marker for early diagnosis. However, sICAM-1 in sera may still be worthwhile to measure for monitoring hematogenous metastasis. PMID- 10741750 TI - Characterization of signaling cascades triggered by human interleukin-6 versus Kaposi's sarcoma-associated herpes virus-encoded viral interleukin 6. AB - Kaposi's sarcoma-associated herpes virus (KSHV) is associated with Kaposi's sarcoma, multicentric Castleman's disease, and body cavity-based lymphomas, settings in which human interleukin-6 (hIL-6) acts as a growth factor. The KSHV open reading frame K2 encodes for viral IL-6 (vIL-6), a protein with 25% amino acid identity to hIL-6, which can promote the growth of hIL-6-dependent cell lines. In the present study, we characterized biological sequelae and signaling cascades triggered by hIL-6 versus vIL-6 in the hIL-6-dependent MH60 and B9 cell lines. Both hIL-6 and vIL-6 induced significant increases (P < 0.01) in DNA synthesis in these cell lines in a dose-dependent fashion. Neutralizing anti-hIL 6 antibody (Ab) inhibited DNA synthesis triggered by hIL-6, without similarly affecting proliferation in response to vIL-6. On the other hand, antimouse IL-6 receptor (mIL-6R) Ab blocked response to vIL-6, but not that to hIL-6. Both hIL-6 and vIL-6 activated gp130, Janus kinase 1, signal transducers and activators of transcription-3, and mitogen-activated protein kinase in both MH60 and B9 cells. Proliferation of these cell lines in response to both hIL-6 and vIL-6 was blocked by PD98059, an inhibitor of MEK1 activation. These data suggest that MEK1 activation mediates the proliferative response to both cytokines. Finally, both hIL-6 and vIL-6 also maintained viability of serum-starved MH60 and B9 cells and blocked dexamethasone-induced apoptosis of MM.1S human myeloma cells. Further characterization of the signaling cascades mediating the growth and antiapoptotic effects of vIL-6 versus hIL-6 may help identify their unique roles in disease pathogenesis in Kaposi's sarcoma and other KSHV-associated neoplasms. PMID- 10741751 TI - TabBO: a model reflecting common molecular features of androgen-independent prostate cancer. AB - We established two human prostate cancer cell lines, MDA PCa 2a and MDA PCa 2b, the TabBO model system, that reflect common features of human androgen independent prostate cancer that are not present in other model systems: bone origin, prostate-specific antigen production, androgen receptor expression, and androgen sensitivity. We therefore hypothesized that molecular pathways in our model system reflect common alterations responsible for the progression of a subset of human prostate cancer. Progression to androgen independence has been hypothesized to be largely associated with impairment of the regulation of cell growth or apoptosis of prostate cancer cells. Therefore, in this study, we examined molecular markers known or suspected to be important in prostate cancer progression and key regulators of cell growth and apoptosis: p53, p21WAF1/CIP1, Bcl-2, Bax, retinoblastoma (Rb), and p16INK4A/MITS1. We analyzed the expression of these markers in the cell lines, their tumor of origin, and tumors derived from the cell lines by s.c. inoculation into nude mice. DNA sequencing of the entire open reading frames of the p53 and p21 genes revealed no mutations. Additionally, accumulation of the p53 protein was not found by Western blot analysis, nor was overexpression of the Bcl-2 oncoprotein detected. Bax expression was detected in MDA PCa 2a cells, whereas it was absent in MDA PCa 2b. Rb and p16 protein expression was normal as measured by both Western blot and immunochemical analyses. Immunohistochemical studies of p53, p21, Bcl-2, and Rb in both samples from the original human cancer from which the lines were derived and mouse xenografts derived from the lines revealed similar levels of protein. These results are consistent with reports indicating that 40-50% of bone metastases of prostate cancer have wild-type p53, 50-70% do not overexpress the Bcl-2 protein, and mutations in the p21 gene are rare. Therefore, we conclude that MDA PCa 2a and MDA PCa 2b reflect molecular pathways in a common subset of human androgen-independent prostate cancer and that important molecular players in apoptosis (namely, p53 and Bcl-2) seem to be intact in this subset of androgen independent prostate cancer. Understanding the signal-transduction pathways operating in these cell lines may help to identify therapeutic targets for prostate cancer. PMID- 10741752 TI - Lovastatin augments apoptosis induced by chemotherapeutic agents in colon cancer cells. PMID- 10741753 TI - Intra-atrial communication and control of atrial natriuretic factor (ANF) release. AB - Atrial natriuretic factor (ANF) release was studied in isolated perfused atria prepared from rats. When the vein-atrial junction (VAJ) was distended with an inflatable balloon, ANF release into the perfusate was greater in intact atria than in appendectomized atria. It was concluded that distention of the VAJ causes ANF release from the atrial appendage. A cascade experiment was then prepared whereby buffer from one isolated atrium perfused a second atrium. Although the VAJ of the first atrium could be distended by balloon, the atrial appendage was ligated so ANF was not secreted into the perfusate. The second atrium was intact, but no balloon was inserted. Despite the fact that there were no changes in intraluminal pressure, ANF secretion from the second atrium increased when the VAJ of the first atrium was distended. This response was blocked by the endothelin (ET) A receptor antagonist BQ-123. However, no distention-induced changes in ET-1 levels could be found in the perfusate from the first atrium. It is proposed that, in response to changes in distention of the VAJ, ANF is released remotely from the atrial appendage. The mediator does not appear to be ET-1 itself, but rather some factor that stimulates ET-1-induced ANF release within the tissue of the atrial appendage. PMID- 10741754 TI - Dopamine D3 receptor binding by D3 agonist 7-OH-DPAT (7-hydroxy dipropylaminotetralin) and antipsychotic drugs measured ex vivo by quantitative autoradiography. AB - Because the dopamine D3 receptor is primarily expressed in regions of the limbic system of brain, it was proposed that it may represent a target for antipsychotic drugs that is free of extrapyramidal side effects. An ex vivo receptor binding technique employing [3H]7-OH-DPAT was used to evaluate in vivo occupancy of dopamine D3 receptors in the rat nucleus accumbens by selective D3 agonist 7-OH DPAT (7-hydroxy-dipropylaminotetralin) and various antipsychotic drugs. With an ID50 value of 0.07 mg/kg, the selective D3 agonist (+)-7-OH-DPAT had the most potent inhibitory effect on ex vivo binding of [3H]7-OH-DPAT among all drugs tested. Clinical doses of phenothiazine drugs, such as chlorpromazine and levomepromazine, induce binding to D3 receptors in vivo, while atypical antipsychotic drugs, such as clozapine, pimozide, and sulpiride, are very weak in inhibiting ex vivo binding of [3H]7-OH-DPAT, indicating that the role of D3 receptors as targets of antipsychotic drugs free of extrapyramidal side effects may not be important. PMID- 10741755 TI - Effects of diabetes and hypertension on myocardial Na+-Ca2+ exchange. AB - Abnormalities in cardiac function have been extensively documented in experimental and clinical diabetes. These aberrations are well known to be exaggerated when hypertension and diabetes co-exist. The objective of the present study was to examine whether alterations in the activity of the myocardial Na+ Ca2+ exchanger (NCX) can account for the deleterious effects of diabetes and (or) hypertension on the heart. To this aim, the following experimental groups were studied: (i) control; (ii) diabetic; (iii) hypertensive; and (iv) hypertensive diabetic. Wistar rats served as the control group (C) while Wistar rats injected with streptozotocin (STZ, 55 mg/kg) served as the diabetic (D) group. Spontaneously hypertensive (SH) rats were used as the hypertensive group (H) while SH rats injected with STZ served as the hypertensive-diabetic (HD) group. Sarcolemma was isolated from the ventricles of the C, D, H, and HD groups and NCX activity was examined using rapid quenching techniques to study initial rates over a [Ca2+]o range of 10-160 microM. The Vmax of NCX was lower in the D group when compared with the C group (D, 2.96 +/- 0.26 vs. C, 4.0 +/- 0.46 nmol x mgprot(-1) x s(-1), P < 0.05), however combined diabetes and hypertension (HD) did not affect the Vmax of NCX activity (HD, 3.84 +/- 0.88 vs. H, 3.59 +/- 0.24 nmol x mgprot(-1) x s(-1), P > 0.05). However, analysis of the Km values for Ca2+ indicated that both the D and HD groups exhibited a significantly lower Km when compared with their respective control groups (D, 42 +/- 4 vs. C, 56 +/- 4 microM, P < 0.05; HD, 33 +/- 7 vs. H, 51 +/- 8 microM, P < 0.05). Immunoblotting using polyclonal antibodies (against canine cardiac NCX) exhibited the typical banding of 160, 120, and 70 kDa. The 120 kDa band is believed to represent the native exchanger with its post-translational modifications. Examination of the blots revealed a lower intensity of the 120 kDa band in the D group when compared with the C group, however, no significant difference in the HD group was observed. We speculate that the lower Vmax in the D group may be due to a reduced concentration of exchanger protein in the membrane. The absence of this defect in the HD group may be a result of compensatory mechanisms to the overall hemodynamic overload, however, this remains to be determined. The increased affinity for Ca2+ in both the D and HD groups (determined by the lower Km values) is an interesting finding and may be due to changes in sarcolemmal lipid bilayer composition secondary to diabetes-induced hyperlipidemia. PMID- 10741756 TI - Three different vasoactive responses of rat tail artery to nicotine. AB - The vasoactive effects of nicotine on isolated rat tail artery tissues were studied. Nicotine transiently contracted rat tail artery tissues (EC50, 55.6 +/- 2 microM) in an extracellular Ca2+ dependent and endothelium-independent fashion. The blockade of alpha1-adrenoceptors, but not alpha2-adrenoceptors or P2X purinoceptors, inhibited the nicotine-induced contraction by 38 +/- 7% (p < 0.05). Nicotine (1 mM) depolarized membrane by 13 +/- 3 mV, but did not affect L type Ca2+ channel currents, of the isolated rat tail artery smooth muscle cells. The phenylephrine-precontracted tail artery tissues were relaxed by nicotine (EC50, 0.90 +/- 0.31 mM), which was significantly inhibited after the blockade of nicotinic receptors. Simultaneous removal of phenylephrine and nicotine, after a complete relaxation of the phenylephrine-precontracted tail artery strips was achieved by nicotine at accumulated concentrations (> or =10 mM), triggered a Ca2+-dependent rebound long-lasting vasoconstriction (n = 20). This rebound contraction was abolished in the absence of calcium or in the presence of tetracaine in the bath solution. Pretreatment of vascular tissues with a nicotinic receptor antagonist did not affect the nicotine-induced vasoconstriction or nicotine withdrawal induced rebound contraction. The elucidation of the triphasic vascular effects of nicotine and the underlying mechanisms is important for a better understanding of the complex vascular actions of nicotine. PMID- 10741757 TI - Results of simulated mastication suggest existence of a periodontogastric motility reflex. AB - Various reflexes inhibit gastric motor activity. Might a contrary one permit the oral region to increase gastric motility? Ten fasted rats were allowed to feed for 15 min. Following anesthesia and cannula insertion, antral pressure was recorded during three consecutive 5-min intervals: the baseline, procedure, and postprocedure periods. The procedure involved manually lowering and raising the mandible about once per second, causing repetitive molar occlusion. Doing this when food is in the stomach resembles conditions as the latter part of a meal is consumed. Gastric motor events increased from 1.10 +/- 1.67 (mean +/- SD) to 5.50 +/- 4.12 per 5 min during the procedure (p < 0.05) and 5.80 +/- 3.97 in the ensuing period (p < 0.05). The findings suggest an excitatory reflex following stimulation of mechanoreceptors in one or more sites related to mastication: the periodontium, temporomandibular joints, or masticatory muscles. Because rubbing the maxillary molars while the mouth remained constantly open also increased motor events, the periodontium is the most likely location of the receptors. They and associated trigeminal neurons would comprise the reflex's afferent arm. The vagi, perhaps with intermediaries, are its likely efferent arm. In these recently fed rats this reflex acts despite receptive relaxation and enterogastric reflexes to increase distal gastric motor activity. PMID- 10741758 TI - Nitric oxide inhibits norepinephrine-induced hepatic vascular responses but potentiates hepatic glucose output. AB - We previously reported that sympathetic nerve-induced vasoconstriction in the intestine resulted in shear stress induced release of nitric oxide (NO) that led to presynaptic inhibition of transmitter release. In contrast, studies in the liver suggested a postsynaptic inhibition of vascular responses, thus leading to the hypothesis tested here that maintained catecholamine release in the liver would result in maintained metabolic catecholamine action in the face of inhibition of vascular responses. In rats, norepinephrine (NE) induced elevations in arterial glucose content were inhibited by NO synthase antagonism (N(omega) nitro-L-arginine methyl ester (L-NAME), 10 mg/kg, intraportal) but potentiated by NO donor administration (3-morpholinosydnonimine (SIN-1), 0.2 mg/kg, intraportal). The potentiated effect of SIN-1 was abolished by indomethacin (7.5 mg/kg, intraportal). To confirm the hepatic site of metabolic effect, cats were used so that blood flow and hepatic glucose balance could be determined. SIN-1 potentiated NE-induced glucose output from the liver from 5.0 +/- 0.4 to 7.2 +/- 0.6 mg x min(-1) x kg(-1). The potentiation was blocked by methylene blue, a guanylate cyclase inhibitor. Contrary to the glucose response, L-NAME potentiated but SIN-1 attenuated NE-induced portal vasoconstriction. Thus NO is shown to produce differential modulation of vascular and metabolic effects of NE. Vasoconstriction of the hepatic vasculature is inhibited by NO, whereas the glycogenolytic response to NE is potentiated, responses that are probably mediated by prostaglandin. PMID- 10741759 TI - Chronic blockade of brain "ouabain" prevents sympathetic hyper-reactivity and impairment of acute baroreflex resetting in rats with congestive heart failure. AB - In rats with congestive heart failure (CHF) post myocardial infarction (MI) acute blockade of brain "ouabain" reverses sympathetic hyperactivity and chronic blockade prevents the desensitization of baroreflex function. This study was conducted to determine: i) if chronic blockade of brain "ouabain" maintains normal sympathetic reactivity; and ii) if acute baroreflex resetting (another parameter of baroreflex function) also becomes impaired, and if so, does brain "ouabain" contribute to impairment in acute baroreflex resetting. CHF post MI was induced by acute coronary artery ligation in Wistar rats. Animals were treated with 200 microg x day(-1) i.c.v. or i.v. Fab fragments (which bind brain "ouabain" with high affinity), or treated with 200 microg x day(-1) i.c.v. gamma globulins (control group). The length of treatment was 0.5-8 weeks or 4-8 weeks post MI. At 8 weeks mean arterial pressure (MAP), central venous pressure (CVP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were recorded in concious rats at rest and in response to: i) air-jet stress, ii) i.c.v. guanabenz (an alpha2-adrenoceptor agonist), and iii) a 30 min i.v. infusion of nitroprusside (NP). Excitatory responses to air stress and inhibitory responses to guanabenz of MAP, HR, and RSNA were significantly enhanced in rats with CHF versus the sham-operated treated group. This enhancement was prevented in the CHF group treated with i.c.v., but not i.v., Fab. Nitroprusside induced a sustained decrease in MAP (approximately 25 mmHg) and a transient decrease in CVP. Heart rate and RSNA increased significantly within 1 min of beginning the infusion. The peak increases as well as the product of changes in MAP-HR and RSNA-HR were significantly smaller in rats with CHF treated with gamma-globulins versus sham rats and versus CHF rats treated with i.c.v. Fab. In sham-operated rats and CHF rats treated with i.c.v. Fab, RSNA and HR began to decrease within 3-4 min of beginning the NP infusion and had returned to baseline by 20 min. In contrast, RSNA and HR remained increased throughout the infusion in the CHF rats treated with gamma-globulins. These data indicate that in rats with CHF acute resetting of the arterial baroreflex in response to a lower BP becomes impaired, and chronic blockade of brain "ouabain" prevents both this change in baroreflex resetting as well as sympathetic hyperactivity. PMID- 10741760 TI - Baroreceptors, alpha1-adrenergic receptors, and regulation of mesenteric blood flow. AB - In the mesenteric circulation of the rat a myogenic autoregulatory system operates at 0.1-0.15 Hz. Negative admittance phase in the region above 0.2 Hz suggested operation of an arterial baroreflex. The present study was designed to test this interpretation and to identify the neurotransmitter involved. In rats anesthetized with isoflurane, blood pressure and mesenteric blood flow (transit time ultrasound) were measured with central mechanisms intact, after sinoaortic denervation, and after denervation of the mesenteric bed. Sinoaortic denervation abrogated the negative phase in the band from 0.3 to 0.6 Hz and increased admittance gain in this region. Subsequent mesenteric denervation had no further effect on the pressure-flow transfer function. In a separate experiment, alpha1 adrenergic blockade reduced, but did not remove, the negative admittance phase in the 0.2- to 0.5-Hz band without altering admittance gain. It is concluded that the baroreflex acting on the mesenteric circulation can be identified by admittance phase, but that admittance gain is uninformative. Part of the response is mediated by alpha1-adrenergic transmission. PMID- 10741761 TI - Specific binding of [3H]ketanserin to hypothalamus membranes of juvenile rainbow trout, Oncorhynchus mykiss. AB - This study examines the existence and pharmacological specificity of [3H]ketanserin binding in hypothalamus of juvenile rainbow trout. Hypothalamic membranes were incubated with [3H]ketanserin (selective 5HT2 antagonist) under several experimental conditions; reactions were terminated by filtration and bound radioactivity was counted by liquid scintillation spectroscopy. Tissue dilution experiments revealed that specific [3H]ketanserin binding (B(sp)) was tissue dependent; 1 hypothalamus equivalent per tube (1100 +/- 115 cpm/mg protein) was subsequently used throughout the rest of this study. In association experiments, B(sp) increased progressively with time, achieved equilibrium binding levels (1192 +/- 120 cpm/mg protein) within 80 min, and remained stable for at least 60 min thereafter; k(obs), and k(+1) were 0.032 and 0.048 min(-1) x nM(-1), respectively. In dissociation experiments, B(sp) completely dissociated within 20 min following addition of excess ketanserin; k(-1) and t1/2 were 0.0803 min(-1) and 8.7 min, respectively. B(sp) was saturable (2500 +/- 256 cpm/mg protein); Scatchard-calculated values for the equilibrium dissociation constant (K(D)) and capacity (Bmax) were 0.48 nM, and 125 fmol/mg protein, respectively. B(sp) was differentially displaced by structurally related competitors, with a rank order of potency of ketanserin = mianserin > ritanserin > serotonin (5HT) = spiperone >> methiothepin mesylate > metergoline = DOI ((+/-)-2-5-dimethoxy-4 iodoamphetamine hyrobromide) > 2-methyl-5HT > alpha-methyl-5HT >>>> 5HIAA (5 hydroxyindole acetic acid) = reserpine. These findings provide pharmacological evidence for the presence of a 5HT2-like receptor subtype in the trout hypothalamus. PMID- 10741762 TI - Effect of hyperoxia and hypoxia on leg blood flow and pulmonary and leg oxygen uptake at the onset of kicking exercise. AB - The purpose of this study was to examine the interactions of adaptations in O2 transport and utilization under conditions of altered arterial O2 content (CaO2), during rest to exercise transitions. Simultaneous measures of alveolar (VO2alv) and leg (VO2mus) oxygen uptake and leg blood flow (LBF) responses were obtained in normoxic (FiO2 (inspired fraction of O2) = 0.21), hypoxic (FiO2 = 0.14), and hyperoxic (FiO2 = 0.70) gas breathing conditions. Six healthy subjects performed transitions in leg kicking exercise from rest to 48 +/- 3 W. LBF was measured continuously with pulsed and echo Doppler ultrasound methods, VO2alv was measured breath-by-breath at the mouth and VO2mus was determined from LBF and radial artery and femoral vein blood samples. Even though hypoxia reduced CaO2 to 175.9 +/- 5.0 from 193.2 +/- 5.0 mL/L in normoxia, and hyperoxia increased CaO2 to 205.5 +/- 4.1 mL/L, there were no differences in the absolute values of VO2alv or VO2mus across gas conditions at any of the rest or exercise time points. A reduction in leg O2 delivery in hypoxia at the onset of exercise was compensated by a nonsignificant increase in O2 extraction and later by small increases in LBF to maintain VO2mus. The dynamic response of VO2alv was slower in the hypoxic condition; however, hyperoxia did not affect the responses of oxygen delivery or uptake at the onset of moderate intensity leg kicking exercise. The finding of similar VO2mus responses at the onset of exercise for all gas conditions demonstrated that physiological adaptations in LBF and O2 extraction were possible, to counter significant alterations in CaO2. These results show the importance of the interplay between O2 supply and O2 utilization mechanisms in meeting the challenge provided by small alterations in O2 content at the onset of this submaximal exercise task. PMID- 10741763 TI - Effect of blood flow and muscle contraction on noradrenaline spillover in the canine gracilis muscle. AB - Many authors have reported that, during exercise, noradrenaline spillover increases and fractional extraction decreases. It has been suggested that the increase in blood flow to active muscles may contribute to these effects. Muscle contraction also causes changes in many factors that may affect noradrenaline spillover and fractional extraction. In this experiment, we studied the effect of muscle contraction and blood flow on noradrenaline and adrenaline spillover and fractional extraction in the in situ canine gracilis muscle. The low intensity stimulation protocol enabled us to have muscle contractions without any effect on the local concentration of noradrenaline, as measured by microdialysis, and noradrenaline spillover. Fractional extraction of both noradrenaline and adrenaline was unaffected by increasing blood flow three and four times its resting value. In addition, noradrenaline spillover was increased by the higher blood flow, from 188 to 452 pg x min(-1) at rest and from 246 to 880 pg x min(-1) during stimulation. Stimulation of muscle contraction caused a significant increase in fractional extraction of noradrenaline and a nonsignificant increase in adrenaline extraction. In addition, an adrenaline spillover was observed in certain conditions. In light of our results, it seems that blood flow may not be the main factor decreasing fractional extraction of noradrenaline during exercise. However, blood flow could contribute to the increase in noradrenaline spillover observed in the active muscles during exercise. PMID- 10741764 TI - Proteinase-activated receptors (PARs): activation of PAR1 and PAR2 by a proteolytic fragment of the neuronal growth associated protein B-50/GAP-43. AB - The neuronal growth associated protein B-50/GAP-43 has been localized in synaptosomes both as an intact protein and as a partial proteolysis product (termed B-60) that has an N-terminal sequence SFRGHITR.... Because of the relationship of this amino acid sequence to those of the tethered ligand for the human proteinase activated receptors PAR1 (SFLLRN...) and PAR2 (SLIGKV...), we wished to determine whether the B-50/GAP-43-derived proteolytic fragment SFRGHITR (SFR(B60)) might function as a PAR-activating peptide (PAR-AP) to stimulate either PAR1 or PAR2. With the use of a newly developed PAR1/PAR2 receptor activation-desensitization assay, employing PAR1/PAR2-bearing cultured human embryonic kidney (HEK293) cells, we found that SFR(B60) could activate both PAR1 and PAR2 so as to elevate intracellular calcium with EC50 values of approximately 200 and 50 microM, respectively. We also showed that trypsin can rapidly degrade B-50 to smaller fragments that would include the sequence SFR(B60). Because PAR1 and PAR2 are present on neurones, our data raise the possibility that in certain circumstances in vivo, B-50/GAP-43 may play a signalling role by serving as a precursor for proteolytically generated PAR-activating peptides. PMID- 10741765 TI - Obstructive, occupational, and environmental diseases. PMID- 10741766 TI - Computed tomographic diagnosis of chronic obstructive pulmonary disease. AB - Recently, two-dimensional high-resolution computed tomography (2D-HRCT) and volumetric high-resolution computed tomography (VHRCT) have allowed great progress to be made in estimating small regions of hypoattenuation in the lung. VHRCT allows minimum-intensity projection (MINIP) imaging, which seems to be much more suitable than conventional 2D-HRCT for detecting subtle hypoattenuating regions, although the reliability of MINIP imaging data has not been conclusively shown. Three-dimensional computed tomography (3D-CT) images reconstructed from helical computed tomography (CT) acquisition over the whole lung field have been increasingly used to determine the absolute volume occupied by emphysematous changes. However, depending on the threshold CT values used for image reconstruction, whole-lung 3D-CT images may be distorted by considerable differences between the x-ray beam thickness and the effective section thickness, resulting in erroneous quantitation of emphysematous lesions by helical 3D-CT. PMID- 10741767 TI - Bronchodilator therapy in chronic obstructive pulmonary disease. AB - This paper reviews new developments in bronchodilator therapy for chronic obstructive pulmonary disease (COPD). Most patients with COPD respond to bronchodilators, but we have no reliable way to predict which patients will respond. When responsiveness is assessed, changes in lung volume as well as improvements in FEV1 should be considered. The combination of a beta-agonist and an anticholinergic agent produces greater improvement than either agent alone. Anticholinergic agents have few adverse side effects in patients with COPD, but concern remains about the possible cardiac side effects of beta-agonists. No clear answer exists about whether new, long-acting beta-agonists, such as salmeterol, should supplant anticholinergic agents as "first-line" therapy in COPD. PMID- 10741768 TI - Inhaled steroid therapy in chronic obstructive pulmonary disease. AB - Chronic obstructive pulmonary disease (COPD) remains difficult to manage. Patients with COPD present with progressive dyspnea; difficulty in stopping smoking; recurrent exacerbations; and, ultimately, respiratory failure. Because of the lack of proven treatments for COPD and because inhaled corticosteroids can prevent airway inflammation and permanent lung damage in patients with asthma, it has become common practice to prescribe inhaled corticosteroids for patients with COPD despite a lack of data suggesting that these agents have any long-term benefit in these patients. In the past 12 months, three randomized, double-blind, placebo-controlled clinical trials (the European Respiratory Society Study on Chronic Obstructive Pulmonary Disease, the Copenhagen City Lung Study, and the Inhaled Steroids in Obstructive Lung Disease study) designed to assess the long term effect of inhaled corticosteroids in patients with varying severity of airway obstruction have been presented. The results of these studies have been disappointing; they show little to suggest that any long-term benefit is gained from using inhaled corticosteroids in most patients with COPD, whether they continue to smoke or not. PMID- 10741769 TI - Nutrition in chronic obstructive pulmonary disease. AB - Weight loss is a frequently occurring complication in patients with chronic obstructive pulmonary disease (COPD) and is a determining factor of functional capacity, health status, and mortality. Weight loss in COPD is a consequence of increased energy requirements unbalanced by dietary intake. Both metabolic and mechanical inefficiency contribute to the elevated energy expenditure. A disbalance between protein synthesis and protein breakdown may cause a disproportionate depletion of fat-free mass in some patients. Nutritional support is indicated for depleted patients with COPD because it provides not only supportive care, but direct intervention through improvement in respiratory and peripheral skeletal muscle function and in exercise performance. A combination of oral nutritional supplements and exercise or anabolic stimulus appears to be the best treatment approach to obtaining significant functional improvement. Patients responding to this treatment even demonstrated a decreased mortality. Poor response was related to the effects of systemic inflammation on dietary intake and catabolism. The effectiveness of anticatabolic modulation requires further investigation. PMID- 10741770 TI - Short- and long-term results after lung volume reduction surgery. AB - Lung volume reduction surgery (LVRS) is a palliative surgical procedure for patients with severe emphysema. Resection of nonfunctional emphysematous lung tissue has been reported to relieve breathlessness and to improve quality of life for many patients by improving lung elastic recoil, respiratory muscle function, and ventilation-perfusion matching. However, the risks and benefits of LVRS remain controversial, as mainly short-term data are available for carefully selected groups of LVRS patients and no prospective, randomized trials for LVRS with pulmonary rehabilitation versus optimal medical therapy plus pulmonary rehabilitation have been reported. Bilateral staple resection for LVRS appears to be superior to use of a laser or unilateral approach in the short term, but relatively little data exist on long-term outcomes. Additional clinical investigation is required to determine whether LVRS should be a widely accepted therapy for severe emphysema. PMID- 10741771 TI - Management of acute exacerbations in chronic obstructive pulmonary disease. AB - An acute exacerbation of chronic obstructive pulmonary disease (COPD) is characterized by an acute worsening of symptoms accompanied by lung infection. In severe cases, an acute exacerbation may cause respiratory failure and death. Successful management of acute exacerbation of COPD in either the inpatient or outpatient setting requires attention to a number of key issues. In this review, issues regarding the management of acute exacerbations of COPD are discussed. An inhaled beta-2 agonist along with the inhaled anticholinergic bronchodilator are recommended. Antibiotic therapy has been demonstrated to improve clinical recovery and physical outcomes. It should be directed against the most commonly occurring pathogens and, in more severe cases, coverage against Gram-negative bacteria is considered. Short course of systemic steroids does provide benefit in hospitalized patients. Supplemental oxygen is appropriate for all patients with hypoxemia. Ventilatory support treatment may be necessary, noninvasive ventilatory assistance being preferable early in the course of the acute episode. In a high number of cases, endotracheal intubation may be avoided. Promoting smoking cessation and the use of influenzae and pneumococcal vaccination may help decrease frequency of episodes of these exacerbations. PMID- 10741772 TI - Clinical course and prognosis of patients with chronic obstructive pulmonary disease. AB - Chronic obstructive pulmonary disease (COPD) is a basically benign disease, but the prognosis is so poor that the mortality rate is similar to some malignant diseases. Depending on the disease severity, the 5-year mortality rate of patients with COPD varies from 40 to 70%. The three major causes of death have been identified as COPD itself, lung cancer, and cardiovascular disease. The following factors have been reported to be related to survival: FEV1 (especially the maximal attainable lung function), age, gender, PaO2, PaCO2, body weight, and comorbidity. There have been several large-scale randomized clinical trials to examine the prophylactic effects of inhaled anti-cholinergics and inhaled corticosteroids on the annual decline in FEV1. However, unfortunately, in all of the published studies, these drugs had no effect on the annual decline in FEV1. PMID- 10741773 TI - Obliterative bronchiolitis after lung transplantation. AB - Despite marked improvements in early survival, long-term outcome after lung transplantation is still threatened by obliterative bronchiolitis (OB). Thought to be a manifestation of chronic allograft rejection, OB affects up to 65% of patients at 5 years after surgery and produces a relentless airflow obstruction. Early and late acute rejection are the primary risk factors for OB, but cytomegalovirus infection and airway ischemia may also play a role. In most patients, OB responds poorly to augmented immunosuppression and eventually leads to infectious complications and terminal respiratory failure. Because early diagnosis is associated with better prognosis, every effort should be made to detect OB in a preclinical stage. This may be best achieved by combining several techniques, such as surveillance transbronchial biopsy and bronchoalveolar lavage, measurements of ventilation distribution and exhaled nitric oxide, and expiratory computed tomography. PMID- 10741774 TI - Pathology and pathophysiology of pneumoconiosis. AB - Cellular and molecular mechanisms, as well as associated gene expressions, in silicosis and asbestosis are widely investigated, and compound mechanisms involved in initiating inflammation and progression to fibrosis are comprehensively studied, though not yet totally understood. Recent advances in this field, especially concerning pathophysiology of these pneumoconioses, are reviewed in this article. Silicosis and asbestosis are two major types of pneumoconiosis. Although the clinico-pathologic features presented are apparently different, silicosis and asbestosis are both interstitial lung diseases caused by chronic exposure to airborne inorganic dusts, and the pathology of these two diseases is essentially a fibrosis. PMID- 10741775 TI - Recent advances in occupational asthma. AB - In the past year, many advances were made in occupational asthma (OA). As in previous years, several new causes of OA were described and the prevalences of some causes of OA were studied. Animal studies suggested possible mechanisms for the induction of sensitization to low-molecular-weight chemicals, and a study in humans indicated the ability of diisocyanates to bind with lung epithelial cells and stimulate mononuclear cells in sensitized patients with OA. New diagnostic and research methods, such as induced-sputum assessment and measurement of exhaled nitric oxide, were evaluated. The effectiveness of protective respiratory devices in patients with OA was shown to be suboptimal (although these devices are necessary in some cases), and the need to prevent OA by reducing or eliminating exposures remains paramount. PMID- 10741776 TI - The past and present of pneumoconioses. AB - Pneumoconioses are still a common cause of chronic lung disease. In industrialized countries, improvements in working conditions and dust control measures have led to a decrease in the incidence of severe forms of silicosis, coal worker pneumoconiosis, and parenchymal asbestosis. However, the diversity of settings in which silica and asbestos are used fuels a continued input of cases, and the burden of cases related to remote exposures is still considerable. Overall, the clinical picture of the classic pneumoconioses and their complications has not changed substantially. However, their limits and links have expanded toward systemic and connective tissue disease, idiopathic pulmonary fibrosis, and antineutrophil cytoplasmic autoantibody-positive vasculitides. Immunologically mediated occupational lung diseases have emerged, such as berylliosis and hard-metal disease. Advances in imaging, mineralogic analysis of bronchoalveolar lavage fluid, and immunologic techniques have been instrumental in describing new patterns of disease and are helpful in litigious or difficult cases. PMID- 10741777 TI - Mesothelioma: new concepts in diagnosis and management. AB - Malignant pleural mesothelioma is an uncommon, but no longer rare, cancer that is frequently difficult to diagnose and poorly responsive to therapy. Because of the difficulties distinguishing mesothelioma from metastatic adenocarcinoma and reactive pleural inflammation, thoracoscopy or open lung biopsy are usually required to obtain adequate samples for pathologic evaluation. Staging of mesothelioma remains a controversial area. Because none of the six staging systems used in the past was found to be predictive, a TNM-based staging system was recently proposed and is awaiting universal acceptance. Generally perceived as a death sentence, this cancer is associated with a median survival of 9 months from the time of diagnosis in most series, but newer therapeutic strategies show promise for improved and even long-term survival in select cases. Randomized trials are awaited to determine if the improvements in survival reported are not simply due to patient selection. PMID- 10741779 TI - Bibliography. Current world literature. Obstructive, occupational, and environmental diseases. PMID- 10741778 TI - New phosphodiesterase inhibitors as therapeutics for the treatment of chronic lung disease. AB - Phosphodiesterase 4 (PDE4) is a member of the growing family cyclic AMP and cyclic GMP. Earliest described inhibitors of PDE4, such as rolipram, demonstrate marked anti-inflammatory and bronchodilatory effects in vitro and in vivo. The clinical utility of these earlier compounds was limited by their propensity to elicit gastrointestinal side effects. This has led to an extensive effort to identify novel PDE4 inhibitors that maintain the anti-inflammatory activity and bronchodilatory activity of rolipram but with a reduced potential to produce side effects. This article summarizes the evidence supporting the utility of selective PDE4 inhibitors in the treatment of asthma and chronic obstructive pulmonary disease, discusses the recent results obtained in clinical trials with second generation inhibitors, and presents two approaches designed to identify additional novel selective PDE4 inhibitors. PMID- 10741780 TI - Endovascular coil embolization of residual or recurrent aneurysms after surgical clipping. AB - PURPOSE: Treatment of residual or recurrent aneurysms after surgical clipping is a challenge and most surgeons prefer to avoid a second surgical attempt. We present treatment of 4 residual or recurrent aneurysms after surgical clipping with electrolytically detachable coils. MATERIAL AND METHODS: In 3 of 4 patients, recurrent aneurysms were diagnosed with angiography 2 months, 5 years and 14 years after surgery, although the domes of the aneurysms were opened following clipping during the surgery. In the 4th patient, an early postoperative angiogram revealed filling of a residual aneurysm secondary to the incomplete neck clipping. Guglielmi detachable coils were used to occlude the residual or recurrent aneurysm. RESULTS: The endovascular approach was successful in all patients and the control angiograms showed complete obliteration of the aneurysms with no recanalization. CONCLUSION: The endovascular approach is a good treatment option for patients in whom complete obliteration of the aneurysm cannot be achieved by surgical clipping. Opening of the aneurysm sac after clipping does not necessarily preclude aneurysm regrowth from a neck remnant proximal to the clip. PMID- 10741781 TI - Effect of CT digital image compression on detection of coronary artery calcification. AB - PURPOSE: To test the effect of digital compression of CT images on the detection of small linear or spotted high attenuation lesions such as coronary artery calcification (CAC). MATERIAL AND METHODS: Fifty cases with and 50 without CAC were randomly selected from a population that had undergone spiral CT of the thorax for screening lung cancer. CT image data were compressed using JPEG (Joint Photographic Experts Group) or wavelet algorithms at ratios of 10:1, 20:1 or 40:1. Five radiologists reviewed the uncompressed and compressed images on a cathode-ray-tube. Observer performance was evaluated with receiver operating characteristic analysis. RESULTS: CT images compressed at a ratio as high as 20:1 were acceptable for primary diagnosis of CAC. There was no significant difference in the detection accuracy for CAC between JPEG and wavelet algorithms at the compression ratios up to 20:1. CT images were more vulnerable to image blurring on the wavelet compression at relatively lower ratios, and "blocking" artifacts occurred on the JPEG compression at relatively higher ratios. CONCLUSION: JPEG and wavelet algorithms allow compression of CT images without compromising their diagnostic value at ratios up to 20:1 in detecting small linear or spotted high attenuation lesions such as CAC, and there was no difference between the two algorithms in diagnostic accuracy. PMID- 10741782 TI - Intravascular ultrasound angioplasty in peripheral arterial occlusion. Preliminary experience. AB - PURPOSE: To determine if ultrasound angioplasty in a percutaneous approach was capable of recanalizing occluded arteries in acute and subacute peripheral arterial occlusions. MATERIALS AND METHODS: We applied an ultrasound angioplasty device in a percutaneous approach in 9 patients with peripheral arterial occlusions. All patients suffered from severe leg ischemia due to subacute thrombotic occlusions. RESULTS: The ultrasound transmitter easily created a channel within the occlusive material. To further reduce the mass of the occlusive material, an aspiration thrombectomy was performed in all cases, leading to a complete recanalization in 7 cases. In 2 cases, a remaining stenosis was successfully dilated. CONCLUSION: Intravascular ultrasonic devices can be useful for recanalization of occluded peripheral arteries. In particular, if thrombolytic therapy of longer peripheral arterial occlusions fails or is contraindicated, ultrasound angioplasty may be a new approach for recanalization. PMID- 10741783 TI - Imaging of aortoiliac arterial disease. Duplex ultrasound and MR angiography versus digital subtraction angiography. AB - PURPOSE: To evaluate and compare the diagnostic accuracy of duplex ultrasound (US) and MR angiography (MRA) at 1.0 T in aortoiliac arterial disease using digital subtraction angiography (DSA) as the reference standard. In addition, a comparison of the 2D time-of-flight (TOF) and 3D contrast-enhanced MRA (CE MRA) techniques was performed. MATERIAL AND METHODS: Prospectively, 39 patients with symptoms of lower-extremity arterial occlusive disease were examined using US, TOF MRA, CE MRA and DSA. Significant lesions (stenosis > or =50%) and occlusions were evaluated blindly for each method. RESULTS: For all segments, the sensitivity for US, TOF MRA and CE MRA with regard to significant lesions was 0.72, 0.81 and 0.81, respectively, and the specificity for each was 0.97, 0.91 and 0.92, respectively. For significant lesions above the inguinal ligament the corresponding sensitivity was 0.84, 0.89 and 0.94 and the specificity 0.93, 0.82 and 0.73, respectively. The specificity was higher when the two MRA methods were combined. TOF MRA overgraded 7 segments as occluded. In most cases, the length of the occlusions was correctly determined on CE MRA, overestimated on TOF MRA and uncertain on US. CONCLUSION: Neither US nor MRA were sufficiently accurate to fully replace angiography. MRA was preferable to US as a non-invasive test when vascular intervention was contemplated. Although CE MRA was superior to TOF MRA, the most accurate results were achieved when the two methods were combined. PMID- 10741784 TI - Symptomatic peripheral vascular tree stenosis. Comparison of subtracted and nonsubtracted 3D contrast-enhanced MR angiography with fat suppression. AB - OBJECTIVE: To determine the clinical feasibility of contrast-enhanced three dimensional (3D) MR angiography (MRA) with fat suppression spectral inversion at lipids and to compare subtracted and nonsubtracted images. MATERIAL AND METHODS: In 13 patients suspected of having lower extremity ischemia, 3D dynamic contrast enhanced MRA of the iliac and femoral arteries was performed using the fat suppression technique. The validity of the MRA interpretations of the degree of stenoses was evaluated by comparing with conventional angiography as the gold standard. The contrast-to-noise (C/N) ratios of both subtracted and nonsubtracted maximum-intensity-projection (MIP) images were calculated. RESULTS: In the arteries with stenosis of 50% or greater, the sensitivity and specificity of the subtracted MR angiograms were 100% and 96.5%, respectively, not significantly different from the corresponding values in nonsubtracted MRAs (100% and 94.1%, respectively). The C/N ratios of the subtracted images were significantly higher than those of the nonsubtracted images (p<0.001). CONCLUSION: With this technique, fat suppression can be performed with only a slight increase in the examination time. A lower dose of contrast material can be used in MRA with fat suppression than without it. Subtracted MRA improves the ease of interpretation compared to nonsubtracted MRA. PMID- 10741785 TI - Improved peripheral MRA using multi-velocity-encoding phase contrast-enhanced MRA techniques. AB - Phase contrast MR angiography (PC-MRA) depends on phase shifts caused by blood flow. Generally, PC sequences employ one VENC (velocity-encoding) value for each encoded spatial direction to optimize the signal in major vessels during peak systolic flow. We compared a mono-(30) with a multi-(20/30/45) VENC-PC-MRA technique in 10 patients with peripheral arterial occlusive disease. In all patients, the multi-VENC-PC sequence enhanced the vascular signal in vessels with very different flow velocities in one measurement. Large fields-of-view can be measured in a relatively short examination time to obtain an overview of the peripheral arterial system of the patient when contrast-enhanced MRA is not possible. PMID- 10741786 TI - Patient radiation exposure during coronary angiography and intervention. AB - PURPOSE: To prospectively register fluoroscopic and cine times in a random fashion, and to measure patient radiation exposure from routine coronary angiography and coronary balloon angioplasty. We also evaluated an optional dose reduction system used during interventions. MATERIAL AND METHODS: The incident radiation to the patient was measured as kerma area product (KAP) in Gycm(2), obtained from an ionisation chamber mounted on the undercouch tube during 65 coronary angiography procedures and another 53 percutaneous transluminal coronary angioplasties (including 29 stent procedures), mostly directly following complete coronary angiography. RESULTS AND CONCLUSION: The values from coronary angiography were comparable to other reports with a mean fluoroscopic time of 4.4 min and a mean KAP value of 62.6 Gycm(2). The corresponding figures from coronary balloon angioplasty without stenting were lower than otherwise reported, with 8.2 min and 47.9 Gycm(2), respectively. The use of coronary stents did prolong the mean fluoroscopic time (10.5 min) but did not significantly enhance the patient mean radiation dose (51.4 Gycm(2)). The dose reduction technique resulted in a significant KAP value reduction of 57%. In conclusion, with regard to radiation exposure, coronary angiography and balloon angioplasty are considered safe procedures. PMID- 10741787 TI - Bread and barium. Diagnostic value in patients with suspected primary esophageal motility disorders. AB - PURPOSE: To evaluate the significance of bread and barium studies as a diagnostic tool as well as a supplement to manometric investigation of the esophagus in patients with suspected esophageal motility disorders. MATERIAL AND METHODS: Eighty-nine patients suspected for primary esophageal motility disorders were examined. All patients were interviewed before the investigation to determine the prevalence of symptoms like heartburn, chest pain, and dysphagia. The patients underwent simultaneous roentgenologic and manometric investigations in the supine position during wet and solid barium swallow, and during continuous drinking, followed by investigation for gastroesophageal reflux (GER). The manometric examination was performed with triple-lumen catheters connected to a hydraulic capillary infusion system and external transducers. RESULTS: All patients with normal esophageal clearing (n=31) had normal manometry. Patients with delayed esophageal clearing (n=58) required manometry for identifying concommittant motility disorders; achalasia and diffuse esophageal spasms were found only in patients with delayed liquid and solid emptying. GER and/or esophageal rings was demonstrated in 31 patients. CONCLUSION: We suggest bread and barium as the first diagnostic step in patients with clinical suspicion of primary esophageal motility disorders. PMID- 10741788 TI - CT and MR imaging of the liver. Clinical importance of nutritional status. AB - PURPOSE: In an experimental study in rats a correlation between nutritional status and hepatic attenuation in CT and signal intensities in MR imaging was shown. Is physiological nutritional status of importance in clinical CT and MR imaging? MATERIAL AND METHODS: In a cross-over study including 12 healthy volunteers (6 women and 6 men, mean age 34 years), CT and MR imaging of the liver were performed with nutritional status at three different levels, i.e., normal, fasting and after glycogen-rich meals. CT and MR were performed on clinical imaging systems and hepatic attenuation and signal intensity, respectively, were assessed. In MR, T1-weighted, proton density-weighted and T2-weighted pulse sequences were used. RESULTS: In CT there were significantly (p<0.01) higher liver attenuations in normal nutritional status and after glycogen rich-meals compared to the fasting condition. The difference between fasting and glycogen rich meals were 10.5 HU for men, 7.4 for women and mean 8.8 HU for all 12 volunteers. In MR imaging the differences were small and non-significant. The results of this study are in accordance with an earlier experimental study in rats. CONCLUSION: In CT it may be of importance not to have patients in a fasting condition as it lowers the attenuation in normal liver tissue. The findings are important for planning of clinical studies where hepatic attenuation will be assessed and may be of some importance in clinical CT. In MR imaging the results indicate that the nutritional status is of less importance. PMID- 10741789 TI - Ischemic complications of transcatheter arterial chemoembolization in liver malignancies. AB - OBJECTIVE: To determine the frequency, character, methods of treatment, and outcome of ischemic complications after transcatheter hepatic artery chemoembolization (TACE). MATERIAL AND METHODS: Between 1985 and 1998, 827 sessions of TACE with Doxorubicin mixed with iodized oil, and gelatin sponge particles were performed in 282 patients with primary and metastatic liver cancer. Post-TACE monitoring included clinical observation, US and CT. RESULTS: Ischemic complications appeared in 13 (4.6%) and included the following: hepatic (n=6) and splenic abscess (n= 1), cholecystitis (n=3), and bile duct necrosis (n=3). The treatment was US-guided drainage in 12 cases and systemic antibacterial therapy in 1. No negative influence of these complications on survival of patients was detected. CONCLUSION: Serious ischemic complications of TACE occur in about 5% of patients and can be successfully managed without open surgery. These complications do not worsen the survival of patients. PMID- 10741790 TI - Infantile hemangioendothelioma. A case report. AB - The case of a 3-month-old boy with a hepatic infantile hemangioendothelioma is reported. There was no previous history of disease and no symptoms, only an incidentally found abdominal mass. The case is presented as an example of establishing the diagnosis, deciding upon the treatment, and performing the follow-up using only non-invasive imaging techniques. PMID- 10741791 TI - Delayed presentation of traumatic left-sided diaphragmatic avulsion. A case report. AB - We describe the case of a 35-year-old man who had suffered a severe multitrauma with blunt thoracic injury, left scapula and humerus fractures 5 years earlier. At the time of the trauma, a diaphragmatic lesion went unnoticed. Five years later, the patient had a 24-h history of increasingly severe abdominal pain with repeated vomiting. Helical CT showed a portion of the left hemidiaphragm avulsed from its insertions on the ribs with large-bowel loop obstruction herniated in the left hemithorax. The preoperative CT diagnosis was confirmed by surgery: reduction of the hernia and reinsertion of the hemidiaphragm to the lumbocostal arch were performed. PMID- 10741792 TI - Factors influencing patient radiation doses from barium enema examinations. AB - PURPOSE: To analyse factors behind the variation of patient doses from barium enema (BE) examinations. MATERIAL AND METHODS: The patients' (n=89) organ and effective doses (E) due to BE examinations were computed with the ODS-60 program. An average risk factor for BE examinations was derived using the BEIR V schema. The correlation of E with several independent variables was analysed. RESULTS: Median Es at five hospitals were 4.4, 6.1, 7.1, 13 and 16 mSv. The E of the female patients (median 9.2 mSv) was higher than that of the males (median 5.4 mSv) (p<0.001) due to the higher female doses to the gonads, bladder and uterus, resulting from different body structure. An average fatal risk factor of 0.02%. per one BE examination was derived. Factors controlled by the radiologist (screening time, number of exposures) explained 40% and patient-related factors explained 16% of the total variation of E. The equipment-related factors are included in the residual 44%. CONCLUSION: Due to the large contribution of the radiologists' examination technique in the value of E, an optimal examination technique is essential in reducing doses and the stochastic risk to patients. PMID- 10741793 TI - Localization of insulinomas. Comparison of conventional arterial stimulation with venous sampling (ASVS) and superselective ASVS. AB - PURPOSE: To examine the value of superselective arterial stimulation venous sampling (ASVS) to localize insulinomas. MATERIAL AND METHODS: Superselective ASVS (SS-ASVS) was performed in 9 patients with insulinoma. Injection of secretagogue (calcium gluconate: 0.01 mEq Ca++/kg) was performed into the gastroduodenal, splenic (proximal and distal), and superior mesenteric arteries in 9 patients and additionally into the dorsal pancreatic artery in 6 patients. Sampling from the hepatic vein was performed to measure serum insulin concentrations at 30, 60 and 120 s after each injection of secretagogue into these arteries. SS-ASVS results were correlated with surgical findings, compared to those of conventional ASVS. RESULTS: Insulinomas were correctly localized to the head, body or tail of the pancreas by SS-ASVS in 8 patients (89%). Conventional ASVS detected insulinomas in 7 patients (78%), although it could not distinguish whether the insulinoma was located in the pancreatic body or tail in 4 of the 7 patients. There were eight-fold or more increases in serum insulin levels in hepatic venous samples related to the artery supplying the tumor in 8 patients. Localization of the insulinomas was verified at surgery in all patients. CONCLUSION: SS-ASVS is a useful method for detailed evaluation of overproduction of insulin from pancreatic insulinomas and their localization. When the pancreatic insulinoma is situated in the pancreatic body or tail, the localization is more accurately made by SS-ASVS than by conventional ASVS. PMID- 10741794 TI - Evaluation of new sclerotic bone metastases in breast cancer patients during treatment. AB - PURPOSE: According to the World Health Organization (WHO) criteria for response of bone metastases to therapy, new lesions indicate progressive disease. We intended to prove that a new sclerotic lesion on conventional radiography may also be a sign of a positive therapeutic response in a previously undetectable lytic metastasis. MATERIAL AND METHODS: In a previous placebo-controlled clinical trial of clodronate (Ostac) therapy, 139 breast cancer patients with bone metastases underwent both conventional radiography and bone scan every 6 months for 2 years with 99mTc before and during clodronate treatment. WHO criteria were applied for therapy response evaluation. RESULTS: In 24 patients, 52 new sclerotic lesions observed during therapy were selected for re-evaluation of conventional radiographs and bone scans. In 8 of the 24 patients, 17 of 52 new sclerotic lesions (33%) had showed positive uptake on previous bone scans. These lesions were possibly misinterpreted as new when applying WHO criteria. CONCLUSION: For better assessment of new sclerotic lesions during treatment, more sensitive techniques, e.g. bone scan, are needed as a complement to conventional radiography. PMID- 10741795 TI - Quantitative bone scintigraphy. A methodological evaluation in prostate cancer. AB - PURPOSE: To evaluate a simple method for quantification of focal activity in bone scintigraphy (BS). MATERIAL AND METHODS: The gamma camera was calibrated using a phantom. Quantitative bone scintigraphy (QBS) was performed on 11 men recently diagnosed with prostate cancer (PCa), for whom routine BS showed involvement of the skeleton. Following endocrine therapy for 4 to 8 months, a second QBS was performed. Changes in QBS values were then compared to changes in serum levels of prostate-specific antigen (PSA). RESULTS: PSA response indicating regression of PCa was accompanied by a decrease in the QBS value in 8 of the 11 patients. The overall mean error of the QBS values was 15%. CONCLUSION: QBS according to this method is a relatively simple procedure that might contribute to objective evaluation of therapeutic effects in skeletal metastases, although its validity must be tested in a larger clinical material. PMID- 10741796 TI - Activity-based costing in radiology. Application in a pediatric radiological unit. AB - PURPOSE: To get an informative and detailed picture of the resource utilization in a radiology department in order to support its pricing and management. MATERIAL AND METHODS: A system based mainly on the theoretical foundations of activity-based costing (ABC) was designed, tested and compared with conventional costing. The study was performed at the Pediatric Unit of the Department of Radiology, Oulu University Hospital. The material consisted of all the 7,452 radiological procedures done in the unit during the first half of 1994, when both methods of costing where in use. Detailed cost data were obtained from the hospital financial and personnel systems and then related to activity data captured in the radiology information system. RESULTS: The allocation of overhead costs was greatly reduced by the introduction of ABC compared to conventional costing. The overhead cost as a percentage of total costs dropped to one-fourth of total costs, from 57% to 16%. The change of unit costs of radiological procedures varied from -42% to +82%. CONCLUSION: Costing is much more detailed and precise, and the percentage of unspecified allocated overhead costs diminishes drastically when ABC is used. The new information enhances effective departmental management, as the whole process of radiological procedures is identifiable by single activities, amenable to corrective actions and process improvement. PMID- 10741797 TI - CT scores of emphysema and oxygen desaturation during low-grade exercise in patients with emphysema. AB - PURPOSE: We evaluated the usefulness of CT for assessing oxygen desaturation during walking in patients with emphysema. MATERIAL AND METHODS: The study comprised 32 patients with emphysema (mean age 67+/-6 years). Serial CT images of 5 mm were obtained from the apex to the basal regions of the lung during deep inspiration. The severity of emphysema was scored by four physicians according to a visual method. A six-minute walking test and oxygen desaturation (pSO2) measurements were performed. RESULTS AND CONCLUSION: The mean CT score of the four observers was significantly correlated with the nadir pSO2 and deltapSO2, but did not correlate with the total distance walked. These results suggest that CT may be used for the assessment of oxygen desaturation during low-grade exercise in patients with emphysema. PMID- 10741798 TI - Subdiaphragmatic location of costal chondrosarcoma. PMID- 10741799 TI - The ethical challenge in the new millennium. PMID- 10741800 TI - Intravenous catheters for hemodialysis: historical perspective. PMID- 10741801 TI - Stroke in patients undergoing dialysis. PMID- 10741802 TI - Effect of adequacy of dialysis and nutrition on morbidity and working rehabilitation of patients treated by maintenance hemodialysis. AB - Numerous studies have demonstrated a correlation between adequacy of hemodialysis (HD) and patient mortality. In this study we evaluated the effect of adequacy of dialysis and nutrition on morbidity and working rehabilitation. Single center experience carried out in 1998 in an outpatient university-affiliated dialysis facility was presented. A total of 181 patients, on HD over 3 months, was included in the study; mean age 54.5 years, mean duration of HD 5.4 years. Thirty two patients were hospitalized in 1998. Patients with an eKt/V < 0.8 had significantly more frequent and extended hospitalizations than patients with eKt/V > 1.2. In the regression model eKt/V had a significant effect on hospital days. Serum albumin, as an index of nutrition, was also found to significantly influence hospitalizations. Patients with a BMI < 20.0 kg/m2 were found to have significantly more frequent hospitalizations, more hospital days/patient, and hospital days/hospitalized patient/year than those with a BMI > 25.0 kg/m2. BMI < 20.0 kg/m2 was associated with a significantly lower dialysis adequacy (eKt/V and URR) and more severe anemia. However, regression model failed to confirm a statistically significant association of BMI with hospital days. The best working rehabilitation (stage 1), according to the EDTA criteria, was obtained in patients with significantly (p<0.05) higher eKt/V, URR, albumin, nPNA, and body fat than in patients at stage 6 (not able to work, and not able to take care of themselves). This study has established that dialysis adequacy and nutrition have an effect on morbidity (estimated through hospitalizations) and patient rehabilitation. PMID- 10741803 TI - Correlation between plasma carnitine, muscle carnitine and glycogen levels in maintenance hemodialysis patients. AB - Chronic hemodialysis (HD) may lead to losses of carnitine from plasma and muscle. Plasma carnitine does not reflect the body content of carnitine. The purpose of this study was the evaluation of total and free plasma and muscle carnitine concentrations (TPC, FPC, TMC, FMC), muscle glycogen and the relationship between plasma and tissue carnitine content and the basic indices of lipid metabolism in HD patients. The studies were conducted in two groups: the first one consisted of 37 HD patients (19 F, 18 M), the second one served as the control and was composed of 29 (10 F, 19 M) patients with healthy kidneys. Tissue specimens in HD patients were taken during surgery on arterio-venous fistula from brachioradial muscle. Carnitine and glycogen measurements were performed using enzymatic methods according to Cederblad and Huijng respectively. Total cholesterol (CH), HDL-CH, and triglycerides were assayed by enzymatic commercial test system (Boehringer-Mannheim, Germany). To summarise, we found the following phenomena in our HD patients in comparison with the controls: 1) In plasma: similar TPC but decreased FPC levels and FPC/TPC ratio which may suggest free carnitine deficiency. 2) In muscle: significantly lower TMC and FMC levels but normal FMC/ITMC ratio. 3) Negative correlation between TMC and FMC levels and duration of dialysis treatment. 4) No correlation between plasma and muscle camitine concentration. 5) Significantly higher concentration of muscle glycogen which could be explained by the changes in the structure of muscle fibres in HD patients and/or lower physical activity. 6) A positive correlation between FPC/APC or FPC/TPC ratio and HDL-CH in HD patients which may suggest that an appropriate proportion between free and acylcarnitines may influence HDL-CH levels in that population. PMID- 10741804 TI - ANCA in dialysis patients: a role for bioincompatibility? AB - BACKGROUND. Anti-neutrophil cytoplasmic autoantibodies (ANCA) have been described in patients suffering from systemic vasculitis such as Wegener granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome and other pathological conditions. In this paper we report a greater incidence of ANCA in hemodialysis patients as compared to peritoneal dialysis patients, pre-dialytic uremic patients and non-renal patients; a possible role for dialysis bioincompatibility in ANCA generation was also investigated. METHODS: A total of 335 uremics in substitutive treatment (176 in hemodialytic treatment and 159 in peritoneal dialysis) were examined for ANCA positivity. A total of 189 patients with advanced renal failure in conservative treatment and 100 healthy subjects were used as control. The dialysis techniques were standard hemodialysis (n = 119), low volume hemodiafiltration (n = 26) and hemofiltration (n = 31). ANCA positivity was examined by immunofluorescence (IF): diffuse finely granular staining was considered as classical positive reaction (C-ANCA) and P-ANCA was diagnosed if a perinuclear staining was observed. EIA for proteinase-3 (anti PR 3) and myeloperoxidase-antibodies (anti-MPO) were also performed. RESULTS: In non renal patients and in patients with pre-dialytic renal insufficiency none were found ANCA positive. In peritoneal dialysis patients all but one were ANCA negative with IF, with all EIA test resulting negative. In hemodialytic patients, a positive IF test was found in 26 (14.7%) for P-ANCA and in 5 (2.8%) for C-ANCA; using the EIA test 23 (13%) patients were positive for MPO and 12 (6.8%) for PR 3. CONCLUSIONS: No correlation with age, primary renal diseases, dialytic age, dialysis membrane materials was found; regarding the different extracorporeal dialytic techniques a higher incidence (p < 0.02) was detected in patients undergoing HDF Backfiltration of contaminated dialysate may induce ANCA via an increased cytokine generation. PMID- 10741805 TI - Comparative analysis of metabolism of medium- and plasma perfused primary pig hepatocytes cultured around a 3-D membrane network. AB - Culture media are frequently used in the evaluation of metabolical functions of hepatocytes in hybrid liver support systems (hLSS). However, media compositions differ substantially from those of plasma. Therefore, our study was designed to investigate whether current in vitro studies with medium are suitable to assess the metabolical competence of hLSS-cultures during clinical application as well as to explore whether the cell nutrition with medium provides a suitable modus operandi for stand by cultivation. Paired bioreactor cultures were perfused with either Williams' Medium E (MPB) or human plasma (PPB). About 6x108 primary pig hepatocytes (>97% viability) were cultured in three laboratory scale bioreactors designed according to Gerlach's bioreactor-concept. Different perfusion protocols were initiated after a standardised period allowing for cell attachment and reorganisation in aggregates. Whereas patterns of enzyme release were similar in both protocols the metabolical behaviour was different between MPB (anabolic state) and PPB (catabolic state). Furthermore, compared to MPB the lidocaine-MEGX tests for PPB demonstrated lower MEGX-concentrations and a different reaction pattern. We conclude that the nutrition of hepatocytes with medium during the stand by period itself might influence the cell function and subsequently the efficacy of the hLSS-treatment during clinical application. PMID- 10741806 TI - Nephrotic syndrome and plasmapheresis. AB - We studied 15 patients suffering from nephrotic syndrome (NS) in bioptically accertained primary and secondary glomerulopathies responding poorly to the common pharmacological treatment. They were monitored for one year by assessing their immunological and kidney functionality parameters, especially proteinuria. The patients underwent 3 apheretic sittings using the cascade double-filtration technique. After the third apheretic sitting the patients received metilprednisolone 300 mg/m2. This therapy was repeated every month for 6 months. At the end of the apheretic cycle it was shown that all patients had responded well to the therapy with a dramatic decrease in proteinuria, maintenance and/or recuperation of kidney functionality and improvement of lipidic asset; these data remained unchanged over time. Considering our preliminary results, we believe that therapeutic apheresis has a precise function in refractory nephrotic syndrome and, in particular, we propose the use of the cascade double-filtration technique. PMID- 10741807 TI - A preliminary survey of the apheresis activity in Italy: a perspective for the Italian Registry of apheresis. AB - Data collection on the apheresis activity in Italy throughout 1997 was performed by means of a standardized questionnaire. These data, provided by 96 Apheresis Units from 13 Italian regions, albeit rough, are sufficiently informative. In 1997, a total number of 170,373 apheresis sessions was carried out, with a clear cut prevalence of productive apheresis (92%) that was performed by all Apheresis Units. Lombardy, Venetia and Latium were the most active regions for therapeutic apheresis (56% of total activity). PMID- 10741808 TI - Blood profile during and after hemoglobin substitute administration. AB - The in vivo effects of Diaspirin Crosslinked Hemoglobin (DCLHb, Baxter Healthcare Corp.) on hematology and biochemistry are unknown. This study includes 6 calves (71.2+/-1.3 kg). In each animal a total of 2 litres of blood was exchanged for the same amount of hydroxylethyl starch (Haes, Fresenius) (n=3) or DCLHb (n=3), which is equivalent to 28cc/kg of blood substitute, over a period of 5 hours. The animals were allowed to survive 7 days. Blood samples were taken hourly during the perfusion protocol, at postoperative day (POD) 1, 2 and 7. ANOVA test was used for repeated measurements. Blood cell profiles were similar in both groups. Peak methemoglobinemia was 4.2% in the DCLHb group. Osmolarity was significantly higher in the DCLHb group with the greatest difference at POD 1 and 2. Postmortem analysis of the major organs did not show any sign of hemoglobin deposit in the DCLHb group. In the given setup DCLHb can be administered in a large quantity with good hematological tolerance and without any deposits in major organs. A prolonged plasma expander effect was observed. PMID- 10741809 TI - Blood flow distribution in sorbent beds: analysis of a new sorbent device for hemoperfusion. AB - A new polymer-based sorbent cartridge has been recently developed for enhancing middle molecule removal during hemodialysis. The cartridge (Betasorb, Renaltech, New York, USA) has been designed to be placed in series with the dialyzer in the blood circuit. It is therefore important to evaluate the distribution of flow into the blood compartment of the device in order to assess if the surface of the sorbent is utilized to the best. For this purpose, a special imaging technique was utilized. Cartridges were analyzed during a simulated in vitro circulation at 250 and 350 ml/min of blood flow and 25% and 40% hematocrit. Cartridges were placed in vertical position and a cross longitudinal section 1 cm thick was analyzed in sequence by a helical scanner. Dye was injected into the arterial inlet and the progressive distribution was evaluated by sequential densitometrical measures carried out automatically by the machine. The sequential images analyzed by the scanner demonstrated excellent distribution of the flow in the blood compartment with minimal difference between the central and the peripheral regions of the compartment. In particular the following flow velocity pattern could be observed under the different experimental conditions tested. We may conclude that the cartridge design is adequate and no channelling effects could be detected in the blood compartment. The flow distribution is slightly affected by changes in flow rate and hematocrit showing an optimal utilization of the available surface for molecule adsorption. PMID- 10741810 TI - Extracorporeal blood oxygenation and ozonation (EBOO) in man. preliminary report. AB - Autohemotherapy with ozone has been used for four decades with encouraging results but, owing to the lack of clinical studies, it has never been adopted by orthodox medicine. Confident of the valid principles of ozone therapy, we have endeavoured to increase its therapeutic efficacy. Over a ten-year period we have developed an apparatus that makes it possible to treat large quantities of blood with ozone in extracorporeal circulation (extracorporeal blood oxygenation and ozonation EBOO). One of us volunteered to test the system and after six treatments noted the disappearance of two lipomas. This prompted us to treat a patient with Madelung disease and several patients with atherosclerotic vasculopathy. Besides showing therapeutic effects, the preliminary results indicate that EBOO is clinically valid, without side-effects and worthy of testing in various diseases. PMID- 10741811 TI - The relation between cervical discographic pain responses and radiographic images. AB - OBJECTIVE: The purpose of this study was to investigate the relation between cervical discographic pain responses and radiographic images. DESIGN: Records were reviewed for a series of patients who had undergone cervical discography. SETTING: All patients were being treated at a spine specialty clinic. PATIENTS: A total of 269 discs were studied in 161 discographic procedures in patients with neck, shoulder, or arm pain. All patients underwent other diagnostic procedures before discography, including magnetic resonance imaging, computed tomography (CT), and CT/myelography. INTERVENTIONS: During the injection of contrast in each disc evaluated, the patient was asked if any pain was felt, and if so, was it similar or dissimilar to the pain typically experienced. OUTCOME MEASURES: Results were determined by analyzing the pain responses during disc injection with respect to imaged pathology seen on the axial CT discographic image of the disc. Results were further analyzed based on patient age. RESULTS: There was a significant relation between the radiographic image of the disc and the results of clinical pain provocation (p < 0.01; chi2). Among the 35 discs appearing as normal, clinical pain was provoked in only 14.3%. Among the 234 discs appearing as abnormal, clinical pain was provoked in 77.8%. The mean age of the patients with painless radiographically abnormal discs was significantly greater than that of the patients in the other subgroups of the study population. CONCLUSIONS: There was good agreement between the radiographic appearance of the disc and the pain provocation results. Discs that were painless but disrupted were found among older patients. Among such patients, discography may be particularly helpful in differentiating clinically significant abnormalities from those associated with aging. PMID- 10741812 TI - Efficacy of sustained-release bupropion in neuropathic pain: an open-label study. AB - OBJECTIVE: The purpose of this study was to assess the analgesic potential of sustained-release (SR) bupropion for neuropathic pain. DESIGN: Open-label treatment design. SETTING: Outpatient pain clinic, University of Arizona Health Sciences Center, Tucson, Arizona. PATIENTS: Twenty-two patients with a diagnosis of neuropathic pain. INTERVENTIONS: Patients with neuropathic pain received 1 week of 150 mg SR bupropion once daily followed by 7 weeks of 150 mg SR bupropion twice daily. OUTCOME MEASURES: Treatment effects were assessed by daily ratings of pain intensity, posttreatment global ratings of pain relief, depression scores (Hamilton Depression Scale), and daily ratings of side effects. RESULTS: Fifteen patients (68%) reported that their pain relief was improved or much improved with bupropion. The mean average pain score at week 1 was 6.7, which decreased at the end of week 8 to 5.3 (paired t test, t[df = 21]3.327; p = 0.003) in all patients studied and to 3.8 (paired t test, t[df = 14]3.754; p = 0.002) in the patients who improved. Pain relief was statistically significant at week 5 (paired t test, t[df = 21]3.816; p = 0.001) and continued throughout weeks 6, 7, and 8. Most patients were not depressed, and analgesia was observed to occur without change in depression ratings in most patients who responded. Side effects were rated as mild and consisted primarily of insomnia (8 patients), tremor (3 patients), and gastrointestinal upset (2 patients). These symptoms had a tendency to recede with continuation of therapy. CONCLUSIONS: This uncontrolled pilot study suggests that bupropion may be an effective and tolerated treatment for some patients with neuropathic pain. Blockade of norepinephrine reuptake may mediate this effect. The role of dopamine reuptake blockade is uncertain. A larger randomized, double blind, placebo-controlled study is currently underway to confirm these preliminary results. PMID- 10741813 TI - Effects of presurgical local infiltration of bupivacaine in the surgical field on postsurgical wound pain in laparoscopic gynecologic examinations: a possible preemptive analgesic effect. AB - OBJECTIVE: A randomized, double-blind, controlled study was designed to evaluate the effect of presurgical local infiltration of bupivacaine in the surgical field on postsurgical wound pain relief and analgesic requirements in 28 healthy patients scheduled for laparoscopic gynecologic examinations. INTERVENTIONS: After induction of general anesthesia by routine methods, the patients were randomly divided into two groups. In the bupivacaine (B) group (n = 15), patients were injected with 5 ml of 0.25% bupivacaine at each incisional area (four sites, total of 20 ml) approximately 15 minutes before skin incision. In the control (C) group (n = 13), the surgical field was injected with an equal volume of physiologic saline solution (four sites, total of 20 ml). OUTCOME MEASURES: Postsurgical wound pain at rest was evaluated by a 10-cm visual analog pain scale at 1, 10, 24, and 72 hours and 1 month after surgery. The patients were interviewed via telephone 1 month after hospital discharge for re-evaluation of resting pain. RESULTS: The results indicated that the incidence of postsurgical wound pain for up to 10 hours after surgery in group B was significantly lower (p < 0.05) than in group C. Pain intensity ranged from mild to moderate (2-5 cm). In addition, the mean visual analog pain scale pain intensity was significantly less for group B (0.31 +/- 0.85 cm) than for group C (2.62 +/- 2.06 cm) for up to 10 hours after surgery (p < 0.05). The number of patients who requested analgesics and complained of sleep disturbances was significantly higher in group C (p < 0.05). The mean cumulative dose of diclofenac sodium at 24 hours was significantly (p < 0.05) lower in group B (6.67 +/- 17.6 mg) than in group C (30.8 +/- 25.3 mg). Prolonged postsurgical wound pain persisting 1 month after surgery was observed in one patient in group C. CONCLUSIONS: It is concluded that presurgical infiltration of 0.25% bupivacaine in the surgical field is a useful method for decreasing postsurgical wound pain for up to 10 hours and analgesic consumption for up to 24 hours after laparoscopic gynecologic examination. PMID- 10741814 TI - Computerized tomographic localization of clinically-guided sacroiliac joint injections. AB - OBJECTIVE: The goal of this study was to use computed tomographic (CT) scanning to localize clinically guided sacroiliac (SI) joint injections and identify other structures affected by this procedure. DESIGN: A prospective, double-blind, correlational outcome study design was used. Injection of 39 SI joints with a mixture of bupivacaine (0.25%), methylprednisolone (40 mg), and iohexol (Omnipaque; 180 mg/dl) using a clinically guided technique, (i.e., no image guidance) was performed. Patients had CT scans obtained both immediately after needle placement and after contrast injection. Neither the patients nor their clinicians were aware of the CT findings at the time of injection. SETTING: Academic multidisciplinary pain center. PATIENTS: Patients with SI disease by clinical criteria. RESULTS: Intra-articular injection was accomplished in 8 of 37 (22%) patients. Injected material was identified within 1 cm of the joint 68% of the time. Epidural (spinal canal) injected material was seen 24% of the time. CONCLUSIONS: The low rate of intra-articular injection seen with this clinically guided technique suggests restraint in its use for injection therapy. Some image guidance (e.g., fluoroscopy, CT) is probably necessary to reliably inject the SI joint. Perhaps in clinical settings, where image guidance is not readily available, a clinically-guided technique could initially be tried in patients at low risk for complications from such injections. This study also provides an anatomic explanation for the occasional weakness observed after SI joint injection. PMID- 10741815 TI - A comparison of pain rating scales by sampling from clinical trial data. AB - OBJECTIVE: The goals of this study were to examine agreement and estimate differences in sensitivity between pain assessment scales. DESIGN: Multiple simultaneous pain assessments by patients in acute pain after oral surgery were used to compare a four-category verbal rating scale (VRS-4) and an 11-point numeric rating scale (NRS-11) with a 100-mm visual analog scale (VAS). The sensitivity of the scales (i.e., their ability [power] to detect differences between treatments) was compared in a simulation model by sampling from true pairs of observations using varying treatment differences of predetermined size. RESULTS: There was considerable variability in VAS scores within each VRS-4 or NRS-11 category both between patients and for repeated measures from the same patient. Simulation experiments showed that the VAS was systematically more powerful than the VRS-4 in all simulations performed. The sensitivity of the VAS and NRS-11 was approximately equal. CONCLUSIONS: In this acute pain model, the VRS-4 was less sensitive than the VAS. The simulation results demonstrated similar sensitivity of the NRS-11 and VAS when comparing acute postoperative pain intensity. The choice between the VAS and NRS-11 can thus be based on subjective preferences. PMID- 10741816 TI - Comorbid fibromyalgia accounts for reduced fecundity in women with myofascial face pain. AB - OBJECTIVE: This study examined factors related to reduced fecundity among women with myofascial face pain (MFP) arising from hypotheses concerning the role of neurohormonal factors in MFP and associated conditions. DESIGN: Fecundity rates among 162 MFP cases and 173 demographically equivalent acquaintance female controls were compared. OUTCOME MEASURES: Fecundity indicators and factors underlying differential fecundity rates were investigated. RESULTS: It was determined that female cases with MFP had significantly fewer children and were more likely to have never been pregnant. Although women with MFP were more likely than controls to indicate that volitional factors related to their health discouraged them from any or additional pregnancies, these factors did not account for lower rates of fecundity. MFP cases also did not differ from controls on self-reported indicators of infertility. Moreover, we show that reduced fecundity was restricted to the subgroup of MFP cases who reported a history of fibromyalgia. CONCLUSIONS: Reduced fecundity in women with MFP is restricted to those who self-report a history of fibromyalgia. Possible mechanisms for reduced fecundity in fibromyalgia are discussed. These findings highlight the need to screen for widespread pain among women with regional myofascial pain syndromes. PMID- 10741817 TI - Are twitches, startles, and body movements pain indicators in extremely low birth weight infants? AB - OBJECTIVE: The goal of this study was to examine whether body activity such as postural, trunk, and limb movements may be potential pain cues in preterm infants. DESIGN: Convenience sample. SETTING: Level III neonatal intensive care unit (NICU). PATIENTS: Extremely low birth weight (< or = 1,000 g) preterm infants (n = 64) undergoing routine NICU medical care. OUTCOME MEASURES: Procedures likely to differ in evoking distress (i.e., endotracheal suctioning, chest physical therapy, diaper change, or nasogastric feed) were observed. Behaviors were recorded at bedside using the Neonatal Individualized Developmental Care and Assessment Program system. RESULTS: Changes in heart rate and sleep/waking state were related to the procedures, supporting the assumption of differing relative disruption to the infant. Arching, squirming, startles, and twitching were not observed significantly more during procedures than at baseline. After controlling for background variables, finger splay and leg extension were significantly related to ongoing procedures. Facial brow raising was a function of the number of invasive procedures in the past 24 hours; thus, it may be a useful cue of sensitization. CONCLUSIONS: Some extensor movements seemed to be distress signals, whereas tremors, startles, and twitches were not related to discomfort during the observation period. These behaviors may differ qualitatively during longer lasting tissue invasive events. The results of this study indicate the need for more in-depth study of patterns of motor activity in preterm infants over longer observation periods to evaluate potential signs of stress and pain in babies undergoing NICU medical care. PMID- 10741818 TI - Sex-specific effects of pain-related anxiety on adjustment to chronic pain. AB - OBJECTIVE: Considerable research indicates that both high levels of anxiety and female sex are associated with increased sensitivity to experimental pain and greater experience of clinical pain. In general, however, previous research has not investigated the joint effects of sex and anxiety on pain responses. A single previous laboratory-based study indicated that anxiety was inversely related to pain thresholds among men but not among women. The present study examined the relation between pain-related anxiety and adjustment to chronic pain in a sex dependent manner. DESIGN AND SETTING: A total of 215 (114 women, 101 men) chronic pain patients referred to a multidisciplinary treatment center completed questionnaires assessing anxiety and adjustment to chronic pain. RESULTS: Results generally supported the previous laboratory-based finding indicating that an inverse relation between anxiety and adjustment to chronic pain was present only among male patients. Although male patients with high pain-related anxiety reported greater pain severity, greater interference of pain, and lower levels of daily activity than male patients with low anxiety, this effect was not present among female patients. Moreover, the effects of pain-related anxiety on adjustment to chronic pain were not attributable to either hypervigilance or use of passive coping strategies. Potential explanations and implications for the present findings are discussed. PMID- 10741819 TI - Measuring movement-exacerbated pain in cognitively impaired frail elders. AB - OBJECTIVE: Prior research examining the utility of nonverbal measures of pain in persons with cognitive impairments has focused on acute procedurally-induced phasic pain (i.e., venipuncture and needle injections). The goal of the current project was to examine the utility of both self-report and nonverbal measures of pain in frail elders experiencing exacerbations of chronic musculoskeletal pain. These were assumed to be more representative of the day-to-day pain experience of elderly patients. DESIGN: Participants were 58 frail elders, 29 of whom had been found to have significant cognitive impairments. All were filmed as they undertook a series of structured activities (e.g., walking and reclining), and pain was assessed using self-report. Trained coders identified the incidence of pain-related behaviors using the videotapes. The various pain measures (i.e., self-report and nonverbal indices) were compared across both patient groups and the several activities. RESULTS: Consistent with our hypotheses, more pain was identified (using both self-report and nonverbal measures) when patients engaged in more physically demanding activities. Facial reactions varied as a function of patient cognitive status, with those participants who were cognitively impaired more responsive. Of the various nonverbal indices that we examined, guarded behavior appeared to be especially sensitive. The various pain indices were only modestly correlated with one another. CONCLUSIONS: This study supports the validity of self-report and behavioral measures of pain in frail elders with and without cognitive impairments. Each of the measures used contributed different information to pain assessment, suggesting that investigations of pain in elders with cognitive impairments should employ varying types of pain assessment tools. PMID- 10741820 TI - Signs and symptoms of the myofascial pain syndrome: a national survey of pain management providers. AB - OBJECTIVE: The goal of this study was to assess clinical consensus regarding whether myofascial pain syndrome (MPS) is a legitimate and distinct diagnosis as well as the signs and symptoms characterizing MPS. DESIGN: A standardized mailed survey with return postage provided. SUBJECTS: A total of 1,663 American Pain Society members in medically related disciplines listed in the 1996/1997 directory. OUTCOME MEASURES: A standardized survey assessing clinical opinion regarding whether MPS is a legitimate diagnosis, whether MPS is a clinical entity distinct from fibromyalgia, and the signs and symptoms believed to be "essential to," "associated with," or "irrelevant to" to the diagnosis of MPS. RESULTS: Of the 403 surveys returned, 88.5% respondents reported that MPS was a legitimate diagnosis, with 81% describing MPS as distinct from fibromyalgia. The only signs and symptoms described as essential to the diagnosis of MPS by greater than 50% of the sample were regional location, presence of trigger points, and a normal neurologic examination. Regarding the signs and symptoms considered to be essential or associated with MPS, more than 80% of respondents agreed on regional location, trigger points, normal neurologic examination, reduced pain with local anesthetic or "spray and stretch," taut bands, tender points, palpable nodules, muscle ropiness, decreased range of motion, pain exacerbated by stress, and regional pain described as "dull," "achy," or "deep." Sensory or reflex abnormalities, scar tissue, and most test results were considered to be irrelevant to the diagnosis of MPS by a large proportion of the respondents. CONCLUSIONS: There was general agreement across specialties that MPS is a legitimate diagnosis distinct from fibromyalgia. There was a high level of agreement regarding the signs and symptoms essential or associated with a diagnosis of MPS. Differences across specialties are discussed. This survey provides a first step toward the development of consensus-based diagnostic criteria for MPS, which can then be validated empirically. PMID- 10741821 TI - What is the quality of the implemented meta-analytic procedures in chronic pain treatment meta-analyses? AB - OBJECTIVE: Meta-analysis (MA) is the application of quantitative techniques for the purposes of summarizing data from individual studies. This type of review has many advantages over traditional reviews. However, different investigators performing MAs on the same data set have reached different conclusions. These reliability problems have been attributed to differences in the quality of the implemented meta-analytic procedures. We, therefore, examined the chronic pain treatment meta-analytic literature for MA procedure quality and for the consistency of conclusions. DESIGN, SETTING, PARTICIPANTS, OUTCOME MEASURES: Chronic pain treatment MAs were isolated according to inclusion/exclusion criteria. Data from these MAs were abstracted into structured tables. Table format reflected eight meta-analytic procedures identified previously as being important to MA implementation quality. These were: adequacy of retrieval, publication bias, inclusion/exclusion criteria, abstraction of data, quality, homogeneity/heterogeneity, independence, and statistical versus clinical interpretation. Each meta-analytic procedure was then independently rated by two raters. Rating results were then analyzed by procedure for each individual MA for percentage scores out of 100%, and mean scores. For MAs addressing the same topic area (pain facility treatment, antidepressant treatment, manipulation treatment) direction of effect size was noted. Mean effect sizes were calculated for these subgroups. RESULTS: Sixteen chronic pain treatment MAs fulfilled inclusion/exclusion criteria. Mean procedure ratings indicated that four procedures may not be implemented adequately. These were publication bias, abstraction of data, quality, and homogeneity/heterogeneity. There was wide MA implementation score variability, with 37.5% scoring less than 50%. The effect sizes of the MA subgroups demonstrated replicate nonvariability. CONCLUSIONS: Some meta-analytic procedures could be interpreted to be implemented inadequately in some chronic pain treatment MAs. There is wide variability between individual chronic pain treatment MAs on adequacy of implementation of these procedures. However, the effect sizes of the different MA subgroups demonstrated consistency. This finding indicates that for these MA subgroups, MA results are consistent between authors. In addition, chronic pain MAs, as compared with other groups of MAs, appear to address some of the procedures in a more adequate fashion. Future chronic pain MAs should concentrate on improving the quality of their methods with particular emphasis on the above four procedures. Because of potential validity problems with these results, these data cannot and should not be used to make administrative decisions about previous MAs. PMID- 10741822 TI - Long-term opioid therapy for refractory lumbar pain. Pain Research Foundation journal club. PMID- 10741823 TI - Construction of a branched chain at C-3 of a hexopyranoside. Synthesis of miharamycin sugar moiety analogs. AB - Synthesis of the conveniently protected epimer at C-3' of the miharamycin sugar moiety was accomplished starting from the corresponding 3,3'-spiroepoxide. Reaction of the epoxide with lithium cyanide, followed by hydrolysis and spontaneous cyclization, afforded the intermediate deoxylactone methyl 4,6-O benzylidene-3-C-(carboxymethyl)-alpha-D-glucopyranoside-3',2-lacto ne (8). Stereoselective hydroxylation with MoO5 x py x HMPA, reduction with lithium aluminum hydride and cyclization with diethyl azodicarboxylate-triphenylphosphine gave the target molecule methyl 2,3''-anhydro-4,6-O-benzylidene-3-C-[(R)-1,2 dihydroxyethyl]-alpha -D-glucopyranoside (5). Direct reduction of 8 gave other analogs having no C-3' hydroxyl group together with having a C-3'' hydroxyl group (hemiacetal). In addition, C-3' epimers were also synthesized through C-3', C-3'' dihydroxy analogs. Wittig reaction of an appropriate ketosugar with [(ethoxycarbonyl)methylene]triphenylphosphorane leading to a 7:3 Z/E mixture, followed by hydroxylation with osmium tetroxide, reduction and cyclization afforded the target molecule 5 and the miharamycin sugar moiety methyl 2,3'' anhydro-4,6-O-benzylidene-3-C-[(S)-1,2-dihydroxyethyl]-alpha -D-glucopyranoside. Examination of X-ray data for 5 and its NMR spectroscopy data allowed us to explain a contradiction reported in the literature. PMID- 10741824 TI - Sulfuric acid-catalyzed acetolysis of anomeric methyl 2,3,4,6-tetra-O-acetyl-D mannopyranosides: kinetics and mechanism. AB - The kinetics of the acetolysis and accompanying anomerization of methyl 2,3,4,6 tetra-O-acetyl-alpha- and -beta-D-mannopyranosides at different concentrations of sulfuric acid in acetic anhydride-acetic acid mixtures were studied. The progress of the reactions was followed by gas chromatography, and the rate constants of the partial reactions were calculated on the basis of the time-dependent product distribution obtained. The mechanisms of the reactions involved are discussed. The involvement of unstable ionic intermediates is taken into account in the evaluation of the kinetic results, and simplified and extended models are used in the mathematical treatment of the results. A fourth-order Runge-Kutta algorithm is used to calculate rate constants. Acetolysis was found to be faster for mannosides than for glucosides relative to their anomerization. The beta mannopyranoside prefers endocyclic CO-bond rupture, while in the alpha anomer the endocyclic and exocyclic cleavages are comparatively rapid. PMID- 10741825 TI - Synthesis of 4-deoxy analogues of 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2 deoxy-D-xylose and their effects on glycoconjugate biosynthesis. AB - 4-Deoxy analogues of 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D xylose were synthesized and evaluated as inhibitors of glycoconjugate biosynthesis. Methyl 2-acetamido-2,4-dideoxy-beta-D-xylo-hexopyranoside (11) showed a reduction in [3H]GlcN and [14C]Leu incorporation into hepatocyte cellular glycoconjugates by 89 and 88%, of the control cells, respectively, at 20 mM, whereas the free sugars, 2-acetamido-2,4-dideoxy-alpha,beta-D-xylo hexopyranoses (15), showed a reduction of [3H]GlcN and [14C]Leu incorporation by 75 and 64%, respectively, at 20 mM. The acetylated analogues of 11 and 15, namely methyl 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-beta-D-xylo-hexopyranoside and 2 acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-alpha,beta-D-xylo-hexopyra noses, showed a greater inhibition of [3H]GlcN and [14C]Leu incorporation at 1 mM compared with their non-acetylated counterparts, but were toxic to hepatocytes at concentrations of 10 and 20 mM. Corresponding derivatives of 2-acetamido-2,4 dideoxy-L-threo-pentopyranose showed no biological effect up to 20 mM, suggesting that the C-6 substituent is important for the biological activity. PMID- 10741826 TI - Activity coefficients for NaCl-monosaccharide (D-glucose, D-galactose, D-xylose, D-arabinose)-water systems at 298.15 K. AB - Electrochemical cells with a sodium ion selectivity electrode (Na-ISE) versus a chloride ion selectivity electrode (Cl-ISE) as a reference electrode were used to determine the activity coefficients for NaCl-monosaccharide (D-glucose, D galactose, D-xylose, and D-arabinose) systems in water at 298.15 K. A comparison of the results thus obtained was made with those determined by another electromotive force (emf) method. It is shown that agreement is excellent. The Gibbs free energy parameters of the interactions between these sugars and NaCl in water were evaluated together with the parameter C1(CHOH, exo), indicating the interaction of the exocyclic CHOH group of saccharide molecules and NaCl. The results suggested that the interactions of these monosaccharides with NaCl are controlled mostly by the dominant conformer of their molecules in water. PMID- 10741827 TI - Synthesis and structure of 6-amino-2,3,6-trideoxy-D-erythro-hexono-1,6-lactam and 6-amino-3,6-dideoxy-D-xylo-hexono-1,6-lactam. AB - Solid-state conformations of 6-amino-2,3,6-trideoxy-D-erythro-hexono-1,6-lactam (3a) and 6-amino-3,6-dideoxy-D-xylo-hexono-1,6-lactam (7a) were determined using X-ray diffraction. Conformations of the compounds 3a, 7a, and their per-O-acetyl derivatives 4,5-di-O-acetyl-6-amino-2,3,6-trideoxy-D-erythro-hexono-1,6-lactam (3b) and 2,4,5-tri-O-acetyl-6-amino-3,6-dideoxy-D-xylo-hexono-1,6-lactam (7b) in solutions were deduced from the analysis of NMR spectra using a modified Karplus equation and compared with the results of circular dichroism measurement of lactams 3a and 7a. Conformation 4C(1,N) was revealed for solid lactams 3a and 7a and for lactams 7a and 7b in solution, while lactams 3a and 3b in solution exist in the approximately 1:1 equilibrium of the conformers 4C(1,N) and (1,N)C4. PMID- 10741828 TI - Thermal C-glycosylation of D-glucal with trimethylsilyl cyanide. AB - The treatment of 3,4,6-tri-O-acetyl-D-glucal and unprotected D-glucal with trimethylsilyl cyanide, under thermal conditions in the absence of catalyst, afforded the corresponding 2,3-unsaturated glycosyl cyanides in high yield. PMID- 10741829 TI - Synthesis of antioxidative and anti-inflammatory drugs glucoconjugates. AB - Glucoconjugates of (+/-)-ibuprofen, (+/-)-alpha-tocopherol (vitamin E), gentisic acid, gallic acid, 2,6-bis(tert-butyl)-4-thiophenol, and N-acetyl-L-cysteine were prepared with the objective of increasing the bioavailability of such antioxidant and anti-inflammatory drugs. The O-glucosides were synthesized using benzylated alpha-D-glucopyranosyl trichloracetimidate as glycosyl donor. For the synthesis of the S-glucosides, the glycosyl donor 1,2,3,4,6-penta-O-acetyl-beta-D glucopyranose provided higher yields than the corresponding O-acetylated imidate. PMID- 10741830 TI - 1999 USPHS/IDSA guidelines for the prevention of opportunistic infections in persons infected with human immunodeficiency virus. U.S. Public Health Service (USPHS) and Infectious Diseases Society of America (IDSA). PMID- 10741831 TI - An archaeal chaperonin-based reactor for renaturation of denatured proteins. AB - We describe an original chaperonin-based reactor that yields folded and active proteins from denatured materials. We used the 920-kDa chaperonin of the archaeon Sulfolobus solfataricus, which does not require any protein partner for its full activity and assists in vitro folding with low substrate specificity. The reactor consists of an ultrafiltration cell equipped with a membrane that retains the chaperonin in a functional state for folding in solution and permits the flowthrough of the folded substrates. By studying the ATP-dependent functional cycle of the chaperonin, we were able to use the reactor for repeated refolding processes. The scale-up of the reactor is made possible by the overproduction of chaperonin in Sulfolobus solfataricus cells that acquired thermotolerance upon appropriate heat shock. PMID- 10741832 TI - Thermosipho japonicus sp. nov., an extremely thermophilic bacterium isolated from a deep-sea hydrothermal vent in Japan. AB - A novel barophilic, extremely thermophilic bacterium was isolated from a deep-sea hydrothermal vent chimney at the Iheya Basin, in the Okinawa area, Japan. The cells were found to be rod shaped and surrounded by a sheath-like outer structure; the organism did not possess flagella and was not motile. Growth was observed between 45 degrees and 80 degrees C (optimum, 72 degrees C, 45 min doubling time), pH 5.3 and 9.3 (optimum, pH 7.2-7.6), 6.6 and 79g/l sea salts (optimum, 40g/l), and 0.1 and 60MPa (optimum, 20MPa). Strain IHB1 was found to be a strictly anaerobic chemoorganotroph capable of utilizing yeast extract and proteinaceous substrates such as peptone and tryptone. Elemental sulfur or thiosulfate acted as electron acceptors improving growth. The isolate was able to utilize casein as a sole carbon and energy source in the presence of thiosulfate. The G + C content of the genomic DNA was 31.4 mol%. Phylogenetic analysis based on 16S rDNA sequences and DNA-DNA hybridization analysis indicated that the isolate is closely related to Thermosipho africanus; however, it represents a species distinct from the previously described members of the genus Thermosipho. On the basis of the physiological and molecular properties, we propose that the new isolate represents a new species, which we name Thermosipho japonicus sp. nov. (type strain: IHB1; JCM10495). PMID- 10741833 TI - Reidentification of facultatively alkaliphilic Bacillus firmus OF4 as Bacillus pseudofirmus OF4. AB - With a view toward verifying the original classification of alkaliphilic Bacillus firmus OF4, physiological and biochemical characteristics were more extensively catalogued than in original studies, and this catalog was supplemented with 16S rDNA sequence homology and more extensive DNA-DNA hybridization analyses. Phylogenetic analysis of this alkaliphile based on the comparison of multiple 16S rDNA sequences from Bacillus species indicated that this strain is most closely related to Bacillus pseudofirmus. Consistently, in the DNA-DNA hybridization analysis of the alkaliphile and Bacillus reference strains, the highest level of DNA-DNA relatedness (96%) was found between the alkaliphile and the B. pseudofirmus type strain (DSM 8715(T)). The findings support the conclusion that this alkaliphile strain is more closely related to B. pseudofirmus than to B. firmus, and we propose the future use of the designation B. pseudofirmus OF4. PMID- 10741834 TI - The structure of the alpha-galactosidase gene loci in Thermus brockianus ITI360 and Thermus thermophilus TH125. AB - The Thermus thermophilus TH125 alpha-galactosidase gene, agaT, and flanking sequences were cloned in Escherichia coli and sequenced as well as flanking sequences of the previously cloned agaT from Thermus brockianus ITI360. Different structures of putative alpha-galactosidase operons in the two Thermus strains were revealed. Downstream of and overlapping with the alpha-galactosidase genes of both strains, a gene was identified that is similar to the galactose-1 phosphate uridylyltransferase gene (galT) of E. coli and Streptomyces lividans. Upstream of the agaT of T. brockianus ITI360, four open reading frames were observed. The deduced translation products displayed similarity to components of bacterial binding protein-dependent transport systems and a beta-galactosidase. No galactoside utilization genes were identified upstream of agaT in T. thermophilus TH125. The inactivation of the alpha-galactosidase genes of both strains by insertional mutagenesis led to an inability to use melibiose or galactose as a single carbohydrate source. An attempt was made to isolate a gene encoding the enzyme responsible for para-nitrophenyl-(pNP-) beta-galactoside hydrolyzing activity in T. thermophilus TH125. A gene designated bglT was cloned and expressed in E. coli. The inactivation of the bglT gene led to 55% reduction of the pNP-beta-galactoside hydrolyzing activity in the mutant strain in comparison to the wild type. PMID- 10741835 TI - Halocins: are they involved in the competition between halobacteria in saltern ponds? AB - Many representatives of the family Halobacteriaceae ("halobacteria") excrete halophilic bacteriocins (halocins) that inhibit the growth of other halobacteria. In spite of the fact that halocin production is widespread among the Halobacteriaceae, no information is available on their ecological significance. To test whether halocins may play a role in the interspecies competition between different types of halobacteria in saltern crystallizer ponds inhabited by dense communities of these red halophiles, we assayed for halocins active against a variety of halobacteria in salterns from different locations worldwide. Detection of halocin activity was based on the inhibition of growth of indicator organisms on agar plates, the decreased incorporation of radiolabeled substrates, and microscopic examinations. No halocin activity was detected in any of the brines examined, in spite of the fact that halocin production was demonstrated in cultures of most microorganisms isolated from these brines. Thus, the contribution of halocins in the competition between different halobacteria in hypersaline aquatic environments is probably negligible. PMID- 10741836 TI - MJ1647, an open reading frame in the genome of the hyperthermophile Methanococcus jannaschii, encodes a very thermostable archaeal histone with a C-terminal extension. AB - All archaeal histones studied to date have similar lengths, 66 to 69 amino acid residues that form three alpha-helices separated by two beta-strand loop regions which together constitute a histone fold. In contrast, the eukaryal nucleosome core histones are larger, 102 to 135 residues in length, with N-terminal and C terminal extensions flanking the histone fold that participate in gene regulation and higher-order chromatin assembly. In the Methanococcus jannaschii genome, MJ1647 was annotated as an open reading frame predicted to encode an archaeal histone with an approximately 27-amino-acid C-terminal extension, and we here document the DNA binding and assembly properties and thermodynamic stability parameters of the recombinant product of MJ1647 synthesized in Escherichia coli with (rMJ1647) and without (rMJ1647delta) the C-terminal extension. The presence of the C-terminal extension did not prevent homodimer formation or inhibit DNA binding, but the complexes formed by rMJ1647, presumably archaeal nucleosomes containing a (rMJ1647)4 tetramer, were apparently less stable than those formed by (rMJ1647delta)4. The presence of the C-terminal extension increased the thermostability of rMJ1647 when compared with rMJ1647delta in 0.2 M KCl at pH 4 but not in the absence of KCl at pH 1. Based on thermal unfolding transitions, rMJ1647 and rHAfB generated by expression of AF0337 cloned from the genome of the related hyperthermophile Archaeoglobus fulgidus in E. coli were found to have higher thermodynamic stabilities than all previously studied archaeal histones. PMID- 10741837 TI - Halomonas magadii sp. nov., a new member of the genus Halomonas, isolated from a soda lake of the East African Rift Valley. AB - A number of novel alkaliphilic organotrophic bacteria have been isolated from several saline and alkaline East African soda lakes. The new isolates grow at pH values between 7.0 and 11.0, with pH optima for growth between 9.0 and 10.0. Growth occurs at total salts concentration between 0% and 20% (w/v) with optimum at 0%-7% (w/v). Phylogenetic analyses based on 16S rDNA sequence comparison indicate that these isolates are related (>96% similarity) to members of the Halomonadaceae within the gamma-3 subdivision of the Proteobacteria. These analyses indicate that existing species within the Halomonadaceae fell within three main groups, one group comprising the type species of Halomonas, Halomonas elongata, and a number of other known species including one soda lake isolate. A second group constituting most of the remaining known species of Halomonas and related Chromohalobacter spp. includes 3 soda lake isolates with high DNA-DNA homologies. The third group included Halomonas halodenitrificans, Halomonas desiderata, Halomonas cupida, and 13 soda lake isolates. Phenotypic comparisons indicated that the majority of soda lake strains shared similar morphological, phenotypic, and chemotaxonomic properties to known strains of Halomonas but grew under alkaline conditions. The 3 soda lake isolates with high DNA-DNA homologies were, however, significantly different in antibiotic sensitivity pattern and in the utilization of several substrates, were unable to reduce nitrite, and showed low DNA-DNA homologies with known halomonads in the same group. We propose that these isolates comprise a new species of the genus Halomonas that we name Halomonas magadii sp. nov. The type strain is strain 21 MI (NCIMB 13595). PMID- 10741838 TI - Microbial diversity at 83 degrees C in Calcite Springs, Yellowstone National Park: another environment where the Aquificales and "Korarchaeota" coexist. AB - The use of molecular phylogenetic approaches in microbial ecology has revolutionized our view of microbial diversity at high temperatures and led to the proposal of a new kingdom within the Archaea, namely, the "Korarchaeota." We report here the occurrence of another member of this archaeal group and a deeply rooted bacterial sequence from a thermal spring in Yellowstone National Park (USA). The DNA of a mixed community growing at 83 degrees C, pH 7.6, was extracted and the small subunit ribosomal RNA gene (16S rDNA) sequences were obtained using the polymerase chain reaction. The products were cloned and five different phylogenetic types ("phylotypes") were identified: four archaeal phylotypes, designated pBA1, pBA2, pBA3, and pBA5, and only one bacterial phylotype, designated pBB. pBA5 is very closely related to the korarchaeotal phylotype, pJP27, from Obsidian Pool in Yellowstone National Park. The pBB phylotype is a lineage within the Aquificales and, based on 16S rRNA sequence, is different enough from the members of the Aquificales to constitute a different genus. In situ hybridization with bacterial-specific and Aquificales-specific fluorescent oligonucleotide probes indicated the bacterial population dominated the community and most likely contributed significantly to biogeochemical cycling within the community. PMID- 10741839 TI - Housing and health--current issues and implications for research and programs. AB - This article provides an overview of the ways in which the home environment can affect human health, describes how specific health hazards in housing are related, and considers implications of these concerns for research and programs to address the health-housing connection. The widespread availability of decent housing has contributed greatly to improvements in health status in developed countries through, for example, provision of safe drinking water, proper sewage disposal, and protection from the elements. However, a lack of decent housing and homelessness among a significant number of Americans remains a significant public health concern. In addition, a number of specific health hazards can be found even in housing that is in good condition and provides all basic amenities. Specific health hazards related to housing include unintentional injuries, exposure to lead, exposure to allergens that may cause or worsen asthma, moisture and fungi (mold), rodent and insect pests, pesticide residues, and indoor air pollution. A number of these specific hazards share underlying causes, such as excess moisture, and all may be influenced by factors in the community environment or by occupant behaviors. We make recommendations for developing programs and research efforts that address multiple housing problems in an integrated way, rather than categorically, and for closer collaboration between housing and public health programs. PMID- 10741840 TI - Childhood injuries and deaths due to falls from windows. AB - BACKGROUND: Unintentional injuries are the leading cause of death in children, and falls are the most common type of unintentional injury in the US. The incidence of falls from windows, a common cause of death in urban areas, has not been described outside major cities such as New York and Chicago, and rates in urban and suburban areas have not been compared. OBJECTIVE: To estimate the incidence and identify the population at risk for falls from windows among children in Hamilton County, Ohio. DESIGN: Retrospective case series identified using Cincinnati Children's Hospital Medical Center (CHMC) Trauma Registry. SETTING: Hamilton County, Ohio, which has urban and nonurban areas. PARTICIPANTS: Children less than 15 years old residing in Hamilton County, Ohio, presenting to CHMC in Cincinnati, Ohio, after a fall from a window between January 1, 1991, and December 31, 1997. OUTCOME MEASURE: Annual incidence by age, race, gender, and residence of those who fell from windows. RESULTS: Over the 7-year study period, 86 (6.3%) of 1,363 falls were from windows. The mortality rate for falls from windows was 4.7%, compared to 0.07% for all other falls presenting to CHMC (P<.0001). Children 0-4 years old had a higher rate of falls than children aged 5 14 (14.6/100,000 vs. 2.0/100,000) (P<.0001). Males were twice as likely to fall as females (P<.016), and black children were three times more likely to fall than non-black children (P<.002). The incidence of falls in the city of Cincinnati was four times that of the non-urban area (P<.0002). CONCLUSIONS: Injuries from falls from windows are a public health problem in Hamilton County, Ohio, especially for young, urban children. PMID- 10741841 TI - Injury-producing events among children in low-income communities: the role of community characteristics. AB - STUDY PURPOSE: Injury remains the leading cause of death in children aged 1 to 4 years. Past studies of determinants of injuries among young children have most often focused on the microlevel, examining characteristics of the child, parent, family, and home environments. We sought to determine whether and how selected neighborhood economic and physical characteristics within these low-income communities are related to differences in risk of events with injury-producing potential among infants and young children. METHODS: Our study used both individual-level data and information on the characteristics of the neighborhood of residence to describe the prevalence of events with injury-producing potential among infants and young children in three low-income communities in Baltimore City, Maryland. Our sample was 288 respondents who participated in a random household survey. Information on respondent (age, employment, and length of residence in the neighborhood) and neighborhood characteristics (average per capita income, rate of housing violations, and crime rate) were available. Methods of multilevel Poisson regression analysis were employed to identify which of these characteristics were associated with increased risk of experiencing an event with injury-producing potential in the month prior to the interview. RESULTS: Although all three communities were considered low income, considerable variation in neighborhood characteristics and 1-month prevalence rates of events with injury-producing potential were observed. Younger age of respondent and higher rates of housing violations were associated significantly with increased risk of a child under 5 years old in the household experiencing an event with injury-producing potential. CONCLUSIONS: Information on community characteristics was important to understanding the risks for injuries and could be used to develop community-based prevention interventions. PMID- 10741844 TI - Opportunities lost: NIH research funding to New York's medical schools. PMID- 10741842 TI - Asthma and the home environment of low-income urban children: preliminary findings from the Seattle-King County healthy homes project. AB - OBJECTIVES: Childhood asthma is a growing public health concern in low-income urban communities. Indoor exposure to asthma triggers has emerged as an important cause of asthma exacerbations. We describe indoor environmental conditions related to asthma triggers among a low-income urban population in Seattle/King County, Washington, as well as caregiver knowledge and resources related to control of these triggers. METHODS: Data are obtained from in-person, structured, closed-end interviews with the caretakers of children aged 4-12 years with persistent asthma living in households with incomes less than 200% of poverty. Additional information is collected during a home inspection. The children and their caregivers are participants in the ongoing Seattle-King County Healthy Homes Project, a randomized controlled trial of an intervention to empower low income families to reduce exposure to indoor asthma triggers. We report findings on the conditions of the homes prior to this intervention among the first 112 enrolled households. RESULTS: A smoker was present in 37.5% of homes. Mold was visible in 26.8% of homes, water damage was present in 18.6% of homes, and damp conditions occurred in 64.8% of households, while 39.6% of caregivers were aware that excessive moisture can increase exposures to allergens. Dust-trapping reservoirs were common; 76.8% of children's bedrooms had carpeting. Cockroach infestation in the past 3 months was reported by 23.4% of caregivers, while 57.1% were unaware of the association of roaches and asthma. Only 19.8% of the children had allergy-control mattress covers. CONCLUSIONS: Many low-income urban children with asthma in King County live in indoor environments that place them at substantial risk of ongoing exposure to asthma triggers. Substandard housing and lack of resources often underlie these exposures. Initiatives involving health educators, outreach workers, medical providers, health care insurers, housing agencies, and elected officials are needed to reduce these exposures. PMID- 10741843 TI - Evaluation of Stachybotrys chartarum in the house of an infant with pulmonary hemorrhage: quantitative assessment before, during, and after remediation. AB - Stachybotrys chartarum is an indoor mold that has been associated with pulmonary hemorrhage cases in the Cleveland, Ohio, area. This study applied two new quantitative measurements to air samples from a home in which an infant developed PH. Quantitative polymerase chain reaction and a protein synthesis inhibition assay were used to determine the level of S. chartarum spores and their toxicity in air samples taken before, during, and after a remediation program was implemented to remove the fungus. Initial spore concentrations were between 0.1 and 9.3 spores/m3 of air, and the toxicity of air particulates was correspondingly low. However, the dust in the house contained between 0.4 and 2.1 x 10(3) spores/mg (as determined by hemocytometer counts). The remediation program removed all contaminated wallboard, paneling, and carpeting in the water damaged areas of the home. In addition, a sodium hypochlorite solution was used to spray all surfaces during remediation. Although spore counts and toxicity were high during remediation, air samples taken postremediation showed no detectable levels of S. chartarum or related toxicity. Nine isolates of S. chartarum obtained from the home were analyzed for spore toxicity, hemolytic activity, and random amplified polymorphic DNA banding patterns. None of the isolates produced highly toxic spores (>90 microg T2 toxin equivalents per gram wet weight spores) after growth for 10 and 30 days on wet wallboard, but three isolates were hemolytic consistently. DNA banding patterns suggested that at least one of these isolates was related to isolates from homes of infants with previously investigated cases. PMID- 10741845 TI - Office-based methadone prescribing: acceptance by inner-city practitioners in New York. AB - In the US, methadone maintenance is restricted by federal and state regulations to large specialized clinics that serve fewer than 20% of the heroin-dependent population. In Europe, Canada, and Australia, primary health care providers already are utilized widely as methadone prescribers. In preparation for a limited study of office-based methadone treatment in New York City, 71 providers from 11 sites were surveyed about their willingness to prescribe methadone in their office-based practices. Of the 71, 85% had methadone-maintained patients who came to their practice for other care. One-third felt knowledgeable enough to prescribe methadone, and 66% said they would if given proper training and support (88% among AIDS care providers). Half expressed concern that they might be unable to meet the multiple needs of these patients. With additional training and ancillary support, the 47 providers willing to become methadone providers could serve, at 10-20 patients each, 470-940 patients, a population the size of 3-5 average methadone clinics. PMID- 10741846 TI - Volunteer bias in nonrandomized evaluations of the efficacy of needle-exchange programs. AB - OBJECTIVE: Nonrandomized comparisons of the incidence of HIV and hepatitis B and C between injection drug users (IDUs) who do and do not attend voluntary needle exchange programs may be subject to bias. To explore possible sources of bias, we examined characteristics associated with voluntarily beginning or ceasing to participate in the Seattle needle exchange. METHODS: In a cohort of 2,879 IDUs, a standardized questionnaire measured characteristics present at enrollment. We examined the relation of these characteristics to the proportion of IDUs who began to use the program during the ensuing 12-month follow-up period and to the proportion of current exchangers who dropped out during that period of time. RESULTS: Of the 494 never-exchangers at baseline, 32% attended the exchange program during follow-up; those who reported sharing syringes or who were homeless at enrollment were more likely to become new exchange users (adjusted risk ratio [ARR] for becoming an exchange user = 1.8 for those who shared syringes, and ARR = 2.2 for those who were homeless). Of 1,274 current exchangers, 16% stopped using the exchange during followup, with daily injectors (ARR = 0.6) and those who reported backloading (ARR = 0.6) being relatively less likely to drop out of the exchange. CONCLUSIONS: The analysis suggests that IDUs participating in needle-exchange programs at a given point in time may include a particularly high proportion of those injectors whose pattern of drug use puts them at elevated risk of blood-borne viral infections. PMID- 10741848 TI - Eighty years of public health in New York City. 1947. PMID- 10741847 TI - Telephone survey of Alaskan pharmacists' nonprescription needle-selling practices. AB - OBJECTIVES: To determine the availability of nonprescription needles and syringes (NS) through pharmacy sales and to assess the impact of pharmacy policies and municipal paraphernalia laws on pharmacists' selling practices. DESIGN: Telephone survey of all pharmacies in Alaska's four largest cities. SETTING: Anchorage, Fairbanks, Juneau, and Ketchikan, Alaska. SUBJECTS: A single pharmacist from each pharmacy represented in the cities' phone books. MAIN OUTCOME MEASURES: Reports of (1) pharmacies' policies and individual pharmacists' procedures regarding the nonprescription sale of NS, (2) pharmacists' selling practices, and (3) identification of conditions that may affect pharmacists' decisions to sell nonprescription NS. RESULTS: Response rate of 86% (37/43); 77% of pharmacists reported selling at least one nonprescription NS in the last month. Store policy was related to selling practices; however, there was no difference in selling practices between a city with a paraphernalia law and cities without such laws. Logistic regression revealed pharmacists were more likely to sell NS if they worked in chain pharmacies and estimated that a high number of other local pharmacists sell nonprescription NS. CONCLUSION: NS are available through nonprescription sales in Alaskan pharmacies. The majority of pharmacies have store policies that permit pharmacists to sell nonprescription NS, either in all cases or at their discretion. Municipal paraphernalia laws do not determine the selling practices of individual pharmacists. PMID- 10741849 TI - Asthma hospitalization in New York City 1988-1997. PMID- 10741850 TI - Regulation of gamma-glutamylcysteine synthetase subunit gene expression: insights into transcriptional control of antioxidant defenses. AB - Gamma-glutamylcysteine synthetase (GCS; also referred to as glutamate-cysteine ligase, GLCL) catalyzes the rate-limiting reaction in glutathione (GSH) biosynthesis. The GCS holoenzyme is composed of a catalytic and regulatory subunit, each encoded by a unique gene. In addition to some conditions which specifically upregulate the catalytic subunit gene, expression of both genes is increased in response to many Phase II enzyme inducers including oxidants, heavy metals, phenolic antioxidants and GSH-conjugating agents. Electrophile Response Elements (EpREs), located in 5'-flanking sequences of both the GCSh and GCSl subunit genes, are hypothesized to at least partially mediate gene induction following xenobiotic exposure. Recent experiments indicate that the bZip transcription factor Nrf2 participates in EpRE-mediated GCS subunit gene activation in combination with other bZip proteins. An AP-1-like binding sequence and an NF-kappaB site have also been implicated in regulation of the catalytic subunit gene following exposure to certain pro-oxidants. Potential signaling mechanisms mediating GCS gene induction by the diverse families of Phase II enzyme inducers include thiol modification of critical regulatory sensor protein(s) and the generation of the reactive oxygen species. This review summarizes recent progress in defining the molecular mechanisms operative in transcriptional control of the genes encoding the two GCS subunits, identifying areas of agreement and controversy. The mechanisms involved in GCS regulation might also be relevant to the transcriptional control of other components of the antioxidant defense battery. PMID- 10741851 TI - Regulation of prostaglandin E2 production by the superoxide radical and nitric oxide in mouse peritoneal macrophages. AB - The purpose of this study was to elucidate the role of NO and O2 on enzymatic components of cyclooxygenase (COX) pathway in peritoneal macrophages. Activation of murine peritoneal macrophages by lipopolysaccharides (LPS) resulted in time dependent production of nitric oxide (NO) and prostaglandin E2 (PGE2). This stimulation was also accompanied by the production of other reactive oxygen species such as superoxide (O2-), and by increased expression of COX-2. Our results provide evidence that O2- may be involved in the pathways that result in arachidonate release and PGE2 formation by COX-2 in murine peritoneal macrophages stimulated by LPS. However, we were not able to demonstrate that NO participates in the regulation of PG production under our experimental conditions. PMID- 10741852 TI - Reduction of ferrylmyoglobin by the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) in aqueous solution and during freezing. AB - The hypervalent muscle pigment ferrylmyoglobin, formed by activation of metmyoglobin by hydrogen peroxide, was found to be reduced in a second-order reaction by N-tert-butyl-alpha-phenylnitrone (PBN, often used as a spin trap). In acidic aqueous solution at ambient temperature, the reduction is relatively slow (deltaH++ = 65+/-2kJ x mol(-1) and deltaS++ = -54+/-7 J x mol(-1). K(-1) for pH = 5.6), but phase transitions during freezing of the buffered solutions accelerates the reaction between ferrylmyoglobin and PBN. In these heterogenous systems at low temperature (but not when ice-formation was inhibited by glycerol), a PBN derived radical intermediate was detected by ESR-spectroscopy, identified as a nitroxyl radical by a parallel nitrogen hyperfine coupling constant of 31.8 G, and from microwave power saturation behavior concluded not to be located in the heme-cleft of the protein. The acceleration of the reaction is most likely caused by a lowering of the pH during the freezing of the buffered solutions whereby ferrylmyoglobin becomes more oxidizing. PMID- 10741853 TI - Controlled oxidation of calf thymus DNA to produce standard samples for 8 oxodeoxyguanosine analysis; effects of freeze-drying, storage and hydrolysis conditions. AB - Calf thymus DNA containing defined levels of 8-hydroxy-2'-deoxyguanosine (8 oxodG) was prepared by treatment with visible light in the presence of photosensitiser Ro 19-8022. The DNA was checked for stability; after freeze drying, the amount of 8-oxodG did not increase during 6 weeks' storage at room temperature. However, freeze-drying itself can introduce additional oxidative damage. Two enzymic hydrolysis regimes (DNase I, phosphodiesterases I and II, and alkaline phosphatase; or P1 nuclease and alkaline phosphatase) give similar values for 8-oxodG. PMID- 10741854 TI - Comparison of different methods of measuring 8-oxoguanine as a marker of oxidative DNA damage. ESCODD (European Standards Committee on Oxidative DNA Damage). AB - We are attempting to resolve some of the problems encountered in measuring 8 hydroxy-2'-deoxyguanosine (8-oxodG) in human cellular DNA as a marker of oxidative stress. Samples of authentic 8-oxodG were distributed, and participating laboratories undertook to analyse this material within a specified period. Most HPLC procedures gave values for 8-oxodG within +/-40% of the target, as did two of four GC-MS procedures, and both LC-MS-MS methods. Calf thymus DNA samples containing increasing amounts of 8-oxodG were also distributed for analysis. Fewer than half the procedures tested were able to detect the dose response; those that were successful tended to be procedures with low coefficients of variation. For the analysis of 8-oxodG in human cells, where it is likely to be present at much lower concentrations than in the calf thymus DNA, it is crucial to reduce analytical variation to a minimum; a coefficient of variation of less than 10% should be the aim, to give reasonable precision. HPLC with amperometric electrochemical detection is not recommended, as it is less sensitive than coulometric detection. Immunological detection, 32P-postlabelling and LC-MS-MS are alternative approaches to measurement of 8-oxodG in DNA that, on the grounds of precision and detection of dose response, cannot at present be recommended. PMID- 10741855 TI - Iron mobilization by succinylacetone methyl ester in rats. A model study for hereditary tyrosinemia and porphyrias characterized by 5-aminolevulinic acid overload. AB - Accumulation of 5-aminolevulinic acid (ALA) is an event characteristic of porphyrias that may contribute to their pathological manifestations. To investigate effects of ALA independent of porphyrin accumulation we treated rats with the methyl ester of succinylacetone, an inhibitor of 5-aminolevulinic acid dehydratase that accumulates in the porphyric-like syndrome hereditary tyrosinemia. Acute 2-day treatment of fasted rats with succinylacetone methyl ester (SAME) promoted a 27% increase in plasma ALA. This increase in plasma ALA was accompanied by augmentation of the level of total nonheme iron in liver (37%) and brain (20%). Mobilization of iron was also indicated by 49% increase in plasma iron and a 77% increase in plasma transferrin saturation. Liver responded with a mild (12%) increase in ferritin. Under these acute conditions, some indications of oxidative stress were evident: a 15% increase in liver reactive protein carbonyls, and a 42% increase in brain subcellular membrane TBARS. Brain also showed a 44% increase in CuZnSOD activity, consistent with observations in treatment with ALA. Overall, the data indicate that SAME promotes ALA-driven changes in iron metabolism that could lead to increased production of free radicals. The findings support other evidence that accumulation of ALA in porphyrias and hereditary tyrosinemia may induce iron-dependent biological damage that contributes to neuropathy and hepatoma. PMID- 10741856 TI - The steady-state levels of oxidative DNA damage and of lipid peroxidation (F2 isoprostanes) are not correlated in healthy human subjects. AB - Oxidative damage to DNA in human tissues can be determined by measuring multiple products of oxidative damage to the purine and pyrimidine bases using gas chromatography-mass spectrometry (GC-MS). Oxidative damage to lipids (lipid peroxidation) can be quantitated by the mass spectrometry-based determination of F2-isoprostanes, specific end-products of the peroxidation of arachidonic acid residues in lipids. For both DNA base damage products and 8-epi prostaglandin F2alpha (PGF2alpha), there is a wide variation in levels between different healthy human subjects. We measured multiple products of oxidative damage to DNA bases in white cells, and 8-epi PGF2alpha in plasma, from blood samples obtained from healthy human subjects in the UK and in Portugal. No correlation of 8-epi PGF2alpha levels with levels of any modified DNA base (including 8 hydroxyguanine) was observed. We conclude that no single parameter can be measured as an index of "oxidative stress" or "oxidative damage" in vivo. PMID- 10741857 TI - Inhibition of inducible nitric oxide synthase lowers the cochlear damage by lipopolysaccharide in guinea pigs. AB - Endotoxin-treated cochleas of the guinea pig were examined electrophysiologically and immunohistochemically concerning the expression of inducible nitric oxide synthase (iNOS/NOS II). One mg of bacterial lipopolysaccharide (LPS, 5 mg/ml) or mixed solution of 1 mg of LPS plus 1 mg of N(G)-nitro-L-arginine methyl ester (L NAME, 5 mg/ml) (L-NAME/LPS) was injected into the middle ear of guinea pigs transtympanically. The electrocochleograms were measured prior to, immediately and 48 h after the injection. Immunohistological studies for iNOS followed after fixation, embedding and sectioning of the temporal bones. The threshold and amplitude of the compound action potential (CAP) became significantly worse in the LPS treated group. In contrast, the changes of the threshold and amplitude of CAP were decreased in the L-NAME/LPS group. iNOS was expressed in the stria vascularis, the spiral ligament, the organ of Corti and the spiral ganglion in the LPS group. These immunoreactivities in the L-NAME/LPS group were less intense than that in the LPS group. These results indicate that LPS has an ototoxic effect on the cochlea and that this effect could be mediated by iNOS produced high nitric oxide under inflammatory conditions. PMID- 10741858 TI - One-electron reduction of selenomethionine oxide. AB - Experimental evidence is provided that selenomethionine oxide (MetSeO) is more readily reducible than its sulfur analogue, methionine sulfoxide (MetSO). Pulse radiolysis experiments reveal an efficient reaction of MetSeO with one-electron reductants, such as e(aq)-, (k = 1.2x10(10) M(-1) s(-1)), CO2*- (k = 5.9x10(8) M( 1) s(-1)) and (CH3)2 C*OH (k = 3.5x10(7) M(-1) s(-1)), forming an intermediate selenium-nitrogen coupled zwitterionic radical with the positive charge at an intramolecularly formed Se(three-electron bond)N 2sigma/1sigma* three-electron bond, which is characterized by an optical absorption with lambda(max) at 375 nm, and a half-life of about 70 micros. The same transient is generated upon HO* radical-induced one-electron oxidation of selenomethionine (MetSe). This radical thus constitutes the redox intermediate between the two oxidation states, MetSeO and MetSe. Time-resolved optical data further indicate sulfur-selenium interactions between the Se(three-electron bond)N transient and GSH. The Se(three electron bond)N transient appears to play a key role in the reduction of selenomethionine oxide by glutathione. PMID- 10741859 TI - Complement receptor type two (CR2,CR21): a target for influencing the humoral immune response and antigen-trapping. AB - Cellular receptors for complement C3 fragments deposited on antigens are important bricks in the wall defending against microbial pathogens. The part of complement receptor type 2 (CR2; CD21) deals with enhancing humoral immune responses and with long-term trapping of C3d-coated antigen by follicular dendritic cells. CR2 is also pivotal for Epstein-Barr virus (EBV) infection. Here, the current understanding, how CR2 interacts with its ligands C3d, EBV, and CD23 is summarized. The potential to target CR2 for clinical therapy or immunization purposes are discussed. PMID- 10741860 TI - HLA-DM and the MHC class II antigen presentation pathway. AB - The MHC class II antigen processing pathway provides a mechanism to selectively present peptides generated in the endosomal compartments of antigen presenting cells to CD4+ T cells. Transport of newly synthesized class II molecules to the endosomal pathway requires the function of an accessory protein, invariant chain, which contains a region that interacts directly with the class II peptide binding site. Release of invariant chain and peptide loading by class II molecules are facilitated by a second accessory protein, HLA-DM. This MHC-encoded membrane protein catalyzes peptide exchange reactions, influencing the repertoire of peptides that are available for recognition by T cells. PMID- 10741861 TI - Interleukin 12 and innate molecules for enhanced mucosal immunity. AB - Recent strategies for understanding the mechanisms underlying mucosal immune responses and subsequent development of mucosal vaccines for induction of targeted immunity now include cytokines and molecules of innate immunity. These studies have shown that cytokines influencing the development of T helper (Th) cells differentially affect the outcome of mucosal vs. systemic immune responses to mucosal vaccines. Serum antigen-specific antibody (Ab) responses were enhanced when either IL-6 or IL-12 was mucosally administered with a protein antigen, while only IL-12 induced antigen-specific mucosal IgA Ab responses. Mucosal IL-6 and IL-12 also affected the type of Th cell responses induced by CD4+ T cells from mice that received IL-12 secreted larger amounts of IFN-gamma and IL-6 when compared with mice nasally treated with IL-6. Discrepancies in the ability to enhance mucosal or systemic immune responses were also observed when human neutrophil peptide (HNP) defensins or lymphotactin were nasally coadministered with protein antigens. Only lymphotactin promoted mucosal secretory IgA (S-IgA) Ab responses while both lymphotactin and defensins enhanced systemic immunity to mucosally co-administered protein antigens. Mixed antigen-specific Th1 -and Th2 type CD4+ T cell responses were induced in the systemic compartment by both lymphotactin and the mixture of HNP-1, HNP-2, and HNP-3 defensins. However, HNPs failed to significantly enhance cytokine secretion by mucosally derived, antigen specific CD4+ T cells relative to those isolated from the systemic compartment. In summary, these studies clearly show that IL-12 and lymphotactin are able to trigger S-IgA Ab responses and provide new avenues for the design of safe and targeted mucosal vaccines. PMID- 10741863 TI - Oligoclonal Th2-biased betabeta T cells induce murine inflammatory bowel disease. AB - A population of CD4+ T cells with TCR beta-chain without TCR alpha-chain (CD4+, betabeta T cells) producing Th2-type cytokines increased in the mucosal and peripheral tissues of TCR alpha-chain deficient mice with inflammatory bowel disease (IBD). Analysis of TCR-beta immunoprecipitates by two-dimensional electrophoresis and RT-PCR revealed TCR of the CD4+ T cells was a homodimer of TCR beta-chains. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analyses of TCR Vbeta-chain transcripts of the betabeta T cells revealed monoclonal to oligoclonal accumulation of the cells in the colon, suggesting clonal expansion of the mucosal betabeta T cells upon the stimulation with gut-derived antigens. The homodimer of TCR beta-chains on the betabeta T cells was a biologically functional receptor that transduced activation signals provided by MHC-class II-associated peptidic antigens and superantigens. Treatments of the mutant mice with mAb against TCR beta or IL-4 suppressed the onset of IBD. These findings suggest that the generation of oligoclonal Th2-type betabeta T cells plays a critical role for the development of IBD. PMID- 10741862 TI - The interaction of intestinal epithelial cells and intraepithelial lymphocytes in host defense. AB - Intestinal intraepithelial lymphocytes (i-IEL) are located at the basolateral surfaces of intestinal epithelial cells (i-EC) and play important roles in the homeostasis of intestinal microenvironment. i-IEL comprise unique T cell populations including CD4-CD8alphaalpha+ T cells expressing T cell receptor (TCR)alphabeta or TCRgammadelta and CD4+ CD8alphaalpha+ T cells expressing TCR alphabeta. We show here that CD4+ CD8alphaalpha+ i-IEL belongs to Th1 type T cells capable of responding to self-MHC class I on i-EC and that a significant fraction of i-IEL expressed Fas ligand (Fas-L) and induced apoptosis in the i-EC via Fas-dependent pathway. i-IEL may recognize and eliminate the effete i-EC for homeostatic regulation of intestinal epithelia. The interaction of i-EC and i-IEL through E-cadherin/alphaEbeta7 integrin is important for homing and maintenance of i-IEL in intestine. Listeria monocytogenes are also known to interact with E cadherin on i-EC and invade into the epithelial cells. Invasion of L. monocytogenes into i-EC activated NFkappa-B and subsequently up-regulated the expression of IL-15 gene, which has a NFkappa-B binding site at the promoter region. i-IEL, especially gammadelta T cells, were significantly activated to produce Th1 type cytokines at the early stage after oral infection with L. monocytogenes in mice and rats. The activation of i-IEL coincided with a peak response of IL-15 production by i-EC after infection. Taken together, mutual interaction of i-IEL and i-EC may be important not only for homeostatic regulation but also host defense against microbial infection in intestine. PMID- 10741864 TI - New gut associated lymphoid tissue "cryptopatches" breed murine intestinal intraepithelial T cell precursors. AB - Numerous mouse intraepithelial T cells (IEL) bearing either TCR-alphabeta or TCR gammadelta have been shown to develop somewhere in the intestinal mucosa without passing through the thymus. However, just where these T cells develop has been much less clear and has remained an open question to date. In an effort to investigate this issue, we carried out immunohistochemical study on the murine gastrointestinal tract and identified numerous tiny lymphoid tissues (approximately 1,650 tissues/intestine) in the cryptal region of the small and large intestinal mucosa except for the stomach in which clusters of c-kit+ IL-7R+ Thy1+ lympho-hemopoietic progenitors accumulated (cryptopatches). The cryptopatch cells isolated from the small intestine, which were c-kit positive (c-kit+) but lineage marker negative (Lin-), gave rise to TCR-alphabeta and TCR-gammadelta IELs following in vivo transfer or tissue engraftment into 2 Gy-irradiated severe combined immunodeficient mice. In contrast, cells isolated from Peyer's patches and mesenteric lymph nodes, which belong in the same intestinal immune compartment but lack c-kit+Lin- cells, failed to do so. These results in conjunction with the findings of electron microscopic analysis provide direct evidence of a local intestinal T cell precursor that develops in the cryptopatches. PMID- 10741865 TI - Mucosal IL-7-mediated immune responses in chronic colitis-IL-7 transgenic mouse model. AB - We have demonstrated that intestinal epithelial cells produce interleukin-7 (IL 7) and IL-7 serves as a regulatory factor for proliferation of mucosal lymphocytes. To clarify the mechanism by which locally produced IL-7 regulates mucosal lymphocytes and the role of mucosal IL-7 in colonic inflammation, we investigated IL-7 transgenic (IL-7 Tg) mice and demonstrated that IL-7 Tg mice developed acute and chronic colitis with histopathological similarity to ulcerative colitis in humans. In concert with our recent findings that IL-7 stimulates the proliferation of inactivated mucosal lymphocytes but eliminates activated lymphocytes in the inflamed mucosa of human ulcerative colitis. These findings suggest that chronic inflammation in the colonic mucosa is mediated by dysregulation of epithelial cell-derived IL-7 system. PMID- 10741867 TI - Regulation of the ryanodine receptor calcium release channel of the sarcoplasmic reticulum in skeletal muscle. AB - In striated muscle contraction is under the tight control of myoplasmic calcium concentration ([Ca2+]i): the elevation in [Ca2+]i and the consequent binding of calcium to troponin C enables, while the decrease in [Ca2+]i prevents the actin myosin interaction. Calcium ions at rest are stored in the sarcoplasmic reticulum (SR) from which they are rapidly released upon the depolarisation of the sarcolemmal and transverse (T-) tubular membranes of the muscle cell. The protein responsible for this controlled and fast release of calcium is the calcium release channel found in the membrane of the terminal cisternae of the SR. This review focuses on the physiological and pharmacological modulators of the calcium release channel and tries to draw an up-to-date picture of the events that occur between T-tubular depolarisation and the release of calcium from the SR. PMID- 10741868 TI - Distribution of cadmium in selected organs of mice: effects of cadmium on organ contents of retinoids and beta-carotene. AB - Cadmium was administered to 32 adult ICR mice i.p. in two single doses (0.25 and 0.5 mg CdCl2, per kg of b.w.). After 48 hours concentrations of cadmium in kidneys, liver, spleen, muscle (m. quadriceps femoris), ovaries and testes and the concentration of retinyl palmitate, retinol and beta-carotene in kidney, liver and testes were determined. Significantly higher cadmium concentration was found in liver, kidney and ovary in both experimental groups in comparison with the control group (p<0.001). In muscle, spleen and testis the cadmium level was higher, however not significantly. No significant differences in the concentration of retinyl palmitate, retinol and alpha-carotene in liver were found. Concentration of alpha-carotene in kidney and testis was significantly decreased in both groups administered with cadmium (p<0.001). Concentration of retinyl palmitate was significantly lower in testis in the group with higher cadmium level (p<0.001) and the concentration of retinol significantly decreased in kidney and testis of mice after an administration of 0.5 mg CdCl2/kg b.w. PMID- 10741869 TI - The influence of sound stimulation during hatching on the mortality of ducks. AB - In the work the influence of the artificial sound stimulation during incubation on the speed of the Pekin duck breed hatching as well as on their mortality during that period was observed. The set eggs were hatched in four hatcheries. The eggs of the first two hatcheries (control groups Ka and Kb) were not sound stimulated. In the other two groups the set eggs were, from the very first hour of hatching, stimulated by the "knocking" sound from an electronic sound generator (experimental groups "a" and "b"). For the stimulation, the acoustic signal with intensity of 25 dB and frequency 5.68 Hz was applied. The fastest hatching process was recorded in the groups with sound stimulation. The "a" experimental group was the fastest, the ducks were hatched after 640.75+/-10.15 hours, in the "b" experimental group the ducks were hatched after 656.50+/-1.70 hours of incubation. In both sound stimulated experimental groups (a, b) the increased embryonic mortality resulted in the decrease of the percentage of hatchability--to 68.09+/-1.77% and 76.44+/-2.68%, respectively. This is statistically evident (P<0.01) when compared with the respective control groups which reached the 88.81+/-1.86% and 89.35+/-0.81% hatchability. PMID- 10741866 TI - Eosinophils, ribonucleases and host defense: solving the puzzle. AB - The eosinophil ribonucleases eosinophil-derived neurotoxin (EDN/RNase 2) and eosinophil cationic protein (ECP/RNase 3) are among the major secretory effector proteins of human eosinophilic leukocytes, cells whose role in host defense remains controversial and poorly understood. We have recently described the unusual manner in which this ribonuclease lineage has evolved, with extraordinary diversification observed in primate as well as in rodent EDNs and ECPs. The results of our evolutionary studies suggest that the EDN/ ECP ribonucleases are in the process of being tailored for a specific, ribonuclease-related goal. With this in mind, we have begun to look carefully at some of the intriguing associations that link eosinophils and their ribonucleases to disease caused by the single-stranded RNA viral pathogen, respiratory syncytial virus (RSV). Recent work in our laboratory has demonstrated that eosinophils can mediate a direct, ribonuclease-dependent reduction in infectivity of RSV in vitro, and that EDN can function alone as an independent antiviral agent. The results of this work have led us to consider the possibility that the EDN/ECP ribonucleases represent a heretofore unrecognized element of innate and specific antiviral host defense. PMID- 10741870 TI - Changes in rat muscle with compensatory overload occur in a sequential manner. AB - The present study was initiated to determine the time course of changes in the profile of selected skeletal muscle myofibril proteins during compensatory overload. Whole muscle isometric contractile properties were measured to assess the physiological consequences of the overload stimulus. Compensatory overload of plantaris muscle of rats was induced by surgical ablation of the synergistic soleus and gastrocnemius muscles. Myosin light chain (LC) and tropomyosin (TM) compositions of control (CP) and overloaded plantaris (OP) muscles were determined by electrophoresis and myofibrillar ATPase assays were performed to assess changes in contractile protein interactions. Within one week of overload decreases in the alpha:beta TM ratio and myofibrillar ATPase activity were observed. Following 30 days of overload, a transition in type II to type I fibres was associated with an increase in slow myosin LC1. Interestingly, after 77 days of overload, the TM subunit ratio returned to one resembling a fast twitch muscle. It is proposed that the early and transitory changes in the TM subunits of OP, as well as the rapid initial depression in maximum tetanic isometric force and myofibrillar ATPase activity may be explained as a result of muscle fibre degeneration-regeneration. We propose that alterations in protein expression induced by compensatory overload reflect both degenerative-regenerative change and increased neuromuscular activity. PMID- 10741871 TI - Changes in the activity of some lysosomal enzymes and in the fine structure of submandibular gland due to experimental diabetes. AB - The aim of this study was to establish and quantify changes in the activities of the some lysosomal enzymes and to determine the type of changes in the ultrastructure of the submandibular gland in rabbits caused during progression of diabetes. The experiment was conducted on 89 New Zealand rabbit males. Diabetes was induced by the intravenous administration of 10% alloxan solution at a dose of 10-mg/kg-body weight. On the seventh day after alloxan administration, the level of glucose in blood was determined. Rabbits were divided into five groups: intact (n=18), 21-day diabetes (n=18), 42-day diabetes (n=17), 90-day diabetes (n=19) and 180-day diabetes (n=17). From killed animals in each group, the submandibular glands were removed and fixed or stored. Enzyme activities were assayed by spectrophotometric methods using substrates (Sigma) which release 4 methyloumbeliferol when they react with the proteases. Fixation procedure was done according to standard methods. Semi-thin and ultra-thin specimens were prepared by use of clearly visible after 42 days of diabetes. Mitochondria were damaged, accumulation of large amounts of lipids in the intracellular spaces was observed. After 90 days the presence of vacuoli and swollen lysosomes were observed, some cells also contained myelin figures. After 180 days the greatest changes were observed in the blood vessels, which had thickened walls and were often occluded. We concluded that the total activity of acid phosphatase and beta N-acetyl-glucosaminidase in the submandibular gland was correlated with the level of glucose but there was no correlation between total beta-galactosidase activity and the serum concentration level of glucose has been detected during course of diabetes. The activities of the free fractions of acid phosphatase, beta galactosidase and beta-N-acetyl-glucosaminidase in the submandibular gland were higher than the bound fractions in all groups of rabbits. The changes in the ultrastructure of the submandibular gland were correlated with changes in serum glucose level and with lysosomal enzymes activities during progression of experimental diabetes in rabbits. PMID- 10741872 TI - Blood flow of the right and left submandibular gland during unilateral carotid artery occlusion in rat: role of nitric oxide. AB - The aim of the present study was to investigate the effect of unilateral carotid artery occlusion on the blood flow of submandibular gland in anesthetized rats and identify the role of nitric oxide (NO) in blood flow changes after the artery occlusion. L-NAME (N omega-nitro-L-arginine-methyl-ester; 10 mg/kg/day, per os) dissolved in tap water was used to block nitric oxide synthase. Glandular blood flow was measured using Sapirstein's indicator (86Rb) distribution technique. In the control animals the blood flow of left (ligated side) submandibular gland was lower than in the right (unligated side) one (right: 76.4+/-15.4 ml/min/100 g, 64.1+/-13.4 ml/min/100 g, p<0.01). The blood flow of submandibular glands decreased in NOS blocked group versus control. The vascular resistance after L NAME treatment was elevated (control: 11+/-2.3 R/kg, L-NAME: 17.5+/-4.1 R/kg, p<0.001). In L-NAME group the difference between blood flow value of the left and right submandibular gland was significantly lower than in the control group (control: -16%, NAME: -8%, p<0.01). CONCLUSION: The maintenance of the blood flow in the left submandibular gland during ligation of the left common carotid artery could be due to the good vascular anastomotic system at these regions and adaptation of the submandibular vessels to the decreased perfusion pressure. Nitric oxide may have a role in the regulation of blood flow tinder this condition. PMID- 10741873 TI - Identification of acute intermittent porphyria carriers by molecular biologic methods. AB - Acute intermittent porphyria (AIP) is an autosomal dominant disease caused by mutations of the gene coding for hydroxymethylbilane synthase. Acute attack of AIP is a potentially life-threatening condition precipitated by certain drugs, alcohol, fasting and stress. Biochemical diagnosis before the manifestation of the symptoms is problematic, and genetic screening is required to identify asymptotic carriers. The aim of this study was to establish a fast, reproducible and reliable genetic method to detect mutations causing AIP. Exon 10 of one healthy individual and 12 AIP patients was studied using a recently developed method, temporal temperature gradient electrophoresis (TTGE). Mutation of exon 10 was detected using TTGE in one patient, DNA sequence analysis confirmed the presence of a heterozygous point mutation causing substitution of the arginine in position 173 of the gene with glutamine. These results were also confirmed using restriction enzyme analysis, and this method and TTGE identified a child of this patient as an asymptotic carrier of AIP. PMID- 10741874 TI - Effects of bradykinin in the cerebral circulation. AB - All components of an intracerebral kallikrein-kinin system have been described. Thus, bradykinin (BK) acting from the parenchymal side as well as from the blood side may influence cerebral microcirculation. BK is a potent dilator of extra- and intraparenchymal cerebral arteries when acting from the perivascular side. The vasomotor effect of BK is mediated by B2 receptors which appear to be located at the abluminal membrane of the endothelial cell. Signal transmission from the endothelial to the smooth muscle cell is mediated by NO, prostanoids, free radicals or H2O2 depending on the animal species and on the location of the artery. Selective opening of the blood-brain barrier for small tracers (Na+ fluorescein: MW, 376) has been found in cats during cortical superfusion or intraarterial application of BK. This leakage is mediated by B2 receptors located at the luminal and abluminal membrane of the endothelial cells and probably mediated by an opening of tight junctions. Formation of brain edema has been found after ventriculo-cisternal perfusion or interstitial infusion of BK. This can be explained by increase of vascular permeability and cerebral blood flow due to arterial dilatation thus enhancing driving forces for the extravasation. An increase of the BK concentration in the interstitial space of the brain up to concentrations which induce extravasation, dilatation and edema formation has been found under several pathological conditions. Thus, BK may be involved in edema and necrosis formation after cold lesion, concussive brain injury, traumatic spinal cord and ischemic brain injury. PMID- 10741875 TI - Selective inhibition of neuronal nitric oxide synthase fails to alter the resting tension and the relaxant effect of bradykinin in isolated rat middle cerebral arteries. AB - The role of the neuronal isoform of the nitric oxide (NO) synthase (nNOS) in the regulation of the cerebrovascular tone was studied in vitro. Selective inhibition of nNOS by 7-nitro indazole monosodium salt (7-NINA) failed to alter the resting tension and the relaxant effect of bradykinin in isolated rat middle cerebral arteries. These results indicate that 1./ 7-NINA is selective for nNOS and 2./ cerebrovascular nNOS is involved neither in the resting NO production nor in the mediation of the relaxant effect of bradykinin. Therefore, nNOS-derived NO that contributes to the maintenance of the resting cerebral blood flow in vivo appears to be released from neurons and/or glial cells. PMID- 10741876 TI - Childhood asthma: causes, epidemiological factors and complications. AB - Asthma is common in children and its prevalence in this age group is increasing. While the reasons for this reported increase, and indeed the true magnitude of the increase, remain unclear, there can be no doubt that asthma is now a major health problem in children worldwide. Fortunately, our knowledge of the pathophysiology of asthma is also increasing. It is now known that asthma is a chronic inflammatory disease regulated by a variety of mediators, of which perhaps the leukotrienes are among the most important. This new understanding of the pathophysiology of the disease has spurred the development of the antileukotriene agents, which can be expected to play an increasingly important role in the management of childhood asthma. PMID- 10741877 TI - Inhaled corticosteroids in childhood asthma: growing concerns. AB - Inhaled corticosteroids (ICS) are an established treatment for asthma in childhood. Recent data bring to light growing concerns that ICS may have significant effects on growth velocity in children. The Food and Drug Administration (FDA) recently convened a joint meeting to review these data, and to release new class labelling for ICS that notes this potential adverse effect. Additional concerns regarding ICS are also discussed, including other potential adverse effects, difficulty of use, noncompliance, and patient and parental concerns with the safety of ICS. The aim of this article is as follows: to describe the rationale for the use of ICS in children with asthma; to delineate the association of ICS with potential growth suppression in children; to discuss recent FDA class labelling for use of ICS in children; to describe other potential long term effects of ICS in children; and to detail compliance issues in children with asthma treated with ICS. PMID- 10741878 TI - Corticosteroid-sparing options in the treatment of childhood asthma. AB - During the last 30 years, a significant rise in wheezing illness has occurred in the child population. Despite its high prevalence there is no clear definition of the disease, which includes a heterogeneous group of syndromes ranging from transient wheezing in infancy to atopic asthma with persistence into adult life. Molecular advances and further epidemiological information from well characterised individuals and their families are likely to clarify the different subtypes of wheezing illness and inform therapeutic options. With the recognition that chronic airway inflammation is a feature of persistent disease, at least in adults, there has been a trend towards the early introduction of anti inflammatory treatment and particularly inhaled corticosteroids (ICS). However, the natural resolution of much wheezing illness, particularly in young children and in children with viral-induced episodes, suggests that newly presenting children should remain on symptomatic therapy alone while the severity of the disease is being assessed. Although ICS have become a cornerstone of management of chronic persistent disease, their ability to protect against exacerbations in young and mildly affected children is questionable. Alongside concerns about long term use of ICS and possible systemic adverse effects, there remains a need for alternative approaches to the control of the disease in children. Extrapolation of the findings of large multicentre adult studies into childhood, particularly for doubling the doses of ICS and long-acting beta2-agonists, may be unsound. Other approaches include the early introduction of inhaled cromones, use of second generation antihistamines, low dose theophyllines and, more recently, leukotriene modifiers. As the majority of preschool children will become asymptomatic by mid-childhood, there is an urgent need to identify those in whom chronic airway inflammation is developing, as it is in this group that early introduction of ICS may be of maximum benefit. In the remainder, other approaches, including use of corticosteroid-sparing longacting P2-agonists and leukotriene modifying drugs, may be more appropriate. Safe and effective oral preparations such as leukotriene modifying drugs are likely to establish a significant role in the management of symptoms in children of all ages and with all types of asthma and wheezing illness. PMID- 10741879 TI - Guidelines update: where do the new therapies fit in the management of asthma? NHLBI and WHO Global Initiative for Asthma. AB - The new leukotriene (LT) modifiers have been shown to be effective, safe and convenient 'controller' medications in patients with asthma. However, their use in asthma is recommended only in the most recent US guidelines, and then only as alternative long term controllers in patients with mild persistent asthma. In fact, as has now been shown in a number of studies, LT modifiers are effective in a variety of other asthma settings, and it is expected that expanded roles for these agents will be described in future asthma guidelines. Until then, clinicians aware of the advantages of individualising asthma therapy might consider LT modifiers for the diverse range of patients with asthma who are likely to benefit from the use of these agents. PMID- 10741880 TI - Clinical evidence with montelukast in the management of chronic childhood asthma. AB - OBJECTIVE: The aim of this article is to review data on the efficacy and safety of montelukast in the treatment of children with asthma. METHODOLOGY: Available published literature, including published abstracts, is reviewed. RESULTS: In patients aged 6 to 14 years with asthma (n = 27), montelukast 5mg demonstrated a significant decrease in exercise-induced bronchoconstriction 20 to 24 hours postdose after 2 days of treatment. For children with chronic asthma, only one study of the regular use of a leukotriene receptor antagonist has been published. The efficacy and safety of montelukast in children aged 6 to 14 years with asthma (n = 336) were studied during an 8-week, double-blind, placebocontrolled trial. There was a significantly greater improvement in forced expiratory volume in 1 second (FEV1) from baseline for the montelukast group (8.23%) compared with the placebo group (3.58%). There was a significant decrease in the use of a 3-agonist for symptom relief, as well as in the percentage of days and percentage of patients with asthma exacerbations. An asthma specific quality-of-life (QOL) questionnaire revealed a significant overall improvement in QOL and a significant improvement in the QOL domains for symptoms, activity and emotions in montelukast recipients. There was no significant difference between montelukast and placebo recipients in the frequency of adverse events, with the exception of allergic rhinitis, which was more prevalent in the placebo group. An open label follow-up of patients from the above study was undertaken. The effect of montelukast on FEV1 was consistent for up to 1.4 years, with the increase in FEV1 being not significantly different from that in a small control group treated with inhaled beclomethasone dipropionate. QOL remained significantly improved during the open treatment period. CONCLUSIONS: Montelukast appears effective and safe for the treatment of children with asthma. PMID- 10741881 TI - Tolerability of montelukast. AB - The tolerability of a medication, especially in children with asthma, is linked to a number of key factors. These include clinical effectiveness, adverse effects, frequency of drug regimen, ease and route of administration. and taste. Montelukast is unusual in that, in most countries, a licence for children aged > or =6 years was granted at the same time as the adult licence. This is related to a variety of evidence. which includes pharmacological and adult studies suggesting the specificity and safety of the drug at many times the licensed dose, and a tolerability profile similar to that with placebo or inhaled corticosteroids in both adult and paediatric studies. The most common adverse effects in paediatric studies were headache, asthma and upper respiratory tract infection at rates not statistically significantly different from those with placebo. Up to July 1999, more than 2 million patients worldwide have received montelukast, of whom nearly 220,000 have received the paediatric formulation. In the UK, one prescribing database suggests that, of children who commenced montelukast therapy, less than 25% discontinued the drug. This implies that montelukast is effective and well tolerated in most children. Adverse effect monitoring by regulatory bodies has revealed little that would not be expected on the basis of the results of clinical trials. Montelukast has been associated with Churg-Strauss syndrome in a very small number of adults. In most. the syndrome was associated with corticosteroid withdrawal, which may have unmasked the condition. Churg-Strauss syndrome has not been reported in children. Its clinical effectiveness, lack of major adverse effects, oral route of administration, palatability and the once-daily regimen combine to make montelukast a generally well tolerated medication in children. PMID- 10741882 TI - Psychopathology of stable and unstable mixed states: a historical view. AB - The concept of mixed states was proposed at the end of last century by Kraepelin and Weygandt to confer unity to manic-depressive illness and to better differentiate this disease from dementia praecox. The concept has been further elaborated by the Hamburg and Vienna schools (Mentzos and Berner, respectively), and dichotomized in stable and unstable mixed states. Stable mixed states represent a condition of the synchronic copresence of symptoms of both polarities; unstable mixed states, or "mixed pictures," are characterized by the rapid cycling of these symptoms. The former represent clinical expressions of bipolar disorder, and the latter represent trans-nosological conditions with multiple outcomes. Current North American nosology retains the original Kraepelinian concept of stable mixed states and ignores the concept of unstable mixed states, which is very close to the concept of ultradian cycling proposed by Kramlinger and Post in 1996. PMID- 10741883 TI - The role of gender in mixed mania. AB - This article reviews the literature regarding possible gender differences in adults with mixed mania. Studies examining gender differences in the prevalence of mixed mania, biological abnormalities, suicidality, long-term outcome, and treatment response were analyzed. Data from these studies suggest that mixed mania may occur more commonly in women than in men, especially when defined by narrow criteria. There were no significant differences between men and women with mixed mania in biological abnormalities, suicidality, outcome, and treatment response. PMID- 10741884 TI - Outcomes associated with delirium in acutely hospitalized acquired immune deficiency syndrome patients. AB - The study demonstrates that delirium in acquired immune deficiency syndrome (AIDS) patients is associated with mortality, the need for long-term care, and an increased length of hospitalization. Data were collected prospectively on human immunodeficiency virus (HIV)/AIDS patients admitted to a teaching hospital from January 1996 through December 1996. The data included demographic characteristics of the participants, medical diagnoses, CD4 cell count, Karnofsky functional assessment, mortality during admission, length of stay, and discharge placement. Participants were evaluated throughout their hospital stay for evidence of delirium. The presence of delirium was determined using DSM-IV diagnostic criteria. There were no significant differences between delirious and nondelirious patients with respect to demographic characteristics or markers of medical morbidity. Patients with delirium were more likely to die during admission (chi-square [chi2] = 39.1, df = 1, P<.0010), to stay longer in hospital (t = 3.50, df = 12.9, P<.0041), or to need long-term care if discharged alive (chi2 = 12.8, df = 2, P<.0021). Delirium is associated with adverse outcomes in hospitalized AIDS patients. More research is needed to characterize the nature of this association. PMID- 10741885 TI - The specificity of epidemiological correlates of major depression. AB - Some of the variables associated with major depression in community samples may be nonspecific correlates of mental ill-health. The study objective was to evaluate this hypothesis by comparing two groups of psychiatric patients with the general population. Subjects were recruited from psychiatric inpatients at a general hospital in Calgary, Alberta, Canada, by dissemination of a consent form. Consenting subjects completed the mood disorders section of the Composite International Diagnostic Interview (CIDI). All subjects were interviewed to evaluate a set of variables that may be risk factors for major depression. The measurement instruments were identical to those used in a national survey so that comparisons to population-based data were possible. The psychiatric inpatients differed dramatically from community subjects in terms of stress, traumatic life events, recent life events, and social support. However, differences were not observed between inpatients with major depression (according to the CIDI) and inpatients without major depression. Associations between certain variables and major depressive disorders in community populations may reflect nonspecific associations with mental ill-health. This may occur because of nonspecific impacts of these variables on the etiology or prognosis of mental disorders or some nonspecific impact of mental illness itself. PMID- 10741886 TI - Anxiety disorders in major depression. AB - The prevalence and clinical impact of anxiety disorder comorbidity in major depression were studied in 255 depressed adult outpatients consecutively enrolled in our Depression Research Program. Comorbid anxiety disorder diagnoses were present in 50.6% of these patients and included social phobia (27.0%), simple phobia (16.9%), panic disorder (14.5%), generalized anxiety disorder ([GAD] 10.6%), obsessive-compulsive disorder ([OCD] 6.3%), and agoraphobia (5.5%). While both social phobia and generalized anxiety preceded the first episode of major depression in 65% and 63% of cases, respectively, panic disorder (21.6%) and agoraphobia (14.3%) were much less likely to precede the first episode of major depression than to emerge subsequently. Although comorbid groups were not distinguished by depression, anxiety, hostility, or somatic symptom scores at the time of study presentation, patients with comorbid anxiety disorders tended to be younger during the index episode and to have an earlier onset of the major depressive disorder (MDD) than patients with major depression alone. Our results support the distinction between anxiety symptoms secondary to depression and anxiety disorders comorbid with major depression, and provide further evidence for different temporal relationships with major depression among the several comorbid anxiety disorders. PMID- 10741887 TI - Premenstrual exacerbation and suicidal behavior in patients with panic disorder. AB - This study examined the relationship between premenstrual exacerbation and suicidal behavior in patients with panic disorder. Twenty-eight patients who reported a premenstrual exacerbation of panic disorder and 42 patients who reported never having an exacerbation were included in the study. The subjects completed a self-rating instrument assessing the 13 DSM-III-R panic symptoms on a 5-point scale (0 = absent, 1 = mild, 2 = moderate, 3 = severe, and 4 = very intense). They also completed the severity scale of the Clinical Global Impression. The Schedule for Affective Disorders and Schizophrenia (SADS) suicide subscale was used to rate the severity of active suicidality. We found that patients with premenstrual exacerbation of panic disorder had higher SADS suicidality scores than patients with panic disorder without premenstrual exacerbation. They were also more likely to be classified as suicidal than the others. It may be that premenstrual exacerbation is a risk factor for suicidal behavior independent of major depression in patients with panic disorder. PMID- 10741888 TI - Borderline personality disorder and bipolar II disorder in private practice depressed outpatients. AB - Bipolar II disorder (BDII) may be confused with borderline personality disorder (BPD) when it is cyclothymic between episodes. The aim of the present study was to determine the prevalence of BPD and to test whether BDII can be distinguished from BPD without difficulty in private practice mood disorder outpatients. Private practice was chosen because it is often the first or second line of treatment of mood disorders in Italy, and many "soft" patients can be found in this setting. Among 63 consecutive unipolar and 50 bipolar II major depressive episode (MDE) outpatients interviewed with the Structured Clinical Interviews for DSM-IV axis I/II disorders (SCIDs), the prevalence of BPD was 6.1% and was significantly higher in BDII patients (12% v. 1.5%). Overall, the rate of BPD diagnosis was very low. BDII was distinguished from BPD without difficulty by DSM IV criteria. The results suggest that there may be a subgroup of BDII patients with a relatively stable course between episodes (or at least not so unstable as to suggest a BPD diagnosis or comorbidity) and a low comorbidity with BPD, in a setting closer to community patients than university settings. The "usual" BDII patient can be distinguished from the BPD patient. PMID- 10741889 TI - Screening for binge eating disorder in obese outpatients. AB - The prevalence of binge eating disorder (BED) in clinical samples of obese patients is controversial, and sensitive diagnostic protocols for use in routine clinical practice need to be further defined. Three hundred forty-four obese (body mass index [BMI] > or =30 kg/m2) patients were studied with the Structured Clinical Interview for DSM-III-R to investigate the lifetime prevalence of mental disorders. The current prevalence of BED was assessed using DSM-IV criteria. Eating attitudes and behavior were investigated with the Bulimic Investigation Test, Edinburgh (BITE) and the Binge Eating Scale (BES). The Beck Depression Inventory (BDI) and Spielberg's State-Trait Anxiety Inventory (STAI) were also applied. The prevalence of BED was 7.5%. Patients with BED had a higher BMI compared with obese patients without BED. Differences in the lifetime prevalence of mental disorders in patients with and without BED were not statistically significant. Using the BES as a screening instrument for BED with a threshold of 17, the sensitivity was 84.8%, specificity 74.6%, positive predictive value 26.2%, and negative predictive value 97.9%. Using the BITE with a threshold of at least 10, the sensitivity was 91%, specificity 51.4%, positive predictive value 71.8%, and negative predictive value 98.2%. The BITE can be a valid alternative to the BES as a screening method for BED in obese patients. PMID- 10741890 TI - Irritable bowel syndrome in a psychiatric patient population. AB - The prevalence of irritable bowel syndrome (IBS) in psychiatric practice was studied in 41 consecutive psychiatric outpatients. Different criteria for IBS were applied to the data set to determine the effects on the rates of IBS obtained. Depending on an option in the Rome criteria, IBS rates varied from 13% using the "and" requirement for combining abdominal pain and altered bowel function symptoms, to 41% using the "and/or" option described in the formal definition statement in 1990. The resultant prevalence rates of IBS varied greatly according to which published criteria were applied, with a maximum of 71%. This wide variation in rates depending on the criteria underscores the critical importance of standardizing diagnostic research criteria for IBS. An exemplary model of empirically based validation has been developed for psychiatric disorder criteria which, like IBS, are symptom-based and lack physiological determinants. Validated diagnostic criteria for IBS await similar study. PMID- 10741891 TI - Parental representations in drug-dependent patients and their parents. AB - The Parental Bonding Instrument (PBI), a measure of perceived parental care and protection, was administered to drug-dependent patients and their parents with the aim to assess the reliability of the instrument in such samples and to compare the parental representations across generations. Ninety drug-dependent patients and 44 mothers and 35 fathers participated. Reliability indices were calculated, and parental representations of parents and their offspring were compared. Linear regression analyses were performed with the patient's PBI score as the dependent variable and the mother's and father's PBI scores as predictor variables. The reliability indices were highly satisfactory and varied between 0.61 and 0.91. The parental bonding of patients, fathers, and mothers was similar. All three groups reported high maternal and paternal control and low maternal care, a pattern characteristic of an "affectionless control" rearing style. Maternal care received by the fathers and paternal protection received by the mothers predicted the care and protection they themselves gave to their drug dependent offspring. PMID- 10741892 TI - Personality traits of women with a history of childhood sexual abuse. AB - This study examined relationships between specific dimensions of childhood sexual abuse and personality traits in adulthood. Study participants were 74 hospitalized female psychiatric patients with a self-reported history of childhood sexual abuse. Characteristics of childhood sexual abuse were obtained from a structured life-events interview. Personality was measured with the NEO Five-Factor Inventory (NEO-FFI), which yields scores on neuroticism, extraversion, openness to experience, agreeableness, and conscientiousness. We hypothesized that parental abuse, intercourse, and the combination of these two childhood sexual abuse characteristics would be associated with personality traits. Supporting this hypothesis, women who were abused by a parent had lower scores on openness to experience than women who were abused by someone else. Patients whose abuse history included both parental abuse and intercourse had very low extraversion scores. Our findings suggest that there are associations between personality traits and childhood sexual abuse characteristics in psychiatric patients. Specifically, women who experienced intercourse by a parent may be more introverted and less open to experience than women whose sexual abuse history does not include parental incest. PMID- 10741893 TI - Identifying personality disorders: towards the development of a clinical screening instrument. AB - The study objective was to identify a set of personality disorder (PD) criteria from the DSM PD diagnostic sets that can be used to detect subjects with an increased likelihood of having a PD diagnosis. In a series of outpatients evaluated systematically in two waves for every criteria item for 12 DSM-III-R PDs, stepwise logistic regression identified 45 criteria as discriminative for their specific PDs, which are selected for further analysis to assess their ability to discriminate for any PD. Receiver operating characteristic (ROC) analysis is used to evaluate their discriminative power in an independent conjoined sample (N = 1,342) from six centers that assessed every PD criteria item by structured instrument (Structured Clinical Interview for DSM-III-R PDs [SCID-II, Personality Disorder Examination [PDE], and Structured Interview for DSM-III-R PDs [SIDP-R]). The cutoff that maximizes information gain is used to determine the diagnostic threshold (DT). Initially, 15 of 45 criteria are identified. At the 0.43 PD prevalence, a DT of 2 or more of the 15 PD criteria across samples is optimal. The maximum information gain (MIG) is .42 bits, and the AUR is 0.94+/-.007. Other performance indices at this cutoff are .90 sensitivity, .84 specificity, .81 positive predictive power (PPP), .91 negative predictive power (NPP), and .86 hit rate (HR). Taken collectively, the 15 PD criteria selected by the data reduction techniques suggest a narrowed set to be assessed in screening for the presence or absence of any PD with comparable or better psychometric properties than other tests routinely used for diagnosing medical and psychiatric disorders. If specific PD categorization is needed, a second-step comprehensive assessment should follow. PMID- 10741894 TI - The catatonia rating scale I--development, reliability, and use. AB - The study objective is to present reliability data on the Catatonia Rating Scale (CRS). The CRS is a 21-item clinician-administered rating scale. Items are individually defined and are scored from 0 (absent) to 4 (severe). A definition for each degree of severity is provided for each item. The diagnostic threshold for catatonia is 4 symptoms rated at least 2 (moderate). The CRS requires, at most, 45 minutes to administer. Reliability data were evaluated in a sample of 120 inpatients, 71 of whom were catatonic. Ratings were performed by 4 pairs of raters. Interrater reliability was high (Pearson correlation coefficient = .91 to .99, intraclass correlation coefficient = .94 to .99). Internal consistency was very good (Cronbach's alpha = .8890). Analysis of individual items showed that all were frequently endorsed and occurred across a wide range of severity. The CRS is a reliable rating scale for the diagnosis of catatonia. Advantages of the scale, areas of use, and clinical implications are discussed. PMID- 10741895 TI - Laboratory assessment as a critical component of the appropriate diagnosis and sub-classification of von Willebrand's disease. AB - von Willebrand's disease (VWD) is now recognized to be most common inherited bleeding disorder. It arises from defects or deficiencies in a protein called von Willebrand factor (VWF). VWD is a heterogeneous disorder, and patients are typed according to pathophysiology. The correct diagnosis and sub-classification of a patient's VWD is crucial because the presenting biological activity of VWF determines the haemorrhagic risk, and since subsequent clinical management will differ accordingly. Although clinical assessment of the propositus will provide the initial clue to, or an index of clinical suspicion for, a diagnosis of VWD, it is the laboratory process that will confirm or discount the diagnosis. A variety of assays may be employed by the laboratory undertaking the investigation, and these will not necessarily be restricted to an assessment of VWF. Due to the limitations of each potential laboratory assay, and because of VWD heterogeneity, no single test procedure is sufficiently 'robust' to permit detection of all VWD variants. This situation often leads to some clinical confusion in the process of laboratory interpretation regarding the likelihood of VWD, and the subtype of VWD. Classically, the test panel might include any combination of the following: (i) determination of (skin) bleeding times, (ii) VWF antigen (VWF:Ag) levels, (iii) 'functional' activity of Factor VIII (i.e. FVIII:coagulant or FVIII:C), (iv) 'functional' activity of VWF (e.g. Ristocetin Cofactor [VWF:RCof] assay), and/or Ristocetin induced platelet aggregation [RIPA] analysis), and (v) assessment of the VWF molecular weight or structural profile (i.e. VWF multimeric analysis or VWF:Multimers). There have also been a number of new diagnostic developments, and these are beginning to significantly influence the overall clinical VWD-diagnostic process. These include automation of existing assay procedures, a relatively new functional VWF assay called the Collagen Binding Assay (VWF:CBA), new automated platelet function analysers such as the PFA-100 and the Xylum Clot Signature Analyser, and specific VWF:FVIII binding assays. The current report focuses on the recommended laboratory process for investigation of VWD. An analysis of this process shows that selection of an appropriate test panel is a critical component for the proper diagnosis and classification. This review also outlines those new and emerging technologies that will help streamline the diagnostic process. Because VWD is just one manifestation of a 'bleeding' disorder (albeit the most common), the review also briefly mentions other related diagnostic processes and general approaches to the investigation of 'bleeding disorders'. The review also provides two algorithms to assist clinicians in making appropriate diagnostic choices in response to the clinical findings. A number of summary tables describing each laboratory assay in detail, and summarising the likely diagnostic findings for each Type of VWD, are also provided. This review should be of value to both haemostasis scientists and clinical specialists involved in VWD diagnosis. PMID- 10741896 TI - Prothrombin and the prothrombin 20210 G to A polymorphism: their relationship with hypercoagulability and thrombosis. AB - Polymorphisms of several clotting factors have been associated during the past few years with an increased risk of both venous or arterial thrombosis. However, final proof for the existence of a pathogenetic relationship between a given polymorphism and an increased risk for thrombosis is still lacking. Particular emphasis has been placed recently on a 20210 G to A prothrombin polymorphism. A critical review of available data indicates that such an abnormality may be associated with an increased risk of venous thrombosis but not arterial thrombosis (with a possible exception for myocardial infarction). However, this conclusion is based only on retrospective cohort studies which compared the prevalence of the abnormality in a group of patients with past venous or arterial thrombosis with a normal group (with no thrombosis). No prospective study has yet to show that patients with the abnormality, given similar additional acquired risk factors, have a higher incidence of thrombotic complications as compared with controls. The mechanism whereby the abnormality might cause thrombosis has been assumed to be an increase in prothrombin levels. Since an association between two phenomena does not necessarily mean that a causal relationship exists between the same events, it is important to be cautious before claiming that such abnormality is responsible for thrombosis. Therefore, although included commonly in the investigation profile, the search for the 20210 G to A prothrombin abnormality should not be considered yet to be an essential component in the routine study of hypercoagulable and/or thrombotic conditions. PMID- 10741897 TI - Biology of BCR-ABL. PMID- 10741898 TI - Current concepts: large granular lymphocyte leukemia. AB - Clonal diseases of large granular lymphocyte (LGL) disorders can arise from a CD3+ T-cell lineage or from a CD3- NK-cell lineage. CD3+ LGL leukemia is the most frequent form of LGL leukemia. T-LGL leukemia usually affects elderly people. Approximately 60% of patients are symptomatic; recurrent infections secondary to chonic neutropenia, anemia, and rheumatoid arthrititis are the main clinical manifestations. The most common phenotype is CD3+, alphabeta+, CD8+, CD57+. Clonality is detected by clonal rearrangement of the T-cell receptor gene. NK cell LGL proliferative disorders include NK LGL leukemia which is a very aggressive disease and NK chronic lymphocytosis. Serologic findings show frequent reactivity to the BA21 epitope of HTLV-I env p21e, suggesting that a cellular or retroviral protein with homology to BA21 may be important in pathogenesis of these diseases. Clonal expansion may be facilitated by IL12 and IL15 cytokines expressed by leukemic LGL, and also by a defective Fas (CD95) apoptotic pathway. Leukemic LGL constitutively express Fas and Fas-Ligand but they are resistant to Fas-induced apotosis. Neutropenia could be due to soluble Fas-Ligand which is highly secreted in the patient's sera. Clinical and molecular remission can be obtained with oral low-dose methotrexate. Leukemic LGL express a multi-drug resistance phenotype (PgP+/LRP+) that could partly explain the chemoresistance observed in aggressive cases. It is suggested that LGL leukemia can serve as a useful model of dysregulated apoptosis as an underlying mechanism for both malignancy and autoimmune disease. PMID- 10741899 TI - Heroin modulates the expression of inducible nitric oxide synthase. AB - The use of heroin (diacetylmorphine) is associated with a high incidence of infectious disease, and the immunologic alterations responsible for heroin induced changes in resistance to infection have not been well characterized. The present study tests the hypothesis that expression of inducible nitric oxide synthase (iNOS) is modulated by the administration of heroin. The initial study using rats showed that heroin administration (0, 0.01, 0.1, or 1.0 mg/kg s.c.) results in a pronounced reduction in lipopolysaccharide (LPS)-induced expression of iNOS mRNA in spleen, lung, and liver tissue as measured by RT-PCR. Heroin also produced a reduction in the level of plasma nitrite/nitrate, the more stable end product of nitric oxide degradation. In a subsequent study, administration of the opioid receptor antagonist, naltrexone (0.1 mg/kg) prior to the injection of heroin (1.0 mg/kg) blocked the heroin-induced reduction of iNOS expression and plasma nitrite/nitrate levels indicating that the effect is mediated via the opioid-receptor. This study provides the first evidence that heroin induces an alteration of iNOS expression, and suggests that a reduction in nitric oxide production may be involved in the increased incidence of infectious diseases amongst heroin users. PMID- 10741900 TI - Phenotypic and functional assessments of immune status in the rat spleen following acute heroin treatment. AB - Heroin use is associated with an increased incidence of several types of infections, including HIV. Yet few studies have assessed whether heroin produces pharmacological alterations of immune status that might contribute to the increased rate of infections amongst heroin users. The present study investigated whether a single administration of heroin to rats produces dose-dependent alterations in functional measures of immune status and in the distribution of leukocyte subsets in the spleen. The results showed that heroin produces a dose dependent, naltrexone-reversible suppression of the concanavalin A-stimulated proliferation of T cells, lipopolysaccharide-stimulated proliferation of B cells, production of interferon-gamma and cytotoxicity of natural killer (NK) cells in the spleen. Heroin's suppressive effect on NK cell activity results in part from a heroin-induced decrease in the relative number of NKR-P1A(hi) CD3- NK cells in the spleen. Heroin also decreases the percent of a splenic granulocyte subset, the CD11b/c+ HIS48(hi) cells, whose function currently is unknown. In contrast, heroin does not alter relative numbers of CD4+ CD3+ T cells, CD8+ CD3+ T cells, CD45+ B cells, NKR-P1A(lo) CD3+ T cells, CD11b/c+ ED1+ (or CD11b/c+ HIS48-) monocytes/macrophages or CD11b/c+ ED1- (or CD11b/c+ HIS48+) total granulocytes in the spleen. Collectively, these findings demonstrate that heroin produces pharmacological effects on functional and phenotypic measures of immune status. PMID- 10741901 TI - C3a and C5a enhance granulocyte adhesion to endothelial and epithelial cell monolayers: epithelial and endothelial priming is required for C3a-induced eosinophil adhesion. AB - Effects of the anaphylatoxins C3a and C5a on eosinophil and neutrophil adhesion to HUVEC and to primary culture human bronchial epithelial cells (HBEC) were investigated. Activities on both leukocytes and on structural cells were examined. C3a upregulated beta2 integrin expression and caused shedding of L selectin on eosinophils, but had no effect on neutrophil adhesion molecule expression. C5a upregulated beta2 integrins and caused shedding of L-selectin on both eosinophils and neutrophils. The potency of C5a was equivalent on both cell types; however, the magnitude of the changes in each of these adhesion molecules was significantly greater in neutrophils than eosinophils. Neither C3a nor C5a altered expression of ICAM-1, VCAM-1, E-selectin or P-selectin on either HUVEC or HBEC. C5a induced adhesion of both neutrophils and eosinophils to unstimulated HUVEC or HBEC, and adhesion was further enhanced when HUVEC and HBEC were "primed" with TNF-alpha and IFN-gamma, respectively. C3a failed to enhance adhesion of either eosinophils or neutrophils to unprimed HUVEC or HBEC, and enhanced only eosinophil adhesion to cytokine-primed HUVEC or HBEC. Similar to C3a, C3a(desArg) and a C3a-analog peptide E7 also enhanced eosinophil adhesion only to cytokine-primed HUVEC and HBEC. These results support the traditional view of anaphylatoxins as leukocyte-specific mediators. The specificity of C3a for eosinophils implicates this molecule as a potential participant in allergic inflammation. The pro-adhesive effects of C3a(desArg) suggest that this molecule, previously characterized as a spasmogenically inactive derivative of C3a, may also alter leukocyte dynamics and migration. Finally, activation of endothelium may represent an important control mechanism for C3a-mediated adhesion preventing unchecked eosinophil adhesion to uninflamed systemic vasculature. PMID- 10741902 TI - An assessment of the acute effects of the serotonin releasers methylenedioxymethamphetamine, methylenedioxyamphetamine and fenfluramine on immunity in rats. AB - The purpose of the present study was to examine the effect of the serotonin releasing amphetamine derivatives methylenedioxymethamphetamine (MDMA), methylenedioxyamphetamine (MDA) and fenfluramine (FEN) on immunity in rats. Similar to MDA and MDMA, FEN reduced the number of circulating lymphocytes, provoked a suppression of Con A-stimulated lymphocyte proliferation and total IFN gamma and IL-10 production in diluted whole blood cultures. Thus the non psychostimulant amphetamine derivative FEN, shares the ability of the psychostimulant methylenedioxy-substituted amphetamine derivatives to alter these indices of immune function in the rat. However, when Con A-stimulated cytokine production was normalised for the number of lymphocytes in culture in order to examine cytokine production at a cellular level, the effect of the amphetamine derivatives begins to diverge. FEN shares with MDMA and MDA the ability to suppress production of the Th2 type cytokine IL-10. However the effect of these drugs on Th1 type cytokine secretion was much more complex. While the methylendioxy-substituted amphetamines increases the secretion of the Th1 type cytokine IL-2 without altering the related Th1 type cytokine IFN-gamma, FEN did not alter IL-2 secretion, but suppressed IFN-gamma secretion. In addition to these effects on T-cell responses, all three drugs inhibited LPS-induced TNF alpha secretion from diluted whole blood cultures suggesting that macrophage activity is impaired following treatment. In all, these data extend our previous findings concerning the effects of MDMA on the immune system and demonstrate that the related serotonin releasers MDA and FEN also provoke immunological changes in rats. PMID- 10741903 TI - Angiotensin converting-like enzymes from urine of untreated renovascular hypertensive and normal patients: purification and characterization. AB - Angiotensin converting-like enzymes (ACE) were isolated from urine of normal (P0N, P1N and P2N) and untreated renovascular hypertensive (P0, P1 and P2) patients. The urine were submitted to ion exchange chromatography. Enzymes P0 and P0N were eluted with the equilibrium buffer (0.02 M Tris-HCl, pH 7.0), while P1, P1N, P2 and P2N with ionic strength linear gradient of 0.02-0.5 M Tris-HCl, pH 7.0 in 0.7 mS and P2 and P2N in 1.2 mS conductance. The active fractions were submitted to gel filtration in Sephadex G-150, equilibrated and performed with 0.05 M Tris-HCl/0.15 M NaCl buffer, pH 8.0. All enzymes were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (molecular mass: P0, P2 and P2N about 60 kDa; P1, 95 kDa and P21N 170 kDa). The enzymes were recognized by Y1 polyclonal antibody raised against human renal ACE. The K(M) values were in millimolar order for hippuryl-L-His-Leu (HHL) while for benzyloxycarbonyl-Phe-L-His-Leu (ZFHL) they were in 10(-4) M order. The enzymes were able to hydrolyze angiotensin I (AI) (P0 and P0N about 25%, P1 and P1N about 70%, P2 100% and P2N 66%) and bradykinin (BK) (P0N 22%, P1N 81%, P2N 62%, P0 and P1 50% and P2 35%), and their activities were inhibited by captopril. PMID- 10741904 TI - The effects of St. John's Wort (Hypericum perforatum) on NK cell activity in vitro. AB - St. John's Wort (Hypericum perforatum; H. perforatum) is a popular herbal supplement used to treat mild to moderate depression. H. perforatum possesses serotonergic properties such as inhibition of serotonin (5-hydroxytryptamine; 5 HT) reuptake. Serotonergic pharmacotherapy is associated with amelioration of depression as well as increases in natural killer (NK) cell activity (NKCA). Also, 5-HT and 5-HT analogs augment NKCA in vitro. Considering the serotonergic properties of H. perforatum, the effects of H. perforatum on NKCA were assessed in vitro. Mononuclear cells (MNCs) from normal donors were exposed in vitro to an extract of H. perforatum (LI160s) or established 5-HT stimulators of NKCA. After an overnight incubation, cells were washed and a standard 51Cr-release cytotoxicity assay performed to assess NKCA. LI160s at all concentrations failed to augment NKCA. However, in corroboration of previous studies, 5-HT, the selective serotonin reuptake inhibitors (SSRIs), paroxetine and norfluoxetine, and alpha-interferon augmented NKCA above control levels. Though an efficacious treatment for mild to moderate depression, H. perforatum differs from commonly prescribed serotonergic antidepressants insofar as H. perforatum fails to enhance NKCA in vitro. Therefore, the present results are consistent with pharmacologic studies indicating that H. perforatum possesses, at best, weak serotonergic activity. PMID- 10741905 TI - Pentoxifylline inhibits ICAM-1 expression and chemokine production induced by proinflammatory cytokines in human pulmonary epithelial cells. AB - Airway epithelium participates in inflammatory reactions by producing chemokines and expressing cell-surface adhesion molecules which aid in the selective recruitment of effector cells. Previous studies showed that proinflammatory cytokines, interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF alpha), induce surface expression of intercellular adhesion molecule 1 (ICAM-1) and the production of the chemokines interleukin 8 (IL-8) and monocyte chemoattractant protein (MCP-1) on pulmonary epithelial cell lines in vitro. In this study, the dose response of four cytokines, IL-1alpha, IL-1beta, TNF alpha and TNF beta, in inducing ICAM-1 expression and production of IL-8 and MCP-1 on pulmonary A549 epithelial cells was examined. Both IL-1alpha and IL-1beta induced ICAM-1 expression and IL-8 and MCP-1 production at lower doses than TNF alpha or TNF beta. Pentoxifylline, an anti-inflammatory agent used to treat vascular diseases, was tested for its ability to inhibit the activation of airway epithelial cells by these cytokines. Pentoxifylline completely inhibited the surface expression of ICAM-1 and the production of IL-8 and MCP-1 by cytokine-activated epithelial cells. As elevated levels of chemokines are often present in bronchial lavage fluids of patients suffering from various acute respiratory diseases, pentoxifylline may be useful for preventing the rapid development of immune reactions leading to lung injury. PMID- 10741906 TI - Long-term administration of galactoside-specific mistletoe lectin in an animal model: no protection against N-butyl-N-(4-hydroxybutyl)-nitrosamine-induced urinary bladder carcinogenesis in rats and no induction of a relevant local cellular immune response. AB - Aqueous extracts from leaves of the European mistletoe (Viscum album L.) are postulated to exert an anticancer efficacy by cytotoxic and/or immunological mechanisms of action. Although popular as an unconventional therapy modality, no controlled randomized clinical trials are available, reliably documenting a clinically beneficial antineoplastic potential of the various commercial mistletoe preparations. Since previous investigations have focused on the purified galactoside-specific lectin (Viscum album L. agglutinin, VAA) as major biological response modifier in the low-dose range, the objective of the present experimental study was to examine its effect on N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced carcinogenesis in the urinary bladder of rats, a suitable animal model for human disease. The carcinogen was fed by gavage in three fractionated low doses (150 mg/kg body weight each) to obtain low-grade and low-stage transitional cell carcinomas. From the onset of the experiment VAA was injected subcutaneously twice a week (1 ng/kg body weight) continuously for either 6 or 15 months. Following an experimental period of 6 months the incidence of bladder carcinomas was 10.2% in rats given exclusively BBN and 6.7% in those additionally treated with VAA. After an experimental time of 15 months 25.8% of the rats fed BBN only and 19.7% of the animals additionally receiving VAA had developed urothelial carcinomas. The differences of the tumor incidences did not reach the level of statistical significance, neither after an experimental duration of 6 (P = 0.88) nor of 15 months (P = 0.71). A difference was found in the size of the transitional cell carcinomas. They proved to be significantly larger (P = 0.02) in the rats additionally treated with VAA for 15 months (mean maximum diameter: 3.31 mm) than in those without lectin treatment (mean maximum diameter: 1.88 mm). Quantitative immunocytochemistry analyzing a panel of immune cells yielded no evidence for the ability of the lectin to provoke a substantial, biologically relevant local cellular immune response in the wall of tumor-free and tumor-bearing bladders. From the current experiment it is obvious that galactoside-specific mistletoe lectin failed to protect against, inhibit, delay or reduce development of chemically induced urothelial carcinomas of the urinary bladder even after long-term administration in the clinically recommended schedule. It seems highly unlikely that adjuvant treatment with mistletoe extracts or VAA might favorably influence bladder cancer in patients by immunological effector mechanisms. PMID- 10741907 TI - Growth suppression of human ovarian carcinoma OV-MZ-2a and OV-MZ-32 cells mediated by gene transfer of wild-type p53 enhanced by chemotherapy in vitro. AB - PURPOSE: The aim of this work was to observe the growth and chemosensitivity of human ovarian cancer OV-MZ-2a and OV-MZ-32 cells following adenovirus-based wild type p53 (Ad-p53) gene transfer alone or combined with chemotherapeutic agents. METHODS: Transduction efficiency was determined with a reporter construct of adenovirus galactosidase by staining with 5-bromo-4-chloro-3-indolyl beta-D galactoside. For growth inhibition, OV-MZ-2a or OV-MZ-32 cells were infected with Ad-p53 particles at a multiplicity of infection (m.o.i.) of 0.2-20, alone or combined with the chemotherapeutic agents taxol, cisplatin, doxorubicin or mitomycin C. Growth inhibition (assayed by trypan blue exclusion), target gene expression (by Western blotting) and clonogenicity (by soft-agar assay) were determined following Ad-p53 transfer. RESULTS: High transduction efficiency was observed following adenovirus galactosidase gene transfer; 94% of OV-MZ-2a cells and 69% of OV-MZ-32 cells expressed the transgene. Following transfer of Ad-p53 into the two cell lines, a high level of p53 expression was detected after 12, 24, 48, 72 and 96 h in OV-MZ-2a cells. At a m.o.i of 20, 96% and 90% growth inhibition were achieved in OV-MZ-2a cells and OV-MZ-32 cells respectively. Clonogenicity was lost completely in both cell lines following wild-type p53 transfer. Meanwhile, Ad-p53 gene transfer combined with taxol, cisplatin, doxorubicin or mitomycin C was shown to be even more effective in suppressing growth in the two cell lines. CONCLUSIONS: Our results may suggest that wild-type p53 gene transfer mediated by an adenoviral vector is a potential strategy for treating ovarian cancer, and a combination of Ad-p53 gene transfer and chemotherapeutic agents may be an even better treatment of the cancer. PMID- 10741908 TI - Expression of the angiogenic factor thymidine phosphorylase in human astrocytic tumors. AB - Thymidine phosphorylase (TP) has been implicated as a potent angiogenic factor and a prognostic factor in various human solid tumors. We investigated the expression of TP in a series of human astrocytic tumors using immunohistochemistry, enzyme-linked immunosorbent assay, and reverse transcriptase/polymerase chain reaction (RT-PCR) analysis. A total of 63 astrocytic tumors [27 glioblastomas (GBM), 19 anaplastic astrocytomas (AA), 17 low-grade astrocytomas (LGA)] and 5 normal brain tissues were immunohistochemically stained with antibodies to TP, vascular endothelial growth factor (VEGF), p53, MIB-1, and factor-VIII-related antigen. They were also evaluated for the degree of apoptosis by a ApopTag kit. Ten tumors (5 GBM, 2 AA, 3 LGA) and 3 normal brain tissues were evaluated for their expression of VEGF and TP by RT-PCR analysis. TP was constantly localized in the cytoplasm of astrocytic tumor cells, less intensely in the cytoplasm of vascular endothelial cells, but not in the normal brain. Some of the TP-positive cells were of macrophage origin, but most positive cells were the tumor cells themselves. Vascular density, MIB-1 positivity, p53 positivity, VEGF expression, and the apoptotic index were significantly higher in the TP-positive tumors than in TP-negative tumors. There was a significant correlation between TP and VEGF mRNA expression. In a limited number of glioblastoma cases, the apoptotic index was significantly higher in TP positive glioblastomas than in TP-negative glioblastomas. In human astrocytic tumors, TP was expressed in the tumor, macrophage, and endothelial cells. TP was a potent angiogenic factor closely associated with cell proliferation and tumor apoptosis. PMID- 10741909 TI - Expression of vascular endothelial growth factor in surgical specimens of hepatocellular carcinoma. AB - PURPOSE: Vascular endothelial growth factor (VEGF) has been reported to play an important role in angiogenesis in hepatocellular carcinoma (HCC). However, there is great variation in reports on the distribution of VEGF expression, especially in non-carcinoma liver cells. Furthermore, some reports have mentioned that endothelial cells were positive for VEGF antibody but have not evaluated its significance. In this study, we focused our attention to these problems and try to solve them. We also analyzed the factors influencing VEGF expression and evaluated the prognostic potential of VEGF protein in HCC. METHODS: We examined the VEGF expression in specimens surgically removed from 46 HCC patients and 3 patients with liver cancer metastatic from the colon, and in 4 specimens of liver tissue with benign disease, by immunohistochemical methods. RESULTS/CONCLUSIONS: VEGF was expressed in HCC cells and hepatocytes and on vascular endothelial cells. Our finding that about seven times more endothelial cells were positive for VEGF antibody in carcinoma areas than in non-carcinoma areas (P < 0.001) suggests that VEGF is a very important angiogenesis factor for HCC growth. VEGF expression in HCC cells and non-carcinoma liver cells and on endothelial cells did not closely correlate with the disease recurrence rate (P > 0.05), suggesting that VEGF expression may not be useful as an individual factor for estimating the prognosis of HCC. A statistical analysis of the relationships between VEGF expression and clinicopathological variables revealed the following: preoperative transcatheter arterial embolization enhanced VEGF expression in both HCC cells and non-carcinoma liver cells. The histological grade of HCC and the level of alanine aminotransferase was related to VEGF expression in non-carcinoma liver cells and on endothelial cells in HCC areas. Tumor size and the histological status of the accompanying chronic hepatitis also influenced the VEGF expression on endothelial cells. Our findings concerning not only HCC but also the surrounding liver and endothelial cells may provide useful information for further research on the role of VEGF expression in HCC patients. PMID- 10741910 TI - Radioimmunodetection of residual, recurrent or metastatic germ cell tumors using technetium-99 anti-(alpha-fetoprotein) Fab' fragment. AB - PURPOSE: The majority of patients with germ cell tumors are cured by multimodality therapy that consists of cisplatin-based chemotherapy and/or surgical resection. Serum tumor markers and conventional radiographs are utilized to stratify patients into treatment categories. Efforts to individualize chemotherapy or minimize surgical interventions without compromising outcome are important. Immunomedics (Morris Plains, New Jersey) developed an anti-(alpha fetoprotein) (anti-AFP) monoclonal antibody IMMU-30 labeled with 15-20 mCi technetium-99, and the purpose of this study is to determine the sensitivity and specificity of radioimmunoscintigraphy using 99mTc anti-AFP antibody for the diagnosis of active germ cell tumors. METHODS: A group of patients with germ cell tumors were enrolled in a non-prospective fashion and 48 AFP scans using 99Tc anti-AFP Fab' fragment were obtained. At the time of the AFP scan, serum AFP was elevated in 40 measurements with a median level of 21 ng/ml (1.6-66, 210.0 ng/ml). AFP scans were obtained at the initial staging, during treatment, at relapse or at long-term follow-up and compared with conventional radiographs done within 4 weeks of the AFP scans. RESULTS: An overall diagnostic sensitivity of 89% and specificity of 58% were obtained. CONCLUSIONS: AFP scanning appears useful and to be sufficiently sensitive to justify prospective studies comparing the procedure with conventional imaging. PMID- 10741911 TI - Modulation of cisplatin sensitivity by taxol in cisplatin-sensitive and resistant human ovarian carcinoma cell lines. AB - PURPOSE: The aim of this study was to determine whether taxol can circumvent cisplatin resistance, using a KF28 cell line derived from human ovarian carcinoma and a cisplatin-resistant line, KFr13, derived from the parental cell line, KF28, and taxol-resistant cell lines, KF28TX and KFr13TX, derived from the respective parental counterpart. METHODS: KF28 is a single-cell clone of the human ovarian carcinoma cell line KF. The cisplatin-resistant KFr13 subline was established by repeated exposure of the parent KF28 cell line to escalating doses of cisplatin. Similarly, KF28TX and KFr13TX were established by repeated exposure of the KF28 and KFr13 cell lines to escalating doses of taxol. A cytotoxicity assay was performed using a crystal violet staining method. Platinum and taxol accumulation were assayed by atomic absorption and reverse-phase high-performance liquid chromatography. The quantitative assay of MDR1 mRNA used polymerase chain reaction. RESULTS: KFr13 cells were about 4.8-fold more resistant to cisplatin and about 1.8-fold more sensitive to taxol than were KF28 cells. When taxol resistance was induced in KF28 and KFr13 cells, sensitivity to cisplatin rose about 1.3- and 1.6-fold respectively. Elevation of sensitivity was correlated with platinum uptake by both KF28TX and KFr13TX cells. Expression of multidrug resistance (MDR1) mRNA, which was not observed in KF28 and KFr13 cells, was observed after induction of taxol resistance. CONCLUSIONS: These results may suggest rational therapeutic strategies for patients with cisplatin-resistant or refractory ovarian carcinoma. PMID- 10741912 TI - Nephrotoxicity of ifosfamide, carboplatin and etoposide (ICE) alone or combined with extracorporeal or radiant-heat-induced whole-body hyperthermia. AB - Although whole-body hyperthermia combined with specific genotoxic chemotherapy can be shown to enhance neoplastic cell killing without a concomitant rise in bone marrow toxicity, nephrotoxicity can become treatment-limiting. This study compares the kidney toxicity to the kidney of ifosfamide, carboplatin and etoposide (ICE) chemotherapy alone, and ICE chemotherapy combined with either extracorporeal (e-WBH) or radiant-heat-induced hyperthermia (r-WBH) in 43 patients with refractory sarcoma. Within 3 days of ICE chemotherapy treatment there was a significant increase in urinary protein excretion and a reduction of the glomerular filtration rate. These effects were more pronounced if WBH was added. The use of immunoluminometric assays revealed a predominance of low molecular-mass proteins. This increase in protein excretion persisted in the e WBH-treated group, whereas it vanished within 3 weeks in both the group treated with ICE alone and that treated with r-WBH. Our findings suggest that ICE chemotherapy causes transient tubular and glomerular damage, which is enhanced by WBH. In terms of long-term nephrotoxicity e-WBH was more nephrotoxic than r-WBH. This finding is consistent with our clinical observations. PMID- 10741913 TI - Renal cell cancer correlated with occupational exposure to trichloroethene. PMID- 10741914 TI - Multiple myeloma--current concepts for an interdisciplinary management. A one-day course in the continuing education program organized by the Tumor Center Heidelberg/Mannheim. 24 April 1999, Heidelberg, Germany. PMID- 10741915 TI - Spontaneous proliferative megakaryocytopoiesis and platelet hyperreactivity in essential thrombocythemia: is thrombopoietin the link? AB - Essential thrombocythemia (ET) is one of the less rare variants of the chronic myeloproliferative disorders (MPD). The present review questions the possible link between spontaneous megakaryocytopoiesis, platelet hyperreactivity, and the occurrence of platelet-mediated vascular manifestations in acquired and hereditary ET. In acquired ET, the role of thrombopoietin (TPO) is crucial to the observed hypermegakaryocytopoiesis, which is characterized by an increased proliferation of megakaryocyte (MK) progenitors, even in conditions of culture without addition of any known megakaryocyte colony-stimulating factors. An increased reactivity of megakaryocyte progenitors to TPO remains to be precisely delineated. A defective clearance of TPO by megakaryocytes and platelets because of a reduced number of TPO receptors is possible. TPO is able to enhance platelet aggregation induced by ADP, thrombin, and collagen. A point mutation in the TPO gene as the cause of increased TPO production in hereditary ET can readily explain both spontaneous megakaryocytopoiesis and platelet-mediated microvascular manifestations simulating the phenotype of acquired ET. Nevertheless, to date, no mutation of the TPO structural gene, as shown in two families with hereditary ET, and no mutations in the TPO receptor have been found in patients with acquired ET. There is good evidence that the microvascular circulation disturbances in ET are caused by intravascular activation and aggregation of hypersensitive platelets, with sludging or occlusion of the endarterial microvasculature. In this process, the generation of platelet-derived products, endothelial cell damage, fibromuscular intimal proliferation, and platelet thrombi are essential and can be inhibited by a platelet-specific regimen of aspirin, thus providing a rationale for using low-dose aspirin as an antithrombotic agent in thrombocythemia. In contrast, the generation of thrombin appears not to be essential for the formation of platelet thrombi, thereby explaining the inefficacy of coumadin derivatives and heparin in the treatment and prevention of microvascular circulation disturbances in hereditary and acquired ET. PMID- 10741916 TI - Blood-derived macrophage layers in the presence of hydrocortisone support myeloid progenitors in long-term cultures of CD34+ cord blood and bone marrow cells. AB - Monocytes/macrophages secrete various cytokines that induce proliferation of colony-forming unit granulocyte-macrophage (CFU-GM) in short-term assays. To determine whether macrophages also support proliferation of more primitive progenitors, i.e., cells that give rise to colony forming cells in a 5-week long term culture (LTC), we established plastic-adherent macrophage layers from human peripheral blood (PB) and filgrastim (G-CSF)-mobilized progenitor cell collections in the presence of hydrocortisone, and compared these layers with bone marrow (BM) stroma regarding their suitability to support proliferation and differentiation of CD34+ BM and cord blood (CB) cells in 5-week LTCs. CD34+ cells were seeded onto irradiated macrophage and BM stromal layers, as well as without any preformed layer. After 5 weeks, colony formation (CFU-GM, BFU-E/CFU-E) and cell expansion were determined. CD34+ cells from BM and CB yielded more CFU-GM and total nucleated cells at 5 weeks in the presence of both types of adherent layer compared with cultures without a layer (p<0.05). For CD34+ BM cells, macrophage layers were superior to BM stroma in enhancing CFU-GM and CFU-E/BFU-E output (p < 0.05). In contrast, BM stroma was favorable compared with macrophages concerning nucleated cell expansion from CD34+ CB cells (p = 0.027). The macrophage nature of PB-derived adherent cells was confirmed immunocytochemically by positive staining for CD68, Ki-Mlp, CD31, CD54, inconstant staining for CD14, and negative staining for CD1a, CD3, CD15, CD34, and CD62E. Cytochemical reactions were positive for alpha-naphthyl acetate esterase and negative for peroxidase and periodic acid-Schiff, consistent with the immunophenotype. In conclusion, the results show that blood-derived macrophages support CFU-GM generation from CD34+ CB and BM progenitors for 5 weeks in vitro. Differential effects on proliferation and maturation of BM versus CB progenitors are discussed. PMID- 10741917 TI - Thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS): treatment outcome, relapses, prognostic factors. A single-center experience of 48 cases. AB - The thrombotic thrombocytopenic purpura/ hemolytic uremic syndrome (TTP/HUS) is a rare disorder characterized by microangiopathic hemolysis and thrombocytopenia. We have undertaken a retrospective analysis of the clinical characteristics, treatment outcome, and prognosis of 48 patients diagnosed and treated in our institution during a 13-year period. Among our patients 22 (46%) had fever, 35 (73%) neurological abnormalities, and 22 (46%) renal impairment at presentation of the syndrome. All patients were treated with a multimodality regimen including plasma exchange, steroids, antiplatelet agents, and IgG infusion. Of the 48 patients, 41 achieved complete remission, two had a partial response, and five had no response and died of progressive disease. Within a median follow-up period of 40 months, ten of the 41 patients who had achieved remission relapsed, most of them within the first 2 years, and nine of these responded promptly to plasma exchange therapy. Eight deaths were observed, seven of refractory disease and one in fourth relapse. The analysis of prognostic factors revealed advanced age and severe renal impairment (creatinine levels above 2 mg%) as the only parameters associated with treatment failure and poor outcome. However, none of the pretreatment characteristics proved to be of prognostic value regarding the probability of relapse. In conclusion, TTP/HUS represent a syndrome of variable clinical expression and aggressiveness. The use of a multimodality regimen in our series produced a high response rate. Nevertheless, the early identification, based on clinical characteristics, of poor-prognosis cases that probably need more or alternative forms of treatment is an issue that remains to be elucidated in prospective trials. PMID- 10741918 TI - How to restrict liver biopsy to high-risk patients in early-stage Hodgkin's disease. German Hodgkin's Lymphoma Study Group. AB - Liver biopsy is an invasive diagnostic method for detecting liver involvement (LI) in Hodgkin's disease (HD). The aim of this retrospective study was to determine and evaluate a method for restricting liver biopsy to a subset of patients. Between 1988 and 1994, a total of 2,016 patients with HD were treated within the HD4-6 study protocol of the German Hodgkin's Lymphoma Study Group (GHSG). We investigated the predictive power of abdominal ultrasound (US) and computed tomography (CT), as well as of various clinical factors related to LI, using univariate and multivariate methods. LI occurred in 4.9% of all patients (99/2,016) and in 3.0% of those who, if LI were disregarded, would have been included in clinical stages I and II. In multivariate analysis the presence of LI was significantly associated with splenic involvement or infradiaphragmatic involvement, absence of mediastinal involvement, serum alkaline phosphatase (SAP) level over 230 units/l, and age over 40 years. We used these factors to define a risk score for LI. LI is very rare in patients who would otherwise be in clinical stages I or II, but knowledge of LI is important because it has therapeutic consequences. With our risk score, liver biopsy is indicated for approximately one quarter of these patients otherwise in clinical stages I or II. PMID- 10741919 TI - Conventional salvage chemotherapy vs. high-dose therapy with autografting for recurrent or refractory Hodgkin's disease patients. AB - Despite progress that has been made in curing Hodgkin's disease (HD), patients whose first remission is brief and those resistant to first-line chemotherapy still have a poor outcome. We retrospectively reviewed data from 29 patients with HD in first relapse or refractory to first-line chemotherapy. Following failure, all patients received three cycles of ifosfomide, epirubicin, and etoposide (IEV); moreover, 11 patients received a conditioning regimen followed by autografting. Of the 18 patients treated with IEV, eight (44%) are alive; nine died of disease progression, and one died of hematologic toxicity. The 24-month overall survival (OS), relapse-free survival (RFS), and event-free survival (EFS) are 18%, 44%, and 22%, respectively. Of the 11 patients treated with IEV and autografting, ten are alive (90%) and one patient died of progressive disease. The 29-month OS, RFS, and EFS are 91%, 71%, and 56%, respectively. Our results confirm data showing that patients with relapsed or resistant HD achieve a significantly better OS and EFS if treated with high-dose therapy and autografting. PMID- 10741920 TI - Ceramide stimulates the uptake of neutral red in human neutrophils, monocytes, and lymphocytes. AB - Neutral red is a vital stain known to be accumulated in the lysosomes of neutrophils and monocytes. It is used mainly to identify and detect the activated state of these cells. We have found that the extracellular application of physiological ceramide, i.e., a product of sphingomyelin hydrolysis and a newly defined intracellular second-messenger substance, increased the uptake of neutral red in a dose-dependent manner in human neutrophils, monocytes, and lymphocytes, as demonstrated by flow cytometry. Staurosporine was able to totally block this phenomenon, suggesting the involvement of protein kinase C in the process. These results indicate that the flow-cytometric analysis of ceramide-induced uptake of neutral red can be a new method for the evaluation of lysosome-related activation processes in both phagocytes and lymphocytes. PMID- 10741921 TI - Bilateral breast MALT lymphoma: a case report and review of the literature. AB - Breast lymphoma is a rare disease. Both primary and secondary breast involvement have been reported. Most primary breast lymphomas are high-grade malignant neoplasms, mainly large cell and Burkitt type. Low-grade lymphomas of the breast, particularly mucosa-associated lymphoid tissue (MALT) lymphomas, have been exceedingly rare. In this report we present a patient with bilateral breast involvement by MALT lymphoma. Our patient developed localized MALT lymphoma in both breasts in a sequential fashion. She was treated with bilateral lumpectomy, followed by radiation therapy to both breasts. The patient is alive and well more than 1 year after therapy with no recurrence. We believe this is the first such case described in detail in the literature. PMID- 10741922 TI - Cerebral dural sinus thrombosis in acute lymphoblastic leukemia with early diagnosis by fast fluid-attenuated inversion recovery (FLAIR) MR image: a case report and review of the literature. AB - Cerebral dural sinus thrombosis (CDST) is a very rare complication of acute lymphoblastic leukemia (ALL) in adult patients. A 23-year-old man with ALL developed dizziness, headache, diplopia, limb weakness, and a sensation of fullness in his head after his second induction chemotherapy with doxorubicin, prednisolone, and vincristine. Examinations of the peripheral blood, bone marrow, and cerebrospinal fluid showed no recurrent leukemic cells. Magnetic resonance (MR) imaging of the brain disclosed unexpected CDST at the left transverse sinus, which was seen only on the fast fluid-attenuated inversion recovery (FLAIR) sequence. His symptoms were relieved soon after treatment with heparin. MR imaging with FLAIR performed a second time 7 days later showed complete disappearance of the thrombosis. The patient was treated continuously with oral anticoagulant therapy and the symptoms did not recur. CDST can be diagnosed in its early phase by MR studies with FLAIR images. Anticoagulant therapy can be administered safely without precipitating the occurrence of infarction hemorrhage at such an early stage of CDST. PMID- 10741923 TI - Complete haematological and cytogenetic response to interferon alpha-2a of a myeloproliferative disorder with eosinophilia associated with a unique t(4;7) aberration. AB - A female patient with eosinophilia and cardiac symptoms was found to have a unique chromosomal aberration [t(4;7)(q11;p13)] of bone-marrow precursors. The disorder was classified as a chronic myeloproliferative syndrome with eosinophilia. Due to a significant increase in the white blood cell and eosinophil count during initial treatment with prednisone and hydroxyurea, Interferon alpha-2a was administered at a dose of 3-5 x 10(6) I.U. s.c., five times per week, and induced a long-term complete haematological and cytogenetic response. The clinical features of this case are presented and discussed in the context of the current literature. PMID- 10741924 TI - Transient eosinophilia by HIV infection. AB - We describe a case of early human immunodeficiency virus infection characterized by transient eosinophilia without an elevated immunoglobulin E concentration, allergic symptoms, or atopic dermatitis. Possible mechanisms of the eosinophilia are discussed. PMID- 10741925 TI - Chemoembolization of hepatic metastases from intestinal neuroendocrine tumours. AB - Gastrointestinal neuroendocrine tumours, i.e. carcinoids and islet cell tumours, often metastasize diffusely into the liver, rendering complete surgical resection impossible. Unlike other malignancies, prolonged survival may occur even in advanced disease. Chemotherapy of these rare tumours is not only aimed at size reduction (objective response) but also at symptom reduction (biological response). Systemic therapy, using 5-fluorouracil, doxorubicin, cisplatin, cyclophosphamide, somatostatin-analogue, interferon or streptozotocin yielded response rates of up to 69%. Since neuroendocrine tumours are generally hypervascular locoregional chemotherapy and chemoembolization also have been used successfully. However, even though severe complications are rare, some degree of pain and the post-embolization syndrome are almost inevitably present. The expected therapeutic efficacy must be weighed against possible side effects. The best result that can be hoped for in the chemoembolization of intestinal neuroendocrine metastatic disease is, as yet, only palliation. PMID- 10741926 TI - Safety of proton-pump inhibitors: the acid test. AB - It is well established that atrophic gastritis, pernicious anaemia and partial gastrectomy are associated with an increased risk of gastric carcinoma. These conditions all result in a low acid output state. Although the mechanism of gastric carcinogenesis is not known, the model proposed by Correa has provided the most attractive explanation to date. The suggestion is that prolonged hypochlorhydria predisposes to gastric carcinoma by an increase in the production of carcinogenic N-nitroso compounds. This hypothesis has led to concerns about the safety of long-term acid suppression in the management of a variety of acid related gastrointestinal conditions. PMID- 10741927 TI - Undernutrition and function: another piece in the jigsaw [commet]. AB - The stomach, small intestine and pancreas share in the progressive bodily dysfunction associated with undernutrition and weight loss. All these changes are reversed by nutritional support whether given enterally or parenterally, emphasizing that parenteral and enteral nutrition are the treatment of gastrointestinal failure as dialysis is of renal failure or ventilation is of respiratory failure. The observations described by Winter and colleagues have important implications for our understanding of the relationship between nutrition and physiology. They also have important implications for the care of patients with gastrointestinal disease. PMID- 10741928 TI - Hepatic arterial chemoembolization with streptozotocin in patients with metastatic digestive endocrine tumours. AB - BACKGROUND: Hepatic arterial chemoembolization (CE) with anthracyclines is an effective treatment for progressive liver metastases of digestive endocrine tumours. Streptozotocin (STZ) is widely used for systemic chemotherapy, but its efficacy by the hepatic arterial route has not been evaluated. PATIENTS AND METHODS: Fifteen consecutive patients, mean age 57.8 years, were prospectively included between July 1993 and January 1997. All patients had progressive liver metastases from either a carcinoid tumour (eight patients) or an islet cell carcinoma (ICC) (seven patients) that had increased in size (> or = 25%) before CE. Five patients had the carcinoid syndrome. STZ was administered, as an emulsion with iodized oil, into the hepatic artery before embolization with gelatin sponge particles. Two to six procedures (median, 3) were performed in 12 patients (one in three patients). Changes in the size of the liver metastases were evaluated by CT scan or MRI according to WHO criteria. The median follow-up was 15 months (1-50). RESULTS: An objective response was achieved in 8/15 patients (53%; median duration of 10.5 months) whatever the primary tumour (carcinoid or ICC). The carcinoid syndrome disappeared in 3/5 patients for 10, 11 and 17 months, respectively. CE effectively controlled hypoglycaemic attacks (decrease of > 50%) in the patient with insulinoma. The biological response was complete in four patients for a median duration of 7 months. CE induced minor side effects, namely nausea, fever and abdominal pain. Acute and reversible tubular necrosis due to CE was observed in one patient who had previously undergone a nephrectomy. CONCLUSION: Hepatic arterial chemoembolization with STZ is an effective treatment for patients with liver metastases caused by digestive endocrine tumours. PMID- 10741929 TI - Plasma nitrites/nitrates in HCV infection and hepatocellular carcinoma. AB - OBJECTIVE: Nitric oxide (NO) is produced in response to inflammatory and mitogenic stimuli and may have a role in carcinogenesis. However, the role of NO in hepatitis C-associated hepatocellular carcinoma (HCC) is unclear. In this study, we investigated the potential role of NO in HCC complicating hepatitis C virus (HCV) infection. METHOD: We measured plasma nitrites/nitrates as being representative for NO release in blood of patients with chronic hepatitis C without cirrhosis (n = 20), cirrhosis of different aetiologies (n = 30) including HCV, HCC (n = 22) and in healthy controls (n = 8), by an enzyme-linked immunosorbent assay. RESULTS: Plasma NO levels in patients with chronic hepatitis C without cirrhosis (32.3+/-8.94 micromol/l) were not significantly different from those in healthy control subjects (35.5+/-15.12 micromol/l). Also, there were no statistical differences between plasma NO levels in patients on alpha interferon (alpha-IFN) therapy (n = 10) (31.60+/-10.55 micromol/l) and in non treated patients (n = 10) (33.00+/-7.51 micromol/l) within the group of chronic hepatitis C. Plasma NO levels in patients with cirrhosis (42.36+/-26.86 micromol/l) were significantly higher than those with chronic hepatitis C (P < 0.001). The cause of cirrhosis had no effect on plasma NO levels. Plasma NO levels in patients with HCC (49.40+/-49.11 micromol/l) were significantly higher than those with liver cirrhosis (P < 0.03). No significant correlation was found between plasma NO and serum ALT (alanine aminotransferase) levels. There were positive correlations between plasma NO levels and alkaline phosphatase (r = 0.528) (P = 0.0001), bilirubin (r = 0.244) (P = 0.039) and GGT (gamma glutamyltransferase) (r = 0.255) (P = 0.030). CONCLUSION: The results of this study demonstrate that patients with chronic hepatitis C without cirrhosis have the same plasma NO levels as controls, and that alpha-IFN therapy had no effect on NO production in these patients. However, patients with HCC have elevated plasma NO levels compared with patients with cirrhosis. These data support the concept that NO is elevated in cirrhosis and HCC, but HCV infection does not appear to be responsible for the increase of NO in these patients. The severity of liver disease may be an important factor. PMID- 10741930 TI - The effect of intra-gastric acidity and flora on the concentration of N-nitroso compounds in the stomach. AB - BACKGROUND: Correa's hypothesis proposes that gastric carcinogenesis is due to atrophic gastritis and hypochlorhydria which permit gastric bacterial colonization, the reduction of dietary nitrates to nitrites and the formation of potentially carcinogenic N-nitroso compounds (NOCs). OBJECTIVE: To test the hypothesis that omeprazole-induced hypochlorhydria is associated with increased intra-gastric concentrations of nitrate-reducing bacteria (NRB), nitrites and NOCs. DESIGN: Single-blind study in healthy volunteers. PARTICIPANTS: Fourteen healthy subjects (seven female, mean age 24 years), free of Helicobacter pylori infection, received a one-week course of placebo followed by a two-week course of omeprazole, 20 mg daily. METHODS: Fasted gastric samples, aspirated using a sterile double-lumen nasogastric tube at the end of the 1 st week (placebo) and the 2nd and 3rd weeks (omeprazole), were cultured aerobically and anaerobically; gastric pH and intra-gastric concentrations of nitrates, nitrites and NOCs were also determined. RESULTS: After weeks 1, 2 and 3, the intra-gastric concentrations of nitrate-reducing bacteria exceeded 10(5) colony-forming units (c.f.u.)/ml in 3, 7 and 9 subjects, respectively (P > 0.05). A gastric pH greater than 4.0 was associated with increased NRB (P < 0.05); however, neither increased gastric pH nor increased NRB, alone or in combination, was associated with increased intra-gastric concentrations of nitrites or NOCs (P > 0.05). CONCLUSIONS: A two-week increase in gastric pH in healthy, H. pylori-negative subjects was associated with increased intra-gastric concentrations of nitrate reducing bacteria but not of nitrites or N-nitroso compounds. These data suggest that reduced gastric acid secretion is not a necessary precursor to the formation of carcinogenic N-nitroso compounds and that other mechanisms should be invoked to explain gastric carcinogenesis. PMID- 10741931 TI - Epidemiology and course of acute upper gastro-intestinal haemorrhage in four French geographical areas. AB - OBJECTIVE: To compare incidence rates and epidemiological characteristics of acute upper gastrointestinal haemorrhage (AUGIH) in France with those of other European studies. DESIGN: Population-based multi-centre prospective survey. SETTING: 29 public hospitals and 96 private specialists in gastroenterology in four administrative areas in France during 1996. SUBJECTS: A total of 2133 AUGIH patients 18 years and over were included in the six-month study. OUTCOME MEASURES: Incidence and mortality. RESULTS: The overall incidence in France was 143 cases per 100000 persons per year, classified as out-patients (16%), emergency admissions (59%) and in-patients (25%). The incidence rates increased with age except for in-patients, and were higher in males. Peptic ulcer (36.6%), varices (13.7%) and erosive disease (12.3%) were the most frequent diagnoses. In 677 patients (31.7%), aspirin, antiinflammatory drugs or corticosteroids were taken on the 7 days before bleeding. The overall mortality (out-patients excluded) was 14.3% (10.7% for emergency patients and 23% for in-patients). Mortality was associated with comorbidities (especially malignancies, cirrhosis, asthma or respiratory deficiency), was lower in emergency patients using non steroid anti-inflammatory drugs, and higher in in-patients using corticosteroids. CONCLUSIONS: In France, patients with AUGIH are frequently managed as out patients. Gastrotoxic drug use is frequently associated with AUGIH and constitutes a strategic opportunity for preventive treatment. Discrepancies between countries are not clearly explained either by demographic factors or by drug use, but this may be related to the emphasis on AUGIH in in-patients. PMID- 10741932 TI - An audit of percutaneous endoscopic gastrostomy in a general hospital in Singapore. AB - OBJECTIVE: We conducted an audit on 50 percutaneous endoscopic gastrostomies (PEGs) performed by physician endoscopists from January 1996 up to November 1997. DESIGN: A retrospective cohort study was conducted. RESULTS: The mean age of the patients was 68.5 years (range 20-101) and the main indications were cerebrovascular accident in 40 (80%), neurological dysphagia in six (12%) and head injury in three (6%). The interval between the diagnosis of dysphagia and PEG was > 60 days in 19 patients (38%), 31-60 days in eight (16%) and < or = 30 days in 23 (46%). The commonest reason for PEG insertion was intolerance to nasogastric tube in 49 patients (98%). Twelve patients had antibiotics given concurrently for other infections and two had antibiotics given specifically to cover PEG insertion. There was evidence of post-PEG infection in two of 14 patients given antibiotics (14%) and in 14 of 36 patients not given antibiotics (39%). By Cox regression, the adjusted relative risk of infection in patients receiving antibiotics versus those not receiving was 0.6927 (95% CI 0.3396 1.4130; not significant). The 30-day mortality was 7/48 patients (15%), with two patients lost to follow-up before 30 days. There were no deaths directly attributable to PEG. CONCLUSIONS: PEG is still not adequately requested by doctors for patients needing enteral feeding for more than 30 days. The use of antibiotics in this retrospective cohort study failed to show any benefit in reducing the rate of infective complications. PMID- 10741933 TI - Efficacy of low and standard midazolam doses for gastroscopy. A randomized, double-blind study. AB - AIM: To evaluate the efficacy and safety of two different doses of intravenous midazolam (35 and 70 microg/kg) compared to placebo in patients undergoing gastroscopy. PATIENTS AND METHODS: Sixty patients scheduled for diagnostic gastroscopy were selected according to factors previously reported to affect tolerance (Eur J Gastroenterol Hepatol 1999; 11:201-204) and were randomly assigned to receive premedication with midazolam 35 microg/kg iv, midazolam 70 microg/kg iv or placebo iv. Oxygen saturation was continuously monitored during the procedure. Patients' tolerance, time to discharge and post-sedative inconvenience were evaluated using visual analogue scales and a questionnaire. RESULTS: Patients receiving either dose of midazolam showed better tolerance of gastroscopy than those receiving the placebo. Fewer patients receiving 70 or 35 microg/kg of midazolam were reluctant to undergo a further gastroscopy compared to those receiving the placebo (2, 1 and 9 patients respectively, P = 0.01). Compared to patients receiving midazolam 70 microg/kg, those receiving midazolam 35 microg/kg were discharged earlier (29.3+/-14.4 versus 43.1+/-12.4 min respectively, P < 0.001), experienced less post-sedative inconvenience (8 versus 15 patients slept for > 1 h at home respectively, P = 0.02), and suffered fewer clinically relevant desaturation episodes (< 90%) (0 versus 5 patients respectively, P = 0.04). CONCLUSIONS: Low doses of intravenous midazolam (35 microg/kg) are adequate and safe when sedation is indicated for gastroscopy. PMID- 10741934 TI - The effect of severe undernutrition, and subsequent refeeding on digestive function in human patients. AB - BACKGROUND: Severe undernutrition may adversely affect gut function. AIMS: To investigate the effects of severe undernutrition and subsequent refeeding on human digestive function. METHODS: Severely undernourished patients (body mass index (BMI) < 17 kg/m2) were studied before, and after a period of intensive nutritional support. Standard intestinal absorption tests (faecal fat and urinary xylose excretion), pentagastrin-stimulated acid secretion, and cholecystokinin octapeptide (CCK-8)-stimulated pancreatic enzyme secretion tests were performed. In addition, duodenal biopies were taken to assess gut mucosal morphology. Findings were evaluated in comparison to a group of normal healthy volunteers. RESULTS: Mean BMI of the patients prior to nutritional support was 13.41 kg/m2, with improvement to 16.12 kg/m2 after. Duodenal histology showed evidence of villous atrophy in six of 14 (43%) undernourished patients. Mean xylose excretion following a 5 g oral dose was 0.62 g/5 h in the group of undernourished patients prior to nutritional support (normal > 1 g/5 h), with improvement to 1.40 g/5 h (P < 0.01) after feeding. Maximal gastric acid output was significantly impaired in the undernourished group, as compared to the controls (6.94 mEq/l vs 25.53 mEq/l, P < 0.02), with a significant improvement to 12.30 mEq/l (P < 0.05) following nutritional support. Pancreatic enzyme output was significantly reduced (amylase 830.9 U/h vs 2304 U/h, P < 0.01; lipase 38.0 U/h vs 118.6 U/h, P < 0.01; trypsin 119.7 U/h vs 341.4 U/h, P < 0.01). Following a period of nutritional support there was a significant improvement in amylase and lipase outputs to 1819 U/h and 85.5 U/h, respectively (P < 0.01). These levels were not significantly different from the normal controls. Trypsin output, however, remained significantly impaired at 174.3 U/h (P < 0.01). CONCLUSIONS: Severe undernutrition is associated with significant impairment of digestive function, with improvement occurring following nutritional support. These changes may affect initial tolerance to enteral feeding, particularly in those patients with co-existent gut disease. PMID- 10741935 TI - Efficacy and tolerability of 20 mg pantoprazole versus 300 mg ranitidine in patients with mild reflux-oesophagitis: a randomized, double-blind, parallel, and multicentre study. AB - BACKGROUND AND AIM: The aim of this study was to compare the efficacy and tolerability of low dose pantoprazole (20 mg) (a gastric proton pump inhibitor) with standard dose ranitidine (300 mg) (a histamine-receptor antagonist), in their ability to relieve symptoms and heal oesophageal lesions associated with gastrooesophageal reflux disease (GORD). METHODS: Patients with endoscopically established mild GORD (stage I, modified Savary-Miller classification) were enrolled into a multicentre, randomized, double-blind, parallel-group comparison study (intention-to-treat population, n = 201; age range, 18-82 years). Patients took either oral pantoprazole 20 mg in the morning (n = 101) or ranitidine 300 mg in the evening (n = 100) once daily for 4 weeks or, if the healing was not complete, 8 weeks. Relief from key symptoms (heartburn, acid regurgitation, pain on swallowing) was assessed after 2, 4, and if applicable, 8 weeks. Healing of lesions was confirmed endoscopically after 4 and, if applicable, 8 weeks. RESULTS: Complete relief from key symptoms was noted after 2 weeks in 70/88 (80%) patients treated with pantoprazole vs 45/89 (51%) patients treated with ranitidine ('per-protocol and key-point available' populations, P < 0.001); the corresponding results after 4 weeks were 77/88 (88%) vs 51/88 (58%) (P < 0.001). Complete healing of lesions after 4 weeks of treatment was seen in 74/88 (84%) vs 49/89 (55%) in the pantoprazole and ranitidine group, respectively (P < 0.001, per-protocol); by week 8 the cumulative healing rates were 84/88 (95%) vs 69/89 (78%) in the pantoprazole and ranitidine group, respectively (P < 0.001). For the intention-to-treat populations, the corresponding values for healing after 4 and 8 weeks were 73% vs 49% (P < 0.001) and 83% vs 69% (P < 0.05), respectively. Both study medications were well tolerated. CONCLUSION: Compared to ranitidine 300 mg, the regimen with pantoprazole 20 mg provides faster relief from symptoms and is significantly more effective in healing of oesophageal lesions in patients with mild reflux-oesophagitis. Thus, the low dose of pantoprazole offers a treatment approach which minimizes drug exposure and costs while retaining high efficacy. PMID- 10741936 TI - Evaluation of the efficacy and safety of rifaximin in the treatment of hepatic encephalopathy: a double-blind, randomized, dose-finding multi-centre study. AB - OBJECTIVE: To determine the efficacy, tolerability and safety of oral rifaximin given at three dose levels in patients with cirrhosis and mild to moderate hepatic encephalopathy (HE). DESIGN: Prospective, double-blind, randomized, parallel-group study. SETTING: Multi-centre trial in four university teaching hospitals. PARTICIPANTS: Fifty-four patients with cirrhosis and mild to moderate HE. INTERVENTION: Seven days treatment with rifaximin, 600, 1200 or 2400 mg/day in three divided doses. MAIN OUTCOME MEASURE: Change in the portal-systemic encephalopathy (PSE) index between baseline and day 7, calculated on the basis of mental state, asterixis, number connection test time, EEG mean cycle frequency and blood ammonia concentrations. RESULTS: Treatment with rifaximin was associated with an improvement in the PSE index. There was a trend towards a greater treatment effect of rifaximin with the highest dose of 2400 mg/day. Rifaximin was well tolerated; the few treatment-related adverse events showed no consistent pattern or dose relationship. CONCLUSION: Rifaximin may be useful as alternative or adjuvant therapy for grade I-III hepatic encephalopathy in patients with cirrhosis at a dose of 1200 mg/day. PMID- 10741937 TI - Long-term gallbladder stone recurrence and risk factors after successful lithotripsy. AB - OBJECTIVE: The risk of recurrence has limited the acceptability of conservative therapies of gallbladder stones. The aim of the present study was to determine the rate of stone recurrence and its risk factors, after successful shock-wave lithotripsy. DESIGN: Prospective ultrasound follow-up at yearly intervals or whenever biliary pain was reported. METHODS: One hundred and fifty-eight consecutive patients (single stone, n = 130; two or three stones, n = 28) were followed up to 70 months (median, 33 months) after stone disappearance and discontinuation of ursodeoxycholic acid. RESULTS: Forty-three patients developed recurrent stones. By actuarial analysis, the recurrence rates (as a percent) at 1, 2, 3, 4 and 5 years were, respectively: 6+/-2, 14+/-3, 27+/-4, 35+/-5, and 44+/-6 (observed +/- SE). Cox's regression analysis identified high body mass indexes to be a risk factor (P = 0.02) for newly formed stones. Having had a single primary stone did not seem to be protective. Fourteen of the 43 patients (33%) had early symptoms of recurrence. Thirty-eight patients (89%) chose to undergo oral dissolution again, which was complemented by lithotripsy in eight patients (19%). CONCLUSION: The 5-year gallbladder stone recurrence after lithotripsy and ursodeoxycholic acid is not substantially smaller than that reported by post-bile acid studies. Obesity is a risk factor for recurrent stones as it is for primary stones. Most patients with secondary stones choose to have conservative therapy again. Gallbladder stone recurrence still is one of the major drawbacks of these treatments and cost-effective strategies are needed to prevent it. PMID- 10741938 TI - Crohn's disease and intestinal endometriosis: an intriguing co-existence. AB - OBJECTIVES: We present a series of eight female patients who came to surgery for complicated Crohn's disease of the terminal ileum (n = 7) or colon (n = 1). Indications for surgery were medically intractable disease in three, steroid dependence in four and ileal perforation in one. RESULTS: Histological examination using routine haematoxylin-eosin stained sections revealed the presence of intestinal endometriosis of the ileum (n = 6), colon (n = 1) or ileum and rectum (n = 1) in addition to the typical features of Crohn's disease. In particular, chronic transmural inflammation was observed in locations other than the endometriotic deposits, which were confined to the serosa in three, the muscularis propria in two, both the serosa and the muscularis propria in one and the serosa, muscularis propria and submucosa in two. In none of these patients had the diagnosis of intestinal endometriosis been suspected pre-operatively based on clinical (gynaecological) or radiological tests. CONCLUSION: Intestinal endometriosis and Crohn's disease can occur simultaneously. The diagnosis is often only made after surgical resection of the diseased segment. In Crohn's disease, endometriosis of the terminal ileum seems more common. PMID- 10741939 TI - Cyclo-oxygenase-2 inhibitors ameliorate the severity of experimental colitis in rats. AB - BACKGROUND: Both in experimental colitis and in inflammatory bowel disease, colonic eicosanoid generation is enhanced and may contribute to the pathogenesis of the inflammatory response. AIMS: To evaluate the effect of selective cyclo oxygenase-2 (COX-2) inhibitors on the extent and severity of two models of experimental colitis. METHODS: Colitis was induced by intra-caecal administration of 2 ml 5% acetic acid or intra-colonic administration of 0.1 ml 3% iodoacetamide. Rats were treated intra-gastrically with nimesulide 2 x 10 mg/kg/day, or once with SC-236 6 mg/kg, and killed 1 or 3 days after damage induction. The colon was isolated, weighed, macroscopic damage was measured, and mucosal samples were obtained for histology and for determination of myeloperoxidase (MPO) and nitric oxide synthase (NOS) activities and eicosanoid generation. The serum levels of thromboxane B2 (TXB2), tumour necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) were determined. RESULTS: Nimesulide significantly decreased the extent of colitis induced by acetic acid. Both nimesulide and SC-236 significantly decreased the extent of iodoacetamide induced colonic damage. The decrease in the extent of colitis induced by nimesulide was accompanied by a significant decrease in mucosal MPO and NOS activities. Nimesulide and SC-236 decreased the enhanced colonic eicosanoid generation in acetic acid and iodoacetamide-induced colitis, and, in iodoacetamide-treated rats, nimesulide also decreased the elevated serum TNF alpha and IL-1beta levels. CONCLUSIONS: The effective nimesulide and SC-236 induced amelioration of the severity of the colitis in acetic acid and iodoacetamide-treated rats confirms the role of eicosanoids in their pathogenesis and suggests that COX-2 inhibitors may be of value in the treatment of inflammatory bowel disease. PMID- 10741940 TI - Cholangiocarcinoma arising in Von Meyenburg complex associated with hepatocellular carcinoma in genetic haemochromatosis. AB - A 61-year-old man had a liver resection for a bilobar mass thought to be, by imaging techniques, an hepatocellular carcinoma. He had been treated for the last 12 years by venesections for genetic haemochromatosis complicated by well compensated cirrhosis. At surgery, prothrombin time and platelet count were normal, as was alpha-fetoprotein. On the resected specimen, the non-tumoral liver was not cirrhotic; septal fibrosis was present as well as mild iron overload and numerous Von Meyenburg complexes. The bilobar tumour was composed of two different parts: one was a cholangiocarcinoma arising from Von Meyenburg complexes, the other was a moderately differentiated hepatocellular carcinoma with a partially invaded capsule. The two tumours, in close proximity, did not communicate. This observation raises three questions: the relative risk of primary liver cancer including both hepatocellular carcinoma and cholangiocarcinoma in haemochromatosis without cirrhosis; the development of cholangiocarcinoma from Von Meyenburg complexes; the reversibility of cirrhosis in treated patients. PMID- 10741941 TI - Hepatocellular carcinoma arising in an elderly male with primary biliary cirrhosis. AB - We report the case of an elderly male with asymptomatic primary biliary cirrhosis (PBC) who developed a hepatocellular carcinoma (HCC). The 89-year-old man, who was otherwise healthy, was admitted for investigation of mild hepatic dysfunction, which had been detected during a routine physical check-up. Serum chemistry, positive anti-mitochondrial antibody (M2) and liver biopsy results led to a diagnosis of PBC. Three years later, at age 92, computed tomography (CT) and ultrasound scans of his abdomen revealed a large hepatic tumour, which was confirmed on liver biopsy to be HCC. The tumour ruptured 3 months after diagnosis and the patient was successfully stabilized by coil embolization of his right hepatic artery. We believe that, to date, this is the oldest reported patient to have had interventional radiology for the management of HCC. PMID- 10741942 TI - Biliary migration of hepatocellular carcinoma fragment after transcatheter arterial chemoembolization therapy. AB - Occasional side-effects of transcatheter arterial chemoembolization therapy in hepatocellular carcinoma are essentially related to tissue necrosis. We report the case of a patient with hepatocellular carcinoma who experienced an acute common bile duct obstruction a few weeks after such a procedure, in the absence of obvious biliary tract invasion. An endoscopic sphincterotomy relieved the obstruction. At histology, the intra-biliary material was identified as a fragment of hepatocellular carcinoma. We discuss the causes of obstructive jaundice in the setting of hepatocellular carcinoma as well as in the specific situation of transcatheter arterial chemoembolization therapy. PMID- 10741943 TI - Severe strongyloidiasis during interferon plus ribavirin therapy for chronic HCV infection. AB - Ribavirin is a nucleoside analogue, recently introduced in hepatitis C virus (HCV) therapy, that has postulated immunomodulatory and immunosuppressive action. Strongyloidiasis is an helmintic infection caused by Strongyloides stercoralis, endemic in tropical countries. Severe strongyloidiasis has been demonstrated after immunosuppression by corticosteroids evolving some fatal cases. Here, we describe two cases of severe strongyloidiasis coincident with ribavirin plus interferon therapy for treating HCV infection. The review of our monotherapy protocol with interferon did not disclose any case of symptomatic strongyloidiasis pointing to a possible role of ribavirin in modifying immune response to S. stercoralis. We propose a careful screening for S. stercoralis before initiating ribavirin therapy or even empiric antihelmintic treatment. PMID- 10741944 TI - Inflammatory cloacogenic polyp mimicking anorectal malignancy. AB - Inflammatory cloacogenic polyp is a rare condition arising from the transitional zone of the anorectal junction. It is benign but may macroscopically resemble anorectal malignancy. Inflammatory cloacogenic polyp was diagnosed in two patients with rectal bleeding and polypoid growths at the anorectal junction. The lesions were best visualized with an endoscope retroflexed in the rectum. Accurate diagnosis was dependent on histological examination. The histological similarities to solitary rectal ulcer syndrome and other mucosal prolapse conditions are discussed, and the literature on inflammatory cloacogenic polyp and its management reviewed. PMID- 10741945 TI - Benefit of interferon-alpha2b in a patient with unresectable hepatoma and chronic infection with hepatitis C virus. AB - Only in a small proportion of patients is advanced hepatocellular carcinoma (HCC) resectable, so the need for effective non-surgical treatments is obvious. We present details of a 72-year-old woman with inoperable HCC and chronic infection with hepatitis C virus, proved by the presence of antibodies directed against hepatitis C virus and positive polymerase chain reaction. The patient was treated with subcutaneous recombinant human interferon-alpha-2b. Within a few weeks, a partial tumour remission, paralleled by a decrease in serum levels of tumour markers and liver enzymes, was observed. In addition, polymerase chain reaction became negative. This observation facilitates the hypothesis that the anti-viral effects of interferon might have been jointly responsible for the anti-tumour activity observed. Interferon-alpha might serve as a treatment option in patients with unresectable hepatoma and chronic active viral hepatitis, but prospective studies are warranted. PMID- 10741946 TI - Charles McBurney: reflecting upon his life's work. PMID- 10741947 TI - A new technique to create an artificial stenosis in the native LAD using a hemoclip. AB - The study of hemodynamics associated with coronary atherosclerotic stenosis has been limited due to the lack of a safe, accurate, and reliable technique to create an artificial stenosis in an animal model. Existing techniques have often resulted in myocardial infarction (MI) or severe injury to the vessel and have been difficult to accurately quantify and reproduce. We developed a new technique to create an artificial stenosis in the native left anterior descending (LAD) coronary artery using a hemoclip in an experimental off-pump coronary artery bypass graft (CABG) animal model that overcomes these limitations. The native LAD was dissected at its proximal end and a hemoclip was applied to create varying degrees of artificial stenosis in 30 mongrel dogs during experimental off-pump CABG procedures. The precise application of the hemoclip was predetermined using a mathematical formula to calculate the reduced circumference required to create a specific stenosis. Using these calculations, artificial stenoses of 25%, 50%, 75%, and 90% were created in the LAD. Postoperative angiography demonstrated only 5-10% error between the true (angiography) and estimated (hemoclip technique) stenosis values. In all cases, the vessel remained intact without any apparent external trauma, and no indications of MI were present during electrocardiograph (ECG) monitoring. The creation of an artificial coronary stenosis using the hemoclip technique was safe, reliable, easy, and accurate. PMID- 10741948 TI - Transmyocardial laser revascularization: current status. AB - Transmyocardial laser revascularization (TMLR) has been widely evaluated for treatment of the ischemic myocardium either in conjunction with coronary artery bypass grafting or as sole therapy. Clinically, it has shown significant improvement for angina symptoms, but the mechanism by which this modality works is unknown at this time. The original premise on which transmyocardial revascularization was established depended on its ability to essentially generate channels that would directly carry blood from the ventricle into the ischemic myocardium. This theory, however, has not been substantiated, so other mechanisms have been postulated. This article gives a historical perspective on the advent of transmyocardial revascularization and the many animal and human studies that have paved the way for its clinical use. Current controversies are examined, along with the new advances in laser technology and where the future of TMLR is headed. PMID- 10741949 TI - Modification of disaccharidase activity measurement in rat bowel. AB - This study describes a modification of the existing disaccharidase assay in rat small bowel in which whole bowel, rather than mucosa, is utilized. In addition, the use of total vs. specific activity as a more accurate unit of measurement of disaccharidase activity is discussed. PMID- 10741950 TI - Effects of intestinal ischemia-reperfusion on major conduit arteries. AB - Intestinal ischemia-reperfusion (I-R) is a common and serious clinical condition associated with simultaneous remote organ dysfunction. The purpose of this study was to investigate the effects of intestinal I-R on the vasomotor functions of major conduit arteries. Anesthetized rabbits were randomly assigned to one of three groups: sham-operated controls (Group I), and one-hour intestinal ischemia with two-hour reperfusion (Group II) or four-hour reperfusion (Group III). The following mechanisms of vasomotor functions were studied in abdominal aorta, superior mesenteric, renal, pulmonary, and carotid arterial rings: (1) endothelial-dependent vasodilation response to acetylcholine, (2) endothelial independent vasodilation response to nitroprusside, (3) beta-adrenergic vasodilation response to isoproterenol, and (4) phenylephrine-induced vasoconstriction. Intestinal injury was quantified using malondialdehyde (MDA) concentration and wet-to-dry intestine weight ratio. Intestinal I-R did not affect the maximal responsiveness or the sensitivity to acetylcholine, nitroprusside, and isoproterenol in all the vessels studied. The maximal contractile response to phenylephrine increased significantly in mesenteric artery in Group II, (227.1+/-15.1% vs. 152.8+/-11.7% in controls) (p<0.05). Intestinal MDA concentration, a marker of oxidant injury, increased from 39.87+/ 9.41 nmol/g to 67.8+/-8.8 nmol/g in group II (p<0.01), and to 94.8+/-7.56 nmol/g in Group III (p<0.001). Wet-to-dry intestine weight ratio increased from 3.62+/ 0.12 to 4.28+/-0.17 in Group II (p<0.01), to 4.62+/-0.14 in Group III (p<0.001). These data indicate that although the intestines of the animals subjected to intestinal I-R are seriously injured, the smooth muscle relaxation of major conduit arteries was not affected. PMID- 10741951 TI - A canine model of multiple portosystemic shunting. AB - The objective of this study was to develop and describe an experimental canine model of multiple acquired portosystemic shunts (PSS) similar in nature to spontaneously occurring PSS. Sixteen dogs were used and were divided into a control (n = 6) and a diseased group (n = 10). Dogs of the diseased group were administered dimethylnitrosamine (2 mg/kg of body weight, po) twice weekly, and clinicopathologic, ultrasonographic, and hepatic scintigraphic findings were recorded during the development of hepatic disease and PSS. Surgery was then performed to permit visual verification of multiple shunts, catheter placement for portography examination, and biopsy of the liver. All diseased dogs developed severe hepatic disease and multiple PSS as documented visually at surgery and on portography. Based on this study, dimethylnitrosamine-induced portosystemic shunting appears to be an appropriate model for spontaneously occurring multiple PSS secondary to portal hypertension. PMID- 10741952 TI - A new model for the assessment of lung allograft ischemia/reperfusion injury. AB - Lung edema is the main clinical manifestation of reperfusion injury following lung transplantation. The evaluation of strategies to prevent this injury is of high clinical importance. Therefore we developed a large-animal model to study the mechanisms of ischemia/reperfusion injury including dynamics of posttransplant reperfusion edema and their prevention. Left lung allotransplantation was performed in 6 weight-matched pigs (25-31 kg). Donor lungs were flushed with 1.5 L low-potassium dextran (LPD) solution (4 degrees C) and preserved for 20 h at 1 degrees C. One hour after reperfusion the recipient contralateral right lung was excluded from perfusion and ventilation to assess graft function only. Extravascular lung water index (EVLWI), intrathoracic blood volume (ITBV), and cardiac output (CO) were assessed (q = 30 min) with a lung water computer (Cold Z-021, Partig, Munich, Germany) by the thermo-dye technique during a 5-h observation period. Gas exchange (FIO2 = 1.0) was measured hourly, and hemodynamics were monitored continuously. The EVLWI of the recipient contralateral lung together with the donor left lung at the time of reperfusion was 6.5+/-1.1 ml/kg, increasing to 7.1+/-1.0 ml/kg at 60 min after reperfusion. After occlusion of the recipient right lung, EVLWI in the graft further increased within 80 min from 8.1+/-0.5 ml/kg to a peak of 11.4+/-1.3 ml/kg, followed by a decrease to 8.5+/-0.8 ml/kg at 5 h after reperfusion in 5 of 6 animals. In 1 animal a severe alveolar edema developed with subsequent deterioration of gas exchange and death 4.5 h after reperfusion. In this animal, peak EVLWI reached 16.8 ml/kg, PaO2 deteriorated from 60.1 to 7.8 kPa, and CO decreased from 3.1 to 1.4 L/min. In all other animals, ITBV (515+/-51 ml), left atrial pressure (LAP), central venous pressure (CVP), and CO (2.9+/-0.3 L/min) were stable during the 5 h assessment period. We conclude that EVLWI measurement is a reliable and very sensitive method to quantify lung allograft reperfusion edema. It may prove useful in early assessment of lung allograft reperfusion injury in the clinical setting and in experimental models. PMID- 10741953 TI - Identification of a locus on chromosome 1q44 for familial cold urticaria. AB - Familial cold urticaria (FCU) is a rare autosomal dominant inflammatory disorder characterized by intermittent episodes of rash with fever, arthralgias, conjunctivitis, and leukocytosis. These symptoms develop after generalized exposure to cold. Some individuals with FCU also develop late-onset reactive renal amyloidosis, which is consistent with Muckle-Wells syndrome. By analyzing individuals with FCU from five families, we identified linkage to chromosome 1q44. Two-point linkage analysis revealed a maximum LOD score (Zmax) of 8.13 (recombination fraction 0) for marker D1S2836; multipoint linkage analysis identified a Zmax of 10. 92 in the same region; and haplotype analysis defined a 10.5-cM region between markers D1S423 and D1S2682. Muckle-Wells syndrome was recently linked to chromosome 1q44, which suggests that the two disorders may be linked to the same locus. PMID- 10741954 TI - Mapping of a locus for a familial autosomal recessive idiopathic myoclonic epilepsy of infancy to chromosome 16p13. AB - Myoclonic epilepsies with onset in infancy and childhood are clinically and etiologically heterogeneous. Although genetic factors are thought to play an important role, to date very little is known about the etiology of these disorders. We ascertained a large Italian pedigree segregating a recessive idiopathic myoclonic epilepsy that starts in early infancy as myoclonic seizures, febrile convulsions, and tonic-clonic seizures. We typed 304 microsatellite markers spanning the 22 autosomes and mapped the locus on chromosome 16p13 by linkage analysis. A maximum LOD score of 4.48 was obtained for marker D16S3027 at recombination fraction 0. Haplotype analysis placed the critical region within a 3.4-cM interval between D16S3024 and D16S423. The present report constitutes the first example of an idiopathic epilepsy that is inherited as an autosomal recessive trait. PMID- 10741958 TI - Giant birefringent optics in multilayer polymer mirrors AB - Multilayer mirrors that maintain or increase their reflectivity with increasing incidence angle can be constructed using polymers that exhibit large birefringence in their indices of refraction. The most important feature of these multilayer interference stacks is the index difference in the thickness direction (z axis) relative to the in-plane directions of the film. This z-axis refractive index difference provides a variable that determines the existence and value of the Brewster's angle at layer interfaces, and it controls both the interfacial Fresnel reflection coefficient and the phase relations that determine the optics of multilayer stacks. These films can yield optical results that are difficult or impossible to achieve with conventional multilayer optical designs. The materials and processes necessary to fabricate such films are amenable to large-scale manufacturing. PMID- 10741955 TI - Comparative genomic hybridization in combination with flow cytometry improves results of cytogenetic analysis of spontaneous abortions. AB - More than 50% of spontaneous abortions (SAs) have abnormal chromosomes; the most common abnormalities are trisomy, sex chromosome monosomy, and polyploidy. Conventional cytogenetic analysis of SAs depends on tissue culturing and is associated with a significant tissue culture failure rate and contamination by maternally derived cells. Comparative genomic hybridization (CGH), in combination with flow cytometry (FCM), can detect numerical and unbalanced structural chromosomal abnormalities associated with SAs while avoiding the technical problems associated with tissue culture. Routine cytogenetic and CGH analysis was performed independently on tissue from 301 SAs. Samples shown to be chromosomally balanced by CGH were analyzed by FCM to determine ploidy. Of 253 samples successfully analyzed by both approaches, there was an absolute correlation of results in 235 (92.8%). Of the 18 cases with discrepancies between cytogenetic and CGH/FCM results, an explanation could be found in 17. Twelve samples produced a 46,XX karyotype by cytogenetics, whereas CGH/FCM demonstrated aneuploidy/polyploidy or a male genome, indicating maternal contamination of the tissue cultures. In two cases, where tetraploidy was demonstrated by cytogenetics and diploidy by FCM, tissue culture artifact is implied. In three cases, CGH demonstrated an aneuploidy, and cytogenetics demonstrated hypertriploidy. In one unexplainable case, aneuploidy demonstrated by CGH could not be detected by repeat CGH analysis, conventional cytogenetic, or FISH analysis. These results demonstrate that CGH supplemented with FCM can readily identify chromosomal abnormalities associated with SAs and, by avoiding maternal contamination and tissue culture artifacts, can do so with a lower failure rate and more accuracy than conventional cytogenetic analysis. PMID- 10741959 TI - Dynamic variations at the base of the solar convection zone AB - We have detected changes in the rotation of the sun near the base of its convective envelope, including a prominent variation with a period of 1.3 years at low latitudes. Such helioseismic probing of the deep solar interior has been enabled by nearly continuous observation of its oscillation modes with two complementary experiments. Inversion of the global-mode frequency splittings reveals that the largest temporal changes in the angular velocity Omega are of the order of 6 nanohertz and occur above and below the tachocline that separates the sun's differentially rotating convection zone (outer 30% by radius) from the nearly uniformly rotating deeper radiative interior beneath. Such changes are most pronounced near the equator and at high latitudes and are a substantial fraction of the average 30-nanohertz difference in Omega with radius across the tachocline at the equator. The results indicate variations of rotation close to the presumed site of the solar dynamo, which may generate the 22-year cycles of magnetic activity. PMID- 10741960 TI - Isolating the spectroscopic signature of a hydration shell with the use of clusters: superoxide tetrahydrate AB - Cluster spectroscopy, aided by ab initio theory, was used to determine the detailed structure of a complete hydration shell around an anion. Infrared spectra of size-selected O(2)-. (H(2)O)(n) (n = 1 to 4) cluster ions were obtained by photoevaporation of an argon nanomatrix. Four water molecules are required to complete the coordination shell. The simple spectrum of the tetrahydrate reveals a structure in which each water molecule is engaged in a single hydrogen bond to one of the four lobes of the pi* orbital of the superoxide, whereas the water molecules bind together in pairs. This illustrates how water networks deform upon accommodating a solute ion to create a distinct supramolecular species. PMID- 10741961 TI - Hot and dry deep crustal xenoliths from tibet AB - Anhydrous metasedimentary and mafic xenoliths entrained in 3-million-year-old shoshonitic lavas of the central Tibetan Plateau record a thermal gradient reaching about 800 degrees to 1000 degrees C at a depth of 30 to 50 kilometers; just before extraction, these same xenoliths were heated as much as 200 degrees C. Although these rocks show that the central Tibetan crust is hot enough to cause even dehydration melting of mica, the absence of hydrous minerals, and the match of our calculated P-wave speeds and Poisson's ratios with seismological observations, argue against the presence of widespread crustal melting. PMID- 10741962 TI - Global carbon sinks and their variability inferred from atmospheric O2 and delta13C. AB - Recent time-series measurements of atmospheric O2 show that the land biosphere and world oceans annually sequestered 1.4 +/- 0.8 and 2.0 +/- 0.6 gigatons of carbon, respectively, between mid-1991 and mid-1997. The rapid storage of carbon by the land biosphere from 1991 to 1997 contrasts with the 1980s, when the land biosphere was approximately neutral. Comparison with measurements of delta13CO2 implies an isotopic flux of 89 +/- 21 gigatons of carbon per mil per year, in agreement with model- and inventory-based estimates of this flux. Both the delta13C and the O2 data show significant interannual variability in carbon storage over the period of record. The general agreement of the independent estimates from O2 and delta13C is a robust signal of variable carbon uptake by both the land biosphere and the oceans. PMID- 10741963 TI - On the presence of liquid in Earth's inner core AB - Seismological studies indicate that the inner core of Earth is anisotropic for compressional waves (P waves), and has low shear wave (S wave) velocity, and high seismic attenuation. Using an effective medium theory for composite materials, we show that the presence of a volume fraction of 3 to 10% liquid in the form of oblate spheroidal inclusions aligned in the equatorial plane between iron crystals is sufficient to explain the aforementioned seismic phenomena. Variation of S-wave velocity between the polar axis and equatorial plane is more sensitive to the addition of liquid than that of P waves. The liquid could arise from the presence of dendrites or a mixture of elements other than iron that exist in liquid form under inner-core conditions. PMID- 10741964 TI - Insect population control using a dominant, repressible, lethal genetic system. AB - A major modification to the sterile insect technique is described, in which transgenic insects homozygous for a dominant, repressible, female-specific lethal gene system are used. We demonstrate two methods that give the required genetic characteristics in an otherwise wild-type genetic background. The first system uses a sex-specific promoter or enhancer to drive the expression of a repressible transcription factor, which in turn controls the expression of a toxic gene product. The second system uses non-sex-specific expression of the repressible transcription factor to regulate a selectively lethal gene product. Both methods work efficiently in Drosophila melanogaster, and we expect these principles to be widely applicable to more economically important organisms. PMID- 10741965 TI - Bicoid-independent formation of thoracic segments in Drosophila. AB - The maternal determinant Bicoid (Bcd) represents the paradigm of a morphogen that provides positional information for pattern formation. However, as bicoid seems to be a recently acquired gene in flies, the question was raised as to how embryonic patterning is achieved in organisms with more ancestral modes of development. Because the phylogenetically conserved Hunchback (Hb) protein had previously been shown to act as a morphogen in abdominal patterning, we asked which functions of Bcd could be performed by Hb. By reestablishing a proposed ancient regulatory circuitry in which maternal Hb controls zygotic hunchback expression, we show that Hb is able to form thoracic segments in the absence of Bcd. PMID- 10741966 TI - Necessity for afferent activity to maintain eye-specific segregation in ferret lateral geniculate nucleus. AB - In the adult mammal, retinal ganglion cell axon arbors are restricted to eye specific layers in the lateral geniculate nucleus. Blocking neuronal activity early in development prevents this segregation from occurring. To test whether activity is also required to maintain eye-specific segregation, ganglion cell activity was blocked after segregation was established. This caused desegregation, so that both eyes' axons became concentrated in lamina A, normally occupied only by contralateral afferents. These results show that an activity dependent process is necessary for maintaining eye-specific segregation and suggest that activity-independent cues may favor lamina A as the target for arborization of afferents from both eyes. PMID- 10741967 TI - Structure of the RNA polymerase domain of E. coli primase. AB - All cellular organisms use specialized RNA polymerases called "primases" to synthesize RNA primers for the initiation of DNA replication. The high-resolution crystal structure of a primase, comprising the catalytic core of the Escherichia coli DnaG protein, was determined. The core structure contains an active-site architecture that is unrelated to other DNA or RNA polymerase palm folds, but is instead related to the "toprim" fold. On the basis of the structure, it is likely that DnaG binds nucleic acid in a groove clustered with invariant residues and that DnaG is positioned within the replisome to accept single-stranded DNA directly from the replicative helicase. PMID- 10741968 TI - Mitotic misregulation and human aging. AB - Messenger RNA levels were measured in actively dividing fibroblasts isolated from young, middle-age, and old-age humans and humans with progeria, a rare genetic disorder characterized by accelerated aging. Genes whose expression is associated with age-related phenotypes and diseases were identified. The data also suggest that an underlying mechanism of the aging process involves increasing errors in the mitotic machinery of dividing cells in the postreproductive stage of life. We propose that this dysfunction leads to chromosomal pathologies that result in misregulation of genes involved in the aging process. PMID- 10741969 TI - Similar requirements of a plant symbiont and a mammalian pathogen for prolonged intracellular survival. AB - Brucella abortus, a mammalian pathogen, and Rhizobium meliloti, a phylogenetically related plant symbiont, establish chronic infections in their respective hosts. Here a highly conserved B. abortus homolog of the R. meliloti bacA gene, which encodes a putative cytoplasmic membrane transport protein required for symbiosis, was identified. An isogenic B. abortus bacA mutant exhibited decreased survival in macrophages and greatly accelerated clearance from experimentally infected mice compared to the virulent parental strain. Thus, the bacA gene product is critical for the maintenance of two very diverse host bacterial relationships. PMID- 10741970 TI - Genetic requirements for inheritance of RNAi in C. elegans. AB - In Caenorhabditis elegans, the introduction of double-stranded RNA triggers sequence-specific genetic interference (RNAi) that is transmitted to offspring. The inheritance properties associated with this phenomenon were examined. Transmission of the interference effect occurred through a dominant extragenic agent. The wild-type activities of the RNAi pathway genes rde-1 and rde-4 were required for the formation of this interfering agent but were not needed for interference thereafter. In contrast, the rde-2 and mut-7 genes were required downstream for interference. These findings provide evidence for germ line transmission of an extragenic sequence-specific silencing factor and implicate rde-1 and rde-4 in the formation of the inherited agent. PMID- 10741971 TI - Single-molecule study of transcriptional pausing and arrest by E. coli RNA polymerase. AB - Using an optical-trap/flow-control video microscopy technique, we followed transcription by single molecules of Escherichia coli RNA polymerase in real time over long template distances. These studies reveal that RNA polymerase molecules possess different intrinsic transcription rates and different propensities to pause and stop. The data also show that reversible pausing is a kinetic intermediate between normal elongation and the arrested state. The conformational metastability of RNA polymerase revealed by this single-molecule study of transcription has direct implications for the mechanisms of gene regulation in both bacteria and eukaryotes. PMID- 10741973 TI - Cash bonanza for NHS. The price is centralisation. PMID- 10741972 TI - T cell-independent rescue of B lymphocytes from peripheral immune tolerance. AB - Autoimmunity arises when immune tolerance to specific self-antigens is broken. The mechanisms leading to such a failure remain poorly understood. One hypothesis proposes that infectious agents or antigens can break B or T lymphocyte self tolerance by expressing epitopes that mimic self. Using a transgenic immunoglobulin model, we show that challenge with self-mimicking foreign antigen rescues B cells from peripheral tolerance independent of T cell help, resulting in the accumulation of self-reactive cells in the lymph nodes and secretion of immunoglobulins that bind to a liver-expressed self-antigen. Therefore, our studies reveal a potentially important mechanism by which B lymphocytes can escape self-tolerance. PMID- 10741974 TI - Cancer trends in England and Wales. Good data and analysis are vital to improving survival. PMID- 10741975 TI - Dilemmas and choices in facing the drugs problem. "Being truthful about drugs...must remain the foundation of drug policy". PMID- 10741976 TI - The new primary care organisations: one year on. Some successes-but many issues remain to be resolved. PMID- 10741977 TI - Helping refugee doctors. The new asylum act may make it easier to help small numbers of refugee doctors. PMID- 10741978 TI - Cycling and health promotion. A safer, slower urban road environment is the key. PMID- 10741980 TI - Blair demands reform of the NHS PMID- 10741979 TI - Health spending in UK to rise to 7.6% of GDP. PMID- 10741981 TI - UK government wants GMC to be given stronger powers PMID- 10741982 TI - NICE issues guidelines on wisdom teeth PMID- 10741983 TI - In brief PMID- 10741984 TI - Spain faces massive decline in population PMID- 10741985 TI - NHS bill for negligence set to soar again PMID- 10741987 TI - NHS board appointments criticised PMID- 10741986 TI - Study explores internet as a tool for care of diabetic patients PMID- 10741989 TI - Mrs clinton aims to reduce psychoactive drugs in young children PMID- 10741988 TI - More services needed for bereaved children PMID- 10741990 TI - Twenty countries pledge to wipe out TB PMID- 10741992 TI - Australia to consider a class action against tobacco industry PMID- 10741991 TI - Supreme court rules that FDA cannot regulate tobacco industry PMID- 10741993 TI - How many deaths have been avoided through improvements in cancer survival? AB - OBJECTIVE: To estimate how many deaths from cancer have been avoided in England and Wales because of recent improvements in survival. DESIGN: Analysis of national statistics. SETTING: England and Wales. SUBJECTS: 1.5 million adults with diagnosis of one of 47 different cancers during 1981-5 or 1986-90. MAIN OUTCOME MEASURES: Reduction in number of cancer deaths within five years of diagnosis among patients with cancer diagnosed during 1986-90 compared with patients with cancer diagnosed during 1981-5. RESULTS: 17 041 deaths were avoided within five years of diagnosis among patients with cancer diagnosed during 1986 90. This represents 3.3% of the cancer deaths that would have been expected if survival had been the same as for patients with cancer diagnosed during 1981-5. Two thirds of the avoided deaths arose from improvements in survival for just five cancers: female breast cancer (4822), cancers of the colon (2560), rectum (1090), and bladder (1157), and melanoma of the skin (1098). The largest proportionate reductions in excess deaths were for melanoma of the skin (23%) and cancers of the testis (17%) and bone (17%). About 12 000 (70%) of the avoided deaths arose among adults aged under 75 at death. Improvements in survival from cancers of lung, prostate, stomach, ovary, and brain were small: they accounted for 33% of all cancers but only 11% of avoided deaths. CONCLUSIONS: Small gains in survival from common cancers save more lives than larger gains for uncommon cancers. If recent rates of improvement in cancer survival continue, about 24 000 deaths within five years of diagnosis would be avoided in patents aged under 75 by the year 2010, representing about a quarter of the government's overall target of 100 000 fewer cancer deaths. PMID- 10741994 TI - Relation between income inequality and mortality in Canada and in the United States: cross sectional assessment using census data and vital statistics. AB - OBJECTIVE: To compare the relation between mortality and income inequality in Canada with that in the United States. DESIGN: The degree of income inequality, defined as the percentage of total household income received by the less well off 50% of households, was calculated and these measures were examined in relation to all cause mortality, grouped by and adjusted for age. SETTING: The 10 Canadian provinces, the 50 US states, and 53 Canadian and 282 US metropolitan areas. RESULTS: Canadian provinces and metropolitan areas generally had both lower income inequality and lower mortality than US states and metropolitan areas. In age grouped regression models that combined Canadian and US metropolitan areas, income inequality was a significant explanatory variable for all age groupings except for elderly people. The effect was largest for working age populations, in which a hypothetical 1% increase in the share of income to the poorer half of households would reduce mortality by 21 deaths per 100 000. Within Canada, however, income inequality was not significantly associated with mortality. CONCLUSIONS: Canada seems to counter the increasingly noted association at the societal level between income inequality and mortality. The lack of a significant association between income inequality and mortality in Canada may indicate that the effects of income inequality on health are not automatic and may be blunted by the different ways in which social and economic resources are distributed in Canada and in the United States. PMID- 10741995 TI - A useful radiology report PMID- 10741996 TI - Effect of patients seeing a general practitioner in accident and emergency on their subsequent reattendance: cohort study. PMID- 10741997 TI - Private funding of elective hospital treatment in England and Wales, 1997-8: national survey. PMID- 10741998 TI - Doctors are just people PMID- 10742000 TI - Cancer patients' information needs and information seeking behaviour: in depth interview study. AB - OBJECTIVES: To explore why cancer patients do not want or seek information about their condition beyond that volunteered by their physicians at times during their illness. DESIGN: Qualitative study based on in-depth interviews. SETTING: Outpatient oncology clinics at a London cancer centre. PARTICIPANTS: 17 patients with cancer diagnosed in previous 6 months. MAIN OUTCOME MEASURES: Analysis of patients' narratives to identify key themes and categories. RESULTS: While all patients wanted basic information on diagnosis and treatment, not all wanted further information at all stages of their illness. Three overarching attitudes to their management of cancer limited patients' desire for and subsequent efforts to obtain further information: faith, hope, and charity. Faith in their doctor's medical expertise precluded the need for patients to seek further information themselves. Hope was essential for patients to carry on with life as normal and could be maintained through silence and avoiding information, especially too detailed or "unsafe" information. Charity to fellow patients, especially those seen as more needy than themselves, was expressed in the recognition that scarce resources-including information and explanations-had to be shared and meant that limited information was accepted as inevitable. CONCLUSIONS: Cancer patients' attitudes to cancer and their strategies for coping with their illness can constrain their wish for information and their efforts to obtain it. In developing recommendations, the government's cancer information strategy should attend to variations in patients' desires for information and the reasons for them. PMID- 10741999 TI - Biochemical diagnosis of ventricular dysfunction in elderly patients in general practice: observational study. AB - OBJECTIVE: To investigate the usefulness of measuring plasma concentrations of B type natriuretic peptide in the diagnosis of left ventricular systolic dysfunction in an unselected group of elderly people. DESIGN: Observational study. SETTING: General practice with four centres in Poole, Dorset. PARTICIPANTS: 155 elderly patients aged 70 to 84 years. MAIN OUTCOME MEASURES: Diagnostic characteristics of plasma B type natriuretic peptide measured by radioimmunoassay as a test for left ventricular systolic dysfunction assessed by echocardiography. RESULTS: The median plasma concentration of B type natriuretic peptide was 39.3 pmol/l in patients with left ventricular systolic dysfunction and 15.8 pmol/l in those with normal function. The proportional area under the receiver operator curve was 0.85. At a cut-off point of 18.7 pmol/l the test sensitivity was 92% and the predictive value 18%. CONCLUSIONS: Plasma concentration of B type natriuretic peptide could be used effectively as an initial test in a community screening programme and, possibly, using a low cut off point, as a means of ruling out left ventricular systolic dysfunction. It is, however, not a good test to "rule in" the diagnosis, and access to echocardiography remains essential for general practitioners to diagnose heart failure early. PMID- 10742001 TI - Survey of active verbs in the titles of clinical trial reports. PMID- 10742002 TI - Not wanted anywhere PMID- 10742004 TI - Email submissions from outside the united kingdom PMID- 10742006 TI - A full examination is always useful PMID- 10742003 TI - Ectopic pregnancy. PMID- 10742005 TI - ABC of arterial and venous disease. Acute stroke. PMID- 10742008 TI - Recommendations for using MMR vaccine in children allergic to eggs PMID- 10742007 TI - Treatment by in vitro fertilisation with surrogacy: experience of one British centre. PMID- 10742009 TI - Rational or rationed medicine? The promise of genetics for improved clinical practice. PMID- 10742011 TI - Wise words PMID- 10742010 TI - The war in south africa: Mr Kipling's hospital sketches PMID- 10742012 TI - Funding of long term care for older people needs to be publicly debated. PMID- 10742013 TI - Future directions for geriatric medicine. Red rag but no bull? PMID- 10742014 TI - Care of newborn infants in delivery room. Two clarifications. PMID- 10742015 TI - Managing acute anaphylaxis. Intravenous adrenaline should be considered because of the urgency of the condition. PMID- 10742016 TI - Unexpected beneficial effects of measles immunisation. Measles vaccination may be marker for other health seeking behaviours. PMID- 10742017 TI - Making decisions about screening for ovarian cancer. Who chooses when an operation is worth having? PMID- 10742018 TI - More trials on early treatment with corticosteroids are needed. PMID- 10742019 TI - Maternal nutrition and birth weight. These factors are related. PMID- 10742020 TI - Assessing palliative care is difficult. PMID- 10742021 TI - Hospitals must become "focused factories". PMID- 10742022 TI - Hyponatraemia can be caused by standard fluid regimens. PMID- 10742023 TI - Time could be the active ingredient in post-trauma debriefing. PMID- 10742024 TI - Obituaries PMID- 10742025 TI - Appraisal could provide information for revalidation PMID- 10742026 TI - Waveguide: an EEG atlas on CD-ROM PMID- 10742027 TI - PDQ evidence-based principles and practice (Book with CD-Rom for windows and macintosh) PMID- 10742028 TI - Trust me, I'm a surgeon PMID- 10742030 TI - Treating colorectal cancer: chances in front of goal PMID- 10742029 TI - Surrogate parents PMID- 10742032 TI - The NHS: last act of a greek tragedy? PMID- 10742031 TI - Why do grownups go to work? PMID- 10742033 TI - Trends in cancer deaths are not heading towards government target PMID- 10742034 TI - Income inequality and mortality are not associated in canada PMID- 10742035 TI - One in seven patients pays for elective hospital treatment PMID- 10742036 TI - GPs in A and E do not prevent reattendance PMID- 10742038 TI - Patients have good reasons for not wanting further information about their cancer PMID- 10742037 TI - Blood test is useful adjunct in diagnosing heart failure PMID- 10742039 TI - Phylogeny reconstruction using duplicate genes. AB - In this paper, we propose a new method (uninode coding) for coding duplicate (paralogous) genes to infer species trees. Uninode coding incorporates data from duplicated and unduplicated gene copies in phylogenetic analyses of taxa. Uninode coding utilizes global parsimony through the inclusion of both duplicated and unduplicated gene copies, allows one to code all data sources from a taxon into a single terminal, and overcomes problems of character dependence among duplicated and unduplicated gene copies. We present an example of uninode coding using the phytochrome A and phytochrome C data from a study by Donoghue and Mathews. PMID- 10742040 TI - mtDNA tandem repeats in domestic dogs and wolves: mutation mechanism studied by analysis of the sequence of imperfect repeats. AB - The mitochondrial (mt) DNA control region (CR) of dogs and wolves contains an array of imperfect 10 bp tandem repeats. This region was studied for 14 domestic dogs representing the four major phylogenetic groups of nonrepetitive CR and for 5 wolves. Three repeat types were found among these individuals, distributed so that different sequences of the repeat types were formed in different molecules. This enabled a detailed study of the arrays and of the mutation events that they undergo. Extensive heteroplasmy was observed in all individuals; 85 different array types were found in one individual, and the total number of types was estimated at 384. Among unrelated individuals, no identical molecules were found, indicating a high rate of evolution of the region. By performing a pedigree analysis, array types which had been inherited from mother to offspring and array types which were the result of somatic mutations, respectively, could be identified, showing that about 20% of the molecules within an individual had somatic mutations. By direct pairwise comparison of the mutated and the original array types, the physiognomy of the inserted or deleted elements (indels) and the approximate positions of the mutations could be determined. All mutations could be explained by replication slippage or point mutations. The majority of the indels were 1-5 repeats long, but deletions of up to 17 repeats were found. Mutations were found in all parts of the arrays, but at a higher frequency in the 5' end. Furthermore, the inherited array types within the mother-offspring pair were aligned and compared so that germ line mutations could be studied. The pattern of the germ line mutations was approximately the same as that of the somatic mutations. PMID- 10742041 TI - Extensive homoplasy, nonstepwise mutations, and shared ancestral polymorphism at a complex microsatellite locus in Lake Malawi cichlids. AB - Recent studies have suggested that size homoplasy is a prevalent feature of microsatellites and is expected to increase with time of divergence among populations and taxa. In this study, we performed sequence analysis of alleles from a complex microsatellite locus (Pzeb4, initially isolated from Pseudotropheus (Maylandia) zebra) from 1 midwater-feeding and 10 rock-dwelling cichlid fish species from Lake Malawi, East Africa, to investigate how widespread size homoplasy is among closely related taxa at this locus. All cichlid fishes endemic to this lake are believed to have originated within the last 700,000 years, and some species may be less than 200 years old. The number of eletromorphs found per species varied from 3 to 13. Sequence analysis of 95 cloned Pzeb4 PCR products (representing 18 electromorphs) revealed 13 new alleles. Ten of the 13 electromorphs (77%) were found to show size homoplasy due to either single nucleotide substitutions/indels or large indels. To investigate how well this locus fits the single-step mutation model (SMM), the minimum number of mutations required to explain the length differences between pairs of alleles was plotted against their size differences. Of the 300 comparisons, 166 (55.3%) corresponded to SMM expectations and 86 (28.7%) required a smaller number of mutations, and for 48 (16.0%) pairwise comparisons, a larger number of mutations were required to explain the length differences as compared with SMM expectations. Finally, a large deletion in the microsatellite sequence observed in the three rock-dwelling species Pseudotropheus lucerna, Pseudotropheus (Tropheops) 'band,' and Pseudotropheus (Tropheops) 'rust' and the midwater feeding species Copadichromis sp. is believed to represent a shared ancestral polymorphism. PMID- 10742042 TI - On the origin and domestication history of Barley (Hordeum vulgare). AB - Remains of barley (Hordeum vulgare) grains found at archaeological sites in the Fertile Crescent indicate that about 10,000 years ago the crop was domesticated there from its wild relative Hordeum spontaneum. The domestication history of barley is revisited based on the assumptions that DNA markers effectively measure genetic distances and that wild populations are genetically different and they have not undergone significant change since domestication. The monophyletic nature of barley domestication is demonstrated based on allelic frequencies at 400 AFLP polymorphic loci studied in 317 wild and 57 cultivated lines. The wild populations from Israel-Jordan are molecularly more similar than are any others to the cultivated gene pool. The results provided support for the hypothesis that the Israel-Jordan area is the region in which barley was brought into culture. Moreover, the diagnostic allele I of the homeobox gene BKn-3, rarely but almost exclusively found in Israel H. spontaneum, is pervasive in western landraces and modern cultivated varieties. In landraces from the Himalayas and India, the BKn-3 allele IIIa prevails, indicating that an allelic substitution has taken place during the migration of barley from the Near East to South Asia. Thus, the Himalayas can be considered a region of domesticated barley diversification. PMID- 10742043 TI - Early fixation of an optimal genetic code. AB - The evolutionary forces that produced the canonical genetic code before the last universal ancestor remain obscure. One hypothesis is that the arrangement of amino acid/codon assignments results from selection to minimize the effects of errors (e.g., mistranslation and mutation) on resulting proteins. If amino acid similarity is measured as polarity, the canonical code does indeed outperform most theoretical alternatives. However, this finding does not hold for other amino acid properties, ignores plausible restrictions on possible code structure, and does not address the naturally occurring nonstandard genetic codes. Finally, other analyses have shown that significantly better code structures are possible. Here, we show that if theoretically possible code structures are limited to reflect plausible biological constraints, and amino acid similarity is quantified using empirical data of substitution frequencies, the canonical code is at or very close to a global optimum for error minimization across plausible parameter space. This result is robust to variation in the methods and assumptions of the analysis. Although significantly better codes do exist under some assumptions, they are extremely rare and thus consistent with reports of an adaptive code: previous analyses which suggest otherwise derive from a misleading metric. However, all extant, naturally occurring, secondarily derived, nonstandard genetic codes do appear less adaptive. The arrangement of amino acid assignments to the codons of the standard genetic code appears to be a direct product of natural selection for a system that minimizes the phenotypic impact of genetic error. Potential criticisms of previous analyses appear to be without substance. That known variants of the standard genetic code appear less adaptive suggests that different evolutionary factors predominated before and after fixation of the canonical code. While the evidence for an adaptive code is clear, the process by which the code achieved this optimization requires further attention. PMID- 10742044 TI - The complete nucleotide sequence of the mitochondrial DNA of the agnathan Lampetra fluviatilis: bearings on the phylogeny of cyclostomes. AB - There are two competing theories about the interrelationships of craniates: the cyclostome theory assumes that lampreys and hagfishes are a clade, the cyclostomes, whose sister group is the jawed vertebrates (gnathostomes); the vertebrate theory assumes that lampreys and gnathostomes are a clade, the vertebrates, whose sister group is hagfishes. The vertebrate theory is best supported by a number of unique anatomical and physiological characters. Molecular sequence data from 18S and 28S rRNA genes rather support the cyclostome theory, but mtDNA sequence of Myxine glutinosa rather supports the vertebrate theory. Additional molecular data are thus needed to elucidate this three-taxon problem. We determined the complete nucleotide sequence of the mtDNA of the lamprey Lampetra fluviatilis. The mtDNA of L. fluviatilis possesses the same genomic organization as Petromyzon marinus, which validates this gene order as a synapomorphy of lampreys. The mtDNA sequence of L. fluviatilis was used in combination with relevant mtDNA sequences for an approach to the hagfish/lamprey relationships using the maximum-parsimony, neighbor-joining, and maximum likelihood methods. Although trees compatible with our present knowledge of the phylogeny of craniates can be reconstructed by using the three methods, the data collected do not support the vertebrate or the cyclostome hypothesis. The present data set does not allow the resolution of this three-taxon problem, and new kinds of data, such as nuclear DNA sequences, need to be collected. PMID- 10742045 TI - The performance of several multiple-sequence alignment programs in relation to secondary-structure features for an rRNA sequence. AB - The performances of five global multiple-sequence alignment programs (CLUSTAL W, Divide and Conquer, Malign, PileUp, and TreeAlign) were evaluated using part of the animal mitochondrial small subunit (12S) rRNA molecule. Conserved sequence motifs derived from an alignment based on secondary structural information were used to score how well each program aligned a data set of five vertebrate and five invertebrate taxa over a range of parameter values. All of the programs could align the motifs with reasonable accuracy for at least one set of parameter conditions, although if the whole sequence was considered, similarity to the structural alignment was only 25%-34%. Use of small gap costs generally gave more accurate results, although Malign and TreeAlign generated longer alignments when gap costs were low. The programs differed in the consistency of the alignments when gap cost was varied; CLUSTAL W, Divide and Conquer, and TreeAlign were the most accurate and robust, while PileUp performed poorly as gap cost values increased, and the accuracy of Malign fluctuated. Default settings for the programs did not give the best results, and attempting to select similar parameter values in different programs did not always result in more similar alignments. Poor alignment of even well-conserved motifs can occur if these are near sites with insertions or deletions. Since there is no a priori way to determine gap costs and because such costs can vary over the gene, alignment of rRNA sequences, particularly the less well conserved regions, should be treated carefully and aided by secondary structure and conserved motifs. Some motifs are single bases and so are often invisible to alignment programs. Our tests involved the most conserved regions of the 12S rRNA gene, and alignment of less well conserved regions will be more problematical. None of the alignments we examined produced a fully resolved phylogeny for the data set, indicating that this portion of 12S rRNA is insufficient for resolution of distant evolutionary relationships. PMID- 10742046 TI - Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. AB - The use of some multiple-sequence alignments in phylogenetic analysis, particularly those that are not very well conserved, requires the elimination of poorly aligned positions and divergent regions, since they may not be homologous or may have been saturated by multiple substitutions. A computerized method that eliminates such positions and at the same time tries to minimize the loss of informative sites is presented here. The method is based on the selection of blocks of positions that fulfill a simple set of requirements with respect to the number of contiguous conserved positions, lack of gaps, and high conservation of flanking positions, making the final alignment more suitable for phylogenetic analysis. To illustrate the efficiency of this method, alignments of 10 mitochondrial proteins from several completely sequenced mitochondrial genomes belonging to diverse eukaryotes were used as examples. The percentages of removed positions were higher in the most divergent alignments. After removing divergent segments, the amino acid composition of the different sequences was more uniform, and pairwise distances became much smaller. Phylogenetic trees show that topologies can be different after removing conserved blocks, particularly when there are several poorly resolved nodes. Strong support was found for the grouping of animals and fungi but not for the position of more basal eukaryotes. The use of a computerized method such as the one presented here reduces to a certain extent the necessity of manually editing multiple alignments, makes the automation of phylogenetic analysis of large data sets feasible, and facilitates the reproduction of the final alignment by other researchers. PMID- 10742047 TI - Evidence for directional selection acting on pheromone-binding proteins in the genus Choristoneura. AB - Patterns of nucleotide variation consistent with the action of natural selection have been discovered at a number of different gene loci. Here, pheromone-binding proteins (PBPs) are examined to determine if selection has acted to fix amino acid changes in PBPs in lineages in which pheromone changes have occurred. PBPs from five different species of moths in the genus Choristoneura were sequenced, along with the PBP of Argyrotaenia velutinana, which serves as an outgroup. Three independent major pheromone changes are represented within this group of five Choristoneura species. Two different lineages show evidence for selection based on polymorphism and divergence comparisons and comparisons of rates of replacement evolution to silent and noncoding evolution. Along one of these lineages, leading to Choristoneura fumiferana, there has been a change to an aldehyde pheromone from an acetate pheromone. The second branch does not appear to be associated with a major pheromone change. Other branches in the tree show a trend toward greater replacement fixation than expected under neutrality. This trend could reflect undetected selective events within this group of PBPs. Selection appears to have acted to fix amino acid changes in the PBP of moths from the genus Choristoneura, but it is not clear that this selection is due to pheromone changes between species. PMID- 10742048 TI - The evolution of an alpha-esterase pseudogene inactivated in the Drosophila melanogaster lineage. AB - Previous analyses of the alpha-esterase cluster of Drosophila melanogaster revealed 10 active genes and the DmalphaE4a-Psi pseudogene. Here, we reconstruct the evolution of the pseudogene from the sequences of 12 alleles from widely scattered D. melanogaster populations and single alleles from Drosophila simulans and Drosophila yakuba. All of the DmalphaE4a-Psi alleles contain numerous inactivating mutations, suggesting that pseudogene alleles are fixed in natural populations. Several lines of evidence also suggest that DmalphaE4a is now evolving without selective constraint in the D. melanogaster lineage. There are three polymorphic indels which result in frameshifts; a key nucleotide of the intron splice acceptor is polymorphic; the neutral mutation parameter is the same for replacement and silent sites; one of the nonsilent polymorphisms results in a stop codon; only 1 of the 13 replacement polymorphisms is biochemically conservative; residues that are conserved among active esterases have different states in DmalphaE4a-Psi; and there are about half as many transitional polymorphisms as transversional ones. In contrast, the D. simulans and D. yakuba orthologs DsalphaE4a and DyalphaE4a do not have the inactivating mutations of DmalphaE4a-Psi and appear to be evolving under the purifying selection typical of protein- encoding genes. For instance, there have been more substitutions in the introns than in the exons, and more in silent sites than in replacement sites. Furthermore, most of the amino acid substitutions that have occurred between DyalphaE4a and DsalphaE4a are located in sites that typically vary among active alpha-esterases rather than those that are usually conserved. We argue that the original alphaE4a gene had a function which it has lost since the divergence of the D. melanogaster and D. simulans lineages. PMID- 10742049 TI - A nucleomorph-encoded CbbX and the phylogeny of RuBisCo regulators. AB - Chloroplasts contain proteins that are encoded by different genetic systems, the plastid genome and the nuclear chromosomes. By comparing the gene content of plastid genomes of different taxa, some predictions about nuclear-encoded genes for plastid proteins are possible. However, early in evolution, many genes were transferred from the plastid to the cell nucleus and are therefore missing from all known plastid genomes and escape such predictions. By sequencing the miniaturized chromosomes of the nucleomorph of the cryptophyte Guillardia theta, as well as the plastid genome, we uncovered two genes encoding CbbX which are predicted to be involved in plastid function. Our findings suggest that (1) red type plastid rbcLS genes evolved together with cbbX, which is related to cbbX genes of purple bacteria; (2) early in rhodoplast evolution, the cbbX gene was duplicated and transferred into the nucleus; (3) the plastid-encoded LysR transcriptional activator gene, rbcR, is homologous to rbcR and cbbR transcriptional activator genes of purple bacteria and cyanobacteria; and (4) the ancestral plastid probably harbored both types of form I RuBisCo. PMID- 10742050 TI - Molecular evolution and phylogenetic utility of Wolbachia ftsZ and wsp gene sequences with special reference to the origin of male-killing. AB - A detailed assessment of the evolution and phylogenetic utility of two genes, ftsZ and wsp, was used to investigate the origin of male-killing Wolbachia, previously isolated from the ladybird Adalia bipunctata and the butterfly Acraea encedon. The analysis included almost all available sequences of B-group Wolbachia and two outgroup taxa and showed that (1) the two gene regions differ in phylogenetic utility, (2) sequence variation is here correlated with phylogenetic information content, (3) both genes show significant rate heterogeneity between lineages, (4) increased substitution rates are associated with homoplasy in the data, (5) wsp sequences of some taxa appear to be subject to positive selection, and (6) only a limited number of clades can be inferred with confidence due to either lack of phylogenetic information or the presence of homoplasy. With respect to the evolution of male-killing, the two genes nevertheless seemed to provide unbiased information. However, they consistently produce contradictory results. Current data therefore do not permit clarification of the origin of this behavior. In addition, A. bipunctata was found to be a host to two recently diverged strains of male-killing Wolbachia that showed increased substitution rates for both genes. Moreover, the wsp gene, which codes for an outer membrane protein, was found to be subject to positive selection in these taxa. These findings were postulated to be the product of high selection pressures due to antagonistic host-symbiont interactions in this ladybird species. In conclusion, our study demonstrates that the results of a detailed phylogenetic analysis, including characterization of the limitations of such an approach, can serve as a valuable basis for an understanding of the evolution of Wolbachia bacteria. Moreover, particular features of gene evolution, such as elevated substitution rates or the presence of positive selection, may provide information about the dynamics of Wolbachia-host associations. PMID- 10742051 TI - Evolution of nucleotide substitutions and gene regulation in the amylase multigenes in Drosophila kikkawai and its sibling species. AB - In order to determine evolutionary changes in gene regulation and the nucleotide substitution pattern in a multigene family, the amylase multigenes were characterized in Drosophila kikkawai and its sibling species. The nucleotide substitution pattern was investigated. Drosophila kikkawai has four amylase genes. The Amy1 and Amy2 genes are a head-to-head duplication in the middle of the B arm of the second chromosome, while the Amy3 and Amy4 genes are a tail-to tail duplication near the centromere of the same chromosome. In the sibling species of D. kikkawai (Drosophila bocki, Drosophila leontia, and Drosophila lini), sequencing of the Amy1, Amy2, Amy3, and Amy4 genes revealed that the Amy1 and Amy2 gene group diverged from Amy3 and Amy4 after duplication. In the Amy1 and Amy2 genes, the divergent evolution occurred in the flanking regions; in contrast, the coding regions have evolved in concerted fashion. The electrophoretic pattern of AMY isozymes was also examined. In D. kikkawai and its siblings, two or three electrophoretically different isozymes are encoded by the Amy1 and Amy2 genes (S isozyme) and by the Amy3 and Amy4 genes (F (M) isozymes). The S and F (M) isozymes show different patterns of band intensity when larvae and flies were fed in different media. Amy1 and Amy2, which encode the S isozyme, are more strikingly regulated than Amy3 and Amy4, which encode the F (M) isozyme. The GC content and codon usage bias were higher for the Amy1 and Amy2 genes than for the Amy3 and Amy4 genes. Although the ratio of synonymous and replacement substitutions within the Amy1 and Amy2 gene group was not significantly different from that within the Amy3 and Amy4 gene group, the synonymous substitution rate in the lineage of Amy1 and Amy2 was lower than that of Amy3 and Amy4. In conclusion, after the first duplication but before speciation of four species, the synonymous substitution rate between the two lineages and the electrophoretic pattern of the isozymes encoded by them changed, although we do not know whether there was any evolutionary relationship between the two. PMID- 10742052 TI - LINE-1 (L1) lineages in the mouse. AB - Recently, a rapidly amplifying family of mouse LINE-1 (L1) has been identified and named T(F). The evolutionary context surrounding the derivation of the T(F) family was examined through phylogenetic analysis of sequences in the 3' portion of the repeat. The Mus musculus domesticus T(F) family was found to be the terminal subfamily of the previously identified L1Md4 lineage. The L1Md4 lineage joins the other prototypical mouse LINE-1 lineage (the L1MdA2 lineage) approximately 1 MYA at about the time of the common ancestor of M. m. domesticus, Mus spicilegus, and Mus spretus. However, the T(F) family from M. m. domesticus was found to join to the previously reported M. spretus Ms475 and Ms7024 LINE-1 families at just 0.5 MYA, indicating horizontal transfer. The T(F) family from M. m. domesticus was then found to be even more recently related to LINE-1's from another species, M. spicilegus. A separate spretus A2 lineage was found through a directed search of a PCR library. This lineage, in contrast to the spretus T(F) lineage, does join domesticus at about 1 MYA, as would be expected in the absence of horizontal transfer. A third major family was also found that splits off from the L1Md4 lineage shortly after its departure from the L1MdA2 lineage. The new family, named the Z family, was found to contain the de novo LINE-1 inserts causing the beige and med mutations. Whether the split with the Z family was before or after the recombination that introduced the F-type promoters and defined the inception of T(F) as a lineage is unclear. In enumerating copies of the various LINE-1 families, we found that T(F) 3' ends were not much more numerous than the reported number of 5' ends, suggesting that T(F) may not be subjected to the 90% truncation pattern typical of LINE-1 as a whole. PMID- 10742053 TI - Interkingdom host jumping underground: phylogenetic analysis of entomoparasitic fungi of the genus cordyceps. AB - Most members of the ascomycetous genus Cordyceps are endoparasitic fungi of insects and other arthropods, but about 20 of the 300 described species are parasitic to hart's truffles, Elaphomyces spp. In order to understand the evolution of host specificity and the process of interkingdom host jumping in Cordyceps, we investigated the phylogenetic relationships of 22 representatives, including 4 truffle parasites and 18 insect parasites, based on nuclear and mitochondrial rDNA sequences. Five monophyletic groups were identified in both nuclear and mitochondrial phylogenies. In three of the five clades, the members utilized hosts from the same insect group, suggesting that the endoparasite-host connections have been conserved to some extent. On the other hand, it was also shown that major host shifts between distantly related insects must have occurred repeatedly. Notably, phylogenetic analyses strongly suggested that parasites of hart's truffles originated from parasites of cicada nymphs during the evolution of the CORDYCEPS: The common habitats of cicada nymphs and hart's truffles, deep underground and associated with tree roots, suggest that the interkingdom host jumping from Animalia to Fungi might have been promoted by the overlapping ecological niche of the unrelated hosts. This finding provides an impressive case of a drastic host shift in favor of the host habitat hypothesis. PMID- 10742054 TI - Dramatic mitochondrial gene rearrangements in the hermit crab Pagurus longicarpus (Crustacea, anomura). AB - The entire mitochondrial gene order of the crustacean Pagurus longicarpus was determined by sequencing all but approximately 300 bp of the mitochondrial genome. We report the first major gene rearrangements found in the clade including Crustacea and Insecta. At least eight mitochondrial gene rearrangements have dramatically altered the gene order of the hermit crab P. longicarpus relative to the putatively ancestral crustacean gene order. These include two rearrangements of protein-coding genes, the first reported for any nonchelicerate arthropod. Codon usage and amino acid sequences do not deviate substantially from those reported for other crustaceans. Investigating the phylogenetic distribution of these eight rearrangements will add additional characters to help resolve decapod phylogeny. PMID- 10742055 TI - Contrasting patterns of nucleotide polymorphism at the alcohol dehydrogenase locus in the outcrossing Arabidopsis lyrata and the selfing Arabidopsis thaliana. AB - Nucleotide variation at the alcohol dehydrogenase locus (Adh) was studied in the outcrossing Arabidopsis lyrata, a close relative of the selfing Arabidopsis thaliana. Overall, estimated nucleotide diversity in the North American ssp. lyrata and two European ssp. petraea populations was 0.0038, lower than the corresponding specieswide estimate for A. thaliana at the same set of nucleotide sites. The distribution of segregating sites across the gene differed between the two species. Estimated sequence diversity within an A. lyrata population with a large sample size (0.0023) was much higher than has previously been observed for A. thaliana. This North American population has an excess of sites at intermediate frequencies compared with neutral expectation (Tajima's D = 2.3, P < 0.005), suggestive of linked balancing selection or a recent population bottleneck. In contrast, an excess of rare polymorphisms has been found in A. thaliana. Polymorphism within A. lyrata and divergence from A. thaliana appear to be correlated across the Adh gene sequence. The geographic distribution of polymorphism was quite different from that of A. thaliana, for which earlier studies of several genes found low within-population nucleotide site polymorphism and no overall continental differentiation of variation despite large differences in site frequencies between local populations. Differences between the outcrossing A. lyrata and the selfing A. thaliana reflect the impact of differences in mating system and the influence of bottlenecks in A. thaliana during rapid colonization on DNA sequence polymorphism. The influence of additional variability-reducing mechanisms, such as background selection or hitchhiking, may not be discernible. PMID- 10742056 TI - Selective constraints, amino acid composition, and the rate of protein evolution. AB - What are the major forces governing protein evolution? A common view is that proteins with strong structural and functional requirements evolve more slowly than proteins with weak constraints, because a stringent negative selection pressure limits the number of substitutions. In contrast, Graur claimed that the substitution rate of a protein is mainly determined by its amino acid composition and the changeabilities of amino acids. In this paper, however, we found that the relative changeabilities of amino acids in mammalian proteins are different for transmembranal and nontransmembranal segments, which have very distinct structural requirements. This indicates that the changeability of a given residue is influenced by the structural and functional context. We also reexamined the relationship between substitution rate and amino acid composition. Indeed, the two kinds of segments exhibit contrasting amino acid compositions: transmembranal regions are made up mainly of hydrophobic residues (a total frequency of approximately 60%) and are very poor in polar amino acids (<5%), whereas nontransmembranal segments have frequencies of 30% and 22%, respectively. Interestingly, we found that within a given integral membrane protein, nontransmembranal segments accumulate, on average, twice as many substitutions as transmembranal regions. However, regression analyses showed that the variability in amino acid frequencies among proteins cannot explain more than 30% of the variability in substitution rate for the transmembranal and nontransmembranal data sets. Furthermore, transmembranal and nontransmembranal segments evolving at the same rate in different proteins have different compositions, and the compositions of slowly evolving and rapidly evolving segments of the same type are similar. From these observations, we conclude that the rate of protein evolution is only weakly affected by amino acid composition but is mostly determined by the strength of functional requirements or selective constraints. PMID- 10742057 TI - Conservation within artiodactyls of an AATA interrupt in the IGF-I microsatellite for 19-35 million years. AB - Occurrence of an AATA interrupt in the IGF-I microsatellite was investigated in a number of Artiodactyl species, namely pigs, camels, deer, cattle, goats, and sheep. Comparison of DNA sequences in the 5' flank of the microsatellite in these species revealed that the interrupt within the microsatellite is conserved in deer, cattle, sheep, and goats but is absent from camels and pigs. The interrupt was introduced into the Artiodactyl phylogeny after the divergence of the Camelidae but before the divergence of the Cervidae, and thus its time of origin can be estimated to be 19-35 MYA. In contrast to the repeat units which are hypermutable, the interrupt has been conserved for a very long time and may even have suppressed microsatellite variation by inhibiting replication slippage. A 12 bp deletion in the 5' flank of the microsatellite in camels corresponds to a consensus reversed repeat in deer, cattle, sheep, and goats with unknown functional significance. Apart from this deletion, the 5' flank of the microsatellite is highly conserved in Artiodactyl species. PMID- 10742058 TI - Metaiodobenzylguanidine assessment of metastatic neuroblastoma: observer dependency and chemosensitivity evaluation. The SFOP Group. AB - BACKGROUND: In children over 1 year of age with metastatic neuroblastoma, clearance of metaiodobenzylguanidine (MIBG) skeletal uptake after four courses of induction chemotherapy is one of the most powerful prognostic factors. How subjective is quantification of MIBG uptake, and can earlier MIBG scintigraphy separate good and bad responders? PROCEDURE: The data from 47 patients who received uniform induction therapy were reviewed. A novel scoring system of MIBG update intensity was proposed. Initial, intermediate (after two courses), and final (after four courses) intensities were scored (0 to 21 points) independently by six different observers. The initial global score and the relative score (calculated by dividing the global score after two courses by the initial score) were compared to the final score. Good responders were those who scored 0 at final MIBG. RESULTS: Between two observers, the correlation coefficient for the global score was superior to 0.80, in nine of ten comparisons established between observers 1-5. The initial score did not predict the final score insofar as only nine of fourteen patients with low initial scores were good responders. The relative score also failed to predict outcome; only six of ten patients with favorable relative score (i.e., <20%), were good responders. CONCLUSIONS: This scoring system is reliable and may be used in multicentric trials. However, both initial and relative scores failed to predict final outcome. Thus, intermediate MIBG may be omitted during induction therapy assessment. PMID- 10742059 TI - Assessment of systemic toxicity in children receiving chemotherapy with cyclosporine for sarcoma. AB - BACKGROUND: Overexpression of P-glycoprotein in malignant tumors has been associated with poor responses to chemotherapy. It appears biologically plausible that addition of the P-glycoprotein inhibitor cyclosporine (CsA) to standard chemotherapy may improve the outcome. The protective functions of P-glycoprotein in healthy tissues, however, have not been fully elucidated. Addition of CsA may lead to increased systemic chemotherapy toxicity, so we compared the rate and severity of chemotherapy-associated systemic toxicity in the presence and absence of CsA. PROCEDURE: Standard chemotherapy consisted of etoposide/ifosfamide (VP16/IFOS) cycles, alternating with vincristine/dactinomycin/cyclophosphamide (VAC) cycles. CsA was given at a median dose of 20 mg/kg with unaltered doses of the antineoplastic drugs. The analysis of toxicity was performed by comparing clinically significant toxicity events recorded during and after chemotherapy cycles with and without CsA. RESULTS: Toxicity-related hospital admissions occurred after 93% of VAC cycles with CsA compared to 40% of the cycles without CsA (P < 0. 0001); 29% of VP16/IFOS cycles with CsA led to admissions vs. 12% with non-CsA cycles (P = 0.04). Infections or fever and neutropenia were the main reasons for these admissions. Thirty-seven percent of the VAC cycles with CsA were complicated by culture-proved sepsis, which did not occur in cycles without CsA (P < 0.0001). Requirements for blood and platelet transfusions were greatly increased after VAC cycles with CsA compared to VAC cycles without CsA. CONCLUSIONS: The chemosensitizer CsA increases the systemic toxicity of VAC chemotherapy in patients with sarcomas. Future trials of chemotherapy with chemosensitizers will have to take into account a potential increase in systemic toxicity. Careful monitoring of chemotherapy-related toxicity becomes mandatory in such studies. PMID- 10742060 TI - Congenital malformations and childhood cancer. AB - BACKGROUND: We investigated the epidemiology of congenital malformations and childhood cancer. PROCEDURE: By employing the cases of the Registry of Childhood Malignancies in Hokkaido Prefecture, Japan, from 1969 to 1996, the numbers of malignancies in cases (diagnosis at 0-14 years of age) with Down syndrome (DS), mental retardation (MR) excluding DS, luxatio coxae congenita (LCC), congenital heart disease (CHD) excluding DS, undescended testicle (UT), and cleft palate-lip (CPL) were calculated. Using the percentages of malignancies in the 2,349 cases without malformation, expected numbers of malignancies in the cases with the malformations were calculated. The observed numbers were statistically compared to expected ones. RESULTS: In the DS cases, leukemia developed with a significantly high frequency, but no UT cases developed leukemia. No brain tumor was preceded by DS. This could not be explained only by early death from coexisting diseases such as CHD, insofar as the CHD cases without DS developed brain tumors more frequently than expected. The ratio of acute lymphoblastic leukemia (ALL) to acute nonlymphoblastic leukemia (ANLL) was different among the malformations. The MR cases developed ANLL more frequently than expected, whereas the CPL cases developed ALL more frequently. The distribution of the age at diagnosis for Wilms' tumor was different according to the underlying malformation. CONCLUSIONS: Malformations might have some factors that inhibit or delay as well as promote the development of certain malignancies. PMID- 10742061 TI - New editor for statistical reviews PMID- 10742062 TI - Model for long-term follow-up of survivors of childhood cancer. PMID- 10742063 TI - Guidelines for integrated activity between pediatric hematology-oncology nurses and physicians. PMID- 10742064 TI - The importance of the pediatric oncologist-nurse partnership in the delivery of total care in pediatric oncology. PMID- 10742065 TI - Intrathecal analgesia for children with cancer via implanted infusion pumps. PMID- 10742066 TI - Care of terminally ill children with cancer. PMID- 10742067 TI - Regarding guidelines for assistance to terminally ill children with cancer: report of the SIOP working committee on psychosocial issues in pediatric oncology. PMID- 10742068 TI - Letter to the editor: care of terminally ill children with cancer PMID- 10742069 TI - Extramedullary relapse in a child with mixed lineage acute lymphoblastic leukemia: chylous pleuropericardial effusion. PMID- 10742070 TI - Double-blind crossover study of chlorpromazine and lorazepam in the treatment of behavioral problems during treatment of children with acute lymphoblastic leukaemia receiving glucocorticoids. PMID- 10742071 TI - Cutaneous side effects of medium dose methotrexate in children with acute lymphoblastic leukaemia. PMID- 10742072 TI - Meningeal leukemia with cerebrospinal fluid block. PMID- 10742073 TI - Pseudotumor cerebri induced by all-trans-retinoic acid in a child treated for acute promyelocytic leukemia. PMID- 10742074 TI - Gastrointestinal relapse of leukemia, mimicking acute graft vs. host disease, following a stem cell transplant. PMID- 10742075 TI - Thyroid carcinoma in a newborn: clinical challenges in managing the first recorded case. PMID- 10742076 TI - Cytogenetic abnormalities in a case of botryoid rhabdomyosarcoma. PMID- 10742077 TI - Dramatic response of adult wilms tumor to paclitaxel and cisplatin. PMID- 10742078 TI - Wilms tumor and autoimmune neutropenia. PMID- 10742079 TI - Severe hypercalcemia in association with a juvenile granulosa cell tumor of the ovary. PMID- 10742080 TI - Down syndrome and Hodgkin disease in childhood. PMID- 10742081 TI - Efficacy of cefepime and amikacin in the empiric treatment of febrile neutropenic children with cancer. PMID- 10742082 TI - Systemic inflammatory response syndrome associated with amifostine. PMID- 10742083 TI - Cyclo-oxygenase-2 in the osteolytic lesions of Langerhans cell histiocytosis. PMID- 10742084 TI - Repair affairs. PMID- 10742085 TI - Fly meets shotgun: shotgun wins. PMID- 10742086 TI - Oct-4, scene 1: the drama of mouse development. PMID- 10742087 TI - A hole in the head. PMID- 10742088 TI - Do all SINEs lead to LINEs? PMID- 10742089 TI - Getting a foothold in nephrotic syndrome. PMID- 10742090 TI - Touchingbase. PMID- 10742091 TI - A systematic, high-resolution linkage of the cytogenetic and physical maps of the human genome. PMID- 10742092 TI - ISIS, the intron information system, reveals the high frequency of alternative splicing in the human genome. PMID- 10742093 TI - MSX1 mutation is associated with orofacial clefting and tooth agenesis in humans. PMID- 10742094 TI - Mutations of SCN1A, encoding a neuronal sodium channel, in two families with GEFS+2. PMID- 10742095 TI - Dolly part one: the end of the beginning in mammalian cloning? PMID- 10742096 TI - NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome. AB - Familial idiopathic nephrotic syndromes represent a heterogeneous group of kidney disorders, and include autosomal recessive steroid-resistant nephrotic syndrome, which is characterized by early childhood onset of proteinuria, rapid progression to end-stage renal disease and focal segmental glomerulosclerosis. A causative gene for this disease, NPHS2, was mapped to 1q25-31 and we report here its identification by positional cloning. NPHS2 is almost exclusively expressed in the podocytes of fetal and mature kidney glomeruli, and encodes a new integral membrane protein, podocin, belonging to the stomatin protein family. We found ten different NPHS2 mutations, comprising nonsense, frameshift and missense mutations, to segregate with the disease, demonstrating a crucial role for podocin in the function of the glomerular filtration barrier. PMID- 10742097 TI - Robustness against mutations in genetic networks of yeast. AB - There are two principal mechanisms that are responsible for the ability of an organism's physiological and developmental processes to compensate for mutations. In the first, genes have overlapping functions, and loss-of-function mutations in one gene will have little phenotypic effect if there are one or more additional genes with similar functions. The second mechanism has its origin in interactions between genes with unrelated functions, and has been documented in metabolic and regulatory gene networks. Here I analyse, on a genome-wide scale, which of these mechanisms of robustness against mutations is more prevalent. I used functional genomics data from the yeast Saccharomyces cerevisiae to test hypotheses related to the following: if gene duplications are mostly responsible for robustness, then a correlation is expected between the similarity of two duplicated genes and the effect of mutations in one of these genes. My results demonstrate that interactions among unrelated genes are the major cause of robustness against mutations. This type of robustness is probably an evolved response of genetic networks to stabilizing selection. PMID- 10742098 TI - Human LINE retrotransposons generate processed pseudogenes. AB - Long interspersed elements (LINEs) are endogenous mobile genetic elements that have dispersed and accumulated in the genomes of higher eukaryotes via germline transposition, with up to 100,000 copies in mammalian genomes. In humans, LINEs are the major source of insertional mutagenesis, being involved in both germinal and somatic mutant phenotypes. Here we show that the human LINE retrotransposons, which transpose through the reverse transcription of their own transcript, can also mobilize transcribed DNA not associated with a LINE sequence by a process involving the diversion of the LINE enzymatic machinery by the corresponding mRNA transcripts. This results in the 'retroposition' of the transcribed gene and the formation of new copies that disclose features characteristic of the widespread and naturally occurring processed pseudogenes: loss of intron and promoter, acquisition of a poly(A) 3' end and presence of target-site duplications of varying length. We further show-by introducing deletions within either coding sequence of the human LINE-that both ORFs are necessary for the formation of the processed pseudogenes, and that retroviral-like elements are not able to produce similar structures in the same assay. Our results strengthen the unique versatility of LINEs as genome modellers. PMID- 10742099 TI - Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the pattern of DNA methylation. AB - A goal of molecular genetics is to understand the relationship between basic nuclear processes, epigenetic changes and the numerous proteins that orchestrate these effects. One such protein, ATRX, contains a highly conserved plant homeodomain (PHD)-like domain, present in many chromatin-associated proteins, and a carboxy-terminal domain which identifies it as a member of the SNF2 family of helicase/ATPases. Mutations in ATRX give rise to characteristic developmental abnormalities including severe mental retardation, facial dysmorphism, urogenital abnormalities and alpha-thalassaemia. This circumstantial evidence suggests that ATRX may act as a transcriptional regulator through an effect on chromatin. We have recently shown that ATRX is localized to pericentromeric heterochromatin during interphase and mitosis, suggesting that ATRX might exert other chromatin mediated effects in the nucleus. Moreover, at metaphase, some ATRX is localized at or close to the ribosomal DNA (rDNA) arrays on the short arms of human acrocentric chromosomes. Here we show that mutations in ATRX give rise to changes in the pattern of methylation of several highly repeated sequences including the rDNA arrays, a Y-specific satellite and subtelomeric repeats. Our findings provide a potential link between the processes of chromatin remodelling, DNA methylation and gene expression in mammalian development. PMID- 10742100 TI - Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. AB - Cell fate during development is defined by transcription factors that act as molecular switches to activate or repress specific gene expression programmes. The POU transcription factor Oct-3/4 (encoded by Pou5f1) is a candidate regulator in pluripotent and germline cells and is essential for the initial formation of a pluripotent founder cell population in the mammalian embryo. Here we use conditional expression and repression in embryonic stem (ES) cells to determine requirements for Oct-3/4 in the maintenance of developmental potency. Although transcriptional determination has usually been considered as a binary on-off control system, we found that the precise level of Oct-3/4 governs three distinct fates of ES cells. A less than twofold increase in expression causes differentiation into primitive endoderm and mesoderm. In contrast, repression of Oct-3/4 induces loss of pluripotency and dedifferentiation to trophectoderm. Thus a critical amount of Oct-3/4 is required to sustain stem-cell self-renewal, and up- or downregulation induce divergent developmental programmes. Our findings establish a role for Oct-3/4 as a master regulator of pluripotency that controls lineage commitment and illustrate the sophistication of critical transcriptional regulators and the consequent importance of quantitative analyses. PMID- 10742101 TI - In vivo modulation of Hmgic reduces obesity. AB - The HMGI family of proteins consists of three members, HMGIC, HMGI and HMGI(Y), that function as architectural factors and are essential components of the enhancesome. HMGIC is predominantly expressed in proliferating, undifferentiated mesenchymal cells and is not detected in adult tissues. It is disrupted and misexpressed in a number of mesenchymal tumour cell types, including fat-cell tumours (lipomas). In addition Hmgic-/- mice have a deficiency in fat tissue. To study its role in adipogenesis and obesity, we examined Hmgic expression in the adipose tissue of adult, obese mice. Mice with a partial or complete deficiency of Hmgic resisted diet-induced obesity. Disruption of Hmgic caused a reduction in the obesity induced by leptin deficiency (Lepob/Lepob) in a gene-dose-dependent manner. Our studies implicate a role for HMGIC in fat-cell proliferation, indicating that it may be an adipose-specific target for the treatment of obesity. PMID- 10742102 TI - Large-scale discovery and genotyping of single-nucleotide polymorphisms in the mouse. AB - Single-nucleotide polymorphisms (SNPs) have been the focus of much attention in human genetics because they are extremely abundant and well-suited for automated large-scale genotyping. Human SNPs, however, are less informative than other types of genetic markers (such as simple-sequence length polymorphisms or microsatellites) and thus more loci are required for mapping traits. SNPs offer similar advantages for experimental genetic organisms such as the mouse, but they entail no loss of informativeness because bi-allelic markers are fully informative in analysing crosses between inbred strains. Here we report a large scale analysis of SNPs in the mouse genome. We characterized the rate of nucleotide polymorphism in eight mouse strains and identified a collection of 2,848 SNPs located in 1,755 sequence-tagged sites (STSs) using high-density oligonucleotide arrays. Three-quarters of these SNPs have been mapped on the mouse genome, providing a first-generation SNP map of the mouse. We have also developed a multiplex genotyping procedure by which a genome scan can be performed with only six genotyping reactions per animal. PMID- 10742103 TI - Functional haploinsufficiency of the human homeobox gene MSX2 causes defects in skull ossification. AB - The genetic analysis of congenital skull malformations provides insight into normal mechanisms of calvarial osteogenesis. Enlarged parietal foramina (PFM) are oval defects of the parietal bones caused by deficient ossification around the parietal notch, which is normally obliterated during the fifth fetal month. PFM are usually asymptomatic, but may be associated with headache, scalp defects and structural or vascular malformations of the brain. Inheritance is frequently autosomal dominant, but no causative mutations have been identified in non syndromic cases. We describe here heterozygous mutations of the homeobox gene MSX2 (located on 5q34-q35) in three unrelated families with PFM. One is a deletion of approximately 206 kb including the entire gene and the others are intragenic mutations of the DNA-binding homeodomain (RK159-160del and R172H) that predict disruption of critical intramolecular and DNA contacts. Mouse Msx2 protein with either of the homeodomain mutations exhibited more than 85% reduction in binding to an optimal Msx2 DNA-binding site. Our findings contrast with the only described MSX2 homeodomain mutation (P148H), associated with craniosynostosis, that binds with enhanced affinity to the same target. This demonstrates that MSX2 dosage is critical for human skull development and suggests that PFM and craniosynostosis result, respectively, from loss and gain of activity in an MSX2-mediated pathway of calvarial osteogenic differentiation. PMID- 10742104 TI - Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. AB - The composite structure of the mammalian skull, which forms predominantly via intramembranous ossification, requires precise pre- and post-natal growth regulation of individual calvarial elements. Disturbances of this process frequently cause severe clinical manifestations in humans. Enhanced DNA binding by a mutant MSX2 homeodomain results in a gain of function and produces craniosynostosis in humans. Here we show that Msx2-deficient mice have defects of skull ossification and persistent calvarial foramen. This phenotype results from defective proliferation of osteoprogenitors at the osteogenic front during calvarial morphogenesis, and closely resembles that associated with human MSX2 haploinsufficiency in parietal foramina (PFM). Msx2-/- mice also have defects in endochondral bone formation. In the axial and appendicular skeleton, post-natal deficits in Pth/Pthrp receptor (Pthr) signalling and in expression of marker genes for bone differentiation indicate that Msx2 is required for both chondrogenesis and osteogenesis. Consistent with phenotypes associated with PFM, Msx2-mutant mice also display defective tooth, hair follicle and mammary gland development, and seizures, the latter accompanied by abnormal development of the cerebellum. Most Msx2-mutant phenotypes, including calvarial defects, are enhanced by genetic combination with Msx1 loss of function, indicating that Msx gene dosage can modify expression of the PFM phenotype. Our results provide a developmental basis for PFM and demonstrate that Msx2 is essential at multiple sites during organogenesis. PMID- 10742105 TI - The direction of microsatellite mutations is dependent upon allele length. AB - Microsatellites, comprising tandemly repeated short nucleotide sequences, are ubiquitous in eukaryotic genomes. Mutations within microsatellites are frequent, altering their overall length by insertion or deletion of a small number of repeat units, with a rate as high as 10(-3) in humans. Despite their high mutability, stable allele frequency distributions are typically observed for microsatellites in humans as well as other primates, although the mechanism maintaining these stable distributions remains unclear. Previous studies have suggested that microsatellite mutations occur more frequently in longer alleles and favour expansion. Generalizing these results has been hindered because the sample sizes were small, only a small subset of alleles for any marker was studied and the direction of mutation (expansion or contraction) was not rigorously determined. Here we examine 236 mutations at 122 tetranucleotide repeat markers and find that the rate of contraction mutations increases exponentially with allele size, whereas the rate of expansion mutations is constant across the entire allele distribution. The overall rate of expansion mutations does not differ from that of contractions. Our findings offer an explanation for the stationary allele distribution of microsatellites. PMID- 10742106 TI - Heterogeneous mutation processes in human microsatellite DNA sequences. AB - Although microsatellite polymorphisms are one of the most commonly used tools in genetic analyses, it remains to be understood how microsatellite DNA has evolved as a ubiquitous and highly abundant class of repetitive sequences in eukaryotic genomes. On the basis of analyses of spontaneous human microsatellite mutations of germline origin, I show here that different mutation biases underlie the evolution of microsatellite repeats. The within-locus mutation rate increases with allele length, but is not affected by the size difference between an individual's two alleles (allele span). Within loci, long alleles tend to mutate to shorter lengths, thereby acting to prevent infinite growth. Expansions are more common than contractions among dinucleotide repeats, whereas no such trend is evident among tetranucleotide repeats. This observation is consistent with the longer repeat lengths and higher frequency of di- compared with tetranucleotide repeats. An excess of paternally transmitted mutations (male-to-female ratio of 4.9) supports a male-biased mutation rate in the human genome. PMID- 10742107 TI - Abnormal adaptations to stress and impaired cardiovascular function in mice lacking corticotropin-releasing hormone receptor-2. AB - The actions of corticotropin-releasing hormone (Crh), a mediator of endocrine and behavioural responses to stress, and the related hormone urocortin (Ucn) are coordinated by two receptors, Crhr1 (encoded by Crhr) and Crhr2. These receptors may exhibit distinct functions due to unique tissue distribution and pharmacology. Crhr-null mice have defined central functions for Crhr1 in anxiety and neuroendocrine stress responses. Here we generate Crhr2-/- mice and show that Crhr2 supplies regulatory features to the hypothalamic-pituitary-adrenal axis (HPA) stress response. Although initiation of the stress response appears to be normal, Crhr2-/- mice show early termination of adrenocorticotropic hormone (Acth) release, suggesting that Crhr2 is involved in maintaining HPA drive. Crhr2 also appears to modify the recovery phase of the HPA response, as corticosterone levels remain elevated 90 minutes after stress in Crhr2-/- mice. In addition, stress-coping behaviours associated with dearousal are reduced in Crhr2-/- mice. We also demonstrate that Crhr2 is essential for sustained feeding suppression (hypophagia) induced by Ucn. Feeding is initially suppressed in Crhr2-/- mice following Ucn, but Crhr2-/- mice recover more rapidly and completely than do wild type mice. In addition to central nervous system effects, we found that, in contrast to wild-type mice, Crhr2-/- mice fail to show the enhanced cardiac performance or reduced blood pressure associated with systemic Ucn, suggesting that Crhr2 mediates these peripheral haemodynamic effects. Moreover, Crhr2-/- mice have elevated basal blood pressure, demonstrating that Crhr2 participates in cardiovascular homeostasis. Our results identify specific responses in the brain and periphery that involve Crhr2. PMID- 10742108 TI - Mice deficient for corticotropin-releasing hormone receptor-2 display anxiety like behaviour and are hypersensitive to stress. AB - Corticotropin-releasing hormone (Crh) is a critical coordinator of the hypothalamic-pituitary-adrenal (HPA) axis. In response to stress, Crh released from the paraventricular nucleus (PVN) of the hypothalamus activates Crh receptors on anterior pituitary corticotropes, resulting in release of adrenocorticotropic hormone (Acth) into the bloodstream. Acth in turn activates Acth receptors in the adrenal cortex to increase synthesis and release of glucocorticoids. The receptors for Crh, Crhr1 and Crhr2, are found throughout the central nervous system and periphery. Crh has a higher affinity for Crhr1 than for Crhr2, and urocortin (Ucn), a Crh-related peptide, is thought to be the endogenous ligand for Crhr2 because it binds with almost 40-fold higher affinity than does Crh. Crhr1 and Crhr2 share approximately 71% amino acid sequence similarity and are distinct in their localization within the brain and peripheral tissues. We generated mice deficient for Crhr2 to determine the physiological role of this receptor. Crhr2-mutant mice are hypersensitive to stress and display increased anxiety-like behaviour. Mutant mice have normal basal feeding and weight gain, but decreased food intake following food deprivation. Intravenous Ucn produces no effect on mean arterial pressure in the mutant mice. PMID- 10742109 TI - Deletion of crhr2 reveals an anxiolytic role for corticotropin-releasing hormone receptor-2. AB - Corticotropin-releasing hormone (Crh), a 41-residue polypeptide, activates two G protein-coupled receptors, Crhr1 and Crhr2, causing (among other transductional events) phosphorylation of the transcription factor Creb. The physiologic role of these receptors is only partially understood. Here we report that male, but not female, Crhr2-deficient mice exhibit enhanced anxious behaviour in several tests of anxiety in contrast to mice lacking Crhr1. The enhanced anxiety of Crhr2 deficient mice is not due to changes in hypothalamic-pituitary-adrenal (HPA) axis activity, but rather reflects impaired responses in specific brain regions involved in emotional and autonomic function, as monitored by a reduction of Creb phosphorylation in male, but not female, Crhr2-/- mice. We propose that Crhr2 predominantly mediates a central anxiolytic response, opposing the general anxiogenic effect of Crh mediated by Crhr1. Neither male nor female Crhr2 deficient mice show alterations of baseline feeding behaviour. Both respond with increased edema formation in response to thermal exposure, however, indicating that in contrast to its central role in anxiety, the peripheral role of Crhr2 in vascular permeability is independent of gender. PMID- 10742110 TI - The SH2 tyrosine phosphatase shp2 is required for mammalian limb development. AB - The tyrosine phosphatase Shp2 is recruited into tyrosine-kinase signalling pathways through binding of its two amino-terminal SH2 domains to specific phosphotyrosine motifs, concurrent with its re-localization and stimulation of phosphatase activity. Shp2 can potentiate signalling through the MAP-kinase pathway and is required during early mouse development for gastrulation. Chimaeric analysis can identify, by study of phenotypically normal embryos, tissues that tolerate mutant cells (and therefore do not require the mutated gene) or lack mutant cells (and presumably require the mutated gene during their developmental history). We therefore generated chimaeric mouse embryos to explore the cellular requirements for Shp2. This analysis revealed an obligatory role for Shp2 during outgrowth of the limb. Shp2 is specifically required in mesenchyme cells of the progress zone (PZ), directly beneath the distal ectoderm of the limb bud. Comparison of Ptpn11 (encoding Shp2)-mutant and Fgfr1 (encoding fibroblast growth factor receptor-1)-mutant chimaeric limbs indicated that in both cases mutant cells fail to contribute to the PZ of phenotypically normal chimaeras, leading to the hypothesis that a signal transduction pathway, initiated by Fgfr1 and acting through Shp2, is essential within PZ cells. Rather than integrating proliferative signals, Shp2 probably exerts its effects on limb development by influencing cell shape, movement or adhesion. Furthermore, the branchial arches, which also use Fgfs during bud outgrowth, similarly require Shp2. Thus, Shp2 regulates phosphotyrosine-signalling events during the complex ectodermal mesenchymal interactions that regulate mammalian budding morphogenesis. PMID- 10742111 TI - Stereocilia defects in the sensory hair cells of the inner ear in mice deficient in integrin alpha8beta1. AB - The mammalian inner ear contains organs for the detection of sound and acceleration, the cochlea and the vestibule, respectively. Mechanosensory hair cells within the neuroepithelia of these organs transduce mechanical force generated by sound waves or head movements into neuronal signals. Defects in hair cells lead to deafness and balance defects. Hair cells have stereocilia that are indispensable for mechanosensation, but the molecular mechanisms regulating stereocilia formation are poorly understood. We show here that integrin alpha8beta1, its ligand fibronectin and the integrin-regulated focal adhesion kinase (FAK) co-localize to the apical hair-cell surface where stereocilia are forming. In mice homozygous for a targeted mutation of Itga8 (encoding the alphabeta8 subunit), this co-localization is perturbed and hair cells in the utricle, a vestibular subcompartment, lack stereocilia or contain malformed stereocilia. Most integrin alpha-8beta1-deficient mice die soon after birth due to kidney defects. Many of the survivors have difficulty balancing, consistent with the structural defects of the inner ear. Our data suggest that integrin alpha8beta1, and potentially other integrins, regulates hair-cell differentiation and stereocilia maturation. Mutations affecting matrix molecules cause inherited forms of inner ear disease and integrins may mediate some effects of matrix molecules in the ear; thus, mutations in integrin genes may lead to inner-ear diseases as well. PMID- 10742112 TI - The homeobox gene mirror links EGF signalling to embryonic dorso-ventral axis formation through notch activation. AB - Recent studies in vertebrates and Drosophila melanogaster have revealed that Fringe-mediated activation of the Notch pathway has a role in patterning cell layers during organogenesis. In these processes, a homeobox-containing transcription factor is responsible for spatially regulating fringe (fng) expression and thus directing activation of the Notch pathway along the fng expression border. Here we show that this may be a general mechanism for patterning epithelial cell layers. At three stages in Drosophila oogenesis, mirror (mirr) and fng have complementary expression patterns in the follicle-cell epithelial layer, and at all three stages loss of mirr enlarges, and ectopic expression of mirr restricts, fng expression, with consequences for follicle-cell patterning. These morphological changes are similar to those caused by Notch mutations. Ectopic expression of mirr in the posterior follicle cells induces a stripe of rhomboid (rho) expression and represses pipe (pip), a gene with a role in the establishment of the dorsal-ventral axis, at a distance. Ectopic Notch activation has a similar long-range effect on pip. Our results suggest that Mirror and Notch induce secretion of diffusible morphogens and we have identified TGF-beta (encoded by dpp) as such a molecule in germarium. We also found that mirr expression in dorsal follicle cells is induced by the EGF-receptor (EGFR) pathway and that mirr then represses pip expression in all but the ventral follicle cells, connecting EGFR activation in the dorsal follicle cells to repression of pip in the dorsal and lateral follicle cells. Our results suggest that the differentiation of ventral follicle cells is not a direct consequence of germline signalling, but depends on long-range signals from dorsal follicle cells, and provide a link between early and late events in Drosophila embryonic dorsal-ventral axis formation. PMID- 10742113 TI - Mutations in Sox18 underlie cardiovascular and hair follicle defects in ragged mice. AB - Analysis of classical mouse mutations has been useful in the identification and study of many genes. We previously mapped Sox18, encoding an SRY-related transcription factor, to distal mouse chromosome 2. This region contains a known mouse mutation, ragged (Ra), that affects the coat and vasculature. Here we have directly evaluated Sox18 as a candidate for Ra. We found that Sox18 is expressed in the developing vascular endothelium and hair follicles in mouse embryos. Furthermore, we found no recombination between Sox18 and Ra in an interspecific backcross segregating for the Ra phenotype. We found point mutations in Sox18 in two different Ra alleles that result in missense translation and premature truncation of the encoded protein. Fusion proteins containing these mutations lack the ability to activate transcription relative to wild-type controls in an in vitro assay. Our observations implicate mutations in Sox18 as the underlying cause of the Ra phenotype, and identify Sox18 as a critical gene for cardiovascular and hair follicle formation. PMID- 10742114 TI - Mutations in the human delta homologue, DLL3, cause axial skeletal defects in spondylocostal dysostosis. AB - Spondylocostal dysostosis (SD, MIM 277300) is a group of vertebral malsegmentation syndromes with reduced stature resulting from axial skeletal defects. SD is characterized by multiple hemivertebrae, rib fusions and deletions with a non-progressive kyphoscoliosis. Cases may be sporadic or familial, with both autosomal dominant and autosomal recessive modes of inheritance reported. Autosomal recessive SD maps to a 7.8-cM interval on chromosome 19q13.1-q13.3 that is homologous with a mouse region containing a gene encoding the Notch ligand delta-like 3 (Dll3). Dll3 is mutated in the X-ray-induced mouse mutant pudgy (pu), causing a variety of vertebrocostal defects similar to SD phenotypes. Here we have cloned and sequenced human DLL3 to evaluate it as a candidate gene for SD and identified mutations in three autosomal recessive SD families. Two of the mutations predict truncations within conserved extracellular domains. The third is a missense mutation in a highly conserved glycine residue of the fifth epidermal growth factor (EGF) repeat, which has revealed an important functional role for this domain. These represent the first mutations in a human Delta homologue, thus highlighting the critical role of the Notch signalling pathway and its components in patterning the mammalian axial PMID- 10742115 TI - Is the UK ready for an NCI? PMID- 10742116 TI - What is a functional recovery after spinal cord injury? PMID- 10742117 TI - Global cooperation in higher education: 'supercourse'. PMID- 10742118 TI - Reply to "What is a functional recovery after spinal cord injury?" PMID- 10742120 TI - Partial UK genetic database planned. PMID- 10742119 TI - UCSF whistle-blower alleges gender discrimination. PMID- 10742121 TI - Parliamentary committee examines UK cancer research efforts. PMID- 10742122 TI - Investigation into CDC employee's industrial move. PMID- 10742123 TI - Gene patent revisions to remove some controversies. PMID- 10742124 TI - Record gift adds another US neuroscience center. PMID- 10742125 TI - UNAIDS releases long-awaited HIV vaccine guidelines. PMID- 10742126 TI - GMO controversy has adverse effects on UK research PMID- 10742127 TI - Hate-mail author trapped by DNA. PMID- 10742129 TI - Survey shows secrecy among scientists PMID- 10742128 TI - $1 million study renews HIV/transplant research. PMID- 10742130 TI - W.D. Hamilton, 1936-2000 PMID- 10742132 TI - Cancer goes prime time PMID- 10742133 TI - Defending the cavewoman and other tales of evolutionary neurology PMID- 10742134 TI - Virus: the Co-discoverer of HIV tracks its rampage and charts the future PMID- 10742131 TI - Can neural stem cells be used as therapeutic vehicles in the treatment of brain tumors? PMID- 10742135 TI - Monoclonal antibody therapies-a 'constant' threat to cancer. PMID- 10742136 TI - De-mystifying the mechanism(s) of maspin. PMID- 10742137 TI - Linking Helicobacter pylori to gastric cancer. PMID- 10742138 TI - Will therapeutic peptides be kryptonite for superantigens? PMID- 10742139 TI - Keeping in touch with angiogenesis. PMID- 10742140 TI - Varicella-zoster vaccine: the bad news may be good. PMID- 10742141 TI - Sniffing out new approaches to spinal cord repair. PMID- 10742142 TI - Neuroprotective autoimmunity-a double-edged sword? PMID- 10742143 TI - Caspase cleavage of APP results in a cytotoxic proteolytic peptide. PMID- 10742144 TI - Research news PMID- 10742145 TI - Mechanisms of angiogenesis and arteriogenesis. AB - Endothelial and smooth muscle cells interact with each other to form new blood vessels. In this review, the cellular and molecular mechanisms underlying the formation of endothelium-lined channels (angiogenesis) and their maturation via recruitment of smooth muscle cells (arteriogenesis) during physiological and pathological conditions are summarized, alongside with possible therapeutic applications. PMID- 10742146 TI - A second cytotoxic proteolytic peptide derived from amyloid beta-protein precursor. AB - The amyloid beta-protein precursor gives rise to the amyloid beta-protein, the principal constituent of senile plaques and a cytotoxic fragment involved in the pathogenesis of Alzheimer disease. Here we show that amyloid beta-protein precursor was proteolytically cleaved by caspases in the C terminus to generate a second unrelated peptide, called C31. The resultant C31 peptide was a potent inducer of apoptosis. Both caspase-cleaved amyloid beta-protein precursor and activated caspase-9 were present in brains of Alzheimer disease patients but not in control brains. These findings indicate the possibility that caspase cleavage of amyloid beta-protein precursor with the generation of C31 may be involved in the neuronal death associated with Alzheimer disease. PMID- 10742147 TI - Favorable effect of VEGF gene transfer on ischemic peripheral neuropathy. AB - Ischemic peripheral neuropathy is a frequent, irreversible complication of lower extremity vascular insufficiency. We investigated whether ischemic peripheral neuropathy could be prevented and/or reversed by gene transfer of an endothelial cell mitogen designed to promote therapeutic angiogenesis. Intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor (VEGF) simultaneously with induction of hindlimb ischemia in rabbits abrogated the substantial decrease in motor and sensory nerve parameters, and nerve function recovered promptly. When gene transfer was administered 10 days after induction of ischemia, nerve function was restored earlier and/or recovered faster than in untreated rabbits. These findings are due in part to enhanced hindlimb perfusion. In addition, however, the demonstration of functional VEGF receptor expression by Schwann cells indicates a direct effect of VEGF on neural integrity as well. These findings thus constitute a new paradigm for the treatment of ischemic peripheral neuropathy. PMID- 10742148 TI - Superantigen antagonist protects against lethal shock and defines a new domain for T-cell activation. AB - Superantigens trigger an excessive cellular immune response, leading to toxic shock. We have designed a peptide antagonist that inhibits superantigen-induced expression of human genes for interleukin-2, gamma interferon and tumor necrosis factor-b, which are cytokines that mediate shock. The peptide shows homology to a b-strand-hinge-a-helix domain that is structurally conserved in superantigens, yet is remote from known binding sites for the major histocompatibility class II molecule and T-cell receptor. Superantigens depend on this domain for T-cell activation. The peptide protected mice against lethal challenge with staphylococcal and streptococcal superantigens. Moreover, it rescued mice undergoing toxic shock. Surviving mice rapidly developed protective antibodies against superantigen that rendered them resistant to further lethal challenges, even with different superantigens. Thus, the lethal effect of superantigens can be blocked with a peptide antagonist that inhibits their action at the beginning of the toxicity cascade, before activation of T cells takes place. PMID- 10742149 TI - Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. AB - The stress-inducible protein heme oxygenase-1 provides protection against oxidative stress. The anti-inflammatory properties of heme oxygenase-1 may serve as a basis for this cytoprotection. We demonstrate here that carbon monoxide, a by-product of heme catabolism by heme oxygenase, mediates potent anti inflammatory effects. Both in vivo and in vitro, carbon monoxide at low concentrations differentially and selectively inhibited the expression of lipopolysaccharide-induced pro-inflammatory cytokines tumor necrosis factor alpha, interleukin-1beta, and macrophage inflammatory protein-1beta and increased the lipopolysaccharide-induced expression of the anti-inflammatory cytokine interleukin-10. Carbon monoxide mediated these anti-inflammatory effects not through a guanylyl cyclase-cGMP or nitric oxide pathway, but instead through a pathway involving the mitogen-activated protein kinases. These data indicate the possibility that carbon monoxide may have an important protective function in inflammatory disease states and thus has potential therapeutic uses. PMID- 10742150 TI - The tyrosine kinase p56lck is essential in coxsackievirus B3-mediated heart disease. AB - Infections are thought to be important in the pathogenesis of many heart diseases. Coxsackievirus B3 (CVB3) has been linked to chronic dilated cardiomyopathy, a common cause of progressive heart disease, heart failure and sudden death. We show here that the sarcoma (Src) family kinase Lck (p56lck) is required for efficient CVB3 replication in T-cell lines and for viral replication and persistence in vivo. Whereas infection of wild-type mice with human pathogenic CVB3 caused acute and very severe myocarditis, meningitis, hepatitis, pancreatitis and dilated cardiomyopathy, mice lacking the p56lck gene were completely protected from CVB3-induced acute pathogenicity and chronic heart disease. These data identify a previously unknown function of Src family kinases and indicate that p56lck is the essential host factor that controls the replication and pathogenicity of CVB3. PMID- 10742151 TI - HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine. AB - Here, we demonstrate a previously unknown function for the 70-kDa heat-shock protein (HSP70) as a cytokine. HSP70 bound with high affinity to the plasma membrane, elicited a rapid intracellular calcium flux, activated nuclear factor (NF)-kappaB and upregulated the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in human monocytes. Furthermore, two different signal transduction pathways were activated by exogenous HSP70: one dependent on CD14 and intracellular calcium, which resulted in increased IL-1beta, IL-6 and TNF-alpha; and the other independent of CD14 but dependent on intracellular calcium, which resulted in an increase in TNF-alpha but not IL-1beta or IL-6. These findings indicate that CD14 is a co-receptor for HSP70-mediated signaling in human monocytes and are indicative of an previously unrecognized function for HSP70 as an extracellular protein with regulatory effects on human monocytes, having a dual role as chaperone and cytokine. PMID- 10742152 TI - Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. AB - Inhibitory receptors have been proposed to modulate the in vivo cytotoxic response against tumor targets for both spontaneous and antibody-dependent pathways. Using a variety of syngenic and xenograft models, we demonstrate here that the inhibitory FcgammaRIIB molecule is a potent regulator of antibody dependent cell-mediated cytotoxicity in vivo, modulating the activity of FcgammaRIII on effector cells. Although many mechanisms have been proposed to account for the anti-tumor activities of therapeutic antibodies, including extended half-life, blockade of signaling pathways, activation of apoptosis and effector-cell-mediated cytotoxicity, we show here that engagement of Fcgamma receptors on effector cells is a dominant component of the in vivo activity of antibodies against tumors. Mouse monoclonal antibodies, as well as the humanized, clinically effective therapeutic agents trastuzumab (Herceptin(R)) and rituximab (Rituxan(R)), engaged both activation (FcgammaRIII) and inhibitory (FcgammaRIIB) antibody receptors on myeloid cells, thus modulating their cytotoxic potential. Mice deficient in FcgammaRIIB showed much more antibody-dependent cell-mediated cytotoxicity; in contrast, mice deficient in activating Fc receptors as well as antibodies engineered to disrupt Fc binding to those receptors were unable to arrest tumor growth in vivo. These results demonstrate that Fc-receptor-dependent mechanisms contribute substantially to the action of cytotoxic antibodies against tumors and indicate that an optimal antibody against tumors would bind preferentially to activation Fc receptors and minimally to the inhibitory partner FcgammaRIIB. PMID- 10742153 TI - Gene therapy of experimental brain tumors using neural progenitor cells. AB - Glioblastomas, the most frequent and malignant of primary brain tumors, have a very poor prognosis. Gene therapy of glioblastomas is limited by the short survival of viral vectors and by their difficulty in reaching glioblastoma cells infiltrating the brain parenchyma. Neural stem/progenitor cells can be engineered to produce therapeutic molecules and have the potential to overcome these limitations because they may travel along the white matter, like neoplastic cells, and engraft stably into the brain. Retrovirus-mediated transfer of the gene for interleukin-4 is an effective treatment for rat brain glioblastomas. Here, we transferred the gene for interleukin-4 into C57BL6J mouse primary neural progenitor cells and injected those cells into established syngeneic brain glioblastomas. This led to the survival of most tumor-bearing mice. We obtained similar results by implanting immortalized neural progenitor cells derived from Sprague-Dawley rats into C6 glioblastomas. We also documented by magnetic resonance imaging the progressive disappearance of large tumors, and detected 5 bromodeoxyuridine-labeled progenitor cells several weeks after the injection. These findings support a new approach for gene therapy of brain tumors, based on the grafting of neural stem cells producing therapeutic molecules. PMID- 10742154 TI - Varicella vaccination: evidence for frequent reactivation of the vaccine strain in healthy children. AB - Wild-type varicella zoster virus (VZV) causes chickenpox, a common childhood illness characterized by fever and a vesicular rash and rare serious complications. Wild-type VZV persists in a latent form in the sensory ganglia, and can re-activate to cause herpes zoster. More than 10 million American children have received the live attenuated Oka strain VZV vaccine (OkaVZV) since its licensure in 1995. Pre-licensure clinical studies showed that mean serum anti VZV levels among vaccinees continued to increase with time after vaccination. This was attributed to immunologic boosting caused by exposure to wild-type VZV in the community. Here, we examine the alternative, that large-scale asymptomatic reactivation of OkaVZV might occur in vaccinees. We analyzed serum antibody levels and infection rates for 4 years of follow-up in 4,631 children immunized with OkaVZV. Anti-VZV titers decreased over time in high-responder subjects, but rose in vaccinees with low titers. Among subjects with low anti-VZV titers, the frequency of clinical infection and immunological boosting substantially exceeded the 13%-per-year rate of exposure to wild-type varicella. These findings indicate that OkaVZV persisted in vivo and reactivated as serum antibody titers decreased after vaccination. This has salient consequences for individuals immunized with OkaVZV. PMID- 10742155 TI - New multi-determinant strategy for a group A streptococcal vaccine designed for the Australian Aboriginal population. AB - Infection with group A streptococci can result in acute and post-infectious pathology, including rheumatic fever and rheumatic heart disease. These diseases are associated with poverty and are increasing in incidence, particularly in developing countries and amongst indigenous populations, such as Australia's Aboriginal population, who suffer the highest incidence worldwide. Immunity to group A streptococci is mediated by antibodies against the M protein, a coiled coil alpha helical surface protein of the bacterium. Vaccine development faces two substantial obstacles. Although opsonic antibodies directed against the N terminus of the protein are mostly responsible for serotypic immunity, more than 100 serotypes exist. Furthermore, whereas the pathogenesis of rheumatic fever is not well understood, increasing evidence indicates an autoimmune process. To develop a suitable vaccine candidate, we first identified a minimum, helical, non host-cross-reactive peptide from the conserved C-terminal half of the protein and displayed this within a non-M-protein peptide sequence designed to maintain helical folding and antigenicity, J14 (refs. 8,9). As this region of the M protein is identical in only 70% of group A streptococci isolates, the optimal candidate might consist of the conserved determinant with common N-terminal sequences found in communities with endemic group A streptococci. We linked seven serotypic peptides with J14 using a new chemistry technique that enables the immunogen to display all the individual peptides pendant from an alkane backbone. This construct demonstrated excellent immunogenicity and protection in mice. PMID- 10742156 TI - Angiopoietin-1 protects the adult vasculature against plasma leakage. AB - Pathological increases in vascular leakage lead to edema and swelling, causing serious problems in brain tumors, in diabetic retinopathy, after strokes, during sepsis and also in inflammatory conditions such as rheumatoid arthritis and asthma. Although many agents and disease processes increase vascular leakage, no known agent specifically makes vessels resistant to leaking. Vascular endothelial growth factor (VEGF) and the angiopoietins function together during vascular development, with VEGF acting early during vessel formation, and angiopoietin-1 acting later during vessel remodeling, maturation and stabilization. Although VEGF was initially called vascular permeability factor, there has been less focus on its permeability actions and more effort devoted to its involvement in vessel growth and applications in ischemia and cancer. Recent transgenic approaches have confirmed the profound permeability effects of VEGF (refs. 12-14), and have shown that transgenic angiopoietin-1 acts reciprocally as an anti-permeability factor when provided chronically during vessel formation, although it also profoundly affects vascular morphology when thus delivered. To be useful clinically, angiopoietin-1 would have to inhibit leakage when acutely administered to adult vessels, and this action would have to be uncoupled from its profound angiogenic capabilities. Here we show that acute administration of angiopoietin-1 does indeed protect adult vasculature from leaking, countering the potentially lethal actions of VEGF and inflammatory agents. PMID- 10742157 TI - Allogeneic bone marrow transplantation with co-stimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment. AB - Allogeneic bone marrow transplantation (in immunocompetent adults) has always required cytoreductive treatment of recipients with irradiation or cytotoxic drugs to achieve lasting engraftment at levels detectable by non-PCR-based techniques ('macrochimerism' or 'mixed chimerism'). Only syngeneic marrow engraftment at such levels has been achieved in unconditioned hosts. This requirement for potentially toxic myelosuppressive host pre-conditioning has precluded the clinical use of allogeneic bone marrow transplantation for many indications other than malignancies, including tolerance induction. We demonstrate here that treatment of naive mice with a high dose of fully major histocompatibility complex-mismatched allogeneic bone marrow, followed by one injection each of monoclonal antibody against CD154 and cytotoxic T-lymphocyte antigen 4 immunoglobulin, resulted in multi-lineage hematopoietic macrochimerism (of about 15%) that persisted for up to 34 weeks. Long-term chimeras developed donor-specific tolerance (donor skin graft survival of more than 145 days) and demonstrated ongoing intrathymic deletion of donor-reactive T cells. A protocol of high-dose bone marrow transplantation and co-stimulatory blockade can thus achieve allogeneic bone marrow engraftment without cytoreduction or T-cell depletion of the host, and eliminates a principal barrier to the more widespread use of allogeneic bone marrow transplantation. Although efforts have been made to minimize host pre-treatment for allogeneic bone marrow transplantation for tolerance induction, so far none have succeeded in eliminating pre-treatment completely. Our demonstration that this can be achieved provides the rationale for a safe approach for inducing robust transplantation tolerance in large animals and humans. PMID- 10742158 TI - Functional deficits in basal ganglia of children with attention deficit/hyperactivity disorder shown with functional magnetic resonance imaging relaxometry. AB - Attention-deficit/hyperactivity disorder is a highly heritable and prevalent neuropsychiatric disorder estimated to affect 6% of school-age children. Its clinical hallmarks are inattention, hyperactivity and impulsivity, which often respond substantially to treatment with methylphenidate or dextroamphetamine. Etiological theories suggest a deficit in corticostriatal circuits, particularly those components modulated by dopamine. We developed a new functional magnetic resonance imaging procedure (T2 relaxometry) to indirectly assess blood volume in the striatum (caudate and putamen) of boys 6-12 years of age in steady-state conditions. Boys with attention-deficit/hyperactivity disorder had higher T2 relaxation time measures in the putamen bilaterally than healthy control subjects. Relaxation times strongly correlated with the child's capacity to sit still and his accuracy in accomplishing a computerized attention task. Daily treatment with methylphenidate significantly changed the T2 relaxation times in the putamen of children with attention-deficit/hyperactivity disorder, although the magnitude and direction of the effect was strongly dependent on the child's unmedicated activity state. There was a similar but nonsignificant trend in the right caudate. T2 relaxation time measures in thalamus did not differ significantly between groups, and were not affected by methylphenidate. Attention deficit/hyperactivity disorder symptoms may be closely tied to functional abnormalities in the putamen, which is mainly involved in the regulation of motor behavior. PMID- 10742160 TI - On the market PMID- 10742159 TI - Development of an in vitro organ culture model to study transmission of HIV-1 in the female genital tract. PMID- 10742161 TI - Orphan amyloid diseases. PMID- 10742162 TI - New perch for the winged helix. PMID- 10742163 TI - mRNA processing: the 3'-end justifies the means. PMID- 10742164 TI - Recognizing misfolded proteins in the endoplasmic reticulum. PMID- 10742165 TI - The plot thickens: how thrombin modulates blood clotting. PMID- 10742166 TI - Working with paul sigler PMID- 10742167 TI - Picture story. Freeze frame. PMID- 10742168 TI - Paul sigler: A structural biologist with passion PMID- 10742169 TI - Crystal structure of the S15-rRNA complex. AB - In bacterial ribosomes, the small (30S) ribosomal subunit is composed of 16S rRNA and 21 distinct proteins. Ribosomal protein S15 is of particular interest because it binds primarily to 16S rRNA and is required for assembly of the small subunit and for intersubunit association, thus representing a key element in the assembly of a whole ribosome. Here we report the 2.8 ? resolution crystal structure of the highly conserved S15-rRNA complex. Protein S15 interacts in the minor groove with a G-U/G-C motif and a three-way junction. The latter is constrained by a conserved base triple and stacking interactions, and locked into place by magnesium ions and protein side chains, mainly through interactions with the unique three-dimensional geometry of the backbone. The present structure gives insights into the dual role of S15 in ribosome assembly and translational regulation. PMID- 10742170 TI - Recognition of local glycoprotein misfolding by the ER folding sensor UDP glucose:glycoprotein glucosyltransferase. AB - The endoplasmic reticulum (ER) contains a stringent quality control system that ensures the correct folding of newly synthesized proteins to be exported via the secretory pathway. In this system UDP-Glc:glycoprotein glucosyltransferase (GT) serves as a glycoprotein specific folding sensor by specifically glucosylating N linked glycans in misfolded glycoproteins thus retaining them in the calnexin/calreticulin chaperone cycle. To investigate how GT senses the folding status of glycoproteins, we generated RNase B heterodimers consisting of a folded and a misfolded domain. Only glycans linked to the misfolded domain were found to be glucosylated, indicating that the enzyme recognizes folding defects at the level of individual domains and only reglucosylates glycans directly attached to a misfolded domain. The result was confirmed with complexes of soybean agglutinin and misfolded thyroglobulin. PMID- 10742171 TI - Crosstalk between the catalytic and regulatory domains allows bidirectional regulation of Src. AB - The catalytic activity of Src family tyrosine kinases is inhibited by intramolecular interactions between the regulatory SH3 and SH2 domains and the catalytic domain. In the inactive state, the critical alphaC-helix in the catalytic domain is positioned such that the formation of the Glu 310-Lys 295 salt bridge is precluded, Tyr 416 in the activation loop is unphosphorylated, and the SH2 and SH3 domains are unavailable for interactions with other proteins. We found that phosphorylation of the activation loop or mutation of the loop preceding the alphaC-helix activates Src and increases the accessibility of the SH3 domain for ligands. Interaction of the alphaC-helix with the activation loop is a central component of this regulatory system. Our data suggest a bidirectional regulation mechanism in which the regulatory domains inhibit Src activity, and Src activity controls the availability of the regulatory domains. By this mechanism, Src family kinases can be activated by proteins phosphorylating or changing the conformation of the catalytic domain. Once active, Src family kinases become less prone to regulation, implying a positive feedback loop on their activity. PMID- 10742172 TI - The structure of the ferric siderophore binding protein FhuD complexed with gallichrome. AB - Siderophore binding proteins play a key role in the uptake of iron in many gram positive and gram-negative bacteria. FhuD is a soluble periplasmic binding protein that transports ferrichrome and other hydroxamate siderophores. The crystal structure of FhuD from Escherichia coli in complex with the ferrichrome homolog gallichrome has been determined at 1.9 ? resolution, the first structure of a periplasmic binding protein involved in the uptake of siderophores. Gallichrome is held in a shallow pocket lined with aromatic groups; Arg and Tyr side chains interact directly with the hydroxamate moieties of the siderophore. FhuD possesses a novel fold, suggesting that its mechanisms of ligand binding and release are different from other structurally characterized periplasmic ligand binding proteins. PMID- 10742173 TI - Structure of the elk-1-DNA complex reveals how DNA-distal residues affect ETS domain recognition of DNA. AB - SAP-1 and Elk-1 are members of a large group of eukaryotic transcription factors that contain a conserved ETS DNA binding domain and that cooperate with the serum response factor (SRF) to activate transcription of the c-fos protooncogene. Despite the high degree of sequence similarity, which includes an identical amino acid sequence for the DNA recognition helix within the ETS domain of these proteins, they exhibit different DNA binding properties. Here we report the 2.1 ? crystal structure of the ETS domain of Elk-1 bound to a high affinity E74 DNA (E74DNA) site and compare it to a SAP-1-E74DNA complex. This comparison reveals that the differential DNA binding properties of these proteins are mediated by non-conserved residues distal to the DNA binding surface that function to orient conserved residues in the DNA recognition helix for protein-specific DNA contacts. As a result, nearly one-third of the interactions between the protein recognition helix and the DNA are different between the SAP-1 and Elk-1 DNA complexes. Taken together, these studies reveal a novel mechanism for the modulation of DNA binding specificity within a conserved DNA binding domain, and have implications for how highly homologous ETS proteins exhibit differential DNA binding properties. PMID- 10742174 TI - Structural insights into substrate binding by the molecular chaperone DnaK. AB - How substrate affinity is modulated by nucleotide binding remains a fundamental, unanswered question in the study of 70 kDa heat shock protein (Hsp70) molecular chaperones. We find here that the Escherichia coli Hsp70, DnaK, lacking the entire alpha-helical domain, DnaK(1-507), retains the ability to support lambda phage replication in vivo and to pass information from the nucleotide binding domain to the substrate binding domain, and vice versa, in vitro. We determined the NMR solution structure of the corresponding substrate binding domain, DnaK(393-507), without substrate, and assessed the impact of substrate binding. Without bound substrate, loop L3,4 and strand beta3 are in significantly different conformations than observed in previous structures of the bound DnaK substrate binding domain, leading to occlusion of the substrate binding site. Upon substrate binding, the beta-domain shifts towards the structure seen in earlier X-ray and NMR structures. Taken together, our results suggest that conformational changes in the beta-domain itself contribute to the mechanism by which nucleotide binding modulates substrate binding affinity. PMID- 10742175 TI - Structural basis for the function of Bacillus subtilis phosphoribosyl pyrophosphate synthetase. AB - Here we report the first three-dimensional structure of a phosphoribosylpyrophosphate (PRPP) synthetase. PRPP is an essential intermediate in several biosynthetic pathways. Structures of the Bacillus subtilis PRPP synthetase in complex with analogs of the activator phosphate and the allosteric inhibitor ADP show that the functional form of the enzyme is a hexamer. The individual subunits fold into two domains, both of which resemble the type I phosphoribosyltransfereases. The active site is located between the two domains and includes residues from two subunits. Phosphate and ADP bind to the same regulatory site consisting of residues from three subunits of the hexamer. In addition to identifying residues important for binding substrates and effectors, the structures suggest a novel mode of allosteric regulation. PMID- 10742176 TI - Tethered blockers as molecular 'tape measures' for a voltage-gated K+ channel. AB - The propagation of electrical signals in excitable cells is orchestrated by a molecular family of voltage-dependent ion channel proteins. These K+, Na+, and Ca++ channels are all composed of four identical or similar units, each containing six transmembrane segments (S1-S6) in a roughly four-fold symmetric structure. The S5-S6 sequences fold into a central pore unit, which is surrounded by a voltage-gating module composed of S1-S4. The recent structure of KcsA, a two transmembrane bacterial K+ channel, illuminates the physical character of the pore unit, but little is known about the arrangement of the surrounding S1-S4 sequences. To locate regions of this gating module in space, we synthesized a series of compounds of varying length that function as molecular 'tape measures': quaternary ammonium (QA) pore blockers that can be tethered to specific test residues. We show that in a Shaker K+ channel, the extracellular ends of S1 and S3 are approximately 30 ? from the tetraethylammonium (TEA) blocking site at the external opening of the pore. A portion of the S3-S4 loop is, at 17-18 ?, considerably closer. PMID- 10742177 TI - Rational design of potent human transthyretin amyloid disease inhibitors. AB - The human amyloid disorders, familial amyloid polyneuropathy, familial amyloid cardiomyopathy and senile systemic amyloidosis, are caused by insoluble transthyretin (TTR) fibrils, which deposit in the peripheral nerves and heart tissue. Several nonsteroidal anti-inflammatory drugs and structurally similar compounds have been found to strongly inhibit the formation of TTR amyloid fibrils in vitro. These include flufenamic acid, diclofenac, flurbiprofen, and resveratrol. Crystal structures of the protein-drug complexes have been determined to allow detailed analyses of the protein-drug interactions that stabilize the native tetrameric conformation of TTR and inhibit the formation of amyloidogenic TTR. Using a structure-based drug design approach ortho trifluormethylphenyl anthranilic acid and N-(meta-trifluoromethylphenyl) phenoxazine 4, 6-dicarboxylic acid have been discovered to be very potent and specific TTR fibril formation inhibitors. This research provides a rationale for a chemotherapeutic approach for the treatment of TTR-associated amyloid diseases. PMID- 10742178 TI - Structure of a novel leech carboxypeptidase inhibitor determined free in solution and in complex with human carboxypeptidase A2. AB - Leech carboxypeptidase inhibitor (LCI) is a novel protein inhibitor present in the medicinal leech Hirudo medicinalis. The structures of LCI free and bound to carboxypeptidase A2 (CPA2)have been determined by NMR and X-ray crystallography, respectively. The LCI structure defines a new protein motif that comprises a five stranded antiparallel beta-sheet and one short alpha-helix. This structure is preserved in the complex with human CPA2 in the X-ray structure, where the contact regions between the inhibitor and the protease are defined. The C terminal tail of LCI becomes rigid upon binding the protease as shown in the NMR relaxation studies, and it interacts with the carboxypeptidase in a substrate like manner. The homology between the C-terminal tails of LCI and the potato carboxypeptidase inhibitor represents a striking example of convergent evolution dictated by the target protease. These new structures are of biotechnological interest since they could elucidate the control mechanism of metallo carboxypeptidases and could be used as lead compounds for the search of fibrinolytic drugs. PMID- 10742179 TI - The NMR structure of the 38 kDa U1A protein - PIE RNA complex reveals the basis of cooperativity in regulation of polyadenylation by human U1A protein. AB - The status of the poly(A) tail at the 3'-end of mRNAs controls the expression of numerous genes in response to developmental and extracellular signals. Poly(A) tail regulation requires cooperative binding of two human U1A proteins to an RNA regulatory region called the polyadenylation inhibition element (PIE). When bound to PIE RNA, U1A proteins also bind to the enzyme responsible for formation of the mature 3'-end of most eukaryotic mRNAs, poly(A) polymerase (PAP). The NMR structure of the 38 kDa complex formed between two U1A molecules and PIE RNA shows that binding cooperativity depends on helix C located at the end of the RNA binding domain and just adjacent to the PAP-interacting domain of U1A. Since helix C undergoes a conformational change upon RNA binding, the structure shows that binding cooperativity and interactions with PAP occur only when U1A is bound to its cognate RNA. This mechanism ensures that the activity of PAP enzyme, which is essential to the cell, is only down regulated when U1A is bound to the U1A mRNA. PMID- 10742180 TI - Kinetics, thermodynamics and evolution of non-native interactions in a protein folding nucleus. AB - A lattice model with side chains was used to investigate protein folding with computer simulations. In this model, we rigorously demonstrate the existence of a specific folding nucleus. This nucleus contains specific interactions not present in the native state that, when weakened, slow folding but do not change protein stability. Such a decoupling of folding kinetics from thermodynamics has been observed experimentally for real proteins. From our results, we conclude that specific non-native interactions in the transition state would give rise to straight phi-values that are negative or larger than unity. Furthermore, we demonstrate that residue Ile 34 in src SH3, which has been shown to be kinetically, but not thermodynamically, important, is universally conserved in proteins with the SH3 fold. This is a clear example of evolution optimizing the folding rate of a protein independent of its stability and function. PMID- 10742182 TI - Ion channels: doing hard chemistry with hard ions. AB - For years, the mechanisms and structural underpinnings of ion selectivity by ion channel proteins have been inferred by indirect experiments. The recently determined structure of a K(+) channel has put flesh on these bones, revealing the coordination chemistry responsible for the unusual combination of high selectivity and high transport rates in ion channels. PMID- 10742183 TI - Magnetite biomineralization and ancient life on Mars. AB - Certain chemical and mineral features of the Martian meteorite ALH84001 were reported in 1996 to be probable evidence of ancient life on Mars. In spite of new observations and interpretations, the question of ancient life on Mars remains unresolved. Putative biogenic, nanometer magnetite has now become a leading focus in the debate. PMID- 10742184 TI - Calcium pumps: structural basis for and mechanism of calcium transmembrane transport. AB - Eukaryotic cells remove calcium from the cytosol using P-type pumps in the plasma membrane and in the sarco(endo)plasmic reticulum. These pumps share membrane topography and general mechanism of action, but differ in regulatory properties. Recent advances in the field include the three-dimensional structure of the sarco(endo)plasmic reticulum and further understanding of the transcriptional regulation of the plasma membrane P-type pump by calcium. PMID- 10742185 TI - Zinc-dependent protein folding. AB - Studies of classic zinc-finger peptides over the past 15 years have offered insights into the coupled processes of metal binding and protein folding. Within the past two years, this insight has been used to increase our understanding of the importance of first and second shell contributions (i.e. contributions from direct and indirect metal ligands) to metal binding and protein-folding stability, and led to advances in de novo protein design and protein redesign. PMID- 10742186 TI - Metal ions in ribozyme folding and catalysis. AB - Current research is reshaping basic theories regarding the roles of metal ions in ribozyme function. No longer viewed as strict metalloenzymes, some ribozymes can access alternative catalytic mechanisms depending on the identity and availability of metal ions. Similarly, reaction conditions can allow different folding pathways to predominate, with divalent cations sometimes playing opposing roles. PMID- 10742187 TI - Structure and chemistry of the copper chaperone proteins. AB - Major advances have been made in the past year towards an understanding of the structure and chemistry of copper chaperone proteins. Three-dimensional structures of Atx1, CopZ, yCCS, and hCCSdII were determined, and reveal a remarkable structural similarity between chaperones and target proteins. In addition, biochemical studies of CCS suggested that chaperones are required in vivo because intracellular copper concentrations are extremely low and also indicated that copper transfer occurs via a direct protein-protein interaction. PMID- 10742188 TI - Methane monooxygenase and its related biomimetic models. AB - The past year has seen significant advances in the understanding of the dioxygen activating chemistry of non-heme diiron enzymes, such as methane monooxygenase. Recent spectroscopic and structural studies on various biomimetic model compounds have provided new and valuable insights into this enzyme's mechanism of action and the important dioxygen-activation process. PMID- 10742189 TI - Metallothioneins: new functional and structural insights. AB - In the past few years, tissue-specific mammalian metallothioneins (i. e. metallothionein-3 and metallothionein-4) have been discovered that possess distinct functional properties. Other recent developments include an insight into the role of the widely expressed mammalian metallothionein-1 and metallothionein 2 isoforms in zinc homeostasis and apoptosis. The three-dimensional structure of the evolutionary distant sea urchin Cd(7)-metallothionein A, which contains two metal-thiolate clusters, supports previous conclusions regarding the functional importance of this structural motif. Despite correlated efforts involving techniques of structural biochemistry and molecular biology, the primary function of metallothioneins remains enigmatic. PMID- 10742190 TI - Probing DNA charge transport with metallointercalators. AB - A wide range of experiments have emerged recently regarding charge transport through DNA, including spectroscopic studies of rates of DNA-mediated electron transfer and biochemical studies of DNA base oxidation over long distances. These experiments have, in turn, led to new proposals about the way in which charge moves through DNA and have prompted the consideration of physiological roles for DNA electron transfer. Importantly, metallointercalators have been key players in many of these experiments. Metallointercalators provide critical probes to examine the migration of charge through the DNA base stack. PMID- 10742191 TI - Biomimetic modeling of copper oxidase reactivity. AB - Bioinspired copper-model-complexes that react with O(2) provide an opportunity to probe biological reactivity at a small-molecule level of detail. Biological structural information combined with appropriate ligand design has proven sufficient to create Cu:O(2) complexes capable of external substrate oxidation. Most notable developments during the review period are the bioinspired catalysts capable of aerobic alcohol-oxidation. The extension of this oxidative reactivity to other important organic transformations beyond the scope of the inspiring system completes a modeling paradigm. PMID- 10742193 TI - Organometallic iron: the key to biological hydrogen metabolism. AB - X-ray crystallography of iron-hydrogenases reveals that the active-site H-cluster contains an unmistakably organometallic dinuclear iron subcluster. The nickel hydrogenases, which in general play different metabolic roles, have a distinct but related active-site structure. The new structural definition, combined with chemical analogs and theoretical treatment, points toward mechanistic understanding of iron-hydrogenases and the possibility of a unified mechanism for all hydrogenases. PMID- 10742192 TI - Electron tunneling pathways in proteins. AB - Long-range electronic interactions between electron donors and acceptors in proteins depend on the structure of the intervening polypeptide. Several methods have been developed for calculating these weak couplings. New challenges in protein electron-transfer research include identifying the role of protein dynamics, and characterizing multistep tunneling over very long distances. PMID- 10742194 TI - The mechanism of heme oxygenase. AB - Major advances have been made in determining the structure of heme oxygenase and the relationship between its structure and catalytic activity. The nature of the first step in the reaction sequence, heme alpha-meso-hydroxylation, is now clear, although the mechanisms that control the alpha-regiospecificity remain elusive. Hypothetical mechanisms can be written for the steps that convert alpha-meso hydroxyheme to biliverdin, but these mechanisms must be validated before this complex reaction sequence can be fully understood. The salient conclusion appears to be that the heme-oxygenase reaction reflects the absence of interactions that channel the reaction towards a ferryl species, rather than the presence of interactions that specifically promote heme oxidation. PMID- 10742195 TI - Metals and neuroscience. AB - Data are now rapidly accumulating to show that metallochemical reactions might be the common denominator underlying Alzheimer's disease, amyotrophic lateral sclerosis, prion diseases, cataracts, mitochondrial disorders and Parkinson's disease. In these disorders, an abnormal reaction between a protein and a redox active metal ion (copper or iron) promotes the formation of reactive oxygen species or radicalization. It is especially intriguing how the powerful catalytic redox activity of antioxidant Cu/Zn-superoxide dismutase can convert into a pro oxidant activity, a theme echoed in the recent proposal that Abeta and PrP, the proteins respectively involved in Alzheimer's disease and prion diseases, possess similar redox activities. PMID- 10742196 TI - A food-grade cloning system for industrial strains of Lactococcus lactis. AB - We have previously reported the construction of a food-grade cloning vector for Lactococcus using the ochre suppressor, supB, as the selective marker. This vector, pFG1, causes only a slight growth inhibition in the laboratory strain MG1363 but is unstable in the industrial strains tested. As supB suppresses both amber and ochre stop codons, which are present in 82% of all known lactococcal genes, this undesirable finding may result from the accumulation of elongated mistranslated polypeptides. Here, we report the development of a new food-grade cloning vector, pFG200, which is suitable for overexpressing a variety of genes in industrial strains of Lactococcus lactis. The vector uses an amber suppressor, supD, as selectable marker and consists entirely of Lactococcus DNA, with the exception of a small polylinker region. Using suppressible pyrimidine auxotrophs, selection and maintenance are efficient in any pyrimidine-free medium including milk. Importantly, the presence of this vector in a variety of industrial strains has no significant effect on the growth rate or the rate of acidification in milk, making this an ideal system for food-grade modification of industrially relevant L. lactis strains. The usefulness of this system is demonstrated by overexpressing the pepN gene in a number of industrial backgrounds. PMID- 10742197 TI - Molecular diversity within Lactobacillus helveticus as revealed by genotypic characterization. AB - Lactobacillus helveticus is a homofermentative thermophilic lactic acid bacterium that is used in the manufacture of Swiss type and long-ripened Italian cheeses, such as Emmental, Grana, and Provolone cheeses. Substantial differences in several technologically important characteristics are found among L. helveticus strains isolated from natural dairy starter cultures. In the present study we investigated the genotypic diversity of 74 strains isolated from different dairy cultures used for manufacturing Grana and Provolone cheeses and six collection strains. A restriction fragment length polymorphism analysis of both total genomic DNA and the 16S rRNA gene (ribotyping) was used as genotypic fingerprinting. A multivariate statistical analysis of the data enabled us to identify significant genotypic heterogeneity in L. helveticus. We found that genotypic fingerprinting could be used to distinguish strains; in particular, it was possible to associate the presence of specific strain genotypes with dairy ecosystem sources (e.g., Grana or Provolone cheese). Our data contribute to the description of microbial heterogeneity in L. helveticus and provide a more solid basis for understanding the functional and ecological significance of the presence of different L. helveticus biotypes in natural dairy starter cultures. PMID- 10742198 TI - A spore counting method and cell culture model for chlorine disinfection studies of Encephalitozoon syn. Septata intestinalis. AB - The microsporidia have recently been recognized as a group of pathogens that have potential for waterborne transmission; however, little is known about the effects of routine disinfection on microsporidian spore viability. In this study, in vitro growth of Encephalitozoon syn. Septata intestinalis, a microsporidium found in the human gut, was used as a model to assess the effect of chlorine on the infectivity and viability of microsporidian spores. Spore inoculum concentrations were determined by using spectrophotometric measurements (percent transmittance at 625 nm) and by traditional hemacytometer counting. To determine quantitative dose-response data for spore infectivity, we optimized a rabbit kidney cell culture system in 24-well plates, which facilitated calculation of a 50% tissue culture infective dose (TCID(50)) and a minimal infective dose (MID) for E. intestinalis. The TCID(50) is a quantitative measure of infectivity and growth and is the number of organisms that must be present to infect 50% of the cell culture wells tested. The MID is as a measure of a system's permissiveness to infection and a measure of spore infectivity. A standardized MID and a standardized TCID(50) have not been reported previously for any microsporidian species. Both types of doses are reported in this paper, and the values were used to evaluate the effects of chlorine disinfection on the in vitro growth of microsporidia. Spores were treated with chlorine at concentrations of 0, 1, 2, 5, and 10 mg/liter. The exposure times ranged from 0 to 80 min at 25 degrees C and pH 7. MID data for E. intestinalis were compared before and after chlorine disinfection. A 3-log reduction (99.9% inhibition) in the E. intestinalis MID was observed at a chlorine concentration of 2 mg/liter after a minimum exposure time of 16 min. The log(10) reduction results based on percent transmittance-derived spore counts were equivalent to the results based on hemacytometer-derived spore counts. Our data suggest that chlorine treatment may be an effective water treatment for E. intestinalis and that spectrophotometric methods may be substituted for labor-intensive hemacytometer methods when spores are counted in laboratory-based chlorine disinfection studies. PMID- 10742199 TI - Survival and filamentation of Salmonella enterica serovar enteritidis PT4 and Salmonella enterica serovar typhimurium DT104 at low water activity. AB - In this study we investigated the long-term survival of and morphological changes in Salmonella strains at low water activity (a(w)). Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 survived at low a(w) for long periods, but minimum humectant concentrations of 8% NaCl (a(w), 0. 95), 96% sucrose (a(w), 0.94), and 32% glycerol (a(w), 0.92) were bactericidal under most conditions. Salmonella rpoS mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. At a lethal a(w), incubation at 37 degrees C resulted in more rapid loss of viability than incubation at 21 degrees C. At a(w) values of 0.93 to 0.98, strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium formed filaments, some of which were at least 200 microm long. Filamentation was independent of rpoS expression. When the preparations were returned to high-a(w) conditions, the filaments formed septa, and division was complete within approximately 2 to 3 h. The variable survival of Salmonella strains at low a(w) highlights the importance of strain choice when researchers produce modelling data to simulate worst-case scenarios or conduct risk assessments based on laboratory data. The continued increase in Salmonella biomass at low a(w) (without a concomitant increase in microbial count) would not have been detected by traditional microbiological enumeration tests if the tests had been performed immediately after low-a(w) storage. If Salmonella strains form filaments in food products that have low a(w) values (0.92 to 0.98), there are significant implications for public health and for designing methods for microbiological monitoring. PMID- 10742200 TI - Evaluation of F-specific RNA bacteriophage as a candidate human enteric virus indicator for bivalve molluscan shellfish. AB - Escherichia coli is a widely utilized indicator of the sanitary quality of bivalve molluscan shellfish sold for human consumption. However, it is now well documented that shellfish that meet the E. coli standards for human consumption may contain human enteric viruses that cause gastroenteritis and hepatitis. In this study we investigated using F-specific RNA bacteriophage (FRNA bacteriophage) to indicate the likely presence of such viruses in shellfish sold for consumption. FRNA bacteriophage and E. coli levels were determined over a 2 year period for oysters (Crassostrea gigas) harvested from four commercial sites chosen to represent various degrees of sewage pollution. Three sites were classified as category B sites under the relevant European Community (EC) Directive (91/492), which required purification (depuration) of oysters from these sites before sale. One site was classified as a category A site, and oysters from this site could be sold directly without further processing. Samples were tested at the point of sale following commercial processing and packaging. All of the shellfish complied with the mandatory EC E. coli standard (less than 230 per 100 g of shellfish flesh), and the levels of contamination for more than 90% of the shellfish were at or below the level of sensitivity of the assay (20 E. coli MPN per 100 g), which indicated good quality based on this criterion. In contrast, FRNA bacteriophage were frequently detected at levels that exceeded 1,000 PFU per 100 g. High levels of FRNA bacteriophage contamination were strongly associated with harvest area fecal pollution and with shellfish associated disease outbreaks. Interestingly, FRNA bacteriophage contamination exhibited a marked seasonal trend that was consistent with the trend of oyster associated gastroenteritis in the United Kingdom. The correlation between FRNA bacteriophage contamination and health risk was investigated further by using a reverse transcription-PCR assay for Norwalk-like virus (NLV). NLV contamination of oysters was detected only at the most polluted site and also exhibited a seasonal trend that was consistent with the trend of FRNA bacteriophage contamination and with the incidence of disease. The results of this study suggest that FRNA bacteriophage could be used as viral indicators for market ready oysters. PMID- 10742201 TI - Isolation and characterization of a new denitrifying spirillum capable of anaerobic degradation of phenol. AB - Two kinds of phenol-degrading denitrifying bacteria, Azoarcus sp. strain CC-11 and spiral bacterial strain CC-26, were isolated from the same enrichment culture after 1 and 3 years of incubation, respectively. Both strains required ferrous ions for growth, but strain CC-26 grew better than strain CC-11 grew under iron limited conditions, which may have resulted in the observed change in the phenol degrading bacteria during the enrichment process. Strain CC-26 grew on phenol, benzoate, and other aromatic compounds under denitrifying conditions. Phylogenetic analysis of 16S ribosomal DNA sequences revealed that this strain is most closely related to a Magnetospirillum sp., a member of the alpha subclass of the class Proteobacteria, and is the first strain of a denitrifying aromatic compound-degrading bacterium belonging to this group. Unlike previously described Magnetospirillum strains, however, this strain did not exhibit magnetotaxis. It grew on phenol only under denitrifying conditions. Other substrates, such as acetate, supported aerobic growth, and the strain exhibited microaerophilic features. PMID- 10742202 TI - Electricity generation in microbial fuel cells using neutral red as an electronophore. AB - Neutral red (NR) was utilized as an electron mediator in microbial fuel cells consuming glucose to study both its efficiency during electricity generation and its role in altering anaerobic growth and metabolism of Escherichia coli and Actinobacillus succinogenes. A study of chemical fuel cells in which NADH, NR, and ferricyanide were the electron donor, the electronophore, and the electron acceptor, respectively, showed that electrical current produced from NADH was proportional to the concentration of NADH. Fourfold more current was produced from NADH in chemical fuel cells when NR was the electron mediator than when thionin was the electron mediator. In microbial fuel cells in which E. coli resting cells were used the amount of current produced from glucose when NR was the electron mediator (3.5 mA) was 10-fold more than the amount produced when thionin was the electron mediator (0.4 mA). The amount of electrical energy generated (expressed in joules per mole of substrate) and the amount of current produced from glucose (expressed in milliamperes) in NR-mediated microbial fuel cells containing either E. coli or A. succinogenes were about 10- and 2-fold greater, respectively, when resting cells were used than when growing cells were used. Cell growth was inhibited substantially when these microbial fuel cells were making current, and more oxidized end products were formed under these conditions. When sewage sludge (i.e., a mixed culture of anaerobic bacteria) was used in the fuel cell, stable (for 120 h) and equivalent levels of current were obtained with glucose, as observed in the pure-culture experiments. These results suggest that NR is better than other electron mediators used in microbial fuel cells and that sludge production can be decreased while electricity is produced in fuel cells. Our results are discussed in relation to factors that may improve the relatively low electrical efficiencies (1.2 kJ/mol) obtained with microbial fuel cells. PMID- 10742203 TI - Characterization and cloning of the genes encoding enterocin 1071A and enterocin 1071B, two antimicrobial peptides produced by Enterococcus faecalis BFE 1071. AB - The pH-neutral cell supernatant of Enterococcus faecalis BFE 1071, isolated from the feces of minipigs in Gottingen, inhibited the growth of Enterococcus spp. and a few other gram-positive bacteria. Ammonium sulfate precipitation and cation exchange chromatography of the cell supernatant, followed by mass spectrometry analysis, yielded two bacteriocin-like peptides of similar molecular mass: enterocin 1071A (4.285 kDa) and enterocin 1071B (3.899 kDa). Both peptides are always isolated together. The peptides are heat resistant (100 degrees C, 60 min; 50% of activity remained after 15 min at 121 degrees C), remain active after 30 min of incubation at pH 3 to 12, and are sensitive to treatment with proteolytic enzymes. Curing experiments indicated that the genes encoding enterocins 1071A and 1071B are located on a 50-kbp plasmid (pEF1071). Conjugation of plasmid pEF1071 to E. faecalis strains FA2-2 and OGX1 resulted in the expression of two active peptides with sizes identical to those of enterocins 1071A and 1071B. Sequencing of a DNA insert of 9 to 10 kbp revealed two open reading frames, ent1071A and ent1071B, which coded for 39- and 34-amino-acid peptides, respectively. The deduced amino acid sequence of the mature Ent1071A and Ent1071B peptides showed 64 and 61% homology with the alpha and beta peptides of lactococcin G, respectively. This is the first report of two new antimicrobial peptides representative of a fourth type of E. faecalis bacteriocin. PMID- 10742204 TI - Physiological analysis of the expression of the styrene degradation gene cluster in Pseudomonas fluorescens ST. AB - The effects of different carbon sources on expression of the styrene catabolism genes in Pseudomonas fluorescens ST were analyzed by using a promoter probe vector, pPR9TT, which contains transcription terminators upstream and downstream of the beta-galactosidase reporter system. Expression of the promoter of the stySR operon, which codes for the styrene two-component regulatory system, was found to be constitutive and not subject to catabolite repression. This was confirmed by the results of an analysis of the stySR transcript in P. fluorescens ST cells grown on different carbon sources. The promoter of the operon of the upper pathway, designated PstyA, was induced by styrene and repressed to different extents by organic acids or carbohydrates. In particular, cells grown on succinate or lactate in the presence of styrene started to exhibit beta galactosidase activity during the mid-exponential growth phase, before the preferred carbon sources were depleted, indicating that there is a threshold succinate and lactate concentration which allows induction of styrene catabolic genes. In contrast, cells grown on glucose, acetate, or glutamate and styrene exhibited a diauxic growth curve, and beta-galactosidase activity was detected only after the end of the exponential growth phase. In each experiment the reliability of the reporter system constructed was verified by comparing the beta galactosidase activity and the activity of the styrene monooxygenase encoded by the first gene of the styrene catabolic operon. PMID- 10742205 TI - Properties of engineered poly-3-hydroxyalkanoates produced in recombinant Escherichia coli strains. AB - To prepare medium-chain-length poly-3-hydroxyalkanoates (PHAs) with altered physical properties, we generated recombinant Escherichia coli strains that synthesized PHAs with altered monomer compositions. Experiments with different substrates (fatty acids with different chain lengths) or different E. coli hosts failed to produce PHAs with altered physical properties. Therefore, we engineered a new potential PHA synthetic pathway, in which ketoacyl-coenzyme A (CoA) intermediates derived from the beta-oxidation cycle are accumulated and led to the PHA polymerase precursor R-3-hydroxyalkanoates in E. coli hosts. By introducing the poly-3-hydroxybutyrate acetoacetyl-CoA reductase (PhbB) from Ralstonia eutropha and blocking the ketoacyl-CoA degradation step of the beta oxidation, the ketoacyl-CoA intermediate was accumulated and reduced to the PHA precursor. Introduction of the phbB gene not only caused significant changes in the monomer composition but also caused changes of the physical properties of the PHA, such as increase of polymer size and loss of the melting point. The present study demonstrates that pathway engineering can be a useful approach for producing PHAs with engineered physical properties. PMID- 10742206 TI - Degradation of pectins with different degrees of esterification by Bacteroides thetaiotaomicron isolated from human gut flora. AB - A complete human fecal flora and cultures of defined species obtained from fecal flora were investigated in vitro to determine their ability to ferment the dietary fiber pectin. Bacteroides thetaiotaomicron was tested as a pectin degrading microorganism alone and in coculture with Escherichia coli. Macromolecular pectins with different degrees of esterification were used as substrates in microbial degradation studies. The levels of oligogalacturonic acids formed in batch cultures were estimated during a 24- or 48-h incubation period by using high-performance thin-layer chromatography and high-performance anion-exchange chromatography. The spectrum and the amount of unsaturated oligogalacturonic acids formed as intermediate products of pectin fermentation changed permanently in the culture media during incubation with the complete fecal flora. After 24 h, no oligogalacturonic acids were detected. The pectin degrading activities of pure cultures of B. thetaiotaomicron were lower than the pectin-degrading activity of a complete fecal flora. Cocultures of B. thetaiotaomicron and E. coli exhibited intermediate levels of degradation activity. In pure cultures of E. coli no pectin-degrading activity was found. Additionally, the rate of pectin degradation was affected by the degree of esterification of the substrate. Saturated oligogalacturonic acids were not found during pectin fermentation. The disappearance of oligogalacturonic acids in the later stages of fermentation with both the complete fecal flora and B. thetaiotaomicron was accompanied by increased formation of short-chain fatty acids. PMID- 10742207 TI - rRNA operon copy number reflects ecological strategies of bacteria. AB - Although natural selection appears to favor the elimination of gene redundancy in prokaryotes, multiple copies of each rRNA-encoding gene are common on bacterial chromosomes. Despite this conspicuous deviation from single-copy genes, no phenotype has been consistently associated with rRNA gene copy number. We found that the number of rRNA genes correlates with the rate at which phylogenetically diverse bacteria respond to resource availability. Soil bacteria that formed colonies rapidly upon exposure to a nutritionally complex medium contained an average of 5.5 copies of the small subunit rRNA gene, whereas bacteria that responded slowly contained an average of 1.4 copies. In soil microcosms pulsed with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), indigenous populations of 2,4-D-degrading bacteria with multiple rRNA genes ( = 5.4) became dominant, whereas populations with fewer rRNA genes ( = 2.7) were favored in unamended controls. These findings demonstrate phenotypic effects associated with rRNA gene copy number that are indicative of ecological strategies influencing the structure of natural microbial communities. PMID- 10742208 TI - Effect of three factors in cheese production (pH, salt, and heat) on Mycobacterium avium subsp. paratuberculosis viability. AB - Low pH and salt are two factors contributing to the inactivation of bacterial pathogens during a 60-day curing period for cheese. The kinetics of inactivation for Mycobacterium avium subsp. paratuberculosis strains ATCC 19698 and Dominic were measured at 20 degrees C under different pH and NaCl conditions commonly used in processing cheese. The corresponding D values (decimal reduction times; the time required to kill 1 log(10) concentration of bacteria) were measured. Also measured were the D values for heat-treated and nonheated M. avium subsp. paratuberculosis in 50 mM acetate buffer (pH 5.0, 2% [wt/vol] NaCl) and a soft white Hispanic-style cheese (pH 6.0, 2% [wt/vol] NaCl). Samples were removed at various intervals until no viable cells were detected using the radiometric culture method (BACTEC) for enumeration of M. avium subsp. paratuberculosis. NaCl had little or no effect on the inactivation of M. avium subsp. paratuberculosis, and increasing NaCl concentrations were not associated with decreasing D values (faster killing) in the acetate buffer. Lower pHs, however, were significantly correlated with decreasing D values of M. avium subsp. paratuberculosis in the acetate buffer. The D values for heat-treated M. avium subsp. paratuberculosis ATCC 19698 in the cheese were higher than those predicted by studies done in acetate buffer. The heat-treated M. avium subsp. paratuberculosis strains had lower D values than the nonheated cells (faster killing) both in the acetate buffer (pH 5, 2% [wt/vol] NaCl) and in the soft white cheese. The D value for heat-treated M. avium subsp. paratuberculosis ATCC 19698 in the cheese (36.5 days) suggests that heat treatment of raw milk coupled with a 60-day curing period will inactivate about 10(3) cells of M. avium subsp. paratuberculosis per ml. PMID- 10742209 TI - Simultaneous detection and differentiation of Escherichia coli populations from environmental freshwaters by means of sequence variations in a fragment of the beta-D-glucuronidase gene. AB - A PCR-based denaturing-gradient gel electrophoresis (DGGE) approach was applied to a partial sequence of the beta-D-glucuronidase gene (uidA) for specific detection and differentiation of Escherichia coli populations according to their uidA sequence variations. Detection of sequence variations by PCR-DGGE and by PCR with direct sequencing correlated perfectly. Screening of 50 E. coli freshwater isolates and reference strains revealed 11 sequence types, showing nine polymorphic sites and an average number of pairwise differences between alleles of the uidA gene fragments (screened fragment length, 126 bp) of 2.3%. Among the analyzed strains a range of dominating to more rarely and/or uniquely observed E. coli sequence types was revealed. PCR-DGGE applied to fecally polluted river water samples simultaneously detected E. coli and generated a fingerprint of the mixed populations by separating the polymorphic uidA amplicons. No significant differences between non-cultivation-based and cultivation-based profiles were observed, suggesting that at least some members of all occurring sequence types could be cultivated. As E. coli is frequently used as a fecal indicator, this work is considered an important step towards a new, practical tool for the differentiation and tracing of fecal pollution in all kinds of waters. PMID- 10742210 TI - Development of a single-reaction multiplex PCR toxin typing assay for Staphylococcus aureus strains. AB - We describe here the development of a single-reaction multiplex PCR assay for the enterotoxin genes from Staphylococcus aureus that utilizes a universal toxin gene primer in combination with toxin-specific primers to amplify characteristic toxin gene products. In combination with a new DNA purification method, the assay can detect enterotoxin genes A to E from a pure culture within 3 to 4 h. The test was used to characterize a diverse set of environmental S. aureus isolates, and a 99% correlation with toxin typing using standard immunological tests was found. The design of the assay allows it to be extended to include both newly characterized and as-yet-unknown toxin genes. PMID- 10742211 TI - Expression of a heterologous glutamate dehydrogenase gene in Lactococcus lactis highly improves the conversion of amino acids to aroma compounds. AB - The first step of amino acid degradation in lactococci is a transamination, which requires an alpha-keto acid as the amino group acceptor. We have previously shown that the level of available alpha-keto acid in semihard cheese is the first limiting factor for conversion of amino acids to aroma compounds, since aroma formation is greatly enhanced by adding alpha-ketoglutarate to cheese curd. In this study we introduced a heterologous catabolic glutamate dehydrogenase (GDH) gene into Lactococcus lactis so that this organism could produce alpha ketoglutarate from glutamate, which is present at high levels in cheese. Then we evaluated the impact of GDH activity on amino acid conversion in in vitro tests and in a cheese model by using radiolabeled amino acids as tracers. The GDH producing lactococcal strain degraded amino acids without added alpha ketoglutarate to the same extent that the wild-type strain degraded amino acids with added alpha-ketoglutarate. Interestingly, the GDH-producing lactococcal strain produced a higher proportion of carboxylic acids, which are major aroma compounds. Our results demonstrated that a GDH-producing lactococcal strain could be used instead of adding alpha-ketoglutarate to improve aroma development in cheese. PMID- 10742212 TI - Cloning, characterization, controlled overexpression, and inactivation of the major tributyrin esterase gene of Lactococcus lactis. AB - The gene encoding the major intracellular tributyrin esterase of Lactococcus lactis was cloned using degenerate DNA probes based on 19 known N-terminal amino acid residues of the purified enzyme. The gene, named estA, was sequenced and found to encode a protein of 258 amino acid residues. The transcription start site was mapped 233 nucleotides upstream of the start codon, and a canonical promoter sequence was identified. The deduced amino acid sequence of the estA product contained the typical GXSXG motif found in most lipases and esterases. The protein was overproduced up to 170-fold in L. lactis by use of the nisin controlled expression system recently developed for lactic acid bacteria. The estA gene was inactivated by chromosomal integration of a temperature-sensitive integration vector. This resulted in the complete loss of esterase activity, which could then be recovered after complementation of the constructed esterase deficient strain with the wild-type estA gene. This confirms that EstA is the main enzyme responsible for esterase activity in L. lactis. Purified recombinant enzyme showed a preference for short-chain acyl esters, surprisingly also including phospholipids. Medium- and long-acyl-chain lipids were also hydrolyzed, albeit less efficiently. Intermediate characteristics between esterases and lipases make intracellular lactococcal EstA difficult to classify in either of these two groups of esterolytic enzymes. We suggest that, in vivo, EstA could be involved in (phospho)lipid metabolism or cellular detoxification or both, as its sequence showed significant similarity to S-formylglutathione hydrolase (FGH) of Paracoccus denitrificans and human EstD (or FGH), which are part of a universal formaldehyde detoxification pathway. PMID- 10742213 TI - 16S rRNA gene-based detection of tetrachloroethene-dechlorinating Desulfuromonas and Dehalococcoides species. AB - Members of the genera Desulfuromonas and Dehalococcoides reductively dechlorinate tetrachloroethene (PCE) and trichloroethene. Two primer pairs specific to hypervariable regions of the 16S rRNA genes of the Dehalococcoides group (comprising Dehalococcoides ethenogenes and Dehalococcoides sp. strain FL2) and the acetate-oxidizing, PCE-dechlorinating Desulfuromonas group (comprising Desulfuromonas sp. strain BB1 and Desulfuromonas chloroethenica) were designed. The detection threshold of a nested PCR approach using universal bacterial primers followed by a second PCR with the Desulfuromonas dechlorinator-targeted primer pair was 1 x 10(3) BB1 cells added per gram (wet weight) of sandy aquifer material. Total community DNA isolated from sediments of three Michigan rivers and six different chloroethene-contaminated aquifer samples was used as template in nested PCR. All river sediment samples yielded positive signals with the BB1- and the Dehalococcoides-targeted primers. One chloroethene-contaminated aquifer tested positive with the Dehalococcoides-targeted primers, and another contaminated aquifer tested positive with the Desulfuromonas dechlorinator targeted primer pair. Restriction fragment analysis of the amplicons could discriminate strain BB1 from other known Desulfuromonas species. Microcosm studies confirmed the presence of PCE-dechlorinating, acetate-oxidizing Desulfuromonas and hydrogenotrophic Dehalococcoides species in samples yielding positive PCR signals with the specific primers. PMID- 10742214 TI - Selective accumulation may account for shellfish-associated viral illness. AB - From 1991 through 1998, 1,266 cases of shellfish-related illnesses were attributed to Norwalk-like viruses. Seventy-eight percent of these illnesses occurred following consumption of oysters harvested from the Gulf Coast during the months of November through January. This study investigated the ability of eastern oysters (Crassostrea virginica) to accumulate indicator microorganisms (i.e., fecal coliforms, Escherichia coli, Clostridium perfringens, and F(+) coliphage) from estuarine water. One-week trials over a 1-year period were used to determine if these indicator organisms could provide insight into the seasonal occurrence of these gastrointestinal illnesses. The results demonstrate that oysters preferentially accumulated F(+) coliphage, an enteric viral surrogate, to their greatest levels from late November through January, with a concentration factor of up to 99-fold. However, similar increases in accumulation of the other indicator microorganisms were not observed. These findings suggest that the seasonal occurrence of shellfish-related illnesses by enteric viruses is, in part, the result of seasonal physiological changes undergone by the oysters that affect their ability to accumulate viral particles from estuarine waters. PMID- 10742215 TI - Characterization of a novel beta-galactosidase from Bifidobacterium adolescentis DSM 20083 active towards transgalactooligosaccharides. AB - This paper reports on the effects of both reducing and nonreducing transgalactooligosaccharides (TOS) comprising 2 to 8 residues on the growth of Bifidobacterium adolescentis DSM 20083 and on the production of a novel beta galactosidase (beta-Gal II). In cells grown on TOS, in addition to the lactose degrading beta-Gal (beta-Gal I), another beta-Gal (beta-Gal II) was detected and it showed activity towards TOS but not towards lactose. beta-Gal II activity was at least 20-fold higher when cells were grown on TOS than when cells were grown on galactose, glucose, and lactose. Subsequently, the enzyme was purified from the cell extract of TOS-grown B. adolescentis by anion-exchange chromatography, adsorption chromatography, and size-exclusion chromatography. Beta-Gal II has apparent molecular masses of 350 and 89 kDa as judged by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating that the enzyme is active in vivo as a tetramer. Beta Gal II had an optimal activity at pH 6 and was not active below pH 5. Its optimum temperature was 35 degrees C. The enzyme showed highest V(max) values towards galactooligosaccharides with a low degree of polymerization. This result is in agreement with the observation that during fermentation of TOS, the di- and trisaccharides were fermented first. Beta-Gal II was active towards beta galactosyl residues that were 1-->4, 1-->6, 1-->3, and 1 <--> 1 linked, signifying its role in the metabolism of galactooligosaccharides by B. adolescentis. PMID- 10742216 TI - Poly(3-hydroxyvalerate) depolymerase of Pseudomonas lemoignei. AB - Pseudomonas lemoignei is equipped with at least five polyhydroxyalkanoate (PHA) depolymerase structural genes (phaZ1 to phaZ5) which enable the bacterium to utilize extracellular poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate) (PHV), and related polyesters consisting of short-chain-length hxdroxyalkanoates (PHA(SCL)) as the sole sources of carbon and energy. Four genes (phaZ1, phaZ2, phaZ3, and phaZ5) encode PHB depolymerases C, B, D, and A, respectively. It was speculated that the remaining gene, phaZ4, encodes the PHV depolymerase (D. Jendrossek, A. Frisse, A. Behrends, M. Andermann, H. D. Kratzin, T. Stanislawski, and H. G. Schlegel, J. Bacteriol. 177:596-607, 1995). However, in this study, we show that phaZ4 codes for another PHB depolymeraes (i) by disagreement of 5 out of 41 amino acids that had been determined by Edman degradation of the PHV depolymerase and of four endoproteinase GluC-generated internal peptides with the DNA-deduced sequence of phaZ4, (ii) by the lack of immunological reaction of purified recombinant PhaZ4 with PHV depolymerase-specific antibodies, and (iii) by the low activity of the PhaZ4 depolymerase with PHV as a substrate. The true PHV depolymerase-encoding structural gene, phaZ6, was identified by screening a genomic library of P. lemoignei in Escherichia coli for clearing zone formation on PHV agar. The DNA sequence of phaZ6 contained all 41 amino acids of the GluC generated peptide fragments of the PHV depolymerase. PhaZ6 was expressed and purified from recombinant E. coli and showed immunological identity to the wild type PHV depolymerase and had high specific activities with PHB and PHV as substrates. To our knowledge, this is the first report on a PHA(SCL) depolymerase gene that is expressed during growth on PHV or odd-numbered carbon sources and that encodes a protein with high PHV depolymerase activity. Amino acid analysis revealed that PhaZ6 (relative molecular mass [M(r)], 43,610 Da) resembles precursors of other extracellular PHA(SCL) depolymerases (28 to 50% identical amino acids). The mature protein (M(r), 41,048) is composed of (i) a large catalytic domain including a catalytic triad of S(136), D(211), and H(269) similar to serine hydrolases; (ii) a linker region highly enriched in threonine residues and other amino acids with hydroxylated or small side chains (Thr-rich region); and (iii) a C-terminal domain similar in sequence to the substrate binding domain of PHA(SCL) depolymerases. Differences in the codon usage of phaZ6 for some codons from the average codon usage of P. lemoignei indicated that phaZ6 might be derived from other organisms by gene transfer. Multialignment of separate domains of bacterial PHA(SCL) depolymerases suggested that not only complete depolymerase genes but also individual domains might have been exchanged between bacteria during evolution of PHA(SCL) depolymerases. PMID- 10742217 TI - Glutathione-dependent conversion of N-ethylmaleimide to the maleamic acid by Escherichia coli: an intracellular detoxification process. AB - The electrophile N-ethylmaleimide (NEM) elicits rapid K(+) efflux from Escherichia coli cells consequent upon reaction with cytoplasmic glutathione to form an adduct, N-ethylsuccinimido-S-glutathione (ESG) that is a strong activator of the KefB and KefC glutathione-gated K(+) efflux systems. The fate of the ESG has not previously been investigated. In this report we demonstrate that NEM and N-phenylmaleimide (NPM) are rapidly detoxified by E. coli. The detoxification occurs through the formation of the glutathione adduct of NEM or NPM, followed by the hydrolysis of the imide bond after which N-substituted maleamic acids are released. N-ethylmaleamic acid is not toxic to E. coli cells even at high concentrations. The glutathione adducts are not released from cells, and this allows glutathione to be recycled in the cytoplasm. The detoxification is independent of new protein synthesis and NAD(+)-dependent dehydrogenase activity and entirely dependent upon glutathione. The time course of the detoxification of low concentrations of NEM parallels the transient activation of the KefB and KefC glutathione-gated K(+) efflux systems. PMID- 10742218 TI - Mechanism of the incidental production of a melanin-like pigment during 6 demethylchlortetracycline production in Streptomyces aureofaciens. AB - The secondary metabolite 6-demethylchlortetracycline (6-DCT), which is produced by Streptomyces aureofaciens, is used as a precursor of semisynthetic tetracyclines. Strains that produce 6-DCT also produce a melanin-like pigment (MP). The correlation between MP production and 6-DCT production was investigated by using S. aureofaciens NRRL 3203. Production of both MP and 6-DCT was repressed by phosphate or ammonium ions, suggesting that syntheses of these compounds are controlled by the same regulators. Ten chlortetracycline-producing recombinants were derived from 6-DCT-producing mutant NRRL 3203 by gene replacement. All of the recombinants produced chlortetracycline but not MP, indicating that MP production is the results of a defect in the 6-methylation step and suggesting that the polyketide nonaketideamide is a common intermediate leading to MP as well as 6-DCT. To further examine the possibility that MP might be synthesized via the 6-DCT-biosynthetic pathway, mutants defective in 6-DCT biosynthesis were derived from a 6-DCT producer. Some of these mutants were able to produce MP, while others, including mutants with mutations in the gene encoding anhydrotetracycline oxygenase, an enzyme catalyzing the penultimate step in the pathway, produced neither 6-DCT nor MP. Production of 6-DCT and production of MP were restored simultaneously by integrative transformation with the corresponding 6-DCT-biosynthetic genes, indicating that some of 6-DCT-biosynthetic enzymes are indispensable for MP production. These findings suggest that a defect in the 6 methylation step results in redirection of carbon flux from a certain intermediate in the 6-DCT-biosynthetic pathway to a shunt pathway and results in MP production. PMID- 10742219 TI - Influence of catalase and superoxide dismutase on ozone inactivation of Listeria monocytogenes. AB - The effects of ozone at 0.25, 0.40, and 1.00 ppm on Listeria monocytogenes were evaluated in distilled water and phosphate-buffered saline. Differences in sensitivity to ozone were found to exist among the six strains examined. Greater cell death was found following exposure at lower temperatures. Early stationary phase cells were less sensitive to ozone than mid-exponential- and late stationary-phase cells. Ozonation at 1.00 ppm of cabbage inoculated with L. monocytogenes effectively inactivated all cells after 5 min. The abilities of in vivo catalase and superoxide dismutase to protect the cells from ozone were also examined. Three listerial test strains were inactivated rapidly upon exposure to ozone. Both catalase and superoxide dismutase were found to protect listerial cells from ozone attack, with superoxide dismutase being more important than catalase in this protection. PMID- 10742220 TI - The complete amino acid substitutions at position 131 that are positively involved in cold adaptation of subtilisin BPN'. AB - To ascertain whether position 131 of a mesophilic protease, subtilisin BPN', is a potential critical site for cold adaptation as screened by evolutionary engineering (S. Taguchi, A. Ozaki, and H. Momose, Appl. Environ. Microbiol. 64:492-495, 1998), a full set of subtilisin BPN' mutants with mutations at position 131 was constructed by site-saturation mutagenesis. All mutated enzymes were measured for specific activity at 10 degrees C by the quantitative titer microplate assay system using polyclonal antibody against subtilisin BPN' and a synthetic chromogenic substrate. All the mutants exhibited proteolytic activities almost the same as or higher than that of the wild-type enzyme, suggesting that position 131 may be important for cold adaptation. In comparison with the wild type, purified mutants G131F, G131R, G131M, and G131W were found to acquire proteolytic activities (k(cat)/K(m)) at 10 degrees C that were 150, 94, 84, and 50% higher, respectively. In particular, for the G131F mutant, temperature dependency in enzyme activity was shown by an increase in k(cat) and a decrease in K(m). All of these amino acid substitution mutants, G131F, G131R, G131M, and G131W, acquired increased proteolytic activities at 10 degrees C for three different synthetic peptide substrates but no increase in caseinolytic activity. Furthermore, they all conferred thermolability on the enzyme to differing extents in terms of the half-life of enzyme inactivation at 60 degrees C. No significant correlation was found between the amino acids preferred for cold adaptation surveyed here and those present at position 131 of subtilisin of psychrophilic cells naturally occurring in cold environments. Based on these findings, position 131 is a contributor in artificial evolution for acquiring a cold-active character and may not be related to physiological requirements for subtilisin producing cells living in cold environments. Therefore, saturation mutagenesis would be effective in achieving rapid improvement in protein properties via evolutionary engineering. PMID- 10742221 TI - Isolation of bacteriophages specific to a fish pathogen, Pseudomonas plecoglossicida, as a candidate for disease control. AB - Two types of bacteriophage specific to Pseudomonas plecoglossicida, the causative agent of bacterial hemorrhagic ascites disease in cultured ayu fish (Plecoglossus altivelis), were isolated from diseased ayu and the rearing pond water. One type of phage, which formed small plaques, was tentatively classified as a member of the family Myoviridae, and the other type, which formed large plaques, was classified as a member of the family Podoviridae. All 27 strains of P. plecoglossicida examined, which were isolated from diseased ayu from geographically different areas in 1991 to 1999, exhibited quite similar sensitivities to either type of phage. One strain of P. plecoglossicida was highly virulent for ayu, and the 50% lethal dose (LD(50)) when intramuscular injection was used was 10(1.2) CFU fish(-1); in contrast, phage-resistant variants of this organism were less virulent (LD(50), >10(4) CFU fish(-1)). Oral administration of phage-impregnated feed to ayu resulted in protection against experimental infection with P. plecoglossicida. After oral administration of P. plecoglossicida cells of this bacterium were always detected in the kidneys of control fish that did not receive the phage treatment, while the cells quickly disappeared from the phage-treated fish. Bacterial growth in freshwater was lower in the presence of phage, and the number of phage PFU increased rapidly. These results suggest that it may be possible to use phage to control the disease caused by P. plecoglossicida. PMID- 10742222 TI - Quantification of Clostridium botulinum toxin gene expression by competitive reverse transcription-PCR. AB - Clostridium botulinum produces a characteristic botulinum neurotoxin which can cause an often fatal neuroparalytic condition known as botulism. Although food borne botulism is rare, critical screening by food companies is necessary to ensure that food products are safe. At present, the food industry assesses the risks of botulinum neurotoxin production by challenge testing to check any new food products and to check the efficacy of new storage regimes. Challenge testing involves artificial introduction of defined strains of microorganisms into food, and microbial growth and possible toxin production are then monitored. Botulinum toxin is normally analyzed by using the mouse bioassay. However, the mouse bioassay is expensive, slow, and politically sensitive because of animal rights issues. In this paper we describe adaptation of a new assay, competitive reverse transcription-PCR (RT-PCR), to monitor botulinum neurotoxin production. This method accurately measures the level of toxin-encoding mRNA in C. botulinum cells. Measurement of mRNA should provide a good indication of gene expression as mRNA is turned over rapidly in bacterial cells. In addition, the method is rapid, specific, and sensitive. The competitive RT-PCR method was developed to examine C. botulinum E VH toxin gene expression and was used to investigate the level of toxin production by C. botulinum E VH when the organism was grown in two different types of broth. The results which we obtained with the competitive RT PCR method demonstrated that this method is more rapid and more sensitive than the mouse bioassay. PMID- 10742224 TI - Efficient improvement of silage additives by using genetic algorithms. AB - The enormous variety of substances which may be added to forage in order to manipulate and improve the ensilage process presents an empirical, combinatorial optimization problem of great complexity. To investigate the utility of genetic algorithms for designing effective silage additive combinations, a series of small-scale proof of principle silage experiments were performed with fresh ryegrass. Having established that significant biochemical changes occur over an ensilage period as short as 2 days, we performed a series of experiments in which we used 50 silage additive combinations (prepared by using eight bacterial and other additives, each of which was added at six different levels, including zero [i.e. , no additive]). The decrease in pH, the increase in lactate concentration, and the free amino acid concentration were measured after 2 days and used to calculate a "fitness" value that indicated the quality of the silage (compared to a control silage made without additives). This analysis also included a "cost" element to account for different total additive levels. In the initial experiment additive levels were selected randomly, but subsequently a genetic algorithm program was used to suggest new additive combinations based on the fitness values determined in the preceding experiments. The result was very efficient selection for silages in which large decreases in pH and high levels of lactate occurred along with low levels of free amino acids. During the series of five experiments, each of which comprised 50 treatments, there was a steady increase in the amount of lactate that accumulated; the best treatment combination was that used in the last experiment, which produced 4.6 times more lactate than the untreated silage. The additive combinations that were found to yield the highest fitness values in the final (fifth) experiment were assessed to determine a range of biochemical and microbiological quality parameters during full-term silage fermentation. We found that these combinations compared favorably both with uninoculated silage and with a commercial silage additive. The evolutionary computing methods described here are a convenient and efficient approach for designing silage additives. PMID- 10742223 TI - The function of cytoplasmic flavin reductases in the reduction of azo dyes by bacteria. AB - A flavin reductase, which is naturally part of the ribonucleotide reductase complex of Escherichia coli, acted in cell extracts of recombinant E. coli strains under aerobic and anaerobic conditions as an "azo reductase." The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (fre) was transferred to Sphingomonas sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in the Sphingomonas strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains, E. coli, and wild-type Sphingomonas sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic "azo reductases," which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds. PMID- 10742225 TI - Imaging the enzymatic digestion of bacterial cellulose ribbons reveals the endo character of the cellobiohydrolase Cel6A from Humicola insolens and its mode of synergy with cellobiohydrolase Cel7A. AB - Dispersed cellulose ribbons from bacterial cellulose were subjected to digestion with cloned Cel7A (cellobiohydrolase [CBH] I) and Cel6A (CBH II) from Humicola insolens either alone or in a mixture and in the presence of an excess of beta glucosidase. Both Cel7A and Cel6A were effective in partially converting the ribbons into soluble sugars, Cel7A being more active than Cel6A. In combination, these enzymes showed substantial synergy culminating with a molar ratio of approximately two-thirds Cel6A and one-third Cel7A. Ultrastructural transmission electron microscopy (TEM) observations indicated that Cel7A induced a thinning of the cellulose ribbons, whereas Cel6A cut the ribbons into shorter elements, indicating an endo type of action. These observations, together with the examination of the digestion kinetics, indicate that Cel6A can be classified as an endo-processive enzyme, whereas Cel7A is essentially a processive enzyme. Thus, the synergy resulting from the mixing of Cel6A and Cel7A can be explained by the partial endo character of Cel6A. A preparation of bacterial cellulose ribbons appears to be an appropriate substrate, superior to Valonia or bacterial cellulose microcrystals, to visualize directly by TEM the endo-processivity of an enzyme such as Cel6A. PMID- 10742226 TI - Development of a fluorogenic probe-based PCR assay for detection of Bacillus cereus in nonfat dry milk. AB - A fluorogenic probe-based PCR assay was developed and evaluated for its utility in detecting Bacillus cereus in nonfat dry milk. Regions of the hemolysin and cereolysin AB genes from an initial group of two B. cereus isolates and two Bacillus thuringiensis isolates were cloned and sequenced. Three single-base differences in two B. cereus strains were identified in the cereolysin AB gene at nucleotides 866, 875, and 1287, while there were no species-consistent differences found in the hemolysin gene. A fluorogenic probe-based PCR assay was developed which utilizes the 5'-to-3' exonuclease of Taq polymerase, and two fluorogenic probes were evaluated. One fluorogenic probe (cerTAQ-1) was designed to be specific for the nucleotide differences at bases 866 and 875 found in B. cereus. A total of 51 out of 72 B. cereus strains tested positive with the cerTAQ 1 probe, while only 1 out of 5 B. thuringiensis strains tested positive. Sequence analysis of the negative B. cereus strains revealed additional polymorphism found in the cereolysin probe target. A second probe (cerTAQ-2) was designed to account for additional polymorphic sequences found in the cerTAQ-1-negative B. cereus strains. A total of 35 out of 39 B. cereus strains tested positive (including 10 of 14 previously negative strains) with cerTAQ-2, although the assay readout was uniformly lower with this probe than with cerTAQ-1. A PCR assay using cerTAQ-1 was able to detect approximately 58 B. cereus CFU in 1 g of artificially contaminated nonfat dry milk. Forty-three nonfat dry milk samples were tested for the presence of B. cereus with the most-probable-number technique and the fluorogenic PCR assay. Twelve of the 43 samples were contaminated with B. cereus at levels greater than or equal to 43 CFU/g, and all 12 of these samples tested positive with the fluorogenic PCR assay. Of the remaining 31 samples, 12 were B. cereus negative and 19 were contaminated with B. cereus at levels ranging from 3 to 9 CFU/g. All 31 of these samples were negative in the fluorogenic PCR assay. Although not totally inclusive, the PCR-based assay with cerTAQ-1 is able to specifically detect B. cereus in nonfat dry milk. PMID- 10742227 TI - Superoxide dismutase activity in Pseudomonas putida affects utilization of sugars and growth on root surfaces. AB - To investigate the role of superoxide dismutases (SOD) in root colonization and oxidative stress, mutants of Pseudomonas putida lacking manganese-superoxide dismutase (MnSOD) (sodA), iron-superoxide dismutase (FeSOD) (sodB), or both were generated. The sodA sodB mutant did not grow on components washed from bean root surfaces or glucose in minimal medium. The sodB and sodA sodB mutants were more sensitive than wild type to oxidative stress generated within the cell by paraquat treatment. In single inoculation of SOD mutants on bean, only the sodA sodB double mutant was impaired in growth on root surfaces. In mixed inoculations with wild type, populations of the sodA mutant were equal to those of the wild type, but levels of the sodB mutant and, to a great extent, the sodA sodB mutant, were reduced. Confocal microscopy of young bean roots inoculated with green fluorescent protein-tagged cells showed that wild type and SOD single mutants colonized well predominantly at the root tip but that the sodA sodB double mutant grew poorly at the tip. Our results indicate that FeSOD in P. putida is more important than MnSOD in aerobic metabolism and oxidative stress. Inhibition of key metabolic enzymes by increased levels of superoxide anion may cause the impaired growth of SOD mutants in vitro and in planta. PMID- 10742228 TI - Interspecific variability in sensitivity to UV radiation and subsequent recovery in selected isolates of marine bacteria. AB - The interspecific variability in the sensitivity of marine bacterial isolates to UV-B (295- to 320-nm) radiation and their ability to recover from previous UV-B stress were examined. Isolates originating from different microenvironments of the northern Adriatic Sea were transferred to aged seawater and exposed to artificial UV-B radiation for 4 h and subsequently to different radiation regimens excluding UV-B to determine the recovery from UV-B stress. Bacterial activity was assessed by thymidine and leucine incorporation measurements prior to and immediately after the exposure to UV-B and after the subsequent exposure to the different radiation regimens. Large interspecific differences among the 11 bacterial isolates were found in the sensitivity to UV-B, ranging from 21 to 92% inhibition of leucine incorporation compared to the bacterial activity measured in dark controls and from 14 to 84% for thymidine incorporation. Interspecific differences in the recovery from the UV stress were also large. An inverse relation was detectable between the ability to recover under dark conditions and the recovery under photosynthetic active radiation (400 to 700 nm). The observed large interspecific differences in the sensitivity to UV-B radiation and even more so in the subsequent recovery from UV-B stress are not related to the prevailing radiation conditions of the microhabitats from which the bacterial isolates originate. Based on our investigations on the 11 marine isolates, we conclude that there are large interspecific differences in the sensitivity to UV B radiation and even larger differences in the mechanisms of recovery from previous UV stress. This might lead to UV-mediated shifts in the bacterioplankton community composition in marine surface waters. PMID- 10742229 TI - 2,4,6-trinitrotoluene reduction by carbon monoxide dehydrogenase from Clostridium thermoaceticum. AB - Purified CO dehydrogenase (CODH) from Clostridium thermoaceticum catalyzed the transformation of 2,4,6-trinitrotoluene (TNT). The intermediates and reduced products of TNT transformation were separated and appear to be identical to the compounds formed by C. acetobutylicum, namely, 2-hydroxylamino-4,6-dinitrotoluene (2HA46DNT), 4-hydroxylamino-2,6-dinitrotoluene (4HA26DNT), 2, 4-dihydroxylamino-6 nitrotoluene (24DHANT), and the Bamberger rearrangement product of 2,4 dihydroxylamino-6-nitrotoluene. In the presence of saturating CO, CODH catalyzed the conversion of TNT to two monohydroxylamino derivatives (2HA46DNT and 4HA26DNT), with 4HA26DNT as the dominant isomer. These derivatives were then converted to 24DHANT, which slowly converted to the Bamberger rearrangement product. Apparent K(m) and k(cat) values of TNT reduction were 165 +/- 43 microM for TNT and 400 +/- 94 s(-1), respectively. Cyanide, an inhibitor for the CO/CO(2) oxidation/reduction activity of CODH, inhibited the TNT degradation activity of CODH. PMID- 10742230 TI - Sediment microbial community structure and mercury methylation in mercury polluted Clear Lake, California. AB - Spatial and temporal variations in sediment microbial community structure in a eutrophic lake polluted with inorganic mercury were identified using polar lipid fatty acid (PLFA) analysis. Microbial community structure was strongly related to mercury methylation potential, sediment organic carbon content, and lake location. Pore water sulfate, total mercury concentrations, and organic matter C/N ratios showed no relationships with microbial community structure. Seasonal changes and changes potentially attributable to temperature regulation of bacterial membranes were detectable but were less important influences on sediment PLFA composition than were differences due to lake sampling location. Analysis of biomarker PLFAs characteristic of Desulfobacter and Desulfovibrio groups of sulfate-reducing bacteria suggests that Desulfobacter-like organisms are important mercury methylators in the sediments, especially in the Lower Arm of Clear Lake. PMID- 10742231 TI - Ionizing-radiation resistance in the desiccation-tolerant cyanobacterium Chroococcidiopsis. AB - The effect of X-ray irradiation on cell survival, induction, and repair of DNA damage was studied by using 10 Chroococcidiopsis strains isolated from desert and hypersaline environments. After exposure to 2.5 kGy, the percentages of survival for the strains ranged from 80 to 35%. In the four most resistant strains, the levels of survival were reduced by 1 or 2 orders of magnitude after irradiation with 5 kGy; viable cells were recovered after exposure to 15 kGy but not after exposure to 20 kGy. The severe DNA damage evident after exposure to 2.5 kGy was repaired within 3 h, and the severe DNA damage evident after exposure to 5 kGy was repaired within 24 h. The increase in trichloroacetic acid-precipitable radioactivity in the culture supernatant after irradiation with 2.5 kGy might have been due to cell lysis and/or an excision process involved in DNA repair. The radiation resistance of Chroococcidiopsis strains may reflect the ability of these cyanobacteria to survive prolonged desiccation through efficient repair of the DNA damage that accumulates during dehydration. PMID- 10742232 TI - Effects of acid adaptation of Escherichia coli O157:H7 on efficacy of acetic acid spray washes to decontaminate beef carcass tissue. AB - Exposure to low pH and organic acids in the bovine gastrointestinal tract may result in the induced acid resistance of Escherichia coli O157:H7 and other pathogens that may subsequently contaminate beef carcasses. The effect of acid adaptation of E. coli O157:H7 on the ability of acetic acid spray washing to reduce populations of this organism on beef carcass tissue was examined. Stationary-phase acid resistance and the ability to induce acid tolerance were determined for a collection of E. coli O157:H7 strains by testing the survival of acid-adapted and unadapted cells in HCl-acidified tryptic soy broth (pH 2.5). Three E. coli O157:H7 strains that were categorized as acid resistant (ATCC 43895) or acid sensitive (ATCC 43890) or that demonstrated inducible acid tolerance (ATCC 43889) were used in spray wash studies. Prerigor beef carcass surface tissue was inoculated with bovine feces containing either acid-adapted or unadapted E. coli O157:H7. The beef tissue was subjected to spray washing treatments with water or 2% acetic acid or left untreated. For strains ATCC 43895 and 43889, larger populations of acid-adapted cells than of unadapted cells remained on beef tissue following 2% acetic acid treatments and these differences remained throughout 14 days of 4 degrees C storage. For both strains, numbers of acid-adapted cells remaining on tissue following 2% acetic acid treatments were similar to numbers of both acid-adapted and unadapted cells remaining on tissue following water treatments. For strain ATCC 43890, there was no difference between populations of acid-adapted and unadapted cells remaining on beef tissue immediately following 2% acetic acid treatments. These data indicate that adaptation to acidic conditions by E. coli O157:H7 can negatively influence the effectiveness of 2% acetic acid spray washing in reducing the numbers of this organism on carcasses. PMID- 10742233 TI - Characterization of the protocatechuic acid catabolic gene cluster from Streptomyces sp. strain 2065. AB - Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) catalyzes the ring cleavage step in the catabolism of aromatic compounds through the protocatechuate branch of the beta-ketoadipate pathway. A protocatechuate 3,4-dioxygenase was purified from Streptomyces sp. strain 2065 grown in p-hydroxybenzoate, and the N-terminal sequences of the beta- and alpha-subunits were obtained. PCR amplification was used for the cloning of the corresponding genes, and DNA sequencing of the flanking regions showed that the pcaGH genes belonged to a 6. 5-kb protocatechuate catabolic gene cluster; at least seven genes in the order pcaIJFHGBL appear to be transcribed unidirectionally. Analysis of the cluster revealed the presence of a pcaL homologue which encodes a fused gamma carboxymuconolactone decarboxylase/beta-ketoadipate enol-lactone hydrolase previously identified in the pca gene cluster from Rhodococcus opacus 1CP. The pcaIJ genes encoded proteins with a striking similarity to succinyl-coenzyme A (CoA):3-oxoacid CoA transferases of eukaryotes and contained an indel which is strikingly similar between high-G+C gram-positive bacteria and eukaryotes. PMID- 10742234 TI - Genetic and biochemical approach for characterization of resistance to Bacillus thuringiensis toxin Cry1Ac in a field population of the diamondback moth, Plutella xylostella. AB - Four subpopulations of a Plutella xylostella (L.) strain from Malaysia (F(4) to F(8)) were selected with Bacillus thuringiensis subsp. kurstaki HD-1, Bacillus thuringiensis subsp. aizawai, Cry1Ab, and Cry1Ac, respectively, while a fifth subpopulation was left as unselected (UNSEL-MEL). Bioassays at F(9) found that selection with Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai gave resistance ratios of >95, 10, 7, and 3, respectively, compared with UNSEL-MEL (>10,500, 500, >100, and 26, respectively, compared with a susceptible population, ROTH). Resistance to Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai in UNSEL-MEL declined significantly by F(9). The Cry1Ac-selected population showed very little cross-resistance to Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai (5-, 1-, and 4-fold compared with UNSEL-MEL), whereas the Cry1Ab-, B. thuringiensis subsp. kurstaki-, and B. thuringiensis subsp. aizawai-selected populations showed high cross-resistance to Cry1Ac (60-, 100-, and 70-fold). The Cry1Ac-selected population was reselected (F(9) to F(13)) to give a resistance ratio of >2,400 compared with UNSEL-MEL. Binding studies with (125)I-labeled Cry1Ab and Cry1Ac revealed complete lack of binding to brush border membrane vesicles prepared from Cry1Ac-selected larvae (F(15)). Binding was also reduced, although less drastically, in the revertant population, which indicates that a modification in the common binding site of these two toxins was involved in the resistance mechanism in the original population. Reciprocal genetic crosses between Cry1Ac-reselected and ROTH insects indicated that resistance was autosomal and showed incomplete dominance. At the highest dose of Cry1Ac tested, resistance was recessive while at the lowest dose it was almost completely dominant. The F(2) progeny from a backcross of F(1) progeny with ROTH was tested with a concentration of Cry1Ac which would kill 100% of ROTH moths. Eight of the 12 families tested had 60 to 90% mortality, which indicated that more than one allele on separate loci was responsible for resistance to Cry1Ac. PMID- 10742235 TI - Novel scheme for biosynthesis of aryl metabolites from L-phenylalanine in the fungus Bjerkandera adusta. AB - Aryl metabolite biosynthesis was studied in the white rot fungus Bjerkandera adusta cultivated in a liquid medium supplemented with L-phenylalanine. Aromatic compounds were analyzed by gas chromatography-mass spectrometry following addition of labelled precursors ((14)C- and (13)C-labelled L-phenylalanine), which did not interfere with fungal metabolism. The major aromatic compounds identified were benzyl alcohol, benzaldehyde (bitter almond aroma), and benzoic acid. Hydroxy- and methoxybenzylic compounds (alcohols, aldehydes, and acids) were also found in fungal cultures. Intracellular enzymatic activities (phenylalanine ammonia lyase, aryl-alcohol oxidase, aryl-alcohol dehydrogenase, aryl-aldehyde dehydrogenase, lignin peroxidase) and extracellular enzymatic activities (aryl-alcohol oxidase, lignin peroxidase), as well as aromatic compounds, were detected in B. adusta cultures. Metabolite formation required de novo protein biosynthesis. Our results show that L-phenylalanine was deaminated to trans-cinnamic acid by a phenylalanine ammonia lyase and trans-cinnamic acid was in turn converted to aromatic acids (phenylpyruvic, phenylacetic, mandelic, and benzoylformic acids); benzaldehyde was a metabolic intermediate. These acids were transformed into benzaldehyde, benzyl alcohol, and benzoic acid. Our findings support the hypothesis that all of these compounds are intermediates in the biosynthetic pathway from L-phenylalanine to aryl metabolites. Additionally, trans-cinnamic acid can also be transformed via beta-oxidation to benzoic acid. This was confirmed by the presence of acetophenone as a beta-oxidation degradation intermediate. To our knowledge, this is the first time that a beta oxidation sequence leading to benzoic acid synthesis has been found in a white rot fungus. A novel metabolic scheme for biosynthesis of aryl metabolites from L phenylalanine is proposed. PMID- 10742236 TI - Molecular identification of species from the Penicillium roqueforti group associated with spoiled animal feed. AB - The Penicillium roqueforti group has recently been split into three species, P. roqueforti, Penicillium carneum, and Penicillium paneum, on the basis of differences in ribosomal DNA sequences and secondary metabolite profiles. We reevaluated the taxonomic identity of 52 livestock feed isolates from Sweden, previously identified by morphology as P. roqueforti, by comparing the sequences of the ribosomal internal transcribed spacer region. Identities were confirmed with random amplified polymorphic DNA analysis and secondary metabolite profiles. Of these isolates, 48 were P. roqueforti, 2 were P. paneum, and 2 were Penicillium expansum. No P. carneum isolates were found. The three species produce different mycotoxins, but no obvious relationship between mold and animal disease was detected, based on medical records. P. roqueforti appears to dominate in silage, but the ecological and toxicological importance of P. carneum and P. paneum as feed spoilage fungi is not clear. This is the first report of P. expansum in silage. PMID- 10742237 TI - Increased growth of the microalga Chlorella vulgaris when coimmobilized and cocultured in alginate beads with the plant-growth-promoting bacterium Azospirillum brasilense. AB - Coimmobilization of the freshwater microalga Chlorella vulgaris and the plant growth-promoting bacterium Azospirillum brasilense in small alginate beads resulted in a significantly increased growth of the microalga. Dry and fresh weight, total number of cells, size of the microalgal clusters (colonies) within the bead, number of microalgal cells per cluster, and the levels of microalgal pigments significantly increased. Light microscopy revealed that both microorganisms colonized the same cavities inside the beads, though the microalgae tended to concentrate in the more aerated periphery while the bacteria colonized the entire bead. The effect of indole-3-acetic acid addition to microalgal culture prior to immobilization of microorganisms in alginate beads partially imitated the effect of A. brasilense. We propose that coimmobilization of microalgae and plant-growth-promoting bacteria is an effective means of increasing microalgal populations within confined environments. PMID- 10742238 TI - Sequencing and expression of additional xylanase genes from the hyperthermophile Thermotoga maritima FjSS3B.1. AB - Two genes, xynB and xynC, coding for xylanases were isolated from Thermotoga maritima FjSS3B.1 by a genomic-walking-PCR technique. Sequencing of the genes showed that they encode multidomain family 10 xylanases. Only XynB exhibited activity against xylan substrates. The temperature optimum (87 degrees C) and pH optimum (pH 6.5) of XynB are different from the previously reported xylanase, XynA (also a family 10 enzyme), from this organism. The catalytic domain expressed without other domains has a lower temperature optimum, is less thermostable, and has optimal activity at pH 6.5. Despite having a high level of sequence similarity to xynB, xynC appears to be nonfunctional since its encoded protein did not show significant activity on xylan substrates. PMID- 10742239 TI - Production of pectate lyases and cellulases by Chryseomonas luteola strain MFCL0 depends on the growth temperature and the nature of the culture medium: evidence for two critical temperatures. AB - Several extracellular enzymes that are responsible for plant tissue maceration were detected in culture supernatant of the psychrotrophic bacterium Chryseomonas luteola MFCL0. Isoelectrofocusing experiments showed that pectate lyase (PL) activity resulted from the cumulative action of three major isoenzymes, designated PLI, PLII, and PLIII. Cellulolytic activity was also detected in culture supernatants. These enzymes exhibited different behaviors with respect to growth temperature. PLII was not regulated by temperature, whereas PLI and PLIII were regulated similarly by growth temperature. Maximal levels of PLI and PLIII were produced at 14 degrees C when cells were grown in polygalacturonate containing synthetic medium and at around 20 to 24 degrees C in nutrient broth. In contrast, thermoregulation of cellulolytic activity production differed from thermoregulation of PL. The level of cellulolytic activity was low in all media at temperatures up to 20 degrees C, and then it increased dramatically until the temperature was 28 degrees C, which is the optimal temperature for growth of C. luteola. Previously, we defined the critical temperature by using the modified Arrhenius equation to characterize bacterial behavior. This approach consists of monitoring changes in the maximal specific growth rate as a function of temperature. Our most striking result was the finding that the temperature at which maximum levels of PLI and PLIII were produced in two different media was the same as the critical temperature for growth observed in these two media. PMID- 10742240 TI - Diversity of Salmonella strains isolated from the aquatic environment as determined by serotyping and amplification of the ribosomal DNA spacer regions. AB - Salmonella species are pathogenic bacteria often detected in sewage, freshwater, marine coastal water, and groundwater. Salmonella spp. can survive for long periods in natural waters, and the persistence of specific and epidemic strains is of great concern in public health. However, the diversity of species found in the natural environment remains unknown. The aim of this study was to investigate the diversity of Salmonella strains isolated from different natural aquatic systems within a Mediterranean coastal watershed (river, wastewater, and marine coastal areas). A total of 574 strains isolated from these natural environments were identified by both conventional serotyping and the ribosomal spacer heteroduplex polymorphism (RS-HP) method (M. A. Jensen and N. Straus, PCR Methods Appl. 3:186-194, 1993). More than 40 different serotypes were found, and some serotypes probably mobilized from widespread animal-rearing activities were detected only during storm events. These serotypes may be good indicators of specific contamination sources. Furthermore, the RS-HP method based on the PCR amplification of the intergenic spacer region between the 16S and 23S rRNA genes can produce amplicon profiles allowing the discrimination of species at both serotype and intraserotype levels. This method represents a powerful tool that could be used for rapid typing of Salmonella isolates. PMID- 10742241 TI - A binding site for Bacillus thuringiensis Cry1Ab toxin is lost during larval development in two forest pests. AB - The insecticidal activity and receptor binding properties of Bacillus thuringiensis Cry1A toxins towards the forest pests Thaumetopoea pityocampa (processionary moth) and Lymantria monacha (nun moth) were investigated. Cry1Aa, Cry1Ab, and Cry1Ac were highly toxic (corresponding 50% lethal concentration values: 956, 895, and 379 pg/microl, respectively) to first-instar T. pityocampa larvae. During larval development, Cry1Ab and Cry1Ac toxicity decreased with increasing age, although the loss of activity was more pronounced for Cry1Ab. Binding assays with (125)I-labelled Cry1Ab and brush border membrane vesicles from T. pityocampa first- and last-instar larvae detected a remarkable decrease in the overall Cry1Ab binding affinity in last-instar larvae, although saturable Cry1Ab binding to both instars was observed. Homologous competition experiments demonstrated the loss of one of the two Cry1Ab high-affinity binding sites detected in first-instar larvae. Growth inhibition assays with sublethal doses of Cry1Aa, Cry1Ab, and Cry1Ac in L. monacha showed that all three toxins were able to delay molting from second instar to third instar. Specific saturable binding of Cry1Ab was detected only in first- and second-instar larvae. Cry1Ab binding was not detected in last-instar larvae, although specific binding of Cry1Aa and Cry1Ac was observed. These results demonstrate a loss of Cry1Ab binding sites during development on the midgut epithelium of T. pityocampa and L. monacha, correlating in T. pityocampa with a decrease in Cry1Ab toxicity with increasing age. PMID- 10742242 TI - Bacillus thuringiensis delta-endotoxin Cry1C domain III can function as a specificity determinant for Spodoptera exigua in different, but not all, Cry1 Cry1C hybrids. AB - In order to test our hypothesis that Bacillus thuringiensis delta-endotoxin Cry1Ca domain III functions as a determinant of specificity for Spodoptera exigua, regardless of the origins of domains I and II, we have constructed by cloning and in vivo recombination a collection of hybrid proteins containing domains I and II of various Cry1 toxins combined with domain III of Cry1Ca. Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ea, and Cry1Fa all become more active against S. exigua when their domain III is replaced by (part of) that of Cry1Ca. This result shows that domain III of Cry1Ca is an important and versatile determinant of S. exigua specificity. The toxicity of the hybrids varied by a factor of 40, indicating that domain I and/or II modulate the activity as well. Cry1Da-Cry1Ca hybrids were an exception in that they were not significantly active against S. exigua or Manduca sexta, whereas both parental proteins were highly toxic. Incidentally, in a Cry1Ba-Cry1Ca hybrid, Cry1Ca domain III can also strongly increase toxicity for M. sexta. PMID- 10742243 TI - Comparison of methods for quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples. AB - Two PCR primer sets were developed for the detection and quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples. The specificity and sensitivity of these primers were tested. Both primer sets were suitable for detection, but only one set, cd3F-cd4R, was suitable for the quantification and enumeration of the functional community using most-probable number PCR and competitive PCR techniques. Quantification of cytochrome cd(1) denitrifiers taken from marine sediment and water samples was achieved using two different molecular techniques which target the nirS gene, and the results were compared to those obtained by using the classical cultivation method. Enumerations using both molecular techniques yielded similar results in seawater and sediment samples. However, both molecular techniques showed 1,000 or 10 times more cytochrome cd(1) denitrifiers in the sediment or water samples, respectively, than were found by use of the conventional cultivation method for counting. PMID- 10742244 TI - Compatible-solute-supported periplasmic expression of functional recombinant proteins under stress conditions. AB - The standard method of producing recombinant proteins such as immunotoxins (rITs) in large quantities is to transform gram-negative bacteria and subsequently recover the desired protein from inclusion bodies by intensive de- and renaturing procedures. The major disadvantage of this technique is the low yield of active protein. Here we report the development of a novel strategy for the expression of functional rIT directed to the periplasmic space of Escherichia coli. rITs were recovered by freeze-thawing of pellets from shaking cultures of bacteria grown under osmotic stress (4% NaCl plus 0.5 M sorbitol) in the presence of compatible solutes. Compatible solutes, such as glycine betaine and hydroxyectoine, are low molecular-weight osmolytes that occur naturally in halophilic bacteria and are known to protect proteins at high salt concentrations. Adding 10 mM glycine betaine for the cultivation of E. coli under osmotic stress not only allowed the bacteria to grow under these otherwise inhibitory conditions but also produced a periplasmic microenvironment for the generation of high concentrations of correctly folded rITs. Protein purified by combinations of metal ion affinity and size exclusion chromatography was substantially stabilized in the presence of 1 M hydroxyecotine after several rounds of freeze-thawing, even at very low protein concentrations. The binding properties and cytotoxic potency of the rITs were confirmed by competitive experiments. This novel compatible-solute-guided expression and purification strategy might also be applicable for high-yield periplasmic production of recombinant proteins in different expression systems. PMID- 10742245 TI - Role of molybdate and other transition metals in the accumulation of protochelin by Azotobacter vinelandii. AB - Both molybdate and iron are metals that are required by the obligately aerobic organism Azotobacter vinelandii to survive in the nutrient-limited conditions of its natural soil environment. Previous studies have shown that a high concentration of molybdate (1 mM) affects the formation of A. vinelandii siderophores such that the tricatecholate protochelin is formed to the exclusion of the other catecholate siderophores, azotochelin and aminochelin. It has been shown previously that molybdate combines readily with catecholates and interferes with siderophore function. In this study, we found that the manner in which each catecholate siderophore interacted with molybdate was consistent with the structure and binding potential of the siderophore. The affinity that each siderophore had for molybdate was high enough that stable molybdo-siderophore complexes were formed but low enough that the complexes were readily destabilized by Fe(3+). Thus, competition between Fe(3+) and molybdate did not appear to be the primary cause of protochelin accumulation; in addition, we determined that protochelin accumulated in the presence of vanadate, tungstate, Zn(2+), and Mn(2+). We found that all five of these metal ions partially inhibited uptake of (55)Fe-protochelin and (55)Fe-azotochelin complexes. Also, each of these metal ions partially inhibited the activity of ferric reductase, an enzyme important in the deferration of ferric siderophores. Our results suggest that protochelin accumulates in the presence of molybdate because protochelin uptake and conversion into its component parts, azotochelin and aminochelin, are inhibited by interference with ferric reductase. PMID- 10742246 TI - Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes. AB - We describe a new PCR-based method for distinguishing human and cow fecal contamination in coastal waters without culturing indicator organisms, and we show that the method can be used to track bacterial marker sequences in complex environments. We identified two human-specific genetic markers and five cow specific genetic markers in fecal samples by amplifying 16S ribosomal DNA (rDNA) fragments from members of the genus Bifidobacterium and the Bacteroides Prevotella group and performing length heterogeneity PCR and terminal restriction fragment length polymorphism analyses. Host-specific patterns suggested that there are species composition differences in the Bifidobacterium and Bacteroides Prevotella populations of human and cow feces. The patterns were highly reproducible among different hosts belonging to the same species. Additionally, all host-specific genetic markers were detected in water samples collected from areas frequently contaminated with fecal pollution. Ease of detection and longer survival in water made Bacteroides-Prevotella indicators better than Bifidobacterium indicators. Fecal 16S rDNA sequences corresponding to our Bacteroides-Prevotella markers comprised closely related gene clusters, none of which exactly matched previously published Bacteroides or Prevotella sequences. Our method detected host-specific markers in water at pollutant concentrations of 2.8 x 10(-5) to 2.8 x 10(-7) g (dry weight) of feces/liter and 6.8 x 10(-7) g (dry weight) of sewage/liter. Although our aim was to identify nonpoint sources of fecal contamination, the method described here should be widely applicable for monitoring spatial and temporal fluctuations in specific bacterial groups in natural environments. PMID- 10742247 TI - Anaerobic naphthalene degradation by microbial pure cultures under nitrate reducing conditions. AB - Pure bacterial cultures were isolated from a highly enriched denitrifying consortium previously shown to anaerobically biodegrade naphthalene. The isolates were screened for the ability to grow anaerobically in liquid culture with naphthalene as the sole source of carbon and energy in the presence of nitrate. Three naphthalene-degrading pure cultures were obtained, designated NAP-3-1, NAP 3-2, and NAP-4. Isolate NAP-3-1 tested positive for denitrification using a standard denitrification assay. Neither isolate NAP-3-2 nor isolate NAP-4 produced gas in the assay, but both consumed nitrate and NAP-4 produced significant amounts of nitrite. Isolates NAP-4 and NAP-3-1 transformed 70 to 90% of added naphthalene, and the transformation was nitrate dependent. No significant removal of naphthalene occurred under nitrate-limited conditions or in cell-free controls. Both cultures exhibited partial mineralization of naphthalene, representing 7 to 20% of the initial added (14)C-labeled naphthalene. After 57 days of incubation, the largest fraction of the radiolabel in both cultures was recovered in the cell mass (30 to 50%), with minor amounts recovered as unknown soluble metabolites. Nitrate consumption, along with the results from the (14)C radiolabel study, are consistent with the oxidation of naphthalene coupled to denitrification for NAP-3-1 and nitrate reduction to nitrite for NAP-4. Phylogenetic analyses based on 16S ribosomal DNA sequences of NAP-3-1 showed that it was closely related to Pseudomonas stutzeri and that NAP-4 was closely related to Vibrio pelagius. This is the first report we know of that demonstrates nitrate-dependent anaerobic degradation and mineralization of naphthalene by pure cultures. PMID- 10742248 TI - Role of tfdC(I)D(I)E(I)F(I) and tfdD(II)C(II)E(II)F(II) gene modules in catabolism of 3-chlorobenzoate by Ralstonia eutropha JMP134(pJP4). AB - The enzymes chlorocatechol-1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, and maleylacetate reductase allow Ralstonia eutropha JMP134(pJP4) to degrade chlorocatechols formed during growth in 2,4 dichlorophenoxyacetate or 3-chlorobenzoate (3-CB). There are two gene modules located in plasmid pJP4, tfdC(I)D(I)E(I)F(I) (module I) and tfdD(II)C(II)E(II)F(II) (module II), putatively encoding these enzymes. To assess the role of both tfd modules in the degradation of chloroaromatics, each module was cloned into the medium-copy-number plasmid vector pBBR1MCS-2 under the control of the tfdR regulatory gene. These constructs were introduced into R. eutropha JMP222 (a JMP134 derivative lacking pJP4) and Pseudomonas putida KT2442, two strains able to transform 3-CB into chlorocatechols. Specific activities in cell extracts of chlorocatechol-1,2-dioxygenase (tfdC), chloromuconate cycloisomerase (tfdD), and dienelactone hydrolase (tfdE) were 2 to 50 times higher for microorganisms containing module I compared to those containing module II. In contrast, a significantly (50-fold) higher activity of maleylacetate reductase (tfdF) was observed in cell extracts of microorganisms containing module II compared to module I. The R. eutropha JMP222 derivative containing tfdR tfdC(I)D(I)E(I)F(I) grew four times faster in liquid cultures with 3-CB as a sole carbon and energy source than in cultures containing tfdR tfdD(II)C(II)E(II)F(II). In the case of P. putida KT2442, only the derivative containing module I was able to grow in liquid cultures of 3-CB. These results indicate that efficient degradation of 3-CB by R. eutropha JMP134(pJP4) requires the two tfd modules such that TfdCDE is likely supplied primarily by module I, while TfdF is likely supplied by module II. PMID- 10742249 TI - Genetic diversity and biological control activity of novel species of closely related pseudomonads isolated from wheat field soils in South Australia. AB - Rhizobacteria closely related to two recently described species of pseudomonads, Pseudomonas brassicacearum and Pseudomonas thivervalensis, were isolated from two geographically distinct wheat field soils in South Australia. Isolation was undertaken by either selective plating or immunotrapping utilizing a polyclonal antibody raised against P. brassicacearum. A subset of 42 isolates were characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA), BIOLOG analysis, and gas chromatography-fatty acid methyl ester (GC-FAME) analysis and separated into closely related phenetic groups. More than 75% of isolates tested by ARDRA were found to have >95% similarity to either Pseudomonas corrugata or P. brassicacearum-P. thivervalensis type strains, and all isolates had >90% similarity to either type strain. BIOLOG and GC-FAME clustering showed a >70% match to ARDRA profiles. Strains representing different ARDRA groups were tested in two soil types for biological control activity against the soilborne plant pathogen Gaeumannomyces graminis var. tritici, the causative agent of take-all of wheat and barley. Three isolates out of 11 significantly reduced take-all-induced root lesions on wheat plants grown in a red-brown earth soil. Only one strain, K208, was consistent in reducing disease symptoms in both the acidic red-brown earth and a calcareous sandy loam. Results from this study indicate that P. brassicacearum and P. thivervalensis are present in Australian soils and that a level of genetic diversity exists within these two novel species but that this diversity does not appear to be related to geographic distribution. The result of the glasshouse pot trial suggests that some isolates of these species may have potential as biological control agents for plant disease. PMID- 10742250 TI - Expanding the known diversity and environmental distribution of an uncultured phylogenetic division of bacteria. AB - Culture-independent molecular phylogenetic methods were used to explore the breadth of diversity and environmental distribution of members of the division level "candidate" phylogenetic group WS6, recently discovered in a contaminated aquifer and with no cultivated representatives. A broad diversity of WS6 affiliated sequences were cloned from 7 of 12 environments investigated: mainly from anaerobic sediment environments. The number of sequences representing the WS6 candidate division was increased from 3 to 60 in this study. The extent of phylogenetic divergence (sequence difference) in this candidate division was found to be among the largest of any known bacterial division. This indicates that organisms representing the WS6 phylogenetic division offer a broad diversity of undiscovered biochemical and metabolic novelty. These results provide a framework for the further study of these evidently important kinds of organisms and tools, the sequences, with which to do so. PMID- 10742251 TI - Inactivation of a novel gene produces a phenotypic variant cell and affects the symbiotic behavior of Xenorhabdus nematophilus. AB - Xenorhabdus nematophilus is an insect pathogen that lives in a symbiotic association with a specific entomopathogenic nematode. During prolonged culturing, variant cells arise that are deficient in numerous properties. To understand the genetic mechanism underlying variant cell formation, a transposon mutagenesis approach was taken. Three phenotypically similar variant strains of X. nematophilus, each of which contained a single transposon insertion, were isolated. The insertions occurred at different locations in the chromosome. The variant strain, ANV2, was further characterized. It was deficient in several properties, including the ability to produce antibiotics and the stationary-phase induced outer membrane protein, OpnB. Unlike wild-type cells, ANV2 produced lecithinase. The emergence of ANV2 from the nematode host was delayed relative to the emergence of the parental strain. The transposon in ANV2 had inserted in a gene designated var1, which encodes a novel protein composed of 121 amino acid residues. Complementation analysis confirmed that the pleiotropic phenotype of the ANV2 strain was produced by inactivation of var1. Other variant strains were not complemented by var1. These results indicate that inactivation of a single gene was sufficient to promote variant cell formation in X. nematophilus and that disruption of genetic loci other than var1 can result in the same pleiotropic phenotype. PMID- 10742252 TI - A homokaryotic derivative of a Phanerochaete chrysosporium strain and its use in genomic analysis of repetitive elements. AB - Analysis of complex gene families in the lignin-degrading basidiomycete Phanerochaete chrysosporium has been hampered by the dikaryotic nuclear condition. To facilitate genetic investigations in P. chrysosporium strain BKM-F 1767, we isolated a homokaryon from regenerated protoplasts. The nuclear condition was established by PCR amplification of five unlinked genes followed by probing with allele-specific oligonucleotides. Under standard nitrogen-limited culture conditions, lignin peroxidase, manganese peroxidase, and glyoxal oxidase activities of the homokaryon were equivalent to those of the parental dikaryon. We used the homokaryon to determine the genomic organization and to assess transcriptional effects of a family of repetitive elements. Previous studies had identified an insertional mutation, Pce1, within lignin peroxidase allele lipI2. The element resembled nonautonomous class II transposons and was present in multiple copies in strain BKM-F-1767. In the present study, three additional copies of the Pce1-like element were cloned and sequenced. The distribution of elements was nonrandom; all localized to the same 3.7-Mb chromosome, as assessed by segregation analysis and Southern blot analysis of the homokaryon. Reverse transcription-PCR (RT-PCR) showed that Pce1 was not spliced from the lipI2 transcript in either the homokaryon or the parental dikaryon. However, both strains had equivalent lignin peroxidase activity, suggesting that some lip genes may be redundant. PMID- 10742253 TI - Nitrile hydratase and amidase from Rhodococcus rhodochrous hydrolyze acrylic fibers and granular polyacrylonitriles. AB - Rhodococcus rhodochrous NCIMB 11216 produced nitrile hydratase (320 nkat mg of protein(-1)) and amidase activity (38.4 nkat mg of protein(-1)) when grown on a medium containing propionitrile. These enzymes were able to hydrolyze nitrile groups of both granular polyacrylonitriles (PAN) and acrylic fibers. Nitrile groups of PAN40 (molecular mass, 40 kDa) and PAN190 (molecular mass, 190 kDa) were converted into the corresponding carbonic acids to 1.8 and 1.0%, respectively. In contrast, surfacial nitrile groups of acrylic fibers were only converted to the corresponding amides. X-ray photoelectron spectroscopy analysis showed that 16% of the surfacial nitrile groups were hydrolyzed by the R. rhodochrous enzymes. Due to the enzymatic modification, the acrylic fibers became more hydrophilic and thus, adsorption of dyes was enhanced. This was indicated by a 15% increase in the staining level (K/S value) for C. I. Basic Blue 9. PMID- 10742254 TI - Biodegradation of cis-1,4-polyisoprene rubbers by distinct actinomycetes: microbial strategies and detailed surface analysis. AB - Several actinomycetes isolated from nature were able to use both natural rubber (NR) and synthetic cis-1,4-polyisoprene rubber (IR) as a sole source of carbon. According to their degradation behavior, they were divided into two groups. Representatives of the first group grew only in direct contact to the rubber substrate and led to considerable disintegration of the material during cultivation. The second group consisted of weaker rubber decomposers that did not grow adhesively, as indicated by the formation of clear zones (translucent halos) around bacterial colonies after cultivation on NR dispersed in mineral agar. Taxonomic analysis of four selected strains based on 16S rRNA similarity examinations revealed two Gordonia sp. strains, VH2 and Kb2, and one Mycobacterium fortuitum strain, NF4, belonging to the first group as well as one Micromonospora aurantiaca strain, W2b, belonging to the second group. Schiff's reagent staining tests performed for each of the strains indicated colonization of the rubber surface, formation of a bacterial biofilm, and occurrence of compounds containing aldehyde groups during cultivation with NR latex gloves. Detailed analysis by means of scanning electron microscopy yielded further evidence for the two different microbial strategies and clarified the colonization efficiency. Thereby, strains VH2, Kb2, and NF4 directly adhered to and merged into the rubber material, while strain W2b produced mycelial corridors, especially on the surface of IR. Fourier transform infrared spectroscopy comprising the attenuated total reflectance technique was applied on NR latex gloves overgrown by cells of the Gordonia strains, which were the strongest rubber decomposers. Spectra demonstrated the decrease in number of cis 1,4 double bonds, the formation of carbonyl groups, and the change of the overall chemical environment, indicating that an oxidative attack at the double bond is the first metabolic step of the biodegradation process. PMID- 10742255 TI - Development and evaluation of a model predicting the survival of Escherichia coli O157:H7 NCTC 12900 in homemade eggplant salad at various temperatures, pHs, and oregano essential oil concentrations. AB - Homemade eggplant salad, a traditional Greek appetizer, was inoculated with Escherichia coli O157:H7 NCTC 12900 supplemented with different concentrations of oregano essential oil (0.0, 0.7, 1. 4, and 2.1% [vol/wt]) and stored at different temperatures (0, 5, 10, and 15 degrees C). The product's pH was adjusted to 4.0, 4.5, or 5. 0 with lemon juice. For each combination of the environmental factors, the bacterial counts were modeled, using the Baranyi model, as a function of time to estimate the kinetic parameters of the pathogen. A reduction of more than 1 log unit in E. coli O157:H7 counts was observed in all cases, and the death rate depended on the pH, the storage temperature, and the essential oil concentration. Separate quadratic models were developed with natural logarithms of the shoulder period and death rate as estimated by the growth model, as a function of temperature, pH, and oregano essential oil concentrations. These were further used to predict the population of E. coli O157:H7 NCTC 12900 from other inoculated eggplant salads at random conditions of temperature, pH, and oregano oil concentration. The predicted values were compared with viable-count measurements for validation. PMID- 10742256 TI - Phylogenetic relationships of butyrate-producing bacteria from the human gut. AB - Butyrate is a preferred energy source for colonic epithelial cells and is thought to play an important role in maintaining colonic health in humans. In order to investigate the diversity and stability of butyrate-producing organisms of the colonic flora, anaerobic butyrate-producing bacteria were isolated from freshly voided human fecal samples from three healthy individuals: an infant, an adult omnivore, and an adult vegetarian. A second isolation was performed on the same three individuals 1 year later. Of a total of 313 bacterial isolates, 74 produced more than 2 mM butyrate in vitro. Butyrate-producing isolates were grouped by 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphism analysis. The results indicate very little overlap between the predominant ribotypes of the three subjects; furthermore, the flora of each individual changed significantly between the two isolations. Complete sequences of 16S rDNAs were determined for 24 representative strains and subjected to phylogenetic analysis. Eighty percent of the butyrate-producing isolates fell within the XIVa cluster of gram-positive bacteria as defined by M. D. Collins et al. (Int. J. Syst. Bacteriol. 44:812-826, 1994) and A. Willems et al. (Int. J. Syst. Bacteriol. 46:195-199, 1996), with the most abundant group (10 of 24 or 42%) clustering with Eubacterium rectale, Eubacterium ramulus, and Roseburia cecicola. Fifty percent of the butyrate producing isolates were net acetate consumers during growth, suggesting that they employ the butyryl coenzyme A-acetyl coenzyme A transferase pathway for butyrate production. In contrast, only 1% of the 239 non-butyrate-producing isolates consumed acetate. PMID- 10742257 TI - The viable-but-nonculturable state induced by abiotic stress in the biocontrol agent Pseudomonas fluorescens CHA0 does not promote strain persistence in soil. AB - The effects of oxygen limitation, low redox potential, and high NaCl stress for 7 days in vitro on the rifampin-resistant biocontrol inoculant Pseudomonas fluorescens CHA0-Rif and its subsequent persistence in natural soil for 54 days were investigated. Throughout the experiment, the strain was monitored using total cell counts (immunofluorescence microscopy), Kogure's direct viable counts, and colony counts (on rifampin-containing plates). Under in vitro conditions, viable-but-nonculturable (VBNC) cells of CHA0-Rif were obtained when the strain was exposed to a combination of low redox potential (230 mV) and oxygen limitation. This mimics a situation observed in the field, where VBNC cells of the strain were found in the water-logged soil layer above the plow pan. Here, VBNC cells were also observed in vitro when CHA0-Rif was subjected to high NaCl levels (i.e., NaCl at 1.5 M but not 0.7 M). In all treatments, cell numbers remained close to the inoculum level for the first 12 days after inoculation of soil, regardless of the cell enumeration method used, but decreased afterwards. At the last two samplings in soil, VBNC cells of CHA0-Rif were found in all treatments except the one in which log-phase cells had been used. In the two treatments that generated high numbers of VBNC cells in vitro, VBNC cells did not display enhanced persistence compared with culturable cells once introduced into soil, which suggests that this VBNC state did not represent a physiological strategy to improve survival under adverse conditions. PMID- 10742258 TI - Use of length heterogeneity PCR and fatty acid methyl ester profiles to characterize microbial communities in soil. AB - In length heterogeneity PCR (LH-PCR) a fluorescently labeled primer is used to determine the relative amounts of amplified sequences originating from different microorganisms. Labeled fragments are separated by gel electrophoresis and detected by laser-induced fluorescence with an automated gene sequencer. We used LH-PCR to evaluate the composition of the soil microbial community. Four soils, which differed in terms of soil type and/or crop management practice, were studied. Previous data for microbial biomass, nitrogen and carbon contents, and nitrogen mineralization rates suggested that the microbial characteristics of these soils were different. One site received two different treatments: no-till and conventional till perennial ryegrass. The other sites were no-till continuous grass plots at separate locations with different soil types. Community composition was characterized by assessing the natural length heterogeneity in eubacterial sequences amplified from the 5' domain of the 16S rRNA gene and by determining fatty acid methyl ester (FAME) profiles. We found that LH-PCR results were reproducible. Both methods distinguished the three sites. The most abundant bacterial community members, based on cloned LH-PCR products, were members of the beta subclass of the class Proteobacteria, the Cytophaga-Flexibacter-Bacteriodes group, and the high-G+C-content gram-positive bacterial group. Strong correlations were found between LH-PCR results and FAME results. We found that the LH-PCR method is an efficient, reliable, and highly reproducible method that should be a useful tool in future assessments of microbial community composition. PMID- 10742259 TI - Design and application of a biosensor for monitoring toxicity of compounds to eukaryotes. AB - Here we describe an alternative approach to currently used cytotoxicity analyses through applying eukaryotic microbial biosensors. The yeast Saccharomyces cerevisiae was genetically modified to express firefly luciferase, generating a bioluminescent yeast strain. The presence of any toxic chemical that interfered with the cells' metabolism resulted in a quantitative decrease in bioluminescence. In this study, it was demonstrated that the luminescent yeast strain senses chemicals known to be toxic to eukaryotes in samples assessed as nontoxic by prokaryotic biosensors. As the cell wall and adaptive mechanisms of S. cerevisiae cells enhance stability and protect from extremes of pH, solvent exposure, and osmotic shock, these inherent properties were exploited to generate a biosensor that should detect a wide range of both organic and inorganic toxins under extreme conditions. PMID- 10742260 TI - Engineering desiccation tolerance in Escherichia coli. AB - Recombinant sucrose-6-phosphate synthase (SpsA) was synthesized in Escherichia coli BL21DE3 by using the spsA gene of the cyanobacterium Synechocystis sp. strain PCC 6803. Transformants exhibited a 10,000-fold increase in survival compared to wild-type cells following either freeze-drying, air drying, or desiccation over phosphorus pentoxide. The phase transition temperatures and vibration frequencies (P==O stretch) in phospholipids suggested that sucrose maintained membrane fluidity during cell dehydration. PMID- 10742261 TI - Bacterial symbiont transmission in the wood-boring shipworm Bankia setacea (Bivalvia: Teredinidae). AB - The Teredinidae (shipworms) are a morphologically diverse group of marine wood boring bivalves that are responsible each year for millions of dollars of damage to wooden structures in estuarine and marine habitats worldwide. They exist in a symbiosis with cellulolytic nitrogen-fixing bacteria that provide the host with the necessary enzymes for survival on a diet of wood cellulose. These symbiotic bacteria reside in distinct structures lining the interlamellar junctions of the gill. This study investigated the mode by which these nutritionally essential bacterial symbionts are acquired in the teredinid Bankia setacea. Through 16S ribosomal DNA (rDNA) sequencing, the symbiont residing within the B. setacea gill was phylogenetically characterized and shown to be distinct from previously described shipworm symbionts. In situ hybridization using symbiont-specific 16S rRNA-directed probes bound to bacterial ribosome targets located within the host gill coincident with the known location of the gill symbionts. These specific probes were then used as primers in a PCR-based assay which consistently detected bacterial rDNA in host gill (symbiont containing), gonad tissue, and recently spawned eggs, demonstrating the presence of symbiont cells in host ovary and offspring. These results suggest that B. setacea ensures successful inoculation of offspring through a vertical mode of symbiont transmission and thereby enables a broad distribution of larval settlement. PMID- 10742262 TI - Natural assemblages of marine proteobacteria and members of the Cytophaga Flavobacter cluster consuming low- and high-molecular-weight dissolved organic matter. AB - We used a method that combines microautoradiography with hybridization of fluorescent rRNA-targeted oligonucleotide probes to whole cells (MICRO-FISH) to test the hypothesis that the relative contributions of various phylogenetic groups to the utilization of dissolved organic matter (DOM) depend solely on their relative abundance in the bacterial community. We found that utilization of even simple low-molecular-weight DOM components by bacteria differed across the major phylogenetic groups and often did not correlate with the relative abundance of these bacterial groups in estuarine and coastal environments. The Cytophaga Flavobacter cluster was overrepresented in the portion of the assemblage consuming chitin, N-acetylglucosamine, and protein but was generally underrepresented in the assemblage consuming amino acids. The amino acid consuming assemblage was usually dominated by the alpha subclass of the class Proteobacteria, although the representation of alpha-proteobacteria in the protein-consuming assemblages was about that expected from their relative abundance in the entire bacterial community. In our experiments, no phylogenetic group dominated the consumption of all DOM, suggesting that the participation of a diverse assemblage of bacteria is essential for the complete degradation of complex DOM in the oceans. These results also suggest that the role of aerobic heterotrophic bacteria in carbon cycling would be more accurately described by using three groups instead of the single bacterial compartment currently used in biogeochemical models. PMID- 10742263 TI - A tetrodotoxin-producing Vibrio strain, LM-1, from the puffer fish Fugu vermicularis radiatus. AB - Identification of tetrodotoxin (TTX) and its derivatives produced from a Vibrio strain in the intestine of the puffer fish Fugu vermicularis radiatus was performed by thin-layer chromatography, electrophoresis, high-performance liquid chromatography, and gas chromatography-mass spectrometry, together with a mouse bioassay for toxicity. It was demonstrated that the isolated bacterium produced TTX, 4-epi-TTX, and anhTTX during cultivation, suggesting that Vibrio strains are responsible for the toxification of the puffer fish. PMID- 10742264 TI - Chlorine, chloramine, chlorine dioxide, and ozone susceptibility of Mycobacterium avium. AB - Environmental and patient isolates of Mycobacterium avium were resistant to chlorine, monochloramine, chlorine dioxide, and ozone. For chlorine, the product of the disinfectant concentration (in parts per million) and the time (in minutes) to 99.9% inactivation for five M. avium strains ranged from 51 to 204. Chlorine susceptibility of cells was the same in washed cultures containing aggregates and in reduced aggregate fractions lacking aggregates. Cells of the more slowly growing strains were more resistant to chlorine than were cells of the more rapidly growing strains. Water-grown cells were 10-fold more resistant than medium-grown cells. Disinfectant resistance may be one factor promoting the persistence of M. avium in drinking water. PMID- 10742265 TI - Significance of inoculum size in the lag time of Listeria monocytogenes. AB - The lag time of Listeria monocytogenes growing under suboptimal conditions (low nutrient concentrations, pH 6, and 6.5 degrees C) was extended when the inoculum was severely stressed by starvation and the inoculum size was very small. Predictive microbiology should deal with bacterial stress and stochastic approaches to improve its value for the agro-food industry. PMID- 10742266 TI - Expression of the antifeeding gene anfA1 in Serratia entomophila requires rpoS. AB - The rpoS gene of Serratia entomophila BC4B was cloned and used to create rpoS mutant strain BC4BRS. Larvae of the New Zealand grass grub Costelytra zealandica infected with BC4BRS became amber colored but continued to feed, albeit to a lesser extent than infected larvae. Subsequently, we found that expression of the antifeeding gene anfA1 in trans was substantially reduced in BC4BRS relative to that in the parental strain BC4B. Our data show that a functional rpoS gene is vital for full expression of anfA1 and for development of the antifeeding component of amber disease. PMID- 10742268 TI - An in vitro study of the effect of fluoridated milk on oral bacterial biofilms. AB - Microcosmic dental plaques were grown in artificial saliva and supplemented with either milk or fluoridated milk. The presence of fluoride in the milk increased the pH of the biofilms and reduced the proportions of streptococci, demonstrating that in this model, fluoridation of milk produces biofilms with reduced cariogenic potential. PMID- 10742267 TI - Prokaryotic diversity in Zostera noltii-colonized marine sediments. AB - The diversity of microorganisms present in a sediment colonized by the phanerogam Zostera noltii has been analyzed. Microbial DNA was extracted and used for constructing two 16S rDNA clone libraries for Bacteria and Archaea. Bacterial diversity was very high in these samples, since 57 different sequences were found among the 60 clones analyzed. Eight major lineages of the Domain Bacteria were represented in the library. The most frequently retrieved bacterial group (36% of the clones) was delta-Proteobacteria related to sulfate-reducing bacteria. The second most abundant group (27%) was gamma-Proteobacteria, including five clones closely related to S-oxidizing endosymbionts. The archaeal clone library included members of Crenarchaeota and Euryarchaeota, with nine different sequences among the 15 analyzed clones, indicating less diversity when compared to the Bacteria organisms. None of these sequences was closely related to cultured Archaea organisms. PMID- 10742269 TI - Effect of sample holding time on recovery of Cryptosporidium oocysts and Giardia cysts from water samples. AB - U.S. Environmental Protection Agency methods for analysis of water for Cryptosporidium and Giardia stipulate maximum sample holding times which are not always practical to comply with. A spiking experiment indicated that holding times of up to 2 weeks had no significant effect on recovery of these parasites from 10-liter samples of raw water in plastic carboys. PMID- 10742270 TI - Effects of acid adaptation, product pH, and heating on survival of Escherichia coli O157:H7 in pepperoni. AB - The thermotolerance of E. coli O157:H7 cells (strain 380-94) heated in pepperoni is reported. Information on the pattern of thermal inactivation of E. coli O157:H7 in pepperoni was applied in the development of heating processes designed to reduce E. coli O157:H7 numbers therein by 5 log(10) units. PMID- 10742271 TI - Effect of copper speciation on whole-cell soluble methane monooxygenase activity in Methylosinus trichosporium OB3b. AB - Soluble methane monooxygenase (sMMO) activity in Methylosinus trichosporium OB3b was found to be more strongly affected as copper-to-biomass ratios changed in a newly developed medium, M2M, which uses pyrophosphate for metal chelation, than in nitrate mineral salts (NMS), which uses EDTA. When M2M medium was amended with EDTA, sMMO activity was similar to that in NMS medium, indicating that EDTA-bound copper had lower bioavailability than pyrophosphate-bound copper. EDTA did not limit the association of copper with the cells; rather, copper was sequestered in a form which did not affect sMMO activity. PMID- 10742272 TI - Genetic and biochemical characterization of a highly thermostable alpha-L arabinofuranosidase from Thermobacillus xylanilyticus. AB - The gene encoding an alpha-L-arabinofuranosidase from Thermobacillus xylanilyticus D3, AbfD3, was isolated. Characterization of the purified recombinant alpha-L-arabinofuranosidase produced in Escherichia coli revealed that it is highly stable with respect to both temperature (up to 90 degrees C) and pH (stable in the pH range 4 to 12). On the basis of amino acid sequence similarities, this 56, 071-Da enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. However, substrate specificity analysis revealed that AbfD3, unlike the majority of F51 members, displays high activity in the presence of polysaccharides. PMID- 10742273 TI - Involvement of two plasmids in fenitrothion degradation by Burkholderia sp. strain NF100. AB - A bacterium capable of utilizing fenitrothion (O,O-dimethyl O-4-nitro-m-tolyl phosphorothioate) as a sole carbon source was isolated from fenitrothion-treated soil. This bacterium was characterized taxonomically as being a member of the genus Burkholderia and was designated strain NF100. NF100 first hydrolyzed an organophosphate bond of fenitrothion, forming 3-methyl-4-nitrophenol, which was further metabolized to methylhydroquinone. The ability to degrade fenitrothion was found to be encoded on two plasmids, pNF1 and pNF2. PMID- 10742274 TI - Cloning, sequencing, and expression in Escherichia coli of the new gene encoding beta-1,3-xylanase from a marine bacterium, Vibrio sp. strain XY-214. AB - The Vibrio sp. strain XY-214 beta-1,3-xylanase gene cloned in Escherichia coli DH5alpha consisted of an open reading frame of 1, 383 nucleotides encoding a protein of 460 amino acids with a molecular mass of 51,323 Da and had a signal peptide of 22 amino acids. The transformant enzyme hydrolyzed beta-1,3-xylan to produce several xylooligosaccharides. PMID- 10742275 TI - Method for rapid purification of class IIa bacteriocins and comparison of their activities. AB - A three-step method was developed for the purification of mesentericin Y105 (60% yield) from the culture supernatant of Leuconostoc mesenteroides Y105. The same procedure was successfully applied to the purification of five other anti Listeria bacteriocins identified by mass spectrometry. Specific activities of the purified bacteriocins were compared. PMID- 10742276 TI - Characterization of a novel integrative element, ICESt1, in the lactic acid bacterium Streptococcus thermophilus. AB - The 35.5-kb ICESt1 element of Streptococcus thermophilus CNRZ368 is bordered by a 27-bp repeat and integrated into the 3' end of a gene encoding a putative fructose-1,6-biphosphate aldolase. This element encodes site-specific integrase and excisionase enzymes related to those of conjugative transposons Tn5276 and Tn5252. The integrase was found to be involved in a site-specific excision of a circular form. ICESt1 also encodes putative conjugative transfer proteins related to those of the conjugative transposon Tn916. Therefore, ICESt1 could be or could be derived from an integrative conjugative element. PMID- 10742277 TI - Efficient heterologous expression in Aspergillus oryzae of a unique dye decolorizing peroxidase, DyP, of Geotrichum candidum Dec 1. AB - Efficient expression of the dye-decolorizing peroxidase, DyP, from Geotrichum candidum Dec 1 in Aspergillus oryzae M-2-3 was achieved by fusing mature cDNA encoding dyp with the A. oryzae alpha-amylase promoter (amyB). The activity yield of the purified recombinant DyP (rDyP) was 42-fold compared with that of the purified native DyP from Dec 1. No exogenous heme was necessary for the expression of rDyP in A. oryzae. From the N-terminal amino acid sequence analyses of native DyP and rDyP, the absence of a histidine residue in both DyPs, which was considered to be important for heme binding of DyP, was confirmed. These results suggest that rDyP without a typical heme-binding region produced by A. oryzae exhibits a function similar to that of native DyP. PMID- 10742278 TI - Pathogenic role of SEF14, SEF17, and SEF21 fimbriae in Salmonella enterica serovar enteritidis infection of chickens. AB - Very little is known about the contribution of surface appendages of Salmonella enterica serovar Enteritidis to pathogenesis in chickens. This study was designed to clarify the role of SEF14, SEF17, and SEF21 fimbriae in serovar Enteritidis pathogenesis. Stable, single, defined sefA (SEF14), agfA (SEF17), and fimA (SEF21) insertionally inactivated fimbrial gene mutants of serovar Enteritidis were constructed. All mutant strains invaded Caco-2 and HT-29 enterocytes at levels similar to that of the wild type. Both mutant and wild-type strains were ingested equally well by chicken macrophage cell lines HD11 and MQ-NCSU. There were no significant differences in the abilities of these strains to colonize chicken ceca. The SEF14(-) strain was isolated in lower numbers from the livers of infected chickens and was cleared from the spleens faster than other strains. No significant differences in fecal shedding of these strains were observed. PMID- 10742279 TI - Identification of motile Aeromonas strains with the MicroScan WalkAway system in conjunction with the combo negative type 1S panels. AB - This study was performed to compare the MicroScan WalkAway automated identification system in conjunction with the new MicroScan Combo Negative type 1S panels with conventional biochemical methods for identifying 85 environmental, clinical, and reference strains of eight Aeromonas species. PMID- 10742281 TI - Negative inotropic effects of isoprenaline on isolated left atrial assays from aged transgenic mice with cardiac over-expression of human beta(2)-adrenoceptors. AB - The action of isoprenaline has been evaluated in an isolated, left atrial assay, from aged transgenic mice with cardiac-specific over-expression of the beta(2) adrenoceptor. In the assay, isoprenaline produced a negative inotropic concentration-response curve that was not altered by incubation with CGP-20712A (1 microM), a beta(1)-adrenoceptor antagonist. However, after incubation with ICI 118,551 (300 nM), a selective beta(2)-adrenoceptor antagonist, isoprenaline produced a positive inotropic concentration-effect curve that was located to the left of the negative inotropic curve. This suggests that the negative inotropic effect was mediated by a homogenous population of negatively-coupled beta(2) adrenoceptors. In the presence of CGP-20712A (300 nM), the positive curve was shifted to the right, suggesting that the positive inotropic effect was mediated, at least in part, by beta(1)-adrenoceptors. These results differ substantially from those previously obtained in young transgenic mice. An outline of an explanatory model, based on a concept of over-expressed receptors 'stealing' G proteins, is suggested. PMID- 10742282 TI - The hypocretins are weak agonists at recombinant human orexin-1 and orexin-2 receptors. AB - The pharmacology of the orexin-like peptides, hypocretin-1 and hypocretin-2, was studied in Chinese hamster ovary (CHO) cells stably expressing orexin-1 (OX(1)) or orexin-2 (OX(2)) receptors by measuring intracellular calcium ([Ca(2+)](i)) using Fluo-3AM. Orexin-A and orexin-B increased [Ca(2+)](i) in CHO-OX(1) (pEC(50)=7. 99+/-0.05 and 7.00+/-0.10 respectively, n=8) and CHO-OX(2) (pEC(50)=8.30+/-0.05 and 8.21+/-0.07 respectively, n=5). However, hypocretin-1 and hypocretin-2 were markedly less potent, with pEC(50) values of 5.31+/-0.04 and 5.41+/-0.04 respectively in CHO-OX(2) cells (n=5). In CHO-OX(1) cells 10 microM hypocretin-1 only elicited a 37.5+/-3.4% response whilst 10 microM hypocretin-2 elicited a 18.0+/-2.1% response (n=8). Desensitisation of OX(1) or OX(2) with orexin-A (100 nM) abolished the response to orexin-A (10 nM) and the hypocretins (10 microM), but not to UTP (3 microM). In conclusion, the hypocretins are only weak agonists at the orexin receptors. PMID- 10742280 TI - Pharmacology of nociceptin and its receptor: a novel therapeutic target. AB - Nociceptin (NC), alias Orphanin FQ, has been recently identified as the endogenous ligand of the opioid receptor-like 1 receptor (OP(4)). This new NC/OP(4) receptor system belongs to the opioid family and has been characterized pharmacologically with functional and binding assays on native (mouse, rat, guinea-pig) and recombinant (human) receptors, by using specific and selective agonists (NC, NC(1 - 13)NH(2)) and a pure and competitive antagonist, [Nphe(1)]NC(1 - 13)NH(2). The similar order of potency of agonists and affinity values of the antagonist indicate that the same receptor is present in the four species. OP(4) is expressed in neurons, where it reduces activation of adenylyl cyclase and Ca(2+) channels while activating K(+) channels in a manner similar to opioids. In this way, OP(4) mediates inhibitory effects in the autonomic nervous system, but its activities in the central nervous system can be either similar or opposite to those of opioids. In vivo experiments have demonstrated that NC modulates a variety of biological functions ranging from nociception to food intake, from memory processes to cardiovascular and renal functions, from spontaneous locomotor activity to gastrointestinal motility, from anxiety to the control of neurotransmitter release at peripheral and central sites. These actions have been demonstrated using NC and various pharmacological tools, as antisense oligonucleotides targeting OP(4) or the peptide precursor genes, antibodies against NC, an OP(4) receptor selective antagonist and with data obtained from animals in which the receptor or the peptide precursor genes were knocked out. These new advances have contributed to better understanding of the pathophysiological role of the NC/OP(4) system, and ultimately will help to identify the therapeutic potential of new OP(4) receptor ligands. PMID- 10742283 TI - Augmentation of SR Ca(2+) release by rapamycin and FK506 causes K(+)-channel activation and membrane hyperpolarization in bladder smooth muscle. AB - 1. The immunosuppressants rapamycin and FK506 are known to relax smooth muscle despite facilitating Ca(2+) release through ryanodine-receptors of the sarcoplasmic reticulum (SR). The apparent contradiction was studied in isolated guinea-pig urinary bladder myocytes. 2. Modulation of spontaneous SR Ca(2+) release was monitored by means of spontaneous transient outward currents (or STOCs) in isolated smooth muscle cells voltage-clamped to -20 mV. Rapamycin (10 microM, n=18) significantly increased amplitude (50+/-12%, mean+/-s.d.), life time (77+/-19%), and time integral of STOCs (113+/-22%), and it reduced the interval between STOCs (20+/-7%). FK506 (20 microM, n=24) increased amplitude (15+/-7%), life time (50+/-7%), time integral (104+/-26%). Cyclosporin A (20 microM, n=18) had no significant effects on STOCs. 3. The basal cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) measured by Indo1-fluorescence was insensitive to rapamycin or FK506. Pretreatment with rapamycin (20 microM, 2 min) did not impair the SR Ca(2+) load as can be concluded from caffeine-induced Ca(2+) transients. 4. As it was expected from the enhanced STOC activity, the non clamped membrane was hyperpolarized by rapamycin (15+/-2 mV) or by FK506 (15+/-3 mV). 5. The data are consistent with the idea that rapamycin and FK506 augment spontaneous SR Ca(2+) release by removal of FK-binding proteins from the RyR complex. Smooth muscle relaxation is interpreted as negative Ca(2+) feedback: augmented Ca(2+) activation of STOCs induces membrane hyperpolarization that reduces Ca(2+) influx through voltage gated channels. PMID- 10742284 TI - Differential effects of 17beta-estradiol and testosterone on the contractile responses of porcine coronary arteries. AB - 1. We investigated the effects of short-term exposure to physiological levels of 17beta-estradiol and testosterone on vasocontractile responses in porcine coronary artery rings. 2. Concentration-response curves to endothelin-1, 5 hydroxytryptamine, the thromboxane analogue U46619 and KCl were constructed in endothelium-intact and endothelium-disrupted artery rings. 3. Thirty minutes exposure to 17beta-estradiol (1 and 30 nM) significantly attenuated vasoconstriction to endothelin-1, 5-hydroxytryptamine and U46619. Conversely, the same concentrations of testosterone significantly potentiated responses elicited by these contractile agents. These inhibitory effects of 17beta-estradiol and enhancing actions of testosterone on contractions were endothelium-independent. KCl-mediated contractions were unaffected by the presence of either sex hormones. 4. The oestrogen receptor antagonists, tamoxifen (10 microM) and ICI 182,780 (10 microM), were unable to reverse the inhibitory influence 1 nM 17beta-estradiol had on the agonist-mediated contractile responses. Similarly, the androgen receptor antagonists, flutamide (10 microM) and cyproterone acetate (10 microM), failed to affect the potentiating activities of 1 nM testosterone. The alteration in vasoconstrictive responses observed following acute exposure to either 1 nM 17beta-estradiol and 1 nM testosterone were apparent even in the presence of the protein synthesis inhibitor cycloheximide (10 microM) and the transcription inhibitor actinomycin D (10 microM). 6. In conclusion, we report a unique type of sex hormone action on the coronary vasculature. These events occur at low nanomolar concentrations of 17beta-estradiol and testosterone, are insensitive to conventional sex hormone receptor antagonists, are not blocked by de novo protein synthesis inhibitors and have rapid time-courses that are uncharacteristic of classical genomic activities. PMID- 10742285 TI - The role of lipopolysaccharide injected systemically in the reactivation of collagen-induced arthritis in mice. AB - 1. We investigated the role of bacterial lipopolysaccharide (LPS) in the reactivation of autoimmune disease by using collagen-induced arthritis (CIA) in mice in which autoimmunity to the joint cartilage component type II collagen (CII) was involved. 2. CIA was induced by immunization with CII emulsified with complete Freund's adjuvant at the base of the tail (day 0) followed by a booster injection on day 21. Varying doses of LPS from E. coli were i.p. injected on day 50. 3. Arthritis began to develop on day 25 after immunization with CII and reached a peak on day 35. Thereafter, arthritis subsided gradually but moderate joint inflammation was still observed on day 50. An i.p. injection of LPS on day 50 markedly reactivated arthritis on a dose-related fashion. Histologically, on day 55, there were marked oedema of synovium which had proliferated by the day of LPS injection, new formation of fibrin, and intense infiltration of neutrophils accompanied with a large number of mononuclear cells. The reactivation of CIA by LPS was associated with increases in anti-CII IgG and IgG2a antibodies as well as various cytokines including IL-12, IFN-gamma, IL-1beta, and TNF-alpha. LPS from S. enteritidis, S. typhimurium, and K. neumoniae and its component, lipid A from E. coli also reactivated the disease. Polymyxin B sulphate suppressed LPS- or lipid A-induced reactivation of CIA. 4. These results suggest that LPS may play an important role in the reactivation of autoimmune joint inflammatory diseases such as rheumatoid arthritis in humans. PMID- 10742286 TI - Nitrergic relaxation of the mouse gastric fundus is mediated by cyclic GMP dependent and ryanodine-sensitive mechanisms. AB - 1. Ryanodine-sensitive, Ca(2+) release ('Ca(2+) sparks') from the sarcoplasmic reticulum (SR) can activate plasmalemmal Ca(2+)-activated K(+) channels (K(Ca)) to cause membrane hyperpolarization and smooth muscle relaxation. Since cyclic guanosine monophosphate (cyclic GMP) can modulate Ca(2+) spark activity, the aim of the present study was to determine if Ca(2+) spark-like events are involved in NO-dependent, NANC relaxations to electrical field stimulation (EFS) of mouse, longitudinal smooth muscle of the gastric fundus in isolated strips contracted to approximately 40% of their maximum contraction. 2. NANC relaxations to EFS were almost abolished by both the NO synthase inhibitor, N(G)-nitro-L-arginine (L NOARG; 100 microM) and the guanylate cyclase inhibitor, 1-H-oxodiazol-[1,2,4] [4,3-alpha] quinoxaline-1-one (ODQ; 10 microM). Also, ODQ abolished relaxations to the NO donor, sodium nitroprusside (SNP; 1 nM - 30 microM). NANC relaxations and SNP-evoked relaxations were both partly ryanodine (10 microM)- and nifedipine (0.3 microM)-sensitive, but in each case, the inhibitory effects of ryanodine and nifedipine were additive. 3. Apamin (1 microM), charybdotoxin (0.1 microM), iberiotoxin (0.1 microM), tetraethylammonium (TEA; 1 mM), glibenclamide (10 microM) and 4-aminopyridine (1 mM) had no effect on either NANC- or SNP-evoked relaxations, the latter of which were also unaffected by high extracellular K(+) (68 mM). 4. Caffeine (0.1 - 1 mM) caused concentration-dependent relaxations of gastric fundus which were inhibited by ryanodine but unaffected by L-NOARG. 5. Relaxation to ATP (30 microM) was abolished by nifedipine, partly inhibited by apamin and ryanodine, but was unaffected by L-NOARG. 6. In conclusion, the results of the present study show that nitrergic relaxations in the mouse longitudinal gastric fundus occur via a cyclic GMP-activated ryanodine-sensitive mechanism, which does not appear to involve activation of K(+) channels. PMID- 10742287 TI - Pharmacology of human sulphonylurea receptor SUR1 and inward rectifier K(+) channel Kir6.2 combination expressed in HEK-293 cells. AB - 1. The pharmacological properties of K(ATP) channels generated by stable co expression of the sulphonylurea receptor SUR1 and the inwardly rectifying K(+) channel Kir6.2 were characterized in HEK-293 cells. 2. [(3)H]-Glyburide (glibenclamide) bound to transfected cells with a B(max) value of 18.5 pmol mg( 1) protein and with a K(D) value of 0.7 nM. Specific binding was displaced by a series of sulphonylurea analogues with rank order potencies consistent with those observed in pancreatic RINm5F insulinoma and in the brain. 3. Functional activity of K(ATP) channels was assessed by whole cell patch clamp, cation efflux and membrane potential measurements. Whole cell currents were detected in transfected cells upon depletion of internal ATP or by exposure to 500 microM diazoxide. The currents showed weak inward rectification and were sensitive to inhibition by glyburide (IC(50)=0.92 nM). 4. Metabolic inhibition by 2-deoxyglucose and oligomycin treatment triggered (86)Rb(+) efflux from transfected cells that was sensitive to inhibition by glyburide (IC(50)=3.6 nM). 5. Diazoxide, but not levcromakalim, evoked concentration-dependen decreases in DiBAC(4)(3) fluorescence responses with an EC(50) value of 14.1 microM which were attenuated by the addition of glyburide. Diazoxide-evoked responses were inhibited by various sulphonylurea analogues with rank order potencies that correlated well with their binding affinities. 6. In summary, results from ligand binding and functional assays demonstrate that the pharmacological properties of SUR1 and Kir6.2 channels co-expressed in HEK-293 cells resemble those typical of native K(ATP) channels described in pancreatic and neuronal tissues. PMID- 10742288 TI - Characterization of human recombinant alpha(2A)-adrenoceptors expressed in Chinese hamster lung cells using extracellular acidification rate changes. AB - 1. Human alpha(2A)-adrenoceptors heterologously expressed in Chinese hamster lung (CHL) fibroblasts have been characterized pharmacologically using a cytosensor microphysiometer to measure ligand-induced extracellular acidification rate changes. 2. In untransfected CHL cells, noradrenaline had no effect at concentrations up to 100 microM. In alpha(2A)-adrenoceptor transfected cells the rank order of agonist potency was A-54741 (mean pEC(50)=8.96)>dexmedetomidine (8.88)>UK-14304 (8.42)>B-HT 920 (7.05)>noradrenaline (6.92). A-54741, UK-14304 and noradrenaline had the same maximum response while dexmedetomidine and B-HT 920 behaved as partial agonists. 3. The selective alpha(2)-adrenoceptor ligand rauwolscine antagonized acidification rate changes with an affinity independent of the agonist used; the affinity (mean pK(B)) against noradrenaline was 8.43. 4. The selective alpha(1)-adrenoceptor ligands prazosin and doxazosin (each 3 microM) had no effect on noradrenaline responses. 5. Acidification rate changes induced by each agonist were abolished by pre-treatment of cells with pertussis toxin. 6. These data suggest that agonist-induced acidification rate responses in CHL cells transfected with the human alpha(2A)-adrenoceptor are mediated exclusively by the recombinant protein, via pertussis toxin sensitive G(i/o) proteins. PMID- 10742289 TI - Characterization of human recombinant alpha(2A)-adrenoceptors expressed in Chinese hamster lung cells using intracellular Ca(2+) changes: evidence for cross talk between recombinant alpha(2A)- and native alpha(1)-adrenoceptors. AB - 1. Human alpha(2A)-adrenoceptors expressed in Chinese hamster lung (CHL) fibroblasts have been pharmacologically characterized by measuring intracellular calcium (Ca(2+)(i)) changes using the Ca(2+)-sensitive dye Fluo3-AM, in conjunction with a fluorometric imaging plate reader (FLIPR). 2. Several alpha adrenoceptor agonists were examined including the alpha(2)-adrenoceptor agonists UK-14304, B-HT 920, dexmedetomidine and A-54741, the selective alpha(1) adrenoceptor agonist phenylephrine and the non-selective adrenergic agonist noradrenaline. Of these only noradrenaline (mean pEC(50)=6.49) and A-54741 (6.90) evoked changes in Ca(2+)(i); A-54741 was a partial agonist relative to noradrenaline, achieving only 33% of the noradrenaline maximum. 3. Ca(2+)(i) changes induced by noradrenaline and A-54741 were antagonized by the alpha(2) selective antagonist rauwolscine (10 nM) and by the alpha(1)-selective antagonists prazosin (0.1 nM) and doxazosin (1.0 nM). 4. Phenylephrine (100 microM) and UK-14304 (10 microM) alone were ineffective in causing Ca(2+)(i) increase. In the presence of a fixed concentration of UK-14304 (3.0 microM), phenylephrine induced concentration-dependent increases in Ca(2+)(i) (mean pEC(50)=5.33). In the presence of phenylephrine (30.0 microM) UK-14304 induced Ca(2+)(i) release (pEC(50)=6.92). The effects of phenylephrine were abolished by prazosin (1.0 nM) or rauwolscine (100 nM). 5. In saturation radioligand binding experiments using membranes of parental (non-transfected) CHL cells there was a small, specific binding of [(3)H]-prazosin (B(max)=24 fmol mg protein(-1); pK(D)=10. 24). 6. Collectively, these data suggest that alpha-adrenoceptor agonist-induced Ca(2+)(i) release in CHL fibroblasts transfected with the human alpha(2A)-adrenoceptor is dependent upon co-activation of the recombinant receptor and a native alpha(1)-adrenoceptor. PMID- 10742290 TI - Effects of protein tyrosine kinase inhibitors on voltage-operated calcium channel currents in vascular smooth muscle cells and pp60(c-src) kinase activity. AB - 1. Tyrosine kinases have been proposed as regulators of voltage-operated calcium channels. The effects of a range of structurally different inhibitors of protein tyrosine kinases (PTK) were examined on voltage-operated calcium channel currents (I(Ba)) and pp60(c-src) kinase (c-src) activity in vitro. 2. I(Ba) was measured in single myocytes isolated from rabbit ear artery by conventional whole cell voltage-clamp techniques. The activity of purified human c-src was measured in vitro using a non-radioactive assay. 3. Bath application of tyrphostin-23 and genistein (non-selective PTK inhibitors), bistyrphostin (a receptor-PTK-selective inhibitor) and PP1 (a src family-selective inhibitor) inhibited I(Ba) in a concentration-dependent manner over a range of test membrane potentials. Intracellular application of peptide-A, a peptide inhibitor of c-src also inhibited currents. Inhibitor potency series against I(Ba) was PP1 > genistein > tyrphostin 23 > bistyrphostin. 4. Tyrphostin-23, genistein, PP1, and peptide-A shifted the steady-state inactivation curves in a hyperpolarized direction without altering their slope. The inhibitors had no significant effects on I(Ba) activation calculated from current-voltage relationships. 5. The agents inhibited c-src activity in a concentration-dependent manner. The order of potency was PP1 > genistein > peptide-A > tyrphostin-23 > bistyrphostin. The IC(50) for inhibition of c-src activity was similar to the IC(50) for inhibition of I(Ba) in all cases. 6. Western blot analysis with a specific antibody to c-src showed the presence of this cytoplasmic tyrosine kinase in rabbit ear artery cells. 7. A range of structurally dissimilar inhibitors of PTKs inhibit I(Ba) and c-src activity with similar potency. These data provide further evidence implicating endogenous c-src in the modulation of L-type calcium channels in vascular smooth muscle cells. PMID- 10742291 TI - The effects of central aromatic amino acid DOPA decarboxylase inhibition on the motor actions of L-DOPA and dopamine agonists in MPTP-treated primates. AB - 1. Endogenous L-DOPA may act as a neuromodulator contributing to the production of motor activity. We now investigate the effects of the centrally acting aromatic amino acid dopa decarboxylase (AADC) inhibitor NSD-1015 (3-hydroxybenzyl hydrazine) on the motor actions of L-DOPA and dopamine agonist drugs in MPTP treated common marmosets. 2. Pretreatment with NSD-1015 (10 - 50 mg kg(-1); i.p.) worsened baseline motor deficits in MPTP-treated common marmosets. Similarly, it abolished L-DOPA (5 - 18 mg kg(-1) s.c.) induced locomotor activity and reversal of disability. NSD-1015 pretreatment inhibited dopamine formation and elevated L DOPA levels in plasma. 3. The increase in locomotor activity and improvement in disability produced by the administration of the D-1 agonist A-86929 (0.03 - 0. 04 mg kg(-1) s.c.) or the D-2 agonist quinpirole (0.05 - 0.3 mg kg(-1) i.p.) was abolished by NSD-1015 (25 mg kg(-1) i.p.) pretreatment. While the effects of a low dose combination of A-86929 (0.04 mg kg(-1) s.c.) and quinpirole (0.05 mg kg( 1) i.p.) were inhibited by NSD-1015 (25 mg kg(-1) i.p.), there was little effect on the action of a high dose combination of these drugs (0.08 mg kg(-1) A-86929 and 0.1 mg kg(-1) quinpirole). 4. Following central AADC inhibition with NSD-1015 (25 mg kg(-1) i.p.), locomotor behaviour induced by administration of high dose combinations of A-86929 (0.08 mg kg(-1) s.c.) and quinpirole (0.1 mg kg(-1) i.p.) was unaffected by L-DOPA (5 mg kg(-1) s.c.) pretreatment. 5. These results do not support a role for endogenous L-DOPA in spontaneous or drug induced locomotor activity. Rather, they strengthen the argument for the importance of endogenous dopaminergic tone in the motor actions of dopamine agonists. PMID- 10742292 TI - Distinct Ca(2+) signalling mechanisms induced by ATP and sphingosylphosphorylcholine in porcine aortic smooth muscle cells. AB - 1. The increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) following repetitive stimulation with ATP or sphingosylphosphorylcholine (SPC) in single porcine aortic smooth muscle cells was investigated using the Ca(2+) indicator, fura-2. 2. The ATP-induced [Ca(2+)](i) increase resulted from both Ca(2+) release and Ca(2+) influx. The former was stimulated by phospholipase C activation, while the latter occurred predominantly via the receptor-operated Ca(2+) channels (ROC), rather than the store-operated Ca(2+) channels (SOC) or the voltage operated Ca(2+) channel (VOC). Furthermore, the P2X(5) receptor was shown to be responsible for the ATP-induced Ca(2+) influx. 3. A reproducible [Ca(2+)](i) increase was induced by repetitive ATP stimulation, but was abolished by removal of extracellular Ca(2+) or inhibition of intracellular Ca(2+) release using U 73122 or thapsigargin, and was restored by Ca(2+) readdition in the former case. 4. SPC only caused Ca(2+) release, and the amplitude of the repetitive SPC induced [Ca(2+)](i) increases declined gradually. However, a reproducible [Ca(2+)](i) increase was seen in cells in which protein kinase C being inhibited, which increased the SPC-induced Ca(2+) influx, rather than IP(3) generation. 5. In conclusion, although the amplitude of the ATP-induced Ca(2+) release, measured when Ca(2+) influx was blocked, or of the Ca(2+) influx when Ca(2+) release was blocked, progressively decreased following repetitive stimulation, the overall [Ca(2+)](i) increase for each stimulation under physiological conditions remained the same, suggesting that the Ca(2+) stores were replenished by an influx of Ca(2+) during stimulation. The SPC-induced [Ca(2+)](i) increase resulted solely from Ca(2+) release and decreased gradually following repetitive stimulation, but the decrease could be prevented by stimulating Ca(2+) influx, further supporting involvement of the intracellular Ca(2+) stores in Ca(2+) signalling. PMID- 10742293 TI - Rapid non-genomic activation of cytosolic cyclic AMP-dependent protein kinase activity and [Ca(2+)](i) by 17beta-oestradiol in female rat distal colon. AB - 1. In this study, the effect of 17beta-oestradiol on adenosine 3' : 5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase (PKA) activity was investigated. 2. Rapid (within 15 min) activation of basal PKA activity was observed in cytosolic fractions by 17beta-oestradiol but not by 17alpha oestradiol, progesterone or testosterone. This stimulation was abolished by the specific PKA inhibitor PKI but not by the classical oestrogen receptor antagonist tamoxifen. 3. 17beta-Oestradiol did not stimulate basal PKA activity in membrane fractions or in cytosolic fractions from male rats. 4. The increase in cytosolic PKA activity was indirect as (i) it was inhibited by the adenylyl cyclase inhibitor SQ22536, (ii) it was mimicked by forskolin and (iii) 17beta-oestradiol did not cause a stimulation of basal PKA activity in either type I or type II commercially available PKA holoenzymes. 5. Protein kinase Cdelta (PKCdelta) was directly activated by 17beta-oestradiol. The specific PKC inhibitor, bisindolylmaleimide I (GF 109203X), abolished the 6. 17beta-oestradiol-induced PKA activation. 17beta-Oestradiol stimulate an increase in free intracellular calcium ion concentration ([Ca(2+)](i)) in isolated female but not male rat colonic crypts. This was inhibited by verapamil, nifedipine and zero extracellular [Ca(2+)] but unaffected by tamoxifen. 17alpha-Oestradiol, testosterone and progesterone failed to increase [Ca(2+)](i). 7. PKC and PKA inhibitors abolished the 17beta-oestradiol-induced increase in [Ca(2+)](i). 8. These results demonstrate the existence of a novel 17beta-oestradiol-specific PKA and Ca(2+) signalling pathway, which is both sex steroid- and gender-specific, in rat distal colonic epithelium. PMID- 10742294 TI - Histamine H(3) receptors mediate inhibition of noradrenaline release from intestinal sympathetic nerves. AB - 1. The present study investigates whether presynaptic histamine receptors regulate noradrenaline release from intestinal sympathetic nerves. The experiments were performed on longitudinal muscle-myenteric plexus preparations of guinea-pig ileum, preincubated with [(3)H]-noradrenaline. 2. In the presence of rauwolscine, electrically-induced [(3)H]-noradrenaline release was inhibited by histamine or R-alpha-methylhistamine, whereas it was unaffected by pyridylethylamine, impromidine, pyrilamine, cimetidine, thioperamide or clobenpropit. The inhibitory effects of histamine or R-alpha-methylhistamine were antagonized by thioperamide or clobenpropit, but not by pyrilamine or cimetidine. In the absence of rauwolscine, none of these drugs modified the release of [(3)H] noradrenaline. 3. The modulatory action of histamine was attenuated by pertussis toxin and abolished by N-ethylmaleimide. Tetraethylammonium or 4-aminopyridine enhanced the evoked tritium outflow and counteracted the inhibitory effect of histamine. However, the blocking effects of tetraethylammonium and 4 aminopyridine were no longer evident when their enhancing actions were compensated by reduction of Ca(2+) concentration in the superfusion medium. 4. Histamine-induced inhibition of tritium output was enhanced by omega-conotoxin or low Ca(2+) concentration, whereas it was not modified by nifedipine, forskolin, rolipram, phorbol myristate acetate, H7 or lavendustin A. 5. The present results indicate that presynaptic H(3) receptors, located on sympathetic nerve endings, mediate an inhibitory control on intestinal noradrenergic neurotransmission. It is suggested that these receptors are coupled to G(i)/G(o) proteins which modulate the activity of N-type Ca(2+) channels through a direct link, thus reducing the availability of extracellular Ca(2+) at the level of noradrenergic nerve terminals. PMID- 10742295 TI - Involvement of nitric oxide in the modulation of dural arterial blood flow in the rat. AB - 1. Nitric oxide (NO) has been proposed to be a key molecule in the pathogenesis of migraine pain and other headaches that are linked to vascular disorders. Several lines of evidence indicate that the meningeal vascularization is crucially involved in the generation of these headaches. In an experimental model in the rat a dominating role of calcitonin gene-related peptide (CGRP) in causing neurogenic vasodilatation and increased blood flow has been shown. The aim of the present study was to clarify the role of NO in this model with regard to the meningeal blood flow. 2. The blood flow in and around the medial meningeal artery (dural arterial flow) was recorded in the exposed parietal dura mater encephali of barbiturate anaesthetized rats using laser Doppler flowmetry. Local electrical stimulation of the dura mater (pulses of 0.5 ms delivered at 7.5 - 17.5 V and 5 or 10 Hz for 30 s) caused temporary increases in dural arterial flow for about 1 min that reached peaks of 1.6 - 2.6 times the basal flow. The effects of NO synthase (NOS) inhibitors on the basal flow and the electrically evoked increases in flow were examined. 3. Systemic (i. v.) administration of N(omega)-nitro-L arginine methyl ester (L-NAME) at cumulative doses of 10 and 50 mg kg(-1) lowered the basal flow to 87 and 72%, respectively, of the control and reduced the evoked increases in blood flow to 82 and 44% on an average. Both these effects could partly be reversed by 300 mg kg(-1) L-arginine. The systemic arterial pressure was increased by L-NAME at both doses. Injection of the stereoisomer D-NAME at same doses did not change basal flow and evoked increases in flow. 4. 4. Topical application of L-NAME (10(-4) - 10(-2) M) was effective only at the highest concentration, which caused lowering of the basal blood flow to 78% of the control; the evoked increases in flow were not changed. Topical application of 2 amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), a specific inhibitor of the inducible NOS, at concentrations of 10(-4) - 10(-2) M lowered the basal flow to 89, 87.5 and 85%, respectively, but did not significantly change the evoked flow increases. Same concentrations of 7-nitroindazole monosodium salt (7-NINA), a specific inhibitor of the neuronal NOS, had no significant effects on basal flow and evoked increases in flow. 5. It is concluded that NO is involved in the maintenance of the basal level of dural arterial blood flow as well as in the electrically evoked flow increases, which have been shown to be mainly mediated by CGRP released from dural afferent fibres. The most important source of NO is probably the endothelium of dural arterial vessels. The synergistic effect of NO and CGRP on the stimulated blood flow may be in part due to a NO mediated facilitation of the CGRP release. PMID- 10742296 TI - Biphasic effect of bimoclomol on calcium handling in mammalian ventricular myocardium. AB - 1. Concentration-dependent effects of bimoclomol, the novel heat shock protein coinducer, on intracellular calcium transients and contractility were studied in Langendorff-perfused guinea-pig hearts loaded with the fluorescent calcium indicator dye Fura-2. Bimoclomol had a biphasic effect on contractility: both peak left ventricular pressure and the rate of force development significantly increased at a concentration of 10 nM or higher. The maximal effect was observed between 0.1 and 1 microM, and the positive inotropic action disappeared by further increasing the concentration of bimoclomol. The drug increased systolic calcium concentration with a similar concentration-dependence. In contrast, diastolic calcium concentration increased monotonically in the presence of bimoclomol. Thus low concentrations of the drug (10 - 100 nM) increased, whereas high concentrations (10 microM) decreased the amplitude of intracellular calcium transients. 2. Effects of bimoclomol on action potential configuration was studied in isolated canine ventricular myocytes. Action potential duration was increased at low (10 nM), unaffected at intermediate (0.1 - 1 microM) and decreased at high (10 - 100 microM) concentrations of the drug. 3. In single canine sarcoplasmic calcium release channels (ryanodine receptor), incorporated into artificial lipid bilayer, bimoclomol significantly increased the open probability of the channel in the concentration range of 1 - 10 microM. The increased open probability was associated with increased mean open time. The effect of bimoclomol was again biphasic: the open probability decreased below the control level in the presence of 1 mM bimoclomol. 4. Bimoclomol (10 microM - 1 mM) had no significant effect on the rate of calcium uptake into sarcoplasmic reticulum vesicles of the dog, indicating that in vivo calcium reuptake might not substantially be affected by the drug. 5. In conclusion, the positive inotropic action of bimoclomol is likely due to the activation of the sarcoplasmic reticulum calcium release channel in mammalian ventricular myocardium. PMID- 10742297 TI - Cardiovascular profile of the calcium sensitizer EMD 57033 in open-chest anaesthetized pigs with regionally stunned myocardium. AB - 1. Ca(2+) sensitizers enhance systolic function, but may impair relaxation in vitro; these effects may differ in stunned and normal myocardium. We therefore studied the effect of EMD 57033 on systolic and diastolic function of normal and stunned porcine myocardium in vivo. 2. Myocardial stunning by 15 min coronary occlusion and 30 min reperfusion abolished systolic shortening (SS) (baseline 13+/-1%) and decreased end-systolic elastance (E(es)) from 67+/-7 to 47+/-5 mmHg mm(-1) (both P<0.05). Maximum rate of fall of myocardial elastance (dE/dt(min)) decreased from -850+/-100 to -320+/-30 mmHg mm(-1) s(-1), while the time constant tau(e) of the decay of elastance increased from 58+/-3 to 68+/-6 ms (both P<0.05). End-diastolic elastance (E(ed)) was unchanged although the zero pressure intercept (L(0,ed)) had increased. 3. In the stunned region, EMD 57033 (0.2 mg kg(-1) min(-1) for 60 min, i.v., n=7) increased SS to 19+/-2%, E(es) to 287+/-40 mmHg mm(-1), dE/dt(min) to -3630+/-640 mmHg mm(-1) s(-1) and decreased tau(e) to 50+/-3 ms, while E(ed) remained unchanged. In the normal region, 4. EMD 57033 increased SS from 14+/-2 to 18+/-3%, E(es) from 59+/-4 to 263+/-23 mmHg mm(-1), dE/dt(min) from -480+/-70 to -2280+/-700 mmHg mm(-1) s(-1) and decreased tau(e) from 91+/-12 to 61+/-3 ms (all P<0.05), while E(ed) remained unchanged. These responses were minimally affected by adrenoceptor blockade (n=7). Vehicle (n=7) had no effect on either region. EMD 57033 increased cardiac output (up to 27+/ 8%) and LVdP/dt(max) (86+/-19%). Mean aortic pressure decreased (19+/-7%) due to systemic vasodilation that was not amenable to blockade of adrenoceptors or NO synthesis. 5. In conclusion, EMD 57033 restored systolic and diastolic function of stunned myocardium, and produced similar improvements in systolic and diastolic function in normal myocardium. PMID- 10742298 TI - Selective cyclo-oxygenase-2 inhibition with celecoxib elevates blood pressure and promotes leukocyte adherence. AB - 1. Selective inhibitors of cyclo-oxygenase-2 have been shown to be effective anti inflammatory drugs with reduced gastrointestinal toxicity relative to conventional nonsteroidal anti-inflammatory drugs (NSAIDs). In the present study, we examined the possibility that selective COX-2 inhibition, by blocking prostacyclin synthesis, would increase blood pressure and cause leukocyte adherence and platelet aggregation. 2. Normal rats and rats with hypertension induced by chronic administration of Nomega-nitro-L-arginine methylester were given celecoxib (10 mg kg(-1)) daily for 3 weeks. Celecoxib significantly elevated of blood pressure in both the normal and hypertensive rats (mean increase of >33 mm Hg after 3 weeks). 3. In normal rats, celecoxib had no effect on serum 6-keto prostaglandin (PG)F(1alpha) levels. Hypertensive rats exhibited a significant increase (82%) in serum 6-keto PGF(1alpha) levels, and this was reduced to the levels of normal rats by treatment with celecoxib. 4. Rats treated with celecoxib exhibited significant increases in weight gain (20%), plasma arginine-vasopressin levels (148%) and plasma urea (69%) relative to vehicle treated controls. Plasma creatinine levels were unaffected by treatment with celecoxib, while plasma renin levels were significantly decreased (30%) relative to controls. 5. Superfusion of mesenteric venules with celecoxib (3 microM) in vivo resulted in significant increases in leukocyte adherence to the endothelium in both normal and hypertensive rats. 6. These studies suggest that suppression of COX-2 significantly influences vascular and/or renal function, leading to elevated blood pressure and leukocyte adherence. PMID- 10742299 TI - Compounds that block both intermediate-conductance (IK(Ca)) and small-conductance (SK(Ca)) calcium-activated potassium channels. AB - 1. Nine bis-quinolinyl and bis-quinolinium compounds related to dequalinium, and previously shown to block apamin-sensitive small conductance Ca(2+)-activated K(+) channels (SK(Ca)), have been tested for their inhibitory effects on actions mediated by intermediate conductance Ca(2+)-activated K(+) channels (IK(Ca)) in rabbit blood cells. 2. In most experiments, a K(+)-sensitive electrode was employed to monitor the IK(Ca)-mediated net loss of cell K(+) that followed the addition of the Ca(2+) ionophore A23187 (2 microM) to red cells suspended at an haematocrit of 1% in a low K(+) (0.12 - 0.17 mM) solution. The remainder used an optical method based on measuring the reduction in light transmission that occurred on applying A23187 (0.4 or 2 microM) to a very dilute suspension of red cells (haematocrit 0.02%). 3. Of the compounds tested, the most potent IK(Ca) blocker was 1,12 bis[(2-methylquinolin-4-yl)amino]dodecane (UCL 1407) which had an IC(50) of 0.85+/-0.06 microM (mean+/-s.d. mean). 4. The inhibitory action of UCL 1407 and its three most active congeners was characterized by (i) a Hill slope greater than unity, (ii) sensitivity to an increase in external [K(+)], and (iii) a time course of onset that suggested use-dependence. Also, the potency of the nonquaternary compounds tested increased with their predicted lipophilicity. These findings suggested that the IK(Ca) blocking action resembles that of cetiedil rather than of clotrimazole. 5. Some quaternized members of the series were also active. The most potent was the monoquaternary UCL 1440 ((1-[N-[1-(3, 5 dimethoxybenzyl)-2-methylquinolinium-4-yl]amino]-10-[N'-(2-me thylqu inolinium 4yl)amino] decane (trifluoroacetate) which had an IC(50) of 1.8+/-0.1 microM. The corresponding bisquaternary UCL 1438 (1, 10-bis[N-[1-(3,5-dimethoxybenzyl)-2 methylquinolinium-4-yl]amino] decane bis(trifluoroacetate) was almost as active (IC(50) 2.7+/-0.3 microM). 6. A bis-aminoquinolium cyclophane (UCL 1684) had little IK(Ca) blocking action despite its great potency at SK(Ca) channels (IC(50) 4.1+/-0.2 nM). 7. The main outcome is the identification of new intermediate-conductance Ca(2+)-activated K(+) channel blockers with a wide range of IK(Ca)/SK(Ca) selectivities. PMID- 10742300 TI - The in vivo pharmacological profile of CS-747, a novel antiplatelet agent with platelet ADP receptor antagonist properties. AB - 1. CS-747 is a novel antiplatelet agent that generates an active metabolite, R 99224, in vivo. CS-747 itself was totally inactive in vitro. This study examined in vivo pharmacological profiles of CS-747 after single oral administration to rats. 2. Orally administered CS-747 (0.3 - 10 mg kg(-1)) partially but significantly decreased [(3)H]-2-methylthio-ADP binding to rat platelets. CS-747 (3 mg kg(-1), p.o.) treatment neutralized ADP-induced decreases of cyclic AMP concentrations induced by prostaglandin E(1), suggesting that metabolites of CS 747 interfere with G(i)-linked P2T receptor. 3. CS-747 (0.3 and 3 mg kg(-1), p.o.) markedly inhibited ex vivo washed platelet aggregation in response to ADP but not to thrombin. CS-747 also exhibited a marked inhibition of ADP-induced ex vivo platelet aggregation in PRP with a rapid onset (<0.5 h) and long duration (>3 days) of action (ED(50) at 4 h=1.2 mg kg(-1)). 4. R-99224 (IC(50)=45 microM) inhibited in vitro PRP aggregation in a concentration-related manner. 5. CS-747 prevented thrombus formation in a dose-related manner with an ED(50) value of 0.68 mg kg(-1). CS-747 was more potent than clopidogrel (6.2 mg kg(-1)) and ticlopidine (>300 mg kg(-1)). 6. CS-747, clopidogrel, and ticlopidine prolonged the bleeding time. The order of potency of these agents in this activity was the same as that in antiaggregatory and antithrombotic activities. 7. These findings indicate that CS-747 is an orally active and a potent antiplatelet and antithrombotic agent with a rapid onset and long duration of action, and warrants clinical evaluations of the agent. PMID- 10742301 TI - Investigation of the role of intracellular Ca(2+) stores in generation of the muscarinic agonist-induced slow afterdepolarization (sADP) in guinea-pig olfactory cortical neurones in vitro. AB - 1. Intracellular recordings were made from guinea-pig olfactory cortical brain slice neurones to assess the possible role of intracellular Ca(2+) stores in the generation of the slow post-stimulus afterdepolarization (sADP) and its underlying tail current (I(ADP)), induced by muscarinic receptor activation. 2. Caffeine or theophylline (0.5 - 3 mM) reduced the amplitude of the I(ADP) (measured under 'hybrid' voltage clamp) induced in the presence of the muscarinic agonist oxotremorine-M (OXO-M, 10 microM) by up to 96%, without affecting membrane properties or muscarinic depolarization of these neurones. 3. The L-type Ca(2+) channel blocker nifedipine (1, 10 microM) also inhibited I(ADP) (by up to 46%), while ryanodine (10 microM) (a blocker of Ca(2+) release from internal stores) produced a small ( approximately 10%) reduction in I(ADP) amplitude; however, neither 10 microM dantrolene (another internal Ca(2+) release blocker) nor the intracellular Ca(2+) store re-uptake inhibitors thapsigargin (3 microM) or cyclopiazonic acid (CPA, 15 microM) affected I(ADP) amplitude. 4. IBMX (100 microM), a phosphodiesterase inhibitor, also had no effect on I(ADP). Furthermore, inhibition of I(ADP) by caffeine was not reversed by co-application of 100 microM adenosine. 5. Caffeine (3 mM) or nifedipine (10 microM) reduced the duration of presumed Ca(2+) spikes revealed by intracellular Cs(+) loading. When applied in combination, nifedipine and caffeine effects were occlusive, rather than additive, suggesting a common site of action on L-type calcium channels. 6. We conclude that Ca(2+)-induced Ca(2+) release (CICR) from internal stores does not contribute significantly to muscarinic I(ADP) generation in olfactory cortical neurones. However caffeine and theophylline, which enhance CICR in other systems, blocked I(ADP) induction. We suggest that this action might involve a combination of L-type voltage-gated Ca(2+) channel blockade, and a direct inhibitory action on the putative I(ADP) K(+) conductance. PMID- 10742302 TI - Functional role of muscarinic M(2) receptors in alpha,beta-methylene ATP induced, neurogenic contractions in guinea-pig ileum. AB - 1. The muscarinic acetylcholine receptors mediating the contractile response elicited to endogenous acetylcholine released by the selective P2X receptor agonist alpha,beta-methylene ATP (mATP) were investigated in guinea-pig ileum. 2. mATP (0.1 - 30 microM) elicited a concentration-dependent neurogenic contractile response inhibited by tetrodotoxin (TTX) and antagonized by the non-selective muscarinic receptor antagonist N-methylscopolamine (NMS). 3. The contractile response to mATP was pertussis toxin-insensitive, irreversibly antagonized by N (2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard), and unaffected by the muscarinic M(2)/M(4) receptor selective antagonist AF-DX 116 (1 microM). 4. When measured in the presence of histamine and isoproterenol after treatment with 4-DAMP mustard, mATP elicited a pertussis toxin-sensitive contractile response potently antagonized by AF-DX 116. 5. Collectively, our data suggest that endogenous acetylcholine released by mATP can elicit a direct contractile response through the muscarinic M(3) receptor and an indirect contractile response through the muscarinic M(2) receptor by antagonizing the relaxant effects of isoproterenol on histamine induced contraction. PMID- 10742303 TI - The stimulant effects of caffeine on locomotor behaviour in mice are mediated through its blockade of adenosine A(2A) receptors. AB - 1. The locomotor stimulatory effects induced by caffeine (1,3, 7 trimethylxanthine) in rodents have been attributed to antagonism of adenosine A(1) and A(2A) receptors. Little is known about its locomotor depressant effects seen when acutely administered at high doses. The roles of adenosine A(1) and A(2A) receptors in these activities were investigated using a Digiscan actimeter in experiments carried out in mice. Besides caffeine, the A(2A) antagonist SCH 58261 (5-amino-7-(beta-phenylethyl)-2-(8-furyl)pyrazolo[4,3-e]-1,2, 4 triazolo[1,5-c]pyrimidine), the A(1) antagonist DPCPX (8-cyclopentyl-1,3 dipropylxanthine), the A(1) agonist CPA (N(6)-cyclopentyladenosine) and A(2A) receptor knockout mice were used. 2. Caffeine had a biphasic effect on locomotion of wild-type mice not habituated to the open field, stimulating locomotion at 6.25 - 25 mg kg(-1) i.p. doses, while depressing it at 100 mg kg(-1). In sharp contrast, caffeine dose-dependently decreased locomotion in A(2A) receptor knockout mice over the whole range of tested doses. 3. The depressant effects induced by high doses of caffeine were lost in control CD1 mice habituated to the open field. 4. The A(1) agonist CPA depressed locomotion at 0.3 - 1 mg kg(-1) i.p. doses. 5. The A(1) antagonist DPCPX decreased locomotion of A(2A) receptor knockouts and CD1 mice at 5 mg kg(-1) i.p. and 25 mg kg(-1) i.p. respectively. 6. DPCPX (0.2 - 1 mg kg(-1) i.p.) left unaltered or even reduced the stimulant effect of SCH 58261 (1 - 3 mg kg(-1) i.p.) on CD1 mice. 7. These results suggest therefore that the stimulant effect of low doses of caffeine is mediated by A(2A) receptor blockade while the depressant effect seen at higher doses under some conditions is explained by A(1) receptor blockade. PMID- 10742304 TI - Blockade of the HERG human cardiac K(+) channel by the antidepressant drug amitriptyline. AB - 1. Amitriptyline has been known to induce QT prolongation and torsades de pointes which causes sudden death. We studied the effects of amitriptyline on the human ether-a-go-go-related gene (HERG) channel expressed in Xenopus oocytes and on the rapidly activating delayed rectifier K(+) current (I(Kr)) in rat atrial myocytes. 2. The amplitudes of steady-state currents and tail currents of HERG were decreased by amitriptyline dose-dependently. The decrease became more pronounced at more positive potential, suggesting that the block of HERG by amitriptyline is voltage dependent. IC(50) for amitriptyline block of HERG current was progressively decreased according to depolarization: IC(50) values at -30, -10, +10 and +30 mV were 23.0, 8.71, 5.96 and 4.66 microM, respectively. 3. Block of HERG by amitriptyline was use dependent: exhibiting a much faster block at higher activation frequency. Subsequent decrease in frequency after high activation frequency resulted in a partial relief of HERG blockade. 4. Steady-state block by amitriptyline was obtained while depolarization to +20 mV for 0.5 s was applied at 0.5 Hz: IC(50) was 3.26 microM in 2 mM [K(+)](o). It was increased to 4. 78 microM in 4 mM [K(+)](o), suggesting that the affinity of amitriptyline on HERG was decreased by external K(+). 5. In rat atrial myocytes bathed in 35 degrees C, 5 microM amitriptyline blocked I(Kr) by 55%. However, transient outward K(+) current (I(to)) was not significantly affected. 6. In summary, the data suggest that the block of HERG currents may contribute to arrhythmogenic side effects of amitriptyline. PMID- 10742305 TI - P2Y(2) receptor-mediated proliferation of C(6) glioma cells via activation of Ras/Raf/MEK/MAPK pathway. AB - 1. Extracellular purine and pyrimidine nucleotides have been implicated in the regulation of several cellular functions including mitogenesis. In this study, experiments were conducted to characterize the P2Y receptor on C(6) glioma cells responsible for stimulating cell proliferation associated with mitogen-activated protein kinase (MAPK) activation. 2. UTP and ATP produced a similar effect on [(3)H]-thymidine incorporation in a time- and concentration-dependent manner, suggesting the involvement of P2Y(2) receptor in mediating proliferation of C(6) glioma cells. 3. In response to UTP, both p42 and p44 MAPK were activated in a time- and concentration-dependent manner using Western blot analysis with an anti phospho-p42/p44 MAPK antibody. The phosphorylation reached maximal levels after 5 min and declining by 30 min. 4. Pretreatment with pertussis toxin (PTX) did not change these responses to UTP. Both DNA synthesis and phosphorylation of MAPK in response to UTP were attenuated by tyrosine kinase inhibitors, genistein and herbimycin A, protein kinase C (PKC) inhibitors, staurosporine and GF109203X, and removal of Ca(2+) by addition of BAPTA/AM plus EGTA. 5. UTP-induced [(3)H] thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2). Furthermore, we showed that overexpression of dominant negative mutants of Ras (RasN17) and Raf (Raf-301) completely suppressed MEK1/2 and p42/p44 MAPK activation induced by ATP and UTP. 6. These results conclude that the mitogenic effect of UTP mediated through a P2Y(2) receptor that involves the activation of Ras/Raf/MEK/MAPK pathway. UTP-mediated MAPK activation was modulated by Ca(2+), PKC, and tyrosine kinase associated with cell proliferation in cultured C(6) glioma cells. PMID- 10742306 TI - Effects of inhibitors of small- and intermediate-conductance calcium-activated potassium channels, inwardly-rectifying potassium channels and Na(+)/K(+) ATPase on EDHF relaxations in the rat hepatic artery. AB - 1. In the rat hepatic artery, the SK(Ca) inhibitors UCL 1684 (300 nM) completely blocked, and scyllatoxin (1 microM) and d-tubocurarine (100 microM) partially inhibited EDHF relaxations when each of them was combined with charybdotoxin (300 nM). 2. The IK(Ca) inhibitors clotrimazole (3 microM) and 2-chlorophenyl bisphenyl-methanol (3 microM) strongly depressed EDHF relaxations when each of them was combined with apamin (300 nM). The cytochrome P450 mono-oxygenase inhibitor ketoconazole (10 microM) had no effect in the presence of apamin. 3. Ciclazindol (10 microM), which abolishes EDHF relaxations in the presence of apamin, almost completely prevented the calcium ionophore (A23187) stimulated (86)Rb(+) influx via the Gardos channel (IK(Ca)) in human erythrocytes. 4. The Na(+)/K(+) ATPase inhibitor ouabain (500 microM) and the K(IR) blocker Ba(2+) (30 microM) neither alone nor in combination inhibited EDHF relaxations. Ba(2+) was also without effect in the presence of either apamin or charybdotoxin. 5. In contrast to EDHF, an increase in extracellular [K(+)] from 4.6 mM to 9.6, 14.6 and 19.6 mM inconsistently relaxed arteries. In K(+)-free physiological salt solution, re-admission of K(+) always caused complete and sustained relaxations which were abolished by ouabain but unaffected by Ba(2+). 6. The present study provides pharmacological evidence for the involvement of SK(Ca) and IK(Ca) in the action of EDHF in the rat hepatic artery. Our results are not consistent with the idea that EDHF is K(+) activating Na(+)/K(+) ATPase and K(IR) in this blood vessel. PMID- 10742308 TI - Diadenosine polyphosphates as antagonists of the endogenous P2Y(1) receptor in rat brain capillary endothelial cells of the B7 and B10 clones. AB - 1. Diadenosine polyphosphates (Ap(n)As, n=2 - 7) are considered as stress mediators in the cardiovascular system. They act both via identified P2 purinoceptors and via yet to be characterized receptors. This study analyses the actions of Ap(n)As in clones of rat brain capillary endothelial cells that express P2Y(1) receptors (B10 cells) or both P2Y(1) and P2Y(2) receptors (B7 cells). 2. B10 cells responded to Ap(3)A with rises in intracellular Ca(2+) concentration ([Ca(2+)](i)). This response was prevented by adenosine-3' phosphate-5'-phosphate, an antagonist of P2Y(1) receptors. It was largely suppressed by a treatment with apyrase VII or with creatine phosphokinase/creatine phosphate to degrade contaminating ADP. 3. Ap(n)As inhibited ADP induced increases in [Ca(2+)](i) mediated by P2Y(1) receptors by shifting ADP concentration-response curves to larger concentrations. Apparent K(i) values were estimated to be 6 microM for Ap(4)A, 10 microM for Ap(5)A and 47 microM for Ap(6)A. Ap(2)A and Ap(3)A were much less active. 4. Ap(n)As were neither agonists nor antagonists of the endogenous P2Y(2) receptor in B7 cells. 5. Ap(n)As are neither agonists nor antagonists of the G(i)-coupled, ADP receptor in B10 cells. 6. The results suggest that most actions of Ap(n)As in B7 and B10 cells can be accounted for by endogenous P2Y(1) receptors. Ap(4)A, Ap(5)A and Ap(6)A are specific antagonists of endogenous Ca(2+)-coupled P2Y(1) receptors. PMID- 10742307 TI - Beta-3 adrenergic stimulation of L-type Ca(2+) channels in rat portal vein myocytes. AB - 1. The effects of beta(3)-adrenergic stimulation were studied on the L-type Ca(2+) channel in single myocytes from rat portal vein using the whole-cell mode of the patch-clamp technique. 2. Reverse transcription-polymerase chain reaction showed that beta(1)-, beta(2)- and beta(3)-adrenoceptor subtypes were expressed in rat portal vein myocytes. Application of both propranolol (a non-selective beta(1)- and beta(2)-adrenoceptor antagonist) and SR59230A (a beta(3) adrenoceptor antagonist) were needed to inhibit the isoprenaline-induced increase in L-type Ca(2+) channel current. 3. L-type Ca(2+) channels were stimulated by CGP12177A (a beta(3)-adrenoceptor agonist with potent beta(1)- and beta(2) adrenoceptor antagonist property) in a manner similar to that of isoprenaline. The CGP12177A-induced stimulation of Ca(2+) channel current was blocked by SR59230A, cyclic AMP-dependent protein kinase inhibitors, H-89 and Rp 8-Br-cyclic AMPs, but was unaffected by protein kinase C inhibitors, GF109203X and 19-31 peptide. This stimulation was mimicked by forskolin and 8-Br-cyclic AMP. In the presence of okadaic acid (a phosphatase inhibitor), the beta(3)-adrenoceptor induced stimulation was maintained after withdrawal of the agonist. 4. The beta(3)-adrenoceptor stimulation of L-type Ca(2+) channels was blocked by a pretreatment with cholera toxin and by the intracellular application of an anti Galpha(s) antibody. This stimulation was unaffected by intracellular infusion of an anti-Gbeta(com) antibody and a betaARK(1) peptide. 5. These results show that activation of beta(3)-adrenoceptors stimulates L-type Ca(2+) channels in vascular myocytes through a Galpha(s)-induced stimulation of the cyclic AMP/protein kinase A pathway and the subsequent phosphorylation of the channels. PMID- 10742309 TI - A redox-based mechanism for nitric oxide-induced inhibition of DNA synthesis in human vascular smooth muscle cells. AB - 1. The current study explored potential redox mechanisms of nitric oxide (NO) induced inhibition of DNA synthesis in cultured human and rat aortic smooth muscle cells. 2. Exposure to S-nitrosothiols, DETA-NONOate and NO itself inhibited ongoing DNA synthesis and S phase progression in a concentration dependent manner, as measured by thymidine incorporation and flow cytometry. Inhibition by NO donors occurred by release of NO, as detected by chemiluminescence and judged by the effects of NO scavengers, haemoglobin and cPTIO. 3. Co-incubation with redox compounds, N-acetyl-L-cysteine, glutathione and L-ascorbic acid prevented NO inhibition of DNA synthesis. These observations suggest that redox agents may alternatively attenuate NO bioactivity extracellularly, interfere with intracellular actions of NO on the DNA synthesis machinery or restore DNA synthesis after established inhibition by NO. 4. Recovery of DNA synthesis after inhibition by NO was similar with and without redox agents suggesting that augmented restoration of DNA synthesis is an unlikely mechanism to explain redox regulation. 5. Study of extracellula interactions revealed that all redox agents potentiated S-nitrosothiol decomposition and NO release. 6. Examination of intracellular NO bioactivity showed that as opposed to attenuation of NO inhibition of DNA synthesis by redox agents, there was no inhibition (potentiation in the presence of ascorbic acid) of soluble guanylate cyclase (sGC) activation judged by cyclic GMP accumulation in rat cells. 7. These data provide evidence that NO-induced inhibition of ongoing DNA synthesis is sensitive to redox environment. Redox processes might protect the DNA synthesis machinery from inhibition by NO, in the setting of augmented liberation of biologically active NO from NO donors. PMID- 10742312 TI - Pediatric Milliman and Robertson length-of-stay criteria: are they realistic? AB - OBJECTIVE: Guidelines for inpatient length of stay (LOS) have been developed by several groups; among the most widely applied are those published by Milliman and Robertson (M&R). Few published reports have examined the relationship of actual practice to such guidelines, none in pediatric populations. This study was designed to compare pediatric practice in a large and defined population to M&R LOS criteria. METHODS: Administrative data from New York State in 1995 were used to examine LOS for discharges corresponding to 16 selected pediatric diagnoses for which M&R publishes guidelines. Outliers, defined as the 2% of discharges with the longest LOS, were eliminated. The distribution of LOS for each diagnosis was compared with M&R LOS guidelines. RESULTS: In New York State during 1995, pediatric LOS was markedly divergent from M&R guidelines. In general, the percentage of discharges in excess of the criterion LOS was less for nonmandatory admissions (croup: 23%, gastroenteritis: 44%, and pneumonia: 48%) than for those requiring surgery (uncomplicated appendectomy: 67%, pyloromyotomy: 62%, and major but noncritical burns: 64%) or prolonged treatment with antibiotics (bacterial meningitis: 91% and osteomyelitis: 86%). CONCLUSIONS: In New York State during 1995, LOS for selected pediatric conditions was generally in excess of published M&R guidelines. This raises concern about the potential effects of such guidelines on both patients and the hospitals caring for them. While endorsing the need for cost-effective practice, we call attention to the methods used to develop and validate guidelines.length of stay, pediatrics, managed health care, administrative data, practice guidelines. PMID- 10742311 TI - Anti-NO action of carvedilol in cell-free system and in vascular endothelial cells. AB - 1. Carvedilol, an adrenoceptor blocker with antioxidant activity, was studied for its ability to interact with NO in a cell-free condition and in an endothelial cell line (ECV304). 2. In a cell-free system, carvedilol attenuated NO-dependent reduction of carboxy-2-phenyl-4,4, 5,5-tetramethyl-imidazoline-1-oxyl-3-oxide induced by a NO donor, 1-hydroxy-2-oxo-3-(aminopropyl)-3-isopropyl-1-triazene (NOC5), which was determined by electron paramagnetic resonance (EPR) spectrometry. The EPR study also showed that nitrosylhaemoglobin formation in rat red blood cells by the addition of NO-saturated solution was attenuated by prior incubation with 0.1 - 10 microM carvedilol. 3. NO-induced fluorescence in 4,5 diaminofluorescein-2 diacethyl (DAF-2DA)-loaded ECV304 cells was attenuated by carvedilol but not by labetalol. The IC(50) of carvedilol for NOC5 or sodium nitroprusside-induced fluorescence of DAF-2DA in ECV304 cells was 1. 0x10(-7) M, which was similar to the reported IC(50) of carvedilol for the antioxidant effect. 4. Cell toxicity induced by a NO donor determined by the number of viable cells after 24 h treatment with 2-2'(hydroxynitrosohydrazino)bis-ethanamine was significantly attenuated by pretreatment with 1 microM carvedilol. 5. Both free and cell-associated carvedilol quenched NO. Because NO mediates both physiological and pathophysiological processes, NO quenching by the drug may have diverse clinical implications depending upon specific functions of local NO in tissues where carvedilol is distributed. PMID- 10742310 TI - Platelet-activating factor increases mucosal permeability in rat intestine via tyrosine phosphorylation of E-cadherin. AB - 1. Platelet-activating factor (PAF), an inflammatory mediator, plays an important role in mediating intestinal injury. However, it remains unclear whether PAF has a function in the intestine. The production of PAF by normal intestine and by unstimulated intestinal epithelial cell lines suggests that PAF may have a regulatory function in the normal bowel. 2. In this study we investigated the role of PAF in modulating intestinal mucosal permeability in rats. Lumen-to-blood transit of FD-4 (dextran 4400), (an index of intestinal permeability), was assessed in sham-operated rats and rats injected with PAF (1.25 microg kg(-1), i.v., a dose insufficient to induce intestinal injury). 3. PAF-induced villus cytoskeletal changes were examined by staining the intestine for F-actin. The effect of PAF on tyrosine phosphorylation of the junctional protein E-cadherin was examined by immunoprecipitation. Some rats were pretreated with AG1288 (a tyrosine kinase inhibitor) before PAF injection, and mucosal permeability change was assessed. 4. To investigate the role of endogenous PAF upon mucosal permeability, we studied the effect of PAF antagonists on (intraluminal) glucose induced increase in mucosal permeability. 5. We found that low dose PAF: (a) alters the cytoskeletal structure of intestinal epithelium, (b) causes the influx of FD4 from intestinal lumen to systemic circulation, (c) induces tyrosine phosphorylation of E-cadherin and cadherin-associated proteins. Glucose-induced mucosal permeability increase is abolished by using two structurally different PAF antagonists. 6. These results suggest that endogenous PAF modulates macromolecular movement across the intestinal mucosal barrier, probably via tyrosine phosphorylation of E-cadherin and cytoskeletal alteration of enterocytes. PMID- 10742313 TI - Effect of infection control measures on the frequency of upper respiratory infection in child care: a randomized, controlled trial. AB - BACKGROUND: Acute upper respiratory infections are common in children who attend child care, and preventing transmission of disease in this setting depends on actions by child care staff. We set out to discover whether transmission of respiratory infections in child care could be reduced by improved infection control procedures. METHODS: We performed a cluster, randomized, controlled trial of an infection control intervention conducted in child care centers in 1 city in Australia. The intervention was training of child care staff about transmission of infection, handwashing, and aseptic nosewiping technique. Implementation of the intervention was recorded by an observer. Illness was measured by parent report in telephone interviews every 2 weeks. RESULTS: There were 311 child-years of surveillance for respiratory symptoms. By multivariable analysis, there was no significant reduction in colds in intervention center children across the full age range. However, a significant reduction in respiratory illness was present in children 24 months of age and younger. When compliance with infection control practices was high, colds in these children were reduced by 17%. CONCLUSIONS: This trial supports the role of direct transmission of colds in young children in child care. The ability of infection control techniques to reduce episodes of colds in children in child care was limited to children 24 months of age and under. PMID- 10742314 TI - Effect of infection control measures on the frequency of diarrheal episodes in child care: a randomized, controlled trial. AB - BACKGROUND: Diarrheal infections are common in children who attend child care, and preventing transmission of disease in this setting depends on actions by child care staff. We set out to discover whether transmission of gastrointestinal infections in child care could be reduced by improved infection control procedures. METHODS: We performed a cluster randomized, controlled trial of an infection control intervention conducted in child care centers for 1 city in Australia. The intervention was training of child care staff about transmission of infection and handwashing and focused on both staff and child behavior. Implementation of the intervention was recorded by an observer. Illness was measured by parent report in telephone interviews every 2 weeks. RESULTS: There were 311 child-years of surveillance for diarrheal episodes. The rate of episodes of diarrhea was 1.9 per child-year in intervention centers and 2.7 per child-year in control centers. Multivariable analysis showed that diarrheal episodes were significantly reduced in intervention center children by 50%. However, the impact of the intervention was confined to children over 24 months of age. For those centers in which children's compliance with handwashing was high, diarrheal episodes were reduced by 66%. CONCLUSIONS: This trial supports education about infection control, for staff and children in child care, as a means of reducing transmission of diarrhea. Reduction in episodes of diarrhea in children in child care was limited to children over 24 months of age. PMID- 10742315 TI - Symptoms associated with infant teething: a prospective study. AB - CONTEXT: Studies of infant teething have been retrospective, small, or conducted on institutionalized infants. OBJECTIVES: To conduct a large, prospective study of healthy infants to determine which symptoms may be attributed to teething and to attempt to predict tooth emergence from an infant's symptoms. DESIGN: Prospective cohort. Setting. Clinic-based pediatric group practice. PATIENTS: One hundred twenty-five consecutive well children of consenting Cleveland Clinic employees. OUTCOME MEASURES: Parents daily recorded 2 tympanic temperatures, presence or absence of 18 symptoms, and all tooth eruptions in their infants, from the 4-month well-child visit until the child turned 1 year old. RESULTS: Daily symptom data were available for 19 422 child-days and 475 tooth eruptions. Symptoms were only significantly more frequent in the 4 days before a tooth emergence, the day of the emergence, and 3 days after it, so this 8-day window was defined as the teething period. Increased biting, drooling, gum-rubbing, sucking, irritability, wakefulness, ear-rubbing, facial rash, decreased appetite for solid foods, and mild temperature elevation were all statistically associated with teething. Congestion, sleep disturbance, stool looseness, increased stool number, decreased appetite for liquids, cough, rashes other than facial rashes, fever over 102 degrees F, and vomiting were not significantly associated with tooth emergence. Although many symptoms were associated with teething, no symptom occurred in >35% of teething infants, and no symptom occurred >20% more often in teething than in nonteething infants. No teething child had a fever of 104 degrees F and none had a life-threatening illness. CONCLUSIONS: Many mild symptoms previously thought to be associated with teething were found in this study to be temporally associated with teething. However, no symptom cluster could reliably predict the imminent emergence of a tooth. Before caregivers attribute any infants' signs or symptoms of a potentially serious illness to teething, other possible causes must be ruled out.teething, tooth eruption, teeth, deciduous dentition. PMID- 10742316 TI - Outcomes after judicious antibiotic use for respiratory tract infections seen in a private pediatric practice. AB - BACKGROUND: Most respiratory tract infections (RTIs) in children have a viral cause, they resolve on their own, and antibiotics need not be prescribed. OBJECTIVE: We sought to provide evidence that judicious antibiotic use can be accomplished in private pediatric practice without observing an increase in return office visits or in the rate of bacterial infections that may follow. STUDY DESIGN: This was a prospective 12-month study from July 1, 1996 through June 30, 1997. On the same 1 day each week, a representative convenience sample of acute respiratory tract illness patients was enrolled, and laboratory studies performed as appropriate, including viral cultures on all. Children were then followed for 30 days to ascertain the outcomes of not prescribing antibiotics except when specific bacterial infections were present at the initial visit. RESULTS: Three hundred eighty-three children were enrolled; 293 (77%) did not receive antibiotics at the enrollment visit. Ninety children (23%) received antibiotics based on a diagnosis of acute otitis media (n = 53), acute streptococcal tonsillopharyngitis (n = 18), or other presumed or documented bacterial infections (n = 19). An unscheduled return visit related to the initial visit occurred for 86 (29%) of the 293 children not receiving antibiotics initially and in 40 (44%) of 90 children receiving antibiotics initially. Eighty seven children (23%) had positive viral culture results. The most frequently isolated viruses were adenovirus, enterovirus, parainfluenzae virus, and influenza virus. CONCLUSION: Children with RTIs without a concomitant presumed or proven bacterial infection do not require antibiotics. In this busy office practice, >75% of the children presenting with an RTI did not have a presumed or proven bacterial infection. These children did not have a higher rate of return office visits or an increase in bacterial infections. This reinforces the judicious use of antibiotics in managing children with RTIs.outcomes, antibiotic, respiratory infections. PMID- 10742317 TI - Access to health care for children with special health care needs. AB - OBJECTIVE: To assess the role health insurance plays in influencing access to care and use of services by children with special health care needs. METHODS: We analyzed data on 57 553 children younger than 18 years old included in the 1994 1995 National Health Interview Survey on Disability. The survey obtained information on special health care needs, insurance status, and access to and use of health services. Bivariate and multivariate analyses were used to assess the association of insurance with several measures of access and utilization, including usual source of care, site of usual care, missed or delayed care, and use of ambulatory physician services. RESULTS: Using the federal Maternal and Child Health Bureau definition of children with special health care needs, we estimate that 18% of US children under 18 years old had an existing special health care need in 1994-1995. An estimated 89% of these children had some form of health insurance coverage, most often private health insurance. Insured children were more likely than uninsured children to have a usual source of care (96.9% vs 79.2%). Among those with a usual source of care, insured children were more likely than uninsured children to have an identified regular clinician (87. 6% vs 80.7%). Insured children were less likely to report unmet health needs, including medical care (2.2% vs 10.5%), dental care (6. 1% vs 23.9%), prescriptions, and/or eyeglasses (3.1% vs 12.3%), and mental health care (.9% vs 3.4%). Insured children were also more likely to have a physician contact in the past year (89.3% vs 73.6%) and have more physician contacts on an annual basis (8.5 vs 4.1 contacts). Unexpectedly, no differences were found between insured and uninsured children in availability of after hours medical care (evenings and weekends) or satisfaction with care. We also found some modest differences in access between publicly and privately insured children. Privately insured children were more likely to have a usual source of care (97.6% vs 95.3%) and a regular clinician (91.0% vs 81.1%). Privately insured children were also less likely to report dissatisfaction with care at their usual site of care (14. 9% vs 21.0%) and have access to care on evenings and weekends (6.8% vs 13.4%). No substantial differences were found between privately and publicly insured children in prevalence of unmet health needs or delays in obtaining care due to cost. CONCLUSIONS: This study illustrates the importance of health insurance for children with special health care needs. Continued efforts are needed to ensure that all children with special health care needs have insurance and that remaining access and utilization barriers for currently insured children with special health care needs are also addressed. PMID- 10742318 TI - A guideline implementation system using handheld computers for office management of asthma: effects on adherence and patient outcomes. AB - OBJECTIVE: To evaluate effects on the process and outcomes of care brought about by use of a handheld, computer-based system that implements the American Academy of Pediatrics guideline on office management of asthma exacerbations. DESIGN: A before-after trial with randomly selected, office-based Connecticut pediatricians. In both the control and intervention phases, physicians collected data from 10 patient encounters for acute asthma exacerbations. During the intervention phase, the computer provided for structured encounter documentation and offered recommendations based on the guideline of the American Academy of Pediatrics. Patients were contacted by telephone 7 to 14 days after the visit to assess outcomes. RESULTS: Nine study-physicians enrolled 91 patients in the control phase and 74 in the intervention phase. Follow-up information was available for 93% of encounters. Use of the intervention was associated with increased mean frequency/visit of: 1) measurements of peak expiratory flow rate (2.18 vs 1.57) and oxygen saturation (1.12 vs.42), and 2) administration of nebulized beta2-agonists (1.25 vs.71). Visits in the intervention phase lasted longer and fees were higher ($145.61 vs $103.11). There were no significant differences in immediate disposition or subsequent emergency department visits, hospitalizations, missed school, or caretaker's missed work during the 7 days post visit. CONCLUSION: Use of handheld computers that provide guideline-based decision support was associated with increased physician adherence to guideline recommendations; however, visits were prolonged, fees were higher, and no improvement could be demonstrated with regard to the observed intermediate-term patient outcomes. Guideline implementers (and users) should be cautious about putting unvalidated recommendations into practice. PMID- 10742319 TI - Potential to prevent carbon dioxide rebreathing of commercial products marketed to reduce sudden infant death syndrome risk. AB - OBJECTIVE: Rebreathing of exhaled air is one proposed mechanism for the increased risk for sudden infant death syndrome among prone sleeping infants. We evaluated how carbon dioxide (CO(2)) dispersal was affected by a conventional crib mattress and 5 products recently marketed to prevent prone rebreathing. SETTING: Infant pulmonary laboratory. EQUIPMENT: An infant mannequin with its nares connected via tubing to an 100-mL reservoir filled with 5% CO(2). The sleep surfaces studied included: firm mattress covered by a sheet, Bumpa Bed, Breathe Easy, Kid Safe/Baby Air, Halo Sleep System, and Sleep Guardian. The mannequin was positioned prone face-down or near-face-down. The sleep surfaces were studied with the covering sheet taut, covering sheet wrinkled, and with the mannequin arm positioned up, near the face. MEASUREMENTS: . We measured the fall in percentage end-tidal CO(2) as the reservoir was ventilated with the piston pump. The half time for CO(2) dispersal (t(1/2)) is an index of the ability to cause or prevent rebreathing. RESULTS: Compared with the face-to-side control, 5 of 6 surfaces allowed a significant increase in t(1/2) in all 3 prone scenarios. The firm mattress and 4 of the 5 surfaces designed to prevent rebreathing consistently allowed t(1/2) above thresholds for the onset of CO(2) retention and lethal rebreathing in an animal model (J Appl Physiol. 1995;78:740). CONCLUSIONS: With very few exceptions, infants should be placed supine for sleep. For infants placed prone or rolling to the prone position, significant rebreathing of exhaled air would be likely on all surfaces studied, except one. PMID- 10742320 TI - An elementary school-based health clinic: can it reduce medicaid costs? AB - OBJECTIVE: School-based health services have evolved from primarily controlling communicable disease to comprehensive programs with direct services, education, and improvement of the school environment. School-based health clinics (SBHCs), currently 1157 in number, are used to reach children for preventive and other routine care. Although several studies have examined the costs and effects of such programs, few, if any, have examined their potential to save Medicaid program outlays. The objective of this study was to assess the effect of the Whitefoord Elementary School-Based Health Clinic (WESBHC), located in Atlanta, Georgia, on health care costs paid by Georgia Medicaid over the 1994-1996 period. This clinic has been in operation since late 1994. DESIGN: The analysis uses 1994 1996 claims data for Medicaid-enrolled children 4 through 12 years old served by the WESBHC and similar children in a comparison school district without such a clinic. Descriptive and multivariate analyses are used to discern the differences in the changes in Medicaid expenses per child-year enrolled for these 2 groups of children. Both those who only used the WESBHC sporadically and those for whom it was their medical home were identified for analysis. RESULTS: The descriptive analysis shows that although there were no significant differences in the Medicaid expenses for the WESBHC and comparison children in 1994, before the operation of the WESBHC, by 1995, the WESBHC children had significantly lower emergency department expenses. In addition, they had higher Early Periodic Screening Diagnosis and Treatment preventive care expenses. By 1996, the WESBHC children had significantly lower inpatient, nonemergency department transportation, drug, and emergency department Medi- caid expenses. Multivariate analysis confirmed the effect of the WESBHC on lowering emergency department expenses. CONCLUSIONS: The results strongly suggest that the operation of a SBHC can have effects on the child's use of services and health care expenses. Given that these clinics serve all those who come for care and many of these are low income children, these savings are likely to accrue to the Medicaid program of the state. As states continue to implement Medicaid-managed care for their child populations, they will need to consider the ability of SBHCs to participate in and receive Medicaid revenues through health maintenance organization networks. PMID- 10742321 TI - Newborn circumcision decreases incidence and costs of urinary tract infections during the first year of life. AB - OBJECTIVE: To assess the effect of newborn circumcision on the incidence and medical costs of urinary tract infection (UTI) during the first year of life for patients in a large health maintenance organization. SETTING: Kaiser Permanente Medical Care Program of Northern California (KPNC). PATIENTS: The population consisted of members of KPNC. The study group consisted of a cohort of 28 812 infants delivered during 1996 at KPNC hospitals; of the 14 893 male infants in the group, 9668 (64.9%) were circumcised. A second cohort of 20 587 infants born in 1997 and monitored for 12 months was analyzed to determine incidence rates. DESIGN: Retrospective study of all infants consecutively delivered at 12 facilities. OUTCOME MEASURES: Diagnosis of UTI was determined from the KPNC computerized database using the International Classification of Diseases, Ninth Revision code for inpatients and KPNC Outpatient Summary Clinical Record codes for outpatients. A sample of 52 patient charts was reviewed to confirm the International Classification of Diseases, Ninth Revision and KPNC Outpatient Summary Clinical Record codes and provide additional data. RESULTS: Infants <1 year old who were born in 1996 had 446 UTIs (292 in females; 154 in males); 132 (86%) of the UTIs in males occurred in uncircumcised boys. The mean total cost of managing UTI was 2 times as high in males ($1111) as in females ($542). This higher total cost reflected the higher rate of hospital admission in uncircumcised males with UTIs (27.3%) compared with females (7.5%); mean age at hospitalization for UTI was 2.5 months old for uncircumcised boys and 6.5 months old for girls. In 1996, total cost of managing UTI in uncircumcised males ($155 628) was 10 times higher than for circumcised males ($15 466) despite the fact that uncircumcised males made up only 35.1% of the male patient base in 1996, reflecting the more frequent occurrence of UTI in uncircumcised males (132 episodes) than in circumcised males (22 episodes), and the larger number of hospital admissions in uncircumcised males (38) than in circumcised males (4). The incidence of UTI in the first year of life was 1:47 (2.15%) in uncircumcised males, 1:455 (.22%) in circumcised males, and 1:49 (2. 05%) in females. The odds ratio of UTI in uncircumcised:circumcised males was 9.1:1. CONCLUSIONS: Newborn circumcision results in a 9. 1-fold decrease in incidence of UTI during the first year of life as well as markedly lower UTI-related medical costs and rate of hospital admissions. Newborn circumcision during the first year of life is, thus, a valuable preventive health measure, particularly in the first 3 months of life, when uncircumcised males are most likely to be hospitalized with severe UTI. PMID- 10742322 TI - Hypothalamic-pituitary-adrenal function and glucocorticoid sensitivity in atopic dermatitis. AB - OBJECTIVES: Topical glucocorticoids (GCs) fail to produce a clinical response in some children with atopic dermatitis (AD), suggesting that GC resistance may be present. To determine whether such resistance is generalized or specific to diseased skin, hypothalamic-pituitary-adrenal (HPA) axis function has been assessed in children with moderate to severe AD, who showed a variable response to treatment with topical GC. STUDY DESIGN: Thirty-five patients (.7-18.7 years old; median: 9.3 years) with AD requiring topical GCs from infancy underwent a low-dose adrenocorticotrophin hormone (ACTH; Synacthen) test (LDST) (500 ng/1.73 m(2) ACTH). Groups 1 (7 patients), 2 (17 patients), and 3 (4 patients) used mild, moderate, or potent/very potent topical preparations, respectively. Group 4 (7 boys with severe, treatment-resistant disease) had received GC in at least 1 form (inhaled +/- intranasal +/- oral) in addition to varying potencies of topical GC. Fourteen healthy subjects (3.8-17.3 years old) served as control subjects. Group 4 patients had a daytime plasma cortisol profile and 08.00 hours measurement of plasma ACTH and its precursors. RESULTS: The response to ACTH for groups 1 and 2 did not differ from that of control subjects. Group 3 had lower peak, increment, and area under curve cortisol responses than those in controls, whereas group 4 had lower baseline, peak, and area under curve cortisol responses. Eight patients failed the LDST (peak cortisol <500 nmol/L and increment <200 nmol/L): controls = 0/14, group 1 = 0/7, group 2 = 1/17, group 3 = 4/4, and group 4 = 3/7. Treatment score (based on GC potency, area treated, and duration) was the only factor to influence peak cortisol response on LDST (r(2) = 24%). In group 4, only 1 of 7 patients had a cortisol profile within the normal range but he failed the LDST. In the 5 subjects with an 08.00 hours cortisol <300 nmol/L, the matched ACTH level was inappropriately low. CONCLUSIONS: HPA suppression was rarely found in children or adolescents with moderate to severe AD who used mild or moderately potent topical GCs over many years. However, HPA suppression was common in those receiving potent topical GC preparations or a combination of GC routes of administration. In those with severe AD, evidence of HPA suppression but lack of clinical response to GC treatment excluded significant generalized GC resistance. This would suggest that localized resistance to GCs within the diseased skin may be part of the aetiopathogenesis of severe AD. PMID- 10742323 TI - Sibling vesicoureteral reflux in multiple gestation births. AB - BACKGROUND: Vesicoureteral reflux (VUR) is the most commonly inherited disease of the genitourinary tract. Although the majority of evidence supports a genetic cause, the tendency for this condition to spontaneously improve over time has made it difficult to determine the actual mode of transmission. We report the incidence of VUR in siblings of multiple gestation births and for the first time compare the relative incidence of reflux between identical and fraternal twins. METHODS: A database consisting of all radionuclide cystograms and voiding cystourethrograms performed between the years 1986 and 1996 was searched for multiple gestation births. The medical records of each patient were evaluated for age at presentation, zygosity, reflux grade, and time to resolution. Children with secondary causes of VUR (eg, posterior urethral valves) were excluded. Triplets were treated as 2 pairs of twins for statistical analysis. RESULTS: Forty-six pairs met the inclusion criteria (31 dizygotic and 15 monozygotic). Overall, 23 (50%) of 46 siblings of index cases had demonstrable VUR. Comparison of VUR prevalence between identical and nonidentical twins was revealing with 80% (12/15) of identical twins and 35% (11/31) of fraternal twins having VUR. When only the youngest individuals in each group were considered, 100% (7/7) of the monozygotics and 50% (5/10) of the dizygotics demonstrated this trait. CONCLUSIONS: High concordance for VUR in identical twin siblings supports a genetic basis for the transmission of this disease. Results obtained from fraternal twin siblings provides convincing evidence that this trait is transmitted in an autosomal dominant fashion. PMID- 10742324 TI - Adverse sedation events in pediatrics: a critical incident analysis of contributing factors. AB - OBJECTIVE: Factors that contribute to adverse sedation events in children undergoing procedures were examined using the technique of critical incident analysis. METHODOLOGY: We developed a database that consists of descriptions of adverse sedation events derived from the Food and Drug Administration's adverse drug event reporting system, from the US Pharmacopeia, and from a survey of pediatric specialists. One hundred eighteen reports were reviewed for factors that may have contributed to the adverse sedation event. The outcome, ranging in severity from death to no harm, was noted. Individual reports were first examined separately by 4 physicians trained in pediatric anesthesiology, pediatric critical care medicine, or pediatric emergency medicine. Only reports for which all 4 reviewers agreed on the contributing factors and outcome were included in the final analysis. RESULTS: Of the 95 incidents with consensus agreement on the contributing factors, 51 resulted in death, 9 in permanent neurologic injury, 21 in prolonged hospitalization without injury, and in 14 there was no harm. Patients receiving sedation in nonhospital-based settings compared with hospital based settings were older and healthier. The venue of sedation was not associated with the incidence of presenting respiratory events (eg, desaturation, apnea, laryngospasm, approximately 80% in each venue) but more cardiac arrests occurred as the second (53.6% vs 14%) and third events (25% vs 7%) in nonhospital-based facilities. Inadequate resuscitation was rated as being a determinant of adverse outcome more frequently in nonhospital-based events (57.1% vs 2.3%). Death and permanent neurologic injury occurred more frequently in nonhospital-based facilities (92.8% vs 37.2%). Successful outcome (prolonged hospitalization without injury or no harm) was associated with the use of pulse oximetry compared with a lack of any documented monitoring that was associated with unsuccessful outcome (death or permanent neurologic injury). In addition, pulse oximetry monitoring of patients sedated in hospitals was uniformly associated with successful outcomes whereas in the nonhospital-based venue, 4 out of 5 suffered adverse outcomes. Adverse outcomes despite the benefit of an early warning regarding oxygenation likely reflect lack of skill in assessment and in the use of appropriate interventions, ie, a failure to rescue the patient. CONCLUSIONS: This study-a critical incident analysis-identifies several features associated with adverse sedation events and poor outcome. There were differences in outcomes for venue: adverse outcomes (permanent neurologic injury or death) occurred more frequently in a nonhospital-based facility, whereas successful outcomes (prolonged hospitalization or no harm) occurred more frequently in a hospital based setting. Inadequate resuscitation was more often associated with a nonhospital-based setting. Inadequate and inconsistent physiologic monitoring (particularly failure to use or respond appropriately to pulse oximetry) was another major factor contributing to poor outcome in all venues. Other issues rated by the reviewers were: inadequate presedation medical evaluation, lack of an independent observer, medication errors, and inadequate recovery procedures. Uniform, specialty-independent guidelines for monitoring children during and after sedation are essential. Age and size-appropriate equipment and medications for resuscitation should be immediately available regardless of the location where the child is sedated. All health care providers who sedate children, regardless of practice venue, should have advanced airway assessment and management training and be skilled in the resuscitation of infants and children so that they can successfully rescue their patient should an adverse sedation event occur. PMID- 10742325 TI - Echocardiographic diagnosis of heart disease in apparently healthy adolescents. AB - OBJECTIVE: Comprehensive data are currently unavailable on the prevalence of cardiac abnormalities in children after the newborn/infant period. The present report describes the prevalence of echocardiographically detected cardiac disease in a cohort of randomly selected healthy junior high school children. METHODS: The cohort for this report consists of 357 children (mean age: 13 years) randomly selected after blood pressure screening of 12 043 fifth through eighth grade students and having an echocardiographic examination as part of a study of insulin resistance in childhood. RESULTS: A physical examination performed by a board-certified pediatrician reported no cardiac abnormalities. However, echocardiography and Doppler studies identified 13 (3.6%) children (7 males and 6 females), with previously unknown cardiac abnormalities, as follows: abnormal mitral valve with mitral regurgitation (4), bicuspid aortic valve (2), atrial septal defect (2), coronary artery to pulmonary artery fistula (1), patent ductus arteriosus (1), pulmonary hypertension (1), cardiomyopathy (1), and pulmonary artery stenosis (1). Physical examination performed by a pediatric cardiologist detected abnormal cardiac findings in 7 (54%) of the children. Cardiac catheterization was required in 3 for additional diagnostic evaluation and in 2 for therapeutic intervention; 1 patient underwent open-heart surgery. Bacterial endocarditis prophylaxis was recommended in 8 (62%) of the 13 children. CONCLUSIONS: The results suggest that: 1) clinically significant cardiac disease in childhood is more prevalent than previously reported; and 2) improved screening methods should be considered to detect asymptomatic but significant cardiac abnormalities that may result in long-term complications. echocardiography, prevalence, incidence, heart disease, children. PMID- 10742326 TI - Evaluation of emergency department referrals by telephone triage. AB - OBJECTIVE: Telephone triage programs are becoming very common at children's hospitals across the nation. One of the proposed benefits of these programs is the more efficient use of health care resources by triaging patients to the appropriate level of health care. The purpose of this study is to examine the appropriateness of referrals to a pediatric emergency department (ED) by the Pediatric Health Information Line (PHIL), a hospital-based telephone triage program, versus all other sources of referrals. METHODS: A blinded Delphi rating system was used to review the physician's sheets of 133 consecutive ED referrals by PHIL for medical appropriateness. A total of 260 randomly selected control patients seen in the ED during the same period were similarly reviewed. If 2 of 3 pediatric emergency medicine physicians agreed that an ED visit was appropriate, then it was considered appropriate. A comparison of the 2 groups' ED appropriateness was made using a contingency table chi(2) test. An odds ratio with confidence limits was also calculated. Demographic data were collected for both groups including age, race, gender, and insurance status. RESULTS: The PHIL group had an appropriateness rate of 80.2%, compared with 60.5% for the control group (chi(2) = 14.6369; odds ratio = 2.65; 95% confidence interval [1.5759,4.5008]). CONCLUSIONS: This demonstrated that for the period studied, PHIL referrals to the ED had a 33% higher rate of appropriateness than controls. This evidence supports telephone triage as an efficient gatekeeper for health care resources. PMID- 10742327 TI - Neonatal assisted ventilation: predictors, frequency, and duration in a mature managed care organization. AB - OBJECTIVES: Reference data are lacking on the frequency and duration of assisted ventilation in neonates. This information is essential for determining resource needs and planning clinical trials. As mortality becomes uncommon, ventilator utilization is increasingly used as a measure for assessing therapeutic effect and quality of care in intensive care medicine. Valid comparisons require adjustments for differences in a patient's baseline risk for assisted ventilation and prolonged ventilator support. The aims of this study were to determine the frequency and length of ventilation (LOV) in preterm and term infants and to develop models for predicting the need for assisted ventilation and length of ventilator support. METHODS: We performed a retrospective, population-based cohort study of 77 576 inborn live births at 6 Northern California hospitals with level 3 intensive care nurseries in a group-model managed care organization. The gestational age-specific frequency and duration of assisted ventilation among surviving infants was determined. Multivariable regression was performed to determine predictors for assisted ventilation and LOV. RESULTS: Of 77 576 inborn live births in the study, 11 199 required admission to the neonatal intensive care unit and of these, 1928 survivors required ventilator support. The proportion of infants requiring assisted ventilation and the median LOV decreased markedly with increasing gestational age. In addition to gestational age, admission illness severity, 5-minute Apgar scores, presence of anomalies, male sex, and white race were important predictors for the need for assisted ventilation. The ability of the models to predict need for ventilation was high, and significantly better than birth weight alone with an area under the receiver operating characteristic curve of.90 versus.70 for preterm infants, and.88 versus.50 for term infants. For preterm infants, gestational age, admission illness severity, oxygenation index, anomalies, and small-for-gestational age status were significant predictors for LOV, accounting for 60% of the variance in the length of assisted ventilation. For term infants, oxygenation index and anomalies were significant predictors but only accounted for 29% of the variance. CONCLUSIONS: Considerable variation exists in the utilization of ventilator support among infants of closely related gestational age. In addition, a number of medical risk factors influence the need for, and length of, assisted ventilation. These models explain much of the variance in LOV among preterm infants but explain substantially less among term infants.neonatal intensive care, assisted ventilation, Score for Neonatal Acute Physiology, resource consumption, prematurity. PMID- 10742328 TI - Effect of seating position and restraint use on injuries to children in motor vehicle crashes. AB - OBJECTIVE: To determine the effect of restraint use and seating position on injuries to children in motor vehicle crashes, with stratification by area of impact. METHODS: Children <15 years old involved in serious automobile crashes in Utah from 1992 through 1996 were identified from statewide motor vehicle crash records. Serious crashes are defined as those resulting in occupant injuries with broken bones or significant bleeding or property damage exceeding $750. Probabilistic methods were used to link these records with hospital records. Analysis used logistic regression controlling for age, restraint use, occupant seating position, and type of crash. RESULTS: We studied 5751 children and found 53% were rear seat passengers. More than 40% were unrestrained. Sitting in the rear seat offered a significant protective effect (adjusted odds ratio: 1.7; 95% confidence interval: 1.6-2.0), and restraint use enhanced this effect (adjusted odds ratio: 2.7; 95% confidence interval: 2.4-3.1). Mean hospital charges were significantly greater for front seat passengers. CONCLUSIONS: Rear seat position during a motor vehicle crash provides a significant protective effect, restraint use furthers this effect, and usage rates of restraint devices are low. The rear seat protective effect is in addition to and independent of the protection offered from restraints. PMID- 10742329 TI - Neonatal and pediatric posttransfusion hepatitis C: a look back and a look forward. PMID- 10742330 TI - Use of telemedicine for children with special health care needs. AB - OBJECTIVE: In 1995, the Children's Medical Services (CMS) of the State of Georgia contracted with the Department of Pediatrics of the Medical College of Georgia (MCG) and the MCG Telemedicine Center to develop telemedicine programs to provide subspecialty care for children with special health care needs. This article presents project statistics and results of client evaluation of services, as well as physician faculty attitudes toward telemedicine. DESIGN: A demonstration project using telemedicine between a tertiary center and a rural clinic serving children with special health care needs was established. Data were collected and analyzed for December 12, 1995 to May 31, 1997, during which 333 CMS telemedicine consultations were performed. RESULTS: Most CMS telemedicine consultations (35%) involved pediatric allergy/immunology. Other subspecialties included pulmonology (29%), neurology (19%), and genetics (16%). Overall, patients were satisfied with the services received. Initially, physician faculty members were generally positive but conservative in their attitudes toward using telemedicine for delivering clinical consultation. After a year's exposure and/or experience with telemedicine, 28% were more positive, 66% were the same, and only 4% were more negative about telemedicine. The more physicians used telemedicine, the more positive they were about it (r =.30). CONCLUSIONS: In terms of family attitudes and individual care, telemedicine is an acceptable means of delivering specific pediatric subspecialty consultation services to children with special health care needs, living in rural areas distant to tertiary centers. Telemedicine is more likely to be successful as part of an integrated health services delivery than when it is the sole mode used for delivery of care. PMID- 10742331 TI - Differences are voiced by two Lyme camps at a Connecticut public hearing on insurance coverage of Lyme disease. PMID- 10742332 TI - Milliman and Robertson-going in the wrong direction. PMID- 10742333 TI - Patient care guidelines in pediatrics. AB - The increase in availability of patient care guidelines is consistent with the expansion of managed care health care systems and enhanced efforts of health care providers to deliver evidence-based patient care. When used effectively, guidelines support: 1) the reduction of unexplained variation in health care practice; 2) education of practitioners and health care delivery systems; and 3) the continuous improvement in processes that improve patient outcomes. It is with these goals in mind that Milliman and Robertson released its latest publication of Health Care Management Guidelines entitled Pediatric Health Status Improvement and Management (HSIM). This publication contains comprehensive information on many aspects of pediatric care, from the prenatal period through adolescence, from preventive medicine through chronic care. Like other Milliman and Robertson Health Care Management Guidelines publications, Pediatric HSIM offers both treatment guides and benchmarks that enable health care professionals to provide optimal levels of care and to measure their performance against peers. The primary focus of Pediatric HSIM is on wellness across a child's lifetime, an objective that fits well with our goal as providers of care to children of keeping patients healthy through preventive medicine. PMID- 10742334 TI - The prepuce, urinary tract infections, and the consequences. PMID- 10742335 TI - The critical role of population-based epidemiology in cost-effectiveness research. PMID- 10742336 TI - What the multimodal treatment study of children with attention deficit/hyperactivity disorder did and did not say about the use of methylphenidate for attention deficits. PMID- 10742337 TI - Adverse sedation events in pediatrics: a critical incident analysis of contributing factors. PMID- 10742338 TI - Recurrent clitoral tourniquet syndrome. PMID- 10742339 TI - Swimming programs for infants and toddlers. Committee on Sports Medicine and Fitness and Committee on Injury and Poison Prevention. American Academy of Pediatrics. AB - Infant and toddler aquatic programs provide an opportunity to introduce young children to the joy and risks of being in or around water. Generally, children are not developmentally ready for swimming lessons until after their fourth birthday. Aquatic programs for infants and toddlers have not been shown to decrease the risk of drowning, and parents should not feel secure that their child is safe in water or safe from drowning after participating in such programs. Young children should receive constant, close supervision by an adult while in and around water. PMID- 10742340 TI - Suicide and suicide attempts in adolescents. Committee on Adolescents. American Academy of Pediatrics. AB - Suicide is the third leading cause of death for adolescents 15 to 19 years old.(1) Pediatricians can help prevent adolescent suicide by knowing the symptoms of depression and other presuicidal behavior. This statement updates the previous statement(2) by the American Academy of Pediatrics and assists the pediatrician in the identification and management of the adolescent at risk for suicide. The extent to which pediatricians provide appropriate care for suicidal adolescents depends on their knowledge, skill, comfort with the topic, and ready access to appropriate community resources. All teenagers with suicidal symptoms should know that their pleas for assistance are heard and that pediatricians are willing to serve as advocates to help resolve the crisis. PMID- 10742341 TI - School health assessments. Committee on School Health. American Academy of Pediatrics. AB - Comprehensive health assessments often are performed in school-based clinics or public health clinics by health professionals other than pediatricians. Pediatricians or other physicians skilled in child health care should participate in such evaluations. This statement provides guidance on the scope of in-school health assessments and the roles of the pediatrician, school nurse, school, and community. PMID- 10742342 TI - Do not resuscitate orders in schools. Committee on School Health and Committee on Bioethics. American Academy of Pediatrics. AB - Increased medical knowledge and technology have led to the survival of many children who previously would have died of a variety of conditions. As these children with continuing life-threatening problems reach school age, families, professionals, and paraprofessionals have to deal with the challenges involved in their care. Some children may be at high risk of dying while in school. When families have chosen to limit resuscitative efforts, school officials should understand the medical, emotional, and legal issues involved. PMID- 10742343 TI - Use of psychoactive medication during pregnancy and possible effects on the fetus and newborn. Committee on Drugs. American Academy of Pediatrics. AB - Psychoactive drugs are those psychotherapeutic drugs used to modify emotions and behavior in the treatment of psychiatric illnesses. This statement will limit its scope to drug selection guidelines for those psychoactive agents used during pregnancy for prevention or treatment of the following common psychiatric disorders: schizophrenia, major depression, bipolar disorder, panic disorder, and obsessive-compulsive disorder. The statement assumes that pharmacologic therapy is needed to manage the psychiatric disorder. This decision requires thoughtful psychiatric and obstetric advice. PMID- 10742344 TI - Firearm-related injuries affecting the pediatric population. Committee on Injury and Poison Prevention. American Academy of Pediatrics. AB - This statement reaffirms the 1992 position of the American Academy of Pediatrics that the absence of guns from children's homes and communities is the most reliable and effective measure to prevent firearm-related injuries in children and adolescents. A number of specific measures are supported to reduce the destructive effects of guns in the lives of children and adolescents, including the regulation of the manufacture, sale, purchase, ownership, and use of firearms; a ban on handguns and semiautomatic assault weapons; and expanded regulations of handguns for civilian use. In addition, this statement reviews recent data, trends, prevention, and intervention strategies of the past 5 years. PMID- 10742345 TI - Clinical practice guideline: early detection of developmental dysplasia of the hip. Committee on Quality Improvement, Subcommittee on Developmental Dysplasia of the Hip. American Academy of Pediatrics. AB - Developmental dysplasia of the hip is the preferred term to describe the condition in which the femoral head has an abnormal relationship to the acetabulum. Developmental dysplasia of the hip includes frank dislocation (luxation), partial dislocation (subluxation), instability wherein the femoral head comes in and out of the socket, and an array of radiographic abnormalities that reflect inadequate formation of the acetabulum. Because many of these findings may not be present at birth, the term developmental more accurately reflects the biologic features than does the term congenital. The disorder is uncommon. The earlier a dislocated hip is detected, the simpler and more effective is the treatment. Despite newborn screening programs, dislocated hips continue to be diagnosed later in infancy and childhood,(1-11) in some instances delaying appropriate therapy and leading to a substantial number of malpractice claims. The objective of this guideline is to reduce the number of dislocated hips detected later in infancy and childhood. The target audience is the primary care provider. The target patient is the healthy newborn up to 18 months of age, excluding those with neuromuscular disorders, myelodysplasia, or arthrogryposis. PMID- 10742346 TI - Improving the implementation of State Children's Health Insurance Programs for adolescents. Report of an invitational conference sponsored by the American Academy of Pediatrics, Section on Adolescent Health*, September 26-27, 1999. PMID- 10742347 TI - Letter from the editor PMID- 10742348 TI - Tribute to the reviewers PMID- 10742349 TI - Literacy promotion in primary care pediatrics: can we make a difference? AB - BACKGROUND: Reading skills are critical to children's success in school and the increasingly technologic workforce. Children from low-income families are at risk for home environments that fail to promote emergent literacy and for reading failure. A home environment that encourages learning and parents who are involved in their children's education are important factors in school achievement. OBJECTIVE: To evaluate the effects of a literacy promoting intervention delivered by pediatric providers as part of well-child care on parent attitudes and behaviors and on child language. DESIGN/METHODS: A multicultural group of 205 low income families with 5- to 11-month-olds were prospectively enrolled, interviewed, and randomized to intervention (n = 106) or control (n = 99) groups. Families in the intervention group received developmentally appropriate children's books and educational materials and advice about sharing books with children, while those in the control group received no books or materials relevant to literacy. After an average of 3.4 well-child visits in both groups, 153 (75%) were reinterviewed and the children's receptive and expressive vocabulary was tested using a modified version of the MacArthur Communication and Development Inventory (Short Form). Parents were asked if their child understood (receptive vocabulary) or said (expressive vocabulary) each of 100 words, half of which were in the books given. Families were found to have a Child-Centered Literacy Orientation if they mentioned reading aloud as one of their child's favorite activities or as one of their own favorite joint activities or if they usually read together at bedtime. At follow-up toddlers were 18.4 months old on average. RESULTS: Intervention and control groups had similar literacy related characteristics at baseline. There was a 40% increase in Child-Centered Literacy Orientation among intervention families compared with 16% among controls. Intervention families read more with their toddlers (4.3 vs 3.8 days/week). Both receptive and expressive vocabulary scores were higher in older intervention toddlers (18-25 months old; n = 88), but not in younger intervention toddlers (13 17 months old; n = 62). This significant effect of the intervention on vocabulary scores in older toddlers was found for both the 50 words in the books and those not in the books. After parent education, foreign birth and language proficiency, and child age were statistically controlled, the intervention remained significantly associated with higher language outcomes in older toddlers. However, when reading aloud was added to the multivariate analysis, the influence of the intervention was no longer evident, suggesting the intervention's effect on child language was mediated through increased shared reading with these toddlers. CONCLUSION: This simple and inexpensive intervention, delivered as part of well-child care, changed parent attitudes toward the importance of reading with their infants and toddlers. These intervention parents and their children read more together and this was associated with enhanced language development in older toddlers in this diverse group of low-income families. PMID- 10742350 TI - Behavioral and self-concept changes after six months of enuresis treatment: a randomized, controlled trial. AB - BACKGROUND: Previous studies have suggested changes in self-concept with successful treatment of primary nocturnal enuresis (PNE), but behavioral changes have not been reported as a consistent associated finding. OBJECTIVE: To determine if self-concept and behavior change after 6 months of treatment of monosymptomatic PNE by conditioning alarm or desmopressin acetate (DDAVP). DESIGN: Randomized, controlled trial in an inner-city hospital clinic. Subjects were 182 children referred or recruited through media publicity, randomly assigned both to 1 of 8 pediatricians and 1 of 3 treatment groups (alarm, DDAVP, or placebo). Included were children >7 years old with PNE, no daytime symptoms, bladder capacity >50% expected, and wetting >3 times a week. Excluded were children with central nervous system disorders or developmental delays, and those currently on DDAVP or alarm. Subjects completed the Piers-Harris Children's Self Concept Scale and Harter's Perceived Competence Scale for Children (PCSC) at initial visit and after 6 months of treatment. Parents completed the Achenbach Child Behavior Checklist (CBCL) at the same times. RESULTS: After 6 months of treatment the Piers-Harris total score showed a highly significant treatment by period interaction effect for DDAVP, a significant effect for alarm, and no effect for placebo. For children who achieved 75% dryness the CBCL showed a treatment by improvement interaction effect that was highly significant for DDAVP and placebo with no effect for alarm. For the PCSC there were no treatment or outcome interaction effects. After 6 months of treatment there were significant changes over time unrelated to outcome or treatment in the Piers-Harris Subscales and in the CBCL Internalizing and Externalizing Scores, and the Social Thought and Attention Problems Subscales. The PCSC was more stable with no changes in total score, and positive changes over time in only 2 Subscales, Scholastic and Social. CONCLUSION: Children's self-concept improved with the type of treatment and amount of success. Parents' perceptions of behavior improve with type of treatment and amount of success. Children rate their self-concept and some physical attributes better after treatment with any of DDAVP, alarm, or placebo regardless of outcome. Frequent follow-up with emotional support and encouragement appear to be important components of an efficacious intervention for children with nocturnal enuresis. PMID- 10742351 TI - On pins and needles? Pediatric pain patients' experience with acupuncture. AB - INTRODUCTION: Despite its increasing use as a complementary therapy to treat pain, acupuncture is rarely considered by pediatricians, in part due to perceptions that it will not be acceptable to pediatric patients. We wished to describe pediatric pain patients' experience with acupuncture treatment for chronic pain. DESIGN: Retrospective case series. METHODS: Subjects were pediatric pain patients referred by the Pain Treatment Service at Children's Hospital in Boston, who went to a pediatric acupuncturist. A research assistant not involved in the patient's care conducted the survey by telephone. Data were analyzed qualitatively and descriptively. RESULTS: Of 50 eligible patients, 47 families were reached by telephone; all agreed to be interviewed. Patients had a median age of 16 years at the time of referral, 79% were female, and 96% were white. The most common three diagnoses were migraine headache (n = 7), endometriosis (n = 6), and reflex sympathetic dystrophy (n = 5). Patients had a median of 8 treatments (range: 0-60) within 3 months (range: 0-48 months); 85% of families paid out-of-pocket. Acupuncture therapies included needle insertion (98%), heat/moxa (85%), magnets (26%), and cupping (26%). Most patients and parents rated the therapy as pleasant (67% children/60% parents), and most (70% children/59% parents) felt the treatment had helped their symptoms; only 1 said that treatment made symptoms worse. CONCLUSION: Pediatric patients with chronic, severe pain found acupuncture treatment pleasant and helpful. Additional, prospective studies are needed to quantify the costs and effectiveness of acupuncture treatment for pediatric pain. PMID- 10742352 TI - Reliabilities of short substance abuse screening tests among adolescent medical patients. AB - OBJECTIVE: To determine the internal consistency and 1-week test-retest reliability of the Simple Screening Instrument for Alcohol and Other Drug Abuse (SSI-AOD), the CAGE-AA (CAGE questions adapted for adolescents), and 4 modified items from the Drug and Alcohol Problem QuickScreen (DAP-4) among adolescents. METHODS: Fifteen- to 18-year-old medical patients (n = 173) completed screening tests during a routine medical visit and then again 1 week later. Internal consistency for each test and retest was calculated using Cronbach's alpha, and 1 week test-retest reliability was calculated by using Winer's unbiased estimate of the intraclass correlation coefficient (r). RESULTS: The SSI-AOD has good internal consistency (alpha =.83) and the CAGE-AA questions acceptable internal consistency (alpha =.60). Alpha varied with gender and race, and item analysis indicated the CAGE-AA test could be improved. As expected, the DAP-4 had a lower alpha score (.46). All screening instruments studied had high 1-week test-retest reliabilities (range r =.82-.90). CONCLUSIONS: The SSI-AOD is a reliable substance abuse screening instrument among adolescent medical patients. The CAGE AA questions must be further revised and tested before their use can be recommended. The DAP-4 questions are likely measuring different, but important, constructs. PMID- 10742353 TI - The associations among pediatricians' knowledge, attitudes, and practices regarding emergency contraception. AB - OBJECTIVES: To quantify practitioner administration of the emergency contraceptive pill (ECP) among adolescent patients, and to determine if such administration is associated with physician knowledge and attitudes regarding efficacy, side effects, and appropriate use. DESIGN: Survey of pediatricians. SETTING: The survey address list was generated from a database of active Fellows of the American Academy of Pediatrics in the District of Columbia metropolitan area. MAIN OUTCOMES MEASURES: Prescription of the ECP in the previous 12 months, or counseling of an adolescent patient about the ECP. RESULTS: Of the 236 questionnaires distributed, 143 (61%) were returned and 121 (51%) were usable. Twenty-four pediatricians (20%) reported prescribing the ECP, and 29 (24%) had counseled adolescent patients about the ECP. Of the practice-related variables surveyed, both the number of adolescents seen per week and the practice setting were significantly associated with these outcomes. Of the knowledge-related variables surveyed, knowledge of the timing and the Food and Drug Administration labeled status of the ECP were significantly associated with outcomes. None of the attitude-related variables surveyed were associated with outcomes. CONCLUSIONS: This study demonstrates that knowledge deficits, not attitude related variables, are significantly associated with the low level of ECP administration and counseling among District of Columbia pediatricians. Because knowledge deficits are amenable to educational interventions, our data suggest that informing pediatricians about the ECP may increase its administration among their adolescent patients.emergency contraceptive pill, pediatricians, adolescents. PMID- 10742354 TI - Primary care role in the management of childhood depression: a comparison of pediatricians and family physicians. AB - OBJECTIVE: To provide a self-described assessment of pediatricians' and family physicians' management of childhood depression. DESIGN: Mail survey of 595 general pediatricians and 557 family physicians in North Carolina. RESULTS: The response rate was 66%. Most primary care physicians used referral (65%) and counseling (61%) for management of childhood depression. Family physicians used medications more commonly (18% vs 9%), and pediatricians referred patients more commonly (77% vs 48%). In logistic regression analysis, physicians comfortable with management of depression (odds ratio [OR], 4.8: 2.7-8.4), physicians who believed that antidepressants are more effective than counseling (OR, 2.6: 1.4 4.8), and family physicians (OR, 2.2: 1.9-4.1) were more likely to have used medications for childhood depression. CONCLUSIONS: Most primary care physicians refer pediatric patients with depression; however, practice patterns vary by specialty and other factors. Future studies must consider the role of primary care and evaluate how interspecialty variations affect costs and outcomes of childhood depression. PMID- 10742355 TI - Primary care providers and childhood mental health conditions. PMID- 10742356 TI - Effective evaluation of residency education: how do we know it when we see it? PMID- 10742357 TI - Does a health maintenance curriculum for pediatric residents improve performance? AB - OBJECTIVE: To assess the knowledge and skills of residents in health maintenance and to measure the effectiveness of a new curriculum. DESIGN: Longitudinal cohort study of first- and second-year pediatric residents over 2 academic years (AY). Residents in AY 1995-1996 (n = 32) comprised a control group. Residents in AY 1996-1997 (n = 36) served as a study group who completed a structured 12-week program of reading materials, small group discussions, and case scenarios. METHODS: A needs assessment identified 7 topic areas in health maintenance: health screening, immunizations, nutrition, development, behavior, injury prevention, and dental health. Control and study residents' cognitive knowledge, chart documentation, and clinical performance were assessed using a 50-item multiple choice examination, medical records audit, and standardized patient evaluations. RESULTS: Mean examination scores, chart documentation, and clinical performance did not differ significantly between first- and second-year control residents. In the study group, mean examination scores of first-year residents were not significantly better than controls. Second-year residents significantly improved their knowledge about health screening, nutrition, and dental health compared with controls but showed no improvement in other areas. Medical record documentation increased significantly among study residents compared with control residents. Clinically, first-year residents significantly improved in the task of health screening (relative risk [RR] 6.33) and markedly improved (36.8% vs 66.7%) their injury prevention counseling compared with controls. Second-year residents showed significant increases in health screening (RR 6.09) and taking a nutritional history (RR 1. 44) compared with controls. Assessment of the curriculum by residents showed a high degree of satisfaction with the program. CONCLUSIONS: Pediatric residents who had clinical experience and completed the curriculum demonstrated significant improvements in their medical record documentation and selective gains in knowledge and clinical performance of health maintenance compared with residents with clinical experience only. A structured curriculum in health maintenance and a multifaceted assessment system can identify and enhance the skills of pediatric residents. PMID- 10742358 TI - Structured clinical observations: a method to teach clinical skills with limited time and financial resources. AB - OBJECTIVE: To develop and implement a methodology to teach clinical skills to medical students in busy clinical settings. METHODS: The Structured Clinical Observation (SCO) program with guidelines and observation sheets for history taking, physical examination, and information-giving skills was created. Faculty development preceded SCO implementation for pediatric clerkship students at Jefferson Children's Health Center. SCO observation sheets were tabulated and faculty and student questionnaires were administered. RESULTS: The mean number of observations per student was 6, with 368 observations done for 63 students. SCOs were highly rated as an educational tool by faculty and students. The impact of the SCO program on faculty ability to perform clinical duties was initially minimal, but increased over the year. Observations were used primarily for feedback, but did influence outpatient clinical faculty's evaluation of two thirds of the students. Only 50% of students reported being observed in other rotations. CONCLUSIONS: SCOs are a feasible, inexpensive, qualitatively effective method of teaching clinical skills. The quantitative effect of SCOs on performance needs to be evaluated. PMID- 10742359 TI - Impact of an encounter-based prompting system on resident vaccine administration performance and immunization knowledge. AB - OBJECTIVES: To evaluate an encounter-based immunization prompting system on resident performance in administering vaccines and knowledge of immunization guidelines. DESIGN/METHODS: Prospective randomized, controlled trial. Subjects were first- and second-year pediatric residents in a hospital-based continuity clinic. The intervention group received manual prompts of immunizations due. Postclinic chart review compared immunizations due with those administered. Acceptable and unacceptable reasons for not administering vaccines were assigned. Resident knowledge was measured by a 70-item examination. RESULTS: The intervention group had significantly less missed opportunities/vaccine administration errors (11.4% vs 21.6%). The most common reason for unacceptable errors in the intervention group: vaccine was given too early; in the control group: vaccine was postponed to next visit. Pre- and postintervention knowledge scores were similar: intervention group (75.5% vs 80.7%, control group; 76.5% vs 81.3%). CONCLUSION: An immunization prompting system in a hospital-based pediatric resident continuity clinic reduced missed opportunities/vaccine administration errors without significantly impacting resident knowledge of immunization guidelines.immunization schedule, vaccination, immunization, prompting systems, resident education. PMID- 10742360 TI - Does being a chief resident predict leadership in pediatric careers? AB - OBJECTIVE: Many organizations make efforts to identify future pediatric leaders, often focusing on chief residents (CRs). Identifying future leaders is an issue of great importance not only to the ultimate success of the organization but also to the profession. Because little is known regarding whether completing a CR predicts future leadership in medicine, we sought to determine if former pediatric CRs when compared with pediatric residents who were not CRs reported more often that they were leaders in their profession. DESIGN/METHODS: Twenty four pediatric training programs stratified by resident size (<18, 18-36, and >36) and geography (East, South, Midwest, and West) were selected randomly from the Graduate Medical Education Directory (American Medical Association, Chicago, IL). Program directors were contacted by mail and telephone and asked to provide their housestaff rosters from 1965-1985. The resulting resident sample was surveyed by questionnaire in 1995. RESULTS: Fifteen of 17 program directors (88%) who possessed the requested data provided 1965-1985 rosters yielding a sample of 963 residents. Fifty-five percent of the resident sample (533) responded. Fifty eight of the respondents had not completed a pediatric residency, leaving a survey sample of 475. Thirty-four percent (163) were CRs. The sample had a mean age of 47, 67% were male and 87% married. Fellowships were completed by 51%. More former CRs compared with non-CRs (75% vs 64%), more former fellows than non fellows (75% vs 60%) and more males than females (74% vs 55%) reported they were professional leaders. These associations persisted in a logistic regression that controlled for CR status, gender, marital status, and fellowship status as leadership predictors. Former CRs, former fellows, and men were, respectively, 1.8, 2.3, and 2.3 times more likely to report professional leadership. CONCLUSIONS: Pediatric residents who were former CRs and/or fellows, and males were more likely to report professional leadership. Although men were more likely to report professional leadership, with more women entering pediatrics the reported gender differences will likely disappear over time. PMID- 10742361 TI - The unmet health needs of America's children. AB - OBJECTIVE: Unmet need for health care is a critical indicator of access problems. Among children, unmet need for care has special significance inasmuch as the failure to obtain treatment can affect health status and functioning in the near- and long-term. The purpose of this study was to present current prevalence estimates and descriptive characteristics of children with unmet health needs using nationally representative household survey data. METHODS: We analyzed 4 years of National Health Interview Survey data spanning 1993 through 1996. Our analysis included 97 206 children <18 years old. Measures of unmet need for medical care, dental care, prescription medications, and vision care were obtained from an adult household member (usually the mother) responding for the child. Bivariate and multivariate analyses were used to assess the degree to which unmet need was related to the demographic and socioeconomic characteristics of the child and family. RESULTS: Overall, 7.3% (4.7 million) of US children experienced at least 1 unmet health care need. Dental care was the most prevalent unmet need. After adjustment for confounding factors, near-poor and poor children were both about 3 times more likely to have an unmet need as nonpoor children (adjusted odds ratio [95% confidence interval] = 2.89 [2.52, 3.32], 3.0 [2.53, 3.56], respectively). Uninsured children were also about 3 times more likely to have an unmet need as privately insured children (adjusted odds ratio [95% confidence interval] = 2. 92 [2.58, 3.32]). CONCLUSIONS: Despite the nation's great wealth, unmet health needs remain prevalent among US children. A combined public policy that addresses financial and nonfinancial barriers to care is required to reduce the prevalence of unmet need for health care. PMID- 10742362 TI - Measuring consumer experiences with primary care. AB - OBJECTIVE: To assess the adequacy of the Primary Care Assessment Tool-Child Edition (PCAT-CE) for evaluating the attainment of the key characteristics of primary care services for children and youth. DESIGN: Community-based telephone survey. SETTING: Specific political subdivision in Washington, DC. PARTICIPANTS: Four hundred fifty parents/guardians of offspring 18 years of age or less. MEASURES: Reliability, validity and principal component analysis of 5 scales representing key aspects of the 4 cardinal domains of primary care included in the PCAT-CE. In addition, 2 subdomains (first contact use and extent of affiliation with a primary care source) were included as indices to describe overall patterns of use and affiliation with the particular source of care. RESULTS: Most scales had adequate internal consistency, test-retest reliability, and construct validity. The principal components factor analysis yielded 5 separate factors. These corresponded to the subdomains of first contact accessibility; coordination of care; characteristics of the professional-patient relationship over-time; and comprehensiveness (both services available and indicated services received). CONCLUSIONS: Psychometric assessment supported the integrity and general adequacy of the PCAT-CE for assessing the characteristics and quality of primary care for children and youth. Testing of revised versions in a variety of different settings is underway. A major component of this testing is to explore the possibility of reducing the number of items while retaining sufficient detail about each component of primary care to make judgements about people's experiences with that care. PMID- 10742363 TI - Commentary: monitoring expanded health insurance for children: challenges and opportunities. PMID- 10742364 TI - Chiropractors and vaccination: A historical perspective. AB - Although there is overwhelming evidence to show that vaccination is a highly effective method of controlling infectious diseases, a vocal element of the chiropractic profession maintains a strongly antivaccination bias. Reasons for this are examined. The basis seems to lie in early chiropractic philosophy, which, eschewing both the germ theory of infectious disease and vaccination, considered disease the result of spinal nerve dysfunction caused by misplaced (subluxated) vertebrae. Although rejected by medical science, this concept is still accepted by a minority of chiropractors. Although more progressive, evidence-based chiropractors have embraced the concept of vaccination, the rejection of it by conservative chiropractors continues to have a negative influence on both public acceptance of vaccination and acceptance of the chiropractic profession by orthodox medicine. PMID- 10742365 TI - Systemic corticosteroids in infant bronchiolitis: A meta-analysis. AB - OBJECTIVE: To determine whether corticosteroids are efficacious in treating bronchiolitis in hospitalized infants. METHODS: Online bibliographic databases (Medline, Embase, and Cochrane Clinical Trials Registry) were searched for: 1) bronchiolitis or respiratory syncytial virus, and 2) corticosteroid or glucocorticoid or steroidal antiinflammatory agents or adrenal cortex hormones. Reference lists from all selected articles were also examined. Randomized, placebo-controlled trials of systemic corticosteroids in treatment of infants hospitalized with bronchiolitis were selected by 2 investigators. Of 12 relevant publications identified in the literature search, 6 met the selection criteria and had relevant data available. Investigators independently extracted data for 3 outcomes: length of stay (LOS), duration of symptoms (DOS), and clinical scores. RESULTS: In the pooled analysis, infants who received corticosteroids had a mean LOS or DOS that was.43 days less than those who received the placebo treatment (95% confidence interval: -.81 to -.05 days). The effect size for mean clinical score was -1.60 (95% confidence interval: -1.92 to -1.28), favoring treatment. Secondary analyses of mean LOS or DOS were performed on 5 trials that had clearly identified methods of randomization, 5 trials that measured LOS, and 4 trials that clearly excluded infants with previous wheezing. The estimates of effect were similar to the primary analysis but were not statistically significant. CONCLUSIONS: Combined, published reports of the effect of systemic corticosteroids on the course of bronchiolitis suggest a statistically significant improvement in clinical symptoms, LOS, and DOS. PMID- 10742366 TI - A randomized, double-blind, placebo-controlled trial of the effects of prophylactic theophylline on renal function in term neonates with perinatal asphyxia. AB - BACKGROUND: The kidney is the most damaged organ in asphyxiated full-term infants. Experiments in rabbits and rats have shown that renal adenosine acts as a vasoconstrictive metabolite in the kidney after hypoxemia and/or ischemia, contributing to the fall in glomerular filtration rate (GFR) and filtration fraction. Vasoconstriction produced by adenosine can be inhibited by the nonspecific adenosine receptor antagonist, theophylline. Gouyon and Guignard performed studies in newborn and adult rabbits subjected to normocapnic hypoxemia. Their results clearly showed that the hypoxemia-induced drop in GFR could be avoided by the administration of low doses of theophylline. OBJECTIVE: This study was designed to determine whether theophylline could prevent and/or ameliorate renal dysfunction in term neonates with perinatal asphyxia. SETTING: Buenos Aires, Argentina. STUDY DESIGN: We randomized 51 severe asphyxiated term infants to receive intravenously a single dose of either theophylline (8 mg/kg; study group: n = 24) or placebo (control group: n = 27) during the first 60 minutes of life. The 24-hour fluid intake and the urine volumes formed were recorded during the first 5 days of life. Daily volume balances (water output/input ratio and weights) were determined. Severe renal dysfunction was defined as serum creatinine elevated above 1.50 mg/dL, for at least 2 consecutive days after a fluid challenge, or rising levels of serum creatinine (.3 mg/dL/day). The GFR was estimated during the second to third days of life by endogenous creatinine clearance (mL/minute/1.73 m2) and using Schwartz's formula: GFR (mL/minute/1.73 m2) =.45 x length (cm)/plasma creatinine (mg/100 mL) during the first 5 days of life. Tubular performance was assessed as the concentration of beta2-microglobulin (beta2M) determined by enzyme immunoassay, on the first voided urine 12 hours after theophylline administration. The statistical analysis for the evaluation of the differences between the groups was performed with Student's t and chi(2) tests as appropriate. RESULTS: During the first day of life, the 24-hour fluid balance was significantly more positive in the infants receiving placebo compared with the infants receiving theophyline. Over the next few days, the change in fluid balance favored the theophyline group. Significantly higher mean plasma values were recorded in the placebo group from the second to the fifth days of life. Severe renal dysfunction was present in 4 of 24 (17%) infants of the theophylline group and in 15 of 27 (55%) infants of the control group (relative risk:.30; 95% confidence interval:.12-.78). Mean endogenous creatinine clearance of the theophylline group was significantly increased compared with the creatinine clearance in infants receiving placebo (21.84 +/- 7.96 vs 6.42 +/- 4.16). The GFR (estimated by Schwartz's formula) was markedly decreased in the placebo group. Urinary beta2M concentrations were significantly reduced in the theophylline group (5.01 +/- 2.3 mg/L vs 11.5 +/- 7.1 mg/L). Moreover, 9 (33%) patients of the theophylline group versus 20 (63%) infants of the control group had urinary beta2M above the normal limit (<.018). There was no difference in the severity of the asphyxia between infants belonging to the theophylline and control groups in regards of Portman's score. Except for renal involvement, a similar frequency of multiorganic dysfunction, including neurologic impairment, was observed in both groups. The theophylline group achieved an average serum level of 12.7 microg/mL (range: 7.5-18.9 microg/mL) at 36 to 48 hours of live versus traces (an average serum level of .87 microg/mg) in the placebo group. CONCLUSIONS: Our data suggest that prophylactic theophylline, given early after birth, has beneficial effects on reducing the renal dysfunction in asphyxiated full-term infants. (ABSTRACT TRUNCATED) PMID- 10742367 TI - Ketogenic diet for the treatment of refractory epilepsy in children: A systematic review of efficacy. AB - OBJECTIVES: To systematically review and synthesize the available evidence on the efficacy of the ketogenic diet in reducing seizure frequency for children with refractory epilepsy. DATA SOURCES: Medline searches were performed using the keywords epilepsy/therapy, dietary therapy, and epilepsy, and the text word ketogenic diet. The Cochrane Library of clinical trials was searched using the term ketogenic diet. Bibliographies of recent review articles and relevant primary research reports, as well as Current Contents were reviewed for additional relevant citations. STUDY SELECTION: Studies were selected for inclusion in the review that reported the reduction of seizure frequency following treatment with the ketogenic diet in children with refractory epilepsy. The outcome measures used were the percentage of patients with: 1) complete elimination of seizures, 2) >90% reduction in seizures, and 3) >50% reduction in seizures. RESULTS: The evidence consists entirely of uncontrolled studies. Of 11 studies identified for this review, 9 are retrospective series of patients from a single institution. Two studies are prospective, 1 of which is a multicenter trial. The results of these studies are consistent in showing that some children benefit from the ketogenic diet, demonstrated by a significant reduction in seizure frequency. Estimates of the rates of improvement by combined analysis (confidence profile method) are complete cessation of all seizures in 16% of children (95% confidence interval [CI]: 11.0-21.7); a greater than 90% reduction in seizures in 32% (95% CI: 25.3-39.8); and a greater than 50% reduction in seizures in 56% (95% CI: 41.2-69.7). It is unlikely that this degree of benefit can result from a placebo response and/or spontaneous remission. CONCLUSIONS: Although controlled trials are lacking, the evidence is sufficient to determine that the ketogenic diet is efficacious in reducing seizure frequency in children with refractory epilepsy. PMID- 10742368 TI - Fixed 50% nitrous oxide oxygen mixture for painful procedures: A French survey. AB - OBJECTIVE: Although the equimolecular mixture of oxygen and nitrous oxide (EMONO) seems a good choice to relieve procedure-related pain in children, it has not been accepted everywhere. In France, the rapid spread of its use has elicited suspicion and doubts regarding its safety. To assess the use and the safety of this gas mixture in the pediatric settings in France, we conducted a national survey. METHODOLOGY: Centers that had accepted a nation- wide invitation to participate in the survey filled out a questionnaire after each EMONO administration during a 2-month study. Procedure and inhalation characteristics, as well as pain evaluations and side effects, were reported. RESULTS: One thousand nineteen EMONO inhalations from 31 centers that agreed to participate in this 2-month survey were analyzed. Median (range) age was 6.4 (0-18) years. Four percent (46) of children were 12 months old or younger, 29% (295) were 5 years old or younger, 45% (459) were 6 to 10 years old, and 26% (265) were older than 10 years of age. The procedures performed with EMONO inhalation were: lumbar punctures (286), bone marrow aspirations (BMA; 231), laceration repairs (215), minor procedures (75), minor surgery (53), punctures (49), fractures (45), dental care (43), and pulmonary endoscopy (22). Nine percent of procedures were undertaken without the presence of a physician; the child being observed only by the attending nurse. A drug association was noted in 182 (17.9%) of procedures: midazolam (63%), acetaminophen (18%), nalbuphine (8.5%), hydroxyzine (5%), flunitrazepam (2%), chlorazepate (2%), morphine (1%), and lorazepam (.5%). EMLA cream (Astra) was applied in 98.6% of lumbar punctures, 93.7% of BMA, and 54.2% of punctures including lymph nodes, hematoma, or renal biopsies. Lidocaine infiltration was performed in 51% of minor surgery procedures, 40% of laceration repairs, and 28% of BMA. The inhalation system included a whistle, a scented mask, and a nonrebreathing respiratory valve in 48.9%, 71.2%, and 78.3% of the patients, respectively. Initial physical restraint was needed in 18. 2% of all the patients. Inhalation refusal was noted in 129 (12.7%) children; of these, 53 had an alternative method of analgesia (EMLA or lidocaine infiltration), 15 had no other analgesia, and in the remaining 61, EMONO inhalation was maintained against the child's will. Median (interquartile) inhalation length was 4 (3-5) minutes before starting the procedure and 6 (6-15) minutes for the total inhalation. Median (interquartile) procedural pain evaluations were 9 (0-30) for children on a 0 to 100 visual analog scale, 1 (0-3) for both nurses and parents on a 0 to 10 numerical scale. Median (interquartile) procedural pain as evaluated by nurses for the 3 most frequent procedures were 0 (0-2) for lumbar punctures, 2 (0-4) for bone marrow aspiration, and 2 (0-4) for laceration repair. Comparison of pain assessed by nurses in children 3 years old or younger and those older than 3 years of age showed a median (range) score of 2 (0-10) versus 1 (0-10), respectively. Pain self-assessment was completed in 647 children 6 years of age or older. Median (interquartile) children pain assessments were as follows: lumbar puncture (5; 0-20), bone marrow aspiration (12.5; 0-40), laceration repair (12; 0-40), minor procedures (18; 0-32), minor surgery (10; 0-35), punctures (0; 0-18), fracture (15; 0-30), dental care (20; 0-40), and pulmonary endoscopy (15; 0-30). Ninety-three percent of the 647 children who were able to answer the question said they would accept EMONO analgesia if a new procedure were to be performed. Behavioral reactions during procedures varied with age of the child; cry was observed in 44.1%, 24.4%, 12.9%, and 11.2% of children 3 years or younger, 4 to 6 years, 7 to 10 years, and 11 years or older, respectively. Physical restraint was necessary in 34.2%, 22%, 13.5%, and 8.4% of children aged 3 years or younger, 4 to 6 years, 7 to 10 years, and 11 years or old PMID- 10742369 TI - Cause and clinical characteristics of rib fractures in infants. AB - OBJECTIVE: Rib fractures are uncommon in infancy and, when diagnosed, often raise the suspicion of child abuse. However, the prevalence of other causes of rib fractures has not been well defined. The purpose of this study was to determine the causes and clinical presentations of rib fractures in infants <12 months old. METHODS: Retrospectively, we identified all infants with rib fractures under 12 months old over a 3-year period using computerized databases at the Children's Hospital Medical Center in Cincinnati, Ohio and at the Children's Hospital, Winnipeg, Manitoba, Canada. Data extracted from the individual patient charts included: age, sex, chief complaint, number and location of rib fractures, associated injuries, birth history, history of cardiopulmonary resuscitation, and any evidence of bone dysplasia. After the chart review and a review of the radiographs by a pediatric radiologist, all fractures were determined to be attributable to one of the following causes: child abuse, birth injury, bone fragility, or accidental trauma. A determination of abuse was made when there were other injuries indicative of abuse, there was no clinical or radiographic evidence of bone fragility, there was a confession of abuse, when no reasonable history of trauma was provided, or when the history was not plausible to explain the rib fractures. Standard practice at these hospitals involves obtaining skeletal surveys on all children <2 years old when abuse is suspected. The child abuse team, which consists of physicians, nurses, and social workers, conducts these investigations and works closely with police in evaluating these children. RESULTS: Thirty-nine infants with rib fractures were identified. Thirty-two (82%) were caused by child abuse. Three (7. 7%) were attributable to accidental injuries, 1 (2.6%) was secondary to birth trauma, and 3 (7.7%) were attributable to bone fragility. All 3 infants with fractures from accidental injury had sustained notable trauma (a motor vehicle collision, a forceful direct blow, and a fall from a height). Of the 3 infants with fractures secondary to bone fragility, 1 infant had osteogenesis imperfecta, 1 infant had rickets, and 1 infant, who was born at 23 weeks' gestation, had fragile bones attributable to prematurity. CONCLUSIONS: Most rib fractures in infants are caused by child abuse. Although much less common, rib fractures can also occur after serious accidental injuries, birth trauma, or secondary to bone fragility. A thorough clinical and imaging evaluation is mandatory. PMID- 10742370 TI - Soothing pain-elicited distress in Chinese neonates. AB - OBJECTIVE: To test the effect of nonnutritive sucking (NNS), music therapy (MT), and combined NNS and MT (NNS + MT), versus no intervention, on heart rate, transcutaneous oxygen (TcPaO(2)) levels, and pain behavior of neonates in intensive care units having blood taken by a heel-stick procedure. METHODOLOGY: A within-subjects, counter-balancing, repeated-measures design conducted in a government-funded hospital in Hong Kong, comparing TcPaO(2) levels, heart rate, and pain behavior outcomes in 27 neonates. RESULTS: Repeated-measures multivariate analysis of variance revealed statistically significant differences in outcomes across all interventions (Wilk's lambda =.142; F [3,27] = 31.82; eta2 =.47). One-way analysis of variance revealed that the 3 comfort interventions significantly reduced neonates' heart rate (Wilk's lambda =.647; F [2,27] = 18.93; eta2 =.35), improved their TcPaO(2) levels (Wilk's lambda =.481; F [2,27] = 37.42; eta2 =.51), and reduced their pain behavior (Wilk's lambda =.312; F [2,27] = 76.42; eta2 =.68). Posthoc scheffe tests revealed that NNS + MT had the strongest effect on neonates' TcPaO(2) levels and pain behavior; MT alone had the strongest effect on neonates' heart rate. CONCLUSIONS: Health professionals using NNS + MT when doing heel-sticks can improve the TcPaO(2) levels of neonates and reduce their pain. Using MT alone can improve the heart rate of neonates. PMID- 10742371 TI - Voiding cystourethrograms and urinary tract infections: how long to wait? AB - OBJECTIVES: Many authorities recommend an interval of at least 3 to 6 weeks after a urinary tract infection (UTI) before performing a voiding cystourethrogram (VCUG). However, such an interval may reduce the likelihood of completing the procedure. This study was performed to investigate whether the length of the interval between a UTI and the performance of the VCUG influences the presence or severity of reflux, and whether it influences the likelihood of actually having the study performed. DESIGN: We reviewed 352 admissions of children under 10 years old whose discharge diagnoses indicated UTIs. These admissions occurred over a 27-month period between October 1994 and December 1996 at the Children's Hospital of Austin, Texas. We identified 213 patients with confirmed UTIs and no other previously defined urinary tract pathology. These patients were divided into 2 groups according to whether they had a VCUG scheduled to be performed either within 1 week after the diagnosis of a UTI (the early group), or later than 1 week after the diagnosis (the late group). We compared the presence and severity of reflux in the 2 groups as well as the proportion of scheduled VCUGs that were actually performed. RESULTS: Reflux was present in 19% of the patients studied within 1 week after UTI (95% confidence interval [CI]: 12.9-26.4) and in 18% of those studied after 1 week (95% CI: 6. 7-34.5). This difference was not statistically significant (chi(2) =. 034; DF = 1). However there was a substantial difference between the 2 groups with regard to the number of scheduled VCUGs actually performed. Whereas 100% of the scheduled VCUGs in the early group were performed, only 48% (95% CI: 35.9-60.1) of those scheduled in the late group were performed. This difference is statistically significant (chi(2) = 89.6; DF = 1). CONCLUSIONS: In the hospitalized children who underwent VCUGs within a week after diagnosis of UTI, the presence of reflux is not significantly different from those studied later. Furthermore, late scheduling of VCUGs resulted in failure to perform the procedure in more than half of the patients. Some of the patients who were not evaluated would be expected to have vesicoureteral reflux and thus be at risk for chronic renal disease. Therefore, the traditional recommendation to perform the VCUG at 3 to 6 weeks after the diagnosis of UTI should be reconsidered, especially for hospitalized children. PMID- 10742372 TI - Poorer behavioral and developmental outcome more than 10 years after treatment for iron deficiency in infancy. AB - OBJECTIVE: To determine the long-term effects of iron deficiency in infancy. DESIGN: Longitudinal follow-up study of children who had been tested and treated for iron deficiency as infants. SETTING: Periurban community near San Jose, Costa Rica. PARTICIPANTS: Of the original 191 participants, 87% were reevaluated at 11 to 14 years old (average age: 12.3 years). The children were free of iron deficiency and growing normally by US standards. Those who had chronic, severe iron deficiency in infancy (n = 48) were compared with those who had good iron status before and/or after iron therapy in infancy (n = 114). OUTCOME MEASURES: Comprehensive set of cognitive, socioemotional, and motor tests and measures of school functioning. RESULTS: Children who had severe, chronic iron deficiency in infancy scored lower on measures of mental and motor functioning. After control for background factors, differences remained statistically significant in arithmetic achievement and written expression, motor functioning, and some specific cognitive processes (spatial memory, selective recall, and tachistoscopic threshold). More of the formerly iron-deficient children had repeated a grade and/or been referred for special services or tutoring. Their parents and teachers rated their behavior as more problematic in several areas, agreeing in increased concerns about anxiety/depression, social problems, and attention problems. CONCLUSIONS: Severe, chronic iron deficiency in infancy identifies children who continue at developmental and behavioral risk >10 years after iron treatment. PMID- 10742373 TI - Adverse effects of nicotine and interleukin-1beta on autoresuscitation after apnea in piglets: implications for sudden infant death syndrome. AB - OBJECTIVES: Maternal cigarette smoking is established as a major dose-dependent risk factor for sudden infant death syndrome (SIDS). Both prenatal and postnatal exposures to constituents of tobacco smoke are associated with SIDS, but no mechanism of death attributable to nicotine has been found. Breastfeeding gives a substantial increase in absorbed nicotine compared with only environmental tobacco smoke when the mother smokes, because the milk:plasma concentration ratio of nicotine is 2.9 in smoking mothers. Furthermore, many SIDS victims have a slight infection and a triggered immune system before their death, thus experiencing a release of cytokines like interleukin-1beta (IL-1beta) that may depress respiration. Because apneas in infancy are associated with SIDS, we have tested the hypothesis that postnatal exposure to tobacco constituents and infections might adversely affect an infant's ability to cope with an apneic episode. This is performed by investigating the acute effects of nicotine and IL 1beta on apnea by laryngeal reflex stimulation and on the subsequent autoresuscitation. DESIGN: Thirty 1-week-old piglets (+/-1 day) were sedated with azaperone. A tracheal and an arterial catheter were inserted during a short halothane anesthesia. The piglets were allowed a 30-minute stabilization period before baseline values were recorded and they were randomized to 4 pretreatment groups (avoiding siblings in the same group): 1) immediate infusion of 10 pmol IL 1beta intravenously/kg (IL-1beta group; n = 8); 2) slow infusion of 5 microg nicotine intravenously/kg 5 minutes later (NIC group; n = 8); 3) both IL-1beta and NIC combined (NIC + IL-1beta group; n = 6); or 4) placebo by infusion of 1 ml .9% NaCl (CTR group; n = 8). Fifteen minutes later, apnea was induced by insufflation of .1 ml of acidified saline (pH = 2) in the subglottic space 5 times with 5-minute intervals, and variables of respiration, heart rate, blood pressure, and blood gases were recorded. RESULTS: Stimulation of the laryngeal chemoreflex by insufflation of acidified saline in the subglottic space produced apneas, primarily of central origin. This was followed by a decrease in heart rate, a fall in blood pressure, swallowing, occasional coughs, and finally autoresuscitation with gasping followed by rapid increase in heart rate, rise in blood pressure, and (in the CTR group) an increase of respiratory rate. Piglets pretreated with nicotine had more spontaneous apneas, and repeated spontaneous apneas caused an inability to perform a compensatory increase of the respiratory rate after induced apnea. This resulted in a lower SaO(2) than did CTR at 2 minutes after apnea (data shown as median [interquartile range]: 91% [91-94] vs 97% [94-98]). The pretreatment with IL-1beta caused prolonged apneas in piglets and an inability to hyperventilate causing a postapneic respiratory rate similar to the NIC. When nicotine and IL-1beta were combined, additive adverse effects on respiratory control and autoresuscitation compared with CTR were observed: NIC + IL-1beta had significantly more spontaneous apneas the last 5 minutes before induction of apnea (2 [.3-3] vs 0 [0-0]). Apneas were prolonged (46 seconds [39 51] vs 26 seconds [22-31]) and followed by far more spontaneous apneas the following 5 minutes (6.6 [4.0-7.9] vs.5 [.2- .9]). Instead of normal hyperventilation after apnea, a dramatic decrease in respiratory rate was seen (at 20 seconds: -45% [-28 to -53] vs +29% [+24-+50], and at 60 seconds: -27% [-23 to -32] vs +3% [-2-+6), leading to SaO(2) below 90% 3 minutes after end of apnea: 89% (87-93) versus 97% (95-98). These prolonged adverse effects on ventilation were reflected in lowered PaO(2), elevated PaCO(2) and lowered pH 2 minutes, and even 5 minutes, after induction of apnea. CONCLUSIONS: Nicotine interferes with normal autoresuscitation after apnea when given in doses within the range of what the child of a smoking mother could receive through environmental t PMID- 10742374 TI - Preventable childhood tuberculosis in Alabama: implications and opportunity. AB - Childhood tuberculosis (TB) cases indicate recent community transmission and thus reflect the effectiveness of TB control efforts, particularly the contact investigation. OBJECTIVE: To evaluate all preventable childhood TB cases and implications in the context of TB morbidity trends. DESIGN: Statewide morbidity trends are presented from 1983 to 1997. Since 1992, each child TB case is classified as either preventable or not preventable, based on a standard definition. MAIN OUTCOME MEASURES: Case characteristics (preventable and not preventable), TB disease rates over time, and reasons for preventable case classification. SETTING: Alabama TB control program, from January 1, 1983 through December 31, 1997. RESULTS: For the period 1983-1997, nonwhite children had a higher disease rate (rate ratio: 5.7; 95% confidence interval: 4.3,7.6) than white children. Since 1990, the overall child rate has increased significantly despite a decline in the adult rate. Among 120 child cases diagnosed from 1992 to 1997, 25 (21%) were classified as preventable. The causes were contact investigation interview failure (12/25 = 48%), delay to evaluation (16%), source case noncompliance with previously prescribed preventive therapy (16%), and source case diagnosed out of state (16%) with no initial investigation performed in Alabama. All preventable cases identified were black children; the proportion of preventable cases did not vary by age group or sex. During 1996, the case rate for nonwhite children exceeded that of adult whites. CONCLUSIONS: Childhood TB in Alabama for nonwhites is rising despite a national downward trend. TB is clearly a disproportionate disease burden for the state's African American population, and the median case age is falling. Additional research and improved training in contact investigation are required to assess this situation and effectively intervene. PMID- 10742375 TI - Cost-effectiveness of universal compared with voluntary screening for human immunodeficiency virus among pregnant women in Chicago. AB - OBJECTIVES: To determine and compare the cost-effectiveness of implementing 3 screening strategies to detect human immunodeficiency virus (HIV) infection among pregnant women in Chicago, Illinois: no screening, voluntary screening, and universal screening. METHODS: A decision-analysis model was developed, using standard cost-effectiveness analysis from a societal perspective. Reference case estimates were derived from a surveillance project conducted by the Illinois Department of Public Health and studies were published in the medical literature. Costs included direct and indirect medical costs associated with identification of pregnant women infected with HIV and identification, prevention, and treatment of perinatally HIV-infected newborns. Specifically, for each screening option, the cost per pregnant woman screened, the resulting number of pediatric HIV infections, and the number of newborn life-years were calculated. All costs were adjusted to the 1997 dollar value and discounted at 3%. Sensitivity analyses were determined for all variables included in the decision model. RESULTS: The estimated prevalence of HIV infection among pregnant women in Chicago is .41%. For every 100,000 pregnant women, it is estimated that 104.6 children would be infected with HIV if no screening strategy were implemented and 44.8 children would be infected if voluntary HIV testing (assuming a 92.7% acceptance rate) were available. In comparison, if universal HIV testing was performed, the number of children infected with HIV would decrease to 40 cases. Sensitivity analysis across a maternal HIV prevalence rate of.01% to 2.2% found that universal screening would be cost-saving in communities where the seroprevalence is.21%. In Chicago, it would take an estimated 5. 2 months of screening pregnant women to avert 1 case of pediatric HIV. Taking into consideration the lifetime costs of treating a child with HIV infection, universal HIV testing of 100,000 pregnant women would result in a cost-savings of $3.69 million when compared with no screening, and $269,445 when compared with voluntary screening. We estimated that it would cost $11.1 million to screen 100,000 pregnant women in Chicago. The cost savings produced with increased screening are the direct result of reduced cases of newborns infected with HIV. A 2-way sensitivity analysis was performed to examine how costs vary as a function of the voluntary rates for HIV-positive and HIV-negative women. When screening falls below 50% for HIV-positive mothers, universal screening becomes cheaper than voluntary screening even if no HIV negative mothers were screened. CONCLUSION: Reference case analyses showed that universal HIV screening of pregnant women in Chicago would both decrease the number of HIV-infected newborns and save money in comparison to voluntary or no testing strategies. Sensitivity analysis was robust across all variables for the conclusion that universal screening was more effective than voluntary screening. For many communities that have HIV prevalence rates for mothers of >.21%, universal screening would also save money in comparison to voluntary screening. For communities with prevalence rates <.21%, the benefits of universal screening may outweigh the costs for screening as we found that desirable incremental cost effectiveness ratios were found for prevalence rates as low as.0075%. PMID- 10742376 TI - Prepubertal gynecomastia: indirect exposure to estrogen cream. AB - OBJECTIVE: To describe the clinical course of 3 prepubertal boys who presented with gynecomastia resulting from indirect exposure to a custom-compounded preparation of estrogen cream used by each child's mother. METHODOLOGY: Each child was initially referred to the Children's Medical Center of Dallas' Endocrinology Center and followed for over 1 year. RESULTS: All 3 boys presented with gynecomastia and elevated estradiol levels. Two had accelerated growth and advanced bone ages. Within 4 months after each child's mother discontinued use of the topical estrogen preparation, each child's gynecomastia regressed and estradiol levels returned to normal. CONCLUSION: Indirect exposure to excessive amounts of topical estrogen may cause gynecomastia, rapid changes in growth, and advanced bone age in prepubertal children. Because custom-compounded topical estrogen preparations are not regulated by the Food and Drug Administration and may contain high concentrations of estrogen, we recommend that women requiring estrogen use an alternate form of estrogen delivery if they are in frequent close contact with children. PMID- 10742377 TI - Activity, dietary intake, and weight changes in a longitudinal study of preadolescent and adolescent boys and girls. AB - OBJECTIVE: To examine the role of physical activity, inactivity, and dietary patterns on annual weight changes among preadolescents and adolescents, taking growth and development into account. STUDY DESIGN: We studied a cohort of 6149 girls and 4620 boys from all over the United States who were 9 to 14 years old in 1996. All returned questionnaires in the fall of 1996 and a year later in 1997. Each child provided his or her current height and weight and a detailed assessment of typical past-year dietary intakes, physical activities, and recreational inactivities (TV, videos/VCR, and video/computer games). METHODS: Our hypotheses were that physical activity and dietary fiber intake are negatively correlated with annual changes in adiposity and that recreational inactivity (TV/videos/games), caloric intake, and dietary fat intake are positively correlated with annual changes in adiposity. Separately for boys and girls, we performed regression analysis of 1-year change in body mass index (BMI; kg/m(2)). All hypothesized factors were in the model simultaneously with several adjustment factors. RESULTS: Larger increases in BMI from 1996 to 1997 were among girls who reported higher caloric intakes (.0061 +/-.0026 kg/m(2) per 100 kcal/day; beta +/- standard error), less physical activity (-.0284 +/-.0142 kg/m(2)/hour/day) and more time with TV/videos/games (.0372 +/-.0106 kg/m(2)/hour/day) during the year between the 2 BMI assessments. Larger BMI increases were among boys who reported more time with TV/videos/games (.0384 +/ .0101) during the year. For both boys and girls, a larger rise in caloric intake from 1996 to 1997 predicted larger BMI increases (girls:.0059 +/-.0027 kg/m(2) per increase of 100 kcal/day; boys:.0082 +/-.0030). No significant associations were noted for energy-adjusted dietary fat or fiber. CONCLUSIONS: For both boys and girls, a 1-year increase in BMI was larger in those who reported more time with TV/videos/games during the year between the 2 BMI measurements, and in those who reported that their caloric intakes increased more from 1 year to the next. Larger year-to-year increases in BMI were also seen among girls who reported higher caloric intakes and less physical activity during the year between the 2 BMI measurements. Although the magnitudes of these estimated effects were small, their cumulative effects, year after year during adolescence, would produce substantial gains in body weight. Strategies to prevent excessive caloric intakes, to decrease time with TV/videos/games, and to increase physical activity would be promising as a means to prevent obesity. PMID- 10742378 TI - Developmental dysplasia of the hip practice guideline: technical report. Committee on Quality Improvement, and Subcommittee on Developmental Dysplasia of the Hip. AB - OBJECTIVE: To create a recommendation for pediatricians and other primary care providers about their role as screeners for detecting developmental dysplasia of the hip (DDH) in children. PATIENTS: Theoretical cohorts of newborns. METHOD: Model-based approach using decision analysis as the foundation. Components of the approach include the following: PERSPECTIVE: Primary care provider. OUTCOMES: DDH, avascular necrosis of the hip (AVN). OPTIONS: Newborn screening by pediatric examination; orthopaedic examination; ultrasonographic examination; orthopaedic or ultrasonographic examination by risk factors. Intercurrent health supervision based screening. PREFERENCES: 0 for bad outcomes, 1 for best outcomes. MODEL: Influence diagram assessed by the Subcommittee and by the methodology team, with critical feedback from the Subcommittee. EVIDENCE SOURCES: Medline and EMBASE search of the research literature through June 1996. Hand search of sentinel journals from June 1996 through March 1997. Ancestor search of accepted articles. EVIDENCE QUALITY: Assessed on a custom subjective scale, based primarily on the fit of the evidence to the decision model. RESULTS: After discussion, explicit modeling, and critique, an influence diagram of 31 nodes was created. The computer-based and the hand literature searches found 534 articles, 101 of which were reviewed by 2 or more readers. Ancestor searches of these yielded a further 17 articles for evidence abstraction. Articles came from around the globe, although primarily Europe, British Isles, Scandinavia, and their descendants. There were 5 controlled trials, each with a sample size less than 40. The remainder were case series. Evidence was available for 17 of the desired 30 probabilities. Evidence quality ranged primarily between one third and two thirds of the maximum attainable score (median: 10-21; interquartile range: 8-14). Based on the raw evidence and Bayesian hierarchical meta-analyses, our estimate for the incidence of DDH revealed by physical examination performed by pediatricians is 8.6 per 1000; for orthopaedic screening, 11.5; for ultrasonography, 25. The odds ratio for DDH, given breech delivery, is 5.5; for female sex, 4.1; for positive family history, 1.7, although this last factor is not statistically significant. Postneonatal cases of DDH were divided into mid-term (younger than 6 months of age) and late-term (older than 6 months of age). Our estimates for the mid-term rate for screening by pediatricians is 0.34/1000 children screened; for orthopaedists, 0.1; and for ultrasonography, 0.28. Our estimates for late-term DDH rates are 0.21/1000 newborns screened by pediatricians; 0.08, by orthopaedists; and 0.2 for ultrasonography. The rates of AVN for children referred before 6 months of age is estimated at 2.5/1000 infants referred. For those referred after 6 months of age, our estimate is 109/1000 referred infants. The decision model (reduced, based on available evidence) suggests that orthopaedic screening is optimal, but because orthopaedists in the published studies and in practice would differ, the supply of orthopaedists is relatively limited, and the difference between orthopaedists and pediatricians is statistically insignificant, we conclude that pediatric screening is to be recommended. The place of ultrasonography in the screening process remains to be defined because there are too few data about postneonatal diagnosis by ultrasonographic screening to permit definitive recommendations. These data could be used by others to refine the conclusions based on costs, parental preferences, or physician style. Areas for research are well defined by our model-based approach. PMID- 10742379 TI - Technical aspects and clinical impact of hematopoietic progenitor subset quantification. AB - As high-dose therapy for malignancies is now being applied to newly diagnosed patients as adjuvant therapy, it has become a requirement that quality and safety assessment of hematopoietic stem cell grafts be evidence-based. This process has developed a new institution in medicine, the stem cell laboratory. In most cases this speciality has evolved from or within hematological research laboratories. However, the increased routine technologies applied in quality evaluation, ex vivo manipulation and safety assessment in stem cell handling naturally places this activity in transfusion medicine. Multiparametric flow cytometry can identify progenitor subsets in normal human bone marrow and peripheral blood, and such subset quantification has been used retrospectively to predict three-lineage engraftment following high-dose therapy for malignancies. Published single center data have suggested an impact on clinical outcome, and a standardized technique for subset enumeration needs to be established before prospective multicenter trials can be initiated to document the prognostic value of such quality assessment in autografting. Based on experiences of CD34 enumeration, which we consider to be the first step in quality assessment of hematopoietic stem cell grafts, this review discusses flow cytometry subset identification by lineage specific differentiation markers, stromal-dependent adherence molecules, and regulatory growth factor receptors from a technical point of view. The aim of this review is:To recommend a simple method based on the experiences of the Nordic workshop III on subset identification; To present new molecular genetic based methods for future use in quality assessment; and To propose new endpoints necessary for validation of the likely clinical impact of subsets in prospective trials. As sample differences between blood and marrow result in technical difficulties, this review only focuses on the methodology of identifying subsets in blood and leukapheresis products. Methods for subset analysis in diagnostic bone marrow samples will be covered in a forthcoming review. PMID- 10742380 TI - Clinical applications of CD34(+) peripheral blood progenitor cells (PBPC). AB - Recently, a number of devices have been developed for the positive selection of CD34(+) peripheral blood progenitor cells (PBPC) for clinical use in autologous or allogeneic transplantation. The rationale for CD34(+) selection is based on clinical studies showing a two- to five-log reduction of contaminating tumor cells in patients with breast cancer, multiple myeloma and low-grade lymphoma. In addition, a three- to five-log reduction of T cells can be obtained by CD34(+) selection in both autologous grafts for patients with autoimmune disease resistant to conventional therapy and allogeneic grafts to reduce the incidence and severity of acute graft-versus-host disease. Transplantation of positively selected autologous CD34(+) PBPC results in a rapid and stable neutrophil and platelet engraftment in patients who received an infused dose of at least 2.0 x 10(6) CD34(+) cells/kg. Results from randomized trials suggest that time to engraftment is not different compared to unmanipulated PBPC autografts. However, close monitoring for infectious complications (e.g., cytomegalovirus disease) is required. Allogeneic CD34(+) PBPC have also been successfully transplanted and, using novel technologies, megadoses of purified CD34(+) PBPC can be obtained and used to overcome histocompatibility differences betweeen allogeneic donor and patient resulting in stable engraftment, even in a haploidentical setting. Additional randomized phase III trials are required to determine whether tumor cell purging or lymphocyte depletion by CD34(+) cell selection will have a significant impact on progression-free and overall survival in both autologous and allogeneic transplantation. PMID- 10742381 TI - Reduction of marrow hematopoietic progenitor and stem cell content is not sufficient for enhanced syngeneic engraftment. AB - The mechanisms regulating long-term engraftment of primitive stem cells are largely unknown. Most conditioning strategies use myeloablative agents for experimental or clinical hematopoietic stem cell transplantation. Host conditioning regimens, in part, have been designed on the assumption that transplanted cells home to specific marrow sites and if these sites are occupied by host stem cells, engraftment will not take place. However, there is now evidence that stable long-term syngeneic engraftment may occur in the absence of host marrow stem cell depletion. To further study the association of engraftment with stem cell depletion, we investigated whether the marked egress of hematopoietic progenitor and stem cells from the marrow into the peripheral blood in C57BL6 mice following a single dose of cyclophosphamide (day 1) and four days of G-CSF (days 3-6) afforded an increased opportunity for long-term syngeneic donor engraftment. During and after mobilization, glucose phosphate isomerase (GPI)-1(b) mice received 30 x 10(6) GPI-1(a) marrow cells without further myeloablation. The level of donor/recipient chimerism was assessed in cell lysates after six months. Increased long-term syngeneic donor engraftment was observed prior to mobilization (before day 6), during a period of active hematopoietic regeneration following the administration of cyclophosphamide. Hematopoietic regeneration was evidenced by a reduced but rapidly increasing marrow cellularity and an increased proportion of hematopoietic progenitors in S phase. In contrast, long-term syngeneic donor engraftment was not increased over controls during the period of maximum progenitor and stem cell mobilization (after day 5). At this time there were minimal numbers of progenitor and stem cells in the marrow. These data suggest that in the absence of host stem cell ablation, maximal engraftment does not occur during marrow progenitor or stem cell depletion, suggesting that the presence of "open" marrow sites is not a prerequisite for engraftment. The mechanisms for increased engraftment during progenitor cell regeneration following cyclophosphamide need further investigation. Understanding the mechanisms for engraftment without host stem cell ablation may allow strategies for improved long-term engraftment of syngeneic or autologous stem cells with reduced post-transplant toxicity. PMID- 10742382 TI - Release from quiescence of primitive human hematopoietic stem/progenitor cells by blocking their cell-surface TGF-beta type II receptor in a short-term in vitro assay. AB - Genetic alterations of the signaling cascade of transforming growth factor-beta (TGF-beta) are often associated with neoplastic transformation of primitive cells. This demonstrates the key role for this pleiotropic factor in the control of quiescence and cell proliferation in vivo. In the high proliferative potential quiescent cell (HPP-Q) in vitro assay, the use of TGF-beta1 blocking antibodies (anti-TGF-beta1) allows the detection within two to three weeks of primitive hematopoietic cells called HPP-Q, which otherwise would not grow. However, the possibility of triggering cell proliferation by blocking the cell-surface TGF beta receptors has not been investigated until now. We have tested here the efficiency of a blocking antibody against TGF-betaRII (anti-TGF-betaRII) on CD34(+)CD38(-) hematopoietic cells, a subpopulation enriched in primitive stem/progenitor cells, and compared its effect with that of anti-TGF-beta1. About twice as many HPP colony-forming cells were detected in the presence of anti-TGF beta1 or anti-TGF-betaRII, compared to the control (p < 0.02). Moreover, anti-TGF betaRII was as efficient as anti-TGF-beta1 for activating multipotent HPP granulocyte erythroid macrophage megakaryocyte and HPP-Mix, bipotent HPP granulocyte-macrophage (GM) and unipotent HPP-G, HPP-M and HPP-BFU-E. We therefore propose the use of anti-TGF-betaRII to release primitive cells from quiescence in the HPP-Q assay. This strategy could be extended to nonhematopoietic tissues, as TGF-beta1 may be a pleiotropic regulator of somatic stem cell quiescence. PMID- 10742383 TI - Effect of thrombopoietin on proliferation of blasts from patients with myelodysplastic syndromes. AB - Thrombopoietin (TPO), a major cytokine involved in megakaryocytopoiesis/thrombopoiesis, may be effective for treatment of the thrombocytopenia associated with myelodysplastic syndromes (MDS). However, it has been unclear whether TPO stimulates proliferation of MDS blasts, as observed in de novo acute myeloid leukemia. This study examined this concern. When marrow cells from 37 MDS cases were cultured with or without recombinant human PEGylated TPO, TPO increased the blast number (stimulation index > or =1.5) in 9 of 16 high risk MDS cases (refractory anemia with excess blasts [RAEB] and RAEB in transformation) and 4 of 10 cases with MDS transformed to acute leukemia (MDS AL), but none of 11 cases with low-risk MDS (RA and RA with ringed sideroblasts). When the cell cycle of cultured cells was determined by three-color flow cytometry, TPO activated the cell cycle of MDS cells (causing a decrease in G(0) phase cells) in most of the cases whose blast number increased in response to TPO. Reverse transcriptase-polymerase chain reaction analysis detected TPO receptor messenger RNA in purified blasts from all six cases examined, irrespective of the response of their blasts to TPO in culture. Analysis of the patients' characteristics identified a high-serum lactate dehydrogenase (LDH) value as being associated with blast proliferation in high-risk MDS cases (p = 0.0036). We conclude that TPO stimulates in vitro proliferation of blasts from a fraction of MDS patients. High-risk MDS patients, especially those who have a high-serum LDH value, and MDS-AL patients should be monitored with particular care in clinical trials of TPO for MDS. PMID- 10742384 TI - Granulocyte-colony stimulating factor impedes recovery from damage caused by cytotoxic agents through increased differentiation at the expense of self renewal. AB - G-CSF is routinely used to hasten recovery from chemotherapy-induced neutropenia. We have recently shown that G-CSF, when combined with stem cell-damaging cytotoxic agents, results in enhanced stem cell damage and loss of marrow reserve. To investigate the mechanisms of stem cell damage caused by G-CSF, we gave C57BL/6 (B6) mice repeated doses of cyclophosphamide ([CY] 84 mg/kg) or carmustine ([BCNU] 13.2 mg/kg) and G-CSF (250 microg/kg/day) for either four days or eight days. Two different regimens of G-CSF were chosen to study the influence of increased proliferation on hematopoiesis which was measured at the end of the first, third and sixth 14-day cycle of each cytotoxic agent and 7 and 20 weeks after completion of all cycles. A spectrum of hematopoietic indices was measured including WBC, bone marrow cellularity, granulocyte/macrophage-colony-forming cells (GM-CFC), colony-forming cells with high proliferative-potential (HPP-CFC), cobblestone area-forming cells ([CAFC]-day 7 and CAFC-day 28), and long-term marrow repopulating ability in vivo. Despite the absence of differences in peripheral blood cell counts or bone marrow cellularity 14 days after each dose, progenitor cell levels (HPP-CFC, GM-CFC, and CAFC-7) were increased up to 2.5 fold with cytotoxic agent and G-CSF administration compared with cytotoxic agent administration alone. Mice given G-CSF for eight days had the greatest number of progenitors suggesting a dose-response relationship for G-CSF administration. G CSF resulted in a decrease in hematopoietic stem cell (CAFC-28) content when measured two weeks after each cycle of saline, CY, and BCNU. Twenty weeks after six cycles of BCNU, the reduction in stem cell levels persisted and was further decreased when G-CSF was added to BCNU for four or eight days. Data from this study suggest that the most likely explanation for the damaging effects of G-CSF is that G-CSF directly or indirectly induces stem cells to differentiate into more committed hematopoietic cells resulting in a loss of marrow reserve. This effect is enhanced in animals with an already compromised hematopoietic stem cell compartment as seen with repeated doses of BCNU. PMID- 10742385 TI - The role of HIV-related chemokine receptors and chemokines in human erythropoiesis in vitro. AB - In order to better define the role of HIV-related chemokines in human erythropoiesis we studied: A) the expression of chemokine receptors, both on human CD34(+) cells which include erythroid progenitors and on more mature erythroid cells; B) the functionality of these receptors by calcium flux, chemotaxis assay and phosphorylation of mitogen-activated protein kinases (MAPK) p42/44 (ERK1/ERK2) and AKT, and finally C) the influence of chemokines on BFU-E formation. We found that HIV-related chemokine receptor CXCR4, but not CCR5, is detectable on human CD34(+) BFU-E cells. CXCR4 surface expression decreased during erythroid maturation, although CXCR4 mRNA was still present in cells isolated from differentiated erythroid colonies. SDF-1, a CXCR4 ligand, induced calcium flux and phosphorylation of MAPK (p42/44) and AKT in CD34(+)KIT(+) bone marrow mononuclear cells which contain BFU-E, as well as chemotactic activity of both human CD34(+) BFU-E progenitors and erythroid cells isolated from day 2-6 BFU-E colonies. Responsiveness to SDF-1 decreased when the cells differentiated to the point of surface expression of the erythroid-specific marker Glycophorin A. In contrast, the CCR5 ligands (macrophage inflammatory protein-1alpha [MIP 1alpha], MIP-1beta, and RANTES) did not activate calcium flux, MAPK and AKT phosphorylation or chemotaxis of CD34(+)KIT(+) cells or cells isolated from the BFU-E colonies. Interestingly, none of the chemokines tested in this study had any effect on BFU-E colony formation. In conclusion, only CXCR4 is functional, and its specific ligand SDF-1 may therefore play an important role in the homing and/or retention of early erythroid precursors in the bone marrow environment. PMID- 10742387 TI - The molecular perspective: DNA. PMID- 10742386 TI - Cytokine-driven differentiation of blasts from patients with acute myelogenous and lymphoblastic leukemia into dendritic cells. AB - We investigated the ability of both acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) blasts to differentiate into dendritic cells (DC) in vitro. Cytokine-supplemented suspension cultures of leukemic blasts in 98 patients with AML and five patients with ALL (normal karyotype, n = 2; BCR/ABL, n = 3) were performed. Mononuclear cells out of peripheral blood or bone marrow containing between 60% and 90% leukemic blasts were cultured for eight days using different growth factor combinations. The highest yield of CD1a(+)/CD14(-) cells could be obtained with stem cell factor, transforming growth factor-beta, tumor necrosis factor-alpha, GM-CSF, and FLT-3-ligand. In the AML samples the median content of CD1a(+)/CD14(-) cells after eight days of culture was 3.5% (r = 0% 82%). In five informed patients CD1a(+)/CD14(-) cells were sorted by fluorescence activated cell sorting or immunomagnetic separation. Cytogenetic and polymerase chain reaction analyses showed known primary chromosomal aberrations (monosomy 7 and inversion 16) in the sorted fractions, respectively. Dendritic cells (DC) could be generated out of leukemic blasts in 68% of AML patients. Leukemic DC showed no phagocytosis of latex beads, but stimulated allogeneic naive cord blood derived T cells more efficiently than did uncultured blasts. In ALL patients the median percentage of CD1a(+)/CD14(-) cells was 1.2% (r = 0.7%-3.8%) after culture. The sorted CD1(+)/CD14(-) fractions were BCR/ABL-negative when analyzed with fluorescence in situ hybridization, indicating their nonleukemic origin. Leukemic DC can be generated out of leukemic progenitors in patients with AML. These cells might become relevant for autologous and allogeneic immunotherapy in selected patients. BCR/ABL-positive lymphoblasts could not be transformed into cells with an early dendritic phenotype with the cytokines used in our experiments. PMID- 10742388 TI - Gene therapy in clinical applications: overview: how do we translate gene therapy to clinical trials? PMID- 10742389 TI - The human sheep xenograft model for the study of the in vivo potential of human HSC and in utero gene transfer. PMID- 10742390 TI - Transduction of mouse hematopoietic stem cells is more efficient with 10A1 retrovirus vectors than with amphotropic vectors. PMID- 10742391 TI - Unrelated placental blood in marrow transplantation. PMID- 10742392 TI - Role of serum-free medium in the ex vivo expansion of human cord blood hematopoietic stem cells. PMID- 10742393 TI - Use of serum-free medium with fibronectin fragment enhanced transduction in a system of gas permeable plastic containers to achieve high levels of retrovirus transduction at clinical scale. PMID- 10742394 TI - Curt I. Civin, M.D. STEM CELLS editor-in-chief PMID- 10742395 TI - Meeting of the Australian Scanned Probe Microscope Society, University of Sydney, February 16-19, 1999: Introduction. PMID- 10742396 TI - Soft-contact Atomic Force Microscopy Imaging of Adsorbed Surfactant and Polymer Layers. AB - The technique of atomic force microscopy (AFM) soft-imaging is outlined with respect to characterizing the adsorption of surfactants and polymers at the solid/liquid interface. This method utilizes the electrostatic and steric repulsion forces between the scanning probe and the sample to allow sensitive placement of the imaging probe near to the delicate surface layer. Specifically, the mixed adsorption of sodium dodecylsulfate (SDS) and poly(vinyl pyrrolidone) (PVP) on graphite is examined. Unlike the adsorbed layer in a solution of either component, the adsorbed layer in the mixture does not cover the substrate uniformly until equilibrium is reached (often hours later). The interesting kinetic and coverage effects observed are significant to the many applications reliant on adsorption from polymer-surfactant mixtures, especially to the flocculation of dispersions. PMID- 10742397 TI - Ultra Flat Gold Surfaces for Use in Chemical Force Microscopy: Scanning Probe Microscopy Studies of the Effect of Preparation Regime on Surface Morphology. AB - The preparation of ultra flat gold surfaces for use in chemical force microscopy (CFM) has been studied. The surfaces were studied in terms of substrate effects by comparing mica, Si (110) wafer and glass slides. The effect of different annealing regimes was also investigated. Measurements on these surfaces were made by both atomic force microscopy (AFM) (in contact and tapping mode) and by scanning tunneling microscopy (STM). The films contain different morphologies with respect to grain size and topography. Calculations of surface roughness present values less than 2.5 nm for all surfaces studied, making the choice of the "flattest" surface difficult if based on criteria of surface roughness alone. Additionally, it is shown that different acquisition parameters can produce dissimilar images that have stability and reproducibility. PMID- 10742398 TI - Modification of a Commercial Atomic Force Microscope for Nanorheological Experiments: Adsorbed Polymer Layers. AB - The atomic force microscope (AFM) has previously been applied to the measurement of surface forces (including adhesion and friction) and to the investigation of material properties, such as hardness. Here we describe the modification of a commercial AFM that enables the "stiffness" of interaction between surfaces to be measured concurrently with the surface forces. The stiffness is described by the rheological phase difference between the response of the AFM tip to a driving oscillation of the substrate. We present the interaction between silica surfaces bearing adsorbed polymer, however, the principles could be applied to a wide variety of materials including biological samples. PMID- 10742399 TI - Characterization of High Resolution Resists and Metal Shims by Scanning Probe Microscopy. AB - Technologies such as compact disc (CD) manufacturing, hologram embossing, and security printing rely on the reproduction of micro-patterns generated on surfaces by optical or electron-beam lithographic writing onto electron-beam or photoresists. The periodicity of such patterns varies from sub-micron to several microns, with depths up to 0.5 um. The scanning probe microscope (SPM) is becoming a routine tool for analysis of these micro-patterns, to check on depths and lateral dimensions of features. Direct scanning of resist-covered plates is now possible, without damage, using resonant low-contact force SPM with etched silicon cantilevers. Metal shims produced from the master resist plates can also be scanned and checked for defects prior to production of embossed foils. The present article discusses examples of the use of a Digital Instruments 3100 microscope in analysis of production electron-beam lithography plates with a 0.5 um resist thickness. We also examine features of nickel replicas ("father" and "mother" shims) produced by electroforming from the original plate. With SPM measurements of the development profile of a particular plate, corrections can be made to exposures and development times during production to correct errors. An example is given of such a feedback process. PMID- 10742400 TI - Anisotropy of Surface Roughness on Aluminium Sheet Studied by Atomic Force Microscopy. AB - Atomic force microscope images of aluminium sheet are used to calculate the mean roughness Ra and the root mean square roughness Rq for different directions (theta) across the samples. The angle determined values of Ra(theta) and Rq(theta) are significantly lower in the rolling direction than in any other direction. The results provide a clear demonstration of the vector nature of surface roughness. Local features in an angle determined Ra(theta) profile are also compared with the positions of peaks and valleys in the matching topographical cross-section of the surface running at right angles to theta. It is further found that the distribution of z heights for an imaged sample is usually not Gaussian in shape unless the z heights are measured about mean lines constructed across the surface parallel with the rolling direction. The results are relevant for controlling lubrication performance in the aluminium rolling process. PMID- 10742401 TI - Approaches to "Standardless" Wavelength Dispersive Analysis. AB - In truly standardless electron microprobe analysis, generated X-ray intensities calculated from first principles are combined with a detector efficiency model. Though already used for energy dispersive (ED) analysis, the application of this concept to wavelength dispersive (WD) analysis is problematic, mainly because the reflectivity of spectrometer crystals is not well known. However, the need to carry out standard measurements with every batch of WD analyses can be avoided by using stored intensity data, and interpolation may be used when no standard is available. An empirical adjustment factor allowing for changes in spectrometer efficiency with time can be applied as necessitated by the variability of the spectrometer characteristics and the accuracy required. A similar approach to background corrections, based on measured continuum intensities, can be used. While the convenience of standardless WD analysis is attainable only at the expense of reduced accuracy, it can have a useful role where high accuracy is not needed or as a preliminary to applying a more rigorous routine using standards. PMID- 10742402 TI - Localization of Rat Liver Group IIA Phospholipase A(2) in Secretory Pathways: Green Fluorescent Protein Approach. AB - Rat liver group IIA phospholipase A(2) (PLA(2), Enzyme Commission Number [E.C.] 3.1.1.4) was initially isolated from mitochondria. Since then it has been thought that it resides in mitochondria and that mitochondria are its main subcellular target. We cloned the cDNA sequence of rat group IIA PLA(2) and fused its presequence (signal peptide) to enhanced green fluorescent protein (EGFP). In baby hamster kidney (BHK) cells transiently and stably expressing the fusion protein, the fluorescence does not localize in mitochondria, but rather in the secretory pathway. These results suggest that the plasma membrane, and not mitochondria, is the primary target of rat group IIA PLA(2). PMID- 10742403 TI - Limiting Factors on Image Quality in Imaging through Turbid Media under Single photon and Two-photon Excitation. AB - The effect of multiple scattering in a turbid medium on single-photon and two photon fluorescence microscopy is experimentally investigated for different scattering characteristics including scattering anisotrophy and optical thickness of a turbid medium. It is demonstrated that two-photon excitation can provide significant improvement in penetration depth through turbid media, due to reduced scattering experienced by the excitation beam. It is also shown that the limiting factor in obtaining high-quality images under single-photon excitation is the fast degradation of image resolution caused by multiple scattering, while for two photon excitation it is limited by the degradation of image contrast due to the reduction in fluorescence strength. PMID- 10742404 TI - Quantitative Energy-filtering Transmission Electron Microscopy in Materials Science. AB - Energy-filtered transmission electron microscopy (EFTEM) can be used to acquire elemental distribution images at high lateral resolution within short acquisition times. In this article, we present an overview of typical problems from materials science which can be preferentially solved by means of EFTEM. In the first example, we show how secondary phases in a steel specimen can be easily detected by recording jump ratio images of the matrix element under rocking beam illumination. Secondly, we describe how elemental maps can be converted into concentration maps. A Ba-Nd-titanate ceramics serves as a typical materials science example exhibiting three different compounds with varying composition. In order to reduce diffraction and/or thickness variation effects which may be a problem for quantification of crystalline specimens, we calculated atomic ratio maps by dividing two elemental maps and subsequent normalizing by the partial ionization cross-sections (or k-factors). Additionally, the atomic ratio maps are correlated using the scatter diagram technique thus leading to quantitative chemical phase maps. Finally, we show how the near-edge structures (electron energy-loss near edge fine structures, or ELNES) can be used for mapping chemical bonding states thus differentiating between various modifications of an element. In order to distinguish between diamond and non-diamond carbon in diamond coated materials, we have investigated a diamond layer on a substrate with the help of ELNES mapping utilizing the pi*-peak of the C-K ionization edge. PMID- 10742405 TI - News and Commentary. PMID- 10742406 TI - Ewing's family of tumors involving structures related to the central nervous system: a review. AB - This review consolidates information gleaned from several case reports and larger series on Ewing's sarcoma family of tumors (EFT) involving structures related to and found in the central nervous system (CNS). These tumors involve the skull, the spinal column, adjacent soft tissues, the meninges, and the brain. We have separated the cases by skull region and spinal column level, and we discuss the attendant differences in prognosis following treatment by neurosurgery, radiation, and chemotherapy. Light and electron microscopic features can be used to differentiate EFT from other small round blue cell tumors that involve the CNS (central primitive neuroectodermal tumor, lymphoma, etc.). Recent molecular and genetic findings in EFT provide new diagnostic methods. We conclude that EFT involving the CNS and adjacent structures is not so rare as previously stated and that the prognosis is more favorable, as a rule, than for the more common examples arising in the long bones and pelvis. PMID- 10742407 TI - Childhood Lichen scrofulosorum revisited. AB - Lichen scrofulosorum (LS), an uncommon cutaneous tuberculous reaction, has been described in children and young adults. In the last three decades, there has been a dearth of literature on the entity in children, despite a global increase in tuberculosis. It is usually associated with localized cervical, hilar, or mediastinal lymphadenopathy or with osseous tuberculosis. The occurrence of LS in association with pulmonary tuberculosis is rare and its occurrence with generalized lymphadenopathy is unrecognized. We report LS in two children and highlight its occurrence with pulmonary tuberculosis and generalized lymphadenopathy. PMID- 10742408 TI - Circumferential distribution of ganglion cells in the transition zone of children with Hirschsprung disease. AB - We prospectively studied the circumferential distribution of ganglion cells in the transition zone from a study population of 21 patients with Hirschsprung disease (HD) undergoing a pull-through procedure over a 26-month period. Twelve cases were satisfactory for examination, in that the transition zone was contained within a single surgical specimen and specimen distortion was minimal. Ganglion cells in the submucosa were counted in all 12 cases. In seven of the cases, the transition zone was proximal to the rectal sleeve and myenteric plexus ganglion cells were also counted. We found an uneven circumferential distribution of ganglion cells in both myenteric plexus and submucosa of the transition zone, resulting in a "leading edge" of ganglion cells extending into aganglionic distal bowel. The maximum length of this leading edge was 2.4 cm and 2.1 cm in the myenteric plexus and submucosa, respectively. Ganglion cells at the tip of the leading edge were present in clusters of up to six ganglion cells, in marked contrast to an absence of ganglion cells for most of the remainder of the circumference. Closely spaced myenteric plexus ganglia were seen at the tip of the leading edge in some cases. The leading edge was more frequently observed along the antimesenteric side, but this was not statistically significant. Our findings have relevance in the interpretation of intraoperative biopsies at the time of pull-through surgery and subsequent biopsies of neorectum in patients with surgically corrected HD. PMID- 10742409 TI - Fetal obstructive uropathy: patterns of renal pathology. AB - Fetal obstructive uropathy (FOU) is characterized by obstruction of the urethra, renal anomalies, ureterovesical dilatation, oligohydramnios, cryptorchidism, and abdominal muscle wall changes. The main objective of the present study was to better understand the relationship between FOU and renal pathology using a series of 15 male autopsy cases. A total of 11 cases with patent anus and 4 with imperforate anus were analyzed. Of the first group, most cases showed obstruction at the level of prostatic urethra. Seven cases showed obstruction at the level of the prostatic urethra and histologic study revealed scarring and partial or complete absence of the prostate, while in the remaining four cases the prostate was present. Of the cases with imperforate anus, two showed obstruction at the level of prostatic urethra, one showed posterior urethral valves, and one was obstructed at the proximal urethra. In all cases the kidneys showed mixed (dysplastic and cystic) changes with no significant differences between the two groups. An inverse correlation was observed between degree of renal dysplasia and gestational age, whereas the opposite was true for cystic changes. Distal and collecting tubules were more intensely immunoreactive to the anti-cytokeratin antibody when compared to proximal tubules. Moreover, anti-cytokeratin immunoreactivity was more prominent in tubules displaying cystic dilatation. DNA fragmentation analysis of renal tissue revealed a higher apoptosis of mesenchymal and tubular cells in the FOU cases, compared to gestational aged-matched controls. These results suggest that renal anomalies in FOU might be related to the gestational age at which the injury occurred and to the duration of the obstruction. PMID- 10742410 TI - Escherichia coli O157:H7-induced hemolytic-uremic syndrome: histopathologic changes in the colon over time. AB - The 1993 E. coli O157:H7 epidemic in the Western United States has provided a unique opportunity to evaluate the histopathologic temporal progression of disease in the colon in children who developed hemolytic uremic syndrome (HUS). In this report we briefly summarize the clinical courses of eight patients and then discuss the colonic pathology observed in specimens obtained at surgery or at the time of autopsy. The patients were divided into two groups: group 1 consisted of six subjects whose colonic samples were obtained during the acute phase of disease, and group 2 consisted of two subjects whose samples were obtained late in their disease. Both the gross and microscopic findings showed that the most severely affected as well as the earliest affected regions of the colon were the left and transverse portions. Only later in the disease progression was there right-sided colon involvement. These findings are in contrast to the distribution described in E. coli O157:H7 hemorrhagic colitis without HUS, thus suggesting a different mechanism of injury. PMID- 10742411 TI - Congenital sacrococcygeal teratomas: effect of gestational age on size, morphologic pattern, ploidy, p53, and ret expression. AB - Prognosis of infants born with sacrococcygeal teratomas (SCTs) correlates with gestational age (GA). The survival rate after 30 weeks of gestation is 75%, compared to 7% before 30 weeks of gestation. Studies correlating GA with size, morphologic composition of teratomas, ploidy or expression of cell cycle control proteins such as p53, and ret [a tyrosine kinase receptor of the GDNF (glial cell line-derived neurotrophic factors)] receptor family may provide information explaining differences in survival. Seven SCTs (GA 21 to 41 weeks), ranging in size from 5 to 15 cm, were evaluated for morphologic composition. DNA ploidy was assessed in mature and immature neural elements. Immunohistochemical reactivity with monoclonal antibodies recognizing p53, and ret was quantitated and correlated with morphological pattern and GA. Relative size of teratomas to infants' weight and content of immature neural tissues correlated inversely with advancement of GA. Yolk sac tumor (YST) and immature tissues showed aneuploid cell populations. Nuclear p53 reactivity was apparent in the teratoma with YST in the microcystic patterns, the neuroectodermal rosettes, and the glandular patterns. Ret reactivity was seen in osteoclasts adjacent to bone formation surrounding developing teeth in an immature teratoma, and in rare mature neural cells of one SCT of 35 weeks GA. The rapid growth of SCT (GA <30 weeks) correlates with increase in immature neural tissues. Our study confirms aneuploidy in YST and suggests aneuploid populations within immature tissues. p53 accumulates in a variety of patterns of YST and may be seen in immature components of SCTs. To understand the possible role of ret, further studies comparing ret expression in immature human tissues are needed. PMID- 10742412 TI - Detection of trisomy 18 on formalin-fixed and paraffin-embedded material by fluorescence in situ hybridization. AB - Formalin-fixed and paraffin-embedded autopsy material from 10 fetuses and infants with unknown karyotype and anomalies suggestive of trisomy 18 were subjected to fluorescence in situ hybridization (FISH). Nuclei were extracted from the tissues and hybridized with a chromosome 18-specific centromere probe. The hybridization was successful in 9 of 10 cases. Two cases showed three hybridization signals in most of the nuclei (74% and 85%). These had anomalies frequently occurring with trisomy 18 (congenital heart defect, omphalocele, and horseshoe kidney). Two cases showed a mixture of two and three signals (47%/49% and 59%/36%), suggesting the possibility of mosaicism. One of these cases had anomalies consistent with a trisomy 18 phenotype. In the other case intrauterine growth retardation and syndactylies suggested triploidy. Hybridization with a chromosome 8-specific probe gave a distribution of two and three signals (34% and 62%, respectively). This result strengthened the suspicion of a possible triploid mosaicism. In five of the cases most of the nuclei showed two signals (85% to 88%). However, as only one type of tissue was examined for enumeration of chromosome 18, the possibility of organ mosaicism or other chromosome aberrations cannot be excluded. The FISH technique is applicable on macerated and autolysed formalin-fixed tissue, making it possible to retrospectively analyze autopsy material from aborted and stillborn fetuses and infants. This analysis contributes to a better quality of perinatal autopsies and is helpful in parental counseling. PMID- 10742413 TI - Anomalous costochondral cartilage in fetal anencephaly. AB - Anencephaly is a human fetal malformation with absence of brain and calvarium superior to the orbits. The consequent absence of hypothalamus provides a unique model for studying human development, and therefore skeletal growth, in the absence of hypothalamic hormones and their regulatory functions. To assess the influence of hypothalamic insufficiency on cartilage development, we studied costochondral cartilage sections from eight anencephalic fetuses (18-22 weeks old) and seven controls (16-22 weeks old) with pathologies not directly related to skeletal growth. We found a previously undescribed anomalous organization of the cartilage in the anencephalic. The proliferative chondrocytes showed a disordered appearance with an increased proliferative zonal length (156 +/- 28 microm in anencephalic fetuses vs. 103 +/- 14 microm in controls, p = 0.006) and a concomitant decrease in the maturing portion, where cells form ordered isogenic groups (58 +/- 13 microm in anencephalic fetuses vs. 93 +/- 19 microm in controls, p = 0.003). In addition, cell density was significantly decreased in the proliferating and maturing zones in the anencephalic cases (84 +/- 21 vs. 130 +/- 21 cells/40 microm(2) in proliferating zone; 53 +/- 8 vs. 94 +/- 8 in maturing portion, p < 0.005). These alterations in the developing cartilage of the anencephalic may contribute to the observed growth retardation in these fetuses and reflect modifications in pituitary hormones and growth factors resulting from reduction in hypothalamopituitary function. PMID- 10742414 TI - Nijmegen breakage syndrome-associated T-cell-rich B-cell lymphoma: case report. AB - In 1981 Weemaes et al. first described the Nijmegen breakage syndrome (NBS), a rare autosomal recessive disorder characterized by stunted growth, microcephaly, immunodeficiency, spontaneous chromosome instability, and a peculiar predisposition to cancer development. Most NBS-related malignancies are lymphomas, but their pathologic features have rarely been specified. We report here the case of a northern Italian 8-year-old child who, 2 years after the diagnosis of NBS, developed a diffuse large B-cell lymphoma (T cell-rich B-cell lymphoma variant). The histological and immunobiological features of the lymphoma population are analyzed and discussed in detail. PMID- 10742415 TI - Congenital misalignment of pulmonary veins with alveolar capillary dysplasia causing persistent neonatal pulmonary hypertension: report of two affected siblings. AB - Misalignment of pulmonary vessels with alveolar capillary dysplasia is a rare cause of persistent pulmonary hypertension of the newborn. Most of the reported cases have been sporadic. We present two consecutive affected siblings with this disorder. This is the fifth reported family occurrence of this condition. In addition to the pulmonary abnormality, one of our cases had duodenal atresia. PMID- 10742416 TI - Extensive gastric heterotopia of the small intestine resulting in massive gastrointestinal bleeding, bowel perforation, and death: report of a case and review of the literature. AB - Gastric heterotopia of the small intestine is a rare occurrence outside of Meckel's diverticulum and intestinal duplication. The vast majority of cases of gastric heterotopia occur as polypoid or tumorous lesions in the duodenum. These lesions have been associated with clinical symptoms including diarrhea, obstruction, dyspepsia, ulceration, and gastrointestinal bleeding. We present a case of gastric heterotopia that is unique because the lesions occurred as multiple, carpet-like, nonpolypoid areas throughout a large portion of the small intestine. A review of the literature is included. PMID- 10742417 TI - New case of Beemer-Langer syndrome. AB - We present the case of a male infant born at 37 weeks gestation with multiple congenital anomalies, including hydrops fetalis, facial and visceral abnormalities, short ribs, and short limbs without polydactyly. We believe that this represents a further case of the Beemer-Langer syndrome, a relatively recently described form of lethal osteochondrodysplasia with an autosomal recessive mode of inheritance. This case also showed some less frequently described anomalies, including arachnoid cysts of the brain and short intestines. PMID- 10742418 TI - Castleman's disease of the left triceps in a child suspected to be a small round cell tumor of childhood. AB - Castleman's disease (CD) is histologically characterized by a proliferation of polyclonal small lymphocytes and plasma cells. The clinical presentation varies widely, but most commonly manifests as a solitary mediastinal mass, incidentally found on radiographic examination. We present a case of a 10-year-old girl who exhibited a left arm mass which, preoperatively and on frozen section, was diagnosed as a small round cell tumor of childhood (SRCT). This report emphasizes the unusual location of CD in the soft tissue and as a rare entity to be considered in the differential diagnosis of SRCT. PMID- 10742419 TI - Clinicopathologic study of ectomesenchymomas from Intergroup Rhabdomyosarcoma Study Groups III and IV. AB - Ectomesenchymomas (EM) are rare malignant neoplasms usually consisting of rhabdomyosarcoma (RMS) with a neural component. Only 21 cases have been previously reported. Here we extend the clinicopathologic spectrum of EM by describing our findings in 15 cases. Only 5 patients were infants; 10 were < or =3 years old and 5 were > or =6 years old. No male predilection was observed; 7 were female. The originating institutional diagnoses were; RMS (12), undifferentiated sarcoma (1), or EM (2), suggesting underdiagnosis of this entity. The primary tumor sites included external genital (5), pelvis/abdomen (6), head and neck (3), and extremity (1). The size of the primary neoplasm was usually > or =5 cm at diagnosis but dissemination only occurred in a minority. Local infiltration was not uncommon. These neoplasms were typically multilobate, thinly encapsulated, hemorrhagic, and necrotic. Light microscopic features were highly variable, but embryonal RMS with scattered or clustered ganglion cells, often in lacunae, was characteristic. In some cases, primitive neuroblastic or neuroectodermal areas were found and/or a component of alveolar RMS was seen. Focal anaplasia was occasionally observed. Mitotic activity appears higher than previously appreciated and some necrosis was invariably present. Electron microscopy was performed in 11 cases, which confirmed skeletal muscle +/- neural differentiation. Cytogenetic studies performed in five cases revealed no specific abnormality. Monoclonal neuron-specific enolase was the best marker of ganglion cells and primitive neural elements. MIC-2 (CD99) membrane expression was not definitively present in any of the six cases examined. A number of the above parameters appear to be of some prognostic significance, but overall, these neoplasms appear to have a similar outcome as would be predicted for their RMS element alone (exclusive of any neural component), with respect to the RMS subtype, age of the patient, and anatomic location of the neoplasm. PMID- 10742420 TI - Sturge-Weber syndrome. PMID- 10742421 TI - Combined revascularization and microvascular free tissue transfer for limb salvage: a six-year experience. AB - Atherosclerotic vascular disease causing extensive tissue loss of the lower extremities often results in primary amputation. Combined revascularization and free tissue transfer has been described as a method of extending limb salvage to these patients. The durability of this combined procedure remains unknown, thus the objective of this report is to describe the immediate and long-term results in a series collected over 6 years. From 1992 to 1998, 15 patients with a mean age of 60 years underwent combined revascularization and free tissue transfer. Mean ulcer size measured 45 cm(2) for a mean duration of 7.4 months preoperatively and 12 patients had exposed bone or tendon. Vascular reconstruction included popliteal (3), tibial (6), and pedal (6) bypass with concomitant myocutaneous free flap, using mostly rectus abdominis or latissimus dorsi muscle. There were no perioperative deaths. One patient suffered a nonfatal myocardial infarction. Two patients had a postoperative wound hematoma and one required vascular graft revision. Patients were followed for 4 to 75 months (mean = 23 months). Four patients have required amputations (3 early, 1 late), three of whom had preoperative renal failure. The limb salvage rate has been 72% at 36 months, PMID- 10742422 TI - Failure of nonoperative management of isolated superior mesenteric artery dissection. AB - A case of isolated dissection of the superior mesenteric artery is presented here. This rare condition was confirmed angiographically in a 46-year-old man with persistent abdominal pain. He was treated initially with anticoagulation alone. One year later, he developed recurrent symptoms and had radiologic documentation of progression of the condition. Operative repair was performed and recovery was uneventful. This case demonstrates a failure of the nonoperative approach to this rare condition and suggests that disease progression may be inevitable. Early surgical correction may ease operative management. PMID- 10742423 TI - Arterial injury in uncomplicated upper extremity dislocations. AB - The purpose of this study is to analyze causes and consequences of arterial injury complicating simple dislocations of the upper extremity. This is a multicenter, 5-year, retrospective analysis of vascular injury in patients sustaining shoulder or elbow dislocations without associated fractures. Foci of the study were mechanisms of injury, preoperative imaging, incidence of closed reductions, types of arterial injury, methods of surgical repair, and ultimate outcome. Arterial injury in this patient cohort occurred in 0.74% of patients. Axillary artery injury occurred in 0. 97% and brachial in 0.47%. Both elbow and shoulder dislocations may cause severe arterial damage and strenuously test surgical expertise to achieve successful repair. PMID- 10742424 TI - Staged thoracic and abdominal aortic aneurysm repair using stent graft technology and surgery in a patient with acute renal failure. AB - A 52-year-old male presented with severe hypertension and acute renal failure. Carbon dioxide (CO(2)) angiography identified a saccular thoracic aortic aneurysm, right renal artery stenosis, left renal artery occlusion, an infrarenal aortic aneurysm, celiac artery, and inferior mesenteric artery (IMA) orificial stenoses. Via an anterior retroperitoneal approach, bilateral renal artery thromboendarterectomy, infrarenal aortic aneurysmectomy, and IMA reimplantation were performed. The patient's tortuous iliac arteries were straightened to permit future passage of a thoracic stent graft by mobilizing the aortic bifurcation and anastomosing it to a Dacron graft within 4 cm of the renal vessels. Two weeks later, a stent graft was placed via a femoral incision utilizing CO(2) angiography, successfully excluding the saccular thoracic aneurysm. Recovery from both procedures was quick, with rapid return of renal function, and alleviation of the hypertension. At 8 months follow-up, his renal arteries and aorta are patent. PMID- 10742425 TI - The use of colorflow duplex scanning to detect significant renal artery stenosis. AB - The purpose of this study was to review our institutional experience with colorflow duplex scanning in detecting significant renal artery stenosis and to validate the criteria used: renal artery peak systolic velocity (PSV) >/=200 cm/sec and renal-to-aortic peak systolic ratio (RAR) >/=3.5. The results of renal artery duplex and arteriography in 58 patients (107 kidneys) who underwent both exams were reviewed. Arteriography revealed 32 main renal arteries with >/=60% stenosis. The PSV criterion detected 29, for a sensitivity of 91%, specificity of 75%, positive predictive value (PPV) of 60%, negative predictive value (NPV) of 95%, and accuracy of 79%. Using RAR >/=3.5 provided a sensitivity of 72%, specificity of 92%, PPV of 79%, NPV of 88%, and accuracy of 86%. In a subset of 36 kidneys that had hilar scans, the criteria of acceleration time (AT) >/=100 cm/sec and index (AI) 0.6. Failure to accomplish this puts the patient at risk for continued steal syndrome. PMID- 10742429 TI - Carotid endarterectomy: a comparison of regional versus general anesthesia in 500 operations. AB - The outcomes of 489 patients undergoing 500 carotid endarterectomies were reviewed to compare results from using general anesthesia with those from using regional (cervical block) anesthesia. From September 1982 to May 1998, 228 procedures (221 patients) were performed under general anesthesia and 272 procedures (268 patients) were performed under local anesthesia. A detailed comparison of the two groups revealed close similarity in age, sex, presenting symptoms, and associated risk factors. Overall, perioperative mortality was 0.8%. In comparison to regional anesthesia, the general anesthesia group had greater overall morbidity (23.3 vs. 13. 6%, p < 0.008) and more frequent use of postoperative vasoactive drugs to control blood pressure (40.4 vs. 26.1%, p < 0.001). Furthermore, anesthesia time, operative time, and frequency of shunt use were significantly greater in the general anesthesia group (p < 0.03). PMID- 10742430 TI - Aortic wall tension as a predictive factor for abdominal aortic aneurysm rupture: improving the selection of patients for abdominal aortic aneurysm repair. AB - Aortic wall tension was determined in 40 patients to assess its predictive value in abdominal aortic aneurysm (AAA) rupture. A 3-year retrospective analysis of 243 patients with ruptured AAAs and 45 patients with intact AAAs was conducted. The 288 patient sample was limited to the 40 patients with an abdominal CT scan investigation. Aortic wall tension was calculated using blood pressure data and measurements from computerized tomographic (CT) images of 26 patients with intact AAAs and 14 patients with ruptured AAAs in accordance with LaPlace's Law for wall tension: P x R/W, where P = mean arterial pressure (MAP), R = radius of the vessel, and W = wall thickness of the vessel. The wall tension was approximated with the more readily accessible patient parameters of AAA diameter, MAP, height, and weight. This approximation was termed the body mass index (BMI)-pressure approximation for tension (BPAT), which is AAA diameter/BMI x MAP. Data were analyzed using one-sided t-tests, chi-squared tests, and a regression analysis for the relationship between aortic wall tension and the BPAT. AAA wall tension is a significant predictor of pending rupture. BPAT used to approximate the actual tension in the AAA wall is a more sensitive predictor of rupture than aneurysm diameter alone. A prospective study has been initiated to validate these conclusions. PMID- 10742431 TI - Survival in patients with chronic lower extremity ischemia: a risk factor analysis. AB - This is a prospective cohort comparison study aiming to determine the mortality of patients with peripheral arterial occlusive disease (PAOD) and identify the risk factors affecting their survival. Data regarding demographic and biochemical risk factors, and lower limb disease severity classified by vascular laboratory criteria were collected prospectively from 665 consecutive patients presenting with symptoms of peripheral arterial occlusive disease. The effect of patient and disease risk factors on survival was analyzed by the life-table method and independent significant variables examined by a multivariate Cox regression model. The cumulative survival for all patients at 1, 3, and 5 years were 86.1, 71.2, and 55.8%, respectively, with a median survival of 72.2 months. Female sex, age, smoking, heart disease, renal disease, respiratory disease, stroke, critical ischemia, lowest anklebrachial index, no vascular reconstruction, and major amputation were associated with higher mortality. Lipid and biochemical variables were not significant determinants. Using multivariate Cox regression, age (>70), disease severity, anklebrachial index (<0.5), no vascular reconstruction, diabetes mellitus, and renal and cardiorespiratory diseases were identified as independent risk factors affecting patient survival. The survival of patients with PAOD is poor compared with the general population. Significant patient related variables were largely coexisting diseases and advanced age, whereas the other risk factors for atherosclerosis are less influential. Disease severity may bear a direct relationship to mortality, and patients with critical ischemia have the worst prognosis. Early disease detection and timely vascular reconstruction may lead to an improvement in overall survival. PMID- 10742432 TI - Primary lower limb varicosities arising directly from normal deep venous systems: a series report. AB - Primary lower limb varicosities classically arise from incompetence of the junction of the superficial and the deep venous systems with retrograde flow into the saphenous veins. However, some patients with superficial varicosities have no demonstrable incompetence of the saphenofemoral or saphenopopliteal junctions. In this study, we examined 52 limbs with primary varicose disease in whom saphenofemoral and saphenopopliteal incompetence had been excluded (clinically and with the hand-held Doppler) using a duplex ultrasound scan. Seventeen (33%) of the limbs had superficial varicosities despite normal long and short saphenous veins. The varicosities in 12 of these legs originated from groin veins, while those in the remaining 5 limbs communicated directly with normal deep veins. In this latter group of limbs, the superficial varicosities were found on the lateral aspect of the thighs only. Primary varicosities arising from normal deep venous systems have not been previously described, and are relatively rare (1.0% of patients referred to our clinic). As this complex venular anatomy was only detected on duplex scanning, we conclude that this study provides further evidence of the need for this imaging modality in patients with varicose disease of uncertain origin and/or those with an unusual distribution of superficial varicosities. PMID- 10742433 TI - Iliac arteriovenous fistula secondary to iliac aneurysm rupture associated with pulmonary embolism and anuria. AB - We present a case of iliac aneurysm rupture that started with high-output cardiac failure and anuria and later presented as a pulmonary embolism that needed a preoperatory filter for the cava vein. PMID- 10742434 TI - Chyloperitoneum: a rare complication after abdominal aortic aneurysm repair. AB - We report a case of chylous ascites as a rare complication following elective aortic aneurysm repair in a 66-year-old male. After early development of this condition on the second postoperative day, relaparotomy was performed with ligation of fistulae as well as omentoplasty. After recurrence of chylous ascites, conservative treatment consisting of parenteral nutrition and a low-fat diet for 3 months along with continuous peritoneal drainage finally led to successful resolution of this complication. PMID- 10742436 TI - Assessing the role of gene therapy in the treatment of vascular disease. PMID- 10742435 TI - Surgical repair of infected peripheral graft and abdominal aortic aneurysm using arterial homograft. AB - We report a case of combined surgical repair including lower limb revascularization (below-knee bypass) and abdominal aortic aneurysm repair using cryopreserved arterial homograft. The patient experienced lower limb ischemia due to repeated thrombosis of a long-infected polytetrafluoroethylene (PTFE) graft, and was also shown to have a complicating abdominal aortic aneurysm. Infection was eradicated with total graft excision and intravenous antibiotics. Two-year patency of the in situ arterial homograft revascularization was demonstrated with hemodynamic and tomographic controls; no degenerations have been found to date. Benefits of the use of in situ arterial homograft for arterial reconstruction may include improved hemodynamics and greater resistance to infection compared to when alloplastic materials are used. Because of the risk of allograft deterioration, close follow-up of the patient is required. PMID- 10742437 TI - Endovascular management of brachiocephalic arterial occlusive disease. PMID- 10742438 TI - Effect of avicatonin (chicken carbocalcitonin) on galvanic skin response: a randomized, prospective, double-blind, controlled study for an objective assessment of pain. AB - In an attempt to objectively evaluate the analgesic effect of avicatonin (chicken carbocalcitonin), galvanic skin response (GSR) was recorded in 18 patients with osteoporosis or osteopenia and backache in a randomized, prospective, double blind, controlled study. Backache on examination and in daily living was assessed weekly by scores utilizing a questionnaire. After two measurements 1 week apart on induction of backache with a maximum anterior flexion of the back from a supine position, either 20 units of avicatonin or inactive placebo was intramuscularly injected once a week for 4 consecutive weeks. In the avicatonin group but not in the placebo group, the area under the curve (AUC) of GSR tracing was decreased, giving a significant difference between the avicatonin and placebo groups after the second week. The pain score obtained by questionnaire decreased in both groups, suggesting a placebo effect. Galvanic skin response may provide a breakthrough to the objective and reliable evaluation of the biological response to pain which could not be accomplished by questionnaires based on subjective impression markedly influenced by emotional and psychological factors. PMID- 10742439 TI - Influence of physical activity on ultrasound and dual-energy X-ray absorptiometry bone measurements in peripubertal girls: a cross-sectional study. AB - The aim of this cross-sectional study was to investigate whether two types of physical exercise affect the growing skeleton differently. We used calcaneal quantitative ultrasound measurements (QUS) and dual-energy X-ray absorptiometry (DXA) for measurement of bone mineral density (BMD), and to test how QUS values reflect the axial DXA values in these various study groups. A total of 184 peripubertal Caucasian girls aged 11-17 years (65 gymnasts, 63 runners, and 56 nonathletic controls) were studied. Weight, height, stage of puberty, years of training, and the amount of leisure-time physical activity were recorded. Broadband ultrasound attenuation (BUA) and sound of speed (SOS) through the calcaneus were measured. The BMD of the femoral neck and the lumbar spine were measured by DXA. The differences in mean values of bone measurements among each exercise group were more evident in pubertal than prepubertal girls. The mean BUA and SOS values of the pubertal gymnasts were 13.7% (77.8 dB/MHz versus 68.4 dB/MHz, P < 0.05) and 2.2% (1607.7 m/s versus 1572.4 m/s, P < 0.001) higher than of the controls, respectively. The mean BMD of the femoral neck in the pubertal gymnasts and runners was 20% (0.989 g/cm2 versus 0.824 g/cm2, P < 0.001) and 9.0% (0.901 g/cm2 versus 0.824 g/cm2, P < 0.05) higher than in the controls, respectively. The amount of physical activity correlated weakly but statistically significantly with all measured BMD and ultrasonographic values in the pubertal group (r = 0.19-0.35). The correlation between ultrasonographic parameters and BMD were weak, but significant among pubertal runners (r = 0.47-0.55) and controls (r = 0.39-0.42), whereas the DXA values of the femoral neck and the ultrasonographic parameters of the calcaneus did not correlate among highly physically active gymnasts. By stepwise regression analysis, physical activity accounted for much more of the variation in the DXA values than the ultrasonographic values. We conclude that the beneficial influence of exercise on bone status as measured by ultrasound and DXA was evident in these peripubertal girls. In highly active gymnasts the increase of the calcaneal ultrasonographic values did not reflect statistically significantly the BMD values of the femoral neck. PMID- 10742440 TI - Ossification centers of human femur. AB - The purpose of the present work was to investigate the appearance of ossification centers in human femur by Dual-energy X-ray Absorptiometry (DXA), comparing densitometric data with morphogenetic events. Posteroanterior scans were performed on 31 dried femora (from 11.5 weeks of conceptual age to 1 year of postnatal life) by a Hologic QDR 1000 X-ray densitometer with Ultra-Hi-Resolution software. The results were expressed as bone mineral content (BMC, g) and bone mineral density (BMD, g/cm2). The analyses were performed on a rectangular area corresponding to the minimum width and total length of the shaft. The rectangular area was divided into five equal sections along its longitudinal axis, and BMC was calculated on each section. To distinguish the ossification area of the lesser trochanter with respect to the area of the greater trochanter, the proximal femoral end was divided into two portions, medial and lateral, respectively; BMC and BMD were calculated on each portion. Our data show that the ossification center of the femoral shaft extends prevalently in the proximal direction. A denser area was recognizable on the densitometric images at the level of the lesser trochanter from the 19th week of prenatal life and at the level of the greater trochanter at term. During development, the trends of BMC and BMD are similar in both trochanteric areas, but these parameters are fairly constantly higher at the level of the lesser trochanter. Our findings disagree with the data of the current literature on the postnatal appearance of the trochanteric ossification centers and suggest a different biomechanical interpretation of the secondary ossification of the femur. PMID- 10742441 TI - Reference data of vertebral morphometry by X-ray absorptiometry (MXA) in Argentine women. AB - Anterior, middle, and posterior heights and A/P and M/P ratios were determined from T5 to L4 in 111 normal Caucasian Argentine women from 20 to 70 years of age using dual energy X-ray absorptiometry (DXA) densitometry (Expert XL). Scanning time was less than 1 minute and the semiautomatic analysis requires approximately 5 minutes. The precision error for the measurements ranged from 2.2% to 4.6%. The absolute precision error for heights was 0.6 mm. The vertebral bodies tended to be significantly larger in younger women than older women, especially for anterior and middle heights and the A/P and M/P ratios of the midthoracic vertebrae (T6-T10). There were no significant differences between pre- and postmenopausal women in the lumbar vertebral heights. It does not appear that this was a cohort effect because stature was identical in both age groups, and there was no age difference in posterior height. The Expert-XL software normalized the vertebral height based on the average height of the L2-L4 segment in order to minimize the influence of interindividual variation of body size. The average Z-scores for vertebral heights and ratios provided by the software were close to zero indicating that the normalization procedure appropriately corrected for smaller stature in Argentine women. Consequently, the reference values for morphometry X-ray absorptiometry (MXA) were appropriate for our population. In summary, we found that (1) in "normal" women the anterior heights of the thoracic vertebrae (and therefore the A/P ratio) were higher in premenopausal than in postmenopausal women; and (2) the normalization approach corrected for differences of vertebral height and allowed utilization of the manufacturers software in our population. PMID- 10742442 TI - Bone mineral density and biochemical markers of bone turnover in peri- and postmenopausal women. AB - Bone mineral density (BMD) measured by densitometry is the elective parameter for the diagnosis of osteopenia and osteoporosis. Biochemical markers have been proposed as sensitive indicators of high bone turnover and for monitoring response to antiresorptive treatment. We conducted a retrospective study to investigate the values of biochemical markers of bone metabolism with a view to early diagnosis of osteoporosis and monitoring of hormone replacement and calcitonin therapy. The subjects were 415 women, mean age 51+/-8 years (43-62 years) in peri- and postmenopause, recruited at the Menopause Center of Obstetrics and Gynecology Department of Siena University and divided in five groups. Bone densitometry was performed in all subjects and blood samples were taken for assayed biochemical markers, that is, [osteocalcin (OC), parathyroid hormone (PTH), type 1 procollagen (PICP), and calcitonin (CT)]. Three groups of women were divided into two subgroups: those with normal and those with low BMD (<1 SD). Basal concentrations of PCP1, OC, PTH, and CT were compared in the various groups. Two groups of postmenopausal women with BMD below the normal were treated with estrogen replacement therapy and unmodified eel calcitonin. We evaluated whether some of these biochemical markers of bone turnover could help identify women with low BMD and whether they could be useful for monitoring the results of antiresorptive therapies. Markers of bone formation (PICP and OC) make it possible to distinguish women with high turnover who are at risk for osteoporosis from women with low turnover in menopause. A good correlation was also found between changes in levels of these markers and changes in BMD during treatments, which suggests that the PICP and OC would be useful for monitoring response to antiresorptive therapy. PMID- 10742443 TI - Associations of the collagen type Ialpha1 Sp1 polymorphism with five-year rates of bone loss in older adults. AB - The collagen type Ialpha1 Sp1 (ColIA1) polymorphism has been associated with reduced bone mineral density (BMD) and increased prevalence of osteoporosis. This study examines associations of the ColIA1 genotype with BMD and 5-year rates of change in BMD in elderly men and women. The 243 subjects, aged 65 years and older, were participants in two consecutive studies lasting a total of 5-years. BMD of the total body, femoral neck, and lumbar spine were made by dual-energy X ray absorptiometry (DXA). The distribution of the genotypes (155 in the SS genotype, 79 in Ss, and 9 in ss) was proportionately similar to those reported by others. Baseline BMD did not differ significantly at any skeletal site. Unadjusted 5-year percent changes in BMD differed significantly by genotype only at the total body (P = 0.009), where the change was -0.29+/-0.21 (SEM) in the SS genotype, -0.60+/-0.25 in the Ss genotype, and -3.01+/-0.72 in the ss genotype. This 9.4% increase in bone loss of the ss genotype relative to the SS genotype was reduced to an 8.9% increase after adjustment for sex, age, weight, and supplementation group. Results at the femoral neck were directionally similar, but not statistically significant. No effect of genotype on change in spine BMD was observed. In conclusion, bone loss from the total body was significantly greater in elderly men and women who were homozygous for the s allele compared with heterozygotes and SS homozygotes. This finding suggests a possible explanation for the association of the ColIA1 polymorphism with increased rates of osteoporotic fracture, but should be interpreted with caution because of the small number of subjects in the unfavorable ss genotype. PMID- 10742444 TI - Expression of Indian hedgehog during fracture healing in adult rat femora. AB - Indian hedgehog (Ihh) has recently been shown to be expressed in prehypertrophic and hypertrophic chondrocytes during embryonic development, and it has been implicated in the regulation of terminal differentiation of chondrocytes. In this paper we examined the expression of Ihh during fracture healing in an adult rat model. A transverse diaphyseal fracture was made in the right femur, and the expression of Ihh in the fracture callus was examined at 1, 2, and 3 weeks after fracture. Northern blot analysis demonstrated the expression of Ihh mRNA in these tissues. Immunohistological analysis detected hedgehog protein in prehypertrophic chondrocytes in the fracture callus at 1 week after fracture. From 2 weeks and on, positive staining was observed in hypertrophic chondrocytes as well. At 3 weeks, some of the osteoblasts close to the endochondral ossification front were also stained positive for hedgehog protein. Our data indicate that Ihh is expressed in chondrocytes and osteoblasts during the process of fracture healing in adult rat femora, suggesting that Ihh, a regulator of endochondral ossification in embryonic development, may also play a role in the regulation of bone formation during fracture repair in adult animals. PMID- 10742445 TI - Specialized basement membrane of monkey maturation stage ameloblasts mediates firm ameloblast-enamel association by its partial calcification. AB - A basement membrane-like structure associated with the maturation stage ameloblasts of the monkey (Macaca fuscata) tooth germ was examined with high resolution electron microscopy. The tissue was prepared either with or without demineralization. This structure was composed of a lamina lucida-like (lamina lucida) and lamina densa-like (lamina densa) structure. The latter was made up of a fine "cord" network, the major constituent of the basement membrane. It was closely associated with the third layer of a 200 nm wide looser cord network. In specimens without demineralization the third layer and a part of the lamina densa were calcified, and it formed the edge of the enamel. This particular area had a higher electron density, and the size, shape, and arrangement of mineral crystals were different from those of the rest of the enamel. Also, mineralization appeared to be proceeding along the cords. These observations indicate that this dense layer is a highly specialized basement membrane which mediates the firm association of maturation stage ameloblasts with the enamel by means of the mineralization of a part of this basement membrane itself which becomes integrated as a part of the enamel. Also, this highly specialized manner of association is favorable with the reported control of the loss of organic substances in the maturing enamel by maturation stage ameloblasts. PMID- 10742446 TI - Effect of age on the expression of Pex (Phex) in the mouse. AB - Pex is a newly discovered gene (also called Phex) whose mutation is the cause of X-linked hypophosphatemia. Other members of this gene family encode endopeptidases that activate or inactivate endocrine and paracrine factors. Though embryonic bone expresses mRNA for the Pex gene at relatively high levels, we have found Pex expression to be widespread in adult organs and to be poorly expressed in adult bone. This led to the hypothesis that Pex mRNA expression changes with age. To test this, genetically normal mice of the B6C3H hybrid strain were studied at 0 (newborn), 2, 3, 10, and 72 weeks of age. Organs known to express Pex were collected, and RNA was extracted from them. Following reverse transcription, cDNA was amplified by the polymerase chain reaction with primers for Pex and G3PDH, a housekeeping gene. The amplimers were separated by electrophoresis, blotted onto nylon membranes, and hybridized with radioactively labeled internal oligonucleotide probes. The radioactivity was quantified, and the data were analyzed as the Pex/G3PDH ratio. The brain samples had high levels of Pex mRNA expression that rose slightly with age. Calvaria, kidney, and lung samples had the highest Pex mRNA expression at birth. In these organs Pex mRNA expression fell with age to undetectable or barely detectable levels. Thymus, heart, and skeletal muscle samples had low Pex mRNA expression at birth that did not change with age. Some organs showed a decline in G3PDH levels with age, but Pex expression decreased more, leading to a reduced Pex/G3PDH ratio. The widespread expression of mRNA for Pex suggests a role beyond that of phosphate homeostasis. The high level of expression in newborn animals suggests a role in growth and development. This seems to occur in addition to its role for the endocrine regulation of phosphate homeostasis by as yet unknown humoral agents that must occur throughout life. In summary, Pex mRNA expression is high in brain and bone at birth. Expression remains high in brain with age but falls with age in bone, kidney, and lung. PMID- 10742447 TI - Comparison of radiographic and pQCT analyses of healing rat tibial fractures. AB - Fracture healing and callus formation have traditionally been evaluated by using X-ray radiography. Here we compared X-ray radiography and peripheral quantitative computed tomography (pQCT) in evaluating the healing callus of standardized tibial fractures in 141 female rats after a 4- or 8-week follow-up. The results were compared with the tensile (4-week) and compressive (8-week) failure load of the callus. The projectional size of callus, as defined from lateral ex vivo radiographs, correlated significantly with the pQCT-defined cross-sectional area (CSA) of midcallus. This relationship was dependent on the pQCT attenuation threshold, being higher for the CSA of compact bone (r = 0.85, P < 0.0001) than for the total bone CSA (r = 0.68, P < 0.0001). Radiographically defined callus projectional area also correlated strongly with bone mineral content (BMC) (r = 0.84-0.86, P < 0.0001). The mean optical density of the callus analyzed from the radiographs had only a weak correlation with the pQCT-defined bone mineral density (BMD) of callus. A weak negative relationship was found between CSA and BMD. The optical density analyzed from lateral radiographs did not correlate with the tensile or compressive failure load of callus. Callus size, BMC, and BMD were associated with the compressive failure load, whereas both radiographs and pQCT were poor in explaining the failure load in tension. PMID- 10742448 TI - Matrix vesicles mediate mineralization of human thyroid cartilage. AB - Mineralization and ossification of human thyroid cartilage first starts after the end of adolescence when the previously cartilaginous human skeleton has become ossified and the epiphyseal discs are in the process of closing. However, the mechanisms involved in mineralization and ossification of human thyroid cartilage are not well understood. Ultrastructural analysis of human thyroid cartilage revealed that mineralization started close to cartilage canals in a matrix containing gigantic collagen fibers (asbestoid fibers). Matrix vesicles were detected in mineralized areas and were often associated with needle-like crystals. For the first time we were able to isolate matrix vesicles from human thyroid cartilage by mild enzymatic digestions and ultracentrifugation. These particles were oval and varied in size; some were heavily calcified. They were enriched in alkaline phosphatase, calcium, and inorganic phosphate, suggesting that the particles contain Ca2+-Pi complexes. Immunoblot analysis of these vesicles revealed the presence of annexins II, V, and VI, membrane-associated, channel-forming proteins, which allow influx of Ca2+ into the vesicles and intralumenal crystal growth. In addition, the vesicles were associated with types II and X collagen, suggesting that this association not only anchors the vesicles to the extracellular matrix, but, as shown previously, also stimulates Ca2+ influx into these particles. In conclusion, matrix vesicles isolated from human thyroid cartilage contain all the components, enabling them to initiate and mediate the mineralization process in human thyroid cartilage. PMID- 10742449 TI - Exercise and mechanical loading increase periosteal bone formation and whole bone strength in C57BL/6J mice but not in C3H/Hej mice. AB - To identify the genes, and the mechanisms that account for the 53% higher peak bone density in C3H/HeJ (C3H) mice compared with C57BL/6J (B6) mice, we are performing quantitative trait locus and phenotypic analyses. The phenotypic studies revealed differences in bone formation and resorption, and showed that hindlimb immobilization (by sciatic neurectomy) caused a greater increase in endosteal resorption in the tibiae of B6 compared with C3H mice. The current studies were intended to examine the hypothesis that the bones of C3H mice are less sensitive to mechanical loading than the bones of B6 mice. To increase mechanical loading, 9-week-old female B6 and C3H mice (n = 10-13 mice/group) were subjected to a jumping exercise (20 jumps/day, 5 days/week, to heights of 20-30 cm) for a total of 4 weeks. Control mice did not jump. Osteocalcin, alkaline phosphatase (ALP) activity, and IGF-I were measured in serum. The left tibiae were used for histomorphometry (ground cross-sections prepared at the tibiofibular junction) and the right tibiae and femora were used for determinations of bone breaking strength (3-point bending). The results of these studies revealed (1) significant effects of both mouse strain (B6 and C3H) and the jumping exercise on tibial strength; (2) an exercise-dependent increase in serum IGF-I in C3H, but not B6 mice; and (3) no effects on serum ALP or osteocalcin. The histomorphometric analyses showed no effect of exercise on C3H tibiae, but significant exercise-dependent increases in total bone area, periosteal perimeter, periosteal mineral apposition rate (MAR), and periosteal bone formation (P < 0.02 for each) in B6 tibiae. There were no effects of exercise on periosteal resorption or any endosteal measurement in either C3H or B6 mice. Since the jumping exercise was designed to cause a two-three fold increase in muscular-skeletal loading at the tibio-fibular junction, and the calculated stress (g/mm2) at this sampling site was only 16% greater for B6 compared with C3H mice, we had anticipated that both strains of mice would show exercise-dependent increases in periosteal bone formation, with a greater response in the B6 mice. The lack of a response in the C3H tibiae demonstrates that the bones of C3H mice are less sensitive to mechanical loading (and unloading) than the bones of B6 mice. PMID- 10742450 TI - The stimulation of vertebral and tibial bone growth by the parathyroid hormone fragments, hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1 30)NH2. AB - The native human parathyroid hormone, hPTH-(1-84), and certain carboxyl truncated analogs such as hPTH-(1-34) and even smaller fragments such as hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-30)NH2 stimulate femoral trabecular and cortical bone growth in ovariectomized (OVX) rats. Here we show that when injected once daily for 6 weeks starting 2 weeks after OVX in doses of 1 or 2 nmol/100 g of body weight, hPTH-(1-31)NH2, [Leu27] cyclo(Glu22-Lys26)hPTH (1-31)NH2, and hPTH-(1-34)NH2 prevented the loss of trabecular volume in the L5 vertebrae induced by OVX. In fact, by the end of the sixth week of injections (i.e., the eighth week after OVX) the fragments had increased the volume and trabecular thickness significantly above the values in vehicle-injected sham operated rats. hPTH-(1-30)NH2 can stimulate vertebral bone growth as much as the larger fragments, but 10-25 times more of it was needed to do so. The same daily doses of hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1 34)NH2 also raised the trabecular volume and thickness in the L5 vertebrae of rats well above the values in vehicle-treated animals when the injections were started 9 weeks after OVX. This restoration of trabecular bone in the L5 vertebrae in estrogen-deprived animals was accompanied by a significant increase in the bone mineral density (BMD) of the L1-L4 vertebrae and tibias. However, there was no significant drop in the pelvic BMD in the estrogen-deprived animals and the effects of hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-(Lys) hPTH-(1-31)NH2, and hPTH-(1-34)NH2 on the pelvic BMD were equivocal. PMID- 10742451 TI - Low dietary protein and low bone density. PMID- 10742452 TI - European Calcified Tissue Society. PMID- 10742453 TI - Partners in protection: interdependence of cytoskeleton and plasma membrane in adaptations to applied forces. AB - In mechanically active environments mammalian cells must cope with potentially injurious forces to survive, but the most proximal mechanosensors are largely unknown. How mechanoprotective responses to applied forces are generated and regulated is still a mystery. We consider recent evidence that suggests cellular mechanoprotective adaptations involve a coordinated remodeling of the cell membrane and the associated cytoskeleton. The plasma membrane "protects" the cytoskeleton by maintenance of intracellular ionic balance and can modulate force induced cytoskeletal rearrangements by stretch-activated (e.g., Ca(2+)) ion channels and mechanosensitive enzymes (e.g., Phospholipase A(2) and Phospholipase C). Conversely, the cytoskeleton protects the plasma membrane by providing structural support, reinforcement of the cortical framework at sites of force application, modulation of mechanosensitive ion channels and by potentially contributing to the membrane resealing process after mechanical rupture. We suggest that the plasma membrane and the cytoskeleton are partners in the cytoprotective response to physical forces. PMID- 10742454 TI - Effect of dicyclohexylcarbodiimide (DCCD) on transport parameters in the frog cornea epithelium. AB - Dicyclohexylcarbodiimide (DCCD) is a carboxyl group modifier and it is an inhibitor of various ATPases. Present experiments, using an in vitro preparation, were designed to study whether DCCD affected the transporters of the bullfrog cornea epithelium, specifically, the Na(+)/K(+) ATPase pump located in the basolateral membrane. For this purpose, corneas were impaled with microelectrodes and experiments were done under short-circuit current (I(sc)) conditions. Addition of DCCD to a concentration of 10(-4) m to the tear solution gave a marked decrease in I(sc); a marked depolarization of the intracellular potential, V(o); and a significant decrease in the apical membrane fractional resistance, fR(o). There were small and variable although significant changes in the transepithelial conductance, g(t). The effects may be explained by a decrease in the basolateral membrane K(+) conductance, in combination with a partial inhibition of the Na(+)/K(+)-ATPase pump located in the basolateral membrane. There is also evidence for an increase in the apical membrane Cl(-) conductance. PMID- 10742455 TI - Modulation of the calmodulin-induced inhibition of sarcoplasmic reticulum calcium release channel (ryanodine receptor) by sulfhydryl oxidation in single channel current recordings and [(3)H]ryanodine binding. AB - The modulation of the calmodulin-induced inhibition of the calcium release channel (ryanodine receptor) by two sulfhydryl oxidizing compounds, 4 (chloromercuri)phenyl-sulfonic acid (4-CMPS) and 4, 4'-dithiodipyridine (4,4' DTDP) was determined by single channel current recordings with the purified and reconstituted calcium release channel from rabbit skeletal muscle sarcoplasmic reticulum (HSR) and [(3)H]ryanodine binding to HSR vesicles. 0.1 microm CaM reduced the open probability (P(o)) of the calcium release channel at maximally activating calcium concentrations (50-100 microm) from 0.502 +/- 0.02 to 0.137 +/ 0.022 (n = 28), with no effect on unitary conductance. 4-CMPS (10-40 microm) and 4,4'-DTDP (0.1-0.3 mm) induced a concentration dependent increase in P(o) (> 0.9) and caused the appearance of longer open states. CaM shifted the activation of the calcium release channel by 4-CMPS or 4,4'-DTDP to higher concentrations in single channel recordings and [(3)H]ryanodine binding. 40 microm 4-CMPS induced a near maximal (P(o) > 0.9) and 0.3 mm 4,4'-DTDP a submaximal (P(o) = 0.74) channel opening in the presence of CaM, which was reversed by the specific sulfhydryl reducing agent DTT. Neither 4-CMPS nor 4,4'-DTDP affected Ca-[(125)I]calmodulin binding to HSR. 1 mm MgCl(2) reduced P(o) from 0.53 to 0.075 and 20-40 microm 4 CMPS induced a near maximal channel activation (P(o) > 0.9). These results demonstrate that the inhibitory effect of CaM or magnesium in a physiological concentration is diminished or abolished at high concentrations of 4-CMPS or 4,4' DTDP through oxidation of activating sulfhydryls on cysteine residues of the calcium release channel. PMID- 10742456 TI - Mechanically induced calcium movements in astrocytes, bovine aortic endothelial cells and C6 glioma cells. AB - Forces applied to resting primary astrocytes, bovine aortic endothelial cells and C6 glioma cells with collagen-coated magnetite particles produce a fast transient change of intracellular Ca(2+). It peaks in the micromolar range as measured by Fura-2. This mechanical response adapts within seconds so that repeated stimulation causes smaller responses requiring >10 min for recovery. When cytoplasmic Ca(2+) is high after treating with ATP, cyclopiazonic acid and thapsigargin, stimulation causes a transient decrease in Ca(2+). In these three cell types, no influx of ions is required for Ca(2+) elevation showing the response is not caused by activation of plasmalemmal mechanosensitive channels. Approximately half the cells tested showed similar behavior, while the other half, such as fibroblasts, required extracellular Ca(2+). The Ca(2+) response is not temperature sensitive suggesting the possible involvement of intracellular mechanosensitive channels. We tested a number of second messenger reagents and were only able to block the response in BAECs, but not C6 glioma cells, with Xestospongin C, a blocker of IP(3)-activated channels. Despite the lack of a causal involvement of plasmalemmal mechanosensitive channels, mechanical stimulation immediately activates a persistent Mn(2+) influx pathway. This Mn(2+) pathway may be mechanosensitive channels, Ca(2+)-activated cation channels or depletion-activated Ca(2+) channels. PMID- 10742457 TI - The melibiose carrier of Escherichia coli: cysteine substitutions for individual residues in helix XI. AB - The melibiose carrier from Escherichia coli is a sugar-cation cotransport system. Previously evidence was obtained that this integral membrane protein consists of 12 transmembrane helices. Starting with the cysteine-less melibiose carrier, cysteine has been substituted individually for amino acids 374-396, which includes all of the residues in the proposed helix XI. The carriers with cysteine substitutions were studied for their transport activity and the effect of the water soluble sulfhydryl reagent p-chloromercuribenzenesulfonic acid (PCMBS). Studies were carried out on both intact cells and inside out vesicles. Cysteine substitution caused loss of transport activity in seven of the mutants (K377C, G379C, A383C, F385C, L391C, G395C and Y396C). PCMBS produced more than 50% inhibition in six of the mutants (S380C, A381C, A384C, F387C, A388C and L391C). Preincubation of the cells with melibiose protected five of these residues from the inhibitory action of PCMBS. It was concluded that the residues whose cysteine derivatives were inhibited by PCMBS probably faced the aqueous channel. PMID- 10742458 TI - Polyamine triggering of exocytosis in Paramecium involves an extracellular Ca(2+)/(polyvalent cation)-sensing receptor, subplasmalemmal Ca-store mobilization and store-operated Ca(2+)-influx via unspecific cation channels. AB - The polyamine secretagogue, aminoethyldextran (AED), causes a cortical [Ca(2+)] transient in Paramecium cells, as analyzed by fluorochrome imaging. Our most essential findings are: (i) Cortical Ca(2+) signals also occur when AED is applied in presence of the fast Ca(2+) chelator, BAPTA. (ii) Extracellular La(3+) application causes within seconds a rapid, reversible fluorescence signal whose reversibility can be attributed to a physiological [Ca(2+)](i) transient (while injected La(3+) causes a sustained fluorescence signal). (iii) Simply increasing [Ca(2+)](o) causes a similar rapid, short-lived [Ca(2+)](i) transient. All these phenomena, (i-iii), are compatible with activation of an extracellular "Ca(2+)/(polyvalent cation)-sensing receptor" known from some higher eukaryotic systems, where this sensor (responding to Ca(2+), La(3+) and some multiply charged cations) is linked to cortical calcium stores which, thus, are activated. In Paramecium, such subplasmalemmal stores ("alveolar sacs") are physically linked to the cell membrane and they can also be activated by the Ca(2+) releasing agent, 4-chloro-m-cresol, just like in Sarcoplasmic Reticulum. Since this drug causes a cortical Ca(2+) signal also in absence of Ca(2+)(o) we largely exclude a "Ca(2+)-induced Ca(2+) release" (CICR) mechanism. Our finding of increased cortical Ca(2+) signals after store depletion and re-addition of extracellular Ca(2+) can be explained by a "store-operated Ca(2+) influx" (SOC), i.e., a Ca(2+) influx superimposing store activation. AED stimulation in presence of Mn(2+)(o) causes fluorescence quenching in Fura-2 loaded cells, indicating involvement of unspecific cation channels. Such channels, known to occur in Paramecium, share some general characteristics of SOC-type Ca(2+) influx channels. In conclusion, we assume the following sequence of events during AED stimulated exocytosis: (i) activation of an extracellular Ca(2+)/polyamine sensing receptor, (ii) release of Ca(2+) from subplasmalemmal stores, (iii) and Ca(2+) influx via unspecific cation channels. All three steps are required to produce a steep cortical [Ca(2+)] signal increase to a level required for full exocytosis activation. In addition, we show formation of [Ca(2+)] microdomains (20 mmHg) activate not only cardiopulmonary and arterial baroreflexes, but also intramuscular mechanoreflexes, while 6 degrees HDT only activates cardiopulmonary baroreflexes. We conclude that LBPP is not a suitable model for simulated weightlessness in humans. PMID- 10742549 TI - Corrigendum to "Sympathetic skin responses in psoriasis and vitiligo". PMID- 10742548 TI - A deficiency of interstitial cells of Cajal in Chagasic megacolon. AB - Disordered gut motor activity is a feature of patients with Chagas' disease: colonic involvement leads to the development of megacolon and symptoms of constipation. Interstitial cells of Cajal are thought to modulate gut motility. The aim of this study was to test the hypothesis that there is an abnormality of the density of distribution of interstitial cells of Cajal in Chagasic megacolon. Interstitial cells of Cajal were identified by immunohistochemistry using an anti c-kit antibody. Six patients with Chagasic megacolon were compared with normal controls. The density of distribution of interstitial cells of Cajal was assessed in the longitudinal and circular muscle layers, and in the intermuscular plane of the Chagasic and normal colon. Statistical analysis was performed using Fisher's exact test. The interstitial cells of Cajal density in Chagasic megacolon was much reduced in comparison to normal colonic tissue in the longitudinal muscle layer (P=0.0084), intermuscular plane (P<0.0001), and circular muscle layer (P=0.0051). The lack of interstitial cells of Cajal may play a role in the pathophysiology of the disease, leading to the development of megacolon and symptoms of constipation. PMID- 10742550 TI - Interleukin-4-dependent induction of preproenkephalin in antigen-specific T helper-type 2 (Th2) cells. AB - Naive Th cells obtained from OVA(323-339)-specific DO11.10 TCR-Tg mice did not express preproenkephalin (PPE) mRNA. However, culture of naive Th cells with OVA(323-339) peptide (OVA-pep) plus IL-2 under Th2-inducing conditions for 7 days resulted in an induction of PPE mRNA. The PPE mRNA was also induced by culturing with OVA-pep plus IL-2 (neutral condition). However, PPE mRNA induction under neutral conditions was totally abrogated by addition of anti-IL-4 mAb. The existence of methionine-enkephalin was also demonstrated in peptidase-digested peptides derived from Th2 cell lysate. These results demonstrate that IL-4 is a critical factor for the induction of PPE mRNA in freshly expanded antigen specific Th2 cells. PMID- 10742551 TI - Modulation of fas-ligand (Fas-L) on human microglial cells: an in vitro study. AB - The expression of Fas-Ligand (Fas-L) on microglia could be relevant in multiple sclerosis immunopathology. The present study was performed to evaluate in vitro the expression of Fas-L in human microglial cells both unstimulated and after stimulation with IFN-gamma, beta-IFN-1b and beta-IFN-1b+IFN-gamma. Cells were stimulated for 6,12, 24 and 48 h. Surface Fas-L was evaluated by flow cytometry, total Fas-L by Western blot, whereas mRNA for Fas-L was measured by RT-PCR. We also evaluated the capacity of microglial cells to induce, in vitro, apoptosis on Fas-positive T leukemia Jurkat cells. Our results showed a constitutive expression of Fas-L on microglia. IFN-gamma downregulated the expression of the molecule, while beta-IFN-1b and beta-IFN-1b+IFN- gamma did not. The amount of surface Fas-L was related to the ability of microglial cells to induce apoptosis in Fas-positive target cells, which was partly inhibited by blockade of the Fas Fas-L pathway. PMID- 10742552 TI - Monocyte-derived IL-10-secreting dendritic cells in choroid plexus epithelium. AB - Choroid plexuses form an interface between peripheral blood and cerebrospinal fluid. Dendritic-like cells have been reported in a few studies of choroid plexuses in man. Here we used electron microscopy and immunophenotyping to precise the morphologic features and phenotype of these cells. Examination of 10 human choroid plexuses evidenced intra-epithelial dendritic cells with a clear cytoplasm, reniform nucleus and long expansions. These cells express MHC Class II, CD11b, CD14, CD32, CD68 and IL-10, but not CD40, CD80 or CD86, suggesting an immunosuppressive role for these dendritic cells. Their sentinel position could make them participate to the immunological silence of the brain. PMID- 10742553 TI - Enhancement of Th2 response in IL-6-deficient mice immunized with myelin oligodendrocyte glycoprotein. AB - To investigate the mechanism for the resistance of IL-6-deficient mice to experimental autoimmune encephalomyelitis (EAE), we examined the production of cytokines in lymph nodes (LNs) of wild-type and IL-6-deficient mice immunized with myelin oligodendrocyte glycoprotein (MOG) by reverse transcriptase polymerase chain reaction analysis. Significant up-regulation of IL-4 production and down-regulation of IFN-gamma production were found in LNs from IL-6-deficient mice as compared to LNs from wild-type mice. Administration of IL-6, which caused typical EAE in IL-6-deficient mice immunized with MOG, reduced IL-4 production but did not restore IFN-gamma production in LNs of IL-6-deficient mice. These results implied that the resistance of IL-6-deficient mice to EAE might be mainly due to enhancement of Th2 response. PMID- 10742554 TI - The role of complement anaphylatoxin C5a in neurodegeneration: implications in Alzheimer's disease. AB - There is evidence that the complement system, a major component of inflammatory responses, may play an important role in neurodegenerative conditions such as Alzheimer's disease (AD). Work from our lab demonstrated that mice genetically deficient in the complement component C5 are more susceptible to hippocampal excitotoxic lesions (Pasinetti et al., 1996) and that the C5-derived ana;hylatoxin C5a may protect against excitotoxicity in vitro and in vivo (Osaka et al., 1999). Potential mechanisms identified in C5a-mediated neuroprotection include activation of mitogen activated protein (MAP)-kinase (Osaka et al., 1998; Osaka et al., 1999). This novel neuroprotective role of C5a complicates current theories that complement proteins augment beta-amyloid (Abeta) toxicity in AD. In view of the fact that the complement system represents a target for therapeutic interventions in AD, further characterization of the complex role of complement proteins is essential. Towards this aim, we have characterized a transgenic C5a receptor (C5aR) knockout (KO) mouse. Recent studies in the lab using C5aR-KO mice show that disruption of C5aR alters calcium calmodulin kinase (CaM-KII) signal transduction in brain cells. We are presently using C5aR-KO mice to study the role of C5a in caspase mediated apoptotic neuronal death. In this review we will attempt to delineate possible neuroprotective roles for C5a in mechanisms of neurotoxicity pertaining to AD. PMID- 10742556 TI - Evidence for antigenic cross-reactivity between herpesvirus and the acetylcholine receptor. AB - Herpes simplex virus (HSV) is neurotropic and can pass from neuron to neuron at nerve terminals. During the long evolutionary relationship between HSV and vertebrates, this virus may have evolved surface ligands that mimic nerve cell receptors. The present study was undertaken to determine if herpes simplex virus type 1 (HSV-1) has an antigenic relationship with the acetylcholine receptor (AChR). Mice immunized with HSV-1 antigens or an AChR-expressing cell line were tested for antibodies directed against the AChR. By flow cytometry and ELISA, mouse anti-HSV-1 sera were found to contain antibodies that would bind to an epitope on the plasma membrane of AChR-expressing cells. Mice immunized with the AChR-expressing cells were tested for their resistance to HSV-1 infection. Statistically significantly more of the animals immunized with AChR-expressing cells resisted infection and fatal encephalitis, compared to control animals immunized with a cell line not expressing the AChR. Sera from AChR-immunized mice were tested for anti-HSV antibody by ELISA and were found to contain antibodies cross-reactive with HSV-1 antigens. These sera also neutralized virus in a plaque inhibition assay. The results indicate that there are one or more antigenic epitopes shared by herpesvirus and the AChR. Studies are in progress to define the pathogenetic significance of this molecular mimicry. PMID- 10742555 TI - Suppression of experimental myasthenia gravis by monoclonal antibodies against MHC peptide region involved in presentation of a pathogenic T-cell epitope. AB - We have prepared monoclonal antibodies (mAbs) against an antigen-binding region of I-A, region 62-76 of I-Abeta(b), which is involved in the T-cell participation in the pathogenesis of EAMG. The mAbs reacted with its parent molecules and inhibited the proliferation of disease-related T-cells. Passive transfer of these mAbs suppressed the occurrence of clinical EAMG, which was accompanied by decreased T-cell and Ab responses to tAChR. The results indicated that blocking the function of disease-related MHC by targeting a disease-associated region on MHC molecules could be an effective, straightforward and feasible strategy for immunointervention in MG. PMID- 10742557 TI - Modulation of type I interleukin-1 receptor messenger RNA followed by one and repeated endotoxin treatment in the mouse. AB - The expression of transcripts for type 1 interleukin-1 receptors (IL-1R1) were investigated utilizing reverse transcription-polymerase chain reaction (RT-PCR) techniques in male C57BL/6 mice. First, we measured IL-1R1 mRNA 1, 2, 6, and 12 h after a single injection of endotoxin lipopolysaccharide (LPS; 30 microg/mouse). IL-1R1 mRNA was measured in relevant tissues utilizing semiquantitative RT-PCR. IL-1R1 mRNA levels were significantly increased 2 and 6 h after one LPS injection group in comparison with the saline injection group in the hippocampus and testis. Next, we investigated the effect of repeated injections of LPS on IL-1R1 expression. LPS or saline were injected at 24-h intervals for 4 consecutive days. The mice were divided into four groups: (1) LPS injections for 4 days and LPS injection on day 5 (LPS-LPS); (2) LPS injections for 4 days and saline injection on day 5 (LPS-saline); (3) saline injections for 4 days and LPS injection on day 5 (saline-LPS); (4) saline injections for 4 days and saline injection on day 5 (saline-saline). The mice were sacrificed by decapitation 2 h after the last injection. In the hippocampus and pituitary gland, significant increases of IL 1R1 mRNA levels were observed in the saline-LPS group, however, the increases were attenuated in LPS-LPS group. IL-1R1 mRNA levels in the hypothalamus were unchanged in all four groups. In the adrenal gland and testis, IL-1R1 mRNA levels were significantly increased in the LPS-LPS group as well as saline-LPS group. These data demonstrate differential regulation of IL-1R1 mRNA by one and repeated endotoxin treatment and suggest the importance of the cytokines in mediating the effects of infectious challenge on brain, endocrine and immune function during endotoxemia. PMID- 10742558 TI - Inefficient T cell memory in the brain of mice infected with Candida albicans. AB - We compared the contribution of T cell memory to the clearance of the fungus Candida albicans from the liver, kidneys and brain of Balb/c mice in a model of secondary systemic infection. In secondary infection, the fungi were more rapidly eliminated from the liver and kidneys than during primary infection. This was most pronounced in the liver where the fungi were eliminated at day 14 of infection. In contrast, in the brain, cultivable yeasts were still detectable 35 days after infection. Although both CD4(+) and CD8(+) cells could be detected in the brain with immunohistology, these cells appeared later in infection and in lower numbers than in the liver, and there were no significant differences in the numbers of T cells detected in the brain between primary and secondary infection. In contrast to the liver and the kidneys where an effect of T cells on the fungal load could be demonstrated, depletion of neither CD4(+) nor CD8(+) nor Thy-1.2(+) cells resulted in a significant increase of the amount of fungi in the brain above levels measured in secondarily infected mice treated with irrelevant antibodies. We conclude that the contribution of CD4(+) and CD8(+) cells to the clearance of C. albicans in secondary infection is organ-dependent and that T cell memory is inefficient in the brain. PMID- 10742559 TI - Antigen-specific T cell trafficking into the central nervous system. AB - The initiation step of cell-mediated immune responses in the central nervous system (CNS) involves the trafficking of the antigen-specific T cells into the brain. To study this trafficking, we developed an in vivo system for studying antigen-specific responses in the CNS. In this assay, T cell receptor (TCR) transgenic mice having 95% of T cells specific for a defined antigen-pigeon cytochrome c (PCC) were cannulated intraventricularly for PCC antigen infusion and cerebrospinal fluid (CSF) sampling. Upon PCC infusion into the CNS, the number of alpha/beta TCR(+) Vbeta3(+) Mac1(-) cells in the CSF was characterized. We found that infusion of antigen into the CSF induced an increased number of antigen-specific T cells in the CNS and activation of antigen-specific T cells in the peripheral blood. Hence, the drainage of CNS antigen into the periphery might play an important role in sustaining autoimmune reactivity in CNS inflammatory diseases. PMID- 10742560 TI - Normal human plasma contains antibodies that specifically block neuropathy associated human anti-GM1 IgG-antibodies. AB - Intravenous immunoglobulin (IVIg) is used in the treatment of a variety of autoimmune diseases. The blocking of disease-associated antibodies by anti idiotype antibodies present in IVIg has been proposed as an action mechanism. Anti-GM1 antibodies have been implicated in motor neuropathies. Although IVIg is frequently applied for these diseases, the presence in IVIg or in human plasma of anti-idiotype antibodies that recognize anti-GM1 antibodies has not been clearly demonstrated. Here we present evidence that normal human plasma contains antibodies that inhibit the binding of anti-GM1 IgG-antibodies from neuropathy patients but do not inhibit anti-GM1 IgG-antibodies of rabbit origin with the same fine specificity. The significance of these findings in the course of acute and chronic neuropathies is discussed. PMID- 10742561 TI - Genetic variation in the B7-1 gene in patients with multiple sclerosis. AB - Co-stimulation of T cells by B7-1, a protein that is expressed on antigen presenting cells, favors a T helper type1 (Th1) cellular response. Th1/Th2 bias may influence disease susceptibility or course of MS. We screened the entire coding sequence as well as the 5'- and 3'-untranslated regions of the B7-1 gene using a mutation scanning technique, dideoxyfingerprinting, in DNA from 111 patients with MS from Olmsted County, Minnesota. We identified five genetic variants. None alter protein structure nor have apparent functional significance. Selected variants of sufficient frequency were tested for an association with course and severity of MS and one was tested for an association with susceptibility; none of the association tests were positive. PMID- 10742563 TI - IgG antiganglioside antibodies in Guillain-Barre syndrome with bulbar palsy. AB - Some patients with Guillain-Barre syndrome (GBS) develop bulbar palsy, which may lead to serious complications during the acute phase of the illness. A serological marker that could predict the occurrence of bulbar palsy would be valuable for the treatment of acute GBS. We examined the serum levels of various IgG antiganglioside antibodies in the sera of 16 patients with GBS with bulbar palsy [GBS-BP(+)] and 72 patients with GBS without bulbar palsy [GBS-BP(-)]. Anti GT1a antibodies were detected in a higher percentage of the GBS-BP(+) patients (10/16, 63%) than the GBS-BP(-) patients (2/72, 3%). In addition to GT1a, a new disialosylganglioside antigen was recognized by the sera of four GBS-BP(+) patients. Anti-GM1b antibodies were also frequently detected in the sera of the GBS-BP(+) cases. However, anti-GM1 and anti-GalNAc-GD1a antibodies, which are highly associated with acute axonal motor neuropathy (AMAN), were not detected in any of the GBS-BP(+) cases, while anti-GM1 antibodies were detected in 29% (21/72) and anti-GalNAc-GD1a antibodies were detected in 8% (6/72) of the GBS-BP( ) cases. These findings suggest that the presence of particular antiganglioside antibodies might be related with certain clinical manifestations of GBS. In patients who are diagnosed with GBS, the presence or absence of anti-GT1a and anti-GM1b antibodies should be tested at the early stage of GBS so that appropriate therapies that prevent the development of bulbar palsy and improve the outcome of GBS, may be initiated. PMID- 10742562 TI - Association of a myeloperoxidase promoter polymorphism with multiple sclerosis. AB - Myeloperoxidase (MPO) generates hypochlorous acid and other reactive oxygen intermediates leading to tissue damage. MPO is expressed in macrophages-microglia in multiple sclerosis (MS) lesions. A G-->A substitution that abolishes an SP1 transcription factor consensus sequence in the promoter reduces gene expression. We studied the association of the genetic variant with MS. We did not find an association with gender, age at onset, susceptibility to, or the course and severity of MS in a population-based sample of 122 patients from Olmsted County. PMID- 10742564 TI - Introduction PMID- 10742565 TI - The application of electroencephalographic techniques to the study of human olfaction: a review and tutorial. AB - The use of a variety of electrophysiological techniques to determine the effects of odor on the nervous system is reviewed. Methods and problems associated with the collection of on-going EEG, chemosensory event-related potentials, and contingent negative variation data are discussed in depth as is the use of odors as modulators of brain potentials produced by other senses. In addition, the advantages of several seldom used EEG analysis techniques are discussed with respect to the unique problems of understanding olfaction. PMID- 10742566 TI - Chemosensory event-related potentials (CSERP) as a key to the psychology of odors. AB - The present article gives a critical overview of how the components N1, N2, especially the mismatch negativity (MMN), and P3 have been investigated and interpreted in the context of 'chemosensory event-related potential' (CSERP) research. In order to integrate the respective CSERP results, findings and theoretical models from other modalities are briefly described for each component. It is suggested that all components found so far within the CSERP strongly depend on the psychological state of the individual. In particular the dominant positivity of the CSERP has been focused on by investigating the specificity of olfactory emotional processing in comparison to emotional and neutral stimuli from the visual modality. The results reveal that the late positive complex within the CSERP consists of two subcomponents, one of which has a frontal and the other a parietal dominance. The posterior positivity seems to reflect the features of the P3 component and varies with the valence of odors, whereas the anterior positivity seems to be similar to the Novelty-P3. A link between olfactory and emotional processing has been confirmed by the finding that the P3 elicited by visual stimuli shows similar valence effects. PMID- 10742567 TI - The effect of odour priming on cortical EEG and visual ERP responses. AB - Two studies are reported that were designed to examine the way in which subjects, when primed with an olfactory stimulus, make perceptual judgements regarding subsequent congruent/incongruent information. The paradigm used was based on a modified version of a standard ERP semantic priming procedure. Subjects watched a computer screen and made a decision as to whether an image was congruent or incongruent with a priming olfactory stimulus. Results of the two studies showed significant effects in the N400 component of the visual ERP between congruent and incongruent stimuli are reported when a complex malodour accord was used as a prime but not when a complex pleasant odour accord was used as a prime. PMID- 10742568 TI - Olfactory event-related potentials and aging: normative data. AB - Unlike the clinical usages of evoked potentials (e.g. brain stem auditory evoked potentials for the assessment of auditory function), normative data for the olfactory event-related potential (OERP) have been unavailable. The principal objective was to establish normative data across the human life span for OERPs with a given set of parameters. Participants were 140 persons from seven age groups (16-19, 20-29, 30-39, 40-49, 50-59, 60-69 and 70-79 years of age), with equal numbers of males and females, screened for nasal health and dementia. The odor stimulus was amyl acetate, presented at nasal temperature in a humidified airstream delivered by an air-dilution olfactometer at a constant flow rate, using a 60-s inter-stimulus interval. OERPs were recorded at Fz, Cz, and Pz electrode sites, amplified and averaged over trials. Amplitudes of the N1/P2 and P3 and latencies of the P2 and P3 were analyzed. Processing speed decreased at a constant rate over decades for the sensory (P2 latency) as well as cognitive (P3 latency) components. Decline in amplitude over decades was also apparent. Normative data will be useful in research on olfactory function and in clinical assessment of olfactory functional status. PMID- 10742569 TI - Assessment of intranasal trigeminal function. AB - Intranasal trigeminal function is more and more understood as an integral part of human chemosensory perception. Sensations like burning, stinging, warmth, coolness, or itching are produced by almost all odorants so that they can be perceived by anosmics. Electrophysiological responses to trigeminal stimuli allow the specific assessment of trigeminally mediated information at different levels of processing including the periphery or the cortex. Information regarding the localization of these processes can be derived from magnetoencephalographic recordings or functional imaging data. When using these techniques in combination with psychophysical measures, it seems to be possible to specifically describe how and where the processing of irritation takes place, how it may interact with olfactory mediated sensations, and how it is modulated, e.g. by environmental influences or analgesic drugs. PMID- 10742570 TI - Olfactory functional imaging and physiology. AB - Over the last few years, several different neuroimaging techniques have been applied to get a better insight into cortical regions activated after olfactory stimulation. Here we report on data obtained by recent magnetic source imaging (MSI) and functional magnetic resonance imaging (fMRI) studies during well defined olfactory stimulation and compare the results obtained with these two techniques. PMID- 10742572 TI - Microencapsulation and characterization of tramadol-resin complexes. AB - Tramadol was complexed with a sulfonic acid cation-exchange resin by a column method. Microencapsulation of tramadol-resin complexes (TRC) was carried out by the spray-drying method. The effect of ethylcellulose (EC) of various viscosities, different solvent systems and addition of plasticizers on microencapsulation were investigated. The morphology of microcapsules was characterized by scanning electron microscopy (SEM). Selected solvents having similar toxicity levels while various physico-chemical properties resulted in greatly different microcapsules. Regular particle morphology and sustained release behavior were obtained when dichloromethane or ethyl acetate was used as the solvent system, whereas ethanol produced a substantial number of coalesced microcapsules and acetone produced apparently surface-smooth monomicrocapsules with faster release behavior. Three kinds of plasticizers affected the drug release rate similarly. On the addition of plasticizer (DEP), the drug release rate from the microcapsule obtained from low viscosity-grade EC reduced, while there was no alternation for those obtained from middle viscosity-grade EC. Compared to EC with a viscosity of 100 cP, which produced multi-microcapsules and released 60% of the drug within 1 h, microcapsules prepared with 10, 20 and 45 cP viscosity-grade EC showed slower drug release and regular and smooth morphology. PMID- 10742571 TI - Functional neuroimaging of the olfactory system in humans. AB - Positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) have begun to provide unique information regarding the neural underpinnings of olfactory functioning in humans. We review the relative strengths and weaknesses of PET and fMRI techniques for studying olfaction. We then review PET and fMRI studies relating to the olfactory functions of the pyriform cortex, orbitofrontal cortex, amygdala and the entorhinal/hippocampal region. A pixelwise correlational analysis of PET data is also presented in order to clarify the relationship between blood flow in the medial temporal lobes and psychoperceptual variables. PMID- 10742573 TI - Vitamin A-loaded solid lipid nanoparticles for topical use: drug release properties. AB - Burst release as well as sustained release has been reported for SLN suspensions. For dermal application, both features are of interest. Burst release can be useful to improve the penetration of a drug. Sustained release becomes important with active ingredients that are irritating at high concentrations or to supply the skin over a prolonged period of time with a drug. Glyceryl behenate SLN were loaded with vitamin A and the release profiles were studied. Franz diffusion cells were used to assess the release kinetic over a period of 24 h. Within the first 6 h retinol SLN displayed controlled release. After longer periods (12-24 h) the release rate increased and even exceeded the release rate of comparable nanoemulsions. Pure SLN dispersions are characterised by low viscosity. In contrast to membranous vesicles, SLN can also be stably incorporated in convenient topical dosage forms like hydrogels or creams. In the Franz diffusion cell these preparations showed a controlled release over 12-18 h. Similar to SLN dispersions an increase in release rate over a 24-h period was found. A good correlation between polymorphic transitions and increased drug release was observed in this study. Sustained release was often related to the metastable beta' polymorph. Drug expulsion is explained by a reduction of amorphous regions in the carrier lattice due to a beta'-->beta(i) polymorphic transition. This transformation can be controlled with surfactant mixtures or, in the case of the hydrogel and oil/water cream, with humectants or gelling agents. Thus, the release rate for the topical route of application is adjustable. PMID- 10742574 TI - The effect of electroporation on iontophoretic transdermal delivery of calcium regulating hormones. AB - Electrically-assisted delivery by iontophoresis and/or electroporation was used in vitro to deliver the calcium regulating hormones, salmon calcitonin (sCT) and parathyroid hormone (1-34) (PTH) through human epidermis. Such delivery could be useful for chronic treatment of post-menopausal osteoporosis and other clinical indications as a superior alternative to parenteral delivery. sCT (50 microg/ml) or PTH (1-34) (100 microg/ml) formulation was prepared in citrate buffer (pH 4.0 or 5.0, respectively). Epidermis separated from human cadaver skin was used. Iontophoresis was applied using a constant current power source and electroporation with an exponential pulse generator. Silver/silver chloride electrodes were used. A combination of electroporation and iontophoresis resulted in higher transdermal permeation than either one technique alone. Electroporation also shortened the lag time of iontophoretic transdermal delivery of salmon calcitonin. Pulsing at lower voltages followed by iontophoresis did not result in increased transport (over iontophoresis alone), perhaps because the transdermal voltage was very low. The transdermal transport of salmon calcitonin by pulsing with 15 pulses (1 ppm) of 500 V (200 ms) followed by iontophoresis led to a quick input and high flux. The average transdermal voltage was only about 50 V for a 500 V study. PMID- 10742575 TI - Evaluation of matrices containing molecularly imprinted polymers in the enantioselective-controlled delivery of beta-blockers. AB - Granules and beads of methacrylic acid (MAA) and granules of N-acryloyl-alanine polymer (NAA) were produced using ethylene glycol dimethacrylate as cross-linking monomer either by bulk (in the case of granules) or suspension (in the case of beads) polymerization. Either R- or S-propranolol, were used as an imprint molecule, acting as a template, with a view to conferring enantioselectivity of release upon the polymer. The molecularly imprinted polymers (MIPs) or nonMIPs (control) were formulated with racemic propranolol and other excipients and compressed to form matrix tablets. Enantioselective release of propranolol in vitro was monitored using a stereoselective HPLC assay. The influence of the method of polymer synthesis, drug: polymer ratio, pH and temperature on the release of the two enantiomers was determined. Stereoselectivity of release was identified in tablets containing either MAA or NAA granules or MAA beads, with the latter showing the greatest differences between enantiomers. Release of the enantiomer used as the print was always faster than the release of the nonprint enantiomer. In the case of S-propranolol-MIP bead matrices composed of MAA, greater differences in the release of enantiomers could be promoted by increasing the polymer: drug ratio of the tablet. Differences in the release rate of the two propranolol enantiomers was still apparent as the pH was varied between 3 and 7.4 and when the temperature was decreased from 37 to 25 degrees C. S-Propranolol-MIP bead matrices demonstrated cross-reactivities of stereoselective dissolution for enantiomers of pindolol and oxprenolol, both of which have structural similarities to the imprint molecule. It is concluded that polymers of this type may have great potential in controlling, via means of formulation, the release of drug eutomer whilst enhancing retention of distomer in the dosage form. PMID- 10742576 TI - Analysis of skin permeation-enhancing mechanism of iontophoresis using hydrodynamic pore theory. AB - The effects of constant DC iontophoresis (0-1.5 mA/0.966 cm(2)) on the permeation of three hydrophilic compounds, antipyrine (ANP, M.W. 188.23), sucrose (SR, M.W. 342.30) and 1-kestose (KT, M.W. 506.73), through excised hairless rat skin were evaluated using hydrodynamic pore theory. The electro-osmotic flow caused by iontophoresis was measured using deuterium oxide (D(2)O). The penetration enhancing mechanism of iontophoresis was found to increase solvent flow through electro-osmosis and pore enlargement and/or new pore production in the skin barrier, together with enhancement of electrochemical potential difference across the skin. These effects were closely related to the strength of the current applied. The electro-osmotic flow of D(2)O (J(D(2)O)) greatly enhanced the skin permeation clearance of all hydrophilic penetrants (CL(drug)). Pore production was classified into reversible and irreversible processes, which resulted from lower (0-0.5 mA/0.966 cm(2)) and higher (0.5-1. 5 mA/0.966 cm(2)) currents, respectively. Thus, the enhancing effects of iontophoresis on skin permeation of nonionic hydrophilic compounds can be explained by increase in pore size and higher solvent flow. PMID- 10742577 TI - Pharmacokinetics of prolonged-release CPT-11-loaded microspheres in rats. AB - CPT-11-containing microspheres composed of poly-D,L-lactic acid or poly (D,L lactic acid-co-glycolic acid) copolymers were prepared by an oil-in-water evaporation method. The size and shape of the microspheres were examined, and the drug release rates were analyzed from the in vitro release profiles. CPT-11 aqueous solution was intravenously or intraperitoneally injected at 10 mg/kg, and microspheres were intraperitoneally administered at 50 mg eq CPT-11/kg in rats. The microspheres had an average diameter of around 10 microm and their shape was spherical. All the microspheres contained CPT-11 in a lactone form, and their drug contents and release profiles were basically similar to those of previous microspheres. After i.v. injection of CPT-11 solution, the CPT-11 plasma concentration decreased quickly, SN-38 decreased slowly at a much lower level, and SN-38 glucuronide (SN-38G) declined very slowly at a higher level than SN-38. The plasma concentration of CPT-11 reached a maximum at 30 min after i.p. administration of CPT-11 solution. The area under the plasma concentration-time curve (AUC) of CPT-11 after i.p. administration was somewhat lower compared with that after i.v. administration, but the plasma concentration-time profiles of SN 38 and SN-38G were nearly identical between i.v. and i.p. administration. An i.p. administration of the microspheres resulted in gradually increasing or almost constant CPT-11 levels. The levels of SN-38 were also stable during the observation period (4 days) except for the slowest releasing microsphere in which SN-38 was not detected after 24 h following administration. Intraperitoneal administration of any of the microspheres resulted in stable and similar levels of SN-38G after 24 h following administration. When judging from apparent simple pharmacokinetic analysis, an inconsistency was found between the in vitro drug release and the plasma level to a fair extent, but overall the in vivo drug release rate from microspheres was considered parallel to the in vitro one. The microspheres showing a faster release of CPT-11 exhibited higher plasma levels of CPT-11 and SN-38, explaining the previous results that efficacy was better when the in vitro release rate was higher. That the SN-38 level could be attained to a certain extent even at the range of modest or low plasma concentration of CPT-11 in each administration may be related to the non-linear metabolic conversion from CPT-11 to SN-38. PMID- 10742578 TI - Active growth factor delivery from poly(D,L-lactide-co-glycolide) foams prepared in supercritical CO(2). AB - A method for the production of microporous poly(D, L-lactide-co-glycolide) foams containing encapsulated proteins using supercritical carbon dioxide is described. Foams generated as aqueous protein emulsions in a polymer-solvent solution were saturated with carbon dioxide at supercritical conditions, and then suddenly supersaturated at ambient conditions causing bubble nucleation and precipitation of the polymer. Proteins contained in the water phase of the emulsion were encapsulated within the foams, including basic fibroblast growth factor (bFGF), an angiogenic factor of interest in tissue engineering applications. The release and activity of bFGF from these foams was determined in vitro and compared with similar porous scaffolds prepared by traditional solvent casting-salt leaching techniques. Total protein release rate was greater from structures made in CO(2) than those made by the salt leaching technique, however a large initial burst of bFGF was released from the salt leached structures. This initial burst was not observed from the polymer foams processed in CO(2) and active bFGF was released at a relatively constant rate. Residual methylene chloride levels were measured in the foams made with CO(2) and were found to be above the limits imposed by the US Pharmacopoeia implying that further solvent removal would be required prior to in vivo use. PMID- 10742579 TI - Linear type azo-containing polyurethane as drug-coating material for colon specific delivery: its properties, degradation behavior, and utilization for drug formulation. AB - A segmented polyurethane containing azo aromatic groups in the main chain was synthesized by reaction of isophorone diisocyanate with a mixture of m,m' di(hydroxymethyl)azobenzene, poly(ethylene glycol) (Mn = 2000), and 1,2 propanediol. This polyurethane was soluble in various solvents and showed a good coating and film-forming property. A solution-cast film of this polyurethane was found to be degraded in a culture of intestinal flora with the azo group reduction to hydrazo groups, not to amino groups. The film degradation, therefore, was attributed to the decreased cohesive energy in the hydrazo polymer compared with that in the original azo polymer. Then, the drug pellets containing water-soluble drugs (ONO-3708 and OKY-046) were undercoated with (carboxymethyl)(ethyl)-cellulose and overcoated with the azo polymer in order to examine the drug-releasing profiles in the culture of intestinal flora. The releasing rate of drugs from these double-coating pellets was found to depend on the molecular weight and the composition of the polyurethane used as the overcoat as well as the hydrophilicity of the incorporated drugs. Since the polyurethane was glassy and its segment motion or conformational change is frozen, the structure change should be retarded even after partial reduction of the azo groups, resulting in the effective prevention of the drug leakage. These data suggested that the present azo-containing polyurethanes are applicable as coating material of drug pellets in a colon-targeting delivery system. PMID- 10742580 TI - Cutaneous vaccination: the skin as an immunologically active tissue and the challenge of antigen delivery. AB - Vaccination is one of the major achievements of modern medicine. As a result of vaccination, diseases such as polio and measles have been controlled and small pox has been eradicated. However, despite these successes there are still many microbial diseases that cause tremendous suffering because there is no vaccine or the vaccines available are inadequate. In addition, even if vaccines were available for all infectious diseases there is no guarantee that people would use them routinely. One of the major impediments to ensuring vaccine efficacy and compliance is that of delivery. Presently most vaccines are given by intramuscular administration. Unfortunately this is often traumatic, especially in infants. Thus, if it was possible to replace intramuscular immunization by mucosal (oral/intranasal) or transdermal delivery it may be possible to both enhance mucosal immunity as well as improve overall compliance rates. The transdermal route has been used by the pharmaceutical industry for the delivery of various low molecular weight drugs. Some of the approaches used for smaller compounds may also have potential for delivery of either protein or polynucleotide vaccines. However, there is a greater challenge to delivering large molecular weight molecules through the skin due to size, charge and other physicochemical properties. This review will describe the recent advances that have been made in dermal and topical delivery as related to vaccines. PMID- 10742581 TI - Evaluation of starch-maltodextrin-Carbopol 974 P mixtures for the nasal delivery of insulin in rabbits. AB - In this study insulin was administered nasally to rabbits as a dry powder formulation. The powders consisted of drum-dried waxy maize starch (DDWM) or maltodextrins with different DE values and Carbopol 974 P. The powders were prepared by freeze-drying a dispersion of these excipients with insulin. Bioavailabilities obtained with the powder formulations containing DDWM/Carbopol 974 P (5 and 10%) were significantly higher (p<0.05) than those containing maltodextrins-Carbopol 974 P mixtures. The bioavailability of the powder formulation containing DDWM and 10% Carbopol 974 P was significantly higher (14.4%) than the bioavailability of the same mixture containing 5% Carbopol 974 P (9.9%). The bioavailability, t(max) and C(max) values of the formulation with 5% Carbopol 974 P were significantly higher in comparison with the formulation without Carbopol 974 P. 10% Carbopol 974 P was required when maltodextrins were used in order to obtain a significantly higher bioavailability compared with the formulations without Carbopol 974 P. Freeze-drying seemed a prerequisite for a good bioavailability from the powder formulation as well as the ratio of insulin versus bioadhesive powder (1 IU and 2 IU/mg of bioadhesive powder). PMID- 10742582 TI - In vivo EPR of topical delivery of a hydrophilic substance encapsulated in multilamellar liposomes applied to the skin of hairless and normal mice. AB - In vivo low frequency EPR was used to measure the enhancement of topical delivery of hydrophilic substances by use of multilamellar liposomes. The contribution of transepidermal or/and transfollicular routes of transport was investigated using hairless and normal mice. Two liposome dispersions that previously had been shown to have different enhancement properties on ex vivo skin were used. The kinetics of the reduction of hydrophilic spin probe GluSL (N-(1-oxyl-2,2,6,6-tetramethyl-4 piperidinyl)-2,3,4,5, 6-pentahydroxy-hexaneamide) applied to the skin encapsulated into the liposomes was measured. To distinguish the reduction of GluSL on the skin surface from its reduction inside the skin, the oxidizing agent potassium ferricyanide (KFeCN) was used. This does not penetrate into the skin and therefore it oxidizes hydroxylamines back to nitroxide only on the surface of the skin. We observed significant differences in the properties of the two types of liposomes with respect to their stability when in contact with skin and their transport characteristics. The results measured in vivo are consistent with those obtained ex vivo, indicating that in vivo L-band EPR is a powerful technique for following pharmacokinetics in the skin of live animals. The results also show that clearance by blood flow and possible alterations of skin after sacrifice of animal do not influence the results of penetration of liposome entrapped substances into the skin during the time of our experiment (typically around 60 min). The reduction of GluSL in the skin of hairless vs. normal mice was similar, indicating that the transfollicular penetration was not of major importance in vivo in this experimental model. PMID- 10742583 TI - Formulation of poly(D,L-lactic-co-glycolic acid) microparticles for rapid plasmid DNA delivery. AB - An optimised water-in-oil-in-water double emulsion process for the microencapsulation of plasmid DNA in poly(D,L-lactic-co-glycolic acid) (PLGA) was used to prepare microparticles from a range of different PLGA formulations. This process has been developed using pharmaceutically accepted solvents and is potentially scaleable. Incorporation of plasmid DNA in the microparticles of up to 11 microg/mg was obtained and the retention of plasmid DNA integrity was considerably greater than previously reported. Microparticle structure was determined, by scanning electron microscopy, to be hollow and size distribution characteristics were found to be independent of polymer formulation. The ability to vary the plasmid DNA release profile by changing the PLGA formulation and polymer concentration used in the encapsulation process was also demonstrated. This ability to control the release profile of the microparticles was shown to be especially important as the physical integrity of the encapsulated plasmid DNA was found to deteriorate with extended release times in vitro. PMID- 10742584 TI - Influence of three synthetic membranes on the release of caffeine from concentrated W/O emulsions. AB - We measured the release rate characteristics of caffeine from concentrated emulsions using three different sources of synthetic membranes. The formulations tested included, on the one hand, two stable cosmetic concentrated W/O emulsions (90% w/w) - one with a non ionic surfactant and one with a silicone surfactant - and on the other hand, a commercially available hydroalcoholic gel. All formulations contained 5% caffeine. In vitro diffusion measurements (24 h) were performed with static diffusion Franz cells. A silicone membrane could not allow us to differentiate the two concentrated emulsions (CE), but the two other membranes, not rate limiting, showed difference in the release profile of caffeine from the two CE. Results with the cellulose and polysulfone membrane showed that in vitro release of caffeine is influenced by the nature of the emulsifier in the concentrated emulsion, the non ionic surfactant being more efficient than the silicone surfactant. The polysulfone membrane was the only one that allows statistical differentiation of the three products. For further studies the polysulfone membrane will be use to make screening on concentrated emulsions. PMID- 10742585 TI - Effect of DNA/liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/cationic liposome complexes and subsequent gene expression. AB - In order to identify the important factors involved in cationic liposome-mediated gene transfer, in vitro transfection efficiencies by plasmid DNA complexed with DOTMA/DOPE liposomes at different DNA/liposome mixing ratios were evaluated using four types of cultured cells with respect to their physicochemical properties. Significant changes were observed in the particle size and zeta potential of the complexes as well as in their structures, assessed by atomic force microscopy, which depended on the mixing ratio. In transfection experiments, except for RAW 264.7 cells (mouse macrophages), efficient gene expression was obtained in MBT-2 cells (mouse bladder tumor), NLH3T3 cells (mouse fibroblasts) and HUVEC (human umbilical vein endothelial cells) at an optimal ratio of 1:5, 1:7.5 or 1:5, respectively. On the other hand, cellular uptake of the [32P]DNA/liposome complexes increased in all cell types with an increase in the mixing ratio, which was not reflected by the transfection efficiency. The cellular damage determined by MTT assay was minimal even at the highest DNA/liposome ratio (1:10), indicating that the lower gene expression level at the higher ratio was not due to cytotoxicity induced by the complex. An ethidium bromide intercalation assay showed that the release of plasmid DNA from the complex, following the addition of negatively charged liposomes, was restricted as the mixing ratio increased. Furthermore, confocal microscopic studies using HUVEC showed that the 1:5 complexes exhibited a dispersed distribution in the cytoplasm whereas a punctuate intracellular distribution was observed for the 1:10 complexes. This suggests that there was a significant difference in intracellular trafficking, probably release from the endosomes or lysosomes, of the plasmid DNA/cationic liposome complexes between these mixing ratios. Taken together, these findings suggest that the DNA/liposome mixing ratio significantly affects the intracellular trafficking of plasmid DNA complexed with the cationic liposomes, which is an important determinant of the optimal mixing ratio in cationic liposome-mediated transfection. PMID- 10742586 TI - Controlled release of a water-soluble drug, captopril, by a combination of hydrophilic and hydrophobic cyclodextrin derivatives. AB - Parent beta-cyclodextrin (beta-CyD) and 2-hydroxypropyl-beta-CyD (HP-beta-CyD) form 1:1 solid complexes with an orally active angiotensin-converting enzyme inhibitor, captopril, while hydrophobic perbutanoyl-beta-CyD (TB-beta-CyD) forms a solid dispersion or solid solution with the drug. The binary system of captopril/HP-beta-CyD or captopril/TB-beta-CyD and the ternary system of captopril/TB-beta-CyD/HP-beta-CyD in different molar ratios were prepared by the kneading method, and the release behavior of the drug was investigated. The release rate of captopril from the binary HP-beta-CyD system was rather fast, whereas that from the binary TB-beta-CyD system was comparatively slower, the retarding effect being dependent on the amounts of TB-beta-CyD. The release rate from the ternary captopril/TB-beta-CyD/HP-beta-CyD system was slowed down by the addition of small amounts of HP-beta-CyD, whereas the rate became faster as the molar ratio of HP-beta-CyD further increased (>.25 molar ratio). Both water penetration studies and microscopic observation suggested that the retarding effect is attributable to a gel formation of HP-beta-CyD in the TB-beta-CyD hydrophobic matrix. It was difficult to prolong plasma levels of captopril by administering orally either the binary HP-beta-CyD or TB-beta-CyD system in dogs. On the other hand, the ternary captopril/TB-beta-CyD/HP-beta-CyD system (molar ratio of 1:0.5:0.5) gave a plasma profile comparable to that of a commercially available sustained release preparation (Captoril R). Therefore, a combination of HP-beta-CyD and TB-beta-CyD is useful for the controlled release of water-soluble drugs such as captopril. PMID- 10742587 TI - Preparation and evaluation of the in vitro drug release properties and mucoadhesion of novel microspheres of hyaluronic acid and chitosan. AB - Rapid mucociliary clearance of intranasally administered drugs is often a key factor in determining the bioavailability of such therapeutic agents. The use of mucoadhesive microparticles provide a potential strategy for improving retention of drugs within the nasal cavity, and thereby improve the resultant pharmacokinetic profile. This study describes the comparison of a number of novel, potentially mucoadhesive microspheres, prepared by solvent evaporation, composed of hyaluronic acid (HA), chitosan glutamate (CH) and a combination of the two with microcapsules of HA and gelatin prepared by complex coacervation. The microspheres had a mean particle size of 19.91+/-1.57 microm (HA), 28.60+/ 1.34 microm (HA/CH), 29.47+/-3.58 microm (CH). The incorporation of a model drug, gentamicin sulphate (%) was 46.90+/-0.53 (HA), 28.04+/-1.21 (HA/CH) and 13.32+/ 1.04 (CH). The in vitro release profiles of microsphere formulations prepared by solvent evaporation were determined. The release of gentamicin from HA and HA/CH was 50% longer than CH and was best modelled as a release from a matrix. The degree of mucoadhesion of each formulation was investigated by determining the mucociliary transport rate (MTR) of the microparticles across an isolated frog palate. Acacia/gelatin microcapsules were used as a positive control. The rank order of mucoadhesion for the microspheres and the microparticles was HA=HA/CH>CH>HA/gelatin>CHins. The entrapment of gentamicin did not affect the mucoadhesive properties (P>0.05, Mann--Whitney U-test). The combination of HA with chitosan may afford additional advantages in combining the mucoadhesive potential of HA with the penetration enhancing effect of chitosan. PMID- 10742588 TI - A particulate pulse-release system and mathematical description with the Maxwell Stefan theory. AB - In this contribution both the development of a multi-particulate delayed release system with release properties dependent on the swelling of an UV crosslinked coating and a mathematical model to describe its release properties are presented. The formulation consists of a water-soluble core coated with a copolymer of methacrylic acid and ethyl acrylate. Incorporating a network of crosslinked pentaerythritol triacrylate decreases the water-solubility of the coating. After immersing the formulation in water the coating will take up water and subsequently swell in such a degree that the diffusion coefficient of water in the coating will increase. This makes the coating permeable to the dissolved components present in the core. The swelling kinetics of the coating are such that the formulation has a pulse-release profile, i.e. a fast release of the contents is obtained after a pre-determined lag-time. Both the coating thickness and the duration of the UV crosslinking time can be used to adjust the lag-time. The experimental results are used to estimate the Maxwell-Stefan diffusion coefficients of water in the coating. The relation between the Maxwell-Stefan diffusion coefficient and the mole fraction of water in the coating differs from results found in the literature. However, the prediction of the release time based on the presented model is in good agreement with the experimental findings. PMID- 10742589 TI - Control of protein delivery from amphiphilic poly(ether ester) multiblock copolymers by varying their water content using emulsification techniques. AB - Protein-containing films and microspheres, based on poly(ethylene glycol) poly(butylene terephthalate) (PEG-PBT) multiblock copolymers, were prepared from water-in-oil (w/o) emulsions. The properties of the matrices could be controlled by the water-to-polymer ratio (w/p) in the w/o emulsion. A linear increase in water uptake of the matrices was observed with increasing emulsion w/p. This could be explained by an increase in the number of dispersed water-rich domains in the polymer matrix. At low volume fraction of the dispersed phase (epsilon), lysozyme release was mainly dependent on the permeability of the swollen polymer bulk. Above a critical volume fraction (the percolation threshold epsilon(c)), the dispersed water-rich phase formed an interconnected network, which largely enhanced the permeability of the matrix. Determination of the permeability of PEG PBT matrices for vitamin B(12) as a function of epsilon confirmed the formation of such an interconnected network. This interconnected network could be used to achieve controlled release of a large protein (bovine serum albumin, BSA) from PEG-PBT films and microspheres. Due to its hydrodynamic diameter, BSA was screened by the polymer network when epsilon was low. However above epsilon(c), the fraction released BSA increased with increasing volume fraction of the dispersed phase. A very rapid BSA release could be obtained, with the majority of the incorporated BSA released within 1 day, as well as a slow and continuous release, lasting for over 150 days. When BSA-containing microspheres were prepared with a volume fraction just below the percolation threshold, a delayed release was observed. This was attributed to the effect of polymer degradation on matrix permeability. PMID- 10742590 TI - Diversity and functions of glycosaminoglycan sulfotransferases. AB - Sulfate residues attached to the specific position of the component sugar residues of glycosaminoglycans play important roles in the formation of functional domain structures. The introduction of a sulfate group is catalyzed by various sulfotransferases with strict substrate specificities. A rapid development achieved in the cloning of various glycosaminoglycan sulfotransferases has allowed us to study the biological functions of glycosaminoglycan sulfotransferases and their products, sulfated glycosaminoglycans. PMID- 10742591 TI - The myosin motor: muscle contraction and in vitro movement. AB - The molecular mechanism of in vitro movement is assumed, by most investigators, to be identical to that of muscle contraction. We discuss this view, which raises various problems. We believe there are mechanisms for muscle contraction (in this case considerable forces are developed, with small displacements) and other mechanisms for in vitro movement (giving large displacements, without necessarily generating substantial forces). Hybrid models may explain muscle contraction. The traditional swinging-crossbridge model may explain in vitro movement. For muscle contraction, movement may result partly from the swinging-crossbridge mechanism and partly from other factors. Comparisons of different fibres at different moments of the Mg-ATPase cycle suggest that both the value of the isometric force in muscle and in vitro and that of the Mg-ATPase activity used in vitro need to be reconsidered. The recently reported dependence of the isometric active tension of smooth skinned fibres on temperature appears to be weaker than predicted by the swinging-crossbridge theory alone. This recent observation is compatible with the existence of other forces (electrostatic repulsions) decreasing with temperature as has been known for some years. From recent experimental data, we think the biochemistry of myosin and actomyosin should be reassessed, to try to find new details of the mechanisms of muscle contraction and in vitro motility. PMID- 10742592 TI - Mechanisms of phase behaviour and protein partitioning in detergent/polymer aqueous two-phase systems for purification of integral membrane proteins. AB - Detergent/polymer aqueous two-phase systems are studied as a fast, mild and efficient general separation method for isolation of labile integral membrane proteins. Mechanisms for phase behaviour and protein partitioning of both membrane-bound and hydrophilic proteins have been examined in a large number of detergent/polymer aqueous two-phase systems. Non-ionic detergents such as the Triton series (polyoxyethylene alkyl phenols), alkyl polyoxyethylene ethers (C(m)EO(n)), Tween series (polyoxyethylene sorbitol esters) and alkylglucosides form aqueous two-phase systems in mixtures with hydrophilic polymers, such as PEG or dextran, at low and moderate temperatures. Phase diagrams for these mixtures are shown and phase behaviour is discussed from a thermodynamic model. Membrane proteins, such as bacteriorhodopsin and cholesterol oxidase, were partitioned strongly to the micelle phase, while hydrophilic proteins, BSA and lysozyme, were partitioned to the polymer phase. The partitioning of membrane protein is mainly determined by non-specific hydrophobic interactions between detergent and membrane protein. An increased partitioning of membrane proteins to the micelle phase was found with an increased detergent concentration difference between the phases, lower polymer molecular weight and increased micelle size. Partitioning of hydrophilic proteins is mainly related to excluded volume effects, i.e. increased phase component size made the hydrophilic proteins partition more to the opposite phase. Addition of ionic detergent to the system changed the partitioning of membrane proteins slightly, but had a strong effect on hydrophilic proteins, and can be used for enhanced separation between hydrophilic proteins and membrane protein. PMID- 10742593 TI - Ion-protein dissociation predicts 'windows' in electric field-induced wound-cell proliferation. AB - There are many experiments showing that weak, non-thermal electric fields influence living tissues. In many cases, biological effects display 'windows' in biologically effective parameters of electric fields: most dramatic is the fact that relatively intense electric fields sometimes do not cause appreciable effect, while smaller fields do. Linear resonant physical processes do not explain frequency windows in this case. Both frequency and amplitude windows are evident from experiments on human dermal fibroblasts in a collagen matrix. For this in vitro model of skin, exposure to extremely low frequency (ELF) electric fields in the frequency range 10-100 Hz and the amplitude range of 0-130 microA/cm(2) macroscopic current density demonstrates such unusual 'window' behavior. Amplitude window phenomena suggest a non-linear physical mechanism. We consider non-linear quantum-interference effects on protein-bound substrate ions: These ions experience, due to electric fields in the media or biological tissue as small as 1 mV/m, electric gradients produced by polarized binding ligand atomic shells. The electric gradients cause an interference of ion quantum states. This ion-interference mechanism predicts specific electric-field frequency and amplitude windows within which fibroblast proliferation occurs. PMID- 10742594 TI - Radical mediated indirect oxidation of a PEG-coupled polycyclic aromatic hydrocarbon (PAH) model compound by fungal laccase. AB - A high molecular model compound of polycyclic aromatic hydrocarbon was synthesised by coupling pyrene to PEG(5000). The pyrene-PEG was used for the study of a laccase-mediator-system. To prevent direct contact between the substrate and the enzyme the two were kept in their own compartments separated by a membrane. The low molecular mediators, 1-hydroxybenzotriazole and 2, 2'-azino bis-(3-ethylbenzothiazoline-6-sulphonic acid), which were oxidised by laccase to the corresponding radicals or cations permeated the membrane and reacted with the pyrene-PEG model compound. Oxidation of the model compound resulted in an alpha oxidation of the alkyl-chain leading to two main oxidation products. The same oxidation products were obtained in the reaction system without a membrane. PMID- 10742595 TI - Carotenoid:methyl-beta-cyclodextrin formulations: an improved method for supplementation of cultured cells. AB - BC. Two days after supplementation with 5 microM BC in MbetaCD, cellular BC levels reached a maximum of 140+/-11 pmol/microg DNA, leveling off to 100+/-15 pmol/microg DNA until day 8. Incubation with BC dissolved in THF/DMSO resulted in a lower BC uptake of 105+/-14 pmol/microg DNA and 64+/-20 pmol/microg DNA respectively. No cytotoxic effects of these formulations were detected. The results show that the MbetaCD formulation is an improved method for investigations of carotenoids and other lipophilic compounds in in vitro test systems compared to methods using organic solvents. PMID- 10742596 TI - Regulation of copper uptake and transport in intestinal cell monolayers by acute and chronic copper exposure. AB - Adaptation to high and low copper intake in mammals depends on the cellular control of influx, efflux and storage mechanisms of cellular copper concentrations. In the present study, we used an intestinal cell line (Caco-2), grown in bicameral chambers to study the effect of equilibrium loading with copper. We analyzed (64)Cu uptake from the apical surface, intracellular metal (Cu, Zn, Fe) content, (64)Cu transport into the basal chamber, and total copper, zinc and iron in the basal chamber. We found that the (64)Cu uptake is saturable, shows a linear response phase up to 1.5 microM reaching a plateau at 4-6 microM extracellular Cu. Intracellular copper increased 21.6-fold, from 1.5 to 32.4 mM (at 0.2-20.2 microM extracellular copper respectively). The time course for (64)Cu uptake and transport was linear when the cells were incubated with different copper concentrations. Uptake increased 10-fold when intracellular copper concentration was raised. Fluxes were lowest at 1.5 mM and highest at 32.4 mM Cu intracellular copper (2.03 and 20. 98 pmole (64)Cu insert(-1) h(-1), respectively). The apical-to-basolateral copper transfer rate was lower at 32.4 mM as compared to 1.5 mM intracellular copper (0.55-1.95 pmole (64)Cu insert(-1) h(-1), respectively). The total copper in the basal chamber increased 4.2-fold (from 3.04 to 12.85 pmole Cu insert(-1) h(-1)) when the intracellular copper concentration was raised. If cells are preincubated in a low copper medium most of the newly incorporated copper (64%) is transferred to the basolateral compartment. In contrast, under preloading with high copper concentration, only 4% of the fresh copper is transferred to the basal chamber; however, the intracellular copper contribution to this chamber increases by 4.2-fold. Thus, the process results in an increase in both storage and intracellular-to basolateral flux of copper. In summary, our results indicate that copper fluxes from apical-to-cell and apical-to-basolateral domains are affected by intracellular copper concentration suggesting that mechanisms of copper transport involved in cellular adaptation to low and high copper exposure are different. PMID- 10742597 TI - Fluorescence quenching of dipyridamole associated to peroxyl radical scavenging: a versatile probe to measure the chain breaking antioxidant activity of biomolecules. AB - Dypiridamole is a highly efficient chain breaking antioxidant (Iuliano et al., Free Radic. Biol. Med. 18 (1995) 239-247) with an aromatic ring system responsible for an intense absorption band in the 400-480-nm region and for an intense fluorescence. Dipyridamole fluorescence is quantitatively quenched upon reaction with peroxyl radicals. In the presence of a flux of peroxyl radicals generated by thermal dissociation of azo-initiators, dipyridamole fluorescence decays linearly, showing a first-order reaction with respect to peroxyl radicals, and zero-order with respect to dipyridamole. The pH optimum for the fluorescence quenching is in the 7-8 range, from pH 7 to 6, the decay of fluorescence rapidly decreases to became negligible below pH 5.5. Dipyridamole consumption is blocked in the presence of an added chain breaking antioxidant for a time that is proportional to the antioxidant concentration. This effect is shown for ascorbic acid, trolox, vitamin E, uric acid, and N, N'-diphenyl-p-phenylenediamine. The slope of the linear correlation relative to trolox allows calculation of the bimolecular rate constant for a given molecule and peroxyl radicals. Comparison of data obtained by the dipyridamole consumption are comparable to values obtained by the oxygen consumption method. PMID- 10742598 TI - Docosahexaenoic acid reverses cyclosporin A-induced changes in membrane structure and function. AB - The use of a fish oil vehicle for cyclosporin A (CsA) can decrease the toxic effects of CsA but the mechanism is unclear. Here we examine the mechanism by which docosahexaenoic acid (DHA), a fish oil-derived polyunsaturated fatty acid, can alter the toxic effects of CsA on mouse organ function, endothelial macromolecular permeability, and membrane bilayer function. Mice given CsA and fish oil showed increased liver toxicity, kidney toxicity, incorporation of DHA, and evidence of oxidized fatty acids compared to control animals. We hypothesized that the toxic effects of CsA were primarily a result of membrane perturbation, which could be decreased if DHA were not oxidized. The presence of CsA (10 mol%) alone increased dipalmitoylphosphatidylcholine membrane permeability by seven fold over control (no CsA, no DHA). However, if non-oxidized DHA (15 mol%) and CsA were added to the membrane, the permeability returned to control levels. Interestingly, if the DHA was oxidized, the antagonistic effect of DHA on CsA was completely lost. While CsA alone increased endothelial permeability to albumin, the combination of non-oxidized DHA and CsA had no effect on endothelial macromolecular permeability. However the combination of oxidized DHA and CsA was no different than the effects of CsA only. CsA increased the fluorescence anisotropy of DPH in the liquid crystalline state of DPPC, while DHA decreased fluorescence anisotropy. However the combination of CsA and DHA was no different than DHA alone. We conclude that non-oxidized DHA can reverse the membrane perturbing effects of CsA, and the increases in endothelial macromolecular permeability, which may explain how fish oil is capable of decreasing the toxicity of CsA. PMID- 10742599 TI - Selective 'in synthesis' labeling of peptides with biotin and rhodamine. AB - A new method is described for the selective 'in synthesis' labeling of peptides by rhodamine or biotin at a single, predetermined epsilon-amino group of a lysine residue. The alpha-amino group and other lysyl residues of the peptide remain unmodified. Peptides are assembled by the Fmoc approach, which requires mild operative conditions for the final deprotection and cleavage, and ensures little damage of the reporter group. The labeling technique involves the previous preparation of a suitable Lysine derivative, easily obtained from commercially available protected amino acids. This new derivative, where the reporter group (biotin, or rhodamine) acts now as permanent protection of lysyl side chain functions, is then inserted into the synthesis program as a conventional protected amino acid, and linked to the preceding residue by aid of carbodiimide. A simpler, alternative method is also described for the selective 'in synthesis' labeling of peptides with N-terminal lysyl residues. Several applications of labeled peptides are reported. PMID- 10742600 TI - Molecular cloning and functional expression of human ST6GalNAc II. Molecular expression in various human cultured cells. AB - A cDNA clone encoding a human Galbeta1-3GalNAc alpha2, 6-sialyltransferase (designated hST6GalNAc II) was identified employing the PCR with degenerated primers to the sialylmotifs, followed by BLAST analysis of databanks. This sialyltransferase sequence is similar to that of previously cloned ST6GalNAc II (chicken and mouse) and shows the sialylmotifs that are present in all eukaryotic members of the sialyltransferase gene family. The predicted amino acid sequence encodes a putative type II transmembrane protein as found for other eukaryotic sialyltransferases and shows significant similarity to chicken (56. 8% identity) and mouse (74.6% identity) enzymes. Expression of a secreted form of hST6GalNAc II in COS-7 cells showed that the gene product had Galbeta1-3GalNAc (sialyl to GalNAc) alpha2, 6-sialyltransferase activity. In vitro analysis of substrate specificity revealed that the enzyme required a peptide aglycone fraction to be active and used both Galbeta1-3GalNAc and Neu5Acalpha2-3Galbeta1-3GalNAc as acceptor substrates. Northern analysis revealed a restricted expression pattern of two hST6GalNAc II transcripts, a 2.0 kb mRNA found mainly in skeletal muscle, heart and kidney and a 1.8 kb mRNA found in placenta, lung and leukocytes. No transcriptional expression was detected in brain, thymus or spleen. Transcriptional expression of the ST6GalNAc II gene was followed in various human cell lines and found to be expressed in almost all cell types with notable exceptions for several myeloid and lymphoid cell lines. PMID- 10742601 TI - Glycine prevents the phenotypic expression of streptococcal glucan-binding lectin. AB - Glycine has been used extensively in bacterial cell surface research. Some researchers employ glycine in growth media so as to increase the transformability of streptococci during electroporation. Others have found that glycine, similar to wall antibiotics, 'weakens' peptidoglycan. It is now shown that when glycine is incorporated into the growth medium, Streptococcus sobrinus exhibits a diminished ability to aggregate with high molecular weight alpha-1,6-glucan. Growth of the bacteria in either a rich or a chemically defined medium results in a cell population with full lectin (glucan-binding) fidelity. Incorporation of glycine, but not serine or other amino acids, at concentrations of 100-200 mM gives rise to bacteria with lowered lectin activities. Bacteriolytic enzymes were able to lyse bacteria from glycine-grown cultures more readily than from cultures without the glycine supplement. The bacteria produce glucan-binding proteins, including glucosyltransferases, but they do not readily aggregate with added dextran. Furthermore, SDS-PAGE gels of supernatants of growth media (+/-glycine) are similar, suggesting the bacteria do not produce a unique set of proteins. Western blotting with a fluorescein isothiocyanate-labeled dextran probe reveals normal amounts of glucan-binding proteins in glycine-grown streptococci. Glycine may be acting as a type of antibiotic, reducing wall integrity upon which glucan promoted cellular aggregation depends. PMID- 10742602 TI - In vitro inhibition of the activity of phosphorylase kinase, protein kinase C and protein kinase A by caffeic acid and a procyanidin-rich pine bark (Pinus marittima) extract. AB - Caffeic acid (CA) is a common constituent of human diet while pine bark extract (PBE) is utilized either as nutritional supplement or as phytochemical remedy for different diseases. CA and PBE, are reported as efficient antioxidants and more recently have been described to modulate cellular response to oxidative challenge and to possess many other biological activities, i.e. anti-inflammatory, antimutagenic, antitumoral effects. In order to investigate in depth the mechanism of action of these polyphenols, the effects of CA and PBE on the activity of some protein kinases involved in the regulation of fundamental cellular processes were studied in vitro: phosphorylase kinase (PhK), protein kinase A (PKA), protein kinase C (PKC). PBE at the concentration of 20 microg/ml (corresponding to 69 microM catechin equivalents) inhibited PKA, PhK and PKC by about 90, 59, 57%, respectively, while 100 microM CA inhibited by 37, 52 and 54%, respectively. Considerable inhibitions have been still observed at even lower concentrations of CA and PBE. For PhK and PKA, the inhibition follows a non competitive mechanism. CA also inhibits PKC activity in a partially purified cellular extract. The results suggest a possible involvement of CA and PBE in modulation of cellular functions. PMID- 10742603 TI - Primary structures of two hemagglutinins from the marine red alga, Hypnea japonica. AB - As the first examples among marine algal hemagglutinins, the primary structures of two hemagglutinins, named hypnin A-1 and A-2, from the red alga Hypnea japonica, were determined by Edman degradation. Both hemagglutinins were single chain polypeptides composed of 90 amino acid residues including four half cystines, all of which were involved in two intrachain disulfide bonds, Cys(5) Cys(62) and Cys(12)-Cys(89). Hypnin A-1 and A-2 had calculated molecular masses of 9146.7 and 9109.7 Da which coincided with determined values, 9148 and 9109 Da, by electrospray ionization-mass spectrometry, respectively. Both hemagglutinins only differed from each other at three positions; Pro(19), Arg(31) and Phe(52) of hypnin A-1 as compared with Leu(19), Ser(31), and Tyr(52) of hypnin A-2. Approximately 43% of total residual numbers consisted of three kinds of amino acids: serine, glycine and proline. The hemagglutination activities were lost by reduction and alkylation of the disulfide bonds. The nature of the small-sized polypeptides, including disulfide bonds, may contribute to the extreme thermostability of the hemagglutinins. Sequence having overall similarity to hypnin A-1 or A-2 was not detected in databases. Unexpectedly, however, hypnins contained a motif similar to the alignment of the C-terminal conserved amino acids within carbohydrate-recognition domains of C-type animal lectins. Furthermore, interestingly, the hemagglutination activities were inhibited by a protein, phospholipase A-2 besides some glycoproteins, suggesting that hypnins may possess both a protein-recognition site(s) and a carbohydrate-recognition site(s). PMID- 10742604 TI - A versatile assay to study cellular uptake of gene transfer complexes by flow cytometry. AB - In this study, we present a simple and reliable method to analyse the first steps of DNA-based gene delivery into eucaryotic cells, i. e. binding and internalisation of transfection complexes. Taking advantage of flow cytometry, it is possible to discriminate quantitatively between total and internal DNA on a single-cell level. Here, we use two fluorescent dyes with high specificity and affinity to double-stranded DNA that cannot penetrate the extracellular membrane of living cells. Total DNA is stained prior to complexation with the first dye and complexes are added to cells. After the incubation, only extracellular DNA remains accessible to the second dye. Cell associated fluorescence is measured simultaneously using a flow cytometer and data are analysed using a computer program capable of calculating the ratio of fluorescence intensities on a single cell level. These ratios are indicative of the binding and internalisation kinetics of gene transfer complexes. PMID- 10742605 TI - Transfection using synthetic peptides: comparison of three DNA-compacting peptides and effect of centrifugation. AB - Positively charged peptides have been shown to allow efficient transfection in vitro, especially when mixed with lipids. We have compared the ability of three positively charged peptides both to compact DNA and to increase the transfection efficiency of the cationic lipid DOTAP. The peptides are: a polymer of 17 lysines (pK17), YKAWK8WK (peptide K8) and SPKRSPKRSPKR (peptide P2). Peptides pK17 and K8 compact DNA efficiently in a gel retardation assay and protect DNA efficiently against DNase I degradation. Peptide P2, on the other hand, interacts weakly with DNA and provides poor protection. In order to compare their transfection efficiency, the three peptides were mixed with DNA (plasmid pEGFP-N1) at different charge ratios (+/-) and DOTAP (at a charge ratio of 2). The transfection efficiency was measured by FACS analysis at different times post transfection. With NIH-3T3 cells, peptide P2 provides the highest transfection efficiency (about 40%), when compared with peptides pK17 (29%) and K8 (31%) and DOTAP alone (21%) under optimal conditions. Finally, we showed that centrifugation of the complexes onto the cells increased the transfection efficiency by a factor 1.5 to 2 with the various cell lines tested (ECV, primary human keratinocyte, CFT-2, NT-1). PMID- 10742606 TI - End-to-end distance distribution in bradykinin observed by Forster resonance energy transfer. AB - Forster resonance energy transfer (FRET) was used to study the conformational dynamics of bradykinin related peptides. The fluorescent probe aminobenzoic acid (Abz) bound to the amino terminal of bradykinin maintained its fluorescence characteristics, like high quantum yield and excited state decay dominated by a lifetime of 8.3 ns. The binding of the acceptor group N-[2, 4-dinitrophenyl] ethylenediamine (EDDnp) to the carboxy terminal of Abz labeled bradykinin resulted in a drastic decrease of the fluorescence intensity and in a fastening of the excited state decay. The change of the decay kinetics to an heterogeneous process, precludes the use of energy transfer models based on a single fixed distance between donor and acceptor. The computational package CONTIN was employed to the analysis of time-resolved fluorescence data, allowing the recovery of a distance distribution between donor and acceptor corresponding to the end-to-end distance of the labeled peptide. The distance distribution reflects the occurrence of distinct conformations for the peptide, that coexist in equilibrium during the fluorescence lifetime. We observed three distance populations for bradykinin in water, that merged to two populations when the solvent was trifluoroethanol (TFE). The results were consistent with those obtained from circular dichroism spectroscopy, that showed structural flexibility in water and the presence of more defined secondary structure in TFE. We also studied several peptides related to bradykinin, and the results emphasized the formation of turns involving the proline residues and the decrease of conformational flexibility induced by using TFE as the solvent. PMID- 10742607 TI - Identification of sialic acids on the cell surface of Candida albicans. AB - The cell-surface expression of sialic acids in two isolates of Candida albicans was analyzed by thin-layer and gas chromatography, binding of lectins, colorimetry, sialidase treatment and flow cytofluorimetry with fluorescein labeled lectins. N-acetylneuraminic acid (NANA) was the only derivative found in both strains of C. albicans grown in a chemically defined medium. Its identification was confirmed by mass spectrometry in comparison with an authentic standard. The density of sialic acid residues per cell ranged from 1. 6x10(6) to 2.8x10(6). The surface distribution of sialic acids over the entire C. albicans was inferred from labeling with fluorescein-Limulus polyphemus and Limax flavus agglutinins and directly observed by optical microscopy with (FITC)-Sambucus nigra agglutinin (SNA), abrogated by previous treatment of yeasts with bacterial sialidase. Sialidase-treated yeasts generated beta-galactopyranosyl terminal residues that reacted with peanut agglutinin. In C. albicans N-acetyl-neuraminic acids are alpha2,6- and alpha2,3-linked as indicated by yeast binding to SNA and Maackia amurensis agglutinin. The alpha2,6-linkage clearly predominated in both strains. We also investigated the contribution of sialic acids to the electronegativity of C. albicans, an important factor determining fungal interactions in vivo. Adhesion of yeast cells to a cationic solid phase substrate (poly-L-lysine) was mediated in part by sialic acids, since the number of adherent cells was significantly reduced after treatment with bacterial sialidase. The present evidence adds C. albicans to the list of pathogenic fungi that synthesize sialic acids, which contribute to the negative charge of fungal cells and have a role in their specific interaction with the host tissue. PMID- 10742608 TI - Dietary essential fatty acids, long-chain polyunsaturated fatty acids, and visual resolution acuity in healthy fullterm infants: a systematic review. AB - BACKGROUND: Biologically active neural tissue is rich in docosahexaenoic acid (DHA), an omega-3 long-chain polyunsaturated fatty acid (LCPUFA). We conducted a systematic review to examine the nature of discordant results from studies designed to test the hypothesis that dietary DHA leads to better performance on visually-based tasks in healthy, fullterm infants. We also conducted a meta analysis to derive combined estimates of behavioral- and electrophysiologic-based visual resolution acuity differences and sample sizes that would be useful in planning future research. STUDY DESIGN AND METHODS: Twelve empirical studies on LCPUFA intake during infancy and visual resolution acuity were identified through bibliographic searches, examination of monograph and review article reference lists, and written requests to researchers in the field. Works were reviewed for quality and completeness of information. Study design and conduct information was extracted with a standardized protocol. Acuity differences between groups consuming a source of DHA and groups consuming DHA-free diets were calculated as a common outcome from individual studies; this difference score was evaluated against a null value of zero and then used, with the method of DerSimonian and Laird (Meta-analysis in clinical trials. Control Clin Trials 1986;7:177-188), to derive combined estimates of visual resolution acuity differences within seven age categories. RESULTS OF RANDOMIZED COMPARISONS: The combined visual resolution acuity difference measured with behaviorally based methods between DHA supplemented formula fed groups and DHA-free formula fed groups is 0.32+/-0.09 octaves (combined difference+/-S.E.M., P=0.0003) at 2 months of age. The direction of this value indicates higher acuity in DHA-fed groups. RESULTS OF NON RANDOMIZED STUDY DESIGNS: The combined visual resolution acuity difference measured with behaviorally based methods between human milk fed groups and DHA free formula fed groups is 0.49+/-0.09 octaves (P< or =0.000001) at 2 months of age and 0.18+/-0.08 octaves (P=0.04) at 4 months of age. Acuity differences for electrophysiologic-based measures are also greater than zero at 4 months (0.37+/ 0.16 octaves, P=0.02). CONCLUSION: Some aspect of dietary n-3 intake is associated with performance on visual resolution acuity tasks at 2, and possibly, 4 months of age in healthy fullterm infants. Whether n-3 intake confers lasting advantage in the development of visually based processes is still in question. PMID- 10742609 TI - Baby, souls, name and health: traditional customs for a newborn infant among the Hmong in Melbourne. AB - In this paper, I discuss childrearing beliefs and practices in Hmong culture. In particular I focus on issues related to souls and ceremonies for a newborn infant in Hmong society. The Hmong believe that each living body has three souls. For a newborn infant, the first soul enters his or her body when he or she is conceived in the mother's womb. The second soul enters when the baby has just emerged from the mother's body and taken its first breath. The third soul, however, will have to be called on the third morning after birth, as will be discussed in this paper. If all three souls are secured in the infant's body, he or she will be healthy and hence thrive well. On the contrary, the infant may become ill and eventually die if all three souls do not reside in his or her body. This, therefore, makes a soul calling ceremony on the third morning after birth essential in Hmong culture. I will show that for Hmong society to survive, the Hmong strongly adhere to their cultural beliefs and practices related to a newborn infant. These beliefs and practices tie the Hmong with not only their family and their society at large, but also the supernatural world. PMID- 10742610 TI - Evidence of endothelium involvement in the pathophysiology of hydrops fetalis? AB - A fetus with signs of hydrops is at high risk of intrauterine death. The pathophysiology of hydrops fetalis is still unclear. We found decreased concentrations of cyclic guanosine monophosphate in fetal plasma of alloimmunized pregnancies complicated by hydrops fetalis, which might suggest reduced nitric oxide production due to injury of fetal vascular endothelial cells. PMID- 10742611 TI - Measurement of peripheral oxygen utilisation in neonates using near infrared spectroscopy: comparison between arterial and venous occlusion methods. AB - The aim of this study was to develop an arterial occlusion method and compare it with the venous occlusion method for measurement of peripheral oxygen utilisation in neonates using near infrared spectroscopy (NIRS). Twenty healthy neonates were studied. Arterial occlusion was produced by inflating a neonatal blood pressure cuff to 100 mmHg for 30-40 s and oxygen utilisation (VO(2)) was calculated using the HbO(2) decrement slope following occlusion. Venous occlusion was produced by inflating the cuff to 30 mmHg for 15-20 s and VO(2) was calculated by: VO(2)=HbTx4x(SaO(2)-SvO(2)), where SaO(2) is the arterial oxygen saturation measured by pulse oximetry and SvO(2) is the venous oxygen saturation measured by NIRS. Each baby had a minimum of three arterial and three venous occlusions. Criteria were developed for acceptance/rejection of an occlusion. Using the arterial method, the mean VO(2) was 1.12 mM cm(-1) O(2)/min (S.D.=0.25), (95% CI=1.00-1.24 mM cm(-1) O(2)/min). The coefficient of variation was 6.6+/-4.1%. Using the venous method, the mean VO(2) was 1.60 mM cm(-1) O(2)/min (S.D.=0.48), (95% CI=1. 38-1.82 mM cm(-1) O(2)/min). The coefficient of variation was 12. 6+/ 5.7%. The correlation between the two methods was weak (r=0.28 and r(2) was 0.08). The mean difference between the two methods was 0. 47 mM cm(-1) O(2)/min (S.D.=0.51). The limits of agreement were -0. 53 to 1.47 mM cm(-1) O(2)/min. The arterial method gives more consistent results. PMID- 10742612 TI - High frequency ventilation trial. Nine year follow up of lung function. AB - The high frequency ventilation (HIFI) trial for hyaline membrane disease (HMD) showed no advantage of high frequency over conventional ventilation in pulmonary outcomes after 24 months. The present study tested the hypothesis that there would be no significant difference in childhood lung function between patients who had been ventilated by either method. Thirty-two children aged 8-9 years who completed the HIFI trial were asked to return for pulmonary function tests. For purposes of analysis, the patient population was divided according to mode of ventilation, and by diagnosis of bronchopulmonary dysplasia (BPD) or HMD. Results were compared to those of 15 term-born, matched, controls. Lung function tests showed a mildly obstructive pattern in prematurely born children. More severe obstruction was seen in those children who had physician-diagnosed asthma or who had used bronchodilators in the past. The prevalence of mild obstructive pattern on pulmonary function testing in preterm infants with HMD or BPD was similar in those who received high frequency vs. conventional ventilation. Factors other than the mode of ventilation exert greater influence on pulmonary outcome in survivors of lung disease of prematurity. PMID- 10742614 TI - Neonatal society abstracts PMID- 10742613 TI - Abstracts of the papers presented at the neonatal society meeting 5 november 1999 PMID- 10742615 TI - Neonatal society abstracts PMID- 10742616 TI - Neonatal society abstracts PMID- 10742618 TI - Neonatal society abstracts PMID- 10742617 TI - Abstracts PMID- 10742619 TI - Neonatal society abstracts PMID- 10742620 TI - Neonatal society abstracts PMID- 10742621 TI - Dermatologic therapy for the 21st century PMID- 10742623 TI - Atopic dermatitis: unapproved treatments or indications. PMID- 10742622 TI - Pruritus: unapproved treatments or indications. PMID- 10742624 TI - Psoriasis, lichen planus, and disorders of keratinization: unapproved treatments or indications. PMID- 10742625 TI - Acne, perioral dermatitis, flushing, and rosacea: unapproved treatments or indications. PMID- 10742626 TI - Alopecia: unapproved treatments or indications. PMID- 10742627 TI - Collagen vascular diseases: unapproved treatments or indications. PMID- 10742628 TI - Bullous diseases: unapproved treatments or indications. PMID- 10742630 TI - Skin neoplasias including cutaneous lymphoma, melanoma, and others: unapproved treatments or indications. PMID- 10742629 TI - Oral disorders: unapproved treatments or indications. PMID- 10742632 TI - Products in dermatologic surgery: unapproved treatments or indications. PMID- 10742631 TI - Wound healing and leg ulcers: unapproved treatments or indications. PMID- 10742633 TI - Treatment services received by supplemental security income drug and alcoholic clients. AB - This study examined self reported problems and treatment services received by 237 recipients of Supplemental Security Income (SSI) benefits for "drug abusers and alcoholics" who had been assigned and entered into substance abuse treatment. All were administered the Treatment Services Review (TSR), a brief interview in which patients describe treatment services they have received during the past month and substance-related problems they are currently experiencing. In addition to describing serious alcohol and drug problems, SSI recipients reported a need for treatment for medical and psychiatric problems. The TSR data revealed that these clients primarily received drug and alcohol services and more limited medical, psychiatric, and employment services during treatment. There was relatively little indication of the provision of legal or family/social treatment services. The data are consistent with other findings that indicate that treatment for substance dependence provides only limited services other than those for alcohol and drug abuse. PMID- 10742634 TI - Effectiveness of communication and relationship skills training for men in substance abuse treatment. AB - Although the importance of gender-sensitive programming for women has been acknowledged, few studies have examined outcomes from male-centered interventions in substance abuse treatment programs. Data were collected from 122 male clients in a court-mandated residential treatment program who participated in a study of a psychoeducational group intervention for men. The intervention (Time Out! For Men) addressed communication skills, sexuality, gender socialization, and intimacy. Participation in the training resulted in significant increases in knowledge and social conformity, along with reductions in attitudes that may be associated with rigid socialization and gender-role conflict. The results provide support for the utility of male-targeted programming in substance abuse treatment settings. PMID- 10742635 TI - Measuring treatment process variables in Alcoholics Anonymous. AB - Alcoholism treatment research has traditionally focused on direct questions of efficacy, such as is a particular intervention better than no treatment or is one treatment more effective than another. Recent projects, however, have also attempted to identify variables explaining why treatments vary in their effects. Many of these variables relate to the process of treatment itself or changes that may occur within the patients. Clinicians also need to continuously monitor progress of patients in engaging in behaviors supportive of long-term sobriety and how well the values and behaviors fostered by the particular treatment regimen are being incorporated into daily life. Measurement of process variables may assist in both regards. In the last decade several psychometric instruments have been developed to elucidate the processes involved in Alcoholic Anonymous (AA), a key adjunct of most formal alcoholism programs in the United States. These instruments measure dimensions such as involvement in AA, completion of steps, and adoption of values encouraged by AA. Six such measures are summarized here and several fruitful topics for future research on the measures are suggested. PMID- 10742637 TI - Factors associated with level of care assignment in substance abuse treatment. AB - Managed-care approaches to controlling expenditures for publicly funded substance abuse treatment have proliferated in the past decade. As many as 40 states now have some type of utilization review mechanism. Many have adopted the American Society on Addiction Medicine placement criteria to assign persons to a level of care. This study examined the use of those criteria with 3,000 consecutive admissions to publicly funded treatment in Kansas and identified variables that predicted level of care assignment. For the most part, the placement domains predicted level of care as expected. However, even when controlling for the contribution of the placement domains, housing status and employment were among the best predictors of placement. These findings suggest a need for criteria that address social service as well as clinical concerns when working with vulnerable populations. PMID- 10742636 TI - Women's steps of change and entry into drug abuse treatment. A multidimensional stages of change model. AB - The Transtheoretical, or Stages of Change Model, has been applied to the investigation of help-seeking related to a number of addictive behaviors. Overall, the model has shown to be very important in understanding the process of help-seeking. However, substance abuse rarely exists in isolation from other health, mental health, and social problems. The present work extends the original Stages of Change Model by proposing "Steps of Change" as they relate to entry into substance abuse treatment programs for women. Readiness to make life changes in four domains-domestic violence, HIV sexual risk behavior, substance abuse, and mental health-is examined in relation to entry into four substance abuse treatment modalities (12-step, detoxification, outpatient, and residential). The Steps of Change Model hypothesizes that help-seeking behavior of substance abusing women may reflect a hierarchy of readiness based on the immediacy, or time urgency, of their treatment issues. For example, women in battering relationships may be ready to make changes to reduce their exposure to violence before admitting readiness to seek substance abuse treatment. The Steps of Change Model was examined in a sample of 451 women contacted through a substance abuse treatment-readiness program in Los Angeles, California. A series of logistic regression analyses predict entry into four separate treatment modalities that vary. Results suggest a multidimensional Stages of Change Model that may extend to other populations and to other types of help-seeking behaviors. PMID- 10742638 TI - Behavioral couples therapy versus individual-based treatment for male substance abusing patients. An evaluation of significant individual change and comparison of improvement rates. AB - Fals-Stewart, Birchler, and O'Farrell (1996) found that married or cohabiting substance-abusing men (n = 40) who participated in behavioral couples therapy (BCT) in addition to individual-based treatment (IBT) for substance abuse had fewer days of substance use and, along with their partners, reported higher levels of dyadic adjustment during and 1-year after treatment than husbands who received IBT only (n = 40). In the present study, significant individual change in posttreatment frequency of substance use and dyadic adjustment was evaluated and comparisons of the proportions of participants receiving IBT and BCT who were improved, unchanged, or deteriorated in these domains of functioning were made using data from Fals-Stewart et al. (1996). Growth curve analysis revealed that a larger proportion of husbands in the BCT condition showed significant reductions in substance use (n = 33, 83%) than husbands who received IBT (n = 24, 60%). Also, a larger proportion of couples who participated in BCT showed improvements in dyadic adjustment (n = 24, 60%) than couples whose husbands received IBT only (n = 14, 35%). PMID- 10742639 TI - Community reinforcement approach for combined opioid and cocaine dependence. Patterns of engagement in alternate activities. AB - We compared outcomes for agonist-maintained patients with combined opioid and cocaine dependence who were treated in an earlier clinical trial with group drug counseling (DC; n = 57) or in a current trial with the Community Reinforcement Approach (CRA; n = 60). The association between engagement in nondrug-related activities and abstinence was also evaluated. There were no significant differences between the treatments in retention or drug use. The total number of hours and average hours per week engaged in nondrug-related activities was significantly higher for CRA-treated patients who achieved abstinence from opioids, cocaine, or both combined than for those who never achieved abstinence. Although CRA was not more effective overall than DC, the finding that engagement in reinforcing community activities unrelated to drug use (e.g., planned pleasurable events or parenting activities) was associated with abstinence suggests that the planning and reinforcement of specific nondrug-related social, vocational, and recreational activities is a crucial component of CRA. PMID- 10742640 TI - Provision of influenza and pneumococcal vaccines to injection drug users at a syringe exchange. AB - The objective of this study was to evaluate the feasibility, acceptance, and utility of administering influenza and pneumococcal vaccines to active injection drug users at a syringe exchange program (SEP) in New York City. Influenza and pneumococcal vaccines were offered for 1 month. Data on demographics, health status, vaccine awareness, and prior vaccination status were collected using a staff-administered questionnaire. Of 199 participants interviewed 167 (86%) agreed to one or both vaccinations; 24% of study participants had a chronic condition for which vaccination was indicated and 53% had no regular source of medical care; 95% were aware of influenza vaccine while 25% were aware of pneumococcal vaccine (p <.0001). Of those offered the influenza vaccine, 86% accepted it and 70% of those offered pneumococcal vaccine accepted it (p <.001). Influenza and pneumococcal vaccinations were well-accepted by active drug users at a syringe exchange although there was both greater awareness of and acceptance of influenza. Many SEP participants with chronic medical conditions for which these vaccines are indicated did not have a regular source of health care. Syringe exchange programs may be valuable sites to administer respiratory vaccines and other public health interventions to drug injectors not engaged in medical care in other settings. PMID- 10742641 TI - Family risk factors versus peer risk factors for drug abuse. A longitudinal study of an African American urban community sample. AB - This study compared the influence of family problems with influence of deviant and delinquent social behavior and peer relationships up to the time of the 16th birthday as risk factors for substance use, for lifetime up to age 26. Control variables for the analysis were available from the National Collaborative project's longitudinal data file, collected from time of birth, on the African American community study sample (N = 380). A key finding was that the social behavior and peer relationship problems accounted for 18.8% of the additional variance in later degree of substance use, whereas the family problems accounted for only 5.1% of the additional variance in later degree of substance use. PMID- 10742642 TI - An open-label study of a functional opioid kappa antagonist in the treatment of opioid dependence. AB - Several lines of evidence, including the well-established observation that kappa opiate agonists produce dysphoria and psychotomimetic effects in humans, suggest that dysfunction of the endogenous kappa opioid system may contribute to opioid and cocaine addiction. The objective of this open-label study was to determine the effectiveness of a functional kappa antagonist as a treatment for opioid dependence. This was accomplished by combining a partial mu agonist/kappa antagonist (buprenorphine, 4 mg, sublingual) with a mu antagonist (naltrexone, 50 mg by mouth), theoretically leaving kappa antagonism as the major medication effect. Subjects were treatment-seeking heroin-dependent (as per Diagnostic and Statistical Manual of Mental Disorders, 4th ed.) men (41 +/- 7 years old; 19 +/- 8 years heroin use) eligible for methadone maintenance. After inpatient detoxification and a naloxone-challenge test to verify that they were not physically dependent on opioids, subjects received naltrexone. Starting on the fourth day, patients also received liquid buprenorphine. All patients received medication at the clinic 6 days per week and a full program of psychosocial treatment. The major endpoints of the study were: pupil diameter to determine if the mu agonist effects of buprenorphine were blocked by naltrexone, urine toxicology, and retention in treatment. Five patients (33%) completed the 3-month study. Four were abstinent from opioids and cocaine for the entire study, and one was abstinent from opioids and cocaine for the last 9 weeks. Six subjects dropped out due to either minor side effects or disliking the sensation of sublingual buprenorphine. There were no significant changes in pupillary diameter. The positive response to treatment exceeds that expected from naltrexone alone (90% dropout). These promising results suggest that controlled studies of this medication combination should be conducted. PMID- 10742643 TI - Ohio's Bill 167 fails to increase prenatal referrals for substance abuse. PMID- 10742644 TI - Health status of employed and unemployed methadone patients. PMID- 10742645 TI - Intentions to quit smoking in substance-abusing teens exposed to a tobacco program. AB - In 1993-94, fifty-five clients at an adolescent residential drug treatment facility with an innovative tobacco prevention, education and cessation program reported their tobacco attitudes, intentions, and behavior at admission and discharge. Of entering clients, 93% were current smokers and 93% felt the facility should help clients quit smoking. Clients interested in quitting increased from 61% at admission to 87% at discharge, as measured by the precontemplation/contemplation ladder of Rustin and Tate (1993). Clients who wanted to immediately quit smoking increased from 15% to 29%. Sixty-five percent of the teens studied said the tobacco-free activities were extremely helpful. At discharge, 16% of the smokers reported having quit tobacco and all four nonsmokers remained smoke-free. During the preceding year there was a naturally occurring quit rate of 1%. As a result of this work, the facility required residential clients to be nicotine-free as of July 1996. PMID- 10742646 TI - Increased morbid risk for schizophrenia in families of in-patients with bipolar illness. AB - BACKGROUND: It has been reported that relatives of probands with severe, psychotic forms of bipolar illness have increased rates of schizophrenia but not the relatives of individuals with milder, non-psychotic forms of disorder. In this study, we examined the prevalence of psychiatric disorders in the first degree relatives of a sample of 103 inpatients with bipolar disorder and in a matched control sample of 84 healthy individuals. METHOD: Relatives of cases and controls were interviewed using the FH-RDC to determine familial morbid risk for schizophrenia and bipolar disorder. Age- and sex-adjusted morbidity risks were calculated in both samples according to the method of Stromgren. RESULTS: The morbid risks for both bipolar disorder (4.9%) and schizophrenia (2.8%) were higher in relatives of patients than in relatives of controls (0.3% and 0.6% respectively). The relative risks were 14.2 [95% confidence interval (CI)=3.1 64.2] for bipolar disorder and 4.9 (95% CI=1.3-18.8) for schizophrenia. Relatives of women with early onset of bipolar illness had the highest morbid risks for both bipolar illness and schizophrenia. The presence of more than one patient with bipolar disorder in a family increased the risk for schizophrenia nearly fourfold (RR=3.5, 95% CI=1.2-10.2). There was no additional effect of presence of psychotic features. CONCLUSION: Our results suggest that the transmission of psychosis is not disorder-specific. Bipolar illness characterised by a high familial loading is associated with increased risk of schizophrenia in the relatives. PMID- 10742647 TI - Can we improve the diagnostic efficiency and predictive power of prodromal symptoms for schizophrenia? AB - Prodromal symptoms and other variables for a sample of 200 young people who had experienced a first-onset functional psychosis, were analyzed to examine their diagnostic efficiency and predictive power in relation to a diagnosis of schizophrenia. Two different techniques were utilized to generate optimal cut-off points for a number of prodromal symptoms, and optimal decision rules to maximize diagnostic efficiency. The product of the chance-corrected sensitivity and specificity, or the area under the QROC curve, was used to assess the predictive efficiency of a number of prodromal variables, DSM-III-R prodromal variables, pre psychotic deterioration, pre-morbid functioning, and prodromal duration. The SPAN technique generated a decision rule that performed equivalently to the single variable 'duration of prodrome'. Implications of these results for future research are discussed. PMID- 10742648 TI - Auditory working memory and verbal recall memory in schizotypy. AB - Deficits on verbal memory tasks, as well as on spatial and auditory working memory tasks, have been observed in schizophrenia patients. A useful strategy in the determination of the premorbid indicator status of specific cognitive and memory deficits observed in patients is to examine those persons at increased biological risk for schizophrenia (e.g. first-degree relatives), schizotypal personality disorder patients, and/or psychometrically identified schizotypes for comparable deficits, though perhaps less profound than those seen in actual patients. We examined verbal memory and auditory working memory functioning in 31 schizotypic and 26 normal control subjects from a large randomly ascertained non clinical university population. Schizotypy status was determined psychometrically using the well-known Perceptual Aberration Scale. Contrary to our theory-guided expectations, noteworthy deficits in verbal memory and auditory working memory were not observed in the schizotypic subjects and the two experimental groups did not differ significantly on any of the memory measures. These results were discussed in light of prior results obtained using the spatial delayed response task (i.e. spatial working memory) and Wisconsin Card Sorting Test performance on these same subjects. The theoretical implications of these findings are discussed in relation to the putative processes involved in the working memory system, as well as in relation to the schizotypy construct. PMID- 10742649 TI - Two-point discrimination thresholds and schizotypy: illuminating a somatosensory dysfunction. AB - The current study sought to determine if somatosensory deviations, assessed by determining two-point discrimination thresholds, were associated with schizotypic features in non-psychotic subjects (N=100). It was hypothesized that elevated two point discrimination thresholds would be associated with an increased likelihood of higher levels of schizotypy features. The results of the study suggested that at the level of individual differences, higher two-point discrimination thresholds (ascending series assessments) were associated with higher levels of schizotypy as operationalized by several prominent measures of schizotypy. A deviant subgroup analysis clearly suggested that, for ascending series two-point threshold assessments, those persons requiring the greatest distance (i.e. highest thresholds) to detect two-point stimulation were substantially more schizotypic than a contrast group. Control analyses that focused on psychological state (depression, anxiety) and intellectual functioning variables revealed that these factors were, more or less, unrelated to two-point discrimination thresholds and they did not account for the observed significant relations between the schizotypy scales and two-point threshold values. The results of this study are discussed within a neuropsychological context implicating parietal cortex involvement with schizotypy and schizophrenia. PMID- 10742650 TI - Executive functions in adolescents with schizotypal personality disorder. AB - Adolescents meeting diagnostic criteria for schizotypal personality disorder (SPD) are presumed to be at risk for developing schizophrenia in adulthood, making them an important group for exploring the developmental trajectory of the disease. Deficits in executive functioning have been documented in schizophrenia patients and adults with SPD. The present study examined executive functions in adolescents with SPD. It was predicted that the SPD group would score below comparison groups (normals and adolescents with other disorders) on measures of executive function, and that those with greater 'negative' signs of SPD would show more pronounced performance deficits. Analyses revealed that the performance of the SPD subjects was impaired relative to the other groups on the modified Wisconsin Card Sorting Test (MCST), but not on the Tower of London or the Controlled Oral Word Association Test. Consistent with prediction, regression analyses indicated that MCST deficits were associated with greater negative signs of SPD, but not positive signs. PMID- 10742651 TI - Neuropsychological deficits in pediatric patients with childhood-onset schizophrenia and psychotic disorder not otherwise specified. AB - OBJECTIVE: Children with transient psychotic symptoms and serious emotional disturbances who do not meet current criteria for schizophrenia or other presently recognized diagnostic categories commonly present diagnostic and treatment problems. Clarifying the connections between children with narrowly defined schizophrenia and children with a more broadly defined phenotype (i.e., Psychotic Disorder Not Otherwise Specified, PD-NOS) has implications for understanding the pathophysiology of schizophrenia. In this study, the neuropsychological test performance of a subgroup of children with atypical psychosis was compared with that of patients with childhood-onset schizophrenia (COS). METHOD: Cognitive function was assessed with neuropsychological test battery regimens in 51 neuroleptic-nonresponsive patients within the first 270 at NIMH testing (24 PD-NOS, 27 COS) were included in this analysis. Seventeen (39%) of 44 COS subjects were unavailable for this study as their IQ tested <70. The PD NOS patients were younger than the COS patients at the time of testing (12.0+/ 2.8 vs 14.4+/-1.8years, respectively, p<0.004). The test levels of these groups were compared with each other. RESULTS: The neuropsychological test results for the PD-NOS and COS patients were 1-2standard deviations below normative data across a broad array of cognitive functions. There were no overall differences in the test levels for the six summary scales (F=2.82, df=1, 36, p=0.10) or in the profile shape (F=1.70, df=5, 180, p=0.14) between the PD-NOS and COS groups. For the COS patients, there was a significant difference between their mean full scale WISC IQ (84.7+/-16.2) and their average standard scores for both the spelling (97.7+/-16.1, n=23, t=4.0, p=0.001) and reading decoding subtests (97.7+/-13.7, n=23, t=3.7, p=0.001) of the Kaufman Test of Educational Achievement. CONCLUSIONS: Treatment-refractory PD-NOS and COS patients share a similar pattern of generalized cognitive deficits, including deficits in attention, learning and abstraction which are commonly observed in adult patients with schizophrenia. These data support a hypothesis that at least some of the PD NOS cases belong within the schizophrenic spectrum, which is of importance for future genetic studies planned for this cohort. PMID- 10742652 TI - Theory of mind abilities of children with schizophrenia, children with autism, and normally developing children. AB - Theory of mind (ToM) abilities of children with schizophrenia, children with high functioning autism, and normally developing children, matched on mental age (MA), verbal MA, and performance MA, were compared. Both clinical groups were matched on chronological age as well, whereas the normally developing children were younger. A fact belief task, a value belief task, a deception task, and a false belief task were administered. The three groups did not differ on the fact belief task. Children with autism performed more poorly than normally developing children on value belief and false belief tasks, and more poorly than individuals with schizophrenia on the deception task. Children with schizophrenia performed more poorly than normally developing children only on the false belief task. Overall, the group with autism passed significantly fewer tasks compared to the normally developing group. ToM abilities correlated with verbal abilities for individuals with autism. The ToM abilities of children with paranoid schizophrenia and children with undifferentiated or disorganized schizophrenia did not differ. Findings strengthen the notion of a limited understanding of ToM in schizophrenia, and support the notion that ToM deficits, although more severe in autism, are not unique to autism. PMID- 10742653 TI - Immunosuppressive effects of clozapine and haloperidol: enhanced production of the interleukin-1 receptor antagonist. AB - In schizophrenic patients, multiple immune abnormalities have been reported, including increased production of proinflammatory cytokines. There is some evidence that antipsychotic drugs may have immunosuppressive effects. The aim of this study was to examine the in-vitro effects of different concentrations of antipsychotic agents on cytokine production by human whole blood. We examined the effects of clozapine and haloperidol, 10(-4), 10(-6) and 10(-8)M, on the unstimulated and stimulated (lipopolysaccharide+phytohemagglutinin) production of interleukin-6 (IL-6), IL-10, interferon-gamma (IFNgamma), and the IL-1 receptor antagonist (IL-1RA). Clozapine, 10(-6) and 10(-8)M, and haloperidol, 10(-4), 10( 6), and 10(-8)M, significantly increased the unstimulated and stimulated production of IL-1RA. Clozapine 10(-6)M significantly increased the stimulated production of IFNgamma. Clozapine 10(-4)M significantly suppressed the unstimulated production of IL-6 and IL-1RA and the stimulated production of IL-6, IL-10, IFNgamma and IL-1RA. The results suggest that both clozapine and haloperidol, at concentrations within the therapeutic range, may exert immunosuppressive effects through an enhanced production of IL-1RA. PMID- 10742654 TI - Is the neuropeptide urocortin, a member of the corticotropin-releasing factor family, involved in schizophrenia? PMID- 10742655 TI - Antioxidant lipoate and tissue antioxidants in aged rats. AB - Oxidative metabolism produces free radicals that must be removed from the cellular environment for the cell to survive. The levels of nonenzymic antioxidants involved in the elimination of free radicals were investigated in an attempt to correlate any changes in the levels of enzymic antioxidants during aging with changes in free radical mediated cellular damage. Antioxidants were measured in liver and kidney of young and aged rats with respect to DL-alpha lipoic acid supplemented rats. In both organs lipid peroxidation damage (a marker of free radical mediated damage) increased with age, and a significant decrease in antioxidant systems was observed. Moreover, DL-alpha-lipoic acid treated aged rats showed a decrease in the level of lipid peroxides and an increase in the antioxidant status. The results of this study provide evidence that DL-alpha lipoic acid treatment can improve antioxidants during aging and minimize the age associated disorders in which free radicals are the major cause. PMID- 10742656 TI - Isolation and characterization of free radical scavenging activities peptides derived from casein. AB - A peptide having the strong free radical scavenging activities was separated from casein protein hydrolysate by chromatographic analyses such as ion-exchange and gel filtration. SP-II fraction obtained by SP-Sephadex C-25 chromatography showed the most potent superoxide anion scavenging activity (SOSA), and it was further separated into a peptide using an octadecylsilano-high performance liquid chromatography. The amino acid sequence of the peptide was Tyr-Phe-Tyr-Pro-Glu Leu (YFYPEL). The concentration of the test compound required to reduce the produced superoxide anion to one-half (IC(50)) value for SOSA was 79.2 microM using tetrazolium salt 3'-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis(4 methoxy-6-nitro)benzenesulfonic acid hydrate method. The IC50 value for the 1,1 diphenyl-2-picrylhydrazyl radical and hydroxyl radical scavenging activities were 98 and 251 microM, respectively, based on the electron spin resonance method. We characterized SOSA of the C-terminal sequence using EL, PEL, YPEL, and FYPEL. The activities preferred sequences were EL>YFYPEL>FYPEL>YPEL>PEL, suggesting that the Glu-Leu sequence is important for the activity. PMID- 10742657 TI - Zinc deficiency and the activities of lipoprotein lipase in plasma and tissues of rats force-fed diets with coconut oil or fish oil. AB - The present study was performed to investigate the effect of zinc deficiency on the activities of lipoprotein lipase in postheparin serum and tissues of rats fed diets containing either coconut oil or fish oil as dietary fat, using a bifactorial experimental design. To ensure an adequate food intake, all the rats were force-fed by gastric tube. Experimental diets contained either 0.8 mg zinc/kg (zinc-deficient diets) or 40 mg zinc/kg (zinc-adequate diets). The effects of zinc deficiency on the activities of lipoprotein lipase in postheparin serum and postprandial triglyceride concentrations and distribution of apolipoproteins in serum lipoproteins depended on the type of dietary fat. Zinc deficient rats fed the coconut oil diet exhibited a reduced activity of lipoprotein lipase in postheparin serum and adipose tissue, markedly increased concentrations of triglycerides in serum, and a markedly reduced content of apolipoprotein C in triglyceride-rich lipoproteins and high density lipoproteins compared with zinc-adequate rats fed coconut oil. By contrast, zinc-deficient rats fed the fish oil diet did not exhibit reduced activities of lipoprotein lipase in postheparin serum and adipose tissue and increased concentrations of serum lipids compared with zinc-adequate rats fed the fish oil diet. This study suggests that a reduced activity of lipoprotein lipase might contribute to increased postprandial concentrations of serum triglycerides observed in zinc deficient animals. However, it also demonstrates that the effects of zinc deficiency on lipoprotein metabolism are influenced by dietary fatty acids. PMID- 10742658 TI - Nutritional deprivation reduces the transcripts for transcription factors and adipocyte-characteristic proteins in porcine adipocytes. AB - For an organism to survive during nutritional deprivation, it must be able to regulate the genes involved in energy metabolism. White adipose tissue is an energy source during fasting conditions. In adipose tissue, transcription factors regulate several adipocyte-characteristic proteins involved in differentiation and energy metabolism. We investigated the transcript concentrations of two key transcription factors, as well as the transcript concentrations of several adipocyte-characteristic proteins, and genes involved in adipocyte energy metabolism in the adipose tissue of pigs fasted for 72 hours. Nutritional deprivation resulted in decreased transcript concentrations of the transcription factors, peroxisome proliferator-activated receptor gamma, and CCAAT-enhancer binding protein alpha. The transcript concentrations of several adipocyte characteristic proteins, fatty acid synthase, glucose transporter 4, lipoprotein lipase, leptin, and adipocyte fatty acid binding protein were also significantly reduced. The insulin receptor transcript concentration did not change. We conclude that these transcript concentration changes are aimed collectively at adjusting energy partitioning to conserve energy during nutritional deprivation, thereby enabling survival. PMID- 10742659 TI - Digestion of plant monogalactosyldiacylglycerol and digalactosyldiacylglycerol in rat alimentary canal. AB - We investigated digestion of orally fed galactoglycerolipids such as monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) from wheat flour in the rat alimentary canal, especially focusing on the digestive fates of deacylated galactosylglycerol structures. After a single oral administration of MGDG (20 mg/rat), monogalactosylmonoacylglycerol and monogalactosylglycerol (MGG) were found to be major digestion products in the intestinal tract. Similarly, digalactosylmonoacylglycerol and digalactosylglycerol (DGG) were confirmed to be present in the intestinal tract after DGDG ingestion (20 mg/rat). In rats fed wheat flour glycolipids (42 mg MGDG and 81 mg DGDG per rat), completely deacylated galactosylglycerols (MGG and DGG) were not detected in portal plasma. Although the deacylated galactosylglycerols were not significantly decomposed by intestinal mucosa in vitro, they were hydrolyzed by cecal contents. The results demonstrated that orally ingested plant galactoglycerolipids in the rat alimentary canal are rapidly hydrolyzed into constituent fatty acids and that hydrophilic galactosylglycerols and the hydrophilic backbone galactosylglycerols are not absorbed from intestine or degraded into galactose and glycerol in the intestinal tract. Therefore, the presence of deacylated galactosylglycerols may affect the fermentative activity of enterobacteria in the cecum and colon. PMID- 10742660 TI - Effect of sterols and fatty acids on growth and triglyceride accumulation in 3T3 L1 cells. AB - Epidemiologic studies suggest a role of dietary fat in the development of obesity. Populations that consume Western diets have a higher incidence of obesity than do those that consume a vegetarian type diet such as Asians. Because dietary fats are made up mostly of triglyceride with minor lipids such as sterols, the objective of this study was to examine the effect of different fatty acids, the main component of triglycerides, and sterols on cell growth and triglyceride accumulation in 3T3-L1 cells. These cells are being used as an in vitro model for studying obesity because upon differentiation in culture they accumulate triglycerides. Cells were seeded at 5,000 cells/cm(2) and supplemented with 0, 3, 10, or 30 microM of oleic acid, elaidic acid, or docosahexaenoic acid (DHA). Similarly, cells were supplemented with 0, 2, 8, or 16 microM of cholesterol, beta-sitosterol (SIT), or campesterol. Cell growth was measured by cell counting. Cellular triglycerides were measured by the Oil Red O method. In some experiments, fatty acids were combined with sterols and growth and triglyceride content were assessed as described. Both DHA and SIT had inhibitory effects on 3T3-L1 cell growth. However, SIT was more potent than DHA in this regard. The combination of SIT and oleic acid was the most potent in inhibiting cell growth and increasing cellular triglyceride content. It is concluded that cell growth and triglyceride accumulation in 3T3-L1 cells is influenced by fatty acid and sterols. When used alone, DHA and SIT inhibit cell growth. SIT was more effective in this process than was DHA. There was an interaction between fatty acids and sterols. The most effective combination inhibiting cell growth and triglyceride concentration was the combination of SIT and oleic acid. This combination reduced cell growth and increased triglyceride accumulation. These data suggest that diets rich in both monounsaturated fatty acids and phytosterols may play a role in controlling obesity. PMID- 10742661 TI - Ibotenic acid-induced lesions of the medial septum increase hippocampal membrane associated protein kinase c activity and reduce acetylcholine synthesis: prevention by a phosphatidylcholine/vitamin B12 diet. AB - Ibotenic acid infusion into the medial septum (MS) results in biochemical alterations in the hippocampus. The biochemical events involved in this neuronal lesion are poorly understood. We investigated the effect of a purified diet supplemented with egg phosphatidylcholine (PC) and vitamin B(12) on ibotenic acid medicated biochemical changes in the rat hippocampus and crude synaptosomal membranes. Male Wistar rats with this MS lesion were fed a purified diet (control diet) or a purified diet supplemented with 5.7 g PC and 125 microg vitamin B(12) per 100 g (experimental diet) for 18 days. Sham-operated rats were fed the control diet. Compared with the sham-operated rats, MS-lesioned rats fed the control diet showed increased activity of membrane-bound protein kinase C (PKC), decreased activity of choline acetyltransferase, and decreased concentrations of acetylcholine in the hippocampus. The ratio of cholesterol to phospholipid in the crude synaptic membrane was lower in the lesioned rats than in the sham-operated rats, but this was not accompanied by any alteration in membrane lipid fluidity. MS-lesioned rats fed the experimental diet showed lowered PKC activity and elevated acetylcholine concentrations than did rats fed the control diet, but there were no significant effects on choline acetyltransferase activity and the lipid ratio. The ibotenic acid-mediated elevation of PKC activity was observed as early as 2 days postinjury in the control diet-fed rats but not in the experimental diet-fed rats. We propose that ibotenic acid mediates pathophysiologic actions through the activation of PKC and that PC combined with vitamin B(12) ameliorates the second messenger-mediated injury. PMID- 10742662 TI - Effects of dietary zinc deficiency on homocysteine and folate metabolism in rats. AB - In rats, zinc deficiency has been reported to result in elevated hepatic methionine synthase activity and alterations in folate metabolism. We investigated the effect of zinc deficiency on plasma homocysteine concentrations and the distribution of hepatic folates. Weanling male rats were fed ad libitum a zinc-sufficient control diet (382.0 nmol zinc/g diet), a low-zinc diet (7.5 nmol zinc/g diet), or a control diet pair-fed to the intake of the zinc-deficient rats. After 6 weeks, the body weights of the zinc-deficient and pair-fed control groups were lower than those of controls, and plasma zinc concentrations were lowest in the zinc-deficient group. Plasma homocysteine concentrations in the zinc-deficient group (2.3 +/- 0.2 micromol/L) were significantly lower than those in the ad libitum-fed and pair-fed control groups (6.7 +/- 0.5 and 3.2 +/- 0.4 micromol/L, respectively). Hepatic methionine synthase activity in the zinc deficient group was higher than in the other two groups. Low mean percentage of 5 methyltetrahydrofolate in total hepatic folates and low plasma folate concentration were observed in the zinc-deficient group compared with the ad libitum-fed and pair-fed control groups. The reduced plasma homocysteine and folate concentrations and reduced percentage of hepatic 5-methyltetrahydrofolate are probably secondary to the increased activity of hepatic methionine synthase in zinc deficiency. PMID- 10742663 TI - Impaired glucose tolerance in vitamin D deficiency can be corrected by calcium. AB - Vitamin D(3), via its active metabolite 1alpha,25-dihydroxyvitamin D(3), helps maintain normal calcium levels in the body. Apart from the maintenance of calcium homeostasis, the active form of vitamin D(3) is now known to be involved in a number of other functions including that of pancreatic beta cells. Low serum insulin levels and impaired glucose tolerance in a vitamin D-deficient state have been reported in experimental animals. Hypocalcemia is a major consequence of vitamin D deficiency. Whether the impairment observed is due to vitamin D deficiency per se or is secondary to low calcium is still a matter of controversy. The present study was conducted to delineate the roles of vitamin D and calcium in glucose intolerance associated with vitamin D deficiency in vivo. It was found that supplementation with either vitamin D(3) or high calcium alone to vitamin D-deficient rats could correct the defects. In addition, insulin sensitivity was found to be enhanced in the vitamin D-deficient group compared with vitamin D control or calcium-supplemented groups. Hence the present study demonstrates that calcium per se in the absence of vitamin D increases insulin secretion and normalizes intolerance to glucose seen in vitamin D deficiency. PMID- 10742664 TI - Dietary oxidized linoleic acid enhances liver cholesterol biosynthesis and secretion in rats. AB - Based on studies showing that excretion of cholesterol is elevated in rats fed oxidized linoleic acid, we hypothesized that cholesterol metabolism is enhanced under such oxidative stress. Liver cholesterol biosynthesis and secretion and fecal cholesterol excretion were studied in rats fed for 4 weeks diets containing 10% oxidized linoleic acid. Incubation of liver slices with 1-(14)C acetate and intraperitoneal injection of 5-(3)H-mevalonate showed the occurrence of enhanced hepatic cholesterol biosynthesis and elevated liver cholesterol secretion in animals subjected to oxidative stress. In addition, impaired liver cholesterol uptake was suggested. Higher levels of excreted cholesterol observed in the experimental animals were accompanied by augmented levels of liver phospholipids, primarily phosphatidylcholine, which most likely increased to enable the excessive cholesterol excretion. This study thus demonstrates that ingestion of oxidized lipids causes profound alterations in cholesterol metabolism. PMID- 10742665 TI - Pediatric adolescent gynecologists--is there a union in your future? PMID- 10742666 TI - Impact of adolescent pregnancy as we approach the new millennium. AB - Between 1990 and 1997 teen pregnancy and birth rates have fallen dramatically, especially in black teens. However, the teen birth rate in the United States remains the highest of any industrialized nation: four times higher than Germany, six times higher than France. Contraception use has also increased. Condoms were used during last intercourse approximately 55% of the time in 1997 vs. only approximately 38% in 1991. Likewise, the percentage of teens having intercourse has dropped, and the number of abortions is down. The negative social and economic impact of early teenage pregnancy is tremendous. It is estimated to cost the nation about $21 billion annually (in 1993 dollars). The long-term productive life prospects are also lower for teenage mothers and their offspring. Many programs have been utilized to reduce the incidence of teen pregnancy. While some have had modest success, no single or simple solution is on the horizon. PMID- 10742667 TI - Epilepsy in adolescents: hormonal considerations. AB - A correlation between seizure disorders and reproductive hormones has been recognized for centuries. Through basic scientific and clinical studies, an understanding of the interrelationships has become clearer. Puberty does not increase the incidence of seizures. Catamenial seizures, when diagnosed, may be improved with hormonal manipulation. Therapeutic regimens for those treated unsuccessfully with antiepileptic therapy need continued validation. Patients with seizure disorders have an increased incidence of menstrual disorders. Hormonal contraception is not contraindicated in this population, yet requires a more cautious approach to avoid failure. PMID- 10742668 TI - Adolescents' knowledge of human papillomavirus and cervical dysplasia. AB - STUDY OBJECTIVE: This study examined adolescents' knowledge of human papillomavirus (HPV) and cervical dysplasia (CD). Factors associated with knowledge and self-reported change in health-related behaviors were identified. DESIGN: Interviews were conducted at an average of 2.5 years following the diagnosis of HPV/CD. Medical charts were reviewed. SETTING: The study was conducted at a university-based adolescent dysplasia clinic. PARTICIPANTS: Fifty females, ages 15-23 participated in the study: 88% African-American, 12% Caucasian. RESULTS: On average, participants responded correctly to 86% of the questions regarding HPV/CD. However, the following key points were routinely missed: 52% did not know cigarette smoking increased the risk for cervical cancer; 42% believed that HPV/CD was always symptomatic; and 22% did not know condoms decreased the transmission of HPV. According to participants, their health care provider explained the diagnosis and treatment of HPV/CD using words they understood "some" or "most of the time." Higher academic skills significantly correlated with greater knowledge of HPV/CD. Forty-one percent of participants with a smoking history reportedly increased their smoking since the diagnosis, and only 40% used condoms "most of the time." However, 90% had maintained or increased their frequency of Pap tests. CONCLUSIONS: Adolescent girls had knowledge of most factors related to HPV/CD, but many did not understand the risks of cigarette smoking and failure to use condoms. To improve understanding and compliance, health care providers should tailor educational strategies to the functional level of adolescents. PMID- 10742670 TI - Laparoscopic gonadectomy in a patient with testicular feminization syndrome. AB - A laparoscopic technique is reported as an operative approach for gonadectomy in a case of testicular feminization syndrome. A 17-year-old phenotypic female presented with primary amenorrhea and was evaluated through a diagnostic protocol that included clinical, cytogenetic, and hormonal examinations. The findings supported the diagnosis of testicular feminization syndrome. The left gonad was found at the internal opening of the inguinal canal and removed laparoscopically. No gonad was found at the contralateral side. No complications occurred during the operation and the patient left the hospital the same day. A long-term hormonal replacement treatment was initiated postoperatively. PMID- 10742669 TI - Surgicel in the management of labial and clitoral hood adhesions in adolescents with lichen sclerosus. AB - STUDY OBJECTIVE: Lichen sclerosus (LS) is an inflammatory dermatosis of the vulva with potentially destructive consequences to the young woman's perineum. Long term sequelae include atrophy of the labia minora, scarring of the clitoral hood, and labial and/or clitoral hood adhesions. This study aims to find techniques for preventing these devastating effects. PARTICIPANTS: Three young women, ages 14 and 15 years, with a long history of lichen sclerosus and labial adhesions, presented with recurrent labial and periclitoral adhesions. Increasing pain prior to presentation, exquisite enough to inhibit even walking, necessitated urgent operative intervention. Scarring of the clitoral hood with an area of firmness beneath the scarring was present. Sharp dissection of the clitoral hood was performed with the entrapped keratinaceous debris and hair expressed. The adherent labia were separated. Surgicel, oxidized regenerated cellulose gauze (Johnson & Johnson, Arlington, TX), was sutured to the exposed clitoral hood and labial surfaces with vicryl suture. Complete dissolution of the Surgicel occurred between postoperative day 4-6 without recurrence of adhesions. One-year follow-up did not reveal any evidence of recurrence in any of the three patients. CONCLUSION: The recurrence of labial and clitoral hood adhesions in young women with a history of LS was prevented for at least 1 year by surgical lysis and application of Surgicel to the affected area. This technique has prevented the recurrence during the interval when these surfaces are at highest risk of re agglutination. PMID- 10742671 TI - Understanding partner notification (Patient self-referral method) by young women. AB - STUDY OBJECTIVE: To understand the communication process involved in the patient self referral method among adolescent females with chlamydia and gonococcal infection. DESIGN: A cross-sectional descriptive study using a convenience sample was conducted in 54 predominantly African-American females, 13 to 20 years-old, with gonococcal and/or chlamydia cervicitis at an urban hospital based reproductive health clinic. Subjects interviewed at their treatment visit were asked what method of notification they used to tell their partner(s). Subjects who had not notified their partner were asked about their intended communication method and what they envisioned they would say to their partner. Coding methodology was used to analyze the information. In addition to qualitative information, outcome measures were the proportion of subjects who notified their partner(s), their communication method, style, and barriers to communication. RESULTS: According to the treatment visit, 57% (31/54) of subjects reported notifying their partner. Most had notified their partner by phone or face-to face, stated basic facts about the infection, and used a "direct" and "sensitive" communication style. Of the subjects who had not notified their partner (23/54), several barriers to notification were reported, but 82% said they intend to notify their partner(s). CONCLUSIONS: Strategies to promote the patient-self referral method among young women who do not notify their partner(s) need further assessment. PMID- 10742672 TI - An unusual presentation of Tourette's syndrome. AB - STUDY OBJECTIVE: To report an unusual complex motor tic as the presenting symptom in a patient diagnosed with Tourette's syndrome. METHODS: This case report was compiled via interview with a 9-year-old white female who presented to the private gynecologic practice of the senior author. Additional history was gained from the patient's mother and follow up information was obtained from written and verbal communication with neurology colleagues. MAIN OUTCOME MEASURES: Referral to neurology. Diagnosis of persistent perineal touching. RESULTS: Diagnosis of Tourette's syndrome. CONCLUSION: In young patients with nonspecific vulval symptoms, Tourette's syndrome should be considered in the differential diagnosis particularly if persistent self touching is a feature of the complaint. Referral to a physician experienced with the syndrome is important since the diagnosis is made on the basis of the clinical symptoms and signs. PMID- 10742675 TI - Obstetric and gynecologic dermatology. Edited By martin M. Black, marilynne McKay, and peter braude. mosby-wolfe, 1995. Reviewed By betsy schroeder, MD, director, division of pediatric and adolescent gynecology, department of obstetrics and gynecology, allegheny general hospital, pittsburgh, pennsylvania PMID- 10742674 TI - Perspectives on pediatric and adolescent gynecology from the allied health care professional. Teens, periods, and pregnancy. PMID- 10742673 TI - Flunitrazepam: more than a date rape drug. AB - STUDY OBJECTIVE: To evaluate the independent relationship between depressive symptoms. self-esteem. and drug resistance self-efficacy, and future intentions to use flunitrazepam. DESIGN: Cross-sectional survey. SETTING: Community-based family planning clinics. PARTICIPANTS: 865 sexually active women who self identified as Caucasian. African-American. or Mexican American. denied using flunitrazepam in the last 12 months and reported intentions to use or not use this substance in the next 12 months. INTERVENTIONS: None. MAIN OUTCOME MEASURE: An anonymous self-report measure assessed the patient's intentions to use flunitrazepam in the next 12 months: other lifetime drug use: and standardized measures of depression. self-esteem, and drug resistance self-efficacy. We hypothesized that future potential users of flunitrazepam would exhibit an increased number of depressive symptoms, lowered self-esteem, and limited drug resistance self-efficacy. RESULTS: Of the 865 subjects. 16 (1.8%) reported using flunitrazepam in their lifetime but not in the last 12 months. and 46 (5.3%) were identified as potential users. Logistic regression analyses controlling for confounding factors found that potential to use flunitrazepam was significantly associated with limited drug resistance self-efficacy (adjusted odds ratio [AOR] = 9.3) and the presence of both severe depressive symptoms and lowered self esteem (AOR = 3.2). CONCLUSIONS: These data suggest that young women with severe depressive symptoms and diminished self-esteem are at high risk for future flunitrazepam use and may use this drug to self-medicate psychological distress. PMID- 10742676 TI - FDG-PET and CT in Evaluation of Chemotherapy in Advanced Head and Neck Cancer. AB - Purpose: To compare [18F]2-deoxy-2-fluoro-D-glucose-positron emission tomography (FDG-PET) and computed tomography (CT) scans in assessment of response to neoadjuvant chemotherapy in advanced head and neck cancer.Materials and Methods: In a prospective clinical study, advanced head and neck cancer patients were enrolled in a neoadjuvant organ preservation protocol and received CT and FDG-PET scans prior to and after 2 or 3 rounds of chemotherapy. All patients had prechemotherapy and postchemotherapy tissue biopsies within the tumor region. Patients were then classified as pathologic complete response (PCR) or residual disease (RD) based on biopsies. Analysis of the tumor activity, using FDG-PET, was performed using standardized uptake ratios (SUR) in the region of the primary tumor. Analysis of the tumor size, using contrast enhanced CT, was performed using measurements of the primary tumor in 3 dimensions.Results: Nineteen of the 28 patients with stage III and IV cancer of the head and neck enrolled between December 1994 and May 1996 completed the study. Three patients were PCR and had a mean SUR reduction of 82% by positron emission tomography (PET) and volume reduction of 80% by CT. Sixteen patients had RD after chemotherapy, their SUR and volume reductions were 32% and 41%, respectively. Reduction in SUR with PET was significant. The mean tumor volume reduction by CT approached statistical significance. There was a positive correlation between the percent reduction in tumor volume and SUR (P < 0.004).Conclusion: FDG-PET and CT imaging are at least equivalent in correctly assessing tumor response to chemotherapy with a trend toward better performance by PET. PMID- 10742677 TI - Clinical Utility of FDG-PET in Detecting Head and Neck Tumors. A Comparison of Diagnostic Methods and Modalities. AB - Positron emission tomography (PET) using F-18-fluoro-2-deoxy-d-glucose(F-18-FDG) is gaining acceptance as a useful imaging method for head and neck tumors. Results of 59 PET scans done on 45 patients with head and neck tumors were evaluated retrospectively. Thirty-six patients had prior treatment consisting of surgery, radiation therapy, chemotherapy, or a combination of these modalities. PET results were compared to computed tomography and magnetic resonance imaging results and validated by histologic findings or survival. PET identified 36 out of 37 tumors (sensitivity 97%) and 18 of 22 benign processes (specificity 82%). Tumor was ruled out in 18 out of 19 patients. For CT, the sensitivity was 80% (20/25) and specificity was 31% (4/13). FDG-PET facilitates differentiation of recurrent head and neck tumors from treatment related changes sometimes difficult to characterize by CT or MRI and may have a significant impact on the management of patients reducing morbidity and costs. PMID- 10742678 TI - Intraindividual Comparison of F-18-Fluorodeoxyglucose and Tc-99m-Tetrofosmin in Planar Scintimammography and SPECT. AB - Since the comparative value of several nuclear medicine investigations for breast cancer has not been established, the rationale of this study was to examine the diagnostic performances of F-18-fluorodeoxyglucose (FDG) and Tc-99m-tetrofosmin both in single photon emission computed tomography (SPECT) and planar technique in an intraindividual comparative study.A total of 54 patients with suspected breast cancer (48 malignant and 9 benign lesions) was investigated by planar and SPECT imaging with both FDG and tetrofosmin within the last preoperative week. Pathohistological confirmation of the diagnosis was obtained in all patients.2 cm 81%/67%, 92%/67%, 75%/50%, and 75%/100%. Sensitivity was higher with FDG SPECT than with the other imaging modalities and significantly lower for tumors 5 ms/mmHg), in 17 moderately impaired (1.5-5 ms/mmHg) and in 22 severely depressed (<1.5 ms/mmHg). Baroreflex function was relatively preserved in patients in NYHA class I (5.1+/-2.5) in comparison to patients in NYHA class II and III (2.1+/-2.3 and 2.08+/-1.9 ms/mmHg, respectively). Of the 52 patients who entered the study at the end of follow-up 15 died of cardiac cause and 5 underwent heart transplantation. Survival free from heart transplantation was 62% in patients with normal baroreflex function, 62% in patients with moderate impairment of baroreflex and 66% in patients with major derangement. NYHA class, LVEF, LVFS and LVEDD were significantly associated with event free survival while baroreflex function was not. CONCLUSIONS: Our results suggest that evaluation of BRS impairment by phase IV Valsalva manoeuvre has limited prognostic value in patients with heart failure. PMID- 10742703 TI - Ventilatory capacity and exercise tolerance in patients with chronic stable heart failure. AB - BACKGROUND: Patients with chronic heart failure complain of breathlessness. This is associated with an increase in the ventilatory response to carbon dioxide production (VE/VCO(2) slope), yet a reduction in the maximal ventilation achieved at peak exercise. We analysed ventilatory capacity in heart failure in relation to exercise capacity. METHODS: We analysed data from 74 patients with chronic stable heart failure [age (S.D.) 50.6 (8.8) years; left ventricular ejection fraction 30 (15)%] and 36 controls [48.9 (11.5) years]. Subjects undertook maximal incremental exercise testing with metabolic gas exchange measurements to derive peak oxygen consumption (VO(2)), the VE/VCO(2) slope and ventilation. Spirometry was used to measure FEV(1) and FVC. Maximal voluntary ventilation (MVV) was calculated as FEV(1)x 35. RESULTS: Peak VO(2) was lower in patients [20.9 (7.5) ml min(-1) kg(-1) vs. 34.5 (10.1); P<0.001] and VE/VCO(2) greater [33.4 (10.7) vs. 26.0 (4.7); P<0.001]. Ventilation at peak exercise was lower in patients [63.5 (20.4) l/min vs. 86.9 (29.5); P<0.001], as was MVV [110.1 (37.9) l/min vs. 136.2 (53.1); P<0.001], but ventilation at peak as a proportion of MVV was the same in patients [60.0 (19.0)%] as controls [65.7 (12.4)%)]. There was an inverse relation between peak VO(2) and VE/VCO(2) slope (r=-0. 62; P<0.001). Percentage predicted FEV(1) correlated with ventilation at peak (r=0.62; P<0.001) and inversely with VE/VCO(2) slope (r=-0.32; P<0.001). There was no relation between percentage of MVV achieved and peak VO(2), or VE/VCO(2) slope. CONCLUSIONS: Although ventilation at peak exercise is lower in patients with heart failure than normal subjects, ventilation is the same proportion of maximal voluntary ventilation. These findings suggest that ventilatory capacity does not limit exercise capacity in heart failure. PMID- 10742705 TI - Effect of an exercise programme for elderly patients with heart failure. AB - AIMS: Benefit from exercise training programmes for patients with chronic heart failure has been demonstrated in relatively young (mean age 60 years) and predominantly male subjects. This study was undertaken to assess the effect of an exercise programme for older subjects more representative of the general heart failure population. METHODS AND RESULTS: Twenty-two patients with chronic heart failure of mean age 81+/-4 years were recruited into the study. Twenty-five percent were female and 31% were in chronic atrial fibrillation. A crossover study design was employed. The programme consisted of once weekly exercise sessions tailored to the abilities of elderly subjects and lasted for 12 weeks. Fifteen subjects completed the exercise component and nine the control component. The programme resulted in a 20% relative increase in 6-min walk test distance (ANCOVA: P<0.012). There was no improvement in quality of life as measured by the Living With Heart Failure Questionnaire, but the majority of subjects reported subjective improvement in wellbeing. CONCLUSION: Elderly patients with chronic heart failure can benefit from an appropriately designed exercise programme and should not be excluded from future large scale trials. PMID- 10742704 TI - The effect of physical training on skeletal muscle in patients with chronic heart failure. AB - BACKGROUND: The improvement of exercise capacity in patients with chronic heart failure (CHF) by physical training has been connected with reversal of the abnormalities in muscle fiber distribution and with the reduced activity of the enzymes of oxidative metabolism in skeletal muscle. However, the change in fiber type distribution induced by training is controversial and in previous studies the activities of the rate-limiting enzymes of the metabolic pathways have not been measured. AIMS: To examine the effect of dynamic training on percentage distribution of muscle fibers, on activities of the rate-limiting enzymes of the metabolic pathways and on electrophysiology in skeletal muscle. METHODS: A total of 27 patients with stable CHF (NYHA class II-III) were randomized to a training (N=12) or a control (N=15) group. The training group exercised on a bicycle ergometer for 30 min three times a week for 3 months using a load corresponding to 50-60% of their peak oxygen consumption. This was followed by a 3-month training period at home according to personal instructions. The control group did not change its physical activities. We studied muscle histology and measured the activities of the rate-limiting enzymes of anaerobic glycolysis (phosphofructokinase, PFK), glycogenolysis (phosphorylase), citric acid cycle (alpha-ketoglurate dehydrogenase, KGDH) and fatty acid oxidation (carnitinepalmitoyl transferase I and II, CPT I and II) from biopsies of the vastus lateralis muscle at baseline and after 3 and 6 months. Muscle strength and strength endurance with surface EMG and macro EMG of the right knee extensors were also determined. RESULTS: Exercise capacity, particularly submaximal, improved in the training group. The activity of PFK rose significantly but that of the other enzymes did not when compared with the change in the controls. Training had no effect on the percentage distribution of slow-twitch and fast twitch muscle fibers or on capillary density around these fibers in skeletal muscle. Maximum voluntary force, strength endurance and the function of motor units remained unaffected. CONCLUSIONS: Dynamic training results in improved exercise endurance in CHF. In skeletal muscle, the capacity of anaerobic glycolysis is increased but that of the citric acid cycle and fatty acid oxidation is not. Furthermore, the improvement in exercise endurance seems to be independent of changes in the percentage distribution of muscle fibers, capillarity or electrophysiological factors. PMID- 10742706 TI - Combination therapy with carvedilol and amiodarone in patients with severe heart failure. AB - BACKGROUND: Carvedilol and at least in some studies, amiodarone have been shown to improve symptoms and prognosis of patients with heart failure. There are no reports on the outcome of combined treatment with both drugs on top of angiotensin-converting enzyme inhibitors (ACEI), diuretics and digitalis. METHODS AND RESULTS: In 109 patients with severe heart failure submitted for heart transplantation at one single center between the years 1996 and 1998 [left ventricular ejection fraction (LVEF) 24.6+/-11%, 85% males, 52% idiopathic dilated cardiomyopathy (DCM), mean observation time 1. 9+/-0.4 years] a therapy with low-dose amiodarone (1000 mg/week) plus titrated doses of carvedilol (target 50 mg/day) was instituted. In addition, patients received a prophylactic dual chamber pacemaker (PM) in order to protect from bradycardia and for continuous holter monitoring. The devices were programmed in back-up mode with a basal rate of 40 i.p.m. with a hysteresis of 25%. Significantly, more patients were in sinus rhythm after 1 year than at study entry (85% vs. 63%, P<0.01). In 47 patients, under therapy over at least 1 year, the resting heart rate fell from 90+/-19 to 59+/-5 b.p.m. (P<0.001). Ventricular premature contractions in 24-h holter ECGs were suppressed from 1.0+/-3 to 0.1+/-0.3%/24 h (P167 b.p.m. detected by the pacemaker (1.2+/-2.8 episodes/patient/3 months vs. 0.3+/-0.8 episodes/patient/3 months after 1 year (P<0.01). The LVEF increased from 26+/-10 to 39+/-13% (P<0.001). NYHA class improved from 3. 17+/-0.3 to 1.8+/-0.6 (P<0.001) as well as right heart catheterization data. From the total cohort, seven patients (6%) developed symptomatic documented bradycardic rhythm disturbances requiring reprogramming of their pacemakers to DDD(R)/VVI(R) mode with higher basic rates. Two of these patients developed AV block, four sinu-atrial blocks or sinus bradycardia and one patient had bradycardic atrial fibrillation. During the observation period five patients died (3 sudden, 1 due to heart failure and 1 due to mesenteric infarction). Two patients had undergone heart transplants. The 1 year survival rate (Kaplan-Meier) without transplantation was 89%. Compared to historic control patients with amiodarone only (n=154) or without either agent (n=283) this rate was 64 and 57% (P<0.01). CONCLUSIONS: Heart failure patients benefit from a combined therapy with carvedilol and amiodarone resulting in a markedly improved NYHA stage, an increase in LV ejection fraction, a stabilization of sinus rhythm, a significant reduction in heart rate, a delay of electrical signal conduction and a suppression of ventricular ectopies. Approximately 6% of patients under such a regime became pacemaker-dependent in the first year. Compared to historic controls prognosis was better and the need for heart transplantation was lower. The exact role of either agent in combination or alone should be clarified in larger randomized studies. PMID- 10742707 TI - Celiprolol augments the effect of physical exercise on insulin sensitivity and serum lipid levels in chronic heart failure. AB - PURPOSE: Impaired insulin sensitivity has been linked with chronic heart failure (CHF). Exercise has a beneficial effect on insulin sensitivity in healthy subjects. It is used also as an adjunctive therapy in patients with CHF. We studied the effect of randomized treatment with celiprolol, a vasodilating beta(1)-adrenoceptor antagonist, 200 mg once daily (n=20) or placebo (n=11) on serum lipid levels and insulin sensitivity in patients with CHF. In addition, all subjects participated in a 6-month exercise training protocol. Thirteen subjects in the celiprolol and eight subjects in the control group were on additional beta(1)-adrenoceptor antagonist as part of their tailored CHF therapy. Insulin sensitivity was determined using the hyperinsulinemic euglycemic clamp test (diabetic subjects excluded, n=11 for the celiprolol group and n=8 for the placebo group). RESULTS: Insulin sensitivity index (ISI) increased by 33% (P<0.05) in the celiprolol group and by 17% (NS) in the control group. The mean increase in the whole group was 20% [from 68.2+/-11.5 to 81.7+/-10.7 ml/min/kg (mU/l), P<0.05]. No change was found in the total cholesterol level. HDL cholesterol levels increased by 12% (from 0.98+/-0.05 to 1.10+/-0.05 mmol/l, P<0. 005), and HDL/total cholesterol and HDL/LDL cholesterol ratios by 15% and 16%, respectively (P<0.005). The increase in serum fasting HDL cholesterol level was greater in the celiprolol-treated group (P<0.05). At baseline ISI correlated with maximal oxygen uptake (r=0. 65, P<0.0001) and body mass index (r=-0.55, P<0.001). The change in ISI correlated weakly with the improvement in muscle exercise capacity (r=0.53, P<0.05). CONCLUSIONS: Insulin sensitivity and serum lipid levels may be favorably affected by exercise training in subjects with mild-to moderate CHF. Celiprolol, a vasodilating beta1- selective adrenoceptor antagonist, potentiates this effect. PMID- 10742708 TI - A comparison of intervention with losartan or captopril in acute myocardial infarction. AB - AIM OF STUDY: Angiotensin-converting enzyme (ACE) inhibitors prolong life, lower the progression of heart failure, and decrease the need for hospitalizations in patients after myocardial infarctions. It is still unclear whether these effects could also be achieved by blocking the angiotensin II (ATII) type 1 receptor. METHODS AND RESULTS: We randomized 201 patients with acute myocardial infarction treated with either direct angioplasty, thrombolysis, or heparin alone to the ACE inhibitor captopril or the ATII antagonist losartan. The primary endpoints were safety, tolerability, and left ventricular parameters. The patients were followed for at least 15 days. The incidence of severe adverse events was similar in both groups, although cough presented less often in the losartan group. Captopril failed to prevent an increase in end-diastolic volume and did not influence left ventricular end-systolic volume. This effect led to an increase in the left ventricular ejection fraction (P<0. 001) without a change in wall-motion index. Losartan did not affect end-diastolic volume but decreased end-systolic volume (P<0.001), resulting in a significant increase in left ventricular ejection fraction (P<0.001) and a decrease in wall-motion index (P<0.001). CONCLUSION: This study suggests that losartan is safe and well tolerated in patients after myocardial infarction. ATII antagonists seem to have a more pronounced effect on left ventricular remodeling than ACE inhibitors. PMID- 10742709 TI - The basics of heart failure management: are they being ignored? AB - BACKGROUND: Advances have been made in the medical management of congestive heart failure. However, there is concern that these changes may not be transmitted to the heart failure population in the community. Other impediments to improved prognosis, such as failure to apply non-pharmacological strategies and poor patient comprehension may also be prevalent in the community. AIMS: The purpose of this study was to assess physician practice and patient knowledge in a heart failure population admitted to a University Hospital in Ireland. METHODS: Patients admitted with a primary diagnosis of heart failure were studied. Estimation of ejection fraction was used to subdivide the population into heart failure with impaired and normal systolic function. Patients' course in hospital was noted with reference to management by cardiology or internal medicine, use of angiotensin-converting enzyme inhibition therapy and digoxin and application of dietary and rehabilitative services. Patient knowledge was assessed by questionnaire. RESULTS: Eighty patients were included in this study. Two-thirds of the population had impaired systolic function. The majority of patients were managed by internal medicine physicians, and this population was older and more likely to have normal systolic function. Prescription of converting enzyme inhibitor therapy was more frequently used in cardiology-managed patients (96 vs. 70%, P<0.05). Neither group applied dietary or rehabilitative advice to a significant level. Patient comprehension was poor, especially with regard to understanding of medicine and the value of weight measurement. CONCLUSION: The above data demonstrate a lack of use of rehabilitative and dietary services and poor patient knowledge. These deficiencies may play a role in determining outlook and may impede the expected improvement in prognosis that has been witnessed in large randomised studies. PMID- 10742710 TI - Update of ELITE-II, BEST, CHAMP, and IMPRESS clinical trials in heart failure. AB - The ELITE-II, BEST and CHAMP Trials were reported for the first time at the American Heart Association in November 1999. These trials provide valuable new information to guide clinical practice in the management of heart failure and of myocardial infarction, although none mandate a major change from current clinical practice. The IMPRESS trial of the vasopeptidase inhibitor, omapatrilat, indicated a promising new treatment for the management of heart failure. PMID- 10742711 TI - Management of heart failure in Switzerland. AB - Similar to other countries, heart failure is a major cause of morbidity and mortality in Switzerland. Among heart failure patients admitted to a Swiss university hospital in 1998, admission therapy included: ACE inhibitors/AT-II blockers in approximately two-thirds; diuretics in approximately 70%; and beta blockers in approximately one-third. Easy access to diagnostic tests and limited results of surveys suggest that quality of care of heart failure patients is satisfactory in Switzerland. However, results from ongoing studies are required to assess more reliably the quality of diagnosis and therapy of this high-risk population in Switzerland. PMID- 10742712 TI - Expression of NMDA receptor subunit mRNAs in neurochemically identified projection and interneurons in the human striatum. AB - N-methyl-D-aspartate (NMDA) receptors are composed of subunits from two families: NR1 and NR2. We used a dual-label in situ hybridization technique to assess the levels of NR1 and NR2A-D messenger ribonucleic acid (mRNA) expressed in projection neurons and interneurons of the human striatum. The neuronal populations were identified with digoxigenin-tagged complementary RNA probes for preproenkephalin (ENK) and substance P (SP) targeted to striatal projection neurons, and somatostatin (SOM), glutamic acid decarboxylase 67 kD (GAD(67)), and choline acetyltransferase (ChAT) targeted to striatal interneurons. Intense NR1 signals were found over all striatal neurons. NR2A signals were high over GAD(67) positive neurons and intermediate over SP-positive neurons. ENK-positive neurons displayed low NR2A signals, whereas ChAT- and SOM-positive neurons were unlabeled. NR2B signals were intense over all neuronal populations in striatum. Signals for NR2C and NR2D were weak. Only ChAT-positive neurons displayed moderate signals, whereas all other interneurons and projection neurons were unlabeled. Moderate amounts of NR2D signal were detected over SOM- and ChAT positive neurons; GAD(67)- and SP-positive striatal neurons displayed low and ENK positive neurons displayed no NR2D hybridization signal. These data suggest that all human striatal neurons have NMDA receptors, but different populations have different subunit compositions that may affect function as well as selective vulnerability. PMID- 10742713 TI - Neurons involved in nitric oxide-mediated cGMP signaling in the tobacco hornworm, Manduca sexta. AB - Recently, both nitric oxide synthase (NOS), and nitric oxide (NO)-sensitive guanylyl cyclase were cloned in Manduca sexta and implicated in several cellular, developmental, and behavioral processes (Nighorn et al. [1998] J Neurosci 18:7244 7255). However, NO is a highly diffusive gas, and little is known about the range and specificity of its actions on neurons. To begin examining the role of NO as a neurotransmitter in the central nervous system (CNS) of larval Manduca, we have mapped potential NO-producing neurons using fixation-resistant NADPH-diaphorase staining and antisera that recognize a NOS-specific epitope. In addition, to detect NO-responsive neurons, we treated the CNS with NO donors and used antibodies that recognize elevated levels of cyclic 3;,5;-guanosine monophosphate (cGMP). Many potential NO-producing neurons were mapped, including the ventral unpaired median cells and three pairs of lateral cells in each abdominal ganglion. Additional neurons in the dorsal midline of ganglia A5-7 (PM2) appear to express NOS in a segment-specific manner. At the larval-to-pupal transition, this staining pattern changes; the PM2 neurons stain weakly or are undetectable and there is novel expression of NOS in cell 27. In response to NO donors, a small number of neurons produce detectable cGMP accumulation in a segment specific pattern. These include a pair of posteriodorsally positioned interneurons (IN505) in all the abdominal ganglia, PM2 neurons in A5, and PM1 and PM2 neurons in A7. Hence, PM2 neurons in A5 and A7 are potentially capable of producing and responding to NO. These identified NO-producing and responding neurons provide a tractable model system for studying the dynamics and specificity of NO signaling in the CNS. PMID- 10742714 TI - Anatomical characteristics of tonic and phasic postganglionic neurons in guinea pig bronchial parasympathetic ganglia. AB - Anatomical characteristics of principal parasympathetic ganglia neurons on the guinea pig primary bronchus were analyzed, and the procedure for localizing the ganglia without the aid of staining for in vitro physiological studies is described. The neurons were tightly packed within a perineural sheath, and the cell bodies formed a homogeneous population based on size and shape. By using intracellular electrophysiological recordings, unstained neurons within these ganglia were characterized with suprathreshold depolarizing stimuli as having either accommodating action potential patterns (phasic neurons) or repetitive action potential patterns (tonic neurons). After determining whether a cell was tonic or phasic, it was injected with either horseradish peroxidase or Neurobiotin for characterization of its dendrites. There were no differences between tonic and phasic neurons, and both exhibited the following: (1) dendrites were multiple and branching; (2) all processes (axon and dendrites) arose from a circumscribed area on the somatic surface; (3) the initial direction of the processes was usually toward the center of the ganglion, creating a very dense intraganglionic neuropil; (4) tapering processes (presumed dendrites) extended beyond the border of the perineural sheath; and (5) many processes terminated with bouton-like swellings near the somatic surfaces of neighboring neurons within the same ganglion. Electron microscopic examination of dendritic and cell body membranes revealed that greater than 90% of the synapses occurred on dendrites. Based on immunohistochemical staining, all neurons were calbindin negative. These results indicate a relatively homogeneous population of neurons in bronchial parasympathetic ganglia displaying dendritic characteristics compatible with complex integrative properties. PMID- 10742715 TI - Localization of NADPH diaphorase/nitric oxide synthase and choline acetyltransferase in the spinal cord of the frog, Rana perezi. AB - The localization of nitrergic cells and fibers and cholinergic cells has been analyzed in the spinal cord of the anuran amphibian Rana perezi. Histochemistry for nicotinamide adenine dinucleotide phosphate-diaphorase and nitric oxide synthase immunohistochemistry revealed a concurrent pattern of labeled structures. A large population of nitrergic spinal neurons was found from the level of the obex to the filum terminale. They are abundant in the dorsal horn and intermediate gray matter, but also occur in territories of the ventral horn and, only occasionally, in somatic motoneurons. Numerous nitrergic fibers were present in the spinal white matter, particularly in the dorsal and dorsolateral funiculi. A special arrangement of nitrergic axons is present in Lissauer's tract, where a collateral system is formed. Cholinergic cells, revealed by choline acetyltransferase immunohistochemistry, were observed throughout the spinal cord. The somatic motoneurons were the most conspicuously immunoreactive cells. A large population of cholinergic cells forms a discontinuous column in the intermediate gray, from the third spinal segment to lumbar segments. These cells were organized in a medially located or intercalated cell group, and a laterally located intermediolateral group. Numerous scattered cholinergic cells were present in the central zone of the ventral horn and were absent in the dorsal horn. Double-labeling experiments revealed a high degree of codistribution of nitrergic and cholinergic cells, mainly in the intermediate gray, but colocalization of both markers in the same neurons was not found. This result contrasts with the situation found in mammals and raises the question of whether coexpression of both substances was acquired in spinal cord neurons through evolution only in amniotes or, even, only in mammals. PMID- 10742716 TI - Ultrastructural localization of the binding fragment of tetanus toxin in putative gamma-aminobutyric acidergic terminals in the intermediolateral cell column: a potential basis for sympathetic dysfunction in generalized tetanus. AB - Tetanus toxin (TeTx) causes sympathetic hyperactivity, a major cause of mortality in generalized tetanus, apparently by obstructing the inhibition of sympathetic preganglionic neurons (SPNs). Neuroanatomic tracing and immunohistochemistry were used to investigate whether axon terminals in the intermediolateral cell column (IML) that synapse on SPNs and use the inhibitory neurotransmitter gamma aminobutyric acid (GABA) may be infected transsynaptically with TeTx. The binding fragment of TeTx (TTC; an atoxic surrogate of TeTx) and the cholera toxin B subunit (CTB; a retrograde tracer) were injected into the rat superior cervical ganglion and, over 16-48 hours, were transported to the ipsilateral IML in the caudal half of the last cervical and first three thoracic spinal cord segments. With light microscopy, diffuse CTB immunolabeling extended throughout SPN perikarya and dendrites. Punctate TTC and GABA immunolabeling were accumulated densely in the neuropil between and surrounding SPN processes. With electron microscopy, 54% of the axon terminals in the IML (n = 1,337 terminals) were TTC immunolabeled (TTC(+)), and 25% contained putative neurotransmitter levels of GABA immunolabeling (GABA(+)). On average, GABA(+) terminals had a 76% chance of also being TTC(+) and a 62% greater chance of being TTC(+) than GABA(-) terminals (P < 0.000001). Axon terminals were just as likely to be TTC(+) and/or GABA(+) regardless of whether the dendrites they synapsed on were large (>1 microM) or small in cross-sectional area or were labeled retrogradely. Sympathetic hyperactivity in tetanus may involve 1) retrograde and transsynaptic transport of TeTx by SPNs and 2) at least in part, an infection of GABAergic terminals in the IML. PMID- 10742717 TI - Development of the mammillothalamic tract in normal and Pax-6 mutant mice. AB - The mammillary bodies represent important relay stations for one of the major neuronal circuits in the brain: the limbic circuit. Mammillary projections traveling through the principal mammillary tract are established early during development, forming the mammillotegmental bundle, which appears fully developed by embryonic day 15 (E15). The mammillothalamic tract develops later, around E17 E18, forming a compact system of collateral fibers originating from the principal mammillary tract and reaching the thalamus by E20. The Pax-6 gene is expressed in various regions of the developing brain, among which the border separating the ventral thalamus from the dorsal thalamus, known as the zona limitans intrathalamica, is especially significant. In this report, the development of the efferent mammillary system of fibers was studied in wild type and Pax-6 mutant mice by using carbocyanine tracers and Golgi preparations. In mutant mice, the mammillotegmental bundle developed normally; however, the mammillothalamic tract was missing. By using anti-Pax-6 antibodies in wild type mice, the existence of an immunoreactive cell cluster is described surrounding the bifurcation point of the principal mammillary tract. The results of this study suggest that there is a correlation of these cells with a particular type of Golgi impregnated neuron. PMID- 10742718 TI - Midbrain acoustic circuitry in a vocalizing fish. AB - The mapping of auditory circuitry and its interface with vocal motor systems is essential to the investigation of the neural processing of acoustic signals and its relationship to sound production. Here we delineate the circuitry of a midbrain auditory center in a vocal fish, the plainfin midshipman. Biotin injections into physiologically identified auditory sites in nucleus centralis (NC) in the torus semicircularis show a medial column of retrogradely filled neurons in the medulla mainly in a dorsomedial division of a descending octaval nucleus (DO), dorsal and ventral divisions of a secondary octaval nucleus (SO), and the reticular formation (RF) near the lateral lemniscus. Biotin-filled neurons are also located at midbrain-pretectal levels in a medial pretoral nucleus. Terminal fields are identified in the medulla (ventral SO, RF), isthmus (nucleus praeeminentialis), midbrain (nucleus of the lateral lemniscus, medial pretoral nucleus, contralateral NC, tectum), diencephalon (lateral preglomerular, central posterior, and anterior tuber nuclei), and telencephalon (area ventralis). The medial column of toral afferent neurons is adjacent to and overlapping the positions of DO and SO neurons shown previously to be linked to the vocal pacemaker circuitry of the medulla. Midshipman are considered "hearing generalists" because they lack the peripheral adaptations of "specialists" that enhance the detection of the pressure component of acoustic signals. Whereas the results indicate a general pattern of acoustic circuitry similar to that of specialists, they also show central adaptations, namely, a vocal-acoustic interface in DO and SO related to this species' vocal abilities. PMID- 10742719 TI - Presynaptic and postsynaptic relations of mu-opioid receptors to gamma aminobutyric acid-immunoreactive and medullary-projecting periaqueductal gray neurons. AB - The ventrolateral portion of the periaqueductal gray (PAG) is one brain region in which ligands of the mu-opioid receptor (MOR) produce analgesia. In the PAG, MOR ligands are thought to act primarily on inhibitory [e.g., gamma-aminobutyric acidergic (GABAergic)] neurons to disinhibit PAG output rather than directly on medullary-projecting PAG neurons. In this study, the ultrastructural localization of MOR immunolabeling was examined with respect to either GABAergic PAG neurons or PAG projection neurons that were labeled retrogradely from the rostral ventromedial medulla. Immunoreactivity for MOR and GABA often coexisted within dendrites. Dual-labeled profiles accounted for subpopulations of dendrites containing immunoreactivity for either MOR (65 of 145 dendrites; 45%) or GABA (65 of 183 dendrites; 35%). In addition, nearly half of PAG neuronal profiles (148 of 344 profiles) that were labeled retrogradely from the ventromedial medulla contained MOR immunoreactivity. MOR was distributed equally among retrogradely labeled neuronal profiles in the lateral and ventrolateral columns of the caudal PAG. With respect to the presynaptic distribution of MOR, approximately half of MOR-immunolabeled axon terminals (35 of 69 terminals) also contained GABA. Some MOR and GABA dual-immunolabeled axon terminals contacted unlabeled dendrites (11 of 35 terminals), whereas others contacted GABA-immunoreactive dendrites (15 of 35 terminals). Furthermore, axon terminals synapsing on medullary-projecting PAG neurons sometimes contained immunoreactivity for MOR. These data support the model that MOR ligands can act by inhibiting GABAergic neurons, but they also provide evidence that MOR ligands may act directly on PAG output neurons. In addition, MOR at presynaptic sites could affect both GABAergic neurons and output neurons. Thus, the disinhibitory model represents only partially the potential mechanisms by which MOR ligands can modulate output of the PAG. PMID- 10742720 TI - Molecular epidemiology of a nosocomial outbreak due to Enterobacter cloacae and Enterobacter agglomerans in Campinas, Sao Paulo, Brazil. AB - A total of 73 isolates (57 Enterobacter cloacae and 16 Enterobacter agglomerans), recovered during an outbreak of bacteremia in the Campinas area, Sao Paulo, Brazil, were studied. Of these isolates, 61 were from parenteral nutrition solutions, 9 from blood cultures, 2 from a sealed bottle of parenteral nutrition solution, and one was of unknown origin. Of the 57 E. cloacae isolates, 54 were biotype 26, two were biotype 66 and one was non-typable. Of 39 E. cloacae isolates submitted to ribotyping, 87.2% showed the same banding pattern after cleavage with EcoRI and BamHI. No important differences were observed in the antimicrobial susceptibility patterns among E. cloacae isolates exhibiting the same biotype, serotype and ribotype. All E. agglomerans isolates, irrespective of their origin, showed same patterns when cleaved with EcoRI and BamHI. The results of this investigation suggest an intrinsic contamination of parenteral nutrition solutions and incriminate these products as a vehicle of infection in this outbreak. PMID- 10742721 TI - Asymptomatic infections by diarrheagenic Escherichia coli in children from Misiones, Argentina, during the first twenty months of their lives. AB - Diarrheagenics Escherichia coli are the major agents involved in diarrheal disease in developing countries. The aim of this study was to evaluate the time of appearance of the first asymptomatic infection by the different categories of diarrheagenic E. coli in 44 children since their birth and during the first 20 months of their lives. In all of the children studied, we detected at least one category of diarrheagenic E. coli through the 20 months of the study. 510 diarrheagenic E. coli (33.5%) were obtained from the 1,524 samples collected from the 44 children during the time of the study (31.4% EAggEC, 28.8% EPEC, 27.1% DAEC, and 12.7% ETEC). Neither EHEC nor EIEC were identified. The median age for diarrheagenic E. coli colonization was 7.5 months. The mean weaning period was 12.8 months and the mean age for introduction of mixed feeding (breast fed supplemented) was 3.8 months. A significantly lower incidence of diarrheal disease and asymptomatic infections was recorded among the exclusively breast-fed rather than in the supplemented and non breast-fed infants. For ETEC, EPEC and EAggEC the introduction of weaning foods and complete termination of breast feeding were associated with an increase of asymptomatic infections. PMID- 10742722 TI - A clinico-epidemiological study of bites by spiders of the genus Phoneutria. AB - From January, 1984 to December, 1996, 422 patients (ages 9 m-99 y, median 29 y) were admitted after being bitten by spiders which were brought and identified as Phoneutria spp. Most of the bites occurred at March and April months (29.2%), in the houses (54.5%), during the day (76.5%), and in the limbs (feet 40.9%, hands 34.3%). Upon hospital admission, most patients presented only local complaints, mainly pain (92.1%) and edema (33.1%) and were classified as presenting mild (89.8%), moderate (8.5%) and severe (0.5%) envenomation. Few patients (1.2%) did not present signs of envenomation. Severe accidents were only confirmed in two children (9 m, 3 y). Both developed acute pulmonary edema, and the older died 9 h after the accident. Patients more than 70 year-old had a significantly greater (p<0.05) frequency of moderate envenomations compared to the 10-70-year-old individuals. Proceedings to relief local pain were frequently performed (local anesthesia alone 32.0%, local anesthesia plus analgesics 20.6% and oral analgesics alone 25. 1%). Only 2.3% of the patients (two cases classified as severe and eight as moderate, eight of them in children) were treated with i.v. antiarachnid antivenom. No antivenom early reaction was observed. In conclusion, accidents involving the genus Phoneutria are common in the region of Campinas, with the highest risk groups being children under 10 years of age and adults over 70 years of age. Cases of serious envenomation are rare (0.5%). PMID- 10742723 TI - Nutritonal evaluation of pemphigus foliaceus patients on long term glucocorticoid therapy. AB - Our objective was to compare food intake and nutritional status of Pemphigus Foliaceus patients (PG) on long term glucocorticoid therapy to a Control Group (CG). Fourteen PG female inpatients receiving prednisone (0.33 +/- 0.22mg/kg) for at least 12 months and twelve CG subjects were submitted to nutritional evaluation, including anthropometry, urinary creatinine determination and serum biochemical measurements, besides 48-h-based food intake records. Groups were compared by Chi-square, Mann-Whitney and "t" tests. PG patients and CG were paired, respectively, in relation to age (24.7 +/- 14.1 vs. 22.0 +/- 12.0 years), body mass index (25.8 +/- 6.4 vs. 24.0 +/- 5.6kg/m2), daily protein intake (132.9 +/- 49.8 vs. 95.2 +/- 58.9g), and serum albumin (median; range) (3.8; 3.5-4.1 vs. 3.8; 3.6-5.0g/dl). However, PG patients had lower height-creatinine index (64.8 +/- 17.6 vs. 90.1 +/- 33.4%), and higher daily energy (3080 +/- 1099 vs. 2187 +/- 702kcal) and carbohydrate (376.8 +/- 135.8 vs. 242.0 +/- 80.7g) intakes. Despite high food, protein and energy consumption, PG patients on long term glucocorticoid therapy had lower body muscle mass than controls, while showing high body fat stores. These findings are possibly related to combined metabolic effects of long term corticotherapy and inflammatory disease plus corticosteroid induced increased appetite. PMID- 10742725 TI - Paracoccidioidomycosis of the male genital tract. Report of eleven cases and a review of Brazilian literature. AB - Eleven cases of involvement of the genital tract in paracoccidioidomycosis were collected in a retrospective study of the clinical records of 683 patients seen in Porto Alegre, Rio Grande do Sul, Brazil. These cases are herein summarily reported. Eighteen similar cases were gathered in review of the Brazilian literature. Obtained data are discussed. PMID- 10742724 TI - Randomized, double-blind trial comparing indinavir alone, zidovudine alone and indinavir plus zidovudine in antiretroviral therapy-naive HIV-infected individuals with CD4 cell counts between 50 and 250/mm3. AB - Treatment with indinavir has been shown to result in marked decreases in viral load and increases in CD4 cell counts in HIV-infected individuals. A randomized double-blind study to evaluate the efficacy of indinavir alone (800 mg q8h), zidovidine alone (200 mg q8h) or the combination was performed to evaluate progression to AIDS. 996 antiretroviral therapy-naive patients with CD4 cell counts of 50-250/mm3 were allocated to treatment. During the trial the protocol was amended to add lamivudine to the zidovudine-containing arms. The primary endpoint was time to development of an AIDS-defining illness or death. The study was terminated after a protocol-defined interim analysis demonstrated highly significant reductions in progression to a clinical event in the indinavir containing arms, compared to the zidovudine arm (p<0. 0001). Over a median follow up of 52 weeks (up to 99 weeks), percent reductions in hazards for the indinavir plus zidovudine and indinavir groups compared to the zidovudine group were 70% and 61%, respectively. Significant reductions in HIV RNA and increases in CD4 cell counts were also seen in the indinavir-containing groups compared to the zidovudine group. Improvement in both CD4 cell count and HIV RNA were associated with reduced risk of disease progression. All three regimens were generally well tolerated. PMID- 10742726 TI - Hantavirus pulmonary syndrome in Brazil: clinical aspects of three new cases. AB - Hantavirus pulmonary syndrome (HPS) has been recognized recently in Brazil, where 28 cases have been reported as of September 1999. We report here the clinical and laboratory findings of three cases whose diagnoses were confirmed serologically. All the patients were adults who presented a febrile illness with respiratory symptoms that progressed to respiratory failure that required artificial ventilation in two of them. Laboratory findings were most of the time consistent with those reported in the United States in patients infected with the Sin Nombre virus, and included elevated hematocrit and thrombocytopenia; presence of atypical lymphocytes was observed in one patient. The chest radiological findings observed in all the patients were bilateral, diffuse, reticulonodular infiltrates. Two patients died. Histopathological examination of the lungs of these patients revealed interstitial and alveolar edema, alveolar hemorrhage, and mild interstitial pneumonia characterized by infiltrate of immunoblasts and mononuclear cells. In the epidemiologic investigation of one of the cases, serologic (ELISA) tests were positive in 3 (25%) out of 12 individuals who shared the same environmental exposure. HPS should be included in the differential diagnosis of interstitial pneumonia progressing to acute respiratory failure. PMID- 10742727 TI - Systematic reviews on leptospirosis. AB - OBJECTIVES: To find the existing clinical evidence on interventions for leptospirosis. The objective is to evaluate the effectiveness and safety of any intervention on leptospirosis through systematic reviews of randomized controlled trials (RCTs). DATA SOURCE: The sources of studies used (where there were no limitations concerning language, date, or other restrictions) were: EMBASE, LILACS, MEDLINE, the Cochrane Controlled Clinical Trials Database, and the Cochrane Hepato-Biliary Group Randomized Trials register. SELECTION OF STUDIES: TYPE OF STUDY: All systematic reviews of randomized controlled trials. PARTICIPANTS: patients with clinical and/or laboratorial diagnosis of leptospirosis, and subjects potencially exposed to leptospirosis as defined by the authors INTERVENTIONS: any intervention for leptospirosis (as antibiotics or vaccines for prevention or treatment). DATA COLLECTION: The assessment will be independently made by the reviewers and cross-checked. The external validity was assessed by analysis of: studies, interventions, and outcomes. DATA SYNTHESIS: Located 163 studies using the search strategy described above, at the electronic databases above. Only 2 hits were selected, which are protocols of systematic reviews of Cochrane Collaboration, and not full reviews. One of the protocols evaluates antibiotics for treatment, and the other evaluates antibiotics for prevention of leptospirosis. CONCLUSIONS: There were not complete systematic reviews on interventions for leptospirosis. Any interventions for leptospirosis, such as prevention and treatment remains unclear for guidelines and practice. PMID- 10742728 TI - Parasitological and immunological diagnoses of strongyloidiasis in immunocompromised and non-immunocompromised children at Uberlandia, State of Minas Gerais, Brazil. AB - Parasitological and immunological diagnoses were part of a study conducted among 151 children, 83 immunocompromised (IC) and 68 non-immunocompromised (non-IC) aged from zero to 12, seen at the University Hospital, Universidade Federal de Uberlandia, State of Minas Gerais, Brazil, from February, 1996, to June, 1998. Three fecal samples from each child were analyzed for the parasitological diagnosis by Baermann-Moraes and Lutz methods. The immunological diagnosis to detect IgG and IgM antibodies was carried out by the indirect immunofluorescence antibody test (IFAT) with cryo-microtome sections of Strongyloides stercoralis and Strongyloides ratti larvae as antigens and by the ELISA test with an alkaline extract of S. ratti as the antigens. Of the 151 children 5 (3.31%) were infected with larvae of S. stercoralis (2 cases IC, 2.41%, and 3 cases non-IC, 4.41%). The IFAT-IgG detected 7 (8.43%) serum samples positive among IC, and 2 (2.94%) cases among non-IC. The ELISA-IgG test detected 10 (12.05%) serum samples positive among IC, and 1 (1.47%) case among non-IC. The IFAT-IgM detected 6 (7.22%) positive cases among IC, and 3 (4.41%) cases among non-IC. ELISA-IgM test detected 10 (12.05%) positive cases among IC, and 3 (4.41%) cases among non-IC. It was concluded that the immunological tests can help in the diagnosis of strongyloidiasis in immunocompromised children. PMID- 10742729 TI - Frequency of suspected cases of neurocysticercosis detected by computed skull tomography in Santa Maria, RS, Brazil. AB - Due to the lack of studies about neurocysticercosis in the South of Brazil, an investigation was conducted to determine the percentage of suspected cases of neurocysticercosis in computed tomography diagnoses in Santa Maria, RS, from January 1997 to December 1998. Of 6300 computed tomographies (CT) of the skull performed at the private Hospital de Caridade Astrogildo de Azevedo, 80, i.e., 1.27% were suspected of neurocysticercosis. Fifty were women (62.5%) and 30 were men (37.5%). The most frequent radiological manifestation indicating neurocysticercosis was the presence of calcifications (isolated or associated), with a 95% rate (76 cases), while the presence of hypodense lesions reached a 5% rate (4 cases). After routine analysis, each CT was evaluated again and the suspected cases were confirmed. The percentage of suspected cases of neurocysticercosis detected by CT in the present study carried out in Santa Maria was considered low (1.27%). This can be explained by the fact that tomography is not accessible to the economically underprivileged population of Santa Maria. We hope that the present study can alert the population and the professionals to the fact that neurocysticercosis is a more frequent disease than indicated by the few diagnoses made. PMID- 10742730 TI - Is India ready for the integrated management of childhood illness strategy? PMID- 10742731 TI - Evaluation of the WHO/UNICEF algorithm for integrated management of childhood illness between the age of two months to five years. AB - OBJECTIVE: To evaluate the utility of the "WHO/UNICEF algorithm for integrated management of childhood illness (IMCI) between the age of 2 months to 5 years. DESIGN: Prospective observational. SETTING: The Outpatient Department and Emergency Room of a medical college hospital. METHODS: 203 children presenting to Outpatient Department (n=101) or Emergency Room (n=102) were assessed and classified as per 'IMCI' algorithm and treatment required was identified. A detailed evaluation with all relevant investigations was also done for these subjects. The final diagnoses made and therapies instituted on this basis served as 'gold standard'. The diagnostic and therapeutic agreements between the 'gold standard' and the IMCI and vertical (on the basis of primary presenting complaint) algorithms were computed. RESULTS: More than one illness was present in 135 (66.5%) of subjects as per 'gold standard'. The mean (SD) numbers of morbidities as per the gold standard and IMCI- low and high malaria risks were 2.1 (1.1), 1.8 (1.0) and 2.2 (1.1), respectively. Subjects having any referral criteria as per IMCI module had a greater co-existence of illnesses (mean 2.6 vs. 1.6 illnesses per child, respectively). The referral criteria proved useful in predicting hospitalization and a combination of hospitalization and observation; their sensitivity and specificity were 81% and 69% and 74% and 85%, respectively. IMCI algorithms covered majority (92%) of the recorded illnesses. A total agreement with IMCI (malaria low risk) was found in 129 (64%) cases while in 43 (22%) cases, there was partial agreement. Corresponding figures for vertical (split IMCI) program were 93 (46%; p<0.001) and 41 (25%). The difference was primarily due to underdiagnoses (30%). Diagnostic discordance of IMCI algorithm and gold standard was evident for the cough category due to underdiagnosis of bronchial asthma and bronchiolitis and an overdiagnosis of pneumonia whereas the discordance for fever was due to an overdiagnosis of malaria. Identical results were found for broad treatment categories. The IMCI algorithm had a provision for preventive services of immunization (16.3% possibility of availing missed opportunities) and feeding advice. CONCLUSIONS: There is a sound scientific basis for adopting the IMCI approach since: (i) co-existence of morbidities is frequent; (ii) severe illness is assessed with good sensitivity and specificity; and (iii) the IMCI algorithm is diagnostically and therapeutically superior to the vertical disease specific algorithms. The generic IMCI algorithm needs adaptation to reflect the regional morbidity profile. PMID- 10742732 TI - Cardiac function in hypothyroid children: effect of replacement therapy. AB - OBJECTIVE: To evaluate prospectively cardiac function in children with primary hypothyroidism before and after replacement therapy with L-thyroxine. DESIGN: Prospective clinical and laboratory cardiac evaluation of children with hypothyroidism before and after therapy. SETTING: Hospital based. SUBJECTS: 20 consecutive children aged 6 months - 14 years with primary hypothyroidism. METHODS: Assessment of cardiac status by clinical, radiological, ECG, echocardiography, M-mode and 2 dimensional echo-Doppler study and phonocardiography for systolic and diastolic functions and structural anomalies. RESULTS: Indices of myocardial contractility like ejection fraction (EF), velocity of circumferential fibre shortening (VCF) did not change with therapy. However, systolic time intervals, both left ventricular (before therapy 0.32+/ 0.03 msec; after therapy 0.25+/-0.03 msec; p <0.001) showed a significant change. In diastolic functions, isovolumic relaxation time fell from 62+/-9 msec before therapy to 50+/-5 msec after therapy (p <0.001). Pericardial effusion was found in 10 children before treatment which disappeared in 7 following therapy. CONCLUSION: Subtle evidence of alteration of myocardial function is thus seen in children with primary hypothyroidism which reverses with treatment. PMID- 10742733 TI - Update on the recommendations of the Academy to other agencies on immunization. PMID- 10742734 TI - Genetic counseling in pediatric practice. PMID- 10742735 TI - Nutrient intake and consumption of supplementary nutrition by severely malnourished children in two ICDS projects in Rajasthan state. PMID- 10742736 TI - Experiences of neonatal care in a secondary level hospital. PMID- 10742737 TI - Prolonged diarrhea in pre-school children: an endoscopic evaluation. PMID- 10742738 TI - Myoclonic epilepsy with red ragged fibres. PMID- 10742739 TI - Inflammatory linear verrucous epidermal nevus syndrome with bilateral vertebral artery occlusion. PMID- 10742740 TI - Idiopathic hypereosinophilic syndrome. PMID- 10742741 TI - Multiple lentigines: a case with new associations. PMID- 10742742 TI - Klippel Feil syndrome. PMID- 10742743 TI - Dandy Walker malformation. PMID- 10742744 TI - Immunization in cancer. PMID- 10742745 TI - Immunization in cancer - reply PMID- 10742746 TI - Should a sick child be immunized. PMID- 10742747 TI - Should A sick child Be immunized - reply PMID- 10742748 TI - Pulseoximetric pseudobradycardia in ventilated newborns with pneumothorax. PMID- 10742749 TI - Drug resistant infections: solution lies in regulated antibiotic use. PMID- 10742750 TI - Drug resistant infections: solution lies in regulated antibiotic use - reply PMID- 10742751 TI - Is it possible to achieve hundred per cent vaccination coverage for children below 5 years of age? PMID- 10742752 TI - Careless disposal of disposable needles: a less recognized risk factor for transmission of blood borne diseases. PMID- 10742754 TI - Immature gastric teratoma in an infant. PMID- 10742753 TI - Factors influencing nutritional status of children in Bihar. PMID- 10742755 TI - Weighty Reporting. PMID- 10742756 TI - Gallbladder Findings after Cholecystectomy in Morbidly Obese Patients. AB - Morbidly obese patients constitute a high risk group for the development of gallbladder disease. In our series 70 consecutive patients underwent vertical gastroplast in an effort to manage morbid obesity. The mean age was 37 years (range 20-60), and the mean excess body weight was 92 kg (range 52-265). Six patients (8.5%) had undergone cholecystectomy before bariatric surgery because of symptomatic cholelithiasis. The remaining 64 patients underwent cholecystectomy at the time of vertical gastroplasty. Ninety-seven percent of the removed gallbladders had gross or histologic abnormalities, including cholelithiasis 18.5% (13 patients), and cholesterolosis 31% (22 Patients). Histologically, chronic cholecystitis was present in all patients with cholelithiasis and cholesterolosis. Chronic cholecystitis alone was found in 27 patients (38.5%) and only two patients (3%) had normal findings. The mean excess body weight of the patients with cholesterolosis (96 kg) was not significantly greater than that of patients with cholelithiasis (89 kg) or chronic cholecystitis (88 kg). Our findings suggest that cholecystectomy should be performed in all morbidly obese patients concomitant with vertical gastroplasty. PMID- 10742757 TI - Laparoscopic Cholecystectomy after Vertical Banded Gastroplasty. AB - Following vertical gastroplasty, laparoscopic cholecystectomy has been performed in 27 consecutive patients who developed symptomatic gallstones. Dissection identified structures without difficulty in these patients, and problems were not encountered in these procedures with the laparoscopic technique PMID- 10742758 TI - Bariatric Surgery vs. Conventional Dieting in the Morbidly Obese. AB - Weight loss and psychosocial events have been compared between low calorie conventional diet (n = 11) or following obesity surgery (n = 17). Interviews were >/= 9 months following initiation of treatment. After surgery significantly less hunger was experienced (surgery 76% [13/17] vs diet 18% [2/11] p < 0.01) and less will-power was required to stop eating (surgery 88% [15/17] vs diet 27% [3/11] p < 0.001). More dieters stopped eating because of 'figure and health' (surgery 12 % [2/17] vs diet 64 % [7/11 ] p < 0.01) whereas postoperative patients stopped due to vomit avoidance (surgery 53% [9/17] vs diet 0% [0/11] p /= 9 months following surgery or beginning a conventional diet, the morbidly obese have a more positive response to surgery. PMID- 10742759 TI - Taste Acuity of the Morbidly Obese before and after Gastric Bypass Surgery. AB - Obese individuals have an increased preference for high caloric foods, such as sweets and fats. However, following gastric bypass (GBP) surgery, morbidly obese patients tend to avoid these foods. We hypothesize that this aversion may occur, in part, from permutations in taste acuity. To test this hypothesis, taste detection and recognition thresholds for the four basic tastes (salt, sweet, sour, and bitter) were assessed using a modification of the Henkin forced choice three stimulus technique. Taste acuity measurements were obtained at baseline and at 30, 60, and 90 days post-operative for six morbidly obese GBP women and ten non-surgical, lean female controls. We found nonsignificant differences in taste detection and recognition thresholds between morbidly obese and lean control study subjects at baseline, and no significant correlation between taste acuity and body size. Furthermore, in our study population of lean and obese women, ages 26 to 52, there were no significant interrelationships between baseline taste thresholds and known effectors of taste acuity, i.e., zinc levels, glycemic status, liver and kidney function, or age. Following GBP surgery, a significant up-regulation in taste acuity for bitter and sour was observed along with a trend toward a reduction in salt and sweet detection and recognition thresholds. These findings would suggest the following: (1) taste acuity does not influence taste preferences of the obese individual who has not had bariatric surgery; (2) taste effectors such as zinc, when within the range of normal values, do not alter thresholds of the 4 basic tastes; and (3) weight loss following gastric bypass surgery is associated with an up-regulation in taste acuity in the morbidly obese. Studies are currently under investigation at our center to identify the specific etiology of taste acuity upregulation in the morbidly obese following GBP surgery. PMID- 10742760 TI - A Study on Predicting Weight Loss Following Surgical Treatment for Obesity. AB - To date, longitudinal weight loss analyses (curve-fitting) have been complicated by non-linear weight patterns, incomplete follow-up, and varied follow-up times. Therefore, the cross-sectional design (one time point survey) was chosen to study predictors of weight loss at yearly postoperative time intervals (+/- 6 months). Mean values for the initial cohort of 7,540 patients were: age 37.4 years (+/- 9.4), weight 124.0 kg (+/- 25.5), height 165.4 cm (+/- 8.5). Females comprised 87.7% of the data set. Follow-up was 62.5% at year one, 32.0% at year 2, 20.6% at year 3, and 15.4% at year 4. The multiple regression model used included 20 explanatory variables and was performed separately for four yearly time points. Only operative weight, initial visit height, age, and operative type were consistent predictors of weight loss (p < 0.05) at all time points examined and accounted for 40-50% of weight loss variation. PMID- 10742761 TI - Long-term Follow-up of Jejunoileal Bypass Patients with Preoperative Cirrhosis of the Liver. AB - Though obesity, alcohol abuse and jejunoileostomy are often a dangerous combination, exceptions do exist. This paper reports on the long-term outcome of 12 patients who had hepatic cirrhosis prior to jejunoileostomy. The six patients who refrained from or significantly restricted alcohol intake following jejunoileostomy all survived a minimum of 2 years following surgery. Four of these patients, now 6 to 25 years post surgery, are still living as of August 1993. PMID- 10742762 TI - Laparoscopic Ileogastrostomy. AB - Laparoscopic ileogastrostomy was successfully performed on two of three morbidly obese persons. In our first case, access ports proved too short and the patient was converted to an open procedure. Aims were to carry out surgery for the morbidly obese patient through a laparoscope and, as a consequence, increase ambulation, while reducing pain, morbidity, and the chance of apnea (due to impaired breathing in the first 24 h following conventional surgery). Length of laparoscopic surgeries for the second and third patients were 5 and 4 h, respectively, while hospital stays were 10 days and 5 days. Pulmonary function tests at 24 h were carried out and showed a great advantage in favor of the laparoscopic approach. Response of the medical team to this procedure was that it was more time-consuming and demanding than open surgery. Although gastric banding and gastric stapling have been accomplished laparoscopically, we believe these to be the first procedures with anastomoses carried out on morbidly obese patients. PMID- 10742763 TI - Laparoscopic Vertical Banded Gastroplasty with Complete Transection of the Staple Line. AB - The authors describe a vertical banded gastroplasty with complete division of the staple-line performed via the laparoscope PMID- 10742764 TI - Endoscopically Assisted Gastric Stomal Dilation for Reflux and Vomiting after Gastric Banding. AB - Stomal stenosis after gastric banding is one the troublesome problems that can lead to eventual reoperation. Several publications refer to this complication and to reoperation. The author describes a method of dealing a short or long-term basis by dilatation of the stoma. Stomal stenosis is probably more common with non-rigid gastric bands such as Dacron; the author postulates this is due to contracture of the pseudo-capsule which develops over the band in time. By repeated dilatations, this capsule can be stretched, and thereby the stenosis managed. The passage of regulated dilators, placed over an endoscopically guided wire, facilitates this. The author further hypothesizes that rigid bands do not contract after the inevitable pseudo-capsule formation. PMID- 10742765 TI - Gastroscopically Assisted Balloon Tamponade for Massive Bleeding after Silastic Ring Vertical Gastroplasty. AB - We report a case of massive upper gastrointestinal bleeding after silastic ring vertical gastroplasty. The bleeding originated from the staple-line at the insertion point of the silastic ring. A Sengstaken-Blakemore tube tamponaded the area of bleeding, by a new technique whereby the gastroscope was inserted through the gastrostomy orifice and used to guide the placement of the occluding balloon. PMID- 10742766 TI - Standards for Reporting Results. PMID- 10742767 TI - Surgery for Severe Obesity: Information for Patients. PMID- 10742768 TI - Motivating the Bariatric Patient. AB - Bariatric surgeries can fail. Whether it be the fault of patient or the procedure, an evaluation of the reason for failure should be done. If the fault is with the procedure, there are revisions. If it is with the patient, how do we revise human behavior? A judgment must be made preoperatively if the patient will be an acceptable operative risk, and if the individual is an acceptable personality risk. Behavior modification, and a focus on positive factors of weight loss are what are believed to be characteristics for successful weight loss and long-term success. A multidisciplinary team should work together preoperatively and postoperatively to educate, encourage and support the bariatric patient so that he or she will be able to focus on motivation and compliance. The goal is to see that each individual will internally develop a sense of personal confidence and self-worth. PMID- 10742769 TI - Mechanisms of Weight Loss following Intestinal Bypass Surgery. AB - Dramatic weight loss has been observed following intestinal bypass surgery. Initially, malabsorption was recognized as the major cause of weight loss. Only in recent years have investigators noted that the intestinal bypass could significantly reduce caloric intake, which largely or almost fully explains the weight loss following this procedure. The reduced energy intake as well as malabsorption are probably the major causes of weight loss after intestinal bypass surgery. Increased energy output has been proposed as a factor for weight loss, and more recently, it has been suggested that certain hormonal changes may play a role in regulating satiety and metabolic processes. This article examines these proposed factors that may impact on weight loss following intestinal bypass surgery. PMID- 10742770 TI - Metabolic Implications of Obesity, Weight Loss and Energy Expenditure following Gastric Restrictive Surgery. AB - This review examines the energetics and metabolic parameters which influence the effectiveness of gastric restrictive surgery in achieving weight loss in the clinically severely obese patient. Among the subjects discussed are the metabolic determinants and consequences of obesity, energy expenditure and its components, factors other than dietary restriction and weight loss which affect energy expenditure, and the metabolic risk factors for weight gain. The role of exercise is reviewed, including the effects of exercise on energy balance and the thermic effect of food. The value of combining exercise with diet restriction, the effect of caloric restriction on the capacity to exercise and the place of exercise in the obese diabetic are examined. Finally, the metabolic consequences of gastric restrictive surgery, the adaptive response to surgically-induced weight loss and nutritional recommendations following gastric restrictive surgery are reviewed. PMID- 10742771 TI - Utility of Hydrogen and Methane Breath Tests in Combination with X-Ray Examination after a Barium Meal in the Diagnosis of Small Bowel Bacterial Overgrowth after Jejuno-Ileal Bypass for Morbid Obesity. AB - To study why the symptoms of abdominal bloating occurring in a number of patients after jejuno-ileal bypass for morbid obesity become resistant to antibiotics, we used a method which combined a hydrogen breath test after lactulose with an X-ray examination of the abdomen after barium. Ten operated patients with bloating symptoms resistant to antibiotics, ten operated patients without symptoms or with pre-existing symptoms, that had remitted after antibiotic treatment and ten nonoperated obese controls were investigated. There was a significant correlation between post-surgical symptoms persisting after antibiotics and the exhalation of large amounts of hydrogen of colonic origin (> 100 parts per million) after lactulose. Furthermore, symptomatic patients had high prevalence of colonic motility disorders (slow transit). In these patients, treatment with a prokinetic (cisapride 40 mg/kg/day for 10 days) reduced colonic transit time, colonic hydrogen production and bloating symptoms. Abdominal symptoms in these patients may therefore have other causes than small bowel bacterial overgrowth alone. All operated patients with persistent abdominal bloating should therefore be investigated before starting empirical treatment with antibiotics. PMID- 10742772 TI - Vertical Banded Gastroplasty: Stable Trends in Weight Control at 10 or More Years. AB - Whether long-term weight loss outcomes will succumb to adaptations in patients after gastric restrictive procedures has yet to be determined. The relationship, if any, of post-operative weight loss and maintenance to follow-up by support group and/or by surgeon, at least long-term, is uncertain. These questions have prompted review of my patients at 10-12 and at 5-10 years after vertical banded gastroplasty (VBG). Of the 118 patients (43 at 10-12 years), 65 were able to be contacted (55%). Successes were defined as patients who at the time of study interview, without operative revision, had lost at least 25% of their pre operative excess weight. All known revisions (7) are reported as failures; only one of the seven was discovered in the study interviews. The mean body mass index of the entire study group (65) pre-operatively was 49.7, with 32% (21/65) super obese. Patient pre-operative profiles of successes and failures of both interval groups are remarkably similar. Successes were 65% in the >/= 10 year group and 68% in the 5-10 year group. Respectively, mean percent excess weight loss was 46% and 54%. Six (of seven) failures, and ten (of 13) successes, in the >/= 10 year group had 5.5 cm circumference outlets; all patients in the 5-10 year group were at 5.0 cm. Marked reduction in the follow-up pattern in the 5-10 year group failures was not significant but appears predictive. This experience suggests little deterioration in success rate, weight loss among successes, or revision requirement in VBG patients between 5 and > 10 years. PMID- 10742773 TI - Vertical Banded Gastroplasty with Adjustable Silicone Band: Preliminary Experience. AB - A series of ten patients operated on with vertical banded gastroplasty (VBG) with an adjustable silicone band at the outlet is presented. The loss of body weight and complication rate is evaluated. Preoperative mean excess overweight of the patients was 94% and mean BMI was 42.6. The loss of body weight at one year's follow-up was 38 kg or 59% of excess weight. Complications were one case of infection at the subcutaneous injection port and one case of a nonfatal pulmonary embolus. The results so far are thus comparable with VBG with a conventional fixed band, but the adjustable band actually simplifies the operative procedure since no exact calibration of the collar size is necessary at the time of surgery and should diminish the need for reoperations due to misalignment of collar size. The possibility of better weight control in the long-term perspective remains to be proven. PMID- 10742774 TI - Adjustable Gastric Loop: An Effective Method of Treatment for Morbid Obesity- Preliminary Report. AB - During a 2-year period, the adjustable gastric loop gastroplasty was performed on more than 100 patients, all more than 100 lb (45 kg) over ideal weight. Although a small sampling in a short-time period, the initial follow-up records of weight loss have been very satisfactory. The significant advantages of this procedure over others are: (1) the procedure is fairly simple; (2) without the use of staples, this procedure does not carry the common complications of infection and follow-up surgeries for morbid obesity; (3) follow-up adjustments can be performed in the office with local anesthesia. Overall, the savings for patients, in both time and money, are another important factor in making the adjustable gastric loop a procedure of choice. PMID- 10742775 TI - Normalization of Insulin Sensitivity in the Obese Patient after Stable Weight Reduction with Biliopancreatic Diversion. AB - Insulin resistance is a common feature in obese patients. To evaluate the modifications in insulin sensitivity after a bariatric operation such as Bilio pancreatic diversion (BPD), three groups of subjects (14 normal controls (N); seven ex-obese patients (X) with at least 2 years at weight-stable conditions after BPD surgery; and eight morbidly obese patients (0)) were studied with intravenous (IVGTT) and oral (OGTT) glucose tolerance tests. The ratio of the area under the curve (AUC) for glucose over that of insulin was used as a measure of insulin sensitivity. All the following tests were conducted as Bonferroni corrected pairwise t-tests, in case overall ANOVA was significant. No significant difference was found between N and X subjects, while obese patients showed a reduced AUCg/AUCI ratio with respect to the normal controls (O vs N: 0.01164 +/- 0.00039 vs 0.02392 +/- 0.0039, p < 0.05). IVGTT, AUCS: significant differences were found in each case: N vs X: 0.0591 +/- 0.0075 vs 0.1402 +/- 0.0399, p < 0.05; N vs 0:0.0591 +/- 0.0075 vs 0.0223 +/- 0.0031, p < 0.01; X vs 0:0.1402 +/- 0.0399 vs 0.0223 +/- 0.0031, p < 0.05. IVGTT-derived data were also analyzed using the minimal model of glucose kinetics; with this method, glucose effectiveness was significantly different between normal subject and obese subjects (0.0248 +/- 0.00288 Vs 0.00906 +/- 0.00135 per min, p < 0.001). The insulin sensitivity index was not significantly different between normal and ex obese subjects, while both of these groups were significantly different from obese patients (N vs 0: 12.04 x 10&sup5; +/- 2.61 x 10&sup5; vs 3.29 x 10&sup5; +/- 0.61 x 10&sup5;, p < 0.066; X vs 0: 16.42 x 10&sup5; +/- 4.23 x 10&sup5; vs 3.29 x 10(1)+/- 0.61 x 10&sup5; per min per pM, p < 0.02). In conclusion, the present study indicates that, after a body weight reduction operation capable of almost re-establishing ideal body weight like BPD, obese individuals with a family history of obesity show a normalization of insulin response to glucose load. PMID- 10742776 TI - Whither Obesity? PMID- 10742777 TI - Motility Disorders after Roux-en-Y Gastrojejunostomy. AB - About 30% of patients who have a Roux-en-Y gastrojejunostomy after gastrectomy suffer from abdominal pain, nausea, vomiting of food and bloating made worse by eating. This syndrome, called the Roux stasis syndrome, is caused, in part, by a motility disorder of the Roux limb. Transection of the jejunum during the construction of the limb separates the limb from the natural small intestinal pacemaker located in the duodenum. Ectopic pacemakers then appear in the limb and trigger retrograde contractions in its proximal portion. These contractions slow transit through the limb and result in Roux stasis. Current nonsurgical treatment of the syndrome includes the use of prokinetic agents and intestinal pacing, neither of which has demonstrated long-term benefits. A near-total gastrectomy may speed upper gastrointestinal transit somewhat, but stasis in the Roux limb often persists. Our current approach alms at preventing the syndrome by the use of an 'uncut' Roux limb, an operation which preserves myoneural continuity between the duodenal pacemaker and the Roux limb and so prevents the appearance of ectopic pacemakers and stasis in the limb. PMID- 10742778 TI - Improvement in Obesity-associated Medical Conditions following Vertical Banded Gastroplasty and Gastrointestinal Bypass. AB - Thirty-nine patients undergoing either Silastictrade mark ring vertical banded gastroplasty (SRVG, n = 23) or Roux-en-Y gastrointestinal bypass (RGB, n = 16) over a 1-year period were analyzed retrospectively. Weight loss averaged 40 kg (89 lb). Clinical diabetes mellitus was markedly improved in seven out of nine patients (p = 0.023). Shortness of breath resolved in all 26 patients who had this condition preoperatively (p < 0.001). Orthopedic problems, high blood pressure, and self-assessment of general health and quality of life were also dramatically improved. We conclude that bariatric surgery serves as an effective method to alleviate a multitude of conditions associated with morbid obesity. PMID- 10742779 TI - Clinical Benchmark for Gastric Stapling Procedures. AB - To help answer the call to cut costs of surgical care, hospitals and physicians have joined to compare methods of care for the more common Diagnosis Related Group (DRG) diagnoses to form a Benchmark. Since many bariatric surgeons are the only ones performing this surgery in their primary hospitals, they do not have two or more surgical routines for comparison. This presentation compares data for the preoperative work-up, operating-room, and methods of postoperative care used by 29 members of the American Society for Bariatric Surgery (ASBS). There was representation of both academic and private surgeons and hospitals. To target areas for possible savings, the hospital bills of 16 patients without complication were compared. The synthesis of this information revealed significant differences in the extent and cost of preoperative work-up, antibiotic coverage, other postoperative care, and length of stay. These differences are examined under the assumption that patient outcome was the same. PMID- 10742780 TI - Can a Bariatric Surgery Program Succeed Without Close Patient Proximity? The Experience in a Military Medical Center. AB - Many centers advocate close patient follow-up and a multidisciplinary approach as necessary ingredients for the success of a bariatric surgery program. The military medical environment is not suitable for these conditions. Many patients are referred from great distances to the large regional medical centers, thereby preventing such close follow-up and the ability to create active support groups. A review of the 4-year experience with bariatric surgery at a major military medical center was conducted to determine if the program could be successful, considering that 60% of its patients came from out of state. Hospital records of all 92 patients and the bariatric registry were reviewed. A comprehensive survey to update weight data and assess patient satisfaction was sent to the first 72 patients to undergo surgery. There were no deaths and a perioperative complication rate of 18%. By I year after surgery, 67% of patients lost greater than 50% of their excess weight (mean = 56.6%). Sixty-eight percent of patients responded to the survey; 87% felt they were better off and satisfied with their quality of life since surgery, and 75% reported improved energy levels. If given a chance to rethink their decision, 86% of responders would choose surgery again. A total of 91% were satisfied with their follow-up. Patient proximity to the medical center did not influence weight loss or patient satisfaction. These results suggest that a bariatric surgery program can succeed in a medical environment such as the military where patients are likely to live at great distances from the hospital. PMID- 10742781 TI - Redo Gastric Restriction: A Higher Risk Procedure. AB - Twenty-one patients have undergone redo gastric restrictive procedures, three after gastric bypass, 18 after gastroplasty. Weight at the time of the redo procedure was 101 kg +/- 32.4 SD (222 lb +/- 71.2) (178% IBW +/- 33.8), down from 137 kg +/- 65.5) (217% ISW +/- 33.9) at the time of initial procedure. Sixteen of 21 (76%) initial procedures failed because of staple-line dehiscence, at least in part. Redo procedures were vertical banded gastroplasty (VBG) in ten, gastric bypass (GB) in ten, and silico ring banded gastroplasty in one. Early complications were three gastric leaks, one enterocutaneous fistula, one gastroplasty outlet obstruction, and one wound infection occurring in three of ten VBG and three of ten GB. All three leaks and one fistula were not seen on gastrograffin/barium swallow done 2-4 days after the redo procedure. Late complications were failure of seven redo procedures, one small bowel obstruction, and one recurrent enterocutaneous fistula, with two lost to follow-up. We conclude that redo gastric restriction procedures carry relatively high perioperative risks. Radiologic contrast studies done in the early postoperative period do not necessarily detect leaks, which are presumably due to gastric ischemia and subsequent infarction. Ischemia is thought to be due to devascularization from the initial procedure. Late failure is unfortunately common, possibly reflecting behaviors which lead to the initial staple line dehiscence and failure. Higher risk of perioperative complications and late failure must be considered prior to revisional surgery. PMID- 10742782 TI - Long-term Results of Gastroplasty for Morbid Obesity: Binge-Eating as a Predictor of Poor Outcome. AB - We investigated the long-term results after vertical banded gastroplasty (VBG) for morbid obesity and evaluated some predictors of poor outcome. Twenty-seven morbidly obese patients (mean BMI 50 kg/m(2)) who underwent VBG a mean of 5.4 years earlier were studied. The mean weight loss was 31 kg or 40.5% of excess weight. Ten patients (37%) were within 50% of their ideal weight. The patients were classified as bingers (n = 10) and non-bingers (n = 17) according to the modified Binge Eating Scale. One-year postoperative weight losses were 55% and 57% respectively of excess weight, but at the time of the study weight losses were 24% and 50% of excess weight (p = 0.04) in the bingers and non-bingers, respectively. The mean weight regain was 23 kg in the bingers vs 8 kg in the non bingers, p = 0.01. Two bingers had excellent weight loss results, but otherwise an unsuccessful outcome. Weight reduction did not correlate with age, sex or age of onset of obesity. These results show that binge-eating strongly predicts poor weight loss maintenance after gastroplasty in the long-term, though initial weight reduction is good. We suggest that binge-eating should be evaluated before gastroplasty and taken into consideration in the design of treatment. PMID- 10742783 TI - Staple-line Disruptions in Vertical Banded Gastroplasty Related to Different Stapling Techniques. AB - We performed one or more upper G.I. barium single-contrast studies on 125 out of 166 Mason vertical banded gastroplasty (VBG) operated patients (total: 226 X-ray examinations during a 3 month-10 year postoperative period). Forty four patients had a staple-line performed by double application of a 2-row stapler with manual reinforcement (group 1); 12 had a single application of a 4-row stapler with reinforcement (group 2); the last 69 patients had a partition with a 4-row stapler without reinforcement (group 3). A staple-line disruption was observed in 34 cases (27.2%); 17/44 (38.6%) cases belong to group 1, 6/12 cases (50%) to group 2 and 11/69 cases (15.9%) to group 3. The range of breakdowns diameter was 2-30 mm (nine cases double, one case quadruple). In 16 out of 34 cases we observed a preferential contrast pathway through the perforations. In 23 cases we noted a weight regain and in one case an initial failure on weight loss; in 12 cases the excess weight loss (EWL) was less than 30%. In group 3, we found two tiny perforations at the top of the partition, but another nine along with the staple-lines. In our experience, staple line disruptions are only reduced using the 4-row stapler without reinforcement; even with this stapling technique late breakdowns along the staple-line, not only at the apex of the partition, can occur. PMID- 10742784 TI - The Double Application of the TA-90B Four-row Stapler and Pouch Formation: Eight Rows are Safe and Effective in Roux-en-Y Gastric Bypass. AB - Staple-line failure in gastroplasty means eventual weight re-gain in most cases. In gastric bypass this can be disastrous, with a high risk of stomal ulcer formation and reflux esophagitis, as well as weight re-gain. Because of this, total transaction of the two pouches has become more popular, eliminating the staple-line altogether, and thereby preventing any possibility of staple-line failure. However, there is a higher risk of leaks and the two pouches can fistulize into one another, producing the equivalent of a staple-line failure. From July 1987 to August 1991, 350 patients had a Roux-en-Y gastric bypass (RYGBP) with a single application of the TA-90B Autosuturetrade mark instrument, with four known staple-line failures. From August 1991 to May 1993, 210 patients received a double application of the TA-90B four row Autosuturetrade mark stapler. There are no known staple-line failures to date and no leaks, with quite adequate weight loss. This technique has proven safe and effective in the short run. PMID- 10742785 TI - Marlex Mesh Gastric Banding: A 7-12 Year Follow-up. AB - This paper presents a 7-12 year (mean 9.8 years) follow-up of 92 extremely obese patients treated with Marlextrade mark mesh gastric banding (GB). The follow-up rate was 92% (85 patients). Weight loss was initially good (an average reduction in BMI of 13 during the first year), but late weight gain has been a common complaint and a reason for reoperation. Other complications that necessitated reoperation were severe vomiting and esophagus not amenable to medical treatment. Four patients have developed signs of Barrett's esophagus at late follow-up. Forty-six patients (50%) were reoperated 70 times for correction of the band or conversion to vertical banded gastroplasty (VBG). The most common reoperative procedure was conversion to VBG (38 patients). Only 25 (31%) of the 80 patients with long-term follow-up have an intact band. Our results show the need of long follow-up and that this GB cannot be recommended for the treatment of morbid obesity. PMID- 10742786 TI - Stoma Adjustable Silicone Gastric Banding: Results in 111 Consecutive Patients. AB - From April 1990 through December 1992, 111 patients (80 females, 31 males, mean age 38 years, range 16-60) underwent stoma adjustable silicone gastric banding (SASGB) at the Department of Surgery, University Hospital, Padua, Italy. Patients' characteristics were: mean height 166 +/- 8 cm; mean body weight (BW) 129.1 +/- 21.6 kg; mean body mass index (BMI) 46.4 +/- 6.3 kg/m(2); mean percentage of ideal body weight (%IBW) 206.2 +/- 27. Eighty-eight patients were morbidly obese and 23 superobese. All patients were available for follow-up. Median follow-up was 18.8 months (range 12-44). At I year (I 03 patients), mean postoperative BW, BMI, %IBW and excess weight lost (%EWL) were 101.5 +/- 20 kg, 36.5 +/- 6 kg/m(2), 164 +/- 30 and 40.8 +/- 19 respectively; at 2 years (58 patients) 92.3 +/- 19 kg, 33.1 +/- 6 kg/m(2), 148.8 +/- 28, 52 +/- 23, respectively, and at 3 years (26 patients) 86.9 +/- 14 kg, 31.4 +/- 5 kg/m(2), 141.5 +/- 25 and 63.6 +/- 20 respectively. The overall postoperative mortality rate was zero and the early morbidity rate 9%. Late complications were band slippage (two patients), stoma stenosis with pouch dilatation (seven patients), band erosion (one patient), reservoir leakage (three patients) and reservoir infection (two patients). Surgical revision was performed in ten (9%) patients, two of whom required band removal. Most complications occurred in patients who underwent SASGB during our initial experience. Our findings confirm that SASGB is a safe and effective surgical means of achieving weight reduction. PMID- 10742787 TI - Gastric Bypass Distal Roux-en-Y Jejunal Interposition with Selective Proximal Vagotomy and Posterior Truncal Vagotomy. AB - Selective proximal vagotomy and posterior truncal vagotomy have been performed in 71 consecutive gastric bypass (GBP) patients from June 1991 to December 1992. Vagotomy was used to prevent or diminish the incidence of marginal ulcer in GBP patients. Anterior and posterior highly selective proximal vagotomy with circular instrument stapled gastrojejunostomy in patients undergoing GBP distal Roux-en-Y with jejunal interposition had no marginal ulcer complications (minimal follow-up 18 months). PMID- 10742788 TI - Prevention and Reversal of Liver Damage following Biliopancreatic Diversion for Obesity. AB - Liver damage following biliopancreatic diversion (BPD) for obesity is an infrequent, dangerous and, when left untreated, even potentially fatal complication. The laboratory expression of developing liver damage is the progressive deterioration of the liver enzymes over 70 i.u., persisting and progressing beyond 6 months after surgery. Until recently, the treatment for this complication consisted of hospitalization for total parenteral nutrition and then, if this proved unsatisfactory, surgical reversal. In this report, a new therapeutic approach to this condition is presented. Four patients who, following a BPD for obesity, developed deterioration of the liver enzymes (AST, ALT over 70 i.u.) which persisted and progressed beyond the first 6 months after surgery, were given a treatment consisting of the ambulatory administration of 'Viokase' tablets, an uncoated pancreas extract with a high protease content (30,000 i.u.) taken with protein-rich meals. After 2-4 months of Viokase treatment, in all four patients the liver enzymes returned to normal levels: AST from 160, 86, 120 and 89 i.u. to 26, 20, 24 and 27 i.u. Two patients who complained of diarrheic stools (ten per day) received metronidazole for several days, dramatically improving their condition and returning their daily stool count to normal. PMID- 10742789 TI - Invited Commentary. PMID- 10742790 TI - Helicobacter pylori-induced Diarrhea Ileogastrostomy. AB - A retrospective study of all ileogastrostomy procedures (n = 26) performed in 1993 by one surgeon (IGMC) was carried out to investigate the hypothesis that Helicobacter pylori may be implicated in certain severe cases of postoperation nausea and diarrhea. Ten of 26 persons (38.5%) displayed nausea and notable diarrhea (greater than or equal to ten bowel movements per day), seven of which warranted upper GI investigation. One hundred per cent (seven of seven) of these persons were found to possess H. pylori upon C-14 breath test. In four of six cases eradication therapy (1 g amoxicillin b.i.d./20 mg omeprazole b.i.d. for 2 weeks) corresponded with a resolution of severe nausea and diarrhea (one additional case involved omeprazole use only), suggesting that H. pylori should be considered as a possible cause of these symptoms post-ileogastrostomy. Additionally, in four of seven cases persons were re-tested (C-14 breath analysis) at least 1 month post-therapy and in this group three persons were found to be free of the organism. All three cases of notable diarrhea and nausea resolved with treatment, providing the strongest evidence for a possible association between infection and these symptoms. PMID- 10742791 TI - Jujunoileal Bypass with Ileogastrostomy for Relief of Undesirable Side-effects of Jejunoileal Bypass. AB - Jejunoileal (JI) bypass with ileogastrostomy was performed on eleven patients who previously had JI bypass and were having undesirable side-effects. Short-term (17 44 months) follow-up observation reveals good to complete relief of undesirable side-effects in ten of the eleven. In the one case of total failure the author felt that the patient's symptoms were not caused by her JI bypass. The author suggests that JI bypass with ileogastrostomy be considered for JI bypass patients who are having undesirable side-effects, gastric stapling failures, and as original surgery in morbidly obese patients. PMID- 10742792 TI - Invited Commentary. PMID- 10742793 TI - Invited Commentary. PMID- 10742794 TI - The Challenge of Teaching Behavior Modification. AB - In March 1991 the National Institutes of Health claimed surgery as a viable means of treatment for clinically severe obesity once other conventional methods had failed. Also stated in the same report was that postoperative care, nutritional counseling and surveillance should continue for an indefinitely long period. Many surgeons are choosing regular office examinations and group behavior modification sessions for this surveillance. This educational process often poses insurmountable challenges for the professional who chooses to follow the patients postoperatively. As the doctor continues to monitor vital signs and systems, the group facilitator must monitor the patient's emotional ups and downs, signs of low self-esteem, and evidences of self-defeating behavior. Considering this, the ultimate challenge is that those who teach must understand what needs to be taught. The educational process could be divided into three areas, including lessons of the mind, the body, and the 'self.' The group leader will not always find the material for these lessons in books. Many will be developed through heartfelt experiences, but the most important resource for information will be the patient. PMID- 10742795 TI - The Use of Pneumoperitoneum in the Repair of Giant Hernias. AB - Preoperative pneumoperitoneum is used to re-establish the right of domain for abdominal viscera before repair of otherwise inoperable giant abdominal hernias. The aim of this study was to evaluate the use and safety of preoperative pneumoperitoneum in the repair of giant hernias in relation to surgical treatment of obesity. The medical records of patients who underwent preoperative pneumoperitoneum in the treatment of giant hernias between 1953-1993 were reviewed. There were 27 patients (11 males, 16 females; mean age: 56 years) whose mean preoperative weight was 99 kg (range: 69-183). Hernias were predominantly in the midline (17). Other locations were right lower quadrant (5), right upper quadrant (3) and groin (2). The mean duration of preoperative pneumoperitoneum was 28 days (3-100). Subcutaneous emphysema developed in three patients with no sequelae. Primary repair of the giant hernia without Marlex mesh was possible in 19 patients (70%). Marlex mesh was used in seven (26%). One patient had a fascia late graft. Operative complications were one pulmonary embolus and one hematoma. There were no deaths. We conclude that preoperative pneumoperitoneum is a useful adjunct to giant hernia repair. Severe obesity should be corrected before preoperative pneumoperitoneum and hernia repair. Some patients may need mesh to replace insufficient abdominal wall or to reinforce repair. PMID- 10742796 TI - Significant Changes in the Lipid-Lipoprotein Status of Premenopausal Morbidly Obese Females following Gastric Bypass Surgery. AB - The morbidly obese premenopausal female may be more dyslipoproteinemic and at greater risk for developing coronary heart disease than her lean or less seriously obese counterparts. The purpose of the present study was to examine the effects of weight loss with Roux-en-Y gastric bypass surgery on the lipid lipoprotein status of morbidly obese, premenopausal females. Anthropometrics and blood samples for lipid-lipoprotein analyses were obtained before surgery and 6 - 12 months postoperatively. Following surgery, patients lost 30% of their initial body weight, along with a 40% decline (p < 0.01) in total triglyceride and a 20% decrease (p < 0.01) in total cholesterol. Levels of cholesterol in the high density lipoprotein (HDL) fraction were unaffected by weight loss, but there was a significant (p < 0.05) increase in the proportion of HDL in its more buoyant and anti-atherogenic form, i.e. HDL-L. The apolipoprotein B-containing lipoproteins, very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL), were reduced up to 70% following surgery. There were no significant changes in VLDL or IDL particle composition, i.e. cholesterol/triglyceride, cholesterol/ protein, but there was a significant (p < 0.01) increase in the ratio of cholesterol/apolipoprotein B in LDL, suggesting a shift from the small, dense atherogenic LDL to a larger, less atherogenic particle. We conclude that weight loss following gastric bypass surgery markedly improves the lipid-lipoprotein status of morbidly obese premenopausal females and, thereby, significantly reduces the risk of coronary disease. PMID- 10742797 TI - Psychosocial Outcome and Long-term Weight Loss after Gastric Restrictive Surgery for Morbid Obesity. AB - Forty-five of 60 consecutive morbidly obese patients who had a vertical banded gastroplasty carried out by the one surgeon between 1982 and 1988 were assessed by questionnaire at long-term follow-up in 1993. Eighteen patients (40%) had maintained their BMI at close to the lowest achieved levels. Twenty-seven patients (60%) had had a significant rise in BMI, and 14 of these (31%) had gained weight to return close to or above their pre-surgery BMI levels. No reliable predictors of successful long-term weight loss were detected in the pre operative data. Forty-eight patients (84%) were satisfied with their surgical treatment. Twenty patients (44%) reported improved social life after surgery. Twenty-one patients (46%) reported a similar social life and only four patients (9%) a worse social life. At follow up five patients (12%) reported emotional problems related to their weight loss surgery and two of this group had had psychiatric counseling for depression. Pre-operative psychiatric assessment appeared to have facilitated intervention by the psychiatrist with these patients. Gastric restrictive surgery, however, remains unpredictable in its long term weight loss effect. PMID- 10742798 TI - Initial Experience with the Surgical Management of Morbid Obesity Associated with Symptomatic Gastro-esophageal Reflux: A Comparison between Gastroplasty Alone and Gastroplasty with Anterior Fundoplication. AB - The optimal management of patients with morbid obesity and gastro-esophageal reflux disease (GERD) remains an unresolved issue. We have performed a vertical banded gastroplasty combined with an anterior fundoplication (VBG + AF) in 28 selected morbidly obese patients with moderate or severe heartburn. The patients who underwent VBG +/- AF are compared to patients who had similar heartburn symptoms and underwent gastroplasty alone during this period. In the VBG + AF group there were two treatment failures (7%). In the gastroplasty group there were 63 patients with 15 treatment failures (24%). These differences were independent of demographic and weight loss variables. These results suggest that VBG +/- AF may provide a superior option for the management of morbidly obese patients with GERD. PMID- 10742799 TI - Vertical Ligated Gastroplasty by Clamp, Cut and Suture: A Series of 504 Cases Dating Back to 1977. AB - A restrictive operation for obesity makes a small pouch above a reinforced stoma. This series of 504 patients of one surgeon dating back to February 1977 has developed the same end result as the vertical banded gastroplasty, but does not involve stapling. The stomach is divided from the angle of His downwards for 11 or 12 cm, and the right channel is ligated with two ligatures made of Teflon coated braided stainless steel wire. The pouch is made snug on a 30-French (9.5 mm) bougie, and the stoma is accurately gauged to 43 mm circumference, by matching marks made on the ligature. Making the pouch actually starts from the middle of the stomach and progresses upwards by clamp, cut and suture. The sutured anastomosis would appear to be more reliable than a stapled one. Weight loss is similar to other restrictive procedures, and two series of 30 consecutive cases each are presented with a 100% follow-up: 80% lost at least 60% of their excess weight, and 60% lost more than 60% of their excess weight. There has been no mortality for the primary gastroplasty operation. PMID- 10742800 TI - Vertical Gastroplasty with Artificial Pseudopylorus: A Modification in the Treatment of Morbid Obesity. Early Results. AB - A new technique of vertical gastroplasty was applied in 50 morbidly obese patients. Eight of them were male and 42 female with a mean age of 34 years (range 2058). The mean excess body weight was 77 kg (range 52-133) and the mean Body Mass Index 51 kg/m(2) (range 42-81). Under direct vision the hepatogastric and gastrophrenic ligaments were divided and two TA-90 doublerow staplers were passed together through the lesser sac vertically downwards from His angle parallel to the lesser curvature of the stomach. An additional TA-90 stapler was applied between the two double rows. The pseudopylorus was constructed 3-4 cm below the stomach angle by the use of two bands of silk No. 0 which were covered by stomach serosa, thus creating a reinforced outlet having the shape of a pylorus which we called 'artificial pseudopylorus'. The circumference of pseudopylorus was 4.8 cm and the total volume of the vertical pouch of stomach 15 20 ml. The percentage excess weight loss on the 3rd, 6th, 12th, 18th and 24th month postoperatively was 35, 51, 68, 80, and 82 respectively. One patient died 12 days after operation. One other patient was re-operated because of staple-line breakdown and acute abdomen formation. It is concluded that vertical gastroplasty with artificial pseudopylorus is a simple and safe method that avoids complications of other forms of gastroplasty, and is of value in the treatment of morbidly obese patients. PMID- 10742801 TI - Laparoscopic Gastric Bypass, Roux-en-Y: Preliminary Report of Five Cases. AB - The technique of laparoscopic gastric bypass, Roux-en-Y, has been developed, and performed in five patients. The detailed technique and instrumentation is described. Early case results show comparable weight loss, and reduced morbidity and disability. Laparoscopic gastric bypass is a feasible alternative to the open operative procedure. PMID- 10742802 TI - Laparoscopic Ileogastrostomy for Morbid Obesity. AB - This paper describes the technique of laparoscopic ileogastrostomy which we developed during the summer of 1993. The procedure is identical to that of our 'open' ileogastrostomy except that it is performed laparoscopically. The aim of the surgery is to increase ambulation of the patient, while reducing pain, morbidity, and the chance of apnea (due to impaired breathing in the first 24 h following conventional surgery), by carrying out surgery for the morbidly obese person through a laparoscope. This form of laparoscopic surgery may be completed within 4 h and, as our staff gains more experience with laparoscopic ileogastrostomy, we expect patient stays to be 2-3 days in length. Pulmonary function tests at 24 h show a great advantage in favor of the laparoscopic approach. Response of the medical team to this procedure was that it was more time-consuming and demanding than open surgery. PMID- 10742803 TI - The Psychology of Bariatric Surgery. AB - Our discussion focuses on theoretical and applied considerations for psychological care of the morbidly obese patient presenting for surgical correction of this refractory condition. Observations are gleaned from a biomedical literature review and our clinical experiences in the design and implementation of the psychological component in our extant multidisciplinary bariatric surgery program at Universal Medical Center, Plantation, Florida. The clinical sample includes 401 patients receiving vertical banded gastroplasty, Roux-en-Y gastric bypass and silastic ring gastroplasty from September of 1986 to May of 1994. Beyond a consideration of the psychological sequelae of depression, anxiety and addictions, we discuss the specific impact of psychosocial stress and implications of the morbid obesity archetype, with particular emphasis on iratrogenic medical stereotype. Owing to the nature of this report, and the literature review, our impressions are anecdotal and clinical. However, when considered against the undeserved and misconceived needs of the morbidly obese patient, we propose that a comprehensive multidisciplinary program incorporating psychological service is not only of critical benefit in enhancing patient co operation, but also in securing patients' dramatic quality of life change. Recommendations for future research and the development of standardized psychological care models for the morbidly obese patient conclude the discussion. PMID- 10742804 TI - Erosive Esophagitis following Horizontal Gastroplasty for Morbid Obesity: Treatment by Gastric Bypass. AB - We describe a patient who underwent horizontal gastroplasty for morbid obesity while exhibiting mild symptoms of gastroesophageal reflux disease. The patient lost 34.4 kg, but reflux symptoms progressed to the point of becoming almost incapacitating, associated with erosive esophagitis. The patient subsequently underwent vertical Roux-en-Y gastric bypass, becoming asymptomatic and demonstrating complete healing of the esophageal erosions. Post-operative esophageal pH monitoring demonstrated continuing pathologic reflux in this patient despite her being asymptomatic. PMID- 10742805 TI - [Postmastectomy locoregional radiotherapy for breast cancer: literature review]. AB - Postoperative radiotherapy is controversial after radical mastectomy. Recent clinical trials have shown an increase in survival with this irradiation and conclusions of previous meta-analyses should be reconsidered. The results of a large number of randomized clinical trials in which women received post mastectomy radiotherapy or not have been reviewed. These trials showed a decrease in locoregional failure with the use of postoperative radiotherapy but survival advantages have not been clearly identified. A larger number of randomized clinical trials compared postoperative radiotherapy alone, chemotherapy alone and the association of the two treatments. They showed that chemotherapy was less active locally than radiotherapy and that radiotherapy and chemotherapy significantly increased both disease-free and overall survival rates in the groups which received postoperative radiotherapy. These favourable results were, however, obtained with optimal radiotherapy techniques and a relative sparing of lung tissue and cardiac muscle. Many retrospective clinical analyses concluded that results obtained in locoregional failure rate were poor and that these failures led to an increase in future risks. Both radiotherapy and systemic treatment should be delivered after mastectomy, reserved for patients with a high risk of locoregional relapses, particularly of nodes and/or tumors with a diameter > or = 5 cm. However, radiotherapy could produce secondary effects, and techniques of radiotherapy should be optimal. PMID- 10742806 TI - [Germ cell tumors of the testes: state of the art]. AB - Germ cell tumors of the testes are rare tumors occurring in young men, the incidence of which increases continuously. They are curable in more than 80% of the cases. The treatment of stage I seminoma is lomboaortic radiotherapy, and that of stage I non-seminomatous tumors is either surveillance, retroperitoneal lymph node dissection or adjuvant chemotherapy according to the risk factors of extra-testicular involvement (pure embryonal carcinoma, vascular invasion). For advanced diseases, the standard treatment is three cycles of bleomycin, etoposide, cisplatin (BEP regimen) or four cycles of the same association without bleomycin (EP regimen) and four cycles of the BEP regimen for patients with good risk and poor risk prognostic characteristics, respectively. The five-year overall survival rates are 90% and 50% for patients with good risk and poor risk factors respectively. It is recommended to resect all residual masses after chemotherapy. The standard salvage treatment is four cycles of vinblastin, ifosfamide, cisplatin (VelP regimen). New associations of drugs are under study in order to improve the overall survival rate for the poor-risk and relapsed tumors patients. PMID- 10742807 TI - [Retrospective analysis of results of treatment of 91 oral cavity cancers from 1982 to 1992]. AB - PURPOSE: To analyse retrospectively the results of different treatment regimens of carcinomas of the floor of the mouth and tongue. MATERIALS AND METHODS: Between 1982 and 1992, 61 patients with carcinoma of the floor of the mouth and 30 with tongue cancer (25 stage I, nine stage II, 28 stage III, 29 stage IV) were treated in the radiotherapy department of Poitiers. Nine patients with stage I tumours were treated with 70 Gy low-dose rate brachytherapy only, without nodal dissection. Stages II and III were treated with combined surgery with neck dissection; and radiotherapy of stage II with nodal metastasis and for all stage III cases. Stage IV cases were treated either surgically if possible, or with combined chemotherapy and radiation. RESULTS: The five-year overall survival rate was 87.3% for stage I, 68.5% for stage II, 45.3% for stage III, and 0% for stage IV patients. Most relapses appeared in the first two years after treatment. Eight patients (32%) with stage I cancer developed nodal relapses, isolated in five cases. Complications of radiotherapy were acceptable. Four cases of osteonecrosis were observed after radiotherapy. All of these appeared simultaneously with a local relapse. CONCLUSION: These results are comparable with reports in the literature. The remarkable observation of our study is the high incidence of nodal recurrences after local treatment of stage I tumours. Therefore, local treatment is insufficient for early-stage tumours. The question of neck dissection for the early stage is discussed. PMID- 10742808 TI - [Evolution of the use of the portal imaging device: prospective study over three years]. AB - PURPOSE: To describe the evolution of the use of the electronic portal imaging device (EPID) over three periods. MATERIAL AND METHODS: From 1990, as part of the quality assurance research programs, the radiotherapy department of the G.-F. Leclerc Centre of Dijon used EPID systems in a prospective fashion. During the first of the three periods (PER 1:1990-1993), the study consisted of analysis criteria determination, software efficiency improvement and a selection of patients who could benefit from the method. Eight hundred and forty-five images of 40 patients were analysed qualitatively and quantitatively. Two verifications per week were planned, and the action level for correction was 10 mm. Head and neck images were also displayed in 'cinema' presentation for internal movements analysis. From 1994 to 1995 (PER 2), off-line procedure (OLP) based upon early correction of the systematic error and the rules calculated from our previous experience were tested for checking the brain, head and neck (LOC 1: 396 images) and many of the pelvic irradiations (LOC 2: 260 images). A double-exposure procedure and/or movie loop presentation was reserved for other patients. During the last period (PER 3: 1996-1997), the OLP procedure was routinely performed in 54 patients (images: 321 LOC 1, 680 LOC 2). RESULTS: LOC 1: deviations of < 3 mm increased from 75.5% during PER 1 to 81% during PER 2 to 83% during PER3. Conversely, deviations of 3-5 mm dropped from 19.5 to 13%, while deviations of more than 5 mm remained stable, around 5%. The actual standard error of the mean deviation observed was 2 mm. LOC 2: deviations of < 5 mm were observed in 81% of the cases during PER 1 and in 91% during PER 3 (89.5% in PER 2). These good results led to a decrease in deviation of 5 to 7 mm (11 to 6%) and also to a significant drop in deviations of more than 7 mm, 8 to 3% respectively. The actual precision obtained was 2.5 mm +/- 3 mm SD. CONCLUSIONS: The OLP based upon the early correction of the systematic error led to a significant increase of setup accuracy of patients irradiated for the brain, head and neck, and especially for pelvic lesions. PMID- 10742809 TI - [An anthropological study of radiotherapy care experience]. AB - An anthropological study has been carried out in order to evaluate the need expressed by patients undergoing radiotherapy treatment. The study was mostly qualitative and based on the radiotherapy experiences of 13 women with breast cancer and six men with head and neck cancer. A 24-year-old female anthropologist spent one year in the department of radiotherapy at the Bergonie Institute in Bordeaux. She collected data on patients' needs through the observation of their experience of treatment and personal interviews. These were put in context, analyzed both by qualitative and quantitative methods. The results point out the need for more information on the different steps of treatment and the patient's need 'for a smile' from the medical team; in other words, emphatic support. PMID- 10742811 TI - [41st Reunion of the American Society for Therapeutic Radiology and Oncology, San Antonio, Texas, October 31-November 4 1999]. PMID- 10742810 TI - [Concomitant radiochemotherapy for cancer of the cervix: critical analysis based on the Standards, Options and Recommendations methodology]. AB - INTRODUCTION: The "Standards, Options and Recommendations" (SOR) project, started in 1993, is a collaboration between the National Federation of the French Cancer Centres (FNCLCC), the 20 French Cancer Centres (CRLCC) and specialists from French public universities, general hospitals and private clinics. The main objective is the development of clinical practice guidelines to improve the quality of health care and outcome for cancer patients. The methodology is based on literature review and critical appraisal by a multidisciplinary group of experts, with feedback from specialists in cancer care delivery. OBJECTIVES: To update, according to the methodology of SOR, the Standards, Options and Recommendations for the management of patients with cancer of the cervix, and in particular, the place of concomitant radiochemotherapy. METHODS: Data have been identified by a literature search using Medline (to April 1999) and the personal reference lists of experts. Once the guidelines were defined, the document was submitted for review to independent national and international reviewers and to the medical committees of the CRCC. RESULTS: The principle recommendations concerning the place of radiochemotherapy in the treatment of cancer of the cervix are 1/ the available data shows a significant increase in local control (level of evidence A) and of overall survival (level of evidence B1) following concomitant radiochemotherapy as compared to radiotherapy alone or the combination of radiotherapy-hydroxyurea. For stages IB, IIA, proximal IIB with bad prognostic factors (tumour size greater than 4 cm and/or invasion of pelvic nodes and/or microscopic invasion of the parametrium) and without lumbo-aortic nodal invasion, concomitant radiochemotherapy can be considered as standard treatment. This benefit is less clear for stages distal IIB, III and IVA without para-aortic nodal invasion (level of evidence C) and must be confirmed (expert agreement). 2/ the toxicity of radiochemotherapy is essentially haematologic and gastrointestinal (level of evidence B1) and is greater than that of radiotherapy alone (level of evidence B1). 3/ these results have been obtained by the combination of chemotherapy based oncisplatin alone, or in combination with 5-FU. Although of equal benefit, the toxicity of the cisplatin/5-FU/ hydroxyurea combination was greater than that of cisplatin alone in a trial comparing the two protocols. A significantly longer survival have also been obtained by the combination of chemoradiation and adjuvant chemotherapy with epirubicin (level of evidence C). These results must be confirmed. 4/ the exact means of delivering the chemotherapy has not been clearly established. In fact, in these trials, some protocols use cisplatin weekly at a dose of 40 mg/m2 and others every three or four weeks at doses ranging from 50 to 75 mg/m2. Subsequent randomised studies are likely to establish optimal schema for the delivery of chemotherapy when combined with external radiotherapy and brachytherapy. PMID- 10742812 TI - [Stereotactic radiosurgery plus whole brain radiotherapy versus radiotherapy alone for patients with multiple brain metastasis]. PMID- 10742813 TI - [Timely intervention to prevent late diabetic sequelae]. PMID- 10742814 TI - [The quality of the treatment of diabetics in kidney failure in Germany]. AB - BACKGROUND AND OBJECTIVE: In recent years there has been a rapid increase in the number of dialysis-dependent diabetics in Germany. Survival on dialysis is not satisfactory and damage acquired in the preterminal stage of renal failure is thought to play an important role. Late referral to a nephrologist and insufficient quality of medical management are thought to contribute importantly to poor outcome. This hypothesis was evaluated in the present study. PATIENTS AND METHOD: The data of all 173 diabetic patients (16 with type 1, 157 with type 2 diabetes, 90 men, 83 women, mean age 63.3 [31-95] years), who had been referred in 1996 for the first time to five renal units, were retrospectively assessed using a structured protocol. RESULTS: Patients were usually referred in advanced renal failure (median creatinine clearance 29 ml/min, range 1-216) with insufficient control of systolic (170 [120-260] mmHg) and diastolic blood pressure (90 [60-180] mmHg), insufficient antihypertensive therapy (without treatment 32 of 173 patients; median number of classes of antihypertensive agents used 2 [range 1-6]; ACE inhibitors 79 of 173 patients), high HbA1c (7.9 [4.9 15.7]%) and LDL cholesterol (176 [67-307] mg/dl). Immediate dialysis was required in 45 patients. CONCLUSION: The data document insufficient quality of treatment and late incorporation of a nephrologist into the medical team involved in the care of diabetic patients. Changes in the structure of diabetes care are necessary to improve treatment quality. PMID- 10742815 TI - [The walking-counting test. A simple test for assessing the risk of falling]. AB - BACKGROUND AND OBJECTIVE: Falls and fractures caused by falls are common in the elderly. Interventions to prevent such falls and/or fractures are available. But there are no appropriate tests in general practice for the targeted use of preventative measures. This prospective study was undertaken to validate a simple "walking and counting" test for assessing the risk of falling. PATIENTS AND METHODS: 92 patients of a general practice, aged between 70 and 96 years (64 women, 28 men) were included in the study. All patients were given the "walking and counting" test in which they would walk as quickly as possible along a stretch of 4 m, repeated immediately in a 3-step sequence while counting backwards from 100. The walking speed without counting was measured initially (vo), as was the speed during distraction by counting (vD). The difference between the two values, delta v%, measured the percentage change under distraction. In 80 patients it was possible to record the number of falls over the entire subsequent period of 12 months. All data were assessed by relative operating characteristics (ROC) and Bayesian analysis. RESULTS: 40 patients had falls in the follow-up period of 12 months, falls being more frequent the higher the age and if there had been previous falls. While all patients were able to do the test, individual values differed considerably. There was a statistical correlation between the calculated change in walking speed and the risk of falling. Using a threshold value of 20% slowing of the walking speed, the test sensitivity was 95% (0.51-0.81: 95% confidence interval) and the specificity 0.85 (0.71-0.93), with a positive predictive value of 0.77 (0.59-0.84). CONCLUSION: The walking and counting test is a simple and informative test, more reliable than other parameters, to predict for an individual patient with danger of falling over a 12-months period. It provides the general practitioner with a tool for demanding appropriately targeted preventative measures. PMID- 10742816 TI - [Metformin-induced lactic acidosis]. AB - HISTORY AND ADMISSION FINDINGS: A 62-year-old woman had been found unconscious on her bed. She had to be resuscitated several times in the ambulance on the way to hospital. On admission her pupils were dilated and fixed, the cardiovascular system was unstable. Her rectal temperature was 28 degrees C. She was a diabetic being treated with metformin and glimepiride and was in incipient renal failure (serum creatinine 1.5 mg/dl). She was also in heart failure due to coronary heart disease and was in a debilitated state. INVESTIGATIONS: She had marked lactic acidosis (lactate 45.3 mmol/l; pH 6.6). Toxicological screening tests were negative. TREATMENT AND COURSE: In the absence of a history she was at first treated symptomatically. Conventional management of the lactic acidosis neither corrected the acidosis nor stabilized the circulatory system. Continuous veno venous haemodialysis with bicarbonate-buffered solutions succeeded in reducing the need for catecholamines. Neurological examination was supplemented by recording acoustic and sensory evoked potentials. Suspected metformin-induced lactic acidosis was confirmed by appropriate tests. Three weeks after admission she was well enough to be transferred to a normal medical ward and ultimately discharged without further complications. CONCLUSION: Metformin should only be prescribed if the contraindications, in particular renal failure are carefully monitored. Severe lactic acidosis should be treated early with continuous veno venous haemodialysis with bicarbonate-buffered substituting fluids. The good neurological results in this case are probably largely due to the marked hypothermia. PMID- 10742817 TI - [Obesity--the new challenge]. PMID- 10742818 TI - [Current drug formulations in obesity therapy]. PMID- 10742819 TI - [The cardiovascular risk factor in obesity]. PMID- 10742820 TI - [The attendance and residency duty of the staff physician. The judgement of the Higher Social Court of Baden-Wurttemberg of 14 July 1999]. PMID- 10742821 TI - [The therapy of acute myeloid leukemia in patients of advanced age]. PMID- 10742822 TI - [The legal significance of a patient's testament for the physician]. PMID- 10742823 TI - [Percutaneous interventions combined with carotid artery stenting]. AB - BACKGROUND AND OBJECTIVE: Although the value of interventional treatment of arterial stenosis has not been confirmed for all sites by randomized studies, these methods are used more and more, often for several arteries simultaneously. This study reports results of percutaneous carotid artery stenting combined with simultaneous interventions in other central arteries. PATIENTS AND METHODS: Among 90 patients who had undergone percutaneous carotid artery stenting, 13 had simultaneous intervention in other arteries: contralateral carotid artery (n = 4), ipsilateral common carotid artery near its aortic origin (n = 1), left subclavian artery (n = 1), coronary artery (n = 6) and one of both carotid arteries and a coronary artery. RESULTS: Primary success (restenosis < 30%) was achieved in all cases. Additional carotid artery stenting was done in 18. Stents were also implanted in eight coronary arteries, angioplasty without stenting in two. Mean stenosis of the carotid arteries was reduced from 85 +/- 10% to 3 +/- 6%, that of the coronary arteries from 90 +/- 10% to 9 +/- 10%. Serious complications, a major stroke, occurred in one of the 13 patients (7.7%). Minor complications were seen in two patients: transitory ischaemic attack in one, small myocardial infarction in the other. CONCLUSION: Carotid artery stenting combined with simultaneous intervention in other central arteries can be done with a high rate of success and relatively few complications. This form of treatment should be considered in selected patients. PMID- 10742824 TI - [Hydroxyethyl starch accumulation in the skin with special reference to hydroxyethyl starch-associated pruritus]. AB - BACKGROUND AND OBJECTIVE: Hydroxyethyl starch (HES) is a colloidal infusion fluid that has for a long time been used in emergency situations and to improve impaired blood perfusion. In the last few years there have been numerous reports about treatment resistant pruritus, often persisting for months, after HES infusion. We investigated the intracellular uptake of HES in the skin, special attention being focused on associated pruritus. PATIENTS AND METHODS: Skin biopsies were obtained from 120 patients (120 men, 35 women) and examined immunohistochemically and for their ultrastructure. Three patients had received various HES preparations, while 22 had been given dextran. Five patients who had received no infusions served as controls. RESULTS: All patients given HES had lysosomal deposits in the histiocytes, some of them also in cutaneous epithelium and endothelium. The extent of lysosomal storage correlated with the amount of infused HES and the interval between biopsy and last HES infusion. Consecutive biopsies in some cases demonstrated a definite decrease over the years of HES deposits in the vacuoles. This suggests that HES is regularly metabolized in the skin. Pruritus after high cumulative doses of HES was closely correlated with HES deposition in cutaneous nerves. CONCLUSIONS: These results emphasize the need for starch derivatives that can be better metabolized and for better adapted infusion schedules to reduce the high incidence of pruritus. PMID- 10742825 TI - [Cardiac pacemaker therapy for optimizing brain circulation. A possible prevention for cerebrovascular diseases?]. AB - HISTORY AND CLINICAL FINDINGS: A 78-year-old patient experienced dizziness, impairment of mnemic and cognitive function, chronic fatigue and recurrent syncope. INVESTIGATIONS: Hypertensive heart disease, reduced left ventricular function, and ventricular ectopia classification Lown IVb was documented. Computed tomography showed minimal brain atrophia. Stenoses of the brain supplying arteries and of other intracranial diseases were excluded. A distinct correlation between cardiac output and cerebral blood flow in correspondence to changes of heart rate were found (cardiac output 4.2 l/min during sinus rhythm, 7.4 l/min during temporary atrial pacing--AAI-Mode with a pacing rate of 90/min; 4.8 l/min--AAI-Mode with a pacing rate of 120/min; cerebral blood flow: 70, 74 and 62 ml/100 g per minute, respectively). Thus, impairment of cerebral blood flow autoregulation can be assumed. TREATMENT AND COURSE: After implantation of a permanent pacemaker the patient was without any complaints. The mnemic and cognitive function improved, dizziness and fatigue disappeared. Synopsis did not occur. 14 months later a sudden onset of complaints occurred caused by atrial fibrillation (heart rate 120/min). Cardiac output and cerebral blood flow were now 4.0 l/min and 35 ml/100 g per minute. After antiarrhythmic drug therapy and restoration of sinus rhythm cardiac output and cerebral blood flow increased and the complaints disappeared again. CONCLUSION: In patients with impaired capacity of cerebral autoregulation a reduced cardiac function and output can induce a reduction of cerebral blood flow. Thus, impairment of mnemic and cognitive function as well as other unspecific neurological deficits can be caused. In these cases pacemaker therapy has to be discussed as an effective therapeutical concept. PMID- 10742826 TI - [Friedreich ataxia. 3 years after the identification of the gene a glimmer of hope for therapy]. PMID- 10742827 TI - [Current guidelines for cardiopulmonary resuscitation]. PMID- 10742828 TI - Biological terrorism: understanding the threat, preparation, and medical response. AB - The thought of an outbreak of disease caused by the intentional release of a pathogen or toxin in an American city was alien just 10 years ago. Many people believed that biological warfare was only in the military's imagination, perhaps to be faced by soldiers on a far-away battlefield, if at all. Political factors- and possibly biotechnology--have changed that. As we enter the new millennium, national, state, and local governments in the United States are preparing for what is now called "not if, but when" biological terrorism. In contrast to the acute onset and first-responder focus with a chemical attack, in a bioterrorist attack, the physician and the hospital will be at the center of the fray. Whether the attack is a hoax, a small food-borne outbreak, a lethal aerosol cloud moving silently through a city at night, or the introduction of contagious disease, the physician who understands threat agent characteristics and diagnostic and treatment options and who thinks like an epidemiologist will have the greatest success in limiting the impact of the attack. As individual health-care providers, we must add the exotic agents to our diagnostic differentials. Hospital administrators must consider augmenting diagnostic capabilities and surveillance programs and even making infrastructure modifications in preparation for the treatment of victims of bioterrorism. Above all, we must all educate ourselves. If done correctly, preparation for a biological attack will be as "dual use" as the facility that produced the weapon. A sound public health infrastructure, which includes all of us and our resources, will serve this nation well for the control of disease, no matter what the cause of the disease. PMID- 10742829 TI - Review of the U.S. Army's health risk assessments for oral exposure to six chemical-warfare agents. Introduction. AB - The U.S. Army is under a congressional mandate and the Chemical Weapons Convention of January 1993 to destroy its entire stockpile of chemical munitions. In addition to stockpiled munitions, nonstockpile chemical materiel (NSCM) has been identified for destruction. NSCM includes a host of lethal wastes from past disposal efforts, unserviceable munitions, chemically contaminated containers, chemical-production facilities, newly located chemical munitions, known sites containing substantial quantities of buried chemical weapons and wastes, and binary weapons and components. There are eight stockpile sites located in the continental United States and one on an island in the Pacific Ocean, and 82 NSCM locations have been identified. There are concerns, based on storage and past disposal practices, about soil and groundwater contamination at those sites. Six of the most commonly found chemical-warfare agents at stockpile and NSCM sites are the nerve agents GA, GB, GD, and VX and the vesicating (blistering) agents sulfur mustard and lewisite. To ensure that chemical contamination is reduced to safe concentrations at stockpile and NSCM sites before they are used for residential, occupational, or wildlife purposes, the U.S. Army requested that health-based exposure limits for GA, GB, GD, VX, sulfur mustard, and lewisite be developed to protect the public and the environment. Oak Ridge National Laboratory (ORNL) was asked to conduct the health risk assessments and propose chronic oral reference doses (RfDs) and, where appropriate, oral slope factors (SFs) for the six agents. RfDs are toxicological values developed for noncancer effects and used as reference points to limit human oral exposure to potentially hazardous concentrations of chemicals thought to have thresholds for their effects. RfDs are estimates (with uncertainty spanning an order of magnitude or greater) of daily oral chemical exposures that are unlikely to have deleterious effects during a human lifetime. For chemicals identified as carcinogens (e.g., sulfur mustard), SFs are also calculated. SFs are estimates of upper-bound lifetime cancer risk from chronic exposure to an agent. The Army's Surgeon General adopted the proposed RfDs and SFs developed by ORNL as interim values to ensure that consistent health-based criteria were applied in ongoing initiatives requiring decisions on the safety of contaminated sites. The Army's Surgeon General also requested that the National Research Council (NRC) independently review the scientific validity of these values. The NRC assigned this task to the Committee on Toxicology (COT), and a multidisciplinary subcommittee of experts was convened to assess the scientific validity of the interim RfDs developed for GA, GB, GD, VX, sulfur mustard, and lewisite and the SF developed for sulfur mustard. Specifically, the subcommittee was asked to (1) determine whether all the relevant toxicity data were considered appropriately; (2) review the uncertainty, variability, and quality of the data; (3) determine the appropriateness of the assumptions used to derive the RfDs (e.g., the application of uncertainty factors); and (4) identify data gaps and make recommendations for future research. Although multiple agents are present at stockpile and NSCM sites, the subcommittee was asked to evaluate the agents only on an individual basis. Furthermore, although the most likely routes of exposure to chemical warfare agents at these sites are the inhalation and dermal routes, the subcommittee was only asked to evaluate toxicological risk from the oral route at this time. The Army is in the process of developing inhalation exposure guidelines. The subcommittee was also not asked to address issues related to risk management, such as technology, detection, and feasibility. PMID- 10742830 TI - Influence of treatment technique on dose-volume histogram and normal tissue complication probability for small bowel and bladder. A prospective study using a 3-D planning system and a radiobiological model in patients receiving postoperative pelvic irradiation. AB - PURPOSE: A prospective study was undertaken to evaluate the influence of pelvic irradiation techniques on the dose-volume histograms of organs at risk and to analyze its possible clinical relevance using radiobiological models. PATIENTS AND METHODS: For 20 patients receiving postoperative pelvic irradiation because of rectal cancer a 3-field technique (3-FT), a 4-field technique (4-FT) and an opposing field technique (OFT) were designed by a 3-D planning system (Helax, TMS). Dose-volume histograms (DVH) of small bowel, urinary bladder and planning target volume (PTV) were analyzed. The normal tissue complication probability (NTCP) was determined by the radiobiological model of Lyman and Kutcher using the tolerance data of Emami. RESULTS: Median dose to the PTV did not differ between the 3 techniques (3-FT/4-FT/OFT: 99.2%/98.6%/98.1% of the prescribed dose; p > 0.05). Although the median dose to the urinary bladder was lower for the 3-FT than for the 4-FT (44.7% vs 60.3%; p < 0.001), there was no difference in the calculated NTCP (0.0% vs 0.0%; p > 0.05). Using multiple field techniques both the dose to and the treated volume of the urinary bladder were significantly lower than using the OFT. As a consequence of this the late NTCP of the bladder was higher for the OFT (5.46%). Although the median dose to the small bowel was lower for the 3-FT than for the 4-FT (30.8% vs 54.5%; p < 0.005), the fractional part of small bowel within the high-dose region (90% isodose) was higher for the 3-FT (10.6% vs 8.2%; p > 0.05). Thus the calculated NTCP was higher for the 3-FT (0.79 vs 0.44) than for 4-FT. For the OFT the median dose to small bowel was 69.9% and the small bowel volume within the high-dose region was 57.8% resulting in a late NTCP of 9.36% (OFT vs 3-FT/4-FT: p < 0.05). CONCLUSION: Using multiple field techniques both the dose to the organs of risk and the fractional part of risk organs within the high-dose region can be reduced significantly. As a consequence of this a lower NTCP was calculated for the 3-FT and the 4-FT than for the OFT. Using the biological model a small, but significant difference between a 3-FT and a 4-FT was demonstrated in favor to the 4-FT. PMID- 10742831 TI - The postoperative adjuvant radiation therapy and radiochemotherapy for UICC stage II and III rectal cancer. A retrospective analysis. AB - AIM: This analysis was undertaken to review the outcome and toxicity of postoperative adjuvant therapy for Stage II and III rectal cancer. PATIENTS AND METHODS: We reviewed 112 patients treated with radiotherapy (44 patients) and radiochemotherapy (68 patients) after potentially curative (R0) surgery for rectal cancer (UICC Stages II and III), between 1983 and 1994 at the University Clinic of Wurzburg. Median radiation dose was 56 Gy (range: 45 to 66 Gy). Chemotherapy consisted of 4 to 6 courses of 5-fluorouracil (5-FU) (420 mg/m2/d) and leucovorin (200 mg/m2/d). Median follow-up was 37 months. RESULTS: The overall survival was 84% for patients with UICC Stage II and 45% for patients with UICC Stage III disease (p = 0.0045). There were no statistically significant differences between patients treated with radiochemotherapy vs radiotherapy in terms of 5-year survival (63% after radiochemotherapy vs 53% after radiotherapy, p = 0.16), relapse-free survival (52% vs 50%) and locoregional control (69% vs 67%). UICC Stage III disease was associated with high failure rates (40% pelvic recurrences and 53% distant metastases). There was a statistically significant difference in terms of the incidence of distant metastases between the 2 treatment modalities for patients with Stage III disease (49% 5-year probability for developing distant metastases after radiochemotherapy vs 66% after radiotherapy, p = 0.047). In a multivariate analysis, the addition of chemotherapy, lymph node stage and grading were independent prognostic factors for survival. Severe late toxicity was documented in 5% of treated patients. CONCLUSIONS: Prognosis of patients with UICC Stage III rectal cancer remains poor after "standard" surgery followed by postoperative adjuvant treatment (pelvic radiotherapy and bolus intravenous injection of 5-FU and leucovorin). Major efforts should be made in order to improve prognosis for these patients, including optimization of surgical treatment and systemic treatment. More effective multimodality treatment strategies should be investigated in prospective randomized trials. PMID- 10742832 TI - CT-image-based conformal brachytherapy of breast cancer. The significance of semi 3-D and 3-D treatment planning. AB - PURPOSE: To compare the conventional 2-D, the simulator-guided semi-3-D and the recently developed CT-guided 3-D brachytherapy treatment planning in the interstitial radiotherapy of breast cancer. PATIENTS AND METHODS: In 103 patients with T1-2, N0-1 breast cancer the tumor bed was clipped during breast conserving surgery. Fifty-two of them received boost brachytherapy after 46 to 50 Gy teletherapy and 51 patients were treated with brachytherapy alone via flexible implant tubes. Single, double and triple plane implant was used in 6, 89 and 8 cases, respectively. The dose of boost brachytherapy and sole brachytherapy prescribed to dose reference points was 3 times 4.75 Gy and 7 times 5.2 Gy, respectively. The positions of dose reference points varied according to the level (2-D, semi-3-D and 3-D) of treatment planning performed. The treatment planning was based on the 3-D reconstruction of the surgical clips, implant tubes and skin points. In all cases the implantations were planned with a semi-3-D technique aided by simulator. In 10 cases a recently developed CT-guided 3-D planning system was used. The semi-3-D and 3-D treatment plans were compared to hypothetical 2-D plans using dose-volume histograms and dose non-uniformity ratios. The values of mean central dose, mean skin dose, minimal clip dose, proportion of underdosaged clips and mean target surface dose were evaluated. The accuracy of tumor bed localization and the conformity of planning target volume and treated volume were also analyzed in each technique. RESULTS: With the help of conformal semi-3-D and 3-D brachytherapy planning we could define reference dose points, active source positions and dwell times individually. This technique decreased the mean skin dose with 22.2% and reduced the possibility of geographical miss. We could achieve the best conformity between the planning target volume and the treated volume with the CT-image based 3-D treatment planning, at the cost of worse dose homogeneity. The mean treated volume was reduced by 25.1% with semi-3-D planning, however, it was increased by 16.2% with 3-D planning, compared to the 2-D planning. CONCLUSION: The application of clips into the tumor bed and the conformal (semi-3-D and 3-D) planning help to avoid geographical miss. CT is suitable for 3-D brachytherapy planning. Better local control with less side effects might be achieved with these new techniques. Conformal 3-D brachytherapy calls for new treatment planning concepts, taking the irregular 3-D shape of the target volume into account. The routine clinical application of image-based 3-D brachytherapy is a real aim in the very close future. PMID- 10742833 TI - [The age-specific results of the adjuvant radiotherapy of breast carcinoma]. AB - BACKGROUND: Aim of our work was to evaluate the standards of treatment in elder women with breast cancer and their results of radiation therapy. PATIENTS AND METHODS: In our hospital 218 breast cancer patients were treated in the years 1990 and 1991. Forty-three women were younger than 50 years of age (group I), 92 between 50 and 64 years (group II), 83 elder than 64 years (group III). One hundred and forty-nine patients underwent mastectomy, 65 patients had conservative treatment (Table 5). Four patients were not operable. All of the patients were irradiated uniformly loco-regionally with 50 Gy Co-60-photons, followed by a boost to the tumor bed with 10 Gy with 6- to 12-MeV electrons. Forty-four patients had an additional chemotherapy. RESULTS: The 5-year survival rate was 79.8% (n = 174), the disease free survival was 59.1% (n = 129) (Table 6). The mean rate of local recurrence was 3.6% (n = 8), 4% after mastectomy (n = 6) and 3% (n = 2) with breast conservation. Age group specific 5-year survival rates were 72% (I), 85.6% (II) and 77.1% (III), respectively, disease free survival rates were 48.8% (I), 65.2% (II) and 57.8% (III), respectively. The rates of local recurrence were 9.3% (I), 3.2% (II) and 1.2% (III), respectively. Significant age group specific differences in surgical and radiotherapeutical treatment could not be found. CONCLUSION: The curative chance of treatment has to be used in every age. A treatment of elder patients below the actual valid standards of treatment is not justified. PMID- 10742834 TI - [Conventional and virtual simulation in retrobulbar irradiation]. AB - BACKGROUND: Radiotherapy is commonly used in Grave's ophthalmopathy. The target volume encompasses the ocular muscles and the orbital tissue. The result of conventional simulation can be examined by means of CT simulation. PATIENTS AND METHODS: Twenty-five planning CTs with Grave's ophthalmopathy were studied. The conventional simulation of 4 x 4 cm2 lateral portals confined anteriorly by the fleshy canthus was performed on a CT-simulator using the observer's eye view (OEV) and digitally reconstructed radiographs (DRR). The coverage of the target volume and sparing of the eye lenses were studied on axial CT slices and multiplanar reconstructions (MPR). The distances between the apex of the orbita and cornea, fleshy canthus, and bony canthus were measured as well as the distance between cornea and posterior face of the lens. RESULTS: The pituitary gland and the ocular lenses were spared in each case (25/25). The orbita was entirely covered in 24 cases (96%). However, anterior parts of the external eye muscles were not completely encompassed in 7 cases (28%). The distance from the apex of the orbita to the cornea was 54.6 mm (53.3 to 55.8 mm, 95% confidence interval), to the fleshy canthus 40.3 mm (39.4 to 41.2 mm), and to the bony canthus 31.4 mm (30.2 to 32.5 mm). The distance between cornea and posterior face of the lens was 8.3 mm (7.9 to 8.7 mm). The distance between cornea and canthus differed significantly from normal eyes while the distance between cornea and posterior face of the lens was very similar to normal eyes. CONCLUSIONS: Conventional simulation of orbital irradiation with lateral fields confined anteriorly by the fleshy canthus ensures protection of the ocular lenses and the pituitary gland. However, anterior parts of the eye muscles may occasionally not completely be covered. The fleshy canthus and the cornea are more reliable landmarks as compared to the bony canthus. PMID- 10742835 TI - Optimization of tumor radiotherapy. Part VI: Modification of tumor glucose metabolism for increasing the bioavailability of 2-deoxy-D-glucose (2-DG) in a murine tumor model. AB - AIM: Differential radiomodification induced by 2-deoxy-D-glucose (2-DG) is proving to be a feasible modality for optimizing tumor radiotherapy. Our earlier work on Ehrlich ascites tumor cells has shown that pretreatment with hematoporphyrin derivatives increases the uptake and phosphorylation of 2-DG. Moreover, the alteration induced in bioenergetic profile was more drastic and less reversible. The promising combination of hematoporphyrin derivatives and 2 DG has been further evaluated in the Ehrlich ascites tumor bearing mice for determining the effects on radiotherapeutic response. MATERIALS AND METHODS: Solid tumors (average volume = 0.9 +/- 0.1 cm3) implanted in Swiss-albino strain "A" mice were focally irradiated (10 Gy) using 60Co teletherapy. Drugs were administered intravenously. Tumor bioenergetics was assessed by 31P MR spectroscopy. RESULTS: The uptake and phosphorylation of 2-DG was observed to be increased following pretreatment with hematoporphyrin derivatives. Upon hematoporphyrin derivatives + 2-DG treatment followed by irradiation, the intracellular pH reduced and a remarkable increase in glycerophosphorylcholine and inorganic phosphate levels was observed. CONCLUSION: The present study demonstrates the potential of hematoporphyrin derivative pretreatment in increasing the bioavailability of 2-DG in a mice Ehrlich ascites tumor model. The finding may have interesting clinical implications in the form of increased manifestation of the radiation-induced damage in the case of use of these drugs as a potential adjuvant in radiotherapy of tumors. PMID- 10742836 TI - Soft tissue sarcomas after radiation treatment for breast cancer. Three case studies and review of literature. AB - AIMS: By means of 3 cases with infield soft tissue carcinomas after radiotherapy for breast cancer, symptoms and therapy are described. Consequences for treatment planning and patient's information before radiotherapy for breast cancer are discussed. PATIENTS: Three of 1,025 patients with breast cancer irradiated from 1984 to 1997 suffered from infield secondary soft tissue sarcomas. The latency periods were 61, 49 and 59 months. Two patients had been treated with breast conserving therapy (computerized planning, 50 Gy to reference point, 5 times 2 Gy/week, 5-MV photons), 1 patient received a local boost dose of 15 Gy (10-MeV electrons), patient 3 radiotherapy of the thoracic wall and regional lymph nodes after mastectomy using 12-MeV electrons (thoracic wall) and 5-MV photons (lymph node areas) to 50 Gy, 5 times 2 Gy/week. No adjuvant chemotherapy was given. All sarcomas were very extensive, all patients died from local progression and/or distant failure after 17, 13 and 12 months. RESULTS: The incidence of spontaneous sarcomas of the breast is about 0.06%, after operation and radiotherapy 0.09 to 0.45%. No correlations to radiotherapy technique and no risk factors were found. Radiation dose could play a role, but there are very sparse data about this. CONCLUSIONS: Secondary soft tissue sarcomas are very rare, but familiar complications of radiotherapy. Only early diagnosis leads to a chance for cure. Because of unclear correlations to the treatment parameters and rareness of this event, in our opinion no regular information to the patient receiving radiotherapy for breast cancer is mandatory. PMID- 10742837 TI - [A retrospective comparison of radical retropubic prostatectomy and of iodine-125 brachytherapy in localized prostatic carcinoma]. PMID- 10742838 TI - [18F-FDG whole-body positron-emission tomography (PET) in patients with unknown primary tumors]. PMID- 10742839 TI - [Can additional maintenance chemotherapy improve the results of simultaneous adjuvant radiochemotherapy in rectal carcinoma even more?]. PMID- 10742840 TI - [The importance of the interval between preoperative radiotherapy and the operation in the treatment of rectal carcinoma]. PMID- 10742841 TI - Distinguishing between memory illusions and actual memories using phenomenological measurements and explicit warnings. AB - Previous research has demonstrated that the false memory effect is robust and that false memories are essentially indistinguishable from memories for events that actually occurred. The current study used several techniques intended to eliminate false memories (source monitoring decisions, confidence ratings, remember/know judgments, and explicit warnings). A robust false memory effect was found in each experiment. However, participants were able to differentiate false memories and actual memories when using specific phenomenological tasks. The current findings provide insight into basic human memory processes. PMID- 10742842 TI - The aesthetics of rectangle proportion: effects of judgment scale and context. AB - Two experiments examined preference for rectangle proportion, using a method that allowed participants to adjust 1 dimension of a rectangle to achieve the most preferred proportion. Instructing participants to use an interestingness response scale resulted in a preference for rectangles having more extreme sides ratios, compared with pleasingness or preferability scales. Instructing participants to produce a rectangle representing the preferred proportions of a painting or a kitchen tile shifted the preferred sides ratio toward a less extreme value than did a no-context rectangle instruction. Implications of the results for the findings of experiments that seek evidence for the special significance of particular ratios, such as the golden section, are discussed. PMID- 10742844 TI - Further considerations regarding inhibitory processes, working memory, and cognitive aging. AB - The present study explored the relationship between inhibitory processing (as indexed by identity negative priming in a letter-naming task), working memory, discourse processing, and cognitive aging effects. Consistent with several other recent reports, this study found evidence of intact inhibitory processing, as measured by negative priming, in older adults as well as younger adults. This intact negative priming occurred in conjunction with diminished working memory span and impaired memory in the discourse processing task in the same sample of older adults, further arguing against the likelihood that declines in these areas are caused by impairments in the inhibitory processes measured by negative priming. The implications of these results for theories of cognitive aging and possible reasons for inconsistent findings regarding negative priming effects among the elderly are discussed. PMID- 10742843 TI - Musical preferences during and after relaxation and exercise. AB - Effects of the listening context on responses to music largely have been neglected despite the prevalence of music listening in our everyday lives. This article reports 2 studies in which participants chose music of high or low arousal potential during (Experiment 1) or immediately after (Experiment 2) exercise or relaxation. In Experiment 1, participants preferred appropriate arousal-polarizing music over arousal-moderating music. In Experiment 2, participants preferred arousal-moderating music over arousal-polarizing music, such that their listening times contrasted clearly with those in the first study even though the same music and methods were used. Thus musical preferences interact with the listening situation, and participants' music selections represent an attempt to optimize their responses to that situation. When motivated to maintain a state of polarized arousal, listeners use music to achieve this; when they have no such goal, they use music to moderate arousal. PMID- 10742845 TI - Does the generation effect occur for pictures? AB - The generation effect is the finding that self-generated stimuli are recalled and recognized better than read stimuli. The effect has been demonstrated primarily with words. This article examines the effect for pictures in two experiments: Subjects named complete pictures (name condition) and fragmented pictures (generation condition). In Experiment 1, memory was tested in 3 explicit tasks: free recall, yes/no recognition, and a source-monitoring task on whether each picture was complete or fragmented (the complete/incomplete task). The generation effect was found for all 3 tasks. However, in the recognition and source monitoring tasks, the generation effect was observed only in the generation condition. We hypothesized that absence of the effect in the name condition was due to the sensory or process match effect between study and test pictures and the superior identification of pictures in the name condition. Therefore, stimuli were changed from pictures to their names in Experiment 2. Memory was tested in the recognition task, complete/incomplete task, and second source-monitoring task (success/failure) on whether each picture had been identified successfully. The generation effect was observed for all 3 tasks. These results suggest that memory of structural and semantic characteristics and of success in identification of generated pictures may contribute to the generation effect. PMID- 10742846 TI - [Postangioplasty arterial restenosis. Achilles heel or nemesis?]. PMID- 10742847 TI - [Anatomopathologic-embryologic correlation in atrioventricular connection absence]. AB - In order to offer a pathogenetic explanation for the absence of atrioventricular connexion, a correlation was made between the pathologic anatomy of this cardiac malformation and the embryonic processes which take part in the septation of the atrioventricular canal and the development of atrioventricular connections. The correspondence was made between the development of the canal's atrioventricular cushions, the septation of the common atrioventricular canal, the right and left atrioventricular canals and the morphogenesis of the mitral valve, all these processes were correlated with the anatomic elements derived from them. This led to infer that the malposition of the atrioventricular cushions divide the common atrioventricular canal unequally, giving rise to a narrow canal becoming atretic and a big canal where the mitral valve is evolved. The extreme lateralization of the atrioventricular septum to the right side would led to the absence of the right atrioventricular connection and the same process but to the left side, would give rise to the left absence of the atrioventricular connection. This ectopic septation process is supported by similar ones which can occur in other segments of the heart such as in tetralogy of Fallot and the transposition of the great arteries. This hypothesis explains sufficiently the pathologic anatomy of this type of congenital heart disease. PMID- 10742848 TI - The effects of body position, controlled breathing and exercise on the heart rate variability parameters in healthy subjects. AB - The behavior of temporal and spectral parameters of the heart rate variability was determined during 5 different maneuvers in order to characterize the level of sympathetic-vagal activity. The discriminating capacity of two spectral parameter estimation schemes were compared, and the respiratory influence was evaluated. One hundred and ten records of instantaneous heart rate and respiratory amplitude were analyzed, both in temporal and spectral perspectives. The records were obtained from 22 healthy subjects, under a five-stage protocol: supine, controlled breathing, standing, exercise, and recovery. A discriminating and characteristic behavior was found among the maneuvers, specifically for the dispersion parameters, low and intermediate partial components. The two integration-normalization procedures used in the estimation of the spectral components showed similarities, producing a functional interpretation independent of the selected procedure. The division of the low frequency component in two separate bands, allowed an improved discriminating capacity. The respiration had an important influence in the controlled breathing maneuver while being lower in the rest of the stages. In conclusion, the maneuvers determined a typical behavior of the dispersion and spectral parameters (low and intermediate partial components) of the heart rate variability, showing adequate distinctive levels in the sympathetic-vagal activity for each maneuver. PMID- 10742849 TI - [Heterotopic heart transplantation: 13-year experience at the Methodist Hospital of the Baylor Medical College]. AB - In order to evaluate our experience in heterotopic cardiac transplantation, we conducted a retrospective analysis of the clinical files of patients who underwent this procedure. RESULTS: A total of 405 heart transplants were performed in our institution. In 24 (5.9%), the grafts were placed heterotopically. In group I (12 patients), the indication was irreversible pulmonary hypertension and in group II (remaining 12 patients), it was marginal grafts or size mismatch. Both groups demonstrated similar demographics and the survival rate was slightly better in group I. Nine patients from group I demonstrated an early reduction in pulmonary pressures which normalized in one year. CONCLUSIONS: The heterotopic heart supports the function of the native ventricles. In 9 patients, the heterotopic heart enables the reversal of a state of pulmonary hypertension previously thought to be irreversible. This finding supports the use of pulmonary vaso-dilators on a chronic basis or the use of a left ventricular assist device pre-transplant with the intention of normalizing pulmonary pressures and allowing the patients to become candidates for orthotopic cardiac transplantation and thereby avoiding the necessity of heterotopic cardiac transplantation. PMID- 10742850 TI - Myocardial perfusion scintigraphy (SPECT) in the evaluation of patients in the emergency room with precordial pain and normal or doubtful ischemic ECG. Study 60 cases. AB - OBJECTIVES: To evaluate the diagnostic utility of myocardial perfusion by SPECT and Gated-SPECT in the diagnosis of acute coronary syndrome in patients with precordial pain associated with normal or doubtful ischemic ECG within the first 6 hrs of the last episode of pain. METHODS: Sixty such patients who sought attention in the Emergency room were included. Myocardial perfusion SPECT and Gated-SPECT (GSPECT) was performed in all patients using two distinct protocols. All patients underwent resting and pharmacological stress test. In 30 cases coronary angiogram were performed. RESULTS: Resting myocardial perfusion was abnormal or positive in 25 patients (42%) and normal or negative in 35 patients (58%). In the latter group perfusion became abnormal in 15 patients (43%) under stress with dipyridamole, while it remained normal in 19 (54%). The last subgroup presented no coronary events during the 12 months following their hospital discharge. In the group of 25 patients with resting perfusion abnormalities acute myocardial infarction was diagnosed in 7 patients, ischemia in 12 and reverse reversibility in 6. Myocardial perfusion scintigraphy showed in the resting phase a low sensitivity of 61% (95% CI 39-74%), and negative predictive value of 71% (95% CI, 58-82%). During the stress phase, the utility of the test increased significantly, with a sensitivity of 97% (95% CI, 83-99%), specificity of 79% (95% CI, 57-92%), positive predictive value of 87% (95% CI, 72-95%) and, most outstanding, a negative predictive value of 95% CI, 73-99%). CONCLUSIONS: Myocardial perfusion studies have a sensitivity of 97% for identifying patients with acute coronary syndrome, with precordial pain and normal or doubtful ischemic ECG. For the intermediate or low risk patients with acute coronary syndrome the non-invasive diagnostic techniques of SPECT and GSPECT systems of evaluating myocardial perfusion achieve a high degree diagnostic accuracy, safety and reduces unnecessary admissions and costs. PMID- 10742851 TI - [Myocardial ischemia detection with adenosine administration and SPECT. Initial experience in Mexico]. AB - Myocardial perfusion SPECT has a high sensitivity for the diagnosis of myocardial ischemia. Adenosine has been recently used to induce myocardial ischemia in the United States and Europe. At the present time there is not published experience using adenosine in Mexico. METHOD: We studied 22 patients with suspected myocardial ischemia. Coronary angiography was performed in 17 patients. We used a 8 mCi rest Tc-99m sestamibi followed by a 6 minute infusion of adenosine at a dose of 140 ug/kg/min; 24 mCi of Tc-99m were injected after the third minute of adenosine infusion. Patients returned 2 or 3 days later for a new stress study using physical stress or dipiridamole and the images were read using a 20 segments analysis and each segment was scored using a 5 points scale (0 = normal to 4 = absent uptake). The results were then compared with the adenosine images. RESULTS: The segmental score agreement between adenosine and physical or dipyridamole stress were good with 90% exact correlation. The side effects experienced by patients who received dipyridamole and adenosine were similar. CONCLUSION: Adenosine is a good alternative to induce myocardial ischemia. It showed a good correlation with physical or dipyridamole stress test. PMID- 10742853 TI - Kearns-Sayre syndromes an absolute indication for prophylactic implantation of definitive pacemaker? AB - Kearns-Sayre syndrome is a mitochondrial cytopathy characterized by chronic progressive external ophthalmoplegia, retinitis pigmentosa and heart block, the last of which determines the survival of these patients. The case of a 23 year old man with Kearns-Sayre syndrome, conduction disturbances and mitral valve prolapse is presented. The characteristics of this syndrome are described and the criteria for prophylactic installation of a pacemaker discussed. PMID- 10742852 TI - [Gianturco-Grifka device in the percutaneous closure of patent ductus arteriosus]. AB - We present our early experience with the Gianturco-Grifka device for transcatheter closure of patent ductus arteriosus. Grifka occlusion was attempted in 13 patients (7 female, 6 male), median patient age was 6.5 years. Echocardiographic evaluation showed a mean ductal diameter of 5.47 +/- 1.09 mm, and the morphology was type C in 7 patients, type A in 5 and type E in 1 patient. The angiographic ductal morphology was 6 type A, 5 type C and 2 type E. Mean PDA diameter was 5.69 +/- 1.15 mm. Three patients had pulmonary hypertension. They were given supplemental oxygen (100%), decreasing their pulmonary pressures. A total of 13 devices were implanted, 5 of 7 mm and 8 of 9 mm. Twelve patients had complete ductal occlusion documented by aortic angiography (92%), one patient presented residual shunt (8%). Color echocardiography at 24 hrs documented complete occlusion in 12 cases. One device embolized to the descending aorta 2 hours after closure, and it was successfully retrieved in the catheterization lab. We conclude that the Gianturco-Grifka device is an appropriate alternative for transcatheter closure of the PDA. This technique can be performed in ductus arteriosus type C, D and E, with diameter < 9 mm. More clinical trials are needed to establish the long-term results of this technique. PMID- 10742854 TI - [Does heart sarcoma mimick lupus erythematosus and vasculitis? Report of 2 cases]. AB - Primary heart tumors are rare. The most common one is myxoma. It may synthetize IL-6 and frequently shown systemic clinical manifestations that confuse the diagnosis. Primary heart sarcomas are even more rare, as far as we know they have been not associated with systemic illnesses. We observed two cases with a presumptive diagnosis of systemic lupus erythematosus who were identified as primary sarcomatous heart neoplasms after surgical excision. PMID- 10742855 TI - [Pharmacologic thrombolysis in acute myocardial infarction: lessons learned after ISIS-2 and GUSTO-I. Part II]. PMID- 10742856 TI - [Overview of cardiology textbooks in New Spain (at the 460 anniversary of the press in Mexico, 1539-1999)]. PMID- 10742857 TI - On the acquisition of a human cardiomyocyte cell line. PMID- 10742858 TI - Organization ethics in healthcare. PMID- 10742860 TI - Business ethics, stakeholder theory, and the ethics of healthcare organizations. PMID- 10742859 TI - Ethics and the structures of healthcare. PMID- 10742861 TI - Managed care, cost control, and the common good. PMID- 10742862 TI - Conflicts of interest and management in managed care. PMID- 10742863 TI - Trust and transforming medical institutions. PMID- 10742864 TI - Organizational ethics programs and the law. PMID- 10742865 TI - Bioethics activities in rural hospitals. PMID- 10742866 TI - Deregulating the genetic supermarket: preimplantation screening, future people, and the harm principle. PMID- 10742867 TI - Dubious premises--evil conclusions: moral reasoning at the Nuremberg trials. PMID- 10742868 TI - The complexities of organ allocation policies. PMID- 10742869 TI - Changing economics and clinical ethical decision-making: a view from the trenches. PMID- 10742870 TI - Psychosomatic medicine. PMID- 10742871 TI - Heinz Lehmann introduces imipramine. PMID- 10742872 TI - Recent developments in alexithymia theory and research. AB - OBJECTIVE: To review recent developments in alexithymia theory and research that are relevant to the field of psychosomatic medicine. METHOD: Articles were selected from the alexithymia literature published over the past decade that describe advances in the theoretical understanding of alexithymia or report empirical investigations of the relationships of the construct with emotion regulation and with somatic illness and disease. Empirical investigations of the neural correlates of alexithymia were reviewed also, as were studies that explore therapeutic attempts to modify alexithymic characteristics. RESULTS: The salient features of the alexithymia construct are now thought to reflect deficits in the cognitive processing and regulation of emotions. This is supported by studies showing that alexithymia is associated with maladaptive styles of emotion regulation, low emotional intelligence, a bidirectional interhemispheric transfer deficit, and reduced rapid eye movement (REM) density (number of eye movements divided by number of REM periods). Although empirical evidence demonstrates that alexithymia is associated with several somatic disorders, more prospective studies are required to establish the direction of causality. Preliminary data suggest that psychotherapies involving specific techniques to enhance emotional awareness and integrate symbolic and subsymbolic elements of emotion schemas may be effective in reducing alexithymic characteristics. CONCLUSION: Alexithymia is proving to be a heuristically useful construct for exploring the role of personality and emotions in the pathogenesis of certain somatic illnesses and diseases. PMID- 10742873 TI - Psychiatry and terminal illness. AB - OBJECTIVE: To provide an overview of the palliative care literature salient to the psychiatric aspect of end-of-life care. METHOD: A literature review was conducted, targeting primarily empirical studies that addressed the following topics: 1) psychological issues pertaining to life-threatening conditions; 2) family issues in the context of palliative care; 3) psychological issues and challenges faced by end-of-life health care providers; and 4) psychiatric disorders, including depression, anxiety, and organic mental disorders, in people with terminal illness. RESULTS: There is a small but emerging literature that can guide psychiatrists in their role of providing care to dying patients. CONCLUSIONS: While psychiatry has made tremendous inroads toward providing care to patients throughout the life cycle, its presence is only just beginning to be felt in end-of-life care. Within the domain of palliative care, psychiatry has an expanded and important role to play. PMID- 10742874 TI - Olanzapine use in the elderly: a retrospective analysis. AB - OBJECTIVE: To evaluate the efficacy and safety of olanzapine in a hospitalized geriatric population that had previously failed to respond to, or tolerate, numerous trials with other antipsychotic medications. METHOD: A retrospective chart analysis was conducted on 58 elderly patients with psychotic symptoms who were given a trial on olanzapine. Data was collected regarding patients' psychiatric diagnoses, duration of illness, duration of hospitalization, prior response to psychotropic therapies, concomitant psychotropic agents, side effects, and clinically determined changes over time. RESULTS: Results indicated that 60.3% of this refractory group of patients improved on olanzapine. Side effects were reported for 38% of the patients, with delirium, extrapyramidal symptoms (EPS), and drowsiness or lethargy being the most common. CONCLUSIONS: The reported level of improvement in this group of refractory elderly psychotic patients indicates that olanzapine can make an important contribution to the mental health of elderly patients with similar characteristics. PMID- 10742875 TI - [Factor analysis of french translation of the Barratt impulsivity scale (BIS 10)]. AB - Though the concept of impulsiveness is controversial, there are many attempts being made to measure this dimension. In this context, only psychometric measures are widely considered valid and are routinely in use. Barratt developed the first scale that specifically measured impulsiveness. Subsequently, various refinements have improved the validity of results. We have translated, without any significant problems, the tenth validated version of this scale (BIS 10) into French, and we have completed a factorial analysis. The scale was coupled with a self-administered questionnaire designed to assess anxiety. A sample of 280 subjects between the ages of 18 and 79 years (average age, 36.9) were recruited from the general population. Subject age was found to have a weak but nevertheless significant correlation with the impulsiveness rating. A principal component analysis (PCA) resulted in the first 9 factors explaining 55.6% of the variance. Another PCA of these factors allowed the identification of a second tier of 3 second order factors; these were closely related to Barratt's ranking. Our study confirms results from the scale's initial analysis--results which could not be subsequently reproduced. To our knowledge, this is the first French translation of an instrument that specifically measures impulsiveness and the first in which a factorial structure has been tested in the general population. PMID- 10742877 TI - Comparison of family therapy and family group psychoeducation in adolescents with anorexia nervosa. AB - OBJECTIVE: To compare the effects of 4 months of 2 family-oriented treatments, family therapy and family psychoeducation, on female adolescents with newly diagnosed restrictive eating disorders. METHOD: Twenty-five female adolescents requiring hospitalization were randomized into either family therapy or family group psychoeducation. Outcome measures included medical (body weight) and psychosocial (specific and nonspecific eating disorder psychopathology) variables at baseline and after 4 months of treatments every 2 weeks. RESULTS: A significant time effect was found in both treatment groups for the restoration of body weight (percentage of ideal body weight, P < 0.00001). The group averages ranged from 75% to 77% ideal body weight before treatment to 91% to 96% after it. A time effect was also seen on the Family Assessment Measure (P < 0.018), in that the patients of both groups acknowledged more family psychopathology at the end of treatment. No significant group differences were found on any of the self report measures of specific and nonspecific eating disorder pathology. CONCLUSIONS: Weight restoration was achieved following the 4-month period of treatment in both the family therapy and family psychoeducation groups, but no significant change was reported in psychological functioning by either adolescents or parents. Family group psychoeducation, the less expensive form of treatment, is an equally effective method of providing family-oriented treatment to newly diagnosed, medically compromised anorexia nervosa patients and their families. PMID- 10742876 TI - Differentiating DSM-IV anxiety and depressive disorders in the general population: comorbidity and treatment consequences. AB - OBJECTIVE: To attempt, for the first time, to apply a positive and differential diagnosis process in the general population during interviews using DSM-IV classification to ascertain the profile and occurrence of concomitant mental disorders. METHOD: A representative sample of 1832 individuals aged 15 years or older living in the metropolitan area of Toronto were interviewed by means of telephone interviews. The participation rate was 72.8%. RESULTS: Overall, 13.2% (n = 242) of the sample had either a mood disorder (n = 127; 6.9%) or an anxiety disorder (n = 170; 9.3%) at the time of their interview. The prevalence was higher among women (16.5%) than among men (9.7%), with an odds ratio of 1.8. The comorbidity of mood and anxiety disorders was found in 3% (n = 55) of the sample. Less than one-third of respondents with a mood and/or anxiety disorder were being treated by a physician for a mental disorder. However, these individuals were greater consumers of health care services. Most of them consulted a physician an average of 5 times in the past year. Individuals on medication diagnosed with a mood and an anxiety disorder consulted a physician an average of 12 times in the past year. Only 13% of them were treated with antidepressants and under 9% with anxiolytics. CONCLUSIONS: More than 70% of subjects with a mood disorder also complained of insomnia. With the differential process, 12% of the subjects manifesting a full-fledged anxiety disorder were diagnosed with only a mood disorder because the anxiety occurred only in the course of the mood disorder. About two-thirds of the subjects diagnosed in this study were undiagnosed and untreated by their physician. PMID- 10742878 TI - Tertiary mental health services: I. Key concepts. AB - There are some individuals with severe and persistent mental illnesses who cannot be managed by primary and secondary services and who require tertiary care. Such clients are characterized by aggressiveness, noncompliance with medication, and dangerousness. Tertiary care program elements include psychosocial rehabilitation, sophisticated medication management, and behavioural approaches. Tertiary care may be delivered through assertive community treatment and/or specialized outreach teams, community residential programs, or hospital-based services. Increasingly, organized systems have been developed to ensure that individuals meet criteria for tertiary care and receive the most appropriate level of care. Most importantly, the delivery of tertiary care must not be tied to particular settings or time frames, and level of care must be delinked from model or location of care in order to create flexible, efficient, effective mental health services. PMID- 10742879 TI - Tertiary mental health services: II. Subpopulations and best practices for service delivery. AB - Tertiary care subpopulations are characterized by having more than one significant condition, each of which has been traditionally dealt with by different systems of care. They experience severe and persistent mental illness and one or more of the following: age-related physical or medical conditions, substance use disorders, developmental handicaps, and acquired brain injury. This paper provides estimates of prevalence for each of these subgroups and discusses best practices which have developed in response to their special needs. PMID- 10742880 TI - Paroxetine and tardive akathisia. PMID- 10742882 TI - A quick rating scale for depression. PMID- 10742881 TI - Obsessive-compulsive disorder with onset in old age. PMID- 10742883 TI - Clozapine weight gain, plus topiramate weight loss. PMID- 10742885 TI - New indices for transcultural comparisons in somatization. PMID- 10742884 TI - Visual and auditory hallucinations with the association of bupropion and valproate. PMID- 10742886 TI - Bupropion treatment-related sexual side effects: a case report. PMID- 10742887 TI - Increased susceptibility to seasonal cardiovascular death in the elderly and seasonal mood changes. PMID- 10742888 TI - A case of partial lycanthropy. PMID- 10742889 TI - Tardive dyskinesia from low-dose risperidone. PMID- 10742890 TI - Surgical treatment of T3 lung cancer invading the chest wall. AB - Lung tumors invading the chest wall are classed as belonging to the T3 group and are considered potentially resectable. Their management, however, is controversial, and extrapleural resection, when possible, is preferred to en bloc resection which is regarded as a far more invasive and dangerous operation. Five year survival rates for completely resected cases range in the literature from 25 to 35%, but survival rates are much worse if lymph node metastases are present. These poor outcomes have prompted the development of combined surgical approaches: preoperative radiation therapy, with or without chemotherapy, has been used with an improvement in resectability rates, but only modest results in terms of median survival; in a number of case series, increased operative morbidity and mortality have been reported with this approach. The present report relates to 122 patients treated by en bloc (20 cases) or extrapleural (102 cases) resection, 31 of whom also received neoadjuvant treatment. The operative mortality was 4.6%. Median survival was 17 months after en bloc resection and 19 months after extrapleural resection. Though no statistically significant difference was found, extrapleural resection would appear to yield better results than the en bloc procedure. PMID- 10742891 TI - [Tumor samples as a tool for large-scale monitoring of antigen-specific lymphocytes in gastric and colorectal neoplasms]. AB - The characterization of tumor-associated antigens has enabled to direct the host immune response towards the autologous tumor through appropriate loading and presentation of the antigen. In vivo conditions that generate large numbers of tumor antigens would be an important step in vaccine strategies. In this study we have therefore tested the ability of freshly isolated gastric and colorectal cancer cells to induce a specific anti-tumor response in autologous T lymphocytes. Because dendritic cells (DC) are critically involved in both initiating and boosting host immune responses, they have been used to present apoptotic bodies generated by irradiated tumor cells. Results show that these native antigens stimulate T cytotoxic response against tumor, but not peritumor normal tissues. Induction of IFN-gamma secreting cell activity, which is a standard readout in current cancer vaccine protocols, was also demonstrated by Elispot single-cells assay. These data show the antigenicity of gastric and colorectal tumor cells and open new perspectives in immunotherapy. PMID- 10742892 TI - [Hartmann's operation in acute perforated diverticulitis]. AB - Acute perforated diverticulitis of the colon is still a serious clinical event that requires an emergency treatment which is based upon clinical staging and pathological characteristics. Surgical treatment, performed in Hinchey's stages III and IV, is correlated with the presence of infection in the peritoneal cavity: it is always necessary to remove the septic focus, but there are different reconstruction strategies. The resection of the diseased colonic segment can be performed with primary anastomosis or Hartmann's operation with reconstruction in a later time. In our experience, based on 97 patients (33 of which, Hinchey's III and IV, underwent emergency surgical treatment) we preferred Hartmann's operation which carries a low risk of mortality in seriously ill patients. PMID- 10742893 TI - [The use of synthetic prosthesis in the treatment of hernia]. AB - The authors report their experience on synthetic prosthesis used in 36 patients with abdominal hernias. Thirty six cases of incisional hernias have been analyzed by Institute of General Surgery of Messina University from 1992 to 1998. Among the 33 cases with median parietal defect, in 7 cases concerning great incisional hernias group a PTEF-e prosthesis, which was positioned in an intraperitoneal site, was employed. In 21 cases of medium size incisional hernias they used a polypropylene prosthesis, which was positioned in an intraparietal or preperitoneal site. In 5 cases of small size incisional hernias, polypropylene prosthesis was placed in a superfascial site. In the 3 cases of medium size lateral laparocele++, polypropylene prosthesis was positioned in a intraparietal site. There was no operative mortality. Among postoperative complications, 4 seromas, 1 parietal and 1 subcutaneous haematoma and 2 haematomas in retroperitoneal site appeared. Clinical-instrumental controls, which have continued until two or three years after the operation were performed which pointed out a recurrence in just one case where the prosthesis was positioned in a superfascial site. To conclude, we consider that nowadays the use of synthetic prosthesis lets us face and solve problems concerning surgical treatment of incisional hernias with confidence. PMID- 10742894 TI - [Colo-anal anastomosis in tumors of the lower rectum]. AB - The Authors report their experience about totally mechanical coloanal anastomoses. Seven patients affected by rectal cancer and submitted to proctosigmoidectomy and, following totally mechanical coloanal anastomoses as described by Goligher are taken in consideration. The distance of the neoplasm from the anal verge goes from 5 to 7 cm. Using Dukes classification 6 patients were classified in B stage, only 1 in A stage and nobody in C stage. No patient received adjuvant radiotherapy. In the patients in B stage has been performed an extended ilio-pelvic lynfoadenectomy. Particular attention was put on the complete remotion of the mesorectum. In no case has been provided with a creation of a diverting colostomy. No mortality in the post-operative period nor anastomotic leakages haven't been recorded. The follow-up goes from a minimum of 8 months to a maximum of 7 years. Have been reported 2 cases of recurrence, at pelvic level after 18 months and at empathic level after 8 months from intervention. In 6 of the 7 patients have been valued the functional results that have put in evidence a satisfying gas and fecal continence. Only during the first months following the intervention has been recorded an increasing of the number of defecation that have reduced in the long term. PMID- 10742895 TI - [Minimally invasive percutaneous ultrasonography-guided treatment of postoperative splenic abscess. Personal experience with 5 clinical cases]. AB - INTRODUCTION: Splenic abscesses are very rare pathologies encountered in daily clinical practice. The treatment, after failure of other medical treatments, has basically been surgical, in spite of large diffusion of percutaneous echo and CT techniques. The initial hesitation in applying this sort of method was caused by an unfounded fear of difficult management of haemorrhagic complications. METHODS: In our Clinic we treated 6 postoperative abscesses, observed in 5 patients. One of these (16%) was double and 3 concomitant with other intraperitoneal abscessual septic collections, which received priority in percutaneous treatment. In four cases (67%), Van-Sonnemberg drainage catheters were placed which caliber varied from 12 to 14 F. In the remaining two cases, we evacuated the abscess using an echo-guided needle puncture. In two cases we applied fibrinic glue; in one case as a haemostatic agent and in the other, to protect the wide abscessual cavity. RESULTS: We recorded the resolution of the symptomatology and instrumental findings in all patients. One patient died two weeks later due to unrelated causes. We recorded one minor complication which involved an intra-abscessual haemorrhage, which was then treated by fibrinic glue (Tissucol). CONCLUSION: Echo guided percutaneous treatment, at present, is the treatment of choice for both multiple or single splenic abscesses. This treatment is not used for the miliaric form because we believe that in this case, the most appropriate choice is intensive medical treatment, which if fails, may require splenectomy. PMID- 10742896 TI - [Gastrointestinal stromal tumors: clinical and instrumental diagnosis. Analysis of a review of the literature]. AB - AIM: From a personal experience of 23 treated gastrointestinal stromal tumor (GISTs), this study analyzed both clinical and diagnostic problems of this quite new nosological category. MATERIALS AND METHODS: A this literature review provides a rigid selection of papers (scientific basis, statistic inference, type/quality of the journal, etc.); only numerous series have been included (case reports were excluded) and only those published after 1990. Three-hundred-seventy five cases have therefore been selected. Starting and late symptoms/signs and diagnostic tests employed were analyzed. RESULTS: Results show 1) a relevant GIST quantity (approx. 30%) is casually discovered, during operations carried out for other pathologies or diagnostic tests for other indications; 2) poor correlation between site of the tumor and clinical manifestations; 3) a positive correlation between tumor diameter and presence of symptoms/signs seems to exists. The accuracy of different diagnostic tests is reported. CONCLUSION: No specific symptoms/signs have been isolated; this kind of tumor is often accidentally found. An analysis of different diagnostic tests available today shows the very important role of endoscopic ultra-sound, together with CT and MRI. PMID- 10742897 TI - Laparoscopic robot-assisted right adrenalectomy and left ovariectomy (case reports). AB - BACKGROUND: Laparoscopic robot-assisted surgery has been created to reduce the patient risk of inappropriate scope movements by an assistant and to perform operations quicker and with greater ease. The Authors report their experience in laparoscopic robot-assisted right adrenalectomy for Conn's syndrome and right ovariectomy for benign ovarian mass. MATERIAL AND METHODS: Case 1. CT scan: solid right adrenal mass (diam. 2 cm). An anterior transperitoneal approach was used to perform the right adrenalectomy. The surgeon was placed at the ventral side of the patient and robotic-device was placed at the backside. HISTOLOGY: adrenocortical adenoma (diam. 3 x 2.5 x 1.5 cm). Case 2. CT scan: left iliac mass (diam. 3.5 cm) with origin in the left ovary. The patient was positioned in the gynecological position. The surgeon was positioned on right side of the patient and robot-device on left side. Left ovariectomy was performed. HISTOLOGY: ovarian serous cyst. RESULTS: Operating time was 180 min. for the adrenalectomy and 25 min. for the ovariectomy. No blood loss or complications for both operations were encountered. Image was steady and lens cleaning was unnecessary. CONCLUSIONS: The robot device (AESOP 2000) facilitated the procedures by enhancing stability of the image and reducing the need for lens cleaning. We believe that this method is feasible and could be advantageous especially for cholecystectomy, Nissen funduplication or ovariectomy but at the moment there are no comparative studies to establish the real value of this device. PMID- 10742898 TI - [Aorto-esophageal fistula caused by foreign body]. AB - Aortoesophageal fistula is a rare but fatal cause of upper gastrointestinal bleeding. AEF develop progressively from the esophageal perforation caused by foreign body. Clinically, there is a medial chest pain, followed by hematemesis and finally terminal exsanguination. Diagnosis must be achieved during the free intervals in this triad of often rapidly succeeding signs. PMID- 10742899 TI - Strangulated traumatic hernia of the diaphragm. A report of two cases. AB - The herniation of abdominal viscera in the thorax can immediately follow diaphragmatic rupture or be delayed even years after the injury. The herniated viscera can strangulate; this consequence may lead to a dangerous misdiagnosis which could be lethal for the patient. Radiological procedures, serial chest X ray studies, CT and MRI scans are mandatory to confirm diagnosis. The insertion of a naso-gastric tube is a very helpful method in ruling out hypertensive pneumothorax in the presence of an air-fluid level in the thorax. We report 2 cases of strangulated traumatic hernia of the diaphragm occurring just a few hours (case 1) and 18 months (case 2) after the trauma. During thoracotomy, a rupture of the left diaphragmatic cupola was demonstrated with herniation of the stomach in case 1, the stomach, spleen and transverse colon in case 2. No postoperative mortality or morbidity were detected. PMID- 10742900 TI - [Emergency surgery: suppurating urachal cyst producing intestinal occlusion]. AB - Urachal cysts are anomalies related to the persistence of urachal remnants after the birth. They are seldom asymptomatic, but in some cases they mimic acute abdominal disease. We report a case of a 67-year-old man with relapsing episodes of intestinal sub-occlusion caused by the presence of an infected urachal cyst. The surgical procedure we used consisted of a viscerolysis and excision of the infected cyst, avoiding partial resection of the dome of the urinary bladder. Our article also includes a review of the literature on urachal infected cyst and its management. PMID- 10742901 TI - [Unusual cause of upper digestive hemorrhage: gastric lipoma. Report of a case]. AB - Lipomas are benign tumors that are common in other sites but rare in the stomach. They are usually submucosal and, when symptomatic, are most often accompanied by gastrointestinal bleeding from ulceration of gastric mucosa overlying the mass or by symptoms of obstruction. A 55-year-old male with epigastric abdominal pain and upper gastrointestinal hemorrhage due to a gastric lipoma is presented; endoscopic and x-ray examinations revealed a submucosal tumor on the posterior wall of the gastric corpus, with ulceration in the mucosa covering it. Biopsies of the mass were nondiagnostic. The patient underwent a complete submucosal excision: the intraoperative histologic study established the diagnosis of gastric lipoma. The post operative period was uneventful. A review of the clinical, diagnostic and therapeutic aspects of this rare disease is reported. PMID- 10742902 TI - [Mixed carcinoid-adenocarcinoma tumor of the appendix: a case report and diagnostic and therapeutic considerations]. AB - The layout of the work has stayed motivated from the recent observation of mixed carcinoid, located to the appendix and wide to the peritoneal cable, joint to our observation in an elderly patient with aspecific demonstration clinical symptomatologic. The study of the endocrine tumors is in continuous evolution also if, to the actual state, there is a better comprehension of this neoplasm, variegated and complex and, under some appearances, still not known well. Jejunoileal carcinoid tumors differ, under many appearances, from those occurring in other sites of the gut. They have relatively high rate of transmural invasion and aggressive clinical behavior, contrasted by a scarce objectivity; the demonstration of symptoms generally implicates the presence of an illness in advanced stage. Also pharmacological therapy made important progress, with the possibility of administer composed able to interfere with the development and the neoplastic growth. PMID- 10742903 TI - [Butterfly mesh for the treatment of hernia]. AB - Incisional hernias (IH) occur with an incidence of 2 to 11% after laparotomy and represent a huge social and economical problem. Polypropylene meshes remarkably decreased the incidence of recurrence after first repair. This paper reports a personal method that allows a strong, safe and quick replacement of the abdominal wall with a double layer of polypropylene mesh. This method is reliable for IHs bigger than 4 cm. The sac and the surrounding fascia are cleaned from fat and scarred tissue: peritoneum is dissected up to at least 3 cm all around the edges of the hernial sac and under the fascia, as far as possible. Two sheets of PM, 3 cm larger than the defect, are sutured together with non-absorbable running suture (polypropylene 2-0) from the center to 2 cm from the extremities at the bank. The inferior mesh is extended and fixed under the fascia with polypropylene mattress stitches. When the peritoneum is not present, the edges of the mesh are refolded and sutured to the fascia in order to avoid trauma to the loops. The superior mesh is sutured directly onto the fascia. Closed aspiration drainage is positioned. We performed this method on 20 IHs (from 5 to 25 cm long). At 24 months follow-up we never observed recurrence. This method is similar to abdominal wall suture, and permits tension free repair. PMID- 10742904 TI - [Local anesthesia in the treatment of inguinal hernia]. AB - Local anesthesia is the most common technique used in the surgical treatment of inguinal hernia. The introduction of synthetic prosthesis, which are resistant to infection, has to the development of surgical techniques used in local anesthesia, in a day hospital setting. These techniques permit a lowering of hospital costs and a reduction of the incidence of complications and recurrences. Over the last few years the authors have been performing a modified Lichtenstein repair or the Rives technique in local anesthesia. In this study they present their data on 52 patients surgically treated in the period 1997-1998, and discuss the advantages of their technique. PMID- 10742905 TI - Acute colonic pseudo-obstruction affects predisposed patients. PMID- 10742906 TI - Pattern formation in integrative biology--a marriage of theory and experiment. AB - The interdisciplinary challenge to discover the underlying mechanisms in the generation of biological pattern and form are central issues in development. In this review we briefly discuss the philosophy of such an integrative biology approach. We then describe one pattern formation approach which has intimate ties to experiment, namely the mechano-chemical theory. We discuss, by way of example, the successful use of such a framework in the formation of cell-matrix networks, intimately associated with angiogenesis. All of the model parameters are estimated from experiment and the results of the model analysis compare well with experiment. We conclude with some general views on the use of models in biology. PMID- 10742907 TI - The life of form. Emergent patterns of morphological transformation. AB - The problem of biological form remains unresolved despite the known details of gene activities in embryonic development. Segmentation is discussed in connection with observed gene activities and the question of generic regularities across phyla. The sciences of complexity and nonlinear dynamics are considered as contexts for the exploration of constraints at both the level of genetic networks and morphogenesis. A new model of morphogenetic constraints coupled to dynamic attractors is proposed as an approach to the question--how to unite the contingencies of evolutionary genetic changes with the emergent regularities of organismic morphologies? PMID- 10742908 TI - Models for organizer and notochord formation. AB - In the development of higher organisms, small groups of cells can play an important role by directing the fate of the surrounding cells. Models are discussed that account for the generation of such organizing regions. The generation of local high concentrations of signalling substances was proposed to depend on local self-enhancement combined with a long-range inhibition. The model accounts for pattern regulation, for instance, for the formation of multiple embryos after fragmentation of the early blastodisc in chickens or for head regeneration in the fresh water polyp Hydra. The model has found support from more recently discovered interactions involved in organizer formation. The mutual down-regulation of noggin/chordin and BMP-4 is proposed to function as an indirect self-enhancement, establishing in this way an essential prerequisite for primary pattern formation. Self-enhancement and long-range inhibition is also crucial for the generation of substructures such as bristles or tracheae. A poisoning of an organizing region by a second antagonistic reaction of a short range but a long time constant can lead to its displacement. Long extended structures can be formed as a trace behind the moving organizer. The notochord and the tracheae of insects are discussed as examples. PMID- 10742909 TI - Mechanical signalling and angiogenesis. The integration of cell-extracellular matrix couplings. AB - In vitro angiogenesis assays have shown that the couplings between fibrin gel and cell traction forces trigger biogel pre-patterning, consisting, in the formation of lacunae which evolve toward capillary-like structures (CLS) networks. Depending on the experimental conditions (number of seeded cells, gel elasticity,...), this pre-patterning can be enhanced or inhibited. A theoretical model based on a description of the cell-biogel biochemical and mechanical interactions is proposed as a basis for understanding how integrating these interactions can lead to the pre-patterning of the biogel. We showed that the critical parameter values corresponding to the bifurcation of the model solutions correspond to threshold values of the experimental variables. Furthermore, simulations of the mechanocellular model give rise to dynamic remodelling patterns of the biogel which are in good agreement both with the lacunae morphologies and with the time and space scales derived from the in vitro angiogenesis assays. Special attention has been paid in the simulations to cell proteolytic activity and to the amplitude of cell traction forces. We finally discussed how modelling guided experiments can be inferred from these results. PMID- 10742910 TI - Geometrical models of the renewal of the epidermis. AB - We give a review of the different models developed recently that describe the renewal of the epidermis. These models, based on concepts borrowed from statistical mechanics, geometry and topology, shed new light on the understanding of the organization and the dynamics of the system. We discuss in detail a topological model of the dynamics of the inner-most layer of the epidermis: the basal layer. PMID- 10742911 TI - Theoretical models for circadian rhythms in Neurospora and Drosophila. AB - We examine theoretical models proposed for the molecular mechanism of circadian rhythms in Drosophila. The models are based on the negative feedback exerted by a complex between the PER and TIM proteins on the expression of the per and tim genes. We show that a similar model can account for circadian oscillations in Neurospora, where the protein FRQ negatively regulates the expression of the frq gene. The effect of light on the circadian rhythms is included by considering that it elicits a rise in the rate of TIM degradation in Drosophila, whereas in Neurospora it enhances the rate of frq transcription. The models account for the occurrence of sustained circadian oscillations in continuous darkness in Drosophila and Neurospora. Numerical simulations further indicate that the periodic forcing of circadian oscillations by light-dark cycles can result either in the entrainment to the external periodicity or in aperiodic oscillations (i.e. chaos), depending on the magnitude of the periodic changes in the light controlled parameter. PMID- 10742912 TI - Positive feedback circuits and memory. AB - The concept of regulatory feedback circuit refers to oriented cyclic interactions between elements of a system. There are two classes of circuits, positive and negative, whose properties are in striking contrast. Positive circuits are a prerequisite for the occurrence of multiple steady states (multistationarity), and hence, they are involved in all processes showing hysteresis or memory. Endogenous or exogenous perturbations can lead the system to exhibit or to evoke one particular stable regime. The role of positive circuits in cell differentiation and in immunology is well documented. Negative circuits are involved in homeostatic regulation, with or without oscillations. The aim of this paper is to show: a) that positive circuits account for many features of memory stricto sensu (i.e., neural memory and mnesic evocation) as well as largo sensu (e.g. differentiation or immunological memory); and b) that simple combinations of positive and negative circuits provide powerful regulatory modules, which can also be associated in batteries. These entities have vast dynamical possibilities in the field of neurobiology, as well as in the fields of differentiation and immunology. Here we consider a universal minimal regulatory module, for which we suggest to adopt the term 'logical regulon', which can be considered as an atom of Jacob's integron. It comprises a positive and a negative circuit in its interaction matrix, and we recall the main results related to the simultaneous presence of these circuits. Finally, we give three applications of this type of interaction matrix. The first two deal with the coexistence of multiple stable steady states and periodicity in differentiation and in an immunological system showing hysteretic properties. The third deals with the dual problems of synchronization and desynchronization of a neural model for hippocampus memory evocation processes. PMID- 10742913 TI - Long-distance transport, storage and recall of morphogenetic information in plants. The existence of a sort of primitive plant 'memory'. AB - An asymmetrical treatment of Bidens seedlings (pricking one of the seedling cotyledons) causes the cotyledonary buds to grow asymmetrically after release of apical dominance by decapitation of the seedlings. The symmetry-breaking signal propagates within the seedlings at a rate of at least a fraction of a millimetre per second. This information may be 'stored' (STO function) within the seedlings, without taking effect, for at least 2 weeks; then the information may be 'recalled' (RCL function), thus permitting transduction of the signal into the final response (differential growth of the buds), as a consequence of subjecting the seedlings to various symmetrical or asymmetrical treatments. A similar behaviour was observed with stimuli other than pricking (including non-traumatic stimuli), with plants other than Bidens (flax, tomato), and with responses other than cotyledonary-bud growth (hypocotyl elongation, induction of meristems, thigmomorphogenesis). There are indications that storage may involve the activation of elements implicated in cell cycle control, and that the last steps of the final response involve genes such as tch1 and hsp70. The adaptive advantage for plants in possessing STO/RCL functions is discussed. Manipulating the STO/RCL functions may have interesting practical applications, e.g. in the resistance of plants to natural stresses. The existence of the STO/RCL functions in plants constitutes an elementary form of 'memory' which may provide an experimental system simpler than the animal brain to test the validity of the theoretical models of interpretation of important features such as memory storage and evocation. PMID- 10742914 TI - A mathematical model for storage and recall functions in plants. AB - In plantlets of Bidens pilosa L., under severely limiting environmental conditions the growth of the buds at the axil of the cotyledons (cotyledonary buds) is asymmetric (i.e. one of the buds starts growing before the other one), this asymmetry being oriented by the pricking of one of the cotyledons (i.e. pricking one cotyledon increases the probability that the bud at the axil of the other cotyledon be the first to start to grow). As long as the plant apex (i.e. the terminal bud) is present, the growth of the cotyledonary buds is inhibited (apical dominance), but the souvenir of the asymmetric message caused by sub optimal environmental conditions and the orientation given by the cotyledon pricking is always present in the plant and can be revealed by removing the apex. Depending on the conditions for removing the plant apex and/or on the application of a variety of symmetrical treatments (e.g. thermal treatment, symmetrical pricking treatments, etc.) the stored asymmetry will either take effect (the bud at the axil of the non-pricked cotyledon will be the first to start to grow more often than the other one) or not (both buds will have equal chance to be the first to start to grow). This has been termed 'recalling' the stored asymmetry. By combining several successive symmetrical treatments, it is possible to reversibly switch on and off the recall function several times. This recall of the stored plant-asymmetry is analogous to the evocation function of a memory system. In this paper, we will present first a discrete logical version of the observed interaction structure between the main components of the bud growth system, then a continuous differential version, taking into account the main features of the observed experimental reality and trying to explain this phenomenology. The interaction structure of both the discrete and the continuous models presents similar positive and negative feedback circuits, necessary condition for observing multistationarity and stability. PMID- 10742915 TI - The role of intracellular components in food chain dynamics. AB - The dynamics of a simple food web, including multiple substrates and predator prey interactions, is studied. An individual-based model is presented that describes the intracellular composition of the biomass of each population with two components: reserves and structural biomass. The model describes the simultaneous uptake of multiple substrates via specific carriers and their assimilation into reserve energy via multiple assimilation pathways. The available energy is used for maintenance and growth. Parameters are estimated by curve-fitting data from the literature under the condition that the elemental balances and the enthalpy balances are met. The proposed model provides an adequate description of the macromolecular composition of biomass. The model is not too complicated to be of use in the study of food webs. The consequences of the presence of intracellular components in a food web on its long-term dynamics are investigated with bifurcation analysis. PMID- 10742916 TI - Modelization of the role of currents and turbulence on the growth and dispersion of marine phytoplankton. AB - This note introduces a model of growth and dispersion of marine phytoplankton, focusing on the effects of currents (3D) and vertical mixing. Our method consists in describing these effects as the product of the horizontal current, which is solved along characteristic lines, and the coupled action of vertical current and vertical diffusion, restricted on each characteristic line of the horizontal current. One thus obtains explicit formulae, which it will be possible to use in the study of the phytoplankton distribution. PMID- 10742917 TI - Emergence of individual behaviour at the population level. Effects of density dependent migration on population dynamics. AB - The aim of this work is to study the effects of different individual behaviours on the overall growth of a spatially distributed population. The population can grow on two spatial patches, a source and a sink, that are connected by migrations. Two time scales are involved in the dynamics, a fast one corresponding to migrations and a slow one associated with the local growth on each patch. Different scenarios of density-dependent migration are proposed and their effects on the population growth are investigated. A general discussion on the use of aggregation methods for the study of integration of different ecological levels is proposed. PMID- 10742918 TI - The emergence of diversity in plant communities. AB - The diversity of functional forms and strategies in plant communities is essential to the maintenance of the services that ecosystems provide humanity, and ultimately to the homeostasis of the biosphere. This diversity emerges from evolutionary forces operating at lower levels; these exploit the opportunities for specialization presented by exogenous and endogenous spatial and temporal heterogeneity. Two major theoretical approaches have been taken to understand how strategies arise and are maintained: optimization models, which consider the fitnesses of types in isolation, and game-theoretic methods, which take frequency dependence into account. The game-theoretic approach is more powerful, but also more challenging to apply. For some relatively simple problems in the study of biodiversity, we show how the game-theoretic formulation can be translated into an equivalent problem in optimization. More generally, however, new techniques will be needed to explore the dynamics of multiple coexisting types and strategies. PMID- 10742919 TI - Update on nuclear medicine. PMID- 10742920 TI - Lead poisoning: a disease for the next millennium. AB - The decline in the prevalence of childhood lead poisoning is a public health success story. However, nearly a million preschool-aged children in the United States alone have elevated BPb levels. Toxicity correlates with BPb concentrations and progresses from biochemical and subclinical abnormalities at levels around 10 micrograms/dL to coma and death at levels over 100 micrograms/dL. Treatment consists of the elimination of exposure, interruption of the pathway into the child, modification of diet to ensure adequate essential metal intake (calcium, iron), and on occasion, chelation therapy. The identification of children with the most lead poisoning depends on screening for exposure (questionnaire) or evidence of increased absorption (BPb test). Follow up is crucial to maximize the effectiveness of any intervention. PMID- 10742921 TI - Iron poisoning. PMID- 10742922 TI - Mercury poisoning. PMID- 10742923 TI - Quantitative image analysis of lung connective tissue in murine silicosis. AB - Pulmonary fibrosis is a disabling consequence of many lung diseases but is difficult to quantify. Lucifer yellow CH fluorescent dye (LY) appears to stain connective tissue matrix macromolecules selectively. Laser scanning confocal microscopy can quantify the intensity of fluorescence and determine the area of fluorescent material. We hypothesized that the abundance of lucifer yellow stained matrix macromolecules in lung tissue sections could be measured by laser scanning confocal microscopy, would reflect differences between varying degrees of pulmonary fibrosis, and could be compared directly to biochemical measurements of lung collagen. We exposed C57B1/6 and 129 strains of mice by aerosol to cristobalite silica (70 mg/m3, 12 days, 5 hours/day) or sham-air and examined them 2 and 16 weeks after exposure. The area of LY-stained matrix in tissue sections was quantitated by laser scanning confocal microscopy, and total lung collagen was measured biochemically as hydroxyproline (OH-proline). The LY stained connective tissue matrix appeared as bright linear bands in the alveolar septae, and was increased significantly by image analysis in C57B1/6 and 129 mice with silicosis 16 weeks after exposure. Total lung OH-proline was significantly increased in silica-exposed mice from both stains at both time points. Comparing all 8 groups, there was a significant linear correlation between the average area of connective tissue measured by LY stain and the total OH-proline per lung measured by chemical analysis (r = .72, P = .042). LY staining and confocal microscopy with image analysis offers a rapid technique for quantitative measurements of the extent of pulmonary fibrosis. PMID- 10742924 TI - Angiocentric recruitment of lymphocytes into the lung after the intrabronchial instillation of antigen. AB - The pathogenesis of acute lymphocytic inflammation in the lower respiratory tract appears to involve the recruitment of lymphocytes out of the blood stream and into the extravascular lung tissue. To investigate the membrane molecules regulating this process, we used the intrabronchial instillation of cellular antigen to trigger lymphocyte recruitment into the lower respiratory tract. Sheep presensitized 6 to 10 weeks earlier at a remote site were intrabronchially challenged with 1-5 x 10(7) cells from a B lymphoblastoid cell line. The cells were instilled into a subsegmental bronchus through a bronchial catheter. The stimulated and contralateral control segments were studied at a peak of inflammation, approximately 72 hours after antigen stimulation. Gross and microscopic studies of the stimulated segment demonstrated localized inflammation characterized by the perivascular infiltration of lymphocytes. In contrast, control areas of the lung demonstrated only scattered perivascular lymphocytes. Immunohistochemistry of the stimulated lung showed that the majority of these perivascular cells were CD3+ CD4+ lymphocytes. The T lymphocytes expressed high levels of the cell adhesion molecules beta 1 integrin and LFA-1, but low levels of the L-selectin membrane molecule. Immunohistochemistry of the endothelial cells associated with the lymphocyte infiltrates demonstrated intense staining of the ICAM-1, and beta 1 integrin adhesion molecules. Electron microscopic studies of the endothelial cells in the antigen stimulated areas of the lung confirmed morphologic changes consistent with endothelialitis. These results suggest that the intrabronchial instillation of cellular antigen stimulates an angiocentric T cell infiltration regulated by activated pulmonary endothelial cells. The histologic and morphologic findings are remarkably similar to those observed during acute lung transplant rejection. PMID- 10742925 TI - Changes in lung structure and cellular division induced by tracheal obstruction in fetal sheep. AB - Increased expansion of the fetal lung, caused by obstruction of the fetal trachea, is a potent stimulus for growth and structural development of the fetal lung. Our aim was to analyze the changes in lung structure induced by fetal tracheal obstruction and to identify cell types that contribute to the growth response. Fetal sheep were exposed to 2, 4, or 10 days of tracheal obstruction (TO) and on day 128 of gestation (term "147 d"), were injected with 3H-thymidine 8 hours before tissues were collected. The right lung was fixed at 20 cm H2O and prepared for stereological and autoradiographic analysis. Alveolar wall thickness (7.8 +/- 0.3 microns vs 5.5 +/- 0.4 microns) and percent tissue space (27.9 +/- 0.9% vs 21.4 +/- 2.8%) were increased at 2 days of TO, but were not different from control at 4 and 10 days. The luminal surface area of the right lung increased gradually from 2.4 +/- 0.2 m2/kg in control fetuses to 3.6 +/- 0.4 m2/kg following 10 days of TO and this increase was accompanied by an increase in alveolar number (control: 808 x 10(6) +/- 81.9 x 10(6) vs 10d obstruct: 1254 x 10(6) +/- 63 x 10(6). Alveolar diameter increased at 2 days of TO (51.8 +/- 1.4 microns vs 43.8 +/- 1.9 microns), but was not increased further at 4 or 10 days. The percentage of dividing cells was increased at 2 days of TO (12.64 +/- 3.39% vs 1.73 +/- 0.31%), remained elevated at 4 days (5.01 +/- 0.27%), but had returned to control by day 10. The increase at 2 days was due to division of type II epithelial cells, fibroblasts, and endothelial cells. We conclude that increased expansion of the fetal lung induces time-dependent changes in lung structure and cell division rates; these include a transient increase in alveolar wall thickness, a rapid increase in alveolar number, and a gradual increase in luminal surface area. The latter is probably caused by an increase in alveolar number rather than an increase in the alveolar size. PMID- 10742927 TI - Views retrospective and prospective. PMID- 10742928 TI - Emotions in solution-focused therapy: a re-examination. AB - This article re-examines whether and how emotions are an aspect of solution focused therapy. A major theme in the article focuses on the usual ways that therapists define and discuss emotions in solution-focused and other therapies. We argue that these discussions are a source of much confusion about emotions and about solution-focused therapy, including the confusing idea that emotions are neglected in solution-focused therapy. The second major theme describes an alternative approach to these issues, one that we believe better fits with the assumptions and concerns of solution-focused therapy. The approach is based on Wittgenstein's writings about language games, private experience, and how emotions are rule-following activities. Viewed from this perspective, solution focused therapists take account of their clients' emotions by helping clients to create new emotion rules to follow. PMID- 10742929 TI - Does couple and family therapy have emotional intelligence? PMID- 10742926 TI - Interaction of tobacco-specific toxicants with nicotinic cholinergic regulation of fetal pulmonary neuroendocrine cells: implications for pediatric lung disease. AB - Pulmonary neuroendocrine cells (PNEC) produce neuropeptides and biogenic amines which regulate pulmonary vasoconstriction and bronchoconstriction. Increased numbers of PNEC along with elevated levels of their products have been consistently observed in a variety of pediatric pulmonary diseases, some of which are etiologically linked with prenatal exposure to cigarette smoke, and all of which are exacerbated by passive smoking. The objective of our studies was to characterize the autonomic regulation of these cells and to explore a potential direct interaction of tobacco-specific toxicants with these regulatory pathways. Using reverse transcription polymerase chain reaction (RT-PCR), radioreceptor assays, flow cytometry, enzyme-linked immunosorbent assay (ELISA), and cell proliferation assays, we found that the release of serotonin from fetal PNEC is regulated by a neuronal alpha 7-nicotinic acetylcholine receptor (alpha 7-nAChR) via a Ca(2+)-dependent mechanism. The tobacco-specific toxicants nicotine and 4 (methylnitrosamino)-1-(-3-pyridine)-1-butanone (NNK) bound with high affinity to this receptor, resulting in influx of Ca2+, release of serotonin, and stimulation of DNA synthesis. Chronic stimulation of the alpha 7-nAChR in the fetal lungs by prenatal exposure to cigarette smoke may contribute to the development of smoking related pediatric pulmonary disease, whereas postnatal chronic exposure to environmental smoke may exacerbate these pediatric diseases via direct interaction with this receptor. PMID- 10742930 TI - Uncommon strategies for a common problem: addressing racism in family therapy. AB - Race and racism have a profound effect on our daily lives and the practice of family therapy. Whether individual or institutional level, overt or covert, intentional or unintentional, there are a variety of ways in which racism can infiltrate the therapeutic process. Before therapists can take steps to address racism effectively within the context of family therapy, it is important to attend to the development of their racial awareness and racial sensitivity. These provide the critical foundation upon which specific skills and strategies associated with effectively identifying and responding to racism in therapy are based. This article defines racial awareness and sensitivity and provides suggestions for enhancing both. In the section that follows, three major ways in which racism can infiltrate the therapeutic process are described. Skills and strategies for addressing each of these in family therapy are presented. PMID- 10742931 TI - Using a feminist lens in contextual therapy. PMID- 10742932 TI - Lesbians, gay men, and their parents: family therapy for the coming-out crisis. AB - It is considered psychologically healthy for lesbians and gay men to come out and live outside of the closet. However, parents tend to react with shock, disappointment, and shame when they learn of a son's or daughter's gay sexual orientation. Disclosure often precipitates a painful family crisis, which can lead to cutoffs between members. This article describes family therapy theories and interventions that can aid therapists in sheparding families through the initial stages of the coming-out crisis. Family therapists are advised to acknowledge and address the distinct emotional needs of coming-out individuals and their parents. Parents must grieve and obtain accurate information about gay lifestyles. Lesbians and gay men need support as they struggle to cope with their parents' negative reactions. Family members should be coached to maintain non combative communication following the disclosure, even if contacts are initially brief and superficial. Case examples, drawn from the author's clinical work, will demonstrate how to address the separate needs of lesbians, gay men, and their parents while maintaining (or rebuilding) family relationships and ultimately guiding families toward successful resolution of this crisis. PMID- 10742933 TI - Multisystemic therapy: monitoring treatment fidelity. AB - The challenges of specifying a complex and individualized treatment model and measuring fidelity thereto are described, using multisystemic therapy (MST) as an example. Relations between therapist adherence to MST principles and instrumental and ultimate outcome variables are examined, as are relations between clinical supervision and therapist adherence. The findings provide modest support for the associations between MST adherence measures and instrumental and ultimate outcomes. Results also show that adherence can be altered when clinical supervision and adherence monitoring procedures are fortified. The modest associations between adherence measures and youth outcomes argue for further refinement and validation of the MST adherence measure, especially in light of the well-established effectiveness of MST with challenging clinical populations and the increasing dissemination of MST programs. PMID- 10742935 TI - Dissecting life with a Jewish scalpel: a qualitative analysis of Jewish-centered family life. AB - This article highlights findings from a qualitative analysis of the ways in which Jewish families identify how Judaism influences their lives. A theoretical sample of two religious and two cultural families were chosen from a larger sample of 48 Jewish families in Central New York. The qualitative part of this study, which was part of a larger multimethod investigation, was done in order to gather inductively any data that would allow the researchers to build a theory about a particular type of ethnic identity--Jewish identity--and how it affects family dynamics. Eleven categories emerged from this study, which suggest that this particular type of ethnic identity influences many family dynamics, for example, styles of communicating, parenting, values, and family rituals. The participants seem to dissect the world with a "Jewish scalpel." This "scalpel" informs their daily interactions, their parenting styles, and their childrens' self perceptions. PMID- 10742934 TI - Difficulty in implementing a family intervention for bipolar disorder: the predictive role of patient and family attributes. AB - Family affect was examined as a predictor of difficulty implementing a 9-month, manual-based, psychoeducational family therapy for recently manic bipolar patients. Prior to therapy, family members were administered measures to assess both their expressed emotion and affective behavior during a family interaction task. Following family treatment, both therapists and independent observers rated the overall difficulty of treating the family, and therapists also rated each participant's problem behaviors during treatment, in the areas of affect, communication, and resistance. Therapists regarded affective problems among relatives and resistance among patients as central in determining the overall difficulty of treating the family. Relatives' critical behavior toward patients during the pretreatment interaction task predicted both independent observers' ratings of overall treatment difficulty and therapists' perceptions of relatives' affective problems during treatment. Moreover, patients' residual symptoms predicted independent observers' ratings of overall difficulty and therapists' perceptions of patients' resistance to the family intervention. Results suggest that difficulties in conducting a manual-based family intervention can be predicted from systematic, pretreatment family and clinical assessment. PMID- 10742936 TI - "Factor analyses of the family assessment device," by Ridenour, Daley, & Reich. AB - Ridenour, Daley, and Reich conducted a series of factor analyses using the correlational matrix of the subscale scores of the Family Assessment Device (FAD), published in Family Process, December, 1999. They conclude that "the FAD subscales be reorganized from their current seven-subscale format" (p. 507). We propose that this suggestion for reorganization is premature and based on the inappropriate application of an "internal consistency" model of scale construction to the FAD. We further suggest that the most important criteria regarding an assessment instrument are those of reliability, validity, and clinical utility. In the absence of this kind of data regarding alternative organizations of the FAD, we believe that the original subscales remain the best choice. PMID- 10742937 TI - The Oregon report: neutrality at OHD? PMID- 10742938 TI - The Oregon report: neutrality at OHD? PMID- 10742939 TI - The Oregon report: neutrality at OHD? PMID- 10742940 TI - The contributions of sociology to medical ethics. PMID- 10742941 TI - Moral teachings from unexpected quarters. Lessons for bioethics from the social sciences and managed care. PMID- 10742942 TI - Beneficence today, or autonomy (maybe) tomorrow? PMID- 10742943 TI - America as pattern and problem. PMID- 10742944 TI - Centering bioethics. PMID- 10742945 TI - Why gender matters to the euthanasia debate. On decisional capacity and the rejection of women's death requests. PMID- 10742946 TI - Universalism & particularism. Fighting to a draw. PMID- 10742947 TI - Appropriations: the Sargasso Sea of Congress. PMID- 10742948 TI - Reinventing the delivery of health care services in Puerto Rico. PMID- 10742949 TI - Academic and ethical implications of health care reforms based on managed care: some critical reflections. PMID- 10742950 TI - Confidentiality dilemmas in clinical education. AB - Through an analysis of three cases, this article illustrates many of the subtleties of ethical discernment involved during clinical fieldwork rotations when allied health students decide to withhold information about their disabilities. By law in the United States, educators are bound to uphold students' confidentiality in this regard. However, this analysis examines many of the complexities that clinical coordinators, preceptors, and students must grasp when faced with conflicting ethical duties. PMID- 10742951 TI - Health care professionals' attitudes toward licensure, national certification, and continuing education requirements: results of a Texas survey. AB - Health care professionals are challenged to not only maintain their skills but take them to new levels. Continuing education (CE), mandated by licensure and national certification, is one means of working toward this goal. This study examined the opinions and attitudes of professionals in regard to these issues. The majority of the survey respondents supported the need for licensure and certification and perceived that they benefited personally. A significant percentage indicated that existing CE systems might not meet their intended purposes and goals and might not be the best criteria for renewal. Many had difficulty finding appropriate and convenient programs, and some programs did not deliver as advertised. The results suggest an opportunity to improve both CE systems and CE programs offered to health care professionals. PMID- 10742953 TI - The decline of clinical laboratory science programs in colleges and universities. AB - Enrollment in clinical laboratory science (CLS) programs has declined over 50% since 1980. This has led to the closure of many CLS programs while putting others at risk. This article examines the environmental changes that have occurred since the early 1980s and their impact on CLS programs and enrollment. Recommendations for CLS programs and Schools of Allied Health for adaptation to these changes are also made. PMID- 10742952 TI - Genetic technology and testing: perceptions of allied health professional education departmental chairs. AB - This study elicited a preliminary report that reflected the perspectives and interest of health professional academicians in regard to the need for and appropriateness of genetic technology and testing information in their curricula. Of the original attainable sample of 135 departmental chairs of allied health professional education programs nationwide, 88 responded to a mailed questionnaire, for a response rate of 65%. Of the 88, 11 (13%) saw no need for such information to be provided for allied health students at the undergraduate or graduate level or for currently practicing allied health professionals (AHPs). Consequently, the elicited results were based on the responses of the other respondents who perceived a need for genetic technology and testing information to be made available to both AHP students (86% undergraduate and 92% graduate) and currently practicing AHPs (91%). In regard to preferences for required and/or recommended student exposures to and participation in genetic technology information and coursework, their responses varied. PMID- 10742954 TI - Inclusion of medical-legal issues in entry-level occupational and physical therapy curricula. PMID- 10742955 TI - Final report of a study to assess the validity and reliability of the standards of eight allied health professions. PMID- 10742956 TI - Characterization of the YbBa2Cu3O7-y and YBa2Cu3O7-y thin superconducting films prepared by chemical solution deposition on MgO(001) substrate. AB - Thin superconducting films of the YbBa2Cu3O7-y (Yb123) and YBa2Cu3O7-y (Y123), prepared by post-deposition annealing of the metal naphthenates gels spin-coated on MgO(001) substrate, have been characterized by cross-sectional high-resolution electron microscopy and X-ray diffraction. It was found that the c-axis Yb123 films were epitaxially grown on the MgO(001) substrate at the temperature range from 700 degrees C to 775 degrees C in a gas mixture containing Ar and O2 with the oxygen partial pressure of p(O2) = 10(-4) atm. In contrast to the Yb123 films, it was found that Y123 films could be derived at a wider temperature range from 750 degrees C to 950 degrees C. Randomly oriented Y123 films were also grown on the MgO(001) substrate besides the majority of in-plane c-axis oriented growth. PMID- 10742957 TI - Ultrastructural verification of anchoring role of lamina fibroreticularis of dental basement membrane in odontogenesis. AB - In a previous study of the developing tooth a characteristic fibrillar layer associated with the basement membrane of the inner enamel epithelium was found to be a highly specialized lamina fibroreticularis of the basement membrane which is unusually rich in basotubules, 10 nm wide microfibril-like structures. In this study this layer was further examined in detail in the hope of ultrastructurally elucidating its role in odontogenesis. Tooth germs of the monkey (Macaca fuscata) were processed for thin section observations. Dental papilla cell processes were inserted into the lamina fibroreticularis and their surface was closely associated with numerous parallel basotubules. With high-resolution observations the space between the surface and nearest basotubules as well as the spaces between neighbouring basotubules were bridged by 1.5-3 nm wide filaments running perpendicular to the axis of basotubules. These results indicate that the processes of dental papilla cells are linked to groups of basotubules by means of 1.5-3 nm wide filaments. Immunoperoxidase staining showed the presence of fibronectin along basotubules as well as within the space between the process and basotubule. This result, together with the comparison of these filaments with microfibril-associated 1.2-3 nm wide fibronectin filaments and the reported abundance of fibronectin in the basement membrane area during odontogenesis, indicates that these 1.5-3 nm wide filaments are composed of fibronectin. After immunostaining for amyloid P component, done with the rat tissue because of the nature of an available antiserum, basotubules in the lamina fibroreticularis were positively stained, as has been shown in basotubules/microfibrils in other locations. Microfibrils function as anchoring rods by interlinking connective tissue components to one another and to the cells. Basotubules, thought to be basement membrane-incorporated microfibrils, in the lamina fibroreticularis in this study are also likely to function as an anchoring device to immobilize dental papilla cells along the basement membrane. Such an arrangement of mesenchymal cells is known to be crucial for the successful differentiation of odontoblasts in the developing tooth. PMID- 10742958 TI - Periciliary structure of developing rat photoreceptor cells. A deep etch replica and freeze substitution study. AB - The purpose of this study is to identify the morphological machinery for selective transport of proteins required in the outer segments of the rat photoreceptor cell. As a first step, the three-dimensional architecture of the periciliary region and its developmental changes were examined. Freeze-deep etching and freeze-substitution methods combined with rapid freezing technique were used. The apical surface of the inner segment was swollen and partially enclosed the base of the connecting cilium in early postnatal stages, so that the basal region of the connecting cilium was inevitably surrounded by a groove. However, a specialized periciliary ridge complex as seen in frog photoreceptor cells has never been observed in rat photoreceptor cells. The cytoplasmic surface of the plasma membrane of the apical inner segment in the vicinity of the connecting cilium was covered with loose fine filaments. However, it was unlikely to be a possible structural candidate for selective transport of membrane proteins. This study also revealed the interior structure of the connecting cilium. Actin filaments in the distal axonem formed a complicated meshwork together with an unknown substance. Since S1 decorated filaments were not detected in the middle region of the connecting cilium, actin filaments at the base of outer segment seem to be independently polymerized locally from G-actin that is transported from the inner segment. PMID- 10742959 TI - Poxvirus virions: their surface ultrastructure and interaction with the surface membrane of host cells. AB - Virions of vaccinia and orf viruses were examined by ultrahigh-resolution scanning electron microscopy using a non-coating method. Intracellular mature particles of vaccinia virus appeared to be covered with a net and ultrastructurally their surface consists of many fine ridges and globules, while the surfaces of orf virus mature particles recovered from infected cells consist of spirally running protrusions. The ridge-like structures of vaccinia virus were presumed to correspond to surface tubules shown by negative staining of this virus, while the spiral protrusions of orf virus were presumed to correspond to spiral threads having a criss-cross appearance by the same staining. Using scanning electron microscopy in which the samples were prepared by the conventional method, we observed: (i) many virions, i.e. one or two hundreds, or occasionally more reaching about one thousand particles, of the IHD strain of vaccinia virus, (ii) many or a moderate number of virions, i.e. about one hundred or fewer particles, of the 58 strain of cowpox virus and (iii) rather few virions, i.e. several tens or fewer particles, of the Iwate strain of orf virus on the free surface of each cell infected with these viruses. It must be noted that the number of virions detected considerably differed in respective cells examined. Virus budding was frequently observed at the cell surface of monolayer cells infected with vaccinia virus but it was never detected with cowpox or orf virus, indicating a difference in the mechanism of virus release between vaccinia and the other two viruses. When whole cells infected with vaccinia virus were examined by a combination of high-voltage and scanning electron microscopies, virions on the cell surface and those inside the cells were clearly differentiated. All virions on the cell surface had an envelope, and some of the envelopes had a slack and/or one or more bulges. PMID- 10742960 TI - Ultrastructure of taste receptor cells in active and hibernating ground squirrels. AB - Differences in taste bud ultrastructure between active and low-temperature hibernation states were investigated in the Siberian ground squirrel, Citellus undulatus. Compared to active summer animals, taste bud volume and the morphology of the taste pore showed little change while an animal was hibernating. However, impressive differences were observed in nuclei and cytoplasmic organelles of all cells within taste buds. In low-temperature hibernation the nucleoli of elongated, non-dark cells had few, but large, fibrillar centres, a sign of inactivity. In the cytoplasm, the number of free polyribosomes (polysomes) was sharply decreased; ribosomes were seen mainly as (non-synthesizing) monosomes and more seldom as membrane-associated ribosomes. Profiles of endoplasmic reticulum (ER) were reduced, to the extent that only separate and rare cisternae of smooth and rough ER remained. The large vacuoles, which are typically found in many taste cells of active animals, were absent during hibernation, and the Golgi apparatus appeared to be disassembled into numerous vesicles. Of these, many had small diameters near 60 nm, while a few had larger diameters near 300 nm. Secretory organelles (dense granules in dark cells and dense-cored vesicles in type III cells) were rare during hibernation, the lateral plasma membrane was smooth, and signs of exocytosis were not found. These changes suggest reduced protein synthesis and reduced sensory function during hibernation. PMID- 10742961 TI - High-dose therapy for follicular lymphoma. AB - Most patients with advanced-stage follicular non-Hodgkin's lymphoma (NHL) are not cured with conventional therapy. The use of high-dose therapy and autologous stem cell transplantation in patients with relapsed follicular NHL has received increasing attention. Several large studies suggest a disease-free survival rate of approximately 40% among patients transplanted during sensitive relapse, although the role of autologous transplantation in first remission remains controversial. Patients with histologic transformation from low-grade to diffuse large B-cell lymphoma whose disease remains sensitive to conventional therapy have a similar disease-free survival rate. Allogeneic transplantation has achieved relapse, overall survival, and treatment-related death rates of approximately 15%, 50%, and 40%, respectively, in patients with follicular NHL. Studies of minimal residual disease suggest that the presence of lymphoma cells in the autologous graft and within the patient before clinically apparent relapse is predictive of later recurrence. Therefore, treatment of minimal residual disease may improve the outcome of high-dose therapy. Use of a tumor-free stem cell product through improved purging or allogeneic stem cells is one approach, although the morbidity and mortality of allogeneic transplantation remain high. Immunomodulatory strategies with monoclonal antibodies, vaccines, or adoptive immunotherapy may be particularly well suited to patients at high risk for relapse following high-dose therapy. PMID- 10742962 TI - Survival rates significantly worse for African-Americans with endometrial cancer. PMID- 10742963 TI - Regional strategies for managing hepatocellular carcinoma. AB - Hepatocellular carcinoma is a common, difficult-to-treat cancer that has a variable natural history depending on patient demographics and the etiology and extent of underlying liver disease. Resection is the preferred treatment option but is only possible in the rare patient who has adequate hepatic reserve and limited-stage cancer. Systemic chemotherapy is mostly inactive. Because most patients with hepatocellular cancer succumb to hepatic failure, this is a disease that appears to be amenable to regional treatments. For this reason, numerous intratumoral, ablative techniques are available. Other routinely used regional treatment modalities include intraarterial chemotherapy, chemoembolization, Lipiodol chemoembolization, and internal radiation. However, the benefits of these interventions have been difficult to ascertain given the variable clinical course of the disease. Regional delivery may prove to be most valuable as a route for administering newer agents. PMID- 10742964 TI - Prostate surgeries: average charges throughout the United States, 1997. PMID- 10742965 TI - Photodynamic therapy in lung cancer. AB - Photodynamic therapy (PDT) involves the use of photosensitizing agents that are selectively retained within tumor cells. The agents remain inactive until exposed to light of the proper wavelength. When activated by light, these compounds generate toxic oxygen radicals that result in tumor necrosis. In lung cancer, PDT can be used for both carcinoma in situ and for the treatment of unresectable disease with endobronchial obstruction. For patients with advanced disease, careful patient selection and integration of PDT with other interventional techniques are critical. Limited data suggest that PDT is comparable in efficacy to neodymium-yttrium-aluminum garnet (Nd-YAG) laser therapy, and some evidence indicates that it may be superior in terms of duration of response. For PDT to be used effectively, it should be integrated into a multimodality approach with chemotherapy and radiation. The optimal sequencing of these treatment modalities remains an area for further investigation. PMID- 10742966 TI - Clinical trials referral resource. Current clinical trials in prostate cancer. PMID- 10742968 TI - Preserving fertility in young women with ovarian cancer does not decrease survival. PMID- 10742967 TI - Management of venous thromboembolism in cancer patients. AB - Venous thromboembolism is a common complication in patients with cancer. The management of deep-vein thrombosis and pulmonary embolism can be a considerable challenge in these patients. Diagnosing venous thrombosis requires objective testing, and noninvasive investigations may be less accurate in patients who have cancer than in those who do not. Treatment of acute venous thrombosis at home with low-molecular-weight heparin is an attractive option in patients with malignant disease, in whom quality of life is especially important. Comorbid conditions, warfarin resistance, difficult venous access, and a potentially high bleeding risk are some of the factors that often complicate the prolonged course of anticoagulant therapy needed in this group. In addition, the use of central venous catheters is increasing, but the optimal treatment of catheter-related thrombosis remains controversial. This article reviews the current diagnostic and treatment approaches to venous thromboembolism in patients with cancer and provides several clinical scenarios to illustrate and discuss some common management problems. PMID- 10742969 TI - Meta-analysis: methods, strengths, and weaknesses. AB - Meta-analysis is a systematic, quantitative approach to the combination of data from several clinical trials that address the same question. This analytic approach can help resolve questions that remain unclear from the results of individual trials. Meta-analysis is of particular interest in oncology because of the small differences in efficacy between therapeutic alternatives. The large number of patients included in meta-analyses permit small to moderate benefits of a treatment to be reliably detected and larger treatment benefits to be quantified more accurately. Despite these apparent benefits, the use of meta analysis has met with a great deal of resistance and has generated much controversy in clinical journals. After a brief description of the basic methods of conducting meta-analyses, this article will explore both their advantages and disadvantages. PMID- 10742970 TI - Overview of membrane transport. AB - Pharmaceutical scientists increasingly utilize transporters for drug delivery and targeting. The biological barriers to drug delivery can basically be divided into epithelial, endothelial, elimination, and target cell barriers. Membrane transporters play an important role in drug entrance and exit from the body. In addition, it is possible to utilize transporters for drug delivery, e.g., improving oral absorption via the peptide transporter. Identification, a better understanding of their transport characteristics, and the regulation of the membrane transporters will allow the development of better drug delivery strategies. PMID- 10742971 TI - Classification of membrane transporters. PMID- 10742972 TI - Drug transport and targeting. Intestinal transport. AB - A wide variety of transporters are found in the intestine, and are involved in the membrane transport of daily nutrients as well as drugs. These intestinal transporters are located in the brush border membrane as well as basolateral membrane. Each transporter exhibits its own substrate specificity, and some have broader specificities than others. In addition, the distribution and characteristics of the intestinal transporters exhibit regional differences along the intestine, implying diverse physiologic functions and in some cases pathologic responses. Indeed several genetic disorders have been shown to result from deficient intestinal transporters. The development of prodrugs that target to intestinal transporters has been successful in improving oral absorption. For example, the intestinal peptide transporter is utilized in order to increase the bioavailability of several classes of peptidomimetic drugs, especially ACE inhibitors and beta-lactam antibiotics. The bioavailability of poorly absorbed drugs can be improved by utilization of the transporters responsible for the intestinal absorption of various solutes and/or by inhibiting the transporter involved in the efflux system. Recent advances in gene cloning and molecular biology techniques make it possible to study the characteristics and distribution of transporters at the molecular level. Based on molecular characterizations of membrane transporters and accumulated biochemical data on their specificities and kinetics, structural modification and targeting of a specific transporter is a promising strategy for the design of drugs that improve bioavailability and tissue distribution. PMID- 10742973 TI - The molecular basis for hepatobiliary transport of organic cations and organic anions. PMID- 10742974 TI - Affinity of drugs to the different renal transporters for organic anions and organic cations. PMID- 10742975 TI - Drug disposition and targeting. Transport across the blood-brain barrier. PMID- 10742976 TI - The mammalian facilitative glucose transporter (GLUT) family. PMID- 10742977 TI - Cationic amino acid transporters (CATs). Targets for the manipulation of NO synthase activity? PMID- 10742978 TI - Electrophysiological analysis of renal Na(+)-coupled divalent anion transporters. PMID- 10742979 TI - Dipeptide transporters. PMID- 10742980 TI - Antigenic peptide transporter. PMID- 10742982 TI - Multidrug-resistance transporters. AB - P-glycoprotein was initially isolated due to its role in multidrug resistance to cancer chemotherapeutics. Recent work, however, makes it increasingly apparent that this transporter is also involved in the pharmacokinetics of many drugs. P gp is strategically expressed in the luminal epithelial cells of organs often associated with drug absorption and disposition, for example, hepatocyte canalicular membrane, renal proximal tubules, and the intestinal mucosa. P-gp is also expressed in the endothelial cells comprising the blood-brain barrier. This localization clearly suggests the potential for this protein to serve as a protective mechanism against entry of toxic xenobiotics and also suggests that P gp is well situated to participate in the removal of therapeutic agents. Numerous investigations with drugs such as digoxin, etoposide, cyclosporine, vinblastine, Taxol, loperamide, dom-peridone, and ondansteron demonstrate that P-gp has an important role in determining the pharmacokinetics of substrate drugs. Pharmacological modulation of P-gp function to increase drug bioavailability, both on a organismal and a cellular level, is one approach currently being explored to enhance therapeutic effectiveness. This approach is not without potential collateral consequences given the wide tissue distribution of P-gp. While animals deficient in P-gp are viable and without obvious abnormalities, the pharmacokinetics and toxic consequences of several compounds are significantly altered in these animals. Thus blockade of the protective P-gp barrier in humans may have adverse effects on substrate drugs. In particular, this situation may arise when several compounds which may be substrates compete for P-gp-mediated transport. Additional multidrug transporters, notably MRP and family members, have been identified and may also determine the fate of pharmaceuticals. Further understanding the physiological role of each of the multidrug transporters is critical for determining their role in pharmacokinetics and for evaluating the consequences of modification of their activities. Such information is also important in the development of novel drugs which may be substrates for these transporters. PMID- 10742981 TI - Nucleoside transporters of mammalian cells. AB - In this review, we have summarized recent advances in our understanding of the biology of nucleoside transport arising from new insights provided by the isolation and functional expression of cDNAs encoding the major nucleoside transporters of mammalian cells. Nucleoside transporters are required for permeation of nucleosides across biological membranes and are present in the plasma membranes of most cell types. There is growing evidence that functional nucleoside transporters are required for translocation of nucleosides between intracellular compartments and thus are also present in organellar membranes. Functional studies during the 1980s established that nucleoside transport in mammalian cells occurs by two mechanistically distinct processes, facilitated diffusion and Na(+)-nucleoside cotransport. The determination of the primary amino acid sequences of the equilibrative and concentrative transporters of human and rat cells has provided a structural basis for the functional differences among the different transporter subtypes. Although nucleoside transporter proteins were first purified from human erythrocytes a decade ago, the low abundance of nucleoside transporter proteins in membranes of mammalian cells has hindered analysis of relationships between transporter structure and function. The molecular cloning of cDNAs encoding nucleoside transporters and the development of heterologous expression systems for production of recombinant nucleoside transporters, when combined with recombinant DNA technologies, provide powerful tools for characterization of functional domains within transporter proteins that are involved in nucleoside recognition and translocation. As relationships between molecular structure and function are determined, it should be possible to develop new approaches for optimizing the transportability of nucleoside drugs into diseased tissues, for development of new transport inhibitors, including reagents that are targeted to the concentrative transporters, and, eventually, for manipulation of transporter function through an understanding of the regulation of transport activity. PMID- 10742983 TI - Transporters for bile acids and organic anions. PMID- 10742984 TI - Molecular and functional characteristics of cloned human organic cation transporters. PMID- 10742985 TI - Organic anion transporters. PMID- 10742986 TI - Vitamin B12 transporters. AB - The uptake of vitamin B12 from the intestine into the circulation is perhaps the most complex uptake mechanism of all the vitamins, involving no less than five separate VB12-binding molecules, receptors and transporters. Each molecule involved in uptake has a separate affinity and specificity for VB12 as well as a separate cell receptor. Thus VB12 is initially bound by haptocorrin in the stomach, then by IF in the small intestine. An IF receptor is then involved in uptake of the IF-VB12 complex by the intestinal epithelial cell, with the subsequent proteolytic release of VB12 and subsequent binding to TcII. The TcII receptor then transports the TcII-VB12 complex across the cell, whence it is released into the circulation. It is surprising, then, that despite its complexity, it has been possible to harness the vitamin VB12 uptake mechanism to enhance the oral uptake of peptides, proteins, and nanoparticles. PMID- 10742987 TI - [The assessment of working memory by Mini-Mental State Examination]. AB - INTRODUCTION: Delayed recall items of the Mini-Mental State Examination (MMSE) and the Mini Cognitive Examination are based on a simultaneous processing of the storage of the material to be remembered while other test's items are executed. This function is probably mediated by the working memory (WM) system. OBJECTIVE: A new protocol of WM assessment during the Mini Cognitive Examination application is presented. PATIENTS AND METHODS: The standard Mini Cognitive Examination is modified with two new items of registration and recall. Criteria to introduce three new words in registration 2 and 3 is a complete recall of the three previous words. The score of the three recall trials, until a 27 points maximum, represents WM. A sample of 44 patients with probable Alzheimer's disease (AD), 19 with possible AD, 39 patients with cognitive impairment without dementia and 14 control subjects were evaluated. RESULTS: Patients with probable AD and possible AD showed the minimum WM scores and the greatest percentage of words repetitions (WM 3.2 +/- 3.6 and 7 +/- 6, with 91 and 83% of repetitions, respectively); control subjects scored a WM of 22 +/- 6 with 14% of repetitions; patients with cognitive impairment without dementia scored a WM of 11 +/- 4 and 50% of repetitions. CONCLUSIONS: We propose that delayed recall items of the MMSE and the Mini Cognitive Examination are an index of WM capacity. This new method reinforces the assessment of delayed recall during Mini Cognitive Examination or MMSE application, and can facilitate an early detection of anterograde amnesia. PMID- 10742988 TI - [Side effects of antithrombotic treatment in the secondary prevention of cerebrovascular disease]. AB - OBJECTIVE: To determine the incidence of adverse effects of antithrombotic drugs (platelet anti-aggregants and anticoagulants) in patients with transient ischemic attacks (TIA) and mild ischemic strokes (IL). PATIENTS AND METHODS: Clinical follow-up data on the patients with TIA and IL included in a community-based observational study undertaken in the province of Segovia. Adverse effects which led to suspension of treatment or were a potential danger for the patient's life were recorded. RESULTS: The groups studied included 235 patients and the average initial age was 70.8 years. The average follow-up time was 3.6 years. The treatment initially given included: aspirin (75.7%), ticlopidine (12.8%) and acenocumarol (9.4%). Adverse effects led to suspension of treatment in 6% (5% with aspirin and acenocumarol, 17% with ticlopidine). The adverse effects of ticlopidine were mild and patients did not need admission to hospital (cutaneous exanthema, diarrhoea and reversible leucopenia). Aspirin was associated with digestive tract bleeding (2.8%) and cerebral hemorrhage (1.7%) which required hospital admission in most cases. One patient treated with acenocumarol presented with a retroperitoneal hematoma. CONCLUSIONS: In general antithrombotic drugs are well-tolerated by patients with TIA and LI. Hemorrhagic complications, particularly of the digestive tract, associated with aspirin limit tolerance to it. PMID- 10742989 TI - [The electroencephalographic evolution of a group of patients on a ketonic diet]. AB - INTRODUCTION: A ketogenic diet is an old method for the treatment of resistant epilepsy in children, which has been revived in recent years because of its proven efficacy. OBJECTIVE: To correlate electroencephalographic findings and clinical changes observed in a group of patients on the classical ketogenic diet, to determine the validity of the electroencephalogram in following-up treatment. PATIENTS AND METHODS: We made a prospective evaluation of 11 patients aged between 1 and 19 years for an average period of 22 months. The patients had different types of resistant epilepsy according to Schmidt's score as modified by Aicardi, with a sleep encephalogram prior to treatment and 3, 6, 12 and 24 months later, after following the protocol of the classical ketogenic diet. RESULTS: The sleep encephalograph recordings showed changes in all patients. In 9% of cases they became normal, there was great improvement in 45.45% and some improvement in 45.45%. No patient had worsening of the recording. There was over 50% reduction in the number of seizures seen in 72.7% of the children and complete control was achieved in 27.3%. CONCLUSIONS: Normalization or improvement of sleep encephalogram recordings was correlated with improvement in seizures. Electroenchephalogram follow-up is a useful method for prediction of response to treatment with a ketogenic diet. PMID- 10742990 TI - [Cerebral malaria in children]. AB - INTRODUCTION: Malaria is one of the main health problems in the Third World. Plasmodium falciparum infects as many as 300 million people, causing up to three million deaths each year, most of which occur in African children. Cerebral malaria is the most common lethal complication of P. falciparum infection in children and is defined by three criteria: disturbances of consciousness, presence of P. falciparum parasitaemia and absence of other causes of acute encephalopathy. Cerebral malaria is a medical emergency and parenteral quinine is the most recommended treatment because of the frequency of chloroquine-resistant strains. Mortality is as high as 50 per cent and residual disability is present in about 20 per cent of survivors. OBJECTIVE: We want to warm Spaniard neuropaediatricians about the existence of cases of cerebral malaria in our country in order to get a better diagnose and treatment for those children. PATIENTS AND METHODS: A retrospective medical scores review of 20 hospitalised children diagnosed of malaria from 1990 to 1998. We selected three cases with neurological signs and we analysed clinical onset, EEG, neuroimaging, and permanent sequels. RESULTS: All patients had acute encephalopathy with fever, obtundation and seizures. They all presented residual disability (mainly hemiparesis). CONCLUSION: We must know better about cerebral malaria because of an increasing incidence of imported malaria due to emigration from African countries and Spaniard tourism to areas of endemic paludism. PMID- 10742991 TI - [Risk factors in cerebrovascular disease in childhood]. AB - INTRODUCTION: The etiopathogenesis of cerebrovascular diseases in paediatrics is little known and very varied. Review of the literature gives little practical information about how to investigate a paediatric patient who presents with an acute cerebrovascular illness. OBJECTIVES: To identify the risk factors for cerebrovascular accidents in the paediatric age group in our setting and establish guidelines as to how best to act. PATIENTS AND METHODS: A retrospective study was made of the patients admitted to the Hospital San Joan de Deu in Barcelona between January 1984 and December 1995 with the diagnosis of ischemic or hemorrhagic cerebral infarct. RESULTS: A total of 141 cases were identified. Sixty specific causes (43.7%) were detected, of which cardiopathies made up the biggest etiological group. Risk factors for cerebrovascular disease were found in 66% of the cases. The risk factors most often identified were association with non-specific (22) and specific (varicella: 7 cases) viral infections; moderate dehydration (10); mild head injuries (10) and situations of stress (9). There were prethrombotic factors in 32.8% of the patients and anaemia in 29%. The high percentage of the anti Cytomegalovirus (67.1%) was of doubtful significance, and the percentage rose to 82.8% in the patients who died (29/35). In this study the role of hyperhomo-cysteinemia and resistance to C activated protein could not be assessed. CONCLUSIONS: In this study we identified the risk factors for cerebrovascular disease in the paediatric age group of our population. Stroke in the paediatric age group is probably due to a combination of several risk factors which will need structured protocols for identification. It is important to recognize these causes so as to plan strategies for action during the acute phase and to prevent recurrence, as are currently available to the adult population. PMID- 10742992 TI - [Subarachnoid hemorrhage, cerebral ischemia and endothelin-1]. AB - INTRODUCTION: Cerebral vasospasm is involved in the development of delayed ischemic lesions in patients with subarachnoid hemorrhage. We developed an integral theoretical model to explain the pathophysiology of cerebral vasospasm, in which endothelin-1 has a pivotal role in the development of both cerebral vasospasm and delayed ischemic neurological deficits (DIND). OBJECTIVE: The objective of this study is to analyze the relationship between temporal profile of plasma endothelin-1 levels and the development of cerebral vasospasm and DIND. PATIENTS AND METHODS: We analyzed sequentially plasma endothelin-1 levels in 17 patients with aneurysmatic subarachnoid hemorrhage. All the patients had complete clinical and neuroradiological studies. Patients were classified according to Fisher's score. RESULTS: Patients (4 males and 13 females, aged 48.1 +/- 20.3 years) had a good clinical condition (Hunt-Hess < 4, GCS > 10). Two weeks after bleeding, patients had higher plasma endothelin-1 levels than healthy volunteers (p = 0.024). Patients who developed DIND had higher plasma endothelin-1 levels (p = 0.034) and a different evolution (p = 0.0146) than patients without DIND. There is a significant correlation (p = 0.02) between basal plasma endothelin-1 levels and GOS score. Multiple regression analysis shows a significant dependence between plasma endothelin-1 levels and Fisher's score (p = 0.0195), development of DIND (p = 0.0095), and GOS score (p = 0.0319). Logistic regression analysis finds a predictive relation between Fisher's score and plasma endothelin-1 levels for the development of DIND (overall predicted = 74.24%; p = 0.0148). CONCLUSIONS: Plasma endothelin-1 levels are increased in patients after subarachnoid hemorrhage and are associated with the development of cerebral vasospasm and DIND. PMID- 10742993 TI - [Revista de Neurologia: the challenge of spreading the neurosciences in Spanish]. PMID- 10742994 TI - [Sturge-Weber syndrome: differential diagnosis of neurocysticercosis]. AB - INTRODUCTION: The Sturge-Weber syndrome is characterized by facial cutaneous angioma associated with leptomeningeal and cerebral angioma, typically ipsilateral to the facial lesion, which is accompanied by convulsions, mental retardation, contralateral hemiparesia, hemiatrophy, homonymous hemianopsia and glaucoma. Most of the patients with radiographic evidence of intracranial angioma develop convulsive crises, but only half have severe mental retardation. The image of calcification on cranial tomography often leads to confusion in diagnosis, especially with neurocysticercosis, particularly in places where this is endemic and the patients present with minimal skin lesions or these are at atypical sites. CLINICAL CASE: We present the case of a 13 year-old boy hospitalized with status epilepticus who, since the age of 1 year and 3 months, had had convulsive seizures which were of generalized tonic-clonic type and partially complex with secondary generalization, treated with carbamazepine at a dose of 400 mg per day. Neurocysticercosis was diagnosed on a tomogram showing calcification of the left parieto-occipital gyrus. Following physical examination and complementary tests the diagnosis of Sturge-Weber syndrome was made. CONCLUSION: We emphasize the importance of the diagnosis of Sturge-Weber syndrome, its clinical picture and treatment. PMID- 10742995 TI - [Pseudotumor cerebri: the malignant end of the clinical spectrum]. AB - INTRODUCTION: The pseudotumor cerebri syndrome (PTC) is a clinical entity of unknown etiology affecting mainly young, obese women. The major morbidity factor in this condition is the possibility of vision loss, initially considered to be low, but now known to affect a significant proportion of patients. CLINICAL CASE: We describe a case of PTC in a young, obese black women who presented with headache, transient visual blurring and diplopia. Besides obesity, there where no other relevant clinical findings, namely other pathologies and history of medication use. An extensive analytical workup was negative and the CSF showed no abnormalities, aside from increased pressure. Brain MRI was also normal. The clinical course was progressively worse, consisting of rapid vision loss associated with an intracranial pressure of over 850 mmH2O, despite treatment with diuretics (acetazolamide, furosemide and spironolactone) in therapeutic dosage. Eventually, the patient needed the rapid implantation of a lumboperitoneal shunt to prevent blindness, and went on to make a nearly full recovery. CONCLUSIONS: In all major series of PTC, loss of vision has been reported to occur in a significant percentage of patients, and cases have been described in which a catastrophic course with rapid progression to near blindness has led to the introduction of the designation of 'malignant' pseudotumor cerebri. We believe that such cases, although rare, need to be recognized as the malignant end of the spectrum of PTC, and that their management constitutes a neurological emergency. PMID- 10742996 TI - [Miller-Dieker syndrome: a new case with cerebral echographic study]. AB - INTRODUCTION: Miller-Dieker syndrome is characterized for type 1 lissencephaly associated with facial dysmorphism. In 90-95% of the cases, deletion of the distal fragment of chromosome 17 is seen. Nevertheless, this is difficult to confirm in about 50% of the cases, if we don't resort to special technics of molecular genetics. CLINICAL CASE: We show a 3 years old patient diagnosed of lissencephaly and with peculiar facial features in whom the cytogenetic study was normal, but by in situ hybridization deletion of 17p13.3 fragment was showed. CONCLUSION: We want to emphasize the ultrasonography findings and we want to suggest this easy imaging method and useful technic in the study of neuronal migrational disorders. PMID- 10742997 TI - [Respiratory form of onset of motor neuron disease]. AB - OBJECTIVE: To present a case of respiratory failure as the form of onset of amyotrophic lateral sclerosis, to review the main clinical findings, data of the investigations done which suggest the presence of this disorder and describe its therapeutic management. CLINICAL CASE: A 68 year old man presented with a subacute illness characterized by a sleep disorder with sleep fragmentation, snoring of increasing intensity, without clear pauses of apnea, progressive diurnal hypersomnia accompanied by progressive dyspnea followed by respiratory failure with respiratory acidosis and difficulty in manipulating things with his hands. Diagnostic investigations showed a restrictive pattern without pulmonary fibrosis, due to paralysis of the diaphragm, and the presence of electromyographic signs compatible with motorneuron disease. The patient was treated with riluzole 100 mg/day and non-invasive mechanical ventilation and maintained an acceptable quality of life. CONCLUSIONS: Motorneuron disease may start with acute or progressive respiratory failure without a clear etiological cause and may appear to be similar to obstructive sleep apnea syndrome. The treatment of choice for this respiratory problem is non-invasive mechanical ventilation. Absence of symptoms of bulbar involvement is essential for a favourable prognosis. PMID- 10742998 TI - [Alpha interferon and Alzheimer's disease]. AB - INTRODUCTION: Alzheimer's disease (AD) is the commonest cause of dementia; its aetiology is unknown and there is non-specific treatment to detain the course of the disease. The interferons (IFN) are proteins which have antiviral, antiproliferative and immuno-modulating effects, and in the central nervous system these effects are mediated through the opiate receptors and the dopaminergic system. There is evidence that AD may be related to certain prion diseases and certain viruses, and that the IFN system has become deteriorated in this condition. OBJECTIVE: We present a review of patients with AD treated with alpha interferon. DEVELOPMENT: The first known case in the literature was that of a 69 year old man with definite AD (NINCDS-ADRDA) who was given alpha leucocytic IFN intrathecally and who initially was suspected of having Creutzfeldt-Jakob disease; after his treatment with IFN-alpha, his neurological signs were observed to have stabilized. Subsequently, the results of a controlled, randomized, clinical trial were analyzed for 16 patients with probable EA (NINCDS-ADRDA) treated with recombinant IFN-alpha 2b intramuscularly, in whom no changes in clinical and neurophysiological assessment were observed after a year of treatment. However, there was a significant improvement in one of the variables used to measure quality of life, together with a certain reduction in mortality in the patients treated with IFN-alpha. CONCLUSION: These results should be investigated in future studies in the light of current findings concerning the fact that, in the neurophysiological changes in AD, the pro-inflammatory cytokines, of which some of their numerous actions are blocked by IFN-alpha, may produce a deleterious effect on the course of AD. PMID- 10742999 TI - [Mechanical ventilation in patients with amyotrophic lateral sclerosis]. AB - INTRODUCTION AND DEVELOPMENT: In the final stage of amyotrophic lateral sclerosis, the majority of patients develop chronic respiratory failure. If these patients are not informed about the acute respiratory insufficiency, they run the risk of having to be intubated and stay in Intensive Care Unit over a long term; many of them die of respiratory failure without being informed about the available options. Prognosis and treatment options should be discussed with the patient and family. Their informed consent to prolonged mechanical ventilation of the lungs and stopped it under certain circumstances would be obtained before the condition progress to critical. The non invasive artificial home ventilation has led to advances in the management of patients with amyotrophic lateral sclerosis, as a longer period of survival with a higher level of quality of life. We also have analyzed ethical and social aspects and the results of long term mechanical ventilation in the literature. PMID- 10743000 TI - [Electrodiagnosis in disorders of neuromuscular transmission]. AB - INTRODUCTION: Electrophysiological studies are of recognized use in the confirmation of alterations of neuromuscular transmission in further determining their physiopathological characteristics, helping to differentiate them from other conditions with secondary effects on their function. DEVELOPMENT AND CONCLUSIONS: In our study we review the physiopathology of these disorders which compromise the safety factor of the neuromuscular junction, by presynaptic or postsynaptic alterations, and forms the basis of the results of electrophysiological studies. We describe the techniques currently most used: repetitive stimulation and single fibre electromyography complemented by conventional electromyography. Their application and findings in the commonest syndromes are discussed. Finally, their sensitivity, specificity and difficulties are considered. PMID- 10743001 TI - [The genetics of type 1 Charcot-Marie-Tooth disease, the hereditary focal neuropathies and the hereditary distal motor neuropathies]. AB - INTRODUCTION: Charcot-Marie-Tooth disease (CMTD) or hereditary motor and sensory neuropathy shows great genetic heterogeneity. The type 1 (CMT-1) or demyelinating form and the type 2 (CMT-2) or neuronal form are two clinically and genetically distinct forms. DEVELOPMENT: Apart from the above mentioned motor and sensory forms, there is another type of neuropathy in which the clinical and neurophysiological involvement is exclusively motor and is known as distal spinal atrophy or hereditary distal motor neuropathy (HDMN), which is a syndrome to be included among the CMTD. The CMT-1 is the most prevalent form and the best understood from the genetic point of view. At least four genes have been reported to be involved: the gene for 22 kDa peripheral myelin protein (PMP-22), situated on the 17p11.2 chromosome (locus CMT-1A); the gene for myelin 0 protein situated on chromosome 1q23 (locus CMT-1B); the gene for conexine 32 (Cx32), found on chromosome Xq13 (locus CMT-X) and the gene EGR2 found on chromosome 10q21.1 q22.1. The genes PMP-22 and PO are related to autosomal dominant forms, the gene Cx32 is transmitted linked to chromosome X and transmission of gene EGR2 has been shown in both autosomal dominant and autosomal recessive forms. In the demyelinating forms with autosomal recessive inheritance, positive linkage has been found on four different loci: 8q21.1 (CMT-4A), 11q23 (CMT-4B), 5q23-33 (CMT 4C) and 8q24 in a form related to deafness (CMT-Lom). Two loci have been identified for the autosomal dominant forms of the HDMN. A large family initially and predominantly of the upper limbs is mapped on chromosome 7p (HDMN-V) and another with the classical phenotype mapped on chromosome 12q24. In the familial neuropathy with liability to pressure palsies in most families, deletion of the locus CMT-1A was found, and in rare cases mutation precisely of the PMP-22 gene. In familial amyotrophic neuralgia, genetic molecular analysis has shown linkage with markers on the chromosome 17q24-25. PMID- 10743002 TI - [Type 2 Charcot-Marie-Tooth disease?]. AB - INTRODUCTION AND DEVELOPMENT: Type 2 Charcot-Marie-Tooth disease (CMTD-2) is of similar prevalence to CMTD-1. The age of onset is very variable (ranging between the second and seventh decades). It is impossible to clinically differentiate CMTD-1 from type 2, but for equivalent degrees of muscle weakness, amyotrophia is more marked in the latter. The speed of motor and sensory nerve conduction are normal or minimally slower (> or = 38 m/s) and accompanied by a fall in the nerve potentials of the lower limbs, but not always in the upper limbs. These electrophysiological anomalies do not occur early and therefore do not permit rapid, presymptomatic diagnosis of carriers at risk. The histopathological changes are compatible with primary atrophy of the motor neurons of the anterior horns and of the sensory neurons of the ganglia of the posterior roots, with secondary degeneration of the distal axons of the peripheral nerves. Autosomal dominant transmission is the main mode of inheritance, but only a minority of families have genetic loci situated on chromosomes 1, 3 and 7. CONCLUSION: The first step in rapid molecular diagnosis and genetic treatment is to identify the genes situated in the currently known loci and discover new loci. PMID- 10743003 TI - [Historical review of the most outstanding neurological institutions]. AB - OBJECTIVE: To present a historical review of the main Institutions of Neurological Sciences which have existed from the mid XIX century to the present day. DEVELOPMENT: We remember the founders and collaborators of these Institutions who with dedication contributed to the maintenance of their prestige and the advance of the neurosciences. These names are no doubt well known to those of us who have worked in any of the branches of the neurosciences. Unfortunately, some of the Institutions described no longer exist. Others, although somewhat overlooked continue their scientific work while some still maintain the prestige which was always theirs. The description follows the chronological order of their foundation. We have given a more extensive description of those which we consider of greater importance due to their contribution to advances in the neurosciences, and which we have had the opportunity to visit and study their function and organization at first hand. We state the site of an institution of a given neurological speciality, independently of whether or not it forms part of a general hospital complex, but maintaining its own autonomy under the direction of a neuroscientist, contributing to establish closer collaboration with neurologists, neurosurgeons, neuropathologists etc., which undoubtedly helps to raise the level of teaching and investigation in the neurosciences. PMID- 10743004 TI - [A perspective of neurology in Iberoamerica as seen from the United States]. AB - INTRODUCTION: Neurology in Iberoamerica is out-of-date as compared to that of North America and Europe. There is little basic scientific or clinical research and the academic institutions are weak. Specialists in neurology take little part in academic activities as a result of lack of institutional support and little corporate spirit. DEVELOPMENT AND CONCLUSIONS: In this article we recommend the establishment of Institutes of Neurology to foment work as a team and the creation of a Panamerican Academy of Neurology to bridge gaps between Panamerican congresses. The Academy would also stimulate the development of neurology in Iberoamerica in the aspects of investigation, teaching and clinical services. PMID- 10743005 TI - [Lumbosacral plexopathy 15 years after radiotherapy for carcinoma of the cervix]. PMID- 10743006 TI - [TIA in a patient with Lyme disease: cause or coincidental association?]. PMID- 10743007 TI - [Rapidly progressive myelopathy due to human lymphotropic virus (HTLV-1)]. PMID- 10743008 TI - [Hemangioblastomas in the von Hippel-Lindau disease]. PMID- 10743009 TI - [Apoptosis and Alzheimer disease. Contribution of cellular and transgenic models]. PMID- 10743010 TI - [Neuronal and synaptic properties: fundamentals of network plasticity]. AB - Neurons, within the nervous system, are organized in different neural networks through synaptic connections. Two fundamental components are dynamically interacting in these functional units. The first one are the neurons themselves, and far from being simple action potential generators, they are capable of complex electrical integrative properties due to various types, number, distribution and modulation of voltage-gated ionic channels. The second elements are the synapses where a similar complexity and plasticity is found. Identifying both cellular and synaptic intrinsic properties is necessary to understand the links between neural networks behavior and physiological function, and is a useful step towards a better control of neurological diseases. PMID- 10743011 TI - [Right parietal lesions, spatial neglect and egocentric reference]. AB - Using a proprioceptive "straight-ahead" pointing task, we determined the position of the subjective sagittal middle in thirty unselected patients with unilateral vascular lesions in the right hemisphere and twenty-two normal controls. Patients with extensive right parietal damage (n = 16) showed an ipsilesional (rightward) deviation of their egocentric reference, whereas patients with lesions that substantially spared the right parietal lobe (n = 14) showed a contralesional (leftward) deviation. No significant correlation emerged between the position of the egocentric reference and the performance on a neglect battery. These results can help explain some dissociations between left neglect signs and ipsilesional deviation of the egocentric reference, and raise some questions about the links among lesion location, neglect signs and egocentric frame of reference. PMID- 10743012 TI - [Study of hemispheric lateralization of language regions by functional MRI. Validation with the Wada test]. AB - Language hemispheric dominance can be assessed by the Wada test before temporal lobectomy, in patients with refractory epilepsy of the mesial temporal lobe. Functional MR is a less invasive technique which recently demonstrated the ability to localize some language areas. The aim of this study was to determine if functional MR can be substituted to the Wada test in the determination of language dominance. Seven patients with a refractory epilepsy of the mesial temporal lobe were included. The MR protocol was carried out using a 1.5 T system including 12 axial gradient echo BOLD EPI images. A semantic fluency task, interspersed with rest periods, was used as activation paradigm. An activation of middle and inferior frontal region was found in all patients. It predominated in the left hemisphere in 6 cases and in the right hemisphere in 1 case. The hemispheric and frontal lobe lateralization of the functional MR activation was correlated with the hemispheric language dominance as assessed by the Wada test. In conclusion, functional MR is a promising technique in the determination of language dominance. PMID- 10743013 TI - [Ring chromosome 20: an identifiable epileptic syndrome]. AB - Ring chromosome 20 (RC20) is a rare chromosomopathy characterized by slight mental deficiency, behavioral disorders and epilepsy. We report two cases of ring chromosome 20, who exhibited non convulsive status epilepticus lasting minutes to hours, often triggered by emotional events, with associated typical electroencephalographic features. Our report highlights the features of the electroclinical epilepsy syndrome associated with RC20, which represents a distinct and recognizable entity. The genetic basis of this disorder is discussed. PMID- 10743014 TI - [Cerebral venous thrombosis after lumbar puncture and intravenous steroids in two patients with multiple sclerosis]. AB - Two patients affected with a multiple sclerosis developed cerebral venous thrombosis after lumbar puncture and treatment with intravenous methylprednisolone. In one case, the course was favorable. The second patient died in spite of intracerebral thrombolysis. The autopsy confirmed the diagnosis of cerebral venous thrombosis and multiple sclerosis. We discuss the relationship between lumbar puncture, steroid treatment and cerebral venous thrombosis. PMID- 10743015 TI - [Miyoshi distal myopathy: specific signs and incidence]. AB - We report 21 French patients (12 males and 9 females), presenting a distal myopathy of Miyoshi type. The main clinical features of these patients were 1) onset in late adolescence or early adulthood (mean age: 20.3 years), 2) early and predominant involvement of the posterior compartment muscles of legs, 3) marked elevation of serum CK (from 10 to 50 times the normal value), 4) dystrophic features with a necrotic regeneration pattern without vacuole in muscle biopsy. All cases were sporadic and a consanguinity of parents was found in five cases. The clinical course was relatively mild: twelve patients could walk without aid; However four patients were severely disabled. Four patients were initially considered as having polymyositis; corticosteroids and immunosuppressive drugs were always inefficient. A genetic linkage to chromosome 2 was ascertained in five cases. In our experience the Miyoshi distal myopathy is the most common form of distal myopathy, particularly in young patients. PMID- 10743016 TI - [Probable Miller Fisher syndrome during Dengue fever type 2]. AB - A 57 year-old-man developed a left ophthalmoplegia associated with ataxia and areflexia while he had fever (39 degrees C) for two days. Dengue fever (DF) was diagnosed by definite criteria, i.e.: IgM seroconversion, positive culture from serum and positive PCR both from serum and CSF. Within one week, he fully and spontaneously recovered. To our knowledge, this neurological picture has never been reported in DF. Considering the immune-mediated nature of this condition, its pathogenesis in DF is proposed in reviewing the literature. PMID- 10743017 TI - [Aspergillus spondylodiscitis]. AB - Four weeks after an Amanita phalloides poisoning, a 29-year-old man presented an ocular aspergillosis. One month later he developed a bilateral L5-S1 radicular syndrome. The diagnosis of aspergillar spondylodiscities was established on the basis of x-ray, CT-scan, MRI and pathology findings. Clinical and radiological improvement was obtained with fluconazole. PMID- 10743018 TI - [Pseudometabolic distrophinopathy without immunohistochemical anomaly]. AB - A 9 year old boy suffering from migraines, vomiting and exercise intolerance was hospitalized. Clinical examination revealed calf hypertrophy only. There was no muscular deficit and cardiac examination was normal. Creatine kinase and transaminase were elevated. Muscle biopsy revealed fibers of various sizes, centrally located nuclei, occasional necrotic and regenerative fibers. Interstitial tissue was normal. Immunohistochemistry with various antibodies directed against the membranous dystrophin complex was normal. Western Blot analysis revealed dystrophin of abnormal size, and multiplex PCR confirmed the dystrophinopathy showing an absence of exon 43 and 44. This observation highlights the occurrence of unusual dystrophinopathies revealed by exercise intolerance and pseudo-metabolic syndrome. Normal anti-dystrophin immunostaining does not rule out the diagnosis which may only be made by Western Blot analysis or genetic studies. PMID- 10743019 TI - [Cranial nerve palsies in a multifocal demyelinating neuropathy with persistent nerve block associated with hemorrhagic ulcerative colitis]. PMID- 10743020 TI - [Fulminant multiple sclerosis]. PMID- 10743021 TI - [Behavior disorders due to acute vigabatrin intoxication]. PMID- 10743022 TI - [Classification of headache by the International Headache Society (IHS)]. PMID- 10743023 TI - [The concept of neuroprotection]. PMID- 10743024 TI - [In Process Citation] PMID- 10743025 TI - [Social class, life satisfaction and health assessment. Results of a representative study of the German population]. AB - The supposition that a higher risk of morbidity and mortality is connected with the belonging to a particular social class has been confirmed by numerous national and international researches. But still unsolved is the question of how objective social situations do lead to individual processes of disease. In order to solve this question in a representative assessment of the german population, data of 2948 individuals, referring to aspects of life-satisfaction, personality, subjective complaints and attitudes to current state of health, has been ascertained. In nearly all scales of the chosen instruments significant gradients connected to social class have been found. Persons belonging to a lower social class show a minor life satisfaction, significant differences in personality, higher subjective complaints and a poor state of health. This persons also seem to be convinced that they could do less to care for their health. This self assessment indicates processes, that stand before the state of manifestation of disease. They can be seen as an expression of persons mental reflection of their social situation. Within the context of this analysis of social inequality and disease we argue to include psychological theories and stress theory in sociological theories. They achieve an important contribution to the clarification of the interface between social class, respectively life circumstances, experiences of distress, available resources of coping with the situation and the individual process of development of disease. PMID- 10743026 TI - Diet in residents of East and West Germany in 1991-1992 as ascertained by a retrospective food frequency questionnaire. AB - In this study, we compared dietary habits of residents in East (N = 76) and West Germany (N = 266) using results obtained in 1992-1993 from a retrospective semi quantitative food frequency questionnaire referring to 1991-1992. Nutrient intakes were calculated based on the German Federal Food Code. Univariate and multivariate logistic regression was used to determine whether dietary intakes varied according to residence in East and West Germany. At the food level, East German subjects reported a higher consumption of bread, spreadable fat, and sausage, whereas West German participants reported a higher intake of fruit, vegetables, and pasta and rice. At the macronutrient level, energy intake did not differ significantly between groups, nor did the percent contribution of protein, carbohydrate, fat, and alcohol to total energy intake. East German participants had a lower total water and fibre intake, the latter significant only after adjustment for confounders. At the micro-nutrient level, East German participants had a higher intake of cobalamin, retinol and retinol-equivalents (but not of beta-carotene). There were no differences in the intake of vitamins C, D, and E between groups. Less salt and more potassium, calcium, magnesium and zinc were consumed by West than by East German subjects. Overall, both groups showed disadvantageous dietary patterns. The results are discussed in the context of an overview of other dietary surveys performed in the two parts of Germany before and after reunification. In general our results are consistent with other observations showing that dietary habits in East Germany rapidly approached those in West Germany after reunification, although some residual differences seemed to persist. PMID- 10743027 TI - [Effect of occupationally related stress on diagnosis-specific work incapacity]. AB - Sickness absence data of approx. 50,000 employees were gathered from the health insurance funds of five companies from the metal processing and retail trade. According to the job characteristics the employees were grouped in 83 different job types. Each job type was accessed with respect to the occurrence of risk factors (70 items). Finally, adjusted relative risks for disease-specific sickness absence were calculated. With respect to all diseases studied "low job control" turned out to be the risk factor highest associated with sickness absence. E.g., for back disorders a relative risk of 4.7 was seen for employees whose jobs were highest characterised by "low job control" compared to employees without. In contrast, relative risks concerning "high job demands" were well below one. In general, associations between physical work bad (e.g. heavy work, vibrations) and sickness absence from various diseases were observed also, but those of psychosocial factors were more consistent and dominant. PMID- 10743028 TI - Patterns of health-related behaviour and their cross-cultural validity--a comparative study on two populations of young people. AB - The study aimed at exploring health-related behaviour patterns among young people. The approach was cross-cultural and the study was focused on identifying culturally valid behaviour patterns in two countries, Finland (F) and Switzerland (CH). Data came from two surveys and included 16- and 18-year-old respondents (F: study year 1993, n = 280, CH: study year 1992/3, n = 272). Three intake behaviours--eating, drinking and smoking--were analysed by means of cluster and logistic regression analyses. Three cross-culturally valid behaviour clusters emerged: a healthy, an unhealthy and a mixed pattern where unhealthy eating behaviours were combined with non-smoking and low alcohol use. The determinants of the patterns were tested by comparing the two countries in relation to the socio-demographic characteristics of the young people that belonged in the same cluster. In both countries the structure of the determinants was almost identically indicating high cross-cultural stability. The study suggests that among young people of this age there is clear and cross-culturally consistent patterning of health-related behaviours. PMID- 10743029 TI - Cardiovascular mortality patterns in Turkey: what is the evidence? AB - In Turkey, reliable cause-specific mortality data are not available. It is thus unknown whether ischaemic heart disease (as in western Europe and the US) or stroke (as in the Far East) is the prevailing cause of cardiovascular death. This information, however, is required for planning cardiovascular prevention programmes. We analyse available Turkish national cause-of-death data as well as patterns of cardiovascular mortality in a hospital in Ankara and among Turkish migrants in Germany. According to national statistics, the ischaemic heart disease-to-stroke ratio would be 0.3 among men aged 45-64 years, lower than that in Japan. Hospital and migrant data show this ratio to be 2-4. We demonstrate the implausibility of the national data by assessing the precision of cause-of-death assignment. We then discuss to what degree mortality experience among migrants is representative for their country of origin. Our findings suggest that the pattern of cardiovascular mortality in Turkey is closer to that in western Europe and the US than to that in the Far East. Finally, we discuss options for improving cardiovascular surveillance in Turkey. PMID- 10743030 TI - [Differential learning effects in preventive drug education by peer group members]. AB - In the drug prevention programme studied here, peers were acting as teachers for their colleagues. We focused on the contribution of student characteristics and of teaching quality to the gain in knowledge about drugs. 165 8th- and 9th graders in eight classes answered a questionnaire three times: before, immediately after and five months after the intervention. There was a significant increase in knowledge immediately after the intervention (mean m = 6.2) and after five months (m = 6.1) compared to the score before (m = 4.5). Furthermore, non German speaking students of the "Sekundarschule" (higher level) learned the most, non-German speaking students of the "Realschule" (lower level) learned nothing at all. We found a positive association between teaching quality as assessed by each class and knowledge gain in the same class. Multiple linear regression analysis showed that five months after the intervention student characteristics explained an important part of the variance in knowledge gain (25% of the total variance), while teaching quality explained another 5%. Consequently, these characteristics can serve as predictors of knowledge gain in such programmes. PMID- 10743031 TI - [Measures for allogeneic blood conservation in surgery]. AB - The aim of all efforts to reduce the need of allogeneic blood transfusions is to avoid associated risks. There should particularly be a favourable effect according to the rate of transfusion-transmitted virus infections and immunological side-effects. The acceptance of an individually adjusted lowest haematocrit level and the minimisation of intra-operative blood loss by the application of optimal surgical techniques are among the most essential strategies to reduce or even avoid allogeneic blood transfusions. In addition the following interventions are generally accepted: Preoperative autologous blood donation, where appropriate supported by erythropoietin Preoperative haemodilution, where appropriate supported by erythropoietin Intra- and postoperative blood salvage Topical or systemic pharmacologic interventions to accelerate haemostasis Controlled hypotension Efficacy and indication of the different measures always depend on the individual circumstances of the specific patient. Therefore one should develop an individual approach for every case. In this context the most important subjects are an optimal coordination and if required an appropriate combination of the discussed methods. Algorithms which preoperatively allow approximate calculation of expected transfusion need may be a meaningful tool to facilitate blood conservation planning. However, at the same time one must consider that all strategies to reduce allogeneic transfusion needs are also associated with particular risks. Therefore one has to weigh carefully the pros and cons prior to their application, including the possible alternative of allogeneic transfusion in one's decision making process. PMID- 10743032 TI - [Immunonutrition with omega-3-fatty acids. Are new anti-inflammatory strategies in sight?]. AB - In the early phase of sepsis and SIRS an overwhelming activation of humoral and cellular mediator systems can alter vascular resistance and causes capillary leakage increasing the risk of organ dysfunction. omega-6-arachidonic acid is released from lipid pools of cellular membranes during inflammation and is metabolized to pro-inflammatory prostaglandins and leukotriens, which are key mediators in the pathogenesis of organ dysfunction. omega-3-eicosapentaenoic acid derived lipid mediators present altered biologic effects. Thus, omega-3-fatty acid application enables anti-inflammatory intervention on the level of lipid mediators. The current article reviews experimental and clinical data on omega-3 fatty acids. Besides the decrease of pro-inflammatory mediators, fish oil supplementation lowered post operative infection rates and showed a tendency to reduce hospital stay in surgical patients. It is believed that the decreased formation of LTB4 and TXA2 during sepsis after administration of omega-3-fatty acids accounts for improved microcirculatory perfusion and declined lactate acidosis. PMID- 10743033 TI - [Therapeutic management of postoperatively diagnosed bilateral recurrent laryngeal nerve paralysis]. AB - Bilateral vocal cord paralysis is a rare but potentially dangerous postoperative complication in thyroid gland surgery. There is a controversial discussion about therapeutic management of postoperative bilateral vocal cord paralysis. METHODS: We analysed the frequency of bilateral nerve palsy in 985 operations. The disease of thyroid gland, the operative procedure, the exposure of laryngeal nerve, the mobility of vocal cord detected by an otolaryngologist, clinical symptoms and therapy of patients with bilateral paralysis were analysed. All patients were examined immediately postoperatively and 5 days, 14 days, 6 and 12 months after resection. RESULTS: The overall transient bilateral palsy rate was 0.7%, the permanent 0.3%. The palsy rate depended on the disease of thyroid gland. After resection of simple goitre we found a 0.2% transient injury rate (0.1% permanent), after operation of thyroid cancer 2.0% transient (1.0% permanent) and in cases of recurrent goitre 5.9% transient (1.9% permanent) palsies. The immediate postoperative symptoms are also very different. There are patients suffering from stridor and dyspnoea, patients with dysphonia without dyspnoea and those without any symptoms. These different clinical symptoms are due to the different grade of laryngeal nerve damage and the resulting position of vocal cords. The bilateral paralysis was completely temporary in 4 cases. 12 months later 4 patients suffered from dysphonia. Only in 3 patients with thyroid cancer and a preoperative unilateral vocal cord paralysis tracheostomy was necessary after operation. The vocal cord mobility did not recover in these 3 cases after 12 months and the patients are not decannulated. DISCUSSION: Bilateral paralysis is only relevant in thyroid cancer and recurrent goitre. The symptoms varies and no patient should leave the hospital without examination of the vocal cords by an otolaryngologist. Because vocal cord paralysis is temporary in most cases an emergency tracheostomy is seldom indicated. PMID- 10743034 TI - [Diagnosis and therapy of non-occlusive mesenteric ischemia (NOMI)]. AB - Pathophysiologically, the non-occlusive mesenteric ischemia (NOMI) results from reduced blood supply to the intestine, caused by "low cardiac output syndrome", or the use of certain drugs leading to intestinal vasoconstriction and stasis of the microcirculation. Regardless of the aetiopathogenesis, the patient's prognosis crucially depends on rapid diagnosis and initiation of adequate medical or surgical intervention. In a 10-year retrospective chart analysis (1989 to 1998) we identified a total of 62 patients that demonstrated classical features of NOMI. The investigation focused on patients' history, risk factors, clinical symptoms, diagnostic procedures and patient's clinical outcome. The most important associated risk factors and concomitant diseases were reduced cardiac output (caused by preexisting heart failure), renal diseases, diabetes and the use of some specific drugs (digitalis, furosemide, ergotamine). Except for leucocytosis, elevated serum lactate and an increased CK/CK-MB level, all laboratory findings were unspecific. Using abdominal ultrasound and plain abdominal x-ray, 80% of the cases showed positive signs of ileus, subileus and free intraabdominal fluid. The angiographic diagnostics (mesentericography) of non-occlusive mesenteric ischemia showed the typical signs of peripheral vasoconstriction in 90% of the cases. Fifty three patients (86%) presenting with peritoneal signs underwent operative bowel exploration. Necrotic bowel had to be resected in 37 cases (60%). The overall letality was 58%. The progress made in better understanding the pathophysiology of NOMI has led to differential treatment of the disease. Close cooperation between surgeons and radiologists, coupled with early diagnosis and prompt treatment are necessary to optimize the clinical outcome. PMID- 10743035 TI - [Experiences with reconstruction of large abdominal wall cicatricial hernias using Stoppa-Rives pre-peritoneal mesh-plasty]. AB - BACKGROUND: The observed recurrence-rate up to 20-50% of conventional incisional hernia repair might be improved by alloplastic hernia repair in "sublay technique" described by Stoppa and Rives. METHODS: From 12/94 to 12/97 122 pt. underwent surgery of incisional hernia of whom 50 had a very large abdominal wall defect so that hernia repair by alloplastic technique was necessary. All 50 pt. had mesh-implantation in subfascial plane, 28 pt. by ePTFE-patch (Gore-Tex) and 22 pt. by polypropylen-mesh (Prolene). RESULTS: The implantation-procedure required a mean time of 165 min. (range 55-345 min.). There were pulmonary complications in 2 patients (4%) of whom one had to be ventilated over 34 days, prosthesis-infection occurred in 3 pt. (6%). Follow-up on average 19 months (range 5-44 months) postoperatively revealed 5 hernia-recurrences (10.4%) in 48 patients. CONCLUSION: Mesh-implantation for hernia repair in sublay-technique requires intensive preparation of preperitoneal plane. Thus it took approximately 3 hours for mesh-implantation in this study. From our data Stoppa-Rives-procedure seems to be effective for 4-repair of large incisional hernias and allows immediate abdominal wall stress. PMID- 10743036 TI - [Postoperative wound healing in wound-water contact]. AB - The avoiding of water-contact to the freshly closed operation-wound is an unwritten law in surgery. In general, taking a bath or shower is not allowed before wound stitch removal under the idea that early water-contact may lead to a higher wound-infection rate. In a prospective trial 170 patients, operated in our short-stay surgery-unit, were allowed to take a shower 24 hours after surgery. Full water contact of the fresh uncovered wound was accepted. The wound-infection rate in this group was compared with the infection-rate of an historical group from our department (n = 956). RESULTS: In the water-contact group no case of wound-infection was observed while we were observing a wound-infection-rate of 0.6% in the other group. The difference between the two groups wasn't statistically significant (p = 0.125). CONCLUSION: Water-contact of the fresh operation wound 24 hours after surgery did not increase the post-operative wound infection risk in this study. PMID- 10743037 TI - [Plastic surgery of the anorectal area. Indications, technique and outcome]. AB - Anal canal stenosis with alteration of the sensoric continence or mucosal ectropion may occur after anorectal operations. Island flaps with perianal skin or the VY-anoplasty are simple plastic methods to reconstruct the anorectal region and cure patients--who often have suffered for a long time--from anal strictures or mucosal ectropion. In the period from 1994-1998 we reconstructed the anodermal region of seven patients using one of the above mentioned anorectal plastic procedures. Three patients complained of an anal stenosis and one patient suffered from an ectropion of the rectal mucosa after an improperly performed Whitehead hemorrhoidectomy. Three patients had a sensomotoric incontinence twice due to a congenital anal atresia and in one case caused by an accident. All patients were highly pleased after the operation--no complication occurred. PMID- 10743038 TI - [Differential diagnosis and therapy of acute abdomen in sickle cell crisis. A rare case in visceral surgery]. AB - Surgical therapy of the acute abdomen often allows only limited time for differential diagnosis to confirm the indication for surgery. Under consideration of clinical aspects and case history both common and rare causes of an acute abdomen should be investigated without undue loss of time. Differential diagnostic considerations and eventual therapy are presented in the following case of a 25-year-old Afro-american who developed multiorgan failure after an initial course of lower-back pain. In addition to the clinical setting of an acute abdomen the patient presented with acute respiratory failure and laboratory signs of severe hemolysis in combination with newly detected splenomegaly. The indication for splenectomy was made following CT-proven complete splenic infarction due to repeated acute squestration. Histologic examination of the spleen together with hemoglobin electrophoresis confirmed the clinical assumption of unusually late primary manifestation of a sickle cell crisis. In the underlying case, the hemoglobinopathy was in fact the less common form of combined sickle-cell-beta-thalassemia. A ten-day course of intensive care therapy was necessary to treat ongoing multiorgan failure due to persistent sickle cell crisis. Current diagnostic and therapeutic procedures in connection with sickle cell crisis as a rare cause of an acute abdomen with the necessity for surgical intervention are presented. PMID- 10743039 TI - [Acute upper gastrointestinal hemorrhage as a complication of hepatic artery port catheter]. AB - We report a case of a 56-year-old patient with an acute upper gastrointestinal bleeding following penetration of an aneurysm of the gastroduodenal artery in the duodenal bulb. The patient received an i.a. portsystem five month before the reported acute gastrointestinal bleeding. The portsystem was implanted for treatment of multiple liver metastases of an neuroendocrine tumor. This life threatening situation could not controlled endoscopically. Also an embolisation was impossible so we carried out a laparotomy with ligation of the proper hepatic artery. The postoperative course was uneventful. Due to her tumor disease the patient died 13 month after surgery. PMID- 10743040 TI - [Tube obstruction in operation of esophageal atresia. Brief review of intraoperative complications based on a case report]. AB - In this case report we discuss the anaesthetic management of newborns with esophageal atresia classified as Vogt III b. This type is characterised by an upper esophageal pouch which ends blindly and a distal tracheoesophageal fistula. Commonly associated diseases are cardiac, renal, vertebral and anal anomalies. The most important intraoperative anaesthesiological complications are acidosis, hypoxaemia, gastric distension, endotracheal tube obstruction, tracheal compression, cardiac arrhythmias and atelectasis. In the presented case an endotracheal tube obstruction with hypercapnia occurred which required a change of the airway. After changing the endotracheal tube the newborn could be ventilated sufficiently. Further postoperative course was uneventful. PMID- 10743041 TI - [Intraoperative cholangiography in laparoscopic cholecystectomy for acute cholecystitis]. AB - In our prospective study we wanted to prove whether the safety of laparoscopic treatment of acute cholecystitis could be improved by intraoperative cholangiography. From July 1993 to June 1998 210 patients with acute cholecystitis underwent a laparoscopic cholecystectomy. In 23 cases (10.9%) a conversion was necessary. 189 patients underwent a laparoscopic cholangiography. In 2 cases (1.1%) an incision of the common bile duct was detected which had been mistaken for the cystic duct. So the cutting of the common bile duct could be prevented. In 12 patients (6.3%) unknown common bile duct stones were found. The complication rate was 9.5% without any mortality or major injury of the common bile duct. PMID- 10743042 TI - [Contribution of Berlin clinics and especially the Charite to the development of modern anesthesia in Germany]. AB - Soon after announcement of the first American ether anesthesias in 1846 one started to anesthetize with ether in Europe. The first ether anesthesia in Germany took place on January 24th, 1947, and was given by Johann Ferdinand Heyfelder in Erlangen. Two weeks later the first ether anesthesia in Berlin was given on February 6th, 1847, by the orthopaedic surgeon Heimann-Wolff Berend. On February 12th, 1847, the surgeon Johann Friedrich Dieffenbach used ether to anesthetize a patient in the Berlin university hospital. Still in 1847 he published his book "The Ether against Pain" which used to be the standard textbook of anesthesia in Germany for many years to come. On February 15th, 1847, Johann Christian Jungken operated his first patient under ether anesthesia in the surgical department of the Charite hospital assisted by Rudolf Virchow. Since then several famous Berlin surgeons influenced the development of modern anesthesia until the middle of the 20th century. The nitrous oxide bottle was introduced into practice by the Barth company in cooperation with the dentist Carl Sauer and Kurt Schimmelbusch introduced his mask for ether anesthesia. Carl Ludwig Schleich reported at the German congress of surgery in 1894 about his first experience with infiltration anesthesia. Spinal anesthesia developed by August Bier in Kiel was improved during his time in Berlin. PMID- 10743043 TI - [Surgery of chronic pancreatitis (2)]. PMID- 10743044 TI - [Analyses of variation trend and short-term detection of Chinese malignant tumor mortality during twenty years]. PMID- 10743045 TI - [Estrogen receptor expression and its relationship with biologic feature in hepatocellular carcinoma]. AB - OBJECTIVE: For understanding the clinicopathologic significance of estrogen receptor (ER) in hepatocellular carcinoma (HCC) ER of the surgical resected species was detected and its relation to biologic feature was analysis. METHODS: ER was detected in 62 HCC species with Dextran coated charcoal method. Some pathologic characteristics were studied with gross and microscopic examination and proliferating cell nuclear antigen (PCNA) was determined with immunohistochemistry using monoclonal antibody PC-10. RESULTS: ER positive HCC had higher incidence of single nodule (83.3%) and complete encapsulation (58.3%) compared to ER negative HCC (42.1% and 21.0%). The difference was significant (P < 0.05). In small liver cancer ER positive rate was 62.5% which was higher than 30.4% in large liver cancer (P < 0.05). The PCNA labeled index was (26.2 +/- 18.7)% in ER positive HCC and (43.6 +/- 32.0)% in ER negative HCC. The difference was significant (P < 0.05). CONCLUSION: The results suggest that ER positive HCC has less malignant biologic behavior and better prognosis than ER negative HCC. PMID- 10743046 TI - [Study on the synthesis and secretion rate of transferrin in hepatoma and peritumor hepatocytes]. AB - OBJECTIVE: To study the changes of synthesis and secretion of transferrin (Tf) in human hepatocellular carcinoma (HCC) cells and search the causes of reduced membrane-bond transferrin in HCC tissue. METHODS: By radioisotope incorporation and immunoprecipitation to determine the synthesis and secretion rate of transferrin in human HCC cells and peritumor hepatocytes. RESULTS: When compared with peritumor hepatocytes the quantity of transferrin synthesized was not changed in HCC cells, but its secretion rate was accelerated significantly while the transferrin storage was significantly decreased. CONCLUSION: The results suggest that HCC growth might be enhanced by increased Tf secretion and help explain the decreased membrane-bond form of Tf in HCC tissue. PMID- 10743047 TI - [Sequence alterations in p53 gene of hepatocellular carcinoma from high aflatoxin risk area in Guangxi]. AB - OBJECTIVE: Check on the sequence alterations of the p53 gene of hepatocellular carcinoma (HCC) in some areas of Guangxi. METHODS: 40 cases of archival HCC samples were divided into two groups. One includes 26 cases from Fusui--an area notorious for high risk in HCC, AFB1 and HBV; The other includes 14 cases from Yulin where both HCC and HBV were high but AFB1 may be low because rice low in AFB1 was the staple food. Exon 7 were sequenced by DNA direct sequencing. p53 mutational protein and HBsAg were examined by immunostaing. RESULTS: In the Fusui group, 16/26 (61.5%) cases showed mutation at codon 249 third position G to T transversion to form a hot-spot which is generally considered to be caused by AFB1. In the Yulin group, only one single place-Guigang showed p53 mutation clustered at the hot-spot like Fusui in 3/5 cases (60%), while the other cases demonstrated mutational points in various positions. CONCLUSION: Besides Qidong and South Africa, a third AFB1 high risk area was demonstrated where HCC is accompanied by p53 gene specific mutation. PMID- 10743048 TI - [The expression of mutant p53 (mp53) protein in rat hepatic preneoplastic lesions induced by diethylnitrosamine (DEN)]. AB - OBJECTIVE: To study p53 genes related to hepatocarcinogenesis. METHODS: Using immunohistochemistry techniques, the relation between expression of mp53 protein, placental glutathione S-transferase (GST-P) and the cell proliferative activity was investigated in rat hepatic preneoplastic lesions induced by DEN and the control liver. RESULTS: Two of five rats with hepatic prenoplastic lesions had mp53 protein expression which was seen only in a portion of large-sized hyperplastic nodules (HN) or GST-P positive foci at 5th week. The bromodeoxyuridine labeling indeX (BrdU-LI) in p53-positive HN or GST-P foci was significantly higher than that of p53-negative HN or GST-P-negative foci respectively (P < 0.05). CONCLUSIONS: Mutation of p53 gene occurs in the early stage of hepatocarcinogenesis, and is responsible for the hepatocellular proliferations and hepatocarcinogenesis, and might become an additional tumor marker. PMID- 10743049 TI - [Mutation analysis of transforming growth factor-beta type II receptor gene in human gastric cancer tissues]. AB - OBJECTIVE: Mutations which cause abarrent expression of transforming type II receptor (TGF-beta RII) are responsible for the escape of some tumor cells from growth control midiated by TGF-beta. Two mutation hot spots (cDNA 709-718, 1931 1936) have been reported on human colon cancer cell lines. To elucidate the situation of this gene in human gastric cancer, 44 gastric cancer tissues were examined for mutation at the two hot spots. METHODS: Genomic DNA extracted from the cancer tissues was PCR amplified and analysed by silver stain for single stranded conformation polymorphism. Positive samples were sequenced by dry-primer automatic sequencing method. RESULTS: The data revealed a deletion rate of 6.8% (3/44) of the 1st hot spot; CA insertion of the 2nd hot spot was not found in all 44 gastric cancer tissues. CONCLUSION: The mutation incidence of TGF-beta R II in human gastric cancer is low. PMID- 10743050 TI - [Study on tumor necrosis factor receptor of human gastric cancer cells]. AB - OBJECTIVE: To explore the relationship between the number of tumor necrosis factor receptors (TNFR) and the degree of differentiation of gastric cancer cells, and the relationship between the number of TNFR and the cytotoxicity of tumor necrosis factor mutant (TNF-m). METHODS: With 125I-TNF-m, the radio-ligand binding assay was used to detect the TNFR on three gastric cancer cell lines (MKN28, SGC7901 MKN45) with from high to low degrees of differentiation. MTT colorimetric method was used to study the cytotoxicity of TNF-m on the three gastric cancer cell lines in vitro. RESULTS: The number of TNFR of the three gestric cancer cells was 9.8 x 10(-12) nmol, 5.6 x 10(-12) nmol and 3.2 x 10(-12) nmol per cell respectively it differed significent from each other (P < 0.05) but the dissociation constant was basically the same. The rate of TNF-m internalization of the three gastric cancer cells was nearly the same and temperature dependent. The half-life of TNFR was about 90 mins. The ratio of membrane receptors to cytoplasm receptors was about 0.5. The maximum killing rate of TNF-m was 86%, 60% and 34% for MKN28, SGC7901, MKN45 cells, respectively, which were significantly different (P < 0.05). Killing rate of TNF-m to these cells at 39 degrees C was higher than at 37 degrees C. CONCLUSIONS: The number of TNFR on the surface of gastric cancer cells was associated with the degree of differentiation of gastric cancer cells. The cytotoxicity of TNF-m was related to the number of TNFR and the rate of internalization of TNF-m. PMID- 10743051 TI - [Scanning electron microscopic observation of the three-dimensional structure of laryngeal carcinoma using freeze-cracking method]. AB - OBJECTIVE: To investigate the relation between the morphology and the biological characteristics of laryngeal carcinoma. METHODS: The freeze-cracking method was used to examine 126 specimens of the larynx under scanning electron microscope (SEM). The specimens were obtained from 12 patients suffering from laryngeal cancer and 4 patients with normal larynx. RESULTS: (1) In the normal larynx, the squamous-cell epithelium showed marked keratinization and the intracellular bridges were clear and regular. (2) In areas adjacent to cancer, the structure of the squamous-cell epithlium could be identified, but the cells were deranged and paramorphic. (3) There were two types in the marginal area of the carcinoma. Along the areas adjacent to cancer, infiltrating lymphocytes were usually observed and the surface of the lymphocytes had many leading lamella. CONCLUSION: The results indicate that the three-dimensional observation with SEM using the freeze-cracking technique is helpful for studying the relation between morphology and biologic characteristics of laryngeal carcinoma. PMID- 10743052 TI - [Loss of heterozygosity on chromosome 13 in squamous cell carcinomas of the larynx]. AB - OBJECTIVE: To locate lost region of tumor suppressor gene on chromosome 13q in squamous-cell carcinoma of the larynx (LSCC) and to provide clues and evidence for discovering and locating new suppressor gene. METHODS: Loss of heterozygosity (LOH) on chromosomes 13 was analyzed in 58 LSCC patients. By microsatellite polymorphic sequences in loci D13S765 (13q13), RB 1.20 (13q14.2), D13s133 (13q14.3) and D13S318 (13q21) on chromosome 13 by PCR. RESULTS: There weren't any LOH on chromosome 13q in 3 cases with preinvasive LSCC. Forty-five percentage (24/53) of the 53 invasive LSCC cases showed LOH at one or more loci on chromosome 13q region. The highest perentage of LOH on chromosome 13q was 52% (22/53) at D13S765 locus. CONCLUSION: The deletion region on chromosome 13q was located near by D13S765 locus which is centrimeric to RB1. In this region there is suppressor gene, which related to the genesis and developement of LSCC, possibly including RB1. The inactivation of these suppressor gene may be related to the genesis and developement of invasive LSCC. PMID- 10743053 TI - [Criteria of measuring mdr-1 gene expression level in breast cancer by RT-PCR]. AB - OBJECTIVE: To formulate criteria of judjing multidrug resistance gene(mdr-1 gene) expression level and to provide basis for predicting chemotherapy response and prognosis. METHODS: Using reverse transcription-polymerase chain reaction (RT PCR) assay, the expression of mdr-1 gene in 82 breast cancer samples was detected. The data were treated by statistic analysis system (SAS)-singlevariate analysis. RESULTS: The level of mdr-1 gene expression clearly deviated from normal to right distribution (P < 0.0001), and thus might be divided by quantiles P50(mdr-1/beta 2-MG = 0.2) and P75 (mdr-1/beta 2-MG = 0.6), which were taken as the criteria for comparing 56 patients' chemosensitivity to ADM, VDS, VCR in vitro and 32 relapsed metastatic patients' chemotherapy response in vivo, seperately. When mdr-1/beta 2-MG < 0.2, the ratio of coincidence was lower between expression of mdr-1 and drug resistance in vitro and in vivo; When mdr 1/beta 2-MG > or = 0.2-< 0.6, the ratio of coincidence elevated slightly, but in 30%-50% of the cases drug resistance in vitro and in vivo did not correlate. When mdr-1/beta 2-MG > or = 0.6, the ratio of coincidence elevated significantly. According to the above-mentioned results, criteria of evaluating mdr-1 gene expression level was formulated: the mdr-1/beta 2-MG ratio < 0.2(P50) was considered as negative expression, the ratio > or = 0.2-< 0.6(P75) was weakly positive expression, > or = 0.6 was strongly positive expression. CONCLUSIONS: The criteria of mdr-1 gene expression may reflect objectively drug resistance in vitro and chemotherapy response in vivo. The method may also be applicable to other tumours. PMID- 10743054 TI - [Recombinant human GM-CSF retroviral vector construction and expression in human breast cancer cells]. AB - OBJECTIVE: To provide an effective GM-CSF gene transferring vector mediated by retrovirus and a basis for study of GM-CSF gene-modified tumor cell vaccines. METHODS: The recombinant retroviral vector pDORGM was constructed by cloning the GM-CSF cDNA into the replication defective retroviral vector pDORneo, and transferred into packaging cell line PA317 by Lipofectin method. The viral titer was determined with the NIH3T3. Using the NIH3T3 amplification assay, replication competent retroviral particles were detected. A high titer of retrovirus was used to infect the human breast cancer cell line MCF-7. RESULTS: The retrovirus containing GM-CSF gene had a highest titer of virus of 1 x 10(6) colony-forming units/ml. No replication competent retrovirus were detectable in the vector preparation from the packaging cells. GM-CSF gene-modified MCF-7 cell lines stably released 500 to 800 units/10(6) cell of GM-CSF within 24h in vitro. CONCLUSION: The GM-CSF gene transferring vector mediated by retrovirus was effective and safe. These results provied a basis for study of GM-CSF gene used in cancer gene therapy. PMID- 10743055 TI - [Interleukin-6 functions as an autocrine growth stimulator for a human lung adenocarcinoma cell line PAa]. AB - OBJECTIVE: To study the effect of interleukin-6(IL-6) on the growth of a human lung adenocarcinoma cell line PAa in vitro. METHODS: IL-6 receptor (IL-6R) mRNA level was detected by means of reverse transcription polymerase chain reaction (RT-PCR) and IL-6 mRNA was analyzed using Northern blotting hybridization. The production of IL-6 was detected by bioassay. The biological effect of IL-6 released by PAa cells was assessed with anti-IL-6 neutralizing antibodies. RESULTS: PAa cells expressed IL-6R mRNA. Recombinant human IL-6 stimulated the proliferation of PAa cells. PAa cells also expressed IL-6 mRNA and produced bioactive IL-6. Treatment of PAa cells with anti-IL-6 antibodies resulted in decreased growth of PAa cells. CONCLUSION: These results demonstrate that IL-6 acts as an autocrine growth stimulator for PAa cells in vitro. PMID- 10743056 TI - [Epstein-Barr virus infection in midline malignant reticulosis]. AB - OBJECTIVE: To observe the presence of Epstein-Barr virus (EBV) infection in a group of patients with midline malignant reticulosis (MMR). METHOD: DNA-RNA in situ hybridization was used to detect EBER and immunohistochemical staining was used to examine the expression of latent membrane protein (LMP) and immunophenotype of atypical lymphoid cells (ALCs) in 37 cases of MMR; polymerase chain reaction (PCR) was used to detect EBV-DNA in 15 cases. RESULTS: (1) ALCs were positive for EBER 1/2 in 31/37(83.8%) cases; EBV-DNA was detected in 12/15(77.8%) cases; LMP was expressed in some ALCs in 5/19(26.3%) cases. (2) ALCs in 20/37(54.1%) gave both positive reactions to CD3 and CD56 antigens. CONCLUSION: A higher percentage of EBV infection was detected in this group of MMR, its ALCs expressed not only T-cell markers, but also NK-associated antigen, CD56. The presence of EBV infection in different kinds of lymphoproliferative disorders is reviewed and discussed. PMID- 10743057 TI - [Effects of icariin on the differentiation of HL-60 cells]. AB - OBJECTIVE: In order to search for new tumor differentiation inducer, the effects and mechanism of Icariin were studied. METHODS: Icariin is an monomer purified from Epimedium Koreanum Nakai. The differentiation of induction effects of Icariin were observed on the human promyelocytic leukemic cell (HL-60) by NBT reduction test, 125I-cAMP and 125I-cGMP double antibody radioimmune methods, scanning electronic microscopy technique. RESULTS: After treating HL-60 cells with Icariin at concentration 0.1 g/L, reduction of NBT and cAMP/cGMP ratio were increased. There were many rugosities and ball-like processes on the cell surface. CONCLUSION: Icariin had the effects of induction of differentiation on HL-60 cells, the mechanism might be related to elevating the cAMP/cGMP ratio. PMID- 10743058 TI - [CT analysis of hepatic metastases]. AB - OBJECTIVE: To describe the manifestations of the hepatic metastases. To investigate the value of CT examination in diagnosis of hepatic metastases. METHOD: CT radiographs of 90 patients with hepatic metastases were studied retrospectively. The tumor number, size, density, shape, spreading in the hepatic parenchyma were analyzed. The size of liver and spleen was measured. RESULTS: Three or more metastatic nodules spreading in the hepatic parenchyma were found in 65 (72%) cases. Two nodules were shown in 6 (7%) cases. 19(21%) cases had only single nodule. 29 of the metastatic deposits located in the hepatic parenchyma near the liver edge. The size of metastatic nodules varied from 0.5 cm to 14 cm. In 87 cases, the density of metastatic nodules was lower than the surround liver parenchyma. In 3 cases of diffuse fatty liver, the metastatic deposits appeared as higher CT density nodules. Central necrosis of the tumor foci was seen in 44 cases. Striking hepatomegaly was noted in 31 cases with widespread nodules. In 26 cases which had one and two nodules, the shape of the liver was normal. In 45 cases, the primary tumour was found by CT at the same time. CONCLUSION: The CT findings of hepatic metastases differ. Multiple nodules disseminating in liver parenchyma is the general sign. Tiny nodules without other clinical signs can be detected by CT. PMID- 10743059 TI - [The study of chronobiology of CT features of liver cancer]. AB - OBJECTIVE: To explore the biological cycle of malignant biological behavior of liver cancer, and to provide a research basis for the excepically specific phase of drugs for clinical treatment of liver cancer. METHODS: 24 kinds of CT features reflected varying degree of malignant biological behavior. There were 360 cases altogether from randam sampling 30 cases each month. The analytic method is each CT feature fitting a group data of yearly cycle using cosine curve made the analysis of the cyclic sequence character of each CT feature (cosinor). RESULTS: With 95% confidence, there were 10 CT features which showed biologic rhythm (P < 0.05). The acrophase of CT features for invasive growth concentrated between -60 degrees and -120 degrees (March and April). But for non-invasive growth, the CT features were relatively low and concentrated between -180 degrees and -270 degrees (July and September). No acrophase was shown in between -120 degrees and 180 degrees (May and June) and between -270 degrees and -330 degrees (October and November). CONCLUSION: Between CT features of the invasive and non-invasive growths of liver cancer, a sequential difference in biologic cycles can be observed. PMID- 10743060 TI - [The study of relations of cellular DNA content and proliferative activity to lymph node metastasis in gastric carcinoma]. AB - OBJECTIVE: To evaluate DNA/Cytokeratin(CK) multiparametric method in detection to the gastric cancer and to study the relations of cellular DNA content, S-phase fraction (SPF) and G2/M-phase fraction (G2/MPF) to lymph node (LN) metastasis. METHODS: 50 cases of fresh tumor samples, were examined by using single (DNA) and two-colored DNA/CK flow cytometry respectively. RESULTS: The rate of aneuploid, the mean and the range of SPF and G2/MPF were higher in the multiparametric method than that in single one. In aneuploid carcinoma, the incidence of LN metastasis was significantly higher, the G2/MPF values with LN metastasis were higher than those without metastasis; values were higher in metastasis to n2 or n3 than that to n1. The SPF or G2/MPF values correlated with the number of LN metastasis, being significantly higher in metastasis to 5 LN than to 1-4 LN. CONCLUSION: Multiparametric DNA/CK cytometric analysis is better than single DNA one; correlations between cellular DNA content, SPF, G2/MPF and the extent of LN matastasis exist. PMID- 10743061 TI - [Evaluation of a novel adjuvant chemotherapy for non-resectable advanced gastric cancer]. AB - OBJECTIVE: To evaluate the effects of a novel adjuvant chemotherapy for the patients with advanced gastric cancer. METHODS: Preoperative chemotherapy with 5 Fluorouracil and cisplatin (5-FU/CDDP) was performed on 11 cases of nonresectable for advanced gastric cancer. The therapy comprised of either intravenous or arterial infusion or intraperitoneal administration of 5-Fu 750 mg/m2 and CDDP 60 mg/m2 or (CBDCA 400 mg/m2), for 1-2 courses. RESULTS: There was no CR, the PR, NC occurred in 8, and 3 cases respectively. There was no disease progression seen. The overall response rate was 72.7%(8/11) and ascites disappeared in being 85.7%(6/7) of the treated cases. 7 of the 8 PR cases were operated. Histologically the effects of the chemotherapy was considered as slight, moderate and marked in 3, 3 and 1 cases, respectively. Side effects were mainly bone marrow suppression which recovered in two to three weeks. These 7 cases recieved additional 1-2 courses of chemotherapy postoperatively, one patients died of intestinal obstruction. CONCLUSION: The adjuvant chemotherapy reported here as a part of combination therapy plays a significant role in the effective treatment of otherwise nonresectable gastric cancer patients. PMID- 10743062 TI - [Detection of occult tumor cells in resected lymph nodes of patients with stage I carcinoma and its clinico-pathological significance]. AB - OBJECTIVE: Detection of tumor micrometastases in resected lymph nodes of cancer patients to obtain more reliable information concerning nodal status and its clinicopathological importance. METHODS: Paraffin blocks of 3,715 resected lymph nodes from 350 Stage I cancer patients, suffering from NSCLC (n = 94), breast cancer (n = 112), esophageal cancer (n = 115), and vulvar cancer (n = 29), were re-examined by immunohisto-chemical staining, using monoclonal anti-cyokeratins (AE1/AE3), anti-EMA, and polyclonal anti-keratins antibodies. RESULTS: Occult nodal metastases was observed in 113 of 350 cancer patients (32.5%), and in 203 of 3,715 lymph nodes examined. The positive rates both in patients and in lymph nodes were higher in NSCLC than in others. Occult nodal metastasis was seen in 58% of pulmonary squamous-cell carcinomas and 53.8% of adenocarcinomas, while it was seen in 22.5% of esophageal and 10.3% of vulval squamous-cell carcinomas, and in 27.7% of breast adenocarcinomas (P < 0.05). Follow up of a fraction of breast cancer patients showed that the prognosis of patients with positive nodes was worse than that in patients with negative nodes (P < 0.05). CONCLUSION: The data suggested that the immunohistochemical technique can significantly facilitate the detection of micrometastatic tumor cells in lymph nodes. The frequency of occult lymph node metastasis may have significant impact on the prognosis of cancer patients. PMID- 10743063 TI - [Clinical significance of multidrug resistance gene(mdr1) expression in patients with acute leukemia]. AB - OBJECTIVE: To evaluate relationship between drug resistance of leukemia cells and prognosis. METHODS: The expression of multidrug resistance gene(mdr1) mRNA was measured in 85 patients with acute leukemia and in 20 normal controls by using reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The mdr1 positive rate in untreated group was 44.7%. The CR rate differed significantly between mdr1+ (23.9%) and mdr1-(88.5%)(P < 0.005). The mdr1 positive rate of refractory-relapsed group was higher than that in CR group (P < 0.001). The mdr1 mRNA expression level of refractory-relapsed group was higher than that in CR group. A gradually increasing level of mdr1 mRNA expression in CR patients indicated early relapse. The mdr1 positive rate in normal contral and long-term surviver patients was very low. The mdr1 expression was correlated with French American-Birtish Cooperative Group (FAB) classification. CONCLUSION: The mdr1 expression correlated with chemotherapeutic effect and prognosis. It is an unfavorable prognostic factor for patients with acute leukemia. PMID- 10743064 TI - [Teniposide, cyclophosphamide and dexamethasone combination treatment for stage III multiple myeloma: a report of 21 cases]. AB - OBJECTIVE: To evaluate the efficacy and toxicites of combination chemotherapy of teniposide, cyclophosphamide, and dexamethasone (VCD) for stage III multiple myeloma (MM). METHODS: Twenty-one patients with stage III MM were treated with VCD regimen. Among 21 evaluable patients, 16 were in stage IIIA; 5 in stage IIIB. Teniposide was given 30 mg/m2 by intravenous(i.v.) infusion on days 1-3; cyclophosphamide 200 mg/m2 i.v. on day 1, 3, 5; dexamethasone 10-20 mg daily p.o. on days 1-7. The treatment was recycled every 28 days. RESULTS: The overall response rate was 76%(16/21), including partial response 38%(8/21); improvement 38%(8/21) and failure 24%(5/21). The survival time was 3-31 months; medium survival time (MST) 13 months; MST of MM patients in stage IIIA, IIIB were 11, 12 months, respectively. The medium duration of response was 5 months; the medium cycle for initial response was 2. Among 21 cases, leukopenia and thrombocytopenia were detected respectively in 10%, 54%, 31%, 5% and 67%, 17%, 13%, 3% according to WHO grade 0, I, II, III criteria. The gastrointestinal reaction was mild. No cardiovascular, renal and hepatic toxicites were observed. CONCLUSION: VCD regimen is an effective treatment for stage III multiple myeloma. PMID- 10743065 TI - [Closely relationship between expression of endoplasmic reticulum molecular chaperone Grp94 and c-myc oncogene in human colorectal carcinoma cell lines]. AB - OBJECTIVE: To study the biological significance of Grp94 deleted product (Grp94 beta) expressed in human colorectal carcinoma cells. METHODS: The relationship between molecular chaperone Grp94 and c-myc oncogene expression in the human colorectal carcinoma cell line CCL229, CX-1 and retinoic acid (RA) induced CCL229 by both of digoxin labelled c-myc cDNA probe spot by-bridization and RT-PCR with endoplasmic reticulum molecular chaperone Grp94 proximal 3' end oligonucletide primers was investigated. RESULTS: In CCL229 cells, the expression of Grp94 beta and c-myc oncogene are significantly higher than those in CX-1 cells. Along with the RA inducing, both decreased with one accord, and to the lowest level after six days induction. CONCLUSION: The Grp94 normal expression (Grp94 alpha), which was undetectable in CCL229 cells increased after RA induction. The results showed that the expression of Grp94 beta may be closely related to that of c-myc oncogene. It is suggested that the Grp94 be related to some biological characteristics of cancer, for example, the invasiveness. PMID- 10743066 TI - [Immunohistochemical study of hepatitis C virus core antigen and HBxAg in liver cirrhosis and hepatocellular carcinoma tissues]. AB - OBJECTIVE: To study the distribution and significance of hepatitis C virus core antigen in liver cirrhosis and hepatocellular carcinoma tissues. METHODS: Hepatitis C virus antigen and HBx-Ag were detected in liver cirrhosis (LC) and hepatocellular carcinoma (HCC) tissues with immunohistochemical methods. RESULTS: In some cases, not only was HCV core antigen positive stain distributed in the liver cells and nuclei of the cancer cell but it was also found in the cytoplasm. In different cases, it may be predominantly cytoplasm positive or nuclear positive or both. In liver cancer tissues, the HCV core antigen cytoplasmic positive cells were focally distributed and whereas the nuclear positive cells were diffusely distributed. The HCV core antigen nuclei positive cells were often observed in HCC tissues, but the cytoplasmic positive cells were often observed in the pericancerous liver tissue. The detection rates of HCV core antigen in LC, HCC and pericancerous liver tissues were 67.3% (66/98), 75.0% (78/104) and 48.1% (25/52), respectively. Statistical analysis suggested that: HCV core antigen nuclei positive rate in HCC be much higher than that in LC and pericancerous liver tissues (P < 0.01) and HCV core antigen nuclei positive rate be much higher than the cytoplasm positive rate in HCC tissues (P < 0.01). CONCLUSION: HCV, of which the infection being very common in LC and HCC of our country, may play an important role in the development of LC and HCC except HBV infection. PMID- 10743067 TI - [Influence of transfection of sense- and antisense-TGF beta 1 gene on human pulmonary giant cell carcinoma cell proliferation]. AB - OBJECTIVE: To investigate the effect of human sense- and antisense-transforming growth factor beta 1 (TGF beta 1) gene on human pulmonary giant cell carcinoma cell (PLA-801D) proliferation. METHODS: PLA-801D cells were transfected with an expression vector containing a full length human sense- or antisense-TGF beta 1 gene by gene transfer. The changes of cell proliferation, expressions of nm23 mRNA and c-myc mRNA and the feature of apoptosis were studied on transfected cells and untransfected cells. RESULTS: The cells transfected by sense-TGF beta 1 gene showed higher TGF beta 1 activity in the conditioned media, growth inhibition in vitro and decreased coloning formation in anchorage-independent assay. The tumorigenic rate was reduced about 47%, the metastasis was inhibited in athymic nude mice as compared with untransfected cells group. The mechanism of such changes described may be associated with the higher expression of nm23 mRNA, and the lower expression of c-myc mRNA as well as the apoptosis in sense-TGF beta 1 gene transfected cells. However, opposing changes were observed in the antisense-TGF beta 1 gene transfected cells. CONCLUSION: TGF beta 1 acts as a negative growth regulator of the PLA-801D in vitro as well as in vivo. PMID- 10743068 TI - [TGF beta 1 on murine tumor growth following direct intratumoral injection of plasmid DNA]. AB - OBJECTIVE: To investigate TGF beta 1 gene expression and its effect on murine tumor growth following direct intratumoral injection of naked plasmid DNA encoding human TGF beta 1. METHODS: LM3 murine lung adenocarcinoma cells, were injected subcutaneously to T739 mice and grew to tumor nodules in 2 weeks, Multiple direct intratumoral injections of plasmid DNA, PMAM-neo-TGF beta 1, were given and were compared with saline or vector plasmid administration groups. The growth of tumor was observed till the 8th week when the mice were killed for Northern blot analysis and histopathological study of tumoral tissue. RESULTS: The growth of tumor was boosted in the TGF beta 1 gene treated mice as compared with the control groups, whereas there was no significant difference in the metastatic behavior. Northern blot showed efficient expression of TGF beta 1 mRNA in the treated group. CONCLUSION: TGF beta 1 may stimulate tumor growth in vivo through certain mechanisms. Direct intratumoral injection of nude plasmid DNA may be a promising gene transfer strategy in vivo. PMID- 10743069 TI - [Comparison of total N-nitrosamides in fasting gastric juice from subjects in high and low risk areas for gastric cancer]. AB - OBJECTIVE: The status of human exposure to N-nitrosamides (NAD) is almost unknown, although they are probably carcinogenic to human stomach. This study is to elucidate the relationship between NAD exposure and causes of gastric carcinomas (GC). METHODS: The total amount of natural NAD was detected with a photohydrolysis-TEA method (184 nmole/l, detection limit) in fasting gastric juice samples from subjects aged 35-68 by fibroendoscope both in a high and low risk area for GC with the same geographical and socioeconomic conditions in Shangdon Province, China. RESULTS: A) More NAD-positive samples in the high risk area (Linqu County, n = 176) were observed than in the low risk area (Chongshan County, n = 99), the difference was significantly after age-adjustment (40.9% vs. 30.3%, P = 0.0324). B)84% of samples was pH < or = 3 in Chongshan, whereas only 48% in Linqu (P < 0.001). In the acidic samples more NAD-positive ones in Linqu were found than in Chongshan (46.2% vs 27.4%, P < 0.01). NAD-positive rate was higher in samples with pH > or = 5 than those with pH < or = 3 only in the low but not in the high risk area. C)NAD-positive rate was also higher in samples from subjects with chronic atrophic gastritis (CAG) with or without dysplasia than those from normal subjects and from those with superficial gastritis or CAG with intestinal metaplasia both in the high and low risk areas. CONCLUSION: The results suggest that human endogenous exposure to NAD in the high risk area for GC be higher than that in the low risk area; CAG and dysplasia may play a role in human gastric carcinogenesis by N-nitrosamides. PMID- 10743070 TI - [In situ DNA labeling apoptosis in breast cancer as related to prognosis]. AB - OBJECTIVE: This study was undertaken to determine the expression of apoptosis in breast cancer and to evaluate it's significance as a prognostic marker. METHODS: A series of 91 invasive breast cancer was analysed for the expression of apoptosis by using the 3-end-labeling method of DNA in tissue sections. The apoptotic indexes were the percentages of apoptotic cells among tumor cells. RESULTS: The end-labeling method allowed a precise evaluation of the expression of apoptosis. Apoptosis occurred in 91.2% of breast cancer patients, and apoptotic indexes were divided into two groups, 0-0.21 and 0.28-0.62. Low apoptotic index was related to axillary lymph node metastasis (P < 0.01). In survival analysis, higher apoptotic index was related to disease free survival (P = 0.0095) and overall survival (P = 0.0348) in the entire cohort. Cox's analysis showed apoptotic index had no independent prognostic value. CONCLUSION: The apoptosis was a spontaneous phenomenon in breast cancer tissue, and the expression was different from each other. Further analysis was needed to clarify the relationship between apoptosis and prognosis, especially the response to adjuvent therapy. PMID- 10743071 TI - [Mechanism of taxol-induced apoptosis in human breast cancer cells]. AB - OBJECTIVE: To investigate the mechanism by which taxol induces apoptosis in human breast cancer cells. METHODS: Cell morphology, agarose gel electrophoresis, flow cytometry, video time-lapse monitor and Western blot were performed for taxol induced apoptosis in human breast cancer cells (Bcap 37). RESULTS: BCap 37 cells treated with taxol (100 nm) underwent the arrests of cell mitosis at metaphase of mitosis and induction of apoptosis. Apoptotic cells demonstrated cell shringkage, condensation and fragmentation of chromosomes. Nuclear DNA of apoptotic cells displayed ladder bands characteristic of internucleosomal DNA fragmentation. The expression of bcl-2, inhibitor of apoptosis, was decreased with modification, while that of bax, inducer of apoptosis, increased only at early stage of the apoptotic pathway and decreased later. CONCLUSION: In human breast cancer cells the induction of apoptosis by taxol was closely associated with mitotic arrest of cell cycle, and altered expression of bcl-2 and bax gene, possibly playing an important role in regulating taxol-induced apoptosis. PMID- 10743072 TI - [Ara-c induced apoptosis in human myeloid leukemia cell line HL-60]. AB - OBJECTIVE: To elucidate the pattern of chemotherapy drugs induced apoptosis and its role in chemotherapy of acute leukemia. METHODS: Apoptosis induced by Ara-c in human myeloid leukemia cell line HL-60 was investigated by applying light microscopy, electron microscopy combined with DNA electrophoresis and flow cytometry analysis techniques. RESULTS: Apoptosis persisted throughout 36 hours following addition of Ara-c with a gradual augmentation. Efficiency of apoptosis was enhanced in a dose-dependent pattern, HL-60 treated with six other chemotherapy drugs and perpherial white blood cells from a AML case undergoing DA protocol chemotherapy exhibited typical DNA ladder pattern. Further investigation indicated that chemotherapy drugs induced apoptosis came into being possibly by downregulation the oncogenes expression of c-myc and bcl-2. CONCLUSION: Chemotherapy induced apoptosis is a primary mechanism in chemotherapy. PMID- 10743073 TI - [Study on antitumor drug-induced apoptosis in human cancer cells by terminal deoxynucleotidyl transferase assay]. AB - OBJECTIVE: Considerable evidence has showed that apoptosis is involved in both cancer development and inhibition. A new assay (terminal deoxynucleotidyl transferase assay, TdT assay) was recently reported to have advantages in the detection of apoptosis. In this study, this assay was used to investigate antitumor drug-induced apoptosis in human cancer cells. METHODS: TdT assay, DNA gel electrophoresis, electron and light microscopy were used to observe apoptosis. RESULTS: Our results showed that cisplatin-induced apoptosis in both HL-60 and SV40T-transformed human bronchial epithelial cells was detected with a good dose and time response. The occurrence of the apoptosis was preceded by the decrease of bcl-2 mRNA expression. With TdT assay, apoptotic cells were observed in the ovarian tumor of patients treated with carboplatin. CONCLUSION: TdT assay may be applicable to moniter apoptosis in human cancers induced by chemotherapy, and to evaluate tumor cell response during treatment. PMID- 10743074 TI - [Antitumor and immunological activities of oxalysine]. AB - OBJECTIVE: To study the antitumor and immunological activities of oxalysine (OXL). METHODS: Growth of mouse ascites hepatoma-22 (H-22) cells and splenocyte proliferation were investigated in vitro by MTT colorimetric assay. RESULTS: Suppression of OXL (6.3-200 micrograms/ml) on H-22 cell growth was weaker when used singly. But a marked synergetic inhibition was observed when OXL was used in combination with 5-fluorouracil (5-Fu), with suppression percentage on hepatoma growth increased to 43.1% from 13.8% (25 micrograms/ml of OXL alone) or 18.9% (3.1 micrograms/ml of 5-Fu alone). OXL (6.3-100 micrograms/ml) in vitro produced an inhibitory action on splenocyte proliferation induced by concanvilian A (Con A, 5 micrograms/ml) in the normal mice, but was able to enhance obviously the proliferative response in mice bearing H-22. CONCLUSION: OXL inhibits H-22 cell growth. The immunoregulatory action of OXL may contribute to its antihepatoma activity. PMID- 10743075 TI - [Genistein suppresses growth stimulatory effect of growth factors in HCE 16/3 cells]. AB - OBJECTIVE: The role of genistein in the effect of growth factors (KGF and EGF) on the growth of HPV 16 DNA-immortalized human cervical epithelial cells (HCE 16/3 cells) was studied. METHODS: Genistein affected the modulation effect of the growth factors on the growth of HCE 16/3 cells using [3H]-thymidine incorporation, immunocapture assay of uPA and tyrosine kinase assay. RESULTS: The growth factors stimulated growth of HCE 16/3 cells in serum-free medium. However, genistein suppressed the growth stimulatory effect of growth factors on HCE 16/3 cells and also reduced KGF-enhanced uPA secretion in this cell line. The inhibitory effects might be associated with its attenuation of activity of tyrosine kinases of growth factors. CONCLUSION: The results suggest that genistein have potential value in the prevention and treatment of some tumors in vivo. PMID- 10743076 TI - [Inhibition effect of p53 antisense RNA on malignant phenotype of colorectal cancer cells]. AB - OBJECTIVE: Inhibition effect of p53 antisense RNA on malignant phenotype of colorectal cancer cells was studied. METHODS: For the purpose of inhibiting tumorigenecity of mutant p53 gene in colorectal cancer cells, a 2.1 kb human p53 full length cDNA was inserted into a mammalian expression vector pREP9 to make a p53 antisense RNA expression vector pREP9-p53 (AS). pREP9-p53 (AS) was then introduced into human colorectal cancer cell line SW1116 (with mutated endogenous p53). MTT method and FCM analysis were performed to measure the effect of p53 antisense RNA expressed by pREP9-p53 (AS) on SW1116 cell cycle progression. RESULTS AND CONCLUSION: It was shown that the growth rate of SW1116 cells with pREP9-p53 (AS) was significantly suppressed by the expression of p53 antisense RNA as compared to the control SW1116 cells and SW1116 cells with pREP9 vector alone. FCM analysis showed that SW1116 cells with pREP9-p53 (AS) were arrested at G0/1 phase, whereas no significant influence can be observed on control SW1116 cells with pREP9. PMID- 10743077 TI - [CT-guided percutaneous transthoracic fine needle aspiration biopsy of small peripheral pulmonary lesions]. AB - OBJECTIVE: CT-guided percutaneous transthoracic fine needle aspiration biopsy (PTFNAB) was performed in small peripheral pulmonary lesions, which are not easily diagnosed or accessible to fibrobronchoscopes. METHODS: Twenty-four sputum negative cases of small pulmonary lesions smaller than 2 cm in diameter were studied. After thin-slide scanning and localization with Somatom DR-H computed tomorgraphy, the pulmonary nodules were biopsied with a 20 Gauge needle guided by a ruler especially designed. Biopsy materials were used for cyto- and histopathologic examination. The diagnosis accuracy of biopsy was evaluated by the result of postoperative pathology and/or observation for 2 years. RESULTS: Sixteen pulmonary lesions were proved to be malignant and 8 benign. The sensitivity rate was 75.0% (12/16), specitivity rate 8/8 and accuracy rate 83.3% (20/24) according to cytopathology and 87.5% (14/16), 8/8, 91.7% (22/24) by histopathology, respectively. Two cases of early-stage peripheral lung cancer were detected and proved by operation. CONCLUSION: CT-guided PTFNAB is safe and quick for diagnosis with high accuracy for small peripheral pulmonary lesions which are usually negative in sputum examination and bronchoscopy. PTFNAB should be widely used as routine in the diagnosis of lung neoplasms. PMID- 10743078 TI - [Imaging findings of malignant fibrous histiocytoma in the lung]. AB - OBJECTIVE: To show the characteristic findings of the image of primary malignant fibrous histiocytoma (PMFH) in the lung. METHODS: Fifty-one cases of pulmonary PMFH were reviewed, 7 from our hospital and 44 from the literature including chest radiograms 51 cases; CT scans 9 cases and MRI 1 case. RESULTS: Primary MFH comprises 0.01% of all pulmonany malignant tumors seen in the same period. The mean age was 55 years. The most common complaints were cough, hemoptysis and chest pain. In 40 cases who were followed, recurrence/metastasis developed in 42.9% with 80% appearing within one year. The sites of the lesion were: right lobe in 34 cases and left lobe in 17 cases. It was peripheral in 49 (96.1%) cases and central in 2 (3.9%). The finding of the image was a large solitary mass (> 5 cm, 68.6%) with regular or irregular margin. Most of the tumors were well circumscribed (56.9%) or lobulated (43.1%) showing homogeneous attenuation (64.7%). Cystic changes or cavitation was observed in a few cases. On CT, the lesion shows low attenuation and involves the adjacent structures. CONCLUSION: PMFH gives images which are not pathognomonic. Yet, its regular, well defined border, less lobulated configuration could differentiate itself from the common malignant and benign lesionsin the lung. CT and MRI can provide useful information in demonstrating the nature and extent of invasion of the tumor. PMID- 10743079 TI - [Multi-operations for non-small cell lung cancer]. AB - OBJECTIVE: Experiences and results of multi-operations in order to raise the survival rate of advanced non-small cell lung cancer are presented. METHODS: Fifty-six operations were performed in 26 patients with recurrent, metastatic or second primary non-small cell lung cancer. The operative procedure and the postoperative complications were observed and survival was analysed with Kaplan Meier of SPSS. RESULTS: The postoperative complication rate of the first operation was 3.8%. The 1-, 3- and 5-year survival rates were 83.5%, 58.4% and 35.7% respectively with a median survival of 46 months. The second postoperative complication rate was 26.9% and operative mortality was 11.5%. 1-, 3- and 5-year cumulative survival rates were 55.8%, 17.1% and 17.1% respectively with a median survival of 15 months. The third for 2 cases survived 18 and 17 months respectively. The forth and fifth were carried out for the same patient all in the sixth month. The fifth operation gave a survival of 8 months. CONCLUSION: Multi-operations for non-small cell lung cancer are still effective for recurrent, metastatic or second primary lung cancer. Simple spirometry is far from being satisfactory to evaluate pulmonary function for these patients. Meticulous attention must be paid to complication of respirative failure and infra-thoracic hemorrhage. PMID- 10743080 TI - [Long-term results of small cell lung cancer treated by surgery combined with postoperative chemotherapy: a report of 65 patients]. AB - OBJECTIVE: To analyse the long term results of small cell lung cancer (SCLC) treated by surgery combined with postoperative chemotherapy. METHODS: From 1983 to 1994, 65 patients with SCLC were treated by surgery combined with postoperative chemotherapy, and 22 of them received prophylactic cranial irradiation. RESULTS: In 64 evaluable patients, the overall 1-, 3- and 5-year survival rates were 87.5%, 53.2% and 36.3%. Patients treated by radical operation plus chemotherapy gave 1-, 3- and 5-year survival rates of 92.5%, 58.5% and 43.8%. The prognostic surviving factors as analysed showed that statistical differences were observed between stage I and II, I and III A (P < 0.03), N0 and N1 (P < 0.021) in 3-year survival rate, between 2-3 and 4-6 cycle chemotherapy in 1-year survival rate (P < 0.007). CONCLUSION: Surgery combined with postoperative chemotherapy and radiotherapy is preferred for stages I and II, N0 and N1 lesions of SCLC. PMID- 10743081 TI - [Apoptosis-antagonizing protein Bcl-2 expression, distribution in non-Hodgkin's lymphoma and its clinical significance]. AB - OBJECTIVE: Apoptosis-antagonizing protein Bcl-2 expression, distribution in non Hodgkin's lymphoma (NHL) and its clinical significance were studied. METHODS: Four-stage peroxidase antiperoxidase complex (PAP) method was applied to comparatively investigate the expression and distribution of Bcl-2 protein in formalin-fixed, paraffin-embedded lymph node tissue comprising 12 cases of reactive follicular hyperplasia and 71 cases of NHL. RESULTS: (1) Bcl-2 protein was detected in 93.0% cases of NHL with T- or B-lymphocytic phenotype. (2) Bcl-2 protein was restricted in centrocytes but not in centroblasts in 8 of 9 cases of NHL stemed from germinal centers. (3) A significant increase in Bcl-2 protein expression was found in 5 of 10 cases of diffuse large B-lymphocytic NHL showing plasmacytoid differentiation. (4) In 7 cases of follicular NHL the neoplastic nodules were strongly labeled for Bcl-2 protein and only scattered positive cells were seen in its periphery; On the contrary, in reactive lymphoid follicles Bcl-2 protein was predominantly anchored in mantle zones whereas germinal center cells were unstained. CONCLUSION: (1) Bcl-2 protein is expressed in NHL of T- or B-cell origin. (2) Bcl-2 protein expression appears to be associated with differentiation stages of NHL. (3) Immunohistochemical staining for Bcl-2 protein conduces to differentiate between reactive and neoplastic follicles. PMID- 10743082 TI - [Detection of MLL gene rearrangement in acute myelomonocytic and monocytic leukemia]. AB - OBJECTIVE: To reveal the incidence of MLL gene rearrangement and its clinical, features in acute myelomonocytic leukemia (M4) and acute monocytic leukemia (M5). METHODS: 10 patients with M4 and 27 cases with M5 were studied with the Southern blot technique to detect MLL gene abnormalities. RESULTS: 5 M5 patients were found to have MLL rearrangement, and these patients were characterized clinically by: (1) Younger at onset, (2) a higher white blood cell count (often > 100 x 10(9)/L), (3) frequent extramedullary involvement with hepatosplenomegaly and central nervous system leukemia (CNSL), and (4) a lower remission rate with a short survival time. CONCLUSION: There is a high incidence of MLL rearrangement in M5, and this gene abnormality stands for a marker of poor prognosis. PMID- 10743083 TI - [Radioimmunologic targeting and therapy with antihuman primary hepatic cancer monoclonal antibodies (Hepama I) in patients]. AB - OBJECTIVE: Effects of monoclonal antibody Hepama-I on targeting and therapy for primary hepatic cancer (PHC) were studied. METHODS: Thirty patients with veritified unresectable PHC were included in this study. For radioimmunoimaging (RII), 131I hepama was administered to 18 patients at a mean dose of 9.25 GBq. For radioimmunotherapy (RIT), 12 patients received a mean dose of 18.5 GBq by Seldinger's method via the hepatic artery. RESULTS: The optimum imaging time was 96 hours after injection. A decline in AFP level was found in 6/8 (75.0%). Reduction of tumor volume was observed as PR 66.6% and the survival time was prolonged in groups with 131I-Hepama-I treatment. CONCLUSION: Hepama I can recognized PHC cells in vivo and intra-hepatic arterial infusion of 131I-Hepama I is one of the acceptable methods of multimodality treatment for unresectable primary hepatic cancer in man. PMID- 10743084 TI - [Significance of c-erbB-2 and PCNA expression in adenocarcinoma of uterine cervix]. AB - OBJECTIVE: Significance of c-erbB-2 and PCNA expression in adenocarcinoma. METHODS: Expression of c-erbB-2 and PCNA in 74 cases of adenocarcinoma of the uterine cervix was examined by immunohistochemistry. RESULTS: The expression of c erbB-2 was detected in 34 cases. The positive staining of c-erbB-2 was associated with increased lymph node metastasis (57.1% Vs 24.0%, P = 0.041) and lower 5-year survival rate (32.4% Vs 58.9%, P = 0.008). The average PCNA labelling index (PCNALI) was 40.6% (0.1%-91.4%). High PCNALI was associated with lymph node metastasis (56.4% Vs 38.5%, P = 0.016) and lower 5-year survival rate (28.7% Vs 64.4%, P = 0.005). Positive staining of c-erbB-2 were associated with high PCNALI (44.7% Vs 34.6%, P = 0.003). CONCLUSION: c-erbB-2 and PCNALI were associated with malignant biological behavior and poor prognosis. They may be potential prognostic factors in cervical adenocarcinoma. PMID- 10743085 TI - [Predictive chemotherapy of breast cancer directed by MTT assay in vitro]. AB - OBJECTIVE: The predictive value of in vitro MTT assay for chemotherapy to breast cancer patients was studied. METHODS: Of the 156 advanced breast cancer patients, 83 had had MTT assay before chemotherapy. According to the result of the MTT assay, 73 patients in MTT sensitive group received chemotherapy. The other 10 patients in MTT resistant group and 73 patients in the control group were given chemotherapy according to clinician' experience. RESULTS: The response rate in MTT sensitive group was 76.7% (56/73). There was statistically significant difference as compared with 0(0/10) in the resistant group and 43.8% (32/73) in the control group. Between MTT sensitive group and control group, similar results were obtained at different therapeutic phases, in the subgroups of most lesions and chemotherapeutic schemes. Both the relapse rate and mortality rate were reduced remarkably in the MTT sensitive group. But there was no difference in the median remission and survival of relapsed patients in the two groups. Between in vitro and in vivo, the overall coincident rate was 79.5% [(56 + 10)/83]. CONCLUSION: The in vitro MTT assay can predict chemotherapy for breast cancer giving satisfactory results. PMID- 10743086 TI - [Clinical significance of galactose oxidase-Schiff reaction in the detection of carcinoma and precancerous lesions of large intestine]. AB - OBJECTIVE: To study the clinical value of galactose oxidase-Schiff (GO-S) reaction to test carcinoma and precancerous lesions of large intestine. METHODS: 127 patients who had symptoms and signs of digestive tract diseases and 10 normal controls were studied, with their samples of rectum mucus smears stained. The histological sections of 73 cases of carcinoma and precancerous lesions of large intestine were stained with GO-S reaction. RESULTS: The positive rates of GO-S reaction in carcinoma and precancerous lesions of large intestine were over 80%. The specificity and sensitivity of GO-S reaction to detect carcinoma and precancerous lesions of large intestine were 98% and 86%, respectively. The positive predictive value was 90%. CONCLUSION: GO-S reaction, being satisfactory and sensitive marker of carcinoma and precancerous lesions of large intestine, can be used for screening and general survey of these lesions. PMID- 10743087 TI - [Research on basilar membrane vibration of guinea pigs elicited by direct current pulse]. AB - To investigate the electromotility of basilar membrane (BM) of guinea pigs in vivo, a couple of Platinum-iridium wire electrodes were placed into holes drilled in the scala vestibuli and scala tympani on the basal turn of cochlea separately. The organ of Corti was stimulated with rectangular direct current pulses. The displacement and velocity of BM were measured with Laser Doppler velocimeter. The results indicated that the displacement of BM elicited by current moved toward the scala where positive current was injected. The waveform of BM displacement was corresponding to the rectangular pulse of electric current step. Ringing responses could be seen at the onset and offset of current pulse that was the transient responses of outer hair cells. The frequency of the ringing was the same as that of characteristic frequency of partition in BM. It is supposed that the ringing of BM is an active process of depletive energy and participate with cochlear amplifier. In the insensitive cochlea or dead animal, direct current can still elicit a BM displacement but the ringing response is decreased or disappeared. This phenomenon is probably because of metabolic disturbance in damaged outer hair cells. The BM vibration induced by direct current is similar to that of induced by acoustic stimulation, which can transfer to other partition of BM by traveling wave. This characteristic has laid a foundation of electromotile hearing and electrically evoked otoacoustic emission. PMID- 10743088 TI - [Effect of monaural cochlear ablation on cell areas of ventral cochlear nucleus neurons in neonatal and adult guinea pigs]. AB - The changes of the cross-sectional areas of anteroventral cochlear nucleus (AVCN) and posteroventral cochlear nucleus (PVCN) were studied in neonatal and adult guinea pigs after monaural cochlear ablation with computer imaging analysis system. The cross-sectional areas of neurons in AVCN and PVCN were not significantly changed after 24 h of monaural cochlear ablation in neonatal guinea pigs. But the cell areas of AVCN neurons of ablated side were respectively reduced by 20.93%, 25.70% and 28.72% compared to non-ablated side in 4 d, 7 d, 60 d after cochlear ablation, the cell areas of PVCN neurons were reduced by 17.58%, 20.30% and 38.55% respectively. The area reduction of AVCN and PVCN neurons of ablated side in 60 d group with monaural cochlear ablation were 51.00% and 32.75% relative to normal control group. Our results showed that cochlear ablation could results in rapid cell area reduction of VCN neurons in guinea pigs. Our investigation implied that it is important to stimulate the auditory nerve early in the patients with hearing loss. PMID- 10743089 TI - [Effect of white noise on activity and gene expression of nitric oxide synthase in cochlear nuclei of guinea pigs]. AB - NADPH-diaphorase (NADPH-d) histochemistry method and semiquantitative polymerase chain reaction (PCR) technology were adopted to study nitric oxide synthase (NOS) neurons and to investigate the content change of NOS mRNA and the relationship between it and auditory threshold and to explore the possible role of NOS neurons in cochlear nuclei of guinea pigs after exposed to white noise. The results showed that the quantity and staining intensity of positive NOS neurons in cochlear nuclei increased obviously after exposed to noise. It reached the peak 2 week later and remained 5 week than normal. The regulation of NOS mRNA was consistent with that of morphological observation. NOS mRNA content in 2 w group was the highest and was 4.02 times higher than normal. After exposed to noise, ABR threshold shift was in positive correlation with NOS mRNA content in cochlear nuclei (r = 0.9655, P < 0.01). It is suggested that positive NOS neurons in cochlear nuclei might be involved in controlling injury and repair of cochlear nerves. The high expression of NOS gene might be an important factor in pathogenic mechanism of noise deafness. PMID- 10743090 TI - [Recurrent acoustic neuroma: report of 11 cases]. AB - To improve the therapeutic effect of acoustic neuroma, 158 patients with acoustic neuroma treated in our hospital were retrospectively reviewed. Among them, 11 recurrent acoustic neuroma were found. The clinical analysis demonstrated that the recurrence of acoustic neuroma had close relationship with the size of the tumor, operative approach and extent of excision. All recurrent acoustic neuroma was large-sized when first operated. The more the tumor was excised, the lower the recurrent rate was. The recurrent rates were 19.4% for partial resection, 13.2% for near-total resection, 0% for total resection respectively. Retrolabyrinthine approach was more likely to recur than other approaches. The key to decrease recurrent rate was to excise as much tumor as possible. According to growth rate and double time of acoustic neuroma measured in this study, regular follow-up with CT or MRI at interval of 6 months was emphasized. X-knife was effective for early-diagnosed recurrent acoustic neuroma. PMID- 10743091 TI - [Sudden hearing loss in acoustic neuroma]. AB - To avoid misdiagnosis and mistherapy of acoustic neuroma, 104 cases of acoustic neuroma treated between 1986 and 1995 were retrospectively reviewed. Among them, 23 patients (24 ears, 23%) presented with sudden hearing loss as the starting symptoms. The clinical and audiological analysis demonstrated that 54.2% of the ears showed hearing loss over 71 dB HL, and all ears had deteriorated ABRs. SP/AP was measured in nine ears, seven showed -SP/AP greater than 0.4. Acoustic reflex was negative in all 11 ears tested, and 94.4% of the ears showed abnormal ENG. The rate of positive CT scan was 88.8%. The diagnosis was made with CT pneumo-encephalography or MRI in those with negative CT result. It was concluded that for patients with sudden deafness, ABR should be used as a routine test. When ABR was abnormal, CT scan of the internal auditory meatus was needed. PMID- 10743092 TI - [Interleukin-6 and tumor necrosis factor-alpha in middle ear effusions]. AB - Using enzyme-linked immunosorbant assay (ELISA), we measured levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) in middle ear effusions (MEE) from patients with secretory otitis media (SOM) undergoing auripuncture to study the role of cytokines in the pathogenesis of the disease. Significant levels of IL-6 (> 62.5 ng/L) were found in 19 (86.36%) of 22 MEEs and those of TNF-alpha (> 37.5 ng/L) in 19 (70.37%) of 27 MEEs. The mean (+/- s) levels of IL-6 and TNF-alpha were 507.68 +/- 813.11 ng/L and 186.86 +/- 166.93 ng/L, respectively. It revealed that the shorter the course, the higher the concentration of IL-6 in MEE: the longer the course, the higher the concentration of TNF-alpha in MEE. These findings suggested that during the early stages of SOM, IL-6 might participate in the defensive reaction of organism, while TNF alpha might be closely related to the persistence of SOM, resulting in an excessive inflammatory reaction with a potential for pathological changes. It is concluded that the immunological mechanisms probably play a significant role in the pathogenesis of SOM. PMID- 10743093 TI - [Measurement of minimal cross-sectional area of the nasal cavity and distance of minimal cross-section area from the nostril in healthy Chinese by acoustic rhinometry]. AB - 1,355 healthy Chinese, 3-86 years old, were measured with acoustic rhinometry. The range of normal value of minimal cross-sectional area (MCA) of the nasal cavity and distance of minimal cross-section area from the nostril (DCA) were obtained and the association of MCA and DCA with age in children and juvenile were discussed. The results showed that the range of MCA of unilateral nasal cavity was 0.192-0.915 cm2, DCA was 0.300-2.554 cm. The changes of MCA and DCA were highly associated with age in children and juvenile (P < 0.01). In adults, there was significant difference of MCA and DCA between different sexes in the different groups. It was demonstrated that these data could provide available information for the study of nasal physiology and pathophysiology, as well as for the diagnosis and judgement of therapeutic effectiveness of nasal diseases. PMID- 10743094 TI - [The study of effects of immunologic modifier on proliferation and cytotoxicity against HEP-2 of LAK cells in vitro]. AB - Adoptive immunotherapy as a new approach has been used to treat advanced carcinoma for almost ten years, and gained some good results. However, the application of this method is largely restricted by the side effects along with high dose of IL-2 and lack of sufficient amount of LAK cells. Mycobacterial Polysaccharides (MPS) and anti-CD3 antibodies (CD3Ab) have been demonstrated to be effective immunologic modifiers capable of proliferating lymphocytes and increasing cytotoxity against tumor cells. In this article we discussed the effects of MPS and CD3Ab on proliferation and cytotoxity against HEP-2 cells of LAK cells. Our results showed that: 1. MPS group (incubating IL-2 2 x 10(5)/L plus MPS 0.4 mg/L) or CD3 group (IL-2 1 x 10(6)/L plus CD3Ab 4 mg/L) were more proliferative than LAK group (only incubated with IL-2 1 x 10(6)/L). MPS group is the most proliferative in the three groups, P < 0.05. 2. Cytotoxity against HEP-2 in MPS or CD3 group was also better than in LAK group. CD3 group could maintain good cytotoxity in the longest period. These results could provide guidance for adoptive immunotherapy assisted by MPS or CD3Ab. PMID- 10743095 TI - [The effect of the expression of BHRF1 gene of EB virus on the proliferation of the cells of nasopharyngeal carcinoma]. AB - In order to learn the effect of the expression of BHRF1 of Epstein-Barr virus on the growth of the cell of nasopharyngeal carcinoma, high BHRF1 expressive vector was set up and transfected in low grade cell line of nasopharyngeal carcinoma, CNE2. Then the cells were cultivated under the condition of insufficient nutrition. The results show that the BHRF1 expression can prohibit the expression of PCNA, and enhance the survival of tumor cells under conditions of nutrition deprivation. So, it suggests that BHRF1 of EB virus could be involved in the development of nasopharyngeal carcinoma. PMID- 10743096 TI - [Long-term effect of voice rehabilitation in 125 patients following total laryngectomy]. AB - One hundred and twenty-five patients received voice rehabilitation operations following total laryngectomy over the period from August 1977 to December 1994. Among them, 119 were male and 6 were female, the eldest was 70 years old and the youngest 30 years old, and the average age was 55.3 years. Clinical staging was as follows: stage II, 2 cases; stage III, 45 cases; stage IV, 66 cases; and recurrence, 12 cases. The voice rehabilitation operations included pharynx trachea anastomosis 18 cases and trachea-esophagus fistulization 107 cases. One hundred and fourteen patients were followed-up for 13 months to 18 years. The 3, 5, and 10 year survival rates were 78.2%, 61.4%, and 40.5% respectively. The effective rates of voice rehabilitation according to hearing voice distance were 84.6%, 81.1%, and 67.5% 3 weeks, 1 year, and 5 years respectively after operation. The causes of failure were closure of fistula 15 cases; food aspiration 8 cases and unknown 3 cases. The clinical value, advantages and disadvantages of voice rehabilitation, and the time to evaluate the effect of voice rehabilitation were discussed. PMID- 10743097 TI - [Improvements of laryngectomy cricopharyngeal anastomosis laryngeal reconstruction (report of 27 cases)]. AB - To alleviate the deglutition and increase decannulation rate in Arslan's operation, 27 patients who had undergone laryngectomy cricopharyngeal anastomosis reconstruction from 1989 to 1995 in our hospital were reported. There were T1bN0 8 cases, T2N0 17 cases, T3N0 2 cases according to UICC staging in 1987. All lesions located in vocale cords bilaterally. On the side of the mild lesion, over half of the vocale cord was invaded. The main points of surgical procedure were that the epiglottic and cricoid cartilage were restored. Hyoid bone and arytenoid cartilages were resected and cricoid cartilage anastomosis with the base of the tongue and submental muscles. Phonation was restored in all postoperative patients. Deglutition was alleviated significantly. Decannulation rate was 92.6% (25/27). The follow-up period of 14 cases was over 3 years. Of the 14 cases, 1 died. The 3 years survival rate was 92.86% (13/14). The follow-up period of 6 cases was over 5 years. Of the 6 cases, 1 was lost, the 5 years survival rate was 83.33% (5/6). The operation has great effects on alleviating deglutition and increasing decannulation rate. PMID- 10743098 TI - [The expression of P16, Rb and cyclin D1 and biological behavior of laryngeal cancer]. AB - To determine the effect of gene of carcinorma on the proliferation and regulation in primary laryngeal cancer, P16, Rb, Cyclin D1 were examined with immunohistochemical SABC method in 36 cases of primary laryngeal cancer. The results showed: P16 was positive in 11 of 36 in carcinoma specimens (30.6%), 22 of 36 in the specimens surrounding the carcinoma (61.1%) were positive. The difference was significant (P < 0.05). The positive rates of Rb and Cyclin D1 were 61.1% and 47.2% respectively. Comparing the positive rate of P16 to Rb was more significantly (P < 0.05). In most circumstances, when Rb was positive, P16 was negative and vice versa. The positive rate of Rb correlated with the one of Cyclin D1 positively by rank correlation. The expression of P16 and Rb was significantly different between well and poor tissues differenciation. The expression had no significant difference in the same antibody in various clinical stage and position or in the same stage and position but different antibody. The results suggest that the feedback regulation chain consisted of P16 plays an important role in the proliferation and regulation. PMID- 10743099 TI - [Reconstruction of laryngotracheal stenosis: experience of twenty years]. AB - In order to improve the curative effects of laryngotracheal stenosis, 261 patients treated for laryngotracheal stenosis during the last twenty years were retrospectively reviewed. Glottic reconstruction, fence-form laryngotracheal reconstruction and laryngotracheotomy with autogenous tissue or hydroxylapatite rings were selectively used for one-stage reconstruction. Our results demonstrated that among 261 patients, 9 failed in decannulation, 5 were lost for follow-up, 247 (94.6%) patients were successfully decannulated. Among 192 patients followed-up from 1 to 18 years, 4 had restenosis 3 years after operation, 3 were successfully retreated and 10 failed. 182 (94.7%) patients had stable airways. It is concluded that there was no fixed treatment for laryngotracheal stenosis. The choice of surgical procedure and grafting must be decided on the pathologic condition of the larynx and trachea. The advantages of fence-form laryngotracheal reconstruction are simple technique and good result. Reconstruction with combined bipedicled myocutaneous flap and costal cartilage or artificial materials are better for severe laryngotracheal stenosis and large tracheal defect. The usage of stent is important for laryngotracheal reconstraction. PMID- 10743100 TI - [Nerve growth factor family and peripheral auditory nerve system]. PMID- 10743101 TI - [Therapy of head and neck neoplasms: surgery, surgery ... and others?]. PMID- 10743102 TI - [Clinical and pathological study on cervical lymph node metastasis of supraglottic cancer]. AB - AIM: To study pathological features of cervical lymph node metastasis and the relationship between supraglottic cancer and cervical lymph node metastasis. METHOD: Observation of 100 whole-organ serial sections of the larynges and 170 sides of the radical neck dissection specimens. RESULTS: 1. Cervical lymph node metastasis 55 cases, metastatic rate 55%; 2. Four series of cervical metastasis: clinico-pathologic metastases (29 cases), pathologic metastases (26 cases), clinical metastases (5 cases), no metastasis (40 cases). 3. Four stages of metastatic lymphnode: early stage, growth stage, fullness stage and capsular perforative stage; 4. Three types of metastases: single type (21 cases), multiple type (10 cases) and aggregated (24 cases) type. 5. Metastatic rates of supraglottic cancer of different regions: aryepiglottic fold 85.7%, arytenoid cartilage area 66.6%, epiglotto-ventricular fold 56.8%, epiglottic 46.4%, ventricular fold 45.4%. 6. When the tumour spread to the perilaryngeal region, the metastatic rate was 80% to 84%. CONCLUSION: Metastatic rate of cervical lymph node is higher in supraglottic cancers, the aggregated type and capsular perforative stage are more in metastasis lymph nodes. Analysis the original sites of supraglottic cancer is conductive to the prediction of the cervical lymph node metastasis. PMID- 10743103 TI - [Histopathological study of local invasion and cervical lymph node metastasis in transglottic carcinoma]. AB - To study the histopathological characteristics of local invasion and cervical lymph node metastasis in transglottic carcinoma and provide theoretical guide to clinical practice, 30 laryngectomy and 35 neck dissection specimens were studied by serial section, HE staining and observation under light microscope. The results showed that transglottic carcinomas originated from the ventricle invaded supraglottic and glottic areas equally. The metastatic rate of cervical lymph nodes was 43.3%; the latent metastatic rate was 30%. The metastatic and extracapsular perforation rates as well as the number of the aggregated fusion lymphnodes increased with the increase in the size of the lymphonodes. It was concluded that the transglottic carcinoma should be defined as one with the main lesion locates in the ventricle, or the ventricle being the center with transglottic spread. Since the barrier of ventricle was weak, partial laryngectomy should be performed with care, and ipsilateral neck dissection shoud be included during operation. PMID- 10743104 TI - [Apoptosis of nasopharyngeal carcinoma cells induced by topoisomerase I inhibitor]. AB - PURPOSE: To determine if the topoisomerase I inhibitor Camptothecin (CPT) can induce apoptosis in vitro in a human nasopharyngeal carcinoma (NPC) cell line with low differentiation (CNE-2Z). METHODS: The light microscopy, flow cytometry and agarose gel electrophoresis were used to examine the morphological changes, cell cycle distribution, hypodiploid cells and DNA fragmentation. RESULTS: After exposure to CPT for a certain period, CNE-2Z cells underwent obvious morphological changes with characteristics of apoptosis such as decrease in cell volumes condensation of chromatin and the formation of apoptotic bodies. Flow cytometry (FCM) test showed that when CNE-2Z cells were treated with 2-10 mumol/L CPT for 12 or 24 hours, hypodiploid cells accounted for 30% and 50% respectively. Cell cycle analysis by FCM revealed that changes in CNE-2Z cell cycle distribution were apparent 24 hours after treatment with various doses of CPT, showing no obvious dose-dependent relationship. Compared with controls, the percentage of cells in G2/M phase decreased markedly while those in G1 and S phases increased moderately. CONCLUSION: The results demonstrated that DNA topoisomerase I inhibitor Camptothecin can induce apoptosis in CNE-2Z cells in vitro. PMID- 10743105 TI - [Detection of P-glycoprotein in squamous cell carcinoma of larynx]. AB - The multidrug resistance (MDR) of malignant tumor is a troublesome problem in the chemotherapy. One established mechanism of multidrug resistance is elevated expression of the P-glycoprotein (PGP). JSB1 (which is a special monoclonal antibody of human MDR, PGP) was used to examine the expression of PGP by immunohistochemistry in 23 specimens of squamous cell carcinoma of larynx. The expression of JSB1 in 10 normal laryngeal tissue specimens was also studied, no positive result was found. Of the 23 laryngeal cancer specimens, positive reaction to JSB1 was showed in 15, and there was no relationship between the positive expression, and the tumor differentiation, T staging and lymph-node metastasis. However, by using another newly developed monoclonal antibody C219, a different result was obtained from specimens of the same patients. PMID- 10743106 TI - [Reconstruction of larynx with epiglottis and strip muscular flap after extended partial laryngectomy with T3 category]. AB - Fourty patients with T3 glottic cancer were treated by extended partial laryngectomy with reservation of epiglottis and posterior part of thyroid cartilage. The operative defect was covered by bilateral strip muscular flap to reconstruct the sphincter function of the glottis. The 3-year survival rate was 82.5% (32/40) and 5-year survival rate 72.9%(27/37). The function of the reconstructed laryngx was satisfactory. The authors believe that the strip muscular flap is a good material for the reconstruction of the new laryngx because of its good blood supply and wide-range repairing capability. PMID- 10743107 TI - [Partial laryngectomy for treatment of the laryngeal cancer of T3 and T4 categories]. AB - Between 1980 to 1993, 31 patients (27 men and 4 women) with squamous cell carcinoma of the larynx with T3,4 categories undergone partial largngectomy. The 3- and 5-year survival rates were 64.5% and 59.3% respectively. The extubation rate was 71.0% and recurrence rate 19.4%. After opration, the 31 patients have acquired speech function of different degrees and the swallow function returned to normal. The authors concluded that in selected patients with T3,4 laryngeal cancers, partial laryngectomy is feasible and acceptable, which preserves all laryngeal functions and improves life quality. PMID- 10743108 TI - [Human papillomavirus (HPV) and DNA test in inverted papillomas of the nasal cavities and paranasal sinuses]. AB - To understand the relationship between inverted papillomas and human papilloma virus, 38 cases (44 specimens) of inverted papillomas of nasal cavities and paranasal sinuses were examined for the presence of human papilloma virus (HPV) DNA using polymerase chain reaction (PCR). The genetic studies were performed on the formalin fixed and parafin embeded specimens. The results showed that 30 cases were infected by HPV-DNA, in which HPV11 positive was noted for 30 times (68.2%), HPV16 positive for 18 times (40.9%), and HPV18 positive for 2 time (4.5%). 18 HPV11,16 (40.9%) and 2 were HPV11,18 (4.5%). The result suggests that HPV plays a role in the etiology of nasal and paranasal inverted papillomas. PMID- 10743109 TI - [Accumulation sites of kanamycin in the organ of Corti by microautoradiography]. AB - The accumulation sites of the 3H-labelled kanamycin in the organ of Corti were observed with electron microscope. The results showed that the accumulation of kanamycin was mainly in the mitochondria, cell membrane and stereocilia of the hair cells. The possible mechanism of the kanamycin accumulation in the mitochondria is their similarity with bacteria in the protein synthesis, i.e. the way that kanamycin might inhibit the protein synthesis in the mitochondria is similar to that of bacteria. The kanamycin accumulation in the cell membrane may indicate the entrance of kanamycin into the hair cells. The kanamycin deposition in the stereocilia was most likely related to its high chemical affinity with glycocalyx. PMID- 10743110 TI - [Effects of nimodipine on calcium currents in isolated outer hair cells of guinea pig cochlea]. AB - The effects of nimodipine on calcium currents in outer hair cells isolated from guinea pig cochlea were investigated using a whole-cell recording patch-clamp technique. External application of nimodipine (10 mumol/L) caused a decrease of voltage-dependent calcium currents and an elevation of current-voltage curve. The half-maximal inhibition concentration of nimodipine inhibiting the voltage dependent calcium currents was 60.14 nmol/L, and the maximal inhibition was 34.16%. The present results demonstrate that nimodipine reversiblly and partially blocks the voltage-dependent calcium currents in isolated outer hair cells of guinea pig cochlea in a concentration-dependent manner. It is suggested that nimodipine may be used in the treatment of Meniere disease through decreasing the influx of Ca2+ into the outer hair cells. PMID- 10743111 TI - [Treatment effects of fibroblast growth factors on blast-induced hearing loss]. AB - Nineteen guinea pigs were chosen to measure the compound action potential (CAP) using the silver-ball electrode before and after explosion and 48 h after perfusion of acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF). After explosion the average CAP thresholds in the group perfused with bFGF and aFGF were 88.7 dB and 93.2 dB SPL respectivery, the CAP threshold in the control group was 119.4 dB SPL. The difference was significant. The results of the hair cell count from the surface preparation of the cochlea showed that hair cell damage in the control group was more severe than that in the group perfused with aFGF and bFGF. It suggests that aFGF and bFGF perfused to the cochlea may facilitate recovery processes of hearing loss and help to repair the hair cell following acoustic trauma. PMID- 10743112 TI - [A study on monoclonal antibodies against epithelial cell of endolymphatic sac in guinea pigs]. AB - Using a microdissected endolymphatic sac of the guinea pig (n = 35) as an initial antigen preparation, serials of monoclonal antibodies were established which were used to label the epithelial cells of endolymphatic sac. The antibodies showed strong immunoreactivity with kidney, but not with other organs. It also showed isotopes IgG1, IgG2b and IgM. The results of Western blotting and SDS-PAGE indicated that the epitopes of monoclonal antibodies were proteins or glyoprotein with a molecular weight of approximately 7,400. The locations of the epitopes in epithelial cells suggest that it may play some roles in construction and function of the endolymphatic sac. PMID- 10743113 TI - [Long-term effects of stapes elevation]. AB - To assess the clinical value of stapes elevation, 38 cases had been followed-up for 1-12 years (average 6.7 years). The results revealed that hearing improvement rate (over 10 dB) was 94.7% in short term and 86.8% in long term follow-up. The long term regression rate was similar to that in stapedectomy, and no severe sensorineural hearing loss occurred. In addition, the vestibular reaction was slight, especially in reoperated cases. The stapes elevation was easy to perform as compared with stapedectomy. Therefore, this technique may be the first choice for stapes surgery. PMID- 10743114 TI - [The uncinate process: ultrastructural and CT studies]. AB - To provide further understanding of uncinate process, 15 uncinate processes were examined under scanning electron microscope, and 32 high-resolution coronal CT scans were studied in patients with septal deformity or sinusitis. It was found that(1)pathological ultrastructural changes of uncinate process were noted in patients with chronic sinusitis; (2) medially deviated, laterally deviated, pneumatization and hypertrophy of uncinate process were demonstrated. The following conclutions were drawn(1)the anatomic variations in ostiomeatal complex is responsible for the formation of chronic sinusitis; (2)for patients with chronic sinusitis, uncinatectomy is the treatment of choice; (3)preoperative and intraoperative studies of CT scans are mandatory for avoidance of complications. PMID- 10743115 TI - [Current status of otorhinolaryngology and its nomenclature]. PMID- 10743116 TI - [Partial stapedectomy with CO2 laser]. AB - For the efficacy and safety of partial stapedectomy, Co2 laser was used in 44 patients with otosclerosis. Postoperative follow-up (3-12 months) demonstrated that the average hearing gain was 31.7 +/- 8.2 dB, and air-bone conduction gap was within 11.2 +/- 6.6 dB. There was no significant hearing change at 4.000 Hz. The advantages of CO2 laser technique are summarized as follows: convenience for cutting posterior crus, less time needed for penetration of thick footplate or removal of occlusive sclerotic plaque in oval window niche, avoidance of footplate floating, rapid coagulation of residual stapedial artery and absence of instrumentation difficulties in narrow spaces. Our results indicate that CO2 laser provides essential benefits in stapedectomy for otosclerosis. PMID- 10743117 TI - [Diagnosis and management of Mondini malformation]. AB - Owing to the development of imaging technology and audiology, some of the sensorineural hearing loss cases that were previcusly considered to be of unknown cause have been found to be inner ear malformation. Five cases of Mondini malformation are reviewed in this paper. CSF otorhinorrhea occurred in four cases, Klippel-Feil syndrome in three, and concurrent otosclerosis in one patient. In discussion, the authors point out that: 1) CT scanning is an supplement to audiologic tests for such patients; 2) perilymph fistula in Mondini malformation is often found at the oval window and its vicinity, the foot plate and the round window; 3) the fistula can be treated by plugging with fascial tissue via tympanoplastic approach with endaural incision. The mucosa around the fistula should be stripped away and the plugging tissue should be of dumb-bell shape. PMID- 10743118 TI - [Facial nerve protection during surgery for congenital malformation of the external and middle ear (review of 519 cases)]. AB - The prevention of facial nerve injury in otoplasty for ear malformation is important. This paper analysed 519 cases of ear malformation collected during 10 years from 1985 to 1995. The hearing of 428 cases was improved, of which the hearing of 221 cases did recover to available level. A long-term follow-up of 198 cases showed that 152 cases had kept the same hearing level as that immediately after surgery. In 130 cases with single middle ear malformation the malformation rate of facial nerve was 60.8%(79/130), and that for chorda tympani 5.4% (7/130). In 75 cases of middle ear malformation with stricture of external acoustic meatus the malformation rate of facial nerve was 60.0% (45/75), and that for chorda tympani 13.3%(10/75). In 314 cases of middle ear malformation with atresia of external acoustic meatus the malformation rate of facial nerve was 42.5%(142/314), and that for chorda tympani nerve 100.0%(314/314). Two cases(0.4%) were of surgical facial nerve paralysis. For protection of facial nerve, it was important to master the anatomy of temporal bone and have a high resolution computed tomographic scanning to identify facial nerve. It was also important to use electroneurographic monitoring when necessary. PMID- 10743119 TI - [Clinical and pathological observation of recurrent nasal polyp formation]. AB - To investigate the mechanism of nasal polyp recurrence, 19 patients with recurrent nasal polyp were retrospectively analysed. In 4 patients, nasal polyp specimens had been sent for pathological examination more than three times, and in 6 patients, bacteria culture was performed. There were two types of recurrent nasal polyp: water-cyst type (fast recurrence) and edematous granulation type (slow recurrence). Both types were the result of inflammatory reaction, perhaps related to trauma of operation. Water-cyst formation indicated stronger inflammatory reaction. During follow-up, 10 patients were cured and 9 recurred. Preventive measures against nasal polyp recurrence were discussed. PMID- 10743120 TI - [Detection of antigen specificities of HLA-A, B loci in perennial allergic rhinitis]. AB - In order to elucidate whether there are any features of HLA haplotype in perennial allergic rhinitis and the essentials of its genetic background, the antigen specificities of HLA-A, B loci in 31 non-kindred adult cases of southern Chinese of Han nationality were detected and calculated the antigen frequency, gene frequency and relative risk value of different alleles evaluated by serological methods. The results were compared with 78 healthy adult individuals of Guizhou Han (control group) and revealed that RR (relative risk) of HLA-B27 was as high as 8.25, RR of A31 (2.68), A28 (2.57), B12 (2.57) and A33 (2.27) were also higher than the control group. It is suggested that the presence of the above mentioned antigens are positively associated with the genetic liability of the patients. The RR of A9, B60, A24, B22, B51, B15, B16, B48, B46, B7, A11 and A2 were all lower than 1 representing the genetic stability to allergic rhinitis. The antigen frequency of B27 and A9 were significantly different from that of the control group. PMID- 10743121 TI - [Quantitative observation of expression of proliferation cell nuclear antigen in nasopharyngeal carcinoma]. AB - Eighty-two cases of nasopharyngeal squamous cell carcinoma and twenty-five cases of nasopharyngitis were studied by means of LAS-SP immunohistochemical method with monoclonal anti-PCNA antibody and using image pattern analysis technique; correlation analysis was performed on expression of PCNA and five-year survival rate. It was shown: 1. The cell proliferative index and pathological grading of NPC were positively correlated, and the cell proliferative index and five-year survival rate negatively correlated. 2. PCNA expression level and pathological grading of NPC were positively correlated, and PCNA expression levels and five years survival rate negatively correlated in all pathological gradings of NPC except the vesicular nucleus cell carcinoma. The results indicate that PCNA affects proliferation activity of NPC and there are bright prospects for their application in the clinic. Expression of PCNA may also become a good indicator of the prognosis of NPC and the choice of treatment. PMID- 10743122 TI - [Ongoing production of sensory epithelium cells in the avian inner ear]. AB - Does the ongoing proliferation of sensory cells occur in the postnatal avian inner ear? This question still awaits further investigation. 5-bromodeoxyuridine (BrdU) is a thymidine analogue that is incorporated into DNA during S phase of the cell cycle. Chickens were given intraperitoneal injections of BrdU at 100 ml/kg body weight. Six hours after injection, they were sacrificed and the temporal bones were removed and prepared for BrdU immunohistochemistry to examine mitotic activity in the inner ear. A small number of labeled hair cells and supporting cells were seen in the basilar papilla (BP). There is a low rate of ongoing production in the postnatal avian vestibular epithelium. The ongoing, postnatal proliferation of vestibular epithelial cell suggests that this epithelium has the potential for repair after degeneration, trauma or ototoxic damage. PMID- 10743123 TI - [Experimental study on the effect of nitric oxide on cochleas of guinea pigs]. AB - The effects of nitric oxide (NO), 1-monoarginine (L-Arg), sodium nitroprusside (SNP) and N-methyl-1-monoarginine (NMLA) (which is an antagonist of nitric oxide synthase) on endocochlear potential, compound action potential(CAP) and cochlear microphonic(CM) of cochleas were studied by means of perilymphatic perfusion of the agents. The results showed that NMLA reduced EP, CAP and CM, L-Arg perilymphatic perfusion could reverse the change caused by NMLA. Continuous NO perilymphatic release restored the changes of EP, CAP and CM back to normal, sometimes above normal, followed by rapid reduction. After SNP perilymphatic perfusion, EP, CAP and CM went up transiently and then declined gradually and remained at a lower level. Addition of NMLA did not reverse the changes. The change of the latent period of CAP N1 and drift of CM was consistent with their amplitude changes. It was suggested that NO might help maintaining inner ear function under physiological condition and participate in controlling the micromechanical character and sensitivity of cochlear hair cells, undue expression of NO may cause toxicity to cochleas. PMID- 10743124 TI - [The vascular pattern of the endolymphatic duct, endolymphatic sac and its anatomic differences in guinea pigs]. AB - In order to study the vascular pattern of the endolymphatic duct and sac, endolymphatic duct and sac were examined with vascular Indian ink injection and image analysis. The Results were as follows: 1. In the 20 temporal bones, 17 (85%) had posterior meningeal artery (PMA) and posterior vestibular artery (PVA) supply and the rest (3 specimens, 15%) had no PVA supply; 2. The distribution frequency of PMA in the endolymphatic sac was much higher than that of PVA(P < 0.01), but the distribution of PMA and PVA in the endolympatic duct were not different (P > 0.05). The conclusions is that there are anatomic differences in vascular supply and pattern of the endolymphatic duct and sac, PMA is the main vascular structure in the endolymphatic sac. PMID- 10743125 TI - [A preliminary study on the effect of estrogen on nasal mucosal hyperreactivity]. AB - To understand the mechanism of the effect of estrogen on nasal mucosal hyperreactivity, animal models (guinea pigs) with different levels of estradiol (E2) were observed as follows: 1. effect of the change of E2 levels on E2 receptors (E2R) content in nasal mucosa; 2. effect of E2 level changes on histamine content in nasal mucosa and on nasal mucosal eosinophil and mast cell counts; 3. effect of E2 on release of cytokine IL-6 from macrophages. The results showed that high level of E2 might reduce the E2R content and eoinophil count but no significant effect on mast cells count and histamine content. The secretion of IL-6 from the macrophages was suppressed by E2 within concentration range of 3.13 50 micrograms/L. It was suggested that estradiol may play a complicated role in nasal mucosal hyperreactivity. PMID- 10743126 TI - [Evaluation of the function of palatine tonsil with regard to phagocytosis]. AB - Trypan blue and Indian ink made in Fuzhou as vital staining dyes were injected into the abdominal cavities of rabbits. Nine days later, the tonsil, thymus, spleen and lymph nodes were examined. Numerous pigment granules were observed in the macrophages of the spleen and lymph node. However, no phagocytosis of pigment granules was found in the palatine tonsil and thymus. It indicated that the function of palatine tonsil as a lymph organ is to contact exogenous antigens and to take part in immune response. It is important for children, especially infants, to establish a complete immune mechanism. The Tonsils reach their maximum development in childhood and thereafter decline. Once the palatine tonsils become chronic foci, surgical removal of the diseased tonsils is necessary. PMID- 10743127 TI - [Effects of short-term tone exposure on DPOAEs]. AB - Test parameter combinations are known to affect DPOAE measurement. The present study was attempted to find out if different test parameter combinations affect DPOAEs' utility in reflecting the change of cochlear function. Pure tones of moderate intensity (82 dB SPL) and short duration (3 min) were introduced into rabbit's ear canal to cause temporary change of cochlear mechanics, and the change of amplitude of 2f1-f2 DPOAE representing 10 kHz over 10 minutes after the exposure was monitored using equal- and different primary level combinations. It was found that, under the same exposure condition, 2f1-f2 DPOAEs induced by primaries of different levels (L2 = L1 - 12 dB) had showed more initial reduction in amplitude and slower recovery than that induced by primaries of equal levels (L1 = L2). The difference may indicate that there are multiple physiological processes that contribute to the generation of 2f1-f2 DPOAE and that change of DPOAE test parameters may selectively reveal different DPOAE generating processes. PMID- 10743128 TI - [Ten-year experiences of epiglottic laryngoplasty after partial laryngectomy]. AB - In order to objectively evaluate the long-term result of epiglottic laryngoplasty (ELP), ten-year experience of ELP after partial laryngectomy in 100 patients with glottic cancer was analysed. Our program included 1. complete excision of laryngeal carcinoma; 2. correct selection of material for laryngoplasty (LP); 3. technical modification of LP. The 3, 5 and 10 year survival rates were 87%, 79.5% and 66.7% respectively. It is believed that modified ELP is better than original K-S-T technique in the aspect of preserving all laryngeal functions. PMID- 10743129 TI - [Clinical analysis of bilateral neck dissection in patients with laryngeal cancer]. AB - The clinical materials of 54 cases with laryngeal cancer treated between 1980 and 1995 were retrospectively analysed. All patients had received bilateral neck dissection. The results indicated that if one internal jugular vein was preserved, bilateral neck dissection was safe and no fatal complications occur. In this series, most patients were of T3 and T4. The 3 and 5 year survival rates were 62% and 52% respectively. Cervical recurrence was the main reason for the deaths. It is suggested that for patients with advanced supraglottic laryngeal carcinoma, bilateral neck dissections are often needed, functional neck dissection, with preservation of at least one internal jugular vein is the best choice. PMID- 10743130 TI - [PCR detection of Chlamydia pnumoniae DNA in nasopharyngolaryngeal swab samples from patients with rhinitis and pharyngolaryngitis]. AB - A PCR assay was used to detect Chlamydia pneumoniae specific Pst I 474 fragment DNA in swabs from patients with acute or subacute pharyngolaryngitis or rhinitis and nasosinusitis. C. pneumoniae specific antibodies in sera were also assayed with microimmuno-fluoresence (MIF). About 28% of the patients (49/175) were PCR positive and 25.7% (45/175) were MIF antibodies positive. The accordance rate of the two methods was 91.8%. It is suggested that the C. pneumoniae Pst I 474 specific PCR is sensitive and specific for detecting C. pneumoniae in pharyngolaryngitis or rhinitis and nasosinusitis. PMID- 10743131 TI - [Audiologic measure in Meniere's disease]. AB - To determine some audiologic phenomena of Meniere's disease before and after ingestion of dehydration agent and evaluate its diagnostic value, 48 ears of Meniere's disease, 40 ears of other vertigo disease and 40 normal ears were investigated. The measures were undertaken before and after glycerol or urea ingestion, which included threshold of pure tone audiometry, peak static acoustic admittance (Ya) and conductance(Ga) at probetone signal of 678 Hz, peak compensate static admittance(delta Y) and conductance(delta G) at resonance frequency, -SP/AP, and width of SP-N1 in ECochG. Results showed that abnormal rates of glycerol group and urea group were similar, and there was no significant statistical difference. So these two groups were combined together as one in statistics. The rate of totally abnormal ears were 17/32 in seven measures. Abnormal rates of dehydration test were: pure audiometry 66.67%, Ga 62.50%, SP/AP 52.08%, width of SP-AP N1 66.67%. chi 2 test showed no significance statistical difference among these results. It indicated that these measures had same diagnostic value for the Meniere's disease. The shift value of G a and delta G of dehydration test might be as the objective diagnostic methods of Meniere's disease. PMID- 10743132 TI - [The value of the ratio of--SP/AP in diagnosing Meniere's disease]. AB - To further explore the diagnostic value of the ratio of--SP/AP (> 0.4) in Meniere's disease, the EcochG results from 70 (76 ears) cases with Meniere's disease were compared to that of 49 cases with sudden hearing loss(SHL), noise induced hearing loss(NIHL), ototoxic hearing loss, and 20 normal hearing person. The effects of different stimulus intensities and the degree of hearing loss on the detectable rate of--SP, the ratio of--SP/AP and its feature in the above mentioned disease were analysed. The results indicated: 1. The mean ratio of- SP/AP was 0.39 +/- 0.02 in normal person on 75 dB nHL stimulus level, while it was larger than 0.45 in SHL and NIHL on 75 dB and 65 nHL stimulus level respectively. 2. There was a rapid descending trend of the ratio of--SP/AP with stimulus level from 65 to 55 dB nHL in all ears except Meniere's cases, which was correlated with stimulus level as well as the degree of hearing loss. 3. The mean ratio of--SP/AP presented linear increase when stimulus level was < or = 75 dB nHL, and when stimulus level was > 75 dB nHL, the mean ratio of--SP/AP no longer presented linear increase and could even decrease, it suggested stimulus level on 75 dB nHL could be used as the diagnostic stimulus level. 4. If the degree of hearing loss exceeded 80 dB HL, the ratio of--SP/AP > 0.4 had no practical significance in diagnosis. PMID- 10743133 TI - [Sudden deafness: stepwise regression analysis on the correlation factors with prognosis]. AB - To further investigate the correlation factors of the prognosis and therapeutic efficiency of sudden deafness, 83 cases (91 ears) from 1993. 1 to 1996. 4 were studied with stepwise regression analysis. The results showed that the prognosis is correlate with the age, the hearing threshold levels of both speech and high frequencies, and the types of the audiometric curve, but not with the illness duration, sex, vertigo, ipsi or bilateral, recurrence, virus infection, cardiac or cerebral vascular diseases, and the medicine or the duration of the treatment. By comparing the results obtained from different statistical analysis, it was suggested that the unification of the criteria of the diagnosis and the therapeutic efficiency as well as the application of the multiple factors regression analysis are essential for the research on sudden deafness. PMID- 10743134 TI - [Application of electronystagmography glycerine test for diagnosing Meniere's disease]. AB - To evaluate electronystagmography (ENG) glycerine test (GT) for diagnosing Meniere's disease (MD), ENG GT were performed in 44 patients (48 ears) with MD and 20 normal subjects (40 ears). In normal subjects, the increase or decrease in percentage of maximum velocity of caloric nystagmus were calculated before and 1, 2, 3 hours after administration of glycerin (1.5 g/kg). According to the statistical analysis, 95% of normal subjects fell below +15% at 44 degrees C and +10% at 30 degrees C, therefore the value of +15% at 44 degrees C and +10% at 30 degrees C were taken as the upper limit of normal range. Increase in maximum velocity of caloric nystagmus beyond the normal range was referred to as positive. In the patients with MD, positive reaction were observed in 86.7% of typical MD, 77.8% of vestibular type MD. The rate of positive response was much higher in stage of attack of hydrolabyrinth than that of remission. The results indicated that this test is another diagnostic method for patient with MD and is particularly suited for diagnosing vestibular type of atypical MD. PMID- 10743135 TI - [Effects of white noise exposure on nonlinearities of outer hair cell]. AB - The technique of microelectrode has been used to record receptor potentials of outer hair cell (OHC) of seven guinea pigs' cochlea basing on the programmable signal processing system. The nonlinearies of input-output function of the receptor potential recorded from OHC are compared before and after with noise exposure. Results indicated that the input-output functions of the receptor potential of OHC reveal linear behavior increased in linearity in low level and turned to be nonlinear when the sound level increased to 50 dB SPL, and significant saturation was seen in 80 dB SPL. After the white noise exposure (100 dB SPL , 10-20 minutes), the phenomenon of nonlinearity disappeared with the amplitude of receptor potential decreased, which suggested that nonlinearity was easy affected by noise exposure. OHC response recruitment has shown after noise exposure. These results together with preceding clinical observation of loudness recruitment, suggest that loudness recruitment occurs at least in part at the hair cell level. PMID- 10743136 TI - [Effect of hyaluronan perfusion through the round window on the function and morphology of the inner ear in guinea pigs]. AB - In order to study the effect of the hyaluronan (HA) on the inner ear, twenty-six guinea pigs were divided into experimental and control groups, the experimental group were perfused with 2% HA 0.2 ml through the left round window. Auditory and vestibular function were investigated before and 5, 14, 28 days after perfusion. The threshold of action potential (AP) response to filtered clicks (0.25-10 kHz) were elevated at 5 days and were improved (at 1-4 kHz) at 14 days after perfusion (P < 0.05). At 28 days all AP response thresholds had returned to normal (P > 0.05). The nystagmus duration induced by caloric test (ENG) had no obviously change comparing with that of preoperation (P > 0.05). All guinea pigs were sacrificed 28 days after operation. The histology of inner ear were examined by means of membranous labyrinth mapping and temporal bone section after celloidin embedding. The morphology of Corti's and vestibular organs were not significantly damaged after perfusion. IN CONCLUSION: 1) 2%HA caused reversible and temporary changes in auditory function of inner ear; 2) 2%HA lacked ototoxic adverse effects, whereas exerted considerable osmotic effects and a slow dehydration of inner ears. PMID- 10743137 TI - [Histological study of experimental reconstructive materials for lateral skull base and dural defect in dogs]. AB - In order to observe the reconstructive effects of fascia lata, superficial fascia and bone morphogenic protein (BMP) in skull base surgery, a lateral skull base bone and dural defect model in dogs was established. Fascia was selected alone or combined with BMP as repairing materials and BMP was evaluated in reconstructing large cranial defect model. Forty dogs underwent a 3.0 x 4.0 cm full-thickness excision of the parietal bone were divided into different groups of five dogs each, and were reconstructed by 1. fascia lata; 2. fascia lata plus BMP; 3. nonreconstructed controls; 4. fascia lata plus BMP and exposed. The implants were harvested at 2-15 weeks and examined histologically. The new bone was found in the BMP. Implants treated and untreated were quite different. The unreconstructed controls demonstrated only a bridge of fibrovascular connective tissue. BMP are defined as osteoinductive by their ability to produce bone formation by stimulating mesenchymal cells to transform into bone-producing osteo-blasts. The results suggested the combination of BMP and reconstructive material in the treatment of bone defects. PMID- 10743138 TI - [Differentiation-inducing effect of stepholidine and retinoic acid on human head and neck carcinoma]. AB - To observe the differentiation-inducing effect of Stepholidine (ST) and Retinoic Acid (RA) on laryngeal and nasopharyngeal carcinoma, the expression of oncogene (C-myc, bcl-2) protein and tumor suppressor gene (p53) was done by using flow cytometry and ABC immunohistochemical methods combined with image analysis technique. The results indicated that after treated with ST and RA, the cells became well-differentiated, the cell growth was suppressed, contact suppress was partially recovered, colony forming was decreased, protooncogenes (C-myc bcl-2) protein was decreased, tumor suppressor gene (p53) protein was increased. No significant difference was observed between the cells treated with ST and TA. We believed that the Chinese medicine-Stephania might be expected to become a new prospective differentiation inducer in carcinoma of head and neck. PMID- 10743139 TI - [Transmission electron microscopic study on eosinophil degranulation in nasal mucosa in allergic rhinitis]. AB - To observe the ultrastructure of eosinophil (EOS) degranulation in nasal mucosa in allergic rhinitis, nasal mucosa biopsies from patients with perennial allergic rhinitis (PAR, n = 13) pollinosis (PS, n = 9) subjects and normal controls (n = 3) were processed and the ultrastructure changes of EOS degranulation were examined under transmission electron microscope and routine histopathological observation. The ultrastructure of EOS degranulation in nasal mucosa collected from PAR and PS patients in their remission period differed from that in their attacking period, though there was no distinct differences in the distribution patterns of EOS. Increased cell membrane projections, eminent cytoplasm vacuolization, inconsistent density of granules with its crystalloid being lost, plus tissue edema and damage of collagen fibers in adjacent areas of degranulating EOS were among the most important ultrastructural changes of EOS in attacking period. The interrelations of EOS, mast cells (MC) and T-lymphocytes play important roles in the pathogenesis of allergic rhinitis. PMID- 10743140 TI - [Transsphenoidal microscopic removal of invading pituitary adenoma]. AB - Eighteen cases of invading pituitary adenoma were removed via the approach of transsphenoidal microscopical operation. Among them 4 cases had insulin dependent diabetes. The pituitary adenoma were near-totally removed in 15 cases, partially removed in 3, and 10 cases were treated by supplement radiotherapy postoperatively and 5 cases by oral bromocriptin postoperatively. The average time of follow-up was 3.8 years. There were 3 cases(16.6%) had postoperative recurrence of the tumor which were finally cured by transfrontal craniotomy. The feasibility of the transsphenoidal microscopic approach for the invading pituitaty adenoma was discussed. PMID- 10743141 TI - [Relationship between nasal airflow sensation and nasal patency]. AB - The relationship between nasal airflow sensation and nasal patency was evaluated by means of visual analogue test and anterior rhinomanometry. It was demonstrated that there is no significant correlation between the subjective sensation of nasal airflow and objective assessment of nasal airflow resistance. The site responsible for sensing airflow is located in the nasal vestibule. The volatile agents used in traditional Chinese medicine for nasal obstruction, such as camphor and menthol, could only improve the nasal sensation of airflow without alteration of nasal airway resistance. Our results suggested that the nasal sensation of airflow could not reflect real patency completely. Therefore, assessment of subjective sensation of airflow and measurement of nasal airway resistance combined must be used in the diagnosis and treatment of nasally obstructed patients. PMID- 10743142 TI - [The effects of menthol, borneolum and moschus on nasal airflow sensation and nasal resistance]. AB - Menthol and borneolum have been used widely in traditional Chinese medicine for the treatment of nasal obstruction, but the mechanism has been still unknown. The effects of inhalation of menthol, borneolum, moschus on nasal resistance and airflow sensation were investigated in 52 subjects. All of these medicines could cause a highly significant enhancement of nasal airflow sensation but had no effect on nasal resistance. Therefore this sort of medicine is regarded as pseudo nasal decongestant. PMID- 10743143 TI - [Surgery of substernal thyroid goiter]. AB - A retrospective study on 87 substernal thyroid nodules operated between 1965 and 1994 was made. Among them 73.6% were goiters, 14.9% adenoma and 11.5% malignancy. Resection via cervical collar incision was performed in 70% of all cases, sternotomy in 16% and thoracotomy in 14%. A cervical collar incision was adequate in 91% of 43 cases after 1985, only one goiter was completed by sternotomy. Morbidity rate of complications was 41.4%, while the rate of recurrent laryngeal nerve paralysis was 20.7%. Surgery via cervical collar incision for all retrosternal thyroid nodules was advised and sternotomy or thoracotomy are suggested. PMID- 10743144 TI - [A report of 50 cases of transglottic carcinoma]. AB - 50 cases of transglottic carcinoma were chosen and the collodion-embedded specimens of whole-organ serial section were observed. We found that: 1. Transglottic carcinoma was of laryngeal ventricle origin; 2. There were 1 case in T1 and 2 cases in T2 category; 3. Deep infiltration was the main local growth pattern (90%, 45/50), and paraglottic space and thyroid cartilage were easily involved (82%, 41/50 and 65%, 32/50 respectively), clinical stage was not parallel to pathological stage (48%, 24/50). PMID- 10743145 TI - [Symptoms of depressive state in otolaryngology]. AB - A psychiatric study was carried out to seek correct diagnosis and effective therapy for patients with various pain symptoms in ear, nose and throat area, and the unknown cause of pain and invalid treatment. There were 12 patients who coincided with the diagnostic criteria of depression. The therapeutic effect was satisfactory with antidepression medicine. It is suggested that the practicing ENT doctors should have some knowledge of psychiatry, understand the symptoms of depressive state, avoid misdiagnosis and offer correct management. PMID- 10743146 TI - [Clinical uses should be emphasized in current research of head and neck neoplasms]. PMID- 10743147 TI - [Current status and problems in nasal surgery in China]. PMID- 10743148 TI - [On the mechanism of hemilaryngeal fixation in pyriform sinus carcinoma and its clinical significance]. AB - To approach the mechanism of hemilaryngeal fixation in pyriform sinus carcinoma (PSC), surgical specimens from total laryngectomy and partial hypopharyngectomy in 43 cases of pyriform sinus carcinoma were studied using organ subserial sections. Invasion of intralaryngeal muscles and the upper and outer aspects of arytenoid cartilage via paraglottic space was found to be the main cause of hemilaryngeal fixation in these cases. Presence of tumor on the medial wall of the pyriform sinus makes involvement of intralaryngeal structures easy, and thus is more frequently seen with hemilaryngeal fixation. Clinical T3 lesion of the sinus may infiltrate laryngeal cartilages and adjacent soft tissue to become pathologic T4 lesion. For PSC appearing hemilaryngeal fixation, total laryngectomy is safer to thoroughly remove cancer. PMID- 10743149 TI - [Study on the relationship between intratumor microvessel density and neck metastasis of laryngeal and hypopharyngeal squamous cell carcinomas]. AB - Sixty-one laryngeal and hypopharyngeal squamous cell, carcinoma (LC, HPC) tissue slides were immunochemically stained using LSAB method to study epithelium cells. The results demonstrated that (1) intratumor microvessel density (ITMD) in LC and HPC group was higher than that of the benign group (P < 0.05). ITMD was higher in the subgroup of LC and HPC with positive lymph node positive than that with negative lymph nodes. This result suggest that ITMD is relevant not only to the nature of the tumor, but also to lymph node metastasis. The level of ITMD is an important predictive sign of metastasis. (2) The relationship between ITMD and the clinical staging had no statistic significance (P > 0.05). (3) The analysis on the relationship between ITMD and pathologic differentiation indicated that the level of ITMD raised gradually with the lowering of the pathologic differentiation. PMID- 10743150 TI - [Validity of partial laryngectomy for glottic carcinoma]. AB - To evaluate the effect of partial laryngectomy for the treatment of glottic carcinoma, 126 patients with glottic carcinoma were reviewed. Between 1974 and 1994, 132 patients with glottic carcinoma were treated. Up to Dec. 1994, 126 cases (95%) had been followed-up and analyzed statistically in this paper. According to TNM classification (UICC 1987), 98 (77.8%) of them were T1.2, 28 (22.2%), T3.4. One patient was T4N1, and the rest 125 were No. All patients were M0 on admission. A total of 126 patients underwent partial laryngectomy. Among these patients, 89 were treated with surgical procedures alone, and 4 received pre-operative 33 post-operative, radiotherapy. Survival curves were plotted by the method of Kaplan and Meier. The results showed that three-year, five year and ten-year survival rates for 126 patients were 94.7%, 89% and 86.1% respectively; Those for group of T1.2 were 97.8%, 95.1% and 93.3%; For T3.4 were 83.2%, 62.7% and 52.3%. All patients preserved their voice and were able to communicate by speech after operation. The function of larynx was satisfactory. It was concluded that partial laryngectomy is preferable choice for the treatment of glottic carcinoma. PMID- 10743151 TI - [Evidence for a major role of genetic factors in the etiology of laryngeal and hypopharyngeal carcinomas]. AB - Several investigations revealed that lymphocytes from patients with head and neck cancers showed a high sensitivity to mutagens. However, whether lymphocytes from healthy blood relatives of the patients were sensitive to mutagen or not had not been reported. This study was aimed to investigate the unknown problem. Mutagen sensitivity was investigated in 116 laryngeal and 38 hypopharyngeal cancer patients, 107 healthy controls, and 30 blood relatives of laryngeal or hypopharyngeal cancer patients. Chromosome damage was measured by enumerating mutagen-induced chromatid breaks, the average number of chromatid breaks per cell (b/c value) was used as marker which reflects variable host susceptibility to the action of environmental carcinogens. The b/c values were 0.61 +/- 0.27, 0.66 +/- 0.31, 0.28 +/- 0.12, and 0.45 +/- 0.26 for patients with laryngeal cancer, hypopharyngeal cancer, healthy controls, and blood relatives respectively. A significant difference of b/c value was found between patients and controls (P < 0.01), but not between patients and their blood relatives (P > 0.05). Our findings suggest that genetic predisposition plays an important role in the eitiology of laryngeal and hypopharyngeal cancers. Both patients with laryngeal or hypopharyngeal cancers and their blood relatives should be considered as in high cancer risk. PMID- 10743152 TI - [Clinical significance of beta 2-microglobulin content in nasopharyngeal secretion]. AB - To study its clinical significance, beta 2-microglobulin (beta 2-MG) in nasopharyngeal secretion was examined using radioimmunoassasy methods in 73 patients with nasopharyngeal carcinoma (NPC), 30 with chronic nasopharyngitis and 30 normal control subjects. In 31 NPC patients, the examination was repeated before and after radiotherapy. The average concentrations of beta 2-MG were 4.87 +/- 2.51 mg/L in NPC group, 1.05 +/- 0.64 mg/L in chronic nasopharygitis group, and 0.85 +/- 0.32 mg/L in the control. The beta 2-MG concentration was higher in the NPC patients than in both chronic pharyngitis patients and normal subjects (P < 0.01). The concentration was lowered down in NPC patients after radiotherapy, and remained high when the treatment failed to control the condition. It seems that beta 2-MG level in nasopharyngeal secretion is closely related to the prognosis and recurrence of nasopharyngeal carcinoma. PMID- 10743153 TI - [Application of electronic larynx in speech rehabilitation after laryngectomy]. AB - An electronic larynx has been developed at this center and used among laryngectomees for speaking rehabilitation. The phonation rate was 98% in 4,520 patients from all over the country. The frequency characteristics of the artificial larynx is close to that of human sound. Other advantages of the electronic larynx are: the best sound conducting point at the neck; natural phonation actions; and on/off switch synchronized with speaking attempts. The electronic larynx is easy to use and worth to be recommended as an alternative to surgical intervention in speech rehabilitation after laryngectomy. PMID- 10743155 TI - [Topographic mapping of slow cortical response in guinea pigs]. AB - Auditory slow cortical responses (SCR) were studied in 10 awake guinea pigs using topographic mapping techniques. Fourteen electrodes were installed through the guinea pig skull and fixed with dental cement. Auditory SCRs were recorded via 13 leads and brain maps were realized on a concerto system. The results showed that SCRs were relatively stable at the temporal lobe, with a stable positivity (31.25 37.50 ms TP) followed by a negativity (62.50-72.50 ms TN). Variability was seen at other leads. The foci of maximum positivity and negativity were seen at the temporal cortex. The results suggest that there are multiple contributions to SCRs which partially overlap in time, and that auditory cortex contribute significantly more than other sources. SCRs also received auditory information in parallel from the brainstem. The focus of AM-evoked SCRs was seen located at temporal lobe while that of FM-evoked SCRs at temporal and frontal lobes. It suggests that intensity analysis is essentially completed at the temporal cortex whereas precise frequency discrimination relies on function of higher integrating centers. PMID- 10743154 TI - [Regeneration and functional restoration of vestibular hair cells in guinea pigs after gentamicin damage]. AB - To investigate the possibility of regeneration and functional restoration of vestibular hair cells after toxic destruction, gentamicin (150 mg/kg per day) was given to guinea pigs subcutaneously for 10 days to induce damage of vestibular hair cells. The control animals received the same volume of saline. At 2, 4, 12 and 24 weeks after the treatment, animals were sacrificed and their vestibular organs prepared for scanning electronic microscopic study. Change of the hair cell density was studied using quantitative analysis techniques. Electronystagmogram, induced with ice water, was taken before and at 2, 4, 12, and 24 weeks after the treatment. Immature stereocillia bundles were noticed in regions with heavy hair cell loss in the utricle and crista ampullaris 2 weeks after treatment and were still visible 24 weeks after the treatment. Hair cell density (number of hair cells/100 microns basilar membrane length) decreased significantly at 12 weeks after treatment as compared with the control (P < 0.01), and showed partial recovery at 24 weeks after treatment as compared with 12 weeks after treatment (P < 0.05) which was still below normall level (P < 0.05). Vmean showed significant reduction at 4 and 12 weeks after treatment (P < 0.01), and recovery at 24 weeks (P < 0.05), which was still below the normal level (P < 0.01). SEM results indicated the potential of repair of vestibular hair cells in guinea pigs after damage which may occur 24 weeks after the damage. Quantitative analysis of hair cell density further suggests that regeneration is the mechanism of repair. The regenerated hair cells seen in this study may play a role in the restoration of vestibular function. PMID- 10743156 TI - [Effect of perilymphatic fistula on distortion product otoacoustic emissions in guinea pigs]. AB - Perilymphatic fistula was induced in 11 healthy guinea pigs to study its effects on distortion product otoacoustic emissions (DPOAEs). DPOAE amplitudes decreased significantly right after formation of the fistula (P < 0.01), and recurred to near pretreatment level in animals whose fistula healed. The amplitude remained below pretreatment level 18 days after fistulization in those animals whose fistula failed to heal. Under light microscope, the organ of Corti was seen normal. The authors feel that DPOAEs may be helpful in detecting perilymphatic fistula. PMID- 10743158 TI - [Study on cochlear microphonic potentials obtained by adaptive filtering technique]. AB - In order to delete the contamination of acoustic stimulus artefact in average evoked cochlear microphonics by surface recording, an application of adaptive filtering technique for cancelling the stimulus artefact from the cochlear microphonics was proposed. The results demonstrated that in normal hearing ears, the cochlear microphonic potentials (CM) which was obtained by adaptive filtering technique was later than the ipsilateral response, whereas the AP response could be seen in the ipsilateral response to 2 kHz, 4 kHz and 8 kHz stimuli. In 31(77.5%) ears with profound sensorineural hearing loss and 29(27.5%) ears with moderate to severe sensorineural hearing loss, the CM was absent, but in the other ears the CM was as clear as in ears with normal hearing. It suggests that by this technique sensorineural hearing loss can be further differentiated into two kinds: neural hearing loss and sensory hearing loss. PMID- 10743157 TI - [Control of blood flow in nasal mucosa by electro-stimulation of sphenopalatine ganglion]. AB - The effects of control of blood flow on cat nasal mucosa by stimulating sphenopalatine ganglion (SPG) were studied using laser Doppler flowmetry in order to investigate the role of SPG. The study showed that the stimulation on one side of SPG could induce vasodilation, with increased blood flow, as well as protective reflexes on both sides. The reactivity depended on the stimulation intensity, frequency and duration. The stimulation on one side of postganglionic parasympathetic nerves, in which the stellate ganglion was resected, caused vasodilation only ipsilaterally. The results indicated that the vasodilation induced by stimulating SPG might be mediated via central reflex arc. The "reverse blood regulation" phenomenon was observed during stimulation of SPG as well. The mechanisms of "reverse blood regulation" induced by stimulating SPG need further investigation. PMID- 10743159 TI - [Surgical treatment of traumatic facial paralysis]. AB - The paper reviews 27 cases of traumatic facial paralysis undergone surgical treatment. Most of the cases showed injuries to the horizontal and perigeniculate segments of the facial nerve. All cases have been followed up for 6 months to 2 years. Complete recovery occurred in 20 of the cases and partial recovery in 7 cases (II in 2, III in 5). Trans-mastoid/attic approach was used which yielded exposure of the geniculate ganglion and distal part of the labyrinthine segment of the nerve. Indications and surgical approach were discussed. PMID- 10743160 TI - [Influence of intercellular adhesion molecule-1 transcription on nasal epithelial cell by airborne allergenic pollens]. AB - Eleven patients with Artemisia allergic rhinitis were evaluated. Among them 8 were studied during pollen season and 3 out of pollen season. Intercellular adhesion molecule-1(ICAM-1) was detected on nasal epithelial cells by reverse transcription polymerase chain reaction (RT-PCR). The results showed that ICAM-1 was detectable from all samples in the pollen season. However, during off-pollen season 2 of the 3 samples were negative, 1 was positive (who was also positive to house dust). It is suggested that ICAM-1 is detectable on nasal epithelial cells during exposure to specific allergen. PMID- 10743162 TI - [Comments on advances in otorhinolaryngology and its nomenclature]. PMID- 10743161 TI - [Rhinometry of 176 normal adults with acoustic rhinometer]. AB - RH-2000 acoustic rhinometer was used to measure nasal parameters in 176 normal adults. The results showed that nasal minimal cross-sectional area (NMCA) was 0.55 +/- 0.13 cm2 (x +/- s), and nasal cavity volume (NCV) 6 cm beyond the nostrils was 7.16 +/- 1.82 cm3. NMCA was located in the anterior edge of the inferior turbinate and the distance from the nostrils was 2.22 +/- 0.35 cm in 82.7% of the subjects; and the distance from the nostrils to the ostium internum was 0.79 +/- 0.25 cm in 17.3% of them. Our findings indicated that the narrowest segment of the nasal cavity was located in the anterior part, from the ostium internum to the anterior edge of the inferior turbinate. NMCA and NCV were larger in male than in female (P < 0.05). There was significant difference in NMCA and NCV between two sides(P < 0.001). The width of the nasal cavity between two sides might also be different. The measured NMCA and NCV was larger in subjects above 60 years old(P < 0.01, P < 0.05). Acoustic rhinometry is a new objective method to assess the geometry of nasal cavity. NMCA is a valuable objective mark to evaluate nasal patency. PMID- 10743163 TI - [Effect of eosinophil major basic protein on histamine release from nasal epithelial mast cells]. AB - Eosinophils and nasal epithelial mast cells (NEMCs) are closely related in nasal allergy. The activity of the eosinophil cationic protein, major basic protein (MBP) on histamine release from human NEMCs has not been documented. We examined the effects of MBP on histamine release from NEMCs. Nasal scrapings (containing NEMCs) from 59 patients with house dust mite nasal allergy were incubated with MBP or alkylated MBP. Specimens were also pre-incubated with MBP followed by incubation with house dust mite allergen (HDM) or preincubated with HDM followed by incubation with MBP or HDM. Histamine released in incubated media and remained in incubated mast cells was quantified by radio-immunoassay. MBP at the concentration of 10(-5) mol/L induced mild but statistically significant histamine release from the NEMCs. Preincubation with MBP at the concentration of 1.57 x 10(-5) mol/L produced slight inhibition of subsequent HDM-induced histamine release but preincubation with HDM showed no effect on the subsequent MBP-induced or HDM-induced histamine release from NEMCs. These results suggest that accumulated and activated eosinophils may release MBP resulting in clinical symptoms by interaction with NEMCs and inhibit of histamine release from NEMCs by subsequent interaction with allergen. PMID- 10743164 TI - [Determination of soluble interleukin-2 receptor levels in patients with allergic rhinitis]. AB - To explore the regulatory mechanism of the soluble interleukin-2 receptor (SIL 2R) in patient allergic rhinitis, SIL-2R levels in serum and nasal secretion were detected in 39 patients with allergic rhinitis, 19 patients with chronic rhinitis and 15 normal controls, The determination of SIL-2R was done by a sandwich ELISA method. The results showed that the SIL-2R concentrations in patients with allergic rhinitis were significantly higher than those of the controls (P < 0.001). These results suggest that SIL-2R may play an important role in allergic rhinitis. PMID- 10743165 TI - [Endoscopic and CT evaluation of three types of intranasal ethmoidectomies]. AB - To evaluate the curative effects and complications of conventional, microscopic and endoscopic intranasal ethmoidectomuies, 130 cases of sinusitis and polys (stage II) were operated on with intranasal ethmoidectomies. There were 40 conventional, 40 microscopic, and 50 endoscopic ethmoidectomies, followed-up with endoscopy and CT scanning. The results showed that 1. the relieve of symptoms in the endoscopic group(94.0%) was obviously better than the conventional (60.0%) and the microscopic groups (70.0%, P < 0.01): 2. the clinical cure rate in the endoscopic group (86.0%) was obviously higher than the conventional (40.0%) and the microscopic groups (52.5%, P < 0.01); the CT scans showed a normal ratio in the endoscopic group(82.0%) obviously higher than those in the conventional (22.5%) and microscopic groups (37.5%, P < 0.01); 3. the complication ratio in the endoscopic group(12.0%) was slightly higher than those in the conventional(5.0%) and the microscopic groups (2.5%, P > 0.05). The intranasal endoscopic ethmoidectomy was more successful than the conventional and microscopic ethmoidectomies, but attention should be paid to the possibility of complications. PMID- 10743166 TI - [Scanning electron microscopic changes of maxillary sinus mucosa before and after functional endoscopic sinus surgery]. AB - We attempted to evaluate postoperative maxillary sinus mucosal changes in 20 maxillary sinuses of 16 patients who underwent functional endoscopic sinus surgery (FESS). Ciliated area of the maxillary sinus mucosa and ostium of maxillary sinus were quantitatively observed by scanning electron microscope and image analysis before and after FESS. In the present study, the number of preoperative ciliated cells of maxillary sinus mucosa and ostium was significantly reduced, goblet cells and microvillous cells were increased, and squamous cell metaplasia observed in part of the maxillary sinuses. Many damaged ciliated cells tend to recover and ciliated area significantly increased at 6-12 months after the surgery. The results showed that the damaged ciliated epithelium could return to normal by the improvement of ventilation and drainage of the maxillary sinus following FESS. PMID- 10743167 TI - [CT diagnosis of maxillary sinus diseases]. AB - To evaluate the diagnostic value of CT scan in maxillary sinus diseases, CT findings in 100 cases of maxillary sinus diseases, which included 64 inflammatory diseases; 14 benign tumors; 17 malignant tumors and 5 fractures The result showed that the accordance rate between CT scan and operative diagnosis was 95.0%; between CT scan and pathology was 92.6%(88/95). It is concluded that CT scan showed a better visualization of maxillary sinus than X-ray tomogram. PMID- 10743168 TI - [Changes in the levels of artemisia pollen specific antibodies during rush immunotherapy for pollinosis]. AB - Artemisa pollen-specific immumoglobulins (IgG4, IgG and IgE) were evaluated by means of ELISA during rush immunotherapy in 49 patients with pollinosis. The results showed that six weeks after the beginning of immunotherapy, the levels of specific IgG4(P < 0.05) and IgG (P < 0.001) antibodies were significantly higher than those of patients prior to receiving rush immunotherapy. These results suggest that rush immunotherapy may result in a rapid increase in IgG antibody levels, and high specific IgG level may implicate a clinical improvement in allergic patients due to the rush immunotherapy. PMID- 10743169 TI - [Rapid functional plasticity of the neurons in the inferior colliculus and dorsal cochlear nucleus after brief exposure to intense pure tone]. AB - The changes of response properties were observed from the single neuron in inferior colliculus (IC) and dorsal cochlear nucleus (DCN) located in the area bordering the lesion created by intense pure tone. The frequencies of traumatizing tone were chosen higher than the characteristic frequency (CF) of the neurons and beyond their excitatory response area so that the traumatizing tone would not influence the excitatory input to the neurons under study. Different types of disinhibition effects were found in more than half of the neurons after the traumatizing tone. The results indicated a subcortical origination of cortical functional reorganization and the involvement of lateral inhibition in the formation of inhibitory neural networks in both IC and DCN. PMID- 10743170 TI - [Delayed damage of ionizing radiation on the inner ear]. AB - To investigate the feature and mechanism of delayed effect of ionizing radiation on the inner ear, the function and structure of the inner ear of the guinea pigs were observed during 8 month's period after exposure to fractional doses of gamma radiation (2 Gy per day) with a total dose of 60 Gy. Morphological study was performed using light microscopy, transmission and scanning electron microscopy while the function was measured with electrocochleography (ECochG) and electronystagmography (ENG). The results showed that the mean CAP response threshold was 35.67 +/- 6.78 dB before radiation, and 41.17 +/- 7.76 dB, 47.00 +/ 8.82 dB and 71.00 +/- 7.63 dB at 1st day, 3rd month and 8th months after completion of treatment, respectively. No significant change was demonstrated in the auditory responses at the 1st day of radiation (P > 0.05), but there was obvious hearing loss at 3rd and 8th month (P < 0.01). The nystagmus duration induced by ice water (ENG) was 40.00 +/- 5.44 seconds before radiation, 22.71 +/- 7.93 seconds at 8th month after radiation (P < 0.01). Histologic examination at 8th month after radiation revealed atrophy and degeneration of stria vascularis, a reduced number of capillaries, degeneration of endotheliocyte in vessels, outer hair cell and supporting cell of Corti's organ. The percentage of damaged hair cells in basal turn of the cochlea was significantly higher than those of the upper turn. Degeneration of vestibular hair cell was found. It is suggested that radiation from routine radiotherapy doses may result in a delayed impairment of the inner ear. The main mechanism of the radiation damage may be interference of oxygen supply and metabolism of the inner ear by radiation vascular lesion. PMID- 10743171 TI - [Histopathological studies of laryngeal allotransplantation in rats]. AB - The establishment of an animal model for laryngeal allotransplantation in rat has enabled us to correlate clinical impressions of laryngeal allograft rejection with histologic data. In this study we used both clinical and histopathologic criteria to define the sequence and time parameters of rejection in histoincompatible vascularized rat laryngeal allografts. Of the 31 rats receiving vascularized laryngeal allografts, 18 served as experimental group, in which allogeneic transplantations were performed from inbred SD to outbred Wistar rats, 13 rats used as controls receiving laryngeal histocompatible transplants. The clinical phenomena of rejection aas started at 3 days post-operation, characterized by noticeable dermal swelling and lymphadenectasis of the recipient's neck. At this time, the graft appeared slightly edematous. These changes progressed and were more pronounced by 1 week. Two weeks after operation, the recipients exhibited an inflammatory scar tissue encasing the graft, the normal laryngeal architecture of grafts was invisible. Gross clinical evidence of rejection was well correlated with pathologic findings. Three days following transplantation, there was a slight histologic difference between the experimental group and the control group. Five days later, the surface respiratory epithelium exhibited progressive squamoi metaplasia, and minor salivary glands were mostly atrophic, afterwards extensive loss of acini emerged, with predominant lymphocyte and polymorphonuclear leukocyte infiltration throughout the lamina propria, sub-mucosa and the laryngeal adventitia. The arteriolar walls showed fibrinoid changes, with reactive intimal proliferation, and thickening of vessel wall. Focal thrombosis were frequently found. By 2 weeks, the normal architecture of the laryngeal allografts had nearly completely been replaced by fibrovascular connective and granulation tissues. Definition of the time sequence and histopathology of rejection will allow future determination of the efficacy of various immunosuppressive agents. PMID- 10743172 TI - [Experimental study of anti-tumour effect of rhTNF alpha on the laryngeal squamous cell carcinoma]. AB - The anti-tumor effect of rhTNF alpha (recombination human tumor necrosis factor) with three different doses on laryngeal squamous cancer was investigated in nude mice. The results indicated that the higher and the middle doses of rhTNF alpha had significant antitumour effect against laryngeal cancer in BALB/C nude mice. The inhibitory rate of tumor growth was 54.4%(P < 0.05). The lower dose of rhTNF alpha also had antitumour effect but the cancer grew again when rhTNF alpha was stopped. Ultrastructural changes showed that the plasma membrane and the nucleus envelope of the tumor cell were destroyed and the number of lysosomes increased, fibroid material could be seen in the cancer capillary. PMID- 10743173 TI - [Selection of surgical methods for Meniere's disease]. AB - 119 cases (cars) of Meniere's disease operated on with different surgical methods were reevaluated with the criteria (Chinese ENT Association, 1996). 48 cases in second stage underwent endolymphatic sac shunt (ESS); 32 cases in third stage underwent scarpa ganglionectomy (SGN) and 12 cases in the same stage ESS; 16 cases in fourth stage vestibulocochlear neurectomy(VCN) and others ESS(7 cases) and SGN(4 cases). The results showed that 1. ESS successfully relieved vertigo in 79.2 per cent of the patients in second stage (reversible stage), and their hearing tended to stabilize(35.4%); 2. SGN was successful in controlling the vertigo of those patients in third stage who may retain some usable hearing; 3. Patients underwent VCN in fourth stage experienced a rapid elimination of tinnitus. The postoperative results were highly dependent on careful staging and selection of the surgical method. PMID- 10743174 TI - [Preliminary study on erythrocyte immunity during sensorineural hearing loss of unknown etiology]. AB - Red cell immune function was determined in 50 patients with sensorineural deafness and 30 healthy persons as control. The results showed that in patients with sensorineural deafness, the red cell C3b receptor (RBC-C3bR) rosette formation rate (15.10 +/- 1.66) was lowered markedly than controls (18.95 +/- 1.16, P < 0.001) and the red cell surface immune complex (RBC-IC 9.94 +/- 1.68) was elevated markedly than controls (7.44 +/- 1.17, P < 0.001). The results indicate that the red cell immunity in the patients with sensorineural deafness of unknown etiology is lowered. PMID- 10743175 TI - [Experimental and clinical studies on sternocleidomastoid myoperiosteal flap for laryngotracheal reconstruction]. AB - In order to explore the value of sternocleidomastoid myoperiosteal flap for laryngotracheal reconstruction, eight dogs were included in the experimental study. After resecting the anterior body of the cricoid cartilage and tracheal tissue, the sternocleidomastoid myoperiosteal flap was used to close the defect immediately. All dogs were killed at two, four, six and eight months after operation. It was found that the sternocleidomastoid myoperiosteal flap had incorporated completely into the defect. Histologic studies by light and scanning electron microscopies showed complete epithelialization with no granuloma formation on surface of the flap and apparent metaplastic new bone formed in the periosteum which was crucial to the stability of the air way and long-term success of the procedure. Five patients who underwent subglottic and tracheal reconstruction with sternocleidomastoid myoperiosteal flap were successfully decannuated and had normal exercise tolerance. The results indicate that the sternocleidomastoid myoperiosteal flap is an ideal transplant for laryngotracheal reconstruction. PMID- 10743176 TI - [Voice rehabilitation with double-valve voice prosthesis after laryngectomy]. AB - Between March 1993 and November 1996, 97 laryngectomized patients underwent voice rehabilitation with prostheses designed by ourselves. Of these patients, 84 were males and 13 females. The age ranged from 35 to 78 years. 20 cases had the prosthesis inserted during the laryngectomy operation (first stage), while other 77 cases were inserted after the laryngectomies (second stage). The results showed that all of the patients had clear voice with their own local dialect. 34 of them could even sing songs. 68 cases had been followed-up for 2 years. All could speak during the effective time of the prosthesis and no misswallow occurred. PMID- 10743177 TI - [Investigation of the infectious route of peritonsillar abscess]. AB - One hundred and thirty-one cases of peritonsillar abscess were studied to investigate the pathogenesis and the infectious route. The involved tonsils were resected in 83 patients. The surface of the resected tonsils was smooth and intact, neither ulceration nor pustular sinus was found on the surface of the upper part of these tonsils. There were some inflammatory and/or fibrotic minor salivary glands (Weber's glands) on the top of the quinsy tonsils. The glands adjacent to the tonsils of non-quinsy patients had normal appearance. It suggests that the origin of peritonsillar infection might be the Weber's glands rather than extension from acutely inflamed tonsil. PMID- 10743178 TI - [Advances in the study of the fruits of Rosa davurica Pall]. PMID- 10743179 TI - [Computer image analysis of 20 tiny medicinal seeds]. AB - Twenty tiny medicinal seeds were determined by computer image analysis. The diameter, perimeter, area, volume and irregular parameter of the seeds are presented. PMID- 10743180 TI - [Mineral medicinal material cinnabar in Hunan Province]. AB - Presenting the distribution characteristics and formation types of the mineral medicinal material cinnabar in Hunan Province, this paper expounds in detail the mineralogical features of the material through chemical analysis, X ray diffraction analysis etc. PMID- 10743181 TI - [Comparative study on the erect and fallen types of Platycodon grandiflorum (Jacq.) DC]. AB - Based on botanica characters, phenophase, yield and content of active constituents, the erect and fallen types of platycodon grandiflorum were compared. The results show that the comprehensive characters of erect type are better than those of fallen type and the erect type is therefore good for popularization. Some specific characters of the fallen type are better than those of the erect type, and the fallen type is therefore to be preserved as one of the germ plasm resources. PMID- 10743182 TI - [Optimal high-yield agronomic measures for Curcuma longa L]. AB - Regression method of orthogonal conic substitution with factors was employed to build up a tuber yield simulation model. Three main measures (sowing time, plant population, application rate of fertilizer) affecting the tuber yield were analyzed by the mathematical model of three unknown second order orthogonal rotative regression. The optimal agronomic measures were obtained. The results indicate that sowing time and plant population play a important role in raising the tuber yield. Sowing time is clearly interrelated with plant population, and likewise, plant population application rate of fertilizer. PMID- 10743183 TI - [Effect of processing on toxcicity and analgesia of radix Aconiti coreani]. AB - The experimental results showed that the Radix Aconiti Coreani and its processed products did not have remarkable toxic effect in p.o. But there was significant toxicity before processing and weakened toxicity after processing in i.p. In the skin test, the Radix Aconiti Coreani and its processed products showed no stimulating effect. The analgesic experiment showed that the Radix Aconiti Coreani and its steamed products could inhibit the pain. The products processed with bean curd and ginger-KAl(SO4)2.12H2O also could alleviate the pain induced by acetic acid, but no remarkable effect was observed in mice on the pain induced by hot plate. PMID- 10743184 TI - [Effect of processing and storage time on content of hesperidin in green tangerine peel]. AB - The contents of hesperidin in Green Tangerine Peel (Citrus reticulata), in its processed products and in commecial samples were determined by HPLC. The results indicate that the contents of hesperidin in the processed products from Chengdu and Huangyan are 14.28% and 10.75% less than shose of the crude drugs respectively. It has been found out that the content of hesperidin in Green Tangerine Peel before and after processing reduces with the passage of storage time. PMID- 10743185 TI - [Assay of terpenes in the leaves of Ginkgo biloba extract and its preparations by in situ fluorometric TLC]. AB - A new TLC method for the assay of terpene lactones in Ginkgo biloba extract and its preparations has been established by means of optimized development condition and post-chromatographic thermal fluorescence derivatization. Satisfactory results can be obtained through polynomial regression calibration. The data obtained by this method have been proved ten times higher in sensitivity than those obtained by HPLC-refracto-detector. PMID- 10743186 TI - [Chemical constituents of Pedicularis muscicola Maxim]. AB - From an ethanolic extract of whole plants of Pedicularis muscicola four glycosidic compounds were isolated. On the basis of chemical and spectal methods, their structurdes were determined to be one lignan glycosied and three iridoid glycosides: syringaresinol-4"-O-beta-D-glucoside (1), sesamoside (2), phloyoside II (3) and caryoptoside (4). All the four compounds were isolated from P. muscicola for the first time, and the compounds 2 and 3 were obtained for the first time from Pedicularis genus. PMID- 10743187 TI - [Chemical constitutents of Podocarpus imbricatus BI. (II)]. AB - From Podocarpus imbricatus five compounds were isolated and identified as 2"-O rhamnosylvitexin (I), hinokiflavone (II), ecdysterone (III), beta-sitosteryl heptadecoate (IV) and daucosterol(V) on the basis of spectral studies (UV, IR, 1HNMR, 13CNMR, MS) and chemical reactions. I, II, III, IV are obtained from this plant for the first time, and IV is obtained as beta-sitosteryl odd number carboxylic ester for the first time. PMID- 10743188 TI - [Chemical constituents of Cistanche tubulosa (Schenk) Wight]. AB - From the dry bulbs of Cistanche tubulosa six compounds were isolated and identified as beta-sitosterol, D-mannitol, daucosterol, succinic acid, D-glucose and D-fructose by spectral and chemical analyses. All of them were isolated from the plant for the first time. PMID- 10743189 TI - [Active constituents reducing side-effects of prednisone acetate in leaves of Panax ginseng C.A.Mey]. AB - The rise of total lipid, triglyceride and total cholesterol, and the drop of cortisol in serum induced by PA can be significantly inhibited by total ginsenosides in the leaves of Panax ginseng [GSL, 60 mg/(kg.d)]. From GSL ten compounds have been isolated and identified as ginsenoside-Rb2, -Rc, -Rd, -Re, Rg1 -F3, F2, -Rg2, 20(R)-Rg2 and -Rh1, respectively. Pharmacological study has proved ginsenoside-Re to be the chief active constituent of GSL. PMID- 10743190 TI - [Protective action of Acanthopanax giraldii harms polysaccharides against experimental liver injury in rats and mice]. AB - The experimental data indicate that Acanthopanax giraldii polysaccharides (AGP) significantly inhibit the elevation of serum ALT in CCl4-, D-galactosamine- and thioacetamide-intoxicated rats and mice. In CCl4-intoxicated mice, the retention of serum BSP was also reduced obviously by AGP. AGP was found to shorten the pentobarbital sleeping time in both normal and CCl4-intoxicated mice. PMID- 10743191 TI - [Pharmacokinetics of Caulis Erycibes injection in rabbits]. AB - The pharmacokinetics of single i.m. doses of Caulis Erycibes injection in ten rabbits was studied. The concentration of scopoletin-one of the effective components in Caulis Erycibes injection in serum was analyzed by high performance liquid chromotography with fluorescence monitor. In eight of ten rabbits a second peak was found on the serum concentration-time curve. The absorption of scopoletin was quick with Tmax 1 = 8.08 +/- 3.88 min, Cmax 1 = 145.45 +/- 47.65 ng/ml. The time to the second peak concentration (Tmax 2) was 2.45 +/- 1.79 h, Cmax 2 = 48.66 +/- 41.66 ng/ml. The other parameters were as follows: Ke = 0.56 +/- 0.37 h-1, T1/2 = 1.81 +/- 1.14 h, MRT = 156.61 +/- 98.41, CL/F = 220.54 +/- 109.35 ml/min, Vd/F = 30.49 +/- 19.34 L, AUC = 8.52 +/- 5.15 micrograms.min/ml. The reason for the appearance of double peaks was discussed. PMID- 10743192 TI - [Effect of Yang-warming and Qi-tonifying natural products on neuroendocrine of deficiency-cold rats]. AB - The rat fed with the compound drug Zhi Mu and Shi Gao was made into an animal model of deficiency cold syndrome. The deficiency cold state was kept for more than 7 days after stopping the drug. So the animal model is good for studing the therapeutic effect of products of heat nature. After 3 therapeutic days with Fu Jiang and Shen Qi compounds, the D beta H activity in serum, cortisone in adrenal gland and NE, DA amount in brain were increased, but serotonin was decreased, and after 7 days the effect was increased significantly. Both compounds could excite the sympathetic nerve and endocrine system in the peripheral and central nervous systems. Shen Qi compound acts quickly, but does not last as long as Fu Jiang. The effect will be better if the two compounds are used together. PMID- 10743193 TI - [Microscopic identification of the powder of roots of genus Adenophora: II. The roots of Sect. Remotiflorae and Sect. Adenophora]. AB - This paper deals with the microscopic characteristics of the powder of the roots of 11 species and subspecies of Genus Adenophora. The results show that Sect. Basiphyllae, Sect. Pachydiscus, Sect. Remotiflorae and Sect. Adenophora can be identified based on the amount and thickness of the wall as well as the characteristics of the pit of sclerified cork cells, and 29 species, subspecies and varieties can be identified based on the amount, shape and thickness of the walls of the sclerified cork cells, the diameter and pit of the vessels, the number and shape of the secretion of the laticifers, and the number of inulin crystals. PMID- 10743194 TI - [Identification of the Chinese herbs of Indigoferae L. by RAPD analysis]. AB - Eight Chinese herbs of Indigoferae were identified randomly amplified polymorphic DNA by (RAPD) analysis. The results has shown that RAPD analysis is a new and effective method for the identification of Chinese herbs. PMID- 10743195 TI - [Treatment of hard seeds of some plants with sulphuric acid]. AB - By soaking seed in sulphuric acid, the hard seeds of some medicinal plants featuring low germination rate can be softened so as to acclerate the germination. Some relevant physiological processes are analysed in this paper. The study provides a theoretical basis for the artificial raising of medicinal plants in the field. PMID- 10743196 TI - [Effect of cultivating measures on the tuber yield of Curcuma longa L]. AB - The effect of cultivating measures on the tuber yield of Curcuma longa was observed. dibble planting has been proved move effective in raising the tuber yield than drill culture, and the dibble planting quantity has no effect on the tuber. The rational producing area, soil, seed tuber and so on were selected according to the experiments. PMID- 10743197 TI - [Quality of processed samples of Daphne genkwa Sieb. et Zucc]. AB - Content determination has been made for the genkwanin, yuanhuacine, agueous and ethanolic extracts, total ash and acid-insoluble ash in thirteen samples of Daphne genkwa (processed by the authors, processed in pilot plant and marketed in seven regions), as well as for the acetic acid in thirteen vinegar-processed samples of Daphne genkwa. Studies have also been conducted on the characteristics, microscopic and physico-chemical identification of the herb, so as to provide objective, scientific criteria for quality control of the herb in small slices for medical use. PMID- 10743198 TI - [Quality of baked fructus Aurantii immaturus with bran of different storage periods]. AB - Studies have been made on baked Fructus Auantii Immaturus with bran of different storage periods by physico-chemical and TLC identification methods. The contents of synephrine were determined by HPLC, and the volatile oil and soaked constituents were analysed. The experimental result shows that the contents of samples get reduced as the storage is prolonged. PMID- 10743199 TI - [Preliminay studies on polyvinyl alcohol gel and its transderman therapeutic system]. AB - By pouring-cooling method, five kinds of hydrophilic gels (starch, agar, gelatin, CMC-Na and PVA) were prepared. The characteristics of preparation, tenacity and elasticiey were compared. Taking the PVA gel which features better characteristics as the carrier a plaster of verapamine hydrochloride and a traditional Chinese antitussive and expectorant in the form of plaster were prepared. The behavior of drug-releasing in vitro and curative effect were preliminarily studied. The results show that the PVA gel may be a fine carrier for transdermal therapeutic system. PMID- 10743200 TI - [Experimental studies on the quality control of Qing'e pills]. AB - TLC was used in the qualitative study of Qing'e Pills. The contents of psoralen and isopsoralen in these pills were determined by TLC-densitometry. The proposed method can be used to control the quality of Qing'e Pills. PMID- 10743202 TI - [Chemical constituents of Aster poliothamnus Diels]. AB - Seven compounds were isolated from Aster poliothamnus. By means of spectral evidences and crystal analysis, their structures were elucidated as beta sitosterol, beta-amyrin, daucosterol, spinasterol, stigmasterol, dammara-20, 24 dien-3 beta-ol and epifriedlinol. PMID- 10743201 TI - [Isolation and structure elucidation of cirensenosides Q and R]. AB - Two new saponins (cirensenoside Q and R) were isolated from the leaves of Oplopanax elatus. Their structures were elucidated as follows: Q, gypsogenin 3-O beta-glucopyranosyl-28-O-alpha-L-rhamnopyranosyl(1-->4)-beta-D- glucopyranosyl(1- >6)-beta-D-glucopyranoside; R, 3 beta-hydroxylup-20(29)-ene-23-al-28-oic acid 3-O beta-D-glucopyranosyl-28-O-alpha-L-rhamnopyranosyl(1-->4)-beta-D- glucopyranosyl(1-->6)-beta-D-glucopyranoside, respectively. PMID- 10743203 TI - [Chemical constituentes of Teucrium bidentatum Hemsl]. AB - Two diterpenes were isolated from the herb Teucrium bidentatum and identified as teucvin and teupernin A on the basis of chemical properties and spectral data. They were obtained from T. bidentatum for the first time. PMID- 10743204 TI - [Chemical constituents of the leaves of Ginkgo biloba]. AB - Four compounds were isolated from ethyl acetate extract of the leaves of Ginkgo biloba. Compounds II, III, IV were identified as ginkgolides A, B and C respectively, Compound I, which was obtained from the leaves of G. biloba for the first time, was identified as 3,3'-dimethoxy-4,4'-dihydroxy-stilbene by spectroscopic methods. PMID- 10743205 TI - [Determination of chrysophanol in semen Cassiae by HPLC]. AB - A HPLC method for the determination of chrysophanol in Semen Cassiae was investigated. The result showed that this method was simple, specific and accurate. The recovery was 99.0% and relative standard deviation was 1.8%. The method is useful for the quality control of this medicinal meaterial. PMID- 10743206 TI - [Mechanism of protective action of Phyllanthus urinaria L. against injuries of liver cells]. AB - It has been found out that the carbon tetrachloride (CCl4)-induced increase of serum glutamic-pyruvic transaminase (ALT) and elevation of MDA in liver of mice are significantly lowered by Phyllanthus urinaria in vivo, and the coincubation of isolated rat hepatocytes with Phyllanthus urinaria in vitro significantly inhibits CCl4-induced decrease of mobility of membrane of liver cells and increase of intracellular free Ca2+ ([Ca2+]i) concentrations of liver cells. These results suggest that the anti-lipid peroxidation effect and protective action of membrane of Phyllanthus urinaria may be related to its protective action against CCl4-induced liver injuries. PMID- 10743207 TI - [Anticarcinogenic effect and hormonal effect of Cordyceps militaris Link]. AB - Cordyceps militaris (CML) has been found good for inhibiting the growth of tumor, prolonging the survival period of mice implanted with S180, inhibiting the growth and metastasis of Lewis pneumonic cancer in the implanted mice, increasing the plasm content of cortisol and testosterone in normal rats, and elevating the weight of sexual organs in normal and castrated rats. CML exerts a malehormone like effect. PMID- 10743208 TI - [Effect of chicken bile on heart and blood pressure in animals]. AB - In this paper, the effect of chicken bile on heart and blood pressure in animals was studied. The result shows that chicken bile(50 mg/kg, i.v.) can significantly reduce the normal blood pressure and hypertension induced by ephedrine in white rats. Chicken bile(1000 ppm) markedly inhibits myocardial contractive force, frequency and cardiac output of the isolated frog heart. But no influence was observed on the ECG in rabbits after administration of the bile (50 mg/kg, i.v.). PMID- 10743209 TI - [Identification of herba taraxaci and its adulterants in Hong Kong market by DNA fingerprinting with random primed PCR]. AB - DNA fingerprinting for Taraxacum mongolicum and its adulterants of six species of Compositae was demonstrated with random-primed polymerase chain reaction (PCR) including arbitrarily-primed PCR (AP-PCR) and random-amplified polymorphic DNA(RAPD). Distinctive genomic fingerprints from DNA of Taraxacum mongolicum and its adulterants were generated with two long (20-24 mer) and one short (10 mer) random-chosen primers Taraxacum mongolicum can be distinguished from six adulterants according to the banding patterns of their amplified DNA on agarose gels. Moreover, that Taraxacum mongolicum and its adulterant are unrelated. The Similarity Indexes values of the genomic fingerprints. Results showed AP-PCR and RAPD might be used for identifying Chinese drugs. PMID- 10743210 TI - [Effect of light colours and illumination intensity on the sexual behaviour of Hepialus gonggaensis Fu et Huang]. AB - The sexual behaviour of Hepialus gonggaensis has close relations with ecological environment and varies with its ecotypes. The more intense the illumination, the faster the sexual drive of Hepialus gonggaensis begins and the longer its sexual intercourse continues, light colours have a bearing on its sexual intercourse time. This study has provided an evidence for the artificial cultivation of Hepialus gonggaensis. PMID- 10743211 TI - [Correlativity among biological characteristics of Gardenia jasminoides Eills]. AB - The relationship among certain biological characteristics, fruit yield and quality of G. jasminoides was studied, and the special features, closely correlateied with fruit production and quality, as well as with the steadiness of heredity were found out. According to these features the G. jasminoides produced in Sichuan province has been divided into 6 types. PMID- 10743212 TI - [Effect of processing on chemical composition and medical activity of rhizoma Anemarrhenae]. AB - A comparative study was conducted on the chemical composition and medical activity of different kinds of processed drug of Rhizoma Anemarrhenae. It was found that the content of effective components of zhimu was affected obviously by processing: the rhizome should not be barked when used as drug. It was also observed that different kind of processed drug should be used in line with different particular circumstance. PMID- 10743213 TI - [Pharmacological of cortex moutan and core]. AB - Comparative studies have been made on the medicinal efficacy of core and cortex moutan. The result shows: no marked difference between the cortex and core in reducing inflammation, promoting thrombocyte coagulation, inhibiting convulsion, depressing blood pressure and strengthening antiseptic activity. The toxicity of the core is 38.5 percent of the cortex. This study provides ascientific evidence that the core can be used as medicine. PMID- 10743214 TI - [Sample pretreatment method for assay of chlorogenic acid in Chinese medicinal preparations]. AB - This paper introduces a sample pretreatment method for assaying chlorogenic acid D101-column separation-precipitation method. The method features thorough removal of impurities, complete separation of chlorogenic acid and simple operational procedures. The experimental result of four samples from three preparations shows that this method is good in reliability and applicability. PMID- 10743215 TI - [Chemical constituents of seeds of Camellia sinensis var. assamica]. AB - Five compounds were isolated from the acid-hydrolytic products of n-butanol extract and ethanol extract of the seeds of Camellia sinensis var. assamica. Their structures were elucidated by chemical and spectroscopic analyses as octacosane, three new acyl-sapogenins: 22-O-angeloyl theasapogenol B, 22-O angeloyl theasapogenol E and 22-O-angeloyl theasapogenol A, and naringenin respectively. PMID- 10743216 TI - [Chemical constituents of fructus Amomi]. AB - Seven compounds have been isolated from Fructus Amomi. They were identified as bornyl acetate, camphor, borneol, beta-sitosterol, vanillic acid, stearic acid and palmitic acid. The vanillic acid was found in this medicine for the first time. Fifty-seven components were identified from the volatile oil of Fructus Amomi on the basis of GC-MS analysis. Eight components in the volatile oil over 1% in content are bornyl acetate, camphor, borneol, limonene, camphene, myrecene, carene-3 and alpha-terpeneol. PMID- 10743217 TI - [Determination of maesopsin in shengdeng (Rhamnella gilgitica Mansf. et Melch) by HPLC]. AB - A reversed-phase HPLC method was developed for the determination of maesopsin, a fliavonoid, in the xylem parts of the traditional Tibetan medicinal herb Shengdeng (Rhamnella gilgitica). An ODS column was used and the mobile phase was methanol-water (28:27) with 0.05 mol/L KH2PO4 (pH4). The detection wavelength was UV 290 nm. The recovery was 98.9% (RSD = 2.1%, n = 6) and the content of maesopsin in Shengdeng was 1.02% (RSD = 0.52%, n = 3). PMID- 10743218 TI - [Determination of camptothecine in the fruit of Camptotheca acuminata Decne. by RP-HPLC]. AB - A RP-HPLC method wsa developed for the determination of camptothecine in the fruit of Camptotheca acuminata, using YWG-C18 column and methanol-water(6:4) as the mobile phase with UV detection at 254 nm. The average recovery was 98.67% and RSD 1.71%. PMID- 10743219 TI - [Preventive and therapeutic effects of radix Salviae miltiorrhizae on glycerol induced acute renal failure in rats]. AB - Three substances obtained from Radix Salviae Miltiorrhizae were administered to rats with acute renal failure (ARF) induced by intramuscular injection of glycerol. The rats treated with the substances had significantly lower serum urea and creatinine levels than the untreated animals. In the treated group, the survival percentage was higher than that in the untreated group. Examination of kidneys showed that the treated group had significantly lighter renal damage than the untreated group. These results suggest that the three substances exert preventive and therapeutic effects on the ARF in rats and the effects are probably the result of adenosine antagonism. PMID- 10743220 TI - [Preventive effect of gubao on hydrocortisone-induced osteoporosis in rats]. AB - The antagonistic effect of Gubao (consisting of Herba Epimedii, Radix Astragali, etc.) on hydrocortisone-induced osteoporosis in rats was observed in quantitative bone histomorphometry study with stereological method. The results of experiment show that Gubao helps to reduce trbecular bone resorption and increase bone formation in ratsuith hydrocortisone-induced osteoporosis. It is suggested that Gubao is useful in preventing glucocorticoid-induced osteoporosis. PMID- 10743221 TI - [Hemostatic effect of Dracaena cochinensis (Lour.) S.C. Chen]. AB - Dracaena cochinensis helps to decrease the clotting time in mice, the recalcification time and eugloblinlysis time (ELT) of plasma in rabbits, but has no effect on the prothrombin time in rabbits. PMID- 10743222 TI - [Antitussive expectorant and antiasthmatic actions of Cynanchum Komarovii Ai. Iljinski]. AB - The water and ethanol extracts of Cynanchum Komaroviiu could obviously relieve the cough caused by the irritation of ammonia water in mice, increase secretion of the respiratory tract and promote expectoration. They could also alleviate the asthma induced by histamine and acetylcholine chloride in guinea pigs. PMID- 10743223 TI - [Discussion on application of traditional Chinese analgesic medicine]. AB - When using traditional chinese analgesic medicines for aching-syndromes, it is advisable to choose herbal analgesics on the basis of diseases degree of seriousness of the root causes and symptoms, by the principle of treating both the root causes and symptoms, or treating symptoms under urgency. It is also advisable to put emphasis on mediating qi (vital energy) and blood, select herbal drugs according to different positions of pain and make use of compatibility to enlarge the working scope of traditional chinese analgesic medicines. PMID- 10743224 TI - [Current status and prospect of the study of Lonicera japonica Thunb]. PMID- 10743225 TI - [Nucleotide sequence analysis of a species specific probe by an inserted fragment from recombinant plasmid pCX7 of L. interrogans sensu stricto serovar lai]. AB - The etiological agents of leptospirosis are the pathogenic leptospires (L. interrogans sensu lato) which can be divided into 223 serovars organized into 23 serogroups. The serovar remains the basic taxon, but serotyping may now be accomplished and recognized by acceptable methods. Complementary molecular approaches are being used extensively to assess genetic relatedness amongst leptospires with restriction endonuclese analysis (REA), pulse field gel electrophoresis (PFGE) and DNA-DNA hybridization as well established tools. However, the method is cumbersome and unsuitable for routine application. To develop a sensitive and specific method for identification of pathogenic leptospires, a genomic library of L. interrogans sensu stricto serovar lai was constructed with the plasmid vector pUC9. A recombinant plasmid, designated pCX7 which has homologous fragment of pathogenic leptospires was screened from the bank. pCX7 could recognize pathogenic leptospiral DNA fragment 1.7 kb of strain 017 without cross hybridization to nonpathogenic leptospiral DNA. Inserted fragment of pCX7 DNA sequencing was performed by Dr. Yan Zhengxin (Max-Plank Institut fur Biology, Tubingen, Germany). Insert fragment was cloned into pBluescript and sequenced by using ABI(Applied Bio. Systems, Model 373A). Nucleotide sequences were analyzed by Dr. Xiao Jianguo (Texas University Medical School and School of Public Health, Center for Infectious Diseases) using a suit of computer program (NIH). One open reading frame of 306 nucleotids were identified. There were identifiable initiation codons, terminators, pribnow box and sextama box within the sequenced regions. These results further confirmed that the little homology between L. interrogans sensu strito and L. borgpeterseni serovar javanica, L. inadai serovar ranarun and serovar manhao (L. genomospecies 2), L. biflexa serovar patoc, L. illini. pCX7 DNA probe could provide a base for identification and classification of leptospires. PMID- 10743226 TI - [Cloning, characterization and expression in E. coli of the flagellin gene from Leptospira interrogans Serovar lai]. AB - To obtain a large amount of recombinant endoflagellin protein of leptospsira, we have cloned the fla B gene from Leptospira interrogans serovar lai strain 017, which encodes the core flagellar protein. The entire open reading frame was amplified by polymerase chain reaction, which was digested with endonucleases Sal I and Cla I, then was orientationally cloned into the expression vector, pT7-7. The recombinant plasmid was transformed into Escherichia coli JM109 (DE3) for expression. After induction with IPTG, a 34 kd expression protein was detected by SDS-PAGE. The amount of recombinant protein was estimated as 11.8% of the whole bacterial lysate proteins. Probed with insert fragments, Southern blot analysis indicated that possibly two gene related to fla B were present in the genome of Leptospira interrogans serovar lai stain 017. Western blot analysis showed that the 34 kd expression protein reacting with rabbit polyclonal antiserum raised against leptospira endflagellin. PMID- 10743227 TI - [Mutation analysis of the p15 gene exon 2 in human primary hepatocarcinoma]. AB - To investigate the role p15 gene plays in the pathogenesis of human primary hepatocarcinoma, 35 human hepatocarcinomas, 35 cases of adjacent non-cancerous liver cirrhosis and the blood cells of 10 normal human were analyzed for somatic mutation in p15 gene with PCR-SSCP. One case of adjacent non-cancerous liver cirrhosis showed abnormal migration single strand. In the hepatocarcinomas and in the other cases of adjacent non-cancerous liver cirrhosis, no mutation was found. Cloning and sequencing of the amplified abnormal migration single strand DNA revealed that it contained a wild type exon 2 of p15 gene in 345 bp length. The results indicate that the inaction of p15 gene by point mutation is a very uncommon event in human hepatocarcinoma. PMID- 10743228 TI - [Slot blot analysis of metastasis suppression gene nm23 expression in human lung cancer]. AB - To investigate the role of metastasis suppression gene (nm23) in the development and progression of human lung cancer, the mRNA expressions of nm23-H1 and nm23-H2 genes in a series of pulmonary tissues collected at various sites and with different properties were studied with slot blot hybridization. According to the observations on the normal pulmonary tissues, benign lesions, para- and non cancer tissues, primary cancer and metastatic lymph nodes, there was a tendency of mRNA expression reduction of nm23-H1 and nm23-H2 genes. Among them, nm23-H2 mRNA expressions in lung cancer tissues were significantly decreased when compared with normal pulmonary tissues (P < 0.05), and nm23-H1 and nm23-H2 gene expressions in metastatic lymph nodes were both reduced, compared with those in normal pulmonary tissues (P < 0.05). There was no significant correlation between nm23 gene expression and lymph node metastasis in lung cancer. The results implied that reduction of nm23 gene expression might be associated with the development of lung cancer, but no evidence of metastasis suppression by nm23 gene was revealed in this study. PMID- 10743229 TI - [Effects of the new compound-XW630 on osteoblast]. AB - This study aimed to observe the influence of the new compound-XW630 on the proliferation of osteoblast, ALP action, and the forming of osteoclast. The MTT method and the alkaline phosphatase method were adopted to investigate the influence of XW630 on ALP action and the proliferation of osteoblast cultured from grown rat's skull. The Tartrate-resistant acid phosphatase dyeing method was used to observe the influence of XW630 on forming of cultured osteoclast in vitro. The result showed that, when XW630 concentration was 10(-6)-10(-8) mol/L, it obviously enhanced the proliferation of osteoblast and improved the activity of ALP, and it evidently provented PTH from stimulating the forming of osteoclast. It is concluded that XW630 is obviously effective for stimulating the proliferation of cultivated osteoblast in vitro and for inhibiting the forming of osteoclast. PMID- 10743230 TI - [Change of level of basic fibroblast growth factor in serum of rats with chronic hypoxic pulmonary hypertension]. AB - To evaluate the role of basic fibroblast growth factor (bFGF) in the development of hypoxic pulmonary hypertension, 30 wistar rats were divided into hypoxic group and control group. Rat models with chronic hypoxia induced pulmonary hypertension were established. The mean pulmonary artery pressure and the ratio of free wall of the right ventricular weight to the weight of the left ventricle septum were measured. The Level of bFGF in serum of rats with chronic hypoxic pulmonary hypertension was determined quantitatively by ELISA. Expresion of bFGF mRNA in lung, heart, brain and kidney of rats was observed by in situ hybridization using digoxigenin-labeled oligonucleotide probe. The result showed that the mPAP, the ratio of free wall of the right ventricular weight to the weight of the left ventricle plus septum, and the concentration of bFGF in serum were 3.96 +/- 0.47 kPa, 0.33% +/- 0.023%, 35.9 +/- 23.5 pg.ml-1 in rats exposed to hypoxia for 3 weeks, respectively; all were significantly higher than those in control group. Expression of bFGF mRNA in the wall pulmonary arterioles of rats with hypoxia was stronger than that of control group, but changes of expression of bFGF mRNA in heart, brain and kindey of rats were not observed. These suggest that bFGF takes part in reconstruction of pulmonary arterioles wall in chronic hypoxic pulmonary hypertension and bFGF in serum may be one of mechanism of modulating the structure remodeling of pulmonary arterioles. PMID- 10743231 TI - [The change of plasma level of endothelin in patients with acute attack of asthma]. AB - In order to assess the interaction of endothelin (ET) and acute attack of asthma, the plasma levels of ET in 23 patients with acute attack of asthma and 12 normal controls were measured by radioimmunoassay. The partial pressure of oxygen in arterial blood (PaO2) and peak flow (PEF) were measured synchronously. The results showed that the plasma ET level in asthmatic patients (89.2 +/- 8.4 ng/L) was much higher than that in normal controls (52.2 +/- 6.5 ng/L), P < 0.001. There was a significant negative correlation between the plasma level of ET and the PaO2 (r = -0.7893, P < 0.01) or PEF (r = -0.7124, P < 0.01). The results suggest that ET may play a role in acute attack of asthma, and its level may stand for the degree of the disease. PMID- 10743233 TI - [Research on bending strength and fracture toughness of alumina-glass composite]. AB - To develop a new ceramic material that can be machined and infiltrated with glass, a porous alumina blank sintered at 1350 degrees C was made of high purity, super fine alpha-alumina and then infiltrated with glass in this study. The density, bending strength and fracture toughness of the partially sintered alumina and alumina-glass composite were determined. The results indicated that the porous alumina density was 2.12 g/cm3, the three point bending strength 102 MPa, the fracture toughness 1.61 MPam1/2; that the alumina-glass composite density was 3.85 g/cm3, the three point bending strength 385 MPa, and the fracture toughness 4.05 MPam1/2. By SEM and EDXA analysis, lanthanum boroslicate glass was completely infiltrated into the 3 mm thick porous alumina blank for 6 h at 1150 degrees C. These suggest that the new developed alumina blank is suited for clinical use. PMID- 10743232 TI - [The inhibitory effect of wild-type p53 gene on the growth and proliferation of lingual squamous cell carcinoma cell line Tca8113 in nude mice]. AB - The aim of this study was to observe the growth and proliferation of the Tca8113 cell line in nude mice after the wild-type p53 gene has been transfered by electroporation. The experiment consisted of four groups: the wild-type p53 gene, blank control plasmid, the mutant p53 gene, and blank control into which no gene was transfered. After the gene was transfected, the cells were planted into 16 nude mice respectively. When the tumor formed, the nude mice were killed and the tumors were studied by regular pathology, stereology and immunohistochemistry. The results showed that in the wild-type p53 gene group, the tumors weighed less heavy, the pathological mitosis decreased markedly, and stereologically, the ratio of the area of necrosis to the total tumorous area was smaller. Similarly, the count of the proliferation cell nuclear antigen in non-necrotic area went down. The above findings were statistically significant (P < 0.05). These indicate that when the tumor cells re-obtain the well functional exogenic wild type p53 gene, the gene expresses and produces new negative cell-growth regulator to replace the abnormal regulation system partially, then the growth and proliferation of the tumors can be inhibited; therefore, it is suggested that the p53 tumor suppressor gene may possibly be used as a gene therapy agent to treat oral squamous cell carcinoma. PMID- 10743234 TI - [Effect of crystalling temperatures on microstructure of dental glass-ceramic]. AB - To know well the effect of crystalling temperature on the microstructure of dental glass-ceramic, the parameters of microstructure such as the type of crystal, crystal percentage, crystal size and crystal form were investigated by altering crystalling temperatures and using scanning electronic microscope, energy dispersive X-ray analysis and X-ray diffraction techniques. The results showed that the crystalline phase was identified to be tetrasilicic fluromica, and the promotion of crystalling temperature contributed to the increased crystal percentage, crystal size and the transformation of crystal form from 830 degrees C to 950 degrees C. PMID- 10743235 TI - [An immunohistochemical study of the proliferating activity of ameloblastic fibroma and ameloblastic fibrosarcoma]. AB - Six cases of ameloblastic fibroma (AF) and three cases of ameloblastic fibrosarcoma (AFS) were immunohistochemically investigated with a proliferating cell nuclear antigen (PCNA) monoclone antibody. Data on the PCNA labeling index (LI) of both epithelial and mesenchymal components were analyzed. The results showed that the PCNA LI of mesenchymal component of AFS (40.8%) was significantly higher than that of AF (3.2%) (P < 0.01) and that of the epithelial component of AFS (5.3%) was significantly lower than that of the mesenchymal one of AFS (P < 0.01). The results suggested that PCNA LI be a useful marker for the differentiation diagnosis of AF and AFS. PMID- 10743236 TI - [Effect of Ox-LDL on cell cycling phases and PCNA, P53, P27 and c-erbB-2 expression in cultured human arterial smooth muscle cells]. AB - It was found in our previous study that oxidative modification LDL (Ox-LDL) could stimulate the proliferation of cultured human arterial smooth muscle cells (SMC). Yet, the mechanism responsible for the SMC proliferation induced by Ox-LDL is not clear. Proliferating cell nuclear antiger (PCNA), P53 and P27 are the key regulatory factors of cell replication. In order to observe the effects of Ox-LDL on cell cycling phase and PCNA, P53, P27 and c-erb B-2 expression in SMC, we used the flow cytometric method in the present study on the proliferation of cultured human SMC induced by Ox-LDL. The results showed a relation between the Ox-LDL mediated SMC proliferation and the cycling phase shifting. The relative number of S phase cells in the Ox-LDL group was higher than that of the control group (22.9% vs 15.7%). Ox-LDL mediated SMC proliferation was accompanied with the increasing expression of PCNA. The percentage of specific PCNA positive FITC cells in the Ox-LDL group was significantly higher than that of the control group (12.6% vs 6.5%). PMA, an activitor of protein kinase C (PKC), stimulated SMC proliferation and increased the PCNA exression in cultured SMC, while the PKC inhibitor, F109203X, significantly decreased the PCNA expression in SMC (PCNA positive cells 13.4% vs 0.4%). No changes were observed in the expression of P53, P27 and c-erb B-2 in the cultured proliferating SMC induced by Ox-LDL. In all, the results suggest that the OX-LDL mediated SMC proliferation is related to increasing S Phase Cells and involved in the PCNA expression which might undergo the PKC cellular signal transduction pathway. PMID- 10743237 TI - [Expression of P62c-myc protein in breast cancer and its clinical significance]. AB - The objective of this study was to determine the clinical significance of P62c myc protein expression in breast cancer. P62c-myc protein was detected in 107 patients with breast cancer by immunohistochemical techniques (LSAB). The results showed that the positive rate of P62c-myc protein expression was 63.55% (68/107). The overexpression of P62c-myc protein related negatively with survival. 94.00% of the cases with overexpression of P62c-myc protein survived < or = 5 years, 65.00% survived > 5 years-< 10 years, and 21.62% survived > or = 10 years. There were significant associations of P62c-myc expression with advance clinical stage, high histological grade, and positive axillary node status in breast cancers. All of these findings suggested that overexpression of P62c-myc might be an important prognostic factor, and the detection of P62c-myc protein might be arranged as a regular pathological examination in the cases of breast cancer. PMID- 10743238 TI - [Effect of vasectomy on apoptosis in spermatogenic cells of the male rat]. AB - To explore the effect of vasectomy on apoptosis of spermatogenic cells, we have detected apoptosis in the testis of vasectomized and sham-operated adult rats within a period of 12 postoperative weeks by means of flow cytometry and TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuride triphosphate nick-end labeling) method. Flow cytometry found that, in vasectomized rat, the sub-haploid peak was higher and the percentage of cells with sub-haploid was increased. The total number of positive TUNEL cells in the vasectomized group was greater than that in the sham-operated group (P < 0.001). Compared with the same time-phased sham-operated subgroups, the number of apoptotic spermatogonia and primary spermatocytes in the testes of the subgroups 2 weeks, 6 weeks and 10 weeks after vasectomy as well as the number of spermatogonia of the subgroup 12 weeks after vasectomy were significantly higher (P < 0.05). These findings demonstrate that vasectomy could enhance the apoptosis of spermatogenic cells. PMID- 10743239 TI - [MMC conditioning regimen (Melphalan, MeCCNU and cyclophosphamide) followed by allogeneic peripheral blood stem cells transplantation for chronic myeloid leukemia]. AB - This paper reports 3 cases of allogeneic peripheral blood stem cell transplantation (Allo-PBSCT) for the patients with chronic myeloid leukemia (CML). The patients received MMC preparative regimen with high dose chemotherapy (Melphalan 170 mg/m2, p.o. on Day-5, MeCCNU 400 mg/m2, p.o. on Day-4, and Cyclophosphomide 60 mg/kg/day, i.v. on Days-3 and -2). The HLA-identical sibling donors received filgrastim (rhG-CSF) for mobilization at a dose of 300 micrograms/day for 6 days. Leukaphereses were done at the 6th day of mobilization. A median of 8000 ml (2 times total blood volume) of blood was processed the collecting: 2.5-4.5 x 10(8)/kg MNC, 12.8-20.0 x 10(6)/kg CD34+ cells (including 4.8-7.5 x 10(6)/kg CD34+CD33-, 8.0-13.0 x 10(6)/kg CD34+CD33+), and 3.5-4.3 x 10(5)/kg CFU-GM. Cyclosporin A and methotrexate were used for GVHD prophylaxis. Hematopoitic function recovered as for 14-20 days to > 0.5 x 10(9)/L of neutrophil count, and for 16-34 days to > 20 x 10(9)/L of platelet count. At day + 100, chromosome analysis of bone marrow cells showed that complete chimera without ph1 positive chromosome in Cases 1 and 3, and a partial chimera with 73% donor karyotype in Case 2. All patients now are in disease free survival. No episode of acute graft versus host disease (GVHD) developed. It was concluded that HLA matched sibling allogeneic PBSCT result in rapid hematopoitic reconstitution and the MMC conditioning regimen is effective both in leukemic cells eradication and in immunosuppression for stem cells engraftment, and the drug related toxicity could be tolerated by patients. PMID- 10743240 TI - [Prethrombotic state in patients with chronic obstructive pulmonary disease and treatment with heparin]. AB - Several clinical reports have evidenced that chronic obstructive pulmonary disease (COPD) may be complicated by thrombosis in pulmonary vessels. The aim of this study was to investigate the putative prethrombotic state of COPD. Highly sensitive molecular markers of hemostasis, plasma levles of prethrombin F1 + 2 fragment (F1 + 2), granule membrane protein-140 (GMP-140), von Willebrand factor (vWF), Fibrinogen (Fg), and D-dimer fragments (DD) were simultaneously measured in 37 COPD patients and in 20 controls matched for sex and age. The results showed that COPD patients had significantly higher values of F1 + 2(P < 0.001), GMP-140 (P < 0.001), vWF (P < 0.001), Fg (P < 0.005) and DD (P < 0.001) than controls. In 16 patients with stable COPD and F1 + 2 greater than 1.65 nmol/L (mean +2s of control), intra-venose heparin therapy (100 mg qd for 10 days) significantly reduced F1 + 2(P < 0.005), vWF (P < 0.05), GMP-140 (P < 0.05), and PaCO2 (P < 0.05). This study has confirmed that COPD patients have an ongoing prethrombotic state which potentially accounts for thrombosis occurring in pulmonary vessels. The effects of heparin on clotting system activation and blood gas indicate that such treatment may be beneficial to COPD patients. PMID- 10743241 TI - [The expression of CD11a and CD11b on leucocytes in cerebral thrombosis]. AB - This study sought to understand the mechanism for the increased adhesion of leucocytes and endothelial cells in ischemic stroke. 20 patients with acute cerebral thrombosis and 20 healthy subjects as controls for expression of CD11a and CD11b (adhesion molecules on surface of leucocytes) were tested in vitro by flow cytometry (FCM) method. The results showed that compared with the control group, the patient group had significantly higher rates for expression of CD11a on monocytes, granulocytes and lymphocytes (P < 0.05). The CD11b expression in the patient group was positively elevated on monocytes and granulocytes (P < 0.05), but it was of lower positive rate on lymphocytes and no statistical difference was noted between the patient and control groups. These indicate that the expression of CD11a and CD11b on leucocytes increases in cerebral ischemic damage; thus adhesion of leucocytes and endothelial cells obviously increases. This change may aggravate post-ischemic delayed neuronal death. PMID- 10743242 TI - [Genetic polymorphisms of the D19S400 locus in Chinese Zhuang, Bai, Tibetan, Mongolian populations]. AB - Four Chinese populations have been studied by the polymerase chain reaction (PCR) for obtaining the data on genetic polymorphisms of D19S400 locus in four populations in China and for evaluating its use in forensic practice. EDTA-blood samples were collected from 356 unrelated individuals in Nanning (Zhuang), Dali (Bai), Lasa (Tibetan); Hailaer (Mongolian), DNA samples were extracted by Phenol/Chloroform method. The PCR products were analyzed by PAG vertical electrophoresis. Nine alleles and 42 genotypes were found at D19S400 locus in these four populations. The heterozygosity was between 0.75 and 0.89 and the discriminating power between 0.9274 and 0.9625. Significant difference was observed in the distribution of allele frequencies among the four Chinese populations. According to the results obtained in this study, D19S400 locus is a useful marker for individual indentification, paternity testing and for genetics study. PMID- 10743243 TI - [Determination of airway resistance with the interruption technique in 300 normal adolescents]. AB - The airway resistance (Raw) was determined with interruption technique in 300 normal adolescents aged 12 to 19 years on the RM-6000 polygraph system. FVC, V75, V50, V25 were also measured. The regression equations of expiratory and inspiratory resistances were built with multiple stepwise regression analysis on the IBM-PC/XT computer with age, height and FVC as independent variables. The results showed that both expiratory and inspiratory resistances decreased with age and negatively correlated with height and FVC, but FVC, V75, V50, V25 increased with age. These reflected that Raw declined with the growth and development of the lung and airway proportionally. This fact is correspondent with some international investigations. The rules and regression equations of Raw variation during the growth of adolescents for Chinese presented in this paper can be put to use in public health and clinical work. PMID- 10743244 TI - [Simultaneous assay of Ca(2+)-ATPase and Na+, K(+)-ATPase activities of osteoblast rat by malachite green colorimetic method]. AB - The aim of this study was to find a method to measure simultaneously the activities of Ca(2+)-ATPase and Na+, K(+)-ATPase of rat osteoblast membrane. In this test, Ca(2+)-ATPase and Na+, K(+)-ATPase released inorganic phosphate (Pi) from ATP, and Pi was determined by colorimetric method using malachite green dye to show ATPase activities. Ca2+ concentration and osteoblast culture generation were important factors affecting ATPase activities. Ca2+ could elevate membrane ATPase activity. When Ca2+ concentration reached 40 mumol/L, ATPase activities were improved. Ca(2+)-ATPase activity decreased with the increase of osteoblast culture generation. Ca(2+)-ATPase activity was lower than Na+, K(+)-ATPase activity in osteoblast membrane. This method is simple, sensitive and reliable. PMID- 10743245 TI - [Investigation of the Zeta reference values by Chinese-built Zetafuge and preliminary application]. AB - We determined the Zeta sedimentation ratio (ZSR) for 326 normal subjects and 106 inpatients by Chinese-built Zetafuge. The mean ZSR for normal subjces was 52.5 +/ 5.05% and that for inpatients 68.52 +/- 5.54% (P < 0.001). ZSR can be determined by using directly the blood sample with the anticoagulant EDTA-K2; the determination is faster and requires less volume of blood sample; it is not affected by anemia and need not be corrected for age and sex. The results of ZSR are expressed in a new unit, %, not in mm/h. The reference value we established is: < or = 62%. We also discussed the problem of reproducibility, the speed of centrifuge, the height of blood, and compared this method with the westergren method. The agreement between the two methods is 86.6%. PMID- 10743246 TI - [Rapid microdose 14C-urea breath test for detection of Helicobacter pylori infection]. AB - The purpose of this study was to develop and evaluate a rapid microdose 14C-urea breath test (14C-UBT) with a simplified protocol for detecting the infection of hilicobacter pylori (HP). 157 fasting patients who underwent endoscopy with histological examination and rapid urease test (RUT) were given a drink of 37 kBq of 14C-urea. Samples of breath carbon dioxide (1 mmol) were collected at baseline and 10, 20 and 30 min after administration by trapping in hyamine solution. 14C activity was measured by liquid scintillation counting. Results were expressed as cpm. Histolal examination and RUT were used as gold standard for the detection of HP infection. The cutoff value was selected as 200 cpm at 10 min. The results showed that the sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 94.79%, 90.16%, 93.81%, 91.60% and 92.99% respectively. In this study, a 10 min, single sample, 37 kBq 14C-urea breath test for detection HP was developed. The test has good diagnostic accuracy with minimal radiation exposure and low cost. Thus, the authors considered the test to be reliable, safe, convenient and cost-effective to clinical use. PMID- 10743247 TI - [Isolation, purification and activity measurement of proteolytic enzymes in dental plaque]. AB - The purpose of this study was to investigate the types and activities of proteolytic enzymes in human root surface plaque. Sephadex G-200 gel filtration, fluorometry and spectrometry were used to isolate, purify and demonstrate the activities of proteolytic enzymes. The results indicated that three enzymes were present in the root surface plaque, namely leucine amino peptidase, dipeptidyl peptidase i.v. and trypsin-like proteinase, and their activities were different. PMID- 10743248 TI - [XVII Annual meeting of the Argentine Society of Protozoology. 1st Argentine Congress and VI annual meeting of Laboratory Networks. Colon, Argentina, September 30-October 3, 1999. Abstracts]. PMID- 10743249 TI - John Watson's paradoxical struggle to explain Freud. AB - John Watson was fascinated by the discoveries of psychoanalysis, but he rejected Freud's central concept of the unconscious as incompatible with behaviorism. After failing to explain psychoanalysis in terms of William James's concept of habit, Watson borrowed concepts from classical conditioning to explain Freud's discoveries. Watson's famous experiment with Little Albert is interpreted not only in the context of Pavlovian conditioning but also as a psychoanalytically inspired attempt to capture simplified analogues of adult phobic behavior, including the "transference" of emotion in an infant. Watson used his behavioristic concept of conditioned emotional responses to compete with Freud's concepts of displacement and the unconscious transference of emotion. Behind a mask of anti-Freudian bias, Watson surprisingly emerges as a psychologist who popularized Freud and pioneered the scientific appraisal of his ideas in the laboratory. PMID- 10743250 TI - Conditioned emotional reactions. 1920. PMID- 10743251 TI - The psychology laboratory at the turn of the 20th century. AB - The author provides a brief history of the psychology laboratory from 1879 to 1900, discusses its crucial role in the founding of scientific psychology, and describes how it enabled psychology's separation from philosophy. The laboratory model is described as a research and graduate training enterprise that operated with K. Danziger's (1990) concept of a "community of scholars" and was eventually extended to the training of undergraduate students. PMID- 10743252 TI - Psychological instruments at the turn of the century. AB - Psychology at the turn of the last century was primarily a natural science in its approach. Its goals paralleled those of experimental physiology and physics that had been so successful earlier in the 19th century. The use of scientific instruments to produce stimuli and record responses in psychological experiments became the hallmark for this new psychology of the laboratory. The origins, role, and significance of some of these instruments are discussed as well as the role of instrument makers and their patrons. PMID- 10743253 TI - A social justice agenda. PMID- 10743254 TI - What ought psychology to do? PMID- 10743255 TI - Misassumptions, misadventures, and the misuse of psychology. AB - The history of psychology reveals tragic episodes of the misuse of psychological concepts and methods. Some of these misassumptions continue to influence the psychology practices of today. The contributions of people who differ from male, Eurocentric norms are not often appreciated, nor are the cultural differences of these individuals or groups either understood or respected. In this address, the author notes some of the pernicious effects and serious implications of misconceptions about difference within contemporary psychology. PMID- 10743256 TI - Psychology, time, and culture. PMID- 10743257 TI - Applying science to human behavior. PMID- 10743258 TI - Thorndike's science of values. PMID- 10743259 TI - Is suffering part of being human? PMID- 10743260 TI - The forces that will shape general practice. PMID- 10743261 TI - Testing head lice. PMID- 10743262 TI - Tricyclic antidepressants in the treatment of depression. PMID- 10743263 TI - Somatisation. What is it? AB - BACKGROUND: Patients with a strong somatic conviction despite an absence of objective measures of physical disease are vexing and frustrating for doctors. OBJECTIVE: This paper describes the phenomenon of somatisation and the common forms of presentation. DISCUSSION: Somatisation--the expression of distress in somatic form--is perhaps the commonest of the psychiatric phenomena seen in general practice. It is usually accompanied by degrees of depression and anxiety. General practitioners have a special opportunity for recognition and early intervention in order to prevent the development of chronic somatizing states. PMID- 10743264 TI - Management of somatoform disorders. AB - BACKGROUND: Physical illness, where there is no apparent disease, is a problem for doctors and the health system. Such an illness is also a cause of distress and anguish for patients and families. General practitioners are inevitably involved. OBJECTIVE: This paper describes the technique of re-attribution used to reduce patients' somatic focus, and presents principles for the management of chronic somatizers. DISCUSSION: General practitioners have a critical role to play with somatizing patients. For those patients presenting occasionally, or for the first time, with somatoform symptoms, GPs have the opportunity, through education, reassurance and re-attribution, to prevent reinforcement of the somatic perception and to reduce illness behaviour. In the care of patients with chronic somatizing disorders, GPs have an equally important role as case managers and carers, in order to prevent unnecessary medical interventions and to help maximise patient functioning. PMID- 10743265 TI - Depression in young people. A growing challenge for primary care. AB - BACKGROUND: Recent research indicates that 27% of young people aged 18-24 have a mental health problem involving mood, anxiety, personality and/or substance abuse disorders and 15-40% report depressive symptomatology. A proportion of these young people will suicide. The morbidity associated with depressive illness in this age group is high, with those experiencing a depressive episode having reduced vocational and life prospects and being highly vulnerable to further episodes in later life. OBJECTIVE: To outline the clinical features of depression in young people and to provide strategies for appropriate management. DISCUSSION: Presentation of depression in young people is likely to vary from accepted diagnostic criteria with non specific symptoms such as boredom, anxiety, failing adjustment and sleep disturbance predominating. Management includes attending to key social problems, ensuring a safe environment and counselling, which may be supportive counselling or more specific treatments of cognitive behavioural therapy or interpersonal psychotherapy. Medication is indicated for more severe depression or with failure of response to psychological strategies. Early case identification and intensive treatment of first episodes of depression is important in reducing prevalence, cost and morbidity. PMID- 10743267 TI - Patient education. Groin pain. PMID- 10743266 TI - New developments in schizophrenia. AB - BACKGROUND: Over 2% of the population suffer from schizophrenia and related psychoses. Virtually all sufferers now live in the community, and go to see their general practitioners. OBJECTIVE: To overview recent developments in the presentation and causes of schizophrenia and its treatment with medication and psychosocial intervention. DISCUSSION: The diagnostic boundaries of schizophrenia have become less distinct as aetiological and therapeutic similarities between schizoaffective and affective psychoses have emerged. The importance of specific symptom groups (positive, negative, cognitive, manic, depressed) with respect to brain mechanisms and response to treatment has been recognised. It is now thought that recognition of early phase illness and vigorous treatment aimed at relapse prevention may improve long term prognosis. Availability of new medications has improved outcomes. Patients with schizophrenia living in the community need a range of services best delivered through collaborative care between GPs, community and specialist services. PMID- 10743268 TI - Clinical audit activity. Prescribing for common conditions--hyperlipidaemia. PMID- 10743269 TI - Sudden onset of visual disturbance. PMID- 10743270 TI - Is it asthma? PMID- 10743271 TI - Does hepatitis B serology mean infection or immunity? PMID- 10743273 TI - Ends and means. PMID- 10743272 TI - Footballer with groin pain. PMID- 10743274 TI - Euthanasia--almost. PMID- 10743275 TI - Do guidelines aid diabetes care? PMID- 10743276 TI - The use and perceived value of diabetes clinical management guidelines in general practice. AB - OBJECTIVE: To assess attitudes to and use of NSW Health Department (NSWHD) guidelines for the clinical management of diabetes mellitus by divisions of general practice, general practitioners, patients and allied health professionals. METHOD: A mail cross sectional survey of divisions of general practice and a series of focus groups of GPs, patients and allied health professionals, on extent of use and perceived usefulness of the NSWHD guidelines. RESULTS: Forty divisions had or recently concluded a diabetes shared care project. Thirteen out of the 31 diabetes projects with guidelines reported using the NSWHD guidelines. Nineteen divisions were aware of the NSWHD guidelines. While the general response to the guidelines in both the survey and the focus groups was positive, there were suggestions for modifications. Many respondents were not aware of the guidelines. CONCLUSION: Optimal use of the guidelines in general practice requires improved dissemination and an implementation strategy based not only on GP education but also systems to reduce barriers to implementation and to support better quality of GP care. PMID- 10743278 TI - Immunization safety: a global priority. PMID- 10743277 TI - Collaboration between general practitioners and a child and adolescent mental health service. AB - OBJECTIVE: The aim of this project was to develop a collaborative working relationship between general practitioners and a child and adolescent mental health service (CAMHS). METHODS: The project focused on changing the policies and culture within CAMHS to be more GP aware and inclusive. The project also provided GPs with an accredited training package on common mental health problems. The impact of the project was determined through pre and post project surveys and interviews with GPs and mental health workers (case managers) respectively. RESULTS: There was a two-fold increase in communication between the two sectors and a 120% increase in shared care arrangements. There was a 144% improvement in GPs' perceptions of the helpfulness of public CAMHS. DISCUSSION: Implications for future collaboration are discussed. The results of this pilot study are encouraging and warrant replication and validation using standardised instruments. CONCLUSION: To create, enhance and extend the working relationships between GPs and public CAMHS requires strategies for change in both sectors. PMID- 10743279 TI - New challenges in assuring vaccine quality. AB - In the past, quality control of vaccines depended on use of a variety of testing methods to ensure that the products were safe and potent. These methods were developed for vaccines whose safety and efficacy were based on several years worth of data. However, as vaccine production technologies have developed, so have the testing technologies. Tests are now able to detect potential hazards with a sensitivity not possible a few years ago, and an increasing array of physicochemical methods allows a much better characterization of the product. In addition to sophisticated tests, vaccine regulation entails a number of other procedures to ensure safety. These include characterization of starting materials by supplier audits, cell banking, seed lot systems, compliance with the principles of good manufacturing practices, independent release of vaccines on a lot-by-lot basis by national regulatory authorities, and enhanced pre- and post marketing surveillance for possible adverse events following immunization. These procedures help assure vaccine efficacy and safety, and some examples are given in this article. However, some contaminants of vaccines that can be detected by newer assays raise theoretical safety concerns but their presence may be less hazardous than not giving the vaccines. Thus risk-benefit decisions must be well informed and based on scientific evidence. PMID- 10743280 TI - Safety of immunization injections in Africa: not simply a problem of logistics. AB - In 1995, the WHO Regional Office for Africa launched a logistics project to address the four main areas of immunization logistics: the cold chain, transport, vaccine supply and quality, and the safety of injections in the countries of the region. The impact of this logistic approach on immunization injection safety was evaluated through surveys of injection procedures and an analysis of the injection materials (e.g. sterilizable or disposable syringes) chosen by the Expanded Programme on Immunization (EPI) and those actually seen to be used. Re use of injection materials without sterilization, accidental needle-stick injuries among health care workers, and injection-related abscesses in patients were common in countries in the WHO African Region. Few health centres used time steam saturation-temperature (TST) indicators to check the quality of sterilization and, in many centres, the injection equipment was boiled instead of being steam sterilized. Facilities for the proper disposal of used materials were rarely present. Although the official EPI choice was to use sterilizable equipment, use of a combination of sterilizable and disposable equipment was observed in the field. Unsafe injection practices in these countries were generally due to a failure to integrate nursing practices and public awareness with injection safety issues, and an absence of the influence of EPI managers on health care service delivery. Holistic rather than logistic approaches should be adopted to achieve safe injections in immunization, in the broader context of promoting safe vaccines and safety of all injections. PMID- 10743281 TI - Developing a national system for dealing with adverse events following immunization. AB - Although vaccines are among the safest of pharmaceuticals, the occasional severe adverse event or cluster of adverse events associated with their use may rapidly become a serious threat to public health. It is essential that national monitoring and reporting systems for vaccine safety are efficient and adequately coordinated with those that conventionally deal with non-vaccine pharmaceuticals. Equally important is the need for an enlightened and informed national system to be in place to deal with public concerns and rapid evaluation of the risk to public safety when adverse events occur. Described in this article is the outcome of efforts by the WHO Global Training Network to describe a simple national system for dealing with vaccine safety and with emergencies as they arise. The goals of a training programme designed to help develop such a system are also outlined. PMID- 10743282 TI - Monitoring signals for vaccine safety: the assessment of individual adverse event reports by an expert advisory committee. Advisory Committee on Causality Assessment. AB - Monitoring vaccine safety is a complex and shared responsibility. It can be carried out in many ways, one of which is the reporting of individual cases of adverse reactions thought to be due to vaccination. The task is difficult because ascribing causality to an individual case report is fraught with challenges. A standardized evaluation instrument--known as the causality assessment form--was therefore developed for use by an expert advisory committee to facilitate the process. By following the several sections in this form, the members of the committee are taken through a series of points to establish causality. These points include the basic criteria for causation such as biological plausibility, the time elapsed between the vaccine administration and the onset of the adverse event, and whether other factors (drugs, chemicals or underlying disease) could account for the adverse symptoms. The form concludes with a consensus assessment of causality, a commentary about the assessment, and advice for further study or follow-up. This method of assessing the more serious cases of adverse reaction reported to vaccination has proven useful in evaluating ongoing safety of vaccines in Canada. Through analyses such as this, new signals can be identified and investigated further. PMID- 10743283 TI - The Vaccine Safety Datalink: immunization research in health maintenance organizations in the USA. AB - The Vaccine Safety Datalink is a collaborative project involving the National Immunization Program of the Centers for Disease Control and Prevention and several large health maintenance organizations in the USA. The project began in 1990 with the primary purpose of rigorously evaluating concerns about the safety of vaccines. Computerized data on vaccination, medical outcome (e.g. outpatient visits, emergency room visits, hospitalizations, and deaths) and covariates (e.g. birth certificates, census data) are prospectively collected and linked under joint protocol at multiple health maintenance organizations for analysis. Approximately 6 million persons (2% of the population of the USA) are now members of health maintenance organizations participating in the Vaccine Safety Datalink, which has proved to be a valuable resource providing important information on a number of vaccine safety issues. The databases and infrastructure created for the Vaccine Safety Datalink have also provided opportunities to address vaccination coverage, cost-effectiveness and other matters connected with immunization as well as matters outside this field. PMID- 10743284 TI - Simian virus 40, poliovirus vaccines, and human cancer: research progress versus media and public interests. AB - From 1955 through early 1963, millions of people were inadvertently exposed to simian virus 40 (SV40) as a contaminant of poliovirus vaccines; the virus had been present in the monkey kidney cultures used to prepare the vaccines and had escaped detection. SV40 was discovered in 1960 and subsequently eliminated from poliovirus vaccines. This article reviews current knowledge about SV40 and considers public responses to reports in the media. SV40 is a potent tumour virus with broad tissue tropism that induces tumours in rodents and transforms cultured cells from many species. It is also an important laboratory model for basic studies of molecular processes in eukaryotic cells and mechanisms of neoplastic transformation. SV40 neutralizing antibodies have been detected in individuals not exposed to contaminated poliovirus vaccines. There have been many reports of detection of SV40 DNA in human tumours, especially mesotheliomas, brain tumours and osteosarcomas; and DNA sequence analyses have ruled out the possibility that the viral DNA in tumours was due to laboratory contamination or that the virus had been misidentified. However, additional studies are necessary to prove that SV40 is the cause of certain human cancers. A recently published review article evaluated the status of the field and received much media attention. The public response emphasized that there is great interest in the possibility of health risks today from vaccinations received in the past. PMID- 10743285 TI - Clinical safety issues of measles, mumps and rubella vaccines. AB - The clinical safety of measles and measles-mumps-rubella vaccines has been questioned in recent reports that propose a possible link between measles virus or measles vaccines and the occurrence of juvenile Crohn disease and autism. This article reviews the outcomes of several laboratory investigations which were carried out independently to identify the presence or absence of measles virus in the intestinal tissues derived from cases of inflammatory bowel disease. One research group reported the presence of measles virus particles and genomic RNA in inflammatory bowel disease tissues, but this could not be confirmed by other groups, despite use of techniques that are highly specific and sensitive for the detection of measles virus nucleic acid in clinical specimens down to the molecular level. Based on the published data reviewed here, it can be concluded that there is no direct association between measles virus or measles vaccines and the development of Crohn disease, a conclusion which is supported by most epidemiological findings. PMID- 10743286 TI - Vaccine adverse events in the new millennium: is there reason for concern? AB - As more and more infectious agents become targets for immunization programmes, the spectrum of adverse events linked to vaccines has been widening. Although some of these links are tenuous, relatively little is known about the immunopathogenesis of even the best characterized vaccine-associated adverse events (VAAEs). The range of possible use of active immunization is rapidly expanding to include vaccines against infectious diseases that require cellular responses to provide protection (e.g. tuberculosis, herpes viral infections), therapeutic vaccines for chronic infections (e.g. human immunodeficiency virus (HIV) infection, viral hepatitis B and C), and vaccines against non-infectious conditions (e.g. cancer, autoimmune diseases). Less virulent pathogens (e.g. varicella, rotavirus in the developed world) are also beginning to be targeted, and vaccine use is being justified in terms of societal and parental "costs" rather than in straightforward morbidity and mortality costs. In the developed world the paediatric immunization schedule is becoming crowded, with pressure to administer increasing numbers of antigens simultaneously in ever simpler forms (e.g. subcomponent, peptide, and DNA vaccines). This trend, while attractive in many ways, brings hypothetical risks (e.g. genetic restriction, narrowed shield of protection, and loss of randomness), which will need to be evaluated and monitored. The available epidemiological and laboratory tools to address the issues outlined above are somewhat limited. As immunological and genetic tools improve in the years ahead, it is likely that we shall be able to explain the immunopathogenesis of many VAAEs and perhaps even anticipate and avoid some of them. However, this will only happen if the human and financial resources needed for monitoring and studying vaccine safety stay in step with the accelerating pace of vaccine development. Failure to make such a commitment would put all immunization programmes at risk. PMID- 10743287 TI - Presenting the case for an immunization safety surveillance system. PMID- 10743288 TI - A view from the media on vaccine safety. PMID- 10743289 TI - Evaluating vaccine safety before and after licensure. PMID- 10743290 TI - Vaccination benefits, risks and safety: the need for a complete picture. PMID- 10743291 TI - Vaccine adverse events in the new millennium: is the sky really falling? PMID- 10743292 TI - Concern, but not with surveillance. PMID- 10743293 TI - Inactivated measles vaccine and the risk of adverse events. PMID- 10743295 TI - Health inequalities and social inequalities in health. PMID- 10743294 TI - Measles immunization with killed virus vaccine. Serum antibody titers and experience with exposure to measles epidemic. 1965. PMID- 10743296 TI - Reducing HIV/AIDS risk, impact and vulnerability. PMID- 10743298 TI - Prevalence of use of complementary/alternative medicine: a systematic review. AB - Reported are the results of a systematic review of the prevalence of use of complementary/alternative medicine. Computerized literature searches were carried out in four databases. Twelve surveys thus found were selected because they dealt with the utilization of complementary/alternative medicine in random or representative samples of the general population. Data were extracted in a predefined, standardized way. Prevalence of use of complementary/alternative medicine ranged from 9% to 65%. Even for a given form of treatment such as chiropractic, as used in the USA, considerable discrepancies emerged. The data suggest that complementary/alternative therapies are used frequently and increasingly. Prevalence of use seemed to depend critically on factors that were poorly controlled in surveys of complementary/alternative medicine. The true prevalence of use of complementary/alternative medicine in the general population remains uncertain. PMID- 10743297 TI - Directly observed treatment, short-course strategy and multidrug-resistant tuberculosis: are any modifications required? AB - Multidrug-resistant tuberculosis (MDRTB) should be defined as tuberculosis with resistance to at least isoniazid and rifampicin because these drugs are the cornerstone of short-course chemotherapy, and combined isoniazid and rifampicin resistance requires prolonged treatment with second-line agents. Short-course chemotherapy is a key ingredient in the tuberculosis control strategy known as directly observed treatment, short-course (DOTS). For populations in which multidrug-resistant tuberculosis is endemic, the outcome of the standard short course chemotherapy regimen remains uncertain. Unacceptable failure rates have been reported and resistance to additional agents may be induced. As a consequence there have been calls for well-functioning DOTS programmes to provide additional services in areas with high rates of multidrug-resistant tuberculosis. These "DOTS-plus for MDRTB programmes" may need to modify all five elements of the DOTS strategy: the treatment may need to be individualized rather than standardized; laboratory services may need to provide facilities for on-site culture and antibiotic susceptibility testing; reliable supplies of a wide range of expensive second-line agents would have to be supplied; operational studies would be required to determine the indications for and format of the expanded programmes; financial and technical support from international organizations and Western governments would be needed in addition to that obtained from local governments. PMID- 10743299 TI - Preventing trachoma through environmental sanitation: a review of the evidence base. AB - A review of the available evidence for the associations between environmental sanitation and transmission of trachoma was undertaken with a view to identifying preventive interventions. The WHO Global Alliance for the Elimination of Trachoma by the Year 2020 (GET2020) has adopted the "SAFE" strategy, consisting of four components: Surgery, Antibiotic treatment, promotion of Facial cleanliness and initiation of Environmental changes. This review of 19 studies selected from the 39 conducted in different parts of the world shows that there is clear evidence to support the recommendation of facial cleanliness and environmental improvements (i.e. the F and E components of the SAFE strategy) to prevent trachoma. Person-to-person contact and flies appear to constitute the major transmission pathways. Improvement of personal and community hygiene has great potential for a sustainable reduction in trachoma transmission. Controlled clinical trials are needed to estimate the relative contribution of various elements to the risk of transmission of trachoma and the effectiveness of different interventions. These could show the relative attributable risks and effectiveness of interventions to achieve improvement of personal hygiene and fly control by environmental improvements, alone or in combination, and with or without antibiotic treatment. PMID- 10743300 TI - Biomedical and development paradigms in AIDS prevention. AB - In the fight against the HIV/AIDS pandemic different approaches can be distinguished, reflecting professional backgrounds, world views and political interests. One important distinction is between the biomedical and the development paradigms. The biomedical paradigm is characterized by individualization and the concept of "risk". This again is related to the concept of the market where health is a product of services and progress a series of new discoveries that can be marketed. The development paradigm is characterized by participation of the different stakeholders and by community work. The concept "vulnerability" is important in the development paradigm and emphasis is placed on efforts to decrease this vulnerability in a variety of sustainable ways. Biomedical technology is definitely one of the tools in these efforts. In the beginning of the pandemic the biomedical approach was important for the discovery of the virus and understanding its epidemiology. Later, stakeholders became involved. In the light of absence of treatment or vaccines, the development paradigm became more important and the two approaches were more in balance. However, since the reports about effective treatment of AIDS and hope of development of vaccines, the biomedical paradigm has become a leading principle in many HIV/AIDS prevention programmes. There is a need for a better balance between the two paradigms. Especially in developing countries, where it is not realistic to think that sustainable biomedical interventions can be organized on a short-term basis, it would be counterproductive to base our efforts to deal with HIV/AIDS exclusively on the biomedical approach. PMID- 10743301 TI - Cercarial dermatitis in India. PMID- 10743302 TI - Should everyone over age 75 take a multivitamin? PMID- 10743303 TI - In preventive cardiology there is no 'one true thing'. AB - Prescribing exercise, dietary changes, or stress management regimens separately and in the short term will ultimately fail to prevent coronary events or bring about regression of existing coronary disease. Only a multifactorial approach that focuses on permanent changes in all three areas works. PMID- 10743304 TI - New therapies for allergic rhinitis. AB - New therapies for allergic rhinitis are more effective and have fewer side effects than older medications. Antihistamines, decongestants, and cromolyn sodium nasal sprays are often tried first. Second generation prescription antihistamines have fewer side effects than over-the-counter ones. Steroid nasal sprays are extremely effective and safe for the entire range of allergy symptoms. Immunotherapy requires a lengthy course of injections, but it can bring long-term relief for severe allergies. PMID- 10743305 TI - Where to draw the line using statins: lessons from 4S to AFCAPS/TexCAPS. PMID- 10743306 TI - Primary pulmonary hypertension: update on pathogenesis and novel therapies. AB - Primary pulmonary hypertension is a progressive and fatal disease that chiefly affects young adults. Although its etiology is a mystery, recent research has uncovered several biochemical derangements in this disorder, and new therapies are prolonging survival. PMID- 10743307 TI - How new technology is changing mammography and breast cancer management. AB - Breast-imaging technology has improved in ways that allow one not only to detect breast cancer earlier, but also to distinguish benign from malignant lesions better. These capabilities are influencing the approach to breast cancer. We review current trends and issues for the non-radiologist. PMID- 10743308 TI - Adjunctive medical therapy for acute coronary syndromes. AB - Adjunctive therapy is critical in treating acute coronary syndromes. Aspirin, nitrates, beta-blockers, ACE inhibitors, HMG-CoA reductase inhibitors (statins), and intra-aortic balloon pumps all have important roles and should be considered on a case-by-case basis. PMID- 10743309 TI - Eradicating H pylori in nonulcer dyspepsia: 7 reasons for vs 7 reasons against. PMID- 10743310 TI - Salmonella perils of pet reptiles. PMID- 10743311 TI - 'All the history that you can remember' from the twentieth century. PMID- 10743312 TI - Microbiological standards for water and their relationship to health risk. AB - Maintenance of the microbiological quality of water has been used as an important means of preventing waterborne disease throughout the twentieth century. The commonest microbiological tests done on water are for coliforms and Escherichia coli (or faecal coliform). This paper reviews the legislative and other guidance for microbial standards in drinking and bathing waters and considers evidence for the relationship between the microbiological quality of water and risk to human health. In the past measures of the microbiological quality of water correlated well with risks of acquiring gastrointestinal disease. More recent work suggests that gastrointestinal disease is more strongly associated with the presence of enterococci than of E. coli. New diseases such as cryptosporidiosis have been shown to cause outbreaks of waterborne disease when levels of conventional microbiological parameters are satisfactory. In response to this, and because of failure of prosecution in one outbreak, the United Kingdom (UK) Government has introduced new legislation that requires water providers to perform a risk assessment on their water treatment facilities and to implement continuous monitoring for cryptosporidium. A new European directive on drinking water has been introduced and legislation on cryptosporidium in drinking water has been proposed in the UK. PMID- 10743313 TI - Guidelines for the control of infection with Vero cytotoxin producing Escherichia coli (VTEC). Subcommittee of the PHLS Advisory Committee on Gastrointestinal Infections. AB - Increasing numbers of cases of Vero cytotoxin producing Escherichia coli (VTEC) O157 infection, well published incidents, and new scientific evidence make it appropriate to produce new guidelines for their control. This document reviews the clinical and epidemiological features of VTEC O157 infection, describes the principles of microbiological investigation and laboratory safety, and presents recommendations for the prevention of spread of VTEC) O157. The recommendations consider direct spread of infection from animals, foodborne spread, the institutions in which spread is more likely to occur (nursing homes, schools, and children's day nurseries), and groups at particular risk of acquiring and transmitting infection (in essence, food handlers, and those unable to maintain high standards of hygiene for themselves and their carers). PMID- 10743314 TI - Advice on the response from public and environmental health to the detection of cryptosporidial oocysts in treated drinking water. PHLS Advisory Committee on Water and the Environment. AB - New water quality regulations in the United Kingdom (UK) will lead to the regular sampling of water supplies for the presence of cryptosporidial oocysts. It is likely therefore that consultants in communicable disease control (CCDCs) and environmental health officers (EHOs) will be informed of the presence of oocysts in water supplies rather more often. These guidelines were developed, by an ad hoc working group of the PHLS Advisory Committee on Water and the Environment, to help CCDCs and EHOs respond to such reports. The implications for public health of oocysts in treated water supplies is still not fully understood. Nevertheless, on the basis of the available evidence, these guidelines suggest an approach to planning, to deciding what information is required on initial contact, and an approach to health risk assessment. The guidelines also suggest possible strategies for managing such situations. It is accepted, however, that the vast majority of reports of positive samples will require no intervention. It is essential that CCDCs and EHOs work well with their colleagues in the water companies in undertaking a health risk assessment and responding appropriately. PMID- 10743316 TI - The contribution of influenza to combined acute respiratory infections, hospital admissions, and deaths in winter. AB - The impact of influenza is assessed by comparing events during epidemics with those expected outside epidemic periods (defined from incidence data collected by the Weekly Returns Service of the Royal College of General Practitioners from 1989 to 1998 for influenza-like illness, acute otitis media, acute bronchitis, and all respiratory infections combined in the community and virus isolate data). Estimates of the consulting populations for each condition in England and Wales were derived by extrapolating the difference between observed and baseline incidence rates to the total population. Similar methods were applied to data on hospital admissions for cardiac and respiratory diseases and to deaths. Each year an average of 422,000 extra people consulted and were diagnosed with influenza like illness during the epidemic period; among 1.1 million extra people who presented with acute respiratory infections. There were 3028 excess respiratory admissions (England only) in the age group 65 to 74 years and 6049 who were aged over 75 years, but no excess cardiac admissions. An average of 12,554 deaths occurred in England and Wales during influenza epidemic periods each year. Age specific national data are needed to interpret the economic impact of an illness in relation to the setting for health care delivery. PMID- 10743315 TI - Factors influencing the distribution of tuberculosis cases in an inner London borough. AB - The incidence of tuberculosis (TB) has increased throughout London, especially in inner city boroughs. Ethnicity, poverty, and the success of TB control measures all affect the distribution of cases between boroughs. This study was conducted to see which factors affect the distribution of cases between electoral wards within an inner London borough. The Borough of Newham in the East End of London is made up of 24 electoral wards and has one of the highest notification rates in the United Kingdom. Our analysis showed that the percentage of the population who were non-white made a strong contribution to the variance in TB rates between wards (p < 0.0001), but the age distribution of the population and Jarman scores for poverty did not, the latter because the entire borough is deprived. Measures to control TB should be targeted at those areas where members of the community are most at risk. PMID- 10743317 TI - Implementation of influenza immunisation policy in general practice: 1997 to 1998. AB - Influenza immunisation policy and practice in primary care in central southern England was surveyed in early 1998, when national guidelines advised immunisation only for people with specified 'higher risk' medical conditions or who lived in long stay facilities such as nursing or residential homes. Three hundred and one questionnaires were returned from 441 general practices (68%). Between 71% and 82% of all respondents stated that they 'always' offered influenza immunisation to adults with the specified 'higher risk' medical conditions. For children the corresponding range was 33% to 43%. We estimated that 11.5% of the combined practice population of two million had received influenza immunisation late in 1997. The age data supplied suggested that 64% of those aged over 75 years had been immunised. Seventy-four per cent of respondents said they would be willing to follow an age based policy. The total coverage figures mask suboptimal uptake in target groups, coupled with immunisation of those outside the target groups. This could be partly addressed by rewarding high coverage of target groups such as elderly people aged over 75 years and by clarifying the guidelines for immunisation of children with 'higher risk' medical conditions. PMID- 10743318 TI - A cluster of cases of legionnaires' disease associated with exposure to a spa pool on display. AB - Interviews with four apparently sporadic cases of legionnaires' disease identified by laboratory reporting in one health district revealed that three of the four cases had been exposed to a new design of spa pool on display at a retail outlet. The fourth case had been exposed to the same design of spa pool in a private home. None had bathed in the spa pool. Legionella pneumophila serogroup 1 mAB subgroup 'Philadelphia' was isolated from samples taken from both spa pools. The display pool had not been maintained as recommended for a public spa pool in use by bathers. Revised maintenance instructions were issued to all purchasers of these spa pools in the United Kingdom (UK) and overseas. Patients may not remember specific exposures; scrutiny of activity diaries helped to identify exposure to a common location. Even when not in use by bathers, spa pools on display can be a source of legionella infection and should be maintained according to current hygiene guidelines. PMID- 10743319 TI - Cyclospora infections in England and Wales: 1993 to 1998. AB - The coccidian protozoon Cyclospora cayetanensis is a treatable cause of prolonged, watery diarrhoea in humans. Microbiology laboratories in England and Wales often restrict testing to those who have recently travelled abroad. Only 44 to 66 laboratory reports of C. cayetanensis are made in England and Wales each year and a large proportion are found to have visited developing countries. Large foodborne outbreaks of infection have arisen in North America among people who have not travelled abroad but no such outbreaks have been identified in the United Kingdom. Public health laboratories in England and Wales were surveyed in 1998 to investigate their procedures for identifying C. cayetanensis. Sixty-eight per cent actively looked for the protozoon, but only half used a recommended method of direct microscopy of formol ether concentrates. National external quality assurance results for all participating UK laboratories were reviewed to assess laboratory proficiency in identification. C. cayetanensis was correctly identified in a wet preparation by 58% of laboratories, the lowest rate for specimens containing a single parasite species. Cyclosporiasis could be acquired in the UK from imported food, but current laboratory procedures might fail to identify it. Ascertainment must improve and awareness needs to be raised among food handlers, public and environmental health workers, laboratory staff, and general practitioners. We recommend that laboratories test all patients with watery diarrhoea for > 1 week for cyclospora, use formol ether concentration and microscopy with a calibrated eyepiece graticule, and confirm diagnoses with the help of a reference laboratory. PMID- 10743320 TI - Foodborne outbreaks of cyclosporiasis have arisen in North America. Is the United Kingdom at risk? AB - Cyclospora cayetanensis is a parasitic protozoon that causes prolonged watery diarrhoea. It is endemic in some developing countries, and recent foreign travel is often used as a selection criterion for screening in the United Kingdom (UK). Epidemiological investigations of outbreaks of cyclosporiasis among people in the United States and Canada who had not travelled abroad showed the infection to be foodborne and often associated with foods eaten raw. These included raspberries imported from Guatemala, and pesto (made with basil) and lettuce from other sources. Such foods are also being imported in increasing amounts to the UK, but no outbreaks have been documented, perhaps because none has occurred or because of poor ascertainment. This paper reviews the outbreaks reported from North America, evaluates the risks to the UK population, and suggests how surveillance could be improved. PMID- 10743321 TI - Effect of a boil water notice on behaviour in the management of a water contamination incident. AB - In late 1998, the water supply of 878 households was affected by possible sewage contamination. A notice was issued to advise residents of the need to boil their water. This provided an opportunity to assess to what extent the boil water notice led people to avoid activities that might put them at risk of waterborne infection. A postal questionnaire sent to 350 randomly selected households in the affected area asked about timing and mode of receipt of the notice, risk behaviour (boiling water, brushing teeth, washing dishes, drinks for pets, preparation of food), and subsequent changes in drinking water consumption habits. Eighty-one per cent of the households surveyed engaged in behaviour likely to increase the risk of waterborne infection. Comments were collected from consumers on how to improve the management of future water contamination incidents. PMID- 10743322 TI - Outbreak of hepatitis A spread by contaminated drinking glasses in a public house. AB - Ten cases of hepatitis A were associated with a public house whose barman had chronic non-infectious diarrhoea and had served drinks while incubating hepatitis A himself. Eight cases had drunk in the public house in the two weeks before the barman became ill, had no other risk factors for hepatitis A infection, and were regarded as primary cases. Two other cases had visited the pub during the same period, but were classified as secondary cases because they became ill later and were contacts of primary cases. Sexual transmission was excluded. Fomite transmission by contamination of glasses was a more likely route of spread of the infection among the eight primary cases than food or contaminated surfaces in the toilet. PMID- 10743323 TI - Direct communication with general practitioners improves management of infants at risk of hepatitis B. AB - The National Screening Committee has recommended that all women presenting for antenatal care be offered routine screening for hepatitis B by the year 2000. In a low prevalence area a survey of the management of neonates whose mothers were positive for hepatitis B surface antigen showed that management of this group was better among general practitioners who had received advice on treatment. PMID- 10743324 TI - Is infection control in residential homes neglected? AB - We investigated the potential for cross infection during the cleaning of commodes in residential homes in one health authority in order to investigate why residential institutions are the commonest setting for outbreaks of infectious intestinal disease. Most homes used commodes, but half lacked a designated sluice area for cleaning them after use and in half soiled commodes were cleaned in bathroom or toilet washbasins, baths, showers, or toilet pans, creating a risk of cross infection. PMID- 10743325 TI - Infantile Salmonella meningitis associated with gecko-keeping. AB - A serotype found mainly in reptiles was isolated from cerebrospinal fluid from a 2 month old baby with meningitis. A related salmonella was isolated from gecko faeces from the floor of the tank in the baby's home, suggesting a possible source of infection, and indicating the need for hygienic precautions in homes where reptiles are kept as pets. PMID- 10743326 TI - Targeted mailing of information to improve uptake of measles, mumps, and rubella vaccine: a randomised controlled trial. AB - Uptake of the measles, mumps, and rubella vaccine in the United Kingdom has declined to levels that will allow outbreaks of these preventable diseases to occur. A leaflet sent with a personalized reminder did not increase vaccine uptake in children who had not been immunised at 21 months of age. PMID- 10743327 TI - Meningococcal disease requires an 'out of hours' public health response. PMID- 10743328 TI - DNA microarrays. PMID- 10743329 TI - Ergonomics and accident behaviour. PMID- 10743330 TI - Legionellosis associated with a hotel in South Wales. PMID- 10743331 TI - Outbreak of Listeria monocytogenes serovar 4b infection in France. PMID- 10743332 TI - [Animal protection during transport in Hungary]. AB - To assess animal welfare during transport in Hungary, a questionnaire study was carried out in 1998. The questionnaire consisted of 39 questions addressing some general characteristics as well as specific ones regarding means of transport, fulfillment of the animals' needs on board and others. Three-hundred and ninety one questionnaires were analysed representing about 0.1% of all yearly transports in Hungary. The most significant problems encountered were with conditions of transport that are more relevant for longer transports, especially those exceeding eight hours. Such are, inter alia, weather protection, ventilation, means to tie or separate animals and the provision of food and water. Even though there are several different types of vehicle available for transporting animals in Hungary, vehicles that are not appropriate for the species, distance or other conditions of the transport are sometimes used. Our results apply to the preparatory phase before the introduction of new, EU conform legislation on the protection of animals during transport. They enable more specific investigations or corrective measures to be undertaken by the competent authority responsible for the enforcement of such regulations in Hungary. PMID- 10743334 TI - [Mycobacterium avium infections in poultry--a risk for human health or not?]. AB - Avian tuberculosis is an animal disease notifiable for statistical purposes in Germany. Cases notified (between 130 and 230 annually) were primarily related to private flocks of pedigree poultry and layers consisting of less than 20 animals and individual animals in game enclosures and zoological gardens. Mycobacterium (M.) avium infection does not play any role in modern intensive poultry husbandry. Human M. avium infections have considerably gained in importance in the last two decades, mainly in HIV-infected patients. Due to the ubiquitous character of MAIC (Mycobacterium avium intracellulare-Complex), it is difficult to establish confirmed epidemiological associations with infections in humans. Surface and drinking water, soil and also foods as well as direct contact with infected birds (pet birds) have been discussed as possible sources of infection. Recently, strains of the serovars 1, 2 and 3 which have often been isolated from birds (bird-type strains) could be defined as a taxon on its own right among MAIC by using molecular-biological methods for MAIC typing (RFLP--restriction fragment length polymorphism and PFGE-pulsed field gel elektrophoresis). In exceptional cases only, strains of this character have been isolated from humans. Consequently, poultry-to-man transmission of M. avium appears to be a very improbable event. In contrast, extensive conformity has been found to exist between M. avium isolates of human origin and isolates from pigs. This fact has rightly given rise to assumptions of either the presence of epidemiological links between pigs and humans or of infection from common sources. In a summarizing view, it can be stated that M. avium infection of farm poultry is hardly of any importance for poultry production as well as for human disease. The importance of MAIC for infections in other farm animals (cattle and swine) is outlined and discussed. PMID- 10743333 TI - [Demonstration of Chlamydia from an equine abortion]. AB - The isolation and identification of a chlamydial agent from an equine fetus is reported. The fetus was aborted by a mare with respiratory disease and fever in the 9th month of pregnancy. The serum of the mare was investigated by the compliment fixation test. Specific antibodies were detected for chlamydial antigen in a titer of > 1:40 and for equine herpes virus 1 antigen in a titer of 1:32. Pathological lesions were not found in the organs of the fetus. Chlamydiae were detected in the placenta by ELISA and subsequently isolated by cell culture. Using PCR technique the agent was identified as Chlamydophila psittaci. PMID- 10743335 TI - [Reactions of dairy calves exposed to transport stress at 60 days of age. Effects of various rearing conditions in the developmental quality of calves on physiological variables and their changes]. AB - Dairy calves reared in groups of constant (Gk, N = 16, m 8, w 8) and changing composition (Gw, N = 16, m 8, w 8) and single box reared calves (E, N = 15, m 8, w 7) were exposed to transport stress lasting around 60 minutes at 60 days of age. Long term measurements of heart rate (HF) were combined with blood sampling (V. jugularis) before and immediately after transport and at the following day as well. Mean differences of HF, Hb, Hk, lactate and blood urea between groups were found at all sampling points. After transport mean values of HF, MetHb, pO2, O2SAT and O2CONT were increased in all calves and such of HHb were decreased. Measures of acid-base balance and lactate changed differently in calves of the three rearing groups concerning direction and degree. Changing of the variables after transport comparing the starting values was different between rearing groups in 16 out of 25 possible cases. Changing of variables immediately after transport showed strong negative correlations with starting values in case of pH, HCO3, base excess, COHb, total protein, albumin, lactate, glucose and Ca in all groups, in all other cases not in all groups. Strong correlations between sampling points of Hb, Hk, MCHC, O2CONT, O2CAP, creatinine, blood urea and of the minerals indicate a high individual specificity of these measures. Results proof variation components of physiological variables in calves caused by husbandry conditions and their effects on reactions of animals in case of transport stress. PMID- 10743336 TI - Transient classical swine fever virus infection in wild boar piglets partially protected by maternal antibodies. AB - An experimental study was conducted to investigate the clinical course of classical swine fever (CSF) in wild boar piglets partially protected by maternal antibodies. Five healthy wild boar piglets with a low serum titre of colostral antibodies against CSF virus were challenged with virulent CSF virus at the age of three months. Apart of reduced food intake and diarrhoea no major clinical symptoms were noticed after challenge. These signs were seen during the second and third week of infection, afterwards the piglets recovered completely. CSF virus could be re-isolated from blood samples taken on day 12 and day 19 post challenge. From blood samples taken later on and from the organ material taken at post mortem examinations no CSF virus could be isolated anymore. It can be concluded that the presence of maternal antibodies influences the clinical course of CSF in terms that the outcome is rather transient than lethal. Such wild boar could play a crucial role in the spread of CSF virus and might contribute to the maintenance of long lasting epizootics. PMID- 10743337 TI - Extracellular presence of reactive oxygen species (ROS) in fresh and frozen thawed canine semen and their effects on some semen parameters. AB - The objective of this study was to determine the presence of reactive oxygen species (ROS), especially superoxide anion (O2-), in fresh and frozen-thawed canine semen, its effect on other seminal parameters and whether the freezing thawing process influences the O2- production rate. A total of 56 mature male domestic dogs were used in this study. Their semen was examined before and after freezing-thawing process. Sperm motility, hypo-osmotic swelling, sperm morphology and O2- extracellular production were measured. The dogs were divided in two groups, with the criterion of the absence or presence of O2- in fresh semen (groups A and B, respectively). Values of group A were compared with the respective values of group B. A significant decrease in motility and percentage of swollen spermatozoa and an increase in morphological abnormalities and O2- production in group B were found in comparison with group A after freezing thawing process (p < 0.05). It seems that the freezing-thawing process leads to an increase of the O2- production. However, it remains to be documented if the increased concentrations of O2- in canine semen are responsible for the alterations in the seminal parameters observed in this study. PMID- 10743338 TI - Prevalence of Toxocara spp. eggs in the soil of public parks in Ankara, Turkey. AB - One hundred and seventy soil samples from forty-six public parks in Ankara area were examined to determine the level of contamination with Toxocara spp. eggs. 30.6% of the 170 soil samples were contaminated with Toxocara spp. eggs. The number of eggs in the positive soil samples varied from 1 to 10. A high proportion of the eggs was fully embryonated. The findings demonstrate the common occurrence of these eggs in locations likely to be important in the transmission of visceral larva migrans. Of 19 fecal collected, 5 (26.3%) contained Toxocara spp. eggs. Eggs of Ancylostomidae, Toxascaris leonina, Trichuris spp., Taenia spp. and Enterobius vermicularis were also recovered from 17.6, 4.1, 2.4, 1.8 and 1.2% of soil samples, respectively. PMID- 10743339 TI - Imperialism, colonial identity, and race in Algeria, 1830-1870. The role of the French Medical Corps. AB - During the military administration of Algeria, which lasted for forty years, the foundation of the French colony was laid. Indispensable to the military in Algeria was its sizable medical corps. While the ostensible reason for its presence was to maintain the soldiers' health and thus the army's efficiency, it role extended beyond this primary objective. Starting from the intellectual and political influences that shaped the training in France of the members of the medical corps, this essay examines the ways in which they contributed to the creation of a French colonial space in Algeria. It traces how their involvement in the intellectual, cultural, and political life of the colony enabled them both to further their own ambitions and to influence wider developments. It explores how colonial physicians and surgeons, deemed to be among the most efficient agents of the civilizing mission owing to their humane contacts with the indigenous population, in fact contributed to that population's categorization and marginalization. PMID- 10743340 TI - Foot complications in people with diabetes: a community-based study in Taiwan. AB - BACKGROUND AND PURPOSE: This comparative cross-sectional and community-based study was aimed at defining foot complications in diabetic patients. These data have not yet been reported for Asian societies. METHODS: Of a population of 3,602 subjects aged 35 years or more in Chin-Shan, Taipei, 309 diabetic patients were identified. Two hundred and nineteen (71%) of those patients were compared to 100 individuals randomly selected for the nondiabetic control group in the same community. RESULTS: Three diabetic patients underwent lower extremity amputation and four had skin ulcerations. Diabetic patients had a significantly higher prevalence of peripheral neuropathy (32.4% vs 16%), arterial insufficiency (12.6% vs 3.0%), and medial arterial calcification (13.6% vs 5.0%), when compared to the nondiabetic controls. The age and sex-adjusted rates of hallux valgus, loss of skin hair on the dorsum of the foot, tinea unguium, arterial insufficiency, medial artery calcification, and peripheral neuropathy were significantly higher in diabetic than nondiabetic subjects. Aging and hyperglycemia (> 140 mg/dL) increased the prevalence of foot complications in both groups. Foot complications were also remarkably associated with the duration of diabetes (p = 0.003). CONCLUSIONS: This study shows that diabetes mellitus is associated with an increased likelihood of foot complications in this geographically defined Taiwanese population. Patient age and diabetic duration are associated with the significantly higher prevalence of foot complications. PMID- 10743341 TI - Physical activity, physical fitness, and osteopenia in postmenopausal Taiwanese women. AB - BACKGROUND AND PURPOSE: The purpose of this cross-sectional study was to investigate whether physical activity level and physical fitness parameters differ between postmenopausal Taiwanese women with normal and subnormal bone mineral density (BMD). METHODS: Seventy-six postmenopausal women aged from 42 to 65 years participated in this study. Women taking medication that might influence BMD measurements were not included. The BMDs of the lumbar spine (L2-4) and right femoral neck were measured using dual energy x-ray absorptiometry. Thirty-one women with both BMD values within the normal ranges (1.055 +/- 0.092 g/cm2 for the spine and 0.845 +/- 0.088 g/cm2 for the right femoral neck) of premenopausal Chinese women served as the normal density group. Another 43 women with both BMD values more than one standard deviation below the normal value (0.760 +/- 0.089 g/cm2 for the spine and 0.656 +/- 0.052 g/cm2 for the femoral neck) were recruited as the osteopenic group. Physical activity level was assessed with a 7 day recall questionnaire. Physical fitness assessment included tests of flexibility, muscular strength, endurance, body composition, and cardiopulmonary fitness. A multiple linear regression model adjusted for age, body weight, height, and years since menopause was used. RESULTS: The results revealed that energy expenditure and maximal oxygen consumption were significantly lower in the osteopenic group than in the non-osteopenic group (p < 0.05), while flexibility, body composition, muscle strength and muscular endurance did not differ significantly between the two groups (p > 0.05). CONCLUSIONS: These findings indicate that physical activity may play a major role in BMD levels in postmenopausal women in Taiwan. Future studies should emphasize the effect of physical exercise training on BMD in postmenopausal women. PMID- 10743342 TI - Pneumonia caused by penicillin-nonsusceptible Streptococcus pneumoniae: clinical characteristics, prognostic factors, and outcomes. AB - BACKGROUND AND PURPOSE: Pneumococcal pneumonia caused by penicillin nonsusceptible Streptococcus pneumoniae (PNSSP) is increasing worldwide. To better understand this infection, patients with pneumococcal pneumonia treated at National Taiwan University Hospital during a 5-year period were evaluated to determine the clinical characteristics, prognostic factors, and outcomes of the infection. METHODS: Eighty-one patients with 81 episodes of pneumococcal pneumonia diagnosed from January 1993 to December 1997 were analyzed retrospectively. Patients were categorized into two groups according to susceptibility results. Differences between groups were evaluated with Student's t-test and the chi-square test. Univariate analysis was used to identify factors associated with mortality. RESULTS: Infections were caused by penicillin susceptible S. pneumoniae (PSSP) in 57 (70%) patients and by PNSSP in the remaining 24 (30%). Nosocomial acquisition was more common in PNSSP (25%) than in PSSP patients (10%) (p = 0.05), while the frequency of bacteremia (54% vs 60%) and the mortality rate (46% vs 32%) did not differ significantly between the PNSSP and PSSP groups. Among the 24 patients with pneumonia caused by PNSSP, those 65 years of age or older had a lower incidence of bacteremia (4/14, 29%) than those who were younger (9/10, 90%) (p = 0.03). In patients with pneumonia caused by PNSSP, the outcome was not significantly related to the treatment regimen (penicillin vs nonpenicillin beta-lactam antibiotics or vancomycin, 33% vs 47%; p = 0.4). The only factors associated with mortality from these infections were shock (p = 0.003) and multilobar consolidation (p = 0.01) at the time of admission. CONCLUSIONS: These data suggest that the clinical outcome of pneumococcal pneumonia is more closely related to the clinical condition at presentation than the susceptibility status of the pneumococcus. PMID- 10743343 TI - Immunoglobulin M and G immunoblots in the diagnosis of parvovirus B19 infection. AB - BACKGROUND AND PURPOSE: To identify parvovirus B19 infection by means of immunoglobulin (Ig) G and IgM immunoblots among immunocompetent patients who tested negative or had low-titer B19 IgM antibodies in enzyme-linked immunosorbent assays (ELISA). METHODS: Serum samples were obtained from 20 patients with parvovirus B19 infection. Another 130 study subjects presumed to be without B19 infection (40 medical personnel and 90 prisoners) were also included. All sera from the patient and study groups tested positive for IgG or IgM with ELISA and were further evaluated using the immunoblot method. Detection of B19 DNA by nested polymerase chain reaction (PCR) was also performed on IgG and IgM positive sera. RESULTS: IgM immunoblots disclosed one false positive IgM ELISA result in the patient group and three false positive results in the study group. In the patient group, four patients were in the latter stage of antibody response to B19 infection as suggested by the low titer of anti-B19 IgM, incomplete IgM immunoblots, with only a weak viral capsid protein VP-N reaction band, and fading but still strong reaction bands on IgG immunoblots. Strong reaction bands on IgG immunoblots comparable to these four patients were found in three of the 130 study group sera. Furthermore, B19 DNA was detected in three of the four patients and one of the three study subjects by means of nested-PCR. A serum sample from one study subject showed strong IgG but no IgM reactivity to viral capsid protein VP2; nested PCR identified B19 DNA in this serum sample. CONCLUSIONS: Immunoblots and nested PCR should be applied in the diagnosis of B19 infection for patients with low-titer anti-B19 IgM tested by means of ELISA. For diagnosis of B19 infections in certain clinical entities such as chronic arthritis of recent onset and hydrops fetalis, B19 IgM antibodies may have disappeared but B19 infection can still be recognized by the intensity of the reaction bands on IgG immunoblots. The correlation between chronic B19 infection and persistence of antilinear VP2 epitopes requires further study. PMID- 10743344 TI - Seroprevalence of Coxiella burnetii infection in southern Taiwan. AB - BACKGROUND AND PURPOSE: The emergence of infection with Coxiella burnetii, the causative organism of Q fever, has been only recently recognized in Taiwan. Several cases of acute Q fever infection have been described, but the prevalence of antibodies to C. burnetii in the general population in Taiwan has not been reported. Thus, we studied the seroprevalence of C. burnetii infection in southern Taiwan. METHODS: We conducted a retrospective serosurvey to examine the prevalence of C. burnetii infection among subjects admitted to a rural hospital in Taiwan for various reasons, and among presumably healthy attendees of a routine physical examination clinic of an urban public hospital. The diagnosis of C. burnetii infection required the presence of antibodies to both phase I and II antigens (titer > or = 1:16) or only to phase II antigens (titer > or = 1:256), as detected by indirect immunofluorescence assay. RESULTS: The prevalence of C. burnetii infection was 4.2% in both the in-patient (15/357) and physical examination participant (11/259) populations. None of these subjects had signs compatible with acute Q fever (febrile illness within the past 3 months). The antibody prevalence rate was higher in males than in females, and peaked in persons aged 61 to 70 years. CONCLUSIONS: These data suggest that C. burnetii infection is not rare in southern Taiwan and does not cause clinical symptoms in all infected patients. PMID- 10743345 TI - Vertical alignment axis for transtibial prostheses: a simplified alignment method. AB - BACKGROUND AND PURPOSE: To evaluate a simplified method of alignment for fitting a transtibial prosthesis known as the vertical alignment axis method. METHODS: We measured the relationship between the vertical projection of the prosthetic socket center (geometric center of the cross section of the transtibial prosthetic socket at the level of the patella tendon bar) and the alignment reference center (junction of the middle and posterior thirds of the longitudinal axis of the shoe) in 36 dynamically aligned transtibial prostheses. The prostheses were fabricated by students, and the fit (proper vs improper) was assessed by the faculty. RESULTS: The faculty members found 26 of the 36 prostheses to be properly fitted. The vertical projections of the socket centers of the properly-fitted prostheses were concentrated within a small area of the shoe tracing (a mean value of 1.3 mm medially and 6.4 mm anteriorly from the alignment reference center). Conversely, in the improperly-fitted prostheses, the vertical projections of the socket center were scattered well away from the alignment reference center; the mediolateral deviation was significantly greater for improperly-fitted prostheses than for those that were properly fitted (p < 0.001). CONCLUSIONS: These results demonstrate the usefulness of the vertical alignment axis procedure. This procedure may help clinicians to better understand the alignment of transtibial prostheses and gait deviations associated with improper prosthesis fit. PMID- 10743346 TI - Modulation transfer function and critical flicker frequency in high-myopia patients. AB - BACKGROUND AND PURPOSE: Critical flicker frequency (CFF) decreases due to optic nerve and retinal damage in patients with optic neuritis or glaucoma. Because retinal degeneration is also found in high myopia, we investigated whether the modulation transfer function (MTF) and CFF are altered in patients with high myopia. METHODS: Forty-six patients with high myopia (< -8 diopters, D) were recruited from our outpatient department. The control group comprised 21 young volunteers with myopia of 0 to -2 D. The myopic and control groups were similar in terms of age and sex. RESULTS: At all frequencies tested, the myopic group had lower CFF and higher modulation values than the control group. The mean CFF was significantly lower in myopic patients (46.8 +/- 9.0 Hz) than in control subjects (52.5 +/- 4.4 Hz). Patients with extremely high myopia (< -10 D) had significantly greater modulation values at 5, 10, 15, 55 and 60 Hz than those with moderately high myopia (-8 D to -10 D). The CFF did not differ significantly between those with extremely high (46.3 +/- 8.8 Hz) and those with moderately high (49.0 +/- 6.9 Hz) myopia. CONCLUSIONS: The findings of this study support that the CFF decreases and MTF increases in patients with high myopia, and that the alternation of MTF is related to the degree of myopia. CFF and MTF may, therefore, have potential as indexes to evaluate the severity of retinal degeneration in patients with high myopia. PMID- 10743347 TI - Cloning a novel metallophosphoesterase gene from a kidney cDNA library of hypertensive rat. AB - BACKGROUND AND PURPOSE: Genetic and environmental factors may contribute to the pathogenesis of essential hypertension. To facilitate genetic studies of hypertension and renal disorders, we sought to clone novel genes from a modified, equalized kidney (MEK) cDNA library of a spontaneously hypertensive rat (SHR). METHODS: A kidney cDNA library of an SHR was synthesized using the modified equalization method. Inserts of 350 random clones were amplified by polymerase chain reaction (PCR) and sequenced, of which 246 were presumably unknown after being compared against a nonredundant database in the GenBank. The cDNA ends of clone 38S were obtained by rapid amplification of cDNA ends, sequenced, and then analyzed with Translate, Prosite, Profile, SignalP, and TMpred programs. RESULTS: The full-length cDNA was 938 bp, and translated into a 182-amino acid protein. The deduced protein had a metallophosphoesterase domain, a signal peptide at its amino end, a protein kinase C phosphorylation site, and a transmembrane domain. Northern blot analysis revealed that this gene was expressed in the heart, brain, spleen, lungs, liver, skeletal muscles, kidneys and testes of Sprague-Dawley rats. A putative protein of Arabidopsis thaliana shares 62% homology with protein 38S, but the two proteins differ in terms of function and structure. CONCLUSIONS: Our results support that protein 38S is a novel membrane metallophosphoesterase, although its function in the kidneys remains to be elucidated. This study also demonstrates the feasibility of using PCR to clone novel genes from our MEK cDNA library. PMID- 10743348 TI - Simple bone cyst in cervical vertebral spinous process and laminae: report of a case. AB - Simple (unicameral or solitary) bone cysts are the only true cysts of primary intraosseous origin. These cysts involve the metaphysis of long tubular bones such as the proximal humerus and proximal femur and, occasionally, the proximal tibia, distal femur, patella, ilium, rib, and calcaneus. They are very rarely found in vertebrae, especially in the cervical vertebral spinous process and laminae. We report a 14-year-old boy with an expansile, bubbly cystic lesion in the spinous process and laminae of the seventh cervical vertebra. Histopathologic examination showed a cyst surrounded by thin fibrous or fibro-osseous wall without any lining epithelial cells, compatible with a simple bone cyst. As far as we know, this is the first case of simple bone cyst involving the spinous process and laminae of a cervical vertebra reported in Taiwan. PMID- 10743349 TI - Fibrinofibrous pericarditis mimicking a pericardial tumor. AB - Primary pericardial disease is rare in children. The clinical features usually reflect limited venous return and cardiac output. Tuberculous pericarditis is the leading cause of pericardial disease in developing nations. A definitive diagnosis in children is frequently difficult and the manifestations are protean. We report a 10-month-old girl with fibrinofibrous pericarditis that manifested as constrictive pericarditis with prolonged fever, hepatomegaly, edema, and poor appetite. Echocardiography showed a solid mass that originated from the thickened pericardium and compressed the whole heart. In contrast, computed tomography revealed pericardial thickening with fluid collection. The symptoms and signs dramatically improved after surgical pericardiectomy. Pathologic analysis confirmed the diagnosis of tuberculous fibrinofibrous pericarditis. The patient received a 1-year course of antituberculosis therapy and has remained symptom free for 2 years. We suggest that a discrepancy between echocardiography and computed tomography (CT) findings might indicate a diagnosis of fibrinofibrous pericarditis. PMID- 10743350 TI - Ischemic stroke in a teenage girl with primary antiphospholipid antibody syndrome. AB - The diagnosis of stroke in children and young adults can be difficult, and the causes of ischemic stroke often remains unexplained even after extensive evaluation. We present a 13-year-old girl who had two ischemic strokes within a period of 3 weeks. A carotid duplex study and magnetic resonance angiography confirmed total occlusion of the left internal carotid artery. Serum immunologic examination revealed a high titer of immunoglobulin G anticardiolipin antibody (45.7 GPL U/mL; normal range, 0-16.2 GPL U/mL). The patient received long-term anticoagulation treatment with warfarin 5 to 7.5 mg per day, and international normalized ratios were maintained between 2.4 and 4.0. She recovered completely 5 weeks after the first stroke and has been well during the 6 months of follow-up after the onset. This is the first reported case of childhood ischemic stroke secondary to primary antiphospholipid antibody syndrome in Taiwan. This case emphasizes the importance of evaluating antiphospholipid antibody levels in patients with unexplained ischemic strokes. PMID- 10743351 TI - Sinus node dysfunction in a patient with lithium intoxication. AB - Lithium can suppress sinus node function, especially when it is used concomitantly with carbamazepine. We describe a 42-year-old woman who took lithium and carbamazepine for manic-depressive psychosis and seizure disorders, and developed marked sinus node dysfunction. Drug screening showed a toxic serum lithium level of 3.38 mmol/L and a normal serum carbamazepine level of 22.1 mumol/L. An electrophysiologic study showed prolongation of the corrected sinus node recovery time (CSNRT) of up to 9,708 msec. After three sessions of hemodialysis, normal sinus rhythm was resumed. The serum lithium level was 0.1 mmol/L 2 weeks later, and the CSNRT shortened to 309 msec. Because the combination of lithium and carbamazepine in psychiatric patients is not uncommon, recognition of the potential complication of severe bradyarrhythmia is essential in the emergency care of such patients. PMID- 10743352 TI - The Asian Taenia and the possibility of cysticercosis. AB - In certain Asian countries, a third form of human Taenia, also known as the Asian Taenia, has been discovered. This Asian Taenia seems to be an intermediate between Taenia solium and T. saginata since in morphological terms it is similar to T. saginata, yet biologically, as it uses the same intermediate host (pigs), it is more akin to T. solium. Taenia solium causes human cysticercosis, while T. saginata does not. It is not known whether the Asian taeniid is able to develop to the larval stage in humans or not. The arguments proposed by those authors who consider it unlikely that the Asian Taenia causes human cysticercosis are: (a) its molecular similarities with T. saginata; (b) the absence of cases of human cysticercosis in populations where the Asian adult is highly prevalent; and (c) the unsupporting results derived from an experimental infestation study. These three arguments are debated, although bearing in mind that at present there is still no clear scientific data to support that human cysticercosis can be caused by the Asian Taenia. PMID- 10743353 TI - Surface ultrastructure of Metagonimus takahashii metacercariae and adults. AB - A scanning electron microscopic study was performed on the surface ultrastructure of metacercariae and adults of Metagonimus takahashii. Metacercariae were collected from the scale of crucian carp (Carassius auratus), and adult flukes were harvested 1-4 weeks after infection to rats. In excysted metacercariae, the oral sucker had type I (numerous) and type II (seven in total) sensory papillae. Tegumental spines were dense and digitated into 5-7 points on the surface anterior to the ventral sucker, but became sparse and less digitated posteriorly toward the end of the body. In adults, seven type II sensory papillae were characteristically arranged around the lip of the oral sucker, and on the inner side of the lip four small and two large type I sensory papillae were symmetrically seen on each side (12 in total). Tegumental spines on anterior two thirds of the body, were digitated with 9-12 tips ventrally and 8-13 tips dorsally. Sperms entering into the Laurer's canal were observed. The results show that the surface ultrastructure of M. takahashii is generally similar to those of M. yokogawai and M. miyatai except for the digitation of tegumental spines. PMID- 10743355 TI - A human case of Stellantchasmus falcatus infection in Korea. AB - In an attempt to find the worm producing unidentified egg, one minute fluke was collect from a Korean patient after praziquantel administration. The fluke was identified to be Stellantchasmus falcatus by the expulsor. Brackish water fish was suggested to be a probable source of the infection. PMID- 10743354 TI - Excretory-secretory product of newly excysted metacercariae of Paragonimus westermani directly induces eosinophil apoptosis. AB - Eosinophils are important effector cells in host defense against parasites. Excretory-secretory product (ESP) produced by helminthic worms plays important roles in the uptake of nutrients, migration in the host tissue, and in immune modulation. However, little is known about the ability of the ESP to directly trigger eosinophil apoptosis. This study investigated whether the ESP of newly excysted metacercariae of Paragonimus westermani could induce apoptosis in human eosinophils. Apoptosis was assayed by staining the cells with FITC-annexin V, and the cells were analyzed by flow cytometry. It was found that the ESP of newly excysted metacercariae of P. westermani induced a direct time- and concentration dependent increase in the rate of constitutive apoptosis in mature human eosinophils. Eosinophil apoptosis was first apparent 3 hr after treatment with the ESP and continued to increase after 6 hr of incubation with respect to the cells cultured in the absence of the ESP. While only 2.8% of the eosinophils incubated in the medium for 3 hr were apoptotic, 7.6%, 10.9% and 22.6% of the eosinophils treated with 10, 30 and 100 micrograms/ml ESP were apoptotic, respectively. This result suggests that the ESP of newly excysted metacercariae of P. westermani directly induce eosinophil apoptosis, which may be important for the survival of the parasites and the reduction of eosinophilic inflammation in vivo. PMID- 10743356 TI - A toxoplasmic uveitis case of a 60-year-old male in Korea. AB - A toxoplasmic uveitis case was reported on the focus of impairment of pathological findings and serological antibody titers after chemotherapy. A chief complaint of a 60-year-old male was a decreased and blurred vision in his right eye for 2 weeks after experiencing tremendous stress and fatigue. A steroid therapy for 3 weeks was not effective and the retinal lesion became necrotic. Anti-Toxoplasma gondii antibody titer was checked to be a strong positive by both ELISA and indirect latex agglutination assay (ILA). He was treated with Fansidar F for 8 weeks. His vision improved as the necrotic lesion healed by scarring, but the antibody titers still remained very high without any signs of negative conversion. It is suggested to be a recurrent case of the past asymptomatic infection by presumed immune suppression caused by excessive stress. PMID- 10743357 TI - High endemicity of Metagonimus yokogawai infection among residents of Samchok shi, Kangwon-do. AB - A small-scale epidemiological survey was undertaken during 1997-1998 on the residents along the Osib-chon (Stream), Samchok-shi (City), Kangwon-do (Province), to evaluate the status of Metagonimus yokogawai infection. A total of 165 fecal samples was collected and examined by cellophane thick smear and formalin-ether sedimentation techniques. The egg positive rate of M. yokogawai was 29.7%, showing a remarkable difference between males (46.6%) and females (16.3%). To obtain the adult flukes of M. yokogawai, 11 egg positive persons were treated with praziquantel and purged with magnesium sulfate. A total of 242,119 adult flukes (average 22,010 per person, 367-119,650 in range) was collected from diarrheic stools, all of which were identified as M. yokogawai. The results show that M. yokogawai is still highly endemic in this area. PMID- 10743358 TI - Infection status with trematode metacercariae in pond smelts, Hypomesus olidus. AB - Many Koreans usually eat raw pond smelts, Hypomesus olidus, in the winter. This study was performed to evaluate the infection status with trematode metacercariae in pond smelts from January 1998 through February 1999. Among 1,305 fish collected, 459 were purchased from wholesale dealers in Chinchon-gun, Chungchongbuk-do, and the rest of them were caught with a casting net in Soyangho (Lake), Taehoman (Bay) and Paekkokchosuchi (Pond). Seven species of trematode metacercariae including two unidentified ones were detected from 1,305 pond smelts. The number of detected trematode metacercariae according to the species are as follow: Clonorchis sinensis 8, Holostephanus nipponicus 7, Cyathocotyle orientalis 24, Diplostomum sp. 14, and Metorchis orientalis 7. From the above results, it was confirmed that H. olidus plays a role as the second intermediate host of some kinds of trematode including C. sinensis in Korea. Our report shows possible clonorchiasis caused by eating raw pond smelts. PMID- 10743359 TI - Infestation status of head louse and treatment with lindane shampoo in children of primary school and kindergarten in Chinju-shi, Kyongsangnam-do, Korea. AB - The infestation status of head louse among children attending primary schools and kindergartens in Chinju-shi, Kyongsangnam-do, Korea, was investigated between June and July 1999. Out of 2,288 children examined, 3.9% of boys (48/1,242) and 23.5% of girls (246/1,046) were infested with nits or adult/nymphs of lice. The effectiveness of lindane shampoo (1% gamma benzene hexachloride solution) was evaluated after one or two time applications to all the children infested. The negative conversion rate of pediculosis was 93.5%. Effective control measures are needed to control and prevent such ectoparasite infestation amongst children. PMID- 10743360 TI - Isozyme electrophoresis patterns of the liver fluke, Clonorchis sinensis from Kimhae, Korea and from Shenyang, China. AB - An enzyme analysis of the liver fluke, Clonorchis sinensis from Kimhae, Korea and from Shenyang, China was conducted using a horizontal starch gel electrophoresis in order to elucidate their genetic relationships. A total of eight enzymes was employed from two different kinds of buffer systems. Two loci from each enzyme of aconitase and esterase (alpha-Na and beta-Na); and only one locus each from six enzymes, glucose-6-phosphate dehydrogenase (G6PD), alpha-glycerophosphate dehydrogenase (GPD), 3-hydroxybutyrate dehydrogenase (HBDH), malate dehydrogenase (MDH), phosphoglucose isomerase (PGI), and phosphoglucomutase (PGM) were detected. Most of loci in two populations of C. sinensis showed homozygous monomorphic banding patterns and one of them, GPD was specific as genetic markers between two different populations. However, esterase (alpha-Na), GPD, HBDH and PGI loci showed polymorphic banding patterns. Two populations of C. sinensis were more closely clustered within the range of genetic identity value of 0.998-1.0. In summarizing the above results, two populations of C. sinensis employed in this study showed mostly monomorphic enzyme protein banding patterns, and genetic differences specific between two populations. PMID- 10743361 TI - Yeah or nay? This congressman supports doctors and patients. PMID- 10743362 TI - Patients suffer when the Medicaid program doesn't work. PMID- 10743364 TI - New MSMS reimbursement service assists in meeting today's stringent payer guidelines. PMID- 10743363 TI - We must look closely at responsibilities before returning any budget surplus. PMID- 10743365 TI - Ask yourself this question from the past: is it good medicine? PMID- 10743366 TI - Hypertension control: challenges and opportunities. PMID- 10743367 TI - Private entry into health insurance: what does it mean for India? PMID- 10743369 TI - Prevalence, awareness, treatment and control of hypertension in an elderly community-based sample in Kerala, India. AB - BACKGROUND: Hypertension is one of the most important causes of cardiovascular morbidity and mortality in the elderly. With the increase in the number of elderly in India, hypertension is likely to emerge as an important public health problem. METHODS: We performed a cross-sectional survey of a random sample of 357 community-dwelling elderly individuals (191 women, 166 men; mean age 70 years) in Kerala. We measured blood pressure on all study participants using a standardized technique to assess the prevalence of hypertension. We compared the variations in prevalence, awareness, treatment and control of hypertension according to age, sex and place of residence of the subjects, and examined the socio-demographic correlates of hypertension using sex-specific multiple logistic regression. RESULTS: The overall prevalence of hypertension in our sample was 51.8% (95% CI: 46.8%-56.8%), which did not vary with sex but increased with age. Fewer than half of the hypertensive subjects were aware of their condition or were on treatment, and only a quarter of the treated hypertensives achieved adequate control of blood pressure. Rural elderly subjects were especially less likely to be aware of, and on treatment for hypertension. Smoking status and rural residence (in men) and marital status (in women) were important correlates of hypertension. CONCLUSIONS: Our findings emphasize the public health importance of hypertension in the elderly in Kerala at present, and point to a likely increase in burden of this problem in India in the near future. A National Hypertension Programme, targeted to meet the imminent public health challenge posed by hypertension seems warranted. PMID- 10743368 TI - Risk factors for cancer nasopharynx: a case-control study from Nagaland, India. AB - BACKGROUND: A high incidence of nasopharyngeal carcinoma has been reported from Nagaland, though it is considered to be a rare neoplasm in India. No case-control study to identify the risk factors of cancer nasopharynx has been conducted in this region. This study was undertaken to identify dietary and environmental risk factors for nasopharyngeal carcinoma relevant to this region. METHODS: A matched case-control study using neighbourhood controls was conducted. For each of the 47 cases identified, 2 apparently healthy neighbourhood controls were matched for age, sex and ethnicity. All information on dietary, environmental, social and demographic factors was collected. Univariate and multivariate logistic regression analysis using maximum likelihood method was used to analyse data. RESULTS: Consumption of smoked meat was found to be the risk factor for nasopharyngeal carcinoma (adjusted odds ratio = 10.8; 95% CI 3.0-39.0). History of using herbal nasal medicine was also found to be associated with nasopharyngeal carcinoma (OR = 21.9, CI = 6.8-71.4). However, exposure to a smoky atmosphere, betel-nut chewing, use of smokeless tobacco products, smoking and drinking habits were not found to be associated with nasopharyngeal carcinoma. CONCLUSION: This study reveals an association of nasopharyngeal carcinoma with consumption of smoked meat in Nagaland. The use of herbal nasal medicine seems to be an additional risk factor for nasopharyngeal carcinoma in Nagaland and needs further assessment. PMID- 10743370 TI - Adverse drug reactions among inpatients in a north Indian referral hospital. AB - BACKGROUND: Monitoring spontaneous adverse drug reactions is one of the epidemiological methods for assessing the safety of drugs in a hospital setting. METHODS: Data on adverse drug reactions (ADRs) were collected over a 3-year period among inpatients in a north Indian referral hospital using the spontaneous ADR monitoring system. RESULTS: A total of 317 ADRs were reported. Cutaneous reactions (38.8%) and gastrointestinal disturbances (28.4%) made up a large proportion of reported ADRs. Antimicrobial agents, including those used for antituberculosis therapy, were responsible for 47.3% of the events. Radiocontrast dyes, and antineoplastic and psychoactive agents were the other important drugs causing ADRs. No age- or sex-related differences were found in the overall rate of ADRs, though toxic epidermal necrolysis was more frequent in the elderly. CONCLUSION: The pattern of ADRs and drugs involved were largely similar to those reported in the western literature. PMID- 10743371 TI - High-tech imaging: impact on clinical medicine. PMID- 10743372 TI - Sleep apnoea and road traffic accidents. PMID- 10743373 TI - Calcium and the treatment of nutritional rickets. PMID- 10743374 TI - Basic counselling skills for medical practice. PMID- 10743375 TI - Sampling techniques, confidence intervals, and sample size. PMID- 10743376 TI - Teenage pregnancies in Scotland. PMID- 10743377 TI - Caesarean sections on the rise. PMID- 10743378 TI - Medical students: their attitudes to organ donation and transplantation. PMID- 10743379 TI - Convulsions in non-epileptics due to mefloquine-fluoroquinolone co administration. PMID- 10743380 TI - Provide water, health and education--not nuclear weapons. PMID- 10743381 TI - Yet to surface problems of corneal refractive surgery. PMID- 10743382 TI - Untrained speakers' use of prosody in syntactic disambiguation and listeners' interpretations. AB - We investigated how naively produced prosody affects listeners' end interpretations of ambiguous utterances. Non-professional speakers who were unaware of any ambiguity produced ambiguous sentences couched in short, unambiguous passages. In a forced-choice task, listeners could not tell which context the isolated ambiguous sentences came from (Exp. 1). However, listeners were able to correctly paraphrase the least ambiguous subset of these utterances, showing that prosody can be used to resolve ambiguity (Exp. 2). Nonetheless, in everyday language use, both prosody and context are available to interpret speech. When the least ambiguous sentences were cross-spliced into contexts biasing towards their original interpretations or into contexts biasing towards their alternative interpretations, answers to content questions about the ambiguous sentence, confidence ratings, and ratings of naturalness all indicated that prosody is ignored when context is available (Exp. 3). Although listeners can use prosody to interpret ambiguous sentences, they generally do not, and this makes sense in light of the frequent lack of reliable prosodic cues in everyday speech. PMID- 10743383 TI - The cataphoric use of spoken stress in narratives. AB - This study investigated the effects of introducing a concept into discourse either marked with spoken stress or in an unstressed form using a story continuation paradigm. Participants first listened to informal narratives and then continued them. The final phrase of the narratives introduced a new concept. If that concept had been introduced in a stressed form, participants referred to it more often and sooner. Also, there was a tendency towards using a higher proportion of pronominal references as opposed to NP references. These results closely resemble the findings of Gernsbacher and Shroyer, who, using the same experimental procedure, investigated the effect of marking a concept with the unstressed, indefinite article this as opposed to a/an. The two studies together support the notion of a common cataphoric function of the two linguistic devices; they mark concepts the speaker is likely to refer to again. PMID- 10743384 TI - Patterns, chunks, and hierarchies in serial reaction-time tasks. AB - The impact of relational structures (i.e., the systematicity of relations between successive items) on incidental sequence learning was investigated in a serial reaction-time (SRT) task while keeping constant the statistical structure. In order to assess the influence of relational structures in stimulus and response sequences separately, the strength of relational patterns in sequences of digits as stimuli and of keystrokes as responses was orthogonally varied. In Exps. 1 and 2, the variation of relational patterns was mainly effective in the keystroke sequence. In Exp. 2, in addition to the variation of relational patterns, the presentation of stimuli was delayed at serial positions that were incongruent with the relational structure. The results show that these incongruent pauses reduced the learning of strongly structured sequences of keystrokes but improved the learning of weakly structured sequences. Experiment 3 suggests that even higher-order relations between elementary patterns are utilized to accelerate responses. The data are interpreted as evidence for the impact of relational patterns, in addition to statistical redundancies, on the formation of chunks. Reasons are discussed for the finding that relational chunking was more pronounced in the keystroke than in the digit sequences. PMID- 10743385 TI - Temporal, but not spatial, context modulates a masked prime's effect on temporal order judgment, but not on response latency. AB - Three experiments investigated a dissociation originally described by Neumann, Esselmann, and Klotz. Stimuli were geometric shapes, preceded by similar shapes that were masked by metacontrast. Each experiment consisted of three parts. In the reaction time (RT) part, participants saw an array of geometric shapes, one of which was marked by bars, and had to respond to the marked shape's position by pressing an appropriate button. A prime (a similar, but smaller stimulus) preceded either the marked or an unmarked stimulus. In the temporal order judgment (TOJ) part, the task was to judge the temporal order of the marked and the unmarked stimulus. In the detection part, detectability of the prime was tested. Although its detectability was zero or close to zero, the prime affected both RT and the apparent onset as measured by TOJ. The effect on RT was significantly larger than the effect on TOJ (Exp. 1). Increasing the spatial context (number of non-target stimuli in the display) did not affect this pattern (Exp. 2). By contrast, reducing the temporal context (range of stimulus onset asynchronies) abolished the prime's effect in the TOJ task, although the prime affected RT under identical conditions. It is concluded that partially different mechanisms mediate the prime's effect in the two tasks and that the effect of stimulus context on TOJ found in the Neumann et al. study was due to temporal, not spatial context. PMID- 10743386 TI - Depth motion sensitivity functions. AB - Functions reliably describing perception of motion in depth have been established experimentally by using psychophysical methods of size and distance estimations and threshold measurements. The stimuli were generated with a new hybrid technique yielding an image refresh rate of 1667 Hz. In this way it was possible to generate rapid expansions and contractions of the moving checkerboard pattern constituting the stimulus for depth motion perception. The results showed that perceived size constancy as well as depth impression varied with oscillation frequency. Under the conditions of slow motions (oscillation frequencies around 2 Hz), perfect size constancy was obtained. Above that limit, size constancy systematically decreased, and with oscillation frequencies of about 5 Hz the perceived size constancy was close to zero when small-sized patterns were used. Under the conditions of wide field stimulation (when the pattern subtended 66 degrees of visual angle), the cut-off limit increased to 16 Hz. Since the perception of depth motion amplitudes as well as perceived velocities of the visual object are related to perceived size constancy, the findings have certain implications for theoretical explanations of depth motion perception. PMID- 10743387 TI - Temporal stability of rhythmic tapping "on" and "off the beat": a developmental study. AB - This study, following a dynamic pattern approach, examines age-related differences in the stability of unimanual rhythmic perception-action patterns. Thirty-six children, aged 7, 9, and 11 years, attempted to synchronize their finger tapping to the beats of an auditory metronome, either "on the beat" (i.e., in-phase coordination), or "off the beat" (i.e., antiphase coordination). The temporal stability of these perception-action patterns was measured by the variability of the relative phase between taps and auditory events and by the critical frequency, that is, the frequency at which a loss of stability was observed when the metronome frequency was increased. Age-related differences in stability were found for both relative phase variability and critical frequency. These findings suggest that the relative phase dynamics underlying perception action coordination patterns change with age in the direction of an increased temporal stability. PMID- 10743388 TI - Comfort constrains graphic workspace: test results of a 3D forearm model. AB - Human movement performance is subject to many physical and psychological constraints. Analyses of these constraints may not only improve our understanding of the performance aspects that subjects need to keep under continuous control, but may also shed light on the possible origins of specific behavioral preferences that people display in motor tasks. The goal of the present paper is to make an empirical contribution here. In a recent simulation study, we reported effects of pen-grip and forearm-posture constraints on the spatial characteristics of the pen tip's workspace in drawing. The effects concerned changes in the location, size, and orientation of the reachable part of the writing plane, as well as variations in the computed degree of comfort in the hand and finger postures required to reach the various parts of this area. The present study is aimed at empirically evaluating to what extent these effects influence subjects' graphic behavior in a simple, free line-drawing task. The task involved the production of small back-and-forth drawing movements in various directions, to be chosen randomly under three forearm-posture and five pen-grip conditions. The observed variations in the subjects' choice of starting positions showed a high level of agreement with those of the simulated graphic-area locations, showing that biomechanically defined comfort of starting postures is indeed a determinant of the selection of starting points. Furthermore, between condition rotations in the frequency distributions of the realized stroke directions corresponded to the simulation results, which again confirms the importance of comfort in directional preferences. It is concluded that postural rather than spatial constraints primarily affect subjects' preferences for starting positions and stroke directions in graphic motor performance. The relevance of the present modelling approach and its results for the broader field of complex motor behavior, including the manipulation of tools, is indicated briefly. PMID- 10743389 TI - [Comments about the article on taeniasis and cysticercosis caused by Taenia solium]. PMID- 10743390 TI - Comments about the article on the clinico-epidemiologic characteristics of cholera patients in the metropolitan area. PMID- 10743392 TI - [Challenges and updating in public health]. PMID- 10743391 TI - [Comments about the article on the perception of the body image as a qualitative approximation to the nutritional status]. PMID- 10743393 TI - [Prevention and control of the smoking world epidemic: an integral strategy]. PMID- 10743394 TI - [Prevalence of malnutrition in Tarahumara children under 5 years of age in the municipality of Guachochi, Chihuahua]. AB - OBJECTIVE: To determine the prevalence of malnutrition among Tarahumara children under 5 years of age in the municipality of Guachochi, Chihuahua, Mexico. MATERIAL AND METHODS: During the summer of 1996, we interviewed a representative sample of 450 children from 62 communities, stratified by size of population. The sample represents 12.2% of the total number of Tarahumaras in the municipality. For each of three anthropometric indices (weight-for-age, weight-for-height and height-for-age), we calculated the Z-score in reference to NCHS population values. Results are presented as means and standard deviations as well as prevalence of malnutrition at different cut-off-points, stratified by age, sex, and size of the community. RESULTS: Prevalence figures of malnutrition (< -2Z) were: weight-for-age: 36.4% (mean SD +/- -1.66 +/- 1.1); weight-for-height: 3.5% (mean SD +/- -0.43 +/- 0.9); and height-for-age: 57.1% (mean SD +/- -2.15 +/- 1.3). Children aged 12-23 months were the most affected, both in weight-for height and weight-for-age (10.3% and 52.6%, respectively). Children over one year of age presented prevalences of height-for-age < -2Z over 60%. Significant sex differences (p < 0.05) were observed, favoring girls. CONCLUSIONS: Malnutrition among Tarahumara children reaches its peak during the second year of life (12-23 months old), affecting boys more than girls. This information may be useful for planning and targeting nutrition intervention programs for this underprivileged indigenous group. PMID- 10743395 TI - [Strategies for teaching self-examination of the breast to women in reproductive age]. AB - OBJECTIVE: To develop an effective strategy to inform Mexican women between 12 and 47 years of age about breast cancer (BC) and train them to perform breast self-examination (BSE). MATERIAL AND METHODS: Between April, 1996 and July, 1998, two different strategies to teach BSE were designed and evaluated in Cuernavaca, State of Morelos, Mexico. These strategies included teaching materials and were based on opinions of women participating in the first stage of this study. Both strategies consisted of the presentation of a leaflet and a video. The first strategy was delivered by a public health nurse in charge of leading the session. The second one was presented by a BC survivor. This second strategy included slides and silicon breast models. These two strategies were then compared to a third one, consisting of a primary care nurse handling out a leaflet and showing a video, based on Health Secretariat's guidelines. In total, 149 women were trained. They were randomly assigned to any one of these three strategies. The effectiveness of the teaching strategies was determined by assessing the changes in the women's knowledge of BC and BSE, as well as in their ability to identify the largest number of lumps on a natural size silicon breast, before and after training. Statistical analysis included Student's t test, variance analysis, McNemar's chi 2, Pearson's chi 2, and multiple linear regression. RESULTS: With all three strategies, there was an increase of approximately 30% in women's knowledge of BC and BSE as well as in their ability to detect lumps. Although at the beginning of the training only one out of every 20 women were able to identify more than three lumps in the silicon model, by the end between 3 and 4 of every 10 women were able to do this, with values of p < 0.05. Some factors determine women's learning of both BSE and information about BC as well as their ability to identify lumps. CONCLUSIONS: Teaching BSE with any of the strategies evaluated could be included in an integral program to educate Mexican women on breast cancer prevention and early detection. The appropriate and monthly practice of BSE is linked both to a decrease in the amount of time elapsing between the identification of suspect signs and the demand for medical care, such as requesting a clinical breast examination, which entails identifying small tumors when BC is present. PMID- 10743396 TI - Risk factors of breast cancer in Mexican women. AB - OBJECTIVE: To investigate the association between family history (FH) of neoplasia, gyneco-obstetric factors and breast cancer (BC) in a case-control study. In cases, to analyze those variables in relation with early onset of BC, the manner of detection (self-examination, prompted by pain, or casual), the size of tumor, and the elapsed time to seek medical attention. MATERIAL AND METHODS: Data from 151 prevalent BC cases and 235 age-matched controls were analyzed by multiple logistic regression, to assess the influence of BC risk factors. RESULTS: Ten per cent of patients and 1% of controls had first-degree relatives (FDR) with BC. Family history of FDR with BC (OR, 11.2; 95% CI 2.42-51.92) or with gastric or pancreatic cancer (OR, 17.7; 95% CI 2.2-142.6) was associated with BC risk. Breastfeeding at or under 25 years of age was protective against BC (OR, 0.40; 95% CI 0.24-0.66). The manner of tumor detection did not influence its size at the time of diagnosis. CONCLUSIONS: Our study confirms that FH of BC and/or of gastric or pancreatic carcinoma are risk factors for BC, while lactation at 25 years of age or earlier is protective. PMID- 10743397 TI - [Knowledge of health professionals about the prevention of cancer the cervix. Alternatives to medical education]. AB - OBJECTIVE: Several studies have shown the importance of health care professionals as predictors of the use of cervical cancer screening (CCS). MATERIAL AND METHODS: A cross-sectional study of 520 health care professionals in the State of Morelos during 1998, in order to evaluate and quantify their level of knowledge on the impact, etiology, screening, diagnosis and treatment of cervical cancer. A 1 to 10 scale questionnaire was given, and the sample included family medicine specialists, general practitioners, specialist and general nurses, and social workers. Statistical analysis included analysis of variance and 95% confidence intervals. RESULTS: A knowledge mean of 4.74 (95% CI 4.57-4.88) was observed for a scale of 10; specialist physicians scored higher (mean 5.21, 95% CI 4.81-5.60) than social workers (mean 3.07, 95% CI 2.31-3.82). Periodicity of the Pap test in most cases was less than 1 year and there was poor consensus about the age period during which the Pap test should be obtained. The knowledge level was lower when trying to identify etiologic aspects and treatment perspectives. CONCLUSIONS: The results of this study show that, besides the improvement of undergraduate academic programs, it is necessary to improve educational interventions for health care professionals through the updating, recycling, training, health education, and continuing medical education, among others, to promote professional competence and thus improve the quality of medical care. PMID- 10743398 TI - [Prevalence of Chlamydia trachomatis infection in registered prostitutes in the city of Durango, Mexico]. AB - OBJECTIVE: To determine the prevalence of Chlamydia trachomatis infection among registered prostitutes of Durango City and to establish whether there is a correlation between epidemiological factors and infection. MATERIAL AND METHODS: Two-hundred-and-forty-seven registered prostitutes of Durango city were studied. Endocervical samples and epidemiological data were obtained. C.trachomatis antigen was detected with the Chlamydiazyme test (Abbott Laboratories, USA.) RESULTS: Forty-one (16.6%) out of 247 prostitutes were positive to C. trachomatis. Thirty-seven out of the 41 positive women had had sexual activity on several States of Mexico (95.1%), as compared to only 109 out of 206 negative women (53.0%) (p < .0001). Prostitutes positive to C. trachomatis (39/41, 95.1%) were more likely to belong to low socioeconomic level than negatives (171/206, 83%) (p = 0.05). CONCLUSIONS: The prevalence of C. trachomatis infection was 16.6%. C. trachomatis infection was associated with sexual activity in multiple States of Mexico, and had a tendency to be associated with low socioeconomic level. PMID- 10743399 TI - [Tuberculosis in health personnel: importance of surveillance and control programs]. AB - OBJECTIVE: To describe tuberculosis surveillance results among healthcare workers of a tertiary care center. MATERIAL AND METHODS: All medical records of workers from 1992-1998 were reviewed. Demographics, labor, medical history, previous testing, PPD, booster shots and follow-up were analyzed. Statistical analysis was performed with odds ratios, p-values, and 95% confidence intervals. Subgroup analysis were done with chi 2. Kaplan-Meier estimates were used to analyze times to conversion. RESULTS: Surveillance was done in 1617 workers (68% female and 32% male). Mean age was 26.9 +/- 7.6 (15-68) years. Job positions were 30.5% nurses, 14.6% residents and 14.1% interns. Place of origin was Mexico City in 65.8%. BCG vaccination was present in 71.6% and 15.1% had previous PPD. Admission PPD was positive in 39.6%, negative in 48.3% and 12.1% were lost to follow-up. On negatives, 483 booster shots were applied, and 49 additional positives were found. Follow-up was done in 231 workers, of which 100 (43.3%) converted. The mean time for conversion was 22.8 +/- 12.4 months. The conversion rate at twelve months was 20%. Fifty workers received/accepted isoniazid prophylaxis. CONCLUSIONS: A high percentage of workers were PPD-positive; booster shots allowed the detection of an additional 10%. A high conversion rate underscores the need to organize tuberculosis control programs in Mexico. PMID- 10743400 TI - [Acute pesticide poisoning]. AB - OBJECTIVE: To describe the epidemiologic pattern of acute pesticide poisoning (APP) in a general hospital in Merida, Yucatan, Mexico. MATERIAL AND METHODS: From 1994 to 1998, 33 patients 13 years of age or older with diagnosis of APP were studied. Descriptive statistics were used to analyze information. RESULTS: Males were frequently affected (82%), specially those coming from rural areas (60%). The mean age of the group was 34 +/- 15.8 years. In 79% of the cases, pesticides were used to commit suicide and 33% of poisoning cases were due to organophospate pesticides. The mortality rate was 12%. CONCLUSIONS: In this small sample, acute poisoning from pesticides in the agricultural setting may be underestimated, since it was less frequent than in the general population. APP was more commonly used by indigent people to commit suicide. PMID- 10743402 TI - [1999 balance]. PMID- 10743401 TI - [Myths about the prevention and control of non-communicable diseases in Latin America]. AB - Given the increase of the burden of non-communicable diseases (NCD) and the possibility to avoid it, it is urgent to implement or strengthen NCD preventive programs in Latin America. However, many myths hinder the implementation of NCD programs. Myths on NCD include: a) NCD are degenerative and incurable; b) they are diseases of the elderly; c) they are diseases of the rich. Like wise there are myths about NCD preventive programs are: a) difficult to implement, b) expensive, and c) ineffective. We present data that demonstrate how these myths are untrue and discuss the challenges to find a balanced health policy that emphasizes the importance of NCD without overlooking other diseases. PMID- 10743403 TI - [Basic information about resources and services of the national health system]. PMID- 10743405 TI - Forecasting in communicable diseases. WHO Eastern Mediterranean Region. Part I. PMID- 10743404 TI - Dracunculiasis eradication. Certification of absence of transmission. AB - Dracunculiasis transmission is now confined to the African continent. Eradication efforts must be intensified in the countries that are still endemic: Benin, Burkina Faso, Central African Republic, Cote c'Ivoire, Ethiopia, Ghana, Mali, Mauritania, Niger, Nigeria, Sudan, Togo and Uganda. In these countries provision of safe drinking-water, containment of cases and health education for the exposed populations remain key measures for the interruption of transmission++ and for success. The Commission felt that particular attention should be given to Ghana, Nigeria and Sudan where eradication activities have been facing difficulties. In countries where transmission has recently ceased (Chad, Senegal and Yemen) or where no indigenous cases were reported in 1999 (Cameroon, Kenya), surveillance must be intensified over a period of 3 consecutive years and should include coverage of non-endemic regions at risk. In addition, reporting should continue and be reinforced to ensure the provision of monthly reports to WHO. PMID- 10743406 TI - Priority setting for nursing research. PMID- 10743407 TI - Determinants of psychological well-being in Irish immigrants. AB - Immigration accounts for nearly half of the U.S. population growth in the past 20 years, and this trend is expected to continue. The purpose of this study is to examine the relationship between demographics, resilience, life satisfaction, and psychological well-being among Irish immigrants. Employing a cross-sectional design, a sample of 100 Irish immigrants completed the Demographic and Migration Questionnaire, the Resilience Scale, the Migration Quality of Life Scale, and the General Well-Being Schedule. Hierarchical regression analyses were used to test the association of psychological well-being with demographics, resilience, and life satisfaction. Findings revealed that number of annual health care appointments, higher resilience, and greater life satisfaction were the strongest predictors of psychological well-being. PMID- 10743408 TI - Refugee experiences and Southeast Asian women's mental health. AB - The wars in Southeast Asia displaced thousands of families from Cambodia, Laos, and Vietnam. The upheavals led to a number of waves of immigration to the United States. Current research supports hypotheses of post-traumatic stress disorder diagnoses in refugees from the wars in Vietnam but omits pertinent cultural factors. This phenomenological study of 19 women from Southeast Asia examines the meanings of their refugee experiences. Open-ended interviews with these women reveal themes of survival, despair, and isolation. Health care providers may notice cultural bereavement as opposed to post-traumatic stress disorder, reflecting a psychological resilience not extensively explored previously. Developing empathetic interactions and including important ethnic identity factors in caring for refugee women appear essential in providing appropriate health care. PMID- 10743410 TI - Folklore linked to pregnancy and birth in Nigeria. AB - As a part of a safe motherhood project implemented in eastern Nigeria between 1992 and 1996, in-house interviews were conducted with rural women and traditional birth attendants in the seven states of eastern Nigeria. The overall project was designed to contribute toward the reduction of maternal mortality and morbidity through the involvement of community leaders and women's organizations in women's health activities in rural Nigeria. It also focused on identifying and addressing some of the underlying cultural factors in maternal mortality and morbidity in Nigeria. Findings from the interview show that women in rural eastern Nigeria still hold many folklore beliefs about pregnancy and childbirth, and some of these beliefs lead to delay in the referral of complications to hospitals. PMID- 10743409 TI - An ethnographic study of a day care center for Iranian immigrant seniors. AB - Late-in-life immigrants are often at risk for psychological stress and social isolation because of language problems, small social networks, and cultural differences from the host society. Community intervention programs can reduce such stress and isolation. In this article, the authors describe a Swedish municipality's culturally appropriate intervention program for elderly Iranian immigrants based on ethnographic data gathered during a 12 month period. The description includes the activities provided by the program and the experiences of the elderly Iranian immigrants who participate regularly in these activities. The findings document the positive impact of regular participation in the center's activities on the elders' well-being and health. PMID- 10743411 TI - Mexican Americans' perceptions of culturally competent care. AB - Although the literature abounds with authors' discussions of the need for nurses and other health care providers to deliver safe and optimal care to patients of diverse ethnic groups, little work has focused on defining and measuring the dimensions of culturally competent care. The purpose of this research was to identify culturally competent concepts from the perspective of Mexican Americans. Focus group interviews with Mexican American registered nurses and Mexican American lay recipients of health care were used to explore the participants' subjective perceptions regarding the indicators of culturally competent care. For this group of Mexican American registered nurses, the influence of culture remained strong despite nursing professional experience and knowledge of Western biomedical system. The predominance of themes emphasizing respect, caring, understanding, and patience in health care encounters support the critical importance of personal processes of health care with Hispanics. PMID- 10743413 TI - Accessing vulnerable populations for research. PMID- 10743414 TI - Managed care--what potential for export? PMID- 10743412 TI - A rural perspective on home care communication about elderly patients after hospital discharge. AB - Little is known about home health agencies (HHA) and their integration in the continuum of care in rural areas. The aims of this study are to describe the amount and type of patient-related data transferred by discharging hospitals to rural HHAs and to explore the influence of selected organizational factors on that communication process using a previously tested model of interorganizational communication. In this study, 446 closed-case, elderly patient records at three rural HHAs were reviewed using the Referral Data Inventory. Rural HHAs receive about half of the literature-recommended referral data, characterized primarily by background data, some medical data, and almost no psychosocial or nursing-care data. Referrals transmitted by telephone and written data were superior to referrals transferred by a telephone call only. Hospital-affiliated HHAs received significantly greater amounts and richer types of referral data than did free standing HHAs. Findings suggest that cost-saving measures in the referral process need investigation. PMID- 10743415 TI - Development of the course in applied basic sciences for the new MRCS examination. PMID- 10743416 TI - The Raven Department of Education: a strategic perspective. PMID- 10743417 TI - The CSiG examination. PMID- 10743418 TI - Short-listing for specialist registrars: consultants versus medical personnel. PMID- 10743419 TI - The Sheffield basic surgical training scheme. PMID- 10743420 TI - Transition for the dental specialties. PMID- 10743421 TI - Measuring and modelling surgical bed usage. AB - Surgical departments treat two groups of inpatients--the simple and the complex- consequently a single average fails to describe the use being made of the occupied beds. Using decision support techniques, we show why indicators such as the average length, the average occupancy and the average admissions mislead. Furthermore, by analysing the fluctuating pattern of weekly admissions we show how weekends and the Christmas holiday periods impact on bed usage. Next, we demonstrate that flow process models can be used to describe how the in-patient workload concerns two groups of patients. On an average day, 71.4% of the beds contained patients who will have an average (exponential) stay of 4.8 days, and the other beds, 28.6%, contain patients who will have an average (exponential) stay of 22.8 days. The article concludes by demonstrating the short and long-term impact on daily admissions of a 10% change in four different parameters of the model. The data used come from a surgical department in Adelaide, as UK data sets report finished consultant episodes rather than completed in-patient spells. PMID- 10743422 TI - Human reliability assessment in surgery--a new approach for improving surgical performance and clinical outcome. AB - Surgical operative performance is currently assessed by audit of morbidity, mortality and, especially in patients with cancer, in terms of long-term outcome. Its chief merit is the identification of problems and sub-optimal results by individual surgeons/centres. There is one aspect of audit that constitutes its intrinsic weakness, the verdict on performance it gives is always retrospective- the problem is identified because of the bad results thrown up by analysis of the data. As a result, there is a distinct possibility that surgeons might exclude patients with potentially curative conditions because of increased operative risk due to co-morbid disease from major surgery because of a fear, conscious or otherwise, of comparative under-performance. There is a methodology in established use by industry that is both prospective and prescriptive in ensuring optimal performance--human reliability assessment (HRA), which can be translated into clinical practice. This paper explains the nature of HRA and reports on its initial use in surgery. PMID- 10743423 TI - The role of simulation in surgical training. AB - Surgical training has undergone many changes in the last decade. One outcome of these changes is the interest that has been generated in the possibility of training surgical skills outside the operating theatre. Simulation of surgical procedures and human tissue, if perfect, would allow complete transfer of techniques learnt in a skills laboratory directly to the operating theatre. Several techniques of simulation are available including artificial tissues, animal models and virtual reality computer simulation. Each is discussed in this article and their advantages and disadvantages considered. PMID- 10743424 TI - A regional survey of emergency surgery: the trainees' perspective. AB - The reduction of junior doctors' hours and the 'Calmanisation' of higher surgical trainees have led to an inevitable decrease in clinical experience. The development of subspecialisation within general surgery limits the diversity of elective operative experience, while the resident surgical registrar continues to be faced by the same range of emergencies. Procedures such as tracheostomy, thoracotomy and emergency burr hole, although rare in an emergency setting, are seldom seen by surgical trainees outside ENT, cardiothoracic and neurosurgical departments, respectively. However, these life saving procedures continue to be within the remit of the general surgeon, and were considered as essential knowledge in the operative viva of the FRCS examination. PMID- 10743425 TI - Traumatic diaphragmatic rupture: associated injuries and outcome. AB - A retrospective case note analysis was performed on all patients treated for traumatic diaphragmatic rupture (TDR) at a major teaching hospital between January 1990 and August 1998. Patients were identified from the prospectively maintained UK Trauma and Research Network Database. Of the 480 cases of torso trauma admitted during the study period, 16 (3.3%) had TDR. Blunt trauma accounted for 13 (81%) of the injuries. A radiological pre-operative diagnosis was made in 10 (62.5%) patients. Seven of these were made on initial chest radiography, two on ultrasound scan and one on computed tomography. All patients underwent a midline laparotomy and TDR was subsequently diagnosed at operation in 6 patients. The left hemidiaphragm was ruptured in 14 (87.5%) patients and there was visceral herniation in 8 (50%). Twelve patients with blunt trauma had associated abdominal and extra-abdominal injuries, but only one of the three patients with penetrating trauma had other injuries. The median Injury Severity Score (range) was 21 (9-50). The median time (range) spent on the intensive care unit was 2 days (0-35 days). Pulmonary complications occurred in 7 (44%) patients. Two (12.5%) patients died from associated head injuries. TDR results from blunt and penetrating torso trauma, is uncommon, rarely occurs in isolation and is associated with a high morbidity and mortality. A high index of suspicion makes early diagnosis more likely as initial physical and radiological signs may be lacking. PMID- 10743426 TI - Early diagnosis of traumatic rupture of the right hemidiaphragm. AB - A case of traumatic rupture of the right hemidiaphragm, which was diagnosed early on the basis of clinical and radiographic suspicions, is discussed. PMID- 10743427 TI - Presentation of blunt small intestinal and mesenteric injuries. AB - During the 10 year period from 1988 to 1997, 64 patients with blunt small bowel and mesenteric injuries were treated at two trauma centres. The majority (52 cases) were victims of motor vehicle accidents, and 54% of them wore seat belts at the time of the accident. There were 22 small bowel injuries (17 full thickness and 5 seromuscular) and 42 mesenteric injuries (7 with and 35 without a devascularised bowel segment). Shock on admission was present in 34% of the patients and generalised abdominal tenderness in 75%. Diagnostic peritoneal lavage was positive for blood in 25 out of 36 cases in which it was performed (69%), and positive for bowel content in 4/6 patients (67%) with full-thickness bowel perforations or transactions. Emergency room ultrasound was positive for blood in 13/25 cases (52%), and CT scan in 7/17 (41%). It is concluded that blunt small bowel and mesenteric injuries including patients with perforated or ischaemic bowel are difficult to diagnose using currently available diagnostic tools, and require a low threshold for exploration based on clinical suspicion in order to reduce the complications following delayed treatment of these injuries. PMID- 10743429 TI - Inguinal hernia: medicolegal implications. AB - Repair of an inguinal hernia is one of the commonest operations undertaken by surgeons but the role of trauma in causing inguinal hernia is not well understood. This paper does not attempt to discuss the cause of inguinal hernia but seeks to analyse the cases which may be accepted by the Courts as being due to trauma. PMID- 10743428 TI - Successful treatment of Rhodococcus equi pulmonary infection in a renal transplant recipient. AB - The rhodococcus is a mycobacterium-like organism which is normally a pathogen in foals. It usually spreads by direct contact or by aerosol from horse faeces and causes pyogranulomatous pulmonary infections. Occasionally, it acts opportunistically to infect immuno-compromised human hosts, most commonly those with the acquired immune deficiency syndrome (AIDS). Here we report a pulmonary infection by Rhodococcus equi in a renal transplant recipient who was successfully treated. The literature on this infection in transplant recipients is also reviewed with respect to manifestations and treatment. PMID- 10743430 TI - Analgesic requirements for appendicectomy: the differences between adults and children. AB - AIM: The analgesia provided for children is often less than for adults with the same underlying pathology. This paper attempts to quantify the postoperative analgesic requirements of patients undergoing appendicectomy. METHODS: Patients between 6 and 30 years of age who underwent an unscheduled appendicectomy were prospectively recruited. Regular non-opiate analgesia, calculated according to weight, was administered. Hourly visual analogue pain scores and morphine patient controlled analgesia (mPCA) usage were recorded for 24 h following surgery. RESULTS: 19 children (6-16 years) and 23 adults (17-30 years) were recruited. There was no significant difference in the pain scores following appendicectomy between the two groups. Significantly more mPCA was demanded (t = 2.02, P < 0.02) and morphine received (t = 2.02, P < 0.005) by adults than children following appendicectomy. CONCLUSION: Children appear to require and do demand less analgesia than adults following appendicectomy to maintain similar postoperative pain scores. Acceptable pain scores may be achieved by the administration of regular analgesia to these patients. PMID- 10743431 TI - Mesh repair of a coccygeal hernia via an abdominal approach. AB - We report on the presentation and management of a patient with herniation of the rectum following a coccygectomy. We used an abdominal approach and careful pelvic dissection to define the defect in the pelvic floor at the site where coccyx used to be. Prolene mesh repair resulted in the reduction of the hernia. To our knowledge, this is the first report on the mesh repair of the coccygeal hernia via an abdominal approach. PMID- 10743432 TI - Tourniquet use during varicose vein surgery: a survey of current practice among Wessex surgeons. AB - The use of a tourniquet during varicose vein surgery, has been shown, through previous randomised trials, to result in a significant reduction in blood loss, superior post-operative cosmesis with no increase in operating time. Nonetheless, it would seem that few surgeons use this technique. Using postal questionnaires (n = 107), we have assessed the views and current practice among general surgeons (consultants and higher surgical trainees) in Wessex where the method was first proposed, to see how widely it has been adopted. We find that the majority (69.5%) of general surgeons in Wessex never use a tourniquet during varicose vein surgery. Possible reasons for this include the belief that it is time consuming, inconvenient, compromises the sterility of the operative field and confers no advantage. We conclude, however, that by not using a tourniquet during varicose vein surgery, surgeons are overlooking an important, evidence-based technique. Given that in the UK over 50,000 patients per year undergo operative varicose vein procedures, this can only adversely affect the delivery of quality healthcare to a large group of patients. PMID- 10743433 TI - Varicose veins made easy! AB - A novel device that is more convenient than other tourniquet systems is described. PMID- 10743434 TI - Pre-operative evaluation of the lower extremity prior to microvascular free fibula flap harvest. AB - The microvascular free fibula flap, is currently one of the preferred methods for reconstruction of the oromandibular defect. The patency of the major vessels in the donor limb should be evaluated before the fibula is harvested because the blood supply can be inadequate to safely utilise this flap. The best method of evaluating, pre-operatively, the lower limb vasculature is controversial. Femoral angiography has been considered as the gold standard, however, the current literature advocates less invasive methods of assessment such as magnetic resonance angiography and colour flow Doppler. A postal questionnaire was sent to all members of The British Association of Head and Neck Oncologists asking details of the preferred method of lower limb vascular assessment prior to fibula flap harvest. Of 137 responses, 48 performed free fibula flaps. Of these 48 surgeons, the preferred method for evaluation was palpation of pulses combined with either angiography (40%) or Doppler on the ward (38%). None of this subgroup of surgeons utilised colour flow Doppler as a first line investigation despite this being available to 67% of responders. This survey highlights the diversity in pre-operative assessment amongst surgeons performing fibula flaps for head and neck malignancy. Few relied on clinical examination alone; however, the less invasive methods of vascular imaging were seldom utilised. PMID- 10743435 TI - Lavate vestras manus. Handwashing Liaison Group. AB - Hospital acquired infection has a direct effect on the quality of patient care and is therefore, a major issue in the context of clinical governance. The role of hand washing by health care workers in hospital acquired infection is discussed and recommendations made. PMID- 10743436 TI - Percutaneous (Portex) tracheostomy: an audit of the Newcastle experience. AB - The purpose of this study was to audit the results of percutaneous tracheostomies performed by ENT surgeons in Newcastle. During a 3-year period, 298 tracheostomies: 196 percutaneous and 102 open were studied. A complication rate for percutaneous tracheostomy was found to be 10% compared to 8% for open, this difference was not statistically significant chi 2 = 0.279, P = 0.598 (DF = 1). The indications for percutaneous tracheostomy were mainly limited to respiratory support for intensive care unit patients, whereas the majority of open tracheostomies were performed in the operating theatre. The results of this audit suggest that percutaneous tracheostomy is the method of choice for respiratory support in the intensive care unit and is as safe as the open technique. PMID- 10743437 TI - Laryngeal mask airway and fibre-optic tracheal inspection in thyroid surgery: a method for timely identification of tracheomalacia requiring tracheostomy. AB - Use of the laryngeal mask airway combined with fibre-optic laryngoscopy in thyroid surgery was first described in 1991. In this unit, it has been successfully used in over 130 cases. The advantages in identification and preservation of the recurrent laryngeal nerves using this technique have been demonstrated. However, to date, no report exists of a further advantage, namely the management of tracheomalacia. PMID- 10743438 TI - McIndoe's scissors--a simple technique to aid distal locking of an intramedullary nail. AB - Distal locking of intramedullary nails may be difficult and time consuming. We describe a simple technique using a readily available instrument (McIndoe's scissors) to aid start of this procedure and to locate both distal locking holes at one time. By holding the scissors out of the operative field and facilitating rapid incisions, the operative procedure time and, importantly, the radiation exposure time may be reduced. PMID- 10743439 TI - An audit of emergency abdominal aortic aneurysm repair to establish the necessity for an emergency vascular surgical rota. PMID- 10743440 TI - Prevalence of abdominal aortic aneurysms in urology patients referred for ultrasound. PMID- 10743441 TI - Adenocarcinoma within a rectal duplication: case report and literature review. PMID- 10743442 TI - Fractured femur in the elderly: intensive perioperative care is warranted. PMID- 10743443 TI - Anaesthesia and the nitric oxide-cyclic GMP pathway in the central nervous system. PMID- 10743444 TI - Effect of continuous epidural 0.2% ropivacaine vs 0.2% bupivacaine on postoperative pain, motor block and gastrointestinal function after abdominal hysterectomy. AB - We have investigated the effect of 24-h postoperative continuous epidural infusion of 0.2% ropivacaine or 0.2% bupivacaine 8 ml h-1 on pain, request for supplementary analgesics, motor block and gastrointestinal function, in a double blind, randomized study in 60 patients undergoing open hysterectomy. There were no significant differences between groups in pain, number of patients requesting supplementary analgesics, motor block, ability to walk or time to first flatus or stool. In the subgroup of patients who received supplementary analgesics, patients in the ropivacaine group received significantly more ketorolac than patients in the bupivacaine group. Time to discharge from hospital was similar with ropivacaine and bupivacaine. PMID- 10743445 TI - Effect of meloxicam on postoperative pain after abdominal hysterectomy. AB - We studied 36 patients, allocated randomly to receive meloxicam 15 mg rectally (n = 18) or placebo suppository (n = 18) before total abdominal hysterectomy in a double-blind study. Visual analogue scores for pain at rest (P < 0.005), on movement (P < 0.05) and on coughing (P < 0.05) were significantly decreased in the meloxicam group during the first 24 h after surgery. Mean 24-h PCA morphine requirements were 33.2 (SD 16.9) mg and 38.2 (20.8) mg in the meloxicam and placebo groups, respectively (ns). There was no difference in the incidence of nausea, vomiting or sedation between groups. PMID- 10743446 TI - Differential effects of systemically administered ketamine and lidocaine on dynamic and static hyperalgesia induced by intradermal capsaicin in humans. AB - We have examined the effect of systemic administration of ketamine and lidocaine on brush-evoked (dynamic) pain and punctate-evoked (static) hyperalgesia induced by capsaicin. In a randomized, double-blind, placebo-controlled, crossover study, we studied 12 volunteers in three experiments. Capsaicin 100 micrograms was injected intradermally on the volar forearm followed by an i.v. infusion of ketamine (bolus 0.1 mg kg-1 over 10 min followed by infusion of 7 micrograms kg-1 min-1), lidocaine 5 mg kg-1 or saline for 50 min. Infusion started 15 min after injection of capsaicin. The following were measured: spontaneous pain, pain evoked by punctate and brush stimuli (VAS), and areas of brush-evoked and punctate-evoked hyperalgesia. Ketamine reduced both the area of brush-evoked and punctate-evoked hyperalgesia significantly and it tended to reduce brush-evoked pain. Lidocaine reduced the area of punctate-evoked hyperalgesia significantly. It tended to reduce VAS scores of spontaneous pain but had no effect on evoked pain. The differential effects of ketamine and lidocaine on static and dynamic hyperalgesia suggest that the two types of hyperalgesia are mediated by separate mechanisms and have a distinct pharmacology. PMID- 10743447 TI - Effect of alkalinization of lidocaine on median nerve block. AB - Median nerve blocks were performed in 10 volunteers in a randomized, double blind, crossover study to compare the effects of 1% plain lidocaine with 1% lidocaine in sodium bicarbonate 0.1 mmol litre-1. Sensations of hot, cold, pinprick and light touch, compound motor and sensory nerve action potentials, and skin temperature were assessed at 2-min intervals. pH was 6.4 +/- 0.1 for plain lidocaine and 7.7 +/- 0.2 for alkalinized lidocaine (P < 0.001). Alkalinized lidocaine produced more rapid inhibition of compound motor action potentials than plain lidocaine (median 4 (range 2-6) vs 9 (2-14) min) (P = 0.039). Alkalinized lidocaine also produced more rapid onset of inhibition of compound motor than sensory nerve action potentials (4 (2-6) vs 8 (4-12) min) (P = 0.0039). There was no significant difference in any other sensory modality between alkalinized and plain lidocaine. These data suggest that addition of bicarbonate to lidocaine for median nerve block significantly increased the rate of motor block without changing the onset or extent of sensory block. PMID- 10743448 TI - Postoperative pain management and recovery after remifentanil-based anaesthesia with isoflurane or propofol for major abdominal surgery. Remifentanil Study Group. AB - We have assessed if recovery times after morphine or fentanyl, given before terminating remifentanil anaesthesia with isoflurane or propofol, are compromised. We studied patients undergoing elective, major abdominal surgery, allocated randomly to receive remifentanil and isoflurane (n = 277) or remifentanil and propofol (n = 274) anaesthesia. Twenty-five minutes before the end of surgery, patients received fentanyl 0.15 mg or morphine 15 mg in a randomized, double-blind manner followed by a second dose (fentanyl 0.05 mg, morphine 7 mg) for moderate or severe pain in recovery. Recovery was rapid and at an Aldrete score > or = 9 (median 12-15 min), 42-51% of patients reported none or mild pain. However, 26-35% of patients reported severe pain and > 90% required a second dose of opioid within 21-27 min after anaesthesia. PMID- 10743449 TI - Comparison of the incidence of complications at induction and emergence in infants receiving oral atropine vs no premedication. AB - We studied 120 patients less than 1 yr of age, allocated randomly to receive atropine 40 micrograms kg-1 orally 1 h before operation (group A) or no premedication (group B). All patients underwent a standardized anaesthetic, including inhalation induction with halothane followed by atracurium 0.5 mg kg-1, tracheal intubation and positive pressure ventilation. Monitoring during anaesthesia included heart rate, arterial oxygen saturation, temperature and airway conditions at induction and emergence. The incidence of a decrease in arterial oxygen saturation to 94% or less at induction and recovery was similar in both groups (30.5% at induction, 39% at extubation in group A; 31% at induction, 41% at extubation in group B). There were significantly more airway complications in group B both at induction and emergence (25% and 49%, respectively, compared with 9% and 25% in group A; P < 0.015). Mean heart rate at induction and in the peroperative period was significantly higher in the group receiving atropine (P < or = 0.001). There was an increased incidence of bradycardia (decrease in heart rate of > or = 20%) at induction in the non premedicated group (23% in group B compared with 10% in group A), but this was not statistically significant. We conclude that the incidence of airway complications at induction and emergence was reduced by orally administered atropine premedication. PMID- 10743450 TI - Anaesthetic complications of acromegaly. AB - The anaesthetic risks of acromegaly include difficulties in airway management, hypertension, and cardiac, gastrointestinal and renal problems. To estimate the incidence of major complications in this rare group of patients, we reviewed 28 patients with acromegaly who had pituitary tumour excision over a 10-yr period. Each patient was matched for age, weight and sex to a non-acromegalic patient undergoing transsphenoidal pituitary surgery. Acromegalic patients received significantly more fentanyl and midazolam and less thiopental and succinylcholine than controls (all P < 0.05). Mean arterial pressure (baseline, minimal and maximal values) was higher in acromegalic patients than in controls. There was no difference between groups in the use of vasoactive drugs. PaO2, FIO2 and PaCO2 were similar in both groups. Arterial pH was significantly lower (P = 0.015), blood glucose was higher (P < 0.001) and fluid intake minus output was higher (P = 0.04) in acromegalic patients than in controls. Airway difficulty and tongue enlargement were encountered more often in acromegalic patients (P = 0.002 and P = 0.001, respectively). Our data confirm that in acromegalic patients: airway difficulties occurred more frequently; severe haemodynamic instability did not typically occur during surgery for acromegaly; pulmonary gas exchange was not altered during operation; glucose intolerance may be an intraoperative problem; and fluid regulation may be altered. PMID- 10743451 TI - NOx- concentrations in the rat hippocampus and striatum have no direct relationship to anaesthesia induced by ketamine. AB - Using microdialysis, we have examined the effects of ketamine on concentrations of total nitric oxide oxidation products (NOx-) in the rat hippocampus and striatum in vivo to investigate the relationship between anaesthesia and NOx- production in the brain. Ketamine 25, 50 and 100 mg kg-1 i.p. increased NOx- concentrations to mean 125 (SD 13)%, 165 (11)% and 193 (13)% of basal, respectively, in the hippocampus and to 122 (12)%, 147 (7)% and 177 (14)% of basal in the striatum. Local perfusion with ketamine 50 and 100 mumol litre-1 into the hippocampus or striatum increased NOx- concentrations to 212 (32)% and 291 (17)% of basal, respectively, in the hippocampus and to 148 (20)% and 201 (18)% of basal in the striatum. Ketamine 50 and 100 mg kg-1 i.p. caused dose dependent prolongation of loss of the righting reflex (LRR) and 100 mg kg-1 i.p. also caused loss of the corneal reflex (LCR). Local perfusion of ketamine did not provoke LRR or LCR. Inhibition of NOS by L-NAME 100 mg kg-1 i.p. decreased hippocampal NOx- concentrations to 58 (7)% of basal and did not provoke LRR or LCR. Although the effect of ketamine-induced increases in hippocampal NOx- concentrations was significantly depressed by L-NAME, LRR was not affected. These data imply that NOx- concentrations in the hippocampus or striatum have no direct relationship to the anaesthetic efficacy of ketamine, although this requires further investigation. PMID- 10743452 TI - The neuronal nitric oxide synthase inhibitor 7-nitroindazole (7-NI) and morphine act independently on the control of breathing. AB - Inhibitors of nitric oxide synthase (NOS) have analgesic properties and reduce opioid tolerance and dependency. To investigate a possible interaction of NOS inhibitors with the respiratory depressant action of morphine, we determined the effects of the neuronal NOS inhibitor 7-nitroindazole (7-NI) on the ventilatory carbon dioxide response curve; subsequently, we studied the effects of additional morphine application. Finally, using naloxone, we investigated a possible interaction (at the opioid receptor) between the effects of 7-NI and morphine. The effects of 7-NI 50 mg kg-1 i.p., morphine 0.1 mg kg-1 i.v. and naloxone 0.1 mg kg-1 i.v. were studied using dynamic end-tidal carbon dioxide forcing in eight cats under alpha-choralose-urethane anaesthesia. Data analysis was performed using a two-compartment model comprising a fast peripheral and a slow central component characterized by carbon dioxide sensitivities and a single offset B (apnoeic threshold). 7-NI decreased the mean apnoeic threshold from 4.27 (SD 0.87) to 2.59 (1.71) kPa. Peripheral and central carbon dioxide sensitivities were reduced from 0.56 (0.22) to 0.26 (0.09) litre min-1 kPa-1 and from 0.09 (0.05) to 0.04 (0.03) litre min-1 kPa-1, respectively. Morphine increased the apnoeic threshold by 0.5 kPa and reduced carbon dioxide sensitivity by a further 35%. Naloxone reversed the ventilatory effects of morphine but not those induced by 7-NI. We conclude that the respiratory effects of 7-NI and morphine are mediated independently and that the effects of 7-NI do not result from interaction with opioid receptors. PMID- 10743453 TI - Biologically variable ventilation prevents deterioration of gas exchange during prolonged anaesthesia. AB - We have studied the time course of changes in gas exchange and respiratory mechanics using two different modes of ventilation during 7 h of isoflurane anaesthesia in pigs. One group received conventional control mode ventilation (CV). The other group received biologically variable ventilation (BVV) which simulates the breath-to-breath variation in ventilatory frequency (f) that characterizes normal spontaneous ventilation. After baseline measurements with CV, animals were allocated randomly to either CV or BVV (FIO2 1.0 with 1.5% end tidal isoflurane). With BVV, there were 376 changes in f and tidal volume (VT) over 25.1 min. Ventilation was continued over the next 7 h and blood gases and respiratory mechanics were measured every 60 min. The modulation file used to control the ventilator for BVV used an inverse power law frequency distribution (I/fa with a = 2.3 +/- 0.3). After 7 h, at a similar delivered minute ventilation, significantly greater PaO2 (mean 72.3 (SD 4.0) vs 63.5 (6.5) kPa) and respiratory system compliance (1.08 (0.08) vs 0.92 (0.16) ml cm H2O-1 kg-1) and lower PaCO2 (6.5 (0.7) vs 8.7 (1.5) kPa) and shunt fraction (7.2 (2.7)% vs 12.3 (6.2)%) were seen with BVV, with no significant difference in peak airway pressure (16.3 (1.2) vs 15.3 (3.7) cm H2O). A deterioration in gas exchange and respiratory mechanics was seen with conventional control mode ventilation but not with BVV in this experimental model of prolonged anaesthesia. PMID- 10743454 TI - Fentanyl protects the heart against ischaemic injury via opioid receptors, adenosine A1 receptors and KATP channel linked mechanisms in rats. AB - We have investigated if fentanyl protects against myocardial ischaemic injury and if so, if the mechanism of this protection is mediated via opioid and adenosine A1 receptors, and KATP channels. Langendorff rat hearts were subjected to global ischaemia (30 min) and reperfusion (60 min). The drugs were administered before induction of ischaemia and maintained throughout the experiment. Treatment with fentanyl 740 nmol litre-1 improved post-ischaemic mechanical function, assessed as developed pressure, +dP/dtmax and -dP/dtmin, compared with controls after 60 min of reperfusion. These effects were abolished by naloxone 1 mumol litre-1, DPCPX 10 mumol litre-1, a selective adenosine A1 antagonist and sodium 5 hydroxydecanoate 100 mumol litre-1, a K+ATP channel blocker. We conclude that fentanyl protected the heart against post-ischaemic injury by a mechanism which was blocked by an opioid and an adenosine A1 receptor antagonist and also by a KATP channel antagonist. PMID- 10743455 TI - Halothane attenuates the endothelial Ca2+ increase and vasorelaxation of vascular smooth muscle in the rat aorta. AB - Isolated spiral strips of rat thoracic aorta with endothelium were suspended for isometric tension recordings in a physiological salt solution. Endothelium dependent vasorelaxation was elicited by carbachol 10(-6) and 10(-5) mol litre-1 during norepinephrine-induced contractions, and the effects of 1.5% and 3% halothane were evaluated with concomitant measurement of [Ca2+]i using fura-2 Ca2+ fluorescence. The effects of halothane on endothelium-dependent relaxation were compared with those of nitro G-L-arginine methyl ester 10(-4) mol litre-1 (L NAME: an inhibitor of nitric oxide synthase). Carbachol reduced norepinephrine induced contractions in a concentration-dependent manner, but augmented the norepinephrine-induced increase in [Ca2+]i in endothelium intact strips. In contrast, carbachol did not influence muscle tension or [Ca2+]i when the endothelium was completely denuded. Although 3% halothane and L-NAME 10(-4) mol litre-1 inhibited carbachol-induced vasorelaxation in a similar manner, halothane inhibited carbachol-induced increases in [Ca2+]i. These results indicate that halothane inhibited a carbachol-induced increase in [Ca2+]i in the endothelium, which subsequently attenuated the decrease in muscle tension. PMID- 10743456 TI - Effects of xenon on cerebral blood flow and autoregulation: an experimental study in pigs. AB - We have investigated the effects of xenon on regional cerebral blood flow (rCBF) and autoregulation in pigs sedated with propofol 4 mg kg-1 h-1. Balloon-tipped catheters were placed into the descending aorta and inferior vena cava of 15 Gottingen Minipigs for manipulation of arterial pressure and blood sampling. rCBF was measured using the sagittal sinus outflow technique. Xenon was adjusted randomly to end-tidal fractions (FE'Xe) of 0, 0.30, 0.50 and 0.70. After baseline measurements of heart rate (HR), mean arterial pressure (MAP), rCBF, sagittal sinus pressure (SSP) and calculation of regional cerebrovascular resistance (rCVR) at each respective FE'Xe, autoregulation was tested in the MAP range 60 120 mm Hg. Increasing FE'Xe had no effect on HR, MAP, rCBF or SSP. rCVR increased with increases in MAP, regardless of FE'Xe. Autoregulation was not impaired. We conclude that xenon inhalation had no effect on rCBF and autoregulation in our model, which could suggest that xenon is an adequate adjunct for neurosurgical anaesthesia. PMID- 10743457 TI - Measurement of blood volume in surgical and intensive care practice. AB - Clinical studies to assess the benefits of blood transfusion or haemodilution in critical illness should take account of measured CBV before, during and after intervention. As mentioned above, surrogate measures of CBV are inadequate and studies based on these must be considered incomplete, because they cannot distinguish between effects of changes in haemoglobin concentration and changes in blood volume. The choice of a suitable technique for measuring CBV depends on the facilities available locally. In general, methods based on labelled red cells are more reliable but are technically demanding and time consuming. Those based on albumin are likely to yield false high values and this is particularly true in all patients with impaired capillary integrity. The most promising plasma marker is hydroxyethyl starch which may be particularly useful when the polysaccharide is labelled with a fluorescent dye. Attaching fluorescein to hydroxyethyl starch is not difficult and, should demand be sufficient, it may well become available from manufacturers who are already capable of providing other fluorescent polysaccharides. The clinical benefits of such a development would include more rational schedules of i.v. fluid and blood transfusion management in surgical and intensive care patients. PMID- 10743458 TI - EEG controlled rapid opioid withdrawal under general anaesthesia. AB - We performed rapid opioid detoxification under propofol anaesthesia in 30 opioid addicts, using the opioid receptor antagonist naltrexone. Two strategies to obtain a sufficient depth of anaesthesia and to avoid anaesthetic overdose were evaluated. Patients were allocated randomly to one of two groups. In group 1, the effects of propofol were monitored by observing clinical signs, and in group 2, depth of anaesthesia was controlled using an EEG threshold method. Withdrawal symptoms and post-anaesthetic recovery time were assessed. All patients remained stable and no anaesthetic complications were noted. There were significant differences in the total dose of propofol given (group 1, mean 72 (SD 9) mg kg-1; group 2, 63 (8) mg kg-1; P < 0.01), duration of anaesthesia (318 (53) min vs 309 (42) min; P < 0.05), duration of recovery time (49 (13) min vs 40 (12) min; P < 0.01) and frequency of withdrawal symptoms between groups. In addition, the incidence of side effects was different between groups (62 vs 29 points on a withdrawal symptom scale; P < 0.01). To obtain a sufficient depth of anaesthesia but to avoid inappropriately large doses of anaesthetic, we consider that EEG monitoring is valuable during rapid opioid detoxification. PMID- 10743459 TI - Use of microalbuminuria as a predictor of outcome in critically ill patients. AB - We have investigated the use of microalbuminuria as a predictor of outcome in a pilot study involving 50 critically ill patients in a six-bed hospital intensive care unit (ICU). Urinary microalbumin:creatinine (M:Cr) ratios measured only 6 h after admission to the ICU demonstrated a significant difference (P = 0.01) between survivors and non-survivors, allowing rapid identification of patients at increased risk of developing organ failure and at greater risk of death. This work suggests that earlier identification of these patients using a rapid, simple, inexpensive biochemical test is possible; if confirmed in a larger study, it may be that clinical interventions can be targeted at those most likely to benefit. PMID- 10743461 TI - Adenosine potentiation of neuromuscular blocking agents in guinea-pigs. AB - We have investigated the effects of adenosine i.v. on neuromuscular block induced by rocuronium, vecuronium and pipecuronium in an in vivo guinea-pig sciatic nerve tibialis anterior preparation. The ED50 of each neuromuscular blocker was determined from cumulative log dose-response regression lines (n = 14). In separate experiments, adenosine 0.1 mg kg-1 min-1 or the same volume of 0.9% NaCl was given i.v. via a constant infusion and the ED50 of each neuromuscular blocking agent was then administered (n = 24). Adenosine 0.1 mg kg-1 min-1 increased significantly maximal block induced by the ED50 of these neuromuscular blockers (55-72%, 49-73% and 60-96%, respectively, for rocuronium, vecuronium and pipecuronium; P < 0.05). Time to maximal block after rocuronium was significantly prolonged by adenosine (1.4-2.1 min; P < 0.05) and time to maximal block after vecuronium and pipecuronium was unchanged by adenosine. Time to maximal recovery of twitch tension after administration of the ED50 of all neuromuscular blocking agents was prolonged significantly by adenosine (4.5-10.7 min, 8.2-15.8 min and 47.0-128.7 min, respectively, for rocuronium, vecuronium and pipecuronium; P < 0.05). We conclude that continuous infusion of adenosine 0.1 mg kg-1 min-1 potentiated the effects of neuromuscular blocking agents in this in vivo guinea pig preparation. PMID- 10743462 TI - Infiltration of the abdominal wall with local anaesthetic after total abdominal hysterectomy has no opioid-sparing effect. AB - We have measured the effect of infiltration of the deep and superficial layers of the abdominal wound on morphine consumption and pain for 48 h after operation, in 40 patients undergoing total abdominal hysterectomy, in a double-blind randomized study. Patients received wound infiltration with 0.9% normal saline 40 ml or 40 ml of 0.25% bupivacaine with epinephrine 1:200,000. There were no significant differences between groups in morphine consumption, linear analogue scores for pain at rest or on movement, nausea or sedation during the first 48 h after operation. We conclude that infiltration of the deep and superficial layers of the wound of a Pfannenstiel incision with local anaesthetic solution did not confer additional analgesia in patients undergoing major gynaecological surgery. PMID- 10743460 TI - Do blood concentrations of neurone specific enolase and S-100 beta protein reflect cognitive dysfunction after abdominal surgery?ISPOCD Group. AB - Neurone specific enolase (NSE) and S-100 beta protein have been used as markers of brain damage. We hypothesized that blood concentrations of NSE and S-100 beta protein reflect cognitive dysfunction after abdominal surgery. We studied 65 elderly patients in whom neuropsychological testing was performed before abdominal surgery, at discharge from hospital and after 3 months. Serum concentrations of NSE and S-100 beta protein were measured before surgery and after 24, 48 and 72 h. Serum concentrations of S-100 beta protein increased significantly while NSE concentrations decreased significantly. The increase in S 100 beta protein concentration after 48 h was significantly greater in patients with delirium. No correlation was found between cognitive dysfunction and S-100 beta protein or NSE concentration. We conclude that blood concentrations of S-100 beta protein increase after abdominal surgery and may be related to postoperative delirium. PMID- 10743463 TI - Propofol decreases stimulated dopamine release in the rat nucleus accumbens by a mechanism independent of dopamine D2, GABAA and NMDA receptors. AB - Although propofol (2,6-di-isopropylphenol) is a popular i.v. general anaesthetic, it has been suggested to have abuse potential. As many drugs of abuse act preferentially via release of dopamine in the limbic system, we investigated the action of propofol on stimulated dopamine release in the rat nucleus accumbens. Nucleus accumbens slices were superfused (1.6 ml min-1) with artificial cerebrospinal fluid at 32 degrees C. Dopamine release was evoked by electrical stimulation (10 pulses, 0.1 ms, 10 mA, 10 Hz, every 10 min) and monitored by fast cyclic voltammetry. Propofol 100 mumol litre-1 reduced stimulated dopamine release over the 2 h after administration, relative to intralipid controls, to mean 30 (SEM 2)% (P < 0.01). The dopamine D2 receptor antagonist metoclopramide 0.3 mumol litre-1 increased dopamine release but did not block the effect of propofol (38 (3)%). The selective GABAA antagonist bicuculline 24 mumol litre-1 also failed to antagonize the action of propofol (45 (3)%). The NMDA receptor antagonist dextromethorphan 10 mumol litre-1 decreased dopamine release to 57 (6)% (P < 0.01) but failed to block the inhibitory effect of propofol (46 (6)%). Although propofol has been reported to bind to D2, GABAA and NMDA receptors, failure of metoclopramide and bicuculline to block its effects suggests that an agonist action at D2 or GABAA receptors does not mediate the effects of propofol on dopamine release in the rat nucleus accumbens. The lack of effect of dextromethorphan makes an NMDA receptor antagonist action unlikely. PMID- 10743464 TI - The intubating laryngeal mask airway does not facilitate tracheal intubation in the presence of a neck collar in simulated trauma. AB - Tracheal intubation must be performed with great care in the multiply injured patient when it must be assumed that the cervical spine may be damaged. Use of conventional direct laryngoscopy usually requires removal of the neck collar and manual in-line stabilization of the head and neck. The intubating laryngeal mask (ILMA) has been designed to facilitate tracheal intubation in the neutral position. We used the ILMA to intubate the trachea in 10 patients wearing a neck collar and with cricoid pressure applied in a simulated trauma scenario. The ILMA was difficult to insert and ventilation proved difficult. In only two patients was intubation successful. These problems were probably caused by the neck collar strap under the chin lifting up and tipping the larynx anteriorly. On the basis of these findings, ILMA use in a subject wearing a neck collar cannot be recommended. PMID- 10743465 TI - Retrograde nasotracheal intubation with a new tracheal tube: a feasibility study. AB - We have assessed the feasibility of retrograde nasotracheal intubation using a flexometallic tracheal tube with a detachable pilot balloon and connector in a study of 20 consecutive adult patients undergoing oropharyngeal surgery. The technique consisted of: (1) laryngoscope-guided orotracheal intubation; (2) insertion of an 18-gauge Foley catheter through the nose and retraction into the mouth; (3) detachment of the anaesthesia circuit, pilot balloon and connector; (4) insertion of the Foley catheter tip into the proximal end of the tracheal tube and inflation of the Foley catheter cuff; (5) withdrawal of the Foley catheter and attached tracheal tube back through the nose; (6) deflation of the Foley catheter cuff; and (7) re-attachment of the pilot balloon, connector and anaesthesia circuit. The technique was successful at the first attempt in all patients. Mean time taken to insert the Foley catheter and retract it into the mouth was 19 (range 12-30) s. Mean time taken from disconnection to reconnection of the anaesthesia circuit was 8 (6-10) s. Heart rate increased after intubation, but there were no significant changes in arterial pressure. Nasal bleeding, airway problems and hypoxic events did not occur. No anatomical abnormalities or nasal trauma were detected at rhinoscopy. We conclude that retrograde nasotracheal intubation is feasible using a flexometallic tracheal tube with a detachable pilot balloon and connector. PMID- 10743466 TI - Surgery for a fractured femur and elective ICU admission at 113 yr of age. AB - Disability and medical dependence increase with age and as the percentage of the population over 80 yr old increases, demands on our healthcare budget will likewise grow. Clinical decision making should always balance the physiological state of the patient against the likely prognosis of the pathological state. Age may influence this decision-making process in an indirect way, as a perceived measure of physiological status, but whether age alone may be used as criteria for the type or degree of clinical intervention is a contentious point. In light of these issues, we present a woman of 113 yr, admitted to hospital with a fractured shaft of femur. The patient underwent surgery and was electively admitted to the intensive care unit for postoperative treatment. She survived surgery and several complications and was subsequently returned to the community where she celebrated her 114th birthday. PMID- 10743467 TI - Thoracic paravertebral block: radiological evidence of contralateral spread anterior to the vertebral bodies. AB - We report contralateral spread of contrast medium anterior to the vertebral bodies after injection of contrast through a thoracic paravertebral catheter that was used to manage pain in a patient with multiple fractured ribs. We review the literature and propose that the anatomical basis for this observation is spread in the extrapleural compartment of the thoracic paravertebral space along the subserous fascial plane. PMID- 10743468 TI - Self-citations in six anaesthesia journals and their significance in determining the impact factor. AB - Self-citation of a journal may affect its impact factor. We investigated self citations in the 1995 and 1996 issues of six anaesthesia journals by calculating the self-citing and self-cited rates for each journal. Self-citing rate relates a journal's self-citations to its total number of references. We defined self-cited rate as the ratio of a journal's self-citations to the number of times it is cited by the six anaesthesia journals. We also correlated self-citing rates with the impact factor of the six journals for 1997. Citations among the six journals differed significantly (P < 0.0001). Anesthesiology had the highest self-citing rate (57%). Anaesthesia, Anesthesia and Analgesia, British Journal of Anaesthesia, Canadian Journal of Anaesthesia and the European Journal of Anaesthesiology had self-citing rates of 28%, 28%, 30%, 11% and 4% respectively. The self-cited rates were 31%, 35%, 34%, 27%, 31% and 17% for Anaesthesia, Anesthesiology, Anesthesia and Analgesia, British Journal of Anaesthesia, Canadian Journal of Anaesthesia and the European Journal of Anaesthesiology, respectively. North America journals cited the North America literature. This also occurred, to a lesser extent, in the European anaesthesia journals. A significant correlation between self-citing rates and impact factors was found (r = 0.899, P = 0.015). A high self-citing rate of a journal may positively affect its impact factor. PMID- 10743469 TI - Post-thoracotomy epidural vs paravertebral analgesia. PMID- 10743470 TI - Appropriate size of laryngeal mask. PMID- 10743471 TI - Local anaesthetic infiltration and natural killer cell cytotoxicity. PMID- 10743472 TI - Double-lumen tube placement: protecting the good lung. PMID- 10743473 TI - Paediatric resuscitation guidelines. PMID- 10743474 TI - Which is better in children: edrophonium or neostigmine? PMID- 10743475 TI - Bacterial contamination of needles used for spinal and epidural anaesthesia. PMID- 10743476 TI - Bacterial contamination of needles used for spinal and epidural anaesthesia. PMID- 10743477 TI - Cholesterol-lowering effects of high-protein soya milk. PMID- 10743478 TI - The need for nitrogen. PMID- 10743479 TI - Low-dose folic acid supplementation does not influence plasma methionine concentrations in young non-pregnant women. AB - An elevated plasma total homocysteine (tHcy) concentration is a risk factor for cardiovascular disease and for having offspring with a neural-tube defect. Folate is a methyl donor in the remethylation of homocysteine into methionine. Although folic acid supplementation decreases tHcy concentrations, effects of folic acid supplementation on plasma methionine concentrations are unclear. There is also concern that folic acid supplementation negatively affects vitamin B12 status. We studied effects of low-dose folic acid supplementation on methionine and vitamin B12 concentrations in plasma. We also investigated whether baseline plasma methionine and tHcy concentrations correlated with the baseline folate and vitamin B12 status. For a period of 4 weeks, 144 young women received either 500 micrograms folic acid each day, or 500 micrograms folic acid and placebo tablets on alternate days, or a placebo tablet each day. Plasma methionine, tHcy and plasma vitamin B12 concentrations were measured at start and end of the intervention period. Folic acid supplementation had no effect on plasma methionine or plasma vitamin B12 concentrations although it significantly decreased tHcy concentrations. Plasma methionine concentrations showed no correlation with either tHcy concentrations (Spearman rs-0.01, P = 0.89), or any of the blood vitamin variables at baseline. Baseline tHcy concentrations showed a slight inverse correlation with baseline concentrations of plasma vitamin B12 (rs 0.25, P < 0.001), plasma folate (rs-0.24, P < 0.01) and erythrocyte folate (rs 0.19, P < 0.05). In conclusion, low-dose folic acid supplementation did not influence plasma methionine or plasma vitamin B12 concentrations. Furthermore, no correlation between plasma methionine concentrations and the blood folate and vitamin B12 status was shown. PMID- 10743480 TI - Double-blind study of the addition of high-protein soya milk v. cows' milk to the diet of patients with severe hypercholesterolaemia and resistance to or intolerance of statins. AB - Total substitution of soyabean protein for animal protein in the diet has been repeatedly shown to lower plasma cholesterol levels in hypercholesterolaemic individuals. A new, highly palatable, high-protein soya drink may allow replacement of a significant percentage of animal protein in the diet. The soya drink was given, within a crossover design v. a cows' milk preparation of similar composition and taste, to twenty-one severely hypercholesterolaemic patients (mean baseline plasma cholesterol 8.74 mmol/l) with a history of resistance to or intolerance of statin treatment. Each dietary supplement was given for 4 weeks, with a 4-week interval between treatments, Plasma lipid levels were monitored every 2 weeks during each dietary sequence. The concomitant dietary treatment, which had been followed for a long time by all patients, was carefully monitored throughout the study. The soya supplementation reduced plasma total cholesterol level by 6.5%, when given first, and by 7.4% when given after cows' milk. When given first, cows' milk resulted in a small, non-significant reduction of plasma cholesterol level (-3.9%), and when given after soya, it changed total plasma cholesterol to a minimal extent (-1.6%). Changes in total and LDL-cholesterol levels after 2 and 4 weeks of soya v. cows' milk treatment were, thus, respectively -6.1, -7.0 and -6.2, -7.8% (both P < 0.05). These first data from a double-blind study confirm a significant cholesterol-lowering effect of soyabean protein, even when only partly replacing animal protein in the diet, in individuals with extreme plasma cholesterol elevations. PMID- 10743481 TI - Postprandial lipoprotein, glucose and insulin responses after two consecutive meals containing rapeseed oil, sunflower oil or palm oil with or without glucose at the first meal. AB - There is increasing evidence that the degree of postprandial lipaemia may be of importance in the development of atherosclerosis and IHD. Postprandial lipid, lipoprotein, glucose, insulin and non-esterified fatty acid (NEFA) concentrations were investigated in eleven healthy young males after randomized ingestion of meals containing rapeseed oil, sunflower oil or palm oil with or without a glucose drink. On six occasions each subject consumed consecutive meals (separated by 1.75 h) containing 70 g (15 g and 55 g respectively) of each oil. On one occasion with each oil 50 g glucose was taken with the first meal. One fasting and fifteen postprandial blood samples were taken over 9 h. There were no statistically significant differences in lipoprotein and apolipoprotein responses after rapeseed, sunflower and palm oils, whereas insulin responses were lower after sunflower oil than after rapeseed oil (ANOVA, P = 0.04). The NEFA and triacylglycerol concentrations at 1.5 h were reduced when 50 g glucose was taken with the first meal (ANOVA, P < 0.0001 and P < 0.05 respectively), regardless of meal fatty acid composition. In conclusion, the consumption of glucose with a mixed meal containing either rapeseed, sunflower or palm oil influenced the immediate triacylglycerol and NEFA responses compared with the same meal without glucose, whereas no significant effect on postprandial lipaemia after a subsequent meal was observed. The fatty acid composition of the meal did not significantly affect the lipid and lipoprotein responses, whereas an effect on insulin responses was observed. PMID- 10743482 TI - Effect of modified dairy fat on fasting and postprandial haemostatic variables in healthy young men. AB - It has been suggested that milk fat, due to its content of saturated fatty acids, may have a thrombogenic effect. In the present study the fatty acid profile of milk fat was modified by changing the feeding regimens of cows and the effect on haemostatic variables of a diet containing the modified milk fat (M) was compared with that of a diet containing milk fat of typical Danish composition (D). In the modified fat 16% of the saturated fatty acid (C12-C16) content was replaced mainly by oleic acid. Eighteen subjects were fed on two strictly controlled isoenergetic diets containing 40% energy from total fat (30% energy from the test fats) for periods of 4 weeks in a study with a crossover design. Fasting samples were taken in the last week of each study period. Postprandial samples were taken on day 21, 3 h after lunch (n 18), and on the last day of the study 2, 4, 6 and 8 h after a fat load containing 1.2 g of one of the milk fats/kg body weight (n 8). After 4 weeks' dietary intervention fasting plasma factor VII coagulant (FVIIc) activity, tissue-type plasminogen activator (t-PA) activity, plasminogen activator inhibitor (PAI-1) antigen and beta-thromboglobulin did not differ between diets M and D. Postprandially FVIIc and t-PA activities increased (P < 0.001) and PAI-1 antigen and PAI-1 activity decreased (P < 0.001) as compared with fasting values, regardless of diet. After the fat load, the postprandial increase in FVIIc was marginally lower after diet M than diet D (diet effect, P < 0.05). In conclusion, the modified milk fat obtained by the applied feeding strategy had virtually the same effects on haemostatic variables as conventional milk fat. PMID- 10743483 TI - Effects of red pepper on appetite and energy intake. AB - Two studies were conducted to investigate the effects of red pepper (capsaicin) on feeding behaviour and energy intake. In the first study, the effects of dietary red pepper added to high-fat (HF) and high-carbohydrate (HC) meals on subsequent energy and macronutrient intakes were examined in thirteen Japanese female subjects. After the ingestion of a standardized dinner on the previous evening, the subjects ate an experimental breakfast (1883 kJ) of one of the following four types: (1) HF; (2) HF and red pepper (10 g); (3) HC; (4) HC and red pepper. Ad libitum energy and macronutrient intakes were measured at lunch time. The HC breakfast significantly reduced the desire to eat and hunger after breakfast. The addition of red pepper to the HC breakfast also significantly decreased the desire to eat and hunger before lunch. Differences in diet composition at breakfast time did not affect energy and macronutrient intakes at lunch-time. However, the addition of red pepper to the breakfast significantly decreased protein and fat intakes at lunch-time. In Study 2, the effects of a red pepper appetizer on subsequent energy and macronutrient intakes were examined in ten Caucasian male subjects. After ingesting a standardized breakfast, the subjects took an experimental appetizer (644 kJ) at lunch-time of one of the following two types: (1) mixed diet and appetizer; (2) mixed diet and red-pepper (6 g) appetizer. The addition of red pepper to the appetizer significantly reduced the cumulative ad libitum energy and carbohydrate intakes during the rest of the lunch and in the snack served several hours later. Moreover, the power spectral analysis of heart rate revealed that this effect of red pepper was associated with an increase in the ratio sympathetic: parasympathetic nervous system activity. These results indicate that the ingestion of red pepper decreases appetite and subsequent protein and fat intakes in Japanese females and energy intake in Caucasian males. Moreover, this effect might be related to an increase in sympathetic nervous system activity in Caucasian males. PMID- 10743484 TI - Inverse association between coffee drinking and serum uric acid concentrations in middle-aged Japanese males. AB - Consumption of caffeine-rich beverages, which have diuretic properties, may decrease serum uric acid concentrations. We examined cross-sectionally the relationship of coffee and green tea consumption to serum uric acid concentrations in 2240 male self-defence officials who received a pre-retirement health examination at four hospitals of the Self-Defence Forces between 1993 and 1994. The mean levels of coffee and green tea consumption were 2.3 and 3.1 cups/d respectively. There was a clear inverse relationship between coffee consumption and serum uric acid concentration. When adjusted for hospital only, those consuming less than one cup of coffee daily had a mean serum uric acid concentration of 60 mg/l, while that of those drinking five or more cups of coffee daily was 56 mg/l (P < 0.0001). No such relationship was observed for green tea, another major dietary source of caffeine in Japan. The relationship between coffee consumption and serum uric acid concentration was independent of age, rank in the Self-Defence Forces, BMI, systolic blood pressure, serum creatinine, serum total cholesterol and serum HDL-cholesterol concentrations, smoking status, alcohol use, beer consumption and intake of dairy products. These findings suggest that coffee drinking may be associated with lower concentrations of serum uric acid, and further studies are needed to confirm the association. PMID- 10743485 TI - A comparison of methods of assessment of dietary selenium intakes in Otago, New Zealand. AB - The aims of the present study were (1) to compare three methods of assessment of dietary Se intake, i.e. chemical analysis of duplicate diets, diet records and a food-frequency questionnaire (FFQ) designed specifically for Se, and (2) to determine dietary Se intakes of residents of Otago, New Zealand. The FFQ was completed by 110 free-living adults. Diet records (3 d) and duplicate diet collections were carried out by forty-three of these subjects chosen on the basis of low blood Se concentration, and during a period when consumption of the high Se foods fish, kidney, liver and Brazil nuts was discouraged. Mean Se intakes were similar for duplicate diet analysis (29 (SD 13) micrograms/d) and diet record assessments (28 (SD 15) micrograms/d). Estimates of intakes from the FFQ for the subgroup of forty-three subjects were higher (51 (SD 26) micrograms/d) than those from duplicate diets and diet records. Values from duplicate diet analysis and diet record assessments were strongly correlated (r 0.7, P = 0.0001), but difference plots indicated a lack of agreement between the two methods. Thus, diet record assessment was not adequate for predicting dietary Se intakes of individuals. Significant correlations were found for relationships between Se intake from duplicate diets (microgram/kg body weight per d) and plasma Se, Se intake from diet records (microgram/d and microgram/kg body weight per d) and plasma Se; and Se intake from the FFQ and whole-blood Se. Se intakes from duplicate diets and diet records were similar to those reported previously for New Zealanders, but lower than the recommended intakes in the USA (National Research Council, 1989), Australia (Truswell et al. 1990) and the UK (Department of Health, 1991) and the World Health Organization/Food and Agriculture Organization/International Atomic Energy Agency (1996) normative requirement. PMID- 10743486 TI - Ileal amino acid digestibilities in pigs of barley-based diets with inclusion of lucerne (Medicago sativa), white clover (Trifolium repens), red clover (Trifolium pratense) or perennial ryegrass (Lolium perenne). AB - Two experiments were performed with post-valve T-cannulated growing pigs, using five animals in each experiment in a change-over design to evaluate the effect of inclusion of four different dried forage meals on ileal crude protein (CP) and amino acid (AA) digestibilities. The control diets (C1 and C2) were barley-based and the experimental diets were formulated by replacing the barley with 100 or 200 g/kg of either lucerne (Medicago sativa) or white clover (Trifolium repens) meal in Expt 1 and red clover (Trifolium pratense) or perennial ryegrass (Lolium perenne) meal in Expt 2. A decrease (P < 0.05) in the apparent ileal digestibility of CP and most of the essential and nonessential AA was found with the inclusion of luceme, white clover and perennial ryegrass meal in the barley based diets. When red clover meal was included, only the apparent ileal digestibilities of CP, leucine, phenylalanine, tyrosine and glutamic acid were found to decrease (P < 0.05). The estimated apparent ileal digestibilities of most essential AA in the forage meals were lower than in the barley-based diets. The ileal flow of glucosamine and ornithine was found to increase (P < 0.05) with increasing proportion of fibre in the diet, suggesting an increase in endogenous N secretions and small-intestinal microbial activity. With the minor changes found for ileal essential AA digestibilities with forage meal inclusion in the diet the present data confirm the potential of forage meals as a source of protein in pig diets. PMID- 10743487 TI - Effect of supplementing a fibre basal diet with different nitrogen forms on ruminal fermentation and microbial growth in an in vitro semi-continuous culture system (RUSITEC) AB - Incubation trials were carried out with the rumen simulation technique (RUSITEC) to study the effects of four forms of N on the growth of ruminal micro-organisms and the fermentation variables when an all-fibre basal diet was incubated. The basal diet consisted of 10 g neutral-detergent fibre (NDF) from grass hay plus 2 g NDF from sugarbeet pulp. N forms were isolated soyabean protein, soyabean peptides, amino acids blended to profile soyabean protein and NH3 as NH4Cl. Half of the daily N supply was infused as NH4Cl and the other half was infused as each of the four treatments described. Non-NH3 N (NAN) forms increased NDF (P = 0.006), acid-detergent fibre (P = 0.003) and cellulose (P = 0.015) disappearance after 48 h incubation, CO2 (P < 0.001), CH4 (P = 0.002) and total volatile fatty acids production (P < 0.001), as well as the molar percentages of isobutyrate, isovalerate and valerate, which reflected the fermentation of amino acid C skeletons. NAN treatments also increased microbial N flow (P < 0.001) compared with NH3, with peptides and protein supporting more (P = 0.036) than amino acids. The proportion of microbial N derived from NH3 decreased successively (P < 0.05) with NH3 > amino acids > peptides > protein treatments, indicating preferential uptake of peptides without passage through the NH3 pool. Microbial efficiency (g microbial N/kg organic matter apparent disappearance) was greater (P = 0.002) for the NAN forms than for the NH3 treatment, with peptides and protein treatments supporting higher (P = 0.009) values than amino acid treatment. These results indicate that N forms other than NH3 are required for optimal fibre digestion and microbial growth. PMID- 10743488 TI - Nutritional status and energy expenditure in elderly patients with recent hip fracture during a 2-month follow-up. AB - A 2-month follow-up of nutritional status was performed in forty elderly patients with recent hip fracture. Patients were nutritionally assessed on admission to our rehabilitation unit (day 0), then monthly (day 30, day 60) by measurement of resting energy expenditure (REE), anthropometric, impedance and biological variables. Patients were defined as undernourished (n 13) or normally nourished (n 27) on the basis of mid-arm circumference (MAC) and triceps skinfold thickness (TST) measurements. Seven patients recovered a walking autonomy and were discharged from the hospital before day 30 (group I) whereas thirteen patients were discharged after day 30 (group II); twenty patients remained in the study at day 60 (group III). MAC and TST decreased in normally nourished patients from group III throughout the study whereas they did not change in group II or in undernourished patients from group III. REE values in relation to fat-free mass were increased compared with normal values and were similar in the three groups on day 0; they did not change during the study. Daily energy intake in relation to body weight was higher in group I and increased in group II and in undernourished patients from group III throughout the study. In contrast, it was below the recommended value at day 0 and it did not significantly improve in normally nourished patients from group III. Serum albumin, transthyretin and transferrin levels on day 0 were below reference intervals in the three groups. Albumin levels increased in group III throughout the study. Inflammatory proteins decreased in groups II and III, with C-reactive protein levels returning to normal values in group II by day 30 and in group III at day 60, while orosomucoid levels did not become completely normal over this period. Our findings indicate no improvement in nutritional status in undernourished patients after surgery for recent hip fracture, despite an adequate energy intake. An insufficient spontaneous energy intake for normally nourished patients was associated with a delayed favourable outcome resulting in a prolonged duration of hospitalization. A hypermetabolic state persisted during the 3 months after surgery. PMID- 10743490 TI - Evaluation of the novel Tanita body-fat analyser to measure body composition by comparison with a four-compartment model. AB - The Tanita body-fat analyser is a novel device to estimate body fat, based on the principles of bioelectrical impedance. It differs from other impedance systems which use surface electrodes in that the subjects stand bare-footed on a metal sole-plate which incorporates the electrodes, hence impedance is measured through the legs and lower trunk. In 104 men and 101 women (16-78 years and BMI 16-41 kg/m2) the mean bias in body-fat mass measured using the Tanita body-fat analyser was 0.8 (2SD 7.9) kg relative to a four-compartment model. This is comparable to the other prediction techniques tested (conventional tetrapolar impedance -1.3 (2SD 6.9) kg, skinfold thicknesses 0.3 (2SD 7.4) kg, and BMI-based formulas -0.2 (2SD 9.0) kg and -0.6 (2SD 8.5) kg), but the agreement was poorer than for 'reference' methods to measure body fat (density 0.2 (2SD 3.7) kg, total body water -0.9 (2SD 3.4) kg and dual-energy X-ray absorptiometry 0.1 (2SD 5.0) kg). The present paper also describes the derivation of a new prediction equation for the calculation of body composition from the Tanita body-fat analyser. The equation incorporates sex, age, and a log-transformation of height, weight and the measured impedance to predict body fat measured by a four-compartment model. This approach is recommended in the derivation of other prediction equations in body composition analysis. Using this novel prediction equation the residual standard deviations were 4.8% for men and 3.3% for women. A similar analysis using data collected with a conventional tetrapolar system yielded residual standard deviations of 4.3% for men and 3.1% for women. This demonstrates that the practical simplicity of the novel Tanita method is not associated with a clinically significant decrement in performance relative to a traditional impedance device. PMID- 10743489 TI - Effects of flavour amplification of Quorn and yoghurt on food preference and consumption in relation to age, BMI and odour perception. AB - Since the food habits of many elderly people are inadequate, the first experiment of the present study tested whether flavour amplification induces changes in preferences for and consumption of food and thus might result in a nutritional benefit. Two panels, one of 260 and one of 120 subjects, aged 19-98 years, took part in the study in which preferences for flavour-amplified yoghurt and Quorn were measured. For both products, only a few of the young subjects (20%) preferred the high flavour level; the percentage of subjects preferring the high flavour levels increased with age. These changes were highly significant. In a second experiment, participants received, under ad libitum conditions over 2 d in random order, a dish of yoghurt with either a high or a low flavour level. When adjusted for total consumption quantity, consumption of the highly flavoured yoghurt was not significantly correlated with age (r -0.03, P = 0.35). In a third experiment, odour perception was measured by determining the detection threshold for isoamylacetate. BMI values were obtained and the relationships between BMI and odour perception, age, preference and consumption were assessed in the age group 40-65 years. A significant correlation was observed between age and BMI (r 0.51, P < 0.0005). No significant correlation was observed between BMI and relative consumption of highly flavoured yoghurt (r -0.14, P = 0.14). A significant correlation was observed between BMI and preference for flavour amplified yoghurt (r 0.35, P < 0.001). However, no significant correlation was observed between BMI and odour perception (r 0.07, P = 0.32). With increasing age, a combined influence of age, sex, BMI and odour perception on food preference is to be expected. According to our multiple regression analysis, BMI showed a significant partial regression coefficient (standardized beta 0.36, P = 0.03). In conclusion, flavour amplification of food for older adults deserves attention, but specific approaches, which are tailored to the candidate food systems and older adult target groups, are needed. PMID- 10743491 TI - Comparison of the isotope dilution method for determination of the ileal endogenous amino acid losses with labelled diet and labelled pigs. AB - The aims of the present study were first to compare the amino acid dilution method performed using labelled animals with that using labelled diets, and second to determine real digestibilities and total ileal endogenous losses of N and amino acids. Two diets containing pea cultivars (Solara and Amino) and a protein-free diet were compared in a 3 x 3 Latin-square experiment. The three pigs were each prepared with an ileo-rectal anastomosis and were continuously infused with [1-13C]leucine. For each cultivar, 15N-labelled and unlabelled diets were formulated. The real digestibility and endogenous losses of leucine were higher when obtained by labelling the pig than by labelling the foodstuff. This was due either to the inadequate estimation of the endogenous protein enrichment in the first case or to the importance of dietary N recycling in the second case. However, in both cases the ileal endogenous losses of N and amino acids were higher than the basal losses determined with the protein-free diet. There were significant differences between the two pea cultivars in terms of phenylalanine and leucine when measured with labelled diets. It is suggested that, although ileal endogenous losses may be underestimated, using labelled feedstuffs is of great interest due to the direct estimation of the individual amounts of amino acids. PMID- 10743492 TI - Estimating the extent of degradation of ruminant feeds from a description of their gas production profiles observed in vitro: comparison of models. AB - An evaluation of general models that describe gas production profiles is presented. The models are derived from first principles by considering a simple three-pool scheme and permit the extent of ruminal degradation to be calculated, as described in the companion paper. The models evaluated were the generalized Mitscherlich, simple Mitscherlich, generalized Michaelis-Menten, simple Michaelis Menten, Gompertz, and logistic. Five sets of gas production data consisting of 216 curves, obtained using a wide range of feeds (including straw, hay, silage, grain and various byproducts), were analysed to study the performance of these gas production models. Application of the non-sigmoidal models (simple Mitscherlich and Michaelis-Menten) to the data resulted in convergence problems and these models were found to be inadequate in many cases. Based on results of a pairwise comparison between models (variance ratio test), ranking of residual mean squares, lack-of-fit test, and of analyses of residuals, the generalized Mitscherlich and the generalized Michaelis-Menten models seemed particularly suited because of their flexibility to encompass sigmoidal and non-sigmoidal shapes of gas production profiles, whether symmetrical or not. PMID- 10743493 TI - Estimating the extent of degradation of ruminant feeds from a description of their gas production profiles observed in vitro: derivation of models and other mathematical considerations. AB - Equations to describe gas production profiles, obtained using manual or automated systems for in vitro fermentation of ruminant feeds, were derived from first principles by considering a simple three-pool scheme. The pools represented were the potentially degradable and undegradable feed fractions, and accumulated gases. The equations derived and investigated mathematically were the generalized Mitscherlich, generalized Michaelis-Menten, Gompertz, and logistic. They were obtained by allowing the fractional rate of degradation to vary with time. The equations permit the extent of ruminal degradation (hence the supply of microbial protein to the duodenum) to be evaluated, thus linking the gas production technique to animal production. PMID- 10743494 TI - Decreasing dietary fat saturation lowers HDL-cholesterol and increases hepatic HDL binding in hamsters. AB - In order to study the mechanism by which increasing unsaturation of dietary fat lowers HDL-cholesterol levels, we studied various measures of HDL metabolism in hamsters fed with fats with different degrees of saturation. Hamsters were fed on a cholesterol-enriched (1 g/kg) semipurified diet containing 200 g/kg of maize oil, olive oil, or palm oil for 9 weeks. Increasing saturation of dietary fat resulted in increasing concentrations of total plasma cholesterol (4.29 (SD 0.51), 5.30 (SD 0.67) and 5.58 (SD 0.76) mmol/l respectively, n 12) and HDL cholesterol (3.31 (SD 0.50), 3.91 (SD 0.12) and 3.97 (SD 0.43) mmol/l) and these concentrations were significantly higher (P < 0.05) in the palm-oil and olive-oil fed hamsters compared with the maize-oil group. Total plasma triacylglycerol levels also increased with increasing fat saturation (1.01 (SD 0.59), 1.56 (SD 0.65) and 2.75 (SD 1.03) mmol/l) and were significantly higher (P < 0.05) in the palm-oil group compared with the olive-oil and maize-oil-fed hamsters. The three diets did not have differential effects on plasma activity levels of lecithin: cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP). Levels of phospholipid transfer protein (PLTP) tended to be higher with increasing fat saturation but this effect was not significant. The capacity of liver membranes to bind human HDL3 was significantly higher (P < 0.05) in the hamsters fed with maize oil (810 (SD 100) ng HDL3 protein/mg membrane protein, n 4) compared with those fed on palm oil (655 (SD 56) ng/mg), whereas the olive-oil group had intermediate values (674 (SD 26) ng/mg). The affinity of HDL3 for the binding sites was not affected by the type of dietary fat. Hepatic lipase (EC 3.1.1.3) activity, measured in liver homogenates, increased with increasing fat saturation. We conclude that dietary maize oil, when compared with either olive oil or palm-oil, may lower HDL-cholesterol concentrations by enhancing HDL binding to liver membranes. PMID- 10743495 TI - Dietary L-carnitine supplementation increases antigen-specific immunoglobulin G production in broiler chickens. AB - The usefulness of supplementary dietary L-carnitine as an immunomodulator to increase antigen-specific antibody levels was analysed in 2-6-week-old broilers. The chickens received commercial feeds either unsupplemented (starter feed 17.8 mg carnitine/kg, finisher diet 22.9 mg carnitine/kg) or supplemented with L carnitine (100 mg carnitine/kg added to feed). At 14 d of age, both groups were distributed in equal numbers and sex ratios over two environmentally controlled chambers where temperature (28 degrees) was either reduced immediately to 20 degrees, or gradually to 22 degrees at 36 d of age. Antigen-specific immunoglobulin (Ig)M, IgG, IgA and total Ig responses were measured following two immunizations with bovine serum albumin (BSA). The typical BSA-specific IgM responses followed by IgG responses to the primary immunization were boosted by the secondary immunization. The kinetics of these responses were not altered by L carnitine treatment. However, BSA-specific total Ig and IgG, but not IgM, responses were significantly increased by dietary L-carnitine supplementation, after both the primary and the secondary immunization. No significant influence of the sex of the chicks or the imposed environmental temperature on Ig responses was found. Temperature treatment and sex, but not L-carnitine supplementation, did significantly influence body-weight gain: cockerels were heavier than females and this became most evident in the second half of the rearing period. Further, lowering the temperature increased body weight. In conclusion, dietary L carnitine supplementation appeared to be beneficial in enhancing specific humoral responses on vaccination. PMID- 10743496 TI - Enhancement of natural and acquired immunity by Lactobacillus rhamnosus (HN001), Lactobacillus acidophilus (HN017) and Bifidobacterium lactis (HN019). AB - Consumption of lactic acid bacteria (LAB) has been suggested to confer a range of health benefits including stimulation of the immune system and increased resistance to malignancy and infectious illness. In the present study, the effects of feeding Lactobacillus rhamnosus (HN001, DR20), Lactobacillus acidophilus (HN017) and Bifidobacterium lactis (HN019, DR10) on in vivo and in vitro indices of natural and acquired immunity in healthy mice were examined. Mice were fed daily with L. rhamnosus, L. acidophilus or B. lactis (10(9) colony forming units) and their immune function was assessed on day 10 or day 28. Supplementation with L. rhamnosus, L. acidophilus or B. lactis resulted in a significant increase in the phagocytic activity of peripheral blood leucocytes and peritoneal macrophages compared with the control mice. The proliferative responses of spleen cells to concanavalin A (a T-cell mitogen) and lipopolysaccharide (a B-cell mitogen) were also significantly enhanced in mice given different LAB. Spleen cells from mice given L. rhamnosus, L. acidophilus or B. lactis also produced significantly higher amounts of interferon-gamma in response to stimulation with concanavalin A than cells from the control mice. LAB feeding had no significant effect on interleukin-4 production by spleen cells or on the percentages of CD4+, CD8+ and CD40+ cells in the blood. The serum antibody responses to orally and systemically administered antigens were also significantly enhanced by supplementation with L. rhamnosus, L. acidophilus or B. lactis. Together, these results suggest that supplementation of the diet with L. rhamnosus (HN001), L. acidophilus (HN017) or B. lactis (HN019) is able to enhance several indices of natural and acquired immunity in healthy mice. PMID- 10743498 TI - Intestinal, hepatic, and circulating vitamin K levels at low and high intakes of vitamin K in rats. AB - The aim of the present study was to assess how high doses of dietary vitamin K influence the intestinal profile of K-vitamins in vitamin K-deficient rats, and whether the induced changes are reflected in the hepatic vitamin K store. Vitamin K-deficient rats were fed for 10 d on diets containing different forms of vitamin K, and it was determined how these diets affected the vitamin K concentration at various sites of the instestine, serum, and the liver. It was found that the absorption of phylloquinone from standard food is not more than 10%, while the absorption of pharmacological doses of oil-solubilized phylloquinone and menaquinone-4 was also far from complete (18 and 55% respectively). High intakes of phylloquinone suppress the colonic production of all higher menaquinones. High menaquinone-4 intake induces very high menaquinone-8 concentrations, both in the colonic contents as well as in the liver. These data suggest that menaquinone-4 may be converted into menaquinone-8 (but not into other menaquinones) via a metabolic pathway which has not been reported previously. PMID- 10743497 TI - High-dose folic acid supplementation in rats: effects on gestation and the methionine cycle. AB - There is new evidence that a good folate status may play a critical role in the prevention of neural-tube defects and in lowering elevated homocysteine concentrations. This adequate folate status may be achieved through folic acid dietary supplementation. Folate is a water-soluble vitamin with a low potential toxicity. However, the possible consequences of long-term high-dose folic acid supplementation are unknown, especially those related to the methionine cycle, where folate participates as a substrate. With the aim of evaluating such possible effects, four groups of Wistar rats were classified on the basis of physiological status (virgin v. pregnant) and the experimental diet administered (folic-acid-supplemented, 40 mg/kg diet v. control, 2 mg folic acid/kg diet). Animals were fed on the diets for 3 weeks. Results showed that gestation outcome was adequate in both groups regardless of the dietary supplementation. However, there were reductions (P < 0.001) in body weight and vertex-coccyx length in fetuses from supplemented dams v. control animals. Folic acid administration also induced a higher (P < 0.01) S-adenosylmethionine: S-adenosylhomocysteine value due to increased S-adenosylmethionine synthesis (P < 0.01). However, hepatic DNA methylation and serum methionine concentrations remained unchanged. Serum homocysteine levels were reduced in supplemented dams (P < 0.05). Finally, pregnancy caused lower serum folate, vitamin B6 and vitamin B12 levels (P < 0.05). Folic acid administration prevented the effect of pregnancy and raised folate levels in dams, but did not change levels of vitamins B12 and B6. These new findings are discussed on the basis of potential benefits and risks of dietary folic acid supplementation. PMID- 10743499 TI - Whole small fish as a rich calcium source. AB - The present rat balance study investigated Ca availability from the whole indigenous small fish species, mola (Amblypharyngodon mola) from Bangladesh and from skimmed milk. Four groups of six young male rats each were fed ad libitum with diets containing either small fish or milk, at 100 or 160 g crude protein/kg diet dry matter. The study was conducted for 28 d and faeces and urine samples were collected in two balance periods: day 8-14 and 22-28. The Ca and N contents of the diets, faeces and urine were analysed. Ca intake, fractional Ca absorption and Ca retention in relation to N retention were measured. Digestibility and protein utilization of the experimental diets, weight gain of the rats, femur bone weight and mineral composition as well as bone alkaline phosphatase activity were also determined. It was concluded from the values of these variables that Ca from small fish with bones was available and appeared to be a useful Ca source in rats, though perhaps not as available as Ca from milk. This study suggests that small fish with bones may be an important source of Ca in human diets. Promotion of the production and consumption of small fish in population groups with low intakes of milk and milk products should therefore be encouraged. PMID- 10743500 TI - Effects of the mycelial extract of cultured Cordyceps sinensis on in vivo hepatic energy metabolism and blood flow in dietary hypoferric anaemic mice. AB - The beneficial effects of a traditional Chinese medicine, Cordyceps sinensis (Cs), on mice with hypoferric anaemia were evaluated by NMR spectroscopy. Experimental hypoferric anaemia was induced in mice by feeding with an Fe-free diet for 6 weeks. They were then given extract from cultured Cs (200 mg/kg body weight daily, orally) and were placed on an Fe-containing recovery diet (35 mg Fe/kg diet) for 4 weeks. In vivo 31P and 2H NMR spectra acquired noninvasively and quantitatively at weekly intervals were used to evaluate hepatic energy metabolism and blood flow in the mice. During the 4-week Cs-extract treatment, consistent increases were observed in liver beta-ATP: inorganic phosphate value by liver 31P NMR spectroscopy, representing the high energy state, and in blood flow rate as determined by 2H NMR spectroscopy of deuterated water (D2O) uptake after intravenous injection of D2O. The haematological variables (the packed cell volume and the haemoglobin level) and the hepatic intracellular pH, which was determined from the NMR chemical shift difference between the inorganic phosphate peak and the alpha-phosphate peak of ATP, were not significantly different between Cs-extract-treated and control mice. As blood flow and energy metabolism are thought to be linked, the Cs-extract-increased hepatic energy metabolism in the dietary hypoferric anaemic mice was concluded to be due to increased hepatic blood flow. PMID- 10743501 TI - Whole body hyperthermia cytokine induction: a review, and unifying hypothesis for myeloprotection in the setting of cytotoxic therapy. AB - Whole Body Hyperthermia (WBH) enhancement of chemotherapy and/or radiation without a concomitant increase in myelosuppression has been documented in clinical trials. We propose that the biological basis for this phenomena relates in part to the previously reported induction of peripheral cytokines by WBH, that is, granulocyte colony stimulating factor (G-CSF), interleukin (IL)-1 beta, IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha), and the regulatory cytokine IL-10. To further explain this myeloprotection and the additional clinical observation that WBH promotes early engraftment of bone marrow (when used as part of an allogenic bone marrow transplant preconditioning regimen) we developed a hypothesis: WBH increases peripheral IL-1 beta, IL-6, and TNF-alpha resulting in a secondary induction of IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF) in the bone marrow, for which supportive data also exists. Taken collectively, these data provide an increased understanding of the biological sequelae of fever, as well as a testable unifying hypothesis, for future antineoplastic treatment strategies. PMID- 10743502 TI - Latent transforming growth factor-beta binding proteins (LTBPs)--structural extracellular matrix proteins for targeting TGF-beta action. AB - Growth factors of the transforming growth factor-beta family are potent regulators of the extracellular matrix formation, in addition to their immunomodulatory and regulatory roles for cell growth. TGF-beta s are secreted from cells as latent complexes containing TGF-beta and its propeptide, LAP (latency-associated peptide). In most cells LAP is covalently linked to an additional protein, latent TGF-beta binding protein (LTBP), forming the large latent complex. LTBPs are required for efficient secretion and correct folding of TGF-beta s. The secreted large latent complexes associate covalently with the extracellular matrix via the N-termini of the LTBPs. LTBPs belong to the fibrillin-LTBP family of extracellular matrix proteins, which have a typical repeated domain structure consisting mostly of epidermal growth factor (EGF)-like repeats and characteristic eight cysteine (8-Cys) repeats. Currently four different LTBPs and two fibrillins have been identified. LTBPs contain multiple proteinase sensitive sites, providing means to solubilize the large latent complex from the extracellular matrix structures. LTBPs are now known to exist both as soluble molecules and in association with the extracellular matrix. An important consequence of this is LTBP-mediated deposition and targeting of latent, activatable TGF-beta into extracellular matrices and connective tissues. LTBPs have a dual function, they are required both for the secretion of the small latent TGF-beta complex as well as directing bound latent TGF-beta to extracellular matrix microfibrils. However, it is not known at present whether LTBPs are capable of forming microfibrils independently, or whether they are a part of the fibrillin-containing fibrils. Most LTBPs possess RGD-sequences, which may have a role in their interactions with the cell surface. At least LTBP-1 is chemotactic to smooth muscle cells, and is involved in vascular remodelling. Analyses of the expressed LTBPs have revealed considerable variations throughout the molecules, generated both by alternative splicing and utilization of multiple promoter regions. The significance of this structural diversity is mostly unclear at present. PMID- 10743503 TI - Dual role of tumor necrosis factor alpha in brain injury. AB - Brain injury (ischemia, trauma) is among the leading cause of mortality and disability in the western world. It induces increased production of tumor necrosis factor (TNF alpha) by brain resident cells. There is conflicting evidence on the role of this response in the injured brain, showing its potential effect in both processes of repair and of damage. This review presents data from clinical and experimental studies on the stimulation of TNF alpha production in brain injury and on the deleterious consequence of this acute response. Its inhibition by pharmacologic agents, neutralizing antibodies or soluble receptors has protective effects. In contrast, there are reports (from in-vitro studies or knock-out mice) on the beneficial effects of TNF alpha. To reconcile these apparently conflicting reports, the exact timing and extent of TNF alpha activation must be taken into account, as well as the presence of other mediators such as reactive oxygen species. It is suggested that the appropriate context of mediators, at any given time after brain injury may well determine whether the effect of TNF alpha is protective or toxic. PMID- 10743504 TI - Stat5a and Stat5b: fraternal twins of signal transduction and transcriptional activation. AB - Stat5a and Stat5b are discretely encoded transcription factors that mediate signals for a broad spectrum of cytokines. Their activation is often an integral component of redundant cytokine signal cascades involving complex cross-talk and pleiotropic gene regulation by Stat5 has been implicated in cellular functions of proliferation, differentiation and apoptosis with relevance to processes of hematopoiesis and immunoregulation, reproduction, and lipid metabolism. Although Stat5a and Stat5b show peptide sequence similarities of > 90%, targeted gene disruptions in mice yield distinctive phenotypes. Prolactin-directed mammary gland maturation fails without functional Stat5a, while disruption of Stat5b in males mitigates growth hormone effects on hepatic function and body mass. The molecular basis for this biologic dichotomy is probably multifaceted. Limited structural dissimilarities between the Stat5a and Stat5b transactivation domains, or subtle differences in the DNA-binding affinities of Stat5 dimer pairs undoubtedly influence gene regulation, but cell-dependent asymmetries in availability of phosphorylated Stat5 can be an underlying factor. Differences in serine phosphorylation(s) of Stat5a and Stat5b, or Stat5 associations with adaptor proteins or co-transcription factors are other potential sources of functional disparity and the signal amplitude, frequency or duration also can be significant. In addition to Stat5 signal attenuation by phosphatase actions or classical feedback inhibition, truncated forms of Stat5 lacking in transactivation capacity may compete upstream for activation and diminish access of full length molecules to DNA binding sites. PMID- 10743505 TI - Jerusalem of cytokines. AB - The Second Joint Meeting of the International Cytokine Society and the International Society for Interferon and Cytokine Research was held on October 25 30, 1998 in Jerusalem, Israel. The nature of this Joint Meeting dictated that it was intensive and covered topics that included receptor-ligand interactions, signal transduction, transcriptional regulation, antiviral action and apoptotic pathways induced by cytokines such as interferons, interleukines and chemokines. Their roles in infectious diseases and cancers were considered. This overview is by no mean comprehensive and covers only part of the many topics and subjects that were presented in the many plenary talks, symposia and poster sessions. The meeting was held in an excellent scientific atmosphere, that was probably affected by the "divine presence" in Jerusalem, and special thanks for the excellent organization are owed to Drs. Kaempfer, Revel, Wallach and Witz. PMID- 10743506 TI - Stopping vertical transmission of HIV: global strategies. PMID- 10743507 TI - National HIV strategy in Australia. PMID- 10743508 TI - Common head and neck manifestations of AIDS. AB - Head and neck manifestations of acquired immunodeficiency syndrome (AIDS) can involve the skin, ear, upper aerodigestive tract, and neck. Several head and neck manifestations of AIDS may be the only initial sign of this disease process and therefore primary-care physicians and otolaryngologists must be able to recognize and understand the management of these lesions. Cystic enlargement of the parotid gland and Kaposi's sarcoma are increasingly being encountered in the head and neck exam of HIV-infected patients. An example of each of these disease processes is presented with full discussion about the various treatment methods. PMID- 10743509 TI - Thoracic lymphadenopathy in HIV patients: spectrum of disease and differential diagnosis. AB - To evaluate the etiology and differential features of intrathoracic lymphadenopathy (LAD) in HIV patients, chest computed tomography (CT) records from an 18-month period were reviewed to identify all HIV-positive patients with intrathoracic LAD (nodal size > or = 1 cm). Medical records were reviewed for the documentation of specific diseases causing LAD and the CD4 count at the time of imaging. Of 45 HIV-positive patients with LAD, 40 had specific diagnoses including 22 (55%) infections and 17 (43%) tumors; one patient had both (3%). Mycobacterial disease accounted for 78% of infections; five cases were secondary to bacterial pneumonia and sepsis. Of tumors, lymphoma (7 cases, 39%) was most common, followed by lung cancer, germ cell tumors, and Kaposi's sarcoma. Mean CD4 cell count in patients with tumors was much higher than in patients with infections (314 vs. 62, p < .01). Patients with tumors were somewhat more likely than patients with infections to demonstrate axillary adenopathy (29 vs. 5%, p = .068). Cavitary disease was only observed in patients with infections (27%, p < .03). CT and clinical findings may help direct the differential diagnosis of LAD in AIDS, and promote expedient definitive diagnosis and therapy. PMID- 10743510 TI - Epidemiology of pediatric AIDS in Puerto Rico: 1981-1998. AB - The objective of this study was to describe the AIDS-defining conditions (ADC) and survival experience of pediatric AIDS cases (< 13 years age) reported in Puerto Rico. A descriptive analysis of the data gathered by the Puerto Rico AIDS Surveillance System was performed. Data for the 377 pediatric AIDS cases reported from January 1981 through June 1998 were reviewed. Survival curves following AIDS diagnosis were estimated using the Kaplan--Meier method and differences between curves were assessed by the Wilcoxon test. The majority (61%) of the cases were diagnosed before 2 years of age, and nearly 94% of them acquired the infection through perinatal transmission. The most common ADC were Pneumocystis carinii pneumonia (PCP) (23%), wasting syndrome (19.4%), and esophageal candidiasis (19.1%). The overall median survival time during the study period was 53.5 (95% CI: 38.0-106.2) months. Children < 1 year of age had a significantly shorter median survival time compared with older ages (p < 0.05). The survival experience in children diagnosed with PCP, pulmonary candidiasis, cytomegalovirus, and lymphocytic interstitial pneumonia (LIP) was significantly different (p < 0.05) to those children not diagnosed with these conditions. Although patients diagnosed after 1990 showed a median survival time longer than those diagnosed prior to 1990, the difference did not reach statistical significance (p > 0.05). The frequency of several ADC and median survival time of Puerto Rican children differed from those reported in the United States. This may reflect differences in diagnostic procedures or reporting practices. PMID- 10743511 TI - Service delivery for patients with HIV in a rural state: the Vermont model. AB - The incidence of human immunodeficiency virus (HIV) infection is increasing rapidly in rural areas of the United States. Barriers to health-care delivery for this patient population include the complexity of this rapidly changing field, inexperienced rural physicians, long travel distances to receive expert care, lack of psychosocial support systems, and concerns about confidentiality. Models of HIV care for rural areas have not been developed that remove these barriers. We present the philosophy, structure, implementation, and services of a model of care in Vermont that is designed to remove many of these barriers and bring HIV expertise into the rural areas of the state. Three HIV specialty clinics have been developed in regional hospitals throughout the state. The clinic team includes an HIV-trained nurse practitioner and social worker from the hospital, a client consultant from the regional AIDS service organization, and an infectious disease specialist who travels to each of the clinics monthly. Patient care will be centralized in these regionally located clinics. The dispersion of HIV care among numerous and inexperienced rural providers will be obviated. Confidentiality will be emphasized within the hospital environment. The model has the potential to provide a complete continuum of medical care and psychosocial case management, integrate patient care and regional provider education, and increase community awareness. Patients will be able to receive their care in their own community, avoiding long travel distances. This may encourage patients to seek care earlier in their illness. The model may be adaptable to other rural areas of the United States. PMID- 10743513 TI - HIV/AIDS case histories: diagnostic problems. Hemophagocytic syndrome. PMID- 10743512 TI - Demographic and attitudinal variables related to high-risk behaviors in Asian males who have sex with other men. AB - Although AIDS is spreading rapidly in minority communities, little is known about attitudes, knowledge, and behavior related to AIDS and HIV in the Asian community. The purpose of this study was to examine these variables in a sample of gay Asian males, as well as to investigate the relationship between knowledge, sources of information, culturally influenced attitudes and high-risk behaviors in this population. Results from a sample of 60 young Asian men who self identified as "having sex with other men" indicated they were generally knowledgeable about methods of transmission and prevention, and appeared linked to a large information network that included informal sources such as peers and formal sources. However, significant percentages held culturally biased views of AIDS, such as believing race of partner or one's own gender role in the sexual encounter determined level of risk; and one third of the sample did not use condoms regularly. Having been tested for HIV was associated with holding less traditional cultural beliefs and a higher sexual activity level. Open communication about safer sexual practices was associated with monogamous as opposed to multiple relationships and with decreased tendency to engage in alcohol-related unprotected sex. Variance in overall risk was predicted by demographic variables such as education, age, and level of sexual activity, rather than by the attitudinal factors measured. Limitations of this exploratory study include a time-limited subject recruitment period and consequent small sample size, a homogeneous sample weighted toward young, well-educated, and middle-class respondents, and the exclusion of non-English speaking individuals. Nevertheless, study findings suggest that educational outreach targeting Asians who have sex with other men needs to address inaccurate cultural beliefs about HIV/AIDS, emphasize consistent condom use, and encourage models of open communication with partners about safer sexual practices. PMID- 10743514 TI - Merck to launch vaccine trial. PMID- 10743515 TI - Potato vaccine for hepatitis. PMID- 10743516 TI - Idiopathic esophageal ulcers in an AIDS patient resulting in a stricture. PMID- 10743517 TI - Sexual issues in older adults. PMID- 10743518 TI - Presentation, diagnosis, and management of renal failure in patients with HIV infection. AB - HIV infected patients are at increased risk of developing renal failure when compared to the general population. Renal disease occurs both as a primary manifestation of HIV infection and secondary to complications such as intercurrent illness or drug therapy. Examples of primary HIV renal disease, including HIV associated nephropathy (HIV AN) and immune complex glomerulonephronephritis, and secondary causes of renal failure are reviewed with full discussion about investigation and treatment methods. PMID- 10743519 TI - Role of CD4:CD8 ratio in predicting HIV co-infection in patients with newly diagnosed tuberculosis. AB - Because of the clinical implications of Human Immunodeficiency Virus (HIV) status on treatment of tuberculosis (TB) and in view of the low percentage of patients in whom HIV testing is performed, we evaluated immunological features of 54 patients with newly diagnosed TB and its ability to predict HIV co-infection. All 54 patients had initially unknown HIV status and had no other Acquired Immunodeficiency Syndrome (AIDS) defining illnesses. Twenty-two patients were found to be HIV seropositive and 32 were seronegative. The median CD4 and CD8 counts were statistically different between the HIV seropositive and seronegative patients, however, there was overlap between the two groups. The median CD4:CD8 ratio was 0.17 in HIV seropositive patients and 1.95 in the seronegative patients and had minimal overlap (p < 0.0001). A CD4:CD8 ratio < or = 0.7 gave a sensitivity of 100%, specificity of 94%, positive-predictive value of 92% and a negative-predictive value of 100% in predicting HIV co-infection. In conclusion, HIV-co-infection in patients with newly diagnosed TB could be predicted on the basis of the CD4:CD8 ratio. PMID- 10743520 TI - Kaposi's sarcoma of the breast. AB - Kaposi's sarcoma of the breast is extremely rare. We report a case of a patient with AIDS presenting with Kaposi's sarcoma localized to the breast and review another published case of a patient with AIDS and disseminated cutaneous Kaposi's sarcoma with breast involvement. Although rare, the diagnosis should be considered in patients with breast lumps. PMID- 10743521 TI - Oral manifestations of pediatric vertical HIV infection. AB - To assess the prevalence and prognostic significance of the history of oral manifestations in children with human immunodeficiency virus infection (HIV), a cohort study of 73 children with vertical HIV infection was conducted. The study subjects were examined every 6 months for oral manifestations. The period prevalence of oral manifestations ranged from a low of 1% for submandibular enlargement and 3% for hairy leukoplakia to a high of 36% for xerostomia and 51% for cervical lymphadenopathy. The occurrence of oral manifestations did not change significantly over time from 1995 to 1998. Finally, the odds of occurrence of cervical lymphadenopathy, xerostomia, and oral candidiasis were greater among children in whom these manifestations had been diagnosed in the preceding 6-18 months than in children without prior diagnosis. Oral manifestations are significant clinical outcomes in pediatric vertical HIV infection, particularly for children diagnosed previously with an oral manifestation. PMID- 10743523 TI - Pancreatic warning for ddI. PMID- 10743522 TI - Association between delirium and death in AIDS patients. AB - The purpose of this study was to examine the relationship between delirium and death in AIDS patients. Forty-one patients admitted to a combination skilled nursing and assisted-living facility in 1994 were included in the retrospective chart review. Patients were grouped according to the presence versus absence of delirium during the first week of admission. Demographic characteristics and medical morbidity of the two groups were compared using the Chi-square statistic. Kaplan-Meier survival analysis was used to estimate survival functions during the study period for the delirious and nondelirious groups. Nine patients (22%) were found to have an episode of delirium in the first week of admission. There were no significant differences in demographic characteristics or medical morbidity between the delirious and nondelirious groups. Median days from admission to death for those with delirium (10 days) versus those without delirium (135 days) was significantly different (log rank = 19.03; p < 0.0001). Authors concluded that delirium is a marker for decreased survival in this sample of AIDS patients. Future research needs to demonstrate whether improved care of AIDS patients can prevent delirium or limit adverse outcomes associated with it. PMID- 10743524 TI - Compassionate use for tenofovir. PMID- 10743525 TI - Ritonavir and anemia in women. PMID- 10743526 TI - Drinking teenagers more sexual. PMID- 10743527 TI - 5-FU for cervical intraepithelia neoplasia. PMID- 10743528 TI - Less frequent TMP-SMX is effective. PMID- 10743529 TI - Hepatitis B vaccine safe. PMID- 10743530 TI - "Amplified perversity": trends in heterosexual HIV. PMID- 10743531 TI - Using print media for STD prevention: reflections of a journalist/public health researcher. PMID- 10743532 TI - Genital HIV shedding in women. AB - Heterosexual transmission of HIV is the predominant transmission mode among adults world-wide, while mother-to-child transmission accounts for the majority of HIV infections in children. Factors that affect genital tract shedding of HIV virus or cell-associated provirus in women are probably important determinants of infectiveness, and hence of transmission risk during sexual contact or delivery. This review discusses the genital HIV RNA and DNA loads in relation to those in the blood and outlines some of the parameters influencing genital tract shedding of HIV. PMID- 10743533 TI - Sexually transmitted infections in female rape victims. AB - Sexual assault is a common occurrence that carries significant physical and mental health morbidity. Some of the most common concerns expressed by sexual assault victims can be allayed by addressing the risks of rape-related sexually transmitted infections. Although exact risks cannot be accurately quantified, it is important that health-care professionals provide counseling as well as prophylaxis and/or treatment as indicated. Screening and treatments should follow established guidelines, and follow-up care is indicated to confirm successful prevention or treatment. Continued support, surveillance, and medical attention should be offered as needed. This article will address important clinical issues regarding the prevalence and consequences of sexual assault, and will summarize current recommendations for the prophylaxis and treatment of sexually transmitted infections in adolescent and adult rape victims. Some of the logistical issues surrounding HIV testing, counseling, and prevention for rape victims will also be addressed. PMID- 10743534 TI - Female genital mutilation: complications and risk of HIV transmission. AB - There are over 100 million girls and women who have undergone female genital mutilation (FGM). The World Health Organization (WHO) estimates that another 2 million are subject to it every year. FGM is practiced in many countries, especially Africa and parts of the Middle East. Various degrees of FGM are prevalent, the most mutilating one being infibulation (pharaonic). With infibulation there are numerous life-long health problems such as hemorrhage, infection, dyspareunia, genital ulcers, and gynecological and obstetrical complications. It has been postulated that FGM may also play a significant role in facilitating the transmission of HIV infection through numerous mechanisms. In this article several of the most common complications are discussed and helpful suggestions for management during pregnancy and delivery are explored. Included are the legal and ethical ramifications. PMID- 10743535 TI - Heterosexual anal intercourse: prevalence, cultural factors, and HIV infection and other health risks, Part I. AB - Studies of heterosexual HIV transmission have consistently found anal intercourse to be a highly predictive risk factor for seroconversion. Yet most AIDS prevention messages targeted at heterosexuals, presumably influenced by cultural taboos against acknowledging this sexual practice, continue to emphasize vaginal and, increasingly, oral sex transmission. The health risks of anal sex appear to be severely underestimated by a substantial proportion of sexually active women and men in North and Latin America as well as parts of South Asia, Africa, and other regions. Among heterosexuals reported rates of condom use are nearly universally lower for anal than for vaginal intercourse. This review examines anal sex among the general population, including its prevalence in various world regions, related sociocultural factors, and other associated health problems including anorectal STDs, Hepatitis B infection, and HPV-related anal cancer in women. U.S. survey and other data suggest that, in terms of absolute numbers, approximately seven times more women than homosexual men engage in unprotected receptive anal intercourse. Research among higher risk subpopulations, including bisexual men, injecting drug users, female sex workers, inner-city adolescents, and serodiscordant heterosexual couples, indicates that persons particularly at risk of being infected by or transmitting HIV are also more likely to practice anal sex. Considering this finding, along with the much greater efficiency for HIV infection as well as lower rates of condom usage, a significant proportion of heterosexual transmission in some populations is due to anal intercourse. This typically stigmatized and hidden sexual practice must be given greater emphasis in AIDS/STD prevention, women's care, and other health promotion programs. PMID- 10743536 TI - Condom availability for HIV/STD prevention. AB - Condom availability is a structural intervention that increases access to condoms. It is the most important strategy for the prevention of the sexual transmission of HIV/STDs in developing countries. While it is an integral component of many prevention programs, it is considered controversial in high school settings and its role in a variety of HIV/STD prevention interventions has been understudied, understated, and/or unacknowledged. Condom availability as an HIV/STD prevention strategy needs to be reprioritized and should be considered a critical foundation of all programs to prevent the sexual transmission of HIV. PMID- 10743537 TI - Contraceptive use and pregnancy decision making among women with HIV. AB - HIV is a growing epidemic among women in the United States. This study seeks to determine if knowledge of HIV infection and of the benefits of prenatal zidovudine (ZDV) to decrease vertical HIV transmission is related to decisions about pregnancy planning, contraceptive and condom use, and pregnancy termination among noninjection drug using (IDU) women with HIV. Eighty-two HIV-infected women were interviewed about their pregnancy decisions, contraceptive and condom use, and pregnancy outcome. Data was verified by structured chart review where available. Awareness of HIV infection or knowledge of the benefits of prenatal ZDV use did not significantly influence pregnancy planning, contraceptive choice, use of contraception, or consideration of pregnancy termination. Condom use was extremely low (14.6% consistent use), the majority of pregnancies (68.0%) were unplanned, contraceptive use was low (50.9%), and few pregnancies were terminated (6.3%). Women on Medicaid were significantly less likely than women with private or no insurance to terminate their pregnancy (2/20, 10% vs. 3/5, 60%, p = 0.04, two-tailed Fisher's exact test). Most women (70.0%) reported the most important reason for carrying the pregnancy to term was the desire for a child. In conclusion, among women in this non-IDU, Midwestern cohort, knowledge of HIV infection was not associated with decisions to plan a pregnancy, use contraception if not planning pregnancy, or terminate an unplanned pregnancy. PMID- 10743538 TI - Potential role of lactobacilli as prophylactic agents against genital pathogens. AB - The characteristics required of lactobacilli as probioties are the following: beneficial function, easy cultivation, nonpathogenicity, adhesion, and population stability. Several studies have assessed the potential of lactobacilli in the prevention or treatment of certain genitourinary tract infections such as bacterial vaginosis, vaginitis, or urinary tract infections. The main goal of therapy with biotherapeutic agents should be to prevent overgrowth of a pathogen until such a time that the normal microbiota can be reestablished. The possibility of using lactobacilli is promising, especially in pregnant women and in the case of patients with recurrent genitourinary tract infections produced by strains with resistance to several antibiotics. In addition, probiotic therapy is considered as "natural" and without side effects in contrast with conventional pharmaceutical treatments, but there is a limited array of tested biotherapeutic agents and a lack of pharmacokinetic data. PMID- 10743539 TI - Calcium may help with diarrhea. PMID- 10743540 TI - T-20 continues to look promising. PMID- 10743541 TI - Ketoconazole may improve PI penetration. PMID- 10743542 TI - Phase III trial of IL-2. PMID- 10743543 TI - Reformulated ritonavir. PMID- 10743544 TI - Trial studies PI combination. PMID- 10743545 TI - Amprenavir study results released. PMID- 10743546 TI - Drug combination treats brain cancer. PMID- 10743547 TI - Phase I results of herpes vaccine. PMID- 10743548 TI - Stopping PCP prophylaxis. PMID- 10743549 TI - Gel condoms being developed. PMID- 10743550 TI - New contraceptive for men. PMID- 10743551 TI - STD treatment and incidence. PMID- 10743552 TI - The Second Georgia Tech International Conference on Bioinformatics: Sequence, Structure and Function. November 11-14, 1999, Atlanta, Georgia, USA. Proceedings. PMID- 10743553 TI - FramePlus: aligning DNA to protein sequences. AB - MOTIVATION: Automated annotation of Expressed Sequence Tags (ESTs) is becoming increasingly important as EST databases continue to grow rapidly. A common approach to annotation is to align the gene fragments against well-documented databases of protein sequences. The sensitivity of the alignment algorithm is key to the success of such methods. RESULTS: This paper introduces a new algorithm, FramePlus, for DNA-protein sequence alignment. The SCOP database was used to develop a general framework for testing the sensitivity of such alignment algorithms when searching large databases. Using this framework, the performance of FramePlus was found to be somewhat better than other algorithms in the presence of moderate and high rates of frameshift errors, and comparable to Translated Search in the absence of sequencing errors. AVAILABILITY: The source code for FramePlus and the testing datasets are freely available at ftp.compugen.co.il/pub/research. CONTACT: raveh@compugen.co.il. PMID- 10743554 TI - Finding prokaryotic genes by the 'frame-by-frame' algorithm: targeting gene starts and overlapping genes. AB - MOTIVATION: Tightly packed prokaryotic genes frequently overlap with each other. This feature, rarely seen in eukaryotic DNA, makes detection of translation initiation sites and, therefore, exact predictions of prokaryotic genes notoriously difficult. Improving the accuracy of precise gene prediction in prokaryotic genomic DNA remains an important open problem. RESULTS: A software program implementing a new algorithm utilizing a uniform Hidden Markov Model for prokaryotic gene prediction was developed. The algorithm analyzes a given DNA sequence in each of six possible global reading frames independently. Twelve complete prokaryotic genomes were analyzed using the new tool. The accuracy of gene finding, predicting locations of protein-coding ORFs, as well as the accuracy of precise gene prediction, and detecting the whole gene including translation initiation codon were assessed by comparison with existing annotation. It was shown that in terms of gene finding, the program performs at least as well as the previously developed tools, such as GeneMark and GLIMMER. In terms of precise gene prediction the new program was shown to be more accurate, by several percentage points, than earlier developed tools, such as GeneMark.hmm, ECOPARSE and ORPHEUS. The results of testing the program indicated the possibility of systematic bias in start codon annotation in several early sequenced prokaryotic genomes. AVAILABILITY: The new gene-finding program can be accessed through the Web site: http:@dixie.biology.gatech.edu/GeneMark/fbf.cgi CONTACT: mark@amber.gatech.edu. PMID- 10743555 TI - Evaluation of gene prediction software using a genomic data set: application to Arabidopsis thaliana sequences. AB - MOTIVATION: The annotation of the Arabidopsis thaliana genome remains a problem in terms of time and quality. To improve the annotation process, we want to choose the most appropriate tools to use inside a computer-assisted annotation platform. We therefore need evaluation of prediction programs with Arabidopsis sequences containing multiple genes. RESULTS: We have developed AraSet, a data set of contigs of validated genes, enabling the evaluation of multi-gene models for the Arabidopsis genome. Besides conventional metrics to evaluate gene prediction at the site and the exon levels, new measures were introduced for the prediction at the protein sequence level as well as for the evaluation of gene models. This evaluation method is of general interest and could apply to any new gene prediction software and to any eukaryotic genome. The GeneMark.hmm program appears to be the most accurate software at all three levels for the Arabidopsis genomic sequences. Gene modeling could be further improved by combination of prediction software. AVAILABILITY: The AraSet sequence set, the Perl programs and complementary results and notes are available at http://sphinx.rug.ac.be:8080/biocomp/napav/. CONTACT: Pierre.Rouze@gengenp.rug.ac.be. PMID- 10743556 TI - An automated comparative analysis of 17 complete microbial genomes. AB - MOTIVATION: As sequenced genomes become larger and sequencing becomes faster, there is a need to develop accurate automated genome comparison techniques and databases to facilitate derivation of genome functionality; identification of enzymes, putative operons and metabolic pathways; and to derive phylogenetic classification of microbes. RESULTS: This paper extends an automated pair-wise genome comparison technique (Bansal et al., Math. Model. Sci. Comput., 9, 1-23, 1998, Bansal and Bork, in First International Workshop of Declarative Languages, Springer, pp. 275-289, 1999) used to identify orthologs and gene groups to derive orthologous genes in a group of genomes and to identify genes with conserved functionality. Seventeen microbial genomes archived at ftp://ncbi.nlm.nih.gov/genbank/genomes have been compared using the automated technique. Data related to orthologs, gene groups, gene duplication, gene fusion, orthologs with conserved functionality, and genes specifically orthologous to Escherichia coli and pathogens has been presented and analyzed. AVAILABILITY: A prototype database is available at ftp://www.mcs.kent.edu/arvind/intellibio / orthos.html. The software is free for academic research under an academic license. The detailed database for every microbial genome in NCBI is commercially available through intellibio software and consultancy corporation (Web site: http://www.mcs.kent.edu/arvind/intellibio . html). CONTACT: arvind@mcs.kent.edu. PMID- 10743557 TI - Genome rearrangement with gene families. AB - MOTIVATION: The theory and practice of genome rearrangement analysis breaks down in the biologically widespread contexts where each gene may be present in a number of copies, not necessarily contiguous. In some of these contexts it is, however, appropriate to ask which members of each gene family in two genomes G and H, lengths lG and lH, are its true exemplars, i.e. which best reflect the original position of the ancestral gene in the common ancestor genome. This entails a search for the two exemplar strings of same length n (= number of gene families, including singletons), having the smallest possible rearrangement distance: the exemplar distance. RESULTS: A branch and bound algorithm calculates these distances efficiently when based on easily calculated traditional rearrangement distances, such as signed reversals distance or breakpoint distance, which also satisfy a property of monotonicity in the number of genes. Simulations show that in two random genomes, the expected exemplar distance/n is sensitive to the number and size of gene families, but approaches 1 as the number of singleton families increases. When the basic rearrangement distance is just the number of breakpoints, the expected cost of computing the exemplar breakpoints distance (EBD), as measured by total calls to the underlying breakpoint distance routine, is highly dependent on both n and the configuration of gene families. On the other hand, basing exemplar distance on exemplar reversals distance (ERD), the expected computing cost depends on the configuration of gene families but is not sensitive to n. AVAILABILITY: Code for EBD and ERD is available from the author or may be accessed at http://www.crm.umontreal.ca/viart/exemplar_di s. html CONTACT: sankoff@ere.umontreal.ca. PMID- 10743558 TI - Structural basis for triplet repeat disorders: a computational analysis. AB - MOTIVATION: Over a dozen major degenerative disorders, including myotonic distrophy, Huntington's disease and fragile X syndrome, result from unstable expansions of particular trinucleotides. Remarkably, only some of all the possible triplets, namely CAG/CTG, CGG/CCG and GAA/TTC, have been associated with the known pathological expansions. This raises some basic questions at the DNA level. Why do particular triplets seem to be singled out? What is the mechanism for their expansion and how does it depend on the triplet itself? Could other triplets or longer repeats be involved in other diseases? RESULTS: Using several different computational models of DNA structure, we show that the triplets involved in the pathological repeats generally fall into extreme classes. Thus, CAG/CTG repeats are particularly flexible, whereas GCC, CGG and GAA repeats appear to display both flexible and rigid (but curved) characteristics depending on the method of analysis. The fact that (1) trinucleotide repeats often become increasingly unstable when they exceed a length of approximately 50 repeats, and (2) repeated 12-mers display a similar increase in instability above 13 repeats, together suggest that approximately 150 bp is a general threshold length for repeat instability. Since this is about the length of DNA wrapped up in a single nucleosome core particle, we speculate that chromatin structure may play an important role in the expansion mechanism. We furthermore suggest that expansion of a dodecamer repeat, which we predict to have very high flexibility, may play a role in the pathogenesis of the neurodegenerative disorder multiple system atrophy (MSA). CONTACT: pfbaldi@ics.uci.edu, yves@netid.com, brunak@cbs.dtu.dk, gorm@cbs.dtu.dk. PMID- 10743559 TI - A simple algorithm for detecting circular permutations in proteins. AB - MOTIVATION: Circular permutation of a protein is a genetic operation in which part of the C-terminal of the protein is moved to its N-terminal. Recently, it has been shown that proteins that undergo engineered circular permutations generally maintain their three dimensional structure and biological function. This observation raises the possibility that circular permutation has occurred in Nature during evolution. In this scenario a protein underwent circular permutation into another protein, thereafter both proteins further diverged by standard genetic operations. To study this possibility one needs an efficient algorithm that for a given pair of proteins can detect the underlying event of circular permutations. A possible formal description of the question is: given two sequences, find a circular permutation of one of them under which the edit distance between the proteins is minimal. A naive algorithm might take time proportional to N3 or even N4, which is prohibitively slow for a large-scale survey. A sophisticated algorithm that runs in asymptotic time of N2 was recently suggested, but it is not practical for a large-scale survey. RESULTS: A simple and efficient algorithm that runs in time N2 is presented. The algorithm is based on duplicating one of the two sequences, and then performing a modified version of the standard dynamic programming algorithm. While the algorithm is not guaranteed to find the optimal results, we present data that indicate that in practice the algorithm performs very well. AVAILABILITY: A Fortran program that calculates the optimal edit distance under circular permutation is available upon request from the authors. CONTACT: ron@biocom1.ls.biu.ac.il. PMID- 10743560 TI - Exploiting the past and the future in protein secondary structure prediction. AB - MOTIVATION: Predicting the secondary structure of a protein (alpha-helix, beta sheet, coil) is an important step towards elucidating its three-dimensional structure, as well as its function. Presently, the best predictors are based on machine learning approaches, in particular neural network architectures with a fixed, and relatively short, input window of amino acids, centered at the prediction site. Although a fixed small window avoids overfitting problems, it does not permit capturing variable long-rang information. RESULTS: We introduce a family of novel architectures which can learn to make predictions based on variable ranges of dependencies. These architectures extend recurrent neural networks, introducing non-causal bidirectional dynamics to capture both upstream and downstream information. The prediction algorithm is completed by the use of mixtures of estimators that leverage evolutionary information, expressed in terms of multiple alignments, both at the input and output levels. While our system currently achieves an overall performance close to 76% correct prediction--at least comparable to the best existing systems--the main emphasis here is on the development of new algorithmic ideas. AVAILABILITY: The executable program for predicting protein secondary structure is available from the authors free of charge. CONTACT: pfbaldi@ics.uci.edu, gpollast@ics.uci.edu, brunak@cbs.dtu.dk, paolo@dsi.unifi.it. PMID- 10743561 TI - Local multiple sequence alignment using dead-end elimination. AB - MOTIVATION: Local multiple sequence alignment is a basic tool for extracting functionally important regions shared by a family of protein sequences. We present an effectively polynomial-time algorithm for rigorously solving the local multiple alignment problem. RESULTS: The algorithm is based on the dead-end elimination procedure that makes it possible to avoid an exhaustive search. In the framework of the sum-of-pairs scoring system, certain rejection criteria are derived in order to eliminate those sequence segments and segment pairs that can be mathematically shown to be inconsistent (dead-ending) with the globally optimal alignment. Iterative application of the elimination criteria results in a rapid reduction of combinatorial possibilities without considering them explicitly. In the vast majority of cases, the procedure converges to a unique globally optimal solution. In contrast to the exhaustive search, whose computational complexity is combinatorial, the algorithm is computationally feasible because the number of operations required to eliminate the dead-ending segments and segment pairs grows quadratically and cubically, respectively, with the total number of sequence elements. The method is illustrated on a set of protein families for which the globally optimal alignments are well recognized. AVAILABILITY: The source code of the program implementing the algorithm is available upon request from the authors. CONTACT: alex_lukashin@biogen.com. PMID- 10743562 TI - Importance of the Sgs1 helicase activity in DNA repair of Saccharomyces cerevisiae. AB - The Saccharomyces cerevisiae Sgs1 protein, together with Schizosaccharomyces pombe Rqh1 and the human Bloom and Werner proteins, is a DNA helicase of the Escherichia coli RecQ family. Mutation of SGS1 causes premature aging in yeast cells, including the accumulation of extrachromosomal rDNA circles. We have recently shown that Sgs1p interacts with the DNA repair Rad16p protein and is epistatic to Rad16p for UVC, 4-NQO and H2O2 lesions. Therefore we tested sgs1 strains containing mutations in the helicase and C-terminal domains. We demonstrate here that the helicase activity of the Sgs1 is important for most elements of the sgs1 mutation phenotype, including sensitivity to UVC, 4-NQO, H2O2, MMS and hydroxyurea. PMID- 10743563 TI - The cadmium-resistant gene, CAD2, which is a mutated putative copper-transporter gene (PCA1), controls the intracellular cadmium-level in the yeast S. cerevisiae. AB - Yeast cells carrying the CAD2 gene exhibit a resistance to cadmium. We cloned this gene and demonstrated that it was a mutated form derived from the gene of a putative copper-transporting ATPase (PCA1). By site-directed mutagenesis, it appeared that the mutation conferring cadmium resistance was a R970G-substitution in the C-terminal region of Pca1 protein. The intracellular cadmium level of cells carrying CAD2 was lower than that of cells carrying either PAC1 or delta cad2. Furthermore, cells with overexpression of CAD2 showed a much lower intracellular cadmium level than that of cells with a single-copy CAD2. From these results, we conclude that the Cad2 protein controls the intracellular cadmium level through an enhanced cadmium efflux system. PMID- 10743564 TI - Disruption of the MRP-L23 gene encoding the mitochondrial ribosomal protein L23 is lethal for Kluyveromyces lactis but not for Saccharomyces cerevisiae. AB - The Kluyveromyces lactis nuclear gene, MRP-L23, encodes a polypeptide of 155 amino acids that shares 70% and 43% identity to the ribosomal proteins L23 and L13 of Saccharomyces cerevisiae and Escherichia coli. The deduced protein, designated K1L23, is a likely component of the large subunit of mitochondrial ribosomes as it can complement the respiratory deficient phenotype of a S. cerevisiae mrp-L23 mutant. As in S. cerevisiae, KlMRP-L23 is essential for respiratory growth of K. lactis because disruption of the gene in a "petite positive" strain carrying a rho o-lethality suppressor atp mutation rendered cells unable to grow on a nonfermentable carbon source. However, in contrast to S. cerevisiae, disruption of MRP-L23 in wild type K. lactis is lethal. Meiotic segregants of K. lactis with a disrupted MRP-L23 allele form microcolonies with cell numbers varying from 32 to 300. These data clearly indicate an essential role of mitochondrial protein synthesis for viability of the petite-negative yeast K. lactis. PMID- 10743566 TI - Directed deletions in the aflatoxin biosynthesis gene homolog cluster of Aspergillus oryzae. AB - To investigate the structure of the aflatoxin gene cluster in Aspergillus oryzae, 39 strains belonging to this species were examined for the existence of pksA, fas1A, aflR and vbs, and the results compared with those for ver-1 obtained previously. These five genes are involved in aflatoxin biosynthesis in Aspergillus parasiticus. The strains examined were categorized into three groups; group 1, having the five homologs; 2, having ver-1 and vbs; and 3, having vbs homologs. Long-PCR analysis of the regions between the five homologs in A. oryzae IFO 4135, coupled with Southern-hybridization analysis, shows that those homologs are clustered with the same arrangement as in A. parasiticus. These results suggest that directed deletions of the cluster occur in A. oryzae strains. The possible breakpoints of the deletions in the strains of group 2 and 3 were estimated. PMID- 10743565 TI - A gene associated with filamentous growth in Ophiostoma novo-ulmi has RNA-binding motifs and is similar to a yeast gene involved in mRNA splicing. AB - The COL1 gene was isolated from Ophiostoma novo-ulmi using the techniques of insertional mutagenesis and plasmid rescue. Sequence analyses suggest that the COL1 gene encodes a unique protein of 826 amino acids with consensus-type RNA binding domains, most similar to a putative protein from Schizosaccharomyces pombe which resembles the C-terminus of the Saccharomyces cerevisiae U4/U6 splicing factor PRP24. Disruption of the COL1 gene produced the yeast-like col1 mutant. The inability of the mutant to synthesize the COL1 gene product was confirmed by transcript analysis. Transformation of the col1 mutant with the COL1 gene restored the wild phenotype and production of the 4.0-kb mRNA. The results from this study demonstrate that the COL1 RNA-binding protein is associated with filamentous growth in O. novo-ulmi. PMID- 10743567 TI - The plasma membrane H(+)-ATPase gene family in the arbuscular mycorrhizal fungus Glomus mosseae. AB - To identify genes that encode plasma membrane H(+)-ATPases in the arbuscular mycorrhizal fungus Glomus mosseae two sets of degenerate primers matching highly conserved motifs present in all plant and fungal ATPases were designed. Nested PCR-amplification of G. mosseae genomic DNA using the designed degenerate primers was carried out. Sequence analysis of the cloned PCR products identified five different clones (GmHA1, GmHA2, GmHA3, GmHA4 and GmHA5) encoding putative plasma membrane H(+)-ATPases. Comparison of the deduced amino-acid sequences of GmHA1 GmHA5 indicate that GmHA1, GmHA3 and GmHA4 are highly identical, while GmHA2 and GmHA5 are more divergent. The evolutionary and functional significance of the divergence found among the different members of the H(+)-ATPase gene family in G. mosseae is discussed. PMID- 10743568 TI - Characterization and expression of a Neurospora crassa ribosomal protein gene, crp-7. AB - We have isolated and characterized a Neurospora crassa cytoplasmic ribosomal protein gene, named crp-7, which is found upstream of the photolyase gene. The deduced amino-acid sequence of this gene is highly homologous to the YS25 ribosomal protein of Saccharomyces cerevisiae. The crp-7 ORF consists of two exons which are separated by a short intron. The deduced polypeptide contains 87 amino acid residues and has a calculated molecular weight of 9.7 kDa. RFLP mapping showed that the crp-7 gene is located on the right arm of linkage group I. Southern blot hybridization analyses indicated that there is only one copy of the crp-7 gene in the N. crassa genome. Transcriptional elements, the Dde box, the Taq box and the CG element, that have been identified in other N. crassa ribosomal protein genes are observed in the promoter region of the crp-7 gene. The crp-7 mRNA levels were low in conidia and highest in young mycelia during vegetative growth. The mRNA levels of four r-protein genes, including the crp-7 gene, as well as the tef-1 gene encoding translational elongation factor 1 alpha, were raised following the treatment of mycelia with a low concentration of cycloheximide. This indicates that the expression of r-protein genes is under the control of so-called super-induction. PMID- 10743569 TI - In vitro DNA fragmentation of mitochondrial DNA caused by single-stranded breakage related to macroplasmodial senescence of the true slime mold, Physarum polycephalum. AB - We found that mitochondrial DNA (mtDNA) isolated from Physarum polycephalum fragmented itself in weak ionic solutions. The mtDNA was dissolved in STE (saline Tris-EDTA: 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA), TE (10 mM Tris-HCl, 1 mM EDTA) and DW, and then electrophoresed in an agarose gel. The intact 86-kbp mtDNA band was seen in STE, but several novel bands appeared in TE and DW. In TE, two discrete bands appeared at 6.7-kbp (alpha-band) and 5.0-kbp (beta-band), whereas at least 17 discrete bands were observed in distilled water (DW). These fragmentation patterns were not stoichiometric, as seen when using restriction endonucleases, but were clearly different from the degradation of DNA caused by a physical shearing force or a contaminating nuclease. In this paper, we characterize this in vitro fragmentation of mtDNA from P. polycephalum. We located 19 fragments, including the alpha and beta fragments, on a mtDNA restriction map, and demonstrated that these cleavage sites were S1 nuclease sensitive regions, which are single-stranded DNA regions such as nicks and gaps in the mtDNA. The alpha and beta fragments are derived from the region encoding ribosomal RNAs (rRNAs) and the ATP synthase (atpA) gene, while the other 17 fragments are not derived from any specific region, but the cleavage sites are located throughout the mtDNA molecule. In P. polycephalum, it is well known that the growth rate of macroplasmodia decreases with aging. Equal amounts of mtDNA from juvenile and aged macroplasmodia were electrophoresed and the frequency of the beta fragment in each sample was measured. The ratio of the beta band to the total signal including background was estimated to be 3.3-4.0% in juvenile macroplasmodia, whereas it increased to 8.3-28.2% in aged macroplasmodia. This result suggests that the in vitro fragmentation of mtDNA is associated with macroplasmodial senescence. The single-stranded breakage of mtDNA of P. polycephalum may accumulate with age. PMID- 10743570 TI - Mutations at three different nuclear loci of Chlamydomonas suppress a defect in chloroplast psbD mRNA accumulation. AB - In the photosynthetic chloroplast mutant PRB2A of Chlamydomonas reinhardtii the psbD mRNA is unstable. Three strains were isolated, in which the underlying site directed mutation within the psbD 5' UTR (untranslated region) is suppressed. In all three suppressors, psbD RNA levels and RNA 5' maturation are restored to a varying extent, suggesting a tight coupling of RNA stabilization and 5' processing. Expression of the psbA gene is not compromised in these strains. Genetic crosses revealed that the suppressor mutations affect three unlinked nuclear loci, which may encode new factors involved in psbD gene expression. PMID- 10743571 TI - Isolation, characterization, and chromosomal mapping of an ftsZ gene from the unicellular primitive red alga Cyanidium caldarium RK-1. AB - The FtsZ protein is involved in eukaryote plastid division, but there is little information on its involvement in the plastid-dividing apparatus. To investigate the relationship between FtsZ and the plastid-dividing ring, the ftsZ gene was isolated from the unicellular primitive red alga Cyanidium caldarium RK-1. Comparison of several prokaryotic and eukaryotic FtsZ proteins shows that there are six highly conserved domains in the core region of FtsZ. To determine the chromosomal location of ftsZ, we first determined the electrophoretic karyotype of C. caldarium RK-1. Southern-hybridization analysis combined with CHEF revealed the chromosomes on which the ftsZ gene exist. Northern-hybridization analysis indicated that the C. caldarium RK-1 ftsZ gene is transcribed as a 1.9-kb molecule, and that the transcripts specifically accumulate just before plastid division. Phylogenetic analysis indicated that C. caldarium RK-1 and other eukaryotic ftsZ genes are the descendants of cyanobacterial ftsZ genes, supporting the current agreement that FtsZ is involved in plastid division. PMID- 10743572 TI - Regulation of endopolygalacturonase gene expression in Botrytis cinerea by galacturonic acid, ambient pH and carbon catabolite repression. AB - The phytopathogenic fungus Botrytis cinerea produces a set of endopolygalacturonases (endoPGs) which are involved in the enzymatic degradation of pectin in plant cell walls. The endoPG-encoding genes of B. cinerea are differentially expressed when the fungus is grown in liquid culture on different carbon sources. A basic constitutive expression level was observed for two genes, Bcpg1 and Bcpg2, which encode basic isozymes. Galacturonic acid was shown to induce the expression of Bcpg4 and Bcpg6. Low pH of the culture medium resulted in induced expression of the Bcpg3 gene. Expression of the Bcpg5 gene was inducible; however the inducing factors could not be identified. Finally, galacturonic acid-induced expression of the Bcpg4 gene was repressed by the presence of more-favourable carbon sources, such as glucose. PMID- 10743573 TI - [Microchimera. New thought process for pathogenesis of systemic scleroderma]. AB - Persistent cellular microchimerism might play a role in the pathogenesis of systemic sclerosis (SSc). Microchimerism results from the traffic of fetal cells through the placenta during pregnancy into the maternal circulation and their survival due to HLA class II compatibility. In female SSc patients the presence of fetal CD3-positive T-cells in the maternal circulation and of fetal cells in the affected skin tissue has been identified through y chromosome specific DNA sequences. The persistent microchimerism might cause SSc in certain patients by initiating a fetal anti-maternal Graft-versus-host-like response. PMID- 10743574 TI - [Mycophenolate mofetil: a new immunosuppressive drug in dermatology and its possible uses]. AB - Mycophenolate mofetil represents a new immunosuppressive drug. Several case reports have shown that mycophenolate mofetil has a good therapeutic effect in patients with psoriasis and autoimmune dermatoses. Mycophenolate mofetil is a morpholine ester of mycophenolic acid which was shown in the 1970s to be effective in patients with severe psoriasis. Mycophenolate mofetil has an increased bioavailability as compared to mycophenolic acid and thereby an improved therapeutic window. Currently, mycophenolate mofetil is indicated for the prevention of organ rejection in transplant patients. The mode of action and the pharmacology of mycophenolate mofetil are reviewed and the current clinical experience with this immunosuppressive drug for dermatological disorders is summarized. PMID- 10743575 TI - [Determining the severity of venous insufficiency with duplex sonography]. AB - OBJECTIVE: The volume flow in the common femoral vein (SV) as a representative parameter of the venous hemodynamics of the leg can be measured using duplex sonography. A direct correlation between the SV-data and the clinical grade of the venous disease was postulated. PATIENTS/METHODS: SV was measured in 36 patients (62 limbs) with complete varicosis of the long saphenous vein, 18 patients (24 limbs) with an incomplete form of varicosis and 40 healthy persons (78 limbs). The assessments were done under standardized conditions. We found a significant difference of the SV-data between the three groups. RESULTS: The values of SV were significantly elevated in varicosis compared with the healthy limbs. The data determined in legs with complete varicosis were significantly raised compared to incomplete varicosis. The means were 0.38 l/min in complete varicosis, 0.26 l/min in incomplete varicosis and 0.13 l/min in healthy limbs. During a time course of 30 minutes the values were stable. CONCLUSIONS: The results demonstrate a significant correlation between the measured SV data and the grade of venous disease. PMID- 10743576 TI - [Scabies epidemic in a sheltered workshop--what should be done?]. AB - Scabies is an infectious parasitic skin disease with a notable rising incidence in Germany. The disease is usually transmitted by close physical contact, but indirect spread e.g. by bedding is also possible. Due to its contagiousness, introduction of scabies into crowding living facilities, such as dormitories or kindergartens, can easily cause an epidemic outbreak. We describe an epidemic of scabies in a workshop for handicapped people in February 1998. A worker with severe scabies reported that numerous colleagues in both workshop and the associated hostel had complained of pruritus for months and that some of them already had undergone scabicide treatment. The number of contacts (staff, colleagues, friends, attendants, family) of our patient and the other already affected people was more than 460. The management of the workshop asked for help in handling the epidemic. We describe the cooperative efforts of the management, as well as hospital and private dermatologists, to evaluate all potential contacts and present a concept of treatment for the termination of such an epidemic outbreak of scabies. PMID- 10743577 TI - [The UV record. Document for quality control, therapy planning and risk assessment in dermatologic photo- and photochemotherapy]. AB - Ultraviolet (UV-) radiation therapy as a mono- or combination therapy (UV-A, UV A1, UV-B, SUP, UV-B311) or as photochemotherapy with photosensitization (systemic PUVA-, bath PUVA-, topical PUVA-therapy) are successfully used for the treatment of several dermatological disorders. Long-term side effects of natural UV (sun light) include photoaging and induction of skin tumors. At present, the relevance of in-vitro findings of potential tumor induction in animals through therapeutic levels of UV radiation is a matter of debate. To assess these risks, information on treated location, kind of UV radiation and cumulative UV doses are required. Practically this information is barely accessible. This makes decisions on possible therapies difficult. To solve this problem we propose to use an "UV pass". At the end of each UV radiation cycle, the body location, the type of radiation and the cumulative dose are documented and this pass is given to the patient. This will improve the information transfer if the doctor is changed, as well as facilitating decisions about certain therapies and calculation of long term risks of UV radiation. Finally it will improve the quality of UV photo- and photochemotherapy. PMID- 10743578 TI - [Combination of antimalarial drugs mepacrine and chloroquine in therapy refractory cutaneous lupus erythematosus]. AB - Antimalarials represent the first line in treatment of cutaneous lupus erythematosus (LE). However, some patients show no improvement on monotherapy with chloroquine or hydroxychloroquine. A 30-year-old female patient had treatment-resistant cutaneous LE exhibiting features of both LE tumidus and subacute cutaneous LE. Previously, the patient had been unsuccessfully treated with chloroquine, hydroxychloroquine, dapsone, and azathioprine, each in combination with variable doses of prednisolone. However, the LE lesions increased during these therapeutic regimens. A combination of chloroquine and mepacrine therapy led to improvement and then total clearing after 4 months of treatment. PMID- 10743579 TI - [Perianal streptococcal dermatitis]. AB - Two boys aged 6 and 9 years presented with persistent sharply circumscribed perianal erythema. One boy had no other skin findings; the other had additional lesions on the genitalia and extremities. The diagnosis of perianal streptococcal dermatitis was made after streptococci were cultured from skin swabs. This primarily childhood disease should be distinguished from candidiasis and perianal atopic dermatitis. As in our cases, topical therapy is usually ineffective and oral penicillin remains the treatment of choice. PMID- 10743580 TI - [Interstitial granulomatous dermatitis with arthritis]. AB - Interstitial granulomatous dermatitis with arthritis is a rare dermatologic disorder seen in patients suffering from diseases in which circulating immune complexes occur. The typical cutaneous signs are linear cords usually located on the lateral aspect of the trunk. The characteristic, although not specific, histology reveals a dense diffuse infiltrate composed mostly of histiocytes, accompanied by neutrophils and eosinophils, and degenerated collagen surrounded by palisades of histiocytes. We discuss this disorder and its differential diagnosis. PMID- 10743581 TI - [Dubowitz syndrome and atopic eczema. Case report of monozygotic twins]. AB - Autosomal recessive inheritance, intrauterine growth retardation, short stature, microcephaly, distinct facial dysmorphism, psychomotoric retardation, and often uncharacterized eczematous skin lesions distinguish the rare Dubowitz syndrome. Here a pair of monozygotic twins with Dubowitz syndrome and clear-cut atopic eczema is presented. PMID- 10743582 TI - [Andy Warhol's plastic nose revision reflected in his work]. AB - Andy Warhol underwent dermabrasion in 1957 because of a nasal skin lesion, which is best diagnosed as a rhinophyma on the basis of the available biographical literature. Throughout his creative life, the artist worked on different aspects of plastic surgery especially in the nasal area. As one of the founders of Pop Art, Warhol left essential marks on the modern society and influenced our standards of aesthetic shapes. PMID- 10743583 TI - [Response to the comment on the contribution "Successful therapy of Lyell syndrome with intravenous immunoglobulins"]. PMID- 10743584 TI - [Annual Meeting of the German-Speaking Mycology Society and the European Confederation of Medical Mycology in Dresden, June 4-6 1999]. PMID- 10743585 TI - [Hans Holzmann (1929-1999)]. PMID- 10743586 TI - [Photodiagnostic test methods. 1: Stepwise light exposure and the photopatch test]. PMID- 10743587 TI - [Pathophysiology of chronic heart failure]. PMID- 10743588 TI - [Heart failure in primary non-cardiac diseases]. PMID- 10743589 TI - [Diagnosis of heart failure]. PMID- 10743590 TI - [Therapy of acute heart failure. Emergency therapy]. PMID- 10743591 TI - [Current status of therapy of chronic heart failure]. PMID- 10743592 TI - [Arterial hypertension. 2: Therapy]. PMID- 10743593 TI - [Intermittent claudication in a 58-year-old man without risk factors]. PMID- 10743594 TI - [66-year-old patient with myxedema and polyserositis]. PMID- 10743595 TI - [Police ordered blood specimen collection]. PMID- 10743596 TI - [Legal aspects of medical care]. PMID- 10743597 TI - [Antibiotic therapy in cholecystitis, cholangitis and pancreatitis]. PMID- 10743598 TI - A generalized transport model for biased cell migration in an anisotropic environment. AB - A generalized transport model is derived for cell migration in an anisotropic environment and is applied to the specific cases of biased cell migration in a gradient of a stimulus (taxis; e.g., chemotaxis or haptotaxis) or along an axis of anisotropy (e.g., contact guidance). The model accounts for spatial or directional dependence of cell speed and cell turning behavior to predict a constitutive cell flux equation with drift velocity and diffusivity tensor (termed random motility tensor) that are explicit functions of the parameters of the underlying random walk model. This model provides the connection between cell locomotion and the resulting persistent random walk behavior to the observed cell migration on longer time scales, thus it provides a framework for interpreting cell migration data in terms of underlying motility mechanisms. PMID- 10743599 TI - Mathematical modelling of the intravenous glucose tolerance test. AB - Several attempts at building a satisfactory model of the glucose-insulin system are recorded in the literature. The minimal model, which is the model currently mostly used in physiological research on the metabolism of glucose, was proposed in the early eighties for the interpretation of the glucose and insulin plasma concentrations following the intravenous glucose tolerance test. It is composed of two parts: the first consists of two differential equations and describes the glucose plasma concentration time-course treating insulin plasma concentration as a known forcing function; the second consists of a single equation and describes the time course of plasma insulin concentration treating glucose plasma concentration as a known forcing function. The two parts are to be separately estimated on the available data. In order to study glucose-insulin homeostasis as a single dynamical system, a unified model would be desirable. To this end, the simple coupling of the original two parts of the minimal model is not appropriate, since it can be shown that, for commonly observed combinations of parameter values, the coupled model would not admit an equilibrium and the concentration of active insulin in the "distant" compartment would be predicted to increase without bounds. For comparison, a simple delay-differential model is introduced, is demonstrated to be globally asymptotically stable around a unique equilibrium point corresponding to the pre-bolus conditions, and is shown to have positive and bounded solutions for all times. The results of fitting the delay differential model to experimental data from ten healthy volunteers are also shown. It is concluded that a global unified model is both theoretically desirable and practically usable, and that any such model ought to undergo formal analysis to establish its appropriateness and to exclude conflicts with accepted physiological notions. PMID- 10743600 TI - Traveling waves and pulses in a one-dimensional network of excitable integrate and-fire neurons. AB - We study the existence and stability of traveling waves and pulses in a one dimensional network of integrate-and-fire neurons with synaptic coupling. This provides a simple model of excitable neural tissue. We first derive a self consistency condition for the existence of traveling waves, which generates a dispersion relation between velocity and wavelength. We use this to investigate how wave-propagation depends on various parameters that characterize neuronal interactions such as synaptic and axonal delays, and the passive membrane properties of dendritic cables. We also establish that excitable networks support the propagation of solitary pulses in the long-wavelength limit. We then derive a general condition for the (local) asymptotic stability of traveling waves in terms of the characteristic equation of the linearized firing time map, which takes the form of an integro-difference equation of infinite order. We use this to analyze the stability of solitary pulses in the long-wavelength limit. Solitary wave solutions are shown to come in pairs with the faster (slower) solution stable (unstable) in the case of zero axonal delays; for non-zero delays and fast synapses the stable wave can itself destabilize via a Hopf bifurcation. PMID- 10743601 TI - Disclosing the diagnosis of pediatric HIV infection: mothers' views. AB - ISSUES AND PURPOSE: The stigma of HIV infection creates barriers to disclosure. The purpose of this study was to identify to whom biological and foster mothers disclose the diagnosis of HIV infection, discuss their rationale, and describe the recipient's reactions. DESIGN AND METHODS: A descriptive, qualitative study included biological (n = 9) and foster (n = 11) mothers of children with HIV infection. RESULTS: Three themes emerged from the data: Telling for support, determining who should know, and telling children. These themes were present for both biological and foster mothers. PRACTICE IMPLICATIONS: It is important for nurses to realize that parental disclosure of the diagnosis of HIV infection is a long-term, age-appropriate process that will take place over many discussions and time, and should have the support of the interdisciplinary team. Additional psychological support also should be available. PMID- 10743602 TI - Risky sexual behavior among adolescent women. AB - ISSUES AND PURPOSE: To review the epidemiology and etiology of risky sexual behavior in adolescent women, and to discuss implications for primary prevention. CONCLUSION: Adolescent women who participate in risky sexual behavior are at risk for sexually transmitted infections, including HIV. Black, Hispanic, and out-of home adolescent women, however, are at greatest risk. Factors contributing to risky sexual behavior include early initiation of sexual intercourse, inconsistent use of condoms and other barrier contraception, and unprotected sexual intercourse. Identified protective factors for early initiation of sexual activity include the development of healthy sexuality, family and school connectedness, and the presence of caring adults. PRACTICE IMPLICATIONS: Effective clinical interventions target high-risk adolescent women; incorporate environmental and cognitive-behavioral components; use social learning theories; address differences in regards to culture, developmental stage, and sexual experience; and support family and school involvement. PMID- 10743603 TI - Topics of conflict between parents and young adolescents. AB - ISSUES AND PURPOSE: Parents often examine and question interactions with their young teen and may ask the advice of healthcare professionals. Topics, frequency, and intensity of conflicts between young adolescents and parents were therefore examined. DESIGN AND METHODS: A descriptive survey using the 44-item Issues Checklist (Robin, 1975) with 163 parent and young adolescent (ages 11-14) dyads. RESULTS: Parents and teens were congruent about their reports of the topics, frequency, and intensity of conflict. Discussion of the topics generally was not angry. Mothers reported the greatest quantity of issues. Potentially sensitive topics such as substance use, dating, and sex were rarely approached by either parent or young adolescent. Sociodemographic characteristics did not distinguish or were not associated with IC scores. PRACTICE IMPLICATIONS: Conflict is a common component of the parent-young adolescent relationship. Families with children entering adolescence can expect conflict about issues that recur but usually are not that "hot". Anticipating topics may put conflict in perspective. Nurses help families resolve conflicts associated with day-to-day conflicts as a first step toward opening up larger, potentially sensitive topics. PMID- 10743604 TI - 'Tis the season. PMID- 10743605 TI - Genetic discoveries: challenges for nurses who care for children and their families. AB - Genetic discoveries emerging from the HGP are revolutionizing health care. With the potential to promote health and minimize disease through these discoveries, pediatric nurses will need to incorporate the health, ethical, and social aspects of this knowledge into their care of children and families. Genetic advancements are creating new challenges not only for nursing practice, but also for educational curricula. While genetic information and technology bring great potential for new ways to detect and manage a wide range of pediatric health problems, nurses must ensure that this information is managed in ways that will maximize benefit and minimize potential harm to children and their families. Educational preparation is critical to the application of genetic information and testing in health care of all clients. Pediatric nurses with adequate educational preparation in genetics now have an exciting opportunity to develop new skills and roles and make a significant contribution to genetic services and the advancement of nursing knowledge of children. PMID- 10743606 TI - Metabolic control analysis of glycolysis in trypanosomes as an approach to improve selectivity and effectiveness of drugs. AB - Glycolysis is the only ATP-generating process in bloodstream form trypanosomes and is therefore a promising drug target. Inhibitors which decrease significantly the glycolytic flux will kill the parasites. Both computer simulation and experimental studies of glycolysis in bloodstream form Trypanosoma brucei indicated that the control of the glycolytic flux is shared by several steps in the pathway. The results of these analyses provide quantitative information about the prospects of decreasing the flux by inhibition of any individual enzyme. The plasma membrane glucose transporter appears the most promising target from this perspective, followed by aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and glycerol-3-phosphate dehydrogenase. Non-competitive or irreversible inhibitors would be most effective, but it is argued that potent competitive inhibitors can be suitable, provided that the concentration of the competing substrate cannot increase unrestrictedly. Such is the case for inhibitors that compete with coenzymes or with blood glucose. PMID- 10743607 TI - Competition and protease sensitivity assays provide evidence for the existence of a hydrogenosomal protein import machinery in Trichomonas vaginalis. AB - Hydrogenosomes are double membrane bounded redox organelles found in a number of amitochondriate protists and fungi. They are involved in carbohydrate metabolism and ATP synthesis and thus resemble mitochondria. Molecular analysis of the hydrogenosomal heat shock proteins Hsp70, Hsp60 and Hsp10 in Trichomonas vaginalis, one of the deepest-branching eukaryotes known to date, has revealed that these group exclusively with mitochondrial heat shock proteins. This finding indicates strongly that a progenitor organelle which gave rise to contemporary mitochondria and hydrogenosomes existed early in eukaryotic life. This hypothesis is further supported by similarities of hydrogenosomal and mitochondrial biogenesis. It was shown that T. vaginalis hydrogenosomal proteins are synthesized on free ribosomes in the cytosol with an N-terminal presequence that carries targeting information and is cleaved upon import into the organelle. Furthermore, as in mitochondrial import, hydrogenosomal protein import requires ATP, an electrochemical transmembrane potential and cytosolic protein factor(s). Here we demonstrate that inhibition of hydrogenosomal protein import occurs (i) in the presence of a synthetic presequence peptide and (ii) after pretreatment of hydrogenosomes with the protease trypsin. Trypsin pretreatment affects two hydrogenosomal membrane proteins of 31 and 70 kDa, respectively. Thus, we present evidence that import is saturable and that proteinaceous hydrogenosomal import receptor(s) exist. These results are a first step towards a characterization of the hydrogenosomal import machinery which should provide further insights into the relationship of hydrogenosomes and mitochondria and the evolution of protein targeting into organelles of endosymbiotic origin. PMID- 10743608 TI - Cell-cycle and developmental regulation of TbRAB31 localisation, a GTP-locked Rab protein from Trypanosoma brucei. AB - Rab proteins are small GTPases that control the direction and timing of vesicle fusion during intracellular trafficking between membraneous compartments. Genome sequencing and EST analysis of Trypanosoma brucei indicates that the trypanosome Rab (TbRAB) gene family, and hence complexity of intracellular transport pathways, is intermediate between Saccharomyces cerevisiae and mammals. TbRAB31 is a constitutively expressed T. brucei Rab protein (formerly Trab7p) and is the product of one of two closely linked TbRAB genes, the other being TbRAB2 (TbRab2p, in: Field H, Ali BRS, Sherwin T, Gull K, Croft SL, Field MC. TbRab2p, a marker for the endoplasmic reticulum of Trypanosoma brucei, localises to the ERGIC in mammalian cells. J Cell Sci 1999; 112:147-156), involved in ER to Golgi transport. TbRAB31 has high homology to members of the Sec4/Ypt1 subfamily of Rab proteins from S. cerevisiae and to Rab13 and Rab11 from higher eukaryotes. Recombinant TbRAB31 binds GTP but, unusually for a Rab protein, has undetectable GTPase activity resulting in a constitutively GTP-bound protein. Antibodies against TbRAB31 recognise a discrete structure located between the kinetoplast and nucleus in interphase procyclic cells; by contrast the structure is morphologically more complex in bloodstream form (BSF) parasites, consisting of at least two foci. TbRAB31 behaviour was also studied during the cell cycle; TbRAB31 always localised to a discrete structure that duplicated very early in mitosis and relocated to daughter cells in a coordinate manner with the basal body and kinetoplast, suggesting the involvement of microtubules. Additional evidence suggests that TbRAB31 localises to the trypanosome Golgi complex. Firstly, the interphase position of TbRAB31 is consistent with a Golgi location. Secondly, the TbRAB31 structure is also recognised by cross-reacting antibodies to mammalian beta-coatomer protein (beta-COP), which localises to the Golgi in mammalian cells. Thirdly, the fluorescent ceramide analogue, BODIPY-TR-ceramide, a reliable marker of the mammalian Golgi apparatus, exhibited overlapping distribution with TbRAB31. The location of BODIPY-TR-ceramide was confirmed at the trypanosome Golgi by histochemistry with diaminobenzidine and electron microscopy. PMID- 10743609 TI - Molecular characterization of a Litomosoides sigmodontis protein involved in the development of the microfilarial sheath during embryogenesis. AB - A cDNA clone Ls110 was isolated from a female Litomosoides sigmodontis expression library using an antiserum raised against the microfilarial sheath. The complete cDNA encodes a protein (Ls110) of 382 amino acids. Southern and PCR analyses revealed the presence of Ls110 in L. sigmodontis as a single copy gene. The transcription of the Ls110 gene was limited to female worms. In these worms the transcription was confined to the epithelial cells of the uterus. The protein Ls110 was detected not only in the epithelial layer of the uterus but also secreted in the lumen of the uterus. All the intra-uterine embryonic stages showed the protein bound to their egg shell/sheath, except the early multicellular embryonic stages and fully developed microfilariae. The transient occurrence of Ls110 on these structures of intra-uterine stages besides the presence of a cysteine-rich N-terminal region (SXC-like domain) suggest that the protein may play a role in the formation of the microfilarial sheath during embryogenesis. PMID- 10743610 TI - Loss of matrix metalloproteinase 9 activity in Theileria annulata-attenuated cells is at the transcriptional level and is associated with differentially expressed AP-1 species. AB - The schizont stage of the protozoan parasite Theileria annulata reversibly transforms bovine monocytes into an immortalised and metastatic state. We have been studying T. annulata induction of host matrix metalloproteinases (MMP) which are involved in parasite dissemination and pathogenesis. We have observed that prolonged in vitro culture of T. annulata-infected cell lines results in their attenuation and this process is associated with alterations in both host and parasite gene expression. In particular, a loss in bovine MMP expression in later passage cultures suggests that these parasite-induced MMPs are virulence factors. As a means to further our understanding of the attenuation process we examine in detail the parasite-induced differential expression of one particular bovine proteinase, MMP9, in non-attenuated (p58) and attenuated (p158) passage levels of the Ode vaccine line. We show here that MMP9 expression is regulated at the transcriptional level and we suggest that a particular parasite-induced AP-1 recognition transcription factor present in the Ode non-attenuated line may have a role to play in the expression of this host gene. PMID- 10743611 TI - Stage-specific isoforms of Ascaris suum complex. II: The fumarate reductase of the parasitic adult and the succinate dehydrogenase of free-living larvae share a common iron-sulfur subunit. AB - Complex II of adult Ascaris suum muscle exhibits high fumarate reductase (FRD) activity and plays a key role in anaerobic electron-transport during adaptation to their microaerobic habitat. In contrast, larval (L2) complex II shows a much lower FRD activity than the adult enzyme, and functions as succinate dehydrogenase (SDH) in aerobic respiration. We have reported the stage-specific isoforms of complex II in A. suum mitochondria, and showed that at least the flavoprotein subunit (Fp) and the small subunit of cytochrome b (cybS) of the larval complex II differ from those of adult. In the present study, complete cDNAs for the iron-sulfur subunit (Ip) of complex II, which with Fp forms the catalytic portion of complex II, have been cloned and sequenced from anaerobic adult A. suum, and the free-living nematode, Caenorhabditis elegans. The amino acid sequences of the Ip subunits of these two nematodes are similar, particularly around the three cysteine-rich regions that are thought to comprise the iron-sulfur clusters of the enzyme. The Ip from A. suum larvae was also characterized because Northern hybridization showed that the adult Ip is also expressed in L2. The Ip of larval complex II was recognized by the antibody against adult Ip, and was indistinguishable from the adult Ip by peptide mapping. The N-terminal 42 amino acid sequence of Ip in the larval complex II purified by DEAE-cellulofine column chromatography was identical to that of the mature form of the adult Ip. Furthermore, the amino acid composition of larval Ip determined by micro-analysis on a PVDF membrane is almost the same as that of adult Ip. These results, together with the fact, that homology probing by RT-PCR, using degenerated primers, failed to find a larval-specific Ip, suggest that the two different stage-specific forms of the A. suum complex II share a common Ip subunit, even though the adult enzyme functions as a FRD, while larval enzyme acts as an SDH. PMID- 10743612 TI - Proteolysis of Plasmodium falciparum surface antigen, Pfs230, during gametogenesis. AB - During Plasmodium falciparum gametogenesis, proteolysis of Pfs230, a 360 kDa gametocyte surface protein, generates two large polypeptides, 307 and 300 kDa, that remain associated with the surface of the newly formed gamete. Using peptide specific antibodies, the amino termini of the 307 and 300 kDa forms have been mapped to between aa 477-487 and aa 523-555, respectively, which is the region between the glutamate rich repeats and the cysteine motif domains. Concomitantly, two peptides, 47 and 35 kDa, corresponding to the region upstream from the cleavage site are released into the medium. The membrane permeant cysteine protease inhibitor, E64d, blocks production of the 300 and 35 kDa forms of Pfs230, but does not alter the formation of the 307 or 47 kDa forms. In contrast, E64, which has been shown to inhibit the development of P. falciparum trophozoites, does not block proteolytic processing of Pfs230. Production of both the 307 and 300 kDa forms was reduced by a metallo-protease inhibitor, 1,10 phenanthroline, whereas the rest of the protease inhibitors tested had no effect on Pfs230 processing. This is the first study of proteolysis during gametogenesis and it demonstrates that the two large forms of Pfs230 produced are generated by proteases with different specificities. The data also suggest that Pfs230 undergoes proteolytic processing prior to emergence from the red blood cell. PMID- 10743613 TI - Comparative study of Leishmania mexicana and Trypanosoma brucei NAD-dependent glycerol-3-phosphate dehydrogenase. AB - The NAD-dependent glycerol-3-phosphate dehydrogenases (G3PDH, EC 1.1.1.8) of Trypanosoma brucei and Leishmania mexicana are thought to have different roles in carbohydrate metabolism. Here the physicochemical and kinetic properties of natural G3PDH from T. brucei with the recombinant homologue of L. mexicana which share 63% positional identity are compared. Despite their supposed different functions in energy metabolism of the parasites the two G3PDHs have remarkably similar properties, including pH optima and K(m) value for dihydroxyacetone phosphate (DHAP) and NADH in the formation of glycerol 3-phosphate (G3P) and for NAD+ and G3P in the reverse reaction. Both enzymes are subject inhibition by dihydroxyacetone phosphate at concentrations above 0.2 mM and are inhibited by the trypanocidal drugs suramin and melarsen oxide at sub-micromolar concentrations. PMID- 10743614 TI - Differential regulation of nucleoside and nucleobase transporters in Crithidia fasciculata and Trypanosoma brucei brucei. AB - The regulation of the activity of purine transporters in two protozoan species, Crithidia fasciculata and Trypanosoma brucei brucei, was investigated in relation to purine availability and growth cycle. In C. fasciculata, two high-affinity purine nucleoside transporters were identified. The first, designated CfNT1, displayed a K(m) of 9.4 +/- 2.8 microM for adenosine and was inhibited by pyrimidine nucleosides as well as adenosine analogues; a second C. fasciculata nucleoside transporter (CfNT2) recognized inosine (K(m) = 0.38 +/- 0.06 microM) and guanosine but not adenosine. The activity of both transporters increased in cells at mid-logarithmic growth, as compared to cells in the stationary phase, and was also stimulated 5-15-fold following growth in purine-depleted medium. These increased rates were due to increased Vmax values (K(m) remained unchanged) and inhibited by cycloheximide (10 microM). In the procyclic forms of T. b. brucei, adenosine transport by the P1 transporter was upregulated by purine starvation but only after 48 h, whereas hypoxanthine transport was maximally increased after 24 h. The latter effect was due to the expression of an additional hypoxanthine transporter, H2, that is normally absent from procyclic forms of T. b. brucei and was characterised by its high affinity for hypoxanthine (K(m) approximately 0.2 microM) and its sensitivity to inhibition by guanosine. The activity of the H1 hypoxanthine transporter (K(m) approximately 10 microM) was unchanged. These results show that regulation of the capacity of the purine transporters is common in different protozoa, and that, in T. b. brucei, various purine transporters are under differential control. PMID- 10743615 TI - Characterization of a Trypanosoma brucei SR domain-containing protein bearing homology to cis-spliceosomal U1 70 kDa proteins. AB - The protozoan parasite Trypanosoma brucei relies on trans-splicing of a common spliced leader (SL) RNA to maturate mRNAs. Using the yeast two-hybrid system a protein (TSR1IP) was identified that interacts with the T. brucei serine-arginine (SR) protein termed TSR1. TSR1IP shows homology to U1 70 kDa proteins, and contains an SR rich domain as well as an acidic/arginine domain homologous to the U1 70 kDa poly(A) polymerase inhibiting domain. This protein is localized in the nucleoplasm and excluded from the nucleolus in trypanosomal bloodstream and procyclic forms. Based on structural modelling predictions and on the identification of a RNA recognition motif (RRM), it was possible to demonstrate by the yeast three-hybrid system that TSR1IP interacts with the 5' splice region of the SL RNA. All the above characteristics suggest that TSR1IP could be involved in trans-splicing. PMID- 10743616 TI - Molecular cloning, expression analysis and iron metal cofactor characterisation of a superoxide dismutase from Toxoplasma gondii. AB - A genomic region of 12 kb encompassing the gene encoding the superoxide dismutase (SOD) of Toxoplasma gondii has been cloned. The gene contains four exons of 121, 42, 381 and 59 bp which are separated by three introns of 321, 202, and 577 bp, respectively. The open reading frame can be translated into a protein of 201 amino acids with a molecular mass of 22.6 kDa. Alignment indicated that it is a FeSOD, a type only found in bacteria, protozoa and chloroplast of higher plants. Recombinant SOD was expressed in a Escherichia coli double mutant lacking both MnFeSOD and FeSODs. The presence of iron as metal cofactor was confirmed by measurements of iron by absorption mass spectrometry and electron paramagnetic resonance studies. Semi-quantitative reverse transcribed polymerase chain reaction experiments showed a similar amount of SOD transcripts in two developmental stages of T. gondii. Antibodies raised against the purified recombinant protein detected SOD protein in both bradyzoite and tachyzoite forms suggesting this SOD might be essential for the intracellular growth of both developmental stages. Southern blot analysis indicated that SOD occured as a single copy gene in T. gondii genome. PMID- 10743617 TI - Evidence for vesicle-mediated trafficking of parasite proteins to the host cell cytosol and erythrocyte surface membrane in Plasmodium falciparum infected erythrocytes. AB - Plasmodium falciparum malaria parasites actively remodel the host cell cytosol and plasma membrane during the erythrocytic cycle. The focus of this investigation was to characterize intra-parasitic and -erythrocytic secretory pathways. Electron-dense vesicles, similar in appearance to mammalian secretory vesicles were detected in proximity to smooth tubo-vesicular elements at the periphery of the parasite cytoplasm in mature parasites by transmission electron microscopy. Vesicles (60-100 nm diameter), which appeared to be coated, were visualized on the erythrocytic side of the parasite vacuolar membrane and in the erythrocyte cytosol. The vesicles seemed to bind to and fuse with the erythrocyte membrane, giving rise to cup-shaped electron-dense structures, which might be intermediates in knob structure formation. Treatment of mature parasites with aluminum tetrafluoride, an activator of GTP-binding proteins, resulted in the accumulation of the vesicles with an electron-dense limiting membrane in the erythrocyte cytosol into multiple vesicle strings. These vesicle complexes were often associated with and closely abutted the erythrocyte membrane, but were apparently prevented from fusing by the aluminum fluoride treatment. The parasite proteins PfEMP1 and PfEMP3 were found by immunoelectron microscopy to be associated with these vesicles, suggesting they are responsible for transporting these proteins to the erythrocyte membrane. PMID- 10743618 TI - Phosphatidylcholine formation is the predominant lipid biosynthetic event in the hemoparasite Babesia bovis. AB - This work examines the lipid composition and metabolism of bovine red blood cells infected by apicomplexan Babesia parasites, organisms closely related to Plasmodium sp. We found that erythrocytes infected with Babesia bovis (i-RBC) accumulate lipids and show striking increases in phosphatidylcholine, phosphatidic acid, diacylglycerol and cholesteryl esters as compared to uninfected erythrocytes cultured under the same conditions (n-RBC). A similar pattern was observed in cultures of erythrocytes infected with Babesia bigemina. The lipid profile of purified B. bovis merozoites showed that phosphatidylcholine is the most abundant phospholipid in this parasite (31.8% +/- 2.8 of total phospholipid), markedly differing from bovine n-RBC, in which it is only a minor component (4.8% +/- 0.6). B. bovis cultures incorporate radiolabeled choline into complex lipids, especially phosphatidylcholine, with minor amounts recovered in sphingomyelin and lysophosphatidylcholine. When [14C] stearate was used as precursor, the labeling pattern again gave the highest incorporation into phosphatidylcholine, with lesser incorporation in sphingomyelin, phosphatidylinositol, phosphatidylethanolamine and phosphatidic acid. Diacylglycerol and small amounts of cholesteryl esters were the only labeled neutral lipids found. B. bovis also incorporates [3H] myo-inositol into phosphatidylinositol. Parallel incubations with n-RBC as a control yielded no incorporation into either polar or neutral lipids with any precursor. These results indicate that the lipid changes observed in i-RBC can be explained on the basis of the lipid biosynthetic activities of the babesial parasite. Gas chromatography-mass spectrometry (GC-MS) analysis of fatty acid methyl esters from phospholipids of i-RBC and n-RBC showed the same qualitative composition in both. However, i-RBC had higher ratios of saturated to unsaturated fatty acids and B. bovis cultures did not desaturate [14C] stearate. Cholesterol was the only sterol detected by GC-MS. Phospholipase A2 treatment of i-RBC and n-RBC revealed no enhanced hemolytic effects in i-RBC, suggesting that the erythrocyte membrane phospholipid composition is essentially unaltered by the parasite. Labeling of i RBC or n-RBC with [125I] Bolton-Hunter resulted in an enhanced phosphatidylserine labeling in i-RBC. This study provides the first data on B. bovis lipid constitution and biosynthesis. They show that phosphatidylcholine formation is the main biosynthetic process in these cells. The striking differences in the contents of phosphatidylcholine between host erythrocytes and the parasite suggests that it may be a useful target for both chemotherapy and immunoprophylaxis against bovine babesiosis. PMID- 10743619 TI - Mobilization of intracellular calcium upon attachment of Toxoplasma gondii tachyzoites to human fibroblasts is required for invasion. PMID- 10743620 TI - Non-autonomous transposable elements in the genome of the parasitic nematode Haemonchus contortus. PMID- 10743621 TI - Genomic organisation and chromatin structure of Plasmodium falciparum chromosome ends. PMID- 10743622 TI - Plasmodium falciparum clones resistant to (S)-9-(3-hydroxy-2-phosphonylmethoxy propyl)adenine carry amino acid substitutions in DNA polymerase delta. PMID- 10743623 TI - Further evidence for a link between melarsoprol resistance and P2 transporter function in African trypanosomes. PMID- 10743624 TI - A novel developmentally regulated galectin of Onchocerca volvulus. PMID- 10743625 TI - [Tissue engineering in orthopedics--a gravid future concept]. PMID- 10743626 TI - [TGF-beta-1 gene transfer in joint cartilage cells. Stimulating effect in extracellular matrix synthesis]. AB - TGF beta-1 has been shown to upregulate matrix synthesis in articular chondrocytes. TGF beta-gene transfer to chondrocytes has the potential to increase the local production of this key component within regenerating cartilage after trauma and could support the repair process in articular cartilage lesions. Primary rabbit articular chondrocytes were cultured and retrovirally transfected with the experimental TGF beta-1 and the lacZ marker gene for control purposes. After radioactive labeling of new synthesized matrix proteins results were compared with normal primary chondrocytes. After TGF beta-1 gene transfer the endogenous growth factor concentration was doubled compared to normal chondrocytes and decreased in the lacZ control group. The proteoglycan synthesis in TGF beta-1 transfected chondrocytes showed a 96% increase compared to the basal production of normal chondrocytes. The LacZ transfected group revealed the opposite effect by a 44% decrease. The collagen synthesis of TGF beta-1 transfected chondrocytes was 304% compared to normal chondrocytes, predominantly type II collagen. The lacZ group collagen production was reduced by 35%. We conclude that TGF beta-1 gene transfer overcomes the decreasing effect observed by transfection with the LacZ marker gene and increases matrix synthesis in articular chondrocytes. Genetically altered chondrocytes might improve the repair of cartilage lesions by stimulating matrix synthesis and supporting the expression of the hyaline phenotype. PMID- 10743627 TI - [Effect of ion channel modulators on the membrane potential of human chondrocytes]. AB - As all living cell systems, human chondrocytes are provided with a membrane potential. For its origin the existence of ion channels at the cell membrane is an essential prerequisite. In non-human chondrocytes, different ion channels could already be identified. A connection between the potassium channel activity and the proliferation has been detected in different human cell systems. Whereas, the proof of a connection between ion channel activity of human chondrocytes and the proliferation has yet to be established. In this study the concentration dependent influence of the ion channel modulators tetraethylammonium (TEA), 4 amino-pyridin (4-AP), 4',4'diisothiocyanatostilbene-2,2'disulfonic acid (DIDS), 4 acetamido-4'isothiocyano-2,2'disulfonic acid stilbene (SITS) and verapamil on the membrane potential and the proliferation of human chondrocytes was investigated using flow cytometry. The results show an effect of the used ion channel modulators causing a change of the membrane potential of human chondrocytes. The decrease of the membrane potential by 18% was measured with 0.25 mmol/l verapamil meaning the maximal measurable effect compared with a control group. When measuring DNA distribution, it became apparent that the human chondrocytes are diploid cells with a very low proliferation tendency. These results allow the conclusion of an influence of ion channel modulators on chondrocyte proliferation. PMID- 10743628 TI - [In vitro cell behavior of human osteoblasts after physiological dynamic stretching]. AB - The cell activity of human bone derived cell cultures was studied after mechanical stimulation by cyclic strain at a magnitude occurring in physiologically loaded bone tissue. Monolayers of subconfluently grown human bone derived cells were stretched in rectangular silicone dishes with cyclic uniaxial movement along their longitudinal axes. Strain was applied over two days for 30 min per day with a frequency of 1 Hz and a strain magnitude of 1000 mustrain. Cyclic stretching of the cells resulted in an increased proliferation (10-48%) and carboxyterminal collagen type I propeptide release (7-49%) of human cancellous bone derived osteoblasts while alkaline phosphatase activity and osteocalcin release were significantly reduced by 9-25% and 5-32% respectively. These results demonstrate that cyclic strain at physiologic magnitude leads to an increase of osteoblast activities related to matrix production while those activities which are characteristic for the differentiated osteoblast and relevant for matrix mineralization are decreased. PMID- 10743629 TI - [Redifferentiation of dedifferentiated joint cartilage cells in alginate culture. Effect of intermittent hydrostatic pressure and low oxygen partial pressure]. AB - One of the goals in the field of tissue engineering is the development of artificial cartilage for the treatment of cartilage defects. Therefore autologous chondrocytes are seeded on different artificial matrices to test their possible use as implants (resorption, antigenicity, toxicity and their integration in the tissue). One of the main problems in these experiments is that usually the amount of available chondrocytes is too low for treating large-scale defects or for comparing different matrices. An in-vitro-multiplication of the cells is needed which causes the chondrocytes to dedifferentiate and become fibroblast-like. Therefore parameters which induce a redifferentiation are of great interest. The objective of this study was to determine the influence of intermittent hydrostatic pressure and low oxygen partial pressure on the redifferentiation of dedifferentiated bovine articular chondrocytes in monolayer and three-dimensional alginate bead culture. The redifferentiation process was monitored by immunocytochemical detection of newly synthesized collagen type II. The viability of the cells was determined by the trypanblue exclusion test. The chondrocytes were dedifferentiated by a two week culture in plastic flasks with an oxygen level of 20%. After this they were subcultured in monolayer or three-dimensional alginate culture and subjected to three different stimuli for three weeks in order to redifferentiate: 1.) 20% O2 (= 20.26 kPa PO2) + 5% CO2 + 75% N2; 2.) 5% O2 (= 5.07 kPa PO2) + 5% CO2 + 90% N2; 3.) 5% O2 (= 5.07 kPa PO2) + 5% CO2 + 90% N2 + 8 h/d of intermittent hydrostatic pressure (frequency: 3 bar absolute for 30 min and 1 bar absolute for 2 min). In the monolayer there was no detectable collagen type II found by immunocytochemistry under either of the three culture conditions. Therefore a redifferentiation of dedifferentiated chondrocytes was not possible in monolayer cultures with the tested parameters. In the three dimensional alginate culture there was no immunocytochemical staining of collagen type II found in the beads cultured with 20% oxygen. With 5% oxygen we found a strong collagen type II-production by chondrocytes throughout the whole bead. The intermittent hydrostatic pressure combined with 5% oxygen lead to a decreased collagen type II-production compared to cells subjected to 5% oxygen only. Also chondrocytes closer to the edge of these beads were more often immunopositive and seemed to produce more immunoreactive collagen type II. The viability of the chondrocytes in the alginate culture was close to 90% after three weeks. Our experiments showed that oxygen partial pressure is an important parameter in the cultivation of articular chondrocytes. Reduced partial oxygen pressure promoted or induced the redifferentiation of dedifferentiated chondrocytes in alginate culture. PMID- 10743630 TI - [Adult human chondrocytes in alginate culture. Preservation of the phenotype for further use in transplantation models]. AB - Successful and cartilage-specific cultivation of chondrocytes requires a stable phenotype during the in vitro culture period. This is based on a differentiated extracellular matrix synthesis. The alginate system as a three-dimensional support is a useful system to culture chondrocytes and to analyze the biochemical processes in this system. Talar cartilage from the talocrural joints of 40 different donors were obtained through the Regional Organ Bank of Illinois within 24 hours of death. In 65% of the tissues the cartilage was classified as being undamaged. In these studies we were interested in the results of short-term culture over 14 days. Cell proliferation, total collagen content and total proteoglycan content were measured in the different matrix compartments and were visualized by histology and immunohistochemistry. Already after 7 days in culture the adult human chondroctes looked intact and formed a stable and cell-associated cartilage-specific extracellular matrix in the presence of 10% calf serum. This could be also demonstrated in the presence of IGF-I. With regards to the collagen content IGF-I at a concentration of 50 ng/ml seemed to induce an equal effect to 10% serum; with regards to the proteoglycan content IGF-I at a concentration of 20 ng/ml was equivalent. These encouraging preliminary results may lead to a new approach in tissue engineering for chondrocyte transplantation in combination with their extracellular matrix. PMID- 10743631 TI - [Stimulation of primary osteoblast cultures with rh-TGF-beta, rh-bFGF, rh-BMP 2 and rx-BMP 4 in an in vitro model]. AB - Bone metabolism is influenced by systemic and local acting hormons. Bone morphogenetic proteins (BMPs) as representatives of the latter substances are known to have the ability for ectopic bone formation. Within this study, we investigated the influence of different growth factors on the proliferation- and differentiation rate of osteoblast-like cells. For that purpose, human osteoblast like cells (HPOC) were incubated in the presence of either recombinant BMP-4 of the genome of xenopus laevis (rxBMP-4), recombinant human BMP 2 (rhBMP-2), transforming growth factor-beta (TGF-beta) or basic fibroblast growth factor (rh bFGF) in two different concentrations each (10 ng/ml and 50 ng/ml). Cell proliferation was measured within a MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5 Diphenyltetrazolium Bromid] assay, the amount of cell differentiation by the activity of alcaline phosphatase. Rx-BMP-4 induced a differentiation of HPOC to almost the same extent as rhBMP-2, whereas the addition of rh-bFGF, applied at the same concentration, failed to have any influence on cell differentiation. However, rh-bFGF provoked an increase in cell proliferation when compared with unstimulated HPOC, while rhBMP-2 and rxBMP-4 showed no effect on proliferation. TGF-beta influenced bone proliferation as well as differentiation significantly. The equipotent effect of recombinant human BMP-2 and recombinant BMP-4 obtained from Xenopus laevis with regard to differentiation and proliferation of human primary osteoblast-like cells originates either in the fact that target cells have receptors for BMP 2 as well as BMP 4, or that both BMP's link to the same receptor with almost the same affinity. PMID- 10743632 TI - [Chondrocyte transplantation in PGLA/polydioxanone fleece]. AB - The transplantation of chondrogenic cells in a supportive carrier structure proved to be a promising alternative for the treatment of cartilage defects. In the study presented we focused on the transplantation of allogeneic chondrocytes in a biodegradable polymer scaffold (PGLA/Polydioxanon) in articular cartilage defects in a rabbit defect model. Isolated allogeneic chondrocytes embedded in a PGLA polymer scaffold were transplanted into osteochondrogenic defects of the patellar groove and compared with empty defects and transplants of polymer scaffolds without cells. The histological and histochemical analysis was performed after 4 and 12 weeks. The transplant integration and the architecture of the newly formed cartilage were evaluated with a semiquantitative score. After 4 weeks the development of a hyaline-like cartilage tissue of the cell-polymer transplants was observed, after 12 weeks the defects were nearly completely filled with hyaline-like cartilage. The biodegradation of the polymer construct did not affect the histological structure of the transplant area. Defects of the groups with empty defect and polymer transplants without cells revealed no or insufficient healing indices. The study demonstrated that biodegradable polymers served as suitable carriers for the chondrocyte transplantation, which is due to the in-vitro establishment of a semi-solid cartilage transplant and the resulting effective transplant fixation into the defect. In-vivo the polymer cell transplants seem to provide a supportive microenvironment for the development of hyaline cartilage. The controlled release of morphogenic factors or bioactive molecules and the use of pluripotent mesenchymal progenitor cells opens new perspectives for the optimization of cartilage repair procedures. PMID- 10743634 TI - [In vitro culture of human autologous osteoblast cells on natural bone mineral]. AB - Different methods are available for the treatment of osseous defects. In recent years the use of autologous bone was established as the golden standard. However, significant disadvantages are limited availability of the bone graft and its harvest implies additional morbidity for the patient. Alternatives to the use of autologous bone, as allogeneic bone from bone banks or biomaterials like hydroxyapatite are therefore of special interest. However, the currently available methods have severe disadvantages; allogenic bone carries a high risk of transmitting infectious diseases, most biomaterials show an unsatisfying osseous integration as well as prolonged healing with disability for the patient. Therefore, the aim has to be the development of a biomaterial that is as close as possible to human bone. In this in vitro study the natural bone mineral Bio Oss/Orthos was used as a matrix for human osteoblast-like cells isolated from bone marrow of healthy patients. Even after three months the cell showed typical osteblast-like behaviour. Histologic evaluation demonstrated the ability of Bio Oss/Orthos to guide cell growth within its matrix structure and therefore mimics in vivo situation of the healthy bone. The results show that culturing human osteoblast-like cells under standardised conditions is possible and that the combination of human osteoblast-like cell with an appropriate matrix may have the potential for a new treatment option of osseous defects. PMID- 10743633 TI - [Degree of differentiation of chondrocytes and their pretreatment with platelet derived-growth factor. Regulating induction of cartilage formation in resorbable tissue carriers in vivo]. AB - Current methods for articular cartilage repair are unpredictable with respect to clinical success. In the present study, we investigated the ability of cells from articular cartilage, perichondrium, and costochondral resting zone to form new cartilage when loaded onto biodegradable scaffolds and implanted into calf muscle pouches of nu/nu mice. Prior in vitro studies showed that platelet derived growth factor-BB (PDGF-BB), but not transforming growth factor beta-1 (TGF-beta 1), basic fibroblast growth factor, or bone morphogenetic protein-2 promoted proliferation and extracellular matrix sulfation of resting zone chondrocytes without causing the cells to exhibit a hypertrophic chondrocyte phenotype. TGF beta 1 has also been shown to stimulate chondrogenesis by multipotent chondroprogenitor cells like those in the perichondrium. In addition, PDGF-BB has been shown to modulate chondrogensis by resting zone cells implanted in poly(D,L lactide-co-glycolide) (PLG) scaffolds. In the present study we examined whether the cartilage formation is dependent on state of chondrocyte maturation and whether the pretreatment of chondrocytes with growth factors has an influence on the cartilage formation. Scaffolds were manufactured from 80% PLG with a 75:25 lactide:glycolide ratio and 20% modified PLG with a 50:50 lactide:glycolide ratio (PLG-H scaffolds). For each experimental group, four nude mice received two identical implants, one in each calf muscle resulting in an N = 8 implants: PLG-H scaffolds alone; PLG-H scaffolds with cells derived from either the femoral articular cartilage, costochondral periochondrium, or costochondral resting zone cartilage of 125 g male Sprague-Dawley rats; PLG-H scaffolds with either articular chondrocytes or resting zone chondrocytes that were pretreated with 37.5 ng/ml rhPDGF-BB for 4 h or 24 h before implantation, or with perichondrial cells treated with PDGF-BB plus 0.22 ng/ml rhTGF beta-1 for 4 h and 24 h. At 4 or 8 weeks after implantation, samples were harvested and analyzed histomorphometrically for new cartilage formed, area of residual implant and area of fibrous connective tissue. Only resting zone cells showed the ability to form new cartilage at a heterotopic site in this study. There was no neocartilage found in nude mice with implants loaded with either articular chondrocytes or perichondrial cells. Pretreatment of resting zone chondrocytes for 4 h prior to implantation significantly increased the amount of newly formed cartilage after 8 weeks and suppressed chondrocyte hypertrophy. The amount of fibrous connective tissue around implants containing either articular chondrocytes or perichondrial cells decreased with time, whereas the amount of fibrous connective tissue around implants containing resting zone chondrocytes pretreated with PDGF-BB was increased. The results showed that resting zone cells can be successfully incorporated into biodegradable porous PLG scaffolds and can induce new cartilage formation in a nonweight-bearing site. Articular chondrocytes as well as perichondrial cells did not have the capacity for neochondrogenesis when implanted heterotopically in this model. PMID- 10743635 TI - [Diagnostic imaging after autologous chondrocyte transplantation. Correlation of magnetic resonance tomography, histological and arthroscopic findings]. AB - Autologous chondrocyte transplantation (ACT) is a newly therapy option for treatment of cartilage damage. Since 1996 we performed the ACT in 10 patients with 14 cartilage damages. Magnetic resonance imaging (MRI) is a non-invasive method for postoperative controlling the transplantation area. The MRI showed in the first 3-6 months postoperatively signal irregularities with partial gadolinium uptake at the transplantation site. Arthroscopically the transplantation site was spongy, when it was probed and at the histological examination there was a hyalinlike appearance of the cells. 1 year postoperatively it seemed that process of reparation was finished. There was no more Gadolinium-uptake at the transplantation site and the borders of the transplant were hardly visible. The histological examination revealed hyalinlike repair tissue with a more columnar appearance of the cells. The Lysholm Score improved from 78 to 92 points, 1 year postoperatively. PMID- 10743636 TI - [Autologous meniscus replacement with rib perichondrium. Experimental results]. AB - The purpose of our study was to examine the potential of autologous perichondrial tissue to form meniscal replacements. Eighteen mature sheep were used. In 12 animals a complete meniscal resection was performed; replacement was formed using strips of autologous perichondrial tissue explanted from the lower rib; six animals with a complete meniscal resection but without any replacement served as controls. In all animals restriction from weight-bearing was achieved by means of transsection and partial achillestendon resection. Six animals each, 4 of group T and 2 of group C, were sacrificed after 3, 6 and 12 months. Perichondrial grafts and the underlying articular cartilage were removed and investigated by gross macroscopic examination, by means of light and scanning electron microscopy, polarized light examination, and biomechanical tests evaluating the failure stress and tensile modulus. In all transplanted animals a new perichondrial meniscus developed. After 3 months the transplants resembled in size and thickness normal menisci, while in the control animals only small rims of spontaneously grown tissue were detectable. Microscopically, the perichondrial menisci exhibited similarity to normal collagen fiber orientation and cellular characteristics, but, in their central region, areas of calcification disturbed the regular tissue differentiation. In contrast, spontaneously grown tissue in control animals lacked normal fiber orientation and cellularity. Scanning electron-microscopy of perichondrial menisci revealed surface characteristics similar to normal sheep menisci without fissures and lacerations, while the control specimens exhibited such defects. The femoral and tibial cartilage being in contact with the new menisci showed normal surface characteristics apart from one animal with slight surface irregularities. Control animals showed superficial lesions after 3 months, and the extent increased from 6 to 12 months postoperatively. Exemplary microangiographies of the newly grown tissue exhibited a less intense vascularisation after three months when compared to normal menisci with an improving tendency after 6 and 12 months. Biomechanically, values of the failure stress as well as of the tensile modulus of perichondrial menisci were significantly lower than those of normal contralateral menisci. But, spontaneously regenerated tissue in meniscectomized animals exhibited even smaller values also with significant differences towards original menisci. There were no significant differences in values of newly grown perichondrial menisci and spontaneously grown tissue. PMID- 10743637 TI - [A new resorbable bone-cartilage replacement transplant. Results of an animal experiment study]. AB - Hyaline cartilage is thought to be unable to regenerate. All efforts so far- including autologous chondrocyte cell transplantation--to reconstruct cartilage defects in joints have not been totally convincing. However, mesenchymal cells are able to differentiate into chondrocytes under mechanical pressure conditions. In this study, an open porous resorbable two-layer "bioimplant" was constructed in which mechanical pressure was exerted onto mesenchymal cells when migrated into the open porous structure of the bioimplant. Differentiation of the cells into chondrocytes was thus induced. The bioimplants were implanted into the medial condyles of nine rabbits and left in place for eight or twelve weeks, respectively. In seven of these cases, cartilage formation was found, in contrary to the controls in which only connective tissue and bone had grown into the empty holes. The new bioimplants have proven their effectiveness in cartilage defect repair and might evolve in the future as a new alternative treatment of full thickness defects of joint surfaces. PMID- 10743638 TI - [Value of synovial analysis for prognosis of matrix synthesis of transplanted chondrocytes]. AB - Successful transplantation of autologous chondrocytes for repair of articular cartilage defects requires an undisturbed matrix-synthesis of the transplanted cells. This, in turn, is dependent on the composition of the synovial fluid (SF) of the respective joint. We addressed the question whether analysis of a patient's SF can predict the rate of matrix-synthesis of articular cartilage exposed to this SF in vitro. SF was obtained from 115 patients with disorders of the knee, including gonarthrosis (n = 44), meniscal tears (n = 10), rheumatoid arthritis (n = 53), and reactive arthritides (n = 8). In the SF, the following parameters were determined: Interleukin-1 beta, IL-6, IL-8, IL-1-RA, TNF alpha, Insulin-like growth-factor I (IGF-I), IGF-II, IGF-binding protein-2 (IGFBP-2), IGFBP-3 as well as total proteinase activity and total collagenase activity. To assess the effect of SF on the matrix synthesis of articular chondrocytes, bovine cartilage was incubated in the presence of SF, and the rate of proteoglycan synthesis subsequently determined. In some cases, a monoclonal antibody directed against IGF-I was added. SF from patients with OA or trauma, respectively, stimulated PG-synthesis of bovine cartilage more markedly than did SF from patients with rheumatic arthritides. On the average, 60 percent of the SF-induced increase of cartilage matrix synthesis could be titrated out by an anti-IGF-I-AB. The best predictor for the SF-effects on PG-synthesis of exposed cartilage was the proportion of free IGF-I (r = 0.573, p < 0.001, Spearman rank correlation) followed by the SF-concentrations of IGF-I (with a positive sign), IGFBP-3, IL-1 beta, and TNF alpha (all with a negative sign). According to our data, IGF-I is the most important anabolic factor in human SF with respect to cartilage PG synthesis. The proportion of free IGF-I seems to be of special importance in this regard. Low SF-levels of free IGF-I could be identified as a possible risk-factor for a sub-optimal protoeglycan synthesis of chondrocytes exposed to this synovial milieu. PMID- 10743639 TI - [Radiosynoviorthesis in inflammatory joint diseases]. AB - The use of beta-(beta) emitting radionuclides for radiosynoviorthesis (RSO) has been a well established therapy for inflammed joints for more than 40 years. Radionuclides are often applied to patients with rheumatoid arthritis as local therapy. The choice of the radionuclide is empirical. The evaluation of the therapeutic success is mainly based on the improvement of the joint motion. MRI and other radiological examinations have not been proved to be reliable for diagnosis of the therapeutic success. The aims of this manuscript are to familiarize physicians with the different radionuclides, to explain the therapeutic success which can be expected due to dosimetric data, and to present the three phase bone scintigraphy as a valuable instrument for the evaluation of the therapeutic success. PMID- 10743640 TI - [The upper ankle joint]. PMID- 10743641 TI - Anti-Toxoplasma activity of vegetal extracts used in West African traditional medicine. AB - Both Toxoplasma gondii and Plasmodium are Apicomplexan protozoa that share common metabolic pathways and potential drug targets. The objective of this study was to examine the anti-Toxoplasma activity of nine West African plants with known activity against P. falciparum. The extracts were obtained from parts of plant commonly used, by most traditional healers, in the form of infusion or as water decoction. The in vitro activity of plant extracts on T. gondii was assessed on MRC5 tissue cultures and was quantified by enzyme-linked immunoassay. Aqueous extracts from Vernonia colorata were found to be inhibitory for Toxoplasma growth at concentrations > 10 mg/L, with an IC50 of 16.3 mg/L. A ten-fold gain in activity was obtained when organic solvents such as dichloromethane, acetone or ethanol were used to extract V. colorata's active principles. These extracts were inhibitory at concentrations as low as 1 mg/L, with IC50 of 1.7, 2.6 and 2.9 mg/L for dichloromethane, acetone and ethanol extracts respectively. These results indicate a promising source of new anti-Toxoplasma drugs from V. colorata and African medicinal plants. PMID- 10743642 TI - Local hepatic immune response in rats during primary infection with Fasciola hepatica. AB - The distribution of lymphocyte subpopulations (TCD4+, TCD8+, TCD43+ and Ig+ cells), macrophages and eosinophils were analysed in the inflammatory infiltrates associated with hepatic lesions and in hepatic lymph nodes (HLN) from rats experimentally infected with F. hepatica and necropsied 1, 2, 3, 4, 6 and 8 week post infection (WPI). We also investigated the fixation of immunoglobulin isotypes on migrating flukes in the liver. As early as 1WPI, portal tract areas surrounding migratory tunnels were infiltrated with immune and inflammatory cells. The dominant cells were eosinophils and to lesser extent, macrophages and lymphocytes (TCD4+, TCD8+ and B). Most of the inflammatory and immune cells reached the posterior part of flukes, whereas in front of the parasites these cells were fewer in number. Except for eosinophils, no immune cells penetrated through granuloma consisting of hepatic necrotic cells. As early as 1WPI, IgM could be detected in the liver, and to a lesser extent IgA, IgG2a and IgG2b. At 2WPI, IgE and IgG1 began being detected. IgG2c was detectable at 3WPI. In HLN, we observed numerous microscopic follicles in the cortical zone with proliferation of germinal centres and medullary cords. The protective role of infiltrating cell populations and immunoglobulin isotypes and possible mechanisms of immune evasion by the parasite are discussed. PMID- 10743643 TI - Ultrastructure and function of mitochondria in gametocytic stage of Plasmodium falciparum. AB - Morphological properties of the mitochondrial organelles in the asexual and sexual gametocytic stages of Plasmodium falciparum have been analyzed and found to be markedly different. From in vitro cultures of both stages in human erythrocytes, it has been demonstrated that the asexual stages contained a defined double-membrane organelle having a few tubular-like cristae. The numbers of mitochondria in the gametocytes were found to be approximately 6 organelles per parasite, and they showed a greater density of the cristae than that of the asexual stage parasite. The organelles of the gametocytes were successfully purified by differential centrifugation following Percoll density gradient separation with the results of approximately 7% yields and approximately 5 folds. The gametocytic organelles contained much more activities of mitochondrial electron transporting enzymes (i.e., cytochrome c reductase, cytochrome c oxidase) than the asexual stage organelles. Mitochondrial function as measured by oxygen consumption were found to be different between these two stages organelles. Their rates of oxygen consumption were relatively low, as compared to those of human leukocyte and mouse liver mitochondria. In contrast to the coupled mammalian mitochondria, the gametocytic organelles were in the uncoupling state between oxidation and phosphorylation reactions during their respiration. However, they were sensitive to inhibitors of the electron transport system, e.g., antimycin A, cyanide. Our results suggest that the mitochondria of the gametocytic stages are metabolically active and still underdeveloped, although their inner membranes are extensively folded. The biochemical significance of the unique structure of the mitochondria in these developing stages in host erythrocytes remains to be elucidated. PMID- 10743644 TI - Schulzia chiribita n. sp. (Nematoda, Trichostrongylina, Molineoidea) parasite of Leptodactylus rhodonotus (Amphibian) from Peru. AB - A third species of the genus Schulzia Travassos, 1937 a parasite of Leptodactylus rhodonotus (Amphibian, Leptodactylidae) originating from Peru is described. By the pattern of the caudal bursa, the specimens are closely related to the two other species. They are distinguished from Schulzia uzu Lent & Santos, 1989, parasite from Atelopus oxyrhynchus in Venezuela, by the shape of the ovejector and from Schulzia travassosi Durette-Desset, Baker & Vaucher, 1985, parasite from Bufo crucifer in Brasil, Bufo granulosus and Leptodactylus bufonius in Paraguay, by the shape of the spicules. The presence of a new species in Peru points out the wide geographic distribution of the genus in the Neotropical region. PMID- 10743645 TI - From isolates to a synthetic laboratory population: maintenance of variability in the nematode Haemonchus contortus. AB - Haemonchus contortus isolates were collected from goats of five locations with different climatic characteristics in Guadeloupe archipelago. They were investigated for morphology, morphometrics and allozyme diversity after passage in immunosuppressed lambs using long acting corticoids. The basic aim of the work was to construct a synthetic strain in laboratory conditions which was representative of the isolates. The isolates were only slightly different although climatic conditions were very different. The resemblance of isolates might be due to the practice of goat exchanges between farms or to their insular origin. However the isolate from a smaller island (Les Saintes) was different (mostly on morphometrics) from all the others originating from Guadeloupe main Island. The first assemblage in laboratory resulting from the installation from a mixture of the five isolates was not very representative, whereas the next generation (synthetic strain) resembled all the isolates as shown from allozyme study. Female fecundity and length in established synthetic strain were lower than that recorded in isolates, indicating a decrease in fitness, possibly due to the stability of experimental environment. The representativity of the synthetic strain was good but the strain could still evaluate on further passages and should be evaluated on a large number of generations maintained in laboratory. PMID- 10743646 TI - Toxoplasma encephalitis: influence of the vehicle on the efficacy of different doses of 2',3'-dideoxyinosine in mice. AB - In this study we investigated the effect of the antiretroviral molecule 2',3' dideoxyinosine (Videx) against cerebral cysts in a murine model of toxoplasmic encephalitis caused by a wild cystic strain of Toxoplasma gondii. The role of the vehicle was also studied. Three doses were used: 50, 100 and 150 mg/kg of body weight/day. The doses of 50 and 150 mg/kg were prepared by dissolving pure 2',3' dideoxyinosine powder in Maalox suspension before gavaging the mice; the dose of 100 mg/kg was prepared by grinding tablets of Videx that were suspended in water. A decrease in the number of cysts and a morphological modification of them were noted from day 15 with the lowest dose. The most important decrease could be observed with the dose of 100 mg/kg/d. After 30 days of treatment with this dose, 65% of the cysts were destroyed compared to controls. For the doses of 50 and 150 mg/kg/d prepared with Maalox, 36% and 51% of the cysts were destroyed respectively. So ddI has an effect on the cerebral cysts of T. gondii even at a low dose. The galenic formulation influences its action since the doses prepared with Maalox were less efficient than those prepared from ground tablets. PMID- 10743647 TI - Trypanocidal activity of organotin chlorides on Trypanosoma brucei-infected mice. AB - The organotin compounds dibutyltin (DBTC) and diphenyltin dichlorides (DPTC) were tested for trypanocidal activity on a Trypanosoma brucei-infected mice model. At a dose of 10 mg DBTC and 15 mg DPTC/kg/day for five consecutive days, they cleared the parasites from the peripheral blood of the infected mice. Subinoculation of some healthy mice with the homogenates of liver, spleen, kidney, cerebrospinal fluid and blood from the mice considered cured, showed a few cases of relapse. The LD50 of DBTC and DPTC are 90 mg/kg and 75 mg/kg respectively. PMID- 10743649 TI - Occurrence of Thelazia lacrymalis (Nematoda, Spirurida, Thelaziidae) in native horses in Abruzzo region (central eastern Italy). AB - A survey on the prevalence of Thelazia spp. in Abruzzo region (Italy) in slaughtered native horses was conducted from August 29, 1997 to August 28, 1998. Both eyes from 128 eight-month to 11 year-old native animals were examined. 50 horses (39.06%) were found parasitized by Thelazia lacrymalis. 502 specimens (371 females, 88 males and 12 larvae) were collected. In the infected horses the numbers of T. lacrymalis ranged from 1 to 48, with a mean count burden of 3.92 per head (SD = 7.79). T. lacrymalis specimens were mainly in the excretory ducts of the Harderian gland, and also in the ducts of the lacrimal glands, free in the conjunctiva and behind the nictitancte. Gross examination showed a conjunctivitis, more frequently a follicular conjunctivitis, in the 58% of the infected horses. PMID- 10743648 TI - Presence of cholinesterases in Echinococcus granulosus protoscolices. AB - Cholinesterases were detected in protoscolices of Echinococcus granulosus spectrophotometrically and electrophoretically. To characterize these activities as acetylcholinesterases or pseudocholinesterases, BW284C51 and the organophosphate anthelmintic Neguvon were assayed as specific inhibitors of acetylcholinesterases, while Iso-OMPA was employed as specific inhibitor of pseudocholinesterases. We concluded that these cholinesterase (ChE) activities would be considered as possible targets in chemotherapy. PMID- 10743650 TI - A case from India of neurocysticercosis responsive to albendazole treatment. PMID- 10743651 TI - [A case of gastric myiasis due to larva of Eristalis tenax (Linne, 1758) (Insecta: Diptera)]. PMID- 10743652 TI - The Landau and Chabaud's phenomenon. PMID- 10743653 TI - The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. AB - The Chlamydomonas reinhardtii HSP70A promoter can be induced by both heat shock and light. Several characteristics of this promoter suggest its usefulness as a tool for improved transgene expression in this alga. (i) It may by itself confer high inducibility to a transgene. Fusion of the HSP70A promoter to reporter genes HSP70B or ARS yields high levels of transgene product that, as shown for ARS, may accumulate when repeated cycles of heat shock induction are applied. (ii) It activates other promoters. Using HSP70B as a reporter gene, we show that the HSP70A promoter serves as a transcriptional activator when placed upstream of the promoters RBCS2, beta 2 TUB and HSP70B. Activation of these promoters was observed both under basal conditions and upon light induction. In addition, transformation rates obtained for the eubacterial resistance gene aadA were significantly increased, when expression of this gene was controlled by the HSP70A-RBCS2 promoter fusion as compared to the RBCS2 promoter alone. PMID- 10743654 TI - Identification and analysis of proteins that interact with the Avena fatua homologue of the maize transcription factor VIVIPAROUS 1. AB - The Avena fatua (wild oat) homologue of VIVIPAROUS 1 (AfVP1) has been implicated in controlling the maintenance of embryo dormancy in mature imbibed seeds, but the detailed mechanisms by which this transcription factor family activates embryo maturation pathways and simultaneously represses germination are not known. A two-hybrid screen in yeast identified three proteins that interacted specifically with AfVP1 (AfVP1 interacting proteins; AfVIPs). AfVIPs 2 and 3 interacted with the C-terminus of AfVP1, which contains the B2 + B3 domains, previously shown to bind DNA, whereas AfVIP1 interacted with the isolated B3 domain. Using purified proteins in in vitro experiments, all three AfVIPs were shown also to interact with the Arabidopsis homologue ABSCISIC ACID INSENSITIVE 3 (ABI3). The three AfVIPs were expressed in both dormant and non-dormant embryos, but the abundance of AfVIP1 and 3 transcripts was greater in germinated than dormant seeds, whereas transcripts of AfVIP2 (and AfVP1) were more highly expressed in dormant embryos. The AfVIP3 protein has homology to a human cell crisis gene with a predicted role in the cell cycle; AfVIP2 contains a ring-type zinc finger motif. These homologies, together with analysis of expression studies, suggest that these proteins may play specific roles in AfVP1-mediated regulation of the dormancy to germination transition in A. fatua seeds. PMID- 10743655 TI - Interactions of the developmental regulator ABI3 with proteins identified from developing Arabidopsis seeds. AB - The ABI3 locus is a major regulator of embryo development in Arabidopsis and is essential for the simultaneous activation of the maturation pathway, as well as repression of germination and seedling development. We used a two-hybrid screen in yeast in order to identify proteins that interact with ABI3. Four ABI3 interacting proteins (AIPs) were identified which showed specific in vivo and in vitro interactions with the C-terminal region of ABI3 that contains the B2 and B3 domains, previously shown to have DNA binding activity. The expression characteristics of the genes encoding the AIPs have also been analysed in wild type and abi3, lec1 and fus3 embryo mutants. This analysis demonstrated differential expression of these genes during normal embryo development and in the mutant lines. All the AIPs show homology to existing transcription factors and therefore they may function with ABI3 within the network of transcriptional regulators that control embryo development in Arabidopsis. PMID- 10743656 TI - Movement of plant viruses is delayed in a beta-1,3-glucanase-deficient mutant showing a reduced plasmodesmatal size exclusion limit and enhanced callose deposition. AB - Susceptibility to virus infection is decreased in a class I beta-1,3-glucanase (GLU I)-deficient mutant (TAG4.4) of tobacco generated by antisense transformation. TAG4.4 exhibited delayed intercellular trafficking via plasmodesmata of a tobamovirus (tobacco mosaic virus), of a potexvirus (recombinant potato virus X expressing GFP), and of the movement protein (MP) 3a of a cucumovirus (cucumber mosaic virus). Monitoring the cell-to-cell movement of dextrans and peptides by a novel biolistic method revealed that the plasmodesmatal size exclusion limit (SEL) of TAG4.4 was also reduced from 1.0 to 0.85 nm. Therefore, GLU I-deficiency has a broad effect on plasmodesmatal movement, which is not limited to a particular virus type. Deposition of callose, a substrate for beta-1,3-glucanases, was increased in TAG4.4 in response to 32 degrees C treatment, treatment with the fungal elicitor xylanase, and wounding, suggesting that GLU I has an important function in regulating callose metabolism. Callose turnover is thought to regulate plasmodesmatal SEL. We propose that GLU I induction in response to infection may help promote MP-driven virus spread by degrading callose. PMID- 10743657 TI - Differential regulation of three functional ammonium transporter genes by nitrogen in root hairs and by light in leaves of tomato. AB - To elucidate the role of NH4+ transporters in N nutrition of tomato, two new NH4+ transporter genes were isolated from cDNA libraries of root hairs or leaves of tomato. While LeAMT1;2 is closely related to LeAMT1;1 (75.6% amino acid identity), LeAMT1;3 is more distantly related (62.8% identity) and possesses two short upstream open reading frames in the 5' end of the mRNA and a particularly short N-terminus of the protein as unique features. When expressed in yeast mutants defective in NH4+ uptake, all three genes complemented NH4+ uptake. In roots of hydroponically grown plants, transcript levels of LeAMT1;2 increased after NH4+ or NO3- supply, while LeAMT1;1 was induced by N deficiency coinciding with low glutamine concentrations, and LeAMT1;3 was not detected. In aeroponic culture, expression of LeAMT1;1 and LeAMT1;2 was higher in root hairs than in the remaining root fraction. Growth of plants at elevated CO2 slightly decreased expression of LeAMT1;2 and LeAMT1;3 in leaves, but strongly repressed transcript levels of chloroplast glutamine synthetase and photorespiratory serine hydroxymethyl-transferase. Expression of LeAMT1;2 and LeAMT1;3 showed a reciprocal diurnal regulation with highest transcript levels of LeAMT1;3 in darkness and highest levels of LeAMT1;2 after onset of light. These results indicate that in tomato at least two high-affinity NH4+ transporters, LeAMT1;1 and LeAMT1;2, are differentially regulated by N and contribute to root hair mediated NH4+ acquisition from the rhizosphere. In leaves, the reciprocally expressed transporters LeAMT1;2 and LeAMT1;3 are supposed to play different roles in N metabolism, NH4+ uptake and/or NH3 retrieval during photorespiration. PMID- 10743658 TI - RPP13 is a simple locus in Arabidopsis thaliana for alleles that specify downy mildew resistance to different avirulence determinants in Peronospora parasitica. AB - Disease resistance (R) genes are found in plants as either simple (single allelic series) loci, or more frequently as complex loci of tandemly repeated genes. These different loci are likely to be under similar evolutionary forces from pathogens, but the contrast between them suggests important differences in mechanisms associated with DNA structure and recombination that generate and maintain R gene diversity. The RPP13 locus in Arabidopsis represents an important paradigm for studying the evolution of an R gene at a simple locus. The RPP13 allele from the accession Nd-1, designated RPP13-Nd, confers resistance to five different isolates of the biotrophic oomycete, Peronospora parasitica (causal agent of downy mildew), and encodes an NBS-LRR type R protein with a putative amino-terminal leucine zipper. The RPP13-Rld allele, cloned from the accession Rld-2, encodes a different specificity. Comparison of three RPP13 alleles revealed a high rate of amino acid divergence within the LRR domain, less than 80% identity overall, compared to the remainder of the protein (> 95% identity). We also found evidence for positive selection in the LRR domain for amino acid diversification outside the core conserved beta-strand/beta-turn motif, suggesting that more of the LRR structure is available for interaction with target molecules than has previously been reported for other R gene products. Furthermore, an amino acid sequence (LLRVLDL) identical in an LRR among RPP13 alleles is conserved in other LZ NBS-LRR type R proteins, suggesting functional significance. PMID- 10743659 TI - Molecular cloning, functional expression and characterisation of RCC reductase involved in chlorophyll catabolism. AB - Red chlorophyll catabolite (RCC) reductase (RCCR) and pheophorbide (Pheide) a oxygenase (PaO) catalyse the key reaction of chlorophyll catabolism, porphyrin macrocycle cleavage of Pheide a to a primary fluorescent catabolite (pFCC). RCCR was purified from barley and a partial gene sequence was cloned (pHvRCCR). The gene was expressed at all stages of leaf development and in roots. By comparison with different databases, genomic sequences and expressed sequence tags similar to RCCR were found in phylogenetically diverse species, and activity of RCCR was demonstrated in two of them, Arabidopsis thaliana and Marchantia polymorpha. The gene of A. thaliana (AtRCCR) was employed for molecular cloning, heterologous expression and the production of polyclonal antibodies. With recombinant RCCR, the major product of RCC reduction was pFCC-1, but small quantities of its C1 epimer, pFCC-2, also accumulated. The reaction required reduced ferredoxin and was sensitive to oxygen. AtRCCR encoded a 35 kDa protein which was used for chloroplast import experiments. Upon transport, it was processed to a mature form of 31 kDa. The significance of cloning of RCCR is discussed in respect to the evolution of chlorophyll catabolism and to the cloning of PaO. PMID- 10743660 TI - Allene oxide synthases of barley (Hordeum vulgare cv. Salome): tissue specific regulation in seedling development. AB - Allene oxide synthase (AOS) is the first enzyme in the lipoxygenase (LOX) pathway which leads to formation of jasmonic acid (JA). Two full-length cDNAs of AOS designated as AOS1 and AOS2, respectively, were isolated from barley (H. vulgare cv. Salome) leaves, which represent the first AOS clones from a monocotyledonous species. For AOS1, the open reading frame encompasses 1461 bp encoding a polypeptide of 487 amino acids with calculated molecular mass of 53.4 kDa and an isoelectric point of 9.3, whereas the corresponding data of AOS2 are 1443 bp, 480 amino acids, 52.7 kDa and 7.9. Southern blot analysis revealed at least two genes. Despite the lack of a putative chloroplast signal peptide in both sequences, the protein co-purified with chloroplasts and was localized within chloroplasts by immunocytochemical analysis. The barley AOSs, expressed in bacteria as active enzymes, catalyze the dehydration of LOX-derived 9- as well as 13-hydroperoxides of polyenoic fatty acids to the unstable allene oxides. In leaves, AOS mRNA accumulated upon treatment with jasmonates, octadecanoids and metabolizable carbohydrates, but not upon floating on abscisic acid, NaCl, Na salicylate or infection with powdery mildew. In developing seedlings, AOS mRNA strongly accumulated in the scutellar nodule, but less in the leaf base. Both tissues exhibited elevated JA levels. In situ hybridizations revealed the preferential occurrence of AOS mRNA in parenchymatic cells surrounding the vascular bundles of the scutellar nodule and in the young convoluted leaves as well as within the first internode. The properties of both barley AOSs, their up regulation of their mRNAs and their tissue specific expression suggest a role during seedling development and jasmonate biosynthesis. PMID- 10743661 TI - Axillary meristem development in Arabidopsis thaliana. AB - Axillary shoot apical meristems initiate post-embryonically in the axils of leaves. Their developmental fate is a main determinant of the final plant body plan. In Arabidopsis, usually a single axillary meristem initiates in the leaf axil even though there is developmental potential for formation of multiple branches. While the wild-type plants rarely form multiple branches in the leaf axil, tfl1-2 plants regularly develop two or more branches in the axils of the rosette leaves. Axillary meristem formation in Arabidopsis occurs in two waves: an acropetal wave forms during plant vegetative development, and a basipetal wave forms during plant reproductive development. We report here the morphological and anatomical changes, and the STM expression pattern associated with the formation of axillary and accessory meristems during Arabidopsis vegetative development. PMID- 10743662 TI - Hyperpolarisation-activated calcium currents found only in cells from the elongation zone of Arabidopsis thaliana roots. AB - Calcium currents across the plasma membrane of plant cells allow transduction of environmental signals as well as nutritive calcium uptake. Using transgenic Arabidopsis plants with cell-specific expression of green fluorescent protein (GFP), we analyzed whole cell calcium currents in epidermal cells of the rapidly growing root apex, mature epidermal cells, cortical and epidermal cells from the elongation zone, and mature pericycle cells. In cells only from the rapidly growing root apex, a hyperpolarization-activated calcium current was identified. This current was irreversibly inhibited by 10 microM Al3+, as well as being inhibited by 1 mM Co2+ and 100 microM verapamil. In no cells could a depolarisation-activated current be attributed to calcium influx. In the growing root apex, the hyperpolarization-activated calcium current may function to allow constitutive uptake of calcium for rapid cell division and elongation. PMID- 10743663 TI - c-MYB oncogene-like genes encoding three MYB repeats occur in all major plant lineages. AB - Since the identification of the first plant MYB-like protein, the Zea mays factor C1, the number of MYB-related genes described has greatly increased. All of the more than 150 plant MYB-like proteins known so far contain either two or only one sequence-related helix-turn-helix motif in their DNA-binding domain. Animal c-MYB genes contain three such helix-turn-helix motif-encoding repeats (R1R2R3 class genes). It has therefore been concluded that R2R3-MYB genes are the plant equivalents of c-MYB and that there are significant differences in the basic structure of MYB genes of plants and animals. Here, we describe expressed R1R2R3 MYB genes from Physcomitrella patients++ and Arabidopsis thaliana, designated PpMYB3R-1 and AtMYB3R-1. The amino acid sequences of their DNA-binding domains show high similarity to those of animal MYB factors, and less similarity to R2R3 MYB proteins from plants. In addition, R1R2R3-MYB genes were identified in different plant evolutionary lineages including mosses, ferns and monocots. Our data show that a DNA-binding domain consisting of three MYB repeats existed before the divergence of the animal and plant lineages. R1R2R3-MYB genes may have a conserved function in eukaryotes, and R2R3-MYB genes may predominantly regulate plant-specific processes which evolved during plant speciation. PMID- 10743664 TI - Ultrasound evaluation of TIPS placement. AB - Sonography is a valuable tool for evaluating shunts used to treat variceal bleeding due to portal hypertension. This article defines the protocol for ultrasound imaging of the transjugular intrahepatic portosystemic shunt (TIPS) and abnormal findings associated with TIPS placement. It also discusses common pitfalls in TIPS imaging, including artifacts, echoes and incomplete visualization. PMID- 10743665 TI - Autonomy and satisfaction among mammographers. AB - This article reports results of a study assessing the relationship between decision autonomy and task satisfaction in Texas mammographers. As hypothesized, the results indicate a positive correlation between autonomy and satisfaction. The authors also found that no independent variable (age, years of experience, employment status, position or type of imaging facility) had predictive value for mammographers' autonomy and task satisfaction. PMID- 10743666 TI - Image-guided percutaneous needle biopsy: an overview. AB - Image-guided percutaneous needle biopsy (IGPNB) is a valuable tool for diagnosis of nonpalpable or deep-seated lesions that cannot be characterized definitively with diagnostic imaging alone. The procedure entails inserting a needle through the skin into an area of suspected pathology under the guidance of various imaging techniques to retrieve a sample of tissue or fluid for analysis. This article discusses the historical development, indications, contraindications, patient preparation, equipment and possible complications of IGPNB, with particular attention to biopsies of the thorax, breast, abdominal organs, head and neck and the musculoskeletal system. PMID- 10743667 TI - Survey research techniques. AB - This article is an introduction to the basics of survey research. It discusses the strengths and weaknesses of different types of surveys, explains basic statistical concepts and offers guidance in preparing a research article for publication. PMID- 10743668 TI - Looking inside an x-ray tube. PMID- 10743669 TI - DXA's role in diagnosing osteoporosis. PMID- 10743670 TI - Adult learning: classroom to workplace. PMID- 10743671 TI - Y2K hysteria and the ARRT. PMID- 10743672 TI - More respect, more money: specialize! PMID- 10743673 TI - Reader corrects MR facts. PMID- 10743674 TI - Spotting the problem. PMID- 10743675 TI - Toshiba system muffles MR noise. PMID- 10743676 TI - Effect of simvastatin therapy on blood and tissue ATP levels and erythrocyte membrane lipid composition. AB - 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors decrease mevalonate and subsequently cholesterol synthesis competitively. Mevalonate is also the precursor of ubiquinone. Ubiquinone is an important component of electron transport chain. We therefore investigated the effect of simvastatin on rat blood and tissue ATP concentrations and the lipid composition of red blood cell membranes after 4 weeks of therapy. Significant reductions in rat plasma cholesterol, triglyceride, and blood ATP concentrations were detected. Tissue ATP levels were not affected. Membrane phospholipids increased, while cholesterol and the cholesterol to phospholipid ratio decreased (P < 0.05). A positive correlation between the plasma cholesterol concentration and the cholesterol to phospholipid ratio was noted (P < 0.05, r = 0.851). Our results show that HMG-CoA reductase inhibitors change the composition and probably also the functions of cell membrane lipids and blood ATP concentration. PMID- 10743677 TI - Protective effect of myristic acid on renal necrosis occurring in rats fed a methyl-deficient diet. AB - Weanling rats fed a methyl-deficient diet develop acute renal failure, the morphological features of which vary from focal tubular necrosis to widespread cortical necrosis. We and others have shown that coconut oil, rich in saturated fatty acids, has a renal protective effect in this experimental model. In the experiment we are reporting now, we studied which fatty acid is involved in the protection afforded by coconut oil by feeding five groups of methyl-deficient rats a mixture of corn oil and hydrogenated vegetable oil, C6-C8-C10 fatty acids, C12 fatty acid, C14 fatty acid and C16-C18 fatty acids. Five groups of rats receiving the same diets supplemented with choline chloride were used as controls. The group of methyl-deficient rats fed C14 fatty acid (myristic acid) showed a greater percentage of surviving animals and lower renal damage than the other groups of methyl-deficient rats, indicating that the protective effect of coconut oil found in previous experiments is due to its high content of myristic acid. PMID- 10743678 TI - Effect of EGb-761 on vasospasm in experimental subarachnoid hemorrhage. AB - Based on the previously suggested hypothesis that the generation of free radicals leading to lipid peroxidation is involved in the genesis of vasospasm and vasculopathy following subarachnoid hemorrhage, the therapeutic effect of EGb 761 as an antioxidant on experimental vasospasm and vasculopathy was evaluated in a double hemorrhage dog model of chronic cerebral vasospasm. For this study 14 dogs were randomly assigned to two groups, a control and a Ginkgo biloba group. The control group was only administered saline in a volume equivalent to a dose of 100 mgEGb 761/kg while the treatment group was given 100 mg EGb 761/kg. The diameter of the basilar artery decreased from 1.95 +/- 0.16 mm at day 0 to 1.11 +/- 0.07 mm at day 8 in the control group, while in the treatment group the vessel diameter decreased from 2.01 +/- 0.17 mm at day 0 to 1.72 +/- 0.16 mm at day 8. These results correspond a decrease in vessel diameter of 15.1% in the treatment group and of 43.1% in the control group (P < 0.05). Histopathological studies of the specimens obtained from basilar arteries showed that pathological signs of proliferative vasculopathy, including narrowing of the vessel lumen, corrugation of the lamina elastica and subendothelial thickening, were present in all the animals in the control group, while they could not be demonstrated in the Ginkgo biloba group. These results suggest that Ginkgo biloba may have a protective effect against subarachnoid hemorrhage-induced vasospasm and vasculopathy as a result of antioxidants. PMID- 10743679 TI - Simple quantitative method for determining the amount of blood-borne tumor cells: initial in vitro results. AB - In metastasis research it would be useful to determine the number of blood borne tumor cells which are released from a primary tumor into the blood circulation. One way to quantify the number of released tumor cells could be to take blood from a vessel which is located close to a primary tumor and is draining the tumor. The number and viability of tumor cells released into the blood stream at any given time could be measured in cancer patients, especially those known to bear a primary, hematogenous metastasizing tumor. Plating efficiency is a precise method for the quantitative determination of the number of colony-forming cells in an adherent cell population. We performed initial in vitro experiments using plating efficiency to count adherent tumor cells within whole human blood. Exploiting the difference in adherence properties of colon carcinoma cells and blood cells in standard cell culture medium, these initial investigations show that it is possible to determine the plating efficiency of colon carcinoma cells within fresh whole human blood. PMID- 10743680 TI - Lipid peroxidation and glutathione levels after cortical injection of ferric chloride in rats: effect of trimetazidine and deferoxamine. AB - Intracortical injection of iron salts causes seizures. Oxidation of lipids in neural membranes by reactive oxygen species is involved in the mechanism responsible for iron-induced seizures as a model of posttraumatic epilepsy. In this study, we examined the effect of trimetazidine (TMZ) and deferoxamine (DFO) on lipid peroxidation after cortical injection of 5 microliters of an aqueous solution containing 100 mM of ferric chloride (FeCl3) in rats. Animals were divided into four groups (n = 7 each) and treated as follows: group 1, saline injection into the cortex (control group); group 2, iron injection into the cortex (injury group); group 3, iron injection into the cortex plus TMZ; group 4, iron injection into the cortex plus DFO. The animals were killed 3 h after injections, and the levels of malondialdehyde (MDA), a lipid peroxidation product, and reduced glutathione (GSH) were measured. A significant elevation of MDA was observed in group 2 (P < 0.05). MDA levels were found to be lower in both the TMZ-treated (P < 0.05) and DFO-treated (P < 0.05) groups than in the injury group. Tissue GSH levels were significantly decreased in group 2 (P < 0.05). GSH levels were increased in the TMZ-treated (P < 0.05) and DFO-treated (P < 0.05) groups compared to the injury group. The results of our study suggest that lipid peroxidation is a critical event in iron-induced epilepsy and that treatment with TMZ and DFO is effective in preventing the formation of free radicals and reducing lipoperoxides in brain tissue. PMID- 10743681 TI - Therapeutic effect of tirilazad mesylate (U-74006F), mannitol, and their combination on experimental ischemia. AB - The effect of tirilazad mesylate (U-74006F), mannitol, and their combination was investigated on focal cerebral ischemia induced by permanent middle cerebral artery (MCA) occlusion in rabbits. Rabbits were divided into four groups receiving vehicle, U-74006F, mannitol, and U-74006F plus mannitol. Hematocrit (hct), glucose, mean arterial blood pressure (MABP), pH, PCO2, and PO2 were measured both before and after occlusion. Seventy-two hours following the permanent MCA occlusion, the neurological outcome was assessed and a quantitative neuropathologic examination was performed in all rabbits. The neurological outcome was better in the rabbits treated with U-74006F plus mannitol than in the other groups. The size of infarction of the affected hemisphere following MCA occlusion was 49.7% in the control group, 30.6% in the U-74006F group, 47.6% in the mannitol group, and 24.1% in the U-74006F plus mannitol group. There was a statistically significant reduction in infarct size in the U-74006F plus mannitol group compared with the other groups (P < 0.05). The ratio of ischemic neurons to total neurons in the cortex was smaller in the U-74006F plus mannitol group than in the other groups. The ratio of ischemic neurons to total neurons in the subcortex was significantly lower in the U-74006F plus mannitol group than in the other groups (P < 0.05). Our data provide evidence for the beneficial effects of both U-74006F and U-74006F plus mannitol in promoting neurological recovery and preservation of the ischemic area. PMID- 10743682 TI - Effect of alpha-lipoic acid supplementation on oxidative protein damage in the streptozotocin-diabetic rat. AB - An increase in oxidative stress may contribute to the development of oxidative protein damage in streptozotocin diabetic rats. In the present study, the influence of alpha-lipoic acid supplementation on plasma protein carbonyl, plasma thiol, and plasma lipid hydroperoxide levels was examined in order to characterize the relationship between the oxidative stress and the oxidative protein damage. Rats were randomly divided into three groups of equal body weight. Chronic hyperglycemia was induced by intravenous streptozotocin injection in both the group of male Wistar rats to be supplemented with alpha-lipoic acid and the group that was not to receive alpha-lipoic acid. Nondiabetic rats formed the control group and received a saline injection. In streptozotocin diabetic rats with and without alpha-lipoic acid supplementation, plasma carbonyl levels were significantly increased, while plasma thiol levels were significantly decreased compared with those of the control group. Plasma lipid hydroperoxide levels were significantly increased in diabetic rats without alpha-lipoic acid supplementation compared with those of the controls, but the lipid hydroperoxide levels in the alpha-lipoic acid supplemented group were no different from those of the controls. In streptozotocin-diabetic rats, oxidative stress was significantly decreased in the alpha-lipoic acid-supplemented group. The results of this study suggest that alpha-lipoic acid, by decreasing oxidative stress, may be effective in preventing oxidative protein damage, which may contribute to the development of diabetic complications. PMID- 10743683 TI - Functional, histological, and inflammatory changes in chronically rejecting small bowel transplants. AB - Our aim was to develop a model of chronic rejection (CR) in small bowel allografts, and to study the changes occurring in these grafts. Small bowel transplantation was performed using the DA to AS rat strain combination. Short term (5 mg/kg intramuscular, from days -2 to +9), or long-term cyclosporin treatment (5 mg/kg, 3 times a week until day 50) was given to prevent acute rejection. Controls were untreated allografts, DA isografts with and without cyclosporin, and normal DA and AS rats. They were followed for 50 and 100 days after transplantation. Recipients of a syngeneic graft lost weight during the first week after transplantation, but started to regain weight and kept growing thereafter. Histology showed normal bowel architecture with normal mesenteric lymph nodes and Peyers patches. Vigorous acute rejection occurred in the untreated allografts. Animals had persistent weight loss, and were killed between 6-13 days after transplantation. No clinical signs of graft-versus-host disease were seen. Histology showed end-stage acute rejection. In both cyclosporin treated allografted groups the postoperative course was as in the isografted animals. However, all animals had histologic signs of CR by 50 and 100 days after transplantation. Changes were most prominent in the mesentery. Serositis with increased vascularity, inflammation with sclerosis, and patchy myointimal proliferation with endothelialitis of the mesenteric vessels were found. Changes in the bowel were patchy and included some thickening of the muscle coat, crypt hyperplasia, scattered necrotic cells in the crypts, slight blunting of villi and loss of goblet cells. Infiltrating cells in the mesentery and bowel consisted mainly of CD 4+ cells, CD 8- T-cells and monocytes/macrophages. Lactulose mannitol urinary excretion ratio was significantly increased in short-term cyclosporin treated allografts at days 50 and 100 posttransplant. Serum albumin levels were significantly lowered in this group at both time points examined. We developed two models in which CR occurs after small bowel transplantation. Long term cyclosporin treatment delayed the development of CR, since functional abnormalities were only seen in the animals that were treated with short-term cyclosporin. PMID- 10743684 TI - Donor-derived alloantigen-presenting cells persist in the liver allograft during tolerance induction. AB - The predictive value of chimerism was evaluated in three different transplantation models in the rat without immunosuppression: small bowel- (SBTx), liver- (LTx), and liver/small bowel transplantation (LSBTx) were performed in the Brown Norway (BN)-to-Lewis-(LEW) strain combination. Immunohistochemistry and flow cytometry were used to identify donor cells in the recipient's spleen. Their number did not change significantly during transient rejection or tolerance after LTx and LSBTx. However, the amount of donor-derived nonparenchymal cells within the liver allograft including antigen-presenting cells (APCs), such as dendritic and Kupffer cells, clearly mirrored the recipient's immune status: as expected, their number decreased during rejection, but recovered considerably during and after tolerance induction. We conclude that donor cells in the periphery of the recipient correlate with the presence of the allograft, but do not seem to influence graft acceptance actively. However, the kinetics of the detected donor APC population in the liver suggests their important role in modifying the recipient's immune response towards tolerance. PMID- 10743685 TI - Transforming growth factor-beta 1 derived from biliary epithelial cells may attenuate alloantigen-specific immune responses. AB - Immune response to liver allografts may be different from that to other organ transplants since immunological manipulation easily attenuates immune-response to liver allografts. Numerous studies on the alloantigen-specific immune response have been carried out, however, the precise mechanisms involved in this attenuation are not clear yet. Here we suggest the attenuation of alloantigen specific immune response by TGF-beta 1 derived from biliary epithelial cells. The transforming growth factor-beta 1 (TGF-beta 1) expression in rat liver was examined immunohistologically. Rat biliary epithelial cells (BEC) were purified from the perfused liver and added to allogeneic mixed lymphocyte reaction (allo MLR) to assess their attenuating potential on allo-MLR and alloantigen-specific cytotoxic T lymphocyte (allo-CTL) induction. Immunohistological investigation revealed the expression of TGF-beta 1 in biliary epithelial cells. Both purified biliary epithelial cells and TGF-beta 1 attenuated allo-MLR and allo-CTL induction in a dose-dependent manner, and anti-TGF-beta 1 antibody partially relieved this attenuation. This study reveals that biliary epithelial cells, the major target cells of allo-antigen specific immune response, contain TGF-beta 1 and that they have a capacity to attenuate allo-MLR and allo-CTL induction. PMID- 10743686 TI - Pre- and early postoperative risk factors for death after cardiac transplantation: a single center analysis. AB - Due to the limited number of donor organs, death on the waiting list and waiting time for cardiac transplantation have markedly increased. A pressing need of appropriate selection criteria for patients who would benefit most from transplantation is apparent. The purpose of this study is to identify pre- and early postoperative risk factors that influence long term survival after cardiac transplantation. 702 consecutive patients who underwent cardiac transplantation between 3/1984 and 12/1997 were analyzed retrospectively for the influence of different pre- and early postoperative risk factors on early (30 days) and late death (5 years). Univariate and multivariate regression analysis revealed risk factors for early as well as late death. Predictors of early death were higher preoperative PVR, retransplantation, longer ischemic time, postoperative acute kidney failure and longer intubation time. Risk factors for late death were early transplant era, previous cardiac surgery, patients awaiting transplantation in a hospital, prolonged stay in an intensive care unit, and any rejection during the first month after transplantation. These results demonstrate that pre- and early postoperative risk factors have significant influence on early and long term survival. PMID- 10743687 TI - Laparoscopic and open live donor nephrectomy: a cost/benefit study. AB - Recently, laparoscopic liver-donor nephrectomy has been developed in order to increase organ donation. In this study we compare and review the records of 10 donors operated by open extraperitoneal approach and of 10 donors operated by a laparoscopic transperitoneal approach (LSC). Results show less use of postoperative parenteral narcotics in the LSC group (109 mg vs 272 mg; P < 0.0005) than in the extraperitoneal group. Morbidity was similar in both groups. There was no difference in postoperative stay. Allograft kidney function was similar in both groups until 6 months after donation. The use of disposable laparoscopic material bears an extra cost of 900 US$. We can thus conclude that laparoscopic live-donor nephrectomy is a safe procedure that significantly reduces postoperative pain, and is not detrimental to the allograft. The total cost of the laparoscopic procedure will be lower than that of the open approach if the length of postoperative stay is cut by 3 days. PMID- 10743688 TI - Ogilvie's syndrome associated with acute cytomegaloviral infection after liver transplantation. AB - Ogilvie's syndrome, or acute colonic pseudo-obstruction, is a rare complication following liver transplantation. We describe two cases in which the onset of Ogilvie's syndrome is strongly temporally associated with acute cytomegaloviral (CMV) infection in immunosuppressed liver transplant recipients. The pseudo obstruction resolved rapidly in both cases following treatment with intravenous ganciclovir. Acute CMV infection therefore appeared to be causally linked to pathogenesis of Ogilvie's syndrome in these two cases. This association has not been described previously to our knowledge, and should be considered in any transplant patient presenting with Ogilvie's syndrome. PMID- 10743689 TI - Serum antibodies to heat shock proteins are of no diagnostic value for human kidney allograft rejection. AB - Because of the prevailing evidence that heat shock proteins (hsp) are involved in transplantation immunology, we investigate in this study the serum levels of anti hsp60, and anti-hsp70 antibodies in human kidney allograft recipients. We analyzed 67 sera from 20 patients immediately before and 2 weeks after receiving a kidney allograft, and from 27 healthy age-matched controls with an ELISA. Eleven kidneys had normal allograft function, six had a mild rejection episode, all of which could be reversed successfully; three kidneys had to be removed later on because of resistant rejection. Hsp antibody frequency and titres were the same for transplant recipients and for healthy controls. In patients receiving a kidney allograft, no difference in the level of hsp-antibodies before and after transplantation was observed. Additionally, anti-hsp60 and anti-hsp70 antibody titres were found to be independent of the clinical course. These data suggest that the determination of anti-hsp60 and 70 antibody titers are of no diagnostic value for renal allograft rejection. PMID- 10743690 TI - Increased apoptosis of hepatocytes in vascular occlusion after orthotopic liver transplantation. AB - Early vascular occlusion is liable to cause graft failure, and differential diagnosis between this condition and primary nonfunction (PNF) caused by preservation injury may be difficult. Apoptosis has been detected in immunomediated cytotoxicity and is known to be triggered by mild ischemia. In a retrospective analysis we investigated the role of apoptosis in vascular occlusion, PNF, and acute allograft rejection to improve the differential diagnosis of early graft failure. The liver graft histology of 75 patients (46 male, 29 female) a median 47 (1-64) years of age was screened semiquantitatively for the rate of apoptosis on the hematoxylin-eosin stain (HE) and by the in situ end nick labeling technique (TUNEL). This cohort included all patients who developed PNF (n = 9) or vascular occlusion (n = 11) after orthotopic liver transplantation (OLT) in the years 1992 to 1996. Within this period of time we performed 205 OLTs on 189 patients. We further included 22 patients with early acute rejection and 11 controls. The highest rates of apoptotic hepatocytes were seen in vascular occlusion (P < 0.001). Grafts with PNF were explanted 1-3 days after OLT and showed hepatocytes that were 100% necrotic. Cases of acute early rejection showed a significantly higher apoptotic cell count than did normal controls (P < 0.003), increasing in direct proportion to the severity of rejection. Screening biopsies for the rate of apoptosis can improve the efficacy and accuracy of differential diagnosis of early graft failure. PMID- 10743691 TI - Effects of cytomegalovirus infection and prolonged cold ischemia on chronic rejection of rat renal allografts. AB - Previous studies have demonstrated that both cytomegalovirus (CMV) infection and prolonged cold ischemia of the allograft (CI) are associated with chronic rejection of renal transplants. The purpose of this study is to investigate the effect of CMV infection, of CI and of the combination of both, on the progression of chronic rejection, and to obtain a more detailed insight in their effects on the expression of adhesion molecules. Therefore, a rat transplantation model was used. Lewis recipients of renal allografts (with and without CI) from MHC incompatible Brown Norway rats were inoculated with rat CMV or left uninfected. CMV infection alone resulted in an increased influx of CD4+ cells and macrophages early after infection, and in an increase in glomerular sclerosis and intima proliferation. CI caused an increase in infiltrating NK cells and an effect on intimal proliferation, glomerular sclerosis, and tubular atrophy. When CMV infection and CI were combined, an additive effect could be measured. This was however not the case for the function of the kidney. The creatinin showed a synergistic effect of the two influencing factors. Due to the CMV infection, an increase in CD49d cells was detected. CI resulted in an increase in CD18 cells and an increase in the expression of CD62P on vessels, and CD54 and CD44 on tubules. When CMV infection and CI were combined, all the effects caused by CMV and CI alone were present in an additional way. The results of the present study suggest that special attention should be paid to the recipient of an ischemically injured graft when either the donor or the recipient is CMV-infected. The patterns seen in histology, the infiltration of leukocytes and the expression of adhesion molecules, suggest that CI and CMV infection both have an effect on rejection, but act by different mechanisms. PMID- 10743692 TI - Effects of portal versus systemic venous drainage in kidney-pancreas recipients. AB - A randomized study of combined kidney-pancreas transplantation was performed on 30 insulin-dependent diabetic patients with end-stage renal disease to compare the consequences of pancreas transplantation with portal venous (PV) and systemic venous (SV) drainage. Fourteen patients (SV) group) received systemically drained and sixteen (PV group) portally drained pancreas allografts. Enteric drainage was performed in both groups. The routine follow-up included documentation of the clinical course and detailed endocrine studies. At 1 year after transplantation, the patient survival rate was 92% for the SV group and 96% for the PV group; the graft survival rate was 78% and 82%, respectively. Endocrine studies indicated no difference in fasting and stimulated glucose or in glycosylated hemoglobin between the two groups. In addition, no hyperinsulinemia and lipidic abnormalities were evidenced in either group Long-term studies are required to conclude whether PV and SV drainage in pancreas transplantation are equivalent in terms of patient and graft survival as well as metabolic consequences. PMID- 10743693 TI - Post-transplant diabetic ketoacidosis--a possible consequence of immunosuppression with calcineurin inhibiting agents: a case series. AB - Post-transplant diabetes mellitus, a complication due to corticosteroids and the calcineurin inhibitors, cyclosporine and tacrolimus (FK506), is commonly regarded as a form of type-2 (adult-onset) diabetes mellitus. Diabetic ketoacidosis, which requires relative insulin deficiency to impair fatty acid metabolism, is a complication of type-1 diabetes mellitus. We report three patients who presented with diabetic ketoacidosis post-transplant. All three patients presented with severe hyperglycemia, significant ketosis and metabolic acidosis of variable severity. One patient was a renal transplant recipient on a cyclosporine-based regimen. The other two patients were liver transplant recipients receiving either cyclosporine or tacrolimus-based immunosuppression. Both of the liver transplant recipients were found to have moderate to high serum levels of calcineurin inhibitors on presentation. The liver recipient on cyclosporine (Neoral) had a 4 hour post-dose level of 388 ng/ml and the patient on tacrolimus was found to have a trough level of 21.2 ng/ml. Our experience suggests that post-transplant diabetes mellitus, in association with calcineurin inhibition, may result in ketoacidosis either secondary to relative beta cell dysfunction, peripheral insulin resistance, or a combination of the two effects. Post-transplant diabetes mellitus can be an atypical form of adult-onset diabetes with features of both type I and type II diabetes mellitus. PMID- 10743694 TI - Treatment of tacrolimus-related adverse effects by conversion to cyclosporine in liver transplant recipients. AB - When tacrolimus side effects persist despite dose reduction, conversion to cyclosporine-based immunosuppression (CyA) is necessary. We characterized tacrolimus side effects that warranted discontinuation of the drug, and outcomes after conversion. Of 388 liver recipients who received tacrolimus as primary immunosuppression, 70 required conversion to CyA. We recorded indication for conversion, whether conversion was early or late after transplantation, tacrolimus dose and trough blood level at conversion, and incidence of rejection after conversion. Conversion was early in 29 patients (41.4%) and late in 41 (58.6%). Indications for early conversion were neurotoxicity (20), (insulin dependent) diabetes mellitus (IDDM) (5), nephrotoxicity (3), gastrointestinal (GI) toxicity (6), and cardiomyopathy (1), and for late conversion were neurotoxicity (15), IDDM (12), nephrotoxicity (3), GI toxicity (5), hepatotoxicity (6), post-transplant lmphoproliferate disease (PTLD) (2), cardiomyopathy (1), hemolytic anemia (1), and pruritus (1). All early-conversion patients showed improvement/resolution of symptoms. Among late-conversion patients, 37 (90.2%) had improvement/resolution; in 4 (9.8%), adverse effects persisted. The overall rejection rate was 30%. Sixty-two patients (88.6%) are alive with functioning grafts 686 +/- 362 days (range, 154-1433 days) after conversion. When tacrolimus side effects are unresponsive to dose reduction, conversion to CyA can be accomplished safely, with no increased risk of rejection and excellent long-term outcome. PMID- 10743695 TI - Influenza pneumonia in a paediatric lung transplant recipient. AB - Although a common cause of morbidity and mortality in the general population, influenza infections are uncommon in lung transplant recipients and, to date, have only been associated with transient declines in pulmonary function and a relatively benign clinical course. This paper describes severe influenza pneumonia in a 13-year-old paediatric lung transplant recipient (5 months after double lung transplantation). Influenza pneumonia was diagnosed by direct fluorescent antibody testing and viral culture of bronchoalveolar lavage fluid. The patient required mechanical ventilation for 2 days due to respiratory failure and fatigue. Since his recovery from this pneumonia, he has developed obliterative bronchiolitis and currently awaits re-transplantation. PMID- 10743696 TI - Successful endoluminal thrombo-aspiration of renal graft venous thrombosis. AB - Renal transplant vein thrombosis is an unusual event occurring in 0.3-3% of renal transplantations. Prognosis is uniformly poor with graft loss in nearly every case. We report here the first three cases of renal graft vein thrombosis successfully treated by percutaneous endoluminal thromboaspiration. After an initially uneventful course all recipients developed anuria and required hemodialysis. In two cases, an ultrasound examination suggested a diagnosis of venous thrombosis. Emergency arteriography and phlebography were performed, confirming the complete thrombosis of the graft veins. Thromboaspiration was carried out with full heparinization and led to renal function improvement in all cases. Grafts are still functioning 6 months after the procedure, with serum creatinine levels of 176 mumol/l, 120 mumol/l and 184 mumol/l, respectively. Thus, this procedure avoids surgical and anaesthetic risks and allows, if performed at an early stage, restoration of graft function. Great care must be taken to avoid vein wall damage, vascular suture line rupture, or pulmonary embolism. PMID- 10743697 TI - Urinary tract infection in diabetic patients. AB - AIM: The aim of the study was to compare the epidemiological, microbiological and clinical features of diabetic patients with urinary tract infection (UTI) to those of nondiabetic ones. METHODS: A prospective study was performed on 490 consecutive patients with proven UTI. The patients were studied on the basis of a specific questionnaire and hospital records. RESULTS: Of 490 enrolled patients, 89 (18.1%) had diabetes mellitus. The mean age of diabetics and nondiabetics was respectively 64.9 +/- 13.2 (SD) and 54.4 +/- 23.3 years. Most diabetics had asymptomatic bacteriuria and had undergone bladder catheterization more frequently than the nondiabetics. The most frequent causative agents of UTI in diabetics and nondiabetics were: E. coli (respectively, 56.1 vs. 56.8%), Proteus sp. (7.9% vs. 7.2%), Pseudomonas sp. (6.7 vs. 8.2%), Enterococcus sp. (6.7 vs. 7.2%). More than 50% of the isolated Pseudomonas sp. strains in both groups were resistant to gentamicin, piperacillin and norfloxacin. Both diabetics (52.8%) and nondiabetics (42.2%) had recurrent UTI during the follow-up period; the difference in the incidences did not reach statistical significance. CONCLUSION: No significant differences in epidemiological, clinical and microbiological evaluated features of diabetics and nondiabetics were pointed out, except for the higher frequency of bladder catheterization of diabetics than nondiabetics. However, the eradication of UTI seemed to be more difficult in diabetics than in nondiabetics. PMID- 10743699 TI - Tc-99m MAG3 renal studies: renogram and effective renal plasma flow in spinal cord injury patients. AB - To determine whether years since injury, level of injury, degree of injury, gender, or age influence renal function in 66 spinal cord injuries (SCI) patients without urological complications, individual kidney effective renal plasma flow (ERPF) and radionuclide renogram (RR) were measured using technetium-99m mercaptoacetyltriglycine (Tc-99m MAG3). The 176 individual kidneys included 84 kidneys with normal ERPF and RR, 48 kidneys with normal ERPF but abnormal RR, and 44 kidneys with abnormal ERPF and RR. The incidence of abnormal Tc-99m renal study results was higher in the kidneys of female patients (37/54, 68%) than of male patients (55/122, 45%) (p < 0.05). We found that Tc-99m MAG3 renal studies including ERPF and RR are safe and noninvasive urological screening tests for SCI patients. PMID- 10743698 TI - Effectiveness of oral L-arginine in first-line treatment of erectile dysfunction in a controlled crossover study. AB - BACKGROUND AND AIMS: Relaxation of cavernous smooth muscle is a parasympathetic and non-adrenergic, non-cholinergic mediated process which requires nitric oxide (NO). NO is synthesized from L-arginine by NO synthase (NOS). Some studies report good clinical results under oral L-arginine medication in the treatment of erectile dysfunction. We examined the effectiveness and safety of L-arginine in the treatment of mixed-type impotence. METHODS: 32 patients (mean age 51.6 years) with mixed-type impotence diagnosed according to the results of sexual history and urological examination were enrolled in a randomized, placebo-controlled, crossover comparison of an oral placebo with 3 x 500 mg L-arginine/day. A validated questionnaire (KEED) was used to define the grade of impotence with a score. The treatment consisted of two 17-day courses (50 tablets). After a 7-day washout period the patients who initially received the placebo for 17 days were switched to L-arginine and vice versa. We assessed the efficacy with the validated questionnaire at the end of each drug period. RESULTS: 30 patients (94%) completed the whole treatment schedule. Five (17%) patients reported a significant improvement in erectile function at the end of the L-arginine phase and 6 (20%) patients after the placebo period. 17 (56%) patients showed little improvement with L-arginine and 13 (43%) with placebo. In 8 patients (27%) of the verum group there was either no change in the ED score or even a slight worsening. No statistical difference in the impotence scores were found. No drug related adverse effects occurred with L-arginine treatment. CONCLUSION: Oral L arginine 3 x 500 mg/day is not better than placebo as a first-line treatment for mixed-type impotence. PMID- 10743700 TI - Management of staghorn calculus: analysis of combination therapy and open surgery. AB - OBJECTIVE: To assess the role of primary open surgery versus the recommended combination approach (percutaneous and lithotripsy) to treat staghorn calculi in a developing country. PATIENTS AND METHODS: Available records (n = 91) of patients with staghorn managed during the last 4 years were retrieved. Patients were placed in two groups, open surgery and combination group, according to the primary procedure chosen by the patient. Demographic data in two groups was comparable in most of the respects except that renal failure patients were more in the combination group. Stone clearance, major residue, auxiliary procedures, morbidity, hospital stay and the cost were studied in the two groups for comparison. RESULTS: Complete clearance could be obtained in 66 and 59% with open and combination method respectively. Major residue (> 16 mm2) was present in 21% of open and 38% of the combination group. In patients with primary stone burden < 900 mm2, the total clearance rates were 66 and 60% in open and combination group, respectively. Total clearance was not affected by caliceal dilatation, total stone burden, pelvic and caliceal bulk separately or their ratio, as arrived by logistic regression analysis. The incidence of haematuria in the combination group was marginally higher, probably due to more renal failure patients in this group. Hospital stay in the two groups was comparable (13.0 days in combination vs. 12.1 days for open). The cost of treatment with combination group was significantly higher. CONCLUSION: Open surgery for staghorn is still an economically viable option for difficult stone disease, specially in a developing country, with comparable efficacy, favourable morbidity and hospital stay. PMID- 10743701 TI - Standardization and potential use of HPLC for detection of cellular placental alkaline phosphatase using established tumour cell lines and fresh tumour biopsies. AB - Placement alkaline phosphatase (PLAP) is one of the cellular phosphatases (ALP) expressed in patients with testis cancers, particularly in seminomas. Using various techniques including Western blot and high-performance liquid chromatography (HPLC) systems and ATC2, a newly developed specific anti-PLAP monoclonal antibody (Mab), the presence of active form of PLAP in lysates prepared from testis tumour fragment and tumour cell lines, was studied. This was carried out following isolation of PLAP from biological samples using CNBr Sepharose-conjugated ATC2 beads. The results showed that: (1) The target for the newly developed Mab ATC2 was PLAP. (2) The ATC2-conjugated bead system was an efficient method for isolating pure PLAP. (3) Diethylamine (DEA), in contrast to urea and glycine, was the most efficient for separation of PLAP from ATC2 conjugated beads, as the isolated molecule did not lose any phosphatase activity and there was very little uncoupling of the ATC2 Mab from the beads. (4) ATC2 conjugated CNBr beads could pick up PLAP from a solution containing standard PLAP and lysates prepared from tumour cell lines or testis tissue fragments positive for the PLAP. (5) HPLC profile of testis tumour lines and testis tumours showed two distinct peaks with ALP activity, one at retention time 7-8 min (corresponding to 95 kDa molecule) and one at 12-13 min corresponding to 70 kDa molecule). These data demonstrated the potential use of various biochemical methods in combination with HPLC for isolation of the fully functional molecules with ALP activity from different samples including lysates prepared from patients with testis cancer. The nature of ALP activity at 95 kDa is being investigated as no such molecule has been reported previously. These techniques might have an important implication for an early detection of germ cell tumours, particularly in patients with equivocal ultrasound. PMID- 10743702 TI - Xanthine stone in the urinary bladder of a male child. AB - Urinary tract calculi composed primarily of xanthine are rate both in adults and children. We describe the clinical presentation and management of a 3.5-year-old boy with hereditary xanthinuria (an autosomal recessive disorder of purine metabolism) and primary bladder calculus formed from xanthine. To our knowledge this case demonstrates a previously undescribed form of xanthinuria in childhood. Xanthine stones, although rare, should be considered in the diagnosis of urolithiasis. PMID- 10743703 TI - Ureterocele arising from a lower-pole moiety. AB - We report on an uncommon case of a ureterocele arising from the lower-pole ureter in a duplex system. To our knowledge, this represents the 3rd such case reported in the English literature. Ultrasonography and retrograde pyelogram established the diagnosis. The patient underwent left upper-pole nephroureterectomy with excision of the ureterocele and cross-trigonal ureteral reimplantation. PMID- 10743704 TI - Life-threatening spontaneous retroperitoneal bleeding: a rare complication of oral anticoagulation. AB - In a patient on oral anticogulation with sudden onset of loin pain, the possibility of spontaneous bleeding in the retroperitoneum must be considered in the differential diagnosis of renal colic. This rare pathology can be life threatening, and rapid diagnosis with ultrasound and computerized tomography of the abdomen must be made. If the patient is hemodynamically stable, a conservative approach is justified. Otherwise, emergency nephrectomy may be needed. As patients become older and indications for anticoagulant therapy become more common, we will probably have to face more of these rare bleeding complications in the future. We report such a case. PMID- 10743705 TI - Lower ureteral obstruction due to a persistent umbilical artery. AB - A 32-year-old lady presented with primary infertility and a 1-year history of recurrent left-flank pain. She was found to have left lower ureteric obstruction on intravenous urography. No specific cause for the obstruction could be determined preoperatively. The patient underwent open extraperitoneal surgery to determine the cause and to treat the obstruction. A 4-mm vessel was seen crossing over the ureter at the site of narrowing. It was the persistent umbilical artery traced in continuity from the internal iliac artery. The vessel was resected, and the ureter was reimplanted into the bladder. Extrinsic obstruction of the distal ureter because of aberrant or persistent vessels has been infrequently reported. Such reports predominantly refer to children, and the diagnosis is usually made at laparotomy, frequently following previous failed attempts at endourological management. PMID- 10743706 TI - Bilateral emphysematous pyelonephritis with autosomal-dominant polycystic kidney disease successfully treated by conservative method. AB - Emphysematous pyelonephritis (EPN) is an uncommon and potentially life threatening necrotizing inflammation of the renal parenchyma. EPN associated with autosomal dominant polycystic kidney disease (ADPCK) is extremely rare. We report such a case of bilateral EPN with ADPCK that was successfully treated with conservative methods. To our knowledge, our case is only the second to document bilateral EPN occurring with ADPCK and the first one to be treated successfully with conservative methods. PMID- 10743707 TI - Spontaneous regression of inflammatory pseudotumor of the urinary bladder. AB - Inflammatory pseudotumor of the urinary bladder is a benign proliferative lesion of the submucosa and its treatment has not yet been established. Here we present a case of spontaneously regressed inflammatory pseudotumor of the urinary bladder. PMID- 10743708 TI - Inverted papilloma of the urinary bladder in a girl. AB - We report a case of inverted papilloma of the urinary bladder in a 10-year-old girl. She was referred to us with intermittent asymptomatic gross hematuria. A polypoid, pedunculated mass, which had a thin and long stalk, approximately 6 x 20 mm in diameter, was observed by cystscopic examination at the right paratrigone. The lesion was resected endoscopically. Histological findings were compatible with the trabecular type of inverted papilloma. There have been only 3 cases reported of inverted papilloma in children, and no report has previously been published concerning a girl. Since the biological potential of pediatric inverted papilloma remains unclear, we believe that our patient should undergo periodic and detailed urological examinations. PMID- 10743709 TI - Primary renal malignant fibrous histiocytoma. A case report and review of the literature. AB - Primary renal malignant fibrous histiocytoma is a rare tumor of the kidney. We report an additional case noting, as other authors have, the similarity in clinical presentation with renal cell carcinoma. We also review the 23 documented cases of the international literature, emphasizing the current diagnostic approaches and therapeutic management. PMID- 10743710 TI - Diffuse metastasized testicular teratoma and paraneoplastic thyreotoxicosis. Case report and literature review. AB - Paraneoplastic syndromes are rare entities in patients with excessive HCG production. This has been attributed to the thyroid-stimulating hormone-like activity of HCG. In most cases hyperthyroidism remains subclinical. We report on a patient with diffuse metastasized malignant teratoma and thyrotoxicosis due to excessive levels of tumor-derived HCG. This complication of nonseminomatous germ cell tumors is associated with potential morbidity and the need for prompt recognition is stressed. Treatment consists of standard antithyroid medication and immediate cytoreductive chemotherapy to reduce the paraneoplastic stimulus. Evaluation of thyroid hormone levels in all patients with serum HCG levels exceeding 20,000 U/l is recommended. PMID- 10743711 TI - Pyelic metastasis from a renal cell carcinoma following partial nephrectomy. AB - The majority of ureteropelvic metastatic tumors are of non-urologic origin. A case of metastatic clear cell adenocarcinoma to the renal pelvis in a patient with a bilateral, asynchronous renal tumor is presented. Conservative surgical treatment involved open pyelotomy and local resection of the tumor. At the 12 month follow-up, the patient is alive, asymptomatic and not on dialysis. The hypothesis for the mechanisms of dissemination and the relevance of conservative treatment are discussed. PMID- 10743712 TI - An infiltration technique for reduction mammaplasty: results in 192 consecutive breasts. AB - The use of local anaesthetic infiltration with adrenaline is now considered safe in reduction mammaplasty. However, the technique of infiltration by those who support its use is often unclear. Any technique must take account of the neurovascular anatomy of the breast if it is to be effective. We propose the use of a large volume of dilute local anaesthetic (20 ml of 1% lignocaine and 1 mg of adrenaline made up to 400 ml with 0.9% saline) which is placed judiciously in the retroglandular space 15 minutes prior to surgery. The results in 96 consecutive patients (192 breasts) who had an inferior pedicle technique were analysed. The breast complication rate was 9.36% and the patient complication rate 19.79%. Postoperative blood loss ranged from 0 to 305 ml with a mean of 56.03 ml and a median of 50 ml. The described method could be considered a variation of the tumescent technique used in liposuction. The results is an almost bloodless dissection with minimal postoperative blood loss. It should be possible to dispense with the use of drains in most cases. PMID- 10743713 TI - Precision grip with congenital hypoplasty or hypofunction of the thumb. AB - We tested the grip in four patients with congenital defects of the hand and either a hypoplastic thumb or a thumb with impaired inervation. Small objects were taken by a scissors grip between the fingers. In a hand with radial duction in the manus vara congenita, during strengthening of the wrist, the grip from the ulnar side between the fourth and fifth fingers was changed to the radial side between the second and third fingers. Large objects were gripped by all the three phalanx fingers into the palm in a horizontal position. In case 4 with hypoplasia of the thumb grade IIIC by the classification of Blauth and Buck-Gramcko, we describe a transposition of the index finger to the site of the thumb and the hypoplastic thumb to the site of the index finger. It is obvious that the precision grip is affected by the thumb length and strengthening of the ulnar side of the wrist. We assume that the scissors grip is the earliest precision grip in the evolution of the primate hand. PMID- 10743714 TI - Changing pattern of infection in the Bratislava Burn Center. AB - Infection still remains one of the major problems in burn treatment. The authors investigated the occurrence of burn wound pathogens in burn wound biopsies and/or semiquantitative wound surface off-prints. As the results have shown, trends of a decreased contribution of "classical pathogens", like Staphylococcus aureus and Pseudomonas aeruginosa, to burn wound infections were observed. The role of "other pathogens", like Enterobacter, Acinetobacter, etc., which were quite rare in the past, is on the opposite, increasing. One of the explanations can be the increasing rate of early surgical treatment methods of deep burns. The results were in accordance with similar studies from other burn centres. PMID- 10743715 TI - Spherical dilators to resolve arterial spasm. AB - Arterial spasms developing during microsurgical vascular anastomoses can be a difficult problem. We have been using specially developed spherical dilators in clinical practice to remove spasm that do not respond to the application of warm saline with lidocain. Dilating the spastic segment of the vessel two or three times with the instrument will usually relieve the spasm. Experimental testing of the method on the rat carotid artery has shown that using the dilator three times causes only moderate damage to the endothelium, which is largely overcome within one week. Thrombosis of the dilated artery was never seen in experimental studies and clinical use. PMID- 10743716 TI - A modified staining method for connective tissue in semi-thin histological sections of peripheral and cranial nerves. AB - A simple staining method for collagen in semi-thin sections of nerves is described. This technique consists of applying basic fuchsin (0.05%) solution for 2 min. to +/- 1.25 microns plastic resin-embedded sections after routine toluidine blue staining. This staining method clearly demonstrates the amount and orientation of collagenous connective tissue in the nerve, both in transverse and longitudinal sections. PMID- 10743717 TI - Noninvasive measurement of early atherosclerosis by high-resolution B-mode ultrasonography. AB - Development of sophisticated noninvasive methods permits the identification of asymptomatic subjects with early atherosclerosis. High-resolution B-mode ultrasound measurement of the intima-media thickness (IMT) is a safe and reproducible way of quantifying early changes in the arterial wall, mainly in the carotid arteries. Increased carotid IMT is associated with major cardiovascular risk factors and seems to be a powerful predictor of cardiovascular events, especially coronary artery disease. PMID- 10743719 TI - Selected prognostic factors of long-term renal graft function. AB - Kidney transplantation is a method of choice as a treatment for end-stage renal disease in indicated cases. However, the long graft survival represents only about 50% due to various types of rejection as a leading cause of graft loss in renal transplant recipients. The early dg. of rejection and especially acute rejection, it's adequate management, decreased risk for the future chronic rejection nephropathy. This is a primary goal of the clinician caring for these patients. We use several methods in order to make diagnosis of acute rejection. Urine cytology and urine flow cytometry have been found highly sensitive specific for the early diagnosis of acute rejection, provide us useful information in differentiation from others causes of graft dysfunction. Urine analysis have some advantage over other diagnostic methods and can facilitate the observation of a graft over time. PMID- 10743718 TI - Recent approaches in the treatment of Hodgkin's lymphoma. AB - An aim has been to find the optimal therapeutic approach for each patient suffering from Hodgkin's lymphoma. The disease with 80% curability rate presents the great success of clinical oncology. There is no doubt that the development and improvement of therapeutic approaches described in this article has contributed to the excellent results achieved in the last twenty years. But a lot of problems still remain to be solved by haemato-oncologists despite such progress in the treatment. The most important tasks are: 1. to increase the curability of advanced stages, 2. to improve therapeutic results in refractory and relapsing patients, 3. to eliminate or decrease the late toxicity particularly in the favourable early and intermediate stages. PMID- 10743720 TI - Processing of mRNA from human leukocytes by biomagnetical separation: comparison with current methods of RNA isolation. AB - The article refers to the technique of biomagnetical separation (BS) of mRNA using "Dynabeads mRNA DIRECT". After a brief survey of RNA isolation methods authors describe a general procedure of BS as well as its concrete application for mRNA isolation from bronchoalveolar cells. Authors report results of specific experiments to assess the efficiency and accuracy of mRNA isolation by BS. The applicability of BS technique for the gene expression studies is discussed in the end of the article. PMID- 10743721 TI - Electrophoretic patterns of Trichophyton mentagrophytes mutants with changed growth rate at different cultivation temperatures. AB - Five mutants of Trichophyton mentagrophytes with changed optimum cultivation temperature and the wild strain were used in this study. The growth rates, the keratinolytic activities and the 1-D electrophoretic patterns of these mutants were examined repeatedly. The differences among the individual mutants and the wild strain can be useful for a better understanding of the participating individual proteins for thermotolerance of this fungus. PMID- 10743722 TI - An ultrastructural study of the biostimulative effect of He-Ne laser radiation on the early postnatal differentiation of the rat submandibular gland. AB - Submandibular gland of the rat during the first week of postnatal development was used for the study of the ultrastructural changes after the He-Ne laser radiation. In the experimental group we observed an increase of the dark cells with greater quantity of the granular endoplasmic reticulum and an increased number of specific secretory granules. These changes show positive biostimulative effect of He-Ne laser radiation on the morphologic differentiation of the submandibular gland of the rat in the early postnatal period. PMID- 10743723 TI - Nuclear channel system (NCS) in normal endometrium and after hormonal stimulation. AB - The ultrastructure of NCS was investigated in two groups of patients (15 in each) during the secretory phase of the normal menstrual cycle and after the hormonal stimulation performed in the IVF programme by GnRH-a, FSH and hCG. Three developmental stages of the NCS were classified. The most numerous were the NCS in the mature stage (type 2), less in the early developmental stage (type 1) and least in the regression stage (type 3). The simultaneous occurrence of all three types does not conform the statement of Spornitz. The proportions of the three types were identical in both investigated groups of patients. The number of NCS (per 100 of nuclei) was higher in the group after hormonal stimulation than in the normal cycle. The quantitative measurement of the NCS showed its larger size in the group after hormonal stimulation than in the normal cycle, but the differences in the shape were not significant. In the serial ultrathin sections we demonstrated the relation of NCS to the nucleolus and to the invaginations of the nuclear envelope, the formation of the electron lucent center and the spiral arrangement of the tubules. In the early regression stage we described the relation of the internal row of tubules to the formed myelin figures. We suppose that the higher number and larger size of NCS can be taken as a sign of the increased activity of the endometrium during the early secretory phase after the hormonal stimulation. PMID- 10743724 TI - Ultrastructure of endometrial surface relief in normal menstrual cycle and after hormonal stimulation. AB - Ultrastructure of the superficial relief of the endometrial epithelium was investigated under the scanning (SEM) and transmission electron microscopes (TEM) in a group of patients after the hormonal stimulation, performed in the program of in vitro fertilization and embryotransfer. The results were compared with those obtained in a group of patients studied during the secretory phase of normal menstrual cycle. Total 26 bioptic samples were subjected to the investigation, 13 in each group. Different types of the epithelial surface were classified in TEM and SEM and their relative amount was evaluated quantitatively in TEM. Special attention was paid to the pinopodes, considered as specific markers of the nidation window. Differences in the number of pinopodes were observed under the SEM in two groups of patients after different schemes of the hormonal treatment. These differences were not manifested under the TEM due to the small frequency of pinopodes in the ultrathin sections. The ultrastructural changes observed in the two groups of patients after different schemes of hormonal stimulation can be considered as signs of the changed metabolic activity of the endometrial epithelium and they can reflect the different endometrial receptivity for nidation. PMID- 10743725 TI - A report on anomalies of digastric muscle. AB - The anterior belly of the digastric muscle varies greatly in shape and size. In this report, two cases of anomalies in the digastric muscle found in necropsy material belonging to the Institute of Anatomy are described. In the first case, we found a bilateral anomaly--a lateral widening of the muscle belly as an adaptation; this was shown by separation of the anterior muscle belly of digastric muscle into a medical and lateral head. The latter of which passing through the intermediate tendon and fixing partially on the hyoid bone. In the second case, a bilateral asymmetrical anomaly of the anterior belly was described. This involved the separation of the anterior belly into medial and lateral parts and the occurrence of accessory bundles; these bundles run from the left part of the mandible to the intermediate tendon of the right side of digastric muscle and to the hyoid bone. PMID- 10743726 TI - Expression of BCL-2 in the developing kidney of human embryos and fetuses qualitative and quantitative study. AB - Twelve human embryos and fetuses aged of 7-30 weeks of intrauterinal life were examined to determine the expression of bcl-2 gene in the developing kidney. Tissue samples were routinely processed and three-step indirect immunohistochemical method was used for the detection of Bcl-2 protein. End-point cytophotometry was performed with computer-controlled microscope photometer with a scanning table and the mean relative absorbance of the final product of peroxidase reaction was determined and taken as a measure of Bcl-2 expression. The morphometric evaluation was carried out from the TV display using Weibel s universal hexagonal raster and we determined the relative volume of Bcl-2 positive structures in the various zones of the embryonal kidney. The aim of our research was mapping of the Bcl-2 occurrence in the developing kidney of human embryos and fetuses. The Bcl-2 protein is involved in the regulation of apoptosis and its effect is antiapoptotic. The highest Bcl-2 expression was proved in the cells of metanephrogenic blastema. The lower occurrence of Bcl-2 positive cells was demonstrated in proximal tubules analges+ and it was almost on the borderline of detection in branches of ureteral bud. In the fetal period the marked Bcl-2 expression was maintained in the epithelial cells of proximal tubules analges. PMID- 10743727 TI - Expression of apoptosis in the developing human cephalic region during the embryonic and early fetal period. AB - The aim of our work was to study the expression of apoptosis in the cephalic region in human embryonic and fetal material. Twelve human embryos and fetuses were processed by routine histological technique and then studied using the Boehringer-Mannheim Company kit for TUNNEL technique. Rich appearance of apoptotic cells was typical for mesenchyme and for tissues of the mesenchymal origin. Dispersed or focal occurrence of apoptotic cells was described in nervous system and some orofacial structures during the embryonic period. During the early fetal period occurrence of apoptosis decreased and only rare apoptotic nuclei were visible in studied areas. Often they failed at all. PMID- 10743728 TI - Occurrence of vancomycin-resistant enterococci in relation to the administration of glycopeptide antibiotics. AB - Occurrence of vancomycin-resistant enterococci and its relationship to the administration of glycopeptide antibiotics (vancomycin, teicoplanin) was described. A total number of 628 strains of the genus Enterococcus was isolated in the Hemato-oncologic Department of the University Hospital in Olomouc in 1997. Seven strains (1.1%) were found to be resistant to vancomycin. Five of them were identified as Enterococcus faecalis of phenotype VanB, and two strains as E. faecium of phenotype VanA. The administration of vancomycin and teicoplanin in the Hemato-oncologic Department amounted to 85.7% and 89.2%, respectively, of the total consumption of these drugs at the University Hospital. PMID- 10743729 TI - Characteristics of Acinetobacter strains (phenotype classification, antibiotic susceptibility and production of beta-lactamases) isolated from haemocultures from patients at the Teaching Hospital in Olomouc. AB - A total of 85 strains of the genus Acinetobacter were isolated from haemocultures at the Institute of Microbiology of the Teaching Hospital in Olomouc over the period January 1993 to June 1997. Sixty-two (73.0%) strains of the Acinetobacter calcoaceticus-baumannii complex (Acb complex) were the most frequent. In 3 (3.5%) strains it was impossible to decide whether they belonged to the Acb complex. Other acinetobacter species were represented by 20 (23.5%) strains. The greatest amount (28.2%) of these strains was collected from the Clinic of Internal Medicine. Leukemias, lymphomas and myelodysplastic syndromes were the most frequent clinical diagnoses (20.0%) of the patients with a positive haemoculture. The most effective antimicrobial preparations tested were as follows: meropenem (98.8% of susceptible strains), colistin (94.1%), quinolones (90.6-94.1% according to the type of agent) and amikacin (91.8%). The Acb complex strains were less susceptible to antimicrobial agents than other acinetobacters. Production of inductive chromosomal beta-lactamases AmpC was proved in 42 (49.4%) strains whilst no occurrence of extended-spectrum beta-lactamases (ESBL) in the isolated organisms was recorded. PMID- 10743731 TI - Single photon bone densitometry in multiple myeloma. AB - The study was performed in 45 patients with multiple myeloma. There was found significant correlation between diminished bone mineral density of forearm and duration of the disease. No correlation between bone mineral density and biochemical markers of osteolysis (pyridinoline and deoxypyridinoline in urine) and serum levels of cytokines was found. PMID- 10743730 TI - Short-term heart rate variability changes after exercise training in subjects following myocardial infarction. AB - Heart rate variability reflects the activity of autonomic nervous system. The aim was to evaluate the value of short-term spectral analysis of heart rate for monitoring the effects of exercise training on the autonomic nervous system in subjects following myocardial infarction. Short-term spectral analysis of heart rate variability was performed during standardised supine-standing-supine test in a group of 29 clinically stable patients after myocardial infarction and the same test was repeated after 2 months of exercise training (ergometry or rapid walking). Each subject exercised at 60-80% of his maximal performance for 30 minutes once a day. Short-term spectral analysis of heart rate variability was assessed in the frequency range of 0.02-0.5 Hz involving very low frequency (VLF, 0.02-0.05 Hz), low frequency (LF, 0.05-0.15 Hz) and high frequency (HF, 0.15-0.5 Hz) spectral bands. The influence of exercise training on the autonomic nervous system was registered in the physically active group (n = 16) as a decrease in sympathetic response to standardised orthostatic load. In conclusions the regular exercise influenced an altered neural autonomic regulation of heart rate in post infarction patients shown by a decrease in sympathetic activity in the short-term heart rate variability. This method enables monitoring of the effects of physical training on the autonomic nervous system in patients following myocardial infarction. PMID- 10743732 TI - Some manoeuvres for releasing the hypertonus of spastic and shortened muscles. AB - We describe some physiotherapeutic techniques and manoeuvres releasing the increased tone of spastic and shortened muscles. The techniques can be generally characterised and summed up into three groups as follows: I. First types of manoeuvres consists in setting up the extremity or segment into the position usually opposed or antagonistic to the limited direction. This position is held for several tens of seconds. After this, partial of the originally limited range of motion could be observed. A modification of this approach is effective for persistent extensor spasticity of the lower extremities. Extremity is taken up into the internal rotation and flexion of the hip joint, into the maximal flexion of knee joint and into dorsiflexion of ankle joint, and this position is held at least for 30 seconds. After this, the extensor spasticity is usually diminished and also the voluntary movements and gait pattern improved. II. Short active jerk (not exceeding 1/2 sec) in the free direction, alternatively against mild resistance, followed by 3-4 second release and slow stretch into the restricted range. It is essentially important that the initial jerk is as brisk as possible but performed with no special effort. In case the resistance is applied, it should be only slight. Then the therapist must grasp a "melting" of the hypertonus and cautiously guide the segment into the slow and gentle stretch. III. Pressure stimulation of the particular active zones. The application of these manoeuvres and its combinations are demonstrated in case reports. PMID- 10743733 TI - Determination of leptin receptor in the serum and relations to laboratory and anthropological parameters in patients with atherosclerotic complications. AB - Leptin receptors are supposed to have signal effects and are located in most tissues in the organism but we failed to find literary data on concentration (measurement) of leptin receptors in the system circulation. We examined by the method of randomized selection the group of 20 patients with manifested atherosclerosis in whom BMI was calculated. Then we analyzed concentration of leptin receptor (double sandwich ELISA, standard recombinant human leptin), leptin, glucose, insulin, proinsulin, CRP and uric acid in the serum. The control group consisted of 103 probands without signs of atherosclerosis or other manifested diseases. The control group was subjected to determination of BMI, leptin and leptin receptor in the serum. Concentration of leptin receptor does not differ significantly between the patients with atherosclerosis and normal population. Probands with atherosclerosis showed a very close negative correlation between concentration of leptin receptor and leptinemia which is absent in normal population. PMID- 10743734 TI - The value of examining blood supply in prostate cancer using Doppler frequency analysis. AB - Stating the contribution of Doppler ultrasound examination (DU) of prostatic vascularisation towards timely diagnosing prostate cancer. 87 patients with bioptically proven prostate cancer and 10 healthy young men of control group were examined by transrectal ultrasound in B mode, colour flow mapping (CFM) and Doppler frequency analysis. (DFA) The distribution of vessels in prostate is not specific for carcinoma and using CFM, it is impossible to distinguish normal tissue from carcinoma. DU combined with other methods as rectal palpation, PSA, transrectal sonography in B mode may, however, increase carcinoma suspicion, and quicken the indication of considered bioptic puncture. PMID- 10743735 TI - Our experience with 3D CT. AB - The authors present the results of 9-year work with 3D CT in orthopaedics and musculoskeletal traumatology. In the years 1990-1998, the method was used in the examination of 59 patients (27 males, 20 females, and 12 children). The place of 3D CT in clarifying areas of complex musculoskeletal anatomy for operative treatment is discussed and presented as a contribution to the indication conference. According to the authors' experience, in many cases 3D CT increases the quality of diagnostics. Most of all, it helps the surgeon in operation planning. 3D reconstructions are particularly valuable in proximal humerus fractures, spine fractures and tumours, acetabulum fractures, as well as in conditions after hip dysplasia, LCPD and coxa vara adolescent. PMID- 10743736 TI - [Hematologic findings and value of value of endogenous creatinine in patients following kidney transplantation]. AB - We have studied serum erythropoietin (EPO) levels, serum creatinine and blood hemoglobin and hematocrit levels in the group of 140 patients after kidney transplantation. All patients received conventional immunosuppressive therapy consisting of cyclosporine (Sandimmune Neoral), azathioprin (Imuran) and steroids (Prednison). The aim of the study was to investigate the relationship between serum EPO, serum creatinine and hematologic parameters. Complete restoration of hemoglobin and hematocrit levels was obtained in 99 patients (70.7%), moderate degree of anaemia with average hemoglobin level 114.7 +/- 12.3 g/l was seen in 37 patients (26.4%) and four patients (2.9%) had posttransplant erythrocytosis. Normal hemoglobin values are generally restored during 3 months after successful renal transplantation. Statistical analysis confirmed significant correlation (p < 0.001) between graft function and hematologic parameters, but none correlation was found neither between serum EPO levels and serum creatinine and not between serum EPO levels and hemoglobin/hematocrit values. Plasma creatinine is not reliable indicator of renal EPO secretory capacity. The degree of correction of anemia is limited by the stage of graft function and correlates with the serum creatinine concentration. The results suggest, that the restoration of normal excretory graft function after transplantation improves bone marrow response to EPO and leads to normalization of erythropoietic activity. PMID- 10743737 TI - Aneurysm of the subclavian artery. AB - In this case report about a female patient with a true aneurysm of the left subclavian artery (SAA), the authors would like to present the symptoms, possibilities of diagnostics and management of this rarely appearing pathology. The diagnosis of a randomly found aneurysm in a symptom free patient was confirmed by spiral AGCT. Classical surgical procedure was indicated for the patient. With a combined supra- infraclavicular approach the partial resection of the aneurysm and its revascularization by a ePTFE prosthetic interposition were performed. A histological investigation showed nonspecific changes in the aneurysm wall. Two years after the operation the patient is without any complications and with complete functional vascular reconstruction and optimal limb function. PMID- 10743738 TI - [Contribution to the correction of central venous hypertension after the creation of an arteriovenous (AV) fistula for hemodialysis]. AB - Stenosis or thrombosis of the central venous tract (axillaris or subclavian vein) may be after creation of arteriovenous fistula for hemodialysis the cause of manifestation of venous hypertension. The authors evaluate positive bypass to internal jugular vein in the therapy of this complication. PMID- 10743739 TI - Anatomic prerequisites of the surgical therapy of vaginal prolapse after hysterectomy. AB - The prolapse of vaginal stub occurs in about 4% of hysterectomised patients. Suspension of vaginal stub on the ligamentum sacrospinale dextrum is an effective method of the surgical management. The vaginal approach puts increased demands on handiness and orientation in the operation field. Using a proper operation technique and perfect knowledge of the anatomic situation in fossa ischiorectalis can minimize the increased risk of bleeding during the fixation, which results from frequent and varied anastomoses of pelvic vessels. The authors measured several parameters on the group of pelvic girdles of 32 females and found following results: the distance between the base and the apex of spina ischiadica (17.1 mm); the distance between spina ischiadica and the lateral margin of the os sacrum in the axis of central fibres of ligamentum sacrospinale (29.8 mm); the length of axial fibres of ligamentum sacrospinale (37.2 mm); the longest (11.2 mm) and the shortest (1.3 mm) diameter of 15 mm from the top of spina ischiadica. In this site the thickness of muscular layer of musculus coccygeus (3.2 mm) has also been measured. The anatomic study can facilitate the introduction and implementation of more complex surgical techniques. PMID- 10743740 TI - Is it reasonable to use faecal occult blood test for colorectal cancer screening? AB - There is a high prevalence and incidence of colorectal cancer in industrial countries. It is the commonest cancer in women after breast cancer and in men after prostate and lung cancer and one of the most common causes of death from malignant disease. The cause of colorectal cancer is unknown, thus primary prevention programs could not be elaborated and the only occasion to reduce mortality is the use of screening to detect this cancer early. Faecal occult blood test (FOBT) was chosen for screening. Large population based trials included thousands of inhabitants of the United States and Europe to demonstrate: 1. Screening with FOBT Hemoccult II reduced mortality from colorectal cancer. 2. In the screening group the incidence of advanced stages of colorectal cancer decreased (Dukes' D stage). 3. The ideal balance between sensitivity and specificity revealed HemeSelect (immunodiagnosis of human haemoglobine) and combined test (Hemoccult II Sensa + HemeSelect). These two screening tests for colorectal cancer have not been verified by large population trials. 4. More sensitive guaiac tests decrease their own specificity and also lead to increase rate of expensive colon examinations. 5. The high-risk population for colorectal cancer ought to be submitted to screening. The problem is the detection of this group. 6. Patients' compliance to screening differs in various trials, but the percentage of patients undergoing FOBT evaluation decreases as the number of screenings increases. Therefore faecal occult blood test could be recommended for national programs of the screening for colorectal cancer. PMID- 10743741 TI - Miniinvasive approach to the treatment of liver cysts. AB - During the period from 1995 to 1997 we followed and treated six patients for liver cysts. Diagnostics was based not only on clinical examination but also on ultrasonography and computational tomography (CT). Surgery is applied in solving larger cysts damaging liver parenchyma and causing pressure symptomatology. Nowadays most cysts can be solved by means of laparoscopy. PMID- 10743742 TI - Analysis of chemokine gene expression in lung cells by polymerase chain reaction. AB - The article describes a polymerase chain reaction (PCR) based method for semiquantitative assessment of mRNA expression for chemotactic cytokines in the lower respiratory tract cells. Cytokine mRNA was detected using PCR preceded by reverse transcription of mRNA into cDNA. The procedures of RNA extraction and of reverse transcription-PCR assay are described in the first part of the article. The second part demonstrates optimalisation experiments performed to ensure specificity, accuracy and reproducibility of the RT-PCR assay and discuss the approach to mRNA quantification using normalisation of cytokine mRNA expression to the expression of beta-actin mRNA. PMID- 10743743 TI - Single photon bone densitometry in hemodialysis patients. AB - Renal osteodystrophy is a common finding in patients with renal insufficiency. The maximum of its intensity is found in hemodialysis patients. Bone densitometry is so far the best method for non-invasive assessment of the extent of the illness. Some densitometric studies in hemodialysis patients have already been published but their results differ in prevalence and intensity of renal osteodystrophy. They also demonstrated a slight relationship between intensity of renal osteodystrophy and duration of the dialysis treatment. Opinions vary on the relationship between bone mineral density and markers of bone turnover. This cross-sectional study found high prevalence of renal osteodystrophy (Z-score below -1 in 57% of patients) as well as high a number of severely damaged patients (T-score below -2.5 in 40% of patients). It also showed some correlation between bone demineralisation and the duration of dialysis. None from evaluated markers of bone turnover correlated with bone mineral density. PMID- 10743744 TI - The sequence of reticularisation of epithelium of human palatine tonsil: scanning electron microscopic study. AB - The changes appearing during the development of the human palatine tonsil, both in the surface and in the tonsillar crypt epithelium, were studied from samples of the 16th to 40th prenatal weeks. From the 18th week, we observed areas of mesh like epithelium in an originally homogeneous stratified squamous epithelium, i.e. the reticularisation began in the tonsillar crypt epithelium. The important role of the epithelial basal membrane in this process is discussed. The diffuse spongy form type of reticularisation as well as the formation of the intraepithelial passages between the epithelial columns are described. The lymphoid cells and macrophages migrated through these microcrypts and they were frequently seen near the crypt epithelial micropores on the surface of the reticularised epithelium. PMID- 10743745 TI - Miniinvasive approaches in varicose vein surgery. AB - Authors present their own experience with the treatment of chronic venous insufficiency. Between August 1995 and November 1998 they operated on 318 patients for the disease of venous system. In 28 patients (i.e. 9%) they performed 30 miniinvasive interventions--8x retroperitoneoscopic lumbar sympathectomy and 22x endoscopic subfascial dissection of vein perforators. The rest of patients (91%) were treated by means of classical operations on superficial venous system. PMID- 10743746 TI - Perinatal regionalization: old lessons for the new millennium. PMID- 10743747 TI - Osteoporosis: a factor in mortality following cardiac surgery. AB - Osteoporosis may be a significant factor in unfavorable results following open cardiac surgery. This brief case report describes the fatal outcome of mitral valve replacement in an elderly woman with congestive heart failure complicated by severe osteoporosis. Profound collapse of the thoracic spine resulted in pulmonary insufficiency. The discussion includes substantiation that this phenomenon is not unusual but rather can be expected to occur in postmenopausal women who are inactive, not receiving calcium and vitamin D supplements, and are suffering from congestive heart failure for which loop diuretics are administered. Bone mineral density testing is essential to determine and track the efficacy of treatment. PMID- 10743748 TI - Glaucoma and race: a case for screening in Connecticut. AB - This study is a review of the current literature regarding the relationship of primary open angle glaucoma and race. There is strong evidence that blacks have a much higher prevalence rate of this disease. They also tend to have a more severe clinical course. Glaucoma is the leading cause of irreversible blindness in blacks. The number of blacks in Connecticut with glaucoma is estimated to be between 14,000 and 21,000, and it is estimated that up to half of these cases are currently undetected. New technology has made screening for glaucoma more practical and cost-effective. This study suggests that glaucoma screening would be beneficial for the black population of Connecticut. PMID- 10743749 TI - Diagnosis and management of spinal epidural space extravasation complicating percutaneous central venous line placement in a premature infant: case report and review of literature. AB - Percutaneous central venous lines are commonly used to establish long-term venous access in the care of premature infants. Misplacement of these catheters can occur and may lead to significant morbidity and mortality. Here we report a very low-birth-weight premature infant whose percutaneous central venous line was inadvertently placed into the spinal epidural space. The anatomical basis of this complication as well as a comprehensive review of literature are provided. PMID- 10743750 TI - Golf course injuries. PMID- 10743751 TI - How much care is good for us? Surely there are limits! PMID- 10743752 TI - Unfair trade practices. PMID- 10743753 TI - Protecting medicine's professionalism. PMID- 10743754 TI - Radical mastoidectomy cavity with debris and exposed middle ear mucosa. PMID- 10743755 TI - Vocal fold cyst, hemorrhage, and scar in a professional singer. PMID- 10743756 TI - Polypoid change of the uncinate process. PMID- 10743757 TI - Electronystagmography in a patient with a peripheral vestibular disorder. PMID- 10743758 TI - Magnetic resonance imaging in lymphomatous adenopathy of the neck. PMID- 10743759 TI - A method for detecting errors in discounted fee-for-service payments by insurance companies. AB - In order to discover and correct inaccurate payments received from insurance companies, a practice requires a plan for the systematic, efficient, and prompt monitoring of receipts. When any such discrepancies are detected, the physician can query the insurance company and correct the problem. Once the system is in place, it behooves each physician to look at the results at least weekly. Monitoring receipts requires interest, know-how, and persistence in the face of ever-confusing payment methodologies. This paper explains one such plan for detecting and correcting inaccurate insurance company payments. PMID- 10743760 TI - Otorhinolaryngology emergency unit care: the experience of a large university hospital in Italy. AB - We recorded the symptoms and disposition of every patient who visited the otorhinolaryngology emergency department at our hospital in Rome, Italy, during 1996. During that time, our ENT specialists saw 21,548 patients. Only 311 of these patients (1.4%) required immediate hospitalization, while another 2,391 patients (11.1%) received treatment and were released. The other 18,846 patients (87.5%) did not have any pathology or condition that qualified as an actual emergency, and they were examined and released, often with a prescription or instructions for home care. These patients could have easily been treated by a family physician. The fact that emergency care in Italy is rendered free of charge (unlike ambulatory care, for which fees are charged) provides patients with a strong incentive to misuse the system. Such overutilization drives up the cost of health care and stretches the capacity of the medical staff. Steps need to be taken to redirect patients who misuse emergency services to seek medical care in ambulatory care centers. PMID- 10743761 TI - Granular cell tumors arising nearly simultaneously in the larynx and subcutaneous cervical region. AB - Granular cell tumors are benign, relatively rare lesions that can occur in any organ in the body. Tumors are known to occasionally arise in two or more different organs. We report the case of a 60-year-old woman in whom granular cell tumors arose almost simultaneously in her subcutaneous cervical region and her larynx. The two tumors were surgically resected and examined histopathologically, and they exhibited the same histopathologic features. The tumor of the larynx was resected with the help of a carbon dioxide laser. Immunohistochemical staining revealed that both were positive for S-100 protein and neuron-specific enolase. Cases of the nearly simultaneous appearance of granular cell tumors in the neck and larynx are rather unusual. The results of immunohistochemical staining suggested that this tumor originates in nerve tissue. PMID- 10743763 TI - Description of an office technique for laser ventilation of the ears. AB - The author presents a description of a technique he developed called laser office ventilation of ears (LOVE). LOVE, an office-based procedure performed under local anesthesia, can provide intermediate-term ventilation for middle ear disease. This procedure has potential to change the standard of care for otitis media. Such changes might include a decrease in the need for antibiotics (and thus antibiotic resistance), a decrease in hearing loss associated with otitis media, and a decrease in the overall cost of treatment. PMID- 10743762 TI - Acanthamoeba sinusitis with subsequent dissemination in an AIDS patient. AB - Otolaryngologists can play an important role in the care of patients with acquired immunodeficiency syndrome (AIDS) and/or human immunodeficiency virus infection. We present the case of an AIDS patient who was hospitalized for dehydration and who was soon found to have sinusitis and subsequent disseminated infection caused by Acanthamoeba. To treat the Acanthamoeba infection, the patient was started on oral itraconazole and intravenous metronidazole; i.v. pentamidine was added 2 days later. Despite aggressive therapy, on the eleventh day of hospitalization, the patient was obtundent and provided minimal response to noxious stimuli. He died on the sixteenth day of hospitalization. This case is one of only six reported cases of Acanthamoeba associated with sinusitis. Current therapeutic regimens have not been successful for most of these patients, and the prognosis is poor. PMID- 10743764 TI - Spondyloepiphyseal dysplasia congenita associated with conductive hearing loss. AB - Spondyloepiphyseal dysplasia is a disorder characterized by abnormalities of growth. Previous studies of this disorder have identified a significant incidence of associated hearing loss. Hearing loss has been reported to occur in 25 to 30% of affected patients. To date, all reports of associated hearing loss have indicated the presence of a sensorineural component. In this article, we report the case of a child who was diagnosed with spondyloepiphyseal dysplasia congenita and who was found to have a significant conductive hearing loss with a Carhart's notch, indicating the likely presence of stapes footplate fixation. We also review the diagnosis of this condition and the literature associated with hearing loss as it occurs with this disorder. PMID- 10743765 TI - Yolk sac tumor of the temporal bone: report of a case. AB - Yolk sac tumor (endodermal sinus tumor) is rarely encountered in the temporal bone. Facial nerve paralysis can be a primary manifestation of this condition. Histologically, the tumor can be difficult to diagnose, although elevated levels of alpha fetoprotein can facilitate its identification. In this report, we describe the case of an 18-month-old girl who developed peripheral VIIth nerve palsy and a polypoid mass in the left external ear canal 3 months following myringotomy. Computed tomography and magnetic resonance imaging revealed that the tumor involved the left external ear canal, middle ear space, and mastoid air cells. Biopsies were consistent with a yolk sac tumor. Special staining demonstrated that only a very few tumor cells were positive for alpha fetoprotein, despite the markedly elevated level of alpha fetoprotein in her serum. The patient was treated with chemotherapy, which included cisplatin, etoposide, and bleomycin. Within a period of weeks, she experienced a complete reversal of her left VIIth nerve palsy, a marked decrease in her serum alpha fetoprotein levels, and a dramatic resolution of the tumor as demonstrated radiographically. Such a successful chemotherapeutic response in this case argues against surgical intervention in other cases, particularly in view of the risk of serious complications with surgery. PMID- 10743766 TI - Kimura's disease: a case report. AB - Kimura's disease is a rare, idiopathic condition that usually affects young men of Asian descent. The decrease is characterized by swelling and lesions in the head and neck region, with involvement of the subcutaneous soft tissue, major salivary glands, and lymph nodes. Patients almost always have eosinophilia and elevated serum immunoglobulin E levels. The diagnosis is established by biopsy. Kimura's disease is usually self-limiting. Its etiology is unknown but is thought to be a manifestation of an aberrant allergic response. In this paper, we describe the case of a 30-year-old patient who was diagnosed with Kimura's disease at our institution. PMID- 10743767 TI - Basal cell carcinoma of the head and neck: identification of predictors of recurrence. AB - The goal of this retrospective study was to identify factors that predict the recurrence of basal cell carcinoma of the head and neck. We reviewed the medical records of 165 patients who had undergone a wide surgical excision (negative margins) of one or more basal cell carcinomas of the head and neck. Univariate analysis revealed that recurrence was significantly influenced by the patient's gender (p < 0.01), the presence of preoperative risk factors (p < 0.05), the presence of multiple lesions (p < 0.01), and their histopathologic subtype (p < 0.05). Multivariate analysis revealed that the best predictors of recurrence were the presence of preoperative risk factors and the presence of multiple lesions (p < 0.01); the lesion's histologic subtype approached but did not reach statistical significance in predicting recurrence (p = 0.06). PMID- 10743768 TI - Peritonsillar abscess: the rationale for interval tonsillectomy. AB - Although peritonsillar abscess (quinsy) and peritonsillitis are common ENT emergencies, management strategies in the United Kingdom still vary among otolaryngologists. In order to obtain data on the success of the various strategies, we conducted two surveys--one concerned itself with patient outcomes, while the other sought information on physician preferences. The survey of 571 practicing ENT surgeons revealed that 83% advise interval tonsillectomy only for patients who have a history of tonsillitis; they prefer to take a wait-and-see approach for a single attack of quinsy. Conversely, 15% advise a routine interval tonsillectomy following even a single isolated attack of quinsy/peritonsillitis. Only 6.8% still perform a quinsy tonsillectomy in selected cases. Survey responses from 192 adults and 15 children who had been hospitalized for the treatment of quinsy/peritonsillitis revealed that the vast majority of patients who did not undergo an interval tonsillectomy were still asymptomatic 2 to 8 years later. These results indicate that a wait-and-see policy is indeed suitable for most patients who present with an isolated attack of quinsy/peritonsillitis without a history of tonsillitis. We recommend that tonsillectomy be performed as a definitive treatment for quinsy/peritonsillitis in patients who have a history of tonsillitis. Such a history is a reliable indicator of recurrent quinsy or tonsillitis following an attack of quinsy/peritonsillitis in both children and adults. Quinsy tonsillectomy should be reserved for those few patients who do not respond to conservative measures. PMID- 10743769 TI - A high-yield microassembly structure for three-dimensional microelectrode arrays. AB - This paper presents a practical microassembly process for three-dimensional (3-D) microelectrode arrays for recording and stimulation in the central nervous system (CNS). Orthogonal lead transfers between the micromachined two-dimensional probes and a cortical surface platform are formed by attaching gold beams on the probes to pads on the platform using wire-free ultrasonic bonding. The low-profile (150 microns) outrigger design of the probes allows the bonding of fully assembled high-density arrays. Micromachined assembly tools allow the formation of a full 3 D probe array within 30 min. Arrays having up to 8 x 16 shanks on 200-micron centers have been realized and used to record cortical single units successfully. Active 3-D probe arrays containing on-chip CMOS signal processing circuitry have also been created using the microassembly approach. In addition, a dynamic insertion technique has been explored to allow the implantation of high-density probe arrays into feline cortex at high-speed and with minimal traumatic injury. PMID- 10743770 TI - Long-term maintenance of patterns of hippocampal pyramidal cells on substrates of polyethylene glycol and microstamped polylysine. AB - For neurons to attach and remain in precise micropatterns for weeks in culture, background molecules that remain nonpermissive for extended culture durations need to be identified. Nonpermissive background molecules of either polyethylene glycol (PEG) or the amino acid serine (C3H7NO3) were evaluated. The foreground regions were microstamped with 3-, 5-, or 10-micron lines of poly-D-lysine (PDL), which promotes neural attachment and growth. After 29 days in culture the foreground compliance, or the fraction of all live somata which rested on the desired PDL surface, averaged 86% for serine and 90% for PEG, with only a small decline. The background compliance, or the fraction of square areas in the pattern background which were free of neurite extension, declined from highs of 40% and 55% (midculture) to 5.5% and 12% (29 days) for serine and PEG, respectively. Images of the cultures suggest that PEG is significantly more effective as a nonpermissive substrate. We conclude that these materials, especially PEG, are adequate for the maintenance of long-term patterned cultures of neurons. We believe that this is the first report of high-quality long-term patterning of cultured neurons. PMID- 10743771 TI - Laser induced fluorescence attenuation spectroscopy: detection of hypoxia. AB - The development of a new laser-induced fluorescence (LIF) spectroscopy technique for the measurement of the attenuation spectrum of tissue is described. The technique, termed laser-induced fluorescence attenuation spectroscopy (LIFAS), has been applied to study the effects of hypoxia on the in vivo optical properties of renal and myocardial tissue in the 350-600-nm band. Excimer laser (Xe-Cl) is used to excite a small volume of the tissue (rabbit model, N = 20) and induce autofluorescence. The emitted LIF is monitored fiberoptically at two locations that are unevenly displaced about the fluorescing volume. The optical attenuation of the tissue is calculated from the dual LIF measurements by assuming an exponential decay of the fluorescence with distance. The results indicate that hypoxia modulates the attenuation spectrum leading to characteristic changes in its shape. Primarily, the spectral profile becomes more concave between 455 nm and 505 nm and two spectral peaks at about 540 and 580 nm disappear leaving in their place a single peak at about 555 nm. The attenuation spectra of normoxic and hypoxic tissue are used to train partial least squares multivariate model for spectral classification. The model detected acute renal and myocardial hypoxia with an accuracy greater than 90% (range: 90%-96%) and 74% (range: 74%-90%), respectively. PMID- 10743772 TI - Effect of length on the fundamental resonance frequency of arterial models having radial dilatation. AB - The pressure wave moving along an elastic artery filled with blood was examined as a moving Windkessel having a natural oscillation angular frequency nu 0 and a damping coefficient b. The radial directional motion for an element of the wall segment and the adherent fluid was considered. This equation was solved with conditions at both ends of an artery of length L. An external impulse force was applied at one end and a static pressure Po at the other. Analytic solution allowed only certain oscillation modes of resonance frequencies fn, where fn2 = a + cnL-2 with [formula: see text] and V infinity is the high frequency phase velocity. The relationship between f0 and L was examined experimentally for tubes constructed of latex, rubber, or dissected aorta. The effect of raising the static pressure P0 or increasing the tension in the tube was consistent with the prediction. The hypertension that accompanies an augmentation in arterial wall and the association between the heart rate and the mean blood pressure were discussed. PMID- 10743773 TI - Hemodynamic mechanisms of penile erection. AB - A model of penile hemodynamics was developed to explain the process of erection that is not yet fully understood. Contradicting observations regarding blood flow and vessels occlusion during erection were examined. The model that was based on the physical structure and physiological function of the system was validated by comparing its predictions to clinical and experimental observations. Simulation of the process for both normal and pathological conditions indicates that pressure buildup in the corpus cavernosum during erection depends mainly on the interaction between the arterial inflow system and the venous draining system and that the venous draining vessels do not fully collapse and flow through the penis continues throughout the erection period. In pathological conditions, the model predicts that tumescence can be obtained without functional rigidity and demonstrates that small increases in vessel stiffness can result in such behavior. PMID- 10743774 TI - Spatial regularization of the electrocardiographic inverse problem and its application to endocardial mapping. AB - Numeric regularization methods for solving the inverse problem of electrocardiography in realistic volume conductor models have been mostly limited to uniform regularization in the spatial domain. A method of spatial regularization (SR) was developed and tested in canine, where each spatial spectral component of the volume conductor model was considered separately, and a SR operator was selected based on explicit a posteriori criterion at each time instant through the heartbeat. The inverse problem was solved in the left ventricle by reconstructing endocardial surface electrograms based on cavitary electrograms measured with the use of a noncontact, multielectrode probe. The results were validated based on electrograms measured in situ at the same endocardial locations using an integrated, multielectrode basket-catheter. A probe-endocardium three-dimensional model was determined from multiplane fluoroscopic images. The boundary element method was applied to solve the boundary value problem and derive the relationship between endocardial and probe potentials. Endocardial electrograms were reconstructed during both normal and paced rhythms using SR as well as standard, uniform, zeroth-order Tikhonov (ZOT) regularization. Compared to endocardial electrograms measured by the basket, electrograms reconstructed using SR [relative error (RE) = 0.32, correlation coefficient (CC) = 0.97, activation error = 3.3 ms] were superior to electrograms reconstructed using ZOT regularization (RE = 0.59, CC = 0.79, activation error = 4.9 ms). Therefore, regularization based on spatial spectral components of the model improves the solution of the inverse problem of electrocardiography compared to uniform regularization. PMID- 10743775 TI - Pulse oximetry: an improved in vitro model that reduces blood flow-related artifacts. AB - Artifacts may occur in many in vitro models of pulse oximetry due to the optical effects of synchronously oriented and/or deformed erythrocytes. Although these artifacts are most likely negligible in living superficial tissues, they are demonstrated to have considerable influence on the calibration curve obtainable from the in vitro simulation of pulse oximetry in such models, especially at low oxygen saturations. Therefore, we have developed a modified in vitro model which reduces the effect of these artifacts. This is achieved by excluding data obtained during pressure transients and by raising the blood flow velocity. As a result, the model more closely approximates in vivo pulse oximetry, particularly under clinically important conditions of low blood oxygen saturation levels. PMID- 10743776 TI - Effects of mechanical stress/strain and estrogen on cancellous bone structure predicted by fuzzy decision. AB - A theoretical model was developed on the basis of fuzzy decision to predict cancellous bone structure following changes in mechanical stress/strain and estrogen. The model was validated experimentally by simulation of the normal structure of a rat distal femur, and further used to predict the structural alterations following ovarian hormone deficiency. The results show that net bone resorption after ovariectomy occurs in the metaphysis at locations where trabecula were subjected to the lowest mechanical stress/strain. These findings, consistent with experimental results, suggest that estrogen deficiency increase the mechanostat set point at which bone cells perceive mechanical stress/strain. Additionally, the results show that changes in bone architecture which are due to alterations in bone remodeling can be simulated by fuzzy decision without precise mathematical description, and multiple factors can also be readily incorporated into the model. PMID- 10743777 TI - Bispectrum analysis of focal ischemic cerebral EEG signal using third-order recursion method. AB - In this paper, a model for Sprague-Dawley (SD) rat focal ischemic cerebral injury is presented. Based on this experimental model, the electroencephalogram (EEG) from the ischemic region and from a normal region are collected during the first 30 min of ischemia. The EEG bispectrum analysis is carefully investigated by using the third-order recursion method. We found that some characteristics of the bispectrum are very sensitive to focal ischemic cerebral injury. The maximum magnitude and the weighted center of EEG bispectrum (WCOB) change according to the extent and the place of the injury region. The bispectrum analysis results have been verified by the heat shock protein (HSP) test. The study indicates that the EEG bispectrum analysis may be useful to distinguish the ischemic region from the normal one and to estimate the ischemic extent. PMID- 10743778 TI - Wavelet-based noise removal for biomechanical signals: a comparative study. AB - The purpose of this paper is to present wavelet-based noise removal (WBNR) techniques to remove noise from biomechanical acceleration signals obtained from numerical differentiation of displacement data. Manual and semiautomatic methods were used to determine thresholds for both orthogonal and biorthogonal filters. This study also compares the performance of WBNR approaches with four automatic conventional noise removal techniques used in biomechanics. The conclusion of this work is that WBNR techniques are very effective in removing noise from differentiated signals with sharp transients while leaving these transients intact. For biomechanical signals with certain characteristics, WBNR techniques perform better than conventional methods, as indicated by quantitative merit measures. PMID- 10743779 TI - Detection of characteristic waves of sleep EEG by neural network analysis. AB - In psychiatry, the sleep stage is one of the most important evidence for diagnosing mental disease. However, doctors require much labor and skill for diagnosis, so a quantitative and objective method is required for more accurate diagnosis since it depends on the doctor's experience. For this reason, an automatic diagnosis system must be developed. In this paper, we propose a new type of neural network (NN) model referred to as a sleep electroencephalogram (EEG) recognition neural network (SRNN) which enables us to detect several kinds of important characteristic waves in sleep EEG which are necessary for diagnosing sleep stages. Experimental results indicate that the proposed NN model was much more capable than other conventional methods for detecting characteristic waves. PMID- 10743780 TI - Noninvasive estimation of motor unit conduction velocity distribution using linear electrode arrays. AB - Determining the conduction velocity of motor unit action potentials is one of the most important problems in surface electromyography. The estimate of one average conduction velocity value depends on a variety of uncontrollable factors. More meaningful information is obtained from the estimation of the distribution of the different delays in the myoelectric signals. A solution to the problem is the separation and characterization of the individual components propagating at different velocities. A technique, based on surface electrode array recording, is proposed to estimate motor unit conduction velocity distribution. The method consists in the identification of the single action potentials in the time scale domain (with the continuous wavelet transform) and in the estimation of their conduction velocities based on the beamforming algorithm. The performances of the technique have been evaluated using simulated and real myoelectric signals. The results demonstrate that the technique is accurate and reliable. The method may be useful for the diagnosis of neuromuscular disorders, for the monitoring of muscle fatigue and for noninvasive investigation of individual motor units. PMID- 10743781 TI - Nonlinear adaptive filtering of stimulus artifact. AB - Noninvasive measurements of somatosensory evoked potentials have both clinical and research applications. The electrical artifact which results from the stimulus is an interference which can distort the evoked signal, and introduce errors in response onset timing estimation. Given that this interference is synchronous with the evoked signal, it cannot be reduced by the conventional technique of ensemble averaging. The technique of adaptive noise cancelling has potential in this regard however, and has been used effectively in other similar problems. An adaptive noise cancelling filter which uses a neural network as the adaptive element is investigated in this application. The filter is implemented and performance determined in the cancelling of artifact for in vivo measurements on the median nerve. A technique of segmented neural network training is proposed in which the network is trained on that segment of the record time window which does not contain the evoked signal. The neural network is found to generalize well from this training to include the segment of the window containing the evoked signal. Both quantitative and qualitative measures show that significant stimulus artifact reduction is achieved. PMID- 10743782 TI - Variability in EMF permittivity values: implications for SAR calculations. AB - Digital anatomical models of man and animals are available for use in numerical calculations to predict electromagnetic field (EMF)-induced specific absorption rate (SAR) values. To use these models, permittivity values are assigned to the various tissues for the EMF frequencies of interest. There is, as yet, no consensus on what are the best permittivity data. This study analyzed the variability in published permittivity data and investigated the effects of permittivity values that are proportional on SAR calculations. Whole-sphere averaged and localized SAR values along the diameter of a 4-cm sphere are calculated for EMF exposures in the radio frequency range of 1 MHz to 1 GHz. When the dimensions of a sphere are small compared to the wavelength (i.e., wavelength inside the material is greater than ten times the dimensions of the object), the whole-sphere averaged SAR is inversely proportional to the permittivity of the material composing the sphere. However, the localized SAR values generally do not have the same relation and, as a matter of fact, vary greatly depending on the location within the sphere. These results indicate that care must be taken in choosing the permittivity values used in calculating SAR values and some estimate of the dependence of the calculated SAR values on variability in permittivity should be determined. PMID- 10743783 TI - A box coil for the stimulation of biological tissue and cells in vitro and in vivo by pulsed magnetic fields. AB - An alternative coil system to the Helmoholtz coil-pair is described for the stimulation of biological tissue and cells: a relatively large box coil made of copper or aluminum sheet stock. The design is based on the principal determinant of the induced electric field, namely, the magnetic vector potential (A), in the equation, [formula: see text]. The second term in the equation is needed when boundaries of the conducting medium are in close proximity to the region of interest, such as in a culture dish. An electric surface charge builds up on the boundaries to generate an electric field which cancels [formula: see text] at the surface. The effectiveness of the new coil is demonstrated in a study of the outgrowth enhancement of axons from rat embryonic dorsal root ganglia. PMID- 10743784 TI - Noninvasive feature-based detection of delayed gastric emptying in humans using neural networks. AB - Radioscintigraphy is currently the gold standard for gastric emptying test which involves radiation exposure and is considerably expensive. We present a feature based detection approach using neural networks for the noninvasive diagnosis of delayed gastric emptying from the cutaneous electrogastrogram (EGG). Simultaneous recordings of the EGG and scintigraphic gastric emptying test were made in 152 patients with symptoms suggestive of delayed gastric emptying. Spectral analyses were performed to derive EGG parameters which were used as the input of the neural network. The result of scintigraphic gastric emptying was used as the gold standard for the training and testing of the neural network. A correct classification of 85% (a specificity of 89% and a sensitivity of 82%) was achieved using the proposed method. PMID- 10743785 TI - A new video-synchronized multichannel biomedical data acquisition system. AB - This data acquisition system records video frames onto a video tape, and simultaneously acquires biomedical data along with video time codes onto a computer hard disk to achieve a 30-min video-synchronized data recording with a summed data rate of 2.16 Mbit/s. A time-code-bridge-file created during acquisition matches each video frame-start with the corresponding index number of the acquired data. The mean synchronization accuracy of the system is 0.22 ms. PMID- 10743786 TI - Multivariate dynamic analysis of cerebral blood flow regulation in humans. AB - The contributions of beat-to-beat changes in mean arterial blood pressure (MABP) and breath-by-breath fluctuations in end-tidal CO2 (EtCO2) as determinants of the spontaneous variability of cerebral blood flow velocity (CBFV) were studied in 16 normal subjects at rest. The two input variables (MABP and EtCO2) had significant cross-correlations with CBFV but not between them. Transfer functions were estimated as the multivariate least mean square finite impulse response causal filters. MABP showed a very significant effect in explaining CBFV variability (p < 10(-11), Fisher's aggregated-p test) and the model mean square error was significantly reduced (p < 0.001) by also including the contribution of EtCO2. The estimated mean CBFV step response to MABP displayed the characteristic return to baseline caused by the cerebral autoregulatory response. The corresponding response to EtCO2 showed a gradual rise taking approximately 10 s to reach a plateau of 2.5%/mmHg. This study demonstrated that spontaneous fluctuations in EtCO2 can help to explain the CBFV variability at rest if appropriate signal processing techniques are employed to address the limited power and bandwith of the breath-by-breath EtCO2 signal. PMID- 10743787 TI - Is rheumatoid arthritis a form of reactive arthritis? PMID- 10743788 TI - Infection and rheumatoid arthritis: guilt by association? PMID- 10743789 TI - Friction and lubrication in synovial joints. PMID- 10743790 TI - Does smoking influence disease outcome in patients with rheumatoid arthritis? PMID- 10743791 TI - The predictive value of the HLA shared epitope for severity of radiological joint damage in patients with rheumatoid arthritis. A 10 year observational prospective study. AB - OBJECTIVE: To evaluate the clinical importance of the RA "shared epitope" (SE) as a prognostic marker of radiological severity and extension of the disease to large joints in patients with rheumatoid arthritis (RA). METHODS: Eighty-two patients who met the American College of Rheumatology criteria for RA with a disease duration < 2 years at presentation were included in the study. Radiographs of hands, wrists, feet, shoulders, elbows, hips, and knees were taken at study entry and 8-10 years later. HLA-DRB1 genotypes were determined by polymerase chain reaction-sequence-specific oligonucleotide probes. Radiological severity was assessed in small joints of hands, wrists, and feet. Extension of the disease to large joints was evaluated in radiographs of shoulders, elbows, hips, and knees. RESULTS: At the end of the study, erythrocyte sedimentation rate (ESR), rheumatoid factor (RF) titer, and percentage of maximum radiological score at baseline were significantly associated with RA severity (p < 0.01, p < 0.01, p < 0.05, respectively). Extension of RA to large joints was related to a higher ESR (p < 0.001) and a lower hemoglobin level (p < 0.01) at baseline. Neither at entry nor at the end of the study was the RA shared epitope predictive for radiologic severity or extension of disease to large joints (p = 0.8, p = 0.3, respectively). CONCLUSION: Presence of the SE in patients with RA is not a good prognostic marker of radiological severity after a mean followup period of 9 years. PMID- 10743792 TI - IgG antibodies to type II collagen reflect inflammatory activity in patients with rheumatoid arthritis. AB - OBJECTIVE: To determine the clinical significance of IgG antibodies to type II collagen (CII) and to define any correlation of antibodies to CII with the inflammatory response in patients with rheumatoid arthritis (RA). METHODS: IgG antibodies to native human type II collagen (IgG anti-CII) were measured in sera and synovial fluid (SF) from patients with RA, patients with osteoarthritis (OA), and healthy controls by an improved ELISA. Demographic, clinical, and laboratory data including tumor necrosis factor-alpha (TNF-alpha) and interleukin 6 (IL-6) levels were also obtained at the time of sampling in patients with RA. RESULTS: The median level and positivity for circulating IgG anti-CII were higher in patients with RA (n = 297) than patients with OA (n = 34) and healthy controls (n = 50) (p < 0.001). The titers of IgG anti-CII in SF were also higher in RA (n = 45) than in OA (n = 16) (p < 0.001). In paired samples, the levels of IgG anti CII were significantly higher in SF compared to the sera in patients with RA (n = 45) (p < 0.001), but levels were not different in patients with OA (n = 16). Circulating IgG anti-CII converted from positive to negative in 13 patients (10.7%) and from negative to positive in 18 patients (14.8%) among 122 patients with RA in whom IgG anti-CII were monitored sequentially at a mean interval of 12.2 months. IgG anti-CII positive patients (n = 98) had shorter disease duration (p = 0.04) and less frequent deformity (p = 0.013), and higher median erythrocyte sedimentation rate (ESR) (p = 0.001) and C-reactive protein (CRP) (p < 0.001) than IgG anti-CII negative patients (n = 120). The levels of IgG anti-CII correlated with CRP (r = 0.270) and ESR (r = 0.253). CRP decreased significantly in patients (n = 13) who converted from IgG anti-CII positive to negative (p = 0.013). IgG anti-CII positive patients (n = 40) had higher levels of TNF-alpha and IL-6 than negative patients (n = 40) (p < 0.001). Levels of IgG anti-CII correlated well with TNF-alpha (r = 0.617) and IL-6 (r = 0.347). CONCLUSION: Increased IgG anti-CII in sera and SF in RA correlated directly with acute phase reactants and the proinflammatory cytokines TNF-alpha and IL-6. Our data suggest that IgG anti-CII could reflect inflammatory activity with a potential to destroy cartilage in the early stages of RA. PMID- 10743793 TI - Taxol (paclitaxel) involution of articular cartilage destruction in collagen induced arthritis: an ultrastructural demonstration of an increased superficial chondroprotective layer. AB - OBJECTIVE: To determine the ultastructural changes of Taxol (paclitaxel) involution of articular cartilage destruction in collagen induced arthritis (CIA) and to compare with articular cartilage from normal rats. METHODS: Forty-five Louvain rats were randomized to one of 3 protocols for structural analysis: (1) control group, (2) CIA group, and (3) Taxol treated CIA group. The latter group received 10 mg/kg body weight of Taxol at Days 10, 12, and 14 and 7.5 mg/kg body weight of Taxol on Days 16, 18, and 20 postimmunization with collagen type II. Eight days later, each group was examined by light microscopy and scanning and transmission electron microscopy. RESULTS: In Taxol treated rats, the morphology of the articular cartilage reverted to that observed in naive rats except for a striking increase in the thickness of the superficial amorphous layer covering the articular surface. CONCLUSION: The involution of CIA by Taxol suggests that this agent may be useful in the clinical treatment of RA. PMID- 10743794 TI - Accumulation of T cells reactive to type II collagen in synovial fluid of patients with rheumatoid arthritis. AB - OBJECTIVE: To determine if type II collagen (CII) reactive T lymphocytes selectively accumulate in the inflamed joint of patients with rheumatoid arthritis (RA) and to study the specificity of the CII reactive cells. METHODS: Synovial fluid (SF) cells or peripheral blood lymphocytes were cultured with interleukin 2 (IL-2) for 24 h and then cultured at limiting dilution concentrations in the presence of filler cells and IL-2. The outgrowing cell lines were screened for their responses to CII. The percentages of the CII reactive T cells from SF were compared with those from peripheral blood of patients with RA. The CII reactive T cell lines were tested for their responses to different types of collagen. RESULTS: CII reactive T cell lines were identified in the SF of 3 RA patients; the frequencies were 5.0% (11/219), 3.7% (5/134), and 3.5% (3/86), respectively. In contrast, none of CII-specific T cell lines were identified in peripheral blood of the patients. The T cell lines recognized both human and bovine CII and, to a lesser extent, type I collagen. CONCLUSION: CII reactive T cells are present in high frequency in the inflamed joint of patients with RA, where they may play an important role in the pathogenesis of RA. PMID- 10743795 TI - Lubricin is a product of megakaryocyte stimulating factor gene expression by human synovial fibroblasts. AB - OBJECTIVE: The boundary lubricating ability of human synovial fluid has been attributed to lubricin, a mucinous glycoprotein. We investigated the primary structure of lubricin and its cellular origin. METHODS: Lubricin was purified from pooled synovial fluid aliquots with normal lubricating activity obtained from patients with osteoarthritis. Lubricating ability of lubricin was assayed in a friction apparatus that oscillates natural latex against a ring of polished glass. Native and lubricin deglycosylated with O-glycosidase DS and NANase III were trypsinized and sequenced by liquid chromatography mass spectrometry. Sequence results were compared to known structures in GenBank. Sequence data from strong matches were used in creating cDNA primers for reverse transcription polymerase chain reaction (RT-PCR) with RNA from human synovial fibroblasts obtained intraoperatively. RESULTS: Purified lubricin possesses an apparent molecular weight of 280 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Deglycosylation decreased the apparent molecular weight on SDS-PAGE to 120 kDa. Sequences specific for megakaryocyte stimulating factor precursor (MSF) were identified in GenBank. A 100% match was observed for exons 6 though 9 of MSF. Lubricin/MSF reduced the coefficient of friction (m) in the latex:glass bearing from 0.131 to 0.047. MSF is 1404 amino acids in size with multiple functional domains similar to vitronectin. The reported structure of MSF contains a centrally located mucin (exon 6) with 76 repeats of the degenerate motif of KEPAPTT, the presumed site of extensive O-linked glycosylation. RT-PCR with primers complementary for Pro214- Ala307 in exon 6 and RNA from human synovial fibroblasts produced the predicted product size of 280 bp. CONCLUSION: Lubricin is secreted by synovial fibroblasts via expression of the MSF gene. Lubricin is constructed of MSF exons 6 through 9 but the presence of other exons cannot be excluded. Lubricin/MSF is the only lubricating component in the final lubricating fraction of human synovial fluid. PMID- 10743796 TI - Phase I/II trial of recombinant methionyl human tumor necrosis factor binding protein PEGylated dimer in patients with active refractory rheumatoid arthritis. AB - OBJECTIVE: To evaluate the safety, immunogenicity, pharmacokinetics, and efficacy of intravenous administration of tumor necrosis factor binding protein (TNFbp) dimer in patients with rheumatoid arthritis (RA). METHODS: This phase I/II study was a multicenter, randomized, double blind, placebo controlled, ascending dose study evaluating TNFbp dimer administered by i.v. infusion. Thirty-three patients with RA divided into 3 cohorts received TNFbp dimer (30, 100, 300 microg/kg) or placebo during a 5 min infusion at baseline and at 3 and 6 weeks; patients were followed at routine intervals after each infusion through 77 days postinfusion. Pharmacokinetics were analyzed using a log-linear regimen and comparisons were made between half-life after first, 2nd, and 3rd doses. Plasma TNFbp dimer concentrations and serum antibody levels were used in the measurement of pharmacokinetics. RESULTS: Administration of 30 microg/kg of TNFbp dimer was generally well tolerated; the maximum tolerated dose was 100 microg/kg. No serious adverse events were reported. A significant antibody response affected the half-life and clearance of TNFbp dimer at each dose group. Anti-TNFbp antibodies were noncytotoxic and nonagonistic. Clinical evaluations provided evidence of in vivo activity of TNFbp dimer in these patients. CONCLUSION: TNFbp dimer administered to patients with long standing RA resulted in significant antibody production to the study drug. This effect reduced the half-life and clearance of the TNFbp. This TNFbp will not be a viable option for treating patients with RA secondary to immunogenicity. PMID- 10743797 TI - Use of antikeratin antibodies to distinguish between rheumatoid arthritis and polyarthritis associated with hepatitis C infection. AB - OBJECTIVE: To investigate whether antikeratin antibodies (AKA) could be useful in the differential diagnosis of patients with rheumatoid arthritis (RA) compared to patients with hepatitis C virus (HCV) associated polyarthritis, who are seropositive for rheumatoid factor (RF). METHODS: AKA were assayed in 3 different groups of patients; all were RF seropositive: Group 1: 25 patients with HCV associated polyarthralgia or arthritis. Group 2: 33 patients with RA. Group 3: 13 patients with autoimmune disorders other than RA. Fifteen healthy individuals served as controls. RESULTS: AKA were detected in 20/33 patients with RA (60.6%) compared to only 2/25 patients (8%) with HCV associated arthritis (p < 0.0001). AKA were observed in 2/13 patients of Group 3 (15.3%). These results were also statistically different from those of patients with RA (p = 0.008). AKA were not found in the sera of the healthy controls. CONCLUSION: AKA is a useful marker to differentiate patients with RA from those with hepatitis C arthritis. PMID- 10743798 TI - Disability and handicap in rheumatoid arthritis and ankylosing spondylitis- results from the German rheumatological database. German Collaborative Arthritis Centers. AB - OBJECTIVE: To describe indicators of disability and handicap in the 2 major inflammatory rheumatic diseases rheumatoid arthritis (RA) and ankylosing spondylitis (AS) and to estimate the burden of illness in terms of functional status, pain, and global well being, as well as with regard to unemployment and early retirement. METHODS: Data from the German rheumatological database on 52,444 patients with RA and 8,776 patients with AS seen at 21 collaborative arthritis centers in Germany between 1993 and 1997 were analyzed. To estimate the burden of the 2 diseases at different biographical phases, age and sex matched groups of patients were compared for functional disability, pain, global assessment of health status, education level, and employment status. RESULTS: For comparable ages, disability in female patients with AS or RA was rated similarly by the physicians (e.g., 61-70 years: 42% severe disability in RA and 44% in AS), whereas men with AS were rated more disabled than men with RA (61-70 years: 35% in RA and 48% in AS). Patients' self-ratings of disability were generally worse for women than for men (age 61-70: women 37% severe disability in RA and 35% in AS, men: 24% in RA and 19% in AS). Pain intensity was rated similarly by women and men with RA and AS at ages < 51 years (33% severe pain at ages 41-50 in men and 34% in women in both diseases). It increased with age in women (> 70 years: 41% severe pain in RA and 44% in AS) and remained stable in men (27% RA and 29% AS > 70 years). At 71.3% the employment rate in AS was clearly higher than in RA (49.5%). There was significant influence of the education level (men 51-60 years with AS: low education 56% employment rate, high education 79%) and the labor market (men 51-55 years with AS: 80% employment rate under good, 59% under bad overall labor market conditions). CONCLUSION: Age and sex matched groups of patients with RA and AS in tertiary rheumatological care show similar amounts of disability, pain, and reduction in well being. Therefore, the offer of comprehensive care and pain management to both groups should be comparable. PMID- 10743799 TI - Consequences of delayed therapy with second-line agents in rheumatoid arthritis: a 3 year followup on the hydroxychloroquine in early rheumatoid arthritis (HERA) study. AB - OBJECTIVE: To assess the longterm effect of delaying therapy with second-line agents in patients with early rheumatoid arthritis (RA). METHODS: One hundred nineteen patients who participated in a 9 month placebo controlled randomized trial of hydroxychloroquine sulfate (HCQ) were followed prospectively for an additional 3 years. Those randomized to HCQ are referred to as the early treatment group and those randomized to placebo as the delayed treatment group. Participants were assessed annually for pain [Arthritis Impact Measurement Scales (AIMS) and Stanford Health Assessment Questionnaire (HAQ)], physical disability (AIMS and HAQ), and the RA global well being scale (AIMS). Conversion of results into standard deviation (SD) units permitted defining a substantial difference as per Felson as > 0.30 SD units and a clinically indistinguishable difference as < or = 0.06 SD units. RESULTS: One hundred fifteen patients (97%) participated and complete data were available on 104 (87%). Compared to the early treatment group, the delayed group remained worse for both the pain and the physical disability outcomes over the additional 3 year followup. The difference in the RA global well being score became clinically indistinguishable for the early and delayed groups only after the 2 year post-trial assessment. The between-group differences were not explained by post-trial therapy with corticosteroids, other second-line agents, or nonsteroidal antiinflammatory drugs and analgesic preparations. CONCLUSION: These findings show that a delay in instituting therapy with second line agents, even a 9 month delay in instituting a moderately powerful second line agent such as HCQ, has significant effects on longterm patient outcome, and provides strong evidence in support of early therapy in RA. PMID- 10743800 TI - The effect of smoking on clinical, laboratory, and radiographic status in rheumatoid arthritis. AB - OBJECTIVE: (1) To determine the degree to which rheumatoid factor (RF) positivity is associated with smoking; (2) to determine the quantitative effect of smoking and smoking length on the concentration of RF in all patients, and, in the seropositive patients separately, determining if a "dose-response" effect exists; (3) to investigate these relationships in men and women to clarify whether the effect of smoking is similar in both sexes; (4) to determine the effect of smoking, controlling for RF, on a variety of measures of disease status, severity, and activity. METHODS: Six hundred ten consecutive patients with rheumatoid arthritis seen for routine clinical care provided information on their smoking history. All underwent a complete joint examination, completed a series of health status questionnaires, provided information concerning pulmonary illnesses, underwent determinations for RF and erythrocyte sedimentation rate (ESR), and had hand radiographs. RESULTS: RF concentration was linearly related to the number of years smoked. This association was present in both sexes, but was stronger in men. Smoking was similarly related to rheumatoid nodule formation. A nonlinear relationship was found between smoking and radiographic abnormalities as determined by the Larsen method, even controlling for RF. Similarly, pulmonary illness was independently related to smoking and RF. No effect of smoking was seen on disease process variables such as ESR, pain, joint count, global severity, or functional ability. CONCLUSION: Quantitative relationships exist between smoking extent and RF positivity, RF concentration, nodule formation, radiographic progression, and pulmonary disease. These 3 latter effects are independent of RF positivity or concentration. Smoking does not contribute to alterations in disease activity measures, but appears to play a role in overall severity of disease. PMID- 10743801 TI - Effect of age on 3 year outcome in early rheumatoid arthritis. AB - OBJECTIVE: To investigate the effect of age on clinical and radiological outcome and on efficacy and tolerance of antirheumatic therapy in early rheumatoid arthritis (RA). METHODS: In a prospective 3 year study 113 patients (83 women, 30 men) were divided into 2 groups according to age at onset of disease: before (n = 55) and after 55 years of age (n = 58). For clinical outcome, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor, Ritchie index, and number of swollen joints were measured. Radiological progression was analyzed by Larsen score. The principles of the "sawtooth" strategy were applied in the treatment of all patients. RESULTS: At baseline, inflammatory activity (ESR, CRP) and the Larsen score for hands were significantly higher in patients with late onset RA (LORA) and they also developed more extraarticular symptoms compared to patients with early onset RA (EORA). However, no differences were found in Ritchie index, number of swollen joints, or CRP values between the groups. Also during the followup there was a trend toward increased inflammatory activity (ESR) among LORA patients. After the initiation of antirheumatic therapy a parallel improvement in clinical activity was observed in the 2 groups. The frequencies of remissions, side effects, and withdrawals due to drug inefficacy did not differ significantly between the 2 groups. The radiological progression was also comparable. CONCLUSION: The onset of RA was more active in patients with LORA. However, the clinical course and the radiological progression were parallel in LORA and EORA patients. The "sawtooth" therapy was equally tolerated in both patient groups. PMID- 10743802 TI - Modeling therapeutic strategies in rheumatoid arthritis: use of decision analysis and Markov models. AB - OBJECTIVE: The management of patients with rheumatoid arthritis (RA) is controversial, with a number of different proposed treatment strategies based on different conceptions of the natural history of the disease and different interpretations of the efficacy and effectiveness of the drugs used for treatment. We attempted to develop a theoretical framework to assess the effectiveness of different treatment regimens for RA. METHODS: We used decision analysis to structure the problem of comparing sequential monotherapy to a combination strategy. Subsequently, we used 3 different estimates of drug effectiveness: one from expert rheumatologists; a metaanalysis; and a recent nationwide survey of American rheumatologists, in a Markov model. Last, we utilized published duration of therapy data to model drug treatment over time. RESULTS: Estimates of drug effectiveness differed substantially among rheumatologists, but regardless of the estimates and the treatment strategy used, the model predicted over 90% of patients improved by the 3rd drug trial. Over time, treatment patterns in our model resemble the "sawtooth" pattern previously observed. CONCLUSION: Treatment strategies in RA are difficult to model because of uncertainty in both the structure of the model and the data needed to perform the analysis. These models tend to overestimate the effectiveness of drug sequences because of nonindependence between therapies, probably due to sequence effects, a change in responsiveness over time, or resistant subgroups. Our preliminary analysis suggests that the most effective agent, possibly methotrexate, should be used first if the objective is to get as many patients into remission as quickly as possible. PMID- 10743804 TI - Novel roles of complement in systemic lupus erythematosus--hypothesis for a pathogenetic vicious circle. AB - We propose that impaired complement function enhances a pathogenetic vicious circle in SLE. In this process, induction and clearance of apoptotic cells is central. Apoptosis could be triggered by various etiological factors, such as infection, UV light, and drug reactions. Clearance of apoptotic material is reduced when complement function is impaired. Apoptosis leads to increased exposure of nuclear antigens to the immune system, to which estrogenic hormones could contribute. This could in turn lead to activation of autoreactive B cells, autoantibody production, and immune complex formation, all of which is accelerated by hypocomplementemia. Immune complexes may, at least partly via complement dependent mechanisms, accelerate apoptosis. PMID- 10743803 TI - Effects of sulfasalazine and its metabolites on steady state messenger RNA concentrations for inflammatory cytokines, matrix metalloproteinases, and tissue inhibitors of metalloproteinase in rheumatoid synovial fibroblasts. AB - OBJECTIVE: To determine the effects of sulfasalazine (SASP) and its metabolites sulfapyridine (SP) and 5-amino salicylic acid (5ASA) on steady state mRNA levels of inflammatory cytokines [interleukin 1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha)], matrix metalloproteinases [collagenase (MMP1), stromelysin (MMP3), gelatinase 72 kDa (MMP2)], tissue inhibitors of metalloproteinase (TIMP 1 and TIMP 2), and the TNF-alpha receptor in rheumatoid synovial fibroblasts. METHODS: Cells were dosed with each compound for 24 h in the presence or absence of PMA inducer and messenger RNA (mRNA) extracted and subjected to Northern blot analysis. Messenger RNA levels were quantitated by densitometry and normalized to GAPDH or 18S rRNA. RESULTS: We observed some modest effects of sulfasalazine and its metabolites on steady state mRNA levels including: (1) repressed mRNA levels for TNF-alpha [approximately 40% with 3x (drug median serum concentration) all 3 drugs], stromelysin (approximately 24% with 3x all 3 drugs and approximately 31% with 3x 5ASA), and collagenase (approximately 27% with 3x 5ASA); (2) elevated mRNA levels for TIMP 2 (3.5 kb transcript) (51% with 3x SP and 44% with 3x 5ASA), gelatinase (approximately 20% with 3x SP and 3x 5ASA), stromelysin (approximately 40% with 3x and 1x SASP), IL-1beta (approximately 31% with 0.1x 5ASA); and (3) no effect on mRNA levels for TNF-alpha receptor and TIMP 1. CONCLUSION: (1) SASP and its metabolites showed varied effects on steady state mRNA concentrations for gene transcripts that fell into 3 categories: (a) repressed, (b) elevated, (c) no effect on mRNA levels. (2) No apparent linear dose response effect was observed for SASP or its metabolites, although a generalized suppression of mRNA levels at all doses was seen in some cases. (3) No predominant suppressive effect (> or = 50%) of mRNA levels by any of the drugs was observed for any of the genes studied; however, TIMP 2 mRNA levels increased 51% with 3x SP and 44% with 3x 5ASA. PMID- 10743805 TI - Comparison of the validity and sensitivity to change of 5 activity indices in systemic lupus erythematosus. AB - OBJECTIVE: To compare the construct validity and sensitivity to change of the Systemic Lupus Activity Measure (SLAM), Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), Lupus Activity Index (LAI), British Isles Lupus Assessment Group index (BILAG), and the European Consensus Lupus Activity Measure (ECLAM). METHODS: Twenty-three patients with systemic lupus erythematosus (SLE) were examined prospectively every 2 weeks for up to 40 weeks. Nineteen patients completed all 20 assessments. At each assessment, each of the 5 activity indices was scored, along with physicians' and patients' global assessments of SLE activity. Construct validity was determined by the strength of correlations between changes over time in each activity index and changes in physician and patient global assessments. Sensitivity to change was determined by the magnitude of change in each index over the 2 week interval of greatest change in the physician or patient global assessments, and calculated as standardized response means (SRM; mean change/standard deviation of change). Thirteen patients were also examined monthly by a second physician who was blinded to previous scores on the activity indices and to the patient global assessments. RESULTS: Patients had substantial changes in SLE activity during the study. Changes in each activity index were correlated with changes in the physician global assessment (SLAM r = 0.54; SLEDAI r = 0.52; LAI r = 0.75; BILAG r = 0.61; ECLAM r = 0.65; all p < 0.0001). Correlations were somewhat lower with the blinded physician assessment (SLAM r = 0.42; SLEDAI r = 0.12; LAI r = 0.30; BILAG r = 0.28; ECLAM r = 0.32). The SLAM was the only index that was positively correlated with changes in the patient global assessment (r = 0.22; p < 0.0001). Sensitivity to change was greatest for the LAI (SRM = 0.74) and the ECLAM (SRM = 0.75) and smallest for the SLEDAI (SRM = 0.48) when the physician global assessment was used as the standard. Sensitivity to change was greatest for the SLAM (SRM = 0.61) and the BILAG (SRM = 0.57) and smallest for the SLEDAI (SRM = -0.01) when the patient global assessment was used as the standard. CONCLUSION: Each index is a valid measure of SLE activity. The SLAM captures patients' assessments better than the other indices, perhaps because it assesses the patients' subjective complaints to a greater extent. Estimates of sensitivity to change varied with the standard used, but the SLEDAI was least sensitive to change. Larger studies are indicated to further compare the sensitivity to change of these indices. PMID- 10743806 TI - Association of coping responses with fibromyalgia tender points in patients with systemic lupus erythematosus. AB - OBJECTIVE: To determine the association between fibromyalgia (FM) tender points (TP) and psychological constructs in patients with systemic lupus erythematosus (SLE). METHODS: One hundred seventy-three patients with SLE were examined for FM TP, and asked to complete 2 questionnaires at the same visit, the Health-Related Hardiness Scale (HRHS), and the Mishel Uncertainty in Illness Scale (MUIS). RESULTS: The examination of FM TP showed that 38.2% had no TP, 44.5% had 1-10 TP, and 17.3% had > or = 11 TP. The mean +/- SD score of the HRHS was 155.6 +/- 19.7 (range 105.0-198.0; higher scores indicate greater hardiness), and the MUIS was 85.3 +/- 18.7 (range 41.0-132.0; higher scores indicate uncertainty). There were significant associations between FM TP and HRHS (no TP 161.2 +/- 20.2, 1-10 TP 152.5 +/- 19.7, > or = 11 TP 151.0 +/- 15.8; p = 0.0108) and between FM TP and MUIS (no TP 78.2 +/- 20.2, 1-10 TP 86.9 +/- 17.6, > or = 11 TP 95.8 +/- 14.7; p = 0.0001). CONCLUSION: This study shows a strong association between FM TP and uncertainty or lack of "hardiness." We conclude that SLE patients with FM TP are less likely to be good "copers." Prospective studies to determine if "poor coping" predicts FM in SLE are recommended. If the association between coping and FM is causal, it will justify interventions to improve coping and similar constructs, such as self-efficacy. PMID- 10743807 TI - Learning from discordance in patient and physician global assessments of systemic lupus erythematosus disease activity. AB - OBJECTIVE: Differences have been described between patient and physician assessments of well being in several chronic illnesses, and these differences may affect outcome. Disagreement may lead to dissatisfaction and to behaviors with dangerous consequences. We describe and identify predictors of patient-physician differences on ratings of disease activity in systemic lupus erythematosus (SLE). METHODS: Data collected on 154 patients included age, education, disease duration, and patient and physician global assessments of lupus activity on a 10 cm visual analog scale (VAS), the Health Assessment Questionnaire (HAQ), the Medical Outcome Study Short-Form 36 (SF-36), the Systemic Lupus Disease Activity Index (SLEDAI), the Systemic Lupus Activity Measure (SLAM-R), and the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SDI). Multiple linear regression models were performed using patient VAS scores, physician VAS scores, and patient minus physician VAS scores as the dependent variables, and age, disease duration, selected SF-36 and SLAM-R subscales, and SDI as independent variables. RESULTS: Patients were 90% female and 80% Caucasian, with a mean education of 13 +/- 2.8 years and a mean age of 43.1 +/- 13.6 years. The overall mean disease duration was 10.5 +/- 7.8 years. Physicians overscored patients by 2.5 cm in 6% of the cases and patients overscored physicians in 16% of the cases. The best multivariate model to predict overall differences included SF-36 mental health and SLAM-R kidney scores. CONCLUSION: Patient-physician differences may result from a divergence in focus. Patients score lupus activity based on their psychological status, while physicians rely more heavily on the physical effect of the disease. PMID- 10743808 TI - Association between clinical factors, socioeconomic status, and organ damage in recent onset systemic lupus erythematosus. AB - OBJECTIVE: To determine the prevalence and socioeconomic and clinical predictors of early organ damage in a cohort of patients with systemic lupus erythematosus (SLE) of 2-7 years' duration randomly sampled at 5 centers and balanced by socioeconomic status and race. METHODS: The Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) Damage Index was measured in 200 patients who met the ACR criteria for SLE with a mean disease duration of 3.8 years. The SLICC/ACR scores for each organ system and the prevalence of damage within organ systems were assessed. Logistic regression analyses evaluated the simultaneous effects of age at diagnosis, disease duration, disease activity, and sociodemographic factors. RESULTS: Sixty-one percent of the patients had damage within 7 years of onset (mean 3.8 yrs). Neuropsychiatric (20.5%) and musculoskeletal (18.5%) systems were the most frequently involved, followed by renal (15.5%) and skin (12.5%) systems, all with median SLICC/ACR organ system scores of 1. In multivariate models, African American race was associated with skin damage but not with damage in other specific organ systems. Socioeconomic status was not associated with organ system damage. Older age at diagnosis correlated with cardiovascular, musculoskeletal, gastrointestinal, ocular, and pulmonary damage. Clinical factors such as longer disease duration correlated with higher renal and cardiovascular damage, and greater disease activity at diagnosis of SLE correlated with greater renal, musculoskeletal, and pulmonary damage. CONCLUSION: There is evidence of organ system damage in SLE within a mean of 3.8 years after onset. We found little evidence for differences in early organ damage according to race or socioeconomic status. Damage to most organ systems was related to age at diagnosis of SLE and clinical factors such as disease duration. PMID- 10743809 TI - Incidence studies of systemic lupus erythematosus in Southern Sweden: increasing age, decreasing frequency of renal manifestations and good prognosis. AB - OBJECTIVE: To identify all new cases of systemic lupus erythematosus (SLE) within a defined area in Southern Sweden with validated methods of retrieval, and to compare 2 cohorts assembled during 1981-86 and 1987-91. METHODS: The catchment area, the health care district of Lund-Orup, had during 1981-91 a mean adult population (> 15 years of age) of 172,300 individuals. During 1987-91 we identified 379 individuals with potential SLE diagnosis from diagnosis registers and from central laboratory databases. Out of these, 121 had a previously known SLE diagnosis. All patient records were reviewed and patients with possible SLE not already known at the SLE unit were invited and examined. Organ damage was recorded as the Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) damage index. RESULTS: Forty-one new SLE cases were diagnosed during 1987-91, giving a median annual incidence of 4.8/100,000 inhabitants, with a median age at diagnosis of 47 years. The incidence is similar to that found 1981-86 (4.5/100,000/year) in the same population using the same methods for retrieval. Age and sex-specific incidence 1981-91 was notably highest at the age of 65-74 (14.1/100,000/year) in women and age 65-74 (3.2/100,000/year) in men. The point prevalence on December 31, 1986, was a 42/100,000 and on December 31, 1991, 68/100,000. The 5 year survival was 93% and 10 year survival 83%. While overall survival was not decreased, 10 year survival was slightly reduced compared with an age and sex matched healthy population (p = 0.03). In the 1987-91 cohort the sensitivity of the American Rheumatism Association criteria was 92.7% and the specificity was 94%. The frequency of renal manifestations was decreased in the latter cohort. The damage rate was highest during the first year and then constant during a 5 year followup, and was similar in the 2 cohorts. Damage that related to atherosclerosis was common and cardiovascular disease was the most common cause of death. CONCLUSION: The incidence of SLE in Sweden was notably constant during the 11 years 1981-91. Mortality was low and only late mortality (> 10 years disease duration) exceeded that in an age and sex matched control population. Atherosclerosis was the main cause of damage and mortality. Specificity and sensitivity of the ACR classification criteria are high in this epidemiologically recruited cohort. PMID- 10743810 TI - Cardiovascular autonomic function, autoantibodies, and esophageal motor activity in patients with systemic sclerosis and mixed connective tissue disease. AB - OBJECTIVE: To study cardiovascular autonomic nerve function and presence of autoantibodies in relation to esophageal motor activity in patients with systemic sclerosis (SSc) and mixed connective tissue disease (MCTD). METHODS: Twenty-five patients with SSc (13 limited, 12 diffuse cutaneous disease; disease duration 1 19 yrs) and 6 patients with MCTD (disease duration 1-10 yrs) were studied. Cardiovascular autonomic function was assessed using 5 standard tests and autoantibody status determined. Esophageal motor activity and lower and upper esophageal sphincter pressures were recorded manometrically. RESULTS: Five patients with SSc had definite, 7 borderline, and 13 no autonomic dysfunction; 23 had antinuclear. 9 anti-Sc170, 4 anticentromere, and 1 U1snRNP antibodies. Contraction amplitudes in the smooth muscle as well as the striated muscle esophagus and lower esophageal sphincter pressures were significantly lower and autonomic dysfunction more frequent in patients with than in those without anti Sc170 (6 of 9 vs 6 of 16 patients); upper esophageal sphincter pressures did not differ. All patients with MCTD had antinuclear antibodies, 5 had definite autonomic dysfunction; their lower esophageal sphincter pressures were significantly lower than in SSc patients without anti-Sc170 and anti-U1snRNP. CONCLUSION: Esophageal motor dysfunction may be associated with the presence of anti-Sc170 and anti-U1snRNP autoantibodies and prevail in patients with cardiovascular autonomic neuropathy. PMID- 10743811 TI - Circulating levels of soluble CD30 are increased in patients with localized scleroderma and correlated with serological and clinical features of the disease. AB - OBJECTIVE: To determine serum soluble CD30 (sCD30) levels in patients with localized scleroderma. METHODS: Serum soluble CD30 levels were assayed by a sensitive ELISA in 55 patients with localized scleroderma, in 15 with systemic sclerosis (SSc), and in 20 healthy controls. RESULTS: Serum levels of sCD30 were significantly higher in patients with localized scleroderma than in healthy controls. Serum levels of sCD30 were correlated with the number of sclerotic lesions, the number of involved areas, levels of anti-histone antibody IgM, and levels of interleukin 6. sCD30 levels in sera from patients with SSc were also significantly higher than in healthy controls. CONCLUSION: These results suggest possible involvement of Th2 cells in the immunopathogenesis in localized scleroderma, and serum sCD30 levels may be helpful in following the outcome of the disease. PMID- 10743812 TI - Epidemiologic and clinical aspects of Behcet's disease in a defined area of Northwestern Spain, 1988-1997. AB - OBJECTIVE: To assess the frequency and clinical manifestations of Behcet's disease (BD) during the past 10 years in a defined area of Northwestern Spain. METHODS: The charts of all the patients diagnosed as having BD at the Hospital Xeral-Calde from 1988 through 1997 were reviewed. All the patients were residents of Lugo. Patients were followed from the time of diagnosis until either the patient's death or July 1, 1998. By that time patients were classified as having complete BD if they fulfilled the International Study Group Criteria (ISG) for BD. Patients with recurrent oral ulcerations plus one of the remaining 4 criteria of the ISG were classified as having an incomplete form of BD. RESULTS: Sixteen patients (9 men/7 women) were diagnosed as having complete BD and 7 (3 men) as having incomplete BD. The average annual incidence rate of complete BD in the Lugo region of Northwestern Spain was 0.66/100,000. Oral ulcerations with or without genital ulcerations were the most common initial manifestations. Neurological involvement was relatively frequent in men with complete BD. In contrast, a positive pathergy test was less common. Two patients with neurological involvement and 3 with uveitis had severe sequelae. However, there was a single death, apparently not related to BD. CONCLUSION: In Northwestern Spain, BD is more common than was expected and is a nonfatal disease. However, central nervous system involvement and severe ocular complications overshadow the good prognosis observed in the majority of patients. PMID- 10743814 TI - Clinical efficacy of mesalamine in the treatment of the spondyloarthropathies. AB - OBJECTIVE: Sulfasalazine (SSZ) has been found to have beneficial effects in the treatment of patients with spondyloarthropathy (SpA) with active disease. The effectiveness of SSZ is limited by both idiosyncratic and dose related side effects when treating SpA. Mesalamine is a drug used to treat inflammatory bowel disease. Case reports have suggested potential efficacy in SpA. We investigated the efficacy and safety of the Pentasa formulation of mesalamine in treating SpA. METHODS: Thirty patients with SpA as defined by the European Spondylarthropathy Study Group were recruited from a rheumatology specialty clinic. All subjects began 16 week open label therapy with mesalamine 1500 mg/day. Dose escalation for lack of efficacy was permitted after 8 weeks of therapy. Clinical, physical, and laboratory data were collected at baseline, at 8 weeks, and at the conclusion of the study at 16 weeks. RESULTS: Twenty-nine of the 30 patients completed the study. There was clinically and statistically significant improvement in all clinical measures (morning stiffness, night awakenings, quality of sleep, severity of stiffness, severity of pain, Dougados functional index, patient and physician global indices). Joint counts and enthesitis counts improved, but measures of axial flexibility did not. Erythrocyte sedimentation rate and C reactive protein also improved over the duration of the study. CONCLUSION: In this population of patients with SpA, mesalamine was well tolerated in the dose range 1500 to 4000 mg/day. Improvements in clinical, physical, and laboratory measures indicate the efficacy of mesalamine in treating SpA. PMID- 10743813 TI - T cell responses to 60/65 kDa heat shock protein derived peptides in Turkish patients with Behcet's disease. AB - OBJECTIVE: Sequence homology and cross reactivity between microbial and human heat shock proteins (HSP) led to the concept that HSP might be involved in the etiopathogenesis of Behcet's disease (BD). We investigated T cell responses to 8 synthetic peptides derived from the mycobacterial 65 kDa and homologous human 60 kDa HSP in patients with BD. METHODS: T cell proliferative responses to synthetic peptides were studied in 49 patients with BD and 46 disease (DC) and 34 healthy controls (HC) with 3H-thymidine uptake test. RESULTS: Positive T cell responses to one or more of the mycobacterial peptides were observed in 52% (12/23) of patients with BD compared with 17% (3/18) of DC (p = 0.02) and to homologous human peptides in 57% (13/23) of BD and 11% (2/18) of DC (p < 0.01). Responses to the mixtures of 4 mycobacterial peptides were also significantly higher in BD compared with controls (stimulation index in BD 4.7 +/- 3.5 vs DC 2.0 +/- 1.2, HC 1.6 +/- 0.4; BD vs DC and HC, p < 0.001). Similar elevated responses to the mixture of 4 human peptides was also observed in patients with BD (BD 3.4 +/- 2.3; DC 1.9 +/- 0.8; HC 1.4 +/- 0.6; BD vs DC, p < 0.01; BD vs HC, p < 0.001). CONCLUSION: These results suggest that cellular immunity against the 65 kDa mycobacterial and 60 kDa human HSP derived peptides is significantly increased in Turkish patients with BD compared to controls, as observed in the UK and Japan. PMID- 10743815 TI - Efficacy of sacroiliac corticosteroid injections in patients with inflammatory spondyloarthropathy: results of a 6 month controlled study. AB - OBJECTIVE: To evaluate changes in articular symptoms, spinal mobility, and global function over 6 months after intraarticular injections of long acting corticosteroid into the sacroiliac (SI) joints of patients with inflammatory low back pain (ILBP). METHODS: Nineteen patients with symptoms of ILBP were studied. Thirteen (68%) had radiographic evidence of sacroiliitis. The remaining 6 patients (32%) had normal imaging studies and thus were considered to have mechanical low back pain. All patients received bilateral SI joint injections of triamcinolone hexacetonide (40 mg/joint) under computer tomographic guidance. Outcome variables included the duration of low back morning stiffness, back pain (by visual analog scale, McGill Pain Questionnaire), spinal mobility (chest expansion, Schober test, 10 cm segments test, finger-fibula distance), and self report health status (SF-36). RESULTS: Both groups of patients showed a transient improvement in stiffness and pain, spinal mobility, and general health status that was most pronounced at 1-3 months after intraarticular therapy. This did not reach statistical significance (p > 0.05) and by 6 months, followup all outcome variables had reverted to pretherapy levels in both groups. CONCLUSION: These preliminary observations suggest that SI corticosteroid injections are ineffective in the management of patients with inflammatory spondyloarthropathy. PMID- 10743816 TI - Treatment of spondyloarthropathy with 5-aminosalicylic acid (mesalazine): an open trial. AB - OBJECTIVE: Ankylosing spondylitis (AS) and spondyloarthropathy (SpA) are inflammatory diseases of unknown etiology. Various exogenous and endogenous (inherited) factors play a role in their development. Sulfasalazine (SSZ) is generally accepted as a disease modifying drug in the treatment of AS and SpA. Which part of SSZ, 5-acetylsalicylic acid (5-ASA, mesalazine) or sulfapyridine (SP), is the effective moiety is unknown. As the bowel, colon, and the ileum play an important role in the development of AS and SpA, it may be possible that 5-ASA is the effective moiety, with a similar mode of action as in the treatment of inflammatory bowel disease. To determine the efficacy of 5-ASA an open pilot study was done in 2 groups of patients with SpA. METHODS: Twenty patients with SpA, who were taking SSZ, were switched to 5-ASA (Pentasa), and 19 patients with active SpA were treated with 5-ASA without previous administration of SSZ. RESULTS: In the first group, 17 (85%) patients responded with respect to the physician global clinical assessment compared to the previous SSZ treatment period; whereas in the second patient group a statistically significant improvement was obtained in erythrocyte sedimentation rate. CONCLUSION: The results support our hypothesis that 5-ASA might be the active moiety of SSZ in the treatment of SpA. PMID- 10743817 TI - Expression of vitronectin and its integrin receptors in the synovial membrane like interface tissue from aseptic loosening of total hip replacement. AB - OBJECTIVE: To investigate expression of vitronectin (VN) and its integrin (Int) receptors in synovial membrane-like interface tissue (SMLIT) in aseptic loosening of total hip replacement (THR), and the potential role of VN-Int interaction in production of collagenase-3. METHODS: Avidin-biotin-peroxidase complex (ABC) staining was used to detect distribution of VN and Int alphaV, beta3, and beta5 subunits. Immunofluorescence labeling with FITC and TRITC conjugated IgG was used to localize Int beta3 subunit and matrix metalloproteinase (MMP-13) double positive cells in SMLIT. RESULTS: Intensive VN immunoreactivity was found in the lining-like layers, sublining area, and endothelium of SMLIT. Statistical analysis of the VN staining score revealed a significant difference between SMLIT and control synovial membrane. All 3 Int subunits appeared in the lining-like layers and sublining area. The Int beta3 subunit was also detected in giant cells of SMLIT. Int beta5 subunit staining was relatively weak and rarely found in vascular endothelium. Immunofluorescence labeling showed many double positive cells in the lining-like layer and sublining area of SMLIT. CONCLUSION: Expressions of VN and Int alphaVbeta3 and alphaVbeta5 are increased in SMLIT compared with that in OA synovial membrane. Int alphaVbeta3 engagement with VN might play a potential role in local MMP-13 production in SMLIT. PMID- 10743818 TI - Bone mineral density varies as a function of the rate of joint space narrowing in the hip. AB - OBJECTIVE: To determine whether patients with a rapid rate of joint space narrowing (JSN) in the hip have higher initial bone mineral density (BMD) in the proximal femur and/or lumbar spine than corresponding patients with a slow rate of JSN. METHODS: Twenty-eight patients undergoing unilateral total hip replacement (THR) for osteoarthritis (OA), but whose contralateral hips were asymptomatic and had minimal or no radiographic OA, were evaluated. The contralateral proximal femur (i.e., non-operated hip) and lumbar spine were scanned by dual energy x-ray absorptiometry at baseline (prior to THR) and at 2 years. The rate of JSN was determined by serial longitudinal quantification of the joint spaces over the 2 year period following THR from conventional radiographs, and the patients were divided into a group with a slow rate of JSN (< or = 0.2 mm/yr, n = 20) and a group with a rapid rate of JSN (> 0.2 mm/yr, n = 8). RESULTS: The baseline BMD z and t scores at the femoral neck, Ward's triangle, and lumbar spine of the patients with subsequent rapid rates of JSN were significantly higher than those of patients with slower rates (p < 0.05). There was no difference between the rapid and slow narrowers at the greater trochanter (p > 0.2). Age, sex, weight, height, body mass index, Kellgren- Lawrence scores, and initial joint space width were not significantly different between the 2 groups. CONCLUSION: Patients with a rapid rate of JSN of the asymptomatic hip after unilateral THR are characterized by elevated local and remote BMD. The local elevation in BMD implies that increased local bone density may contribute to or serve as a marker for increased risk of development of OA (assuming that JSN can be used as a predictive marker). The presence of elevated BMD in the spine suggests that there are systemic as well as local aspects of OA pathogenesis, at least in patients with one THR and rapid JSN in the contralateral hip. PMID- 10743820 TI - The effects of freeze/thawing on human synovial fluid observed by 500 MHz 1H magnetic resonance spectroscopy. AB - OBJECTIVE: To investigate the effect of freeze/thaw and low temperature storage on the biomolecular profile of human synovial fluid (SF) using high resolution (500 MHz 1H) magnetic resonance spectroscopy (MRS). METHODS: SF was collected from 12 patients undergoing arthroscopic debridement for treatment of moderate osteoarthritis (OA). Six of the larger samples were divided into 5 parts and treated as follows: the first was analyzed with spin-echo MRS soon after arthroscopy (< 24 h); the 2nd, 3rd, and 4th parts were frozen (-75 degrees C) and thawed for a total of one, 5, and 10 freeze/thaw cycles, respectively, followed by MRS analysis; the 5th part was kept in -75 degrees C storage for > or = 1 year before MRS processing. The 6 smaller samples were divided into 2 parts, the first analyzed shortly after extraction (< or = 24 h), while the 2nd was processed after storage at -75 degrees C for > or = 1 year. Changes in measured metabolite levels were tested for significance using paired t tests. RESULTS: Freeze-thaw cycling had no statistically significant effect on the relative concentrations of endogenous metabolites measured by MRS, though it did alter individual sample results. Prolonged low temperature storage resulted in a significant drop (p < 0.05) in the signal intensities of glucose (45%), N-acetyl glycoproteins (39%), CH2-chain and CH3-terminal and resonances of lipoproteins (46 and 37%, respectively), valine (43%), leucine (35%), and isoleucine (43%). CONCLUSION: This study raises questions about routine procedures that may inadvertently affect the outcomes of quantitative SF analyses. Extended low temperature storage should be avoided as it permanently alters the biochemical profile of SF, possibly leading to erroneous conclusions about the nature of OA related changes in metabolite levels with disease progression. PMID- 10743819 TI - Excitatory amino acid profiles of synovial fluid from patients with arthritis. AB - OBJECTIVE: Previous studies in an experimental synovitis model in rats determined that administration of glutamate and aspartate into the joint produces hyperalgesic responses, while their receptor antagonists provide protection against the development of a hyperalgesic state. We examined concentrations of amino acids in synovial fluid (SF) to determine if increases might be relevant to human joint pathology. METHODS: One hundred forty-four repository SF samples from patients undergoing diagnostic or therapeutic arthrocentesis and 14 SF samples from 7 cadavers were analyzed by high pressure liquid chromatography and compared as arthritic and control cohorts. RESULTS: Compared to the average concentrations from the autopsy cases, the excitatory amino acids (EAA) glutamate and aspartate in SF from patients with synovitis were 54 and 28 times higher, respectively. Increases for all other amino acids ranged from 3 to 18-fold. The values for glutamate and aspartate were significantly higher than the mean increase for other amino acids compared using unpaired t tests (p < 0.0001). The mean ratio of glutamate and aspartate elevations over the mean increase for other amino acids was 4-fold and 2-fold, respectively. The EAA were highest in Reiter's, infectious arthropathies, and systemic lupus erythematosus, but did not appreciably segregate to diagnosis or SF white blood cell count. CONCLUSION: Our data provide evidence of increased glutamate and aspartate in the SF of humans with active arthritis, suggesting that glutamate mediated events may contribute to the pathogenesis of human arthritic conditions. PMID- 10743821 TI - Synovial fluid glycosaminoglycan concentration does not correlate with severity of chondropathy or predict progression of osteoarthritis in a canine cruciate deficiency model. AB - OBJECTIVE: Considerable interest exists today in biochemical or immunochemical tests for monitoring the progression of osteoarthritis (OA). It has been suggested that measurements made on synovial fluid (SF) will more accurately reflect the magnitude of cartilage destruction in an index joint than those performed on serum. However, we have shown that the synovitis that occurs in OA affects the rate of protein clearance from the joint. We tested the hypothesis that if adjusted for clearance rate, the SF concentration of cartilage proteoglycans (PG) estimates severity of chondropathy and predicts progression of cartilage damage more accurately than if clearance is not taken into account. METHODS: Clearance of radioiodinated serum albumin (RISA), a surrogate for the clearance of PG, was measured in 19 adult dogs at baseline and again 16 weeks and 32 weeks after anterior cruciate ligament transection (ACLT). Severity of chondropathy was determined arthroscopically after 16 weeks of instability and at postmortem 32 weeks after ACLT. RESULTS: Adjustment for the RISA clearance rate showed that the SF PG concentration markedly underestimated the quantity of PG released from the OA cartilage. Regardless of whether the concentration was adjusted for clearance, no correlation existed between the SF PG level and the severity of chondropathy. Further, the SF concentration of PG 16 weeks after ACLT failed to predict severity of cartilage damage at postmortem. CONCLUSION: SF concentration of a cartilage derived molecule is unlikely to predict the course of cartilage damage in an OA joint over time or in response to treatment with a potential disease modifying OA drug. PMID- 10743822 TI - Double blind randomized placebo control trial of controlled release codeine in the treatment of osteoarthritis of the hip or knee. AB - OBJECTIVE: Pain is the cardinal feature of osteoarthritis (OA), and with advancing disease there is loss of function and increasing pain even at times of joint rest. Few studies have evaluated the role of opioid analgesics in treating the pain of OA. METHODS: This randomized, double blind, parallel group study compared the efficacy and safety of a 12 hourly controlled release codeine formulation (Codeine Contin) with placebo in patients with chronic pain due to OA of the hips and/or knees. The 4 week treatment period, following an analgesic washout phase of 2-7 days, included weekly clinic evaluations, at which the dose was escalated as appropriate, and daily patient diary completion. Pain (daily), stiffness, and physical function (weekly) were assessed using the multidimensional, self-administered WOMAC (visual analog scale version) questionnaire. RESULTS: Sixty-six eligible patients completed the study. The mean initial and final daily doses of controlled release codeine were 50 mg every 12 h at baseline and 159 mg every 12 h at the final assessment. All variables in the efficacy analysis indicated superiority of controlled release codeine over placebo. The WOMAC pain scale showed an improvement of 44.8% over baseline in the controlled release codeine group compared with 12.3% taking placebo (p = 0.0004). For the WOMAC stiffness and physical function scales the improvements over baseline on controlled release codeine were 47.7% and 49.3%, respectively compared with 17.0% and 17.0%, respectively, with placebo (p = 0.003; p = 0.0007). Controlled release codeine was also significantly better than placebo on measures of sleep quality and requirement for supplemental acetaminophen. CONCLUSION: Single entity controlled release codeine is an effective treatment for pain due to OA of the hip or knee. PMID- 10743823 TI - Efficacy of tramadol in treatment of chronic low back pain. AB - OBJECTIVE: To evaluate the efficacy and safety of tramadol in the treatment of chronic low back pain. METHODS: A 3 phase trial: (1) a washout/screening phase; (2) a 3 week, open label, run-in phase; and (3) a 4 week, randomized, placebo controlled, double blind treatment phase. Three hundred eighty outpatients between 21 and 79 years with chronic low back pain with no or a distant history of back surgery enrolled in the open label phase and were treated with tramadol up to 400 mg/day. At the end of the open label phase, patients who tolerated tramadol and perceived benefit from it were randomized to continue treatment with tramadol or to convert to placebo in the double blind phase. Reasons for discontinuing from the open label phase included adverse events, 78 patients (20.5%); drug ineffective, 23 patients (6.1%); and other reasons, 25 patients (6.6%). Two hundred fifty-four patients entered the double blind phase, during which the daily dose was maintained within the range 200-400 mg tramadol or equivalent amount of placebo. The primary outcome measure in the double blind phase was the time to discontinuation due to inadequate pain relief. RESULTS: The distribution of time to therapeutic failure was significantly (p < or = 0.0001) different in the tramadol group compared to placebo. Kaplan-Meier estimate of the cumulative discontinuation rate due to therapeutic failure was 20.7% in the tramadol group and 51.3% in the placebo group. There were significantly lower (p < or = 0.0001) mean pain visual analog scores (10 cm scale) among tramadol patients (3.5 cm) compared to placebo patients (5.1 cm) at the final visit of the double blind phase. Tramadol patients scored significantly better on the McGill Pain Questionnaire (p = 0.0007) and the Roland Disability Questionnaire (p = 0.0001). Five of 127 tramadol treated patients and 6/127 placebo treated patients discontinued treatment during the double blind phase due to an adverse event. Commonly reported adverse events with tramadol included nausea, dizziness, somnolence, and headache. CONCLUSION: Among patients who tolerated it well, tramadol was effective for the treatment of chronic low back pain. PMID- 10743824 TI - Association analysis of the vitamin D receptor gene, the type I collagen gene COL1A1, and the estrogen receptor gene in idiopathic osteoarthritis. AB - OBJECTIVE: Evidence has accumulated supporting a role for genes in the etiology of osteoarthritis (OA). Several candidates have been targeted as potential susceptibility loci including genes that are involved in the regulation of bone density. Genetic association analysis has suggested a role for the vitamin D receptor gene (VDR) and the estrogen receptor gene (ER) in susceptibility. Such findings must be tested in additional independent cohorts. We tested for association of these 2 genes, plus a third gene implicated in bone density, COL1A1, with idiopathic OA. METHODS: A case-control cohort of 371 affected probands and 369 unaffected spouses was used. Association was tested using 4 intragenic single nucleotide polymorphisms (SNP), one each for the VDR and COL1A1 genes, and 2 for the ER gene. The VDR and ER SNP are the same SNP that have been associated with OA. All 4 SNP affect restriction enzyme sites and were genotyped using polymerase chain reaction and enzyme digestion. Allele and genotype distributions for each SNP were compared between cases and controls and analyzed using Fisher's exact test. RESULTS: There was no evidence of association of the VDR or the ER gene SNP to OA. There was weak evidence of association of the COL1A1 SNP in female cases (p = 0.017), reflected by a difference in the distribution of genotypes at this SNP between female cases and controls (p = 0.027). However, when corrected for multiple testing, these results were not significant. CONCLUSION: If the VDR, ER, or COL1A1 genes do encode predisposition to OA then the 4 SNP tested are not associated with major susceptibility alleles at these 3 loci. PMID- 10743825 TI - Quality of well being in patients with fibromyalgia. AB - OBJECTIVE: The Quality of Well-being Scale (QWB) is a generic measure of health related quality of life that can be used for population monitoring, measurement of clinical outcomes, or cost effectiveness analysis. We report data on the validity of the QWB for patients with fibromyalgia (FM) and compare the effect of FM to that of other chronic diseases. METHODS: The participants were 594 people recruited from a private health maintenance organization with a confirmed diagnosis of FM. The QWB was administered, along with measures of self-rated health status, physical functioning, pain, stiffness, anxiety, sleep, and depression. The QWB places levels of wellness on a continuum ranging from 0.0 (for death or the equivalent of being dead) to 1.0 (for optimum functioning without symptoms). RESULTS: Patients with FM had mean QWB scores of 0.559 (SD 0.074), which is lower than scores reported for patients in most other chronic disease categories. QWB was significantly correlated with measures of physical functioning, stiffness, anxiety, depression, pain, and sleep quality. CONCLUSION: Evidence supports the validity of the QWB for patients with FM. Patients with FM obtain lower scores on the QWB than patients with diagnoses of chronic obstructive pulmonary disease, rheumatoid arthritis, atrial fibrillation, advanced cancer, and several other chronic diseases. Although FM is generally considered a syndrome rather than a disease, substantial disability is experienced by people with this diagnosis. PMID- 10743826 TI - Perspectives on posttraumatic fibromyalgia: a random survey of Canadian general practitioners, orthopedists, physiatrists, and rheumatologists. AB - OBJECTIVE: To determine which factors physicians consider important in patients with chronic generalized posttraumatic pain. METHODS: Using physician membership directories, random samples of 287 Canadian general practitioners, 160 orthopedists, 160 physiatrists, and 160 rheumatologists were surveyed. Each subject was mailed a case scenario describing a 45-year-old woman who sustained a whiplash injury and subsequently developed chronic, generalized pain, fatigue, sleep difficulties, and diffuse muscle tenderness. Respondents were asked whether they agreed with a diagnosis of fibromyalgia (FM), and what factors they considered to be important in the development of chronic, generalized posttraumatic pain. RESULTS: More-recent medical school graduates were more likely to agree with the FM diagnosis. Orthopedists (28.8%) were least likely to agree, while rheumatologists (83.0%) were most likely to agree. On multivariate analysis, 5 factors predicted agreement or disagreement with the diagnosis of FM: (1) number of FM cases diagnosed by the respondent per week (p < 0.0001); (2) patient's sex (p < 0.0001); (3) force of initial impact (p = 0.003); (4) patient's pre-collision psychiatric history (p = 0.03); and (5) severity of initial injuries (p = 0.03). The force of initial impact and the patient's pre collision psychiatric history were both negatively correlated with agreement in diagnosis. Patient related factors (personality, emotional stress, pre-collision physical, mental health) were considered more important than trauma related factors in the development of chronic, widespread pain. CONCLUSION: Future studies of the association between trauma and FM should identify potential cases outside of specialty clinics, and baseline assessments should include some measurement of personality, stress, and pre-collision physical and mental health. PMID- 10743827 TI - The influence of age on nailfold capillary dimensions in childhood. AB - OBJECTIVE: To identify the influence of age on nailfold capillary dimensions in children between 6 and 15 years. METHODS: Capillary density (the number of capillaries in a 3 mm length of the distal row) and capillary dimensions were measured in 110 healthy children using the technique of nailfold video microscopy. The age groups studied were as follows: 6 to 7 years (17 children), 8 to 9 years (15 children), 10 to 11 years (34 children), 12 to 13 years (24 children), and 14 to 15 years (20 children). RESULTS: There was a significant trend for the arterial and venous diameters to rise with age. However, this trend was not present for apical or loop diameters, nor for the capillary density. Results did not differ between males and females. CONCLUSION: In studies incorporating capillary dimensions in children, results should ideally be age adjusted, and dimensions should not be categorized as normal or abnormal without taking the child's age into account. PMID- 10743828 TI - Apoptosis of peripheral blood mononuclear cells in Kawasaki disease. AB - OBJECTIVE: Kawasaki disease (KD) is an acute systemic vasculitis characterized by marked depletion of peripheral lymphocytes in the acute stage of the disease. We evaluated the degree of apoptosis of peripheral blood mononuclear cells (PBMC) in KD and investigated whether the Fas-Fas ligand system is involved in inducing apoptosis in KD. METHODS: Fifteen patients with KD were classified into 2 groups, Group KW1 (initial stage of disease, or fever < 5 days) and Group KW2 (fulminant stage of disease, or fever > 5 days). PBMC were isolated and after 5, 24, and 48 h incubation stained and analyzed by flow cytometry. The levels of serum soluble Fas were measured by ELISA. mRNA expression of Fas ligand was evaluated by reverse transcription polymerase chain reaction. RESULTS: The degree of apoptosis at 24 h was 20.2 +/- 5.6% in Group KW1, higher than that of normal (p < 0.05) and febrile controls (p < 0.01); and 10.8 +/- 2.8% in Group KW2, lower than that of normal (p < 0.01) and febrile controls (p < 0.01). At 48 h the degree of apoptosis was 37.7 +/- 4.2% in Group KW1, significantly higher than normal (p < 0.01) and febrile controls (p < 0.01); and 17.1 +/- 5.6% in Group KW2, lower than normal (p < 0.01) and febrile controls (p < 0.01). The mean level of serum soluble Fas in the acute stage of KD was 0.45 +/- 0.26 ng/ml, significantly lower versus the subacute stage (1.06 +/- 0.40 ng/ml) (p < 0.01). FasL of mRNA was expressed in PBMC of all patients with acute stage KD. whereas no expression was noted in the subacute stage of disease. CONCLUSION: Marked decrease of peripheral blood lymphocytes in acute stage KD may be caused by the upregulated apoptosis of lymphocytes; Fas-Fas ligand may be involved in this upregulation. PMID- 10743829 TI - Neonatal lupus erythematosus in triplets. AB - Neonatal lupus erythematosus (NLE) is an inflammatory syndrome in the fetus or neonate associated with the presence of anti-Ro(SSA) and anti-La(SSB) antibodies in the mother. It is characterized by a combination of dermatologic, hematologic, hepatic, and cardiac manifestations. NLE has been reported in twins; we describe neonatal lupus erythematosus occurring in triplets. PMID- 10743830 TI - Reversible methotrexate associated lymphoproliferative disease evolving into Hodgkin's disease. AB - We describe a case of nodular sclerosing Hodgkin's disease (NSHD) developing in a 61-year-old woman with seropositive rheumatoid arthritis treated with oral methotrexate (MTX) 5 to 15 mg/week for 5 years. Computed tomography (CT) of the abdomen revealed splenomegaly and marked abdominal and retroperitoneal lymphadenopathy. MTX was discontinued; several weeks later prednisone 10 mg/day was added to control worsening polyarthralgia. Repeat CT at 3 months showed almost complete regression of the splenomegaly and lymphadenopathy. However, CT studies at 10 months showed asymptomatic progression of lymphadenopathy, which on biopsy revealed NSHD. Patients with apparently reversible MTX associated lymphoproliferative disorder require periodic monitoring for asymptomatic development of malignant lymphoma. PMID- 10743832 TI - Evaluating the safety of self-injection of gold and methotrexate in patients with inflammatory arthritis. PMID- 10743831 TI - Hepatitis C virus infection with and without cryoglobulinemia as a case of Churg Strauss syndrome. AB - Chronic hepatitis C virus (HCV) infection may be associated with numerous immune disorders, with vasculitis including polyarteritis nodosa, or with both. Cryoglobulinemia, which is often present, can also be expressed by vasculitis. We describe 2 cases of Churg-Strauss syndrome (CSS) in patients with HCV infection. We found no previous case of CSS accompanying HCV infection in the literature. The current patients were women aged 40 and 66 years. In both cases, a clinical and laboratory pattern suggesting CSS was found before the HCV infection was discovered. One patient had cryoglobulinemia. One patient was successfully treated with interferon (IFN). The other was treated for 18 months with IFN and corticosteroids. Second-line therapy consisting of IFN with ribavirin was successful. The emergence of HCV infection may have led to an induced form of CSS. The relationship among HCV, cryoglobulinemia, and CSS is not clear, but may be similar to that existing between polyarteritis nodosa and hepatitis B virus. These observations suggest that IFN-alpha therapy may be effective against CSS in HCV infected patients with or without cryoglobulinemia. PMID- 10743833 TI - Henoch-Schonlein purpura associated with Toxocara canis infection. PMID- 10743834 TI - Hiccups with betamethasone dipropionate. PMID- 10743835 TI - A subset of rheumatoid factors crossreacts with cardiolipin in patients positive for IgM rheumatoid factor and anticardiolipin antibodies. PMID- 10743836 TI - Evidence of activation of 2 herpesviruses, Epstein-Barr virus and cytomegalovirus, in systemic sclerosis and normal skins. PMID- 10743837 TI - Antiphospholipid antibody syndrome associated with hepatitis C infection. PMID- 10743838 TI - Accuracy of self-reported family history in psoriatic arthritis. PMID- 10743839 TI - The childhood roots of bipolar disorder. PMID- 10743840 TI - Introduction. Current issues in childhood bipolarity. PMID- 10743841 TI - Prepubertal and early adolescent bipolarity differentiate from ADHD by manic symptoms, grandiose delusions, ultra-rapid or ultradian cycling. AB - BACKGROUND: In contrast to differential diagnosis (ddx) of older adolescent and adult bipolarity (BP), which includes schizophrenia and substance use disorders, the main ddx of prepubertal and early adolescent BP is attention-deficit disorder with hyperactivity (ADHD). To address this ddx issue, and to provide prepubertal mania manifestations, interim baseline data are presented from the National Institute of Mental Health (NIMH)-funded study 'Phenomenology and Course of Pediatric Bipolarity'. METHODS: Data are from the first 60 BP and the first 60 ADHD cases from 270 consecutively ascertained subjects (90 BP, 90 ADHD and 90 community controls). Comprehensive assessments included the Washington University at St. Louis Kiddie and Young Adult-Schedule for Affective Disorders and Schizophrenia--Lifetime and Present Episode Version-DSM-IV (WASH-U-KSADS) blindly administered by nurses to mothers about their offspring and to children/adolescents about themselves. Caseness was established by consensus conferences that included diagnostic and impairment data, teacher and school reports, agency records, videotapes and medical charts. RESULTS: Mean baseline age of BP cases was 11.0+/-2.7 years and the mean age at onset of BP was 8.1+/ 3.5 years. Elated mood, grandiosity, hypersexuality, decreased need for sleep, racing thoughts and all other mania items except hyperenergetic and distractibility were significantly and substantially more frequent among BP than ADHD cases (e.g., elation: 86.7% BP vs. 5.0% ADHD; grandiosity: 85.0% BP vs. 6.7% ADHD). In the BP group, 55.0% had grandiose delusions, 26.7% had suicidality with plan/intent and 83.3% were rapid, ultra-rapid or ultradian cyclers. LIMITATIONS: Sites for consecutive case ascertainment from the lowest socioeconomic status classes were unavailable due to current health care policies. CLINICAL RELEVANCE: Prepubertal and early adolescent BP cases differentiate from ADHD by mania specific criteria and commonly present with ultra-rapid or ultradian cycling. PMID- 10743842 TI - Prepubertal and young adolescent bipolarity versus ADHD: assessment and validity using the WASH-U-KSADS, CBCL and TRF. AB - BACKGROUND: This addendum to 'Prepubertal and early adolescent bipolarity differentiate from ADHD by mania criteria; grandiose delusions; ultra-rapid or ultradian cycling' (in this volume) provides (1) a description of Washington University at St. Louis Kiddie Schedule for Affective Disorders and Schizophrenia (WASH-U-KSADS) with sample sections (hypersexuality, rapid cycling); (2) a comparison of WASH-U-KSADS to KSADS-P/L and KSADS-1986 and (3) a comparison of WASH-U-KSADS to Child Behavior Checklist (CBCL) and Teachers Report Form (TRF) data. METHODS: Data were from the first 60 bipolar (BP) and first 60 ADHD subjects of 270 consecutively ascertained cases (90 BP, 90 ADHD and 90 community controls) in the NIMH funded 'Phenomenology and Course of Pediatric Bipolarity' study. Comprehensive assessments included the WASH-U-KSADS (administered blindly to mothers and separately to children), CBCL and TRF. RESULTS: As reported elsewhere in this volume, WASH-U-KSADS data significantly differentiated BP and ADHD groups. Significant differences were also found with the parent-rated CBCL and the teacher-rated TRF, thereby providing cross-modality and cross-informant validation of the WASH-U-KSADS. Because of the close agreement with published CBCL data from another investigator, cross-site validation also occurred. LIMITATIONS: Venues for consecutive ascertainment from the lowest socioeconomic status classes were unavailable due to current health care policies. CLINICAL RELEVANCE: CBCL and TRF data separated BP from ADHD groups, largely by non specific externalizing dimensions (e.g., hyperactivity, aggressivity). Clinically relevant differentiation by categorical mania-specific criteria (e.g., elated mood, grandiosity, racing thoughts) occurred with WASH-U-KSADS data. Both types of data are crucial for genetic and neurobiological studies. PMID- 10743843 TI - Clinical features of children with both ADHD and mania: does ascertainment source make a difference? AB - OBJECTIVE: We evaluated the structural diagnostic results of children ascertained through an ADHD diagnosis with comorbid mania to determine if they have the same phenotype as children ascertained through a mania diagnosis with comorbid ADHD. METHOD: We compared a sample of children participating in a family genetic study of ADHD to a sample of children ascertained through a study of childhood mania. RESULTS: Similar correlates of ADHD and mania were observed in children satisfying criteria for both disorders irrespective of ascertainment source. CONCLUSIONS: Findings suggest that children with mania and ADHD have two disorders, their features not varying with the primary diagnostic focus. LIMITATIONS: The results may have been limited by small sample size. CLINICAL RELEVANCE: Because the coexistence of ADHD and mania seriously complicates the course and treatment of children, understanding the compatibility of these disorders has important clinical implications in the management of this population. PMID- 10743844 TI - Young referred boys with DICA-P manic symptoms vs. two comparison groups. AB - A total of 23 boys met DICA-P manic symptom and clustering criteria in a diagnostic investigation of 233 outpatient boys between ages 6 and 10. In this manic-symptom group, the most frequently endorsed of an average of five manic symptoms were extreme mood changes, difficulty concentrating, feeling too 'up' to sit still, and racing thoughts. Comparison groups were 23 non-manic boys seen next in the investigation and 23 non-manic boys matched to the manic-symptom boys on symptoms of three comorbid disruptive disorders (ADHD, ODD and CD). Manic symptom boys differed significantly from next-seen boys, but not from matched comorbid boys, in number of oppositional symptoms and pervasiveness of problems. Manic-symptom boys differed significantly from next-seen boys on six of eight mother-rated RCBCL factors. In contrast, manic-symptom and matched comorbid boys did not differ on any of eight RCBCL factors, which suggests that the RCBCL differences can be attributed to shared ADHD, ODD and/or CD. However, manic symptom and matched comorbid boys tended to differ on RCBCL Anxiety/Depression. On the teacher-rated TRF, manic-symptom boys were rated higher than next-seen boys on four internalizing factors, and higher than matched comorbid boys on two of those factors, including Anxiety/Depression. Thus, manic symptomatology also predicted substantial emotionality, which was not a controlled comorbidity. The findings of this and other studies suggest that there is a mania dimension or syndrome, which may be an indicator of true bipolar disorder--or simply a marker for disruptive comorbidity, behavioral and emotional multimorbidity, or general severity of psychopathology. PMID- 10743845 TI - Manic symptoms in psychiatrically hospitalized children--what do they mean? AB - OBJECTIVE: To examine the clinical implications of manic symptoms in psychiatrically hospitalized children aged 5-12. METHODS: DSMIIIR manic symptoms, along with symptoms of other psychiatric disorders, were rated by parents and teachers on the Child Symptom Inventory IIIR prior to hospitalization. The Child Behavior Checklist (CBCL; was also completed. During hospitalization children were evaluated by structured interview (K-SADS-E), and numerous rating scales weekly. Children with symptoms of mania (mania criteria with/without episodes) were compared to those without mania. Severity of attention deficit hyperactivity disorder (ADHD), oppositional defiant disorder (ODD), depression, CBCL factors, and comparable factors from teacher and parent inpatient rating scales were examined. Finally, a subgroup of both groups of children treated with stimulants were compared at baseline and at least two weeks of treatment. RESULTS: Children with manic symptoms had more severe ADHD, ODD and depression symptoms. CBCL scores on aggression, social and thought problems were higher. Teachers and nursing staff made similar observations. Time in hospital was greater for children with manic symptoms. Both groups improved significantly on stimulant medication though reduction in overall psychopathology was often modest. CONCLUSIONS: Manic symptoms, regardless of whether or not they represent bipolar disorder, are a marker of serious psychopathology and treatment resistance. PMID- 10743846 TI - Premorbid functioning in adolescent onset bipolar I disorder: a preliminary report from an ongoing study. AB - BACKGROUND: This study reports on premorbid academic and peer functioning and psychiatric illness in a rigorously diagnosed sample (N = 28) of adolescent onset bipolar I patients. METHODS: Premorbid functioning was assessed by parental report and review of the Ontario School Record (OSR). Premorbid psychiatric diagnoses were assigned on the basis of all information gathered. RESULTS: Overall, findings suggest that this cohort demonstrates good to excellent peer and academic functioning prior to illness onset. Rates of premorbid psychiatric illnesses were similar to that described in epidemiologic samples. CONCLUSIONS: Results are discussed in relation to current understanding of early onset bipolar illness and directions for future research. PMID- 10743847 TI - Early childhood attention deficit hyperactivity disorder predicts poorer response to acute lithium therapy in adolescent mania. AB - We compared the response to acute lithium therapy in 30 adolescents, 13-17 years of age, with mania and a prior history of early childhood attention deficit hyperactivity disorder (ADHD) to a sex- and age-matched control group of adolescent manics without premorbid psychiatric illness. Response to treatment was assessed daily over the course of 28 days using measures of global clinical improvement and severity ratings on the Bech-Rafaelsen Mania Scale (BRMS). BRMS scores decreased by a mean of 24.3 in the subgroup without prior ADHD compared to 16.7 in patients with ADHD (P = 0.0005). The average percent drop in BRMS scores over the study period in these two subgroups was 80.6% and 57.7%, respectively (P = 0.0005). Time to onset of sustained global clinical improvement was also assessed using Kaplan-Meier survival methods and possible covariates of time to improvement were tested in a Cox proportional hazards model. Median time to onset of sustained improvement was lengthened significantly in patients with early ADHD (23 days) compared to those without it (17 days; log rank chi2 = 7.2, P = 0.007). The results suggest that early childhood ADHD defines an important source of heterogeneity in bipolar illness with developmental, clinical, and neuropharmacogenetic implications. PMID- 10743849 TI - Lithium for prepubertal depressed children with family history predictors of future bipolarity: a double-blind, placebo-controlled study. AB - BACKGROUND: Because of negative studies of TCAs for prepubertal major depressive disorder (PMDD) and because of the potentially high switch rate of PMDD to prepubertal bipolarity (BP), it was hypothesized that lithium would be efficacious treatment for PMDD in children who also had family history (FH) predictors of future BP. METHODS: A double-blind, placebo-controlled, and pharmacokinetically dosed study of lithium for PMDD with FH predictors of future BP was performed. Random assignment was stratified by FH of BP-I or mania versus loaded/multigenerational (L/M) FH of MDD without BP-I or mania. Comprehensive assessments were done during a six week outpatient protocol that included weekly serum lithium levels. RESULTS: Mean age was 10.7+/-1.2 years; 17 subjects were randomized to active and 13 to placebo; 80% had FH of BP-I or mania (40% of parents had BP-I or mania); and 20% had FH of L/M MDD. Using both intent to treat with last observation carried forward (n = 30) and completer (n = 24) analyses, there were no significant differences on continuous or categorical measures between active and placebo groups. Mean serum lithium level was 0.99+/-0.16 mEq/l. There were no significant differences between mean total daily dose or mean serum lithium levels between responders and non-responders. LIMITATIONS: Four subjects on active drug were discontinued because of dose-limiting side effects (three were cognitive impairment). Future studies of treatment for PMDD should consider alternative drugs. CLINICAL RELEVANCE: Lithium was not significantly more efficacious than placebo for PMDD with FH predictors of future BP. PMID- 10743848 TI - Are childhood psychiatric histories of bipolar adolescents associated with family history, psychosis, and response to lithium treatment? AB - OBJECTIVE: To examine if childhood psychiatric diagnoses are associated with family history, psychosis, age, and lithium response. METHOD: Associations among variables, and their contributions to explaining lithium response were examined in 48 bipolar adolescents enrolled in a study of lithium. RESULTS: Presence of a childhood diagnosis was not associated with family psychiatric history or lithium response. Subjects with psychotic features, however, were less likely to have a childhood psychiatric diagnosis, were older, and had a poorer response rate to lithium than subjects without psychosis. DISCUSSION: Heterogeneity within bipolar adolescents may be based on clinical features such as psychosis rather than childhood or family history alone. PMID- 10743850 TI - Mania and ADHD: comorbidity or confusion. AB - The frequency of occurrence of prepubertal mania is contingent on how much adherence to episodic disorder with separate periods of mania and depression is required. While manic symptoms superimposed on other psychiatric disorders is not uncommon, non-comorbid bipolar disorder is rare. A number of developmental, phenomenological and assessment considerations may complicate simple extrapolation of adult criteria onto young children. Nevertheless, it is clear that a significant number of preadolescents found in outpatient and inpatient samples meet at least symptom criteria for bipolar disorder. Such children have significant comorbidity and impairment. It is likely that some may develop classical bipolar disorder, some will continue to have substantial affective and behavioral comorbidity as do some complicated bipolar adults, and some will continue to have affective lability superimposed on their other, primary psychiatric disorders. Further research and follow-up will be necessary to determine who develops which outcome. PMID- 10743851 TI - A rose is a rose is a rose. AB - This young man presented initially and repeatedly over a period of twenty years with symptoms of irritability, depressed and withdrawn mood, impulsivity, out-of control behavior, and suicidal ideation and behavior. These symptoms occurred with disabling intensity at ages 3 year, 6 1/2 years, and 8 1/2 years, when he had not yet been adequately diagnosed and treated with appropriate psychopharmacology. Symptoms re-occurred in early adulthood after medication had been discontinued by the patient. While he had symptoms characteristic of ADHD before age seven, these were present episodically and only in the context of significant mood disturbance and behavior disruption, which were more characteristic of mood disorder than oppositional behavior associated with ADHD. His reaction to stimulants alone was significant. His response to Ritalin suggested dysphoria and/or neurovegetative slowing. On Dexedrine, he developed frank visual hallucinations. Both reactions can be typically seen in youths with underlying mood or psychotic disorders. This patient's symptoms and their repeated occurrence, in the face of significant family history of mood disorder, demonstrate the utility of DSM-IV criteria and longitudinal history taking in making the diagnosis of bipolar disorder in prepubertal children. PMID- 10743852 TI - "George". PMID- 10743853 TI - A prospective study of stability and change over 2 years of affective temperaments in 14-18 year-old Italian high school students. AB - OBJECTIVE: It is generally accepted that temperament is not entirely stable, and that it changes with development, particularly in juvenile subjects; also, some temperaments are believed to be inherently more unstable. There is a great deal of current interest in Kraepelin's thesis that temperamental dysregulation in juvenile subjects represents the constitutional foundation from which the more florid episodes of manic-depressive illness arise; the cyclothymic, hyperthymic, depressive and irritable temperaments under consideration might represent the first observable phenotypes of the genetic diathesis for bipolarity. The analyses on the temperamental attributes in juvenile subjects were undertaken within this theoretical framework. METHOD: We evaluated 206 Italian high school students (14 18 years old) by means of a semi-structured affective temperament interview (TEMPS-I) at T0 and T1 two years later. Age, sex and psychometric properties of TEMPS-I raw scale score and weighted cut-off (as specially weighted linear combination of items) were used as predictive variables of stability. RESULTS: Affective temperaments had a low to moderate level of stability, reaching 60% in the case of subjects with dominant cyclothymic temperament. The stability of the depressive temperament was primarily related to its weighted cut-off. The stability of the hyperthymic temperament appeared related to male sex, young age, and total scale score. Male sex represented the best stability predictor for the cyclothymic temperament as well. The group of subjects with an unstable depressive temperament showed a change toward the dominant cyclothymic temperament, whereas individuals with unstable hyperthymic temperamental traits moved on towards the dominant cyclothymic and depressive temperaments. The irritable construct was the least stable. LIMITATIONS: The infeasibility of a multiwave design represents the main limitation in evaluating the predictors of stability. Furthermore, in the present analyses, the size of the cyclothymic subsample was small. CONCLUSION: Our data indicate considerable fluctuation and instability in depressive and hyperthymic temperaments in mid-adolescence. The cyclothymic temperament appears to be the most stable. Interestingly, cyclothymic moodiness appears more persistent in juvenile males; likewise persistent hyperthymic traits appear more of a "male" attribute. CLINICAL AND PUBLIC HEALTH IMPLICATIONS: We submit that these sex-relevant traits could be important in the risk of developing juvenile bipolarity. Literature review indicates that clinical studies, albeit on small samples, have already provided some support for this thesis. Larger studies on epidemiological samples could be more informative from a public health perspective. A user-friendly affective temperament questionnaire, which is under development, is critical for the methodology of such studies. Our study indicates that the present version of the Akiskal-Malya questionnaire can be easily used post-pubertally. Age adjustment must be considered for younger subjects. PMID- 10743855 TI - Palaearctic species of the genus Confluaria Ablasov (Cestoda, Hymenolepididae): redescriptions of C. podicipina (Szymanski, 1905) and C. furcifera (Krabbe, 1869), description of C. pseudofurcifera n. sp., a key and final comments. AB - Redescriptions are provided of Confluaria podicipina (Szymanski, 1905) (specimens from Podiceps nigricollis and Tachybaptus ruficollis from Bulgaria) and C. furcifera (Krabbe, 1869) (syntypes from P. grisegena from Denmark and specimens from P. grisegena, P. nigricollis and T. ruficollis from Bulgaria). C. pseudofurcifera n. sp. is described from P. cristatus from Switzerland and Bulgaria. The previous records of C. furcifera from P. cristatus in Switzerland (Joyeux & Baer, 1950), Poland (Jarecka, 1958; Korpaczewska, 1960), Czech Republic (Rysavy & Sitko, 1995) and Baltic Coast (Galkin, 1986) are recognised as belonging to C. pseudofurcifera. Confluaria Ablasov in Spasskaya, 1966 is recognised as a valid genus and Dimorphocanthus Maksimova, 1989 is confirmed as its synonym. A key to the Palaearctic species of Confluaria is presented. PMID- 10743854 TI - Re-validation of Echinostoma miyagawai Ishii, 1932 (Digenea: Echinostomatidae) on the basis of the experimental completion of its life-cycle. AB - The life-cycle of Echinostoma miyagawai, a Eurasian species closely related to E. revolutum, was completed in the laboratory, and the morphology of the larval stages and the adults obtained experimentally was studied. Planorbis planorbis and Anisus vortex were the first intermediate hosts in the brackish Lake Durankulak on the Bulgarian Black Sea coast. Characteristic features of the cercaria include: a prominent collar with 37 spines; a tail as long as the body and with seven conspicuous fin-folds, the two ventral fin-folds being very close to each other; and a specific number and distribution of both the para oesophageal gland-cell outlets and sensilla. The adult is characterised by: a very elongate body with a constriction at the posterior border of the ventral sucker; a large head collar with relatively small spines; a spherical ventral sucker which is only about half the maximum body width; a long cirrus-sac reaching posteriorly dorsal to the middle of the ventral sucker; indented subglobular testes; and a vitellarium forming two lateral fields of follicles which are almost confluent in the post-testicular space. The species described in this study resembles E. miyagawai, as described by Kosupko, in the morphology of larval stages and both the site and the general morphology of the adults. It differs from both E. revolutum, as described by both Kanev and Nasincova, and E. echinatum (also referred to as E. lindoense and E. barbosai by Kanev). The re examination of Kanev's voucher specimens from his experimental studies used in his delimitation of E. revolutum and E. echinatum showed that the specimens identified by him as E. revolutum represent two distinct forms which consistently differ both from each other and from the redescription of E. revolutum which was based upon them. It also revealed that a number of specimens were wrongly identified and erroneously treated as E. echinatum by Kanev and co-workers; these include members of different genera (Hypoderaeum and Echinoparyphium) and an Echinostoma species of the group possessing 47 collar spines. The relative merits of the features used by Kanev and co-workers in discriminating the closely related Echinostoma spp. are discussed in detail with respect to the experimental evidence provided by these authors. PMID- 10743856 TI - Description of Acanthotrema armata n. sp. (Trematoda: Heterophyidae) from Larus audouinii (Aves: Laridae), with an amended diagnosis of the genus Acanthotrema Travassos, 1928. AB - Acanthotrema armata n. sp. (Trematoda: Heterophyidae) is described from the gut of Larus audouinii Payraudeau (Aves: Laridae) caught on the Chafarinas Islands in the southwestern Mediterranean (Spain). This trematode differs from other species of Acanthotrema Travassos, 1928 in having two spined, sclerotised pieces in the ventrogenital sac and in the position of the genital pore. Parastictodora Martin, 1950 is considered a synonym of Acanthotrema. As the diagnosis of the genus Acanthotrema was based only on its type-species, it is amended to include the characteristics of the new species and those attributed to Parastictodora. PMID- 10743857 TI - Morphological studies of Ancyracanthopsis winegardi Wong & Anderson, 1990 (Nematoda: Acuarioidea) and larval stages of acuariid nematodes parasitic in Larus dominicanus Lichtenstein (Aves: Laridae) from Argentina. AB - Ancyracanthopsis winegardi Wong & Anderson, 1990 (Nematoda: Acuarioidea) is described from Larus dominicanus Lichtenstein (Aves: Laridae) on the Southwest Atlantic coast (38 degrees 42 minutes S, 59 degrees 47 minutes W). The main character used to distinguish species of Ancyracanthopsis is the morphology of the ptilina. Thus, although the specimens described here have some differences in the morphology and size of the spicules and in the female genitalia, they were referred to A. winegardi because they have a very similar ptilina. This is the first record of a member of Ancyracanthopsis from larid birds and for A. winegardi in the Southwest Atlantic coast. We have also studied acuariid larvae found inhabiting the gizzard alongside adult specimens of A. winegardi. Among those larvae, two morphological groups were clearly distinguished. The first group was characterised by the absence of ptilina and the presence of spicular primordia and rectal cells (third-stage larvae). The second group could be distinguished by the presence of ptilina and partly-developed genitalia (fourth stage larvae). In order to identify the larvae, a Principal Component Analysis was applied to morphometric data taken from the third-stage larvae. These results and the morphology of the partly-developed ptilina of the fourth-stage larvae indicated that the larval stages found in L. dominicanus appear to belong to Sciadiocara haematopodi, Cremonte, Navone & Etchegoin, 1999. PMID- 10743858 TI - The status of Plasmodium corradettii Laird, 1998 (Haemosporida: Plasmodiidae): a malarial parasite of birds. AB - The type-material of Plasmodium corrcadettii Laird, 1998, a malarial parasite of birds, has been examined and compared with the original description. It is concluded that the validity of P. corradettii as a distinct species is questionable and is herein declared to be a nomen dubium. PMID- 10743859 TI - Pseudorhabdosynochus spp. (Monogenea: Diplectanidae) from the gills of Epinephelus spp. in Brazilian waters. AB - Pseudorhabdosynochus sulamericanus n. sp. from the gills of Epinephelus niveatus has a reniform proximal region of the cirrus-bulb which is divided in four chambers and contains a large, round reservoir of the male accessory glands, a partly sclerotised vagina which is enclosed in a muscular funnel cap and squamodiscs with 15-16 open concentric rows of elements. P. beverleyburtonae (Oliver, 1984) is redescribed from E. marginatus with additional morphological data. These are the first reports of Pseudorhabdosynochus spp. in South American Atlantic waters, where the potentiality for the mariculture of Epinephelus spp. is currently being evaluated. Pseudorhabdosynochus hargisi (Oliver & Paperna, 1984) n. comb. is proposed for Diplectanum hargisi. PMID- 10743860 TI - Detection of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2(PAI-2) in gingival crevicular fluid from healthy, gingivitis and periodontitis patients. AB - BACKGROUND: The regulation of plasminogen activation is a key element in controlling proteolytic events in the extracellular matrix. Our previous studies had demonstrated that in inflamed gingival tissues, tissue-type plasminogen activator (t-PA) is significantly increased in the extracellular matrix of the connective tissue and that interleukin 1beta(IL-1beta) can up regulate the level of t-PA and plasminogen activator inhibitor-2 (PAI-2) synthesis by human gingival fibroblasts. METHOD: In the present study, the levels of t-PA and PAI-2 in gingival crevicular fluid (GCF) were measured from healthy, gingivitis and periodontitis sites and compared before and after periodontal treatment. Crevicular fluid from106 periodontal sites in 33 patients were collected. 24 sites from 11 periodontitis patients received periodontal treatment after the first sample collection and post-treatment samples were collected 14 days after treatment. All samples were analyzed by enzyme-linked immunosorbent assay (ELISA) for t-PA and PAI-2. RESULTS: The results showed that significantly high levels of t-PA and PAI-2 in GCF were found in the gingivitis and periodontitis sites. Periodontal treatment led to significant decreases of PAI-2, but not t-PA, after 14 days. A significant positive linear correlation was found between t-PA and PAI 2 in GCF (r=0.80, p<0.01). In the healthy group, different sites from within the same subject showed little variation of t-PA and PAI-2 in GCF. However, the gingivitis and periodontitis sites showed large variation. These results suggest a good correlation between t-PA and PAI-2 with the severity of periodontal conditions. CONCLUSION: This study indicates that t-PA and PAI-2 may play a significant role in the periodontal tissue destruction and tissue remodeling and that t-PA and PAI-2 in GCF may be used as clinical markers to evaluate the periodontal diseases and assess treatment. PMID- 10743861 TI - Effect of an essential oil-containing antiseptic mouthrinse on plaque and salivary Streptococcus mutans levels. AB - BACKGROUND: Clinical studies in which antimicrobial mouthrinses were shown to have significant antiplaque activity most frequently have used gingivitis as the clinically relevant endpoint. However, there is evidence to suggest that mouthrinses containing active agents effective against Streptococcus mutans, such as chlorhexidine, may also have a role in inhibiting dental caries. This clinical study was conducted to determine the effect of 2x-daily rinsing with an essential oil-containing antiseptic mouthrinse (Listerine Antiseptic) on levels of recoverable S. mutans and total streptococci in supragingival interproximal plaque and in saliva. Additionally, a follow-up in vitro study is reported which determined whether a differential susceptibility to the antiseptic mouthrinse exists among different strains of streptococci. METHOD: Following baseline saliva and plaque sampling for quantification of recoverable S. mutans and total streptococci, 29 qualifying subjects were randomly assigned either the essential oil mouthrinse or a sterile water control. They rinsed with 20 ml for 30s 2 x daily for 11 days and once on the 12th day, in addition to their usual oral hygiene procedures. On day 12, saliva and plaque samples were again collected and microbiological quantification performed. The procedures were repeated with the alternate rinse after a 1-week washout period. RESULTS: The essential oil mouthrinse produced respective reductions of 69.9% and 75.4% in total recoverable streptococci and in S. mutans in plaque, and corresponding reductions of 50.8% and 39.2% in saliva. The in vitro study revealed that streptococci from the mutans group were more susceptible to the bactericidal activity of the essential oil mouthrinse than streptococci from the mitis group. CONCLUSIONS: As antimicrobial mouthrinses are most frequently recommended to patients whose mechanical oral hygiene procedures are not adequate for the control of supragingival plaque and gingivitis, this study provides an additional rationale for the inclusion of the essential-oil mouthrinse as an adjunct to daily oral hygiene procedures. PMID- 10743862 TI - Regenerative periodontal surgery in interproximal intrabony defects with biodegradable barriers. AB - AIM: The comparison of the effects of guided-tissue regeneration (GTR) using 2 different biodegradable barriers (polylactide acetyltributyl citrate; polydioxanon) in 3- and 2-wall intrabony defects. METHOD: The polydioxanon barrier is an experimental membrane for GTR therapy that consists of an continuous occlusive barrier that has a layer of slings on the side that is meant to face the mucoperiosteal flap. 15 patients provided 15 pairs of similar contralateral periodontal defects: 12 predominantly 2-wall and 18 predominantly 3 wall intrabony defects. Each defect was randomly assigned to treatment with either polylactide acetyltributyl citrate (control [c]) or polydioxanon (test [t]) devices. At baseline and 6 months after surgery, clinical measurements (P1I, GI, PPD, PAL-V) were performed. RESULTS: Barrier exposure was commonly observed in both groups (control/test): 5/4 after 7 days, 9/11 after 14 days and 11/12 after 28 days postsurgically. 4 weeks after surgery, 77% of all barriers were exposed to some extent. However, both treatments revealed a significant GI reduction (p<0.05), PPD reduction [-4.63+/-1.85 mm (t), -4.17+/-1.89 mm (c); p<0.001] and PAL-V gain [3.97+/-1.17 mm (t), 3.40 mm+/-1.40 (c); p<0.001] 6 months after surgery. Regarding GI and PPD reduction as well as PAL-V gain, there were neither statistically significant nor clinically relevant differences between test and control: similar clinical results were found 6 months after surgical treatment using both biodegradable barriers. CONCLUSIONS: Based on the results of the present study, the use of both biodegradable barriers in GTR therapy may be recommended. PMID- 10743863 TI - Treatment of intrabony defects with resorbable materials, non-resorbable materials and flap debridement. AB - BACKGROUND: Different types of barriers are used in guided tissue regenerative procedures. AIM: This prospective study compared resorbable citric acid ester softened polylactic acid membranes (RM) and non-resorbable expanded polytetrafluoroethylene (ePTFE) barriers (NRM) in GTR treatment of intrabony defects. METHODS: 29 subjects were randomly assigned to the RM group or NRM group. Each patient received one GTR procedure. An open flap debridement (FD) was performed at another site 2 weeks later to evaluate healing potential. Clinical treatment outcomes were finally evaluated 12 months after surgery for changes of pocket depth PD, probing attachment level PAL, and probing bone level PBL, and radiographically for bone change using standardised radiographs. RESULTS: No differences in healing patters after surgery were found between patients in the 2 study groups as evaluated from the FD surgical procedures. NRM treated sites showed less signs of post-surgical inflammation during the 1st 4 weeks of healing than did RM treated sites (p<0.05). GTR-treated defects in the RM group, initially 7.0+/-2.2 mm deep, showed PD reduction of 3.3+/-2.2 mm, PAL gain of 2.4+/-1.8 mm, PBL gain of 2.4+/-3.7 mm (28%) and a radiographic bone fill of 2.3+/-2.4 mm. Defects treated with the NRM exhibited PD reduction of 3.1+/-2.1 mm, PAL gain of 2.4+/-0.8 mm, PBL gain of 2.2+/-1.7 mm (25%) and a radiographic bone fill of 3.3+/-2.2 mm. All improvements were statistically significant (p<0.01) but there was no difference between RM and NRM treatments for any of the efficacy variables. The results of this study indicated that there was no clinically significant difference in treatment outcomes following GTR treatment of intrabony defects with citric acid ester softened polylactic acid membranes as compared to ePTFE barriers. The overall mean inter-proximal vertical bone defect fill at 12 months as assessed from intra-oral radiographs was 44% of the original mean defect depth. CONCLUSIONS: Thus, no clinically significant difference in treatment outcomes was observed following GTR treatment of intrabony defects with citric acid ester softened polylactic acid membranes or ePTFE barriers. PMID- 10743864 TI - Comparisons of clinical and radiographic measurements of inter-proximal vertical defects before and 1 year after surgical treatments. AB - BACKGROUND: Radiographic measurements are often used as a substitute for direct clinical measurements requiring re-entry surgery for follow-up outcome studies. AIMS: (1) To assess the reliability of clinical and radiographic measurements of periodontal defects as compared to direct bone measurements during surgical procedures, and (2) to assess the associations between selected clinical and radiographic measurements of periodontal inter-proximal defects. METHODS: 57 inter-proximal periodontal defects were measured at baseline and at 12 months after surgical treatment. Direct measurements during surgery of the distance between the CEJ to the bottom of defects (ABL) were compared with probing to bone (PB), probing attachment level (PAL), and radiographic measurements. RESULTS: Probing to bone is an accurate measure to assess inter-proximal bone level as compared to ABL (mean difference: 0.1 mm) and that intra-oral standardized radiographs underestimate bone level and defect depth by approximately 1.4 mm. The interpretation of periodontal changes between baseline and 12 months after treatment by probing to bone, or PAL measurements, or from radiographic images yield almost identical results (mean difference< or =0.2 mm). For the assessments of changes over time using PB change as the standard, intra-class correlation (ICC) coefficients varied between 0.52 to 0.90. The best ICC coefficient was found for relative attachment level change assessed by the Florida probe (0.90), and with an ICC value of 0.61 for changes assessed from intra-oral radiographs. Two-way analysis of variance failed to demonstrate differences between sets of comparisons. CONCLUSIONS: Both radiographic interpretations of changes over time, and measurements of attachment level changes are reliable in assessing the treatment outcome of inter-proximal intra-bony defects when compared to probing to bone changes as the standard method. PMID- 10743865 TI - Periodontal flap surgery with 25% metronidazole gel. (1). Clinical outcomes. AB - OBJECTIVES: The aim of this 12-month, randomised controlled, single-blind, parallel-arm study, was to test whether local application of a slow-release antimicrobial might exert an adjunctive effect on healing following periodontal surgery. MATERIALS AND METHODS: Following non-surgical therapy, 43 patients with moderate to advanced periodontitis, had at least 1 pocket > or =6 mm with bleeding on probing (BOP). Treatment consisted of modified Widman surgery with no osseous resection. The test group (T) received gel application over the exposed root surface and the control group (C) received no gel, followed by flap closure. RESULTS: 38 patients completed the study: 5 patients did not attend at 12 months and could not be traced. Surgery was very successful in both groups in improving probing depths (mean baseline: T=7.4 mm, C=7.1 mm; 12 months T=3.7 mm, C=3.5 mm) and relative attachment levels (baseline: T=10.3 mm, C=10.1 mm; 12 months T=8.2 mm, C=8.8 mm). Differences between groups were not significant at 12 months, although the improvements within groups between baseline and 12 months were highly statistically significant (p<0.01). Plaque and BOP levels were low in both groups after treatment. CONCLUSION: MWF surgery was effective in establishing the periodontal health of sites with advanced disease and this study was unable to detect an additional benefit to using a 25% metronidazole gel. PMID- 10743866 TI - Periodontal flap surgery with 25% metronidazole gel. (2). Effect on gingival crevicular fluid PGE2. AB - OBJECTIVES: The aim of this study was to examine the effect of periodontal surgery on PGE2 levels in patients treated for advanced adult periodontitis, with and without application of a 25% metronidazole gel over 12 months follow-up. MATERIAL AND METHODS: The trial employed a randomised controlled, single-blind, parallel arm design on 43 patients with at least 1 pocket probing > or =6 mm with bleeding on probing (BOP). RESULTS: The results showed that GCF PGE2 levels showed no significant change following surgical therapy despite marked improvements in clinical periodontal health (p=0.653). No significant differences in the reaction of sites to treatment were detected between test and control groups throughout the study (p=0.709). However, PGE2 levels in GCF were significantly higher in experimental sites compared with healthy untreated sites (negative controls) in both surgery only (p= 0.001) and surgery+gel (p=0.023) groups throughout the study. CONCLUSION: Whilst clear differences were observed between healthy control sites and sites with periodontal breakdown, no effect of treatment on PGE2 levels was discernible in this study. PMID- 10743867 TI - Periodontal healing after intentional auto-alloplastic reimplantation of injured immature upper front teeth. AB - BACKGROUND/AIMS: Conventional endodontic treatment results in high complication quota when performed in immature teeth. Intentional reimplantation with extraoral insertion of an endodontic implant (auto-alloplastic reimplantation) is an alternative. METHOD: In a retrospective study, the healing of 40 teeth reimplanted intentionally according to this method in patients aged 7 to 15 years was evaluated. Clinical tests (palpation, percussion sound, periotest values) and radiographical examinations were used to determine the type of periodontal healing (inflammatory resorption/periodontitis apicalis; replacement resorption/ankylosis; normal healing). RESULTS: Mean lifetime of the replanted teeth was 59.2+/-42.5 months, estimated survival time on the basis of the Kaplan Meier analysis was 99.5 months. 17 teeth (42.5%) were classified as failures, mostly due to inflammatory resorption or periodontitis apicalis. Further investigations demonstrated that success rate and retention period of intentionally replanted teeth depend on the preoperative condition of the pulp. Teeth with preoperative infection suffered frequently from inflammatory resorption or periodontitis apicalis after being replanted (14 of 28 teeth). Estimated survival time according to Kaplan-Meier was 75.5 months. In contrast, inflammations or progressive resorptions were not observed in teeth without preoperative infection of the pulp. All these 12 teeth showed normal periodontal healing and regular tooth mobility. In the absence of any pathology in clinical or radiological findings after an average functional period of 72.3 months, the prognosis can be presumed excellent. Estimated survival time of 148.3 months according to Kaplan-Meier differs significantly from survival time of teeth infected preoperatively. CONCLUSIONS: From the results of this investigation, it may be concluded that an infection of the pulp - due to delay of treatment or attempts at endodontic therapy - should be avoided before intentional replantation of immature front teeth with pulp necrosis. Periodontal healing of the autologous root is not impaired by the insertion of posts made of Al2O3 ceramics or titanium. The inserted posts do not ankylose. Orthodontic movement of auto-alloplastically replanted teeth is possible. PMID- 10743868 TI - Attributions to dental and diabetes health outcomes. AB - Previous studies have proposed common psychological factors between oral health behavior and diabetes self-care. The aim here was to describe and analyse more comprehensively the relationships between dental and diabetes health behavior on the basis of attribution theory. The likeness between subjects' own assessments, similarities of the causes given to success and failure, and the predictive power of own dental assessments concerning the metabolic balance of diabetes were studied. The research population was composed of 149 IDDM patients. Data were collected by means of a quantitative questionnaire, a clinical oral examination and from patient records. It was found that from the patients reporting success with avoiding gingivitis 82% also reported success with metabolic status and they also had lower mean HbA1c levels than patients assessing failure with gingivitis. There were some correlations between causes of failure: not bothering to clean approximal surfaces correlated with non-adherence to diabetes treatment instructions, and laziness as the cause of caries correlated with non-adherence to diabetes treatment instructions and with poor motivation for diabetes care. It can be concluded that there are some common determinants for both dental health behavior and diabetes self-care. This connection should be taken into account in health education by health care professionals. PMID- 10743869 TI - Plaque removal by worn toothbrush. AB - BACKGROUND/AIMS: Assessment of the influence of toothbrush wear on plaque (PI) and gingival (GI) indexes. METHOD: 20 university students were recruited. PI and GI were recorded at the starting point (T0). Each subject received a toothbrush and toothpaste. 5 measurements of toothbrush were recorded to establish the initial size. The subjects were instructed to brush 3 x a day and to refrain from using other plaque removal aids. After 1 month (T1), the subjects were randomly divided into 2 groups: group no. 1 substituted the toothbrush at each monthly visit; group no. 2 brushed only with the toothbrush provided at T0. Recalls were scheduled after 1 (T1), 2 (T2) and 3 months (T3). PI, GI and an index of wear (WI) were calculated using 5 measurements of the toothbrush head and were recorded at recalls. RESULTS: From T0 to T3, a significant increase of PI was found within both groups. Non-significant differences, but very close to the significant level (p= 0.063), in group no. 1 and significant differences in group no. 2 of GI were found. No significant differences of PI and GI were found between groups. Increase of the WI was registered from T0 to T3 (p<0.001). CONCLUSION: Each individual is capable of maintaining low PI, even if using a toothbrush that shows evidence of wear. PMID- 10743870 TI - Beyond valence in the perception of likelihood: the role of emotion specificity. AB - Positive and negative moods have been shown to increase likelihood estimates of future events matching these states in valence (e.g., E. J. Johnson & A. Tversky, 1983). In the present article, 4 studies provide evidence that this congruency bias (a) is not limited to valence but functions in an emotion-specific manner, (b) derives from the informational value of emotions, and (c) is not the inevitable outcome of likelihood assessment under heightened emotion. Specifically, Study 1 demonstrates that sadness and anger, 2 distinct, negative emotions, differentially bias likelihood estimates of sad and angering events. Studies 2 and 3 replicate this finding in addition to supporting an emotion-as information (cf. N. Schwarz & G. L. Clore, 1983), as opposed to a memory-based, mediating process for the bias. Finally, Study 4 shows that when the source of the emotion is salient, a reversal of the bias can occur given greater cognitive effort aimed at accuracy. PMID- 10743871 TI - The impact of positive mood and category importance on crossed categorization effects. AB - Four studies examined the effect of positive versus neutral affect on preference among potential discussion partners who were members of two in-groups, two out groups, or both an in-group and an out-group (crossed targets). The importance of targets' category memberships was manipulated by idiographically based selection. Positive affect elevated evaluation of crossed targets with a dominant (differentially important) in-group (Study 1). When categories were made equally important, positive affect had no impact (Studies 2 and 3). Study 4 presented crossed targets with both equally and differentially important group memberships and showed that differential category importance (dominance) is necessary for positive affect to influence judgments about them. These results are explained by the broadened categorization induced by positive affect. PMID- 10743872 TI - Cultural variation in the use of current life satisfaction to predict the future. AB - Three studies examined cultural and situational influences on the tendency for people to use their current life satisfaction to predict future life events. On the basis of the self-enhancement literature, it was predicted that either writing about a positive personal experience or reading about another's negative experience would lead European Americans to focus their attention on internal attributes and thus would lead them to use their current life satisfaction in predicting the future. Conversely, on the basis of the self-criticism literature, it was predicted that these same conditions would lead Asian Americans to focus their attention on external factors and, therefore, would decrease their likelihood of using their current life satisfaction to predict the future. Studies 1 and 2 supported these hypotheses. Study 3 showed that these patterns could be obtained by subliminally priming concepts associated with individualism and collectivism. PMID- 10743873 TI - Running from the shadow: psychological distancing from others to deny characteristics people fear in themselves. AB - Four experiments tested the hypothesis that people distance themselves from others who display characteristics they fear in themselves. In Study 1, participants were given false feedback that they were high or low in repressed anger and were given information about a person who became angry and responded in a violent or nonviolent manner. High anger feedback participants distanced themselves only from the violent person. In Study 2, high anger feedback led to distancing from a violent other but not a dishonest other, whereas dishonesty feedback led to distancing from a dishonest other but not a violent other. The results of Studies 3 and 4 replicated and extended the distancing effect with an anger induction: Participants who were insulted distanced themselves from an angry/violent person, and verbalizing their emotions about being insulted eliminated this effect. Implications for understanding defenses against undesirable self-attributions are discussed. PMID- 10743874 TI - Assessing perceived social inequity: a relative deprivation framework. AB - The set of studies presented here describes a theoretically based method of assessing perceived social inequity and illustrates the approach through the development of an instrument assessing this experience in women's lives. The Perceived Social Inequity Scale-Women's Form (PSIS-W) is grounded in relative deprivation theory (Davis, 1959), which states that discontent results from recognition of an unfair discrepancy between one's own situation and that of others. The psychometric quality of the 26-item PSIS-W is supported by consistent results across two factor analyses, strong temporal stability over 1- and 4-month intervals, and relationships found between it and a number of predicted variables. Implications for theory and research in the areas of social inequity, social stigma, and stereotype perception are discussed. PMID- 10743875 TI - Self-esteem and the quest for felt security: how perceived regard regulates attachment processes. AB - The authors proposed that personal feelings of self-esteem foster the level of confidence in a partner's regard critical for satisfying attachments. Dating and married couples described themselves, their partners, how they thought their partners saw them, and how they wanted their partners to see them on a variety of interpersonal qualities. The results revealed that low self-esteem individuals dramatically underestimated how positively their partners saw them. Such unwarranted and unwanted insecurities were associated with less generous perceptions of partners and lower relationship well-being. The converse was true for high self-esteem individuals. A longitudinal examination of the dating couples revealed that the vulnerabilities of lows were only exacerbated over time. A dependency regulation model is proposed, wherein felt security in a partner's perceived regard is suggested as a prime mechanism linking self-esteem to relational well-being. PMID- 10743876 TI - Talking facilitates cognitive-emotional processes of adaptation to an acute stressor. AB - The authors examined the influence of talking and the social context of talking on cognitive-emotional processes of adjustment to stressors. Two hundred fifty six undergraduates viewed a stressful stimulus and were then assigned to a no talk control condition or 1 of 3 talk conditions: talk alone, talk to a validating confederate, or talk to an invalidating confederate. Two days later, they were reexposed to the stressor. Compared with individuals in the no-talk condition, those in the talk alone and validate conditions had a lower level of intrusive thoughts in the 2-day interim, and they had lower perceived stress when reexposed to the stressor. The effects of talking and validation on perceived stress appeared to be mediated by lowered intrusions. The benefits of talking were diluted when disclosures were invalidated. These findings suggest that talking about acute stressors can facilitate adjustment to stressors through cognitive resolution. PMID- 10743877 TI - Stress and accessibility of proximity-related thoughts: exploring the normative and intraindividual components of attachment theory. AB - Three studies examine the effects of stress on the accessibility of proximity related thoughts. In all the studies, participants reported on their attachment style, and the accessibility of proximity themes and worries in a lexical decision task was assessed upon the priming of a stress or neutral word. In Study 2, the primed stress word was semantically related to attachment themes. In Study 3, lexical decisions were made under low or high cognitive load conditions. Overall, the priming of a stress word led to increased accessibility of proximity themes, regardless of attachment style. Anxious-ambivalent people also showed high accessibility to proximity themes and worries in both neutral and stress contexts. In most conditions, avoidant persons' reactions were similar to those of secure persons. However, they showed no accessibility to proximity worries even after the priming of a semantically related word and reacted with high accessibility to these worries upon the addition of cognitive load. PMID- 10743878 TI - Genetic and environmental influences on sexual orientation and its correlates in an Australian twin sample. AB - We recruited twins systematically from the Australian Twin Registry and assessed their sexual orientation and 2 related traits: childhood gender nonconformity and continuous gender identity. Men and women differed in their distributions of sexual orientation, with women more likely to have slight-to-moderate degrees of homosexual attraction, and men more likely to have high degrees of homosexual attraction. Twin concordances for nonheterosexual orientation were lower than in prior studies. Univariate analyses showed that familial factors were important for all traits, but were less successful in distinguishing genetic from shared environmental influences. Only childhood gender nonconformity was significantly heritable for both men and women. Multivariate analyses suggested that the causal architecture differed between men and women, and, for women, provided significant evidence for the importance of genetic factors to the traits' covariation. PMID- 10743880 TI - Self-other agreement in personality and affectivity: the role of acquaintanceship, trait visibility, and assumed similarity. AB - Self- and other-ratings on the Big Five and a comprehensive inventory of trait affect were obtained from 74 married couples, 136 dating couples, and 279 friendship dyads. With the exception of Surprise, all scales showed significant self-other agreement in all 3 samples, thereby establishing their convergent validity. Consistent with the trait visibility effect, however, the Big Five consistently yielded higher agreement correlations than did the affectivity scales. Conversely, the affective traits consistently showed stronger evidence of assumed similarity (i.e., the tendency for judges to rate others as similar to themselves) than did the Big Five. Cross-sample comparisons indicated that agreement was significantly higher in the married sample than in the other 2 groups; however, analyses of 3 potential moderators in the dating and friendship samples failed to identify the source of this acquaintanceship effect. PMID- 10743879 TI - Do individual differences in sociosexuality represent genetic or environmentally contingent strategies? Evidence from the Australian twin registry. AB - Although men are substantially more interested than women in casual sex, there is ample variation in this trait (sociosexuality) within both sexes. One theory hypothesizes that within-sex sociosexual variation results from genetic variation maintained by frequency-dependent selection. If so, sociosexuality should be substantially heritable. A competing theory is that children acquire their mating strategy after observing their parents' relationship. By this theory, sociosexuality should reveal a strong shared environmental component. The authors studied genetic and environmental influences on sociosexuality using a large, representative volunteer twin sample. Parental marital instability was modestly associated with sociosexuality, but this could have been due to either genetic or environmental factors. Consistent with genetic theory, familial resemblance appeared primarily due to additive genetic rather than shared environmental factors. PMID- 10743881 TI - Social motives and cognitive power-sex associations: predictors of aggressive sexual behavior. AB - The present study investigated whether implicit social motives and cognitive power-sex associations would predict self-reports of aggressive sexual behavior. Participants wrote stories in response to Thematic Apperception Test pictures, which were scored for power and affiliation-intimacy motives. They also completed a lexical-decision priming task that provided an index of the strength of the cognitive association between the concepts of "power" and "sexuality." For men, high levels of power motivation and strong power-sex associations predicted more frequent aggression. There was also an interaction: Power motivation was unrelated to aggression for men with the weakest power-sex associations. For women, high levels of affiliation-intimacy motivation were associated with more frequent aggression. Strong power-sex associations were also predictive for women but only when affiliation-intimacy motivation was high. PMID- 10743882 TI - On the invalidity of validity scales: evidence from self-reports and observer ratings in volunteer samples. AB - Because of the potential for bias and error in questionnaire responding, many personality inventories include validity scales intended to correct biased scores or identify invalid protocols. The authors evaluated the utility of several types of validity scales in a volunteer sample of 72 men and 106 women who completed the Revised NEO Personality Inventory (NEO-PI-R; P. T. Costa & R. R. McCrae, 1992) and the Multidimensional Personality Questionnaire (MPQ; A. Tellegen, 1978/1982) and were rated by 2 acquaintances on the observer form of the NEO-PI R. Analyses indicated that the validity indexes lacked utility in this sample. A partial replication (N = 1,728) also failed to find consistent support for the use of validity scales. The authors illustrate the use of informant ratings in assessing protocol validity and argue that psychological assessors should limit their use of validity scales and seek instead to improve the quality of personality assessments. PMID- 10743883 TI - Carotid endarterectomy using the eversion technique. AB - Carotid endarterectomy by the eversion technique allows for all of the benefits of conventional endarterectomy but obviates the need for a distal suture line on the smaller internal carotid artery, and thus batching. Carotid artery reanastomosis onto the bifurcation can be quickly and simply performed with almost no risk of closure-related restenosis, given the anastomosis is on the larger of 2 arteries. In our experience of over 3,000 eversion carotid artery endarterectomies, the restenosis rate has been less than 1% judged by rigorous duplex follow-up. In this article, the technique and utility of eversion carotid endarterectomy is discussed. PMID- 10743884 TI - Approach to the confluence of the subclavian and internal jugular veins without claviculectomy. AB - A new technique is described to approach the confluence of the subclavian vein and the internal jugular, as well as to expose the innominate vein from its origin to the superior vena cava. The operation accomplishes decompression of the subclavian vein and allows direct approach of the subclavian and the innominate veins. It avoids dividing the clavicle or disarticulating the sternal-clavicular joint. It also has the advantage of accomplishing the procedure without entering the pleural cavity. Its reconstruction is explained in detail and achieves solid stability of the sternum and of the shoulder girdle. This approach also prevents deformity that other operations proposed in the past may cause. PMID- 10743885 TI - Arteriovenous fistulas as an adjunct to venous surgery. AB - The objectives of the temporary arteriovenous fistula (AVF) are to increase blood flow in the thrombectomized segment to prevent immediate thrombosis and to allow time for healing of the endothelium. After complete thrombectomy of the iliac vein, confirmed by intraoperative venogram, the AVF is constructed using the divided long saphenous vein end-to-side to the superficial femoral artery. After 6 to 8 weeks, the patency of the AVF and the deep venous system is assessed by duplex scan, followed by an arteriovenogram from the opposite femoral artery, evaluating the anatomy of the AVF, common femoral vein, iliac vein, and the inferior vena cava (IVC). The AVF is closed using coils. PMID- 10743886 TI - Harvesting the superficial femoral vein as an autograft. AB - The superficial femoral-popliteal vein (SFPV) is an excellent conduit for reconstructing large arteries and veins. Herein we describe the anatomy, preoperative evaluation, and exposure technique of the SFPV. Harvesting the SFPV from the level of the knee joint to the confluence with the profunda femoris vein is associated with minimal mid- and late-term venous morbidity. These vein grafts have proved to be durable conduits, particularly in the aortofemoral position. PMID- 10743887 TI - Video-assisted endoscopic saphenous vein harvest: an evolving technique. AB - Saphenous vein harvest is a necessary step in many cardiac and peripheral vascular reconstructions. However, saphenectomy is not inconsequential and can lead to short- and long-term morbidity. To avoid the morbidity, new technology has been developed to remove the saphenous vein with limited incisions using video-assisted, or endoscopic, techniques. Although a number of manufacturers have developed equipment for this purpose, we have developed expertise with the Ethicon Endo-Surgery (Cincinnati, OH) system. We have used this technique for more than 100 lower-extremity bypass operations with minimal wound complications and satisfactory revascularization. The technical features of this operation are presented. PMID- 10743888 TI - Tourniquet occlusion technique for tibial artery reconstruction. AB - The pneumatic compression tourniquet for tibial artery reconstruction reduces the necessary dissection, decreasing the risk of arterial and venous injury. It provides effective control of side branches, often expedites the operation, and provides a bloodless operative field with elimination of the need for clamps or vessel loops and their risk of arterial injury. They are easy to use, readily available, and, when used after leg elevation and esmarch compression, very effective. Despite the theoretical risks of muscle injury, nerve damage, or reperfusion syndrome, complications are rare. Overall results with the tourniquet in all recent reports have been excellent, and it can greatly facilitate tibial artery bypass. PMID- 10743889 TI - Increasing use of autogenous fistulas: selection of dialysis access sites by duplex scanning and transposition of forearm veins. AB - Although the autogenous radiocephalic fistula is considered to be the ideal access for hemodialysis, significant changes in the demographics of patients undergoing angioaccess have occurred since its initial description in the 1960s. Patients presenting for initial access evaluation in contemporary practice are less likely to have arteries and veins suitable for autogenous fistula (AF) formation in the classic location. Physical examination of the upper extremity alone may be inadequate for selection of arteries and veins that will mature into a functioning AF. The authors have used duplex ultrasound (DU) to assess upper extremity vasculature for planning of dialysis access procedures. Criteria for selection of arteries and veins and a detailed description of our DU examination protocol are reviewed. Routine use of upper-extremity DU has identified many patients with forearm veins that are suitable for use, but in locations remote from the optimal arterial inflow or too deep to facilitate easy needle cannulation. Modifications to the single-incision radiocephalic fistula that allow for expanded use of forearm veins identified by DU are described, and the technique of superficial venous transposition of forearm veins in particular is reviewed in detail. On the basis of our experience, we recommend the combination of upper-extremity planning DU and superficial venous transposition in the forearm to increase use of AF and reduce reliance on prosthetic bridging grafts (BG) in patients requiring access for hemodialysis. PMID- 10743890 TI - Obturator bypass: technical considerations. AB - The obturator bypass is a useful method for revascularizing the lower extremity in the presence of a hazardous groin dissection. The most common indication for employing this method of reestablishing arterial continuity is in circumventing infected vascular prostheses. One- and 5-year patency rates of 72.7% and 56.9% have been described. This article reviews the indications for obturator bypass and discusses technical considerations regarding the procedure. PMID- 10743891 TI - Venous boot construction for a distal prosthetic bypass. AB - Autologous vein should be used at all costs for infrainguinal grafting. There is, nevertheless, a need for other materials in tertiary referral centres whose practice is becoming more complex. Polytetrafluoroethylene (PTFE) has been the prosthesis of choice for most surgeons, but results were poor for grafts to crural arteries. The use of a venous boot, cuff, or collar appeared to improve these results, but only one randomized trial has been performed. This article describes the St Mary's venous boot, which purports to combine the advantages of the other techniques. PMID- 10743892 TI - Compression therapy for acute iatrogenic femoral pseudoaneurysms. AB - The marked increase in performance of percutaneous catheter-based vascular interventions combined with aggressive anticoagulation regimens has resulted in a greater frequency of iatrogenic femoral artery pseudoaneurysms. Of the available treatment modalities, ultrasound-guided compression has been shown to be widely available, easy to perform, noninvasive, effective, safe, and inexpensive. It is currently the procedure of choice for treatment of these iatrogenic femoral artery injuries. PMID- 10743893 TI - Constructing muscle flap coverage for vascular grafts in the groin. AB - Groin wound infections can jeopardize patency and function of vascular bypass grafts anastomosed to the femoral artery. Attempts to salvage vascular reconstructions threatened by perigraft infection often fail because of inadequate debridement of surrounding infected soft tissue or because of an inability to cover an exposed graft with sufficient healthy tissue. In selected circumstances, use of rotational myocutaneous flaps makes it possible to salvage vascular grafts threatened by localized wound infection. PMID- 10743894 TI - Thoracofemoral bypass: proximal exposure and tunneling. AB - Although axillobifemoral bypass is the usual alternative to the standard aortobifemoral bypass (ABF) when the latter is contraindicated because of comorbid operative risk or when the transabdominal approach is considered hazardous, a more proximal aortic inflow source is desirable in selected low-risk patients. The results with these more proximal aortic procedures are more durable than with axillobifemoral bypass and approach those that can be achieved with an ABF. This article reviews some of the specific technical details of descending thoracofemoral bypass, specifically, the techniques for proximal exposure and tunneling. PMID- 10743895 TI - Vertebral-carotid transposition. AB - In the early years of evaluating patients with cerebral disease, the origins of the vertebral and subclavian arteries were not routinely visualized. Surgical intervention was more formidable then, and most symptoms were thought to be relieved by corrective carotid surgery. The continued occurrence of nonhemispheric symptoms in some patients, coupled with better techniques of diagnosis and improved surgical techniques, prompted the complete angiographic evaluation of total cerebral flow in all patients evaluated for cerebral symptoms. When carotid endarterectomy did not relieve hind brain symptoms, whether there was significant decreased vertebral flow, and whether surgical was indicated, could then be determined. Most vertebral artery occlusive disease is located near its origin and can be relieved by vertebral carotid transposition. The operative technique is described in this article along with pertinent anatomy and historical perspectives. PMID- 10743896 TI - Axillary artery anastomosis to avoid axillofemoral bypass disruption. AB - Acute disruption at or adjacent to axillary anastomoses of axillofemoral grafts has been sporadically reported. The cause of this serious complication is believed to be attributable to mechanical stresses on the proximal portion of the graft and anastomosis. A modification in the proximal tunneling of the axillofemoral graft, which appears to have effectively reduced the occurrence of this vexing complication, is described in this report. PMID- 10743897 TI - Distal revascularization-interval ligation for maintenance of dialysis access and restoration of distal perfusion in ischemic steal syndrome. AB - Ischemic steal syndrome after hemodialysis access challenges the clinician to reconcile the dichotomy of maintenance of access patency and restoration of distal limb perfusion. Results from traditional procedures directed toward increasing the resistance in the fistula (eg, banding, lengthening) have yielded unreliable results and frequently eventuate in fistula thrombosis. The recently described technique of distal revascularization, interval ligation (DRIL) provides a more physiological approach. Based on several recent series, application of the technique has provided excellent resolution of ischemic symptoms and superior preservation of fistula patency. PMID- 10743898 TI - Total posterior approach for femoropopliteal bypass. AB - Standard approaches to the femoral and popliteal arteries are used in most extremity arterial reconstructions. In unusual circumstances, such as infection, reoperation, or variant anatomy, novel approaches to infrainguinal bypass may be useful, particularly in reoperative or infected cases. One such approach involves exposure of the femoral and popliteal arteries through posterolateral incisions with the patient prone. The major advantage of this exposure is the increased accessibility to the distal above-knee popliteal artery, which is not easily reached through either medial or lateral incisions. This approach also can be useful in cases of significant groin sepsis. The details of this exposure and its application in an illustrative case are presented. PMID- 10743899 TI - Stent-assisted vascular anastomoses. AB - A stent can be used to rapidly create a laminar end-to-end vascular anastomosis. To construct a stent-assisted vascular anastomosis (SAVA), 2 traction sutures 180 degrees apart are used to pull 1 conduit into another so that they overlap. A stent is balloon expanded to create a seal in the area of overlap. A SAVA takes 5 to 15 minutes to create. Essential to the procedure is proper sizing of the graft, stent, and balloon. The SAVA becomes reinforced internally by a neointima and externally by periadventitial scar tissue. No anastomotic leaks or pseudoaneurysms were observed in 13 patients (1 to 2.5 years; mean, 14 months), 6 adult mongrel dogs (6 months), and 5 growing pigs (5.5 months). Three of 9 upper extremity basilic vein SAVAs (dialysis shunts) occluded (3, 9, and 13 months). Stent deformation was seen in all 3 despite protection by external graft rings. Within our follow-up interval, a balloon-expandable stent is an effective tool to use to rapidly join a vessel to a graft. Because the stent is deformable, a SAVA is not recommended in the extremity. PMID- 10743900 TI - Stimulus compounding enhances conditioned suppression produced by cocaine-paired stimuli. AB - When 2 stimuli that occasion cocaine self-administration are presented in compound, their ability to increase cocaine-reinforced operant responding is substantially enhanced. The goal of the present experiment was to determine whether stimulus compounding could produce analogous enhancements of a classically conditioned drug effect. Food-maintained responding in rats was suppressed by a tone and a light that were individually paired with response independent cocaine (3 mg/kg iv). This conditioned suppression was significantly enhanced when the stimuli were presented together in a stimulus-compounding test. The magnitude of this enhancement was similar to that in previous studies in which responding was suppressed by shock-paired stimuli. These results demonstrate that multiple drug-related cues interact in a predictable manner to influence both operant and classically conditioned behavior. PMID- 10743901 TI - A verification of psychostimulant-induced improvement in sustained attention in rats: effects of d-amphetamine, nicotine, and pemoline. AB - Previous studies from this laboratory have demonstrated that a variety of psychostimulant drugs can improve the performance of rats trained in a 2-choice stimulus detection task in which the correct responses are indicated by a briefly illuminated light. To enhance the construct validity of the task for assessing sustained attention, the procedure was modified so that the precue interval across trials varied unpredictably between 3, 7, and 11 s. After training rats (N = 17) so that their baseline accuracy levels stabilized between 75% and 88% correct, their performance was assessed after administration of d-amphetamine (0.125-0.75 mg/kg sc), nicotine (0.25-0.75 mg/kg sc), and pemoline (5.0-30.0 po). At certain doses all 3 drugs induced performance improvements in mean choice accuracy and choice response time. Because the precue intervals varied unpredictably and the cue durations used to maintain the rats' baseline accuracy levels were typically short (range = 70-500 ms), the task conforms to most conditions typically required for assessing sustained attention. Results verify the proposal that psychostimulant drugs can enhance the attentiveness of animals in a fashion similar to that documented in humans. PMID- 10743903 TI - Effects of amphetamine on food and fruit drink self-administration. AB - The effects of oral d-amphetamine (0.12-1.0 mg/kg) on the responding of adult baboons were examined during choice sessions. In Experiment 1, responding on 1 lever was reinforced with 1 food pellet, and responding on a 2nd lever was reinforced with 4 food pellets. The response requirement (fixed ratio [FR]) on the latter lever was 4 times the FR value; that is, the unit price (responses/g) was the same. Amphetamine decreased responding on both levers similarly under all conditions. In Experiment 2, responding on 1 lever was reinforced with 1 pellet, and responding on a 2nd lever was reinforced with a sweet fruit drink. Amphetamine decreased responding reinforced by food to the greatest extent when the FR value was large and fruit drink was available. Findings indicate that choice procedures can provide baselines that allow the evaluation of the specificity of a manipulation on intake of a commodity. PMID- 10743902 TI - Discriminative-stimulus effects of zolpidem, triazolam, pentobarbital, and caffeine in zolpidem-trained humans. AB - Six non-drug-abusing humans were trained to discriminate 15 mg zolpidem in the present experiment. After participants acquired discrimination, a range of doses of zolpidem (2.5-15.0 mg), triazolam (0.0625-0.3750 mg), pentobarbital (25-150 mg), caffeine (100-600 mg), and placebo were tested to determine whether they shared discriminative-stimulus effects with 15 mg zolpidem. The participant-rated and performance-impairing effects of zolpidem, triazolam, pentobarbital, and caffeine were assessed concurrently. Triazolam and pentobarbital dose dependently increased zolpidem-appropriate responding. Caffeine occasioned low levels of zolpidem-appropriate responding. Zolpidem, triazolam, and pentobarbital, but not caffeine, generally produced a similar constellation of participant-rated drug effects (e.g., increased scores for the Pentobarbital, Chlorpromazine, and Alcohol Group subscale on the Addiction Research Center Inventory) and dose dependently impaired performance. These results suggest that humans can reliably discriminate zolpidem. Despite its unique benzodiazepine-receptor binding profile, the discriminative-stimulus, participant-rated, and performance impairing effects of zolpidem are similar to those of the barbiturates and benzodiazepines. PMID- 10743904 TI - A clinical laboratory model for direct assessment of medication-induced antihyperalgesia and subjective effects: initial validation study. AB - Analgesic medications are often tested in clinical laboratory studies by observing their ability to reduce the pain produced by noxious stimuli presented to healthy skin. These medications may then be used clinically to reduce disease related hyperalgesia. This article describes a clinical laboratory model useful for testing a medication's ability to reduce hyperalgesia in humans. Results demonstrate that ultraviolet (UV) light induces hyperalgesia, commonly prescribed analgesic medications reduce UV-induced hyperalgesia, and this UV-induced hyperalgesia model can be used to assess the time course of a medication's antihyperalgesia effects. Coupled with participant-rated measures of drug liking and mood, this model may prove useful for predicting the clinical efficacy and side-effect profile of novel analgesic medications in cost-efficient and statistically powerful laboratory studies. PMID- 10743905 TI - The role of formalin-induced pain in morphine tolerance, withdrawal, and reward. AB - The effect of a commonly used experimental pain-induction procedure (formalin injection into a hindpaw site) on morphine tolerance, withdrawal, and reward was examined in rats. Results suggest that the effects of morphine are different in the organism that is experiencing pain at the time it receives the drug than in the organism that is pain free. The presence of pain at the time of each morphine administration decreased analgesic tolerance, decreased naloxone-precipitated withdrawal, and enhanced the rewarding effect of the opiate. These findings, together with those of previous studies, suggest that theories of opiate tolerance, withdrawal, and reward should incorporate the effects of pain. PMID- 10743906 TI - The impact of imagining completed versus interrupted smoking on cigarette craving. AB - A brief imagery procedure was used to determine the effects of imagining completed versus interrupted smoking on self-reported craving, mood, autonomic functioning, and reaction time to an auditory probe. Cigarette smokers (N = 60) imagined actively participating in 3 types of scenarios in which they (a) engaged in smoking behavior, (b) attempted to smoke but were interrupted by a lack of cigarettes, and (c) were not confronted with smoking cues. Imagining both completed and interrupted smoking produced equivalent increases in craving compared with imagining neutral scenarios. Imagery of interrupted smoking was associated with higher heart rate and increased negative mood relative to the other scenario types, whereas imagery of completed smoking was associated with slower reaction time on the probe reaction-time task. Theoretical and methodological implications of the results are discussed. PMID- 10743907 TI - Tobacco craving: intensity-related effects of imagery scripts in drug abusers. AB - Two experiments were conducted to determine whether active imagery would elicit tobacco craving in smokers with histories of drug abuse who were not interested in quitting smoking. In Experiment 1, the authors used scripts that contained positive, negative, or neutral affective content with and without descriptions of smoking urge. Scripts with urge content and negative affect scripts increased subjective reports of tobacco craving. An interaction between affective manipulation and urge content was observed on self-reported mood. In Experiment 2, positive affect scripts that varied in amount of urge content produced an orderly increase in tobacco craving as a function of urge intensity, suggesting that changes were specific to the imagery manipulation. In both experiments, increases in tobacco craving were positively correlated with craving for drug of choice, suggesting that stimuli that engender smoking urges may occasion craving for other drugs of abuse. PMID- 10743908 TI - Tobacco withdrawal signs and symptoms among women with and without a history of depression. AB - A past history of depression is associated with a decreased likelihood of quitting smoking. Tobacco withdrawal may be a mechanism through which depression history impedes smoking cessation. This research examined the influence of depression history on unmedicated tobacco withdrawal signs (polysomnographic measures of sleep) and symptoms (self-reported urge, negative affect, hunger, and sleep) among women (N= 13). Depression history was associated with differential withdrawal-induced changes in several REM sleep parameters. Self-report and other polysomnography (sleep fragmentation, slow-wave sleep) measures displayed statistically significant withdrawal effects but did not discriminate between depression history groups. These results suggest that REM sleep parameters may be sensitive to differential tobacco withdrawal responses that are not readily apparent through self-reported symptoms. PMID- 10743910 TI - The effects of cigarette smoking on negative priming. AB - Nicotine administration has been found to enhance performance on tasks of selective attention. It has been proposed that efficient attentional filtering depends on the successful inhibition of distracting information. In the work reported here, a negative priming paradigm was adopted to test whether smoking enhanced the inhibition of irrelevant information. Thirty-six minimally deprived smokers, half of whom smoked and half of whom sham smoked, completed the negative priming task. A significantly larger negative priming effect was found in participants who had smoked in comparison with those who sham smoked. These results support the hypothesis that nicotine enhances the inhibition of distracting information and thus suggest a possible mechanism by which smoking may enhance selective attention. PMID- 10743909 TI - Associations between tobacco smoking and illicit drug use among methadone maintained opiate-dependent individuals. AB - Tobacco chippers are individuals who smoke regularly yet are not nicotine dependent. In the present study, the authors examined the prevalence of tobacco chipping among methadone-maintained opiate abusers. Furthermore, the authors examined associations between tobacco and illicit substance use by comparing heavy smokers, tobacco chippers, and nonsmokers. Results demonstrate that tobacco chipping occurs among methadone-maintained individuals. Illicit substance use, measured through urine toxicology, was found to increase in a stepwise fashion from nonsmokers, to chippers, to heavy smokers. Smoking status (nonsmoker, chipper, heavy smoker) proved a more powerful predictor of cocaine and opiate use than daily methadone dose. Findings lend support to existing evidence suggesting associations between tobacco and opiate and cocaine use and strongly suggest that smoking cessation should be offered to all methadone-maintained individuals. PMID- 10743911 TI - Dose-response analyses of associative tolerance to nicotine analgesia in the rat: tail-flick and hot-plate tests. AB - This study is the first to demonstrate associative tolerance to nicotine's analgesic effects as a shift in the dose-response curve (DRC) to the right. The subjects were 43 experimentally naive, male Sprague Dawley rats (Rattus norvegicus) randomly assigned to 2 groups. Home cage rats (HC; n = 21) received a series of 1 mg/kg nicotine doses explicitly unpaired with the distinctive context, whereas distinctive context rats (DC; n = 22) were injected with nicotine explicitly paired with the distinctive context. Rats in each of the 2 groups were randomly assigned to 1 of 3 nicotine testing doses to construct DRCs. The DRC of the rats that received nicotine in the distinctive context was shifted to the right of the DRC of rats that had had as much exposure to nicotine but had never experienced nicotine in the distinctive context. DC rats required nearly twice as much nicotine as HC rats to produce the same DRC. The discussion describes the implications of the results for theories of drug tolerance and nicotine addiction. PMID- 10743912 TI - Comparison of the reinforcing and anxiogenic effects of intravenous cocaine and cocaethylene. AB - People report that ethanol improves the experience produced by cocaine. This effect may be attributable to cocaethylene (CE), a cocaine metabolite formed only in the presence of ethanol. To test this, rats were trained to run an alley for a single intravenous dose of either cocaine (0.5-2.0 mg/kg) or an equimolar dose of CE (0.75-2.88 mg/kg). The rats' start latency and running speed measured the reinforcing effects of the drugs and the number of times rats approached but failed to enter the goal box (i.e., approach-avoidance retreats) indexed anxiety. Rats reinforced with CE had shorter start latencies and faster running speeds and exhibited fewer "retreats" than cocaine-reinforced rats. These results suggest that CE is more reinforcing and less anxiogenic than cocaine and hence may account for the combined effects of cocaine and ethanol in humans. PMID- 10743913 TI - Carbamazepine reduces dopamine-mediated behavior in chronic neuroleptic-treated and untreated rats: implications for treatment of tardive dyskinesia and hyperdopaminergic states. AB - Chronic treatment with neuroleptic drugs such as haloperidol (HAL) can result in a syndrome of abnormal involuntary movements known as tardive dyskinesia (TD). The authors have obtained evidence that TD in humans is reduced in patients also taking anticonvulsant drugs, primarily carbamazepine (CBZ). To test for a causal role of CBZ in this effect, the authors quantified abnormal movements elicited by dopamine (DA) receptor stimulation in rats (Rattus norvegius) withdrawn from chronic treatment with HAL or CBZ alone or in combination. The expected increased behavioral responsiveness to combined D1/D2 stimulation in rats treated with HAL for 8 weeks was significantly attenuated by chronic CBZ, which also attenuated behavioral responsiveness in otherwise untreated rats. Striatal D2 DA receptor density was elevated in rats treated chronically with HAL but unaffected by CBZ. Striatal D1 DA receptor density was elevated by chronic CBZ but unaffected by HAL. These findings suggest that by reducing DA supersensitivity, CBZ may be useful in treating TD and other hyperdopaminergic states. PMID- 10743916 TI - Looking to the future of behavioral pharmacology while being attentive to its past. PMID- 10743915 TI - Effects of dose and interdose interval on conditioned heart rate tolerance to smoking. AB - The effects of an environmental cue and smoking administration on heart rate (HR) responses to smoking were investigated in 2 studies. The 1st study was performed without smoking, to rule out the possibility that the cue manipulations alone could produce HR habituation. Thirty-six male nonsmokers were exposed to 6 trials of a changing or repeating cue (segments of a story on audiotape), followed by a paced-breathing period. HR habituation was not found. In the 2nd study, 40 male smokers smoked 4 puffs every 10 min (small-dose/long-interdose interval [IDI]) or 6 puffs every 5 min (large-dose/short-IDI) in 6 trials. The same repeating or changing cue preceded smoking. Only the repeating-cue, small-dose/long-IDI group developed HR tolerance. Modifying the cue on Trial 6 did not reverse tolerance. The results indicated that (a) tolerance to smoking appears to be subject to conditioning, (b) this effect depends on the dose and IDI, and (c) the observed tolerance is not likely to be a result of the effects of the cue alone. Nonassociative tolerance to smoking a high-dose/short-IDI did not occur in this study. PMID- 10743914 TI - Antidepressant drugs appear to enhance cocaine-induced toxicity. AB - It has been shown that cocaine-induced convulsions and lethality appear to be mediated by serotonin and dopamine neurotransmission, respectively. However, many antidepressants considered for treatment of cocaine addiction target these monoamine systems and may thus amplify these toxic effects during relapse. In this study, the authors assessed whether pretreatment with antidepressants influences cocaine-induced toxicity in mice as well as the potency of these medications at cocaine-binding sites previously shown to be associated with cocaine toxicity. Overall, selective serotonin reuptake inhibitors (SSRIs) facilitated cocaine-induced convulsions but not lethality. Dopamine uptake inhibition facilitated cocaine-induced lethality, but not convulsion. The SSRI sertraline enhanced neither convulsions nor lethality and may be unique due to its high affinity for sigma receptors. These results have important implications for safe and effective addiction treatments. PMID- 10743917 TI - The operation was a success, but the patient died. PMID- 10743918 TI - Fourier analysis of irregular astigmatism after trabeculectomy. AB - OBJECTIVE: To examine irregular astigmatism following trabeculectomy using Fourier analysis of videokeratography data. PATIENTS AND METHODS: Forty eyes of 40 glaucoma patients, undergoing primary trabeculectomy, underwent videokeratographic examinations preoperatively, and at 2 weeks and 1, 3, 6, and 12 months postoperatively. The dioptric data of the central cornea were decomposed into spherical, regular astigmatic, and irregular astigmatic (decentration and higher-order irregularity) components using Fourier analysis. RESULTS: The mean spherical equivalent did not change throughout the observation period. The regular astigmatic component from 2 weeks to 6 months post operatively was greater than the preoperative diopter, but the increase was not significant. In contrast, the decentration component increased significantly after surgery, but returned to the preoperative level by 12 months. The higher order irregularity component also presented a transient increase postoperatively. CONCLUSIONS: Irregular astigmatism, particularly decentration component, increases significantly after trabeculectomy, but returns to the preoperative level 12 months after surgery. PMID- 10743919 TI - Clinical results of Ahmed glaucoma valve implantation in refractory glaucoma with adjunctive mitomycin C. AB - OBJECTIVE: To study the surgical outcome and complications of Ahmed glaucoma valve (AGV) implantation with mitomycin C (MMC) in refractory glaucoma. PATIENTS AND METHODS: The authors retrospectively reviewed the records of 40 eyes in 37 consecutive patients who underwent combined AGV with MMC (0.4 mg/mL; 5 minutes). All patients had a minimum of 6 months of follow-up. Outcome measures were best corrected visual acuity, intraocular pressure (IOP), the number of medications, success rates, and the incidence of complications. RESULTS: The Kaplan-Meier life table analysis revealed that the probabilities of surgical success based on our success criteria at 1 year and 2 years postoperatively were 80% and 77%, respectively. Postoperative IOP was significantly lower at all follow-up intervals. At the final visit fewer glaucoma medications were used (2.5 vs 0.7, preoperative vs postoperative). The most frequent complication was hypotony (17%). CONCLUSION: In this retrospective case series with relatively short follow ups, AGV implantation with mitomycin C in refractory glaucoma appears to be effective without increased complication rates. PMID- 10743920 TI - Does laser trabeculoplasty prevent steroid glaucoma? AB - OBJECTIVE: To investigate the effect of laser trabeculoplasty in the normal myopic eye on steroid intraocular pressure responsiveness in patients undergoing radial keratotomy. MATERIALS AND METHODS: Diode laser trabeculoplasty was performed on 94 patients with myopia undergoing radial keratotomy who were then started on dexamethasone drops. Intraocular pressures were measured by applanation tonometry compensated for corneal flattening. RESULTS: Twenty-eight percent of patients had an intraocular pressure increase of 6 mm Hg or more by the fourth week. CONCLUSION: Laser trabeculoplasty does not prevent steroid intraocular pressure increase in patients with myopia. PMID- 10743921 TI - Effectiveness of LASIK to correct refractive error after penetrating keratoplasty. AB - OBJECTIVE: Refractive errors may invalidate the good results of penetrating keratoplasty (PK). The Authors evaluate the effectiveness of excimer laser in situ keratomileusis (LASIK) in the correction of refractive error after PK. MATERIALS AND METHODS: Four patients, a 26-year-old woman, a 54-year-old man, a 19-year-old man, and a 51-year-old woman, showed refractive errors: -11 = -4.5 x 85 ; -8, -4.5 = -11 x 95 ; and -4.5 = -4 x = 1200, with a clear graft at least 20 months after penetrating keratoplasty secondary to keratoconus. However, they underwent the LASIK procedure with a nasal-hinged flap of 160 um. No sutures were placed. RESULTS: At follow-up, 24, 18, 12, and 12 months, respectively, the graft remained clear and the endothelial cells were unchanged. The uncorrected visual acuities were 20/50, 20/25, 20/50, and 20/25, respectively with an unchanged best corrected visual acuity (20/20) for all patients. No significant complications were observed. CONCLUSIONS: LASIK procedure seems to be an effective technique to correct refractive error after successful penetrating keratoplasty. PMID- 10743923 TI - Human lens effect on in vivo scanning laser polarimetric measurements of retinal nerve fiber layer thickness. AB - OBJECTIVE: To determine the effect of the human lens on retinal nerve fiber layer measurements by scanning laser polarimetry. This technique uses the form birefringence of the retinal nerve fiber layer to estimate its thickness. The GDx Nerve Fiber Analyzer contains an optical retarder element to compensate for the birefringence of the human cornea. PATIENTS AND METHODS: Fourteen nonglaucomatous pseudophakic or aphakic eyes were matched by age and race to 14 nonglaucomatous phakic eyes. Intraocular pressure, optic nerve head evaluation by slit lamp biomicroscopy and visual fields were performed to establish the absence of glaucoma. The nerve fiber layer thickness measurements by the GDx Nerve Fiber Analyzer were compared between the two groups. RESULTS: The average nerve fiber layer thickness in the phakic group was 67.2+/-11.9 ,m and in the nonphakic group was 66.1 +/- 11.8 microm (P=0.80). CONCLUSION: The human lens has negligible influence on in vivo nerve fiber layer thickness measured by the GDx Nerve Fiber Analyzer. PMID- 10743922 TI - Retinal detachment following laser in situ keratomileusis. AB - OBJECTIVE: To report the clinical characteristics, surgical management and outcome of retinal detachment following laser in situ keratomileusis (LASIK) in myopic patients. PATIENTS AND METHODS: Retrospective review of 10 eyes of 10 myopic patients with retinal detachment who had previously undergone LASIK surgery were analyzed. Included in the study were 7 males and 3 females, aged 22 to 68 years (35.2+/-2.8). RESULTS: Mean spherical equivalent refraction was 10.51+/-3.90 D (ranging from -6.37 D to -17.00 D) before surgery. The time interval between the LASIK procedure and the development of retinal detachment varied from two months to nine months (5.2 +/- 2.78 months). The number of retinal breaks was one in 7 patients, two in 2 patients and three in 1 patient. The type of retinal breaks included 7 patients with horse shoe tears, 1 patient with a retinal hole, 1 patient with a giant retinal tear, and 1 patient with retinal dialysis. Retinal breaks were located anterior to the equator in 9 patients and posterior to the equator in 1 patient. Retinal reattachment was achieved with one operation in 8 eyes (80%) and the remaining 2 eyes required a second surgery for the reattachment of the retina. CONCLUSION: This study suggests the possible association between retinal detachment and LASIK procedure in patients with myopia. Clinicians should be aware of retinal pathology predisposing to retinal detachment in patients undergoing LASIK. PMID- 10743924 TI - Alpha-tocopherol derivatives and wound healing in an experimental model of filtering surgery. AB - OBJECTIVE: To describe the effect of alpha-tocopherol derivatives (acetate and acid-succinate) on the histopathological characteristics of the surgical fistula in an experimental model of filtering surgery. MATERIALS AND METHODS: Thirty pigmented rabbits were divided into 3 groups. Twenty-four hours before surgery the animals were injected subconjunctivally with 0.5 mL of solution that depended on the group of treatment: Group 1 (n = 10) 0.75% ethanol in balanced salt solution (BSS); Group 2 (n = 10) 100 microg alph-tocopherol-acetate in 0.75% ethanol in BSS; Group 3 (n = 10) 100 microg alpha-tocopheryl-acid-succinate in 0.75% ethanol in BSS. Histological findings were evaluated 30 days after surgery. RESULTS: The groups treated with alpha-tocopherol derivatives showed a higher percentage of persistence of the fistula and better intraocular pressure (IOP) control. CONCLUSIONS: Alpha-tocopherol derivatives showed antiproliferative properties in this experimental model of filtering surgery. PMID- 10743927 TI - Combined hamartoma of the retina and retinal pigment epithelium as the presenting sign of neurofibromatosis-1. AB - The authors report a case of combined harmartoma of the retina and retinal pigment epithelium in a 6-year-old child as the presenting sign of neurofibromatosis-1. The patient was followed closely for three years but received no treatment. Observation over these years revealed no significant change in the patient's visual acuity. A referral to the University of California San Francisco (UCSF) Neurofibromatosis Clinic resulted in a diagnosis of neurofibromatosis (NF)-1, and she continues to be observed for further systemic manifestations of this disease. Combined hamartoma of the retina and retinal pigment epithelimicrom, a rare and benign tumor, can be easily mistaken for malignant processes such as retinoblastoma or choroidal melanoma. Ophthalmologists should also be aware of the association with neurofibromatosis and consider this diagnosis when such retinal findings are observed in a child. PMID- 10743925 TI - Threshold power levels for NPe6 photodynamic therapy. AB - OBJECTIVE: To determine the threshold power levels for producing retinal and choroidal vascular occlusion using mono-L-aspartyl chlorin e6 (NPe6) photodynamic therapy; to evaluate its efficacy with longer intervals between photosensitizer injection and laser application; to determine the elapsed time between light application and appearance of angiographic changes. METHODS: Pigmented and nonpigmented rabbits were injected intravenously with 2 mg/kg of NPe6 before laser irradiation of the retina-choroid. Group 1 was treated at increasing power levels; fluorescein angiograms were obtained at each fluence. Group 2 animals were exposed to laser irradiation at 5 minutes, and 1 and 3 hours postinjection to determine (by fluorescein angiography 24 hours post-treatment) if increasing the interval affected outcome. Group 3 animals underwent fluorescein angiography at 30 minutes, 1 hour, 2 hours, and 24 hours posttreatment to document the time between laser application and subsequent vessel closure. RESULTS: Choroidal vessel occlusion was angiographically evident in all lesions at fluences of > or = 2.65 J/cm2 in pigmented rabbits and at > or = 0.88 J/cm2 in nonpigmented rabbits. Lesion diameter decreased as the time between injection and treatment increased. Vessel occlusion was documented at least 2 hours after treatment. CONCLUSION: Choroidal vessel occlusion can occur at very low fluence. PMID- 10743926 TI - Silicone oil emulsification of the retinal surface. AB - A 35-year-old patient with cytomegalovirus (CMV) retinitis secondary to acquired immunodeficiency syndrome (AIDS) underwent pars plana vitrectomy and fluid-gas silicone exchange for retinal detachment. Three weeks following surgery and additional laser photocoagulation, extensive retinal surface emulsification was noted. It masked the retinal surface and demonstrated shifting in subsequent examinations. This case stresses the importance of careful and close follow-up after pars plana vitrectomy and silicone oil injection. Silicone oil emulsification may in some cases require removal of the emulsified fluid or silicone oil exchange, but in others such as our case, shifting of the emulsified silicone oil may allow evaluation of the nonobscured retina. PMID- 10743928 TI - Improved retinal capillary perfusion following treatment of severe proliferative diabetic retinopathy. AB - The authors report a photodocumented case of improved retinal capillary perfusion accompanied by visual acuity improvement after treatment of severe proliferative diabetic retinopathy. PMID- 10743929 TI - Combined use of an amniotic membrane and tissue adhesive in treating corneal perforation: a case report. AB - We report a new method combining the use of an amniotic membrane and cyanocrylate tissue adhesive to seal a corneal perforation. A 47-year-old male suffered from an alkali injury complicated with corneal melting and perforation in the left eye. We placed an amniotic membrane of optimal size in the anterior chamber directly under the corneal perforation lesion. The cyanocrylate tissue adhesive was then applied over the perforation site and sealed successfully. Three weeks later, the tissue adhesive had dislodged. The amniotic membrane had sealed the perforated lesion and was well adhered to the surrounding corneal tissue with complete epithelial covering. Vision was 20/25 six months after the operation. The combined use of an amniotic membrane and tissue adhesive is a promising method in the treatment of corneal perforation. PMID- 10743930 TI - Nonsmall cell lung adenocarcinoma metastatic to the vitreous without fundus lesions. AB - A 76-year-old man presented with vitritis in the absence of visible fundus lesions. Vitreal cytologic analysis revealed adenocarcinoma; immunostaining was consistent with a pulmonary origin. This case highlights the need to exclude metastasis to the vitreous in a patient with an oncologic history, even in the absence of visible posterior segment lesions. It also illustrates the utility of immunostaining to better determine the tissue of origin when a metastatic lesion is encountered. PMID- 10743931 TI - Excision of multiple eyelid apocrine hidrocystomas via an en-bloc lower eyelid blepharoplasty incision. AB - A 39-year-old man presented with upper and lower eyelid apocrine hidrocystomas that had recurred after each of three prior attempts at excision. These multiple, cystic tumors were adherent to the epidermis, thus precluding complete dissection and excision of each individual lesion. We report a surgical technique using a lower eyelid blepharoplasty incision to remove the confluent tumors of the lower eyelids en-bloc. We paid careful attention to both removing the lesions without rupturing the cysts and to achieving adequate depth of excision. Histology revealed features typical of apocrine hidrocystomas. The patient has remained free of recurrence three years since the en-bloc excision of the lesions. PMID- 10743932 TI - Retinal surgery for treatment of central retinal vein occlusion. AB - Vitreous surgery with retinal vein cannulation and injection of tissue plasminogen activator was performed in 8 eyes of 8 patients with longstanding visual loss secondary to central retinal vein occlusion. A modest improvement in visual acuity was demonstrated in 4 of the 8 eyes, and 3 of the 8 eyes maintained the preoperative vision. Retinal vein cannulation may offer a new treatment option for patients with central retinal vein occlusion. PMID- 10743933 TI - A technique for performing ultrasound biomicroscopy in the sitting and prone positions. AB - To develop a reliable method for performing ultrasound biomicroscopy (UBM) in the sitting and prone positions. The probe suspended from an articulated arm was removed and reattached to the arm upside-down for use in the prone position and horizontally for use in the sitting position. A latex Tono-Pen tip cover was attached to the edge of the eyecup and was placed over the transducer through the small hole on the tip of the Tono-Pen tip cover. UBM was successfully performed in the prone and sitting positions with no loss of image quality. This method can expand the ability of the UBM to examine alterations in anatomic relationships among anterior segment structures between the supine, sitting, and prone positions. PMID- 10743934 TI - Petalloid phacoemulsification. AB - Hard grade IV nuclei are difficult to emulsify without causing additional stress on the zonular apparatus. We herein, describe a method to accomplish successful phacoemulsification in hard cataracts. This technique of "petalloid phacoemulsification" consists of partial central debulking, followed by sequential chopping to create petal-shaped nuclear fragments emanating from the partially debulked center. The rim of the petals are emulsified followed by the emulsification of the central disc and the base of the petal. Twenty eyes with grade IV nuclei underwent uncomplicated petalloid phacoemulsification. The mean phaco time was 1.02 +/- 0.06 minutes and mean percentage endothelial loss was 4.2 +/- 0.8%. Petalloid phacoemulsification is a safe and useful technique in cases of hard cataracts. PMID- 10743935 TI - Laser peripheral iridotomy in chronic primary angle closure glaucoma. PMID- 10743936 TI - Irreversible inhibition of type I dehydroquinase by substrates for type II dehydroquinase. AB - Mechanistic differences between type I and type II dehydroquinases have been exploited in the design of type specific inhibitors. (2R)-2-Bromo-3-dehydroquinic acid (3), (2R)-2-fluoro-3-dehydroquinic acid (5) and 2-bromo-3-dehydroshikimic acid (4), all excellent substrates for type II dehydroquinase, are shown to be irreversible inhibitors of type I dehydroquinase. PMID- 10743937 TI - Effects of isosteric pyridone replacements in androgen receptor antagonists based on 1,2-dihydro- and 1,2,3,4-tetrahydro-2,2-dimethyl-6-trifluoromethyl-8 pyridono[5,6-g]quin olines. AB - A series of nonsteroidal human androgen receptor (hAR) antagonists based on 8 substituted 1,2-dihydro- and 1,2,3,4-tetrahydro-2,2-dimethyl-6 trifluoromethylpyrido[3,2-g]quin olines was synthesized. Compounds in this series were tested for the ability to bind to hAR and inhibit hAR-dependent transcription in a mammalian cellular background. PMID- 10743938 TI - Nonsteroidal progesterone receptor antagonists based on 6 thiophenehydroquinolines. AB - Synthesis and biological evaluation of 6-thiophene 1,2-dihydro or 1,2,3,4 tetrahydroquinoline derivatives resulted in a number of potent nonsteroidal antiprogestins. PMID- 10743939 TI - Synthesis and biological activities of NB-506 analogues modified at the glucose group. AB - A new indolocarbazole compound, NB-506 (1), modified at the glucose group yielded a beta-D-glucopyranoside, J-107,088 (2), which showed potent anticancer activity. A beta-D-ribofuranoside, J-109,534 (3), was found to be 6 times more potent than J-107,088 at inhibiting topoisomerase I. PMID- 10743940 TI - DNA aptamers that bind to chitin. AB - We have succeeded in the acquisition of DNA aptamers that recognize chitin using in vitro selection. The obtained DNA aptamers have the stem-loop or bulge loop structures with guanine rich loop clusters and the clockwise B-form stems. PMID- 10743941 TI - Pyrrolidine as a cogwheel-like scaffold for the deployment of diverse functionality through cycloaddition reactions of metallo-1,3-dipoles in aqueous media. AB - The reaction of glycinatocopper complexes with cinnamaldehydes under mildy basic aqueous conditions, affords polysubstituted prolines, which can be systematically modified in a number of chemoselective transformations. PMID- 10743942 TI - Design and synthesis of mimics of S-adenosyl-L-homocysteine as potential inhibitors of erythromycin methyltransferases. AB - A series of indanotriazine C-ribosides were prepared as SAH mimics, and tested for their ability to inhibit erythromycin resistance methylases Erm AM and Erm C'. A carbocyclic analogue derived from quinic acid was also synthesized and tested. PMID- 10743943 TI - Design and synthesis of functionalized glycomers as non-peptidic ligands for SH2 binding and as inhibitors of A-431 human epidermoid and HT-29 colon carcinoma cell lines. AB - A set of O-substituted aryl beta-D-glucopyranosides were prepared and found to have inhibitory activity on the growth of two carcinoma cell lines. PMID- 10743944 TI - Potent antagonists of gonadotropin releasing hormone receptors derived from quinolone-6-carboxamides. AB - SAR studies which focused upon the C-6 position of a recently described series of quinolone gonadotropin releasing hormone antagonists are reported. Synthetic access to diverse quinolone-6-carboxamides was achieved via the palladium catalyzed amino-carbonylation reactions of iodide 4 with various amines. Amides related to 9y were especially potent, functional antagonists of rat and human GnRH receptors. PMID- 10743945 TI - Ring constrained analogues of beta-alanine-containing GPIIb/IIIa receptor antagonists. AB - A series of ring constrained analogues of the GPIIb/IIIa receptor antagonist XR299 (1) was investigated as potential inhibitors of glycoprotein IIb/IIIa, a platelet receptor that plays a key role in platelet aggregation and platelet adhesion. Ring size was found to have a large effect on in vitro potency. Selected compounds showed good in vitro activity, a preference for binding to activated platelets, and modest duration of action when dosed i.v. as a racemate in a canine model. PMID- 10743946 TI - Synthesis of neurotensin(9-13) analogues exhibiting enhanced human neurotensin receptor binding affinities. AB - Recent evidence is consistent with neurotensin (NT)(8-13) adopting a Type I beta turn conformation while binding the NT receptor, which would place the cationic side-chains of Arg(8) and Arg(9) in close proximity. This was the basis for the design, synthesis and analysis of truncated NT(9-13) analogues 1-5 with dicationic position 9 side-chains to emulate the functions of the 8 and 9 side chains of NT(8-13). PMID- 10743947 TI - Synthesis and characterization of a novel class of protein tyrosine phosphatase inhibitors. AB - Nonpeptidyl aryloxymethylphosphonates were prepared and evaluated as protein tyrosine phosphatase inhibitors. The results suggest that aryloxymethylphosphonates are effective nonhydrolyzable phosphotyrosine surrogates and provide further insight into the molecular mechanisms by which phosphate mimics inhibit phosphatase function. PMID- 10743948 TI - 5-Arylamino-2-methyl-4,7-dioxobenzothiazoles as inhibitors of cyclin-dependent kinase 4 and cytotoxic agents. AB - 5-Arylamino-2-methyl-4,7-dioxobenzothiazoles were synthesized as inhibitors of cyclin-dependent kinase 4 (CDK4) and cytotoxic agents. Most of the 4,7 dioxobenzothiazoles exhibited selective inhibitory activities for the CDK4 and cytotoxic potential against human cancer cell lines. PMID- 10743949 TI - (1,4-Benzothiazinyloxy)alkylpiperazine derivatives as potential antihypertensive agents. AB - A series of compounds having a piperazine moiety variously linked to the benzothiazine nucleus were synthesized and evaluated for their in vitro alpha adrenoceptor affinity by radioligand receptor binding assays. Some compounds bearing a oxyalkyl-(2-methoxyphenyl)piperazine side chain were good alpha1 adrenoreceptor ligands. PMID- 10743950 TI - Antimitotic activity of moroidin, a bicyclic peptide from the seeds of Celosia argentea. AB - A unique bicyclic peptide, moroidin (1), from the seeds of Celosia argentea (Amaranthaceae) strongly inhibited the polymerization of tubulin. The stereostructure of moroidin (1) was reinvestigated by spectroscopic data, chemical degradation, and molecular dynamics simulation. PMID- 10743951 TI - Synthesis of enantiomerically pure (+)- and (-)-18-methoxycoronaridine hydrochloride and their preliminary assessment as anti-addictive agents. AB - Chemical resolution of racemic 18-methoxycoronaridine (18-MC) was achieved by the formation of its diastereomeric sulfonamides with either (R)-(-)- or (S)-(+) camphorsulfonyl chloride. Preliminary assessment of (+)-, (-)-, and (+/-)-18-MC x HCl showed similar effects on morphine self-administration in a rat model, and similar affinities at the kappa opioid receptors. PMID- 10743952 TI - The design, synthesis and activity of non-ATP competitive inhibitors of pp60(c src) tyrosine kinase. Part 1: hydroxynaphthalene derivatives. AB - A series of hydroxynaphthalene pp60(c-src) non-peptide inhibitors was designed, using the crystal structure of the insulin receptor tyrosine kinase as a qualitative model, to target the peptide substrate binding site. Representative inhibitors were shown to bind non-competitively with respect to ATP. PMID- 10743953 TI - The design, synthesis and activity of non-ATP competitive inhibitors of pp60(c src) tyrosine kinase. Part 2: hydroxyindole derivatives. AB - As part of a continuing effort to identify novel scaffolds that inhibit the pp60(c-src) protein tyrosine kinase, a series of hydroxyindole amides was rationally designed and synthesized. The most potent derivative was found to bind non-competitively with respect to ATP. PMID- 10743954 TI - Rational design, synthesis and structure-activity relationships of antitumor (E) 2-benzylidene-1-tetralones and (E)-2-benzylidene-1-indanones. AB - Novel substituted 6,7-dimethoxy-1-tetralones and 5,6-dimethoxy-1-indanones have been synthesized and evaluated for their cytotoxicity. Compounds with 3' lipophilic, 3',5'-dilipophilic, or 3',5'-dilipophilic-4'-hydrophilic substituents on (E)-2-benzylidene moiety showed highly cytotoxic effects. The unique structure of 42 possibly matches the pharmacophore features for these cytotoxic compounds. PMID- 10743956 TI - Bifunctional alkylating agents derived from duocarmycin SA: potent antitumor activity with altered sequence selectivity. AB - The series of four dimers derived from head to tail coupling of the two enantiomers of the duocarmycin SA alkylation subunit are described. PMID- 10743955 TI - Selection of a potent inhibitor of trihydroxynaphthalene reductase by sorting disease control data. AB - Compounds that control rice blast, but not other crop diseases, were selected for testing as inhibitors of trihydroxynaphthalene reductase of the fungal melanin biosynthetic pathway. A potent inhibitor of the enzyme (2) (Ki = 25 nM) was identified. An X-ray structure of the enzyme-NADPH-2 complex was determined at 2.1 A resolution. PMID- 10743957 TI - Protease inhibitors: synthesis of clostridium histolyticum collagenase inhibitors incorporating sulfonyl-L-alanine hydroxamate moieties. AB - A series of hydroxamates was obtained by the reaction of N-(4-nitrobenzyl)-L alanine with alkyl/arylsulfonyl halides, followed by conversion of the COOH group into CONHOH. Structurally-related compounds were prepared similarly by using arylsulfonyl isocyanates, aryl isocyanates or arylsulfenyl halides instead of the sulfonyl halides. Many of the new compounds showed nanomolar affinity for the bacterial collagenase isolated from the pathogen Clostridium histolyticum. PMID- 10743958 TI - Solid phase synthesis of diamides as potential bone resorption inhibitors. AB - Unsymmetrical diamide libraries have been prepared by a general and versatile solid phase route, using diacid templates in combination with aromatic and aliphatic amines chosen with the help of statistical experimental design. The compounds were tested as potential inhibitors of osteoclast vacuolar ATPase. PMID- 10743959 TI - N-[[1-(2-phenylethyl)pyrrolidin-2-yl]methyl]cyclohexanecarboxamides as selective 5-HT1A receptor agonists. AB - A series of benzamides was synthesized as selective agonists for the 5-HT1A receptor. It was found that (S)-N-[[1-(2-phenylethyl)pyrrolidin-2 yl]methyl]cyclohexanecarb oxamide(7-(S)) has potent and selective agonistic activity for the 5-HT1A receptor (5-HT1A; Ki 0.49 nmol/L, D2; IC50 = >1000 nmol/L, 5-HT2; Ki = 240 nmol/L). PMID- 10743960 TI - Antitumour benzothiazoles. Part 10: the synthesis and antitumour activity of benzothiazole substituted quinol derivatives. AB - The synthesis of a series of new antitumour agents, the benzothiazole substituted quinol ethers and esters, is reported via the hypervalent iodine mediated oxidation of hydroxylated 2-phenylbenzothiazoles. The products were found to be active in vitro against human colon and breast cancer cell lines with IC50 values in the nanomolar range. PMID- 10743961 TI - Synthesis and biological evaluation of novel 9-functional heterocyclic coupled 7 deoxy-9-dihydropaclitaxel analogue. AB - Novel 9-functional heterocyclic coupled 7-deoxy-9-dihydropaclitaxel analogues 17 and 22-24 synthesized from a natural taxoid 5-cinnamoyltriacetyltaxicin-I (3) and their biological evaluation in tubulin assembly activity and cytotoxicity in vitro against several human tumor cell lines are first presented. The biologically tested results show that 17, 22 and 23 are inactive in tubulin assembly assay and have no more remarkable cytotoxicities against human tumor cell lines SK-OV3, WIDR and MCF-7, though 22 and 23 exhibit more potent cytotoxicity against human liver cancer and human esophagus cancer cell lines (BEL-7402 and ECa-109) than paclitaxel. PMID- 10743962 TI - Isoxsuprine: more questions than answers. PMID- 10743963 TI - Postexercise and positional variation in mechanical properties of the radius in young horses. AB - The metacarpal of the horse is severely loaded during vigorous exercise. Metacarpal specimens have a greater impact strength in young horses that have been exercised than in those that have only been walked. We did not find a corresponding difference in the radius of the same horses. We show that cranial (anterior) cortical bone from the radius, which is loaded in tension during locomotion, has a greater Young's modulus, and tensile and bending strength, than bone from the caudal (posterior) cortex, which is loaded in compression. Caudal bone is, however, stronger in compression. The differences can be explained by differences in the histological structure developed by the 2 cortices and are presumably adaptive. This work confirms the work of others. Furthermore, we demonstrate that the impact energy absorption of cranial bone is nearly twice as great as that of caudal bone. The caudal cortex has apparently paid a heavy price in its reduction in resistance to accidental impact loading for being stronger than the cranial cortex in compressive loading. PMID- 10743964 TI - Analysis of the right ventricular function in the exercising horse: use of the Fourier Transform. AB - The objective of this study was to develop and test a technique to allow dynamic cardiac function to be studied during exercise in the horse. Blood pressure waveforms in the exercising horse are difficult to interpret because of the large influence of stride and respiration. A method has been devised to study dynamic right ventricular variables during high-speed exercise in the horse. A Fast Fourier Transform was performed on the digitised pressure waveforms and the frequency components associated with stride and respiration were removed. An inverse Fourier Transform was then performed to generate a time-domain pressure signal. Several dynamic right ventricular variables were calculated using the derived signal. Various parameters associated with removing frequencies from the frequency-domain pressure signal were changed to determine their influence on the variables. Most of the variables were not sensitive to these parameters. When compared during separate exercise bouts, some variables differed among runs, while others were not significantly different. Using the signal separation technique described here, right ventricular function of an exercising horse can be critically analysed. PMID- 10743965 TI - Ultrasonography of the equine neonatal kidney. AB - Ultrasonography is important in the clinical examination of the foal. The ultrasonographic appearance and size of the neonatal kidneys were defined and an imaging protocol established in 6 normal Thoroughbred foals (mean age +/- s.d. 5.0 +/- 3.2 days). Characteristically, in both the heart-shaped right kidney and bean-shaped left kidney, the renal cortex was more echogenic than the medulla. The terminal recesses, renal crest and pelvis were identified, as was the ureter, which contained anechoic urine in its lumen. The renal, interlobar and arcuate vessels were seen. For the right kidney, the ultrasonographic probe was placed at the 14-17th intercostal spaces and paralumbar fossa. For the left kidney, the probe was at the 16th or 17th intercostal spaces and paralumbar fossa. Perirenal structures, including the caudate lobe of the liver, the dorsal extremity of the spleen, the adrenals, the aorta and caudal vena cava were also identified. An understanding of the ultrasonographic appearance of the normal neonatal kidney, accompanied by a routine imaging protocol to ensure that all regions of each kidney are examined, permit a more informed interpretation of renal images in the first few days postpartum. PMID- 10743966 TI - Melanin affinity: a possible explanation of isoxsuprine retention in the horse. AB - Isoxsuprine is used in veterinary medicine as a vasodilating agent. The drug has been detected in the urine of horses up to 6 weeks after the cessation of administration. In the present study, the distribution pattern of 3H-isoxsuprine was investigated using whole body autoradiography in mice to find a possible site of retention. Melanin was the only place of retention identified. Additional in vitro studies showed an affinity of isoxsuprine to both melanin and keratin. The K(d) values were 0.02 mmol/l and 1 mmol/l, and the B(max) values were 0.2 micromol/mg and 2 micromol/mg, respectively. A low affinity site with approximately the same K(d) and B(max) as keratin was also detected for melanin. 3H-isoxsuprine was found to have affinity to pigmented horse skin after incubation in vitro and microautoradiography. We believe that affinity to melanin and possibly also to keratin can cause retention of the drug in the body and therefore explain the prolonged excretion of low levels of isoxsuprine in the horse. PMID- 10743967 TI - Affinity of isoxsuprine for adrenoreceptors in equine digital artery and implications for vasodilatory action. AB - We used isolated equine digital arteries to study the vasodilatory mechanism of isoxsuprine, and fowl caecum preparations to investigate the affinity of the drug for beta-adrenoceptors. Isoxsuprine is a potent vasodilator of arterial smooth muscle that has been precontracted by an alpha-adrenoceptor agonist such as noradrenaline (log EC50 = -6.33 [-5.98; -6.68]). The present study indicates that its effect is due to alpha-adrenoceptor blockade since: (1) after a long lasting exposure to cumulative doses of isoxsuprine the vasoconstricting action of noradrenaline cannot be restored; (2) isoxsuprine does not promote relaxation on preparations precontracted by PGF2alpha; (3) isoxsuprine shifts the dose-response curve of noradrenaline to the right; and (4) its affinity (pK(B) = 6.90 [6.60; 7.20]) in this experiment is comparable to that in noradrenaline-precontracted preparations and is 14 times lower than that of the selective alpha1-adrenergic antagonist prazosin [pK(B) = 8.04 (7.40; 8.68]). The affinity of isoxsuprine for beta-adrenoceptors was 100 times lower than that of isoprenaline when tested on fowl caecum. This preparation has a large beta-adrenoceptor and negligible alpha adrenoceptor population concerned with the control of smooth muscle motility. Our data suggest that the alpha-mediated effect of isoxsuprine on horse arterial smooth muscle is due to higher affinity of the drug for alpha- than beta adrenoceptors rather than low concentration or functionality of beta-sites at this site. According to these data, pure beta2-agonists seem to be more profitable tools to determine vasodilation of the arterial bed in horses legs. PMID- 10743968 TI - Variability of Doppler ultrasound measurements of hindlimb blood flow in conscious horses. AB - Ultrasonography of the left and right femoral artery and vein was performed in 5 conscious horses. Velocity waveforms, recorded using low pulse repetition frequency, were used to calculate time-averaged mean velocity (TAV), velocity of component a (TaVa), velocity of component b (TaVb) and volumetric flow. Waveform analysis included calculation of early diastolic deceleration slope (EDDS) and pulsatility index (PI) and subjective description of the waveform. Measurements were recorded at monthly intervals for 6 months. The repeated measurements were used to determine the within-horse variation. Horses were then anaesthetised with halothane and the same measurements recorded 60 min after induction. Differences between measurements recorded in conscious and anaesthetised horses were determined. Within-horse variability for diameters of femoral arteries and veins was small (CV<10%). Within variability for all other measurements was marked (CV>11%), with within-horse variability being the largest for femoral venous flow (CV>35%). Source of variability was measurement error and biological variation. Despite variability in conscious horses it was possible to detect changes in blood flow during anaesthesia. In the femoral arteries, volumetric flow was lower and EDDS and PI was higher in anaesthetised horses. In the femoral veins, volumetric flow was also lower in anaesthetised horses. Therefore Doppler ultrasound appears to be a useful technique for studying blood flow in conscious and anaesthetised horses. PMID- 10743969 TI - Nitric oxide inhibits aggrecan degradation in explant cultures of equine articular cartilage. AB - Arthroses are debilitating diseases of articular joints which result in erosion of the cartilage extracellular matrix. Nitric oxide (NO) is a major component of the inflammatory response, and has been implicated as a mediator of some of the effects of the proinflammatory cytokine, interleukin-1 (IL-1). In this study, we investigated the role of NO in the regulation of proteoglycan degradation in equine articular cartilage. NO fully mediated the suppressive effect of IL-1 on proteoglycan synthesis. However, NO was also antagonistic to proteoglycan degradation, irrespective of whether degradation was initiated by 10 ng/ml IL-1 or 1 micromol/l all-trans retinoic acid (RA) which (unlike IL-1) does not elevate NO production. This was confirmed using the NO donor 2,2' (hydroxynitrosohydrazono) bis-ethanamine (DETA-NONOate) and the iNOS inhibitor L N5-iminoethyl ornithine (dihydrochloride) (L-NIO). The G1 fragments of aggrecan were detected in the media and extracts of cartilage explant cultures treated with all-trans RA, DETA-NONOate and L-NIO. The presence of exogenous NO in culture resulted in a decrease in the appearance of the 'aggrecanase' cleavage epitope. Therefore, changes in the appearance of the G1 fragment expressing the 'aggrecanase' cleavage epitope in the media emulated the glycosaminoglycan loss from the tissue. These results lend further support to the hypothesis that NO has an anticatabolic role in equine cartilage proteoglycan degradation, and suggest that this may be mediated by the regulation of 'aggrecanase' activity. Therefore, any pharmacological intervention using NO as a target must take into account both its catabolic and anticatabolic roles in joint tissue turnover. PMID- 10743970 TI - Spontaneous production of nitric oxide (NO), prostaglandin (PGE2) and neutral metalloproteinases (NMPs) in media of explant cultures of equine synovial membrane and articular cartilage from normal and osteoarthritic joints. AB - Nitric oxide (NO), prostaglandin E2 (PGE2), and the activity of neutral metalloproteinases (NMPs) were measured in conditioned media of equine synovial membrane and articular cartilage explant cultures from horses with normal joints (n = 7) and from horses affected with moderate (n = 7) or severe osteoarthritis (n = 14) as judged by macroscopic appearance. Normal articular cartilage appeared glossy and bluish-white, was of normal thickness and showed no evidence of discolouration, fibrillation or other cartilage discontinuity. Slight discolouration and fibrillation or minor clefts of the cartilage were considered as moderate OA, whereas erosions of articular cartilage down to the subchondral bone were considered as cases of severe OA. Explant cultures of equine synovial membrane and articular cartilage released the local mediators, NO and PGE2, as well as detectable levels of NMP activity into culture media. Concentrations of NO were higher in articular cartilage explants compared to synovial membrane explants, whereas concentrations of PGE2 were higher in synovial membrane explants. The NMPs with collagenolytic activities were similar in both explant cultures, whereas gelatinolytic activities were higher in synovial membrane explant cultures and caseinolytic activities were generally higher in articular cartilage explant cultures. Furthermore it was shown that concentrations or enzyme activities increased according to the severity of disease of the joints. Concentrations for NO, collagenolytic and gelatinolytic NMPs were relatively stable, whereas PGE2 and caseinolytic NMP concentrations increased over time in culture. PMID- 10743971 TI - The effect of oxytocin on contractility of the equine oesophagus: a potential treatment for oesophageal obstruction. AB - This study was performed to determine the effect of administration of i.v. oxytocin on the contractility of the musculature associated with the equine oesophagus. Nine clinically normal horses were fitted with a nasogastric tube modified with inflatable latex cuffs. These cuffs were connected to piezoelectric pressure recording devices. Oxytocin in 3 different doses or saline controls were administered i.v. in a randomised block pattern. Systolic blood pressure, ECG, heart rate and nasogastric tube cuff pressures were then measured for 60 min. Administration of oxytocin i.v. at 0.11 and 0.22 iu/kg bwt, resulted in a short term statistically significant relaxation of the musculature of the equine oesophagus. When oxytocin was administered at 0.11, 0.22 and 0.44 iu/kg bwt, no clinically significant cardiovascular changes were seen. In approximately 5% of the oxytocin administrations, signs of mild short-term abdominal discomfort were observed. In clinical cases of noncomplicated oesophageal obstruction, it is suggested that reduction in tone of oesophageal musculature may result in passage of oesophageal obstructions with reduced risk of oesophageal injury when compared to other traditional treatments. PMID- 10743972 TI - Ossification of the cartilages in the front feet of young Norwegian coldblooded horses. AB - The purpose of the present study was to evaluate the nature of ossification of the cartilages in the front feet of young, about 2-year-old Norwegian coldblooded horses, and to compare offspring of different sires in this respect. Dorsopalmar radiographs of the front feet of 392 horses (187 female and 205 male) were evaluated for ossification at the base of the cartilage and for separate centres of ossification. The horses were offspring of 45 different sires. Ossification extending above the navicular bone and separate centres of ossification were considered as significant. Minimal to mild ossification at the base of the cartilages was commonly seen, and significant ossification was present in one or more of the cartilages in 11.5% of the horses. The lateral compared to medial cartilages had more ossification and females had more ossification and more separate centres of ossification than males. The prevalence of horses with significant ossifications was significantly higher (46.3%) among offspring of one frequently used stallion than in the group consisting of offspring of 4 other popular stallions (3.5%) and in another group consisting of offspring of other, less frequently used stallions (9.6%). Ossification of the cartilages is considered to have a hereditary background in Norwegian coldblooded horses. PMID- 10743973 TI - Exercise-induced changes in proteoglycan metabolism of equine articular cartilage. PMID- 10743974 TI - Effect of infusion volume and sperm numbers on persistence of uterine inflammation in mares. PMID- 10743975 TI - Polycystic kidneys as a cause of chronic renal failure and secondary hypoparathyroidism in a horse. PMID- 10743976 TI - Mitral valve dysplasia in a foal. PMID- 10743977 TI - Splenic infarction and splenectomy in a jumping horse. PMID- 10743978 TI - Cefaclor revisited. AB - OBJECTIVE: This paper describes the rationale for choosing cefaclor for the management of respiratory tract infections. BACKGROUND: Since 1979, cefaclor has established a record of efficacy in the management of respiratory tract infections. Factors contributing to the efficacy and tolerability of this drug include its molecular stability, activity against the most prevalent gram positive and gram-negative respiratory tract pathogens, rapid absorption, >90% bioavailability, and good penetration into respiratory mucosa. After 2 decades of widespread use, this agent remains clinically effective in patients with respiratory tract infections, making it competitive with other cephalosporins and with macrolides and fluoroquinolones, including many newer agents used for respiratory tract infections. Cefaclor extended-release tablets, the newest formulation, retain the positive efficacy and tolerability attributes of immediate-release cefaclor, varying mainly in the rate of dissolution. The approved indications for extended-release cefaclor include bacterial bronchitis, pharyngitis, and skin infections. METHODS: A MEDLINE search showed that the few adverse effects related to therapy with cefaclor are usually minor and transient and that drug-drug interactions involving cefaclor are rare. CONCLUSIONS: Multiple clinical trials have shown that extended-release cefaclor in 375-mg and 500-mg doses BID demonstrates tolerability and efficacy comparable to those of immediate-release cefaclor 250 mg TID. Extended-release cefaclor is indicated for BID dosing, which should encourage greater compliance. PMID- 10743979 TI - Antiglaucoma medications: a review of safety and tolerability issues related to their use. AB - BACKGROUND: Much experience has been gained with the use of older classes of antiglaucoma agents--topical beta-adrenergic-receptor antagonists, nonselective adrenergic-receptor agonists, oral carbonic anhydrase inhibitors, and cholinergic agents. In the past decade, new drugs and classes of drugs used to treat glaucoma have become available, including topical carbonic anhydrase inhibitors, prostaglandin analogues, and alpha2-adrenergic-receptor agonists. Extensive community-based use of antiglaucoma medications has led to an increased understanding of the acute and long-term safety and tolerability issues associated with their use. OBJECTIVE: This paper reviews the side effects associated with the various classes of topical antiglaucoma drugs, with a particular focus on long-term safety issues. PMID- 10743980 TI - Efficacy of oral long-term N-acetylcysteine in chronic bronchopulmonary disease: a meta-analysis of published double-blind, placebo-controlled clinical trials. AB - OBJECTIVE: This meta-analysis was performed to assess the possible prophylactic benefit of prolonged treatment with oral N-acetylcysteine (NAC) in chronic bronchitis (CB) based on qualifying clinical trials. Treatment of acute exacerbations with NAC was not investigated. BACKGROUND: Prolonged treatment with oral NAC has been investigated in a number of studies of patients with CB. NAC prevented acute exacerbations and symptoms of CB in some but not all trials. METHODS: The trials included in this analysis were selected from a MEDLINE search of the period from January 1, 1980, through June 30, 1995; references in the articles retrieved in the initial search; and consultation with 2 experts. Selection was based on the following criteria: published, double-blind, placebo controlled, chronic bronchopulmonary disease, duration of therapy > or =2 months, and data sufficient to calculate an outcome variable permitting direct comparison of studies (effect size) for both NAC and placebo groups. The primary end point was the incidence of acute exacerbations in 7 of 8 trials and clinical assessment in the other. In 7 studies, inclusion criteria were based on Medical Research Council criteria for CB, with an additional criterion in some trials. For the meta-analysis, the end points of individual trials were transformed into an effect size as a common outcome. RESULTS: Of 21 trials initially identified, 8 qualified for inclusion. References from the 8 papers and consultation with the experts produced 8 additional publications, 1 of which qualified for inclusion. NAC was administered orally at a daily dose of 400 mg (1 study), 600 mg (5 studies), or 1200 mg (1 study). One other trial used a dose of 600 mg 3 times per week. The duration of treatment was 3 months (1 study), > or =5 months (2 studies), or 6 months (7 studies). The results of this meta-analysis showed a statistically significant effect size for NAC compared with placebo. The overall value of effect size was -1.37 (95% CI, -1.5 to -1.25). Sensitivity analyses did not significantly alter these results. In a subset analysis of trials with the number of acute exacerbations as a clinical end point, a mean difference of -0.32 clinical event (95% CI, -0.50 to -0.18) was found (ie, a 23% decrease in the number of acute exacerbations compared with placebo). CONCLUSION: These findings suggest that a prolonged course of oral NAC prevents acute exacerbations of CB, thus possibly decreasing morbidity and health care costs. PMID- 10743981 TI - Improved postprandial glycemic control with Humalog Mix75/25 after a standard test meal in patients with type 2 diabetes mellitus. AB - OBJECTIVE: This double-blind study was designed to compare the postprandial glucodynamic profile of Humalog Mix75/25, a new premixed insulin analogue containing 75% neutral protamine lispro and 25% insulin lispro with that of human insulin 70/30 (70% neutral protamine Hagedorn insulin and 30% regular human insulin) in patients with type 2 diabetes mellitus. BACKGROUND: Insulin lispro Mix75/25 (Mix75/25) is the first available insulin formulation in which both the rapid-acting and basal components are insulin analogues. METHODS: This randomized, multicenter, double-blind, crossover study monitored patients' postprandial glucodynamic response to Mix75/25 and human insulin 70/30 (70/30) after a standard test meal. Eighty-four patients with type 2 diabetes participated in this study and were randomly assigned to 1 of 2 treatment sequence groups. Patients received an identical test meal on 4 occasions, completing 2 test meals for each treatment. Equal doses of Mix75/25 or 70/30 were administered 5 minutes before each of the 2 test meals, with doses individualized for each patient. Blood samples were collected for 4 hours after the meal for measurement of plasma glucose. From these plasma glucose measurements, fasting plasma glucose, 2-hour postprandial glucose (2pp), 2-hour postprandial glucose excursion (2pp(ex)), maximum glucose excursion (Gex(max)), the area under the glucose concentration versus time curve from 0 to 4 hours (AUC4), and the area under the glucose excursion versus time curve from 0 to 4 hours (AUCex4) were calculated. RESULTS: Because of significant differences in the baseline fasting plasma glucose levels between Mix75/25 and 70/30 (Mix75/25: 8.9+/-2.2 mmol/L [160.2+/-39.6 mg/dL]; 70/30: 8.6+/-1.9 mmol/L [154+/-34 mg/dL), analyses of the excursion parameters provide a truer comparison of the glucodynamic response between insulin formulations. Mix75/25 resulted in significantly lower values for 2pp(ex) (3.35+/-2.28 vs 4.13+/-2.26 mmol/L), Gex(max) (4.51+/-1.88 vs 5.19+/-1.98 mmol/L), and AUCex4 (8.01+/-7.02 vs 10.6+/-6.47 mmol x h/L) compared with 70/30. CONCLUSIONS: In patients with type 2 diabetes mellitus, premeal injection of Mix75/25 resulted in better postprandial glycemic control than did premeal injection of 70/30 in the 4 hours after a standard meal. Mix75/25 is a valuable option for managing postprandial blood glucose in patients with type 2 diabetes mellitus who require insulin. PMID- 10743982 TI - Effect on bioavailability of admixing the contents of lansoprazole capsules with selected soft foods. AB - OBJECTIVE: This study was designed to compare the bioavailability of lansoprazole when administered as an intact capsule and when the contents are admixed with various soft foods. BACKGROUND: Patients sometimes cannot swallow or have difficulty swallowing intact capsules such as lansoprazole. To enable them to ingest the drug, the contents of the capsule can be admixed with small amounts of soft foods. METHODS: Twenty-four healthy adult volunteers participated in this single-dose, 4-period crossover study by ingesting the contents of a 30-mg lansoprazole capsule that had been emptied into either a tablespoon of yogurt (regimen A), Ensure pudding (regimen B), or cottage cheese (regimen C), or given as an intact capsule (regimen D) during the first study period. The regimen assignments were rotated at weekly intervals so that each subject received each regimen. Blood samples were obtained over the 12-hour period after administration of each regimen, and pharmacokinetic parameters were determined. RESULTS: Of the 23 subjects who completed all 4 periods of the study, 18 were male and 5 were female. Their mean (+/- SD) age was 33.3+/-11.6 years, and their ages ranged from 19 to 52 years. No statistically significant differences between regimens were detected in mean maximum concentration, area under the curve (AUC) from time zero to the last measurable concentration, and AUC from time zero to infinity (AUC0 infinity) using analysis of variance. A statistically significant difference was detected in the time to maximum concentration between regimens C and D at 2.1 and 1.5 hours, respectively (P < or = 0.05). Bioavailability was assessed by the two 1-sided tests procedure using a 90% CI for the AUC0-infinity ratio of test-to reference regimens. The 90% CIs were all within an acceptable equivalence range of 0.80 to 1.25. CONCLUSION: These results indicate similar bioavailabilities between the regimen in which the lansoprazole capsule was emptied and administration of the intact capsule. However, they may have limitations in predicting the results in ill, elderly, or very young patients. PMID- 10743983 TI - Use of hydrofluoroalkane propellant delivery system for inhaled albuterol in patients receiving asthma medications. AB - OBJECTIVE: This study was undertaken to assess drug-use patterns associated with albuterol delivery via a new propellant device compared with conventional chlorofluorocarbon (CFC) metered-dose inhalers (MDIs) in patients taking asthma medications in a population with pharmacy benefits. BACKGROUND: In addition to their ozone-depleting properties, conventional CFC inhalers often deliver inconsistent doses because of loss of prime and temperature instability. A new propellant, hydrofluoroalkane (HFA), incorporates a re-engineered delivery system associated with dosing reproducibility throughout the life of the canister. METHODS: Drug markers associated with management of asthma were used to identify a study cohort of new users of inhaled albuterol from a geographically diverse pharmacyclaims database from July 1, 1997, through December 31, 1997. A population of 282,879 members was identified over the 20-month follow-up period. In addition, a subset of chronic albuterol inhaler users (> or = 12 months; n = 96,879) was also identified to support a longitudinal analysis. Disease severity was controlled for by use of inhaled corticosteroids (ICS). To control for canisters received via physician office samples, HFA patient use was corrected by a physician-based canister adjustment based on HFA sample data. RESULTS: A total of 53.1% of participants were women and 46.1% were men; most of the population (72.5%) was <65 years of age. Canister use for HFA patients was consistently lower (2.7+/-3.2 vs 5.4+/-6.7) than for CFC MDIs for the entire cohort over the 20-month assessment period. This difference was consistently observed for albuterol canister use in patients with and without concomitant ICS use (3.3+/ 3.8 HFA vs 7.2+/-7.5 CFC for ICS users and 2.1+/-2.1 HFA vs 4.1+/-5.7 CFC for non ICS users). Time to next prescription also was longer for HFA patients than for CFC patients (61.6+/-50.9 days HFA vs 47.3+/-40.8 days CFC). When duration of therapy and physician samples associated with product launch were controlled for, similar differences were consistently observed. CFC patients used, on average, 1.3 more canisters per year than did HFA patients (P < 0.001), averaging 10.7 canisters (95% CI, 10.6 to 10.7), compared with 9.4 canisters used by HFA patients (95% CI, 8.9 to 9.9). Further analyses indicated that this finding was consistent when ICS use was controlled for (CFC plus ICS mean, 11.9 canisters vs HFA plus ICS mean, 10.4 canisters; P < 0.001). CONCLUSION: This study provides useful information about the effect of use of a new albuterol delivery system on asthma inhaler management. These data suggest that CFC patients use an average of 1.3 more canisters per year compared with HFA patients independent of asthma severity as measured by ICS use. This improvement in dosing characteristics has the potential to translate into enhanced economic outcomes. PMID- 10743984 TI - Economic assessment of the community-acquired pneumonia intervention trial employing levofloxacin. AB - OBJECTIVE: The purpose of this study was to assess use of a critical pathway designed to manage community-acquired pneumonia more efficiently than its management with conventional therapy. METHODS: Economic outcomes were assessed in conjunction with a cluster-design, randomized, controlled trial. Nineteen participating Canadian hospitals were randomized to implement the critical pathway (n = 9) or conventional therapy (n = 10). The critical pathway included a clinical prediction rule to guide the admission decision, treatment with levofloxacin, and practice guidelines. Patient data on medical resource use, lost productivity, and quality of life were collected prospectively for > or =6 weeks after treatment. Costs were calculated from the government, health care system, and societal perspectives, with imputation of missing outpatient costs and the costs of lost productivity when necessary. Bootstrapping was used to identify 95% CIs for the total cost per patient. RESULTS: The analysis included all eligible patients in the critical pathway (n = 716) and conventional therapy (n = 1027) arms. There were fewer hospital admissions in the critical pathway arm than in the conventional therapy arm, both overall (46.5% vs 62.2%; P = 0.01) and in low risk patients (33.2% vs 46.8%; P < 0.001). Compared with conventional therapy, hospitals in the critical pathway arm had 1.6 fewer bed days per patient managed (P = 0.05) and used fewer inpatient medical resources. The 2 study arms had similar outpatient, readmission, and lost-productivity costs, and similar quality of-life outcomes. The critical pathway produced cost savings from all 3 perspectives that ranged from $457 to $994 per patient. CONCLUSIONS: The critical pathway employing levofloxacin resulted in cost savings compared with conventional therapy and did not compromise health outcomes. PMID- 10743985 TI - The Bone and Joint Decade 2000-2010: an update. PMID- 10743986 TI - The Swedish Knee Arthroplasty Project. PMID- 10743987 TI - Mortality after total hip replacement: 0-10-year follow-up of 39,543 patients in the Norwegian Arthroplasty Register. AB - We have studied the mortality after total hip replacement (THR) of 39,543 patients, having a mean age of 69 years, who were reported to the Norwegian Arthroplasty Register. The median follow-up time was 5.2 (0-10.4) years. 323 of 6201 deaths occurred during the first 60 postoperative days. The patient mortality was compared with the mortality in the Norwegian population, using standardized mortality ratios (SMR). The SMRs were compared and adjusted for age, gender, and other possible confounders in a Cox regression model incorporating the population mortality. We observed a lower mortality in patients with THR than in the Norwegian population (8-year patient mortality was 25%, versus 30% in the corresponding Norwegian population. SMR = 0.81). There was an increased standardized mortality ratio in patients less than 50 years (SMR = 2.50), patients 50-59 years (SMR = 1.16), patients with THR due to rheumatoid arthritis (SMR = 1.48), and patients with femoral neck fracture (SMR = 1.11). The SMR decreased with increasing age at the time of THR surgery. After revision surgery, the SMR was similar to that after the first primary operation, whereas a second primary operation in the opposite hip was associated with a further reduction in the SMR (SMR = 0.65). During the first 60 postoperative days, all patient categories had a higher mortality than the general population (0.8% mortality, SMR = 1.39). PMID- 10743988 TI - Degeneration of the acetabular cartilage in osteonecrosis of the femoral head: histopathologic examination of 15 hips. AB - Acetabular cartilage with subchondral bone was taken from the superior dome from 15 hips of 13 patients undergoing total hip arthroplasty due to osteonecrosis of the femoral head. The mean age of the patients was 40 years. There were 10 hips ARCO stage IIIA, and 5 hips stage IIIB. 3 of the cases were mild, 12 moderate, and 1 had severe arthrosis. The degree of collapse of the femoral head was significantly related to the degeneration of the acetabular cartilage on histological examination. Our observations support the view that patients with an ARCO Stage III hip do not benefit from head-preserving procedures. They may also explain why bipolar prosthesis gives poorer results than total hip arthroplasty, in cases of osteonecrosis of the femoral head. PMID- 10743989 TI - Complications of liner locking system in Micro-structured Omnifit acetabular components: a radiographic evaluation of 887 hips followed for 5-10 years. AB - We evaluated 887 hips in 672 patients with uncemented MicroStructured Omnifit acetabular components for liner locking complications. We found 2 types of radiographic signs of liner locking system complications in 7 hips, developing between 2 and 4 years postoperatively. The incidence of liner locking system complications was 0.8% using this modular acetabular component. We recommend that a patient who has received a total hip arthroplasty including a MicroStructured Omnifit acetabular component should be monitored frequently for radiographic signs of liner locking system complications, especially with a polyethylene thickness of less than 8 mm. PMID- 10743990 TI - 11-year results in 2,846 patients of the Peterborough Hip Fracture Project: reduced morbidity, mortality and hospital stay. AB - 3,025 consecutive patients presenting over an 11-year period with an acute hip fracture were prospectively studied to determine the effectiveness of a designated hip fracture service. After the introduction of the service, the mean length of hospital stay per patient was reduced from 51 days to 21 days. This reduction was accomplished by a progressive increase in the proportion of patients discharged directly home from the admission ward (50%-86%) and a reduction in the numbers of patients transferred to care of the elderly wards (28%-6%) and other outlying wards (15%-3%). This change was accompanied by a reduction in the 30-day mortality rate from 22% to 7%. There was no significant increase in the re-admission rate or proportion of patients requiring institutional care. We conclude that the provision of designated staff and treatment regimes for hip fracture patients can result in a significant reduction in both mortality and morbidity, combined with substantial savings in patient bed days. PMID- 10743991 TI - Validity and reliability of Swedish WOMAC osteoarthritis index: a self administered disease-specific questionnaire (WOMAC) versus generic instruments (SF-36 and NHP). AB - We assessed whether a disease-specific, self-administered questionnaire could replace a generic instrument as an outcome tool after total hip replacement, and tested the validity and reliability of the Swedish WOMAC osteoarthritis index. 58 patients operated on with total hip arthroplasty 2-10 years ago were randomized to the study. All patients were asked to answer one disease-specific questionnaire (WOMAC osteoarthritis index) and two generic instruments (NUP and SF-36) in the same week. The procedure was repeated after 4 weeks. We tested content validity, construct validity, criterion validity, test-retest reliability and internal consistency reliability according to total score, domains and items. We found that both the generic instruments (NHP and SF-36) and the disease specific test (WOMAC) had a high validity, i.e., measuring what they were supposed to measure (high validity). The questionnaires were also reproducible over time (high reliability). We suggest the Swedish WOMAC to be used after total hip arthroplasty in future studies. PMID- 10743992 TI - Late occurring clinical deep vein thrombosis in joint-operated patients. AB - In a prospective study of 4,840 patients, we determined the annual incidence of clinical deep vein thrombosis (DVT) in mobilized, discharged orthopedic-operated "high-risk" patients (hip replacement surgery, knee replacement surgery, nailed hip fracture) and assumed "low-risk" patients (diagnostic knee arthroscopy). In addition, the time from the operation to the time when the patients were readmitted with clinically suspected DVT and the distribution of radiologically confirmed DVT were recorded. Thromboprophylaxis was routinely given for about 10 days to the high-risk groups during the hospital stay but not to patients undergoing knee arthroscopy. During 9 years, the annual incidence of DVT following major procedures was 2.1% (95% CI 1.6-2.6) vs. 0.6% (95% CI 0.2-1.1) after diagnostic knee arthroscopy. Symptoms appeared, on average, 27 (3-150) days after total hip replacement surgery, 36 (3-150) days after nailed hip fracture, 17 (6-30) days after total knee replacement and 1 (1-6) day after knee arthroscopy. In hip-operated patients, 50% of the DVTs were found in the proximal veins vs. 40% following knee arthroplasty. PMID- 10743993 TI - Reduced bleeding through temporary balloon occlusion in hip and knee revision surgery. AB - We used temporary balloon occlusion of the iliac or femoral artery to reduce blood loss in major hip or knee operations in 15 cases in 13 patients. The balloon was introduced by an interventional radiologist in the afternoon of the day before surgery or in the morning before. A latex occlusion balloon was inserted via the transfemoral ipsi- or contralateral route. The patients received two 40 mg doses of low molecular weight heparin. At the beginning of the operation, saline was injected into the predetermined volume to inflate the balloon. The balloons were inflated during 1-6 hours. In each case, the balloon occlusion clearly reduced bleeding in the surgical field and facilitated surgery. The perioperative bleeding was reduced by half, as compared to a retrospective control group. We measured the intraarterial blood pressures distally to the balloon in 2 patients. They decreased from 120 to 40 mm Hg and 155 to 50 mm Hg, respectively, after inflation. Two complications occurred, one bleeding due to catheter dislocation the night before surgery and one postoperative necrosis of the tip of a toe in a patient in whom the deflated balloon was not extruded until the day after surgery and the dose of heparin was too low. PMID- 10743994 TI - The psychological and social functioning of 14 children and 12 adolescents after Ilizarov leg lengthening. AB - We studied the psychological and social impact of the Ilizarov leg lengthening procedure in 26 patients (aged 6-17 years), who had completed the treatment at least 2 months previously. Measurements of depression and anxiety were performed preoperatively and at follow-up. Questions about functioning at school, daily life activities and treatment-related experiences were answered by the patient and his/her parents. The school or work results showed no significant decline during the lengthening procedure. Serious sleeping problems occurred during the procedure. There was no subjective improvement in physical skill after the procedure and almost one-fourth of the children still had complaints about their leg. We conclude that the Ilizarov procedure caused no serious psychological disturbances. PMID- 10743995 TI - Quantitative assessment of intravascular volume of the human Achilles tendon. AB - The pathogenesis of Achilles tendon rupture remains unclear, but vascular patterns may play an important role. We determined the intravascular volume of the Achilles tendon using a new method with injection of radioisotopes. A solution of Tc-99m and gelatin-ink was injected into the lower limbs of body donors. The intravascular volume of each 1 cm section of the Achilles tendon was measured using a gamma well counter. We found that the distal part of the Achilles tendon (0-2 cm above the calcaneus) had an intravascular volume of 59-98 microL/g tendon tissue. In the middle part of the tendon (3-6 cm above the calcaneal insertion), the intravascular volume was much less: 2743 microL/g tissue. The proximal part of the tendon (7-9 cm) had an intravascular volume between 51-100 microL/g tendon tissue. The reduced vascularization in the middle part of the human Achilles tendon may play a role in degeneration and spontaneous rupture of the tendon. PMID- 10743996 TI - Function after Achilles tendon rupture in the elderly: 25 patients older than 65 years followed for 3 years. AB - We retrospectively analyzed the function after Achilles tendon rupture in 25 patients older than 65 years, 3 (1-5) years after the initial treatment. The patients' median age at the time of injury was 71 (65-86) years. The initial management was surgical in 14 patients and non-surgical (8-week immobilization) in 10, 1 patient was not treated. The ratio of the number of heel-raises on the injured to the uninjured side was median 0.64 (0-1.14), showing a reduction in performance. However, in both surgically- and non-surgically-treated patients, the subjective impairment was mild, and the patients were able to perform most walking activities. Only 9 patients reached their previous activity level. Co morbidity was frequent: 17 patients had other diseases that affected their performance. 14 complications occurred in 11 patients. 5 patients sustained a rerupture (4 following initial closed treatment with plaster), 1 a deep venous thrombosis and 4 had superficial infections requiring antibiotic treatment. 1 patient sustained a fibular nerve injury following compression by the plaster cast and another a sural nerve injury during the operation. 2 patients had symptoms due to adhesions between the tendon and the skin. We conclude that Achilles tendon rupture in patients older than 65 years reduces lower limb function and that complications are common following surgical and non-surgical treatment. PMID- 10743997 TI - Unfavorable effect of knee immobilization on Achilles tendon healing in rabbits. AB - This study was undertaken to assess the effect of knee immobilization on the treatment of Achilles tendon rupture. After their Achilles tendons were severed, rabbits were divided into 2 groups. In Group A, only the ankle joint was immobilized. In Group B, both the knee and ankle joints were immobilized. At 4 weeks after surgery, both the ultimate tensile force and stiffness of the severed tendons were significantly greater in Group A than in Group B. In Group A, dense collagen fibers were seen in the repaired tendons, and the bundles of collagen fibers were parallel to one another along the axis of the tendons. In contrast, in Group B, dilated veins and capillaries were seen in the repaired tendons, and the proliferation of connective tissue containing collagen fibers was severely reduced around these veins and capillaries and was in general irregular and uneven. These results suggest that knee immobilization retards the healing of a ruptured Achilles tendon without suture, due to congestion and tension deprivation produced by keeping the tendon static. PMID- 10743998 TI - Time-dependent sensory nerve ingrowth into a bone conduction chamber. AB - We studied time-dependent ingrowth of sensory nerve fibers into a bone defect in a rat bone conduction chamber model. In 10 male Sprague Dawley rats, a titanium chamber was implanted bilaterally in the proximal tibiae, representing an experimental bone defect. To mimic a clinical situation, the chambers were filled with a fresh blood clot After 1, 2, 4, 6 and 8 weeks, 2 rats were fixed in vivo at each time before removal of specimens, and histological and immunohistochemical analyses. We used antisera against protein gene product 9.5, neural growth-associated protein 43/B-50, calcitonin gene-related peptide, and substance P, to locate regenerating sensory nerve fibers in the chamber. During bone defect healing, hematoxylin/eosin sections showed that new bone grew in through the ingrowth openings in the chamber, gradually filling it and replacing the blood clot. At 1 and 2 weeks after implantation, no nerve fibers could be detected. At 4, 6 and 8 weeks, however, small numbers of nerve fibers were seen in 8 of 11 specimens. The nerve fibers were located mainly in the dense fibrous tissue in close proximity to the new bone, and in some cases within the new forming bone. In this chamber model, the periosteum is not in contact with the bone ingrowth openings, and all ingrowing nerve fibers thus originated from the cortical bone, endosteum or bone marrow. We speculated that these late ingrowing sensory nerve fibers may actively participate in bone repair. PMID- 10743999 TI - Cancellous bone as an antibiotic carrier. AB - We compared the release characteristics of antibiotics from in vivo and in vitro processed morselized cancellous bone. The bone was impregnated with 7 antibiotics and compressed into a wire-mesh cylinder. In vitro, the bone was processed by daily transfer of the cylinder with its contents into test tubes with broth. The amount of antibiotic eluted from the bone was measured after 1, 3 and 7 days. In vivo, the cylinder was implanted intramuscularly in the interscapular region in rats. After 1, 3 and 7 days, the cylinder was removed and the amount of antibiotic eluted in broth was measured. The results showed that morselized cancellous bone can act as a carrier of antibiotics in vitro and in vivo. The elution profiles of netilmicin-, vancomycin-, clindamycin- and rifampicin impregnated cancellous bone processed in vitro and in vivo were similar. PMID- 10744000 TI - Surgical exposure of the stiff knee. PMID- 10744001 TI - Distal femoral allograft for massive proximal femoral deficiency. PMID- 10744002 TI - Fractured coracoid with anterior shoulder dislocation and greater tuberosity fracture--report of a bilateral case. PMID- 10744003 TI - Morselized bone allografting in revision total knee replacement--a case report with a 4-year histological follow-up. PMID- 10744004 TI - Accurate accuracy assessment: review of basic principles. PMID- 10744005 TI - Relationship between physical activity recall and free-living daily physical activity in older claudicants. AB - The purposes of this study were to determine the relationship between the physical activity values obtained from the peripheral arterial disease-physical activity recall (PAD-PAR) questionnaire and (1) the free-living daily physical activity obtained from the doubly labeled water technique and (2) clinical measures of PAD severity. Fifty-one older PAD patients (age= 70 +/- 6 years) were recruited from the Vascular Clinic at the Baltimore Veterans Affairs Medical Center and from radio and newspaper advertisements. Energy expenditure of physical activity (EEPA) was determined by using doubly labeled water and indirect calorimetry techniques. PAD severity was measured by ankle/brachial index (ABI) and walking distance to maximal claudication pain determined during a graded treadmill test. In addition, patients were also characterized on body composition and total daily energy expenditure. The physical activity values obtained from the PAD-PAR questionnaire (113 +/- 37 MET-hr/wk) were not related to EEPA (542 +/- 260 kcal/day; r= -0.057, p=0.690), ABI (0.64 +/- 0.19; r=0.032, p=0.826), or distance to maximal claudication pain (376 +/- 229 m; r=-0.054, p=0.731). The authors conclude that the PAD-PAR questionnaire is not an accurate measurement of free-living daily physical activity when compared to EEPA by use of the criterion method of doubly labeled water, and the activity questionnaire measures were poorly correlated with clinical measures of PAD severity. PMID- 10744006 TI - Plasma homocysteine and lipoprotein profile in patients with peripheral arterial occlusive disease. AB - Several studies have identified moderate hyperhomocysteinemia (HCy) as an independent risk factor for atherosclerosis. The purpose of this case control study was to determine lipoprotein profile and homocysteine concentration in serum of 85 male patients with peripheral arterial occlusive disease (PAOD) and in 51 normolipidemic age-matched male controls. Cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol as well as subfractions HDL2 and HDL3 cholesterol, low-density lipoprotein (LDL) cholesterol, apo B, apo A-I, and lipoprotein particles LpA-I and LpA-I:A-II were measured in serum. Homocysteine, folic acid, and vitamins B6 and B12 were determined with the help of high pressure liquid chromatography. The 677 C --> T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene was analyzed in PAOD patients. Patients with peripheral arterial occlusive disease showed a significantly higher mean concentration of homocysteine than control subjects (p<0.001). There was a negative correlation between the levels of homocysteine and vitamin B12 as well as folic acid (for vitamin B12: r=-0.40 and for folic acid: r=-0.38). The prevalence of hyperhomocysteinemia (Hcy >16 micromol/L) in the patients was 45% in contrast to 8% in controls. HDL cholesterol, HDL3 cholesterol, Apo A-I, and Lp A-I were significantly reduced in patients and triglycerides were elevated. The elevated plasma homocysteine concentration is frequently seen in homozygous carriers of a point mutation (677 C --> T) in the methylenetetrahydrofolate reductase gene, as the product of this gene is an enzyme, participating in homocysteine remethylation. The homozygous state for the 677 C --> T mutation was found in 13.3% of PAOD patients. PMID- 10744007 TI - Three treatments for chronic venous insufficiency: escin, hydroxyethylrutoside, and Daflon. AB - Escin, hydroxyethylrutoside (HR), and Daflon have been shown to be safe and effective for the treatment of chronic venous insufficiency (CVI). They seem to work differently than compression therapy, suggesting that they would usefully augment this therapy. All three phlebotonics attenuate the drop in adenosine triphosphate in venous endothelial cells during hypoxia. This attenuates (1) the inflammation response, (2) the attraction of neutrophils, (3) damage to the veins, and (4) the release of growth factors. These factors otherwise would perpetuate venous insufficiency and contribute to varicose veins. Additional independent effects that would be useful for the treatment of CVI are that they reduce permeability and fragility; HR, Daflon, and perhaps escin increase venous tone; escin inhibits hyaluronidase; Daflon and probably HR are attracted to the veins. With regard to similarity, no differences in effect have been established among these phlebotonics. PMID- 10744008 TI - Brachial artery blood flow velocity pattern in patients with congestive heart failure: duplex Doppler ultrasonographic assessment. AB - The purpose of this study was to determine whether there are abnormalities in blood flow velocity pattern of the brachial artery in patients with congestive heart failure (CHF). Brachial artery blood flow velocities were measured with duplex Doppler ultrasonography in 12 normal subjects, 31 patients with congestive heart failure (CHF), and 26 patients with coronary artery disease (CAD). None of the patients had clinical evidence of arterial disorders at upper extremities. In both patient groups, the presence of hypertension was correlated with the peak systolic velocity (r=0.48, p<0.05). Patients with heart failure had significantly larger (p<0.0001) peak reverse velocity (20 +/- 6 m/sec) than healthy subjects (5 +/- 4 m/sec) and patients with CAD (7 +/- 3 m/sec). Peak reverse velocity did not differ significantly between normal subjects and CAD group. These data indicate that the blood flow velocity pattern at brachial artery is abnormal in CHF. The simple measurement of brachial artery flow velocity suggests changes in peripheral vasculature related to CHF. PMID- 10744009 TI - Echocardiographic, morphologic, and geometric variations of the left ventricular outflow tract: possible role in the pathogenesis of discrete subaortic stenosis. AB - Although the clinical features and natural course of discrete subaortic stenosis (DSS) are well defined, the etiology remains speculative. The purpose of this study was to identify the echocardiographic, morphologic, and geometric variations of the left ventricular outflow tract associated with DSS in children and to determine whether these variations have a role in the pathogenesis of DSS. The aortoseptal angle (ASA), mitral-aortic valve separation (MAS), and the size of the aortic annulus were determined in two groups of children. Group 1 comprised 11 patients with isolated DSS, who were compared with an age- and body surface area- (BSA) matched healthy children (Group 1A, n: 20). Group 2 comprised 10 patients with DSS and ventricular septal defect (VSD). Group 2 was compared with an age- and BSA-matched patients with isolated perimembranous VSD (Group 2A, n: 22). Measurements were carried out from previously recorded echocardiographic studies. The ASA was steeper (119.3 +/- 6.1 degrees vs 137.5 +/- 5.6 degrees , p < 0.001), and the MAS was wider (6.1 +/- 1.6 vs 3.2 +/- 0.7 mm, p < 0.001) in patients with isolated DSS than in healthy control subjects. Similar differences were found between patients in Group 2 and Group 2A; the ASA was steeper (122.2 +/- 6.5 degrees vs 141.3 +/- 5.0 degrees, p < 0.001), and the MAS was wider (5.8 +/- 1.5 vs 3.8 +/- 1.1 mm, p < 0.001). The size of the aortic annulus was not different among the four study groups. Although the MAS was significantly wider in patients with DSS, there was significant overlap in MAS between patients and controls. However, if an ASA < or = 130 degrees was chosen as a predictive variable, it was found to be a highly sensitive, specific, and positive predictive marker for the development of DSS. This study demonstrates that DSS is associated with a steeper ASA, and a wider MAS, in patients with or without associated VSD. These morphologic abnormalities, especially a steeper ASA, may be risk factors for the development of DSS. PMID- 10744010 TI - Sensitive detection of myocardial contraction abnormality in chronic hemodialysis patients by ultrasonic tissue characterization with integrated backscatter. AB - Since acoustic properties of the myocardium are sensitive to the myocardial structure and the contractile conditions of myocyte, the authors evaluated cardiac dysfunction based on the integrated ultrasonic backscatter in 18 hemodialysis (HD) patients (duration: 102 +/- 84 months, mean age: 57.6 +/- 9.7 years) and 11 age-matched normals. The cyclic variation of integrated backscatter (CV-IB) at interventricular septum (IVS) and left ventricular posterior wall (PW) was measured and compared with percent fractional shortening (%FS) and percent wall thickening (%Th). The CV-IB of HD patients was smaller than that of control subjects (IVS: 6.2 +/- 1.1 dB vs 8.2 +/- 1.1 dB, p = 0.0003 and PW: 8.4 +/- 2.2 vs 10.3 +/- 1.3, p= 0.025). No significant difference was observed in %FS and %Th between HD patients and control subjects. In HD, the ratio of velocities of early diastolic inflow (E) to late atrial inflow was decreased (0.7 +/- 0.2 vs 1.1 +/- 0.7, p = 0.049) and deceleration time of E was prolonged significantly (200 +/- 28 msec vs 159 +/- 30 msec, p = 0.0082). In the absence of overt cardiac systolic dysfunction, myocardial damage indicated as a decrease in CV-IB and diastolic dysfunction identified on transmitral velocity waveform were detected, which may reflect from the myocardial fibrosis. As a mechanism, pressure overload, hyperparathyroidism, and anemia were neglected, and the other humoral factors may contribute to the myocardial damage in chronic renal failure. PMID- 10744011 TI - Pulsatile venous insufficiency in severe tricuspid regurgitation: does pulsatility protect against complications of venous disease? AB - Prior observations showed that the consequences of venous hypertension depend not only on the magnitude of the venous pressure but also on the efficiency of compensatory mechanisms that protect against the effects of excessive pressures on the microcirculation. Pulsatile venous insufficiency (PVI) associated with severe tricuspid regurgitation (TR) provides the opportunity to investigate the effect of the pulsatile shear stress on the outcome of venous insufficiency. The authors conducted a study to assess the flow characteristics and clinical outcome of PVI associated with TR. Five patients were evaluated, presenting venous insufficiency associated with ectasia, varices, and visible systolic pulsations of the leg veins. Characteristics of the venous flow were assessed by duplex ultrasound. In two patients, flow in the distal calf veins was evaluated by power Doppler sonography, and the supine-to-sitting leukocyte trapping was calculated. Results of the latter measurements were compared with measurements in five control patients who presented chronic nonpulsatile venous insufficiency. A survey of complications of PVI was conducted. On follow-up for 6 to 15 years (average 9.4 years) none of the patients developed venous thrombosis, phlebitis, or cutaneous ulcer. Flow in the distal calf vessels was increased in PVI (12-20 vessels/field) as compared with nonpulsatile venous insufficiency (0-7 vessels/field). Leukocyte trapping in the upright position was diminished in PVI (0.8-3%) as compared with nonpulsatile venous insufficiency (7-22%). In conclusion, PVI is characterized by increased flow in the distal calf veins, diminished leukocyte trapping, and a benign clinical course. These data are in agreement with experimental studies showing that pulsatile shear stress enhances secretion of cytokines by venous endothelial cells and, consequently, counteracts a predisposition to platelet aggregation, hypercoagulability, and white cell adhesion and promotes healing of leg ulcers. PMID- 10744012 TI - Evaluation of effective aortic regurgitant orifice area and its effect on aortic regurgitant volume with Doppler echocardiography. AB - The authors measured the aortic regurgitant orifice area (ROA) using Doppler echocardiography and attempted to clarify how important the ROA is in determining the regurgitant volume (RV) in 22 patients with chronic aortic regurgitation (AR). The RV was calculated from the difference between the left ventricular ejection flow volume and transmitral inflow volume as measured by Doppler echocardiography. The ROA was obtained by two methods: RV/time velocity integral of AR jet measured by continuous wave Doppler (calculation method) and manual tracing of minimum cross-sectional area of short-axis color Doppler. The RV ranged from 10 to 90 mL/beat and the ROA by calculation method was from 0.05 to 0.35 cm2, which showed a strong correlation (r = 0.93, p < 0.001). The time velocity integral of aortic regurgitant jet showed a poor correlation with the RV (r = 0.45, p < 0.05). The values of ROA by the two methods showed a good correlation (r = 0.93, p < 0.001). Thus, the authors conclude that the ROA is a basic determinant of the RV in AR and that color Doppler can be employed to precisely assess the ROA. PMID- 10744013 TI - "No-reflow" phenomenon following percutaneous coronary intervention: an uncommon complication. AB - The no-reflow phenomenon has been recognized as an uncommon complication after reperfusion therapy (thrombolytic or mechanical) for acute myocardial infarction and after percutaneous coronary intervention. As management and outcomes differ, early diagnosis and angiographic exclusion of other causes of impaired blood flow are important. The authors describe a case report of a patient with no-reflow following emergent stenting of the left circumflex artery (LCX). Pathophysiology and management of the no-reflow phenomenon are described along with the case report. PMID- 10744014 TI - Deep venous thrombosis associated with acute brucellosis--a case report. AB - Vascular complications, including arteries and veins associated with Brucella infection, have rarely been reported. To date, only three cases of deep venous thrombosis (DVT) of the lower extremities associated with brucellosis have been previously reported. The authors describe another case of DVT of the right leg in association with acute Brucella infection. Since infection with Brucella may be asymptomatic, and the manifestations of acute brucellosis are exceedingly nonspecific, and in view of the potential for Brucella to cause unexplained or unusual illness involving almost any organ including blood vessels, the authors' case and those previously reported suggest that brucellosis should be included among the causes and infections taken into account in patients suffering from DVT, particularly in those coming from Brucella-endemic areas. PMID- 10744015 TI - Transient global amnesia and amaurosis fugax in a patient with common carotid artery occlusion--a case report. AB - The etiology of transient global amnesia (TGA) is debatable. The hypothesis of a thromboembolic cause of TGA has been questioned by recent case control studies. Occlusion of the common carotid artery (CCA) is rare. Although amaurosis fugax (AF) is a hallmark of ipsilateral internal carotid artery disease, its occurrence in CCA occlusion is less known. Association of these three conditions in a patient may imply pathophysiologic significance. Here, the authors report a 76 year-old man who suffered from a spell of TGA and then several attacks of AF of the right eye. Progressive occlusion of the right CCA was documented by repeat carotid duplex scans and was finally confirmed by cerebral angiography. In this patient, the occurrence of CCA occlusion and AF implicates a vascular etiology for the event of TGA. A noninvasive carotid ultrasonographic screen may, therefore, be worthwhile for patients with TGA. PMID- 10744016 TI - Neurally mediated syncope induced by lung cancer--a case report. AB - The authors present hemodynamic and autonomic features of recurrent and episodic neurally mediated syncope in a man with lung cancer involving afferent vagus. He revealed extreme hypotension with bradycardia occurring during sitting or standing. A head-up tilt test also induced syncope. However, syncope attacks no longer occurred 2 weeks after admission. Alternatively, the paralyses of the left recurrent laryngeal nerve and the left phrenic nerve developed. It is suggested that the lung cancer involved upper rootlets of the left vagus and caused transient hypersensitivity of baroreceptor function that resulted in neurally mediated syncope. PMID- 10744017 TI - An unusual structure formed by antisense-target RNA binding involves an extended kissing complex with a four-way junction and a side-by-side helical alignment. AB - The antisense RNA CopA binds to the leader region of the repA mRNA (target: CopT). Previous studies on CopA-CopT pairing in vitro showed that the dominant product of antisense RNA-mRNA binding is not a full RNA duplex. We have studied here the structure of CopA-CopT complex, combining chemical and enzymatic probing and computer graphic modeling. CopI, a truncated derivative of CopA unable to bind CopT stably, was also analyzed. We show here that after initial loop-loop interaction (kissing), helix propagation resulted in an extended kissing complex that involves the formation of two intermolecular helices. By introducing mutations (base-pair inversions) into the upper stem regions of CopA and CopT, the boundaries of the two newly formed intermolecular helices were delimited. The resulting extended kissing complex represents a new type of four-way junction structure that adopts an asymmetrical X-shaped conformation formed by two helical domains, each one generated by coaxial stacking of two helices. This structure motif induces a side-by-side alignment of two long intramolecular helices that, in turn, facilitates the formation of an additional intermolecular helix that greatly stabilizes the inhibitory CopA-CopT RNA complex. This stabilizer helix cannot form in CopI-CopT complexes due to absence of the sequences involved. The functional significance of the three-dimensional models of the extended kissing complex (CopI-CopT) and the stable complex (CopA-CopT) are discussed. PMID- 10744018 TI - RNA folding at elementary step resolution. AB - We study the stochastic folding kinetics of RNA sequences into secondary structures with a new algorithm based on the formation, dissociation, and the shifting of individual base pairs. We discuss folding mechanisms and the correlation between the barrier structure of the conformational landscape and the folding kinetics for a number of examples based on artificial and natural sequences, including the influence of base modification in tRNAs. PMID- 10744019 TI - RNA motifs mediating in vivo site-specific nonhomologous recombination in (+) RNA virus enforce in vitro nonhomologous crossovers with HIV-1 reverse transcriptase. AB - There are several lines of evidence that both RNA viruses and retroviruses recombine according to a copy choice mechanism. Using the brome mosaic virus (BMV)-based system, we recognized elements in the RNA structure that enhance nonhomologous crossovers within or near the local heteroduplex formed by recombining molecules. The same structural motifs were employed in vitro to test the ability of human immunodeficiency virus reverse transcriptase (HIV-RT) to switch templates during DNA synthesis. We demonstrated that a specific combination of the local double-stranded region with short homologous sequences and a hairpin structure allows template switching by HIV-RT. In contrast to BMV replicase, HIV-RT does not mediate the detectable level of recombination using only the heteroduplex structure, though local hybridization between RNA molecules efficiently pauses primer extension. Moreover, the presented data suggest that a proper arrangement of identified structural motifs can ensure site specificity of RNA-RNA recombination. These results indicate that HIV-RT utilizes the same or a very similar mechanism as BMV replicase to change nonhomologous RNA templates in a site-specific manner. PMID- 10744020 TI - Splicing enhancement in the yeast rp51b intron. AB - Splicing enhancement in higher eukaryotes has been linked to SR proteins, to U1 snRNP, and to communication between splice sites across introns or exons mediated by protein-protein interactions. It has been previously shown that, in yeast, communication mediated by RNA-RNA interactions between the two ends of introns is a basis for splicing enhancement. We designed experiments of randomization selection to isolate splicing enhancers that would work independently from RNA secondary structures. Surprisingly, one of the two families of sequences selected was essentially composed of 5' splice site variants. We show that this sequence enhances splicing independently of secondary structure, is exportable to heterologous contexts, and works in multiple copies with additive effects. The data argue in favor of an early role for splicing enhancement, possibly coincident with commitment complex formation. Genetic compensation experiments with U1 snRNA mutants suggest that U1 snRNP binding to noncanonical locations is required for splicing enhancement. PMID- 10744021 TI - A novel mechanism for inhibition of translation by pokeweed antiviral protein: depurination of the capped RNA template. AB - Pokeweed antiviral protein (PAP) is known to inactivate ribosomes by removal of a specific adenine from the sarcin/ricin (S/R) loop of the large rRNA, thereby inhibiting translation. We demonstrate here that in addition to the previously identified adenine (A4324), PAP removes another adenine (A4321) and a guanine (G4323) from the eukaryotic large rRNA. Recent results indicate that the antiviral activity of PAP may not be due to depurination of host ribosomes. Using PAP mutants that do not depurinate either tobacco or reticulocyte lysate rRNA, we show that PAP inhibits translation of brome mosaic virus (BMV) and potato virus X (PVX) RNAs without depurinating ribosomes. Furthermore, translation of only capped, but not uncapped, luciferase transcripts is inhibited by PAP, providing evidence that PAP and PAP mutants are able to distinguish between capped and uncapped transcripts. Translation inhibition of BMV RNAs is overcome by treatment with PAP in the presence of increasing concentrations of the cap analog m7GpppG, but not GpppG or GTP, indicating that PAP recognizes the cap structure. Incubation of BMV RNAs or the capped luciferase transcripts with PAP results in depurination of either RNA. In contrast, uncapped luciferase transcripts are not depurinated after incubation with identical concentrations of PAP. These results demonstrate for the first time that PAP can inhibit translation by a mechanism other than ribosome depurination, by recognizing the cap structure and specifically depurinating the capped RNAs. PMID- 10744022 TI - Translation termination in eukaryotes: polypeptide release factor eRF1 is composed of functionally and structurally distinct domains. AB - Class-1 polypeptide chain release factors (RFs) trigger hydrolysis of peptidyl tRNA at the ribosomal peptidyl transferase center mediated by one of the three termination codons. In eukaryotes, apart from catalyzing the translation termination reaction, eRF1 binds to and activates another factor, eRF3, which is a ribosome-dependent and eRF1-dependent GTPase. Because peptidyl-tRNA hydrolysis and GTP hydrolysis could be uncoupled in vitro, we suggest that the two main functions of eRF1 are associated with different domains of the eRF1 protein. We show here by deletion analysis that human eRF1 is composed of two physically separated and functionally distinct domains. The "core" domain is fully competent in ribosome binding and termination-codon-dependent peptidyl-tRNA hydrolysis, and encompasses the N-terminal and middle parts of the polypeptide chain. The C terminal one-third of eRF1 binds to eRF3 in vivo in the absence of the core domain, but both domains are required to activate eRF3 GTPase in the ribosome. The calculated isoelectric points of the core and C domains are 9.74 and 4.23, respectively. This highly uneven charge distribution between the two domains implies that electrostatic interdomain interaction may affect the eRF1 binding to the ribosome and eRF3, its activity in the termination reaction and activation of eRF3 GTPase. The positively charged core of eRF1 may interact with negatively charged rRNA and peptidyl-tRNA phosphate backbones at the ribosomal eRF1 binding site and exhibit RNA-binding ability. The structural and functional dissimilarity of the core and eRF3-binding domains implies that evolutionarily eRF1 originated as a product of gene fusion. PMID- 10744023 TI - p62, a novel Xenopus laevis component of box C/D snoRNPs. AB - U16 belongs to the family of box C/D small nucleolar RNAs (snoRNAs) whose members participate in ribosome biogenesis, mainly acting as guides for site-specific methylation of the pre-rRNA. Like all the other members of the family, U16 is associated with a set of protein factors forming a ribonucleoprotein particle, localized in the nucleolus. So far, only a few box C/D-specific proteins are known: in Xenopus laevis, fibrillarin and p68 have been identified by UV crosslinking and shown to require the conserved boxes C and D for snoRNA interaction. In this study, we have identified an additional protein factor (p62), common to box C/D snoRNPs, that crosslinks to the internal stem region, distinct from the conserved box C/D "core motif," of U16 snoRNA. We show here that, although the absence of the core motif and, as a consequence, of fibrillarin and p68 binding prevents processing and accumulation of the snoRNA, the lack of the internal stem does not interfere with the efficient release of U16 from its host intron and only slightly affects snoRNA stability. Because this region is likely to be the binding site for p62, we propose that this protein plays an accessory role in the formation of a mature and stable U16 snoRNP particle. PMID- 10744024 TI - A hierarchy of RNA subdomains in assembly of the central domain of the 30 S ribosomal subunit. AB - Beginning with the framework that has been developed for the assembly of the 30 S ribosomal subunit, we have identified a series of RNAs that are minimal binding sites for proteins S15, S6, S18, and S11 in the central domain from Thermus thermophilus. The minimal binding RNA for proteins S15, S6, and S18 consists of helix 22 and three-way junctions at both ends composed of portions of helices 20, 21, and 23. Addition of the remaining portion of helix 23 to this construct results in the minimal site for S11. Surprisingly, almost half of the central domain (helices 24, 25, and 26) is dispensable for binding the central domain proteins. Thus, at least two classes of RNA elements can be identified in ribosomal RNA. A protein-binding core element (such as helices 20, 21, 22, and 23) is required for the association of ribosomal proteins, whereas secondary binding elements (such as helices 24, 25, and 26) associate only with the preformed core RNP complex. Apparently, there may be a hierarchy of ribosomal RNA elements similar to the hierarchy of primary, secondary, and tertiary binding ribosomal proteins. PMID- 10744025 TI - Contribution of the intercalated adenosine at the helical junction to the stability of the gag-pro frameshifting pseudoknot from mouse mammary tumor virus. AB - The mouse mammary tumor virus (MMTV) gag-pro frameshifting pseudoknot is an H type RNA pseudoknot that contains an unpaired adenosine (A14) at the junction of the two helical stems required for efficient frameshifting activity. The thermodynamics of folding of the MMTV vpk pseudoknot have been compared with a structurally homologous mutant RNA containing a G x U to G-C substitution at the helical junction (U13C RNA), and an A14 deletion mutation in that context (U13CdeltaA14 RNA). Dual wavelength optical melting and differential scanning calorimetry reveal that the unpaired adenosine contributes 0.7 (+/-0.2) kcal mol( 1) at low salt and 1.4 (+/-0.2) kcal mol(-1) to the stability (deltaG(0)37) at 1 M NaCl. This stability increment derives from a favorable enthalpy contribution to the stability deltadeltaH = 6.6 (+/-2.1) kcal mol(-1) with deltadeltaG(0)37 comparable to that predicted for the stacking of a dangling 3' unpaired adenosine on a G-C or G x U base pair. Group 1A monovalent ions, NH4+, Mg2+, and Co(NH3)6(3+) ions stabilize the A14 and deltaA14 pseudoknots to largely identical extents, revealing that the observed differences in stability in these molecules do not derive from a differential or specific accumulation of ions in the A14 versus deltaA14 pseudoknots. Knowledge of this free energy contribution may facilitate the prediction of RNA pseudoknot formation from primary nucleotide sequence (Gultyaev et al., 1999, RNA 5:609-617). PMID- 10744026 TI - Requirements for mini-exon inclusion in potato invertase mRNAs provides evidence for exon-scanning interactions in plants. AB - Invertases are responsible for the breakdown of sucrose to fructose and glucose. In all but one plant invertase gene, the second exon is only 9 nt in length and encodes three amino acids of a five-amino-acid sequence that is highly conserved in all invertases of plant origin. Sequences responsible for normal splicing (inclusion) of exon 2 have been investigated in vivo using the potato invertase, invGF gene. The upstream intron 1 is required for inclusion whereas the downstream intron 2 is not. Mutations within intron 1 have identified two sequence elements that are needed for inclusion: a putative branchpoint sequence and an adjacent U-rich region. Both are recognized plant intron splicing signals. The branchpoint sequence lies further upstream from the 3' splice site of intron 1 than is normally seen in plant introns. All dicotyledonous plant invertase genes contain this arrangement of sequence elements: a distal branchpoint sequence and adjacent, downstream U-rich region. Intron 1 sequences upstream of the branchpoint and sequences in exons 1, 2, or 3 do not determine inclusion, suggesting that intron or exon splicing enhancer elements seen in vertebrate mini exon systems are absent. In addition, mutation of the 3' and 5' splice sites flanking the mini-exon cause skipping of the mini-exon, suggesting that both splice sites are required. The branchpoint/U-rich sequence is able to promote splicing of mini-exons of 6, 3, and 1 nt in length and of a chicken cTNT mini exon of 6 nt. These sequence elements therefore act as a splicing enhancer and appear to function via interactions between factors bound at the branchpoint/U rich region and at the 5' splice site of intron 2, activating removal of this intron followed by removal of intron 1. This first example of splicing of a plant mini-exon to be analyzed demonstrates that particular arrangement of standard plant intron splicing signals can drive constitutive splicing of a mini-exon. PMID- 10744027 TI - In vivo selection of lethal mutations reveals two functional domains in arginyl tRNA synthetase. AB - Using random mutagenesis and a genetic screening in yeast, we isolated 26 mutations that inactivate Saccharomyces cerevisiae arginyl-tRNA synthetase (ArgRS). The mutations were identified and the kinetic parameters of the corresponding proteins were tested after purification of the expression products in Escherichia coli. The effects were interpreted in the light of the crystal structure of ArgRS. Eighteen functional residues were found around the arginine binding pocket and eight others in the carboxy-terminal domain of the enzyme. Mutations of these residues all act by strongly impairing the rates of tRNA charging and arginine activation. Thus, ArgRS and tRNA(Arg) can be considered as a kind of ribonucleoprotein, where the tRNA, before being charged, is acting as a cofactor that activates the enzyme. Furthermore, by using different tRNA(Arg) isoacceptors and heterologous tRNA(Asp), we highlighted the crucial role of several residues of the carboxy-terminal domain in tRNA recognition and discrimination. PMID- 10744028 TI - The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo. AB - The yeast superkiller (SKI) genes were originally identified from mutations allowing increased production of killer toxin encoded by M "killer" virus, a satellite of the dsRNA virus L-A. XRN1 (SKI1) encodes a cytoplasmic 5' exoribonuclease responsible for the majority of cytoplasmic RNA turnover, whereas SKI2, SKI3, and SKI8 are required for normal 3'-degradation of mRNA and for repression of translation of poly(A) minus RNA. Ski2p is a putative RNA helicase, Ski3p is a tetratricopeptide repeat (TPR) protein, and Ski8p contains five WD-40 (beta-transducin) repeats. An xrn1 mutation in combination with a ski2, ski3, or ski8 mutation is lethal, suggesting redundancy of function. Using functional epitope-tagged Ski2, Ski3, and Ski8 proteins, we show that Ski2p, Ski3p, and Ski8p can be coimmunoprecipitated as an apparent heterotrimeric complex. With epitope-tagged Ski2p, there was a 1:1:1 stoichiometry of the proteins in the complex. Ski2p did not associate with Ski3p in the absence of Ski8p, nor did Ski2p associate with Ski8p in the absence of Ski3p. However, the Ski3p/Ski8p interaction did not require Ski2p. In addition, ski6-2 or ski4-1 mutations or deletion of SKI7 did not affect complex formation. The identification of a complex composed of Ski2p, Ski3p, and Ski8p explains previous results showing phenotypic similarity between mutations in SKI2, SKI3, and SKI8. Indirect immunofluorescence of Ski3p and subcellular fractionation of Ski2p and Ski3p suggest that Ski2p and Ski3p are cytoplasmic. These data support the idea that Ski2p, Ski3p, and Ski8p function in the cytoplasm in a 3'-mRNA degradation pathway. PMID- 10744029 TI - A fluorescence-based assay for 3' --> 5' exoribonucleases: potential applications to the study of mRNA decay. AB - A cell-free mRNA decay assay has been adapted to permit the kinetics of 3' --> 5' exoribonuclease activities to be monitored in real time. RNA probes containing 5' caps and 3' poly(A) tails generated by transcription in vitro are 3' labeled using fluorescein-N6-ATP and poly(A) polymerase. Release of fluorescein conjugated adenosine residues from the 3' end of the RNA substrate is monitored by a time-dependent decrease in fluorescence anisotropy in the presence of cytosolic proteins. To demonstrate the utility of the assay, an RNA probe was constructed containing a fragment of the c-myc 3' untranslated region and an 85 base poly(A) tail. Following 3' fluorescein labeling, the rate of 3'-terminal adenosine excision was monitored in the presence of an S100 cytosolic extract prepared from K562 erythroleukemia cells. Removal of the fluorescein-tagged A residues resolved to a first-order decay function, allowing the rate constant and enzyme-specific activity to be determined in this extract. Further applications and advantages of this technology are discussed. PMID- 10744030 TI - Temporal lobe epilepsy associated up-regulation of metabotropic glutamate receptors: correlated changes in mGluR1 mRNA and protein expression in experimental animals and human patients. AB - Aberrant axonal reorganization and altered distribution of neurotransmitter receptor subtypes have been proposed as major pathogenic mechanisms for hippocampal hyperexcitability in chronic temporal lobe epilepsies (TLE). Recent data point to excitatory class I metabotropic glutamate receptors (mGluR1 and mGluR5) as interesting candidates. Here, we have analyzed the hippocampal distribution and mRNA expression of mGluR1 and mGluR5 in two rat models of limbic seizures, i.e. electrical kindling and intraperitoneal kainate injections, as well as in human TLE. Quantitative RT-PCR analysis detected a significant increase of hippocampal mGluR1 gene transcript levels in kainate treated and kindled rats. In addition, microdissected hippocampal tissue samples localized this increase to the dentate gyrus. Using immunohistochemistry with mGluR1alpha subtype specific antibodies, increased labeling was observed within the dentate gyrus molecular layer (DG-ML). A similar pattern of increased mGluR1alpha neuropil staining was found within the DG-ML of epilepsy patients (n = 42) compared with peritumoral hippocampus specimens obtained from nonepileptic patients (biopsy controls, n = 3). This increase was detected in TLE patients with segmental hippocampal cell loss, as well as in TLE patients with focal lesions but no histopathological alterations of the hippocampus. In contrast, mGluR5 immunoreactivity and mRNA expression were not significantly altered in the DG-ML. Our data demonstrate a striking regional induction of mGluR1alpha in the hippocampal dentate gyrus of experimental animals with limbic seizures as well as in human patients with chronic, intractable TLE. This increase corresponds to functional alterations of class I mGluRs observed in seizure models and may significantly contribute to hippocampal hyperexcitability in focal human epilepsies. PMID- 10744031 TI - In vivo reversal of amyloid-beta lesions in rat brain. AB - Cerebral amyloid-beta (Abeta) deposition is central to the neuropathological definition of Alzheimer disease (AD) with Abeta related toxicity being linked to its beta-sheet conformation and/or aggregation. We show that a beta-sheet breaker peptide (iAbeta5) dose-dependently and reproducibly induced in vivo disassembly of fibrillar amyloid deposits, with control peptides having no effect. The iAbeta5-induced disassembly prevented and/or reversed neuronal shrinkage caused by Abeta and reduced the extent of interleukin-1beta positive microglia-like cells that surround the Abeta deposits. These findings suggest that beta-sheet breakers, such as iAbeta5 or similar peptidomimetic compounds, may be useful for reducing the size and/or number of cerebral amyloid plaques in AD, and subsequently diminishing Abeta-related histopathology. PMID- 10744032 TI - Transforming growth factor-beta1 stimulates contraction of human glioblastoma cell-mediated collagen lattice through enhanced alpha2 integrin expression. AB - Rapid invasiveness is a feature of the highly malignant glioblastoma tumor and is closely related to patient prognosis. The interaction between extracellular matrix (ECM) and cell surface receptors such as integrin heterodimers play a key role in the process of tumor invasion. We investigated the effects of transforming growth factor-beta1 (TGF-beta1), which is a mitogenic factor for glial cells, on integrin expression in T98G human glioblastoma cells using an in vitro model 3-dimensional collagen lattice. Exogenously applied TGF-beta1 dose dependently enhanced collagen lattice contraction. Among the inhibitory antibodies tested against alpha integrin subunits, the anti-alpha2 antibody, P1 E6, alone prevented the enhanced contractile response by TGF-beta1, whereas any alpha integrin antibody (including P1-E6) had little effect on lattice contraction when cultured without TGF-beta1. RT-PCR analysis revealed that TGF beta1 strongly increased alpha2 integrin transcript level. Furthermore, pretreatment with antisense phosphorothioate oligodeoxynucleotides against human alpha2 integrin using hemagglutinating virus of Japan (HVJ) liposome-mediated transfer prevented the effects of TGF-beta1 and also reduced the lattice contraction even in the absence of TGF-beta1. This data indicates that increased expression of alpha2 integrin is responsive to enhanced collagen lattice contraction by TGF-beta1. We suggest that TGF-beta1 exerts its effects on the invasive property of glioblastoma cells via upregulation of the alpha2 integrin subunit expression. PMID- 10744033 TI - In vitro evidence that beta-amyloid peptide 1-40 diffuses across the blood-brain barrier and affects its permeability. AB - Beta amyloid peptides are major insoluble constituents of amyloid fibrils in senile plaques and cerebrovascular deposits, both characteristic of Alzheimer disease (AD). Low concentrations of soluble forms of amyloid peptides are also present in normal CSF. We previously demonstrated that the 40 amino acid form of soluble beta-amyloid peptide (sAbeta) is rapidly cleared from rat CSF into blood. Herein we hypothesized that a saturable, outwardly directed flux of this peptide occurs at the blood-brain barrier (BBB) and tested whether supraphysiological (possibly pathological) concentrations of sAbeta could alter the permeability of this barrier to a paracellular tracer, polyethylene glycol (PEG). Using an in vitro model of BBB, we showed that influx and efflux of sAbeta were equal, modest (60%-160% greater than that of PEG), and not saturable. These observations suggest that sAbeta moved across the monolayer by a diffusional process, and not via a transporter. PEG flux was doubled immediately after the luminal concentration of cold sAbeta was raised to 5 microM, and was doubled 150 min after the abluminal concentration of sAbeta was increased to 5 microM. Pathological elevations of sAbeta concentration in plasma or brain interstitial fluid may, therefore, alter the permeability of brain capillaries in vivo. PMID- 10744034 TI - Filamentous tau pathology in nerve cells, astrocytes, and oligodendrocytes of aged baboons. AB - Intracellular filamentous inclusions containing abnormally phosphorylated tau protein are hallmarks of several human neurodegenerative disorders. This study reveals tau-positive cytoskeletal abnormalities in neurons and glial cells of aged baboons. The brains of four baboons (Papio hamadryas, 20-30 yr of age) were examined using the Gallyas silver technique for neurofibrillary changes and phosphorylation-dependent anti-tau antibodies (AT8, AT100, AT270, PHF-1, TG-3). Conspicuous changes were noted in two animals, 26 and 30 yr of age. In both animals, a combination of neuronal and glial cytoskeletal pathology was seen preferentially affecting limbic brain areas, including the hippocampal formation. In the 30-yr-old animal, numerous tau-positive inclusions were seen in the granule cells of the fascia dentata. These cells even exhibited an accumulation of argyrophilic neurofibrillary tangles. The glial changes affected both astrocytes and oligodendrocytes. Tau-positive astrocytes were seen in perivascular, subpial, and subependymal locations. Tau-positive oligodendrocytes preferentially occurred in limbic fiber tracts including the entorhinal perforant path. Ultrastructurally, tau-positive straight filaments (10-14 nm) in both neurons and glial cells were revealed by anti-tau immunoelectron microscopy. This study thus indicates the potential usefulness of aged baboons for experimental investigation of neuronal and glial filamentous tau pathology. This nonhuman primate species may provide valuable information pertinent to the broad spectrum of human tauopathies. PMID- 10744035 TI - Enhanced N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice deficient in CuZn-superoxide dismutase or glutathione peroxidase. AB - Administration of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mammals causes damage to the nigrostriatal dopaminergic pathway similar to that observed in Parkinson disease (PD). Reactive oxygen species (ROS) are thought to be involved in the pathogenesis of MPTP-mediated dopaminergic neurodegeneration. To further clarify the role of superoxide anion radical (*O2-) and to study the possible involvement of hydroperoxides in MPTP-mediated neurodegeneration, MPTP neurotoxicity was induced in mice deficient in either CuZn superoxide dismutase (SOD), a scavenger enzyme for *O2-, or cellular glutathione peroxidase (GSHPx-1), a scavenger enzyme for hydroperoxides. Littermate control and homozygous deficient mice were injected intraperitoneally with a total cumulative dose of 0, 75, or 150 mg/kg of MPTP delivered over 5 d. All mice were killed 5 d after the last injection and the brains were processed for immunohistological analysis for tyrosine hydroxylase (TH) in the striatum and the substantia nigra pars compacta (SNc), as well as for direct measurements of dopamine concentrations in the striatum. The intensity of TH immunoreactivity in the striatum was evaluated by measuring the relative optical density (OD) with NIH IMAGE, and expressed as Log (OD of striatum)/Log (OD of white matter). Degeneration of TH-containing neurons was assessed by counting TH-positive neurons in the SNc. We found that this MPTP exposure protocol produced dose-dependent depletion of TH immunoreactivity and dopamine in the striatum in littermate control mice and both strains of knockout mice; however. reduction in TH immunoreactivity and dopamine content were significantly greater in CuZn-SOD or GSHPx-1 deficient mice compared with littermate controls. MPTP exposure did not significantly alter the number of TH positive neurons in the SNc in littermate control or knockout mice. These data suggest that some of the deleterious effects of MPTP on striatal dopaminergic nerve terminals are mediated by both *O2- and hydroperoxides, and that they occur prior to dopaminergic neurodegeneration in the SNc. The similarity between the MPTP model and PD raises the possibility that both types of ROS may play a significant role in the early pathogenesis of dopaminergic neurodegeneration in PD. PMID- 10744036 TI - Postmortem neuropathological features secondary to boron neutron capture therapy for glioblastoma multiforme. AB - This postmortem study of 12 patients with glioblastoma multiforme (GBM) treated with boron neutron capture therapy (BNCT) employing an epithermal neutron beam and p-boronophenylalanine describes the neuropathological findings in patients receiving a relatively high radiation dose to the tumor, but a relatively low radiation dose to the normal brain. In addition to a standardized neuropathology panel of sections, we used individual treatment dosimetry maps to select sections along the projected maximum radiation beam pathway. We found that the normal neuroparenchyma exposed to the highest radiation dose exhibited a single instance of radiation-induced focal venular fibrinoid necrosis and a single instance of multifocal demyelination. Semiquantitative analysis of pretreatment neurosurgical and postmortem tumor samples revealed only two radiation ascribed histopathological findings to be particular to therapy, fibrinoid necrosis and vascular hyalinization. In this relatively small series of cases we found an unexpectedly high frequency of cases (3 of 12) with neurodegenerative histopathology (Lewy bodies, neurofibrillary tangles, and neuritic senile plaques), which appeared, by distribution, to be independent of the radiation beam. Two of these patients were over 70 yr of age. One was only 41. Our findings suggest an acceptable radiation-induced level of neurotoxicity at the lower doses employed, but raise the possibility of unexpected boron neurodegenerative toxicity. PMID- 10744038 TI - Reproducibility of diagnosis and its influence on the distribution of lung cancer by histologic type in Osaka, Japan. AB - The histologic types of lung cancer cases diagnosed in 1979-1980 (n=799) and 1987 (n=587) were independently reviewed by two pathologists in order to investigate the reproducibility of the diagnosis of the histologic type when the WHO classification (1981) was used. The specimens from 354 surgical cases and biopsy or cytology specimens from 1032 non-surgical cases were reviewed. The inter observer agreement was 87.9% (kappa=0.79) for surgical cases and 81.4% (kappa=0.72) for non-surgical cases. When compared to the original diagnosis, the agreement was 86.8% (kappa=0.78) for surgical and 86.4% (kappa=0.79) for non surgical cases in 1979-1980 and the agreement was 92.8% (kappa=0.87) for surgical and 89.1% (kappa=0.83) for non-surgical cases in 1987. By histologic type, no difference in the agreement was observed except for large cell carcinoma. The distribution of histologic types after the review differed only slightly (less than 6%) from the original distribution. This suggests that in Osaka, Japan, the diagnosis based on the WHO classification (1981) had only a limited influence on the distribution of histologic types, and is not a major reason for the changing trends in lung cancer incidence by histologic type. PMID- 10744037 TI - Tumor necrosis factor alpha and human Schwann cells: signalling and phenotype modulation without cell death. AB - The aim of the study was to evaluate the biological response of human Schwann cells (SC) to tumor necrosis factor alpha (TNFalpha) in vitro and to the inflammatory milieu of chronic inflammatory demyelinating polyradiculoneuritis (CIDP). By immunocytochemical and functional assays, we found that SC expressed TNF receptors and that TNFalpha promoted in SC cultures transient activation of transcription factors NFkappaB and c-jun in the absence of apoptosis. In addition, TNFalpha significantly increased the proportion of non-myelin-forming SC expressing the p75 nerve growth factor receptor. Such phenotypic effect was dose-dependent and partially mediated by NFkappaB, as assessed by functional blockage with acetylsalicylic acid. We then extended our study to a human disease in which SC are exposed to TNFalpha. Increased signals for NFkappaB, but not c jun, molecules were observed by immunohistochemistry on SC nuclei in nerve biopsies from patients with CIDP, as compared with controls. Irrespective of the presence of nerve inflammation, SC showed no evidence of apoptosis. Taken together, our results suggested that SC are potential targets of TNFalpha and that this cytokine exerted no cytotoxic effects either in vivo or in vitro. Rather, TNFalpha may influence the fate of SC by activating transcriptional pathways and modulating their phenotype. PMID- 10744039 TI - Lack of evidence of association of p21WAF1/CIP1 polymorphism with lung cancer susceptibility and prognosis in Taiwan. AB - An association between the Arg allele of the p21WAF1/CIP1 codon 31 polymorphism and lung cancer has been reported. However, the genotype distribution of the p21 codon 31 polymorphism, as well as the association of this polymorphism with lung cancer risk and prognosis, remain undefined in the Taiwanese population. Therefore, we investigated the genotype distribution of the p21 codon 31 polymorphism in 155 lung cancer patients and 189 non-cancer controls. The genotype frequencies in the Taiwanese non-cancer controls were 0.51 (Ser) and 0.49 (Arg). Chi2 analysis indicated significant differences in Taiwanese genotype distribution of p21 from those reported for Swedes (P=0.001), Caucasians (P=0.001), Indians (P=0.001), and African-Americans (P=0.001). However, our data did not demonstrate an association of the Arg allele of the p21 polymorphism with lung cancer risk in Taiwan. Lung cancer patients with Ser/Arg and Arg/Arg genotypes were at a nonsignificant 1.15-fold increased risk of lung cancer when compared to individuals with the Ser/Ser genotype (95%CI, 0.70-1.86). In addition, although p21 is a downstream target of p53, we found no significant correlation of the p21 polymorphism with the p53 polymorphism and p53 gene mutation in lung cancer patients. We further investigated the association of the p21 polymorphism with prognosis in 154 lung cancer patients. Patients with the Ser/Ser genotype tended to have a poorer prognosis than those with the Ser/Arg and Arg/Arg genotypes (P=0.097, by the log rank test). Our data suggest that the p21 codon 31 polymorphism may not play a significant role in cancer susceptibility and the prognosis of lung cancer patients in Taiwan. PMID- 10744040 TI - Low susceptibility of Long-Evans Cinnamon rats to N-butyl-N-(4-hydroxybutyl) nitrosamine-induced urinary bladder carcinogenesis and inhibitory effect of urinary copper. AB - We studied the susceptibilities to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) induced urinary bladder carcinogenesis of male Long-Evans Cinnamon (LEC), F344 and Long-Evans Agouti (LEA) rats. Male rats (n=21) were given 0.1% BBN in their drinking water from week 6, 8 and 10 for one week, and killed in week 56. The incidences of transitional cell tumors (papillomas plus carcinomas) in BBN treated LEC and F344 rats were 12% and 76%, respectively (P<0.001, experiment 1), and those in LEC and LEA rats were 11% and 95%, respectively (P<0.001, experiment 2). When male LEC and F344 rats were given 0.1% BBN in their drinking water for 7 days, the intake of BBN and the urinary concentration of its active metabolite, N butyl-N-(3-carboxypropyl)nitrosamine (BCPN), were higher in the LEC rats (P<0.01). The urinary pHs of untreated LEC and F344 rats were similar between week 6 and 30. The urinary copper concentration was lower in LEC rats before jaundice than in F344 rats, but its concentrations in 28- and 50-week-old LEC rats were 1.7 and 2.3 times those in F344 rats. In a two-stage carcinogenesis study using F344 rats, i.p. injections of cupric nitrilotriacetate increased urinary copper excretion, and inhibited BBN-induced bladder carcinogenesis. In a two-stage carcinogenesis study using LEC rats, oral administration of D penicillamine decreased urinary copper excretion, and increased BBN-induced bladder cancer, although the difference was not significant. These data show that LEC rats are resistant to bladder carcinogenesis and suggest that urinary copper has a significant role in their resistance. PMID- 10744041 TI - Chemopreventive effect of bovine lactoferrin on 4-nitroquinoline 1-oxide-induced tongue carcinogenesis in male F344 rats. AB - The modifying effects of dietary feeding of bovine lactoferrin (bLF) on tongue carcinogenesis initiated with 4-nitroquinoline 1-oxide (4-NQO) were investigated in male F344 rats. The activities of phase II detoxifying enzymes, glutathione S transferase (GST) and quinone reductase (QR), polyamine content and ornithine decarboxylase (ODC) activity in the tongue were also examined for mechanistic analysis of possible modifying effects of bLF on carcinogenesis. At 7 weeks of age, all animals except those treated with bLF alone and untreated rats were given 20 ppm 4-NQO in drinking water for 8 weeks to induce tongue neoplasms. Starting 7 days before 4-NQO exposure, experimental groups were fed experimental diets containing bLF (0.2% and 2%) for 10 weeks ("initiation feeding"). Starting 1 week after the cessation of exposure to 4-NQO, the other experimental groups given 4-NQO and a basal diet were fed the experimental diets for 22 weeks ("postinitiation feeding"). At week 32, the incidence and multiplicity of tongue neoplasms in the "initiation feeding" groups of 0.2% and 2% bLF and the "post initiation feeding" group of 0.2% bLF were lower than those of the 4-NQO alone group, but without statistical significance. However, "post-initiation feeding" of 2% bLF caused a significant reduction in the incidence (20% vs. 55%, P=0.02418) and multiplicity (0.25+/-0.54 vs. 0.70+/-0.71, P<0.05) of tongue squamous cell carcinoma (by 64%, P=0.02418). bLF treatment elevated liver and tongue GST activities and liver QR activity. The "post-initiation feeding" with 2% bLF significantly decreased QR activity, proliferating cell nulcear antigen positive index and ODC activity in the tongue. In addition, feeding with bLF decreased tongue polyamine content. These results suggest that bLF, when given at the 2% dose level during the post-initiation phase, exerts chemopreventive action against tongue tumorigenesis through modification of cell proliferation activity and/or the activities of detoxifying enzymes. PMID- 10744042 TI - Green tea polyphenols induce apoptosis in vitro in peripheral blood T lymphocytes of adult T-cell leukemia patients. AB - Green tea polyphenols (TEA) are known to exhibit antioxidative activity as well as tumor-suppressing activity. In order to examine the tumor-suppressing activity of TEA against adult T-cell leukemia (ATL), we cultivated peripheral blood T lymphocytes of ATL patients (ATL PBLs), an HTLV-I-infected T-cell line (KODV) and healthy controls (normal PBLs) for 3 days in the presence of TEA and its main constituent, epigallocatechin-3-gallate (EGCg), to measure cell proliferation and apoptosis, and to quantitate mRNAs of HTLV-I pX and beta-actin genes of the cultured cells. Growth of ATL PBLs was significantly inhibited by 9-27 microg/ml of TEA and EGCg, in contrast to minimal growth inhibition of T cells of normal PBLs. Inhibition of KODV was intermediate between ATL PBLs and normal PBLs. The ATL PBLs and KODV treated with 27 microg/ml of either TEA or EGCg induced apoptotic DNA fragmentation, producing terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL)-positive cells, while the normal PBLs treated with the same concentration of TEA or EGCg produced a negligibly small number of TUNEL-positive cells, in which apoptotic DNA fragmentation was not detectable. Expression of HTLV-I pX mRNA was suppressed more than 90% in ATL PBLs by treatment with 3-27 microg/ml of either TEA or EGCg, while expression of beta-actin mRNA was much less suppressed by treatment with the same concentration of TEA or EGCg. These results indicate that TEA and EGCg inhibit growth of ATL PBLs, as well as HTLV-I-infected T-cells, by suppressing HTLV-I pX gene expression and inducing apoptotic cell death. PMID- 10744043 TI - Adenovirus-mediated gene transduction of IkappaB or IkappaB plus Bax gene drastically enhances tumor necrosis factor (TNF)-induced apoptosis in human gliomas. AB - Tumor necrosis factor-alpha (TNF), which was initially supposed to be a promising cancer therapeutic reagent, does not kill most types of cancer cells partly due to the activation of an anti-apoptotic gene, NF-kappaB. NF-kappaB forms an inactive complex with the inhibitor kappa B alpha (IkappaBalpha), which is rapidly phosphorylated and degraded in response to various extracellular signals. To disrupt this protective mechanism, we introduced an inhibitor kappa B alpha (IkappaBdN) gene, a deletion mutant gene lacking the nucleotides for the N terminal 36 amino acids of IkappaBalpha, into human glioma cells (U251, T-98G, and U-373MG) via an adenoviral (Adv) vector in addition to treatment of the glioma cells with recombinant TNF. Immunohistochemical analysis revealed that NF kappaB was translocated to nuclei by TNF treatment in U251 and T-98G cells, but not in U-373MG cells. Neither transduction of IkappaBdN nor treatment with TNF protein alone induced apoptosis in U251 and T-98G cells, whereas both cell lines underwent drastic TNF-induced apoptosis after transduction of IkappaBdN. On the other hand, U-373MG cells were refractory to TNF-induced apoptosis even when they were transduced with the IkappaBdN gene. U-373MG cells underwent drastically increased apoptosis when co-transduced with the IkappaBdN and Bax gene in the presence of TNF. Adv-mediated transfer of IkappaBdN or IkappaBdN plus Bax may be a promising therapeutic approach to treat gliomas through TNF-mediated apoptosis. PMID- 10744044 TI - Immunohistochemical detection of carcinogen-DNA adducts in normal human prostate tissues transplanted into the subcutis of athymic nude mice: results with 2-amino 1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 3,2'-dimethyl-4-aminobiphenyl (DMAB) and relation to cytochrome P450s and N-acetyltransferase activity. AB - Human prostate tissue transplanted into nude mice was examined immunohistochemically for DNA adducts formed after administration of 3,2' dimethyl-4-aminobiphenyl (DMAB) or 2-amino-1-methyl-6-phenylimidazo[4,5 b]pyridine (PhIP). Positive staining for DMAB- or PhIP-DNA adducts was evident in 70-95% of both epithelial and stromal cells in human prostate xenografts. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed a normal human prostate epithelial cell line (PrEC) to express both cytochrome P450 1A2 (CYP1A2) and N-acetyltransferase 2 (NAT2) mRNA, while a normal human prostate fibroblast cell line (NHPF) expressed NAT2, but not CYP1A2 mRNA. In addition, NAT2 and to a lesser extent CYP1A2 mRNAs were also found in four cases of normal human prostate tissues. The results suggest that initial activation of chemicals by liver CYP1A2 and subsequent metabolism by prostate NAT2 is a major pathway of DNA adduct formation in human prostate cells. Thus, the data suggest that human prostate has the potential to be targeted by environmental carcinogens. PMID- 10744045 TI - Preferential activity of wild-type and mutant tumor necrosis factor-alpha against tumor-derived endothelial-like cells. AB - Tumor-derived endothelial-like cells (tEC) were prepared by culturing human umbilical vein endothelial cells (HUVEC) in the presence of HT1080 human fibrosarcoma-conditioned medium. tEC showed higher permeability and less cell adhesion activity than normal HUVEC (nEC). Tumor necrosis factor-alpha (TNF) is known to have tumor-vasculature disrupting activity. tEC showed higher cytotoxicity to recombinant human TNF (rhTNF) than nEC, and was not observed using HUVEC cultured with WI38 human diploid cell-conditioned medium as a medium control. These results demonstrate that tEC acquire physiological properties of tumor-associated vasculature, and may be a useful model system for the study of the mechanisms of TNF antitumor action. The TNF-mutant RGD-V29 (code No. F4614), which has an inserted 4Arg-Gly-Asp sequence and an 29Arg-->Val replacement, was found to induce greater preferential destruction of tEC compared to rhTNF. When the preferential activities were evaluated in terms of 30% cytotoxicity (IC30) ratio (nEC/tEC), the ratio was 460 for RGD-V29 compared to 4.2 for rhTNF. RGD-V29 also exhibited cell-adhesive function and bound preferentially to the p55 TNF receptor. Both these properties of RGD-V29 contributed to the tEC selective cytotoxicity, indicating that the RGD ligands and selective p55 receptor binding on the cells, although uncharacterized, are involved in tEC targeting. Therefore, the TNF mutant RGD-V29 may show greater selectivity toward tumor vasculature than wild-type TNF. PMID- 10744046 TI - Vitamin K contents in liver tissue of hepatocellular carcinoma patients. AB - Serum protein induced in vitamin K absence-II (PIVKA-II) is used as a tumor marker because it increases at a notably higher rate in patients with hepatocellular carcinoma. To clarify the mechanism causing the elevation of serum PIVKA-II, we measured the contents of vitamins K1 (phylloquinone, PK) and K2 (menaquinone, MK) (MK-4, MK-5, MK-6, MK-7, MK-8, MK-9, MK-10) in liver tissue resected from 21 hepatic cancer patients (12 patients with hepatocellular carcinoma and 9 patients with metastatic hepatic cancer), using HPLC combined with coulometric reduction and fluorometric detection. In the cancerous tissue of hepatocellular carcinoma patients, PK, MK-7, MK-8, and MK-10 were significantly lower than that found in the noncancerous tissue. Furthermore, MK-6, MK-7, MK-8, and MK-10 in the cancerous tissue of hepatocellular carcinoma patients were significantly lower than that in the cancerous tissue of metastatic hepatic cancer patients. These data suggested that one of the mechanisms of the elevation of serum PIVKA-II levels in hepatocellular carcinoma patients is a vitamin K deficiency in the local cancerous tissue. PMID- 10744047 TI - Annexin VII as a novel marker for invasive phenotype of malignant melanoma. AB - Both F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. While examining the genetic difference between the two sublines, we found a marked reduction of annexin VII expression in BL6 cells. In addition, fusion cell clones of both sublines were as poorly metastatic as F10 cells after subcutaneous injection, and contained the annexin VII message as abundantly as F10 cells. Hence, we examined whether the annexin VII expression was correlated with the less malignant phenotype of clinical cases by immunohistochemistry. Immunoreactivities to anti-annexin VII antibody in melanoma cells were evaluated quantitatively by using skin mast cells as an internal positive control. Eighteen patients with malignant melanoma were divided into two groups: lymph node metastasis-negative and positive groups. The ratio of numbers of patients positive versus negative to the antibody was significantly larger in the former than in the latter group. These results not only indicated that annexin VII serves as a marker for less invasive phenotype of malignant melanoma, but also suggested a possible role of annexin VII in tumor suppression. PMID- 10744048 TI - Expression of drug resistance-related genes in head and neck squamous cell carcinomas and normal mucosa. AB - We examined the expression levels of mRNA for multidrug resistance 1 (MDR1), multidrug resistance-associated protein (MRP), human canalicular multispecific organic anion transporter (cMOAT), lung resistance-related protein (LRP), topoisomerase IIalpha, beta (Topo IIalpha, beta) and topoisomerase I (Topo I) genes in human head and neck squamous cell carcinoma (HNSCC) specimens and mucosa (HNM) specimens, to elucidate their roles in relation to the biological characteristics and drug resistance in vivo. Fifty-eight samples (45 head and neck carcinomas and 13 head and neck mucosa) obtained during surgical resection or biopsy from 38 patients were analyzed using the quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. MDR1, MRP, LRP, Topo IIalpha, Topo IIbeta, and Topo I gene transcripts were detected in all the samples tested, but cMOAT mRNA was not detected in them. Comparisons of the expression levels in HNSCC with those in HNM showed that the Topo IIalpha gene expression level was higher in HNSCC than in HNM (P=0.0298). Moreover, the Topo IIalpha mRNA level was significantly higher in metastatic lymph node samples of HNSCC than in HNM samples (P=0.0205). There were no significant differences in the six genes' expression levels between samples exposed to platinum drugs and those not exposed to platinum drugs. These results suggest that it may be effective in anticancer therapy to use topoisomerase-targetting drugs against HNSCC, especially metastatic neck tumors, and that the expression of these genes in HNSCC is not associated with platinum drug exposure. PMID- 10744050 TI - In vitro antitumor activity, intracellular accumulation, and DNA adduct formation of cis-[((1R,2R)-1,2-cyclohexanediamine-N,N')bis(myristato)] platinum (II) suspended in lipiodol. AB - SM-11355, cis-[((1R,2R)-1,2-cyclohexanediamine-N,N')bis(myristato)] platinum (II), is a lipophilic platinum complex under clinical development that targets primary hepatocellular carcinoma using Lipiodol as a carrier. SM-11355 was compared with cisplatin (CDDP) using an in vitro evaluation system capable of examining the release characteristics and the cytotoxicity of drugs suspended in Lipiodol. SM-11355 suspended in Lipiodol (SM-11355/Lipiodol) and CDDP suspended in Lipiodol (CDDP/Lipiodol) showed cytotoxic activity against rat ascites hepatoma AH-109A cells in a dose-dependent manner. Their IC50 values following 7 day exposure were 22.3 and 0.40 microg/ml, respectively. Following the subsequent 7-day exposure, from day 7 to day 14 after preparation of the suspension, SM 11355/Lipiodol showed an almost equivalent activity, but CDDP/Lipiodol did not show any activity at all. SM-11355/Lipiodol showed a sustained release into the culture medium over the course of a 14-day exposure. Following the exposure to CDDP/Lipiodol, the platinum concentration in the medium was at its maximum on the first day and remained constant thereafter. Intracellular platinum uptake and formation of platinum-DNA adducts were dependent on the release characteristics of each drug suspension. For SM-11355/Lipiodol, the drug release, intracellular drug uptake, and formation of platinum-DNA adducts over the course of the subsequent 7-day exposure were similar to those observed during the first 7 days. DPC, one of the compounds released from SM-11355/Lipiodol, was taken up by cells and showed formation of platinum-DNA adducts. Thus, this study suggests that SM 11355/Lipiodol may release active platinum compound(s) that bind to nuclear DNA and mediate the cytotoxic activity of SM-11355/Lipiodol. PMID- 10744049 TI - HeLa cell transformants overproducing mouse metallothionein show in vivo resistance to cis-platinum in nude mice. AB - Plasmid pSV2MT-I encoding mouse metallothionein-I (MT-I) designed to be expressed under the control of an SV40 promoter was introduced into human HeLa S3 cells. Several transformants (HeLa/MTH) carrying multi-copies of mouse MT-I cDNA in their genomes were isolated. These transformants produced 4 to 20-fold larger amounts of MT than their parent cells. The MT levels in HeLa/MTH were well correlated with the extent of resistance to cadmium, but not with that to cis platinum (cis-DDP) in vitro. To study the role of MT in resistance to cis-DDP in vivo, nude mice were inoculated subcutaneously with two independent HeLa/MTH clones. MT levels in these tumors were about 3-fold higher than those in the parental cells. The growth of tumors derived from either HeLa/MTH clone was not inhibited in the presence of 15 micromol/kg of cis-DDP, which completely inhibited the growth of tumors derived from the parental HeLa cells. These data strongly suggest that the elevated level of MT confers resistance to cis-DDP in vivo but not in vitro. Thus, the results of this study indicate that in vitro determinations of the influence of MT on cis-DDP resistance may underestimate its importance in in vivo situations. PMID- 10744051 TI - Dihydropyrimidine dehydrogenase and messenger RNA levels in gastric cancer: possible predictor for sensitivity to 5-fluorouracil. AB - We investigated the correlation between tumor sensitivity to 5-fluorouracil (5 FU) and enzymatic activities of thymidylate synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) in human gastric cancer specimens. Forty-one patients with advanced gastric cancer gave informed consent and were enrolled in the study. Biopsy specimens of gastric cancer were obtained preoperatively through gastrofiberscopy and used to determine TS and DPD messenger RNA (mRNA) levels. TS and DPD enzyme activity and mRNA levels were also measured in resected tumor tissue samples obtained after surgical resection. TS and DPD activity were measured using the TS-binding assay and a radioenzymatic assay, respectively, while mRNA levels were measured by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), with co-amplification of glyceraldehyde-3 phosphate dehydrogenase (GAPDH) as an internal standard. 5-FU sensitivity of resected tumor specimens was measured by the tetrazolium-based colorimetric assay (MTT assay). Both TS and DPD mRNA levels correlated well between biopsied and resected tumor specimens. A statistically significant correlation was also observed between mRNA levels in biopsied specimens and enzymatic activities in resected specimens. DPD levels significantly correlated with 5-FU sensitivity, such that high DPD activity and high DPD mRNA levels resulted in low sensitivity to 5-FU. In contrast, no correlation was observed between TS activity or TS mRNA levels and 5-FU sensitivity. We conclude that tumor DPD mRNA level, as assessed from biopsy specimens obtained by gastrofiberscopy, may be a useful indicator in predicting tumor sensitivity to 5-FU in patients with gastric cancer. PMID- 10744052 TI - Apoptosis in human hepatoma cell line SMMC-7721 induced by water-soluble macromolecular components of Artemisia capillaris Thunberg. AB - The aim of this study was to investigate the effect of water-soluble macromolecular components of Artemisia capillaris Thunberg (ACT) on human hepatoma cell line SMMC-7721 (SMMC-7721). The morphological changes of SMMC-7721 were observed under a light microscope and an electron microscope. Inhibition of proliferation was measured with a colorimetric MTT assay. It was discovered that ACT extract-treated cells exhibit morphological changes typical of apoptosis, including condensed chromatin and a reduction in volume. ACT extract at 25-200 microg/ml dose-dependently inhibited the proliferation of SMMC-7721. The 50% effective dose, evaluated on day 3 of exposure to the extract, was 64.52+/-3.53 microg/ml. Upon gel electrophoresis, the fragmented DNA showed a characteristic ladder pattern. Cell cycle analyses revealed that ACT induced cell cycle arrest at the G0/G1 phase. PMID- 10744053 TI - Targeting chemotherapy to solid tumors with long-circulating thermosensitive liposomes and local hyperthermia. AB - The effectiveness of the combination of long-circulating, thermosensitive liposomes and hyperthermia is described. Small-sized, thermosensitive liposomes that encapsulate doxorubicin (DXR-PEG-TSL (SUV)) have a prolonged circulation time and are extravasated to targeted solid tumors in vivo, where they preferentially release the agent in an anatomical site subjected to local hyperthermia. Liposomes were prepared by the incorporation of amphipathic polyethyleneglycol (PEG) to prolong their circulation time. DXR-PEG-TSL (SUV) was retained longest and was accumulated most efficiently in solid tumors in Balb/c mice. The combination of DXR-PEG-TSL (SUV) and hyperthermia at the tumor sites 3 h after injection, gave high concentrations of doxorubicin in tumor tissue and resulted in more effective tumor retardation and increased survival time. A large amount of DXR-PEG-TSL (SUV) was extravasated into the tumors during circulation for 3 h after injection, suggesting that the encapsulated drug was released into the interstitial spaces of the lesions by local hyperthermia. This system is expected to be clinically valuable for the delivery of a wide range of chemotherapeutic agents in the treatment of solid tumors. PMID- 10744055 TI - Teleconferences between the Gustave-Roussy Institute (Villejuif, France) and the National Cancer Center (Tokyo, Japan) as a new bilateral cooperative activity. PMID- 10744054 TI - Detection of apoptosis and expression of apoptosis-associated proteins as early predictors of prognosis after irradiation therapy in stage IIIb uterine cervical cancer. AB - We investigated the proportion of apoptotic cells and the expression of apoptosis associated proteins after the delivery of the first week of irradiation for stage IIIb uterine cervical cancer. Thirty patients with stage IIIb squamous cell carcinoma of the uterine cervix who received only irradiation therapy were registered in this study. Specimens were obtained before irradiation therapy and at the end of the first week of irradiation. The apoptotic index (AI) of each tissue specimen was calculated by counting the apoptotic cells and expressed as a percentage. Immunohistochemical evaluation for apoptosis-related proteins, p53, Bcl-2, Bax, caspase-1 and caspase-3 was also performed. The AI was 0.8+/-0.9% (mean+/-SD) before irradiation and 1.7+/-1.3% at the end of the first week of irradiation. We observed that the patients who survived more than 5 years had AI levels of 2.1+/-1.3% at the end of their first week of therapy. This rate was significantly higher than the rate of 1.1+/-0.8% (P=0.02) of the patients who died within 5 years. When the cut-off value of the AI was set at 1.7%, the sensitivity, specificity, positive predictive value, and negative predictive value for the prediction of patients' prognosis after irradiation therapy were 73.4%, 72.4%, 82.4%, and 61.5%, respectively. In 17 of the AI-positive cases, expressions of Bax (P=0.006), caspase-1 (P=0.045), and caspase-3 (P=0.013) at the end of the first week were significantly higher than before irradiation. The proportion of apoptotic cells and the expression of apoptosis-associated proteins, Bax, caspase-1, and caspase-3, at the end of the first week of irradiation could be useful predictors of the prognosis in stage IIIb squamous cell carcinoma of the uterine cervix treated by irradiation therapy. PMID- 10744056 TI - Local neurochemicals and site-specific immune regulation in the CNS. AB - Although it is often described as "immunologically privileged," the brain can display vigorous immune activity, both clinically and experimentally. The underlying control mechanisms are under active study. Here we shift attention from the brain as a whole to its diverse microenvironments. We review evidence that immune regulation in the brain is site-specific, and that local neurochemicals contribute to the site-specific control. Key points are illustrated by recent work from a rat model in which local injection of the proinflammatory cytokine, IFN-gamma, was used to modulate 2 essential aspects of the cell-mediated immune response: T cell entry from the blood, and expression of the MHC proteins that are needed to present antigen to the newly entered T cells. A growing number of neurologic disorders are known to be exacerbated by the immune/inflammatory network. Understanding the factors that influence local immune function may help explain the distribution of localized CNS damage and, more importantly, may suggest new therapeutic approaches for both desirable and unwanted responses. PMID- 10744057 TI - Novel cerebral lesions in the Eker rat model of tuberous sclerosis: cortical tuber and anaplastic ganglioglioma. AB - The Eker rat is a model for human tuberous sclerosis (TSC) caused by a mutation in the Tsc2 gene. We describe here histological and immunohistochemical findings of the brain lesions in Eker rats, with emphasis on 2 novel lesions found in this study: a cortical tuber and an anaplastic ganglioglioma. The rat cortical tuber resembled those of humans, and further confirmed the value of this animal model as a tool for investigating the molecular pathology of tuberous sclerosis. On the other hand, the rat anaplastic ganglioglioma had features of a malignant neoplasm that are absent from human subependymal giant cell astrocytomas. PMID- 10744058 TI - Myoblast transplantation in whole muscle of nonhuman primates. AB - The goal of the present study was to determine the feasibility, success, and toxicity of myoblast transplantation (MT) in the whole muscle of primates. Allogenic myoblasts transduced with the beta-galactosidase (beta-Gal) gene were transplanted in the whole Biceps brachii of 5 monkeys immunosuppressed with FK506. Myoblast injections were spaced at every 1 to 1.5 mm in 7 muscles, as well as at every 5 mm in 2 muscles. Myoblasts were resuspended in HBSS, notexin 1 microg/ml or notexin 5 microg/ml. Depending on the number of beta-Gal labeled myoblasts and the injection protocol, biopsies of transplanted muscles exhibited 7% to 74% beta-Gal+ fibers 1 month after MT. Beta-Gal+ fibers were present in muscle biopsies made 3, 8, and 12 months after MT. Myoglobinuria and hyperkalemia, the risk factors after extensive muscle damage and notexin toxicity, were not observed. The withdrawal of immunosuppression led to histological evidences of cellular rejection of the graft. We concluded that MT can be successfully performed in large primate muscles without toxicity due to muscle damage. An effective immunosuppression allowed the maintenance of beta Gal+ fibers up to 1 year after MT. These results suggest parameters that may allow effective MT in humans. PMID- 10744059 TI - Vascular factors are critical in selective neuronal loss in an animal model of impaired oxidative metabolism. AB - Thiamine deficiency (TD) models the cellular and molecular mechanisms by which chronic oxidative deficits lead to death of select neurons in brain. Region- and cell-specific oxidative stress and vascular changes accompany the TD-induced neurodegeneration. The current studies analyzed the role of oxidative stress in initiating these events by testing the role of intercellular adhesion molecule-1 (ICAM-1) and endothelial nitric oxide synthase (eNOS) in the selective neuronal loss that begins in the submedial thalamic nucleus of mice. Oxidative stress to microvessels is known to induce eNOS and ICAM-1. TD increased ICAM-1 immunoreactivity in microvessels within the submedial nucleus and adjacent regions 1 day prior to the onset of neuronal loss. On subsequent days, the pattern of ICAM-1 induction overlapped that of neuronal loss, and of induction of the oxidative stress marker heme oxygenase-1 (HO-1). The intensity and extent of ICAM-1 and HO-1 induction progressively spread in parallel with the neuronal death in the thalamus. Targeted disruption of ICAM-1 or eNOS gene, but not the neuronal NOS gene, attenuated the TD-induced neurodegeneration and HO-1 induction. TD induced ICAM-1 in eNOS knockout mice, but did not induce eNOS in mice lacking ICAM-1. These results demonstrate that in TD, an ICAM-1-dependent pathway of eNOS induction leads to oxidative stress-mediated death of metabolically compromised neurons. Thus, TD provides a useful model to help elucidate the role of ICAM-1 and eNOS in the selective neuronal death in diseases in which oxidative stress is implicated. PMID- 10744060 TI - Chronic prenatal exposure to carbon monoxide results in a reduction in tyrosine hydroxylase-immunoreactivity and an increase in choline acetyltransferase immunoreactivity in the fetal medulla: implications for Sudden Infant Death Syndrome. AB - Maternal cigarette smoking during pregnancy is associated with a significantly increased risk of Sudden Infant Death Syndrome (SIDS). This study investigated the effects of prenatal exposure to carbon monoxide (CO), a major component of cigarette smoke, on the neuroglial and neurochemical development of the medulla in the fetal guinea pig. Pregnant guinea pigs were exposed to 200 p.p.m CO for 10 h per day from day 23-25 of gestation (term = 68 days) until day 61-63, at which time fetuses were removed and brains collected for analysis. Using immunohistochemistry and quantitative image analysis, examination of the medulla of CO-exposed fetuses revealed a significant decrease in tyrosine hydroxylase immunoreactivity (TH-IR) in the nucleus tractus solitarius, dorsal motor nucleus of the vagus (DMV), area postrema, intermediate reticular nucleus, and the ventrolateral medulla (VLM), and a significant increase in choline acetyltransferase-immunoreactivity (ChAT-IR) in the DMV and hypoglossal nucleus compared with controls. There was no difference between groups in immunoreactivity for the m2 muscarinic acetylcholine receptor, substance P- or met-enkephalin in any of the medullary nuclei examined, nor was there evidence of reactive astrogliosis. The results show that prenatal exposure to CO affects cholinergic and catecholaminergic pathways in the medulla of the guinea pig fetus, particularly in cardiorespiratory centers, regions thought to be compromised in SIDS. PMID- 10744061 TI - Expression of the neurotrophin receptor p75NTR in medulloblastomas is correlated with distinct histological and clinical features: evidence for a medulloblastoma subtype derived from the external granule cell layer. AB - Medulloblastomas (MBs) are primitive neuroectodermal tumors (PNET) of the cerebellum. They represent the most frequent malignant pediatric brain tumors, but their origin still remains unresolved and controversial. MB cells correspond to different stages of neural development and differentiation as illustrated by their expression of neuronal and glial markers. In the present study, we examined the expression pattern of the common low-affinity neurotrophin receptor p75NTR in a series of 167 MBs by immunohistochemistry. While p75NTR was present in only 17% of classic MBs (CMB), we found expression of p75NTR in all desmoplastic (nodular) MBs (DMB) examined, and in 71% of those MBs with a significant desmoplastic component. Furthermore, both desmoplastic histology and p75NTR expression were present preferentially in those tumors of adolescents and adults that are frequently located laterally in the cerebellar hemispheres. In DMBs, p75NTR was expressed predominantly in the proliferative, reticulin-rich areas, which may show coexpression of GFAP. In the pale islands of DMB, p75NTR was expressed only weakly or was absent. The expression pattern showed an inverse relation to that of the synaptic vesicle protein synaptophysin that was predominant in p75NTR negative classic MBs. Since the neurotrophin receptor p75NTR is expressed in cells of the external granule cell layer (EGL) of the fetal cerebellum, our findings suggest that progenitor cells of the EGL are the cellular origin of a distinct subset of MB, namely the desmoplastic variant and MBs with a significant desmoplastic component. PMID- 10744062 TI - Selective neuronal survival and upregulation of PCNA in the rat inner retina following transient ischemia. AB - In this study we investigated the extent and time course of neuronal cell death and the regulation of the proliferating cell nuclear antigen (PCNA) in the different retinal cell layers following ischemia-reperfusion injury. Retinal ischemia was induced by controlled elevation of the intraocular pressure for a duration of 60 min. Changes in thickness and cell numbers in the retinal cell layers were analyzed at various time points (1 h to 4 weeks) after reperfusion. In parallel, apoptotic cell death was determined by the TUNEL method and the expression of PCNA analyzed by immunocytochemistry. In addition, we tested whether PCNA is expressed in neurons by double immunocytochemistry. The reduction in thickness was found to be less pronounced in the inner nuclear layer (INL). Correspondingly, cell numbers decreased by only 33% in the inner retina, but by more than 80% in the outer nuclear layer (ONL). Alterations in glial cell numbers did not contribute significantly to postischemic changes in the INL and ONL as assessed by using immunocytochemical markers for microglial and Muller cells. The time course of cell death determined by the TUNEL technique also differed markedly in the retinal layers being rapid and transient in the inner retina but delayed and prolonged in the ONL. PCNA immunoreactivity was undetectable in the normal retina, but was specifically induced in neurons of the inner retina within 1 h after reperfusion and was sustained for at least 4 weeks. We conclude that in contrast to photoreceptors in the ONL, a significant proportion of inner retinal neurons is resistant to ischemic insult induced by transiently increased intraocular pressure and that PCNA may possibly play a role in the selective postischemic survival of these cells. PMID- 10744063 TI - Derangement of Purkinje cells in the rat cerebellum following prenatal exposure to X-irradiation: decreased Reelin level is a possible cause. AB - It has been reported that prenatal X-irradiation of rats during the late gestation period causes heterotopic Purkinje cells in the internal granular layer (IGL) of the abnormally foliated cerebellum. The present study was designed to demonstrate the process of X-ray-induced derangement of Purkinje cells and their surrounding cells. In addition, the expression of some morphoregulatory molecules was examined to determine which molecules are involved in the abnormal pattern of Purkinje cells. Pregnant rats (n = 22) were exposed to 2.5 Gy X-radiation on gestation day 21 and the cerebellum of progeny was examined histologically and by immunohistochemistry to identify Purkinje and Bergmann cells. At 12 h after exposure, extensive cell death was observed in the external granular layer (EGL). By postnatal day (P) 9, while Purkinje cells with well-developed dendrites aligned underneath the EGL in the control cerebellum, Purkinje cells with shorter and abnormally oriented dendrites failed to align and remained in the heterotopic location in the IGL. Bergmann cells and their fibers were also disoriented but later recovered in their proper position. Abnormal folia developed in the irradiated rats. Using immunohistochemistry, we next examined the levels of the neural cell adhesion molecule (NCAM), fibronectin, tenascin, and Reelin. Among them, only the level of Reelin was affected significantly. Reelin decreased strikingly in the premigratory zone of the EGL and IGL in the irradiated cerebellum on P1, and the decrease continued until P9. Decreased Reelin expression was demonstrated quantitatively by Northern blot analysis and the correlation between the mRNA and protein levels was well presented. The expression of reelin mRNA decreased significantly by irradiation from P0, being almost one third of the level in controls on P4, and tended to recover up to P9. It is thus indicated that X-irradiation causes a marked decrease in the level of Reelin at the critical stage for the alignment of Purkinje cells. Since Reelin has been shown to play an important role in the migration of neural cells, it is suggested that the decrease in Reelin by X-irradiation is an important factor for the derangement of Purkinje cells. PMID- 10744064 TI - Molecular evolution and intratumor heterogeneity by topographic compartments in muscle-invasive transitional cell carcinoma of the urinary bladder. AB - Superficial transitional cell carcinomas (TCC) of the urinary bladder have been shown to be monoclonal. However, no combined study of clonality and tumor suppressor genes (TSG) is available to date for muscle-invasive TCC. Forty-four muscle-invasive TCC of the urinary bladder selected from women were included in this study. Tumor cells located above and below the muscularis mucosa zone were systematically microdissected and used for DNA extraction. Hha-I digested and undigested samples were used to study the methylation pattern of androgen receptor alleles and undigested samples were used for microsatellite analysis of TSG (TP53, RB1, WT1, and NF1). Both loss of heterozygosity (LOH) and single nucleotide polymorphism (SNP) analyses were performed using optimized denaturing gradient gel electrophoresis. The expression of p53, pRB, and p21WAF1 was assessed by immunohistochemistry. Appropriate controls were run in every case. All except two TCC showed a monoclonal pattern with the same allele inactivated in both compartments. Microsatellite analysis of TSG revealed the same LOH/SNP pattern in both tumor compartments in 30 cases (involving more than 1 TSG locus in 8) and genetic heterogeneity in 14 cases. From the latter group, 9 cases expressed more genetic changes in the deep compartment (involving TP53 gene in all cases, WT1 gene in 2, and NF1 in 1), whereas in 4 cases the superficial compartment showed more genetic changes (three involving NF1 and one involving both RB and TP53). No statistical difference in the immunoexpression was detected, although it tended to be higher in the superficial compartment than in the deep compartment. These concordant data in polymorphic DNA regions indicate that bladder-muscle-invasive TCC are monoclonal proliferations with homogeneous tumor cell selection. Heterogeneous tumor cell selection by topography defined two different genetic compartments: superficial, NF1-defective, and deep, TP53 defective. No differences in the immunohistochemical expression were observed, precluding a more extensive clinical application. PMID- 10744065 TI - Genetic heterogeneity in ductal carcinoma of the breast. AB - Genetic heterogeneity in breast cancer has been observed both by cytogenetic and loss of heterozygosity (LOH) analyses; however, the frequency with which genetically heterogeneous clones arise is unknown. In this study, a panel of 115 breast carcinomas was analyzed to determine the extent of clonal divergence in tumor foci at progressive stages of tumor evolution. Intraductal, infiltrating, and metastatic tumor components were microdissected from each tumor and tested for LOH at 20 microsatellite markers on seven chromosomal arms. Of these cases, 24 (21%) demonstrated genetically divergent clones during tumor progression. Clonal divergence, inferred from discordant LOH patterns, was observed most commonly between intraductal and infiltrating tumor (18 cases), but was also demonstrated between infiltrating and metastatic tumor (11 cases). Discordant LOH was observed with markers on one chromosomal arm in 16 cases, on two in 7 cases, and on four in 1 case, and was observed most commonly with markers on 17p, 17q, and 16q. More detailed microdissection of four cases provided evidence for a specific chronology of genetic alterations occurring during the progression of each tumor. The results indicate that the different tumor components observed microscopically in breast cancer specimens often represent genetically divergent clones. PMID- 10744066 TI - Epstein-Barr virus infection to Epstein-Barr virus-negative nasopharyngeal carcinoma cell line TW03 enhances its tumorigenicity. AB - Almost all nasopharyngeal carcinomas (NPCs) are infected by Epstein-Barr virus (EBV), but most ex vivo NPC cells lose EBV genomes during passages. In this study, an EBV-negative NPC cell line, TW03, established from EBV-carrying NPC was reinfected with EBV by cocultivation with irradiated Akata cells carrying recombinant EBV containing a neomycin-resistant gene. The reinfected EBV (+) TW03 cells expressed EBERs and EBNA1, but not EBNA2, lytic proteins (ZEBRA and EA-D), or LMP1. They had an epithelial appearance similar to that of EBV (-) TW03 cells. The doubling times of EBV (+) and EBV (-) TW03 cells were almost identical. However, the EBV (+) TW03 cells formed larger colonies with ragged contours in anchorage-independent cultures. An in vitro invasion assay showed that EBV (+) TW03 cells had a higher invasive activity than EBV (-) TW03 cells (p < 0.01). Both EBV (-) and EBV (+) TW03 cells formed poorly differentiated squamous cell carcinomas in SCID and nude mice. EBV (+) TW03 cells showed a higher tumorigenicity to nude mice (12 of 13) than EBV (-) TW03 cells (1 of 9) (p < 0.001). In the severe combined immunodeficiency (SCID) tumors of EBV (+) TW03 cells, not all of the tumor cells were EBER-1 positive. EBER-1 was more frequently detected in the peripheral regions and daughter nodules of the tumors than in the central areas. The microdissection polymerase chain reaction showed that the EBER-1-negative TW03 cells in the EBV (+) TW03 SCID tumors lost EBV genomes. EBER-1-negative cells showed as high a rate of Ki-67 positivity as EBER 1-positive cells, indicating that the former were proliferating rather than dead or dying. In horny pearls, keratinizing cells were ZEBRA-positive and EBER negative. Loss of EBV genomes was not associated with squamous differentiation. These data indicated that reinfection of EBV promotes the tumorigenicity of EBV ( ) TW03 cells by enhancing the invading activity. PMID- 10744067 TI - Inhibition of experimental allergic encephalomyelitis with an antibody that recognizes a novel antigen expressed on lymphocytes, endothelial cells, and microglia. AB - Experimental allergic encephalomyelitis (EAE) is a frequently employed animal model of the human disease multiple sclerosis. EAE can be induced by adoptive transfer of CD4+ T cells that are specific for central nervous system (CNS) antigens, typically myelin proteins. Although the pathogenic mechanism or mechanisms responsible for the clinical signs and histological changes in EAE and multiple sclerosis are not fully defined, the entry of T lymphocytes and antigen recognition within the CNS are required. The present study describes the participation of a novel cell surface molecule with properties suggesting a role in cell-cell adhesion or co-stimulation, or both, in the development of EAE in the rat. The molecule is defined by the unique monoclonal antibody (mAb) TLD-4A2. The TLD-4A2 antigen is present on resting and activated T lymphocytes, activated CNS endothelial cells, and microglia. The antigen is normally distributed in many tissues including lymph node, thymus, and spleen, as well as in the inflamed CNS. Both its pattern of tissue distribution and immunoprecipitation and immunoblotting studies suggest that the TLD-4A2 antigen is a novel molecule. Treatment of rats with the purified 4A2 mAb resulted in the inhibition of the clinical signs of EAE and also decreased the number T cells and macrophages accumulating in the CNS parenchyma. TLD-4A2 antibody did not seem to directly interfere with T cell viability in vivo, as demonstrated by the ability to recover and stimulate CD4+ encephalitogenic T cells from cervical lymph nodes of 4A2-treated animals. In vitro, the antibody partially blocked T cell proliferation assays. These data suggest that the TLD-4A2 mAb recognizes a novel molecule expressed on lymphocytes, endothelial cells, and macrophages that may play a role in hematogenous cell traffic and the initiation of CNS inflammation. PMID- 10744068 TI - Immunostaining and laser-assisted cell picking for mRNA analysis. AB - Isolation of single cells or cell clusters from complex tissue sections has become possible by microdissection techniques. Employing laser-assisted cell picking, cell-specific mRNA analysis of a few isolated cell profiles may be performed. However, microscopic discrimination of different cell types in routinely stained tissue sections is limited, whereas immunostaining enables a more precise access to cells of interest. This approach was noted to interfere with mRNA recovery. To define optimal conditions for mRNA amplification from immunodetected cells, we systematically investigated several potential affectors. Kind of fixation, antibodies and staining reagents, incubation and total processing time, and digestion with proteinase K turned out to influence mRNA stability. We present rapid protocols for immunohistochemistry and immunofluorescence with total incubation times of approximately 25 to 40 minutes and 10 to 20 minutes, respectively, and suggest mRNA amplification without a preceding extraction step. Applying these protocols to oligocellular clusters containing approximately 20 cell profiles and nuclei each from lung and kidney tissue, the highest efficiency rates of mRNA amplification were obtained when combining short-term formalin fixation, reduction of antibody incubation time, application of immunofluorescence, and digestion with proteinase K. Thus, the successful combination of immunostaining and laser-assisted cell picking remarkably improves cell type-specific analysis of gene expression within complex tissues. PMID- 10744069 TI - A possible role of CD46 for the protection in vivo of human renal tumor cells from complement-mediated damage. AB - It is still unclear which membrane-bound regulatory proteins (mCRP) are important in vivo to protect tumor cells from complement-mediated damage. To address this question, the expression levels of CD46, CD55, and CD59 were measured semi quantitatively in situ on renal cell carcinomas and compared with the expression level and cellular distribution of these mCRP in proximal tubuli within each patient (n = 31). It was also determined whether the expression of mCRP on tumor cells is associated with deposition of C3d and C5b-9. CD46 expression was decreased on tumor cells; in contrast, CD55 was expressed on tumor cells (12 out of 31 samples), while it was not detected on proximal tubular epithelial cells (PTEC). Also, expression of CD59 on tumor cells was increased as compared with its expression on PTEC. Furthermore, the localization on the cell surface of mCRP as observed on PTEC was altered on tumor cells. Because expression of mCRP may limit a complement-mediated anti-tumor response, we determined whether complement deposition was associated with the expression level of CD46, CD55, and CD59. The presence of C3d on tumor cells was associated with a low expression level of CD46 (p < 0.02). The expression level of CD46 was also associated with a low tumor stage (p < 0.04). The results suggest that in vivo CD46 plays a role in the protection of human renal tumor cells from complement-mediated injury. PMID- 10744070 TI - Enhanced expression of caspase-3 in hypertrophic scars and keloid: induction of caspase-3 and apoptosis in keloid fibroblasts in vitro. AB - Recent studies have suggested that the regulation of apoptosis during wound healing is important in scar establishment and development of pathological scarring. To examine the phenomenon of apoptosis and its involvement in the process of pathological scarring, we immunohistochemically quantified differential levels of expression of caspase-3 and -2, which are activated during apoptosis in vitro, in surgical resected scar tissues. We divided 33 cases of normally healed flat scars and 18 cases of pathological scars (15 cases of hypertrophic scars and 3 cases of keloid) into three groups (S1 = <10 months' duration; S2 = 10 to 40 months' duration; and S3 = >40 months' duration) according to the duration of scar. In all three groups examined, the semiquantitative scores for caspase-3 staining were significantly higher for the combination of hypertrophic scars and keloid as a group compared with normally healed flat scars, suggesting reduced cell survival and increased apoptotic cell death in hypertrophic scars and keloid. Apoptosis and caspase proteolytic activities were examined in vitro using two flat scar-derived fibroblast lines (FSFB-1 and -2) and two keloid-derived fibroblast lines (KFB-1 and -2). After 24 hours of serum deprivation, apoptotic cells were significantly increased in both KFB lines, whereas serum deprivation of FSFB-1 cells did not result in a significant increase in apoptotic cell number. After serum deprivation, significant increases in caspase-3 proteolytic activities were detected in both KFB lines compared with both FSFB lines. In contrast, no significant differences with caspase-8 activity were observed between similarly treated KFB and FSFB lines. Furthermore, serum deprivation-induced apoptosis of KFB-2 cells was significantly inhibited by the caspase-3 inhibitor Ac-Asp-Glu-Val-Asp fluoromethyl ketone (DEVD-FMK), indicating that caspase-3 is important for serum deprivation-induced apoptosis in KFB-2 cells. Considering the role of caspase-3 as a key effector molecule in the execution of apoptotic stimuli, our results suggested that enhanced expression of caspase-3 in hypertrophic scars and keloid induces apoptosis of fibroblasts, which may play a role in the process of pathological scarring. PMID- 10744071 TI - HMGI-C and HMGI(Y) immunoreactivity correlates with cytogenetic abnormalities in lipomas, pulmonary chondroid hamartomas, endometrial polyps, and uterine leiomyomas and is compatible with rearrangement of the HMGI-C and HMGI(Y) genes. AB - High-mobility group (HMG) proteins are nonhistone nuclear proteins that play an important role in the regulation of chromatin structure and function. HMGI-C and HMGI(Y) are members of the HMGI family of HMG proteins, and their expression in adult tissues generally correlates with malignant tumor phenotypes. However, HMGI C and HMGI(Y) dysregulation as a result of specific rearrangements involving 12q15 and 6p21, the respective chromosomal sites in which the HMGI-C and HMGI(Y) genes are located, is also identified in a variety of common benign mesenchymal tumors, such as lipomas and uterine leiomyomata. The general prevalence of HMGI-C and HMGI(Y) protein expression and its correlation with chromosomal alterations in these benign tumors are unknown. We analyzed 95 human tumors (20 lipomas, 21 pulmonary chondroid hamartomas, 26 uterine leiomyomata, and 28 endometrial polyps) representing a selection of the benign lesions in which karyotypic alterations involving the chromosomal regions 12q15 and 6p21 are frequently detected. All cases were successfully karyotyped and some of them analyzed by fluorescent in situ hybridization with probes spanning the HMGI-C and HMGI(Y) genes. The results of this study demonstrate that expression of HMGI-C or HMGI(Y) is a common occurrence in lipomas, pulmonary chondroid hamartomas, leiomyomata, and endometrial polyps; that it correlates with 12q15 and 6p21 chromosomal alterations (p < 0.001); and that it is compatible with rearrangement of the HMGI C and HMGI(Y) genes. The expression pattern and cellular localization of the immunoreactivity support the view that in biphasic lesions composed of a mixture of both stromal and epithelial cells, such as pulmonary chondroid hamartoma and endometrial polyps, the mesenchymal component is the site of the HMGI genetic alterations. PMID- 10744072 TI - Cytoprotection by metallothionein against gastroduodenal mucosal injury caused by ethanol in mice. AB - Metallothionein (MT) is a small, cysteine-rich protein that can act as a free radical scavenger at least in vitro. To test the hypothesis that MT participates in gastroduodenal cytoprotection, we studied sensitivity to gastroduodenal mucosal injury caused by ethanol in MT-null mice that have null mutations in MT-I and MT-II genes. MT-null mice and wild-type mice were orally treated with ethanol (60% or 99.5%, 0.2 ml/mouse). The macroscopic gastric lesion indices were significantly higher in MT-null mice than in wild-type mice 90 minutes after ethanol treatment. Histopathological examination in ethanol-treated MT-null mice showed vacuolar degeneration, necrosis of the epithelial cells, and hemorrhage throughout the tunica mucosa. Moreover, the duodenum also showed morphologic changes, including marked degeneration and coagulative necrosis of the entire villi, desquamation of the degenerated epithelial cells, and hemorrhage. In contrast, histopathologic changes were less prominent in the wild-type mice treated with ethanol. MT was not detected either in the stomach or duodenum of MT null mice, whereas gastric and duodenal zinc contents were not significantly different between MT-null mice and wild-type mice. These results provide direct evidence that intrinsic MT plays a cytoprotective role in gastroduodenal mucosal injury caused by ethanol. PMID- 10744073 TI - Ethylnitrosourea induces neoplasia in zebrafish (Danio rerio). AB - The zebrafish (Danio rerio) has been successfully used to discover hundreds of genes involved in development and organogenesis. To address the potential of zebrafish as a cancer model, it is important to determine the susceptibility of zebrafish to tumors. Germ line mutations are most commonly induced for zebrafish mutant screens by exposing adult male zebrafish to the alkylating agent, ethylnitrosourea (ENU). To determine whether ENU induces tumors, we compared the incidence of tumors in ENU-treated fish with untreated controls. Interestingly, 18 of 18 (100%) fish mutagenized with either 2.5 or 3.0 mM ENU developed epidermal papillomas, which numbered 1 to 22 per fish, within 1 year of treatment. The induced epidermal lesions included epidermal hyperplasia, flat papillomas (0.2 to 1.2 mm), and pedunculated papillomas (1.2 to 8 mm in greatest dimension), but no skin cancers. Angiogenesis was evident in papillomas larger than approximately 1 mm. All but two papillomas contained the three cell types (keratinocytes, club, and mucous cells) of normal zebrafish epidermis; histologic variants lacked either club cells or mucous cells. Two cavernous hemangiomas and a single malignant peripheral nerve sheath tumor were also found in the treated fish. None of five untreated controls developed tumors. These studies establish the feasibility of the zebrafish as an experimental model for the study of skin tumors. PMID- 10744074 TI - Loss of cell-cell contact is induced by integrin-mediated cell-substratum adhesion in highly-motile and highly-metastatic hepatocellular carcinoma cells. AB - The cadherin-mediated cell-cell adhesion system plays a critical role in normal development and morphogenesis. Inactivation of this system is thought to be responsible for cancer invasion and metastasis. A human hepatocellular carcinoma (HCC) cell line, KYN-2, was observed to have great potential for intrahepatic metastasis when orthotopically implanted into the liver of SCID mice. In vitro cultures of KYN-2 cells showed that they formed trabecular structures in suspension but lost tight cell-cell adhesion and became scattered when attached to a substratum such as collagen or fibronectin. In response to adhesion to the substratum, subcellular colocalization of E-cadherin and actin filaments were shown to be reduced, and a significant amount of alpha-catenin was dissociated from the E-cadherin-catenin complex in KYN-2 cells. These changes of cell-cell adhesion were blocked by inhibitory monoclonal antibodies against beta1 and beta5 integrins. We found that c-Src was coimmunoprecipitated with E-cadherin-catenin complex and was tyrosine-dephosphorylated and activated in the adherent cells. The tyrosine dephosphorylation of c-Src was induced by cell adhesion to the substratum and inhibited by addition of inhibitory monoclonal antibodies against beta1 and beta5 integrins. These findings indicate that integrin-mediated cell substratum adhesion inhibits cadherin-mediated cell-cell adhesion, possibly through c-Src activation, and suggest that this cross-talk mediates transient inactivation of the cadherin system and plays an important role in intrahepatic metastasis of human HCC. Modulation of this interaction might provide a new approach to prevent metastasis and recurrence of HCC. PMID- 10744075 TI - Lamellar body formation in normal and surfactant protein B-deficient fetal mice. AB - Surfactant protein B (SP-B) -/- mice die of lethal respiratory distress syndrome shortly after birth. Alveolar type II epithelial cells in SP-B-deficient mice are characterized by a complete absence of lamellar bodies, the intracellular storage form of pulmonary surfactant, and the presence of inclusions containing numerous small vesicles and electron-dense masses. The present study was undertaken to characterize the formation of these inclusions during fetal lung development and clarify their relationship to lamellar bodies. In wild-type and SP-B +/- mice, small lamellar bodies with loosely organized lamellae and distinct limiting membranes were first detected on day 16 to 16.5 of gestation. SP-B -/- mice were readily identified on day 16 by the absence of immature lamellar bodies, the appearance of vesicular inclusions similar to those previously described in late gestation SP-B -/- mice, and the accumulation of misprocessed SP-C protein. Vesicular inclusions were rarely detected in SP-B +/- mice and were never detected in wild-type littermates. Classical multivesicular bodies were observed fusing with lamellar bodies in wild-type mice, and with the vesicular inclusions in SP-B -/- mice that occasionally contained a few membrane lamellae. On day 18, the airways of SP-B -/- mice lacked tubular myelin and were filled with vesicles and electron-dense masses, suggesting that the contents of the vesicular inclusions were secreted. Taken together, these observations suggest that vesicular inclusions in SP-B -/- mice are disorganized lamellar bodies in which the absence of SP-B leads to failure to package surfactant phospholipids into concentric lamellae. PMID- 10744076 TI - Microsatellite analysis of microdissected tumor cells and 6p high density microsatellite analysis in head and neck squamous cell carcinomas with down regulated human leukocyte antigen class I expression. AB - Down-regulated human leukocyte antigen (HLA) class I expression is frequently correlated with allelic loss at 6p21.3, which is the location of the HLA coding sequence, in head and neck squamous cell carcinomas (HNSCCs). Previously, we have demonstrated loss of heterozygosity (LOH) at 6p21.3 for at least one locus in 49% of the HNSCCs using 5 microsatellite markers spanning the 4 megabase HLA region. In the present study, the detection threshold (25%) to assign LOH was addressed by laser-assisted microdissection of tumor cells from tumors containing marginal loss. In addition, we describe high density microsatellite analysis of chromosome 6p21.3 in HNSCC with down-regulated HLA class I expression. The purpose of this study was to refine the identification of genetic alterations at 6p21.3 and to pinpoint allelic loss to individual HLA class I genes, using additional markers closely located to the HLA-A, -B, and -C loci and the transporter associated with antigen processing (TAP) genes. LOH analysis by amplification of microsatellite markers and subsequent fluorescent detection is a rapid and sensitive technique to predict HLA class I loss phenotypes in tumors. LOH can be identified at 25% relative signal reduction. Analysis of heterogeneous tumor samples and samples containing a small amount of tumor cells is facilitated by laser-assisted microdissection of tumor cells. In addition, we showed that accurate HLA LOH analysis requires application of microsatellite markers in close proximity to HLA class I and TAP genes. PMID- 10744077 TI - Expression of C-C chemokines is associated with portal and periportal inflammation in the liver of patients with chronic hepatitis C. AB - Inflammation of the portal and periportal areas is a common feature of chronic hepatitis C. Antigen-presenting dendritic cells are located in the portal area, and infiltrating T cells are initially exposed to infected hepatocytes in the periportal area. Thus, these areas could be sites of the initial processes of the immune response in chronic hepatitis C. C-C chemokines (dendritic-cell-derived C C chemokine [DC-CK1] and regulated upon activation, normal T-cell expressed and secreted [RANTES])-attracting T cells may play a role in the portal inflammatory changes. The relationship between the expression of these C-C chemokines, which attract T cells and the infiltration of T cells into the liver of patients with chronic hepatitis C, was examined by in situ hybridization and reverse transcription-polymerase chain reaction. T-cell activation was examined by immunostaining T-cell subsets. Specific signals were detected for DC-CK1 mRNA in mononuclear cells mainly in the portal area and for RANTES mRNA in the portal area and at sites of piecemeal necrosis in the liver of patients with chronic hepatitis C. Naive T cells were located mainly in the portal area, whereas active T cells were found mainly at sites of piecemeal necrosis in the periportal area. In addition, hepatic DC-CK1- and RANTES-mRNA levels were significantly correlated with serum alanine aminotransferase levels (p < 0.001). These results suggest that the local production of DC-CK1 and RANTES participates in immune responses by attracting naive and active T cells to the portal and periportal areas, respectively. PMID- 10744078 TI - Localization of inducible nitric oxide synthase to mast cells during ischemia/reperfusion injury of skeletal muscle. AB - Nitric oxide contributes to tissue necrosis after ischemia-reperfusion (IR). A biochemical and immunohistochemical study was made of the amounts and localization of both Ca++-independent nitric oxide synthase (NOS) II and Ca++ dependent (NOS I and NOS III) in rat skeletal muscle after ischemia and 0.5, 2, 8, 16, and 24 hours reperfusion. NOS II was not detectable in control muscle or during ischemia, was first detected after 2 hours reperfusion, increased further by 8 hours, and remained elevated at 24 hours. Both NOS II and nitrotyrosine, a marker of peroxynitrite formation, were localized exclusively to mast cells except after 24 hours reperfusion when some macrophages and neutrophils also showed positive immunoreactivity. Mast cells underwent extensive degranulation during reperfusion. NOS I was not detected in injured or control muscle. The level of NOS III, which was localized to the endothelium of blood vessels of all sizes in control muscle, decreased progressively during ischemia and reperfusion to reach undetectable levels after 16 hours reperfusion. These findings indicate that most of the nitric oxide formed during IR injury is generated by NOS II located almost exclusively in mast cells. PMID- 10744079 TI - Expressional patterns of cytokines in a murine model of acute myocarditis: early expression of cardiotrophin-1. AB - To determine the role of cytokines in acute myocarditis, we examined expressional patterns of cardiotrophin-1 (CT-1), TNF-alpha, and IL-1alpha in a murine model of acute myocarditis. Ten-day-old Institute of Cancer Research mice were injected with Coxsackievirus B3 and killed on Days 1, 2, 3, 4, 5, 7, 10, 14, and 28 of injection. TNF-alpha and IL-1alpha expressions were investigated on histological sections from each heart, and mRNA expression of TNF-alpha, IL-1alpha, and CT-1 in the heart was examined by reverse transcription-polymerase chain reaction and RNase protection assay. To determine myocardial regeneration, cardiomyocytic DNA synthesis was investigated using bromodeoxyuridine on Days 3, 5, 7, and 10, and the labeling index was calculated in each heart. Age-matched uninfected mice were used as controls. TNFalpha and IL-1alpha expression was first detected in the cardiomyocytes on Day 3 and reached the maximum level on Day 7, when inflammatory changes were most prominent. Although an increased expression of TNFalpha and IL 1alpha mRNA was also detected on Day 3, CT-1 mRNA expression was distinctly augmented on Day 2. The labeling indices in the hearts with myocarditis were significantly higher than in those of the controls in all of the time points examined. CT-1 expression preceded TNF-alpha and IL-1alpha expressions and active DNA synthesis in a murine model of acute myocarditis. All CVB3-infected mice with anti-glycoprotein-130 antibody treatment died within 6 days. CT-1 may exert a protective role by modulating cytokine production and by inducing cardiomyocytic proliferation in CVB3-infected murine hearts. PMID- 10744080 TI - Prescriptions benefits for America's elderly. PMID- 10744081 TI - Influence of origin of ovarian cancer on efficacy of screening. PMID- 10744082 TI - No role for NO in asthma? PMID- 10744083 TI - Neurotransmitter antagonism in management of irritable bowel syndrome. PMID- 10744084 TI - Tyrosine-kinase inhibition in treatment of chronic myeloid leukaemia. PMID- 10744085 TI - Does autonomic nervous impairment have a role in pathophysiology of Sjogren's syndrome. PMID- 10744086 TI - From star signs to trial guidelines. PMID- 10744087 TI - Aids to navigation. PMID- 10744088 TI - Efficacy and safety of alosetron in women with irritable bowel syndrome: a randomised, placebo-controlled trial. AB - BACKGROUND: Irritable bowel syndrome (IBS) is a common gastrointestinal disorder with symptoms of abdominal pain, discomfort, and altered bowel function. Antagonists of the type 3 serotonin receptor (5-HT3) have shown promising results in the relief of IBS-associated symptoms. We aimed to confirm these findings by doing a randomised, placebo-controlled trial. METHODS: We studied 647 female IBS patients with diarrhoea-predominant or alternating bowel patterns (diarrhoea and constipation). 324 patients were assigned 1 mg alosetron and 323 placebo orally twice daily for 12 weeks, followed by a 4-week post-treatment period. Adequate relief of abdominal pain and discomfort was the primary endpoint; secondary endpoints included improvements in urgency, stool frequency, and stool consistency. Analysis was by intention to treat. FINDINGS: 79 (24%) of patients in the alosetron group and 53 (16%) in the placebo group dropped out. The difference in the drop-out rate between groups was mainly due to a greater occurrence of constipation in the alosetron group. A greater proportion of alosetron-treated patients than placebo-treated patients (133 [41%] vs 94 [29%], respectively) reported adequate relief for all 3 months of treatment (difference 12% [4.7-19.2]). Alosetron also significantly decreased urgency and stool frequency, and increased stool firmness. Constipation occurred in 30% and 3% of patients in the alosetron and placebo groups, respectively. INTERPRETATION: Alosetron was well tolerated and clinically effective in alleviating pain and bowel-related symptoms in this population of women with IBS. PMID- 10744089 TI - Irinotecan combined with fluorouracil compared with fluorouracil alone as first line treatment for metastatic colorectal cancer: a multicentre randomised trial. AB - BACKGROUND: Irinotecan is active against colorectal cancer in patients whose disease is refractory to fluorouracil. We investigated the efficacy of these two agents combined for first-line treatment of metastatic colorectal cancer. METHODS: 387 patients previously untreated with chemotherapy (other than adjuvant) for advanced colorectal cancer were randomly assigned open-label irinotecan plus fluorouracil and calcium folinate (irinotecan group, n=199) or fluorouracil and calcium folinate alone (no-irinotecan group, n=188). Infusion schedules were once weekly or every 2 weeks, and were chosen by each centre. We assessed response rates and time to progression, and also response duration, survival, and quality of life. Analyses were done on the intention-to-treat population and on evaluable patients. FINDINGS: The response rate was significantly higher in patients in the irinotecan group than in those in the no irinotecan group (49 vs 31%, p<0.001 for evaluable patients, 35 vs 22%, p<0.005 by intention to treat). Time to progression was significantly longer in the irinotecan group than in the no-irinotecan group (median 6.7 vs 4.4 months, p<0.001), and overall survival was higher (median 17.4 vs 14.1 months, p=0.031). Some grade 3 and 4 toxic effects were significantly more frequent in the irinotecan group than in the no-irinotecan group, but effects were predictible, reversible, non-cumulative, and manageable. INTERPRETATION: Irinotecan combined with fluorouracil and calcium folinate was well-tolerated and increased response rate, time to progression, and survival, with a later deterioration in quality of life. This combination should be considered as a reference first-line treatment for metastatic colorectal cancer. PMID- 10744090 TI - QTc-interval abnormalities and psychotropic drug therapy in psychiatric patients. AB - BACKGROUND: Sudden unexplained death in psychiatric patients may be due to drug induced arrhythmia, of which lengthening of the rate-corrected QT interval (QTc) on the electrocardiogram is a predictive marker. We estimated the point prevalence of QTc lengthening in psychiatric patients and the effects of various psychotropic drugs. METHODS: Electrocardiograms were obtained from 101 healthy reference individuals and 495 psychiatric patients in various inpatient and community settings and were analysed with a previously validated digitiser technique. Patients with and without QTc lengthening, QTc dispersion, and T-wave abnormality were compared by logistic regression to calculate odds ratios for predictive variables. FINDINGS: Abnormal QTc was defined from the healthy reference group as more than 456 ms and was present in 8% (40 of 495) of patients. Age over 65 years (odds ratio 3.0 [95% CI 1.1-8.3]), use of tricyclic antidepressants (4.4 [1.6-12.1]), thioridazine (5.4 [2.0-13.7]), and droperidol (6.7 [1.8-24.8]) were robust predictors of QTc lengthening, as was antipsychotic dose (high dose 5.3 [1.2-24.4]; very high dose 8.2 [1.5-43.6]). Abnormal QT dispersion or T-wave abnormalities were not significantly associated with antipsychotic treatment, but were associated with lithium therapy. INTERPRETATION: Antipsychotic drugs cause QTc lengthening in a dose-related manner. Risks are substantially higher for thioridazine and droperidol. These drugs may therefore confer an increased risk of drug-induced arrhythmia. PMID- 10744091 TI - Neurological manifestations of dengue infection. AB - BACKGROUND: Severe forms of dengue, the most important arboviral infection of man, are associated with haemorrhagic disease and a generalised vascular leak syndrome. The importance of dengue as a cause of neurological disease is uncertain. METHODS: During 1995, all patients with suspected CNS infections admitted to a referral hospital in southern Vietnam were investigated by culture, PCR, and antibody measurement in serum and CSF for dengue and other viruses. FINDINGS: Of 378 patients, 16 (4.2%) were infected with dengue viruses, compared with four (1.4%) of 286 hospital controls (odds ratio [95% CI] 3.1 [1.7-5.8]). Five additional dengue positive patients with CNS abnormalities were studied subsequently. No other cause of CNS infection was identified. Seven infections were primary dengue, 13 secondary, and one was not classified. Ten patients had dengue viruses isolated or detected by PCR, and three had dengue antibody in the CSF. 12 of the 21 had no characteristic features of dengue on admission. The most frequent neurological manifestations were reduced consciousness and convulsions. Nine patients had encephalitis. No patient died, but six had neurological sequelae at discharge. Phylogenetic analysis of the four DEN-2 strains isolated mapped them with a DEN-2 strain isolated from a patient with dengue haemorrhagic fever, and with other strains previously isolated in southern Vietnam. INTERPRETATION: In dengue endemic areas patients with encephalitis and encephalopathy should be investigated for this infection, whether or not they have other features of the disease. PMID- 10744092 TI - Benign ovarian cysts and ovarian cancer: a cohort study with implications for screening. AB - BACKGROUND: Whether some benign ovarian cysts can develop into cancerous cysts is not known. If a large proportion of ovarian cancers arose in this way, it might be possible to remove the benign cysts in a screening programme before they became malignant. We used follow-up data from a cohort of 5479 self-referred women without symptoms, who participated in a ultrasonographic-screening trial for early ovarian cancer between June, 1981, and August, 1987. We assessed whether the removal of persistent ovarian cysts from these women was associated with a reduction in the expected number of deaths from ovarian cancer in the cohort as a whole. METHODS: The expected number of deaths from all causes, all cancers, and ovarian, breast, and colorectal cancers were calculated for the study cohort by the standard life-table method. The actual number of deaths and each cause were obtained and the proportional mortality ratio was calculated for each cause of death. FINDINGS: 5135 (95%) of the participants in the original trial were traced. During the screening, five of these women were found to have stage I epithelial ovarian cancer and 88 had benign epithelial ovarian tumours. The number of reported deaths from all causes (387 [50% of expected]), all cancers (221 [71%]), and ovarian cancer (22 [90%]) was lower than expected because of the "healthy-volunteer effect". Proportional mortality ratios were 100% (by definition) for all cancers, 141% for breast cancer, 128% for ovarian cancer (95% CI 87.7-187.6, p=0.19), 84% for colorectal cancer, and 48% for lung cancer. INTERPRETATION: The removal of persistent ovarian cysts was not associated with a decrease in the proportion of expected deaths from ovarian cancer relative to other cancers during follow-up. For population-based screening of healthy women without a family history of ovarian cancer, a screening test is required that is specific and sensitive to early malignant disease, and inexpensive. PMID- 10744093 TI - Subgroup analysis and other (mis)uses of baseline data in clinical trials. AB - BACKGROUND: Baseline data collected on each patient at randomisation in controlled clinical trials can be used to describe the population of patients, to assess comparability of treatment groups, to achieve balanced randomisation, to adjust treatment comparisons for prognostic factors, and to undertake subgroup analyses. We assessed the extent and quality of such practices in major clinical trial reports. METHODS: A sample of 50 consecutive clinical-trial reports was obtained from four major medical journals during July to September, 1997. We tabulated the detailed information on uses of baseline data by use of a standard form. FINDINGS: Most trials presented baseline comparability in a table. These tables were often unduly large, and about half the trials inappropriately used significance tests for baseline comparison. Methods of randomisation, including possible stratification, were often poorly described. There was little consistency over whether to use covariate adjustment and the criteria for selecting baseline factors for which to adjust were often unclear. Most trials emphasised the simple unadjusted results and covariate adjustment usually made negligible difference. Two-thirds of the reports presented subgroup findings, but mostly without appropriate statistical tests for interaction. Many reports put too much emphasis on subgroup analyses that commonly lacked statistical power. INTERPRETATION: Clinical trials need a predefined statistical analysis plan for uses of baseline data, especially covariate-adjusted analyses and subgroup analyses. Investigators and journals need to adopt improved standards of statistical reporting, and exercise caution when drawing conclusions from subgroup findings. PMID- 10744094 TI - Neck lumps: expect the unexpected. PMID- 10744095 TI - High-dose therapy and autologous haematopoietic stem-cell transplantation for HIV 1-associated lymphoma. AB - We describe the results of autologous haematopoietic stem-cell transplantation in eight patients with HIV-1-associated lymphoma. Collection and grafting of stem cells is feasible and this treatment seems appropriate in chemotherapy-sensitive HIV-1-associated lymphoma. PMID- 10744096 TI - Melanocortin-1-receptor gene and sun sensitivity in individuals without red hair. AB - Susceptibility to sunburn, photoageing, and skin cancer is inversely related to an individual's ability to tan after sun exposure. We examined variants in the melanocortin-1-receptor (MC1R) gene in individuals from Ireland and the UK. We found evidence of an association between the degree of tanning after repeated sun exposure, and the number of variant alleles present. Heterozygotes were intermediate between wild-type individuals and those with two variant alleles. We suggest that MC1R gene status therefore determines sun sensitivity in people without red hair. PMID- 10744097 TI - Transcranial magnetic stimulation and auditory hallucinations in schizophrenia. AB - 12 patients with schizophrenia and auditory hallucinations received 1 Hz transcranial magnetic stimulation of left temporoparietial cortex. In a double blind crossover trial, active stimulation significantly reduced hallucinations relative to sham stimulation. PMID- 10744098 TI - Circulating plasma platinum more than 10 years after cisplatin treatment for testicular cancer. AB - We have shown in patients cured from metastatic testicular cancer that up to 20 years after administration of cisplatin-containing chemotherapy, circulating platinum is still detectable in plasma. This finding may influence the development of long-term, treatment-related side-effects. PMID- 10744099 TI - Patients' perceptions of pain with spinal, intramuscular, and venous injections. AB - Most of 46 patients believed that spinal injection would be more painful than intramuscular or intravenous injections. The reverse proved true: spinal injections were perceived to be less painful than the other two procedures. Intramuscular injections should be avoided. Local anaesthesia should be given for lumbar punctures and intravenous cannulation. PMID- 10744100 TI - Scientific expert witnesses in legal cases go on trial. PMID- 10744101 TI - Artificial-vision research comes into focus. PMID- 10744102 TI - Health spending is an economic and social investment. PMID- 10744103 TI - UK necropsy guidelines updated in wake of organ-retention scandals. PMID- 10744104 TI - Human rights in Pakistan remain as bad as they ever were. PMID- 10744105 TI - Chiral switches. AB - Developments in synthetic and analytical chemistry have provided the tools to differentiate between two enantiomers (mirror images) of drugs or between the parent compound and metabolite(s) with respect to desired and undesired pharmacological effects. Several drugs are now marketed or being developed as single enantiomers in place of a previous racemic mixture, a process known as "chiral switching". It is easier to understand "pure" as opposed to "mixture" pharmacology but whether the promise of chiral (and metabolite) switches will translate into real clinical advances remains to be seen. PMID- 10744106 TI - Lessons from the 1800s: tuberculosis control in the new millennium. PMID- 10744107 TI - Iraqi medical education under the intellectual embargo. PMID- 10744108 TI - Surveillance after colorectal cancer resection. PMID- 10744109 TI - Surveillance after colorectal cancer resection. PMID- 10744110 TI - Toxicity of nucleoside-analogue reverse-transcriptase inhibitors. PMID- 10744111 TI - Impact of sex on inheritance of ankylosing spondylitis. PMID- 10744112 TI - Impact of sex on inheritance of ankylosing spondylitis. PMID- 10744113 TI - Intestinal side-effects of docetaxel/vinorelbine combination. PMID- 10744114 TI - Epinephrine-resistant food anaphylaxis. PMID- 10744115 TI - Surgery for biliary atresia. PMID- 10744116 TI - Maternal dysphoria and postnatal environmental stress adaptation. PMID- 10744117 TI - Regulatory mechanisms in prostate cancer. PMID- 10744118 TI - Infant formula supplementation. PMID- 10744119 TI - Raynaud's syndrome in systemic lupus erythematosus. PMID- 10744120 TI - Leakage of oncologists from clinical oncology in Norway. PMID- 10744121 TI - Limiting multiple births. PMID- 10744122 TI - Michelangelo's gouty knee. PMID- 10744123 TI - The Nobel Chronicles. 1989: John Michael Bishop (b 1936) and Harold Eliot Varmus (b 1939). PMID- 10744124 TI - The Eighth AACR American Cancer Society Award lecture on cancer epidemiology and prevention. Genetically tailored preventive strategies: an effective plan for the twenty-first century? American Association for Cancer Research. AB - A preventive strategy of considerable appeal would offer specific lifestyle changes and preventive interventions to people at increased cancer risk because of inherited susceptibility. The hope for such a strategy stems in part from the variation in risk among carriers of cancer-predisposing mutations. For example, despite the high risks of cancers of the breast and ovary among BRCA1 and BRCA2 mutation carriers, some 30% of these women are estimated to reach age 70 years without developing either cancer. We need to know what protects these women, compared with carriers who do develop these malignancies. Apart from chance, possible explanations include variation in type of mutation, in genotypes at other loci, or in potentially modifiable lifestyle characteristics. This article reviews our present knowledge about risks for cancers of the colorectum, female breast, and ovary in carriers of highly penetrant germ-line mutations of susceptibility genes. The paper also reviews our present knowledge about options for reducing these risks through changes in lifestyle, chemopreventive agents, and prophylactic surgery. It concludes with a discussion of what we need to know to offer those with inherited susceptibility safe, effective options for preventing these cancers and to provide a framework for deciding among the options. PMID- 10744125 TI - Colorectal adenomas and the C677T MTHFR polymorphism: evidence for gene environment interaction? AB - 5,10-Methylene-tetrahydrofolate reductase (MTHFR), an enzyme in folate metabolism, may play a role in the etiology of colorectal adenomas via effects on DNA methylation and nucleotide synthesis. We investigated the association between a common polymorphism (C677T, reduced MTHFR activity) and colorectal adenomas within the Minnesota CPRU case-control study. Cases (n = 527) were diagnosed with colonoscopically confirmed adenomas; controls (n = 645) were derived from the same gastroenterology practice and were polyp free at colonoscopy. Dietary intakes were obtained from a self-administered food-frequency questionnaire prior to colonoscopy. Age- and sex-adjusted odds ratios (ORs) and 95% confidence intervals for the MTHFR genotype were 0.9 (0.7-1.2; CT versus CC wild-type) and 0.8 (0.6-1.3; TT versus CC). The associations between dietary intakes of folate, vitamin B12, vitamin B6, or methionine and risk of adenomas showed consistent patterns dependent upon MTHFR genotype. Individuals with the TT genotype and intakes of any of these nutrients in the lowest tertile were at elevated risk for adenomas (about 2-3-fold when compared with TT genotype with high intakes). These trends were more pronounced among individuals over age 60, resulting in a 3-6 fold increase for low intakes of folate, B12, and B6. An increased risk with increasing alcohol consumption was observed only among those with the CC genotype (P-trend = 0.005); among those with the TT genotype, those with moderate alcohol consumption were at lowest risk (P for interaction P = 0.02). In conclusion, nutrients involved in the MTHFR metabolic pathway may modify the relationship between the MTHFR C677T polymorphism and colorectal adenomas. Low intakes of folate, vitamin B12, and vitamin B6 increase risk among those (particularly the elderly) with the MTHFR TT genotype. PMID- 10744126 TI - hOGG1 Ser326Cys polymorphism and lung cancer susceptibility. AB - The human homologue of the yeast OGG1 gene, hOGG1, has been cloned, and its genetic structure has been determined. Several polymorphisms in the hOGG1 gene were detected in the Japanese populations, and among them, the Ser-Cys polymorphism at codon 326 has been shown to have a functional difference in complementation of mutant Escherichia coli that is defective in the repair of 8 hydroxyguanine. Activity in the repair of 8-hydroxyguanine is greater in hOGG1 Ser326 protein than in hOGG1(326) protein. Because many environmental carcinogens produce 8-hydroxyguanine residue and mismatching to this modified base potentially causes oncogenic mutations, the capacity to repair these lesions can be involved in cancer susceptibility in human beings. We, therefore, examined allele distributions of the Ser326Cys polymorphism in a case-control study of male lung cancer in Okinawa. The analyses based on 241 cases and 197 hospital controls disclosed the following findings. (a) Those with the Cys/Cys genotype were at an increased risk of squamous cell carcinoma and nonadenocarcinoma compared to those with the Ser/Cys and those with the Ser/Ser genotypes combined. The odds ratios adjusted for age and smoking history were 3.01 (95% confidence interval, 1.33-6.83) and 2.18 (95% confidence interval, 1.05-4.54), respectively. (b) The odds ratios for other histological subtypes of lung cancer or those in total were not significant. Those for Cys/Cys or Ser/Cys genotype against Ser/Ser did not reach statistical significance in any cell type. (c) The distributions of this polymorphism varied for different populations (Chinese, Japanese, Micronesians, Melanesians, Hungarians, and Australian Caucasians), with much less prevalence of Cys allele in the latter three populations. Although our sample size was limited, these results indicate that the Ser326Cys variant may be related to squamous cell lung cancer susceptibility. The Cys/Cys genotype appears to be more susceptible to squamous cell carcinoma, although the risk is less than that previously reported to be associated with the CYP1A1 gene. Further studies are needed to assess the importance of the interpopulation variation to cancer susceptibility. PMID- 10744127 TI - Glutathione S-transferase M1 status and lung cancer risk: a meta-analysis. AB - Interindividual differences in lung cancer susceptibility may be mediated in part through polymorphic variability in the bioactivation of procarcinogens. GSTM1 status has been extensively studied in this context as a lung cancer risk factor, although published studies have produced conflicting results. To clarify the impact of GSTM1 status on lung cancer risk a meta-analysis of 23 case-control studies from the literature has been carried out using a random effects model. The principal outcome measure was the odds ratio for the risk of lung cancer. There was heterogeneity between the studies attributable to differences in the methods of assigning GSTM1 status. Pooling the studies that were based on phenotyping methods, the overall odds ratio of lung cancer risk associated with GSTM1 deficiency was 2.12 (95% confidence interval, 1.43-3.13). The risk of lung cancer risk associated with GSTM1 deficiency derived from the studies based on genotyping methods was, however, lower. The overall odds ratio was 1.13 (95% confidence interval, 1.04-1.25). These findings suggest that the estimates of lung cancer risk associated with GSTM1 deficiency in the early studies, based on phenotyping, were overinflated. Moreover, it is conceivable, given publication bias, that GSTM1 status has no effect on the risk of lung cancer per se. A major concern in case-control studies of polymorphisms and cancer risk is bias. A review of the 23 case-control studies indicates that greater attention should, therefore, be paid to the design of future studies. PMID- 10744128 TI - Distribution and concordance of N-acetyltransferase genotype and phenotype in an American population. AB - Polymorphic arylamine N-acetyltransferase 2 (NAT2) status varies widely between individuals and ethnic groups and has been associated with susceptibility to several cancers. Few studies have reported the distribution of NAT2 status for Caucasian-American populations or evaluated the concordance between methods of assessment for cancer cases and controls. In our study, distribution of NAT2 status was classified by genotype and phenotype measurements in PANCAN, a population-based case-control study of pancreatic cancer, and concordance between measurements was evaluated for 33 cases and 222 controls. Major genotypes and alleles among controls were *5B/*6A, *5B/*5B, *4/*6A, and *5B/*4. One putative new allele was found in a single individual. Genotypes and phenotypes were classified as rapid or slow, according to a bimodal model. Presence of the *4 (wild-type) allele defined a NAT2 genotype as rapid. The NAT2 phenotype was analyzed by the caffeine assay. Ratios of 5-acetylamino-6-formylamino-3 methyluracil to 1-methylxanthine were determined, and individuals with values of > or =0.66 were identified as having a rapid phenotype. In our population, 58.1 and 59.5% of control subjects were classified as slow acetylators by phenotype and genotype, respectively. Concordance of NAT2 genotype and phenotype classification was 97.8% in the bimodal model. A similar analysis was completed for a trimodal model. Concordance of genotype and phenotype was high in cases (90.9%) and similar to controls; genotyping alone provided an efficient, accurate method of analysis for acetylator status. A comparison with two previous reports revealed subtle differences in genotype and allele distribution but exhibited overall similarity with other Caucasian-American populations. PMID- 10744129 TI - Alpha-tocopherol dietary supplement decreases titers of antibody against 5 hydroxymethyl-2'-deoxyuridine (HMdU). AB - This study evaluated the effects of vitamin E (alpha-tocopherol) on oxidative DNA damage in a randomized double-blind Phase II chemoprevention trial. Oxidative DNA damage was measured by the level of auto-antibody (Ab) against 5-hydroxymethyl-2' deoxyuridine (HMdU) in plasma. After the baseline screening, eligible subjects (n = 31; plasma samples from 28 subjects were available for this study) were randomized to receive 15, 60, or 200 mg of alpha-tocopherol per day for 28 days. Biomarkers were measured twice at baseline--on day 1 (visit 1) and day 3 (visit 2)--and twice after intervention--on day 17 (visit 3) and day 31 (visit 4). At baseline, there was a highly significant inverse correlation between anti-HMdU Ab titer and plasma vitamin E level (r = -0.53; P = 0.004; n = 28). Smoking did not affect baseline anti-HMdU Ab titer; however, anti-HMdU Ab titer levels at baseline were significantly lower in subjects with above-median (0.75 ounce/day) alcohol consumption (P = 0.008). No significant change in anti-HMdU Ab level occurred at either visit 3 or visit 4 for subjects on the lowest dose, 15 mg alpha-tocopherol per day. Subjects receiving 60 mg of alpha-tocopherol per day had a significant decrease in anti-HMdU Ab level at visits 3 and 4 compared with baseline (P = 0.049 and P = 0.02, respectively). However, subjects receiving the highest dose, 200 mg/day, had less consistent results: a significant decrease in anti-HMdU Ab level was seen at visit 4 (P = 0.04) but not at visit 3. Our results demonstrate an inverse relationship between alpha-tocopherol and anti-HMdU Abs in plasma; oxidative DNA damage can be modulated by short-term dietary supplementation of alpha-tocopherol in some subjects. PMID- 10744130 TI - Urinary isoflavonoid and lignan excretion on a Western diet: relation to soy, vegetable, and fruit intake. AB - Dietary isoflavone and lignan phytoestrogens are potential chemopreventive agents. This has led to a need to monitor exposure to these compounds in human populations and to determine which components of a mixed diet contribute to the exposure. Typically, urinary isoflavonoid excretion is associated with soy consumption and that of lignans is associated with whole grains. However, other plant foods are known to contain phytoestrogen precursors. The purpose of this study was to examine the association between urinary isoflavonoid and lignan excretion and intakes of vegetables and fruits (V&F). Isoflavonoids (genistein, daidzein, O-desmethylangolensin, and equol) and lignans (enterolactone, enterodiol, and matairesinol) were measured in urine collected for 3 days from 49 male and 49 female volunteers (age, 18-37 years) reporting a wide range of habitual V&F intakes. Dietary intakes were assessed using 5-day diet records and a food frequency questionnaire. V&F groupings (total V&F, total V, total F, soyfoods, and V&F grouped by botanical families) were used to assess the relationship between V&F intake and urinary isoflavonoid and lignan excretion. Pearson correlations were performed. Intake of soyfoods was correlated significantly with urinary genistein (r = 0.40; P = 0.0001), O desmethylangolensin (r = 0.37; P = 0.0002), daidzein (r = 034; P = 0.0007), and the sum of isoflavonoids (r = 0.39; P = 0.0001). There was no association between equol excretion and soy intake or between the isoflavonoids and any other V&F groupings. In addition, isoflavonoid excretion was correlated positively with intake of high-fat and processed meats, particularly among men who did not consume soy. This suggests that, even in the United States, on a Western diet, soyfoods are the primary contributors to isoflavone intake; however, additional "hidden sources" of soy may also contribute to exposure. In contrast, a variety of fiber-containing foods contributed to lignan excretion; the sum of the urinary lignans, enterodiol, enterolactone, and matairesinol, was associated with intake of total F (r = 0.27; P = 0.008), total V&F (r = 0.25; P = 0.01), soyfoods (r = 0.28; P = 0.006), and dietary fiber (r = 0.36; P = 0.0003). Overall, urinary phytoestrogens (isoflavonoids + lignans) were significantly higher in "high" compared with "low" V&F consumers. Compared with the "low" V&F group, the "high" group consumed diets that were, on average, higher in fiber and carbohydrate and soyfoods and lower in fat; thus, the urinary phytoestrogens may also be a useful marker of healthier dietary patterns. PMID- 10744131 TI - Ingestion of green tea rapidly decreases prostaglandin E2 levels in rectal mucosa in humans. AB - The objective of this Phase I/II study was to assess the potential for green tea to be used as a colorectal cancer chemopreventive agent. This study measured the dose-related biological effects of administration of a single dose of green tea on the rectal mucosa of normal volunteers. Volunteers were admitted to the Robert Wood Johnson Medical School Clinical Research Center for 24 h. Baseline blood and rectal biopsy samples were obtained before the volunteers drank 0.6, 1.2, or 1.8 g of green tea solids dissolved in warm water. Blood samples were taken 2, 4, 8, and 24 h after the tea administration. Rectal biopsies were obtained at 4, 8, and 24 h. Prostaglandin E2 (PGE2) levels were analyzed by ELISA. Tea polyphenol levels in the blood, urine, and rectal tissue were measured by high-performance liquid chromatography using a Coulochem electrode array detection system. Statistical comparisons were made using ANOVA. Decreased levels of PGE2 in rectal mucosa were observed at 4 and 8 h after consumption of green tea. There was no correlation between inhibition of PGE2 and tissue or plasma levels of tea polyphenols. Ten of 14 subjects demonstrated a response to green tea, as evidenced by at least a 50% inhibition of PGE2 levels at 4 h. We conclude that green tea constituents have biological activity in inhibiting PGE2 synthesis. Given the 71% "response rate," we believe these data support the study of green tea as a colorectal chemopreventive agent in more long-term Phase II trials. PMID- 10744132 TI - Familial occurrence of carcinoid tumors and association with other malignant neoplasms. AB - Carcinoid tumors are generally thought to be sporadic, except for a small proportion that occur as a part of multiple endocrine neoplasia syndromes. Data regarding the familial occurrence of carcinoid as well as its potential association with other neoplasms are limited. A chart review was conducted on patients indexed for malignant carcinoid tumor of the gastrointestinal tract seen at the Mayo Clinic between 1988 and 1996. A survey of family history of malignancies and personal history of other tumors was mailed to all eligible patients. Data for 245 patients were analyzed. Observed rates of carcinoids and other malignancies were compared with Surveillance, Epidemiology, and End Results data. Estimates of the cumulative probability for first-degree relatives developing a carcinoid tumor were calculated. Nine (3.7%) patients with carcinoid tumor had at least one first-degree relative with the same malignancy. The rate of carcinoid tumor in first-degree relatives of probands was higher (P < 0.0001) than expected based on the Surveillance, Epidemiology, and End Results population data. Cumulative probability in a first-degree relative for developing a carcinoid was calculated to be 1.5% at age 80. There was an increased risk for developing a carcinoid tumor among first-degree relatives of patients with carcinoid. Neither patients with carcinoid nor their first-degree relatives had an increased incidence of other malignancies. PMID- 10744133 TI - Validity of short food frequency questionnaires used in cancer chemoprevention trials: results from the Prostate Cancer Prevention Trial. AB - Here, we describe the measurement characteristics of a 13-item dietary screener used in the Prostate Cancer Prevention Trial. We used data from 10,913 men who completed the 13-item dietary screener, a food frequency questionnaire (FFQ), and questionnaires on demographic and health-related characteristics and from 146 men who also completed multiple 24-h dietary recalls in a substudy. The analyses in this report focused on percentage energy from fat and saturated fat and used the mean estimates from the dietary recalls as the criterion measures. Absolute nutrient estimates from the screener were about one-third of the estimates from the recalls and the FFQ. Validity was defined as the Pearson correlation of the criterion measures of fat with the corresponding measures from the FFQ and the screener. The FFQ was a statistically significantly more precise measure of percentage energy from fat (r = 0.71) and saturated fat (r = 0.72) than was the screener (r = 0.50 and 0.53, respectively). There were also statistically significant differences in how well these instruments could detect variation in dietary fat across various participant characteristics, suggesting that the screener may not perform as well as the FFQ across demographic strata such as education (P < 0.001). The results from this study suggest that the use of short dietary screeners as the sole assessment instrument may result in a serious loss of information regarding important exposures (e.g., fat intake) and lost opportunities to enhance our knowledge regarding dietary factors and cancer risk. PMID- 10744134 TI - International collaborative study on genetic susceptibility to environmental carcinogens. PMID- 10744136 TI - Factor V HR2: an ancient haplotype out of Africa--reasons for being interested. PMID- 10744135 TI - Correspondence re: J. S. Mandelblatt et al., Is HIV infection a cofactor for cervical squamous cell neoplasia? Cancer Epidemiol. Biomark. Prev., 8: 97-106, 1999. PMID- 10744137 TI - Protein C deficiency and venous thrombosis--the search for the second genetic defect. PMID- 10744138 TI - Mutations in the R2 FV gene affect the ratio between the two FV isoforms in plasma. AB - Molecular genetics and biochemical studies were performed in homozygotes for the R2 allele (4070G) in the factor V gene, most of them affected by coronary artery disease. Novel polymorphisms (G642T, 156Ser; T1328C, 385Met/Thr), among which a functional candidate (A6755G, 2194Asp/Gly) located in the C2 domain of FV, were identified in the R2 gene. In chromatographic studies R2 FV appeared qualitatively identical to normal FV. However, a relative increase of the more thrombogenic and more glycosylated FV isoform (FV1) was observed in plasma of 2194Gly homozygotes (mean FV1/FV2 ratio 0.71, 95% CI 0.66-0.77) as compared to R2 free controls (0.37, 95% CI 0.34-0.40). We conclude that carriership of the R2 FV gene is associated with an imbalance between the two functionally different FV isoforms, and propose that genetically determined differential glycosylation of FV could represent a novel mechanism of thrombotic disease. PMID- 10744139 TI - The G20210A prothrombin polymorphism is not associated with increased thromboembolic risk in a large protein C deficient kindred. AB - Likelihood analysis was used to test the effect of the G20210A prothrombin mutation and the His107Pro protein C mutation (resulting from a C insertion) on thrombosis status and prothrombin level in a large kindred of French Canadian descent with type I protein C deficiency. Genotypes were available on 279 pedigree members or their spouses. Of this total, 36 pedigree members were heterozygous for the G20210A variant and one pedigree member was homozygous for G20210A, while 64 were heterozygous for the His107Pro protein C mutation. The factor V Leiden mutation (Arg506Gln) was observed in only one of 181 tested family members. Objectively verified thrombosis was present in 26 of the 279 pedigree members. Thrombosis was suspected in an additional 19 pedigree members. The transmission disequilibrium test of Spielman, 1996, as extended to pedigrees, was used to test for excess transmission of G20210A or His107Pro to thrombosis cases, with transmission of 0.5 specifying no effect. Although the His107Pro mutation was over transmitted (0.837 +/- 0.075 p <0.001) to thrombosis cases in this pedigree, the G20210A variant was not (0.491 +/- 0.130 NS). Measured genotype analysis was used to examine a total of 184 individuals for the relationship between prothrombin level and both the G20210A variant and thrombosis. The G20210A variant increased prothrombin level from 97 +/- 2% to 124 +/- 4% (p <0.0001), but thrombosis status was not associated with any additional increase in prothrombin level. Thus, in a large thrombophilic, protein C deficient kindred, with the G20210A variant present in a proportion (13%) far higher than the general Caucasian population (approximately 2%), neither the presence of the variant nor the plasma concentration of prothrombin were associated with increased risk for thrombosis. These findings contrast with those of others who have established the G20210A variant as a thrombophilic risk factor; and emphasize the complex nature of the multigenic pathogenesis of thrombophilia. PMID- 10744140 TI - Changes in glycoprotein expression after platelet activation: differences between in vitro and in vivo studies. PMID- 10744141 TI - Effect of the factor V Leiden mutation on the clinical expression of severe hemophilia A. AB - To determine whether the factor V Leiden mutation is associated with decreased bleeding in individuals with severe hemophilia A, factor concentrate utilization, maximum annual number of bleeding episodes, and the prevalence of hemophilic arthropathy between carriers and non-carriers of the factor V Leiden mutation were compared. Heterozygosity for the factor V Leiden mutation was found in 6 of 137 subjects (4.4%). Carriers of the factor V Leiden mutation utilized less factor concentrate (geometric mean: 310 vs. 1185 units/kg/year) and had fewer bleeding episodes than non-carriers (proportion with 10 or fewer bleeding episodes in their worst year: 50 vs. 11%). However, the factor V Leiden mutation was not associated with the absence of arthropathy. The intron 22 inversion mutation of the factor VIII gene was tested for in a subgroup of 80 subjects, but it was not found to be a significant variable for any of the bleeding endpoints. The results of this small study are consistent with the hypothesis that the factor V Leiden mutation imparts a protective effect; however, a larger confirmatory study in which the factor VIII molecular defects can be controlled for is needed. Furthermore, most severe hemophiliacs who used fewer than 200 units/kg/year of factor concentrate or who had experienced 10 or fewer bleeding episodes per year did not carry the factor V Leiden mutation, suggesting that the proportion of severe hemophiliacs whose mild clinical course can be attributed to the factor V Leiden mutation is small. PMID- 10744142 TI - The impact of the glycoprotein Ia collagen receptor subunit A1648G gene polymorphism on coronary artery disease and acute myocardial infarction. AB - Platelet glycoprotein (GP) Ia is the major receptor for collagen and plays an important role in platelet adhesion and aggregation. Different gene polymorphisms have been identified that induce either various expression levels (C807T) or alterations of the tertiary structure (A1648G) of GPIa. Previously, we could demonstrate an association of the GPIa C807T dimorphism with nonfatal myocardial infarction. We have now analysed the influence of the GPIa A1648G (Br, HPA-5) dimorphism on the risk of coronary artery disease (CAD) and acute myocardial infarction (AMI). DNA samples from 2163 male Caucasian patients who underwent coronary angiography were genotyped by polymerase chain reaction and restriction fragment length analysis. The relation of the GPIa A1648G dimorphism to the extent of CAD was determined by multiple regression analysis with adjustment for coronary risk factors. Odds ratios (OR) as an estimate of relative risk of CAD and AMI and two-tailed p-values were calculated by multiple logistic regression. In the total study sample, no association was detected between the A1648G dimorphism and CAD or AMI. However, upon analysis of low-risk patient subgroups we found an association of the GPIa A1648G polymorphism with the risk and the extent of CAD (patients with high apoAI/apoB ratio: OR 0.59, p = 0.0090; non- and ex-smokers: OR 0.66, p = 0.0131; both inclusion criteria: OR 0.44, p = 0.0003). The relative frequency of the A1648 allele was higher in controls whereas the GG1648 genotype was overrepresented in patients with CAD. This association was also detectable when individuals with low expression levels of GPIa (C807 homozygotes) were analysed (patients with high apoAI/apoB ratio: OR 0.44, p = 0.0045; non- and ex-smokers: OR 0.61, p = 0.0370). Our findings indicate that the A1648G polymorphism of the platelet collagen receptor plays a role in CAD in well defined patient groups. PMID- 10744143 TI - Fibrinolytic proteins and progression of coronary artery disease in relation to gemfibrozil therapy. AB - Impaired fibrinolytic function, mainly due to increased plasma plasminogen activator inhibitor-1 (PAI-1) activity, is common in patients with manifest coronary artery disease (CAD) and a predictor of recurrent cardiovascular events. We investigated the relationships of plasma tissue-type plasminogen activator (tPA) and PAI-1 antigen levels, plasma PAI-1 activity and PAI 4/5-guanosine (4G/5G) genotype to CAD progression in 203 middle-aged men participating in the Lopid Coronary Angiography Trial (LOCAT). A higher tPA antigen concentration, whether baseline or on-trial, was associated with a more severe global angiographic response (p < 0.05), an association mainly accounted for by progression of diffuse lesions in graft-affected segments (change in per-patient means of average diameters of segments haemodynamically related to bypass grafts). Plasma PAI-1 activity and mass concentration and 4G/5G PAI-1 genotype were unrelated to angiographic outcome measurements. tPA and PAI-1 antigen increased significantly in the gemfibrozil group (+11.3% and + 16.4%, respectively, p < 0.001), whereas there was no treatment effect on PAI-1 activity (median change 0.0%). It is concluded that fibrinolytic function does not substantially influence progression of CAD as assessed by angiography in middle aged men. Furthermore, pronounced long-term lowering of serum triglycerides by gemfibrozil treatment does not significantly affect the plasma PAI-1 activity level but increases the plasma tPA and PAI-1 antigen concentrations. PMID- 10744144 TI - Prospective analysis after coronary-artery bypass grafting: platelet GP IIIa polymorphism (HPA-1b/PIA2) is a risk factor for bypass occlusion, myocardial infarction, and death. AB - Recently, we have demonstrated that human platelet antigen 1b (HPA-1b or P1A2) is a hereditary risk factor for platelet thrombogenicity leading to premature myocardial infarction in preexisting coronary artery disease. However, HPA-lb does not represent a risk factor for coronary artery disease itself. The aim of our present study was to evaluate the role of HPA-lb on the outcome in patients after coronary-artery bypass surgery. We prospectively determined the HPA-1 genotype in 261 consecutive patients prior to saphenous-vein coronary-artery bypass grafting. The patients were followed for one year. Among patients with bypass occlusion, myocardial infarction, or death more than 30 days after surgery, the prevalence of HPA-lb was significantly higher than among patients without postoperative complications (60 percent, 6/10, vs. 24 percent, 58/241, p <0.05, odds ratio 4.7). Using a stepwise logistic regression analysis with the variables HPA-1b, age, sex, body mass index, smoking (pack-years), hypertension, diabetes, cholesterol and triglyceride concentration, only HPA-lb had a significant association with bypass occlusion, myocardial infarction, or death after bypass surgery (p = 0.019, odds ratio 4.7). This study shows that HPA-1b is a hereditary risk factor for bypass occlusion, myocardial infarction, or death in patients after coronary-artery bypass surgery. PMID- 10744145 TI - Minor events and the risk of deep venous thrombosis. AB - BACKGROUND: Deep venous thrombosis is a common disease, with genetic and acquired risk factors. Many patients have a history of minor events (short periods of immobilisation such as prolonged travel, short illness, minor surgery or injuries) before onset of venous thrombosis. However, the role of these minor events has received little formal study. Also, we do not know how minor events might interact with the presence of genetic prothrombotic defects (factor V Leiden mutation, factor II mutation, protein C, S and antithrombin deficiency). PATIENTS AND METHODS: On the basis of case-control data from a thrombosis service in the Netherlands, we added a follow-up period for a case-cross-over analysis of minor events as risk factors, and a case-only analysis for the interaction with factor V Leiden. A total of 187 patients with first, objectively diagnosed venous thrombosis of the legs, aged 15-70, without underlying malignancies and without major acquired risk factors entered the study. For the analysis of minor events in the case-cross-over analysis, we used a matched odds ratio; in the case-only analysis, we used the multiplicative synergy index. RESULTS: In 32.6% of the 187 patients with deep venous thrombosis who did not have major acquired risk factors, minor events were the only external risk factors. Minor events increased the risk of thrombosis about 3-fold, as estimated in the case-cross-over analysis (odds ratio 2.9, 95% confidence interval 1.5-5.4). The synergy index between minor events and factor V Leiden mutation in the case-only analysis was 0.7 (95% confidence interval 0.3-1.5). Therefore, persons with factor V Leiden mutation who experience a minor event will have an estimated risk increase of about 17 fold, which exceeds the sum of the individual risk factors. CONCLUSIONS: Minor events are likely to play an important role in the development of deep venous thrombosis, especially in the presence of genetic prothrombotic conditions. PMID- 10744146 TI - Value of chest X-ray combined with perfusion scan versus ventilation/perfusion scan in acute pulmonary embolism. AB - BACKGROUND: The main purpose of ventilation scanning, as adjunct to perfusion lung scintigraphy, in acute pulmonary embolism is to allow for the classification of segmental perfusion defects as mismatched, which is generally accepted as proof for the presence of pulmonary embolism. We examined whether this function of the ventilation scan could be replaced by the chest X-ray. METHODS: In 389 consecutive patients with suspected pulmonary embolism and at least one segmental perfusion defect we classified the ventilation/perfusion (V/Q) scan and chest X ray/perfusion (X/Q) scan as either mismatched or matched. Furthermore we analyzed whether this comparison was different in subgroups of patients with concomitant congestive heart failure or chronic obstructive pulmonary disease. RESULTS: Overall agreement between the X/Q and V/Q scan diagnostic category was found in 341 of 389 patients (88%; 95% CI 84-92%). The positive predictive value for obtaining a mismatched V/Q scan result in case of a mismatched X/Q scan result was 86% (95% CI 81-90%). If the X/Q scan yielded only matched defects the V/Q scan resulted in the same classification in 90% (95% CI 85-95%). Analysis of the small subgroup of patients with chronic obstructive pulmonary disease showed that a mismatched X/Q scan was confirmed by V/Q scanning in 21 of 34 cases (62%; 95% CI 45-78%). CONCLUSION: This study shows that in the great majority of patients with clinically suspected acute pulmonary embolism combination of chest X-ray with perfusion scintigraphy reliably replaced ventilation/perfusion scintigraphy in defining (mis)-matching of segmental perfusion defects. These results need confirmation before the chest X-ray can fully obviate the use of ventilation scintigraphy. PMID- 10744147 TI - Derivation of a simple clinical model to categorize patients probability of pulmonary embolism: increasing the models utility with the SimpliRED D-dimer. AB - We have previously demonstrated that a clinical model can be safely used in a management strategy in patients with suspected pulmonary embolism (PE). We sought to simplify the clinical model and determine a scoring system, that when combined with D-dimer results, would safely exclude PE without the need for other tests, in a large proportion of patients. We used a randomly selected sample of 80% of the patients that participated in a prospective cohort study of patients with suspected PE to perform a logistic regression analysis on 40 clinical variables to create a simple clinical prediction rule. Cut points on the new rule were determined to create two scoring systems. In the first scoring system patients were classified as having low, moderate and high probability of PE with the proportions being similar to those determined in our original study. The second system was designed to create two categories, PE likely and unlikely. The goal in the latter was that PE unlikely patients with a negative D-dimer result would have PE in less than 2% of cases. The proportion of patients with PE in each category was determined overall and according to a positive or negative SimpliRED D-dimer result. After these determinations we applied the models to the remaining 20% of patients as a validation of the results. The following seven variables and assigned scores (in brackets) were included in the clinical prediction rule: Clinical symptoms of DVT (3.0), no alternative diagnosis (3.0), heart rate >100 (1.5), immobilization or surgery in the previous four weeks (1.5), previous DVT/PE (1.5), hemoptysis (1.0) and malignancy (1.0). Patients were considered low probability if the score was <2.0, moderate of the score was 2.0 to 6.0 and high if the score was over 6.0. Pulmonary embolism unlikely was assigned to patients with scores < or =4.0 and PE likely if the score was >4.0. 7.8% of patients with scores of less than or equal to 4 had PE but if the D-dimer was negative in these patients the rate of PE was only 2.2% (95% CI = 1.0% to 4.0%) in the derivation set and 1.7% in the validation set. Importantly this combination occurred in 46% of our study patients. A score of <2.0 and a negative D-dimer results in a PE rate of 1.5% (95% CI = 0.4% to 3.7%) in the derivation set and 2.7% (95% CI = 0.3% to 9.0%) in the validation set and only occurred in 29% of patients. The combination of a score < or =4.0 by our simple clinical prediction rule and a negative SimpliRED D-Dimer result may safely exclude PE in a large proportion of patients with suspected PE. PMID- 10744148 TI - Haemostatic and lipid determinants of prothrombin fragment F1.2 and D-dimer in plasma. AB - The determinants of plasma levels of prothrombin fragment F1.2 (F1.2) and D-dimer in different populations are unclear and this may complicate their interpretation as predictors of thrombotic risk, particularly in the case of D-dimer. We therefore measured F1.2 and D-dimer levels together with a number of other haemostatic and lipid variables in a cross-sectional community-based study of 150 healthy adults (73 male, 77 female), age range 23-80 years, identified from the list of a general practice by stratified random sampling within sex and decade of age. Plasma F1.2 was significantly higher in females than males and was independently and positively associated with age, factor VII activity (FVIIc) and C1 inhibitor, and inversely associated with high density lipoprotein (HDL) cholesterol. Plasma D-dimer showed a quadratic association with age (p <0.0001). In those < or =55 years D-dimer was inversely associated with dilute clot lysis time (DCLT) and activated protein C (APC) ratio. In those >55 years it was significantly higher in females than males and associated positively with age, fibrinogen and, in males, activated factor XII (FXIIa). In a multiple-linear model which combined both age groups, F1.2 and D-dimer were independently associated with each other (r = 0.22, p = 0.03). Thus, thrombin generation and fibrin turnover/fibrinolysis are associated in healthy subjects. HDL cholesterol (inversely) and FVIIc are associated with basal thrombin generation (i.e. F1.2). Determinants of D-dimer differ according to age and interpretation of the biological significance of D-dimer levels in epidemiological studies may therefore not be straightforward. PMID- 10744149 TI - Haemostatic factors in human peripheral afferent lymph. AB - Peripheral afferent lymph was obtained by cannulation of a collecting vessel in 17 healthy men (mean age 26 years). Lymph/plasma ratios of all vitamin K dependent factors were lower than expected from molecular weight. Factor VII, factor IX and tissue factor pathway inhibitor (TFPI) lymph/plasma activity ratios were higher than antigen ratios. Activated factor VII (FVIIa) and TFPI-Xa complex concentrations were higher in lymph than plasma, and the raised FVIIa did not appear to be due to cannulation. The fibrinogen lymph/plasma activity (Clauss) ratio averaged about 20% of the antigen ratio. The result of an ELISA for D-dimer was higher in lymph than plasma, often more than five-fold. This high level in lymph was not explored but may indicate proteolysis of fibrinogen and fibrin with release of D-like and D-dimer-like fragments in interstitial fluid. PMID- 10744150 TI - Absence of correlation between X chromosome inactivation pattern and plasma concentration of factor VIII and factor IX in carriers of haemophilia A and B. AB - Haemophilia A and B are X-linked disorders which are due to a reduced activity of coagulation factor VIII or IX, respectively. Female carriers have a wide range of plasma concentration of factor VIII or factor IX, and may in rare cases have an affected phenotype. In order to investigate if this variation is related to X chromosome inactivation, we determined the X inactivation pattern in 31 haemophilia A and 15 haemophilia B carriers, using a PCR in the androgen receptor locus in blood DNA. Seven of the haemophilia A carriers and none of the haemophilia B carriers had a skewed pattern (> or =80:20). One of the skewed haemophilia A carriers had a low plasma concentration of factor VIII (0.15 U/ml), but the remaining 6 carriers did not differ in factor VIII concentration from that of carriers with a random X inactivation pattern. One carrier with a high factor VIII concentration (2.0 U/ml) did not have a skewed pattern. Similarly, for the haemophilia B carriers, there was no tendency to a more skewed X inactivation pattern in the carriers with low or high factor IX concentrations. In addition, we analysed a female with haemophilia B who was heterozygous for the mutation R180W in the factor IX gene. She had a random X chromosome inactivation pattern. We conclude that the wide range in plasma concentration of factor VIII and factor IX in haemophilia A and B carriers cannot in general be explained by the X chromosome inactivation pattern in peripheral blood cells. PMID- 10744151 TI - A novel fibrinogen-clotting enzyme, TL-BJ, from the venom of the snake Bothrops jararaca: purification and characterization. AB - Three chromatographically distinct forms of a novel fibrinogen-clotting serine endopeptidase, TL-BJ1, 2 and 3, were purified from the venom of Bothrops jararaca by a combination of ammonium sulfate precipitation and chromatographic steps. The three forms of TL-BJ have similar amidolytic and plasma coagulating activities. TL-BJ 1, TL-BJ 2 and TL-BJ 3 cause the specific clotting of fibrinogen with release of fibrinopeptide A, the specific activities are 16.8 NIH U/mg (TL-BJ 1), 16.7 NIH U/mg (TL-BJ 2) and 20.8 NIH U/mg (TL-BJ 3). The most sensitive chromogenic substrates for measuring the amidolytic activity of TL-BJ 3 were D Pro-Phe-Arg-pNA, D-Phe-pipecolyl-Arg-pNA and Z-D-Arg-Gly-Arg-pNA. The amidolytic and coagulant activities of TL-BJ were inhibited by phenylmethylsulfonyl fluoride but not by hirudin. Benzamidine derivatives, which are competitive inhibitors of trypsin-like serine endopeptidases, also inhibited the amidolytic activity of TL BJ. In SDS/PAGE the main bands of TL-BJ 1, 2 and 3 showed molecular masses of 30 kDa, 31 kDa and 32 kDa. Upon incubation with N-glycosidase F only TL-BJ 3 remained unchanged, whereas TL-BJ 1 and TL-BJ 2 showed products with molecular masses around 23 kDa. Thus, TL-BJ 3 does not seem to be N-glycosylated. The N terminal amino acid sequences of TL-BJ 2 and TL-BJ 3 are identical while TL-BJ 1 has five substitutions. PMID- 10744153 TI - Cloning of guinea pig tissue factor cDNA: comparison of primary structure among six mammalian species. AB - Tissue factor (TF) is a transmembrane glycoprotein that serves as an essential cofactor for plasma coagulation factor VII. TF procoagulant activity exhibits varying species specificity. In particular, guinea pig (GP) TF is unable to activate clotting in heterologous plasma systems, but the molecular basis for this phenomenon is not yet understood. The full-length GP TF cDNA was cloned and sequenced. The open reading frame encoded a predicted precursor protein of 289 amino acids (aa) which was expressed in a reticulocyte lysate system as a protein of apparent molecular weight of 34 kD. The identity of the predicted aa sequence of mature GP TF with rabbit, human, bovine, rat and mouse TF was 66.4, 64.4, 60.6, 53.2 and 52.2%, respectively. With a focus on sites of potential functional significance, we compared sequences within the known binding regions. The eleven residues at the interface region between the TF1 and TF2 modules, which bind to the EGF domain of VIIa, were perfectly conserved among the six species, with the exception of an isoleucine replacing a lysine in the guinea pig sequence. However, only four of the eleven binding residues in the TF1 module, known to interact with the catalytic domain of factor VII, and three of the five residues in the TF2 module, involved in binding the factor VII Gla domain, were conserved among species. We hypothesize that divergence at these regions contributes to the specificity and non-reciprocity of TF procoagulant activity between species. PMID- 10744152 TI - Measurement of tissue factor activity in whole blood. AB - High circulating levels of the procoagulant molecule tissue factor (TF) are associated with thrombosis in a variety of diseases including unstable angina, cancer, and sepsis. Currently, there are no clinical assays to measure the level of TF activity in whole blood. We present an assay called Tissue Factor Clotting Time ("TiFaCT") that detects fibrin formation in human blood. The mean baseline clotting time in a healthy population was 472 +/- 94 s (mean +/- SD, n = 150). Bacterial lipopolysaccharide (LPS or endotoxin) shortened the clotting time in a time-dependent manner. Inhibitory anti-TF antibodies prolonged the clotting time of LPS-stimulated blood, indicating that the shortened clotting time was due to induction of TF expression. Patients with unstable angina had shortened mean baseline clotting time (284 +/- 86, n = 13) compared with healthy volunteers (474 +/- 98, n = 30), suggesting that these patients had elevated levels of circulating TF. The TiFaCT assay should prove clinically useful in quantifying the levels of circulating TF in patients at risk of thrombosis. PMID- 10744154 TI - Neuroprotection by recombinant thrombomodulin. AB - We examined whether recombinant human soluble thrombomodulin (rhs-TM) reduces compression trauma-induced spinal cord injury through protein C activation in rats. Administration of rhs-TM, either before or after the induction of spinal cord injury (SCI), markedly reduced the resulting motor disturbances. However, neither rhs-TM pretreated with an anti-rhs-TM monoclonal antibody (MAb) F2H5, which inhibits thrombin binding to rhs-TM, nor those pretreated with MAb R5G12, which selectively inhibits protein C activation by rhs-TM, prevented the motor disturbances. Intramedullary hemorrhages, observed 24 h after trauma, were significantly reduced in animals given rhs-TM. The increase in the tissue levels of tumor necrosis factor-alpha (TNF-alpha), TNF-alpha mRNA expression, and the accumulation of leukocytes in the damaged segment of the spinal cord were significantly inhibited in animals receiving rhs-TM, but these effects were not observed following administration of rhs-TM pretreated with MAb R5G12 or MAb F2H5. Leukocytopenia and activated protein C all produced effects similar to those of rhs-TM. These findings suggest that rhs-TM prevents compression trauma induced SCI by inhibiting leukocyte accumulation by reducing the expression of TNF-alpha mRNA and such therapeutic effects of rhs-TM could be dependent on its protein C activation capacity. Findings further suggest that thrombomodulin can be implicated not only in the coagulation system but in regulation of the inflammatory response. PMID- 10744155 TI - The effect of flow on lysis of plasma clots in a plasma environment. AB - Fibrinolysis initially generates channels in an occluding thombus which results in blood flow through the thrombus. Since the impact of flow along the surface of a thrombus on thrombolysis has not been investigated in detail, we studied in vitro how such a flow affects lysis. Compacted and noncompacted plasma clots were used as model thrombi. With compacted clots, fibrin-specific lysis induced by alteplase in the outer plasma was accelerated about 2-fold by strong flow (arterial shear rate). Non-fibrin-specific lysis induced either by a high concentration of alteplase or by streptokinase was slow, was accompanied by rapid depletion of plasminogen in the outer plasma, and was only slightly accelerated by flow. With noncompacted clots, similar acceleration factors were documented, when mild flow (venous shear rate) was applied. Strong flow further accelerated fibrin-specific lysis, up to 10-fold as compared to lysis without flow, but paradoxically retarded non-fibrin-specific lysis. The data suggest that flow accelerates lysis by enhancing transport of plasminogen from the outer plasma to the surface of the clot. Both opposite effects of the strong flow were mediated by forceful intrusion of the outer plasma into the noncompacted clot due to flow irregularities. In the case of non-fibrin-specific lysis this resulted in the replacement of the plasminogen-containing milieu by plasminogen-depleted outer plasma in certain areas of the clot turning them into virtually unlysable fragments. This flow-enforced "plasminogen steal" may contribute to the relatively high percentage of incomplete thrombolysis (TIMI-2 grade flow) documented in a number of trials for non-fibrin-specific thrombolytic agents. In the case of fibrin-specific lysis, the effect of flow on the speed of fibrinolysis is always beneficial. PMID- 10744156 TI - Rapid change of platelet aggregability in acute hyperglycemia. Detection by a novel laser-light scattering method. AB - We examined the alteration of platelet aggregability in acute hyperglycemia during 75-gram oral glucose tolerance tests (OGTT). Twenty subjects underwent 75 gram OGTT and venous blood samples were obtained before (0 min), 60, 120 and 180 min postload. Platelet aggregability shown as the number of small platelet aggregates was measured with a novel laser-light scattering (LS) method. Platelet aggregability increased in parallel with both glucose and immunoreactive insulin (IRI) levels. The number of mean small aggregates at 60 min (12.30 +/- 1.10 X 10(4)) was significantly higher than the one at 0 min (8.32 +/- 0.88 x 10(4), p <0.001), 120 min (10.63 +/- 0.98 x 10(4), p <0.05) and 180 min (8.28 +/- 0.84 x 104, p <0.001) (mean +/- SEM). Small aggregates correlated positively with plasma glucose levels at 60 min postload (r = 0.67, p = 0.001) while not with IRI. It might be important to suppress transient hyperglycemia for preventing the onset of acute coronary syndromes that could be closely related to platelet hyperaggregability. PMID- 10744157 TI - A novel approach to the assessment of variations in the human platelet count. AB - This is the first report of a method to assess the significance of numerical changes in the platelet count based upon a result exceeding the normal intra individual variation in platelet numbers. Serial platelet counts from 3,789 subjects were analysed to determine the intra-individual variation in platelet numbers. A platelet count difference of 98 x 10(9)/L in males was found to represent a change that would occur by chance in less than 1 in 1,000 platelet count determinations. Tables to determine the significance of platelet number variations, given N previous observations, are provided at two probability levels. The repeatability of the platelet count was calculated as 0.871 (males) and 0.849 (females) indicating that the heritability of platelet count is high and that the platelet count is predominantly genetically determined. A seasonal variation in platelet count was found with a 'winter' versus 'summer' difference of 5.10 X 10(9)/L (males) and 5.82 x 10(9)/L (females). PMID- 10744158 TI - Superoxide anion and hydroxyl radical release by collagen-induced platelet aggregation--role of arachidonic acid metabolism. AB - Previous study demonstrated that platelets undergoing anoxia-reoxygenation generate superoxide anion (O2-) and hydroxyl radical (OH ) which in turn contribute to activate arachidonic acid (AA) metabolism. However it has not been clarified if oxygen free radicals (OFRs) are also generated when platelets are aggregated by common agonists. We used two probes, i.e. lucigenin and salicylic acid (SA), to measure platelet release of O2- and OH(0), respectively. Among the agonists used, such as ADP, thrombin and collagen, the release of O2- and OH was observed mainly when platelets were stimulated with collagen. Such release was inhibited in platelets pre-treated by aspirin suggesting that AA metabolism was the main source of O2- and OH(0) formation. To further analyze this relationship, O2- and OH(0) formation was measured during AA-stimulated platelet aggregation (PA); we observed that O2- and OH(0) release were dependent upon AA concentration. Furthermore, we found that the incubation of platelets with AACOCF3, a potent inhibitor of cytosolic phospholipase A2, inhibited collagen induced platelet O2- and OH(0) release. The incubation of platelets with salicylic acid or ascorbic acid, which blunt OH and O2- respectively, inhibited both collagen-induced platelet aggregation and AA-release. This study demonstrated that collagen-induced platelet aggregation is associated with O2- and OH formation, which is dependent upon AA release and metabolism. PMID- 10744160 TI - Inhibition of platelet thrombosis using an activated protein C-loaded stent: in vitro and in vivo results. AB - In high-risk and complicated coronary intervention, the risk of acute closure is unpredictable. Thrombus and platelet deposition at the intervention site may also have further effects on subsequent restenosis. In vivo infusion of activated protein C has previously been shown to achieve potent anticoagulation without any haemostatic side effects. We now evaluated the in vitro and in vivo efficacy of polymer-coated coronary stents loaded with purified rabbit Activated Protein C (APC). By measuring 125I-fibrinogen/fibrin deposition APC-loaded stent-wires were antithrombotic compared to albumin-loaded, inhibited-APC-loaded, plain polymer coated and stainless steel stent-wires. In a balloon injury rabbit iliac artery model, APC-loaded stents did not occlude (0/14) compared to plain stents (9/15) and BSA-loaded stents (2/4). Relative 111In-labelled platelet deposition showed a similarly significant degree of inhibition. In conclusion, APC-loading could render stents significantly less thrombotic. Whether an effective antithrombogenic stent like this effectively reduces restenosis rates warrants further evaluation. PMID- 10744159 TI - Altered platelet shape change in hereditary gelsolin Asp187Asn-related amyloidosis. AB - Hereditary gelsolin-related amyloidosis (AGel amyloidosis) is a systemic disorder caused by a G654A or G654T mutation in the gene coding for gelsolin, an actin modulating protein. Altered platelet shape change has been demonstrated in gelsolin-deficient knock-out mice, but this has not been studied in humans with gelsolin deficiency. We measured platelet shape change, characterized by maximal decrease in light transmission (D) and reaction time (T), and aggregation, associated with stimulation of platelets with different agonists in platelet rich plasma, as well as coagulation factor VIII and ristocetin cofactor activities in 20 patients, 10 healthy sibs and 20 healthy control subjects. Statistically significant alterations of parameters describing platelet shape change (D, T) were observed after stimulation with adenosine diphosphate and collagen in patients when compared to healthy subjects, but not in maximal aggregation responses, platelet counts, coagulation factor VIII or ristocetin cofactor activity levels. Patients had more haemostatic derangements. Our results suggest that, in addition to amyloid deposition, the G654A gelsolin gene defect causes altered gelsolin-mediated cellular mechanisms, which may contribute, e.g., to bleeding tendency in AGel amyloidosis patients. PMID- 10744161 TI - Effect of exercise training on oxidized LDL-mediated platelet function in rats. AB - This study investigates how exercise training affects Oxidized LDL (Ox-LDL) mediated-platelet activation. Five-week-old male Wistar rats were assigned to either control or trained groups. Trained rats were treadmill-trained for 10 weeks after familiarization. The following measurements were taken in both control and trained groups: Ox-LDL-mediated platelet aggregability and [Ca2+]i, plasma and platelet-derived nitric oxide (NO) metabolite (nitrite plus nitrate) levels, and antiaggregating activity of NO derived from endothelial cells. Based on those measurements, major findings in this study can be summarized as follows: 1) the trained group had a higher plasma -NO metabolite level than the control group; 2) the trained group had a lower platelet aggregability and [Ca2+]i elevation and a higher platelet derived-NO metabolite level than the control group; 3) the trained group had lower Ox-LDL-potentiated platelet aggregability and [Ca2+]i elevation and Ox-LDL-attenuated NO metabolite in platelet than the control group; 4) treating the platelet with L-arginine inhibited Ox-LDL potentiated platelet activation in both control and trained groups; 5) Ox-LDL enhances platelet aggregation directly although impairing NO bioactivity but not guanylate cyclase activity in both control and trained groups. Results in this study demonstrate that exercise training decreases Ox-LDL-potentiated platelet activation most likely by enhancing platelet-derived NO release. PMID- 10744162 TI - Factor V Leiden and prothrombin G20210A variant are risk factors for venous thromboembolism in the Argentinean population. PMID- 10744163 TI - Venous thromboembolism and associated high plasma factor VIII levels: linked to cytomegalovirus infection? PMID- 10744164 TI - The C536T transition in the tissue factor pathway inhibitor gene is not a common cause of venous thromboembolic disease in the UK population. PMID- 10744165 TI - An effective route for the isolation of human genetic loci exemplified using the BglI restriction fragment length polymorphism of the factor VIII gene. PMID- 10744166 TI - A novel variant of the platelet glycoprotein Ibalpha macroglycopeptide region lacks any copies of the "perfect" 13 amino acid repeat. PMID- 10744167 TI - Combined von Willebrand factor deficiency and factor XII deficiency. PMID- 10744168 TI - Erythrocyte aggregation and erythrocyte membrane properties in type 2 diabetes mellitus and in vascular atherosclerotic disease. PMID- 10744169 TI - Hyperhomocysteinemia, MTHFR C677T genotype and low folate levels: a risk combination for acute coronary disease in a Portuguese population. PMID- 10744170 TI - Platelet vesiculation in angina patients treated with coronary angioplasty. PMID- 10744171 TI - The contribution of factor VII gene polymorphisms to longevity in Scottish nonagenarians. PMID- 10744172 TI - Physical risk factors for neck pain. AB - To identify physical risk factors for neck pain, a systematic review of the literature was carried out. Based on methodological quality and study design, 4 levels of evidence were defined to establish the strength of evidence for the relationship between risk factors and neck pain. Altogether, 22 cross-sectional studies, 2 prospective cohort studies, and 1 case-referent study were eligible for determining the level of evidence. The results showed some evidence for a positive relationship between neck pain and the duration of sitting and twisting or bending of the trunk. A sensitivity analysis was carried out excluding 3 items of the quality list, the importance of which seemed doubtful. On the basis of this sensitivity analysis, it was concluded that there is some evidence for a positive relationship between neck pain and the following work-related risk factors: neck flexion, arm force, arm posture, duration of sitting, twisting or bending of the trunk, hand-arm vibration, and workplace design. PMID- 10744174 TI - A forward-facilitating influence of cortisol on catecholamines assessed during the work of garbage collectors. AB - OBJECTIVES: In this study, the hypothesis was tested of whether part of the hypothalamic-pituitary-adrenal system can have a facilitating influence on mean adrenomedullary reactivity during a workday. METHODS: Data of a field study on 115 male garbage collectors were used for this purpose. Catecholamines and cortisol were assessed by urinary sampling during a workday and during a day off. The within-subjects baseline levels of the catecholamines, personal factors, physical work load, and work characteristics were controlled for in the analyses. RESULTS: The results showed that there is reason to assume a "forward facilitating influence" between the aforementioned systems. The morning cortisol excretion rate explained a reasonable amount of the mean catecholamine excretion rates during a workday. The morning cortisol level explained more variance in the adrenaline than in the noradrenaline values, and for both catecholamines the influence of cortisol was more pronounced than the influence of psychosocial stress factors like autonomy and job demands. CONCLUSIONS: The morning level of cortisol proved to be a more powerful predictor of the reactivity of adrenaline than that of noradrenaline during work. The association between the pituitary adrenocortical system and the adrenomedullary system could therefore be a "forward-facilitating influence". It is recommended that future research should focus on cortisol as the predictor of neuroendocrine reactivity and on determining whether this predictive power is expandable to work-induced health complaints. PMID- 10744173 TI - Primary osteoarthrosis of the knee in men and women as a result of lifelong physical load from work. AB - OBJECTIVES: This study investigated the effect of lifelong physical load from work on the development of knee osteoarthrosis (OA) leading to prosthetic surgery among men and women. METHODS: In a population-based case-referent study, men and women (N=625) who had had prosthetic surgery due to primary tibiofemoral OA were compared with referents (N=548) as to job titles and exposure to physical load in occupational work, housework, and leisure-time activities from 15 to 50 years of age. RESULTS: Male forestry and construction workers, and both male and female farmers ran the highest risk of knee OA. The men had considerably higher exposure to lifting at work, and also to jumps and vibration, than the women. Among the men there was an association between lifting at work [odds ratio (OR) 3.0, 95% confidence interval (95% CI) 1.6-5.5], squatting or knee bending (OR 2.9, 95% CI 1.7-4.9), kneeling (OR 2.1, 95% CI 1.4-3.3), and jumping (OR 2.7, 95% CI 1.7-4.1) with knee OA. Exposure to physically demanding tasks at home, such as taking care of an elderly or handicapped person, was associated with knee OA among the women (OR 2.2, 95% CI 1.3-3.6). CONCLUSIONS: Working as a farmer or as a construction worker could be associated with the development of knee OA and lead to prosthetic surgery. Men and women differ in the quality and quantity of reported physical load and also in the strength of the risk estimates. A reduction of high physical load at work and at home could probably lower the risk of knee OA later in life. PMID- 10744175 TI - Relationship between vibration dose and the absorption of mechanical power in the hand. AB - OBJECTIVES: The aim of this study was to examine the relationship between calculated vibration dose and the measured absorption of vibration power in the human hand, as well as the measured grip and feed forces applied by the subjects. METHODS: The study was carried out with 10 healthy subjects. A special handle was used during the measurements. The influence of 4 different vibration levels with different durations during a test period of 5 minutes was investigated. The number of exposure intervals varied between 1 and 5. The same overall equivalent acceleration was used in all the experiments. RESULTS: There is a significant difference between the calculated vibration dose and the amount of measured absorption of power. A higher acceleration level leads to significantly higher absorption. Furthermore, the outcome shows that rest periods contribute to a lower absorption of power in the hand and also lower feed forces. Conclusions This study supports the hypothesis that vibration-free rest periods give the human organism an opportunity to recover. PMID- 10744176 TI - Biases in estimating the effect of cumulative exposure in log-linear models when estimated exposure levels are assigned. AB - OBJECTIVES: Exposure-response trends in occupational studies of chronic disease are often modeled via log-linear models with cumulative exposure as the metric of interest. Exposure levels for most subjects are often unknown, but can be estimated by assigning known job-specific mean exposure levels from a sample of workers to all workers. Such assignment results in (nondifferential) measurement error of the Berkson type, which does not bias the estimate of exposure effect in linear models but can result in substantial bias in log-linear models with dichotomous outcomes. This bias was explored in estimated exposure-response trends using cumulative exposure. METHODS: Simulations were conducted under the assumptions that (i) exposure level is assigned to all workers based on the job specific means from a sample of workers, (ii) exposure level and duration are log normal, (iii) the true exposure-response model is log-linear for cumulative exposure, (iv) the disease is rare, and (v) the variance of job-specific exposure level increases with its job-specific mean. Results Assignment of job-specific mean exposure levels from a sample of workers causes an upward bias in the estimated exposure-response trend when there is little variance in the duration of exposure but causes a downward bias when duration has a large variance. This bias can be substantial (eg, 30-50%). CONCLUSIONS: Berkson errors in exposure result in little bias in estimating exposure-response trends when the standard deviation of duration is approximately equal to its mean, which is common in many occupational studies. No bias occurs when the variance of exposure level is constant across jobs, but such conditions are probably uncommon. PMID- 10744177 TI - Comparison of industrial hygienists' exposure evaluations for an epidemiologic study. AB - OBJECTIVES: A study was conducted to determine what level of information is required by industrial hygienists before they can develop exposure estimates comparable with those developed from a more in-depth evaluation. METHODS: Three industrial hygienists evaluated formaldehyde exposures of 300 jobs selected from an earlier epidemiologic study. The jobs were evaluated over the following 6 cycles: (i) job title and industry; (ii) job title, industry, dates; (iii) job and department title and industry; (iv) cycle 3 information with dates; (v) cycle 3 information with a plant report; and (vi) job and department title, industry, dates, and the report. Each hygienist assigned jobs to 1 of 4 exposure categories, which were compared with the categories in the original epidemiologic study. RESULTS: Overall, the mean differences between the hygienists' evaluations and the standard, although small, changed little over the cycles. The kappa statistic was poor to moderate for all the cycles, but the agreement was greater than expected due to chance. There was moderate improvement in overall agreement over the cycles using the weighted kappa statistic, but little improvement in the intraclass correlation coefficients of the hygienists' evaluations, which ranged from 0.4 to 0.5. Department information improved the agreement with the standard by 5--10%, but dates did not the improve agreement. There were some differences by type of plant, job function, exposure level, and date of the estimate. Using a hypothetical exposure-response scenario, this level of misclassification would have resulted in missing an association. CONCLUSIONS: Although there was slight improvement with increasing levels of information, these findings suggest that the subjective categorical assessment of exposures by industrial hygienists will not produce exposure estimates comparable to more in-depth evaluations of exposure. PMID- 10744178 TI - Past occupational exposure to asbestos among men in France. AB - OBJECTIVES: This study aimed at reconstructing changes in the frequency and levels of occupational asbestos exposure in France over the past century. METHODS: Work histories were collected during 11 population-based case-referent studies recently carried out in France, and an asbestos-specific job-exposure matrix including 10 625 jobs was used to estimate indices of past occupational asbestos exposure. The results were estimated from a sample of 4287 subjects, bootstrapped 200 times. RESULTS: The distribution of socioeconomic categories within the sample was compared with that of the general population in 1954, 1962, 1968, 1975, and 1982. The proportion of blue-collar workers was similar. The highest proportion of exposed subjects was found between 1950 and 1980. Around 10% of each 10-year age class was exposed to asbestos. For those born in 1930 1939, 15.2% was exposed between the ages of 20 and 29 years. For each age class born in 190-1939, the proportion exposed at least once by 60 years of age ranged from 18.2% to 24.5 % and, of those exposed, the cumulative duration of exposure ranged from 11.3 to 15.4 years by the age of 60 years. A population exposure index showed that the heaviest exposure occurred between 1960 and 1970 and that the age classes born between 1920 and 1929 were the most heavily exposed. Time trends showed that the mean value of this index for the men aged 20-59 years reached a peak in the 1960s and then decreased. CONCLUSIONS: This study presents data of reasonable validity about occupational asbestos exposure in France and its trends over the past century; the data are being used to forecast the development of male mortality from mesothelioma in France. PMID- 10744179 TI - Analysis of urinary S-phenylmercapturic acid and trans, trans-muconic acid as exposure biomarkers of benzene in petrochemical and industrial areas of Korea. AB - OBJECTIVES: Recently, S-phenylmercapturic acid (S-PMA) and trans,trans-muconic acid (t,t-MA) in urine have been proposed as reliable biomarkers for monitoring occupational exposure to benzene. The aim of this study was to test the applicability of S-PMA and t,t-MA as exposure biomarkers and to monitor the occupational exposure level and the extent of environmental contamination from benzene in Korea. METHODS: The urinary excretion of S-PMA and t,t-MA in rats after the intraperitoneal administration of benzene (0.88-800 mg/kg body weight, 7 days) was examined. These biomarkers were also validated in human urine samples collected from elementary schoolchildren in several industrial areas including chemical manufacturing plants, oil refineries, and natural gas-producing installations in Korea. Urine was collected from elementary schoolchildren in a mountain village with no known occupational exposure to benzene and air pollution as the reference group. RESULTS: In rats, there was a significant relationship between the benzene concentration and the excretion of the urinary S-PMA and t,t MA as a function of concentration, and the excretion of benzene metabolites peaked on the first day after intraperitoneal administration. In human urine, higher levels of S-PMA and t,t-MA were detected more frequently in petrochemical industrial areas than in areas with no known occupational exposure to benzene. CONCLUSIONS: These results show that the quantitative determination of S-PMA and t,t-MA in urine can be used as a reliable exposure biomarker for benzene, and they also suggest that extensive attention to benzene exposure is needed for maintaining the health of the population in Korea. PMID- 10744180 TI - Serum concentrations of hexachlorobenzene in family members of workers in an electrochemical factory. AB - OBJECTIVES: This study analyzed the effect of living in the same household with a worker employed in an electrochemical factory on the hexachlorobenzene (HCB) concentrations of serum in the general population of Flix, Spain. METHODS: A total of 608 subjects from the general population (response rate 42%) completed a questionnaire about residence, occupation, life-styles, and medical history and provided blood samples. Among them, 412 had never worked in the electrochemical factory. Information about the occupation of the family members was completed, and the subjects were classified with the degree of relationship with the worker having been taken into account. A multiple linear regression analysis was conducted to model HCB serum for nonfactory workers. RESULTS: Having a spouse who worked in the factory was associated with elevated HCB concentrations in serum. The adjusted relative increases were 1.28 (P=0.0004) and 1.23 (P=0.0022) times the corresponding value of people not living with workers of the factory, respectively for spouses of current and past workers. Relatives other than spouses did not show any increase. CONCLUSIONS: The results of this study suggest that, among nonworkers, living with a worker of the electrochemical factory in Flix is associated with an increase in concentrations of HCB in blood. These findings may suggest a source of exposure to HCB that has thus far not been studied and that could be important in populations not occupationally exposed to organochlorines. PMID- 10744181 TI - Asbestos-exposed blood monocytes--deoxyribonucleic acid strand lesions in co cultured bronchial epithelial cells. AB - OBJECTIVES: In lungs of asbestos-exposed persons alveolar and interstitial macrophages are able to release genotoxic substances such as reactive oxygen intermediates. It is unknown whether reactive oxygen intermediates released by macrophages are able to induce DNA (deoxyribonucleic acid) strand lesions in neighboring bronchial epithelial cells. METHODS: A co-culture (transwell) system was established which allows exposure of human blood monocytes cultured on a polycarbonate membrane within a distance of 1 mm of a monolayer of the bronchial epithelial cell line BEAS-2B. RESULTS: Exposure of blood monocytes to chrysotile B (100 microg/10(6)cells) caused an up to 2.8-fold increase in DNA strand lesions in co-cultured BEAS-2B cells measured by alkaline elution when compared with the levels of control cells after 1, 3, 24, and 48 hours. The main DNA damage thus occurred as early as within 1 hour of incubation, corresponding to the time course of the release of reactive oxygen intermediates by blood monocytes determined by chemiluminescence. The maximum release of reactive oxygen intermediates (3.2-fold increase over control values) was measured after 30 minutes of exposure of blood monocytes to chrysotile B. The addition of catalase (200 U/ml) or desferoxamine (100 microM) to the culture medium blocked almost completely the induction of DNA strand lesions in this system (maximum 85%). CONCLUSIONS: Exposure of blood monocytes to chrysotile B results in an increase in the release of reactive oxygen intermediates and induces DNA strand lesions in neighboring bronchial epithelial cells. PMID- 10744182 TI - Late asthmatic reaction caused by naphthylene-1,5 diisocyanate. AB - After occupational exposure to napthylene-1,5 diisocyanate (NDI) for 2 years, a 33-year-old female worker developed work-related asthma attacks. Occupational type exposure tests with NDI resulted in a late asthmatic response, whereas a corresponding challenge test with 4,4'-methylenediphenyl diisocyanate was negative. We conclude that NDI exposure in the range of its occupational exposure limit caused occupational asthma in this subject. PMID- 10744183 TI - Tennis elbow. Misconceptions and widespread mythology. PMID- 10744184 TI - Functional assessment and care of children with neurodevelopmental disabilities. AB - OBJECTIVES: To determine the relationship between pediatric assessment scores and ratings by parents and teachers regarding the amount of assistance required to complete basic activities of daily living; and to examine the relationship among scores for three commonly used pediatric assessments. DESIGN: Prospective correlational study. 205 children with developmental disabilities. The children ranged in age from 11 to 87 mo and included 72 females and 133 males of diverse socioeconomic and ethnic backgrounds. The children were evaluated by using the Battelle Developmental Inventory Screening Test, Vineland Adaptive Behavior Scales, Functional Independence Measure for Children (WeeFIM instrument), and the Amount of Assistance Questionnaire. RESULTS: The test-retest reliability coefficients for items on the Amount of Assistance Questionnaire were found to range from 0.82 to 0.97. Correlations among subscale scores and amount of assistance ratings were highest for the WeeFIM instrument and Battelle Developmental Inventory Screening Test. The highest correlation was between WeeFIM total rating and total amount of assistance rating (r = 0.91). CONCLUSION: Total WeeFIM instrument ratings and severity of disability were the best predictors of amount of assistance ratings provided by parents and teachers. PMID- 10744185 TI - Symptom duration and spontaneous activity in lumbosacral radiculopathy. AB - OBJECTIVES: A long-held notion in the electrodiagnostic literature is that paraspinal muscles tend to show spontaneous activity (fibrillations and positive sharp waves) on needle electromyography, early on in a lumbosacral radiculopathy, and that more distal muscles become abnormal later in the disease process. The purpose of this study was to determine whether paraspinal muscle and other major proximal and distal muscle spontaneous activity is related to a lumbosacral radiculopathy symptom duration. METHODS: A multicenter, prospective study that collected standard information on history, physical examination, and electrodiagnostic findings in patients with electrodiagnostically confirmed lumbosacral radiculopathies was undertaken. RESULTS: Multivariate probit analyses of 96 patients identified with a lumbosacral radiculopathy showed no evidence of correlation between spontaneous activity in the paraspinal muscles and symptom duration. Symptom duration was also nonsignificant in nine of the remaining ten lower limb muscles analyzed. CONCLUSION: These findings emphasize the limitations of using symptom duration when interpreting electrodiagnostic findings in lumbosacral radiculopathy. PMID- 10744186 TI - Describing paraspinal EMG findings: inadequacy of the single 0-4+ score. AB - OBJECTIVE: Clinical electrodiagnostic textbooks instruct that lumbar radiculopathies typically have paraspinal abnormalities and that these abnormalities should be recorded using a single 0-4+ scale. Recent work demonstrates that that the innervation of the paraspinal muscles is segmental, not homogenous, and that asymptomatic persons may have reproducible ("1+") positive waves or fibrillation potentials. DESIGN: To assess current use of the 0 4+ scale, a retrospective study of EMG scores in persons diagnosed with radiculopathy at a university laboratory was performed. No specific paraspinal EMG technique was used. Included were 117 consecutive, qualified S-1 radiculopathies and 33 L-5 radiculopathies. RESULTS: When radiculopathy was defined by "more than one limb muscle abnormal," paraspinal scores were recorded as 0 to 1+ in 83% of L-5 and 63% of S-1 cases (chi2, L-5 vs. S-1, not significant). For more severe radiculopathies (at least one limb muscle with a "2+" score), 0-1+ paraspinal scores were recorded in 76% of L-5 and 57% of S-1 cases (chi2, not significant). A total of 82% of S-1 radiculopathies with no overlapping L-5 innervation had 0-1+ scores. CONCLUSIONS: These data demonstrate the common use of equivocal paraspinal scores in patients whom electromyographers believe have radiculopathies. Standardized exploration techniques and a more detailed scoring system for the paraspinal muscles may eliminate this discrepancy. PMID- 10744187 TI - Neoplastic vs. traumatic spinal cord injury: an inpatient rehabilitation comparison. AB - OBJECTIVE: To compare demographics, injury characteristics, and functional outcomes of patients with neoplastic spinal cord compression with those with traumatic spinal cord injuries. DESIGN: A prospective 5-yr comparison was undertaken comparing 34 patients with neoplastic spinal cord compression with 159 patients with traumatic spinal cord injury. RESULTS: Patients with neoplastic spinal cord compression were significantly older, more often female, and unemployed than patients with traumatic spinal cord injury. Neoplastic spinal cord compression presented more often with paraplegia involving the thoracic spine, and injuries were more often incomplete compared with traumatic spinal cord injury. Patients with neoplastic spinal cord compression had a significantly shorter rehabilitation length of stay compared with those with traumatic spinal cord injury. The neoplastic group had significantly lower FIM change scores. Both groups had similar FIM efficiencies and discharge to home rates. CONCLUSIONS: Patients with neoplastic spinal cord compression have different demographic and injury characteristics but can achieve comparable rates of functional gains as their traumatic spinal cord injury counterparts. Although patients with traumatic injuries achieve greater functional improvement, patients with neoplasms have a shorter rehabilitation length of stay and comparable FIM efficiencies and home discharge rates. PMID- 10744188 TI - Deep vein thrombosis after lower limb amputation. AB - OBJECTIVE: The purpose of this study is to prospectively document the incidence of deep vein thrombosis (DVT) in the residual limb after a below-knee amputation. DESIGN: Eight of 13 male patients, admitted to the acute rehabilitation floor after a below-knee amputation, were included in the study. Patients already receiving anticoagulants were excluded. An investigator questioned the patient regarding the patient's risk factors for DVT and history of DVT and pulmonary embolus. A coagulation profile was obtained for all patients. A Doppler ultrasound was completed on the residual limb 2 wk after amputation, and if negative, it was repeated 2 wk later. Patients found to have a DVT were treated appropriately. The incidence of DVT was calculated by a point estimate, and a 95% confidence interval was calculated using simple large sample methods. RESULTS: Four of the eight patients had ultrasound evidence of DVT in the thigh. Two of the four patients had signs or symptoms of a DVT. There were a comparable number of risk factors for DVT in both groups. Laboratory values were not statistically significant in predicting the occurrence of DVT, probably because of the limited number of subjects. CONCLUSIONS: The present study supports the assumption that the diagnosis of lower limb DVT is frequently associated with lower limb amputation. However, a larger sample may be necessary to conclude that a routine screening ultrasound of the lower limbs is indicated after a below-knee amputation. PMID- 10744189 TI - Breathing out into water during subtotal immersion: a therapy for chronic pulmonary emphysema. AB - OBJECTIVE: To determine the importance of a breathing out into water exercise during subtotal immersion for emphysematous patients. DESIGN: Results of a respiratory function test and arterial blood gas analysis were examined to compare the programs of breathing exercise with and without the breathing out into water exercise. The breathing exercises in a pool filled with 38 degrees C water were performed 30 min/day, 5 days/wk, for 2 mo in 17 patients with stable chronic emphysema. Patients were randomly assigned to breathing exercise programs including subtotal water immersion with the nose and mouth either immersed (9 patients) or out of the water (8 patients) during exhalation. RESULTS: The ratio of %forced expired volume in 1 sec to forced vital capacity and %forced vital capacity at rest were significantly increased in the program with the breathing out into water exercise, but they did not change in the program without the breathing out into water exercise. A significant increase in peak flow was observed in the former program, although it did not change in the latter program. The maximal expiratory flow at 25% did not change in either program. Pao2 was significantly increased and Paco2 was significantly decreased in the former program, although a trend for a decrease in Paco2 was observed in the latter program. CONCLUSIONS: These results suggest that the breathing out into water exercise enhances the effects of a breathing exercise during immersion and is useful in rehabilitation for chronic pulmonary emphysema. PMID- 10744190 TI - Physical function and fear of falling after hip fracture rehabilitation in the elderly. AB - OBJECTIVE: The purpose of this study was to determine the relationship between physical function and fall-related self-efficacy in older patients with a hip fracture who are undergoing an intensive rehabilitation program. DESIGN: We used a prospective cohort study over 12 mo to determine the effect of a specialized hip fracture rehabilitation program in a geriatric hospital on physical function and fear of falling. Fifty-six patients were admitted consecutively from acute care. Physical function was assessed using the Functional Independence Measure, and fall-related self-efficacy was measured using two scales: the Falls-Efficacy scale and the Activities-Specific Balance Confidence scale. We also used the Vitality scale to measure quality of life. All measures, represented by change scores, were determined at the beginning and end of the patients' rehabilitation programs. RESULTS: Significant improvement in physical function and fall self efficacy was observed. The Vitality scale was also improved after rehabilitation. The Falls-Efficacy scale appeared to be more sensitive to change than the Activities-Specific Balance Confidence scale, whereas no correlation was found between changes in the fall-related self-efficacy measures and the Functional Independence Measure. CONCLUSIONS: These findings may represent a discrepancy between attention of the rehabilitation program on functional outcomes and less emphasis on confidence building behaviors. Restrictions in function from a fear of falling may negate any gains made through rehabilitation, and this could limit the long-term success of these programs and patient outcomes after hip fracture. PMID- 10744191 TI - Employment satisfaction of individuals with spinal cord injury. AB - OBJECTIVES: Community reentry after a spinal cord injury is a long-term integrative process, because individuals must adjust to an extraordinary new set of circumstances. In this study, quality-of-life and work satisfaction variables associated with employed or unemployed individuals with spinal cord injury were examined. METHODS: Quality-of-life and work satisfaction variables associated with 109 employed or unemployed individuals with spinal cord injury were examined by using an employment satisfaction survey. RESULTS: Most of those employed indicated that they would either change jobs or some employment variable such as duties, supervisor, or hours worked. Participants who were not working attributed unemployment to an array of factors including ongoing health problems, lack of transportation, and impact of and/or lost disability benefits. Both groups offered feedback related to information provided to them by medical professionals about return-to-work resources. CONCLUSIONS: The following three themes emerged from this analysis: (1) more comprehensive provider training about spinal cord injury is needed; (2) increased communication between consumer and family would be beneficial; and (3) an increase in the flexibility and availability of services would help to ensure access to essential care. PMID- 10744192 TI - Dysphagia in patients with brainstem stroke: incidence and outcome. AB - OBJECTIVE: This study was conducted to delineate the incidence and outcome of dysphagia among hospitalized patients who were referred for rehabilitation because of brainstem stroke. DESIGN: We retrospectively reviewed the medical records of 36 patients who were admitted because of brainstem stroke. Information on the patients' clinical features, feeding status, and the results of clinical and videofluoroscopic swallowing examinations were obtained through chart review. Follow-up interviews were conducted via telephone to learn the general medical condition and feeding status of the patients 7-43 mo after hospital discharge. RESULTS: A total of 81% of the patients had dysphagia at the time of initial clinical swallowing evaluation, which was performed 10-75 days after the onset of stroke. A total of 79% of the dysphagic individuals depended on tube feeding at the initial evaluation; 22% of all individuals could not resume oral intake at discharge. Statistical analyses revealed a significant association between poor outcome and disease involving the medulla, the presence of a wet voice during the initial swallowing test, and a delay or absence of the swallowing reflex. The incidence of aspiration pneumonia was 11%. There was a correlation between the detection of aspiration by modified barium meal videofluoroscopy and the development of aspiration pneumonia. Follow-up interviews showed that 88% of the 27 patients who were contacted had resumed full oral intake 4 mo after the onset of stroke. CONCLUSIONS: The incidence of dysphagia was relatively high in our study population. The long-term outcome was favorable. PMID- 10744194 TI - UDS(MR)SM: follow-up data on patients discharged in 1994-1996. Uniform Data System for Medical Rehabilitation. PMID- 10744193 TI - Amputations associated with arteriovenous access. AB - This study was performed to investigate the common characteristics of hemodialysis patients who need upper limb amputations. An index case was identified and involved questioning physicians and reviewing hospital and office records. Hemodialysis patients who have diabetes and leg amputations are at high risk for ischemic episodes that may lead to amputation of the arm, distal to the arteriovenous access site. PMID- 10744195 TI - Standards of care in MDA clinics. Muscular Dystrophy Association. PMID- 10744196 TI - The future of physical medicine and rehabilitation. PMID- 10744197 TI - Interfaces. AB - There are important similarities in molecular composition and structural organization of the interface between the vitreous and retina and that between the retina and retinal pigment epithelium. It is striking that the two most common causes of severe vision loss in the western world involve neovascularization at these interfaces; i.e., proliferative diabetic vitreo retinopathy at the vitreo-retinal interface and exudative age-related macular degeneration at the retina-retinal pigment epithelium interface. Improved knowledge of the physiology of these interfaces will lead to a better understanding of the effects of aging and diseases, especially those that involve neovascularization. Such advances will no doubt result in new treatment strategies offering more effective therapy, and, even more importantly, perhaps providing prevention from these devastating causes of blindness. PMID- 10744198 TI - Distance alternate-letter suppression test for objective assessment of sensorial status in intermittent exotropia. AB - PURPOSE: To investigate the value of distance alternate-letter suppression testing in the objective assessment of fusional control of intermittent exotropia. METHODS: A total of 71 consecutive patients with intermittent exotropia (study group) and 112 normal subjects (control group) underwent a series of measurements including distance alternate-letter suppression testing, Worth 4 Dot test at distance and near, and distance and near stereo acuity tests to see whether sensorial behavior differed in the study and control groups. The distance alternate-letter suppression test and distance stereo acuity tests were performed using the Mentor B-VAT II-SG Video Acuity Tester and Binocular Vision Testing System. The TNO test was used to measure near stereo acuity. RESULTS: Seventeen patients with intermittent exotropia (24%) presented suppression with the distance alternate-letter suppression test, but none of the normal subjects (p<0.001, chi2 test). Only, three patients (4%) showed suppression on the Worth 4 Dot test at distance, and all had fusion on the Worth 4 Dot test near. Patients with intermittent exotropia had significantly diminished distance stereo acuity compared to normal subjects (p<0.001). Normal subjects and patients had good near stereo acuity (p>0.05). Patients with intermittent exotropia who had fusion with the distance alternate-letter suppression test had significantly better distance stereo acuity than patients who had suppression (p<0.001). CONCLUSIONS: Distance alternate-letter suppression testing and distance stereo acuity tests may be useful as objective measures for assessing sensory loss in patients with intermittent exotropia. PMID- 10744199 TI - Tear lactoferrin levels in chronic meibomitis associated with acne rosacea. AB - PURPOSE: To determine tear lactoferrin levels in patients with chronic diffuse meibomitis associated with acne rosacea and to investigate their role in the development and perpetuation of some inflammatory ocular surface complications. METHODS: In this non-randomized, controlled trial performed in a tertiary care center, eight patients with biopsy-proven acne rosacea and blepharitis, and ten patients with seborrheic blepharitis not associated with acne rosacea formed the study groups and ten normal patients were used as controls. Schirmer I test results, tear break-up time and radial immunodiffusion (Lactoplate) tests on tear samples of the three groups were compared. RESULTS: Compared to controls, tear lactoferrin concentrations were lower in both the acne rosacea and seborrheic blepharitis groups. However, this decrease was significant in only the seborrheic blepharitis group (p = 0.026). One patient in the acne rosacea group, with the lowest measurement, developed bacterial keratitis. CONCLUSIONS: Though within the normal range, tear lactoferrin levels in patients with acne rosacea and seborrheic blepharitis were lower than controls. Low concentrations of lactoferrin may play a role in the ocular surface inflammatory components associated with these diseases. PMID- 10744200 TI - A comparison of topical mequitazine and disodium cromoglycate in allergic conjunctivitis induced by a specific conjunctival provocation test. AB - PURPOSE: Twenty-two people with a history of grass pollen allergic conjunctivitis were enrolled outside the pollen season in this study to compare the efficacy of 0.05% mequitazine eyedrops with topical 2% disodium cromoglycate. METHODS: This randomized, double-blind study compared right and left eyes. Subjects received 2% disodium cromoglycate eyedrops in one eye and mequitazine vehicle in the fellow eye 4 times daily for 4 days. For the last instillation, mequitazine vehicle was replaced by one drop of 0.05% mequitazine and 15 min later a conjunctival provocation test (CPT) with grass pollen was performed. RESULTS: The sum of the scores for hyperemia and itching, and total composite score were lower after 0.05% mequitazine than after 2% disodium cromoglycate (p < 0.0001 in each case). The allergen threshold dose which elicited a positive allergic response was higher in the mequitazine group (p < 0.001). Both treatments showed good tolerance upon instillation. No adverse events related to study treatment occurred. CONCLUSIONS: These results demonstrate the superior efficacy of a single drop of 0.05% mequitazine 15 min before CPT compared with 2% disodium cromoglycate 4 times daily for 4 days. PMID- 10744201 TI - Congenital lacrimal fistula. AB - PURPOSE: Congenital lacrimal fistula is a rare abnormality of the lacrimal system. The patients may be asymptomatic but treatment of those with symptoms such as epiphora and dacryocystitis is still controversial. METHODS: The clinical findings, methods of surgical treatment and results were evaluated in seven patients (four male, three female) with congenital lacrimal fistula. Three were asymptomatic, types of surgical treatment were fistula excision with dacryocystorhinostomy (DCR) (two cases), fistula excision with conjunctival DCR (one case) and closed fistula excision alone (one case). RESULTS: Three of the four patients who underwent surgery had relief of symptoms. The fistula recurred in the case that had undergone fistula excision alone. CONCLUSIONS: It is difficult to design controlled studies to detect which method is superior for the treatment of lacrimal fistula, considering the small number of cases in the literature. We recommend treating each case according to its own characteristics. PMID- 10744202 TI - A topical or oral carbonic anhydrase inhibitor to control ocular hypertension after cataract surgery. AB - PURPOSE: To compare the effects of oral acetozolamide and topical 2% dorzolamide to prevent ocular hypertension after cataract surgery. METHODS: This prospective, randomized study comprised 62 consecutive patients who had extracapsular cataract extraction and posterior chamber intraocular lens implantation. Patients received either oral acetozolamide (Diazomide) 250 mg three times daily or topical dorzolamide 2% (Trusopt) three times daily, for three days. Intraocular pressures (IOP) were measured by Goldmann applanation tonometry preoperatively and 16, 40, 64 hours postoperatively. RESULTS: IOP in the dorzolamide group peaked at 16 hours and had returned to preoperative values by 40 hours. In the acetozolamide group mean IOP was significantly higher than preoperative values at 16, 40 and 64 hours (p<0.05). At all three postoperative measurement times, mean IOP was significantly higher in the acetozolamide group (p<0.05). CONCLUSIONS: Topical dorzolamide 2% offers better IOP control than oral acetozolamide to prevent ocular hypertension after cataract surgery. PMID- 10744204 TI - A randomized, comparative study of fluorometholone 0.2% and fluorometholone 0.1% acetate after photorefractive keratectomy. AB - PURPOSE: To compare the efficacy and tolerance of fluorometholone 0.1% acetate and fluorometholone 0.2% eyedrops in the postoperative management of photorefractive keratectomy (PRK). METHODS: A randomised single-blind comparative study was performed on two groups of 30 patients who underwent myopic PRK. The first group was given fluorometholone 0.1% acetate and the second fluorometholone 0.2%. Uncorrected and best corrected visual acuity, haze, IOP and local tolerance were evaluated. Statistical analysis was done using parametric and non-parametric tests. RESULTS: Visual acuity did not differ in the two groups; both were homogeneous as far as refractive error and haze were concerned. Three patients (10%) treated with fluorometholone 0.2% and two patients (6.6%) with fluorometholone 0.1% acetate developed ocular hypertension, but this was not statistically significant. CONCLUSIONS: Fluorometholone 0.1% acetate was effective on inflammation after PRK, with the same efficacy as fluorometholone 0.2%. PMID- 10744203 TI - Prevention of corneal keratocyte apoptosis after argon fluoride excimer laser irradiation with the free radical scavenger ubiquinone Q10. AB - PURPOSE: To assess in vitro the potential of the free radical scavenger ubiquinone Q10 in preventing keratocyte apoptosis after argon fluoride (ArF) excimer laser irradiation. METHODS: Cultured rabbit keratocytes were irradiated at very low single-pulse laser fluences. The cumulative effects generated by three total fluence doses between 12 and 45 mJ/cm2, representative of single pulse subablative doses during photorefractive keratectomy (PRK) in humans, were evaluated. We employed the following parameters to compare pretreated (10 microM ubiquinone Q10) and untreated samples: 1) number and morphology of living cells by Trypan blue test and ultramicroscopy, respectively; 2) level of free-radical formation assessed by malonaldehyde quantitation; 3) cellular energy level evaluated by ATP assay. RESULTS: Excimer laser irradiation kills cultured keratocytes by inducing apoptosis. The effect increases with the cumulative fluence dose. In the samples pretreated with ubiquinone Q10 there were significantly fewer cumulative apoptotic events than in the untreated ones. Quantitative analysis of malonaldehyde cellular levels suggested this protective action of ubiquinone Q10 was connected with its ability to scavenge laser generated free radicals. ATP assay also confirmed that it raised cellular energy levels. CONCLUSIONS: The treatment of corneal keratocytes with relatively low concentrations of ubiquinone Q10 can prevent apoptosis after ArF excimer laser irradiation. If these findings are confirmed on human keratocytes this treatment could be usefully exploited in the PRK surgical procedure. That might lead to a reduction in the occurrence of haze and curvature regression triggered by programmed cell death. PMID- 10744205 TI - The pharmacokinetics and effects of diltiazem in rabbits. AB - PURPOSE: To investigate the effect of diltiazem on wound healing after the creation of conjunctival flaps in rabbit eyes. Also, to investigate the pharmacokinetics of diltiazem in rabbits after subconjunctival and topical administration. METHODS: For the histopathological study, a limbal-based flap was prepared and diltiazem was injected subconjunctivally for five days after the surgery. The rabbits were euthanised 20 days after surgery. The effectiveness of diltiazem on wound healing was evaluated by histopathological examination and measurement of the thickness of subconjunctival fibrous tissue. For the pharmacokinetic study, diltiazem was applied topically or injected subconjunctivally. Aqueous paracenteses were performed 0.5, 1, 2, 4 hours thereafter. RESULTS: The histopathological study found no difference in thickness of the subconjunctival fibrous tissue in control and diltiazem-treated eyes. No significant toxicity was observed in eyes treated with diltiazem. The peak aqueous concentration was 3.8 +/- 0.4 microg/ml after topical application and 15.3 +/- 1.1 microg/ml after subconjunctival injection. The peak aqueous concentration was achieved 1/2 hours after administration in both cases. CONCLUSIONS: Diltiazem did not appear to affect wound healing at the dose tested. Topical and subconjunctival diltiazem successfully penetrated the aqueous humor of rabbit eyes. PMID- 10744206 TI - Experimental retinopathy of prematurity: angiostatic inhibition by nimodipine, ginkgo-biloba, and dipyridamole, and response to different growth factors. AB - PURPOSE: To investigate whether commonly used vasodilating drugs ameliorate angiogenesis in experimental retinopathy of prematurity (ROP), and to study the response of these drugs to different growth factors. METHODS: We used a rat and mouse model of oxygen-induced ischemic retinopathy. Animals were treated with nimodipine, gingko-biloba and dipyridamole intraperitoneally starting the day before exposure to room air (day 1). Controls were injected with vehicle solution only. Eyes were processed histopathologically with serial sections and neovascularization was measured by counting the nuclei within the retinal internal limiting membrane, by a masked observer. Retinal and vitreous tissues were assayed by ELISA for VEGF, PDGF and TGFbeta2. RESULTS: Nimodipine significantly inhibited the growth of new vessels in rats. The number of nuclei was 310 +/- 69 in the control group (n:14) and 121 +/- 53 in the treated ones (n:14), (p<0.0005). Similar results were found with ginkgo-biloba extracts: 344 +/- 53 (n:15) in controls, and 136 +/- 29 (n:11) in treated ones (p<0.0005), and with dipyridamole: 303 +/- 69 (n:13) in controls, and 131 +/- 48.5 in treated rats (p<0.0005). Results were similar in mice. 186 +/- 45 (n:7) nuclei counted in controls against 90 +/- 25 (n:6) for dipyridamole treated (p<0.0005); and 81 +/- 21 for ginkgo-biloba treated animals (p<0.0005). A gradual, very significant increase in VEGF values in response to relative hypoxia (room air) contrasted with the significant inhibition noted both with ginkgo-biloba extracts and dipyridamole. TGFbeta2 and PDGF both showed a gradual increase in relative hypoxia at days 2 and 4 of room air (p<0.0005). Treated animals showed marked inhibition of the three growth factors. CONCLUSIONS: All three drugs markedly inhibited angiogenesis in experimental ROP. Growth factors were elevated in hypoxic conditions. Treated animals showed significant decreases of PDGF, VEGF, and TGFbeta2 in retinal and vitreous tissues. PMID- 10744207 TI - Long-term prognosis after removal of silicone oil. AB - PURPOSE: To investigate surgical and functional results six or more months after silicone oil (SiO) removal in patients undergoing pars plana vitrectomy (PPV) and tamponade for various reasons. METHODS: Retrospective chart review. Inclusion criteria were recurrent retinal detachment with PVR grade C (R-RD), primary PVR grade C longer than 9 hours, recurrent vitreous hemorrhage in PDR (PDR-RVH) with traction RD, giant retinal tears (GRT) with PVR grade C and total RD with vitreous hemorrhage and hypotony in penetrating traumas (PT). Indications for removal of SiO included attached retina and intra-ocular pressure (IOP) more than 10 mmHg after 60 days or IOP more than 30 mmHg despite medication. RESULTS: Of the 212 patients undergoing PPV and SiO tamponade between 1994-1997, 91 met the inclusion criteria, 8 had incomplete charts so 83 eyes were included in the study. The mean interval between PPV and SiO removal was 163.1 +/- 111.0 days and follow-up was 351.5 +/- 148.6 days. At the time of SiO removal, 30.6% of phakic eyes had cataract, 14.4% keratopathy and 8.4% IOP more than 30 mmHg. At the last visit after SiO removal, 43.5% had cataract, 12.0 keratopathy, 6.0% IOP > 30 mmHg and 3.6% IOP < 5 mmHg. After SiO removal, 6.0% eyes developed R-RD. There was no significant difference in SiO duration for patients with and without R-RD. VA was more than 5/200 in 16.8% of eyes preoperatively, 79.5% at the time of SiO removal (p<0.05) and 78.3% at the last visit (n.s.) and better than 20/400 in respectively 2.4%, 51.8% (p<0.05) and 53% (n.s.). There was a tendency for VA to improve after SiO removal (p = 0.011). CONCLUSIONS: SiO is an effective tamponade for complex RD, although its possible benefits must always be weighted carefully against the complications and the need for further intervention. The present series compares favorably with the current literature in terms of complication rates. The optimal timing of SiO removal and precise screening and decision making guidelines before removal are still the main issues and need careful consideration. PMID- 10744208 TI - Comparison of analgesia and akinesia after retrobulbar injections at different speeds. AB - PURPOSE: To assess how the speed of injection of local anesthetic solutions affected pain of injection, bulbar akinesia and analgesia with retrobulbar anesthesia (RBA). METHODS: 70 patients undergoing RBA for cataract surgery were enrolled in a prospective masked trial. They were allocated randomly to receive 5 ml anesthetic solution injected either within 20 seconds (group A) or within 60 seconds (group B). Additionally, akinesia of the orbicularis muscle was created according to O'Brien's technique. The pain of injection was registered on an ordinal analogue scale immediately before and after RBA. The following data were collected before and 20 minutes after retrobulbar injection: eye motility (Kestenbaum test), and corneal sensitivity (0: no sensitivity; 1: sensitivity remaining). Data were also collected on age, sex, and bulbar length, and any side effects of the intervention. RESULTS: Injection pain did not differ in the two groups. After RBA horizontal and vertical eye motility was slightly lower in group A than group B. Persistent motility was found in 18 patients in group A and 16 in group B. Median horizontal and vertical motility was 0 mm in both groups. Four patients in group A and five in group B had corneal sensitivity persisting after RBA. CONCLUSIONS: This comparison of different injection velocities brought to light no significant differences regarding bulbar analgesia and akinesia after RBA. PMID- 10744209 TI - Effect of xyloglucan (tamarind seed polysaccharide) on conjunctival cell adhesion to laminin and on corneal epithelium wound healing. AB - PURPOSE: To explore the role of a natural polysaccharide extracted from tamarind seed (xyloglucan, or tamarind seed polysaccharide, TSP) on the integrin-substrate recognition system and on repair of corneal wounds. METHODS: a) Cultured human conjunctival cells were labeled by addition of a tritiated amino acid mixture. Their adhesion to laminin-coated culture wells in the absence or presence of TSP was checked by radioactivity count. b) The corneal epithelium of albino rabbits was damaged by applying a paper disc soaked with n-heptanol. The eyes were then treated with TSP, with a hyaluronate reference formulation and with normal saline solution (controls). The diameter of corneal wounds was measured daily, after fluorescein staining. RESULTS: Compared to hyaluronate, TSP slightly but significantly increased the wound healing rate. TSP 1.0% exerted a positive influence on cell adhesion to laminin, up to a certain laminin concentration. CONCLUSIONS: The ability of the polysaccharide to promote corneal wound healing might depend on its influence on the integrin recognition system. PMID- 10744210 TI - Visual acuity and color vision deficiency in amblyopia. AB - PURPOSE: To investigate color vision and its relation with the type of amblyopia and visual acuity of amblyopic eyes. METHODS: In this prospective study, 67 amblyopic eyes of 64 patients, aged from 4 to 13 years (mean 6.8 +/- 2.1) and 26 eyes of 13 control subjects aged from 5 to 13 years (mean 7.3 +/- 1.6) were examined with the Farnsworth-Munsell 100 Hue Test (FM-100). Amblyopic eyes were grouped as strabismic (21 eyes) and anisometropic (46 eyes). Each group was subdivided according to their visual acuity, as less than 5/10 and 6/10 or better. The total errors, blue-yellow (B-Y) and red-green (R-G) partial error scores were obtained for each group. One-way ANOVA was used to assess differences between groups. RESULTS: The error scores of all axes were lower in the control group than the amblyopic groups (p<0.001), but the differences within amblyopic groups were not significant (p>0.05). CONCLUSIONS: Deficient color vision in the amblyopic eyes was not related to the visual acuity and type of amblyopia. PMID- 10744211 TI - Anterior segment ischemia and retinal detachment after vertical rectus muscle surgery. AB - PURPOSE: The authors describe the clinical course of a woman who developed two complications following vertical strabismus repair: anterior segment ischemia (ASI) and retinal detachment. METHODS: A 62 year-old woman is described. She presented with new onset proptosis and left hypertropia with significant diplopia in all fields of gaze. This presentation, her 15 year history of thyroid disease, and preoperative computed tomography (CT) of the orbits were consistent with Graves' ophthalmopathy. Vertical strabismus repair was carried out by recessing the left superior rectus muscle and resecting the left inferior rectus muscle. RESULTS: The diplopia was eliminated. The patient developed significant postoperative ASI and iatrogenic rhegmatogenous retinal detachment in the left eye due to unsuspected globe perforation. She was treated with systemic corticosteroids and radial scleral buckling. CONCLUSIONS: Severe ASI following strabismus surgery is a well recognized complication, with age, thyroid ophthalmopathy, and manipulation of the vertical rectus muscles as risk factors. The retinal detachment soon after strabismus surgery was difficult to detect, possibly due to diminished visualization of the posterior segment as a result of ASI. PMID- 10744212 TI - Orbital Kaposi's sarcoma in acquired immunodeficiency syndrome. AB - A 28-year-old white male with AIDS-C3 staging, presented with an extensive hemorrhagic dark mass localized in the left orbit. No other ophthalmic findings were disclosed. Ultrasonography and computed axial tomographic scans showed orbital involvement. Orbital Kaposi's sarcoma is a rare finding and only a few cases have been reported. Systemic examination revealed other lesions suggestive of disseminated mucocutaneous Kaposi's sarcoma, oral candidiasis, membranous esophagitis and granulomatous hepatitis. Eyelid incisional biopsy disclosed Kaposi's sarcoma. Despite intensive chemotherapy progression was aggressive with a fatal outcome. PMID- 10744213 TI - The intercostal to phrenic nerve transfer: an effective means of reanimating the diaphragm in patients with high cervical spine injury. AB - Nerve transfers have been well described for the treatment of congenital and traumatic injuries in the brachial plexus and extremities. This series is the first to describe nerve transfers to reanimate the diaphragm in patients confined to long-term positive pressure ventilation because of high cervical spine injury. Patients who have sustained injury to the spinal cord at the C3 to C5 level suffer axonal loss in the phrenic nerve. They can neither propagate a nerve stimulus nor respond to implanted diaphragmatic pacing devices (electrophrenic respiration). Ten nerve transfers were performed in six patients who met these conditions. The procedures used end-to-end anastomoses from the fourth intercostal to the phrenic nerve approximately 5 cm above the diaphragm. A phrenic nerve pacemaker was implanted as part of the procedure and was placed distal to the anastomosis. Each week, the pacemaker was activated to test for diaphragmatic response. Once diaphragm movement was documented, diaphragmatic pacing was instituted. Eight of the 10 transfers have had more than 3 months to allow for axonal regeneration. Of these, all eight achieved successful diaphragmatic pacing (100 percent). The average interval from surgery to diaphragm response to electrical stimulation was 9 months. All patients were able to tolerate diaphragmatic pacing as an alternative to positive pressure ventilation, as judged by end tidal CO2 values, tidal volumes, and patient comfort. Intercostal to phrenic nerve transfer with diaphragmatic pacing is a viable means of liberating patients with high cervical spine injury from long term mechanical ventilation. PMID- 10744214 TI - Interdental distraction osteogenesis and rapid orthodontic tooth movement: a novel approach to approximate a wide alveolar cleft or bony defect. AB - The closure of a wide alveolar cleft and fistula in cleft patients and the reconstruction of a maxillary dentoalveolar defect in traumatic patients are challenging for both orthodontists and surgeons. This is due to the difficulty in achieving complete closure by using local attached gingiva and the great volume of bone required for the graft. In this article, the authors propose using interdental distraction osteogenesis to create a segment of new alveolar bone and attached gingiva for the complete approximation of a wide alveolar cleft/fistula and the reconstruction of a maxillary dentoalveolar defect. They performed this procedure on one patient with a traumatic maxillary dentoalveolar defect and 10 patients with unilateral or bilateral cleft lips and palates who had varied dentoalveolar clefts/fistulas. Interdental and maxillary osteotomies were performed on one side of the dental arch by the cleft or defect. After a latency period of 3 days, the osteotomized distal segment of the dental arch was then distracted and transported toward the cleft or defect by using a toothborne intraoral distraction device. The alveoli and gingivae on both ends of the cleft or defect were approximated after distraction osteogenesis. The need for extensive alveolar bone grafting was eliminated. A segment of new edentulous alveolus and attached gingiva was created interdentally at a site distant to the cleft or defect. In the cleft patients, teeth were moved orthodontically into the regenerate (newly formed alveolar bone) dental crowding 1 week after distraction. The orthodontic tooth movement was rapidly completed in 3 months, and the edentulous space was eliminated. Interdental distraction osteogenesis minimizes an alveolar cleft/fistula and helps reconstruct a maxillary dentoalveolar defect by approximating the native alveoli and gingivae; it also creates new alveolar bone and gingiva for rapid orthodontic tooth movement. PMID- 10744215 TI - Three-dimensional nasolabial displacement during movement in repaired cleft lip and palate patients. AB - The objective of this study was two-fold: (1) to explore the suitability of a novel modified Procrustes fit method to adjust data for head motion during instructed facial movements, and (2) to compare the adjusted data among repaired unilateral (n = 4) and bilateral (n = 5) cleft lip and palate patients and noncleft control subjects (n = 50). Using a video-based tracking system, three dimensional displacement of 14 well-defined nasolabial landmarks was measured during four set facial animations without controlling for head motion. The modified Procrustes fit method eliminated the contributions of head motion by matching the most stable landmarks of each video-recorded frame of the face during function to frames at rest. Its effectiveness was found to approximate that of a previous method (i.e., use of a maxillary occlusal splint to which stable dentition-based markers were attached). Data from both the unilateral and bilateral cleft lip and palate patients fell outside the normal range of maximum displacements and of asymmetry, and individual patients demonstrated greater right-versus-left asymmetry in maximum displacement than did individual noncleft subjects. It is concluded that the modified Procrustes fit method is fast, is easy to apply, and allows subjects to move the head naturally without the inconvenience of a splint while facial movement data are being collected. Results obtained using this method support the view that facial movements in cleft patients may be severely hampered and that assessment of facial animation should be strongly considered when contemplating surgical lip revisions. PMID- 10744216 TI - A classification schema for the vomeronasal organ in humans. AB - The vomeronasal organ is a chemoreceptive structure located at the base of the nasal septum with direct axonal connections to the accessory olfactory bulb in many terrestrial vertebrates. Pheromones presumably bind to the vomeronasal organ and exert behavioral or physiologic responses, thereby allowing chemical communication between animals of the same species. The presence and function of the vomeronasal organ in humans is debated. A phenotypic classification schema for the human vomeronasal organ is described and applied to 253 human subjects who underwent nasal examination. Of these subjects, only 6 percent possessed a vomeronasal organ with 64 percent unilateral and 36 percent bilateral in appearance. No difference existed in gender, age, or race between those subjects with or without a vomeronasal organ. There is no evidence supporting involutional senescence of this structure. Future investigations should use this phenotypic schema for the vomeronasal organ to allow accurate comparisons of study populations. PMID- 10744217 TI - Lengthening temporalis myoplasty and lip reanimation. AB - The authors report a new surgical technique of myoplasty of the temporalis muscle applied to the treatment of permanent peripheral facial paralysis. It consists of a muscle lengthening, using the totality of the temporalis muscle and the transfer of its tendon attached to the coronoid process directly to the lips, therefore modifying the temporal fixed point and respecting the deep temporal pedicles. Ten cadaver dissections were performed for a better understanding of the muscle fiber redistribution during surgery to measure the distance between coronoid process and lip commissure and to measure the width of the temporal tendon. The results in 10 patients have been very encouraging. This technique was performed on ten cases of permanent facial paralysis of various etiologies, by the same surgeon, with a 4-year follow-up. PMID- 10744218 TI - Long-term restoration of masticatory function with fixed mandibular implants in cases of oral cancer. AB - The results of the present study show that the fixed mandibular implant system enables patients who have operations to treat oral cancer to use a stabilized denture continuously over an extended period of time. PMID- 10744219 TI - Osteocutaneous flap prefabrication based on the principle of vascular induction: an experimental and clinical study. AB - Conventional osteomyocutaneous flaps do not always meet the requirements of a composite defect. A prefabricated composite flap may then be indicated to custom create the flap as dictated by the complex geometry of the defect. The usual method to prefabricate an osteocutaneous flap is to harvest a nonvascularized bone graft and place it into a vascular territory of a soft tissue, such as skin, muscle, or omentum, before its transfer. The basic problem with this method is that the bone graft repair is dependent on the vascular carrier; the bone needs to be revascularized and regenerate. The bone graft may not be adequately perfused at all, even long after the transfer of the prefabricated flap. This study was designed to prefabricate an osteocutaneous flap where simply the bone nourishes the soft tissues, in contrast to the conventional technique in which the soft tissue supplies a bone graft. This technique is based on the principle of vascular induction, where a pedicled bone flap acts as the vascular carrier to neovascularize a skin segment before its transfer. Using a total of 40 New Zealand White rabbits, two groups were constructed as the experimental and control groups. In the experimental group, a pedicled scapular bone flap was induced to neovascularize the dorsal trunk skin by anchoring the bone flap to the partially elevated skin flap with sutures in the first stage. After a period of 4 weeks, the prefabricated composite flaps (n = 25) were harvested as island flaps pedicled on the axillary vessels. In the control group, nonvascularized scapular bone graft was implanted under the dorsal trunk skin with sutures; after 4 weeks, island composite flaps (n = 15) were harvested pedicled on the cutaneous branch of the thoracodorsal vessels. In both groups, viability of the bony and cutaneous components was evaluated by means of direct observation, bone scintigraphy, measurement of bone metabolic activity, microangiography, dye injection study, and histology. Results demonstrated that by direct observation on day 7, the skin island of all of the flaps in the experimental group was totally viable, like the standard axial-pattern flap in the control group. Bone scintigraphy revealed a normal to increased pattern of radionuclide uptake in the experimental group, whereas the bone graft in the control group showed a decreased to normal pattern of radioactivity uptake. The biodistribution studies revealed that the mean radionuclide uptake (percent injected dose of 99mTc methylene diphosphonate/gram tissue) was greater for the experimental group (0.49+/-0.17) than for the control group (0.29+/-0.15). The difference was statistically significant (p<0.01). By microangiography, the cutaneous component of the prefabricated flap of the experimental group was observed to be diffusely neovascularized. Histology demonstrated that although the bone was highly vascular and cellular in the experimental group, examination of the bone grafts in the control group revealed necrotic marrow, empty lacunae, and necrotic cellular debris. Circulation to the bone in the experimental group was also demonstrated by India ink injection studies, which revealed staining within the blood vessels in the bone marrow. Based on this experimental study, a clinical technique was developed in which a pedicled split-inner cortex iliac crest bone flap is elevated and implanted under the medial groin skin in the first stage. After a neovascularization period of 4 weeks, prefabricated composite flap is harvested based on the deep circumflex iliac vessels and transferred to the defect. Using this clinical technique, two cases are presented in which the composite bone and soft-tissue defects were reconstructed with the prefabricated iliac osteomyocutaneous flap. This technique offers the following advantages over the traditional method of osteocutaneous flap prefabrication. Rich vascularity of the bony component of the flap is preserved following transfer (i.e. (ABSTRACT PMID- 10744220 TI - Carpal tunnel syndrome in women undergoing reduction mammaplasty. AB - Women with mammary hypertrophy who present for reduction mammaplasty have several well-described musculoskeletal complaints, but a high prevalence of carpal tunnel syndrome has not been reported. We identified 151 patients from a plastic surgery practice who underwent reduction mammaplasty from 1994 to 1996. To this group we added a convenience sample of 64 women volunteers with relatively smaller breasts (brassiere cup size B or smaller). We questioned the entire group about specific symptoms and examined them using standard provocative tests. Carpal tunnel syndrome was defined as the coexistence of symptoms and at least two physical examination findings. We examined its association with breast size, age, race, and body mass index. Stepwise logistic regression was used to determine which physical characteristics were predictive of the condition. Carpal tunnel syndrome was found in 30 patients (19.9 percent) (95 percent confidence interval, 13.8 to 27.1) and in none of the women in the convenience sample. Breast size and, to a lesser degree, body mass index were found to be highly significant predictors of carpal tunnel syndrome. After controlling for breast size, race was also significant. Breast size displayed an independent risk ratio of 6.67 when comparing the upper quartile of size to the lower quartiles. There is a markedly higher prevalence of carpal tunnel syndrome in women who present for reduction mammaplasty than in those with smaller breasts. Breast size was a significant predictor of carpal tunnel syndrome. PMID- 10744221 TI - Clinical results of TRAM flap delay by selective embolization of the deep inferior epigastric arteries. AB - Preoperative selective embolization of the deep inferior epigastric arteries constitutes a new technique in TRAM flap delay. Whereas surgical ligation of these vessels has proved to be an effective delay procedure in experimental and clinical settings, it requires an additional operative step under general anesthesia. Despite the introduction of the free TRAM leading to improved flap perfusion, this microsurgical technique is not always available because of the requirements of specialized equipment and staff, longer operating hours, and subsequently higher expenses. The search for a minimally invasive, easy, and inexpensive technique to improve perfusion of the pedicled TRAM flap led us to selective embolization of the deep inferior epigastric arteries by an angiographic procedure. After 4 years of experience with this technique, we now present the first clinical results. Breast reconstruction by a delayed pedicled TRAM flap was performed in 40 patients with a mean age of 48.4 years (range, 31 to 66 years). The mean interval between embolization and surgery was 3.6 months. Postoperative data concerning flap survival and complications were available for all patients. Embolization of the deep inferior epigastric arteries was performed bilaterally in 35 patients (87.5 percent) and unilaterally in 5 patients (12.5 percent). Radiotherapy had been applied in 21 patients (52.5 percent) before surgery. Postoperative flap complications consisted of partial necrosis in three (7.5 percent), fat necrosis in one (2.5 percent), impaired wound healing in five (12.5 percent), and postoperative bleeding in two patients (5 percent). Abdominal wound healing complications occurred in six patients (15 percent), abdominal wall weakness in eight (20 percent), and hernia formation in four (10 percent). Surgical corrections were performed at the breast (TRAM flap) in 22 patients (55 percent) and at the abdomen (donor site) in 9 (22.5 percent). Preoperative selective embolization of the deep inferior epigastric arteries constitutes an alternative delay procedure for the pedicled TRAM flap. It is superior to the conventional procedure without delay, offers several advantages compared with surgical ligation of these vessels, and represents an alternative to the free TRAM flap in selected cases. PMID- 10744222 TI - Pfannenstiel incision as an alternative approach for harvesting the rectus abdominis muscle for free-tissue transfer. AB - The rectus abdominis muscle has been one of the most commonly used donor tissues for free-flap reconstruction of defects in the extremities and in selected head and neck patients. The rectus abdominis has provided adequate soft-tissue mass with predictable anatomy and results for the majority of its applications in free flap reconstruction. Harvesting of this muscle has typically been done through a paramedian or midline incision, which has left a lengthy notable scar on a patient's abdomen. To avoid the late aesthetic deformity associated with this typical approach for the rectus abdominis, we began harvesting the muscle through a Pfannenstiel incision. Patients were initially selected based on young age and limited soft-tissue requirements. With additional experience, this technique was extended to include all healthy patients regardless of age. Also, soft-tissue limitations no longer became an issue, as we learned the entire rectus abdominis muscle could be harvested from this approach. An extended Pfannenstiel incision was made from the ipsilateral anterior superior iliac spine to the lateral border of the contralateral rectus abdominis. A superiorly based flap was raised to expose the full length of the anterior rectus sheath from pubis to costal margin. In our earlier patients, a periumbilical incision was made for presumed easier access, but we discovered this was an unnecessary maneuver. With the anterior sheath fully exposed, the muscle was harvested and the sheath repaired in a routine manner. The elevated abdominal flap was returned to its anatomic position and closed over a suction drain. Since 1993, 10 patients have undergone a Pfannenstiel approach for harvesting of the rectus abdominis muscle. The mean age was 16. The areas requiring coverage included a traumatic elbow defect, seven traumatic lower extremity defects, one lower extremity sarcoma defect, and one lower extremity septic joint defect. Mean follow-up for these patients was 12 months. There were no flap failures. One patient developed an arterial thrombosis on postoperative day 5 and was treated with successful revision. There were no abdominal wall complications. Cosmesis was judged as good in all patients. We would recommend avoiding this approach in heavy or moderate smokers, diabetic patients, and patients with significant obesity. The Pfannenstiel approach to the rectus abdominis muscle has allowed for complete harvest of the muscle, improved aesthetic results compared with alternative techniques, and avoidance of donor site morbidityin healthy patients. PMID- 10744223 TI - The extended Segmuller flap. AB - A modification of the homodigital lateral V-Y advancement flaps described by Segmuller in 1976 for the treatment of fingertip injuries is described. The extended flap used in this series makes this flap more versatile in the treatment of injuries with considerable soft-tissue loss from the volar aspect of the fingertip. The Segmuller flap technique, its applications, and the results of 100 flaps are reported and discussed. PMID- 10744224 TI - Extensor digitorum brevis free flap: anatomic study and further clinical applications. AB - The extensor digitorum brevis muscle flap is reliable, safe, and can be used either as a pedicle or as a free flap with minimal donor site morbidity. To increase the existing knowledge of this flap and to establish further anatomic basis for the design and elevation of the extensor digitorum brevis flap, 26 specimens from 13 fresh cadavers were dissected under 3.5x loupes. The lateral tarsal artery was found to be the main blood supply to the muscle. It has an average diameter of 1.83+/-0.35 mm and a length of 1.89+/-0.69 cm. The dorsalis pedis artery has, at the level of the lateral tarsal artery takeoff, a diameter of 3.25+/-0.62 mm. From this point to the origin of the deep plantar branch, the dorsalis pedis artery has minimal branching, and the surgeon has available an artery homogeneous in diameter that is 6.77+/-0.99 cm in length. Related neurovascular structures (anterior tibial artery and the venae comitantes, dorsalis pedis and first dorsal metatarsal artery, and deep peroneal nerve) were also studied. A safe and reliable harvesting technique and the "T interposed extensor digitorum brevis" technique for sparing the anterior tibial artery are presented, as are clinical case examples on the use of this flap as a flow through, extensor digitorum brevis-vascularized nerve graft, a combined extensor digitorum brevis-deep peroneal nerve graft, and a bilobed extensor digitorum brevis-dorsalis pedis fasciosubcutaneous free flap. PMID- 10744225 TI - The venous communication between the gastrocnemius muscle heads. AB - This investigation establishes the venous communication between the gastrocnemius muscle heads. Fourteen fresh (with the exception of one) cadaveric specimens were examined. Anastomotic veins were dissected along the raphe after perfusion of each muscle with 0.1 % methylene blue solution. A distally based musculocutaneous perforating vein of the medial head was the site of perfusion in 13 specimens, whereas one specimen received injection through the accompanying vein of the medial sural cutaneous nerve. Additional perfusion of dye from different sites was required in two specimens. Communicating veins were dissected in all 14 muscles. Direct anastomotic veins were detected in eight specimens; whereas in six the accompanying vein of the medial sural cutaneous nerve that had been included proved to be the intermediate pathway of venous communication between the muscle heads. Obstructive valves were encountered in most cases; nevertheless anastomotic veins were always recognized. The role of valves and the clinical implications are discussed. PMID- 10744226 TI - A new surgical procedure for phallic reconstruction: Istanbul flap. AB - A new surgical procedure is described for phallic reconstruction, which still remains a great challenge in reconstructive surgery. In this procedure, an osteocutaneous radial forearm flap is combined with a radial recurrent fasciocutaneous flap from the anterolateral aspect of the upper arm. While keeping a fasciovascular connection between them, both flaps are elevated as a combined free flap based on the radial artery. The forearm skin island is used solely to construct the outer skin cover of the phallic shaft, and the neourethra is created by using the radial recurrent flap. Over the past 4 years, this surgical procedure, termed the Istanbul flap, has been used successfully for complete phallic reconstruction in five patients. Although more clinical experience with this new technique is needed, it seems to be a useful alternative in phallic reconstruction. It remarkably minimizes the donor-site scar without sacrificing the length of the neopenis. In addition, this technique reduces the risk of a hairy urethra. PMID- 10744227 TI - Effects of pulsed magnetic energy on a microsurgically transferred vessel. AB - This article reports the findings of a study that attempted to elucidate whether pulsed magnetic energy stimulates neovascularization in vivo, using a microsurgically created arterial loop model in a prospective randomized trial of 108 rats (n = 12/group). Pulsed magnetic energies of 0.1 and 2.0 gauss were applied immediately postoperatively and for 4, 8, and 12 weeks, respectively, with a statistically significant increase in neovascularization among the treated animals compared with control rats. The study provides a starting point for future study and evaluation of the stimulation of angiogenesis with the use of pulsed magnetic energy and suggests a possible use of this modality to increase the quality of revascularized tissue. PMID- 10744228 TI - Effect of hyperbaric oxygen on neutrophil CD18 expression. AB - Previous work has shown that treatment with hyperbaric oxygen significantly reduces neutrophil adhesion to postcapillary venules in a rat microcirculation model of ischemia-reperfusion injury. The mechanism of this process is unknown. The purpose of this study was to evaluate the effect of hyperbaric oxygen on neutrophil CD18 adhesion sites by flow cytometry in an animal model of ischemia reperfusion injury. The gracilis muscle flap was raised in three groups of male Wistar rats: (1) a sham group (n = 25), (2) a group that underwent 4 hours of ischemia (n = 25), and (3) a group that underwent 4 hours of ischemia and received hyperbaric oxygen (100% 02, 2.5 atmospheres absolute, during the last 90 minutes of ischemia) (n = 25). Samples from one subgroup of each group (n = 5) were divided into two portions, and one portion was stimulated with phorbol-12 myristate 13-acetate (PMA). Samples from another subgroup of each group (n = 5) were treated in the same manner, and a flap flush was added at the end of reperfusion to determine the number of CD18 adhesion sites on adherent neutrophils remaining in the flap. Venous blood was drawn 10 minutes after the operation, at 5 minutes of reperfusion, and at 90 minutes of reperfusion. Hematocrit and white blood cell count were measured. Samples were analyzed by flow cytometry, and the antibody binding capacity was assessed using microbead standards and linear regression (antibody binding capacity was expressed as the mean number of sites per cell +/- SEM). Microbeads were used to align the flow cytometer and to provide external and internal standards. Ischemia-reperfusion injury increased the expression of CD18 by neutrophils (p < 0.05). Expression of CD18 was not decreased by hyperbaric oxygen treatment. Stimulation with PMA increased the expression of CD18 in all groups (p < 0.01). These results suggest that ischemia-reperfusion injury does increase the expression of CD18 by neutrophils. Hyperbaric oxygen, as administered in this experiment, did not prevent the increase in CD18 expression. PMID- 10744229 TI - Biomolecular mechanisms of calvarial bone induction: immature versus mature dura mater. AB - The ability of newborns and immature animals to reossify calvarial defects has been well described. This capacity is generally lost in children greater than 2 years of age and in mature animals. The dura mater has been implicated as a regulator of calvarial reossification. To date, however, few studies have attempted to identify biomolecular differences in the dura mater that enable immature, but not mature, dura to induce osteogenesis. The purpose of these studies was to analyze metabolic characteristics, protein/gene expression, and capacity to form mineralized bone nodules of cells derived from immature and mature dura mater. Transforming growth factor beta-1, basic fibroblast growth factor, collagen type IalphaI, osteocalcin, and alkaline phosphatase are critical growth factors and extracellular matrix proteins essential for successful osteogenesis. In this study, we have characterized the proliferation rates of immature (6-day-old rats, n = 40) and mature (adult rats, n = 10) dura cell cultures. In addition, we analyzed the expression of transforming growth factor beta-1, basic fibroblast growth factor-2, proliferating cell nuclear antigen, and alkaline phosphatase. Our in vitro findings were corroborated with Northern blot analysis of mRNA expression in total cellular RNA isolated from snap-frozen age matched dural tissues (6-day-old rats, n = 60; adult rats, n = 10). Finally, the capacity of cultured dural cells to form mineralized bone nodules was assessed. We demonstrated that immature dural cells proliferate significantly faster and produce significantly more proliferating cell nuclear antigen than mature dural cells (p < 0.01). Additionally, immature dural cells produce significantly greater amounts of transforming growth factor beta-1, basic fibroblast growth factor-2, and alkaline phosphatase (p < 0.01). Furthermore, Northern blot analysis of RNA isolated from immature and mature dural tissues demonstrated a greater than 9-fold, 8-fold, and 21-fold increase in transforming growth factor beta-1, osteocalcin, and collagen IalphaI gene expression, respectively, in immature as compared with mature dura mater. Finally, in keeping with their in vivo phenotype, immature dural cells formed large calcified bone nodules in vitro, whereas mature dural cells failed to form bone nodules even with extended culture. These studies suggest that differential expression of growth factors and extracellular matrix molecules may be a critical difference between the osteoinductive capacity of immature and mature dura mater. Finally, we believe that the biomolecular bone- and matrix-inducing phenotype of immature dura mater regulates the ability of young children and immature animals to heal calvarial defects. PMID- 10744230 TI - Adhesion of tissue-engineered cartilate to native cartilage. AB - Reconstruction of cartilaginous defects to correct both craniofacial deformities and joint surface irregularities remains a challenging and controversial clinical problem. It has been shown that tissue-engineered cartilage can be produced in a nude mouse model. Before tissue-engineered cartilage is used clinically to fill in joint defects or to reconstruct auricular or nasal cartilaginous defects, it is important to determine whether it will integrate with or adhere to the adjacent native cartilage at the recipient site. The purpose of this study was to determine whether tissue-engineered cartilage would adhere to adjacent cartilage in vivo. Tissue-engineered cartilage was produced using a fibrin glue polymer (80 mg/cc purified porcine fibrinogen polymerized with 50 U/cc bovine thrombin) mixed with fresh swine articular chondrocytes. The polymer/chondrocyte mixture was sandwiched between two 6-mm-diameter discs of fresh articular cartilage. These constructs were surgically inserted into a subcutaneous pocket on the backs of nude mice (n = 15). The constructs were harvested 6 weeks later and assessed histologically, biomechanically, and by electron microscopy. Control samples consisted of cartilage discs held together by fibrin glue alone (no chondrocytes) (n = 10). Histologic evaluation of the experimental constructs revealed a layer of neocartilage between the two native cartilage discs. The neocartilage appeared to fill all irregularities along the surface of the cartilage discs. Safranin-O and toluidine blue staining indicated the presence of glycosaminoglycans and collagen, respectively. Control samples showed no evidence of neocartilage formation. Electron microscopy of the neocartilage revealed the formation of collagen fibers similar in appearance to the normal cartilage matrix in the adjacent native cartilage discs. The interface between the neocartilage and the native cartilage demonstrated neocartilage matrix directly adjacent to the normal cartilage matrix without any gaps or intervening capsule. The mechanical properties of the experimental constructs, as calculated from stress-strain curves, differed significantly from those of the control samples. The mean modulus for the experimental group was 0.74 +/- 0.22 MPa, which was 3.5 times greater than that of the control group (p < 0.0002). The mean tensile strength of the experimental group was 0.064 +/- 0.024 MPa, which was 62.6 times greater than that of the control group (p < 0.0002). The mean failure strain of the experimental group was 0.16 +/- 0.061 percent, which was 4.3 times greater than that of the control group (p < 0.0002). Finally, the mean fracture energy of the experimental group was 0.00049 +/- 0.00032 J, which was 15.6 times greater than that of the control group. Failure occurred in all cases at the interface between neocartilage and native cartilage. This study demonstrated that tissue-engineered cartilage produced using a fibrin-based polymer does adhere to adjacent native cartilage and can be used to join two separate pieces of cartilage in the nude mouse model. Cartilage pieces joined in this way can withstand forces significantly greater than those tolerated by cartilage samplesjoined only by fibrin glue. PMID- 10744231 TI - Prefabricated (expander) capsule-lined transposition and advancement flaps in reconstruction of lower eyelid and oral defects: an experimental study. AB - In six pigs with prefabricated transposition flaps and six pigs with prefabricated advancement flaps, both flap types (lined with an expander capsule) were used to reconstruct wedge excisions of the lower eyelid or defects in the cheek/oral mucosa. The capsules replaced the conjunctiva in eyelid defects and the oral mucosa in cheek defects. Histopathologic studies were performed at 5 to 7 days, 9 to 10 days, 2 weeks, 3 to 4 weeks, and 2 and 3 months after flap reconstructions. Healing was rapid and uneventful, leading to restoration of the conjunctiva/eyelid and oral mucosa between 9 days and 2 weeks. The healing of the eyelid conjunctiva was somewhat faster than of the oral mucosa. The expander capsule acted as a conjunctival/ mucosal substitute, providing a temporary physical shield, an infectious barrier, and a matrix for epithelial regeneration. All reconstructions were successful except one oral reconstruction with early flap necrosis. Flaps lined with an expander capsule could improve and facilitate clinical reconstructions in the eyelid and oral cavity. PMID- 10744232 TI - The role of allopurinol and deferoxamine in preventing pressure ulcers in pigs. AB - Ischemia and reperfusion may be important in the pathogenesis of pressure ulcers. On the basis of this hypothesis, the effects of intermittent pressure and the anti-free radical agents allopurinol and deferoxamine were studied in a pig model in which a pressure of 150 mmHg was applied intermittently to the scapulae. Cutaneous blood flow, transcutaneous oxygen tension, skin and muscle damage, and muscle levels of adenosine triphosphate were quantified. A control group of pigs (n = 6) was untreated, the allopurinol group (n = 6) received oral allopurinol beginning 2 days before the experiment, and the deferoxamine group (n = 6) received an intramuscular injection of deferoxamine 2 hours before the experiment. Pressure (150 mmHg) was applied to the scapulae for 210 minutes, and it was relieved for 30 minutes. This 4-hour cycle was repeated continuously for 48 hours, and it resulted in pressure injuries in all animals. Allopurinol and deferoxamine improved cutaneous blood flow and tissue oxygenation, but only deferoxamine could significantly reduce cutaneous and skeletal muscle necrosis (p < 0.001). This study suggests a future role for anti-free radical agents in the reduction of pressure-induced injury. PMID- 10744233 TI - Lymphatic malformation causing intractable chylorrhagia. AB - Lymphatic malformation is a developmental error usually noticed at birth or in early childhood. Lesions of the upper leg and lower trunk are the most difficult to remove, because they are often the largest ones encountered and also because they tend to extend proximally into the retroperitoneal tissues. Chyle reflux, usually associated with lymphedema of the extremity, has not been reported to be caused by lymphatic malformation. We report a case of intrapelvic retroperitoneal lymphatic malformation with an extension of gluteal-thigh soft-tissue involvement causing intractable chylorrhagia. The tumor was subtotally excised, and the defect was closed by a distally based, peninsular latissimus dorsi myocutaneous flap. The flap served both as a filling material and as a "bridge" between the residual tumor, including abnormal lymphatics, and normal lymph flow. PMID- 10744234 TI - Osteochondroma of the mandibular body. AB - We have treated four cases of osseous ostechondromas arising on the mandibular body, where this tumor rarely presents on the craniomaxillofacial bones. There were no recurrences after simple surgical resection of these tumors. PMID- 10744235 TI - Biocompatibility and oxidative stability of radiolucent breast implants. PMID- 10744236 TI - Replantation of an avulsive amputation of a foot after recovering the foot from the sea. AB - A foot avulsion case, with the dismembered body part submerged in sea water for 1 hour, is presented. This report is unique in that it is the first to document the reattachment of a body part that had been submerged in sea water. It was not known how salt-water exposure would affect wound management. Differences in osmolarity and bacterial flora between the sea water and foot tissues have not caused any problems, and the patient has not suffered any vascular or infectious complications after replantation. Neurotization of the plantar surface by the tibial nerve, which was stripped off during amputation and replaced in its original traces, was the most critical part of convalescence. After management of such an interesting case, we conclude that exposure to sea water of the dismembered part should not be a contraindication for replantation surgery. PMID- 10744237 TI - Rectus abdominis muscle free flap harvest by laparoscopic sheath-sparing technique. AB - Previous reports of endoscopic rectus abdominis muscle harvest have described techniques that are hampered by the need for anterior rectus sheath division or mechanical devices to maintain the optical cavity. The authors report the first successful clinical case of a laparoscopic sheath-sparing rectus abdominis muscle harvest for free tissue transfer. It offers considerable advantages over the traditional open method and, with the help of an experienced laparoscopic surgeon, it should add little to operative time. PMID- 10744238 TI - Technique of percutaneous anchoring suture placement. AB - We describe a technique to assist in the placement of percutaneous anchoring sutures when used in poorly accessible tunnels such as in rhinoplasty by using a sucker tube to guide retrieval and placement of the suture needle. This technique avoids unintended catching of the needle on soft tissue within the tunnel, which could prevent the structure from becoming anchored at other than in its intended location. PMID- 10744239 TI - Correction of congenital microtia using stereolithography for surgical planning. PMID- 10744240 TI - Reconstruction of composite metacarpal defects using a fibula free flap. PMID- 10744241 TI - Functional and aesthetic labia minora reduction. PMID- 10744243 TI - Simplified nomenclature for compound flaps. AB - The unique niche for compound flaps is their potential role for the repair of massive defects that demands the simultaneous restoration of multiple, missing tissue types. These complex flaps can be sorted into two major classes, and their subtypes on the basis of their means of vascularization are described. (1) Solitary vascularization, the composite flap: "multiple tissue components with a single vascular supply and dependent parts." (2) Combined flaps: (a) Siamese flaps: "multiple flap territories, dependent due to some common physical junction, yet each retaining their independent vascular supply"; (b) conjoint flaps: "multiple independent flaps, each with an independent vascular supply, but linked by a common indigenous source vessel"; and (c) sequential flaps: "multiple independent flaps, each with an independent vascular supply, and artificially linked by a microanastomosis." Many technical modifications that have improved or will improve the reliability of these flaps should not be confused as distinct flap types, but rather acknowledged as variations that can be more conveniently classified for the purposes of improved communication and research by using this basic schema as a guideline. PMID- 10744242 TI - The rising level of medical student debt: potential risk for a national default. AB - At the turn of the 20th century, mostly as a result of the Flexner report, medical education changed dramatically by establishing a scientific basis for the study of medicine within the institutions of the major universities. There have been major and dramatic changes in medicine during the past 80 years that have improved medical education in the United States, but these changes have also placed major economic strains on students who have educational debts. If medicine is a social responsibility to the public, then the public should share the responsibility of identifying and supporting new approaches to funding and financially managing the teaching of future physicians. There is no universal solution because there are various approaches institutions may take to structure these financial responsibilities. This article describes trends in medical student educational debt, identifies the financial needs of medical students, and proposes ways of addressing those needs to avert a possible national financial crisis among medical students. We must invest in medical students because they will be the leaders we need to help care for our society and our own families in the next century. PMID- 10744244 TI - So you are board-certified in plastic surgery: what it means in the new millennium. PMID- 10744245 TI - Surgical anatomy of the ligamentous attachments in the temple and periorbital regions. AB - This study documents the anatomy of the deep attachments of the superficial fasciae within the temporal and periorbital regions. A highly organized and consistent three-dimensional connective tissue framework supports the overlying skin and soft tissues in these areas. The regional nerves and vessels display constant and predictable relationships with both the fascial planes and their ligamentous attachments. Knowledge of these relationships allows the surgeon to use the tissue planes and soft-tissue ligaments as intraoperative landmarks for the vital neurovascular structures. This results in improved efficiency and safety for aesthetic procedures in these regions. PMID- 10744246 TI - Evaluation of body contouring surgery today: a 30-year perspective. AB - Concepts of beauty have been continuously evolving throughout the history of mankind. The voluptuous figures that were idealized by artists in the past have been substituted by slimmer forms. Medical advances in this century have permitted safe and efficient surgical correction of contour deformities. Until recently, these alterations were mostly hidden under heavy clothing or were accepted reluctantly. Current fashion trends generally promote body-revealing attire. The media frequently encourage the importance of fitness and good health, linking these qualities with youthfulness and beauty. The subliminal and the overt message is that these are necessary and desirable requirements for social acceptance and professional success. PMID- 10744247 TI - Facial cosmetic surgery: a 30-year perspective. AB - Although traits that are specific to different social and cultural groups may express the uniqueness in what humans regard as beautiful, the quest for beauty itself is universal. It is intrinsically related to the search for harmony as a means of achieving intimate well being. Inevitably, aging brings changes that are undesirable. The face, because of its exposure and expression and the constant action of gravity, is frequently the main focus of anxiety in individuals who have attained a certain age because their face has flaccid skin, marked lines of expression, and fat deposits. Motivations for surgery for the aging face are generally deeply hidden and include personal, social, and professional factors. These motivations might not be perceived or investigated by the surgeon who, in turn, will not have done a complete and precise diagnosis. This evaluation is the most valuable tool for determining the most favorable procedure for each patient. PMID- 10744248 TI - Correction of the cornrow hair transplant and other common problems in surgical hair restoration. AB - Hair on a man's head is an important emblem of health, youth, and vitality. As in all areas of cosmetic surgery, the refinements of surgical technique and instrumentation have improved the results of hair transplantation. The state of the art in hair grafting today produces a result that is undetectable as being a surgical hair transplant. Many earlier techniques of plug hair transplantation are not aesthetically acceptable by today's standards. This is especially true in the face of progressive hair loss, which can unmask previously camouflaged cornrow plugs. A technique to reduce the plugs and recycle the grafts into smaller grafts is described. The recycled hair grafts can be combined with scalp lifting, scalp reductions, and occipital harvesting of grafts to improve the results of cornrow appearing hair transplants and other problems of surgical hair restoration. PMID- 10744249 TI - Textured-surface saline-filled silicone breast implants for augmentation mammaplasty. AB - The earliest silicone breast implants were smooth-surface, silicone rubber devices filled with either silicone gel or saline. Because of persistent problems with capsular contracture, polyurethane-covered silicone implants were developed as an alternative. Particularly in the short run, these alternatives proved highly successful at reducing the incidence of capsular contracture. By 1990, polyurethane-covered implants were rapidly becoming the preferred implant choice of many plastic surgeons, but for legal, regulatory, financial, and safety reasons they were withdrawn from the market by Bristol-Myers in 1991. Meanwhile, during the late 1980s, surface texturing and improved materials became available on other silicone breast implants and expanders. Most studies suggest that textured-surface silicone gel-filled implants, saline-filled implants, and tissue expanders have less frequent capsular contracture than their smooth-surface counterparts. PMID- 10744250 TI - Failure to remove soft tissue injected with liquid silicone with use of suction and honesty in scientific medical reports. PMID- 10744251 TI - How to avoid eyelid ptosis when injecting botulinum toxin into the corrugators. PMID- 10744252 TI - Pyogenic granuloma: a complication of transconjunctival incisions. PMID- 10744253 TI - Cleft lip repair: rub off the sutures, not the smile! PMID- 10744254 TI - The ethics of randomized trials in the context of cleft palate research. PMID- 10744255 TI - Securing skin graft on the nasal area by plaster splint. PMID- 10744256 TI - Intranasal web: a rare cause of nasal obstruction. PMID- 10744257 TI - The effect of betadine on silicone implants. PMID- 10744258 TI - Pectoralis major myospasm resulting from a subpecotoral implant. PMID- 10744259 TI - Lumbar hernia after latissimus dorsi flap dissection. PMID- 10744260 TI - Basal cell carcinoma affecting the areola-nipple complex. PMID- 10744261 TI - A comparison of pedicle and free tram flaps for breast reconstruction in a single institution. PMID- 10744262 TI - An easy method to remove serous fluid from beneath occlusive plastic dressings. PMID- 10744263 TI - Clinical application of free digital artery flap of the hand. PMID- 10744264 TI - An easy method to control the rats in experimental studies. PMID- 10744265 TI - The inverted nipple: its grading and surgical correction. PMID- 10744266 TI - Conversion to digital photography and photo archiving. PMID- 10744268 TI - A new type of needle holder for buried sutures. PMID- 10744267 TI - The clinical outcome of abdominoplasty performed under conscious sedation: increased use of fentanyl correlated with longer stay in outpatient unit. PMID- 10744269 TI - Forked insertion of flexor digitorum profundus to the little finger: an aberration. PMID- 10744270 TI - Danger of using a Padgett dermatome blade in Zimmer dermatome. PMID- 10744271 TI - Traumatic brain injury: patterns of failure of nonoperative management. AB - OBJECTIVE: The circumstances of failure for nonoperative management of blunt traumatic brain injury have been poorly defined. In this study, all trauma patients identified over a 12-year period with progression of neurologic injury requiring craniotomy were retrospectively reviewed. METHODS: Data collected included demographic information, mechanism of injury, field and admission vital signs, and Glasgow Coma Scale score, medications, associated injuries, and coagulopathy. Head computed tomographic scans were reviewed, and anatomic findings were correlated with clinical changes (change in mental status or elevation of intracranial pressure) that led to subsequent CT scan and craniotomy. RESULTS: Of 20,100 patients, there were 852 who had computed tomographic scans with acute intracranial injuries on admission; 462 patients were managed nonoperatively. Fifty-seven patients had progression of neurologic injury (34 < 24 hours = early; 23 > 24 hours = late) that required surgery. CONCLUSION: Of the variables investigated, only anatomic location of injury was found to be predictive of early failure of nonoperative management. Frontal intraparenchymal hematomas are particularly prone to early failure. Clinical examination and intracranial pressure monitoring are equally important in detecting failure and should be an integral part of nonoperative management. PMID- 10744272 TI - Fluid resuscitation of patients with multiple injuries and severe closed head injury: experience with an aggressive fluid resuscitation strategy. AB - BACKGROUND: Despite increasing experimental and clinical evidence to the contrary, a dichotomy of management strategies of the patient with multiple injuries still exists, based on the presence or absence of traumatic brain injury. Many still advocate fluid restriction or small volume resuscitation if traumatic brain injury is present. PURPOSE: To demonstrate results of aggressive fluid resuscitation in a prospective case series of patients with multiple injuries and with severe head injury. METHODS: Thirty-four patients with Glasgow Coma Scale score < or = 8 and Injury Severity Score > or = 16 were enrolled into the study over a period of 18 months. Fluid resuscitation was guided in part by cerebral perfusion pressures (mean cerebral perfusion pressures > 80) as well as by hemodynamic monitoring and evidence of end organ perfusion. Overall fluid intake, intensive care unit fluid balance, presence or absence of hypoxia, hypotension, or both, were analyzed. Ninety- and 180-day Glasgow Outcome Scale and Disability Rating Scale scores were also obtained. RESULTS: By using an aggressive fluid resuscitation strategy, secondary insults were avoided in 74% of the patients. A good functional outcome was achieved in 74% and mortality was impressively low at 6%. CONCLUSION: Fluid restriction is not necessary to achieve good results in the severely injured patient who also has a severe head injury. PMID- 10744273 TI - Anterior ischemic optic neuropathy: a complication after systemic inflammatory response syndrome. AB - BACKGROUND: Patients are surviving previously fatal injuries. Unique morbidities are occurring in these survivors. Anterior ischemic optic neuropathy represents a previously unrecognized cause of blindness in the trauma victim. We hypothesize that this phenomenon is caused by unique characteristics of optic edema/ pressure or decreased blood flow associated with massive resuscitation. METHODS: Between November of 1991 and August of 1998, there were 18,199 admissions to our trauma center. Of this group, 350 patients required massive volume resuscitation (>20 liters infused over first 24 hours). Patients having closed head injuries, facial fractures or direct orbital trauma were excluded from study. The following variables were studied: demographics, injury severity (Injury Severity Score, highest lactate, worst base deficit, and lowest pH) crystalloid and transfusion requirements, ventilator requirements (PEEP) RESULTS: Of the 350 patients with massive resuscitation, 9 patients were diagnosed with anterior ischemic optic neuropathy (2.6%). Of these, seven patients required celiotomy (78%). Six of the seven celiotomy patients had damage control celiotomies and abdominal compartment syndrome (86%). One patient had a repair of a subclavian artery; one had a complex acetabular repair. Blindness was unilateral in five patients and bilateral in four. All nine patients had evidence of global hypoperfusion, systemic inflammatory response, massive resuscitation, and high ventilatory support; one patient required cardiopulmonary resuscitation. CONCLUSION: Prone positioning is known to be associated with an increased intraocular pressure. We postulate that the combination of massive resuscitation and prone positioning will increase the incidence of anterior ischemic optic neuropathy. As such, we recommend that prone positioning for adult respiratory distress syndrome be reserved for only those patients at risk of death. PMID- 10744274 TI - Hypertonic saline attenuation of polymorphonuclear neutrophil cytotoxicity: timing is everything. AB - BACKGROUND: The potential to modulate the inflammatory response has renewed interest in hypertonic saline (HTS) resuscitation of injured patients. However, the effect of the timing of HTS treatment with respect to polymorphonuclear neutrophil (PMN) priming and activation remains unexplored. We hypothesized that HTS attenuation of PMN functions requires HTS exposure before priming and activation. METHODS: Isolated PMN were incubated in HTS (180 mM Na+) before L alpha-phosphatidylcholine, beta-acetyl-gamma-O-alkyl (PAF)/N-formylmethionyl leucyl-phenylalanine (fMLP) priming/activation, after priming, or after priming/activation. Superoxide production was measured by the reduction cytochrome c, elastase release by cleavage of AAPV-pNA, and beta2-integrin expression by flow cytometry. RESULTS: HTS before priming or activation decreased beta2-integrin expression, superoxide production, and elastase release. In contrast, HTS after priming/activation augmented superoxide production and elastase release. CONCLUSION: The timing of HTS is a key variable in the attenuation of PMN cytotoxic functions. Maximal attenuation of cytotoxicity is achieved before priming, whereas HTS exposure after activation augments cytotoxicity. PMID- 10744275 TI - Utility of near-infrared spectroscopy in the diagnosis of lower extremity compartment syndrome. AB - OBJECTIVE: To determine the utility of near-infrared spectroscopy in the diagnosis of lower extremity compartment syndrome (CS). METHODS: Nine patients with CS confirmed by physical examination and elevated compartment pressures (64 +/- 17 mm Hg) were evaluated before and after fasciotomy. Control readings were also performed on 33 surgical patients who had no evidence of CS. The deltoid muscle was used as a reference value. RESULTS: The deltoid muscle oxygen saturation (StO2) readings revealed a mean = 84 +/- 17% prefasciotomy and mean = 83 +/- 12% postfasciotomy in the CS group. The control group had a mean StO2 of 83 +/- 11%. In the CS group, the leg compartment with the highest pressure had a StO2 mean = 56 +/- 27% before fasciotomy. This value was statistically significantly lower (p < 0.05) than either the postfasciotomy mean StO2 in that compartment (82 +/- 16%) or the values found in matched control patients with no evidence of CS (87 +/- 7%). CONCLUSION: Near-infrared spectroscopy-derived StO2 values in the lower extremities of trauma patients with CS were diminished relative to the control patients and usually normalized after fasciotomy. Near infrared spectroscopy evaluation may offer a rapid, noninvasive method of assessing extremities at risk for CS. PMID- 10744276 TI - Diagnosis and management of blunt small bowel injury: a survey of the membership of the American Association for the Surgery of Trauma. AB - BACKGROUND: Blunt small bowel injury (SBI) may be difficult to diagnose accurately. Diagnostic delays are associated with increased morbidity and mortality. METHODS: A cross-sectional survey of members of the American Association for the Surgery of Trauma was conducted. A Likert-type multiple choice scale was used to evaluate use and usefulness of diagnostic and laboratory tests. Data were analyzed by using univariate and multivariate techniques. RESULTS: A total of 461 of the 702 members (68%) surveyed responded, of which 133 members (29%) were excluded because they did not currently manage adult SBI. Of the remaining 328 respondents, 244 members (74%) reported prior experience as the most important influence on their current practice of diagnosing blunt SBI. None of the standard laboratory tests were reported as useful. Seventy-seven percent of respondents use computed tomographic (CT) scan most or all of the time for diagnosis (p < 0.05 compared with other modalities). Most respondents estimated their annual incidence of SBI at 5% with a >15% frequency of delay in diagnosis. Forty-four percent of the respondents estimated the mortality associated with a delay in diagnosis at < or =5%. Respondents varied significantly in their management of the patient with an unreliable abdominal exam and a CT scan finding of isolated free fluid. In patients with head injuries, 28% observe, 12% repeat the CT scan, 42% perform diagnostic peritoneal lavage, and 16% operate. For intoxicated patients, 51% observe, 11% repeat the CT scan, 26% perform diagnostic peritoneal lavage, and 10% operate. A more aggressive approach with diagnostic and operative intervention was significantly (p < 0.05) advocated by respondents practicing without residents, more than 15 years out from residency, or by those with a perception of higher morbidity and mortality from delays in diagnosis. CONCLUSION: There is significant variation in the diagnostic approach to the patient with suspected SBI. The perceived mortality of delayed diagnosis is much less than reported. Those surgeons with more experience or perception of greater morbidity and mortality from a delayed diagnosis are more aggressive. Further investigation into the diagnosis and treatment of this injury is needed. PMID- 10744277 TI - Relatively short diagnostic delays (<8 hours) produce morbidity and mortality in blunt small bowel injury: an analysis of time to operative intervention in 198 patients from a multicenter experience. AB - OBJECTIVE: Blunt small bowel injury (SBI) is uncommon, and its timely diagnosis may be difficult. The impact of operative delays on morbidity and mortality has been unclear. The purpose of this study was to determine the relationship of diagnostic delays to morbidity and mortality in blunt SBI. METHODS: Patients with blunt SBI with perforation were identified from the registries of eight trauma centers (1989-1997). Patients with duodenal injuries were excluded. Data were extracted by individual chart review. Patients were classified as multi-trauma (group 1) or near-isolated SBI (group 2 with Abbreviated Injury Scale score < 2 for other body areas). Time to operation and its impact on mortality and morbidity was determined for each patient. RESULTS: A total of 198 patients met inclusion criteria: 66.2% were male, mean age was 35.2 years (range, 1-90 years) and mean Injury Severity Score was 16.7 (range, 9-47). 100 patients had multiple injuries (group 1). There were 21 deaths (10.6%) with 9 (4.5%) attributable to delay in operation for SBI. In patients with near-isolated SBI, the incidence of mortality increased with time to operative intervention (within 8 hours: 2%; 8-16 hours: 9.1%; 16-24 hours: 16.7%; greater than 24 hours: 30.8%, p = 0.009) as did the incidence of complications. Delays as short as 8 hours 5 minutes and 11 hours 15 minutes were associated with mortality attributable to SBI. The rates of delay in diagnosis were not significantly associated with age, gender, intoxication, transfer status, or presence of associated injuries. CONCLUSION: Delays in the diagnosis of SBI are directly responsible for almost half the deaths in this series. Even relatively brief delays (as little as 8 hours) result in morbidity and mortality directly attributable to "missed" SBI. Further investigation into the prompt diagnosis of this injury is needed. PMID- 10744278 TI - Transmediastinal gunshot wounds: a prospective study. AB - OBJECTIVE: To evaluate admission systolic blood pressure (SBP) in the emergency center (EC) as a means by which patients with transmediastinal gunshot wounds (TM GSWs) can be triaged to the operating room versus further diagnostic evaluation. METHODS: A prospective case series presenting concurrent data collected for 68 consecutive patients with TM-GSWs admitted to one urban trauma center over a 4.5 year period. For purposes of analysis, patients were assigned to the following groups based on SBP in the EC: group I, SBP > 100 mm Hg; group II, SBP from 60 to 100 mm Hg; group III, SBP < 60 mm Hg. RESULTS: The management and outcomes of 68 patients with a mean age of 29 years were evaluated. For patients in group I (n = 20), TM-GSW was diagnosed by findings on x-ray film for 15 patients (75%), at physical examination for 4 patients (20%), and at operation for 1 patient (5%). Indications for immediate operation were found in five patients (25%), whereas further diagnostic evaluation prompted operation for three additional patients. Only one patient developed persistent hypotension from neurogenic shock. There were two deaths from late complications. In patients in group II (n = 16), TM-GSW was diagnosed by findings on x-ray film for 9 patients (56%), at physical examination for 5 patients (31%), and at operation for 2 patients (13%). Six patients with persistent hypotension had indications for immediate operation, whereas further diagnostic evaluation in the remaining patients, who became hemodynamically normal during resuscitation, prompted operation in an additional two patients. There were two intraoperative deaths. For the patients in group III (n = 32), six patients with signs of life underwent immediate operation with one intraoperative death, seventeen patients required EC thoracotomy with 100% mortality, and nine patients were pronounced dead in the EC without an attempt at operation. CONCLUSION: The diagnosis of TM-GSW for patients in groups I and II is confirmed by finding at physical examination and on chest x-ray films in 90% of cases. In the absence of obvious bleeding, patients with TM-GSWs and SBP > 100 mm Hg may safely undergo further diagnostic evaluation. Sixty percent of such patients did not require an operation. All patients with TM-GSWs and SBP < 60 mm Hg (group III) require immediate operation. For patients with TM-GSWs, SBP from 60 to 100 mm Hg (group II), and without obvious bleeding, it is the response to resuscitation and the results of further diagnostic evaluation that determine the need for operation. Fifty percent of such patients did not require operation. PMID- 10744279 TI - Cranial nerve VII region of the traumatized facial skeleton: optimizing fracture repair with the endoscope. AB - OBJECTIVE: Cranial nerve VII (CN VII) is anatomically positioned adjacent to the condylar neck of the mandible and arch of the midface. Fracture treatment of this region of the facial skeleton remains controversial because of difficult surgical access. Conservative management rarely achieves anatomic fracture repair and can result in irreversible structural deformity and dysfunction. Traditional operative methods require access through very visible facial incisions and risk injury to the facial nerve. We report endoscopic methods of facial fracture repair in the region of CN VII that achieve excellent fracture reduction and stabilization, with minimal risk of facial nerve injury by using hidden incisions. METHODS: A consecutive series of 65 endoscopically assisted facial fracture repairs were analyzed. Endoscopic repairs of the condylar neck (n = 40) of the mandible were performed through an intraoral incision. Endoscopic arch repairs (n = 25) of the midface were performed through a preauricular incision. Outcomes were evaluated by postoperative fracture reduction on radiographs, occlusion, interincisal jaw opening, and facial nerve function. RESULTS: Thirty seven of 40 condylar neck mandible fractures went on to anatomic bone union, whereas 3 of 40 had either incomplete fracture reduction or re-fracture through the plate. There was one temporary palsy of CN VII that completely resolved spontaneously. Jaw opening exceeded 40 mm by the 8th postoperative week. Computed tomographic images demonstrated anatomic arch repair in all 25 endoscopically repaired cases. Six of seven endoscopically repaired Le Fort III facial fractures went on to restoration of their premorbid occlusion. One of seven had an excellent restoration of the occlusal interface but a cant to the occlusal plane. Two of seven had improved but incomplete restoration of the malar prominence and enopthalmos ipsilateral to the side of endoscopic arch repair. Eight of 25 endoscopic arch repairs developed temporary paralysis of the frontal branch of CN VII that recovered completely by the 10th postoperative week. CONCLUSION: We have developed novel endoscopically assisted techniques to facilitate repair of facial fractures in the region of CN VII. These techniques have been successfully applied to accurately restore the facial skeleton to its preinjury anatomic position in the region of CN VII with minimal risk of facial paralysis by using limited and well-hidden incisions. PMID- 10744280 TI - Should trauma surgeons do general surgery? AB - OBJECTIVE: Many trauma centers have separated emergency and general surgery from trauma care. However, decreased trauma volume and more frequent nonoperative management may limit operative experience and the economic viability of the trauma service. Trauma surgeons at our Level I trauma center have long provided all emergency surgical care and elective surgery. We sought to determine the impact of this policy. METHODS: We reviewed all admissions to the trauma service from June of 1992 to July of 1998 and cross-referenced this with our trauma registry. The number of major and minor procedures performed was also determined, and we reviewed all operative procedures by the trauma service for June of 1996 to October of 1998. RESULTS: Total admissions by the trauma service averaged 3,003 patients/year (range, 2,798-3,198 patients). Nontrauma patients accounted for 34% of all trauma service admissions (range, 26-40%). During this time period, there was no change in volume of operative or intensive care unit procedures, whereas minor procedures recently decreased from a peak of 141/month to 50/month. This was largely due to decreased use of diagnostic peritoneal lavage (surgeon reimbursable) and an increased use of computed tomographic scan and ultrasound (not presently reimbursed) to evaluate blunt abdominal trauma. During the past 2 years, nontrauma cases accounted for 33% of all operative procedures by the trauma service. CONCLUSIONS: Maintenance of emergency and general surgical care by the trauma service has allowed us to buffer impact of variations in trauma volume and to maintain operative skills in an era of increased nonoperative management of many injuries. PMID- 10744281 TI - Induced hypothermia during emergency department thoracotomy: an animal model. AB - BACKGROUND: Induced hypothermia is used clinically to prevent ischemic injury during elective procedures. We present an animal model of asanguinous hypothermic (10 degrees C) circulatory arrest, induced through a left anterior lateral thoracotomy after exsanguinating uncontrolled hemorrhage. METHODS: Through a left anterior thoracotomy, 26 swine (45-70 kg) sustained a laceration of the descending thoracic aorta, producing exsanguinating uncontrolled hemorrhage. After 5 minutes of severe hypotension (systolic BP <20 mm Hg), a 22 French Foley catheter was directed cephalad through the enlarged aortic wound. A solution (containing 42.5 mmol/L K+ and precooled to 1 degrees C) was infused to arrest/preserve the heart and brain. A second 24 French Foley catheter was then directed caudally through the same wound. The right atrium was opened to drain the venous system. The animal was cooled with a cardiopulmonary bypass pump (>5L/min) through the Foley catheters. Once 10 degree C was reached, a cannula was placed to the aortic root and the aortic laceration repaired. The animal was maintained at 10 degree C for a total of 90 minutes. Before the rewarming process, the circulation was rinsed with a solution containing normal levels of electrolytes followed by infusion of whole blood. Rewarming was performed by maintaining a 10-degree gradient on the heat exchanger. The first 16 animals were used in nonsurvival experiments to develop the technique and to record dural temperatures and electroencephalogram tracings. The last 10 animals were used to determine long-term survival and neurologic outcome. Group I: seven animals were kept at < 10 degrees C with flows less than 2L/min. Group II: three animals underwent 20, 30, and 40 minutes of no flow once they were cooled to 10 degrees C. After 6 weeks of survival and neurologic examinations, the brains were fixed for histologic evaluations. RESULTS: The average time to cool the head to 18 degrees C and 10 degrees C was 6 minutes and 12 minutes, respectively. The hematocrit fell below 2% by the end of the cooling period. A total of 7 of the 10 animals from the long-term study survived. Group I: five of seven animals survived. Four of the survivors had no appreciable neurologic deficits, were fully functional at 6 weeks, and had no evidence of histologic injury. One of the five survivors in this group had moderate neurologic disability. Of the two animals that died, one died from air embolism from the i.v. line. The second death was in an animal for which maximal cooling to 2.7 degrees C was attempted. Group II: The first two animals that had "no flow" for 20 and 30 minutes were fully functional and had normal neurologic examinations. However, the second animal was found to have brain injury on histologic examination. The last animal in this group died of accidental extubation during recovery. CONCLUSION: Induction of hypothermic arrest through the chest after exsanguination is possible. The further development of this technique may provide an extended state of "suspended animation" to allow for repairs of hemorrhaging injuries in trauma patients who require emergency department thoracotomy. PMID- 10744282 TI - Impact of preinjury warfarin use in elderly trauma patients. AB - BACKGROUND: This study examined the hypothesis that elderly trauma patients on warfarin before injury will have increased morbidity and mortality compared with elderly trauma patients not on warfarin. METHODS: From 1993 to 1995, trauma patients were grouped by age and presence or absence of warfarin use before injury. Groups were analyzed with respect to Injury Severity Score, Trauma Registry and Injury Severity Score, Glasgow Coma Scale score, Intensive Care Unit days, hospital days, units of blood transfused, and mortality rates. Statistical analysis was completed by using the Student's t test. RESULTS: Records of 61 patients administered warfarin and 800 patients not administered warfarin were available for analysis. There were no statistically significant differences between patients on prehospital warfarin and those not on prehospital warfarin. CONCLUSION: This study indicates that elderly trauma patients on warfarin before injury do not have increased morbidity and mortality compared with elderly trauma patients not on warfarin. PMID- 10744283 TI - Comparison of poly-N-acetyl glucosamine (P-GlcNAc) with absorbable collagen (Actifoam), and fibrin sealant (Bolheal) for achieving hemostasis in a swine model of splenic hemorrhage. AB - OBJECTIVE: To compare the hemostatic capabilities of poly-Nacetylglucosamine (p GlcNAc) with three currently available products: Actifoam, Surgicel, and Bolheal fibrin glue. This study was conducted in a controlled animal model, with monitoring of hematologic parameters over the course of the study. Two series were conducted, one in unheparinized animals comparing Bolheal fibrin sealant, Actifoam (absorbable collagen, AC), and Surgicel (ORC) with p-GlcNAc, and the second in systemically heparinized animals comparing p-GlcNAc with AC. METHODS: This study was performed in immature female Yorkshire White swine. Splenic lacerations controlled for length and depth of wound were used as sources of bleeding, with one material used per wound to assess hemostatic effectiveness. A total of 97 wounds in 12 animals were created for the study, 74 wounds in unheparinized animals, and 23 wounds in the heparinized animals. In the heparinized animals, hemostatic efficacy was judged by number of applications needed to achieve complete hemostasis. In the unheparinized animals, hemostatic efficacy was judged by length of time required to achieve complete hemostasis (p GlcNAc vs. fibrin sealant) or the number of applications needed to achieve complete hemostasis (p-GlcNAc vs. AC or ORC). RESULTS: In systemically heparinized animals, p-GlcNAc demonstrated greater hemostatic efficacy (72.7 %) in one application than did the control material (0%), p < 0.01. In the unheparinized animals, p-GlcNAc took less time to achieve hemostasis (mean, 22.9 seconds) than fibrin sealant (mean, 172.9 seconds), p < 0.01. p-GlcNAc achieved hemostasis with a greater efficacy (79.2%) in one application than did the AC or ORC (16.7%), p < 0.01, whereas there was no difference in the efficacy of the control materials. CONCLUSION: The results of the previous series in unheparinized animals demonstrated that p-GlcNAc in the form of a membrane is a more effective topical hemostatic agent than Bolheal fibrin glue, AC or ORC. The results in the anticoagulated animals similarly demonstrate that p-GlcNAc is a more effective topical hemostatic agent than the control material AC. These data indicate that p-GlcNAc is a promising hemostatic agent as evaluated in this model. PMID- 10744284 TI - Maximizing tolerance of enteral nutrition in severely injured trauma patients: a comparison of enteral feedings by means of percutaneous endoscopic gastrostomy versus percutaneous endoscopic gastrojejunostomy. AB - BACKGROUND: Intolerance of enteral nutrition interrupts caloric balance and increases hospital costs. This study proposes that enteral feeding by percutaneous endoscopic gastrojejunostomy (PEGJ) provides continuous uninterrupted nutrition with greater consistency than percutaneous endoscopic gastrostomy (PEG). METHODS: This prospective nonrandomly assigned study was conducted at a Level I trauma center from December of 1997 through October of 1998. All feeding tubes were placed by trauma/critical care surgeons for nutritional support. Feeding course was monitored for 14 days from time of tube placement. Demographic data and outcome variables compared were age, sex, Injury Severity Score, Abbreviated Injury Score, hospital length of stay, number of days to reach nutritional goal feedings, caloric goal, protein goal, cc/hr at goal, total parenteral nutrition usage, complications, and hospital charges. Statistical analyses used the independent samples t test, Cox regression, and Pearson chi2 with significance level set at 0.05. RESULTS: Patients receiving enteral nutrition by PEGJ reached nutritional goal sooner than patients who received enteral nutrition by PEG (p = 0.02). Thirty-seven of 46 PEGJ patients (80%) were at goal rate at day 3, whereas 28 of 43 PEG patients (65%) were at goal on day 3. Nine of 43 PEG patients (21%) and 3 of 46 PEGJ patients (7%) failed to reach goal within 14 days. CONCLUSION: This study suggests that enteral nutrition delivered by means of PEGJ is better tolerated than enteral nutrition delivered by means of PEG in trauma patients with no abdominal conditions that preclude percutaneous feeding tube placement. PMID- 10744285 TI - Assessing the need for reintubation: a prospective evaluation of unplanned endotracheal extubation. AB - OBJECTIVE: Unplanned endotracheal extubation (UEE) is a common complication in medical intensive care units but very little data about UEE in surgical populations are available. Our hypothesis is that the surgical intensive care unit (SICU) population requires reintubation less frequently compared with the medical intensive care unit population. We prospectively gathered data on patients in a SICU in an attempt to identify the incidence of UEE and to study the need for reintubation after UEE. METHODS: During an 18-month period, we prospectively identified SICU patients from a quality improvement database who required ventilatory support. All patients who self-extubated were included in the study. RESULTS: Fifty-eight of 1,178 intubated patients experienced unplanned extubation 61 times during the 18-month period. A total of 22 patients (36%) required reintubation, whereas 39 patients (64%) did not. Thirty-three patients self-extubated while being actively weaned from ventilatory support. Of these, only 5 patients (15%) required reintubation and 28 patients (85%) did not (p < 0.01). CONCLUSION: A total of 85% of patients who self-extubate during the weaning process did not require reintubation in our study. Those who have an FiO2 >50%, a lower PaO2/FiO2 ratio, had UEE occur by accident, or were not being weaned when UEE occurred required reintubation more frequently. These data suggest that some of our SICU patients are intubated longer than necessary, which may translate into more ventilator related complications, longer ICU stays and increased cost. PMID- 10744286 TI - Treatment of posttraumatic internal carotid arterial pseudoaneurysms with endovascular stents. AB - BACKGROUND: The sequelae of blunt injury to the carotid arteries are unusual, but pseudoaneurysms causing subsequent strokes are devastating. The utility of treatment of these pseudoaneurysms was examined. METHODS: All patients at a Level I trauma center with previously documented traumatic risk factors were assessed for blunt injury to the carotid arteries and, when a pseudoaneurysm was present, a self-expanding metallic stent was placed across the lesion and the patient placed on anticoagulation. Follow-up arteriograms were obtained in 2 months and every 6 months thereafter. RESULTS: Fourteen patients (7 men, 7 women) with an average age of 27 years, an Injury Severity Score of 38, had formed pseudoaneurysms in 16 extracranial internal carotid arteries. These were stented with metallic endoprostheses. No strokes occurred after the placement of the stents. Mean follow-up period has been 2.5 years. CONCLUSIONS: Use of metallic endoprostheses is an effective method to treat this potentially devastating injury. However, longer follow-up and more patients studied are needed to further examine this promising treatment. PMID- 10744288 TI - Effect of soft-tissue trauma on the early periosteal response of bone to injury. AB - OBJECTIVE: To determine whether the periosteal response to skeletal trauma is impaired when muscle is also injured, thereby providing a possible explanation for why fractures with extensive soft-tissue damage may take longer to heal. METHODS: A bone defect was made in the tibia of male Fisher rats, and the proliferative response, osteoblast concentration, and callus formation that occurred within 7 days were measured in the presence and absence of simultaneously administered model soft-tissue injury (removal of 10% of the anterior tibialis muscle from a region within 2 to 3 mm of the bone defect). Measurements were made by using autoradiography, quantitative histology, and morphometry. RESULTS: Addition of the muscle injury increased proliferation in the cambium and in the fibrous periosteum on day 1, but had no effect thereafter; proliferation of fibroblasts in the loose connective tissue above the periosteum was not affected. Addition of the muscle injury resulted in increased osteoblast levels 2 to 5 days after injury but had no effect on the amount of callus produced. CONCLUSION: The inflammatory milieu created by the muscle injury unexpectedly resulted in an increased periosteal response to skeletal trauma, suggesting that inflammatory mediators generated in response to wounding of soft tissues are unlikely to account for delayed fracture healing. These findings may indicate that surgical trauma associated with internal fixation by using plates and screws may not be as deleterious to the fracture-healing response as previously thought. PMID- 10744287 TI - Treatment of toxic epidermal necrolysis with cyclosporin A. AB - BACKGROUND: Toxic epidermal necrolysis (TEN) is a severe skin disorder characterized by separation of the dermal-epidermal junction, as is observed in second-degree superficial burns. It has been proposed that immunosuppressive treatment may improve prognosis of patients with TEN. METHODS: We report here a case series of patients with TEN treated with cyclosporin A (CSA) without other concomitant immunosuppressive agent. These patients (n = 11) were consecutively admitted to our Intensive Care Burn Unit because of severe TEN, involving a large body surface area (83 +/- 17% [mean +/- SD], median, 90%; range, 35-96%) and were treated with CSA 3 mg/kg per day enterally every 12 hours. We compared the series of patients treated with CSA with a historical series of patients admitted to our Intensive Care Burn Unit before CSA was introduced as part of the treatment protocol These patients (n = 6) were treated with cyclophosphamide (150 mg i.v. every 12 hours) and different doses of corticosteroids (> or =1 mg/kg per day of 6-methyl-prednisolone). Both groups of patients were similar in regard to age, delay from onset of disease to Intensive Care Burn Unit admission, and body surface area involved. RESULTS: Time from the onset of skin signs to arrest of the disease progression (1.4 +/- 0.3 days, vs. 3.6 +/- 1.5 days) and to complete reepithelialization (12.0 +/- 3.6 days, vs. 17.6 +/- 3.1 days) was significantly shorter in patients treated with CSA compared with those treated with cyclophosphamide and corticosteroids (p = 0.0002, and p = 0.0058, respectively). Significantly fewer patients in the CSA group had > or =4 organs failing (2 of 11 vs. 3 of 6, respectively, p = 0.029), had severe leukopenia (<1,000 cells/microL) (0 of 11 vs. 4 of 6, respectively, p = 0.006), or died (3 of 6 vs. 0 of 11, respectively, p = 0.0029). CONCLUSION: We conclude that immunosuppressive treatment with CSA is safe and is associated with a rapid reepithelialization rate and a low mortality rate in patients with severe TEN. Our data suggest that this regimen could be more effective than treatment with cyclophosphamide and corticosteroids. Prospective controlled trials are required to test the hypothesis that CSA is more effective than cyclophosphamide or other immunosuppressive regimens for the treatment of TEN. PMID- 10744289 TI - Limb salvage for chronic tibial osteomyelitis: an outcomes study. AB - BACKGROUND: The decision to undergo a limb salvage procedure is difficult and multifaceted. This study reviews the outcomes of patients with chronic tibial osteomyelitis who underwent limb salvage and hopes to enhance our understanding of the impact this complex procedure has on the patient's ability to have a functional and fulfilling life. METHODS: Forty-six patients, with at least 18 months follow-up, who had undergone limb salvage for chronic, refractory tibial osteomyelitis were evaluated. A modification of the Limb Extremity Outcomes Instrument was utilized emphasizing inquiries pertaining to quality of life. RESULTS: Thirty-nine (85%) of the 46 patients were able to ambulate independently without pain. All patients younger than 45 years of age had successful outcomes. Thirty-one percent of the patients with a positive smoking history were failures, and 71% of all failures were smokers. CONCLUSION: Limb salvage seems to be a satisfactory option for patients with chronic tibial osteomyelitis. A history of smoking and advanced age may have adverse affects and are relative contraindications. PMID- 10744290 TI - Posttraumatic stress reactions after injury. AB - BACKGROUND: Many individuals experience psychological distress after injury. It is unclear whether poor psychological outcome reflects pretrauma variables. METHODS: In a prospective, cross-sectional study, 152 accident and emergency department patients with physical injuries and an acute stress reaction completed trauma and psychometric questionnaires, including the Impact of Event Scale and the Hospital Anxiety and Depression Scale. RESULTS: Although levels of pretrauma social, occupational, and familial functioning were high, and the modal Abbreviated Injury Scale score was 1, there were high levels of psychological distress at 3 weeks. Mean Hospital Anxiety and Depression Scale anxiety and depression scores were 11.8 (SD, 4.4) and 8.7 (SD, 4.4), respectively. The mean total Impact of Event Scale score was 46.0 (SD, 16.1). Stepwise linear regression analysis found unemployment and previous history of trauma to be associated with increased symptoms of traumatic stress. CONCLUSION: Trauma patients with high levels of pretrauma functioning may develop acute psychological distress. Unemployment and previous trauma increase risk. Psychological wellbeing should be considered in routine injury assessments. PMID- 10744291 TI - Continuous versus interrupted suturing of traumatic lacerations: a time, cost, and complication rate comparison. AB - BACKGROUND: The most frequently used techniques in the repair of traumatic lacerations are interrupted and continuous suturing. METHODS: We performed a prospective study of interrupted and continuous suturing techniques evaluating suturing rates, suture economy, and complication rates in the repair of 101 traumatic lacerations. RESULTS: Continuous closure was accomplished at a statistically faster rate (mean, 0.276 cm/min) than interrupted closure (mean, 0.175 cm/min; p = 0.004). Less suture material was used in the continuous closures (0.321 suture packets/cm) than in interrupted closures (0.508 suture packets/cm; p = 0.03). No statistically significant difference existed in the complication rate between the two closure methods (continuous, 1 of 44; interrupted, 1 of 57; p = 0.59). CONCLUSION: Although suturing technique should be selected primarily on the basis of wound characteristics and surgeon preference, continuous suturing warrants consideration for the closure of traumatic lacerations because of its time/material economy and the lack increased complication rates. PMID- 10744292 TI - Hospital-based trauma registries in Uganda. AB - OBJECTIVES: Toward the establishment of an injury surveillance system in Uganda, the first step was to initiate hospital-based trauma registries that generate relevant and timely data on the causes, severity, morbidity, mortality, and outcomes of injuries at Mulago and Kawolo hospitals. This would help establish injury patterns and priorities in these hospital populations. The registries are based on a minimal data set and a new injury severity instrument, the Kampala Trauma Score (KTS). The usefulness of the registry and the qualities of the KTS are presented. METHODS: The Accident and Emergency Department of Mulago, an urban 1,500-bed, tertiary hospital, and the Casualty Unit of Kawolo, a 100-bed district level hospital. Trained staff in the hospitals used a one-page, 19-item registry form to collect data on demographic, injury incident, and outcome data. The registry describes injuries based on cause, frequency, and severity. The inter rater reliability and the predictive validity of the KTS were evaluated. Registry subjects include all injured persons that come to the above hospitals. RESULTS: Results are based on the first 5,210 records. Gender distribution was 27.7% female and 71.3% male. The younger than 5 years old category was 7.4%, whereas 3.9% were older than 55 years old. Admitted patients composed 37.3% of cases, and three of four injuries were unintentional. The KTS is highly predictive of need for admission or death (adults, Az = 0.95 +/- 0.01; children, Az = 0.89 +/- 0.01). CONCLUSION: A trauma registry and injury severity measurement are both possible and useful in sub-Saharan Africa. This minimal data set and the KTS are recommended for investigators with similar resource constraints. PMID- 10744294 TI - Practice management guidelines for prophylactic antibiotic use in penetrating abdominal trauma: the EAST Practice Management Guidelines Work Group. PMID- 10744293 TI - Characteristics and outcome of injured patients treated in urban trauma centers in Iran. AB - BACKGROUND: Because of the need to improve the quality of care of trauma patients in our country, we decided to evaluate the epidemiology and find the most powerful tool for prediction of survival. The Trauma and Injury Severity Score (TRISS) has been known as conventional method for this purpose. We planned to test its ability for prediction of survival of our trauma patients, and also we wanted to compare its ability with the New Injury Severity Score (NISS) in combination with Revised Trauma Score (RTS) and age. We used the most suitable model to evaluate the trauma care in our centers. METHODS: From the Tehran University data registry on trauma patients of three different hospitals during 1 year, we selected trauma patients admitted to hospital for at least 1 day and all those patients who were declared dead at the emergency department. Epidemiologic description of patients has been given and evaluation of TRISS and (NISS + RTS + age) for prediction of survival has been performed. We determined factors affecting mortality and morbidity, evaluated hospitals, and analyzed patients admitted directly and the patients transferred from other hospitals. RESULTS: A total of 2,662 patients had complete data necessary for the calculation of probability of survival based on the TRISS method. The population at risk for trauma was the young, especially students and industrial workers. The major mechanisms of trauma were road traffic crashes and falls. The time expenditure and means of transportation as well as the time of stay in emergency department all seemed to be far less than optimal. We found that TRISS has higher performance than (NISS + RTS + age). CONCLUSION: Based on our descriptive findings, we proposed some suggestions that seem to be necessary for improvement of trauma care in our centers. Among them were improved measures for prehospital service, and emergency department and other health care units of our centers. The findings of this study suggest that conducting trauma surgery training programs and direct transportation to trauma centers can improve the outcome of trauma patients. We conclude that small sample size, mixing penetrating trauma cases with blunt trauma cases, and differences in the mechanism of trauma between study populations may be responsible for the difference between our results and others. PMID- 10744295 TI - Surgical management of cardiac arrest caused by massive pulmonary embolism in trauma patients. PMID- 10744296 TI - Coexistent rupture of the proximal right subclavian and internal mammary arteries after blunt chest trauma. PMID- 10744297 TI - Aortic root trauma: serious injuries requiring early recognition and management. PMID- 10744298 TI - Contre-coup lung injury: evidence of existence. PMID- 10744299 TI - Traumatic scapulothoracic dissociation: case report and literature review. PMID- 10744300 TI - Stenotrophomonas maltophilia and lawn mower injuries in children. PMID- 10744301 TI - Group A streptococcal necrotizing fasciitis subsequent to pectoralis muscle strain. PMID- 10744302 TI - Extending damage-control principles to the neck. PMID- 10744303 TI - Blunt laryngeal fracture: another airbag injury. PMID- 10744304 TI - Concomitant fractures of the ipsilateral femur and tibia with intra-articular extension into the knee joint. PMID- 10744305 TI - Devices for ischemic preconditioning of the pedicled groin flap. AB - BACKGROUND: Early division of the pedicled groin flap can be achieved by using ischemic preconditioning. The goal of this study was to investigate the devices available for ischemic preconditioning and determine which device is the most effective and results in lowest patient discomfort. METHODS: Rubber bands, custom made Orthoplast sheets, long-nose locking pliers, intestinal clamps, and a pneumatic tourniquet device were used for ischemic preconditioning on 13 patients who sustained severe hand injuries with reconstruction of pedicled groin flaps. The devices were compared by using laser Doppler flowmetry and the patient's local pain levels. RESULTS: Twelve of 13 flaps were successfully divided at a mean period of 8.3 days by using a custom-made Orthoplast sheet or a pneumatic tourniquet device. All devices except the rubber bands could result in a biologic zero flow level. CONCLUSION: The pneumatic tourniquet device is the most desirable ischemic preconditioning device, having the advantages of excellent ischemic effect, easy application, and minimal discomfort. PMID- 10744306 TI - Clinical measurement, statistical analysis, and risk-benefit: controversies from trials of spinal injury. AB - BACKGROUND: The National Acute Spinal Cord Injury Studies have been a series of trials assessing the role of pharmacologic agents in the prevention of secondary neuronal damage after acute spinal cord injury. METHODS: The trials were multicenter randomized, controlled studies. RESULTS: Two trials have demonstrated the efficacy of high-dose methylprednisolone in improving neurologic and functional recovery and have shown a reassuring safety profile. CONCLUSION: This study responds to a recent commentary on these trials and examines in particular the roles of clinical measurement, statistical analysis, and risk benefit in assembling evidence for or against innovative therapies. PMID- 10744307 TI - Current clinical options for the treatment and management of acute respiratory distress syndrome. PMID- 10744308 TI - Erectile dysfunction induced by orthopedic trauma managed with a fracture table. PMID- 10744309 TI - Rural trauma. PMID- 10744310 TI - Advanced separation methods for collagen parent alpha-chains, their polymers and fragments. AB - Current techniques used for collagen alpha-chains and their CNBr fragments are reviewed. Ion exchange, gel permeation, reversed-phase and affinity chromatography are discussed mainly from the preparative aspects as these are both the techniques of choice to remove biological matrix contaminants always present in collagen preparations and techniques routinely used for preparative purposes. Among electromigration procedures gel electrophoresis is widely used both for intact collagen alpha-chains and their fragments. Recently this technique was applied also for miniaturised preparations. Immunoblotting techniques serve more specific detection of otherwise hard to distinguish different collagen polypeptide chains. Capillary electromigration techniques brought recently new aspects of understanding the behaviour of collagen proteins upon different separation modes and seem to represent a smart perspective for better quantitation of individual collagen species. PMID- 10744311 TI - Development of an immobilized P-glycoprotein stationary phase for on-line liquid chromatographic determination of drug-binding affinities. AB - The membrane transporter P-glycoprotein (PGP) has been immobilized on an immobilized artificial membrane (IAM) LC stationary phase. The resulting PGP-IAM phase retained the ability of the native PGP to bind the known PGP-ligand vinblastine. Displacement studies using other known PGP ligands, verapamil and cyclosporin A, demonstrated that there was selective binding between vinblastine and the immobilized PGP transporter. The binding affinity (Kd value) of vinblastine for the PGP-IAM was determined to be 19+/-20 and 71+/-11 nM on two separate columns. These values are consistent with previously reported values of 9+/-2, 8+/-2, and 37+/-10 nM, which were obtained using native membranes. The Kd values obtained on the PGP-IAM for cyclosporin A and verapamil were 492+/-21 and 172+/-29 microM, respectively. These results were higher than the corresponding Kd values obtained using native membranes, but the relative affinities vinblastine > cyclosporin A >> verapamil are consistent in both approaches. During several months of experiments and storage, the PGP-IAM was found to be reproducible and stable. The stationary phase appears to be useful in the on-line screening for PGP ligands. PMID- 10744312 TI - High-performance affinity chromatography and immobilized serum albumin as probes for drug- and hormone-protein binding. AB - The binding of drugs and hormones to proteins within the blood is an important process in determining the transport, excretion, metabolism and activity of such agents. This paper discusses the combined use of immobilized serum albumin and high-performance affinity chromatography (HPAC) as tools for the study of such binding processes. The general approaches that are used in such work and are illustrated by several examples taken from previous work in the author's laboratory. The type of qualitative and quantitative information that can be obtained by such work is described, including the comparison of relative binding affinities, competitive displacement by other agents or the measurement of equilibrium and rate constants based on immobilized albumin columns. A comparison is also provided between the results that are obtained by these methods and those that are provided by solution-phase albumin. Some newer advances that are highlighted include use of HPAC to examine the binding of non-polar compounds to albumin, the effects of binding site heterogeneity on HPAC measurements and the use of chemically-modified albumin as a tool to examined the site-specific interactions of solutes with albumin. PMID- 10744313 TI - Chromatography on cells: analyses of solute interactions with the glucose transporter Glut1 in human red cells adsorbed on lectin-gel beads. AB - The affinities of the human red cell glucose transporter Glut1 for D-glucose and cytochalasin B (CB) and the stoichiometry of CB binding vary with the Glut1 environment. In order to study the native state of Glut1 we adsorbed human red cells to wheat germ lectin agarose gel beads for frontal affinity chromatographic analyses. Glut1 showed relatively high affinities for D-glucose (Kd 12+/-1 mM) and CB (Kd 59+/-17 nM). The number of CB-binding sites per Glut1 monomer, 0.46+/ 0.16, was approximately doubled upon coating the cells with polylysine, which induced cell association. PMID- 10744314 TI - Preparation, evaluation and application of new pseudo-affinity chromatographic supports for penicillin acylase purification. AB - New pseudo-affinity chromatographic supports for penicillin acylase were prepared and evaluated with three different samples: pure penicillin acylase, industrial clarified feedstock and crude extract. The different gels were studied for their purification fold (three to six) and their recovery power (80-100%). The best support was characterized by its dynamic capacity, (20 mg/ml) and its recovery power was tested at five flow-rates (30, 150, 300 and 750 cm/h) to determine the optimal flow-rate (300 cm/h). In addition we used cleaning in place to test the resistance to hard conditions of sanitization by 1 M NaOH (90% of recovery for 12 h of contact). These gels may therefore be used on an industrial scale. PMID- 10744315 TI - Polymeric adsorbent for removing toxic proteins from blood of patients with kidney failure. AB - A hypercrosslinked styrenic polymer with an enhanced proportion of mesopores in the range 2-20 nm has been developed. The principle of the synthesis consists of the suspension polymerization of divinylbenzene (or copolymerization of styrene with divinylbenzene) in the presence of a porogen that is a theta-solvent for polystyrene. On the scale of thermodynamic affinity, theta-solvents occupy a border position between good solvents and precipitating media for the growing polymer chains. In this case, microphase separation takes place during the final stages of the polymerization process. The polymer was shown to adsorb 93-98% of beta2-microglobulin from the blood or plasma of patients with chronic kidney failure. At the same time, large essential proteins, like albumin, are not removed to a significant extent, obviously, due to the size-exclusion effect and the difference in the hydrophobicity of the proteins. By replacing surface exposed pendant vinyl groups of the polymer with hydrophilic functional groups, the material was made hemocompatible, according to the standard battery of biocompatibility tests required by ISO 10993 guidelines. No adverse effects such as fever or hypotension were noted in dogs in direct hemoperfusion experiments with the polymer. PMID- 10744316 TI - Improved method for carbohydrate-deficient transferrin determination in human serum by capillary zone electrophoresis. AB - Carbohydrate-deficient transferrin (CDT) is a reliable marker of chronic or repeated alcohol abuse. It indicates a group of isoforms of human transferrin (Tf), the main iron transport serum protein, deficient in sialic acid residues (asialo-, monosialo- and disialo-Tf) in comparison to the main isotransferrin which contains four sialic acid groups (tetrasialo-Tf). The aim of the present work was to develop a capillary electrophoretic method suitable for rapid determination of CDT components in serum. Serum samples (0.1 ml) were saturated with iron by incubation with 10 mM FeCl3 (2 microl) and 500 mM NaHCO3 (3 microl) for 30 min, then diluted 1:10 in water and injected by positive pressure (0.5 p.s.i. for 10 s). Separation was performed with a capillary zone electrophoretic method using bare fused-silica capillaries (57 cm x 20 microm I.D.) and a buffer composed of 100 mM sodium tetraborate adjusted with 6 M HCl to pH 8.3 added with 1.5 mM diaminobutane. Applied voltage was 20 kV and temperature 25 degrees C. Detection was by UV absorption at 200 nm wavelength. Under the described conditions, asialo-, monosialo-, disialo-, trisialo- and tetrasialo-transferrin were baseline separated. The limit of detection (signal-to-noise ratio of 2) was about 0.3% for disialo-Tf, and 0.5% of trisialo-Tf, expressed as percentages of the terasialo-Tf peak area. Day-to-day RSDs of relative migration times were < or = 0.2%. Quantitation showed day-to-day RDSs < or = 6.9% and < or = 10.9% for disialo- and trisialo-Tf, respectively. The results from 79 control subjects, including social drinkers, and 23 alcoholics showed disialo- and trisialo-Tf significantly increased in patients (P<0.0001 and <0.01, respectively). A clear interference from trisialo-Tf in an immunoassay for CDT was demonstrated. The present method is suitable for confirmation of CDT immunoassays by independent technique. PMID- 10744317 TI - Analysis of cyanogenic glycosides by micellar capillary electrophoresis. AB - The separation of amygdalin, prunasin and their isomers neoamygdalin and sambunigrin could be achieved with micellar capillary electrophoresis (MEKC). The two isomers were obtained in alkaline conditions and were produced in less than 15 min at pH 11.0. The developed methods showed a good selectivity in the separation of the isomers only in the presence of SDS micelles. The working pH was optimized to allow best resolution and quantitative analysis of these compounds. With a linear calibration over an injection time from 1 to 20 s, the detection limit was found to be in the range of 5 microM (S/N=3; 20 s injection time). Two pH buffer systems (pH 5.2 and pH 9.1) were chosen to confirm the peak attributions of the compounds in the apple and peach seeds samples. Sambunigrin was found in both apple and peach seeds but could not be quantified because of missing standards. Prunasin and amygdalin were not found in the apple sample, while they were quantified in the peach seeds in concentrations of 50 microg/g and 90 microg/g (dry weight), respectively. PMID- 10744318 TI - Determination of pyrroloquinoline quinone by capillary zone electrophoresis. AB - A new method for the determination of pyrroloquinoline quinone by capillary zone electrophoresis has been developed. Separation conditions have been optimised with the respect to different parameters including pH and ionic strength of the background electrolyte, separation voltage and temperature of the capillary. A buffer consisting of 50 mM beta-alanine-HCl pH 3.0 was found to be the most suitable electrolyte for this separation. An applied voltage of 25 kV (negative polarity) and a temperature of 25 degrees C gave the best analysis of pyrroloquinoline quinone. The linear detection range for concentration versus peak area for the assay is from 5 to 500 microM (correlation coefficient 0.9998) with a detection limit of 0.1-0.2 microM. The inter-day reproducibility of the peak area was 2.5% and the inter-day reproducibility of the migration time was below 0.18%. PMID- 10744319 TI - Application of Pluronic copolymer liquid crystals for the capillary electrophoretic separation of collagen type I cyanogen bromide fragments. AB - A capillary electrophoretic method exploiting the properties of Pluronic copolymer liquid crystals (F127) was developed for the separation of collagen cyanogen bromide (CNBr) fragments. The separations obtained were at least comparable (if not better) to those obtained by other methods applicable to this category of compounds. In the optimized version a bare silica capillary [47 cm (40 cm to the detector) x 75 microm I.D.] was used with 10 mM Tris and 75 mM phosphate buffer (pH 2.5) containing 7.5% Pluronic F127 copolymer. The separation mechanism which involves both the molecular sieving and surfactant properties of the Pluronic F127 gel phase is discussed. PMID- 10744320 TI - One-step purification of R-phycoerythrin from the red macroalga Palmaria palmata using preparative polyacrylamide gel electrophoresis. AB - Phycoerythrin is a major light-harvesting pigment of red algae and cyanobacteria widely used as a fluorescent probe. In this study, phycoerythrin of the red macroalga Palmaria palmata was extracted by grinding the algal sample in liquid nitrogen, homogenisation in phosphate buffer and centrifugation. Phycoerythrin was then purified from this crude extract using preparative polyacrylamide gel electrophoresis (PAGE) with a continuous elution system and detected by its pink colour and fluorescence. The pigment presented a typical spectrum of R phycoerythrin, with three absorbance maxima at 499, 545 and 565 nm, and displayed a fluorescence maximum at 578 nm. The absorbance ratio A565/A280, a criterion for purity, was 3.2. A single protein of relative molecular mass 240,000 was detected on native-PAGE with silver staining. Sodium dodecyl sulphate-PAGE demonstrated the presence of two major subunits with Mr 20,000 and 21,000, respectively, and a very minor subunit of Mr 30,000. These observations are consistent with the (alphabeta)6gamma subunit composition characteristic of R-phycoerythrin. Phycoerythrin of Palmaria palmata was determined to be present in larger amounts in autumn and showed a good stability up to 60 degrees C and between pH 3.5 and 9.5. In conclusion, phycoerythrin of Palmaria palmata was purified in a single step using preparative PAGE. Obtaining pure R-phycoerythrin of Palmaria palmata will allow one to evaluate its fluorescence properties for future applications in biochemical techniques. PMID- 10744321 TI - How to approach substance identification in qualitative bioanalysis. AB - The ultimate goal in qualitative analysis in the biosciences is to demonstrate with acceptable probability that for an unknown constituent in a sample only one substance comes into consideration and that all other substances can be rejected. In the biosciences, identification of relevant substances in complex matrices through database retrieval is frequently required. Yet, despite its importance, the subject has not received much attention, so that progress has been limited and relevant literature is scarce. As a result, one can conclude from many publications and reports that qualitative analysis in practice is often not being addressed properly. In this paper, some fundamental aspects of qualitative analysis will be discussed and a general approach is provided for the correct identification of organic substances in complex matrices through database retrieval. Special attention is given to the choice of proper analytical techniques and their inter-laboratory standard deviations, as well as to match factors and decision criteria based on applying multiple analytical techniques, also if the latter have different dimensions (e.g. retention data and spectral data). In addition, the requirements for suitable databases are outlined and the need for inter-laboratory cooperation is emphasized. PMID- 10744322 TI - Nanotechnology in bio/clinical analysis. AB - Nanotechnology is being exploited now in different fields of analytical chemistry: Single cell analysis; in chip/micro machined devices; hyphenated technology and sampling techniques. Secretory vesicles can be chemically and individually analyzed with a combination of optical trapping, capillary electrophoresis separation, and laser induced fluorescence detection. Attoliters (10(-18) l) can be introduced into the tapered inlets of separation capillaries. Chip technology has come of age in the field of genomics, allowing faster analyses, and will fulfil an important role in RNA and peptide/protein analysis. The introduction of nanotechnology in LC-MS and CE-MS has resulted in new findings in the study of DNA adduct formation caused by carcinogenic substances, including anticancer drugs. Sample handling and introduction also can benefit from nanotechnology: The downscaling of sample volumes to the picoliter level has resulted in zeptomole (10(-21)) detection limits in the single-shot mass spectrum of proteins. PMID- 10744323 TI - Direct high-performance liquid chromatographic determination of (R)- and (S) propranolol in rat microdialysate using on-line column switching procedures. AB - Two different column-switching HPLC systems (CSWs), employing restricted access material for initial pretreatment of biological samples, were developed for the determination of propranolol enantiomers in microdialysate. CSW 1 was a single pump set-up based on an initial sample clean-up step with a RP-18 ADS precolumn coupled with an ovomucoid analytical column for direct drug enantioseparation. For the two-pump column set-up (CSW 2), a teicoplanin analytical column was applied for the enantioselective assay after initial sample pretreatment using a RP-8 ADS precolumn. The inter-day precision of the CSW 1 ranged from 0.5 to 5.1% for (R)-propranolol and from 5.1 to 10.5% for (S)-propranolol. The limit of detection (LOD) was set at 10 ng/ml and 15 ng/ml for (R)- and (S)-propranolol, respectively. Inter-day relative standard deviation values of the CSW 2 ranged from 1.1 to 9.9% for (R)-propranolol and from 1.3 to 9.6% for (S)-propranolol. The LOD of the method was 3.0 ng/ml for (R)-propranolol and 2.5 ng/ml for (S) propranolol. Both approaches were successfully applied for stereoselective monitoring of unbound propranolol levels in rat microdialysates. PMID- 10744324 TI - Molecular imprinting for drug bioanalysis. A review on the application of imprinted polymers to solid-phase extraction and binding assay. AB - Molecularly imprinted polymers have been applied as selective sorbents in several analytical techniques, including liquid chromatography, capillary electrophoresis and capillary electrochromatography, solid-phase extraction, and 'immunoassay'. An advantage of this type of sorbent is the possibility to synthesize polymers with selectivity pre-determined for a particular analyte. This review critically discusses the use of imprinted polymers for analysis of drugs and other compounds in biological samples, with emphasis on their use as highly selective solid-phase extraction sorbents for sample pre-concentration and alternative binding entities in immunoassay type protocols. PMID- 10744325 TI - Determination of lidocaine in plasma by direct solid-phase microextraction combined with gas chromatography. AB - Direct-immersion solid-phase microextraction (SPME) has been used to extract the local anesthetic lidocaine from human plasma. A simplified model shows the relationship between the total amount of drug in plasma and the amount of drug extracted. The model takes into account that the drug participates between the fiber, sample and proteins. Therefore the model can also be used to obtain a good approximation of the drug-protein binding. Extraction yields of lidocaine in plasma are <1%, and the protein binding of lidocaine was found to be about 74% at pH 9.5. A SPME method has been developed for the determination of the total amount of lidocaine in plasma. The protein binding was reduced by acidification and, subsequently, the sample was deproteinized with trichloroacetic acid. With a 100-microm polydimethylsiloxane-coated fiber and addition of sodium chloride to the sample an extraction yield of about 12% at equilibrium (45 min) has been obtained. The relative standard deviation of this method is <10%. A linear range was found from 25 to 2000 ng ml(-1) lidocaine in plasma (r=0.998) with a detection limit of 5 ng ml(-1) in plasma. An extraction yield of about 80% could be obtained after an overnight extraction by use of a 65-microm polydimethylsiloxane-divinylbenzene-coated fiber. If an extraction time of 10 min is used with this fiber, the same yield is obtained as with the single-phase fiber in 45 min. However, the drawback of this mixed-phase fiber is its much shorter lifetime. PMID- 10744326 TI - Determination of isoprene in human expired breath using solid-phase microextraction and gas chromatography-mass spectrometry. AB - An analytical method for determination of isoprene in expired breath as a marker of body cholesterol synthesis was developed with a special emphasis on breath sampling. Patients were breathing controlled air using respiratory masks for 2 min (washout period) and then their expired breath was collected in 8-1 Tedlar bags. The bags were heated to 40 degrees C and the solid-phase microextraction fiber Carboxen-polydimethylsiloxane 75 microm was inserted through the septum. Extraction time was 10 min. Analytes were desorbed in the GC injector for 2 min at 270 degrees C. Analyses were performed on a Q-PLOT column and fragment ions 68, 67 and 53 were quantified. The concentration range was 1-40 nmol/l, limit of detection was 0.25 nmol/l, the calibration curve was linear. Precision, expressed as RSD, was 5.5-12.5%. These tests are non-invasive, feasible and relatively inexpensive. PMID- 10744327 TI - Automated residue analysis of tetracyclines and their metabolites in whole egg, egg white, egg yolk and hen's plasma utilizing a modified ASTED system. AB - An automated analytical method is described allowing simultaneous determination of all tetracyclines and metabolites in whole egg, egg yolk, egg white and blood plasma of the hens. Sample pretreatment is restricted to homogenization and a dilution step. Clean-up is by on-line dialysis and on-line solid-phase extraction utilizing an extended ASTED system, followed by liquid chromatography with UV or fluorescence detection with post-column pH adjustment and confirmational analysis by LC-MS-MS. After feeding oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC) to laying hens, not only residual parent compounds could be found in the eggs but also in vivo formed 4-epimers, isochlortetracycline (ICTC) and tentatively identified N-desmethyl metabolites of OTC, TC and ICTC. PMID- 10744328 TI - Automated liquid chromatographic determination of atenolol in plasma using dialysis and trace enrichment on a cation-exchange precolumn for sample handling. AB - A fully automated method involving dialysis combined with trace enrichment was developed for the liquid chromatographic (LC) determination of atenolol, a hydrophilic beta-blocking agent, in human plasma. The plasma samples were dialysed on a cellulose acetate membrane and the dialysate was reconcentrated on a short trace enrichment column (TEC) packed with a strong cation-exchange material. All sample handling operations can be executed automatically by a sample processor (ASTED system). After TEC conditioning, the plasma sample, to which the internal standard (sotalol, another hydrophilic beta-blocker) was automatically added, was introduced in the donor channel and dialysed in the static/pulsed mode. The dialysis liquid consisted of 4.3 mM phosphoric acid. When the dialysis process was discontinued, the analytes were eluted from the TEC in the back-flush mode by the LC mobile phase and transferred to the analytical column, packed with octyl silica. The LC mobile phase consisted of phosphate buffer, pH 7.0-methanol (81:19; v/v) with 1-octanesulfonate. Atenolol and the internal standard were monitored photometrically at 225 nm. The different parameters influencing the dialysis and trace enrichment processes were optimised with respect to analyte recovery. The influence of two different kinds of cation exchange material on analyte recovery and peak efficiency was also studied. The method was then validated in the concentration range 25-1000 ng/ml. The mean recovery for atenolol was 65% and the limit of quantitation was 25 ng/ml. PMID- 10744329 TI - Analysis of riboflavin and riboflavin cofactor levels in plasma by high performance liquid chromatography. AB - We describe an assay which determines simultaneously riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in plasma, using galactoflavin (GF) as an internal standard. The flavins were extracted on a C18 Sep-Pack cartridge after protein precipitation with 10% trichloroacetic acid, and were analyzed on a C18 RP-HPLC with 85% phosphate-magnesium acetate buffer (pH 3.4) and 15% acetonitrile. FAD, FMN, GF and RF extraction recoveries were 101.0 5.6, 97.0-6.5, 97.0-2.0 and 95.0-4.1%, and reproducibilities were 5.9, 6.8, 2.1 and 4.3%, respectively. FAD, FMN and RF values in infant and adolescent plasma were in the range 53.5-108.2, 9.0-25.1 and 12.7-53.4 nM, and 36.5-157.20, 7.1 24.6 and 8.2-57.8 nM, respectively. Using GF as an internal standard improved the quantification of these B2 vitamers. PMID- 10744330 TI - Biomonitoring of environmental polycyclic aromatic hydrocarbon exposure by simultaneous measurement of urinary phenanthrene, pyrene and benzo[a]pyrene hydroxides. AB - A high-performance liquid chromatographic method with fluorescence detection was developed which enables the simultaneous determination of the urinary polycyclic aromatic hydrocarbon metabolites 3-hydroxyphenanthrene, 1-hydroxypyrene and 3 hydroxybenzo[a]pyrene. The method has small solvent consumption because of the use of a microbore RP C18 column and a relatively short run time. Low detection limits of 0.02 nmol/l for 3-hydroxypyrene to 0.19 nmol/l for 3 hydroxybenzo[a]pyrene were attained. In contrast, the detection limits of alpha naphthol and 9,10-dihydroxy-9,10-dihydrophenanthrene were not adequate for the determination of environmental exposure. The developed method was successfully used for the analysis of urine samples from children. PMID- 10744331 TI - Synthesis and biological activities of lipid A-type pyrancarboxylic acid derivatives. AB - The synthesis of lipid A-type pyrancarboxylic acid derivatives, which have a carboxylic acid group in the anomeric position of the reducing part of the disaccharide instead of the phosphate group in lipid A, is described. One of the compounds thus synthesized, which has an acyl substitution pattern similar to that of Escherichia coli lipid A, showed lipopolysaccharide (LPS)-agonistic activity. The other, which contains four lipid chains in the molecule, exhibited strong LPS-antagonistic activity toward human monoblastic U937 cells. PMID- 10744332 TI - Synthesis and NMR study of a heptasaccharide, epitope of the stage-specific embryonic antigen-1 (SSEA-1). AB - This paper describes an efficient synthesis of the beta-2-trimethylsilylethyl glycoside of lacto-N-fucoheptaose based on a highly stereo- and regioselective glycosylation between a Lewis(x) trisaccharidic donor and a tetraol tetrasaccharidic acceptor. The title compound was characterized by high resolution NMR spectroscopy. PMID- 10744333 TI - Conformational studies of the glycopeptide Ac-Tyr-[Man5GlcNAc-beta-(1-->4)GlcNAc beta-(1-->Ndelta)]-Asn-Leu-Thr-Se r-OBz and the constituent peptide and oligosaccharide. AB - Glycopeptides of desired structure can be conveniently prepared by the coupling of reducing oligosaccharides to aspartic acid of peptides via their glycosylamines formed in the presence of saturated aqueous ammonium hydrogen carbonate. The resulting oligosaccharide chains are N-linked to asparagine as in natural glycoproteins, allowing different peptide oligosaccharide combinations to be analysed for conformational effects. In the present paper, a pentapeptide of ovalbumin was coupled to Man5GlcNAc2 oligosaccharide and the glycopeptide and the two parent compounds compared by NMR ROESY experiments and molecular dynamics simulations. Despite the small size of the peptide, conformational effects were observed suggestive of the oligosaccharide stabilising the peptide in solution and of the peptide influencing oligosaccharide conformation. These effects are relevant to the function of glycosylation and the enzymic processing of oligosaccharide chains. PMID- 10744334 TI - Extracellular polysaccharide of Erwinia chrysanthemi A350 and ribotyping of Erwinia chrysanthemi spp. AB - Erwinia chrysanthemi spp. are gram-negative bacterial phytopathogens causing soft rots in a number of plants. The structure of the extracellular polysaccharide (EPS) produced by the E. chrysanthemi strain A350, which is a lacZ- mutant of the wild type strain 3937, pathogenic to Saintpaulia, has been determined using a combination of chemical and physical techniques including methylation analysis, low-pressure gel-filtration and anion-exchange chromatography, high-pH anion exchange chromatography, partial acid hydrolysis, mass spectrometry and 1- and 2D NMR spectroscopy. In contrast to the structures of the EPS reported for other strains of E. chrysanthemi, the EPS from strain A350 contains D-GalA, together with L-Rhap and D-Galp in a 1:4:1 ratio. Evidence is presented for the following hexasaccharide repeat unit: [structure: see text] All the Erwinia chrysanthemi spp. studied to date have been analyzed by ribotyping and collated into families, which are consistent with the related structures of their EPS. PMID- 10744335 TI - A simple preparation of half N-acetylated chitosan highly soluble in water and aqueous organic solvents. AB - A simple and improved method of preparing highly soluble chitosan (half N acetylated chitosan) was developed using a series of chitosan samples of low molecular weights, and the solubility of the half N-acetylated chitosan in water and organic solvents was investigated in detail. To reduce the molecular weight, chitosan was treated with NaBO3 under the condition that chitosan was homogeneously dissolved in aqueous acetic acid. Weight-average molecular weights of the obtained chitosan samples were determined using a size-exclusion chromatography system equipped with a low-angle laser light-scattering photometer. Each chitosan sample was then N-acetylated with acetic anhydride under the condition that chitosan was homogeneously dissolved in aqueous acetic acid again. The water solubility of the half N-acetylated chitosan thus prepared increased with decreasing molecular weight. From 1H NMR spectroscopy, it was suggested that the sequence of N-acetylglucosamine and glucosamine residues was random. The solubility of the half N-acetylated chitosan of low molecular weight was rather high even in aqueous dimethylacetamide and dimethylsulfoxide. PMID- 10744336 TI - Preparation and characterisation of copper(II) hyaluronate. AB - Amorphous copper complexes of the general composition Cu(C14H20O11N)2 x xH2O have been prepared with high- and low-molecular-weight hyaluronic acid (HA). Optimal conditions for preparation are obtained at pH values from 5.0 to 5.5, with a molar ratio of HA versus Cu2+ of 1:1, and at a mass concentration of 5 and 10 mg/mL for high- (Mw = 1.8 x 10(6) Da) and low-molecular-weight sodium hyaluronate (Mw = 2 x 10(5) Da), respectively. The coordination polyhedron of the copper ion has been elucidated by EXAFS and XANES spectroscopy. Copper atoms are octahedrally coordinated in both cases with four equatorial Cu-O bond lengths of 1.95 A, and two axial Cu-O bonds of 2.46 A. Visible spectra of acidic aqueous solution suggest that substitution of axial oxygens by NH groups occurs at pH 6.5 or higher. If the pH value of the copper(II) hyaluronate solution increases above 6.5, the coordination of copper(II) changes. It is very likely that the N atom coming from the acetamido group enters into the coordination sphere of the copper(II) ion. PMID- 10744337 TI - Size-exclusion chromatography study of the molecular-weight distribution of gamma irradiated pullulan. AB - The polyelectrolyte behavior of gamma-irradiated pullulan in aqueous solutions leads to secondary effects of adsorption on a Shodex OHPak KB 806 column gel during size-exclusion chromatography. Suppression of the polyelectrolyte properties of gamma-irradiated pullulan is achieved by using a 0.05 M aqueous solution of NaH2PO4 (pH 4.95) as the mobile phase. Under these conditions, adequate molecular-weight distributions of gamma-irradiated pullulan samples are obtained. PMID- 10744338 TI - Inhibition of transcription factors by anti-inflammatory and anti-rheumatic drugs: can variability in response be overcome? AB - 1. The drugs used in the treatment of rheumatoid arthritis (RA) form a diverse group with unpredictable adverse effects, mostly weak efficacy and variable responses. Despite their differences, a common feature of many anti-inflammatory and disease-modifying anti-rheumatic drugs (DMARD) is inhibition of pro inflammatory transcription factors, particularly nuclear factor (NF)-kappaB and activator protein (AP)-1. 2. The present brief review identifies those drugs capable of inhibiting transcription factors, particularly steroids, gold salts, D penicillamine, cyclosporine A and possibly salicylates. 3. The newer biological inhibitors of tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta are capable of indirect inhibition of NF-kappaB activation, although even with these potent agents the problem of variability in response has not disappeared. 4. The development of selective inhibitors of the transcription factor NF-kappaB should have the benefit of the anti-inflammatory drugs and DMARD, both new and old. 5. It is hypothesized that this strategy will overcome much of the variability in the therapeutic response and adverse effects that limit the usefulness of the existing drugs in the treatment of RA. PMID- 10744339 TI - Macromolecular crystallography as a tool for investigating drug, enzyme and receptor interactions. AB - 1. Protein crystallography is an essential tool for the discovery and investigation of pharmacological interactions at the molecular level. It allows investigators to directly visualize the three-dimensional structures of proteins, including enzymes, receptors and hormones. 2. Increasingly, knowledge of these interactions is being used in the drug-discovery process. This is popularly called structure-based drug design. The desired drug could be an enzyme inhibitor or an agonist that mimics endogenous transmitters or hormones. 3. Once the 3-D structure of a pharmacologically relevant target is known, computational processes can be used to search databases of compounds to identify ones that may interact strongly with the target. Lead compounds can be improved using the 3-D structure of the complex of the lead compound and its biological target. 4. The present review describes the processes involved in the determination of a structure by means of protein crystallography and the use of structures in the drug-discovery process. A number of successful examples of structure-based drug design are described. The limitations of the techniques are discussed. PMID- 10744340 TI - Chemistry and biological effects of dietary phenolic compounds: relevance to cardiovascular disease. AB - 1. There has been considerable recent interest in the possibility that increased intake of dietary anti-oxidants may protect against cardiovascular disease. This is partly due to the knowledge that oxidative events in vivo may play a role in the pathogenesis of atherosclerosis. 2. While dietary anti-oxidants, such as vitamins E and C, have received considerable attention in this regard, relatively little is known about a similar anti-oxidant role for plant-derived polyphenolic compounds, such as the flavonoids and phenolic acids. A review of the distribution, bioavailability and biological activity of these compounds suggests that they may have a physiological role as anti-oxidants. 3. Human trials on the anti-oxidant effects of beverages rich in polyphenolics, such as red wine, fruit juice or tea, have been limited and results are, at present, inconclusive. This is due, in part, to poor methodologies available to measure oxidative damage in vivo. 4. There is a sound rationale for considering polyphenolics as important contributors to the dietary anti-oxidant intake derived from fruits and vegetables. However, continuing research is needed using appropriate biomarkers of oxidant damage in vivo before these compounds can be conclusively considered as dietary anti-oxidants with nutritional benefit. PMID- 10744341 TI - Oxidative status and anti-oxidant enzyme activity during calcium paradox in the rat isolated heart. AB - 1. The effect of calcium paradox on oxidative status and the activity of anti oxidant enzymes were studied in the rat isolated heart. Glutathione status, sulphydryl group contents and lipid peroxidation in the myocardium, as well as the release of oxidized and reduced glutathione from the heart, were taken as indices of oxidative events. 2. Reperfusion with calcium after calcium-free perfusion induced a significant decrease in the myocardial content of reduced and oxidized glutathione and non-protein sulphydryl groups. At the same time, a significant release of both forms of glutathione from the heart was observed. However, the ratio of oxidized to reduced glutathione remained unchanged and was not different from control. Increased lipid peroxidation was observed only after 30 min of reperfusion with calcium. 3. Increased anti-oxidant activity during the reperfusion period was observed. Mitochondrial Mn-superoxide dismutase (SOD) activity was increased throughout the reperfusion period, while cytoplasmic Cu,Zn SOD and glutathione peroxidase activity showed a transient increase at 5 min reperfusion. 4. The results do not support an important role of oxygen free radicals in cell damage observed during calcium paradox in the rat isolated heart. Production of oxygen free radicals may occur during the reperfusion period, but the quantity produced is insufficient to exceed the anti-oxidant capacity of the heart. PMID- 10744342 TI - Effect of dopamine agonists and antagonists on the lorazepam withdrawal syndrome in rats. AB - 1. The effects of dopaminergic agonists and antagonists were investigated on withdrawal signs in lorazepam-dependent rats. Physical dependence was developed by giving lorazepam admixed with food in the following dose schedules: 10 x 4, 20 x 4, 40 x 4, 80 x 4 and 120 x 7 mg/kg, daily for x days. 2. The parameters observed during the periods of administration of lorazepam and after its withdrawal were spontaneous locomotor activity (SLA), reaction time to pain, foot shock aggression (FSA) and audiogenic seizures. 3. During the withdrawal period, rats were divided into groups of 10 rats each. One group did not receive any drug and served as the control withdrawal groups. Three other groups received, separately, one of the following dopamimetic drugs: (i) 200 mg/kg per day, i.m., L-dihydroxyphenylalanine (DOPA; +50 mg/kg per day, i.m., carbidopa); (ii) 2 mg/kg per day, i.m., amphetamine; or (iii) 1 mg/kg per day, i.m., apomorphine. The remaining groups received one of the following dopamine antagonists: (i) 0.1 mg/kg per day, i.m., SCH 23390; (ii) 0.5 mg/kg per day, i.m., haloperidol; (iii) 0.5 mg/kg per day, i.m., centbutindol; and (iv) either 1 or 20 mg/kg per day, i.m., clozapine. 4. The withdrawal signs observed in the control group were hyperkinesia, hyperaggression and audiogenic seizures. 5. L Dihydroxyphenylalanine (+ carbidopa), amphetamine and apomorphine potentiated hyperaggression and audiogenic seizures. The dopamine D2 receptor antagonists haloperidol, centbutindol and clozapine (at 20 mg/kg, i.m.) blocked all withdrawal signs. The D1 receptor antagonist SCH 23390 inhibited hyperkinesia and hyperaggression. The D4 receptor antagonist clozapine (at 1 mg/kg, i.m.) had no effect on any of the withdrawal signs. 6. It may be concluded that dopamine D2 receptors exert a dominant facilitatory influence, with partial contribution of D1 receptors, on the benzodiazepine withdrawal syndrome. PMID- 10744343 TI - T-0162, a novel free radical scavenger, reduces myocardial infarct size in rabbits. AB - 1. We investigated the effects of 1-(3-tert-butyl-2-hydroxy-5-methoxyphenyl)-3-(3 pyridylmethyl)urea hydrochloride (T-0162), a novel low-molecular weight free radical scavenger, on the generation of superoxide anions and hydroxyl radicals in vitro and in vivo and on myocardial infarct (MI) size in an in vivo model of MI in rabbits. 2. It was found that T-0162 scavenged both superoxide anions and hydroxyl radicals in a concentration-dependent manner in vitro. 3. In an in vivo rabbit model with 30 min coronary occlusion and 30min reperfusion, T-0162 scavenged hydroxyl radicals generated in the myocardium during reperfusion. 4. Anaesthetized open-chest Japanese white male rabbits were subjected to 30 min coronary occlusion and 48 h reperfusion. The control group (n = 10) was infused with 10% lecithin solution for 220 min from 10 min before occlusion to 180 min after reperfusion. The pretreatment group (n = 10) was infused with T-0162 dissolved in 10% lecithin solution for 220 min from 10 min before occlusion to 180 min after reperfusion at a rate of 400 microg/kg per min. The post-treatment group (n = 10) was injected with an i.v. bolus of 10 mg/kg T-0162 and was then infused with 400 microg/kg per min T-0162 for 190 min from 10 min before reperfusion to 180 min after reperfusion. After 48 h reperfusion, infarct size was measured histologically and expressed as a percentage of area at risk (AAR). 5. There was no significant difference in haemodynamic parameters among the three groups throughout the experimental period. The per cent infarct size of the AAR in the T-0162 groups (24.8+/-4.3 and 30.5+/-3.9% for pre- and posttreatment groups, respectively) was significantly reduced compared with control (44.7+/ 4.1%; P<0.05). There was no significant difference in the AAR among the three groups. 6. In conclusion, T-0162 reduces MI size through the inhibition of reperfusion injury. PMID- 10744344 TI - Pharmacokinetics of several subcutaneous doses of erythropoietin: potential implications for blood transfusion. AB - 1. The aim of the present study was to assess the effectiveness of repeated subcutaneous low-dose recombinant human erythropoietin (rHuEPO) on parameters associated with improved procurement of autologous blood; a procedure regularly used to preclude the need for homologous blood transfusion at the time of elective surgery. 2. Three groups of three volunteers each (n = 9) were administered one of three low doses of rHuEPO (30, 60 or 100 IU/kg bodyweight, s.c.) on days 1, 4 and 8. The plasma pharmacokinetic profile of rHuEPO was studied after the first and third injections. Statistical evaluations were intragroup and intraindividual comparisons. 3. There was a linear relationship between maximum plasma concentration (Cmax) and dose. In the overall study group, Cmax and area under the curve (AUC) were significantly decreased, while the mean residence time (MRT) and elimination half-life (t1/2beta) were significantly increased on day 8 relative to day 1. Significant and sustained increases in reticulocytes were observed after rHuEPO administration, which were maintained above the predose values throughout the study period. 4. In conclusion, rHuEPO, by subcutaneous repeat-dose, was eliminated more slowly and remained longer in the circulation, despite lowered plasma concentrations. Repeated low rHuEPO administration at doses > or = 60 IU/kg bodyweight stimulated modest but sustained reticulocyte concentrations, suggesting that cost may be substantially decreased in autologous blood donation or perioperative treatment programmes. PMID- 10744345 TI - Chronotropic and vasodilatory responses to adenosine and isoproterenol in mouse heart: effects of adenosine A1 receptor overexpression. AB - 1. Chronotropic and vasodilatory effects of adenosine receptor activation with 2 chloroadenosine (2-ClAdo) and beta-adrenoceptor activation with isoproterenol were studied in wild-type murine hearts and transgenic hearts overexpressing the A1 adenosine receptor. 2. Treatment of wild-type hearts with 2-ClAdo induced bradycardia (pEC50 6.4+/-0.2) and vasodilatation (pEC50 7.9+/-0.1; minimal resistance 2.2+/-0.2 mmHg/mL per min per g). The A1 receptor-mediated bradycardia was 20-fold more sensitive in transgenic hearts (pEC50 7.7+/-0.2), whereas coronary vasoactivity of 2-ClAdo was unaltered (pEC50 7.6+/-0.1). 3. beta Adrenoceptor stimulation with isoproterenol increased heart rate (pEC50 8.5+/ 0.2; maximal rate 594+/-23 b.p.m.) and produced vasodilation (pEC50 8.7+/-0.1; minimal resistance 1.7 +/-0.2 mmHg/ml, per min per g) in wild-type hearts. Treatment with 10 IU/mL adenosine deaminase increased the magnitude of the tachycardia (maximal rate 653+/-27 b.p.m.) without altering potency (pEC50 8.5+/ 0.1). Antagonism of A1 receptors with 10nmol/L 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) produced a comparable increase in the magnitude of the chronotropic response (maximal rate 695+/-26b.p.m.) without altering potency (pEC50 8.3+/ 0.1). 4. Isoproterenol-mediated vasodilatation was unaltered by transgenic A1 receptor overexpression. Overexpression of A1 receptors significantly reduced the maximal heart rate during beta-adrenoceptor stimulation by 35% (to 381 +/-28 b.p.m.) without altering potency (pEC50 8.4+/-0.2). At 10nmol/L, DPCPX increased the magnitude of the chronotropic response to isoproterenol in transgenic hearts (maximal heart rate 484+/-36 b.p.m.) without altering potency (pECs50 8.3+/-0.2). 5. The data show that transgenic A1 receptor overexpression selectively sensitizes the cardiovascular A1 receptor response and that A1 receptor activation by endogenous adenosine depresses the magnitude, but not potency, of the beta-adrenoceptor-mediated chronotropic response in mouse heart. The A1 receptor-mediated depression of beta-adrenoceptor responsiveness is non competitive (reduced response magnitude with no change in sensitivity). This indicates that A1 receptor activation non-competitively inhibits effector mechanisms activated by beta-adrenoceptors (e.g. adenylate cyclase) and/or A1 receptors activate unrelated but opposing mechanisms. This inhibitory response may have physiological importance during periods of sympathetic stimulation of cardiac work. PMID- 10744346 TI - Role of brain angiotensin II in the somatosensory induced antinatriuresis in the anaesthetized rat. AB - 1. The present study set out to explore the importance of angiotensin (Ang)II in the brain in allowing the somatosensory system to cause a reflex renal nerve mediated reduction in renal sodium and water excretion. 2. In chloralose-urethane anaesthetized rats receiving saline i.c.v. (2 microL + 1 microL/h), the administration of capsaicin (0.5 mg, s.c.) increased blood pressure by 14% (P < 0.001) and, while renal perfusion pressure was regulated at an unchanged level, neither renal blood flow (RBF) nor glomerular filtration rate was changed. However, urine flow and absolute and fractional sodium excretion was reduced between 29 and 38% (P<0.05-0.01). All variables had returned to control levels 30 min later. 3. The administration of captopril (40 microg + 20 microg/h i.c.v.) decreased blood pressure and sodium excretion by 6 and 17%, respectively (both P < 0.05). Under these conditions, capsaicin s.c. increased blood pressure by 9% (P<0.05); however, with renal perfusion pressure regulated at a constant level, neither renal haemodynamics nor water nor sodium excretion were changed. 4. A final group of animals received AngII (100 ng + 50 ng/h) concomitantly with captopril i.c.v., which increased blood pressure, RBF and urine flow, absolute and fractional sodium excretions by 8 (P < 0.05), 22 (P < 0.001 ) and 52-149% (P < 0.05-0.01), respectively. Capsaicin given s.c. under these conditions increased blood pressure by 6% (P < 0.05) and, while renal perfusion pressure was maintained at an unchanged value and renal haemodynamics remained constant, urine flow and absolute and fractional sodium excretion were reduced by 35-38% (all P < 0.05). 5. These data show that for the somatosensory system to induce a reflex increase in renal sympathetic nerve activity sufficient to cause an antinatriuresis and antidiuresis, the presence of AngII is necessary in the brain. How AngII exerts this facilitatory action within the central nervous system remains to be investigated. PMID- 10744347 TI - Nitric oxide production and hepatic dysfunction in patients with postoperative sepsis. AB - 1. Although hepatic function is well known to deteriorate following bacterial infection, the underlying mechanisms remain poorly understood. We have previously reported that nitric oxide (NO) radical leads to a decrease in the ketone body ratio (KBR) and in ATP content due to the inhibition of mitochondrial electron transport in primary cultured rat hepatocytes. 2. To evaluate the effects of NO radical on the liver in patients with postoperative sepsis, we analysed both the stable end-product of nitric oxide radical (NOx) as well as the arterial KBR (AKBR), which reflects liver tissue NAD+/NADH. 3. Twenty patients who had undergone general abdominal surgery and who developed postoperative sepsis were divided into two groups: (i) surviving; and (ii) non-surviving. Blood samples were collected before the development of postoperative sepsis and every 3 days until the patient either died or was discharged from hospital. 4. Plasma NOx levels in seven patients who subsequently died became progressively higher than those in the 13 surviving patients over the clinical course of postoperative sepsis. 5. In the non-surviving group, the AKBR was significantly lower than in surviving patients, indicating impaired hepatic function. In contrast, plasma NOx levels in non-surviving patients were significantly higher than in surviving patients. 6. Decreases in AKBR to levels below 0.7 in non-surviving patients followed high NOx levels. Moreover, plasma NOx levels were closely correlated with the AKBR, indicating that NO radical is associated with mitochondrial dysfunction in the liver. 7. It is likely that the overproduction of NO radical plays an important role in causing fatal metabolic disorders in patients with postoperative sepsis. PMID- 10744348 TI - Effects of beta-adrenoceptor stimulation on pacing-induced failure of dog hypertrophic hearts. AB - 1. We tested the hypothesis that the transition to pacing-induced failure in hypertrophic hearts would result in reduced functional and metabolic responses to beta-adrenoceptor stimulation. 2. Isoproterenol (ISO; 0.1 microg/kg per min) was infused into a coronary artery in five anaesthetized open-chest control, five aortic stenosis-induced left ventricular hypertrophy (LVH) and five LVH pacing induced failure dogs. 3. In both control and LVH dogs, but not in failure dogs, ISO significantly increased local regional work (1,923+/-665 vs 2,656+/-715, 1,185+/-286 vs 1,906+/-562 and 835+/-106 vs 849+/-216g.mm/min, respectively), force (11.1+/-1.4 vs 16.9+/-2.6, 8.6+/-1.5 vs 13.7+/-2.3 and 12.2+/-1.1 vs 11.0+/ 1.8g, respectively) and myocardial O2 consumption (7.3+/-2.0 vs 10.0+/-1.5, 8.2+/ 1.6 vs 11.6+/-2.6 and 4.4+/-1.5 vs 5.5+/-1.8 mL O2/min per 100 g, respectively). 4. Isoproterenol also significantly increased cAMP in control and LVH dogs (474+/ 67 vs 600+/-91 and 473+/-34 vs 619+/-53 pmol/g, respectively). In heart failure, cAMP was significantly lower and there was no significant increase in cAMP in response to ISO (245+/-43 vs 314+/-40pmol/g, respectively). 5. We conclude that there were no significant myocardial functional, O2 consumption or cAMP responses to ISO after the transition from hypertrophy to cardiac failure. PMID- 10744350 TI - Multiple inferences using confidence intervals. AB - 1. In a recent review article, the problem of making false-positive inferences as a result of making multiple comparisons between groups of experimental units or between experimental outcomes was addressed. 2. It was concluded that the most universally applicable solution was to use the Ryan-Holm step-down Bonferroni procedure to control the family-wise (experiment-wise) type 1 error rate. This procedure consists of adjusting the P values resulting from hypothesis testing. It allows for correlation among hypotheses and has been validated by Monte Carlo simulation. It is a simple procedure and can be performed by hand. 3. However, some investigators prefer to estimate effect sizes and make inferences by way of confidence intervals rather than, or in addition to, testing hypotheses by way of P values and it is the policy of some editors of biomedical journals to insist on this. It is not generally recognized that confidence intervals, like P values, must be adjusted if multiple inferences are made from confidence intervals in a single experiment. 4. In the present review, it is shown how confidence intervals can be adjusted for multiplicity by an extension of the Ryan-Holm step-down Bonferroni procedure. This can be done for differences between group means in the case of continuous variables and for odds ratios or relative risks in the case of categorical variables set out as 2 x 2 tables. PMID- 10744349 TI - Discrimination of histamine H1 and muscarinic receptor-mediated signalling pathways by phorbol ester in human astrocytoma cells. AB - 1. Histamine H1 receptor-mediated signalling was compared with muscarinic receptor-mediated signalling in 1321N1 human astrocytoma cells. 2. Short-term (2 min) treatment of cells with phorbol 12-myristate 13-acetate (PMA) resulted in a reduction of increases in intracellular Ca2+ ([Ca2+]i) induced by carbachol or histamine. 3. Carbachol-induced increases in [Ca2+]i were 10-fold more sensitive to PMA than the histamine-induced increases. 4. When cells were treated with PMA for 48 or 72 h (long-term treatment), protein kinase C (PKC) was down-regulated and PMA did not inhibit carbachol-induced increases in [Ca2+]i. 5. Histamine induced increases in [Ca2+]i were significantly reduced by long-term treatment with PMA. 6. These findings suggest that the signalling pathways mediated by histamine H1 and muscarinic receptors can be distinguished by using PKC in 1321N1 human astrocytoma cells. PMID- 10744351 TI - Excitation-contraction coupling in skeletal muscle: comparisons with cardiac muscle. AB - 1. The present review describes the mechanisms involved in controlling Ca2+ release from the sarcoplasmic reticulum (SR) of skeletal muscle, which ultimately regulates contraction. 2. Comparisons are made between cardiac and skeletal muscle with respect to: (i) the role of the dihydropyridine receptors (DHPR) as Ca2+ channels and voltage-sensors; (ii) the regulation of the ryanodine receptor (RyR)/Ca2+-release channels in the SR; and (iii) the importance of Ca2+-induced Ca2+ release. 3. It is shown that the key differences of the skeletal muscle Ca2+ release channel (RyR1), namely the increase in its stimulation by ATP and its inhibition by Mg2+, are critical for its direct regulation by the associated DHPR and, consequently, for the fast, accurate control of skeletal muscle contraction. PMID- 10744352 TI - Skeletal muscle metabolism during exercise in humans. AB - 1. Contracting skeletal muscle is able to use a number of intra- and extramuscular substrates to generate ATP during exercise. These include creatine phosphate (CP), muscle glycogen, blood-borne glucose, lactate and free fatty acids (FFA), derived from either adipose tissue or intramuscular triglyceride stores. 2. During high-intensity short-duration exercise, CP degradation and the breakdown of muscle glycogen to lactate are the major energy yielding pathways, although oxidative metabolism can make a significant contribution. The 'anaerobic' substrates are also important fuels during the transition from rest to steady state exercise. 3. The oxidative metabolism of carbohydrate and lipid supplies most, if not all, of the ATP during prolonged submaximal exercise. Muscle glycogen, blood glucose and FFA are the key fuels. The relative importance of the various substrates for exercise metabolism is primarily determined by exercise intensity and duration, although training status, dietary manipulation and environmental factors can modify the metabolic response to exercise. PMID- 10744353 TI - Imaging and nano-manipulation of single actomyosin motors at work. AB - 1. Muscle contraction is achieved by the sliding movement of myosin and actin using the energy of ATPase. 2. Our research has focused on the question of how chemical energy is used to perform mechanical work. Recent developments of single molecule imaging and manipulation techniques have allowed us to study the chemical and mechanical events at a molecular level. 3. There are many lines of evidence that show that the energy liberated from ATPase is stored in the actomyosin molecules for later use. 4. The displacement produced by a single ATP molecule is made up of several steps, each of 5.3 nm. Sometimes the mechanical event is delayed after the ADP is released from myosin. 5. This storage of energy may be explained by a slow conformational transition between the metastable states of the proteins. This suggestion has been supported by results obtained using single-molecule fluorescence spectroscopy. PMID- 10744354 TI - Persistent improvement of cardiovascular risk factors in spontaneously hypertensive rats following early short-term captopril treatment. AB - This study was designed to determine whether an improvement in cardiovascular risk factors persists in spontaneously hypertensive rats (SHR) following withdrawal of angiotensin converting enzyme inhibitor (ACE-I) treatment. SHR were given deionized drinking water or captopril solution from four to sixteen weeks of age. At twelve weeks of age, rats from each group were instrumented with radiotelemetry devices for continuous monitoring of blood pressure. Mean arterial blood pressure was significantly lower in captopril-treated SHR during treatment (92+/-2 vs 147+/-1 mm Hg), and at twelve weeks after treatment withdrawal (131+/ 2 vs 158+/-2 mm Hg). In addition, proteinuria, renal vascular resistance, plasma triglyceride levels, fasting glucose levels, post-prandial insulin levels, and heart weights were significantly reduced in the treated SHR compared to control SHR, at time-points between three to seven months after captopril withdrawal. Our findings indicate that short-term administration of an ACE-I during the developmental phase of hypertension in the SHR results in a long-term overall improvement of cardiovascular risk factors. PMID- 10744355 TI - Reverse relationship between mitogen activated protein kinase and human platelet aggregation. AB - The role of MAPK in platelets was investigated. In human platelets maintained at 4 degrees C for 2 hr, the MAPK activity increased (approximately 2 fold) when compared to those maintained at 37 degrees C. When aggregation was monitored under these conditions, the platelets maintained at 4 degrees C or 15 degrees C showed an 85% and 71% decrease respectively to thrombin (0.5 U/ml for 1 min) induced aggregation. When the platelet cytosol was maintained at 4 degrees C and assayed for MAPK activity, the MAPK activity decreased significantly, indicating that the observed effects are seen in intact platelets only, and are not due to temperature effects on the assay. When platelets maintained at 4 degrees C or 15 degrees C (for 2 hrs) were transferred to 37 degrees C, the MAPK activity decreased to levels observed in platelets maintained throughout at 37 degrees C and was thus reversible. Therefore, it is concluded that a possible reverse relationship between MAPK and platelet aggregation plays a role in platelet responses. PMID- 10744356 TI - Cardiovascular responses to simulated microgravity in Sprague-Dawley rats. AB - Microgravity is known to induce orthostatic intolerance and baroreflex impairment in astronauts. Cardiovascular responses observed in 30 degrees head-down tilt rat models, whether 24 hr whole body suspension (WBS) or 7 day tail-suspension (TS), mimic observations made during exposure to microgravity. We evaluated the cardiovascular effects of simulated microgravity and the subsequent post suspension in rats using the above models. Mean arterial pressure (MAP) of both WBS and TS rats did not change during suspension. In both models, MAP decreased post-suspension and this response lasted for 6 hrs. Salt-loaded animals did not show a post-suspension reduction in MAP. Plasma ionized calcium was decreased at 2 hr of WBS, with no change in sodium, potassium, magnesium, glucose, or hematocrit. Body weight changes were similar for all animals whether under suspension or control conditions. Both rat models demonstrate post-suspension hypotension and these results support the notion that salt-loading may have some beneficial effects in ameliorating this hypotension. PMID- 10744357 TI - Ischemic preconditioning and infarct mass: the effect of hypercholesterolemia and endothelial dysfunction. AB - In an experimental model of atherosclerosis we investigated whether rabbits fed an atherogenic diet (0.25% cholesterol, 3% coconut oil) develop endothelial dysfunction accompanied with increased infarct mass compared to normal fed rabbits and, whether hypercholesterolemia would interfere with the beneficial outcome of ischemic preconditioning observed in normal rabbits. After four weeks on either a normal or an atherogenic diet, New Zealand White rabbits (n=7 in each group) were subjected to 30 min of myocardial ischemia by occlusion of a branch of the left anterior descending coronary artery (LAD) followed by 2 hours of reperfusion (infarct studies). For ischemic preconditioning experiments, LAD was additionally occluded twice for 5 min followed by 10 min reperfusion before the long-lasting (30 min) ischemia. Infarct mass was evaluated by triphenyl tetrazolium staining. Besides the assessment of aortic endothelium-dependent function and NO-release, aortic and cardiac vessels were inspected for atherosclerotic lesions. Total cholesterol serum levels in rabbits on an atherogenic diet were significantly higher (15.3+/-2.7 mmol/L) than those on a standard diet (0.65+/-0.08 mmol/L). The aortas and heart vessels were without any histological evidence of atherosclerosis, whereas endothelial dysfunction and significantly reduced calcium-ionophore stimulated endothelial NO-release were found in isolated aortic rings of hypercholesterolemic animals. Rabbits on a standard diet showed an infarct mass (related to the area at risk) of 41+/-33%, which was reduced to 21+/-2% by ischemic preconditioning (49% decrease, p<0.05). In rabbits on an atherogenic diet, infarct mass was significantly increased to 63+/-3% (52% increase versus standard diet). Interestingly, hypercholesterolemia did not affect the beneficial influence of ischemic preconditioning; infarct mass (21+/-3%, p<0.05 vs hypercholesterolemia) was similar to rabbits on a standard diet with ischemic preconditioning. Our results show that experimental hypercholesterolemia increases infarct mass in nonpreconditioned hearts but it does not interfere with the reduction of infarct mass elicited by preconditioning. This may suggest that NO produced by the endothelium is not a prime factor in the cardioprotective mechanism of preconditioning. PMID- 10744359 TI - Daily exercise attenuates the development of arterial blood pressure related cardiovascular risk factors in hypertensive rats. AB - This study was designed to test the hypothesis that daily spontaneous running (DSR) attenuates the development of blood pressure-related cardiovascular disease risk factors (BP-related CVD risk factors) in spontaneously hypertensive rats (SHR). After 8 weeks of DSR or sedentary control, rats were chronically instrumented with arterial catheters. Daily exercise attenuated the development of all measures of BP-related CVD risk factors. Specifically DSR attenuated the increase in systolic blood pressure (delta--22 mmHg), systolic blood pressure variability (delta--2.5 mmHg), and systolic blood pressure load (delta--27%). Similarly, DSR attenuated the increase in diastolic blood pressure (delta--15 mmHg), diastolic blood pressure variability (delta--1.19 mmHg), and diastolic blood pressure load (delta--17%). Finally, DSR attenuated the development of tachycardia (delta--63 bpm). These data demonstrate that daily exercise attenuates the development of hypertension and tachycardia in animals predisposed to hypertension. PMID- 10744358 TI - Lipoxygenase-dependent mechanisms in hypertension. AB - This study was designed to examine the contribution of lipoxygenase products to mechanisms of vascular contraction and elevated blood pressure in rats with aortic coarctation-induced hypertension. In cytosolic fractions of aortae taken from hypertensive rats, 12-lipoxygenase protein was increased as compared to normotensive controls. Aortic rings from hypertensive, but not from normotensive rats, exhibited a basal tone which was reduced 74+/-12 and 71+/-22%, respectively, by the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-alpha cyanocinnamate (CDC, 10(-5) mol/L) and 5,8,11-eicosatriynoic acid (ETI, 10(-5) mol/L). CDC (8 mg/kg s.c.) did not affect the blood pressure of normotensive rats but decreased that of hypertensive rats from 182+/-6 to 151+/-10 mm Hg. The blood pressure lowering effect of CDC was blunted in hypertensive rats pretreated with indomethacin or antibodies against 5,6-dihydro-prostaglandin I2. These data suggest contribution of lipoxygenase-derived products to mechanisms underlying aortic smooth muscle basal tone and elevated blood pressure in rats with aortic coarctation-induced hypertension. The vasodepressor effect of CDC depends on a mechanism involving vasodilatory prostaglandins. PMID- 10744360 TI - Cyclooxygenase inhibition reduces blood pressure elevation and vascular reactivity dysfunction caused by inhibition of nitric oxide synthase in rats. AB - In the present study we investigated the role of cyclooxygenase (COX)-dependent vasoconstrictors in the hypertension and altered vascular reactivity following prolonged nitric oxide (NO) synthase inhibition. Male Wistar rats (250-270 g) were divided into four groups and treated for 7 days with Placebo (control), L NAME (48 mg/kg/day), indomethacin (4 mg/kg/day) and L-NAME in combination with indomethacin. L-NAME treatment induced arterial hypertension, in vitro aortic hyperresponsiveness to phenylephrine, impaired vasodilatory response to acetylcholine and no significant change in response to sodium nitroprusside. Indomethacin co-treatment partially prevented blood pressure elevation, restored responsiveness to phenylephrine and improved sensitivity to acetylcholine. Indomethacin treatment alone did not modify blood pressure and aortic vascular reactivity. Both enhanced phenylphrine-induced contraction and impaired acetylcholine-evoked vasodilation induced by acute NO synthase inhibition with L NAME (10(-4) M) in normal rat aortas were not modified by indomethacin (10(-5) M). These results are consistent with the hypothesis that constricting factors, which arise from the COX pathway, contribute to hypertension and altered vascular reactivity following continued inhibition of NO synthase. PMID- 10744361 TI - Comparative evaluation of the MB/BacT and BACTEC 460 TB systems for the detection of mycobacteria from clinical specimens: clinical relevance of higher recovery rates from broth-based detection systems. AB - New broth-based detection systems have higher recovery rates of mycobacteria from clinical specimens than traditional cultures on solid media. The clinical significance of this higher sensitivity rate is largely unknown. We prospectively evaluated the performances of two liquid media detection systems (the MB/BacT system and the BACTEC 460 TB system) and an egg-based Lowenstein-Gruft solid medium (LG) on the recovery rates of mycobacteria from 849 clinical specimens. Mycobacteria (other then M. gordonae) were detected in 51 (6.0%) specimens. In 12/51 (23%) specimens, mycobacteria (five mycobacterium tuberculosis (MtB) and seven non-M tuberculosis complex (MOTT) were recovered only from the broth-based systems. Review of the patients' clinical charts revealed that failure of LG to recover Mtb were due to nonmycobacterial overgrowth and antibiotic treatment. The recovered MOTT were all clinically nonsignificant. Higher sensitivity of broth based mycobacteria detection systems is largely due to their capability to recover mycobacteria from treated tuberculous patients or from partially decontaminated specimens. The high recovery rates of nonclinically significant MOTT could potentially increase inappropriate use of antibiotics. PMID- 10744362 TI - Longitudinal 10-year prospective survey of fungaemia in Slovak Republic: trends in etiology in 310 episodes. Slovak Fungaemia study group. AB - A 10-year prospective survey of fungaemia in the Slovak Republic, involving 31 microbiology laboratories and 71 hospitals, was conducted from 1989-1998 (10 years): 310 fungaemias were analyzed for etiology, clinical characteristics, therapy, and outcome. C. albicans was responsible for 191 (61.6%) fungaemias, non albicans Candida spp. (NAC) for 97 (31.3%), non-Candida yeasts for 18 (5.8%) and moulds (Fulsarium spp.) for four fungaemias. The most frequent NAC isolated from blood cultures were C. parapsilosis--30 (9.7%), C. krusei--18 (5.8%), C. tropicalis--14 (4.5%), and C. glabrata--10 (3.2%). Secular trends in etiology showed a sustaining decrease of C. albicans (from 100% in 1989 to 50.7% in 1998) and increase of NAC (from 0% in 1989-1990 to 46.3% in 1998). Non-Candida yeasts and moulds showed a stable proportion during the investigated period. There were statistically significant differences in etiology of fungaemia various subgroups of patients: non-albicans Candida spp. was significantly more frequent observed among subgroups of patients with pancreatitis and coma (53.3% vs. 31.3%, p < or = 0.02) and less frequently in the subgroup of neonates (15.0% vs. 31.3%, p < or = 0.006). Vice versa, C. albicans appeared more frequently in neonates (85%). PMID- 10744363 TI - Pharmacodynamics of fluconazole, itraconazole, and amphotericin B against Candida albicans. AB - The objective of this study was to evaluate the pharmacodynamic activity of fluconazole, itraconazole, and amphotericin B against Candida albicans. Susceptibilities were determined according to the NCCLS guidelines (M27). Time kill studies were performed using antifungal concentrations of 0.25-32 x MIC. Samples were withdrawn at predetermined timepoints, then plated using a spiral plater. Colony counts were determined after incubation at 35 degrees C for 24 h. The AUKC(0-48) was plotted against the concentration/MIC ratio. Candida isolates (95-2672, 96-15, and 95-2542) were classified as susceptible, susceptible-dose dependent, and resistant to fluconazole and itraconazole (MIC = 0.25 and 0.03 microg/mL, 32 and 0.5 microg/mL, 64 and 1 microg/mL; respectively). All three isolates were susceptible to amphotericin B (MIC = 0.13 microg/mL). Fluconazole inhibited the growth of the susceptible and S-DD isolates and was ineffective at all concentrations against the resistant isolate. Itraconazole, on the other hand, inhibited growth of the susceptible isolate, but was ineffective for the S DD and resistant isolates. Maximal effectiveness was noted at the concentration 8 x MIC and 2 x MIC for fluconazole and itraconazole, respectively. Amphotericin B demonstrated concentration-dependent antifungal activity. The times necessary for the colony counts to fall below the limit of quantification were inversely related to increasing concentrations of amphotericin B. The maximal effect for amphotericin B was recorded at 2 x MIC. In summary, the triazoles inhibit growth of susceptible C. albicans; however, careful consideration should be given to the MIC for S-DD isolates because itraconazole may not be active if the MIC is reported in the higher susceptible-dose dependency range. In reference to amphotericin B, optimal activity may be achieved by maximizing the peak drug concentration/MIC ratio. PMID- 10744364 TI - Antimicrobial activity and spectrum of the new glycylcycline, GAR-936 tested against 1,203 recent clinical bacterial isolates. AB - The in vitro activity of GAR-936, a new semisynthetic glycylcycline, was evaluated in comparison with two tetracyclines and several other antimicrobial agents. A total of 1,203 recent clinical isolates were tested by reference broth or agar dilution methods. Among the members of the family Enterobacteriaceae, GAR 936 was generally two- to four-fold more active than minocycline, and two- to 16 fold more active than tetracycline. All enteric bacilli MIC90 results were < or = 4 microg/mL; the exception being Proteus mirabilis and indole-positive Proteae (> or = 8 microg/mL). GAR-936 demonstrated excellent activity against all gram positive cocci with 90% of the penicillin-resistant Streptococcus pneumoniae isolates inhibited at 0.03 microg/ml, while the same isolates had a MIC90 of 8 and > 8 microg/mL for minocycline and tetracycline, respectively. All Enterococcus spp., including vancomycin-resistant isolates, were inhibited at 0.25 microg/mL of GAR-936 (MIC90, 0.12 or 0.25 microg/mL). Although GAR-936 (MIC50, 0.25 microg/mL) was two-fold less active than minocycline (MIC50, 0.12 microg/mL) against oxacillin-resistant Staphylococcus aureus, all isolates were inhibited at < or = 0.25 microg/mL. GAR-936 demonstrated good activity against nonfermentative bacteria such as Acinetobacter spp. (MIC90, 2 microg/ml) and Stenotrophomonas maltophilia (MIC90, 4 microg/mL), but the compound exhibited only modest activity against Pseudomonas aeruginosa (MIC50, 8 microg/mL). Haemophilus influenzae (MIC90, 1-2 microg/mL), Moraxella catarrhalis (MIC90, 0.12 microg/mL), and various Neisseria spp. (MIC90, 0.12-0.5 microg/mL) were susceptible to GAR-936. These results indicate that GAR-936 has potent in vitro activity against a wide range of clinically important pathogenic bacteria, and that several gram-positive and -negative isolates resistant to older tetracyclines and other drug classes remain susceptible to GAR-936, the newest glycylcycline candidate for clinical use. PMID- 10744365 TI - In-vitro activity of arbekacin alone and in combination with vancomycin against gentamicin- and methicillin-resistant Staphylococcus aureus. AB - In-vitro susceptibility studies were performed on 99 clinical Staphylococcus aureus isolates. A total of 68 of 73 methicillin-resistant S. aureus and 2 of 26 methicillin-susceptible S. aureus were gentamicin-resistant (gentamicin MIC range 16 to 1,024 microg/mL). All 70 gentamicin-resistant isolates contained the aac(6')-Ie-aph(2'')-Ia aminoglycoside resistance gene, and none possessed the aph(2'')-Ic or aph(2'')-Id aminoglycoside resistance genes. The arbekacin MIC for the 70 gentamicin-resistant isolates ranged from 0.25 to 4 microg/mL. The combination of arbekacin plus vancomycin produced synergistic killing against 12 of 13 gentamicin-resistant MRSA isolates. The combination of gentamicin plus vancomycin produced synergistic killing against 7 of the same 13 isolates. Arbekacin may prove useful when used in combination with vancomycin in treating infections caused by gentamicin-resistant MRSA. PMID- 10744366 TI - Susceptibility of Legionella species to five antibiotics and development of resistance by exposure to erythromycin, ciprofloxacin, and rifampicin. AB - The minimal inhibitory concentration (MIC) values of erythromycin, ciprofloxacin, ofloxacin, rifampicin, and clindamycin were determined for 56 strains of Legionella pneumophila (38 patient, 3 environmental, and 15 reference strains) and 37 strains of other Legionella species (7 patient, 2 environmental, and 28 reference strains) using the epsilon-test system on BCYEalpha agar plates. High level resistance (MIC > or = 4 microg/mL) was found only for clindamycin (57%), with MIC values ranging from 0.25-32 microg/mL. Low-level resistance was found for erythromycin (18%) (0.5 < MIC < 8), ciprofloxacin (1%) (1 < MIC < 4), and clindamycin (40%) (0.5 < MIC < 4), but not for ofloxacin and rifampicin. MIC50 for the 45 Danish clinical Legionella strains were 0.25 microg/mL (erythromycin), 0.25 microg/mL (ciprofloxacin), 0.19 microg/mL (ofloxacin), below 0.016 microg/mL (rifampicin), and 4 microg/mL (clindamycin). Of the clinical isolates, 64% were resistant to clindamycin. There were no significant differences between the MIC50 values obtained for clinical and nonclinical Legionella strains. Selected susceptible strains were exposed to increasing concentrations of either erythromycin, ciprofloxacin, or rifampicin to select for resistance. Isolates resistant to erythromycin (MIC 0.75-32 microg/mL) or ciprofloxacin (MIC 2-3 microg/mL) could be selected by a two-step procedure. One single strain recovered from media containing 50 microg/mL of erythromycin had an MIC value higher than 256 microg/mL to erythromycin. In contrast, high-level resistance toward rifampicin with MIC > or = 256 microg/mL developed as a one-step phenomenon in several strains. PMID- 10744367 TI - In vitro activity of quinupristin/dalfopristin (Synercid, RP 59500) against Legionella spp. AB - The activities of quinupristin/dalfopristin (Synercid), erythromycin and azithromycin against 22 Legionella spp. isolates were measured by a microbroth dilution method. The MICs that inhibited 90% of strains tested were 0.5, 0.35, and 0.5 microg/mL for quinupristin/dalfopristin, erythromycin, and azithromycin, respectively. Quinupristin/dalfopristin was only partially active against intracellular L. pneumophila at high (2 microg/mL), but not low (1 microg/mL) concentration. Activity of the drug in a guinea pig model of Legionnaires' disease could not be accurately determined because of drug toxicity for the guinea pig, although there was evidence that the drug has in vivo activity. PMID- 10744368 TI - High incidence of Klebsiella pneumoniae clinical isolates to extended-spectrum B lactam drugs in intensive care units. AB - A prospective study conducted among Jordanian ICU patients in 1997 using Etest identified resistance rates among isolates of E. coli (25%-44%), Enterobacter spp. (54%-62%), and Klebsiella spp. (30%-80%) to extended-spectrum B-lactams (ESBLs): ceftazidime, cefotaxime, ceftriaxone, and aztreonam. All these isolates were susceptible to imipenem and showed low resistance rate to ciprofloxacin (5% 19%) and amikacin (13%-18%). Higher and significant resistance rates of Klebsiella isolates to ceftazidime (80%) and aztreonam (65%) were observed in 1997 compared with a previous study performed in 1994. The majority of Klebsiella pneumoniae (70%) express different ESBL phenotypes that were almost resistant to aztreonam and ceftazidime but susceptible or resistant to cefotaxime and/or ceftriaxone. This prospective study strongly suggests that ESBL production of Klebsiella pneumoniae isolates have been highly disseminated among ICU patients during 1997. PMID- 10744369 TI - Evaluation of a latex agglutination assay for rapid detection of oxacillin resistant Staphylococcus aureus. AB - Oxacillin resistance in Staphylococcus aureus is mediated by the mecA gene, resulting in production of penicillin-binding protein 2a (PBP2a), which is not present in the oxacillin susceptible strains. We evaluated the ability of a 30 min latex agglutination (LA) test (Seiken, Tokyo, Japan) to detect production of PBP2a in 315 clinical isolates of S. aureus. The LA results were compared with results of susceptibility testing using the Vitek GPS-SV test card. The latex test was positive for all 206 isolates determined to be methicillin resistant by Vitek (sensitivity 100%), the latex test was negative for 108 of 109 isolates determined to be oxacillin susceptible by Vitek, and the latex test was positive for 1 isolate determined to be susceptible by Vitek (specificity 99.1%). The discrepant isolate was negative for the mecA gene by polymerase chain reaction (PCR). The LA test is a rapid and reliable method for detecting oxacillin resistant S. aureus. PMID- 10744370 TI - Occurrence of single-point gyrA mutations among ciprofloxacin-susceptible Escherichia coli isolates causing urinary tract infections in Latin America. AB - To detect if isolates susceptible to quinolones already carry mutations in the gyrA and parC genes, we selected 12 ciprofloxacin-susceptible Escherichia coli strains collected from patients with urinary tract infections in Latin America in 1998, as part of ongoing SENTRY Antimicrobial Surveillance Program. The isolates studied exhibited minimal inhibitory concentrations (MICs) for ciprofloxacin between < or = 0.015 microg/mL and 0.5 microg/mL. The molecular characterization of quinolone resistance was determinated by amplification of the gyrA and parC by PCR followed by sequencing of the respective amplicons. We observed that E. coli isolates exhibiting MIC, < or = 0.06 microg/mL for ciprofloxacin did not show mutations in either topoisomerase. On the other hand, all isolates with MIC between 0.12 microg/mL and 0.5 microg/mL demonstrated single mutation in the gyrA gene. The most frequent mutation occurred at position 83, where the amino acid serine was replaced by leucine. No mutations in the parC gene were observed. To preserve the potency and prevent the development of resistance, we suggest that quinolone usage should be rational, especially in the treatment of urinary tract infections, and in the prophylaxis of immunosupressed patient populations. PMID- 10744371 TI - Specific and rapid identification of multiple-antibiotic resistant Pseudomonas aeruginosa clones isolated in an intensive care unit. AB - Seventeen multiple-antibiotic-resistant Pseudomonas aeruginosa isolates were collected from two patients hospitalized in the same intensive care unit. They showed a parallel acquisition of resistance to antibiotics and they were, therefore, thought to have a common clonal origin. These strains were typed by biotyping, serotyping, plasmid profile, three different PCR-based techniques, and macrorestriction analysis to determine their relationship. Only the use of PCR techniques and macrorestriction analysis allowed an accurate identification of the clones and revealed that each patient was infected by A unique multidrug resistant strain. Therefore, there was no cross-infection or reinfection with a new strain. PMID- 10744372 TI - Annual Awards of the Association of American Medical Colleges. Abraham Flexner Award for Distinguished Service to Medical Education. PMID- 10744373 TI - Annual Awards of the Association of American Medical Colleges. David E. Rogers Award. PMID- 10744374 TI - Annual Awards of the Association of American Medical Colleges. Humanism in Medical Education Award. PMID- 10744375 TI - Annual Awards of the Association of American Medical Colleges. Award for Distinguished Research in the Biomedical Sciences. PMID- 10744376 TI - Annual Awards of the Association of American Medical Colleges. Alpha Omega Alpha Robert J Glaser Distinguished Teacher Awards. PMID- 10744378 TI - FAQ re abbreviations. Frequently asked question. PMID- 10744377 TI - A new BDA structure. British Dental Association. PMID- 10744379 TI - British Society for Investigative Dermatology annual meeting. Cardiff, 7-9 April 1999. Abstracts. PMID- 10744380 TI - The full spectrum of persistent fetal vasculature in Aicardi syndrome: an integrated interpretation of ocular malformation. PMID- 10744381 TI - Isolated focal melanocytes collection in the lacrimal sac. PMID- 10744382 TI - Pupillary distortion after contact transscleral diode laser cyclophotocoagulation. PMID- 10744383 TI - Urrets-Zavalia syndrome following trabeculectomy. PMID- 10744384 TI - Fluorescein angiography in altitude retinopathy. PMID- 10744385 TI - Panuveitis as a presenting feature of giant cell arteritis. PMID- 10744386 TI - [Multislice CT angiography of the upper abdomen. Stenosis of the superior mesenteric artery]. PMID- 10744387 TI - Alloantigen-induced anti-HIV activity occurs prior to reverse transcription and can be generated by leukocytes from HIV-infected individuals. PMID- 10744388 TI - Correction of the PNH defect by GPI protein transfer: still an open question. PMID- 10744389 TI - Acute priapism associated with the use of sildenafil in a patient with sickle cell trait. PMID- 10744390 TI - A single tube multiplex PCR method to detect the common alpha+ thalassemia alleles. PMID- 10744391 TI - Protein C inhibitor in platelets? PMID- 10744392 TI - Papers presented at the 24th annual meeting of the British Mass Spectrometry Society. 12-15 September 1999. PMID- 10744393 TI - The Rosen von Rosenstein award. PMID- 10744394 TI - Presentation of Abraham Rudolph when receiving the Rosen von Rosenstein award on 6 May 1999. PMID- 10744395 TI - [1ST Symposium for Photodynamic Diagnosis and Therapy. Vienna, 28 October 1998]. PMID- 10744396 TI - [Mortality associated with oral contraceptive use: 25-year follow-up of a cohort of 46,000 women from Royal College of General Practitioners's Oral Contraception Study. Analysis and comment on the BMJ study of the long-term risk of using oral contraceptives]. PMID- 10744397 TI - Cyanide spill threatens health in Hungary. PMID- 10744398 TI - Management of hypertension. Patients from ethnic minorities are at greater risk. PMID- 10744399 TI - Management of hypertension. Guidelines have serious weaknesses. PMID- 10744400 TI - Management of hypertension. Guidelines do not consider workload implications in primary care. PMID- 10744401 TI - Management of hypertension. Non-medical remedies should be considered first. PMID- 10744402 TI - Management of hypertension. "Mortality" is preferable to "major disease end points". PMID- 10744403 TI - Management of hypertension. Evidence shows that calcium antagonists reduce cardiovascular end points in diabetic patients. PMID- 10744404 TI - Management of hypertension. Optimal target pressure is not supported by strength A evidence. PMID- 10744405 TI - Management of hypertension. Trials showing no reduction in mortality do not receive same exposure. PMID- 10744406 TI - beta Blockade after myocardial infarction. Absence of evidence is failure of research policy. PMID- 10744407 TI - The sexual health of boys and men. Attitudes to adolescent boys' sexual and reproductive health urgently need changing. PMID- 10744408 TI - Doctor, help! My child has cancer. Cancer care is team care, and the GP must be included. PMID- 10744409 TI - Doctor, help! My child has cancer. Patients mustn't expect their GP to read their mind. PMID- 10744410 TI - Doctor, help! My child has cancer. Relatives are often grateful for GP's presence at patient's death. PMID- 10744411 TI - Doctor, help! My child has cancer. Patients get great benefit from their GP visiting them in hospital. PMID- 10744412 TI - Injection with methylprednisolone for carpal tunnel syndrome. Study does not show long term benefits of injection for the syndrome. PMID- 10744413 TI - Injection with methylprednisolone for carpal tunnel syndrome. Study is needed to determine best treatment for this syndrome. PMID- 10744414 TI - Prediction of survival for preterm births. Curve presents information on survival and morbidity more clearly than tables. PMID- 10744415 TI - Prediction of survival for preterm births. Data on the quality of survival are needed. PMID- 10744416 TI - Managing doctors and health care. Health service monitoring in Britain and the United States. PMID- 10744418 TI - Microanalytical Characterisation of Semi-conducting Materials and Devices. Workshop proceedings. Lecce, Italy, 9-11 November 1998. PMID- 10744417 TI - Revalidation in the United Kingdom. Supportive culture must accompany revalidation. PMID- 10744419 TI - Bacterial infection and extent of necrosis are determinants of organ failure in patients with acute necrotizing pancreatitis. PMID- 10744420 TI - Oral nifedipine reduces resting anal pressure and heals chronic anal fissure. PMID- 10744421 TI - Bilirubin and peripheral vascular disease. PMID- 10744422 TI - Measurement of phosphatidylserine exposure in leukocytes and platelets by whole blood flow cytometry with annexin V. AB - Phosphatidylserine (PS) exposure serves as a procoagulant stimulus and a signal for phagocytic clearance of apoptotic cells. In order to measure PS exposure in blood cells, we developed a flow-cytometric procedure to measure annexin V binding to leukocytes and platelets in whole-blood samples. Leukocytes were identified by CD45 and side-scatter gating, and platelets by CD6 1 and side scatter gating. The absolute number of annexin V molecules bound per cell was determined from an independent calibration procedure. Normal populations had the following levels of annexin V binding (in molecules per cell): lymphocytes, 0.53 x 10(3) neutrophils, 1.75 x 10(3) monocytes, 2.45 x 10(3) platelets, 0.14 x 10(3). These levels represent or =60 micromol/L [3.5 mg/dL], factor V level < or =50%, portal hypertension, encephalopathy, ascites, gastrointestinal bleeding, or histologically proven extensive fibrosis or cirrhosis). Contributing factors were assessed by using univariate and multivariate (Cox model) analysis. RESULTS: 58 patients (65%) developed chronic cholestasis after a median of 6 months (range, 3 to 132 months), and 37 (41.5%) developed complicated home parenteral nutrition-related liver disease after a median of 17 months (range, 2 to 155 months). Of these patients, 17 showed extensive fibrosis after 26 months (range, 2 to 148 months) and 5 had cirrhosis after 37 months (range, 26 to 77 months). The prevalence of complicated home parenteral nutrition-related liver disease was 26%+/-9% at 2 years and 50%+/-13% at 6 years. Six patients died of liver disease (22% of all deaths). In multivariate analysis, chronic cholestasis was significantly associated with a parenteral nutrition-independent risk for liver disease, a bowel remnant shorter than 50 cm in length, and a parenteral lipid intake of 1 g/kg of body weight per day or more (omega-6-rich long-chain triglycerides), whereas complicated home parenteral nutrition-related liver disease was significantly associated with chronic cholestasis and lipid parenteral intake of 1 g/kg per day or more. CONCLUSION: The prevalence of complicated home parenteral nutrition-related liver disease increased with longer duration of parenteral nutrition. This condition was one of the main causes of death in patients with permanent intestinal failure. Parenteral intake of omega-6 rich long-chain triglycerides lipid emulsion consisting of less than 1 g/kg per day is recommended in these patients. PMID- 10744589 TI - Interactions between apolipoprotein E and apolipoprotein(a) in patients with late onset Alzheimer disease. AB - BACKGROUND: Apolipoprotein(a) [apo(a)], the distinctive, highly polymorphic glycoprotein of lipoprotein(a), shares a series of common features with apolipoprotein E (apoE), which is implicated in the development of Alzheimer disease. OBJECTIVE: To determine whether apo(a) is associated with Alzheimer disease. DESIGN: Case-control study. SETTING: University hospitals in Europe. PARTICIPANTS: 285 patients with Alzheimer disease and 296 controls. MEASUREMENTS: Plasma lipoprotein(a) levels, size of the apo(a) isoforms, and apoE and apo(a) genotyping. RESULTS: Among carriers of the apoE epsilon4 allele, lipoprotein(a) was associated with a progressive, age-dependent increased risk for late-onset Alzheimer disease (odds ratio for patients >80 years of age, 6.0 [95% CI, 1.2 to 30.8]; P<0.01). Among noncarriers older than 80 years of age, lipoprotein(a) was associated with a reduced risk for Alzheimer disease (odds ratio, 0.4 [CI, 0.2 to 0.91; P<0.05). CONCLUSIONS: In this convenience sample, lipoprotein(a) was an additional risk factor for late-onset Alzheimer disease in carriers of the apoE epsilon4 allele. However, lipoprotein(a) may protect against late-onset Alzheimer disease in noncarriers. PMID- 10744590 TI - Substituting walnuts for monounsaturated fat improves the serum lipid profile of hypercholesterolemic men and women. A randomized crossover trial. AB - BACKGROUND: It has been reported that walnuts reduce serum cholesterol levels in normal young men. OBJECTIVE: To assess the acceptability of walnuts and their effects on serum lipid levels and low-density lipoprotein (LDL) oxidizability in free-living hypercholesterolemic persons. DESIGN: Randomized, crossover feeding trial. SETTING: Lipid clinic at a university hospital. PATIENTS: 55 men and women (mean age, 56 years) with polygenic hypercholesterolemia. INTERVENTION: A cholesterol-lowering Mediterranean diet and a diet of similar energy and fat content in which walnuts replaced approximately 35% of the energy obtained from monounsaturated fat. Patients followed each diet for 6 weeks. MEASUREMENTS: Low density lipoprotein fatty acids (to assess compliance), serum lipid levels, lipoprotein(a) levels, and LDL resistance to in vitro oxidative stress. RESULTS: 49 persons completed the trial. The walnut diet was well tolerated. Planned and observed diets were closely matched. Compared with the Mediterranean diet, the walnut diet produced mean changes of -4.1% in total cholesterol level, -5.9% in LDL cholesterol level, and -6.2% in lipoprotein(a) level. The mean differences in the changes in serum lipid levels were -0.28 mmol/L (95% CI, -0.43 to -0.12 mmol/L) (-10.8 mg/dL [-16.8 to -4.8 mg/dL]) (P<0.001) for total cholesterol level, -0.29 mmol/L (CI, -0.41 to -0.15 mmol/L) (-11.2 mg/dL [-16.3 to -6.1 mg/dL]) (P<0.001) for LDL cholesterol level, and -0.021 g/L (CI, -0.042 to -0.001 g/L) (P = 0.042) for lipoprotein(a) level. Lipid changes were similar in men and women except for lipoprotein(a) levels, which decreased only in men. Low-density lipoprotein particles were enriched with polyunsaturated fatty acids from walnuts, but their resistance to oxidation was preserved. CONCLUSION: Substituting walnuts for part of the mono-unsaturated fat in a cholesterol lowering Mediterranean diet further reduced total and LDL cholesterol levels in men and women with hypercholesterolemia. PMID- 10744591 TI - Urinary incontinence among male veterans receiving care in primary care clinics. AB - BACKGROUND: Male urinary incontinence is not a well-investigated subject. OBJECTIVE: To determine the prevalence and consequences of incontinence in men. DESIGN: Cross-sectional survey. SETTING: 3 primary care clinics in a Kentucky Veterans Affairs facility. PATIENTS: 840 men with a mean age of 59.8 years (range, 25 to 93 years). MEASUREMENTS: Written survey on patient demographic information and continence, including the Incontinence Impact Questionnaire. RESULTS: Among all study patients, 32.3% reported incontinence within the past 12 months and 13.8% (43% of those who were incontinent) reported at least weekly episodes. Age did not correlate with frequency or amount of urine loss. Frequency correlated with emotional health, social relationships, physical activity, and travel. Greater amounts of lost urine had a stronger effect on emotional health, social relationships, and travel. Among men with incontinence, only 32% had discussed incontinence with their medical provider but 75% desired evaluation and treatment. CONCLUSIONS: Urinary incontinence is common among male veterans and affects all age groups. Although incontinence often has unfavorable consequences on quality of life and although men with incontinence desired treatment, they seldom discussed the problem with medical providers. Systematic screening of men for urinary incontinence may be necessary. PMID- 10744592 TI - Heart rate recovery after submaximal exercise testing as a predictor of mortality in a cardiovascularly healthy cohort. AB - BACKGROUND: Abnormal heart rate recovery after symptom-limited exercise predicts death. It is unknown whether this is also true among patients undergoing submaximal testing. OBJECTIVE: To test the prognostic implications of heart rate recovery in cardiovascularly healthy adults undergoing submaximal exercise testing. DESIGN: Population-based cohort study. SETTING: 10 primary care sites. PARTICIPANTS: 5234 adults without evidence of cardiovascular disease who were enrolled in the Lipid Research Clinics Prevalence Study. MEASUREMENTS: Heart rate recovery was defined as the change from peak heart rate to that measured 2 minutes later (heart rate recovery was defined as < or =42 beats/min). RESULTS: During 12 years of follow-up, 312 participants died. Abnormal heart rate recovery predicted death (relative risk, 2.58 [CI, 2.06 to 3.20]). After adjustment for standard risk factors, fitness, and resting and exercise heart rates, abnormal heart rate recovery remained predictive (adjusted relative risk, 1.55 [CI, 1.22 to 1.98]) (P<0.001). CONCLUSION: Even after submaximal exercise, abnormal heart rate recovery predicts death. PMID- 10744593 TI - Identification and management of patients with failed thrombolysis after acute myocardial infarction. PMID- 10744594 TI - Single-therapy androgen suppression in men with advanced prostate cancer: a systematic review and meta-analysis. AB - PURPOSE: To compare luteinizing hormone-releasing hormone (LHRH) agonists with orchiectomy or diethylstilbestrol, and to compare antiandrogens with any of these three alternatives. DATA SOURCES: A search of the MEDLINE, Cancerlit, EMBASE, and Cochrane Library databases from 1966 to March 1998 and Current Contents to 24 August 1998 for articles comparing the outcomes of the specified treatments. The search was limited to studies on prostatic neoplasms in humans. Total yield was 1477 studies. STUDY SELECTION: Reports of efficacy outcomes were limited to randomized, controlled trials. Twenty-four trials involving more than 6600 patients, phase II studies that reported on withdrawals from therapy (the most reliable indicator of adverse effects), and all studies reporting on quality of life were abstracted. DATA EXTRACTION: Two independent reviewers abstracted each article by following a prospectively designed protocol. The meta-analysis combined data on 2-year overall survival by using a random-effects model and; reported results as a hazard ratio relative to orchiectomy. DATA SYNTHESIS: Ten trials of LHRH agonists involving 1908 patients reported no significant difference in overall survival. The hazard ratio showed LHRH agonists to be essentially equivalent to orchiectomy (hazard ratio, 1.1262 [corrected] [95% CI, 0.915 to 1.386]). There was no evidence of difference in overall survival among the LHRH agonists, although CIs were wider for leuprolide (hazard ratio, 1.0994 [CI, 0.207 to 5.835]) and buserelin (hazard ratio, 1.1315 [CI, 0.533 to 2.404]) than for goserelin (hazard ratio, 1.1172 [CI, 0.898 to 1.390]). Evidence from 8 trials involving 2717 patients suggests that nonsteroidal antiandrogens were associated with lower overall survival. The CI for the hazard ratio approached statistical significance (hazard ratio, 1.2158 [CI, 0.988 to 1.496]). Treatment withdrawals were less frequent with LHRH agonists (0% to 4%) than with nonsteroidal antiandrogens (4% to 10%). CONCLUSIONS: Survival after therapy with an LHRH agonist was equivalent to that after orchiectomy. No evidence shows a difference in effectiveness among the LHRH agonists. Survival rates may be somewhat lower if a nonsteroidal antiandrogen is used as monotherapy. PMID- 10744595 TI - Physicians and patient spirituality: professional boundaries, competency, and ethics. AB - Clinical studies are beginning to clarify how spirituality and religion can contribute to the coping strategies of many patients with severe, chronic, and terminal conditions. The ethical aspects of physician attention to the spiritual and religious dimensions of patients' experiences of illness require review and discussion. Should the physician discuss spiritual issues with his or her patients? What are the boundaries between the physician and patient regarding these issues? What are the professional boundaries between the physician and the chaplain? This article examines the physician-patient relationship and medical ethics at a time when researchers are beginning to appreciate the spiritual aspects of coping with illness. PMID- 10744597 TI - Technology transfer and continuity of care by a "consultant". PMID- 10744596 TI - Hormonal therapy for advanced prostate cancer. PMID- 10744598 TI - Reflections on a bone marrow transplant. PMID- 10744599 TI - Serum uric acid and cardiovascular disease risk. PMID- 10744600 TI - Serum uric acid and cardiovascular disease risk. PMID- 10744601 TI - Clinically useful problem statement systems. PMID- 10744602 TI - Pain management in patients with advanced cancer. PMID- 10744603 TI - Medical management of aldosterone-producing adenomas. PMID- 10744604 TI - Medical management of aldosterone-producing adenomas. PMID- 10744605 TI - Nasogastric tubes permit reflux and aspiration: is there any evidence? PMID- 10744606 TI - Calcium carbonate-induced hypothyroidism. PMID- 10744607 TI - Whipple endocarditis without gastrointestinal disease. PMID- 10744608 TI - Ischemic colitis associated with factor V Leiden mutation. PMID- 10744609 TI - Chemoembolization of liver metastases from medullary thyroid carcinoma. PMID- 10744610 TI - Congenital C2 (type I) deficiency associated with common variable immunodeficiency. PMID- 10744611 TI - Sumatriptan-associated mesenteric ischemia. PMID- 10744613 TI - Lamivudine-associated remission of chronic hepatitis delta. PMID- 10744612 TI - Cerivastatin-induced rhabdomyolysis. PMID- 10744614 TI - The Mutter Museum: education, preservation, and commemoration. PMID- 10744615 TI - AORN leaders identify perioperative accomplishments, threats and attributes. PMID- 10744616 TI - New roles and new twists on old roles. PMID- 10744617 TI - Taking the confusion out of the regulation of health care professions. PMID- 10744618 TI - Evaluating patient care programs. PMID- 10744619 TI - Internet resources for total parenteral nutrition. PMID- 10744620 TI - Transgenic mouse models for studying the functions of insulin-like growth factor binding proteins. AB - The insulin-like growth factor-binding proteins (IGFBPs) comprise a family of six related peptides that interact with high affinity with IGFs. IGFBPs compete with IGF receptors for IGF binding, and as a consequence of this competition they can affect cell growth. In addition, IGF-independent regulatory mechanisms of IGFBPs have been described. Despite their common property to interact with IGFs every IGFBP is expressed in a tightly regulated time- and tissue-specific manner suggesting that each protein may have its own distinct functions. Several transgenic mouse models overexpressing IGFBP-1, -2, -3, or -4 were developed in the past few years. Brain abnormalities were a common feature of IGFBP-1 transgenic models. Individual strains showed alterations in glucose homeostasis, reproductive performance, and a reduction of somatic growth as the most prominent phenotypes. The latter was also the main effect observed in IGFBP-2 transgenic mice. The overexpression of IGFBP-3 under the control of an ubiquitous promoter resulted in selective organomegaly, whereas mammary gland-targeted expression of this protein caused an altered involution after pregnancy in this organ. Tissue specific overexpression of IGFBP-4 resulted in hypoplasia and reduced weight of smooth muscle-rich tissues such as bladder, aorta, and stomach. This review summarizes the current knowledge about the actions of IGFBPs in vivo based on the presently established transgenic mice. PMID- 10744621 TI - Inhibitors of poly (ADP-ribose) synthetase reduce renal ischemia-reperfusion injury in the anesthetized rat in vivo. AB - The activation of poly (ADP-ribose) synthetase (PARS) subsequent to DNA damage caused by reactive oxygen or nitrogen species has been implicated in several pathophysiological conditions, including ischemia-reperfusion injury and shock. The aim of this study was to investigate whether PARS inhibitors could provide protection against renal ischemia-reperfusion injury in the rat in vivo. Male Wistar rats were subjected to 45 min bilateral clamping of the renal pedicles, followed by 6 h reperfusion (control animals). Animals were administered the PARS inhibitors 3-aminobenzamide, 1, 5-dihydroxyisoquinoline, or nicotinamide during the reperfusion period. Ischemia, followed by reperfusion, produced significant increases in plasma concentrations of urea, creatinine, and fractional excretion of Na(+) (FE(Na)) and produced a significant reduction in glomerular filtration rate (GFR). However, administration of the PARS inhibitors significantly reduced urea and creatinine concentrations, suggesting improved renal function. The PARS inhibitors also significantly increased GFR and reduced FE(Na), suggesting the recovery of both glomerular and tubular function, respectively, with a more pronounced recovery of tubular function. In kidneys from control animals, histological examination revealed severe renal damage and immunohistochemical localization demonstrated PARS activation in the proximal tubule. Both renal damage and PARS activation were attenuated by administration of PARS inhibitors during reperfusion. Therefore, we propose that PARS activation contributes to renal reperfusion injury and that PARS inhibitors may be beneficial in renal disorders associated with oxidative stress-mediated injury. PMID- 10744622 TI - Bcl-2 overexpression and hypoxia synergistically act to modulate vascular endothelial growth factor expression and in vivo angiogenesis in a breast carcinoma line. AB - We have previously demonstrated that bcl-2 overexpression enhances the metastatic potential of the MCF7 ADR human breast cancer cell line resistant to adriamycin by inducing metastasis-associated properties. To further elucidate the relationship between bcl-2 expression and the metastatic potential of the MCF7 ADR line, we evaluated whether bcl-2 could be also involved in the modulation of the angiogenic phenotype. Four bcl-2-overexpressing clones, a control transfectant clone, and the MCF7 ADR parental line were used for in vitro and in vivo experiments. Bcl-2 overexpression enhanced the synthesis of the hypoxia stimulated VEGF protein and mRNA. Northern blot analysis demonstrated an increased VEGF mRNA expression in bcl-2-overexpressing clones, and reverse transcription-polymerase chain reaction showed higher levels of the VEGF(121) and VEGF(165) mRNA isoforms, which are the most active in eliciting angiogenesis. When incorporated into matrigel, supernatants of bcl-2-transfected cells cultured under hypoxic conditions induced an increased angiogenic response in C57BL/6 mice compared with that of control clone. Tumors from bcl-2 transfectants demonstrated increased VEGF expression and neovascularization as compared to the parental line, whereas the apoptosis in in vivo xenografts was similar in control and bcl 2 transfectants. The effect of bcl-2 on angiogenesis was not mediated by p53 protein. These results demonstrate that bcl-2 and hypoxia can act synergistically to modulate VEGF expression and the in vivo angiogenic response in the MCF7 ADR line. PMID- 10744623 TI - Tumor cell-derived prostaglandin E2 inhibits monocyte function by interfering with CCR5 and Mac-1. AB - The cyclooxygenases (COX)-1 and COX-2 are key enzymes in the conversion of arachidonic acid to prostaglandins and other eicosanoids. Whereas COX-1 is expressed ubiquitously, COX-2 is an immediate-early gene often associated with malignant transformation, and a role for the COX enzymes in tumor initiation and promotion is discussed. Nonsteroidal anti-inflammatory drugs (NSAIDs) like aspirin and indomethacin that block COX-1 and -2 have been shown to have beneficial effects for tumor patients. Therefore, these compounds have gained interest also among oncologists. However, the molecular mechanism by which NSAIDs inhibit carcinogenesis is not clearly understood. The prostaglandin-dependent and -independent effect may both account for their antineoplastic action. We show here that tumor cells derived from different tumors regularly produce prostaglandin E(2) (PGE(2)) interfering with the function of monocytes. In particular, PGE(2) inhibits the potential of monocytes to migrate in the direction of a chemotactic stimulus and to adhere to endothelial cell. This inhibition is most probably due to a modulation of the chemokine receptor CCR5 and the beta2-integrin Mac-1. Both down-regulation of CCR5 and reduced expression of Mac-1 may diminish the potential of peripheral blood monocytes to leave blood vessels and invade target tissues. Since both dysfunctions can be restored with NSAIDs, our findings help to explain the molecular chemopreventive action of NSAIDs on tumor formation and progression. PMID- 10744624 TI - Chronic stretch of engineered heart tissue induces hypertrophy and functional improvement. AB - To examine the influence of chronic mechanical stretch on functional behavior of cardiac myocytes, we reconstituted embryonic chick or neonatal rat cardiac myocytes to a 3-dimensional engineered heart tissue (EHT) by mixing freshly isolated cells with neutralized collagen I and culturing them between two Velcro coated silicone tubes, held at a fixed distance with a metal spacer. After 4 days, EHTs were subjected to a phasic unidirectional stretch for 6 days in serum containing medium. Compared to unstretched controls, RNA/DNA and protein/cell ratios increased by 100% and 50%, respectively. ANF mRNA and alpha-sarcomeric actin increased by 98% and 40%, respectively. Morphologically, stretched EHTs exhibited improved organization of cardiac myocytes into parallel arrays of rod shaped cells, increased cell length and width, longer myofilaments, and increased mitochondrial density. Thus, stretch induced phenotypic changes, generally referred to as hypertrophy. Concomitantly, force of contraction was two- to fourfold higher both under basal conditions and after stimulation with calcium or the beta-adrenergic agonist isoprenaline. Contraction kinetics were accelerated with a 14-44% decrease in twitch duration under all those conditions. In summary, we have developed a new in vitro model that allows morphological, molecular, and functional consequences of stretch to be studied under defined conditions. The main finding was that stretch of EHTs induced cardiac myocyte hypertrophy, which was accompanied by marked improvement of contractile function. PMID- 10744625 TI - Extracellular cyclic ADP-ribose increases intracellular free calcium concentration and stimulates proliferation of human hemopoietic progenitors. AB - Cyclic ADP-ribose (cADPR) is a universal second messenger that regulates many calcium-related cellular events by releasing calcium from intracellular stores. Since these events include enhanced cell proliferation and since the bone marrow harbors both ectoenzymes that generate cADPR from NAD(+) (CD38 and BST-1), we investigated the effects of extracellular cADPR on human hemopoietic progenitors (HP). Exposure of HP to 100 microM cADPR for 24 h induced a significant increase in colony output (P<0.01) and colony size (P<0.003). A horizontal expansion of HP, as demonstrated by a markedly increased replating efficiency in semisolid medium (up to 700 times compared to controls), was also observed, indicating that cADPR priming can affect cell growth for multiple generations over several weeks after exposure. Influx of extracellular cADPR into the cells was demonstrated, and a causal relationship between the functional effects and the increase of intracellular free calcium concentration induced by cADPR on HP was established through the use of specific antagonists. Similar effects on HP were produced by nanomolar concentrations of the nonhydrolyzable cADPR analog 3-deaza-cADPR. These data demonstrate that extracellular cADPR behaves as a cytokine enhancing the proliferation of human HP, a finding that may have biomedical applications for the ex vivo expansion of hemopoietic cells. PMID- 10744627 TI - Occurrence of D-aspartic acid and N-methyl-D-aspartic acid in rat neuroendocrine tissues and their role in the modulation of luteinizing hormone and growth hormone release. AB - Using two specific and sensitive fluorometric/HPLC methods and a GC-MS method, alone and in combination with D-aspartate oxidase, we have demonstrated for the first time that N-methyl-D-aspartate (NMDA), in addition to D-aspartate (D-Asp), is endogenously present as a natural molecule in rat nervous system and endocrine glands. Both of these amino acids are mostly concentrated at nmol/g levels in the adenohypophysis, hypothalamus, brain, and testis. The adenohypophysis maximally showed the ability to accumulate D-Asp when the latter is exogenously administered. In vivo experiments, consisting of the i.p. injection of D-Asp, showed that D-Asp induced both growth hormone and luteinizing hormone (LH) release. However, in vitro experiments showed that D-Asp was able to induce LH release from adenohypophysis only when this gland was co-incubated with the hypothalamus. This is because D-Asp also induces the release of GnRH from the hypothalamus, which in turn is directly responsible for the D-Asp-induced LH secretion from the pituitary gland. Compared to D-Asp, NMDA elicits its hormone release action at concentrations approximately 100-fold lower than D-Asp. D-AP5, a specific NMDA receptor antagonist, inhibited D-Asp and NMDA hormonal activity, demonstrating that these actions are mediated by NMDA receptors. NMDA is biosynthesized from D-Asp by an S-adenosylmethionine-dependent enzyme, which we tentatively denominated as NMDA synthase. PMID- 10744626 TI - Uric acid, a peroxynitrite scavenger, inhibits CNS inflammation, blood-CNS barrier permeability changes, and tissue damage in a mouse model of multiple sclerosis. AB - Peroxynitrite (ONOO(-)), a toxic product of the free radicals nitric oxide and superoxide, has been implicated in the pathogenesis of CNS inflammatory diseases, including multiple sclerosis and its animal correlate experimental autoimmune encephalomyelitis (EAE). In this study we have assessed the mode of action of uric acid (UA), a purine metabolite and ONOO(-) scavenger, in the treatment of EAE. We show that if administered to mice before the onset of clinical EAE, UA interferes with the invasion of inflammatory cells into the CNS and prevents development of the disease. In mice with active EAE, exogenously administered UA penetrates the already compromised blood-CNS barrier, blocks ONOO(-)-mediated tyrosine nitration and apoptotic cell death in areas of inflammation in spinal cord tissues and promotes recovery of the animals. Moreover, UA treatment suppresses the enhanced blood-CNS barrier permeability characteristic of EAE. We postulate that UA acts at two levels in EAE: 1) by protecting the integrity of the blood-CNS barrier from ONOO(-)-induced permeability changes such that cell invasion and the resulting pathology is minimized; and 2) through a compromised blood-CNS barrier, by scavenging the ONOO(-) directly responsible for CNS tissue damage and death. PMID- 10744628 TI - Dopamine transporter proline mutations influence dopamine uptake, cocaine analog recognition, and expression. AB - Analyses of mutation effects can aid in understanding how large proteins act. The dopamine transporter (DAT) mediates complex actions in recognizing cocaine and in recognizing and translocating dopamine, sodium, and chloride. DAT proline residues, especially those in transmembrane (TM) domains, are good candidates for involvement in these DAT actions. We now report production of mutants substituting alanine and/or glycine residues for 16 prolines located in or near putative DAT TM domains. We examine effects of these modifications on DAT expression, dopamine uptake, and cocaine analog binding. Mutants in prolines located in five DAT TM domains and four connecting loops alter apparent DAT membrane targeting. Five mutations decrease dopamine affinities more than threefold without significantly decreasing cocaine analog affinities. One decreases cocaine analog affinity without decreasing dopamine affinity. Two mutations decrease affinities for both dopamine and cocaine analog. P101 is especially implicated in dopamine uptake. Alanine substitution for this proline yields dopamine V(max) values of less than 3% of wild-type values despite dopamine affinities more than fourfold higher than wild-type and normal Na(+) and Cl(-) dependence. These DAT proline mutants identify DAT regions likely for dopamine translocation and for recognition of dopamine and cocaine. PMID- 10744629 TI - Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. AB - Apoptosis inducing factor (AIF) is a novel apoptotic effector protein that induces chromatin condensation and large-scale ( approximately 50 kbp) DNA fragmentation when added to purified nuclei in vitro. Confocal and electron microscopy reveal that, in normal cells, AIF is strictly confined to mitochondria and thus colocalizes with heat shock protein 60 (hsp60). On induction of apoptosis by staurosporin, c-Myc, etoposide, or ceramide, AIF (but not hsp60) translocates to the nucleus. This suggests that only the outer mitochondrial membrane (which retains AIF in the intermembrane space) but not the inner membrane (which retains hsp60 in the matrix) becomes protein permeable. The mitochondrio-nuclear redistribution of AIF is prevented by a Bcl-2 protein specifically targeted to mitochondrial membranes. The pan-caspase inhibitor Z VAD. fmk does not prevent the staurosporin-induced translocation of AIF, although it does inhibit oligonucleosomal DNA fragmentation and arrests chromatin condensation at an early stage. ATP depletion is sufficient to cause AIF translocation to the nucleus, and this phenomenon is accelerated by the apoptosis inducer staurosporin. However, in conditions in which both glycolytic and respiratory ATP generation is inhibited, cells fail to manifest any sign of chromatin condensation and advanced DNA fragmentation, thus manifesting a 'necrotic' phenotype. Both in the presence of Z-VAD. fmk and in conditions of ATP depletion, AIF translocation correlates with the appearance of large-scale DNA fragmentation. Altogether, these data are compatible with the hypothesis that AIF is a caspase-independent mitochondrial death effector responsible for partial chromatinolysis. PMID- 10744630 TI - Cellular dedifferentiation of endothelium is linked to activation and silencing of certain nuclear transcription factors: implications for endothelial dysfunction and vascular biology. AB - We investigated the gene expression of the nuclear transcription factors c/EBPalpha, GATA-2, and the silencer Oct-1 in conjunction with the gene expression of all major cytochrome P450 genes and of eNOS in cultures of endothelial cells of the rat. The purity of cultured endothelial cells was also confirmed by flow cytometry measurements of PECAM-1, a surface antigen of endothelial cells. Taken collectively, the gene expression and flow cytometry studies provide strong evidence for c/EBPalpha, GATA-2, and Oct-1 to play a key role in the cellular dedifferentiation of endothelial cells; gene expression of eight individual CYP genes in conjunction with protein activity could be significantly increased upon treatment with Aroclor 1254, a well-documented chemical inducer of a battery of genes. Nevertheless, the gene expression of c/EBPalpha, GATA-2, and most of the CYP genes was dramatically reduced (up to 90%) in cell cultures lacking PECAM-1 expression; in strong contrast, expression of the silencer Oct-1 was massively increased (approximately 14-fold). We thus conclude activation of the silencer Oct-1 to be strongly correlated with loss of PECAM-1 and eNOS gene expression, e.g., loss of cellular differentiation and endothelial function; in conjunction, gene expression of all major P450 isoforms was dramatically reduced in cultures of dedifferentiated endothelial cells. This process of cellular dedifferentiation and endothelial dysfunction was accompanied by down-regulation of endothelial specific transcription factors. PMID- 10744631 TI - Control of murine hair follicle regression (catagen) by TGF-beta1 in vivo. AB - The regression phase of the hair cycle (catagen) is an apoptosis-driven process accompanied by terminal differentiation, proteolysis, and matrix remodeling. As an inhibitor of keratinocyte proliferation and inductor of keratinocyte apoptosis, transforming growth factor beta1 (TGF-beta1) has been proposed to play an important role in catagen regulation. This is suggested, for example, by maximal expression of TGF-beta1 and its receptors during late anagen and the onset of catagen of the hair cycle. We examined the potential involvement of TGF beta1 in catagen control. We compared the first spontaneous entry of hair follicles into catagen between TGF-beta1 null mice and age-matched wild-type littermates, and assessed the effects of TGF-beta1 injection on murine anagen hair follicles in vivo. At day 18 p.p., hair follicles in TGF-beta1 -/- mice were still in early catagen, whereas hair follicles of +/+ littermates had already entered the subsequent resting phase (telogen). TGF-beta1-/- mice displayed more Ki-67-positive cells and fewer apoptotic cells than comparable catagen follicles from +/+ mice. In contrast, injection of TGF-beta1 into the back skin of mice induced premature catagen development. In addition, the number of proliferating follicle keratinocytes was reduced and the number of TUNEL + cells was increased in the TGF-beta1-treated mice compared to controls. Double visualization of TGF beta type II receptor (TGFRII) and TUNEL reactivity revealed colocalization of apoptotic nuclei and TGFRII in catagen follicles. These data strongly support that TGF-beta1 ranks among the elusive endogenous regulators of catagen induction in vivo, possibly via the inhibition of keratinocyte proliferation and induction of apoptosis. Thus, TGF-betaRII agonists and antagonists may provide useful therapeutic tools for human hair growth disorders based on premature or retarded catagen development (effluvium, alopecia, hirsutism). PMID- 10744632 TI - A Bethlem myopathy Gly to Glu mutation in the von Willebrand factor A domain N2 of the collagen alpha3(VI) chain interferes with protein folding. AB - A single G1679E mutation in the amino-terminal globular domain N2 of the alpha3 chain of type VI collagen was found in a large family affected with Bethlem myopathy. Recombinant production of N2 ( approximately 200 residues) in transfected mammalian cells has now been used to examine the possibility that the mutation interfered with protein folding. The wild-type form and a G1679A mutant were produced at high levels and shown to fold into a stable globular structure. Only a small amount of secretion was observed for mutants G1679E and G1679Q, which apparently were efficiently degraded within the cells. Homology modeling onto the related von Willebrand factor A1 structure indicated that substitution of G1679 by the bulky E or Q cannot be accommodated without considerable changes in the folding pattern. This suggests protein misfolding as a molecular basis for this particular mutation in Bethlem myopathy, in agreement with radioimmunoassay data showing reduced levels of domain N2 in cultured fibroblasts from two patients. PMID- 10744633 TI - Degradation of unassembled soluble Ig subunits by cytosolic proteasomes: evidence that retrotranslocation and degradation are coupled events. AB - Many aberrant or unassembled proteins synthesized in the endoplasmic reticulum (ER) are degraded by cytosolic proteasomes. To investigate how soluble glycoproteins destined for degradation are retrotranslocated across the ER membrane, we analyzed the fate of two IgM subunits, mu and J, retained in the ER by myeloma cells that do not synthesize light chains. Degradation of mu and J is prevented by proteasome inhibitors, suggesting that both chains are retrotranslocated to be disposed of by proteasomes. Indeed, when proteasomes are inhibited, some deglycosylated J chains that no longer contain intrachain disulfide bonds accumulate in the cytosol. However, abundant glycosylated J chains are still present in the ER at time points in which degradation would have been almost complete in the absence of proteasome inhibitors, suggesting that retrotranslocation and degradation are coupled events. This was confirmed by protease protection and cell fractionation assays, which revealed that virtually all mu chains are retained in the ER lumen in a glycosylated state when proteasomes are inhibited. Association with calnexin correlated with the failure of mu chains to dislocate to the cytosol. Taken together, these results suggest that active proteasomes are required for the extraction of Ig subunits from the ER, though the requirements for retrotranslocation may differ among individual substrates. PMID- 10744634 TI - Subtle shifts in the ratio between pro- and antiapoptotic molecules after activation of corticosteroid receptors decide neuronal fate. AB - Glucocorticoid receptor (GR) activation induces apoptosis of granule cells in the hippocampus. In contrast, neuroprotection is seen after mineralocorticoid receptor (MR) activation. To date there is no in vivo evidence for direct interactions between corticosteroids and any of the key regulatory molecules of programmed cell death. In this report, we show that the opposing actions of MR and GR on neuronal survival result from their ability to differentially influence the expression of members of the bcl-2 gene family; specifically, in the rat hippocampus, activation of GR induces cell death by increasing the ratio of the proapoptotic molecule Bax relative to the antiapoptotic molecules Bcl-2 or Bcl x(L); the opposite effect is observed after stimulation of MR. The same results were obtained in both young and aged animals; however, older subjects (which were more susceptible to GR-mediated apoptosis) tended to express the antiapoptotic genes more robustly. Using a loss-of-function mouse model, we corroborated the observations made in the rat, demonstrating Bax to be essential in the GR mediated cell death-signaling cascade. In addition, we show that GR activation increases and MR activation decreases levels of the tumor suppressor protein p53 (a direct transcriptional regulator of bax and bcl-2 genes), thus providing new information on the early genetic events linking corticosteroid receptors with apoptosis in the nervous system. PMID- 10744635 TI - Glutathione S-transferase M1 null genotype is associated with a decreased risk of myocardial infarction. AB - Tobacco smoke is a major cause of both cancer and vascular disease. Although its carcinogenic role via induction of DNA damage and mutation is well established, the mechanisms involved in vascular disease remain unclear. One possibility is that DNA damage causes smooth muscle cell proliferation in the intima of arteries, thereby contributing to atherothrombotic processes. The binding of chemicals to DNA is modulated by detoxification enzymes, including glutathione S transferases (GST) and microsomal epoxide hydrolase (EPXH). We therefore examined whether polymorphisms in these genes influence risk of cardiovascular disease. Blood was obtained from 398 patients admitted for angiographic investigation of chest pain and 196 age- and sex-matched controls. Patients were subdivided into those with and without previous acute myocardial infarction (AMI). DNA was analyzed for deletions in the GSTM1 and T1 genes and for substitutions in EPXH and GSTP1 genes. The GSTM1 null genotype occurred at a significantly lower frequency in the AMI patient group (48%) compared both to patients with no history of AMI (59%) and to the control group (57.2%). When subjects were stratified for smoking status, a significant association was observed only in smokers, suggesting the polymorphism is more important in the presence of tobacco smoke exposure. The association remained significant after adjusting for age, sex, and stenosis (presence or absence). No significant associations were observed between the other genotypes and cardiovascular disease (chi(2) test; P>0.1). The results of this study indicate that the GSTM1 null genotype is protective against AMI, an effect that is more marked in smokers. However, further study is required in order to elucidate the as yet unexplained, mechanisms underlying this association. PMID- 10744636 TI - Heterogeneity of [Ca(2+)](i) signaling in intact rat aortic endothelium. AB - Most existing knowledge about [Ca(2+)](i) signaling in vascular endothelium has been based on studies using endothelial cells cultured in vitro. To examine how endothelial cells behave in situ, we have developed a method to monitor single cell [Ca(2+)](i) from Fura-2-loaded rat aortic segments. Fluorescence ratio images from large numbers of endothelial cells were acquired by using a flow chamber mounted on a dual-wavelength fluorescence microscope. Our results showed that either acetylcholine or histamine reversibly activated the vascular endothelium by eliciting M(3) or H(1) receptor-mediated [Ca(2+)](i) increases, respectively. The acetylcholine-evoked endothelial [Ca(2+)](i) elevation at the branch site (intercostal orifice) was much more pronounced than that at the non branch area. However, endothelium at the branch site was relatively insensitive to histamine. Both acetylcholine-sensitive and histamine-sensitive endothelial cells were arranged in belts aligned along flow lines and were intercalated with each other. Data analyzed from 400 endothelial cells located at the non-branch site showed drastically heterogeneous [Ca(2+)](i) responses to a fixed concentration of either acetylcholine or histamine, differing by two orders of magnitude in individual cells. As a conclusion, vascular endothelial cells appear to have their own characteristic [Ca(2+)](i) 'fingerprint' to various agonists and they may function coordinately in situ. PMID- 10744637 TI - Nitric oxide reduces vascular smooth muscle cell elastase activity through cGMP mediated suppression of ERK phosphorylation and AML1B nuclear partitioning. AB - Nitric oxide (NO) reduces the severity of pulmonary vascular disease in rats as do elastase inhibitors. We therefore hypothesized that NO inhibits elastase by suppressing mitogen-activated protein kinases that trans-activate AML1B, a transcription factor for elastase. We used cultured pulmonary artery smooth muscle cells in which serum-treated elastin (STE) induces a > threefold increase in elastase activity as evaluated by solubilization of [(3)H]-elastin. NO donors (SNAP and DETA NONOate) inhibited elastase in a dose-dependent manner as did a cGMP mimetic (8-pCPT-cGMP). SNAP inhibition of elastase was reversed by coadministration of a cGMP-PKG inhibitor (Rp-8-pCPT-cGMP). The STE-induced increase in phospho-ERK was suppressed by NO donors and the cGMP mimetic, and reversed by cGMP-PKG inhibitor, as was expression of AML1B and DNA binding in nuclear extracts. A concomitant increase in p38 phosphorylation was also inhibited by SNAP, but whereas MEK inhibitor (PD98059) suppressed elastase and AML1B-DNA binding, a p38 inhibitor (SB202190) did not. Our study uniquely links NO with inhibition of elastase-dependent matrix remodeling in vascular disease by suggesting a cGMP-PKG-related mechanism suppressing ERK-mediated partitioning of AML1B in nuclear extracts. PMID- 10744638 TI - NFkappaB decoy oligodeoxynucleotides reduce monocyte infiltration in renal allografts. AB - Monocyte influx secondary to ischemia-reperfusion conditions the renal allograft to rejection by presentation of antigens and production of cytokines. Monocyte influx depends on NFkappaB-dependent transcription of genes encoding adhesion molecules and chemokines. Here we demonstrate that cationic liposomes containing phosphorothioated oligodeoxynucleotides (ODN) with the kappaB binding site serving as competitive binding decoy, can prevent TNF-alpha-induced NFkappaB activity in endothelial cells in vitro. In an allogenic rat kidney transplantation model (BN to LEW), we show that perfusing the renal allograft with this decoy prior to transplantation abolishes nuclear NFkappaB activity in vivo and inhibits VCAM-1 expression in the donor endothelium (P<0.05). At 24 h postreperfusion, periarterial infiltration of monocytes/macrophages was significantly reduced in decoy ODN-treated allografts compared to control allografts (3.7+/-0.7 vs. 9.2+/-1.2 macrophages/vessel; P<0.01). At 72 h, there was a reduction of tubulointerstitial macrophage infiltration in decoy ODN treated kidneys compared to controls (75.6+/-13.9 vs. 120.0+/-11.2 macrophages/tubulointerstitial area; P<0.05). In conclusion, perfusion of the renal allograft with NFkappaB decoy ODN prior to transplantation decreases the initial inflammatory response in a stringent, nonimmunosuppressed allogenic transplantation model. Therefore, the NFkappaB decoy approach may be useful to explore the role of endothelium and macrophages in graft rejection and may be developed into a graft-specific immunosuppressive strategy allowing reduction of systemic immunosuppression on organ transplantation. PMID- 10744639 TI - Modeling of mosaic patterns in chimeric liver and adrenal cortex: algorithmic organogenesis? AB - If organogenesis were a completely deterministic process, then the amount of information required to store the spatial position and fate of every cell in vertebrate organisms would be larger than the total information that could be contained in their genomes. This suggests that the instructions of developmental mechanisms involved in organogenesis, coded in DNA, must be at least in part procedural or algorithmically based. Chimeric mosaic patterns in rat livers have been shown to be isotropic and to have fractal profiles (D approximately 1.3) whereas adrenal gland mosaics show a less irregular radial pattern, with lower fractal dimension (D approximately 1.2) than in the liver. These findings suggested a possible model of parenchyma generation. We propose that during organogenesis in both liver and adrenal cortex, the same basic mechanism is directed to organ mass enlargement, whereas the differences observed in mosaic patterns between the organs could be due to the control of a single parameter, namely, a form of contact inhibition. Computer simulations in two dimensions returned comparable results in both the fractal dimension value of mosaic patches and appearance of the mosaic 'tissues', as observed histologically in chimeras. This suggests that position information and locomotion of cells would not be required to produce the mosaic pattern observed in chimeras. PMID- 10744640 TI - Heterogeneous ectopic RNP-derived structures (HERDS) are markers of transcriptional arrest. AB - We have reviewed the reports in the literature concerning the segregation and clustering of ribonucleoprotein (RNP) -containing nuclear structures in either physiological or experimentally induced conditions (i.e., spermiogenesis and erythropoiesis in mammals, early embryonic development, hibernation, spontaneous and drug-induced apoptosis, treatment with different drugs). Irrespective of the biological system or the experimental conditions, heterogeneous ectopic RNP derived structures (HERDS) are always found. We hypothesize that the common event at the basis of this phenomenon might be the block in transcription; this is also consistent with our own and others' results on cultured cells after actinomycin D treatment. HERDS may therefore be considered as the morphological sign of transcriptional arrest. Based on the evidence that the restructuring/relocation of RNPs may be reversible, we also hypothesize that HERDS may serve as storage sites from which RNPs may be eventually retrieved, as soon as transcription restarts. Under acute stress conditions or during apoptotic cell death, the same reorganization of RNP-containing structures would be an adaptively suitable mechanism to induce an irreversible arrest in RNA processing, thus effectively blocking protein synthesis. PMID- 10744641 TI - Transcriptional regulation of the MHC II gene DRA in untransformed human thyrocytes. AB - MHC class II molecules are heterodimeric, polymorphic transmembrane glycoproteins physiologically expressed on cells of the immune system and pathologically expressed on the affected target cells of autoimmunity. Their function is to present processed peptides to antigen-specific CD4(+) T cells. To understand the molecular mechanism of the regulation of class II genes in autoimmune target cell thyrocytes, we investigated the transcriptional regulation of DRA on untransformed, differentiated human thyroid cells following IFN-gamma stimulation, which is potentially relevant to the inappropriate class II expression found in Graves' disease. Data from this study show that IFN-gamma enhances a promoter Y box binding protein and induces an X box binding protein in untransformed thyrocytes, but not in SV-40-transfected thyrocytes. Initial characterization of the proteins has indicated that the Y box binding protein is approximately 132 kDa in size while the X box binding protein binds to the X2 region and is approximately 116 kDa. The X box binding protein may correspond to poly(ADP-ribose) polymerase, a recently described component of the X2 box binding protein, X2BP. In addition, the signal transducer and activator of transcription 1alpha protein (STAT1alpha) is also induced by IFN-gamma in these cells. These results further suggest that there are differences in class II gene regulation between differentiated cells and transformed cell lines. PMID- 10744642 TI - In vivo cell penetration and intracellular transport of anti-Sm and anti-La autoantibodies. AB - Anti-nuclear autoantibodies (ANA) are the hallmark of systemic autoimmune diseases. Yet, the in vivo function of ANA remains controversial to a large extent due to the intracellular nature of their antigenic targets. It has been reported that a subset of autoantibodies can penetrate live cells and translocate into the subcellular compartments containing the corresponding antigens. The studies presented herein show that murine anti-Sm and anti-La monoclonal autoantibodies can also enter a variety of cell types from different animal species and that the cell penetration activity is not isotype-restricted. Interestingly, only mAb with cross-reactivity against double-stranded DNA did enter cells. Both these autoantibodies rapidly accumulate in the nucleus of viable cells but display different penetration kinetics. In co-localization experiments, monoclonal autoantibodies did not accumulate significantly within endocytic vesicles containing dextran, suggesting that they are internalized by mechanisms distinct from conventional receptor-mediated endocytosis. This report represents the first evidence that anti-La and anti-Sm autoantibodies are capable of entering live cells. Our observations support the notion that the phenomenon of intracellular autoantibodies may have a larger scope than previously reported and are consistent with a potential pathogenic role for ANA. PMID- 10744643 TI - The peripheral CD8 T cell repertoire is largely independent of the presence of intestinal flora. AB - While numerous studies have analyzed the shaping of T cell repertoires by self or foreign peptides, little is known on the influence of commensal self peptides derived from the intestinal flora (IF). Here, we have analyzed naive and immune repertoires in mice devoid of IF [germ-free (GF) mice]. First, by means of an extensive CDR3beta sequencing strategy, we show that the naive peripheral CD8 T cell repertoire does not exhibit a major imprint of IF antigens. Second, using MHC-peptide tetramers, CDR3beta length distribution analyses and TCR sequencing, we show that cytotoxic T lymphocyte (CTL) responses specific for two distinct epitopes are quasi-identical in normal and GF mice. Our findings indicate that, in general, peptides derived from the intestinal microflora have little if any influence on CTL responses in the mouse. PMID- 10744644 TI - Kappa-deficient mice are non-responders to dextran B512: is this unresponsiveness due to specialization of the kappa and lambda Ig repertoires? AB - In the dextran B512 high-responder strain C57BL, the response to dextran is restricted to the preferential expression of the V(H)B512 and the V(kappa)OX1 gene combination. The importance of the heavy chain is suggested by the fact that mice with the Ig C(H) allotype, different from C57BL, are low or non-responders to dextran, but the light chain could also play a role. All anti-dextran B512 mAb described to date (>200) use kappa light chains. No anti-dextran antibody using lambda has ever been observed. To ascertain if the restriction of the use of V(kappa) genes in response to dextran B512 was more stochastic or due to other factors, we have studied the response to dextran B512 in C57BL/6 mice where the C(kappa) domain has been disrupted (C57BL.C(kappa)T). These mice are unable to express kappa light chains and their humoral antibodies bear light chains of the lambda type. We found that C(kappa) knockout mice are unable to respond to dextran given in a thymus-independent or -dependent form. The lack of responsiveness is specifically directed to the dextran epitopes since these mice are fully competent to respond to other antigenic structures present in the same immunogenic molecule. These mice are also apparently normal regarding the expression of V(H) genes. Finally, we tested the response to dextran in C57BL.C(kappa)T mice carrying the lpr mutation that was introduced to favor an increase in the life span and make the response to dextran more easily detectable. The introduction of the lpr mutation was not sufficient to change the pattern of unresponsiveness in the C57BL.C(kappa)T mice. We concluded that there are deficiencies in the light chain repertoire because the V lambda light chain could not reconstitute the response to dextran. We discuss the possible mechanisms for this new type of unresponsiveness to dextran B512. PMID- 10744645 TI - Molecular cloning and characterization of mouse CD97. AB - The EGF-TM7 family (CD97 and EMR1) is a group of class II seven-span transmembrane receptors predominantly expressed by cells of the immune system. Recently, we have identified CD55, a regulatory molecule of the complement cascade, as a cellular ligand of human CD97 (hCD97). In this study, the molecular properties of mouse CD97 (mCD97) are described. Like hCD97, mCD97 has an extended extracellular region with several epidermal growth factor-like (EGF) domains. Due to alternative RNA splicing, isoforms with three and four EGF domains exist, designated mCD97(EGF1,2,4) and mCD97(EGF1,2, 3,4) respectively. All EGF domains, except for the N-terminal one, possess a calcium-binding site. In a third isoform mCD97(EGF1,2,X,3, 4), a sequence of 45 amino acids was found between the second and third EGF domain that does not correspond to any known protein module. Using newly generated mCD97 mAb, we show that analogous to the blood expression pattern of hCD97, mCD97 can be found on lymphoid and myeloid cells. Adhesion of mouse erythrocytes and splenocytes to COS cells expressing mCD97(EGF1,2,4) or mCD97(EGF1,2, 3,4) could be blocked by mouse CD55 (mCD55) antibody, identifying mCD55 as a cellular ligand for mCD97. Consistent with the necessity of directly linked EGF domains for the integrity of the CD55-binding site on hCD97, no adhesion was detected to the largest mouse isoform mCD97(EGF1,2,X,3,4). Remarkably, we found that the interaction between CD97 and CD55 is phylogenetically restricted, as indicated by the selective adhesion of primate erythrocytes to hCD97 transfectants, and of mouse and rat erythrocytes to mCD97 transfectants respectively. PMID- 10744646 TI - Defective p56Lck activity in T cells from an adult patient with idiopathic CD4+ lymphocytopenia. AB - Idiopathic CD4(+) lymphocytopenia (ICL) is defined by a stable loss of CD4(+) T cells in the absence of any known cause of immune deficiency. This syndrome is still of undetermined origin. It affects adult patients, some of them displaying opportunistic infections similar to HIV-infected subjects. The hypothesis that the cellular immune defect may be due to biochemical failures of the CD3-TCR pathway is investigated here in a patient associating a severe selective CD4(+) lymphocytopenia with an increased CD8(+) T cell count discovered in the course of a cryptococcal meningitidis. A 40% reduction of T cell proliferation to CD3-TCR stimulation is observed only in the CD4(+) subpopulation. The early CD3-induced protein tyrosine phosphorylations are conserved in both CD4(+) and CD8(+) subsets, and the levels of the T cell protein tyrosine kinases p56(Lck), p59(Fyn) and ZAP-70 are normal. However, we find a 50% reduction of p56(Lck) kinase activity in the patient's T cells compared to a healthy control donor. p59(Fyn) activity does not appear to be altered. Nevertheless, we do not find any genetic abnormality of p56(Lck). These results thus suggest that a defect of an unknown protein regulating p56(Lck) activity takes place in this patient's T cells. Taken together, these findings reveal p56(Lck) alteration in ICL and confirm the critical role of this kinase in the maintenance of the peripheral CD4(+) T cell subpopulation. PMID- 10744647 TI - Control of CD4 T cell fate by antigen re-stimulation with or without CTLA-4 engagement 24 h after priming. AB - After two consecutive inoculations with Staphylococcus enterotoxin B (SEB) at 24 h intervals in vivo, CD4 T cells became anergic to the antigen challenge in vitro. Administration of anti-CTLA-4 mAb in conjunction with the second SEB inoculation 24 h after antigen priming interfered with anergy and CD4 T cells became T(h)2 cells. However, the anergy induction was not ablated when SEB and anti-CTLA-4 mAb were administered 48 or 72 h after antigen priming. Moreover, anti-CTLA-4 mAb without SEB did not interfere with anergy nor promoted the T(h)2 differentiation. T-antigen-presenting cell (APC) interaction in vitro in the presence of high doses of antigen and anti-CTLA-4 mAb induced a T(h)2-polarizing cytokine IL-6 and IL-10. IL-10 then down-modulated a T(h)1-polarizing cytokine IL 12. The results demonstrate that 24 h after the initial antigen stimulation, CD4 T cells enter the critical activation phase where antigen re-stimulation with or without CTLA-4 engagement alters the fate of the cell, anergy or differentiation respectively. Once anergy is interfered with, T(h)2-polarizing cytokines produced upon prolonged T-APC interaction favor the T(h)2 differentiation. PMID- 10744648 TI - The role of antigen-independent persistence of memory cytotoxic T lymphocytes. AB - We use mathematical models to analyze the role of a memory cytotoxic T lymphocyte (CTL) response in viral infections. The model predicts that antigen-independent persistence of an elevated number of precursor CTL (CTLp) does not protect the host from clinical symptoms upon re-infection. Instead, we find that antigen independent long-term persistence of CTLp is required to clear virus infections. This mechanism also applies to infection in hosts that have never experienced the pathogen before. Requirement of antigen for the long-term maintenance of CTLp results in failure to clear the infection, even in hosts characterized by a high CTL responsiveness. We compare the CTL model to a B cell model. In keeping with experimentally established findings, B cells are efficient in protecting against re-infection, but are unlikely to clear viral infections unless the virus is cytopathic. We conclude that the role of 'memory CTLp' is different from the role of memory B cells in viral infections: antigen-independent long-term persistence of CTLp is a pre-requisite to ensure clearance of infection. PMID- 10744649 TI - Inhibition of the CD8+ T cell-mediated cytotoxicity reaction by hypericin: potential for treatment of T cell-mediated diseases. AB - The cytotoxicity reaction of murine CD8 T lymphocytes has been found to be strongly inhibited by nanomolar concentrations of hypericin, a lipophilic dianthraquinone with photodynamic properties. Cytotoxic T lymphocyte (CTL) induced target cell apoptosis, as well as exocytosis of cytolytic granules from these cells, were ablated by hypericin, administered at the onset of the reaction, without affecting CTL viability. The inhibition of cytolysis occurred without the light irradiation which is essential for photosensitization. The findings suggest that the action of hypericin targets the effector CTL; however, apoptosis induced in murine L-cells with recombinant tumor necrosis factor (TNF) alpha was also prevented by hypericin. Since hypericin is a known inhibitor of protein kinase C, MAP kinase and at least one other tyrosine kinase, this inhibitory activity could play a role in the down-modulation of CTL-induced cytotoxicity. Furthermore, our studies show that the action of hypericin induces rapid dephosphorylation of phospholipids associated with low-density membranes in CTL, but not with membranes of the cytotoxic granules. The ability of hypericin to interfere with cytotoxicity may render it useful in the treatment of T cell mediated diseases. PMID- 10744650 TI - Redistribution of selectin counter-ligands induced by cytokines. AB - Soluble recombinant (r) P-selectin and rP-selectin immobilized on plastic surfaces were tested for their capacity to activate neutrophils to produce superoxide anion. Soluble rP-selectin was incapable of activating leukocytes, whereas immobilized rP-selectin was able to induce leukocyte activation. When neutrophils were pretreated with a low dose of IL-8, granulocyte colony stimulating factor or granulocyte macrophage colony stimulating factor, soluble rP-selectin was no longer inert. These cytokine-primed leukocytes produced superoxide anion in the presence of soluble rP-selectin. During this priming period, sialyl Lewis X (sLe(X)) epitopes redistributed to one end of the leukocytes. Similar polarization of sLe(X) epitopes was observed at the attachment site of cells that adhered to immobilized rP-selectin. Cap formation and superoxide anion production induced by solid-phase P-selectin or by IL-8 and soluble rP-selectin treatment were inhibited by treatment of the leukocytes with cytochalasin B. These observations suggest that the redistribution of the carbohydrate ligands and the polarization of the leukocyte surface through an active process is a prerequisite but not sufficient to leukocyte superoxide production through P-selectin. PMID- 10744651 TI - The motif for peptide binding to the insulin-dependent diabetes mellitus associated class II MHC molecule I-Ag7 validated by phage display library. AB - The MHC class II molecule I-Ag7 is essential for the development of insulin dependent diabetes mellitus (IDDM) in the non-obese diabetic (NOD) mouse but the requirements for peptide binding to I-Ag7 are still controversial. We have now isolated I-Ag7-binding phage from a large phage display library encoding random nonamer peptides. Ninety peptide-encoding regions of phage eluted from I-Ag7 were sequenced and >75% of the corresponding synthetic peptides bound to I-Ag7. Peptide alignment led to the identification of position-specific anchor residues. Hydrophobic (V and P) and positively charged (K) residues were highly enriched at P6 and positively charged (R and K), aromatic (Y) or hydrophobic (L) residues at P9. In addition, small amino acid residues (G and A) were enriched at P7 and G at P8. The primary anchors at P6 and P9 defining the phage-derived motif were present in most high-affinity I-Ag7-binding peptides from IDDM candidate antigens but only in < or =25% of peptides that were low-affinity binders or failed to bind to I-Ag7. A comparison of these results with the proposed motifs for peptide binding to I-Ag7 validates the one we have previously described. PMID- 10744652 TI - The CD45 tyrosine phosphatase regulates Campath-1H (CD52)-induced TCR-dependent signal transduction in human T cells. AB - Campath-1H, a humanized mAb undergoing clinical trials for treatment of leukemia, transplantation and autoimmune diseases, produces substantial lymphocyte depletion in vivo. The antibody binds to CD52, a highly glycosylated molecule attached to the membrane by a glycosylphosphatidylinositol anchor. Cross-linked Campath-1H is known to activate T cells in vitro. We have investigated the molecular basis for these effects by comparing the protein tyrosine phosphorylation signals induced by Campath-1H and the CD3 mAb OKT3 in primary T cells, and in CD45(+)TCR(+), CD45(-)TCR(+) and CD45(+)TCR(-) Jurkat subclones transfected with CD52. Our results show that Campath-1H triggers similar tyrosine phosphorylation events as OKT3 in both primary T cells and in the CD45(+)TCR(+) Jurkat sub-clone, albeit at quantitatively lower levels. However, no phospholipase C gamma 1 activation nor calcium signals were detected in response to CD52 ligation. The CD52-mediated induction of protein tyrosine phosphorylation was absolutely dependent upon the expression of both the TCR and the CD45 phosphotyrosine phosphatase at the cell surface. Cross-linking of Campath-1H was essential for signal transduction in all cells investigated. Fluorescence resonance energy transfer was used to demonstrate CD52 homo-association at the cell surface in Jurkat T cells in a TCR- and CD45-independent manner, and CD52 TCR association in CD45(+)TCR(+) cells. We propose a model to explain the activating effects of Campath-1H in which CD52 mAb cross-linking causes the trapping of TCR polypeptides within molecular complexes at the cell surface, thereby inducing signals via the TCR by a process which depends on the CD45 mediated regulation of the p56(lck) and p59(fyn) tyrosine kinases. PMID- 10744653 TI - Rapid B cell apoptosis induced by antigen receptor ligation does not require Fas (CD95/APO-1), the adaptor protein FADD/MORT1 or CrmA-sensitive caspases but is defective in both MRL-+/+ and MRL-lpr/lpr mice. AB - Antigen receptor ligation-induced apoptosis is thought to play a role in self tolerance by deleting autoreactive lymphocytes. Antigen receptor ligation-induced apoptosis of mature T cells and T cell lines requires autocrine or paracrine activation of Fas (CD95/APO-1). Whether B cell antigen receptor (BCR)-mediated apoptosis requires Fas or related molecules is unclear. Here we demonstrate that expression of either CrmA, the cowpox virus serpin, or an inhibitor of the adapter protein FADD/MORT1 blocks Fas-mediated apoptosis but has no effect on BCR ligation-induced apoptosis of the B cell line WEHI-231. In contrast, expression of Bcl-2 blocks BCR-mediated but not Fas-induced apoptosis in WEHI-231 cells. These results indicate that BCR ligation activates an apoptotic signaling pathway distinct from Fas-mediated apoptosis in WEHI-231 cells, and that BCR-mediated apoptosis of WEHI-231 cells does not require Fas or related molecules such as DR3, DR4 and DR5, as all of these death receptors require FADD/MORT1 and/or CrmA sensitive caspases for induction of apoptosis. Moreover, extensive BCR ligation induces death of mature B cells from C57BL/6-lpr/lpr mice as efficiently as those from C57BL/6 mice, indicating that Fas is not essential for BCR-mediated apoptosis of mature B cells. In contrast, BCR ligation-induced apoptosis is reduced in mature B cells from MRL mice and this is not affected by the lpr mutation. Since MRL-lpr/lpr mice but not C57BL/6-lpr/lpr mice develop severe autoimmune disease, defects in BCR-mediated apoptosis in the MRL background, together with lpr mutation, may contribute to the development of severe autoimmune disease in MRL-lpr/lpr mice by allowing survival of self-reactive B cells. PMID- 10744654 TI - The geometry of synthetic peptide-based immunogens affects the efficiency of T cell stimulation by professional antigen-presenting cells. AB - In the pathway leading to antibody production there are two points at which CD4(+) T(h) cells need to be recruited. The first of these is priming of T cells by their interaction with dendritic cells (DC) bearing antigen presented on MHC class II molecules and the second is the collaborative interaction of these primed T cells with B cells presenting the same antigen. We have previously shown that the configuration of T and B cell determinants within synthetic peptide immunogens can greatly influence the amount of immunogen required to produce an antibody response. Here we investigate whether the difference in potency of different immunogens is related to their ability to be presented by either DC or B cells. We show that determinants in a branched configuration, which are the most efficient at eliciting antibody in vivo, are presented to T cell clones by splenic CD8(-) DC 10-fold more efficiently than the corresponding determinants in a tandem linear arrangement. B cells also showed preferential presentation of branched immunogens to one T cell clone but in contrast to DC, not to a second T cell clone, indicating differences between the two antigen-presenting cell types. We also show that branched immunogens have a greater stability in serum compared to linear peptides, which may further enhance the differences in their in vivo potency. PMID- 10744655 TI - Evidence for in situ expansion of diverse antitumor-specific cytotoxic T lymphocyte clones in a human large cell carcinoma of the lung. AB - We have isolated several cytotoxic T lymphocyte (CTL) clones from lymphocytes infiltrating a human large cell carcinoma (LCC) of the lung. All these clones were found to express a CD3(+), TCRalphabeta(+), CD8(+), CD4(-), CD28(-) phenotype. According to their TCR beta chain variable region expression, they were divided in three major groups. The first group, including the majority of the clones, expressed a unique V(beta)3-J(beta)1.2 TCR. The second group expressed a V(beta)22-J(beta)1.4 rearrangement and the third group, including only two clones, expressed a V(beta)8-J(beta)1.5 TCR. Functional studies showed that all the CTL clones mediated a high cytotoxic activity against the autologous tumor cell line. While the V(beta)3(+) clones showed a weak lysis against few allogeneic non-small cell lung cancer (NSCLC) tumor cell lines, V(beta)8(+) and V(beta)22(+) T cell clones were able to kill a panel of allogeneic NSCLC tumor cell lines. Cytotoxicity-blocking experiments using specific mAb indicated that, while the V(beta)3(+) and V(beta)22(+) CTL clones were HLA-A2 restricted, the V(beta)8(+) clones appeared HLA-B or -C restricted. TCR transcripts expressed in the cloned cells were determined by CDR3 size and sequence analyses, and compared to those present in fresh tumor tissue. Interestingly, our studies demonstrated that the CTL clones identified in vitro were selectively expanded in vivo at the tumor site as compared to autologous peripheral blood lymphocytes. These results further provide evidence that an immune response may take place in NSCLC and that effector T cells may contribute to tumor regression. PMID- 10744656 TI - Stimulation of Fc gamma R receptors induces monocyte chemoattractant protein-1 in the human monocytic cell line THP-1 by a mechanism involving I kappa B-alpha degradation and formation of p50/p65 NF-kappa B/Rel complexes. AB - THP-1 monocytic/macrophage cells were stimulated via their FcgammaR receptors with insoluble aggregates of human IgG and the production of the C-C chemokine monocyte chemoattractant protein (MCP)-1 assayed. A dose- and time-dependent production of MCP-1 comparable to that produced by the most potent agonists could be detected in the culture medium by a sensitive ELISA assay. This was accompanied by a parallel activation of the transcription factor NF-kappaB as judged from both the appearance of kappaB-binding activity containing p50/p65 NF kappaB/Rel complexes in the nuclear extract and the disappearance of the NF kappaB inhibitor IkappaB-alpha in the cell lysate. In contrast, IkappaB-beta and IkappaB-epsilon expression was not modified, thus pointing to the occurrence of a selective degradation of IkappaB-alpha under those conditions. Attempts to modulate MCP-1 production with compounds that display inhibitory effects on the activation of NF-kappaB such as the proteasome inhibitor N-acetyl-leucinyl leucinyl-norleucinal, the antioxidant pyrrolidine dithiocarbamate and the salicylate derivative 2-hydroxy-4-trifluoromethylbenzoic acid showed a parallel effect on both MCP-1 production and NF-kappaB activation, thus pointing to the involvement of kappaB-binding sites on the transcriptional regulation of MCP-1 production. Our findings suggest the existence in monocytic cells of a signaling mechanism initiated by cross-linking of low-affinity FcgammaR, most likely of the FcgammaRII family since THP-1 cells do not express FcgammaRIII receptors, that involves activation of NF-kappaB associated to the proteolytic degradation of IkappaB-alpha and leads to the transcriptional up-regulation of MCP-1. PMID- 10744657 TI - A novel murine anti-human Fas mAb which mitigates lymphadenopathy without hepatotoxicity. AB - Defects in Fas-mediated apoptosis are implicated in autoimmune diseases including rheumatoid arthritis (RA). Although induction of Fas-mediated apoptosis could have therapeutic effects on these diseases, it might cause deleterious effects in liver as Fas ligand or an agonistic anti-murine Fas antibody Jo2 causes severe hepatic injury in mice. We report here on the interesting characteristics of the newly obtained anti-Fas mAb, HFE7A, which cross-reacts with the Fas molecules of various species ranging from human to mouse and mitigates autoimmune symptoms without hepatotoxicity in mice. The administration of HFE7A to mice induced apoptosis in the thymocytes, although administration of HFE7A to mice or to marmosets did not induce any sign of hepatitis. The effect of HFE7A on liver is different from that of anti-murine Fas antibody Jo2, which causes acute and lethal hepatic injury to mice. Administration of HFE7A reduced lymphadenopathy and abnormal T cells in MRL-gld/gld mice. HFE7A induced apoptosis in synovial cells prepared from RA patients. Surprisingly, HFE7A protected mice from fulminant hepatitis induced by Jo2. Therefore, HFE7A is a potential therapeutic antibody not only for autoimmune diseases including RA but also for fulminant hepatitis. PMID- 10744658 TI - Activation of NF-kappa B in human endothelial cells induced by monoclonal and allospecific HLA antibodies. AB - Chronic graft rejection, characterized by a gradual occlusion of grafted vessels, is the most serious complication following heart and kidney transplantation. Although often associated with chronic production of anti-HLA and anti endothelial antibodies, the precise role of antibodies in chronic rejection remains uncertain. Here we have investigated whether HLA-specific antibodies, either monoclonal or derived from patients, cause endothelial cell activation. Thus we investigated tyrosine phosphorlyation, NF-kappaB activation and cell proliferation in human umbilical vein endothelial cells (HUVEC) or microvascular endothelial cells from adult human heart (CMEC). Ligation of monomorphic determinants of MHC class I molecules (using the mAb W6/32) on the surface of HUVEC caused an increase in tyrosine phosphorylation of proteins of mol. wt approximately 75-80 kDa. Similarly, ligation of monomorphic determinants on both CMEC and HUVEC resulted in increased NF-kappaB binding compared to controls (by 74.4 and 52.5%, P = 0.001) and this was enhanced by addition of secondary antibody. Two HLA-specific mAb resulted in a 277 and 170% increase in NF-kappaB binding activity compared to controls. Four patient samples containing HLA antibodies were used against HLA-specific HUVEC and four samples were incubated with HUVEC bearing irrelevant antigens. Patient sera alone enhanced NF-kappaB binding by 27-186%, but only when added to HUVEC bearing relevant antigens. W6/32 and allospecific antibodies from patients significantly enhanced HUVEC proliferation, measured by uptake of [(3)H]thymidine. In conclusion, activation of NF-kappaB by human anti-HLA antibodies demonstrates their potential role in pathogenesis of chronic vascular occlusive disease following transplantation. PMID- 10744659 TI - A BASH/SLP-76-related adaptor protein MIST/Clnk involved in IgE receptor-mediated mast cell degranulation. AB - Cross-linking of the high-affinity IgE receptor (FcepsilonRI) on mast cells by IgE-antigen complex triggers signal transduction cascades leading to the release of inflammatory mediators and production of cytokines, which are critical for the development of allergic reactions. We have identified a novel member of the BASH/SLP-76 immunoreceptor-coupled adaptor family expressed in mast cells, termed MIST (for mast cell immunoreceptor signal transducer), which has later been found to be identical to a recently reported cytokine-dependent hemopoietic cell linker, Clnk. Upon FcepsilonRI cross-linking, MIST/Clnk is tyrosine phosphorylated and associates with signaling proteins, phospholipase Cgamma, Vav, Grb2 and linker for activation of T cells (LAT). Overexpression of a mutant form of MIST/Clnk inhibited FcepsilonRI-mediated degranulation, increase in intracellular Ca(2+), NF-AT activation and phosphorylation of LAT. As a crucial signaling component for FcepsilonRI-induced mast cell degranulation, MIST/Clnk might serve as a target for anti-allergic therapy. PMID- 10744660 TI - A ribonuclease A variant with low catalytic activity but high cytotoxicity. AB - Onconase, a homolog of ribonuclease A (RNase A) with low ribonucleolytic activity, is cytotoxic and has efficacy as a cancer chemotherapeutic. Here variants of RNase A were used to probe the interplay between ribonucleolytic activity and evasion of the cytosolic ribonuclease inhibitor protein (RI) in the cytotoxicity of ribonucleases. K41R/G88R RNase A is a less active catalyst than G88R RNase A but, surprisingly, is more cytotoxic. Like Onconase, the K41R/G88R variant has a low affinity for RI, which apparently compensates for its low ribonucleolytic activity. In contrast, K41A/G88R RNase A, which has the same affinity for RI as does the K41R/G88R variant, is not cytotoxic. The nontoxic K41A/G88R variant is a much less active catalyst than is the toxic K41R/G88R variant. These data indicate that maintaining sufficient ribonucleolytic activity in the presence of RI is a requirement for a homolog or variant of RNase A to be cytotoxic. This principle can guide the design of new chemotherapeutics based on homologs and variants of RNase A. PMID- 10744661 TI - Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility. AB - Myosin light chain phosphatase (MLCP) plays a pivotal role in smooth muscle contraction by regulating Ca(2+) sensitivity of myosin light chain phosphorylation. A smooth muscle phosphoprotein called CPI-17 specifically and potently inhibits MLCP in vitro and in situ and is activated when phosphorylated at Thr-38, which increases its inhibitory potency 1000-fold. We produced a phosphospecific antibody for this site in CPI-17 and used it to study in situ phosphorylation of endogenous CPI-17 in arterial smooth muscle in response to agonist stimulation. In the intact femoral artery, CPI-17 phosphorylation was negligible at the resting state and was not increased during contraction induced by K(+) depolarization. The Ca(2+)-sensitizing agonists histamine and phenylephrine induced nearly equivalent contractions, but histamine generated significantly higher levels of CPI-17 phosphorylation. In alpha-toxin permeabilized strips at pCa 6.7, contractile force and CPI-17 phosphorylation were proportional in response to histamine, guanosine 5'-O-(gamma thiotriphosphate), and histamine plus guanyl-5'-yl thiophosphate, implying that histamine increased CPI-17 phosphorylation through activation of G proteins. Inhibitors of Rho-kinase (Y27632) and protein kinase C (PKC; GF109203X) reduced contraction and CPI-17 phosphorylation in parallel, suggesting that CPI-17 functions downstream of Rho kinases and PKC. The results show that agonists such as histamine signal through phosphorylation of CPI-17 to produce Ca(2+) sensitization of smooth muscle contraction. PMID- 10744662 TI - IHG-2, a mesangial cell gene induced by high glucose, is human gremlin. Regulation by extracellular glucose concentration, cyclic mechanical strain, and transforming growth factor-beta1. AB - We used cloning in silico coupled with polymerase chain reaction to demonstrate that IHG-2 is part of the 3'-untranslated region of gremlin, a member of the DAN family of secreted proteins that antagonize the bioactivities of members of the transforming growth factor (TGF)-beta superfamily. Mesangial cell gremlin mRNA levels were induced by high glucose, cyclic mechanical strain, and TGF-beta1 in vitro, and gremlin mRNA levels were elevated in the renal cortex of rats with streptozotocin-induced diabetic nephropathy in vivo. gremlin expression was observed in parallel with induction of bone morphogenetic protein-2 (BMP-2), a target for gremlin in models of cell differentiation. Together these data indicate that (a) IHG-2 is gremlin, (b) gremlin is expressed in diabetic nephropathy in vivo, (c) both glycemic and mechanical strain stimulate mesangial cell gremlin expression in vitro, (d) high glucose induces gremlin, in part, through TGFbeta-mediated pathways, and (e) Gremlin is a potential endogenous antagonist of BMPs within a diabetic glomerular milieu. PMID- 10744663 TI - Up-regulation of glucosylceramide synthesis upon stimulation of axonal growth by basic fibroblast growth factor. Evidence for post-translational modification of glucosylceramide synthase. AB - We have previously shown that ongoing glucosylceramide (GlcCer) synthesis is required for basic fibroblast growth factor (bFGF) and laminin to stimulate axonal growth in cultured hippocampal neurons (Boldin, S., and Futerman, A. H. (1997) J. Neurochem. 68, 882-885). We now demonstrate that stimulation of axonal growth by bFGF leads to an increase in the rate of GlcCer synthesis. Within minutes of incubation with bFGF, a significant increase in the rate of metabolism of [(14)C]hexanoyl ceramide to [(14)C]hexanoyl GlcCer is detected, but there are no changes in the rate of [(14)C]hexanoyl sphingomyelin synthesis. In vitro analysis of GlcCer synthase activity revealed an approximately 2-fold increase in the rate of [(14)C]hexanoyl GlcCer synthesis upon incubation with either bFGF or laminin; other growth factors, which did not effect the rate of axon growth, had no effect on the rate of [(14)C]hexanoyl GlcCer synthesis. The increased rate of [(14)C]hexanoyl GlcCer synthesis was not affected by preincubation with either cycloheximide or actinomycin, and no elevation of GlcCer synthase mRNA levels was detected, suggesting that GlcCer synthase is up-regulated by a post-translational mechanism. The relevance of these results for understanding the regulation of axonal growth is discussed. PMID- 10744664 TI - Characterization of Staphylococcus aureus cell wall glycan strands, evidence for a new beta-N-acetylglucosaminidase activity. AB - Using sequential digestion with the glycyl-glycine endopeptidase lysostaphin followed by the pneumococcal N-acetylmuramyl-L-alanine amidase (amidase), the glycan strands of the peptidoglycan of Staphylococcus aureus were purified and analyzed by a combination of reverse-phase-high pressure liquid chromatography (HPLC) and mass spectrometry. Reverse-phase-HPLC resolved the glycan strands to a family of major peaks, which represented oligosaccharides composed of repeating disaccharide units (N-acetylglucosamine-[beta-1, 4]-N-acetylmuramic acid) with different degrees of polymerization and terminating with N-acetylmuramic acid residues at the reducing ends. The method allowed separation of strands up to 23 26 disaccharide units with a predominant length between 3 and 10 and an average degree of polymerization of approximately 6. Glycan strands with a higher degree of polymerization (>26 disaccharide units) represented 10-15% of the total UV absorbing glycan material. A unique feature of the staphylococcal glycan strands was the presence of minor satellite peaks that were present throughout the HPLC elution profile eluting either just prior or shortly after the major oligosaccharide peaks. A number of observations including mass spectrometric analysis suggest that the satellites are the products of an N acetylglucosaminidase activity that differs from the atl gene product and that appears to be involved with modification of the glycan strand structure. PMID- 10744665 TI - Phosphorylation-dependent interaction between plant plasma membrane H(+)-ATPase and 14-3-3 proteins. AB - The H(+)-ATPase is a key enzyme for the establishment and maintenance of plasma membrane potential and energization of secondary active transport in the plant cell. The phytotoxin fusicoccin induces H(+)-ATPase activation by promoting the association of 14-3-3 proteins. It is still unclear whether 14-3-3 proteins can represent natural regulators of the proton pump, and factors regulating 14-3-3 binding to the H(+)-ATPase under physiological conditions are unknown as well. In the present study in vivo and in vitro evidence is provided that 14-3-3 proteins can associate with the H(+)-ATPase from maize roots also in a fusicoccin independent manner and that the interaction depends on the phosphorylation status of the proton pump. Furthermore, results indicate that phosphorylation of H(+) ATPase influences also the fusicoccin-dependent interaction of 14-3-3 proteins. Finally, a protein phosphatase 2A able to impair the interaction between H(+) ATPase and 14-3-3 proteins was identified and partially purified from maize root. PMID- 10744666 TI - Role of the N-terminal proline residue in the catalytic activities of the Escherichia coli Fpg protein. AB - The Escherichia coli Fpg protein is a DNA glycosylase/AP lyase. It removes, in DNA, oxidized purine residues, including the highly mutagenic C8-oxo-guanine (8 oxoG). The catalytic mechanism is believed to involve the formation of a transient Schiff base intermediate formed between DNA containing an oxidized residue and the N-terminal proline of the Fpg protein. The importance and the role of this proline upon the various catalytic activities of the Fpg protein was examined by targeted mutagenesis, resulting in the construction of three mutant Fpg proteins: Pro-2 --> Gly (FpgP2G), Pro-2 --> Thr (FpgP2T), and Pro-2 --> Glu (FpgP2E). The formamidopyrimidine DNA glycosylase activities of FpgP2G and FpgP2T were comparable and accounted for 10% of the wild-type activity. FpgP2G and FpgP2T had barely detectable 8-oxoG-DNA glycosylase activity and produced minute Schiff base complex with 8-oxoG/C DNA. FpgP2G and FpgP2T mutants did not cleave a DNA containing preformed AP site but readily produced Schiff base complex with this substrate. FpgP2E was completely inactive in all the assays. The binding constants of the different mutants when challenged with a duplex DNA containing a tetrahydrofuran residue were comparable. The mutant Fpg proteins barely or did not complement in vivo the spontaneous transitions G/C --> T/A in E. coli BH990 (fpg mutY) cells. These results show the mandatory role of N-terminal proline in the 8-oxoG-DNA glycosylase activity of the Fpg protein in vitro and in vivo as well as in its AP lyase activity upon preformed AP site but less in the 2,6 diamino-4-hydroxy-5-N-methylformamidopyrimidine-DNA glycosylase activity. PMID- 10744667 TI - Sequential strand exchange by XerC and XerD during site-specific recombination at dif. AB - Successful segregation of circular chromosomes in Escherichia coli requires that dimeric replicons, produced by homologous recombination, are converted to monomers prior to cell division. The Xer site-specific recombination system uses two related tyrosine recombinases, XerC and XerD, to catalyze resolution of circular dimers at the chromosomal site, dif. A 33-base pair DNA fragment containing the 28-base pair minimal dif site is sufficient for the recombinases to mediate both inter- and intramolecular site-specific recombination in vivo. We show that Xer-mediated intermolecular recombination in vitro between nicked linear dif "suicide" substrates and supercoiled plasmid DNA containing dif is initiated by XerC. Furthermore, on the appropriate substrate, the nicked Holliday junction intermediate formed by XerC is converted to a linear product by a subsequent single XerD-mediated strand exchange. We also demonstrate that a XerC homologue from Pseudomonas aeruginosa stimulates strand cleavage by XerD on a nicked linear substrate and promotes initiation of strand exchange by XerD in an intermolecular reaction between linear and supercoiled DNA, thereby reversing the normal order of strand exchanges. PMID- 10744668 TI - The genomic organization, complete mRNA sequence, cloning, and expression of a novel human intracellular membrane-associated calcium-independent phospholipase A(2). AB - During the sequencing of the long arm of chromosome 7 in the Human Genome Project, a predicted protein product of 40 kDa was identified, which contained two approximately 10-amino acid segments homologous to the ATP and lipase consensus sequences present in the founding members of a family of calcium independent phospholipases A(2). Detailed inspection of the identified sequence (residues 79, 671-109,912 GenBank accession no. AC005058) demonstrated that it represented only a partial sequence of a larger undefined polypeptide product. Accordingly, we identified the complete genomic organization of this putative phospholipase A(2) through analyses of previously published expressed sequence tags, PCR of human heart cDNA, and 5'-rapid amplification of cDNA ends. Polymerase chain reaction and Northern blotting demonstrated a 3.4-kilobase message, which encoded a polypeptide with a maximum calculated molecular weight of 88476.9. This 3.4-kilobase message was present in multiple human parenchymal tissues including heart, skeletal muscle, placenta, brain, liver, and pancreas. Cloning and expression of the protein encoded by this message in Sf9 cells resulted in the production of two proteins of apparent molecular masses of 77 and 63 kDa as assessed by Western analyses utilizing immunoaffinity-purified antibody. Membranes from Sf9 cells expressing recombinant protein released fatty acid from sn-2-radiolabeled phosphatidylcholine and plasmenylcholine up to 10 fold more rapidly than controls. The initial rate of fatty acid release from the membrane fraction was 0. 3 nmol/mg.min. The recombinant protein was entirely calcium-independent, had a pH optimum of 8.0, was inhibited by (E)-6 (bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (IC(50) = 3 microM), and was predominantly present in the membrane-associated fraction. Collectively, these results describe the genomic organization, complete mRNA sequence, and sn-2 lipase activity of a novel intracellular calcium-independent membrane-associated phospholipase A(2). PMID- 10744669 TI - Genuine monovalent ligands of TrkA nerve growth factor receptors reveal a novel pharmacological mechanism of action. AB - Developing small molecule agonistic ligands for tyrosine kinase receptors has been difficult, and it is generally thought that such ligands require bivalency. Moreover, multisubunit receptors are difficult to target, because each subunit contributes to ligand affinity, and each subunit may have distinct and sometimes opposing functions. Here, the nerve growth factor receptor subunits p75 and the tyrosine kinase TrkA were studied using artificial ligands that bind specifically to their extracellular domain. Bivalent TrkA ligands afford robust signals. However, genuine monomeric and monovalent TrkA ligands afford partial agonism, activate the tyrosine kinase activity, cause receptor internalization, and induce survival and differentiation in cell lines and primary neurons. Monomeric and monovalent TrkA ligands can synergize with ligands that bind the p75 subunit. However, the p75 ligands used in this study must be bivalent, and monovalent p75 ligands have no effect. These findings will be useful in designing and developing screens of small molecules selective for tyrosine kinase receptors and indicate that strategies for designing agonists of multisubunit receptors require consideration of the role of each subunit. Last, the strategy of using anti receptor mAbs and small molecule hormone mimics as receptor ligands could be applied to the study of many other heteromeric cell surface receptors. PMID- 10744670 TI - p-hydroxyphenylacetaldehyde, an aldehyde generated by myeloperoxidase, modifies phospholipid amino groups of low density lipoprotein in human atherosclerotic intima. AB - Oxidation of low density lipoprotein (LDL) may be of critical importance in the pathogenesis of atherosclerosis. Recent studies suggest that oxidized phospholipids render LDL atherogenic. However, both the structures and the physiologically relevant pathways for the formation of modified phospholipids in oxidized LDL remain poorly understood. We previously showed that p hydroxyphenylacetaldehyde (pHA) is the major product of L-tyrosine oxidation by the myeloperoxidase/hydrogen peroxide/chloride system of phagocytes. In the current studies, we demonstrate that this reactive aldehyde targets the aminophospholipids of LDL in vitro and in vivo. Activated human neutrophils generated pHA-ethanolamine, the reduced adduct of pHA with the amino group of phosphatidylethanolamine, on LDL phospholipids by a reaction that required myeloperoxidase, H(2)O(2), and L-tyrosine. The cellular system could be replaced by HOCl and L-tyrosine but not by a wide variety of other oxidation systems, indicating that pHA-ethanolamine is a specific marker for covalent modification of aminophospholipids by myeloperoxidase. To determine whether aldehydes modify aminophospholipids in vivo, we quantified levels of pHA-ethanolamine in acid hydrolysates of reduced lipid extracts through isotope dilution gas chromatography/mass spectrometry. Circulating LDL contained undetectable levels of pHA-modified phospholipid (<0.1 mmol/mol). In contrast, the concentration of pHA-ethanolamine in LDL isolated from human atherosclerotic lesions was strikingly elevated (4.5 mmol/mol). Collectively, these results demonstrate a novel, myeloperoxidase-based mechanism for modifying the amino group of LDL phospholipids. They also offer the first evidence that myeloperoxidase may damage LDL lipids in vivo, raising the possibility that aldehyde-modified aminophospholipids play a role in inflammation and vascular disease. PMID- 10744671 TI - Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro. AB - Dengue virus type 2 NS3, a multifunctional protein, has a serine protease domain (NS3pro) that requires the conserved hydrophilic domain of NS2B for protease activity in cleavage of the polyprotein precursor at sites following two basic amino acids. In this study, we report the expression of the NS2B-NS3pro precursor in Escherichia coli as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni(2+) affinity and gel filtration chromatography under denaturing conditions. The denatured precursor was refolded to yield a purified active protease complex. Biochemical analysis of the protease revealed that its activity toward either a natural substrate, NS4B-NS5 precursor, or the fluorogenic peptide substrates containing two basic residues at P1 and P2, was dependent on the presence of the NS2B domain. The peptide with a highly conserved Gly residue at P3 position was 3-fold more active as a substrate than a Gln residue at this position. The cleavage of a chromogenic substrate with a single Arg residue at P1 was NS2B-independent. These results suggest that heterodimerization of the NS3pro domain with NS2B generates additional specific interactions with the P2 and P3 residues of the substrates. PMID- 10744672 TI - A potent new class of reductively activated peptide gene delivery agents. AB - A new class of peptide gene delivery agents were developed by inserting multiple cysteine residues into short (dp 20) synthetic peptides. Substitution of one to four cysteine residues for lysine residues in Cys-Trp-Lys(18) resulted in low molecular weight DNA condensing peptides that spontaneously oxidize after binding to plasmid DNA to form interpeptide disulfide bonds. The stability of cross linked peptide DNA condensates increased in proportion to the number of cysteines incorporated into the peptide. Disulfide bond formation led to a decrease in particle size relative to control peptide DNA condensates and prevented dissociation of peptide DNA condensates in concentrated sodium chloride. Cross linked peptide DNA condensates were 5-60-fold more potent at mediating gene expression in HepG2 and COS 7 cells relative to uncross-linked peptide DNA condensates. The enhanced gene expression was dependent on the number of cysteine residues incorporated, with a peptide containing two cysteines mediating maximal gene expression. Cross-linking peptides caused elevated gene expression without increasing DNA uptake by cells, suggesting a mechanism involving intracellular release of DNA triggered by disulfide bond reduction. The results establish cross linking peptides as a novel class of potent gene delivery agents that enhance gene expression through a new mechanism of action. PMID- 10744673 TI - Tubulin anchoring to glycolipid-enriched, detergent-resistant domains of the neuronal plasma membrane. AB - After incubation of intact living cultured rat cerebellar granule cells at 37 degrees C with a new GM1 ganglioside analog, carrying a diazirine group and labeled with (125)I in the ceramide moiety, followed by photoactivation, a relatively small number of radiolabeled proteins were detected in a membrane enriched fraction. A protein of about 55 kDa with a pI of about 5 carried a large portion of the radioactivity even if incubation and cross-linking were performed at 4 degrees C and in the presence of inhibitors of endocytosis, suggesting that it is cross-linked at the plasma membrane. Immunoprecipitation and Western blotting experiments showed the positivity of this protein for tubulin. Trypsin treatment of intact cells ruled out the involvement of a plasma membrane surface tubulin. Release of radioactivity from cross-linked tubulin after KOH treatment (but not hydroxylamine treatment) suggested that the photoactivated ganglioside reacts with an ester-linked fatty acid anchor of tubulin. Low buoyancy, detergent resistant membrane fractions, isolated from cells after incubation with the GM1 analogue and photoactivation, proved their enrichment in endogenous and radioactive GM1 ganglioside, sphingomyelin, cholesterol, signal transduction proteins, and tubulin. It is noteworthy that radioactive tubulin was also detected in this fraction, indicating the presence of tubulin molecules carrying a fatty acid anchor in detergent-resistant, ganglioside-enriched domains of the plasma membrane. Parallel experiments carried out with a phosphatidylcholine analogue, also carrying a diazirine group and labeled with (125)I in the fatty acid moiety, showed the specificity of tubulin interaction with GM1. Taken together, these results indicate that some tubulin molecules are associated with a lipid anchor to detergent-resistant glycolipid-enriched domains of the plasma membrane. This novel feature of membrane domains can provide a key for a better understanding of their biological role. PMID- 10744674 TI - A stretch of positively charged amino acids at the N terminus of Hansenula polymorpha Pex3p is involved in incorporation of the protein into the peroxisomal membrane. AB - Pex3p is a peroxisomal membrane protein that is essential for peroxisome biogenesis. Here, we show that a conserved stretch of positively charged amino acids (Arg(11)-X-Lys-Lys-Lys(15)) in the N terminus of Hansenula polymorpha Pex3p is involved in incorporation of the protein into its target membrane. Despite the strong conservation, this sequence shows a high degree of redundancy. Substitution of either Arg(11), Lys(13), Lys(14), or Lys(15) with uncharged or negatively charged amino acids did not interfere with Pex3p location and function. However, a mutant Pex3p, carrying negatively charged amino acids at position 13 and 15 (K13E/K15E), caused moderate but significant defects in peroxisome assembly and matrix protein import. Additional changes in the N terminus of Pex3p, e.g. replacing three or four of the positively charged amino acids with negatively charged ones, led to a typical pex3 phenotype, i.e. accumulation of peroxisomal matrix proteins in the cytosol and absence of peroxisomal remnants. Also, in these cases, the mutant Pex3p levels were reduced. Remarkably, mutant Pex3p proteins were mislocalized to mitochondria or the cytosol, depending on the nature of the mutation. Furthermore, in case of reduced amounts of Pex3p, the levels of other peroxisomal membrane proteins, e.g. Pex10p and Pex14p, were also diminished, suggesting that Pex3p maybe involved in the recruitment or stabilization of these proteins (in the membrane). PMID- 10744675 TI - A cell adhesion protein from the crayfish Pacifastacus leniusculus, a serine proteinase homologue similar to Drosophila masquerade. AB - A cDNA encoding a protein resembling masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development in Drosophila melanogaster, was identified in hemocytes of the adult freshwater crayfish, Pacifastacus leniusculus. The crayfish protein is similar to Drosophila masquerade in the following aspects: (a) overall sequence of the serine proteinase domain, such as the position of three putative disulfide bridges, glycine in the place of the catalytic serine residue, and the presence of a substrate-lining pocket typical for trypsins; (b) the presence of several copies of a disulfide-knotted motif in the putative propeptide. This masquerade-like protein is cleaved into a 27-kDa fragment, which could be detected by immunoblot analysis using an affinity-purified antibody against a synthetic peptide in the C terminal domain of the protein. The 27-kDa protein could be immunoaffinity purified from hemocyte lysate supernatant and exhibited cell adhesion activity in vitro, indicating that the C-terminal domain of the crayfish masquerade-like protein mediates cell adhesion. PMID- 10744676 TI - Ischemia-induced STAT-1 expression and activation play a critical role in cardiomyocyte apoptosis. AB - We show here that exposure of cardiac cells to simulated ischemia results in apoptosis and is accompanied by phosphorylation and increased expression and transcriptional activity of STAT-1. Similarly, interferon-gamma, which is known to induce STAT-1 activation, also induced apoptosis in cardiac cells. STAT-1 transfected cells were more susceptible to ischemia-induced cell death than cells transfected with a control plasmid lacking the STAT-1 coding sequence. Furthermore, an antisense STAT-1 vector reduced both ischemia- and overexpressed STAT-1-induced cell death in cardiac cells. Both STAT-1 overexpression and interferon-gamma treatment or exposure to ischemia activated the promoter of the pro-apoptotic caspase-1 gene in cardiomyocytes. Finally, ischemia/reperfusion also induced STAT-1 activation and caspase-1 processing in ventricular myocytes in the intact heart ex vivo. Immunofluorescent staining demonstrated an increase in STAT-1-positive staining in cardiomyocytes in response to ischemia/reperfusion that co-localized with terminal deoxynucleotidyl transferase dVTP nick end labeling-positive apoptotic cells. These results suggest that STAT-1 plays a critical role in the regulation of ischemia/reperfusion-induced apoptosis in cardiac cells, acting at least in part via a caspase-1 activation-dependent pathway. PMID- 10744677 TI - Solution structure and backbone dynamics of long-[Arg(3)]insulin-like growth factor-I. AB - Long-[Arg(3)]insulin-like growth factor-I (IGF-I) is a potent analog of insulin like growth factor-I that has been modified by a Glu(3) --> Arg mutation and a 13 amino acid extension appended to the N terminus. We have determined the solution structure of (15)N-labeled Long-[Arg(3)]-IGF-I using high resolution NMR and restrained molecular dynamics techniques to a precision of 0.82 +/- 0.28 A root mean square deviation for the backbone heavy atoms in the three alpha-helices and 3.5 +/- 0.9 A root mean square deviation for all backbone heavy atoms excluding the 8 N-terminal residues and the 8 C-terminal eight residues. Overall, the structure of the IGF-I domain is consistent with earlier studies of IGF-I with some minor changes remote from the N terminus. The major variations in the structure, compared with IGF-I, occur at the N terminus with a substantial reorientation of the N-terminal three residues of the IGF-I domain. These results are interpreted in terms of the lower binding affinity for insulin-like growth factor-binding proteins. The backbone dynamics of Long-[Arg(3)]IGF-I were investigated using (15)N nuclear spin relaxation and the heteronuclear nuclear Overhauser enhancement (NOE). There is a considerable degree of flexibility in Long-[Arg(3)]IGF-I, even in the alpha-helices, as indicated by an average ((1)H)(15)N NOE of 0.55 for the regions. The largest heteronuclear NOEs are observed in the helical regions, lower heteronuclear NOEs are observed in the C domain loop separating helix 1 from helix 2, and negative heteronuclear NOEs are observed in the N-terminal extension and at the C terminus. Despite these data indicating conformational flexibility for the N-terminal extension, slow amide proton exchange was observed for some residues in this region, suggesting some transitory structure does exist, possibly a molten helix. A certain degree of flexibility may be necessary in all insulin-like growth factors to enable association with various receptors and binding proteins. PMID- 10744678 TI - Precursors bind to specific sites on thylakoid membranes prior to transport on the delta pH protein translocation system. AB - The Delta pH pathway is one of two systems for protein transport to the thylakoid lumen. It is a novel transport system that requires only the thylakoidal DeltapH to power translocation. Several substrates of the Delta pH pathway, including the intermediate precursor form of OE17 (iOE17) and the truncated precursor form of OE17 (tOE17), were shown to bind to the membrane in the absence of the DeltapH and be transported into the lumen when the DeltapH was restored. Binding occurred without energy or soluble factors, and efficient transport from the bound state ( approximately 80-90%) required only the DeltapH. Binding is due to protein protein interactions because protease pretreatment of thylakoids destroyed their binding capability. Precursors are bound to a specific site on the Delta pH pathway because binding was competed by saturating amounts of Delta pH pathway precursor proteins, but not by a Sec pathway precursor protein. These results suggested that precursor tOE17 binds to components of the Delta pathway translocation machinery. Hcf106 and Tha4 are two components of the Delta pH pathway machinery. Antibodies to Hcf106 or Tha4, when prebound to thylakoids, specifically inhibited precursor transport on the Delta pH pathway. However, only Hcf106 antibodies reduced the level of precursor binding. These results suggest that Hcf106 functions in early steps of the transport process. PMID- 10744679 TI - Regulation of FasL by NF-kappaB and AP-1 in Fas-dependent thymineless death of human colon carcinoma cells. AB - Cell death due to thymine (dThd) deficiency, associated with the cytotoxic action of 5-fluorouracil in colon cancer, is regulated in thymidylate synthase-deficient (TS(-)) human colon carcinoma cells via the Fas (CD95, APO-1) death receptor. This was demonstrated by inhibiting the loss in clonogenicity of TS(-) cells by anti-FasL and in enhanced survival of TS(-) clones selected for resistance to Fas mediated apoptosis, following dThd deprivation. During thymineless stress in TS( ) cells, Fas ligand (FasL) is expressed, and its promoter (hFasLPr) is activated. Transactivation of hFasLPr, dependent upon dThd deficiency, was inhibited following mutation of the binding sites for NF-kappaB or AP-1 and by preventing NF-kappaB or AP-1 activation, which inhibited expression of FasL and enhanced clonogenic survival in stable transformants expressing IkappaBalphaM or DN-MEKK, respectively. These results demonstrate the crucial roles for NF-kappaB and AP-1 in the regulation of FasL in Fas-mediated thymineless death of colon carcinoma cells. PMID- 10744680 TI - The two halves of CFTR form a dual-pore ion channel. AB - The cystic fibrosis transmembrane conductance regulator (CFTR) exhibits two conductance states, 9 picosiemens (pS) and 3 pS. To investigate the origin of these two distinct conductance states, we measured the single-channel activity of three truncated forms of CFTR. These include: TNR, which contains the first transmembrane domain, the first nucleotide binding domain, and the R domain; RT2N2, which contains the R domain, the second transmembrane domain, and the second nucleotide-binding domain; and T2N2, which contains only the second transmembrane domain and the second nucleotide-binding domain. The results show that TNR exhibits only the large conductance of 9.2 pS, whereas RT2N2 and T2N2 exhibit only the small conductance (3.8-4.0 pS). Co-expression of TNR with T2N2 resulted in a mixed pattern of two conductance states, which is similar to that observed in wild-type CFTR. In further studies, a "dual-R mutant," R334W and R347P in the transmembrane segment 6 of the first half of CFTR, severely impaired the large conductance channel without affecting the small conductance channel. The ion selectivity and gating behavior of the two conductance channels are different regardless of whether they are measured in wild-type CFTR or in truncated CFTRs. The ion selectivity of the large conductance channel is Br(-) > Cl(-) > I(-), whereas the ion selectivity of the small conductance channel is Br( ) = Cl(-) = I(-). The open probability (P(o)) of the large conductance is about 4 fold higher than that of the small conductance. Transition from closed to open states of the small conductance is not dependent upon the open or closed states of the large conductance. The independent behaviors of the two conductances in CFTR strongly suggest that CFTR may have two distinct pores. Thus, like ClC0, CFTR is likely to be a double-barreled ion channel, with the first half of CFTR forming the large conductance and the second half forming the small conductance. PMID- 10744681 TI - Processing of lysosomal beta-galactosidase. The C-terminal precursor fragment is an essential domain of the mature enzyme. AB - Lysosomal beta-D-galactosidase (beta-gal), the enzyme deficient in the autosomal recessive disorders G(M1) gangliosidosis and Morquio B, is synthesized as an 85 kDa precursor that is C-terminally processed into a 64-66-kDa mature form. The released approximately 20-kDa proteolytic fragment was thought to be degraded. We now present evidence that it remains associated to the 64-kDa chain after partial proteolysis of the precursor. This polypeptide was found to copurify with beta gal and protective protein/cathepsin A from mouse liver and Madin-Darby bovine kidney cells and was immunoprecipitated from human fibroblasts but not from fibroblasts of a G(M1) gangliosidosis and a galactosialidosis patient. Uptake of wild-type protective protein/cathepsin A by galactosialidosis fibroblasts resulted in a significant increase of mature and active beta-gal and its C terminal fragment. Expression in COS-1 cells of mutant cDNAs encoding either the N-terminal or the C-terminal domain of beta-gal resulted in the synthesis of correctly sized polypeptides without catalytic activity. Only when co-expressed, the two subunits associate and become catalytically active. Our results suggest that the C terminus of beta-gal is an essential domain of the catalytically active enzyme and provide evidence that lysosomal beta-galactosidase is a two subunit molecule. These data may give new significance to mutations in G(M1) gangliosidosis patients found in the C-terminal part of the molecule. PMID- 10744682 TI - Association of carboxyl esterase with facilitative glucose transporter isoform 4 (GLUT4) intracellular compartments in rat adipocytes and its possible role in insulin-induced GLUT4 recruitment. AB - Facilitative glucose transporter isoform 4 (GLUT4) in rat adipocytes is largely sequestered in intracellular sites, and insulin recruits GLUT4 from these sites to the cell surface. The process is known to involve multiple intracellular compartments and associated proteins, many of which are yet to be identified. Recently, we purified three distinct insulin-sensitive intracellular GLUT4 compartments (G4T(L), G4H, and G4L) in rat adipocytes and unraveled several new resident proteins in these compartments. Here, we describe one of them, a 62-kDa protein, purified and identified as rat adipose tissue carboxyl esterase (p62/CE) by matrix-assisted laser desorption/ionization time of flight mass spectroscopy, reverse transcription-polymerase chain reaction, gene cloning, and immunological and enzymatic activity measurements. p62/CE in rat adipocytes was 80% cytosolic and 20% microsome-associated. It was found in all of the three insulin-sensitive intracellular GLUT4 compartments, and particularly enriched in G4T(L,) a compartment thought to represent GLUT4 endocytic vesicles. Significantly, an antibody against p62/CE introduced into rat adipocytes completely abolished the insulin-induced GLUT4 recruitment to the plasma membrane in host cells without affecting the basal GLUT4 distribution. Together, these findings suggest that p62/CE plays a key role in insulin-induced GLUT4 recruitment in rat adipocytes, probably by hydrolyzing acylglycerols or acyl-CoA esters to the respective free acids that are required for GLUT4 transport vesicle budding and/or fusion. PMID- 10744683 TI - Phosphorylation of a new brain-specific septin, G-septin, by cGMP-dependent protein kinase. AB - The septins are a family of GTPase enzymes, some of which are required for the cytokinesis stage of cell division and others of which are associated with exocytosis. We purified and cloned the cDNA for a 40-kDa protein from rat brain that is a substrate for type I cGMP-dependent protein kinase (PKG). The amino acid sequences of two tryptic peptides of P40 showed high homology to the septins. Molecular cloning revealed the 358-amino acid P40 to be a new member of the septin family. P40 was named G-septin, as it is phosphorylated in vitro by PKG, but relatively poorly by the related cAMP-dependent protein kinase and not by protein kinase C. Two splice variants of G-septin (alpha and beta) were found with distinct N and C termini, but a common GTPase domain. G-septin lacks the C terminal coiled-coil domain characteristic of all other mammalian septins and uniquely has two predicted phosphorylation site motifs for type I PKG. Photoaffinity labeling with [alpha-(32)P]GTP confirmed that G-septin is a GTP binding protein. Northern blotting showed that G-septin mRNA (5.0 kilobases) is highly expressed in brain and undetectable in 12 other tissues, indicating that the G-septins are primarily neuronal proteins. Very low levels of 6.0-, 3.4-, and 2.6-kilobase transcripts were found in testis. Our results reveal a new class of brain-specific septins that may be regulated by PKG in neurons. PMID- 10744684 TI - Substitution of betaGlu(201) in the alpha(3)beta(3)gamma subcomplex of the F(1) ATPase from the thermophilic Bacillus PS3 increases the affinity of catalytic sites for nucleotides. AB - In the crystal structure of bovine mitochondrial F(1)-ATPase (MF(1)) (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621 628), the side chain oxygen of betaThr(163) interacts directly with Mg(2+) coordinated to 5'-adenylyl beta, gamma-imidodiphosphate or ADP bound to catalytic sites of beta subunits present in closed conformations. In the unliganded beta subunit present in an open conformation, the hydroxyl of betaThr(163) is hydrogen bonded to the carboxylate of betaGlu(199). Substitution of betaGlu(201) (equivalent to betaGlu(199) in MF(1)) in the alpha(3)beta(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 with cysteine or valine increases the propensity to entrap inhibitory MgADP in a catalytic site during hydrolysis of 50 microM ATP. These substitutions lower K(m3) (the Michaelis constant for trisite ATP hydrolysis) relative to that of the wild type by 25- and 10-fold, respectively. Fluorescence quenching of alpha(3)(betaE201C/Y341W)(3)gamma and alpha(3)(betaY341W)(3)gamma mutant subcomplexes showed that MgATP and MgADP bind to the third catalytic site of the double mutant with 8.4- and 4.4-fold higher affinity, respectively, than to the single mutant. These comparisons support the hypothesis that the hydrogen bond observed between the side chains of betaThr(163) and betaGlu(199) in the unliganded catalytic site in the crystal structure of MF(1) stabilizes the open conformation of the catalytic site during ATP hydrolysis. PMID- 10744685 TI - The human transcription factor IID subunit human TATA-binding protein-associated factor 28 interacts in a ligand-reversible manner with the vitamin D(3) and thyroid hormone receptors. AB - Using coexpression in COS cells, we have identified novel interactions between the human TATA-binding protein-associated factor 28 (hTAF(II)28) component of transcription factor IID and the ligand binding domains (LBDs) of the nuclear receptors for vitamin D3 (VDR) and thyroid hormone (TRalpha). Interaction between hTAF(II)28 and the VDR and TR LBDs was ligand-reversible, whereas no interactions between hTAF(II)28 and the retinoid X receptors (RXRs) or other receptors were observed. TAF(II)28 interacted with two regions of the VDR, a 40-amino acid region spanning alpha-helices H3-H5 and alpha-helix H8. Interactions were also observed with the H3-H5 region of the TRalpha but not with the equivalent highly related region of the RXRgamma. Fine mapping using RXR derivatives in which single amino acids of the RXRgamma LBD have been replaced with their VDR counterparts shows that the determinants for interaction with hTAF(II)28 are located in alpha-helix H3 and are not identical to those previously identified for interactions with hTAF(II)55. We also describe a mutation in the H3-H5 region of the VDR LBD, which abolishes transactivation, and we show that interaction of hTAF(II)28 with this mutant is no longer ligand-reversible. PMID- 10744686 TI - Subsenescent telomere lengths in fibroblasts immortalized by limiting amounts of telomerase. AB - Human fibroblasts expressing the catalytic component of human telomerase (hTERT) have been followed for 250-400 population doublings. As expected, telomerase activity declined in long term culture of stable transfectants. Surprisingly, however, clones with average telomere lengths several kilobases shorter than those of senescent parental cells continued to proliferate. Although the longest telomeres shortened, the size of the shortest telomeres was maintained. Cells with subsenescent telomere lengths proliferated for an additional 20 doublings after inhibiting telomerase activity with a dominant-negative hTERT mutant. These results indicate that, under conditions of limiting telomerase activity, cis acting signals may recruit telomerase to act on the shortest telomeres, argue against the hypothesis that the mortality stage 1 mechanism of cellular senescence is regulated by telomere positional effects (in which subtelomeric loci silenced by long telomeres are expressed when telomeres become short), and suggest that catalytically active telomerase is not required to provide a protein capping role at the end of very short telomeres. PMID- 10744687 TI - Overexpression of secretory phospholipase A(2) causes rapid catabolism and altered tissue uptake of high density lipoprotein cholesteryl ester and apolipoprotein A-I. AB - Plasma levels of high density lipoprotein (HDL) cholesterol and its major protein component apolipoprotein (apo) A-I are significantly reduced in both acute and chronic inflammatory conditions, but the basis for this phenomenon is not well understood. We hypothesized that secretory phospholipase A(2) (sPLA(2)), an acute phase protein that has been found in association with HDL, promotes HDL catabolism. A series of HDL metabolic studies were performed in transgenic mice that specifically overexpress human sPLA(2) but have no evidence of local or systemic inflammation. We found that HDL isolated from these mice have a significantly lower phospholipid and cholesteryl ester and significantly greater triglyceride content. The fractional catabolic rate (FCR) of (125)I-HDL was significantly faster in sPLA(2) transgenic mice (4.08 +/- 0.01 pools/day) compared with control wild-type littermates (2.16 +/- 0.48 pools/day). (125)I-HDL isolated from sPLA(2) transgenic mice was catabolized significantly faster than (131)I-HDL isolated from wild-type mice after injection in wild-type mice (p < 0.001). Injection of (125)I-tyramine-cellobiose-HDL demonstrated significantly greater degradation of HDL apolipoproteins in the kidneys of sPLA(2) transgenic mice compared with control mice (p < 0.05). The fractional catabolic rate of [(3)H]cholesteryl ether HDL was significantly faster in sPLA(2)-overexpressing mice (6.48 +/- 0.24 pools/day) compared with controls (4.80 +/- 0.72 pools/day). Uptake of [(3)H] cholesteryl ether into the livers and adrenals of sPLA(2) transgenic mice was significantly enhanced compared with control mice. In summary, these data demonstrate that overexpression of sPLA(2) alone in the absence of inflammation causes profound alterations of HDL metabolism in vivo and are consistent with the hypothesis that sPLA(2) may promote HDL catabolism in acute and chronic inflammatory conditions. PMID- 10744688 TI - Xylene monooxygenase catalyzes the multistep oxygenation of toluene and pseudocumene to corresponding alcohols, aldehydes, and acids in Escherichia coli JM101. AB - Xylene monooxygenase of Pseudomonas putida mt-2 catalyzes the methylgroup hydroxylation of toluene and xylenes. To investigate the potential of xylene monooxygenase to catalyze multistep oxidations of one methyl group, we tested recombinant Escherichia coli expressing the monooxygenase genes xylM and xylA under the control of the alk regulatory system of Pseudomonas oleovorans Gpo1. Expression of xylene monooxygenase genes could efficiently be controlled by n octane and dicyclopropylketone. Xylene monooxygenase was found to catalyze the oxygenation of toluene, pseudocumene, the corresponding alcohols, and the corresponding aldehydes. For all three transformations (18)O incorporation provided stong evidence for a monooxygenation type of reaction, with gem-diols as the most likely reaction intermediates during the oxygenation of benzyl alcohols to benzaldehydes. To investigate the role of benzyl alcohol dehydrogenase (XylB) in the formation of benzaldehydes, xylB was cloned behind and expressed in concert with xylMA. In comparison to E. coli expressing only xylMA, the presence of xylB lowered product formation rates and resulted in back formation of benzyl alcohol from benzaldehyde. In P. putida mt-2 XylB may prevent the formation of high concentrations of the particularly reactive benzaldehydes. In the case of high fluxes through the degradation pathways and low aldehyde concentrations, XylB may contribute to benzaldehyde formation via the energetically favorable dehydrogenation of benzyl alcohols. The results presented here characterize XylMA as an enzyme able to catalyze the multistep oxygenation of toluenes. PMID- 10744689 TI - Phosphorylation of PDE3B by phosphatidylinositol 3-kinase associated with the insulin receptor. AB - Phosphatidylinositol 3-kinase mediates several actions of insulin including its antilipolytic effect. This effect is elicited by the insulin-stimulated serine phosphorylation and activation of cGMP-inhibited phosphodiesterase (PDE3B). In human adipocytes, we found that insulin differentially stimulated phosphatidylinositol 3-kinase activity; the lipid kinase activity was associated with IRS-1, whereas the serine kinase activity was associated with the insulin receptor and phosphorylated a number of proteins including p85, p110, and a 135 kDa protein identified as PDE3B. PDE3B phosphorylation was associated with enzyme activation, thus initiating the antilipolytic effect of insulin. These results show a novel pathway for intracellular signaling through the insulin receptor leading to the serine phosphorylation of key proteins involved in insulin action. PMID- 10744690 TI - The nuclear import of RCC1 requires a specific nuclear localization sequence receptor, karyopherin alpha3/Qip. AB - RCC1 is the only known guanine nucleotide exchange factor for the small GTPase Ran and is normally found inside the nucleus bound to chromatin. In order to analyze in more detail the nuclear import of RCC1, we created a fusion construct in which four IgG binding domains of protein A were fused to the amino terminus of human RCC1 (pA-RCC1). Surprisingly, we found that neither Xenopus ovarian cytosol nor a mixture of recombinant import factors (karyopherin alpha2, karyopherin beta1, Ran, and p10/NTF2) were able to support the import of pA-RCC1 into the nuclei of digitonin-permeabilized cells. Both, in contrast, were capable of supporting the import of a construct containing another classical nuclear localization sequence (NLS), glutathione S-transferase-green fluorescent protein NLS. Subsequently, we found that only one of the NLS receptors, karyopherin alpha3 (Kapalpha3/Qip), would support significant nuclear import of pA-RCC1 in permeabilized cells, while members of the other two main classes, Kapalpha1 and Kapalpha2, would not. Accordingly, in vitro binding studies revealed that only Kapalpha3 showed significant binding to RCC1 (unlike Kapalpha1 and Kapalpha2) and that this binding was dependent on the basic amino acids present in the RCC1 NLS. In addition to Kapalpha3, we found that the nuclear import of pA-RCC1 also required both karyopherin beta1 and Ran. PMID- 10744692 TI - Identification of the mechanism responsible for the increased fibrin specificity of TNK-tissue plasminogen activator relative to tissue plasminogen activator. AB - TNK-tissue plasminogen activator (TNK-t-PA), a bioengineered variant of tissue type plasminogen activator (t-PA), has a longer half-life than t-PA because the glycosylation site at amino acid 117 (N117Q, abbreviated N) has been shifted to amino acid 103 (T103N, abbreviated T) and is resistant to inactivation by plasminogen activator inhibitor 1 because of a tetra-alanine substitution in the protease domain (K296A/H297A/R298A/R299A, abbreviated K). TNK-t-PA is more fibrin specific than t-PA for reasons that are poorly understood. Previously, we demonstrated that the fibrin specificity of t-PA is compromised because t-PA binds to (DD)E, the major degradation product of cross-linked fibrin, with an affinity similar to that for fibrin. To investigate the enhanced fibrin specificity of TNK-t-PA, we compared the kinetics of plasminogen activation for t PA, TNK-, T-, K-, TK-, and NK-t-PA in the presence of fibrin, (DD)E or fibrinogen. Although the activators have similar catalytic efficiencies in the presence of fibrin, the catalytic efficiency of TNK-t-PA is 15-fold lower than that for t-PA in the presence of (DD)E or fibrinogen. The T and K mutations combine to produce this reduction via distinct mechanisms because T-containing variants have a higher K(M), whereas K-containing variants have a lower k(cat) than t-PA. These results are supported by data indicating that T-containing variants bind (DD)E and fibrinogen with lower affinities than t-PA, whereas the K and N mutations have no effect on binding. Reduced efficiency of plasminogen activation in the presence of (DD)E and fibrinogen but equivalent efficiency in the presence of fibrin explain why TNK-t-PA is more fibrin-specific than t-PA. PMID- 10744691 TI - The nonreceptor protein-tyrosine kinase c-Fes is involved in fibroblast growth factor-2-induced chemotaxis of murine brain capillary endothelial cells. AB - Fibroblast growth factor-2 (FGF-2)-induced migration of endothelial cells is involved in angiogenesis in vivo. However, signal transduction pathways leading to FGF-2-induced chemotaxis of endothelial cells are largely unknown. Previous studies have shown that the cytoplasmic protein-tyrosine kinase c-Fes is expressed in vascular endothelial cells and may influence angiogenesis in vivo. To investigate the contribution of c-Fes to FGF-2 signaling, we expressed wild type or kinase-inactive human c-Fes in the murine brain capillary endothelial cell line, IBE (Immortomouse brain endothelial cells). Wild-type c-Fes was tyrosine-phosphorylated upon FGF-2-stimulation in transfected cells, whereas kinase-inactive c-Fes was not. Overexpression of wild-type c-Fes promoted FGF-2 independent tube formation of IBE cells. Tube formation was not observed with endothelial cells expressing kinase-inactive c-Fes, indicating a requirement for c-Fes kinase activity in this biological response. Expression of kinase-defective c-Fes suppressed endothelial cell migration following FGF-2 treatment, suggesting that activation of endogenous c-Fes may be required for the chemotactic response. Expression of either wild-type c-Fes or the kinase-inactive mutant did not affect the tyrosine phosphorylation FRS2, Shc, or phospholipase C-gamma, nor did it influence the kinetics of mitogen-activated protein kinase activation. These results implicate c-Fes in FGF-2-induced chemotaxis of endothelial cells through signaling pathways not linked to mitogenesis. PMID- 10744693 TI - Two 5'-regions are required for nutritional and insulin regulation of the fatty acid synthase promoter in transgenic mice. AB - We previously reported that 2.1 kilobase pairs of the 5'-flanking sequence are sufficient for tissue-specific and hormonal/metabolic regulation of the fatty acid synthase (FAS) gene in transgenic mice. We also demonstrated that the -65 E box is required for insulin regulation of the FAS promoter using 3T3-L1 adipocytes in culture. To further define sequences required for FAS gene expression, we generated transgenic mice carrying from -644, -444, -278, and -131 to +67 base pairs of the rat FAS 5'-flanking sequence fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Similar to the expression observed with -2100-FAS-CAT transgenic mice, transgenic mice harboring -644-FAS CAT and -444-FAS-CAT expressed high levels of CAT mRNA only in lipogenic tissues (liver and adipose tissue) in a manner identical to the endogenous FAS mRNA. In contrast, -278-FAS-CAT and -131-FAS-CAT transgenic mice did not show appreciable CAT expression in any of the tissues examined. When previously fasted mice were refed a high carbohydrate, fat-free diet, CAT mRNA expression in transgenic mice harboring -644-FAS-CAT and -444-FAS-CAT was induced dramatically in liver and adipose tissue. The induction was virtually identical to that observed in -2100 FAS-CAT transgenic mice and to the endogenous FAS mRNA. In contrast, -278-FAS-CAT transgenic mice showed induction by feeding, but at a much lower magnitude in both liver and adipose tissue. The -131-FAS-CAT transgenic mice did not show any CAT expression either when fasted or refed a high carbohydrate diet. To study further the effect of insulin, we made these transgenic mice insulin-deficient by streptozotocin treatment. Insulin administration to the streptozotocin-diabetic mice increased CAT mRNA levels driven by the -644 FAS and -444 FAS promoters in liver and adipose tissue, paralleling the endogenous FAS mRNA levels. In the case of -278-FAS-CAT, the induction observed was at a much lower magnitude, and deletion to -131 base pairs did not show any increase in CAT expression by insulin. This study demonstrates that the sequence requirement for FAS gene regulation employing an in vitro culture system does not reflect the in vivo situation and that two 5'-flanking regions are required for proper nutritional and insulin regulation of the FAS gene. Cotransfection of the upstream stimulatory factor and various FAS promoter-luciferase constructs as well as in vitro binding studies suggest a function for the upstream stimulatory factor at both the -65 and -332 E-box sequences. PMID- 10744694 TI - Lysophospholipids open the two-pore domain mechano-gated K(+) channels TREK-1 and TRAAK. AB - The two-pore (2P) domain K(+) channels TREK-1 and TRAAK are opened by membrane stretch as well as arachidonic acid (AA) (Patel, A. J., Honore, E., Maingret, F., Lesage, F., Fink, M., Duprat, F., and Lazdunski, M. (1998) EMBO J. 17, 4283-4290; Maingret, F., Patel, A. J., Lesage, F., Lazdunski, M., and Honore, E. (1999) J. Biol. Chem. 274, 26691-26696; Maingret, F., Fosset, M., Lesage, F., Lazdunski, M. , and Honore, E. (1999) J. Biol. Chem. 274, 1381-1387. We demonstrate that lysophospholipids (LPs) and platelet-activating factor also produce large specific and reversible activations of TREK-1 and TRAAK. LPs activation is a function of the size of the polar head and length of the acyl chain but is independent of the charge of the molecule. Bath application of lysophosphatidylcholine (LPC) immediately opens TREK-1 and TRAAK in the cell attached patch configuration. In excised patches, LPC activation is lost, whereas AA still produces maximal opening. The carboxyl-terminal region of TREK-1, but not the amino terminus and the extracellular loop M1P1, is critically required for LPC activation. LPC activation is indirect and may possibly involve a cytosolic factor, whereas AA directly interacts with either the channel proteins or the bilayer and mimics stretch. Opening of TREK-1 and TRAAK by fatty acids and LPs may be an important switch in the regulation of synaptic function and may also play a protective role during ischemia and inflammation. PMID- 10744695 TI - neo-inositol polyphosphates in the amoeba Entamoeba histolytica. AB - We have reexamined the structure of inositol phosphates present in trophozoites of the parasitic amoeba Entamoeba histolytica and show here that, rather than being myo-inositol derivatives (Martin, J.-B., Bakker-Grunwald, T., and Klein, G. (1993) Eur. J. Biochem. 214, 711-718), these compounds belong to a new class of inositol phosphates in which the cyclitol isomer is neo-inositol. The structures of neo-inositol hexakisphosphate, 2-diphospho-neo-inositol pentakisphosphate, and 2, 5-bisdiphospho-neo-inositol tetrakisphosphate, which are present in E. histolytica at concentrations of 0.08-0.36 mM, were solved by two-dimensional (31)P-(1)H NMR spectroscopy. No evidence for the co-existence of their myo inositol counterparts has been found. These neo-inositol compounds were not substrates of 6-diphospho-inositol pentakisphosphate 5-kinase, an enzyme purified from Dictyostelium discoideum that phosphorylates 6-diphospho-myo-inositol pentakisphosphate and more slowly also myo-inositol hexakisphosphate, specifically on position 5. Because preliminary data indicate that large amounts of the same neo-inositol phosphate and diphosphate esters are also present in another primitive amoeba, Phreatamoeba balamuthi, the occurrence of high concentrations of neo-inositol polyphosphates may be much more general than previously thought. PMID- 10744696 TI - Vav2 is an activator of Cdc42, Rac1, and RhoA. AB - Vav and Vav2 are members of the Dbl family of proteins that act as guanine nucleotide exchange factors (GEFs) for Rho family proteins. Whereas Vav expression is restricted to cells of hematopoietic origin, Vav2 is widely expressed. Although Vav and Vav2 share highly related structural similarities and high sequence identity in their Dbl homology domains, it has been reported that they are active GEFs with distinct substrate specificities toward Rho family members. Whereas Vav displayed GEF activity for Rac1, Cdc42, RhoA, and RhoG, Vav2 was reported to exhibit GEF activity for RhoA, RhoB, and RhoG but not for Rac1 or Cdc42. Consistent with their distinct substrate targets, it was found that constitutively activated versions of Vav and Vav2 caused distinct transformed phenotypes when expressed in NIH 3T3 cells. In contrast to the previous findings, we found that Vav2 can act as a potent GEF for Cdc42, Rac1, and RhoA in vitro. Furthermore, we found that NH(2)-terminally truncated and activated Vav and Vav2 caused indistinguishable transforming actions in NIH 3T3 cells that required Cdc42, Rac1, and RhoA function. In addition, like Vav and Rac1, we found that Vav2 activated the Jun NH(2)-terminal kinase cascade and also caused the formation of lamellipodia and membrane ruffles in NIH 3T3 cells. Finally, Vav2 transformed NIH 3T3 cells showed up-regulated levels of Rac-GTP. We conclude that Vav2 and Vav share overlapping downstream targets and are activators of multiple Rho family proteins. Therefore, Vav2 may mediate the same cellular consequences in nonhematopoietic cells as Vav does in hematopoietic cells. PMID- 10744697 TI - A comparative study of calf thymus DNA binding to Cr(III) and Cr(VI) ions. Evidence for the guanine N-7-chromium-phosphate chelate formation. AB - Chromium(VI) salts are well known to be mutagens and carcinogens and to easily cross the cell membranes. Because they are powerful oxidizing agents, Cr(VI) reacts with intracellular materials to reduce to trivalent form, which binds DNA. This study was designed to investigate the interaction of calf thymus DNA with Cr(VI) and Cr(III) in aqueous solution at pH 6.5-7.5, using Cr(VI)/DNA(P) molar ratios (r) of 1:20 to 2:1 and Cr(III)/DNA(P) molar ratios (r) of 1:80 to 1:2. UV visible and Fourier transform infrared (FTIR) difference spectroscopic methods were used to determine the metal ion-binding sites, binding constants, and the effect of cation complexation on DNA secondary structure. Spectroscopic results showed no interaction of Cr(VI) with DNA at low anion concentrations (r = 1:20 to 1:1), whereas some perturbations of DNA bases and backbone phosphate were observed at very high Cr(VI) contents (r > 1) with overall binding constant of K = 508 M(-1). Cr(III) chelates DNA via guanine N-7 and the nearest PO(2) group with overall binding constant of K = 3.15 x 10(3) M(-1). Evidence for cation chelate formation comes from major shiftings and intensity variations of the guanine band at 1717 and the phosphate asymmetric stretching vibration at 1222 cm(-1). At low Cr(III) concentration (r = 1:40), the number of Cr(III) ions bound to DNA were 6-7 cations/500 base pairs, and this increased to 30-35 cations/500 base pairs at high metal ion content (r = 1:4). DNA condensation occurred at high cation concentration (r = 1:10). No major alteration of DNA conformation was observed, and the biopolymer remained in the B family structure upon chromium complexation. PMID- 10744698 TI - Signal peptide determinants of SecA binding and stimulation of ATPase activity. AB - A signal peptide is required for entry of a preprotein into the secretory pathway, but how it functions in concert with the other transport components is unknown. In Escherichia coli, SecA is a key component of the translocation machinery found in the cytoplasm and at membrane translocation sites. Synthetic signal peptides corresponding to the wild type alkaline phosphatase signal sequence and three sets of model signal sequences varying in hydrophobicity and amino-terminal charge were generated. These were used to establish the requirements for interaction with SecA. Binding to SecA, modulation of SecA conformations sensitive to protease, and stimulation of SecA-lipid ATPase activity occur with functional signal sequences but not with transport incompetent ones. The extent of SecA interaction is directly related to the hydrophobicity of the signal peptide core region. For signal peptides of moderate hydrophobicity, stimulation of the SecA-lipid ATPase activity is also dependent on amino-terminal charge. The results demonstrate unequivocally that the signal peptide, in the absence of the mature protein, interacts with SecA in aqueous solution and in a lipid bilayer. We show a clear parallel between the hierarchy of signal peptide characteristics that promote interaction with SecA in vitro and the hierarchy of those observed for function in vivo. PMID- 10744699 TI - TAF(II)250-independent transcription can be conferred on a TAF(II)250-dependent basal promoter by upstream activators. AB - TAF(II)250, a component of the general transcription factor, TFIID, is required for the transcription of a subset of genes, including those involved in regulating cell cycle progression. The tsBN462 cell line, with a temperature sensitive mutation of TAF(II)250, grows normally at 32 degrees C, but when grown at 39.5 degrees C, it differentially arrests transcription of many, but not all, genes. The present studies examine the basis for the requirement for TAF(II)250. We show that the basal promoter of a major histocompatibility complex class I gene requires TAF(II)250. This dependence can be overcome by select upstream regulatory elements but not by basal promoter elements. Thus, the coactivator CIITA rescues the basal promoter from the requirement for TAF(II)250, whereas introduction of a canonical TATAA box does not. Similarly, the SV40 basal promoter is shown to require TAF(II)250, and the presence of the 72-base pair enhancer overcomes this requirement. Furthermore, the SV40 72-base pair enhancer when placed upstream of the basal class I promoter renders it independent of TAF(II)250. These data suggest that the assembly of transcription initiation complexes is dynamic and can be modulated by specific transcription factors. PMID- 10744700 TI - Stimulation of phosphatidylinositol-4-phosphate 5-kinase by Rho-kinase. AB - The serine/threonine kinase Rho-kinase was recently identified as a downstream effector of the small GTPase Rho, mediating effects of Rho on the actin cytoskeleton. Also phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) has been implicated in the regulation of actin polymerization. As the synthesis of PI(4,5)P(2) has been suggested to be affected by Rho proteins, we investigated whether Rho-kinase is involved in the control of PI(4,5)P(2) levels. Overexpression of RhoA in HEK-293 cells increased phosphatidylinositol 4 phosphate (PI4P) 5-kinase activity and concomitantly enhanced cellular PI(4,5)P(2) levels, whereas overexpression of the Rho-inactivating C3 transferase decreased both PI4P 5-kinase activity and PI(4,5)P(2) levels. These effects of RhoA could be mimicked by overexpression of wild-type Rho-kinase and of the constitutively active catalytic domain of Rho-kinase, Rho-kinase-CAT. In contrast, a kinase-deficient mutant of Rho-kinase had no effect on PI4P 5-kinase activity. Importantly, the increase in PI4P 5-kinase activity and PI(4,5)P(2) levels by wild-type Rho-kinase, but not by Rho-kinase-CAT, was completely prevented by coexpression of C3 transferase, indicating that the effect of Rho kinase was under the control of endogenous Rho. In cell lysates, addition of recombinant RhoA and Rho-kinase-CAT stimulated PI4P 5-kinase activity. Finally, the increase in PI(4,5)P(2) levels induced by both Rho-kinase-CAT and RhoA was reversed by the Rho-kinase inhibitor HA-1077. Our data suggest that Rho-kinase is involved in the Rho-controlled synthesis of PI(4,5)P(2) by PI4P 5-kinase. PMID- 10744702 TI - Phosphatidylinositol 4,5-bisphosphate and intracellular pH regulate the ROMK1 potassium channel via separate but interrelated mechanisms. AB - ROMK channels are responsible for K(+) secretion in kidney. The activity of ROMK is regulated by intracellular pH (pH(i)) with acidification causing channel closure (effective pK(a) approximately 6.9). Recently, we and others reported that a direct interaction of the channels with phosphatidyl-4,5-bisphosphate (PIP(2)) is critical for opening of the inwardly rectifying K(+) channels. Here, we investigate the relationship between the mechanisms for regulation of ROMK by PIP(2) and by pH(i). We find that disruption of PIP(2)-ROMK1 interaction not only decreases single-channel open probability (P(o)) but gives rise to a ROMK1 subconductance state. This state has an increased sensitivity to intracellular protons (effective pK(a) shifted to pH approximately 7.8), such that the subconductance channels are relatively quiescent at physiological pH(i). Open probability for the subconductance channels can then be increased by intracellular alkalinization to supra-physiological pH. This increase in P(o) for the subconductance channels by alkalinization is not associated with an increase in PIP(2)-channel interaction. Thus, direct interaction with PIP(2) is critical for ROMK1 to open at full conductance. Disruption of this interaction increases pH(i) sensitivity for the channels via emergence of the subconductance state. The control of open probability of ROMK1 by pH(i) occurs via a mechanism distinct from the regulation by PIP(2). PMID- 10744701 TI - Tyrosine kinase activation in response to fungal spores is primarily dependent on endogenous reactive oxygen production in macrophages. AB - Studies from our laboratory (Shahan, T. A., Sorenson, W. G., and Lewis, D. M. (1994) Environ. Res. 67, 98-104) demonstrated that spores from different fungal species differentially activate rat alveolar macrophages as detected by the measurement of superoxide anion and cytokine production (Shahan, T. A., Siegel, P. D., Sorenson, W. G., Kuschner, W. G., and Lewis, D. M. (1998) Am. J. Respir. Cell Mol. Biol. 18, 435-441). Spores from Aspergillus candidus stimulated production of the highest levels of superoxide anion (5.2 nmol/1.0 x 10(6) alveolar macrophages (AMs)/30 min), followed by those from Aspergillus niger (2.4 nmol/1.0 x 10(6) AMs/30 min) and Eurotium amstelodami (0.4 nmol/1.0 x 10(6) AMs/30 min). The mechanism of this differential activation was studied. Our data demonstrate that the tyrosine kinases p56(Hck), p72(Syk), p77(Btk), p62(Yes), p56(Lck), and p59(Fyn) were specifically activated in response to spores from A. candidus, whereas spores from either A. niger or E. amstelodami activated p56(Hck), p72(Syk), and p77(Btk). Kinetic analysis of specific tyrosine kinases demonstrated that p56(Hck), p72(Syk), and p77(Btk) were activated faster and to a greater extent by spores from A. candidus as compared with spores from E. amstelodami. These data suggest a relationship between reactive oxygen species and tyrosine kinase activation. Treatment of AMs with H(2)O(2) (1 mM) caused the activation of p72(Syk) only, whereas treatment with superoxide dismutase and catalase before treatment with the spores had no effect on tyrosine kinase activation. Incubation with NADPH oxidase inhibitors inhibited both superoxide anion production and the activation of p56(Hck), p72(Syk), and p77(Btk) in response to fungal spores. These data indicate that endogenous reactive oxygen species are necessary for the activation of p56(Hck), p72(Syk), and p77(Btk) by spores; they also indicate that some species of spores are capable of activating tyrosine kinases independent of superoxide anion. PMID- 10744703 TI - A protein kinase C site highly conserved in P2X subunits controls the desensitization kinetics of P2X(2) ATP-gated channels. AB - P2X receptors are nonselective cation channels gated by extracellular ATP. Recombinant mammalian P2X subunits assemble in homomeric ionotropic ATP receptors that differ by their agonist sensitivity and desensitization rate in heterologous expression systems. Using site-directed mutagenesis and voltage clamp recording in Xenopus oocytes, we identified the highly conserved protein kinase C site TX(K/R) located in the intracellular N terminus of P2X subunits as a critical determinant of kinetics in slowly desensitizing (time constant, >1 min) rat P2X(2) receptors. Mutant receptors P2X(2)T18A, T18N, and K20T devoid of this consensus site exhibited quickly desensitizing properties (time constant, <1 s). In contrast with wild-type receptors, mutant P2X(2) receptors with truncated C terminus exhibited variable cell-specific kinetics with quickly desensitizing currents converted to slowly desensitizing currents by phorbol ester-mediated stimulation of protein kinase C. Phosphorylation of Thr(18) was demonstrated directly by immunodetection using specific monoclonal antibodies directed against the phosphothreonine-proline motif. Our data indicate that both phosphorylation of the conserved threonine residue in the N-terminal domain by protein kinase C and interaction between the two cytoplasmic domains of P2X(2) subunits are necessary for the full expression of slowly desensitizing ATP-gated channels. PMID- 10744704 TI - Binding relationships of membrane tethering components. The giantin N terminus and the GM130 N terminus compete for binding to the p115 C terminus. AB - By forming a molecular tether between two membranes, p115, giantin, and GM130 may mediate multiple Golgi-related processes including vesicle transport, cisternae formation, and cisternal stacking. The tether is proposed to involve the simultaneous binding of p115 to giantin on one membrane and to GM130 on another membrane. To explore this model, we tested for the presence of the putative giantin-p115-GM130 ternary complex. We first mapped p115-binding site in giantin to a 70-amino acid coiled-coil domain at the extreme N terminus, a position that may exist up to 400 nm away from the Golgi membrane. We then generated glutathione S-transferase (GST) fusion proteins containing either giantin's or GM130's p115 binding site and tested whether such proteins could bind p115 and GM130 or bind p115 and giantin, respectively. Unexpectedly, GST fusions containing either the giantin or the GM130 p115 binding site efficiently bound p115, but the p115 bound to GST-giantin did not bind GM130, and the p115 bound to GST-GM130 did not bind giantin. To explain this result, we mapped the giantin binding site in p115 and found that it is located at the C-terminal acidic domain, the same domain involved in binding GM130. The presence of a single binding site in p115 for giantin and GM130 was confirmed by demonstration that giantin and GM130 compete for binding to p115. These results question a simple tethering model involving a ternary giantin-p115-GM130 complex and suggest that p115-giantin and p115-GM130 interactions might mediate independent membrane tethering events. PMID- 10744705 TI - Physical interaction between Wilms tumor 1 and p73 proteins modulates their functions. AB - The WT1 gene, which is heterozygously mutated or deleted in congenital anomaly syndromes and homozygously mutated in about 15% of all Wilms tumors, encodes tissue-specific developmental regulators. Through alternative mRNA splicing, four main WT1 protein isoforms are synthesized. All isoforms can bind to DNA via their zinc fingers, albeit with different affinities and specificities, and thereby modulate the transcriptional activity of their target genes. Several proteins bind to and alter the transcription regulatory properties of the WT1 proteins, including the product of the tumor suppressor gene p53. Interaction between WT1 and p53 was shown to modulate their ability to regulate the transcription of their respective target genes. Here, we report that all four isoforms of WT1 bind to p73, a recently cloned homologue of p53. p73 binds to the zinc finger region of WT1 and thereby inhibits DNA binding and transcription activation by WT1. Similarly, WT1 inhibits p73-induced transcription activation in reporter assays and counteracts p73-induced expression of endogenous Mdm2. This, taken together with our finding that WT1 also interacts with p63/KET, another p53 homologue, suggests that association between WT1 and the members of the p53 family of proteins may be an important determinant of their functions in cell growth and differentiation. PMID- 10744706 TI - Interleukin-4 mediates cell growth inhibition through activation of Stat1. AB - Interleukin-4 (IL-4) activates Stat6 (signal transducer and activator of transcription 6) and plays multiple roles in regulation of the immune system. IL 4 also triggers phosphorylation of insulin receptor substrate (IRS), leading to stimulation of cell growth. Moreover, IL-4 inhibits proliferation of a variety of cells, but the molecular mechanism of its growth inhibitory effect is not understood. In this study, we demonstrated that IL-4 inhibited cell growth of colon carcinoma cell lines (HT29 and WiDr) but promoted cell growth of Burkitt's lymphoma cell lines (BL30 and BL41) in a dose-dependent manner. The growth inhibition was not dependent on Stat6 activation, because Stat6 was activated at similar levels in all cell lines in response to IL-4. Strikingly, IL-4 activated Stat1 in colon carcinoma cell lines but not in Burkitt's lymphoma cell lines. Therefore, these results suggest that IL-4 induced Stat1 activation, resulting in growth inhibition of colon carcinoma cell lines. Importantly, we present evidence that Stat1 is necessary for IL-4-mediated growth inhibition using Stat1-deficient and Stat1-reconstituted cells. The growth inhibitory effect of IL-4 was diminished in Stat1-deficient cells, whereas it was restored in Stat1 reconstituted cells. In addition, the expression of dominant-negative Stat1 in HT29 cells led to the loss of growth inhibition in response to IL-4. Taken together, our data suggest that IL-4 activates Stat1, leading to cell growth inhibition in colon cancer cells. Thus, this study demonstrates, for the first time, a molecular mechanism by which IL-4 inhibits cell growth. PMID- 10744707 TI - AU-rich elements in the 3'-untranslated region of a new mucin-type gene family of Trypanosoma cruzi confers mRNA instability and modulates translation efficiency. AB - Trypanosoma cruzi has a complex mucin gene family of 500 members with hypervariable regions expressed preferentially in vertebrate associated stages of the parasite. In this work, a novel mucin-type gene family is reported, composed of two groups of genes organized in independent tandems and having very short open reading frames. The structures of deduced proteins share the N and C termini but differ in central regions. One group has repeats with the consensus Lys-Asn Thr(7)-Ser-Thr(3)-Ser(Ser/Lys)-Ala-Pro and the other a Thr-rich sequence of the type Asp-Gln-Thr(17-20)-Asn-Ala-Pro-Ala-Lys-Asp-Thr(5-7)-Asn-Ala-Pro-Ala-L ys. In both cases, expected mature core proteins are around 7 kDa. Both groups, named L and S, respectively, differ in the structure of genomic loci and mRNA, with differential blocks in the 3'-untranslated region. The highest mRNA level for S and L groups are in the epimastigote stage but they show distinct developmentally regulated patterns. Transcripts are short lived and their steady-state abundance is regulated post-transcriptionally with increased mRNA stability in insect stage epimastigote. AU-rich sequences, similar to ARE motives known to cause mRNA instability in higher eukaryotes, are present in the 3'-untranslated region of the transcripts. In transfection experiments this sequence is shown to be functional for the L group destabilizing its mRNA in a stage-specific manner. Furthermore, an effect of this AU-rich region on translation efficiency is shown. To our knowledge, this is the first time that a functional ARE sequence-dependent post-transcriptional regulation mechanism is reported in a lower eukaryote. PMID- 10744708 TI - Identification of a urokinase receptor-integrin interaction site. Promiscuous regulator of integrin function. AB - Adhesion and signaling by integrins require their dynamic association with nonintegrin membrane proteins. One such protein, the glycolipid-anchored urokinase receptor (uPAR), associates with and modifies the function of the beta(2)-integrin Mac-1 (CD11b/CD18). In this study, a critical non-I-domain binding site for uPAR on CD11b (M25; residues 424-440) is identified by homology with a phage display peptide known to bind uPAR. Recombinant soluble uPAR and cells expressing uPAR bound to immobilized M25, binding being promoted by urokinase and blocked by soluble M25, but not a scrambled control or homologous peptides from other beta(2)-associated alpha-chains. Mac-1, but not a mutated Mac 1 in which M25 was replaced with the homologous sequence of CD11c, co precipitated with uPAR. In the beta-propeller model of alpha-chain folding, M25 spans an exposed loop on the ligand-binding, upper surface of alphaM, identifying uPAR as an atypical alphaM ligand. Although not blocking ligand binding to Mac-1, M25 (25-100 microM) inhibited leukocyte adhesion to fibrinogen, vitronectin, and cytokine-stimulated endothelial cells. M25 also blocked the association of uPAR with beta(1)-integrins and impaired beta(1)-integrin-dependent spreading and migration of human vascular smooth muscle cells on fibronectin and collagen. These observations indicate that uPAR associates with integrins directly and that disruption of this association broadly impairs integrin function, suggesting a novel strategy for regulation of integrins in the settings of inflammation and tumor progression. PMID- 10744709 TI - The membrane anchor influences ligand binding two-dimensional kinetic rates and three-dimensional affinity of FcgammaRIII (CD16). AB - Kinetic rates and affinity are essential determinants for biological processes that involve receptor-ligand binding. By using a micropipette method, we measured the kinetics of human Fcgamma receptor III (CD16) interacting with IgG when the two molecules were bound to apposing cellular membranes. CD16 is one of only four eukaryotic receptors known to exist natively in both the transmembrane (TM, CD16a) and glycosylphosphatidylinositol (GPI, CD16b) isoforms. The biological significance of this anchor isoform coexistence is not clear. Here we showed that the anchor influenced kinetic rates; compared with CD16a-TM, CD16a-GPI bound faster and with higher affinities to human and rabbit IgGs but slower and with lower affinity to murine IgG2a. The same differential affinity patterns were observed using soluble IgG ligands. A monoclonal antibody bound CD16a-GPI with higher affinity than CD16a-TM, whereas another monoclonal antibody reacted strongly with CD16a-TM but weakly with CD16a-GPI. No major differential glycosylation between the two CD16a isoforms was detected by SDS-polyacrylamide gel electrophoresis analysis. We suggest a conformational difference as the mechanism underlying the observed anchor effect, as it cannot be explained by the differing diffusivity, flexibility, orientation, height, distribution, or clustering of the two molecules on the cell membrane. These data demonstrate that a covalent modification of an Ig superfamily receptor at the carboxyl terminus of the ectodomain can have an impact on ligand binding kinetics. PMID- 10744710 TI - Stat5a serine phosphorylation. Serine 779 is constitutively phosphorylated in the mammary gland, and serine 725 phosphorylation influences prolactin-stimulated in vitro DNA binding activity. AB - The activity of transcription factors of the Stat family is controlled by phosphorylation of a conserved, carboxyl-terminal tyrosine residue. Tyrosine phosphorylation is essential for Stat dimerization, nuclear translocation, DNA binding, and transcriptional activation. Phosphorylation of Stats on specific serine residues has also been described. We have previously shown that in HC11 mammary epithelial cells Stat5a is phosphorylated on Tyr(694) in a prolactin sensitive manner, whereas serine phosphorylation is constitutive (Wartmann, M., Cella, N., Hofer, P., Groner, B., Xiuwen, L., Hennighausen, L., and Hynes, N. E. (1996) J. Biol. Chem. 271, 31863-31868). By using mass spectrometry and site directed mutagenesis, we have now identified Ser(779), located in a unique Stat5a SP motif, as the site of serine phosphorylation. By using phospho-Ser(779) specific antiserum, we have determined that Ser(779) is constitutively phosphorylated in mammary glands taken from different developmental stages. Stat5a isolated from spleen, heart, brain, and lung was also found to be phosphorylated on Ser(779). Ser(725) in Stat5a has also been identified as a phosphorylation site (Yamashita, H., Xu, J., Erwin, R. A., Farrar, W. L., Kirken, R. A., and Rui, H. (1998) J. Biol. Chem. 273, 30218-30224). Here we show that mutagenesis of Ser(725), Ser(779), or a combination of Ser(725/779) to an Ala had no effect on prolactin-induced transcriptional activation of a beta-casein reporter construct. However, following prolactin induction the Ser(725) mutant displayed sustained DNA binding activity compared with that of wild type Stat5a. The results suggest that Ser(725) phosphorylation has an impact on signal duration. PMID- 10744711 TI - A novel marker for vertebrate embryonic heart, the EH-myomesin isoform. AB - Myomesin is a structural component of the M-band that is expressed in all types of striated muscle. Its primary function may be the maintenance of the thick filament lattice and its anchoring to the elastic filament system composed of titin. Different myomesin isoforms have been described in chicken and mice, but no particular function has been assigned to them. Here we investigate the spatio temporal expression pattern of myomesin isoforms by means of reverse transcriptase-polymerase chain reaction and isoform-specific antibodies. We find that two alternative splicing events give rise to four myomesin isoforms in chicken contrary to only one splicing event with two possible isoforms in mice. A splicing event at the C terminus results in two splice variants termed H-myomesin and S-myomesin, which represent the major myomesin species in heart and skeletal muscle of avian species, respectively. In contrast, in mammalian heart and skeletal muscle only S-myomesin is expressed. In embryonic heart of birds and mammals, alternative splicing in the central part of the molecule gives rise to the isoform that we termed EH-myomesin. It represents the major myomesin isoform at early embryonic stages of heart but is rapidly down-regulated around birth. Thus, the strict developmental regulation of the EH-myomesin makes it an ideally suited marker for embryonic heart. PMID- 10744712 TI - The transit sequence of ferredoxin contains different domains for translocation across the outer and inner membrane of the chloroplast envelope. AB - Deletion mutants in the transit sequence of preferredoxin were used in label transfer cross-linking assays to map the interactions of the transit sequence with the import machinery. The deletion mutants gave distinct cross-linking patterns to the Toc and Tic components of the import machinery, consistent with the binding and import properties obtained in in vitro import assays. The cross linking results revealed two separate properties of the transit peptide: first the presentation of specific binding domains for the initial interaction with outer membrane components, and second the presence of different domains for interaction with the outer and inner membrane components of the transport machinery for full envelope translocation. The N-terminal Delta6-14 deletion blocked import of the precursor at the Toc components, whereas the more internal deletion Delta15-25 blocked import at the Tic components. The information for association with the outer and inner membrane components therefore resides in two separate but partly overlapping domains in the first 25 amino acids of the transit sequence. PMID- 10744713 TI - Cloning and characterization of rat dentin matrix protein 1 (DMP1) gene and its 5'-upstream region. AB - Rat dentin matrix protein 1 (DMP1) is a highly acidic 58-kDa phosphoprotein, and DMP1 was the first gene to be cloned from the mineralized dentin matrix. It exists as a highly phosphorylated protein with a pI of 3 in the dentin matrix and, in that state, might have an important role in the mineralization process. The spatio-temporal distribution during development indicates that the expression of this gene is tightly regulated in the odontoblasts. It is now known that DMP1 is not unique to dentin but is present in other mineralized tissues like long bone, calvaria, and ameloblasts. To study the transcriptional regulation and the function of DMP1 in these tissues, a genomic clone with a functional promoter, introns, and exons was isolated. Sequence analysis showed that the rat DMP1 gene is comprised of six exons and five introns and spans approximately 13 kilobases (kb). Exon 1 contains the 5'-untranslated sequences. Exon 2 encodes a total of 18 amino acids including the 16 amino acids of the signal sequence. Exons 3-5 encode 16, 11, and 15 amino acids, respectively. Exon 6 contains 1.3 kb of the coding sequence with the RGD domain, stop codon, and the 3'-untranslated region (1.1 kb). We have mapped two transcription start sites within the DMP1 promoter that are 280 and 321 base pairs, respectively, from the ATG start codon. The location of functional elements within the 5'-upstream DMP1 DNA fragment was determined by cloning it into a luciferase reporter gene. Transient transfection and luciferase assays revealed that the 3 kb fragment has the ability to drive the luciferase gene. However, this promoter activity was restricted to MC3T3-E1 cells (an osteoblast cell lineage). The promoter was silent in Chinese hamster ovary cells (an epithelial cell lineage), indicating the necessity of tissue-specific factors to drive the transcription. PMID- 10744714 TI - Regulation of mOAT-mediated organic anion transport by okadaic acid and protein kinase C in LLC-PK(1) cells. AB - Organic anion transporters in the kidney proximal tubule play an essential role in eliminating a wide range of organic anions including endogenous compounds, xenobiotics, and their metabolites, thereby preventing their potentially toxic effects within the body. We have previously cloned a cDNA encoding an organic anion transporter from mouse kidney (mOAT) (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J. G., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478; Kuze, K., Graves, P., Leahy, A., Wilson, P., Stuhlmann, H., and You, G. (1999) J. Biol. Chem. 274, 1519-1524). In the present study, we assessed the potential for regulation of this transporter by heterologous expression of mOAT in the pig proximal tubule-like cell line, LLC-PK(1). We report here that both protein phosphatase (PP1/PP2A) inhibitor, okadaic acid, and protein kinase C (PKC) activators down-regulate mOAT-mediated transport of para-aminohippuric acid (PAH), a prototypic organic anion, in a time- and concentrationdependent manner. However their mechanisms of action for this down-regulation are distinct. Okadaic acid modulated PAH transport, at least in part, through phosphorylation/dephosphorylation of mOAT; phosphoamino acid analysis indicated this phosphorylation occurs on serine. In contrast, PKC activation induced a decrease in the maximum transport velocity (V(max)) of PAH transport without direct phosphorylation of the transporter protein. Together these results provide the first demonstration that regulation of organic anion transport by mOAT is likely to be tightly controlled directly and indirectly by phosphatase PP1/PP2A and PKC. Our results also suggest that kinases other than PKC are involved in this process. PMID- 10744715 TI - p21(Waf1/Cip1) is an assembly factor required for platelet-derived growth factor induced vascular smooth muscle cell proliferation. AB - The cyclin-dependent kinase inhibitors interact with cyclin-cdk complexes to arrest mitogen-stimulated transit through the cell cycle, but these proteins have recently been shown to have positive regulatory effects on cyclin-cdk complex activity as well. Most of the previous work in this area has focussed on the finding that overexpressed p21(Waf1/Cip1) causes growth arrest. However, mice lacking p21(Waf1/Cip1) showed normal development with no aberrancy in their cell cycles, and antisense p21(Waf1/Cip1) has only been shown to prevent cell cycle arrest, leading to the conclusion that the cyclin kinase inhibitors may not be required for cell cycle progression. We found that transfection of several lines of vascular smooth muscle cells with antisense oligodeoxynucleotide specific to p21(Waf1/Cip1) correlates with decreased cyclin D1/cdk 4, but not cyclin E/cdk 2, association, yet, unexpectedly, results in dose-dependent inhibition of platelet derived growth factor-BB-stimulated DNA synthesis and cell proliferation. Our finding that p21(Waf1/Cip1) exhibits permissive effects on growth factor-induced vascular smooth muscle cell cycle progression, such that its presence is required for growth factor-induced proliferation, is the first such report and opens up a fertile area of research relevant to diseases involving vascular cell proliferation. PMID- 10744716 TI - Nectin-3, a new member of immunoglobulin-like cell adhesion molecules that shows homophilic and heterophilic cell-cell adhesion activities. AB - We have isolated a novel cell-cell adhesion system localized at cadherin-based adherens junctions (AJs). This system consists of at least nectin, a Ca(2+) independent immunoglobulin-like adhesion molecule, and afadin, an actin filament binding protein, that connects nectin to the actin cytoskeleton. Nectin constitutes a family consisting of two members, nectin-1 and -2. We have isolated here a third member of the nectin family and named it nectin-3. Nectin-3 has three splicing variants, nectin-3alpha (biggest), -3beta (middle), and -3gamma (smallest). Like nectin-1 and -2, nectin-3alpha consists of three extracellular immunoglobulin-like domains, a transmembrane segment, and a cytoplasmic region with the C-terminal consensus motif for binding to the PDZ domain. Nectin-3alpha formed a cis-homo-dimer and showed Ca(2+)-independent trans-homo-interaction to cause homophilic cell-cell adhesion. Nectin-3alpha furthermore showed trans hetero-interaction with nectin-1 or -2 but did not form a cis-hetero-dimer with nectin-1 or -2. Nectin-1 did not show trans-hetero-interaction with nectin-2. The affinity of trans-hetero-interaction of nectin-3alpha with nectin-1 or -2 was higher than that of trans-homo-interaction of nectin-1, -2, or -3alpha. Nectin-2 and -3 were ubiquitously expressed, whereas nectin-1 was abundantly expressed in brain. Nectin-3alpha was colocalized with nectin-2 at cadherin-based AJs and interacted with afadin. These results indicate that the nectin family consists of at least three members, nectin-1, -2, and -3, all of which show homophilic and heterophilic cell-cell adhesion activities and are localized at cadherin-based AJs. PMID- 10744717 TI - Structure-based design of a low molecular weight, nonphosphorus, nonpeptide, and highly selective inhibitor of protein-tyrosine phosphatase 1B. AB - Several protein-tyrosine phosphatases (PTPs) have been proposed to act as negative regulators of insulin signaling. Recent studies have shown increased insulin sensitivity and resistance to obesity in PTP1B knockout mice, thus pointing to this enzyme as a potential drug target in diabetes. Structure-based design, guided by PTP mutants and x-ray protein crystallography, was used to optimize a relatively weak, nonphosphorus, nonpeptide general PTP inhibitor (2 (oxalyl-amino)-benzoic acid) into a highly selective PTP1B inhibitor. This was achieved by addressing residue 48 as a selectivity determining residue. By introducing a basic nitrogen in the core structure of the inhibitor, a salt bridge was formed to Asp-48 in PTP1B. In contrast, the basic nitrogen causes repulsion in other PTPs containing an asparagine in the equivalent position resulting in a remarkable selectivity for PTP1B. Importantly, this was accomplished while retaining the molecular weight of the inhibitor below 300 g/mol. PMID- 10744718 TI - Identification and initial characterization of four novel members of the interleukin-1 family. AB - Interleukin-1 (IL-1), fibroblast growth factors (FGFs), and their homologues are secreted factors that share a common beta-barrel structure and act on target cells by binding to cell surface receptors with immunoglobulin-like folds in their extracellular domain. While numerous members of the FGF family have been discovered, the IL-1 family has remained small and outnumbered by IL-1 receptor homologues. From expressed sequence tag data base searches, we have now identified four additional IL-1 homologues, IL-1H1, IL-1H2, IL-1H3, and IL-1H4. Like most other IL-1/FGFs, these proteins do not contain a hydrophobic leader sequence. IL-1H4 has a propeptide sequence, while IL-1H1, IL-1H2, and IL-1H3 encode only the mature protein. Circular dichroism spectra and thermal stability analysis suggest that IL-1H1 folds similarly to IL-1ra. The novel homologues are not widely expressed in mammals. IL-1H1 is constitutively expressed only in placenta and the squamous epithelium of the esophagus. However, IL-1H1 could be induced in vitro in keratinocytes by interferon-gamma and tumor necrosis factor alpha and in vivo via a contact hypersensitivity reaction or herpes simplex virus infection. This suggests that IL-1H1 may be involved in pathogenesis of immune mediated disease processes. The addition of four novel IL-1 homologues suggests that the IL-1 family is significantly larger than previously thought. PMID- 10744720 TI - Sweet Tooth, a novel receptor protein-tyrosine kinase with C-type lectin-like extracellular domains. AB - A gene encoding a novel type of receptor protein-tyrosine kinase was identified in Hydra vulgaris. The extracellular portion of this receptor (which we have named Sweet Tooth) contains four C-type lectin-like domains (CTLDs). Comparison of the sequences of these domains with the sequences of the carbohydrate recognition domains of various vertebrate C-type lectins shows that Sweet Tooth CTLD1 and CTLD4 have amino acids in common with those shown to be involved in carbohydrate binding by the lectins. Comparison of sequences encoding CTLD1 from the Sweet Tooth genes from different species of Hydra shows variation in some of the conserved residues that participate in carbohydrate binding in C-type lectins. The Sweet Tooth gene is expressed widely in the Hydra polyp, and expression is particularly high in the endoderm of the tentacles. Treatment of polyps with peptides corresponding to sequences in the Sweet Tooth CTLDs results in the disintegration of the animal. These same peptides do not block adhesion or morphogenesis of Hydra cell aggregates. PMID- 10744719 TI - Isolation of a novel family of C(2)H(2) zinc finger proteins implicated in transcriptional repression mediated by chicken ovalbumin upstream promoter transcription factor (COUP-TF) orphan nuclear receptors. AB - Two novel and related C(2)H(2) zinc finger proteins that are highly expressed in the brain, CTIP1 and CTIP2 (COUP TF-interacting proteins 1 and 2, respectively), were isolated and shown to interact with all members of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of orphan nuclear receptors. The interaction of CTIP1 with ARP1 was studied in detail, and CTIP1 was found to harbor two independent ARP1 interaction domains, ID1 and ID2, whereas the putative AF-2 of ARP1 was required for interaction with CTIP1. CTIP1, which exhibited a punctate staining pattern within the nucleus of transfected cells, recruited cotransfected ARP1 to these foci and potentiated ARP1-mediated transcriptional repression of a reporter construct. However, transcriptional repression mediated by ARP1 acting through CTIP1 did not appear to involve recruitment of a trichostatin A-sensitive histone deacetylase(s) to the template, suggesting that this repression pathway may be distinct from that utilized by several other nuclear receptors. PMID- 10744721 TI - Chitinases of the avian malaria parasite Plasmodium gallinaceum, a class of enzymes necessary for parasite invasion of the mosquito midgut. AB - The Plasmodium ookinete produces chitinolytic activity that allows the parasite to penetrate the chitin-containing peritrophic matrix surrounding the blood meal in the mosquito midgut. Since the peritrophic matrix is a physical barrier that the parasite must cross to invade the mosquito, and the presence of allosamidin, a chitinase inhibitor, in a blood meal prevents the parasite from invading the midgut epithelium, chitinases (3.2.1.14) are potential targets of malaria parasite transmission-blocking interventions. We have purified a chitinase of the avian malaria parasite Plasmodium gallinaceum and cloned the gene, PgCHT1, encoding it. PgCHT1 encodes catalytic and substrate-binding sites characteristic of family 18 glycohydrolases. Expressed in Escherichia coli strain AD494 (DE3), recombinant PgCHT1 was found to hydrolyze polymeric chitin, native chitin oligosaccharides, and 4-methylumbelliferone derivatives of chitin oligosaccharides. Allosamidin inhibited recombinant PgCHT1 with an IC(50) of 7 microM and differentially inhibited two chromatographically separable P. gallinaceum ookinete-produced chitinase activities with IC(50) values of 7 and 12 microM, respectively. These two chitinase activities also had different pH activity profiles. These data suggest that the P. gallinaceum ookinete uses products of more than one chitinase gene to initiate mosquito midgut invasion. PMID- 10744722 TI - Caffeine abolishes the mammalian G(2)/M DNA damage checkpoint by inhibiting ataxia-telangiectasia-mutated kinase activity. AB - Recent evidence indicates that arrest of mammalian cells at the G(2)/M checkpoint involves inactivation and translocation of Cdc25C, which is mediated by phosphorylation of Cdc25C on serine 216. Data obtained with a phospho-specific antibody against serine 216 suggest that activation of the DNA damage checkpoint is accompanied by an increase in serine 216 phosphorylated Cdc25C in the nucleus after exposure of cells to gamma-radiation. Prior treatment of cells with 2 mM caffeine inhibits such a change and markedly reduces radiation-induced ataxia telangiectasia-mutated (ATM)-dependent Chk2/Cds1 activation and phosphorylation. Chk2/Cds1 is known to localize in the nucleus and to phosphorylate Cdc25C at serine 216 in vitro. Caffeine does not inhibit Chk2/Cds1 activity directly, but rather, blocks the activation of Chk2/Cds1 by inhibiting ATM kinase activity. In vitro, ATM phosphorylates Chk2/Cds1 at threonine 68 close to the N terminus, and caffeine inhibits this phosphorylation with an IC(50) of approximately 200 microM. Using a phospho-specific antibody against threonine 68, we demonstrate that radiation-induced, ATM-dependent phosphorylation of Chk2/Cds1 at this site is caffeine-sensitive. From these results, we propose a model wherein caffeine abrogates the G(2)/M checkpoint by targeting the ATM-Chk2/Cds1 pathway; by inhibiting ATM, it prevents the serine 216 phosphorylation of Cdc25C in the nucleus. Inhibition of ATM provides a molecular explanation for the increased radiosensitivity of caffeine-treated cells. PMID- 10744723 TI - UCR1 and UCR2 domains unique to the cAMP-specific phosphodiesterase family form a discrete module via electrostatic interactions. AB - The cAMP-specific phosphodiesterases (PDE4) enzymes contain unique "signature" regions of amino acid sequence, called upstream conserved regions 1 and 2 (UCR1 and UCR2). UCR1 and UCR2 are located between the extreme amino-terminal region and the catalytic region of the PDE4 enzymes. The UCR1 of the PDE4D3 isoform was used as a "bait" in a two-hybrid screen, which identified a PDE4D cDNA clone containing UCR2 and the catalytic region but not UCR1. Two-hybrid and "pull down" analysis of constructs incorporating various regions of the PDE4D3 cDNA demonstrated that the carboxyl-terminal region of UCR1 interacted specifically with the amino-terminal region of UCR2. The interaction was blocked by mutations of two positively charged amino acids (Arg-98 and Arg-101 to alanine) located within an otherwise largely hydrophobic region of UCR1. Mutation of three negatively charged amino acids in UCR2 (Glu-146, Glu-147, and Asp-149, all to alanine) also blocked the interaction. The phosphorylation of UCR1 by cAMP dependent protein kinase (PKA) in vitro attenuated the ability of UCR1 to interact with UCR2. Mutation of the PKA substrate site in UCR1 (Ser-54) to aspartic acid, which mimics the activation of PDE4D3 by PKA, profoundly reduced the interaction between UCR1 and UCR2. Our data are consistent with a model in which UCR1 and UCR2 act as independent domains whose interaction is determined by electrostatic interactions and which may be disrupted by PKA phosphorylation. We suggest that the UCR1 and UCR2 domains may form a module that interacts with and regulates the PDE4 catalytic region. PMID- 10744724 TI - A novel pro-Arg motif recognized by WW domains. AB - WW domains mediate protein-protein interactions through binding to short proline rich sequences. Two distinct sequence motifs, PPXY and PPLP, are recognized by different classes of WW domains, and another class binds to phospho-Ser-Pro sequences. We now describe a novel Pro-Arg sequence motif recognized by a different class of WW domains using data from oriented peptide library screening, expression cloning, and in vitro binding experiments. The prototype member of this group is the WW domain of formin-binding protein 30 (FBP30), a p53-regulated molecule whose WW domains bind to Pro-Arg-rich cellular proteins. This new Pro Arg sequence motif re-classifies the organization of WW domains based on ligand specificity, and the Pro-Arg class now includes the WW domains of FBP21 and FE65. A structural model is presented which rationalizes the distinct motifs selected by the WW domains of YAP, Pin1, and FBP30. The Pro-Arg motif identified for WW domains often overlaps with SH3 domain motifs within protein sequences, suggesting that the same extended proline-rich sequence could form discrete SH3 or WW domain complexes to transduce distinct cellular signals. PMID- 10744725 TI - Collagen XI nucleates self-assembly and limits lateral growth of cartilage fibrils. AB - Fibrils of embryonic cartilage are heterotypic alloys formed by collagens II, IX, and XI and have a uniform diameter of approximately 20 nm. The molecular basis of this lateral growth control is poorly understood. Collagen II subjected to fibril formation in vitro produced short and tapered tactoids with strong D-periodic banding. The maximal width of these tactoids varied over a broad range. By contrast, authentic mixtures of collagens II, IX, and XI yielded long and weakly banded fibrils, which, strikingly, had a uniform width of about 20 nm. The same was true for mixtures of collagens II and XI lacking collagen IX as long as the molar excess of collagen II was less than 8-fold. At higher ratios, the proteins assembled into tactoids coexisting with cartilage-like fibrils. Therefore, diameter control is an inherent property of appropriate mixtures of collagens II and XI. Collagen IX is not essential for this feature but strongly increases the efficiency of fibril formation. Therefore, this protein may be an important stabilizing factor of cartilage fibrils. PMID- 10744726 TI - Tumor necrosis factor-alpha-converting enzyme is required for cleavage of erbB4/HER4. AB - HER4 is a member of the epidermal growth factor receptor family and has an essential function in heart and neural development. Identification of two HER4 isoforms, HER4 JM-a and JM-b, which differ in their extracellular juxtamembrane region and in their susceptibility to cleavage after phorbol ester stimulation, showed that the juxtamembrane region of the receptor is critical for proteolysis. We now demonstrate that phorbol ester and pervanadate are effective stimuli for HER4 JM-a processing and that the HER4 JM-b isoform does not undergo cleavage in response to any of the stimuli studied. We also show that HER4 JM-a is not cleaved in cells lacking the metalloprotease tumor necrosis factor-alpha converting enzyme (TACE) and that reexpression of TACE in these cells restores constitutive and regulated processing of HER4 JM-a. Moreover, we show that the sequence specific to the HER4 JM-a juxtamembrane region is sufficient to confer susceptibility to phorbol 12-myristate 13-acetate-induced cleavage of the HER2 receptor. In conclusion, we provide evidence that TACE is essential for the regulated shedding of the HER4 JM-a receptor. PMID- 10744727 TI - Up-regulation of protein-disulfide isomerase in response to hypoxia/brain ischemia and its protective effect against apoptotic cell death. AB - We isolated and identified a stress protein that is up-regulated in response to hypoxia in primary-cultured glial cells. Protein-disulfide isomerase (PDI) was up regulated not only by hypoxia in glia in vitro, but also by transient forebrain ischemia in rats in vivo. To determine whether newly synthesized PDI is involved in tolerance to ischemic stress, we carried out two procedures to induce PDI gene expression in human neuroblastoma SK-N-MC cells, as well as intrahippocampal injection following electroporation of an expression vector capable of overexpressing PDI in rats. Overexpression of this gene resulted in attenuation of the loss of cell viability induced by hypoxia in neuroblastoma SK-N-MC cells and a reduction in the number of DNA-fragmented cells in the CA1 area of the hippocampus in brain ischemic rats, respectively. These findings suggest that up regulated PDI may play a critical role in resistance to ischemic damage, and that the elevation of levels of this protein in the brain may have beneficial effects against brain stroke. PMID- 10744728 TI - Nucleic acid binding properties of the simian immunodeficiency virus nucleocapsid protein NCp8. AB - The nucleocapsid protein of simian immunodeficiency virus (SIV) NCp8 has two copies of conserved sequences (termed zinc fingers, ZF) of 14 amino acids with 4 invariant residues (CCHC) that coordinate Zn(II). Each of its two ZFs has a Trp residue. A significant quenching of NCp8 Trp fluorescence was seen in nucleic acid complexes, suggesting stacking of the indole ring with nucleobases and the simultaneous involvement of both ZFs in the binding process. Both ZFs contribute to the nucleic acid binding free energy of NCp8, albeit in a not additive manner. NCp8 exhibited a base preference analogous to that of NCp7: G approximately I > T > U > C > A. Alternating base sequences that bind HIV-1 NCp7 in a sequence specific manner were also bound selectively by NCp8. Specific sequence recognition required at least five bases and the presence of bound Zn(II). The two ZFs account for the net displacement of 3 out of 4 sodium ions upon binding (2 by the first and one by the second finger), and for most (85%) of the hydrophobic stabilization in complex formation. Based on the sequence and functional similarity of SIV NCp8 and HIV-1 NCp7, and using available structural information for free and oligonucleotide bound NCp7, we propose a structural model for NCp8-oligonucleotide complexes. PMID- 10744729 TI - Curcuminoids inhibit the angiogenic response stimulated by fibroblast growth factor-2, including expression of matrix metalloproteinase gelatinase B. AB - We have studied mechanisms controlling activation of the gelatinase B gene (matrix metalloproteinase-9) by fibroblast growth factor-2 (FGF-2) during angiogenesis, and the effects of the natural product curcuminoids on this process. Using a transgenic mouse (line 3445) harboring a gelatinase B promoter/lacZ fusion gene, we demonstrate FGF-2 stimulation of reporter gene expression in endothelial cells of invading neocapillaries in the corneal micropocket assay. Using cultured corneal cells, we show that FGF-2 stimulates DNA binding activity of transcription factor AP-1 but not NF-kappaB and that AP-1 stimulation is inhibited by curcuminoids. We further show that induction of gelatinase B transcriptional promoter activity in response to FGF-2 is dependent on AP-1 but not NF-kappaB response elements and that promoter activity is also inhibited by curcuminoids. In rabbit corneas, the angiogenic response induced by implantation of an FGF-2 pellet is inhibited by the co-implantation of a curcuminoid pellet, and this correlates with inhibition of endogenous gelatinase B expression induced by FGF-2. Angiostatic efficacy in the cornea is also observed when curcuminoids are provided to mice in the diet. Our findings provide evidence that curcuminoids target the FGF-2 angiogenic signaling pathway and inhibit expression of gelatinase B in the angiogenic process. PMID- 10744730 TI - Inhibition of proliferation and apoptosis by the transcriptional repressor Mad1. Repression of Fas-induced caspase-8 activation. AB - Mad1 is a member of the Myc/Max/Mad network of transcriptional regulators that play a central role in the control of cellular behavior. Mad proteins are thought to antagonize Myc functions at least in part by repressing gene transcription. To systematically examine the function of Mad1 in growth control and during apoptosis, we have generated U2OS cell clones that express Mad1 under a tetracyline-regulatable promoter (UTA-Mad1). Mad1 was induced rapidly and efficiently, localized to the nucleus, and bound to DNA as a heterodimer with Max. The induction of Mad1 reduced cellular growth and, more profoundly, inhibited colony formation of UTA-Mad1 cells. Conditioned medium neutralized this inhibitory effect implying that Mad1 function is regulated by extracellular signals. In addition Mad1 interfered with Fas-, TRAIL-, and UV-induced apoptosis, which coincided with a reduced activation of caspase-8 during Fas-mediated apoptosis in response to Mad1 expression. Furthermore, microinjection of Mad1 expressing plasmids into fibroblasts inhibited apoptosis induced by the oncoproteins c-Myc and E1A. Thus, Mad1 not only interferes with cellular proliferation but also with apoptosis, which defines a novel aspect of Mad1 function. PMID- 10744731 TI - A conserved "hydrophobic staple motif" plays a crucial role in the refolding of human glutathione transferase P1-1. AB - The specific (i, i+5) hydrophobic staple interaction involving a helix residue and a second residue located in the turn preceding the helix is a recurrent motif at the N terminus of alpha-helices. This motif is strictly conserved in the core of all soluble glutathione transferases (GSTs) as well as in other protein structures. Human GSTP1-1 variants mutated in amino acid Ile(149) and Tyr(154) of the hydrophobic staple motif of the alpha6-helix were analyzed. In particular, a double mutant cycle analysis has been performed to evaluate the role of the hydrophobic staple motif in the refolding process. The results show that this local interaction, by restricting the number of conformations of the alpha6-helix relative to the alpha1-helix, favors the formation of essential interdomain interactions and thereby accelerates the folding process. Thus, for the first time it is shown that the hydrophobic staple interaction has a role in the folding process of an intact protein. In P(i) class GSTs, Tyr(154) appears to be of particular structural importance, since it interacts with conserved residues Leu(21), Asp(24), and Gln(25) of the adjacent alpha1-helix which contributes to the active site. Human GSTP1-1 variants L21A and Y154F have also been analyzed in order to distinguish the role of interdomain interactions from that of the hydrophobic staple. The experimental results reported here suggest that the strict conservation of the hydrophobic staple motif reflects an evolutionary pressure for proteins to fold rapidly. PMID- 10744732 TI - Quantitative expression analysis of genes regulated by both obesity and leptin reveals a regulatory loop between leptin and pituitary-derived ACTH. AB - Absence of the hormone leptin leads to dramatic increases in appetite, food intake, and adiposity. The primary site of action, at least with respect to appetite, is the hypothalamus. Leptin also has significant effects on the function(s) of peripheral organs involved in maintaining body composition. Some of these effects are mediated through direct interaction of leptin with its receptor on the target tissue, and some effects are indirectly mediated through secondary hormonal and neural pathways. Few of the genes that are responsible for regulating body composition and the peripheral effects of leptin are known. We have used a new gene profiling technology to characterize gene expression changes that occur in the pituitary, hypothalamus, fat, muscle, and liver in response to both obesity and treatment with exogenous leptin. These differences were then overlaid to allow the identification of genes that are regulated by obesity and at least partially normalized by leptin treatment. By using this process we have identified five genes (POMC, PC2, prolactin, HSGP25L2G, and one novel) that are both abnormally expressed in the pituitaries of obese mice and are sensitive to the effects of leptin. We also show that adrenocorticotropic hormone appears to be involved in a regulatory loop involving leptin. PMID- 10744733 TI - Inhibition of polyglutamine protein aggregation and cell death by novel peptides identified by phage display screening. AB - Proteins with expanded polyglutamine domains cause eight inherited neurodegenerative diseases, including Huntington's, but the molecular mechanism(s) responsible for neuronal degeneration are not yet established. Expanded polyglutamine domain proteins possess properties that distinguish them from the same proteins with shorter glutamine repeats. Unlike proteins with short polyglutamine domains, proteins with expanded polyglutamine domains display unique protein interactions, form intracellular aggregates, and adopt a novel conformation that can be recognized by monoclonal antibodies. Any of these polyglutamine length-dependent properties could be responsible for the pathogenic effects of expanded polyglutamine proteins. To identify peptides that interfere with pathogenic polyglutamine interactions, we screened a combinatorial peptide library expressed on M13 phage pIII protein to identify peptides that preferentially bind pathologic-length polyglutamine domains. We identified six tryptophan-rich peptides that preferentially bind pathologic-length polyglutamine domain proteins. Polyglutamine-binding peptide 1 (QBP1) potently inhibits polyglutamine protein aggregation in an in vitro assay, while a scrambled sequence has no effect on aggregation. QBP1 and a tandem repeat of QBP1 also inhibit aggregation of polyglutamine-yellow fluorescent fusion protein in transfected COS-7 cells. Expression of QBP1 potently inhibits polyglutamine induced cell death. Selective inhibition of pathologic interactions of expanded polyglutamine domains with themselves or other proteins may be a useful strategy for preventing disease onset or for slowing progression of the polyglutamine repeat diseases. PMID- 10744734 TI - Mutational analysis of Gbetagamma and phospholipid interaction with G protein coupled receptor kinase 2. AB - Agonist-dependent regulation of G protein-coupled receptors is dependent on their phosphorylation by G protein-coupled receptor kinases (GRKs). GRK2 and GRK3 are selectively regulated in vitro by free Gbetagamma subunits and negatively charged membrane phospholipids through their pleckstrin homology (PH) domains. However, the molecular binding determinants and physiological role for these ligands remain unclear. To address these issues, we generated an array of site-directed mutants within the GRK2 PH domain and characterized their interaction with Gbetagamma and phospholipids in vitro. Mutation of several residues in the loop 1 region of the PH domain, including Lys-567, Trp-576, Arg-578, and Arg-579, resulted in a loss of receptor phosphorylation, likely via disruption of phospholipid binding, that was reversed by Gbetagamma. Alternatively, mutation of residues distal to the C-terminal amphipathic alpha-helix, including Lys-663, Lys 665, Lys-667, and Arg-669, resulted in decreased responsiveness to Gbetagamma. Interestingly, mutation of Arg-587 in beta-sheet 3, a region not previously thought to interact with Gbetagamma, resulted in a specific and profound loss of Gbetagamma responsiveness. To further characterize these effects, two mutants (GRK2(K567E/R578E) and GRK2(R587Q)) were expressed in Sf9 cells and purified. Analysis of these mutants revealed that GRK2(K567E/R578E) was refractory to stimulation by negatively charged phospholipids but bound Gbetagamma similar to wild-type GRK2. In contrast, GRK2(R587Q) was stimulated by acidic phospholipids but failed to bind Gbetagamma. In order to examine the role of phospholipid and Gbetagamma interaction in cells, wild-type and mutant GRK2s were expressed with a beta(2)-adrenergic receptor (beta(2)AR) mutant that is responsive to GRK2 phosphorylation (beta(2)AR(Y326A)). In these cells, GRK2(K567E/R578E) and GRK2(R587Q) were largely defective in promoting agonist-dependent phosphorylation and internalization of beta(2)AR(Y326A). Similarly, wild-type GRK2 but not GRK2(K567E/R578E) or GRK2(R587Q) promoted morphinedependent phosphorylation of the mu-opioid receptor in cells. Thus, we have (i) identified several specific GRK2 binding determinants for Gbetagamma and phospholipids, and (ii) demonstrated that Gbetagamma binding is the limiting step for GRK2-dependent receptor phosphorylation in cells. PMID- 10744735 TI - A dominant negative Fas-associated death domain protein mutant inhibits proliferation and leads to impaired calcium mobilization in both T-cells and fibroblasts. AB - Death domain-containing members of the tumor necrosis factor (TNF) receptor family ("death receptors") can induce apoptosis upon stimulation by their natural ligands or by agonistic antibodies. Activated death receptors recruit death domain adapter proteins like Fas-associated death domain protein (FADD), and this ultimately leads to proteolytic activation of the caspase cascade and cell death. Recently, FADD has also been implicated in the regulation of proliferation; functional inhibition of FADD results in p53-dependent impairment of proliferation in activated T-cells. In this study we have further analyzed T cells derived from transgenic mice expressing a dominant negative FADD mutant (FADD DN) under control of the lck promoter in vitro so as to identify the signaling pathways that become engaged upon T-cell receptor stimulation and that are regulated by death receptors. FADD DN expression inhibits T-cell proliferation, both at the G(0) --> S transition and in the G(1) phase of continuously proliferating cells. We observe a decrease in the release of calcium from intracellular stores after T-cell receptor stimulation, whereas influx of extracellular calcium seems to be unaffected. FADD DN-expressing fibroblasts show a similarly inhibited cell growth and impaired calcium mobilization indicating that the modulation of proliferation and calcium response by death receptors is not cell type-specific. PMID- 10744736 TI - Mapping of the 5'-2-deoxyribose-5-phosphate lyase active site in DNA polymerase beta by mass spectrometry. AB - The mechanism of the 5'-2-deoxyribose-5-phosphate lyase reaction catalyzed by mammalian DNA beta-polymerase (beta-pol) was investigated using a cross-linking methodology in combination with mass spectrometric analyses. The approach included proteolysis of the covalently cross-linked protein-DNA complex with trypsin, followed by isolation, peptide mapping, and mass spectrometric and tandem mass spectrometric analyses. The 8-kDa domain of beta-pol was covalently cross-linked to a 5'-2-deoxyribose-5-phosphate-containing DNA substrate by sodium borohydride reduction. Using tandem mass spectrometry, the location of the DNA adduct on the 8-kDa domain was unequivocally determined to be at the Lys(72) residue. No additional amino acid residues were found as minor cross-linked species. These data allow assignment of Lys(72) as the sole Schiff base nucleophile in the 8-kDa domain of beta-pol. These results provide the first direct evidence in support of a catalytic mechanism involving nucleophilic attack by Lys(72) at the abasic site. PMID- 10744737 TI - Competition between C-terminal tyrosine and nicotinamide modulates pyridine nucleotide affinity and specificity in plant ferredoxin-NADP(+) reductase. AB - Chloroplast ferredoxin-NADP(+) reductase has a 32,000-fold preference for NADPH over NADH, consistent with its main physiological role of NADP(+) photoreduction for de novo carbohydrate biosynthesis. Although it is distant from the 2' phosphoryl group of NADP(+), replacement of the C-terminal tyrosine (Tyr(308) in the pea enzyme) by Trp, Phe, Gly, and Ser produced enzyme forms in which the preference for NADPH over NADH was decreased about 2-, 10-, 300-, and 400-fold, respectively. Remarkably, in the case of the Y308S mutant, the k(cat) value for the NADH-dependent activity approached that of the NADPH-dependent activity of the wild-type enzyme. Furthermore, difference spectra of the NAD(+) complexes revealed that the nicotinamide ring of NAD(+) binds at nearly full occupancy in the active site of both the Y308G and Y308S mutants. These results correlate well with the k(cat) values obtained with these mutants in the NADH-ferricyanide reaction. The data presented support the hypothesis that specific recognition of the 2'-phosphate group of NADP(H) is required but not sufficient to ensure a high degree of discrimination against NAD(H) in ferredoxin-NADP(+) reductase. Thus, the C-terminal tyrosine enhances the specificity of the reductase for NADP(H) by destabilizing the interaction of a moiety common to both coenzymes, i.e. the nicotinamide. PMID- 10744738 TI - Reciprocal regulation via protein-protein interaction between c-Myc and p21(cip1/waf1/sdi1) in DNA replication and transcription. AB - The c-myc protooncogene product (c-Myc) is a transcription factor and is rapidly induced in resting cells following various mitogenic stimuli. c-Myc is thus suggested to play an important role in the transition from quiescence to proliferation. Despite numerous studies, including those on the connection between cyclin E/cyclin-dependent kinase 2 and c-Myc, little has been clarified about c-Myc in terms of the cell cycle regulation. Here we show that c-Myc can directly bind to the carboxyl-terminal region of the cyclin-dependent kinase inhibitor p21(cip1/waf1/sdi1) and thus partially relieves the p21 of the inhibitory effect on DNA synthesis directed by the proliferating cell nuclear antigen-dependent DNA polymerase delta. As for transcription, on the other hand, the p21 binding to the Myc box II region of c-Myc blocks c-Myc-Max complex formation on the E-box and thereby suppresses the transcriptional activation from the E-box by c-Myc. These results suggest that c-Myc activates DNA replication via inactivation of p21 and that p21, vice versa, represses the transcriptional activity of c-Myc. The balance of the reciprocal inactivation between c-Myc and p21 may determine the course of cellular processes such as cell proliferation, differentiation, and apoptosis. PMID- 10744739 TI - Structure, expression, and function of the Xenopus laevis caspase family. AB - Caspases, a family of cysteine proteases, have been recognized as the central executors of programmed cell death. Nonetheless, the information on the caspase family has been limited to mammals, Drosophila, and nematodes. To examine the structure and characterization of the Xenopus caspase family, we have cloned the cDNAs encoding caspase-2 and -6-10 in addition to caspase-1 and -3, which we characterized previously (Yaoita, Y., and Nakajima, K. (1997) J. Biol. Chem. 272, 5122-5127). First, the existence of these caspases in frog suggests that the caspase cascades clarified in mammals are conserved at least from Amphibia. Interestingly, Xenopus caspase-1, -8, and -10 (especially caspase-8) showed a lower degree of identity to human equivalents than the other caspases. Second, mRNAs of many caspases increased during the climax of metamorphosis in regressing organs, tail, and intestine, where programmed cell death occurs, but not in apoptotic tail-derived cultured cells (XLT-15-11) treated with thyroid hormone, showing that new RNA synthesis of caspases is dispensable to programmed cell death. Third, comparison of human and Xenopus caspase sequences implies that some proposed regulations of human caspases are not conserved in frog. PMID- 10744740 TI - ASC1/RAS2 suppresses the growth defect on glycerol caused by the atp1-2 mutation in the yeast Saccharomyces cerevisiae. AB - To better define the regulatory role of the F(1)-ATPase alpha-subunit in the catalytic cycle of the ATP synthase complex, we isolated suppressors of mutations occurring in ATP1, the gene for the alpha-subunit in Saccharomyces cerevisiae. First, two atp1 mutations (atp1-1 and atp1-2) were characterized that prevent the growth of yeast on non-fermentable carbon sources. Both mutants contained full length F(1)alpha-subunit proteins in mitochondria, but in lower amounts than that in the parental strain. Both mutants exhibited barely measurable F(1)-ATPase activity. The primary mutations in atp1-1 and atp1-2 were identified as Thr(383) -> Ile and Gly(291) --> Asp, respectively. From recent structural data, position 383 lies within the catalytic site. Position 291 is located near the region affecting subunit-subunit interaction with the F(1)beta-subunit. An unlinked suppressor gene, ASC1 (alpha-subunit complementing) of the atp1-2 mutation (Gly(291) --> Asp) restored the growth defect phenotype on glycerol, but did not suppress either atp1-1 or the deletion mutant Deltaatp1. Sequence analysis revealed that ASC1 was allelic with RAS2, a G-protein growth regulator. The introduction of ASC1/RAS2 into the atp1-2 mutant increased the F(1)-ATPase enzyme activity in this mutant when the transformant was grown on glycerol. The possible mechanisms of ASC1/RAS2 suppression of atp1-2 are discussed; we suggest that RAS2 is part of the regulatory circuit involved in the control of F(1)-ATPase subunit levels in mitochondria. PMID- 10744741 TI - Mechanism whereby proliferating cell nuclear antigen stimulates flap endonuclease 1. AB - Human flap endonuclease 1 (FEN1), an essential DNA replication protein, cleaves substrates with unannealed 5'-tails. FEN1 apparently tracks along the flap from the 5'-end to the cleavage site. Proliferating cell nuclear antigen (PCNA) stimulates FEN1 cleavage 5-50-fold. To determine whether tracking, binding, or cleavage is enhanced by PCNA, we tested a variety of flap substrates. Similar levels of PCNA stimulation occur on both a cleavage-sensitive nicked substrate and a less sensitive gapped substrate. PCNA stimulates FEN1 irrespective of the flap length. Stimulation occurs on a pseudo-Y substrate that exhibits upstream primer-independent cleavage. A pseudo-Y substrate with a sequence requiring an upstream primer for cleavage was not activated by PCNA, suggesting that PCNA does not compensate for substrate features that inhibit cleavage. A biotin.streptavidin conjugation at the 5'-end of a flap structure prevents FEN1 loading. The addition of PCNA does not restore FEN1 activity. These results indicate that PCNA does not direct FEN1 to the cleavage site from solution. Kinetic analyses reveal that PCNA can lower the K(m) for FEN1 by 11-12-fold. Overall, our results indicate that after FEN1 tracks to the cleavage site, PCNA enhances FEN1 binding stability, allowing for greater cleavage efficiency. PMID- 10744742 TI - The assembly and secretion of apolipoprotein B-48-containing very low density lipoproteins in McA-RH7777 cells. AB - We have used an extraction procedure, which released membrane-bound apoB-100, to study the assembly of apoB-48 VLDL (very low density lipoproteins). This procedure released apoB-48, but not integral membrane proteins, from microsomes of McA-RH7777 cells. Upon gradient ultracentrifugation, the extracted apoB-48 migrated in the same position as the dense apoB-48-containing lipoprotein (apoB 48 HDL (high density lipoprotein)) secreted into the medium. Labeling studies with [(3)H]glycerol demonstrated that the HDL-like particle extracted from the microsomes contains both triglycerides and phosphatidylcholine. The estimated molar ratio between triglyceride and phosphatidylcholine was 0.70 +/- 0.09, supporting the possibility that the particle has a neutral lipid core. Pulse chase experiments indicated that microsomal apoB-48 HDL can either be secreted as apoB-48 HDL or converted to apoB-48 VLDL. These results support the two-step model of VLDL assembly. To determine the size of apoB required to assemble HDL and VLDL, we produced apoB polypeptides of various lengths and followed their ability to assemble VLDL. Small amounts of apoB-40 were associated with VLDL, but most of the nascent chains associated with VLDL ranged from apoB-48 to apoB-100. Thus, efficient VLDL assembly requires apoB chains of at least apoB-48 size. Nascent polypeptides as small as apoB-20 were associated with particles in the HDL density range. Thus, the structural requirements of apoB to form HDL-like first-step particles differ from those to form second-step VLDL. Analysis of proteins in the d < 1.006 g/ml fraction after ultracentrifugation of the luminal content of the cells identified five chaperone proteins: binding protein, protein disulfide isomerase, calcium-binding protein 2, calreticulin, and glucose regulatory protein 94. Thus, intracellular VLDL is associated with a network of chaperones involved in protein folding. Pulse-chase and subcellular fractionation studies showed that apoB-48 VLDL did not accumulate in the rough endoplasmic reticulum. This finding indicates either that the two steps of apoB lipoprotein assembly occur in different compartment or that the assembled VLDL is transferred rapidly out of the rough endoplasmic reticulum. PMID- 10744743 TI - Two forms of the apoptosis-linked protein ALG-2 with different Ca(2+) affinities and target recognition. AB - The apoptosis-linked gene ALG-2 encodes a Ca(2+)-binding protein of the penta EF hand family. To investigate the Ca(2+) binding properties of the recombinant ALG 2 protein, we have cloned ALG-2 cDNA from mouse liver mRNA. Sequence analysis showed that two types of clones were present. One (named ALG-2,5) corresponds to the published ALG-2 sequence (Vito, P., Lacana, E., and D'Adamio, L. (1996) Science 271, 521-525); the second (named ALG-2,1) is 6 nucleotides shorter, and the corresponding protein lacks the amino acid residues Gly(121) and Phe(122). Both transcripts are present in mouse tissues in the same 2:1 molar ratio. The ALG-2,5 and ALG-2,1 recombinant proteins are fully soluble in the metal-free form but can be precipitated from bacterial lysates by Ca(2+). In the presence of Tween the Ca(2+) binding profiles display two high affinity sites with [Ca(2+)](0.5) values of 1.2 and 3.1 microM for ALG-2,5 and ALG-2,1, respectively, plus one low affinity site. Using the yeast two-hybrid system we demonstrate that both proteins have a strong tendency to form homo- and heterodimers. In contrast to ALG-2, 5, the ALG-2,1 isoform does not interact with the target protein AIP-1, earlier described to play a role in apoptosis (Vito, P., Pellegrini, L., Guiet, C., and D'Adamio, L. (1999) J. Biol. Chem. 274, 1533-1540). We propose that the minor sequence difference between ALG-2,5 and ALG-2,1 affects the Ca(2+) binding properties and function of the proteins. PMID- 10744744 TI - Disruption of hsp90 function results in degradation of the death domain kinase, receptor-interacting protein (RIP), and blockage of tumor necrosis factor-induced nuclear factor-kappaB activation. AB - The death domain kinase, receptor interacting protein (RIP), is one of the major components of the tumor necrosis factor receptor 1 (TNFR1) complex and plays an essential role in tumor necrosis factor (TNF)-mediated nuclear factor kappaB (NF kappaB) activation. The activation of NF-kappaB protects cells against TNF induced apoptosis. Heat-shock proteins (Hsps) are chaperone molecules that confer protein stability and help to restore protein native folding following heat shock and other stresses. The most abundant Hsp, Hsp90, is also involved in regulating the stability and function of a number of cell-signaling molecules. Here we report that RIP is a novel Hsp90-associated kinase and that disruption of Hsp90 function by its specific inhibitor, geldanamycin (GA), selectively causes RIP degradation and the subsequent inhibition of TNF-mediated IkappaB kinase and NF kappaB activation. MG-132, a specific proteasome inhibitor, abrogated GA-induced degradation of RIP but failed to restore the activation of IkappaB kinase by TNF, perhaps because, in the presence of GA and MG-132, RIP accumulated in a detergent insoluble subcellular fraction. Most importantly, the degradation of RIP sensitizes cells to TNF-induced apoptosis. These data indicate that Hsp90 plays an important role in TNF-mediated NF-kappaB activation by modulating the stability and solubility of RIP. Thus, inhibition of NF-kappaB activation by GA may be a critical component of the anti-tumor activity of this drug. PMID- 10744745 TI - Platelet-derived growth factor-induced H(2)O(2) production requires the activation of phosphatidylinositol 3-kinase. AB - Autophosphorylation of the platelet-derived growth factor (PDGF) receptor triggers intracellular signaling cascades as a result of recruitment of Src homology 2 domain-containing enzymes, including phosphatidylinositol 3-kinase (PI3K), the GTPase-activating protein of Ras (GAP), the protein-tyrosine phosphatase SHP-2, and phospholipase C-gamma1 (PLC-gamma1), to specific phosphotyrosine residues. The roles of these various effectors in PDGF-induced generation of H(2)O(2) have now been investigated in HepG2 cells expressing various PDGF receptor mutants. These mutants included a kinase-deficient receptor and receptors in which various combinations of the tyrosine residues required for the binding of PI3K (Tyr(740) and Tyr(751)), GAP (Tyr(771)), SHP-2 (Tyr(1009)), or PLC-gamma1 (Tyr(1021)) were mutated to Phe. PDGF failed to increase H(2)O(2) production in cells expressing either the kinase-deficient mutant or a receptor in which the two Tyr residues required for the binding of PI3K were replaced by Phe. In contrast, PDGF-induced H(2)O(2) production in cells expressing a receptor in which the binding sites for GAP, SHP-2, and PLC-gamma1 were all mutated was slightly greater than that in cells expressing the wild-type receptor. Only the PI3K binding site was alone sufficient for PDGF-induced H(2)O(2) production. The effect of PDGF on H(2)O(2) generation was blocked by the PI3K inhibitors LY294002 and wortmannin or by overexpression of a dominant negative mutant of Rac1. These results suggest that a product of PI3K is required for PDGF-induced production of H(2)O(2) in nonphagocytic cells, and that Rac1 mediates signaling between the PI3K product and the putative NADPH oxidase. PMID- 10744746 TI - The RIPE3b1 activator of the insulin gene is composed of a protein(s) of approximately 43 kDa, whose DNA binding activity is inhibited by protein phosphatase treatment. AB - Glucose-stimulated and pancreatic islet beta cell-specific expression of the insulin gene is mediated in part by the C1 DNA-element binding complex, termed RIPE3b1. In this report, we define the molecular weight range of the protein(s) that compose this beta cell-enriched activator complex and show that protein phosphatase treatment inhibits RIPE3b1 DNA binding activity. Fractionation of beta cell nuclear extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that RIPE3b1 binding was mediated by a protein(s) within the 37-49-kDa ranges. Direct analysis of the proteins within the RIPE3b1 complex by ultraviolet light cross-linking analysis identified three binding species of approximately 51, 45, and 38 kDa. Incubating beta cell nuclear extracts with either calf alkaline phosphatase or a rat brain phosphatase preparation dramatically reduced RIPE3b1 DNA complex formation. Phosphatase inhibition of RIPE3b1 binding was prevented by sodium pyrophosphate, a general phosphatase inhibitor. We discuss how changes in the phosphorylation status of the RIPE3b1 activator may influence its DNA binding activity. PMID- 10744747 TI - Cardiac-specific overexpression of a superinhibitory pentameric phospholamban mutant enhances inhibition of cardiac function in vivo. AB - Phospholamban is a regulator of the Ca(2+) affinity of the cardiac sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) and of cardiac contractility. In vitro expression studies have shown that several mutant phospholamban monomers are superinhibitory, suggesting that monomeric phospholamban is the active species. However, a phospholamban Asn(27) --> Ala (N27A) mutant, which maintained a normal pentamer to monomer ratio, was shown to act as a superinhibitor of SERCA2a Ca(2+) affinity. To determine whether the pentameric N27A mutant is superinhibitory in vivo, transgenic mice with cardiac-specific overexpression of mutant phospholamban were generated. Quantitative immunoblotting revealed a 61 +/- 6% increase in total phospholamban in mutant hearts, with 90% of the overexpressed protein being pentameric. The EC(50) value for Ca(2+) dependence of Ca(2+) uptake was 0.69 +/- 0.07 microM in mutant hearts, compared with 0.29 +/- 0.02 microM in wild-type hearts or 0. 43 +/- 0.03 microM in hearts overexpressing wild-type PLB by 2-fold. Myocytes from phospholamban N27A mutant hearts also exhibited more depressed contractile parameters than wild-type phospholamban overexpressing cells. The shortening fraction was 52%, rates of shortening and relengthening were 46% and 38% respectively, and time for 80% decay of the Ca(2+) signal was 146%, compared with wild-types (100%). Langendorff-perfused mutant hearts also demonstrated depressed contractile parameters. Furthermore, in vivo echocardiography showed a depression in the ratio of early to late diastolic transmitral velocity and a 79% prolongation of the isovolumic relaxation time. Isoproterenol stimulation did not fully relieve the depressed contractile parameters at the cellular, organ, and intact animal levels. Thus, pentameric phospholamban N27A mutant can act as a superinhibitor of the affinity of SERCA2a for Ca(2+) and of cardiac contractility in vivo. PMID- 10744748 TI - Gab-1-mediated IGF-1 signaling in IRS-1-deficient 3T3 fibroblasts. AB - The insulin receptor substrate (IRS) family of proteins mediate a variety of intracellular signaling events by serving as signaling platforms downstream of several receptor tyrosine kinases including the insulin and insulin-like growth factor-1 (IGF-1) receptors. Recently, several new members of this family have been identified including IRS-3, IRS-4, and growth factor receptor-binding protein 2-associated binder-1 (Gab-1). 3T3 cell lines derived from IRS-1 deficient embryos exhibit a 70-80% reduction in IGF-1-stimulated S-phase entry and a parallel decrease in the induction of the immediate-early genes c-fos and egr-1 but unaltered activation of the mitogen-activated protein kinases extracellular signal-regulated kinase-1 and extracellular signal-regulated kinase 2. Reconstitution of IRS-1 expression in IRS-1-deficient fibroblasts by retroviral mediated gene transduction is capable of restoring these defects. Overexpression of Gab-1 in IRS-1-deficient fibroblasts also results in the restoration of egr-1 induction to levels similar to those achieved by IRS-1 reconstitution and markedly increases IGF-1-stimulated S-phase progression. Gab-1 is capable of regulating these biological end points despite the absence of IGF-1 stimulated tyrosine phosphorylation. These data provide evidence that Gab-1 may serve as a unique signaling intermediate in insulin/IGF-1 signaling for induction of early gene expression and stimulation of mitogenesis without direct tyrosine phosphorylation. PMID- 10744749 TI - The human T cell leukemia/lymphotropic virus type 1 Tax protein represses MyoD dependent transcription by inhibiting MyoD-binding to the KIX domain of p300. A potential mechanism for Tax-mediated repression of the transcriptional activity of basic helix-loop-helix factors. AB - The human T cell leukemia/lymphotropic virus type 1 (HTLV-1) Tax protein strongly activates viral and cellular gene transcription. It mainly functions by interacting with cellular transcription factors and the KIX domain of the p300/CBP coactivators. Tax can also repress the transcription of cellular genes through the basic helix-loop-helix (bHLH) protein family. To investigate the molecular mechanisms of this Tax-mediated inhibition, we analyzed its effect on the transcriptional activity of the myogenic MyoD protein, which was used as a paradigm of bHLH factors. In this study, we show that overexpression of the p300 coactivator in transient transfection assays was sufficient to rescue MyoD repression by Tax. Furthermore, an N-terminal domain of p300 (amino acids 379 654) containing the region of KIX serving as the Tax binding site was found, when overexpressed, to potentiate Tax-mediated transactivation of HTLV-1 proviral as well as MyoD-dependent transcription, and to antagonize the inhibition by Tax of the transcriptional activity of MyoD. These results revealing the presence of an N-terminal MyoD binding site were confirmed by in vitro protein-protein interaction assays that demonstrate that MyoD binds to the KIX domain of p300 and that Tax competes with MyoD binding in a nonreciprocal manner. These observations provide evidence that Tax binding to the KIX domain of p300 prevents bHLH proteins from contacting this N-terminal domain of the coactivator, thus resulting in their transcriptional repression. As bHLH proteins are implicated in many developmental fate decisions, especially during thymopoiesis, Tax-mediated inhibition of their transcriptional activity may contribute to the induction of HTLV-1-linked leukemogenesis. PMID- 10744750 TI - Signal transducer and activator of transcription 3 is required for glycoprotein 130-mediated induction of vascular endothelial growth factor in cardiac myocytes. AB - Activation of glycoprotein (gp) 130 transduces hypertrophic and cytoprotective signals in cardiac myocytes. In the present study, we have demonstrated that signals through gp130 increase the expression of vascular endothelial growth factor (VEGF) in cardiac myocytes via the signal transducer and activator of transcription (STAT) 3 pathway. After activation of gp130 with leukemia inhibitory factor (LIF), expression of VEGF mRNA rapidly increased with a peak at 3 h in cultured cardiac myocytes. Cardiotrophin-1 also enhanced VEGF mRNA expression in a dose-dependent manner. VEGF protein production and secretion to the medium were also enhanced by LIF and cardiotrophin-1 but not by interleukin 6. Adenovirus transfer of the dominant-negative form of STAT3 to cultured cardiac myocytes inhibited induction of VEGF expression induced by LIF, but neither PD98059 nor wortmannin was affected. In murine hearts, intravenous administration of LIF augmented expression of VEGF mRNA; however, the hearts of transgenic mice overexpressing dominant-negative STAT3 showed reduced expression of VEGF mRNA that was not induced after LIF stimulation. These data provide the first evidence that a STAT family protein functions as a regulator of angiogenic growth factors and suggest that gp130/STAT signaling in cardiac myocytes can control vessel growth during cardiac remodeling. PMID- 10744751 TI - Regulation of mouse p45 NF-E2 transcription by an erythroid-specific GATA dependent intronic alternative promoter. AB - The erythroid-enriched transcription factor NF-E2 is composed of two subunits, p45 and p18, the former of which is mainly expressed in the hematopoietic system. We have isolated and characterized the mouse p45 NF-E2 gene; we show here that, similar to the human gene, the mouse gene has two alternative promoters, which are differentially active during development and in different hematopoietic cells. Transcripts from the distal promoter are present in both erythroid and myeloid cells; however, transcripts from an alternative proximal 1b promoter, lying in the first intron, are abundant in erythroid cells, but barely detectable in myeloid cells. During development, both transcripts are detectable in yolk sac, fetal liver, and bone marrow. Transfection experiments show that proximal promoter 1b has a strong activity in erythroid cells, which is completely dependent on the integrity of a palindromic GATA-1 binding site. In contrast, the distal promoter 1a is not active in this assay. When the promoter 1b is placed 3' to the promoter 1a and reporter gene, in an arrangement that resembles the natural one, it acts as an enhancer to stimulate the activity of the upstream promoter la. PMID- 10744752 TI - The Arabidopsis thaliana PIN1At gene encodes a single-domain phosphorylation dependent peptidyl prolyl cis/trans isomerase. AB - A homologue of the human site-specific prolyl cis/trans isomerase PIN1 was identified in Arabidopsis thaliana. The PIN1At gene encodes a protein of 119 amino acids that is 53% identical with the catalytic domain of the human PIN1 parvulin. Steady-state PIN1At mRNA is found in all plant tissues tested. We show by two-dimensional NMR spectroscopy that the PIN1At is a prolyl cis/trans isomerase with specificity for phosphoserine-proline bonds. PIN1At is the first example of an eukaryotic parvulin without N- or C-terminal extensions. The N terminal WW domain of 40 amino acids, typical of all the phosphorylation dependent eukaryotic parvulins, is absent. However, triple-resonance NMR experiments showed that PIN1At contained a hydrophobic helix similar to the alpha1 helix observed in PIN1 that could mediate the protein-protein interactions. PMID- 10744753 TI - Extracellular DsbA-insensitive folding of Escherichia coli heat-stable enterotoxin STa in vitro. AB - To study the folding of human Escherichia coli heat-stable enterotoxin STh, we used the major protein subunit of CS31A fimbriae (ClpG) as a marker of STh secretion and a provider of a signal peptide. We established that STh genetically fused to the N or C terminus of ClpG was able to mobilize ClpG to the culture supernatant while still retaining full enterotoxicity. These features indicate that the STh activity was not altered by the chimeric structure and suggest that spatial conformation of STh in the fusion is close to that of the native toxin, thus permitting recognition and activation of the intestinal STh receptor in vivo. In contrast to other studies, we showed that disulfide bond formation did not occur in the periplasm through the DsbA pathway and that there was no correlation between DsbA and secretion, folding, or activity. This discrepancy was not attributable to the chimeric nature of STh since there was no effect of dsbA or dsbB mutations on secretion and activity of recombinant STh from which ClpG had been deleted. Periplasmic and lysate fractions of dsbA(+) and dsbA(-) cells did not have any STh activity. In addition, the STh chimera was exclusively found in an inactive reduced form intracellularly and in an active oxidized form extracellularly, irrespective of the dsbA background. Subsequently, a time course experiment in regard to the secretion of STh from both dsbA(+) and dsbA(-) cells indicated that the enterotoxin activity (proper folding) in the extracellular milieu increased with time. Overall, these findings provide evidence that STa toxins can be cell-released in an unfolded state before being completely disulfide-bonded outside the cell. PMID- 10744754 TI - Functional characterization of five eIF4E isoforms in Caenorhabditis elegans. AB - Recognition of the 5'-cap structure of mRNA by eIF4E is a critical step in the recruitment of most mRNAs to the ribosome. In Caenorhabditis elegans, approximately 70% of mRNAs contain an unusual 2,2,7-trimethylguanosine cap structure as a result of trans-splicing onto the 5' end of the pre-mRNA. The characterization of three eIF4E isoforms in C. elegans (IFE-1, IFE-2, and IFE-3) was reported previously. The present study describes two more eIF4E isoforms expressed in C. elegans, IFE-4 and IFE-5. We analyzed the requirement of each isoform for viability by RNA interference. IFE-3, the most closely related to mammalian eIF4E-1, binds only 7-methylguanosine caps and is essential for viability. In contrast, three closely related isoforms (IFE-1, IFE-2, and IFE-5) bind 2,2, 7-trimethylguanosine caps and are partially redundant, but at least one functional isoform is required for viability. IFE-4, which binds only 7 methylguanosine caps, is most closely related to an unusual eIF4E isoform found in plants (nCBP) and mammals (4E-HP) and is not essential for viability in any combination of IFE knockout. ife-2, ife-3, ife-4, and ife-5 mRNAs are themselves trans-spliced to SL1 spliced leaders. ife-1 mRNA is trans-spliced to an SL2 leader, indicating that its gene resides in a downstream position of an operon. PMID- 10744755 TI - The role of the regulatory protein of glucokinase in the glucose sensory mechanism of the hepatocyte. AB - Glucokinase has a very high flux control coefficient (greater than unity) on glycogen synthesis from glucose in hepatocytes (Agius et al., J. Biol. Chem. 271, 30479-30486, 1996). Hepatic glucokinase is inhibited by a 68-kDa glucokinase regulatory protein (GKRP) that is expressed in molar excess. To establish the relative control exerted by glucokinase and GKRP, we applied metabolic control analysis to determine the flux control coefficient of GKRP on glucose metabolism in hepatocytes. Adenovirus-mediated overexpression of GKRP (by up to 2-fold above endogenous levels) increased glucokinase binding and inhibited glucose phosphorylation, glycolysis, and glycogen synthesis over a wide range of concentrations of glucose and sorbitol. It decreased the affinity of glucokinase translocation for glucose and increased the control coefficient of glucokinase on glycogen synthesis. GKRP had a negative control coefficient of glycogen synthesis that is slightly greater than unity (-1.2) and a control coefficient on glycolysis of -0.5. The control coefficient of GKRP on glycogen synthesis decreased with increasing glucokinase overexpression (4-fold) at elevated glucose concentration (35 mM), which favors dissociation of glucokinase from GKRP, but not at 7.5 mM glucose. Under the latter conditions, glucokinase and GKRP have large and inverse control coefficients on glycogen synthesis, suggesting that a large component of the positive control coefficient of glucokinase is counterbalanced by the negative coefficient of GKRP. It is concluded that glucokinase and GKRP exert reciprocal control; therefore, mutations in GKRP affecting the expression or function of the protein may impact the phenotype even in the heterozygote state, similar to glucokinase mutations in maturity onset diabetes of the young type 2. Our results show that the mechanism comprising glucokinase and GKRP confers a markedly extended responsiveness and sensitivity to changes in glucose concentration on the hepatocyte. PMID- 10744756 TI - Cooperative signaling between alpha(6)beta(4) integrin and ErbB-2 receptor is required to promote phosphatidylinositol 3-kinase-dependent invasion. AB - We previously demonstrated that beta(4) integrin subunit overexpression increases in vitro invasiveness of NIH3T3 cells that have been transformed by ErbB-2 oncogene. We used this model to identify domains within the large beta(4) cytoplasmic domain that are involved in the interaction of alpha(6)beta(4) with ErbB-2, invasion, and phosphatidylinositol 3-kinase (PI3K) activation. For this purpose, we expressed deletion mutants of beta(4) that lacked either all or portions of the beta(4) cytoplasmic domain in NIH3T3/ErbB-2 cells. We also used an ecto-domain mutant in which most of the extracellular domain of beta(4) was replaced with a c-Myc tag. These transfectants were examined for their ability to invade Matrigel and their ability to activate PI3K, as well as for the ability of alpha(6)beta(4) to co-immunoprecipitate with ErbB-2. The results obtained revealed that a region of the beta(4) cytoplasmic domain between amino acids 854 and 1183 is critical for the ability of alpha(6)beta(4) integrin to increase invasion. Interestingly, the extracellular domain of beta(4) is not necessary for alpha(6)beta(4) to stimulate invasion. The association of alpha(6)beta(4) with ErbB-2 is dependent upon the beta(4) cytoplasmic domain and can occur in the absence of alpha(6)beta(4) heterodimerization. Finally, we observed strong activation of PI3K with beta(4) wild type and with those beta(4) deletion mutants that were able to stimulate invasion upon the expression in NIH3T3/ErbB-2 cells. In conclusion, our results establish that there is cooperation between alpha(6)beta(4) and ErbB-2 in promoting PI3K-dependent invasion and implicate a specific region of the beta(4) cytoplasmic domain (amino acids 854-1183) in this event. PMID- 10744757 TI - RNase P ribozymes selected in vitro to cleave a viral mRNA effectively inhibit its expression in cell culture. AB - An in vitro selection procedure was used to select RNase P ribozyme variants that efficiently cleaved the sequence of the mRNA encoding thymidine kinase of herpes simplex virus 1. Of the 45 selected variants sequenced, 25 ribozymes carried a common mutation at nucleotides 224 and 225 of RNase P catalytic RNA from Escherichia coli (G(224)G(225) --> AA). These selected ribozymes exhibited at least 10 times higher cleavage efficiency (k(cat)/K(m)) than that derived from the wild type ribozyme. Our results suggest that the mutated A(224)A(225) are in close proximity to the substrate and enhance substrate binding of the ribozyme. When these ribozyme variants were expressed in herpes simplex virus 1-infected cells, the levels of thymidine kinase mRNA and protein were reduced by 95-99%. Our study provides the first direct evidence that RNase P ribozyme variants isolated by the selection procedure can be used for the construction of gene targeting ribozymes that are highly effective in tissue culture. These results demonstrate the potential for using RNase P ribozymes as gene-targeting agents against any mRNA sequences, and using the selection procedure as a general approach for the engineering of RNase P ribozymes. PMID- 10744758 TI - Purification and characterization of a Neu5Acalpha2-6Galbeta1-4Glc/GlcNAc specific lectin from the fruiting body of the polypore mushroom Polyporus squamosus. AB - A lectin has been purified from the carpophores of the mushroom Polyporus squamosus by a combination of affinity chromatography on beta-D-galactosyl Synsorb and ion-exchange chromatography on DEAE-Sephacel. Gel filtration chromatography, SDS-polyacrylamide gel electrophoresis, and N-terminal amino acid sequencing indicated that the native lectin, designated P. squamosus agglutinin, is composed of two identical 28-kDa subunits associated by noncovalent bonds. P. squamosus agglutinin agglutinated human A, B, and O and rabbit red blood cells but precipitated only with human alpha(2)-macroglobulin, of many glycoproteins and polysaccharides tested. The detailed carbohydrate binding properties of the purified lectin were elucidated using three different approaches, i.e. precipitation inhibition assay (in solution binding assay), fluorescence quenching studies, and glycolipid binding by lectin staining on high-performance thin layer chromatography (solid-phase binding assay). Based on the results obtained by these assays, we conclude that although the P. squamosus lectin binds beta-D-galactosides, it has an extended carbohydrate-combining site that exhibits highest specificity and affinity toward nonreducing terminal Neu5Acalpha2, 6Galbeta1,4Glc/GlcNAc (6'-sialylated type II chain) of N-glycans (2000-fold stronger than toward galactose). The strict specificity of the lectin for alpha2,6-linked sialic acid renders this lectin a valuable tool for glycobiological studies in biomedical and cancer research. PMID- 10744759 TI - Additional N-glycosylation and its impact on the folding of intestinal lactase phlorizin hydrolase. AB - Lactase-phlorizin hydrolase (LPH) is a membrane bound intestinal hydrolase, with an extracellular domain comprising 4 homologous regions. LPH is synthesized as a large polypeptide precursor, pro-LPH, that undergoes several intra- and extracellular proteolytic steps to generate the final brush-border membrane form LPHbeta(final). Pro-LPH is associated through homologous domain IV with the membrane through a transmembrane domain. A truncation of 236 amino acids at the COOH terminus of domain IV (denoted LAC236) does not significantly influence the transport competence of the generated mutant LPH1646MACT (Panzer, P., Preuss, U., Joberty, G., and Naim, H. Y. (1998) J. Biol. Chem. 273, 13861-13869), strongly suggesting that LAC236 is an autonomously folded domain that links the ectodomain with the transmembrane region. Here, we examine this hypothesis by engineering several N-linked glycosylation sites into LAC236. Transient expression of the cDNA constructs in COS-1 cells confirm glycosylation of the introduced sites. The N-glycosyl pro-LPH mutants are transported to the Golgi apparatus at substantially reduced rates as compared with wild-type pro-LPH. Alterations in LAC236 appear to sterically hinder the generation of stable dimeric trypsin resistant pro-LPH forms. Individual expression of chimeras containing LAC236, the transmembrane domain and cytoplasmic tail of pro-LPH and GFP as a reporter gene (denoted LAC236-GFP) lends strong support to this view: while LAC236-GFP is capable of forming dimers per se, its N-glycosyl variants are not. The data strongly suggest that the LAC236 is implicated in the dimerization process of pro LPH, most likely by nucleating the association of the ectodomains of the enzyme. PMID- 10744760 TI - Identification of the DNA binding specificity and potential target genes for the farnesoid X-activated receptor. AB - The farnesoid X-activated receptor (FXR; NR1H4) is a member of the nuclear hormone receptor superfamily and functions as a heterodimer with the 9-cis retinoic acid receptor (RXR). In order to determine the optimal DNA binding sequence for the FXR/RXR heterodimer, we have utilized the selected and amplified binding sequence imprinting technique. This technique identified a number of related sequences that interacted with FXR/RXR in vitro. The consensus sequence contained an inverted repeat of the sequence AGGTCA with a 1-base pair spacing (IR-1). This sequence was shown to be a high affinity binding site for FXR/RXR in vitro and to confer ligand-dependent transcriptional activation by FXR/RXR to a heterologous promoter. Electrophoretic mobility shift assays and transient transfection assays were used to investigate the importance of the core half-site sequences, spacing nucleotide, flanking sequences, and orientation and spacing of the core half-sites on DNA binding and ligand-dependent transcriptional activation by FXR/RXR. These studies demonstrated that the FXR/RXR heterodimer binds to the consensus IR-1 sequence with the highest affinity, although FXR/RXR can bind to and activate through a variety of elements including IR-1 elements with changes in the core half-site sequence, spacing nucleotide, and flanking nucleotides. In addition, FXR/RXR can bind to and transactivate through direct repeats. Three genes were identified that contain IR-1 sequences in their proximal promoters. These elements were shown to bind FXR/RXR in vitro and to confer FXR/RXR-dependent transcriptional activation to a heterologous promoter in response to a bile acid or synthetic retinoid. The endogenous mRNA levels of one of these genes, phospholipid transfer protein, were shown to be induced by FXR and FXR ligands. The identification of the IR-1 and related elements as high affinity binding sites and functional response elements for FXR/RXR and the identification of a target gene for FXR/RXR should assist in the identification of additional genes regulated by FXR/RXR. PMID- 10744761 TI - Characterization of a human angiotensinogen cleaved in its reactive center loop by a proteolytic activity from Chinese hamster ovary cells. AB - Angiotensinogen, the renin (E.C. 3.4.23.15) substrate, belongs to the serpins superfamily and has been classified as a noninhibitory serpin. Using mass spectroscopy, angiotensinogen purified from Chinese hamster ovary cell supernatant shows a broad spectrum. The absence of protease inhibitors throughout the purification leads to an angiotensinogen cleaved within the reactive center loop. This cleavage does not affect the Ang I generation because kinetic parameters are similar to the values of the full-length angiotensinogen. Although cleavage is complete, the cleaved angiotensinogen migrates after deglycosylation on SDS-polyacrylamide gel electrophoresis as a doublet differing by 4 kDa. To test whether the circulating angiotensinogen is cleaved in the reactive center loop, it was purified from a pool of human plasma and was shown to be uncleaved. Its migration was obviously slower than of cleaved angiotensinogen but also consisted of two bands pointing to a so far unexplained residual heterogeneity. We then compared the heat-induced polymerization of full-length- and reactive center loop-cleaved angiotensinogens. Both monomers were able to aggregate, revealing a particular behavior of angiotensinogen distinct from that of reactive center loop-cleaved serpins. Lacking the three-dimensional structure of angiotensinogen, we propose and discuss a structural model of the serpin fold within the renin substrate. PMID- 10744762 TI - Impaired proteasome function rescues thermosensitivity of yeast cells lacking the coatomer subunit epsilon-COP. AB - Formation of COPI-coated transport vesicles requires a cytosolic protein complex consisting of seven subunits: alpha-, beta-, beta'-, gamma-, delta-, epsilon- and zeta-COP, collectively designated coatomer. The yeast Saccharomyces cerevisiae gene encoding the epsilon-COP subunit is known as SEC28/ANU2. anu2 null mutant cells (anu2Delta) are temperature-sensitive, and alpha-COP is rapidly degraded in these cells when they are shifted to the restrictive temperature. We isolated extragenic suppressors that rescue the temperature-sensitive growth defect of anu2Delta cells. Genetic analysis revealed that one of the suppressors is allelic to PRE8 (PRS4), which encodes a 20 S proteasome subunit. In the presence of a proteasome inhibitor, MG132, anu2Delta cells did not cease growth even at the restrictive temperature. Furthermore, MG132 inhibited the rapid decrease of alpha COP levels in anu2Delta cells shifted to the restrictive temperature. However, secretion of certain proteins by these cells was impaired even in the presence of MG132. In conclusion, impairment of proteasome-dependent proteolysis rescued some, but not all, temperature-sensitive defects of anu2Delta cells. These results are discussed in terms of evidence that epsilon-COP plays a critical role in maintaining the structural integrity of alpha-COP. PMID- 10744763 TI - Vascular endothelial growth factor (VEGF)-driven actin-based motility is mediated by VEGFR2 and requires concerted activation of stress-activated protein kinase 2 (SAPK2/p38) and geldanamycin-sensitive phosphorylation of focal adhesion kinase. AB - In endothelial cells, vascular endothelial growth factor (VEGF) induces an accumulation of stress fibers associated with new actin polymerization and rapid formation of focal adhesions at the ventral surface of the cells. This cytoskeletal reorganization results in an intense motogenic activity. Using porcine endothelial cells expressing one or the other type of the VEGF receptors, VEGFR1 or VEGFR2, or human umbilical vein endothelial cells pretreated with a VEGFR2 neutralizing antibody, we show that VEGFR2 is responsible for VEGF-induced activation of the stress-activated protein kinase-2/p38 (SAPK2/p38), phosphorylation of focal adhesion kinase (FAK), and enhanced migratory activity. Activation of SAPK2/p38 triggered actin polymerization whereas FAK, which was phosphorylated independently of SAPK2/p38, initiated assembly of focal adhesions. Both processes contributed to the formation of stress fibers. Geldanamycin, an inhibitor of HSP90 blocked tyrosine phosphorylation of FAK, assembly of focal adhesions, actin reorganization, and cell migration, all of which were reversed by overexpressing HSP90. We conclude that VEGFR2 mediates the physiological effect of VEGF on cell migration and that two independent pathways downstream of VEGFR2 regulate actin-based motility. One pathway involves SAPK2/p38 and leads to enhanced actin polymerization activity. The other involves HSP90 as a permissive signal transduction factor implicated in FAK phosphorylation and assembly of focal adhesions. PMID- 10744764 TI - Stimulation of integrin-mediated cell contractility by fibronectin polymerization. AB - Ligation of integrins with extracellular matrix molecules induces the clustering of actin and actin-binding proteins to focal adhesions, which serves to mechanically couple the matrix with the cytoskeleton. During wound healing and development, matrix deposition and remodeling may impart additional tensile forces that modulate integrin-mediated cell functions, including cell migration and proliferation. We have utilized the ability of cells to contract floating collagen gels to determine the effect of fibronectin polymerization on mechanical tension generation by cells. Our data indicate that fibronectin polymerization promotes cell spreading in collagen gels and stimulates cell contractility by a Rho-dependent mechanism. Fibronectin-stimulated contractility was dependent on integrin ligation; however, integrin ligation by fibronectin fragments was not sufficient to induce either tension generation or cell spreading. Furthermore, treatment of cells with polyvalent RGD peptides or pre-polymerized fibronectin did not stimulate cell contractility. Fibronectin-induced contractility was blocked by agents that inhibit fibronectin polymerization, suggesting that the process of fibronectin polymerization is critical in triggering cytoskeletal tension generation. These data indicate that Rho-mediated cell contractility is regulated by the process of fibronectin polymerization and suggest a novel mechanism by which extracellular matrix fibronectin regulates cytoskeletal organization and cell function. PMID- 10744765 TI - Isolation and functional characterization of the PfNT1 nucleoside transporter gene from Plasmodium falciparum. AB - Plasmodium falciparum, the causative agent of the most lethal form of human malaria, is incapable of de novo purine synthesis, and thus, purine acquisition from the host is an indispensable nutritional requirement. This purine salvage process is initiated by the transport of preformed purines into the parasite. We have identified a gene encoding a nucleoside transporter from P. falciparum, PfNT1, and analyzed its function and expression during intraerythrocytic parasite development. PfNT1 predicts a polypeptide of 422 amino acids with 11 transmembrane domains that is homologous to other members of the equilibrative nucleoside transporter family. Southern analysis and BLAST searching of The Institute for Genomic Research (TIGR) malaria data base indicate that PfNT1 is a single copy gene located on chromosome 14. Northern analysis of RNA from intraerythrocytic stages of the parasite demonstrates that PfNT1 is expressed throughout the asexual life cycle but is significantly elevated during the early trophozoite stage. Functional expression of PfNT1 in Xenopus laevis oocytes significantly increases their ability to take up naturally occurring D-adenosine (K(m) = 13.2 microM) and D-inosine (K(m) = 253 microM). Significantly, PfNT1, unlike the mammalian nucleoside transporters, also has the capacity to transport the stereoisomer L-adenosine (K(m) > 500 microM). Inhibition studies with a battery of purine and pyrimidine nucleosides and bases as well as their analogs indicate that PfNT1 exhibits a broad substrate specificity for purine and pyrimidine nucleosides. These data provide compelling evidence that PfNT1 encodes a functional purine/pyrimidine nucleoside transporter whose expression is strongly developmentally regulated in the asexual stages of the P. falciparum life cycle. Moreover, the unusual ability to transport L-adenosine and the vital contribution of purine transport to parasite survival makes PfNT1 an attractive target for therapeutic evaluation. PMID- 10744766 TI - Poly(ADP-ribose) polymerase is a B-MYB coactivator. AB - B-MYB is implicated in cell growth control, differentiation, and cancer and belongs to the MYB family of nuclear transcription factors. Evidence exists that cellular proteins bind directly to B-MYB, and it has been hypothesized that B-MYB transcriptional activity may be modulated by specific cofactors. In an attempt to isolate proteins that interact with the B-MYB DNA-binding domain, a modular domain that has the potential to mediate protein-protein interaction, we performed pull-down experiments with a glutathione S-transferase-B-MYB protein and mammalian protein extracts. We isolated a 110-kDa protein associated endogenously with B-MYB in the nuclei of HL60 cells. Microsequence analysis and immunoprecipitation experiments determined that the bound protein was poly(ADP ribose) polymerase (PARP). Transient transfection assays showed that PARP enhanced B-MYB transactivation and that PARP enzymatic activity is not required for B-MYB-dependent transactivation. These results suggest that PARP, as a transcriptional cofactor of a potentially oncogenic protein, may play a role in growth control and cancer. PMID- 10744767 TI - Dual role of pseudosubstrate in the coordinated regulation of protein kinase C by phosphorylation and diacylglycerol. AB - The activity of protein kinase C is reversibly regulated by an autoinhibitory pseudosubstrate, which blocks the active site of the enzyme in the absence of activators. However, before it can be allosterically regulated, protein kinase C must first be processed by three ordered phosphorylations, the first of which is modification of the activation loop catalyzed by the phosphoinositide-dependent kinase-1 (PDK-1). Here we use limited proteolysis to show that 1) newly synthesized protein kinase C adopts a conformation in which its pseudosubstrate sequence is removed from the active site, and 2) this exposure is essential to allow PDK-1 to phosphorylate the enzyme. Precursor (unphosphorylated) protein kinase C betaII obtained by 1) in vitro transcription and translation, 2) expression of a phosphorylation-deficient mutant (T500V), or 3) in vivo labeling with a pulse of [(35)S]cysteine/methionine is cleaved at the amino-terminal pseudosubstrate by the endoproteinase Arg-C. In marked contrast to mature (phosphorylated) enzyme, proteolysis occurs in the absence of lipid activators, revealing that precursor protein kinase C has its pseudosubstrate sequence removed constitutively. Additionally, we show that PDK-1 is unable to phosphorylate protein kinase C when the active site is sterically blocked by a peptide substrate. Neither can mature enzyme be dephosphorylated when the active site is blocked by binding either the pseudosubstrate sequence or a heterologous substrate. Thus, the accessibility of the activation loop to both phosphorylation and dephosphorylation requires an exposed pseudosubstrate. In summary, newly synthesized protein kinase C adopts a conformation in which its pseudosubstrate sequence is removed from the active site, rendering the activation loop accessible to phosphorylation by PDK-1. Phosphorylation serves as a conformational switch to position the pseudosubstrate so that it blocks the active site, a conformation that is maintained until stimulus-dependent membrane binding releases it, thus activating the enzyme. PMID- 10744768 TI - Recombinant carboxyltransferase responsive to redox of pea plastidic acetyl-CoA carboxylase. AB - Acetyl-CoA carboxylase regulates the rate of fatty acid synthesis. This enzyme in plants is localized in plastids and is believed to be composed of biotin carboxyl carrier protein, biotin carboxylase, and carboxyltransferase made up of alpha and beta polypeptides, although the enzyme has not been purified yet. Accumulated evidence shows that pea plastidic acetyl-CoA carboxylase is activated by light and the activation is caused by light-dependent reduction of carboxyltransferase, but not of biotin carboxylase, via a redox cascade. To understand the reductive activation of carboxyltransferase at the molecular level here, we obtained the active enzyme composed of decahistidine-tagged (His tag) alpha and beta polypeptides through the expression of the pea plastidic carboxyltransferase gene in Escherichia coli. Gel filtration showed that the molecular size of the recombinant carboxyltransferase is in agreement with that of partially purified carboxyltransferase from pea chloroplasts. The catalytic activity of the recombinant enzyme was similar to that of native carboxyltransferase. These results indicate that the molecular structure and conformation of recombinant carboxyltransferase resemble those of its native counterpart and that native carboxyltransferase is indeed composed of alpha and beta polypeptides. This recombinant enzyme was activated by dithiothreitol, a known reductant of S-S bonds, with a profile similar to that of its native counterpart. The recombinant enzyme was activated by reduced thioredoxin-f, a signal transducer of redox potential in chloroplasts under irradiation. Thus, this enzyme was redox regulated, like that of the native carboxyltransferase. PMID- 10744769 TI - Desferrioxamine-mediated iron uptake in Saccharomyces cerevisiae. Evidence for two pathways of iron uptake. AB - In the yeast Saccharomyces cerevisiae, uptake of iron is largely regulated by the transcription factor Aft1. cDNA microarrays were used to identify new iron and AFT1-regulated genes. Four homologous genes regulated as part of the AFT1-regulon (ARN1-4) were predicted to encode members of a subfamily of the major facilitator superfamily of transporters. These genes were predicted to encode proteins with 14 membrane spanning domains and were from 26 to 53% identical at the amino acid level. ARN3 is identical to SIT1, which is reported to encode a ferrioxamine B permease. Deletion of ARN3 did not prevent yeast from using ferrioxamine B as an iron source; however, deletion of ARN3 and FET3, a component of the high affinity ferrous iron transport system, did prevent uptake of ferrioxamine-bound iron and growth on ferrioxamine as an iron source. The siderophore-mediated transport system and the high affinity ferrous iron transport system were localized to separate cellular compartments. Epitope-tagged Arn3p was expressed in intracellular vesicles that co-sediment with the endosomal protein Pep12. In contrast, Fet3p was expressed on the plasma membrane and was digested by extracellular proteases. These data indicate that S. cerevisiae has two pathways for ferrrioxamine-mediated iron uptake, one occurring at the plasma membrane and the other occurring in an intracellular compartment. PMID- 10744770 TI - Intracellular mechanisms regulating apoB-containing lipoprotein assembly and secretion in primary hamster hepatocytes. AB - We studied the biogenesis of apolipoprotein B (apoB) in primary hepatocytes isolated from hamster liver, an animal model with striking resemblance to humans in lipoprotein metabolism. Hamster hepatocytes were found to assemble and secrete apoB-containing lipoproteins at a density of VLDL. Intracellular mechanisms of apoB biogenesis were investigated in both intact and permeabilized hamster hepatocytes. Translocational status of hamster apoB-100 was examined using trypsin protection assays in permeabilized cells as well as isolated microsomes which revealed that 27-42% of newly synthesized apoB was trypsin accessible as opposed to a control protein, transferrin, which was found to be essentially insensitive to exogenous trypsin. Subcellular fractionation of membrane and lumenal apoB pools indicated, however, that only a minor fraction of hamster apoB was associated with the microsomal membrane. Approximately 40% of newly synthesized apoB was found to be degraded post-translationally in a process sensitive to MG132. Immunoblotting analysis of apoB immunoprecipitates revealed ubiquitination of hamster apoB suggesting the involvement of the proteasome in its intracellular turnover. In addition to MG132, o-phenanthroline, a metalloprotease inhibitor, was also effective in stabilizing hamster apoB. Experiments in permeabilized hamster hepatocytes further confirmed post translational instability of hamster apoB which was degraded over a 3-h chase generating proteolytic fragments including 167, 70, 57, and 46 kDa intermediates. Of these only the 70 kDa fragment was ALLN sensitive. Oleate treatment of hamster hepatocytes provided protection against intracellular apoB degradation, but did not stimulate its extracellular secretion. ApoB was assembled in the microsomal lumen into lipoprotein particles with densities of LDL and VLDL which were subsequently secreted as VLDL with a minor fraction forming HDL-like particles. In summary, hamster hepatocytes appear to efficiently assemble and secrete apoB containing VLDL, although a significant pool of newly synthesized apoB is retained intracellularly and becomes sensitive to proteasome-mediated degradation as well as other proteases in the secretory pathway, generating specific degradative intermediates. PMID- 10744771 TI - Peroxisome proliferator-activated receptor alpha (PPARalpha) and agonist inhibit cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription. AB - Fibrates are widely used hypolipidemic drugs that regulate the expression of many genes involved in lipid metabolism by activating the peroxisome proliferator activated receptor alpha (PPARalpha). The objective of this study was to investigate the mechanism of action of peroxisome proliferators and PPARalpha on the transcription of cholesterol 7alpha-hydroxylase, the rate-limiting enzyme in the conversion of cholesterol to bile acids in the liver. When cotransfected with the expression vectors for PPARalpha and RXRalpha, Wy14,643 reduced human and rat cholesterol 7alpha-hydroxylase gene (CYP7A1)/luciferase reporter activities by 88% and 43%, respectively, in HepG2 cells, but not in CV-1 or CHO cells. We have mapped the peroxisome proliferator response element (PPRE) to a conserved sequence containing the canonical AGGTCA direct repeats separated by one nucleotide (DR1). This DR1 sequence was mapped previously as a binding site for the hepatocyte nuclear factor 4 (HNF-4) which stimulates CYP7A1 transcription. Electrophoretic mobility shift assay (EMSA) showed no direct binding of in vitro synthesized PPARalpha/RXRalpha heterodimer to the DR1 sequence. PPARalpha and Wy14,643 did not affect HNF-4 binding to the DR1. However, Wy14,643 and PPARalpha/RXRalpha significantly reduced HNF-4 expression in HepG2 cells. These results suggest that PPARalpha and agonist repress cholesterol 7alpha-hydroxylase activity by reducing the availability of HNF-4 for binding to the DR-1 sequence and therefore attenuates the transactivation of CYP7A1 by HNF-4. PMID- 10744772 TI - Plasma and vessel wall lipoprotein lipase have different roles in atherosclerosis. AB - Lipoprotein lipase (LPL) is a key enzyme in lipoprotein metabolism, and has been hypothesized to exert either pro- or anti-atherogenic effects, depending on its localization. Decreased plasma LPL activity is associated with the high triglyceride (TG);-low HDL phenotype that is often observed in patients with premature vascular disease. In contrast, in the vessel wall, decreased LPL may be associated with less lipoprotein retention due to many potential mechanisms and, therefore, decreased foam cell formation. To directly assess this hypothesis, we have distinguished between the effects of variations in plasma and/or vessel wall LPL on atherosclerosis susceptibility in apoE-deficient mice. Reduced LPL in both plasma and vessel wall (LPL(+/-)E(-/-)) was associated with increased TG and increased total cholesterol (TC) compared with LPL(+/+)E(-/-) sibs. However despite their dyslipidemia, LPL(+/-)E(-/-) mice had significantly reduced lesion areas compared to the LPL(+/+)E(-/-) mice. Thus, decreased vessel wall LPL was associated with decreased lesion formation even in the presence of reduced plasma LPL activity. In contrast, transgenic mice with increased plasma LPL but with no increase in LPL expression in macrophages, and thus the vessel wall, had decreased TG and TC and significantly decreased lesion areas compared with LPL(+/+)E(-/-) mice. This demonstrates that increased plasma LPL activity alone, in the absence of an increase in vessel wall LPL, is associated with reduced susceptibility to atherosclerosis. Taken together, these results provide in vivo evidence that the contribution of LPL to atherogenesis is significantly influenced by the balance between vessel wall protein (pro-atherogenic) and plasma activity (anti-atherogenic). PMID- 10744773 TI - Microsomal triglyceride transfer protein expression during mouse development. AB - Feto-maternal transfer of lipophilic nutrients is an important factor in the normal development of the fetus and may be mediated by lipoproteins as carriers of these nutrients. Two proteins that may be important in this process are apolipoprotein B (apoB, the major structural protein of secreted lipoproteins) and microsomal triglyceride transfer protein (MTP) whose normal activity is required for the secretion of apoB-containing lipoproteins. Although no abnormalities of conception and embryonic lethality are known in humans who inherit genetic deficiencies of either of these proteins, homozygous mice bearing knockouts of either apoB or MTP show early embryonic lethality. To characterize the ontogeny of MTP expression during embryonic mouse development, we have used in situ hybridization to characterize the pattern of expression. By using microwave heating of tissue sections to optimize hybridization, we show that there is robust MTP expression in the yolk sac tissues followed by expression in the primordial liver cell nests as early as day 9 post-coitum (E9.5). Intestinal expression is detected around E12.5 and attains full adult expression patterns by E14.5. No expression in any other tissues was observed, including developing heart, kidney, placenta, and maternal decidua. Thus the pattern of MTP expression is compatible with a role in the transfer of lipophilic nutrients from the yolk sac, prior to hepatic development and to the liver, once the circulatory system has been established. PMID- 10744774 TI - Long-chain acyl-CoA esters and acyl-CoA binding protein are present in the nucleus of rat liver cells. AB - A detailed analysis of the subcellular distribution of acyl-CoA esters in rat liver revealed that significant amounts of long-chain acyl-CoA esters are present in highly purified nuclei. No contamination of microsomal or mitochondrial marker enzymes was detectable in the nuclear fraction. C16:1 and C18:3-CoA esters were the most abundant species, and thus, the composition of acyl-CoA esters in the nuclear fraction deviates notably from the overall composition of acyl-CoA esters in the cell. After intravenous administration of the non-beta-oxidizable [(14)C]tetradecylthioacetic acid (TTA), the TTA-CoA ester could be recovered from the nuclear fraction. Acyl-CoA esters bind with high affinity to the ubiquitously expressed acyl-CoA binding protein (ACBP), and several lines of evidence suggest that ACBP functions as a pool former and transporter of acyl-CoA esters in the cytoplasm. By using immunohistochemistry, immunofluorescence microscopy, and immunoelectron microscopy we demonstrate that ACBP localizes to the nucleus as well as the cytoplasm of rat liver cell and rat hepatoma cells, suggesting that ACBP may also be involved in regulation of acyl-CoA-dependent processes in the nucleus. PMID- 10744775 TI - Phosphatidylcholine fluidity and structure affect lecithin:cholesterol acyltransferase activity. AB - The purpose of this study was to test the hypothesis that lipid fluidity regulates lecithin:cholesterol acyltransferase (LCAT) activity. Phosphatidylcholine (PC) species were synthesized that varied in fluidity by changing the number, type (cis vs. trans), or position of the double bonds in 18 or 20 carbon sn-2 fatty acyl chains and recombined with [(3)H]cholesterol and apolipoprotein A-I to form recombinant high density lipoprotein (rHDL) substrate particles. The activity of purified human plasma LCAT decreased with PC sn-2 fatty acyl chains containing trans versus cis double bonds and as double bonds were moved towards the methyl terminus of the sn-2 fatty acyl chain. The decrease in LCAT activity was significantly correlated with a decrease in rHDL fluidity (measured by diphenylhexatriene fluorescence polarization) for PC species containing 18 carbon (r(2) = 0.61, n = 18) and 20 carbon (r(2) = 0.93, n = 5) sn 2 fatty acyl chains. rHDL were also made containing 10% of the 18 carbon sn-2 fatty acyl chain PC species and 90% of an inert PC ether matrix (sn-1 18:1, sn-2 16:0 PC ether) to normalize rHDL fluidity. Even though fluidity was similar among the PC ether-containing rHDL, the order of PC reactivity with LCAT was significantly correlated (r(2) = 0.71) with that of 100% PC rHDL containing the same 18 carbon sn-2 fatty acyl chain species, suggesting that PC structure in the active site of LCAT determines reactivity in the absence of measurable differences in bilayer fluidity. We conclude that PC fluidity and structure are major regulators of LCAT activity when fatty acyl chain length is constant. PMID- 10744776 TI - Uptake and activation of eicosapentaenoic acid are related to accumulation of triacylglycerol in Ramos cells dying from apoptosis. AB - The present study investigates the mechanism behind induction of cell death by eicosapentaenoic acid (EPA) in leukemia cells. The PUFA-sensitive cell lines Raji and Ramos, which die by necrosis and apoptosis upon incubation with EPA respectively, had 2- to 3-fold higher uptake rate of EPA than the PUFA-resistant U-698 cell line. Furthermore, Ramos cells contained more lipid bodies and 3-fold more triacylglycerol than U-698 cells after 24 h incubation with 60 microm EPA. The mechanism behind the increased rate of EPA uptake in the PUFA-sensitive cell lines was examined by comparing the expression of 6 different fatty acid binding proteins (FABPs) and 3 acyl-CoA synthetases (ACSs) in U-698 and Ramos cells. Moreover, enzymatic activity of ACS and acyl-CoA:1,2-diacylglycerol acyltransferase (ADGAT) was investigated. The protein expression level of CD36 and p-FABPpm, the mRNA level of FABP, liver-FABP, heart-FABP, intestinal-FABP, ACS1, ACS2, and enzymatic ADGAT activity were similar in the two cell lines. However, an mRNA signal observed with a probe for ACS3 was 1.7 times higher in Ramos than in U-698 cells, and lysate from Ramos cells had a higher capacity to activate EPA to EPA-CoA than U-698 cell lysate. In conclusion, the present findings indicate that cellular uptake, activation and incorporation of EPA into lipids may be related to induction of cell death in leukemia cell lines. PMID- 10744777 TI - Structural characterization of oxidized phospholipid products derived from arachidonate-containing plasmenyl glycerophosphocholine. AB - Plasmenyl phospholipids are a structurally unique class of lipids that contain a vinyl ether substituent at the sn-1 position of the glycerol backbone, imparting unique susceptibility to oxidative reactions that may take place at the cell membrane lipid bilayer. Several studies have supported the hypothesis that plasmalogens may be antioxidant molecules that protect cells from oxidative stress. Because the molecular mechanism for the antioxidant properties of plasmenyl phospholipids is not fully understood, the oxidation of arachidonate containing plasmalogen-glycerophosphocholine (GPC) was studied using electrospray tandem mass spectrometry after exposure to the free radical initiator 2, 2' azobis(2-amidinopropane)hydrochloride (AAPH). Various oxidized GPC products involving the sn-1 position alone (1-formyl-2-arachidonyl lipids and lysophospholipid), oxidation products involving the sn-2 position alone (chain shortened omega-aldehyde radyl substituents at sn-2) as well as products oxidized both at the sn-1 and sn-2 positions were observed and structurally identified. The results of these experiments suggest that oxidation of plasmenyl phospholipids esterified with polyunsaturated fatty acid groups at sn-2 likely undergo unique and specific free radical oxidation at the 1'-alkenyl position as well as oxidation of the double bond closest to the ester moiety at sn-2. PMID- 10744778 TI - A locus conferring resistance to diet-induced hypercholesterolemia and atherosclerosis on mouse chromosome 2. AB - Dietary cholesterol is known to raise total and low density lipoprotein cholesterol concentrations in humans and experimental animals, but the response among individuals varies greatly. Here we describe a mouse strain, C57BL/6ByJ (B6By), that is resistant to diet-induced hypercholesterolemia, in contrast to the phenotype seen in other common strains of mice including the closely related C57BL/6J (B6J) strain. Compared to B6J, B6By mice exhibit somewhat lower basal cholesterol levels on a chow diet, and show a relatively modest increase in absolute levels of total and LDL/VLDL cholesterol in response to an atherogenic diet containing 15% fat, 1.25% cholesterol, and 0.5% cholate. Correspondingly, B6By mice are also resistant to diet-induced aortic lesions, with less than 15% as many lesions as B6J. Food intake and cholesterol absorption are similar between B6By and B6J mice. To investigate the gene(s) underlying the resistant B6By phenotype, we performed genetic crosses with the unrelated mouse strain, A/J. A genome-wide scan revealed a locus, designated Diet1, on chromosome 2 near marker D2Mit117 showing highly significant linkage (lod = 9.6) between B6By alleles and hypo-response to diet. Examination of known genes in this region suggested that this locus represents a novel gene affecting plasma lipids and atherogenesis in response to diet. PMID- 10744779 TI - Functional significance of Sp1, Sp2, and Sp3 transcription factors in regulation of the murine CTP:phosphocholine cytidylyltransferase alpha promoter. AB - The transcription factor Sp1 has been implicated in regulation of the expression of the murine CTP:phosphocholine cytidylyltransferase alpha (CTalpha) gene, Ctpct (M. Bakovic, K. Waite, W. Tang, I. Tabas, and D. E. Vance. 1999. Biochim. Biophys. Acta. 1438: 147;-165). We have utilized transient transfections, mutation analysis, electromobility gel-shifts, and immunoblot analysis to test the hypothesis that expression of the CTalpha gene is controlled in part by the binding of three trans-acting nuclear factors, Sp1, Sp2, and Sp3. Sp1 and Sp3 activate CTalpha gene transcription through sequence specific binding within three promoter domains. In Sp1-mediated transcription, Sp3 acts as an activator in a dose-dependent manner and vice versa. Sp2 represses Sp1- and Sp3-driven transcription in Drosophila SL2 cells, but stimulates transcription in C3H10T1/2 mammalian cells. Our results suggest that the predominant action of Sp proteins is a direct function of local organization of three cis-acting elements in the regions A (-31/-9), B (-88/-50), and C (-148/-128). The ability of distal C ( 148/-128) and proximal A (-31/-9) regions to activate or repress transcription depends upon the cellular background. The multiple binding elements at position B (-88/-50) confer a positive regulation independent of the cell context. However, the effectiveness of Sp proteins at this site is strongly governed by neighboring sites A and C. The results suggest that the level of expression of the CTalpha gene will depend on the cell type, the availability of Sp proteins, and the structure and organization of three cis-acting elements. PMID- 10744780 TI - Relationship between carbohydrate-induced hypertriglyceridemia and fatty acid synthesis in lean and obese subjects. AB - We previously reported that a eucaloric, low fat, liquid formula diet enriched in simple carbohydrate markedly increased the synthesis of fatty acids in lean volunteers. To examine the diet sensitivity of obese subjects, 7 obese and 12 lean volunteers were given two eucaloric low fat solid food diets enriched in simple sugars for 2 weeks each in a random-order, cross-over design (10% fat, 75% carbohydrate vs. 30% fat, 55% carbohydrate, ratio of sugar to starch 60:40). The fatty acid compositions of both diets were matched to the composition of each subject's adipose tissue and fatty acid synthesis measured by the method of linoleate dilution in plasma VLDL triglyceride. In all subjects, the maximum % de novo synthesized fatty acids in VLDL triglyceride 3;-9 h after the last meal was higher on the 10% versus the 30% fat diet. There was no significant difference between the dietary effects on lean (43+/-13 vs. 12+/-13%) and obese (37+/-15 vs. 6+/-6%) subjects, despite 2-fold elevated levels of insulin and reduced glucagon levels in the obese. Similar results were obtained for de novo palmitate synthesis in VLDL triglyceride measured by mass isotopomer distribution analysis after infusion of [(13)C]acetate. On the 10% fat diet, plasma triglycerides (fasting and 24 h) were increased and correlated with fatty acid synthesis. Triglycerides were higher when fatty acid synthesis was constantly elevated rather than having diurnal variation.Thus, eucaloric, solid food diets which are very low in fat and high in simple sugars markedly stimulate fatty acid synthesis from carbohydrate, and plasma triglycerides increase in proportion to the amount of fatty acid synthesis. However, this dietary effect is not related to body mass index, insulin, or glucagon levels. PMID- 10744781 TI - Effect of increasing lipid loads on the ability of the endoplasmic reticulum to transport lipid to the Golgi. AB - We have previously shown (Mansbach, C. M. and P. J. Nevin, 1998. J. Lipid Res. 39: 963;-968) that after the development of a mass steady state with respect to triacylglycerol absorption in rats, the introduction of radiolabeled trioleoylglycerol, while maintaining the input rate of trioleoylglycerol constant at 135 micromol/h, was followed by a slow (60 min) achievement of a radiolabel steady state in the intestinal endoplasmic reticulum (ER). We hypothesized that this was due to the large input load and that the time to steady state would be shorter at lower lipid loads. Rats were infused intraduodenally with 22.5, 45, 90, or 135 micromol trioleoylglycerol/h for 6 h to obtain a mass steady state in the intestine. [(3)H]trioleoylglycerol was added to the infusate and the ER and Golgi were isolated from the proximal intestine after 5;-60 min of radiolabel infusion. The time required to reach a radiolabel steady state in the ER lengthened from 10 min at the 22.5 micromol/h infusion rate to 60 min at the 135 micromol/h rate. Similar data were obtained for the Golgi. Incubation of the ER with lipase reduced the ER;-triacylglycerol amount by 43% and increased its specific activity by 73%. The amount of [(3)H]TG-dpm in the ER was not reduced unless taurocholate, 10 mm, and colipase were added. We conclude that as the rate of triacylglycerol infusion is increased, TG movement from the ER to the Golgi progressively lengthens until finally all the triacylglycerol infused cannot be transported. A portion of this triacylglycerol is disposed on the cytoplasmic face of the ER and thus able to be attacked by lipase whereas another fraction is sequestered in the ER lumen and immune to lipase attack unless the ER membrane is solubilized. PMID- 10744782 TI - Altered immune responses in apolipoprotein E-deficient mice. AB - Apolipoprotein E (apoE) is a 34 kDa glycosylated protein with multiple biological properties. In addition to its role in cholesterol transport, apoE has in vitro immunomodulatory properties. Recent data suggest that these immunomodulatory effects of apoE may be biologically relevant, and apoE-deficient mice have altered immune responses after bacterial inoculation and increased susceptibility to endotoxemia induced by lipopolysaccharide (LPS). To better understand the mechanism by which apoE-modulates immune responses, we tested the role of human apoE isoforms in assays of human T cell proliferation, and analyzed the immune responses of apoE-deficient mice. Both the E3 and E4 isoforms of apoE induced similar suppression of human lymphocyte function in assays of T cell proliferation, including mitogenic responses to phytohaemagglutin (PHA), stimulation of the T cell receptor with alphaCD3, and antigen-specific response to tetanus toxoid. ApoE-deficient mice showed no quantitative differences in thymic, splenic, or bone marrow lymphocyte populations, nor were there in vitro abnormalities in splenocyte proliferation after stimulation with alphaCD3 to suggest an inherent T cell defect in apoE-deficient mice. ApoE deficient animals, however, had significantly higher levels of antigen-specific IgM after immunization with tetanus toxoid, and impaired delayed type hypersensitivity responses as compared to control C57-BL/6 mice. These results support a growing body of evidence demonstrating an interplay between lipid metabolism and immune responses, and suggest that apoE plays a biologically relevant role in regulating humoral and cell-mediated immunity. PMID- 10744783 TI - Induction of aggregation and fusion of cholesterol-containing membrane vesicles by an anti-cholesterol monoclonal antibody. AB - A monoclonal IgM antibody that reacts with cholesterol was able to aggregate small and large unilamellar lipid vesicles. Vesicles aggregated by the antibody could be dispersed by trypsin digestion. Inclusion of unsaturated phosphatidylethanolamine in the vesicle formulation lowered the relative amount of cholesterol necessary for aggregation, and prevented disaggregation by trypsin treatment. Fluorimetric assays indicated that membrane mixing occurred in aggregates resistant to trypsinization, but the vesicles did not mix or leak their aqueous contents. Analysis of the kinetics of lipid-mixing showed an increase in the aggregation and fusion rate constants with increasing antibody concentrations, indicating that the antibody reaction promotes both processes. An apparent inactivation process whose rate increased with antibody dose has been considered. We conclude that the simultaneous binding of antibodies to more than one vesicle at densities that allow the contact of membrane surfaces, induces first aggregation followed by hemifusion, and with excess of antibody also results in inactivation of the latter process. PMID- 10744784 TI - Phytanoyl-CoA hydroxylase: recognition of 3-methyl-branched acyl-coAs and requirement for GTP or ATP and Mg(2+) in addition to its known hydroxylation cofactors. AB - Phytanoyl-CoA hydroxylase is a peroxisomal alpha-oxidation enzyme that catalyzes the 2-hydroxylation of 3-methyl-branched acyl-CoAs. A polyhistidine-tagged human phytanoyl-CoA hydroxylase was expressed in E. coli and subsequently purified as an active protein. The recombinant enzyme required GTP or ATP and Mg(2+), in addition to its known cofactors Fe(2+), 2-oxoglutarate, and ascorbate. The enzyme was active towards phytanoyl-CoA and 3-methylhexadecanoyl-CoA, but not towards 3 methylhexadecanoic acid. Racemic, R- and S-3-methylhexadecanoyl-CoA were equally well hydroxylated. Hydroxylation of R- and S-3-methylhexadecanoyl-CoA yielded the (2S, 3R) and (2R,3S) isomers of 2-hydroxy-3-methylhexadecanoyl-CoA, respectively. Human phytanoyl-CoA hydroxylase did not show any activity towards 2-methyl- and 4 methyl-branched acyl-CoAs or towards long and very long straight chain acyl-CoAs, excluding a possible role for the enzyme in the formation of 2-hydroxylated and odd-numbered straight chain fatty acids, which are abundantly present in brain. In conclusion, we report the unexpected requirement for ATP or GTP and Mg(2+) of phytanoyl-CoA hydroxylase in addition to the known hydroxylation cofactors. Due to the fact that straight chain fatty acyl-CoAs are not a substrate for phytanoyl CoA hydroxylase, 2-hydroxylation of fatty acids in brain can be allocated to a different enzyme/pathway. PMID- 10744785 TI - Cholesterol deficit but not accumulation of aberrant sterols is the major cause of the teratogenic activity in the Smith-Lemli-Opitz syndrome animal model. AB - Low cholesterol and high 7-dehydrocholesterol (7DHC) levels are associated with a blockade of Delta7-reductase in the Smith-Lemli-Opitz syndrome (SLOS) and in the animals treated with the inhibitor AY9944. The impact of the cholesterol deficit and of the accumulation of 7DHC on the embryo were investigated in AY9944-treated pregnant rats receiving an enriched cholesterol or 7DHC diet. Sterol profiling was performed under the various nutritional conditions. AY9944 caused a severe decrease in the maternal and embryo cholesterol. The deficit in the embryo was sustained by the embryonic uptake of the inhibitor. A cholesterol-rich diet was efficient in restoring the maternal and embryonic cholesterol and phenotype but a 7DHC-rich diet did not modify the sterol status compared with dams treated with only AY9944. The offspring phenotype remained deleterious whether or not the dams received 7DHC-rich diet. Over 80% of the 7DHC was absorbed, as was cholesterol, which was not quantitatively influenced by AY9944. When cholesterol and 7DHC were simultaneously administered, a competition for intestinal absorption enhanced the lowering cholesterol effect of AY9944. Whether or not the dams received a 7DHC dietary supplement, the offspring's phenotype became normal when the diet was supplemented with cholesterol. Under conditions in which the ratio of cholesterol/7DHC is substantially varied, the normal development of embryos can be achieved as long as the cholesterol is sufficient. The phenotype is reversed in vivo by cholesterol which contrasts with the irreversible effects manifested in vitro by oxidized 7DHC by-products. PMID- 10744787 TI - Base- and acid-catalyzed interconversions of O-acyl- and N-acyl-ethanolamines: a cautionary note for lipid analyses. AB - The isolation and quantification of ethanolamine containing lipids from animal tissues may expose neutral lipid extracts to acidic or basic conditions during chromatographic separations or derivatization chemistry. While investigating the acid- and base-catalyzed production of anandamide in chromatographic fractions of rat brain extracts not containing anandamide, we observed that O,N-acyl migrations are facile. O,N-acyl migrations are well documented in synthetic organic chemistry literature, but are not well described or recognized with regard to methods in lipid isolation or lipid enzyme studies. We report here the synthesis and characterization of O- and N-acyl (palmitoyl- or arachidonoyl )ethanolamines. Their rearrangements in base and acid are quantified by liquid chromatography;-electrospray ionization mass spectrometry. The rearrangements proceed through a cyclic intermediate that is also formed during chemical reactions commonly used for derivatization of acylethanolamines for gas chromatography-mass spectrometry. The isolation and characterization of N- or O acylethanolamines and their enzymatic formation requires awareness and consideration of the proclivity of these compounds to chemically rearrange. PMID- 10744786 TI - Liver and intestinal fatty acid-binding proteins obtain fatty acids from phospholipid membranes by different mechanisms. AB - Intestinal enterocytes contain high concentrations of two cytosolic fatty acid binding proteins (FABP), liver FABP (L-FABP) and intestinal FABP (I-FABP), which are hypothesized to play a role in cellular fatty acid trafficking. The mechanism(s) by which fatty acids move from membranes to each of these proteins is not known. Here we demonstrate that fluorescent anthroyloxy fatty acid analogues (AOFA) are transferred from phospholipid vesicles to L-FABP versus I FABP by different mechanisms. For L-FABP a diffusion-mediated transfer process is demonstrated. The AOFA transfer rate from phosphatidylcholine-containing vesicles (POPC) to L-FABP is similar to that observed with another diffusional process, namely inter-membrane AOFA transfer. Furthermore, the AOFA transfer rate was modulated by buffer ionic strength and AOFA solubility, while the transfer rate remained relatively unchanged by the presence of anionic phospholipids in vesicles. In contrast, the data for I-FABP suggest that a transient collisional interaction of I-FABP with the phospholipid membrane occurs during AOFA extraction from the vesicles by the protein. In particular, the presence of the anionic phospholipid cardiolipin in donor vesicles increased the rate of AOFA transfer to I-FABP by 15-fold compared with transfer to POPC vesicles. The effects of ionic strength on transfer suggest that the interaction of I-FABP with cardiolipin-containing vesicles is likely to contain an electrostatic component. Finally, based on the regulation of AOFA transfer to I-FABP compared with transfer from I-FABP, it is hypothesized that apo- and holo-I-FABPs adopt conformations which may differentially promote I-FABP-membrane interactions. In summary, the results suggest that I-FABP, but not L-FABP, can directly extract fatty acids from membranes, supporting the concept that I-FABP may increase the cytosolic flux of fatty acids via intermembrane transfer. PMID- 10744788 TI - Specialty care review and reform. PMID- 10744790 TI - The medicine cabinet. PMID- 10744789 TI - Nutrition and cardiovascular disease: from research to practice. PMID- 10744791 TI - Molecular diagnosis and treatment of cardiovascular diseases. PMID- 10744792 TI - The distinct HERG missense mutation L564P causes long QT syndrome in one French Canadian family. AB - BACKGROUND: Long QT syndrome is a congenital abnormality of cardiac repolarization causing syncope and sudden death from ventricular tachyarrhythmias known as torsades de pointes. This hereditary cardiac disorder often shows an increase of the value of the QT interval corrected for heart rate over 0.45 s in a 12-lead electrocardiogram. OBJECTIVE: To find and identify pertinent mutations occurring in French Canadians by extracting genomic DNA from blood samples and performing a combination of polymerase chain reaction (PCR), single-strand conformational polymorphism and DNA sequencing. RESULTS: A novel mutation was identified in the S5 region of the HERG potassium channel. In codon 564 CTA, T was replaced by C, resulting in a leucine to proline substitution. Two family members had the mutation in two distinct generations. A new restriction site was created at this position and therefore enabled the development of a rapid diagnostic test using PCR. HERG wild type and mutant potassium channel mRNAs were then expressed in Xenopus laevis oocytes. CONCLUSION: This electrophysiological study suggests that coexpression of HERG wild type and mutant L564P results in a dominant negative effect of the mutation. PMID- 10744793 TI - Increased levels of basic fibroblast growth factor are found in the cross-clamped heart during cardiopulmonary bypass. AB - BACKGROUND: High concentrations of fibroblast growth factors (FGFs) are found in the heart. Even higher levels are measured during ischemia. Exogenous administration of FGF to ischemic myocardium promotes synthesis of collateral coronary circulation and induces local myocardial hypertrophy. The kinetics and the contribution of the heart and lungs to circulating basic FGF (bFGF) levels during cardiac surgery were characterized. PATIENTS AND METHODS: Plasma bFGF levels were measured in seven adults undergoing coronary artery bypass operations and 11 neonates undergoing congenital cardiac anomaly repair during cardiopulmonary bypass. RESULTS: In both the adult and the neonatal groups, bFGF plasma levels increased significantly immediately after removal of the aortic cross-clamp (adult group 15.43+/-6.3 aorta cross-clamped versus 29+/-4.1 after release, P=0.011; neonatal group 17.09+/-9.43 aorta cross-clamped versus 43.55+/ 14.25 after release, P=0.004) and declined thereafter. In the adult group, higher levels of bFGF were recorded in blood recovered from the coronary sinus than in the aortic root during aortic cross-clamping (63.14+/-14.42 versus 43.86+/-12.05, P=0.011), and in both, levels were significantly higher than the peripheral measurements. CONCLUSIONS: Plasma bFGF levels increase during cardiopulmonary bypass. The source of this elevation is the lungs and heart. PMID- 10744794 TI - Sex differences in cardiac rehabilitation. AB - OBJECTIVE: To determine and compare the baseline characteristics and outcomes between men and women in a Canadian cardiac rehabilitation program. DESIGN: Nonrandomized, retrospective, observational study with a before and after research design. SETTING: The Prevention and Rehabilitation Centre at the University of Ottawa Heart Institute, Ottawa, Ontario, a tertiary cardiac care centre. PATIENTS: Three hundred and eighty-seven patients, 82% male aged 59+/-10 years and 18% female aged 61+/-4 years, who were enrolled in the on-site cardiac rehabilitation program between November 1, 1995 and April 1, 1997. INTERVENTION: A three-month, multifactorial cardiac rehabilitation program that incorporates exercise training, risk factor modification, education and psychosocial support. MAIN RESULTS: Fewer than 20% of all rehabilitation participants (n=70; 18%) were women; most participants were under 65 years of age. More women than men had a primary diagnosis of myocardial infarction (42% versus 28%, respectively), whereas men were more likely than women to have had coronary artery bypass grafting (45% versus 23%, respectively). Men and women had similar mean baseline measures of body mass index, blood pressure and glucose levels, whereas women had significantly higher mean baseline measures of total cholesterol (5.6 mmol/L versus 5.0 mmol/L for men, P 0.001), low density lipoprotein (LDL) cholesterol (3.4 mmol/L versus 3.1 mmol/L, P=0. 012) and high density lipoprotein (HDL) cholesterol (1.2 mmol/L versus 1.0 mmol/L, P 0.001). Baseline LDL to HDL ratios were 3.3 for men and 3.0 for women (not significant), and total cholesterol to HDL ratios were 5.4 and 4.9 for men and women, respectively. Men had a higher exercise capacity than women coming into the program (metabolic equivalent [METs] 6.6 versus 4.9, respectively, P 0.001), had a higher baseline activity level (1114 kcal/week versus 617 kcal/week, P=0.001) and scored higher than women in all health-related quality of life scores. After the program, there were no significant sex differences in improvement in MET level, physical activity or risk factor profile. Although men exercised more than women (increase of 557 kcal/week versus 343 kcal/week, respectively), this was not statistically significant. In health-related quality of life scores, both men and women improved in all scores, although women reported less increase than men in their level of overall vitality (P=0.016). CONCLUSION: Women are the minority of cardiac rehabilitation patients, although they appear to benefit equally well from the program. PMID- 10744795 TI - Perioperative anticoagulation in patients with chronic atrial fibrillation who are undergoing elective surgery: results of a physician survey. AB - OBJECTIVE: To survey physicians' anticoagulation preferences in patients with chronic atrial fibrillation who are undergoing elective surgery. MATERIALS AND METHODS: A survey was performed that asked physicians to provide pre- and postoperative anticoagulation preferences for two clinical scenarios of patients with chronic atrial fibrillation (high stroke risk, low stroke risk) undergoing elective surgery. In addition to the interruption of warfarin therapy, perioperative anticoagulation options were as follows: a) in-hospital full dose intravenous heparin; b) outpatient full dose subcutaneous unfractionated heparin or low molecular weight heparin (LMWH); c) low dose unfractionated heparin or LMWH (postoperative only); d) nothing other than stopping warfarin preoperatively and restarting it postoperatively; or e) another anticoagulant strategy. RESULTS: In the high stroke risk scenario, the proportions of respondents preferring anticoagulation options a, b, d and e in the preoperative period were 24%, 20%, 54% and 2%, respectively; the proportions preferring options a, b, c, d and e in the postoperative period were 35%, 13%, 15%, 35% and 1%, respectively. In the low stroke risk scenario, the proportions of respondents preferring options a, b, d and e in the preoperative period were 7%, 10%, 80% and 3%, respectively; the proportions preferring options a, b, c, d and e in the postoperative period were 11%, 9%, 10%, 68% and 2%, respectively. CONCLUSIONS: In patients with chronic atrial fibrillation who underwent elective surgery, perioperative anticoagulant management preferences varied widely in patients at high risk for stroke, but were more uniform and less aggressive in patients at low risk for stroke. PMID- 10744796 TI - Prolonged distress and clinical deterioration before pericardial drainage in patients with cardiac tamponade. AB - OBJECTIVE: To determine whether patients with cardiac tamponade are subject to delays and clinical deterioration before undergoing echocardiography and pericardial drainage. DESIGN: Retrospective study. SETTING: The Montreal Heart Institute, Montreal, Quebec, a cardiology referral centre. PATIENTS: The charts of 50 patients who presented with tamponade were reviewed. Intervals between the appearance of symptoms, consultation, echocardiography and drainage were noted. The presence of clinical deterioration before drainage was evaluated. Causes for delays were investigated. RESULTS: Previous cardiac surgery (74%) was the most common etiology of tamponade. Symptoms were present 6.6+/-5.8 days before consultation. The delay between consultation and echocardiography was 1.2+/-2.0 days (range 0 to 12), and that between echocardiography and drainage was 0.8+/ 0.9 days (range 0 to four). Patients underwent drainage 1. 9+/-2.5 days (range 0 to 16) after the initial consultation. Deterioration of the clinical status was noted in 34% of patients before pericardial drainage. An error in the initial diagnosis was present in 36% of patients; the majority of these were incorrectly diagnosed with heart failure. Another 44% of patients had no mention of either a working diagnosis in the chart at admission or the desire to rule out tamponade on the request for echocardiography. CONCLUSION: The proper diagnosis does not appear to be initially considered in up to 80% of patients who present with cardiac tamponade. Clinical deterioration occurs in approximately a third of patients during the interval between consultation and pericardial drainage. PMID- 10744797 TI - Comparing angiographic coronary revascularization strategies: a 'natural' experiment. AB - OBJECTIVE: To study the impact of intracoronary stents on clinical restenosis in the 'real world'. DESIGN: Retrospective comparison of the rates of clinical restenosis between two cohorts exposed to different strategies for percutaneous transcatheter intervention. The endpoint was the first of death, myocardial infarction, coronary artery bypass grafting, repeat percutaneous transluminal coronary angioplasty (PTCA) or repeat coronary angiography within nine months. SETTING: Tertiary care cardiac referral centre serving a large, metropolitan population. PATIENTS: Patients undergoing angiographic revascularization from January 1 to February 28, 1996 (the 'restricted' group [R], n=147) were compared with a before and after cohort (the 'usual' group [U], n=232, divided into those who underwent revascularization between November 1 and November 30, 1995, and those who underwent revascularization between April 1 and May 31, 1996). INTERVENTIONS: The R group was revascularized during a period of economic constraint, which imposed a shortage on stent availability. The U cohort underwent revascularization before and after the shortage (an 'unrestricted' environment for stent usage). MAIN RESULTS: There was no difference in clinical restenosis rates between the R (34.7%) and U (37.9%) groups (P=0.524, OR R/U=0.915, 95% CI 0.694 to 1.206). Also, the rate of clinical restenosis was the same among patients who underwent PTCA without stent insertion (34.8%) and those who received a stent (39.4%) (P=0.368, OR=1.13, 95% CI 0.87 to 1.44). CONCLUSIONS: At the authors' institution, a restricted stenting policy did not result in a higher clinical restenosis rate than that of usual practice. PMID- 10744798 TI - Heart rate variability, late potentials and QT dispersion as markers of myocardial involvement in patients with Behcet's disease. AB - BACKGROUND: Myocardial involvement in Behcet's disease has been reported to be relatively rare. OBJECTIVE: To evaluate myocardial involvement noninvasively in patients with Behcet's disease by measuring signal-averaged electrocardiography (SAECG), QT dispersion and heart rate variability (HRV). PATIENTS AND METHODS: The study group comprised 28 eligible patients (16 male, mean age 37+/-13 years) of 33 patients with Behcet's disease, and 25 age- and sex-matched control subjects. RESULTS: The echocardiographic left ventricular measurements were within normal limits and similar in both groups except the E/A ratio, which was significantly lower in patients with the disease than in control patients. Minimal pericardial effusion was detected in four patients. Considering the SAECG recordings, values of root mean square voltage in the last 40 ms were 30+/-18 microV and 38+/-18 microV in patients with Behcet's disease and in the control group, respectively. The number of cases with a value less than 20 microV was seven (25%) and one (4%) in the same groups, respectively. Both QT dispersion and the corrected QT interval dispersion were significantly increased in patients with Behcet's disease compared with the control patients (50.2+/-16.6 versus 20.4+/-18.8, P<0.01). Although all HRV measures appeared to be decreased in the Behcet's group, only the standard deviation of all filtered RR intervals in the entire 24 h ECG recordings and the percentage of differences between adjacent filtered RR intervals that are greater than 50 ms for the whole analysis values differed significantly between the groups (P<0.05). No significant difference was observed in frequency domain parameters. In the Holter ECG recording, grade 2 or greater premature ventricular complexes were observed in seven patients from the Behcet's group (25%) but in only one subject from the control group (4%) (P<0.05). CONCLUSIONS: Patients with Behcet's disease appeared to have significantly increased QT dispersion, a left ventricular diastolic dysfunction pattern in echocardiography, a high incidence of positive late potentials and more complex ventricular arrhythmias, suggesting myocardial involvement and the existence of an arrhythmogenic substrate, whereas the HRV measures do not suggest a clear autonomic abnormality in Behcet's disease. PMID- 10744799 TI - Guidelines for pacemaker follow-up in Canada: a consensus statement of the Canadian Working Group on Cardiac Pacing. AB - A survey on Canadian pacing practices conducted in 1997 revealed a widespread desire for national guidelines on pacemaker follow-up. The present guidelines for pacemaker follow-up are a consensus statement of the Canadian Working Group on Cardiac Pacing. Direct patient follow-up rather than transtelephonic monitoring is desirable. Patients should be assessed at a minimum of within 72 h of implantation, at two to 12 weeks and at six months following implantation, and annually thereafter. More frequent assessments may be required for some patients. This depends on associated cardiovascular problems and specific devices. A typical follow-up visit should include a targeted cardiovascular assessment, interrogation of the pacing system, review of telemetered data, assessment of the underlying rhythm, assessment of pacing and sensing thresholds, and appropriate reprogramming of pacing parameters to optimize device function and longevity. PMID- 10744800 TI - Lignes directrices pour le suivi des cardiostimulateurs au canada : consensus du groupe de travail canadien sur la cardiostimulation AB - Un sondage sur les pratiques de cardiostimulation au Canada effectue en 1997 a revele un profond desir pour des lignes directrices nationales sur le suivi des cardiostimulateurs. Ces lignes directrices sur le suivi des cardiostimulateurs representent une declaration de consensus du Groupe de travail canadien sur la cardiostimulation. Le suivi des patients en personne plutot que par moyens transtelephoniques est preferable. Les patients devraient etre examines au minimum dans les 72 heures suivant l'implantation, 2 a 12 semaines et 6 mois apres l'implantation, et annuellement par la suite. Des examens plus frequents peuvent etre requis pour certains patients. Ceci dependra des problemes cardiovasculaires associes et des appareils en particulier. Une visite de suivi typique devrait comprendre un examen cardiovasculaire oriente, l'interrogation du systeme de cardiostimulation et une revue des donnees de telemetrie, un examen du rythme sous-jacent, un examen des seuils de stimulation et de detection et une reprogrammation adequate des parametres pour optimaliser le fonctionnement et la longevite de l'appareil. PMID- 10744801 TI - Reversible cardiomyopathy associated with Addison's disease. AB - Myocardial involvement in Addison's disease has been described previously. A 36 year-old man presented with cardiomyopathy and was found to have Addison's disease. The patient's myocardial function improved after steroid therapy for Addison's disease. This is the only case report of reversible cardiomyopathy associated with Addison's disease in an adult patient. PMID- 10744802 TI - A left atrial paraganglioma patient presenting with compressive dysphagia. AB - Paragangliomas are rare neoplasms of neural crest origin arising in the chromaffin (pheochromocytoma) and chemoreceptor (chemodectoma) tissues. Only a few cases of paragangliomas have been reported in the heart. Most of the cardiac paragangliomas are located in, or adjacent to, the left atrium. The biological activity indicative of catecholamine production has rarely been shown in cardiac paragangliomas. Patients with cardiac paragangliomas may present with hypertension, or with various obstructive or compressive symptoms, depending on the location of the tumour. A left atrial paraganglioma compressing the esophagus causing dysphagia is presented. A 37-year-old female patient underwent work-up for dysphagia to solids. Transesophageal echocardiography showed the presence of a large mass in the dilated left atrium. The tumour was removed completely intact and was a benign noncatecholamine-secreting paraganglioma. The patient's dysphagia was relieved. The clinical perspectives of cardiac paraganglioma and cardiac dysphagia are discussed. PMID- 10744803 TI - A fistula connecting the right coronary artery to the right atrium: a hitherto undescribed association. AB - A case of double right coronary artery accompanying a fistulous connection is presented. An additional right coronary artery is an extremely rare congenital abnormality. In this case, the second right coronary artery was draining into a cardiac chamber via a fistulous connection. This is the first case in the literature to present these abnormalities coexisting in the same patient. PMID- 10744805 TI - On the loss of colleagues PMID- 10744804 TI - Message from the president. PMID- 10744806 TI - Triple-tissue sampling at ERCP in malignant biliary obstruction. AB - BACKGROUND: Procurement of cytologic samples by brushing is common practice at endoscopic retrograde cholangiopancreatography (ERCP) but has low sensitivity for cancer detection. Limited data are available on other techniques, including endoluminal fine-needle aspiration and forceps biopsy. This series reviews the yield of these three stricture sampling methods. METHODS: In this prospective study, patients with biliary obstruction with a clinical suspicion of malignancy underwent triple-tissue sampling at one ERCP session. Final cancer diagnosis was based on all sampling methods plus surgery, autopsy, and clinical follow-up. Tissue specimens were reported as normal, atypia, or malignant. RESULTS: A total of 133 patients were evaluated: 104 had cancer and 29 had benign strictures. Tissue sampling sensitivity varied according to the type of cancer; the highest yield was seen in ampullary cancers (62% to 85%). The cumulative sensitivity of triple-tissue sampling in the cancer patients was as follows: sensitivity was 52% if atypia was considered benign and 77% if it was considered malignant. The addition of a second or third technique increased sensitivity rates in most instances. No serious complications occurred from the tissue sampling methods. CONCLUSIONS: Tissue sampling sensitivity varied according to the type of cancer. Combining a second or third method increased sensitivity; general use of at least two sampling methods is therefore recommended. PMID- 10744807 TI - Endoscopic transpapillary drainage of pancreatic abscess: technique and results. AB - BACKGROUND: Pancreatic abscess is one of the serious complications of acute pancreatitis. Traditionally, pancreatic abscess has been treated by operative drainage. Based on experience with endoscopic transpapillary drainage of pseudocysts, a similar technique was used in patients with pancreatic abscess. METHOD: Patients were evaluated by endoscopic retrograde cholangiopancreatography. In those with pancreatic abscess communicating with the main pancreatic duct, pancreatic sphincterotomy, saline irrigation of the abscess cavity, and catheter dilation followed by 10F pancreatic stent placement were done. Instillation of gentamicin and nasopancreatic catheter drainage were used in difficult cases. RESULTS: Of 22 patients with pancreatic abscess, 11 underwent endoscopic transpapillary drainage with technical success in 10 patients (90%); 8 patients (74%) had resolution of pancreatic abscess, clinically and radiographically. Intracavitary instillation of gentamicin and nasopancreatic catheter drainage were used in 2 patients. Two patients in whom endoscopic transpapillary drainage failed underwent operative drainage with a favorable outcome, and the one patient in whom endoscopic treatment was technically unsuccessful underwent successful percutaneous drainage. One patient had mild pancreatitis. CONCLUSION: Endoscopic transpapillary drainage is an effective nonoperative therapy for selected cases of pancreatic abscess and is associated with minimal morbidity and no mortality. PMID- 10744808 TI - Therapeutic ERCP in the management of pancreatitis in children. AB - BACKGROUND: The use of diagnostic and therapeutic endoscopic retrograde cholangiopancreatography (ERCP) is increasing in the management of pancreatobiliary diseases in children. METHODS: Over a 32-month period, we performed 34 ERCP procedures for the treatment of pancreatitis in 22 children at two university hospitals. Demographics and clinical data and ERCP findings were documented. Clinical status was assessed 6 months before the first ERCP and 6 months after the last ERCP, according to general condition, severity and frequency of pain, and health care encounters (emergency department visits, clinic visits, and hospital admissions related to the pancreatitis). RESULTS: Mean age of the patients was 10.7 years (range 1.5 to 17 years). Abdominal pain was the main presenting symptoms with hyperamylasemia and hyperlipasemia. Clinical diagnoses included acute pancreatitis (6), recurrent pancreatitis (5), and chronic pancreatitis (11). The mean follow-up was 16.4 months. Nine patients had sphincter manometry, with abnormal results leading to biliary sphincterotomy in 4. Fifteen patients underwent a total of 23 therapeutic ERCP procedures unrelated to sphincter dysfunction. There were 2 complications of 34 procedures (6%), both being mild pancreatitis after sphincter manometry. There were no deaths. There was a significant reduction in frequency (p < 0.01) and severity of pain (p < 0.01) after intervention. Patients without pancreatographic changes of chronic pancreatitis had the most marked clinical improvement (p < 0.05). In those with ductal changes of chronic pancreatitis, clinical improvement was not predicted by the extent of ductal changes. There was a significant decrease in health care encounters (p < 0.05) and improvement in general condition (p < 0.01) after endoscopic therapy, especially in those with a normal pancreatogram. CONCLUSIONS: Therapeutic ERCP is safe in pediatric patients with pancreatitis. Significant clinical improvement is achieved in patients with biliary or pancreatic stone disease. Prospective studies with long-term follow-up are needed to determine the impact of endoscopic therapy in patients with chronic pancreatitis and sphincter of Oddi dysfunction. PMID- 10744809 TI - Can ERCP contrast agents cause pseudomicrolithiasis? Their effect on the final outcome of bile analysis in patients with suspected microlithiasis. AB - BACKGROUND: Microlithiasis has been implicated in the etiology of idiopathic pancreatitis and biliary-type pain in patients with intact gallbladders. Contrast injection at endoscopic retrograde cholangiopancreatography (ERCP) is used to confirm access into the bile duct and bile is also aspirated to look for microlithiasis. It is not known whether contrast agents contain crystals that could mimic true microlithiasis. METHODS: Four mL of 2 contrast agents (Hypaque and Omnipaque) were examined after centrifugation under polarizing microscopy. In the second part of the study, bile aspirated during ERCP with contrast injection was examined for microlithiasis and contrast pseudomicrolithiasis. RESULTS: Contrast agents exhibited pseudomicrolithiasis that mimicked calcium bilirubinate granules. Pathologists participating in the study were not aware of contrast pseudomicrolithiasis. Nine of twelve (75%) patients would have been reported as having microlithiasis and would possibly have undergone an unnecessary cholecystectomy. CONCLUSION: When bile collected during ERCP is to be examined for microlithiasis, it should be collected without contamination by a contrast agent. If this is not possible, pathologists should be aware that contrast can cause pseudomicrolithiasis. PMID- 10744810 TI - Intraductal US in assessing the effects of radiation therapy and prediction of patency of metallic stents in extrahepatic bile duct carcinoma. AB - BACKGROUND: We assessed the local effects of radiation therapy using intraductal ultrasonography (US) to predict the subsequent patency of metallic stents in bile duct carcinoma. METHODS: Data from 16 patients with extrahepatic-suprapancreatic bile duct carcinoma were prospectively analyzed. Thin-caliber US probes (2.0 mm diameter/20 MHz frequency and 2.8 mm diameter/10 MHz frequency) were inserted into the bile duct via a percutaneous transhepatic approach pre- and postradiation therapy to evaluate the effects of treatment. When intraductal US showed a reduction in bile duct wall thickness of 30% or greater or showed a lessening of vessel (portal vein or right hepatic artery) invasion, radiation therapy was judged to be effective. Noncovered metallic stents were then inserted, and their patency was evaluated over time. RESULTS: When radiation therapy was effective (n = 7), the metallic stent was patent for 522 +/- 571 days. When radiation was ineffective (n = 9), the metallic stent was patent for only 188 +/- 159 days. When radiation therapy was ineffective, stent obstruction occurred in 6 of 9 (66.7%) patients during this period, significantly more frequently than when radiation therapy was effective (14.3%, p < 0.05). CONCLUSIONS: Assessment of local radiation effects by intraductal US is useful for predicting patency of metallic stents in bile duct cancer. PMID- 10744811 TI - Palliation of hilar biliary obstruction from colorectal metastases by endoscopic stent insertion. AB - BACKGROUND: In patients with hepatic metastases from colorectal carcinoma there is a distinct subgroup in whom jaundice is not due to hepatic replacement but rather biliary obstruction. We reviewed our experience with stent insertion in patients with malignant proximal biliary obstruction from metastatic colorectal carcinoma. METHODS: Thirty-three patients were treated between July 1992 and December 1996. Placement of a single stent was attempted at initial endoscopic retrograde cholangiopancreatography. Hilar biliary obstruction was classified according to Bismuth's classification. RESULTS: Successful stent placement was possible in 94% overall and at initial endoscopic retrograde cholangiopancreatography in 39% of patients. Successful stent placement occurred significantly more often in patients with a type I stricture. Cholangitis was the principal complication occurring in 24% of patients. The 30-day mortality rate was 24%, with death occurring significantly less often in patients with a type I or II stricture. Overall, 45% of patients had a 30% fall in bilirubin at 1 week. The median survival was 81 days, with significantly longer survival seen in patients with a type I or II stricture. CONCLUSIONS: Endoscopic stent placement offers effective palliation in most patients with hilar obstruction from colorectal metastases. A subset of patients with type III strictures and greater than 3 intrahepatic metastases often do not benefit from stent insertion. PMID- 10744812 TI - Percutaneous treatment of bile duct stones in patients treated unsuccessfully with endoscopic retrograde procedures. AB - BACKGROUND: The preferred treatment for stones in the bile duct is endoscopic sphincterotomy followed by stone extraction. When this fails, percutaneous treatment is an alternative to surgery. The purpose of this study was to evaluate the success and complication rate of percutaneous treatment. METHODS: Between April 1990 and April 1997, a total of 31 consecutive patients (20 men, 11 women, mean age 70.1 years) underwent percutaneous treatment of bile duct stones (average of 2.2 per patient, range 1 to 10). The percutaneous treatment was considered successful if all stones could be removed. Time and number of sessions needed for imaging, percutaneous treatment, and complications were scored. RESULTS: Twenty-seven patients (87%) were free of stones after 2 to 15 sessions (mean 5.6). The median time for treatment was 16 days (3 to 299). Complications occurred in 3 of the 31 patients: one myocardial infarction during extracorporeal shockwave lithotripsy, one pancreatitis, and one bacteremia. None of these complications were life threatening. Four patients (13%) underwent surgery after failed percutaneous treatment. CONCLUSION: Percutaneous treatment of bile duct stones is an alternative with a high success rate when endoscopic stone removal fails. Surgery can be avoided in nearly 90% of cases. PMID- 10744813 TI - The accuracy of diagnosis and procedural codes for patients with upper GI hemorrhage. AB - BACKGROUND: Little is known about the accuracy of diagnostic and procedural codes for common gastrointestinal (GI) conditions and endoscopic procedures. METHODS: Eight hundred eighty-two patients with upper GI hemorrhage admitted in 1994 to 1 of 13 regional hospitals were studied. Based on endoscopy reports, the source of hemorrhage, performance of upper endoscopy and use of endoscopic therapy were determined, and we assessed the sensitivity and positive predictive value of discharge codes for measuring the source of hemorrhage and use of upper endoscopy. RESULTS: The sensitivity and positive predictive value of principal diagnosis coding for source of hemorrhage were typically 85% to 95%. The sensitivity and predictive value of coding for upper endoscopy were 97.7% and 99.9%, respectively, and were 72.3% and 99.4%, respectively, for endoscopic therapy. Accuracy did not differ between the 4 major teaching and 9 other hospitals. CONCLUSIONS: Hospital-based diagnostic and procedural codes are a reasonably accurate source of data for clinical and outcomes analyses of upper GI hemorrhage. In particular, it is possible to discern from these data the source of hemorrhage and the overall use of upper endoscopy. PMID- 10744814 TI - Endoscopy in acquired immunodeficiency syndrome patients with diarrhea and negative stool studies. AB - BACKGROUND: Diarrhea is a frequent gastrointestinal symptom in patients with acquired immuno-deficiency syndrome (AIDS) and is a major source of morbidity and mortality. A stepwise diagnostic approach is often recommended to search for treatable causes. However, whether the stepwise diagnostic approach is adequate for planning treatment and whether specific treatment for infectious etiologies will affect the survival of patients with AIDS remain unknown. METHODS: From March 1996 to September 1997, endoscopy was performed in AIDS patients with diarrhea, the etiology of which was not identified by noninvasive methods. Specific treatment was given according to the identified etiologies and symptomatic treatment was given for those without definite diagnosis. The clinical symptoms, signs, and duration of follow-up were recorded and survival patterns were analyzed. RESULTS: Etiologic diagnoses were made in 26 of 40 patients (65%) who underwent endoscopic studies. Amebic colitis and cytomegalovirus colitis were the 2 leading causes of prolonged diarrhea in patients with AIDS. Thirty-five patients (87.5%) recovered after treatment. The difference in survival time after diarrhea between patients whose symptoms resolved after treatment and those who continued to have diarrhea was statistically significant (p < 0.001). CONCLUSIONS: Endoscopic studies were helpful for the diagnosis of prolonged diarrhea in AIDS patients who had negative stool studies and did not respond to 2 weeks of empiric treatment. Specific treatment according to the results of endoscopy may improve survival in these patients. PMID- 10744815 TI - Adenoma size and number are predictive of adenoma recurrence: implications for surveillance colonoscopy. AB - BACKGROUND: Three-year colonoscopic surveillance after initial polypectomy may not be required for all patients. Those with multiple baseline polyps and large adenomas, implicated as predictors of colon cancer, merit close observation. Conversely, patients with single small adenomas may be subjected to early endoscopic surveillance unnecessarily. METHODS: From our Adenoma Registry we evaluated patient and adenoma characteristics in 697 patients. All had an adenoma recurrence within 3 years of a positive baseline colonoscopy. Potential risk factors studied were age, gender, number of adenomas, size of largest adenoma and histology. We defined a significant outcome as size of 1 cm or greater, tubulovillous or villous histology, high-grade dysplasia, carcinoma in situ, invasive cancer, or 4 or more adenomas. RESULTS: Having 3 or more adenomas on initial colonoscopy with at least 1 measuring 1 cm or larger greatly increased the chance of a significant finding on the first surveillance colonoscopy. Conversely, patients with 1 or 2 adenomas all measuring less than 1 cm were at extremely low risk of an important outcome within 3 years. CONCLUSIONS: Patients with 1 or 2 adenomas all measuring less than 1 cm are an identified low risk group and their first surveillance examination may be delayed beyond the standard 3 years. PMID- 10744816 TI - Safety and efficacy of India ink and indocyanine green as colonic tattooing agents. AB - BACKGROUND: Controversy exists concerning the safety and efficacy of colonic tattooing for the intraoperative identification of polypectomy sites. The purpose of this study was to determine (1) the concentrations of India ink and indocyanine green that resulted in high-visibility tattoos without significant tissue inflammation and (2) the India ink injection volume that produces best visibility at colonoscopy, laparoscopy, and laparotomy. METHODS: Twenty-two New Zealand white rabbits (2 kg) were anesthetized and injected with India ink (undiluted 1:10, 1:50, 1:100, 1:1000, 1:10,000) and indocyanine green as an undiluted, concentrated formulation (25 mL/2 mL solvent) or in a diluted form (25 mg/5 mL solvent) at various concentrations (1:10, 1:50, 1:100). Tuberculin syringes were used to create a 0.1 mL serosal bleb at two injection sites 2 cm apart. Laparotomy was repeated at days 1, 3, and 7 after injection. Additionally, 16 rabbits were injected with India ink at laparotomy and re-explored at 1 and 5 months. Twelve mongrel dogs (20 kg) were injected with 1.0 mL volumes. Re exploration by colonoscopy, laparoscopy, and laparotomy was done at 7 days and 1 month. Tattoo visibility at re-exploration in both animal models was graded on a scale (0 = agent not seen, 1 = seen with difficulty, 2 = easily seen). Histology in the rabbit was judged by degrees of inflammation (0 = no inflammation, 2 = mild inflammation, 4 = moderate inflammation, 6 = severe inflammation). RESULTS: The concentrated indocyanine green solution was easily visible only on day 1 in the rabbit. Injections of both concentrated and diluted indocyanine green caused mucosal ulceration and moderate to severe inflammation. India ink studied at 7 days, 1 month, and 5 months after injection in the rabbit model was visible at all concentrations. The undiluted and 1:10 concentrations were easily seen and showed evidence of mucosal ulceration. Tattoos produced with all other India ink concentrations were visible without gross inflammation. India ink was also studied at 7 days and 1 month in dogs. The tattoo with the 1:100 concentration at 0.5 mL was seen consistently at colonoscopy, laparoscopy, and laparotomy with only a mild submucosal reaction at 7 days. The tattoos produced with the 1:100 and 1:1000 concentrations at 0.5 mL and 1.0 mL injection volumes were easily seen by all methods of intraabdominal visualization at 1 month with similar histology. CONCLUSION: Indocyanine green was an ineffective colonic tattooing agent. India ink was an effective colonic tattooing agent. Dilute concentrations that caused little to no inflammation could be visualized at 7 days and 1 month in rabbits and dogs and at 5 months in rabbits. India ink, at appropriated concentrations, appears to be a safe short- and long-term colonic tattooing agent. PMID- 10744817 TI - Growth patterns of superficially elevated neoplasia in the large intestine. AB - BACKGROUND: The growth pattern and malignant potential of superficially elevated neoplastic lesions remain controversial. A flat adenoma is classified as a superficially elevated neoplasm that characteristically demonstrates high-grade dysplasia despite its small size. In contrast, a nodule-aggregating (NA) tumor, which consists of multiple small aggregated nodules, can also be classified as superficially elevated neoplasia. METHODS: In this prospective study, 2720 consecutive patients undergoing total colonoscopy were examined for superficially for elevated lesions. Clinicopathologic characteristics, Ki-ras mutational status, and overexpression of p53 protein were compared in 25 NA tumors and 55 flat adenomas without a central depression (flat tumor). RESULTS: All flat tumors had a tubular pattern, whereas 21 of 25 NA tumors showed a villotubular or tubulovillous pattern. Ki-ras mutation was observed in 44% of NA tumors but in none of the flat tumors. Overexpression of p53 protein was found in 12% of NA tumors and 7% of flat tumors. CONCLUSIONS: NA tumors and flat tumors have different clinicopathologic and genetic characteristics, although both types of tumor are classified as superficially elevated lesions. PMID- 10744818 TI - Still photography versus videotaping for documentation of cecal intubation: a prospective study. AB - BACKGROUND: Documentation of cecal intubation is important for credentialing and continuous quality improvement. However, convincing cecal photographs can be difficult to obtain. The aims of the study were to determine (1) the anatomic variations and photographic factors associated with convincing cecal photographs; (2) whether a prospective attempt to capture specific features resulted in more convincing photographs; and (3) how a prospective attempt at capturing convincing cecal features during still photography compares with videotaping as a method of documenting cecal intubation. METHODS: A single examiner evaluating 165 consecutive patients photographed the entire cecum from just distal to the ileocecal valve, the appendiceal orifice, the ileocecal valve orifice, and in the last 110 consecutive cases, the terminal ileum, if it could be intubated. The photographs were then scored by 8 experts according to how convinced they were that cecal intubation had occurred. Features associated with high and low scores were determined, and 50 additional consecutive patients were photographed with a specific effort to capture these features, along with videotaping of the cecum. These photographs and videotapes were then scored by 7 of the 8 experts on a scale of 1 to 5, with 5 representing "definitely the cecum." RESULTS: There was marked variation in scores of still photographs among reviewers. A combination of photographs produced the highest mean score and the highest percentage of scores that were either "probably" or "definitely" of the cecum. The photograph of the cecum from just distal to the ileocecal valve was most convincing and the terminal ileum photograph was least convincing. In the 50 cases in which an attempt was made to capture specific features, mean scores improved for the overall cecal photograph by 0.10, the appendiceal orifice by 0.23, the ileocecal valve lips by 0.20, and the terminal ileum by 0. 19. The cecum, appendiceal orifice, valve lips, and terminal ileum photographs for the last 50 patients were scored higher than those from the first 165 patients by 4, 4, 4, and 5 reviewers, respectively. Videotapes were consistently convincing (overall mean score 4.78 out of 5) and were scored more convincing than the combination of still photos from the final 50 cases by 6 of 7 reviewers. CONCLUSIONS: Still photography of the cecum can be improved somewhat by attempting to capture specific cecal features. However, because of anatomic variation among normal individuals, still photography remains inconsistently convincing. A combination of photographs is most convincing. Videotaping of the cecum is consistently convincing and would appear to serve effectively as a means of documenting cecal intubation rates for the purposes of continuous quality improvement and credentialing. PMID- 10744819 TI - Comparison among the perforation rates of Maloney, balloon, and savary dilation of esophageal strictures. AB - BACKGROUND: Traditionally, mercury-filled rubber bougies are used for dilation of simple or mild-to-moderate esophageal strictures, whereas through-the-scope balloon dilators and wire-guided polyvinyl bougies have become standard for more complex strictures. Because few comparative trials are available, the choice of dilator and technique is largely based on the training and experience of the operator. METHODS: We reviewed 348 esophageal dilation procedures performed on a total of 142 patients over a 4-year period (January 1, 1993, to January 1, 1997). The location and cause of stricture, the maximum diameter of the instrument used per session, the rate of perforation, and the rate of fluoroscopy use were recorded. RESULTS: Maloney, balloon (hydrostatic and pneumatic type), and Savary dilations were performed in 102, 156, and 90 sessions, respectively. Perforations occurred in 4 patients. All of these perforations occurred when Maloney dilators were passed blindly into complex strictures (Fisher's exact test, p = 0.011, two tailed). Three of these four patients had undergone endoscopy with conscious sedation immediately before the dilation. The immediate outcome of surgery was good in all 4 patients with no deaths. CONCLUSION: Perforation was most commonly associated with the blind passage of Maloney bougies into complex strictures. PMID- 10744820 TI - Jejunal diverticulum: causing occult bleeding and intestinal obstruction. PMID- 10744821 TI - Hemosuccus pancreaticus: rare complication of chronic pancreatitis. PMID- 10744822 TI - Carcinoid tumor of the appendix. PMID- 10744823 TI - Dieulafoy's lesion. PMID- 10744824 TI - High-resolution imaging of the human esophagus and stomach in vivo using optical coherence tomography. AB - BACKGROUND: Optical coherence tomography is a new, high spatial-resolution, cross sectional imaging technique. We investigated the ability of optical coherence tomography to provide detailed images of subsurface structures in the upper gastrointestinal (GI) tract. METHODS: Optical coherence tomography was performed during routine upper GI endoscopy on 32 patients including 20 patients with Barrett's esophagus. An endoscopic mucosal biopsy was obtained immediately after imaging and was used for histopathologic correlation. RESULTS: Optical coherence tomography provided clear delineation of layers of the normal human esophagus extending from the epithelium to the longitudinal muscularis propria. Gastric mucosa was differentiated from esophageal mucosa, Barrett's esophagus was differentiated from normal esophageal mucosa, and esophageal adenocarcinoma was distinguished from normal esophagus and Barrett's esophagus. CONCLUSIONS: Optical coherence tomography allows visualization of the subsurface architectural morphology of the upper GI tract. The diagnostic information provided by this new imaging modality suggests that it may be a useful adjunct to endoscopy. PMID- 10744825 TI - High-resolution endoscopic imaging of the GI tract using optical coherence tomography. AB - BACKGROUND: Optical coherence tomography (OCT) has demonstrated the microscopic structure of the gastrointestinal (GI) tract mucosa and submucosa in vitro. We evaluated a prototype OCT system and assessed the feasibility of OCT in the human GI tract. METHODS: The 2.4 mm diameter prototype OCT probe, inserted through an endoscope, provides a 360-degree radial scan. Images (6.7 frames/sec) are displayed on a television monitor. Tissue contact is not required. In patients undergoing elective endoscopy, OCT images were obtained of normal mucosa (confirmed by biopsy). RESULTS: Seventy-two sites were imaged (38 patients): esophagus (21), stomach (12), duodenum (11), terminal ileum (4), colon (15), and rectum (9). Varying the distance between the probe and the mucosal surface produced images of the GI wall of varying depth. When held about 1 mm above the mucosal surface, the images consisted of mucosal structures such as colonic crypts, gastric pits, and duodenal villi. With the probe held against the wall, the OCT image comprised several layers interpreted as mucosa, muscularis mucosae, and submucosa. Structures including blood vessels were evident within the submucosa. A probe with a 0.5 mm working distance to the focal point provided the best images. Reducing the frame rate to 4.0 per second facilitated image interpretation. CONCLUSIONS: OCT is feasible in the human GI tract and provides interpretable high-resolution images of mucosa and submucosa. PMID- 10744826 TI - Implantation metastasis after PEG: case report and review. PMID- 10744827 TI - Bleeding gastric ulcer: a complication from gastrostomy tube replacement. PMID- 10744828 TI - A wire-loop technique for removal of migrated and embedded biliary stents. PMID- 10744829 TI - Clip closure of a duodenal perforation secondary to a biliary stent. PMID- 10744830 TI - Endoscopic hemoclip therapy of a bleeding duodenal diverticulum. PMID- 10744831 TI - GI hemorrhage from fistula between right hepatic artery pseudoaneurysm and the duodenum secondary to acute cholecystitis. PMID- 10744832 TI - Fatal acalculous cholecystitis after photodynamic therapy for high-grade dysplasia of the major duodenal papilla. PMID- 10744833 TI - Sigmoid lipoma mimicking carcinoma: case report with review of diagnosis and management. PMID- 10744834 TI - Endoscopic removal of sclerotherapy needle from gastric varix after N-butyl-2 cyanoacrylate injection. PMID- 10744835 TI - Radiation-induced hemorrhagic carditis treated with argon plasma coagulator. PMID- 10744836 TI - Combined endoscopic and laparoscopic approach to remove a sharp gastric foreign body. PMID- 10744837 TI - Interhepatic duct accompanied by cholestasis. PMID- 10744838 TI - Retrieval of an impacted Dormia basket and stone in situ using a novel method. PMID- 10744839 TI - Endoscopic management of post laparoscopic cholecystectomy bile leak in a child. PMID- 10744840 TI - Endoscopic diagnosis of malignant enterocolic fistula caused by ileal lymphoma. PMID- 10744841 TI - Massive hemorrhage from a Dieulafoy lesion in the cecum: successful endoscopic management. PMID- 10744842 TI - Imaging beyond the endoscope. PMID- 10744843 TI - Female athletes and eating problems: a meta-analysis. AB - OBJECTIVE: The relationship between athletic participation and eating problems is examined using meta-analysis. Both the risk and protective elements of athletic participation are considered. METHOD: Data from 34 studies were used to examine the overall relationship between athletic participation and eating problems. Relationships for specific sports, elite athletes, and various age groups are also examined. RESULTS: Athletes appeared to be somewhat more at risk for eating problems than nonathletes. This was especially true of dancers. Significant effects did not emerge for gymnasts. Elite athletes, especially those in sports emphasizing thinness, were at risk. Nonelite athletes, especially in high school, had reduced risk of eating problems compared to controls. Body dissatisfaction was lower in athletes. CONCLUSION: There appear to be circumstances under which sports participation by women constitutes a risk factor for certain elements of eating problems. In other situations, athletic participation may be protective against eating problems. PMID- 10744845 TI - Food refusal and insanity: sitophobia and anorexia nervosa in Victorian asylums. AB - Although anorexia nervosa emerged as a new syndrome in the second half of the 19th century, this clinical picture seemed to be unknown in the psychiatric hospitals or asylums. In asylum medicine, the commonly used concept of sitophobia to designate food refusal in the insane covered a wide variety of mental disturbances and cannot be plainly equated with anorexia nervosa. A major difference is the occurrence of hallucinations and delusions specifically centered around religion and digestion. Most probably, anorectic patients were not treated in asylums, but at home, in the doctor's office, or in general hospitals. This pattern may be partly attributed to the fact that both patients and doctors were focusing on symptoms of self-starvation like emaciation, constipation, and amenorrhea, which were primarily interpreted as referring to somatic diseases. Additionally, wealthy families probably preferred private care in water-cure establishments, sanatoria, and rest homes to the stigmatizing referral of their anorectic daughter to an asylum. Hence, the fact that late 19th century institutionalized psychiatry was only incidentally confronted with anorexia nervosa may explain its lack of interest in the emerging syndrome. PMID- 10744844 TI - One-year use and cost of inpatient and outpatient services among female and male patients with an eating disorder: evidence from a national database of health insurance claims. AB - OBJECTIVE: This study examined rates and cost of inpatient and outpatient treatment among 1,932 patients with an eating disorder. METHOD: One-year (1995) data were available through MarketScan, a national insurance database containing claims for 1,902,041 male patients and 2,005,760 female patients. RESULTS: Female patients (n = 1,756, 0.14% of all females) were significantly more likely to have been treated for an eating disorder than male patients (n = 176, 0.016% of all males), and females received more days of treatment than males. Outpatient treatment was the norm, regardless of gender or type of eating disorder. Average number of days (inpatient or outpatient) was less than the minimum recommended by standards of care. Age-adjusted costs for the treatment of anorexia nervosa and bulimia nervosa were comparable to the cost of treatment for schizophrenia. DISCUSSION: The utilization data are discussed in terms of barriers to care and treatment guidelines for eating disorders. PMID- 10744846 TI - Cognitive avoidance and bulimic psychopathology: the relevance of temporal factors in a nonclinical population. AB - OBJECTIVE: Previous research has provided evidence of both an attentional bias towards, and a cognitive avoidance of threat cues, by individuals with unhealthy eating attitudes and disorders. It has been suggested that an initial attentional bias might be followed by a later avoidance process. The present study assessed the speed of processing of self-directed ego threat words following different interstimulus intervals (500, 1,000, 1,500, and 2,000 ms). METHOD: Fifty female students completed a computer-driven threat processing task and the Eating Disorders Inventory. RESULTS: Women were significantly slower to process ego-self threat cues following the 2, 000-ms interval than following the other intervals (500, 1,000, and 1,500 ms). Those women with increased levels of bulimic attitudes were slower to process threats following the 1,500-ms interval, but not after the longer or shorter intervals. CONCLUSIONS: The results stress the importance of temporal factors in the processing of threat by individuals with high levels of eating psychopathology. The findings are discussed in terms of an appropriate cognitive model. PMID- 10744847 TI - Impact of interpersonal and ego-related stress on restrained eaters. AB - OBJECTIVE: This study examined the impact of different types of stress, one interpersonal and two ego-related versus a control condition, on the eating behavior of individuals with varying degrees of dietary restraint. METHOD: Eighty two females were randomly assigned to one of three manipulations or a control group, and then all groups completed an ice cream taste test. RESULTS: A significant interaction revealed that for participants with higher restraint, those in the stressful manipulations ate significantly more than participants in the control group. Further, the pattern of consumption based on restraint for the interpersonal group differed from the other three conditions. In the interpersonal group, the greater the restraint, the more participants ate, whereas in the other three conditions, the pattern was reversed although not significantly so. DISCUSSION: Findings are discussed in terms of the role that interpersonal stress plays in the eating behavior of dieters and potential implications regarding the development of eating disorders. PMID- 10744848 TI - Dietary restraint and addictive behaviors: the generalizability of Tiffany's cue reactivity model. AB - Two studies are presented, which examine cue reactivity in dieting. METHODS: Experiment 1 investigated whether the presence of a preferred food affected dieters' performance on measures of attention, reaction time, and motor speed. The manipulation did not affect the performance. Experiment 2 investigated the performance of dieters (N = 19), highly restrained non-dieters (N = 18) and low to-medium restrained eaters (N = 34) on two simple reaction time tasks. Subjects were either required to imagine their favorite food or to imagine their favorite holiday while completing a reaction time task. RESULTS: In the food condition, both dieters and restrained nondieters displayed significantly slower reaction times during the first three of five blocks of the task than the low-to-medium restrained eaters. CONCLUSIONS: The results are discussed in terms of Tiffany's (Psychological Review 97:147-168, 1990) model of cue reactivity in that different abstinent states produce comparable effects upon performance. PMID- 10744849 TI - Duration of illness predicts outcome for bulimia nervosa: a long-term follow-up study. AB - OBJECTIVE: The purpose of this study was to investigate long-term outcome and prognosis in a bulimic and subthreshold bulimic sample. METHOD: In a follow-up study, 44 patients diagnosed with bulimia nervosa and subthreshold bulimia nervosa were contacted after an average follow-up period of 9 years. RESULTS: Results revealed that 72.7% (n = 32) of the participants were recovered at the time of follow-up. An investigation of prognostic variables showed that good outcome was associated with a shorter duration of illness, which was defined as the time between onset of symptoms and first treatment intervention. If participants were initially treated within the first few years of the illness, the probability of recovery was above 80%. However, if they were initially treated 15 years or more after the onset of the illness, the probability of recovery fell below 20%. DISCUSSION: This finding suggests that early identification of bulimia nervosa may be a very important factor in preventing a chronic eating disorder. PMID- 10744850 TI - Influence of shape and weight on self-evaluation in bulimia nervosa. AB - OBJECTIVE: To compare three related but different measures of excessive concerns about shape and weight in bulimia nervosa (BN): influence of shape and weight (influence; DSM-IV Criterion D), overconcern with shape and weight (overconcern; DSM-III-R Criterion E), and dissatisfaction with shape and weight (dissatisfaction). METHOD: One-hundred twenty BN patients, 27 restrained eaters (RE), and 28 normal controls (NC) were assessed via the Eating Disorders Examination and self-report measures. RESULTS: Influence and overconcern, but not dissatisfaction, successfully discriminated BNs from NCs but not from REs. A minority of patients with BN obtained low scores on both influence and overconcern. However, there were few differences between those patients with low scores and those with high scores on numerous clinical characteristics. DISCUSSION: Influence and overconcern are equally valid measures of the excessive concerns about shape and weight characteristic of BN. PMID- 10744851 TI - Disgust sensitivity in eating disorders: a preliminary investigation. AB - BACKGROUND: Disgust is a basic emotion that has been relatively neglected in psychiatry in general and in eating disorders in particular. Nevertheless, there are features of disgust and its more complex derivatives (e.g., shame) which suggest that disgust may have a role to play in eating disorders. METHOD: Seventy four patients with a DSM-IV diagnosis of anorexia nervosa, bulimia nervosa, eating disorder not otherwise specified, and obese binge eater were compared with 15 control subjects on their levels of disgust sensitivity. RESULTS: Overall, eating disorder patients did not appear to be more sensitive to disgust-eliciting stimuli than comparison subjects, although there was a tendency for patients to be more disgusted by body products. However, drive for thinness and bulimia scores were related to higher levels of disgust sensitivity to food, death, and magical contagion. General psychopathology did not appear to be related to levels of disgust sensitivity. DISCUSSION: Although patients are not more sensitive than controls to the disgust-eliciting stimuli measured, disgust still has a positive relationship to eating disorder symptoms. Future studies will need to examine more precisely what this relationship might be. PMID- 10744852 TI - Season of birth and bulimia nervosa. AB - OBJECTIVE: Previous studies suggest season of birth variation in eating disorders akin to those of psychoses. We studied season of birth variation in bulimia nervosa. METHOD: Season of birth variation in 935 patients was examined after adjustment for population trends. Variation was also examined for subgroups by age and previous anorexia nervosa. RESULTS: Season of birth did not differ significantly from population norms among bulimics (p >.30), contrasting with studies of other eating disorders. With a history of anorexia nervosa (n = 227), peak season of birth was in March (p <.05). This is consistent with previous studies and also with seasonal birth variation for psychoses. DISCUSSION: Overall, we find no evidence of season of birth variation in bulimia nervosa, and suggest any positive findings be treated with caution. We discuss a number of confounding influences and argue that one explanation remains shared trait vulnerability between anorexia nervosa and psychoses. PMID- 10744853 TI - Maternal child feeding practices and obesity: a discordant sibling analysis. AB - BACKGROUND: The relationship between maternal feeding practices and weight status of 7-12 year-old obese and nonobese siblings was evaluated in 18 families using a discordant sibling design. METHOD: Mothers completed measures of concern and perception of children's weight and eating behavior, their control over child feeding, and maternal eating behavior. RESULTS: Intraclass correlations suggested similarity between obese and nonobese siblings in maternal control over feeding. Mothers perceived differences between their obese and nonobese children's eating regulation. Mothers' weight status was positively associated with disinhibition of their own eating as well as concern about both their obese and nonobese children's weight and health. DISCUSSION: These findings fail to support the hypothesis that maternal control over child feeding is related to childhood obesity, but highlight the impact of maternal weight history and eating habits on her impression of children's future weight and health independent of the child's weight status. PMID- 10744854 TI - Validity of questionnaires for assessing dysfunctional cognitions in bulimia nervosa. AB - OBJECTIVE: This research investigated the validity of the Bulimic Cognitive Distortions Scale and the Mizes Anorectic Cognitions Questionnaire. The ability of these questionnaires to discriminate among individuals with bulimia, restrained eaters, and normals was also investigated. METHOD: Scores on these retrospective self-report questionnaires were compared to cognitions assessed via in vivo thought-sampling. Cognitions of 15 women with bulimia nervosa, 15 restrained eaters, and 15 non-eating disordered women were assessed. RESULTS: Results indicated that both the BCDS and the MAC successfully discriminated among individuals with bulimia, restrained eaters, and normals, and adequately measured in vivo attitudes and beliefs associated with food and eating, weight and body image, and low self-efficacy. DISCUSSION: These findings support the continued use of both the BCDS and the MAC. PMID- 10744855 TI - What is a binge? The influence of amount, duration, and loss of control criteria on judgments of binge eating. AB - OBJECTIVE: We investigated the influence of amount of food eaten, duration of eating episode, and loss of control in judgments of eating episodes as binges. METHOD: Participants rated the degree to which the eating behavior of a female actress qualified as a "binge" after observing eight videotaped vignettes in which the amount of food eaten, apparent duration of eating episode, and loss of control were varied. Binge ratings were stable across a test-retest interval of 3 4 weeks, there was minimal observer drift, and the experimental variables were independently perceived. RESULTS: A repeated measures analysis of variance (ANOVA) on binge ratings revealed significant main effects for quantity and loss of control, and a significant Quantity x Time interaction. DISCUSSION: The results are consistent with the definitional criteria of a binge, underscore the independence of loss of control, and highlight the importance of the violation of dietary standards in judgments of binges. Moreover, they illustrate the reliability and sensitivity of the methodology, and its potential for further investigations of binge eating. PMID- 10744856 TI - Reversal of bone marrow hypoplasia in anorexia nervosa: case report. AB - BACKGROUND: Bone marrow hypoplasia seems to be uncommon in anorexia nervosa. This marrow abnormality is rapidly reversible with intensive nutritional rehabilitation. The patient described in this case report had anorexia nervosa. METHOD: She presented with complete serous atrophy of the bone marrow associated with pancytopenia. RESULTS: She was cured with recombinant human erythropoietin administered subcutaneously. After 25, days, the hematological situation was normalized. DISCUSSION: The possible advantage and practical implications of the administration of erythropoietin are discussed. PMID- 10744857 TI - Blood-letting in anorexia nervosa: a case study. AB - BACKGROUND: Deliberate blood-letting has been characterized as an alternative to purging behavior in bulimia. METHOD: We describe a female healthcare worker with an 8-year history of restrictive anorexia nervosa, who initially presented with anemia, using blood-letting, cold baths, and starvation to control her mental state. RESULTS: In contrast with the previous cases of bulimia, the aim of blood letting in this case of anorexia nervosa was to achieve anemia. She compared the psychic correlates of anemia to emaciation, rather than to deliberate self-harm or purging. DISCUSSION: We note that mainstream 19th century psychiatry prescribed "baths, blood-letting and diet" as a treatment of "madness." PMID- 10744858 TI - Phospho-dependent association of neurofilament proteins with kinesin in situ. AB - Recent studies demonstrate co-localization of kinesin with neurofilament (NF) subunits in culture and suggest that kinesin participates in NF subunit distribution. We sought to determine whether kinesin was also associated with NF subunits in situ. Axonal transport of NF subunits in mouse optic nerve was perturbed by the microtubule (MT)-depolymerizing drug vinblastine, indicating that NF transport was dependent upon MT dynamics. Kinesin co-precipitated during immunoprecipitation of NF subunits from optic nerve. The association of NFs and kinesin was regulated by NF phosphorylation, since (1) NF subunits bearing developmentally delayed phospho-epitopes did not co-purify in a microtubule motor preparation from CNS while less phosphorylated forms did; (2) subunits bearing these phospho-epitopes were selectively not co-precipitated with kinesin; and (3) phosphorylation under cell-free conditions diminished the association of NF subunits with kinesin. The nature and extent of this association was further examined by intravitreal injection of (35)S-methionine and monitoring NF subunit transport along optic axons. As previously described by several laboratories, the wave of NF subunits underwent a progressive broadening during continued transport. The front, but not the trail, of this broadening wave of NF subunits was co-precipitated with kinesin, indicating that (1) the fastest-moving NFs were associated with kinesin, and (2) that dissociation from kinesin may foster trailing of NF subunits during continued transport. These data suggest that kinesin participates in NF axonal transport either by directly translocating NFs and/or by linking NFs to transporting MTs. Both Triton-soluble as well as cytoskeleton-associated NF subunits were co-precipitated with kinesin; these data are considered in terms of the form(s) in which NF subunits undergo axonal transport. PMID- 10744859 TI - Control of ciliary orientation through cAMP-dependent phosphorylation of axonemal proteins in paramecium caudatum. AB - Ciliary reorientations in response to cAMP do not take place after a brief digestion with trypsin in ciliated cortical sheets from Triton-glycerol-extracted Paramecium. In this study, we examined the effects of tryptic digestion on the cAMP-dependent phosphorylation of axonemal proteins to clarify the relationship between phosphorylation and ciliary reorientation. As reported for Paramecium tetraurelia, cAMP stimulated phosphorylations of the 29 kDa and 65 kDa axonemal polypeptides also in Paramecium caudatum. After a brief digestion of axonemes by trypsin, none of the cAMP-dependent phosphorylations occurred. On the other hand, the 29 kDa polypeptide still remained to be labeled after a brief digestion of axonemes that had previously been labeled with (32)P in the presence of cAMP, which indicates that this brief digestion breaks down endogenous cAMP-dependent protein kinases but not phosphorylated proteins. This must be the reason that trypsin-treated cilia on the sheets cannot reorient towards the posterior part of the cell. Our results indicate that cAMP regulates not only the beat frequency but also the ciliary orientation via phosphorylation of dynein subunits in Paramecium. PMID- 10744860 TI - Shigella actin-based motility in the presence of truncated vinculin. AB - Mounting evidence supports the role of truncated vinculin in the intracellular actin-based motility of Shigella flexneri. Vinculin's role was recently questioned by Goldberg [1997: Cell Motil Cytoskeleton 37:44-53] who observed Shigella motility in mouse embryonal carcinoma 5.51 cells, a genetically modified cell line that reputedly lacked vinculin. That challenge implicitly relied on the assumption that 5.51 cells had no detectable vinculin polypeptide and lacked full length vinculin mRNA. Despite the appearance of being an unambiguous test of vinculin's role in Shigella motility, 5.51 cells were shown to contain adequate amounts of truncated vinculin (as well as the corresponding mRNA transcript) to support bacterial locomotion. We also examined Shigella locomotion in gamma229 cells, a related embryonal carcinoma cell line containing approximately one-half the vinculin content found in 5.51 cells. We observed that there was a commensurate twofold decrease in the Shigella motility rate, as compared to 5.51 cells; this finding raises the possibility that vinculin can become a rate limiting factor under some circumstances. Immunofluorescence microscopy using vin 11-5 monoclonal antibody directed against the vinculin head domain showed intense staining of Shigella rocket tails in both gamma229 and 5.51 cells. Our findings clearly demonstrate that motility in 5.51 cells cannot be regarded as a valid criterion for evaluating the role of truncated vinculin in Shigella motility. PMID- 10744861 TI - Association between elongation factor-1alpha and microtubules in vivo is domain dependent and conditional. AB - Although the precise definition for a microtubule-associated protein (MAP) has been the subject of debate, elongation factor-1alpha (EF-1alpha) fits the most basic criteria for a MAP [Durso and Cyr, 1994a]. It binds, bundles, stabilizes, and promotes the assembly of microtubules in vitro, and localizes to plant microtubule arrays in situ. In this study, the in vitro and in vivo association of EF-1alpha with microtubules was further investigated. Analysis of the in vitro binding data for EF-1alpha and microtubules indicates that EF-1alpha binds cooperatively to the microtubule lattice. In order to investigate the interaction of EF-1alpha with microtubules in vivo, GFP fusions to EF-1alpha or to EF-1alpha truncates were transiently expressed in living plant cells. Using this method, two putative microtubule-binding domains on EF-1alpha were identified: one in the N-terminal domain I and one in the C-terminal domain III. The binding of domain I to microtubules in vivo, like the binding of full-length EF-1alpha, is conditional, and requires incubation in weak, lipophilic organic acids. The binding of domain III to microtubules in vivo, however, is not conditional, and occurs under normal cellular regimes. Furthermore, domain III stabilizes cortical microtubules as determined by their resistance to the anti-microtubule herbicide, oryzalin. Because the accumulation of EF-1alpha onto microtubules is unconditional in the absence of domain I, we hypothesize that domain I negatively regulates the accumulation of EF-1alpha onto microtubules in vivo. This hypothesis is discussed in terms of possible regulatory mechanisms that could affect the accumulation of EF-1alpha onto microtubules within living cells. PMID- 10744862 TI - Actin filaments are severed by both native and recombinant dictyostelium cofilin but to different extents. AB - Cofilin has been reported to depolymerize F-actin alternately by either severing filaments to increase the number of depolymerizing ends or by increasing the off rate of monomers from F-actin without increasing the number of filament ends. We have compared directly the ability of native and recombinant cofilins from Dictyostelium to sever F-actin. Our results demonstrate that native cofilin has a higher level of severing activity than recombinant cofilin. Significantly, the measurement of cofilin's severing activity by two independent methods, direct visualization with an improved light microscope assay and by scoring of the number of pointed ends by DNase I binding, clearly shows that both native and recombinant cofilins sever F-actin but to different extents. The severing activity in preparations of recombinant cofilin is variable depending on the method of preparation and, in some cases, is difficult to detect by microscopy assays. This latter point is particularly significant because it may lead to the conclusion that cofilin severs weakly or not at all depending on its method of isolation. PMID- 10744864 TI - Kawasaki disease: a clinical dilemma. PMID- 10744863 TI - Nutritional status of adolescent school children in rural North India. PMID- 10744865 TI - Magnitude of recall bias in the estimation of immunization coverage and its determinants. AB - OBJECTIVE: To study the magnitude of recall bias in the estimation of immunization coverage and to identify its determinants. DESIGN: A follow-up study of cohort of children for one year; followed by a cross-sectional recall survey. SUBJECTS: All live births in two contiguous PHC areas in Villupuram district, Tamil Nadu. METHODS: The prospective data collected from mothers of 774 children was considered as 'Gold Standard' and the retrospective recall data from them was compared with it. This was carried out for individual immunization schedules, namely, DPT, OPV, BCG and measles and for the combined immunization status. Measures such as sensitivity, specificity, positive and negative predictive values were computed. Logistic Regression technique was employed for evaluating the determinants of agreement. RESULTS: Less than 50% of mothers has immunization cards with them and more than 70% of the cards did not have complete information. The sensitivity of the recall method was 41.3% and the specificity was 79.5% when the complete immunization status was considered. For individual immunization schedules, the sensitivity ranged from 95% for BCG to 53% for measles vaccination and the specificity ranged from 30% for BCG to 68% for measles vaccination. Mother's age emerged out as a significant determinant in the agreement of two methods. Maintenance of immunization cards were very poor. CONCLUSIONS: Method of obtaining immunization status through recall survey is not sensitive. Proper maintenance of immunization cards and ensuring the availability of them with mothers for inspection are recommended for obtaining accurate estimation of vaccine coverages. PMID- 10744866 TI - An indigenously developed nitrite kit to aid in the diagnosis of urinary tract infection. AB - OBJECTIVE: To evaluate the utility of an indigenously developed nitrite kit for the rapid diagnosis of urinary tract infection (UTI) METHODS: 1018 urine specimens were collected from all cases where there was clinical suspicion of UTI. Samples were cultured as per standard microbiological protocol. Presence of nitrites was indicated by the development of purple color on addition of color developing solution and compared with the set of graded positive and negative controls also provided in the Kit. RESULTS: The results of the nitrite kit were compared with the semi-quantitative urine culture as the gold standard. The sensitivity, specificity, positive predictive and negative predictive values were 47%, 87%, 31% and 93%, respectively. CONCLUSION: Nitrite kit as a screening test can decrease the work load in the clinical bacteriology laboratory. More importantly in a field set up that is devoid of culture facilities, it can be used to correctly predict the absence of UTI. PMID- 10744867 TI - Evaluation of a child with communication disorder. PMID- 10744868 TI - Ceftibuten. PMID- 10744869 TI - Methods of sampling and data collection. PMID- 10744870 TI - Cardiac status in post-streptococcal acute glomerulonephritis. PMID- 10744871 TI - Peripheral vessel exchange transfusion. PMID- 10744872 TI - Recurrent abdominal pain in Indian children and its relation with school and family environment. PMID- 10744873 TI - FNAC as a diagnostic tool in pediatric head and neck lesions. PMID- 10744874 TI - Hodgkin's disease presenting with bone involvement. PMID- 10744875 TI - CHARGE association. PMID- 10744876 TI - Cystic mesenchymal hamartoma of liver. PMID- 10744877 TI - Congenital cleft foot and hand. PMID- 10744878 TI - Prepenile scrotum with high anorectal malformation. PMID- 10744879 TI - BCG vaccination: some practical dilemmas. PMID- 10744880 TI - BCG vaccination: some practical dilemmas - reply PMID- 10744881 TI - Osteogenesis imperfecta lethalis (Type II). PMID- 10744882 TI - Serial sonographic changes in hydatid cyst following medical therapy. PMID- 10744883 TI - Congenital heart disease: clinical spectrum. PMID- 10744884 TI - Awareness, utilization and cost of newer childhood vaccines in a lower middle and middle class urban population of Vellore Town, South India. Immunization Survey Group. PMID- 10744885 TI - Evaluation of leukocyte esterase reagent strips for rapid diagnosis of pyogenic meningitis. PMID- 10744886 TI - The next twenty years: an editorial perspective. PMID- 10744887 TI - Understanding choice of milk and bread for breakfast among Swedish children aged 11-15 years: an application of the Theory of Planned Behaviour. AB - The present study examined the influences of attitudes, social norms, perceived control and underlying beliefs on 11 to 15-year-olds' breakfast choices of milk with different fat content and high-fibre bread. All pupils in the 5th, 7th and 9th grades in Molndal community (N=1730), Sweden, were asked to complete a questionnaire based on the Theory of Planned Behaviour. Two weeks later, they were asked to fill in a 7-day record of food consumed for breakfast. Consumption of milk and high-fibre bread was predicted from intentions, and for milk also by perceived behavioural control. Intentions were influenced by attitudes, perceptions of significant others' preferences, and perceived control. In addition, perception of the parents' consumption (descriptive norm) of the specific food played an important role. Attitudes, norms and perceived control predicted intentions to a similar extent in each age group. Attitudes to the consumption of milk and high fibre bread were influenced by beliefs about their sensory and health aspects. Females and the oldest children had greater knowledge about the healthier alternatives and the oldest children had a tendency to choose healthier options. PMID- 10744888 TI - Taste adaptation during the eating of sweetened yogurt. AB - Taste adaptation, a gradual decline of taste intensity with prolonged stimulation, is frequently observed in laboratory experiments. However, during normal eating the taste of food does not seem to decrease or disappear. During eating, the presence of saliva, the interactions between tastants and odorants, and mouth movements can influence the time course of taste intensity. Therefore, results from standard laboratory adaptation experiments about adaptation seem of limited relevance to the prediction of the time course of taste intensity when eating real foods. We studied whether taste adaptation occurs when subjects eat yogurt, sweetened with two concentrations of sucrose (3.75 and 7.5%). In addition, we examined whether this adaptation is related to taste adaptation measured with a filter paper method. During the eating of yogurt, sweetness intensity declined with time, whereas sourness intensity did not. As expected, taste adaptation in the "yogurt task" was only slightly correlated to adaptation measured with filter paper. PMID- 10744889 TI - Role of cold receptors and menthol in thirst, the drive to breathe and arousal. AB - Menthol is widely used in candy, chewing gum, toothpastes, cigarettes and common cold medications. Menthol has been shown to stimulate cold receptors in the mouth and nose. The present paper puts forward the hypothesis that menthol, by its effects on oral and nasal cold receptors, may influence thirst, the drive to breathe, and arousal. The satisfying effects of menthol on thirst and breathing, together with an effect on arousal, may explain the popularity of menthol and account for the very large amount of menthol-containing products that are consumed each day. PMID- 10744890 TI - Trends in dietary sources of nutrients among middle-aged men in southern Germany. Results of the MONICA Project Augsburg: dietary surveys 1984/1985 and 1994/1995. MONItoring trends and determinants in CArdiovascular disease. AB - The study analyses 10-year trends (1984/1985-1994/1995) in southern Germany in the food sources of nutrients. In two phases of the MONICA Project in Augsburg, dietary surveys were conducted in subsamples of 45 to 64 year-old men. Responses in 1984/1985 and 1994/1995 were 69.8% and 70.8% with eligible samples of 603 and 607, respectively. Seven-day records were collected and coded with the German food composition database. Percentage contribution of food groups to selected nutrient intakes were calculated. Meat and meat products and eggs decreased in importance as contributors to the intake of energy and several nutrients, whereas fish and fish products, milk and milk products and various food groups of plant origin became more important. In 1984/1985, for instance, meat and meat products delivered 23.4% of energy, 47.5% of protein and 38.1% of fat, whereas in 1994/1995 the respective contributions declined to 21.4%, 44.0% and 34.5%. The new product group of supplements has already become an important contributor to the intake of vitamin C. Both static intakes and changes in intake of certain nutrients can be explained by corresponding fluctuations in patterns of intake of the underlying food sources. Therefore it is recommended that dietary assessments use food source lists derived from the most recent dietary surveys. PMID- 10744891 TI - Irish descent, religion and food consumption in the west of Scotland. AB - Mortality and morbidity of people of Irish descent in Britain is high, including from cardiovascular causes potentially linked with diet. The west of Scotland has long had a pattern of Irish migration, where migrants were poorer than the host population, and their different religious background gave rise to prolonged discrimination. This paper uses data collected in 1987/88 from the west of Scotland Twenty-07 study to test whether dietary differences due to poverty or to other factors have persisted among the descendents of these migrants. Being born of Catholic parents was the index of Irish descent used, these respondents consumed less of a factor represented by fruit, yoghurt and vegetables, and more of one represented by snacks and processed foods than the rest of the sample. The picture for those reporting current Catholic affiliation in adulthood was similar. Differences are largely associated with social class and mediated not by low income but by educational disadvantage. The findings suggest the continuation of a diet affected by limited opportunities for social mobility, and thus by obstacles to sustained educational advancement, among the descendants of Irish migrants even after several generations. PMID- 10744892 TI - Measurement of ethical food choice motives. AB - The two studies describe the development of three complementary scales to the Food Choice Questionnaire developed by Steptoe, Pollard & Wardle (1995). The new items address various ethical food choice motives and were derived from previous studies on vegetarianism and ethical food choice. The items were factor analysed in Study 1 (N=281) and the factor solution was confirmed in Study 2 (N=125), in which simple validity criteria were also included. Furthermore, test-retest reliability was assessed with a separate sample of subjects (N=36). The results indicated that the three new scales, Ecological Welfare (including subscales for Animal Welfare and Environment Protection), Political Values and Religion, are reliable and valid instruments for a brief screening of ethical food choice reasons. PMID- 10744893 TI - Effectiveness of teacher modeling to encourage food acceptance in preschool children. AB - Although Social Cognitive Theory (Bandura, 1997) suggests that teacher modeling would be one of the most effective methods to encourage food acceptance by preschool children, opinions of experienced teachers have not yet been sampled, teacher modeling has rarely been examined experimentally, and it has produced inconsistent results. The present study considers opinions of teachers and conditions under which teacher modeling is effective. Study 1 was a questionnaire in which preschool teachers (N=58) were found to rate modeling as the most effective of five teacher actions to encourage children's food acceptance. Study 2 and Study 3 were quasi-experiments that found silent teacher modeling ineffective to encourage either familiar food acceptance (N=34; 18 boys, 16 girls) or new food acceptance (N=23; 13 boys, 10 girls). Children's new food acceptance was greatest in the first meal and then rapidly dropped, suggesting a "novelty response" rather than the expected neophobia. No gender differences were found in response to silent teacher modeling. Study 4 was a repeated-measures quasi-experiment that found enthusiastic teacher modeling ("Mmm! I love mangos!") could maintain new food acceptance across five meals, again with no gender differences in response to teacher modeling (N=26; 12 boys, 14 girls). Study 5 found that with the addition of a competing peer model, however, even enthusiastic teacher modeling was no longer effective to encourage new food acceptance and gender differences appeared, with girls more responsive to the peer model (N=14; 6 boys, 8 girls). Thus, to encourage children's new food acceptance, present results suggest that teachers provide enthusiastic modeling rather than silent modeling, apply such enthusiastic modeling during the first five meals before children's "novelty response" to new foods drops, and avoid placing competing peer models at the same table with picky eaters, especially girls. PMID- 10744894 TI - Naltrexone does not prevent the weight gain and hyperphagia induced by the antipsychotic drug sulpiride in rats. AB - Few pharmacological tools are currently available to counteract the excessive body weight gain often observed during prolonged administration of antipsychotic drugs. Most antipsychotic drugs block dopamine receptors, and both the brain dopaminergic and opioid systems appear to be involved in initiation and maintenance of feeding behavior, respectively. We evaluated whether the opioid antagonist naltrexone (NAL, 0.5-16 mg/kg/ip for 21 days) (a) affects body weight and food intake in gonadally-intact and drug-free female rats, (b) prevents obesity, hyperphagia, hyperprolactinemia and vaginal cycle disruption induced by long-term administration of the antipsychotic drug sulpiride (SUL, 20 mg/kg/ip for 21 days), or (c) reverses the acute hyperphagia induced by SUL (15 microg bilaterally), when directly applied in the perifornical lateral hypothalamus (PFLH). In drug-free rats, only NAL doses above 4 mg/kg, significantly decreased weight gain and food intake. Even though NAL (1 and 8 mg/kg) significantly attenuated SUL-induced hyperphagia and hyperprolactinemia, it did not reverse at any dose the weight gain and permanent diestrous induced by SUL. In addition, local NAL did not prevent the hyperphagia and polidypsia observed after acute intrahypothalamic SUL. Unexpectedly, the cumulative and 24 h food intake in SUL treated rats was significantly increased by NAL. Collectively, these results do not support a role for endogenous opiates in the neural and endocrine mechanisms involved in weight gain during prolonged antipsychotic drug administration in rats. PMID- 10744895 TI - Pairings of a distinctive chamber with the aftereffect of wheel running produce conditioned place preference. AB - Wheel running reinforces behavior that precedes it. Also, wheel running can produce activity anorexia, a marked suppression of feeding in food-restricted rats. Some authors propose that the activity anorexia effect is produced by activation of the same reward system that mediates the reinforcing effect. One hypothesis is that such activation persists after wheel running stops and results in a rewarding aftereffect that suppresses feeding. Alternatively, such activation may give rise to an opponent process, an aversive aftereffect that suppresses feeding. The method of place conditioning was used to test whether the aftereffect of wheel running is rewarding or aversive. Food-deprived rats received pairings of a distinctive chamber with the aftereffect of wheel running. In Experiment 1, 2 h in a running wheel followed by 30 min in a distinctive chamber produced conditioned place preference. In Experiment 2, 22-22.5 h in a running wheel was followed by 30 min in the chamber and then a 60-min feeding test. Wheel running suppressed feeding and produced conditioned place preference. The conditioned place preference indicates that the aftereffect of wheel running is reinforcing rather than aversive. This finding supports the idea that the activation of the reward system persists after wheel running stops, thereby suppressing food intake. PMID- 10744896 TI - "Pass the ketchup, please": familiar flavors increase children's willingness to taste novel foods. AB - Rozin & Rozin (1981) have suggested that the addition of flavour principles (the distinctive combinations of seasonings which characterize many cuisines) may facilitate the introduction of a new staple food into a culture. That is, the reluctance of omnivores to approach novel foods can be reduced by adding the familiar flavor principle to the unfamiliar food. To test this hypothesis, we "created" flavor principles in the laboratory by exposing children repeatedly to one of two initially novel chip dips. After the exposure phase, they were offered familiar and unfamiliar chips and asked about their willingness to taste each, alone, with the exposed dip, the unexposed dip, and several other dips. The main prediction was that addition of the exposed dip to the unfamiliar chip would increase children's willingness to taste it. Since individuals are not reluctant to taste familiar foods, addition of the exposed dip to the familiar chip was not expected to increase willingness to taste it. The results confirmed this prediction. Practical and theoretical implications of this finding were discussed. PMID- 10744897 TI - Psychophysical advances aid the study of genetic variation in taste. PMID- 10744898 TI - Tool for assessing and reducing an individual's fat intake. PMID- 10744899 TI - Regulation of leptin receptor, POMC and AGRP gene expression by photoperiod and food deprivation in the hypothalamic arcuate nucleus of the male Siberian hamster (Phodopus sungorus). PMID- 10744900 TI - Biological and behavioral consequences of food restriction. PMID- 10744901 TI - The ontogeny of postingestive intake inhibition in rats. PMID- 10744902 TI - Transient hypophagia in rats switched from high-fat diets with different fatty acid pattern to a high-carbohydrate diet. AB - The present study investigates the mechanisms underlying the transient hypophagia occurring when rats adapted to high-fat, carbohydrate-free diets are switched to high-carbohydrate, low-fat diets. The hypophagia after the high-fat, carbohydrate free to high-carbohydrate, low-fat diet shift seems to depend on the amount of carbohydrate in the diet, since an attenuation of hypophagia was observed when high-fat, carbohydrate-free-adapted rats were switched to a medium-carbohydrate, medium-fat diet. A role of glucose intolerance in the hypophagia is supported by the attenuation of carbohydrate anorexia in rats adapted to a high-fat diet containing n -3 polyunsaturated fatty acids from fish oil (60% of fat as fish oil), which has been shown to improve glucose tolerance in rats. Furthermore, the increased plasma glucose concentration in the high-fat, carbohydrate-free diet to high-carbohydrate, low-fat shifted rats despite the suppression in food intake also suggests an involvement of glucose intolerance in the hypophagia. The failure of the inhibitor of hepatic-fatty-acid oxidation mercaptoacetate (400 micromol/kg, i.p.) to counteract carbohydrate anorexia in the HF-adapted rats argues against an involvement of fatty-acids oxidation in the inhibition of eating after high-fat, carbohydrate-free to high-carbohydrate, low-fat diet shift. This is also supported by the failure to demonstrate a relationship between plasma beta-hydroxybutyrate and the severity of the hypophagia. A role of leptin in the hypophagia seems unlikely, since plasma leptin after diet shift was unchanged. Ingestion of the high-carbohydrate, low-fat diet also produced an aversion towards this diet in high-fat, carbohydrate-free-adapted rats. It is concluded that the transient hypophagia induced by switching rats from a high-fat to a high-carbohydrate diet is not related to fatty acid oxidation but to transiently impaired carbohydrate utilization. PMID- 10744903 TI - The vagus nerve is involved in the anorexigenic effect of simmondsin in the rat. AB - Simmondsin, 2-(cyanomethylene)-3 hydroxy 4,5 dimethoxy cyclohexyl beta-D glucoside, from jojoba meal reduces food intake in rats. We investigated the mechanism of action simmondsin, by studying the effects of fasting or of vagotomy on the food intake reduction. The food intake reduction was significantly less in fasted rats than in non-fasted rats. The reduction of food intake was also significantly diminished after vagotomy. The results of the present experiments suggest that simmondsin reduces intake of food in rats through the augmentation of satiety, in part vagally mediated. PMID- 10744904 TI - Activity in the circular alley does not produce the activity anorexia syndrome in rats. AB - The activity anorexia syndrome is characterized by reduced food intake and body weight compared to control levels and increasing levels of physical activity. To induce it, food-restricted rats are confined in running wheels except during the daily meal. We tested whether activity in a flat circular alley also produces the activity anorexia syndrome. In Experiment 1, food-restricted rats were maintained in alleys, wheels, or home cages (control condition). In Experiment 2, they were maintained in alleys, wheels, novel cages, or home cages. The novel cage was added to control for the possibility that the alley might produce an anorectic effect simply because it was a new living space. The alley did not produce the activity anorexia syndrome whereas the wheel did. Although weight loss was greater in the alley than home-cage condition, the alley produced weak, inconsistent suppression of feeding. Moreover, the suppression produced by the alley may have stemmed simply from living in a novel environment. Finally, in contrast to wheel running, alley activity decreased over days. Alley activity, unlike wheel running, may not be reinforcing. Likely, a physical activity must be reinforcing to produce the activity anorexia syndrome. Implications for anorexia nervosa were discussed. PMID- 10744905 TI - Effects of macronutrient content and energy density of snacks consumed in a satiety state on the onset of the next meal. AB - We examined the effects of nutrient composition of a 1 MJ afternoon snack, consumed in a satiety state, on the spontaneous onset of the next meal in 11 young male subjects deprived of any temporal cues. All subjects attended four experimental sessions scheduled 2 weeks apart. The first, baseline, session served to establish: (1) the subjects' ad libitum lunch intake, (2) the latency of the spontaneous request for dinner following lunch, (3) ad libitum food intake at dinner. Lunches provided during the next three sessions were based on baseline lunch intakes. During the following three sessions, conducted in counterbalanced order, subjects were given a high-fat (58% of energy from fat), a high-protein (77%) or a high-carbohydrate (84%) snack to be consumed 240 min after the beginning of lunch. Latency to dinner and the amount of energy consumed at dinner were two dependent variables. Consumption of a high-protein snack delayed the request for dinner by 60 min. In contrast, high-fat snack delayed dinner request by 25 min, whereas high-carbohydrate snack delayed dinner request by 34 min. Snack composition had no impact on energy or macronutrient intakes during dinner. PMID- 10744906 TI - Testing taste sensitivity and aversion in very young children: development of a procedure. AB - Taste perception in 45 3- to 6-year-old children was tested using procedures specifically designed for this age group. Detection thresholds for sucrose and urea were measured by a staircase method and aversion to urea was assessed hedonically, using drawings of facial expressions. All children understood the task and could perform the necessary actions. A subgroup of 20 children participated in a second measurement after a mean interval of 9.5 days: there was a satisfactory degree of stability between the sessions. However, a third measurement, on a subgroup of 13 children after a somewhat longer interval, showed a marked drop in the stability of the urea thresholds. This drop was thought to arise from a decrease in the children's motivation, leading to increased distractibility. Mean threshold estimates were 31 mmol/l for sucrose detection, 59 mmol/l for urea detection and 134 mmol/l for urea aversion, but some children were extremely sensitive to the taste of urea. The findings show that it is possible to study taste perception in very young children if their age is taken into consideration in developing the test procedure. Valid data can be obtained if the procedures are short, easy to understand and intrinsically motivating. PMID- 10744907 TI - Nutritional implications of the cephalic-phase salivary response. AB - The rapid release of saliva following cognitive or sensory stimulation is termed the cephalic phase salivary response. Optimal methods and conditions for measuring the cephalic phase salivary response have not been established. Consequently, determination of the extent to which the large inter and intra individual variability reflect biological or procedural influences is not possible. The nutritional significance of the response may be great due to the multiple roles played by saliva in food selection and digestion. The cephalic phase salivary response has been proposed as an objective index of various individual states (e.g. hunger) and characteristics (e.g. dietary restraint, extroversion), but its value for such purposes requires further verification. PMID- 10744908 TI - Vagal circuitry mediating cephalic-phase responses to food. AB - The dorsal vagal complex in the medulla oblongata is the hub of the central nervous system network that produces vagal cephalic-phase reflexes. The preganglionic motor neurons controlling these cephalic responses of digestion and metabolism are organized topographically in longitudinal columnar subnuclei in the dorsal motor nucleus of the vagus. Gustatory and other visceral afferent inputs project into different subnuclei of the nucleus of the solitary tract capping the dorsal motor nucleus. Descending projections from more rostral stations of the neuroaxis project to the nuclei of the dorsal vagal complex, providing input both from exteroceptive senses, such as olfaction and vision, and from forebrain areas that modulate reflex strength. Recent structural analyses of the dorsal vagal complex, as well as characterizations of the region's inputs and neurochemistry, have provided a more complete understanding of the neural basis of cephalic-phase responses. PMID- 10744909 TI - Nutritional implications of cephalic phase gastrointestinal responses. AB - Palatable cephalic stimuli induce a simultaneous activation of gastrointestinal motility, gastric acid and pancreatic enzyme secretion, as well as, release of the gastrointestinal hormones gastrin and pancreatic polypeptide. Cholinergic neural input is the dominant mediator of these responses with cholecystokinin and gastrin acting as additional stimulatory modulators. Central cholinergic circuits, neuropeptide Y, and thyrotropin releasing hormone are candidate central stimulators of the cephalic phase. There are good arguments for glucagon-like peptide-1 and peptide YY to be physiological inhibitors of cephalic-phase responses with these peptides being released in the intestinal phase of digestion and putatively contributing to termination of the cephalically stimulated pattern. Cephalic-phase responses are used clinically as diagnostic tests to assess completeness of selective proximal vagotomy and to explore autonomic neuropathy. Pancreatic polypeptide secretion with sham feeding is an appropriate test of abdominal vagal function. Cephalically stimulated motor and secretory activity contribute greater than 50% of overall postprandial responses. Pharmacological inhibition of cephalic-vagal stimulation, resulting in reduced food intake, may be a novel approach to obesity management. Glucagon-like peptide 1 is a particular candidate because it inhibits the cephalic phase of digestion, diminishes food intake, and reduces the glycemic excursion after a meal by retarding gastric emptying, stimulating insulin and lowering glucagon release. PMID- 10744910 TI - Cephalic phase of pancreatic secretion. PMID- 10744911 TI - Nutritional implications of the cephalic-phase reflexes: endocrine responses. AB - Cephalic phase hormonal release occurs through activation of vagal-efferent fibers in response to food-related sensory stimuli. Thus, tasting, chewing and expectorating food elicits hormonal release prior to nutrient absorption. Differential sensitivity of cell types within the islet to neural activation determines the profile and magnitude of hormonal release. While the magnitude of cephalic phase insulin release is relatively small (25% above baseline), pancreatic polypeptide, a hormone almost exclusively under vagal control increases 100% above baseline when individuals taste, chew and expectorate food. Thus, the cephalic phase pancreatic polypeptide response is a sensitive indicator of vagal activation to food stimuli. The physiological significance of the cephalic phase hormonal responses is demonstrated by experimental manipulations which inhibit or bypass cephalic phase insulin release. Under these circumstances, hyperglycemia and hyperinsulinemia are evident. Conversely, supplementation of insulin during the preabsorptive time period improves glucose tolerance in certain clinical populations. These data suggest that cephalic phase insulin release plays a role in glucose homeostasis. PMID- 10744912 TI - Nutritional implications of cephalic phase thermogenic responses. AB - The sensory stimulation of feeding produces a cephalic thermogenic response which coincides with an increase of plasma insulin, catecholamines and glucagon. These responses also take place with sham feeding but not with tube feeding. Vagal denervation of the pancreas as well as atropine administration, block the initial release of insulin and reduce by 50% the thermogenic response and norepinephrine release. An adrenergic blocker also reduces this response by 50%. However, with the simultaneous administration of both atropine and an adrenergic beta-blocker, this reduction is not observed. Indirect evidence suggests that this paradox is possibly explained by an enhanced secretion of glucagon. PMID- 10744913 TI - Reproductive hormones and eating behavior in young women. PMID- 10744914 TI - Operationalizing flavor principles in food choice laboratories: comment on Pliner and Stallberg-White. PMID- 10744915 TI - Flavor principles visiting a laboratory: a welcome call, but was it too hasty? PMID- 10744916 TI - The flavor principle: comment on use of the concept by Pliner and Stallberg-White (2000). PMID- 10744918 TI - Introduction to neurobehavioral issues in the neurosurgical treatment of movement disorders: basic issues, thalamotomy, and nonablative treatments. PMID- 10744919 TI - Basal ganglia output and cognition: evidence from anatomical, behavioral, and clinical studies. AB - The traditional view that the basal ganglia are simply involved in the control of movement has been challenged in recent years. Three lines of evidence indicate that the basal ganglia also are involved in nonmotor operations. First, the results of anatomical studies clearly indicate that the basal ganglia participate in multiple circuits or 'loops' with cognitive areas of the cerebral cortex. Second, the activity of neurons within selected portions of the basal ganglia is more related to cognitive or sensory operations than to motor functions. Finally, in some instances basal ganglia lesions cause primarily cognitive or sensory disturbances without gross motor impairments. In this report, we briefly review some of these data and present a new anatomical framework for understanding the basal ganglia contributions to nonmotor function. PMID- 10744920 TI - Comparison of brain activation during word retrieval done silently and aloud using fMRI. AB - Using functional MRI we compared the patterns of activation in an effortful word retrieval task (stem completion) performed both silently and aloud. The silent and overt conditions showed expected differences in activation magnitude in regions such as primary motor cortex. Some regions, such as frontal operculum and dorsolateral frontal cortex, showed similar activation magnitude across conditions. Thalamus was more active on the left in both conditions and showed a symmetric drop in activity in the silent compared with the overt condition. Putamen was also more active in the overt condition and showed a larger decrease in activity on the right than on the left in the silent compared with the overt condition. Thus it appears that silent and overt performance of this task engage the thalamus and putamen in different ways. PMID- 10744921 TI - The role of the human thalamus in language and memory: evidence from electrophysiological studies. AB - The data reviewed here indicate that electrical stimulation of the dominant ventrolateral thalamus can produce deficits in language processing that are not seen after similar stimulation of the nondominant ventrolateral thalamus. The nature of the language deficit produced varies, depending upon the rostrocaudal location of the stimulation site. Stimulation of the anterior left ventrolateral thalamus in right-handed patients resulted in production of a repeated erroneous word, stimulation of the medial ventrolateral thalamus evoked perseveration, and stimulation of the posterior ventrolateral thalamus and anterior pulvinar resulted in misnaming and omissions. Additional studies have examined the effect of electrical thalamic stimulation on verbal and nonverbal short-term memory. Left (but not right) ventrolateral thalamic stimulation during verbal memory input greatly decreased subsequent recall errors, while stimulation during verbal memory retrieval increased recall errors. This finding contrasted with those obtained from studies on nonverbal memory, in which right ventrolateral stimulation during memory input decreased recall errors, while left thalamic stimulation at the same stage increased recall errors. Left pulvinar stimulation disrupted verbal memory processing, while right pulvinar stimulation disrupted nonverbal memory processing. Limited evidence suggests that the effects of thalamic electrical stimulation on verbal memory may persist for several days after the stimulation has ended. The lateralization of thalamic functions also affects the motoric aspects of speech production. Left (but not right) ventrolateral thalamic stimulation disrupted speech articulation and increased the expiratory phase of respiration. The fact that these motor effects were evoked from the same general area of the thalamus that produced the language deficits discussed above raises the possibility that the thalamus is involved in coordinating the cognitive and motoric aspects of language production. A model of thalamic function is discussed in which defined regions of the thalamus operate as a "specific alerting response," increasing the input to memory of category specific material while simultaneously inhibiting retrieval from memory. PMID- 10744922 TI - Neurocognitive correlates of stereotactic thalamotomy and thalamic stimulation in Parkinsonian patients. AB - In the present paper we have reviewed five different studies that relate to neuropsychological consequences of stereotactic thalamotomy and thalamic stimulation in patients with Parkinson's disease. The neuropsychological results are in a strict sense confined to thalamotomy and thalamic stimulation, although the more general message of the importance of investigating cognitive functions before and after surgery applies to other stereotactic techniques for surgical treatment of movement disorders as well. It is argued in the paper that stereotactic thalamotomy provides a unique model for basic research on the neuropsychology of the thalamus, while in return, neuropsychological tests for cognitive dysfunction after surgery may be the most important clinical follow-up. Three general conclusions seem warranted from the data. (1) Parkinsonian patients are impaired on a range of cognitive functions, including language processing, memory, and executive functions. (2) Stereotactic thalamotomy does not further impair the patient; instead, we observed improvement on some tests, particularly verbal memory. (3) In general, there does not seem to be a laterality effect, depending on which side the thalamotomy lesion is applied. An exception to this are dichotically presented simple speech sounds and autonomic responses. In both instances, left-sided brain stimulation produced enhanced performance, while lesioning the left thalamus impaired dichotic listening performance. Finally, we present a new hypothesis for a mechanism behind the thalamotomy effect, based in part on changes in arousal thresholds. PMID- 10744923 TI - Neuropsychological functioning in a patient with essential tremor with and without bilateral VIM stimulation. AB - The effects of deep brain stimulation on motor functions, cognitive abilities, and mood were assessed in an 80-year-old, right-handed male with a chronic history of essential tremor. Electrodes were implanted bilaterally in the ventral intermediate nucleus of the thalamus during a single operation. Upon evaluation at 3 months postsurgery, bilateral stimulation was associated with a clinically significant reduction in tremor ratings and improvement in manual dexterity. At that time, a battery of neuropsychological measures was administered with and without bilateral stimulation. The patient demonstrated comparable performances on measures of visuospatial perception, attention, mental tracking, verbal learning, and verbal recognition memory in both the "on" and "off" conditions. Without stimulation, the patient demonstrated declines of greater than 1 SD on measures of verbal fluency and verbal recall compared to when the stimulators were active. Responses to mood rating scales also indicated greater subjective distress without stimulation. Results are discussed in the context of previous studies of the effects of thalamic stimulation on neurocognitive functioning. PMID- 10744924 TI - Cognitive outcomes after deep brain stimulation for Parkinson's disease: a review of initial studies and recommendations for future research. AB - Modern ablative surgery for movement disorders probably results in less frequent and severe cognitive morbidity than seen in early surgical series. Nonetheless, recent studies indicate that neurobehavioral functions commonly compromised in Parkinson's disease (PD) (e.g., executive functions, verbal fluency, and memory) are negatively impacted in some patients by lesion placement. The potential reversibility of cognitive dysfunction after chronic electrical deep brain stimulation (DBS) for PD has lead some to favor this treatment modality over ablation. This paper reviews the initial studies of the cognitive effects of thalamic, pallidal, and subthalamic DBS. These studies suggest that DBS is relatively safe from a cognitive standpoint and that the benefits of motor improvements probably outweigh the cost of minimal cognitive morbidity. This conclusion must be offered with caution, however, given the small numbers of studies to date and their methodological limitations. Neurobehavioral research has yet to adequately address (1) outcome relative to appropriate control groups; (2) effects of electrode placement versus stimulation; (3) laterality- and site specific effects of DBS; (4) long-term effects of DBS; (5) effects of stimulation parameters; (6) risk factors for cognitive dysfunction with DBS; (7) whether cognitive dysfunction associated with DBS is reversible; and (8) comparative neurobehavioral outcome after DBS and ablation. DBS affords an exciting opportunity to clarify the neurobehavioral role of the basal ganglia. PMID- 10744925 TI - Neuropsychological and behavioral aspects of transplants in Parkinson's disease and Huntington's disease. PMID- 10744926 TI - What is a 'sentinel' lymph node? PMID- 10744927 TI - Tumour micrometastases: the influence of angiogenesis. AB - INTRODUCTION: Many cancer patients have undetected micrometastatic disease at first presentation which ultimately progresses. Angiogenesis-the development of an independent blood supply-is a key event in the growth of metastases. Improved understanding of the influence of angiogenesis on micrometastatic growth may lead to new therapeutic intervention. METHODS: This study examines current concepts of the significance of micrometastases and the role of angiogenesis in their development and destruction. A comprehensive review of the literature on micrometastasis and angiogenesis was performed using the Medline database between 1966 and 1999. CONCLUSIONS: Advances in technology have improved our ability to diagnose metastatic disease, but micrometastases in loco-regional lymph nodes and at distant sites can only be detected by sophisticated histological techniques. While the significance of micrometastases remains controversial, there is increasing evidence that micrometastatic status provides useful prognostic information and should be part of standard staging techniques. Anti-angiogenic therapy has the potential to favourably influence management of certain cancers by manipulating a number of key events in the metastatic process. PMID- 10744928 TI - An update in the palliative management of malignant dysphagia. AB - BACKGROUND: Oesophageal cancer is generally associated with late presentation and poor prognosis. Therefore palliative surgery has been largely superseded by less invasive non-surgical techniques. Once palliation is indicated, the aims of the management should be: the maintenance of oral intake, minimizing hospital stay, relief of pain, elimination of reflux and regurgitation and the prevention of aspiration. METHODS: This study was a review of all published English language data on the palliation of malignant dysphagia between 1994-1999. The Medline and Bids databases were searched and other references were derived from the material perused. RESULTS AND CONCLUSIONS: Palliative treatment for oesophageal cancer should be individualized and relate to tumour stage, size and location, the patient's medical condition and his/her personal wishes. The palliative treatment largely includes self-expanding metal stents (SEMS), laser (including photodynamic therapy (PDT)) or a combination of the two to relieve symptoms, this may be employed with or without other treatments such as radiotherapy/chemotherapy (RT/CT) with the aim of reducing tumour bulk and possibly prolonging survival. A multi-disciplinary approach is vital in patients with advanced oesophageal cancer. PMID- 10744929 TI - Methodological issues in the use of guidelines and audit to improve clinical effectiveness in breast cancer in one United Kingdom health region. AB - AIMS: To develop a system to improve and monitor clinical performance in the management of breast cancer patients in one United Kingdom health region. DESIGN: An observational study of the changes brought about by the introduction of new structures to influence clinical practice and monitor change. SETTING: North Thames (East) Health region, comprising seven purchasing health authorities and 21 acute hospitals treating breast cancer. SUBJECTS: The multi-disciplinary breast teams in 21 hospitals and an audit sample of 419 (28%) of the breast cancer patients diagnosed in 1992 in the region. INTERVENTIONS: Evidence-based interventions for changing clinical practice: regional guidelines, senior clinicians acting as <>, audit of quality rather than cost of services, ownership of data by clinicians, confidential feed-back to participants and education. OUTCOME MEASURES: Qualitative measures of organizational and behavioural change. Quantitative measures of clinical outcomes compared to guideline targets and to results from previous studies within this population. RESULTS: Organizational changes included the involvement, participation of and feedback to 16 specialist surgeons and their multidisciplinary teams in 21 hospitals. Regional clinical guidelines were developed in 6 months and the dataset piloted within 9 months. The audit cycle was completed within 2 years. The pilot study led to prospective audit at the end of 2 years for all breast cancers in the region and a 15-fold increase in high quality clinical information for these patients. Changes in clinical practice between 1990 and 1992 were observed in the use of chemotherapy (up from 17-23%) and axillary surgery (up from 46-76%). CONCLUSIONS: The approach used facilitated rapid change and found a balance between local involvement (essential for sustainability within a hospital setting) and regional standardization (essential for comparability across hospitals). The principles of the approach are generalized to other cancers and to other parts of the UK and abroad. PMID- 10744930 TI - Subcutaneous mastectomy with implant reconstruction: cosmetic outcome and patient satisfaction. AB - AIMS: To assess the cosmetic outcome and satisfaction of patients who have undergone subcutaneous mastectomy (SCM) with silicon implant reconstruction and to investigate the factors influencing cosmesis. METHODS: A total of 101 patients who had had SCM implant reconstruction for treatment of primary breast cancer were assessed for cosmesis by a panel using photographic assessment. Satisfaction and sexuality were studied by a self-evaluation questionnaire. RESULTS: A good to excellent cosmetic result was achieved in 71 (70. 3%) patients (and in 85% of the group who had immediate insertion of the implant). Eighty-one patients (81%) were moderately or very satisfied (96% of the immediate reconstruction group were so). Factors found to influence cosmesis positively were submuscular insertion of the implant (P<0.001), the lateral incision approach (P<0.001) and immediate reconstruction (P<0.001). CONCLUSION: The findings of this study support the use of SCM with implant reconstruction as the simplest option in those advised to undergo mastectomy and who wish for reconstruction. PMID- 10744931 TI - Comparison between low and high pressure suction drainage following axillary clearance. AB - AIM: We report the comparison of low (Exudrain) and high pressure (Redivac) drains in 69 patients following mastectomy and axillary clearance. METHODS: Volume of drainage was recorded daily for 8 days postoperatively. RESULTS: Comparison of daily drainage revealed no statistically significant difference between the two groups of patients (P>0.05). There were no complications specific to the drains and we found similar numbers of infective complications in both groups. Hospital stay was also similar in the two groups (P=0. 70). CONCLUSIONS: We conclude that the two drainage systems are equally effective in their drainage abilities although use of the low pressure may have an advantage over the high pressure system as it obviates the need for bottle changes and therefore requires less time and effort to manage. PMID- 10744932 TI - Sarcoma of the oral and maxillofacial soft tissue in adults. AB - AIMS: Sarcoma occurring in oral and maxillofacial soft tissue is rare. This study was carried out to evaluate the prevalence of oral soft tissue sarcoma and to record its natural history and survival. METHODS: Retrospective analysis of the patients with histologically proven oral and maxillofacial soft tissue sarcoma treated at the Regional Cancer Centre (RCC), Trivandrum, betweeen 1990-1998 was carried out. RESULTS: During this period, ten cases of oral and maxillofacial sarcomas were registered. Three lesions were located on the cheek mucosa, two on the tongue and two on the mandibular alveolus, while there was one lesion each in the parotid region, maxilla and face (NOS). Mean age at presentation was 31.3+/ 14.1 years (range 15-54 years). Seven of the patients (70%) were males. There were three cases of rhabdomyosarcoma (RMS), three cases of spindle cell sarcoma and one case each of angiosarcoma, haemangioendothelioma, malignant schwannoma and malignant fibrous histiocytoma (MFH). All the patients were treated with surgery as a primary modality. Median follow-up time was 30 months (range 5-94 months). An overall srvival of 87.5% at 5 years was observed; however, 5-year disease free survival was 60.0% (95% CI 19.5-85.2). CONCLUSIONS: Soft tissue sarcomas are of comparatively less frequent occurrence in oral and maxillofacial soft tissue than in other tissues. A good survival rate can be achieved by multimodality treatment. PMID- 10744933 TI - Screening women with a family history of breast cancer--results from the British Familial Breast Cancer Group. AB - AIMS: To determine the efficacy of screening women under age 50 with a significant family history of breast cancer. METHODS: Results from 22 Breast Units in the UK identified as being able to provide data were surveyed and pooled through regional data managers or consultant breast specialists. RESULTS: Data relating to 8783 women screened and 9075 woman years of follow-up was analysed. Cancer incidence was 11.3/1000/year. The rate of cancer detection was 4. 78/1000 at prevalent screening and 4.52/1000 at incident screening. Median age at diagnosis was 43 years. Interval cancers presented at a rate of 2.45/1000. Comparison with the National Health Service Breast Screening Programme for women aged 50-64 revealed a similar rate of cancer detection and a similar incidence of ductal carcinoma in situ. The pathological features of screen-detected cancers in this study strongly suggest that prognosis for these women is more favourable than if they had presented symptomatically. CONCLUSIONS: This study provides evidence to suggest that screening young women with a significant family history of breast cancer is effective and that a survival benefit can be expected. As a result the British Familial Breast Cancer Group proposes a co-ordinated prospective observational study. PMID- 10744934 TI - Is dissection of the internerve tissue during axillary lymphadenectomy for breast cancer necessary? AB - AIMS: The study evaluates the necessity of dissecting the tissue between the long thoracic and thoracodorsal nerves (internerve tissue) during axillary dissection in breast cancer surgery. By reviewing the lymph node yield and the metastatic rate in the internerve tissue, we examine whether the internerve tissue could be left in situ to minimize the risk of nerve injury. METHODS: A prospective study was conducted on 30 consecutive women undergoing axillary lymphadenectomy for breast cancer. The internerve tissue remaining was excised separately after a routine axillary dissection and was examined by the same pathologist. RESULTS: Twenty (67%) of 30 internerve specimens contained lymph nodes; the internerve nodes were positive for carcinoma in three cases (10%). In one case the lymph node in the internerve tissue was the only metastatic node in the axilla. CONCLUSIONS: There is a significant incidence of lymph nodes (67%) and axillary node metastases (10%) in the tissue lying between the long thoracic and thoracodorsal nerves. Therefore excision of this internerve tissue is strongly recommended in order to optimize decision making regarding adjuvant treatment and oucome in women with operable breast cancer. PMID- 10744935 TI - Hepatic resection in metastatic breast cancer: results and prognostic factors. AB - AIMS: Breast cancer liver metastases (BCLM) usually indicate the presence of disseminated cancer with a very poor prognosis. However, systemic treatments now allow control of tumour progression in certain cases. We evaluated, in a group of highly selected patients with stabilization or complete response to systemic therapy, a particular management protocol for medically controlled BCLM: <> liver surgery. METHODS: Fifty-two patients underwent surgery between May 1988 and September 1997. Results of this strategy are reported, together with analysis of prognostic factors for survival and recurrence in the remaining liver (RRL). RESULTS: The mean number of cycles of chemotherapy, before surgery, was seven (3-24). Resection was considered to be curative in 86% of cases. The median follow-up was 23 months (1-72 months). The survival after surgery, was 86% at 12 months, 79% at 24 months and 49% at 36 months. The 36-month survival rate differed according to the time to onset of BCLM: 45% before versus 82% after 48 months (P=0.023). The RRL rate at 36 months differed according to the lymph node status of the initial breast cancer: 41% for N0-N1 versus 83% for N1b-N2 (P=0.021). CONCLUSIONS: Adjuvant liver surgery allowed discontinuation of chemotherapy in 46% of cases and, in this highly selected patient group, allowed good quality prolonged survival. It could be included in multicentre treatment protocols for controlled BCLM, one arm with prolonged chemotherapy, one with adjuvant liver surgery. PMID- 10744936 TI - Radical surgery for gallbladder cancer: a worthwhile operation? AB - AIMS: Extended operations are the only chance of a cure for patients with advanced gallbladder carcinoma, but there is no consensus about which subset of patients can benefit. The aim of this retrospective study is to evaluate the results of surgical resection with special reference to the prognostic factors and to long-term survival. METHODS: A retrospective review of 70 patients with a diagnosis of gallbladder cancer treated from 1985-1998 was performed: 33 patients had a curative resection and were included in this study. For stage I disease, simple cholecystectomy was considered curative; in most of the other cases, cholecystectomy was associated with lymph node dissection and liver resection. RESULTS: Hospital mortality and morbidity were 6% and 33%, respectively. Curative resection was associated with an actuarial 5-year survival of 27.4%. Survival of pT1-2 patients was significantly better than that of pT3 (P=0.04) or pT4 patients (P=0.002). Patients with lymph node spread had a poorer prognosis (P=0.06) but four were alive and disease-free with a median survival of 22 months. CONCLUSIONS: Depth of the tumour and lymph node metastases are important prognostic factors. Patients with pT3-4 tumours or regional lymph node spread should be considered for curative resection because long-term survival is possible. PMID- 10744937 TI - Overexpression of p53 protein in gallbladder carcinoma in North India. AB - AIMS: p53 mutations are one of the most frequent genetic alterations in human cancers and are thought to play a role in pathogenesis of several malignancies. Overexpression of p53 in gallbladder cancer has not previously been reported from North India which has one of the highest incidence of this malignancy in the world. The present work is aimed at studying the overexpression of p53 in gallbladder carcinoma occurring in North India. METHODS: p53 overexpression by immunohistochemistry was studied in 20 operative specimens of gallbladder carcinoma. The clinico-pathological observations of these patients were correlated with the extent of p53 overexpression. RESULTS: Seventy per cent (14/20) of specimens of gallbladder carcinoma overexpressed p53 protein. There was a significant correlation between presence of gallstones, T stage, grade of tumour and liver invasion with p53 overexpression. There was no significant correlation with other factors studied viz. age, sex, nodal status and histological type. CONCLUSIONS: The results show a strong association between gallstones and p53 protein overexpression in gallbladder carcinoma. The causal relationship in this association, however, still remains unproven. PMID- 10744938 TI - Management and results of proximal anastomotic leaks in a series of 1114 total gastrectomies for gastric carcinoma. AB - AIMS: The management of anastomotic leakage of the oesophago-jejunostomy after total gastrectomy for gastric carcinoma was evaluated in a retrospective study. PATIENTS AND METHODS: Over a 30-year period, a total of 1114 oesophago jejunostomies were performed during total gastrectomy for gastric cancer. In 83 cases (7.5%) a leak of the oesophago-jejunostomy was diagnosed. RESULTS: Frequency of anastomotic leakage was independent of the type of reconstruction and of surgical radicality. Therapeutic management was conservative in 58 cases (69.9%), with placement of a naso-jejunal tube along the anastomoses and with percutaneous drainage of intraabdominal abscesses. In 25 patients re-operation with resuturing of the anastomoses or surgical drainage of an abscess was performed. Mortality was 11/58 (19%) after conservative treatment of the anastomotic leakage and 16/25 (64%) after re-operation. CONCLUSION: Conservative management with a naso-intestinal tube and percutaneous drainage of intraabdominal abscesses is realistic for anastomotic leaks. Re-operation results in a high morbidity and should only be considered when conservative management is not successful. PMID- 10744940 TI - What should we do? Papillary thyroid carcinoma in a lymph node but normal thyroid tissue--how should we proceed? AB - Papillary cell carcinoma of the thyroid is a relatively indolent disease, usually presenting as an asymptomatic mass in the thyroid gland that is either noted by the patient or diagnosed at routine clinical examination. Although the prognosis is generally good there are a number of controversies in the management of this condition. The significance of age at presentation, size of the tumour and the presence of lymph node metastasis are still disputed. We present the unusual case of a woman found to have papillary cell thyroid carcinoma within a lymph node during a cosmetic thyroid lobectomy. The thyroid tissue removed was not found to contain tumour. We describe the evidence behind the decision to proceed to total thyroidectomy and the finding of a tiny focus of papillary cell thyroid carcinoma within the contralateral lobe. PMID- 10744939 TI - Popliteal lymph node metastasis from primary cutaneous melanoma. AB - AIMS: To document the incidence of popliteal lymph node involvement by metastatic melanoma and to consider the implications of this information for clinical management. METHODS: From the computerized database of the Sydney Melanoma Unit, all patients with primary melanomas located at or distal to the knee were identified and their records were examined. Experience with those patients who developed popliteal node metastases was then reviewed. RESULTS: Thirteen of 4262 patients (0.31%) with primary melanomas of the distal lower limb developed popliteal node metastases. Six of the 13 patients had previous, synchronous or subsequent groin node metastases. CONCLUSIONS: Popliteal lymph node involvement by metastatic melanoma is a rare event. The study results suggest only two indications for full popliteal node clearance-either a histologically positive sentinel node in the popliteal fossa or clinical evidence of metastatic disease in a popliteal node. PMID- 10744941 TI - Modelling in tumour biology part II: modelling cancer therapy. PMID- 10744942 TI - Giant melanoma of the left thumb. AB - A 74-year-old female patient is described with a giant melanoma of the left thenar and concomitant bilateral pulmonary metastases. Palliative treatment consisted of a two-staged procedure in order to save the limb from amputation. Firstly, perfusion with gamma-interferon, tumour necrosis factor- alpha and melphalan was carried out, after which the tumour had been reduced to one third of its initial volume. Secondly, excision of the tumour and coverage of the wound with a split skin graft was done. Remarkably, the extent of the multiple pulmonary metastases was temporary and diminished 1 month after perfusion, although no systemic leakage could be determined during the procedure. The management strategy is discussed with emphasis on this indication for limb perfusion. PMID- 10744943 TI - Second primary malignant melanoma on a skin graft used to cover the defect of the first primary's wide excision. AB - The unique case of a second primary malignant melanoma developing on a split thickness skin graft that had been used for reconstruction of the defect following the wide excision of a first primary malignant melanoma is reported. A review of the literature revealed that no similar case has been reported before. PMID- 10744944 TI - Radical hysterectomy for IB cervical cancer in a patient with aorto-femoral transposition. AB - We present a cervical cancer case in stage IB, according to FIGO classification, treated with radical hysterectomy and pelvic lymphadenectomy. The 48-year-old patient had 4 years previously undergone a Y aorto-bifemoral Dallon transposition as a result of Leriche's syndrome. During the routine investigation invasive cervical cancer was diagnosed. She had radical hysterectomy of Piver III type and partial pelvic lymphadectomy. Radical hysterectomy caused no technical trouble. Pelvic lymphadenectomy was only partially possible because of hard connective tissue around the artificial vessels. This scarred region made safe preparation of the total pelvic lymphatic system impossible. PMID- 10744945 TI - The effect of surgical wounding on tumour development. PMID- 10744947 TI - Reply to Dr beechey-newman PMID- 10744946 TI - The true sentinel node by different injection techniques in breast cancer patients. PMID- 10744949 TI - Reply to messrs leris and mokbel PMID- 10744948 TI - Re: perioperative acute phase response modification by polyadenylic-polyuridylic acid. PMID- 10744951 TI - Alkaline protease from Spilosoma obliqua: potential applications in bio formulations. AB - Some properties of the purified alkaline protease from larvae of the insect Spilosoma obliqua (Lepidoptera) and its potential application as an additive in various bio-formulations are reported. The novel feature of the present study is the use of insect protease. The protease was found to be compatible with some of the commercial detergents tested, and was also effective in cleaving various protein substrates tested, albeit to different extents, implying broader substrate specificity and effectiveness of the protease against a wide variety of stains. This property of the protease can also be exploited by using it as an active component in enzymic debriders in view of its ability to digest various protein substrates. The insect protease appears to be potentially useful as an additive in detergent, stain remover and other bio-formulations. PMID- 10744950 TI - Characterization of Mucor pusillus rennin expressed in Pichia pastoris: enzymic, spectroscopic and calorimetric studies. AB - The aspartic proteinase gene of Mucor pusillus rennin expressed in Pichia pastoris was characterized in terms of structural and conformational stability induced by temperature. This enzyme is 12% glycosylated, with a similar specific activity to the native fungal enzyme. The secondary structure determined by CD is mainly due to beta-sheet structures with an important contribution of aromatic components. The calorimetric studies were carried out in the temperature range in which the enzyme is most stable. The enzyme undergoes an irreversible, highly scan-rate-dependent thermal denaturation under all the experimental conditions investigated. Between pH 3.0 and 7.0, only one endotherm characterized the thermal denaturation of enzyme. At pH 5.0, the most stable condition found, the denaturation can be fitted to the two-state irreversible model. Thus the kinetic constant and activation parameters of the denaturation process could be obtained. Upon reaching pH 7.5, the denaturation is characterized by two endotherms. This evidence indicates the complex tridimensional structure of this enzyme. Finally, taking into account the conservative tertiary structure of the aspartic proteinase family we comment on our results with reference to the crystallographic structure of M. pusillus pepsin [Newman, Watson, Roychowdhury, Badasso, Cleasby, Wood, Tickle and Blundell (1993) J. Mol. Biol. 221, 1295-1309]. PMID- 10744952 TI - High-level expression of tetanus toxin fragment C-thioredoxin fusion protein in Escherichia coli. AB - An insert of Clostridium tetani DNA corresponding to fragment C of tetanus toxin was amplified by PCR. This 1.4 kb fragment was cloned into the high-expression vector pET32a, under control of the T7 promoter. Expression of this plasmid in Escherichia coli BL21(DE3) resulted in the production of a fusion protein ( approximately 62 kDa) consisting of 112 amino acids of thioredoxin and approximately 450 amino acids of fragment C. This fusion protein was recognized by anti-tetanus toxoid antiserum in an ELISA and on immunoblots. The recombinant fragment-C-thioredoxin protein was purified significantly in one step by Ni(2+) chelate Sepharose, the final yield being approximately 35 mg/l. Immunization of animals with the recombinant protein produced antibodies that were able to recognize the tetanus toxin. By using this gene-fusion expression system we produced soluble fragment C of tetanus toxin in a high yield, preventing many problems inherent in the use of other expression systems that produce either insoluble fragment C in inclusion bodies, or a soluble form, but in low yield, using E. coli as the expression host. PMID- 10744953 TI - Yeast protease B-digested skimmed milk inhibits angiotensin-I-converting-enzyme activity. AB - Angiotensin-1-converting-enzyme (ACE) inhibitory activity was identified in skimmed milk digested with cell-free extract of yeast Saccharomyces cerevisiae. Simultaneously, a protease enzyme involved in the production of ACE-inhibition materials in digested skimmed milk was purified to homogeneity from the cell-free extracts of S. cerevisiae by ammonium sulphate fractionation and chromatography in DEAE-Sephacel, D-tryptophan methyl ester-Sepharose 4B, Hiload Superdex G-200 and HPLC Mono-Q chromatography. The purified enzyme was identified as protease B, based on the molecular mass on SDS/PAGE and the N-terminal amino acid sequence of the enzyme. The optimum pH for digestion of skimmed milk and production of ACE inhibition materials was pH 4.8. The IC(50) of the hydrolysate was 0.42 mg of protein/ml when skimmed milk was digested with yeast protease B. PMID- 10744954 TI - Albumin purification from human placenta. AB - Albumin is the human protein used mainly for therapeutic purposes. Besides the traditionally used plasma, blood from placenta is an alternative source for albumin purification. We describe here an industrial process for purification of albumin from human placenta. The proposed albumin-purification process, for 50 kg of placentas, comprises: (i) extraction of haemolysed blood with saline and solid/liquid separation by basket centrifugation; (ii) selective precipitation of haemoglobin by ethanol/chloroform and precipitate removal by filtration in a press filter; (iii) concentration/diafiltration of the filtrate in a 30 kDa cross flow ultrafiltration (CFUF) membrane; (iv) thermo-coagulation at 70 degrees C with sodium octanoate/EDTA; (v) treatment with activated charcoal at pH 3; (vi) concentration/diafiltration of the filtrate in a 30 kDa CFUF membrane; (vii) anion-exchange chromatography Q-Sepharose; (viii) hydrophobic-interaction chromatography with phenyl-Sepharose; and (ix) conditioning and pasteurization. The process yields an average of 4.5 g of albumin/kg of placenta with a purity of 97.1% and A(403) of 0.05 (1% protein). The final product passes pyrogen and toxicity tests in vivo and it does not contain polymers or aggregates, even after the accelerated stability test, as judged by gel filtration, as required by the Brazilian Pharmacopoeia. PMID- 10744955 TI - Direct analysis of single-nucleotide polymorphism on double-stranded DNA by pyrosequencing. AB - Pyrosequencing, a new method for DNA sequencing, is gaining widespread use for many different types of DNA analysis. The method takes advantage of four coupled enzymes in a single tube assay to monitor DNA synthesis in real time using a luminometric detection system. Here, we demonstrate the use of pyrosequencing for direct analysis of single-nucleotide polymorphism on double-stranded PCR product. Pyrosequencing data on the human glutathione peroxidase gene (GPX1) from several individuals were analysed and three different allelic variants were determined and confirmed. The possibility of further simplifying the sequencing and template preparation steps is discussed. PMID- 10744956 TI - Purification and characterization of a highly stable tyrosinase from Thermomicrobium roseum. AB - Tyrosinase, with an isoelectric point at pH 4.9, was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermomicrobium roseum. Gel filtration, N-terminal amino acid sequencing and SDS/PAGE analysis indicate that T. roseum tyrosinase is composed of two identical subunits, each with a molecular mass of 43000 Da. The enzyme exhibited high substrate specificity towards catechol, chlorogenic acid, L-3-(3,4-dihydroxyphenyl)-L-alanine (L-DOPA) and pyrogallol. The K(m) value of the enzyme for L-DOPA was 0.18 mM. beta Mercaptoethanol and sodium diethyldithiocarbamate notably inhibited the enzymic activity. The activity of the enzyme was optimal at pH 9.5 and 70 degrees C, and was increased by addition of 1 mM Mg(2+), K(+) or Cu(2+). The enzyme was highly stable against high temperature and guanidine hydrochloride. The N-terminal amino acid sequence of the enzyme was determined to be Asp-Ile-Asn-Gly-Gly-Gly-Ala-Thr Leu-Pro-Gln-Lys-Leu-Tyr. These facts indicate that T. roseum tyrosinase appears to be distinct from the tyrosinases so far purified from other sources. PMID- 10744957 TI - Effective induction, purification and characterization of Trichoderma koningii G 39 beta-xylosidase with high transferase activity. AB - A beta-xylosidase was induced and purified from the culture filtrate of Trichoderma koningii G-39, grown in a medium containing 1% oat spelts xylan and 0.1% xylose. The presence of xylose unequivocally enhanced the induction of beta xylosidase. The purified enzyme, which exhibited a significant alpha arabinosidase activity, was obtained with high yield simply via ethanol precipitation and a single anion-exchange chromatography and was characterized as a monomeric glycoprotein with an estimated molecular mass of 104 kDa and a pI of 4.6. The K(m) values towards p-nitrophenyl beta-D-xylopyranoside and p nitrophenyl alpha-L-arabinopyranoside are 0.04 and 7.5 mM, respectively. It is stable at pH 2.5-7.4, 37 degrees C. The pH and temperature optima are in the range of 3.5-4.0 and 55-60 degrees C, respectively. Contrary to most beta xylosidases from other sources, Hg(2+) (up to 25 mM) has no effect on enzyme activity. Xylose was shown to inhibit the purified enzyme with a moderate K(i) value of 5 mM. The enzyme exhibited transxylosylation activity and was characterized as a 'retaining' enzyme, catalysing the hydrolysis of substrate with the retention of anomeric configuration. PMID- 10744958 TI - Improvement of Staphylococcus aureus-V8-protease hydrolysis of bovine haemoglobin by its adsorption on to a solid phase in the presence of SDS: peptide mapping and obtention of two haemopoietic peptides. AB - Hydrolysis of bovine haemoglobin by the V8 protease from Staphylococcus aureus (EC 3.4.21.19) was studied in the presence of SDS in a homogeneous-phase and in a solid-phase system. In both cases, hydrolyses were performed at 37 degrees C, in 50 mM phosphate buffer, pH 6.0, containing 0.1% SDS. Solid-phase hydrolyses were carried out with haemoglobin adsorbed on a negatively charged hydrophobic support, namely Amberlyst 15Wet (Rohm and Haas). The peptides were isolated from the hydrolysates by reverse-phase HPLC and analysed for their amino acid composition on a Waters Pico-Tag column, confirmed by second-order derivative spectrometry or by MS. A peptide map of the hydrolysates was drawn up, and numerous new cleavages in haemoglobin chains were observed, especially after Asp. This study showed that SDS permitted a dramatic improvement in the hydrolysis of whole haemoglobin by V8 protease in both homogeneous-phase and solid phase systems after adsorption of haemoglobin on to an anionic support. Moreover, in the heterogeneous phase, all the theoretical cleavage sites of V8 protease, Asp as well as Glu bonds, were hydrolysed, except for four sites which were resistant owing to strong interactions with the support. These results led to us obtain two haemopoietic peptides, namely peptide alpha (Leu(76)-Pro-Gly-Ala-Leu-Ser-Glu(82)) and peptide beta (Lys(94)-Leu-His-Val-Asp-Pro-Glu(100)). These active peptides have never before been prepared from bovine haemoglobin, and they may have great potentialities in biotechnology. PMID- 10744959 TI - Advances in microbial amylases. AB - This review makes a comprehensive survey of microbial amylases, i.e. alpha amylase, beta-amylase and glucoamylase. Amylases are among the most important enzymes and are of great significance in present-day biotechnology. Although they can be derived from several sources, such as plants, animals and micro-organisms, the enzymes from microbial sources generally meet industrial demands. Microbial amylases could be potentially useful in the pharmaceutical and fine-chemical industries if enzymes with suitable properties could be prepared. With the advent of new frontiers in biotechnology, the spectrum of amylase application has widened in many other fields, such as clinical, medicinal and analytical chemistries, as well as their widespread application in starch saccharification and in the textile, food, brewing and distilling industries. In this review, after a brief description of the sources of amylases, we discuss the molecular biology of amylases, describing structures, cloning, sequences, and protoplast fusion and mutagenesis. This is followed by sections on their production and finally the properties of various amylases. PMID- 10744960 TI - Examination of bioaffinity immobilization by precipitation of mannan and mannan containing enzymes with legume lectins. AB - The interaction of four lectins from crops of the legume family with Saccharomyces cerevisiae alpha-mannan, and also with two glycoenzymes containing mainly alpha-mannan moieties, has been studied. The interaction was characterized by a quantitative precipitation assay. The results of precipitation differ with respect to both quality (the point of maximum precipitation) and of the quantity (the amount of aggregated lectin and saccharide). The lectin concanavalin A [Con A, from jack bean (Canavalia ensiformis)] was observed to form more extensive precipitates with Saccharomyces cerevisiae mannan and glycoenzymes than did lectins from Lens culinaris (lentil) and Pisum sativum (garden pea), while in the case of Vicia faba (broad or fava bean) no interaction was found with either the examined mannans or with glycosylated enzymes. The complete precipitation of invertase and glucoamylase with Con A (enzymes and also Con A; up to 100%) was achieved at a Con A glycoenzyme molar ratio of 20.2 and 2.3 respectively, whereby about 85% of precipitated and also of initial activities of glycoenzymes were determined in the aggregates. More valuable results were achieved by the technique of enzyme immobilization called 'multiple bioaffinity layering' which is based on the stepwise biospecific adsorption of the glycosylated enzymes and Con A on a matrix precoupled with Con A. A 3-fold repetition of the layering procedure afforded up to a 10-fold increase in catalytic activity of the immobilized invertase, in contrast with a 2.1-fold increase in catalytic activity of the immobilized glucoamylase. PMID- 10744961 TI - An evaluation of hybridization kinetics in biosensors using a single-fractal analysis. AB - The diffusion-limited hybridization kinetics of analyte in solution to receptor immobilized on a biosensor or immunosensor surface is analysed within a fractal framework. The data may be analysed by a single-fractal analysis. This was indicated by the regression analysis provided by Sigmaplot [Sigmaplot Users Manual (1993) Jandel Scientific, San Rafael, CA]. It is of interest to note that the binding-rate coefficient and the fractal dimension both exhibit changes in the same and in the opposite directions for the single example presented in each case. The binding-rate coefficient(s) expressions developed as a function of the analyte (DNA) concentration in solution and the fractal dimension are of particular value, since they provide a means to better control biosensor or immunosensor performance and provide physical insights into the hybridization process. PMID- 10744962 TI - Cell polarity: Nailing Crumbs to the scaffold. AB - Recent studies suggest that the cytoplasmic tail of the Crumbs protein establishes membrane territories in polarised epithelial cells by stabilising a PDZ-domain protein complex. The isolation of a human Crumbs homologue required for normal vision revives questions about the role of Crumbs in cell-cell interactions. PMID- 10744963 TI - Motor control: Mechanisms of motor equivalence in handwriting. AB - Handwriting is a classic example of how the details of movement can be scale and plane invariant: letter forms reflecting personal style are unchanged, whether one is writing on a piece of paper, on a blackboard or in the sand using the foot. Recent research points to a role for the parietal cortex in such motor equivalence. PMID- 10744964 TI - Synaptic plasticity: Building memories to last. AB - A series of recent studies has provided long-awaited direct evidence that enduring changes in synaptic strength, presumably underlying the formation of persistent memories, may be encoded in a lasting form as a change in synaptic structure. PMID- 10744965 TI - Blood coagulation: The outstanding hydrophobic residues. AB - Newly determined crystal structures suggest that the membrane-binding C2 domains of blood coagulation cofactors Va and VIIIa bind anionic phospholipids through protruding solvent-exposed hydrophobic residues, aided by a crown of positively charged residues and by specific hydrogen-bonding side chains. PMID- 10744966 TI - Visual perception: Learning to see through noise. AB - New studies show that perceptual learning does not reduce the noise inherent to the neuronal mechanisms of perception. Rather, learning boosts the brain's capability to extract and make use of the relevant outside signal - but where and how the neuronal changes occur is still unknown. PMID- 10744967 TI - Transcriptional control: The benefits of selective insulation. AB - In eukaryotes, cis-regulatory sequences are often a long way away from the transcription start site, and interactions between regulatory elements can be blocked by 'insulator' sequences. A novel type of cis-regulatory element has now been found that selectively permits some interactions across insulators. PMID- 10744969 TI - Pattern formation: Wingless on the move. AB - Wingless is a key morphogen in Drosophila. Although it is evident that Wingless acts at a distance from its site of synthesis, there is considerable debate about how the protein travels across a field of cells. Recent studies have provided important new insights into this process, though the issue is still far from being resolved. PMID- 10744968 TI - T cells: A proliferation of costimulatory molecules. AB - The identification and characterization of a newly extended family of molecules related to the T-cell costimulatory proteins CD28 and B7 suggests that a distinct form of costimulatory signals could be important for effector T-cell responses outside of lymphoid tissues. PMID- 10744970 TI - Bacterial division: Finding the dividing line. AB - Division of a cell - whether eukaryotic or prokaryotic - requires accurate spatial coordination. Recent work on the bacterium Escherichia coli has shown that correct placement of the cell division site at the midcell position occurs by a combination of selection against potential polar sites and selection of the midcell site. PMID- 10744971 TI - Microarray analysis of the transcriptional network controlled by the photoreceptor homeobox gene Crx. AB - BACKGROUND: Terminal differentiation of many cell types is controlled and maintained by tissue- or cell-specific transcription factors. Little is known, however, of the transcriptional networks controlled by such factors and how they regulate differentiation. The paired-type homeobox transcription factor, Crx, has a pivotal role in the terminal differentiation of vertebrate photoreceptors. Mutations in the human CRX gene result in either congenital blindness or photoreceptor degeneration and targeted mutation of the mouse Crx results in failure of development of the light-detecting outer segment of photoreceptors. RESULTS: We have characterized the transcriptional network controlled by Crx by microarray analysis of gene expression in developing retinal tissue from Crx(+/+) and Crx(-/-) mice. These data were combined with analyses of gene expression in developing and adult retina, as well as adult brain. The most abundant elements of this network are ten photoreceptor-specific or -enriched genes, including six phototransduction genes. All of the available 5' regulatory regions of the putative Crx targets contain a novel motif that is composed of a head-to-tail arrangement of two Crx-binding-element-like sequences. Analysis of the 5' regions of a set of mouse and human genes suggests that this motif is specific to Crx targets. CONCLUSIONS: This study demonstrates that cDNA microarrays can be successfully used to define the transcriptional networks controlled by transcription factors in vertebrate tissue in vivo. PMID- 10744972 TI - Wingless gradient formation in the Drosophila wing. AB - BACKGROUND: Secreted signaling proteins of the Wingless (Wg)/Wnt, Hedgehog and bone morphogenetic protein (BMP)/Decapentaplegic (Dpp) families function as morphogens to control growth and pattern formation during development. Although these proteins have been shown to act directly on distant cells in the developing limbs of the fruit fly Drosophila, little is known about how ligand gradients form in vivo. Wg protein is found in vesicles in Wg-responsive cells in the embryo and imaginal discs. It has been proposed that Wg may be transported by a vesicle-mediated mechanism. RESULTS: A novel method to visualize extracellular Wg protein was used to show that Wg forms an unstable gradient on the basolateral surface of the wing imaginal disc epithelium. Wg movement did not require internalization by dynamin-mediated endocytosis. Dynamin activity was, however, required for Wg secretion. By reversibly blocking Wg secretion, we found that Wg moves rapidly to form a long-range extracellular gradient. CONCLUSIONS: The Wg morphogen gradient forms by rapid movement of ligand through the extracellular space, and depends on continuous secretion and rapid turnover. Endocytosis is not required for Wg movement, but contributes to shaping the gradient by removing extracellular Wg. We propose that the extracellular Wg gradient forms by diffusion. PMID- 10744973 TI - Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft enriched vesicles through WASP-Arp2/3. AB - BACKGROUND: Phosphatidylinositol 4,5-bisphosphate (PIP(2)) has been implicated in the regulation of the actin cytoskeleton and vesicle trafficking. It stimulates de novo actin polymerization by activating the pathway involving the Wiskott Aldrich syndrome protein (WASP) and the actin-related protein complex Arp2/3. Other studies show that actin polymerizes from cholesterol-sphingolipid-rich membrane microdomains called 'rafts', in a manner dependent on tyrosine phosphorylation. Although actin has been implicated in vesicle trafficking, and rafts are sites of active phosphoinositide and tyrosine kinase signaling that mediate apically directed vesicle trafficking, it is not known whether phosphoinositide regulation of actin dynamics occurs in rafts, or if it is linked to vesicle movements. RESULTS: Overexpression of type I phosphatidylinositol phosphate 5-kinase (PIP5KI), which synthesizes PIP(2), promoted actin polymerization from membrane-bound vesicles to form motile actin comets. Pervanadate (PV), a tyrosine phosphatase inhibitor, induced comets even in the absence of PIP5KI overexpression. PV increased PIP(2) levels, suggesting that it induces comets by changing PIP(2) homeostasis and by increasing tyrosine phosphorylation. Platelet-derived growth factor (PDGF) enhanced PV-induced comet formation, and these stimuli together potentiated the PIP5KI effect. The vesicles at the heads of comets were enriched in PIP5KIs and tyrosine phosphoproteins. WASP-Arp2/3 involvement was established using dominant-negative WASP constructs. Endocytic and exocytic markers identified vesicles enriched in lipid rafts as preferential sites of comet generation. Extraction of cholesterol with methyl beta-cyclodextrin reduced comets, establishing that rafts promote comet formation. CONCLUSIONS: Sphingolipid-cholesterol rafts are preferred platforms for membrane-linked actin polymerization. This is mediated by in situ PIP(2) synthesis and tyrosine kinase signaling through the WASP-Arp2/3 pathway. Actin comets may provide a novel mechanism for raft-dependent vesicle transport and apical membrane trafficking. PMID- 10744974 TI - Phosphorylation and spindle pole body localization of the Cdc15p mitotic regulatory protein kinase in budding yeast. AB - Cdc15p is an essential protein kinase and functions with a group of late mitotic proteins that includes Lte1p, Tem1p, Cdc14p and Dbf2p/Dbf20p to inactivate Cdc28p Clb2p at the end of mitosis in budding yeast [1] [2]. Cdc14p is activated and released from the nucleolus at late anaphase/telophase to dephosphorylate important regulators of Cdc28p-Clb2p such as Hct1p/Cdh1p, Sic1p and Swi5p in a CDC15-dependent manner [3] [4] [5] [6] [7]. How Cdc15p itself is regulated is not known. Here, we report that both the phosphorylation and localization of Cdc15p are cell cycle regulated. The extent of phosphorylation of Cdc15p gradually increases during cell-cycle progression until some point during late anaphase/telophase when it is rapidly dephosphorylated. We provide evidence suggesting that Cdc14p is the phosphatase responsible for the dephosphorylation of Cdc15p. Using a Cdc15p fusion protein coupled at its carboxyl terminus to green fluorescent protein (GFP), we found that Cdc15p, like its homologue Cdc7p [8] in fission yeast, localizes to the spindle pole bodies (SPBs) during mitosis. At the end of telophase, a portion of Cdc15p is located at the mother-bud neck, suggesting a possible role for Cdc15p in cytokinesis. PMID- 10744975 TI - Evidence for the derivation of the Drosophila fushi tarazu gene from a Hox gene orthologous to lophotrochozoan Lox5. AB - The DNA-binding homeobox motif was first identified in several Drosophila homeotic genes but also in fushi tarazu, a gene found in the Hox cluster yet involved in segmentation, not anteroposterior patterning [1]. Homeotic transformations are not seen in insect ftz mutants, and insect ftz genes do not have Hox-like expression except within the nervous system [2] [3]. Insect ftz homeobox sequences link them to the Antp-class genes and Tribolium and Schistocerca orthologs have Antp-class YPWM motifs amino-terminal to the homeobox [2] [3]. Orthologs of ftz cloned from a centipede and an onychophoran [4] show that it predates the emergence of the arthropods, but the inability to pinpoint non-arthropodan orthologs suggested that ftz is the product of a Hox gene duplication in the arthropod ancestor [4] [5]. I have cloned ftz orthologs from a mite and a tardigrade, arthropod outgroups of the insects [6]. Mite ftz is expressed in a Hox-like pattern, confirming its ancestral role in anteroposterior patterning. Phylogenetic analyses indicate that arthropod ftz genes are orthologous to the Lox5 genes of lophotrochozoans (a group that includes molluscs) [7] and, possibly, with the Mab-5 genes of nematodes and Hox6 genes of deuterostomes and would therefore have been present in the triploblast ancestor. PMID- 10744976 TI - The progeny of wingless-expressing cells deliver the signal at a distance in Drosophila embryos. AB - Pattern formation in developing animals requires that cells exchange signals mediated by secreted proteins. How these signals spread is still unclear. It is generally assumed that they reach their target site either by diffusion or active transport (reviewed in [1] [2]). Here, we report an alternative mode of transport for Wingless (Wg), a member of the Wnt family of signaling molecules. In embryos of the fruit fly Drosophila, the wingless (wg) gene is transcribed in narrow stripes of cells abutting the source of Hedgehog protein. We found that these cells or their progeny are free to roam towards the anterior. As they do so, they no longer receive the Hedgehog signal and stop transcribing wg. The cells leaving the expression domain retain inherited Wg protein in secretory vesicles, however, and carry it forwards over a distance of up to four cell diameters. Experiments using a membrane-tethered form of Wg showed that this mechanism is sufficient to account for the normal range of Wg. Nevertheless, evidence exists that Wg can also reach distant target cells independently of protein inheritance, possibly by restricted diffusion. We suggest that both transport mechanisms operate in wild type embryos. PMID- 10744977 TI - The human Rad52 protein exists as a heptameric ring. AB - The RAD52 epistasis group was identified in yeast as a group of genes required to repair DNA damaged by ionizing radiation [1]. Genetic evidence indicates that Rad52 functions in Rad51-dependent and Rad51-independent recombination pathways [2] [3] [4]. Consistent with this, purified yeast and human Rad52 proteins have been shown to promote single-strand DNA annealing [5] [6] [7] and to stimulate Rad51-mediated homologous pairing [8] [9] [10] [11]. Electron microscopic examinations of the yeast [12] and human [13] Rad52 proteins have revealed their assembly into ring-like structures in vitro. Using both conventional transmission electron microscopy and scanning transmission electron microscopy (STEM), we found that the human Rad52 protein forms heptameric rings. A three-dimensional (3D) reconstruction revealed that the heptamer has a large central channel. Like the hexameric helicases such as Escherichia coli DnaB [14] [15], bacteriophage T7 gp4b [16] [17], simian virus 40 (SV40) large T antigen [18] and papilloma virus E1 [19], the Rad52 rings show a distinctly chiral arrangement of subunits. Thus, the structures formed by the hexameric helicases may be a more general property of other proteins involved in DNA metabolism, including those, such as Rad52, that do not bind and hydrolyze ATP. PMID- 10744978 TI - Constitutive expression of stromal derived factor-1 by mucosal epithelia and its role in HIV transmission and propagation. AB - HIV particles that use the chemokine receptor CXCR4 as a coreceptor for entry into cells (X4-HIV) inefficiently transmit infection across mucosal surfaces [1], despite their presence in seminal fluid and mucosal secretions from infected individuals [2] [3] [4]. In addition, although intestinal lymphocytes are susceptible to infection with either X4-HIV particles or particles that use the chemokine receptor CCR5 for viral entry (R5-HIV) during ex vivo culture [5], only systemic inoculation of R5-chimeric simian-HIV (S-HIV) results in a rapid loss of CD4(+) intestinal lymphocytes in macaques [6]. The mechanisms underlying the inefficient capacity of X4-HIV to transmit infection across mucosal surfaces and to infect intestinal lymphocytes in vivo have remained elusive. The CCR5 ligands RANTES, MIP-1alpha and MIP-1beta suppress infection by R5-HIV-1 particles via induction of CCR5 internalization, and individuals whose peripheral blood lymphocytes produce high levels of these chemokines are relatively resistant to infection [7] [8] [9]. Here, we show that the CXCR4 ligand stromal derived factor 1 (SDF-1) is constitutively expressed by mucosal epithelial cells at sites of HIV transmission and propagation. Furthermore, CXCR4 is selectively downmodulated on intestinal lymphocytes within the setting of prominent SDF-1 expression. We postulate that mucosally derived SDF-1 continuously downmodulates CXCR4 on resident HIV target cells, thereby reducing the transmission and propagation of X4-HIV at mucosal sites. Moreover, such a mechanism could contribute to the delayed emergence of X4 isolates, which predominantly occurs during the later stages of the HIV infection. PMID- 10744979 TI - The p150-Spir protein provides a link between c-Jun N-terminal kinase function and actin reorganization. AB - The Jun N-terminal kinase (JNK) is a downstream effector of Rac and Cdc42 GTPases involved in actin reorganization [1-3]. A role of the Drosophila JNK homologue, Basket (DJNK/Bsk), in the regulation of cell shape changes and actin reorganization arises from its function in the process of dorsal closure [4-6]. One potential mechanism for induction of cytoskeletal changes by JNK is via transcriptional activation of the decapentaplegic gene (dpp, a member of the TGFbeta superfamily) [6]. A direct link between JNK signalling and actin organization has not yet been found, however. We have identified a novel DJNK interacting protein, p150-Spir, that belongs to the Wiscott-Aldrich syndrome protein (WASP) homology domain 2 (WH2) family of proteins involved in actin reorganization [7] [8]. It is a multidomain protein with a cluster of four WH2 domains, a modified FYVE zinc-finger motif [9], and a DEJL motif, a docking site for JNK [10], at its carboxy-terminal end. In mouse fibroblasts, p150-Spir colocalized with F-actin and its overexpression induced clustering of filamentous actin around the nucleus. When coexpressed with p150-Spir in NIH 3T3 cells, JNK translocated to and colocalizes with p150-Spir at discrete spots around the nucleus. Carboxy-terminal sequences of p150-Spir were phosphorylated by JNK both in vitro and in vivo. We conclude that p150-Spir is a downstream target of JNK function and provides a direct link between JNK and actin organization. PMID- 10744980 TI - LICOS, a primordial costimulatory ligand? AB - In mammals, the classical B7 molecules expressed on antigen-presenting cells, B7 1 (CD80) and B7-2 (CD86), bind the structurally related glycoproteins CD28 and CTLA-4 (CD152), generating costimulatory signals that regulate the activation state of T cells. A recently identified human CD28-like protein, ICOS, also induces costimulatory signals in T cells when crosslinked with antibodies, but it is unclear whether ICOS is part of a B7-mediated regulatory pathway of previously unsuspected complexity, or whether it functions independently and in parallel. Here, we report that, rather than binding B7-1 or B7-2, ICOS binds a new B7 related molecule of previously unknown function that we call LICOS (for ligand of ICOS). At 37 degrees C, LICOS binds only to ICOS but, at lower, non-physiological temperatures, it also binds weakly to CD28 and CTLA-4. Sequence comparisons suggest that LICOS is the homologue of a molecule expressed by avian macrophages and of a murine protein whose expression is induced in non-lymphoid organs by tumour necrosis factor alpha (TNFalpha). Our results define the components of a distinct and novel costimulatory pathway and raise the possibility that LICOS, rather than B7-1 or B7-2, is the contemporary homologue of a primordial vertebrate costimulatory ligand. PMID- 10744981 TI - G-protein-coupled receptors function as oligomers in vivo. AB - Hormones, sensory stimuli, neurotransmitters and chemokines signal by activating G-protein-coupled receptors (GPCRs) [1]. Although GPCRs are thought to function as monomers, they can form SDS-resistant dimers, and coexpression of two non functional or related GPCRs can result in rescue of activity or modification of function [2-10]. Furthermore, dimerization of peptides corresponding to the third cytoplasmic loops of GPCRs increases their potency as activators of G proteins in vitro [11], and peptide inhibitors of dimerization diminish beta(2)-adrenergic receptor signaling [3]. Nevertheless, it is not known whether GPCRs exist as monomers or oligomers in intact cells and membranes, whether agonist binding regulates monomer-oligomer equilibrium, or whether oligomerization governs GPCR function. Here, we report that the alpha-factor receptor, a GPCR that is the product of the STE2 gene in the yeast Saccharomyces cerevisiae, is oligomeric in intact cells and membranes. Coexpression of receptors tagged with the cyan or yellow fluorescent proteins (CFP or YFP) resulted in efficient fluorescence resonance energy transfer (FRET) due to stable association rather than collisional interaction. Monomer-oligomer equilibrium was unaffected by binding of agonist, antagonist, or G protein heterotrimers. Oligomerization was further demonstrated by rescuing endocytosis-defective receptors with coexpressed wild type receptors. Dominant-interfering receptor mutants inhibited signaling by interacting with wild-type receptors rather than by sequestering G protein heterotrimers. We suggest that oligomerization is likely to govern GPCR signaling and regulation. PMID- 10744982 TI - Biology in pictures. Another cell in the wall. PMID- 10744983 TI - Human genetic diseases. PMID- 10744984 TI - What lies ahead? PMID- 10744985 TI - Energy homeostasis and body weight. PMID- 10744986 TI - The immunological synapse. PMID- 10744987 TI - Protein transport into mitochondria. AB - Mitochondria are made up of two membrane systems that subdivide this organelle into two aqueous subcompartments: the matrix, which is enclosed by the inner membrane, and the intermembrane space, which is located between the inner and the outer membrane. Protein import into mitochondria is a complex reaction, as every protein has to be routed to its specific destination within the organelle. In the past few years, studies with mitochondria of Neurospora crassa and Saccharomyces cerevisiae have led to the identification of four distinct translocation machineries that are conserved among eukaryotes. These translocases, in a concerted fashion, mediate import and sorting of proteins into the mitochondrial subcompartments. PMID- 10744988 TI - RNA polymerase structure-function: insights into points of transcriptional regulation. AB - The crystal structure of Thermus aquaticus RNA polymerase (RNAP) with 3.3 A resolution has recently been described. The high degree of sequence similarity between T. aquaticus RNAP and the prototypical RNAP from Escherichia coli invites comparison of the new structural data with genetic and biochemical results that defined the interaction sites of E. coli RNAP with transcription regulators. PMID- 10744989 TI - Regulatory circuits in Caulobacter. AB - The transcriptional regulator CtrA controls DNA replication, DNA methylation and cell division during the Caulobacter cell cycle. Recent work on this master cell cycle regulator has provided insight into its control mechanisms. Feedback regulation of ctrA transcription together with timed proteolysis of CtrA determine the levels of the regulator during the cell cycle. Multiple phosphorylation pathways regulate CtrA activity by a mechanism that includes dynamic subcellular localization of the corresponding sensor kinases. PMID- 10744990 TI - Cell and nuclear recognition mechanisms mediated by mating type in filamentous ascomycetes. AB - Sexual development in filamentous ascomycetes requires mating-type genes to mediate recognition of compatible cell and nuclear types. Characterization of mating-type genes from various fungi shows that they primarily encode transcriptional regulators. Recent studies on mating-type-specific pheromones and internuclear recognition have shed light on how mating-type genes specify mating and nuclear identity in filamentous ascomycetes. PMID- 10744991 TI - Post-transcriptional control by global regulators of gene expression in bacteria. AB - Several authentic or potential global regulators have recently been shown to act at the post-transcriptional level. This is the case for Hfq (HF-1), which is involved in the regulation of an increasing number of genes in Escherichia coli, and CsrA (RsmA) responsible for controlling the expression of genes for extracellular enzymes and secondary metabolism in Gram-negative bacteria. The cold-shock proteins of the CspA family are able to destabilise mRNA secondary structures at low temperature and, therefore, also seem to act post transcriptionally. These findings illustrate a more general aspect of post transcriptional control which, in the past, was generally restricted to regulators acting at a single target. The expression of several global transcriptional regulators, such as the stationary phase and heat-shock sigma factors and H-NS, have also recently been shown to be themselves under post transcriptional control. These examples underline the importance of this type of control in bacterial gene regulation. PMID- 10744992 TI - U-turns and regulatory RNAs. AB - Conventional antisense RNAs, such as those controlling plasmid replication and maintenance, inhibit the function of their target RNAs rapidly and efficiently. Novel findings show that a common U-turn loop structure mediates fast RNA pairing in the majority of these RNA controlled systems. Usually, an antisense RNA regulates a single, cognate target RNA only. Recent reports, however, show that antisense RNAs can act as promiscuous regulators that control multiple genes in concert to integrate complex physiological responses in Escherichia coli. PMID- 10744993 TI - Microbial circadian oscillatory systems in Neurospora and Synechococcus: models for cellular clocks. AB - Common regulatory patterns have emerged among the feedback loops lying within circadian systems. Significant progress in dissecting the mechanism of clock resetting by temperature and the role of the WC proteins in the Neurospora light response has accompanied documentation of the importance of nuclear localization and phosphorylation-induced turnover of FRQ to this circadian cycle. The long awaited molecular description of a transcription/translation loop in the Synechococcus circadian system represents a quantal step forward, followed by the identification of additional important proteins and interactions. Finally, the adaptive significance of rhythms in Synechococcus and by extension in all clocks nicely ties up an extraordinary year. PMID- 10744994 TI - Transcriptional silencing in bacteria. AB - Transcriptional silencing and repression are modes of negative control of gene expression that differ in specificity. Repressors, when present at promoter specific binding sites, interfere locally with RNA polymerase function. Silencing proteins act by covering a continuous region of DNA, compete with a broader spectrum of proteins and are non-specific with respect to the promoters affected. Studies of transcriptional silencing promise an entree to relatively unexplored areas of prokaryotic biology. PMID- 10744995 TI - Bacterial heme sources: the role of heme, hemoprotein receptors and hemophores. AB - The major mechanisms by which Gram-negative bacteria acquire heme from host heme carrier proteins involve either direct binding to specific outer membrane receptors or release of bacterial hemophores that take up heme from host heme carriers and shuttle it back to specific receptors. The ability to interact with and remove heme from carrier proteins distinguishes heme from conceptually similar siderophore and vitamin B12 receptors. Recent genetic, biochemical and crystallization studies have started to unravel the mechanism and molecular interactions between heme-carrier proteins and components of bacterial heme assimilation systems. PMID- 10744996 TI - Quorum sensing and starvation: signals for entry into stationary phase. AB - Quorum sensing occurs at high cell density in many microorganisms. It regulates specialized processes such as genetic competence, bioluminescence, virulence, and sporulation. However, recent evidence suggests that quorum-sensing may play a more central role in the physiology of bacteria, where quorum-sensing pathways converge with starvation-sensing pathways to regulate cell entry into stationary phase. PMID- 10744997 TI - The biogenesis and assembly of bacterial membrane proteins. AB - Bacterial proteins in the inner and outer membranes differ dramatically in their architecture. Although both types of proteins are transported across the inner membrane through a common pore, recent studies have identified distinct factors that target them to transport sites and catalyze proper folding. PMID- 10744998 TI - Diversity of homology-dependent gene silencing strategies in fungi. AB - The experimental introduction of DNA in fungi has revealed a variety of gene inactivation strategies triggered in response to the presence of homologous nucleic acid sequences. These homology effects reveal distinct classes of gene inactivation resulting from hypermutation or relying on regulatory mechanisms that operate at either the transcriptional or the post-transcriptional level. PMID- 10744999 TI - The Sir proteins of Saccharomyces cerevisiae: mediators of transcriptional silencing and much more. AB - The Sir2, Sir3, and Sir4 proteins of the yeast Saccharomyces cerevisiae elicit transcriptional silencing by forming repressive chromatin structures that are confined to specific chromosomal domains. Recent discoveries establish new and unexpected roles for the proteins in seemingly unrelated arenas of chromosome biology, including double-strand break repair, structure and function of the nucleolus, aging, cell cycle regulation, and checkpoint control. PMID- 10745000 TI - The fungal GATA factors. AB - The DNA-binding domains of eucaryotic GATA factors comprise a four-cysteine Zn finger and an adjacent basic region. Fungal GATA factors regulate nitrogen metabolism, light induction, siderophore biosynthesis and mating-type switching. Hydrophobic interactions determine binding-site specificity. Interactions with other factors may determine promoter specificity. One GATA factor has recently been shown to determine a drastic chromatin rearrangement. PMID- 10745001 TI - Two-component and phosphorelay signal transduction. AB - Two-component and phosphorelay signal transduction systems are the major means by which bacteria recognize and respond to a variety of environmental stimuli. Recent results have implicated these systems in the regulation of a variety of essential processes including cell-cycle progression, pathogenicity, and developmental pathways. Elucidation of the structures of the interacting domains is leading to an understanding of the mechanisms of molecular recognition and phosphotransfer in these systems. PMID- 10745002 TI - Transcription termination control in bacteria. AB - Transcription termination is a dynamic process and is subject to control at a number of levels. New information about the molecular mechanisms of transcription elongation and termination, as well as new insights into protein-RNA interactions, are providing a framework for increased understanding of the molecular details of transcription termination control. PMID- 10745003 TI - Protein folding and unfolding by Escherichia coli chaperones and chaperonins. AB - The folding of proteins from their initial unstructured state to their mature form has long been known to be promoted by other proteins known as chaperones and chaperonins. Recent biochemical and structural discoveries have provided dramatic insight into how these folding proteins work. This review will discuss these findings and suggest future experimental directions. PMID- 10745004 TI - The coiled-coil trigger site of the rod domain of cortexillin I unveils a distinct network of interhelical and intrahelical salt bridges. AB - BACKGROUND: The parallel two-stranded alpha-helical coiled coil is the most frequently encountered subunit-oligomerization motif in proteins. The simplicity and regularity of this motif have made it an attractive system to explore some of the fundamental principles of protein folding and stability and to test the principles of de novo design. RESULTS: The X-ray crystal structure of the 18 heptad-repeat alpha-helical coiled-coil domain of the actin-bundling protein cortexillin I from Dictyostelium discoideum is a tightly packed parallel two stranded alpha-helical coiled coil. It harbors a distinct 14-residue sequence motif that is essential for coiled-coil formation, and is a prerequisite for the assembly of cortexillin I. The atomic structure reveals novel types of ionic coiled-coil interactions. In particular, the structure shows that a characteristic interhelical and intrahelical salt-bridge pattern, in combination with the hydrophobic interactions occurring at the dimer interface, is the key structural feature of its coiled-coil trigger site. CONCLUSIONS: The knowledge gained from the structure could be used in the de novo design of alpha-helical coiled coils for applications such as two-stage drug targeting and delivery systems, and in the design of coiled coils as templates for combinatorial helical libraries in drug discovery and as synthetic carrier molecules. PMID- 10745005 TI - A mutant Shiga-like toxin IIe bound to its receptor Gb(3): structure of a group II Shiga-like toxin with altered binding specificity. AB - BACKGROUND: Shiga-like toxins (SLTs) are produced by the pathogenic strains of Escherichia coli that cause hemorrhagic colitis and hemolytic uremic syndrome. These diseases in humans are generally associated with group II family members (SLT-II and SLT-IIc), whereas SLT-IIe (pig edema toxin) is central to edema disease of swine. The pentameric B-subunit component of the majority of family members binds to the cell-surface glycolipid globotriaosyl ceramide (Gb(3)), but globotetraosyl ceramide (Gb(4)) is the preferred receptor for SLT-IIe. A double mutant of the SLT-IIe B subunit that reverses two sequence differences from SLT II (GT3; Gln65-->Glu, Lys67-->Gln, SLT-I numbering) has been shown to bind more strongly to Gb(3) than to Gb(4). RESULTS: To understand the molecular basis of receptor binding and specificity, we have determined the structure of the GT3 mutant B pentamer, both in complex with a Gb(3) analogue (2.0 A resolution; R = 0.155, R(free) = 0.194) and in its native form (2.35 A resolution; R = 0.187, R(free) = 0.232). CONCLUSIONS: These are the first structures of a member of the medically important group II Shiga-like toxins to be reported. The structures confirm the previous observation of multiple binding sites on each SLT monomer, although binding site 3 is not occupied in the GT3 structure. Analysis of the binding properties of mutants suggests that site 3 is a secondary Gb(4)-binding site. The two mutated residues are located appropriately to interact with the extra betaGalNAc residue on Gb(4). Differences in the binding sites provide a molecular basis for understanding the tissue specificities and pathogenic mechanisms of members of the SLT family. PMID- 10745006 TI - Crystal structure of human branched-chain alpha-ketoacid dehydrogenase and the molecular basis of multienzyme complex deficiency in maple syrup urine disease. AB - BACKGROUND: Mutations in components of the extraordinarily large alpha-ketoacid dehydrogenase multienzyme complexes can lead to serious and often fatal disorders in humans, including maple syrup urine disease (MSUD). In order to obtain insight into the effect of mutations observed in MSUD patients, we determined the crystal structure of branched-chain alpha-ketoacid dehydrogenase (E1), the 170 kDa alpha(2)beta(2) heterotetrameric E1b component of the branched-chain alpha ketoacid dehydrogenase multienzyme complex. RESULTS: The 2.7 A resolution crystal structure of human E1b revealed essentially the full alpha and beta polypeptide chains of the tightly packed heterotetramer. The position of two important potassium (K(+)) ions was determined. One of these ions assists a loop that is close to the cofactor to adopt the proper conformation. The second is located in the beta subunit near the interface with the small C-terminal domain of the alpha subunit. The known MSUD mutations affect the functioning of E1b by interfering with the cofactor and K(+) sites, the packing of hydrophobic cores, and the precise arrangement of residues at or near several subunit interfaces. The Tyr- >Asn mutation at position 393-alpha occurs very frequently in the US population of Mennonites and is located in a unique extension of the human E1b alpha subunit, contacting the beta' subunit. CONCLUSIONS: Essentially all MSUD mutations in human E1b can be explained on the basis of the structure, with the severity of the mutations for the stability and function of the protein correlating well with the severity of the disease for the patients. The suggestion is made that small molecules with high affinity for human E1b might alleviate effects of some of the milder forms of MSUD. PMID- 10745007 TI - Crystal structure of Rab geranylgeranyltransferase at 2.0 A resolution. AB - BACKGROUND: Rab geranylgeranyltransferase (RabGGT) catalyzes the addition of two geranylgeranyl groups to the C-terminal cysteine residues of Rab proteins, which is crucial for membrane association and function of these proteins in intracellular vesicular trafficking. Unlike protein farnesyltransferase (FT) and type I geranylgeranyltransferase, which both prenylate monomeric small G proteins or short peptides, RabGGT can prenylate Rab only when Rab is in a complex with Rab escort protein (REP). RESULTS: The crystal structure of rat RabGGT at 2.0 A resolution reveals an assembly of four distinct structural modules. The beta subunit forms an alpha-alpha barrel that contains most of the residues in the active site. The alpha subunit consists of a helical domain, an immunoglobulin (Ig)-like domain, and a leucine-rich repeat (LRR) domain. The N-terminal region of the alpha subunit binds to the active site in the beta subunit; residue His2alpha directly coordinates a zinc ion. The prenyl-binding pocket of RabGGT is deeper than that in FT. CONCLUSIONS: LRR and Ig domains are often involved in protein-protein interactions; in RabGGT they might participate in the recognition and binding of REP. The binding of the N-terminal peptide of the alpha subunit to the active site suggests an autoinhibition mechanism that might contribute to the inability of RabGGT to recognize short peptides or Rab alone as its substrate. Replacement of residues Trp102beta and Tyr154beta in FT by Ser48beta and Leu99beta, respectively, in RabGGT largely determine the different lipid-binding specificities of the two enzymes. PMID- 10745008 TI - The 'fingerprint' that X-rays can leave on structures. AB - BACKGROUND: Exposure of biomacromolecules to ionising radiation results in damage that is initiated by free radicals and progresses through a variety of mechanisms. A widely used technique to study the three-dimensional structures of biomacromolecules is crystallography, which makes use of ionising X-rays. It is crucial to know to what extent structures determined using this technique might be biased by the inherent radiation damage. RESULTS: The consequences of radiation damage have been investigated for three dissimilar proteins. Similar results were obtained for each protein, atomic B factors increase, unit-cell volumes increase, protein molecules undergo slight rotations and translations, disulphide bonds break and decarboxylation of acidic residues occurs. All of these effects introduce non-isomorphism. The absorbed dose in these experiments can be reached during routine data collection at undulator beamlines of third generation synchrotron sources. CONCLUSIONS: X-rays can leave a 'fingerprint' on structures, even at cryogenic temperatures. Serious non-isomorphism can be introduced, thus hampering multiple isomorphous replacement (MIR) and multiwavelength anomalous dispersion (MAD) phasing methods. Specific structural changes can occur before the traditional measures of radiation damage have signalled it. Care must be taken when assigning structural significance to features that might easily be radiation-damage-induced changes. It is proposed that the electron-affinic disulphide bond traps electrons that migrate over the backbone of the protein, and that the sidechains of glutamic acid and aspartic acid donate electrons to nearby electron holes and become decarboxylated successively. The different disulphide bonds in each protein show a clear order of susceptibility, which might well relate to their intrinsic stability. PMID- 10745009 TI - Structure and function of mutationally generated monomers of dimeric phosphoribosylanthranilate isomerase from Thermotoga maritima. AB - BACKGROUND: Oligomeric proteins may have been selected for in hyperthermophiles because subunit association provides extra stabilization. Phosphoribosylanthranilate isomerase (PRAI) is monomeric and labile in most mesophilic microorganisms, but dimeric and stable in the hyperthermophile Thermotoga maritima (tPRAI). The two subunits of tPRAI are associated back-to back and are locked together by a hydrophobic loop. The hypothesis that dimerization is important for thermostability has been tested by rationally designing monomeric variants of tPRAI. RESULTS: The comparison of tPRAI and PRAI from Escherichia coli (ePRAI) suggested that levelling the nonplanar dimer interface would weaken the association. The deletion of two residues in the loop loosened the dimer. Subsequent filling of the adjacent pocket and the exchange of polar for apolar residues yielded a weakly associating and a nonassociating monomeric variant. Both variants are as active as the parental dimer but far more thermolabile. The thermostability of the weakly associating monomer increased significantly with increasing protein concentration. The X-ray structure of the nonassociating monomer differed from that of the parental subunit only in the restructured interface. The orientation of the original subunits was maintained in a crystal contact between two monomers. CONCLUSIONS: tPRAI is dimeric for reasons of stability. The clearly separated responsibilities of the betaalpha loops, which are involved in activity, and the alphabeta loops, which are involved in protein stability, has permitted the evolution of dimers without compromising their activity. The preserved interaction in the crystal contacts suggests the most likely model for dimer evolution. PMID- 10745010 TI - Structure of the zinc-binding domain of Bacillus stearothermophilus DNA primase. AB - BACKGROUND: DNA primases catalyse the synthesis of the short RNA primers that are required for DNA replication by DNA polymerases. Primases comprise three functional domains: a zinc-binding domain that is responsible for template recognition, a polymerase domain, and a domain that interacts with the replicative helicase, DnaB. RESULTS: We present the crystal structure of the zinc binding domain of DNA primase from Bacillus stearothermophilus, determined at 1.7 A resolution. This is the first high-resolution structural information about any DNA primase. A model is discussed for the interaction of this domain with the single-stranded DNA template. CONCLUSIONS: The structure of the DNA primase zinc binding domain confirms that the protein belongs to the zinc ribbon subfamily. Structural comparison with other nucleic acid binding proteins suggests that the beta sheet of primase is likely to be the DNA-binding surface, with conserved residues on this surface being involved in the binding and recognition of DNA. PMID- 10745011 TI - Crystal structure of cathepsin X: a flip-flop of the ring of His23 allows carboxy monopeptidase and carboxy-dipeptidase activity of the protease. AB - BACKGROUND: Cathepsin X is a widespread, abundantly expressed papain-like mammalian lysosomal cysteine protease. It exhibits carboxy-monopeptidase as well as carboxy-dipeptidase activity and shares a similar activity profile with cathepsin B. The latter has been implicated in normal physiological events as well as in various pathological states such as rheumatoid arthritis, Alzheimer's disease and cancer progression. Thus the question is raised as to which of the two enzyme activities has actually been monitored. RESULTS: The crystal structure of human cathepsin X has been determined at 2.67 A resolution. The structure shares the common features of a papain-like enzyme fold, but with a unique active site. The most pronounced feature of the cathepsin X structure is the mini-loop that includes a short three-residue insertion protruding into the active site of the protease. The residue Tyr27 on one side of the loop forms the surface of the S1 substrate-binding site, and His23 on the other side modulates both carboxy monopeptidase as well as carboxy-dipeptidase activity of the enzyme by binding the C-terminal carboxyl group of a substrate in two different sidechain conformations. CONCLUSIONS: The structure of cathepsin X exhibits a binding surface that will assist in the design of specific inhibitors of cathepsin X as well as of cathepsin B and thereby help to clarify the physiological roles of both proteases. PMID- 10745012 TI - RNA folding: beyond Watson-Crick pairs. AB - Several crystal structures of RNA fragments, alone or in complex with a specific protein, have been recently solved. In addition, the structures of an artificial ribozyme, the leadzyme, and the cleavage product of a human pathogen ribozyme, have extended the structural diversity of ribozyme architectures. The attained set of folding rules and motifs expand the repertoire seen previously in tRNA structures. PMID- 10745013 TI - Human glucose-6-phosphate dehydrogenase: the crystal structure reveals a structural NADP(+) molecule and provides insights into enzyme deficiency. AB - BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first committed step in the pentose phosphate pathway; the generation of NADPH by this enzyme is essential for protection against oxidative stress. The human enzyme is in a dimer<-->tetramer equilibrium and its stability is dependent on NADP(+) concentration. G6PD deficiency results from many different point mutations in the X-linked gene encoding G6PD and is the most common human enzymopathy. Severe deficiency causes chronic non-spherocytic haemolytic anaemia; the usual symptoms are neonatal jaundice, favism and haemolytic anaemia. RESULTS: We have determined the first crystal structure of a human G6PD (the mutant Canton, Arg459-->Leu) at 3 A resolution. The tetramer is a dimer of dimers. Despite very similar dimer topology, there are two major differences from G6PD of Leuconostoc mesenteroides: a structural NADP(+) molecule, close to the dimer interface but integral to the subunit, is visible in all subunits of the human enzyme; and an intrasubunit disulphide bond tethers the otherwise disordered N-terminal segment. The few dimer-dimer contacts making the tetramer are charge-charge interactions. CONCLUSIONS: The importance of NADP(+) for stability is explained by the structural NADP(+) site, which is not conserved in prokaryotes. The structure shows that point mutations causing severe deficiency predominate close to the structural NADP(+) and the dimer interface, primarily affecting the stability of the molecule. They also indicate that a stable dimer is essential to retain activity in vivo. As there is an absolute requirement for some G6PD activity, residues essential for coenzyme or substrate binding are rarely modified. PMID- 10745014 TI - Electrons and X-rays gang up on the ribosome. AB - In all cells, protein synthesis is coordinated by the ribosome, and a number of pivotal structural studies on this complex have been completed during 1999. The combined results of the X-ray crystallography and electron microscopy studies have shed new light on the mechanism of this molecular machine. PMID- 10745015 TI - RNA bulges as architectural and recognition motifs. AB - RNA bulges constitute versatile structural motifs in the assembly of RNA architectures. Three-dimensional structures of RNA molecules and their complexes reveal the role of bulges in RNA architectures and illustrate the molecular mechanisms by which they confer intramolecular interactions and intermolecular recognition. PMID- 10745016 TI - Paul sigler PMID- 10745017 TI - Crystallographic analysis of the specific yet versatile recognition of distinct nuclear localization signals by karyopherin alpha. AB - BACKGROUND: Karyopherin alpha (importin alpha) is an adaptor molecule that recognizes proteins containing nuclear localization signals (NLSs). The prototypical NLS that is able to bind to karyopherin alpha is that of the SV40 T antigen, and consists of a short positively charged sequence motif. Distinct classes of NLSs (monopartite and bipartite) have been identified that are only partly conserved with respect to one another but are nevertheless recognized by the same receptor. RESULTS: We report the crystal structures of two peptide complexes of yeast karyopherin alpha (Kapalpha): one with a human c-myc NLS peptide, determined at 2.1 A resolution, and one with a Xenopus nucleoplasmin NLS peptide, determined at 2.4 A resolution. Analysis of these structures reveals the determinants of specificity for the binding of a relatively hydrophobic monopartite NLS and of a bipartite NLS peptide. The peptides bind Kapalpha in its extended surface groove, which presents a modular array of tandem binding pockets for amino acid residues. CONCLUSIONS: Monopartite and bipartite NLSs bind to a different number of amino acid binding pockets and make different interactions within them. The relatively hydrophobic monopartite c-myc NLS binds extensively at a few binding pockets in a similar manner to that of the SV40 T antigen NLS. In contrast, the bipartite nucleoplasmin NLS engages the whole array of pockets with individually more limited but overall more abundant interactions, which include the NLS two basic clusters and the backbone of its non-conserved linker region. Versatility in the specific recognition of NLSs relies on the modular. PMID- 10745018 TI - Three-dimensional organization of the lamina reticularis in the rat tracheal basement membrane zone. AB - The airway basement membrane zone is a region specialized for the attachment of the epithelium with the matrix. The epithelium is attached to the lamina densa, which, in turn, is connected to types I and III collagen of the lamina reticularis with anchoring fibrils. The purpose of this study was to define the three-dimensional organization of the structural proteins of the lamina reticularis in the rat trachea. We approached this problem by using whole mounts to look down on the flat surface of the basement-membrane zone rather than a cross section of its thin profile. Fluorescent microscopy with long working distance water immersion objectives and scanning electron microscopy revealed that the structural proteins are arranged as a mat of large fibers oriented along the longitudinal axis of the airway. Smaller fibers are crosslinked with the larger fibers to complete this structure. Other small fibers are oriented around the large fibers and an amorphous material covers individual fibers. The large fibers oriented along the longitudinal axis of the airway are consistent with prior descriptions of fibers composed of collagen III with some collagen I and V; small fibers encircling the large fibers may be collagen VI. The crosslinking fibers are made up of elastin and probably elastin-associated microfibrils. The amorphous proteins covering the fibrous framework may contain proteoglycans and other nonstructural proteins reported to be in the lamina reticularis. The present studies demonstrate that the structural proteins of the lamina reticularis in the rat trachea are arranged as fibers in a highly organized manner. PMID- 10745019 TI - Intraepithelial lymphocytes in the lung: a neglected lymphocyte population. PMID- 10745020 TI - Coagulation abnormalities in acute lung injury and sepsis. PMID- 10745021 TI - Human bronchial intraepithelial T lymphocytes as a distinct T-cell subset: their long-term survival in SCID-Hu chimeras. AB - Intestinal intraepithelial T lymphocytes (i-IELs) show features different from those of conventional T cells and play specific roles in the mucosal immunity. To investigate whether human bronchial intraepithelial T lymphocytes (IELs) are a distinct entity, we examined T cells in human bronchial xenografts transplanted on mice with severe combined immune deficiency (SCID). We transplanted human bronchi subcutaneously into mice with SCID, resected the xenografts after various incubation periods (7-174 d), and examined them for CD3(+), CD4(+), CD8(+), and CD45(+) cells by immunohistochemistry. The number of CD3(+) cells in the lamina propria decreased significantly in the first month (from 308.7 +/- 60.2 to 70.9 +/- 49. 4/mm(2); P < 0.05), and xenografts more than 5 mo of age had scant T cells in the lamina propria (5.2 +/- 2.0/mm(2)). However, there was no significant difference between the number of CD3(+) IELs in freshly isolated bronchi and in xenografts maintained for more than 5 mo. In freshly isolated bronchi, the number of CD4(+) IELs was significantly lower than that of CD8(+) cells (2.35 +/- 0.62 versus 4.56 +/- 1.32/mm basement membrane; P < 0.01). After transplantation, the mean CD4-to-CD8 ratio of all xenografts was significantly higher than that of freshly isolated bronchi (5.2 +/- 0.9 versus 0.6 +/- 0.2; P < 0.005). The IELs were positive for CD45, which is specific for human leukocytes, and they were eliminated by irradiation before the transplantation. Almost all IELs (99.5%) in the xenografts expressed alphabeta T-cell receptor, and 35.8% of IELs expressed alpha(e)beta7 integrin. Bronchial epithelial cells in the xenografts expressed interleukin (IL)-7, stem cell factor, intercellular adhesion molecule (ICAM)-1, and human leukocyte antigen-DR (HLA-DR). We conclude that in the SCID-Hu chimera model, human bronchial IELs survive for more than 5 mo, unlike the T cells in the lamina propria, and we suggest that human bronchial IELs may be stimulated by bronchial epithelial cells expressing IL-7, stem cell factor, ICAM-1, and HLA-DR. PMID- 10745022 TI - Priming for enhanced alveolar fibrin deposition after hemorrhagic shock: role of tumor necrosis factor. AB - Hemorrhagic shock due to major trauma predisposes to the development of acute respiratory distress syndrome. Because lung fibrin deposition is one of the hallmarks of this syndrome, we hypothesized that resuscitated shock might predispose to the development of a net procoagulant state in the lung. A rodent model of shock/resuscitation followed by low-dose intratracheal lipopolysaccharide (LPS), a clinically relevant "two-hit" model, was used to test this hypothesis. Resuscitated shock primed the lungs for an increased tissue factor and plasminogen activator (PA) inhibitor-1 gene expression in response to LPS, while the fibrinolytic PA was reduced. These alterations were recapitulated in isolated alveolar macrophages, suggesting their role in the process. LPS induced tumor necrosis factor (TNF) was also augmented in animals after antecedent shock/resuscitation, and studies using anti-TNF antibodies revealed that TNF expression was critical to the induction of the procoagulant molecules and the reduction in PA. By contrast, TNF did not appear to play an important role in neutrophil sequestration in this model, inasmuch as anti-TNF had no effect on lung neutrophil accumulation or chemokine expression. However, treatment prevented albumin leak by preventing alveolar neutrophil activation. The inclusion of the antioxidant N-acetyl-cysteine in the resuscitation fluid resulted in prevention of both the development of the net procoagulant state and lung neutrophil sequestration, suggesting a role for upstream oxidant effects in the priming process. These studies provide a cellular and molecular basis for lung fibrin deposition after resuscitated shock and demonstrate a divergence of pathways responsible for fibrin generation and neutrophil accumulation. PMID- 10745023 TI - Studies on the expression of endothelin, its receptor subtypes, and converting enzymes in lung cancer and in human bronchial epithelium. AB - Lung cancer, particularly small cell lung cancer (SCLC), is characterized by production of numerous peptides and their resulting clinical syndromes. Such peptides can act as autocrine growth factors for these tumors. In this study, we investigated the role of endothelin (ET)-1 in lung cancer. Using reverse transcription/polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay, and immunocytochemistry, we screened a panel of lung cancer cell lines for ET-1, its receptors, and endothelin converting enzyme-1 (ECE-1), which generates the active form of ET-1. ET-1 messenger RNA was expressed in five of seven SCLC, four of four non-small cell lung cancer (NSCLC), and human bronchial epithelial (HBE) cells. The intracellular isoform of ECE-1, important in processing ET-1 if an autocrine growth loop is to function, was downregulated in the lung cancer cell lines as compared with expression of the extracellular isoform. Endothelin A receptor (ETAR), which mediates the mitogenic effects of ET-1 in prostate and ovarian cancer, was upregulated in HBE cells compared with expression in three of seven SCLC and two of four NSCLC cell lines. Endothelin B receptor (ETBR) was more widespread, being expressed in seven of seven SCLC, four of four NSCLC, and the HBE cells. We used flow cytometry to measure mobilization of intracellular calcium as a functional assay for the ETAR. These data concurred with the RT-PCR results, indicating that the ETAR was downregulated or was involved in an alternative signal transduction pathway in lung cancer, and no evidence of functional receptor mediating an autocrine growth loop was found. From our study, the data do not support the putative functional autocrine growth role of ET-1 in lung cancer. We propose instead that ET-1 may act as a paracrine growth factor for surrounding epithelial and endothelial cells via alternative pathways, promoting angiogenesis and stromal growth. PMID- 10745024 TI - Neuregulin-1 and human epidermal growth factor receptors 2 and 3 play a role in human lung development in vitro. AB - The human epidermal growth factor receptor (HER) family consists of four distinct receptors: HER1 (epidermal growth factor receptor), HER2, HER3, and HER4. Their specific activating ligands are collectively known as neuregulins (NRG). We hypothesized that one member of the NRG family, NRG-1, and the HER family would play a role in fetal lung development. To test this hypothesis, we defined NRG-1 and HER gene expression in mid-trimester human fetal lung tissue. HER2 and HER3 messenger RNA and protein were detected in the fetal lung, but HER4 expression was not detected. Immunohistochemical staining of fetal lung tissue localized HER2 and HER3 protein to the developing lung epithelium. NRG-1 expression was not found in freshly isolated human fetal lung, but it was observed in fetal lung explants after 2 d of explant culture. Immunohistochemistry of cultured human fetal lung explants revealed that NRG-1 protein was also expressed in pulmonary epithelial cells. Exposing human fetal lung to recombinant NRG-1 activated the HER receptor complex as measured by approximately 4-fold increases in receptor phosphotyrosine content. In addition, NRG-1 increased explant epithelial cell volume density approximately 2-fold (P < 0. 03); increased epithelial cell proliferation approximately 2-fold, as determined by bromodeoxyuridine labeling (P = 0.002); and reduced surfactant protein-A (SP-A) levels by 53% (P < 0.05). These data are consistent with an autocrine regulatory process mediated by NRG-1 activation of HER2/HER3 heterodimers expressed on developing human fetal lung epithelial cells. Receptor activation results in increased lung epithelial cell proliferation and volume density, and decreased SP-A production, a marker of type II pneumocyte differentiation. PMID- 10745025 TI - Partial liquid ventilation with perfluorocarbon in acute lung injury: light and transmission electron microscopy studies. AB - Liquid ventilation using perfluorocarbon has been shown to improve gas exchange in animal models of acute lung injury as well as in children with acute respiratory distress syndrome. This study was designed to define structural features of lung injury following partial liquid ventilation (PLV) using light and transmission electron microscopy in a rabbit model of acute respiratory distress. Animals were treated with either conventional mechanical ventilation (CMV-gas) (n = 6) or PLV (n = 5) for 4 h after the induction of acute lung injury with saline lavage. Control animals were killed after the lung injury. PLV significantly improved alveolar-arterial oxygen tension and the oxygen index compared with CMV (P < 0.05). Morphometric studies using light microscopy show less alveolar hemorrhage, less edema, and fewer hyaline membranes in the PLV group (P < 0.05). Polymorphonuclear leukocyte sequestration in lung capillaries (11.4 +/- 1.5 versus 19.2 +/- 3 x 10(8)/ml, P < 0.05, PLV versus CMV) and migration into airspaces (3.1 +/- 1.2 versus 4.5 +/- 1.1 x 10(8)/ml, P < 0.05, PLV versus CMV) were lower in the gravity-dependent lung regions. There were fewer alveolar macrophages in the PLV group compared with other groups (P < 0.05). Fluorescence microscopy analysis shows fewer type II alveolar epithelial cells in the CMV group and brighter type II cells in the PLV group. Transmission electron microscopy studies show more alveolar wall damage in the CMV group, with type II cells detached from their basement membrane with fewer surfactant containing lamellar bodies. We conclude that quantitative histologic analysis shows less lung damage and inflammation when perfluorocarbon is combined with CMV in the management of acute respiratory distress syndrome. PMID- 10745026 TI - A novel karyopherin-beta homolog is developmentally and hormonally regulated in fetal lung. AB - To investigate molecular mechanisms of lung organogenesis, we used representational difference analysis to search for glucocorticoid-inducible genes in developing lung in a fetal rat model. Messenger RNA prepared from fetal and adult rat lung was used to prepare "representative amplicons." Adult-lung complementary DNA (cDNA) amplicons were used as "driver" in successive rounds of subtractive hybridization/amplification to isolate target fetal lung-specific cDNAs. A single clone, which was conserved and had near-perfect homology to eight human/rodent expressed sequence tags, was used as template for 5' and 3' rapid amplification of cDNA ends and SPICE (system for polymerase chain reaction amplification of cDNA ends) reactions to obtain the 3.6-kb cDNA, LGL2 (Genbank, AF 110195) encoding a deduced polypeptide (lgl2) of 963 amino acids. Northern analysis confirmed that LGL2 is differentially expressed in fetal lung (maximal during the pseudoglandular stage, gestational Days 14 to 16), induced by glucocorticoid, and enriched in epithelium relative to the mesenchyme. LGL2 was also detected in human fetal lung at gestational Week 16 as well as in human and rat fetal brain, heart, intestine, and kidney. We mapped LGL2 to chromosome 1p33 34.2. Comparison with sequences in the genome database identified lgl2 as a member of the karyopherin-beta family of nuclear import proteins, with greatest homology to transportin SR. Maximal expression of LGL2 in the pseudoglandular stage of development is coordinate with that of key transcription factors that regulate prominent signal transduction pathways in fetal lung organogenesis. We propose a role for lgl2 in nuclear import of transcription factors that regulate signal transduction during fetal lung development. PMID- 10745027 TI - Ceramide path in human lung cell death. AB - Lung epithelium plays a significant role in modulating the inflammatory response to lung injury. Airway epithelial cells are targeted by hydrogen peroxide (H(2)O(2)) and oxygen radicals, which are agents commonly produced during inflammatory processes. The mechanisms and molecular sites affected by H(2)O(2) are largely unknown but may involve the induction of sphingomyelin (SM) hydrolysis to generate ceramide, which serves as a second messenger in initiating an apoptotic response. Here we show that exposure of human airway epithelial (HAE) cells to 50 to 100 microM H(2)O(2) induces within 5 to 10 min a greater than 2-fold activation of neutral sphingomyelinase activity with concomitant SM hydrolysis, ceramide generation, and apoptosis. On the other hand, activation of protein kinase C (PKC) by 12-O-tetradecanoylphorbol-13-acetate inhibits both H(2)O(2)-induced ceramide production and apoptosis. The apoptotic response could be restored by the addition of 25 microM cell-permeant C6-ceramide. These findings indicate that ceramide, the product of SM hydrolysis, plays an important role in H(2)O(2)-induced apoptosis in HAE cells, and that PKC counteracts ceramide-mediated apoptosis in these cells. We suggest that the mediation of epithelial cell apoptosis by ceramide and its inhibition by PKC constitute a central mechanism by which inflammatory processes are modulated in the epithelium of the lung. PMID- 10745028 TI - C/EBPalpha and C/EBPdelta activate the clara cell secretory protein gene through interaction with two adjacent C/EBP-binding sites. AB - The Clara cell secretory protein (CCSP) gene is a cell-specific differentiation marker for the bronchiolar Clara cell. Previous studies suggest that CCAAT/enhancer binding protein (C/EBP)alpha is involved in controlling differentiation-dependent gene expression in the distal lung. In this study, immunofluorescence studies demonstrated high level expression of C/EBPdelta in the bronchiolar epithelium as well as lower levels of C/EBPalpha. Cotransfection studies in the lung epithelial cell line A549 showed that both C/EBPalpha and C/EBPdelta activate the murine CCSP gene and that a C/EBP-response element resides in the proximal CCSP promoter. C/EBPdelta exhibits an approximately 2 fold higher transactivation potential than does C/EBPalpha. DNase I footprint analyses revealed a footprint region located at -100 to -62 bp, corresponding to two C/EBP-binding sites. Mutation of either site resulted in abolished or strikingly reduced transactivation of the CCSP promoter by C/EBPalpha and C/EBPdelta, as well as impaired binding of both factors, indicating that the two C/EBP-binding sites form a compound response element. In electrophoretic mobility shift assays, it was shown that C/EBPalpha and C/EBPdelta can bind to both C/EBP sites, whereas in DNase I footprint analyses, the interaction of C/EBPalpha with the proximal site was weak. Furthermore, electrophoretic mobility shift assays demonstrated that C/EBPalpha and C/EBPdelta preferentially form heterodimers at both binding sites. Cotransfections with C/EBPalpha and C/EBPdelta together resulted in a superinduction of the CCSP promoter, indicating a regulatory role for the C/EBPalpha-C/EBPdelta heterodimers. Our findings demonstrate that C/EBPalpha and C/EBPdelta regulate the CCSP gene through a compound response element and suggest that these factors are important for the differentiation dependent expression of CCSP. PMID- 10745029 TI - Local activation of nonspecific defense against a respiratory model infection by application of interferon-gamma: comparison between rat alveolar and interstitial lung macrophages. AB - Pulmonary macrophages play a crucial role in the defense of inhaled pathogens. We characterized functional properties of alveolar (AM) and interstitial (IM) macrophages from rats. AM exhibited a pronounced microbicidal capacity as shown by an elevated production of reactive oxygen intermediates (ROI), nitric oxide (NO), tumor necrosis factor (TNF)-alpha, and tumor cytotoxicity when compared with IM. In contrast, IM were superior to AM regarding mechanisms mainly involved in the induction and maintenance of specific immune reactions (major histocompatibility complex [MHC] class II expression, interleukin [IL]-1 and IL 6). In this line, we were interested in whether the microbicidal potential of AM could be augmented by treating Lewis rats with rat recombinant interferon (IFN) gamma (5 x 10(2) to 1 x 10(5) U/animal) intratracheally, avoiding infection of interstitial lung macrophages or other organ-associated macrophages. The pulmonary cytokine application resulted in an activation of AM when macrophages from IFN-treated animals were compared with control macrophages from saline treated rats 18 h after the treatment: (1) mediator release (ROI, NO, TNF-alpha, IL-6), (2) tumoricidal activity; (3) dose-dependent increase of MHC class II expression. The local immunomodulation enhanced the resistance of normal and immunosuppressed rats against respiratory infections with Listeria monocytogenes. Taken together, local activation of lung macrophages is a feasible therapeutic strategy against pulmonary infections. PMID- 10745030 TI - Interferon-gamma production by specific lung lymphocyte phenotypes in silicosis in mice. AB - We recently described overproduction of interferon (IFN)-gamma by lung lymphocytes in mice with silicosis (11% of cells in air-control versus 19% of cells from silica-exposed mice; Davis and colleagues, Am. J. Respir. Cell Mol. Biol. 1999;20:813-824). We hypothesized that the increased IFN-gamma production might be due to selective enrichment of one lymphocyte phenotype. To test this hypothesis, small mononuclear cells from lung digest preparations of mice exposed 4 mo previously to cristobalite silica (70 mg/m(3), 12 d, 5 h/d) or to sham-air were stained for intracellular cytokines and surface antigen phenotypes, and examined by flow cytometry. Air-sham mouse lung digests included CD4(+) (16%) and CD8(+) (6%) T cells, gammadelta T-cell antigen receptor (TCR)(+) CD4(-)CD8(-) T cells (3%), natural killer (NK) cells (15%), B cells (6%), and macrophages (12%). The total number of lung lymphocytes was increased 1.7-fold in silicosis, but the phenotype frequencies did not change significantly. In the control lungs IFN gamma was produced by three major phenotypes of lymphocytes: 5% of CD4(+) T cells, 5% of gammadelta-TCR(+) CD4(-)CD8(-) T cells, and 2% of NK cells. The percentage of each type producing IFN-gamma was increased 2- to 3-fold in silicosis. When multiplied by cell number, the increased percentages yielded a 3- to 5-fold increase in the total number of each IFN- gamma-producing phenotype in the lung. Our results demonstrate no selective phenotype enrichment but upregulated IFN-gamma production by at least three lymphocyte phenotypes. IFN gamma may be an important signal driving lymphocyte differentiation and macrophage activation in silicosis. PMID- 10745031 TI - A novel mechanism of retinoic acid-enhanced interleukin-8 gene expression in airway epithelium. AB - A 3- to 8-fold stimulation of interleukin (IL)-8 gene expression by all-trans retinoic acid (ATRA) was demonstrated in primary cultures of human and monkey tracheobronchial epithelial cells and BEAS-2B serum-sensitive cell line. The effect of ATRA on IL-8 gene expression is dose- and time-dependent. Using cycloheximide, it was observed that new protein synthesis was required for the stimulation. ATRA had no effect on IL-8 messenger RNA stability. A difference in nuclear run-on activity suggests that a transcriptional mechanism is involved in ATRA-enhanced IL-8 gene expression. Promoter-reporter gene transfection studies demonstrated ATRA enhanced IL-8 promoter activity, especially when cells were cotransfected with retinoic acid nuclear receptor-alpha expression vector. Deletion and site-directed mutagenesis analysis revealed the involvement of nuclear factor (NF)-kappaB binding site of the IL-8 gene in ATRA-enhanced promoter activity. Electrophoretic mobility shift assay (EMSA) demonstrated that ATRA enhanced DNA-NF-kappaB complex formation, especially with the p65 subunit. Western blot analysis demonstrated that ATRA did not enhance the protein amount of both the p50 and the p65 subunits in the nuclei. Because ATRA also enhances thioredoxin (TRX) gene expression, the effect of TRX on IL-8 gene expression was examined. IL-8 promoter activity was enhanced in transfected cells by the addition of TRX protein. Treatment of nuclear extracts with TRX also enhanced DNA NF-kappaB complex formation as observed by EMSA, particularly the p65 subunit. Taking these data together, a novel mechanism is proposed in which ATRA activates promoter activity of IL-8 gene through TRX-dependent NF-kappaB activation. PMID- 10745032 TI - The DEBRA International Visioning/Consensus Meeting on Epidermolysis Bullosa: summary and recommendations. PMID- 10745033 TI - Anti-melanocyte T cell responses - methodology versus biology. PMID- 10745034 TI - Lack of detection of human herpesvirus (HHV)-8 DNA in lesional skin of German pemphigus vulgaris and pemphigus foliaceus patients. PMID- 10745035 TI - Netherton syndrome is not linked to 18q12, a region homologous to the murine lanceolate hair (lah) locus. PMID- 10745038 TI - Abstracts for the 61st annual meeting of the society for investigative dermatology PMID- 10745037 TI - The society for investigative dermatology minutes of the board of directors meeting PMID- 10745039 TI - Abstracts for the 61st annual meeting of the society for investigative dermatology PMID- 10745040 TI - A locus for primary ciliary dyskinesia maps to chromosome 19q. AB - Primary ciliary dyskinesia is an autosomal recessive condition characterised by chronic sinusitis, bronchiectasis, and subfertility. Situs inversus occurs in 50% of cases (Kartagener syndrome). It has an estimated incidence of 1 in 20 000 live births. The clinical phenotype is caused by defective ciliary function associated with a range of ultrastructural abnormalities including absent dynein arms, absent radial spokes, and disturbed ciliary orientation. The molecular genetic basis is unknown. A genome scan was performed in five Arabic families. Using GENEHUNTER, a maximal multipoint lod score (HLOD) of 4.4 was obtained on chromosome 19q13.3-qter at alpha (proportion of linked families) = 0.7. A 15 cM critical region is defined by recombinations at D19S572 and D19S218. These data provide significant evidence for a PCD locus on chromosome 19q and confirm locus heterogeneity. PMID- 10745041 TI - Localisation of the gene causing diaphyseal dysplasia Camurati-Engelmann to chromosome 19q13. AB - Camurati-Engelmann disease, progressive diaphyseal dysplasia, or diaphyseal dysplasia Camurati-Engelmann is a rare, autosomal dominantly inherited bone disease, characterised by progressive cortical expansion and sclerosis mainly affecting the diaphyses of the long bones associated with cranial hyperostosis. The main clinical features are severe pain in the legs, muscular weakness, and a waddling gait. The underlying cause of this condition remains unknown. In order to localise the disease causing gene, we performed a linkage study in a large Jewish-Iraqi family with 18 affected subjects in four generations. A genome wide search with highly polymorphic markers showed linkage with several markers at chromosome 19q13. A maximum lod score of 4.9 (theta=0) was obtained with markers D19S425 (58.7 cM, 19q13.1) and D19S900 (67.1 cM, 19q13. 2). The disease causing gene is located in a candidate region of approximately 32 cM, flanked by markers D19S868 (55.9 cM, 19q13.1) and D19S571 (87.7 cM, 19q13.4). PMID- 10745042 TI - Mutation screening in Rett syndrome patients. AB - Rett syndrome (RTT) was first described in 1966. Its biological and genetic foundations were not clear until recently when Amir et al reported that mutations in the MECP2 gene were detected in around 50% of RTT patients. In this study, we have screened the MECP2 gene for mutations in our RTT material, including nine familial cases (19 Rett girls) and 59 sporadic cases. A total of 27 sporadic RTT patients were found to have mutations in the MECP2 gene, but no mutations were identified in our RTT families. In order to address the possibility of further X chromosomal or autosomal genetic factors in RTT, we evaluated six candidate genes for RTT selected on clinical, pathological, and genetic grounds: UBE1 (human ubiquitin activating enzyme E1, located in chromosome Xp11.23), UBE2I (ubiquitin conjugating enzyme E2I, homologous to yeast UBC9, chromosome 16p13.3), GdX (ubiquitin-like protein, chromosome Xq28), SOX3 (SRY related HMG box gene 3, chromosome Xq26-q27), GABRA3 (gamma-aminobutyric acid type A receptor alpha3 subunit, chromosome Xq28), and CDR2 (cerebellar degeneration related autoantigen 2, chromosome 16p12-p13.1). No mutations were detected in the coding regions of these six genes in 10 affected subjects and, therefore, alterations in the amino acid sequences of the encoded proteins can be excluded as having a causative role in RTT. Furthermore, gene expression of MECP2, GdX, GABRA3, and L1CAM (L1 cell adhesion molecule) was also investigated by in situ hybridisation. No gross differences were observed in neurones of several brain regions between normal controls and Rett patients. PMID- 10745043 TI - Genetic heterogeneity of Usher syndrome type II: localisation to chromosome 5q. AB - Usher syndrome is a group of autosomal recessive disorders that includes retinitis pigmentosa (RP) with hearing loss. Usher syndrome type II is defined as moderate to severe hearing loss with RP. The USH2A gene at 1q41 has been isolated and characterised. In 1993, a large Usher II family affected with a mild form of RP was found to be unlinked to 1q41 markers. Subsequent linkage studies of families in our Usher series identified several type II families unlinked to USH2A and USH3 on 3q25. After a second unlinked family with many affected members and a mild retinal phenotype was discovered, a genome search using these two large families showed another Usher II locus on 5q (two point lod = 3.1 at D5S484). To date, we have identified nine unrelated 5q linked families (maximum combined multipoint lod = 5.86) as well as three Usher II families that show no significant linkage to any known Usher loci. Haplotype analysis of 5q markers indicates that the new locus is flanked by D5S428 and D5S433. Review of ophthalmological data suggests that RP symptoms are milder in 5q linked families; the RP is often not diagnosed until patients near their third decade. Enamel hypoplasia and severe, very early onset RP were observed in two of the three unlinked families; dental anomalies have not been previously described as a feature of Usher type II. PMID- 10745044 TI - Report of five novel and one recurrent COL2A1 mutations with analysis of genotype phenotype correlation in patients with a lethal type II collagen disorder. AB - Achondrogenesis II-hypochondrogenesis and severe spondyloepiphyseal dysplasia congenita (SEDC) are lethal forms of dwarfism caused by dominant mutations in the type II collagen gene (COL2A1). To identify the underlying defect in seven cases with this group of conditions, we used the combined strategy of cartilage protein analysis and COL2A1 mutation analysis. Overmodified type II collagen and the presence of type I collagen was found in the cartilage matrix of all seven cases. Five patients were heterozygous for a nucleotide change that predicted a glycine substitution in the triple helical domain (G313S, G517V, G571A, G910C, G943S). In all five cases, analysis of cartilage type II collagen suggested incorporation of the abnormal alpha1(II) chain in the extracellular collagen trimers. The G943S mutation has been reported previously in another unrelated patient with a strikingly similar phenotype, illustrating the possible specific effect of the mutation. The radiographically less severely affected patient was heterozygous for a 4 bp deletion in the splice donor site of intron 35, likely to result in aberrant splicing. One case was shown to be heterozygous for a single nucleotide change predicted to result in a T1191N substitution in the carboxy-propeptide of the proalpha1(II) collagen chain. Study of the clinical, radiographic, and morphological features of the seven cases supports evidence for a phenotypic continuum between achondrogenesis II-hypochondrogenesis and lethal SEDC and suggests a relationship between the amount of type I collagen in the cartilage and the severity of the phenotype. PMID- 10745045 TI - Universal primer quantitative fluorescent multiplex (UPQFM) PCR: a method to detect major and minor rearrangements of the low density lipoprotein receptor gene. AB - A method based on quantitative fluorescent multiplex PCR has been developed to detect major rearrangements of the low density lipoprotein receptor gene (LDLR) which account for approximately 5% of mutations. The method involves two PCR reactions; the first (P1) amplifies the selected exons using unique primer sequences tagged with newly designed universal primers, while the second (P2) amplifies the P1 amplicons using the universal primers. One of the P2 universal primers is labelled with a fluorescent dye which is incorporated into the PCR products which are then electrophoresed on an ABI DNA sequencer. The relative amounts of the amplified peak areas are determined and compared to ratios obtained for DNA from four normal controls and known major rearrangements. The multiplex set developed is based on LDLR exons 3, 5, 8, 14, and 17 and 86% of reported major rearrangements would be detectable by this assay as well as any deletions and insertions of greater than 1 bp. The method was evaluated using DNA from 15 reported deletions and duplications which were all correctly identified. Two groups of UK patients with a clinical diagnosis of familial hypercholesterolaemia (FH) and where no mutation had been identified in LDLR or APOB (14 children and 42 adults) were screened for the presence of major LDLR rearrangements by this assay. Three major rearrangements were detected and a 4 bp duplication was identified in a fourth patient. Since it avoids the problems associated with Southern blotting, this method will be useful for detecting gene rearrangements. PMID- 10745046 TI - Parental origin and mechanisms of formation of cytogenetically recognisable de novo direct and inverted duplications. AB - Cytogenetic, FISH, and molecular results of 20 cases with de novo tandem duplications of 18 different autosomal chromosome segments are reported. There were 12 cases with direct duplications, three cases with inverted duplications, and five in whom determination of direction was not possible. In seven cases a rearrangement between non-sister chromatids (N-SCR) was found, whereas in the remaining 13 cases sister chromatids (SCR) were involved. Paternal and maternal origin (7:7) was found almost equally in cases with SCR (3:4) and N-SCR (4:3). In the cases with proven inversion, there was maternal and paternal origin in one case each. Twenty three out of 43 cytogenetically determined breakpoints correlated with common or rare fragile sites. In five cases, including all those with proven inverse orientation, all breakpoints corresponded to common or rare fragile sites. In at least two cases, one with an interstitial duplication (dup(19)(q11q13)) and one with a terminal duplication (dup(8) (p10p23)), concomitant deletions (del(8) (p23p23.3) and del(19)(q13q13)) were found. PMID- 10745047 TI - An unbalanced submicroscopic translocation t(8;16)(q24.3;p13.3)pat associated with tuberous sclerosis complex, adult polycystic kidney disease, and hypomelanosis of Ito. AB - We report on a familial submicroscopic translocation involving chromosomes 8 and 16. The proband of the family had a clinical picture suggestive of a large deletion in the chromosome 16p13.3 area, as he was affected with tuberous sclerosis complex (TSC) and had alpha thalassaemia trait, and his half brother, who also had TSC, may have suffered additionally from polycystic kidney disease (PKD). FISH studies provided evidence for a familial translocation t(8;16)(q24.3;p13.3) with an unbalanced form in the proband and a balanced form in the father and in a paternal aunt. The unbalanced translocation caused the index patient to be deleted for the chromosome 16p13.3-pter region, with the most proximal breakpoint described to date for terminal 16p deletions. In addition, FISH analysis showed a duplication for the distal 8q region. Since the index patient also had hypomelanosis of Ito (HI), either of the chromosomal areas involved in the translocation may be a candidate region for an HI determining gene. Furthermore, it is noteworthy that both carriers of the balanced translocation showed a nodular goitre, while the proband has hypothyroidism. PMID- 10745048 TI - Clinical and radiological assessment of a family with mild brachydactyly type A1: the usefulness of metacarpophalangeal profiles. AB - The brachydactylies are a group of conditions in which various subtypes have been defined based upon the specific pattern of digital bones involved. Type A1 brachydactyly is principally characterised by maximal involvement of the middle phalanges. We report an extended family with a mild brachydactyly A1 which was, except for some short stature, not associated with any of the additional clinical findings reported in several published families. While all the hand bones tended to be small, the principal features of the affected members were shortened middle and distal phalanges, proximal 1st phalanges, and 5th metacarpals. The feet were similarly involved and tended to have a broad, slightly adducted forefoot. The two affected children showed multiple coned epiphyses. This paper provides a detailed description of the family including the radiographic signs and metacarpophalangeal profiles, which proved to be useful in distinguishing the mildly affected persons. PMID- 10745049 TI - Cerebrospinal fluid corticotropin-releasing hormone levels are elevated in monkeys with patterns of brain activity associated with fearful temperament. AB - BACKGROUND: Asymmetric patterns of frontal brain activity and brain corticotropin releasing hormone (CRH) systems have both been separately implicated in the processing of normal and abnormal emotional responses. Previous studies in rhesus monkeys demonstrated that individuals with extreme right frontal asymmetric brain electrical activity have high levels of trait-like fearful behavior and increased plasma cortisol concentrations. METHODS: In this study we assessed cerebrospinal fluid (CSF) CRH concentrations in monkeys with extreme left and extreme right frontal brain electrical activity. CSF was repeatedly collected at 4, 8, 14, 40, and 52 months of age. RESULTS: Monkeys with extreme right frontal brain activity had increased CSF CRH concentrations at all ages measured. In addition, individual differences in CSF CRH concentrations were stable from 4 to 52 months of age. CONCLUSIONS: These findings suggest that, in primates, the fearful endophenotype is characterized by increased fearful behavior, a specific pattern of frontal electrical activity, increased pituitary-adrenal activity, and increased activity of brain CRH systems. Data from other preclinical studies suggests that the increased brain CRH activity may underlie the behavioral and physiological characteristics of fearful endophenotype. PMID- 10745050 TI - Increased cerebrospinal fluid glutamine levels in depressed patients. AB - BACKGROUND: There is increasing evidence for an association between alterations of brain glutamatergic neurotransmission and the pathophysiology of affective disorders. METHODS: We studied the association between cerebrospinal fluid (CSF) metabolites, including glutamine, in unipolar and bipolar depressed patients versus control subjects using a proton magnetic resonance spectroscopy technique. Cerebrospinal fluid samples were obtained from 18 hospitalized patients with acute unmedicated severe depression without medical problems and compared with those of 22 control subjects. RESULTS: Compared with the control group, the depressed patient group had significantly higher CSF glutamine concentrations, which correlated positively with CSF magnesium levels. CONCLUSIONS: These findings suggest an abnormality of the brain glial-neuronal glutamine/glutamate cycle associated with N-methyl-D-aspartate receptor systems in patients with depression. PMID- 10745051 TI - Nocturnal growth hormone secretion studies in adolescents with or without major depression re-examined: integration of adult clinical follow-up data. AB - BACKGROUND: Early sleep is associated with an increased secretion of human growth hormone (GH) through muscarinic inhibition of somatostatin, a GH suppressant. A clinical follow-up was performed approximately 1 decade after depressed and psychiatrically "normal" control adolescents, who were now young adults, had undergone baseline serial GH measurements over a 24-hour period on the third night of sleep polysomnography studies. METHODS: The study population consisted of 77 young adults who had received a diagnosis of adolescent major depressive disorder and had participated in the adolescent sleep and neuroendocrine studies. Alternatively, the young adult subjects were assessed as normal adolescent control subjects free of any psychiatric diagnosis. Blood samples had been collected for GH every 20 min during the 24-hour period coinciding with the third consecutive night of sleep electroencephalography. Subjects, now in young adulthood, were relocated and blindly reinterviewed using the Schedule for Affective Disorders and Schizophrenia (lifetime version). The original adolescent nocturnal GH data were analyzed in light of the information obtained regarding clinical course into adulthood. RESULTS: A substantial proportion of the nominally normal control group developed at least one episode of major depression or dysthymia during the follow-up period. "Latent" depressive subjects differed from depression-free control subjects by having exhibited a significantly more rapid increase of adolescent nocturnal GH secretion following sleep onset. Of the subjects who had experienced at least one lifetime major depressive episode during the follow-up, the subgroup who would go on to make suicide attempts secreted significantly greater amounts of GH during the first 4 hours of sleep. Adults with lifetime depression exhibited significantly reduced levels of GH in the 100 min preceding sleep onset during adolescence. CONCLUSIONS: Assignment of subjects based on longitudinal clinical follow-up into adulthood revealed that the sleep-related GH secretion paradigm has predictive value for future depressive episodes and future suicide attempts. Dysfunction of complex sleep onset mechanisms may be a premorbid marker of depression and suicidal behavior. PMID- 10745052 TI - Ultradian rapid cycling in prepubertal and early adolescent bipolarity is not in transmission disequilibrium with val/met COMT alleles. AB - BACKGROUND: Prepubertal children and early adolescents with bipolar disorders (PEA-BP) who participate in the ongoing study "Phenomenology and Course of Pediatric Bipolar Disorders" have a high prevalence of ultradian (within 24-hour periods) rapid cycling. Based on a case-control finding reported in bipolar (BP) adults of an association between rapid and ultradian rapid cycling with the low activity allele of catechol-O-methyltransferase (l-COMT), study of linkage and linkage disequilibrium of l-COMT in the PEA-BP population seemed warranted. METHODS: Genotypes on a subset of the larger PEA-BP sample, for whom trio blood collection was complete (i. e., probands and both of their biological parents), were used to perform transmission disequilibrium tests (TDTs). Diagnoses were established from a comprehensive battery that included WASH-U-KSADS (Washington University Kiddie Schedule for Affective Disorders and Schizophrenia) given to both mothers and children and from consensus conferences. Probands with PEA-BP (N = 52) were 10.9 +/- 2.8 years old at index episode; had a mean age of BP onset at 8.0 +/- 3.8 years; were severely impaired, with a mean Children's Global Assessment Scale score of 44.5 +/- 8.9; and manifested the cardinal features of BP (84.6% had euphoric mood, 76.9% had grandiosity, and 57.7% had psychosis). Ultradian rapid cycling occurred in 75%. Genotyping of the single nucleotide polymorphism at COMT was performed using automated capillary electrophoresis single-strand conformational polymorphism with detection by laser-induced fluorescence. RESULTS: Transmission disequilibrium tests were not significant for preferential transmission of l-COMT for the ultradian rapid-cycling subgroup or for the entire PEA-BP sample. CONCLUSIONS: The lack of linkage disequilibrium between l-COMT and ultradian rapid cycling in the PEA-BP sample compared to reported findings of an association in case-control studies of adults is discussed in terms of age-specific developmentally relevant phenotypes, anticipatory mechanisms, and heterogeneity. Repeat TDT analyses after these PEA BP probands reach their adult phenotypes will be informative. PMID- 10745053 TI - EEG and subjective sleepiness during extended wakefulness in seasonal affective disorder: circadian and homeostatic influences. AB - BACKGROUND: Seasonal affective disorder (SAD) may reflect a disturbance of circadian phase relationships or a disturbance of sleep-wake dependent processes, both of which change daytime energy and sleepiness levels. METHODS: Under the unmasking conditions of a 40-hour constant routine protocol (CR), self-rated sleepiness and waking electroencephalogram (EEG) power density were assessed in women with SAD (n = 8) and in age-matched healthy control subjects (n = 9). RESULTS: There was no significant effect of season or light treatment in any of the measures. The time course of subjective sleepiness was characterized by a circadian modulation and an overall increase during extended wakefulness in both SAD patients and control subjects. A prominent circadian rhythm of subjective sleepiness was not different in SAD patients and control subjects; however, the progressive buildup of sleepiness, as quantified by nonlinear regression analysis, was significantly reduced in SAD patients, mainly because they were sleepier than control subjects during the first 12 hours of the CR. The time course of waking EEG theta-alpha activity showed a more rapid increase during the first 10 hours of the CR in SAD patients. In contrast to control subjects who showed a progressive increase in the course of the 40-hour episode of extended wakefulness, EEG theta-alpha activity in SAD patients did not further increase over the remainder of the CR. CONCLUSIONS: The data suggest that SAD patients may have a trait (rather than state) deficiency in the homeostatic buildup of sleep pressure during extended wakefulness as indexed by subjective sleepiness and EEG theta-alpha activity. PMID- 10745054 TI - Sleep electroencephalographic coherence abnormalities in individuals at high risk for depression: a pilot study. AB - BACKGROUND: Sleep electroencephalographic (EEG) studies of individuals with major depressive disorder have identified several microarchitectural features associated with the illness. These abnormalities are also found in clinically remitted individuals, raising the question of whether they are vulnerability markers of depression. This study evaluated the sleep EEG in high-risk individuals to see if abnormalities are present in the sleep EEG prior to the onset of illness. METHODS: A total of 26 subjects (13 males and 13 females) were recruited for study on the basis of 1) having a parent or grandparent treated for major depressive or bipolar affective disorder and 2) having no history of personal psychiatric illness. Polysomnographic data were collected and compared with gender- and age-matched healthy control subjects with no personal or family history of psychiatric illness. The primary outcome measures were interhemispheric and intrahemispheric coherence. RESULTS: Period analysis of the sleep EEG showed that beta-delta coherence was lower bilaterally in male high risk subjects. Right-hemispheric theta-delta coherence was also lower in male high-risk subjects, with female high-risk subjects evidencing lower beta coherence. CONCLUSIONS: Sleep-EEG abnormalities associated with major depressive disorder are present in never mentally ill individuals at high risk for the illness. These markers may be useful in the prediction of illness and in family genetic studies of mood disorders. PMID- 10745055 TI - Changes in EEG power density during sleep laboratory adaptation in depressed inpatients. AB - BACKGROUND: The aim of the present study was to evaluate the first-night effect in depressed inpatients, using standard sleep measures as well as all-night spectral analysis of the sleep electroencephalogram (EEG). METHODS: Eighteen drug free, depressed inpatients were studied for 3 consecutive nights in the hospital sleep laboratory. RESULTS: Visual sleep scoring results showed a slight but measurable first-night effect, characterized by a reduction of rapid eye movement (REM) sleep amount and increased wakefulness. Sleep EEG spectral analysis showed significantly reduced delta (p <.01) and theta (p <.05) power density in non-REM (NREM) sleep of the first night compared with that of the second and third nights. These differences were limited to the early part of the sleep period, a time during the night that is particularly vulnerable to the effects of depressive disorder. In contrast to the NREM sleep findings, spectral REM variables studied did not significantly vary across the three nights. CONCLUSIONS: The results obtained suggest that first-night data should not be simply discarded but could be used in subsequent analyses and could be considered useful in the evaluation of the sleep of depressed patients. PMID- 10745056 TI - Effects of noise on different disease states of recurrent affective disorders. AB - BACKGROUND: Nonlinear dynamics are currently proposed to explain the course of recurrent affective disorders. Such a nonlinear disease model predicts complex interactions with stochastic influences, in particular, because both disease dynamics and stochastic influences, such as psychosocial stressors, will vary during the course of the disease. We approach this problem by investigating general effects of noise intensity on different disease states of a nonlinear model for recurrent affective disorders. METHODS: A recently developed neurodynamic model is studied numerically. RESULTS: Noise can cause unstructured randomness or can maximize periodic order. The frequency of episode occurrence can increase with noise but it can also remain unaffected or even can decrease. The observed effects, thereby, depend critically on both the noise intensity and the internal nonlinear dynamics of the disease model. CONCLUSIONS: Our findings indicate that altered stochastic influences can significantly affect the outcome of a dynamic disease. To evaluate the effects of noise, it is essential to know about the underlying dynamics of respective disease states. Therefore, characterization of low-dimensional dynamics might become valuable for disease prediction and control. PMID- 10745057 TI - A relationship between serotonin transporter genotype and in vivo protein expression and alcohol neurotoxicity. AB - BACKGROUND: Genetic variation of the promoter for the serotonin transporter (5 HTT) gene has been associated with its functional capacity. In vitro, carriers of a short allele (s-carriers) of the 5-HTT promoter display significant reduction in 5-HTT capacity. Dysfunction of 5-HTT has been observed in alcoholic individuals. We assessed whether the allelic constitution of the 5-HTT gene is associated with reduced serotonin transporter availability among alcoholic individuals. METHODS: We genotyped the 5-HTT promoter region and measured the availability of serotonin transporter protein with [I-123]beta-CIT SPECT in the raphe area in 14 abstinent male alcoholic subjects and 8 age-matched control subjects of European American descent. RESULTS: Among control subjects, the ratio of in vivo 5-HTT availability for ll-homozygous individuals relative to s carriers was comparable to serotonin uptake ratios measured in vitro. There was a significant interaction of diagnosis and 5-HTT promoter genotype on 5-HTT availability (p <.01). Among controls, ll-homozygous individuals displayed a significant increase as compared with s-carriers. The availability of raphe 5-HTT was significantly reduced in ll-homozygous alcoholic individuals and was negatively correlated with their amount of alcohol consumption. Among s-carriers, 5-HTT availability did not differ significantly between control and alcoholic subjects. CONCLUSIONS: Our preliminary findings suggest an association between 5 HTT allelic constitution and in vivo measurements of human serotonin transporter availability, and a potentially selective susceptibility of ll-homozygous individuals to the neurotoxic effects of chronic excessive alcohol consumption. PMID- 10745058 TI - Testosterone and cognition in elderly men: a single testosterone injection blocks the practice effect in verbal fluency, but has no effect on spatial or verbal memory. AB - BACKGROUND: The relevance of the age-associated decline in testosterone for cognition in elderly men is still poorly understood. One hypothesis is that testosterone enhances spatial abilities, while it might impair verbal skills. METHODS: Thirty elderly men received a single testosterone (250 mg testosterone enanthate) or placebo injection. Cognitive performance was tested before and 5 days after treatment using spatial as well as verbal tests. RESULTS: Five days after injection, testosterone and estradiol levels were still in the supraphysiologic range. In the verbal fluency task, the placebo group, but not the testosterone group, showed a practice effect. Therefore, the testosterone group performed significantly worse than the placebo group after treatment. No effects of testosterone were observed in the other verbal and spatial tasks. CONCLUSIONS: The present finding, that testosterone blocks the practice effect in verbal fluency, partly supports the general idea that sex steroids modulate performance in tests with known gender differences. Moreover it demonstrates that these effects can occur rapidly. However, beneficial effects on spatial cognition or memory might need more time to develop and/or might only occur when a less pronounced testosterone increase is induced. PMID- 10745059 TI - In vitro metabolism of trazodone by CYP3A: inhibition by ketoconazole and human immunodeficiency viral protease inhibitors. AB - BACKGROUND: Pharmacologic treatment of emotional disorders in HIV-infected patients can be more easily optimized by understanding of potential interactions of psychotropic drugs with medications used to treat HIV infection and its sequelae. METHODS: Biotransformation of the antidepressant trazodone to its principal metabolite, meta-chlorophenylpiperazine (mCPP), was studied in vitro using human liver microsomes and heterologously expressed individual human cytochromes. Interactions of trazodone with the azole antifungal agent, ketoconazole, and with human immunodeficiency virus protease inhibitors (HIVPIs) were studied in the same system. RESULTS: Formation of mCPP from trazodone in liver microsomes had a mean (+/- SE) K(m) value of 163 (+/- 21) micromol/L. Ketoconazole, a relatively specific CYP3A inhibitor, impaired mCPP formation consistent with a competitive mechanism, having an inhibition constant (K(i)) of 0.12 (+/- 0.01) micromol/L. Among heterologously expressed human cytochromes, only CYP3A4 mediated formation of mCPP from trazodone; the K(m) was 180 micromol/L, consistent with the value in microsomes. The HIVPI ritonavir was a potent inhibitor of mCPP formation in liver microsomes (K(i) = 0.14 +/- 0.04 micromol/L). The HIVPI indinavir was also a strong inhibitor, whereas saquinavir and nelfinavir were weaker inhibitors. CONCLUSIONS: CYP3A-mediated clearance of trazodone is inhibited by ketoconazole, ritonavir and indinavir, and indicates the likelihood of pharmacokinetic interactions in vivo. PMID- 10745060 TI - Impaired prepulse inhibition of acoustic startle in schizophrenia. AB - BACKGROUND: Schizophrenics show deficits in sensorimotor gating, as measured by prepulse inhibition of acoustic startle (PPI). The goal of this investigation is to further characterize PPI and habituation deficits in schizophrenia, and to examine whether differing subgroups of schizophrenics would show comparable PPI deficits. METHODS: PPI was measured in 24 male schizophrenic subjects (9 acutely decompensated inpatients and 15 stable outpatients) and in 20 age-matched normal control subjects. Schizophrenic subjects were rated for positive and negative symptoms at the time of testing. RESULTS: Schizophrenic subjects showed deficits in prepulse inhibition and habituation as compared to normal subjects. Similar latency facilitation was produced by the prepulse in both groups. Acutely decompensated inpatients and stable outpatients did not differ in percent PPI. PPI did not correlate with severity of positive or negative symptoms. CONCLUSIONS: These results suggest that schizophrenic subjects have impaired central inhibitory mechanisms as measured by PPI, and support the hypothesis that periods of relative clinical remission are not accompanied by normalization of sensorimotor gating. PMID- 10745061 TI - Antipsychoticlike effects of amoxapine, without catalepsy, using the prepulse inhibition of the acoustic startle reflex test in rats. AB - BACKGROUND: The dibenzoxazepine amoxapine was introduced as an antidepressant but has shown antipsychoticlike activity in a number of animal screening tests. A recent positron emission tomography study showed a 5-HT(2)/D(2) receptor occupancy profile of amoxapine that is very similar to that of established atypical antipsychotics. Schizophrenics display deficits in sensory gating mechanisms, such as prepulse inhibition (PPI) of the acoustic startle reflex. A similar deficit can be produced by dopamine (DA) and by 5-HT(2A/C) receptor agonists in rats. Antipsychotic compounds reverse this effect. METHODS: Effects of amoxapine on apomorphine- or 1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced disruption of PPI were studied in adult male Sprague-Dawley rats. The extrapyramidal side effect (EPS) liability of amoxapine was assessed using the inclined grid catalepsy (CAT) test. Statistical analyses were performed by analysis of variance (ANOVA) for fully repeated measures (PPI) and by the Kruskal Wallis one-way ANOVA by ranks (CAT). RESULTS: Apomorphine (0.5 mg/kg) produced a significant reduction in PPI compared with the case of rats in the saline control group. Pretreatment with amoxapine (10 mg/kg) significantly attenuated the apomorphine-induced disruption of PPI. DOI (0.5 mg/kg) significantly reduced PPI compared with saline controls. Pretreatment with amoxapine (5 or 10 mg/kg) produced a significant attenuation of the DOI-induced disruption of PPI. Amoxapine by itself did not alter PPI. Amoxapine (5 or 10 mg/kg) did not produce CAT. CONCLUSIONS: The DA D(2)/5-HT(2) receptor antagonist amoxapine produced an antipsychoticlike reversal of both apomorphine- and DOI-induced disruption of PPI. Furthermore, the same doses of amoxapine that reversed disruption of PPI did not produce CAT. The results confirm and lend further support to the results of previous studies on amoxapine, suggesting that amoxapine might possess antipsychotic activity with little propensity for producing EPS. PMID- 10745062 TI - Dexamethasone metabolism in dexamethasone suppression test suppressors and nonsuppressors. AB - BACKGROUND: Variable dexamethasone kinetics is a possible confound in the dexamethasone suppression test. Modifications to include dexamethasone plasma levels and specific dexamethasone "windows" have been proposed. Our study aims to validate our proposed dexamethasone windows in an independent sample of 121 subjects. METHODS: We performed dexamethasone suppression tests in 162 subjects with mixed psychiatric diagnoses. Dexamethasone levels and beta-phase half-life of dexamethasone were computed for suppressors and nonsuppressors. RESULTS: Dexamethasone levels were lower in nonsuppressors than in suppressors. Dexamethasone levels correlated inversely with cortisol levels in the total sample, but were nonsignificant or weakly associated in those samples restricted to the windows. The beta-phase half-life of dexamethasone was shorter in nonsuppressors. The dexamethasone windows were validated at 3:00 PM and 10:00 PM. We propose 4.0 ng/mL as a revised upper limit of the 8:00 AM dexamethasone window. CONCLUSIONS: The plasma dexamethasone level is confirmed as a confound in the dexamethasone suppression test through more rapid dexamethasone clearance in nonsuppressors. Application of dexamethasone windows will reduce this source of test variance. PMID- 10745063 TI - Thirty years of contraception. PMID- 10745064 TI - The contraceptive revolution. PMID- 10745065 TI - The "boom and bust phenomenon": the hopes, dreams, and broken promises of the contraceptive revolution. AB - The "boom and bust phenomenon" is a pattern that has emerged in the development, introduction, and delivery of a number of significant new contraceptive products in the United States. When a new contraceptive product is introduced with great promise and publicity, it usually experiences a "boom" during which sales, demand, and expectations are high. This boom is often followed by a "bust" phase during which a product does not live up to expectations, initial excitement falls off, and a drop in sales and use ensues. The boom and bust phenomenon goes to the heart of what some have referred to as the failed promise of the contraception revolution by creating obstacles to significant expansion of contraceptive choice in the United States. Case studies of oral contraceptives, intrauterine devices, and Norplant(R) are used to illustrate the boom and bust phenomenon and the effect it has had in shaping the direction of advances in contraceptive technology. PMID- 10745066 TI - Bust without boom. PMID- 10745067 TI - The efficacy of medical abortion: a meta-analysis. AB - Multiple clinical studies demonstrate the efficacy of medical abortion with mifepristone or methotrexate followed by a prostaglandin analogue. However, assessing predictors of success, including regimen, is difficult because of regimen variability and a lack of direct comparisons. This meta-analysis estimates rates of primary clinical outcomes of medical abortion (successful abortion, incomplete abortion, and viable pregnancy) and compares them by regimen and gestational age. We identified 54 studies published from 1991 to 1998 using mifepristone with misoprostol (18), mifepristone with other prostaglandin analogues (23), and methotrexate with misoprostol (13). Data abstracted from studies included regimen details and clinical outcomes by gestational age. We found that efficacy decreases with increasing gestational age (p<0.001), and differences by regimen are not statistically significant except at gestational age > or =57 days. For gestations < or =49 days, mean rates of complete abortion were 94-96%, incomplete abortion 2-4%, and ongoing (viable) pregnancy 1-3%. For gestations of 50-56 days, the mean rate of complete abortion was 91% (same for all regimens), incomplete abortion 5-8%, and ongoing pregnancy 3-5%. For > or =57 days, success was lower for mifepristone/misoprostol (85%, 95% confidence interval 78-91%) than for mifepristone/other prostaglandin analogues 95% (CI 91 98%, p = 0.006). For mifepristone/misoprostol, using > or =2 prostaglandin analogue doses seems to be better than a single dose for certain outcomes and gestational ages. We conclude that both mifepristone and methotrexate, when administered with misoprostol, have high levels of success at < or =49 days gestation but may have lower efficacy at longer gestation. PMID- 10745068 TI - Low-dose mifepristone followed by vaginal misoprostol at 48 hours for abortion up to 63 days. AB - The aim of this study was to compare the effectiveness, side effects, and acceptability of one-third the standard dose of mifepristone, i.e., 200 mg, and vaginal misoprostol 800 microg to induce abortion in subjects < or =56 days pregnant with subjects 57-63 days pregnant. A prospective multicenter trial enrolled healthy women > or =18 years, < or =63 days pregnant, and wanting an abortion. Women received mifepristone 200 mg orally, followed by misoprostol 800 microg vaginally, and returned 1-4 days later for ultrasound evaluation. A second dose of misoprostol was administered, if necessary. Surgical intervention was indicated for continuing pregnancy, excessive bleeding, or persistent products of conception 5 weeks later. Of 1137 subjects, 829 were in the < or =56 days pregnant group and 308 in the 57-63 days pregnant group. In all, 34 subjects had surgical intervention and 16 were lost to follow-up. Complete medical abortions occurred in 97% of subjects < or =56 days pregnant and 96% in the 57-63 days pregnant group. In all, 88% of subjects in the < or =56 days pregnant and 92% in the 57-63 days pregnant group bled within 4 h of using vaginal misoprostol. Comparing subjects < or =56 days pregnant with 57-63 days pregnant, there was less diarrhea (20% vs 29%, p = 0.002) and vomiting (33% vs 44%, p = 0.001), although side effects were acceptable to 82% of subjects in both groups. One subject in the < or =56 day group required a transfusion for delayed excessive bleeding. Although bleeding (p = 0.01) and pain (p = 0.02) were less acceptable in the 57-63 day group, 91% of subjects in both groups reported that the overall procedure was acceptable. In summary, low-dose mifepristone 200 mg and home administration of vaginal misoprostol 800 microg at 48 h were highly effective and acceptable to women < or =63 days pregnant, thereby expanding the number of women who can access a medical abortion. PMID- 10745069 TI - Termination of pregnancies of <6 weeks gestation with a single dose of 800 microg of vaginal misoprostol. AB - This study evaluated the effectiveness of a single dose of the abortifacient effect of vaginal misoprostol followed by prolonged observation. Women with < or =42 days of amenorrhea, pregnancy confirmed by ultrasound, and approved request for termination received 800 microg of vaginal misoprostol once and were observed for 1 week. The gestational sac was measured before misoprostol administration, and 24 h and 7 days afterward. Women reported bleeding, expulsion of sac, and other complaints. After 1 week, those who had not aborted received a second dose of 800 microg. Those who had not aborted by 24 h later were treated by vacuum aspiration of the endometrial cavity. Twenty-four hours after treatment, 71.8% had aborted, and 87.1% aborted 3 days after treatment. After the second dose, 7 days later, the cumulative abortion rate reached 92.1%. None of the subjects who aborted required curettage or vacuum aspiration. The main complaints were pain (84.5%), nausea (21.4%), and headache (17.5%). No clinical differences between responders and nonresponders was found. Vaginal misoprostol, 800 microg, is effective in inducing early termination of pregnancy, and there is no need for an additional dose within 72 h after the first administration of misoprostol. PMID- 10745070 TI - Comparative effects of Lunelle monthly contraceptive injection (medroxyprogesterone acetate and estradiol cypionate injectable suspension) and ortho-Novum 7/7/7 oral contraceptive (norethindrone/ethinyl estradiol triphasic) on lipid profiles. Investigators from the Lunelle Study Group. AB - As part of a 60-week, open-label, nonrandomized, parallel, controlled study comparing a monthly contraceptive injection containing medroxyprogesterone acetate (MPA) 25 mg and estradiol cypionate (E(2)C) 5 mg (Lunelle Monthly Contraceptive Injection) and a norethindrone 0.5, 0.75, 1.0 mg/0.035 mg ethinyl estradiol (NET/EE) triphasic oral contraceptive (Ortho-Novum(R) 7/7/7), a longitudinal examination of lipid profiles was conducted. Lipid parameters were assessed at screening and at weeks 20, 40, and 60 (or the final visit) in 114 women using MPA/E(2)C and 93 using NET/EE (lipid analysis population). Extra blood samples were obtained at weeks 21, 22, and 23 in 61 MPA/E(2)C users and 51 NET/EE users (index-cycle analysis population) to investigate lipid changes during one cycle of use. In the index-cycle population, median changes from screening to week 60 showed a decrease in apolipoprotein (apo) A-I and apo A-II in both groups. MPA/E(2)C users had a decrease in total cholesterol (C), total triglycerides, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C), with maintenance of the total C/HDL-C ratio. NET/EE users showed an increase in total C and LDL-C, with no change in HDL-C or the total C/HDL-C ratio. Within the index cycle (weeks 20 to 23), median changes in lipid values in both MPA/E(2)C and NET/EE users were generally greatest during the first week after the injection or the start of the pill pack. The results of this first longitudinal examination of serum lipids in US women using MPA/E(2)C confirm earlier findings in women in other countries. However, a direct comparison of the effects of MPA/E(2)C and NET/EE on lipid profiles was not possible in this study because of its design and because of the baseline and pharmacokinetic/pharmacodynamic differences between the two contraceptive groups. The results of this analysis showed that, although overall lipid values decreased, including a significant decrease in HDL cholesterol, the maintenance of the total-C/HDL-C ratio suggests that the effect of MPA/E(2)C on lipid parameters may not negatively affect CVD risk over 1 year of use. However, these results warrant further investigation, given the nature of this trial. PMID- 10745072 TI - Evaluation of subchronic (13 weeks) and reproductive toxicity potential of intravaginal gel-microemulsion formulation of a dual-function phenyl phosphate derivative of bromo-methoxy zidovudine (compound WHI-05) in B(6)C(3)F(1) mice. AB - Heterosexual transmission of human immunodeficiency virus (HIV) accounts for 90% of all new infections worldwide and significantly contributes to new acquired immunodeficiency syndrome (AIDS) cases in the United States. In a systematic effort to develop a microbicidal contraceptive capable of preventing HIV transmission as well as providing fertility control, we previously identified novel phenyl phosphate derivatives of 3'-azido-3'-deoxythymidine (zidovudine) which exhibit potent anti-HIV and spermicidal activities. This study reports the preliminary preclinical study of our lead compound WHI-05, 5-bromo-6-methoxy-5, 6 dihydro-3'-azidothymidine-5'-(p-methoxyphenyl) methoxyalaninyl phosphate, for use as a dual-function topical microbicide. Acute toxicity studies have shown that WHI-05 has no detectable adverse effects on laboratory animals. The 13-week subchronic and reproductive toxicity potential of intravaginal gel-microemulsion formulation of WHI-05 were studied in mice to support its further development as a virucidal spermicide. Groups of 10 female B(6)C(3)F(1) mice were exposed intravaginally to a gel-microemulsion formulation containing 0%, 0.5%, 1.0%, or 2.0% WHI-05, 5 days/week for 13 consecutive weeks. On a molar basis, these concentrations represent 300 to 1200 times their in vitro spermicidal potency, and 1.5x10(4) to 6.1x 10(4) times their in vitro anti-HIV activity. After 13 weeks of intravaginal treatment, one half of treated mice were evaluated for toxicity, and the other half were mated with untreated males to evaluate potential reproductive and developmental effects. Repetitive intravaginal application of WHI-05 to yield a local concentration 6.1x10(4) times higher than its in vitro HIV IC(50) value and 1200 times higher than its spermicidal EC(50)96%), or pup development. These findings collectively show that the experimental dual-function anti-HIV and contraceptive agent, WHI-05, did not cause significant acute or subchronic and reproductive toxicity under the test conditions. PMID- 10745071 TI - Gossypol blood levels and inhibition of spermatogenesis in men taking gossypol as a contraceptive. A multicenter, international, dose-finding study. AB - The safety and efficacy of gossypol continues to be controversial. The aim of this study was to evaluate gossypol as a contraceptive pill for men at doses lower than those previously prescribed and in men from various ethnic origin. A total of 151 men from Brazil, Nigeria, Kenya, and China were divided into two groups. Both groups received 15 mg gossypol/day for 12 or 16 weeks to reach spermatogenesis suppression. Subjects were then randomized to either 7.5 or 10 mg/day for 40 weeks. In addition, 51 men were enrolled as a control group. In all, 81 subjects attained spermatogenesis suppression. Only one man discontinued treatment because of tiredness. Potassium levels fluctuated within the normal range. FSH increased consistently. Testicular volume decreased, but after discontinuation, values returned to levels not statistically different from admission. Of 19 subjects on the 7.5 mg/day dose group, 12 recovered sperm counts >20 million/mL within 12 months of discontinuing gossypol. In the 10 mg/day group, sperm counts recovered in only 10 of 24 subjects. Eight of the 43 patients remained azoospermic 1 year after stopping gossypol. All men diagnosed with varicocele failed to reverse spermatogenesis suppression. Gossypol blood levels indicated that sperm suppression occurs independently of concentration, whereas spermatogenesis recovery appears to be concentration-dependent. Gossypol may become a medical alternative to surgical vasectomy when the delay in onset of infertility is acceptable. When taken for 1 year, gossypol causes no reduction in sexual desire or frequency of intercourse. The possibility of reversal, occurring in 51% of the men on this regimen within 1 year after stopping gossypol, is an advantage of this compound as compared with surgical sterilization in many parts of the world. PMID- 10745073 TI - Interaction of a novel cysteine and histidine-rich cytoplasmic protein with galectin-3 in a carbohydrate-independent manner. AB - We have used the yeast two-hybrid system to search for cytoplasmic proteins that might assist in the intracellular trafficking of the soluble beta-galactoside binding protein, galectin-3. We utilised as bait murine full-length galectin-3 to screen a murine 3T3 cDNA library. Several interacting clones were found to encode a partial open reading frame and a full-length clone was obtained by rapid amplification of cDNA ends methodology. In various assays in vitro the novel protein was shown to bind galectin-3 in a carbohydrate-independent manner. The novel protein contains an unusually high content of cysteine and histidine residues and shows significant sequence homologies with several metal ion-binding motifs present in known proteins. Confocal immunofluorescence microscopy of permeabilised 3T3 cells shows a prominent perinuclear, as well as cytoplasmic, localisation of the novel protein. PMID- 10745074 TI - Cytosolic Hsp70s are involved in the transport of aminopeptidase 1 from the cytoplasm into the vacuole. AB - Eukaryotic 70 kDa heat shock proteins (Hsp70s) are localized in various cellular compartments and exhibit functions such as protein translocation across membranes, protein folding and assembly. Here we demonstrate that the constitutively expressed members of the yeast cytoplasmic Ssa subfamily, Ssa1/2p, are involved in the transport of the vacuolar hydrolase aminopeptidase 1 from the cytoplasm into the vacuole. The Ssap family members displayed overlapping functions in the transport of aminopeptidase 1. In SSAI and SSAII deletion mutants the precursor of aminopeptidase 1 accumulated in a dodecameric complex that is packaged in prevacuolar transport vesicles. Ssa1/2p was prominently localized to the vacuolar membrane, consistent with the role we propose for Ssa proteins in the fusion of transport vesicles with the vacuolar membrane. PMID- 10745075 TI - The crystal structure of saporin SO6 from Saponaria officinalis and its interaction with the ribosome. AB - The 2.0 A resolution crystal structure of the ribosome inactivating protein saporin (isoform 6) from seeds of Saponaria officinalis is presented. The fold typical of other plant toxins is conserved, despite some differences in the loop regions. The loop between strands beta7 and beta8 in the C-terminal region which spans over the active site cleft appears shorter in saporin, suggesting an easier access to the substrate. Furthermore we investigated the molecular interaction between saporin and the yeast ribosome by differential chemical modifications. A contact surface inside the C-terminal region of saporin has been identified. Structural comparison between saporin and other ribosome inactivating proteins reveals that this region is conserved and represents a peculiar motif involved in ribosome recognition. PMID- 10745076 TI - Observations of rotation within the F(o)F(1)-ATP synthase: deciding between rotation of the F(o)c subunit ring and artifact. AB - F(o)F(1)-ATP synthase mediates coupling of proton flow in F(o) and ATP synthesis/hydrolysis in F(1) through rotation of central rotor subunits. A ring structure of F(o)c subunits is widely believed to be a part of the rotor. Using an attached actin filament as a probe, we have observed the rotation of the F(o)c subunit ring in detergent-solubilized F(o)F(1)-ATP synthase purified from Escherichia coli. Similar studies have been performed and reported recently [Sambongi et al. (1999) Science 286, 1722-1724]. However, in our hands this rotation has been observed only for the preparations which show poor sensitivity to dicyclohexylcarbodiimde, an F(o) inhibitor. We have found that detergents which adequately disperse the enzyme for the rotation assay also tend to transform F(o)F(1)-ATP synthase into an F(o) inhibitor-insensitive state in which F(1) can hydrolyze ATP regardless of the state of the F(o). Our results raise the important issue of whether rotation of the F(o)c ring in isolated F(o)F(1)-ATP synthase can be demonstrated unequivocally with the approach adopted here and also used by Sambongi et al. PMID- 10745077 TI - Prediction of the tertiary structure of a caspase-9/inhibitor complex. AB - Apoptosis, or programmed cell death, plays a central role in the development and homeostasis of an organism. The breakdown of cellular proteins in apoptosis is mediated by caspases, which comprise a highly conserved family of cysteine proteases with specificity for aspartic acid residues at the P1 positions of their substrates. Multiple lines of evidence show that caspase-9 is critical for an apoptosis pathway mediated via the mitochondria. In this study, the three dimensional structure of the catalytic domain of caspase-9 and its interaction with the inhibitor acetyl-Asp-Val-Ala-Asp fluoromethyl ketone (Ac-DVAD-fmk) have been predicted by a segment matching modeling procedure. As expected, the predicted caspase-9 structure shows both a high similarity in the overall folding topology and remarkable differences in the surface loop regions as compared to other caspase family members such as caspase-1, -3 and -8, for which crystal structures have been determined. This kind of comparative analysis reflects the convergence-divergence duality among the caspases. Moreover, some subtle differences have been observed between caspase-9 and caspase-3 in the subsite contacts with the covalently linked inhibitor Ac-DVAD-fmk. Based on the X-ray structural analysis of caspase-8, a main chain carbonyl oxygen appears to be involved in a catalytic triad with the active site Cys and His residues. The corresponding carbonyl oxygen in caspase-9, together with other expected features of the catalytic apparatus, appears in our model. The predicted structure of caspase-9 can serve as a reference for subsite analysis relative to rational design of highly selective caspase inhibitors for therapeutic application. PMID- 10745078 TI - Leap-dynamics: efficient sampling of conformational space of proteins and peptides in solution. AB - A molecular simulation scheme, called Leap-dynamics, that provides efficient sampling of protein conformational space in solution is presented. The scheme is a combined approach using a fast sampling method, imposing conformational 'leaps' to force the system over energy barriers, and molecular dynamics (MD) for refinement. The presence of solvent is approximated by a potential of mean force depending on the solvent accessible surface area. The method has been successfully applied to N-acetyl-L-alanine-N-methylamide (alanine dipeptide), sampling experimentally observed conformations inaccessible to MD alone under the chosen conditions. The method predicts correctly the increased partial flexibility of the mutant Y35G compared to native bovine pancreatic trypsin inhibitor. In particular, the improvement over MD consists of the detection of conformational flexibility that corresponds closely to slow motions identified by nuclear magnetic resonance techniques. PMID- 10745079 TI - A novel reporter mouse strain that expresses enhanced green fluorescent protein upon Cre-mediated recombination. AB - The success of Cre-mediated conditional gene targeting depends on the specificity of Cre recombinase expression in Cre-transgenic mouse lines. As a tool to evaluate the specificity of Cre expression, we developed a reporter transgenic mouse strain that expresses enhanced green fluorescent protein (EGFP) upon Cre mediated recombination. We demonstrate that the progeny resulting from a cross between this reporter strain and a transgenic strain expressing Cre in zygotes show ubiquitous EGFP fluorescence. This reporter strain should be useful to monitor the Cre expression directed by various promoters in transgenic mice, including mice in which Cre is expressed transiently during embryogenesis under a developmentally regulated promoter. PMID- 10745080 TI - Activation of a capacitative Ca(2+) entry pathway by store depletion in cultured hippocampal neurones. AB - Intracellular Ca(2+) ([Ca(2+)](i)) changes were measured in cell bodies of cultured rat hippocampal neurones with the fluorescent indicator Fluo-3. In the absence of external Ca(2+), the cholinergic agonist carbachol (200 microM) and the sarcoendoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (0.4 microM) both transiently elevated [Ca(2+)](i). A subsequent addition of Ca(2+) into the bathing medium caused a second [Ca(2+)](i) change which was blocked by lanthanum (50 microM). Taken together, these experiments indicate that stores depletion can activate a capacitative Ca(2+) entry pathway in cultured hippocampal neurones and further demonstrate the existence of such a Ca(2+) entry in excitable cells. PMID- 10745081 TI - Differential effects of Cbl and 70Z/3 Cbl on T cell receptor-induced phospholipase Cgamma-1 activity. AB - We demonstrate that the differential effects Cbl and oncogenic 70Z/3 Cbl have on Ca(2+)/Ras-sensitive NF-AT reporters is partially due to their opposing ability to regulate phospholipase Cgamma1 (PLCgamma1) activation as demonstrated by analysis of the activation of an NF-AT reporter construct and PLCgamma1-mediated inositol phospholipid (PI) hydrolysis. Cbl over-expression resulted in reduced T cell receptor-induced PI hydrolysis, in the absence of any effect on PLCgamma1 tyrosine phosphorylation. In contrast, expression of 70Z/3 Cbl led to an increase in basal and OKT3-induced PLCgamma1 phosphorylation and PI hydrolysis. These data indicate that Cbl and 70Z/3 Cbl differentially regulate PLCgamma1 phosphorylation and activation. The implications of these data on the mechanism of Cbl-mediated signaling regulation are discussed. PMID- 10745082 TI - A new endogenous differentiating factor (myelopeptide-4) for myeloid cells. AB - Along with known lymphokines involved in the regulation of hematopoiesis, a new differentiating factor (myelopeptide-4, MP-4) for myeloid cells was found. The peptide (Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro) originally isolated from the culture medium of porcine bone marrow cell culture was examined for its ability to induce differentiation in two human myeloid leukemia cell lines, HL-60 and K-562. Agents with well-known differentiation-inducing activity, such as phorbol myristate acetate, dimethylsulfoxide and the lymphokines were used as a reference. It has been shown that MP-4 significantly influences the integral characteristics of metabolism, expression of surface antigens and morphology of these cells. It decreased the level of chromosomal DNA synthesis and, in parallel, increased the total protein synthesis in both HL-60 and K-562 cells. MP-4 induced the expression of CD14 monocyte-specific surface antigen and the appearance of mature monocytes/macrophages in HL-60 cell cultures. There was a good correlation of cell metabolic/morphological changes and the CD14 marker expression for HL-60 cells. A similar phenomenon was observed in K-562 cells treated with MP-4 when the levels of hemoglobin synthesis were detected in their cytoplasm. Thus, we consider MP-4 as a new endogenous differentiating factor for myeloid cells. PMID- 10745083 TI - Evaluation of inhibition of the carbenicillin-hydrolyzing beta-lactamase PSE-4 by the clinically used mechanism-based inhibitors. AB - Characterization of the biochemical steps in the inactivation chemistry of clavulanic acid, sulbactam and tazobactam with the carbenicillin-hydrolyzing beta lactamase PSE-4 from Pseudomonas aeruginosa is described. Although tazobactam showed the highest affinity to the enzyme, all three inactivators were excellent inhibitors for this enzyme. Transient inhibition was observed for the three inactivators before the onset of irreversible inactivation of the enzyme. Partition ratios (k(cat)/k(inact)) of 11, 41 and 131 were obtained with clavulanic acid, tazobactam and sulbactam, respectively. Furthermore, these values were found to be 14-fold, 3-fold and 80-fold lower, respectively, than the values obtained for the clinically important TEM-1 beta-lactamase. The kinetic findings were put in perspective by determining the computational models for the pre-acylation complexes and the immediate acyl-enzyme intermediates for all three inactivators. A discussion of the pertinent structural factors is presented, with PSE-4 showing subtle differences in interactions with the three inhibitors compared to the TEM-1 enzyme. PMID- 10745085 TI - A gene fusion event in the evolution of aminoacyl-tRNA synthetases. AB - The genes of glutamyl- and prolyl-tRNA synthetases (GluRS and ProRS) are organized differently in the three kingdoms of the tree of life. In bacteria and archaea, distinct genes encode the two proteins. In several organisms from the eukaryotic phylum of coelomate metazoans, the two polypeptides are carried by a single polypeptide chain to form a bifunctional protein. The linker region is made of imperfectly repeated units also recovered as singular or plural elements connected as N-terminal or C-terminal polypeptide extensions in various eukaryotic aminoacyl-tRNA synthetases. Phylogenetic analysis points to the monophyletic origin of this polypeptide motif appended to six different members of the synthetase family, belonging to either of the two classes of aminoacyl tRNA synthetases. In particular, the monospecific GluRS and ProRS from Caenorhabditis elegans, an acoelomate metazoan, exhibit this recurrent motif as a C-terminal or N-terminal appendage, respectively. Our analysis of the extant motifs suggests a possible series of events responsible for a gene fusion that gave rise to the bifunctional glutamyl-prolyl-tRNA synthetase through recombination between genomic sequences encoding the repeated units. PMID- 10745084 TI - Isolation and pharmacological characterisation of delta-atracotoxin-Hv1b, a vertebrate-selective sodium channel toxin. AB - delta-Atracotoxins (delta-ACTXs) are peptide toxins isolated from the venom of Australian funnel-web spiders that slow sodium current inactivation in a similar manner to scorpion alpha-toxins. We have isolated and determined the amino acid sequence of a novel delta-ACTX, designated delta-ACTX-Hv1b, from the venom of the funnel-web spider Hadronyche versuta. This 42 residue toxin shows 67% sequence identity with delta-ACTX-Hv1a previously isolated from the same spider. Under whole-cell voltage-clamp conditions, the toxin had no effect on tetrodotoxin (TTX)-resistant sodium currents in rat dorsal root ganglion neurones but exerted a concentration-dependent reduction in peak TTX-sensitive sodium current amplitude accompanied by a slowing of sodium current inactivation similar to other delta-ACTXs. However, delta-ACTX-Hv1b is approximately 15-30-fold less potent than other delta-ACTXs and is remarkable for its complete lack of insecticidal activity. Thus, the sequence differences between delta-ACTX-Hv1a and -Hv1b provide key insights into the residues that are critical for targeting of these toxins to vertebrate and invertebrate sodium channels. PMID- 10745086 TI - Novel mechanism that Trypanosoma cruzi uses to adhere to the extracellular matrix mediated by human galectin-3. AB - Binding of Trypanosoma cruzi trypomastigotes to laminin is enhanced by galectin 3, a beta-galactoside binding lectin. The galectin-3 enhanced binding of trypanosomes to laminin is inhibited by lactose. Co-immunoprecipitations indicate that galectin-3 binds to the 45, 32 and 30 kDa trypanosome surface proteins. Binding of galectin-3 to the 45, 32 and 30 kDa surface proteins is inhibited by lactose. Polyclonal and a monoclonal antibodies to galectin-3 immunoprecipitated a major 64 kDa trypanosome surface protein. T. cruzi monoclonal antibody to mucin recognized the 45 kDa surface protein. The 45, 32 and 30 kDa surface proteins interact with galectin-3 in order to enhance trypanosome adhesion to laminin. PMID- 10745087 TI - Morphine-related metabolites differentially activate adenylyl cyclase isozymes after acute and chronic administration. AB - Morphine-3- and morphine-6-glucuronide are morphine's major metabolites. As morphine-6-glucuronide produces stronger analgesia than morphine, we investigated the effects of acute and chronic morphine glucuronides on adenylyl cyclase (AC) activity. Using COS-7 cells cotransfected with representatives of the nine cloned AC isozymes, we show that AC-I and V are inhibited by acute morphine and morphine 6-glucuronide, and undergo superactivation upon chronic exposure, while AC-II is stimulated by acute and inhibited by chronic treatment. Morphine-3-glucuronide had no effect. The weak opiate agonists codeine and dihydrocodeine are also addictive. These opiates, in contrast to their 3-O-demethylated metabolites morphine and dihydromorphine (formed by cytochrome P450 2D6), demonstrated neither acute inhibition nor chronic-induced superactivation. These results suggest that metabolites of morphine (morphine-6-glucuronide) and codeine/dihydrocodeine (morphine/dihydromorphine) may contribute to the development of opiate addiction. PMID- 10745089 TI - Localization of riboproteins in a trypanosomatid mitochondrion. AB - There is growing evidence in support of mitochondrial translation in trypanosomes but mitoribosomes have never been characterized or localized in these parasites. On RNA-protein blots we identified several proteins from the trypanosomatid Crithidia fasciculata which bound the parasite's 12S and 9S mitochondrial ribosomal RNAs. Two of these proteins had significant amino acid sequence homology to riboproteins S8 and S21 across phyla. Immunoelectron microscopy revealed that antibodies raised against the two proteins react with matrix components in the C. fasciculata mitochondrion. Our data thus provide, we believe for the first time, evidence for the presence of riboproteins within a trypanosomatid mitochondrion, bound, possibly, to the 12S and 9S RNAs. The proteins were immunologically related to two cytosolic riboproteins which were also of identical size, suggesting the interesting possibility that the same set of riboproteins is shared between the cytosol and the mitochondrion in this parasite. PMID- 10745088 TI - Protein kinase C phosphorylates and regulates UDP-N-acetylglucosamine-2 epimerase/N-acetylmannosamine kinase. AB - UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (UDP-GlcNAc 2 epimerase) is the key enzyme in the de novo synthesis pathway of neuraminic acid, which is widely expressed as a terminal carbohydrate residue on glycoconjugates. UDP-GlcNAc 2-epimerase is a bifunctional enzyme and catalyzes the first two steps of neuraminic acid synthesis in the cytosol, the conversion of UDP-N acetylglucosamine to ManAc and the phosphorylation to ManAc-6-phosphate. So far, regulation of this essential enzyme by posttranslational modification has not been shown. Since UDP-N-acetylglucosamine is a cytosolic protein containing eight conserved motifs for protein kinase C (PKC), we investigated whether its enzymatic activity might be regulated by phosphorylation by PKC. We showed that UDP-GlcNAc 2-epimerase interacts with several isoforms of PKC in mouse liver and is phosphorylated in vivo. Furthermore, PKC phosphorylates UDP-GlcNAc 2-epimerase and this phosphorylation results in an upregulation of the UDP-GlcNAc 2-epimerase enzyme activity. PMID- 10745090 TI - In vivo transfer of hepatocyte growth factor gene accelerates proliferation of hepatic oval cells in a 2-acetylaminofluorene/partial hepatectomy model in rats. AB - To clarify the effect of hepatocyte growth factor (HGF) on proliferation of hepatic oval cells, we transferred HGF gene into liver of the Solt-Farber rat model. Male Fisher 344 rats were infected with a recombinant adenovirus carrying the cDNA for HGF (pAxCAHGF) from tail vein. HGF mRNA showed its peak at 4 days, and diminished thereafter. The total and proliferating cell nuclear antigen positive hepatic oval cells were significantly elevated in HGF-transferred rats, in which stem cell factor and c-kit mRNA increased at each time point. Our results suggest that in vivo transfer of the HGF gene into liver accelerates proliferation of hepatic oval cells in the Solt-Farber model in rats. PMID- 10745091 TI - Angiotensin II type 1 receptor-function affected by mutations in cytoplasmic loop CD. AB - To explore peptide hormone-induced conformational changes, we attempted to engineer a metal-ion binding site between the cytoplasmic loops CD and EF in the angiotensin II type 1 (AT(1)) receptor. We constructed 12 double and six triple histidine mutant receptors, and tested the ability of each mutant and the wild type to activate inositol phosphate (IP) production with and without ZnCl(2). Inhibition by ZnCl(2) in the double and triple His mutant receptors was not significant, but these mutations directly decreased the IP production. Systematic analysis of single His mutants demonstrated that the loop CD-mutants displayed 52 74% inhibition of IP production, whereas the loop EF-mutants did not affect IP production. These results indicate that the cytoplasmic loop CD-segment from Tyr(127) to Ile(130) is important for G(q/11) activation by the AT(1) receptor. PMID- 10745092 TI - Purification and characterization of bovine cone arrestin (cArr). AB - To elucidate the quenching mechanism of phototransduction in vertebrate cone photoreceptors, a cDNA clone encoding cone specific arrestin (cArr) was isolated from a bovine retinal cDNA library using a human cArr cDNA probe. Affinity purified anti-peptide antibody specific to cArr was prepared. Immunohistochemical staining displayed specific labeling of cArr in cone photoreceptors and immunoblotting identified a 46 kDa protein band. We purified cArr from bovine retinas by sequential column chromatography using DEAE-cellulose, gel filtration and mono Q columns. Binding studies revealed no binding of cArr to rhodopsin regardless of whether it was bleached and/or phosphorylated. cArr also failed to bind to heparin-Sepharose under conditions which rod arrestin (rArr) bound to the column. The present data suggest that cArr may play a role in the quenching of phototransduction in cone photoreceptors and that its activity therein is different to that of rArr. PMID- 10745093 TI - Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells. AB - 3,4-dihydroxyphenylethanol (hydroxytyrosol; DPE) is the major phenolic antioxidant present in extra virgin olive oil, either in a free or esterified form. Despite its relevant biological effects, no data are available on its bioavailability and metabolism. The aim of the present study is to examine the molecular mechanism of DPE intestinal transport, using differentiated Caco-2 cell monolayers as the model system. The kinetic data demonstrate that [(14)C]DPE transport occurs via a passive diffusion mechanism and is bidirectional; the calculated apparent permeability coefficient indicates that the molecule is quantitatively absorbed at the intestinal level. The only labelled DPE metabolite detectable in the culture medium by HPLC (10% conversion) is 3-hydroxy-4 methoxyphenylethanol, the product of catechol-O-methyltransferase; when DPE is assayed in vitro with the purified enzyme a K(m) value of 40 microM has been calculated. PMID- 10745094 TI - Three of four pseudoknots in tmRNA are interchangeable and are substitutable with single-stranded RNAs. AB - A novel translation, trans-translation, is facilitated by a highly structured RNA molecule, tmRNA. This molecule has two structural domains, a tRNA domain and an mRNA domain, the latter including four pseudoknot structures (PK1 to PK4). Here, we show that replacement of each of these pseudoknots, except PK1, in Escherichia coli tmRNA with a single stranded RNA did not seriously affect the functions as an alanine tRNA and as an mRNA. Furthermore, these three pseudoknots were interchangeable with only small losses of the two functions. These findings suggest that neither PK2, PK3 nor PK4 interacts in a functional manner with ribosome during the trans-translation process. Together with an earlier study showing the significance of PK1, it is concluded that among the four pseudoknots, PK1 is the most functional. PMID- 10745095 TI - Differential activation of MAP kinase family members triggered by CD99 engagement. AB - The molecular basis for the modulatory properties of CD99 is not well understood. Treatment of human Jurkat T lymphocytes with anti-CD99 antibody led to activation of three mitogen-activated protein kinase (MAPK) members, ERK, JNK, and p38 MAPK, along with homotypic aggregation. While phosphorylation of ERK and JNK was inhibited by the pretreatment of a PKC inhibitor, bisindolylmaleimide I, activation of p38 MAPK was upregulated by the same pretreatment. The signaling pathways to MAPKs by CD99 engagement were independent of PI-3 kinase, distinguishing from those by CD3 engagement. Among MAPKs, ERK pathway was essential for homotypic aggregation together with intracytoplasmic Ca(2+). PMID- 10745096 TI - NB-506, an indolocarbazole topoisomerase I inhibitor, binds preferentially to triplex DNA. AB - A novel competition dialysis method was used to study the structural selectivity of the nucleic acid binding of NB-506, a promising indolocarbazole anticancer agent. A pronounced preference for NB-506 binding to the DNA triplex poly [dA]:(poly[dT])(2) was observed among potential binding to 12 different nucleic acid structures and sequences. Structures included in the assay ranged from single-stranded DNA, through a variety of right-handed DNA duplexes, to multistranded triplex and tetraplex forms. RNA and left-handed Z DNA were also included in the assay. The preferential binding to triplex was confirmed by UV melting experiments. The novel and unexpected structural selectivity shown by NB 506 may arise from a complementary shape between its extended aromatic ring system and the planar triplex stack. PMID- 10745097 TI - Isolation of Ich-1S (caspase-2S)-binding protein that partially inhibits caspase activity. AB - Members of the caspase family are essential executors of apoptosis. Caspase-2 has two messenger RNAs generated by alternative splicing, which encode caspase-2L and caspase-2S. Although caspase-2L induces apoptosis, caspase-2S also has the ability to antagonize cell death. Experiments in caspase-2-deficient mice showed that caspase-2 functions to delay cell death in some neuronal populations, suggesting that caspase-2S dominantly acts for cell survival in the brain. However, the mechanism of caspase-2S-mediated anti-apoptotic effect is still unclear. Here, we isolated a protein that interacts with caspase-2S, designated as Ich-1S (caspase-2S)-binding protein (ISBP), by yeast two-hybrid screening using full-length caspase-2S cDNA as a bait. ISBP is identical to the recently isolated calcium and integrin-binding protein, and a small molecule calcium binding protein with two EF-hand motifs of its C-terminus. In vitro transcribed and translated ISBP interacts specifically with glutathione-S-transferase-fused caspase-2S. Moreover, the interaction between ISBP and caspase-2S was observed in cultured cells. Northern blot analysis indicated that ISBP may be a ubiquitous protein. Interestingly, ISBP can partially inhibit the processing of pro-caspase 2L induced by anti-Fas antibody-treated Jurkat cytosolic lysates. These results suggested that ISBP may be the mediator for the survival function of caspase-2S. PMID- 10745098 TI - The chaperonin-related protein Tcm62p ensures mitochondrial gene expression under heat stress. AB - Tcm62p, distantly related to chaperonins, is required for the assembly of succinate dehydrogenase in mitochondria of Saccharomyces cerevisiae and was proposed to exert chaperone activity. We demonstrate here crucial functions of Tcm62p under heat stress. It ensures mitochondrial gene expression at elevated temperatures and prevents heat-aggregation of the ribosomal subunit Var1p. Similar to chaperonins, Tcm62p forms a high molecular mass protein complex of approximately 850 kDa in the mitochondrial matrix space. These results suggest a more general chaperone function of Tcm62p in mitochondria. PMID- 10745099 TI - Pressurization facilitates adenovirus-mediated gene transfer into vein graft. AB - We investigated whether application of non-distending hydrostatic pressure facilitates gene transfer into vein grafts. An external jugular vein was placed in a chamber with 100 microl adenovirus solution at a titer of 10(10) pfu/ml and was pressurized to up to 8 atm above ambient pressure for 10 min. Histochemical analysis demonstrated a positive transgene expression in all layers of the vessel wall. Gene transfer with 8 atm pressurization resulted in an approximately 50 times higher transgene expression than that without pressurization. Under 8 atm pressurization, the efficiency of gene transfer reached a plateau at 7.5 min. The application of hydrostatic pressure may improve the effectiveness of intraoperative genetic engineering of vein grafts. PMID- 10745101 TI - Corrigendum to: molecular cloning and functional expression of ca(v)3.1c, a T type calcium channel from human brain. PMID- 10745100 TI - Light-induced stimulation of carbonic anhydrase activity in pea thylakoids. AB - Stimulation of the bicarbonate dehydration reaction in thylakoid suspension under conditions of saturating light at pH 7.6-8.0 was discovered. This effect was inhibited by nigericin or the lipophilic carbonic anhydrase (CA) inhibitor ethoxyzolamide (EZ), but not by the hydrophilic CA inhibitor, acetazolamide. It was shown that the action of EZ is not caused by an uncoupling effect. It was concluded that thylakoid CA is the enzyme utilizing the light-generated proton gradient across the thylakoid membrane thus facilitating the production of CO(2) from HCO(3)(-) and that this enzyme is covered from the stroma side of thylakoids by a lipid barrier. PMID- 10745102 TI - Characterization of Trypanozoon isolates using a repeated coding sequence and microsatellite markers. AB - Genetic variation of microsatellite loci is a widely used method for linkage analysis, individual identification or inter-population studies. Here we analyse a repeated DNA coding sequence and eleven new microsatellites identified within the Trypanosoma (Trypanozoon) brucei genome. Ninety-seven isolates belonging to the five species and subspecies Trypanosoma evansi, T. equiperdum, T. brucei brucei, T. b. rhodesiense and T. b. gambiense were compared regarding the genetic patterns of these markers. The results reveal a great heterogeneity of the genotypes related to the repeated coding sequence and five microsatellites, some of which show a high degree of polymorphism. This allows us to define group specific genotypes or alleles; in particular, we show that one specific pattern clearly segregates the human pathogen T. b. gambiense group 1. PMID- 10745103 TI - The application of shannon entropy in the identification of putative drug targets. AB - A major challenge in the field of functional genomics is the development of computational techniques for organizing and interpreting large amounts of gene expression data. These methods will be critical for the discovery of new therapeutic drug targets. Here, we present a simple method for determining the most likely drug target candidates from temporal gene expression patterns assayed with reverse-transcription polymerase chain reaction (RT-PCR) and DNA microarrays. PMID- 10745104 TI - Coding efficiency and information rates in transmembrane signaling. AB - A variety of cell types responds to hormonal stimuli by repetitive spikes in the intracellular concentration of calcium ([Ca(2+)](i)) which have been demonstrated to encode information in their frequency, amplitude, and duration. These [Ca(2+)](i)-spike trains are able to specifically regulate distinct cellular functions. Using a mathematical model for receptor-controlled [Ca(2+)](i) oscillations in hepatocytes we investigate the encoding of fluctuating hormonal signals in [Ca(2+)](i)-spike trains. The transmembrane information transfer is quantified by using an information-theoretic reverse-engineering approach which allows to reconstruct the dynamic hormonal stimulus from the [Ca(2+)](i)-spike trains. This approach allows to estimate the accuracy of coding as well as the rate of transmembrane information transfer. We found that up to 87% of the dynamic stimulus information can be encoded in the [Ca(2+)](i)-spike train at a maximum information transfer rate of 1.1 bit per [Ca(2+)](i)-spike. These numerical results for humoral information transfer are in the same order as in a number of sensory neuronal systems despite several orders of magnitude different time scales of operation suggesting a universal principle of information processing in both biological systems. PMID- 10745105 TI - Modeling of signaling pathways for endocrine disruptors. AB - The so called endocrine disruptors have become an important working hypothesis for a wide range of toxicology researchers. This hypothesis has also attracted those who have worked on designer estrogens or selective estrogen receptor modulators. Already numbers of substances have been identified as such chemicals, but there remain a large number of chemicals waiting to be tested for their endocrine modulating capabilities. Because of the time and costs required for wet lab tests, it is unrealistic to apply these kinds of tests to all such suspicious or probable chemicals. Thus some theoretical methods must be developed for this purpose. However the conventional QSAR (quantitative structure activity relationships) approach is of limited relevance to this problem, because these methods do not take detailed mechanisms of molecular interactions in biological systems into account. Thus we have developed a database complex system that enables one to trace molecular interactions triggered by interaction of receptors with xenobiotic chemicals. The main components of this database complex are a potential endocrine disruptor database, a receptor database, a cell signaling networks database, a transcription factor database, and an affinity binding database based on modes of actions. PMID- 10745106 TI - Control of interaction strength in a network of the true slime mold by a microfabricated structure. AB - The plasmodium of the true slime mold, Physarum polycephalum, which shows various nonlinear oscillatory phenomena, for example, in its thickness, protoplasmic streaming and concentration of intracellular chemicals, can be regarded as a collective of nonlinear oscillators. The plasmodial oscillators are interconnected by microscale tubes whose dimensions can be closely related to the strength of interaction between the oscillators. Investigation of the collective behavior of the oscillators under the conditions in which the interaction strength can be systematically controlled gives significant information on the characteristics of the system. In this study, we proposed a living model system of a coupled oscillator system in the Physarum plasmodium. We patterned the geometry and dimensions of the microscale tube structure in the plasmodium by a microfabricated structure (microstructure). As the first step, we constructed a two-oscillator system for the plasmodium that has two wells (oscillator part) and a channel (coupling part). We investigated the oscillation behavior by monitoring the thickness oscillation of the plasmodium in the microstructure with various channel widths. It was found that the oscillation behavior of two oscillators dynamically changed depending on the channel width. Based on the results of measurements of the tube dimensions and the velocity of the protoplasmic streaming in the tube, we discuss how the channel width relates to the interaction strength of the coupled oscillator system. PMID- 10745107 TI - Is there a biology of quantum information? AB - This paper briefly considers the notion of a biology of quantum information from a number of complementary points of view. We begin with a very brief look at some of the biomolecular systems that are thought to exploit quantum mechanical effects and then turn to the issue of measurement in these systems and the concomitant generation of information. This leads us to look at the internalist stance and the exchange interaction of quantum particles. We suggest that exchange interaction can also be viewed using ecological ideas related to apparatus-object. This can also help develop the important notion of complementarity in biosystems in relation to the nature and generation of information at the microphysical scale. PMID- 10745108 TI - Adaptive information processing in microtubule networks. AB - Microtubule networks provide a wide range of microskeletal and micromuscular functionalities. Evidence from a number of directions suggests that they can also serve as a medium for intracellular signaling processing. The model presented here comprises an empirically motivated representation of microtubule growth dynamics, an abstract representation of signal processing, and a feedback learning mechanism that we refer to as adaptive self-stabilization. The growth model mimics the dynamic instability picture of microtubule formation and decomposition, but as modulated by the binding activity of microtubule associated proteins (or MAPs). The signal processing submodel treats each microtubule as a string of linked discrete oscillators capable of propagating signals that are introduced, manipulated, and extracted by bound MAP activity. Adaptive self stabilization is essentially feedback acting on signal processing capabilities via the growth dynamics. The network is presented with a training set of patterns. If the input-output behavior is satisfactory MAP binding affinity increases, thereby stabilizing the network structure; otherwise the binding affinity decreases, allowing for more structural variation. The results obtained suggest that adaptive capabilities are practically inevitable in microtubule networks, a conclusion strengthened by the fact that the signal processing and growth dynamics mechanisms available in nature are undoubtedly much richer than those represented in the model. PMID- 10745109 TI - A model for neural representation of temporal duration. AB - To address how temporal duration is encoded in neural systems, we put forward a simple model for recurrent neural networks. Particular assumptions are only the following two: (1) neuronal bistability and; (2) environmental effects described by a heat bath. The results of Monte Carlo simulation show that population activity triggered at an initial time continues for a prolonged duration, followed by an abrupt self-termination. This time course seems highly suitable for neural representation of temporal duration. The time scale of this prolonged duration is much longer than the time scale of neuronal firing which is of the order of ms. The former time scale implies that of interval timing in cognition and behaviour. Thus, the model provides a possible explanation for a link between these two separated time scales. The Weber law, a hallmark of humans and animals' interval timing, can also be reproduced in our model. PMID- 10745110 TI - A model for molecular mechanisms of synaptic competition for a finite resource. AB - Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) undergoes Ca(2+)/calmodulin-dependent autophosphorylation of threonine-286/287 (Thr(286/287)). Extremely high concentration of CaMKII in the postsynaptic spine indicates that increase in the Ca(2+) concentration in the spine induced by synaptic activation results in Thr(286/287) autophosphorylation of this enzyme. It has recently been shown that the K(d) value of CaMKII for Ca(2+)/calmodulin (Ca(2+)/CaM) drastically decreases after Thr(286/287) autophosphorylation. Therefore, Ca(2+)/CaM associated with CaMKII becomes tightly bound to this kinase after Thr(286/287) autophosphorylation. This has been called 'Ca(2+)/CaM trapping'. We discussed the functional significance of Ca(2+)/CaM trapping in the neuronal system by a mathematical-modelling approach. We considered neighbouring synapses formed on the same dendrite and different increase in the Ca(2+) concentration in each spine. CaMKII undergoing Thr(286/287) autophosphorylation in each spine is eager to recruit nearby calmodulin in the dendrite for Ca(2+)/CaM trapping. Since the amount of calmodulin is limited, recruiting calmodulin to each spine causes competition among synapses for this finite resource. The results of our computer simulation show that this competition leads to 'winner-take-all': almost all calmodulin is taken by a few synapses to which relatively large increases in the Ca(2+) concentration are assigned. These results suggest a novel form of synaptic encoding of information. PMID- 10745111 TI - Cellular information transfer regarded from a stoichiometry and control analysis perspective. AB - Metabolic control analysis (MCA) allows one to formalize important aspects of information processing in living cells. For example, information processing via multi-level enzyme cascades can be quantified in terms of the response coefficient of a cellular target to a signal. In many situations, control and response coefficients cannot be determined exactly for all enzymes involved, owing to difficulties in 'observing' all enzymes experimentally. Here, we review a number of qualitative approaches that were developed to cope with such situations. The usefulness of the concept of null-space of the stoichiometry matrix for analysing the structure of intracellular signaling networks is discussed. It is shown that signal transduction operates very efficiently when the network structure is such that the null-space matrix can be block diagonalized (which may or may not imply that the network consists of several disconnected parts) and some enzymes have low elasticities to their substrates. PMID- 10745112 TI - Spatio-logical processes in intracellular signalling. AB - Classical models of intracellular signalling describe how small changes in a cell's external environment can bring about major changes in cellular activity. Recent findings from experimental biology indicate that many intracellular signalling systems show a high level of spatial organisation. This permits the modification, by protein kinase or protein phosphatase action, of specific subsets of intracellular proteins - an attribute that is not addressed in classical signalling models. Here we use ideas and concepts from computer science to describe the information processing nature of intracellular signalling pathways and the impact of spatial heterogeneity of their components (e.g. protein kinases and protein phosphatases) on signalling activity. We argue that it is useful to view the signalling ecology as a vast parallel distributed processing network of agents operating in heterogeneous microenvironments, and we conclude with an overview of the mathematical and semantic methodologies that might help clarify this analogy between biological and computational systems. PMID- 10745113 TI - A logic for biological systems. AB - This paper proposes a specification language, hybrid projection temporal logic of modelling, analyzing and verifying biological systems which can be considered, in general, to be hybrid systems consisting of a non-trivial mixture of discrete and continuous components. The syntax and semantics of the logic are presented, and some examples of hybrid systems are modelled to illustrate the formalism. PMID- 10745114 TI - Asynchronous, irregular automata nets: the path not taken. AB - This is a prelude to, and an extension of the original paper Artificial tissue models (Stark, R., 1994. The topology and analysis of asynchronous processes. http://www.math.usf.edu/ approximately stark/documents). However, this exposition is designed for a broader audience - anyone working in biological information processing. A primary objective is to demonstrate that irregular asynchronous automata nets, as opposed to cellular automata, are a realistic approach to modeling biological information processing. Also, new material is presented. Sections 1 and 2 review the early history of von Neumann's attempt explore biological information processing and finally the emergence of cellular automata. The history is guided by the question of why John von Neumann knowingly (we believe) compromised his investigation of biological information processing by falling back to the model we now know as cellular automata. Section 3 defines and explores examples of cellular automata and artificial tissue. Sections 4 and 5 contain philosophical observations which unify our paper, and propose an answer to the original question. A new model for Turing's leopards' spot problem is presented. The asynchronous models are defined by a cell program and a local commumications protocol only. Computational freedom comes from asynchronous activity, while global organization emerges from the entropy reducing nature of the cell programs. PMID- 10745115 TI - Cellular automaton of idealized brain tumor growth dynamics. AB - A novel cellular automaton model of proliferative brain tumor growth has been developed. This model is able to simulate Gompertzian tumor growth over nearly three orders of magnitude in radius using only four microscopic parameters. The predicted composition and growth rates are in agreement with a test case pooled from the available medical literature. The model incorporates several new features, improving previous models, and also allows ready extension to study other important properties of tumor growth, such as clonal competition. PMID- 10745116 TI - Coarse-grained reverse engineering of genetic regulatory networks. AB - We have modeled genetic regulatory networks in the framework of continuous-time recurrent neural networks. A method for determining the parameters of such networks, given expression level time series data, is introduced and evaluated using artificial data. The method is also applied to a set of actual expression data from the development of rat central nervous system. PMID- 10745117 TI - Computational dynamics of gradient bistable networks. AB - We describe a neural-like, homogeneous network consisting of coupled bistable elements and we study its abilities of learning, pattern recognition and computation. The technique allows new possibilities of pattern recognition, including the memorization and perfect recall of several memory patterns, without interference from spurious states. When the coupling strength between elements exceeds a critical value, the network readily converges to a unique attractor. Below this critical value one could perfectly recall all memorized patterns. PMID- 10745118 TI - An artificial immune system for data analysis. AB - We present a simplified view of those parts of the human immune system which can be used to provide the basis for a data analysis tool. The motivation for and reasoning behind such a model is given and the desire for a 'transparent' model and meaningful visualization and interpretation techniques is noted. A minimalist formulation of an artificial immune system and some of its behaviour is described. A simple implementation and a suitable visualization technique are demonstrated using some trivial data and the famous 'iris' data set. PMID- 10745119 TI - A formal model of an artificial immune system. AB - The paper presents a mathematical model based on the features of antigen-antibody bindings in the immune system. In the natural immune system, local binding of immune cells and molecules to antigenic peptides is based generally on the behavior of surface proteins. In particular, immune cells contain proteins on their receptors, and apparently, these proteins play the key role both in immune response and recognition processes. In this work, we consider the immune cells in the form of formal B-cell and formal T-cell and develop a mathematical model of their interactions. We refer this model as the formal immune system (FIS). The paper provides an analysis of a network of bindings (or interactions) among the formal proteins of the FIS. PMID- 10745120 TI - A diffuse biosemiotic model for cell-to-tissue computational closure. AB - The adoption of diffuse rationality creates a practical bridge between biosemiotics and computation in formulating local-to-global self-consistent criteria for cellular-to-tissue interfacing and for the emergence of life and consciousness. Nature is always complex, the more so at biological membranic inter-scalar interfaces. We present an evolutionary model of the relationship between autonomy and dependence across scales, and describe the implications of its alternating complex-rational-complex nature. PMID- 10745121 TI - Limb vs trunk sweat gland recruitment patterns during exercise in humans. AB - The purpose of this study was to examine the sweat gland recruitment pattern, on multiple trunk and limb sites, during exercise. Nineteen male volunteers performed 30 min of exercise on a cycle ergometer at approx. 25, 50 and 75% of their maximal oxygen uptake. The number of active sweat glands (per cm(2)) was determined immediately following each exercise bout at the following six sites: left triceps, chest, back, forearm, thigh and calf. The data showed that increases in rectal temperature during exercise resulted in a linear increase in the absolute number of active sweat glands recruited at all six sites (r=0.60 0.80). However, on a percentage basis, the limb sites increased proportionally more (300-600% increase) than the trunk sites (100-200% increase) with increases in rectal temperature. These data suggest that the absolute number of sweat glands recruited, on both the trunk and the limbs, increases in a linear manner with increases in rectal temperature during exercise. However, on a proportional basis, sweat gland recruitment on the limbs is greater than that found on the trunk during progressive exercise. PMID- 10745122 TI - Size and seasonal temperature in free-ranging Drosophila subobscura. AB - Body size of diverse ectotherms is inversely related to developmental temperature in the laboratory. We monitored seasonal variation in wing length of two populations (Oregon, Washington) of D. subobscura, which was introduced in the Pacific Northwest in the late 1970s. Wing length varied seasonally and was shortest in summer. In Washington, however, wing length was longest in spring, not winter. Wing length was inversely and curvilinearly related to mean ambient temperature, as in a few previous studies of drosophilids. Mid-winter D. subobscura might not be the largest either because extremely low temperatures depress size or because flies collected in winter were in fact born the previous autumn, when developmental temperatures were more moderate. PMID- 10745123 TI - Body temperature regulation in the rat. AB - In loosely-restrained adult conscious rats exposed to stepwise changes in ambient temperature (T(a)) from 25 to 5 degrees C or from 20 to 35 degrees C, we have recorded body and tail temperatures, metabolic rate (VO(2)), shivering and ventilation (V). It was found that VO(2) and V vary with T(a) and show a nadir for a T(a) of 30 degrees C whereas shivering starts at 20 degrees C and increases progressively with cold exposure. T(tail) follows changes in T(a) whereas T(body) decreases slightly in cold and increases markedly in warm exposure. These results suggest that the control of T(body) interacts with the control of breathing in order to increase VO(2) during cold exposure and to facilitate evaporative respiratory heat dissipation during warm exposure. PMID- 10745124 TI - Effects of constant or variable thermal rearing condition: a behavioural approach using an automated tracking system in Blaberus craniifer. AB - An automatic tracking device was used to investigate exploratory behaviour of the cockroach, Blaberus craniifer, with regard to the influence of rearing temperature. Two groups were tested, one under variable rearing temperature, the other under constant rearing temperature. Rearing temperature influenced both the rate of decrease of linear speed and the spatial occupation of an open arena. Linear speed decreased with time and was higher during the first 10 minutes of recording for the animals reared under constant temperature conditions. The decrease fitted a linear model for the variable temperature group, whereas it was steeper than exponential for the constant temperature group. Frequency of occupancy in the peripheral zone of the arena was not affected by time and was always higher for the animals of the constant condition group. The results are interpreted as a function of the different thermal rearing conditions. PMID- 10745125 TI - An ontogenetic shift in the response of heart rates to temperature in the developing snapping turtle (Chelydra serpentina). AB - The affect of acute changes in temperature on heart rates was investigated for the first time in a developing reptile. Heart rates were determined early and late in incubation in snapping turtle (Chelydra serpentina) eggs. Late in incubation heart rates at any given temperature were lower than those observed early in incubation. The results of temperature switching experiments late in incubation were consistent with thermal acclimation. PMID- 10745126 TI - Thick-walled physical models improve estimates of operative temperatures for moderate to large-sized reptiles. PMID- 10745127 TI - Comparison of survival and DNA double strand breaks for mild hyperthermia and low dose rate/pulsed low dose rate irradiation in human cells. AB - Long duration mild hyperthermia (LDMH) has been shown to be an effective radiosensitizer when combined with low dose rate irradiation and pulsed low dose rate irradiation. These protocols are being investigated to determine if these effects can be related to DNA double strand breakage (dsb). In our studies we used human melanoma (SK mel-3) and fibroblasts (AG1522). A low dose rate was given at 0.88 cGy/min while pulsed doses were given at 150 cGy/min. Our results showed that the degree of thermal radiosensitization (TER) increased as the average dose rate decreased. This was seen for both the survival endpoints and the degree of DNA strand breaks. There was a very good correlation between the TER and the degree of DNA strand breaks.In conclusion our data show that LDMH is an effective radiosensitizer for both LDR and PSLDR and this may also be an effective clinical protocol. The quantity of DNA dsb's appears to be related to TER and may be predictive of the degree of radiosensitization. PMID- 10745128 TI - Temperature gradient across the skin's layers has no influence on local skin vasomotor responses. AB - The normal negative temperature gradient within the skin of the cheek was reversed by simultaneously heating the skin externally with an infrared lamp and cooling it internally, inside the mouth, with ice. Cutaneous blood flow was measured locally under four different conditions: negative and positive gradient of local skin temperature in hypothermia and hyperthermia. There were no significant differences between negative and positive skin temperature gradients. Cutaneous blood flow depended on the core body temperature. These results show that the local skin temperature gradient can not induce vasomotor responses. PMID- 10745129 TI - Statistical and procedural issues in the use of heated taxidermic mounts. AB - Studies using mounts have an inherently nested error structure; calibration and standardization should use the appropriate procedures and statistics. One example is that individual mount differences are nested within morphological factors related to species, age, or gender; without replication, mount differences may be confused with differences due to morphology. Also, the sensitivity of mounts to orientation to wind or sun is nested within mount; without replication, inadvertent variation in mount positioning may be confused with differences among mounts. Data on heat loss from a of 1-day-old mallard duckling mount are used to illustrate orientation sensitivity. PMID- 10745130 TI - Quantitative multi-modal functional MRI with blood oxygenation level dependent exponential decays adjusted for flow attenuated inversion recovery (BOLDED AFFAIR). AB - A magnetic resonance imaging (MRI) method is described that allows interleaved measurements of transverse (R(2)(*) and R(2)) and longitudinal (R(1)) relaxation rates of tissue water in conjunction with spin labeling. The image-contrasts are intrinsically blood oxygenation level dependent (BOLD) and cerebral blood flow (CBF) weighted, but each contrast is made quantitative by two echo time (TE) and inversion recovery time (TIR) acquisitions with gradient echo (GE) and spin echo (SE) weighted echo-planar imaging (EPI). The EPI data were acquired at 7 Tesla with nominal spatial resolution of 430 x 430 x 1000 microm(3) in rat brain in vivo. The method is termed as blood oxygenation level dependent exponential decays adjusted for flow attenuated inversion recovery (BOLDED AFFAIR) and allows acquisition of R(2)(*), R(2), and CBF maps in an interleaved manner within approximately 12 minute. The basic theory of the method, associated experimental/systematic errors, and temporal restrictions are discussed. The method is validated by comparison of multi-modal maps obtained by BOLDED AFFAIR (i.e., two TE and TIR values with GE and SE sequences) and conventional approach (i.e., multiple TE and TIR values with GE and SE sequences) during varied levels of whole brain activity. Preliminary functional data from a rat forepaw stimulation model demonstrate the feasibility of this method for functional MRI (fMRI) studies. It is expected that with appropriate precautions this method in conjunction with contrast agent-based MRI has great potential for quantitative fMRI studies of mammalian cortex. PMID- 10745131 TI - Ultrafast magnetic resonance imaging of the brain. AB - The purpose of this study was to compare the diagnostic efficacy of single shot fast spin echo sequence (SSh-FSE), and single shot GRASE-sequence (SSh-GRASE) to the conventional T(2)-weighted fast spin echo-sequence (T(2)-FSE) in the imaging of brain disorders. Thirty three patients with high signal intensity lesions on T(2)-weighted images (n = 28), or intracerebral hemorrhage (n = 5), were examined on a 1.0 T MR scanner, with 23 mT/m gradient strength. The scan time for the conventional T(2)-FSE-sequence was 2 min 57 s, the scan time for the single shot FSE-, and single shot-GRASE-sequences was 11 sec, and 17 sec, respectively. Twenty-one patients remained still during the examination, whereas 12 could not stay still with consecutive marked motion artifacts. Images were reviewed by three radiologists. Lesion conspicuity, image quality, and artifacts were scored on a subjective scale. Signal-to-noise ratios of lesions and normal tissue and contrast-to-noise ratios (CNR) were measured by region of interest (ROI). In the patient group without motion artifacts conspicuity for lesions > or =5 mm did not show a significant difference on conventional T(2)-FSE, single shot-FSE and single shot-GRASE. Detectability of the smaller lesions was significantly inferior on single shot-FSE-, and single shot-GRASE-sequences in artifact free images. For the patient group with motion artifacts SSh-FSE and SSh-GRASE were markedly superior to the conventional T(2)-FSE. Grey-white differentiation was better on conventional T(2)-FSE. Physiologic ferritin as well as pathologic hemosiderin depositions were slightly darker and therefore better visible on SSh GRASE than on SSh-FSE. Conventional T(2)-FSE showed significantly more artifacts. In conclusion, SSh-FSE and SSh-GRASE imaging can be used for rapid imaging of the brain in those patients who are claustrophobic or in patients with involuntary movements due to extrapyramidal disorders, as well as in children in whom anesthesia is contraindicated or sedation is not possible. PMID- 10745132 TI - Spin echo MRI in the evaluation of hearts with a double outlet right ventricle: usefulness and limitations. AB - The surgical approach to a double outlet right ventricle (DORV) is dependent on the spatial relationship of the semilunar valves, outflow tracts and ventricular septal defect (VSD). The purpose of the study was to assess the value of MRI for the evaluation of cardiovascular anatomy in patients before and after surgery for DORV. Spin echo MRI was performed in 12 patients with DORV (eight preoperative and four postoperative patients). Thin-section MRI was performed in three orthogonal planes and selected angulated views were obtained. Conventional imaging by color Doppler echocardiography and cine-angiocardiography and surgical findings, when present, served as the reference standards. The results found that the spatial relationship between semilunar valves and VSD was accurately assessed by MRI in eight out of eight preoperative patients. In the four postoperative cases MRI depicted the morphology of both outflow tracts and provided adequate information on their patency. Of the eight preoperative patients, five have undergone corrective surgery and the MRI findings were confirmed. MRI provided additional information to conventional imaging preoperatively in three cases in which the VSD opened into the outlet portion of the DORV, without there being a direct relation to a semilunar valve. In two preoperative cases in which the VSD was directly committed to the aorta, conventional imaging was conclusive. MRI was unable to depict aberrant chordae tendineae in four out of four cases. We conclude that MRI provides accurate additional anatomic information in patients with DORV, which is helpful in presurgical planning as well as during follow-up. Spin echo MRI does not visualize aberrant chordae tendineae. PMID- 10745133 TI - The diagnostic role of gadolinium enhanced MRI in distinguishing between acute medullary bone infarct and osteomyelitis. AB - The objective of the study was to evaluate the diagnostic utility of contrast enhanced magnetic resonance imaging (MRI) for distinguishing between acute medullary bone infarct and osteomyelitis. There were 11 patients (age 6-34 years) presented to our institution between December 1994 and February 1998 with a clinical differential diagnosis of acute bone infarct versus osteomyelitis and inconclusive radiographs were imaged using MRI. All but one received i.v. gadolinium. Nine of the patients had homozygous Sickle Cell disease (SCD) and two had Systemic Lupus Erythematosus (SLE), the latter requiring chronic methylprednisolone. Osteomyelitis was confirmed either by biopsy alone or by the combination of Gallium(67) scan in conjunction with positive blood cultures and clinical resolution following antibiotics. Infarcts without osteomyelitis were confirmed either by biopsy or resolution of symptoms without antibiotic therapy. All patients had at least six months clinical follow-up. The results found that seven of nine patients with SCD had acute infarct only. One patient with SCD had osteomyelitis only. Three patients (two SLE and one SCD) had both acute-on chronic infarcts and superimposed osteomyelitis, one with an adjacent soft tissue abscess. Accurate distinction between infarct and osteomyelitis was impossible for one patient with SLE who did not receive contrast. All other cases were correctly diagnosed prospectively based on distinct patterns of MRI contrast enhancement. In all adult patients, acute infarcts demonstrated thin, linear rim enhancement on MRI while osteomyelitis revealed more geographic and irregular marrow enhancement. Two of four cases of osteomyelitis also demonstrated subtle cortical defects with abnormal signal traversing marrow and soft tissue. The single pediatric patient demonstrated elongated, serpiginous central medullary enhancement with periostitis. We concluded that the pattern of MR contrast enhancement may allow accurate distinction between acute infarct and osteomyelitis, or recognition of osteomyelitis superimposed on bone infarction. PMID- 10745134 TI - Evaluation of Crohn's disease using half-fourier RARE and gadolinium-enhanced SGE sequences: initial results. AB - To assess the bowel changes in Crohn's disease, 11 consecutive patients underwent magnetic resonance imaging (MRI) study using T(2)-weighted half-Fourier rapid acquisition with relaxation enhancement (RARE) and gadolinium-enhanced standard and fat suppressed spoiled gradient echo (SGE) sequences. Comparison was made between MR findings of disease extent, severity, and complications and clinical data, endoscopic findings and/or surgical specimens in all patients. We found that the half-Fourier RARE images showed bowel wall thickening, dilatation of bowel and bowel obstruction well in all patients, however severity of bowel disease could not be determined as the signal intensity of diseased bowel was comparable to normal bowel in 10/11 patients. Gadolinium-enhanced fat suppressed SGE demonstrated variations of mural enhancement that correlated well with extent of disease severity in 10/11 patients. Complications such as intraperitoneal (i. p.) abscess (2 patients), gastric outlet obstruction (1 patient), bowel obstruction (2 patients), and fistula formation (3 patient), were accurately shown. We conclude that T(2)-weighted half-Fourier RARE and gadolinium-enhanced fat suppressed SGE sequences are complementary techniques that possess different imaging features that are of value for assessing bowel changes in Crohn's disease. PMID- 10745135 TI - Generalised cerebral atrophy following temporal lobectomy for intractable epilepsy associated with mesial temporal sclerosis. AB - Studies of post-operative imaging data have mainly concentrated on brain atrophy following radiotherapy and/or chemotherapy. We have investigated the effect of conventional surgery on the unresected brain tissue based on the comparison of magnetic resonance images acquired pre- and post-operatively in 13 subjects with a history of mesio-temporal epilepsy. The pre- and post-operative scans were co registered prior to volumetric analysis. The total brain volume (TBV) was calculated by semi-automated segmentation, and the total volume loss was the difference between the post-operative and pre-operative TBV. The total volume of resection was determined by manual delineation in the post-operative scan. The atrophy volume in the post-operative scan was calculated as the difference between the total volume loss and the resection volume. In 6 cases, there was generalised cerebral atrophy of the order 4-5% of the total brain volume. In addition to the automated volumetric technique, the images were assessed by two expert neuroradiologists. There was complete correspondence between their assessment and the automated technique. The causes and significance of this phenomenon are unknown but it requires further investigation as it may be related to seizure control and neuropsychological changes following epilepsy surgery. PMID- 10745136 TI - MR phase imaging to quantify bone volume fraction: computer simulations and in vivo measurements. AB - Magnetic resonance phase images can be used to assess trabecular bone by measuring the standard deviation of the phases in a region of interest. The standard deviation of regional phase measurements reflects the degree of magnetic field inhomogeneity caused by susceptibility differences between bone and marrow. A 3D computer model of trabecular bone was developed and then used to explore the influence of bone volume fraction and imaging parameters such as pixel size and slice width on the standard deviation of regional phase measurements. The results from these tests show that with appropriate selection of these parameters, phase spread strongly reflects variations in trabecular bone density (a correlation of R(2) = 0.98 with bone volume fraction between 0 and 10%). The technique was then applied in vivo on the radius of 25 patients who already had a bone density scan with peripheral quantitative tomography and a correlation between phase standard deviation and trabecular bone density was found (R(2) = 0.46). PMID- 10745137 TI - The use of view angle tilting to reduce distortions in magnetic resonance imaging of cryosurgery. AB - Susceptibility artifacts from magnetic resonance (MR)-compatible cryoprobes can distort MR images of iceballs. In this work, we investigate the ability of view angle tilting (VAT) to correct susceptibility induced distortions in MR images of cryosurgery. The efficacy of VAT was tested in an ex vivo bovine liver model of cryosurgery using MR-compatible cryoprobes. Artifacts on high bandwidth fast spin echo images of freezing obtained with and without VAT were compared with photographs of the actual iceball shape and size. In vivo imaging with VAT was demonstrated during percutaneous MR-guided cryosurgery of pig liver and brain. VAT was most successful in reducing probe and iceball distortions when the imaging plane was normal to the cryoprobe, and the cryoprobe was perpendicular to the main magnetic field of the scanner. VAT had the greatest benefit when used to correct MR images of freezing when the surface of the iceball was relatively near to the cryoprobe. For large iceballs, the artifact was small so the VAT correction was less important. We conclude that VAT significantly reduced distortions in the shape of the signal void corresponding to the extent of freezing visualized during MR-guided cryosurgery. This improved ability to visualize the exact location of the cryoprobe, as well as the precise shape of the iceball, particularly during initial freezing when the iceball is small, has potential to significantly improve the accuracy of MR-guided cryosurgery of small lesions, and the accuracy of MR-assisted temperature calculations that are based on precise imaging of the probe location, and boundary geometry of the iceball. PMID- 10745138 TI - Improved MR imaging for patients with metallic implants. AB - Pediatric oncology patients with large metallic prostheses were imaged with one of two MR imaging techniques: 1) the "tilted view-angle" technique, 2) or a higher readout bandwidth technique. The tilted view-angle method uses an additional gradient in the slice selection direction during readout. The high bandwidth technique increases the readout bandwidth and shortens the echo time (TE). High bandwidth and short echo times were implemented in both T(1)-weighted (T(1)W) turbo spin echo and turbo short tau inversion recovery (STIR) sequences. Both imaging techniques reduced the size of metal-induced image artifacts. The tilted view-angle method reduced the artifact to a greater degree but had inherent shortcomings. The reformatted images were blurred and shifted. The area of interest was often moved outside of the field of view, unless parameters were adjusted on the basis of a pre-scan calculation. The high readout bandwidth, short echo technique required no special preparation and reduced metal artifacts without image blurring. The combination of high-bandwidth, shorter echo turbo STIR and T(1)W turbo spin echo sequences with subtraction of pre- from post contrast images allowed effective fat suppression without local field inhomogeneity affects. This greatly improved our ability to evaluate suspected disease near metallic implants in pediatric cancer patients. PMID- 10745139 TI - MRI of liver: a comparison of CNR enhancement using high dose and low dose ferumoxide infusion in patients with colorectal liver metastases. AB - The purpose of this study was to compare the effects of high dose (HD) and low dose (LD) ferumoxides infusions on lesion-to-liver contrast-to-noise ratio (CNR) using four different T(2)-weighted MR sequences. Seventy-three patients with known colorectal liver metastases underwent T(2)-weighted fast spin echo (FSE) imaging before and after ferumoxides. After ferumoxides, T(2)-weighted dual echo (DE) and T(2)-weighted GRE FLASH images were also obtained. To evaluate the relationship between TE length and lesion-to-liver CNR, the same FLASH sequence was repeated in 18 LD patients after lengthening the TE. Ferumoxides was administered at a dose of 15 micromol/kg (HD) and 7.5 micromol/kg (LD) in 45 and 28 patients, respectively. The effects of HD and LD ferumoxides infusions were measured as the percentage signal intensity change (PSIC) in the liver and lesions, lesion-to-liver CNR and the change in lesion-to-liver CNR (DeltaCNR). In both LD and HD groups, all CNR values obtained after SPIO were significantly greater than those observed with unenhanced FSE (p < 0.01). There was no significant difference between the mean CNR values obtained with either dose for any sequence. With the FLASH sequence, CNR increased progressively with longer TE. At the longest TE of 26 ms, mean CNR was higher than that recorded with any of the other sequences. Although mean liver PSIC was significantly greater in the HD group than in the LD group (p < 0.01) because the mean lesion PSIC was also greater in the HD group, the mean DeltaCNR after ferumoxides was not significantly different in the two groups. LD SPIO enhanced MR significantly increases lesion-to-liver CNR compared with unenhanced images. At 1. 0 T, HD and LD ferumoxides infusions produce comparable lesion-to-liver CNR. Our results suggest that at 1.0 T ferumoxides may be administered at a dose of 7.5 micromol/kg without loss of image quality. PMID- 10745140 TI - Ferumoxide-enhanced MRI in patients with colorectal cancer and rising CEA: surgical correlation in early recurrence. AB - The purpose of this study was to evaluate the value of Ferumoxide-enhanced magnetic resonance (MR) imaging in the detection of hepatic metastases in high risk patients treated for colorectal cancer that have rising CEA. We used 19 patients treated previously for colorectal cancer with rising CEA levels underwent an unenhanced T(1)-weighted (T1W), T(2)-weighted (T2W), STIR, and Ferumoxide-enhanced hepatic MRI. Following these studies, a laparotomy was performed and the liver was evaluated by palpation and intraoperative ultrasound. Two observers who were blinded to surgical results evaluated each MR sequence separately. The number of lesions considered highly suspicious for metastatic lesions were determined for each sequence and were compared to the results of surgery. The McNemar test was used to compare the outcomes of the different sequences. MR Imaging was unable to detect small (<5 mm) metastases discovered at surgery. The best non-contrast sequences for detecting metastases were the STIR with 42% sensitivity, 83% specificity and an overall accuracy of 56% and the T1W sequence (sensitivity 38%, specificity 100%, accuracy 57%), which were not significantly different (p 0.4). The noncontrast T2W sequence had a sensitivity of 29% and a specificity of 77% with an overall accuracy of 46%. When all pre contrast scans were grouped together the common sensitivity was 42%, specificity was 77% and accuracy was 54%. The post-ferumoxide T(2)W scans had a sensitivity of 42%, specificity of 85%, and accuracy of 57%, but did not detect any additional lesions. There was no statistical difference between the pre- and post contrast studies with regard to identifying patients with metastatic disease (p 0.1). In conclusion, we found small hepatic metastases in patients with early signs of recurrent colorectal cancer are difficult to detect on MRI. Ferumoxide enhanced MRI was unable to detect additional hepatic metastases and performed no better than unenhanced MRI in detecting small hepatic metastasis. PMID- 10745141 TI - Prediction of gene therapy-induced tumor size changes by the vascularity changes measured using dynamic contrast-enhanced MRI. AB - We studied the changes of tumor size after gene therapy treatment and its relationship with the changes of vascular volume as measured by dynamic contrast enhanced magnetic resonance imaging (MRI), to investigate whether the vascular changes is predictive of tumor regression. The study was carried out using a spontaneously regressing rat tumor model (C6 Glioma grown subcutaneously in rats). Three rats were treated with recombinant adenoviruses expressing three genes, mouse interleukin 1-alpha (IL1-alpha), mouse interferon gamma (IFN-gamma), and human transforming growth factor beta (TGF-beta), one from each kind. Two rats were treated with saline as controls. Longitudinal studies were performed to monitor the changes of tumor volume (based on T(2)-weighted images) and the vascular volume (based on dynamic contrast enhanced images). In untreated animals, tumor regression was preceded by several days with a decrease in vascular volume. When the tumor growth was perturbed by expression of mouse IL 1alpha, the increase in vascular volume was correlated with the continuing growth in size, and the decrease in vascular volume was predictive of the onset of tumor regression. As new advances in immunotherapy in cancer treatment emerge, the ability to determine the efficacy of therapy as early as possible will enable optimization of treatment regiments. The vascularity changes measured by dynamic MRI may provide a means to serve for this purpose. PMID- 10745142 TI - NMR Q-space microscopy of concentrated oil-in-water emulsions. AB - The pulsed field gradient stimulated echo technique with selective excitation is used to probe the diffusion of water in the continuous phase of concentrated oil in-water emulsions. The dependence of the echo amplitude, S(q,Delta), on wavevector, q, and diffusion time, Delta, shows that the water diffusion propagator is sensitive to emulsion microstructure. This is analyzed using a multiple exponential time series expansion of S(q,Delta) in Delta, with wavevector dependent expansion coefficients. These coefficients are compared with predictions from several theoretical models for three types of stable emulsion, each differing in microstructure. The relationship between the nuclear magnetic resonance q-space measurements and bulk rheology for all three types of emulsion is also explored. PMID- 10745143 TI - Characterization of water mobility and distribution in low- and intermediate moisture food systems. AB - The mechanism of water uptake in low moisture cereals and cookies has been studied by NMR relaxometry and solid imaging technology implemented on a low resolution benchtop NMR spectrometer. A comparison between classical MRI and SPRITE imaging are also presented to highlight the benefits of each technology. The spin lattice (T(1)) and spin spin (T(2)) relaxation times, the 1D and 2D SPRITE imaging, were determined on Smacks, corn flakes, chocolate chips cookies, soft caramel candies with a chocolate creme filler, and corn starch/water systems. The Smacks and corn flakes were studied based on the soaking time in milk, and the results showed that T(1) and T(2) decreased in the first 20 sec of soaking and then increased with the soaking time. For Smacks stored at different relative humidity, T(1) decreased during the first day of storage and then was relatively constant over storage time indicating that the system reached an equilibrium. 1D SPRITE profiles indicated an increase in signal intensity over storage time for cookies in 58% RH. However, the moisture uptake was insignificant indicating that the water mobility (and not the amount of water) changed due to various chemical interactions in the system (hydrogen bonding, starch retrogradation, glassy/rubbery equilibrium). The T(1) and T(2) of corn starch/water systems decreased as the concentration in starch increased and temperature increased from 30 degrees C to 60 degrees C. However, for temperatures higher than 60 degrees C, the relaxation times increased showing more mobility and flexibility of the polymer chains during gelatinization. PMID- 10745144 TI - High-resolution dose profile studies based on MR imaging with polymer BANG(TM) gels in stereotactic radiation techniques. AB - High-resolution dose profiles produced by the Leksell Gamma Knife were obtained in BANG(TM) polymer gel, using a 3 T whole-body scanner upgraded by a magnetic resonance microscopy unit. The gel was contained in 22.3 mm diameter flasks that were inserted into a solid, tissue-equivalent head phantom irradiated by fields of by 8 and 14 mm collimators. Dose profiles were obtained from a linear dose response curve (R(2) vs. Dose). Excellent agreement was obtained when the gel data were compared to film dosimetry and calculated data. PMID- 10745145 TI - Reduction of anxiety during MR imaging: a controlled trial. AB - An anxiety reduction protocol was developed and evaluated for routine use with neurology and neuropsychiatry patients undergoing brain or spinal scans. Thirty five patients underwent standard procedures, with limited information given in advance. Twenty nine experimental patients received a booklet giving information about the scanning procedure and advice on cognitive strategies for anxiety reduction, a tape-recorded demonstration of scanner noise, a visit to the control room before entering the scanner, a device to signal for adjustment of music volume, precise timings of each scan, and a clock visible during scanning. Anxiety was measured before, during, and after scanning, using subjective ratings and a retrospective version of the Spielberger State Anxiety Scale. There was no difference in anxiety between groups immediately prior to the scan and immediately after entering the scanner. Patients in the experimental group were significantly less anxious during the scan than control patients, as measured by mean anxiety ratings made during the imaging procedure and by retrospective State Anxiety scores completed immediately after leaving the scanner. The results show that scan-related anxiety can be reduced by introducing these simple changes to MR imaging procedures, with minimal cost, no special training of staff, and no disruption of the running of the MR Unit. PMID- 10745146 TI - MR in vivo imaging of oxygen suppression effect of soft contact lens on the human cornea. AB - The O(2) suppression effect of a soft contact lens on the human cornea was measured using dynamic magnetic resonance imaging (MRI) of the anterior chamber transcorneally exposed to O(2). Dynamic T(1)-weighted fast spin echo imaging of anterior chambers (TR = 2 s, TE = 15 ms, 5-mm slice) was performed both before and during oxygen supply to a full goggle placed on the face of volunteers wearing a soft contact lens on one eye and nothing on the other eye as a control. Within 15 min after O(2) administration, significantly lower intensity changes were obtained in the anterior chambers of the eyes with the contact lens than in those of the eyes without one, suggesting that dynamic MRI of the anterior chamber transcorneally exposed to O(2) can be used to evaluate the O(2) suppression effect of a soft contact lens on the cornea. PMID- 10745147 TI - Hypertension: challenges for the new century. PMID- 10745148 TI - New developments in obesity. AB - Obesity is an important health problem. Worldwide epidemiological data show that its frequency is rising steeply, probably because of a reduction in physical activity and bad eating habits. Health risks are most prominent in the central type of obesity, due to the relatively increased lipolytic activity, which leads to a series of events. The overall results of treatment are not satisfactory. Drugs, such as orlistat, fluoxetine, and ephedrine/caffeine, may be useful. The first results with leptin treatment are encouraging, but not yet optimal. Research on various neuropeptides and beta3-agonists is promising. Prevention of obesity is extremely important but difficult. PMID- 10745149 TI - Risk of cancer in inflammatory bowel disease (IBD). AB - Patients with inflammmatory bowel disease (IBD) have been reported to have an increased risk of colorectal cancer. Yet, the quantitation of the risk varies widely from one study to the next. This is most likely due to biases in the assessment of cancer risk in IBD, namely, a relatively low prevalence of IBD related cancer and the clinically heterogeneous nature of IBD in the population. Total proctocolectomy also changes the natural history of IBD-related cancer. Ulcerative colitis (UC)-related cancer is more probable in total and extensive colitis and occurs approximately a decade after diagnosis. Crohn's disease related colorectal cancer is reported in many, but not all, studies, and the relative risk differs between hospital- and population-based studies. IBD-related cancer is relatively uncommon in childhood; however, this is also a subject of debate. There are no data on the incidence of IBD-related cancer in Europe as a whole; there are only separate studies. A reduced risk for UC-related cancer in patients treated with anti-inflammatory drugs has been reported. The two main strategies for preventing IBD-related cancer are prophylactic colectomy and colonoscopic surveillance. To date, there have virtually been no cost effectiveness analyses and no studies regarding total disease outcome or patient's quality of life with either strategy. No controlled, prospective trials have been reported in the literature. UC patients with familiar predisposition for colorectal cancer and with concomitant primary sclerosing cholangitis are groups at high risk for IBD-related cancer. Cancer risk in IBD has to be reinvestigated and properly estimated with population-based studies in several areas with standard methods already described. PMID- 10745150 TI - Thrombomodulin as a marker of endothelium damage in some clinical conditions. AB - Background: Thrombomodulin (TM) is a membrane glycoprotein in the vascular endothelium. It may be cleaved from endothelial cells and released into the circulation. The plasma TM level depends on the integrity of the endothelium and the clearance of the molecule. The physiological role of soluble TM forms is still unclear. The clinical significance of elevated levels of TM in various pathologic conditions is not well established yet. To analyze variations of plasma TM level in different clinical situations, its concentrations in patients with three groups of diseases were measured and compared with those in healthy subjects. Methods: Plasma samples from 23 patients at risk for development of vascular complications [essential hypertension (EH), stages 1 and 2], 31 patients with inflammatory bowel diseases [Crohn's disease (IBD), mostly in the active stage], and 19 patients with malignant tumors [gastric carcinoma (NEO)], were analyzed for soluble TM with an enzyme immunoassay kit. Results: In the group of patients with the early stages of EH and with non-active IBD, no significant changes were found in comparison to the healthy subjects. In the patients with active IBD and mainly with NEO, soluble TM was significantly increased (P<0.05 and P<0.001, respectively). Conclusions: Our TM levels failed to demonstrate increased endothelial damage in the early stage of EH. This suggests that TM is released into the plasma only by true endothelial cell damage during the development of vascular complications. Probably a certain degree of endothelial injury is necessary for an increase in plasma. In the active stage of IBD and in NEO, soluble TM appears to be derived not only from injured endothelial cells, but may also be proteolytically cleaved from membrane TM by proteases. There may also be increased synthesis of TM in activated and/or transformed cells. PMID- 10745151 TI - Treatment of hematologic disorders other than immune thrombocytopenic purpura with intravenous immunoglobulin (IVIg) - report of seven cases and review of the literature. AB - Background: Intravenous immunoglobulin (IVIg) is a standard therapeutic modality for a few autoimmune diseases, such as immune thrombocytopenic purpura. However, in other hematologic autoimmune conditions its role is still controversial. Methods: Seven patients with either autoimmune hemolytic anemia, Evans' syndrome, aplastic anemia, pure red cell aplasia, thrombotic thrombocytopenic purpura, or acquired factor VIII inhibitors were treated with a single course of high-dose (2 g/kg) IVIg. Results: A good response was observed in all seven patients, except the one with thrombotic thrombocytopenic purpura, and there were no adverse effects related to IVIg use. The literature reports on IVIg therapy in these conditions and the mechanisms of action of IVIg in autoimmune diseases are discussed. Conclusions: IVIg might be a useful agent in the treatment of several hematologic disorders other than immune thrombocytopenic purpura. Future aims of clinical research in this respect would be to identify the patient subgroups that might benefit more from IVIg in these conditions. PMID- 10745152 TI - Leukocyte activation and cytokine production during experimental human endotoxemia. AB - Background: At present, it is unclear whether in experimental endotoxemia, the pro-inflammatory response observed in healthy volunteers is followed by an anti inflammatory response, as observed in patients with sepsis. We studied the evolution of a number of inflammatory parameters during a prolonged period (24 h) after infusion of endotoxin in healthy subjects. Methods: Six healthy male subjects received an infusion of endotoxin (4 ng/kg body weight). Blood was drawn before, and at various intervals up to and including 24 h after, endotoxin infusion. Circulating cytokine levels, leukocyte activation surface markers, plasma lactoferrin, and neopterin levels were measured, and clinical signs and symptoms were noted during a 24-h period. Monocyte and neutrophil activation after endotoxin infusion is investigated in relation to the inflammatory response. The extent of neutrophil and monocyte activation was correlated to clinical markers and blood levels of inflammatory mediators and cytokines. Results: Tumor necrosis factor-alpha appeared 30 min after infusion in the circulation, peaking (5665+/-1910 pg/ml) at 2 h. Interleukin-10 appeared 60 min after infusion, peaking (427+/-348 pg/ml) at 3 h. The expression of leukocyte activation markers increased significantly after infusion. Expression of HLA-DR on monocytes decreased significantly after 3 h (P=0.03). There was a correlation between the TNF-alpha:IL-10 ratio and the CD11b:HLA-DR ratio (P=0.03). Conclusions: During experimental human endotoxemia, an initial pro-inflammatory response is successfully compensated by an anti-inflammatory response, leading to homeostasis. This is in contrast to what happens in septic patients with compensatory anti-inflammatory response syndrome. The inflammatory balance, expressed as the cytokine pro:anti-inflammatory ratio, is reflected at a cellular level. PMID- 10745153 TI - Salmonella endocarditis presenting as cerebral hemorrhage. AB - The case of a 35-year-old male with cerebral hemorrhage as a presenting complication of infective endocarditis of the aortic valve, caused by Salmonella typhimurium, is described. We emphasize the infrequent etiology and review the mechanisms of intracranial hemorrhage in infective endocarditis. PMID- 10745154 TI - Myelofibrosis in systemic lupus erythematosus: a new case. AB - A 29-year-old woman with systemic lupus erythematosus and pancytopenia is reported. The hematological abnormalities were found to be due to marrow fibrosis. Pancytopenia was reversed by treatment with corticosteroids. PMID- 10745155 TI - Congestive heart failure: revealing light chain deposition disease. AB - We report a case of light chain deposition disease associated with multiple myeloma revealed by congestive heart failure. Endomyocardial biopsy showed Congo red negative deposits and lambda light chain deposits by the immunofluorescence method. After chemotherapy with melphalan and prednisone, complete remission of multiple myeloma and major improvement in cardiac condition were observed. PMID- 10745156 TI - A splenic cyst and a high serum CA 19-9: a case report. AB - A 22-year-old man was admitted to our hospital with a palpable mass in the left upper quadrant. He had a history of blunt abdominal trauma. Computer tomography showed a large splenic cyst. Another finding was a markedly elevated concentration of the tumor marker CA 19-9 in his serum and cystic fluid. After surgical removal of the cyst this concentration decreased to a nearly normal level. Because no cellular lining of the cyst was found at histological examination, the cyst could have been regarded as 'false' or a 'pseudocyst'. However, since several cases of epithelial splenic cysts in combination with a high serum CA 19-9 have been described recently, our diagnosis was of an epithelial 'true' cyst. We assume that, for some reason, in primary splenic cysts, the thin cellular lining can be released before histological examination is performed. PMID- 10745157 TI - Susac's syndrome or retinocochleocerebral vasculopathy: a misdiagnosed and overlooked disorder. AB - We report a case of Susac's syndrome (retinocochleocerebral vasculopathy) that was initially successfully treated with steroids. The patient had a recurrence of the disorder 2 years after the onset of the first clinical manifestations; this was treated with steroids and both calcium channel blockers and aspirin. Our findings underline the importance of recognizing patients with retinocochleocerebral vasculopathy at an early stage so that it may be accurately managed and, therefore, result in both decreased morbidity and disability related to severe sequelae. We also stress the need for patients with Susac's syndrome to have both close and long-term follow-up in order to disclose both symptomatic and asymptomatic recurrences of the disorder. PMID- 10745158 TI - Isolation of a cDNA encoding a CHH-family peptide from the silkworm Bombyx mori. AB - The crustacean hyperglycemic hormone (CHH) peptide family includes four types of neuropeptide in decapod and isopod crustaceans, and the ion-transport peptide in orthopteran insects. To identify a new member of this family in Insecta, a PCR based search for cDNAs encoding CHH-family peptides was carried out in the silkworm Bombyx mori. A cDNA, named BmCHHL (Bombyx mori CHH-like protein), with an open reading frame of 110 amino acids was isolated. Sequence analyses suggested that the conceptual protein was a precursor of a peptide of 72 amino acids which was amidated at the carboxy terminus. The BmCHHL sequence exhibited significant similarities to members of the CHH family including the orthopteran ion-transport peptide. BmCHHL expression was detected in five or six cells (per hemisphere) in the frontal area of the brain in day 4 fifth instar larvae. PMID- 10745159 TI - Synthesis and resorption of a humoral chymotrypsin inhibitor, CI-8, by fat body of the silkworm, Bombyx mori. AB - The Bombyx mori hemolymph contains up to 16 chymotrypsin inhibitors (CIs). The present in vitro culture of tissues in Grace's medium indicated that CI-8, which belongs to the largest molecular-size group of CIs with sugar moiety, is synthesized in the fat body and secreted from it during the feeding period. When the fat body from other strain which synthesizes an allelic component (CI-7) instead of CI-8 was incubated in vitro in hemolymph from the strain which has CI 8, the fat body was found to receive CI-8. Thus it was concluded that CI-8, once secreted into the hemolymph, was again sequestered into the fat body after the onset of spinning. Protein granules isolated from the pupal fat body were shown to contain CI-8, indicating that the sequestered CI-8 is present in the protein granules. PMID- 10745160 TI - Substrate-stereoselectivity of a high-affinity glutamate transporter cloned from the CNS of the cockroach Diploptera punctata. AB - A cDNA encoding a Na(+)-dependent glutamate transporter has been cloned from the brain of the cockroach Diploptera punctata. The cDNA encodes a transporter protein of 481 amino acids, designated DipEAAT1, which when expressed in baculovirus infected insect cells, resulted in a 40-50 fold increase in [(3)H]L glutamate uptake. DipEAAT1 mRNA is expressed in the brain, as is the RNA encoding TrnEAAT1, a related transporter recently isolated from the caterpillar Trichoplusia ni. The affinity of these transporters for L-glutamate and several structural analogues was compared. Both have a high affinity for L-glutamate, their presumed primary substrate, but quite different affinities for D-aspartate. TrnEAAT1 was found to be similar to other glutamate transporters in that its ability to transport [(3)H]L-glutamate into cells was inhibited strongly by D- and L- isomers of aspartate and its analogues. DipEAAT1, by contrast, was inhibited weakly by all D- isomers tested. The affinity of DipEAAT1 for [(3)H]D aspartate was found to be an order of magnitude lower than that of TrnEAAT1, revealing an unusual stereoselectivity for aspartate substrates by the cockroach transporter. The activity of DipEAAT1 was also unaffected by the presence of Zn(++) in the bathing solution, despite the presence of a putative Zn(++)-binding motif conferring Zn(++)-sensitivity on some mammalian glutamate transporters. PMID- 10745161 TI - Characterization and developmental regulation of tyramine-beta-hydroxylase in the CNS of the moth, Manduca sexta. AB - Octopamine (OA) is present in insect nervous tissue, but little is known about its biosynthesis. In the CNS of Manduca sexta, OA levels increase markedly during postembryonic adult development. To study this increase, we developed an assay for tyramine-beta-hydroxylase, the putatively rate-limiting enzyme for OA biosynthesis. Tyramine-beta-hydroxylase activity in extracts of M. sexta CNS tissue: (1) was time- and protein-dependent, and with protein concentrations up to 2 microg/microl, was linear for 20 min; (2) had a pH optimum of 7.0 for conversion of tyramine to OA; (3) required ascorbate, copper, and catalase; and (4) had an apparent K(M, tyramine) of 0.22+/-0.04 mM. These characteristics resemble those of the mammalian enzyme dopamine-beta-hydroxylase, suggesting that these two enzymes are functionally related. During adult development, tyramine beta-hydroxylase activity increased 11-fold in the brain and 9-fold in the abdominal ganglia, paralleling increases in OA levels in those CNS structures during metamorphosis. The apparent kinetic constants of tyramine-beta-hydroxylase suggested that the amount of this enzyme present in the tissues increases. The increase in OA levels during adult development thus appears to be due to an increase in the level of enzyme available for OA synthesis and may reflect an increase in the number of octopaminergic neurons. PMID- 10745162 TI - Phylogenetic analysis of Phlebotomus species belonging to the subgenus Larroussius (Diptera, psychodidae) by ITS2 rDNA sequences. AB - In the genealogy of Phlebotomus (Diptera: Psychodidae), morphological analyses have indicated that the subgenus Larroussius is a monophyletic group which is most closely related to the subgenera Transphlebotomus and Adlerius. We conducted a phylogenetic analysis of the relationships among six representative species of the subgenus Larroussius and one species representatitive of the Phlebotomus subgenus, assessing sequences of the Second Internal Transcribed Spacer (ITS2) of the ribosomal RNA (rRNA). Three of the species (P. perniciosus, P. ariasi and P. perfiliewi perfiliewi) were collected in different parts of the Mediterranean area. The trees estimated from parsimony and neighbour-joining analyses supported the monophyly of the Larroussius subgenus inferred from the morphological analysis. According to our data, P. ariasi may be a sister group to the rest of the Larroussius subgenus, although additional sequence data are needed to confirm this observation. Our results suggest that P. perniciosus and P. longicuspis are distinct species, in spite of the fact that there are only slight morphological differences. The strict congruence between the phylogeny of the Larroussius subgenus inferred from the ITS2 sequences and that based on morphological studies further confirmed the ability of the spacer sequence to identify recently-derived affiliations. PMID- 10745164 TI - Characterization of lipophorin binding to the midgut of larval Manduca sexta. AB - Lipophorin binding to the midgut of Manduca sexta larvae was characterized in a midgut membrane preparation, using iodinated larval high-density lipophorin ((125)I-HDLp-L). The iodination procedure did not change the affinity of the preparation for lipophorin. In the presence of increasing concentrations of membrane protein, corresponding increases in lipophorin binding were observed. The time-course of lipophorin binding to the membranes was affected by the lipophorin concentration in the medium, and at a low lipoprotein concentration, a longer time was required for equilibrium to be reached. The specific binding of lipophorin to the midgut membrane was a saturable process with a K(d) = 1.5+/ 0.2x10(-7) M and a maximal binding capacity = 127+/-17 ng lipophorin/microg of membrane protein. Binding did not depend on calcium, was maximal around pH 5.5, was strongly inhibited by an increase in the ionic strength, and abolished by suramin. However, suramin did not completely displace lipophorin that was previously bound to the membrane preparation. The lipid content of the lipophorin did not significantly affect the affinity of the membrane preparation for lipoprotein. PMID- 10745163 TI - Isoenzymes of glutathione S-transferase from the mosquito Anopheles dirus species B: the purification, partial characterization and interaction with various insecticides. AB - Previously we have purified and characterized a major glutathione S-transferase (GST) activity, GST-4a, from the Thai mosquito Anopheles dirus B, a model mosquito for study of anopheline malaria vectors [Prapanthadara, L. Koottathep, S., Promtet, N., Hemingway, J. and Ketterman, A.J. (1996) Insect Biochem. Mol. Biol. 26:3, 277-285]. In this report we have purified an isoenzyme, GST-4c, which has the greatest DDT-dehydrochlorinase activity. Three additional isoenzymes, GST 4b, GST-5 and GST-6, were also partially purified and characterized for comparison. All of the Anopheles GST isoenzymes preferred 1-chloro-2,4 dinitrobenzene (CDNB) as an electrophilic substrate. In kinetic studies with CDNB as an electrophilic substrate, the V(max) of GST-4c was 24.38 micromole/min/mg which was seven-fold less than GST-4a. The two isoenzymes also possessed different K(m)s for CDNB and glutathione. Despite being only partially pure GST 4b had nearly a four-fold greater V(max) for CDNB than GST-4c. In contrast, GST 4c possessed the greatest DDT-dehydrochlorinase specific activity among the purified insect GST isoenzymes and no activity was detected for GST-5. Seven putative GST substrates used in this study were not utilized by An. dirus GSTs, although they were capable of inhibiting CDNB conjugating activity to different extents for the different isoenzymes. Bromosulfophthalein and ethacrynic acid were the most potent inhibitors. The inhibition studies demonstrate different degrees of interaction of the An. dirus isoenzymes with various insecticides. The GSTs were inhibited more readily by organochlorines and pyrethroids than by the phosphorothioates and carbamate. In a comparison between An. dirus and previous data from An. gambiae the two anopheline species possess a similar pattern of GST isoenzymes although the individual enzymes differ significantly at the functional level. The available data suggests there may be a minimum of three GST classes in anopheline insects. PMID- 10745165 TI - Developmental changes in the response of larval Manduca sexta fat body glycogen phosphorylase to starvation, stress and octopamine. AB - Fasting or starvation of 1(st)- and 2(nd)-day fifth instar Manduca sexta larvae leads to rapid activation of fat body glycogen phosphorylase. Under feeding conditions, 21-29% of the phosphorylase was found in the active form. However, after only one hour of starvation, the active form increased to 55-65%. In larvae on the 3(rd)-day there was a slower increase in the activation, requiring three hours of starvation to reach a maximum of 60-65%. No activation was observed in 4(th)-day larvae after three hours of starvation. When 1(st)- or 2(nd)-day larvae were decapitated, the time-course of activation of glycogen phosphorylase was very similar to that observed in intact insects. However, activation of glycogen phosphorylase following decapitation was only observed in 1(st)- and 2(nd)-day larvae. In 2(nd)-day larvae, octopamine promoted activation of glycogen phosphorylase and 100-pmol of octopamine promoted maximum activation. Higher amounts of injected octopamine caused a decrease in activation. The injection of 100 pmol of octopamine caused a 50-55% activation of phosphorylase within 30 minutes. The simultaneous injection of the alpha-adrenergic receptor antagonist phentolamine with octopamine blocked the octopamine effect in 1(st)- and 2(nd) day feeding larvae. However, the activation of glycogen phosphorylase observed in ligated/decapitated larvae on the 1(st)- and 2(nd)-day was not abolished by injection of phentolamine. All of these data suggest that factors other than adipokinetic hormone and octopamine may be involved in the activation of glycogen phosphorylase during fasting or starvation in the early part of the fifth larval stage of M. sexta. PMID- 10745166 TI - Markers of proliferation in normal and leukoplakic oral epithelia. AB - We have reviewed the recent literature on immunohistochemical markers of cell proliferation in normal oral epithelia and leukoplakias. Most findings, including our own, point to an increased proliferation in oral leukoplakias that correlates with the degree of dysplasia. Although the basal layer of normal oral epithelia showed a very low proliferative activity, oral leukoplakias, even those containing low-grade dysplasia, exhibited a very significant increase in proliferation. High-grade dysplasia could be clearly differentiated from both low grade dysplasia and normal oral epithelia by the presence of proliferating cells in the superficial cell strata, i.e. above or superficial to the parabasal layer. These changes were detected with several markers including PCNA, Ki-67 (Mib-1), cyclin D1 and CENP-F as well as with procedures using pulse labeling with BrDU, IrDU and tritiated thymidine. Comparison of all methods showed more similarities than discrepancies. Nevertheless, because of its relative simplicity of use and universal acceptance in many other sites, Ki-67 (Mib-1) seems to be the most reliable immunohistochemical marker for future use in cancer prevention and therapeutic clinical trials. PMID- 10745167 TI - Oral mucosal melanoma: epidemiology and pathobiology. AB - The vast majority of healthy individuals have some form of melanocytic lesions with most having several cutaneous melanocytic nevocellular nevi. The incidence of cutaneous melanoma, despite improved prevention and early diagnosis of precursor melanocytic lesions, is on the increase with a projection that one in 75 persons born in the year 2000 will develop cutaneous melanoma in his/her lifetime. With cutaneous melanoma, the number, location and type of nevi, sun exposure and inability to tan, and presence or absence of dysplastic nevi affect transformation to a malignant process. Certain familial factors, syndromes, cytogenetic abnormalities, and mutations in tumor suppressor genes also influence tumor formation. In contrast, mucosal melanoma involving the oral cavity and head and neck regions is not as well understood or characterized. No doubt, this is due to the fact that this subtype of melanoma accounts for less than 1% of all cases. Mucosal melanomas tend to present at a higher stage, are more aggressive, and in a vertical growth phase of disease. A definitive precursor lesion for mucosal melanoma has not been identified; however, atypical melanocytic hyperplasia may represent a proliferative phase before overt tumorigenesis occurs. Melanoma-related antigens, growth factors, and proliferation markers have been identified in cutaneous melanoma, and allow for development of immunotherapy directed against melanoma-associated entities. It is currently possible to evaluate the cytogenetic make-up of precursor melanocytic lesions and frank melanoma, and the constitutional genetic background of individuals at risk for melanoma. No doubt, as concerted investigations of mucosal melanomas of the oral cavity and head and neck evolve, similar factors will be identified which will direct therapy and predict recurrence and survival. In the not too distant future, innovative retroviral transfection, antibodies against specific melanoma associated factors, vaccination against melanoma, and gene therapy to repair cytogenetic abnormalities and tumor suppressor gene mutations may provide effective therapy and protection against melanomas. PMID- 10745168 TI - Risk of oral cancer associated with tobacco smoking, alcohol consumption and oral hygiene: a case-control study in Madrid, Spain. AB - Oral cancer is a disease whose principal etiological factors are tobacco and alcohol consumption, which if controlled could help avoid many tumors. However, consumption has continued to grow for years. We have studied the risk of the principal factors established in the development of oral cancer and the influence of the oral hygiene level on the appearance of these tumors. Seventy-five cases of oral cancer and 150 controls from the Madrid community were interviewed on tobacco and alcohol consumption habits and their oral hygiene level. The Odds Ratio (OR) for consumption of 6-20 cigarettes/day is 3.1 and 7.96 for more than 20 cigarettes/day. When more than 50 g of alcohol/day is consumed the risk results in an OR of 5.3. Daily brushing is a protective factor (OR, 0.41). In conclusion, the most important risk factor for developing oral cancer is tobacco consumption followed by alcohol consumption. PMID- 10745169 TI - Detection of radiation-induced cervical atheromas by panoramic radiography. AB - Therapeutic irradiation of the neck is frequently used to treat patients with head and neck carcinoma. The irradiation, however, has been implicated as the cause of cervical carotid artery atherosclerotic lesions and subsequent stroke. Panoramic radiography previously shown capable of demonstrating isolated lesions was used to assess their development over time. Individuals with a pre irradiation radiograph free of atheromas were enrolled for study. The prevalence rate of atheroma formation on post-irradiation (bilateral portals at >/=45 Gy) radiographs obtained at an interval of >/=36 months was determined. A control group of non-irradiated patients having similar risk factors for head and neck carcinoma and atherosclerosis and having an initial radiograph free of atheroma formation were likewise enrolled for study. The prevalence rate of atheroma formation on a second radiograph obtained from these individuals at an interval of >/=36 months was determined. The study population consisted of 17 patients, with a mean age of 56.5 (range 21.5-77.8) years who received a mean therapeutic irradiation dose of 53.2 Gy (range 45-71) to each side of their neck. The prevalence rate of atheromas manifested on the post-irradiation radiographs was 53%. These radiographs were obtained, on average, 69.7 (range 37-133) months after completion of radiation therapy. The prevalence rate of atheromas manifested on the second radiograph of patients in the control group was 5.9%. These radiographs were obtained, on average, 53.5 (range 52-55) months after the first. The difference in prevalence rates was statistically significant (p=0.0003). Individuals who have received therapeutic irradiation to the neck are more likely to develop carotid artery atheromas after treatment than are risk matched control patients who have not been irradiated. These lesions can be detected by panoramic radiography. PMID- 10745170 TI - The FHIT gene in oral squamous cell carcinoma: allelic imbalance is frequent but cDNA aberrations are uncommon. AB - The FHIT (fragile histidine triad) gene at chromosome 3p14.2 spans the FRA3B fragile site and encodes for a diadenosine triphosphate hydrolase-type protein. FHIT is frequently abnormal in solid tumours including those of the upper aerodigestive tract (UAT) and has therefore been proposed as a tumour-suppressor gene. This proposition was evaluated here for oral squamous cell carcinoma (SCC) using microsatellite analysis, reverse transcription-polymerase chain reaction (RT-PCR), FHIT exon 5 PCR and direct sequencing. Fifty-eight primary oral SCCs were examined with two FHIT gene microsatellite markers (D3S4103 and D3S1300) and two markers flanking FHIT. Allelic imbalance (AI) occurred in 28 of 52 informative cases (54%) at one or both FHIT markers (D3S4103: 53%; D3S1300: 42%). A significant association was noted between frequency of AI and advanced stage tumours for D3S4103 but not between AI frequency and smoking. AI frequency at D3S1300 and at a flanking marker correlated with low survival. Of eight oral/UAT SCC cell lines examined, six produced abundant wild-type transcript and one yielded mostly truncated transcripts, the most abundant of which lacked exons 5 7. A double deletion was also detected in one of 11 primary oral SCCs. Our microsatellite assay results show that the FHIT gene is frequently disrupted in oral SCC. However, as FHIT was shown to be expressed normally in the great majority of oral/UAT SCCs studied, its likely involvement in the molecular pathogenesis of the disease as a tumour suppressor remains doubtful. PMID- 10745171 TI - FHIT gene mutations and single nucleotide polymorphism in Indian oral and cervical squamous cell carcinomas. AB - Genetic alterations at the FHIT (fragile histidine triad) tumor suppressor gene have been found in various human cancers. We have made an attempt to find point mutations of this gene in two different cancers from India, with entirely different etiologic factors: oral cancer (55 samples) caused by chewing tobacco and cervical cancer (43 samples) caused mainly by HPV (human papilloma virus) infection. Analysis of tumor DNA by the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method was performed on each of FHIT exons 5 9 individually, using exon-flanking primers. Two different mutations were identified in both oral and cervical tumors: one at the second nucleotide 3' to the termination codon (TGA) in exon 9 and the other at the ninth nucleotide upstream to the beginning of exon 9. These results indicate that mutations in the FHIT gene are rare events in these tumors in India (approximately 4%). In addition, we found a single nucleotide FHIT gene polymorphism which is due to T/A replacement at 17 nucleotides upstream to exon 9 where the A allele is 0.6 of the population. PMID- 10745172 TI - Cyclin D1 overexpression correlates with poor prognosis in patients with tongue squamous cell carcinoma. AB - Tongue squamous cell carcinoma makes up a large percentage of head and neck cancers, and the incidence among young patients is increasing. The aim of this study was to reveal the correlation between cyclin D1 (CCND1) expression and clinical and histologic features. We performed an immunohistochemical study on the level of CCND1 expression in tumor specimens obtained from 94 patients with tongue squamous cell carcinoma. The relationship between the expression and the following features such as age, sex, smoking and alcohol intake history, T, N, histologic grade, and multiple primary cancer was analyzed. Eighteen patients (19%) showed CCND1 overexpression (tumor cell nuclei positivity >/=50%). The 5 year survival rate of high CCND1 expressors was 39%, which was significantly poor (p=0.04). N classification correlated with CCND1 expression. CCND1 overexpression is associated with poor survival associated with progression of lymph node spread in patients with tongue squamous cell carcinomas. CCND1 expression may be a useful biologic marker for prognosis. PMID- 10745173 TI - Microsatellite alterations on chromosome 9 in chewing tobacco-induced oral squamous cell carcinomas from India. AB - Genomic instability as reflected by microsatellite alterations in specific target regions is an important characteristic of oral squamous cell carcinoma (OSCC). Microsatellite instability (MSI) and loss of heterozygosity (LOH) on chromosome 9 has been reported as an early event in oral cancers, primarily from patients in the USA and UK. Hence, we examined 77 primary oral cancer tissues and corresponding peripheral blood cell (PBC) DNA from Indian oral cancer patients for LOH and MSI, using a panel of 11 microsatellite markers spanning chromosome 9 on p and q arms. The patients were long-time (minimum 10 years) tobacco chewers. The matched DNA samples were amplified by polymerase chain reaction, resolved on a denaturing polyacrylamide gel and visualized by silver staining. An overall of 62% (48/77 cases) of the patients demonstrated microsatellite alterations including 27% MSI and 52% LOH, although at individual loci MSI was observed in 3 8% patients and LOH in the informative cases ranged from 4 to 41%. A majority of the alterations occurred on the p arm at 9p21-23, with 85% (41/48 cases) genetic alterations concentrated between markers D9S157 and D9S161. Multiple alterations were seen in 56% (27/48) of the affected cases with 17 patients showing microsatellite alterations in three to eight loci. Our data show the incidence of genetic alterations primarily in the chromosomal region 9p21-23, and may be indicative of involvement of p16 (CDKN2) tumor suppressor gene on chromosome 9p21, in a subset of chewing tobacco-induced oral cancers. PMID- 10745174 TI - Expression of matrix metalloproteinase-2 and -9 in oral squamous cell carcinomas with regard to the metastatic potential. AB - In order to evaluate the significance of matrix metalloproteinases (MMPs) in predicting the metastatic potential of oral squamous cell carcinomas (SCCs), we compared MMP-2 and -9 expression in 19 metastasizing oral SCCs with that in 25 non-metastasizing cases by immunohistochemistry and gelatin zymography. Immunohistochemistry showed that increased MMP-2 expression was not significantly related to metastasis; increased MMP-9 expression found in oral SCCs was, however, statistically significant (oral SCCs with metastasis, 73.7%; those without metastasis, 36.0%; P<0.05). Gelatin zymography revealed no significant difference in the activated form of MMP-2 between metastasizing and non metastasizing oral SCCs. In metastasizing SCCs, on the other hand, increases in the activated form of MMP-9 were significant. These results suggest that oral SCCs express MMP-2 and -9, and that MMP-9 may play a more important role than MMP 2 in the metastasis of oral SCCs to adjacent tissue. An analysis of MMP-9 expression may be useful for predicting the metastatic potential of oral SCCs. PMID- 10745175 TI - Strength and specificity of different gene promoters in oral cancer cells. AB - Gene therapy of oral cancer will require expression of genes by promoters that are both powerful and relatively tumor specific. We compared the level of expression of a reporter gene from promoters of human cytomegalovirus (CMV), SV40 virus, mouse mammary tumor virus (MMTV), human papillomaviruses (HPV) types 16 and 18, and the human multi-drug-resistance gene (mdr1), in several lines of oral cancer cells. In the oral cancer cell line 686LN the rank order of expression levels was: CMV > SV40 > HPV > mdr1 > MMTV. Unlike in previous reports the mdr1 promoter was no more active in two cancer cell lines with mutations in the p53 gene than in two other lines with wild-type p53, and its expression level could not be increased by either doxorubicin or taxol. On the other hand, expression from the MMTV promoter was increased over 10-fold by the presence of 1 microM dexamethasone. Thus, by an appropriate choice of promoter and inducer a wide variety of expression levels, over a 3-log range, could be attained in 686LN cells. The oral cancer-specificity of each promoter was judged by comparing expression in the neuroblastoma line IMR32. The most specific promoters were those from papillomaviruses, which were up to 45 times more active in the oral cancer cells, and the least specific was the CMV promoter. In order to find if an HPV-derived promoter was sufficient to produce expression of a suicide phenotype the 686 promoter was cloned adjacent to the thymidine kinase gene of herpes simplex and the construct was expressed from an adenovirus vector. The vector reduced the growth of 686LN cells over a 5-day period by up to 32% when optimal concentrations of virus and ganciclovir were used. These data will be valuable in the design of new constructs for gene therapy of oral cancer. PMID- 10745176 TI - Immunohistochemical expression of inducible nitric oxide synthase in DMBA-induced hamster buccal pouch carcinogenesis. AB - Nitric oxide (NO) plays a key role in the processes of inflammation and carcinogenesis. Three isoforms of NO synthase have been identified: endothelial nitric oxide synthase (NOS), neuronal NOS, and inducible NOS (iNOS). The purpose of this study was to investigate the characteristics of iNOS in 7, 12 dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch carcinogenesis. Thirty outbred young (6-week-old) male Syrian golden hamsters were randomly divided into three groups: DMBA (0.5%) painted group (n=10); mineral oil-treated group (n=10); and non-treated group (n=10). The average number of iNOS positive foci per section in the DMBA-treated group was approximately 12.2+/-4.7. Both cytoplasmic and nuclear stainings were observed in the DMBA-treated pouch keratinocytes. No iNOS activity could be detected in the untreated or mineral oil treated pouches. In conclusion, this study has demonstrated that iNOS is expressed in DMBA-induced hamster pouch carcinomas. This finding suggests that iNOS expression may be associated with the development of chemically induced oral carcinomas. PMID- 10745177 TI - Experimental oral carcinoma of the tongue and buccal mucosa: possible biologic markers linked to cancers at two anatomic sites. AB - The application of the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) can initiate and promote the development of oral squamous cell carcinoma of the tongue and buccal mucosa. In this study the level of expression of various markers related to the development of programmed cell death (PCD) in the respective oral carcinomas was analyzed. Sixteen male and female Syrian hamsters (Mesocrietus auratus) were treated with 0.05% DMBA for 16 weeks. Immunohistochemistry was used to observe the expression of p53, proliferating cell nuclear antigen (PCNA), Bcl-2, and nucleosome formation. Single-strand conformational polymorphism (SSCP) for exons 2-9 and sequence analysis of exon 9 of the p53 gene from normal buccal or tongue mucosa as well as the squamous cell carcinomas from the buccal mucosa or the tongue were determined. p53 (wild type) expression was significantly reduced in the tongue dysplastic mucosa or squamous cell carcinoma. The SSCP disclosed banding shifts or new bands in exons 2/3, 4, 8, and 9 for the tongue or buccal oral carcinomas (five of each). In exon 9 the mutation in codon 307 (ala)GCC-GTC(val) was present in the tongue but not in the buccal carcinoma. Other markers included the level of PCNA. PCNA was initially lower in the premalignant tongue lesions but increased in oral squamous cell carcinoma at both sites. In contrast, the amount of nucleosome formation in the tongue carcinomas was less than the level noted for buccal cancers but premalignant dysplasias in the tongue mucosa exhibited higher levels. The inhibitor of PCD, Bcl-2 was lower for dysplasias and carcinomas of the tongue compared to similar lesions of the buccal mucosa. These results indicate that oral carcinomas of different anatomical sites can exhibit differences in growth, oncogene mutation expression, and the development of PCD. The differences in Bcl 2 and nucleosome formation may signify their influence on oncogene expression and growth potential for developing transformed clones and established oral carcinomas. PMID- 10745178 TI - 5-Fluorouracil-induced apoptosis in cultured oral cancer cells. AB - Chemotherapy is commonly used to treat advanced oral squamous cell carcinoma (SCC) and is known to kill cancer cells through apoptosis. Our hypothesis states that 5-fluorouracil (5FU) also kills cultured oral epithelial cells through programmed cell death or apoptosis. Cultured oral cancer cells were exposed to an optimum dose of 20 mg/ml of 5FU. Cells were analyzed for changes in cell cycle distribution and induction of cell death including apoptosis. Normal control, human papilloma virus-immortalized (PP), ATCC SCC cell line (CA1) and two primary oral SCC cell lines (CA3 and -4) were studied. Inhibition of apoptosis by a pan caspase inhibitor was used. SYTO 11 flow cytometry showed increased apoptosis in all 5FU-treated cell cultures compared to untreated controls. The results show biological variation in apoptotic response. CA1 had the lowest apoptotic rate of the cancer cell lines at 1.5%. Next lowest was CA3, followed by CA4 and PP. In addition, alteration in the G1 and S phase fractions were found. Untreated CA1 showed 28% G1, 53% S compared to 43% G1, and 40% S of treated. We investigated the pathway of apoptosis using the pan-caspase inhibitor IDN-1529 by methylthiazolyl diphenyl tetrazolium bromide (MTT) colorimetric analysis. Results showed mild inhibition of cell death when cells were incubated with 50 microM IDN 1529 for 24 h. This suggests a probable caspase-dependent apoptotic pathway. In conclusion, our data suggest that 5FU induces oral cancer cell death through apoptosis and that biological variation exists between normal and cancer cells and between different types of cancer cells themselves. Our data indicate that cultures of a useful in vitro model for chemosensitivity assays are possible. Our results also suggest a caspase-dependent pathway for chemocytotoxicity in oral SCC. PMID- 10745179 TI - Induction of apoptosis in KB cells by pingyangmycin. AB - Pingyangmycin (PYM; Bleomycin A(5)), an antitumour antibiotic is currently used during anticancer therapy. Previous experiments demonstrated that the therapeutic efficiency of PYM for treatment of malignant tumours is considered to be related to its ability to cause DNA strand breaks in vitro. However, very little is known about the interaction of PYM with the target cells, and it is still unclear how PYM enters the cells. In this study, cell death induced by PYM was studied in a human squamous cell carcinoma cell line (KB cells). In order to determine if cell death occurred by necrosis (reproductive cell death) or apoptosis (programmed cell death), KB cells were exposed to different concentrations of PYM and evaluated by biochemical and morphological criteria. Our results indicate that KB cells displayed an arrest in the G(2)-M phase of the cell cycle and became enlarged and polynucleated before dying at the low concentrations of PYM. In contrast, when cells were exposed to high concentrations of PYM, morphological changes identical to those usually associated with apoptosis were observed as well as internucleosomal digestion of genomic DNA. In conclusion, we demonstrate that PYM is able to induce two distinct modes of cell death depending on the doses of PYM. PMID- 10745180 TI - The role of basement membrane proteins on the expression of neural cell adhesion molecule (N-CAM) in an adenoid cystic carcinoma cell line. AB - We investigated the presence of neural cell adhesion molecule (N-CAM) in the adenoid cystic carcinoma cell line CAC2 using immunofluorescence microscopy. Additionally, we analysed the role of laminin and type IV collagen in N-CAM expression. We demonstrated that cultured adenoid cystic carcinoma cells express N-CAM. Control cells presented a scattered N-CAM expression on cell membrane, and type IV collagen had no effect in N-CAM distribution. CAC2 cells grown on laminin coated coverslips expressed N-CAM concentrated on cell lamellipodia suggesting relationship with migratory activity. Our results showed that cultured adenoid cystic carcinoma cells express N-CAM and this expression is modulated by extracellular matrix. PMID- 10745181 TI - Measurement properties of the Galveston Orientation and Amnesia Test (GOAT) and improvement patterns during inpatient rehabilitation. AB - OBJECTIVES: To determine the measurement properties of the Galveston Orientation and Amnesia Test (GOAT) using the Rasch model and rating scale analysis (RSA). DESIGN: Calibration of data collected weekly during rehabilitation. SETTING: Six inpatient rehabilitation facilities. PARTICIPANTS: 77 patients admitted for their first rehabilitation after traumatic brain injury. RESULTS: Rescoring the items as dichotomies, three strata of posttraumatic amnesia (PTA) were identified. All items cohered to define a single construct and the item hierarchy confirmed their hypothesized ordering. CONCLUSIONS: Equal-interval measures of PTA were developed that exhibited good reliability and validity. A self-scoring key was developed to more efficiently assess PTA. PMID- 10745182 TI - Rating scale analysis of the Agitated Behavior Scale. AB - OBJECTIVE: Evaluate the measurement properties of the Agitated Behavior Scale (ABS) using rating scale analysis. SAMPLES: Sample 1: 900 observations of 100 individuals with traumatic brain injury; Sample 2: 204 observations of 102 persons with dementia; Sample 3: 241 observations of 6 individuals with anoxia. RESULTS: The calibration indicated that the rating scale was used as intended. The hierarchies of item difficulty were similar across samples. Person and item separation values were within the acceptable range for the TBI sample. Generally, the items work well together, however 3 items misfit the measurement model moderately. CONCLUSIONS: Agitation as measured by the ABS is best represented as a unitary construct. Results provide additional support for the reliability and validity of the ABS. PMID- 10745183 TI - Refining a measure of brain injury sequelae to predict postacute rehabilitation outcome: rating scale analysis of the Mayo-Portland Adaptability Inventory. AB - OBJECTIVE: Evaluate the psychometric properties of the Mayo-Portland Adaptability Inventory (MPAI). DESIGN: Rating scale (Rasch) analysis of MPAI and principal component analysis of residuals; the predictive validity of the MPAI measures and raw scores was assessed in a sample from a day rehabilitation program. SETTING: Outpatient brain injury rehabilitation. PARTICIPANTS: 305 persons with brain injury. RESULTS: A 22-item scale reflecting severity of sequelae of brain injury that contained a mix of indicators of impairment, activity, and participation was identified. Scores and measures for MPAI scales were strongly correlated and their predictive validities were comparable. CONCLUSIONS: Impairment, activity, and participation define a single dimension of brain injury sequelae. The MPAI shows promise as a measure of this construct. PMID- 10745184 TI - Rating scale analysis of the Neurobehavioral Cognitive Status Examination. AB - OBJECTIVE: Evaluate the measurement properties of the Neurobehavioral Cognitive Status Examination (NCSE) using rating scale analysis. DESIGN: Calibration of item responses collected as part of a study examining characteristics of case management programs and treatment outcomes. SETTING: Three outpatient rehabilitation facilities. PARTICIPANTS: Convenience sample of 186 community dwelling adults with TBI. RESULTS: Several rating scale analyses were performed to construct a unidimensional measure. Deletion of easy and misfitting items created a better targeted test (generated more spread among individuals) without increased error. CONCLUSIONS: If used with a community-based sample, three strata can be differentiated despite a skewed distribution. Recommended applications are with samples with considerably more cognitive impairment. PMID- 10745185 TI - The construct validity of the readiness to change questionnaire for persons with TBI. AB - OBJECTIVE: To examine the reliability and validity of the Readiness to Change Questionnaire (RTC) among persons with recent traumatic brain injury. DESIGN: Survey. SETTING: Inpatient rehabilitation. PARTICIPANTS: One-hundred twenty-six persons with recent TBI. RESULTS: In the first step of the analyses the RTC measure was rescaled and shortened to produce a coherent linear measure of readiness to change. Subsequent analyses showed that the linear measure fit a three stage model of change and correlated in meaningful ways with independent measures of alcohol problem severity. CONCLUSIONS: The RTC measure can be substantially improved by utilizing results obtained from rating scale analysis. The resulting 10-item linear scale has good internal consistency, a theoretically sound factor structure and meaningful correlations with external variables. Measuring readiness to change may be useful for tailoring treatment and predicting outcomes, though more research is needed in this area. PMID- 10745186 TI - Relationship between cognitive impairments and rated activity restrictions in stroke patients. AB - OBJECTIVE: To investigate the relationship between cognitive impairments and rated activity restrictions. DESIGN: Comparison of neuropsychological tests and activity questionnaires. Activity ratings were made by staff and stroke patients. SETTING: Two neurological rehabilitation hospitals. PARTICIPANTS: Eighty-seven stroke patients. RESULTS: Moderately high correlations were found between cognitive test scores and activity ratings made by staff members. In contrast, correlations between ratings made by patients and cognitive tests were much lower. There was also little agreement between the staffs' ratings and the patients' own ratings. Multiple regression analyses indicate that cognitive impairments account for 28.9% of the variance in the activity rating made by the staff members. The coefficient of determination was slightly higher when age, time since onset of illness, and depression scores were included as predictors. CONCLUSIONS: These findings provide partial support for a hierarchical structure of the "International Classification of Impairment, Activities and Participation" model of the World Health Organization. PMID- 10745187 TI - Measurement of problem-solving deficits in adults with acquired brain damage. AB - OBJECTIVE: To compare the relative utility of conventional neuropsychological and social problem-solving approaches to measuring functional problem solving deficits in individuals with acquired brain damage (ABD). DESIGN: In Study I, scores for individuals with ABD were compared to scores for control and normative samples. In Study II, pre- and posttest scores were compared for individuals with ABD who completed a program of outpatient cognitive rehabilitation. PARTICIPANTS: In Study I, individuals with ABD were compared to healthy controls. In Study II, pre- and posttreatment assessments were obtained for 34 individuals with ABD. MAIN OUTCOME MEASURES: Two approaches were used, conventional neuropsychological (WAIS-R/II Comprehension subtest and Wisconsin Card Sorting Test) and social problem solving (Problem Solving Inventory and Rusk Problem Solving Role Play Test). RESULTS: In Study I, the ABD group demonstrated significant deficits on both social problem solving measures; however, neither conventional neuropsychological measure detected significant deficits in the ABD group, relative to control and normative groups. In Study II, significant treatment gains were demonstrated on both social problem-solving measures, however neither conventional neuropsychological measure was sensitive to improvements in functional problem-solving ability. CONCLUSIONS: In higher-level cognitive rehabilitation settings, the evaluation of functional problem-solving deficits in individuals with ABD can be facilitated by augmenting neuropsychological test data with results from social problem-solving measures. PMID- 10745188 TI - The Center for Outcome Measurement in Brain Injury (COMBI): An Internet resource you should know about. AB - OBJECTIVE: This article describes the Center for Outcome Measurement in Brain Injury (COMBI), an Internet resource that provides information on brain injury outcome measures. Funded by the National Institute on Disability and Rehabilitation Research (NIDRR), the COMBI is a collaborative project of eight Traumatic Brain Injury (TBI) Model System centers. Information the COMBI provides includes rating scales and form(s) syllabus and administration guidelines, descriptions of properties, references in the literature, a frequently asked questions (FAQ) section, training and testing materials, and contact information. CONCLUSIONS: As a dissemination effort, the results of the COMBI project have been outstanding with over 1,500 users accessing information every month. The project has a truly international scope, with 20% of its users being outside the United States. PMID- 10745190 TI - TBI State Demonstration Grants. AB - In July 1996, the Congress enacted Public Law 104-166, "to provide for the conduct of expanded studies and the establishment of innovative programs with respect to traumatic brain injury" (TBI). Under the Law, the Health Resources and Services Administration (HRSA), Maternal and Child Health Bureau (MCHB) is charged with implementing a State Demonstration Grant Program to improve access to health and other services for individuals with TBI and their families. The National Institutes of Health (NIH) and the Centers for Disease Control and Prevention (CDC) have also been delegated responsibilities in the areas of research, surveillance and prevention, respectively. Traumatic brain injury (TBI) is defined as an insult to the brain from some externally inflicted trauma to the head that results in significant impairment to an individual's physical, psychosocial, and/or cognitive functional abilities. Motor vehicle crashes, falls, violence, and sport-related injuries are the major causes of TBI; the number one killer and cause of disability for young people in the United States. The Centers for Disease Control and Prevention has identified that approximately 5.3 million Americans live with the effects of TBI. About half the estimated 1.9 million Americans who experience traumatic brain injuries each year incur at least short-term disability; 52,000 people die as a result of their injuries; and more than 90,000 people sustain severe brain injuries leading to debilitating loss of function. The direct medical costs for treatment of TBI have been estimated at more than $4 billion annually. There are two program categories in the TBI State Demonstration Grant Program: Planning and Implementation. The planning category provides support to those States that need assistance in developing an infrastructure for individuals with TBI in their families. The implementation category supports the development and expansion of activities that will improve or enhance access to services for individuals with TBI and their families, within the current service delivery system. PMID- 10745189 TI - Participant-proxy reliability in traumatic brain injury outcome research. AB - OBJECTIVE: To assess reliability between persons with Traumatic Brain Injury (TBI) and their self-selected proxies. DESIGN: Intraclass Correlation Coefficients were used to assess participant-proxy reliability on the Craig Handicap Assessment and Reporting Technique (CHART), the Community Integration Questionnaire (CIQ), and the Functional Independence Measure (FIM). SETTING: Participants had been discharged to the community from inpatient rehabilitation between six months and approximately five years prior to the study's beginning. PARTICIPANTS: 204 persons with moderate to severe TBI and their self-selected proxies. RESULTS: Eighty-seven percent of the items on the three instruments exhibited moderate to high intraclass correlation (ICC), with strongest participant-proxy agreement for questions assessing concrete, observable information. Participant-proxy agreement was poorest when assessing cognitive and money management capacity as well as out-of-home activities. CONCLUSIONS: For many types of items, participant-proxy reliability is sufficient to merit the use of proxies in TBI outcome research when the participants are allowed to select their own proxy. PMID- 10745191 TI - Engineering of coordinated up- and down-regulation of two glycosyltransferases of the O-glycosylation pathway in Chinese hamster ovary (CHO) cells. AB - Production of O-linked oligosaccharides that interact with selectins to mediate cell-cell adhesion occurs in one segment of a branched glycan biosynthesis network. Prior efforts to direct the branched pathway towards selectin-binding oligosaccharides by amplifying enzymes in this branch of the network have had limited success, suggesting that metabolic engineering to simultaneously inhibit the competing pathway may also be required. We report here the partial cloning of the CMP-sialic acid:Galbeta1,3GalNAcalpha2, 3-sialyltransferase (ST3Gal I) gene from Chinese hamster ovary (CHO) cells and the simultaneous inhibition of expression of CHO cell ST3Gal I gene and overexpression of the human UDP GlcNAc:Galbeta1, 3GalNAc-R beta1,6-N-acetylglucosaminyltransferase (C2GnT) gene. A tetracycline-regulated system adjoined to tricistronic expression technology allowed "one-step" transient manipulation of multiple enzyme activities in the O glycosylation pathway of a previously established CHO cell line already engineered to express alpha1, 3-fucosyltransferase VI (alpha1,3-Fuc-TVI). Tetracycline-regulated co-expression of a ST3Gal I fragment, cloned in the antisense orientation, and of C2GnT cDNA resulted in inhibition of the ST3Gal I enzymatic activity and increase in C2GnT activity which varied depending on the extent of tetracycline reduction in the cell culture medium. This simultaneous regulated inhibition and activation of the two key enzyme activities in the O glycosylation pathway of mammalian cells is an important addition to the metabolic engineering field. PMID- 10745192 TI - Growth-rate-independent production of recombinant glucoamylase by Fusarium venenatum JeRS 325. AB - Most recombinant proteins generated in filamentous fungi are produced in fed batch cultures, in which specific growth rate normally decreases progressively with time. Because of this, such cultures are more suited to the production of products that are produced efficiently at low-growth rates (e.g., penicillin) than to products which are produced more efficiently at high-growth rates (e. g., glucoamylase). Fusarium venenatum A3/5 has been transformed (JeRS 325) to produce Aspergillus niger glucoamylase (GAM) under the control of the Fusarium oxysporum trypsin-like protease promoter. No glucoamylase was detected in the culture supernatant during exponential growth of F. venenatum JeRS 325 in batch culture. In glucose-limited chemostat cultures, GAM concentration increased with decrease in dilution rate, but the specific production rate of GAM (g GAM [g biomass](-1) h(-1)) remained approximately constant over the dilution-rate range 0.05 h to 0.19 h(-1), i.e., the recombinant protein was produced in a growth-rate independent manner. The specific production rate decreased at dilution rates of 0.04 h(-1) and below. Specific production rates of 5.8 mg and 4.0 mg GAM [g biomass](-1) h(-1) were observed in glucose-limited chemostat cultures in the presence and absence of 1 g mycological peptone L(-1). Compared to production in batch culture, and for the same final volume of medium, there was no increase in glucoamylase production when cultures were grown in fed-batch culture. The results suggested that a chemostat operated at a slow dilution rate would be the most productive culture system for enzyme production under this trypsin-like promoter. PMID- 10745193 TI - Physiological studies in aerobic batch cultivations of Saccharomyces cerevisiae strains harboring the MEL1 gene. AB - Physiological studies of Saccharomyces cerevisiae strains harboring the MEL1 gene were carried out in aerobic batch cultivations on glucose-galactose mixtures and on the disaccharide melibiose, which is hydrolyzed by the enzyme melibiase (Mel1, EC 3.2.1.22) into a glucose and a galactose moiety. The strains examined (T200, T256, M24, and TH1) were all derived from the bakers' and distillers' strain of S. cerevisiae, DGI 342. All the strains showed a significant higher ethanol yield when growing on glucose, and half the biomass yield, compared with growth on galactose. The maximum specific uptake rates were 2.5-3.3-fold higher on glucose than on galactose for all the strains examined, and hence, ethanol production was pronounced on glucose due to respiro-fermentative metabolism. The T256 strain and the T200 strain having the MEL1 gene inserted in the HXK2 locus and the LEU2 locus, respectively, hydrolyzed melibiose with low specific hydrolysis rates of 0.03 C-mol/g/h and 0.04 C-mol/g/h, respectively. This resulted in high biomass yields on melibiose in the order of 10 g/C-mol compared with 3.7 g/C-mol for M24 and 1.6 g/C-mol for TH1. The M24 strain, constructed by classical breeding, and the mig1/gal80 disrupted and melibiase-producing strain TH1, were superior in their ability to hydrolyze melibiose into glucose and galactose showing specific melibiose hydrolysis rates of 0.17 C-mol/g/h and 0.24 C-mol/g/h, respectively. Hence, high ethanol yields on melibiose were obtained with these two strains. Growth on the glucose-galactose mixtures showed a reduction of glucose control successfully obtained in the M24 strain and the TH1 strain. PMID- 10745194 TI - Hyperosmotic pressure enhances immunoglobulin transcription rates and secretion rates of KR12H-2 transfectoma. AB - When subjected to hyperosmotic pressure resulting from NaCl addition, KR12H-2 transfectoma, like most hybridomas, displayed a decrease in specific growth rate (mu) and an increase in specific antibody productivity (q(Ab)). Elevation of medium osmolality from 285 to 425 mOsm/kg decreased mu by 20%, while it increased q(Ab) by 376%. Although cell mass also increased at higher osmolality, it was not the main factor in increasing q(Ab). Hyperosmotic pressure was found to enhance transcription levels of immunoglobulin (Ig) mRNAs preferentially, compared with non-IgG mRNA. The transcription levels of both heavy chain (HC) and light chain (LC) mRNAs were enhanced as much as q(Ab). This result suggests that enhanced q(Ab) at higher osmolality was mainly due to enhanced transcription levels of Ig mRNA. However, these increased transcription levels of Ig mRNAs were not due to the enhanced stability of Ig mRNA. In fact, the stability of Ig mRNAs decreased at higher osmolality. Elevation of osmolality from 285 mOsm/kg to 425 mOsm/kg decreased the half-lives of HC and LC mRNAs by 37% and 36%, respectively. A simple mathematical model revealed that transcription rates of Ig mRNAs increased by more than 476% at 425 mOsm/kg. These elevated transcription levels could, in turn, increase the translation rates of Ig polypeptides. However, the translation rates of Ig polypeptides were not enhanced as much as the transcription levels of Ig mRNAs and q(Ab). The elevation of osmolality from 285 mOsm/kg to 425 mOsm/kg increased HC and LC mRNA specific translation rates by 172% and 240%, respectively. Taken together, the data suggest that (1) enhanced q(Ab) of KR12H-2 transfectoma at higher osmolality is due to elevated transcription rates of Ig mRNAs and expedited post-translational processing of Ig, and (2) antibody secretion by KR12H-2 transfectoma is most likely controlled at the level of Ig translation, particularly HC translation. PMID- 10745195 TI - Modeling and optimization of anaerobic digested sludge converting starch to hydrogen. AB - The pH and hydraulic retention time (HRT) of a chemostat reactor were varied according to a central composite design methodology with the aim of modeling and optimizing the conversion of starch into hydrogen by microorganisms in an anaerobic digested sludge. Experimental results from 23 runs indicate that a maximum hydrogen production rate of 1600 L/m(3)/d under the organic loading rate of 6 kg starch m(3)/d obtained at pH = 5.2 and HRT = 17 h. Throughout this study, the hydrogen percentage in the biogas was approximately 60% and no methanogenesis was observed. while the reactor was operated with HRT of 17 h, hydrogen was produced within a pH range between 4.7 and 5.7. Alcohol production rate was greater than hydrogen production rate if the pH was lower than 4.3 or higher than 6.1. Supplementary experiments confirm that the optimum conditions evaluated in this study were highly reliable; while a hydrogen production yield of 1.29 l H(2)/g starch-COD was obtained. An examination of the response surfaces, including hydrogen, volatile fatty acids (VFA) and alcohols production, led us to the belief that clostridium sp. predominated in the anaerobic hydrogen-producing microorganisms in this study. Experiment results obtained emphasize that the response of metabolites was a more useful indicator than hydrogenic activity for obtaining efficient hydrogen production. Furthermore, expressions of contour plots indicate that Response-Surface Methodology may provide easily interpretable advice on the operation of a hydrogen-producing bioprocess. PMID- 10745197 TI - Dynamic biochemical reaction process analysis and pathway modification predictions. AB - Recently, the area of model predictive modification of biochemical pathways has received attention with the aim to increase the productivity of microbial systems. In this study, we present a generalization of previous work, where, using a sensitivity study over the fermentation as a dynamic system, the optimal selection of reaction steps for modification (amplification or attenuation) is determined. The influence of metabolites in the activity of enzymes has also been considered (through activation or inhibition). We further introduce a new concept in the dynamic modeling of biochemical reaction systems including a generalized continuous superstructure in which two artificial multiplicative terms are included to account for: (a) enzyme overexpression or underexpression (attenuation or amplification) for the whole enzyme pool; and (b) modification of the apparent order of a kinetic expression with respect to the concentration of a metabolite or any subset of metabolites participating in the pathway. This new formulation allows the prediction of the sensitivity of the pathway performance index (objective function) with respect to the concentration of the enzyme, as well as the interaction of the enzyme with other metabolites. Using this framework, a case study for the production of penicillin V is analyzed, obtaining the most sensitive reaction steps (or bottlenecks) and the most significant regulations of the system, due to the effect of concentration of intracellular metabolites on the activity of each enzyme. PMID- 10745196 TI - Influence of high biomass concentrations on alkane solubilities. AB - Alkane solubilities were measured experimentally for high-density biomass. The resulting Henry's law constants for propane were found to decrease significantly for both dense yeast suspensions and an actual propane-degrading biofilm consortium. At the biomass densities of a typical biofilm, propane solubility was about an order of magnitude greater than that in pure water. For example, a dense biofilm had a propane Henry's law constant of 0.09+/-0.04 atm m(3) mol(-1) compared to 0.6+/-0.1 atm m(3) mol(-1) measured in pure water. The results were modeled with mixing rules and compared with octanol-water mixtures. Hydrogels (agar) and salts decreased the alkane solubility. By considering a theoretical solubility of propane in dry biomass, estimates were made of intrinsic Henry's law constants for propane in pure yeast and biomass, which were 13+/-2 and 5+/-2 atm kg biomass mol(-1) for yeast and biofilm consortium, respectively. PMID- 10745198 TI - Design of metabolic feed controllers: application to high-density fermentations of Pichia pastoris. AB - High-density cultures of the methylotrophic yeast Pichia pastoris were found to exhibit oscillatory metabolic behavior when fed methanol under closed-loop operations using a dissolved oxygen-based bioreactor feed controller (DOstat). This behavior, if left unattended, led to the irreversible loss of culture productivity, 1 to 2 days after growth on methanol commenced, presumably through the accumulation of incompletely oxidized intermediates. To provide insights into how fermentation operation conditions and strain variations might contribute to this phenomenon a theoretical study was initiated. In this article, a simple mathematical model of the closed-loop DOstat is developed and analyzed with the goal of deriving theoretical stability criteria applicable to the design of metabolic feed controllers during high-cell-density fermentations. The model consists of a system of differential, integral, and algebraic equations describing the biological process and the components of the standard proportional integral (PI) feedback controller. Inputs into the process model include metabolic pathway information, oxidative metabolism stoichiometry, and substrate uptake kinetics. Frequency-response analysis and the Bode stability criterion are applied to derive controller stability criteria with particular emphasis on elucidating the role(s) that model parameters, both biological and operational, have on system stability. The results of this analysis are used to construct a closed-loop fermentation-operating diagram relating fermentation operating parameters to the oxidative capacity of the culture. The utility of this analysis is demonstrated through the application of the results to the design and stabilization of the DOstat during high-density fermentations of P. pastoris growing on methanol or glycerol. From this analysis, it is possible to conclude that, when the rate of oxygen transfer approaches in magnitude the rate of oxygen utilization, the potential for controller destabilization is greatest. Under these conditions, the region of parameter space associated with stable controller operations is further constrained. Because the only system-specific experimental inputs into the model are the routinely measured residual substrate and dissolved oxygen concentrations, the framework presented should provide simple and practical theoretical guidelines relevant to the design of similar industrial fermentation feed controllers independent of the specifics of the biological system at hand. PMID- 10745199 TI - A material-balance approach for modeling bacterial chemotaxis to a consumable substrate in the capillary assay. AB - A mathematical model was developed to simulate the movement of chemotactic bacteria into and within a capillary tube containing a metabolizable chemoattractant. The model was based on a material balance that accounts for the transport of bacteria and chemoattractant as well as consumption of the substrate throughout the capillary assay system. By solving the model with a numerical method, it was possible to predict the concentration of substrate and bacteria at points within the capillary and throughout the chamber. The model was tested for its ability to simulate the results of capillary assay experiments performed with Pseudomonas putida G7 and one of its chemoattractants, naphthalene, under conditions wherein naphthalene consumption was expected to affect the flux of bacteria into the capillary. While variations in the chemotactic responses were evident among different experiments, the model could simulate the accumulation of cells in the capillary using previously determined parameters, including the chemotactic sensitivity and random motility coefficients, chi(0) and mu. In particular, model predictions were consistent with the experimental observation that the chemotactic response in the capillary is diminished under conditions wherein consumption would be expected to be significant. PMID- 10745200 TI - Investigation of the TCA cycle and the glyoxylate shunt in Escherichia coli BL21 and JM109 using (13)C-NMR/MS. AB - Acetate accumulation is a common problem observed in aerobic high cell density Escherichia coli cultures. A previous report has hypothesized that the glyoxylate shunt is active in a low acetate producer, E. coli BL21, and inactive in a high acetate producer, JM109. To further investigate this hypothesis, we now develop a model for the incorporation of (13)C from uniformly labeled glucose into key TCA cycle intermediates. The (13)C isotopomer distributions of oxaloacetate and acetyl-CoA are first determined using NMR and MS techniques. These distributions are next validated by predicting the NMR spectrum of glutamate. Under steady state isotopic conditions, and with knowledge of the full isotopomer distributions of oxaloacetate and acetyl-CoA, the flux ratios through the TCA cycle and the glycoxylate shunt are obtained with respect to the flux through the PPC anaplerotic shunt. We conclude that in BL21, the glyoxylate shunt is active at 22% of the flux through the TCA cycle, and is inactive in JM109. Further, in BL21, the flux through the TCA cycle equals the flux through the PPC shunt, while in JM109 the TCA cycle flux is only third of the flux through the PPC shunt. PMID- 10745202 TI - Structural stability of DNA in nonaqueous solvents. AB - One of the defining physicochemical features of DNA in aqueous solution is its ability to maintain a double-helical structure and for this structure to undergo a cooperative, heat-induced denaturation (melting). Herein we show that a 21-mer synthetic DNA can form and maintain such a duplex structure not only in water but even in 99% glycerol; moreover, this double-helical structure reversibly and cooperatively melts in that solvent, with a T(m) value of some 30 degrees lower than in water. Two much larger, natural DNAs, from calf thymus and salmon testes, exhibit similar behavior in glycerol. All three DNAs can also sustain a double helical structure in 99% ethylene glycol, although its thermostability (as reflected by the melting temperature) is some 20 degrees lower than in glycerol. In contrast, no duplex structure of any of the DNAs was detected in 99% formamide, methanol, or DMSO. This solvent trend resembles that previously observed in studies of protein structure and folding and underscores the importance of hydrophobic interactions in both protein and DNA structure and stability. Our findings suggest that water may not be unique as a suitable medium not only for protein structure but also for that of nucleic acids. PMID- 10745201 TI - Culture of human T cells in stirred bioreactors for cellular immunotherapy applications: shear, proliferation, and the IL-2 receptor. AB - Ex vivo expansion of T cells is a key step of many cellular immunotherapy protocols, which require large numbers of immune cells to eradicate malignant or virally infected cells. The use of stirred culture systems for T cell expansion offers many potential advantages over the static culture systems commonly used today, including homogeneity of culture conditions, ease of sampling, and implementation of control systems. Primary human T cells as well as the transformed TALL103/2 T cell line were cultured in 100-mL spinner flasks as well as 2-L bioreactors to investigate the effects of shear forces produced by agitation and sparging-based aeration on the expansion of T cells. Primary T cells could be successfully grown at agitation rates of up to 120 rpm in the spinner flasks and to 180 rpm in the bioreactors with no immediate detrimental effects on proliferation. Exposure to agitation and sparging did, however, cause a significantly increased rate of downregulation of the interleukin-2 receptor (IL-2R), resulting in lower overall expansion potential from a single stimulation as compared to static controls, with faster IL-2R downregulation occurring at higher agitation rates. For the primary T cells, no significant effects of agitation were found on expression levels of other key surface receptors (CD3, CD28, or CD62L) examined. No significant effects of agitation were observed on primary T cell metabolism or levels of cellular apoptosis in the cultures. The TALL103/2 T cell line was found to be extremely sensitive to agitation, showing severely reduced growth at speeds above 30 rpm in 100-mL spinner flasks. This unexpected increased fragility in the transformed T cell line as compared to primary T cells points out the importance of carefully selecting a model cell line which will accurately represent the characteristics of the cell system of interest. PMID- 10745203 TI - Bacterial cellulose production by Acetobacter xylinum in a 50-L internal-loop airlift reactor. AB - Bacterial cellulose (BC) production was realized in a batch cultivation of Acetobacter xylinum subsp. sucrofermentans BPR2001 in a 50-L internal-loop airlift reactor. When the bacterium was cultivated with air supply, 3.8 g/L of BC was produced after 67 hours. When oxygen-enriched gas was supplied, the concentration of BC was doubled and the production rate of BC was 0.116 g/L. h, which was two times higher than that of air-supplied culture and comparable to that in a mechanically agitated stirred-tank fermentor. Bacterial cellulose produced by the airlift reactor formed a unique ellipse pellet (BC pellet), different from the fibrous form which was produced in an agitated stirred-tank fermentor. The BC-pellet suspension was demonstrated to have a higher volumetric oxygen transfer coefficient than the fibrous BC suspension in a 50-L internal loop airlift reactor. The mixing time of BC-pellet suspension in the airlift reactor was also shorter than that in water. PMID- 10745204 TI - A theoretical study on the effect of surface roughness on mass transport and transformation in biofilms. AB - This modeling study evaluates the influence of biofilm geometrical characteristics on substrate mass transfer and conversion rates. A spatially two dimensional model was used to compute laminar fluid flow, substrate mass transport, and conversion in irregularly shaped biofilms. The flow velocity above the biofilm surface was varied over 3 orders of magnitude. Numerical results show that increased biofilm roughness does not necessarily lead to an enhancement of either conversion rates or external mass transfer. The average mass transfer coefficient and Sherwood numbers were found to decrease almost linearly with biofilm area enlargement in the flow regime tested. The influence of flow, biofilm geometry and biofilm activity on external mass transfer could be quantified by Sh-Re correlations. The effect of biofilm surface roughness was incorporated in this correlation via area enlargement. Conversion rates could be best correlated to biofilm compactness. The more compact the biofilm, the higher the global conversion rate of substrate. Although an increase of bulk fluid velocity showed a large effect on mass transfer coefficients, the global substrate conversion rate per carrier area was less affected. If only diffusion occurs in pores and channels, then rough biofilms behave as if they were compact but having less biomass activity. In spite of the fact that the real biofilm area is increased due to roughness, the effective mass transfer area is actually decreased because only biofilm peaks receive substrate. This can be explained by the fact that in the absence of normal convection in the biofilm valleys, the substrate gradients are still largely perpendicular to the carrier. Even in the cases where convective transport dominates the external mass transfer process, roughness could lead to decreased conversion rates. The results of this study clearly indicate that only evaluation of overall conversion rates or mass fluxes can describe the correct biofilm conversion, whereas interpretation of local concentration or flow measurements as such might easily lead to erroneous conclusions. PMID- 10745205 TI - Effects of ammonia on CHO cell growth, erythropoietin production, and glycosylation. AB - The effect of ammonium chloride was determined on a culture of CHO cells transfected with the human erythropoietin (EPO) gene. Cell growth was inhibited above a culture concentration of 5 mM NH(4)Cl with an IC-50 determined to be 33 mM. The specific production of EPO increased with the addition of NH(4)Cl above 5 mM. At 10 mM NH(4)Cl, the final cell density after 4 days in culture was significantly lower but the final yield of EPO was significantly higher. This appeared to be due to continued protein production after cell growth had ceased. The metabolic effects of added NH(4)Cl included higher specific consumption rates of glucose and glutamine and an increased rate of production of alanine, glycine, and glutamate. The EPO analyzed from control cultures had a molecular weight range of 33-39 kDa and an isoelectric point range of 4.06-4.67. Seven distinct isoforms of the molecule were identified by two-dimensional electrophoresis. This molecular heterogeneity was ascribed to variable glycosylation. Complete enzymatic de-glycosylation resulted in a single molecular form with a molecular mass of 18 kDa. Addition of NH(4)Cl to the cultures caused a significant increase in the heterogeneity of the glycoforms as shown by an increased molecular weight and pI range. Enzymatic de-sialylation of the EPO from the ammonia-treated and control cultures resulted in identical electrophoretic patterns. This indicated that the effect of ammonia was in the reduction of terminal sialylation of the glycan structures which accounted for the increased pI. Selective removal of the N-glycan structures by PNGase F resulted in two bands identified as the O-glycan linked structure (19 kDa) and the completely de-glycosylated structure (18 kDa). The proportion of the O-linked glycan structure was reduced, and its pI increased in cultures to which ammonia was added. Thus, the glycosylation pattern altered by the presence of ammonia included a reduction in terminal sialylation of all the glycans and a reduction in the content of the O-linked glycan. The addition of a sialidase inhibitor to the cultures had no effect on the ammonia-induced increase in EPO heterogeneity. Also, the effect of ammonia on glycosylation could not be mimicked using the weak base chloroquine in our system. PMID- 10745206 TI - Enhanced growth of Sf-9 cells to a maximum density of 5.2 x 10(7) cells per mL and production of beta-galactosidase at high cell density by fed batch culture. AB - Significant improvement in cell growth and protein production has been achieved in Sf-9 insect cell cultures using pulse additions of multicomponent nutrient feed concentrates (Bedard et al., 1994; Chan et al., 1998). The present work focuses on investigating an alternative feeding strategy wherein the nutrients are fed in a semi continuous manner. Fed batch culture experiments were carried out to compare the two different feeding strategies, pulse and semi continuous and a process developed to achieve a cell density of 5.2 x 10(7) cells/mL of Sf-9 cells in a 3.5 L bioreactor. Production of recombinant protein beta-galactosidase was carried out by infecting the cells with baculovirus at a MOI of 10 at cell densities of 17 x 10(6)cells/mL. Specific productivity could be maintained at cell densities as high as 14 x 10(6) cells/mL. The results presented indicate that the feeding method can provide significant improvements in the performance with a reduction in the amount of total nutrients added. On-line monitoring of the culture using the capacitance probe showed that the capacitance probe can be used successfully to monitor the biomass and infection process even at higher cell densities. PMID- 10745207 TI - Glutathione excretion in response to heterologous protein secretion in Saccharomyces cerevisiae. AB - Glutathione is excreted in a dose-dependent, non-stoichiometric fashion from Saccharomyces cerevisiae cells expressing and secreting Bovine Pancreatic Trypsin Inhibitor (BPTI), a small, disulfide-bonded protein. Glutathione excretion commences 40 hours following induction of BPTI synthesis. Expression of several secretory proteins with varying disulfide and cysteine contents results in glutathione excretion with no apparent requirement for protein disulfide content. Glutathione excretion is also triggered by overexpression of Kar2p/BiP, a native ER-resident protein-folding chaperone, indicating that the response is a general one not restricted to overexpression of thiol-containing heterologous proteins. Functional vesicular transport is not required at the time of glutathione excretion, and glutathione excretion requires the presence of molecular oxygen. These data are consistent with a delayed oxidative stress response potentiated by earlier heterologous secretion, but are inconsistent with secretory transport of glutathione spent as oxidizing equivalents for disulfide-bond formation in the endoplasmic reticulum. PMID- 10745208 TI - Single-step nitrification models erroneously describe batch ammonia oxidation profiles when nitrite oxidation becomes rate limiting. AB - Nitrification involves the sequential biological oxidation of reduced nitrogen species such as ammonium-nitrogen (NH(4)(+)-N) to nitrite-nitrogen (NO(2)(-)-N) and nitrate-nitrogen (NO(3)(-)-N). The adequacy of modeling NH(4)(+)-N to NO(3)( )-N oxidation as one composite biochemical reaction was examined at different relative dynamics of NH(4)(+)-N to NO(2)(-)-N and NO(2)(-)-N to NO(3)(-)-N oxidation. NH(4)(+)-N to NO(2)(-)-N oxidation and NO(2)(-)-N to NO(3)(-)-N oxidation by a mixed nitrifying consortium were uncoupled using selective inhibitors allylthiourea and sodium azide. The kinetic parameters of NH(4)(+)-N to NO(2)(-)-N oxidation (q(max,ns) and K(S,ns)) and NO(2)(-)-N to NO(3)(-)-N oxidation (q(max,nb) and K(S,nb)) were determined by a rapid extant respirometric technique. The stoichiometric coefficients relating nitrogen removal, oxygen uptake and biomass synthesis were derived from an electron balanced equation. NH(4)(+)-N to NO(2)(-)-N oxidation was not affected by NO(2)(-)-N concentrations up to 100 mg NO(2)(-)-N L(-1). NO(2)(-)-N to NO(3)(-)-N oxidation was noncompetitively inhibited by NH(4)(+)-N but was not inhibited by NO(3)(-)-N concentrations up to 250 mg NO(3)(-)-N L(-1). When NH(4)(+)-N to NO(2)(-)-N oxidation was the sole rate-limiting step, complete NH(4)(+)-N to NO(3)(-)-N oxidation was adequately modeled as one composite process. However, when NH(4)(+) N to NO(2)(-)-N oxidation and NO(2)(-)-N to NO(3)(-)-N oxidation were both rate limiting, the estimated lumped kinetic parameter estimates describing NH(4)(+)-N to NO(3)(-)-N oxidation were unrealistically high and correlated. These findings indicate that the use of single-step models to describe batch NH(4)(+) oxidation yields erroneous kinetic parameters when NH(4)(+)-to-NO(2)(-) oxidation is not the sole rate-limiting process throughout the assay. Under such circumstances, it is necessary to quantify NH(4)(+)-N to NO(2)(-)-N oxidation and NO(2)(-)-N to NO(3)(-)-N oxidation, independently. PMID- 10745209 TI - Modification of a recombinant GPI-anchored metalloproteinase for secretion alters the protein glycosylation. AB - The N-linked glycans of recombinant leishmanolysin (GP63) expressed as a glycosylphosphatidylinositol (GPI)-anchored membrane protein or modified for secretion in Chinese hamster ovary (CHO) cells were analyzed by fast atom bombardment-mass spectrometry (FAB-MS). The glycans isolated from both membrane and secreted protein were predominantly complex biantennary structures. However other aspects of the glycan profiles showed striking differences. The degree of sialylation of the membrane form was greatly reduced and the core fucosylation of biantennary structures was increased compared to the secreted form. Glycans isolated from membrane expressed protein also contained a higher proportion of lactosamine repeats. Residence times in the secretory pathway were similar for both secreted and membrane protein. Glycosylation differences may therefore be due to differences in protein conformation and accessibility to glycosyltransferases or glycosidases. These differences in glycosylation represent an important factor when considering modifying membrane expressed proteins for secreted production. PMID- 10745210 TI - Endogenous model state and parameter estimation from an extensive batch experiment. AB - In this paper an extensive batch experiment of endogenous process behavior in an aerobic biodegradation process is presented. From these experimental data, comprising measurements of MLVSS (mixed liquor volatile suspended solids) and respiration rate, in a first step the states and unknown parameters in a four compartmental model are reconstructed analytically. Subsequently, for a selected set of states and parameters, using the results of the previous step, a recursive state estimation procedure, in particular an Extended Kalman filter-based observer, is applied to deal with the noise properties of the data appropriately. From this it appears that the initially proposed model structure, and especially the hydrolysis term, has to be modified. PMID- 10745211 TI - Stabilization of D-amino acid oxidase and catalase in permeabilized Rhodotorula gracilis cells and its application for the preparation of alpha-ketoacids*. AB - The cellular D-amino acid oxidase (DAAO) and catalase activities of Rhodotorula gracilis were greatly increased upon the treatment of the cells with cetyltrimethylammonium bromide (CTAB). However, these enzymes, slowly leaks out from the permeabilized cells. The released DAAO was rapidly inactivated in the absence of ethylenediaminotetraacetic acid (EDTA), beta-mercaptoethanol, and glycerol. DAAO within the permeabilized cells did not require these stabilizing agents. Treating the CTAB-permeabilized cells with 0.2% glutaraldehyde (GA) at 4 degrees C for 10 min prevented the leakage of both DAAO and catalase. Alternately, stabilized whole cell DAAO and catalase was prepared by treating the whole yeast cells with 1% GA at 4 degrees C for 60 min, followed by permeabilization with CTAB, a method which was equally efficient but easy to scale up. CTAB-permeabilized cells converted D-phenylalanine to 97% phenylpyruvate and 3% phenylacetate, and these cells were reused up to 3 cycles in a batchwise reaction. On the other hand, GA-treated CTAB-permeabilized cells produced more than 99% phenylpyruvate and the cells could be reused up to 20 cycles. PMID- 10745212 TI - Growth kinetics of Pseudomonas putida in cometabolism of phenol and 4 chlorophenol in the presence of a conventional carbon source. AB - Growth kinetics of Pseudomonas putida (ATCC 49451) in cometabolism of phenol and 4-chlorophenol (4-cp) in the presence of sodium glutamate (SG) were studied. In the ternary substrate mixture, phenol and SG are growth substrates while 4-cp is a nongrowth substrate. Cell growth on phenol was found to follow Andrews kinetics and cells displayed substrate inhibition pattern on sodium glutamate in the range of 0-4 g L(-1) as well. A cell growth model for the ternary substrate system was established based on a simplified cell growth mechanism and subsequently modified by experimental results. Model analysis over a wide range of substrate concentrations shows that the inhibition of SG is much larger than phenol at low phenol concentrations (/=600 mg L(-1)). The nongrowth substrate, 4-cp, inhibits cell growth mainly through inactivation of cells (cell decay) and competitive inhibition to cell growth on phenol. In the absence of SG, 4-cp retards cell growth severely and cells cannot grow at 250 mg L(-1) 4-cp. Addition of sodium glutamate, however, greatly attenuates the toxicity of 4-cp and supports cell growth at 4-cp concentration higher than 250 mg L(-1). By using the proposed cell growth model, we were able to optimize the amount of SG needed to enhance cell growth rate and validate model predictions against experimental data. PMID- 10745213 TI - Influence of mixing and water addition on the removal rate of toluene vapors in a biofilter. AB - The effects of successive mixing (homogenization) of packing material (peat), with or without water addition, on the removal of toluene vapors in a biofilter were studied. Over a period of 50 days, an increase in the Elimination Capacity (EC) of approximately 240% was obtained by successive mixing and water additions. After each mixing, a high EC of toluene was maintained only for a short period of 3-4 days. After this time, decreased biofilter performance was observed, probably associated with the development of dried and/or clogged zones. In the long-term experiments, an attenuation of the EC recovery was observed after successive mixing. In this case, an increase of 110% over 4 months of experiment was obtained. The global reduction of EC over time could be explained by the colonization of the biofilter by filamentous fungi which was facilitated by the mixing of the packing material. The most frequently observed fungi were identified as Scedosporium sp. and Cladosporium sp. PMID- 10745214 TI - Modeling biofilm antimicrobial resistance. AB - A computer model capable of integrating mechanisms of biofilm resistance to disinfection by antimicrobial agents was developed. Resistance mechanisms considered included retarded penetration due to a stoichiometric reaction between the antimicrobial agent and biomass, incomplete penetration due to a catalytic reaction between the antimicrobial agent and the biomass, and the existence of a fraction of the cells in a resistant phenotypic state. Mathematical models of these processes were derived and solved in the computer simulation package MATLAB. Four sets of fitted experimental data on the disinfection of Pseudomonas aeruginosa biofilms were fit to each of the three models. No one model fit all of the data sets adequately. Killing of a 2-day old biofilm by tobramycin was best described by the physiological limitation model. Killing by hypochlorite was best described by the stoichiometric transport model. Killing by hydrogen peroxide was best simulated by the catalytic transport model. These results suggest that multiple mechanisms of biofilm reduced susceptibility are manifested even in biofilms of the same species and that the particular resistance mechanism depends on the biofilm age, antimicrobial agent, and biofilm thickness. The models presented in this article may be useful for diagnosing mechanisms of biofilm resistance from experimental data. PMID- 10745215 TI - Production of medium-chain-length polyhydroxyalkanoates by high-cell-density cultivation of Pseudomonas putida under phosphorus limitation. AB - High-cell-density fed-batch cultures of Pseudomonas putida were carried out for the production of medium-chain-length polyhydroxyalkanoates (PHAs) using oleic acid as a carbon source. By employing an optimal feeding strategy without the limitation of any nutrient, a high cell concentration of 173 g/L was achieved, but the PHA concentration and PHA content were only 32.3 g/L and 18.7 wt%, respectively. To increase the PHA concentration and content, phosphorus limitation was applied during fed-bath culture by reducing the initial KH(2)PO(4) concentration. When the initial KH(2)PO(4) concentration was reduced to 4 g/L, cell concentration, PHA concentration, and PHA content obtained in 38 h were 141 g/L, 72. 6 g/L, and 51.4 wt%, respectively, resulting in a high productivity of 1.91 g PHA/L per hour. PMID- 10745216 TI - Dynamic regulation of BDNF and NT-3 expression during visual system development. AB - Recent studies have proposed roles for neurotrophins in the formation and plasticity of ocular dominance columns as well as in the regulation of dendritic arborization in visual cortex of higher mammals. To assess potential roles for neurotrophins in these processes, we have examined the developmental expression of BDNF and NT-3 mRNA in the cat's visual system using in situ hybridization. BDNF and NT-3 mRNAs are dynamically regulated in many CNS structures during embryonic and postnatal development, and both mRNAs undergo striking developmental changes in laminar specificity and levels of expression within primary visual cortex during the critical period for ocular dominance column formation. Within visual cortex, BDNF mRNA is found in neurons in deep cortical layers (5 and 6) prior to eye opening, and in both deep and superficial layers (2 and 3) shortly afterwards. Within layer 4, the target of thalamocortical axons, BDNF mRNA is low initially and rises to high levels by the end of the critical period for ocular dominance column formation. NT-3 mRNA is first detectable in small stellate neurons at the base of layer 4 (4c) after eye opening, and levels decrease near the end of the critical period. BDNF and NT-3 mRNAs can be detected in the lateral geniculate nucleus at birth, and levels peak during the critical period. In both structures, BDNF mRNA expression is maintained into adulthood, while NT-3 is undetectable in the adult. The presence and dynamic regulation of these neurotrophins in visual structures is consistent with suggested roles for both of these neurotrophins in axonal and dendritic remodeling known to accompany the formation of ocular dominance columns. PMID- 10745217 TI - Neuroanatomical identification of mesencephalic premotor neurons coordinating eyelid with upgaze in the monkey and man. AB - Except during blinks, movements of the upper eyelid are tightly coupled to vertical eye movements. The premotor source for the coordination of lid and eye movements is unknown. The present paper provides the anatomical identification of a new premotor cell group in the rostral mesencephalon of the monkey and human, which lies in close proximity to the premotor center for vertical saccades and is thought to participate in lid-eye coordination. After injections of a retrograde transsynaptic tracer (tetanus toxin fragment C or BII(b)) into the levator palpebrae (LP), the superior rectus (SR), or the inferior oblique (IO) muscle of macaque monkeys, a small circumscribed group of premotor neurons was labeled in the central gray of the rostral mesencephalon, but not after superior oblique or inferior rectus muscle injections. This group lies immediately rostral to the interstitial nucleus of Cajal and medial to the rostral interstitial nucleus of the medial longitudinal fasciculus, each of which contain premotor neurons for vertical saccades, and was termed the M-group. Injections of tritiated leucine into the M-group led to afferent labeling primarily over LP motoneurons. In addition, label was present over the SR- and IO-motoneuron subgroups in the oculomotor nucleus and frontalis muscle motoneurons in the facial nucleus. This projection pattern of the M-group suggests a role in the coordination of the upper eyelid and eyes during upgaze. Double-labeling experiments in macaque monkeys revealed that the M-group is strongly parvalbumin immunoreactive and contains high levels of cytochrome oxidase activity. With these two histochemical markers, the homologue of the M-group was identified in the human brain as well. PMID- 10745218 TI - Dopamine D(3) receptor is selectively and transiently expressed in the developing whisker barrel cortex of the rat. AB - The rodent primary somatosensory cortex (SI) contains a map of the body surface, the most conspicuous part of which are "barrels," neuronal aggregates in layer IV that receive somatotopic projections from whiskers on the rodent's snout. We report that the D(3) dopamine receptor (D(3)R) is selectively and transiently expressed in SI during the first 2 weeks of postnatal development. D(3)R binding sites and mRNA overlap completely and are limited to layer IV of SI. D(3)R/mRNA are organized in a pattern corresponding to somatotopic representations of the body (e.g., whiskers, jaws, paws, etc.) with the highest expression in the barrel field. D(3) mRNA is first detected at postnatal day (P)4, increases rapidly until P7-10, and sharply decreases after P14. D(3)R binding sites are detectable at P6, peak at P14, and decline afterwards. D(1), D(2), D(4), or D(5) mRNAs display dissimilar expression pattern. D(1) mRNA is mostly confined to infragranular layers throughout the cortex. D(4) mRNA expression in layer IV rises by 4 weeks postnatal, when D(3)R expression is virtually undetectable. Quantitative analysis of D(3) mRNA expression demonstrates that the proportion of D(3) mRNA-positive cells decreases between P7 and P14, whereas mRNA concentration per cell remains stable. Moreover, D(3)R number continues to rise, whereas mRNA levels begin to decline. Thus, a process limiting D(3)R expression to fewer cells may occur that also induces changes in post-transcriptional regulation of D(3)R expression in remaining cells. These findings indicate that dopamine acting via D(3)R may play an important role in the development or function of the SI. PMID- 10745219 TI - Intrinsic connections in the caudal subdivision of the dorsolateral visual area (DL(C)) in squirrel monkeys. AB - Patterns of intrinsic connections and features of individual intrinsic axons in the caudal subdivision of the dorsolateral visual area (DL(C)) were investigated in four squirrel monkeys (Saimiri) following extracellular injections of the tracers biocytin, biotinylated dextran amine, and wheat germ agglutinin conjugated to horseradish peroxidase. Injections were defined in DL(C) by reference to architectonic borders and patterns of connections with other cortical areas. Intrinsic connections extended up to 6 mm from an injection and were usually anisotropic, extending farther dorsoventrally than anteroposteriorly. Injections that involved the supragranular layers produced up to 20 mainly supragranular patches of projections that had a width of 285 +/- 8 microm (mean +/- standard error) and area of 0.125 +/- 0.016 mm(2). Seventy-four intrinsic axon segments with a total of 3,290 boutons were drawn and their bouton spacing measured. The sample included axons in layers 1, 2-3, 5, and multiple (>2) layers; horizontally and vertically oriented axons; and axons in an injection halo, patch, or nonpatch region of projections. There were no differences in bouton spacing for axons in halo, patch, or nonpatch regions. Layer 1 axons (n = 7) had a significantly sparser distribution of boutons (median interbouton interval of 45.2 +/- 17.8 microm) than the layers 2-3 (n = 35) and layer 5 axons (n = 26), which did not differ in bouton spacing (median interbouton intervals of 8.1 +/- 0.4 microm and 8.4 +/- 0.8 microm, respectively). Patterns of intrinsic connections in DL(C) are related to neural organization and properties reported for DL or visual area V4, and are compared to intrinsic connections of other areas. PMID- 10745220 TI - Immunolocalization of sodium channel isoform NaCh6 in the nervous system. AB - Sodium channel 6 (NaCh6) is the alpha-subunit of a voltage-gated sodium channel expressed in the rat nervous system. The mRNA for this isoform has been shown to be expressed in both neuronal and glial cells by in situ hybridization. To examine localization of NaCh6 protein, polyclonal antibodies specific for NaCh6 were generated against peptides from two cytoplasmic domains and a fusion protein from an extracellular domain. Affinity-purified antibodies were used to localize NaCh6 in the brain, spinal cord, peripheral nervous system, and neuromuscular junction. There was widespread labeling of neurons in the brain and spinal cord. NaCh6 was present in both sensory and motor pathways. Radial glial cells in the cerebellum were intensely labeled for both GFAP and NaCh6. At the subcellular level, NaCh6 is found in axons, dendrites, and the cell body. Motor neurons and primary sensory neurons in dorsal root ganglia had strong cytoplasmic and axonal staining. Nodes of Ranvier in peripheral nerve and in the spinal cord were also intensely labeled. Motor neuron axons near the neuromuscular junction were labeled up to, but not including, terminal boutons. Dendrites of pyramidal cells in the cortex, hippocampus, and cerebellum were labeled. NaCh6 is the first NaCh subtype to be localized either at the node of Ranvier or to a dendrite. We conclude that NaCh6 is widely distributed in the central and peripheral nervous systems and is likely to be important for the electrical properties of the axon and dendrite. PMID- 10745221 TI - Developmental and regional expression of sodium channel isoform NaCh6 in the rat central nervous system. AB - The sodium channel isoform NaCh6 is abundant in the adult rat brain and is expressed in both neurons and glia (Schaller et al. [1995] J. Neurosci. 15:3231 3242; Krzemien et al. [2000] J. Comp. Neurol. 20:70-83). With reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunolabeling, NaCh6 expression was investigated in the developing rat brain and spinal cord [embryonic day 15 (E15) through postnatal day 28 (P28)]. The relative abundance of the four major central nervous system NaCh subtypes was quantitated with RT-PCR. In all regions that were investigated (olfactory bulb, cortex, hippocampus, cerebellum, and spinal cord), each subtype had a unique pattern of expression. NaCh6 mRNA and protein were not detected in either brain or spinal cord at E15 and E18 by in situ hybridization and immunohistochemistry. Neurons in the hippocampus, cortex, and olfactory bulb began to express NaCh6 mRNA and protein shortly after birth. The mRNA signal peaked at P7-P14, and protein expression increased as development proceeded. NaCh6 mRNA was detected at P1 in the cerebellum, and a nonuniform distribution of NaCh6 immunoreactivity in both Purkinje cells and granule cells was observed by P7-P14. NaCh6 protein was expressed in granule cells as soon as they left the proliferative phase and began to migrate. Both NaCh6 mRNA and protein were detected in the spinal cord at P1 and were expressed clearly at P7 in motor neurons. The time course of appearance of NaCh6 in postnatal development is consistent with the development of neurologic symptoms in med and jolting mice, which have mutations in the mouse ortholog of NaCh6. PMID- 10745222 TI - Synaptic localization of NMDA receptor subunits in the rat retina. AB - The distribution and synaptic clustering of N-methyl-D-aspartate (NMDA) receptors were studied in the rat retina by using subunit specific antisera. A punctate immunofluorescence was observed in the inner plexiform layer (IPL) for all subunits tested, and electron microscopy confirmed that the immunoreactive puncta represent labeling of receptors clustered at postsynaptic sites. Double labeling of sections revealed that NMDA receptor clusters within the IPL are composed of different subunit combinations: NR1/NR2A, NR1/NR2B, and in a small number of synapses NR1/NR2A/NR2B. The majority of NMDA receptor clusters were colocalized with the postsynaptic density proteins PSD-95, PSD-93, and SAP 102. Double labeling of the NMDA receptor subunit specific antisera with protein kinase C (PKC), a marker of rod bipolar cells, revealed very little colocalization at the rod bipolar cell axon terminal. This suggests that NMDA receptors are important in mediating neurotransmission within the cone bipolar cell pathways of the IPL. The postsynaptic neurons are a subset of amacrine cells and most ganglion cells. Usually only one of the two postsynaptic processes at the bipolar cell ribbon synapses expressed NMDA receptors. In the outer plexiform layer (OPL), punctate immunofluoresence was observed for the NR1C2; subunit, which was shown by electron microscopy to be localized presynaptically within both rod and cone photoreceptor terminals. PMID- 10745223 TI - Numbers of meynert and layer IVB cells in area V1: a stereologic analysis in young and aged macaque monkeys. AB - Visual impairments that are not related to optical changes are not uncommon during aging, and a number of psychophysical investigations have documented deficits in motion detection as well as in spatiotemporal contrast sensitivity in elderly people. However, little is known about the extent and nature of age related changes in neural structure and how they may affect visual function in aging. To address this question, the authors analyzed the effect of aging on two well-characterized neuronal populations in the primary visual cortex (area V1) of macaque monkeys. Four young adult (ages, 7-11 years) and four aged (ages, 26-32 years) rhesus monkeys were analyzed. The animals were perfused, and their brains were prepared for immunohistochemistry with an antibody to neurofilament protein. Unbiased stereologic estimates of the total numbers of neurofilament protein containing layer IVB cells and Meynert cells were obtained by using the optical fractionator method for the calcarine cortex and the opercular cortex separately. Stereologic estimates of the volume of these parts of area V1 also were calculated by using the Cavalieri principle. A considerable degree of interindividual variability in neuron numbers and cortical volume was observed among animals of both groups. However, there were no differences in either Meynert cell numbers or layer IVB cell numbers between the aged group and the young group. It is noteworthy that the oldest animal in the sample had the lowest numbers of Meynert cells, indicating that, despite the small size of the available sample, it is possible that some animals have a certain degree of neuronal loss in area V1 during aging. No change in the volume of area V1 was observed as a function of aging. These data suggest that the deficits that occur during aging in the visual system are not due to the loss of highly specific neocortical neuronal populations, such as those analyzed in this study. Rather, it is possible that more subtle alterations in the neurochemical characteristics or synaptic organization of the functional pathways subserving the different visual modalities are responsible for these deficits. PMID- 10745224 TI - Projection of the marginal shell of the anteroventral cochlear nucleus to olivocochlear neurons in the cat. AB - The marginal shell of the anteroventral cochlear nucleus is anatomically and physiologically different from its central core. Previous studies suggest that neurons in the marginal shell are well suited to encode the intensity of acoustic stimuli. To investigate the projections of the marginal shell, a focal injection (<100 nl) of a mixture of biotinylated dextran amine (BDA) and (3)H-leucine was made into the marginal shell of the cat combined with injection of cholera toxin subunit-B (CTB) into the cochleas. Following a 7-day survival, the cats were perfused. Axons and swellings labeled with BDA and olivocochlear neurons labeled with CTB were immunocytochemically stained black and brown, respectively. (3)H leucine labels were visualized by autoradiography. Labeled neural structures were examined via light microscopy. We found that swellings labeled with BDA, sometimes doubly labeled with BDA and (3)H-leucine, were in close apposition with dendrites and/or somata of olivocochlear neurons identified with CTB labeling. Double labeling with BDA and (3)H-leucine signifies that the label was anterogradely transported. The results support the conclusion that the anteroventral cochlear nucleus projects to medial olivocochlear neurons bilaterally and to lateral olivocochlear neurons ipsilaterally. Furthermore, the results are consistent with the interpretation that the marginal shell provides a source of the above-mentioned projections. Together with information in the literature, the present anatomical results support a hypothesis that the marginal shell provides information about stimulus intensity as a part of a reflex (or feedback gain control) system comprising the cochlea, cochlear neurons, cochlear nucleus, medial olivocochlear neurons, and cochlear outer hair cells. PMID- 10745225 TI - Hepatitis B virus surface (S) transactivator with DNA-binding properties. AB - Chronic infection with hepatitis B virus (HBV) in humans is strongly linked to the development of hepatocellular carcinoma (HCC). Activation of growth regulatory genes may play a crucial role in carcinogenesis. Proto-oncogene expression has been shown to be higher in HCC tissue with integrated HBV DNA than in the normal liver. Earlier, we showed that the 3' end of the HBV major surface gene (S) (426-855 nucleotides of the S region) is a transactivator of the X promoter-enhancer regulatory element in co-transfection experiments. This region expresses a truncated carboxy terminal S protein extending from amino acid residues 102 to 226. In this study, the truncated S protein (trc-S) was examined for its enhancing activity on several viral and cellular regulatory elements. The results indicate that trc-S activates rous sarcoma virus long terminal repeat (LTR), human T-lymphotropic virus 2 LTR, human immunodeficiency virus 1 LTR, and the c-jun and c-fos promoters. Electrophoretic mobility shift assays carried out to investigate its DNA-binding properties established that trc-S binds to HBV X promoter and oligonucleotides representing binding sites for the AP1 and TFIID transcription factors. The specificity of this interaction was confirmed by using competition experiments and supershift assays. These experiments suggest that trc S is a transactivator of several cellular and viral promoters and that this activity is mediated by direct interaction with DNA. PMID- 10745226 TI - Unusual hepatitis B surface antigen variation in a child immunised against hepatitis B. AB - Perinatal transmission of and infection with hepatitis B (HBV) in early childhood are observed in a small proportion of the offspring of hepatitis B surface antigen (HBsAg)-positive mothers who are vaccinated against HBV immediately after giving birth. The children may be infected by wild-type HBV or by variants with amino acid substitutions in the "a" determinant of HBsAg, particularly at position 145 and, rarely, at positions 120, 126, 129, 131, 141, and 144. Four hundred and forty-six newborn infants of HBsAg-positive mothers in the northeastern part of the Czech Republic received combined active and passive immunisation against HBV. Only one child became an HBsAg carrier. This followed a mild, acute HBV illness in the beginning of the second year of his life. HBV DNA encoding the "a" determinant and surrounding region of HBsAg was sequenced after amplification from the plasma of the child and his mother. The child was infected with variants of HBsAg with substitutions at residues 137 and 139. The virus of the mother had changes at residues 120 and 121. HBV from both child and mother had an unusual substitution at residue 118 and seemed to be of the ayw subdeterminant. PMID- 10745227 TI - Reduced transcription and progeny virus production of hepatitis B virus containing an 8-bp deletion in basic core promoter. AB - We have demonstrated previously the presence of an 8-bp deletion mutant, spanning from nt. 1768 to nt. 1775 in the basic core promoter region of hepatitis B virus (HBV) in patients with anti-HBe positive asymptomatic phase before developing acute exacerbation after immunosuppressive treatment. The transcription and progeny virus production activities of the mutant were examined by transfection of the recombinant plasmid [pUC Del(2)] containing the head-to-tail dimer DNA of the mutant into HepG2 cells. The amounts of hepatitis B surface antigen (HBsAg) and HBe antigens secreted into the culture medium were markedly reduced. Southern blotting of DNAs extracted from the culture medium also showed reduced mutant activity to produce progeny virus. Northern blotting and RNase protection assay of RNAs extracted from transfected cells demonstrated that the transcription of both precore mRNA and pregenome RNA was reduced significantly compared to that of wild-type HBV. The promoter activity examined by transfection of the CAT plasmid containing deletion mutant DNA was much lower than that of wild type. Co transfection experiments, however, of the CAT plasmid containing wild-type DNA with pUC Del(2) reduced CAT activity induced by wild-type, suggesting that truncated X protein produced by the mutant does not possess a sufficient transactivating activity. Gel shift assay using HepG2 nuclear extract and a probe containing four TA-rich regions in CP and various competitors suggested that the lack of the third TA-rich region was responsible for the transcription reduction of precore mRNA and pregenome RNA. The possible mechanisms are discussed. PMID- 10745228 TI - Properties of hepatitis B virus genome recovered from Vietnamese patients with fulminant hepatitis in comparison with those of acute hepatitis. AB - Among the many mutations found in the hepatitis B virus (HBV) genome, some have been associated with fulminant hepatitis, as exemplified by precore-defective mutations. The aim of this study was to determine whether such mutations also are found in Vietnamese cases of fulminant hepatitis B. The full-genome nucleotide sequence of HBV in three patients with fulminant hepatitis (F-2, F-3, and F-6) and one with acute hepatitis (A-3), who were admitted to Cho Ray Hospital, Ho Chi Minh City, Vietnam was ascertained. Additionally, two patients with fulminant hepatitis (F-1 and F-7) and three with acute hepatitis (A-1, A-2, and A-5) were examined only for the precore/core region of HBV. Remarkably, the nonsense mutation at precore codon 28 (Trp82Stop) was found in four of the five patients with fulminant hepatitis, while all the acute hepatitis patients harbored wild type (one had a mixture of wild and mutant types). The missense mutations within the core region, Ile97Leu and Pro130Ile/Thr/Ser, were also remarkable in fulminant hepatitis. Only F-2 was free from these precore/core mutations, but F-2 was unique in that it possessed a chimeric genotype: it could be classified into genotype C as a whole, but its X region was of genotype B, like the other four fulminant hepatitis isolates (F-1, F-3, F-6, and F-7). The codon 41 of the X protein was Pro in all three fulminant hepatitis cases examined for this region, while it was Ser in the wild-type isolates of genotype B. Of note as negative data, the mutations C1653T and T1753M of the enhancer II (Enh II) and A1762T and G1764A of the precore/core promoter regions, once reported to be relevant to severe or fulminant hepatitis, were not found in the present cases. The results with the Vietnamese cases of fulminant hepatitis corroborated results of previous studies with respect to the mutations Trp28Stop of precore and Ile97Leu and Pro130Ile/Thr/Ser of core, but not for the mutations within Enh II and precore/core promoter region. Whether the Ser41Pro mutation in the X region of genotype B HBV is Vietnam-specific or disease-specific deserves further investigation. PMID- 10745229 TI - Lack of clinical evidence for involvement of hepatitis C virus interferon-alpha sensitivity-determining region variability in RNA-dependent protein kinase mediated cellular antiviral responses. AB - The hepatitis C virus (HCV) interferon-alpha (IFN-alpha) sensitivity-determining region (ISDR) has been shown to suppress double-stranded RNA-dependent protein kinase (PKR) activity in vitro in a yeast PKR expression system. Since variability of ISDR was shown to correlate with nonresponsiveness to IFN-alpha therapy in chronically HCV-infected patients, it has been suggested that prototype ISDR might be a viral inhibitor of cellular PKR. The present study evaluates the biological significance of ISDR variability in situ, relating it to PKR-mediated cellular antiviral responses within the liver. ISDR variability was determined in patients chronically infected with HCV genotypes 1a, 1b, and 3a by direct sequencing using liver-derived RNA preparations as starting material. As surrogate parameters for PKR-mediated cellular responses, hepatic endogenous IFN alpha gene expression as well as MxA expression were analysed by a competitive, quantitative reverse transcription-polymerase chain reaction technique. Irrespectively of intra- or intergenotypic ISDR amino acid substitutions, ISDR variability was found not to correlate with endogenous hepatic IFN-alpha or with hepatic MxA gene expression. The data suggest that at least two prominent PKR mediated cellular responses might be largely unaffected by HCV ISDR variability. PMID- 10745230 TI - Antibody-free virion titer greatly differs between hepatitis C virus genotypes. AB - Hepatitis C virus (HCV) virions have been shown to be bound to antibodies in patients with chronic HCV infection. The sera from patients infected with genotype 1b HCV contained more antibody-free virions than those from patients with genotype 2a/2b HCV. When compared at the same levels of serum HCV RNA, free virion titers of genotype 2a/2b-infected patients were much lower than those of genotype 1b-infected patients, indicating that a larger fraction of HCV virions are bound to antibodies in the former than in the latter. The gene segments encoding the hypervariable region (HVR) 1, a principal neutralization epitope, of HCV were amplified from the patients' sera by reverse transcription-polymerase chain reaction. The majority of genotype 2a/2b-infected patients had very similar HVR 1 sequences to one another, whereas patients infected with genotype 1b HCV had highly heterogeneous sequences. Differences in the amount of antibody-free virion and HVR1 sequence variability between genotypes may have an implication in HCV pathogenesis. PMID- 10745231 TI - Hepatitis C virus infection in French hemodialysis units: a multicenter study. AB - The aims of the study were: (i) to evaluate the prevalence of hepatitis C virus (HCV) antibodies (third generation tests) and RNA (standardized ultrasensitive RT PCR assay) in a large cohort of hemodialysis patients, and (ii) to correlate HCV markers with bioclinical features and alanine-aminotransferase (ALT) activity. Antibodies were assayed by two methods in 1,323 patients (60% men, median age 65 years, median hemodialysis duration 3 years) attending 25 French hemodialysis centers including 9 self-care units. RNA was assayed using the Cobas Amplicor 2.0 method in pooled samples from 10 anti-HCV(-/-) patients and on individual samples from the other patients. Of the 16.3% patients (range 0-44%) tested (+/+) for HCV antibodies (anti-HCV), 2.3% tested (+/-) and 81.4% tested (-/-). 70% of the anti HCV(+/+) patients and 3% of the HCV(+/-) patients were RNA(+). Pooled analysis revealed that 5/1077 anti-HCV(-/-) patients (0.5%) were RNA(+); all 5 displayed subsequently an increase in ALT and became anti-HCV(+/+). Mean ALT was higher (multiple of normal) in anti-HCV(+/+) RNA(+) patients than in anti-HCV(+/+) RNA( ) patients (0.46 +/- 0.08 vs. 0.22 +/- 0.07, P < 0.0001) and similar in all the RNA(-) patients, whatever their HCV antibody status. Multivariate analysis demonstrated that HCV status was linked to hemodialysis duration, previous kidney transplantation and positive anti-HBc. To summarize, the determination of the RNA status of anti-HCV(+/-) patients may have clinical relevance if a policy of isolation is contemplated. Standardized ultrasensitive RT-PCR assay combined with a pooling strategy is a promising method for use in epidemiological studies. PMID- 10745232 TI - Evidence that the GBV-C/hepatitis G virus is primarily a lymphotropic virus. AB - GB virus-C and the hepatitis G virus (GBV-C/HGV) are variants of the same positive sense RNA flavivirus, initially thought to be associated with hepatitis. The tissue tropism of GBV-C/HGV in normal subjects has not been evaluated to date using an extended tissue spectrum. Therefore, the sites of GBV-C/HGV replication were investigated in serum and twenty-three tissues collected during post-mortem examination of four apparently healthy individuals who died accidental deaths, who were infected with GBV-C/HGV. All were anti-HIV and anti-HCV negative and three out of four were HBsAg negative. Tissues were collected carefully to prevent cross contamination. A highly strand-specific RT-PCR assay was employed for the detection of either GBV-C/HGV positive strand RNA (virion) or negative strand RNA (replicative intermediary). Strand specificity of the RT-PCR assay was assessed with synthetic positive-and negative strand GBV-C/HGV RNA generated from a plasmid, using T7 and T3 RNA polymerases. The spleen and bone marrow biopsies were found to be uniformly positive for both negative-and positive strand GBV C/HGV RNA. In addition, one cadaver was positive for both RNA strands in the kidney, and another positive for both in the liver. No negative strand RNA was detected in the following: brain, muscle, heart, thyroid, salivary gland, tonsil, lung, lymph nodes, gall bladder, pancreas, oesophagus, stomach, small bowel, large bowel, adrenal gland, gonad, aorta, skin and cartilage. This preliminary study concludes that GBV-C/HGV is a lymphotropic virus that replicates primarily in the spleen and bone marrow. PMID- 10745233 TI - GBV-C/hepatitis G virus in acute nonA-E hepatitis and in acute hepatitis of defined aetiology in Italy. AB - The role of GBV-C/HGV in the aetiology of acute non A-E hepatitis and its impact on the course of acute hepatitis of defined aetiology were investigated by detecting viral RNA by RT-PCR and antibody to the E2 protein of GB virus C (anti E2) by EIA. Ninety-eight patients with acute nonA-E hepatitis, 35 patients with acute hepatitis A, 63 with acute hepatitis B, 29 with acute hepatitis C and 270 controls were enrolled in this study. The prevalence of GBV-C/HGV RNA was similar among patients with acute nonA-E hepatitis (3.1%), with acute hepatitis A (2.9%), and controls (3.7%), but significantly higher (P < 0.05) among those with hepatitis B or C (19.0% and 48.3%, respectively). Similar figures were obtained considering the total rate of GBV-C/HGV exposure (viral RNA or anti-E2 positivity). The majority (24/30 or 80%) of GBV-C/HGV RNA positive patients reported a parenteral source of exposure whereas the remaining 20% denied having known risk factors. The liver function test values and the rate of chronic hepatitis B and C were similar in patients co-infected and in those not co infected with GBV-C/HGV. This study excludes a significant role of GBV-C/HGV infection in the aetiology of acute nonA-E hepatitis in Italy. Concomitant GBV C/HGV and HBV or HCV infection does not worsen the clinical course of illness among patients with acute hepatitis. PMID- 10745234 TI - Human papillomavirus (HPV) DNA in penile carcinomas in Argentina: analysis of primary tumors and lymph nodes. AB - Among sexually transmitted diseases, infection by human papillomavirus (HPV) has become one of the most important. On the other hand, though epidemiological data show that some HPV types are closely associated with cervical cancer, few reports have been found with reference to penile carcinoma because of its rare occurrence. The aim of this study was to investigate the relationship between HPV infection and penile cancer in Argentina. A retrospective study was carried out on 38 white men with penile squamous-cell carcinoma. Sixty-five archival fixed biopsies taken from 34 primary penile tumors, 25 nodal metastases, 1 skin "satellite" metastasis and 5 histologically normal lymph nodes were used as specimens. HPV detection and typing were carried out by the polymerase chain reaction (PCR) using generic primers, combined with single-stranded conformational polymorphism (SSCP) analysis. HPV DNA was found in 71% patients, corresponding 81% of them to "high risk" types, with predominance of HPV 18. Both primary tumors and metastases showed concordance of HPV occurrence and type in both lesions. In 3 patients, HPV 16 was detected not only in primary tumors and metastases, but also in histologically normal lymph nodes. Our data indicate that most penile carcinomas in Argentine patients are etiologically related to HPV, especially to "high risk" genital types. The agreement in HPV detection between primary tumors and metastases suggests a potential viral role in tumor progression. HPV detection in otherwise histologically normal lymph nodes might be useful as early marker of a metastatic process. PMID- 10745235 TI - High prevalence of human papillomavirus type 16 infection among children. AB - Infection with high-risk human papillomaviruses (HPV), is the most significant risk factor for cervical cancer and it may be possible to prevent this malignancy by immunisation. Before immunisation programmes can be designed, however, it is necessary to know the age of acquisition and all routes of infection for these viruses. Sexual transmission is well documented and vertical transmission has also been demonstrated, although the frequency of transmission remains controversial. We previously showed that vertical transmission frequently results in persistent infection, and now present data on the prevalence of HPV-16 DNA (the most prevalent high-risk HPV type) in healthy children. Buccal samples from 267 healthy children aged 3-11 years were tested for HPV DNA by generic PCR (MY09/MY11), and a HPV-16 specific nested PCR. Reverse transcriptase (RT)-PCR was used to determine the prevalence of transcriptionally active HPV-16 infection in a subset of children. HPV-16 DNA was detected by nested PCR in 138 of 267 (51.7%) samples, whereas HPV DNA was detected in only 45 (16.8%) specimens by generic PCR, that has a lower analytical sensitivity. There were no significant differences in prevalence according to age or sex. Early region mRNA was detected by RT-PCR in six (11.3%) of 53 HPV-16 E5 DNA positive samples. HPV-16 E5 DNA sequences from 10 children confirmed the identity of the sequences detected and identified 13 HPV-16 variants. PMID- 10745237 TI - Estimation of incidence of respiratory syncytial virus infection in schoolchildren using salivary antibodies. AB - An assay for respiratory syncytial virus (RSV)-specific IgG in saliva is described. The assay was used to examine the incidence of RSV infection in schoolchildren 7-10 years old during one RSV season. One hundred and twenty-one volunteer children provided saliva samples in October 1997 and March 1998; 18% of the children showed a fourfold or greater rise in anti-RSV IgG in the second sample. This prevalence of antibody increase is similar to that described in previous studies that measured CFT levels in serum samples. Overall, the children who showed rises in antibody levels, indicating that they had experienced an RSV infection, had lower levels of RSV-specific antibody in their preseason samples than those who showed no increase (P = 0.0018). These results show that saliva is an adequate substitute for serum in some antibody tests and may be useful for community studies. Such studies may provide surrogate markers for susceptibility to infection, which should benefit the planning of vaccination strategies. PMID- 10745236 TI - Clinical and epidemiologic characteristics of respiratory syncytial virus subgroups A and B infections in Santa Fe, Argentina. AB - Respiratory Syncytial Virus (RSV) has two major antigenic groups, A and B. The implications of these variants in the epidemiology and pathogenesis of RSV infection are not well defined. This study was undertaken to compare the two RSV subgroups in patients admitted to hospital. Clinical and epidemiologic features of RSV subgroups in children under 30 months of age with proven RSV acute lower respiratory infections were examined during 4 winters from 1993 to 1996 in Santa Fe, Argentina. RSV typing was carried out with monoclonal antibodies in nasopharyngeal cells by indirect immunofluorescence. Of the 177 RSV positive nasopharyngeal aspirates obtained from 1993 to 1996, 85 (48%) were available for typing. Seventy-three (85.9%) specimens were identified as Subgroup A and 12 (14.1%) as Subgroup B. Except in 1993, in which only Subgroup A was detected, both variants circulated throughout the epidemic season. Subgroup A infections produced more severe disease than Subgroup B infections, as assessed by the length of the hospital stay and the use of respiratory support. This difference was age related, being evident in infants 0-6 months old. Patients with Subgroup B infections were also significantly less frequently breast-fed (95% vs. 75% for A and B subgroups, respectively; P = 0.04). It is concluded that the severity of disease in Argentinian patients admitted with acute RSV infections may be associated with Subgroup A strains as determined by a serogrouping method. PMID- 10745238 TI - Efficacy of influenza vaccination in adult liver transplant recipients. AB - To assess the efficacy of influenza vaccination in immunocompromised adult liver transplant (LTx) recipients, the serum antibody responses of 61 of these patients and 35 liver cirrhosis patients with those of 45 of their healthy spouses were compared, after one and two vaccinations with a commercial trivalent subunit influenza vaccine. In addition, virus-specific proliferative T-cell responses were measured in LTx recipients and their healthy spouses. In all three study groups, significant rises in geometric mean antibody titers were observed for all three antigens after one vaccination. These titers did not continue to increase significantly after the second vaccination in patients with cirrhosis and control subjects but did rise for LTx recipients. The overall antibody response to all three influenza virus strains proved to be significantly lower in the LTx recipients than in the group of healthy subjects after both one and two vaccinations. More than 68% of the LTx recipients developed hemagglutination inhibiting serum antibody titers >/=40 against all three vaccine strains after the first vaccination and more than 80% after the second vaccination. These findings correlated with the T-cell responses determined for the group of LTx recipients and healthy control individuals. Testing of the respective serum samples against influenza virus A/Sydney/5/97, which circulated in the 1997-1998 influenza season and showed a considerable mismatch with the vaccine strain A/Nanchang/933/95, indicated that such a mismatch may have significant consequences for vaccine efficacy, especially for LTx recipients. Collectively the data show that LTx recipients can be vaccinated effectively against influenza despite immunosuppressive therapy. A two-dose vaccination regimen improved vaccination efficacy in LTx recipients. Whether transplant patients generally benefit from a two-dose vaccination regimen should be evaluated further. PMID- 10745239 TI - Mismatch between the 1997/1998 influenza vaccine and the major epidemic A(H3N2) virus strain as the cause of an inadequate vaccine-induced antibody response to this strain in the elderly. AB - The success of influenza vaccination depends largely on the antigenic match between the influenza vaccine strains and the virus strains actually circulating during the season. In the past, this match has proved to be satisfactory in most seasons. In the 1997/1998 season, however, hemagglutination inhibition (HI) assays with ferret antisera indicated a considerable mismatch between the H3N2 vaccine component and the most prevalent epidemic influenza A(H3N2) virus. The results from antigenic analyses using pre- and postvaccination serum samples from volunteers of various ages, including residents of nursing homes who were more than 60 years of age, were in good agreement with the results obtained with ferret antisera. Homologous serum antibody responses to the H3N2 vaccine component as well as the cross-reactivity of the induced antibodies to the epidemic H3N2 strain, declined with increasing age of the vaccinees. As a consequence of these two effects, 84% of the vaccinees over 75 years of age did not develop HI antibody titers >/= 40 against the major H3N2 virus variant of 1997/1998, suggesting that they were not protected against infection with this virus variant. These findings support the current policy of the World Health Organization (WHO), which is to base worldwide influenza virus surveillance on results predominantly obtained by antigenic analyses of influenza virus isolates with ferret antisera in HI tests. If an antigenic mismatch is observed, the protective efficacy of the vaccine, especially for the elderly, may be insufficient. The observations also support the current policy to include the elderly in serologic efficacy trials. PMID- 10745240 TI - Local and systemic immune response in community-dwelling elderly after intranasal or intramuscular immunization with inactivated influenza vaccine. AB - Intramuscular (IM) influenza vaccines are about 50% effective in preventing clinical illness among the elderly and their effectiveness in eliciting mucosal response may be even lower. The aim of the present study was to evaluate the immunological effect of a novel inactivated intranasal (IN) trivalent whole influenza virus vaccine among community-dwelling elderly. Sixty-one subjects were vaccinated with two doses of an IN vaccine and a control group of 31 subjects was vaccinated with a commercial IM vaccine. Viral strains in the 1997/8 vaccine used were A/Nanchang/933/95(H3N2), A/Johannesburg/82/96(H1N1) and B/Harbin/7/94. Serum IgG and nasal IgA were determined by HI and ELISA, respectively. Only a few minor local adverse events were reported after vaccination. Seroconversion for the three antigens tested was higher after IM vaccination, although not statistically significant. Local antibody response to the three antigens tested was detected in 50-53% and 19-26% of IN and IM immunized subjects, respectively. The IN vaccine tested was significantly more effective than the IM vaccine in inducing mucosal IgA response. This may prevent influenza at its early stages and thus contribute to the reduction of complications in the elderly. PMID- 10745241 TI - Hydrogels containing monocaprin prevent intravaginal and intracutaneous infections with HSV-2 in mice: impact on the search for vaginal microbicides. AB - Hydrogel formulations containing the 1-monoglyceride of capric acid (monocaprin) possess potent in vitro microbicidal activity against HIV and HSV, Chlamydia trachomatis and Neisseria gonorrhoeae. These formulations were studied to determine whether they prevent intracutaneous and intravaginal infections of mice with HSV-2, a virus that is in vitro as sensitive to the virucidal action of the compound as is HIV. In mice intravaginal infection with HSV-2 and the associated mortality was prevented completely when the infection was carried out in the presence of a 20 mM monocaprin containing gel formulation. Similarly, virtually complete protection of lesion development and associated mortality was observed when mice were infected intracutaneously with HSV-2 in the presence of gels containing 10 or 20 mM monocaprin. No irritation or toxicity was observed following application of the gel to the skin or the vaginal mucosa. Hydrogel formulations of monocaprin could thus be pursued as vaginal microbicides for the prevention of sexual transmission of HSV, HIV and other infectious pathogens. PMID- 10745242 TI - Human immunodeficiency virus type 2 (HIV-2) in Portugal: clinical spectrum, circulating subtypes, virus isolation, and plasma viral load. AB - The human immunodeficiency virus type 2 (HIV-2) is responsible for 4. 5% of AIDS cases in Portugal. Six HIV-2 subtypes have been described so far, subtype A being proposed as more pathogenic than the rest. The relationship between the clinical status and levels of both cellular and plasma HIV-2 viraemia is not well known, nor their modifications under antiretroviral therapy. Thirty-two consecutive HIV 2 infected persons (17 men, 15 women) attending two different hospitals in Lisbon in 1997 were enrolled prospectively in the study. All but 4 individuals most likely acquired the infection through heterosexual contact. More than half of the study population was of African origin, mainly from Guinea-Bissau. Eleven (34.4%) patients had developed clinical manifestations included within the B or C groups of the CDC classification system for HIV infection, with the rest being asymptomatic. Half of the population was undergoing antiretroviral treatment at the time of the study. HIV-2 subtypes were investigated using a new Nef-based restriction fragment length polymorphism (RFLP) method that allows differentiation of the main two variants, A and B. Plasma viral load was quantified using a new quantitative competitive reverse transcriptase polymerase chain reaction (QcRT-PCR) procedure as well as the Amp-RT assay. Virus isolation was attempted from peripheral blood mononuclear cells. All but one person carried HIV-2 subtype A. Plasma viraemia examined by QcRT-PCR was measurable in 15 (50%) of 30 subjects, yielding in all instances values below 20,000 HIV-2 RNA copies per ml. Plasma RT activity could be detected in only 10 (33%) of 30 subjects, a rate much lower than that seen in HIV-1 infection. Virus was isolated from 16 (53.3%) of 30 patients. A significant correlation was found between CD4+ counts, clinical status, rate of virus isolation, and plasma viral load by both QcRT-PCR and Amp-RT. In conclusion, HIV-2 subtype A is the predominant variant circulating in Portugal among both natives and immigrants. A lower cellular and plasma viral load with respect to HIV-1 was seen in persons without immunosuppression, from whom the rate of virus recovery was extremely low. PMID- 10745243 TI - Seroprevalence and seroincidence of Norwalk-like virus infection among Brazilian infants and children. AB - To determine the importance of Norwalk-like viruses (NLVs) as pediatric pathogens in a developing country, the seroprevalence and seroincidence of this group of viruses in a cohort of children less than 4 years of age in an urban shantytown in northeastern Brazil was examined. Serum samples were collected approximately every 6 months from 135 children who were surveyed three times each week for diarrhea and vomiting. NLV IgG was measured by an enzyme immunosorbent assay (EIA) with recombinant Norwalk virus capsid protein. Overall NLV seroprevalence was 71%, and the overall NLV seroconversion rate was 0.7 seroconversions per child-year. The highest age-specific NLV seroconversion rate (0.8 seroconversions per child-year) was observed in the 13-24-month age group. For all study children, the incidence of diarrhea and vomiting was significantly greater (P < 0.01) during time periods spanned by serum pairs that indicated NLV seroconversion compared with time periods without NLV seroconversion. However, NLV seroconversion was not associated with gastrointestinal symptoms during the first year of life. PMID- 10745244 TI - Genomic characterization of human astrovirus type 6 Katano virus and the establishment of a rapid and effective reverse transcription-polymerase chain reaction to detect all serotypes of human astrovirus. AB - We previously reported that human astrovirus type 6 (HAstV T6) was the etiologic agent of a large-scale outbreak of acute gastroenteritis that occurred in 1991 in Katano City, Osaka, Japan [Oishi et al., 1994]. The two representative strains, Katano virus K23 and K24, have been analyzed by sequencing the open reading frame 2 (ORF2) region after amplification by reverse transcription-polymerase chain reaction (RT-PCR). The ORF2 region of HAstV T6 strains, including K23, was found to be about 20 bp smaller than those of other types. There was 94% nucleotide sequence identity and 95% amino acid sequence identity between K23 and K24, with the Oxford strains belonging to HAstV T6. The high homology of the ORF2 region between the Katano and Oxford strains shows intratype genomic stability, irrespective of time and place of virus isolation. Comparing sequences of ORF2 of different HAstV serotypes, we established a rapid and highly sensitive detection system for HAstV types using RT-PCR with the AC230/AC1' primer set designed from the 5'-terminal end region of ORF2. This RT-PCR system seems very useful in detecting at least two different viruses in a single PCR test tube using AC230/AC1' in addition to the NV81/82, SM82 primer sets. Thus, our rapid and effective detection system may contribute to the epidemiologic characterization of astrovirus infections as well as Norwalk-like viruses. PMID- 10745245 TI - A diagnostic EIA for detection of the prevalent SRSV strain in United Kingdom outbreaks of gastroenteritis. AB - Small round structured viruses (SRSVs) are the major cause of outbreaks of gastroenteritis in the UK. Diagnosis is problematic due to insensitive electron microscopy (EM) or technically demanding reverse transcription polymerase chain reaction (RT-PCR) techniques. We have studied outbreaks of non-bacterial gastroenteritis using an EIA based upon recombinant capsid protein from the currently prevalent circulating strain of SRSV (Lordsdale Genotype II) and compared its performance against EM and RT-PCR assays. Faecal specimens sent to the Bristol Public Health Laboratory for outbreak investigation from December 1996 to December 1997 were applied retrospectively to the SRSV EIA and results compared with the routine EM and RT-PCR that had been carried out prospectively. Overall, the three tests identified SRSVs in specimens from 70% of the outbreaks (213/305) investigated. Of the 213 total positive outbreaks, the EIA identified 71%, that compared favourably with EM (63%) and RT-PCR (84%). The Lordsdale Genotype II SRSV EIA provides a simple cost-effective assay that will for the first time make detection of currently circulating SRSV strains associated with UK outbreaks available to all routine laboratories. The EIA format makes the assay widely applicable to non-specialist laboratories, unlike the RT-PCR assay, and the improved sensitivity over EM will allow successful screening of UK outbreaks alongside commercial EIAs currently available for adenovirus, astrovirus and rotavirus. Furthermore, the assay will allow rapid identification of emerging SRSV strains. PMID- 10745246 TI - High prevalence of renal transplant recipients infected with more than one cytomegalovirus glycoprotein B genotype. AB - A prospective analysis of cytomegalovirus (CMV) glycoprotein B (gB) genotypes was conducted on 34 renal transplant recipients using peripheral blood leukocytes (PBLs) and urine specimens. The CMV gB genotypes were analyzed by polymerase chain reaction (PCR) followed by enzyme digestion. PBLs and urine samples showed almost equal proportions of the 4 known gB genotypes, as well as equal proportions of gB genotype mixtures. The gB genotypes 1, 2 and 3 were equally distributed in the patients. Twenty-four (70.6%) patients had more than one gB genotype during follow-up. There was no association of gB genotypes with the development of symptomatic CMV infection. PMID- 10745247 TI - Genetic characterization of adenovirus strains isolated from patients with acute conjunctivitis in the city of Sao Paulo, Brazil. AB - Genome analysis was carried out on adenovirus strains isolated from patients with acute follicular conjunctivitis in the city of Sao Paulo, Brazil. Eighteen conjunctival scrapings, collected between December 1993 and March 1994, were analyzed by two methods: a combination of polymerase chain reaction with restriction fragment length polymorphism and viral DNA restriction analysis, carried out using 10 restriction endonucleases: BamHI, BglI, BglII, HindIII, KpnI, SacI, SalI, SmaI, XbaI, and XhoI. Among 11 adenovirus detected by cell culture isolation, nine were Ad8, and two were Ad7. By restriction analysis the Ad8 isolates were typed as two new variants-Ad8/D11 (seven of nine samples) and Ad8/D12 (two of nine samples). Ad7 isolates were identified as a subtype of the widespread genome type Ad7b and the virulent type Ad7h, a predominant genome type circulating in Argentina, Chile, and Uruguay but absent in Brazil until 1991. PMID- 10745248 TI - Characterisation of rotaviruses from children treated at a London hospital during 1996: emergence of strains G9P2A[6] and G3P2A[6]. AB - Rotavirus strains from 171 patients treated in 1996 at a children's hospital in London were characterised. Use of a panel of typing monoclonal antibodies for serotypes G1-4 identified 105 (61%) of the strains. The majority, 90 strains (86%), were serotype G1. Characterisation of G (VP7) and P (VP4) types using reverse transcription-polymerase chain reaction was more efficient, and 167 of 171 (98%) of the strains were identified this way. The predominant strains were G1P1A[8] (55%) and G4P1A[8] (17%), which are prevalent throughout the world; however, a significant number of cases were associated with genotypes not recorded previously in the United Kingdom. There were 21 (13%) cases associated with G9P2A[6] and 11 (6%) cases associated with G3P2A[6]. The majority (seven of 10) cases of infection in children older than 3 years of age were caused by these two genotypes. A majority (15/21) of G9P2A[6] strains were recovered from children admitted to the hospital, and five children were sufficiently dehydrated to necessitate intravenous rehydration. PMID- 10745249 TI - Persistent fetal rubella vaccine virus infection following inadvertent vaccination during early pregnancy. AB - Inadvertent immunisation of seronegative women with RA27/3 rubella virus live attenuated vaccine several weeks before and after conception is described. Whereas in 5 cases the vaccine virus was not transmitted vertically, in 1 case vaccination led to the development of persistent fetal infection with prolonged virus shedding for more than 8 months. Sequence analysis carried out on isolates from amniotic fluid, from cord blood leukocytes as well as from infantile urine confirmed an infection by the vaccine strain. At birth, the newborn infant exhibited none of the symptoms compatible with the congenital rubella syndrome and signs indicative for development of late onset disease are not apparent. This observation constitutes the first unequivocal documented case of rubella vaccine virus related to persistent fetal infection. PMID- 10745250 TI - Experimental infection of nonenveloped DNA virus (TTV) in rhesus monkey. AB - Virus fragments homologous to TTV were detected previously from an enterically transmitted outbreak of non-A-E hepatitis [Luo et al., 1999]. To test the susceptibility of the Rhesus monkey to this virus and to establish its transmission routes, 6 Rhesus monkeys were inoculated, 3 orally and another 3 intravenously. The inoculum was prepared by extracting and filtering feces collected from a patient during the incubation period identified in the described outbreak. A second group of 3 monkeys was used for the passage study. The feces and blood samples were collected for detection of the virus by polymerase chain reaction (PCR). Four animals were subjected to liver biopsies and bile aspiration by open surgery for in situ virus detection. Viremia occurred in 4-7 days after intravenous and 7-10 days after oral inoculation. The virus was excreted in feces a few days after oral infection and simultaneously with viremia after intravenous inoculation. The virus was also detected in bile during the viremic phase. There was a prolonged carrier state with persistent viremia and virus excretion in feces for more than 6 months. Serum transaminase levels were not raised during the infection. The virus was present in both the cytoplasm and nuclei of hepatocytes, but no significant pathology was found. Therefore, the Rhesus monkey is susceptible to TT virus infection, but the virus seems nonpathogenic. Infection of the liver may be established either by oral or parenteral inoculation. The virus may be released from liver into the blood or via bile into feces, so it may be transmitted by both blood and fecal routes. PMID- 10745251 TI - Indirect evidence of TTV replication in bone marrow cells, but not in hepatocytes, of a subacute hepatitis/aplastic anemia patient. AB - The presence of a new DNA virus (TTV) has been reported in sera from patients with posttransfusion hepatitis of unknown etiology. The precise replication site of TTV, however, has not been established. In this study, the presence of TTV in liver autopsy material, and in bone marrow biopsy and autopsy samples taken from a subacute hepatitis/aplastic anemia patient was determined by PCR and Southern blot analyses. Liver cells were found to contain only TTV DNA and not mRNA. Bone marrow material, especially that taken at biopsy, contained high levels of TTV DNA. It is suggested that the TTV replication site was in the bone marrow rather than in the liver, and that TTV infection was the cause of this patient's aplastic anemia. The precise etiological association of TTV with hepatitis remains to be established. PMID- 10745252 TI - Chronic graft-versus-host disease: is there an alternative to the conventional treatment? AB - Despite conventional and new therapies for the treatment of chronic GVHD (cGVHD), this syndrome continues to account for significant morbidity and mortality after allogeneic hematopoietic stem cell transplantation. With the expanded use of allogeneic peripheral blood stem cell transplantation, matched unrelated as well as mismatched related donors there is an increased incidence of cGVHD that poses a new clinical challenge. Over the past 10 years some new agents have been used, particularly, as a salvage therapy for the treatment of cGVHD. Many of the new agents discussed in this paper may have a role in the future as a therapy for cGVHD. Randomized clinical trials must be performed earlier in the course of cGVHD to establish the efficacy of these new drugs. PMID- 10745253 TI - A phase I dose escalation study of high-dose thiotepa, melphalan and carboplatin (TMCb) followed by autologous peripheral blood stem cell transplantation (PBSCT) in patients with solid tumors and hematologic malignancies. AB - The purpose of this study was to determine the maximum tolerated dose of carboplatin administered with 500 mg/m2 thiotepa and 100 mg/m2 melphalan followed by autologous peripheral blood stem cell (PBSC) infusion in patients with refractory malignancies. Twenty-eight patients with refractory malignancies received high-dose thiotepa (500 mg/m2, melphalan (100 mg/m2) and escalating doses of carboplatin 900-1500 mg/m2) followed by infusion of cryopreserved autologous PBSCs. The maximum tolerated doses were determined to be 500 mg/m2 thiotepa, 100 mg/m2 melphalan and 1350 mg/m2 carboplatin. Two consecutive patients receiving 1500 mg/m2 carboplatin experienced grade 3 mucositis and colitis. Ten patients were enrolled at the maximum tolerated dose and none had grade 3-4 regimen-related toxicity and mortality. All patients at this level experienced grade 1-2 mucositis, 90% grade 1-2 gastrointestinal toxicity, 30% grade 1-2 cardiac and renal toxicity, and 10% experienced grade 1 hepatic toxicity. The median time to achieve a granulocyte count of 0.5x10(9)/l was 9 days (range 7-12 days) and platelet count of 20x10(9)/l was 10 days (range 7-15 days). Of eight patients with stage IV refractory breast cancer, even were evaluable for response, one patient on day 75 will be evaluated soon. Five of seven (71.5%) evaluable patients achieved a complete remission (CR) and two had no response. Of seven patients with non-Hodgkin's lymphoma (n = 4) or Hodgkin's disease (n = 3), five achieved a CR (71.5%). Thiotepa, melphalan and carboplatin can be administered in high doses with tolerable mucositis as the major side effect. This combination has significant activity in patients with breast cancer, and phase II studies in patients with breast cancer and other chemotherapy sensitive malignancies are warranted. PMID- 10745254 TI - Hematological recovery and peripheral blood progenitor cell mobilization after induction chemotherapy and GM-CSF plus G-CSF in breast cancer. AB - In order to determine the effect of GM-CSF plus G-CSF in combination in breast cancer patients receiving an effective induction regimen, we compared hematological recovery and peripheral blood progenitor cell (PBPC) mobilization according to colony-stimulating factor (CSF) support. Forty-three breast cancer patients were treated by TNCF (THP-doxorubicin, vinorelbine, cyclophosphamide, fluorouracil, D1 to D4) with CSF support: 11 patients received GM-CSF (D5 to D14); 16 patients G-CSF (D5 to D14) and 16 patients GM-CSF (D5-D14) plus G-CSF (D10-D14). Between two subsequent cycles, progenitor cells were assessed daily, from D13 to D17. The WBC count was similar for patients receiving G-CSF alone or GM-CSF plus G-CSF, but significantly greater than that of patients receiving GM CSF alone (P<0.001). The GM-CSF plus G-CSF combination led to better PBPC mobilization, with significantly different kinetics (P<0.001) and optimal mean values of CFU-GM, CD34+ cells and cells in cycle, at D15 compared to those obtained with G-CSF or GM-CSF alone. The significantly greater PBPC mobilization obtained with a CSF combination by D15 could be of value for PBPC collection and therapeutic reinjection after high-dose chemotherapies. PMID- 10745255 TI - Thrombopoietic cytokines in relation to platelet recovery after bone marrow transplantation. AB - In order to evaluate the importance of different thrombopoietic stimulatory cytokines in accelerating platelet recovery after bone marrow transplantation (BMT), we assayed serial plasma concentrations of three cytokines, thrombopoietin (TPO), interleukin-6 (IL-6), and IL-11 through the course of platelet nadir and recovery after BMT. Both mean TPO and IL-6 levels showed a marked rise and later fall preceding or coincident with the platelet nadir and recovery, suggesting their potential role as circulating regulators or stimulators of thrombopoiesis. In contrast, IL-11 levels remained remarkably constant through the whole course suggesting that this cytokine, though capable of stimulating thrombopoiesis, does not serve as a circulating regulator of platelet production. Additionally, we assayed the levels of these three cytokines following initial platelet transfusion to assess the capacity of transfused platelets to adsorb these thrombopoietic cytokines from the plasma and reduce their circulating levels, thus potentially modifying their availability for stimulating megakaryocyte proliferation. No consistent falls in TPO, IL-6 or IL-11 levels were observed following the initial two platelet transfusions. These data support the importance of circulating TPO and IL-6 as hormones capable of stimulating platelet production. Their physiologic relevance as in vivo regulators of thrombopoiesis and clinical utility for therapy of thrombocytopenia need further investigation. PMID- 10745256 TI - Outcome of allogeneic stem cell transplantation for B cell chronic lymphocytic leukemia. AB - The objective of this study was to describe the outcome of allogeneic stem cell transplantation (alloSCT) in a series of patients with B cell chronic lymphocytic leukemia (B-CLL). Twenty-three B-CLL patients were transplanted between 1988 and 1997 using stem cells from a related (n = 20) or an unrelated donor (n = 3). The median age of the patients was 46 years, and the median number of prior chemotherapy regimens received was two. At transplantation, 14 patients had chemorefractory disease and 12 of these were refractory to fludarabine. The preparative regimens included total body irradiation (TBI) in 22 of the 23 cases. All patients received graft-versus-host disease (GVHD) prophylaxis with cyclosporine and methotrexate. Twenty patients (87%) achieved a complete remission (CR). The incidence of grade II-IV acute GVHD was 54%. Fourteen (61%) patients are alive and disease-free, including two with unrelated donors, at a median of 26 months (range, 9-115 months). Nine patients (39%) have died, one of whom had progressive B-CLL. The only favorable prognostic factor for failure-free survival (FFS) and overall survival (OS) after alloSCT was the use of a cyclophosphamide/TBI rather than an etoposide/cyclophosphamide/TBI regimen (P = 0.03). The projected 5-year FFS, OS, and relapse rates after alloSCT were 65% (95% CI, 48-88%), 62% (95% CI, 43-88%), and 5% (95%, CI 0-13%), respectively. These findings demonstrate the potential of high-dose therapy and alloSCT for inducing and maintaining a remission in patients with advanced or chemorefractory B-CLL. The low relapse rate may be due to an allogeneic graft-versus-leukemia effect. PMID- 10745257 TI - Autologous stem cell transplantation in multiple myeloma after VAD and EDAP courses: a high incidence of oligoclonal serum Igs post transplantation. AB - Thirty-seven patients with multiple myeloma (stage II and III, 65% increased beta2-microglobulin level) were prospectively treated with a median of 3.7 VAD courses (range 2-8) followed by cyclophosphamide (6 g/m2) in conjunction with G CSF (5 microg/kg filgrastrim (n = 14), or 3.5 microg/kg lenograstrim (n = 22)), and peripheral stem cell (PSC) isolation. After regeneration this was followed by one EDAP course and high-dose melphalan (HDM, 200 mg/m2) in combination with re infusion of PSC. Adequate stem cell mobilization was obtained with both G-CSF regimens. A median of 41x10(6) CD34+ cells/kg (range 4.5-161) was collected in a median of 1.6 leukapheresis procedures following filgrastrim (n = 14) and 24x10(6) CD34+ cells/kg (range 2. 3-80) in a median of 1.7 leukapheresis procedures following lenograstrim (n = 22) which indicated no significant difference (P = 0.24) between both G-CSF regimens. A rapid hematological recovery was obtained after HDM with reinfusion of a median of 9.3x10(6) CD34+ cells/kg. After the total courses the overall response was 84% with a complete remission rate of 30%. Currently the median overall survival is 44.0 months (95% CI 38.9 49.1) with a median follow-up of 33 months (range 3-51) and a median event-free survival of 29.0 months (95% CI 25.3-32.7) (n = 33). Post transplantation a high incidence of oligloclonal serum immunoglobulins (Igs) was observed. In 73% of the patients new oligoclonal or monoclonal serum bands were noticed 3 months post transplantation. IgG-lambda and IgG-kappa bands predominated. In 48% of the cases the oligoclonal Igs disappeared after a median follow-up of 22 months (range 8 36), whereas in 52% of the cases the oligoclonal Igs persisted with a median follow-up of 31 months (range 21-45), which did not correlate with a significant difference in overall, and event-free survival between both subgroups. PMID- 10745259 TI - Does early treatment with high-dose methylprednisolone alter the course of hepatic regimen-related toxicity? AB - Hepatic regimen-related toxicity (RRT) is a serious complication of stem cell transplantation. Cytokine activation may be involved in the pathogenesis. Corticosteroids are potent inhibitors of cytokine production, and, therefore could play a role in the treatment of hepatic RRT. Between January 1994 and June 1998, 28 of 782 consecutive transplant patients (3.6%) developed hepatic RRT (20 veno-occlusive disease (VOD) and eight liver dysfunction of uncertain etiology (LDUE) as defined by Seattle criteria), and were treated with high-dose methylprednisolone (MP, 500 mg/m2 i.v. every 12 h for six doses), initiated upon increase in serum total bilirubin to > or =4 mg/dl. Other causes of liver dysfunction were excluded. Response to therapy with high-dose MP was defined as reduction in total bilirubin by 50% within 10 days of initiation of MP. Overall, 17 patients (61%) responded to treatment (12 patients with VOD, five patients with LDUE). The bilirubin in responding patients decreased from a mean of 8.6 mg/dl (range, 4-17.9) at the start of MP to 4.1 mg/dl (range, 0.5-17.9) 10 days later. There were no statistically significant differences between responders and non-responders in the day treatment with high-dose MP was initiated (P = 0.38), total serum bilirubin (P = 0.17) and percent weight gain at the time high-dose MP was started (P = 0.10) or the calculated probability of fatal outcome from VOD (18% for responders, 23% for non-responders; P = 0.30). A lower pre-transplant DLCOc was observed among non-responders (P = 0.04). At 100 days post-transplant, hepatic RRT resolved in all 13 survivors who responded to high-dose MP, and in one non-responding patient. No serious toxicities due to high-dose MP were observed. We conclude that resolution of hepatic RRT occurred in the majority of patients treated with high-dose MP in this study; however, randomized controlled trials are required to determine the efficacy of high-dose MP for treatment of hepatic RRT. PMID- 10745258 TI - Quantification of BCR-ABL transcripts in CML patients in cytogenetic remission after interferon-alpha-based therapy. AB - We measured using a competitive quantitative polymerase chain reaction-capillary electrophoresis (PCR-CE)-based assay, the levels of bcr-abl transcripts in 44 patients with chronic myeloid leukemia (CML) after interferon-alpha (IFN-alpha) therapy, who achieved a major (10 patients, MCR group) or complete (34 patients, CCR group) cytogenetic response. All 34 CCR patients had molecular evidence of residual disease detected in bone marrow samples at the time of best karyotypic response. The median number of bcr-abl transcripts of 34 evaluable patients in the CCR group at the time of complete cytogenetic remission was 4/microg RNA (range 3-4600), while the median number of bcr-abl transcripts of 10 patients in the MCR group at the time of best cytogenetic response was 4490/microg RNA (range 600-23 900) (P = 0.000024). In nine CCR and five MCR patients we were able to quantify the amount of bcr-abl transcript both at diagnosis and after interferon therapy: no statistical difference (P = 0.18) was found between the two groups at diagnosis (median bcr-abl transcripts/microg RNA was 30 000 vs. 39 650, respectively). During IFN-alpha therapy, the two groups were evaluable at the time of major karyotypic conversion: at this point, there was a statistical difference of expression of bcr-abl transcript between the CCR group (17 patients) (median 2700; range 76-40 000) and the MCR group (10 patients) (median 4490; range 600-23 900), respectively (P = 0.046). No differences of bcr-abl amount of transcript were found in patients with CCR obtained either by IFN-alpha therapy alone (20 patients) vs. IFN-alpha plus ABMT (13 patients) (P = 0.47). We firstly demonstrated that although the CCR and MCR groups were clinically, cytogenetically and molecularly indistinguishable at diagnosis, the two groups could be recognized successfully during interferon therapy based on the level of bcr-abl transcript. PMID- 10745260 TI - Trichosporon beigelii: a life-threatening pathogen in immunocompromised hosts. AB - Patients undergoing bone marrow transplantation are profoundly immunosuppressed as a result of their intensive myeloablative chemotherapy and are at high risk for opportunistic fungal infections mainly caused by Candida spp and Aspergillus spp. Trichosporon beigelii (T beigelii) has emerged as a life-threatening opportunistic pathogen in granulocytopenic and immunocompromised hosts and there is a marked increase in cases reported in the literature. Response to antifungal agents is poor, mortality is high and immunological recovery is the most important factor for a favorable outcome in patients with trichosporonosis. We present three cases of T. beigelii infection in patients undergoing allogeneic bone marrow transplantation in our center and we review cases described in the literature. PMID- 10745261 TI - Respiratory syncytial virus upper respiratory tract illnesses in adult blood and marrow transplant recipients: combination therapy with aerosolized ribavirin and intravenous immunoglobulin. AB - Respiratory syncytial virus (RSV) is an important cause of serious respiratory illness in blood and marrow transplant (BMT) recipients. In some subsets of these immunocompromised patients, RSV upper respiratory illnesses frequently progress to fatal viral pneumonia. The frequency of progression to pneumonia is higher during the pre-engraftment than during the post-engraftment period. Once pneumonia develops, the overall mortality is 60-80%, regardless of the treatment strategy. We performed a pilot trial of therapy of RSV upper respiratory illnesses using aerosolized ribavirin and IVIG (500 mg/kg every other day), with the goal of preventing progression to pneumonia and death. Two dosages of ribavirin were used: a conventional regimen (6 g/day at 20 mg/ml for 18 h/day) and a high-dose short-duration regimen (6 g/day at 60 mg/ml for 2 h every 8 h). Fourteen patients were treated for a mean of 13 days (range: 7-23 days). In 10 (71%) patients, the upper respiratory illness resolved. The other four (29%) patients, three of whom were in the pre-engraftment period, developed pneumonia, which was fatal in two. The most common adverse effect was psychological distress at being isolated within a scavenging tent. In conclusion, prompt therapy of RSV upper respiratory illnesses in BMT recipients with a combination of aerosolized ribavirin and IVIG was a safe and promising approach to prevent progression to pneumonia and death. PMID- 10745262 TI - Risk factors for treatment failures in patients receiving PCR-based preemptive therapy for CMV infection. AB - PCR-based preemptive therapy with ganciclovir has been shown to reduce the incidence of CMV disease after BMT. Failures of this treatment strategy are CMV disease and secondary non-viral infections. Eighty-six consecutive patients at high risk for CMV disease who received PCR-based preemptive therapy with ganciclovir were assessed for treatment failures and possible risk factors. Ganciclovir was initiated in 57 of 86 patients (66%). Only 28 of 86 (32%) patients received 4 or more weeks of ganciclovir. Recurrence of CMV infection after successful treatment was more frequent among recipients of a BMT from an unrelated compared to a sibling donor (P = 0.004). Three (3.5%) patients developed non-fatal early onset CMV disease and seven of 68 (10.3 %) late onset CMV disease (>100 days post transplant). Risk factors for late onset CMV disease were cGVHD (P = 0.0017) and duration of prior antiviral therapy >4 weeks (P = 0. 0073). The incidence of secondary non-viral infections was 28% with the duration of antiviral treatment being a significant risk factor for secondary bacterial (P = 0.0045) and invasive fungal infections (P = 0.006). Thus, PCR-based preemptive treatment with ganciclovir reduces early onset CMV disease, but the duration of antiviral therapy prior to day +100 is a significant risk factor for late onset CMV disease as well as secondary non-viral infections. PMID- 10745263 TI - Risk-adapted pre-emptive therapy for cytomegalovirus disease in patients undergoing allogeneic bone marrow transplantation. AB - We prospectively evaluated a risk-adapted pre-emptive treatment with ganciclovir for CMV diseases in patients undergoing allogeneic bone marrow transplantation (BMT). High-level CMV antigenemia (10 or more positive cells on two slides) or CMV antigenemia at any level in patients with grade II-IV acute graft-versus-host disease (aGVHD) were chosen as risk factors. We also retrospectively evaluated virus reactivation in plasma using quantitative real-time polymerase chain reaction (PCR). Fifty patients were evaluable. None of the 27 patients with or without grade I aGVHD developed high-level CMV antigenemia or CMV disease. Among the 23 patients with grade II-IV aGVHD, 12 patients (52%) developed CMV antigenemia and were treated pre-emptively, of whom two developed CMV gastroenteritis or retinitis in spite of therapy. Six of the remaining 11 patients developed CMV gastroenteritis before CMV antigenemia was detectable. All of the eight patients with CMV diseases were successfully treated with ganciclovir and no deaths directly related to CMV disease occurred. In four of the seven evaluable patients with CMV gastroenteritis, real-time PCR was able to detect virus reactivation earlier than CMV antigenemia. Although our risk-adapted pre-emptive therapy effectively reduced CMV-related mortality, further refinements of this approach, particularly in the prevention of CMV gastroenteritis, may be achieved by incorporating real-time PCR. PMID- 10745264 TI - An overview of the Welsh bone marrow donor registry: 10 years of bone marrow donor provision. AB - The Welsh Bone Marrow Donor Registry (WBMDR) is in its 11th year of operation and its 4th year as an International 'Hub' participating in Bone Marrow Donors Worldwide. It is operated by the Welsh Regional Tissue Typing Laboratory which is accredited by Clinical Pathology Accreditation (UK) Ltd, and the European Federation for Immunogenetics and, together with the Welsh Blood Service, its donor centre, is ISO 9001 Registered. The active donor panel of over 21 000 regular blood donors are all HLA-A, B, DR, DQ typed (over 95% to the split specificity level or higher). All HLA-DR, DQ and over 50% of HLA-A, B typing has been performed by DNA-based methods. CMV antibody status, now tested on all donors, is known on over 70% of subjects. The WBMDR has over 80% success at obtaining Confirmatory Typing samples and operates a rapid Expanded Typing service on stored donor material. It has provided 174 bone marrow donations (140 for UK and 34 for overseas patients), and 11 lymphocyte donations, since its inception in 1989. PMID- 10745265 TI - Multi-purpose silastic dual-lumen central venous catheters for both collection and transplantation of hematopoietic progenitor cells. AB - Autologous peripheral blood progenitor cell (PBPC) transplantation frequently requires sequential placement and use of two separate central venous catheters: (1) a short-term, large-bore, stiff device inserted for leukapheresis, and after removal of that device, (2) a long-term, multi-lumen, flexible, Silastic catheter for administration of high-dose chemotherapy, re-infusion of hematopoietic cells, and intensive supportive care. We reviewed our recent experience with two dual lumen, large-bore, Silastic multi-purpose ('hybrid') catheters, each of which can be used as a single device for both leukapheresis and long-term supportive care throughout the transplant process. Quinton-Raaf PermCath and Bard-Hickman hemodialysis/apheresis dual-lumen catheters were used as the sole venous access device in 112 consecutive patients who underwent autologous PBPC collection and transplantation. The catheter exit site was monitored three times a week, and lumen patency was assessed using clinical and radiologic techniques. Catheters were removed prematurely for persistent thrombus, positive blood cultures despite appropriate antibiotics, or mechanical dysfunction. There were no intra-operative or immediate post-operative complications relating to insertion. Thirty-two patients experienced catheter occlusion necessitating urokinase instillation. Persistent occlusive problems were noted in 16 patients, and in 10 patients the catheter had to be removed. Two exit site infections and 17 bacteremias occurred. Catheters had to be removed for persistent infection in two subjects and for mechanical problems in five others. Cost analysis comparing the hybrid catheters alone vs conventional devices revealed a charge of $4230 in patients with hybrid catheters vs. $7530 in those requiring a temporary non-Silastic dialysis catheter in addition to a flexible, long-term Silastic catheter. Hybrid, Silastic, dual lumen, large-bore central venous catheters are safe, cost-effective and convenient multi-purpose venous access devices that may be used in the setting of autologous PBPC collection and transplantation. The rate of thrombotic, infectious and mechanical complications appears comparable to other central venous access devices. PMID- 10745266 TI - HLA-mismatched CD34-selected stem cell transplant complicated by HHV-6 reactivation in the central nervous system. AB - We report here a patient who suffered from PCR- confirmed human herpesvirus type 6 (HHV-6) meningoencephalitis after allogeneic purified CD34+ cell transplantation from his HLA-mismatched sibling donor, even though he had been on intense prophylaxis with i.v. ganciclovir (GCV), acyclovir (ACV) and gamma globulin containing a specific antibody against HHV-6. Serological evaluation disclosed that both the donor and recipient had IgG antibody against HHV-6 before transplantation. His blood WBC count started to transiently increase on day 10, and all blood components had decreased by day 20. He then developed a severe headache and high blood pressure, and sporadic abnormal neurological findings including nystagmus and delirium. An analysis of cerebrospinal fluid (CSF) revealed 8 cells/microl, a glucose level of 130 mg/dl and a protein level of 201 mg/dl (normal, 50 mg/dl) on day 26. At the time, HHV-6 was detected only in CSF by a PCR-based method and he was diagnosed as having meningoencephalitis due to the local reactivation of HHV-6. Although he failed to respond to high-dose therapy with ACV (60 mg/kg/day) and gamma-globulin, the DNA of this virus disappeared from the CNS upon treatment with GCV (30 mg/kg/day) combined with the intraventricular infusion of alpha-interferon. His clinical course was further complicated with meningoencephalitis due to staphylococcus epidermidis, and he died of tentorial herniation on day 79 without the recovery of blood components. This experience may indicate that intense prophylaxis to prevent reactivation of HHV-6 in the CNS is essential for the management of such profoundly immunosuppressed patients. PMID- 10745267 TI - MR angiographic diagnosis of cerebral venous sinus thrombosis following allogeneic bone marrow transplantation. AB - Allogeneic bone marrow transplantation is frequently associated with neurological complications, particularly intracerebral bleeds and infections. Cerebral venous sinus thrombosis has only rarely been reported following allogeneic transplants. We report three cases of cortical venous thrombosis following allografting for acute lymphoblastic leukaemia. Two patients received marrow from HLA-identical siblings and one from an unrelated donor. Two of the patients presented with grand mal seizures and one presented with a headache. No neurological abnormalities were found upon clinical examination and lumbar puncture was normal in all three cases. In two of the patients computed tomography (CT) of the brain was normal and in the third showed non-specific abnormalities. Magnetic resonance imaging (MRI) with MR angiography (MRA) demonstrated cerebral venous sinus thrombosis in all three patients. In conclusion, cerebral venous sinus thrombosis should be considered in the differential diagnosis when neurological symptoms occur following allogeneic bone marrow transplantation. We therefore advocate the use of MRA for unexplained neurological symptoms post-allograft since without it cerebral venous sinus thrombosis may easily be missed. PMID- 10745268 TI - Prompt and durable engraftment in two older adult patients with high risk chronic myelogenous leukemia (CML) using ex vivo expanded and unmanipulated unrelated umbilical cord blood. AB - Delayed engraftment, graft failure, and adverse transplant-related events have been observed in unrelated umbilical cord blood (UCB) recipients, particularly in those receiving a low leukocyte cell dose and in CML patients. We report the outcomes of two older adult patients with high risk CML who received a low leukocyte cell dose of unmanipulated UCB cells supplemented with ex vivo expanded (AastromReplicell System) UCB cells. Each engrafted promptly and neither patient experienced GVHD or life-threatening infection. Both remain engrafted with cells exclusively of donor origin and are in cytogenetic remission at 19 and 8 months follow-up. Ex vivo expanded UCB cells appear to facilitate hematopoietic recovery and therefore may increase the number of CML patients eligible for unrelated UCB transplant. PMID- 10745269 TI - p53 and thymic 'death by neglect': thymic epithelial cell-induced apoptosis of CD4+8+ thymocytes is p53-independent. AB - The involvement of the tumor suppressor protein, p53, in thymic epithelial cell induced apoptosis of CD4+8+ (double positive) thymocytes, was studied in an in vitro model consisting of a thymic epithelial cell line (TEC) and thymocytes. p53 expression was not augmented in double positive (DP) thymocytes upon co-culturing with TEC, although extensive apoptosis was observed. In the same cells, p53 expression was upregulated in response to low ionizing irradiation, which was accompanied with massive apoptosis. Moreover, TEC induced apoptosis in two DP thymomas, derived from p53(-/-) mice, and in a double positive thymoma clone expressing mutant p53. The extent and kinetics of TEC-induced apoptosis was not affected by the status of p53 in the thymocytes tested. We conclude that thymic epithelial cell-induced apoptosis of immature DP thymocytes is p53-independent and apparently, involves a different apoptotic pathway than that triggered by DNA damage. PMID- 10745270 TI - Staurosporine-induced neuronal death: multiple mechanisms and methodological implications. AB - To examine whether multiple pathways of cell death exist in sympathetic neurons, we studied the cell death pathway induced by staurosporine (STS) in sympathetic neurons and compared it with the well-characterized NGF deprivation-induced death pathway. Increasing concentrations of STS were found to induce sympathetic neuronal death with different biochemical and morphological characteristics. One hundred nM STS induced metabolic changes, loss of cytochrome c, and caspase dependent morphological degeneration which closely resembled the apoptotic death induced by NGF deprivation. In contrast, sympathetic neurons treated with 1 microM STS showed no loss of cytochrome c but exhibited extensive, caspase independent, chromatin changes that were not TUNEL positive. One microM STS treated sympathetic neurons had greatly reduced metabolic activities and became committed to die rapidly, yet maintained soma structure and appeared viable by other criteria even up to 48 h after STS treatment, illustrating the need to assess cell death by multiple criteria. Lastly, in contrast to the cell death inducing activities of 100 nM STS or 1 microM STS, very low concentrations of STS (1 nM STS) inhibited sympathetic neuronal death by acting either at or prior to c jun phosphorylation in the NGF deprivation-induced PCD pathway. PMID- 10745271 TI - Ceramide-induced cell death is independent of the Fas/Fas ligand pathway and is prevented by Nur77 overexpression in A20 B cells. AB - The role of ceramide in triggering apoptosis is still a matter of debate. While in some experimental systems, ceramide was shown to mediate Fas-induced cell death, in other instances it was claimed to induce the expression of Fas ligand (FasL), killing cells in a caspase-dependent fashion. We found that, in mature A20 B cells, ceramide-induced apoptosis is independent of the caspase pathway, since we observed no ICE-like, CPP32-like and Mch2 activities and no PARP proteolysis. Moreover, we were unable to protect these cells from ceramide induced apoptosis using caspase inhibitors, while they blocked Fas-induced apoptosis and no FasL induction could be detected following ceramide treatment. These results suggest that ceramide does not induce apoptosis through the Fas/FasL pathway. We also found that overexpression of Nur77, a zinc-finger transcription factor described to upregulate FasL, antagonizes ceramide-induced apoptosis, but not Fas-induced apoptosis. This further supports the hypothesis that Fas and ceramide death pathways are independent in A20 cells. Ceramide induced cell death was associated with increased c-myc, p53, Bax and p27kip1 levels; in contrast, cells transfected with Nur77 (A20Nur77), resistant to ceramide-induced apoptosis, showed a marked downregulation of p53 after ceramide treatment, with neither Bax nor p27kip1 induction. In conclusion, our results suggest that, in the A20 B cell line, Fas and ceramide trigger two distinct pathways and that Nur77 overexpression confers protection against ceramide mediated apoptosis which correlates with inhibition of p53, Bax and p27kip1 induction. PMID- 10745272 TI - Evidence that the bifunctional redox factor / AP endonuclease Ref-1 is an anti apoptotic protein associated with differentiation in the developing retina. AB - Retinal cell differentiation leads to resistance to apoptosis induced by inhibition of protein synthesis, suggesting the accumulation of anti-apoptotic proteins. The redox factor/AP endonuclease Ref-1 (APE, APEX, HAP1) affects both DNA repair and the activity of various transcription factors, and controls sensitivity to genotoxic insults. We studied the expression of Ref-1 in the retina and brain of developing rats. Ref-1 immunoreactivity increased progressively within the nucleus of differentiating retinal cells, whereas it decreased in the developing hippocampal formation. During both natural and experimentally-induced cell death, Ref-1 disappeared from the nucleus of apoptotic cells. Degradation of Ref-1 in axotomized ganglion cells preceded the morphological characteristics of apoptosis. The sensitivity to apoptosis triggered by either thapsigargin or okadaic acid was the highest in photoreceptors, that contain the least Ref-1 among differentiated retinal cells. In both these differentiated cell types, inhibition of protein synthesis prevented the loss of Ref-1 and rescued the neurons. The data suggest that Ref-1 is an anti-apoptotic protein associated with cell differentiation in the retina. PMID- 10745273 TI - Oxidative stress induces caspase-independent retinal apoptosis in vitro. AB - Apoptosis is the mode of cell death in retinitis pigmentosa (RP), a heterogeneous group of retinal degenerations. The activation of the caspase proteases forms a pivotal step in the initiation and execution phase of apoptosis in many cells. Inhibition of caspases has been reported to prevent apoptosis in many model systems. However, we demonstrate the absence of caspase activation during retinal cell apoptosis in vitro which involves phosphatidylserine (PS) externalisation, DNA nicking and cell shrinkage. In addition, zVAD-fmk, DEVD-CHO and BD-fmk, inhibitors of the caspases, were unable to alter the characteristics or kinetics of apoptosis, implying that retinal cell death in vitro follows a caspase independent pathway. We have previously demonstrated the ability of reactive oxygen species (ROS) to act as mediators of retinal cell apoptosis in vitro as well as the ability of antioxidants to prevent retinal cell apoptosis. Here we demonstrate the oxidative inactivation of caspases in this model of retinal apoptosis and provide evidence for an oxidative stress driven cell death pathway that does not involve caspase activity and which retains key features of apoptotic cell death. Furthermore, our data indicates that apoptotic events such as PS exposure, DNA nicking and cell shrinkage may occur independently of caspase activity. PMID- 10745274 TI - E1A stimulates FGF-2 release promoting differentiation of primary endothelial cells. AB - Basic Fibroblast Growth Factor (FGF-2) is a growth and survival factor and represents one of the most potent differentiation agents of vascular system. In the present study we describe that adenoviral oncoprotein E1A regulates FGF-2 production and determines the acquisition of a pro-angiogenic phenotype in primary bovine aortic endothelial cells (BAEC). Following their transfection, wild type E1A proteins 12S and 13S (wtE1A) stimulated BAEC to differentiate on reconstituted basement membrane matrix (Matrigel). This outcome was paralleled by invasion and migration enhancement in wtE1A-transfected cells. This stimulating effect was absent with the E1A mutant dl646N. Accordingly, zymography and RT - PCR analyses showed that matrix metalloproteinase-9 protein- and mRNA-levels increased following wtE1A transfection. Interestingly, wtE1A-transfected BAEC showed FGF-2 mRNA- and protein-levels higher than controls. Further, FGF-2 neutralization reduced the amount of MMP-9 released in the supernatant of E1A transfected cells and strongly inhibited BAEC differentiation, thus suggesting that wtE1A activates BAEC by a mechanism, at least partially, dependent on a FGF 2 autocrine/paracrine loop. PMID- 10745275 TI - MAP kinase pathway signalling is essential for extracellular matrix determined mammary epithelial cell survival. AB - Mammary epithelial cells in primary cell culture require both growth factors and specific extracellular matrix (ECM)-attachment for survival. Here we demonstrate for the first time that inhibition of the ECM-induced Erk 1/Erk 2 (p42/44 MAPK) pathway, by PD 98059, leads to apoptosis in these cells. Associated with this cell death is a possible compensatory signalling through the p38 MAP kinase pathway the inhibition of which, by SB 203580, leads to a more rapid onset of apoptosis. This provides evidence for a hitherto undescribed Erk 1/Erk 2 to p38 MAP kinase pathway 'cross-talk' that is essential for the survival of these cells. The cell death associated with inhibition of these two MAP kinase pathways however, occurred in the presence of insulin that activates the classical PI-3 kinase-dependent Akt/PKB survival signals and Akt phosphorylation. Cell death induced by inhibition of the MAP kinase pathways did not affect Akt phosphorylation and may, thus, be independent of PI-3 kinase signalling. PMID- 10745276 TI - The NAD+ precursors, nicotinic acid and nicotinamide protect cells against apoptosis induced by a multiple stress inducer, deoxycholate. AB - The bile salt, sodium deoxycholate (NaDOC), is a natural detergent that promotes digestion of fats. At high physiologic levels, NaDOC activates many stress response pathways and induces apoptosis in various cell types. NaDOC induces DNA damage and activates poly(ADP-ribose) polymerase (PARP), an enzyme that utilizes NAD+ as a substrate to repair DNA. NaDOC also induces oxidative stress, endoplasmic reticulum (ER) stress and contributes to protein malfolding. The NAD+ precursors, nicotinic acid (NA) and nicotinamide (NAM) were found to protect cells against NaDOC-induced apoptosis. NA and NAM also decreased constitutive levels of both activated NF-kappaB and GRP78, two proteins that respond to oxidative stress. However, the mechanism by which NA and NAM protects cells against apoptosis does not involve a reduction in constitutive levels of oxidative stress. NA or NAM treatment increased the protein levels of glyceraldehyde-3-phosphate dehydrogense (GAPDH), a multi-functional enzyme, in the nucleus and cytoplasm, respectively. NAM did not activate the promoter/response elements of 13 stress response genes nor reduce intracellular non-protein thiols, suggesting that it is non-toxic to cells. NAM thus has promise as a dietary supplement to help prevent disorders involving excessive apoptosis. PMID- 10745277 TI - A review of the nutritional needs of Meals on Wheels consumers and factors associated with the provision of an effective meals on wheels service-an Australian perspective. AB - OBJECTIVE: A review of the literature was undertaken to identify the nutritional needs of elderly MOW consumers and factors affecting the ability of existing programs to meet those needs. The focus was on the Australian experience but drawing on the world literature. DESIGN: Keyword search of English language based computer databases of the medical and health literature. RESULTS: Several studies suggest the nutritional intake of MOW consumers is below recommended levels, although the risk of nutritional deficiency has not always been identified. The literature indicates the effectiveness of Meals on Wheels programs are affected by a range of issues including the appropriateness of nutritional standards, menu selection, portion control, level of consumption and customer satisfaction. The literature recommends control of time and temperatures associated with food handling procedures, along with education of providers and customers, to assist in the provision of a safe food supply. CONCLUSIONS: Meals on Wheels is an important service, providing meals to housebound consumers. While the effectiveness of such programs is dependent on a range of variables, the nutritional impact of the service and the standard of food hygiene are fundamental assessment criteria. SPONSORSHIP: This work was supported by a grant from the NSW Meals on Wheels Association, Australia. European Journal of Clinical Nutrition (2000) 54, 275-280 PMID- 10745278 TI - Low energy reporters vs others: a comparison of reported food intakes. AB - OBJECTIVE: To partition the food reports of low energy reporters (LERs) and non LERs into four aspects-tendency to report a given food, frequency of reports per user, portion sizes per mention, and the qualitative (low-fat, low-sugar, low energy) differences of the reports-in order to determine what differentiates them from one another. ASSESSMENT METHOD: Two non-consecutive 24h dietary recalls. Low energy reporting was defined as energy intake lower than 80% of estimated basal metabolic rate. SETTING: In-home personal interviews. SUBJECTS: 8334 adults from a stratified, multi-stage area probability sample designed to be representative of noninstitutionlized persons residing in households in the United States. RESULTS: Across all different types of foods, there are those food groups which LERs are less likely to report (28 of 44 food groups), those which they report less frequently when they do report them (15 of 44 groups), and those for which they report smaller quantities per mention (26 of 44). Qualitative differences in the food choices-that is, differences in fat, sugar, and/or energy content-were not so widespread (4 of 24 food groups). CONCLUSIONS: The practical application of analyses such as these is to improve the methods of gathering dietary data so that this kind of bias can be reduced. Further methodological research is needed to reduce the likelihood of respondents neglecting to mention foods and underestimating portion sizes. PMID- 10745279 TI - Effect of 8 week intake of probiotic milk products on risk factors for cardiovascular diseases. AB - OBJECTIVE: To investigate the effect of a probiotic milk product containing the culture CAUSIDO(R) and of two alternative products on risk factors for cardiovascular disease in overweight and obese subjects. DESIGN: An 8 week randomized, double-blind, placebo- and compliance-controlled, parallel study. SUBJECTS: Seventy healthy, weight-stable, overweight and obese (25.0/=30.0 kg/m2) applied to anthropometric and socio-economic data collected by three comparable household surveys undertaken in the two most populated Brazilian regions in 1975 (n=95,062), 1989 (n=15,585) and 1997 (n=10,680). RESULTS: While previous trends (1975-1989) showed increasing obesity prevalence for all population groups except for men in rural areas, recent trends (1989-1997) have pointed to a much more complex picture where increases in obesity tend to be more intense in men than in women, in rural than in urban settings and in poorer than in richer families. Particularly notable was the fact that, in the recent period, obesity was actually reduced for women belonging to the upper income groups, especially in urban settings. CONCLUSION: Earlier obesity trends in Brazil entirely agree with what has been described for both developed and developing countries where reliable secular trend information exists, but the 1989-1997 trend of a substantial reduction in the prevalence of obesity among upper income urban women (12.8-9.2%, or a 28% reduction), is unique in a developing country and, indeed, up to now has only been detected in Scandinavian populations. It is speculated that this declining obesity trend may be a result of an intense mass media work focused on combating a sedentary life style and promoting better food habits. PMID- 10745287 TI - Adherence to protein restriction in patients with type 2 diabetes mellitus: a randomized trial. AB - OBJECTIVE: To describe the extent to which diet counselling can decrease protein intake, and to identify predictors of adherence. DESIGN: (1) Randomized trial; (2) observational longitudinal study. SUBJECTS: (1) 125 type 2 diabetic patients in primary care; (2) 59 patients in the experimental group. INTERVENTION: For a period of 12 months, dieticians provided guidance on protein restriction (experimental group, n=59) or the usual dietary advice (control group, n=66). OUTCOME MEASURES: Adherence was estimated primarily from urinary urea excretion (UUE), but also from food-frequency questionnaires (FFQ). RESULTS: After 6 months protein intake was, according to the UUE and the FFQ, respectively, 8 g/day (95% CI -2, 13) (8%) and 15 g/day (95%-CI 9, 22) (16%) lower in the experimental than in the control group. After 12 months these differences were smaller. Linear regression analysis indicated that protein restriction was greater in patients who were well satisfied with their pre-existing diet (r=0.32, bper 1/10=3.6 (1, 6) g), in patients who were less overweight (r=0.32, bper kg.m-2=1.1 (0.2, 2. 0) g), and in patients living alone (r=0.22, b=7.7 (-2, 17) g). These combined factors explained only 11% of variation in adherence. Adherence was not predicted by the number of barriers reported by the patients or by coinciding changes in diet satisfaction. CONCLUSIONS: The diet counselling resulted in a very moderate degree of protein restriction only. Predictors of adherence could be identified, but only a few, and their predictive power was limited. PMID- 10745290 TI - Chocolate and cocoa: health and nutrition PMID- 10745288 TI - Risk factors for hypertension in obese women. The role of weight cycling. AB - OBJECTIVE: To study significant factors associated with the risk of hypertension among obese women, with and without a history of weight cycling (WC). DESIGN: Case-control study. SETTING: Obesity Clinic of Chieti University, Italy. SUBJECTS: A group of 258 obese women aged 25-64 y (103 cases with hypertension and 155 controls) were recruited. All obese subjects had the same clinical characteristics, were without a family history for hypertension, were non smokers, had normal lipidemic profiles and normal glucose tolerance, were not taking any medication and were otherwise healthy. INTERVENTION: In the weight cycling women, the history of WC was established on the basis of at least five weight losses in the previous 5 y due to dieting, with a weight loss of at least 4.5 kg per cycle. A logistic regression model adjusted for confounding variables such as waist-to-hip ratio (WHR) and weight cycling history parameters was used and the odds ratio (OR) with 95% confidence intervals was calculated. RESULTS: The risk of hypertension increases in subjects with larger WHR (OR 7.8; 95% CI 3.4-17.9) and with a positive history for WC (OR 4.1; 95% CI 2.4-6.9). Further, in obese patients with WC, the weight cycling index and the sum of the weight regained are also important risk factors for hypertension. CONCLUSIONS: These findings could support the hypothesis that it is the combined exposure of central type obesity and WC that strongly raises the risk of hypertension. SPONSORSHIP: This work has been financially supported by a grant of Ministero dell'Universita e della Ricerca Scientifica e Tecnologica. PMID- 10745289 TI - Age- and sex-adjusted iodine/creatinine ratio. A new standard in epidemiological surveys? Evaluation of three different estimates of iodine excretion based on casual urine samples and comparison to 24 h values. AB - OBJECTIVE: The most accurate way to measure urinary iodine excretion in epidemiological surveys is still debated. We propose a new principle of estimating iodine excretion based on casual urine samples. MATERIAL AND METHODS: A total of 123 24 h urine samples and corresponding casual urine samples were collected from 31 subjects. Iodine excretion was expressed as 24 h iodine excretion and three different estimates: iodine concentration in the casual sample, iodine/gram creatinine in the casual sample, and the new principle iodine/creatinine ratio in the casual sample, adjusted for expected creatinine excretion of the individual. RESULTS: All three estimates based on casual urine samples correlated significantly to 24 h values with a r (Pearson) of 0.37 for iodine concentration, 0. 61 for iodine/creatinine ratio and 0.62 for the age- and sex-adjusted iodine/creatinine ratio. The median iodine excretion in the entire group was 143 microg/day in 24 h samples, 87 microg/l as iodine concentration, 77 microg/g creatinine as iodine/creatinine ratio and 126 microg/day as age- and sex adjusted iodine/creatinine ratio. CONCLUSION: Age- and sex-adjusted iodine/creatinine ratio is a more accurate and unbiased estimate of iodine excretion in epidemiological surveys of adults than the two most frequently used estimated: iodine concentration and iodine/gram creatinine, as these two estimates may introduce a bias depending on the composition of the investigated group. The adjusted iodine/creatinine ratio is superior to the other estimates, especially when individual estimates of 24 h iodine excretion is required or cohorts of selected groups are investigated. SPONSORSHIP: This work was supported by grants from the Medical Research Foundation Region Greater Copenhagen, Faroe Islands and Greenland; the Wedell-Wedellsborg Foundation; Musikforlaeggerne Agnes and Knut Morks Foundation. PMID- 10745291 TI - Safety evaluation of certain food additives PMID- 10745292 TI - Chemical hormesis: its historical foundations as a biological hypothesis. AB - Despite the long history of hormesis-related experimental research no systematic effort to describe its early history has been undertaken. The present paper attempts to reconstruct and assess the early history of such research and to evaluate how advances in related scientific fields affected the course of hormesis-related research. The purpose of this paper is not only to satisfy this gap in current knowledge, but also to provide a foundation for the assessment of how the concept of hormetic dose-response relationships may have affected the nature of the bioassay especially with respect to hazard assessment practices within a modern risk assessment framework. PMID- 10745293 TI - The marginalization of hormesis. AB - Despite the substantial development and publication of highly reproducible toxicological data, the concept of hormetic dose-response relationships was never integrated into the mainstream of toxicological thought. Review of the historical foundations of the interpretation of the bioassay and assessment of competitive theories of dose-response relationships lead to the conclusion that multiple factors contributed to the marginalization of hormesis during the middle and subsequent decades of the 20th century. These factors include: (a) the close association of hormesis with homeopathy lead to the hostility of modern medicine toward homeopathy thereby creating a guilt by association framework, and the carry-over influence of that hostility in the judgements of medically-based pharmacologists/ toxicologists toward hormesis; (b) the emphasis of high dose effects linked with a lack of appreciation of the significance of the implications of low dose stimulatory effects; (c) the lack of an evolutionary based mechanism(s) to account for hormetic effects; and (d) the lack of appropriate scientific advocates to counter aggressive and intellectually powerful critics of the hormetic perspective. PMID- 10745294 TI - Radiation hormesis: its historical foundations as a biological hypothesis. AB - This paper represents the first systematic effort to describe the historical foundations of radiation hormesis. Spanning the years from 1898 to the early 1940's the paper constructs and assesses the early history of such research and evaluates how advances in related scientific fields affected the course of hormetic related research. The present effort was designed to not only address this gap in current knowledge, but to offer a toxicological basis for how the concept of hormetic dose-response relationships may affect the nature of the bioassay and its role in the risk assessment process. PMID- 10745295 TI - Radiation hormesis: the demise of a legitimate hypothesis. AB - This paper examines the underlying factors that contributed to the marginalization of radiation hormesis in the early and middle decades of the 20th century. The most critical factor affecting the demise of radiation hormesis was a lack of agreement over how to define the concept of hormesis and quantitatively describe its dose-response features. If radiation hormesis had been defined as a modest overcompensation to a disruption in homeostasis as would have been consistent with the prevailing notion in the area of chemical hormesis, this would have provided the theoretical and practical means to blunt subsequent legitimate criticism of this hypothesis. A second critical factor undermining the radiation hormesis hypothesis was the generally total lack of recognition by radiation scientists of the concept of chemical hormesis which was markedly more advanced, substantiated and generalized than in the radiation domain. The third factor was that major scientific criticism of low dose stimulatory responses was galvanized at the time that the National Research Council (NRC) was organizing a national research agenda on radiation and the hormetic hypothesis was generally excluded from the future planned research opportunities. Furthermore, the criticisms of the leading scientists of the 1930s which undermined the concept of radiation hormesis were limited in scope and highly flawed and then perpetuated over the decades by other 'prestigious' experts who appeared to simply accept the earlier reports. This setting was then linked to a growing fear of radiation as a cause of birth defects, mutation and cancer, factors all reinforced by later concerns over the atomic bomb. Strongly supportive findings on hormetic effects in the 1940s by Soviet scientists were either generally not available to US scientists or disregarded as part of the Cold War mindset without adequate analysis. Finally, a massive, but poorly designed, US Department of Agriculture experiment in the late 1940s to assess the capacity for low dose plant stimulation by radionuclides failed to support the hormetic hypothesis thereby markedly lessening enthusiasm for research and funding in this area. Thus, the combination of a failed understanding of the hormetic hypothesis and its linkage with a strong chemical hormesis database, flawed analyses by prestigious scientists at the critical stage of scientific research development, reinforced by a Cold War mentality led to marginalization of an hypothesis (i.e., radiation hormesis) that had substantial scientific foundations and generalizability. PMID- 10745296 TI - Tales of two similar hypotheses: the rise and fall of chemical and radiation hormesis. AB - This paper compares the historical developments of chemical and radiation hormesis from their respective inceptions in the late 1880's for chemical hormesis and early 1900's for radiation hormesis to the mid 1930's to 1940 during which both hypotheses rose to some prominence but then became marginalized within the scientific community. This analysis documents that there were marked differences in their respective temporal developments, and the direction and maturity of research. In general, the formulation of the chemical hormesis hypothesis displayed an earlier, more-extensive and more sophisticated development than the radiation hormesis hypothesis. It was able to attract prestigious researchers with international reputations from leading institutions, to be the subject of numerous dissertations, to have its findings published in leading journals, and to have its concepts incorporated into leading microbiological texts. While both areas became the object of criticism from leading scientists, the intensity of the challenge was greatest for chemical hormesis due to its more visible association with the medical practice of homeopathy. Despite the presence of legitimate and flawed criticism, the most significant limitations of both chemical and radiation hormesis and their respective ultimate undoing were due to their: (1) lack of development of a coherent dose-response theory using data of low dose stimulation from both the chemical and radiation domains; (2) difficulty in replication of low dose stimulatory responses without an adequate study design especially with respect to an appropriate number and properly spaced doses below the toxic threshold; (3) modest degree of stimulation even under optimal conditions which was difficult to distinguish from normal variation; and (4) lack of appreciation of the practical and/or commercial applications of the concepts of low dose stimulation. PMID- 10745297 TI - A new method for quantitative ultrasound measurements at multiple skeletal sites: first results of precision and fracture discrimination. AB - We investigated a new multisite quantitative ultrasound device that measures the acoustic velocity in axial transmission mode along the cortex. Using a prototype of the Omnisense (Sunlight Ultrasound Technologies, Rehovot, Israel), we tested the performance of this instrument at four sites of the skeleton: radius, ulna, metacarpal, and phalanx. Intraobserver (interobserver) precision errors ranged from 0.2% to 0.3% (0.3% to 0.7%) for triplicate measurements with repositioning. Fracture discrimination was tested by comparing a group of 34 women who had previously suffered a fracture of the hip, spine, ankle, or forearm to a group of 28 healthy women who had not suffered a fracture. Age-adjusted standardized odds ratios ranged from 1.6 to 4.5. Except for the ulna the sites showed a significant fracture discrimination (p < 0.01). The areas under the receiver operating curves (ROC) curves were from 0.88 to 0.89 for radius, metacarpal, and phalanx. A combination of the results from the three sites showed a significant increase of the ROC area to 0.95 (p < 0. 05). Our results show promising performance of this new device. The ability to measure a large variety of sites and the potential to combine these measurements are promising with regard to optimizing fracture risk assessment. PMID- 10745298 TI - pQCT measurement of bone parameters in young children: validation of technique. AB - Dual-energy X-ray absorptiometry (DXA) measures areal bone mineral density (BMD) and is affected by bone size. Peripheral quantitative computed tomography (pQCT) measures volumetric density and should not be affected by bone size. We hypothesized that pQCT could be used to measure geometric parameters in the tibia and that bone size, not density, would correlate with weight and height in 3 and 4 yr olds. Phantom measurements indicate that accurate results for cortical volumetric bone mineral density can be obtained at cortical thickness > 2 mm with voxel/speed sizes of 0.40/20 mm/s. Correlations between pQCT measured geometric bone parameters and phantom calculations were observed (all r > 0.96). Baseline data from an ongoing trial of 101 preschool children (53 male) were used to correlate bone parameters and anthropometrics. Total cross-sectional area, cortical area, and cortical thickness correlated with weight (r = 0.54, p < 0.001; r = 0.52, p < 0. 001; r = 0.30, p = 0.002) and height (r = 0.41, p < 0.001; r = 0.55, p < 0.001; r = 0.41, p < 0.001). Because of the small cortical thickness at this age (mean = 1.2 mm) cortical density was not analyzed. In a regression model including height and weight, weight was the only predictor of total cross-sectional area (p < 0.001); cortical thickness was predicted by height (p = 0.006). Both height (p = 0.005) and weight (p = 0.05) predicted the cortical area. In summary, pQCT accurately measures total cross-sectional area, cortical area, and cortical thickness in children age 3-4 yr. PMID- 10745299 TI - The effects of cyclical etidronate on early postmenopausal bone loss: an open, randomized controlled study. AB - Cyclical etidronate is well established in the treatment of postmenopausal osteoporosis, but there are less data on its effects on bone loss in the early menopause. The aim of this study was to investigate the effects of cyclical etidronate therapy on bone loss in the lumbar spine and proximal femur in early menopausal women. Seventy-seven women aged over 40 yr who had ceased menstruating 6-36 mo prior to enrollment into the study were recruited into an open, randomized controlled study of cyclical etidronate therapy. Bone mineral density in the lumbar spine and proximal femur was assessed by dual-energy X-ray absorptiometry using a Lunar DPX bone densitometer. Fifty-five women completed the study. At the end of the 104-wk study period, significant treatment effects were observed in both the lumbar spine and the proximal femur. The estimated mean treatment effect in the lumbar spine was 2.79% (95% confidence interval 0.47, 5.10; p = 0.019). Corresponding figures for the femoral neck and greater trochanter were 3.23% (0.63, 5.82; p = 0. 016) and 3.77% (1.09, 6.45; p = 0.007). No significant differences between the groups were demonstrated at Ward's triangle. These results demonstrate that cyclical etidronate therapy prevents bone loss in the spine and proximal femur in early postmenopausal women and provides a safe and effective alternative for women who are unwilling or unable to tolerate hormone replacement therapy. PMID- 10745300 TI - Measurement precision of body composition variables using the lunar DPX-L densitometer. AB - The objective of the study was to determine the precision of total- and regional body composition measurements from a total-body scan using dual-energy X-ray absorptiometry (DXA). This is critical information necessary to determine the smallest change from baseline that could be detected with statistical significance when conducting longitudinal measurements of body composition variables in an individual. Twenty volunteers were scanned once each day for 4 consecutive days using a Lunar DPX-L densitometer and manufacturer-supplied software (version 1.3z). Coefficients of variation (CV, %) derived from data using the (preferred) extended research mode of analysis were 0.62, 1.89, 0.63, 2.0, 1.11, 1.10, and 1.09% for total-body bone mineral density (BMD), total percentage fat, total body tissue mass, fat mass, lean mass, bone mineral content (BMC), and total bone calcium, respectively. Regional measurements (arm, leg, trunk, pelvis, and spine) were less precise than total body measurements, with CVs in the range of 1% to 3% (but fat mass for arms was 4.26%, trunk 3.08%, BMC 3.65%). Small but statistically significant differences in mean values for most body composition variables were found when data were compared between extended and standard modes of analysis. Inconsistent use of analysis mode in a cohort or when following a patient longitudinally may negatively affect precision. We conclude that the measurement precision of total and regional body composition variables was generally comparable to the precision limits typically associated with lumbar spine and proximal femur BMD data. PMID- 10745301 TI - Quantitative ultrasound of the calcaneus: an in vivo comparison with dual-energy X-ray absorptiometry and magnetic resonance imaging. AB - The current study was performed in a clinical setting and aimed to evaluate the relationship between quantitative ultrasound (QUS) of the calcaneus with bone mineral density (BMD) assessed with dual-energy X-ray absorptiometry (DXA) and with variables derived from magnetic resonance imaging (MRI). Thirty-two postmenopausal women (mean age 61 years) were studied at the level of the nondominant calcaneus. QUS was performed using a DTU-one device including parametric imaging and yielded speed of sound (SOS) and broadband ultrasound attenuation (BUA) data. DXA was performed at a matched region of interest (ROI) in the calcaneus, using a Hologic QDR 4500 device. MRI, also performed at a matched ROI, yielded, using a Siemens Magnetom Vision device, the inverse of the transverse relaxation time (1/T(2)(*)) and the phase standard deviation (PSD). The strongest relationship between QUS and the other variables involved BUA and BMD (r &equals: 0.677, p < 0.001); 1/T(2)(*) showed a trend to correlation with SOS (r = 0.359, p = 0. 044) and with BMD (r = 0.364, p = 0.040), while the relationship between 1/T(2)(*) and BUA, PSD and BUA, PSD and SOS, PSD and BMD remained far from significance. Regression analysis of QUS, DXA, and MRI variables against age showed a trend to significant decline only for 1/T(2)(*) (r = -0.409, p = 0.020). In conclusion, this study shows that BUA of the calcaneus has the best correlation with BMD, and that, at least in a clinical setting, the ability of QUS to give information about bone structure is limited. PMID- 10745302 TI - Is there a difference between right and left femoral bone density? AB - Because of the known differences in bone mineral density (BMD) of the dominant and nondominant forearms, it has been customary to measure BMD of the nondominant forearm to reduce variance. However, it is less clear whether such systematic differences exist between BMD of the two hips. Accordingly, we measured BMD of both hips and the spine in 131 consecutive white women who presented to a community based private practitioner for evaluation and advice on osteoporosis. There was a highly significant correlation between BMD of the two hips at the femoral neck, trochanter, and Ward's triangle (r = 0.91, 0.93, and 0.89; p < 0.0001 for all three sites), and also between BMD at various measurement sites in the hip and the spine BMD (p < 0.0001). Although, there was no significant difference between the right and left femoral neck BMD, there were small (approximately 1-2.5%) but significant differences between BMD of the two hips at the trochanter and Ward's triangle (p = 0.008 and 0. 005, respectively). The left hip BMD was consistently higher than the right hip BMD at all measurement sites. Because of the small sample size of the left-handed persons, we were unable to determine the influence of dominant handedness on hip BMD. We concluded the following: (1) BMDs of the two hips are highly correlated at relevant measurement sites; (2) there does not appear to be a dominant hip as there is dominant forearm; (3) because of strong agreement between BMD of the two hips there is little justification to measure both hips in routine clinical practice. PMID- 10745303 TI - Artifact in bone mineral measurements during a very low calorie diet: short-term effects of growth hormone. AB - Short-term effects of growth hormone on bone metabolism and soft tissue collagen metabolism during weight loss in obese subjects on a very low calorie diet were investigated in a double-blind, placebo-controlled design. Twenty healthy obese women (BMI between 33 and 45 kg/m(2)) aged 21-48 yr were followed for 8 wk: half received growth hormone. A 740-kcal diet was administered the first 4 wk, followed by a 1200-kcal diet. Lumbar spine BMC, total-body fat mass, total-body lean body mass, total-body BMC, and total-body bone area were measured by dual energy X-ray absorptiometry along with biochemical markers of bone turnover. Body weight decreased by 5.5% and fat mass by 11.4%. There were no changes in biochemical bone markers in the placebo group despite a marked decrease in BMC (3.1%). Projected total bone area decreased proportional to BMC (r = 0.89) during the weight loss. Growth hormone treatment did not modulate the decrease in lean body mass, body weight, fat mass, or BMC, but increased bone turnover markers. Growth hormone did not change the results concerning BMC, projected bone area, BMD, lean body mass or fat mass. Since 89% of the observed change in BMC could be explained by alterations in projected bone area without changes in biochemical bone markers, it is concluded that a large part of the observed decrease in BMC during weight loss may be due to scanner artifact. PMID- 10745304 TI - A fracture risk profile using single-site bone density assessment and clinical risk factors. AB - The Risk Factor Profile combines single-site bone density assessment and selected clinical risk factors to help estimate the risk of osteoporotic fracture for Caucasian postmenopausal women over the next 5 years. The bone density assessment uses T score cut-off values equivalent to the WHO definitions of osteoporosis and osteopenia at the hip that would identify 16-17% of women over 50 years of age as "high" risk. The clinical risk factors are defined as "major" producing about a doubling of fracture risk independent of bone density, and "minor" where the predictive power is less certain. The indications for a risk factor assessment using the Profile are the same as for any bone density assessment with the exception of monitoring response to treatment. In practice the clinical risk factors can be measured at the time of the bone density estimation taking only a few minutes. The decision to treat, or not to treat, a postmenopausal woman at high or medium risk will ultimately depend on the overall assessment of risks and benefits, costs of treatment and the desires of the patient. The Risk Factor Profile aims to supply the clinician with a simple tool to aid treatment decision making. PMID- 10745305 TI - Etridronate therapy in the treatment and prevention of osteoporosis. AB - Etidronate disodium is an oral bisphosphonate compound known to reduce bone resorption through the inhibition of osteoclastic activity. This article is a review of its efficacy and safety in the treatment and prevention of postmenopausal and corticosteroid-induced osteoporosis. In general, studies of cyclical etidronate therapy (400 mg daily for 2 wk every 3 mo) have found a significant improvement in bone density. These studies have not been powered to study fracture incidence, but a reduced fracture rate has been found in some of the studies reviewed. Studies examining cyclical etidronate in the prevention of osteoporosis indicate it prevents early menopausal bone loss and is free of significant side effects. In both prevention of corticosteroid-induced osteoporosis and treatment of patients who have been on long-term corticosteroid therapy, cyclical etidronate appears to increase bone density and prevent further loss of bone. In summary, a review of available literature pertaining to the use of etidronate in prevention and treatment of primary and secondary osteoporosis has been presented. This review suggests etidronate, used as a cyclical therapy, is a safe and effective therapy. The weight of evidence suggests it is capable of reducing fracture risk in patients with osteoporosis. Increases in bone density at the spine and hip are not as pronounced as with some other bisphosphonates, particularly alendronate, but no direct clinical comparison trials of significant size or duration have been undertaken. PMID- 10745306 TI - Nutritional osteomalacia: substantial clinical improvement and gain in bone density posttherapy. AB - A 52-yr-old white female presented with worsening low back and hip pain, associated with lower limb proximal muscle weakness and a waddling gait. Her laboratory evaluation revealed hypocalcemia, hypophosphatemia, a very low 25 hydroxyvitamin D level of less than 5 ng/mL, and a bone mineral density in the osteoporotic range. Her laboratory studies were consistent with osteomalacia, although this diagnosis was not established by histomorphometry. She avoided dairy products, spent little time outdoors, and when she went out, she covered her face, arms, and legs. She was on no medication. Her workup for malabsorption including sprue was negative. She was treated with calcium plus high-dose vitamin D 600,000 IU intramuscularly twice witihin 2 mo and had an impressive clinical improvement. Her difficulty with ambulation improved within 1 wk of start of therapy. Her bone mineral density increased by 40% at the spine and 35% at the hip at 4 mo of therapy, by 63% and 39% at 10 mo, and by 62% and 52% at 15 mo at these sites, respectively. Treatment of osteomalacia is extremely rewarding, with dramtic clinical improvement and normalization of bone mineral density. PMID- 10745307 TI - Long-term complications following childhood cancer. PMID- 10745309 TI - Demasking of subclinical left ventricular dysfunction in anemic children. AB - OBJECTIVE: To study subclinical left ventricular dysfunction in anemic children. SETTING: Hospital. SUBJECTS: The study population consisted of 52 cases of anemia (16 mild, 14 moderate, 22 severe) and 20 normal age and height matched children aged between 7-12 years. METHODS: These children were subjected to Echocardiography on Ascuson XP model using 3.5 MHz Transducer during rest and immediately after maximum tolerated exercise on Quniton Model Q-5000 treadmill using modified Naughton protocol. Left ventricular end diastolic dimensions, left ventricular systolic dimension, fractional shortening, left ventricular end diastolic volume, systolic volume, stroke volume and ejection fraction were studied on M-mode echocardiography, and E/A ratio on doppler mode. RESULTS: Significant differences were observed in resting heart rate, gain in heart rate at peak exercise compared to basal values and double product, total exercise duration and metabolic equivalents at peak exercise in controls and anemic subjects. Left ventricular dysfunction was present in all severely anemic subjects after maximum tolerated exercise on treadmill. The only evidence of left ventricular diastolic dysfunction was in form of decreased left ventricular end diastolic volume after exercise in moderate and severely anemic subjects. CONCLUSIONS: Exercise is helpful in demasking subtle left ventricular dysfunctions in anemic subjects that are not detected during rest. PMID- 10745308 TI - Micronutrient deficiency disorders amongst pregnant women in three urban slum communities of Delhi. AB - OBJECTIVE: To assess the prevalence of three micronutrient deficiency disorders (MDDs), i.e., iron deficiency, iodine deficiency and vitamin-A deficiency individually and in combination amongst pregnant women. METHODS: A hospital based study was conducted amongst 829 pregnant women of II and III trimester attending antenatal clinic, Rural Health Training Center (RHTC), Najafgarh, New Delhi. Anemia was assessed by the presence of clinical signs and by hemoglobin levels. Iodine deficiency disorders (IDD) were assessed by clinical examination of thyroid gland and by urinary iodine excretion levels. Iodine content of the salt consumed by the pregnant women was also assessed by iodometric titration method. Vitamin A deficiency (VAD) was assessed by the presence of clinical symptoms of nightblindness. Current dietary intake, morbidity conditions on the day of survey and anthropometric measurements of pregnant women were also documented. RESULTS: Prevalence of anemia, IDD and VAD amongst pregnant women was 78.8%, 22.9% and 4.8%, respectively. One per cent of the pregnant women had concomitant presence of all the three MDDs. Pregnant women having combined prevalence of IDD and anemia, IDD and VAD, and VAD and anemia was 15.1%, 0.18% and 2.69%, respecively. Eighty nine per cent of the pregnant women were consuming salt with iodine content of more than 15 ppm which was recommended at household level. Results on dietary intake showed that 18%, 34%, 85% and 57% of the pregnant women were consuming less than 50% of calories, proteins, iron and b-carotene, respectively as compared to their RDA. Forty per cent of the pregnant women were suffering from various morbidity conditions on the day of survey. CONCLUSIONS: The prevalence of micronutrient deficiencies amongst pregnant women of urban slum communities is high. PMID- 10745310 TI - Interventional pediatric cardiology. PMID- 10745312 TI - Painless invasive procedures. PMID- 10745311 TI - Systemic antifungal therapy in pediatric practice. PMID- 10745313 TI - Prevalence of anti-microbial resistance among respiratory isolates of Haemophilus influenzae. PMID- 10745314 TI - Cognitive function and behavior in epileptic children of school going age. PMID- 10745315 TI - Cerebral palsy--clinical profile and predisposing factors. PMID- 10745316 TI - Non surgical treatment of complete inferior vena cava obstruction in a child with Budd-Chiari syndrome. PMID- 10745317 TI - Syndrome of renal magnesium wasting and nephrocalcinosis. PMID- 10745318 TI - Post-traumatic hepatic cyst. PMID- 10745319 TI - Harmful effects of aerosolized bronchodilator therapy in bronchiolitis. PMID- 10745320 TI - Leukemic transformation in Fanconi's anemia. PMID- 10745321 TI - Epidermolysis bullosa. PMID- 10745322 TI - Polio virus isolates from AFP cases. PMID- 10745323 TI - Polio virus isolates from AFP cases - reply PMID- 10745325 TI - Pulse polio campaign - reply PMID- 10745324 TI - Pulse polio campaign. PMID- 10745326 TI - Scurvy persists in the current era. PMID- 10745328 TI - Is colostrum really discarded by Indian mothers? PMID- 10745327 TI - Diclofenac induced fatal anaphylactic reaction. PMID- 10745329 TI - Treatment of tachycardia in infants and children. PMID- 10745330 TI - Investigation of an epidemic of Reye's syndrome in northern region of India. AB - OBJECTIVE: To determine the extent, epidemiological and clinical features of an epidemic of non-inflammatory encephalopathy in northern region of India. DESIGN: Surveillance of referred cases having unconsciousness after a short bout of fever during October and November 1997. Case control study in 7 most affected villages. METHODS: Active case finding was done to assess the extent and severity of the epidemic by interviewing health professionals and by reviewing mortality records in 10 districts of Haryana, Punjab and Chandigarh. A house to house survey was conducted in seven most affected villages. A case was defined as any child of less than 15 years of age, who had prodromal fever followed by vomiting and unconsciousness with subsequent recovery or death. Two age and sex matched controls who had fever without unconsciousness were taken for each case, one from nearby house and another staying furthest from the affected house. These groups were compared for various epidemiologic factors, clinical features and treatment pattern. Residual medicines used by affected patients were tested for presence of salicylate. Local village practitioners were interviewed for their knowledge and attitude towards use of aspirin in a febrile child. RESULTS: Information regarding 129 affected children (M: F=1 : 1) could be obtained. Age ranged between 1 to 12 years (mean 5.8 years). Most were from rural or semi-suburban areas. Attack rate was 5.4/1000 and case fatality rate was 72%. Multiple sibs were affected in 9.3%. History of fever was reported by 83%, vomiting preceding unconsciousness by 83% and abnormal behavior by 65%. Abnormal posturing was reported in 55%. Seventeen (61%) of 28 samples had IgM antibodies in serum/CSF against measles. Twelve (36%) of 33 serum samples tested positive for Varicella zoster virus. None gave history of aspirin intake and 10 samples of residual drugs did not contain salicylate. However, 6 out of 19 blood samples taken from affected patients contained salicylate. Environmental factors were in favor of Japanese encephalitis (JE) but brain biopsy and serology disproved it. Based on earlier report of JE from this area, the cases in present epidemic were being reported as JE before this study was undertaken. Intensive fogging with malathion was being undertaken as antimosquito measure, specially around the affected houses. Local village practitioners (n = 37) were unaware of contraindications of aspirin in a febrile child. CONCLUSION: Measles and varicella zoster emerged as the probable etiologies for the viral prodrome precipitating these cases of Reye's syndrome. Aspirin might have a contributory role. Malathion is another putative cofactor. PMID- 10745331 TI - Fulminant hepatic failure: etiology, viral markers and outcome. AB - OBJECTIVE: To investigate the etiology and outcome of fulminant hepatic failure (FHF) in children. SETTING: Hospital based descriptive. METHODS: 36 children (22 males and 14 females) presenting with FHF over a period of one year were investigated. The ages ranged from 1.5 to 9 years. FHF was defined as occurrence of encephalopathy within eight weeks of onset of jaundice with no evidence of pre existing liver disease. Detailed history, clinical examination, routine biochemical parameters and relevant diagnostic tests were carried out. Viral markers studied were anti HAV-IgM, HBsAg, anti HBc-IgM, anti-HCV and anti HEV IgM. RESULTS: A viral etiology could be established in 22 children (61.1%). Hepatitis A (n = 12), Hepatitis B (n = 3), Hepatitis A and B (n = 2), and Hepatitis A and E (n = 4). Two children had enteric fever (1 with associated HEV), 2 children had Wilson's disease, 1 child had Indian Childhood Cirrhosis (ICC) and 2 children had drug induced hepatitis. Etiological diagnosis was not possible in 8 children (22%). Fourteen children (39%) died. Poor outcome was associated with spontaneous bleeding, raised prothrombin time, lower transaminases and higher bilirubin on admission. CONCLUSION: Viral hepatitis is the commonest cause of FHF in children. HAV alone or in combination is responsible for upto 50% of all FHF in children. Chronic liver disease can also present as FHF. Etiological diagnosis is not possible to upto one-fourth of all cases. PMID- 10745333 TI - Carrier screening and prenatal diagnosis of beta-thalassemia. PMID- 10745332 TI - Multicenter randomized placebo controlled trial of therapy with intravenous immunoglobulin in decreasing mortality due to neonatal sepsis. AB - OBJECTIVE: To determine whether therapy with intravenous immunoglobulin G (IVIG) would decrease mortality in neonatal sepsis. SETTING: Three tertiary care neonatal intensive care units in the city of Bangalore. METHODS: All neonates admitted to the Neonatal Intensive Care Units with the clinical diagnosis of sepsis and having at least C-reactive protein and one other rapid diagnostic criteria positive were enrolled. Neonates with a birth weight of less than 1000 g and those with any major congenital malformation were excluded. The neonates were randomized to receive 1 g/kg of IVIG on three consecutive days or an equivalent amount of placebo. The rest of the treatment including antibiotics and supportive care was as per the treating physician's decision. The main outcome variable was survival. RESULTS: The trial was carried out over a period of 8 months and recruited 58 neonates. Seven neonates who qualified but did not receive either IVIG or placebo were taken into a separate control group, and one baby who received only one dose of IVIG was excluded from the analysis. Twenty-five neonates were enrolled into the IVIG arm and 25 in the placebo arm. The neonates in the therapy and placebo groups were comparable in terms of birth weight (2144+/-675 g vs. 2072+/-682 g), gestation (37.0+/-3.56 vs. 35.8+/-3.52 weeks), sex distribution, duration of stay, and number requiring ventilation. The placebo group had a significantly higher number of babies with positive blood culture. Seven babies in each group died (p>0.05). There was no significant benefit in using IVIG (OR 1.0; 95% CI 0.25-4.07) (p = 0.74). CONCLUSION: In the sample studied therapy with IVIG did not reduce mortality in neonatal sepsis PMID- 10745334 TI - Numerical methods to summarize data. PMID- 10745335 TI - Evaluation of physiological and behavioral response to pain following heel prick with different techniques for dextrostix assessment in healthy preterm neonates. PMID- 10745336 TI - Prevalence and persistence of hepatitis A antibody during the first year of life, in Turkish infants. PMID- 10745337 TI - Clinico-biochemical spectrum of hypokalemia. PMID- 10745338 TI - Factor analysis of acute respiratory infections among under fives in Delhi slums. PMID- 10745339 TI - Acute transverse myelitis due to spinal epidural hematoma-first manifestation of severe hemophilia. PMID- 10745340 TI - Palatal nerve palsy and cervical adenopathy in a probable case with cat scratch disease. PMID- 10745342 TI - Pleural effusion-a rare complication of aluminium phosphide poisoning. PMID- 10745341 TI - Congenital sideroblastic anemia. PMID- 10745343 TI - Atlanto-axial subluxation in JRA. PMID- 10745344 TI - Sequencing of vaccine doses. PMID- 10745345 TI - Sequencing of vaccine doses - reply PMID- 10745347 TI - How to avoid multiple Injections? - reply PMID- 10745346 TI - How to avoid multiple injections? PMID- 10745348 TI - Three nostril nose. PMID- 10745350 TI - Hepatitis B virus infection and pregnancy. PMID- 10745349 TI - Osteogenesis imperfecta tarda type IV. PMID- 10745351 TI - Hepatitis B virus infection and pregnancy - reply PMID- 10745352 TI - Concomitant steroid and albendazole for treating neurocysticercosis. PMID- 10745353 TI - Concomitant steroid and albendazole for treating neurocysticercosis - reply PMID- 10745354 TI - After RCH program be ready for IMCI strategy. PMID- 10745355 TI - IMCI strategy for India. PMID- 10745356 TI - IMCI strategy for india - reply PMID- 10745357 TI - Child and maternal health in rural areas of Chandigarh. PMID- 10745358 TI - Child and maternal health in rural areas of chandigarh - reply PMID- 10745359 TI - Anterior encephalocele mimicking choanal atresia. PMID- 10745360 TI - Tuberculous meningitis associated with diabetic ketoacidosis. PMID- 10745361 TI - Glucose and small for gestational age infants. PMID- 10745362 TI - Epidemiological characteristics of poliomyelitis in Delhi, 1997. AB - OBJECTIVE: To describe the epidemiological characteristics of poliomyelitis in Delhi in 1997 after four consecutive statewide immunization campaigns with oral polio vaccine (OPV). METHODS: Stool samples were collected from 158 cases of acute flaccid paralysis (AFP) along with their age, sex, residential address, immunization history and dates of onset of paralysis, reporting and investigation. The samples were processed for isolation of polioviruses. In addition, historical data on vaccination coverage surveys and OPV testing were reviewed. These data were analyzed to understand the epidemiological patterns of poliomyelitis in Delhi. RESULTS: Of 158 cases of AFP, about 23% were investigated within 2 days of onset of paralysis. Two samples each were collected from 97 (61%) cases, and one each from the remaining cases. Detection of 158 cases of AFP gave an incidence of 1.34 per 100,000 population. About 36% (57/158) of AFP cases excreted poliovirus, mostly (53/158) wild poliovirus. Of the wild poliovirus isolates, 72% (38/53) and 25% (13/53) were serotypes P1 and P3 respectively; 2 isolates were P2. Almost 95% (146/154) of AFP cases and all the laboratory confirmed cases (excreting wild poliovirus) occurred in children below 5 years of age. Only one-third of AFP (55/158) or laboratory confirmed cases (18/53) had received 3 or more doses of OPV before onset of paralysis. About one-fourth of cases in both the categories were totally unvaccinated. AFP cases occurred round the year but peaked in November-December. Peaks were always observed during July August in the past. The cases were widely scattered without any obvious clustering in any locality. CONCLUSIONS: Poliomyelitis has declined substantially in Delhi. The study underscores the need for further efforts to improve vaccine coverage levels, AFP surveillance, and cold chain maintenance to achieve the complete interruption of transmission. PMID- 10745363 TI - Impact of mid day meal program on educational and nutritional status of school children in Karnataka. AB - OBJECTIVE: To assess the effect of the Mid Day Meal (MDM) Program on enrollment, attendance, dropout rate and retention rate in the schools and its impact on nutritional status as well as on school performance. DESIGN: Comparison by multistage random sampling. SUBJECTS: Primary school children, who are attending the school in the MDM and non-MDM areas. RESULTS: A total of 2,694 children (MDM: 1361; Non-MDM : 1333) from 60 schools were covered in the study. Results of the study indicated better enrollment (p<0.05) and attendance (p<0.001), higher retention rate with reduced dropout rate (p<0.001) a marginally higher scholastic performance and marginally higher growth performance of MDM children. CONCLUSION: MDM program is associated with a better educational and nutritional status of school children in Karnataka. PMID- 10745364 TI - Current trends in management of the beta thalassemias. PMID- 10745365 TI - Clinical profile and sero conversion pattern of children with HBsAg positivity. PMID- 10745366 TI - Seroprevalence of antibodies to hepatitis A virus among children in Northern India. PMID- 10745367 TI - HIV infection in children: Indian experience. PMID- 10745368 TI - Current status of prevalence of goiter and iodine content of salt consumed in District Solan, Himachal Pradesh. PMID- 10745369 TI - Pediatric emergencies in otolaryngology in a metropolitan city. PMID- 10745371 TI - Imerslund-Grasbeck syndrome. PMID- 10745370 TI - Mediastinal yolk sac tumor--a rare presentation. PMID- 10745372 TI - Bacteremia due to Gemella morbillorum. PMID- 10745373 TI - Cornelia de Lange syndrome: discordance in twins. PMID- 10745374 TI - Radiological demonstration of ascaris in esophagus. PMID- 10745375 TI - Postexposure prophylaxis in hepatitis B infection. PMID- 10745376 TI - Integrating BCG and HBV into the immunization schedule. PMID- 10745377 TI - Ataxia telangiectasia. PMID- 10745379 TI - Brachioradialis reflex. PMID- 10745378 TI - Iniencephaly in a live born and not Klippel Feil syndrome. PMID- 10745381 TI - Adherence to anti-tuberculosis treatment. PMID- 10745380 TI - Brachioradialis reflex. PMID- 10745382 TI - Caffeine and asthma. PMID- 10745383 TI - Stepping into Y2K PMID- 10745384 TI - Tuberculosis control, without protection from BCG. PMID- 10745385 TI - Effectiveness and cost-effectiveness of albendazole in improving nutritional status of pre-school children in urban slums. AB - OBJECTIVE: To study the clinical efficacy and the incremental cost-effectiveness of albendazole in improving the nutritional status of pre-school children. DESIGN: Single blind, placebo-controlled trial with child as the unit of randomization. SETTING: In the Anganwadi centers of the Integrated Child Development Services situated in the urban slums of Lucknow, North India. METHODS: Thirty-two Anganwadi centers were randomly selected for the trial. Included were registered resident children between 1.5 to 3.5 years of age with informed and written parental consent. The intervention group received 600 mg of albendazole powder every six months while the placebo group received same quantity of calcium powder. Enrolled children were contacted once in six months from January 1995 to 1997 and given treatment. The outcome measure were change in the proportion of underweight (weight for age <-2.00z), stunted (height for age < 2.00z) children and the cost per child prevented from becoming stunted. RESULTS: There were 610 and 451 children in the albendazole and placebo groups, respectively. Mean age at recruitment was 31.8 months (SD: 9.7). Follow-up and compliance in both the groups was >95%. During the 2 year follow-up, the proportion of stunted children increased by 11.44% and 2.06% in the placebo and albendazole groups, respectively, and the difference was 9.38% (95% CI 6.01% to 12.75%; p value <0.0001). Direct fecal smear was positive for the ova of ascaris in 41.2% and 55.3% children in the albendazole and placebo groups, respectively at the end of the study (p value <0.001). The annual family expenditure on illness in the recruited child was Rs. 743 (SD: 662) and Rs. 625 (SD: 609) in the albendazole and the placebo groups, respectively. The incremental cost effectiveness ratio was Rs 543.00 for each case of stunting prevented with albendazole. There was no difference in the various morbidity or cognitive performance, as judged by the revised Denver prescreening questionnaire, in both the groups at enrollment as well as at the end of the study. CONCLUSIONS: Six monthly albendazole reduces the risk of stunting with a small increase in the expenditure on health care from the payer's perspective. Larger trials are needed to study the effect of albendazole on prevention of stunting, cognitive functions and all-cause childhood mortality. PMID- 10745386 TI - Wilson's disease with hepatic presentation in childhood. AB - OBJECTIVE: To evaluate clinical, laboratory findings, treatment and long-term follow-up of children with Wilson's disease with hepatic presentation. DESIGN: Retrospective study with a median follow-up period of 9 years. SETTING: University medical center. SUBJECTS: Thirty-four children with hepatic involvement, ranging in age from three to fifteen years, were diagnosed as Wilson's disease over an eighteen year period. METHODS: The diagnosis was based on the presence of family history and Kayser-Fleischer rings, low serum ceruloplasmin levels and increased urinary and hepatic copper concentrations. RESULTS: Four patients had also neurological manifestations. Eight patients were diagnosed as fulminating hepatic failure resulting in death in a few days. The most common symptoms were abdominal distension and abdominal pain. Hepatomegaly was the predominant physical finding and serum transaminases were elevated in most of the patients. Twenty patients had cirrhosis and six had chronic hepatitis histopathologically. All patients with fulminating hepatic failure had hyperbilirubinemia with normal alkaline phosphatase and higher aspartate aminotransferase than alanine aminotransferase. Patients were treated with D penicillamine and zinc sulphate. Three patients underwent liver transplantation. Four more patients besides patients with fulminating hepatic failure died due to end stage liver disease. Twenty-two patients were followed for median 9 years. Four patients with poor compliance progressed to decompensated cirrhosis and the others were stable. CONCLUSIONS: Liver disease with unknown origin with positive family history and parental consanguinity should imply Wilson's disease strongly. PMID- 10745387 TI - Neonatal diarrhea in a diarrhea treatment center in Bangladesh: clinical presentation, breastfeeding management and outcome. AB - OBJECTIVE: To study the clinical presentation and outcome of neonates admitted with diarrhea, and effect of counseling their mothers for exclusive breastfeeding. DESIGN: Prospective study. SETTING: Inpatient unit. RESULTS: Two hundred and forty-four neonates were studied during 1994-95. Their mean (SD) age was 18 (6.2) days, and body weight and length were 2.18 (0.52) kg and 47.5 (3.2) cm, respectively. More neonates had some dehydration than severe dehydration (78% vs. 11%), with mean (SD) serum bicarbonate values 9.6 (5.1) mmol/1. V. cholerae was isolated from 25 (12%), Shigella spp. from 8 (3%), and Salmonella spp. from 3 (1%) of the patients who had rectal swab cultures. Mean (SD) hospital stay was 3.6 (2.1) days, during which the majority (80%) recovered fully, but 29 (13%) left earlier. Eleven (4%) of the neonates had to be referred elsewhere for treatment of other problems and 7 (3%) died. None of the neonates were exclusively breastfed on admission. Excluding mothers of adopted neonates, breastfeeding counseling enabled 64% of the mothers to convert to exclusive breastfeeding during the hospital stay. CONCLUSIONS: Most of the neonates admitted with diarrhea were small and underweight, and had poor feeding practices. The majority of neonates recovered soon, and were exclusively breastfeeding when discharged from the hospital. Breastfeeding counseling should be included as an integral part of case management at all health facilities. PMID- 10745388 TI - Hemophilia. PMID- 10745389 TI - Graphical methods to summarize data. PMID- 10745390 TI - An approach to neurometabolic disorders by a simple metabolic screen. PMID- 10745392 TI - Immunogenicity of indigenous recombinant hepatitis B vaccine in infants following a 0, 1, 2-month vaccination schedule. PMID- 10745391 TI - Chediak-Higashi syndrome: a report of eight cases from three families. PMID- 10745393 TI - Drowning in childhood: a population based study. PMID- 10745394 TI - Familial neuroblastoma. PMID- 10745395 TI - Dorfman-Chanarin syndrome: a rare neutral lipid storage disease. PMID- 10745397 TI - Inadvertent passage of infant feeding tube into the stomach through a tracheo esophageal fistula. PMID- 10745396 TI - Partial lipodystrophy in a boy. PMID- 10745399 TI - Subcutaneous fat necrosis of newborn. PMID- 10745398 TI - Osteopetrorickets. PMID- 10745400 TI - Primary empty sella syndrome. PMID- 10745401 TI - Should Hib vaccine be given priority over hepatitis B vaccine. PMID- 10745402 TI - Universal immunization with hepatitis B vaccine- what it will cost. PMID- 10745403 TI - Hydatiduria. PMID- 10745404 TI - Hematuria--sole manifestation of hemorrhagic disease of newborn. PMID- 10745405 TI - Level of iodine content in iodized salt. PMID- 10745406 TI - Nutritional status of adolescent school children in rural north India: a study with questionable methodology. PMID- 10745407 TI - Primary nocturnal enuresis--where do we stand today? PMID- 10745408 TI - Intravenous pulse cyclophosphamide--a new regime for steroid resistant focal segmental glomerulosclerosis. AB - OBJECTIVE: A prospective study was conducted to evaluate the role of intravenous pulse cyclophosphamide (IVCP) infusions in the management of children with steroid resistant (SR) idiopathic focal segmental glomerulosclerosis (FSGS). METHODS: The study group comprised of 20 consecutive children with idiopathic nephrotic syndrome secondary to FSGS who were SR. All of them were subjected to standard baseline investigations. They were started on monthly infusions of IVCP in a dose of 500-750 mg/m2. Adjunctive prednisolone was given in a dose of 60 mg/m2/day for 4 weeks followed by 40 mg/m2/alternate day for another 4 weeks and tapered off over next 4 weeks. RESULTS: The study group comprised of 15 boys and 5 girls with mean age of onset of disease of 5.5 +/- 3.5 years. Two of these children had chronic renal insufficiency prior to starting therapy. At the end of the study, after a mean duration of disease (since onset of NS) of 77 +/- 55 months, all 20 children had normal renal functions. After a mean follow up post IVCP therapy of 21.2 +/- 13.4 months, 13 of the 20 children (65%) had attained a complete remission. Of these, 10 children were infrequent relapsers, 2 frequent relapsers and 1 steroid dependent. The mean duration of remission following last dose of IVCP in these children was 12.5 +/- 11.9 months. Of the 7 children who continued to be proteinuric, 3 became edema free and have not required any albumin infusion or diuretics. One other died due to peritonitis 2 years after the last dose of IVCP. The mean total protein and serum albumin levels following the IVCP infusion were significantly higher than those prior to therapy (6.5+/ 1.0 mg/dl Vs 5.0+/-0.8) (p=0.0004) and (3.5+/-0.7 g/dl Vs 2.3+/0.7) (p = 0.000007) respectively. The serum creatinine levels following IVCP therapy (0.8+/ 0.2 mg/dl) were significantly lower than those prior to treatment (1.0+/-0.6 mg/dl) (p=0.02). The only side effects that were observed were transient nausea and vomiting during infusion (n=2) and alopecia (n=1). None of the children developed leukopenia or hemorrhagic cystitis. CONCLUSION: IVCP infusions appear to be safe, effective and economical therapeutic modality in steroid resistant children with idiopathic FSGS. PMID- 10745409 TI - Community studies on prevalence of HBsAg in two urban populations of southern India. AB - OBJECTIVE: To find out prevalence of HBsAg in general population, especially in under-five children. SETTING: Bangalore and Rajahmundry towns in southern India. METHODS: Localities were chosen as the sampling units in each town. About 10-20 households were randomly selected from each locality. Only the youngest but apparently healthy person present in the household was interviewed for age, sex and history of jaundice any time in life. Mothers were interviewed to collect data for children below 15 years of age. Blood samples were collected from these persons on filter paper strips (18-mm diameter disc, Whatman filter paper No. 3) by finger prick method. The samples were tested for HBsAg by Micro ELISA (Ortho Clinical Diagnostics). RESULTS: Overall, 3.3% (95% CI, 2.0-4.5) of 737 persons in Rajahmundry and 4.2% (95% CI, 2.8-5.5) of 816 persons in Bangalore were found carriers of HBsAg. Age-specific or sex specific carrier rates were similar in Rajahmundry as well as in Bangalore. Most of the carriers (96%) denied having jaundice ever in life. CONCLUSIONS: The results from this community based study are in agreement with the historical data from hospital based studies that about 3-5% of persons may be carriers of HBsAg and that the pool of chronic carriers of hepatitis B virus in India is built up in childhood and is then maintained in older children and adults. The results highlight the need of completing hepatitis B immunization during the infancy. PMID- 10745411 TI - Cerebrovascular disease in children. PMID- 10745410 TI - Free oxygen radicals in acute renal failure. AB - OBJECTIVE: To assess the levels of free oxygen radicals in acute renal failure and their predictive value in clinical outcome. DESIGN: Prospective. SETTING: Intensive care unit. METHODS: Study was conducted in 50 children (25 with acute renal failure and 25 age and sex matched controls). Blood urea, serum creatinine, serum protein, uric acid and free oxygen radical markers were estimated in both groups. Superoxide dismutase (SOD), glutathione peroxidase(GPx) and lipid peroxide (LPO) were estimated in blood by standard techniques. RESULTS: Hemolytic uremic syndrome (HUS) was a major cause of acute renal failure (52%), rest were due to acute glomerulonephritis (AGN), septicemia and renal venous thrombosis. In the renal failure group 56% of the patients were dialyzed (peritoneal) and the mortality was 28% (7/25). The levels of SOD, GPx and LPO were significantly raised in renal failure group. Higher values of LPO, SOD and GPx were documented in subjects who expired. The most important independent variable for predicting clinical outcome was LPO with a sensitivity of 89.4%, specificity of 93%, positive predictive value of 95%. CONCLUSION: Levels of free oxygen radicals (SOD, LPO and GPx) are raised in acute renal failure and these enzymes can be used as marker of renal injury. LPO levels are highly sensitivity and specific for predicting the clinical outcome PMID- 10745412 TI - Pediatric tertiary care and subspecialities in the new millennium. PMID- 10745413 TI - Comparison of pain response to venipuncture between term and preterm neonates. PMID- 10745414 TI - Validation of spot testing kit in the assessment of iodine content of salt: a multi-centric study. PMID- 10745415 TI - Pediatric parenteral nutrition: South African model and its relevance to the developing countries. PMID- 10745416 TI - Questionnaire survey on sponsorship of Continuing Medical Education program by pharmaceutical companies. PMID- 10745417 TI - Foreign body aspiration: Manipal experience. PMID- 10745418 TI - Immune thrombocytopenic purpura with multiple auto-antibodies: evolution into systemic lupus erythematosus. PMID- 10745419 TI - Triploidy syndrome. PMID- 10745420 TI - Repeated unifocal seizure in post neonatal infants with hypocalcemia. PMID- 10745421 TI - Pyloric atresia associated with intestinal atresias. PMID- 10745422 TI - Pertussis immunization in children with neurological illnesses. PMID- 10745423 TI - Poliovirus infection in the gastrointestinal tracts of children. PMID- 10745424 TI - Congenital varicella. PMID- 10745425 TI - Epidemiology of poliomyelitis in Delhi, 1997. PMID- 10745426 TI - Ceftibuten in enteric fever. PMID- 10745427 TI - Tetanus in immunized children. PMID- 10745428 TI - Vitamin A levels in plasma and concomitant intestinal parasitosis in Howrah. PMID- 10745430 TI - Neointimal hyperplasia and late pathologic remodeling in a porcine coronary stent model. AB - BACKGROUND: Although coronary stenting has been demonstrated to significantly reduce restenosis compared to conventional angioplasty, occurrence of in-stent stenosis still remains one of the major limitations. This study investigates the influence of stent strut diameter on injury, inflammatory response, thrombosis and neointimal hyperplasia in a porcine coronary artery. METHODS: Coil stents made of either a 0.12 mm, 0.15 mm or 0.18 mm wire were randomly implanted in the right coronary arteries of 30 pigs. Quantitative coronary angiography analysis was performed before, immediately after, and 6 weeks following the stenting procedure. At 6 weeks, histopathology for evaluation of injury, thrombosis and inflammation, as well as morphometry for calculation of the neointimal hyperplasia and internal elastic lamina area were performed. RESULTS: Quantitative coronary analysis showed similar quantitative data before and after stent placement in the three groups. At 6 weeks, however, a significantly bigger MLD was found in the 0.18 mm group. Morphometric analysis at 6 weeks confirmed these results, showing a significantly bigger lumen area in both the 0.18 mm (1.71 +/- 0.66 mm2) and 0.15 mm (1.36 +/- 0.53 mm2) groups compared to the 0.12 mm group (0.71 +/- 0.38 mm2). The calculated neointimal hyperplasia was similar in the three groups (0.12 mm: 1.93 +/- 0.51 mm2; 0.15 mm: 1.68 +/- 0.63 mm2; and 0.18 mm: 2.16 +/- 1.48 mm2). The internal elastic membrane area, however, was significantly bigger in the 0.18 mm (3.87 +/- 1.39 mm2) compared to the 0.12 group (2.65 +/- 0.53 mm2). CONCLUSION: These results suggest that pathologic remodeling can also play an important role in late lumen loss after stent implantation. PMID- 10745429 TI - Influence of radiographic contrast media (Iomeprol 350 versus Iopentol 350) on cutaneous microcirculation: single-center prospective randomized double-blind phase iv study in parallel-group design. AB - OBJECTIVE: This single-center, prospective, randomized, double-blind phase IV study in parallel-group design was carried out to investigate whether either of two different x-ray contrast media (iomeprol 350 or iopentol 350) injected into the axillary artery has any influence on cutaneous microcirculation. METHODS AND RESULTS: The investigation was carried out on two groups of patients (n = 10 in each group) who had to undergo a diagnostic heart catheter angiography. The confirmatory response variable for the study was the mean capillary erythrocyte velocity (mm/sec). Blood flow through the ipsilateral nail-fold capillaries was recorded continuously for 3 minutes before and 6 minutes after the injection of the randomly assigned x-ray contrast medium, and was evaluated off-line. A contrast medium-induced, rheologically determined disturbance of the microcirculation was found, which was due to two different effects. First, the high intrinsic viscosity (iopentol = 12.3 mPa.sec) led to an immediate reduction in capillary blood flow. This did not occur in the case of iomeprol (intrinsic viscosity = 7.5 mPa.sec). Second, the contrast medium molecules cause a morphological change in the erythrocyte membrane; echinocytes are formed and are further desiccated depending on osmolality of the contrast medium. CONCLUSION: The time course of the conversion of erythrocytes into echinocytes leads to a maximum reduction in capillary erythrocyte velocity of 30 seconds after the bolus of contrast medium. For the more viscous contrast medium of higher osmolarity (iopentol), this led to a significant overall reduction of up to 48.6% in capillary blood flow (p < 0.0001) that lasted for up to 150 seconds, while iomeprol did not significantly affect capillary blood flow (p = 0.2759). PMID- 10745431 TI - Evaluation of new 4 French catheters by comparison to 6 French coronary artery images. AB - The use of 4 Fr catheters for diagnostic coronary arteriography improves patient comfort and could potentially reduce costs by decreasing arterial complications and encouraging outpatient examination. We examined the performance of recently developed 4 Fr catheters (100 cm long, 0.042 inch internal diameter) through in vitro flow rate patterns and in vivo image quality. Flow patterns (flow rate calculated from pressure with the Hagen Poiseuille relation) were obtained by hand (10 ml volume; maximal effort, one-handed full injection) and power injection (10 ml volume; 5 ml/sec) in the 4 Fr catheters were compared to 5 Fr (0.047 inch) and 6 Fr (0.057 inch) catheters. In 20 patients, coronary arteriograms were obtained from 4 Fr hand injection (4 HI), 4 Fr power injection (4 PI), 6 Fr hand injection (6 HI ), and 6 Fr power injection (6 PI) were compared after being scored into 5 grades. Coronary diameter was assessed at two sites for all injections in the same group of patients. In power injections, peak flow rates were consistently 5.5 ml/sec in all catheters. Hand injections were much more variable; peak flow rate ranges for 5 different physicians were 3.5-6.0 ml/sec. in 6 Fr, 1.8-3.0 ml/sec. in 5 Fr and 1.4-2.0 ml/sec in 4 Fr catheters. Mean image scores of left arteriograms were significantly higher for 4 PI versus 4 HI and for 6 Fr images compared to 4 PI (p < 0.001). In right arteriograms, 4 HI scored significantly lower than other methods (4 PI, 6 HI and 6 PI; p < 0.001). In 4 HI left coronary arteriograms, the frequency of grade > 2 was 96% and > 3 was 82%. Power injection improved 4 Fr imaging to 99% greater than grade 2 and 93% greater than grade 3. There were no differences in the frequency of images above grade 3 in right coronary arteriograms. Measured coronary artery diameters at 40 normal and 17 stenotic sites by QCA analysis (Acom, Siemens AZ, Erlangen, Germany) did not influence the study data. We concluded that coronary arteriograms by hand injection with 4 Fr catheters were inferior compared to 6 Fr images. Power injection and/or larger catheters may be necessary to ensure the highest quality images are obtained. PMID- 10745432 TI - Spontaneous healing of primary dissection of the coronary artery. PMID- 10745433 TI - Therapeutic angiogenesis for critical limb ischemia to limit or avoid amputation. PMID- 10745434 TI - Elective stenting for subclavian artery total occlusions: use of combined radio femoral approach and a modified sheath delivery system. PMID- 10745435 TI - Vascular brachytherapy for prevention of restenosis: a brief history and overview. PMID- 10745436 TI - Plaque removal prior to stent implantation in native coronary arteries: why? when? and how? PMID- 10745437 TI - Coronary stenting challenges: technique, design and pharmacology. PMID- 10745438 TI - The use of intravascular ultrasound and spot stenting for the treatment of long lesions and small vessels. PMID- 10745439 TI - Stent trials in acute myocardial infarction. PMID- 10745440 TI - Therapeutic angiogenesis in patients with advanced coronary artery disease: hype or hope? PMID- 10745441 TI - Local drug delivery via transvascular injection. AB - BACKGROUND: In this experimental series we tested drug distribution and systemic leakage using local drug delivery with a new transvascular injection system. METHODS: Porcine femoral and carotid arteries (n = 56) underwent local drug application with a new 5 French (Fr) over-the-wire needle-injection catheter system (NIC) using three needles. A radioactive indicator [C14-Carvedilol, 2.0 milliliter (ml); 0.03 milligram (mg)] was injected in two carotid and two femoral vessels in parallel. Serial blood withdrawal was performed thereafter. After randomization to different explantation times, the vessels, perivascular tissue, liver and spleen were removed [0.5, 1, 1.5, 3 and 4 hours after injection, respectively]. Radioactivity was determined in a scintillation counter or with autoradiography. The indicator amount was calculated in relation to total drug amount (100%). RESULTS: Use of the NIC caused vessel texture alteration in non diseased porcine vessels, seen as vessel wall penetration and perivascular edema. After single injection the maximum of the indicator was found in perivascular tissue 0.5 hours at the application site (carotid perivascular tissue: 7.48%; femoral perivascular tissue: 2.56%). Thereafter, radioactivity in the artery increased and perivascular content declined. The maximum in femoral arteries (1 hour; 1.96%) occurred earlier and was significantly lower compared to carotid arteries (2 hours; 7.75%). Four hours post-injection, 1.4% of total drug amount was detectable in the carotid arteries and 0.6% was detected in the femoral arteries. Systemic content was measured after C14-Carvedilol application with a maximum in serum of 28% (10 minutes), liver 30% (0.5 hour) and spleen 0.6% (0.5 hour). After 3 hours, still 5% of the indicator was still measureable in the serum and liver and less than 0.1% was measurable in the spleen. LDD with the NIC system is dependent on the vascular anatomy. The data indicate redistribution from perivascular to vascular space thus allowing a prolonged vascular and perivascular drug delivery. The amount deliverable is lower than expected due to substantial systemic drug contamination with this catheter. PMID- 10745442 TI - Clinical experience with a new biocompatible phosphorylcholine-coated coronary stent. AB - AIMS: The BiodivYsio stent is a new stent coated with phosphorylcholine, a biocompatible molecule designed to reduce the formation of thrombus and potentially the risk of restenosis. The feasibility, safety, and efficacy of elective and urgent implantation of this coated coronary stent were prospectively studied. METHODS AND RESULTS: We studied 224 patients who underwent elective (67%) or bail-out implantation of 303 BiodivYsio stents in 286 lesions. Most lesions (62%) had unfavorable characteristics (type B2 or C) and half of them (50%) had thrombus and/or chronic total occlusion. Clinical follow-up was obtained in all patients at one month and in the first 132 patients at six months. Repeat angiography was undertaken in all patients with recurrent ischemia. Successful stent deployment was achieved in 284 lesions (99.3%). One emergency coronary artery bypass graft (CABG) was required. Angiographic success rate was 98.3% (281/286). There was 1 (0.4%) subacute stent thrombosis associated with Q-wave myocardial infarction and two (0.9%) in-hospital deaths. Reference vessel diameter was 2.82 +/- 0.32 mm. Minimum luminal diameter (MLD) increased from 0.38 +/- 0.25 mm to 2.97 +/- 0.35 mm and diameter stenosis decreased from 83.8 +/- 12.1% to 5.8 +/- 9.7%. Clinical restenosis rate was 6.1% (8/132 patients) at 6-month follow-up. Target vessel revascularization rate at 6-month follow-up was 5.4%. CONCLUSIONS: This initial clinical experience indicates that the implantation of stents coated with phosphorylcholine appears to be safe and efficacious in the treatment of complex coronary lesions and is associated with an extremely low target vessel revascularization rate. PMID- 10745443 TI - Prospects for therapeutic angiogenesis and myogenesis in patients with advanced coronary disease. PMID- 10745444 TI - Growth factor alphabet soup--for what ails you. PMID- 10745445 TI - Local arterial drug delivery: a golden gun without bullets? PMID- 10745446 TI - Successful treatment of coronary artery perforation during angioplasty using a new membrane-coated stent. AB - We report a case of successful treatment of coronary artery perforation and cardiac tamponade, which developed during percutaneous transluminal coronary angioplasty, with a PTFE-coated stent. Intravascular ultrasound was first used to overcome the shortcomings of conventional angiography and overlapping of a conventional stent was not as effective as coated-stent placement in sealing a further leakage. Thus, PTFE-coated stents may be an effective alternative to emergency surgery or autologous venous covered stenting and should be considered when coronary artery perforation occurs. PMID- 10745447 TI - Dilutional acidosis complicating rotational atherectomy. AB - Rotational atherectomy is an important technique for the relief of atherosclerotic disease in both coronary and peripheral arteries. Here, we describe an unusual complication, dilutional metabolic acidosis, due to hydration during rotational atherectomy. PMID- 10745448 TI - A case of type IV dual left anterior descending artery associated with anomalous origin of the left circumflex artery in the presence of coronary atherosclerosis. AB - Double origin of the left anterior descending coronary artery (LAD) from the left and right coronary arteries (type IV dual LAD) is a rare coronary anomaly. We report an unusual case of type IV dual LAD associated with anomalous origin of the left circumflex artery from the right coronary artery in a patient with a recent history of myocardial infarction. The first diagonal branch originating from the short LAD demonstrated 70-80% stenosis and the posterior descending artery was totally occluded. We believe that this unusual variance of the coronary arteries in association with atherosclerosis has not been previously reported. PMID- 10745449 TI - Intravascular ultrasound evaluation of a pseudolesion created by stent placement in the right artery. AB - The creation of a pseudolesion after guidewire placement in tortuous arterial segments is a well recognized phenomenon. Intravascular ultrasound has been useful in assessing deployment of intracoronary stents and equivocal angiographic findings. We present a case in which a pseudolesion was not observed until after placement of an intracoronary stent. Intravascular ultrasound demonstrated no dissection or significant lesion; however, there was focal calcification just distal to the stent providing a substrate for the distorted vessel architecture. The lesion resolved with removal of the guidewire. PMID- 10745450 TI - Angioplasty of pedicled right gastroepiploic artery graft through a six French diagnostic catheter. AB - Pedicled right gastroepiploic artery is increasingly being used as a conduit for coronary artery bypass surgery. We describe an interesting case in which balloon angioplasty for stenosis in such a graft was performed through a 6 French diagnostic catheter. PMID- 10745451 TI - Diagnostic testing in vascular medicine: the foundation for successful intervention. PMID- 10745452 TI - Physician preference items: a surgical vignette. PMID- 10745453 TI - Group purchasing: forcing compliance or organizing the talent? PMID- 10745454 TI - Deformation of coronary stents by cardiopulmonary resuscitation. PMID- 10745455 TI - CPR: The "force" may have a "dark side". PMID- 10745456 TI - Late stent malapposition occurring after intracoronary beta-irradiation detected by intravascular ultrasound. AB - We report a case of late stent malapposition occurring 6 months after intracoronary beta-irradiation detected by three-dimensional intravascular ultrasound, in spite of good apposition immediately after the procedure. Volumetric quantification revealed that stent volume remained unchanged, whereas total vessel volume increased by 13% after 6 months within the stent area. The increase of the vessel volume took place mainly in the proximal part of the stent, where the malapposition was located. PMID- 10745457 TI - The use of a new mechanically advantaged syringe for performing coronary intervention. AB - OBJECTIVE: To determine the utility of a new mechanically advantaged syringe in the performance of percutaneous coronary intervention. BACKGROUND: A new mechanically advantaged syringe has been developed to perform cardiovascular imaging. We wanted to assess the utility of this device in the performance of small catheter percutaneous coronary intervention. Data from coronary interventions performed with the standard technique at our institution, using a 10 ml manual syringe, were compared to the use of a new mechanically advantaged syringe. Contrast utilization during the standard technique was assessed by obtaining data from fifty consecutive successful single-vessel coronary interventions (angioplasty with or without stenting). After an initial learning experience with the mechanically advantaged syringe, fifty consecutive successful single-vessel coronary interventions were assessed. Most interventions performed using the mechanically advantaged syringe were with 6 French catheters. Detailed contrast utilization information was gathered. RESULTS: During the standard method of performing coronary intervention, 216 +/- 114 ml of contrast were used. Utilizing the mechanically advantaged OZ Power Syringe (Cardiovascular Innovations Inc., Athens, Texas), the contrast use was reduced to 66 +/- 39 ml (p < 0.0001) per case. The syringe also allowed excellent visualization despite using a smaller guide catheter system. CONCLUSION: The OZ Power Syringe can be utilized effectively in percutaneous coronary intervention. Our preliminary data suggest that there may be reduced contrast use without sacrificing image quality. PMID- 10745458 TI - Angiographic results and late clinical follow-up after Bard-XT intracoronary stent implantation. AB - OBJECTIVES: The aim of this study is the assessment of immediate clinical and angiographic results and medium- to long-term clinical follow-up after Bard-XT (Bard, Billerica, Massachusetts) coronary stent deployment. METHODS: The implantation of 196 stents of various lengths (11, 15 and 19 mm) was attempted in 181 patients (70.1% male) from January 1997 to January 1998. Lesions were type A in 5.6% of the cases, type B1 in 44.4%, type B2 in 45.9% and type C in 4.1%. Implantation of the stent was elective in 53.6% and for bail-out situations in 46.4% of the lesions. RESULTS: The immediate angiographic success rate was 98.5% and the immediate clinical success rate (angiographic success with no major complications during in-hospital stay) was 96.9%. The minimal lumen diameter increased from 0.82 +/- 0.6 mm to 3.0 +/- 0.71 mm after intervention, relative to an immediate post-procedural residual stenosis of 3.05 +/- 9.12%. During the mean in-hospital stay of 2.6 +/- 1.9 days, 3.8% of the patients had a minor clinical event, and 1.5% had a major clinical event. During late follow-up, 3.9% of the patients died. Clinical follow-up of 89.5% of the eligible patients after 241.1 +/- 73.9 days showed that 80.6% of them had negative stress tests, thus reaching a clinical restenosis rate of 19.4%. In a multivariate analysis, only systemic arterial hypertension (p = 0.0012) and a previous history of myocardial infarction (p = 0.02) had a positive correlation with clinical restenosis. CONCLUSIONS: The preliminary experience with the Bard-XT coronary stent was associated with good immediate and medium- to long-term clinical outcome. The results were comparable to those obtained with slotted-tube stents. PMID- 10745459 TI - Determination of blood flow and endothelial shear stress in human coronary artery in vivo. AB - This paper describes a system that permits, for the first time, the in vivo determination of local velocity and endothelial shear stress in the major human coronary arteries. The purpose of the system is to facilitate the study of plaque growth and the relationships between local hemodynamic factors and atherogenesis and restenosis. The three-dimensional anatomy of a segment of the right coronary artery was determined immediately after directional atherectomy via a combination of intracoronary ultrasound and biplane angiography. The highly irregular geometry of the segment was then represented in curvilinear coordinates and a computational fluid dynamics technique was used to investigate the detailed, intravascular velocity profile and shear stress distribution. We found minor flow reversals, significant swirling and a large variation of local velocity and shear stress, both axially and circumferentially, within the artery, even in the absence of significant luminal obstruction. The flow phenomena exhibit characteristics consistent with the focal nature of atherogenesis and restenosis. It is concluded that the technology now exists to determine luminal geometry and local variations in flow fields and endothelial shear stress, in vivo. PMID- 10745460 TI - Can mathematical modeling be used to determine blood flow and shear stress in vivo? PMID- 10745461 TI - Coronary-subclavian steal associated with severe aortic stenosis treated with combined percutaneous stenting and minimally invasive aortic valve replacement. AB - We describe coronary-subclavian steal restricting flow to the left internal mammary artery (LIMA) associated with critical aortic stenosis treated with combined percutaneous transluminal stenting and minimally invasive aortic valve replacement (AVR). An 86-year-old patient had coronary artery bypass graft placement (CABG) seven years prior with the LIMA anastomosed to the left anterior descending coronary artery (LAD). At the time of CABG, the patient had mild aortic stenosis and normal left ventricular function. By the time of re presentation with refractory angina and heart failure, the patient had developed critical aortic stenosis. Because repeat CABG with median sternotomy risked damaging the LIMA, pre-operative revascularization was planned to minimize the likelihood of peri-operative ischemia. Stenting of the subclavian artery was performed prior to minimally invasive AVR. PMID- 10745462 TI - Therapeutic dissolution of an intracoronary thrombus by prolonged intravenous platelet glycoprotein IIb/IIIa antagonism. AB - This case report describes the therapeutic dissolution of an intracoronary thrombus in a patient with ectatic coronary arteries post-myocardial infarction by prolonged intravenous glycoprotein (GP) IIb/IIIa antagonist administration. The report emphasizes the potential thrombotic complications in patients with ectatic coronary arteries and the beneficial use of GP IIb/IIIa receptor antagonists as direct thrombolytic agents even in partially organized thrombus formation. In addition to the well-documented effects of GP IIb/IIIa blockade in the scenario of percutaneous interventions, unstable angina, and non-Q wave infarction, the use of this new class of drugs in acute myocardial infarction seems to be promising and might also be considered in the setting of persistent thrombotic material within the coronary vasculature. PMID- 10745463 TI - Arteriography of the left internal mammary artery graft utilizing a balloon tipped floatation catheter: an alternative approach. AB - In this report, we describe an alternative method to the conventional arteriographic techniques of the left internal mammary artery (LIMA) graft using a balloon-tipped floatation catheter placed within the left subclavian artery. The floatation catheter will serve as both an occluder of the subclavian artery as well as a port for contrast injection. It may be effectively employed in the rare instances where direct cannulation of the LIMA graft is not possible. PMID- 10745464 TI - Stenting in a patient with a single coronary artery and myocardial infarction. AB - The single coronary artery is an uncommon congenital defect. We report on a patient admitted to our hospital with an acute myocardial infarction and a single coronary artery arising from the left sinus of Valsalva who was treated with coronary angioplasty and stent implantation. We have not found an identical report of stenting in such an anomaly in the medical literature. PMID- 10745465 TI - Rescue left main angioplasty on first post-operative day of coronary artery bypass graft surgery ischemia. AB - Percutaneous revascularization has been used for the treatment of post-coronary bypass graft ischemia. This report illustrates the use of emergency left main coronary artery stenting in protected left main on the first post-operative day for management of perioperative coronary artery bypass graft surgery ischemia. PMID- 10745466 TI - Cardiologists and cardiac surgeons: towards an integrated approach facing the forthcoming scenario of coronary artery disease. PMID- 10745467 TI - Goal assessment and resource planning in developing cardiac databases. PMID- 10745468 TI - Effect of isoflurane on defibrillation threshold in biphasic active-can defibrillation systems. AB - In two patients receiving implantable cardioverter-defibrillators (ICD) for spontaneous and inducible ventricular tachyarrhythmias, the use of isoflurane appeared to significantly lower defibrillation thresholds (DFT) at implantation. In the first, at initial implantation and subsequent revision using isoflurane, adequate DFTs were verified by multiple tests. Post-implantation testing using midazolam and fentanyl revealed significantly higher DFTs, necessitating a third operation without isoflurane to obtain an adequate and subsequently verifiable DFT. During the second case, discontinuation of isoflurane intraoperatively resulted in a DFT rise within 25 minutes. In patients receiving general anesthesia for ICD implantation, this possible DFT effect of isoflurane should be recognized. PMID- 10745469 TI - Effects of external beam radiation on the human heart and great vessels. PMID- 10745470 TI - Outpatient coronary stenting using the femoral approach with vascular sealing. AB - PURPOSE: We report here the use of vascular sealing devices in conjunction with the use of small transfemoral guiding catheters to decrease time to ambulation, decrease cost associated with hospitalization and achieve early discharge. METHODS: Fifty patients were enrolled in this pilot project from two busy interventional practices between May 1997 and February 1999. Patients with stable or unstable angina or positive ETT and with recent myocardial infarction greater than 24 hours from the time of the procedure were included. Excluded patients included those who had received glycoprotein IIb/IIIa platelet inhibitors and those with intra-procedure access site complications. RESULTS: Of the 50 patients originally recruited, 49 underwent vascular sealing for hemostasis and 45 were discharged on the same day, as planned. Early home telephone follow-up was available on 41 of the 45 same-day discharge patients, of whom 30 noted no complaints. One patient, who had been re-accessed in the right femoral artery after a previous intervention, developed a pseudoaneurysm requiring surgical repair. One-month follow-up was available on all patients. No patient suffered a late ischemic event or access site complication requiring treatment. There were no instances of stent loss, acute closure or subacute thrombosis. CONCLUSION: Though limited by small numbers, this pilot study shows that selected patients undergoing coronary stenting via the femoral approach can be safely treated on an outpatient basis using vascular sealing devices. Cost savings may be significant using this strategy. PMID- 10745471 TI - Tirofiban and intracoronary stenting: evaluation of a new pharmacologic regimen. AB - Glycoprotein IIb/IIIa receptor antagonists are effective in decreasing cardiac events in patients with acute coronary syndromes. Abciximab reduces cardiac morbidity in patients receiving coronary stents. We sought to evaluate the use of tirofiban infusion in patients receiving intracoronary stents. We created a prospective registry of 50 consecutive patients in a single catheterization lab receiving a single vial of tirofiban (12.5 mg) and stenting. Successful stent deployment with a 30-day follow-up occurred in all patients. Clinical endpoints included death (0%), procedural infarct (4%), urgent revascularization (0%), stroke (0%), bleeding complication (4%), and vascular injury (0%). Tirofiban appeared to be a safe adjunctive therapy to coronary stenting. PMID- 10745473 TI - We don't have the cure yet: ten-year follow-up after angioplasty. PMID- 10745472 TI - Predictors of adverse outcome 10 years after coronary angioplasty. AB - To determine which factors before percutaneous transluminal coronary angioplasty (PTCA) predict long-term outcome, we evaluated the clinical follow-up data from 535 patients 10 years after single-vessel PTCA. Events were defined as death, myocardial infarction, bypass surgery or repeat PTCA. During the follow-up period 79 patients (15%) died, 59 patients (11%) suffered a myocardial infarction, 107 patients (20%) had coronary artery bypass surgery and 141 patients (26%) underwent a redilatation. To determine the predictors of 10-year follow-up, 12 patient-related and 9 lesion parameters were analyzed by logistic regression analysis. Mortality was independently increased in patients with diabetes, with multi-vessel disease, after a previous myocardial infarction and in smokers. The presence of multi-vessel disease, symptoms of a higher angina class and younger age increased the risk for undergoing bypass surgery. In the statistical model with lesion parameters, the risk of bypass surgery was decreased if the stenosis was located in the distal segment of the coronary vessel and by a higher minimal luminal diameter before PTCA. CONCLUSION: Logistic regression analysis identified multi-vessel disease, diabetes, smoking and a previous myocardial infarction as independent clinical predictors of an adverse outcome 10 years after coronary angioplasty. Lesion parameters before PTCA seem to be less important with regard to the long-term outcome after PTCA. PMID- 10745474 TI - Outpatient coronary stenting with vascular sealing: an idea whose time has come or should you stay just a little bit longer? PMID- 10745475 TI - Balloon angioplasty of long-segment aortic coarctation in the neonate. AB - It is generally believed that balloon angioplasty of diffuse, long-segment aortic coarctation is not effective. In this report, we describe two neonates with diffuse, long-segment coarctation in association with complex congenital heart defects in whom we were successful in effectively treating coarctation with transumbilical artery balloon angioplasty. Based on this experience, it may be concluded that balloon angioplasty of long-segment coarctation in neonates is feasible and effective, but confirmation in a larger group of patients may be necessary prior to general adoption of this concept. PMID- 10745476 TI - Severe spasm of the free radial artery graft in a patient undergoing high-risk angioplasty under percutaneous cardiopulmonary support. AB - This report illustrates a case of percutaneous intervention in a very high-risk patient who already had three previous bypass operations and was not a suitable candidate for further surgical revascularization. Since intra-aortic balloon pump insertion was contraindicated due to the origin of a radial graft from the descending aorta, percutaneous cardiopulmonary support (CPS) was used to perform the procedure. Thus, CPS proved useful in providing adequate hemodynamic support and allowed us to use adequate amount of antispastic drugs to relieve any spasm during the procedure. This case also highlights the need to use antispastic drugs before performing percutaneous interventions on arterial conduits, particularly radial artery grafts which are prone to vasospasm. Since the use of the radial artery for bypass surgery has recently been revived with considerable interest, perhaps more percutaneous interventional procedures may be performed in the future on arterial graft stenosis. We therefore recommend the routine use of vasorelaxant drugs in percutaneous interventions involving arterial grafts. PMID- 10745477 TI - Massive thromboembolism due to transcatheter ASD closure with ASDOS device. AB - Transcatheter occlusion of atrial septal defects (ASD) is currently being investigated as an attractive alternative to surgical correction. Thromboembolic events are rare in both techniques. However, we report a case of massive systemic embolization and residual left atrial thrombus after secundum ASD transcatheter closure by the ASDOS device (Atrial Septal Defect Occlusion System, Dr. Ing Osypka Corporation, Germany). The patient was successfully treated by femoral embolectomies, surgical removal of the device and closure of the ASD without a patch. No thrombophilia was found on subsequent exploration. Transcatheter ASD closure with the ASDOS device may therefore expose the patient to severe embolic complications. Further evaluation is needed before this technique can be safely recommended. PMID- 10745478 TI - Continuous high-dose intracoronary nitroglycerin infusion in refractory coronary vasospasm. AB - This case report illustrates the use of continuous high-dose intracoronary nitroglycerin infusion through a 6 French coronary guiding catheter in the treatment of a patient with cocaine-induced refractory coronary vasospasm. PMID- 10745479 TI - Geographic miss: what is it? PMID- 10745480 TI - Intra-arterial reteplase for the treatment of acute limb ischemia. AB - The removal of urokinase from the market has created a dilemma for interventionists and vascular surgeons treating patients with acute limb threatening ischemia due to arterial thrombosis or embolization. Reteplase is a newer, fibrin-specific thrombolytic agent with properties that make it an attractive alternative to urokinase. We report two cases of successful treatment of acute, limb threatening ischemia with intra-arterial Reteplase therapy. PMID- 10745481 TI - Diagnostic testing in vascular medicine: the foundation for successful intervention II--renal and abdominal aortic disease. PMID- 10745482 TI - Immediate angioplasty for acute myocardial infarction: a community hospital's experience. AB - This study describes prospective outcome data from 100 consecutive patients presenting with acute myocardial infarction and treated with immediate angioplasty in a community hospital setting. Successful angioplasty was achieved in 86% of patients with a mean reperfusion time of 77.5 minutes. Only 4 patients did not survive initial hospitalization; three of these initially presented with cardiogenic shock. The survival rate in noncardiogenic shock patients was 98.9%. Four patients underwent repeat angioplasty of the infarct-related artery and 6 patients were referred for coronary artery bypass surgery during initial hospitalization. During the 6 month follow-up, nine patients required repeat hospitalization. Seven of these patients presented with recurrent ischemia; four underwent repeat angioplasty and 3 coronary artery bypass surgery. There were no subsequent deaths or reinfarctions during the 6 month follow-up. The angioplasty success rate and clinical outcomes in this study compare favorably to previous trials performed in select interventional centers and suggest that immediate angioplasty can be the preferred reperfusion therapy in a community hospital setting. PMID- 10745483 TI - Outpatient coronary angiography with 4 French catheters. AB - The early 4 French (Fr), pre-formed, Judkins coronary catheters had unsatisfactory maneuverability due to reduced torque stability. Technical innovations have led to improved 4 Fr catheters. This prospective study evaluates the safety and feasibility of the 4 Fr Quick Care Infinity catheter (Cordis, Miami, Florida). Within a series of 2,366 patients investigated for suspected coronary artery disease, 302 were selected for outpatient treatment according to the guidelines for outpatient catheterization. Fluoroscopy time was 2.8 +/- 2.5 minutes. It was not necessary to change to larger catheters in any patient. After sheath removal, hemostasis was achieved within 14 +/- 8 minutes. Strict bed rest lasted 181 +/- 65 minutes; patients were discharged 303 +/- 76 minutes after arterial puncture. Sixteen of the 302 patients (5.3%; 95% confidence interval (CI): 3.1-7.9%) changed to inpatient treatment for reasons unrelated to the arterial access. Eight patients (2.6%; CI: 1.1-5.1%) required one night of hospitalization for control of hematoma. The following morning, vessel murmur, pain, and large hematoma (more than 10 cm diameter) occurred in 1 (0.3%), 6 (2.0%), and 6 (2.0%) patients, respectively. This necessitated hospital admission in 2 patients (0.7%; CI: 0.0-2.3%) for up to 2 days. Major entry site complications requiring blood transfusion or surgery did not occur (CI: 0.0 0.3%). It was concluded that outpatient catheterization for coronary artery disease using the new 4 French catheters is feasible and can be rapidly and safely performed in a selected patient population. PMID- 10745484 TI - Utility of bilateral coronary injections during complex coronary angioplasty. AB - We describe a technique useful in complex coronary interventions wherein timed bilateral contrast injections are given in both coronary arteries. This technique is useful in chronic total occlusions in which the distal coronary vessel is not visualized except by collateral filling via the contralateral artery. This technique was applied in 12 patients; 11 with native coronary occlusion and one in whom the target site was visualized by contrast injections into a vein graft supplying competitive flow to an otherwise patent native vessel. With this technique, the distal coronary artery segment can be better visualized, which helps to aim and track the guide wire across the occluded segment. PMID- 10745485 TI - Successful direct stent implantation into a marginal branch of the circumflex artery via the right internal mammary artery in a transfemoral approach. PMID- 10745486 TI - Coronary aneurysm treated with vein on stent. PMID- 10745487 TI - Successful coil embolization and follow-up result of a complex pulmonary arterio venous fistula. PMID- 10745488 TI - Coronary revascularization and aggressive lipid lowering. PMID- 10745489 TI - Endovascular brachytherapy for the prevention of restenosis. PMID- 10745490 TI - Emergency treatment of ischemic stroke. PMID- 10745491 TI - Renal artery intervention: origins and new technical advances. PMID- 10745492 TI - Renal artery stenosis: under-diagnosed and under-treated in the cardiac patient? PMID- 10745493 TI - Acute myocardial infarction: to lyse or to inflate? PMID- 10745494 TI - Outpatient coronary angiography protocols. PMID- 10745495 TI - Is smaller better for outpatient coronary angiography? PMID- 10745496 TI - The impact of outcomes data reporting on access to health care of high-risk patients to interventional cardiologists in the United States. AB - BACKGROUND: Pressure is increasingly being placed on medical sub-specialists to lower procedural costs while improving overall outcomes. Outcomes data reporting has been utilized in an attempt to improve procedural results; however, some negative aspects of this type of reporting have emerged. METHODS: We surveyed 5,229 interventional cardiologists practicing in the United States regarding the effect of outcomes data reporting on their approach to high-risk patients who required interventional procedures. The results were analyzed one month after mailing the survey. RESULTS: One thousand, four hundred and forty-four cardiologists responded to the survey. Ninety-three percent of the respondents indicated that outcomes data reporting would have some effect on their approach to high-risk patients. Only 7% said outcomes reporting would have no effect on their willingness to perform interventions on high-risk patients. The majority of respondents performed between 51 and 100 interventions per year; those who performed more than 500 interventions per year reported that they would be less affected if outcomes data were reported from their results. CONCLUSIONS: Our survey indicates that outcomes data reporting would have a significant effect on the willingness of cardiologists to perform procedures on high-risk patients; this effect may limit access to needed care for this high-risk population. PMID- 10745497 TI - Balloon dilation of discrete subaortic stenosis associated with other cardiac defects in children. AB - Balloon dilation (BD) is reported as an effective treatment for isolated discrete thin membranous subaortic stenosis (SAS). We asked if BD of SAS with associated cardiac defects: 1) is effective; 2) creates or worsens mitral insufficiency in the presence of valvar membrane attachment; and 3) creates or worsens aortic insufficiency. BD was performed on 13 patients (9 females, 4 males with a mean age of 5.8 years and an age range of 1-14 years old) for SAS with the following associated defects: VSD (6 patients), coarctation (4 patients), complete atrioventricular canal (1 patient), tetralogy of Fallot (1 patient), and abnormal mitral valve (4 patient). Prior intracardiac surgical procedures (3 for SAS resection) had been performed in 46% of the study group. Peak gradient was reduced from 53 +/- 40 mmHg to 31 +/- 25 mmHg (p < 0.01). The resulting gradient was related to the predilation gradient (r = 0.71; p =.006). Thin membranes (< 3 mm) were associated with lower initial gradient (p < 0.02), lower resulting gradient (p < 0.001) and a greater percent gradient reduction (76% versus 36%; p < 0.01). Aortic insufficiency, which was present predilation in 77%, did not change and was not created in any patient. Mitral valve membrane attachment was present in 69% and associated with a lower resulting gradient (p < 0.006). Mitral insufficiency was not created in 10 patients, was unchanged in 2 and was improved in 1 patient. Surgical procedures were ultimately required in 8 patients during follow-up. BD of SAS associated with other cardiac defects achieves the following: 1) reduces SAS gradient in selected patients without creating or worsening aortic insufficiency; 2) does not create or worsen mitral insufficiency when mitral valve membrane attachment is present; 3) improves preoperative hemodynamics; and 4) is unlikely to be effective in post-surgical recurrence. PMID- 10745498 TI - Gender-related risk factors and outcomes for non-Q wave myocardial infarction patients receiving in-hospital PTCA. AB - BACKGROUND: We analyzed the risk factors and outcomes associated with non-Q wave myocardial infarction (MI) in females and males. We studied 376 consecutive patients N 275 males (73%) and 101 females (27%) N who presented with non-Q wave MI and had percutaneous transluminal coronary angioplasty (PTCA) prior to discharge during the period between January 1992 and February 1996. RESULTS: Females were significantly older (68 +/- 10 years vs. 61 +/- 11 years; p < 0.001) and had more hypertension (67% vs. 51%; p < 0.01). Males had a slightly lower ejection fraction (47 +/- 11%) compared to females (50 +/- 10%; p < 0.001). Angioplasty was equally successful for women and men (96% vs. 97%; p = NS) with a statistically significant smaller number of lesions dilated per patient in females (1.38 vs. 1.51; p < 0.04). There were no significant differences in unstable angina, prior coronary artery bypass graft (CABG) surgery, saphenous vein graft PTCA, single vessel versus multiple vessel disease or history of prior MI. In-hospital complications (i.e., the need for CABG or repeat PTCA, recurrent MI, and stroke) were not statistically significant for either females or males. There was a trend for a higher in-hospital death rate in females after a non-Q wave MI, but it was not statistically significant (4% vs. 1%; p = 0.058). However, at one-year follow-up females had a significantly worse survival rate than men (89% vs. 95%; p < 0.04), although event-free survival rate was similar (61% female, 66% male; p = NS). CABG was performed less commonly in women by the end of one year (p < 0.02) than in men, while the performance of PTCA was similar. CONCLUSIONS: Although women with non-Q wave MI presented with more risk factors than men, in-hospital revascularization was equally successful with few complications and morbid events and similar event-free outcome at one year. However, one year mortality was worse for women, suggesting a need for more aggressive follow-up evaluation and treatment. For both women and men, this aggressive percutaneous revascularization strategy resulted in much better outcome than previously reported for medical treatment of non-Q wave MI. PMID- 10745499 TI - Stent design favorably influences the vascular response in normal porcine coronary arteries. AB - OBJECTIVES: The purpose of this study was to compare the arterial response following implantation of a stainless-steel, balloon-expandable, tubular slotted stent with that of a novel computer-designed, multi-cellular stent in normal porcine coronary arteries. BACKGROUND: Intracoronary stent placement has evolved into the primary strategy for percutaneous revascularization of symptomatic coronary arterial lesions. Presently there is intense interest in developing new stent designs to improve stent delivery and biocompatability. METHODS: Computer assisted design was utilized to develop a balloon-expandable stent with symmetric expansion properties, uniform arterial wall coverage, longitudinal flexibility and radial strength. Quantitative coronary angiography and histological assessment of the stented arteries was used to evaluate the acute and chronic vascular responses to a stainless-steel, balloon-expandable, tubular slotted stent as compared to the computer-designed BX stent in the normolipemic swine. RESULTS: Forty stents (24 BX, 16 tubular slotted) were implanted in 19 miniature swine at a mean inflation pressure of 9 atm using identical delivery systems. Eight of the BX and none of the tubular slotted stents were post-dilated with a non-compliant balloon at 12-14 atm. The mean stent-to-artery ratio was similar for the BX (1.03 +/- 0.06) and tubular slotted (1.04 +/- 0.11; p = 0.59) designs. Protrusion or asymmetric radial flaring of a strut at the stent margin was present in 1 of 23 BX stents (4.3%) and 10 of 15 tubular slotted stents (66.7%; p < 0.0001). The mean arterial injury score was significantly less for the BX stent (0.2 +/- 0.2) as compared with the tubular slotted stents (0.4 +/- 0.4; p = 0.025). At 3 days, thrombus area was similar for the BX and tubular slotted designs (0.42 +/- 0.16 mm2 versus 0.44 +/- 0.18 mm2, respectively; p = 0.88). The mean neointimal area was significantly less for the BX at 2 months (1.09 +/- 0.25 mm2 versus 2.93 +/- 2.26 mm2 in the tubular slotted stent) and at 6 months (1.10 +/- 0.26 mm2 versus 2.07 +/- 0.65 mm2 in the tubular slotted stent; p = 0.01), resulting in approximately 50% less in-stent stenosis. CONCLUSIONS: The arterial response to a balloon-expandable stent can be favorably influenced by computer assisted modification of design in an experimental model. Further study is warranted to determine the impact of stent design upon clinical in-stent restenosis. PMID- 10745500 TI - Treatment of a refractory chronic total coronary occlusion using the stiff backend of a hydrophilic guide wire. PMID- 10745501 TI - Tirofiban-induced coronary thrombolysis. PMID- 10745502 TI - Percutaneous closure of a coronary artery aneurysm with a bare stent. PMID- 10745503 TI - Post-pericardiotomy syndrome after percutaneous balloon pericardiotomy. PMID- 10745505 TI - Valuation of clinical practices - the right way. PMID- 10745504 TI - Treatment of unstable angina: the role of platelet inhibitors and anticoagulants. AB - Thrombo-occlusive events at sites of atherosclerotic stenosis and plaque rupture are caused by exposure of the sub-endothelial layer of the vasculature to platelets and their subsequent aggregation at the locus of injury. Thrombi are generated by the activation of platelets, which are not always inhibited by aspirin or heparin. Both unstable angina and myocardial infarction are ultimately linked to thrombus formation. In addition, various invasive procedures used to re establish patency of coronary vessels are often followed by thrombotic events that can be life threatening. New antagonists to thrombin and the development of glycoprotein (GP) IIb/IIIa platelet receptor inhibitors have been the main focus of recent large clinical trials that have demonstrated both the efficacy and safety of these new agents. The new antithrombotics include hirudin, hirulog, and the low molecular weight heparins. Representative GP IIb/IIIa receptor blockers include: abciximab, a human-murine chimeric monoclonal antibody; tirofiban, a non peptide tyrosine derivative with a short half-life; and eptifibatide, a cyclic heptapeptide. All three are available for clinical use, but their approved indications vary. The GP IIb/IIIa receptor antagonists are anticipated to gain wide clinical acceptance by both cardiologists and internists and should have a prominent role in the management of acute coronary syndromes, most likely in combination with an antithrombin. PMID- 10745506 TI - Value and valuation: the vital signs of your practice. PMID- 10745507 TI - Percutaneous transluminal myocardial revascularization: an emerging technology. PMID- 10745508 TI - Less invasive heart surgery. PMID- 10745509 TI - Integrated coronary revascularization. PMID- 10745510 TI - Transmyocardial revascularization: an industry perspective. PMID- 10745511 TI - Balloon dilation of discrete subaortic stenosis: are we postponing the inevitable? PMID- 10745512 TI - Balloon angioplasty of fixed subaortic stenosis. PMID- 10745513 TI - Efficacy of cutting balloon angioplasty for lesions at the ostium of the coronary arteries. AB - We evaluated 37 patients (39 lesions, 11 women/26 men, median age of 66 years old) with coronary ostial lesions who underwent cutting balloon angioplasty (CB). Seventy-four patients (78 lesions, 18 women/56 men, median age of 65 years old) who had undergone plain old balloon angioplasty (POBA) for coronary ostial lesions were also enrolled as the control group. At clinical diagnosis, unstable angina was seen in 8.1% of the CB group and in 9.5% of the POBA group. Post myocardial infarction silent ischemia was seen in 28.3% of the POBA group and 32.4% of the CB group. The success rate was 94.8% in the CB group and 84.6% in the POBA group. The frequency of intimal dissection was not significantly different between the two groups. However, all cases of intimal dissection in the CB group were mild (either type A or B), whereas the POBA group included many cases of severe intimal dissection (types B through F). No cardiac event was observed in the CB group. However, in the POBA group, cardiac death occurred in 1.4% and emergency bypass surgery occurred in 0.7% of the patients. Calculating the restenosis rate based on the cumulative percent diameter stenosis curve resulted in 43% for the CB group and 53% of the POBA group. We concluded that the cutting balloon was useful for ostial lesions as compared to plain old balloon angioplasty. PMID- 10745514 TI - Influence of catheter position and equipment-related factors on the accuracy of intravascular ultrasound measurements. AB - BACKGROUND: Intravascular ultrasound (IVUS) is frequently used as an adjunct to coronary angiography to guide revascularization procedures and, more recently, to estimate atherosclerotic plaque volumes. Although accuracy of IVUS imaging and analysis is crucial for these measurements, available data are scarce. The purpose of this in vitro study is to determine the extent to which transducer position and equipment-related factors influence measurement accuracy. METHODS: Cross-sectional views of tubular vessel phantoms (diameter 2-14 mm) were acquired using 3.2 French catheters in coaxially centered, eccentric and oblique positions. Catheters were sequentially connected to two different ultrasound systems (A and B) to estimate equipment-related variability. In system B, two software versions were used to analyze ultrasound images. Longitudinal views of phantom segments were reconstructed to document transducer misplacement. RESULTS: Oblique transducer positioning resulted in a non-linear overestimation of phantom areas that was independent of lumen size and also resulted in dramatic distortions of three-dimensionally reconstructed phantom geometry. Eccentric positioning did not significantly influence measurement accuracy. In coaxial positioning, differences between measured and true areas increased non-linearly from 0.36 to 4.5 mm2 in system B and in a linear fashion from -0.01 to 2.68 mm2 in system A with increasing phantom diameters. Relative differences decreased from 11.4% to 2.9% with increasing reference areas in system B (positive off-set error). When using updated software in system B, the off-set error was negative and relative error diminished from -1.34% to 0.44% with increasing phantom size. CONCLUSION: Transducer position and equipment-related factors influence the accuracy of intravascular ultrasound, which may lead to misinterpretation of vessel size and geometry even in straight vessel segments. Transducer position may be controlled by the reconstruction of longitudinal images. Ultrasound equipment should be calibrated before using it for quantitative measurements. PMID- 10745515 TI - Primary angioplasty in acute myocardial infarction: differences between referred patients and those treated in hospitals with on-site facilities? AB - BACKGROUND: Primary angioplasty for acute myocardial infarction (AMI) can only be performed in a limited number of centers. Therefore, some patients will be referred for this procedure. DESIGN: We analyzed the data of the prospective observational MITRA trial, which took place at 54 hospitals in southwest Germany, to describe current practices and outcomes in referred patients compared to patients treated on-site. RESULTS: Out of 491 patients treated with primary angioplasty, 63 (12.8%) were referred. Out of 46 hospitals without facilities to perform primary angioplasty, 29 (63%) never referred patients. Referred patients were less often male (60.3% versus 75.9%; p = 0.013), suffered more often from anterior wall infarction (62.9% versus 45.8%; p = 0.014), and more often had absolute contraindications against thrombolysis (11.1% versus 2.8%; p = 0.006). In-hospital time to treatment was 80 minutes in patients treated on-site compared to 190 minutes in referred patients (p = 0.001). There was a non-significant difference of in-hospital mortality between the two groups (11.1% for referred versus 7.5% on-site; p = 0.319). Referred patients had a higher hospital morbidity as expressed by a higher proportion of post-infarction angina (28.6% versus 4.2%; p = 0.001), a more frequent need for urgent revascularization (43.8% versus 16.4%; p = 0.001) and a higher proportion of advanced heart failure (11.1% versus 4.4%; p = 0.062). CONCLUSIONS: Referral of patients with AMI for primary angioplasty is used only in a minority of hospitals. The referred patients represent a selected, more ill subgroup, resulting in a higher hospital morbidity compared to patients treated on-site. Continuous efforts should be undertaken to decrease the time delay from admission at the initial hospital to the beginning of revascularization. PMID- 10745517 TI - Balloon aortic valvuloplasty in children. PMID- 10745516 TI - Long-term follow-up results of balloon valvuloplasty for congenital aortic stenosis: predictors of late outcome. AB - AIMS: Long-term follow-up of patients with aortic valve stenosis undergoing balloon valvuloplasty was evaluated with respect to survival, the need for repeat intervention and factors predicting late outcome. METHODS AND RESULTS: Forty-five patients between 3.5 to 23 years old (mean 11.7 +/- 4.5) were followed for 62 +/- 30 months (range 11-122). The transvalvar aortic gradient decreased from 84 +/- 20 to 36 +/- 10 mmHg (p < 0.001) and remained significantly lower (50 +/- 26 mmHg; p < 0.001) at follow-up. At that time, 10 patients (including 4 with significant valve incompetence) had gradients >/= 60 mmHg. The procedure resulted in significant valve incompetence (grade >/= 3) in 8 patients (17.8%). There was a progression of incompetence and 13 patients (28.9%) had significant regurgitation at follow-up. All survived. Fifteen patients (33.3%) required re intervention 51 +/- 24 months after valvuloplasty. The indications were: aortic stenosis in 5 patients; regurgitation in 6 patients; and stenosis with regurgitation in 4 patients. Actuarial freedom from re-intervention at 2, 4, 6 and 8 years was 96%, 88%, 61% and 56% of patients, respectively. The residual post-valvuloplasty gradient was the only predictor of re-intervention for valve stenosis (odds ratio = 3.2 for every 10 mmHg gradient increase; p = 0.017). A residual post-valvuloplasty gradient >/= 40 mmHg increased the relative risk of re-intervention sixfold. The immediate post-valvuloplasty aortic regurgitation grade was the only risk factor of re-intervention for regurgitation (odds ratio = 34 for every incompetence degree increase; p = 0.0019). Incompetence grade >/= 2 increased the risk of re-intervention tenfold. CONCLUSIONS: Valvuloplasty carries the risk of development of valve incompetence, which progresses with time. Some patients develop restenosis. The or =1 level following PTMC, which was sustained in 34 patients at follow-up. At a mean follow-up period of 20 +/- 12 months (range 3-51 months) in 35 patients, 26 patients (74.3%) were in New York Heart Association (NYHA) functional Class I, 8 patients (22.9%) were in NYHA Class II, and 1 patient (2.8%) was in NYHA Class III. The cumulative 4-year cardiac event-free survival rate was 81.8%. However, patients with grade 4+ calcification had only 50% event free survival rate. At follow-up, an increased incidence of cardiac events was seen in female patients as compared with male patients (83.3% versus 16.7%). Restenosis was seen in 3 patients (8.6%). One patient underwent repeat PTMC 37 months after the initial procedure. There was no incidence of death or mitral valve replacement at follow-up. We conclude that the stepwise balloon dilatation technique can be safely and effectively applied for patients with significant calcific mitral stenosis to achieve an optimal mitral valve area with low incidence of significant increase in MR. Favorable long-term benefits also accrue in the form of improved functional status and low incidence of repeat procedures (repeat PTMC or mitral valve replacement). The majority of patients (74.3%) were in NYHA functional class I without medication. Patients with grade 4+ calcification show less benefit from PTMC and may be considered for mitral valve replacement. Cardiac events occur more frequently in female patients than in male patients during follow-up. PMID- 10745547 TI - The arteries are small--the challenge is large. PMID- 10745548 TI - Coronary angioplasty in small vessel disease: should all coronary small vessel lesions be stented? PMID- 10745549 TI - Multi-parametric mapping in the heart in three dimensions. PMID- 10745551 TI - Percutaneous transvenous mitral commissurotomy is an acceptable therapeutic alternative in patients with calcified mitral valve. PMID- 10745550 TI - Percutaneous mitral balloon valvuloplasty in non-ideal patients: go for it without expecting too much. PMID- 10745552 TI - Utility of intracardiac echocardiography (ICE) in electrophysiology: ICEing the CAKE (catheter ablation knowledge enhancement). AB - PURPOSE: Previous studies have demonstrated the utility of intracardiac echocardiography (ICE) during electrophysiologic procedures including radiofrequency catheter ablation. The purpose of this study was to analyze the initial learning experience with ICE during invasive electrophysiologic procedures. METHODS: During a 1-month ICE trial, patients scheduled for radiofrequency catheter ablation underwent concurrent imaging using a 9 French, 9 MHz catheter in the right atrium proximal to important endocardial structures and diagnostic/ablation catheters. The procedure length, fluoroscopy and ICE times were systematically recorded during each procedure. In addition, the images were analyzed and recorded and their utility was evaluated after each case. A case control analysis was also performed. RESULTS: Seven patients underwent ICE (as part of an ICE trial period) during their electrophysiology study between July 21 and August 13, 1998. This study demonstrated the utility of ICE in identifying radiofrequency catheter tip stability and intracardiac and endocardial structures, including the crista terminalis, coronary sinus, and foramen ovale. CONCLUSION: ICE contributes to the electrophysiology arsenal for both diagnostic and therapeutic procedures. In particular, this technique demonstrated a decrease in fluoroscopy time as compared to a case control population. This technique limits radiation to the patient and operator. In addition, endocardial structures, which may be pivotal in diagnosing and treating cardiac arrhythmias, were easily identified. Radiofrequency catheter stability can also be assessed with this technique. PMID- 10745553 TI - Stenting of the right internal mammary artery graft and right coronary artery via a femoral approach. AB - We describe a case of balloon angioplasty and stenting of the right internal mammary artery (RIMA) graft anastomosis and the native right coronary artery through an in situ RIMA graft using two Bard XT stents (USCI Division of C.R. Bard, Inc., Billerica, Massachusetts). This case illustrates the feasibility of transluminal angioplasty and stenting of RIMA grafts and the native coronary artery using a femoral artery approach. PMID- 10745554 TI - Tortuous internal mammary artery angioplasty: accordion effect with limitation of flow. AB - Mechanical straightening of a tortuous vessel during angioplasty has been well described. It can be mistaken for thrombus, dissection or spasm. This report presents a case in which straightening of vessel due to stiff guide wire results in accordion effect and flow limitation. PMID- 10745555 TI - Angioplasty and stenting of solitary supra-aortic artery and aortoplasty by kissing balloon technique. AB - A child presented with symptoms of compromise to cerebral blood flow and cardiac failure. On diagnostic angiography, he was found to have a discrete coarctation and related ostial stenosis of the left subclavian artery, which acted as the sole source of cerebral blood flow. The subclavian lesion was initially dilated with a 6 mm x 50 mm balloon. The discrete coarctation was then dilated with an 8 mm x 50 mm balloon. Since significant residual stenosis was present at the subclavian origin, it was stented with a 20 mm Palmaz-Schatz stent (Cordis Corporation, Miami Lakes, Florida). Since the coarcted segment required further dilatations, the kissing balloon technique was used, wherein the 6 mm balloon was placed extending from the left subclavian lesion distally to the related aortic lesion proximally, along with another 10 mm balloon in the aorta. The end result was acceptable and the patient's symptoms improved significantly after the procedure. PMID- 10745556 TI - The glycoprotein IIb/IIIa receptor inhibitor (abciximab) in ST-segment elevation myocardial infarction. AB - A successful therapy is presented using the antiplatelet agent abciximab, alone, in an ST-segment elevation acute myocardial infarction. The patient was treated in a center with permanent catheterization facilities. Clinical trials are necessary to validate the efficacy and the cost-effectiveness of this clinical decision and therapeutic option. PMID- 10745557 TI - Iatrogenic left anterior descending artery stenosis in response to the nose cone of a directional atherectomy device: a different sort of restenosis. AB - Restenosis after percutaneous transluminal coronary angioplasty (PTCA) is well described and refers to the development of a significant narrowing at the site of the lesion treated. A case is reported of a female patient who underwent directional coronary atherectomy (DCA) and stent insertion at the origin of the left anterior descending artery. This was performed without complication. Repeat angiography was performed because of return of angina after ten weeks; it demonstrated a widely patent stent, but also a significant stenosis distal to the stent where the nose cone of the DCA device had been positioned. This stenosis was successfully stented. The patient remains well at follow-up. PMID- 10745558 TI - The changing role for cardiologists: challenges and opportunities in the 21st century. PMID- 10745559 TI - 'N Sync--a walk among the giants. PMID- 10745560 TI - Resources needed to collect and report data for heart care. PMID- 10745561 TI - The peripheral vessels: more than just a pathway. PMID- 10745562 TI - Detection of angiographic coronary restenosis after percutaneous transluminal coronary angioplasty (PTCA): usefulness of angina combined with rest electrocardiogram. AB - PURPOSE: To evaluate the value of recurrent anginal symptoms combined with seriation of conventional EKG in detecting coronary restenosis (renarrowing > or = 50% at the site dilated) after PTCA. Two hundred and seventy patients (204 men, mean age 53.1 +/- 9.4 years) with angina who submitted to successful PTCA of single-vessel coronary disease were followed and restudied angiographically 5.2 +/- 1.6 months after the procedure. At any moment from PTCA to follow-up: 1) typical angina pectoris was classified as either absent or present; 2) a 12-lead EKG was classified as improved, unchanged or worsened in comparison with the EKG either before or early after PTCA on the basis of ST-T alterations; 3) all patients underwent coronariography and were then classified as either having or lacking restenosis. The sensitivity and predictive positive value of recurrent angina, worsened EKG, or both, in detecting restenosis were respectively: 74.6% and 68.6%, 44.3% and 85.3%, and 55.9 and 91.7%. The specificity and predictive negative value of no recurrence of angina, improved EKG, or both, in detecting the absence of restenosis were respectively: 85.8% and 89.1%; 69.6% and 91.0%; and 75.6% and 93.9%. CONCLUSION: In single-vessel coronary disease, the detection of coronary restenosis after PTCA based on behavior of the symptoms plus rest EKG seriation shows acceptable degrees of sensitivity, specificity and mainly of predictive values when comparison with respective historical values of the ergometric test is considered. PMID- 10745563 TI - Influence of different stent materials on endothelialization in vitro. AB - The objective of our study was to investigate the influence of different stent materials on endothelialization in vitro. Using the non-destructive Alamar Blue assay and scanning electron microscopy, we compared long-term growth and morphology of vascular cells on disks of three prospective stent materials, i.e., 316 L stainless steel, 18 K, and 24 K gold. Our results demonstrate superior human EC proliferative capacity on gold surfaces compared to that on 316 L stainless steel. Thus, both the hyperplasia and thrombotic complications which often follow stenting might be minimized by employing gold stents, which have a greater capacity than steel in supporting a functional neo-endothelium. PMID- 10745564 TI - Immediate femoral sheath removal after 6 French routine coronary angioplasty with a weight-adjusted low-dose heparin: results of a prospective registry. AB - This study assesses the feasibility and safety of immediate sheath removal after coronary angioplasty with the use of 6 French (Fr) guiding catheters by the femoral route and weight-adjusted low-dose heparin (100 IU/kg). We prospectively evaluated such a strategy among a single-center cohort of 261 consecutive patients undergoing routine percutaneous transluminal coronary angioplasty (PTCA). Immediate sheath withdrawal was performed in cases when post-PTCA residual coronary stenosis was less than 30%, with or without stenting. One hundred eighty-two (70%) of the enrolled patients were eligible for immediate sheath removal. When compared with non-eligible patients (sheath removal 4 hours or more post-PTCA), we observed a reduction of hematoma occurrence (15% vs. 30%; p < 0.01), time to manual hemostasis of the puncture site (13.8 +/- 7 vs. 19.7 +/ 12 minutes; p < 0.0001), and time to hospital discharge (2.2 +/- 1.9 vs. 2.8 +/- 1.8 days; p < 0.02), while ischemic event rate was similar (1 vs. 2 non-Q wave myocardial infarction; 2 vs. 1 repeat PTCA for out-of-lab acute vessel closure). In conclusion, a good angiographic result at completion of PTCA using a 6 Fr sheath, even without stenting, makes an immediate sheath removal feasible at no increased risk and with a potential reduction in minor bleeding complications. PMID- 10745565 TI - Rheolytic thrombectomy with the Possis AngioJet: technical considerations and initial clinical experience. AB - Percutaneous revascularization of thrombus-containing lesions has an increased incidence of adverse events such as abrupt vessel closure, periprocedural myocardial infarction and death. Various pharmacologic and mechanical approaches have demonstrated modest angiographic success with significant periprocedural complications. The AngioJet (Possis Medical, Inc., Minneapolis, Minnesota) is a dual lumen catheter that uses the principle of rheolytic thrombectomy to remove unorganized thrombus. A previously unreported scoring system based on clinical and angiographic data may help to select patients most likely to benefit from AngioJet treatment. When compared with urokinase in the randomized VeGAS 2 Trial, AngioJet therapy yields greater angiographic success with a lower incidence of 30 day major adverse events. Technical considerations and complications are reviewed in detail. PMID- 10745566 TI - Recurrent angina and restenosis after successful angioplasty. PMID- 10745567 TI - Single coronary arteries: two cases with distinct and previously undescribed angiographic patterns. AB - Single coronary artery is a rare congenital anomaly, sometimes associated with myocardial ischemia. We present the clinical and angiographic features of two symptomatic patients with documented myocardial ischemia and with distinct and previously undescribed patterns of single right coronary arteries. These cases are new variants of the types R-I and R-II-A, in which the most probable mechanisms of ischemia are the insufficient blood supply, due to the long trajectories of the single arteries and the presence of underdeveloped vessels. Also, our second case presented with a fistulae from the LCX to the left ventricle, which is another determinant of myocardial ischemia. PMID- 10745568 TI - Electrical neuromodulation for disabling angina pectoris related to isolated stenoses of small epicardial coronary arteries. AB - Patients with symptomatic small vessel coronary artery disease may be inadequate candidates for revascularization procedures. They may suffer from refractory angina, which does not respond to maximal anti-anginal drug therapy. In addition to patients with end stage coronary artery disease and syndrome X, this newly defined group of subjects with an isolated stenosis of a small coronary artery may benefit from electrical neurostimulation. We describe two patients with intractable angina caused by a significant narrowing of a diagonal branch. This treatment modality should be considered as an alternative method for unsatisfactory revascularization procedures. PMID- 10745569 TI - Successful intracranial thrombolysis for cerebral thromboembolic complications resulting from cardiovascular diagnostic and interventional procedures. AB - We report successful local thrombolysis to treat intracranial ischemic complications of angioplasty with stenting of a high-grade carotid artery stenosis, angioplasty with thrombolysis of an occluded venous graft, and routine coronary angiography. Intracranial complications occurring during cardiovascular diagnostic or interventional procedures can be reversed with prompt management via rescue thrombolysis. These events should not be viewed as irreversible complications, and the opportunity for intracranial thrombolysis should be available wherever cardiovascular interventional procedures are performed. The ability to quickly recognize and remedy complications occurring during these procedures is another step in the continued advancement of neuroendovascular therapy. PMID- 10745570 TI - Second generation bifurcation stent: the rapid development of device technology. AB - A 57-year-old male with unstable angina and an eccentric 88% diameter stenosis of the left anterior descending artery prior to, and involving the first diagonal branch was treated with the 2nd generation Jomed Sidebranch stent (Jomed, Randingengen, Germany). This case outlines the improvements in this novel stent design and demonstrates the rapid advance of device design. PMID- 10745571 TI - Successful balloon angioplasty of in-stent restenosis of left internal mammary artery. AB - The development of disease in left internal mammary artery bypass grafts is uncommon. Furthermore, development of restenosis following successful angioplasty is very low. We report a patient with in-stent restenosis of left internal mammary graft treated successfully by balloon dilatation. PMID- 10745572 TI - Percutaneous arterial puncture and endoluminal access techniques for peripheral intervention. PMID- 10745573 TI - The effects of intrinsic sympathomimetic activity on beta-blocker efficacy for treatment of neurocardiogenic syncope. AB - To compare the efficacy and side effects of beta-blockers with intrinsic sympathomimetic activity (ISA) to those without ISA, we retrospectively reviewed patients diagnosed with neurocardiogenic syncope (NCS) as determined by head-up tilt table testing. Four hundred and thirty-one patients (mean age of 57 +/- 25 years) underwent head-up tilt table testing for syncope of unknown etiology, of which 120 patients were diagnosed with NCS; 87 of these patients were treated with beta-blocker therapy. Only 56 patients could be contacted during follow-up. Twenty-eight patients were treated with beta-blockers with ISA (acebutolol or pindolol) and 28 received beta-blockers without ISA (atenolol or metoprolol) based on physician preference and followed for up to 2 years. During the follow up period, beta-blockers with or without ISA had comparable clinical efficacy in suppressing recurrent syncope in patients with NCS. However, beta-blockers with ISA were better tolerated and caused less fatigue (32% side effects) as compared to those without ISA (50% side effects; p = 0.23). The benefits of beta-blockers with ISA were more pronounced in patients less than 60 years old (19% side effects with beta-blocker with ISA as compared to 85% side effects with beta blocker without ISA; p = 0.0004). Beta-blockers without ISA appear to be better tolerated and caused less fatigue in patients 65 years old or greater (20%) than in patients less than 65 years old (85%; p = 0.0002). PMID- 10745574 TI - Percutaneous balloon mitral valvuloplasty for patients with calcified mitral valves. PMID- 10745576 TI - Enhanced local intracoronary delivery of heparin with the Infiltrator catheter: a comparative study. AB - The efficacy of local drug delivery in the treatment of coronary artery disease is limited by the relatively low delivery efficiency of the available devices. A unique local drug delivery device, the Infiltrator catheter (InterVentional Technologies, Inc.), has been designed specifically to enhance efficiency by injecting drugs directly into the arterial wall through microports mounted on the balloon surface. The purpose of this study was to assess the efficiency of delivery of this device in the porcine coronary model and to compare it to a previously validated device, the hydrogel balloon (Boston Scientific, Maple Grove, Minnesota). Studies were also performed to assess the pattern of intramural heparin deposition following delivery with the Infiltrator catheter and to assess the effect of the microports on vascular integrity. The efficiency of delivery was significantly greater with the Infiltrator catheter than with the hydrogel balloon (4.5% vs. 0.08%; p = 0.02). Similarly, the absolute amount of intramurally deposited heparin was greater with the Infiltrator (111.3 +/- 38.5 units vs. 2.4 +/- 0.85 units; p = 0.02) despite the fact that more heparin was delivered with the hydrogel catheter. Histologic studies revealed characteristic discrete puncture channels in the vessel wall due to penetration of the microports. Other than this histologic finding, there was no significant difference in the extent of architectural disruption between the Infiltrator and conventional balloon inflations. Fluorescein-labeled heparin studies revealed heparin to be diffusely distributed throughout the vessel wall immediately following delivery with the Infiltrator. We conclude that the Infiltrator catheterOs unique mechanism of delivery improves the efficiency of local drug delivery without excessive vessel wall trauma. PMID- 10745577 TI - Can flow cytometry detect successful ticlopidine treatment in patients with VVI pacemakers? AB - An increased platelet activation status is present in patients with VVI pacemakers. With platelet activation, there is modulation of platelet surface molecule expression. In the current study, the expression of platelet surface markers in VVI patients before and after ticlopidine treatment and control subjects was investigated by means of flow cytometry. The study group consisted of 25 patients with VVI pacemaker, and 15 control subjects. CD42b, CD61, and CD62p expression were significantly increased in VVI patients compared with control subjects (CD42b p < 0.001, CD61 p< 0.005 and CD62p p < 0.001). In addition, after ticlopidine treatment, platelets showed a significant fall in expression of all these markers in VVI patients (CD42b p < 0.001, CD61 p < 0.005 and CD62p p< 0.001). Our data suggest an increase of the surface expression of all these markers on platelets and demonstrate the efficacy of ticlopidine in reducing them. PMID- 10745578 TI - Preclinical evaluation of a rheolytic catheter for percutaneous coronary artery/saphenous vein graft thrombectomy. AB - The presence of thrombus increases the rate of acute complications and restenosis in percutaneous revascularization of native coronary arteries and saphenous vein grafts. Rheolytic thrombectomy uses high velocity saline jets to create a Bernoulli effect for thrombus entrainment, dissociation, and evacuation of debris, providing a novel approach to the treatment of thrombotic lesions. The study objective was to determine the preclinical safety and effectiveness of a 5 French rheolytic thrombectomy catheter designed for use in coronary arteries and saphenous vein grafts. In vitro testing was performed to evaluate catheter effectiveness (clot removal rate) and safety (particle generation and hemolysis). This was followed by acute (n = 6) and chronic (n = 6) canine studies to determine hemodynamic, angiographic, and histopathologic effects of the catheter. The results showed effective clot removal with minimal embolization: 99.4% of the total clot volume was removed with only 0.1% proximal embolization and 0.5% distal embolization. 98.4% of the embolic particles were less than 10 microm. Canine studies revealed no significant angiographic, hemodynamic, histopathologic, or electrocardiographic abnormalities with the exception of transient heart block in one animal. There was transient hemolysis which normalized within 24 hours with no adverse effects. These results demonstrate the effectiveness and safety of coronary rheolytic thrombectomy and provided the basis for clinical trials to further evaluate this promising new approach for coronary thrombectomy. PMID- 10745579 TI - Cleaning up the mess: new approaches to the old problem of thrombus in coronary interventions. PMID- 10745580 TI - Intracoronary thrombus: an ongoing challenge. PMID- 10745581 TI - A step forward in the treatment of thrombotic lesions. PMID- 10745582 TI - Platelet glycoprotein IIb/IIIa integrin inhibition in acute myocardial infarction. PMID- 10745583 TI - Normal electrocardiogram with total occlusion of the left anterior descending coronary artery. AB - Total occlusion of the left anterior descending coronary artery is usually characterized by ST-segment elevation in the anterior leads of the surface electrocardiogram. We report a case of a patient who had a persistently normal electrocardiogram throughout his hospitalization despite the angiographic findings of total occlusion of the left anterior descending coronary artery and no collateral vessels. Percutaneous transluminal coronary angioplasty with stent placement was performed successfully. PMID- 10745584 TI - Stenting of a superior mesenteric artery lesion via the right arm approach. AB - Chronic mesenteric ischemia is rare and commonly presents with abdominal pain and weight loss. Treatment options are limited to surgical or endovascular revascularization. In this report we describe in detail successful stent supported angioplasty of a high-grade superior mesenteric artery stenosis utilizing a right brachial artery approach. A brief review of the literature is provided. PMID- 10745585 TI - Stent implantation in a central aorto-pulmonary shunt. AB - A 5.5-week-old infant with tricuspid atresia presented with severe hypoxemia not responding to the placement of a central shunt (4 mm polytetrafluorethylene). The infant was taken to the catheterization laboratory, where an AVE stent was successfully implanted in a severe postoperative stenosis, at the pulmonary end of the anastomosis. The oxygen saturation improved significantly, however, the infant died due to renal failure. The post-mortem anatomical findings are shown. PMID- 10745586 TI - Stenting of an anomalous left circumflex coronary artery arising from the right coronary artery. AB - Coronary angioplasty can provide excellent means of revascularization of anomalous coronary arteries. Successful application of angioplasty to these vessels requires angiographic knowledge of their course, structure and appropriate equipment selection. Advancement of stent delivery systems in such cases requires good support with the possibility of selective cannulation and deep engagement of the guiding catheter. We report a case of stent placement in an anomalous circumflex artery arising from the right coronary artery. PMID- 10745587 TI - Transluminal extraction catheter atherectomy for the treatment of acute occlusion of an ectatic coronary artery. AB - Thrombotic occlusion of an ectatic coronary artery may not respond to thrombolytic therapy or balloon angioplasty, since the infarct-related vessel contains a significant amount of thrombus. A patient with acute myocardial infarction of an ectatic right coronary artery that was occluded by a heavy clot burden is described. The patient was treated successfully with transluminal extraction catheter atherectomy and results were confirmed by intravascular ultrasound. PMID- 10745588 TI - Physics of vascular brachytherapy. AB - Basic physics plays an important role in understanding the clinical utility of radioisotopes in brachytherapy. Vascular brachytherapy is a very unique application of localized radiation in that dose levels very close to the source are employed to treat tissues within the arterial wall. This article covers basic physics of radioactivity and differentiates between beta and gamma radiations. Physical parameters such as activity, half-life, exposure and absorbed dose have been explained. Finally, the dose distribution around a point source and a linear source is described. The principles of basic physics are likely to play an important role in shaping the emerging technology and its application in vascular brachytherapy. PMID- 10745589 TI - Pacing for paroxsymal atrial fibrillation: where do we stand? PMID- 10745590 TI - Sensible technology. PMID- 10745591 TI - Fractional myocardial flow reserve (FFRmyo) after coronary intervention as a predictor of chronic restenosis. AB - The angiographic assessment of coronary stenosis has many limitations, especially after coronary intervention. To determine the physiologic significance of such lesions, we measured the mean translesional pressure gradients at rest (P1), those during hyperemia induced by intracoronary administration of papaverine (12 mg in the left and 8 mg in the right coronary artery) (P2), and fractional myocardial flow reserve (FFRmyo) which is derived from the ratio of the mean distal coronary pressure and aortic pressure during hyperemia. Our objective was to determine the relations among P1, P2, and FFRmyo and restenosis as potential predictors of chronic restenosis. The grouped study consisted of 32 patients with ischemic heart disease scheduled to undergo intervention. The distal coronary pressure was measured using a 0.014 inch pressure monitoring wire (Pressure Guide, Radi Medical Systems, Uppsala, Sweden). The guidewire was advanced through the lesional segment. The mean percent diameter stenosis (%DS) was 67.9 +/- 13.3% before intervention and 22.9 +/- 15.3% after intervention. The mean P2 (9.9 +/- 5.3 mmHg) was significantly higher than the mean P1 (4.5 +/- 3.6 mmHg; p < 0.0001). There was no correlation of P1, P2 or FFRmyo with the %DS after intervention. Follow-up angiography (after 6 months) performed on all patients revealed restenosis (%DS > or =50%) in four patients (12.5%). There was no correlation between P1 and the %DS at the follow-up angiography, but the relation between P2, FFRmyo and the %DS at the follow-up angiography was significant (r = 0.599, p < 0.01; r = 0.703, p < 0.0001, respectively). As a measurement of P2, FFRmyo is useful for the determination of the endpoint of intervention in consideration of prevention of restenosis. A new endpoint may be established after further evaluation in a greater number of patients. PMID- 10745593 TI - Feasibility of routine transradial coronary angiography: a single operator's experience. AB - The aims of this study were to assess the feasibility of routine transradial coronary angiography in a standard population of patients with presumed coronary artery disease over a period of time long enough to allow for technical evolution and evaluation of a single operator's learning curve, and to provide data for a randomized comparison versus the femoral approach. Between June 1994 and March 1997, transradial angiography was attempted in 1,000 patients. Approximately 25% of these patients were excluded because of an abnormal Allen test. Except in the case of acute myocardial infarction, there was no selection based on symptoms, age, sex, weight or size in the absence of double internal mammary artery bypass graft operation or simultaneous right heart catheterization. Symptoms and angiographic results were typical of a standard population. The right radial approach was used in 95% of the cases for ease of handling and comfort of a right handed operator. Radial artery puncture and catheterization success was obtained in 97.6% of the cases; the left coronary artery was selectively catheterized in 100%, right coronary artery in 98%, left ventricle in 96.9%, mammary artery grafts in 100% and saphenous grafts in 97.2%. Average procedure duration was 18 +/- 9 minutes, and decreased progressively with experience and catheter strategies. The optimal catheter selection would seem to be a single catheter, either left Amplatz or Champ, for both coronary arteries. Two coronary complications and 3 transient neurological complications occurred, but no clinically significant vascular complications requiring surgery or transfusion were reported. Transradial angiography seems to be a routine approach that should now be compared with the femoral approach and supersede the brachial approach whenever possible. PMID- 10745592 TI - Benchmarking cardiac catheterization laboratories: the impact of patient age, gender and risk factors on variable costs, device costs, total time and procedural time in 53 catheterization laboratories. AB - Coronary catheterization laboratories (CCLs) are the cornerstones of the delivery system for many cardiovascular procedures performed in the United States. However, few comprehensive data exist benchmarking physician activities in CCLs. This study benchmarks cost and time data on 82,548 consecutive patient encounters in 53 CCLs for the 18-month period of January 1997 through June 1998. The data are compiled from the OEP program, a relational database developed by Boston Scientific/Scimed (Maple Grove, Minnesota) for use in CCLs. CCL productivity (total time and procedure time) and cost (variable costs and device costs) benchmarks are created for: 1) left heart catheterization; 2) right and left heart catheterization; 3) percutaneous transluminal coronary balloon angioplasty (PTCA); 4) atherectomy; and 5) coronary stents. Results show the variable costs (those costs that vary in direct proportion to changes in CCL activities) for the five procedures are: $308, left heart catheterization; $395, right and left heart catheterization; $841, PTCA; $2,768, atherectomy; and $3,186, coronary stent. These variable costs are lower than the typical average costs reported for these procedures because they do not include hospital, laboratory, and physician costs, only the procedure-specific activity-related costs most directly controlled and/or influenced by CCL physicians or administrators. The total time for the left heart catheterization averaged 64 minutes and 84 minutes for the right and left heart catheterization, respectively, and procedural times averaged 25 and 32 minutes, respectively. For the major interventional procedures N PTCA, atherectomy, and coronary stents, total times averages were 102, 135, and 117 minutes, respectively. Procedural times for these procedures averaged between 60 and 65 percent of the total time. The major implications of these findings are discussed and limitations noted. PMID- 10745594 TI - Worse six-month outcome for patients with multiple stents in a single coronary artery. AB - BACKGROUND: Before the "era" of optimal stent deployment, very few data concerning multiple stents in a single coronary artery showed restenosis rates up to 60%. OBJECTIVE: To evaluate the 6-month outcome of patients receiving multiple Palmaz-Schatz stents (> or =2 stents) in a single coronary artery compared to those receiving single stents. METHODS: Three hundred and forty-eight patients having multiple stents were compared to 174 patients receiving single stents during a 6-month follow-up. RESULTS: Repeat target lesion revascularization (RTLR), either repeat PTCA or CABG, was 10.4% in the single-stent group, 22.6% in the two-stent group, and 23.1% in the > or =2 stent group (p = 0.001, single versus 2 or > or =2 stents). There was not a significant difference between single stent and multiple stent groups in myocardial infarction and death during 6-month follow-up. Multivariate analysis showed multiple stents, diabetes mellitus, and type C lesion to be predictors of RTLR. CONCLUSIONS: Placement of two or more stents was associated with a significantly higher RTLR compared with single stent placement. The optimal approach to diffuse coronary artery disease remains to be defined. PMID- 10745595 TI - The transradial approach: where do we stand? PMID- 10745596 TI - The dilemma of treating complex coronary artery disease. PMID- 10745597 TI - Coil embolization of a giant atherosclerotic coronary artery aneurysm. AB - The incidence of the coronary artery aneurysm varies from 1.5-5%. Atherosclerosis is the most common cause of coronary artery aneurysm in adults. A discrete, giant, saccular atherosclerotic coronary artery aneurysm in an artery without significant proximal stenosis is rare. We report the first such case of a giant atherosclerotic coronary artery aneurysm successfully treated with coil embolization. PMID- 10745598 TI - Angioplasty/bypass combination therapy in an 89-year-old man with left main trunk disease. AB - We report a case of an 89-year-old man presenting with unstable angina and left main trunk disease. Minimally invasive direct coronary artery bypass grafting supplemented by catheter intervention was successfully performed. In view of the increasing elderly population, angioplasty/bypass combination therapy may be an important alternative for elderly coronary artery disease patients. PMID- 10745599 TI - Use of the multifunction probing catheter as an adjunctive device for occluded vein graft intervention. AB - We describe the use of the multifunction probing catheter (Schneider, Bulach, Switzerland) as an adjunct to conventional techniques in the treatment of a recent vein graft occlusion by thrombus, in order to highlight a possible further use for this device. PMID- 10745600 TI - Stent-supported angioplasty of an ostial left main stenosis following replacement of the ascending aorta with reimplantation of the coronary arteries. AB - We describe a patient who underwent replacement of the descending aorta with reimplantation of the coronary arteries for acute type II aortic dissection and developed iatrogenic left main stem stenosis 1 year after operation. The patient was successfully treated by stent implantation in the left main stem. PMID- 10745601 TI - Peripheral vascular disease in Behcet's syndrome. AB - We describe a patient with Behcet's syndrome who had peripheral vascular disease involving the left subclavian artery for which angioplasty with stent placement was performed and reangioplasty done for in-stent restenosis. She presented with recurrence one year after stent placement; angiography revealed diffuse disease of the axillary and radial arteries with mild to moderate restenosis at the site of stent placement. PMID- 10745602 TI - Endovascular grafting of abdominal aortic aneurysms (AAA): historical development and evolving technologies. PMID- 10745603 TI - Compassionate use of intracoronary beta-irradiation for treatment of recurrent in stent restenosis. AB - Recurrent in-stent restenosis after balloon angioplasty poses a serious management problem. Previously g-radiation has been shown to be effective in patients with in-stent restenosis. The aim of the study was to determine the feasibility and safety of b-radiation in patients with recurrent in-stent restenosis. From May 1997 to December 1998, 18 patients were treated with balloon angioplasty (n = 8) or laser (n = 10), followed by intracoronary b-radiation at a prescribed dose of 16 Gray at 2 mm from the source, for reference diameters by quantitative coronary angiography < 3.25 mm or 20 Gray for reference diameters > or =3.25 mm. Vessels treated were as follows: left anterior descending: (n = 5); circumflex: (n = 4); right coronary artery: (n = 6); saphenous vein graft: (n = 3). Average recurrence rate was 2.4 +/- 0.7 and the restenotic length was 16 +/- 7 mm. b-radiation was successfully delivered in all patients. Two patients presented complications related to laser debulking: a non-Q wave myocardial infarction in one and a re-angioplasty due to uncovered distal dissection in another. Geographical miss, defined as an area which has been injured but not covered by the radiation source, was demonstrated in 8 patients. Seventeen patients (94%) completed the 6-month angiographic follow-up. Restenosis (> 50% Diameter Stenosis) was observed in 9 patients (53%), leading to target lesion revascularization in 8 patients (47%). Six of the 9 restenoses were located in areas with geographical miss. Intracoronary b-radiation for recurrent in-stent restenosis appears to be a safe and feasible management strategy. However, the mismatch between injured and irradiated area may lead to failure of this therapy. PMID- 10745604 TI - The role of anti-thrombins and low molecular weight heparins for acute coronary syndromes. PMID- 10745605 TI - Anticoagulation requirements for coronary interventions and the role of low molecular weight heparins. PMID- 10745606 TI - Glycoprotein IIb/IIIa receptor antagonists in acute coronary syndromes. PMID- 10745607 TI - Aspirin, ticlopidine and clopidogrel in and out of the catheterization laboratory. PMID- 10745608 TI - The role of local low molecular weight heparin delivery in prevention of restenosis following coronary intervention. PMID- 10745610 TI - Vasoseal hemostasis following coronary interventions with abciximab. AB - Femoral arteriotomy management using a collagen vascular hemostasis device (VasoSeal, Datascope Corporation, Montvale, New Jersey) was studied in 50 consecutive patients following interventional coronary procedures performed with abciximab (ReoPro, Eli Lilly and Company, Indianapolis, Indiana). Low-dose weight adjusted or no heparin was employed. The first 25 patients were permitted to sit up after 6 hours with ambulation the following day. The second 25 patients were allowed to sit up after 1 hour and ambulate after 6 hours. Despite early activity and ambulation, there were no hemorrhagic complications including hematoma, pseudoaneurysm, blood transfusion, or surgical repair. Hemoglobin and platelet counts remained stable overnight prior to discharge. This pilot study demonstrates the potential efficacy of VasoSeal in achieving early sheath removal and ambulation in patients undergoing interventional procedures using ReoPro. PMID- 10745611 TI - A multicenter randomized trial comparing a percutaneous collagen hemostasis device with conventional manual compression after diagnostic angiography and angioplasty. AB - OBJECTIVES: A new percutaneous collagen hemostasis device was compared with conventional compression techniques after diagnostic catheterization and angioplasty. Background. Peripheral vascular complications after diagnostic catheterization or more complex interventional procedures, as well as the discomfort of manual compression and prolonged bed rest, represent significant morbidity for invasive cardiac procedures. METHODS: A prospective, multicenter, randomized trial was designed to compare the hemostasis time in minutes and the incidence of vascular complications in patients receiving a vascular hemostasis device with those undergoing conventional compression techniques. RESULTS: After diagnostic catheterization, hemostasis time was significantly less with the vascular hemostasis device than with conventional manual compression (4.1 +/- 2.8 min [n=90 patients] vs. 17.6 +/- 9.2 min [n= 75], p < 0.0001). This difference was greater in patients undergoing angioplasty and was unrelated to the anticoagulation status (4.3 +/- 3.7 min [n = 71 not receiving the heparin], 7.6 +/- 11.6 min [n= 85 receiving heparin], 33.6 +/- 24.2 min [n= 134 control patients not receiving heparin], p < 0.0001 vs. control patients). The time from the start of the procedure to ambulation was slightly less after diagnostic catheterization in patients treated with the device (13.3 +/- 12.1 hours vs. 19.2 +/- 17.8 hours, p < 0.05). It was also less in patients who underwent angioplasty when the device was used after discontinuation of anticoagulation (23.0 +/- 11.1 hours, without heparin), as compared with control compression techniques (32.7 +/ 18.8 hours, p < 0.0001). Time to ambulation was even shorter (16.1 +/- 11.1 h, p < 0.0001) in patients in whom the device was placed immediately after angioplasty while they were still fully anticoagulated with a prolonged activated clotting time (336 +/- 85 s). There were no major complications (surgery or transfusion) after diagnostic catheterization and a low incidence of major complications in patients who underwent angioplasty (0.7% in control patients, 1.4% with the device without heparin, 1.2% with the device and heparin, p = NS). After angioplasty, there was a trend toward fewer hematomas when the device was used in the absence of heparin (4.2% vs. 9.7% in control patients, p = 0.14). CONCLUSION: A new vascular hemostasis device can significantly reduce the puncture site hemostasis time and the time to ambulation without significantly increasing the risk of peripheral vascular complications The role of this technology in reducing complications, length of hospital stay and cost remains to be determined. PMID- 10745612 TI - Collagen application for sealing of arterial puncture sites in comparison to pressure dressing: a randomized trial. AB - One-hundred patients undergoing routine diagnostic or interventional catheterization were randomly assigned to receive either percutaneously applied collagen (group A; n = 50) or conventional pressure dressing (group B; n = 50) for sealing of the femoral artery. Clinical variables were comparable in both groups. The heparin dose was 100 IU/kg in 30 patients and 200 IU/kg in 20 patients of either group. The average compression time was 4.3 minutes in group A and 42.3 minutes in group B (p < 0.001). Bleeding was not observed in group A but was observed in 6/50 patients in group B. The time to ambulation was 6.4 hours (range: 4-12 hours) in group A and 21.6 hours (range: 10-48 hours) in group B (p < 0.001). Hematomas with a diameter of > 6 cm developed in 4/50 patients in group A and in 11/50 patients in group B (p < 0.05). Blood transfusion or surgical interventions were not required and there was no loss of ankle pulses in either group. CONCLUSION: Percutaneously applied collagen reduced compression time and duration of bedrest after diagnostic catheterization and PTCA. Despite earlier ambulation, the incidence of bleeding was lower with collagen than with conventional pressure dressing. PMID- 10745614 TI - New platelet glycoprotein IIb/IIIa inhibitor trials in percutaneous coronary intervention. PMID- 10745613 TI - Usefulness of collagen plugging with VasoSeal after PTCA as compared to manual compression with identical sheath dwell times. AB - This study investigated the usefulness of collagen plugging with VasoSeal (Datascope Corporation, Montvale, New Jersey) in patients after PTCA compared to a control group having identical sheath dwell times and therefore comparable levels of anticoagulation. A total of 150 patients were enrolled in this prospective and randomized study. Sheaths were pulled at exactly 5 hours after arterial puncture. Time to hemostasis and local complications were determined. There were no statistical differences in baseline characteristics. The mean time to hemostasis in the collagen group was significantly shorter (3 +/- 3 minutes) than that of the control group (17.4 +/- 7 minutes). At 24 hours, 23% of the collagen group patients had a small, 1% a medium and 4% a large hematoma. In the control group, 32% had a small and 4% a medium-sized hematoma, but no patient a large hematoma. After collagen, one patient developed a pseudoaneurysm needing vascular surgery. In the control group, no major complications occurred. Compared to patients with manual compression at an identical sheath dwell time and an identical level of anticoagulation, there was a significant reduction in time to hemostasis but no statistical difference regarding local complications. Although the incidence of medium or large hematoma was low, the trend towards a decreased risk of smaller hematomas seemed to be counterbalanced by an increased risk of larger hematomas. PMID- 10745615 TI - Tirofiban--an overview of the phase III trials. PMID- 10745616 TI - Economic implications of coronary stenting with adjunctive IIb/IIIa receptor antagonists in a community hospital. AB - To assess the implications of coronary stenting with several IIb/IIIa receptor antagonists, total hospital cost and adverse events were reviewed for 674 elective stent procedures from June 1998 through December 1998. The use of IIb/IIIa receptor antagonism and the agent selected were at the discretion of the interventional cardiologist. In-hospital, 30-day and 6-month adverse cardiac events were similar among the treatment strategies. Target vessel revascularization at six months was similar among the treatment strategies. Patients who received a IIb/IIIa receptor blocker with their stent procedure were less likely to be rehospitalized within 30 days. Multivariate regression analysis identified specific factors responsible for prolongation of hospital stay including adverse cardiac events, physician practice pattern and age greater than 70 years (all p < 0.002). Overall hospital cost for patients receiving tirofiban as an adjunct to coronary stenting was approximately $1,000 less than patients receiving abciximab. Total cath lab expenditures were similar for these groups and the savings in hospital cost was directly attributable to a lower pharmacy cost in the tirofiban group. Multivariate regression analysis identified adverse cardiac events, left ventricular systolic dysfunction, multiple stent placement, physician practice and abciximab as significant contributors to increased hospital cost (all p < 0.002). Tirofiban as an adjunct to coronary stenting was not identified by multivariate analysis as a significant contributor to hospital cost. Bleeding rates were similar among the treatment strategies. Thus, coronary stenting in our community hospital is associated with acceptable outcomes regardless of treatment strategy and hospital cost is significantly influenced by the use of IIb/IIIa blockade with stenting and the type of agent selected. PMID- 10745617 TI - Optimizing antiplatelet therapy in patients with acute coronary syndromes: a role for monitoring platelet reactivity. AB - Platelet activation is seminal in the thrombosis underlying acute coronary syndromes. Furthermore, high levels of platelet reactivity are associated with an increased risk of future cardiovascular events both in apparently healthy subjects and men with overt coronary artery disease. We have developed a sensitive and specific assay of platelet reactivity using minimally altered whole blood to assess activation of glycoprotein IIb/IIIa and expression of P-selectin (a measure of alpha granule degranulation). This assay reliably identifies subjects with increased platelet reactivity who may benefit from more aggressive inhibition of platelet function on a long-term basis. In addition, this assay is sensitive to the effects of antiplatelet agents such as glycoprotein IIb/IIIa inhibitors. Abciximab, tirofiban and eptifibatide all inhibit fibrinogen binding in a concentration of dependent manner. However, marked variation in the extent of inhibition was seen between individuals. We have postulated that greater inhibition of agonist-induced fibrinogen binding in some and limited inhibition in others may account, in part, for tendencies to bleeding and recurrent thrombosis. This type of assay should allow for determination of platelet reactivity and titration of dosage to the needs of each individual. The assay has the potential to improve the care of patients with acute coronary syndromes and of those undergoing percutaneous coronary intervention as well as assist in the design of clinical trials utilizing antiplatelet drugs. PMID- 10745618 TI - Overview of glycoprotein IIb/IIIa clinical trials with abciximab and eptifibatide in acute coronary syndromes and percutaneous coronary intervention. AB - Numerous clinical trials have evaluated the use of glycoprotein IIb/IIIa inhibitors in acute coronary syndromes (ACS) and percutaneous coronary interventions (PCI). Four studies that looked at abciximab in these settings include the EPIC, EPILOG, CAPTURE, and EPISTENT trials. Two studies that looked at eptifibatide are the IMPACT-II and PURSUIT trials. These studies have demonstrated the benefits of these agents in treating these conditions. This manuscript will provide an overview of these clinical trials and comment on the difficulties of comparing these trials, the problems encountered with subgroup analyses, and provide thoughts on future areas of research that need to be explored. PMID- 10745619 TI - Platelet function in the myelodysplastic syndromes. AB - Bleeding in myelodysplastic syndromes (MDS) is often related to thrombocytopenia. However, because MDS is a stem cell disorder, it is conceivable that platelet function (PF) abnormalities may play a role as well. A few studies have been reported, mainly with platelet aggregation (PA). A summary of the information discloses that about 68 MDS patients have been studied. Despite some conflicting data, it can be concluded that PA defects are quite common in MDS. About 75% of MDS patients demonstrated reduced PA with epinephrine, followed by decreased PA with arachidonic acid (54%), ADP (46%), collagen (43%), and ristocetin (22%). Several other PFs were studied, but the data require caution in interpretation. Despite the relatively high incidence of platelet dysfunction, bleeding in MDS patients is uncommon, with episodes tending to be mild, although recurrent. Nevertheless, some correlation between PA abnormalities and the tendency to bleed has been suggested. As patients demonstrate more PA defects, they tend to bleed more. Some authors suggest that PF studies may assist in diagnosing tough cases of MDS, as well as serving as prognostic markers. A large-scale study testing PF in a large number of MDS patients is required and is expected. PMID- 10745620 TI - Hematopoiesis and angiogenesis. AB - Hematopoiesis is closely linked with angiogenesis, because they interact with each other and have common ancestors: hemangioblasts or hematogenic endothelial cells. The relationship is reasonable, because vascular and hematopoietic systems must develop together in order to establish the body's oxygen-delivery system during organogenesis. Hematopoietic stem cells have been shown to originate from the para-aortic splanchnopleural mesoderm region or aorta-gonads-mesonephros at successive stages. We discuss the molecular events in the differentiation of hematopoietic cells and endothelial cells. Transcription factors SCL/tal-1 and AML1; tyrosine kinase receptors Flk-1, Tie-2, Eph, and the sialomucins; CD34; thrombomucin; and AA4 play important biological roles in these lineages. PMID- 10745621 TI - Epstein-Barr virus--associated diseases in humans. AB - It has been known for 30 years that Epstein-Barr virus (EBV), a ubiquitous human herpesvirus, is the etiologic agent of acute infectious mononucleosis and is closely associated with the genesis of Burkitt's lymphoma and undifferentiated nasopharyngeal carcinoma. Recent studies have demonstrated that EBV is also implicated in a variety of other diseases, such as EBV-associated hemophagocytic syndrome, chronic active EBV infection, T-cell lymphoma, natural killer cell leukemia/lymphoma, lymphoproliferative diseases in immunocompromised hosts, Hodgkin's disease, pyothorax-associated B-cell lymphoma, smooth-muscle tumors, and gastric carcinoma. Thus, the virus continues to attract worldwide attention, and it is now appropriate for a reappraisal of the relation between EBV and human diseases. This review summarizes the recent progress in research on EBV and the clinical findings of EBV-associated diseases and provides a basis for the development of new therapeutic strategies. PMID- 10745622 TI - Characteristic features of the genotype and phenotype of hereditary spherocytosis in the Japanese population. AB - Hereditary spherocytosis (HS) is the most common hemolytic anemia of congenital origin in the Japanese population. Among 844 cases of 520 kindred with congenital red cell membrane disorders studied at the Kawasaki Medical School in the last 25 years (1975-1999), 407 cases (48.2%) of 215 kindred had HS. Among the recent 60 kindred with HS, autosomal dominant (AD) transmission was proven in 19. The remaining 41 non-AD HS included 1) homozygous patients with autosomal recessive inheritance, 2) HS patients with de novo gene mutations, and 3) mild HS with AD inheritance. The extent of clinical severity in the non-AD HS cases was nearly identical to that in the AD cases. The incidence of membrane protein abnormalities in our 60 Japanese HS kindred was unique: there were lower ankyrin deficiencies (7%), moderate band 3 deficiencies (20%), and much higher protein 4.2 deficiencies (45%), with 28% of unknown etiology. The incidence of membrane protein deficiencies corresponded to that determined by gene analyses; i.e., mutations mostly in band 3 and/or in protein 4.2 genes and fewer ankyrin gene mutations. In the band 3 gene, 11 mutations pathognomonic for HS were identified (3 frameshift and 8 missense mutations). There were 5 mutations of the protein 4.2 gene (3 missense mutations, 1 nonsense mutation, and 1 splicing mutation) pathognomonic for HS. On the other hand, 2 missense mutations were detected in the ankyrin gene in this study. The genetic abnormalities in our HS patients correlated well with the phenotypic ultrastructural abnormalities of red cell membranes in situ. Ankyrin mutations (ankyrin Marburg and ankyrin Stuttgart with frameshift mutations) were associated mostly with a disrupted cytoskeletal network, and band 3 mutations (band 3 Kagoshima with frameshift mutation) typically demonstrated anomalies of intramembrane particles (IMPs). Protein 4.2 mutations (homozygotes of protein 4.2 Nippon) with complete protein 4.2 deficiency showed abnormalities of both the cytoskeletal network and IMPs. PMID- 10745623 TI - Efficacy of a new formulation of lenograstim (recombinant glycosylated human granulocyte colony-stimulating factor) containing gelatin for the treatment of neutropenia after consolidation chemotherapy in patients with acute myeloid leukemia. AB - The efficacy and safety of a new formulation of lenograstim (recombinant glycosylated granulocyte colony-stimulating factor) prepared by switching the stabilizer from human serum albumin (HSA) to gelatin was investigated for the treatment of neutropenia after consolidation chemotherapy in patients with acute myeloid leukemia (AML). The results obtained in the study using the gelatin containing formulation (gelatin-lenograstim) were retrospectively compared to those obtained from a placebo-controlled double-blind randomized study (AML-DBT) using the HSA-containing formulation (HSA-lenograstim). The median time of neutrophil recovery to > or = 1000/mm3 was significantly shorter in the gelatin lenograstim group (14 days) than in the placebo group (21 days, P = .0001), and there was no significant difference between the gelatin-lenograstim group and the HSA-lenograstim group (14.5 days of AML-DBT, P = .5462). The incidences of febrile neutropenia were significantly reduced in the gelatin-lenograstim group (24/43, 55.8%) compared to the placebo group (58/64, 90.6%, P < .0001). The incidence of fever and antibiotic use was also significantly lower in the gelatin lenograstim group (69.8% and 83.7%, respectively) than in the placebo group (92.2%, P = .0034, and 96.9%, P = .0285, respectively). However, between the 2 groups there were no differences in the number of patients who had infectious episodes. No serious adverse drug reactions ascribed to gelatin-lenograstim were encountered. These results demonstrate that gelatin-lenograstim exerted beneficial effects in the acceleration of neutrophil recovery and in the reduction of fever, febrile neutropenia, and antibiotic use, and its efficacy was equivalent to HSA-lenograstim. Therefore, we concluded that the gelatin lenograstim formulation, which offers no risk of virus contamination and can be stored at room temperature, is more beneficial than the HSA-lenograstim formulation. PMID- 10745624 TI - Therapy-related leukemia and myelodysplastic syndrome: a large-scale Japanese study of clinical and cytogenetic features as well as prognostic factors. AB - It is known that alkylating agents and topoisomerase II inhibitors can cause distinct forms of therapy-related leukemia and myelodysplastic syndrome (TRL/MDS). Although several reports have been made on each of these agents separately, no study has yet been conducted to evaluate the effect of these two types of agents in the same population. In a nationwide, large-scale population study, the clinical and cytogenetic features as well as the prognostic factors in 256 patients with TRL/MDS were assessed. Median age was 61 years, and the median period of latency from primary malignancies was 47.9 months. The latency period was significantly shorter in patients undergoing chemotherapy, especially that of topoisomerase II inhibitors, for primary cancer. The morphological diagnosis of TRL/MDS was acute myeloid leukemia in 59% and MDS in 41% of patients. Chromosome abnormalities that frequently involved chromosomes 5, 7 or 11 were documented in 77% of the 189 patients examined. MLL gene rearrangements were detected in 11 of 58 subjects and were correlated with a borderline significance (P = 0.072) with topoisomerase II inhibitor administration. Overall median survival was only 9.7 months. Survival was similar in cases with or without MLL gene rearrangement. Multivariate analysis identified chromosome 5 abnormalities, hypoproteinemia, poor therapy outcomes for primary cancer, C-reactive protein, and thrombocytopenia as being significantly poor prognostic factors (P < 0.05). This large-population study provided a comprehensive update of TRL/MDS status in Japan, identified significant prognostic factors, and enabled the clinical significance of MLL gene rearrangement to be assessed. PMID- 10745625 TI - Protein expression and constitutive phosphorylation of hematopoietic transcription factors PU.1 and C/EBP beta in acute myeloid leukemia blasts. AB - The transcriptional activity of transcription factors is regulated by phosphorylation. The uncontrolled expression and constitutive activation of transcriptional regulators have been reported to cause malignant diseases. However, little is known about the phosphorylation status of tissue-specific transcription factors in human primary malignancies. Here we present the first insights into both protein expression and phosphorylation of transcription factors in a large-scale study of patients with acute myeloid leukemia (AML). We examined the expression and phosphorylation status of hematopoietic transcription factors PU.1 and C/EBP beta detected by the retarded mobility of the phosphorylated forms of the proteins. The rate of protein expression differed among French-American-British (FAB) subclasses. The expression of C/EBP beta and PU.1 were detectable in 77% and 61%, respectively, of 90 AML samples examined. The expressed PU.1 and C/EBP beta was always accompanied with both phosphorylated and unphosphorylated forms of PU.1 and C/EBP beta, respectively. Statistical significance was observed between PU.1 expression (phosphorylation) and FAB classification (M0, M4, or M5 versus M2 or M3, P < .0001). PU.1 and C/EBP beta were simultaneously detected in all M0, M4, M5 and peripheral blood monocytes, whereas in M2 and M3, the expression of the 2 transcription factors varied among samples. Examination of protein expression and phosphorylation of these lineage specific molecules may help us to understand the functional characteristics of AML. PMID- 10745626 TI - Delineation of the frequently deleted region on chromosome arm 13q in B-cell non Hodgkin's lymphoma. AB - The loss of a specific chromosomal region provides a clue to the elucidation of the putative tumor suppressor gene implicated in the pathogenesis and progression of tumors. To delineate the specific region(s) involved in lymphomagenesis, we performed a survey of loss of heterozygosity for 11 polymorphic microsatellite loci scattered on variable chromosome arms. We examined 20 primary lymphoma samples, including both indolent and aggressive B-cell non-Hodgkin's lymphoma (B NHL) and Hodgkin's disease (HD), and found a significant number of B-NHLs with loss of genetic material on chromosome arm 13q at the RB1 locus (50%; 4 of 8 informative cases for the RB1 locus). To specify the 13q deletion and to narrow the critical deleted region, we examined the same 20 lymphomas by intensive microsatellite mapping analysis using 12 microsatellite markers, mapping from 13q12.3 to 13q14. We confirmed the frequent 13q14 deletion to be in the vicinity of the RB1 locus (50% of the informative NHLs for at least 1 of 12 microsatellite loci; 5 of 10 aggressive NHLs and 2 of 4 indolent NHLs, but none of 6 HDs) and determined a subchromosomal region deleted in lymphoma on 13q14 defined by D13S164-D13S273, which is an overlapped region frequently lost in chronic lymphocytic leukemia. Taken together, our data indicate that the 13q alterations are present in a wide variety of NHLs including both indolent and aggressive B NHLs, suggesting that loss of genetic material at chromosome band 13q14 may play an important role in the formation or development of a wide variety of mature lymphoid malignancies. PMID- 10745627 TI - Blastic transformation of splenic lymphoma with villous lymphocytes after a well controlled chronic phase of more than 10 years. AB - A 30-year-old Japanese man with splenomegaly and lymphocytosis was examined in 1985. Blood analysis revealed that some of the lymphocytes had short-surface villi with polar distribution. The cells showed Ig lambda+, CD5+, CD11c+, CD19+, CD22+, CD23+, CD24+, FMC7+ phenotype. A small M peak was detected in the serum. Splenic lymphoma with villous lymphocytes (SLVL) was diagnosed on the basis of these findings. Remission was induced and was maintained with low-dose chlorambucil for more than 10 years. In 1996, the patient developed splenomegaly and lymphadenopathy with "B" symptoms and a high serum lactase dehydrogenase (LDH) level. Large blastoid cells with prominent nucleoli were observed in the bone marrow; later, a small number appeared in the peripheral blood. The bone marrow cells showed a complex chromosomal abnormality involving del(7)(q32). Southern blot analysis of immunoglobulin gene rearrangements in SLVL cells that had been cryopreserved in 1986 and of bone marrow cells in 1996 showed 2 rearranged bands in each cell sample; 1 band showed identical sizes in the 2 samples, and the other showed different sizes. These findings suggest that the blastoid cells were derived from SLVL cells through transformation. After this transformation, the disease followed a highly aggressive course. Various chemotherapeutic agents had little effect, and the patient died 3 months later. PMID- 10745628 TI - Torsade de pointes associated with hypokalemia after anthracycline treatment in a patient with acute lymphocytic leukemia. AB - Severe dose-dependent anthracycline cardiotoxicity is reported to cause myocardial damage resulting in congestive heart failure. However, torsade de pointes, a life-threatening arrhythmia caused by chronic anthracycline cardiotoxicity, has not been reported previously. A 16-year-old girl who developed torsade de pointes after 6 months of chemotherapy for acute lymphocytic leukemia (French-American-British classification L2) is described. When the patient was readmitted to the hospital because of syncope, peripheral blood and bone marrow analysis indicated a relapse. In addition, the patient was hypokalemic. Twenty-four-hour ambulatory electrocardiographic monitoring demonstrated QT prolongation and an episode of torsade de pointes. The electrocardiographic changes and arrhythmia improved after correction of the hypokalemia. An inverse correlation between leukocyte count and hypokalemia was observed. The patient died from pulmonary hemorrhage. Autopsy examination demonstrated myocardial degeneration consistent with damage induced by antineoplastic antibiotics. The cumulative dose of anthracycline and anthraquinone was less than the conventional dose limit associated with chronic cardiotoxicity, even for children who are more sensitive to anthracyclines. Torsade de pointes can occur in the setting of chronic anthracycline cardiotoxicity. Therefore, children or young adults who are more sensitive to anthracycline need careful observation that includes electrolyte monitoring, especially for potassium. PMID- 10745629 TI - Successful bone marrow transplantation in an adult patient with reactive hemophagocytic syndrome associated with myelodysplastic syndrome. AB - We report an adult case of reactive hemophagocytic syndrome (RHS) associated with myelodysplastic syndrome (MDS) who received emergency bone marrow transplantation (BMT). Despite methylprednisolone pulse therapy, high-dose gamma-globulin, and chemotherapy containing etoposide, the pancytopenia progressed. After informed consent, the patient underwent syngeneic BMT using melphalan as the conditioning regimen. The patient has been well without relapse of RHS and MDS for more than 2 years after BMT. This result suggests that the above strategy, including BMT, should be considered for the treatment of adult RHS associated with hematological malignancy. PMID- 10745630 TI - Chronic relapsing thrombocytopenic purpura with severe neurological manifestations and full recovery. AB - Neurological complications in thrombotic thrombocytopenic purpura (TTP) are associated with poor prognosis and/or permanent damage. We report a young woman in whom the diagnosis of TTP was difficult because cardinal manifestations were absent at presentation. The patient relapsed, showing severe and dramatic neurological manifestations, including coma. She was treated with multiple therapeutic modalities and recovered fully with no neurological sequelae. The difficulties involved in the management of chronic relapsing TTP are discussed. In the absence of clear guidelines, patients are still subjected to different treatment modalities according to the personal opinions and approaches of physicians. Clearly, well-controlled clinical trials to address this problem are required. PMID- 10745631 TI - Third Nagoya International Blood and Marrow Transplantation Symposium: challenge for stem cell therapy. PMID- 10745632 TI - ECG of the month. Sinister implications. Transient cardiac standstill. PMID- 10745633 TI - The journal 150 & 100 years ago. January 1850 and 1900. PMID- 10745634 TI - Blood mercury levels and fish consumption in Louisiana. AB - The primary source of non-occupational exposure to mercury is through the consumption of contaminated fish. Since 1994, the Louisiana Department of Environmental Quality has reported mercury contamination in fish obtained from bodies of water throughout the state and has issued fish consumption advisories accordingly. To determine the extent of mercury intoxication in Louisiana, screening for blood mercury levels was offered to volunteers residing near selected advisory areas. A total of 313 residents participated in the screening; 6 were found to have elevated levels. No level was detected in 48 of the participants, while the remaining participants had normal levels. Significantly higher levels were found in those associated with commercial fishing and those reporting increased fish consumption. For most people, ordinary consumption of fish contaminated with mercury does not currently appear to pose a public health hazard in Louisiana; however, educational efforts regarding the risks of fish consumption in great quantities should be continued. PMID- 10745635 TI - Are you sanitary? AB - The authors present an annotated, condensed synopsis of the fundamental basis of public health practice in Louisiana, the state's Sanitary Code. The "tongue-in cheek" remarks about some of the Code's requirements are just that, poking a bit of fun at the rule and law-making processes, but meant not in the least bit to underestimate the value of the public health rules and laws of the state. PMID- 10745636 TI - Public health education opportunities for physicians in Louisiana. AB - Various options exist for physicians and other mid-career health professionals who want additional education to upgrade their credentials in the public health arena by earning degrees or working toward certification. Potential students can fit distance learning classes into full schedules by participating in flexible curriculums that offer night courses through Internet technology. Other programs offer course material that physician-students work into their own schedules. Some traditional programs bring health professionals into the classroom on weekends or in the evening. PMID- 10745637 TI - Louisiana Parish Health Profiles 1999: using information to drive local action. AB - The Parish Health Profiles 1999, published by the Department of Health and Hospitals Office of Public Health (OPH), are intended to be a source of parish level health information to be used for community-level planning. The third edition of the Profiles uses a broader definition of health to understand the quality of life of communities. The included information represents not only health status, but also other aspects of quality of life, such as the status of local education, economy, environment, and crime and safety. The process of collecting this information yielded two additional results: strengthened relationships amongst information-providing agencies across the state and an orientation and subsequent comprehensive chapter of information on action and resources. In addition, the publication is designed to be reader friendly, with a strong emphasis on the use of the Parish Health Profiles to aid in understanding data. The Office of Public Health recognizes that the Parish Health Profiles will continue to evolve to meet the needs of their audience. In order to ensure continuous quality improvement through future editions, the Profiles are supported by an 18-month, multi-level evaluation process, ensuring consumer and user input and comment at different levels. PMID- 10745638 TI - Louisiana Rural Health Access Program. AB - Louisiana Rural Health Access is part of a Robert Wood Johnson Foundation project to address primary and preventive medical care for indigent, uninsured people residing in underserved rural parishes. This 15-month grant funds the development of a pilot program to improve access to health care in Acadia, Evangeline, Iberia, Lafayette, St Landry, St Martin, St Mary, and Vermilion parishes. Led by representatives from the Department of Health and Hospitals and the Louisiana State University Health Sciences Center, team committees designed the program's innovative use of telemedicine, loan development, network integration, and community involvement. A benefit of the program will be to measure the outcomes of each objective in order to determine which intervention works best. This information will be invaluable for the design of a five-year rural health care development plan. PMID- 10745639 TI - Discrimination among major health plans plague nation's minority physicians and patients. PMID- 10745640 TI - Successful low-dose concurrent chemotherapy and radiation for locally advanced or inoperable non-small cell lung carcinoma: a report of six cases. AB - Many studies now demonstrate high overall response rates with concurrent chemotherapy and radiation (CCR) for locoregionally advanced or inoperable non small cell lung carcinoma (NSCLC) but often with severe toxicity and only modest improvement in survival beyond 3 years. We report a simple CCR protocol for NSCLC that has resulted in long-term disease-free survival with low toxicity. In this retrospective review, 84 patients with NSCLC were seen between 1985 and 1991. Of these, 10 patients had stage IIIa or IIIb NSCLC without effusion or inoperable NSCLC, with no failed prior treatment at the time of referral for oncology evaluation. Six of these were treated with CCR consisting of three cycles of cisplatin and 5-fluorouracil administered concurrently with radiation treatment followed by maintenance chemotherapy for at least five additional cycles. All six patients treated with this protocol had complete response with minimal side effects. Survival times ranged from 4.5 to more than 10 years. Three patients survived in complete remission; three others were in complete remission at the time of death due to unrelated causes. Stage III NSCLC without effusion and inoperable NSCLC can be treated effectively with concurrent local and systemic treatment without significant toxicity. In patients with complete response, maintenance chemotherapy may overcome residual microscopic systemic disease, leading to long-term survival and possible cure. The CCR strategy that resulted in this favorable outcome is noteworthy and should be pursued in larger numbers of patients. PMID- 10745641 TI - Neurobehavioral toxicity. AB - A growing number of agents are known to perturb one or more of the interconnected processes of the central nervous system. At the same time, there is an increase in the incidence of neurobehavioral disorders that are confronting clinicians with baffling symptoms and presentations that seem uncommon. Fundamental to the assessment of the environmental-relatedness of the syndromes is a work and exposure history, including information different from that routinely obtained in the clinical setting. Exposure examples are described to suggest the scope of inquiry necessary to differentiate neurotoxic syndromes from nonneurotoxic illness. PMID- 10745642 TI - Recruitment and retention: the development of an action plan for African-American health professions students. AB - This article presents results of a survey of African-American students enrolled in the colleges of medicine, dentistry, allied health, pharmacy, and nursing at the University of Kentucky. The survey was designed to determine the students' perceptions of factors that affect recruitment, enrollment, and academic progress of African-American students. Fifty-three of seventy students responded to survey questions addressing recruitment; admissions; and financial, social, personal, and academic support. Over 50% of medical students decided by junior high to enter a health career; only 15% of other students decided that early. The influence of a family member was more important in student decisions to enter nursing or medicine than in decisions by other students. Only 17% of medical students reported difficulty in locating sources of financial aid compared to 48% of those from other colleges. Perceptions regarding lack of social outlets were consistent among respondents from all colleges. Findings emphasize the importance of early exposure to the health professions, early outreach strategies, ongoing financial assistance, and the importance of establishing social networks for African-American students enrolled in a majority institution. The survey results were used to develop an action plan for the offices of minority affairs, student services, and academic affairs to address identified problems and concerns. PMID- 10745643 TI - A comparison of smoking cessation efforts in African Americans by resident physicians in a traditional and primary care internal medicine residency. AB - Tobacco use causes significant morbidity and mortality among African Americans. Physicians may inconsistently counsel patients against smoking. This retrospective chart review evaluated smoking cessation efforts in African Americans by internal medicine resident physicians in a traditional and a primary care residency program. One hundred twenty-nine African-American patients were evaluated by resident physicians in the traditional internal medicine residency. A tobacco use history was obtained in 84 patients. Twenty-eight patients smoked and two patients were counseled against smoking. Fifty-two African-American patients were evaluated by resident physicians in the primary care residency. A tobacco use history was obtained in 47 patients. Twenty patients smoked and 12 patients were counseled against smoking. There was a statistically significant difference in the rate at which smoking histories were obtained (p = 0.0011) and frequency of counseling against smoking (p < 0.0001). Gender analysis revealed that African-American women were less frequently asked about their smoking history (p = 0.0058) and counseled against smoking (p = 0.0016) by resident physicians in the traditional residency. African-American men received less counseling against smoking (p = 0.055) by resident physicians in the traditional residency. Resident physicians in the primary care residency program demonstrated greater smoking cessation efforts for African American patients. Smoking cessation should be emphasized in all internal medicine residency training programs. PMID- 10745644 TI - Using cultural beliefs and patterns to improve mammography utilization among African-American women: the Witness Project. AB - Breast cancer and early detection of the disease is a significant issue for all women. Moreover, the sociocultural implications in the differential mortality rates increased interest in possible barriers to screening practices. Recently, a number of studies have investigated African Americans' cultural beliefs associated with breast cancer. This study is based upon qualitative focus group data gathered from 1989 to 1991 and 1996. This article provides focus group data that informed a culturally competent community-based cancer education program for African-American women--the Witness Project. Analysis of the qualitative data along with the quantitative outcome data revealed a direct relationship between cultural beliefs and patterns with mammography utilization. The once perceived cultural barriers can actually be applied as a cultural intervention strategy to improve breast cancer screening initiatives designed specifically for African American women. PMID- 10745645 TI - Primary splenic angiosarcoma: case report and literature review. AB - Angiosarcoma of the spleen is a rare neoplasm. We describe the clinical findings of an index case at our institution and the results of a literature review of the topic. Generally, this cancer has a poor prognosis and prompt splenectomy offers the only cure, as the cancer is poorly responsive to adjuvant chemotherapy. PMID- 10745646 TI - Case presentation. PMID- 10745648 TI - Pharmacy overload. PMID- 10745647 TI - Sarah Loguen Fraser, MD (1850 to 1933): the fourth African-American woman physician. PMID- 10745649 TI - Radiology quiz. Metastatic breast carcinoma. PMID- 10745651 TI - Update in platelet therapy. PMID- 10745650 TI - Trends in chronic obstructive pulmonary disease mortality. AB - This study examines the mortality trends for chronic obstructive pulmonary disease (COPD) among whites and African Americans in Missouri from 1980-1996. Data from the Missouri Center for Health Information Management and Epidemiology were used to calculate mortality rates. Missouri's COPD deaths rose 40.6% from 1980-1996. Projections through the year 2006 predict continued escalation in rates. Much of the growth in COPD can be attributed to heavy tobacco use in the population. PMID- 10745652 TI - The birth, development, and legacy of Roman medical thought. PMID- 10745653 TI - Neuraminidase inhibitors and relief of influenza symptoms. PMID- 10745654 TI - Nodal metastases with unknown primary--an unusual presentation of carcinoid tumor. AB - Carcinoid tumors arise from neuroendocrine cells and produce their symptoms mainly through secreted amines, and by a local desmoplastic response. The critical issue in the management of advanced carcinoid tumors is preservation of quality of life and symptomatic relief. This is because of the characteristic indolent growth which makes survival a secondary consideration, and the relative rarity of the condition. Although systemic therapy has been shown to improve symptoms, true survival benefit has not been established. We present a case of carcinoid tumor that has metastasized to mesenteric nodes, and probably other locations, with an occult primary site. The patient remains completely asymptomatic and continues to enjoy a normal lifestyle. No references were found in the literature concerning the management of asymptomatic, but metastatic carcinoids. This case thus constitutes a unique situation in which further treatment may not be beneficial and may even be harmful. Nevertheless, additional treatment is an important consideration in view of the known poor survival of those with metastatic disease versus localized, resectable tumor. However, the concept of medical therapy to improve survival in an asymptomatic individual, though theoretically attractive, has little precedence. In this article we have tried to address the therapeutic dilemma posed by this unusual clinical scenario. PMID- 10745655 TI - [Reflections after International Year of the Elderly 1999]. PMID- 10745656 TI - [The general practitioner who "prevents". The Danish Society of General Practice]. PMID- 10745657 TI - [Sequencing of the first human chromosome. The Danish Society of Medical Genetics]. PMID- 10745658 TI - [1999--a dynamic year of geriatrics. The Danish Society of Geriatrics]. PMID- 10745659 TI - [Myelomatosis--toward a breakthrough? The Danish Society of Hematology]. PMID- 10745660 TI - [Beta-blockers in chronic heart insufficiency. The Danish Society of Cardiology]. PMID- 10745661 TI - [HIV-associated lipodystrophy syndrome (HALS). The Danish Society of Infectious Diseases]. PMID- 10745662 TI - [Treatment of rheumatoid arthritis. The Danish Society of Rheumatology]. PMID- 10745663 TI - [Pulmonary medicine 1999: Screening for lung cancer and lung volume reducing surgery. The Danish Society of Pulmonary Medicine]. PMID- 10745664 TI - [Cirrhosis, transaminasemia and non-alcoholic fatty liver. The Danish Society of Hepatology]. PMID- 10745665 TI - [The solitary thyroid nodule and the TPO-immunostaining. The Danish Society of Surgery]. PMID- 10745666 TI - [Neurosurgery in the year of 2000. The Danish Society of Neurosurgery]. PMID- 10745667 TI - [Integrated therapy for the victims of violence and rape. The Danish Society of Obstetrics and Gynecology]. PMID- 10745668 TI - [Cerebral paresis and botulinum toxin treatment. The Danish Society of Pediatrics]. PMID- 10745669 TI - [New within child and adolescent psychiatry. The Danish Society of Psychiatry, Section of Child and Adolescent Psychiatry]. PMID- 10745670 TI - [Superficial mycoses]. PMID- 10745671 TI - [Waiting for radiotherapy. Is it of prognostic significance?]. PMID- 10745672 TI - [Chronic inflammatory bowel disease--pathogenic concepts and therapeutic perspectives]. AB - Chronic inflammatory bowel disease (IBD) is considered to be a consequence of inappropriate upregulation of immune reactions evoked by the colonic microflora. Abnormalities observed in IBD may be explained, at least in part, by an unfavourable balance between pro- and anti-inflammatory cytokines. Conventional drug treatment of IBD may soon be replaced by more selective inhibitors that act centrally in the inflammatory process. Immunoneutralisation with chimeric anti tumour necrosis factor-alpha (TNF alpha) antibodies reduces treatment refractory IBD, including fistular Chrons' disease, but recombinant human TNF alpha-receptor fusion proteins may be equally effective with potentially fewer side effects. This view also applies to chimeric antibodies directed against cytokines or adhesion molecules. Potentially more promising are antisense oligonucleotides and matrix-metalloproteinase inhibitors. Whether sustained remission can be achieved probably depends on successful unravelling of the aetiology of IBD. PMID- 10745673 TI - [Malignant neuroleptic syndrome. A review of epidemiology, risk factors, diagnosis, differential diagnosis and pathogenesis of MNS]. AB - Neuroleptic malignant syndrome (NMS) is a rare, potentially life-threatening disorder that results from the use of neuroleptics. NMS was first recognised as a complication of dopamine receptor antagonists characterized by extrapyramidal disturbances, hyperthermia, muscle rigidity, autonomic instability, mental status changes and elevated serum creatine kinase levels. Concepts of NMS have changed because medications other than classic neuroleptic drugs have been implicated as triggering agents. The incidence of NMS is about 0.2% with a mortality between 4 30%, which may be diminished by treatment. The neurochemical key features in all these conditions probably result from disruption of the dopamine system in the brain and the effects of neuroleptics on muscle. Recognition of NMS is the most important step in its management by discontinuation of the causative drugs and applying supportive care and therapeutic measures. Specific therapeutic measures include the application of dopamine receptor agonists, e.g. dantrolene and use of benzodiazepines. The differential diagnosis of NMS comprises an extensive list of disorders presenting with fever and with muscle rigidity. Neuroleptics may be reintroduced in the majority of patients by using an atypical neuroleptic drug such as clozapine. PMID- 10745674 TI - [Percutaneous transluminal septal myocardial ablation in hypertrophic obstructive cardiomyopathy. A new therapeutic option]. AB - We here report on the PTSMA results in four HOCM patients having severe symptoms despite medical treatment and dual chamber pacing. Between two and five ml of ethanol was injected in one or two septal branches from the left coronary artery. Six months after treatment the pressure gradient across the left ventricular outflow tract was reduced between 25-80 mmHg at rest and between 55-180 mmHg at exercise. There was an increase in functional capacity of about two NYHA-classes. Two patients developed permanent right bundle branch block, and one patient with pre-existing left bundle branch block developed persistent total atrioventricular block after the treatment. Serum creatine phosphokinase MB increased on average to 146 U/l. No other complications were seen. In conclusion, our initial PTSMA results seems promising. PMID- 10745675 TI - [Mobile extracorporeal shock wave lithotripsy of urinary calculi performed in the county of Ringkjobing]. AB - Experiences with mobile extracorporeal shock wave lithotripsy (ESWL) of urinary calculi using a third generation lithotriptor are reported. A total of 146 renal units in 132 patients received 204 ESWL treatments with mobile lithotriptor for renal and ureteric stones. Treatments were given once a month at Holstebro Hospital and Herning Hospital alternately, with six to eight treatments per day. The overall success was 82% (53% stone-free). Success with renal stones was 89% (51% stone-free) and that with ureteric stones 66% (59% stone-free). Retrograde manipulation appeared to be a safe and effective treatment for patients with obstructing upper ureteric stones. Treatment of other ureteric stones in situ gave unsatisfactory results (success 52%, stone-free 51%). There were no serious complications. PMID- 10745676 TI - [Variation of Doppler ultrasonography of carotid arteries]. AB - The variation of ultrasound examination of the carotid arteries performed at two different hospitals was examined by retrospective analysis. Eighty-one patients were primarily examined by ultrasound at their local hospital on the suspicion of carotid stenosis. Following referral, they underwent a further scanning at the Dept. of Vascular Surgery, Rigshospitalet. Results for 143 carotid arteries were available for comparison. The overall agreement between the two examinations was only 59%. In nine of 40 (22%) cases an originally diagnosed minor stenosis was found to be significant and 3/19 (16%) with shown occlusion were found to be patent at the later examination. In order to draw clinical conclusions validation of each individual laboratory performing ultrasound examination of extracranial cervical arteries is necessary. PMID- 10745677 TI - [The objective structured clinical examination in postgraduate pediatric training. The first Danish experiences]. AB - To evaluate the usefulness of an objective structured clinical examination (OSCE) for identifying weaknesses of the educational program and for providing feedback to trainees in paediatrics an 8-station OSCE was given. Ten residents on different levels of training participated. Stations covered a wide spectrum of clinical situations and included three video-recordings of patients. Skills in history-taking, examination, listing of differential diagnoses, planning of work up as well as in communication and counselling were assessed. Verbal as well as written feedback was provided to all the trainees. In five trainees skills in examination were relatively weak, and subsequently it was possible to implement improvements in the educational program. Strengths and weaknesses of the educational program can be identified, but the benefits of the OSCE should be balanced with the extra workload and logistical difficulties. PMID- 10745678 TI - [Polyphenolic antioxidants in fruit juice. Urinary excretion and effects on biological markers for antioxidative status]. AB - This intervention study was designed as cross-over (four women, one man) with three doses of black currant/apple (1:1) juice (750, 1000, and 1500 mL) for one week corresponding to an intake of 4.8, 6.4, and 9.6 mg quercetin per day. Urinary excretion of quercetin increased significantly with dose and with time. The fraction excreted in urine was constant 0.29-0.47%. Plasma quercetin did not change with juice intervention. Plasma ascorbate increased during intervention due to ascorbate from the juice. Total plasma malondialdehyde decreased with time during 1500 mL juice intervention. Plasma protein 2-adipic semialdehyde residues, increased with time and dose, and glutathione peroxidase increased with juice dose, whereas other selected markers of oxidative status did not change. These effects might be related to several components of the juice and cannot be attributed solely to its quercetin content. PMID- 10745679 TI - [Nursing home residents in Northern Jutland]. AB - The purpose of the study was to describe a Danish nursing home population. The study consists of 288 nursing home residents (median age 84.6 years, 68% females). The psychiatric morbidity of the residents was diagnosed with the GMS AGECAT. The staff was interviewed about the residents' ADL, disturbing behaviour and therapeutic measures. Seventy-one percent had a psychiatric disorder, with organic disorder as the most frequent (61%). Demented residents received significantly less hypnotics, were more often physically restrained and had lower ADL levels compared to other residents. The situation in Denmark can be seen as a paradox. While substantial effort has been made to increase the possibility of choice and to "deinstitutionalize" nursing homes, the majority of the residents are demented and thus not capable of making valid choices. PMID- 10745680 TI - [Multiple organ failure caused by extraadrenal pheochromocytoma]. AB - Extraadrenal phaeochromocytomas are very rare tumours, commonly associated with hypertensive paroxysms. We report a 20 year-old man with unsuspected extraadrenal phaeochromocytoma, who at admission presented with septicaemia and adult respiratory distress syndrome, suggesting pneumonia. PMID- 10745681 TI - [Polyarteritis nodosa diagnosed in acute cholecystectomy]. AB - Polyarteritis nodosa (PAN) is a serious systemic vasculitis, which untreated has a five year survival rate of less than 15%. In the present case PAN was diagnosticed in a 72-year old Asian woman by microscopy of a gall bladder in acalculous cholecystitis. We conclude that PAN must be considered in patients with symptoms from several different organs and evidence of vascular involvement can be obtained by histological investigation of material from involved organs or mesenterial angiography. PMID- 10745682 TI - [Diagnosis of ACTH-producing tumors. Can sinus petrosus catheterization be avoided?]. AB - A 38-year-old woman with Cushing stigmata and an MRI confirmed pituitary tumour was referred for pituitary surgery. High-dose dexamethasone test had indicated ectopic focus. An additional peripheral CRH test was performed, indicating ectopic focus. To secure the diagnosis inferior petrosal sinus sampling (IPSS) was performed, also indicating ectopic tumour. Operation revealed an ACTH producing tumour in the thorax. We conclude that IPSS is necessary when diagnosing Cushing syndrome. PMID- 10745684 TI - [The Vancouver rules]. PMID- 10745683 TI - [Infantile autism--heredity or environment?]. PMID- 10745686 TI - [In Process Citation] PMID- 10745685 TI - [Why can participation of more than one patient in a randomized trial be ethically justified?]. PMID- 10745687 TI - [Malignant neuroleptic syndrome. Treatment and resumption of the treatment in malignant neuroleptic syndrome]. PMID- 10745688 TI - [The immunological background of the allergen vaccines application]. AB - The review and critical estimation of the studies concerning the mechanism of action of SIT was performed. The presented data give the reasons to view the allergen vaccines as the immunomodulators motivating simultaneously the applicability of these agents as the causative treatment of the allergic diseases. PMID- 10745689 TI - [Hemodynamic changes during cholecystectomy with the use of laparolift method]. AB - The study was carried out in 17 randomly chosen patients who were subjected to cholecystectomy under general anesthesia with the use of laparolift method. Heart rate, systolic and diastolic blood pressure, wedge pressure, mean pulmonary artery pressure, central venous pressure and stroke volume were analyzed in the five following stages of the procedure. The performed examinations showed the transient increase of the pressures in peripheral and pulmonary vascular system as well as heart rate acceleration--during the operation and the increase of cardiac output in postoperative period. The observed changes did not need therapeutic interventions in any case. The obtained results prove that applied operative technique caused no dangerous side effects. PMID- 10745690 TI - [The occurrence of the Enterobacter genus rods in the clinical materials and materials taken from hospital environment]. AB - Due to continuously existing essential meaning of strains of Enterobacter spp. in human infections we investigated the occurrence of Enterobacter spp. in different clinical materials and in materials taken from hospital environment. Amongst 26,025 materials taken from patients there were 249 (1.0%) Enterobacter strains and respectively amongst 954 environmental materials--there were 11 (1.2%) Enterobacter spp. In isolated Enterobacter strains the percentage occurrence was: hospital materials: E. cloacae--65.6%, E. agglomerans--12.1%, E. aerogenes- 6.25%, E. sakazakii--5.4%, E. gergoviae--1.3%, E. amnigenus--0.9%, E. intermedius -0.9% and Enterobacter spp.--7.6%; outpatient materials: E. cloacae--72.0%, E. sakazakii--12.0%, E. agglomerans--8.0%, E. aerogenes--4.0% and E. amnigenus- 4.0%; hospital community: E. cloacae--63.6%, E. agglomerans--18.2% and E. sakazakii--18.2%. PMID- 10745691 TI - [Postop complication of pancreatic resection]. AB - Early complications after partial pancreatic resections and benign pancreatic tumours were analysed. 276 patients with pancreatic tumours were treated in the years 1979-1997. In 77 cases radical operation was performed. 65 patients underwent Whipple and 12--Traverso-Longmire pancreatoduodenectomy. Malignant tumour was found in 55 cases and 22 were benign. In 4 patients tumour's resection along with invaded portal vessels and portal confluence reconstruction was performed. These were the first cases with vessels resection operated in Poland. There were 2 post-operative deaths observed in that group. Complications in our material were as follows: pancreatico-jejunal anastomosis fistulae, haemorrhage from ulcers in gastro-jejunal anastomosis, diffuse peritonitis, haemorrhage from pancreatic stump vessels, ileus, acute renal insufficiency, pneumonia and myocardial infarct. Mortality in whole group was 11%. In the last 5 years due to our growing experience and some technical modifications mortality fell to 4.4%. PMID- 10745692 TI - [Own experience in the application of midazolam (Dormicum) before the diagnostic examinations an short surgical procedures in children]. AB - In this paper the evaluation of midazolam (Dormicum) efficiency in obtaining basic sedative effect in 36 children during diagnostic examinations and short surgical operations was done. In 26 patients good sedative effect was obtained. Forgetfulness was present in 17 from 25 patients above 2 years of life, and partial in the rest 8 patients. Fear reactions were observed in 10 children with lower doses of this medicine. Side effects were observed in 8 patients, and had a transient character and were not dangerous for the patients' life. PMID- 10745693 TI - [Autologous transfusion as a integral element of the patient preparation for the general surgery]. AB - This paper presents our 7-year period of autologic transfusion program. Donation was made before an extensive surgery. IN CONCLUSION: autologic transfusion is a safe (significant decrease in different diseases transmission) and economical method, probably for today a golden standard in a surgical treatment. PMID- 10745694 TI - [The evaluation of hypertensive patients' adherence to the pharmacological treatment of hypertension]. AB - Evaluation of patients' compliance is very important considering possible influence on the effectiveness of antihypertensive treatment. The adherence to the rules of pharmacological therapy was assessed by anonymous questionnaires. 414 patients with hypertension participated in the study. Non-compliance is one of the commonest therapeutic problems. Forgetting is the most frequent reason of irregular administration of medicine. PMID- 10745695 TI - [The pathogenesis of abdominal aortic aneurysm and polymorphism of haptoglobin and inflammatory factor]. AB - 52 patients were operated on for abdominal aortic aneurysm and 38 for aortic occlusion disease. Haptoglobin (Hp) phenotypes were determined in all these patients. Hp and C-reactive protein (CRP) concentrations were determined in each phenotype. On the basis of the statistical analysis the highest percentage of elevated CRP concentrations was found in patients with abdominal aortic aneurysm and phenotype Hp 2-1. PMID- 10745696 TI - [Is a post-cholecystectomy syndrome the result of inappropriate preoperative diagnosis?]. AB - The occurrence of the postcholecystectomy syndrome is very often connected with inappropriate preoperative diagnostics. One of the reasons might be the presence of gastric or duodenal ulcer, what can be connected with peri- and late postoperative complications. The prospective analysis of 250 patients admitted to the Department because of cholelithiasis was performed. In 28.8% of cases endoscopical changes in the upper alimentary tract were recognized. That is why, in patients qualified for cholecystectomy the upper alimentary tract endoscopy should be performed. PMID- 10745697 TI - [Rational empiric antibiotic therapy of nosocomial pneumonia]. AB - Antibiotic therapy is the method of choice for treatment of nosocomial pneumonia. It can be divided into empiric and specific therapy. First of them is based on predicting potential pathogens which may cause pneumonia. Important factors modifying the choice of initial empiric therapy are following: clinical history (e.g. time in hospital, type of hospital department, prior usage of antimicrobial agents), severity of pneumonia, presence of other risk factors and information given by direct microscopic examination of pulmonary secretions. Based on them it is frequently possible to select group of possible pathogens and then choose effective antibiotics with broad coverage. After a few days when specific etiology is found and antibiogram is performed antibiotic with narrow coverage should replace previous one. Specific therapy possesses several advantages when comparing with empiric therapy. It is more effective and cheaper. But necessity to wait a few days for the result of culture and its relatively low reliability are clear disadvantages. PMID- 10745698 TI - [Pathophysiology of collateral coronary circulation]. AB - The study presents the concepts of collateral circulation in the heart. These vessels can reduce the fatal consequences of occlusion of large coronary artery. There are described two mechanisms of its growth: vasculogenesis (mitosis of endothelial cells, and smooth muscle cells), and angiogenesis (migration and mitosis of endothelial cells). These processes are triggered by ischemia and by expression of peptide growth factors. Growth factors are produced by macrophages and often are found in inflammation tissue. Therapeutic angiogenesis remains controversial, however in the near future stimulation of angiogenesis by peptides, drugs and gene therapy will have a place in therapy of ischemic heart disease. PMID- 10745699 TI - [Genetical basis of Holt-Oram syndrome]. AB - On the basis of literature the review of data concerning genetical ground of Holt -Oram syndrome was presented. This disease is characterized by coexistence of congenital heart malformation and osseous abnormalities of upper limbs. It has been known since 1994 that the locus of the gene responsible for Holt--Oram syndrome appearance is situated in the region 12q21-q22 of chromosome 12. In 1997 this gene was defined as the TBX5 belonging to the family T-box which codes the transcription factors during embryonic development. In the cases of familial and sporadic appearance of Holt--Oram syndrome different mutations in the TBX5 gene were described. No correlation was found between mutation type and the clinical features. It is suspected that TBX5 gene is interacting with the other ones during embryonic differentiation processes. PMID- 10745700 TI - [Tuberculosis luposa of the face and gluteal-femoral region in a 79 year old patient]. AB - A case of a 79 year old woman with tuberculosis luposa in whom the first cutaneous changes occurred at her young age and it was properly diagnosed only after 50 years. PMID- 10745701 TI - [Persistent patient urachus associated with chronic abscess and umbilical fistula]. AB - A very rare case of persistent urachus in 45-year-old man was presented. A complication in the form of chronic abscess with umbilical fistula occurred. The treatment consisted of surgical excision after the institution of antimicrobial therapy with drainage. PMID- 10745702 TI - [Hypertrophic pyloric stenosis in twins]. AB - Two cases of the pyloric stenosis in twins were described. Etiology of the illness stays still difficult to explain. It is generally accepted that pyloric stenosis has multifactorial inheritance. Familial occurrence of the illness in twins suggests this etiology. PMID- 10745703 TI - Credentialing and privileging of advanced practice nurses. AB - Credentialing and privileging are increasingly common administrative processes that advanced practice nurses must complete. With the intention of assuring the public of safe health care provided by qualified personnel, several federal and state organizations have developed criteria that licensed practitioners must meet. Managed care organizations are also creating credentialing and privileging guidelines for practitioners who are applying to their provider panels. Understanding the requirements of these processes is essential to advanced practice nurses establishing themselves in practice. This article explains the guidelines from federal, state, and institutional agencies and offers recommendations to practitioners as they prepare for the credentialing process. Also included are sample forms for establishing a practice agreement and a privilege-delineation request. PMID- 10745704 TI - Collaborative practice agreements for advanced practice nurses: what you should know. AB - Advanced practice nurses (APNs) seeking employment are often presented with employment practice agreements. A collaborative practice agreement is a written statement that defines the joint practice of a physician and an APN in a collaborative and complementary working relationship. It provides a mechanism for the legal protection of the APN and sets out the rights and responsibilities of each party involved. All APNs, regardless of practice setting, should be knowledgeable about aspects of a collaborative practice agreement before they sign one. The purposes of this article are to delineate basic guidelines for evaluating and developing a collaborative practice agreement and to identify areas of special concern for APNs. PMID- 10745705 TI - Developing practice protocols for advanced practice nursing. AB - In most states, the role of an advanced practice nurse is dependent on practice protocols that provide an organized method for analyzing and managing a disease or major symptom. They are also used to control the process of medical care and to specify steps in the delivery of that care. Creating appropriate practice protocols is one of the most important precursors to implementing the advanced practice role, because they virtually drive the clinician's ability to treat or manage clinical situations or disease states. This article outlines the steps involved in developing practice protocols and discusses the content that should be included in a protocol, providing an example of narrative and algorithm format protocols. Pros and cons, as well as legal issues related to practice protocols, are also presented. PMID- 10745706 TI - Measuring advanced practice nursing outcomes. AB - The measurement of outcomes has become an important component of evaluating health care. Although it is clear that measuring outcomes is necessary to establish the effectiveness of advanced practicing nursing, which outcome measures to use and how to conduct an effective outcomes assessment remain unclear. The purpose of this article is to present an overview of advanced practice nursing outcomes research, review outcome measures important to advanced practice nurses (APNs), and discuss sources of outcome measures and instruments that can be used by APNs to establish the effectiveness of the role. PMID- 10745707 TI - The historical evolution of bioimpedance. AB - Thoracic electrical bioimpedance is a technology that converts changes in thoracic impedance to changes in volume over time. In this manner, it is used to track volumetric changes such as those occurring during the cardiac cycle. These measurements, which are gathered noninvasively and continuously, have become more sophisticated and more accurate with the development of data signal processing and improved mathematical algorithms. This technology, first used in the 1960s, has benefited from the advent of the microprocessor and the better understanding of the cardiac cycle, thanks to technology such as echocardiography and magnetic resonance imaging. Today, noninvasive methods of measuring of cardiac output are coming into clinical use on a larger scale than ever before and are compared with other methods such as thermodilution and the direct and indirect Fick methods. PMID- 10745708 TI - Electrophysiologic principles and theory of stroke volume determination by thoracic electrical bioimpedance. AB - Thoracic electrical bioimpedance (TEB) is a harmless, noninvasive, user-friendly technology with wide patient acceptance. Stroke volume (SV) determination is important because it helps to define oxygen transport. Measurement of SV by TEB is rooted in concrete, basic electrical theory, as well as in theoretical models of electrical behavior of the human thorax and great thoracic vessels. This article is concerned with basic electrical theory as applied to TEB, signal acquisition, and the origin of the thoracic cardiogenic impedance pulse (delta Z). The appendix of the chapter features a more extensive overview of alternating current theory as applied to electrical bioimpedance. PMID- 10745709 TI - Managing congestive heart failure with thoracic electrical bioimpedance. AB - Because of clinical utility, immediate feedback, absence of risk to the patient, portability, and use in virtually any practice setting, facilities using thoracic electrical bioimpedance monitoring are increasing throughout the world. Specialized centers treating patients with heart failure have shown remarkable benefits, both in health care costs and patient morbidity, attributable to the careful monitoring and management of patients with heart failure. The ability of thoracic bioimpedance monitoring to provide objective data in the management of congestive heart failure provides practitioners with the clinical reassurance to optimize pharmacologic therapy safely. PMID- 10745710 TI - Managing inotrope therapy noninvasively. AB - Intermittent and continuous outpatient and home inotrope infusions have contributed to a decreased mortality rate and improved quality of life in the patient with end-stage congestive heat failure. Before the advent of noninvasive hemodynamic monitoring, available with thoracic electrical bioimpedance, initial dosage and titration were determined solely with invasively acquired data requiring hospital admission or with patients' subjective data. Thoracic electrical bioimpedance monitoring of patients receiving inotrope therapy provides objective documentation of drug efficacy, developing tolerance, and optimal dosage. PMID- 10745711 TI - Bioimpedance in the pacemaker clinic. AB - Thoracic electrical bioimpedance (TEB) provides a rapid, accurate, cost-effective method of optimizing the atrioventricular delay in dual-chamber pacemakers. In addition to measurements of cardiac output, TEB provides other hemodynamic indices such as systolic time interval, left cardiac work index, and end diastolic index. The availability of this additional data can assist the clinician in the objective determination of the optimal atrioventricular delay for individual patients. Thoracic electrical bioimpedance is completely noninvasive and takes only minutes to perform. PMID- 10745712 TI - Cost-effectiveness of noninvasive hemodynamic monitoring. AB - Costs for hemodynamic monitoring can comprise a large segment of an institution's budget. Noninvasive monitoring with thoracic electrical bioimpedance is a cost effective alternative to invasive monitoring. It can decrease not only materials costs but also costs related to patient complications. PMID- 10745713 TI - The relationship of academic variables as predictors of success on the National Council Licensure Examination for Registered Nurses (NCLEX-RN) in a selected associate degree program. AB - Identifying valid variables to predict success of nursing students on the NCLEX RN has captivated the interest of nursing educators for decades. The determination of such variables would enable nursing programs to devise pertinent admission criteria, identify and intervene with students at risk of failing, and provide needed advisement and academic supports to increase the likelihood of passing the NCLEX-RN. This study examined six academic and non-academic variables. Study variables included: pre-admission GPA, failing a clinical nursing course, two NLN test scores, age, and race. These variables have been explored in numerous past studies to predict success on the NCLEX-RN. However, the majority of studies have examined these variables in baccalaureate of science nursing (BSN) student populations. Few studies on associate degree nursing (ADN) student populations were found. The purpose of this study was to examine six academic and non-academic variables, explored in previous studies that mostly looked at BSN students, to determine if these same variables could predict success or failure on the NCLEX-RN for students of an ADN program. Data were obtained from a convenience sample of thirty-eight, May 1997 nursing student graduates from a public urban university's ADN program. Findings from the study indicated that four of the variables had significant relationships with NCLEX-RN success. PMID- 10745715 TI - Reinventing your future. PMID- 10745714 TI - Integrated literature review on effects of exposure to violence upon adolescents. AB - Violence has reached epidemic proportions in the Unites States. The past decade has seen professionals in public health, law enforcement, social sciences, and health care commit more attention to this area. Research and anecdotal articles read by the researcher indicated adolescents exposed to chronic community violence may experience a myriad of problems including; academic failure, the tendency to engage in violent behavior, depression, and a nihilistic, fatalistic orientation to the future, which often leads to increased risk-taking behaviors. The purpose of this integrative literature review is to discover what is known about exposure to violence among adolescents. While the review did find various negative effects of being exposed to violence, a cross-sectional design was often used, with self-reported data. Instruments with established validity and reliability were used to gather the data were often appropriate to the population. Aspects of the methodologies used by the researchers should be replicated in order to add to the knowledge of this topic. PMID- 10745716 TI - Is fee-for-service for you? PMID- 10745717 TI - Should PNPs diagnose adults? PMID- 10745718 TI - Understanding a civil trial. PMID- 10745719 TI - Breast & ovarian cancer. Issues in risk assessment. PMID- 10745720 TI - The gastric demon. An update on Helicobacter pylori infection. PMID- 10745721 TI - Sexually transmitted disease in the U.S. An overview of treatment guidelines. PMID- 10745723 TI - Low back pain. An evidence-based approach. PMID- 10745724 TI - Reducing falls in the elderly. Practical solutions. PMID- 10745722 TI - Getting the 'ouch' out. Topical anesthesia for procedural pain in children. PMID- 10745725 TI - Career resilience. Preparing for change now. PMID- 10745726 TI - Fiscal crunch brings changes. PMID- 10745727 TI - The patient transit assistance scheme: the clerk's perspective. AB - The Patient Transit Assistance Scheme (PTAS) is an important government program designed to ensure equity of access for residents in rural and remote Queensland to essential healthcare services. Where specialist services cannot be provided at recognised hospitals, PTAS ensures equity by accepting the responsibility for assisting with the transport and accommodation of patients and at times their escort, to locations where such services are available. This article presents research findings on the concerns of clerks involved in administering PTAS for patients with leukaemia and associated haematological disorders. The findings indicate that there are many problems with this scheme that need attention and can be remedied. PMID- 10745728 TI - Tyranny of distance? The health of mid-age women living in five geographical areas of Australia. AB - Over 14,000 women aged 45-50 are participating in the Australian Longitudinal Study on Women's Health, which is designed to track the health of Australian women for 20 years, and to understand lifestyle and healthcare factors that influence women's health. The study deliberately overrepresents women from rural and remote areas. This analysis of baseline data from the study compares the responses of women living in urban areas (capital city, other metropolitan), large rural centres, small rural centres, other rural areas and remote areas (remote centres, other remote areas) of Australia. The data show that while women in this age group who live in rural and remote areas have similar levels of self rated health, they have significantly fewer visits to general practitioners and specialists (P < 0.001) and more visits to alternative healthcare providers than women living in urban areas. Rural and remote area women were also more likely to undergo gynaecological surgery than women living in urban areas (P < 0.001). Other results suggest that being overweight is more common among women from rural and remote areas, and that these women also report lower levels of stress than women from urban areas (P < 0.001). Further follow up will allow any divergence in health and healthcare equity to be explored as these women get older. PMID- 10745729 TI - Availability of appropriately packaged information on sexual health for people who are legally blind. AB - Access to information on sexual health by legally blind people is an area of study hitherto neglected. Research carried out during 1997 confirmed an almost complete absence of appropriately packaged information for this subgroup in the community. Rural people are even more disadvantaged than their metropolitan counterparts. PMID- 10745730 TI - A comparison of a hospital outpatients department and general practice in remote Queensland. AB - A survey of all ambulant patients attending the hospital outpatients' department and the private general practice in a remote town in Queensland sought information on patients' reasons for attending. Patients handed the completed questionnaires to their practitioner, who indicated the type of visit and the focus of the encounter. There was little difference between the facilities in terms of patient demographics, except that males accounted for a higher proportion of encounters at the Outpatients' Department (OPD), mainly as emergency encounters. Excluding emergency encounters, patients appeared to perceive the facilities as alternative practices. The practitioners' reports suggested some differences in practice and/or experience between the two facilities, which were in line with commonly held perceptions about OPD and private general practice. The data suggest that the similarities between the two facilities may outweigh the differences. Moreover, the differences between rural hospital practice and rural general practice should be seen as providing the opportunity for more rounded training, rather than mutually exclusive forms of practice. PMID- 10745731 TI - Attitudes to using condoms in a group of Darwin women. AB - This study was a qualitative descriptive interpretive investigation into a group of women's attitudes to the use of condoms in Darwin, Northern Territory, Australia. Twenty women were interviewed using a semi-structured interview schedule. The participants' perception of who should be responsible for providing and ensuring use of condoms varied, as did their attitudes to condom use. Negative attitudes centred around the need to be prepared for sex, the nuisance value and the need to be careful of slip-ups, while positive attitudes centred around the lack of mess. Most women in a committed relationship did not use condoms other than for contraception because they trusted their partners to be faithful. Raising the issue outside of birth control suggested unfaithfulness. These women were on the whole more confident of their ability to negotiate condom use than had been found in previous studies. It was concluded that the safe sex message is not getting through to many women, that health promotion should continue to stress the message about safe sex and that advertising should stress the positive aspects of condom use. PMID- 10745732 TI - At the interface of health and community care: developing linkages between aged care services in a rural context. AB - This paper explores issues concerning the development of linkages across the interface between acute and community aged care services in a small regional Australian city. It addresses a participatory action research project that took place over a 2 year period involving an Aged Care Assessment Team (ACAT). Aged Care Assessment Teams are multidisciplinary teams whose members mediate between hospitals and the aged care system in the community and have a key role in developing networks and linkages between various service providers in the field. In an age of economic rationalist-inspired reform agendas in health and community care, rural infrastructures have been compromised to such a degree that the role of rural ACAT in developing linkages between sectors has never been more important. This paper takes up this issue and addresses the project findings, which highlight a field characterised by ineffective linkages within and between the various sectors, a lack of understanding of the operation of the rural aged care system among nurses working in regional hospitals, and the efficacy of ACAT working collaboratively with nurses to create new and more effective linkages in aged care. PMID- 10745733 TI - Metropolitan researchers undertaking rural research: benefits and pitfalls. AB - Can or should metropolitan residents research their rural counterparts and if they do are there inherent pitfalls or benefits? Throughout the history of social and anthropological research there has been debate on the insider-outsider/native stranger controversy as to who should carry out the field work. This discourse will explore the author's personal experiences in the context of planning a rural health project, entering the field, accessing the informants, interviewing the informants and leaving the field. PMID- 10745734 TI - Therapeutic massage: an education program for rural and remote workers in the palliative care field. AB - This paper, drawing upon data from a Rural Health, Support, Education and Training (RHSET) funded project, focuses on the evaluation of a massage education program developed specifically for rural and remote palliative care workers. An 8 week pilot massage education course was run for palliative care workers in the Albury-Wodonga area and, from this course, an instructional video education package on massage has been developed. The program was evaluated by using a questionnaire, journals and focus group interviews. Feedback from participants was incorporated into both the pilot course and final education package, which was consistent with the action research framework. The major findings of the project were that skill development in the therapeutic use of massage was attained and that job satisfaction was indeed achieved by palliative care workers. PMID- 10745735 TI - Common international themes in rural medicine. PMID- 10745736 TI - Acute myocardial infarction. AB - Current statistics reflect that cardiovascular disease (CVD) continues to be the greatest health problem affecting the Australian population, accounting for approximately 42% of all deaths recorded. Of all cardiovascular disorders, ischaemic heart disease is associated with highest mortality rates. PMID- 10745738 TI - Millenni-mum fever: will it deliver on time? PMID- 10745737 TI - Y2K: another bug for nurses. PMID- 10745739 TI - Coming through slaughter and famine. Interview by Anastasia Salamastrakis. PMID- 10745740 TI - Comparing meatal cleansing. PMID- 10745741 TI - Safety and physical restraint. PMID- 10745742 TI - The art and science of gastroenterology nursing: do we have a science? PMID- 10745743 TI - Pancreatitis Partners: a sharing and educational support group. AB - A team in a digestive disease center at a 550-bed tertiary care center in the Southeast created an interactive, educational patient and family support group for people with pancreatitis. No previous support groups for this population could be located in the United States by us, although pancreatitis is a disease that may progress to include chronic pain, frequent hospitalizations or emergency room visits, narcotic dependence, and depression. The article describes and illustrates the need, planning, inception, implementation, evaluation, and re visioning of an outpatient support and education group for people affected by pancreatitis. The group uses a partnership model as a basis for helping empower patients and their family members to have more understanding of, adaption to, and say in the treatment choices and responsibility for managing symptoms of their chronic illness. Facilitated by a multidisciplinary team, this is the first group of its kind in the United States and was enthusiastically received by those who participated. Group members were surveyed at the conclusion of the first ten group sessions. Results of this survey and modification to the group format and expanded outreach plans to people affected by pancreatitis are outlined and discussed. PMID- 10745744 TI - Endoscopic ultrasound: combining sight and sound for cancer and benign disorders. AB - Endoscopic ultrasound (EUS) is one of the most significant advances in the history of gastrointestinal endoscopy. It is unsurpassed in local and regional staging of cancer of the esophagus, stomach, pancreas, and rectum. Recently, EUS has been applied to a number of benign conditions such as achalasia, difficult-to diagnose gallbladder disease, and submucosal tumors. As EUS gains in popularity and increased clinical application in the United States, it is imperative that gastroenterology nurses and associates be aware of this ground-breaking technology and its indications. PMID- 10745745 TI - Aganglionosis of the small intestine: a rare form of Hirschsprung's disease. AB - Absence of ganglion cells in the small intestine, a rare form of Hirschsprung's disease, is a condition found in newborns and associated with increased morbidity and mortality. Families are faced with numerous challenges, including maintenance of nutrition, the need for surgical interventions, and the potential for complications associated with the medical technologies necessary to maintain children with aganglionosis. Home care needs are not unlike those of other children with severe intestinal dysfunction such as short bowel syndrome. This article gives an overview of Hirschsprung's disease and presents three case studies of small bowel Hirschsprung's. One center's experience with Hirschsprung's disease supports the need for a multidisciplinary approach to long term management and home care. PMID- 10745746 TI - Microscopic colitis. AB - Endoscopy nurses and associates occasionally find themselves assisting with colonoscopies in which biopsy specimens are obtained from normal-appearing mucosa to rule out microscopic colitis. A review of the literature was undertaken to learn more about this uncommon but troublesome cause of chronic diarrhea in patients. The few studies completed have been small and have yielded some conflicting and controversial data regarding many aspects of the two forms of microscopic colitis. Some findings, however, have been consistent and may help to provide a better understanding of this challenging syndrome for gastroenterology nurses. PMID- 10745747 TI - Current status of small bowel transplantation: a case study of transplant between identical twins. AB - Small bowel transplantation is coming of age. Although still considered an experimental procedure, much has been accomplished to bring this surgery closer to routine. This article describes the current status of small bowel transplantation and discusses the first successful small bowel transplantation between identical twins. The issues of workup, surgery, postoperative follow-up, common complications, and the history and future of small bowel transplantation are addressed. PMID- 10745748 TI - Reuse of single-use critical medical devices. Society of Gastroenterology Nurses, Inc. PMID- 10745749 TI - Winning entry: 1999 Gastroenterology Nursing Writers Contest. The challenge. PMID- 10745750 TI - Commentary. "Do scopes spread sickness?". PMID- 10745751 TI - Comparison of stress and stress management strategies between lesbian and heterosexual women. AB - Stress is said to be part of life, but stress may be uniquely experienced by different groups of women. We conducted this study to compare the experiences of stress and the methods of stress management used by lesbian and heterosexual women. A convenience sample of 215 (136 lesbian and 79 heterosexual) urban women was used. All women reported generally good mental health; however, more than 80% of the women reported moderate or severe stress. There were more similarities than differences between the groups, but lesbians reported more stress due to sexual identity, being female, and mental problems, and heterosexual women reported more stress due to parents and children. Both groups used a wide range of stress management strategies, although lesbians more frequently used meditation and therapy. PMID- 10745752 TI - Social support, demands of illness, and depression in chronically ill urban women. AB - In this study we used structural equation analysis to examine the relationship between chronic illness and depression among urban women. The model included the number of chronic illnesses reported, the demands of illness, perceived social support, and salient demographic variables as predictors of depression. The number of chronic illnesses had no direct effect on depression but had a direct impact on the demands of illness which led to decreased social support and increased depression. Being married played a protective role by reducing depression both directly and indirectly through increased social support. Having children under 18 in the home increased depression by increasing the demands of illness. These results provide important information about women with chronic illness and their resultant risk of depression. PMID- 10745753 TI - Women's lived experience of conceptualizing the self: implications for health care practice. AB - In this article we describe a unique qualitative research design in which we used our own lived experiences as the basis for understanding theories of the self. Our purpose in this study was to (a) broaden current understandings of self theory, (b) juxtapose theories of the self with lived experiences of selfhood, and (c) use these new understandings to inform health care practice. The participants were four Canadian middle-aged female academic and health care practitioners. We conducted unstructured, open-ended interviews. Through a collaborative, interpretive process, four recurring themes emerged from the women's narratives: struggling for authenticity, inner knowing, changing over time, and the contextual self. We address the need for practitioners to understand theories of the self--their own and their clients--and how these theories impact their clinical practice. PMID- 10745754 TI - The disconnected self: consequences of abuse in a cohort of low-income white women living with HIV/AIDS. AB - An infrequently acknowledged social factor influencing the health of low-income women is a history of physical/emotional abuse. In this article I address how abusive relationships influenced women's self-care practices. In this grounded theory study, vulnerability to abuse was established before diagnosis of HIV infection and promoted "lingering images" of a damaged self. The damaged self images that grew out of abusive relationships provided a barrier to self-care. Data analysis uncovered a core category of disconnection from self-care. Experiences of being cared for, particularly the care of health professionals, offered supportive relationships that encouraged women to care for themselves through self-care practices. PMID- 10745755 TI - The experience of hospitalization and restraint of women who have a history of childhood sexual abuse. AB - Within psychiatric settings, many female clients report experiences of childhood sexual abuse (CSA). In this paper we explore the experience of 10 women who were hospitalized in psychiatric settings, restrained, and given forced medication (FM). All the women have histories that included CSA. Some authors have suggested that the experience of psychiatric hospitalization may represent an event that reenacts the experience of trauma. The results suggest that from the perspective of these women, the experience of restraint engendered traumatic emotional reactions such as fear, anxiety, and rage, and in no way was viewed as therapeutic even years later. Women felt powerless and unheard. The women wanted nurses who were empathic and responsive to their human needs as clients, but they felt nurses did not want to hear about the abuse or their internal distress. We hope that the perspective of these women will help in the consideration of alternatives and modifications to the use of restraint in psychiatric settings. PMID- 10745756 TI - The power of the collective: battered migrant farmworker women creating safe spaces. AB - In this article I describe the use of a participatory action research (PAR) methodology to address the problem of domestic violence among migrant farmworker (MFW) women in California. The article was generated from a variety of data sources used by the investigator over a 4-year period. These include the investigator's observations, field notes, informal conversations, written stories from the women, and interviews. The "power of the collective" is discussed as the development of a power base for battered MFW women to support and take care of one another. The concepts of liberation, enlightenment, and "conscientizacion" are used to describe the evolution of the collective. PMID- 10745757 TI - Becoming whole: a pastoral story. AB - In my role as parish priest, I am present in the daily life of my community to acknowledge, nurture, celebrate, reflect, witness, and listen to the people around me. Hearing stories, helping people see and live in their stories, find meaning in them is my life work, and this is what happened when I met Eva. Together we will tell you her story of depression, her revelations in therapy, and piecing together the new meanings in her life through creating a quilt to symbolize and reflect her healing process. Eva's life, similar to the lives of each woman who has entered into her own suffering and there found healing, is perpetually becoming whole. Sharing our stories as Eva has opens possibilities for each of us who hears, believes, and witnesses our own pain and that of our sisters, allowing us to shape and give meaning to our stories, our selves, our wholeness, and our holiness. PMID- 10745758 TI - Strategic planning: what's the use? PMID- 10745759 TI - Congress waivers on legislative fixes for home health services. PMID- 10745760 TI - Helping families understand and communicate with family members who suffer from dementia. AB - Most clients suffering from dementia are cared for in their homes by family members or significant others. These caregivers anguish about their family members' mental deterioration. As one caregiver said, "How do we keep our hearts open in hell? And why should we try?" Caregiving is as much about the emotional impact on family caregivers as it the physical. PMID- 10745761 TI - Assessment of peripheral arterial pulses. AB - For the home care provider, determination of adequate cardiac function is vital to the well-being of the patient at home. If the heart fails as a pump, the patient's physical condition will deteriorate rapidly. This article will briefly review the normal blood vessel and blood flow principles and the principles guiding assessment of peripheral arterial pulses. PMID- 10745762 TI - JNC-VI, hypertension, and you. AB - More than 50 million U.S. adults are estimated to have hypertension--defined as systolic blood pressure (SBP) of at least 140 mmHg and/or diastolic blood pressure (DBP) of at least 90 mmHg--or take antihypertensive medication. Hypertension is directly implicated in a range of conditions, including coronary artery disease (CAD), renovascular disease, and cerebrovascular accidents. PMID- 10745763 TI - Helping elderly clients adjust to change and loss. AB - Home care providers must assess clients for any difficulties they may have with adjusting to changes and losses in their lives. They should consider the possibility that clients may be depressed and at risk for suicide. Home care providers' attention to psychologic dynamics may help a client face the losses that accompany growing older with integrity rather than despair. PMID- 10745764 TI - Chest pain. How serious is it? AB - The very thought of chest pain often brings terror to patients and health care providers. Chest pain typically invokes a "worst case" scenario of massive myocardial infarction (MI) resulting from cardiovascular disease (CD) and requiring immediate medical attention. However, chest pain is a common complaint suffered by most people at some time in their lives. People complaining of chest pain may be young, middle-aged, or elderly, and they may or may not be under a health care provider's care. Chest pain may be a symptom of a variety of disorders other than CD. Therefore it is helpful to differentiate quickly between the types of chest pain to determine whether immediate assistance is necessary. PMID- 10745765 TI - Management of patients with venous ulcers in the community setting. AB - Venous ulcers, a chronic disabling condition, present a complex management challenge to the interdisciplinary team in the community setting. The incidence of venous ulcers is increasing as the population ages with such comorbidities as congestive heart failure (CHF), deep vein thrombosis (DVT), obesity, and others. Disability associated with venous ulcers may increase caregiver burden in accomplishing the patient's activities of daily living (ADL), and handicap may exist from difficulty in community participation because of impaired mobility. Venous hypertension, the primary culprit in venous ulcerations, must be managed with an arsenal of strategies to control the underlying condition, heal the wound, and prevent recurrence. PMID- 10745766 TI - Caregiver resources. Central venous catheter complications. PMID- 10745767 TI - Caregiver resources. Central venous catheter site care. PMID- 10745768 TI - The most challenging 1998 home care standards. PMID- 10745769 TI - Who and what drives your practice? PMID- 10745770 TI - A tool to assess homebound status. AB - Although this specific instrument focuses solely on helping the home care nurse determine homebound status, this type of instrument can be developed for the other criteria used in assessing a client for Medicare-eligible home care services. For example, a similar instrument can be developed to assess whether a patient meets the criteria for part-time intermittent care or skilled care using the Medicare definitions as a guide to tool development. As we move into a time when there is more scrutiny of home care for compliance to Medicare regulations and increased productivity to assure cost effectiveness, instruments like this are essential for careful documentation that allows the agency to demonstrate accountability. In addition, home care agencies are expecting new home care nurses to know and be able to apply the Medicare regulations after a brief orientation. Instruments like this can shorten the orientation process and provide support for the home care nurse in the field. PMID- 10745771 TI - Influencing the legislative process. PMID- 10745772 TI - Walking meditation--relaxing step-by-step. PMID- 10745773 TI - An Internet primer. Part I. AB - The Internet provides electronic communications and access to information around the globe. In only one decade of public use this format has emerged as the method of rapid access to information, and projections are for continued growth. The WWW consists of over 40,000,000 servers containing billions of pages of information on virtually every conceivable subject. This doesn't include news servers, FTP servers, bulletin boards, and other online information sources. The home healthcare nurse, now and in the future, needs knowledge about and access to this system, to tap into the most current government recommendations about medications and treatment protocols, to access health statistics, consumer information, and additional educational, professional, and client-centered articles and opportunities. We hope this new Home Healthcare Nurse feature, "Staying Connected," will develop into a source of ongoing, up-to-date information to assist you in accessing and utilizing this valuable resource. Next month, we'll provide some specific directions for navigating the information superhighway. PMID- 10745774 TI - Nursing the terminally ill. Being with people in difficult times. AB - There are many impediments to being present and actively listening to people in difficult times. The authors discuss numerous suggestions nurses can use to "be" with patients experiencing a life-threatening or terminal illness. Methods such as eliminating external intrusions, using meditative practices, and ways to create a therapeutic milieu are presented to help the nurse understand how to listen to and be with patients. PMID- 10745775 TI - Research-based practice implications for patients with diabetes. Part I: Diabetes knowledge. AB - Diabetes knowledge of adults who require insulin is examined in this two-part article. Part I reports on the study findings related to diabetes education and compares documented educational nursing interventions with post-home care knowledge outcomes. Part II, which will appear next month, reports on patients' self-efficacy for diabetes management before and after home care. Strategies for improving patient outcomes are presented. PMID- 10745776 TI - Who supports elderly African Americans in adhering to their healthcare regimen? AB - Knowing who supports elderly African Americans in adhering to their healthcare regimen is vital information. This study found that the majority of elderly individuals discussed their chronic illnesses and problems related to managing their illnesses with family members rather than with formal providers. Additionally, family members were also significant in coordinating transportation; assisting with managing medications, diet, and exercise regimens; and monitoring symptoms of chronic illnesses. These findings provide direction on how to develop effective care plans and affect positive outcomes for elderly African American clients. PMID- 10745777 TI - Preventing Refeeding syndrome. PMID- 10745778 TI - Documentation reminders. PMID- 10745779 TI - Building relationships with discharge planners. PMID- 10745780 TI - Rainy days don't always get you down. PMID- 10745781 TI - Transcultural Patient Care Committee: actualizing concepts and developing skills. AB - This article describes activities of a Transcultural Patient Care Committee whose aim is to develop the knowledge and skills of medical center staff to provide culturally congruent care. The activities include staff development offerings, a transcultural patient care resource manual, and communication and assessment tools. These efforts have provided a foundation to assist staff with meeting the challenge of caring for multicultural patients. PMID- 10745782 TI - Enhancing relationships between nursing faculty and clinical preceptors. AB - Rapid changes in health care and nursing education have necessitated an increased need for educators and clinicians to build alliances. Historically, the economic demand has been placed on clinical agencies to provide quality educational experiences for students. However, clinical agencies are increasingly refusing to precept students because of cost and liability issues. To secure and maintain optimal clinical experiences for students, nurse educators must learn to develop positive relationships with practicing nurses, contribute to staff education, and share educational responsibility in evaluating student learning. PMID- 10745783 TI - Restructuring a competency-based orientation for registered nurses. AB - A competency-based orientation is an orientation program that focuses on a new employee's ability to actually perform the expectations of his or her role in a particular setting. It differs from a traditional model in which the focus is on the cognitive knowledge necessary for the role. The authors will identify key elements educators may consider when evaluating such a program. The process used by the author's organization to evaluate and restructure the competency-based orientation program for registered nurses is also described. PMID- 10745784 TI - The "Party Planner": a novel approach to teaching complex material to all levels of the healthcare team. AB - One of the challenges facing staff development educators today is addressing the needs of a new healthcare team with widely differing education and experience. We developed a novel approach--the Party Planner--to teach a diverse healthcare team about a rather complicated practice model. The Party Planner addresses issues that are central in the current healthcare environment--using resources, balancing cost and quality, eliminating non-value added routines, and achieving critical outcomes. PMID- 10745785 TI - Systematic planning of continuing nursing education. AB - To avoid omissions and to promote efficient use of time, it is important to plan continuing nursing education (CNE) activities systematically. A detailed and comprehensive planning checklist is provided to assist the novice planner to organize and implement a CNE offering competently. PMID- 10745786 TI - Writing objectives and evaluating learning in the affective domain. AB - Staff educators recognize the importance of affective competency for effective nursing practice. Inservice programs must include affective learning with objectives stated in measurable terms. Staff educators often express frustration in developing affective objectives and evaluating the learning outcome because attitudes and feelings are usually inferred from observations. This article presents affective learning objectives for a gerontological nursing inservice program and a rating scale that measures attitudes to evaluate the learning outcome. PMID- 10745787 TI - An F-5 in Oklahoma City begets human kindness. PMID- 10745788 TI - Preoperative diagnosis and postoperative management of adult patients with obstructive sleep apnea syndrome: a review of the literature. AB - Obstructive sleep apnea syndrome (OSAS) is a common yet potentially fatal disorder. Several reports and clinical studies have established anesthetic related occurrences of near-fatal respiratory complications in persons with this syndrome, as well as cases of death reported in the medical literature linking OSAS and anesthesia. The postoperative period is a critical time for patients with OSAS recovering from general anesthesia. The purpose of the literature review is to determine current treatment protocols and standards of care for preoperative diagnosis and postoperative management of adults with OSAS in an effort to prevent respiratory complications. The literature review includes recent studies on diagnosis of patients with OSAS. A treatment protocol for the postoperative management of adults with OSAS is also presented. Conclusions are drawn from a summary of the literature review. Future implications for nursing practice and research are drawn from the summary. PMID- 10745789 TI - Ambulatory surgery: the growth of an industry. AB - Over a short 30 years, ambulatory surgery has grown from a few freestanding facilities to a continuously growing industry. This article gives a historical overview of that growth, while specifically documenting the impact that competition, regulation, capitation, technology, and the consumer have on nursing and a single industry. PMID- 10745790 TI - Anesthesia-assisted rapid opiate detoxification: a new procedure in the postanesthesia care unit. AB - Heroin detoxification by anesthesia-assisted rapid opiate detoxification (AAROD) can be achieved in approximately 1 hour using intravenous administration of midazolam, propofol, and naloxone. Opiate dependence and detoxification techniques are reviewed. Important aspects of patient care and perianesthesia nursing are described using a case study. To assist in caring for the patient undergoing anesthesia-assisted rapid opiate detoxification, the perianesthesia nurse should understand the methodology and controversy of this new procedure. PMID- 10745791 TI - Yet another workplace crisis. AB - Most, if not all, managers have experienced disappointment when an employee who is very bright and technically competent cannot quite get it together, or has what is usually referred to as "interpersonal problems" or a poor attitude. Such employees can cause major problems. At a time when jobs can be effected by mergers, downsizing, and other changes, people who are technically skilled but have an attitude problem can be seen as problems that take up too much of a manager's time. Understanding another dimension of job competency helps develop technically competent staff with "attitude" problems into truly star performers. This dimension of job competency is referred to as emotional intelligence or EQ. It is this competency that makes even those with average technical skills become the type of an employee that is self-directed and a real asset. PMID- 10745792 TI - Sickle cell disease: implications for perioperative care. AB - Sickle cell disease is an inherited hemoglobinopathy that develops from a genetic mutation and the production of a dysfunctional variant of hemoglobin. A number of physiological disturbances encountered during the perioperative period (blood loss, hypotension, acidosis, and hypoxia) may induce "sickling" of the biochemically altered hemoglobin, producing painful microvascular occlusion, hemolytic anemia, and impaired oxygen delivery. The only available curative therapy requires bone marrow transplantation. The purpose of this article is to review the pathophysiology of sickle cell disease and the pertinent preoperative, intraoperative, and postoperative care of patients with the disease. PMID- 10745793 TI - Dementia: a complex symptom. AB - The number of perianesthesia patients with dementia increases yearly. Understanding the physiology and functional impairments of these patients will improve the perianesthesia nurse's ability to create a safe environment, communicate effectively, and reduce discomfort. PMID- 10745794 TI - No simple thing. PMID- 10745795 TI - Stress management--a survival skill in today's workplace. PMID- 10745796 TI - Unique considerations for the pediatric heart transplant recipient: the role of the school nurse. AB - Heart transplantation has become an accepted therapy for terminal heart conditions in children. With the increasing incidence of pediatric heart transplantation, school nurses may find cardiac recipients in their schools. The article discusses physical and psychological considerations for managing these students. Among the physical considerations are work capacity, appearance, organ rejection, and infection. The psychological considerations are emotions, cognitive functioning, quality of life, and behavioral concerns. The school nurse is a key resource in informing staff about the unique concerns of pediatric transplant recipients and in ensuring they are integrated into the school setting. PMID- 10745797 TI - The sleep needs of adolescents. AB - The amount of sleep affects the way adolescents perform, feel, think, learn, and remember. Significant physiological and social changes have profound effects on adolescents' sleeping patterns. Lack of sleep increases the possibility of increased daytime sleepiness, which may result in a tragic automobile or work accident. Other consequences of sleep deprivation include poor school performance, heightened risk of drug and alcohol use, increased irritability, and aggressive behavior, all of which can interfere with relationships with classmates, parents, and teachers. The purpose of this paper is to explore the literature concerning what is known about why adolescents need more sleep and why adolescents do not obtain enough sleep. Nursing interventions targeting adolescents, parents, teachers, schools, and employers are included. If adolescents understand and learn to improve their sleep patterns while they are young, they may have improved sleep habits in adulthood. Teaching adolescents about the importance of sleep is an important task for school nurses. PMID- 10745798 TI - The Health Consultation Program: a model school nurse education program. AB - The Health Consultation Program (HCP) provides educational resources to school nurses throughout the state of Washington. It has several components, including consultation with clinical nurse specialists, a video lending library, health education materials, continuing education seminars, and preceptorships. School nurses access desired services to assist them in a variety of activities, such as developing individualized health care plans or teaching other school personnel about a child's special needs. Quotations from school nurses gathered during HCP evaluations indicate greater self-care abilities by students, improved skills of teachers and other professionals, increased planning for safe and appropriate care, and improved quality of care. PMID- 10745799 TI - The prevention of playground injuries. AB - Playground accidents are the most common cause of injury to children in school. As the health professional who attends to these injured children, school nurses should be aware of safety practices that can help eliminate the risks that children encounter. The National Program for Playground Safety has identified four areas that can reduce the level of risk on school playgrounds: supervision, age-appropriate design of the play area, fall surfaces under and around playground equipment, and equipment and surface maintenance. The article discusses each of these, and recommends leadership methods and practices that nurses can implement that will eliminate or reduce risk factors. In addition, a comprehensive playground risk model is described, and suggestions for policy areas are outlined. Policy areas include assessment, education, and enforcement in relation to each of the risk factors. PMID- 10745800 TI - Never too soon: a pilot first and third grade drug education program. AB - Substance use is a leading national health problem, and school nurses are in pivotal positions to assist with prevention activities to reduce this growing epidemic. In response to increasing rates of parental and youth substance use and abuse, a collaborative research-service-education partnership was established between a college of nursing and an elementary school in a low-income, urban community. A two-part pilot study of parental knowledge and behaviors of drug use, and the evaluation of a first- and third-grade drug education model, are described in this paper. Although the findings cannot be generalized, they yield useful information for parental, youth, and neighborhood teaching and future research. Over half of parents consumed alcohol, and 60% smoked cigarettes. Forty seven percent of parents reported discussing drugs with their child. However, only 22% of the parents who used substances reported talking about drugs with their children. The pilot drug education sessions, two, 30-45 minute sessions a week for 8 weeks, resulted in an average of 30% higher posttest knowledge scores in the first and third graders. Drug education classes should be offered every year for patients and youth, kindergarten through twelfth grade. PMID- 10745801 TI - Legal issues: office management practices. AB - Student health records, whether paper or electronic, are restricted and protected to a greater degree than are educational records. Some school health office software is designed to provide greater data protection than is possible in paper records. However, unless basic district and health office practices are established to prevent access to or corruption of electronic health data, technological safeguards will be useless. This article describes school and health office policies and practices that are necessary for the integrity, confidentiality, and security of student health information. PMID- 10745802 TI - Coping with sickle cell anemia: additional recommendations for school nurses. PMID- 10745803 TI - Fair pay for all nurses. PMID- 10745804 TI - Fair pay for all nurses. PMID- 10745805 TI - Running to stand still. PMID- 10745806 TI - What is the pay round? PMID- 10745807 TI - Four-way split. PMID- 10745808 TI - Is Project 2000 ready for 2000? PMID- 10745809 TI - Mary Hampshire reports from the Festival of Science in Sheffield. PMID- 10745810 TI - Doctors and nurses: is teamwork a myth? PMID- 10745811 TI - Actions speak louder than words. PMID- 10745812 TI - Is the carrot worth a nibble? PMID- 10745813 TI - They can stick it. PMID- 10745814 TI - A positive way to play the system. PMID- 10745815 TI - Sacrificial lambs. PMID- 10745817 TI - Mad for it. PMID- 10745816 TI - Nurse heroes of the century. PMID- 10745818 TI - A nurse-led audit has identified major gaps in preventive care for people at risk of coronary heart disease. PMID- 10745819 TI - The heart, Part Three: Anatomy and physiology. PMID- 10745820 TI - The march of cholera. PMID- 10745821 TI - Using the biographical approach. PMID- 10745823 TI - Are you ready for the flu season? PMID- 10745822 TI - Back to normal: children's care after burn injury. PMID- 10745824 TI - The value of auditing tracheostomy care. PMID- 10745825 TI - Administering a catheter maintenance solution--2. PMID- 10745826 TI - Like an iceberg, racial prejudice can be larger than it seems. PMID- 10745827 TI - Chequered memories. Interview by Anne Gulland. PMID- 10745828 TI - Timed out. PMID- 10745829 TI - Where are our increments? PMID- 10745830 TI - Health visitors must remind everyone that their service is worth paying for. PMID- 10745831 TI - Tales of the river bank. PMID- 10745832 TI - FPA takes a firm stand on sex aid catalogue. PMID- 10745833 TI - Forcing a mother with HIV to have her baby tested for the virus is not necessarily in the child's best interest. PMID- 10745834 TI - Talk your way out of a problem. PMID- 10745835 TI - Making strides after limb loss. PMID- 10745836 TI - When amputation is the only option. PMID- 10745837 TI - High ideals. PMID- 10745838 TI - World mental health day. Song sung blue. PMID- 10745839 TI - Face to face. Interview by Eileen Fursland. PMID- 10745840 TI - Jackie Strudwick was sickened by the care her son received on entering hospital with acute appendicitis. PMID- 10745841 TI - The initial management of lower limb amputations. PMID- 10745842 TI - Nurses' role up for review. PMID- 10745843 TI - Scotland gets in on the act. PMID- 10745844 TI - Whooping cough: how the west was won. PMID- 10745845 TI - Gonorrhoea: the return. PMID- 10745846 TI - Changing times: changing practice. PMID- 10745847 TI - Trouble with latex. PMID- 10745848 TI - Good practice network. The quest for quality. PMID- 10745849 TI - Digital rectal examination--1. PMID- 10745850 TI - America's changing face challenges oncology nurses. PMID- 10745851 TI - Advanced caring: hand in hand into the new millennium. PMID- 10745852 TI - The dressing is only a side dish. PMID- 10745853 TI - Quality care among aging Americans. AB - The unique needs of many elderly Americans are often not heard. However, in the face of an ever-growing aging population, clinicians are more actively planning healthcare for older adults across practice settings. This article examines the home care needs of elderly patients who wish for care, but not aggressive, acute care. The author reviews bioethical considerations that apply to elderly individuals and groups and presents suggestions to promote quality care that is ethically sound. This article also describes clinicians' obligation to identify what quality care means to the very old person and explains the deficiency model of aging along with its impact on medical intervention. PMID- 10745854 TI - Applying electric and electromagnetic energy as adjuvant treatment for pressure ulcers: a critical review. AB - Chronic pressure ulcers are a significant health problem especially in the aging population. National estimated annual treatment costs are in the billions of dollars. Only two treatment-related recommendations receive high ratings for reported experimental evidence of validity: Use of moist wound dressings and adjunctive electrotherapy for unresponsive Stage III and IV and recalcitrant Stage II ulcers. A critical literature review pertaining to electrotherapy reveals a myriad of electrical treatment modalities varying greatly in electric current type, strength, direction, frequency, waveform, and underlying voltage. However, few clinical trials pertaining to electrotherapy exist with almost all of them characterized by a small sample size leading to a biased group assignment with no possibility for stratification by ulcer stage, site, and other important factors. Power analysis shows that a sample size of at least 164 subjects is needed to permit cost-effectiveness evaluation with attention to critical variables. "Time to healing" is recommended as the treatment outcome measure to permit proper efficiency comparisons between the various treatment modalities and controls. These comparisons are crucial in a cost-conscious environment. PMID- 10745855 TI - Randomized controlled trial to determine the safety and efficacy of a multi-cell pulsating dynamic mattress system in the prevention of pressure ulcers in patients undergoing cardiovascular surgery. AB - The purpose of this study was to determine the efficacy and safety of a multi cell pulsating dynamic mattress system in comparison with conventional management for the prevention of pressure ulcers in the operative and postoperative period in patients having cardiovascular surgery. The study was a single center, prospective, randomized, controlled trial. Patients who were having cardiovascular surgery for a duration of at least 4 hours were randomly assigned, prior to surgery, to dynamic mattress system or conventional management--both of which were initiated in the operating room and continued for up to 7 days postoperatively. Patients were assessed daily using a standardized scoring system. The results of the study showed that 198 patients in the dynamic pressure system (n = 98) or conventional management group (n = 100) were similar at baseline. A strong trend of decreased pressure ulcers existed in the dynamic pressure system group (n = 2) compared to the conventional management group (n = 7). The study concluded that a multi-cell pulsating dynamic mattress system is safe and mitigates risks for and decreases incidence of pressure ulcers in patients who undergo cardiovascular surgery. PMID- 10745856 TI - Examining the nutritional status of independently living elderly. AB - As more and more people age, the idea of quality, not just quantity, of life becomes increasingly important. Healthcare professionals and community planners need to look at programs aimed at keeping the elderly as healthy as possible for as long as possible. To accomplish this, having a profile of the needs of this population in all possible settings is important. The purpose of this study was to examine wellness behaviors and nutrition in a group of independently living elderly. Wellness behaviors and health practices were generally positive in this study. However, the author identified several areas of deficiencies including performing breast and testicular self-examinations, staying asleep, medication use, exercising and weight maintenance, and eating (dietary patterns). Healthcare professionals need to take nutrition seriously. Although poor nutrition is a risk factor for pressure ulcers, delays wound healing, and increases mortality when someone is ill, effects of poor nutrition are often overlooked when the person is healthy and active. Results of this study show that health professionals need to include nutritional assessment as an important component of any examination in community, acute care, and long-term care settings. PMID- 10745857 TI - [Interdisciplinary qualification in geriatric rehabilitation]. PMID- 10745858 TI - [Contribution of nursing in oncologic rehabilitation]. PMID- 10745859 TI - [Early rehabilitation of patients with neurologic diseases]. PMID- 10745860 TI - [Contribution of pragmatic ethics to the management of patients dying in hospital]. PMID- 10745861 TI - [Nursing development units]. PMID- 10745862 TI - [Television series about hospitals]. PMID- 10745863 TI - [Current trends in geriatric nursing]. PMID- 10745864 TI - [The trouble with learning, essential for a healthy old age]. PMID- 10745865 TI - [Creating an easier, more modern and more accessible health care system]. AB - After superficially reviewing the advances which have occurred in the public health system over the past twenty years, the author speaks to us about the idea of how it is possible to facilitate improved information regarding health, sanity, and health services in order to help create a general public which is more responsible for their own actions while still accepting as a fact that each individual is a whole world with his/her own particular attitude toward health and illness. As a simple and accessible way to provide information, the author analyzes the method used in England to inform the general public by telephone contact, a method ideal to increase the self-care of the general public and to provide appropriate answers to their voiced concerns. The author concludes his article commenting that soon in Spain we should incorporate new information technologies and systems into the daily lives of our health system in order to make it more modern and accessible and to bring it into closer touch with the general public. PMID- 10745866 TI - [Mushrooms, poisoning and treatment]. AB - How to avoid poisoning due to consumption of the wrong type of mushrooms is the first rule of thumb which mushroom gatherers and health professionals in charge of advising the public about health cares need bear in mind. The objective of this article is to provide a wide spread of information to all interested people and to health professionals so that they know how to act when poisoning occurs, thus helping to prevent and reduce the number of deaths, the secondary effects and the complications caused by different poisons and their accompanying syndromes. PMID- 10745867 TI - [Hemiplegic patients. Nursing at home]. AB - By daily performing the seven easy exercises in the rehabilitation program which the authors of this article propose, a patient afflicted by an "ACV" can prevent the appearance of the typical motor skill limitations which appear a while after the onset of the illness. PMID- 10745868 TI - [Hospital infections. Risky practices of nursing students]. AB - In this study the health habits performed by nursing students during their practices in hospitals are describe in the province of Cadiz, Spain and the relationship between these activities involving risk, and the level of knowledge of Public Health of these students. A total of 397 nursing students were studied, 43% male, with an average age of 21.6 years (SD 2.9); the average practice period per student was 73 days. Washing of hands before and after each intervention is performed by 97% of students, and gloves are used on all the sanitary occasions recommended by only 21%; the groups using masks least are those not vaccinated for influenza and not subjected to a Mantoux test (p < 0.05). The relationship between taking unnecessary risk in hospital practice and the low level of knowledge is positive (collective protection p < 0.05, use of gloves p < 0.05, among others). PMID- 10745869 TI - [Validation of two scales for evaluation of the risk of decubitus ulcer: Gosnell and Nova-4]. PMID- 10745870 TI - [Fear of death. How to help a patient with a nightmare]. PMID- 10745871 TI - [Testicular veins and spermatic cord]. PMID- 10745873 TI - [Stethoscopes]. AB - Traditionally, a phonendoscope has been an instrument by which health professionals have been identified since it has been an absolutely necessary tool through which health professionals carried out a mediate auscultation and could isolate the sounds produced by the different organs of the body. This health technology, tools and instruments article analyzes the origins, usefulness, component parts, and special characteristics of the various types of phonendoscopes available in the market while at the same time states the proper way to use a phonendoscope by explaining how to manipulate it and the basic rules for keeping it clean and caring for it. PMID- 10745872 TI - [Cryotherapy: how to use it and cost analysis]. AB - The authors analyze the results obtained by the use of cryotherapy at the Rochapea Health Clinic from October 1995 until the 30th of June 1997. The authors make known the fundamentals and techniques of cryotherapy to other professionals who are not familiar with it and encourage them to put it into practice. They analyze the treatment given in 203 cases of common warts and plantar papillomas and specifically to the use of cryotherapy in 128 lesions at the Rochapea Health Clinic. The procedure is simple, effective, efficient and easy to learn. It happens to be quicker than conventional surgery and presents very few complications. PMID- 10745874 TI - [Sudden infant death syndrome]. AB - Sudden infant crib syndrome in nursing newborns means the syndrome which affects those babies less than one year old whose death can not be explained even after the conclusion of an in depth investigation. This article explains the causes currently provided as possible factors which cause this syndrome, risk factors supported by epidemiological data and preventive measures including the monitoring of babies deemed to be at risk. The authors also specify what this monitoring consists of and they elaborate a proposed nurses' action plan for the newborn and his/her family for those cases in which monitoring is taking place. The authors also present a proposed nurses' action plan in the event of a newborn's death due to this syndrome. PMID- 10745875 TI - Marketing. PMID- 10745876 TI - Click ... and you shall find. PMID- 10745877 TI - Going from the gut. PMID- 10745878 TI - Is it pertussis or RSV? PMID- 10745879 TI - Violence in the hospital: a response plan. PMID- 10745880 TI - Unconventional surgery yields dramatic results. PMID- 10745881 TI - When spinal cord injury affects the bowel. PMID- 10745882 TI - What's wrong with this patient? PMID- 10745883 TI - How to make patient teaching stick. PMID- 10745884 TI - Yep, that's my job too! PMID- 10745885 TI - A kiss. PMID- 10745886 TI - Nosebleeds: not just kids' stuff. PMID- 10745887 TI - Safer needle devices, Part 2. PMID- 10745888 TI - Diet and herbs for BPH? PMID- 10745889 TI - Serving as an expert witness. PMID- 10745890 TI - Buying supplies online. PMID- 10745891 TI - [Professional writing, a collective experience. From thread to needle]. PMID- 10745892 TI - [Does writing kill or liberate?]. PMID- 10745893 TI - [Not writing, making notes, where is the problem?]. PMID- 10745894 TI - [Professional writing, a collective experience. A professional process at the service of the patient]. PMID- 10745895 TI - [The illusion of "everything is written down"]. PMID- 10745896 TI - [Professional writing wiped out, identity erased]. PMID- 10745897 TI - [Nurses, another effort to be connected. The effect of new information and communication systems on professional writing in the hospital...]. PMID- 10745898 TI - [Professional writing. A collective experience. Archives, how to use them]. PMID- 10745899 TI - [Pedagogy. From theory to practice, the evaluation]. PMID- 10745900 TI - [Pedagogy. Teaching nursing care yesterday, today and tomorrow]. PMID- 10745901 TI - [Psychiatric nursing time study: or when economy questions care]. PMID- 10745902 TI - [Conception and realization of an educational film]. PMID- 10745903 TI - [Working towards independence]. PMID- 10745904 TI - [Documentation personnel in the hospital environment, condition of the facility (part 2)]. PMID- 10745905 TI - [Voyage to the center of a book]. PMID- 10745906 TI - [Legislation. Current regulations]. PMID- 10745907 TI - [Appearance of a European nursing identity. Interview by Jacques Lebas]. PMID- 10745908 TI - ["Tomorrow, the hospital...", for a sick citizen]. PMID- 10745909 TI - [General state of health care, balance and perspectives]. PMID- 10745910 TI - [The nurse and Alzheimer's disease]. PMID- 10745911 TI - [Alzheimer's disease: risk factors and protective factors]. PMID- 10745912 TI - [Treatments of Alzheimer's disease]. PMID- 10745913 TI - [Nutrition in Alzheimer's disease]. PMID- 10745914 TI - [Alzheimer's disease. Psychiatric and behavior complications]. PMID- 10745915 TI - [Alzheimer's disease: language and dementia: importance of upkeep of communication]. PMID- 10745916 TI - [Emergencies. What to do about signs of pain]. PMID- 10745917 TI - [Diets: convenience or necessity]. PMID- 10745918 TI - [Explanation: experience of a memory consultation in a geriatric hospital]. PMID- 10745919 TI - [Nursing diagnosis and the Scale for Autonomy, Gerontology and Group Resources for the Dependent Elderly]. PMID- 10745920 TI - [Respect for the elderly in an institution]. PMID- 10745921 TI - [Living better. Incontinence at home, some technical means of prevention]. PMID- 10745922 TI - [Funeral rites, a collective and individual expression]. PMID- 10745923 TI - [Behavior evolution when facing death]. PMID- 10745924 TI - [Significance of funeral rites]. PMID- 10745925 TI - [The caretaker facing death in the hospital environment]. PMID- 10745926 TI - [Diabetes in the aged]. PMID- 10745927 TI - [Accelerate, refrain or regulate intestinal transit]. PMID- 10745928 TI - [Doloplus-2, a scale for pain measurement]. PMID- 10745929 TI - [Dementia, relations, dignity, accusations, insults, lying]. PMID- 10745930 TI - [Improving the mental effect of working conditions of nursing aides]. PMID- 10745931 TI - [Pacemakers]. PMID- 10745932 TI - [Ergotherapy in palliative care]. PMID- 10745933 TI - [Parkinson disease]. PMID- 10745934 TI - The morselized and impacted bone graft. Animal experiments on proteins, impaction and load. AB - The results of primary hip replacements are good. However, dealing with a loose prosthesis has been problematic, especially when major bone deficiencies are encountered. These problems appear to have been solved by the introduction of the Slooff-Ling method of using morselized and impacted allograft chips. The clinical results are excellent in the hands of the innovators. However, it remains confusing that a thick layer of dead, broken, immunogenic tissue taken from another individual does not resorb and collapse during remodeling. Still harder to understand is the impression, as judged by radiography, that this thick layer seems to incorporate and remodel up to a distance of perhaps 10 mm or more from the host bone, whereas the ingrowth distance into a non-morselized graft is limited to a few mms. To clarify the biological basis of the morselized and impacted grafts better, the present study was stated. Three hypotheses were initially proposed to explain the good clinical results: 1. Morselization releases growth factors present in the graft (osteoinduction). 2. Impaction makes it easier for the ingrowing bone to climb up into the graft (osteoconduction). 3. The compliance or elasticity of the graft allows the load to produce deformations that stimulate bone formation (mechanical load). In the first studies, bone chambers were implanted in rats and the distance of new bone ingrowth into a graft in the chamber was measured. In Paper I, a morselized graft was deproteinized by slow heating under high pressure. Ingrowing bone did not reach so far into the deproteinized graft as into a non-treated one. We concluded that the proteins present in the graft partly determine how far ingrowing new bone will extend into a graft. In Paper II, a cancellous graft was impacted so that the bone volume fraction of the graft rose from 35% in the unimpacted to 65%. The impacted grafts were compared to unimpacted ones and it was shown that impaction reduced the ingrowth of new bone into a graft in the chamber at six weeks. In Paper III, this somewhat unexpected finding was further studied. Syngeneic and allogeneic grafts showed a reduced ingrowth distance at six weeks when impacted, compared to unipacted controls. However, the reduction was not found when the time for ingrowth was extended to 12 weeks, indicating a possible catch-up phenomenon. Moreover, an exogenously applied growth factor, osteogenic protein-1, was found to have increased the ingrowth distance of new bone into impacted grafts at six weeks. In Paper IV, a rabbit knee prosthesis was developed to study the effect of a mechanical load on the remodeling of a morselized and impacted graft. All rabbits had their tibial marrow cavity cleansed of cancellous bone, which was replaced by a morselized and impacted bone graft. Six rabbits received a complete tibial prosthesis with a tibial load-bearing tray and a stem transferring the load to the impacted graft with each step made by the rabbit. Another six rabbits had only the stem, without the tibial tray, inserted into the impacted graft. With this design, the load from walking was not transferred to the graft, since there was no joint surface replacement to transfer the load to the stem and the graft. Thus, the graft was loaded in rabbits receiving a full prosthesis, whereas it was unloaded in the animals receiving only the stem. New bone formation and resorption of the graft were increased in the loaded grafts, and we concluded that a load increases the rate or speed of remodeling. In Paper V, four patients were operated on for vertebral fractures. The fractures were stabilized by plates and the vertebral bodies packed with autogenous morselized graft. After 1.5 years, when the fractures were clinically and radiographically healed, a biopsy was taken. It was found, that even after such a long time, large areas remained unremodeled and sometimes even unrevascularized. In some parts, necrotic graft trabeculae were embedded in fibrous vasc PMID- 10745936 TI - [Epidural lipomatosis]. AB - ONSET: Epidural lipomatosis is a rare disorder defined as a pathological overgrowth of normal epidural fat. It is more often associated with administration of exogenous steroid with variable duration and doses. Furthermore, it may occur in some patients in the absence of exposure to steroids but generally associated with obesity. Whatever the predisposing factor, the majority of these patients are men. The causal effect of epidural lipomatosis in the development of spinal cord or radicular compression is generally well accepted. DIAGNOSIS: The diagnosis of epidural lipomatosis can be established by melography, computed tomography (CT) and magnetic resonance imaging (MRI). MRI is considered the imaging procedure of choice, allowing an assessment of the extent of lipomatosis and, as well as CT, an identification of the lipomatous tissue. Most cases of epidural lipomatosis with corticosteroid use occur in the thoracic region, while most idiopathic cases occur in the lumbar region. TREATMENT: Management of treatment depends on the severity of the neurological signs and the patient's background. The most common treatment for epidural lipomatosis with corticosteroid use consists in surgical decompression but with a high risk of postoperative mortality. In some cases however, medical treatment includes corticosteroid withdrawal or reduction and calorie restriction, leading to clinical improvement. Treatment for idiopathic epidural lipomatosis is more often medical, based on weight loss and physical therapy with generally successful outcome. The pathogenesis of epidural lipomatosis remains unknown but different suggested hypotheses may lead to a metabolic disorder as the underlying cause. PMID- 10745935 TI - [Role of anti-TNF therapy in rheumatoid arthritis]. AB - TUMOR NECROSIS FACTOR: TNF is a cytokine produced by several types of cells, but mainly by monocyte-macrophages, activated endothelial cells, fibroblasts, and joint cartilage chondrocytes. The circulating form of TNF alpha (homotrimere) is derived from its membrane form by cleavage induced by a metalloprotease called TACE. This cytokine plays a pivotal role in the inflammatory reaction in conjunction with IL-1 and IL-6. The effect of TNF alpha is mediated by two membrane receptors carried on the surface of target cells (TNF-RI p55 and TNF-RII p75) which are released into the biological fluids (synovial fluid and plasma). ARGUMENTS FOR A PATHOGENIC ROLE: Transgenic mice carrying the human gene for TNF alpha develop polyarthritis suggesting this cytokine is directly implicated in the pathogenesis. In diverse cell types in rheumatoid joints, TNF alpha and its receptors can be identified by immunohistochemistry techniques as can TNF alpha mRNA by RT-PCR. THERAPEUTIC POSSIBILITIES: Anti-TNF alpha antibodies can effectively attenuate or prevent arthritis in the main experimental models. TNF alpha can also be neutralized with soluble receptors, TNF alpha RI or TNF alpha RII. The efficacy of these two therapeutics has been proven in rheumatoid arthritis, but with a short-term though remnant effect, leading to iterative injections and/or combinations with methotrexate. Short-term side effects are mild but the long-term infectious and oncogenic adverse effects remain to be determined. Is this simply a powerful antiinflammatory treatment or a real curative treatment? A precise examination of radiographic scores will be required to provide the answer to this question. PMID- 10745937 TI - [Joint manifestations related to Clostridium difficile]. AB - BACKGROUND: Reactional joint disease subsequent to digestive tract infections have been well studied for Salmonella, Shigella, Yersinia and Campylobacter. Association with HLA B27 is well documented. A review of the literature since 1976 disclosed 23 cases of articular and extra-articular inflammatory reactions following pseudomembranous Clostridium difficile infection. CASE REPORT: We describe 2 new cases which followed a favorable course. This is the usual outcome although inflammatory manifestations may persist or relapse. DISCUSSION: The pathophysiological interest of this condition lies in the pathogenic potential of Clostridium difficile which appears to be directly related to toxin production. This would suggest an immune mechanism rather than pure infection. PMID- 10745938 TI - [Specific cyclo-oxygenase inhibitors. 2. Gastric toxicity?]. PMID- 10745939 TI - [Local and general treatment of pressure sores in the aged]. PMID- 10745940 TI - [Prolonged fever and Behcet's disease]. PMID- 10745941 TI - [Bone hyperresorption in neoplastic diseases. Foreword]. PMID- 10745942 TI - [Bone hyperresorption in bone metastases]. AB - MECHANISMS OF BONE LOSS: In patients with bone metastases, bone loss is the consequence of a dissociated process combining excessive bone resorption and inhibited bone formation. This destructive process occurs in response to soluble factors secreted by metastatic cells (PTH-rP, cytokines) which activate osteoclasts. These cells also secrete proteases (cathepsin K, metalloprotease MMP 9) which degrade the bone's collagen network. Excessive bone resorption is also favored by direct interaction between the metastatic cells and stromal cells in the bone marrow in response to the activation of membrane receptors (integrins a4 beta 1 and a4 beta 7). Finally, metastatic cells secrete non-identified soluble factors capable of inhibiting osteoblast proliferation in vitro. EFFECT OF BONE LOSS ON THE METASTASIS: Bone is a major reservoir of growth factors (mainly TGF beta and IGF-1). Positive feedback mechanism operates at the site of osteolysis where TGF beta released by bone tissue induces a paracrine stimulation of PTH-rP production by metastatic cells. IGF-1 released by the bone favors grow of metastatic cells present in the marrow. In addition, IGF-1 as well as collagen proteolytic fragments may stimulate recruitment of new metastatic cells at the site of the bone metastasis. This creates a vicious circle of mutual stimulation between bone destruction and tumor proliferation. PMID- 10745943 TI - [Bone hyperresorption in multiple myeloma]. AB - OSTEOLYSIS AND HYPERCALCEMIA: Multiple myeloma is a type B high-grade lymphoproliferative syndrome with bone tropism. Bone-related manifestations- osteolysis and hypercalcemia--are observed in 80 and 30% of cases respectively. Excessive bone resorption subsequent to destruction of the bone matrix by osteoclasts is associated with insufficient bone formation. This process plays a determining role in the development of osteolysis and hypercalcemia in multiple myeloma patients. MECHANISM OF BONE DESTRUCTION: The reality and intensity of bone destruction is clearly demonstrated by histomorphometric studies and more recently by biochemical methods using markers of bone resorption. The excessive bone resorption results from complex interactions between tumor plasma cells, bone cells, and stem cells and involves local factors and adhesion molecules. BISPHOSPHONATES: Bisphosphonates are powerful inhibitors of bone resorption. They constitute a substantial advance in the management of bone manifestation in patients with multiple myeloma. Bisphosphonates not only have a well-established curative effect in patients with tumor-induced hypercalcemia, but also inhibit disease progression in bone. PMID- 10745944 TI - [Idiopathic juvenile arthritis. Current nosological aspects]. PMID- 10745945 TI - [Systemic forms of idiopathic juvenile arthritis: clinical course]. AB - FAVORABLE PROGNOSIS: The prognosis of systemic onset juvenile idiopathic arthritis varies. The most favorable outcome is observed when joint involvement is mild or delayed. No initial clinical or biological feature is indicative of severe prognosis except polyarticular involvement at onset. There is no HLA association. After 10 years follow-up, inflammatory features have disappeared in 50% of the cases, with some disability in 25%. POSSIBLE COMPLICATIONS: Some severe complications can occur during the course of systemic juvenile idiopathic arthritis: secondary amyloidosis (1 to 2%), spontaneous or drug-induced macrophage activation syndrome. Gold salts, sulfasalazine, or nonsteroidal antiinflammatory drugs may be implicated, associated or not with viral infection. Growth retardation is most often secondary to corticosteroid therapy. Long-term prognosis as published in various series mentions 8 to 14% mortality, secondary to iatrogenic accidents, infections or secondary amyloidosis. PMID- 10745946 TI - [Juvenile spondylarthropathy]. AB - EPIDEMIOLOGY: Juvenile spondylarthropathy accounts for about 20% of all cases of chronic juvenile idiopathic arthritis. The spondyloarthropathy concept includes chronic inflammatory rheumatic conditions involving the spine, peripheral joints, and tendon insertions. There is an HLA B27 linkage and the condition predominates in boys, mean age 11 years. CLINICAL PRESENTATION: The usual clinical signs are asymmetrical involvement of the joints of the lower limbs associated in 30 to 50% of the cases with enthesiopathy. The diagnosis is based on the B Amor criteria and ESSG. The clinical course follows an episodic pattern in 80% of the cases. TREATMENT: Nonsteroidal antiinflammatory drugs and local care are used. Sulfasalazine can be useful but its efficacy has not been proven. The functional prognosis is relatively good; spinal ankylosis is uncommon and hip involvement (destructive coxitis) occur in 30% of patients. About 80% of the patients have minor or no disability after a 10-year course. PMID- 10745947 TI - [Juvenile oligoarthritis. A diagnosis not to be ignored]. AB - DIAGNOSIS: Juvenile idiopathic arthritis involving 1 to 4 joints is the most frequent inflammatory chronic rheumatic disorder observed in children. Diagnosis, based on clinical features and laboratory findings may be difficult. Septic arthritis and malignant hematopathy as well as other neoplastic conditions must be ruled out first. Chronic asymptomatic uveitis must be searched for without delay. NATURAL COURSE: The natural course is variable. Prognosis is generally good if the disease remains limited to one joint. Conversely, the development of ocular complications and extension to other joints may lead to less favorable outcome. EARLY MULTIDISCIPLINARY CARE: Rheumatologists, pediatricians, ophthalmologists, pediatric orthopedic surgeons, and physical therapy and psychotherapy specialists all have a role to play. The treatment of choice in case of resistance to nonsteroidal antiinflammatory drugs is local infiltration using long-action fluorinated corticosteroids. Methotrexate is indicated in case of extension to other joints and symmetrical involvement. It is crucial to institute a physical therapy program early because of the risk of induced deformations which may become irreversible. Chronic uveitis, which must be looked for systematically, responds well to local corticosteroids given in a long-term protocol. Early multidisciplinary care is essential for optimal long-term outcome. PMID- 10745948 TI - [Tuberculosis or clear-cell chondrosarcoma of the elbow?]. PMID- 10745950 TI - Doctor migration--Daniel in the lions' den PMID- 10745949 TI - Doctor migration--Daniel in the lions' den. PMID- 10745951 TI - Doctor migration--Daniel in the lions' den. PMID- 10745952 TI - A close, fiery call for Capetonians PMID- 10745953 TI - Pharmaceuticals pull out of SA. PMID- 10745954 TI - New notifiability policy. PMID- 10745955 TI - UN's first health issue. PMID- 10745956 TI - Legalities of the MSA. PMID- 10745957 TI - The laboratory diagnosis of HIV infection. PMID- 10745958 TI - Is CT scan essential in the diagnosis of stroke? PMID- 10745959 TI - Legalizing assistance with dying in South Africa. PMID- 10745960 TI - Perforation of toxic megacolon--the value of the plain radiograph. PMID- 10745961 TI - Effects of DDT pesticide exposure--a reminder! PMID- 10745962 TI - Recommendations pertaining to the use of viral vaccines: influenza. Review of influenza activity--1999. PMID- 10745963 TI - Poverty and health--an open invitation to health professionals. PMID- 10745964 TI - Don't forget the ferruginous bodies. PMID- 10745965 TI - Apolipoprotein E polymorphism in pre-eclampsia. PMID- 10745966 TI - Measles elimination--is it achievable? Lessons from an immunisation coverage survey. AB - OBJECTIVE: To determine routine measles coverage at district level and to explore reasons for immunisation failure in Mpumalanga Province, South Africa. DESIGN: An adaptation of the World Health Organisation (WHO) Expanded Programme on Immunisation (EPI) cluster sampling method was used to make a random selection of 30 clusters in each of 21 health districts, 630 clusters in total. Seven individuals from the age group 12-23 months were randomly selected from each cluster and immunisation status and source were transcribed from their child health records. Where no immunisations were administered, reasons provided by parents or guardians were recorded. SETTING: Mpumalanga Province, South Africa. RESULTS: The weighted valid population coverage with measles vaccine for children aged 12-23 months in Mpumalanga Province was 71.1% (95% confidence interval 64.9 78.5%); this was the lowest of all EPI antigens. There was marked heterogeneity in measles coverage across the province, with a coefficient of variation of 22.2%. Districts with the lowest coverage shared borders with neighbouring provinces. District measles coverage was highly positively correlated with diphtheria, pertussis and tetanus (DPT3) coverage (r = 0.960, P = 0.000). There was a strong negative correlation between ranked measles campaign coverage and routine measles immunisation coverage. Obstacles to immunisation accounted for nearly half (49%) of all reasons for immunisation failure, while lack of information and lack of motivation accounted for 30% and 22%, respectively. CONCLUSIONS: Survey results highlight the need for supplementary immunisation, including non-selective campaigns, if Mpumalanga is to achieve the South African goal of measles elimination by 2002. The value of determining district resolution coverage in order to identify areas with low measles coverage requiring supplementary intervention was also demonstrated. A strong negative correlation between routine and campaign coverage deserves further study in other settings. PMID- 10745967 TI - Lymphoma--histopathology in changing clinical perspective. Non-Hodgkin's Lymphoma Classification Project. AB - BACKGROUND: Lymphoma management has traditionally been dominated by nodal histopathology. Unfortunately, many different classifications coexisted and frequent revisions have often obscured clinical correlations. Some improvement in understanding histogenesis followed the introduction of immunophenotyping, while a number of new entities have been described in the last decade. In addition the whole question of lymphomagenesis is undergoing critical exploration. The use of cellular and molecular biological techniques is therefore shifting focus to the role of oncoproteins and the impact of mutation in the normally modulating suppressor genes. To accommodate these advances the International Lymphoma Study Group has proposed the Revised European-American Lymphoma Classification. While this is an undoubted advance, it has met with persisting concerns regarding applicability to patient management. STUDY SETTING: In determining the extent to which the latter reservation is valid, and at the same time directly testing the clinicopathological value of the new system, a group of acknowledged experts drawn from nine major academic centres worldwide analysed approximately 1,400 previously unreported cases, focusing on outcome. As part of that study 196 consecutive patients seen in Cape Town were separately examined. RESULTS: Findings here were similar to those of the overall experience, although distinct geographical differences emerged. Specifically in the follicular centre-cell lymphomas there was no difference in the 5-year failure-free survival rate, but these neoplasms accounted for 33% of lymphomas, which is similar to North America and London but contrasts with the 14% in the remaining six sites. Also, while mean survival for all types of peripheral T-cell lymphoma was 18% at 5 years, these accounted for 8% of lymphomas locally, as seen also in London and Hong Kong, but exceeding the 3-6% reported elsewhere. Local experience, as in the other eight centres, documented good diagnostic concordance between trained haematopathologists when this classification was used by them all. Furthermore, unusual subtypes were generally well accommodated within this revised system. It should be noted that while histopathological features retain predictive value, they should not be considered the predominant factor. It was concluded that for management decisions to be appropriate, renewed and correct weighting must be assigned to other prognostic variables that include clinical features and markers of tumour biology. SUMMARY: This more enlightened prerequisite is the central goal that underlies optimal treatment outcome, since it determines stratification to appropriate and peer-reviewed protocols. It follows that review of histopathology needs to precede management of all newly diagnosed cases, preferably only by accredited multidisciplinary clinics. The previous anachronism of basing therapy on opinions of non-specialist pathologists, without appropriate review, is unwise. Furthermore, treatment by lone practitioners, or even single specialty groups that lack the discipline to analyse their findings critically and regularly report their updated results, can no longer be considered standard of practice and should be discouraged. PMID- 10745968 TI - Veteran athletes exercise at higher maximum heart rates than are achieved during standard exercise (stress) testing. AB - OBJECTIVE: The stress electrocardiogram (sECG) is routinely used to screen individuals for underlying cardiac pathology before an exercise programme is prescribed. The underlying assumption is that the cardiac responses elicited during the sECG test are similar to those achieved during participation in sporting activities. However, this premise may be incorrect since the physical demands of different modes of exercise vary substantially. DESIGN: Ten veteran league squash players (LSP), 10 social squash players (SSP), 10 league runners (LR), 10 social runners (SR) and 10 sedentary individuals (SED) were recruited for the study. All subjects completed a lifestyle questionnaire, a full medical examination and a routine sECG. Thereafter each subject's heart rate (HR) was monitored on two separate occasions while participating in sporting activity. RESULTS: No sECG exercise-induced abnormalities were observed, although five subjects showed resting abnormalities. Maximal HR during the sECG, and maximal and mean HR during the field tests, were not significantly different between groups. However, maximal HR was significantly higher in all groups during their sporting activities than during stress testing in the laboratory (P < 0.01). CONCLUSIONS: Maximal HR in veteran athletes during specific sporting activities was significantly higher than that attained during a routine sECG. This finding was not sport-specific, nor was it related to the level of competitiveness of the trial participants. These data show that a routine sECG is a submaximal test of exercise performance, and should be interpreted as such. PMID- 10745969 TI - Weight and health status of black female students. AB - OBJECTIVE: To examine black female students for the occurrence of risk factors associated with chronic diseases of lifestyle, namely obesity, hypertension, nicotine usage, dyslipidaemia and compromised mental health (depression). DESIGN: A cross-sectional analytical study design was used. All participants were examined within a period of 3 months during 1994. Weight, height, and hip and waist measurements were taken. Body mass index (BMI), waist-hip ratio (WHR) and waist circumference (WC) were calculated for each subject. Two systolic and diastolic blood pressure readings were taken for each participant. Questionnaires were used to determine specific risk factors related to lifestyle. The Beck Depression Inventory (BDI) was used to measure psychological well-being. Fasting blood samples were collected and analysed for serum lipids and iron status. SETTING: The University of the North in the Northern Province of South Africa. SUBJECTS: A complete data set of sociodemographic information, anthropometric measurements and blood pressure readings, as well as a psychological health test and a medical questionnaire, were obtained from 231 of the 431 first-year female students who attended the university orientation programme. Only students with a complete data set were included in the sample. RESULTS: Eighteen per cent of students were overweight (BMI 25-29.9), 6.5% were obese (BMI > or = 30), and 26.8% were underweight. Mean blood pressure, BMI, WHR and WC increased significantly with age and were highest among the > or = 24-year-olds. Only 1.6% of students had elevated blood pressure, 1.0% smoked and 4.4% took snuff. BMI, WC and WHR were positively correlated with blood pressure and age. Few students had dyslipidaemia (3.8% cholesterol > 5.2 mmol/l). However 14.5% were anaemic (Hb < 11.5 g/dl) and 24.6% had microcytosis (< 80 fl). Nearly one-fifth of students (17.7%) were classified as being moderately to severely depressed. CONCLUSIONS: Black female students younger than 24 years exhibited few risk factors associated with chronic diseases of lifestyle. However in older women (> or = 24 years) there were significant increases in BMI, WHR, WC and blood pressure. A large number of students of all ages exhibited moderate to severe depression and anaemia was prevalent. PMID- 10745970 TI - The impact of a pregnancy confirmation clinic on the commencement of antenatal care. AB - OBJECTIVE: To introduce a pregnancy confirmation clinic as part of antenatal care and to determine whether this would alter the gestational age at which patients commence antenatal care. SETTING: Three municipal antenatal clinics in Atteridgeville and Central Pretoria. METHOD: A pregnancy confirmation clinic was set up at three sites. At the clinic any woman wishing to confirm whether she was pregnant was offered a urine beta-HCG test. If this test was positive, on-site testing for syphilis, anaemia and rhesus status, dipstick testing of the urine, clinical examination and ultrasound examination were performed. Women with abnormal test results were commenced on appropriate treatment immediately and women requiring further medical care or investigation were referred appropriately. RESULTS: The study recruited 382 women, 145 of whom were defaulters from contraception. Half of the women (191) had a positive pregnancy test. The mean presenting gestational age was 12 weeks 4 days (standard deviation 5 weeks, range 5 weeks-25 weeks 2 days). Treatable conditions with the potential to influence pregnancy outcome were identified in 37 of the pregnant women (19.4%) Forty-three of the pregnant women intended to terminate the pregnancy. CONCLUSION: It is possible to shift the commencement of antenatal care to an earlier gestational age. PMID- 10745971 TI - How do South African obstetricians manage hypertensive disorders of pregnancy--a survey. AB - OBJECTIVE: To determine the current management of hypertensive disorders of pregnancy in South Africa. METHOD: A postal questionnaire was sent to 600 South African obstetricians. RESULTS: The response rate was 72% (432/600), with 425 questionnaires suitable for analysis. South African obstetricians disagree on the definitions of various hypertensive disorders of pregnancy. Methyldopa was the antihypertensive used most frequently for the treatment of mild to moderate hypertension (diastolic blood pressure between 90 and 109 mmHg), while intravenous dihydralazine was preferred in severe hypertension (diastolic blood pressure > or = 110 mmHg and proteinuria > or = +2). To stop convulsions in eclampsia, 256 respondents (60%) said they would use diazepam, 28 (11%) said they would continue with a diazepam infusion, and the remaining 228 (89%) preferred magnesium sulphate (MgSO4) to prevent further convulsions. The intramuscular route was the preferred method of administration for MgSO4. In cases of eclampsia, 273 respondents (64%) said they would use intravenous dihydralazine to lower high blood pressure (> or = 160/110 mmHg) and proteinuria; 98 respondents (23%) said they would use methyldopa, 38 (9%) nifedipine, and 8 (2%) apresoline. Eight (2%) said they would not use antihypertensives. In patients with severe pre eclampsia and impending eclampsia, 330 respondents (78%) said they would use MgSO4 as prophylaxis, 46 (11%) diazepam, and 6 (1.4%) phenobarbitone. Forty-three of the respondents did not prescribe prophylactic anticonvulsant therapy. To prevent pre-eclampsia, 247 of the respondents (58%) said they would prescribe low dose aspirin. CONCLUSION: This study demonstrates that South African obstetricians show great uniformity in terms of the treatment of hypertensive disorders of pregnancy. PMID- 10745972 TI - Diagnosis, management and prevention of the common dyslipidaemias in South Africa -clinical guideline, 2000. South African Medical Association and Lipid and Atherosclerosis Society of Southern Africa Working Group. AB - The optimum management of dyslipidaemia requires a comprehensive, diagnostic work up. This, minimally, includes: Characterisation of any hyperlipidaemic disorder present. Identification of additional risk factors so as to assess overall (global) risk of future coronary heart disease (CHD). The global risk is best assessed by a calculation combining the risk factors in the individual. In severe monogenic dyslipidaemias and in patients with confirmed pre-existing CHD the risk is usually high; in most such cases the use of lipid-modifying drugs (LMDs) is indicated. Assessment of psychosocial, economic and educational factors relevant to management. Prevention and cost-effective management of even moderately dyslipidaemic patients require appropriate modification of lifestyle: avoidance of tobacco smoking, participation in regular exercise, and a health-promoting diet. Depending on individual circumstance, vigorous, personalised intervention and expert assistance from dieticians, biokineticists and other health care personnel may determine success. The correct choice of patient for drug treatment is a key therapeutic decision and is best done after full lifestyle modification. Recent evidence confirms that appropriately prescribed LMD therapy can lower morbidity and mortality from CHD as well as all-cause mortality. Patients with the following features are candidates for LMD therapy: have clinical CHD and a low-density lipoprotein cholesterol (LDLC) level > 3.0 mmol/l despite optimum non pharmacological intervention, or suffer from familial hypercholesterolaemia (FH) or equivalent severe, monogenic disorder, or have a 10-year risk of an acute clinical coronary event of > 20% (or > 30% risk if extrapolated to the age of 60 years) owing to the presence of the hyperlipidaemia alone or in combination with contributory risk factors. The ideal target LDLC concentration is < or = 3 mmol/l, but a reduction of at least 45% should be regarded as a minimum target in severe cases who do not reach this goal. Successful therapy requires on-going attention to compliance, therapeutic response and side-effects, and may necessitate adjustment or reinforcement. Concurrent or contributory conditions, such as smoking, hypertension and diabetes mellitus, must also be treated along with the clinically manifest CHD. Severely hyperlipidaemic, complicated or unresponsive high-risk cases should be referred to an appropriate specialist or lipid clinic. Prevention of CHD in the community should be encouraged through public and professional education, the provision of community facilities for exercise and recreation, and legislation directed at reducing the use of tobacco products and ensuring the appropriate labelling of food products. PMID- 10745973 TI - Dietary management of dyslipidaemia clinical guideline. South African Medical Association Dyslipidaemia Nutrition Working Group. PMID- 10745974 TI - Evaluation of the Olympus AU 400 clinical chemistry analyzer. AB - The performance of the Olympus AU 400 clinical chemistry analyzer was evaluated according to the guidelines of the European Committee for Clinical Laboratory Standards. The following analytes were tested: glucose, urea, creatinine, calcium, AST, ALT, CK, LDH, ALP and amylase. The Olympus AU 400 was compared with the Olympus AU 800. Coefficients of correlation showed high correlation between the compared analyzers. Other performances (intra- and inter-assay variation, carry-over and interferences) of the analyzer were satisfactory. PMID- 10745975 TI - Sensitive and specific detection of carcinoembryonic antigen cDNA using the hot start polymerase chain reaction technique. AB - This study evaluated the specificity and sensitivity of hot start polymerase chain reaction (RT-PCR) for identification of carcinoembryonic antigen (CEA) cDNA. Hot Start Storage and Reaction Tubes provide a simple mechanism for synchronizing hot start amplifications without the need for manual intervention. A wax barrier uses a layer of solid wax to separate the retained reagents and the test sample from the bulk of the reagents until the first heating step of automated thermal cycling melts the wax and convectively mixes the two aqueous layers. Wax-mediated hot start PCR greatly increases the specificity and sensitivity of amplifying CEA cDNA. 12 out of 12 samples were positive for CEA cDNA from 10 CEA mRNA positive tumor cells in 10(7) normal cells by hot start CEA PCR, and 8 out of these samples were positive for CEA cDNA by CEA-PCR without hot start. The hot start PCR showed the single specific product band, but PCR without hot start showed the specific product band and the other non-specific product band. We conclude that the hot start PCR technique could sensitively and specifically detect CEA cDNA after reverse transcription of CEA mRNA and minimize primer dimers by amplification. PMID- 10745976 TI - Temperature control management during transport of red blood cells (RBC) over one to several days. PMID- 10745977 TI - Rh blood group antigens--update. PMID- 10745978 TI - Evaluation of chemiluminescence immunoassays for detecting thyroglobulin (Tg) and thyroid peroxidase (TPO) autoantibodies using the IMMULITE 2000 system. AB - The chemiluminescence assays for detection of autoantibodies to thyroid peroxidase (TPO) and thyroglobulin (Tg) implemented on the IMMULITE 2000 system (Diagnostic Products Corporation) were evaluated. These were immunometric assays with antigen-coated beads and monoclonal murine anti-IgG antibodies conjugated with alkaline phosphatase. Precision was satisfactory with an intraassay precision of 5.3-5.5% for anti-Tg and 4.8-5.3% for anti-TPO and an interassay precision of 5.7-7.3% for anti-TPO and 5.2-7.5% for anti-Tg. The lower detection limit was determined as 5 IU/ml for anti-TPO and 2.2 IU/ml for anti-Tg. The average dilution linearities of 102% for anti-TPO and 100% for anti-Tg and the average recovery of 80-127% for anti-TPO and 93-112% for anti-Tg were acceptable. The findings of the tests were compared with the systems from Pharmacia & Upjohn, ORGenTec, Roche Diagnostics, Byk Sangtec Diagnostica and BRAHMS Diagnostica. Taking the respective cutoff value into account, concordance was 87-96% for anti Tg and 87-97% for anti-TPO. Summarizing all results from the different methods revealed a clinical agreement of 95% for anti-TPO and 93% for anti-Tg. A good agreement was found with the IMMULITE anti-TPO and anti-Tg assays, which are closely related as regards method and biochemistry. Regression analysis gave the following results: anti-TPO IMMULITE 2000 vs anti-TPO IMMULITE: anti-TPO IMMULITE 2000 = 0.99 x IMMULITE anti-TPO - 1.43 IU/ml (r = 0.99, n = 144). anti-Tg IMMULITE 2000 vs anti-Tg IMMULITE: anti-Tg IMMULITE 2000 = 0.98 x IMMULITE anti Tg + 1.63 IU/ml (r = 0.99, n = 86). Further age-dependent normal ranges were evaluated. A higher prevalence of elevated autoantibody titers was found for patients older than 50 years. The rate of elevated antibody titer can be reduced by using an age-dependent reference range: < or = 50 years anti-TPO < 35 IU/ml, anti-Tg < 40 IU/ml and > 50 years anti-TPO < 100 IU/ml, anti-Tg < 80 IU/ml. Further samples from clinically diagnosed Hashimoto's thyroiditis and Graves' disease were investigated. The levels of positive anti-Tg values and anti-TPO values accorded with those stated in the literature and were comparable to those measured with a reference assay. In the tested INSTAND e. V. interlaboratory samples there was high-level accordance with the expected clinical results. PMID- 10745979 TI - New PCR primer pairs specific for Candida dubliniensis and detection of the fungi from the Candida albicans clinical isolates in Japan. AB - The genetic patterns of Candida dubliniensis, including type strain, were analyzed by random amplified polymorphic DNA (RAPD) method in comparison with those of reference Candida albicans genotypes A, B and C strains. This RAPD pattern analysis showed that the patterns of two strains of C. dubliniensis including the type strain are very similar to the three oligo PCR primers used, but different from other C. albicans genotypes A, B and C strains with most of the PCR primers tested. The RAPD band considered to be specific for C. dubliniensis in one oligo primer was extracted and sequenced. Based on the sequence information (417-bp), two new PCR primer pairs specific for C. dubliniensis were prepared. The survey of 58 strains of Candida clinical isolates using these newly prepared PCR primer pairs identified as C. albicans in Japan suggested the presence of one strain of C. dubliniensis. The phenotypic characteristics and the sequence analysis of the variable D1/D2 domain of the large subunit (26S) ribosomal DNA of the strain confirmed that it belongs to C. dubliniensis. The usefulness and specificity of these PCR primer pairs for the identification of C. dubliniensis are discussed. PMID- 10745980 TI - Apolipoprotein B-100: employing the electrochemiluminescence technology of the Elecsys systems for the detection of the point mutation Arg(3500)Gln. AB - Apolipoprotein B-100 (apo B-100) plays an essential role in lipoprotein metabolism where it is involved in the clearance of LDL particles from the bloodstream. The mutation Arg3500Gln in the apo B-100 gene impairs the binding of the LDL particles to the LDL receptor, resulting in elevated LDL-cholesterol levels in the blood which, in turn, fuel the development of premature atherosclerosis. Here we describe a rapid, automated test for the detection of the most frequent mutation in the apo B-100 gene. This PCR-based test employs electrochemiluminescence as detection technology and allows the reliable discrimination of all genotypes. The assay has been especially developed for the non-specialized routine clinical chemistry laboratory by employing an analyzer and chemistry often present in this type of labof1tory. Because of its low costs and easy handling the assay can be performed on a daily basis. PMID- 10745981 TI - Precision and comparability of Abuscreen OnLine assays for drugs of abuse screening in urine on Hitachi 917 with other immunochemical tests and with GC/MS. AB - Abuscreen OnLine assays for drugs of abuse screening in urine have recently been developed for use on Hitachi 917 analyzers (Roche Diagnostics GmbH). The assays are based on the kinetic interaction of microparticles as measured by changes in light transmission. Drug in a sample inhibits the formation of particle aggregates and diminishes absorbance change increases. It was the goal of this study to evaluate precision and comparability of the new asssys with CEDIA drugs of abuse tests on Hitachi 917 in different laboratories (three European and three US). The assays were calibrated in the nonlinear mode with four to six standards (semiquantitative application). Initial within-run (21 replicates, four labs) and between-day (10 days, two labs) imprecision studies using Abuscreen OnLine tests and commercial negative (0.5 x cut-off) and positive (1.5 x cut-off) controls revealed the following median CVs [withinrun neg./pos. control/between-day neg./pos. control]: amphetamines 1.9/1.3/3.4/2.4, barbiturates 3.0/1.6/3.9/3.1, benzodiazepines 4.7/1.5/6.3/3.0, cocaine metabolite 1.8/0.9/2.4/1.7, methadone 5.4/1.6/5.5/2.2, opiates 5.5/2.8/5.3/2.7, THC 8.9/4.8/21.8/12.1. CVs < 10% were obtained for the THC test using controls with concentrations closer to the cut off. An identical set of 170 GC/MS analyzed urine samples was distributed to the six laboratories and measured with Abuscreen OnLine tests on Hitachi 917. The median values for each individual sample were calculated and compared with the results obtained on individual Hitachi 917 analyzers by Passing-Bablok regression analysis. A good agreement between the laboratories was found with less than +/- 11% slope deviation and intercepts below 7% of the cut-off except for benzodiazepines (one slope 17%, one slope--26%) and THC (one slope 34%, one slope -18%). The comparability with CEDIA tests was analyzed by concordance plots using randomized routine samples in three laboratories. The following results were obtained in one of the participating laboratories [cut-off ng/mL] (No. of positive/negative/discrepant samples): amphetamines [500] 2/147/0, barbiturates [200] 1/148/0, benzodiazepines [100] 52/91/7, cocaine metabolite [300] 17/129/3, methadone [300] 113/34/2, opiates [300] 31/114/4, THC [50] 66/81/2. GC/MS was performed for clarification of the discrepant results. In summary, Abuscreen OnLine tests on Hitachi 917 give precise results which compare well when analyzed in different laboratories. They can be rated as convenient and flexible methods for drugs of abuse screening in the routine. PMID- 10745982 TI - Reference range and method comparison studies for enzymatic and Jaffe creatinine assays in plasma and serum and early morning urine. PMID- 10745983 TI - The quantitative measurement of autoantibodies to thyroglobulin and thyroid peroxidase by automated microparticle based immunoassays in Hashimoto's disease, Graves' disease and a follow-up study on postpartum thyroid disease. AB - The aim of this study was to investigate the association between various autoimmune thyroid diseases and the presence of anti-TPO and anti-Tg antibodies using two novel automated microparticle based immunoassays developed for the AxSYM analyzer. Serum samples from 65 individuals with Hashimoto's Disease, 38 with Graves' Disease and 80 UK blood donors were assayed. In addition, samples were taken from 50 women known to be positive for TPO antibodies, for up to 24 weeks following delivery. Precision for both assays ranges from 5.7-9.1% CV, while analytical sensitivity was determined to be 1.0 IU/ml for Anti-Tg and 0.3 IU/ml for Anti-TPO. The Anti-TPO test showed positive results in 86% of Hashimoto's Disease and 87% of Graves' Disease. The figures obtained for Anti-Tg were 58% and 73% respectively. Specificity was 94% with Anti-TPO and 99% with Anti-Tg. The postpartum women were divided into 2 groups, group A remained symptomless while group B developed thyroid dysfunction. Within the 2 groups, medians calculated at each time point were compared between and within groups using the Mann-Whitney Rank Sum Test or the Kruskal-Wallis One Way ANOVA on Ranks. Anti-TPO baseline levels (week 6) were statistically different between both groups (median 36 vs. 167 IU/ml, p = 0.002). In group A, the median values increased from 36 to 87 IU/ml within the observation period, although the difference was not statistically significant. In group B, antibody titres showed a statistically significant increase from 168 IU/ml (week 6) up to 676 IU/ml after 20 weeks (p < 0.001). Anti-Tg baseline levels were not statistically different between the two groups. In group A, the median values did not change significantly over time (range: 47-86 IU/ml) whereas antibody titres in group B showed a statistically significant increase from 79 IU/ml (week 6) to 276 IU/ml after 24 weeks (p = 0.002). Results obtained indicate that these assays provide useful tools for the quantitative determination of autoantibodies in both primary diagnosis as shown with the Hashimoto's disease and Graves' disease samples and patient follow-up as demonstrated with the postpartum samples. The automation and high precision of the assays make them perfectly suited to routine diagnostic use. PMID- 10745984 TI - Polymer chromosome models and Monte Carlo simulations of radiation breaking DNA. AB - MOTIVATION: Chromatin breakage by ionizing radiation is relevant to studies of carcinogenesis, tumor radiotherapy, biodosimetry and molecular biology. This article focuses on computer analysis of chromosome irradiation in mammlian cells. METHODS: Polymer physics and Monte Carlo numerical methods are used to develop a coarse-grained computational approach. Chromatin is modeled as a random walk on a cubic lattice, and the radiation tracks hitting the chromatin are modeled as straight lines hitting lattice sites. Each track can make a cluster of DSBs on a chromosome. RESULTS: The results obtained replace conjectured DNA fragment-size distribution functions in the recently developed RLC formalism by more mechanistically motivated distributions. The discrete lattice algorithm reproduces features of current radiation experiments relevant to chromatin on large scales. It approximates the continuous formalism and experimental data with adequate precision. It was also found that assuming either fixed chromatin with correlations among different clusters of DSBs or moving chromatin with no such correlations gives virtually identical numerical predictions. PMID- 10745985 TI - IMAGEne I: clustering and ranking of I.M.A.G.E. cDNA clones corresponding to known genes. AB - MOTIVATION: To enhance the usefulness of the I.M.A.G.E. Consortium (Lennon et al., 1996, Genomics, 33, 151-152) cDNA clone collection by directed analysis and organization of their associated Expressed Sequence Tags (ESTs), thus enabling effective mining of the immense amounts of public cDNA information. RESULTS: This paper introduces the IMAGEne suite of tools, which clusters ESTs around known genes, then ranks each clone within a cluster. IMAGEne filters data from known gene sequence databases and the GenBank's EST database (Boguski and Shuler, 1995, Nature Genet., 10, 369-371). It applies biological criteria in connection with judicious use of the BLAST (Altschul et al., 1990, J. Mol. Biol., 215), FASTA (Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA, 85, 2444-2448; Pearson, 1990, Methods Enzymol., 183, 63-98; Gusfield, 1997, Algorithms on Strings, Trees, and Sequences, Cambridge University Press), and SIM (Huang et al., 1990, Comput. Appl. Biosci., 6, 373-381) tools to form known gene clusters. It then applies criteria derived from experienced biologists to select the best representative I.M.A.G.E. clone for a gene. The tool provides an intuitive Java interface for query and display of the gene and its associated clones, thus directing researchers in selecting a clone that will best enhance their research. An important product is a listing of clones that best represent all known genes. The listing will be used for re-arraying clones into minimally redundant Master Arrays. Both the listings and Master Arrays will be made available to the public, which will be a valuable resource to the genomic community in furthering discovery in the area of gene function. PMID- 10745986 TI - SEGMENT: identifying compositional domains in DNA sequences. AB - MOTIVATION: DNA sequences are formed by patches or domains of different nucleotide composition. In a few simple sequences, domains can simply be identified by eye; however, most DNA sequences show a complex compositional heterogeneity (fractal structure), which cannot be properly detected by current methods. Recently, a computationally efficient segmentation method to analyse such nonstationary sequence structures, based on the Jensen-Shannon entropic divergence, has been described. Specific algorithms implementing this method are now needed. RESULTS: Here we describe a heuristic segmentation algorithm for DNA sequences, which was implemented on a Windows program (SEGMENT). The program divides a DNA sequence into compositionally homogeneous domains by iterating a local optimization procedure at a given statistical significance. Once a sequence is partitioned into domains, a global measure of sequence compositional complexity (SCC), accounting for both the sizes and compositional biases of all the domains in the sequence, is derived. SEGMENT computes SCC as a function of the significance level, which provides a multiscale view of sequence complexity. PMID- 10745987 TI - Segmentation of yeast DNA using hidden Markov models. AB - MOTIVATION: Compositionally homogeneous segments of genomic DNA often correspond to meaningful biological units. Simple sliding window analysis is usually insufficient for compositional segmentation of natural sequences. Hidden Markov models (HMM) with a small number of states are a natural language for description of compositional properties of chromosome-size DNA sequences. RESULTS: The algorithms were applied to yeast Saccharomyces cerevisiae chromosomes (YC) I, III, IV, VI and IX. The optimal number of HMM states is found to be four. The optimal four-state HMMs for all chromosomes are very similar, as well as the reconstructed segmentations. In most cases the models with k + 1 states are obtained by 'splitting' one of the states in the model with k states, and the corresponding increase of the level of detail in segmentation. The high AT states usually correspond to intergenic regions. We also explore the model's likelihood landscape and analyze the dynamics of the optimization process, thus addressing the problem of reliability of the obtained optima and efficiency of the algorithms. PMID- 10745988 TI - Modeling and predicting transcriptional units of Escherichia coli genes using hidden Markov models. AB - MOTIVATION: The hidden Markov model (HMM) is a valuable technique for gene finding, especially because its flexibility enables the inclusion of various sequence features. Recent programs for bacterial gene-finding include the information of ribosomal binding site (RBS) to improve the recognition accuracy of the start codon, using this feature. We report here our attempt to extend the model into the total transcriptional unit, enabling the prediction of operon structures. RESULTS: First, we improved the prediction accuracy of coding sequences (CDSs) by employing the models of 'typical', 'atypical' and 'negative (false-positive)' classes as well as the models of RBS and its downstream spacer. The sensitivity of exactly predicting the 204 experimentally confirmed CDSs reached 90.2% in an objective test. Based on the prediction result of CDSs, the positions of the promoters and terminators were predicted. Our model could exactly recognize 60% of 390 known transcriptional units. Thus, the accuracy and significance of this prediction problem is far from trivial. We would like to propose this problem as an open theme in bioinformatics because the ongoing or planned post-sequencing projects will produce much data for future improvements. PMID- 10745989 TI - On the complexity measures of genetic sequences. AB - MOTIVATION: It is well known that the regulatory regions of genomes are highly repetitive. They are rich in direct, symmetric and complemented repeats, and there is no doubt about the functional significance of these repeats. Among known measures of complexity, the Ziv-Lempel complexity measure reflects most adequately repeats occurring in the text. But this measure does not take into account isomorphic repeats. By isomorphic repeats we mean fragments that are identical (or symmetric) modulo some permutation of the alphabet letters. RESULTS: In this paper, two complexity measures of symbolic sequences are proposed that generalize the Ziv-Lempel complexity measure by taking into account any isomorphic repeats in the text (rather than just direct repeats as in Ziv Lempel). The first of them, the complexity vector, is designed for small alphabets such as the alphabet of nucleotides. The second is based on a search for the longest isomorphic fragment in the history of sequence synthesis and can be used for alphabets of arbitrary cardinality. These measures have been used for recognition of structural regularities in DNA sequences. Some interesting structures related to the regulatory region of the human growth hormone are reported. PMID- 10745990 TI - IMPALA: matching a protein sequence against a collection of PSI-BLAST-constructed position-specific score matrices. AB - MOTIVATION: Many studies have shown that database searches using position specific score matrices (PSSMs) or profiles as queries are more effective at identifying distant protein relationships than are searches that use simple sequences as queries. One popular program for constructing a PSSM and comparing it with a database of sequences is Position-Specific Iterated BLAST (PSI-BLAST). RESULTS: This paper describes a new software package, IMPALA, designed for the complementary procedure of comparing a single query sequence with a database of PSI-BLAST-generated PSSMs. We illustrate the use of IMPALA to search a database of PSSMs for protein folds, and one for protein domains involved in signal transduction. IMPALA's sensitivity to distant biological relationships is very similar to that of PSI-BLAST. However, IMPALA employs a more refined analysis of statistical significance and, unlike PSI-BLAST, guarantees the output of the optimal local alignment by using the rigorous Smith-Waterman algorithm. Also, it is considerably faster when run with a large database of PSSMs than is BLAST or PSI-BLAST when run against the complete non-redundant protein database. PMID- 10745991 TI - Post-processing long pairwise alignments. AB - MOTIVATION: The local alignment problem for two sequences requires determining similar regions, one from each sequence, and aligning those regions. For alignments computed by dynamic programming, current approaches for selecting similar regions may have potential flaws. For instance, the criterion of Smith and Waterman can lead to inclusion of an arbitrarily poor internal segment. Other approaches can generate an alignment scoring less than some of its internal segments. RESULTS: We develop an algorithm that decomposes a long alignment into sub-alignments that avoid these potential imperfections. Our algorithm runs in time proportional to the original alignment's length. Practical applications to alignments of genomic DNA sequences are described. PMID- 10745992 TI - Browsing protein families via the 'Rich Family Description' format. AB - MOTIVATION: Multiple alignments of protein sequences are the basis of structural and functional analysis of protein families. It is however difficult even for an expert biologist to comprehend an alignment of more than 50 to 100 homologous sequences. RESULTS: This paper presents a browser for the analysis of multiple alignments of large numbers of protein sequences. Phylogenetic trees and consensus sequences are computed and used to summarise the alignments; these data are stored in a structure called Rich Family Description. Summary alignments and trees are displayed in HTML pages and can be developed or reduced by the user. This browser is used to display the ProDom domain families on the Web. Its zooming facilities allow extracting information from alignments of more than 1000 homologous sequences. PMID- 10745993 TI - PHYSEAN: PHYsical SEquence ANalysis for the identification of protein domains on the basis of physical and chemical properties of amino acids. AB - MOTIVATION: PHYSEAN predicts protein classes with highly variable sequences on the basis of their physical, chemical and biological characteristics such as diverse hydrophobicity, structural propensity and steric properties. These characteristics, calculated from multiple positions in a sequence, may be conserved even between sequences that fail to produce alignments at any acceptable level of statistical significance. PHYSEAN complements methods that require sequence alignments (BLAST, FASTA, dynamic programming) by adding less residue- and position-specific physicochemical information on the protein or the domain. RESULTS: We predict proteins or their domains like signal peptides using physical, chemical, geometric, and biological properties of the 20 amino acids. This comprehensive set of properties may cover the diagnostic functional and structural aspects of a domain or a protein class. We automatically select and weight a subset of properties so as to discriminate between, e.g., signal peptides and amino-termini of cytosolic proteins with the lowest number of incorrect predictions. This optimal selection of properties and their weights significantly decreases the number of incorrect predictions as compared to any single property or any combination of unweighted properties. Weights have been optimized by high-performance linear programming models that systematically find the optimal solution from among an astronomic number of property/weight combinations. PHYSEAN's performance is demonstrated by highly accurate predictions of signal peptides (the vehicles for protein transport across membranes) and their cleavage sites. The results indicate reliable predictions are possible even in the lack of sequence conservation using an automated physical and chemical analysis of proteins. PMID- 10745994 TI - Decision tree-based formation of consensus protein secondary structure prediction. AB - MOTIVATION: Prediction of protein secondary structure provides information that is useful for other prediction methods like fold recognition and ab initio 3D prediction. A consensus prediction constructed from the output of several methods should yield more reliable results than each of the individual methods. METHOD: We present an approach that reveals subtle but systematic differences in the output of different secondary structure prediction methods allowing the derivation of coherent consensus predictions. The method uses a machine learning technique that builds decision trees from existing data. RESULTS: The first results of our analysis show that consensus prediction of protein secondary structure may be improved both quantitatively and qualitatively. PMID- 10745995 TI - Ontology for immunogenetics: the IMGT-ONTOLOGY. AB - MOTIVATION: IMGT, the international ImMunoGeneTics database (http:@imgt.cines.fr:8104), created by M.-P. Lefranc, is an integrated database specializing in antigen receptors (immunoglobulins and T-cell receptors) and major histocompatibility complex (MHC) of all vertebrate species. IMGT accurate immunogenetics data are based on the standardization of the biological knowledge provided by the 'ImMunoGeneTics' IMGT-ONTOLOGY. The IMGT-ONTOLOGY describes the classification and specification of terms needed for immunogenetics and bioinformatics. IMGT-ONTOLOGY covers four main concepts: 'IDENTIFICATION', 'DESCRIPTION', 'CLASSIFICATION' and 'OBTENTION'. These concepts allow an extensive and standardized description and characterization of immunoglobulin and T-cell receptor data. The controlled vocabulary and the annotation rules are indispensable to ensure accuracy, consistency and coherence in IMGT. IMGT ONTOLOGY allows scientists and clinicians to use, for the first time, identical terms with the same meaning in immunogenetics. It provides a semantic repository that will improve interoperability between specialist and generalist databases. PMID- 10745996 TI - A plant calmodulin-binding motor is part kinesin and part myosin. PMID- 10745997 TI - Shufflet: shuffling sequences while conserving the k-let counts. AB - Shufflet is a program and a web-application that generates fast random shufflings of sequences (DNA, protein or others), conserving the exact k-let counts for a given k. The sequences are sampled uniformly from all the valid permutations. PMID- 10745998 TI - ModBase: a database of comparative protein structure models. AB - ModBase is a database of evaluated and annotated comparative protein structure models. The database also includes fold assignments and alignments on which the models were based. PMID- 10745999 TI - A platform for integrating threading results with protein family analyses. AB - We have developed a package for the interactive visualization of results from different threading programs. Additionally, we have integrated relevant information about protein sequence, function, evolution, and structure into the interface. PMID- 10746000 TI - Sausage: protein threading with flexible force fields. AB - Sausage is a protein sequence threading program, but with remarkable run-time flexibility. Using different scripts, it can calculate protein sequence-structure alignments, search structure libraries, swap force fields, create models from alignments, convert file formats and analyse results. There are several different force fields which might be classed as knowledge-based, although they do not rely on Boltzmann statistics. Different force fields are used for alignment calculations and subsequent ranking of calculated models. PMID- 10746001 TI - Representation of functional information in the SWISS-PROT data bank. AB - Functional information in SWISS-PROT results, primarily, from assessment of articles reporting characterization. Predicted information is labeled with flags describing the evidence level (e.g. potential, probable, by similarity). PMID- 10746002 TI - WebPHYLIP: a web interface to PHYLIP. AB - A web interface to PHYLIP (version 3.57 C) is implemented using CGI/Perl programming. It enables users to do phylogenetic analysis through the Internet. PMID- 10746003 TI - [Type 2 diabetes and large vessel disease]. AB - Meal and post-meal period define the post-prandial state, which overall covers a little bit more than half daytime period. The post-prandial state is characterized by transient plasma variations in carbohydrates, lipids and hormones in direct relation with nutrient intake. These variations have an impact on the metabolism of several tissues. The post-prandial state in type 2 diabetic patients is considered as a crucial period, mainly due to the tissular toxicity of glucose. The established links between hyperglycemia and the rise of triglycerides-enriched particles can favor atherogenesis. PMID- 10746004 TI - [Mechanisms of action of repaglinide at a cellular level]. AB - In type 2 diabetic patients, the important post-prandial hyperglycemic peak combined with the defective insulin secretion emphasizes the need for restoring a physiological insulin profile during meals, characterized by insulinemia peaking within 1 hour and returning to basal levels within 4 hours. During type 1 diabetes mellitus, short acting insulin analogs aim at counteracting on post prandial hyperglycemic peak. During type 2 diabetes mellitus, repaglinide is the first fast-acting oral antidiabetic drug able to stimulate endogenous insulin secretion during meal by mimicking physiological insulin secretion pattern. PMID- 10746005 TI - [Early changes in the function of pancreatic beta cells and insulin pulsatility as predictors of type 2 diabetes]. AB - Type 2 diabetes mellitus is characterized by a defective insulin secretion and a reduction in insulin action on its targets. These possibly genetically-driven primary defects combine with secondary long term hyperglycemia and metabolic abnormalities. Whereas type 1 diabetes mellitus is clearly linked to a reduction in beta-cell mass, type 2 diabetes mellitus appears as secondary to an hereditary beta-cell dysfunction. Indeed, insulin resistance does not stand as the sole cause for type 2 diabetes, as suggested by cases of severe insulin resistance in diabetes-free, non aged, obese or acromegalic patients, as well as in patients treated with steroids. In fact, type 2 diabetes results from an inappropriate insulin secretion in regards with body needs. PMID- 10746006 TI - [Insulin secretion and repaglinide]. AB - The natural history of type 2 diabetes involves a progressive pancreatic beta cell dysfunction leading to quantitative, qualitative and/or temporal abnormalities in insulin secretion and insensitivity to insulin action which predominates in muscles. These abnormalities can be observed during early phases of glucose intolerance, but their determinism remains unclear. The high prevalence of type 2 diabetes, its increasing incidence among developed countries and the huge cost induced by diabetic complications explain why this disease is being viewed as a major public health issue. An earlier diagnosis by general practitioners and more intensive treatments are urged for patient and social beneficial outcome. In addition to non pharmacological (dietary, physical activity) approaches, several drugs were established as efficient therapies for type 2 diabetic patients: sulfonylureas acting by enhancing insulin secretion, metformin improving insulin resistance, or acarbose delaying carbohydrate intestinal absorption. These drugs have been used during the UKPDS trial; nevertheless, their effect was somehow limited, when considering long-term blood glucose control, risk for hypoglycemia, and/or prevention of macroangiopathy. The new generation of insulin secretion enhancers, including repaglinide, by allowing a reduction of total insulinemia, potentiating nutrient-induced insulin secretion, and minimizing risks for hypoglycemia, raises hope for a progress. PMID- 10746007 TI - [Is the regulation of prandial glucose important in type 2 diabetes?]. AB - Blood glucose reaches its highest concentration during the post-prandial period, exposing type 2 diabetic patients to high risks for cellular and tissular damage, as shown by numerous experimental data obtained on tissue cultures and as evidenced by chronic hyperglycemia-induced complications in type 2 diabetic patients. Glucose is the main physiological energy supplier in both animal and vegetal reigns and stands as an indispensable nutrient for the maintenance of life. Its toxicity may look paradoxical, but also characterizes most other cell energy sources, i.e., oxygen, carbon oxide, fatty acids or amino acids. Glucose reactive properties are being used in plasma glucose concentration and non enzymatic glycosylation assays. Most attention in type 2 diabetes care is currently focused on the deleterious effects of post-prandial hyperglycemia peaks on endothelium, as shown in vitro. Current approaches aim at restoring physiological post-prandial insulinemia profile. PMID- 10746008 TI - [The impact of regulation of postprandial glucose in practice]. AB - Meal and post-meal periods define a post-prandial state covering about 66% of day duration and characterized by a glucose and other nutrients influx into the blood flow. Post-prandial hyperglycemia can be an early feature of glucose intolerance, indicating a failure to control for post-prandial glucose load, but mostly characterizes type 2 diabetes, reflecting quantitative and/or qualitative abnormalities in insulin secretion by pancreatic beta cells. Post-prandial hyperglycemia contributes to raise glycated hemoglobin (HbA1c) level, which as an indicator of total glycemic load, is tightly correlated with the incidence of micro- and macroangiopathy in type 2 diabetes. It can induce or deteriorate fasting hyperglycemia and be associated with coagulation activation and/or lipid metabolism abnormalities, the latter being considered as cardiovascular risk factors, even in non diabetic populations. Therefore caring for post-prandial glucose regulation is particularly relevant in glucose intolerant and type 2 diabetic patients. Accordingly, several pharmacological treatments have been designed, among which repaglinide recently emerged as an efficient, safe and convenient regulator of post-prandial glycemia. PMID- 10746009 TI - [Repaglinide in combination therapy in type 2 diabetes]. AB - In addition to dietary and lifestyle changes, standing as the cornerstone of type 2 diabetes care, pharmacological treatments, whether as single or multidrug patterns, are often necessary for an efficient blood glucose control. Besides insulin, four different oral antidiabetic drug categories are available, each of them acting through different and potentially synergistic ways. Oral antidiabetic drugs include: 1) biguanides acting through the reduction of hepatic glucose production and are most efficient in obese patients; 2) alpha-glucosidase inhibitors delaying carbohydrate intestinal absorption; 3) thiazolidinediones or "glitazones", acting as insulin sensitizers; 4) insulin secretion enhancers, mainly including sulfonylureas, which increase insulin secretion and are being credited by a long clinical usage; these are now joined by the new generation of insulin secretion enhancers, led by repaglinide, which can mimic the physiological insulin secretion profile by a specific stimulatory effect on beta cells characterized by its fast onset and short half-life. Obviously, the combination of these different antidiabetic drugs, by targetting different synergistic and additive pathways, can help to further improve blood glucose. PMID- 10746010 TI - Solid-phase synthesis of a combinatorial array of 1,3-bis(acylamino)-2-butanones, inhibitors of the cysteine proteases cathepsins K and L. AB - To more rapidly prepare members of the 1,3-bis(acylamino)-2-butanone class of cysteine protease inhibitors, a solid-phase synthesis was developed. 1-Azido-3 amino-2,2-dimethoxybutane (4), which has the two amino groups differentiated and the ketone protected as a a ketal, served as a surrogate for the 1,3-diamino-2 butanone core. Amine (4) was coupled to the BAL-resin-linked carboxylic acids derived from alpha-amino acid esters. Evaluation of a small combinatorial array by measuring inhibition constants (Ki,appS) against cathepsins K, L, and B provided some structure-activity relationship trends with respect to selectivity and potency. Novel, potent inhibitors of cathepsins K and L were identified. PMID- 10746011 TI - Solid-phase synthesis of cyclic imides. AB - A cyclative cleavage strategy for the synthesis of cyclic imides on a polystyrene resin is described. After optimization of the cleavage conditions, a small array of succinimides and phthalimides was synthesized. The methodology was then applied to a drug discovery project, in which it was used to synthesize a new class of delta-opioid receptor ligand by both automated and manual methods. PMID- 10746012 TI - Parallel personal comments on "classical" papers in combinatorial chemistry. PMID- 10746013 TI - Beyond mere diversity: tailoring combinatorial libraries for drug discovery. AB - Combinatorial library design attempts to choose the best set of substituents for a combinatorial synthetic scheme to maximize the chances of finding a useful compound, such as a drug lead. Initial efforts were focused primarily on maximizing diversity, perhaps allowing some bias by the inclusion of a small, fixed set of pharmacophoric substituents. However, many factors besides diversity impact good library design for drug discovery. A library can be better "tailored" by assigning the candidate substituents to categories such as polar, pharmacophoric, rigid, low molecular weight, and expensive. Stratified sampling by successive steps of D-optimal design generates diverse designs which are also consistent with desirable profiles of these properties. Comparing the diversity scores among design profiles reveals the tradeoffs between diversity, physical property distributions, synthetic difficulty, expense, and pharmacophoric bias. The diversity scores can be calibrated by scoring the best designs from subsets of the candidates made either from specific classes of substituents or by randomly eliminating candidates. This procedure shows how poor random designs are compared even to highly biased optimal designs. Library design requires a synergistic effort between computational and synthetic medicinal chemists, so specialized interactive software has been developed to integrate substructure searching, display, and statistical experimental design to facilitate this interaction for the effective design of well-tailored libraries. PMID- 10746014 TI - A knowledge-based approach in designing combinatorial or medicinal chemistry libraries for drug discovery. 1. A qualitative and quantitative characterization of known drug databases. AB - The discovery of various protein/receptor targets from genomic research is expanding rapidly. Along with the automation of organic synthesis and biochemical screening, this is bringing a major change in the whole field of drug discovery research. In the traditional drug discovery process, the industry tests compounds in the thousands. With automated synthesis, the number of compounds to be tested could be in the millions. This two-dimensional expansion will lead to a major demand for resources, unless the chemical libraries are made wisely. The objective of this work is to provide both quantitative and qualitative characterization of known drugs which will help to generate "drug-like" libraries. In this work we analyzed the Comprehensive Medicinal Chemistry (CMC) database and seven different subsets belonging to different classes of drug molecules. These include some central nervous system active drugs and cardiovascular, cancer, inflammation, and infection disease states. A quantitative characterization based on computed physicochemical property profiles such as log P, molar refractivity, molecular weight, and number of atoms as well as a qualitative characterization based on the occurrence of functional groups and important substructures are developed here. For the CMC database, the qualifying range (covering more than 80% of the compounds) of the calculated log P is between -0.4 and 5.6, with an average value of 2.52. For molecular weight, the qualifying range is between 160 and 480, with an average value of 357. For molar refractivity, the qualifying range is between 40 and 130, with an average value of 97. For the total number of atoms, the qualifying range is between 20 and 70, with an average value of 48. Benzene is by far the most abundant substructure in this drug database, slightly more abundant than all the heterocyclic rings combined. Nonaromatic heterocyclic rings are twice as abundant as the aromatic heterocycles. Tertiary aliphatic amines, alcoholic OH and carboxamides are the most abundant functional groups in the drug database. The effective range of physicochemical properties presented here can be used in the design of drug-like combinatorial libraries as well as in developing a more efficient corporate medicinal chemistry library. PMID- 10746015 TI - Affinity NMR: decoding DNA binding. AB - We have shown that affinity NMR can be used to edit a NMR spectrum so that ligands that have affinity to DNA can be observed in the presence of other nonbinding molecules. Diffusion encoded spectroscopy (DECODES) can be used to identify the binding ligands. We were able to identify Hoechst 33342 as binding to the Drew-Dickerson dodecamer d(CGCGAATTCGCG)2 in the presence of the nonbinding molecules adenine, adenosine, and thiamine. Affinity NMR appears to be readily applicable to DNA systems for the following reasons. (1) The relaxation rate of the DNA oligonucleotides is favorable, thus the signal intensity loss due to relaxation is not severe. (2) A comparison of the patterns of the DNA cross peaks upon binding in the two-dimensional total correlation spectroscopy and correlation spectroscopy spectrum are easily performed, and the ligand signals in the two-dimensional DECODES spectrum can be readily identified. (3) The aromatic part of the DNA spectrum is devoid of 2D cross-peaks in these correlation spectra, greatly facilitating the interpretation of the bound ligand in the DECODES spectrum. PMID- 10746016 TI - Determining affinity-selected ligands and estimating binding affinities by online size exclusion chromatography/liquid chromatography-mass spectrometry. AB - Size exclusion chromatography (SEC) isolation of affinity-selected ligands combined with reverse phase liquid chromatography-mass spectrometry (LC-MS) is an effective means for identifying members of mixtures which form tightly bound noncovalent complexes with target proteins. A potential liability of the approach is that the SEC isolation is carried out under nonequilibrium conditions favoring protein/ligand complex dissociation. At long SEC isolation times and/or for complexes with fast off-rates the extent of dissociation can jeopardize the ability to detect the affinity-selected components. Additionally, equilibrium binding affinities cannot be exactly determined from the measured distribution of isolated ligands. We present here an online SEC/LC-MS system for determining affinity-selected members of active mixtures which reduces this liability. A kinetic model of the SEC isolation process is developed to determine the practical limits for the application of the method and to extrapolate equilibrium binding affinities from the nonequilibrium data. The utility of online SEC/LC-MS for identifying affinity-selected ligands and for estimating binding affinities is demonstrated for a small molecule mixture of compounds with known binding affinities and for a simple combinatorial mixture. PMID- 10746017 TI - A combined parallel synthesis and screening of macrocyclic lanthanide complexes for the cleavage of phospho di- and triesters and double-stranded DNA. AB - A parallel synthesis of macrocyclic lanthanide-ligand complexes 4Ln has been developed in conjunction with a parallel screening of these ligands for catalysis of phosphate ester hydrolysis. Complexes 4Ln were screened on a 96-well plate reader for their ability to catalyze the hydrolysis of a variety of phosphate esters efficiently. The hydrolysis of bis(4-nitrophenyl) phosphate (BNPP) 5 and p nitrophenylethyl phosphate 6 was accelerated by up to 150-fold in the presence of the complex 4cGd. The cleavage of a double-stranded DNA plasmid with this same complex obeyed saturation kinetics following a Michaelian model (K(m) = 7.4 microM, kcat = 4.5 x 10(-3) min-1). Our findings demonstrate how a combination of parallel synthesis and screening can expedite compound access, accelerate catalyst identification, and thereby dramatically increase the speed of finding good ligand-metal combinations. PMID- 10746018 TI - A combinatorial approach to recognition of chirality: preparation of highly enantioselective aryl-dihydropyrimidine selectors for chiral HPLC. AB - A parallel library of 108 4-aryl-1,4-dihydropyrimidine (DHPM) enantiomers, which are potential selectors for chiral HPLC separations, was synthesized using the single-step Biginelli multicomponent condensation. The individual compounds were screened by observing the enantioselectivity for resolution on a "brush-type" L (3,5-dinitrobenzoyl)leucine-based chiral stationary phase, and separation factors alpha up to 12 were achieved. The best candidates from the library contained an ortho-substituted aromatic group at C4 carbon atom of the pyrimidine ring and an alkyl substituent at N1 nitrogen atom. Resolution of the enantiomers of the lead compound, 4-(9-phenanthryl)-DHPM 8, using semipreparative chiral HPLC followed by attachment to monodisperse macroporous aminomethacrylate beads, provided the novel polymer based chiral stationary phase with good enantioselectivities in the resolution of several pi-acidic aryl-dihydropyrimidines and derivatized profens. In addition, 3,5-dinitrobenzamido derivatives of alpha-amino acids could be resolved under normal phase HPLC conditions with separation factors up to 8. PMID- 10746019 TI - [Cerebral ischemia and neuroprotection from the viewpoint of the neurosurgeon]. AB - Cerebral ischemia is a frequent and dangerous consequence of some cerebrovascular diseases. Ischaemia may be used also electively in the course of neurosurgery. Therefore new possible ways are sought how to reduce the danger of ischaemia or to prevent it. In the submitted paper some methods of neuroprotection are described and their potential or actual applicability in clinical neurosurgery are discussed. In addition to influencing the brain cell and pretentious methods in the sphere of molecular biology and genetics the induction of systemic hypertension supplemented alternatively by other methods such as hypothermia, the use of mannitol, haemodilution and hypervolaemia seem natural. A higher blood pressure helps to make leptomeningeal and cortical anastomoses patent and strengthens the collateral circulation from marginal zones of ischaemia, penumbra to the ischaemic centre and to prevent thus cerebral infarction. PMID- 10746020 TI - [Adverse effects of administration of intravenous human immunoglobulins]. AB - Treatment with intravenous human immunoglobulin (IVIG) has become a routine therapeutic method in immunodeficiency states and autoimmune diseases. Although it is a relatively safe therapeutic method it may have serious undesirable effects. Knowledge of these undesirable effects is the prerequisite for coping with them and in some instances it is possible to prevent them. Undesirable effects of IVIG administration can be divided into six groups: 1. Generalized reaction, in particular fever, shiver, nausea, vomiting, tachycardia, dyspnoea, changes of blood pressure are recorded in less than 5% patients, usually during infusion and depend on the rate of administration. 2. Hypersensitivity and anaphylactic reactions may be also severe to fatal and are usually the manifestation of the action of antibodies against IgA; they may be anticipated in particular in patients with deficiency of class A immunoglobulins and in patients with autoimmune diseases. 3. Haematological: rare and usually clinically irrelevant haemolytic anaemia. 4. Neurological: frequent and minor headache, rarely relapsing aseptic meningitis syndrome. 5. Nephrological: renal failure which developed by the mechanism of osmotic nephrosis, relatively very rare, affecting almost exclusively patients with nephropathy present before administration of IVIG. 6. Thrombotic complications manifested by cerebral ischaemia. They are however extremely rare and their relationship to IVIG administration is controversial. At present we can rule out transmission of viral infection by IVIG preparations with the exception of transmission of the hepatitis C virus. PMID- 10746021 TI - [Effect of folates, vitamin B12 and life style factors on mild hyperhomocysteinemia in a population sample]. AB - BACKGROUND: Mild hyperhomocysteinaemia (MHHcy) is a significant and independent risk factor for vascular diseases, however, its causality has not yet been unequivocally confirmed. The total homocysteine (Hcy) blood level is considered a product of genetic and lifestyle interactions, mainly folates, vitamin B12 and pyridoxine intake. In this paper we estimated the influence of these factors on MHHcy in the population. METHODS AND RESULTS: The population sample included 292 males a 251 women, mean age 53.4 years, selected from the population study PILS II. All subjects were examined by a standard protocol for clinical, anthropometrical and laboratory examination. Hcy levels were examined by ion exchange chromatography, all other factors by commercial kits. Statistical analysis was done in quartiles of distribution by Kruskal-Wallis ANOVA, Wilcoxon's un-paired test and multiple logistic regression (stepwise). Serum total Hcy levels were in significant positive associations with age in both sexes, with BMI in males only. Negative associations of Hcy were found with plasma folates and B12 concentrations in both sexes, with alcohol consumption again only in males. Smoking and physical activity and serum methionine concentration were not associated with Hcy levels. The established associations remained significant when adjusted by multiple logistic regression. About 40% of subjects with MHHcy had low folates and/or B12 levels and a deficiency in both vitamins was found in 17% of subjects. In contrast, MHHcy also was assessed in 14% of subjects with high folates and in 17% with high B12 concentration and in as few as in 3.7% of subjects with high concentrations of both vitamins as well. CONCLUSIONS: Nutritional factors, i.e. folates and B12 intake, seem to be the most important ones responsible for Hcy levels. A predominating influence of genetic factors may be assumed in less than one fifth of subjects with MHHcy. PMID- 10746022 TI - [Cystic fibrosis--a disease of adolescents and adults?]. AB - BACKGROUND: Cystic fibrosis (CF) is no longer a childhood disease. Since the identification of the gene in 1989 research has made advances and changed views on the pathogenesis, diagnosis and treatment. The objective of the present work is to make doctors treating adult patients familiar with modern therapeutic methods and their value. METHODS AND RESULTS: In the CF Centre of the Faculty Hospital in Prague Motol 349 patients are followed up on a long-term basis, incl. 95 who died since 1985. Hundred and twenty six (36.1%) patients survived to the age of 18 years, of those 41 died and 85 patients live. Comparison of semilongitudinal data of a group of 83 patients born before 1975 whose treatment during childhood and puberty was inadequate and 196 patients born in 1976-90 treated by modern methods proved the great effect of treatment on the course and prognosis of the disease. The median age at death increased during from 12.2 years in 1985-90 to 18.8 years in 1991-1998 (p = 0.004). The nutritional status of adult patients is satisfactory in 40.4%, poor in 33.3% and marginal in 26.3%. A normal pulmonary function was recorded in 17.5%, 22.8% are severely affected, the majority of patients (59.7%) has values within 40 to 80% of normal levels. CONCLUSIONS: Modern intensive treatment improved the prognosis and quality of live in patients with CF. Critical deterioration of the clinical condition shifted to the threshold of adult age. It is therefore essential that doctors treating such patients should be familiar with this issue. PMID- 10746023 TI - [Postmortem diagnosis of Fabry disease in a female heterozygote leading to the detection of undiagnosed manifest disease in the family]. AB - The authors detected on necropsy in a 63-year-old woman with the clinical diagnosis of hypertension, atherosclerosis of the coronary and peripheral arteries, thromboembolism into the cerebral circulation and impaired cardiac conductivity lysosomal storage identified by histochemical and electronoptic analyses along with lipid chromatography as Fabry's disease. The stored lipids were neutral glycosphingolipids of the globo series globotriaosylceramide) and of the gala- series (galabiosylceramide) which accumulated as a result of deficient activity of the degrading enzyme alpha galactosidase A. Marked accumulation of these specific lipids was found in cardiomyocytes, in smooth muscles (of the media in arteries of the heart, kidneys, liver, spleen, lungs) in podocytes and mesangial cells of renal glomeruli, in epithelia of Henle's loop and in the distal tubules. In the vascular endothelium the storage was at the borderline of detectability. Accumulation did not lead to detectable organ disorders with the exception of the heart where it participated, no doubt, significantly in the cardiocyte hypertrophy. Examination of relatives revealed in the proband's son (age 41 years) a combination of renal, cardiac and skin changes typical for Fabry's disease which, however was not clinically diagnosed. The diagnosis was confirmed by proving of alpha-galactosidase A deficiency in the peripheral leucocytes and point mutation L293X in the VIth exon of the appropriate gene. In a granddaughter (age 15 years) biochemical and molecular genetic methods revealed the heterozygous state of Fabry's disease in preclinical stage. PMID- 10746024 TI - [Percutaneous localization and marking of small solitary pulmonary nodules in CT imaging before video thoracoscopy surgery]. AB - BACKGROUND: The localization of small lung nodules by palpation is not possible when videothoracoscopic surgery is performed. Transparietal fine needle biopsy is frequently not successful in small lung nodules. METHODS AND RESULTS: The authors describe their experiences with percutaneous CT controlled marking of small lung parenchyma around small lung nodules by patent blue and contrast medium mixture. The method was used in 7 patients with nodule size from 7 mm to 25 mm. In all patients the surgeon was able to localize the nodule. Operation followed localization as soon as possible, the interval was from 60 to 120 minutes. Four of 7 nodules were benign, 3 nodules were malignant. CONCLUSIONS: New method of marking of small lung nodules makes a minimal invasive operation possible. The operation stress is lower and the time of hospitalization is shorter. PMID- 10746025 TI - [Treatment and prevention of gastrointestinal complications caused by non steroidal antiphlogistic agents]. AB - The aim of our study is to give a survey of the most efficient methods for treatment and prevention of non-steroidal anti-inflammatory drug (NSAID)-induced gastrointestinal adverse in cases when antiphlogistic treatment cannot be discontinued due to active and progressive joint disease. Analysis of published studies shows that, the proton pump inhibitors (omeprazole) are the most efficient agents in treatment of gastric and duodenal ulcers induced by NSAIDs. The analysis shows a reliable effect of prostaglandin analogues (misoprostol) as well. Prostaglandin analogues (misoprostol) proved the most effective in treatment of gastric erosions. Prophylaxis of adverse gastrointestinal mucosal abnormalities can be primary or secondary. Secondary prevention is intended for patients with gastrointestinal symptoms or those treated for mucosal defects (ulcer, erosions). The standard prevention using H2-antagonists or sucralphate does not provide sufficient protection against NSAID in these patients, but omeprazole reduces the chance of a peptic lesion relapse. Primary prevention is intended for patients with a higher risk of gastrointestinal complications (age above 60, history of peptic ulcer, a higher dose of NSAID, simultaneous treatment with glucocorticoids or anticoagulants). Diclofenac with misoprostol and nabumetone reduce the incidence of gastroduodenal ulcers and their complications in short-term as well long-term studies. Meloxicam reduces the incidence of gastroduodenal mucosal abnormalities is short-term studies. Nimesulide is associated with a lower incidence of adverse gastrointestinal events, but the fact is that, reliable data on gastroduodenal ulcer incidence reduction or their complications are not available. PMID- 10746026 TI - [The Th1/Th2 paradigm. The role of the Th1 and Th2 lymphocyte subpopulations in the regulation of immune processes]. AB - A short report on the interaction of Th1 and Th2 lymphocyte subpopulations in the regulation of immune processes is reviewed. Th1 and Th2 subsets have been characterized on the basis of cytokines they secrete and the immune functions they mediate and also on the basis of cytokine and chemokine receptor expression. The clinical and diagnostic influence of immune processes in pathological stages is also mentioned. PMID- 10746027 TI - [Prevalence of genetic variants of luteinizing hormone in the Czech Republic]. AB - BACKGROUND: The relatively common genetic variant of LH beta-subunit, resulting in changes of the pituitary-gonadal axis, displays different prevalence in various ethnic populations. METHODS AND RESULTS: Frequency of occurrence of the variant allele in our country has been determined by analysis of LH in a randomly selected population group from the Cheb region, comprising of 257 subjects (82 men and 175 women) in the age from 14 to 72 years. The LH variant was determined by a novel immunofluorescence method, based on two assays, of which the first detected the wild type of the hormone and its variant, while the second was specific for the wild type of LH only. The frequency of carriers of the variant allele for LH-beta-subunit amounted to 17.5%, i.e. 12.2% in males and 20.6% in females. CONCLUSIONS: The prevalence of the variant allele for LH-beta-subunit in the investigated region of West Bohemia was close to that of other Antral European and North European populations. PMID- 10746028 TI - [Administration of interferon alpha as post-transplantation immunotherapy in malignant lymphoma. Case control study]. AB - BACKGROUND: A possible cause of relapses in patients with malignant lymphomas after autologous transplantation of haematopoietic cells is among others the absence of an immune reaction. The objective of the present study was to assess the effect of long-term interferon alpha administration after autologous transplantation in malignant lymphomas. METHODS AND RESULTS: A total of 16 patients were followed up (9 men and 6 women, mean age 44 years) 14 with non Hodgkin's lymphoma and two with Hodgkin's disease where after high-dose transplantation with autologous transplantation of haematopoietic cells immunotherapy was started by administration of IFN alpha 3 MU three time per week. The treatment was well tolerated, only in two patients (12.5%) the doses had to be reduced or temporarily discontinued. The results were compared with a control group (16 subjects, 12 men, 4 women, mean age 43 years and similar other characteristics as the followed up group) who were not given IFN alpha after transplantation. The controls were paired with regard to the condition after transplantation, grade of pretreatment, diagnosis and age. In the group treated with IFN alpha five relapses developed and two deaths occurred, in the control group four relapses and three deaths. The probability of survival without signs of progression (PFS) and total survival (OS) within 18 months is in the treated group 67.4% and 88.9%, in the control group 86.5% and 93.8%. The results are not statistically significant. CONCLUSIONS: The authors confirmed in their study the feasibility of long-term administration of IFN alpha after transplantation. On a preliminary basis it may be stated that the results did not differ in the treated and not treated patients, nevertheless the impact of this type of immunotherapy or its modification will have to be evaluated in a randomized study. PMID- 10746029 TI - [Nutritional status of drug addicts in a methadone program]. AB - BACKGROUND: Substitution treatment of drug addicts with methadone has been used in many countries for more than 35 years. In our country it was started in 1997. The objective was above all to evaluate the nutritional status and some biochemical parameters before and after 18 months of methadone substitution. METHODS AND RESULTS: A total of 14 patients were examined (9 men, 5 women), mean age 35 years, range 26-44 years, highly dependent on opiates: heroin, codeine and its derivatives). The examination was made before onset of treatment and after 18 months of treatment. The subjective feeling of all patients improved, they felt more active and efficient. The Kehlet score declined from 5.8 +/- 1.9 to 3.8 +/- 1.5. The body weight increased from 69.3 +/- 14.8 to 78.6 +/- 19.7 kg. BMI from 22.8 +/- 3.2 to 25.5 +/- 5.0 (kg/m2), the skinfold above the triceps muscle from 11.9 to 16.6 mm, prealbumin from 0.17 +/- 0.05 to 0.22 +/- 0.66 g/l). The handgrip strength did not increase, no significant changes were recorded in albumin and transferrin levels. The raised serum aminotransferase levels did not change (due to chronic hepatitis). CONCLUSIONS: Substitution treatment of drug addicts with methadone leads not only to improvement of the subjective state but also to objective signs of a better nutritional status. PMID- 10746030 TI - [Changes in the capacity of T-lymphocytes for spontaneous recovery of selected differentiation antigens in relation to age]. AB - BACKGROUND: During physiological ageing changes of the immune system take place at several levels. The objective of the submitted work was to compare the ability of spontaneous restoration of selected differentiation antigens on lymphocytes in the peripheral blood stream after previous trypsin treatment in a group of healthy elderly and adult subjects. METHODS AND RESULTS: Twenty-four adults were examined (19-59 years) and 36 elderly subjects (60-90 years). Isolated lymphocytes from the peripheral blood stream were treated with trypsin and then incubated in a cultivation medium. The authors investigated the capacity of restoration of differentiation antigens CD2, CD4, CD8 and CD45RA. Antigen CD2 was not restored in any of the investigated groups to original levels. However the difference between its expression on lymphocytes before trypsin treatment and on lymphocytes after 16-hour incubation was higher in the elderly subjects 16% (p < 0.001) than in the group of adults 7% (p < 0.01). Restoration of antigen CD4 was in both investigated groups almost equal. The number of CD8+ T-lymphocytes was in elderly people lower (p < 0.05), spontaneous restoration of antigen CD8 did not differ among the investigated groups and reached in both instances the baseline value. Antigen CD45RA was restored more slowly in elderly subjects, the difference between groups was at borderline of statistical significance (p < 0.0595). CONCLUSION: From the results ensues that during physiological ageing the ability of spontaneous restoration of antigens CD2 and CD45RA declines but not of antigens CD4 and CD8. So far there is no unequivocal explanation why this change occurs, it is probably conditioned by several factors. Investigation of these changes and an attempt to influence them can help to understand age-conditioned immunological dysregulation, its consequences and the possibility to influence them by treatment. PMID- 10746031 TI - [Systemic amyloidosis with renal involvement]. AB - Amyloidosis is a summary of a group of disorders of protein metabolism characterized by infiltration of amorphous substance into the tissues. The diagnosis can rarely be made during life. It is usually manifested by defects of the renale, cardiovascular or peripheral nervous system. Heterogenicity of clinical and histological classification of amyloidosis and clinical course in comparison with autopsy findings are presented by two cases of general amyloidosis--secondary amyloidosis of the kidneys and primary amyloidosis with a fatal prognosis. PMID- 10746032 TI - [Magnetic resonance imaging in the diagnosis of prostatic carcinoma]. AB - The standard diagnostic and staging evaluation of prostate cancer includes digital rectal examination, transrectal ultrasonography (TRUS), serum prostate specific antigen (PSA) level measurement, abdominal pelvic CT and radionuclide bone scan. Magnetic resonance imaging (MRI) (especially endorectal magnetic resonance) opens new possibilities for diagnostic imaging of the prostate. It seems to be better for detecting seminal vesicle invasion. Unfortunately, sensitivity in evaluation of minor capsular and seminal penetration is low and differentiation between haemorrhage, chronic inflammatory, fibrotic changes and tumours is impossible. PMID- 10746033 TI - [Chronic rejection of renal allografts. Part 1. Present knowledge of etiopathogenesis]. AB - Chronic rejection is the most common cause of the long term renal graft loss. It is characterized by luminal thickening and obliteration, interstitial sclerosis, glomerulosclerosis and tubular atrophy development. The pathology is still unclear. Alloantigen-dependent factors (acute rejection, HLA mismatch) and allograft-independent factors (ischaemia-reperfusion, hyperlipidaemia, hypertension, infection, nephrotoxicity, reduced nephron dose) have been implicated in the etiology of chronic rejection. As a result of these factors, endothelial cells are activated and express a variety of adhesion molecules, cytokines and growth factors. Lymphocytes and macrophages infiltrate the areas of local damage and express other cytokines and growth factors (TGF, bFGF, PDGF). In the next step, vascular smooth muscle cells proliferate and migrate from the media into the vascular intima and produce local extracellular matrix. Which factors are the most important and which mechanisms are the key for the development of chronic rejection are in the focus of ongoing research. PMID- 10746034 TI - [Reconstruction of the lower urinary tract in women after cystectomy]. AB - BACKGROUND: The first extirpation of the urinary bladder on account of malignant papilomatosis was carried out by Karel Pawlik in 1889 as the first one in the world. At present cystectomy is indicated usually because of an infiltrating carcinoma of the urinary bladder. The objective of the present paper is, based on anatomical investigations, elaboration of a surgical technique of creating a orthotopic neovesica following cystectomy sparing a functional female urethra. METHODS AND RESULTS: In 1993-1998 32 women were operated within the age bracket of 32-72 years with a confirmed infiltration carcinoma of the urinary bladder. The authors describe in detail their own surgical technique. Day continence was achieved in 20 patients. Twelve patients suffer from stress incontinence. Eight patients have a post-micturition residue of 250-300 ml calling for a combination of medicamentous treatment and autocatheterization. The capacity of the neovesicle is the cause of nycturia: 21 patients must micturate once or twice during the night. Urodynamic studies did not reveal significant differences between patients with chronic post-miction residues and without residues. Also the mean functional length of the urethra was in both groups similar (27 mm in patients with a residue and 26.2 mm in patients without a residue). CONCLUSIONS: The elaborated surgical technique of cystectomy and creation of a neovesica makes a good quality of the patients' life possible. PMID- 10746035 TI - [Lysosomal acid lipase deficiency. Overview of Czech patients]. AB - Lysosomal lipase deficiency is a hereditary autosomal recessive enzymopathy leading to lysosomal storage of triacylglycerols (TAG) and cholesterol esters (CE). In particular cells with a permanently high receptor-mediated LDL endocytosis are affected (liver, kidneys). There are two basic phenotypes. The fatal infantile phenotype (Wolman's disease) with generalized storage of both types of apolar lipids. This form was diagnosed in this country only once. The opposite is the protracted, oligosymptomatic form encountered in all age groups. It is characterized by the storage of CE (which gave this entity the name of cholesteryl storage disease--CESD). Its main sign is affection of the liver (hepatomegaly, hepatopathy), which in some instances may lead to organ failure, directly or after cirrhotic transformation. Furthermore there is permanent hypercholesterolaemia (high LDL cholesterol) due to increased VLDL synthesis by hepatocytes, low HDL cholesterol and variably raised TAG. This constellation of blood lipids is a risk factor for the development of atherosclerosis. In the course of 25 years in the Czech Republic 13 cases of CESD were diagnosed in 11 families. Ten of these cases were characterized by clinically manifest hepatopathy with hepatomegaly, detected incidentally during medical examinations (at the age of 2-14 years). In three adult patients with permanent hypercholesterolaemia the storage process was subclinical and the diagnosis was established quite incidentally by examination of non-specific secondary and tertiary manifestations of the disease. The diagnosis was established in all cases of CESD at the tissue level (liver biopsy), at the biochemical (acid lipase deficiency) and molecular genetic level (mutation in enzyme locus). In all instances mutation of G934A was found leading to reduction and loss of the eighth exon. This mutation was present in five patients in a homozygous state. Six mutations were heterozygous. In one instance for technical reasons only one allele was analyzed. In three instances a point "missense" mutation was found: T323A (Trp74Arg), T4(75)A (Asp124Glu), A210T (Asp36Gl), in one instance a "nonsense" mutation: C233T (Arg44-stop) and twice a deletion mutation delta C673 5 and delta G1068-8 leading to impairment of the reading frame and to premature stop of the codon. PMID- 10746036 TI - [The first year of use of the Leksell gamma knife at the Hospital Na Homolce- results after 5 years]. AB - The first Leksell gamma knife procedure was performed in Prague on 26th October 1992 and during the first year 156 patients were treated at the department of stereotactic and radiation neurosurgery. 42% patients suffered from a vascular lesion, 39% of patients from a benign tumour and 19% patients from a malignant tumour. Radiosurgery using the gamma knife is a non-invasive stereotactic neurosurgical procedure. Radiosurgery, as microsurgery, must be evaluated not only early, but also the late effect after the treatment some years. Evaluation of the results is therefore an ongoing process and results of the first year with the 5-year follow up are presented. At this time radiosurgery has a zero treatment mortality and the present morbidity with regard to the diagnosis 0-10%. PMID- 10746037 TI - [The ovarian hyperstimulation syndrome with pericardial and pleural effusions complicated by supraventricular arrhythmia]. AB - Problems which arise in conjunction with the use of assisted reproduction methods are no longer resolved only by the gynaecologist. The authors demonstrate on the case of a female patient the necessity of interdisciplinary collaboration in the diagnosis and therapy of developed ovarian hyperstimulation syndrome (OHSS)--the most serious complication of assisted reproduction. PMID- 10746038 TI - [Clinical sequelae of mutation of the CBP gene]. AB - Gene CBP codes for a transcriptional coactivator, which can interact with many transcriptional factors. It modifies the process of transcription stimulated by these factors by specific binding to RNA polymerase II holoenzyme or by histone acetylation. CBP gene mutation is the molecular cause of autosomal dominant genetic disease called Rubinstein-Taybi syndrome that is manifested by mental and growth retardations, by typical face malformations and broad thumbs and broad big toes. The CBP gene can be affected by the t(8;16)(p11;p13.3) translocation resulting in production of the MOZ/CBP chimeric protein and in induction of acute myeloblastic leukaemia. Therapy using topoisomerase II inhibitors can induce the t(11;16)(q23;13.3) translocation causing acute myeloid or lymphoid leukaemia or myelodysplasia through production of the MLL/CBP protein chimera. PMID- 10746039 TI - [Urinary continence after radical retropubic prostatectomy]. AB - Urinary incontinence following radical retropubic prostatectomy is a serious medical and psychological complication with a major negative impact on quality of life in patients with localized prostate cancer. The overall incidence of this complication is higher than 2% regardless whether the surgery has been done by the retropubic or perineal approach. The correct diagnosis of incontinence is based on urodynamic investigations. Two main causes of incontinence are detrusor hyperactivity and sphincteric insufficiency. The best prevention for postoperative incontinence of urine is meticulous surgery. Treatment options of incontinence are pharmacological or surgical (injections of collagen, autologous fat or implantation of artificial urinary sphincters). PMID- 10746040 TI - [The effect of partial refeeding on serum levels of leptin and resting energy expenditure in female patients with anorexia nervosa]. AB - BACKGROUND: Serum leptin levels and resting energy expenditure are significantly decreased in patients with anorexia nervosa compared to healthy subjects. Partial realimentation of patients with anorexia nervosa increases both these parameters. However, there so far are no data concerning the relationship of serum leptin levels and resting energy expenditure in patients with anorexia nervosa. The aim of our study was to follow the relationship of serum leptin levels and resting energy expenditure in patients with anorexia nervosa before and after refeeding. METHODS AND RESULTS: It was found that serum leptin levels in patients with anorexia nervosa both before and after partial realimentation were significantly lower compared to healthy subjects (0.86 +/- 0.9 ng.ml-1 and 1.77 +/- 1.9 ng.ml-1 vs 6.85 +/- 3.0 ng.ml-1, p < 0.05). The total resting energy expenditure in patients with anorexia nervosa both before and after realimentation were significantly lower than in the control group (4973 +/- 979 and 5263 +/- 899 vs 6401 +/- 827 kJ/day, p < 0.05). When expressed per body weight the resting energy expenditure in patients with anorexia nervosa was significantly higher in comparison with the control group (122.3 +/- 20.6 and 121.5 +/- 14.2 vs 104.2 +/- 14.6 kJ/day, p < 0.05). The differences in resting energy expenditure in patients with anorexia nervosa before and after refeeding did not reach statistical significance. The body mass and the body fat content increased significantly after realimentation index (14.5 +/- 1.4 vs 15.6 +/- 1.5 kg.(m2)-1 and 14.9 +/- 3.4 vs 16.6 +/- 3.7, p < 0.05) but remained still below of those of control group (BMI 22.3 +/- 2.7, body fat content 26.9 +/- 4.2). CONCLUSIONS: Our study confirmed the reduced serum leptin levels and the total resting energy expenditure in patients with anorexia nervosa compared to healthy age-matched subjects. No statistically significant relationships were found between serum leptin levels and resting energy expenditure either in healthy subjects or in patients with anorexia nervosa before or after partial realimentation respectively. PMID- 10746041 TI - [Equipotency of vitamin D2 and D3 in humans]. AB - BACKGROUND: Vitamin D is an effective substance in the treatment of disorders of phosphorus-calcium homeostasis. The biological activity of vitamin D2 and D3 may however differ. The objective of the investigation is to assess the biological action of both vitamins in man and their possible differences. METHODS AND RESULTS: The group comprises two different subgroups of patients. The first is formed by 3 men, mean age 69.2 years and 9 women mean age 70.8 years. The second one comprises 14 drug addicts (10 men mean age 31.1 years and 4 women mean age 28.9 years). All patients were given for a period of two months 1300 IU vitamin D2 and D3. The vitamin D values in blood were assessed using a kit estimating 25 hydroxyvitamin D produced by Nichols Institute Diagnosis. Other laboratory indicators were assessed photometrically. The data were analyzed by the paired t test. No significant differences of mean 25 hydroxyvitamin D levels were found. CONCLUSIONS: The results did not provide an answer to the different biological effectiveness of vitamins D2 and D3 in man. PMID- 10746042 TI - [Economic aspects of treatment of proximal femoral fractures in the Czech Republic]. AB - BACKGROUND: The number of publications evaluating economic and social aspects of the treatment of proximal femur fractures increased steadily in the last 20 years in the foreign literature. Due to the absence of similar studies in the domestic literature the authors decided to investigate this issue in the Czech Republic and to present outcomes. The aim of the presented study is to inform about the costs of treatment of patients with proximal femur fractures in an orthopaedic department and estimate the costs of such treatment on a nationwide scale. METHODS AND RESULTS: A detailed follow up of 244 patients treated for a proximal femur fracture was carried out at the Orthopaedic Clinic of the 3rd Medical Faculty and Faculty Hospital, Kralovske Vinohrady in 1997, including economic aspects and the fate of patients after their discharge from the hospital in the course of one year after their injury or surgery. The average cost of primary treatment of a patient in this group amounted to 38,000 Kc, on a nation-wide scale this sum totalled 29,000. The obtained results were compared with national data acquired from the Institute of Health Care Informations and Statistics and from the General Health Insurance Company. The monitored group accounted for 2% of the national group of patients with this diagnosis. CONCLUSIONS: Exact data on the costs of primary treatment of patients with a proximal femur fracture were obtained only in the monitored group of our patients, in 1997 these costs amounted to 9,389,848 CZK. The costs in the whole Czech Republic may be only estimated at approximately 450 million CZK. PMID- 10746043 TI - [Dialysis therapy in the Czech Republic in 1998 and in the future]. AB - 1. The number of dialyzation centers has not changed during the past two years. Their steep increase occurred in 1990-1995. This development made this treatment available without former limitations (on account of diagnosis and age). 2. In the dialyzed population during the last five years the number of patients in more advanced age groups is increasing. More than half the patients are older than 60 years. During this period there was a marked increase of diabetic subjects and in 1997 they accounted already for one third of the dialyzed population. 3. In the number of patients treated by haemodialysis we still lag behind the countries of the EU. An adverse indicator of the quality of treatment is the high mortality rate. 4. It is essential to ensure early dispensarization of patients with a creatinine level above 300 mumol/l in the predialyzation ambulatory department. 5. Only a very small percentage of patients is treated by peritoneal dialysis as this is not a traditional method in this country. PMID- 10746044 TI - [Confronting patients about a factitious disorder]. AB - Publications on the technique of confrontation in the treatment of factitious disorder are rare. Two serious cases of factitious disorder are discussed, one of a 25-year-old woman who was admitted after having suffered an epileptic insult in the street. When her general practitioner was contacted it turned out that she had a habit of being admitted to emergency departments presenting with pseudo insults. The neurologist confronted her rather bluntly with these facts and she disappeared, without proper treatment. The other case was a woman aged 40 who suffered from a leg wound that would not heal. She was encouraged to consult a psychiatrist as well as a surgeon. When it was discovered that she kept the wound open herself, and when a second opinion was to amputate the leg, she was told that in order to give the leg a chance to heal, it needed to be emplastered. She consented but continued to see the psychiatrist and was reassured that the consultations would continue even if the leg had healed, which it did, without recurrence. The technique of the confrontation is of crucial importance for the prognosis of patients with factitious disorder. Factors include ample communication between surgeon and psychiatrist before a psychiatric consultation and confrontation are performed, mild confrontation together with the proposal of a comprehensive treatment plan by the same surgeon and psychiatrist who developed a working relationship with the patient, coordination with the nurses, and so on. The cases suggest that with the right confrontation technique, even serious patients can be treated successfully. Somatic specialists should be aware of the importance of communication with the psychiatrist before they attempt any kind of confrontation in patients with factitious disorder. PMID- 10746045 TI - [Gastrointestinal surgery and gastroenterology. VII. Proximal motility disorders in the digestive tract]. AB - Dysphagia and chest pain are the two commonest symptoms of abnormalities of oesophageal motility. Dysphagia is to be distinguished into high or oropharyngeal and low or oesophageal dysphagia. Oropharyngeal dysphagia pertains to dysfunction of the pars cricopharyngea of the M. constrictor pharyngis inferior (M. cricopharyngeus), which is frequently associated with a Zenker diverticulum. Treatment consists of endoscopical or surgical myotomy and diverticulectomy. In achalasia there is incomplete relaxation of the lower esophageal sphincter with aperistalsis. The main treatment modalities are endoscopic pneumodilation and surgical myotomy of this sphincter. In dysphagia or non-cardiac chest pain spastic or hypocontractile abnormalities of the oesophageal motility can be involved, these are often difficult to treat. Disorders of gastric motility are mainly gastroparesis and functional dyspepsia. In diabetic gastroparesis, adequate monitoring of the blood sugar level is also necessary. New insights into the pathophysiology of functional dyspepsia concern abnormal visceral sensitivity and reduced adaptive relaxation of the stomach during intake of food. PMID- 10746046 TI - [Hyponatremia in acute intracranial disorders: cerebral salt wasting]. AB - Hyponatraemia is a frequent finding in the course of an acute intracranial disease, especially after a subarachnoid haemorrhage. The fall in plasma sodium concentration is usually mild and not below 124 mmol/l but may reach dangerously low levels with serious neurological complications. In the early 1950s the cause of the hyponatraemia was believed to be primarily excessive natriuresis and therefore named 'cerebral salt wasting'. After the description of the syndrome of inappropriate antidiuretic hormone secretion (SIADH) this was favoured as the most likely explanation. Only in recent years has it become evident that many hyponatraemic patients with acute brain disease are actually hypovolaemic. This is compatible with the original diagnosis of cerebral salt wasting. The increased plasma concentrations of natriuretic peptides are likely to mediate the increased natriuresis. Cerebral salt wasting can be treated with a simple regimen of water and salt suppletion. If needed a mineralocorticoid like fludrocortisone can be given to increase renal tubular sodium reabsorption. PMID- 10746047 TI - [Protocol for ambulant treatment of deep venous thrombosis in a leg: efficacious and safe in general practice]. AB - OBJECTIVE: To evaluate a standard protocol for ambulant treatment of patients with deep venous thrombosis (DVT) in a leg with low molecular weight heparins (LMWH). DESIGN: Prospective. METHOD: A protocol was established within a regional context which provided for all patients in or around Veghel, the Netherlands, with demonstrated DVT in the period 1 March 1997-31 July 1999 to receive ambulant treatment with LMWH and oral anticoagulants. After diagnosing the treatment was started in the emergency department of the Sint Joseph Hospital in Veghel, the subsequent LMWH injectons were administered by the general practitioner in his practice. RESULTS: Of the 130 patients with demonstrated DVT, six were hospitalized because of pulmonary embolism, carcinoma or pregnancy, while 124 (95%) were eligible for ambulant treatment (56 males with a median age of 57 years (range 16-98) and 68 females with a median age of 51 years (18-89). During the acute period of LMWH treatment, none of the 124 patients developed a recurrence of thrombosis, pulmonary embolism or major haemorrhage. During 3 months' follow-up one patient developed a thrombosis in the other leg, this patient died after 2 months from disseminated carcinoma of the colon. CONCLUSION: Ambulant treatment of patients with DVT of the leg with LMWH in a daily practice setting is feasible and can be carried out effectively and safely in 95% of the patients in a regional organization. PMID- 10746048 TI - [Good results from a multidisciplinary and behavioral program for chronic back pain]. AB - OBJECTIVE: To evaluate the results of a multidisciplinary and behavioral program for chronic back pain. DESIGN: Prospective cohort study. METHOD: In 1996, a total of 143 patients with chronic back pain participated in a 4-week multidisciplinary and behavioral treatment program in an outpatient centre for work reintegration (Rug AdviesCentrum) in Zeist, Eindhoven and Noordwijk, the Netherlands. At the start and six months after termination, data were collected on trunk muscle performance, pain intensity, disability and stress related physical complaints, and after 6 months about return to work, analgesics use, and health care utilization. Score differences between the first and the second measurements were tested on statistical significance using paired t-tests and on clinical relevance using the reliable change-index. RESULTS: The study group comprised 99 males and 44 females with a mean age of 41.6 years (range: 23-58). After 6 months the outcome variables were significantly improved statistically: depending on the outcome variable used, the improvement was clinically relevant in 44-91% of patients. The obtained percentage of full return to work at 6-months follow-up was 87%, while 80% had used no pain killers and 91% had made no use of health care facilities. CONCLUSION: The program resulted in important improvements in patients with chronic back pain. PMID- 10746049 TI - [Reversible memory disorders in an older patient caused by epilepsy]. AB - Memory impairment in a 63-year-old man was the most prominent expression of partial epileptic seizures from the temporal lobe, probably resulting from a head injury. Treatment with antiepileptic drugs resulted in complete recovery. This case illustrates that epilepsy should be considered in the differential diagnosis in elderly patients with an isolated memory disorder. The diagnosis is made by clinical methods and may be confirmed by electroencephalography. PMID- 10746050 TI - [Objective re-evaluation procedures in cases of possible diagnostic error]. AB - Auxiliary diagnostic specialists such as clinical pathologists or radiodiagnosticians may be held liable by injured patients for erroneous diagnoses. Nearly always there will be a reconstruction of the situation in which the alleged misstake was made, and relevant facts will be tested against both professional and legal rules. The Court or one of the parties involved frequently ask an expert for re-evaluation of microscopic preparations, X-rays, etc. This objectivating evaluation requires a procedure that does justice to the original diagnostic situation and therefore should not be made by one single expert familiar with the chain of events. The results of the re-evaluation should be interpreted meticulously and their relative importance established by the Court. PMID- 10746051 TI - [Non-oral routes of administration of psychotropic agents]. PMID- 10746052 TI - [Non-oral routes of administration of psychotropic agents]. PMID- 10746054 TI - [Intracerebral tuberculomas in a pregnant Somalian woman]. PMID- 10746053 TI - [Intracerebral tuberculomas in a pregnant Somalian woman]. PMID- 10746055 TI - [Initial experience with surgical treatment of tracheal stenosis in small children using extracorporeal circulation]. AB - Severe tracheal stenosis represents a life threatening malformation which necessitates early surgery. Two patients aged one and two years, respectively, were followed-up for signs of congenital stridor. Following respiratory infection both children became critically ill with severe dyspnoea necessitating intubation and artificial ventilation. In the first patient, echocardiography, tracheobronchoscopy and other investigations revealed a pulmonary artery sling with tracheal compression and hypoplasia of the whole trachea. In the second child, presence of a short local tracheal stenosis was found, the cause of which could not be clarified. In the patient with the pulmonary sling, resection and reimplantation of the anomalous left pulmonary artery was performed first. In both children, however, surgical reconstruction of the lower airways was necessary. Surgery was performed from a midline sternotomy approach in extracorporeal circulation. The hypoplastic trachea with circular rings in the first child was enlarged with a pericardial patch. In the second child, the local tracheal stenosis was resected and a direct anastomosis of the trachea was performed. In both patients, transient formation of granulations was observed. Both children, however, survived and their clinical condition remains good 18 and 9 months, respectively, after surgery. Tracheobronchoscopic controls show very good result. Our experience confirms the possibility of successful surgical reconstruction of lower airways in young children using extracorporeal circulation. Good interdisciplinary cooperation between the surgeon and other specialists is an important prerequisite of good surgical results. PMID- 10746056 TI - [Atresia of the large intestine]. AB - The authors describe two patients with congenital atresia of the large intestine. Both patients had colostomy when neonates. A double colostomy was performed. The microcolon was dilated across the distal colostomy by physiological saline and semolina pudding to achieve dilatation of the distal portion. Occlusion of the colostomy, tapering and end-to-end colo-colo anastomosis was performed in both children at the age of four months. The passage was restored in both patients on the third day after occlusion of the colostomy. One patient had also other congenital defects, i.e. equinovarus of both feet and a defect of the atrial septum. One patient is four, the other three years old. They are doing well and have no digestive trouble. PMID- 10746057 TI - [An unusual esophageal stenosis]. AB - In the submitted case-history the authors describe the case of a child operated after delivery on account of oesophageal atresia type Vogt IIIb. After a two-year interval the child returned on account of acute dysphagia and endoscopy revealed oesophageal stenosis caused by a mucosal strangulating bridge. The latter was removed endoscopically and during a check-up examination 6 months later the endoscopic picture was negative. PMID- 10746058 TI - [The importance of laparoscopy in children]. AB - The authors submit the evaluation of 720 laparoscopic operations performed at the Clinic of Child Surgery and Traumatology in Brno between the end of 1994 and May 1999. The group is formed by children aged 3 weeks to 18 years. The mean age was 8.4 years. The authors recorded once an early and 4 times a late complication. None of the complications were serious and did not threaten the child's health or life. With regard to ever more frequently performed acute laparoscopy the percentage of conversions (2.5%) is higher. The authors emphasize there is practically no age limit for performing this operation (32 infants and toddlers). On the other hand, perfect technical equipment and adequate training of the whole team is necessary. PMID- 10746059 TI - [Comparison of the results of minimally invasive and open osteosynthesis in treatment of injuries of the proximal radius in children]. AB - The authors present their experience with treatment of injuries of the proximal radius in children. During the period between 1994 and 1998 they treated on account of this injury a total of 59 patients. Ten times they used the open method, 15 times percutaneous fixation and 34 times a conservative approach. They compare the therapeutic results of both surgical methods and demonstrate better results when the miniinvasive approach--percutaneous fixation--is used. The authors recommend this procedure in dislocated injuries type II and III and emphasize the necessity of high standard technical equipment and experience of the working team. PMID- 10746060 TI - [Feminizing genital plasty in patients with intersexuality]. AB - Feminizing genitoplasty (FGP) involves modification of a hypertrophic clitoris, vaginoplasty, and modelling of the labia. FGP is performed during childhood and usually in one stage. In 1996-1999 30 patients with intersex were operated on. 25 girls with female pseudohermaphroditism caused by congenital adrenal hyperplasia (CAH), 3 patients with mixed gonadal dysgenesis (MGD), 1 patient with male pseudohermaphroditism and one female patient with true hermaphroditism (HFV). Seventeen patients were between the ages of 3 and 6 years, 13 patients (all female with CAH) were 13 to 21 years old. One-stage FGP, i.e. partial resection of the clitoris and vaginoplasty (VP) was performed in 11 patients. A two-stage operation, i.e. partial resection of the clitoris and subsequent vaginoplasty, was done in 3 patients (all with VHN with a high-orifice vagina). Vaginoplasty after excision of the clitoris was made in 13 patients (all with CAH, 5 of them with a high-orifice vagina). Three female patients (2 VHN, 1 HFV) had partial resection of the clitoris and wait to have VP. Fourteen patients with partial resection of the clitoris healed p.p. Healing p.s. was recorded in 5/27 patients with vaginoplasty (all CAH). Three of them were older, menstruating and obese. In 4/14 patients after partial resection of the clitoris both modelling and fixation of the glans clitoridis was necessary. Urinary incontinence was not recorded. Stricture of the vaginal orifice was found in one 6-year-old patient with CAH after reconstruction of a high-orifice vagina. Thirteen older patients have a normal vaginal orifice, two of them have sexual intercourse. FGP can be performed already at toddler age, the cosmetic effect is favourable, the glans clitoridis is preserved and the psychosexual development of patients is not impaired. FGP can be used also in adults, severely virilized patients. PMID- 10746061 TI - [Primary lung tumors in childhood]. AB - The authors give an account of their experience with the treatment of primary lung tumours in children during 1989-1998. Primary lung tumours in children are extremely rare and represent a varied spectrum of pathological conditions (pneumoblastoma, rhabdomyosarcoma, mucoepidermoid carcinoma, pulmonary endodermal tumour and benign tumours). During the mentioned period 10 children were operated on account of primary lung tumours, 5 with malignant tumours and 5 with benign tumours. In these patients during the above period 3 pneumonectomies, 5 lobectomies and 3 segmental resections were made. Chemotherapy was indicated in children with pneumoblastoma and rhabdomyosarcoma. Two patients with pneumoblastoma died, 3 children with malignant and 5 children with benign tumours live without symptoms of the disease. PMID- 10746062 TI - [Esophageal reconstruction using the stomach in children]. AB - The authors present an account of their experience with oesophageal replacement by gastric transposition in 10 children during the period between November 1992 and January 1999. Indications for oesophageal replacement was in seven children atresia of the oesophagus at the age of 9-36 months and in three children (2.5, 3 and 14 years) a long stricture of the oesophagus after caustic injury which did not respond to balloon dilatation. Replacement after caustic injury was made in children aged 2.5, 3 and 14 years. In eight patients the replacement was made without thoracotomy, by blunt resection of the mediastinum, in two patients after caustic injury thoracotomy had to be performed. In three patients after surgery a temporary fistula developed at the site of anastomosis which healed spontaneously. Eight patients are in a satisfactory condition after surgery, one patient five months after operation swallows with difficulty, his birth weight was 1500 g. One female patient died one year after oesophageal replacement, apparently due to aspiration and respiratory arrest. Gastric transposition is a considerate and relatively simple method in child age. The functional results are favourable. PMID- 10746063 TI - [Rupture of the pancreas in children--present trends in diagnosis and therapy]. AB - Evaluation of pancreatic injuries is presented in the professional literature in retrospective studies and views on treatment vary. The authors evaluated also in a retrospective study a group of eight child patients with a serious injury of the pancreas. The patients were 3-13 years old and were hospitalized at the Clinic of Paediatric Surgery at the Faculty Hospital Motol in 1985-1999. The group is formed by patients with pancreatic injuries (according to the Japanese classification type II and III). The authors evaluate not only diagnostic but also therapeutic procedures. In the diagnosis they evaluate the ultrasonographic examination (USG) as the examination of first choice which however need not record the extent and severity of the pancreatic injury. Computing tomography (CT) and magnetic resonance (MRI) can make the original suspicion of injury more accurate, ERCP is an examination which reveals in particular type III injuries- i.e. those with affection of the pancreatic duct. Injured children where a serious pancreatic lesion was not diagnosed primarily, had to be subjected to repeated surgical revisions. All children in the presented group recovered, none of the patients died. PMID- 10746064 TI - [Infiltration of the inferior vena cava in metastasizing testicular germ cell tumor]. AB - There exists a small percentage of patients with metastatic germ cell tumours who have direct involvement of the inferior vena cava or tumour thrombosis of the inferior vena cava. The authors describe a case of patient with a bulky metastatic germ cell tumour with involvement of the inferior vena cava. Incidence, surgical management and complications in patients with such extent of disease are reviewed. PMID- 10746065 TI - [Ambulatory treatment of deep venous thrombosis using low-molecular heparin, compression and mobilization]. AB - From data in the literature as well as the authors' own experience ensues that diagnosed and objectively confirmed deep venous thrombosis can be successfully treated in out-patients departments by low-molecular heparin and compression with full mobilization without any major risk of pulmonary embolism and without the need of hospital admission (65 out-patients). Despite the high price of low molecular heparin, treatment is economical as it does not require monitoring of haemocoagulation for hospital admission. Partsch et al. 1997 proved unequivoca lambda y that the rate of new attacks of pulmonary embolism during treatment is significantly lower than during classical treatment with non-fractionated heparin and immobilization. PMID- 10746066 TI - [Current status of liver resection of metastases of colon and rectal carcinoma]. AB - Based on an analysis of a group of patients with resection of the liver on account of secondaries of colorectal carcinoma the authors evaluate their therapeutic method. Between 1978 and June 1999 at the Surgical Clinic of the Third Medical Faculty Charles University, Prague resections of the liver were made in 222 patients, incl. 57 on account of metastases of colorectal carcinoma. The liver was resected on account of metastases by right-sided lobectomy twice, left-sided lobectomy 1x, right-sided lateral bisegmentectomy 5x, left-sided lateral bisegmentectomy 9x, segmentectomy 16x and in 24 patients a wedge-shaped resection was made (incl. 10 with multiple metastases several times). In another eight patients where a resection could not be performed either alcoholization of the tumour was used or adjuvant chemotherapy was administered. Postoperative complications were recorded in 10%, the 30-day mortality rate was 2%. The mean survival period was 23 months. Resection of the liver on account of a secondary of colorectal carcinoma is a safe and effective therapeutic method. PMID- 10746067 TI - [An incorrect diagnosis of an arterial injury]. AB - The authors give an account of the interesting clinical development of a primarily erroneously diagnosed and inadequately treated arterial injury caused by a splint. They emphasize the necessity of angiographic examination and truthful recording in the surgical protocol. PMID- 10746068 TI - [Epistola chirurgica. 3. Paronychia]. PMID- 10746069 TI - [Correct use of anatomic nomenclature in selected Czech professional journals]. AB - The objective was to assess in a qualitative study how correctly anatomical terms are used in four selected Czech medical journals (Acta Chirurgiae Orthopaedicae and Traumatologicae Cechoslovaca, Ceska gynecology, Ceska radiology and Rozhledy v chirurgii, 1990-1996). A total of 1200 articles were evaluated where 145 anatomical terms were detected which differed from P.N.A. and from Nomina Anatomica valid at present. Of these 25 were terms from B.N.A. (4) and from I.N.A. (21). Approximately twice as frequent were anatomical and clinical eponyms (52). The highest ratio (71) was formed by incorrect terms (36 Latin and 35 Czech). The authors feel that in the improvement of the standard of exact anatomical terminology in clinical medicine as well as in the professional literature in future a part should be played by better teaching at medical faculties within the framework of the new curriculum and by higher demands on the standard of manuscripts accepted by editors of professional journals. PMID- 10746070 TI - [Carotid microendarterectomy--surgical technique]. PMID- 10746071 TI - [Surgical interventions in dialysis fistulae]. AB - PURPOSE: To evaluate the efficacy, feasibility and safety of interventional procedures in patients with hemodialysis shunts in retrospective analysis. We report the technical success rate and the midterm primary and secondary patency rate in our patients. MATERIAL AND METHODS: Between April 1996 and May 1999 (35 months) we performed 129 interventional procedures on hemodialysis shunts in 87 patients (40 women and 47 men in age 22-88 years, mean age 67 years). We treated 46 occlusions and 83 stenoses. There were native fistulas--63 radiocephalic fistulas, 7 brachiocephalis fistulas, 1 brachiobasilic fistula, and hemodialysis grafts--14 vein allografts and 2 synthetic PTFE grafts. Indication for fistulography and intervention was decreasing efficacy of hemodialysis (in case of stenosis) or shunt occlusion. Patients were treated by means of percutaneous transluminal angioplasty, thrombolysis and percutaneous mechanical thrombectomy with hydrolyser or with thrombectomy basket. Stents were placed twice. Once in suboptimal result of local thrombolysis, second time in case of central venous stenosis. Follow up ranged from 0.3 to 35 months (mean 11 months). Nine patients were lost for follow up, 15 patients died during follow up. RESULTS: Overall technical success rate was 84% (108 procedures, n = 129), technical success in cases of occlusion was 57% (27 procedures, n = 46), in stenoses treatment was 98% (81 procedures, n = 83). Overall primary patency rate in 6 months is 40% (n = 59), in 12 months 24% (n = 59) and in 2 years 5% (n = 59). The primary assisted patency rate and secondary patency rate is 95% (n = 59) in 6 months, 88% (n = 59) in one year and 73% (n = 59) in two years. CONCLUSION: Repeated percutaneous interventional procedures in patient with hemodialysis shunts can extent shunt patency. Despite relatively low primary patency rate we can achieved good primary assisted or secondary patency rate in patients with stenoses of shunt. PMID- 10746072 TI - [Gastrointestinal stromal tumor--GIST--case report]. AB - Authors describe a newly introduced oncological entity that represents different views on some already well known mesenchymal and neuroectodermal tumours of gastrointestinal tract. These submucosal GIT tumours, showing only few differences in both the clinical and histological pictures, are generally called gastrointestinal stromal tumours. This paper presents a case of 37 year old patient with this disease. PMID- 10746073 TI - [Methods of hemostasis in upper digestive tract hemorrhage]. AB - The authors describe methods of haemostasis used nowadays in haemorrhage from the upper digestive tract. Based on evaluation of their own group of 91 patients with haemorrhage of this type they found that 13.2% of the patients with haemorrhage required surgery, when the haemorrhage was localized, 16.4%. Comprehensive treatment and endoscopic methods of haemostasis predominate while surgical treatment is used when non-surgical methods fail. The results are in favour of revision of the surgeon's position in the treatment of patients with haemorrhage from the GI tract. PMID- 10746074 TI - [Treatment of colonoscopic perforations]. AB - Perforations of the large intestine during colonoscopy are relatively rare but serious complications of this routine examination. In recent years the approach to their treatment has changed in favour of more conservative procedures, in particular in perforations which occurred during polypectomy. In the authors' department during the last five years 12 patients were treated with perforations which occurred during colonoscopy. Ten patients were operated on (4x suture of the perforation, 2x primary anastomosis, 5x derivation of the gut), two patients were successfully treated by conservative methods without surgery. Fifty per cent of the operated patients had complications. Nobody died. The mean period of hospitalization was 27.9 days in the operated patients and 4.5 days in those who did not undergo surgery. From the results ensues that a differentiated approach must be used in perforations and in indicated cases (perforations after polypectomy, satisfactory condition of the patient, minimal assumed contamination of the abdominal cavity) a conservative method can be used. Correct surgical tactics reduce the morbidity and mortality. PMID- 10746075 TI - [Fatal Clostridium sepsis after preoperative hepatic artery embolization]. AB - The authors describe the first case in the Czech literature of a death caused by Clostridium sepsis following embolization of the hepatic artery as the preparatory stage for resection of a hepatic tumour. PMID- 10746076 TI - [Drainage of the abdominal cavity using a simple and reliable system]. AB - Experience with drains is more than 2000 years old, but instructive. In abdominal surgery nowadays indications for prophylactic drainage are diminishing as peroperative antibiotic administration has a similar effect. Therapeutic drainage is however still important and drainage is also useful after extensive operations, surgery of the lesser pelvis and operations with major bacterial contamination. The authors present their practical experience with a simple system of wrapped drains (Medisillen-Rubena, Brno) and a cover with an adhesive plate (B-Braun-Biotrol) which they used in 660 abdominal drainages with a very good effect. PMID- 10746077 TI - [Treatment of fractures in the elbow region in children using the Fragment Fixation System--initial experience]. AB - In child traumatology among implants an irreplaceable place is held by Kirschner wires. An improved version of this implant is the Orthofix Fragment Fixation System (FFS) of Kendall Co. which combines the advantages of Kirschner wires with the compressive properties of a screw. During the past two years at the Clinic of Child Surgery and Traumatology in Brno by osteosynthesis, using FFS implants, a total of 21 patients were treated incl. 19 under the age of 15 years. On account of injuries of the elbow 15 children were treated (radial physis of the humerus- 9x, ulnar epicondyle--4x, fracture of the ulnar olecranon--2x). The authors present an account of the hitherto assembled favourable experience and results of this method in the treatment of injuries of the elbow in children. PMID- 10746078 TI - [Complications of colostomy--how to avoid them]. AB - According to data in the literature complications develop in association with colostomy in 20-66% of patients, occlusion of the colostomy occurs in 10-17%. The authors analyze their own group of 122 patients where in 1994-1998 a colostomy was made. Sixty-two patients were operated with developed ileus, 19 colostomies were later abolished and the passage per vias naturales was reconstructed. The authors recorded 38.5% complications, they were most frequent in axial transversostomies and sigmostomies, wound infections were most frequent. For prevention of complications the authors emphasize that stricter indications for colostomy should be used. The same applies to the selection of the type and site of colostomy and exact surgical technique, ATB prophylaxis and lavage, use of modern stomic appliances, perspectives of using the laparoscopic technique. PMID- 10746079 TI - [The BAR-Valtrac for the colon]. AB - The authors present their experience with biofragmentable anastomosis in the area of the colon in 42 patients where they used a biofragmentable ring Valtrac, Davis Geck Co. to restores continuity after resection. The authors focus their attention on the peroperative technical difficulty of construction of the anastomosis proper and on complications encountered during the immediate postoperative course. The most serious among them is, no doubt, dehiscence of the anastomosis. Based on this experience, the authors express their opinion on the simplicity of the implementation proper of a BAR anastomosis and problems of postoperative complications. PMID- 10746080 TI - [Loss injuries of the biliary tract in laparoscopic cholecystectomy-- possibilities of reconstruction]. AB - Authors analyze occurrence of injuries of external biliary tract relating to the increasing number of laparoscopic cholecystectomies, especially of loss injuries, very demanding for its reconstruction. The early diagnosis of these injuries is emphasized and the necessity of their surgical revision as soon as possible, best still during the primary intervention. Authors present the own reconstruction method by high intrahepatic anastomoses in loss injuries of the common hepaticus and its branches. PMID- 10746081 TI - [Spigelian hernia and cholecystolithiasis treated laparoscopically]. AB - The authors present the laparoscopic treatment of the uncommon Spigelian hernia together with gallstones at a 50-years old woman. PMID- 10746082 TI - [Small aneurysms of the abdominal aorta]. AB - As "small" an aneurysm of the abdominal aorta is defined, the diameter of which is < 5 cm in the maximal anteroposterior or transverse diameter. The authors investigated the development of a small aneurysm of the abdominal aorta in a group of 55 patients for a period of 30 months. They evaluated also the influence of the main risk factors and firmness of the thrombus on the growth rate of the aneurysm. A statistically highly significant factor for the growth of the aneurysm was untreated hypertension (p < 0.0001). Other factors (age, smoking, diabetes, ischaemic heart disease) were not significant for the development of the aneurysm. The firmness of the thrombus correlated with its size but was not directly related to the rate of enlargement of the aneurysm. Based on their own experience and reports in the literature the authors assume that elective surgery or endovascular treatment is indicated in patients where the growth of a small aneurysm exceeds 0.5 cm in six months and also patients where the aneurysm has reached the size of 5 cm during regular check-up examinations. Untreated or inadequately treated hypertonic patients with an aneurysm 4-5 cm in diameter should be indicated for surgery sooner. The question remains whether to indicate for early surgery patients with an unchanging diameter of an aneurysm who are in a good general condition without serious risks. Elective surgery is indicated in these aneurysms in view of the low incidence of ruptures only in departments where the surgical mortality is substantially lower than 5%. PMID- 10746083 TI - [Anti-Xa activity during administration of low-molecular weight heparin in the prevention of postoperative thromboembolic complications]. AB - The authors evaluate the follow-up of the anti-Xa level during the prophylactic administration of two different low molecular heparins (enoxaparin, reviparin) in 40 patients with a planned resection of the large intestine, resection of the pancreas, laparoscopic cholecystectomy and laparotomic cholecystectomy. The assessed amounts of anti-Xa did not reach recommended levels even during the perioperative period. The selection of the preparation and the extent of the operation did not influence the anti-Xa levels. PMID- 10746084 TI - [Delayed surgery in congenital diaphragmatic hernia without drainage of the ipsilateral hemithorax]. AB - The objective of the investigation was to evaluate the success of a new therapeutic protocol in patients with congenital diaphragmatic hernia (CDH). During the period from 1/1994 till 12/1998 41 patients with CDH were admitted. In 36 patients (88%) left-sided CDH was diagnosed, in 4 patients (10%) right-sided CDH and one neonate (2%) bilateral CDH. Fifteen cases (37%) of CDH were assessed prenatally. Twenty-two children (54%) were treated by inhalation of nitric oxide (INO) and 4 patients (10%) by extracorporeal membrane oxygenation. The total incidence of associated developmental defects was 20% and the total mortality 34%. On comparison of the surviving (group S, n = 27) and the non-surviving NS, n = 14) patients statistically significant differences were found in the Apgar score during the first minute (S: 5.9 +/- 0.5 vs. NS: 3.4 +/- 0.7, p < 0.008), in the oxygenation index (OI) two hours after admission (S: 11.9 +/- 2.9 vs. NS: 27.7 +/- 8.3, p < 0.03), in the alveolo-arterial oxygen difference (AaDO2) 2 hours and 12 hours after admission (S: 369 +/- 47 torr and 237 +/- 47 torr resp. vs. NS: 552 +/- 29 torr and 557 +/- 26 torr resp., p < 0.02) and in the need to start extracorporeal membrane oxygenation (S: 3.7% vs. NS: 21.4%, p < 0.009). The investigation confirmed a reduced mortality of neonates with CDH by introducing new therapeutic methods. Risk factors are early prenatal diagnosis, the presence of associated developmental defects, high values of oxygenation and ventilation with the necessity to start nitric oxide inhalation. PMID- 10746085 TI - [Injuries of the ureter treated at the General Medical School Hospital in Prague during the last 5 years]. AB - Injury of the ureter is the most frequent trauma in urological practice. The most important deal concerning a patient's expectancy is early diagnosis and adequate treatment. During 1994-1998 the authors have treated 65 patients with diagnosis an injury of the ureter. The most frequent cause was the iatrogenic injury. Timing of surgery was 5-6 weeks. A percutaneous nephrostomy was the most frequent method of initial treatment (38 patients). Ureterorhaphy (32 patients), ureterocystoneostomy (12 patients) and Boari's flap operation (5 patients) was the most frequent definitive method of the treatment. PMID- 10746086 TI - [Implantation of the T-tube in patients with postintubation tracheal stenosis]. AB - The authors submit an account on their experience with implantation of a Montgomery T-tube in patients with postcannulation stenosis of the trachea. The T tube was implanted in 10 patients during an 11-year period (1987-1997). In three patients the tube was inserted on account of restenosis which developed after resection of the trachea. In three patients resection treatment was not indicated because of the considerable length of the stenosis (60 mm), in two patients because of the finding of two stenoses at different levels of the trachea, in one patient because of a high stenosis of the trachea with penetrating granulation into the subglottic space and in one patient because of a medical contraindication. The diagnosis of stenosis and its character was based on tomograms of the trachea, bronchoscopy and possibly CT. The T-tube was implanted under general anaesthesia after revision of the tracheal stenosis and if necessary dilatation. The length of the tube always extended over the stenotic portion, in three patients it extended from the bifurcation to the stoma. The T tube was very well tolerated by the patients and was renewed usually after one year. Only in one patient it proved possible to perform decannulation and close the stoma by a plastic operation using costal cartilage. In the remaining patients the cannula is gradually reduced in length and in some possible decannulation with closure of the stoma in future is assumed. The authors consider the use of a T-tube in some patients with tracheal stenosis a very good therapeutic method which should be used at present in indicated cases. PMID- 10746088 TI - [Epistola chirurgica. V. Fracture of the metatarsal base]. PMID- 10746087 TI - [Epistola chirurgica. IV. Acute admissions]. PMID- 10746089 TI - [Trauma units or trauma teams?]. PMID- 10746090 TI - Measuring blood pressure in normal and hypertensive pregnancy. AB - The measurement of blood pressure is an integral part of antenatal care. Mercury sphygmomanometry is the gold standard for the determination of indirect blood pressure and has been used in pregnancy from the time blood pressure was first associated with adverse obstetric outcome. However, there is good evidence that current practice using mercury sphygmomanometry is far from perfect. This article will describe the history of blood pressure determination, why blood pressure is important in pregnancy and the problems associated with sphygmomanometry. It will discuss the advantages and disadvantages associated with alternative methods of blood pressure determination in pregnancy. Automated equipment should always be compared with mercury sphygmomanometry following a recognized protocol, such as that published by the British Hypertension Society, before its accuracy can be assumed in clinical practice. There is now good evidence that the accuracy of these devices deteriorates in women who develop pre-eclampsia. Home monitors have the potential to improve surveillance and to reduce in-patient monitoring. PMID- 10746091 TI - Classification of hypertension in pregnancy. AB - In many ways there should be no need to classify hypertensive disorders in clinical practice. The very presence of rising blood pressure should alert the clinician to seek evidence for the development of pre-eclampsia and whether there are any emerging abnormalities of fetal growth and/or maternal renal, cerebral, hepatic or coagulation functions which may necessitate specific treatment, including delivery. While such a view may be appropriate for experienced clinicians with an understanding of the pathophysiology of the hypertensive disorders of pregnancy, it is of little help to junior or less experienced medical staff. Moreover, without an agreed international classification system it is impossible to compare truly clinical outcome, intervention or basic research studies from different units as entry criteria to these studies may differ considerably across individual units and certainly across countries. In this chapter we highlight the limitations of the existing classification systems and propose a system that is based on our present understanding of the pathophysiology of pre-eclampsia. The proposed system is not a radical departure from previous classifications, with grouping of hypertensive subjects into gestational hypertension, pre-eclampsia and chronic (usually essential) hypertension. Proteinuria, while remaining a hallmark of pre-eclampsia, is no longer considered a 'sine qua non' for this disorder to be diagnosed, reflecting our greater understanding of the maternal and fetal abnormalities in pre eclampsia since previous classification systems were developed. This classification system has been compared with the traditional system of diagnosing proteinuric pre-eclampsia in a study of 1183 women with hypertension in pregnancy: diagnosing pre-eclampsia in this new manner still stratifies a high risk group of pregnant women and the proposed diagnosis of gestational hypertension in this system stratifies a group of women at low maternal and fetal risk, provided that continual maternal and fetal monitoring is employed. We hope that this system of classification can be adopted uniformly, permitting appropriate triage of pregnant women into higher and lower clinical risk groups while allowing us to compare 'apples with apples' in future research studies. PMID- 10746092 TI - What causes pre-eclampsia? AB - Pre-eclampsia remains a leading cause of maternal and fetal morbidity and mortality. Despite extensive research, the mechanisms that cause pre-eclampsia are unknown and it has been considered to be the 'disease of theories'. Hippocrates wrote in one of his Aphorisms that 'convulsions take place from either repletion or depletion'. Since then, obstetricians have been divided on the question of which factor accounted for the convulsions observed during childbirth. Some considered that a sudden reduction in intra-abdominal pressure at delivery led to a pooling of blood diverted from the brain, causing collapse of the cerebral blood vessels and convulsions. Others postulated that cerebral congestion, secondary to compression of the abdominal organs by the large uterus, diverted blood to the brain, causing eclamptic convulsions. It is the purpose of this review to examine those theories about the cause of pre-eclampsia for which modern evidence is available. At present, it is believed that the pathological chain of events leading to pre-eclampsia is scheduled in two steps: an absolute or relative placental ischaemia is followed by a diffuse endothelial cell activation, which causes the clinical features of the disease. PMID- 10746093 TI - Maternal pathophysiology in pre-eclampsia. AB - Pre-eclampsia is a common condition, unique to pregnancy, and almost unique among medical conditions in that we have, at best, only a patchy knowledge of the underlying aetiology and pathophysiology. In this chapter, established and new hypotheses will be discussed and the recent supporting evidence for each will be reviewed. The principal systems affected by pre-eclampsia, namely the cardiovascular, coagulation, liver and renal systems, together with cerebral manifestations of this disease will be discussed in turn. PMID- 10746094 TI - Clinical management of established pre-eclampsia. AB - This chapter summarizes the identification, assessment and management of women with established pre-eclampsia. Guidelines for out-patient management and in patient treatment are given and are based on clinical experience and published reports. Drug treatment options are briefly reviewed and the timing of delivery is discussed. A comprehensive regimen for the intrapartum management of severe pre-eclampsia is provided founded on protocols used in the Royal Victoria Infirmary, Newcastle upon Tyne, over recent years. The chapter emphasizes the importance of medical history, clinical examination and investigations when arriving at what are often difficult clinical decisions. Controversial areas are highlighted with reference to recent published reports. PMID- 10746095 TI - Clinical management of established pre-eclampsia and gestational hypertension: an anaesthetist's perspective. AB - Expert and aggressive pre-operative preparation of the woman with severe pre eclampsia will ultimately determine her intraoperative outcome. Such considerations as the effect of endotracheal manipulation on intracranial pressure, of thrombocytopenia on the potential to produce a compressive epidural haematoma following epidural or combined spinal-epidural neuraxial block and of adequacy of invasive monitoring for Caesarean section loom large in the eyes of an anaesthetist preparing such a patient for surgery. Time spent pre-operatively in fluid volume optimization, in assessment of ventricular function, filling pressures and systemic vascular resistance, on aspiration pneumonitis and seizure prophylaxis, on control of hypertension, on correction of coagulopathy and on attenuation of pressor responses is time well spent and will have profound effects on the peri-operative course. The choice of agents and techniques for control of hypertension and reduction of vascular resistance, for induction and maintenance of general anaesthesia, for eclampsia prophylaxis and for regional anaesthesia or analgesia for operative or spontaneous delivery is, likewise, important and, at times, problematic. PMID- 10746096 TI - Care of the woman with hypertension in pregnancy: the viewpoint of the midwife. AB - Key principles underpin good midwifery care of every pregnant woman, including a sound knowledge base, meeting the woman's needs and enhancing care. Normal antenatal care, with regular measurement of blood pressure, remains the mainstay of screening for hypertension in pregnancy. The midwife's role is that of accurate assessment and communication when detecting the first signs of hypertension. The midwife will then play a major role in the ongoing monitoring of the condition, liaison with the medical team and education of the woman and her family. Continuity of care is an important principle in avoiding errors in recording blood pressure between care givers; familiarity of a known midwife may reduce the likelihood of white coat hypertension. Advocacy should be employed by the midwife as a key link between the woman, her family and the obstetric team. Every woman who has experienced pre-eclampsia should be given the opportunity to talk through her care at a later date. This may be at the postnatal appointment or through a debriefing service. PMID- 10746097 TI - Chronic essential and secondary hypertension in pregnancy. AB - Hypertension is a relatively common complication of pregnancy, increasing in frequency in older women. It is not a contraindication to pregnancy, but should be fully investigated, correctable causes addressed and those with specific relevance for pregnancy identified. With close supervision and appropriate management, the majority of hypertensive pregnant women have successful outcomes. Ideally all women with chronic hypertension should be seen prior to a planned pregnancy, for explanation and discussion of the significance, risks and treatment plan and for adjustment of antihypertensive medication as necessary. Those charged with the antenatal and perinatal care of the patient should be familiar with the expected physiological changes in pregnancy and of the risks and benefits of any treatment given. Close communication among the patient, her obstetrician and consultant physician will ensure the most appropriate treatment and facilitate decisions regarding admission to hospital, timing and mode of delivery, and management issues in the early postpartum period. PMID- 10746098 TI - Prediction and prevention of pregnancy-induced hypertensive disorders. AB - Because hypertensive disorders complicating pregnancy are common and constitute a leading cause of maternal, fetal and neonatal morbidity and mortality, prevention attempts appear to be justified. Primary prevention is only possible by avoiding pregnancy. Secondary prevention requires identification of patients at risk. A large number of predictive methods have been published and the majority appear to be of no or limited value. At present only the determination of inactive urinary kallikrein and uteroplacental colour-pulsed Doppler velocimetry show promise and require further assessment. Analysis of the many interventions advocated for prevention of pre-eclampsia reveals that only dietary calcium supplementation and prophylactic low-dose aspirin have shown promise of efficacy in small controlled clinical trials, but the results of large, multicentre trials are disappointing. The disappointing results obtained in large, multicentre trials may be explained, at least in part, by the lack of strict eligibility criteria and end-points and by low patient compliance. Prophylactic low-dose aspirin is recommended in women at high risk because it is associated with a moderate reduction in risk, may reduce the severity of pre-eclampsia if it develops and appears to be safe for mother and infant. The present data do not support any prophylactic intervention in pregnant women at low or medium risk. PMID- 10746099 TI - Nutritional response of rats to faba beans (Vicia faba L. minor black 'Kalimattar') and its fractions. AB - The investigations on protein quality of raw faba bean seed meal (Vicia faba L. minor) and its fractions were carried out using rats in two experiments. Two varieties of faba seeds were used, locally grown faba bean 'Kalimattar' or 'black' (KSM) (492 g/kg diet) and its exotic counterpart Vicia faba L. minor 'White' (VFW) obtained from International Centre for Agricultural Research in the Dry Areas (ICARDA) (540 g/kg diet). The pulverized cotyledons of 'Kalimattar' (KMC; 438 g/kg diet), hull (KMH; 78 g/kg diet) as well as cotyledons of 'White' faba bean (VFWC; 470 g/kg diet) and hull (VFWH; 82 g/kg diet) fractions were tested. All diets were made isocaloric and isonitrogenous, however, without addition of individual amino acids to the target requirements. Performance of rats fed on both locally grown KSM and exotic FBW meal-fed rats were found to be inferior to those obtained from the rats fed on standard protein diet. The net protein utilization (NPU) of the rats fed on this seed meal was comparatively better than those obtained from the VFW. Replacement of standard protein with cotyledons from the either of the seed (KMC and VFWC) depressed food intake and protein utilization. This was due, in part, to the excretion of comparatively higher amount of nitrogen in faeces. Inclusion of KMH in the diet had no adverse effects on any variable studies as compared to the VFWH, which was found to impose severe restriction on the absorption of standard protein added to this diet. As a result, body weight gain, and NPU value were significantly lower than those obtained from the KMH. We conclude that the locally grown faba bean 'Kalimattar' is better than those of exotic 'White' cultivar and may contain less antinutritional substances. However, this warrants further investigations. PMID- 10746100 TI - Critical differences between two low protein diet protocols in the programming of hypertension in the rat. AB - Maternal nutrition has been identified as a factor determining fetal growth and risk of adult disease. In rats, the feeding of a low protein diet during pregnancy retards fetal growth and induces hypertension in the resulting offspring. Rat models of low protein feeding have been extensively used to study the mechanisms that may link maternal nutrition with impaired fetal growth and later cardiovascular disease and diabetes. Low protein diets of differing composition used in different laboratories have yielded inconsistent data on the relationship between maternal protein intake and offsprings' blood pressure. Two separate low protein diet protocols were compared in terms of their ability to programme hypertension during fetal life. Pregnant rats were assigned to receive one of four diets. Two diets were obtained from a commercial supplier and provided casein at 22 or 9% by weight (H22, control; H9, low protein). The other two diets, manufactured in our own facility, provided 18% casein (S18, control) or 9% casein (S9, low protein) by weight. The diets differed principally in their overall fat content, fatty acid composition, methionine content and the source of carbohydrate. Feeding of the experimental diets commenced on the first day of pregnancy and continued until the rats delivered their litters. Following weaning all the offspring had blood pressure determined on a single occasion. Both low protein diets reduced maternal weight gain relative to their corresponding control diets. Despite this litter sizes were unaffected by the dietary protocols. Both low protein diets reduced birthweights of the pups. Systolic blood pressure was significantly elevated in the offspring of rats fed a low protein S9 diet relative to all other groups (P < 0.05). Animals exposed to H9 diet in utero had similar blood pressures to their H22 controls. It is concluded from this work that differing low protein diet manipulations in rat pregnancy elicit different programming effects upon the developing cardiovasculature. The balance of protein and other nutrients may be a critical determinant of the long term health effects of maternal undernutrition in pregnancy. PMID- 10746101 TI - Antibiotic resistance of coagulase-negative staphylococci isolated from artisanal Naples-type salami. AB - In the present paper 42 isolates from Italian salami were specified as Staphylococcus xylosus (30), Staph. capitis (1), Staph. saprophyticus (1), Staph. hominis (1), Staph. simulans (1), Staph. cohnii (1) and as Staph. spp. (7). These strains were coagulase-negative and were examined for resistance/sensitivity against 25 antibiotics including beta-lactams (7), macrolides (3), amynoglicosides (5), glycopeptides, lincosamides (4) and novobiocin, fusidic acid, chloramphenicol, rifampicin, tetracycline, minocycline. More than 64% of the strains were resistant to lincomycin, penicillin G, amoxicillin, fusidic acid and novobiocin. All the strains were multiresistant and displayed at least three resistances. Over 75% had a multiple antibiotic resistance (MAR) index between 0.2 and 0.5. PMID- 10746102 TI - Adequacy of a food composition database to estimate fat and fatty acid intake. AB - This work examined the adequacy of the only food composition database available in Croatia to estimate fat and fatty acid intake. A 7-day food record with 39 participants was carried out. In addition to Croatian food composition tables (FCT1), the Swedish food tables (FCT2) were used to convert food records into quantities of total fat and saturated, monounsaturated and polyunsaturated fatty acids, respectively. The subjects simultaneously collected duplicates of all consumed food. The chemical analysis of duplicates to determine total fat and fatty acids included extraction of fat, followed by methylation of fatty acids and GC analysis. According to the comparison of methods, FCT2 are a better predictor of fat and fatty acid intake than FCT1 because of their closer agreement with analysed values. The correlation coefficients imply that both food tables estimate most of these nutrients similarly to a reference method. However, food records combined with either of the food tables could not predict intake of polyunsaturated fatty acids by men. In order to improve applicability of FCT1 in dietary surveys concerned with fat and fatty acids, the next issue should include changes in respect to an identified systematic error, as well as a greater choice of food items and their varieties. Accuracy of food records based on FCT1 could further be increased with subjects giving details on recipe ingredients, analysis of missing foods, etc. PMID- 10746103 TI - Low dietary cyanogen exposure from frequent consumption of potentially toxic cassava in Malawi. AB - In a cassava-growing area in Malawi, where roots are processed by soaking and water is available throughout the year, we interviewed 176 women farmers regarding their preferences for cassava cultivars and frequency of cassava consumption. Dietary cyanogen exposure was estimated from urinary levels of linamarin, the cyanogenic glycoside in cassava, and urinary thiocyanate, the main cyanide metabolite. Protection against unplanned harvest by family members, theft and animal spoilage were stated to be very important reasons for growing bitter cassava cultivars by 91%, 90% and 74% of the women, respectively. The mean (+/- SD) number of cultivars grown by each woman was 4.6 (+/- 2.4). The correlation between mean taste and mean danger scores for the 25 most grown cultivars was strong (r > 0.98). The scoring indicated that cultivars belonged to two distinct groups, eight to a group referred to as 'cool' and 17 to a group termed 'bitter'. The dumpling-like porridge (kondowole) made from cassava flour from bitter roots was eaten twice daily by 51% and at least weekly by 81%. The mean (+/- SEM) urinary linamarin was 14 (+/- 1) mumol/L and thiocyanate was 50 (+/- 4) mumol/L, less than a tenth of levels reported from populations eating insufficiently processed bitter cassava roots, and in the same range as in a non-smoking Swedish reference population. We conclude that cyanogenesis is a preferred characteristic of cassava by the studied farmers because it enhances food security. The availability of water and their knowledge about toxicity and processing enables these women farmers to provide a safe staple food from bitter cassava roots. PMID- 10746104 TI - Investigation of an in vitro model for predicting the effect of food components on calcium availability from meals. AB - The availability of calcium from semi-synthetic meals and model food systems was studied by an in vitro method using equilibrium dialysis after simulating gastric digestion. Twelve different food components (phytate, oxalate, wheat fibre- and barley fibre-extract, D-sorbitol, xylitol, galactitol, casein, three different casein phosphopeptide preparations, and lactose) were added to the semi-synthetic meal and their effect on the relative index of availability of 45Ca was studied. Phytate, oxalate, wheat fibre-extract and barley fibre-extract, and, surprisingly, casein had a negative effect on availability of calcium. The most pronounced effect was observed for the addition of phytate. The other dietary factors had no effect on calcium availability. Therefore, this rapid and inexpensive in vitro model could be a useful screening method for factors affecting calcium absorption, especially inhibitory factors, in future human calcium bioavailability studies. PMID- 10746105 TI - Effect of gamma irradiation on staphylococcal thermonuclease (TNase). AB - Stability of staphylococcal thermonuclease (TNase) on exposure to irradiation though expected has not been experimentally established. In the present work. TNase in presence of standard growth media as well as dairy and meat products was found to be completely resistant even at a dose of 10 kGy indicating that the TNase can be used as an indicator of Staphylococcal aureus contamination even in foods in which S. aureus have been destroyed by irradiation. This new application of TNase is being reported for the first time. PMID- 10746106 TI - The effects of sugar-free vs sugar-rich beverages on feelings of fullness and subsequent food intake. AB - This study compared the effects of equal volumes of sugar-rich and sugar-free beverages on feelings of hunger and fullness and the ad libitum consumption of a palatable, fat-rich snack. Eleven healthy males consumed equal volumes (375 mL) of three drinks (sugar-rich cola, sugar-free cola, mineral water) in random order on separate mornings. After 20 min, the subjects were able to snack freely on potato crisps during the next 90 min. Each subject's individual bowl of potato crisps was covertly replenished at 15 min intervals while the subjects were completing appetite and mood ratings. After the 110 min experimental period, the subjects' ad libitum food intake from a buffet-style lunch was covertly recorded. On leaving the laboratory, the subjects filled in a weighed food dairy for the rest of the day. The equal-volume preloads initially decreased hunger to a similar degree and potato crisp intake during the first 15 min interval was not significantly different among the three preloads. On average, total energy intakes from the crisps and lunch were not significantly different among the preloads, and by the end of the day, total energy intakes were similar for the three test conditions. Therefore, the low-calorie/low-sugar drinks did not facilitate a reduced energy intake by the lean, non-dieting male subjects. PMID- 10746107 TI - Relationship between knowledge of reasons underlying nutritional messages and reported compliance. AB - Subjects (48 male and 52 female) were shown nutritional messages and asked about their frequency of compliance and how this behaviour is beneficial to health. Scores were calculated for both knowledge and behaviour. The correlation between the two scores, after adjustment for effects of social class, age and gender, was 0.57 (P < 0.001). Although most previous research has failed to relate nutritional knowledge to behaviour, this may depend on the type of knowledge and the method of questioning and this deserves further study. PMID- 10746108 TI - Doctors as judges: the verdict on responsibility for health. PMID- 10746109 TI - Excess mortality from avoidable and non-avoidable causes in men of low socioeconomic status: a prospective study in Korea. AB - STUDY OBJECTIVE: The objective of this study was to evaluate the magnitude and contributory factors of socioeconomic differentials in mortality in a cohort of Korean male civil servants. DESIGN: A prospective observational study of male civil servants followed up for five years after baseline measurement. SETTING: All civil service offices in Korea. PARTICIPANTS AND MEASUREMENTS: The study was conducted on 759,665 Korean male public servants aged 30-64 at baseline examination in 1992. The grade of monthly salary of these participants divided into four groups, a proxy indicator of socioeconomic status (SES), was the main predictive variable. Mortality of the participants was followed up from 1992 to 1996. The causes of deaths were categorised into four groups according to the medical amenability: avoidable, partly avoidable, non-avoidable, and external causes of death. The risk of mortality associated with SES was estimated using the Cox proportional hazard model. MAIN RESULTS: Lowest SES group had significantly higher risk of mortality from most causes compared with the highest SES group in the order of external cause (relative risk (RR): 2.26), avoidable (RR: 1.65), all cause (RR: 1.59), and non-avoidable mortality (RR: 1.54). With the adjustment of known risk factors, significantly higher risks of mortality in lowest SES group were attenuated but persisted. Looking at the deaths from partly avoidable causes, significantly higher risks of mortality in the lowest SES group was observed from cerebrovascular disease but not from coronary heart disease. CONCLUSIONS: Socioeconomic differentials in non-avoidable as well as avoidable mortality, persisting even under the control of risk factors, suggest that mortality is influenced not only by the quality of health care and different distribution of risk factors but also by other aspects of SES that are yet unknown. PMID- 10746110 TI - Diabetes prevalence and socioeconomic status: a population based study showing increased prevalence of type 2 diabetes mellitus in deprived areas. AB - OBJECTIVE: To establish the relation between socioeconomic status and the age-sex specific prevalence of type 1 and type 2 diabetes mellitus. The hypothesis was that prevalence of type 2 diabetes would be inversely related to socioeconomic status but there would be no association with the prevalence of type 1 diabetes and socioeconomic status. SETTING: Middlesbrough and East Cleveland, United Kingdom, district population 287,157. PATIENTS: 4313 persons with diabetes identified from primary care and hospital records. RESULTS: The overall age adjusted prevalence was 15.60 per 1000 population. There was a significant trend between the prevalence of type 2 diabetes and quintile of deprivation score in men and women (chi 2 for linear trend, p < 0.001). In men the prevalence in the least deprived quintile was 13.4 per 1000 (95% confidence intervals (95% CI) 11.44, 15.36) compared with 17.22 per 1000 (95% CI 15.51, 18.92) in the most deprived. For women the prevalence was 10.84 per 1000 (95% CI 9.00, 12.69) compared with 15.48 per 1000 (95% CI 13.84, 17.11) in the most deprived. The increased prevalence of diabetes in the most deprived areas was accounted for by increased prevalence of type 2 diabetes in the age band 40-69 years. There was no association between the prevalence of type 1 diabetes and socioeconomic status. CONCLUSION: These data confirm an inverse association between socioeconomic status and the prevalence of type 2 diabetes in the middle years of life. This finding suggests that exposure to factors that are implicated in the causation of diabetes is more common in deprived areas. PMID- 10746111 TI - Employment grade differences in cause specific mortality. A 25 year follow up of civil servants from the first Whitehall study. AB - STUDY OBJECTIVE: To test the hypothesis that the association between socioeconomic status and mortality rates cuts across the major causes of death for middle aged and elderly men. DESIGN: 25 year follow up of mortality in relation to employment grade. SETTING: The first Whitehall study. PARTICIPANTS: 18,001 male civil servants aged 40-69 years who attended the initial screening between 1967 and 1970 and were followed up for at least 25 years. MAIN OUTCOME MEASURE: Specific causes of death. RESULTS: After more than 25 years of follow up of civil servants, aged 40-69 years at entry to the study, employment grade differences still exist in total mortality and for nearly all specific causes of death. Main risk factors (cholesterol, smoking, systolic blood pressure, glucose intolerance and diabetes) could only explain one third of this gradient. Comparing the older retired group with the younger pre-retirement group, the differentials in mortality remained but were less pronounced. The largest decline was seen for chronic bronchitis, gastrointestinal diseases and genitourinary diseases. CONCLUSIONS: Differentials in mortality persist at older ages for almost all causes of death. PMID- 10746112 TI - Unemployment and psychological distress one year after childbirth in France. AB - STUDY OBJECTIVE: To analyse the relation between unemployment and the psychological distress of mothers one year after childbirth. DESIGN: Multicentric survey concerning births occurring between September 1993 and July 1994. SETTING: In France: two maternity units in the Parisian area and one in Champagne Ardennes, in the east of France, comprising both urban and rural areas. PARTICIPANTS: Primipara and secondipara women were interviewed three times: at birth by a face to face interview, five months and 12 months after the birth, by postal questionnaires, with a 83% response rate for the two postal questionnaires. The analysis includes 632 women who answered all three stages of the survey. MEASUREMENTS: Psychological distress was mainly assessed one year after birth by the 12-item General Health Questionnaire. RESULTS: After adjustment for unwanted pregnancy, marital conflicts, marital status, hospitalisation of the baby during the last year, lack of confiding relationship, depressive or anxious troubles before pregnancy, age, educational level and parity, unemployed women had an excess of psychological distress compared with employed women (OR = 1.87; 95% CI = 1.12, 3.13). The ratios for housewives were very close to those of employed women. Among the unemployed women, 60% had recently been without a job, since a few months before or after the birth. An excess of psychological distress among unemployed compared with employed women was observed in all social groups defined by the current or last occupation, but with various extents. Psychological distress was specially linked to the employment status in the group of women with the more qualified occupations. CONCLUSION: Even after a birth, when women are very much involved in their maternal role, those seeking a job have worse mental health than those in a stable situation, either employed or housewives. In France, the unemployment rate among young women is high. It is specially important that social regulations protecting employment during and after pregnancy are adequately applied. Employers, legislators, such as medical doctors, have to be aware to this situation. PMID- 10746113 TI - Common symptoms in middle aged women: their relation to employment status, psychosocial work conditions and social support in a Swedish setting. AB - STUDY OBJECTIVE: Over the past few decades there has been a growing interest among researchers, in women's overall life circumstances and their relation to women's health status. For example, paid employment has been considered an important part of women's living conditions in Western societies as the number of women entering the labour market has grown constantly over the past decades. When comparing men's and women's health, one of the most consistent findings is a higher rate of symptoms among women. The most commonly reported symptoms in women are depressive symptoms, symptoms of bodily tension and chronic pain from muscles and joints. The aim of this study was to investigate whether socioeconomic factors, employment status, psychosocial work conditions and social network/support are associated with middle aged women's health status in terms of common symptoms. DESIGN: A mailed questionnaire was used in a cross sectional design assessing socioeconomic factors, employment status, psychosocial work conditions according to the demand/control model, social network/support and an index based on the 15 most frequent symptoms presented by middle aged women when seeking health care. SETTING: A rural community with 13,200 inhabitants in the western part of Sweden. PARTICIPANTS: Women were randomly selected from the general population in the study area, 40 to 50 years of age. The response rate was 81.7 per cent. MAIN RESULTS: Women who were non-employed had a significantly increased odds of a high level of common symptoms (OR = 2.82; 95% confidence intervals 1.69, 4.70), as well as women exposed to job strain (OR = 3.27; 1.92, 5.57), independently of the level of social network/support. Furthermore, exposure to low social support, low social anchorage or low social participation independently showed significantly increased odds of a high level of common symptoms (OR = 2.75; 1.71, 4.42; OR = 2.91; 1.81, 4.69 and OR = 1.69; 1.10, 2.61, respectively). CONCLUSIONS: Work related factors, such as non-employment and job strain, and circumstances within the private sphere, such as social network/support, seem equally important for middle aged women's health status. These findings ought to have important policy implications and also to be of major importance in a primary health care setting when meeting women who seek health care because of common symptoms. PMID- 10746114 TI - Disability and health status: ethnic differences among women in the United States. AB - STUDY OBJECTIVES: There are few data describing disability and health status for ethnic groups. The disablement process involves social influences, which may include minority status. Cross sectional data were examined to investigate the relation of ethnicity to disability. DESIGN: A stratified random digit dialled sample of women aged 40 and older. Disability and health status were measured as functional and activity limitations, work disability, and days of poor physical and mental health. SETTING: United States. PARTICIPANTS: Women interviewed by telephone included 774 white, 749 African-American, 660 Hispanic, and 739 Native American women. MAIN RESULTS: The prevalence of disability was higher among minority women when classified by general health status, and the need for personal care assistance. There was a striking excess of work disability: 3.5% of white women compared with 7.1% to 10.3% for minority women. The differences were reduced when adjusted for other risk factors and socioeconomic status. White and minority women reported more similar disability when it was defined by poor mental and physical health days. CONCLUSIONS: Disability is correlated with social and demographic characteristics as well as medical diagnoses. Ethnicity also is associated with disability and may be part of a social context for disablement. Future research should concentrate on the temporal sequence of disability. Consistent definitions of disability will facilitate this research. PMID- 10746115 TI - Income non-reporting: implications for health inequalities research. AB - OBJECTIVES: To determine whether, in the context of a face to face interview, socioeconomic groups differ in their propensity to provide details about the amount of their personal income, and to discuss the likely consequences of any differences for studies that use income based measures of socioeconomic position. DESIGN AND SETTING: The study used data from the 1995 Australian Health Survey. The sample was selected using a stratified multi-stage area design that covered urban and rural areas across all States and Territories and included non institutionalised residents of private and non-private dwellings. The response rate was 91.5% for selected dwellings and 97.0% for persons within dwellings. Data were collected using face to face interviews. Income response, the dependent measure, was binary coded (0 if income was reported and 1 for refusals, "don't knows" and insufficient information). Socioeconomic position was measured using employment status, occupation, education and main income source. The socioeconomic characteristics of income non-reporters were initially examined using sex specific age adjusted proportions with 95% confidence intervals. Multivariate analysis was performed using logistic regression. PARTICIPANTS: Persons aged 15-64 (n = 33,434) who were reportedly in receipt of an income from one or more sources during the data collection reference period. RESULTS: The overall rate of income non-response was 9.8%. Propensity to not report income increased with age (15-29 years 5.8%, 30-49 10.6%, 50-64 13.8%). No gender differences were found (men 10.2%, women 9.3%). Income non-response was not strongly nor consistently related to education or occupation for men, although there was a suggested association among these variables for women, with highly educated women and those in professional occupations being less likely to report their income. Strong associations were evident between income non-response, labour force status and main income source. Rates were highest among the employed and those in receipt of an income from their own business or partnership, and lowest among the unemployed and those in receipt of a government pension or benefit (which excluded the unemployed). CONCLUSION: Given that differences in income non-reporting were small to moderate across levels of the education and occupation variables, and that propensity to not report income was greater among higher socioeconomic groups, estimates of the relation between income and health are unlikely to be affected by socioeconomic variability in income non-response. Probability estimates from a logistic regression suggested that higher rates of income non-reporting among employed persons who received their income from a business or partnership were not attributable to socio-economic factors. Rather, it is proposed that these higher rates were attributable to recall effects, or concerns about having one's income information disclosed to taxation authorities. Future studies need to replicate this analysis to determine whether the results can be inferred to other survey and data collection contexts. The analysis should also be extended to include an examination of the relation between socio-economic position and accuracy of income reporting. Little is known about this issue, yet it represents a potential source of bias that may have important implications for studies that investigate the association between income and health. PMID- 10746116 TI - Individual and occupational determinants of low back pain according to various definitions of low back pain. AB - OBJECTIVES: To test associations between non-specific low back pain and several risk factors when definitions of low back pain vary. DESIGN/SETTING/PARTICIPANTS: A cross sectional study was set up in 1991, 725 workers from four occupational sectors answered a self administrated questionnaire including the Nordic questionnaire and questions about intensity of pain and individual and occupational factors. MAIN RESULTS: Prevalence of low back pain varied from 8% to 45% according to the definition used. Psychosomatic problems, bending or carrying loads were often associated to low back pain, whereas other risk factors were related to some specific dimensions of the disorder. CONCLUSIONS: Risk factors of low back pain vary with the definition. This could explain inconsistencies found in literature reviews. To be able to compare data, it seems important to be precise what definition is used and to use comparable questionnaires. PMID- 10746117 TI - Validation of self diagnosis of high blood pressure in a sample of the Spanish EPIC cohort: overall agreement and predictive values. EPIC Group of Spain. AB - STUDY OBJECTIVE: High blood pressure is a variable related to several chronic conditions whose repeated measurement in large cohort studies is often not feasible having to rely on the self reporting of the subjects. The aim of the study is to validate such self diagnosis in a sample of members from the Spanish EPIC cohort study. DESIGN: Comparison of high blood pressure self diagnosis with the information provided by the personal medical record drawn from the primary health centre of reference for such population. SETTING: A small town near the EPIC-Murcia centre, one of five Spanish EPIC centres located in the south east, where inclusion in the cohort was offered to the general population. PARTICIPANTS: The agreement between self reported high blood pressure status and data from medical records was measured in a representative sample of men and women (n = 248) aged 30-69 years. Medical records were studied for a diagnosis of high blood pressure, an anti-hypertensive pharmacological treatment or subject's inclusion in a hypertension control programme run in the medical centre only for hypertensive people (definite high blood pressure cases). As well, in the absence of such a diagnosis, medical annotations of systolic or diastolic high blood pressure > or = 140/90 mm Hg (possible high blood pressure cases) were considered. Sensitivity, specificity, positive and negative predictive values and kappa scores were calculated for all, definite and possible high blood pressure cases. Variables associated with the probability of having a true positive or negative self report of high blood pressure were also tested. MAIN RESULTS: As expected, sensitivity was higher among definite cases (72.7%) than among possible cases (31.6%). Accordingly, the agreement between self report and medical record was higher for definite cases (kappa = 0.65) than for possible (kappa = 0.29 cases leading to a moderate overall agreement for all cases (kappa = 0.58; 95% CI: 0.47, 0.69). Having some level of education (OR: 0.31; 95% CI: 0.09, 1.05) was negatively associated to a true self report of high blood pressure while being female was positively associated (OR: 4.01; 95% CI 1.04, 16.8). No variable showed any association with having a true self report of being normotensive. CONCLUSIONS: High blood pressure self report shows a moderate agreement with medical information in this cohort allowing it to be used, with caution, as a surrogate variable of actual blood pressure status. However, because of its moderate sensitivity, it is not possible to rule out some underestimation when using self reported high blood pressure information for high blood pressure frequency measurements such as prevalence or incidence rates. This underestimation will be higher among men and educated people. PMID- 10746118 TI - A sustainable programme to prevent falls and near falls in community dwelling older people: results of a randomised trial. AB - STUDY OBJECTIVE: In the causative mechanism of falls among older community dwellers, slips and trips have been found to be significant precursors. The purpose of the two year trial was to assess the effectiveness of multi-component interventions targeting major risk factors for falls in reducing the incidence of slips, trips and falls among the well, older community. DESIGN: Four groups with approximately equal numbers of participants were randomly allocated to interventions. The prevention strategies included education and awareness raising of falls risk factors, exercise sessions to improve strength and balance, home safety advice to modify environmental hazards, and medical assessment to optimise health. The interventions combined the strategies in an add on approach. The first intervention group receiving the information session only was regarded as the control. The outcome of interest was the occurrence of a slip, trip or fall, monitored prospectively using a daily calendar diary. PARTICIPANTS AND SETTING: Two hundred and fifty two members of the National Seniors Association in the Brisbane district agreed to participate. National Seniors clubs provide a forum for active, community dwelling Australians aged 50 and over to participate in policy, personal development and recreation. MAIN RESULTS: Using Cox's proportional hazards regression model, adjusted hazard ratios comparing intervention groups with the control ranged from 0.35 (95% CI 0.17, 0.73) to 0.48 (0.25, 0.91) for slips; 0.29 (0.16, 0.51) to 0.45 (0.27, 0.74) for trips; and 0.60 (0.36, 1.01) to 0.82 (0.51, 1.31) for falls. While calendar monitoring recorded outcome, it was also assessed as a prevention strategy by comparing the intervention groups with a hypothetical nonintervened group. At one year after intervention, reductions in the probability of slips, trips and falls (61 (95% CI 54, 66)%; 56 (49, 63)%; 29 (22, 36)% respectively) were demonstrated. CONCLUSIONS: This study makes an important contribution to the priority community health issue of falls prevention by showing that effective, sustainable, low cost programmes can be introduced through community-based organisations to reduce the incidence of slips, trips and falls in well older people. PMID- 10746119 TI - Reduced risk of low weight births among indigent women receiving care from nurse midwives. AB - STUDY OBJECTIVE: To examine the effect of a comprehensive prenatal and delivery programme administered by nurse-midwives on the risk of low weight births among indigent women. STUDY DESIGN: Historical prospective study. Birth outcomes among the cohort were compared with all county births during the same period, adjusting for maternal age and race. Results are expressed as relative risks with 95% confidence intervals. SETTING: An enhanced Medicaid funded pre-natal programme administered by nurse-midwives from 1992 to 1994 in Westchester County, New York. PARTICIPANTS: Indigent mothers (n = 1443), between the ages of 15 and 44, who were residents of Westchester County and indicated having Medicaid or no health care coverage. RESULTS: There were 1474 live births among cohort mothers. Mean (SD) gestational age was 39.4 (1.9) weeks. Less than 6% of births occurred before 37 weeks gestation. The mean birth weight of cohort infants was 3365.6 (518.6) g. Only 4.1% of the cohort births were less than 2500 g. Compared with all county births, the cohort showed a 41% reduction in the risk of low weight births (RRlbw = 0.59, 95% CI: 0.46 to 0.73, p < .001) and a 56% reduction when compared with county Medicaid births only (RR = 0.44, 95% CI: 0.34 to 0.57, p < .005) adjusting for maternal age and race. Larger reductions were found for very low weight births. CONCLUSIONS: Mothers need not be considered at high risk for adverse pregnancy outcomes based on their socioeconomic status alone. Moreover, a comprehensive prenatal programme administered by nurse-midwives may promote a reduction in adverse pregnancy outcomes among indigent mothers. PMID- 10746120 TI - Adult height is inversely associated with ischaemic stroke. The Caerphilly and Speedwell collaborative studies. PMID- 10746121 TI - Spatio-temporal tuning of VEPs: effect of mode of stimulation. AB - The spatial tuning function of the 8 Hz reversal Visual Evoked Potential (VEP) is frequently bimodal. This amplitude 'notch' largely disappears with 16 Hz pattern onset-offset (on-off) stimulation. To investigate the effect of temporal frequency on this phenomenon, we tested seven subjects using a range of rates centred on 8 Hz (reversal) and 16 Hz (on-off). With reversal, a deep notch was present at all temporal frequencies. A much shallower notch was evident with on off. The on-off and reversal functions differed only in their low spatial frequency branches (below 4.5 c deg-1) where, as temporal frequency decreased, the reversal response was reduced by 50%, whilst the on-off responses doubled in amplitude. Thus, although they behave differently with on-off and reversal, multiple spatial frequency mechanisms are evident with both forms of stimulation. PMID- 10746122 TI - Effects of sodium pentobarbital on the components of electroretinogram in the isolated rat retina. AB - Photovoltages, the fast P3(t) component of electroretinogram (ERG), were registered between two microelectrodes across the rod outer segments. The P2(t) component, obtained by subtracting the ERGs measured before the application of 50 microM APB from those measured after the application of 50 microM APB, was used as an indicator of depolarizing bipolar cell activity. Measurements of the scotopic threshold response (STR) and the oscillatory potentials (OPs) were used as indicators of third order neuron activity. The slow P3*(t) component, obtained by subtracting the photovoltages from the transretinal recording in the APB treated retina was used as an indicator of Muller cell activity. The components of the ERG obtained in normal superfusate medium were compared with those obtained in the presence of 100 microM sodium pentobarbital. We found that sodium pentobarbital slowed the kinetics of the P2(t) component and increased its latency. The fast P3(t) component was not affected by pentobarbital. The slow P3*(t) component was slightly reduced in the presence of pentobarbital. The minor components of the ERG, the STR and the OPs, were strongly suppressed by pentobarbital. These results suggest that in rat retina pentobarbital does not affect photoreceptors, but it does affect bipolar cells and Muller cells, and it suppresses activity of third order neurons. PMID- 10746123 TI - L, M and L-M hybrid cone photopigments in man: deriving lambda max from flicker photometric spectral sensitivities. AB - Using heterochromatic flicker photometry, we have measured the corneal spectral sensitivities of the X-chromosome-linked photopigments in 40 dichromats, 37 of whom have a single opsin gene in their tandem array. The photopigments encoded by their genes include: the alanine variant of the normal middle-wavelength sensitive photopigment, M(A180); the alanine and serine variants of the normal long-wavelength sensitive photopigment, L(A180) and L(S180); four different L-M hybrid or anomalous photopigments, L2M3(A180), L3M4(S180), L4M5(A180) and L4M5(S180); and two variants of the L-cone photopigment, encoded by genes with embedded M-cone exon two sequences, L(M2; A180) and L(M2; S180). The peak absorbances (lambda max) of the underlying photopigment spectra associated with each genotype were estimated by correcting the corneal spectral sensitivities back to the retinal level, after removing the effects of the macular and lens pigments and fitting a template of fixed shape to the dilute photopigment spectrum. Details of the genotype-phenotype correlations are summarized elsewhere (Sharpe, L. T., Stockman, A., Jagle, H., Knau, H., Klausen, G., Reitner, A. et al. (1998). J. Neuroscience, 18, 10053-10069). Here, we present the individual corneal spectral sensitivities for the first time as well as details and a comparison of three analyses used to estimate the lambda max values, including one in which the lens and macular pigment densities of each observer were individually measured. PMID- 10746124 TI - Sparse-sampling of gratings in the visual cortex of strabismic amblyopes. AB - Strabismic amblyopes show losses in positional acuity that cannot be explained by their resolution or contrast sensitivities. One hypothesis for these losses is a reduction in the density of cortical neurons that are driven by the amblyopic eye (cortical undersampling). The question this study addressed was whether the foveal representation of the amblyopic eye is undersampled in the cortex of strabismic amblyopes. In order to assess spatial sampling psychophysically, we recorded the perceived orientation of a stationary grating as a function of grating orientation and frequency in three strabismic amblyopes. To ensure high retinal contrast, the grating was imaged on the fovea of each observer using a laser interferometer. We found that the strabismic amblyopes misperceived the orientation of the grating at spatial frequencies that are a factor of two to six lower than the sampling frequency of the foveal cones. Since the retina and LGN in strabismic amblyopes are presumably normal, this result suggests sparse cortical sampling in the foveal representation of the amblyopic eye. Undersampling by cortical neurons may contribute to the spatial distortions present in strabismic amblyopic eyes. PMID- 10746125 TI - Identification of band-pass filtered letters and faces by human and ideal observers. AB - To better understand how the visual system makes use of information across spatial scales when identifying different kinds of complex patterns, we measured human and ideal contrast identification thresholds to estimate identification efficiency for 1- and 2-octave wide band-pass filtered letters and faces embedded in 2-D dynamic Gaussian noise. Varying stimulus center frequency from 1 to 70 c/object had different effects on letter and face identification efficiency. In the 2-octave conditions, identification efficiencies decreased by 0.25-0.5 log units for letters and 0.5-1.2 log units for faces as center frequency increased from 6.2 to 49.5 c/object, but only letters were identifiable at center frequencies below 6.2 c/object. In the 1-octave conditions, letter identification efficiencies increased by about 0.5 log units as center frequency increased from 1.1 to 2.2 c/object, and were nearly constant from 2.2 to 35 c/object. Letters were unidentifiable by human observers at 70 c/object. Surprisingly, face identification was impossible for human observers at all center frequencies except 8.8 c/object for one observer, and 8.8 and 17.5 c/object for a second observer. Ideal observer thresholds were obtained for both letters and faces in all conditions, so information was always available to perform the task. Thus, the failure to identify faces reflects constraints on visual processing rather than a lack of stimulus information. Selective spatial sampling may account for some of the differences between letter and face identification efficiencies. PMID- 10746126 TI - Chromatic induction with remote chromatic contrast varied in magnitude, spatial frequency, and chromaticity. AB - Chromatic induction from a surround is attenuated by chromatic contrast within a remote region outside of the surround (Shevell & Wei, 1998, Vision Research, 38, 1561-1566). The present study reports hue-cancellation measurements that show the attenuation depends on the magnitude, spatial frequency and chromaticity of remote chromatic contrast. Spatial-frequency tuning is shown by maximal attenuation of induction with remote contrast elements of the same size as the test. Experiments with various chromaticities of remote contrast show that S-cone stimulation within the remote region has a much weaker effect than L-/M-cone chromatic contrast, and does not depend on whether the S-cone stimulation is uniform or uneven across the region. Overall, the results show that remote L/M contrast affects classical chromatic induction, with its effect depending on the spatial frequency and magnitude of contrast. The influence of remote S-cone stimulation, on the other hand, is relatively weak and depends on only the S-cone spatial average, at least when S-cone stimulation by the test and its immediate surround is minimal (as in all experiments here). PMID- 10746127 TI - The neural deficit in strabismic amblyopia: sampling considerations. AB - In order to understand the nature of the neural loss in strabismic amblyopia, we have applied a technique which has been used in the normal periphery to psychophysically probe the sampling properties of the neuronal population. We ask whether there is a 'sampling' deficit and if so whether it is based on either an absolute loss of neurons (i.e. spatial undersampling) or an irregular arrangement of a normal number of neurons (i.e. irregular sampling). Our results suggest that neural pooling restricts the spatial frequency region where sampling considerations are important to a very small part of the visible high spatial frequency range. Within this limited region, irregular sampling rather than spatial undersampling is the greater contributor to the strabismic amblyope deficit. PMID- 10746128 TI - Visual completion of partly occluded grating in infants under 1 month of age. AB - Four groups of eight infants (3 weeks of age on average) were each habituated to one of four displays consisting of a grating of either low (0.4 cpd) or high (1.2 cpd) spatial frequency, whose central portion was covered up with a horizontal occluder which was either narrow (1.33 degrees) or broad (4.17 degrees). These habituation displays are referred to as LN (low spatial frequency grating and narrow occluder), LB (low and broad), HN (high and narrow), and HB (high and broad) displays. Posthabituation-test displays consisted of a complete grating (CG) of the same frequency as the habituated grating along with a separate grating (SG) whose central portion was replaced with a black gap of the same height as the occluder in the habituation displays. Infants habituated to the LN display looked significantly longer at the SG than the CG display during posthabituation-test trials. Infants habituated to the LB and HN displays looked at the CG and SG displays, almost equally. In contrast, infants habituated to the HB display looked longer at the CG than the SG display. These results show that infants under 1 month of age can perceive the continuation of the grating behind the occluder, and that their visual completion on habituation displays can be evoked according to the interaction between the spatial frequency of the grating and the occluder height. PMID- 10746129 TI - Is the size aftereffect direction selective? AB - We investigated whether the size aftereffect (apparent spatial-frequency shift after adaptation to slightly different frequencies) is direction selective; i.e., whether it is stronger for test stimuli moving in the adapting direction than the opposite direction. We used drifting sinusoidal gratings of various spatiotemporal frequencies for both adaptation and test stimuli, and the perceived test frequency was estimated by means of a matching technique with a staircase method. For the purpose of comparison, the post-adaptation threshold elevation was measured in addition to the size aftereffect. The results revealed that the direction of stimuli had no influence on the magnitude of the size aftereffect for a wide range of spatiotemporal frequencies, whereas the post adaptation threshold elevation showed clear direction selectivity. Although there was a significant direction selectivity for the size aftereffect at low spatial and high temporal frequencies, the selectivity was much weaker than that seen in the threshold elevation data. These findings are discussed in relation to the validity of a unified account of selective adaptation at and above threshold contrast and the notion of the separate processing of pattern and motion information. PMID- 10746130 TI - Adapting to expansion increases perceived time-to-collision. AB - We used a tracking method to measure errors in estimating absolute time-to collision caused by adapting to an expanding target. After adaptation, the estimated time-to-collision was longer than in the baseline condition. This was the case whether estimates were based on binocular information alone or monocular information alone. Estimates of time-to-collision were lengthened by 8-16% when based on binocular information alone, and by 18-25% when based on monocular information alone. These findings are consistent with a previous proposal that changing-size and changing-disparity information converge before the stage at which motion-in-depth signals are generated. PMID- 10746131 TI - Predicting the present direction of heading. AB - Humans perceive heading accurately when they rotate their eyes. This is remarkable, because (1) the pursuit eye movement makes the retinal flow more complicated; and (2) the eye rotation causes a continuous change of the heading direction on the retina. The first problem prevents a simple association of the centre of flow on the retina with the heading direction. To solve it, the brain needs to take into account the flow associated with the eye's rotation. But even if this is done correctly, the resulting estimate of the heading is retino centric and changing over time. Thus, the processing time to retrieve the heading from the flow field will cause a lag with respect to the actual heading direction. We investigated the latency for heading perception. We presented step wise changes of the centre of expanding flow to stationary and moving eyes. This mimics the movement of the heading direction across the retina, but avoids the complicating effects of rotational flow. For a stationary eye, we found a bias in perceived heading that corresponds to a latency of 300 ms or more. Yet, errors in heading perception are marginal normally, because we found an opposite bias for the moving eye, which counters the errors due to latency and a changing retino centric heading direction. This suggests that the current heading direction is predicted from the extra-retinal signal and the delayed visual signals. PMID- 10746132 TI - Optimal integration of texture and motion cues to depth. AB - We report the results of a depth-matching experiment in which subjects were asked to adjust the height of an ellipse until it matched the depth of a simulated cylinder defined by texture and motion cues. In one-third of the trials the shape of the cylinder was primarily given by motion information, in another one-third of the trials it was given by texture information, and on the remaining trials it was given by both sources of information. Two optimal cue combination models are described where optimality is defined in terms of Bayesian statistics. The parameter values of the models are set based on subjects' responses on trials when either the motion cue or the texture cue was informative. These models provide predictions of subjects' responses on trials when both cues were informative. The results indicate that one of the optimal models provides a good fit to the subjects' data, and the second model provides an exceptional fit. Because the predictions of the optimal models closely match the experimental data, we conclude that observers' cue-combination strategies are indeed optimal, at least under the conditions studied here. PMID- 10746133 TI - Scanning laser densitometry and color perimetry demonstrate reduced photopigment density and sensitivity in two patients with retinal degeneration. AB - PURPOSE: To test the feasibility of scanning laser densitometry with a modified Rodenstock scanning laser ophthalmoscope (SLO) to measure the rod and cone photopigment distribution in patients with retinal diseases. METHODS: Scanning laser densitometry was performed using a modified Rodenstock scanning laser ophthalmoscope. The distribution of the photopigments was calculated from dark adapted and bleached images taken with the 514 nm laser of the SLO. This wavelength is absorbed by rod and cone photopigments. Discrimination is possible due to their different spatial distribution. Additionally, to measure retinal sensitivity profiles, dark adapted two color static perimetry with a Tubinger manual perimeter was performed along the horizontal meridian with 1 degree spacing. RESULTS: A patient with retinitis pigmentosa had slightly reduced photopigment density within the central +/- 5 degrees but no detectable photopigment for eccentricities beyond 5 degrees. A patient with cone dystrophy had nearly normal pigment density beyond +/- 5 degrees, but considerably reduced photopigment density within the central +/- 5 degrees. Within the central +/- 5 degrees, the patient with retinitis pigmentosa had normal sensitivity for the red stimulus and reduced sensitivity for the green stimulus. There was no measurable function beyond 7 degrees. The patient with cone dystrophy had normal sensitivity for the green stimulus outside the foveal center and reduced sensitivity for the red stimulus at the foveal center. The results of color perimetry for this patient with a central scotoma were probably influenced by eccentric fixation. CONCLUSION: Scanning laser densitometry with a modified Rodenstock SLO is a useful method to assess the human photopigment distribution. Densitometry results were confirmed by dark adapted two color static perimetry. Photopigment distribution and retinal sensitivity profiles can be measured with high spatial resolution. This may help to measure exactly the temporal development of retinal diseases and to test the success of different therapeutic treatments. Both methods have limitations at the present state of development. However, some of these limitations can be overcome by further improving the instruments. PMID- 10746134 TI - Variance in transneuronal retrograde ganglion cell degeneration in monkeys after removal of striate cortex: effects of size of the cortical lesion. AB - The extent of transneuronal retrograde degeneration of ganglion cells in the primate retina depends on the age at which striate cortex was damaged, the survival time, the species, and retinal eccentricity. We here report on the effect of lesion size beyond striate cortex, which we assessed along with retinal ganglion cell degeneration in three groups of macaque monkeys who, in each group, had undergone striate cortical ablation at similar ages and survived for similar periods, which ranged from 302 days to 8 years. Where possible, the number of surviving projection neurones in the degenerated dLGN and its volume were also estimated. Results confirm that both geniculate and retinal degeneration correlate significantly with survival time but that the differences within a group can exceed differences between groups and are best accounted for by the extent of the damage to extra-striate visual cortex and underlying white matter. PMID- 10746135 TI - Anatomical and electrophysiological evidence for GABAergic bipolar cells in tiger salamander retina. AB - Our previous work showed that about 12% of bipolar cells in salamander retina synthesize and take up gamma-aminobutyric acid (GABA), are GABA transporter (GAT) immunoreactive, and respond with a GAT current to extracellularly applied GABA, suggesting that these bipolar cells use GABA, in addition to glutamate, as a neurotransmitter. Further support for this idea was obtained in this study by use of immunogold electron microscopy and whole-cell patch clamp electrophysiology. Ultrastructural analysis showed that amacrine cell and ganglion cell processes were postsynaptic to GABA-immunoreactive synapses made by bipolar cell axon terminals. Whole-cell recordings were obtained from amacrine and ganglion cells in response to activation of bipolar cells by puffing KCl at their dendrites in the outer plexiform layer. Inhibitory postsynaptic currents were observed in several third order neurons, even after blocking the excitatory postsynaptic responses, generated in the inner plexiform layer, with a combined application of NMDA and non-NMDA receptor antagonists, AP-5 and CNQX. These ultrastructural and electrophysiological data support our previous neurochemical results, and suggest that the retinal through-information pathway in salamander includes both inhibitory GABAergic as well as excitatory glutamatergic synaptic mechanisms. PMID- 10746136 TI - Sensitivity and dynamics of rod signals in H1 horizontal cells of the macaque monkey retina. AB - We measured the sensitivity, temporal frequency response, latency, and receptive field diameter of rod input to the H1 horizontal cell type in an in vitro preparation of the macaque retina. The H1 cell has both a cone-connected dendritic tree and a long axon-like process that terminates in a rod-connected arbor. We recorded from the H1 cell body where rod signals were distinguished by sensitivity to short wavelength light after dark adaptation. Receptive fields of rod vs. cone mediated responses were coextensive, indicating that the rod signal is transmitted via rod-cone gap junctions. Sensitivity of the H1 cell rod signal was approximately 1 log unit higher than that of the cone signal. Below cone threshold rod signals were temporally low-pass, with a cutoff frequency below 10 Hz. Rod signals became faster and more transient with increasing light levels. We conclude that the H1 cell rod signal is not sensitive in the low scotopic range and, by comparison with the rod signal recorded directly in cones (Schneeweis & Schnapf (1995) Science, 268, 1053-1056), signal transmission across the cone-H1 synapse does not significantly filter the temporal properties of the rod signal. PMID- 10746137 TI - Maturation of the pattern-reversal VEP in human infants: a theoretical framework. AB - Visual evoked potentials to pattern reversal (PR-VEPs) are used to assess the integrity and maturation of the visual pathways in infants and young children. To establish normal ranges and to facilitate interpolation, we consider the maturation rate of PR-VEPs using published normative data. Curves based on the logistic function (a sigmoid model) are introduced and compared with three other models: (1) the power law function; (2) the sum of two decaying exponentials; and (3) a two-stage linear model. Although methods vary somewhat, remarkable consistency among laboratories is found for the maturation of the major positivity (P1) of PR-VEP. The P1 occurs at approximately 260 ms in neonates and is quite variable. It matures rapidly before 12-14 weeks of age and becomes much less variable. The logistic model provides a parsimonious description of P1 maturation with most rapid maturation at around 6 weeks of age for large patterns and around 9 weeks for small patterns. As inter-laboratory agreement is generally good, the normal ranges based on this model could be used in centres, which do not have their own normative databases for infant VEPs. PMID- 10746138 TI - Vernier acuity with compound gratings: the whole is equal to the better of its parts. AB - To evaluate the relative importance of local feature and spatial filter models for hyperacuity, vernier thresholds were determined for gratings consisting of a fundamental (Fo) and third harmonic (3F) presented alone, and added together in compound gratings where the relative phase offsets of 3F to Fo was 0, 90, 180 and 270 degrees. Thresholds were determined for a range of spatial frequencies of Fo (0.5-16 c deg-1) for abutting and non-abutting stimuli. Compound grating vernier performance was found to be: (i) invariant with relative phase offset for the abutting and non-abutting conditions; and (ii) predictable from the vernier thresholds for the individual grating components making up the compound stimulus. The results support a view that supra-threshold components in a multi-frequency stimulus act independently and it is the spatial frequency content, not the local feature characteristics, which limit vernier performance. PMID- 10746139 TI - Perceptual learning on orientation and direction discrimination. AB - Two experiments were conducted to determine the extent to which perceptual learning transfers between orientation and direction discrimination. Naive observers were trained to discriminate orientation differences between two single line stimuli, and direction differences between two single-moving-dot stimuli. In the first experiment, observers practiced the orientation and direction tasks along orthogonal axes in the fronto-parallel plane. In the second experiment, a different group of observers practiced both tasks along a single axis. Perceptual learning was observed on both tasks in both experiments. Under the same-axis condition, the observers' orientation sensitivity was found to be significantly elevated after the direction training, indicating a transfer of learning from direction to orientation. There was no evidence of transfer in any other cases tested. In addition, the rate of learning on the orientation task was much higher than the rate on the direction task. The implications of these findings on the neural mechanisms subserving orientation and direction discrimination are discussed. PMID- 10746140 TI - The Frohlich effect: a consequence of the interaction of visual focal attention and metacontrast. AB - Usually we assume that the central nervous system preserves temporal sequences. Here we show that moving objects--in the context of behaviour often dangerous ones--are seen with a shorter latency than stationary (flashed) objects. In addition moving objects are deblurred. Two mechanisms contribute to this functional specialisation: cue-induced visual focal attention and metacontrast. Under unnatural conditions these mechanisms lead to an optical illusion first described by Frohlich [Frohlich, F. W. (1923). Uber die Messung der Empfindungszeit. Zeitschrift fur Sinnesphysiologie, 54, 58-78]. PMID- 10746141 TI - Discrimination of the speed and direction of global second-order motion in stochastic displays. AB - The ability to integrate local second-order motion signals over space and time was examined using random-dot-kinematograms (RDKs) in which the dots were defined by spatial variation in the contrast, rather than luminance, of a random noise field. When either the speeds or the directions of the individual dots were selected at random from a range of possible values, globally the stimulus appeared to drift either in a single direction or at a single speed in a manner analogous to that reported previously for first-order (luminance-defined) RDKs. To quantify the precision with which observers could extract the global stimulus motion, speed- and direction-discrimination thresholds were measured using pairs of RDKs, one of which (the comparison) comprised dots whose speeds or directions were assigned stochastically and the other (the standard) comprised dots that all had the same drift direction and speed. Speed-discrimination thresholds were of the order of 8% and changed little as the range of dot speeds (bandwidth) of the comparison increased, in that performance was almost as good when the individual dot speeds were selected at random from a range spanning 3.84 deg/s as when all the dots moved at the same speed. There was a tendency for the perceived global speed of the comparison RDK to decrease as the speed bandwidth was increased and perceived speed tended to coincide with the geometric mean speed of the dots rather than the arithmetic mean speed. Direction-discrimination thresholds were lowest (approximately 4 degrees) when the range of dot directions was less than 90 degrees but increased markedly thereafter. Observers were able to perform both discrimination tasks when the lifetimes of the dots comprising the RDKs was reduced from 25 to 2 frames, a manipulation that prevented observers from determining the overall speed or direction of image motion from the extended trajectories of individual dots within the display. Thresholds under these conditions were somewhat higher but were otherwise comparable to those obtained with a dot lifetime of 25 frames. The similarities between the present results and those of previous studies that have employed first-order RDKs suggest that the extraction of the global speed and direction of each type of motion is likely to be based on computationally similar principles. PMID- 10746142 TI - Perception of coherent motion, biological motion and form-from-motion under dim light conditions. AB - Three experiments investigated several aspects of motion perception at high and low luminance levels. Detection of weak coherent motion in random dot cinematograms was unaffected by light level over a range of dot speeds. The ability to judge form from motion was, however, impaired at low light levels, as was the ability to discriminate normal from phase-scrambled biological motion sequences. The difficulty distinguishing differential motions may be explained by increased spatial pooling at low light levels. PMID- 10746143 TI - Senescent changes in scotopic contrast sensitivity. AB - Scotopic contrast sensitivity functions (CSFs) were measured for 50 observers between the ages of 20 and 88 years. Using a maximum-likelihood, 2-alternative, temporal forced-choice threshold-estimation algorithm, scotopic CSFs were measured at 7 spatial frequencies ranging from 0.2 to 3.0 cpd, with mean retinal illuminance equated for observers at -0.85 log scotopic Trolands. For each stimulus condition, eight cycles of a horizontal sinusoidal grating were presented within +/- 1 S.D. of a 2-D Gaussian-spatial envelope and within a 1-s Gaussian-temporal envelope. Stimuli were centered on the nasal retina along the horizontal meridian 6 degrees from the fovea. Scotopic CSFs were found to be low pass. Statistically significant age-related declines in contrast sensitivities were found for spatial frequencies at or below 1.2 cpd. There was also a statistically significant decrease in the high frequency cut-off with age (P < 0.01). An explanation of these results in terms of optical factors is rejected, while the results are consistent with age-related changes in the magnocellular pathway. PMID- 10746144 TI - Discriminating mirror symmetry in foveal and extra-foveal vision. AB - The ability to discriminate perfect from imperfect mirror symmetry was examined at the fovea and at eccentricities out to 10 degrees in the nasal visual field. A 2-AFC method of constant stimuli was employed in which a bilaterally symmetric pattern was presented in one interval and a degraded version of this symmetric pattern in the other. The subject's task was to decide which interval contained the perfectly symmetric pattern. Pattern size was varied by changing the viewing distance. Probit analysis revealed the degree of asymmetry corresponding to 75% correct performance. Given sufficient size scaling, perfectly symmetric stimuli can be discriminated from degraded symmetric stimuli in extra-foveal vision. Spatial scaling with an E2 value similar to that for positional acuity was successful in removing the eccentricity dependence for the task. PMID- 10746145 TI - The stereoscopic (cyclopean) motion aftereffect is selective for spatial frequency and orientation of disparity modulation. AB - Across two experiments, this study investigated the spatial frequency tuning and orientation tuning (both in the disparity domain) of the stereoscopic (cyclopean) motion aftereffect. In Experiment 1, observers adapted to a moving stereoscopic grating of a given cyclopean spatial frequency and tested for the motion aftereffect with a static grating of the same or different spatial frequency. Robust motion aftereffects were induced only when the spatial frequency of the adapt and test stimuli was the same. In Experiment 2, observers adapted to a moving stereoscopic grating of a given cyclopean orientation and tested for the motion aftereffect with a static grating of the same or different orientation. Robust motion aftereffects were induced only when the orientation of the adapt and test stimuli was the same. Together, these results suggest that the stereoscopic motion aftereffect is tuned for cyclopean spatial frequency and orientation which, in turn, suggest that the stereoscopic motion aftereffect is mediated by low-level oriented spatial-frequency mechanisms. PMID- 10746146 TI - Display symmetry affects positional specificity in same-different judgment of pairs of novel visual patterns. AB - Deciding whether a novel visual pattern is the same as or different from a previously seen reference is easier if both stimuli are presented to the same rather than to different locations in the field of view (Foster & Kahn (1985). Biological Cybernetics, 51, 305-312; Dill & Fahle (1998). Perception and Psychophysics, 60, 65-81). We investigated whether pattern symmetry interacts with the effect of translation. Patterns were small dot-clouds which could be mirror-symmetric or asymmetric. Translations were displacements of the visual pattern symmetrically across the fovea, either left-right or above-below. We found that same-different discriminations were worse (less accurate and slower) for translated patterns, to an extent which in general was not influenced by pattern symmetry, or pattern orientation, or direction of displacement. However, if the displaced pattern was a mirror image of the original one (along the trajectory of the displacement), then performance was largely invariant to translation. Both positional specificity and its reduction in symmetric displays may be explained by location-specific pre-processing of the visual input. PMID- 10746147 TI - Eye and head coordination in reading: roles of head movement and cognitive control. AB - Simultaneous recording of eye and head movements during reading revealed that head movements consisted of two components: a modulatory-velocity component coupled to eye saccades, and a constant-velocity component that was independent of eye saccades. Whereas the constant-velocity component increased as subjects repeatedly read the same text, neither the magnitude of the modulatory-velocity component, nor the amplitude of the eye movement, increased. This outcome could be closely simulated when the head movement command was assumed to be stronger, and issued earlier with repeated reading. These results suggest that higher-level processes related to text familiarity modulate eye-head coordination through head movements. PMID- 10746148 TI - Saccades reduce latency and increase velocity of ocular accommodation. AB - Horizontal vergence can be stimulated binocularly with disparity (disparity vergence) or monocularly with accommodation (accommodative vergence). The latter results from a neural cross-coupling that causes both horizontal vergence and accommodation to respond when either one is stimulated [Alpern, M., & Ellen, P. (1956). American Journal of Ophthalmology, 42, 289-303]. The velocity of disparity and accommodative vergence is enhanced when accompanied by saccades [Enright, J. T. (1984). Journal of Physiology (London) 350, 9-31; Enright, J. T. (1986). Journal of Physiology (London) 371, 69-89]. Based upon the coupling between accommodation and vergence, we predicted that accommodation should also be facilitated by saccades. An SRI Dual Purkinje Eyetracker was used to measure left and right eye position, and the accommodation of the left eye, in response to stimulation. Horizontal saccades were stimulated by targets separated by 2-6 degrees and accommodation was stimulated monocularly over a range of +/- 2 diopters (D). When saccades occurred within 0-400 ms following a monocular step stimulus to accommodation, latency of accommodation decreased and the associated accommodative-vergence response was synchronized with the saccade. Saccades also enhanced the velocity of accommodation and accommodative-vergence, and this facilitation increased with saccade amplitude. Transient vergence responses that are normally associated with saccades [Erkelens, C. J., Steinman, R. M., & Collewijn, H. (1989). Proceedings of the Royal Society of London B. Biological Sciences, 236, 441-465; Maxwell, J. S., & King, W. M. (1992). Journal of Neurophysiology, 68 (4), 1248-1260] did not affect accommodation when it was not stimulated by defocus. Because saccades and accommodation utilize separate plants and final common pathways, the synchronization of saccades and accommodation and the enhanced velocity of accommodation and accommodative-vergence must occur at more central sites. Possibilities include the superior colliculus, which represents both accommodation and saccades [Nagasaka, Y., & Ohtsuka, K., (1998). Investigative Ophthalmology AVRO supplement], vestibular nuclei which project to regions near the oculomotor nuclei [Lang, W., Buttner-Ennever, J. A., & Buttner, U. (1979). Brain Research, 177, 3-17], and interactions between omni pause neurons and near response cells of the mesencephalic reticular formation (MRF) [Mays, L. E., & Gamlin, P. D. R. (1995a). Current Opinions in Neurobiology, 5, 763-768; Mays, L. E., & Gamlin, P. D. R. (1995b). Eye movement research: Mechanisms, processes and applications. New York: Elsevier] which represent both accommodation and vergence [Judge, S. J., & Cumming, B. G. (1986). Journal of Neurophysiology, 55, 915-930; Zhang, Y., Mays, L. E., & Gamli, P. D. R. (1992). Journal of Neurophysiology, 67, 944-960]. PMID- 10746149 TI - Neural dynamics of binocular brightness perception. AB - How does the visual cortex combine information from both eyes to generate perceptual representations of object surfaces? Important clues about this process may be derived from data about the perceived brightness of surface regions under binocular viewing conditions, including data about binocular brightness summation in response to Ganzfelds, the U-shaped data of Fechner's paradox that violates binocular brightness summation, and the effects of different combinations of monocular and binocular contours and surface luminance differences on threshold sensitivity to monocular flashes of light. How to reconcile these apparently contradictory data properties has been a severe challenge to previous models, and none has explained them all. The present article quantitatively simulates them all by further developing the FACADE vision model. Key model processes discount the illuminant and compute image contrasts in each monocular channel using shunting on-center off-surround networks; binocularly fuse these discounted monocular signals using shunting on-center off-surround networks with nonlinear excitatory and inhibitory signals; and use these binocularly fused activities to trigger filling-in of a binocular surface representation that represents perceived surface brightness. Previous models that have suggested explanations of subsets of these data are discussed. PMID- 10746150 TI - A highly sensitive ELISA for the quantification of polymyxin B sulfate in human serum. AB - A highly sensitive ELISA for the determination of polymyxin B sulfate (PMB) was developed which is capable of measuring as low as 32 pg/ml. Anti-PMB antibody was obtained by immunizing rabbits with PMB conjugated with mercaptosuccinyl bovine serum albumin (MS. BSA) using N-(gamma-maleimidobutyryloxy) succinimide (GMBS) as a heterobifunctional coupling agent. An enzyme marker was similarly prepared by coupling PMB with horseradish peroxidase (HRP) employing GMBS. This ELISA showed very low reactivity with the PMB analogue, polymyxin E (0.05%). The values for PMB concentration detected by this assay were comparable with those detected by the bioassay. Moreover, the ELISA was about 10,000 times more sensitive in detecting PMB at lower concentrations. Serum PMB concentration after the oral administration of a PMB tablet to human subjects was determined by the ELISA. PMB was rapidly absorbed from the gastrointestinal tract after the administration, then slowly decreased. These results indicate that the ELISA may be a valuable tool for studies of the pharmacokinetics and pharmacodynamics of the anti endotoxin drug, PMB. PMID- 10746151 TI - 16-Dehydropregnenolone 3-sulfate, its source and metabolism in the feto-placental unit. AB - We have investigated the serum concentration of 16-dehydropregnenolone (3 beta hydroxy-5,16-pregnadien-20-one) 3-sulfate (16-DHPS) in the umbilical artery (U.A.), umbilical vein (U.V.) and maternal vein (M.V.) to discover the origin of 16-DHPS. Although there was no significant difference between the levels of 16 DHPS in U.A. (18 +/- 15 ng/ml, mean +/- SD., n = 28) and U.V. (10 +/- 9 ng/ml, n = 28), these values were significantly higher (U.A., p < 0.001; U.V., p < 0.001) than that in M.V. (2 +/- 3 ng/ml, n = 28). These levels in the U.A. and U.V. did not fall in infants (30 +/- 18 ng/ml, n = 7) during the early neonatal period (2 7 d after birth). A significant correlation between the serum levels of 16-DHPS and 16-hydroxypregnenolone (3 beta, 16 alpha-dihydroxy-5-pregnen-20-one) 3 sulfate (16-OH-PregS), which may be the precursor steroid for 16-DHPS, was observed in the U.A. (r = 0.630, n = 28, p < 0.001), but not in the U.V. Moreover, this significant correlation persisted during the early neonatal period (p < 0.05, r = 0.842, n = 7), although the neonate had been separated from the maternal milieu. These results suggest that 16-DHPS originates in the fetus. To confirm the metabolic pathway of 16-DHPS (i.e. pregnenolone (3 beta-hydroxy-5 pregnen-20-one) 3-sulfate (PregS)-->16-OH-PregS-->16-DHPS), we investigated the correlation between the serum concentrations of the precursor steroid and the product in both the U.A. and U.V. A significant correlation was obtained between the serum concentrations of PregS and 16-OH-PregS both in the U.A. (p < 0.001, r = 0.563, n = 28) and U.V. (p < 0.05, r = 0.476, n = 27). As described above, the serum levels of 16-DHPS and 16-OH-PregS only correlated significantly in the U.A. These findings support the existence of the pathway, PregS-->16-OH-PregS-->16 DHPS, in the fetus. PMID- 10746152 TI - A one-step enzyme-linked immunosorbent assay for a novel osteoblast differentiation-promoting compound, TAK-778 in serum. AB - TAK-778, ((2R,4S)-(-)-N-[(4-diethoxyphosphorylmethyl)phenyl]- 1,2,4,5-tetrahydro 4-methyl-7,8-methylenedioxy-5-oxo-3-benzothiepin-2- carboxamide), is a novel osteoblast differentiation-promoting compound, and its sustained-release formulation is expected to be clinically used for the enhancement of fracture healing. To date, TAK-778 levels in serum have been measured using conventional reverse-phase HPLC with inferior sensitivity and time-consuming procedures. We have produced polyclonal antibodies against TAK-778 by using one of its derivatives coupled with a carrier protein, and developed a one-step ELISA for the determination of TAK-778 in serum. The antibodies had minimal cross reactivities to the biologically inactive metabolites including the oxidized-form (0.36%) and the cleavaged-form (0.00083%). The competitive ELISA was accomplished within three hours with a detection limit of 0.5 ng/ml serum, and the coefficients of variations for samples ranging from 1 ng/ml to 80 ng/ml were 1.1 4.4% in an intra-assay and 3.1-9.6% in an inter-assay. The present ELISA is so rapid, sensitive and selective for TAK-778 that it could be conveniently used in a clinical field for the determination of many serum specimens. PMID- 10746153 TI - Hypericin induces both differentiation and apoptosis in human promyelocytic leukemia HL-60 cells. AB - Hypericin is a unique photosensitizing plant pigment and has been separately reported to induce differentiation and apoptosis in neoplastic cells. In this study, we examined the relationship between activities to induce differentiation and apoptosis in human promyelocytic leukemia HL-60 cells, at a concentration range of 0.15 to 0.2 microM. When treated with hypericin, the cell ratio reducible of nitroblue tetrazolium was significantly increased and the cell size was enlarged by flow cytometry analysis. Hypericin also significantly increased the ratio of the cells, which were of positive alpha-naphthyl acetate esterase activity and phagocytic activity, whereas it hardly influenced the naphthol AS-D chloroacetate esterase activity in the cells, as well as 1 alpha, 25(OH)2D3 (10 nM). In addition, hypericin increased hypodiploid nuclei and caused a nucleosomal ladder. These results indicate that hypericin induces both differentiation toward monocyte/macrophage lineage and apoptosis in HL-60 cells. PMID- 10746154 TI - Ceramide enhances susceptibility of membrane phospholipids to phospholipase A2 through modification of lipid organization in platelet membranes. AB - The effects of ceramide on agonist-stimulated phospholipase A2 (PLA2) activity were studied in platelets. Cell-permeable C6-ceramide (N-hexanoylsphingosine) exogenously added to platelet suspension enhanced U46619-stimulated arachidonic acid release and lysophosphatidylcholine production. Treatment of platelets with sphingomyelinase also led to an enhancement of the release. The enhanced arachidonic acid release by exogenous ceramide was completely inhibited by methyl arachidonyl fluorophosphonate, a cytosolic PLA2 inhibitor. However, U46619 stimulated PLA2 activity was not significantly potentiated by ceramide. These results suggest that enrichment of ceramide in membranes causes modification of intermolecular organization, leading to increased susceptibility of substrate phospholipids to PLA2. PMID- 10746155 TI - The inhibitory effect of the toxic fraction from sea urchin (Toxopneustes pileolus) venom on 45Ca2+ uptake in crude synaptosome fraction from chick brain. AB - The effects of toxic peaks (P-I, P-II and P-III eluted from Sephadex G-200 column) from the sea urchin Toxopneustes pileolus on time-dependent 45Ca2+ uptake in chick P2 fraction (crude synaptosome fraction) were studied under physiological ionic conditions. Time-dependent 45Ca2+ uptake was inhibited by P II and P-III, but not by P-I. P-II had the greatest inhibitory effect. The inhibitory effect of P-II was not due to the inhibition of 45Ca2+ binding on P2 fraction, because P-II did not affect 45Ca2+ binding in osmotic-shocked P2 fraction. P-II did not affect KCl-stimulated 45Ca2+ uptake in P2 fraction, (Ca(2+)-Mg2+)-ATPase activity in the synaptic plasma membrane (SPM) fraction, or (Na(+)-K+)-ATPase and Mg(2+)-ATPase activities in osmotically-shocked P2 fraction. In contrast, the Na+/Ca2+ exchanger blocker 2',4'-dichlorobenzamil (DCB; 100 microM), with a poor specificity, inhibited not only time-dependent 45Ca2+ uptake but also KCl-stimulated 45Ca2+ uptake, (Ca(2+)-Mg2+)-ATPase, Mg(2+) ATPase and (Na(+)-K+)-ATPase. Involvement of Na(+)-Ca2+ exchanger in the time dependent 45Ca2+ uptake was ruled out, since it was not inhibited by replacement of Na+ with Li+ in reaction medium. These results suggested that the inhibition by P-II on time-dependent 45Ca2+ uptake appeared to be more specific than the commercially available Na+/Ca2+ exchanger blocker DCB, although the mechanism is not clear yet. PMID- 10746156 TI - Antiplatelet and antithrombotic activities of NQ301, 2-chloro-3-(4-acetophenyl) amino-1,4-naphthoquinone. AB - The antiplatelet and antithrombotic activities of a newly synthesized NQ301, 2 chloro-3-(4-acetophenyl)-amino-1,4-naphthoquinone, were investigated on human platelet aggregation in vitro and rats ex vivo, and murine pulmonary thrombosis in vivo. NQ301 potently inhibited ADP-, collagen-, epinephrine- and calcium ionophore A23187-induced human platelet aggregation in a concentration-dependent manner in vitro. NQ301 significantly inhibited platelet aggregation in orally administered rats ex vivo. NQ301 prevented death due to pulmonary thrombosis in mice dose-dependently in vivo. NQ301 also showed significant prolongation of tail bleeding time in conscious mice. However, NQ301 did not alter such coagulation parameters as activated partial thromboplastin time, prothrombin time, and thrombin time in human plasma. These results suggest that NQ301 may be a promising antithrombotic agent, and the antithrombotic activity of NQ301 may be due to antiplatelet aggregation activity but not to in vitro anticoagulation. PMID- 10746157 TI - An increase in histone acetylation and IL-2 antagonizing the immunoinhibitory effect are necessary for augmentation by butyrate of in vitro anti-TNP antibody production. AB - We investigated the role of histone acetylation in the promotion of antigen specific antibody production in murine B cells induced by sodium butyrate (NaBu) plus interleukin 2 (IL-2). NaBu dose dependently increased the acetylation levels of histone H4 at concentrations which effectively enhanced anti-trinitrophenyl (TNP) antibody production in the presence of IL-2. Among other short-chain fatty acids and NaBu analogs, propionate, valerate and vinylacetate were effective in the presence of IL-2 in increasing both antibody production and the histone H4 acetylation level, but acetate, alpha-, beta- and gamma-hydroxybutyrates and alpha-, beta- and gamma-aminobutyrates were not effective, even in the presence of IL-2. The effect of the specific histone deacetylase inhibitor trichostatin A (TSA), which enhances anti-TNP antibody production without IL-2, was markedly inhibited by adding NaBu simultaneously. However, the effect of TSA was neither inhibited nor potentiated by NaBu in the presence of IL-2. Splenic B cells treated with NaBu, TSA and both together in the presence or absence of IL-2 showed almost the same increased acetylation level of histone H4. These results suggest that the NaBu-induced enhancement of anti-TNP antibody production in the presence of IL-2 is mediated through a moderate increase in the level of histone acetylation and that NaBu has both stimulating and inhibiting activities for anti TNP antibody production, the latter of which is overcome by IL-2. PMID- 10746158 TI - Regulation of gastric mucosal pepsinogen and intrinsic factor contents, and their mRNA levels during starvation and refeeding in rats. AB - Gastric mucosal pepsinogen and intrinsic factor (IF) contents, and their mRNA levels during starvation and refeeding were studied. During starvation for 4 d, gastric mucosal pepsinogen and IF contents significantly decreased, whereas pepsinogen and IF mRNA levels increased by 30-50%. These results suggested that the mRNAs of pepsinogen and IF could be preserved for a long time so as to prepare for refeeding. After ceasing the starvation for 72 h, gastric mucosal pepsinogen and IF contents were significantly decreased at 1 h after refeeding, and their mRNA levels were increased by 20-30% at 30 min after refeeding. We examined whether the refeeding-induced changes in gastric mucosal pepsinogen and IF contents and their mRNA levels could be reproduced by the exogenous administration of secretagogues. They were not found to be affected by the administration of each secretagogue during starvation for 72 h at 30 min. However, by the simultaneous administration of 2 or 3 secretagogues (carbachol, cholecystokinin octapeptide (CCK-8) or secretin), the contents of pepsinogen and IF decreased to 70-80% and 50-80% of the control, respectively. However, their mRNA levels increased to 140-160% and 120-135% of the control, respectively. Therefore, refeeding-induced changes in pepsinogen and IF contents and their mRNA levels were partially reproduced by exogenously administered secretagogues. This showed that food intake influences huge changes in neural, hormonal and physical conditions on the stomach. It was indicated that the secretagogues stimulated not only pepsinogen and IF secretion, but also had a tendency to increase their mRNA. PMID- 10746159 TI - Exposure to hydrogen peroxide induces cell death via apoptosis in primary cultured mouse hepatocytes. AB - To investigate whether direct oxidant damage induces hepatotoxicity via an apoptotic cell suicide pathway, we exposed primary cultured mouse hepatocytes to pro-oxidant hydrogen peroxide. We demonstrate that brief exposure to a concentration of hydrogen peroxide (3 mM) can induce hepatocyte cell death via apoptosis as shown by toxicity assays, specific DNA staining, and the appearance of DNA laddering on agarose gels. When hepatocytes were treated with N acetylcysteine 15 min prior to hydrogen peroxide exposure, the cells were found to be protected from cytotoxicity and apoptosis. These results suggest that direct oxidative injury serves as a general trigger for apoptosis in the liver, and that other apoptotic stimuli, such as exposure to acetaminophen, also involve oxidative injury. Hydrogen peroxidase-induced apoptosis may serve as a valuable model for further studies of apoptosis in the liver. PMID- 10746160 TI - Protective effects of sodium-L-ascorbyl-2 phosphate on the development of UVB induced damage in cultured mouse skin. AB - The protective effect of sodium-L-ascorbyl-2 phosphate (As-2P), a stable form of ascorbic acid (AsA), against photodamage induced by a single dose of UVB exposure (290-320 nm, Max 312 nm) was investigated using cultured mouse skin. When the cultured skin was treated with various As-2P concentrations, the cutaneous AsA level increased in proportion to the As-2P concentration. After 3 h of incubation, the AsA level in the cultured skin treated with 2, 20 and 100 mM As 2P increased 1.03-, 2.17- and 6.27-fold, respectively, compared with that of the control skin. These results suggest that As-2P was transported into the cultured mouse skin where it was converted to AsA. After 3 h, the cutaneous AsA level in irradiated (20 kJ/m2) skin was depleted to a half of that in the control skin. However, the level in skin pretreated with 20 mM As-2P was maintained within normal limits, even after 24 h. Pretreatment with 20 mM As-2P significantly prevented such photodamage as sunburn cell formation, DNA fragmentation and lipid peroxidation, which were caused by a single dose of UVB irradiation. These results suggest that the protective effect of 20 mM As-2P on UVB-induced cutaneous damage is due to the maintenance of a normal As level by conversion of As-2P to As in skin tissue. PMID- 10746161 TI - Tinospora tuberculata suppresses nitric oxide synthesis in mouse macrophages. AB - We have obtained evidence that co-incubation of thioglycollate-elicited peritoneal macrophages with an aqueous extract of Tinospora tuberculata inhibits lipopolysaccharide-stimulated excessive production of nitric oxide (NO) in vitro. This effect is concentration-dependent and appears to involve suppression of both inducible nitric oxide synthase (iNOS) activity and NADPH-diaphorase activity, thus altering NO production. As NO is one of the critical mediators in various disorders and iNOS inhibitors may have therapeutic potential, these results may explain some aspects of the multifunctional properties of Tinospora tuberculata, which has been used in various folk remedies in southeast Asia and China. PMID- 10746162 TI - Nematocidal activity of picrodendrins against a species of diplogastridae. AB - To develop parasiticides, the nematocidal activity of 22 picrotoxane terpenoids, picrodendrins isolated from the Euphorbiaceae plant Picrodendron baccatum (L.) KLUG et URBAN, was measured using a species of Diplogastridae (Nematoda). Picrodendrin P displayed the most potent nematocidal activity with a minimum lethal concentration (MLC) value of 4.4 x 10(-3) M. The nematocidal activity of picrodendrin P was 9-fold more potent than that of kainic acid (4.0 x 10(-2) M) and santonin (4.0 x 10(-2) M) and 6-fold more potent than that of diethylcarbamazine citrate (3.0 x 10(-2) M). Thus, picrodendrin P may eventually be used as a leading parasiticide. In light of the relationship between the structure of picrodendrins and their nematocidal activities, potent nematocidal activity was found to require the following elements within the picrotoxane skeleton: the gamma-lactone that bridges C-3 and C-5, the epoxide group that bridges between C-6 and C-8, a cis-OCH3 group and a trans-CH2OH group related to carbonyl at alpha-methylene-gamma-lactone ring (C-18), and the absence of 4- and 8-hydroxyl groups. These results are the first demonstration of nematocidal activity for these terpenes and thus are important in furthering our understanding. PMID- 10746163 TI - Metabolism of 6"-O-xylosyltectoridin and tectoridin by human intestinal bacteria and their hypoglycemic and in vitro cytotoxic activities. AB - 6"-O-Xylosyltectoridin and tectoridin isolated from the flowers of Pueraria thunbergiana (Leguminosae), are metabolized to tectorigenin by human intestinal bacteria. Although tectoridin is metabolized to tectorigenin by most intestinal bacteria, 6"-O-xylosyltectoridin is metabolized to tectorigenin via tectoridin by only a few intestinal bacteria, such as Bifidobacterium breve K-110 and Eubacterium A-44. The metabolite, tectorigenin, had more potent hypoglycemic activity as well as in vitro cytotoxic activity against tumor cell lines than 6" O-xylosyltectoridin and tectoridin. These results suggest that 6"-O xylosyltectoridin and tectoridin are prodrugs which can be transformed to the active agents by human intestinal bacteria. PMID- 10746164 TI - Constituents in watercress: inhibitors of histamine release from RBL-2H3 cells induced by antigen stimulation. AB - Histamine release inhibitors in watercress (Nasturtium officinale) were isolated using a monitoring system with antigen-stimulated RBL-2H3 cells. Of the 15 compounds isolated, flavonols and megastigmanes significantly inhibited histamine release. Two flavonols, 3-O-sophorosides of rhamnetin and rhamnazin, were new compounds. To investigate the inhibitory mechanism, the effects of rhamnetin, rhamnetin 3-O-sophoroside and an isolated megastigmane glucoside on the increase in the intracellular free calcium concentration were examined at a concentration providing 60% inhibition of histamine release. The results suggest that these compounds did not affect the calcium influx at that concentration. The structure activity relationships of the megastigmanes on histamine release were also investigated. PMID- 10746165 TI - Beneficial effects of sanguisorbae radix in renal dysfunction caused by endotoxin in vivo. AB - The effect of Sanguisorbae Radix extract, a traditional crude drug, was investigated in renal dysfunction induced by lipopolysaccharide (LPS) endotoxin. Injection of LPS in rats resulted in a sharp rise in the serum levels of urea nitrogen and creatinine (Cr), indicating impairment of renal function. Nitrite and nitrate levels and the activity of inducible nitric oxide synthase (iNOS), an enzyme which participates in NO synthesis, were also significantly increased in the serum of LPS-treated rats compared with normal rats. In rats pretreated with Sanguisorbae Radix extract, renal dysfunction was attenuated and the increases in serum urea nitrogen and Cr induced by LPS were significantly reduced. The administration of Sanguisorbae Radix extract also effectively lowered serum nitrite/nitrate level. A similar effect was observed on the iNOS activity. These results indicate that Sanguisorbae Radix extract contributes to the regulation of renal function under conditions where there is excessive generation of NO. PMID- 10746166 TI - Biodistribution of gadolinium incorporated in lipid emulsions intraperitoneally administered for neutron-capture therapy with tumor-bearing hamsters. AB - Emulsions containing a distearylamide (Gd-DTPA-SA) or a distearylester (Gd-DTPA SE) of Gd (gadolinium)-diethylenetriaminepentaacetic acid (Gd-DTPA) were intraperitoneally injected in Greene's melanoma-bearing hamsters at a dose of 2.0 ml (3.0 or 6.0 mg Gd) per hamster. In the standard-Gd and high-Gd formulations used, the weight ratios of soybean oil, water, Gd-DTPA derivative (Gd-DTPA-SA or Gd-DTPA-SE), hydrogenated L-alpha-phosphatidylcholine from egg yolk (HEPC) and co surfactant (HCO-60, Myrj 53, Myrj 59 or Brij 700) were 7.36:92:1:2:3 and 7.36:92:2:1:3, respectively. When the effects of the co-surfactants on the biodistribution of Gd from Gd-DTPA-SA-containing emulsions in the standard-Gd formulation were compared, the HCO-60 emulsion exhibited the highest Gd accumulation in tumors, possibly resulting from its fast and complete absorption, its small particle size (78 nm) and the stable coat on the particle surfaces with polyoxyethylene. Brij 700 emulsion kept the highest blood Gd concentration for a prolonged period, possibly due to particle properties similar to those of HCO-60. However, it exhibited a slower Gd accumulation in tumors, only reaching an identical level, in comparison with the HCO-60 emulsion. This suggested the tumor to be saturated with lipid particles. When Gd-DTPA-SE was used instead of Gd-DTPA SA, its HCO-60 emulsion exhibited only very poor Gd-accumulation due to its easy degradation. The HCO-60 emulsion particles containing Gd-DTPA-SA in the high-Gd formulation (6.0 mg Gd in 2 ml) exhibited in vivo behavior identical to those in the standard-Gd formulation; then the Gd level in tumors reached 107 micrograms Gd/g tumor (wet), and the tumor:blood (T/B) and tumor:skin (T/Sk) Gd concentration ratios were 13.2 and 5.6, respectively, at 48 h after intraperitoneal administration. These results suggest that when intraperitoneally administered, this HCO-60 emulsion, and possibly also the corresponding Brij 700 emulsion, may be an excellent delivery system for accumulating Gd in tumors in neutron-capture therapy (NCT). PMID- 10746167 TI - Pharmacokinetic/pharmacodynamic analysis of tacrolimus-induced QT prolongation in guinea pigs. AB - Recently, several reports of clinical cases of QT prolongation and torsades de pointes, associated with the use of tacrolimus (FK506), have come to light. We have previously demonstrated FK506-induced QT prolongation in guinea pigs [Minematsu T., et al., Life Sci., 65, PL197-PL202 (1999)]. We now examined the relationship between QTc prolongation and the pharmacokinetics of FK506 in guinea pigs, in order to evaluate the arrhythmogenicity of FK506 when compared with that of quinidine sulfate (QND). Thus, dose-response relationships for FK506 (0.01 or 0.1 mg/h/kg) or QND (30 mg/h/kg) were investigated during and after intravenous infusion and also following intravenous bolus administration of FK506 (0.2 mg/kg). The dose-response relationship between plasma drug concentration and QTc prolongation for FK506 and QND were subsequently analyzed using an effect compartment model. The pharmacodynamic parameters thus obtained were as follows: kE0 2.72 x 10(-4) (min-1), Emax 27.1 (ms), EC50 0.376 (ng/ml) for FK506; and kE0 0.148 (min-1), K 8.41 (ms.ml/microgram) for QND. The anti-clockwise hysteresis observed for FK506-induced QT prolongation was successfully analyzed by the present pharmacokinetic/pharmacodynamic model, which may provide a rational basis for developing a clinical dosing regimen to avoid possible QT prolongation induced by FK506. PMID- 10746168 TI - Prodrugs for systemic bioreductive activation-independent delivery of phyllohydroquinone, an active form of phylloquinone (vitamin K1). 1: Preparation and in vitro evaluation. AB - With the aim of overcoming the delivery problems (water-solubility and bioreductive activation problems) of phyllohydroquinone (PKH), an active form of phylloquinone (PK, vitamin K1), the N,N-dimethylglycine esters of phyllohydroquinone (1-mono, 1; 4-mono, 2; and 1,4-bis, 3) have been synthesized and assessed in vitro as a prodrug for the systemic bioreductive activation independent delivery of PKH. The hydrochloride salts of the esters were found to be quite soluble in water. Hydrolysis of the esters in rat liver S9 fraction, rat plasma and phosphate buffer, pH 7.4, at 37 degrees C, was kinetically studied in the presence and absence of an esterase inhibitor. The hydrolysis was catalyzed by esterases located in rat liver and rat plasma and quantitatively yielded PKH. The enzymatic cleavage and the vitamin K-dependent carboxylation activity of the esters in the rat liver microsome preparation at pH 7.2 and 25 degrees C were studied. The regeneration of PKH from the esters was catalyzed by carboxylesterases located in the rat liver microsome, and the order was as follows: 1 > 3 > 2. The carboxylation was stimulated by selected ester 1 in the absence of dithiothreitol, an activator of the vitamin K cycle. The carboxylation activity of 1 was strongly inhibited in the presence of eserine, a carboxylesterase inhibitor. Compound 1 could also stimulate carboxylase under warfarin-poisoning conditions, where the vitamin K cycle was strongly inhibited. These results indicated that these highly water-soluble and liver-esterase hydrolyzable ester derivatives of PKH are potential candidates for parenteral prodrugs which can thus achieve the systemic bioreductive activation-independent delivery of PKH. PMID- 10746169 TI - Cellular pharmacokinetic aspects of reversal effect of itraconazole on P glycoprotein-mediated resistance of anticancer drugs. AB - The reversal effect of itraconazole on P-glycoprotein (P-gp)-mediated resistance of vinblastine, daunorubicin and doxorubicin was analyzed from a cellular pharmacokinetic point of view, namely by [3H]azidopine photoaffinity labeling, intracellular accumulation and transcellular transport experiments. The LLC-GA5 COL150 cells, which expressed human P-gp selectively on the apical membrane due to transfection of MDR1 cDNA into the porcine kidney epithelial cells (LLC-PK1 cells), was used here, since this cell line constructs the monolayer with tight junction, being able to characterize the cellular pharmacokinetics. In LLC-GA5 COL150 cells, itraconazole caused a reversal from resistance as shown by a growth inhibition assay. [3H]Azidopine photoaffinity labeling demonstrated that itraconazole, vinblastine, daunorubicin and doxorubicin showed higher binding ability for P-gp compared with digoxin, suggesting the following results were via P-gp. The intracellular accumulation of [3H]vinblastine, [3H]daunorubicin and [14C]doxorubicin after their application on the basal and apical sides was increased by itraconazole. These changes were similar to the dose modifying factors determined by the growth inhibition assay. However, their basal-to-apical transport was hardly affected by itraconazole, and this was explained by the fact that itraconazole inhibited P-gp, and subsequently increased their intracellular concentration and then the non-P-gp mediated transport from the intracellular space to apical side. The apical-to-basal transport of [3H]vinblastine, [3H]daunorubicin and [14C]doxorubicin was increased by itraconazole, and this was reasonably explained by the inhibition of P-gp, and partly also by the increase of their intracellular concentration via the inhibition of P-gp. PMID- 10746170 TI - Isolation of bovine serum albumin fragment P-9 and P-9-mediated fusion of small unilamellar vesicles. AB - Fusion peptide P-9 was isolated from bovine serum albumin by controlled pepsin degradation in the presence of caprylic acid, followed by Sephadex G-75 gel filtration and ion-exchange chromatography of CM-cellulose. By this procedure, P 9 could be strictly separated from peptic fragment P-Phe, which has a molecular weight close to that of P-9. P-Phe has no fusogenic activity. The addition of P-9 to phosphatidylcholine (PC) liposomes containing cholesterol (Chol) gave rise to an increase of absorption intensity at around pH 4.0. The increase of turbidity by P-9 addition did not decrease with increasing pH, indicating P-9-mediated fusion of PC liposomes. The extent of the fusion of PC liposomes was strongly dependent on the PC chain length and temperature. The membrane fluidity close to the polar head groups of the fatty acyl chains of PC affected markedly the extent of P-9-mediated liposome fusion. However, there was no correlation between membrane fluidity near the hydrophobic end of the fatty acyl chains and the extent of liposome fusion. The rate of liposome fusion was dependent on both lipid composition and PC chain length. These results suggest that a contact or an interaction of P-9 with liposomal membrane occurs in the rigid regions. The character of the membrane-water interface region in the liposome controls a triggering effect for P-9-mediated fusion. PMID- 10746171 TI - The relationship between gastrointestinal transit and motility in dogs with postoperative ileus. AB - We investigated the relationship between gastrointestinal (GI) transit and motility during postoperative ileus in dogs undergoing a single laparotomy. We combined X-ray radiography for a GI transit study with chronically implanted force transducers (FTs) for a GI motility study. Radio-opaque markers made of polyethylene and steel wires or barium sulfate were used to examine solid substance transit or liquid substance transit. For a while after the end of the operation, postoperative ileus was observed, with weak irregular contractions of the GI tract. Transmission of the contractions to the lower GI tract was then observed. The start point of interdigestive migrating contraction (IMC)-like motility was observed in the order of small intestine (I-IMC), duodenum (D-IMC), and stomach (G-IMC), and IMC proceeded gradually after the operation. The gastric emptying time of a solid marker was 73.6 +/- 2.3 h (n = 5), and depended on the time of first occurrence of G-IMC (r = 0.674, p = 0.006). The gastric emptying of the liquid marker was finished before the time of the first occurrence of G-IMC, and its small intestinal transit time correlated with the time of the first occurrence of G-IMC (r = 0.888, p = 0.018). Using combined X-ray radiography and FTs we found that recovery from postoperative ileus was aided by GI motility in which contractions were transmitted from the stomach to the lower GI tract, like IMC. PMID- 10746172 TI - Escherichia coli O157 strains which caused Japanese outbreaks have residues of bacteriophage sequences. AB - Twelve strains of Escherichia coli O157 which caused outbreaks in Japan were used as DNA sources. The sequences of the gene encoding the Shiga toxin 2 in all 12 strains were almost identical and the sequences downstream of this gene were similar to that of bacteriophage 933W. PMID- 10746173 TI - Pyridazines. XVIII. 6-Aryl-3(2H)-pyridazinones inhibit calcium influx in stimulated platelets. AB - 6-Phenyl-5-hydroxymethyl-4,5-dihydro-3(2H)-pyridazinone (1) and 6-thienyl-5 hydroxymethyl-4,5-dihydro-3(2H)-pyridazinone (2) inhibit platelet aggregation induced by thrombin (IC50 = 0.25 and 0.26 mM, respectively) or by the calcium ionophore ionomycin (IC50 = 0.42 and 0.43 mM, respectively). Pyridazinones 1 and 2 also show concentration-dependent attenuation of the increases in platelet cytosolic free calcium concentration induced by thrombin and ionomycin, suggesting that their antiaggregatory activity may be due to their capacity to inhibit the passage of calcium through the cytoplasmic membrane. This effect may be implicated in other pharmacological activities of 6-aryl-5-substituted pyridazinones. PMID- 10746174 TI - Effect of 3,6-dimethylpyrazine-2-thiol on androstenedione-induced increase of uterine weight in female rats. AB - 3,6-Dimethylpyrazine-2-thiol administered at 10-70 mg/kg, p.o. was found to suppress androstenedione-induced increase of uterine weight in female rats. This action was weaker than that of aminoglutethimide (3-30 mg/kg, p.o.). After administration of androstenedione, increased plasma estradiol levels were reduced by 3,6-dimethylpyrazine-2-thiol. Moreover, in vitro, production of estradiol in the pregnant mare serum gonadotropin (PMSG)-treated ovary was inhibited by 3,6 dimethylpyrazine-2-thiol. These results suggest that 3,6-dimethylpyrazine-2-thiol has an inhibitory action on aromatase activity. PMID- 10746175 TI - Z-335, a thromboxane A2 receptor antagonist, suppresses the progression of arachidonic acid-induced hind limb gangrene in rats. AB - We have developed a new rat model of gangrenous peripheral vascular disease with vascular injury and occlusive thrombi. Rat hind limb gangrene was induced by injecting arachidonic acid (2 mg/leg) into the femoral artery. Using this model, we evaluated the effect of a thromboxane A2 receptor antagonist, Z-335, on the progression of hind limb gangrene. Z-335 (10 mg/kg/d, p.o.) ameliorated arachidonic acid-induced hind limb gangrene. In contrast, daltroban (10 mg/kg/d, p.o.) and cilostazol (100 mg/kg/d, p.o.) tended to improve the hind limb gangrene but their effects failed to reach statistical significance. Z-335 (10 mg/kg, p.o.) inhibited U-46619-induced, but not collagen-induced, platelet aggregation in rat whole blood. Daltroban (10 mg/kg, p.o.) showed a tendency to inhibit U 46619-induced platelet aggregation. Cilostazol (100 mg/kg, p.o.) did not inhibit U-46619- or collagen-induced platelet aggregation. Histopathological examination of the injured paws showed that Z-355 (10 mg/kg, p.o.) had partly inhibited the formation of occlusive thrombi. These results indicate that the thromboxane A2 receptor antagonist Z-335 is effective against arachidonic acid-induced hind limb gangrene in rats. Our experiments suggest that Z-335 may be beneficial in the prevention of gangrenous peripheral vascular disease. PMID- 10746176 TI - Antagonism of saikosaponin-induced prostaglandin E2 release by baicalein in C6 rat glioma cells. AB - There are several Kampo medicines (Chinese herbal medicines) containing both Bupleuri Radix and Scutellariae Radix, which are used for the treatment of inflammation. Saikosaponins are derived from Bupleuri Radix, and baicalein is from Scutellariae Radix. The present study was undertaken to investigate the pharmacological interaction of saikosaponin b1 and baicalein in prostaglandin E2 (PGE2) release from C6 rat glioma cells in vitro. Saikosaponin a, b1 and d potently stimulated PGE2 release, while saikosaponin b2 and c moderately stimulated PGE2 release. Saikosaponin b1 caused an irreversible elevation of intracellular Ca2+ concentration, which was eliminated by removing extracellular Ca2+. On the other hand, baicalein inhibited saikosaponin b1-induced PGE2 release in a concentration-dependent manner. These results suggest that saikosaponins are activators of PGE2 release, and baicalein is one of the functional inhibitors of PGE2 release by saikosaponins. PMID- 10746177 TI - Marked reduction in the minimum inhibitory concentration (MIC) of beta-lactams in methicillin-resistant Staphylococcus aureus produced by epicatechin gallate, an ingredient of green tea (Camellia sinensis). AB - We found that epicatechin gallate, a constituent of an extract of tea leaves (green tea) markedly lowered the minimum inhibitory concentration (MIC) of oxacillin and other beta-lactams, but not of other antibacterial agents tested, in strains of methicillin-resistant Staphylococcus aureus. The antibacterial action of epicatechin gallate plus oxacillin was a bactericidal one. PMID- 10746178 TI - Metabolic pathways and pharmacokinetics of BOF-4272, a sulfoxide-containing drug, in the dog: in vivo and in vitro studies. AB - BOF-4272, (+/-)-8-(3-methoxy-4- phenylsulfinylphenyl)pyrazolo[1,5-a]-1,3,5 triazine-4(1H)-one, is a new drug intended for the treatment of hyperuricemia. This report describes the detailed metabolic pathways of BOF-4272 in the dog. The metabolic pathways were investigated using the metabolites found in plasma, urine, and feces after intravenous or oral administration of BOF-4272, as well as the metabolites found in the liver S9 incubation mixture after the addition of BOF-4272 or BOF-4269. BOF-4269 (the sulfide metabolite of BOF-4272) was the only metabolite detected in plasma and feces after the intravenous or oral administration of BOF-4272. BOF-4269 was detected in dog plasma after a lag time following the oral administration of BOF-4272, and the Cmax and AUC0-t of BOF 4269 were higher in fed dogs than in fasted dogs after the oral administration of BOF-4272. A small amount of BOF-4269 was detected in dog plasma immediately after the intravenous administration of BOF-4272. Only BOF-4276 (the sulfone metabolite of BOF-4272) was detected in the S9 incubation mixture after the addition of BOF 4272. Mainly BOF-4272 was detected and small amounts of BOF-4276 and M-1 (the hydroxy metabolite of BOF-4269) were detected in the S9 incubation mixture after the addition of BOF-4269. These findings suggest that BOF-4272 is mainly metabolized to BOF-4269 by the intestinal flora in dogs, whereas little of this drug is metabolized to BOF-4269 in the dog liver. In conclusion, this work has allowed us to formulate the proposed metabolic pathways of BOF-4272 in the dog. PMID- 10746179 TI - Testosterone 5 alpha-reductase inhibitors, menaquinone 7 produced by a Bacillus and phenazine methosulfate. AB - Menaquinone 7 (MW: 649, C46H64O2), a natural electron acceptor for steroid ring A dehydrogenations, produced by Bacillus sp. SNU-299, was isolated as a rat prostate testosterone 5 alpha-reductase inhibitor with an IC50 value of 4.0 x 10( 5) M from the cultured broth. Phylloquinone was as active as the purified microbial metabolite with an IC50 value of 6.6 x 10(-4) M. On the basis of this evidence, the inhibitory activities of electron carriers, menadione, phenazine methosulfate, and 2,6-dichlorophenolindophenol, for rat prostate testosterone 5 alpha-reductase were tested, and the IC50 values were 3.1 x 10(-6) M, 4.9 x 10( 8) M, 8.9 x 10(-5) M, respectively. A product of the 5 alpha-reductase enzyme reaction and an electron and proton carrier, NADP+, inhibited the 5 alpha reduction by rat prostate testosterone 5 alpha-reductase with an IC50 value of 9.2 x 10(-5) M. However, the inhibition effect of a proton carrier, carbonylcyanide-m-chlorophenylhydrazone, for rat prostate testosterone 5 alpha reductase was substantially inactive. PMID- 10746180 TI - Motor unit number estimate (MUNE) testing in male patients with mild to moderate carpal tunnel syndrome. AB - Carpal tunnel syndrome is a frequent clinical entity with an excellent prognosis if adequately treated. However, in rare cases of treatment failure, patients may suffer significant impairment of hand function with various degrees of disability. Loss of motor units in the hand muscles is the pathophysiological basis for this functional impairment. In our study we utilized motor unit number estimate (MUNE) testing in patients with mild to moderate carpal tunnel syndrome in an attempt to identify a patient population with a "silent" loss of motor units in the affected hand. As a result of the study we identified two groups of such patients; first, the group with an associated cervical radiculopathy, and second, the group with an increased amount of polyphasic motor units in the abductor pollicis brevis muscle on electromyographic (EMG) evaluation. Potentially, these patient populations are predisposed for functional impairment of the hand and, therefore, require a more active diagnostic and therapeutic evaluation. Our study also indicates that morphology of motor unit potentials is a good indicator of the motor unit reserve and correlates well with our MUNE results. MUNE testing proved to be a well tolerated, reliable and practical technique in the diagnosis, treatment and research of neuromuscular disorders. PMID- 10746181 TI - Study of motor and sensory nerve conduction velocities, late responses (F-wave and H-reflex) and somatosensory evoked potential in latent phase of intermittent acute porphyria. AB - Twenty-five patients of intermittent acute porphyria (IAP) in latent period were studied neurophysiologically by assessing motor and sensory nerve conduction velocities, late responses (F-wave and H-reflex) and somatosensory evoked potentials of median nerve and were compared with 15 age and sex matched control group. All the patients had at least one classical attack of IAP during which Watson Schwartz test was positive. None of them had any evidence of neurological defecit at the time of examination. Compared with the controls, patients of AIP had significantly slower motor and sensory conduction velocities of median nerve (P < 0.001) and it was more marked in the distal segment of the nerve. Nineteen out of 25 had one or the other abnormal neurophysiological parameter. Reduced nerve conduction velocity was not found to have any relation with age, sex or duration of the illness or number of attacks. Present study shows evidence of subclinical neuropathy in patients of intermittent acute porphyria in latent phase. PMID- 10746182 TI - Side to side difference of nerve conduction amplitudes. PMID- 10746183 TI - Electrophysiological recording of tendon reflexes in cervical myelopathy. AB - Twenty-six patients with unequivocal, clinically and radiologically confirmed cervical myelopathy were investigated electrophysiogically using needle electromyography (EMG), motor and sensory conduction velocity, F-wave latency and the electromyographic recording of the tendon reflexes (TR) of the biceps brachii, brachioradialis and triceps brachii muscles. Normal latency and amplitude values of the reflex evoked compound muscle potentials (T-wave) were calculated from 74 healthy volunteers. Pathological TR were found in 73.1% of the patients, while EMG--which was the next more effective method--was positive in 38.5% of the cases. Eight pathologically delayed T-waves were recorded from muscles with clinically normal or even exaggerated reflexes. TR recording appears to be a useful method in the assessment of segmental functional disturbance in cervical myelopathy. PMID- 10746184 TI - Intrasession and intersession reliability of the soleus H-reflex in supine and standing positions. AB - The Hoffmann reflex (H-reflex) is a measure of motoneuron pool excitability, which is valuable in determining muscle inhibition caused by joint damage (arthrogenic muscle inhibition). In order to detect changes in H-reflex due to injury, the reliability of such a measurement must be established. The purpose of this study was to establish the intrasession and intersession reliability of soleus H-reflex in a supine and standing position. Thirteen healthy volunteers (age 10 +/- 2.63 yr, height 171.35 +/- 10.19 cm, mass 69.62 +/- 13.03 Kg) with no lower extremity orthopedic or neurological disorders within the past year participated in this study. To determine the intrasession and intersession reliability of this measure in a supine resting position and a one-leg standing position, EMG data were collected from the soleus while the tibial nerve was stimulated in the popliteal space. A high voltage (120-200 V), short duration (1.0 msec) stimulus was automatically triggered, eliciting a reflex twitch detected by surface EMG. Several of these measurements were performed with 20 second rest intervals to find the maximum H-reflex. The maximum H-reflex was located by adjusting the intensity of the stimulus. Once a maximum H-reflex was found, 12 measurements were taken in that position with 20 second rest intervals. These steps were repeated for each position (supine and standing) at the same time for 5 consecutive days. Intrasession reliability was computed using 12 measurement trials (12), 12 measurement trials dropping the high and low score (12x), the first 7 measurement trials dropping the high and low score (7x), and the first 5 measurement trials (5). Intrasession and intersession reliability over five consecutive days was estimated using intraclass correlation coefficients (ICC (3, 1)). The supine intrasession reliability measurements were as follows: 0.932 (12), 0.932 (12x), 0.935 (7x), and 0.932 (5). The standing intrasession reliability was 0.853 (12), 0.852 (12x), 0.865 (7x), and 0.862 (5). The intersession reliability was 0.938 in the supine position and 0.803 in the standing position. These results indicate that the H-reflex measured using our protocol in a supine and standing position is a reliable assessment within sessions and between sessions. Five measurements are sufficient to observe reliable measurements within a single session. Most importantly, this data shows that the H-reflex is a reliable assessment that may be used to measure small changes in motoneuron pool excitability over time. PMID- 10746185 TI - Corneal reflex and blink reflex changes in thalamic hemorrhage. AB - We studied the corneal reflex (CR) with air-puff and direct touch by using a standardized method in patients with thalamic hemorrhage (TH) (n: 15) and in normal control subjects (n: 21). The conventional blink reflex (BR) was also studied. In the TH group: 1--When the cornea on the clinically nonaffected side was stimulated the corneal reflex responses were elicited bilaterally, with normal latency on the clinically normal side and delayed on the affected side. 2- When the cornea on the clinically affected side was stimulated, the corneal responses on both sides were either abnormal or could not be elicited. 3--The ipsilateral R1 and R2 responses recorded by stimulation of the supraorbital nerve on the clinically affected side were abnormal where the contralateral R2 responses were in the normal range. In the normal control and TH groups: 1--No statistical difference could be detected between the responses elicited by air puff or direct touch to cornea (p > 0.05). 2--CR responses were statistically different from the R2 response of the BR (p < 0.005). PMID- 10746186 TI - Prostate electromyography: a new concept. AB - Knowledge of the pharmacological and electrophysiological properties of prostatic smooth and striated muscle myofibres are necessary in order to determine whether the dynamic component of benign prostatic hyperplasia (BPH). To our knowledge prostate electromyography have not previously been applied in any type of prostate disorder. The purpose of the present study is to evaluate the electrical activity of the prostatic muscle fibers. We were able to evaluate the features of prostatic muscle action potentials (PMAP) in 15 of the 16 patients from the area around the prostate capsule. Prostate was palpated in the knee-chest position then concentric needle electrode was inserted into the prostate tissue under digital guidance. Needle tip position in the prostate was confirmed with transabdominal ultrasonography. PMAPs were analysed with multi-motor unit action potential analysis (MMA) algorithm. Whenever the PMAPs were unsuitable for analysing with MMA software the amplitude trigger line method was used. Further studies with a large number of patients are needed to establish the correlation between the electrical activity of prostatic muscle fibers and dynamic component of patients with symptomatic BPH. PMID- 10746187 TI - Transient absence of F-waves in acute myelopathy: a potential source of diagnostic error. AB - BACKGROUND: The frequent absence of F-waves in lesions of the nerve roots and proximal nerve is well known, with absence of F-waves occasionally the only electrophysiologic manifestation of early Guillain-Barre Syndrome. It is less well known that acute central nervous system lesions can cause disappearance of F waves. CASE DESCRIPTION: A 25 year old woman presented with quadriparesis and sensory loss progressive over several days. Hyporeflexia and hypotonia were present. Imaging studies were initially negative. Electrophysiologic testing was normal apart from the diffuse absence of F-waves. This led to strong consideration of the diagnosis of Guillain-Barre Syndrome, and treatment for this diagnosis. However, imaging studies ultimately revealed the diagnosis to be transverse myelitis. F responses normalized 6 weeks after the initial study. CONCLUSIONS: F responses are significantly modulated by central nervous system factors. The relevant experimental and clinical literature is reviewed. The relevance of this to the diagnosis of Guillain-Barre Syndrome has not been previously emphasized, but our experience confirms that the absence of F-waves in a patient with acute weakness accompanied by hyporeflexia and hypotonia does not distinguish between peripheral nerve and central nervous system lesions. PMID- 10746188 TI - Neurobehavioural assessment of newborns. AB - Twenty-one full term appropriate for gestational age (FT-AGA), 18 full term small for gestational age (FT-SGA), 18 preterm appropriate for gestational age (PT-AGA) and 15 babies who suffered birth anoxia constituted study subjects. They were subjected to neurobehavioural assessment using Brazelton neurobehaviour assessment scale. FT-SGA babies performed significantly poorly on motor and interactive processes only. No differences were found in terms of overall state organisation. PT-AGA also performed poorly on interactive and motor processes but were placed better than SGA babies. Here also no definite pattern was observed in organisational dimension. No significant effect of asphyxia on behavioural pattern of FT-AGA babies was observed when compared to FT-AGA non-anoxic babies. PMID- 10746189 TI - Motor neuron presentation of an ulnar neuropathy and Riche-Cannieu anastomosis. AB - A Riche (7)-Cannieu (2) anastomosis (ulnar-to-median anastomosis in the hand) in the setting of an ulnar or median nerve lesion can produce confusing clinical and electrodiagnostic findings. We report a patient with a deep branch ulnar neuropathy complicated by a Riche-Cannieu anastomosis. His clinical presentation led to an initial diagnosis of motor neuron disease. Extensive electrophysiologic studies clarified the extent of the Riche-Cannieu anastomosis and the ulnar neuropathy. PMID- 10746190 TI - Anterior tarsal tunnel syndrome. AB - Three hundred twenty patients complaining of pain and/or numbness of their feet were evaluated in our Clinical Neurophysiology Laboratory. Nerve conduction studies of deep peroneal, superficial peroneal, sural and posterior tibial nerves were studied bilaterally. Needle electromyography (EMG) of anterior tibial, long peroneal, abductor hallucis longus, extensor hallucis longus, gastrocnemius and extensor digitorum brevis muscles were examined bilaterally. Nerve conduction studies of 25 healthy volunteer subjects (16 female, 9 male, age range 36-70, mean age 52.82 +/- 8.8) with no complaint composed the control group. Fourteen of these patients (8 female, 6 male, age range 40-70, mean age 55.73 +/- 12.04) were found to have anterior tarsal tunnel syndrome (anterior TTS) bilaterally or unilaterally. In the patients with anterior TTS, the nerve conduction studies revealed deep peroneal nerve distal latency as 6.5 +/- 1.9 msec; the amplitude as 1.8 +/- 1.3 mV at the ankle level; and the conduction velocity as 41.5 +/- 5.9 m/sec in the distal segment. When these values were compared with the control group statistically, results were found highly significant (p < 0.005). Needle EMG findings in the anterior TTS group showed only in the extensor digitorum brevis muscle. Other nerves and muscles were normal. All patients with anterior TTS were performing Namaz for years, and none of them had the predisposing factor which may cause entrapment neuropathy. So, we suggest that chronic prolonged stretching of the deep peroneal nerve on the dorsum of the foot during Namaz may cause anterior TTS. PMID- 10746191 TI - [Investigation of the antibacterial activity of faropenem against Streptococcus pneumoniae]. AB - We evaluated the antibacterial activity of faropenem against penicillin susceptible Streptococcus pneumoniae (PSSP) and penicillin-resistant S. pneumoniae (PRSP). It was shown that the minimum inhibitory concentrations against 90% of the clinically isolated strains (MIC90) of faropenem, penicillin G, cefaclor, cefcapene, and cefditoren against PSSP were 0.032, 0.063, 2, 0.25, and 0.125 micrograms/ml, respectively. While those against PRSP were 0.5, 2, > 128, 1, and 1 micrograms/ml, respectively. Furthermore, we evaluated the bactericidal activity, at the level of 1/4, 1, and 4 MIC, of faropenem and the above four reference antibacterial agents against PSSP and PRSP. Against PSSP No. 127, a sensitive strain to both penicillin G and cefcapene, faropenem showed almost the same bactericidal activity as those of reference agents. Against PSSP No. 108, a penicillin-susceptible and cephem-resistant strain, and PRSP No. 57, a resistant strain to both of penicillin and cephem, faropenem of 1 MIC showed bactericidal activity, but reference agents needed 4 MIC to show bactericidal activity. PMID- 10746192 TI - [In vitro activities of levofloxacin and other antibiotics against fresh clinical isolates]. AB - In this study, the in vitro activity of levofloxacin (LVFX) against 1,020 fresh bacterial clinical isolates was compared with the activities of a range of ofloxacin, ciprofloxacin (CPFX), ampicillin (ABPC), cefaclor, cefpodoxime, methicillin and benzylpenicillin. The clinical isolates except Vibrio cholerae were collected in Japan during 1998 from patients with infectious diseases. MICs were determined using the agar dilution method according to the recommendations by the Japan Society of Chemotherapy. Some isolates of methicillin resistant Staphylococcus aureus (MRSA) and coagulase negative Staphylococcus were resistant to fluoroquinolones, but the MIC50 of LVFX against MRSA was 6.25 micrograms/ml. LVFX was the most active against MRSA among the antibiotics tested. Most of Staphylococcus epidermidis strains were susceptible to the fluoroquinolones. LVFX showed greater activity against all streptococci strains compared with fluoroquinolones tested. In particular, all Streptococcus pneumoniae strains including PRSP were susceptible to LVFX at < or = 1.56 micrograms/ml. Among Enterococcus, ABPC showed superior activity against Enterococcus faecalis but many isolates of Enterococcus species were resistant to ABPC. LVFX was more active against to these Enterococcus species compared with other fluoroquinolones. On the other hand, LVFX and CPFX showed similar activity against isolates of Enterobacteriaceae. CPFX had an MIC50/90 of 0.20, 0.39 microgram/ml and LVFX showed an MIC50/90 of 0.78, 1.56 micrograms/ml against Pseudomonas aeruginosa. LVFX (MIC50/90 0.10, 0.20 microgram/ml) was more active against Acinetobacter species than CPFX (MIC50/90 0.10, 0.39 microgram/ml). Haemophilus influenzae, Branhamella (Moraxella) catarrhalis and V. cholerae were inhibited by low concentration of the fluoroquinolones tested. The MIC90 of LVFX and CPFX were < or = 0.10 microgram/ml against above three species. Some isolates of Neisseria gonorrhoeae and Campylobacter species were moderately resistant to the fluoroquinolones tested but the MIC50 of LVFX and CPFX were < or = 0.39 microgram/ml. Among anaerobes, Propionibacterium acnes was more susceptible than Peptostreptococcus species, and the MIC90 of beta-lactams and fluoroquinolones tested were < or = 0.78 microgram/ml. In conclusion, this study, performed on large number of strains, confirmed an excellent and wide spectrum antibacterial activity of LVFX compared with the fluoroquinolones and beta-lactams tested. And our results suggest that LVFX may be useful in the treatment of various bacterial infections. PMID- 10746193 TI - [Antibacterial activity of beta-lactam antibiotics against extended-spectrum beta lactamase producing bacteria]. AB - MICs of various beta-lactam antibiotics by themselves and in combination with beta-lactamase inhibitor (clavulanic acid) against extended spectrum beta lactamase (ESBL) producing strains of Escherichia coli and Klebsiella pneumoniae which were isolated from clinical materials were investigated. Furthermore, based on the results obtained, a procedure to detect ESBL producing strains was proposed. The MICs of beta-lactam antibiotics against beta-lactamase producing strains were investigated. At first, beta-lactamase was investigated by the drug sensitivity pattern (MIC) to beta-lactam antibiotics and by the substrate profiles of beta-lactamase extracted from the transconjugant of E. coli K-12 strains. After that, we classified the beta-lactamase producing gene by PCR method. Furthermore, a proposal was made for an antibiotic to be used in the confirmation of mixed type beta-lactamase. The data obtained by the above investigations were compiled and used to determine the limit concentration of each beta-lactam against beta-lactamase producing strains including ESBL. By using beta-lactam antibiotics at the following concentrations, it is considered possible to classify beta-lactamase; ampicillin (64 micrograms/ml), ampicillin/clavulanic acid (32/5 micrograms/ml), piperacillin (64 micrograms/ml), cefotaxime (1 microgram/ml), cefpodoxime (2 micrograms/ml), ceftazidime (1 microgram/ml), cefmetazole (4 micrograms/ml), cefminox (2 micrograms/ml), cefepime (0.5 microgram/ml), aztreonam (1 microgram/ml) and imipeneme (1 microgram/ml). This method may be used as a reference in investigating the prevalence of beta-lactam resistant isolates by ESBL producing E. coli and K. pneumoniae. PMID- 10746194 TI - Influence of cranberry juice on attachment of Escherichia coli to glass. AB - An extract from fresh cranberries was shown to decrease the strength of attachment of Escherichia coli to glass coverslips when incubated together for 2 h. Pre-conditioning of the surface prior to biofilm formation also significantly weakened the strength of attached cells. PMID- 10746195 TI - Molecular cloning, sequencing, expression, and site-directed mutagenesis of the 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase gene from Arthrobacter spec. Ru61a. AB - The ring cleaving enzyme 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD)) of Arthrobacter spec. Ru61a is part of the quinaldine degradation pathway. Carbon monoxide and N-acetyl-anthranilate are the products formed by dioxygenolytic cleavage of two C-C bonds in the substrate's pyridine ring. The gene coding for HOD was cloned and sequenced. An isoelectric point of pH 5.40 and a molecular mass of 31,838 Da was deduced from the sequence. HOD is shown to be remarkably similar to 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) of Pseudomonas putida 33/1, but not to other dioxygenases described so far. Consensus regions indicative for any chromophoric cofactor or any catalytically relevant metal were not detected. Sequence comparisons and secondary structure predictions revealed HOD as a new member of the alpha/beta hydrolase fold family. Expression in E. coli yielded recombinant catalytically active His-tagged HOD. S101A and D233A, two mutants of HOD, were obtained by site-directed mutagenesis. Since their residual activity is 43.1% and 62.6%, respectively, they probably are of no catalytic relevance although they might play a role in the interaction between enzyme and substrate. PMID- 10746196 TI - Evaluation of two reverse transcription polymerase reaction assays (GEN ETI-K HGV RNA and LCx GBV-C assay) for the detection of GB virus C/hepatitis G virus RNA in clinical samples. AB - Our study evaluates the analytical performance of two amplification methods in the detection of GB Virus C/Hepatitis G Virus, GEN ETI-K HGV RNA (GEN) and the LCx GBV-C Assay (LCx). GB Virus C RNA was detected by at least one test in 58/315 samples (18.41%). Fifty-five samples (17.46%) were positive by the GEN method and 51 samples (16.19%) by the LCx method. The same rate of detection was found for 71 haemodialysis patients and 18 non-A non-E hepatitis. Method based differences in prevalence were observed for patient samples from the general population, 8/106 (7.55%) positive by GEN vs 7/106 (6.60%) by LCx; and HIV infected patients, 26/98 (26.53%) vs 23/98 (23.46%). For chronic type C hepatitis 10/22 (45.5%) were positive by both methods, with two samples discordant. Overall, discordance was observed for ten samples, with seven positive only by the GEN ETI-K HGV RNA, and three positive only by the LCx GBV-C Assay. An additional evaluation of serial samples, from chronic type C hepatitis patients under interferon treatment, revealed three samples which were positive only by the GEN method. Results were 100% concordant for patients under haemodialysis and for non-A non-E hepatitis, 95.9% in the HIV positive group, 90.9% in the chronic type C hepatitis group, and 97.1% in the general population group. Overall, a 97.2% of concordance was found between methods. Both tests have a similar diagnostic performance, though in our opinion, LCx GBV-C Assay better suits the requirements of a clinical microbiology diagnostic laboratory. PMID- 10746197 TI - Influence of incubation temperature on activity of ligninolytic enzymes in sterile soil by Pleurotus sp. and Dichomitus squalens. AB - Solid state straw cultures of the white rot fungi Pleurotus sp. and Dichomitus squalens covered with a layer of sterile soil were incubated at 20 degrees C, 25 degrees C, 30 degrees C, and 34 degrees C. The activities of the extracellular enzymes laccase and manganese peroxidase (MnP) in the soil layers were measured over eight weeks. Pleurotus sp. produced high enzyme activity at low temperatures. Laccase was maximum at 25-30 degrees C and manganese peroxidase at 20 degrees C. D. squalens showed uniformly high levels of manganese peroxidase at 20-30 degrees C whereas at 34 degrees C, MnP was low and laccase showed an atypical time pattern. The discrepancy between low activity of the main enzyme, MnP, and high straw decomposition at 34 degrees C by D. squalens warrants further investigation of the enzyme inventory produced at that temperature. PMID- 10746198 TI - Cloning and expression of a plasmid-linked pediocin determinant trait of Pediococcus acidilactici F. AB - Plasmid DNA from Pediococcus acidilactici F was prepared by lysozyme-mutanolysin method and purified by cesium chloride-ethidium bromide (CsCl-EtBr) density gradient ultracentrifugation. Agarose gel electrophoresis of plasmid DNA and plasmid-curing experiments suggested that bacteriocin activity was harboured on a small plasmid of approximately 9.1 kb (kilobasepair) in Pediococcus acidilactici F. Plasmid encoding bacteriocin production in P. acidilactici F was examined for restriction enzyme cleavage patterns and its map has been constructed. An Escherichia coli strain transformed with the recombinant plasmid, pQE322, produced and, most probably, secreted pediocin F. PMID- 10746199 TI - Studies on the role of proteases in the white-rot fungus Trametes versicolor: effect of PMSF and chloroquine on ligninolytic enzymes activity. AB - In the present study we investigated the possibility of proteinases, intracellular and extracellular, being involved in the regulation of ligninolytic activities in cultures of Trametes versicolor during the shift from primary growth (i.e. trophophase) to idiophase triggered by nitrogen or carbon starvation. These studies were performed using specific inhibitors added to the cultures of T. versicolor. Addition of PMSF (irreversible inhibitor of serine proteinases) or chloroquine (the lysosomotropic agent inhibiting intralysosomal degradation of proteins) revealed distinct differences in the activity of ligninolytic enzymes between nutrient-deprived and non-starved cultures. The addition of PMSF during the transfer of mycelia to the nutrient limited media significantly enhanced the activities of laccase (2-7-fold) and of unspecified peroxidases (2-4-fold). The activity of lignin peroxidase decreased with PMSF, both in tropho- and in idiophasic cultures. The enhanced activities of laccase and general peroxidases (horseradish peroxidase-like, HRP-like) were accompanied by markedly altered patterns of both intracellular and extracellular proteolytic activities revealed by electrophoretic analysis with polyacrylamide gels containing the copolymerized substrate (haemoglobin or gelatin, respectively). The experiments with chloroquine added to nutrient-deprived cultures showed that inhibition of vacuolar proteolysis resulted in lowered activities of laccase and peroxidase. Electrophoretic analysis revealed altered patterns of intracellular proteinases upon chloroquine addition to nutrient-starved cultures. Moreover, chloroquine was found to enhance the activity of proteases secreted in carbon starved cultures. From the results it is concluded that both intracellular (including vacuolar) and extracellular proteases are involved in the regulation of laccase and peroxidase activity in cultures of T. versicolor under nutrient limitation. PMID- 10746200 TI - Identification of mating-type dependent genes by non-radioactive, arbitrarily primed PCR in Schizophyllum commune. AB - Using arbitrary primers for a PCR reaction on cDNA from different mating interactions, genes differentially expressed in a mating-type dependent manner have been identified. The cDNAs were prepared from mRNA extracted from liquid grown cultures of the fungus in order to prevent further steps in development and increase the possibility of identifying genes directly under control of the mating type system. A reaction using a monokaryotic strain was compared to a semicompatible mating reaction induced for B-regulated development and a reaction using a fully compatible mating. In the comparison of silver-stained, non denaturing polyacrylamide gel separation of the PCR products bands differentially detected between the samples were cloned and subjected to further analysis. Bands were characterized which were present in both mating reactions but absent in the monokaryon while the third band was specifically absent in the fully compatible mating interaction. Sequence analysis allowed design of specific primers for verification of differential expression in a semi-quantitative PCR. Thus, use of radioactivity was not necessary for the fast and reproducible method identifying genes specifically regulated by mating reactions. PMID- 10746201 TI - Personality disorders in the 21st century. AB - The diagnosis and classification of personality disorders have come a long way since the beginning of the 20th century. Perhaps by this time next century, the diagnostic manual of mental disorders will be using a dimensional model of classification that will provide more reliable and meaningful points of demarcation between normal and abnormal personality functioning. This article offers suggestions for the form, content, and placement of such a dimensional model. The particular model emphasized herein is the five-factor model of personality functioning, but the optimal diagnostic system will probably involve an integration of alternative dimensional models. PMID- 10746202 TI - Charting the future of axis II. AB - Although there is general agreement that personality disorders are best conceptualized dimensionally, reflecting continuity with normal personality types, personality disorder traits, and personality disorders themselves, categorical systems continue to be utilized for clinical diagnostic purposes. The arguments for and against each approach are reviewed, and a proposal is presented for DSM-V that involves elements of both. PMID- 10746203 TI - Reflections on the future of DSM axis II. AB - Several proposals are enumerated in this article: (a) the need to employ a coherent classification-guiding theory; (b) the wisdom of conceptualizing personality patterns as prototypes; (c) a proposed shift away from the Axis I Axis II distinction to a three-part continuum; (d) the utility of specifying relationships among the various classification categories of the nosology; (e) a proposal for refining and differentiating personality disorder subtypes; and (f) the utility of expanding the range and comparability of the diagnostic criteria. PMID- 10746204 TI - Difficulties in interpersonal relationships associated with personality disorders and axis I disorders: a community-based longitudinal investigation. AB - A longitudinal study was conducted to investigate the association between Axis I and Axis II psychiatric disorders, interpersonal relationships, and global functioning among men in the community. Structured clinical interviews assessing Axis I and Axis II psychiatric disorders, global assessments of functioning, and questionnaires assessing social support, social conflict, and loneliness were administered to a community sample of 95 HIV+ and 45 HIV- men. The questionnaires were readministered 1 year later. Results indicated that (a) Personality disorders (PDs) and unipolar depressive disorders were associated with loneliness, social conflict, and low levels of social support after HIV status was controlled statistically; (b) PDs were associated with interpersonal and global impairment after HIV status and co-occurring Axis I disorders were controlled statistically; (c) Axis I disorders were associated with global impairment, but were not associated with interpersonal difficulties after HIV status and PDs were controlled statistically; (d) PDs, but not Axis I disorders, predicted increases in social conflict and global impairment after HIV status was controlled statistically; (e) PDs continued to predict increases in global impairment after both Axis I disorders and HIV status were controlled statistically; and (f) HIV+ men reported more loneliness, less social support, and had a higher prevalence of substance use disorders than HIV- men. The present findings are of particular interest because they suggest that PDs are associated with loneliness, social conflict, and a lack of social support among men in the community, whether or not Axis I disorders are present. PMID- 10746205 TI - Borderline personality and obsessive-compulsive symptoms. AB - Diagnostic criteria for borderline personality disorder (BPD) are diverse, covering a broad range of symptoms. One criterion, self-mutilation, is a behavioral excess that may be considered a predictor of other psychopathological states. The present study sought to determine the extent to which two groups of BPD patients, those who mutilate and those who do not, differed on measures of general psychopathology, depression, anxiety, and obsessive-compulsive symptoms. Results indicate that the only source of significant variation was the level of obsessive-compulsive symptoms, with mutilators exhibiting greater symptomatology. The findings from this study support the idea that self-mutilation is a more severe form of psychopathology relative to the rest of the BPD population. These results are interpreted based on the affect regulation model of self-mutilation, and contrasted with contemporary models of impulse control in relation to obsessive-compulsive disorder. PMID- 10746206 TI - Self-defeating personality disorder reconsidered. AB - The objective of this study was to assess the distinctiveness, incremental validity, and gender bias of self-defeating personality disorder (SDPD) symptoms. A total of 441 nonclinical subjects completed personality disorder questionnaires. Structural equation modeling and regression analyses indicated that SDPD symptoms were distinctive, predicted impairment/distress beyond other personality disorder symptoms, and showed no gender bias. SDPD warrants reconsideration as a valid nosological category. PMID- 10746207 TI - A psychometric study of DSM-IV passive-aggressive (negativistic) personality disorder criteria. AB - The passive-aggressive (negativistic) personality disorder (PAPD) is one of the most controversial personality disorders. In order to assess DSM-IV PAPD psychometric properties and comorbidity pattern in a mixed psychiatric sample, 379 consecutively admitted in- and outpatients were administered SCID-II, Version 2.0. Confirmatory factor analysis showed that DSM-IV PAPD is a unidimensional construct with adequate internal consistency (K-R 20 = .85). A strong, specific association (odds ratio = 10.38, 95% CI = 4.83-22.30) was observed between DSM-IV PAPD and narcissistic personality disorder (NPD). Confirmatory factor analysis showed that DSM-IV PAPD should be considered as a subtype of a broader narcissistic disorder. PMID- 10746208 TI - Standardization of the Hare Psychopathy Checklist-Revised in a Spanish prison sample. AB - This investigation examined the reliability, validity, and factor structure of the Hare Psychopathy Checklist-Revised (PCL-R) in male adult Spanish prison populations (n = 117). The interrater reliability and internal consistency coefficients were high, and similar to those obtained in other countries. This data provides support for the homogeneity and unidimensionality of the psychopathy construct in Spanish male prison samples. The analysis of factor structure also replicated the two factor solution of previous studies. The two factors showed different patterns of intercorrelations with several self-report measures of personality, demographic, and criminal history variables, which confirmed the construct validity of PCL-R. The results confirm the psychometric properties of the PCL-R as a measure of psychopathy in Spanish male inmates, and suggest that psychopathy is a construct also observed in Southern European countries. PMID- 10746209 TI - Commemorating the life and work of Silvano Arieti. Introduction. PMID- 10746210 TI - Memories of the son of a psychiatrist. PMID- 10746211 TI - Interpretation of schizophrenia. PMID- 10746212 TI - Language and psychosis: seeking the poetry of malfunction in the spirit of Silvano Arieti. PMID- 10746213 TI - Evolutionary aspects of Silvano Arieti's work. AB - I would like to say a few personal words before beginning this article. I was a pupil of Arieti's in New York in 1963-1964, and then, for several years, together with my wife, I translated most of his books into Italian, in close cooperation with the author. Arieti was eager for his writings to be known in his country of origin. I met Arieti through Pier Francesco Galli and the "Milanese Group for the Development of Psychotherapy," which Galli had founded and directed. At the beginning of the 1960s, this group introduced the psychotherapy of psychoses into Italy through the translation of books and the personal presence of Benedetti and Arieti--two leaders in this area who originated from Italy. I would like Dr. Galli's merits to be acknowledged in this meeting. He made an essential cultural contribution, which initially--with some exceptions--was met with hostility by Italian psychiatry and psychoanalysis. Also thanks to his initiative, the cultural climate has changed since then. The presence at this meeting not only of representatives of psychiatry and of the traditional psychoanalytic schools, but also of OPIFER, a group of independent analysts that follow the model of Arieti's American Academy, is a testimony to this change. By inviting these various groups, the organizers of the meeting have recognized this change in cultural climate. PMID- 10746214 TI - The history of the psychoanalytic treatment of schizophrenia. PMID- 10746215 TI - Psychological factors in depression. PMID- 10746216 TI - Language shift and bilinguals: transference and countertransference implications. PMID- 10746217 TI - Artists' representations of the impact of family suicide during childhood and adolescence. PMID- 10746218 TI - Memories prompted by emotions--emotions attached to memories: studies of depression and of reading fiction. PMID- 10746219 TI - The limits of biological psychiatry. PMID- 10746220 TI - Concanavalin A-binding cell wall antigens of Sporothrix schenckii: a serological study. AB - Sporothrix schenckii cell wall peptido-rhamnomannan (CWPR) was fractionated by affinity chromatography on Sepharose 4B-Concanavalin A giving rise to S. schenckii concanavalin A-binding (SsCBF) and nonbinding (SsNBF) fractions. The CWPR, SsCBF and SsNBF fractions were probed by enzyme-linked immunosorbent assay (ELISA) with the sera of 35 patients with sporotrichosis from Latin America. Both CWPR and SsNBF, although reacting with sera from patients with sporotrichosis, were also cross-reactive with sera from healthy individuals and from patients with other mycoses. In contrast, SsCBF was shown to react specifically with 100% of the sera from patients with sporotrichosis, compared with control sera and sera from patients with other mycoses or cutaneous leishmaniasis. This antigenic fraction was submitted to mild alkaline hydrolysis to remove O-glycosidically linked oligosaccharides and the resulting components had decreased reactivity with patients' sera. The purified SsCBF-derived O-linked pentasaccharide and tetrasaccharide were potent inhibitors of, respectively 76 and 53% of the reaction between SsCBF and sera from patients with sporotrichosis. Our results suggest that SsCBF is a species-specific antigenic fraction recognized by human sera and that this specificity correlates with the presence of O-linked mannose containing oligosaccharides decorated with the alpha-L-Rha 1-->4 alpha-D-GlcA and alpha-L-Rha 1-->4 [alpha-L-Rha 1-->2] alpha-D-GlcA epitope structures previously described [1]. PMID- 10746221 TI - Comparative morphology of Lacazia loboi (syn. Loboa loboi) in dolphins and humans. AB - Lacazia loboi (syn. Loboa loboi), the etiological agent of lobomycosis, was compared in human and dolphin tissue using light and electron microscopy, and computer-assisted morphometrics. The histological features of the lesions were similar; however, preliminary electron microscopy data indicates that cell wall destruction may vary in the two hosts. Calcofluor stained tissue sections of human and dolphin tissue were examined with UV light microscopy and the images digitized. Measurements of area, minimum and maximum diameters, and perimeter were made. Student's t-test (alpha = 0.01) revealed that L. loboi cells infecting dolphin tissue were significantly smaller than those infecting human tissue. This study represents the first comparative analysis of the morphology of the etiological agent of this disease in its two known natural hosts. The data indicate that the organism may not be identical in the two hosts. PMID- 10746222 TI - Monitoring internalization of Histoplasma capsulatum by mammalian cell lines using a fluorometric microplate assay. AB - We have developed a fluorometric microtiter plate assay to quantify the internalization of Histoplasma capsulatum yeasts by macrophages. The assay utilizes the fluorescent dye Calcofluor White to label the yeast cell wall and the vital dye trypan blue, which does not enter viable macrophages, to quench fluorescence of extracellular labeled yeasts. Murine RAW 264.7 cells showed more efficient internalization of strain G217B yeasts than human U937 cells. Both cell lines exhibited a dependence upon actin, and, to a lesser degree, microtubules, in G217B uptake. PMID- 10746223 TI - Comparison of the minimum fungicidal concentration of amphotericin B determined in filamentous fungi by macrodilution and microdilution methods. AB - Minimum fungicidal concentration (MFC) determination could be useful in severe fungal infections in immunocompromised patients. No reference tests to determine the MFC are available, and both macro- and microdilution methods are commonly used. In this study, discrepancies between minimum inhibitory concentrations (MICs) and MFCs of amphotericin B against 58 isolates of filamentous fungi other than Aspergillus (46 Fusarium spp., six Paecilomyces spp. and six Scopulariopsis spp.), obtained with macro- and microdilution methods, were evaluated. Additionally, the agreement between MFCs obtained by both methods were analyzed. In general MFCs were higher than the corresponding MICs overall using the macrodilution method. MFCs were more than one dilution higher than MICs in 52.3% of the cases in the macrodilution test and in 20.5% of the cases in microdilution test. The degree of agreement between MFCs obtained with the two methods was of 70.4% (Kappa coefficient of 0.5). In general, the macrodilution method showed higher MFC values than the microdilution method. Differences of up to six drug dilutions were observed between MFCs obtained by both methods. PMID- 10746224 TI - Isolation of Trichosporon asahii from environmental materials. AB - Trichosporon asahii is the most clinically important pathogenic yeast in the genus Trichosporon, as this species causes both deep-seated infection and summer type hypersensitivity pneumonitis. We isolated 29 T. asahii colonies from environmental samples using the polymerase chain reaction (PCR) and dichloran rose bengal chloramphenicol (DRBC) medium. Our results suggest that T. asahii is common in nature. PMID- 10746225 TI - Differential expression of chitin synthase genes during temperature-induced dimorphic transitions in Paracoccidioides brasiliensis. AB - Fragments of five genes encoding chitin synthase enzymes were identified in the dimorphic fungus Paracoccidioides brasiliensis by polymerase chain reaction (PCR) amplification of conserved CHS gene domains. These represent several classes of enzyme: PbrCHS1, class I; PbrCHS2, class II; PbrCHS3, class IV; and PbrCHS4 and PbrCHS5, class V. Expression of these genes during the temperature regulated dimorphic transition from yeast to mycelium and from mycelium to yeast was determined by Northern analysis. One gene (PbrCHS3) was not expressed at detectable levels. The others were regulated by morphology and/or by the growth phase of the organism. Despite the fact that yeast cells contain more chitin than hyphal cells, the levels of mRNA for PbrCHS1, PbrCHS2, PbrCHS4, and PbrCHS5 were higher in hyphal cells than in yeast cells. This supports observations in other fungi that transcript levels often do not correlate with chitin content and that post-transcriptional regulation of CHS gene expression is important for morphogenesis. PMID- 10746226 TI - Inhibitors of protein phosphorylation including the retinoblastoma protein induce germination of Candida albicans. AB - It has been previously shown that the induction of germination in Candida albicans occurs following its cessation of growth as a yeast. Similarly, mammalian cells undergo a differentiation process that is preceded by a growth cessation associated with a hypophosphorylation of proteins of the retinoblastoma gene family. It is postulated that a similar type of mechanism may be operative in C. albicans and protein phosphorylation inhibitors: forskolin (stimulates cyclic adenosine monophosphate production), okadaic acid (phosphatase inhibitor) and D-erythro-sphingosine (retinoblastoma protein phosphorylation inhibitor) have been used to further strengthen this hypothesis. Okadaic acid (1-1000 nM) and D erythro-sphingosine (100 microM) significantly inhibited the growth of yeast cells of C. albicans. D-Erythro-sphingosine at 1000 microM was candidicidal. Forskolin did not significantly affect growth. Exponentially grown C. albicans pretreated with forskolin (10 microM), okadaic acid (1000 nM) or D-erythro sphingosine (100 microM) readily germinated. In comparison, when these inhibitors were incorporated in the same medium, germination of exponentially grown cells did not occur. These results suggest that protein dephosphorylation may be necessary at an early stage of the yeast-hyphae transition in C. albicans. PMID- 10746227 TI - Differences among chitin synthase I gene sequences in Trichophyton rubrum and T. violaceum. AB - Nucleotide sequences of chitin synthase 1 (CHS1) gene were analysed for the phylogenetic relation between Trichophyton violaceum and T. rubrum, including two isolates of T. raubitschekii and one isolate of T. rubrum var. nigricans. About 620-bp genomic DNA fragments of the CHS1 gene were amplified from these dermatophytes by polymerase chain reaction (PCR) and sequenced. The CHS1 nucleotide sequences of these dermatophytes showed more than 95% similarity between the species. The phylogenetic analysis of their sequences revealed that T. rubrum was genetically distinct from T. violaceum. The specific restriction endonuclease site for HinfI was present in the CHS1 gene sequence of T. rubrum but not in that of T. violaceum. A molecular analysis of CHS1 genes will provide useful information for the identification of these Trichophyton species and the understanding of their evolution. PMID- 10746228 TI - Experimental paracoccidioidomycosis of the Syrian hamster: fungicidal activity and production of inflammatory cytokines by macrophages. AB - Phagocytic cells play an important role in nonspecific resistance to fungal infection by mediating an inflammatory response and by a direct fungicidal action. In this study, the functional activity of peritoneal macrophages obtained from hamsters experimentally infected with strain Pb18 of Paracoccidioides brasiliensis was evaluated during 16 weeks of infection. The results showed that macrophages had a higher spreading ability associated with increased production of tumor necrosis factor alpha (TNF-alpha) and enhanced fungicidal activity during the early periods of infection. TNF-alpha levels remained elevated during all periods studied, while low levels of interleukin-1 beta (IL-1 beta) were produced during the infection. A necrotic area with dead fungi was observed at the inoculation site and the infection disseminated only to liver and lymph nodes in a few animals. These results suggest that during the early stages of infection with P. brasiliensis, macrophage activation by the high levels of TNF-alpha limited fungal dissemination. In contrast, in the later stages of infection, high levels of TNF-alpha were observed while the fungicidal activity of macrophages was lower and the animals presented loss of vitality resulting in their death. These observations suggest a complex role of TNF-alpha in experimental paracoccidioidomycosis of Syrian hamsters, involving not only resistance but also pathogenesis. PMID- 10746229 TI - Ultrastructural and molecular characterization of Pneumocystis carinii isolated from a rhesus monkey (Macaca mulatta). AB - High levels of heterogeneity have been observed among isolates of Pneumocystis carinii derived from different mammalian host species. We report the characterization of P. carinii isolated from a rhesus monkey (Macaca mulatta), which was immunosuppressed as a result of infection with a chimeric simian-human immunodeficiency virus (SHIVsbg). Histopathological examination showed evidence of severe P. carinii pneumonia with a large predominance of trophozoite forms. Alveolitis consisted of typical foamy, honeycomb exudate, with only a few alveolar macrophages. The lung inflammatory response was rather moderate without type-2 pneumocyte hyperplasia or collagenosis. P. carinii organisms were sometimes observed in the bronchiolar lumen. Ultrastructurally, macaque-derived P. carinii was more similar to human- or rabbit-derived parasites than to mouse derived P. carinii. Molecular studies were carried out on the macaque-derived P. carinii DNA at two genetic loci: the genes encoding the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) and the mitochondrial small subunit ribosomal RNA (mt SSU rRNA). Comparison of the DNA sequences with those from P. carinii isolated from eight other host species demonstrated that the macaque-derived P. carinii was genetically distinct at both loci, and was more closely related to human-derived P. carinii than to P. carinii derived from non-primate sources. We propose that macaque-derived P. carinii be named Pneumocystis carinii f.sp. macacae. PMID- 10746230 TI - Proteinase, phospholipase, hyaluronidase and chondroitin-sulphatase production by Malassezia pachydermatis. AB - The production of four functional enzyme categories was investigated in 30 strains of Malassezia pachydermatis isolated from dogs with otitis or dermatitis. The most appropriate reading intervals for these assays were determined with the aid of statistical comparisons. All strains produced proteinase and chondroitin sulphatase; hyaluronidase and phospholipase were produced by all skin isolates (15/15) and 14 out of 15 ear canal isolates. Strains from ear canals did not differ significantly as a group from skin strains in quantitative production of any of the four enzymes; production of proteinase and chondroitin-sulphatase in particular was markedly uniform. PMID- 10746231 TI - The usefulness of blood culture in diagnosing HIV-related systemic mycoses: evaluation of a manual lysis centrifugation method. AB - The results of 5034 blood cultures, implementing a lysis-centrifugation method with saponin, are summarized in this paper. Three hundred and twenty-two blood samples (6.3%) obtained from a pool of human immunodeficiency virus (HIV) positive patients yielded fungi. Cryptococcus neoformans was isolated in 199 samples (3.95%), Histoplasma capsulatum in 95 (1.89%). Candida parapsilosis in 12 (0.23%), C. albicans in 7 (0.13%), C. tropicalis in 2, C. krusei in 1, C. guillermondii in 1, and Prototheca wickerhamii in 4 (0.07%). Blood cultures were positive for C. neoformans in 76.23% of patients having a diagnosis of cryptococcosis and in 89.65% of those who had histoplasmosis. The blood culture was the first means of confirming the diagnosis in 23.8% of the patients with cryptococcosis and in 54% with histoplasmosis. In the four patients in whom P. wickerhamii was isolated, a diagnosis of disseminated protothecosis was not achieved by other findings. Catheter infections were responsible for the majority of recovered Candida spp. PMID- 10746232 TI - Initial case of Candida dubliniensis infection from Asia: non-mucosal infection. AB - A yeast, repeatedly isolated from a post-surgical abdominal infection of a 75 year-old Japanese man, was genotyped as Candida dubliniensis. This is the first reported case in Asia of this recently described fungus. PMID- 10746233 TI - Persistence of cryptococcal antigenemia in an infected dog and uninfected rabbits. AB - A case of cryptococcosis in a dog is reported. The titers of polysaccharide antigen in sera were monitored by latex agglutination (LA) tests during and after treatment with antifungal therapy. Although the LA titers decreased in response to therapy, it took about 3.5 years to achieve a negative LA test. To model the persistence of cryptococcal polysaccharides, we examined the clearance of antigen from the blood of rabbits injected with sterile, cryptococcal capsular polysaccharide isolated from culture supernatant. The distribution half-lives were between 2.6 and 3.9 days and the elimination half-lives were between 10.4 and 39 days. These results suggest that polysaccharide antigen may be stored in tissues. However, we think that slow clearance of antigen cannot explain the extraordinarily slow clearance found in this dog, and that prolonged survival of Cryptococcus neoformans is a more plausible explanation. PMID- 10746234 TI - Production and characterization of monoclonal antibodies reactive with the mycelial and yeast phases of Penicillium marneffei. AB - A definitive tissue diagnosis of Penicillium marneffei is hampered by a microscopic similarity to the yeast form of Histoplasma capsulatum and Cryptococcus neoformans. In order to obtain a better discrimination for accurate diagnosis, monoclonal antibodies (MAbs) were produced from hybridomas raised from Balb/c mice immunized with mycelial culture filtrate. By indirect immunofluorescent or immunoblot analysis, one immunoglobulin (Ig) G1 (3C2) MAb and three IgM (8B11, 3B9 and 8C3) MAbs were found to react strongly with P. marneffei antigens. In the immunoblots, the MAbs 8B11 and 3B9 reacted most strongly with a high molecular weight component (> 200 kDa) produced during either the mycelial or yeast phase of fungal growth. The immunoreactive epitopes for these two IgM MAbs were most likely associated with carbohydrate moieties, judging from their susceptibility to periodate oxidation and concanavalin A binding. This is in contrast to the immunoreactive epitopes for the MAbs 8C3 and 3C2, which were resistant to destruction by periodate treatment and did not bind to the lectin. Judging from immunofluorescent intensity, the three IgM MAbs could react strongly with the yeast cells present in the tissue biopsies of patients with P. marneffei infection. PMID- 10746235 TI - ["Occupational medicine": summing up of the achievements and prospects of development in the 50th anniversary of the journal]. PMID- 10746236 TI - [Incidence of occupational diseases in Poland]. AB - The paper is aimed at presenting the incidence of occupational diseases in Poland. The analysis was performed on the basis of the information included in 'occupational disease certificates'. All sanitary and epidemiological stations throughout the country are committed to send these certificates to the Central Register of Occupational Medicine in The Nofer Institute of Occupational Medicine in Lodz. The incidence of occupational diseases during the three recent years (1996-97-98) was the subject of a thorough analysis. In all, 11,318, 11,685 and 12,017 cases of occupational diseases, respectively were registered over those three years. The corresponding rates were 116.0, 116.9 and 117.3, respectively per 100,000 people employed. In 1998, diseases of the voice organ predominated (30.4%) of all occupational diseases) and they were followed by hearing impairment (28.2%), infectious and parasitic diseases (9.9%), pneumoconioses (8.2%), dermatoses (6.4%), vibratory syndrome (2.9%) and poisoning (2.5%). These disease categories constituted over 88% of all occupational diseases registered in that year. Diseases of the voice organ which showed the greatest growth dynamic were mainly diagnosed among teachers. Neither in the United States nor in the member states of the European Union, this pathology is included into the list of occupational diseases. In view of high rates of its incidence in our country it has become one of essential problems of occupational medicine. In Poland, particular attention is paid to infectious and parasitic diseases among which hepatitis occupies the first place (65%), mostly among health service workers. The decrease in hepatitis incidence observed in the 1990s has been due to an intensive vaccination programme in this group of workers. The incidence of occupational hepatitis became rather stable and accounted for 940 cases per year, however the incidence of hepatitic C increased at the same time. Lower rates of incidence of 'classic' occupational diseases, such as poisoning, pneumoconioses or vibratory syndrome are a positive observation. It is also interesting to note that the rate of occupational diseases diagnosed in advanced stage has also diminished. When analyzing occupational diseases one should bear in mind that the majority of pathologies already diagnosed result from long-term effect of exposure to harmful factors. In all, 93% of cases of occupational diseases emerged after a ten-year exposure. The highest rates, taking account of sections of activity, were found in mining and quarrying, health service, agriculture, hunting and forestry. PMID- 10746237 TI - [Cooperation between regional occupational health centers and the clinic of occupational diseases in the Nofer Institute of Occupational Medicine in the area of diagnosis and certification of occupational pathology]. AB - Diagnosis and certification of occupational diseases is a statutory responsibility of all regional occupational health centres. An increasing number of certificates issued by these centres has been questioned over last recent years by employees concerned. The Nofer Institute of Occupational Medicine co operates with these centres, particularly if some doubts are reported. Some shortcomings in diagnosis and certification become the subject of thorough analysis and steps are taken to identify the causes of miscertification. PMID- 10746238 TI - [Selected issues of health certification among teachers]. AB - The authors propose a standardized model of phoniatric consultations aimed at certifying occupational diseases of the organ of voice. The study included the administration of an expanded questionnaire, laryngological and phoniatric examinations, stroboscopy and determination of the voice acoustic parameters essential in assessing the phonatory function of the larynx. A four-scale hoarness by Yanagihara was employed in the consulting examination and the measurement was taken for three vowels. The model presented should facilitate the diagnosis of occupational diseases of the voice organ. PMID- 10746239 TI - [Quantitative assessment of cancer risk following epidemiological studies]. AB - The role of data derived from epidemiological studies in the quantitative assessment of cancer risk has been constantly growing. That results from the fact that epidemiological studies are performed directly on humans so that it is possible to avoid bias very often associated with methods of extrapolation between animals and humans. Moreover, a slow but continuous increase in the number of well designed epidemiological studies with results which can be used to estimate dose-response relationship is being observed. In this paper, the methodological foundations of cancer risk modelling on the basis of epidemiological studies are discussed, and the dose-response relationship based on epidemiological study carried out in an occupational cohort of workers employed in a pulp and paper plant was estimated. The epidemiological study was performed by the team of the Department of Epidemiology, The Nofer Institute of Occupational Medicine, Lodz. Lung cancer was adopted as the effect of occupational exposure. In oder to estimate the increase in cancer risk induced by occupational exposure, the risk for the whole occupational life was calculated by employing the methods of competitive risks, essential relative risks were modelled using Poisson means for data divided into groups, and a Cox proportional hazards model was used for single data. The analysis indicated, that the data from epidemiological studies render it possible to carry out a more thorough investigation as compared to data obtained from animal experiments. As to the cohort under study, a certain effect of age at which workers enter into exposure on lung cancer risk was observed. A risk 'outfit' the workers bring in at the beginning of their employment is clearly visible in older age groups. A general conclusion drawn from the study may be formulated as follows: epidemiological studies play a major role in assessing the effect of working conditions on the increase in the morbidity risk, however, they are not a panacea for all the problems emerging in the cancer risk assessment in exposed persons. Therefore, information obtained from epidemiological and toxicological studies should supplement and reinforce one another, and data from both sources should be used to obtain the best possible characteristics of risk cancer in humans. PMID- 10746240 TI - [Bronchial hyperreactivity as a factors of chronic obstructive pulmonary disease development in the population of the Bogdanka coal miners]. AB - The aim of the study was to assess the role of bronchial hyperreactivity in the development of chronic airflow obstruction in the population of the Bogdanka (Lublin Basin) coal miners. The population examined consisted of 1456 coal miners working underground in the Bogdanka colliery. The methods of the study included plethysmographic measurements of total airway resistance (Raw), spirometric measurements of forced expiratory volume in one second (FEV1), and determination of forced expiratory flowa at 75, 50 and 25 per cent of vital capacity (FEF75, FEF50 and FEF25%VC). The measurements were repeated twice--before and after administration of 0.2% solution of methacholine (1 min inhalation). Pulmonary function tests were also performed 8 years after initial measurements. The increase in the Raw value above 0.6 kPa/l/s was observed in 5.5% of the population examined. In this group of subjects the skin prick tests with common allergens, measurements of total immunoglobulin E (IgE) levels and determination of blood eosinophils count were performed. Only 12% of all hyperreactives (Raw increase > 0.6 kPa/l/s) showed features of atopy--positive skin prick tests, raised IgE levels, and increase in blood eosinophils count. Using a survival analysis as a statistical tool we evaluated for how long the pulmonary function tests remained within normal ranges in two groups: hyperreactives and controls. All hyperreactives (atopic and non-atopic) showed significantly higher risk of lung function decrease below the normal values as compared with the control group. PMID- 10746241 TI - [Unemployment during pregnancy: direct or indirect risk factor of prematurity and fetus hypotrophy?]. AB - The question as to whether in a transitional phase in the political and socio economic development in Poland, an increased threat of unemployment may also produce health effects manifested by an elevated risk for prematurity and fetus hypotrophy is one of important challenges. If the answer is positive then this issue should have much stronger impact on political and economic decisions. The data obtained from numerous studies carried out in countries of western Europe and the United States shows that the job performance during pregnancy under conditions free from occupational exposure and arduousness does not directly augment the risk of detrimental effect to the health of pregnant woman or her fetus. Just on the contrary, it was found that job performance during pregnancy, and strictly speaking steady employment during this period may somewhat become a 'protective factor'. In Poland, the highest rate of unemployment is observed among women in the 18-24 age group, that is at the age most suitable for procreation. Nowadays it is common that the decision on child-bearing is postponed until the economic status of the woman or her family comes to be more stable. On the other hand, in the population of women looking for a job but determined to set up a family prematurity is quite common that results only in part from an excessive effect of risk factors for this pathology very typical of this social group, like smoking, single parenthood, early or late child-bearing age. After adjusting these factors, the risk of prematurity decreased considerably but it was still high. It is thought that stress and the sense of threat and uncertainty is mostly responsible for this phenomenon. An increased risk of fetus hypotrophy among unemployed women after considering all confounding factors, was not observed. PMID- 10746242 TI - [Physiological reaction to chronic and traumatic stress]. AB - Occupational stress may be evoked by chronic (e.g. psycho-social factors) or traumatic stressors. Both kinds of stressors induce physiological reactions of the human body. If an occupational health physician is aware of them, it is much easier for him to organize activities with the aim to prevent stress in the work environment. The determination of physiological reactions to chronic stressors may be useful in assessing stressogenic traits of various jobs, understanding morbid effect of stress and recognising persons oversensitive to occupational stress. Physiological examinations of traumatic stress render it possible to identify the mechanism responsible for the development of disorders resulting from traumatic stress, provide indicators for an objective diagnosis of this disorders and help to distinguish and assess their long-term effects which may persist after a subsidence of acute symptoms. PMID- 10746243 TI - [Prevention of detrimental effect of traumatic effect in the workplace]. AB - The workplace may be the source of chronic and traumatic stresses. The latter may lead to the occurrence of disorders which have been listed in the Diagnostic and Statistic Manual (DSM) since 1990. In order to avoid this kind of disorders occupational health service workers should promote preventive activities in the workplace. Depending on the prevalence of traumatic episodes in a given workplace the activities should be carried out according to two models. The first model known as 'debriefing' is adjusted to preventing effects of traumatic stress in special services like the police, fire brigades or emergency services. In workplaces where the risk of traumatic episodes is not so high, the prevention should follow the model of crisis prevention developed by M. Braverman. Both models for preventing stress disorders in the workplace are discussed in this paper. PMID- 10746244 TI - [Environmental exposure to electromagnetic fields and the risk of cancer]. AB - Electromagnetic fields (EMFs) of power-line frequency are very common in the human environment. Electric tractions and transmission lines of high voltage are their major sources, however, household appliances are also involved in generating EMFs. Although their biological effect has not as yet been well documented, they evoke an increasing concern as being suspected of their carcinogenic effect. This concern arises mainly from epidemiological studies, which indicate an increased risk for leukemia in children living in the vicinity of transmission lines of high voltage. The author presents a review of epidemiological and experimental studies on a possible effect of power-line frequency EMFs on the development of neoplastic diseases. Epidemiological data obtained to date shows that power-line frequency EMFs do not induce cancer risk and if this is the case the risk is marginal (relevant risk of 1-4 order). Nevertheless, this risk seems to be more evident in occupational than in environmental exposure. The results of experimental studies carried out on animals disclose a possible effect of EMFs as a factor contributing to the development of cancer induced by a known carcinogen. In order to answer the question put in the title it is necessary to continue a large scale investigations especially experimental ones. PMID- 10746245 TI - [The education of occupational physicians practicing in the European Union countries: problems and proposal for the resolution]. PMID- 10746246 TI - ["Occupational health in the health organization". European conference of the health promotion network in the workplace]. PMID- 10746247 TI - Electrophysiological characteristics and morphological properties of dentate granule--and CA3 pyramidal cells in slices cut from neonatally irradiated rats. AB - Neonatal irradiation reduces the dentate granule cells by 60-80%, and consequently the mossy fiber projection toward the CA3 and hilar areas decreases. The number of hilar cells diminishes. Thorny excrescences on the dendrites of the CA3 pyramidal cells get smaller both in number (from 20-30 per neuron in normal to 1-6 per neuron after irradiation) and in size. In spite of these morphological changes functional efficacy of the mossy-fiber projection to CA3 pyramidal cells remains sufficient to generate monosynaptic action potentials when stimulated electrically. Inhibitory circuits activated by mossy fiber volleys seem to be unaffected by irradiation. Main biophysical properties of CA3 pyramidal and surviving granule cells remain within the normal range. Further work should determine if efficacy of the mossy fiber projection increases to compensate for the substantial decrease of presynaptic input, or the power of transmission far exceeds the level needed to fire postsynaptic cells in normal rats. PMID- 10746248 TI - Distribution of pituitary adenylate cyclase activating polypeptide (PACAP) immunoreactive elements in the brain stem of rats studied by immunohistochemistry. AB - In the present work the distribution of pituitary adenylate cyclase activating polypeptide (PACAP) immunoreactive elements in rat brain stem were described using immunohistochemistry. The following structures were PACAP immunoreactive: 1. The dorsomedial and ventrolateral cell columns of the motor nuclei of cranial nerves. 2. Primary somatosensory cells in the mesencephalic nucleus of the trigeminal nerve and central axons of the branchial cranial nerves in the spinal trigeminal tract. 3. Visceral afferent fibers in the solitary tract and cell bodies in the dorsal motor nucleus of the vagus. 4. Second and third order sensory neurons of the cochlear and vestibular systems. 5. Scattered fibers in various regions of the brain stem and well-defined fiber bundles in the interpeduncular area. 6. Cell bodies in the red nucleus, substantia niga, in some cell groups of the reticular formation and in the raphe nuclei, as well as in the pontine dorsolateral tegmentum. PMID- 10746249 TI - The effects of long-term heparin application on ACh-induced isolated ileum contractility and structure. AB - The present study was designed to investigate the side-effects of long-term (one and two months), overdoses (1000 and 2000 IU/kg/day) heparin (Liquamine) applications on the isolated ileum contractility and the histopathological alterations in the ileal tissues. The histopathology of tissues was investigated by using light microscopy. Isolated ileum contractility was measured by using the conventional organ bath system with standard and Ca-free Tyrode perfusion solutions. Ileum preparations were initially contracted with ACh maximally and these contraction amplitudes were given as% values. The average amplitude of these contractions of all experimental groups were found to be increased significantly with respect to control group, in both perfusion solutions. Under light microscopy, in the preparations treated with 1000 IU/kg/day heparin, we have observed edema in the ileal mucosa and neutrophil infiltration in the villi. In addition, the glandular tissue degeneration was also seen in 2000 IU/kg@day group. We can suggest that most probably, the binding of heparin to the receptor on the cell membrane results Ca-release. PMID- 10746250 TI - Direct connections between dendritic terminals of tectal ganglion cells and glutamate-positive terminals of presumed optic fibres in layers 4-5 of the optic tectum of Gallus domesticus. A light- and electron microscopic study. AB - The principal afferent fibres of the avian optic tectum are the optic fibres of retinal origin. They terminate on the contralateral side, in the external layers (2-7) of the optic tectum (called optic layers) turning into these layers from the external surface. The terminal branchings of the optic fibres develop four densely innervated areas in layers 2, 3, 4-5 and 7. Their terminals are large and of various appearance in the different areas. In the middle third of the optic layer (in layers 4-5), thin dendritic terminal sections of tectal ganglion cells (according to Ramon y Cajal) of layer 13 terminate into bunches. Phaseolus vulgaris lectin immunotracer corroborates these dendritic endings (further: dendritic terminals) of tectal ganglion cells. The direct connections between these dendritic terminals and the supposed optic fibres were studied under electron microscope and it was found that the large terminals of optic fibres containing round synaptic vesicles establish asymmetrical synapses with several dendritic profiles, among them Phaseolus lectin labelled dendritic terminals of ganglion cells. This result morphologically supports the former physiological observation of a direct synaptic transmission between optic fibres and ganglion cells of layer 13. In addition, on the dendritic terminals of ganglion cells, symmetrical synapses established by GABA-positive terminals were found. The optic terminals, the GABA-immunopositive terminals and the dendritic terminals of ganglion cells form complex synaptic units surrounded with glial sheath, and thus they establish glomerulus-like synaptic units. The size of the dendritic tree and the branching pattern of the dendrites of ganglion cells point to divergence and convergence in visual transmission. PMID- 10746251 TI - Receptor components of glutamate-evoked increase in intracellular Ca2+ concentration of neurons in culture. PMID- 10746252 TI - Behavioural effects of selective PDE4 inhibitors in relation to inhibition of catalytic activity and competition for [3H]rolipram binding. PMID- 10746253 TI - Preferential inhibition of acetylcholinesterase molecular forms in normal human brain. PMID- 10746254 TI - Effects of mobile GSM radiotelephone exposure on the auditory brainstem response (ABR). PMID- 10746255 TI - Affinity labeling of opioid receptors in vivo and in vitro. PMID- 10746256 TI - Changes in cell adhesion and cell motility during induced neurogenesis, in vitro. PMID- 10746257 TI - Duration of action of GYKI 52466 and its analogues in antiepileptic, anti ischaemic and muscle relaxant tests. PMID- 10746258 TI - The impact factor: deeds and misdeeds. PMID- 10746259 TI - Parotid gland sarcoidosis. AB - Parotid gland sarcoidosis occurs in 6% of patients with sarcoidosis. It was bilateral in 24 (73%), slightly commoner in women, and presented in the majority in the 20 to 40 year age group. There was widespread involvement of other systems, particularly intrathoracic, peripheral lymph-adenopathy, uveitis, lacrimal gland enlargement and skin disease. Patterns of involvement may be pathognomonic as in Heerfordt's syndrome. This account provides the differential diagnosis, imaging and biopsy techniques, and treatment. PMID- 10746260 TI - New perspectives in pulmonary angiitis. From pulmonary angiitis and granulomatosis to ANCA associated vasculitis. AB - Traditionally clinical and histopathological features were mainly relied on for classification of vasculitis and granulomatosis of the lung. These can be complemented by immunodiagnostic features which contribute to the classification as well as to the understanding of the pathogenesis of these disorders. Previously five conditions were classified together under the heading "pulmonary angiitis and granulomatosis" (PA & G), mainly on the basis of histological similarities. These conditions have in common a granulomatous histopathology together with necrosis of varying degree, pulmonary vasculitis and occasionally systemic vasculitis. The introduction of novel immunodiagnostic methods led to different approaches of classification, specifically a separation between a group of disorders associated with antinuclear antibodies (ANA), referred to as collagen vascular diseases, and a group of systemic autoimmune diseases unrelated to ANA, referred to as primary systemic vasculitides. Among the latter, Wegener's granulomatosis (WG), Churg-Strauss syndrome (CSS) and microscopic polyangiitis (MPA) are associated with a group of autoantibodies (antineutrophil cytoplasmic antibodies--ANCA) which separate them from other members of the PA & G group. The granulomatous and vasculitic disorders WG and CSS together with the non granulomatous small-vessel vasculitis MPA now form a new group of diseases ('ANCA associated vasculitides') which have many clinical, serological and immunohistochemical features in common. Collagen vascular diseases (CVD) are serologically characterized by distinct subspecificities of antinuclear antibodies (ANA), sometimes pronounced hypergammaglobulinaemia, complement consumption and immune deposits (antigen-antibody-complement complexes) which are common in situ in immune-complex vasculitis. In this article newer aspects of the clinical course, the immunodiagnostic procedure, and the immunopathogenesis of the relatively large group of pulmonary angiitis will be described. PMID- 10746261 TI - Altered expression of natural killer cell inhibitory receptors (KIRs) on T cells in bronchoalveolar lavage fluid and peripheral blood of sarcoidosis patients. AB - BACKGROUND AND AIM OF THE WORK: The role for natural killer cell inhibitory receptors (KIRs) on T cells is not fully understood, but signalling through KIRs on T cells may inhibit T cell receptor mediated activation, and KIR expression has been suggested to be one mechanism of controlling T cell mediated immune responses. An aberrant KIR expression on T cells could thus be of importance in autoimmune as well as infectious disorders. Sarcoidosis patients have several immunological impairments that have not been clarified, and we here examined the KIR expression on CD4+ and CD8+ peripheral blood (PBL) and bronchoalveolar lavage (BAL) T cells of sarcoidosis patients and controls. METHODS: We used three KIR specific monoclonal antibodies, namely DX9 (specific for p70), DX27 (p58) and DX22 (specific for CD94, that belongs to another major group of KIRs) and flow cytometry. RESULTS: p70 was expressed lower in patient CD8+ PBL (median 2.3%) compared to controls (6.3%) (p < 0.01). In patients, p58 was expressed by less CD8+ BAL lymphocytes (median 1.2%) compared to PBL (6.8%) (p < 0.01) while CD94 was expressed by more CD8+ BAL lymphocytes (median 14.5%) compared to PBL (9.6%) (p < 0.01). Moreover, in CD8+ PBL, CD94 and p58 were expressed significantly lower in patients with an active vs. inactive disease, and in patients with chest radiographic stage I vs. stage II, respectively. CONCLUSIONS: The significantly altered expression of distinct KIRs on CD8+ T cells in sarcoidosis, especially in patients with signs of an active disease, indicate these cells to be dysregulated and implicate them in the pathogenesis of the disease. PMID- 10746262 TI - Methotrexate is steroid sparing in acute sarcoidosis: results of a double blind, randomized trial. AB - BACKGROUND AND AIM OF THE WORK: Methotrexate has been steroid sparing for some patients with chronic sarcoidosis. We wished to determine whether methotrexate can be steroid sparing in the first year of corticosteroid therapy in sarcoidosis. METHODS: Patients with new onset, symptomatic disease within four weeks of starting on prednisone were randomized to receive either methotrexate or placebo for the next year. They were seen monthly and prednisone dosage was tapered following a pre-determined schedule. RESULTS: Of 24 patients enrolled, only 15 received at least six months of therapy. Since methotrexate appears to take six months to be effective, only patients who completed six or more months of therapy were evaluated. The amount of prednisone per day decreased for both groups: methotrexate (First 6 months: Median 26 (Range 15-37) mg/day); Second 6 months 8 (1-22) mg/day, p < 0.01) and placebo (First 6 Months 28 (24-33) mg/day; Second 6 months 16 (11-22) mg/day, p < 0.02), with less prednisone used for the methotrexate patients versus placebo in the last six months (p < 0.01). There was also less weight gain for those patients receiving methotrexate. There was no difference in toxicity between methotrexate and placebo. The difference between methotrexate versus placebo was not seen when all patients (including the dropouts) were analyzed. CONCLUSIONS: Methotrexate can be a steroid sparing agent in acute sarcoidosis. PMID- 10746263 TI - Diagnostic value of routine radioisotope bone scanning in a series of 63 patients with pulmonary sarcoidosis. AB - BACKGROUND AND AIM: Routine use of diagnostic radioisotope bone scanning in patients with sarcoidosis has not previously been evaluated. The aim of this study was to assess whether routine radioisotope bone scanning might be of value in the detection of osseous lesions in sarcoidosis. METHODS: 63 consecutive Caucasian patients (32 men) with a median age of 39 years (range 17-66) and biopsy proven pulmonary sarcoidosis were included. None had symptoms suggesting osseous sarcoidosis. Extrathoracic, non-osseous sarcoidosis was present in 24 patients; 13 patients were on oral steroids. Radioisotope bone scanning was performed with a gammacamera after intravenous injection of 99mTechnetium methylenediphosphonate. An abnormal bone scan was followed by a radiograph of the region of interest. RESULTS: 39 patients (61.9%) had normal bone scans. Minor bone scan abnormalities were found in 24 patients (38.1%). Of these, 11 patients had bone foci (8 in the vertebral spine, 9 in the ribs, 1 in a finger). Radiographically only one of these 11 patients had a bony lesion being typical of sarcoidosis, located in the second finger. 17 patients had joint foci. Radiographs of the joints showed sequelae after a fracture in 1 patient, and degenerative osteoarthritis in 1 patient. There was no difference between clinical and paraclinical variables in patients with normal and abnormal bone scans. CONCLUSIONS: There appears to be no indication for routine radioisotope bone scanning in patients with sarcoidosis. Scanning should be restricted to patients with clinical suspicion of osseous sarcoidosis. PMID- 10746264 TI - The clinical spectrum of the sarcoid peripheral lymph node. AB - BACKGROUND: The finding of sarcoid-type granulomas in a peripheral lymph node (PLN) without clinical evidence of changes suggestive of sarcoidosis elsewhere poses a diagnostic problem. The long term follow-up of these patients has never been described in adults. AIMS OF THE WORK: 1. To describe in the above population whether and when a definite diagnosis of sarcoidosis was eventually made, and the time required to make the diagnosis. 2. To study the percentage of peripheral lymph node presentation in sarcoidosis. PATIENTS: A peripheral lymph node presentation, with lymph node biopsy demonstrating sarcoid granulomas, was seen in 127 patients over the last 20 years. Detailed investigation permitted the early diagnosis of sarcoidosis in 76, and of sarcoid reaction in 8 patients. The other 43-patients with granulomatous lymph node and no clinical evidence of changes outside the lymphatic system at the onset are the subject of the present study. METHODS: Periodic examination at our Sarcoid Clinic every 2 to 4 months, in a long term median follow-up of 36 months (range 1 to 203) and workup according to clinical need, including chest X ray and Computed Axial Tomography (CAT), pulmonary function tests, total body 67Ga scan, Broncho Alveolar Lavage (BAL) studies, blood cell counts, 24 h calciuria and urine analysis, serological tests for liver function, calcaemia, Angiotensin Converting Enzyme (ACE). RESULTS: The diagnosis of sarcoidosis (chronic in all) could be made in 33 patients (25 pulmonary, 8 extrapulmonary), after a median time from presentation of 5 years (range 3-288 months). In the other 10, in spite of a median duration of the illness of 62 months (range 20-487), our diagnosis has been idiopathic granulomatous disease of peripheral lymph nodes. Thus, we observed 109 patients in 20 years presenting with lymph nodes that were surgically removed and provided the diagnosis of sarcoidosis sooner (76 patients) or later (33 patients). CONCLUSIONS: 1. In patients presenting only sarcoid granulomas in peripheral lymph nodes, sarcoidosis may be diagnosed months or years later, but a subpopulation of them still exists where granulomatous lesions remain unexplained. 2. In our series of patients, peripheral lymph node presentation occurred in 11.7% of cases of sarcoidosis. PMID- 10746265 TI - A case of sarcoidosis presenting as multiple pulmonary nodules, nasopharyngeal and cerebellopontine tumors. AB - A 64-year-old woman presented with multiple pulmonary nodules, and after spontaneous regression of the pulmonary lesions in six months, nasopharyngeal and right cerebellopontine tumors developed. Noncaseous epithelioid cell granulomas were demonstrated histologically in both resected pulmonary and nasopharyngeal tumors. She complained of hearing loss and tinnitus probably due to the cerebellopontine tumors. Corticosteroid therapy resulted in the improvement of these symptoms and the gradual decrease of tumor size. Although histological probe of the cerebellopontine tumors was not diagnostic, this patient was finally diagnosed as having sarcoidosis, based on the clinicopathological features, including systemic granulomatous lesions, MRI findings, and good response to corticosteroid therapy. The diagnosis of sarcoidosis is sometimes difficult when its clinical manifestations are uncommon. PMID- 10746266 TI - Sarcoid choroiditis initially diagnosed as birdshot choroidopathy. PMID- 10746267 TI - Sarcoidlike lung granulomatosis induced by glass fibre exposure. PMID- 10746268 TI - Myasthenia gravis relapse associated with sarcoidosis. PMID- 10746269 TI - Two cases of sarcoidosis of the thyroid. PMID- 10746270 TI - Determinants of health care costs and patterns of care of asthmatic patients in Switzerland. AB - OBJECTIVE: To investigate whether medical resource utilisation, costs and clinical status were influenced by: (1) type of physician (specialist versus GP); (2) treatment modality (on-demand vs long-term continuous therapy); (3) labour force participation; (4) insurance coverage (private vs statutory); (5) geographic area (urban vs rural). MATERIAL AND METHODS: Chart reviews in the last 5 patients seen by treating physicians. Direct medical expenditure included charges for hospitalisation, outpatient services, physicians' services and medication, whereas indirect costs included the value of time lost from work. RESULTS: The overall annual mean cost per patient in 589 patients was CHF 2600 [95% confidence interval: 1956; 3245]. Costs in 117 children were CHF 824 [531; 1116], and CHF 3041 [2244; 3837] in adults. Compared with pulmonologists, adults treated by GPs had a higher rate of hospital admissions, equal specialist referrals and more days off work. Hospital admissions in children were similar in GPs and paediatricians. Total direct costs were highest for internists, followed by GPs. Pulmonologists incurred the lowest direct costs in adults compared to GPs or internists (p < 0.05). Total costs for children were equal for GPs and paediatricians. Differences in costs between on-demand and long-term prophylactic treatment were: CHF 834 [318; 1351] versus CHF 1856 [1488; 2224], (p = 0.002). Highest total costs were observed for unemployed patients and those receiving disability payments. Patients with supplementary insurance cover had a lower overall resource utilisation rate and costs than patients without: CHF 2284 [95% CI: 535; 4034] versus CHF 2670 [1844; 3496] (p = 0.8). Total costs were CHF 2319 [1660; 2979] for patients treated in cities and CHF 3062 [1742; 4382] in rural areas (p < 0.0001) as well as lower in the German-speaking part of the country (CHF 2320 [1743; 2897]) than in the French-speaking region (CHF 3610 [1479; 5740] [p < 0.005]). CONCLUSIONS: Factors determining higher resource utilisation and costs are: treatment by non-specialists, long-term continuous treatment, the absence of supplementary health care insurance, treatment in rural areas, and treatment in the French-speaking cantons. PMID- 10746271 TI - [Telemedicine: perspectives and multidisciplinary approach]. AB - BACKGROUND: Telemedicine is use of the new computer-based communication technologies for medical purposes. It augments the exchange of scientific information, while its applications in the fields of patient care and medical education cover remote diagnosis and therapy as well as remote education and training. METHOD: This article reviews the development of telemedicine and its application to specialties such as anaesthesiology, dermatology, medicine, surgery and pathology at the University Hospital of Basle, Switzerland. RESULTS: Since 1980 the Department of Medicine has held multidisciplinary teleconferences for expert consultation and medical education. Since 1992 the Institute of Pathology has been linked to remote hospitals for real-time biopsy, and, since 1997, remote dermato-histopathological diagnosis has been performed in conjunction with a number of centres and practitioners. International academic teleconferences have been held in the field of surgery since 1986 and there is an interactive education programme via telemedicine in the field of anaesthesiology. The technology in use must be adapted to needs: since few practitioners are currently connected to the Internet, teleconferencing will still be the rule in the Department of Medicine. Remote diagnosis in dermatology and pathology requires high-resolution images transmitted by self-developed software via 64 Kb/s ISDN connection, while surgery works with ISDN teleconferencing at 384 Kb/s to ensure live transmission of surgical procedures with high-quality images. CONCLUSION: Our practice, based on several hundred cases, suggests that telemedicine is useful in simplifying and expanding access to remote interdisciplinary expertise, as well as improving medical education in a number of specialties. Telemedicine's multidisciplinary approach is to be recommended. PMID- 10746272 TI - Hereditary hyperferritinaemia cataract syndrome: does it exist in Switzerland? AB - Recent research in iron metabolism has revealed the existence of iron-responding elements in the 5'UTR of the mRNA of ferritin. Binding of these structures with iron-regulatory proteins regulates ferritin synthesis within the cell, according to the intracellular iron level. Several mutations of the iron-responding elements located at the 5'UTR of the L-ferritin subunit, which lead to the hereditary hyperferritinaemia cataract syndrome, an autosomal dominant hereditary disease, have been described. Patients with congenital bilateral nuclear cataract present high serum ferritin (360-2264 micrograms/l) in the absence of iron overload. The purpose of our study was to look for this syndrome in Switzerland and in particular in the Geneva population. About 3000 cases of cataract operated on during a 4-year period (1995-1998) in the University Clinic of Ophthalmology were screened. We found 135 patients operated on before the age of 51 years. However, only 19 had bilateral nuclear cataract. 15 patients agreed to undergo iron screening. In 2 of them, a slight elevation of ferritin (267 micrograms/l in a female, 416 micrograms/l in a male) was found in the absence of iron overload. In both cases there is a positive family history of cataract. DNA sequencing analysis in these patients showed a normal nucleotide sequence of the whole iron responding elements region. One of them (male) was found to present the codon 63 mutation at HFE gene in the heterozygous state. Our local study indicates that hereditary hyperferritinaemia cataract syndrome is extremely rare in Switzerland. However, similar studies should be carried out in other regions of the country. Iron status evaluation and ferritin level monitoring should become routine examinations in all new cases presenting with bilateral nuclear cataract before the age of 50 years. PMID- 10746273 TI - [How relevant is the trough-to peak ratio?]. AB - A marked blood pressure rise in the early morning hours is associated with an increased prevalence of cardiovascular events. Moreover, it has been reported that the blood pressure fall during the night is inversely correlated with left ventricular mass, peripheral vascular resistance and cerebrovascular insults. Recently, assessment of the so called "trough-to-peak ratio" has been increasingly used in order to evaluate the duration of action of antihypertensive agents. In this review some aspects of this arithmetical index are highlighted, and specifically the fact that this ratio as a surrogate endpoint may in some circumstances influence the choice of an antihypertensive agent but nevertheless does not serve to predict the clinical efficacy of blood pressure lowering drugs. To date the trough-to-peak ratio remains a problematic parameter because of the lack of defined optimal values and the lack of general and standardised methods for its determination. It is thus a purely arithmetical index without major clinical relevance. Evaluation of the clinical efficacy of antihypertensive drugs should rather be based on 24-hour measurements, as a more reproducible parameter. PMID- 10746274 TI - [Lysis of an intracoronary thrombus with a glycoprotein IIb/IIIa antagonist]. PMID- 10746275 TI - [Literature reference: image-producing diagnosis in kidney tumors]. PMID- 10746276 TI - [Dicom and SAP: cornerstones of medical data management]. PMID- 10746277 TI - [Focal echogenic splenic involvement in malignant lymphoma: a diagnostic problem]. AB - AIM: The aim of our study was to describe clinical data, frequency of the findings, sonographic patterns, confirmation of diagnosis, and differential diagnosis of focal echorich splenic lymphoma involvement. PATIENTS: During the last 20 years a focal splenic lesion was found in 178 patients with malignant lymphoma. Echorich splenic lesions were seen in 11 out of 178 cases (6.2%). METHODS: Splenic size, echomorphology of lesions, size and number of lesions, and presentation during sonographic follow-up examination were presented. RESULTS: Echorich infiltrates of the spleen were predominantly seen in patients with low grade Non-Hodgkin-Lymphoma (9 out of 11), the size of lesions mostly was less than 3 cm in diameter (9 out of 11), the underlying splenic size was over 8 x 20 cm in 9 out of 11 patients. Diagnosis was confirmed by sonographic follow-up (n = 11) and autopsy (n = 1). CONCLUSION: Echorich splenic lymphoma involvement is a rare event and is characterised by a variable presentation during follow-up. Up to date the clinic significance of our observation is still unclear. PMID- 10746278 TI - [Ultrasound elastography of the prostate. A new technique for tumor detection]. AB - AIM: Prostate tumours are often of harder consistency than the surrounding tissue. During digital rectal examination, this fact can be used not only to detect hypertrophy but also localized hardenings. The examination by digital palpation is inaccurate and, even in combination with PSA-value and a transrectal ultrasonic examination, the result is often not reliable. Ultrasound elastography enables us to measure and visualize the elastic properties of a tissue region and is a useful supplement to the examination by digital palpation. Ultrasound elastography is able to measure and visualize the elastic properties of a tissue region, therefore it is a useful supplement to commonly used diagnostic procedures. METHOD: We have developed a new system for elastographic prostate diagnosis which can be used during the transrectal ultrasonic examination. During the examination a sequence of ultrasonic images is acquired while the organ is slightly compressed by the ultrasound probe. Using numerical analysis of image pairs for the acquired sequence we calculate the tissue strain which represents the spatial elasticity distribution of a specific cross-section of the organ. This enables us to distinguish hard areas in the tissue. Image artifacts resulting from lateral motion components, i.e., orthogonal to the direction of the applied force, are compensated for by a special approach. RESULTS: We present results obtained from a typical elastography phantom and also the first in vivo images from patients who were undergoing radical prostatectomy. Our images prove that ultrasound elastography has the potential to detect malignant tissue areas, which are inconspicuous in the B-mode image. Our findings are confirmed by the corresponding histological specimens. PMID- 10746279 TI - [Venous cross-sectional area: measured or calculated?]. AB - AIM: The aim of this study was to compare measured versus calculated venous cross sectional area in healthy subjects in a standing and a lying position with normal breath-ing and during Valsalva manoeuvre. METHOD: Measurements were carried out in 30 venous segments (the common femoral vein CFV, the superficial femoral vein SFV, the greater saphenous vein GSV) of 5 healthy volunteers (4 female, 1 male) with a median age of 28.7 years (range 23.4-46.7 years) in supine and standing position, while normally breathing and during a standardised Valsalva manoeuvre. Venous diameters were measured from B-mode in longitudinal view while cross sectional areas were planimetrically assessed from transverse B-mode as recorded on video (S-VHS). The mathematical calculation of areas followed the formula (0.5 diameter)2 x pi. All investigations were performed 3 times; mean values from these 3 measurements were used for further computation. Measurements were performed using the NIH Image 1.6 program. RESULTS: Correlation coefficients r of the calculated versus the measured venous area while normal breathing in standing and in lying subjects were: 0.92 and 0.82 in the CFV, 0.92 and 0.84 in the SFV as well as 0.98 and 0.97 in the GSV, respectively. During Valsalva manoeuvre in standing and lying subjects the correlation coefficients r amounted to: 0.94 and 0.93 in the CFV, 0.92 and 0.94 in the SFV as well as 0.99 and 0.98 in the GSV, respectively. CONCLUSIONS: In healthy volunteers measured and calculated venous cross-sectional area at rest and during Valsalva manoeuvre correlate well. Correlation is numerically better in standing compared to lying subjects while normal breathing. Calculated venous area is accurate and can be used for further calculations. PMID- 10746280 TI - [3D-spectrogram of the doppler signal in real time]. AB - AIM: Doppler signals are normally displayed as an x/y-graph. The frequency is plotted in the y-direction, and the intensity is represented by the brightness. This represents less information than is contained in the acoustic signal: often one can hear more one can see. We are investigating the question as to whether it is feasible to render the complete information visible without the use of expensive additional equipment. METHOD: The sound card (SB16 P&P value edition) in a Pentium-PC (200 MHz 32 MB RAM) was used as a 16 bit dual channel a/d converter and connected to the ultrasound system Sonoline Elegra (Siemens, Erlangen, Germany). We used a standard 32 bit dual channel real-time FFT-software (SpectraPro, Sound Technology, California) to display a 3D-spectrogram on the PC screen in real-time mode. This system was first tested on a flow model both with laminar steady flow and with pulsatile flow. Following this, measurements were made of the arteries and veins in healthy volunteers. RESULTS: In the 3D Spectrogram the character of the flow can easily be seen. The quantitative parameters of velocity (Doppler frequency), direction and intensity of flow can be readily determined. Additional information about quality of flow, erythrocyte clusters and changes occurring in areas of slight stenoses can be used for diagnostic evaluation. The orthograde and retrograde flow are displayed separately using the dual channel technique. The arterial and venous flow in small and closely neighbouring vessels, such as the central retinal artery and vein, can easily be differentiated. The venous flow in particular is more turbulent in the 3D-spectrogram than would be expected from the normal display mode. CONCLUSION: The 3D-spectrogram provides much additional information in comparison with the normal Doppler spectrogram of the ultrasound system. This 3D spectrogram can be easily obtained using inexpensive standard hard- and software. PMID- 10746281 TI - [New possibilities in the diagnosis and documentation using 3D power doppler ultrasound with carcinoma of the floor of the mouth as illustration]. AB - AIM: To evaluate a novel 3D power Doppler ultrasound-technique (3D PDUS) in the diagnosis and documentation of tumours of the floor of the mouth. METHOD: 22 patients with tumours of the floor of the mouth (2 T1-, 5 T2-, 6 T3- and 9 T4 carcinomas) prospectively underwent conventional grey-scale ultrasound combined with power Doppler ultrasound and 3D PDUS (3-Scape). All examinations were performed with the "Sonoline Elegra Advanced" in combination with a 7.5 MHz transducer. Two independent observers compared the results regarding clear tumour margins, midline crossing, infiltration of the mylohyoid muscle, infrahyoidal tumour, contact to the mandible, contact to major vessels (such as the lingual, submental and/or facial artery), intra- and peritumoural vascularisation and judged the value of the 3D reconstruction method for documentation purpose. RESULTS: All parameters relevant for therapy could be gathered from the 3D data (agreement: 98.2%) almost without loss of information; results of both observers were identical (kappa value: 1.0). In the comparison to conventional ultrasound, 3D PDUS also allows for the reconstruction of axial images of the floor of the mouth similar to CT-images, and a 3D image of tumour vascularisation can be obtained. Acquisition and reconstruction of the 3D data only takes a couple of minutes. CONCLUSIONS: "3-Scape" is a novel easy-to-perform method allowing the complete acquisition of 3D data of the entire floor of the mouth, and providing the possibility to digitally store and/or transfer examination results without loss of information. Reconstruction of ultrasound-images in any desirable plane and/or as a 3D presentation is possible. Thus, in the future, additional software will facilitate the determination of the volume and the degree of vascularisation of tumours. PMID- 10746282 TI - [Doppler sonography of the short posterior ciliary artery in patients with primary open angle glaucoma]. AB - AIM: The area of primary damage in glaucomas is the optic nerve head. This region is directly supplied by the peripapillary choroid and the short posterior ciliary arteries (SPCA). This study was designed to evaluate the choroidal and SPCA haemodynamics in high tension (HTG) and normal tension (NTG) primary open angle glaucoma patients, as well as in healthy controls (CTL). METHODS: 20 HTG, 12 NTG, and 32 age matched volunteers (CTL) without clinically relevant extracranial stenosis of the carotid arteries were evaluated for ocular pulse amplitude (OPA; OBF-System OBF Labs, UK) and for temporal SPCA peak systolic flow (PSV), enddiastolic flow (EDV), pulsatility (PI) and resistance indices (RI) using a 9 MHz phased array (Elegra Advanced System; Siemens, Germany). RESULTS: (mean +/- SD): In the control group the PSV (cm/s) of the SPCA was 10.90 +/- 1.53; EDV (cm/s) was 3.24 +/- 0.24, PI was 1.30 +/- 0.39, RI was 0.69 +/- 0.11, OPA (mm Hg) was 2.2 +/- 0.25; IOP (mmHg) was 14.6 +/- 1.5. When compared to CTL matched for age, sex, and refraction, NTG-patients showed the following results: OPA was significantly (p < 0.05) reduced, dopplersonography showed a significant (p < 0.05) reduction in diastolic flow patterns and a significant (p < 0.05) increase in PI and RI. In HTG-patients the parameters investigated where not significantly (p > 0.05) altered. CONCLUSION: These data suggest reduced haemodynamics in the choroid and in the SPCA of NTG patients, which may contribute to progression of the optic neuropathy. In HTG the Doppler parameters and the OPA are not altered compared with the control group. PMID- 10746283 TI - [Value of sonographic diagnosis in retroperitoneal teratoma in adults]. AB - A 56-year old man was admitted with a tumour-like lesion located in the retroperitoneal space with adherence to the left adrenal gland. An extensive diagnostic procedure could neither establish the diagnosis nor differentiate the lesion from the adrenal gland. After tumour biopsy by ultrasound surgery was recommended and performed and the final diagnosis--mature teratoma--was established. In this article we discuss the genesis and therapeutic possibilities of mature teratoma in adults. PMID- 10746284 TI - [Sonographic diagnosis of an unusual recurrence of Chilaiditi syndrome after hemicolectomy]. AB - A 71-year old patient presented with acute abdominal pain, nausea and emesis 3 months after right hemicolectomy for Chilaiditi's syndrome. The initial ultrasound examination revealed a loop of thick walled small intestine between the anterior surface of the right liver lobe and the diaphragm. In addition, small amounts of perihepatic fluid were found. The chest x-ray confirmed a recurrence of Chilaiditi's syndrome with intestinal gas under the right diaphragm. Elongation and flaccid of intestinal and hepatic suspensory ligaments are thought to be the principal predisposing factors. However, in our patient, a wedge-shaped enlarged lobus caudatus served as a guide rail for the bowel and facilitated access to the space under the right diaphragm. Although the patient recovered completely after 3 days of conservative therapy a high risk of recurrence remains. In summary, ultrasound examination can reliably diagnose Chilaiditi's syndrome and should also be used, as the method of choice in the follow-up of this rare syndrome, thus avoiding unnecessary x-ray exposure. PMID- 10746285 TI - [Erectile dysfunction]. PMID- 10746286 TI - [Epidemiology and age-related risk factors of erectile dysfunction]. AB - Erectile dysfunction (E. D.) affects millions of men in the whole world. The prevalence of erectile dysfunction in the best conducted study, the Massachusetts Male Aging Study (MMAS), was 52% of male subjects aged 40 to 70 years. ED is strongly associated with age. Multiple risk factors, such as chronic illness, hypertension, diabetes, medication, operation in the pelvis, smoking and alcohol consumption, have a negative influence on erectile function. PMID- 10746287 TI - [Physiology and pathophysiology of erectile dysfunction]. AB - Erectile dysfunction is an entity associated with age but is not necessarily a consequence of aging. During erection the penis acts as a capacitor--accumulating blood under pressure. Erectile function is, therefore, dependent on the integration and regulation of functional interplay between psychologic, neurologic, endocrine and vasoactive factors. Any imbalance in the integration of these factors will result in erectile dysfunction. Trabecular smooth muscle is an important structure, which contributes to control of detumescence and erection. The smooth muscle tone is mediated by many hormones and vasoactive factors (e.g. norepinephrine, acetylcholine, NO, VIP, prostaglandins etc.) but its functioning is also dependent on a sufficient oxygenation to prevent the fibrosis of this important structure. This oxygenated state is achieved during erections. So we can conclude: Erections are good for erections. PMID- 10746288 TI - [Diagnosis of erectile dysfunction]. AB - The diagnostic view on the erectile dysfunction has clearly changed in the last years. According to the development of new therapy options a better understanding about the physiology of the erectile dysfunction has arisen. This fact implies on one hand to pursue a subtile diagnostics, on the other hand you have also to concern the patient's imagination about the therapies ordered to him. It's useful to divide the clarification into 3 steps: step 1--the non-invasive diagnostics, step 2--the semi-invasive diagnostics, step 3--the invasive diagnostics. A great number of patients can be cleared up by examinations of step 1. A thorough anamnesis of the whole body and a sexual anamnesis, a physical examination, laboratory exams and a first psychological exploration should be obligatorily carried out. With the following drug tests there are some differential-diagnostic statements possible. Only in special cases more invasive diagnostic methods will be used. In this essay the different diagnostic possibilities are introduced and the corresponding indications are explained. PMID- 10746289 TI - [Oral therapy of erectile dysfunction]. AB - Erectile disfunction (E. D.) is more common in older men but may affect younger men too. Diabetes mellitus, coronary heart disease and hypertension are often associated with E. D. The majority of the patients are treated medically for erectile dysfunction and, recently, oral therapy has become most important since Viagra has been approved. New phosphodiesterase blockers are in preclinical evaluation since then. Phentolamine and apomorphine will become available soon for the treatment of E. D. It is important to know about the etiology of E. D. as well as the mechanisms by which drugs may improve erection in order to decide which drug is appropriate for a particular patient. PMID- 10746290 TI - [Topical therapy in erectile dysfunction]. AB - All forms of pharmacological therapy result in a relaxation of the corporeal smooth muscle. Intracorporeal injection of vasoactive drugs was introduced around 15 years ago and still is the most effective therapy in erectile dysfunction. Resulting in a consistent success rate of 70-80% this form of therapy will find numerous applications, even after the introduction of effective oral agents such as sildenafil. Prostaglandin E1 and--less frequently used--the combination of papaverine and phentolamine are the mainstay of intracorporeal injection therapy. Intraurethral prostaglandin (MUSE) has recently become available and is somewhat less effective than injection therapy. Externally applied drugs (nitroglycerin paste on the penile shaft and minoxidil solution on the glans penis) have not succeeded in the long run. Vacuum erection devices represent a form of physical topical therapy that is very versatile and also effective but rather infrequently applied. PMID- 10746291 TI - [Penile implants]. AB - Many patients presenting with erectile dysfunction have been successfully treated with techniques mentioned earlier in this issue. However patients with an insufficient response are still candidates for the treatment with penile implants. For this reason it is mandatory to know about the indications, the systems and the operative techniques of penile implants. In a time of decreasing numbers of implant surgery this method still remains a valuable tool for the therapy of erectile dysfunction. PMID- 10746292 TI - [Other side of the problem: "Systemic solutions of sexual functional disturbances"]. AB - From a systemic constructive point of view it seems to be obvious that symptoms of any kind are to be looked at as definite human communicational behaviour. From this angle the sexual behaviour between two or more autonomous (self-conscious) humans can be defined as dialogue. Therefore, sexual problems can be understood, analysed and dealt with as communication problems. A very important and therapeutic idea is to consider symptoms as intervention for relationships and possibility for problem solutions. Furthermore, these disturbances are not to be considered as deficiency but as efficiency and competence. The consequences of this therapeutic attitude in a setting are explained in theory and practice as follows. PMID- 10746293 TI - [German and European child- and adolescent psychiatry]. PMID- 10746294 TI - [Personality disorders in adolescence. A pilot study on classification and early course]. AB - 72 case histories of former adolescent psychiatric inpatients diagnosed on discharge as suffering from a personality disorder were evaluated. It was possible to re-examine 33 (46%) of them. The discharge diagnosis was made according to ICD 9, the follow-up diagnosis according to ICD 10 and DSM-III-R. Depending on the instrument and criterion, 40% to 87.9% of the discharge diagnoses made could be confirmed after an average follow-up period of 3.4 years. Only in 12.1% of the cases there was no personality disorder established. There was no indication of any change of diagnosis to other psychiatric disorders. The re-diagnosis rates obtained are thus similar to those for adults. The classifications of ICD 10 and DSM-III-R corresponded much better than those of ICD 9 to clinical reality. The classification systems and assessment instruments that have normally been verified only for adults thus also seem suitable for use among adolescents. In both adolescents and adults the avoidant personality disorder is a particularly frequent subtype. However, the current study was unable to resolve the question of what the decisive criteria should be in the case of competing definitions such as puberty crisis, personality development disorder, personality disorder and emotional or affective disorder. This issue requires further investigation. PMID- 10746295 TI - [ESES-syndrome in child- and adolescent psychiatry]. AB - OBJECTIVES: The article describes the theoretical background of the diagnosis and therapy of the ESES syndrome. METHODS: The assessment and treatment of children with ESES in a child-psychiatric setting is shown. The underlying data have been taken from 18 outpatients with or without epileptic seizures. RESULTS: The early diagnosis of non-specific symptoms and the anti-epileptic treatment probably play an important role in preventing the progression of ultrastructural brain damage. Sultiam is the drug of first preference. PMID- 10746296 TI - [Proof of equivalence as a new issue in confirmatory statistics]. AB - A correct confirmatory analysis of studies aiming at demonstration of equivalence requires the use of a class of testing procedures that is still widely unknown among non-specialists and not even mentioned in traditional textbooks on Applied Statistics. The naive approach to equivalence testing, i.e. performing an ordinary two-sided test and inferring equivalence from the absence of a significant difference, is unacceptable since it entails an uncontrolled increase of the risk of false positive conclusions. A technically simple approach to avoiding such fallacies is based on the principle of confidence interval inclusion. However, the latter suffers from the disadvantage of yielding testing procedures that tend to exhibit comparatively poor power properties. For the sake of keeping the numbers of patients or subjects to be recruited for the respective trials as low as possible, application of optimum testing procedures, also for equivalence assessment, has much to recommend it. The rationale of such an optimum test for equivalence is described in some detail for four standard settings frequently encountered in clinical research. PMID- 10746297 TI - [Obsessive-compulsive disorder, frontostriatal system and the effect of the serotonergic system]. AB - OBJECTIVES: Focal issues of recent research on obsessive-compulsive disorders have been the involvement of the frontostriatal system in the patho-physiology, as well as the manner of effect and efficacy of the serotonin reuptake inhibitors that unfold their specific efficacy in the frontostriatal system. The course of treatment among adolescent inpatients with obsessive-compulsive disorder was analyzed with regard to the medications used and their effects upon the course of treatment. METHODS: The data for all adolescents with obsessive-compulsive disorder admitted as inpatients to the Hospital for Child and Adolescent Psychiatry and Psychotherapy in Mannheim since 01.01.1990 were analyzed with regard to medication and the parameters for course and outcome. RESULTS: Five patients dropped out of treatment, five patients received no medication, eight received sulpiride, ten were treated with clomipramine, and three with fluvoxamine. The average length of inpatient stay was longer for the groups treated with sulpiride and clomipramine than for the group that received no medication, but equivalent for these two medication groups. The success of treatment for obsessive-compulsive disorder was rated as higher in these two groups than in the group without medication. On sulpiride, patients gained weight and exhibited fatigue and increased levels of prolactin. On clomipramine, half of the patients exhibited cardiac side effects. CONCLUSIONS: This small, nonrandomized medicated sample analyzed under non-double-blind conditions shows no difference in the efficacy of clomipramine and sulpiride under post hoc analysis. Rather, the side effects that occurred in patients on clomipramine indicate a more frequent use of SSRI to increase compliance as regards medication intake. PMID- 10746298 TI - [Atypical neuroleptics in child- and adolescent psychiatry]. AB - Atypical neuroleptic drugs have enriched our treatment programs, especially in childhood and adolescent schizophrenia. Reviewed here is the use of atypical neuroleptics in children and adolescents with a schizophrenic disorder. The receptor binding profile and pharmacological properties, indications, side effects, clinical application, and trials of atypical neuroleptic drugs are compared to the classical neuroleptic drug haloperidol in the treatment of adolescent schizophrenia. Special attention is paid to the most common atypical neuroleptics clozapine, olanzapine and risperidone since most studies are carried out with these compounds, most often with clozapine. More clinically controlled trials have to be conducted since only one has been performed to date. The place of atypical neuroleptic drugs is discussed and further studies are necessary in order to differentiate, and eventually broaden the spectrum of the indications tested thus far. PMID- 10746299 TI - [Max Isserlin--founder of child psychiatry in Munich]. AB - The early days of child and adolescent psychiatry and psychotherapy at the beginning of the 20th century were governed by the schools of thought of that era and by personalities, who as physicians and researchers, recognized that disorders of mental development constituted a separate field. Among them was Max Isserlin, who established the first hospital for child psychiatry in Munich, the Heckscher Clinic, in 1929. Isserlin's landmark discoveries in psychiatry, above all those pertaining to aphasias, are still of topical interest today. He concentrated particularly upon speech development disorders, as well as upon what we today would refer to as described developmental disorders. From 1933 on Isserlin's personal fate was marked by persecution and displacement. The purpose of this brief biographical sketch is to commemorate his work and to make visible his place in modern thought. PMID- 10746300 TI - Acoustic waves and the study of biochemical macromolecules and cells at the sensor-liquid interface. PMID- 10746301 TI - Determination of nicarbazin in feeds using liquid chromatography-electrospray mass spectrometry. AB - A method is presented for the determination of the 4,4'-dinitrocarbanilide component of the coccidiostat nicarbazin in animal feeds. Samples are extracted by shaking with methanol and analysed, without further clean-up, using liquid chromatography-electrospray mass spectrometry. A deuterated form of the analyte is employed as internal standard to improve the repeatability of the method. The method has been validated at levels between 0.1 and 100 mg kg-1 with internal standard corrected recoveries between 88 and 101% and RSD values < 8%. PMID- 10746303 TI - Determination of equilibrium constant of alkylbenzenes binding to bovine serum albumin by solid phase microextraction. AB - Solid phase microextraction (SPME) coupled with GC has been applied to study the binding properties between bovine serum albumin (BSA) and volatile organic compounds such as benzene, toluene, ethylbenzene, propylbenzene and butylbenzene. Their protein-ligand equilibrium constants have been determined. The measurement of free and bound ligand concentrations in the aqueous solution was based on the equilibrium among the analyte in the fiber coating (Cf), headspace (Ch) and aqueous solution (Cs). The work demonstrated that SPME is a simple and effective method in the study of protein binding to measure the freely dissolved analyte concentration as well as the equilibrium constant. The theoretical aspect of the SPME applied to the equilibrium constant measurement in two-phase (liquid sample fiber coating) and three-phase (liquid sample-headspace-fiber coating) systems has been thoroughly discussed. The results demonstrated that the interpretation of the calibration data is crucial to the determination of freely dissolved analyte concentration and the equilibrium constant especially when the sample volume is small. The error in the experimental system is discussed. It is demonstrated in this study that for the three-phase system the amount of the analyte partitioned in the headspace could be ignored only in certain circumstances, where the Henry's law constant and the ratio between headspace volume and sample volume are sufficiently small. PMID- 10746302 TI - Fractionation of soluble selenium compounds from fish using size-exclusion chromatography with on-line detection by inductively coupled plasma mass spectrometry. AB - Fish accumulate significant amounts of selenium and are an important dietary source of this element. Some studies have however indicated a low bioavailability of the selenium from fish. Since little is known of the selenium forms in fish, we have studied soluble selenium compounds in fish species, and compared different techniques for fractionation of selenocompounds (size-exclusion chromatography, ultrafiltration, and precipitation with trichloroacetic acid). The size-exclusion column (Superdex 200 HR 10/30) was coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS). The limit of detection was 0.20 microgram l-1 and the selenium response was linear in the investigated concentration range of 0-20 micrograms l-1 (r2 = 0.98). For plaice 47% of the selenium was extractable while the extraction efficiency for cod was 23%. The fish extracts were injected onto the column four times each and the variation in the quantitative data for different selenium-containing fractions between the runs was small (RSD < 10%). The recovery of selenium in the chromatographic step was about 70%, indicating some interaction between the fish extracts and the column material. Ultrafiltration using a membrane with a cut-off at M(r) 10,000 gave results similar to the size-exclusion fractionation, for cod about 20% of the soluble selenium had a M(r) < 10,000 and the corresponding value for plaice was 69%. Removal of high-molecular-weight compounds from the sample by trichloroacetic acid precipitation showed a similar proportion of low-molecular weight compounds for plaice (77%), while the obtained value for cod was higher (38%) compared with the other techniques. PMID- 10746304 TI - Comparison of formats for the development of fiber-optic biosensors utilizing sol gel derived materials entrapping fluorescently-labelled protein. AB - The development of fiber-optic biosensors requires that a biorecognition element and a fluorescent reporter group be immobilized at or near the surface of an optical element such as a planar waveguide or optical fiber. In this study, we examined a model biorecognition element-reporter group couple consisting of human serum albumin that was site-selectively labelled at Cys 34 with iodoacetoxy nitrobenzoxadiazole (HSA-NBD). The labelled protein was encapsulated into sol-gel derived materials that were prepared either as monoliths, as beads that were formed at the distal tip of a fused silica optical fiber, or as thin films that were dipcast along the length of a glass slide or optical fiber. For fiber-based studies, the entrapped protein was excited using a helium-cadmium laser that was launched into a single optical fiber, and emission was separated from the incident radiation using a perforated mirror beam-splitter, and detected using a monochromator-photomultiplier tube assembly. Changes in fluorescence intensity were generated by denaturant-induced conformational changes in the protein or by iodide quenching. The analytical parameters of merit for the different encapsulation formats, including minimum protein loading level, response time and limit-of-detection, were examined, as were factors such as protein accessibility, leaching and photobleaching. Overall, the results indicated that both beads and films were suitable for biosensor development. In both formats, a substantial fraction of the entrapped protein remained accessible, and the entrapped protein retained a large degree of conformational flexibility. Thin films showed the most rapid response times, and provided good detection limits for a model analyte. However, the entrapment of proteins into beads at the distal tip of fibers provided better signal-to-noise and signal-to-background ratios, and required less protein for preparation. Hence, beads appear to be the most viable method for interfacing of proteins to optical fibers. PMID- 10746305 TI - Determination of methylmercury and inorganic mercury in whole blood by headspace, cryofocusing gas chromatography and atomic absorption spectrometry. AB - A method for the determination of different mercury species in whole blood is described. Inorganic mercury (InHg) was determined in 2 ml of standard solutions or blood samples using head space (HS) injection coupled to atomic absorption spectrometry (AAS) after treatment with concentrated sulfuric and tin(II) chloride as a reductant agent in a closed HS vial. After stirring, the InHg was converted to elementary mercury and carried with a nitrogen flow through a quartz cell heated at 200 degrees C and the absorbance signal was evaluated by AAS. For the determination of methylmercury (MeHg), 2 ml of a standard solution or a blood sample were treated with 10 mg of iodoacetic acid and 0.4 ml of concentrated H2SO4. Then, the MeHg species were HS-injected into a gas chromatograph (GC), separated on a semicapillary column (AT-1000) with a flow of helium, then carried to the quartz cell heated at 1000 degrees C and detected by AAS. The high content of salts in blood samples, where sodium chloride is the major component (0.14 mol l-1), affected the gas-liquid distribution coefficient of both mercury species in the HS vial. A linear calibration graph was obtained in the ranges 1-20 and 1-125 micrograms Hg l-1 added as InHg and MeHg, respectively. The detection limits for InHg and MeHg were 0.6 and 0.2 microgram Hg l-1, respectively. The relative standard deviations for eleven independent measurements were 5% for both mercury species. Recovery values ranging from 98 to 106% for InHg and from 95 to 105% for MeHg and from 93 to 95% for ethylmercury (EtHg) were obtained. The accuracy of the proposed method was also established by the analysis of certified whole blood samples for InHg and MeHg. No difference between the sum of these two species determined by our procedure and the recommended total mercury concentrations in the certified samples was observed. Results for the determination of MeHg and InHg in 30 controls and 30 dentists are presented to illustrate the practical utility of the proposed method. PMID- 10746306 TI - Flow injection determination of lactate based on a photochemical reaction using photometric and chemiluminescence detection. AB - A photochemical method for the determination of lactate using a flow-injection system is proposed. The method is based on the decomposition of lactate in the presence of UO2(2+) and Fe3+ upon irradiation with UV or visible light. The Fe2+ produced in the photochemical process was monitored by measuring the absorbance after complexation with ferrozine (lambda max = 562 nm) or the chemiluminescence (CL) intensity in a luminol system without added oxidant. The range of measurements depended on the length of the irradiation time and the detection system used. The detection limits using CL and photometric detection were 2 ng ml 1 and 50 ng ml-1, respectively. The sample throughput was 45 samples h-1. The usefulness of the method was demonstrated by determining lactate levels in blood serum, milk, yoghurt, beer and pharmaceutical preparations. PMID- 10746307 TI - Incorporation of selenium into selenoprotein P and extracellular glutathione peroxidase: HPLC-ICPMS data with enriched selenite. AB - The metabolic turnover of selenoprotein P (Sel P) and extracellular glutathione peroxidase (eGPx) in plasma was examined by HPLC-ICPMS, with the use of enriched Se, [82Se]selenite. [82Se]selenite was injected intravenously into rats at a dose of 25 micrograms 82Se kg-1 body weight, and the concentrations of labeled 82Se and naturally occurring 77Se in the serum, liver and kidneys were determined in samples obtained 1, 3, 6, 9, 12, 18 and 24 h after the injection. The distributions of both exogenous (labeled) 82Se and endogenous (naturally occurring) 77Se in serum, and supernatant fractions of the liver and kidneys were determined on a gel filtration column by HPLC-ICPMS. This dose was shown not to affect the constitutive levels of cellular GPx (cGPx), eGPx and Sel P. The labeled Se in Sel P increased soon after the injection, peaked at 6-9 h and then decreased, whereas that in eGPx continued increasing after 6 h post-injection and then throughout the remaining observation period in the bloodstream. These observations demonstrated the rapid and efficient incorporation of Se into Sel P in the liver and excretion into the plasma followed by the slow and steady incorporation of Se into eGPx in the kidneys and excretion into the plasma, with a minimal response of cGPx to selenite injection. PMID- 10746308 TI - Sensitive gas chromatographic-mass spectrometric method for the determination of gacyclidine in rat plasma and spinal cord dialyzates. AB - A sensitive gas chromatographic-mass spectrometric (GC-MS) procedure is described for the selective determination of gacyclidine (a non-competitive N-methyl-D aspartate antagonist) in rat plasma and spinal cord dialyzates. It involves a single-step liquid-liquid extraction of plasma samples and dialyzates with hexane (pH 8.0) and the use of phencyclidine as an internal standard. The compounds were separated on a GC capillary column and specifically detected by MS in the selected-ion monitoring mode. Gacyclidine and its internal standard were monitored by using the fragment ions at m/z 206 and 200, respectively. The method was accurate and reproducible (intra- and inter-day reproducibility < 12%) with a limit of quantification of 1.6 ng ml-1 using 100 microliters plasma of dialyzate samples. The calibration curves for rat plasma and Ringer's solution were linear (r2 > 0.996) over a range from 1.6 to 200 ng ml-1. The extraction efficiency was close to 100%. This simple and rapid assay (total run time < 10 min) was validated for a pilot pharmacokinetic study in healthy rats after intravenous injection of a bolus dose of gacyclidine (2.5 mg kg-1). PMID- 10746309 TI - Study of a molecular imprinting polymer coated BAW bio-mimic sensor and its application to the determination of caffeine in human serum and urine. AB - A bio-mimic bulk acoustic wave (BAW) sensor was fabricated by coating the caffeine (CAF) template molecular imprinting polymer (MIP). This sensor exhibited high selectivity and a sensitive mass response to CAF. The response range of the sensor was between 5.0 x 10(-9) and 1.0 x 10(-4) M with a detection limit of 5.0 x 10(-9) M at pH 8.0. Recoveries were 96.1-105.6%. Influencing factors were investigated in detail and optimized. When employed to detect real samples, the proposed method proved to be a convenient method with the advantages of high sensitivity, good selectivity and ease of handling. PMID- 10746310 TI - Sono-cathodic stripping voltammetry of manganese at a polished boron-doped diamond electrode: application to the determination of manganese in instant tea. AB - Ultrasonically assisted cathodic stripping voltammetry at a boron-doped diamond electrode was developed for the detection of manganese. Differential-pulse voltammetry was used to give the analytical signal from a cathodic strip of electrodeposited MnO2; linearity was observed from 10(-11) M to at least 3 x 10( 7) M, with 10(-11) M being the detection limit for a 2 min deposition. The procedure involves both ultrasonic-anodic deposition of MnO2 and ultrasonic cathodic stripping. This novel analytical tool is robust, reproducible, mercury free, oxygen insensitive and highly specific towards manganese. The differential pulse sono-cathodic stripping voltammetric technique was used to determine successfully the manganese content of two instant tea samples, giving excellent agreement with independent AAS analyses. PMID- 10746311 TI - Complexation study of diclofenac with beta-cyclodextrin and spectrofluorimetric determination. AB - The inclusional complexation between the anti-inflammatory pharmaceutical diclofenac and beta-cyclodextrin (beta-CD) was studied by potentiometry, spectrophotometry and spectrofluorimetry, in both acid and neutral pH. Guest-host 1:1 stoichiometries for the complexes in both media were determined, and their equilibrium constants were calculated. The values obtained from the different methods used are in very good agreement and are in the order of 10(3). From the analysis of the pKa value for diclofenac in both the absence and presence of beta CD (4.84 and 4.90 respectively), it was inferred that in the inclusion complex the carboxylic group is located outside the cavity. Further structural characterization of the inclusate was carried out by means of 1H NMR spectra and AM1 semiempirical calculations. Based on the obtained results, a spectrofluorimetric method for the determination of diclofenac in the presence of beta-CD was developed in the range of 0-5 micrograms ml-1. Better limits of detection (0.03 microgram ml-1) and quantification (0.1 microgram ml-1) were obtained in this latter case with respect to those obtained in the absence of beta-CD. The method was satisfactorily applied to the quantification of diclofenac in pharmaceutical preparations. PMID- 10746312 TI - The spectrophotometric determination of hydroperoxide and peroxide in a lipid pharmaceutical product by flow injection analysis. AB - A sensitive, rapid and automatable flow injection analysis procedure is described for the determination of total hydroperoxides and peroxides in lipid products. All unsaturated lipids are susceptible to degradation by oxidation, and the quantification of these major oxidation products is an essential measure of lipid product stability. In this methodology a lipid emulsion is dispersed and injected into an acidic solution of propan-2-ol, which is then merged with iodide ion in situ in a two-stream manifold. The lipid hydroperoxide oxidises acidified iodide to iodine, which is detected spectrophotometrically at 350 nm. The closed conditions prevent interference from atmospheric oxygen and the short reaction time minimises interference from side reactions. Conditions were optimised, using experimental design, for a lipid product under development at GlaxoWellcome. A two-level half-fractional factorial design was applied to screen for the critical factors, followed by a multi-level central composite design to optimise these variables. The resulting method was fully validated and is linear down to 0.1 nmol ml-1. This approach should be applicable to other lipid formulations and offers significant advantages in terms of speed, automation and precision compared with existing manual procedures. PMID- 10746313 TI - Mechanised flow system for on-line microwave digestion of food samples with off line catalytic spectrophotometric determination of cobalt at ng l-1 levels. AB - A mechanised system for on-line slurry food sample digestion was developed and an off-line cobalt determination was performed. The stabilised slurry sample was introduced into an air carrier stream until reaching the digestion coils located inside a household microwave oven. Software written in Visual Basic 3.0 was developed to permit the transport of the slurry samples and the programming of the microwave oven and also the control of the mineralization valve. The proposed system was optimized for determination of cobalt in certified samples such as mussels, bovine liver and fish and also uncertified fish samples. The digestion parameters were established as 3 mol l-1 HNO3 for mussels, 3 mol l-1 HNO3 plus 0.16% v/v H2O2 for bovine liver and 12 mol l-1 HNO3 for fish employing maximum power for 5 min of microwave actuation. In the subsequent spectrophotometric method for the catalytic determination of cobalt, the Tiron and hydrogen peroxide concentrations were 1.8 x 10(-3) and 3.0 x 10(-4) mol l-1, respectively, and the sample residence time was 300 s as determined by an optimisation process. The proposed method features a linear range from 10 to 200 ng l-1 Co (r > 0.996) with detection and quantification limits of 1.7 and 5.5 ng l-1 Co, respectively. The precision, expressed as RSD, was 2.4% (n = 12) for repeatability and 5.2% (n = 10) for reproducibility and the accuracy of the proposed method was assessed by using certified samples and an alternative technique (ETAAS). PMID- 10746314 TI - Determination of carphedon in human urine by solid-phase microextraction using capillary gas chromatography with nitrogen-phosphorus detection. AB - Carphedon is a phenyl derivative of nootropil and is effective in increasing physical endurance and cold resistance, and is used for amnesia treatment. Carphedon was extracted from human urine samples by solid-phase microextraction with a 65 microns carbowax-divinylbenzene-coated fiber. This analysis was performed by using capillary gas chromatography with nitrogen-phosphorus detection and optimized at pH 9.6, 30% NaCl, immersion time 10 min and desorption in the GC injector at 250 degrees C for 3 min. The regression equation for carphedon showed good linearity in the range from 0.1 to 10 micrograms ml-1 for human urine samples. The limit of detection was 0.01 microgram ml-1. The developed method is more sensitive and simpler in sample preparation than liquid liquid extraction and can be applied to doping analysis for stimulants. PMID- 10746315 TI - Production and characterization of polyclonal antibodies to sulfamethazine and their potential use in immunoaffinity chromatography for urine sample pre treatment. AB - An immunoaffinity chromatographic (IAC) method for isolating sulfamethazine (SMZ) from incurred urine samples was developed. This was achieved by (i) generating polyclonal antibodies that recognize equally well SMZ and its major urinary metabolites, (ii) evaluating in an ELISA procedure the influence of methanol, salt and pH on the antigen-antibody interaction in order to determine the optimum conditions for IAC and (iii) covalent coupling of the IgG fractions of anti-SMZ to CNBr activated Sepharose for the preparation of re-usable immunoaffinity columns, having a high capacity for SMZ (1900 ng SMZ mL-1 gel). For desorbing SMZ from the immunoaffinity column, different elution modes were evaluated, with 40% MeOH-0.1 mol L-1 HOAc-0.5 mol L-1 NaCl being the most efficient combination. Using the IAC column for processing SMZ spiked urine samples resulted in high recoveries, ranging from 92 to 100%. Because of the high cross-reactivity with the major metabolites of SMZ present in urine of treated animals, the antibodies show excellent properties for use in both IAC and ELISA. For the isolation and concentration of the parent drug and its major metabolites, the urine could be applied directly to the IAC column, without the time-consuming step of deconjugation. Moreover, the use of IAC prior to ELISA for the analysis of incurred urine samples showed good efficiency for the elimination of matrix interferences. Owing to the urine-tissue relationship, the urine concentrations can be used to predict the presence of the parent drug in tissues and so possible violations of the maximum residue limit (MRL) can be controlled. PMID- 10746316 TI - Utilization of a scientifically operated charge-coupled device detector for high performance thin-layer chromatographic analysis of tetracyclines. AB - A high-performance thin-layer chromatography (HPTLC) method using a scientifically operated charge-coupled device detector is described for the assay of tetracycline pharmaceutical products. Quantitative information can be obtained for all samples on a TLC plate within a few seconds. The separation efficiency and detection limits were determined on both normal phase and reverse phase TLC plates. Fluorescence detection mode offers higher sensitivity than fluorescence quenching mode. The dynamic range, sensitivity, accuracy and precision of the system were evaluated. Detection limits of the impurities are in the range of 0.1 to 0.5 ng or 0.3 to 1% of tetracycline, depending on the compound, with a recovery percentage over 85%. The existing impurities in tetracycline capsules were determined using both HPLC and HPTLC techniques. All of the impurities were below the regulation level. PMID- 10746317 TI - Determination of dexamethasone in feed by TLC and HPLC. AB - Two detection methods are described for the determination of dexamethasone in feed, which may be used as screening and confirmatory methods. The procedures were developed after studying different extraction and clean-up procedures: feed extraction was optimized using methylene chloride and hexane followed by a tandem solid-phase clean-up step with silica and C18 cartridges. The confirmatory method presented is based on normal-phase high-performance liquid chromatography on a Diol 5 microns column, with hexane-propan-2-ol (90:10 v/v) as mobile phase, followed by diode array detection and confirmation. All parameters are discussed, especially the extraction and clean-up steps. Recovery studies after application of the proposed method to different kinds of feed samples spiked with dexamethasone at levels between 120 and 1600 ng g-1 yielded a mean value of 55.4% with an overall standard deviation of 15.6%. The proposed procedure allows the determination of dexamethasone in feed at levels lower than 50 ng g-1. PMID- 10746318 TI - An HPLC assay for the determination of ketoconazole in common pharmaceutical preparations. AB - An HPLC method is described using octadecylsilica (3 microns) with an acetonitrile phosphate buffer mobile phase containing diethylamine which is capable of separating ketoconazole [(+/-)-cis-1-acetyl-4-(4[2-(dichlorophenyl)-2- (1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl)pi perazine] from four related compounds, (R049223, R063600, R053165 and R039519) and from excipients in tablets, cream and shampoo. The method was validated using an external calibration method for tablets, shampoo and creams and a standard addition method for cream. The limits of detection for the related compounds in the presence of ketoconazole are also reported. PMID- 10746319 TI - Dipicolinic acid (DPA) assay revisited and appraised for spore detection. AB - Delayed gate fluorescence detection of dipicolinic acid (DPA), a universal and specific component of bacterial spores, has been appraised for use in a rapid analytical method for the detection of low concentrations of bacterial spores. DPA was assayed by fluorimetric detection of its chelates with lanthanide metals. The influence of the choice and concentration of lanthanide and buffer ions on the fluorescence assay was studied as well as the effects of pH and temperature. The optimal system quantified the fluorescence of terbium monodipicolinate in a solution of 10 microM terbium chloride buffered with 1 M sodium acetate, pH 5.6 and had a detection limit of 2 nM DPA. This assay allowed the first real-time monitoring of the germination of bacterial spores by continuously quantifying exuded DPA. A detection limit of 10(4) Bacillus subtilis spores ml-1 was reached, representing a substantial improvement over previous rapid tests. PMID- 10746320 TI - Automated stand-alone flow injection immunoanalysis system for the determination of cephalexin in milk. AB - A fully automated stand-alone flow injection immunoanalysis (FIIA) device for the determination of cephalexin in milk is developed with a main focus on the investigation of the influence of the sample matrix. The system is based on principles of flow-through immunoassays and on sequential addition of the assay components to an immunoreactor. Protein G is immobilised on the surface of the immunoreactor serving as affinity matrix for the polyclonal anti-cephalexin antibodies. A cephalexin-alkaline phosphatase conjugate is mixed with the analyte containing sample and binds in a competitve manner to the corresponding antibodies in the immunoreactor. After substrate addition enzymatically generated p-aminophenol is detected at a carbon electrode at +150 mV vs. Ag/AgCl. One assay cycle takes 16 min including regeneration of the immunoreactor. The large excess of protein G allows for more than 150 regenerations without significant loss of signal height. Due to the high specificity of the anti-cephalexin antibodies, other beta-lactam antibiotics like penicillin, amoxicillin and cloxacillin do not interfere in the measurements, even when added at 10 mg l-1. To deactivate alkaline phosphatase present in milk, samples are heat-treated for 3 min prior to measurements. Cephalexin recoveries from two milk samples are 90 and 110%. The detection limit in milk is 1 microgram l-1 (mean relative standard deviation of 3%), less than the maximum residue level of 4 micrograms per kg milk fixed for some beta-lactam antibiotics in the European Union. The device is suitable for fast quantitative data generation from consecutively measured samples and thus adds to analytical screening methods. PMID- 10746321 TI - Production of monoclonal antibody and development of enzyme-linked immunosorbent assay for kanamycin in biological matrices. AB - Monoclonal antibodies (MAbs) against kanamycin were prepared by using a kanamycin bovine gamma-globulin conjugate for the immunization of mice. Splenocytes from BALB/c immunized mice were fused with P3X63Ag8U.1 myeloma cells. This resulted in two hybridoma cell lines. Fifty per cent inhibition concentrations (IC50) for the MAbs were 2 and 5 ng ml-1. One MAb (IC50 = 2 ng ml-1) was named #22 and was used to develop quantitative assays for kanamycin by means of an enzyme-linked immunosorbent assay (ELISA). The detection limit was 0.2 ng ml-1 and the standard deviations were 0.2-4.4% for intra-assay and 0.6-4.7% for inter-assay, respectively. The detection limits using peroxidase were 4 ppb in cattle milk, cattle plasma, cattle urine, swine plasma, swine urine and chicken plasma. Using the MAb #22 produced, a rapid test kit based on an immunochromatographic method was developed. The detection limits using the kit were 50 ppb in cattle milk, cattle plasma, cattle urine and chicken plasma. PMID- 10746322 TI - Construction and analytical application of ion-selective piezoelectric sensor for atropine sulfate. AB - The method describes the use of a piezoelectric quartz crystal (PQC) as a substitute for ion-selective electrodes. The approach is feasible when the membrane materials are electrically non-conductive and membrane potential measurements are consequently not possible. An ion-selective piezoelectric sensor sensitive to atropine sulfate was constructed by coating a PVC membrane containing activant on one the side of a PQC. On the basis of selective adsorption of atropine ions across the modified film and the sensitive mass response of PQC, the method exhibits a sensitive, rapid response and is easy to operate without pretreatment of the sample. The logarithm of the frequency shift gave a linear relationship with the logarithm of atropine sulfate concentration in the 1.0 x 10(-8)-1.0 x 10(-3) M range with a detection limit of 5.0 x 10(-9) M at pH 7.0. Recoveries were from 98.7-102.2%. Two activants, atropine tetraphenylborate and atropine dipicrylaminate, were synthesized and investigated. Influencing factors were also examined and optimized. The results for real samples obtained by the proposed method agreed with those obtained by conventional methods. PMID- 10746323 TI - Adsorptive and electrochemical behaviors of estradiol valerate at a mercury electrode. AB - It was found that estradiol valerate could be adsorbed at a mercury electrode under open circuit. The adsorptive and electrochemical behaviors of estradiol valerate on a static mercury electrode were investigated by cyclic voltammetry, linear scan voltammetry and chronocoulometry. Based on this, a sensitive and selective adsorptive stripping square-wave voltammetric method was developed for the determination of estradiol valerate based on the optimization of solution conditions and electrochemical parameters. It was found that in a Britton Robinson buffer solution containing 18% alcohol (pH 9.5), estradiol valerate gave a sensitive reductive peak at potential -1.29 V (vs. SCE) and the peak current was linear with the concentration of estradiol valerate in the range 2.0 x 10(-8) 2.5 x 10(-6) mol L-1. The detection limit was 1.1 x 10(-8) mol L-1. The interference of some common steroid estrogens was examined and it was found that they did not interfere in the determination of estradiol valerate in the present system. PMID- 10746325 TI - Towards the practical application of recent advances. PMID- 10746324 TI - Experimental design and multivariate calibration in the development, set-up and validation of a differential pulse polarographic and UV spectrophotometric method for the simultaneous plasmatic determination of the therapeutic metronidazole pefloxacin combination. AB - Partial least squares regression (PLS1 and PLS2) and GOLPE variable selection procedures were used for the treatment of differential pulse polarographic and UV spectrophotometric data obtained from the analysis of the therapeutic combination of metronidazole and pefloxacin. The analytical method used for the determination was set up using experimental design strategies (Doehlert's design, full factorial design, fractional face center cube design, etc.) and by involving the simultaneous optimization of several responses (desirability function). Method validation was also performed, determining accuracy, precision, linearity and range, detection and quantification limits and robustness. The quantitative prediction abilities in determining metronidazole and pefloxacin plasma levels of the PLS1 and PLS2 models were tested on spiked plasma samples and good results were obtained (metronidazole, 97.5%, RSD = 4.8%, n = 3; pefloxacin, 100.6%, RSD = 3.6%, n = 3). The use of multivariate calibration was particularly useful for spectrophotometric quantification because of the highly overlapping spectra of the binary mixture. PMID- 10746326 TI - Optimal use of resources for the treatment and prevention of injuries. AB - Injuries are an important cause of mortality and morbidity. Although accidental injury rates have been declining throughout the twentieth century in the UK, this pattern has been variable. For example, in young adults aged 15-24 years there has been no improvement and, when deliberate injuries are included, the picture is worsening. Although there is little evidence that road traffic accident case fatality rates have been improving, there is some evidence that improvements in trauma care have been responsible for reducing injury death rates in children. Thus, although there have been considerable successes in the primary prevention of accidents, and the secondary prevention of injuries in accidents, there is an important role for tertiary prevention, that is in the prevention of avoidable outcomes through good trauma care. PMID- 10746327 TI - Systemic responses to trauma. AB - The systemic responses to trauma can be divided into cardiovascular, immunological, and metabolic. The cardiovascular responses are seen immediately after a traumatic insult. The pattern of response depends on whether the insult is mainly haemorrhagic, tissue damage, or a combination of the two. The response may be quite different for penetrating vascular trauma, compared with a crush injury to a limb. The immunological, or inflammatory, consequences of trauma usually become apparent several hours or days after the initial insult, although it is increasingly clear that they may be triggered by the very early cardiovascular changes. These have been implicated in the development of multiple organ failure. The metabolic responses are of greatest importance in the longer term: after successful resuscitation and after the definitive treatment of the patient's injuries. The metabolic responses need to be taken into account during the recovery from treatment and during the rehabilitation of the patient. PMID- 10746328 TI - Co-morbid factors in trauma patients. AB - A variety of factors influence the survival of trauma patients including the severity and site of injury, and the timing and quality of care. However, host factors including age and gender have also been reported as independent risk factors that adversely influence outcome. In addition, the presence of co-morbid or pre-existing factors has been shown to increase mortality and morbidity after trauma. This chapter reviews the evidence for these associations and considers their impact on assessment and management of the trauma patient. PMID- 10746329 TI - Prehospital care--a UK perspective. AB - In the UK, emergency ambulances are responding to astonishing increases in levels of emergency calls, in the order of a 40% increase nationally in the last 5 years. Pressures in primary care service out-of-hours provision, and increasing community-based care of elderly patients, as well as increased expectation by the public are contributory causes. Services are also being pressed to improve response times, particularly to life-threatening cases. These various aspects are discussed below. PMID- 10746330 TI - Organisation of hospital responses for the trauma epidemic. AB - The caseload of the Department of Surgery (Baragwanath Hospital, Johannesburg, South Africa) is characterised by a singularly heavy trauma component. Penetrating injuries account for the majority of cases. The management of penetrating trauma is not as demanding as that of blunt trauma, yet the sheer number of cases, over the past four decades, has imposed a clinical burden that has never been met by commensurate resources. The organisation of the hospital and departmental responses has manifested itself on two different levels. The first one is the structural and functional deployment of insufficient staff, facilities and equipment to cope as flexibly as possible with the trauma epidemic. The second one is the gradual adaptation of the clinical management philosophy to ensure that the best possible quality of care is provided to the majority of trauma victims, with the full knowledge that better resources would sometimes have elicited a different clinical approach. PMID- 10746331 TI - Resuscitation after high energy polytrauma. AB - The majority of patients with polytrauma seen in the UK are victims of blunt injury. The trauma reception team approach, using a predetermined plan for initial assessment and urgent resuscitation, can improve outcome. It is important, therefore, that each member of the team is familiar with both their own role and that of their colleagues. This chapter reviews the immediate management of the patient with polytrauma, focusing on the importance of the 'ABC' approach. Optimal management of the trauma airway is essential and the benefits of early intubation and alternative techniques for securing the difficult airway are discussed. The early identification and management of life threatening respiratory and circulatory complications is emphasised. Accurate assessment of shock in the victim of trauma is difficult, as the simple clinical indicators are not ideal. Some of the techniques available for advanced assessment of tissue perfusion are discussed in detail. The management of polytrauma provides a considerable clinical challenge, and this chapter emphasises the importance of a team approach. PMID- 10746332 TI - Strategies for ventilatory support. AB - Since the introduction of manual positive pressure ventilation during the Copenhagen polio epidemic of 1952, a range of mechanical devices and methods of ventilatory support have been developed to provide life-saving respiratory therapy to critically ill patients. In addition to the development of the equipment hardware to enable effective mechanical ventilation, there has been a gradual realisation that a single mode of ventilation is not universally applicable to all patients, individual pathologies, or to individual patients at various stages in the evolution of their pulmonary pathology. While mechanical ventilation can undoubtedly be life-saving, it can also cause lung damage as a result of its non-physiological method of promoting effective gas exchange. This iatrogenic problem is known as ventilator induced lung injury and, although it may be a problem in any patient requiring mechanical ventilation for any reason, is of particular importance to those patients requiring mechanical ventilation as a consequence of trauma. This chapter describes the range of ventilatory support techniques available, the problem of ventilator induced lung injury with specific reference to trauma patients and offers a strategy for ventilatory support in the trauma patient. PMID- 10746333 TI - Fluid replacement. AB - Appropriate fluid replacement is an essential component of trauma patient resuscitation. Once haemorrhage is controlled, the restoration of normovolaemia is a priority. In the presence of uncontrolled haemorrhage, aggressive fluid resuscitation may be harmful. The crystalloid-colloid debate continues, but existing clinical practice is more likely to reflect local biases and dogma rather than evidence-based medicine. Colloids vary substantially in their pharmacology and pharmacokinetics and the experimental findings based on one colloid cannot be extrapolated reliably to another. In the initial stages of trauma patient resuscitation, the precise fluid used is probably not important, as long as an appropriate volume is given. Later, when the microcirculation is relatively leaky, there may be some advantages to colloids such as hydroxyethyl starch. Hypertonic saline solutions may have some benefit in patients with head injuries. A number of haemoglobin solutions are under development but one of the most promising of these has been withdrawn recently. It is highly likely that at least one of these solutions will eventually become routine therapy for trauma patient resuscitation. In the mean time, contrary to traditional teaching, recent data suggest that a restrictive strategy of red cell transfusion may improve outcome in some critically ill patients. PMID- 10746334 TI - Blunt abdominal injuries. AB - The management of blunt abdominal injury (BAI) has undergone quite significant changes over recent years. The emphasis is now on the recognition and limitation of the underlying metabolic insult associated with severe abdominal injury. The concepts of damage control and non-operative management while seeming diametrically opposed have both found favour in selected patient groups. The interventional radiologist has opened a new dimension in the control of inaccessible bleeding and is able to contribute to non-operative approaches. The complimentary use of the methods of investigation available for BAI will also improve the accuracy and specificity of diagnosis allowing more appropriate management. Embracing these new concepts of management by all institutions dealing with trauma victims will hopefully reduce the morbidity and mortality of BAI. PMID- 10746335 TI - The biology of fracture healing: optimising outcome. AB - Optimising the results of fracture treatment requires a holistic view of both patients and treatment. The nature of the patient determines the priority targets for outcome, which differ widely between the elderly and the young, and between the victims of high and low energy trauma. The efficacy of treatment depends on the overall process of care and rehabilitation as well as the strategy adopted to achieve bone healing. The rational basis for fracture treatment is the interaction between three elements: (i) the cell biology of bone regeneration; (ii) the revascularisation of devitalized bone and soft tissue adjacent to the fracture; and (iii) the mechanical environment of the fracture. The development of systems for early fracture stabilisation has been an advance. However, narrow thinking centred only on the restoration of mechanical integrity leads to poor strategy--the aim is to optimise the environment for bone healing. Future advances may come from the adjuvant use of molecular stimuli to bone regeneration. PMID- 10746336 TI - Limb reconstruction after high energy trauma. AB - Limb reconstruction techniques rely on stable external fixation to provide early limb function after major long bone injury. Bone may be generated by callus distraction techniques and internal techniques of moving bone segments used to fill bone defects. Soft tissue defects may be treated by acute shortening, although skin defects will also close spontaneously during bone transport as the leading edge of bone is covered with granulation tissue. External fixation is also used to cross joints permitting rest and repair of the joint. Hinges placed within the bars of the fixation frame may be used to correct deformities in the bone and soft tissue contractures using closed distraction techniques. These techniques are appropriate to metaphyseal fractures and diaphyseal fractures with bone loss. A major advantage is the lack of donor site morbidity, associated with skin flaps and large bone grafts. Acceptance of these techniques is growing whilst the methodology continues to improve. In more complicated cases, specialist training and dedicated hospital units with multidisciplinary support is desirable. PMID- 10746337 TI - Burns. PMID- 10746338 TI - Injury in the aging: recovery and rehabilitation. AB - Interest in how victims of traumatic injuries recover is increasing and a number of observational studies have now been done. There are very few intervention studies aimed at enhancing recovery, but there will be more as our knowledge base grows. Older recipients of traumatic injuries differ from the young in the types of injuries they sustain, in the way they respond to their injuries and in the consequences of even relatively minor injuries on their future independence. In this paper, we summarise our understanding of recovery after injury and consider this in more depth for older people with specific injuries. PMID- 10746339 TI - Improving outcome after traumatic brain injury--progress and challenges. AB - This article describes the rapid advances in the head injury field which have taken place within the professional lifetime of many doctors in practice today. These have led to a better understanding of what happens in the injured brain and how these events might be manipulated to achieve better outcomes. Clinical tools we now take for granted, like the CT scanner and the Glasgow Coma Scale, were new developments 25 years ago. They provided a foundation on which clinicians and basic scientists could build what we now know: what to assess in the patient, how to respond to certain findings, what imaging to do, how to plan treatment rationally, how to minimise brain damage at different stages after injury, how to predict and measure outcome, what disabled survivors need, and how to organise the service to do the greatest good for the most people. Some of these topics raise as many questions as answers. The head injury field may be broad but it has essential unity. At one extreme, some patients have a life-threatening illness where the acts and omissions of the clinical team can powerfully influence not only survival but its quality. Later the drama of the acute phase gives way to the 'hidden disabilities' of the long-term deficits which so many survivors have. At the other end of the severity spectrum is the relatively vast number of people who suffer an apparently mild head injury, a few of whom deteriorate and need urgent treatment, and many of whom have unspectacular but, nevertheless, disabling problems. The article attempts to address this broad canvas. Clinicians, neuroscientists, policy makers, and service users must work together to address the major scientific, individual, and population challenges posed by head injury. Much has already been achieved, but much remains to be done, especially in translating 'what we know' into 'what we do'. PMID- 10746340 TI - Rehabilitation after traumatic brain injury. AB - Head injury is a common disabling condition but regrettably facilities for rehabilitation are sparse. There is now increasing evidence of the efficacy of a comprehensive multidisciplinary rehabilitation team compared to natural recovery following brain injury. This chapter outlines some basic concepts of rehabilitation and emphasises the importance of valid and reliable outcome measures. The evidence of the efficacy of a rehabilitation programme is discussed in some detail. A number of specific rehabilitation problems are outlined including the management of spasticity, nutrition, pressure sores and urinary continence. The increasingly important role of assistive technology is illustrated, particularly in terms of communication aids and environmental control equipment. However, the major long-term difficulties after head injury focus around the cognitive, intellectual, behavioural and emotional problems. The complex management of these disorders is briefly addressed and the evidence of the efficacy of some techniques discussed. The importance of recognition of the vegetative stage and avoidance of misdiagnosis is emphasised. Finally, the important, but often neglected, area of employment rehabilitation is covered. PMID- 10746341 TI - Micronutrients in fetal growth and development. AB - The roles that the different vitamins and minerals play in fetal growth and development are reviewed, primarily with respect to growth and differentiation in humans; but, as appropriate, data provided from animal and cellular studies are also considered. PMID- 10746342 TI - The role of micronutrients in psychomotor and cognitive development. AB - The literature on the effects of micronutrients on cognitive, motor and behavioural development is reviewed focusing mainly on children. Iron, zinc, iodine and vitamins are discussed. The review is selective and concentrates on the more recent work and areas of controversy. There are well established associations with poor development and iron and iodine deficiency but the deficiencies usually occur in disadvantaged circumstances and establishing causal relationships is difficult. PMID- 10746343 TI - Selenium and viral virulence. AB - A mouse model of coxsackievirus-induced myocarditis is being used to investigate nutritional determinants of viral virulence. This approach was suggested by research carried out in China which showed that mice fed diets composed of low selenium ingredients from a Keshan disease area suffered more extensive heart damage when infected with a coxsackie B4 virus than infected mice fed the same diet but supplemented with selenium by esophageal intubation. Selenium deficiency in our mice increased the virulence of an already virulent strain of coxsackievirus B3 (CVB3/20) and also allowed conversion of a non-virulent strain (CVB3/0) to virulence. Such conversion of CVB3/0 was accompanied by a change in the viral genome to more closely match that of the virulent virus, CVB3/20. As far as the authors are aware, this is the first report of host nutrition influencing the genetic make-up of an invading pathogen. Nutritionists may need to consider this mechanism of increased viral virulence in order to gain a better understanding of diet/infection relationships. PMID- 10746344 TI - Anaemia and iron deficiency disease in children. AB - Iron deficiency is the single most common nutritional disorder world-wide and the main cause of anaemia in infancy, childhood and pregnancy. It is prevalent in most of the developing world and it is probably the only nutritional deficiency of consideration in industrialised countries. In the developing world the prevalence of iron deficiency is high, and is due mainly to a low intake of bioavailable iron. However, in this setting, iron deficiency often co-exists with other conditions such as, malnutrition, vitamin A deficiency, folate deficiency, and infection. In tropical regions, parasitic infestation and haemoglobinopathies are also a common cause of anaemia. In the developed world iron deficiency is mainly a single nutritional problem. The conditions previously mentioned might contribute to the development of iron deficiency or they present difficulties in the laboratory diagnosis of iron deficiency. PMID- 10746345 TI - Disorders of copper transport. AB - Copper is an essential component of a number of important enzymes. Efficient systems have developed for providing sufficient copper for essential functions, while eliminating excess to avoid tissue toxicity. Copper transport is disrupted in two human diseases: Wilson disease and Menkes disease. Both have defects in copper transporting membrane proteins. Many other proteins are involved in copper transport. Some of these proteins have been identified through a study of the similar copper pathway in yeast. This suggests other copper transport diseases are yet to be discovered. Molecular diagnosis holds promise for reliable diagnosis of patients. Testing of flanking markers is a reliable way to detect presymptomatic sibs of a definite patient. PMID- 10746346 TI - Inborn errors of metabolism: iron. AB - The iron content of the body is normally closely regulated. Despite this, iron deficiency anaemia is common in women because iron losses due to menstruation and childbirth are not always compensated for by iron absorption from the diet. The role of transferrin in delivering iron to cells and of ferritin in storing iron within cells is well understood but the proteins involved in iron transport across membranes are only now being investigated. Relatively few genetic disorders affecting iron metabolism are known and most are rare. This paper briefly describes pyridoxine responsive sideroblastic anaemia, hyperferritinaemia cataract syndrome, atransferrinaemia and genetic haemochromatosis. Rather than rare, the latter is one of the most common inherited disorders in northern European populations. Mutations in genes regulating membrane iron transport causing simple iron deficiency have not yet been described. PMID- 10746347 TI - Effects of antioxidants on vascular health. AB - Substantial in vitro and animal model evidence implicates the free radical mediated oxidation of low density lipoprotein and its subsequent preferential uptake by macrophages in the arterial intima as an important factor in the development of vascular disease. In addition, antioxidants which prevent the oxidation of LDL in vitro also reduce the severity of vascular disease in animal models. Although some epidemiological studies also suggest that inadequate antioxidant status is related to the development of vascular disease, particularly cardiovascular disease, results from intervention trials have been contradictory. Whereas vitamin E may have a role in reducing the incidence of vascular disease, evidence is less strong for vitamin C, flavonoids and beta carotene. Additionally, supplementation with some antioxidants such as beta carotene may increase the incidence of cancer in high risk groups. Although increasing antioxidant intake is generally beneficial for health, this should perhaps be achieved by an increased dietary intake of antioxidant-rich foods rather than by use of supplements. PMID- 10746348 TI - Folic acid deficiency and cancer: mechanisms of DNA instability. AB - Folic acid deficiency in humans has been linked with megaloblastic anaemia, neural tube defects in the neonate, and heart disease. Folate has also been implicated in the development of cancer, especially cancer of the colorectum. There appear to be two principal mechanisms through which low folate status may increase the risk of malignancy. Folate deficiency, by reducing intracellular S adenosylmethionine (SAM), can alter cytosine methylation in DNA, leading to inappropriate activation of proto-oncogenes and induction of malignant transformation. Alternatively, folic acid is crucial for normal DNA synthesis and repair. Folate deficiency may cause an imbalance in DNA precursors, uracil misincorporation into DNA, and chromosome breakage. This chapter briefly describes the epidemiological data supporting the involvement of folic acid in the aetiology of cancer. It also assesses the evidence from cellular, animal and human studies that folic acid can modulate DNA by such mechanisms. PMID- 10746349 TI - Chemoprevention of cancer: a controversial and instructive story. AB - Increased intake of fruits and vegetables seems to be one of the simplest means of decreasing the risk for cancer. Cancer-preventive effects of fruits and vegetables have been observed in epidemiological studies, which could not, however, distinguish the effects of the various ingredients. Antioxidant defence has been proposed as a mechanism of chemoprevention, although inconclusive results have been obtained. The results of randomized intervention trials have shown that beta-carotene supplements are of limited value and may even be deleterious. Vitamins are a good marker of the ingestion of fruits and vegetables, and vitamin E (alpha-tocopherol) is a lipid-soluble antioxidant which can scavenge free radicals. It has no significant effect on the risk for lung cancer of long-term smokers in an intervention trial, but it decreased both the incidence of and mortality from prostate cancer; however, there was a 50% increase in the occurrence of cerebral haemorrhage among the men given vitamin E. Aspirin and aspirin-like drugs appear to decrease the risk for intestinal tumours; the mechanism of action appears to involve diminishing prostaglandin production due to inhibition of cyclooxygenases. Dietary fibre has been linked to a reduced risk for colorectal cancer in many observational studies, but opposite findings were reported recently. In order to resolve these paradoxes, we need to understand better the underlying biology, develop mechanistic hypotheses and test them in clinical trials in humans. Until that time, we should confine any premature enthusiasm for chemopreventive micronutrient supplementation. PMID- 10746350 TI - Problems and approaches in investigating the role of micronutrients in the aetiology of cancer in humans. AB - Observational studies have provided leads regarding a number of micronutrients which may account for the apparent protective effects of high intakes of vegetables and fruit against many types of cancer. In general, these leads have not been confirmed by randomised controlled trials. This apparent conflict raises issues about the timing and duration of a critical period or periods during which micronutrient intake may influence the development of cancer, the dose, possible interaction between high doses of micronutrients and exposures conferring a high risk of cancer and gene-micronutrient interactions. When gene-environmental interaction exists, failure to take both of these sets of factors into account leads to bias in the estimation of disease risk. As a result of recent advances, it is now possible to take measures of genetic susceptibility into account. Therefore, in future studies, the opportunity should be taken to obtain DNA samples to determine genotypes for polymorphisms potentially affecting micronutrient metabolism. PMID- 10746351 TI - Bone health: the role of micronutrients. AB - Development and maintenance of skeletal health is essential since the resultant effect of poor bone health is an increased risk of osteoporotic fracture. Osteoporosis is currently a major public health problem and with predicted demographic changes, its future health and economic impact is likely to be phenomenal. Adult bone health is predominantly governed by two factors: (i) maximum attainment of peak bone mass; and (ii) rate of bone loss which occurs with ageing. Both aspects are determined by a combination of endogenous and exogenous factors and, although genetic influences are believed to account for up to three-quarters of the variation in bone mass, there is still room for the modifiable factors (including nutrition) to play an important role. There is now good evidence to show that calcium is important not only to peak bone mass development but also in reducing bone loss in women who are greater than 5 year postmenopause. Vitamin D and calcium (and possibly vitamin K) are vital to fracture prevention in the elderly. Our knowledge of the influence of other micronutrients on bone health remains limited and further research is required to establish the essential ingredients for optimum bone health. PMID- 10746352 TI - Diagnosis and detection of deficiencies of micronutrients: minerals. AB - Determination of the presence or absence of a deficiency of one or more of the micronutrient minerals (usually described as trace elements) can be a complex problem, frequently requiring the integration of clinical, nutritional and biochemical data. Almost invariably, laboratory investigations are required and this short review describes the more common techniques applied for the various essential trace elements. Using a combination of techniques it is usually possible to determine, with confidence, whether an individual subject or small groups of subjects have a deficiency of a specific trace elements, but simple reliable tests which can be used in population studies are still lacking for several key elements. This problem appears most acute for studies of chromium, copper and zinc, deficiencies of which may have important roles in the pathogenesis of a variety of human disorders. PMID- 10746353 TI - Diagnosis and detection of vitamin deficiencies. AB - Vitamin deficiencies can be detected in several ways, none of which is entirely unambiguous. Evidence of several types is, therefore, required. For instance, it is rare for clinical signs to result from a single cause, except in controlled experiments. Natural diets are rarely deficient in only a single nutrient, and individual requirements vary considerably. Biochemical and functional status indices can help bridge the gap between inadequate diet and resulting pathology. Some indices are very specific for individual vitamins; others, although only semi-specific, are useful if closely linked to tissue malfunction and hence to pathology. Ideally, biochemical indices should separate severe deficiency, mild subclinical deficiency, normal status and overload toxicity. Vitamin concentrations in plasma, serum, red cells, urine, and other accessible tissues have been used, and metabolic products of vitamin-dependent metabolic pathways have been exploited. However, many of the assays are difficult to perform and interpret, and are limited to few laboratories, world-wide. There is a need for simpler and more 'portable' tests, for routine laboratories and for the medical profession. PMID- 10746354 TI - Thyroid function. AB - Normal thyroid status is dependent on the presence of many trace elements for both the synthesis and metabolism of thyroid hormones. Iodine is most important as a component of the hormones, thyroxine and 3,3',5-tri-iodothyronine (T3) and iodine deficiency may affect approximately one billion people throughout the world. Selenium is essential for normal thyroid hormone metabolism being involved with selenium-containing iodothyronine de-iodinases that control the synthesis and degradation of the biologically active thyroid hormone, T3. Additionally, selenoperoxidases and thioredoxin reductase protect the thyroid gland from peroxides produced during the synthesis of hormones. The roles of iron, zinc and copper in the thyroid are less well defined but sub- or supraoptimal dietary intakes of all these elements can adversely affect thyroid hormone metabolism. PMID- 10746355 TI - Brain function in the elderly: role of vitamin B12 and folate. AB - Vitamin B12 (cobalamin) deficiency associated neuropathy, originally called subacute combined degeneration, is particularly common in the elderly. The potential danger today is that with supplementation with folic acid of dietary staples such as flour, that the incidence of this disease could rise as folic acid, as opposed to natural folate (N5CH3HFGlu1), enters the cell and the metabolic cycle by a cobalamin independent pathway. This chapter briefly describes the clinical presentation of the disease, which unless treated will induce permanent CNS damage. The biochemical basis of the interrelationship between folate and cobalamin is the maintenance of two functions, nucleic acid synthesis and the methylation reactions. The latter is particularly important in the brain and relies especially on maintaining the concentration of S adenosylmethionine (SAM) which, in turn, maintains the methylation reactions whose inhibition is considered to cause cobalamin deficiency associated neuropathy. SAM mediated methylation reactions are inhibited by its product S adenosylhomocysteine (SAH). This occurs when cobalamin is deficient and, as a result, methionine synthase is inhibited causing a rise of both homocysteine and SAH. Other potential pathogenic processes related to the toxic effects of homocysteine are direct damage to the vascular endothelium and inhibition of N methyl-D-aspartate receptors. PMID- 10746356 TI - Role of micronutrients in sport and physical activity. AB - Many micronutrients play key roles in energy metabolism and, during strenuous physical activity, the rate of energy turnover in skeletal muscle may be increased up to 20-100 times the resting rate. Although an adequate vitamin and mineral status is essential for normal health, marginal deficiency states may only be apparent when the metabolic rate is high. Prolonged strenuous exercise performed on a regular basis may also result in increased losses from the body or in an increased rate of turnover, resulting in the need for an increased dietary intake. An increased food intake to meet energy requirements will increase dietary micronutrient intake, but athletes in hard training may need to pay particular attention to their intake of iron, calcium and the antioxidant vitamins. PMID- 10746357 TI - Micronutrient antioxidants and smoking. AB - Cigarette smoking is a major risk factor in such human diseases as cardiovascular disease (especially atherosclerosis), lung cancer (the leading world-wide cancer killer), and chronic obstructive pulmonary disease (COPD). An avalanche of studies has suggested that a diet rich in fruit and vegetables is associated with decreased risk for atherosclerosis and cancer. However, the dietary intake of fruits and vegetables, as well as antioxidant micronutrients, is decreased in smokers. This, along with evidence of increased utilization of ascorbic acid and alpha-tocopherol, possibly on the basis of increased oxidative stress, contributes to the low plasma antioxidant concentrations seen in many smokers. This review addresses selected mechanistic considerations of this relationship. PMID- 10746358 TI - Reflections on studying prosody in talk-in-interaction. AB - Rather than focusing on conversation as one context among many in which to study prosody, this paper approaches prosody as one set of resources and practices among many by which participants interactively produce conversation and other talk-in-interaction. Three episodes of conversation are examined, each exemplifying a different order of organization in which prosodic practices may be implicated. The first develops various lines of evidence to show that pitch peaks may be deployed and understood as projecting that a next syntactic possible completion is the designed end of the turn. In the second, the initial turns in the opening of a telephone conversation are examined as the site in which the participants work out the pitch level at which the conversation--or at least its first part--will be conducted, and thereby "negotiate" the tenor of the conversation's launching. The third episode focuses on the central part which prosody can play in the constitution of the action which an utterance is implementing. The paper closes with some reflections on what is needed for students of conversation in dealing with prosody--focusing especially on the need for a relevant way of describing the mediating operations which take the prosody as (partial) input and yield the action (or other conversational feature) being accomplished as outcome. PMID- 10746359 TI - Prosody as an interactional resource: turn-projection and overlap. AB - One aim of current research into talk-in-interaction is to identify the resources that enable recipients to monitor the course of a turn in progress in order to project its upcoming completion. This issue is addressed through analysis of instances of overlapping talk, focusing on their design--that is, their particular prosodic and other linguistic characteristics; their placement--in other words, where precisely they occur in relation to the turn being overlapped; and the subsequent behavior of the coparticipants. Phonetic analysis is combined with interactional techniques developed within Conversation Analysis, to warrant the relevance of categories by reference to the behavior of the participants themselves. As French and Local (1983) found, for an incoming to be treated as turn-competitive, it has to be designed with relatively high pitch and loud volume. These turn-competitive incomings are positioned within the turn in progress, and before the final major accent. By contrast, overlapping incomings positioned after the major accent are not designed as or treated as turn competitive. On the basis of this analysis, we can define transition relevance place (TRP) as the space between the TRP-projecting accent of the current turn and the onset of the next turn. TRP-projecting accents are identifiable on independent grounds, being phonetically distinct from non-TRP-projecting accents. They thus provide a robust resource for participants to monitor the upcoming completion of the turn. PMID- 10746360 TI - An analysis of turn-taking and backchannels based on prosodic and syntactic features in Japanese map task dialogs. AB - In this study, we investigate syntactic and prosodic features of the speaker's speech at points where turn-taking and backchannels occur, on the basis of our analysis of Japanese spontaneous dialogs. Specifically, we focus on features such as part of speech, duration, F0 contour pattern, relative height of the peak F0, energy trajectory pattern, and relative height of the peak energy at the final part of speech segments. We examine, first, the relationship between turn taking/backchannels and each feature of speech segments independently, showing that the features examined in this study are all related to turn-taking or backchannels and that the way they correlate is fairly consistent with previous studies. Next, we explore the inter-relationship among the features with respect to turn-taking and backchannels. We show that in both turn-taking and backchannels, (1) some instances of syntactic features make extremely strong contributions, and (2) in general, syntax has a stronger contribution than any individual prosodic feature, although the whole prosody contributes as strongly as, or even more strongly than, syntax. We also discuss some implications of our results, comparing them with previous models that have mentioned roles of syntax and prosody in turn-taking and backchannels. PMID- 10746361 TI - Collaborative signaling of informational structures by dynamic speech rate. AB - The research reported in this paper is an attempt to elucidate the functions of dynamic speech rates as contextualization cues in conversational Japanese. We examine five spontaneous task-oriented dialogs conducted in Japanese and analyze the potential of speech rate changes in signaling the structure of the information being exchanged in the dialogs. A correlation is found between speech decelerations and the openings of new information, and another is found between speech accelerations and the absence of information openings. These correlations hold not only in the case of a single speaker's speech, but also in the case of multiple speakers' sequential utterances, both with and without turn shifts. On the basis of these findings, we examine the potential of dynamic speech rates as cues to information structures in dialogs, in terms of their precision, recall, and primacy. We claim that changes in the speech rate in conversational Japanese have a definite potential for cuing the structure of information collaboratively constructed by participants of a conversation. PMID- 10746362 TI - Intonational settings as markers of discourse units in telephone conversations. AB - A study of business telephone cells provides quantitative evidence suggesting "intonational settings": Certain attributes of intonation are sustained throughout discourse units in the calls (openings, business transactions, preclosures, and final closures), and differentiate one unit from another, as if phonologically significant aspects of intonation are realized within a space controlled by discourse-related parameters. Two types of parameter emerge, one controlling the midpoint of the F0 contour in frequency space, the other controlling the way it fluctuates. PMID- 10746363 TI - Who's next? The melodic marking of question vs. continuation in Dutch. AB - The present study is concerned with functional differences between the accent lending rise followed by sustained level pitch (GDI-notation "10," cf., 't Hart, Collier, and Cohen, 1990) and the combination of the accent-lending rise and final rise ("12") in Dutch. Theoretical analyses of the meaning of Dutch intonation claim that "10" can be interpreted as a question. In contrast, recent investigations of the relevance of intonation for turn-taking indicate that "10" signals that the speaker wants to keep the turn. The main goal of the present experimental investigation is to find evidence for the idea that speakers may use contour "10" to keep the floor. Thirty subjects were presented with short utterances bearing either a "12" or a "10" contour in three perception experiments. Results support the hypothesis that "10" is not readily interpreted as a question, as opposed to contour "12," which indicates that contour "10" may play a role in signaling turn-keeping in Dutch. PMID- 10746364 TI - Articulatory correlates of prosodic control: emotion and emphasis. AB - This study examines mandibular correlates of prosodic control in nonread dialog exchanges, in which the subject is asked to repeat the same correction of one digit in a three-digit sequence consisting of "five" or "nine" followed by "Pine Street." Articulatory and acoustic data were collected for four speakers of American English at the X-ray Microbeam Facilities at the University of Wisconsin. Jaw opening was measured as vertical jaw position at the time of maximum opening. Middle digits perceived by independent listeners as emphasized generally show jaw opening which is larger than the average jaw opening for the utterances in which they occur. As the speaker repeatedly makes the same correction, not only does jaw opening increase significantly on the corrected digit but also the overall amount of jaw opening on all digits in the corrected exchanges increases. Independent separate perception tests show that listeners also perceive the speakers' answers to be more irritated as the speaker repeats the same correction. The findings suggest a local and global use of the jaw opening gesture to produce both linguistic or paralinguistic and extralinguistic information, that is, word emphasis and the emotional tenor of the dialog itself. PMID- 10746365 TI - Linguistic adaptations during spoken and multimodal error resolution. AB - Fragile error handling in recognition-based systems is a major problem that degrades their performance, frustrates users, and limits commercial potential. The aim of the present research was to analyze the types and magnitude of linguistic adaptation that occur during spoken and multimodal human-computer error resolution. A semiautomatic simulation method with a novel error-generation capability was used to collect samples of users' spoken and pen-based input immediately before and after recognition errors, and at different spiral depths in terms of the number of repetitions needed to resolve an error. When correcting persistent recognition errors, results revealed that users adapt their speech and language in three qualitatively different ways. First, they increase linguistic contrast through alternation of input modes and lexical content over repeated correction attempts. Second, when correcting with verbatim speech, they increase hyperarticulation by lengthening speech segments and pauses, and increasing the use of final falling contours. Third, when they hyperarticulate, users simultaneously suppress linguistic variability in their speech signal's amplitude and fundamental frequency. These findings are discussed from the perspective of enhancement of linguistic intelligibility. Implications are also discussed for corroboration and generalization of the Computer-elicited Hyperarticulate Adaptation Model (CHAM), and for improved error handling capabilities in next generation spoken language and multimodal systems. PMID- 10746366 TI - Can prosody aid the automatic classification of dialog acts in conversational speech? AB - Identifying whether an utterance is a statement, question, greeting, and so forth is integral to effective automatic understanding of natural dialog. Little is known, however, about how such dialog acts (DAs) can be automatically classified in truly natural conversation. This study asks whether current approaches, which use mainly word information, could be improved by adding prosodic information. The study is based on more than 1000 conversations from the Switchboard corpus. DAs were hand-annotated, and prosodic features (duration, pause, F0, energy, and speaking rate) were automatically extracted for each DA. In training, decision trees based on these features were inferred; trees were then applied to unseen test data to evaluate performance. Performance was evaluated for prosody models alone, and after combining the prosody models with word information--either from true words or from the output of an automatic speech recognizer. For an overall classification task, as well as three subtasks, prosody made significant contributions to classification. Feature-specific analyses further revealed that although canonical features (such as F0 for questions) were important, less obvious features could compensate if canonical features were removed. Finally, in each task, integrating the prosodic model with a DA-specific statistical language model improved performance over that of the language model alone, especially for the case of recognized words. Results suggest that DAs are redundantly marked in natural conversation, and that a variety of automatically extractable prosodic features could aid dialog processing in speech applications. PMID- 10746367 TI - Intonation and dialog context as constraints for speech recognition. AB - This paper describes a way of using intonation and dialog context to improve the performance of an automatic speech recognition (ASR) system. Our experiments were run on the DCIEM Maptask corpus, a corpus of spontaneous task-oriented dialog speech. This corpus has been tagged according to a dialog analysis scheme that assigns each utterance to one of 12 "move types," such as "acknowledge," "query yes/no" or "instruct." Most ASR systems use a bigram language model to constrain the possible sequences of words that might be recognized. Here we use a separate bigram language model for each move type. We show that when the "correct" move specific language model is used for each utterance in the test set, the word error rate of the recognizer drops. Of course when the recognizer is run on previously unseen data, it cannot know in advance what move type the speaker has just produced. To determine the move type we use an intonation model combined with a dialog model that puts constraints on possible sequences of move types, as well as the speech recognizer likelihoods for the different move-specific models. In the full recognition system, the combination of automatic move type recognition with the move specific language models reduces the overall word error rate by a small but significant amount when compared with a baseline system that does not take intonation or dialog acts into account. Interestingly, the word error improvement is restricted to "initiating" move types, where word recognition is important. In "response" move types, where the important information is conveyed by the move type itself--for example, positive versus negative response--there is no word error improvement, but recognition of the response types themselves is good. The paper discusses the intonation model, the language models, and the dialog model in detail and describes the architecture in which they are combined. PMID- 10746368 TI - [Analysis of biologic changes in PSA blood concentration and free PSA percentage in patients with normal rectal digital examination]. AB - OBJECTIVE: To study intra-individual variations in serum PSA and percent free PSA in patients with normal digital rectal examination. MATERIAL AND METHODS: Analysis of changes in serum PSA levels and percent free PSA in two blood measurements conducted in 107 non-consecutive patients prior to prostate biopsy, over a period of time ranging from 23 to 60 days. Both total and free PSA were measured with two dual monoclonal antibody assays, Tandem-E and Tandem-R, Hybritech. Diagnosis was benign hyperplasia in 63 patients and cancer in 44. RESULTS: PSA variations ranged between -6.8 and +3.2 ng/ml in BPH patients, and between -2.8 and +9.0 when cancer was detected. The median coefficient of variation was 15.4 and 15.7, respectively. Variations in percent free PSA ranged between -30.7 and +40.9 in the BPH group and between -17.9 and +15.8 in the cancer group. The median coefficient of variation was 32.2 and 32.3%, respectively. Should prostate biopsy had been indicated when percent free PSA was equal to or lower than 25 in the 4 to 10 ng/ml PSA range, 15% patients would have exhibited discrepancies. Sensitivity would have ranged between 100 and 94.4%, with a reduction rate in negative biopsies between 16.6 and 19.4%. CONCLUSIONS: Intra-individual variations in serum PSA levels and percent free PSA may condition the decision of whether to perform a prostate biopsy. PMID- 10746369 TI - [Biochemical control after external radiotherapy in localized prostatic cancer: results of a modern cohort]. AB - PURPOSE: The present study reports biochemical outcomes of a modern series of patients with localised prostate cancer treated with external beam radiation therapy, and analyses the implications of the nadir PSA levels in monitoring outcome after treatment. METHODS: From March 1993 to March 1997, eighty three patients with clinical stages T1-T3 NxM0 prostate cancer received definitive external radiation therapy, median dose 66 Gy (range 60 to 68 Gy). Adjuvant androgen deprivation was associated in 53 high risk patients. Initial response to treatment was defined as a decrease of serum PSA to levels of < or = 1.5 ng/ml, and biochemical failure as three consecutive PSA rises over post-treatment nadir PSA value. RESULTS: The 3-year actuarial BDFS was 78% +/- 7 for the whole series, 74% +/- 12 for patients treated with radiotherapy alone, and 71% +/- 10 for high risk patients treated with combination therapy (p = 0.27). Only nPSA emerged as a potential indicator of biochemical control. The probability of BDFS at 3 years was 82%, 83% and 40% for nPSA of < or = 1 ng/ml, 1-2 ng/ml and > 2 ng/ml respectively (p = 0.0409). In multivariate analysis, this correlation was independent on the effect of other variables and persisted after adjusting for the effect of hormonal therapy (p = 0.0540). CONCLUSION: Radiation therapy is a potentially curative treatment for prostate carcinoma. Our data indicate that the nadir PSA value after radiation can be an excellent early determinant of outcome. PMID- 10746370 TI - [Renal hematomas after extracorporeal lithotripsy with the lithotriptor "lithostar multiline de Siemens"]. AB - OBJECTIVE: Renal haematomas after extracorporeal shockwave lithotrity are an immediate and potentially serious complication. The incidence of post-ESWL renal haematoma with the new Siemens' Lithostar Multiline lithotripter is analyzed in an attempt to know its occurrence, predisposing factors and presentations signs and symptoms. MATERIAL AND METHODS: Between January and December 1998, a total of 1313 extracorporeal shockwave lithotrity sessions were performed on lithiasis located at any level of the urinary tract in 686 patients. Treatment were performed with a Lithostar Multiline lithotripter (electromagnetic generator). The power used ranged from 4 to 9 units, which are power steps based on load tension in Kv, pressure peaks in bars and energy flow density in mJ/mm2, with an average of 3800 pulses/session. A radiological study (ultrasound-tomography) was conducted when faced with clinical suspicion of complications. RESULTS: A total of 7 clinical renal haematomas (1.02%) were diagnosed. A conservative attitude was taken in most cases. One patient presented hemodynamic instability and required surgery to empty the haematoma and haemostasia of the renal unit, though no nephrectomy was finally required. As predisposing factors: presence of HBP, prior ESWL and hypercholesterolemia are all emphasized. In rare cases several factors concurred simultaneously in the same patient. CONCLUSIONS: Renal haematoma should be suspected in the presence of continuous or unjustified pain after ESWL treatment. Any likely coagulation disorder should be corrected, urinary infections ruled out and pressure figures controlled. PMID- 10746372 TI - [Metachronous tumors of the upper urinary tract following primary superficial tumors of the bladder]. AB - OBJECTIVE: To evaluate the long term incidence of upper urinary tract tumours in patients with surface tumours of the bladder. MATERIAL AND METHODS: Analysis of 78 patients with primary tumour of the bladder pTa-pTl diagnosed between 1978 and 1988. These patients were followed for up to 10 to 21 years (mean follow-up 13.4 years) and were treated with transurethral resection and a variety of endovesical cytostatic agents. RESULTS: Of all 78 patients, 9 (11%) developed an upper urinary tract tumour over an average of 9 years (range 4-18); 2 in the first 5 years, 4 between 5 to 10 years, and 3 between 10 and 18 years. The likelihood of finding a tumour in the upper tract if all 20-year old patients were to be followed would be 25.6% (Kaplan-Meier's method). A high percentage of all upper urinary tract tumours are infiltrant, but the cause of death in 44% of these patients is progression of the tumour of the bladder. CONCLUSIONS: The percentage of tumours of the upper urinary tract that become metachronic following a tumour of the bladder, increases with the duration of the follow-up. A high percentage of these cases are already in high stages when diagnosed. It would be convenient to monitor the upper urinary tract with the same periodicity than the bladder in order to establish early diagnoses. These patients show a high mortality rate, in most cases secondary to progression of the tumour of the bladder. PMID- 10746371 TI - [Usefulness of free PSA/total PSA ratio in the diagnosis of prostatic cancer in symptomatic patients with PSA levels ranging from 2.5 to 20 ng/ml]. AB - OBJECTIVE: To evaluate the value of free PSA/total PSA ratio (fPSA/tPSA) to boost total PSA (tPSA) specificity in the diagnosis of prostate cancer in a series of symptomatic patients. MATERIAL AND METHOD: Prospective study on 334 patients presenting symptoms compatible with PBH and tPSA (ProStatus, Wallace Oy, Turku, Finland) between 2.5 and 20 ng/ml, and mean age 67.2 +/- 7 (49-87). Patients were divided into 3 groups: Group I (normal DRE and tPSA between 2.5 and 10 ng/ml)- 189 patients. Group II (normal DRE and tPSA between 10 and 20 ng/ml)--78 patients. Group III (suspicion DRE and sPSA between 2.5 and 20 ng/ml)--67 patients. For each group, the sensitivity, specificity and ROC curves were calculated for several cut-off values of the lPSA/tPSA ratio. RESULTS: Group I- Prostate cancer was diagnosed in 11% (20/189) patients. Significant differences were found only in the lPSA/tPSA ratio (p = 0.01). Using 27% as the cut-off value, cancer would have been diagnosed in 95% (19 of 20) patients, decreasing the total number of negative biopsies by 21% (39 of 189). Group II--Prostate cancer was diagnosed in 19% (15/78) patients. The only significant differences found were in prostate volume (p = 0.02). Using 30% as the cut-off value, 93% (14 of 15) patients with cancer would have been diagnosed, decreasing the total number of biopsies by 6.5% (5 of 78) and the number of negative biopsies by 8% (5 of 63). Group III--Prostate cancer was detected in 72% (48/67) patients. Significant differences were found in lPSA/tPSA ratio (p = 0.003), prostate volume (p = 0.02) and presence of ultrasound nodes (p = 0.004). Using 25% as the cut-off value, 96% (46 of 48) of patients with cancer would have been diagnosed, decreasing the total number of biopsies by 13% (9 of 67) and the number of negative biopsies by 47% (9 of 19). CONCLUSIONS: We advise use of lPSA/tPSA ratio only in patients with normal DRE and tPSA between 2.5 and 10 ng/ml. Biopsing patients with lPSA percent equal to or lower than 27% would have prevented 23% of negative biopsies while still maintaining a diagnostic sensitivity of 95%. PMID- 10746373 TI - [Endoscopic resolution of complications caused by urethrovesical suspension]. AB - We presented a total of ten women which referred a varied syntomatology secondary to a bladder foreign body due to the suture employed in the stress incontinence surgery in which urethrovesical suspension techniques were performed. In these patients, to six a Marshall-Marchetti-Krantz technique was practised, to three modified Burch procedure and to the left a suspension according to Raz method. It is described the varied clinic they presented, the necessity of endoscopic study to confirm and the results after solving the 90% of these cases using endoscopic techniques. It is concluded that is not mandatory the employment of non absorbable suture to perform the suspension techniques and in the opposite of the usual tendency of the authors recurring to open surgery, this complication can be solved endoscopically and in ambulatory regime in the most of these cases. PMID- 10746374 TI - [Pure prostatic leiomyoma]. AB - We present a case of pure prostatic leiomyoma, benign and rare entity, diagnosed in a patient with prostatism. We review the published papers about this disease and the different subjects about etiology, clinic, histopathology, diagnosis and treatment. PMID- 10746375 TI - [Usefulness of Doppler-color ultrasonography and identification of resistance indexes as early indicators of testicular infarction secondary to orchiepididymitis]. AB - The testicular infarction can be a rare complication of the orchiepididymitis. The ultrasonography appearance of the partial infarctions creates confusion with expansive intratesticular processes. We present three cases of focal testicular infarction associated with orchiepididymitis, stressing the usefulness of the Color doppler sonography for detecting the avascular nature o these lesions. The determination of de indeces of resistance (IR) in the doppler spectrum of intratesticular arteries showed a highly increased vascular resistance in respect of the healthy testicle. In patients with grave orchiepididymitis vein drainage can be affected due to compressive edema or to thrombosis, causing the raising of the resistance indeces. Acknowledging the avascular resistance of the focal post orchiepididymitis infarction we can prevent unnecessary orchiectomy. PMID- 10746376 TI - [Cryptorchism and testicular cancer]. AB - OBJECTIVE: This report insists on the higher risk of testicular cancer in cryptorchidism and indicates the more adequate management of this pathology. METHODS/RESULTS: We report a 27 years old patient with history of cryptorchidism, diagnosed of abdominal tumor in urgency. This tumor was a seminoma in intrapelvic testicle. CONCLUSIONS: The risk of developing testicular cancer in cryptorchidism is 3 to 10 times greater than in a patient with normally descended testes, and orchiopexy does not prevent the risk of malignancy. We underscore the need to follow these patients closely and recommend the orchiectomy in postpubertal patients. PMID- 10746378 TI - [Bilateral testicular luxation]. AB - Contribution of a case report of long-standing bilateral testicular luxation, the result of scrotal trauma caused by a soccer ball. Good aesthetic, endocrine and exocrine results were obtained with surgical treatment. Androgenic hormonal profile and spermiogram performed at six months showed values within the normal range. PMID- 10746377 TI - [Versatility of axial penile skin fasciocutaneous flap in reconstruction of pendulous urethra]. AB - PURPOSE: We describe three cases, with different features, of anterior urethral strictures that were treated with the same technique: a circular axial penile fasciocutaneous flap. MATERIAL AND METHODS: We report three cases of patients with urethral strictures that affected the whole glandar and penile urethra. In one case the stricture affected the bulbar urethra. No patient has the preputial skin disposable, because of a previous circumcision, and all has been treated with different techniques. In one patient, a first step urethroplastia was performed previously, open all the penile urethra. All the cases were successfully treated with a circular axial fasciocutaneous penile flap in one stage. In case number one the reconstruction was combined with a scrotal flap. In case number three, the glandar urethra was reconstructed. CONCLUSIONS: The circular axial fasciocutaneous penile flap is a versatile technique that can be use to correct various type of strictures of the whole glandar and penile urethra alone or associated with strictures of the bulbar urethra. PMID- 10746379 TI - [Bladder schistosomiasis. Etiologic diagnosis of detrusor contraction insufficiency]. AB - This paper reports a case of bladder bilharziasis with histopathological exam and different patterns of urodynamic evaluation. The need for urodynamics is emphasized in order to avoid diagnostic and therapeutic mistakes. PMID- 10746380 TI - [Ureterointestinal anastomosis in ileal conduit in single kidney. New surgical technique]. AB - Introduction of a new approach for uretero-ileal implantation when only one kidney is available. The technique follows the same approach as the Wallace-type re-implantation but in this case the uretero-ileal anastomosis plate is formed with the far en 2-3 cm of ureter. All cases performed with this technique have been highly successfull as reported in this clinical account. PMID- 10746381 TI - [Kidney annulment and bladder lithiasis secondary to ureteral catheter]. AB - We reported a case of a kidney annulment and bladder stone secondary a foreign body (ureteral stent). We review all the cases of urinary stone secondary a foreign bodies. PMID- 10746382 TI - [Histiocytoid hemangioma of the epididymis. A very unusual entity]. AB - The presence of vascular origin tumor in the epididymis and testicle, are today considered of low frequency. We report a case of histiocytoid (epithelioid) hemangioma of the epididymis. Its differential diagnosis with the adenomatoid tumor, is discussed. PMID- 10746383 TI - [Subacute intestinal obstruction caused by giant bladder diverticulum]. AB - A case of a patient affected with intestinal obstruction of 4 days of evolution is presented. The diagnosis of giant bladder diverticulum due to Benign Prostatic Hypertrophy was made by abdominal ultrasound at Emergency Room, and afterwards was confirmed by cystography. A trabeculated wall and a diverticulum at the posterior side was seen with the cystography. The diverticulum full of urine couldn't be emptied due to a narrow diverticular neck and the rectum was obstructed. When a urethral catheter was put in, the symptoms of intestinal obstruction disappeared 800 ml of urine were passed through the catheter and the rectum compression was released; the gas was allowed to pass through the bowel and it's seen in plain X-Ray. This case is presented, considering its unusual this way of presentation of a giant bladder diverticulum. We have reviewed the literature en Medline since 1966 and we have not found a similar case. PMID- 10746384 TI - [Current status of the treatment of organic erectile dysfunction]. PMID- 10746385 TI - [The other side of the so-called "cases interesting for publication"]. PMID- 10746386 TI - Adolescent drug use in Zimbabwe assessed by their teachers. AB - OBJECTIVES: To obtain secondary school teachers' assessment of adolescent drug use in Zimbabwe. Secondly, to compare this assessment with some of the results from student drug use surveys undertaken in 1990 and 1994. DESIGN: Cross sectional. SETTING: 17 secondary schools in Harare, Mashonaland East and West and Matabeleland North Provinces (including Bulawayo). SUBJECTS: 442 secondary school teachers, of whom half were males. MAIN OUTCOME MEASURES: Teachers' assessment of how serious a problem adolescent drug use is in Zimbabwe in general and at the teachers' own school. Secondly, whether adolescent drug use is currently on the increase or decrease. RESULTS: The teachers assessed alcohol use as the most serious drug problem among secondary school students in Zimbabwe, followed in descending order by cannabis, tobacco, inhalants and other drugs. Drug use was considered less serious at the teachers' own school as compared to the assessment of the general situation. Student drug use is largely stable or increasing moderately. Alcohol appears to be increasing the most. Although partly corresponding with previous studies, the situation was assessed as more serious than what appeared from the student drug use surveys in 1990 and 1994. The largest gap between teachers' assessment and students' self-reported drug use was found for cannabis and inhalants. CONCLUSIONS: The study among teachers concur to some extent with results from previous studies among students. There are, however, variations in the assessment of drug use among secondary school students that do not correspond completely with the picture drawn by previous student surveys. It is argued that some of the variation in the assessment may in part be explained by sociocultural and normative differences between different sociocultural segments of Zimbabwean society. PMID- 10746387 TI - Analysis of the user fee for health care policy in Eritrea. AB - OBJECTIVES: To assess the macro-context of the user fee policy design and implementation; to critically evaluate the content of the user free policy with respect to its implications for efficiency, equity, quality and utilisation of health care and to scrutinize the implementation of the scheme and identify the successes and problems encountered since the introduction of the user fee policy in 1996. DESIGN: Cross sectional. SETTING: Mekane Hiwot Central Referral Hospital and Semenawi Asmore Health Centre. SUBJECTS: 50 health professionals and support staff; 150 outpatients of which 100 patients were from the hospital and 50 from the health centre and 50 inpatients from the hospital. MAIN OUTCOME MEASURES: Service utilisation. RESULTS: The policy was launched against a conducive macro economic, political and health sector background. Fees reduced attendance at the tertiary referral hospital and reversed the previous trend of sharply declining use of primary level facilities. However, the fee levels could not give an adequate signal to patients to use the appropriate cost effective levels of care and the referral system. The cost recovery ratio for the system as a whole was found to be one of the highest in sub-Saharan Africa. At some of the lowest levels of the health care system, however, the scheme does not seem viable. Fee waivers were infrequent, and the process of means testing was lengthy, thus adversely affecting equity. CONCLUSIONS: The new policy resulted in a cost recovery ratio. There was some indication of allocative efficiency achievement as a result of the price signals. The policy required some refinement in terms of expanding the exemption categories and revenue retention at the facility level. PMID- 10746388 TI - The impact of health education on the knowledge, attitudes and practices of a rural community with regards to schistosomiasis control using a plant molluscicide, Phytolacca dodecandra. AB - OBJECTIVE: To determine the impact of health education on knowledge, attitudes and practices (KAP) with regards to use of a plant molluscicide in snail control. DESIGN: Repeat cross sectional survey. SETTING: Chiweshe communal lands. SUBJECTS: Males and females--age range 13 to 87 years. MAIN OUTCOME MEASURES: Changes in: KAP after health education, water usage pattern, water contact behaviour, willingness to participate in use of P. dodecandra. RESULTS: A high proportion of the community indicated prior knowledge of schistosomiasis during both knowledge, attitudes and practices (KAP) surveys. In the follow up KAP survey changes in the community's water contact behaviour as well as their practices in relation to sanitation were reported. The involvement of the community in the application of P. dodecandra during the course of the study ensured continued support and participation of the community. This was evidenced in the follow up survey when it was apparent that the community's attitude towards schistosomiasis had been influenced by the different activities that had taken place. CONCLUSION: Changes in the community's KAP with regards to schistosomiasis control, can be interpreted as an indication of the impact of the health education delivered during the course of the study. Health education should, therefore, precede programmes that require full participation of the community, as this enables the community to make informed decisions regarding their participation. PMID- 10746389 TI - Diagnostic performance of two antigen capture tests for the diagnosis of Plasmodium falciparum malaria in Zimbabwe. AB - OBJECTIVE: The objective was to compare the diagnostic performance of two antigen capture tests, ParaSight-F test and Immunochromatographic test (ICT), for the diagnosis of Plasmodium falciparum malaria. DESIGN: A comparative study. SETTING: Burma Valley, Mutare District, Manicaland in Zimbabwe. SUBJECTS: Patients attending the local clinic with clinical signs and symptoms associated with malaria infection. A blood film was collected from the patient and at the same time blood for the ParaSight-F test and the ICT was collected too. A total of 123 patients were diagnosed using the ICT test, the ParaSight-F test and microscopy which was used as a "gold" standard. MAIN OUTCOME MEASURES: True positives and negatives, false positives and negatives with reference to microscopy. RESULTS: The results indicate that ICT had a higher sensitivity (100%) than the ParaSight F test (93.94%) but showing a lower specificity. The specificity for ICT (75%) is lower than the ParaSight-F test (81.2%) because of the presence of circulating P. falciparum histidine-rich protein-2 (PfHRP-2) in blood even after two weeks post treatment. Two slides that were negative for ParaSight-F tests showed positive on microscopy. However, 21 and 12 slides which were negative on microscopy, showed positive with ICT and ParaSight-F respectively. The fact that ICT detects very low quantities of PfHRP-2 puts it at a comparative advantage because it detects positives even at very low parasite rates. CONCLUSION: The availability of these two antigen capture tests, which are easy to perform, will reduce the number of severe cases by providing early diagnosis. The tests will also reduce the number of unnecessary treatments and irrational use of antimalarials. PMID- 10746391 TI - "A little reminder does no harm": case report. AB - A case of falsified non-accidental ocular injury of a 29 year old male is presented. The importance of an accurate history and appropriate investigations in the management of ocular injuries is highlighted to promote a high standard of health care delivery in Zimbabwe. PMID- 10746390 TI - Penicillin resistant Streptococcus pneumoniae isolates in Harare, Zimbabwe. AB - OBJECTIVE: To determine the susceptibility of Streptococcus pneumoniae isolates to penicillin and other antimicrobial drugs. DESIGN: This was a laboratory based study. SETTING: Department of Medical Laboratory Sciences, University of Zimbabwe and the Bacteriology Unit, Public Health Laboratories, Harare. SUBJECTS: 71 S. pneumoniae isolates from Parirenyatwa and Harare hospitals. MAIN OUTCOME MEASURES: Penicillin resistance, MIC of penicillin to S. pneumoniae, multi-drug resistance. RESULTS: 71 S. pneumoniae isolates were tested for their susceptibilities to penicillin G, erythromycin, tetracycline, ampicillin, ciprofloxacin and clindamycin. Five (7%) of the isolates were resistant to penicillin G and were also all resistant to erythromycin. Isolates resistant to other antibiotics were; tetracycline (4), ampicillin (3) and ciprofloxacin (2). The five isolates that were resistant to penicillin G showed resistance to two or more antibiotics. Four S. pneumoniae isolates were designated highly resistant to penicillin (MIC > or = 2 micrograms/ml) and one isolate was designated intermediate in resistance to penicillin (MIC between 0.1 and 1.0 microgram/ml). CONCLUSIONS: A low percentage of S. pneumoniae isolates were resistant to penicillin and were also resistant to erythromycin. The penicillin resistant strains showed multi-drug resistance. PMID- 10746392 TI - Burkitt's lymphoma of the ovary. AB - Two cases of a unilateral ovarian Burkitt's lymphoma are presented. One patient is 32 years and the other 15 years old. The case report highlight the clinical presentation and differential diagnosis of unilateral ovarian Burkitt's lymphoma in adults. PMID- 10746393 TI - PVC bags considerably reduce availability of diazepam. Cent Afr J Med 1998;44(7) PMID- 10746394 TI - Pain: friend or foe. AB - Pain, the most urgent of symptoms usually signals the presence of potential or on going injury to tissue which requires attention. The warning that pain provides is, therefore, a good thing and in a way friendly. When pain continues or resumes after the healing process of injury is complete, it is no longer signalling on going tissue damage but becomes a disease in its own right. That, in essence, is the presentation of most chronic pain syndromes referred to Pain Clinics for investigation and treatment. PMID- 10746395 TI - Clinical pharmacologist wanted--where? AB - Many people could define with ease the job of a cardiologist, a paediatrician or an obstetrician. However, only a few would have any clear idea of how a clinical pharmacologist might fill a day. Indeed, within the medical profession, and often within the scientific community, there has been a sneaking suspicion that clinical pharmacologists are themselves unsure of their remit. This article describes the possible role for clinical pharmacologists and different areas where they can make significant contributions to medical services. PMID- 10746396 TI - Trauma and mental health problems of Sudanese refugees in Uganda. AB - OBJECTIVES: To determine the extent of trauma and mental health problems. DESIGN: Screening surveys. SETTING: Sudanese refugee communities in Northern Ugandan camps. SUBJECTS: 100 adult refugees, 44 ex-soldiers, 60 patients at camp outpatient health facilities, 63 traditional healers' patients and 56 refugee children. MAIN OUTCOME MEASURE: Rate of trauma. RESULTS: The most common trauma events experienced by 100 adults were forced isolation from others (94%), forced separation from family members (91%) and lack of food or water (83%). Thirty-two percent of the adults suffered post traumatic stress disorder (PTSD). Among the 56 children, 12 (20%) suffered from a chronic PTSD. Forty-four ex-soldiers scored a median of 74.6 (Q1 = 62.3; Q3 = 79.1) on the depressive scale of the Hopskins Symptom checklist. Out of 60 outpatients at a dispensary, 12 (20%) had psychological disorder. Out of a total of 63 patients attending traditional and faith healers' facilities, 26% suffered from PTSD and 39% from depressive disorder. CONCLUSION: High rates of trauma and psychosocial problems were found among Sudanese refugees in Uganda. PMID- 10746397 TI - Non-traumatic paraplegia [correction of paraplegis] in a Zimbabwean population--a retrospective survey. AB - OBJECTIVES: To ascertain the aetiology of non-traumatic paraplegia in a Zimbabwean population and to compare findings with other studies from Africa. DESIGN: Retrospective survey over a six year period. SETTING: National Rehabilitation Centre, Ruwa, Zimbabwe. SUBJECTS: 159 patients referred to the National Rehabilitation Centre with non-traumatic paraplegia. RESULTS: The commonest causes of non-traumatic paraplegia were neoplasms (28% of cases) of which a third were metastatic, followed by tuberculosis (TB) (27%). Transverse myelopathy of unknown aetiology was diagnosed in 11% of cases and 6% had Guillain Barre syndrome. Miscellaneous conditions included: degenerative bone and joint conditions, degenerative cord disorders and infections. In 7% of cases no diagnosis was reached. On admission all patients were confined to wheelchairs. On discharge 49% had regained some degree of mobility. A year after discharge 48% were known to be alive and 18% had died. The fate of 34% was not known. CONCLUSIONS: The aetiology of non-traumatic paraplegia in a Zimbabwean population is similar to that reported from other African countries, with tumours and tuberculosis accounting for over half the cases. Survival appears to be related not only to the primary condition but also to the severity of the disability. PMID- 10746399 TI - Radical radiotherapy for carcinoma of the cervix--the Zimbabwean experience. AB - OBJECTIVE: Carcinoma of the cervix accounts for over 30% of all female malignancies in Zimbabwe. Patients treated with radiotherapy tend to have advanced disease. This study examines some presentation patterns, radiotherapy treatment parameters, outcomes of treatment and identifies some prognostic factors in this set up. DESIGN: Retrospective study. SETTING/SUBJECTS: The study was undertaken on all patients (273) who presented to the Parirenyatwa Hospital Radiotherapy Centre (RTC) between November 1990 and December 1991 with a diagnosis of cervical carcinoma. Of these patients 93 were suitable for radical treatment and they were studied. MAIN OUTCOME MEASURES: Complete remission rates in relation to total dose given and stage of disease, incidence of acute complications, local recurrence and development of metastases. RESULTS: Of the 93 patients studied 72 (77.4%) were Stages I and II and 21 (22.6%) were stage III. Fifty nine percent of the patients gave a history of three to eight months symptom duration and 21.5% symptom duration longer one year. The mean length of follow up time was 13.4 months. The overall complete remission (CR) rate 49%. Ninety seven percent of patients achieving a CR had received 70 Gy or more as total dose to Point A. Patients with both pelvic wall and lower one third vaginal involvement (Stage A + B) did more poorly than other Stage III patients. Acute complications were mainly mild and seen in patients whose overall treatment time was under seven weeks. Most recurrences and metastases occurred within one year of completion of treatment. CONCLUSION: It is only a minority of patients (34.1%) that present to the Radiotherapy Centre with cervical carcinoma who are suitable for radical treatment. These are patients who become aware of their symptoms early. Treatment achieves a good complete remission rate. Follow up of treated patients is poor. The sub group of patients with Stage III A + B must be recognised for their poorer response compared to other patients with Stage III disease. PMID- 10746398 TI - Lung function status of some Nigerian men and women chronically exposed to fish drying using burning firewood. AB - OBJECTIVE: To determine the lung function status of some Nigerian men and women chronically exposed to fish drying using burning firewood. DESIGN: Case control study. SETTING: Ibaka, Utaewa and Ikanga fishing settlements. SUBJECTS: 183 males and 192 females engaged in fishing, aged 20 to 45 years who have been exposed for a minimum of five years as cases. The control group comprised sex matched male (142) and female (152) Nigerians from the same area who were not exposed to any known air pollutant. MAIN OUTCOME MEASURES: Lung function indices: FVC, FEV1, FEV1% and PEFR. RESULTS: Lung function indices were significantly lower in men engaged in fishing than in their controls: FVC [mean (SD): 2.98 (0.20) vs 3.52 (0.29), p < 0.001]; FEV1 [2.08 (0.18) vs 2.82 (0.25), p < 0.001]; FEV1% [69.8 (3.1) vs 80.2 (6.7), p < 0.001]; and PEFR [335 (22) vs 592 (99), p < 0.001], respectively. Similarly, lung function indices were lower in females in the fishing industry than in their controls; FVC [2.42 (0.17) vs 3.02 (0.24), p < 0.001]; FEV1 [1.70 (0.19) vs 2.55 (0.21), p < 0.001]; FEV1% [72.9 (3.2) vs 84.4 (6.7), p < 0.001]; and PEFR [298 (22) vs 418 (34), p < 0.001]. All the lung function indices (except FEV1%) of the fishermen and women declined significantly (p < 0.001) with their duration of exposure. The results showed a predominantly mixed pattern (restrictive and obstructive) of respiratory defect. There were higher prevalences of respiratory and other symptoms among the cases than the controls. The respiratory symptoms included cough with sputum, chest pain, dyspnoea catarrh and sneezing and unproductive cough. Other symptoms were eye and skin irritation, internal heat and headache. CONCLUSIONS: Chronic exposure to fish drying using burning firewood can impair lung function and cause respiratory and other symptoms. PMID- 10746400 TI - Pneumocystis carinii pneumonia (PCP) at Ga-Rankuwa Hospital. AB - Pneumocystis carinii is recognized as one of the leading causes of death in AIDS patients in developed countries but its role in this regard in developing countries appears to be less prominent. Sub-Saharan African countries, in spite of their high HIV prevalence, have hardly recorded any cases. We report the first microbiologically proven case of PCP in an adult patient at Ga-Rankuwa Hospital. A 37 year old African woman was referred to Ga-Rankuwa Hospital from the local clinic for chest infection with a non productive cough that had not responded to conventional treatment. On admission, she was febrile, emaciated and in respiratory distress with oral thrush. Chest radiography showed diffuse bilateral infiltrations and a preliminary diagnosis of atypical pneumonia and tuberculosis was made. The patient was begun on penicillin, gentamicin, contrimoxazole and anti-tuberculosis therapy. Laboratory investigations revealed a low haemoglobin, positive HIV test (after counselling) and Pneumocystis carinii trophozoites and cytes in the bronchoalveolar larvage specimen. In spite of appropriate treatment the patient died within three days. One wonders whether the outcome for this middle aged woman with advanced HIV infection would have been different had appropriate cotrimoxazole therapy been administered at the primary health care centre. It must be noted that PCP may no longer be a rare disease in sub-Saharan countries and intensive investigations should be carried out to avoid losing patients with treatable infectious diseases. PMID- 10746401 TI - Partial atrioventricular septal defect causing confusion with rheumatic heart disease in children in Harare--case reports. AB - Four cases of partial atrioventricular septal defect (AVSD) [corrected] which had been confused with rheumatic heart disease are presented. The need for a full clinical and echocardiographic assessment to reduce this confusion and avoid potentially harmful therapy is highlighted in the report. PMID- 10746402 TI - The control of sexually transmitted diseases. PMID- 10746403 TI - Laparoscopic presacral neurectomy for chronic pelvic pain. AB - Presacral neurectomy is an effective treatment for chronic pelvic pain and dysmenorrhea. The theory behind presacral neurectomy for pain relief is based upon the anatomy of the sensory pathways from the pelvic viscera through the inferior and superior hypogastric plexus, located in the presacral area, to the spinal columns. The excision of the presacral nerve trunk can obstruct pain sensory pathways. Unfortunately 20-25% of patients treated medically for severe, disabling pelvic pain fail to show improvement. As a result, presacral neurectomy has become an alternative management for those with chronic pelvic pain and dysmenorrhea, especially for those who have failed to respond to medical treatment. Operative endoscopy further facilitates the adaptation of the laparoscopic approach to this pelvic denervation. The efficacy of pain relief by laparoscopic presacral neurectomy in chronic pelvic pain and dysmenorrhea is documented at about 75-80%. Major complications are rare with a rate of less than 1% reported. Laparoscopic presacral neurectomy therefore offers an effective alternative treatment and should also be considered as the initial surgical intervention for chronic pelvic pain. PMID- 10746404 TI - Organ system failures predict prognosis in critically ill patients with acute renal failure requiring dialysis. AB - BACKGROUND: Despite the widespread availability of dialytic and intensive care unit technology, the probability of early mortality in critically ill patients with acute renal failure is still high. Previous efforts to predict the outcome in this population have been limited by small sample sizes. In addition, data obtained decades ago may not apply today owing to changes in the case mix. We have attempted to determine whether organ system failures can be used to predict prognosis. METHODS: The medical records of 100 consecutive intensive care unit patients with acute renal failure who required dialysis from January 1997 through December 1998 were evaluated by a blinded reviewer. RESULTS: Of the 100 patients studied, 65 were men and 35 were women. The mean age of survivors and non survivors was 59.4 +/- 20.3 years and 58.3 +/- 20.0 years, respectively. The overall mortality rate was 71%. There were no significant differences between survivors and non-survivors in age, gender, or indication for dialysis. The cause of death in the majority of patients was related to organ system failure, and they carried mortality rates exceeding 83% with the coexistence of four or more failed organs. CONCLUSION: The mortality rate for intensive care unit patients with acute renal failure continues to be high. Multiple organ system failure occurs frequently and is the major cause of death. The mortality rate increases as the number of failed organs increases. PMID- 10746405 TI - Endotoxemia augments neurogenic plasma exudation in guinea pig lungs. AB - BACKGROUND: Lipopolysaccharide (LPS) is closely associated with the development of infection-induced deleterious pulmonary reactions. In this study, we investigated the enhancement effects of LPS on tachykinin-mediated plasma exudation in the lungs of guinea pigs. The role of oxidants was also explored. METHODS: Intravenous LPS (100 mu kg-1) or its vehicle was administered 0 to 3 hours prior to bilateral electrical or sham stimulation of the cervical vagus nerves in animals anesthetized with urethane and artificially ventilated. Plasma exudation into the lungs was assessed by measurement of extravasated 125I-albumin which had been intravenously administered before stimulation. RESULTS: The plasma exudation in the lungs increased after bilateral cervical vagal stimulation. LPS alone did not induce significant plasma exudation. The vagally-mediated plasma exudation was enhanced by LPS with the peak effect 1 hour after LPS administration. LPS also enhanced exogenous substance P (10(-8) mol kg-1, i.v.) induced plasma exudation. The vagally-induced plasma exudation was abolished by a specific neurokinin-1 (NK-1) receptor antagonist, L-732,138. The LPS-induced enhancement response was also attenuated by L-732,138. The vagally-induced plasma exudation was not affected by superoxide dismutase (SOD, 5000 U kg-1, i.p.) pretreatment. However, SOD significantly inhibited the LPS-enhanced neurogenic plasma leakage. The LPS-induced enhancement was not completely abolished by either L-732,138 or SOD pretreatment alone, but by a combination of both. CONCLUSION: LPS augments neurogenic plasma exudation partly through NK-1 receptors to increase vascular permeability and partly via the generation of oxidative metabolites. Tachykinins released from nerve endings may contribute to endotoxin-related pulmonary inflammatory responses. PMID- 10746406 TI - Caries levels and patterns in the primary dentition of preschool children in Taiwan. AB - BACKGROUND: The specific caries experience and caries patterns have not been systematically studied in Taiwan. The purpose of this study was to determine the prevalence of caries patterns and caries levels for children aged 3 through 6 years in the northern Taiwan area. METHODS: Eight hundred fifty-one children were examined and their dentition classified according to five etiology-oriented caries patterns. RESULTS: The deft was 2.8 for 3-year-olds, 3.2 for 4-year-olds, 3.8 for 5-year-olds, and 3.7 for 6-year-olds. Nearly half the children (44.4%) had the "facial lingual/molar proximal" pattern; this pattern is consistent with bottle caries continuing to the smooth surfaces of molars. Only 15.4% were caries free. CONCLUSION: We interpret the disease to be well established by age 3; prevention programs should thus begin before that age. The influence of bottle caries is interpreted from the high percentage of children with facial-lingual caries by age 3. PMID- 10746407 TI - Retroperitoneoscopic ureterolithotomy for impacted ureteral stones. AB - BACKGROUND: Despite the fact that extracorporeal shock wave lithotripsy and endourological procedures have greatly diminished the need for open ureterolithotomy, invasive open surgery is still necessary in some cases of obstinate ureteral stones when less invasive methods fail. Since Wickham first performed a retroperitoneoscopic ureterolithotomy in 1979, procedures which are less invasive than open ureterolithotomy have been available in some selected cases. METHODS: In 1997, retroperitoneoscopic ureterolithotomy was performed in 5 patients with ureteral stones, in whom treatment with other minimally invasive procedures had failed. Using Gaur's technique of balloon dissection of the retroperitoneal space, retroperitoneoscopic ureterolithotomy was successfully performed in all cases. RESULTS: All the patients were treated successfully with the retroperitoneoscopic maneuver. The average operating time was 125 (90 to 175) minutes, and the average blood loss was 75 (20 to 200) ml. No patient required an analgesic injection postoperatively. The average postoperative hospital stay was 5.5 (4 to 8) days, and there were no immediate operative complications in any case. Patients were followed an average of 18.5 (13 to 24) months. A ureteral stricture developed in one patient and was resolved by ureteroureterostomy 3 months after the first operation. CONCLUSION: As an impacted ureteral stone is always associated with chronic ureteritis, firmness and adhesion of the periureteral tissue make the retroperitoneoscopic procedure difficult. In one case, a late complication of ureteral stricture may have been due to pre-existing severe chronic inflammation. Based on our limited experience, retroperitoneoscopic ureterolithotomy provides a practical alternative for the management of ureteral stones in selected patients. An expert clinician with experience in this procedure will provide the best results. PMID- 10746408 TI - Embolization of a pulmonary arteriovenous fistula by electrolytic detachable coils: case report. AB - Transarterial embolization with detachable coils is a technique commonly used for the treatment of intracranial aneurysms. We report on a patient with a pulmonary arteriovenous fistula (PAVF) treated successfully with this technique. The patient presented with a history of intermittent hemoptysis, nasal bleeding, numbness of the upper extremities, and seizures. Computed tomographic angiography and magnetic resonance angiography demonstrated a single-hole arteriovenous fistular lesion in the left lower lung. Pre-embolization superselective pulmonary angiography revealed multiple fistulae communicating to the venous sac of the lesion. Eleven detachable coils were deployed into the venous sac, with resultant total occlusion of the pulmonary arteriovenous fistula. We conclude that venous sac embolization in treating this kind of patients is effective. The combined use of a microcatheter system and electrolytic detachable coils may be an excellent technique for achieving this kind of embolization. Superselective angiographic evaluation is essential before embolization, because many occult feeders can be present in cases of high-flow PAVF. PMID- 10746409 TI - Adult monteggia fracture with ipsilateral distal radius fracture: case report. AB - Although the adult Monteggia fracture is a well-known injury, the combination of a Monteggia fracture and an ipsilateral distal radius fracture is extremely rare. It is important for the treating physician to recognize that this injury involves not only the bone elements but also their articulations. The distortion of the integrity of both the elbow and the wrist results in the potential for functional compromise, if inadequately treated. The case report presented here describes a 21-year-old woman with this complex injury resulting from a fall. The injury included a Monteggia type II fracture and an ipsilateral distal radius intra articular fracture in the left forearm. An excellent result was obtained by surgical intervention in both the radial and ulnar bones. The factors we believe contributed to the excellent result were early diagnosis, anatomic reduction, stable fixation, and early physical exercise. The mechanism of injury giving rise to this rare combination of fractures is discussed, as well as a review of the literature. PMID- 10746410 TI - Renal dysplasia and situs inversus totalis: an autopsy case report and literature review. AB - Renal dysplasia has rarely been reported to be associated with situs inversus. Only 4 cases were reported previously with a spectrum of anomalies including bilateral renal dysplasia, situs inversus totalis, and pancreatic and hepatic fibrosis. Recently we encountered another case. A 22-year-old mother, gravida 1 para 0, was in her 23rd week of gestation when she was found to have oligohydramnios. A dead baby was delivered by extraovular induction. On autopsy, Potter's sequences including flattened, low-set ears, receding chin, upturned nose, and club hands and feet were noted. The posterior fontanel was obliterated. Internal examination showed situs inversus totalis, bilateral renal dysplasia, and agenesis of bilateral ureters. There was increased interstitial fibrosis in the pancreas. None of the family members was known to have any renal anomalies. Pinar and Rogers suggested a new syndrome when situs inversus totalis was combined with bilateral renal dysplasia as well as multisystem fibrosis. In our case, 2 major abnormalities, i.e., situs inversus totalis and bilateral renal dysplasia, were present though the increase of interstitial fibrosis within the pancreas was not as significant. This case is likely to be within the spectrum of anomalies mentioned by Pinar and Rogers. PMID- 10746411 TI - Aortoduodenal fistula presenting as acute massive gastrointestinal bleeding and recurrent syncope: case report. AB - Aortoenteric fistula is a rare condition that may cause death in patients due to gastrointestinal bleeding. The duodenum is the most frequently involved site, at 78.5% of 191 cases by Nagy and Marshall's meta-analysis. It is characterized by the clinical triad of abdominal pain, gastrointestinal bleeding, and an abdominal mass. Abdominal computed tomography is the most useful tool in detecting an aortoenteric fistula. To prevent a high mortality rate, early diagnosis is necessary. Exploratory laparotomy is required for patients who are highly suspected of having an aortoduodenal fistula. Herein, we report a 60-year-old man who suffered from acute gastrointestinal bleeding, recurrent syncope, and impending shock. Abdominal computed tomography revealed a 6 cm longitudinal aneurysm in the infrarenal aorta. Emergency laparotomy was performed and revealed an aortoduodenal fistula in the fourth portion of the duodenum causing acute duodenal bleeding. The patient survived and has undergone 2 years worth of regular follow-up in our outpatient department. PMID- 10746412 TI - Diagnosis and follow-up of acute promyelocytic leukemia by detection of PML-RAR alpha gene rearrangement. AB - BACKGROUND: Rapid and accurate diagnosis of acute promyelocytic leukemia (APL) is essential for management of the disease, as all-trans retinoic acid (ATRA) therapy only induces complete remission in patients whose leukemic cells harbor a t(15;17) translocation, resulting in promyelocytic-retinoic acid receptor alpha (PML-RAR alpha) fusion transcripts. Moreover, a positive reverse transcriptase polymerase chain reaction (RT-PCR) of PML-RAR alpha is reported to be a sensitive predictor of relapse in APL. This prompted us to use RT-PCR for rapid diagnosis and monitoring of minimal residual disease in APL patients. METHODS: A nested RT PCR technique was applied to detect the unique PML-RAR alpha fusion transcript in 13 APL patients. The test was applied to help clarify the diagnosis and monitor minimal residual disease after treatment. RESULTS: All 13 APL patients had a positive test result: five patients with the S-form, seven patients with the L form and one patient with the V-form of mRNA fusion transcripts. Minimal residual disease was prospectively monitored using this technique in six patients. Although in clinical remission, all four patients treated with ATRA alone were persistently PCR positive. Of the six patients receiving various forms of consolidation chemotherapy, one was persistently PCR positive while in remission and relapsed four months after the positive PCR test. Five patients were PCR negative. One of the five negative patients relapsed six months after a negative PCR test. The other four patients remained in remission, with a follow-up period of 25 to 46 months after the negative test. PCR was performed in two patients who had been in continuous remission for 3.5 and seven years, respectively. They both had negative PCR tests. CONCLUSIONS: Nested RT-PCR is valuable for confirming the diagnosis of APL and in monitoring minimal residual disease. However, we found that negative test cannot absolutely exclude the possibility of future relapse. PMID- 10746413 TI - Surgical management of complete ureteric duplication abnormalities in children. AB - BACKGROUND: A variety of clinical problems due to complete ureteric duplication (CUD) in children may be encountered. Surgical management of CUD varies and is controversial. Therefore, we reviewed the cases of 15 children with CUD operated on at our hospital in an effort to evaluate the various facets of this disorder that influenced our surgical management. METHODS: Fifteen children with abnormalities associated with CUD underwent surgery from 1987 to 1998. There were 14 girls and one boy. Their age at surgery ranged from 15 days to 3.5 years (average, 9.5 months). All children underwent ultrasonography, including 10 intravenous urograms and five prenatal examinations. The anatomic abnormalities of all children were noted according to the Weigert-Meyer rule. RESULTS: Six of 15 ultrasonograms were misinterpreted as hydronephrosis only, while the 10 intravenous urograms were interpreted correctly as CUD. Four patients had recurrent urinary tract infections after surgery. Two of them underwent further surgery, one for stone formation in the residual ureter stump and the other for persistent ureterocele. There was one surgical complication after an upper pole nephroureterectomy. It involved ureteric necrosis with urine leakage. This patient underwent ipsilateral lower pole nephrectomy 28 days after the first operation. All children remained symptom-free during follow-up. CONCLUSIONS: Accurate diagnosis of duplex kidney requires careful imaging studies, especially for fetal renal abnormalities. Management of CUD should be individualized. Usually, upper pole nephroureterectomy is performed for a nonfunctioning moiety. For functioning segments, ureteropyelostomy or ureteric reimplantation can be considered. The results are satisfactory, but long-term follow-up is necessary. PMID- 10746414 TI - Sudomotor abnormalities in reflex sympathetic dystrophy. AB - BACKGROUND: Intractable pain and sweating dysfunction can occur in patients with reflex sympathetic dystrophy (RSD). We, therefore, investigated autonomic function by measuring sympathetic skin response (SSR) in these patients. This method provides useful information regarding the pathophysiologic changes in and clinical evaluation of RSD. METHODS: Twenty-six RSD patients and 22 age-matched normal subjects were involved in our investigation. Three significant stages of RSD were classified through three-phase bone scanning of the wrist, carpal and metacarpal bones. There were 11 patients in stage I RSD, nine in stage II and six in stage III. SSRs were recorded from the bilateral palms of each subject simultaneously using electrical stimulation of the median nerve in the wrist. Latency and amplitude side ratios were calculated and compared with the control group. RESULTS: The affected/normal (A/N) hand amplitude ratios were significantly lower in stage I RSD, and stage II RSD patients exhibited significantly higher A/N amplitude ratios compared with the control group. A/N amplitude and A/N latency ratios in stage III RSD did not change significantly. The A/N latency ratios in stage I RSD were significantly higher than those of the control group and those of stage II RSD. In one stage I patient and three stage III patients, SSRs were bilaterally unobtainable. CONCLUSIONS: It appears that abnormal SSRs may be associated with autonomic disturbance in RSD patients. PMID- 10746415 TI - Splanchnic endotoxin levels in cirrhotic rats induced by carbon tetrachloride. AB - BACKGROUND: Bacterial translocation (passage of intestinal bacteria to mesenteric lymph nodes) observed in cirrhosis may be a source of endotoxin that can stimulate nitric oxide production and participate in the pathogenesis of hyperdynamic circulation. Currently, there are no published data concerning splanchnic endotoxin levels in cirrhotic rats. This study was designed to determine systemic and portal hemodynamics and to detect endotoxins in the portal and systemic circulation. METHODS: Liver cirrhosis was induced by carbon tetrachloride intragastric gavage. Systemic and splanchnic endotoxin levels in control rats and cirrhotic rats with or without ascites were measured using a chromogenic Limulus assay. In addition, systemic and portal hemodynamic data were obtained using a thermodilution technique and catheterization. RESULTS: Cirrhotic rats with ascites had the lowest systemic vascular resistance (2.6 +/- 0.1 mmHg.ml-1.min.100 g body weight, BW) compared with control rats (6.3 +/- 0.3 mmHg.ml-1.min.100 g BW; p < 0.05) and cirrhotic rats without ascites (3.7 +/- 0.3 mmHg.ml-1.min.100 g BW; p < 0.05). Cirrhotic rats with ascites displayed the highest splanchnic levels of endotoxin (10.6 +/- 3.1 pg/ml) compared with cirrhotic rats without ascites (2.0 +/- 0.7 pg/ml; p < 0.05) and control rats (2.0 +/- 0.4 pg/ml; p < 0.05). There was no difference in the splanchnic endotoxin levels between control rats and cirrhotic rats without ascites (p > 0.05). Similar results were observed with systemic endotoxin values (cirrhotic rats with ascites, 10.8 +/- 2.8 pg/ml; cirrhotic rats without ascites, 2.7 +/- 0.6 pg/ml; control rats, 2.5 +/- 0.4 pg/ml; p < 0.05). A significant correlation existed between portal and systemic endotoxin values in cirrhotic rats with or without ascites (r = 0.96, p < 0.001 and r = 0.9, p < 0.05, respectively), whereas this correlation did not exist in control rats (r = 0.5, p > 0.05). CONCLUSIONS: Cirrhotic rats with ascites had the lowest systemic vascular resistance and the highest splanchnic endotoxin levels when compared with cirrhotic rats without ascites and control rats. These results suggest that splanchnic endotoxemia may be involved in the development and/or maintenance of hyperdynamic circulation. PMID- 10746416 TI - Endovascular embolization of intractable epistaxis. AB - BACKGROUND: We undertook this study to define the role of angiography and endovascular embolization in the treatment of patients with intractable epistaxis. METHODS: A series of 19 patients with massive intractable epistaxis, all treated with endovascular embolization were reviewed. There were 15 males and four females ranging in age from 18 to 70 years, with a mean age of 38 years. The predisposing factors of intractable epistaxis were head and neck tumors (n = 11), idiopathic symptoms (n = 3), surgical complications (n = 2), arteriovenous malformation of the face (n = 1), thrombocytopenia (n = 1) and trauma (n = 1). Polyvinyl alcohol (PVA) particles and gelfoam plugs were used as embolic agents in 11 patients for devascularization; detachable balloons with N-butyl-2 cyanoacrylate (NBCA) were used in four patients for vascular occlusion; NBCA and/or coils were used in four patients in the carotid and subclavian arteries for obliteration of pseudoaneurysms. RESULTS: Complete cessation of epistaxis was achieved in all 19 patients immediately after embolization. Seven patients had also undergone surgical tumor removal at two to five days after embolization. Two patients experienced mild to moderate facial pain on the first day after the procedure, but the symptoms later subsided. No significant complication or recurrence was observed in 18 patients. One patient with advanced hypopharyngeal carcinoma died two weeks after embolization due to another episode of massive epistaxis. Clinical follow-up for these patients was 15 days to eight years, with a mean of 41 months. CONCLUSIONS: Therapeutic endovascular embolization of intractable epistaxis is both efficient and safe. It should be considered as the primary treatment modality in intractable epistaxis. PMID- 10746417 TI - Surgical repair of postinfarction ventricular septal defect. AB - BACKGROUND: Rupture of the interventricular septum complicates 1% to 2% of all acute myocardial infarction patients and its natural course is ominous. The purpose of this study is to present our experience with surgical ventricular septal defect (VSD) repair and examine the possible risk factors and explanations for surgical mortality. METHODS: Fourteen patients underwent repair of postinfarction VSD from 1996 to 1998 at the Taipei Veterans General Hospital. Thirteen patients were in New York Heart Association (NYHA) Functional Class IV and one was in Functional Class III. Eleven patients were in cardiogenic shock with intra-aortic balloon pumps (IABPs) prior to surgery. The operative techniques for VSD repair range from extensive infarctectomy with reconstruction of the septum and the right and left ventricular free walls using single or double patches, to minimal or no infarctectomy with closure of the VSD by excluding the infarcted muscle from the left ventricular cavity and leaving the right ventricle intact. RESULTS: Overall surgical mortality occurred in four patients. All deaths occurred in patients with cardiogenic shock, two with anterior VSD and two with posterior VSD. Three late survivors had limited exercise tolerance with NYHA Functional Class II to III. Left ventricular function was moderately impaired in most patients with a mean nuclear scan ejection fraction of 0.32. However, all patients were elderly and adapted to their residual symptoms without significant life-style changes. CONCLUSIONS: The surgical mortality for treating patients with postinfarction VSD has decreased with improvements in surgical technique. Rapid diagnosis, appropriate preoperative management and delicate surgical repair improve the overall results and help to attain long-term survival. PMID- 10746418 TI - Optimal debulking surgery is an independent prognostic factor in patients with FIGO IIIC primary epithelial ovarian carcinoma. AB - BACKGROUND: Ovarian cancer is a well-known disease with a poor prognosis. Due to the relatively small number of cases in Taiwan, the outcome and prognostic factors of patients with primary epithelial ovarian carcinoma are unknown. METHODS: We retrospectively studied patients with proven surgical and pathologic (Federation Internationale de Gynecologie et d'Obstetrique) FIGO IIIC primary epithelial ovarian carcinoma. All patients underwent standard staging surgery, including washing cytology, total abdominal hysterectomy, bilateral salpingo oophorectomy, retroperitoneal lymphadenectomy, infracolic omentectomy and excisional biopsy of all suspicious lesions followed by adjuvant chemotherapy with four to 12 courses of cyclophosphamide, epirubicin and cisplatin (CEP) or cyclophosphamide, adriamycin and cisplatin (CAP) intravenously, every three weeks. To avoid the coeffects of chemotherapy and surgical procedures upon the outcome, patients who received paclitaxel-based regimens or underwent incomplete surgery were excluded. Ninety-eight patients from 1990 to 1996 were identified. RESULTS: The mean follow-up time was 28.7 months, ranging from 5.4 months to 105.9 months. The cumulative five-year disease-free survival rate for all patients was 31.6%. Optimal debulking surgery was completed in 41.8% of patients, which contributed to long-term patient survival (54% vs 16%, p < 0.0001), compared to patients without optimal debulking surgery. Optimal debulking surgery was the only statistically significant independent prognostic factor for five year disease-free survival using multivariate analysis. CONCLUSIONS: To improve survival of patients with FIGO stage IIIC epithelial ovarian carcinoma, optimal debulking surgery should be performed as the initial form of surgical intervention. PMID- 10746419 TI - Acute pancreatitis associated with temporal lobectomy and intractable seizure. AB - We present a rare case of acute pancreatitis associated with temporal lobectomy due to intractable seizure in a 23-year-old man. The patient underwent elective right temporal lobectomy and hippocampectomy. Severe upper abdominal pain occurred just 10 hours after surgery. The diagnosis of acute pancreatitis was based on the elevation of serum amylase and lipase levels, and the findings of abdominal computerized tomography. Other possible causative factors of acute pancreatitis including alcohol, biliary tract stone, hypertriglyceridemia, hypercalcemia, hyperparathyroidism, biliary dysmotility and autoimmune disease were excluded by a series of examinations. The possibility of drug-induced pancreatitis was very low in this patient. The patient was discharged after supportive treatment. No recurrence of seizure or abdominal pain was noted in the three months after discharge. Acute abdominal pain after brain surgery deserves clinical evaluation for acute pancreatitis. PMID- 10746420 TI - Hypohidrotic ectodermal dysplasia. AB - Hypohidrotic ectodermal dysplasia was first described by Thurnam in 1848. It is a rare, X-linked, recessive disorder characterized by anhidrosis or hypohidrosis, hypotrichosis, dental hypoplasia and characteristic facial features. Herein, we report a typical case of hypohidrotic ectodermal dysplasia. A 20-year-old male presented with the above symptoms at birth. When a family history was taken, it was discovered that his uncle (mother's brother) had the same characteristic facial features and hypotrichosis. PMID- 10746421 TI - Down's syndrome with mucosa-associated lymphoid tissue, thyroid lymphoma and cerebral infarction. AB - Primary lymphoma of the thyroid gland is rare. The histopathology of most low grade thyroid lymphomas is of a mucosa-associated lymphoid tissue (MALT) type. A typical feature of this type of lymphoma is a close lymphocyte-epithelium interaction. It tends to appear in patients with a history of autoimmune disease or chronic inflammatory disorders. A clinical picture of hypothyroidism may be present. Hyperthyroidism associated with thyroid lymphoma is also rare. Thyroid lymphoma could be misdiagnosed as lymphocytic thyroiditis or small cell anaplastic carcinoma. It is rarely reported in patients with Down's syndrome. In this report, we describe a Down's syndrome patient with MALT thyroid lymphoma and cerebral infarction. The patient, a 42-year-old man, presented with chest discomfort and bilateral leg weakness of one week's duration. Physical examination of his neck showed a right-sided mass lesion. Neurologic examination revealed decreased muscle power and hyperreflexia in both lower legs. Babinski's sign was present bilaterally. Endocrinologic studies showed subclinical hypothyroidism. A thoracolumbar radiograph showed disc space narrowing. Thyroid sonography revealed a hypoechoic mass lesion in the right lobe of the thyroid gland. Fine needle aspiration cytology of the neck mass demonstrated a large amount of lymphocyte infiltration. An I131 thyroid scan and 24-hour uptake revealed the possibility of thyroid malignancy at the upper poles of both thyroid lobes. Computerized tomography of the brain revealed a lacunar infarct in the posterior aspect of the left putamen. Magnetic resonance imaging of the lumbar spine revealed a healed L4 compression fracture with L4-5 retrolithesis. The patient later underwent a right total thyroidectomy. The pathologic finding showed MALT lymphoma. The patient received steroid suppression therapy, and after nine months of treatment and follow-up, he developed clinical hypothyroidism. Neither local tumor recurrence nor distant metastasis was found. PMID- 10746422 TI - Postoperative seizure outcome after corpus callosotomy in reflex epilepsy. AB - Flickering light and color patterns, reading, language, movement, decision making, eating, tapping and touching, hot water immersion and auditory stimulation can induce seizures in some epileptic patients. These are known as the "reflex epilepsies". The mechanism of reflex epilepsy is not clear. Recently, we performed anterior two-thirds corpus callosotomies in two reflex epilepsy patients (ages 12 and 14 years), with follow-up for more than three years. Patient 1 had Lennox-Gastaut syndrome with auditory-induced generalized atonic or tonic seizures (startle epilepsy), which decreased by 60% after callosotomy. Patient 2 had Lennox-Gastaut syndrome with somatosensory-induced generalized tonic seizures (tap epilepsy). He was seizure-free for one year immediately after callosotomy, but his seizures recurred with the same degree and frequency as before surgery. The nonsignificant postoperative seizure outcome suggests that the corpus callosum only plays a partial role in seizure generation. Our report also discusses the possible mechanisms of generation of reflex seizures. PMID- 10746423 TI - Ruptured metastatic ovarian carcinoma presenting as acute abdomen. AB - Acute abdomen is a challenge to first-line physicians because of frequently missed diagnoses and potential follow-on legal problems. Improving the management of these patients is of paramount importance, not only for saving lives, but also for reducing untoward problems associated with improper management. We present a case of a patient with acute abdomen due to intraperitoneal hemorrhage secondary to rupture of an ovarian tumor. Following emergency surgery, the patient was diagnosed with metastatic ovarian carcinoma. Because of improper preparation of the gastrointestinal tract, the patient underwent repeat exploratory laparotomy for colon carcinoma. Although this situation did not affect the outcome of the patient in this case, we are concerned that the patient did not benefit from a single operation, with primary complete excision of the tumor plus a colostomy. The outcome of patients with pelvic malignancy, especially those with ovarian carcinoma, might be better if initial surgery achieved optimal tumor debulking. This is possible with good preoperative planning and preparation. We emphasize the importance of preoperative preparation in spite of urgently needed care. Furthermore, every first-line physician should communicate the possibility of malignancy to patients and their families. PMID- 10746424 TI - Chronic idiopathic intestinal pseudo-obstruction. AB - Chronic idiopathic intestinal pseudo-obstruction (CIIP) is a rare heterogeneous clinical syndrome characterized by recurrent episodes of symptoms and signs of intestinal obstruction in the absence of a mechanically obstructing lesion. Dilatation of other viscera, such as the renal pelvis, ureter or urinary bladder, is identified in a minority of patients. We report the cases of two patients with CIIP presenting with abdominal fullness and constipation. Radiologic examination of the first patient revealed dilatation of the esophagus, stomach, duodenum and bowel loops up to the ascending colon. The nerve conduction velocity study of the right extremities revealed polyneuropathy and urinary bladder manometry revealed poor sensation. The patient had been admitted to our hospital three times for symptomatic relief within the prior six months. During the last admission, his symptoms persisted without response to medical treatment. Soon after discharge, the patient underwent surgery at another hospital and died of nutritional problems. The second patient was transferred to our hospital after an exploratory laparotomy was performed one month earlier. A radiographic examination revealed distention of the stomach, duodenum, small intestine and ascending colon, as well as bilateral hydronephrosis. Rheumatologic examination revealed no evidence of autoimmune disorder. The patient also had heavy proteinuria due to minimal change disease that was proven by renal biopsy. After receiving prokinetic, cathartic and corticosteroid medication for kidney disease, symptoms improved, but hydronephrosis persisted. PMID- 10746425 TI - Repeated episodes of spontaneous intracystic hemorrhage of hepatic cysts mimicking malignancy. AB - Repeated episodes of spontaneous intracystic hemorrhage in the liver are very rare. We present the case of a patient with spontaneous intracystic hemorrhage mimicking hepatic malignancy, following an episode of spontaneous hemorrhage of a cyst in the left lobe of the liver. Sonography revealed a heterogenous mass with amorphous material in one episode and a cystic mass with internal papillary projections and septations in the other. The patient experienced acute abdominal pain in the first episode and remained asymptomatic during the two-year follow-up period. With an accurate diagnosis, spontaneous hemorrhage of a hepatic cyst may follow a benign course and be treated conservatively. PMID- 10746426 TI - Therapeutic equivalence of alendronate 70 mg once-weekly and alendronate 10 mg daily in the treatment of osteoporosis. Alendronate Once-Weekly Study Group. AB - Dosing convenience is a key element in the effective management of any chronic disease, and is particularly important in the long-term management of osteoporosis. Less frequent dosing with any medication may enhance compliance, thereby maximizing the effectiveness of therapy. Animal data support the rationale that once-weekly dosing with alendronate 70 mg (7 times the daily oral treatment dose) could provide similar efficacy to daily dosing with alendronate 10 mg due to its long duration of effect in bone. In addition, dog studies suggest that the potential for esophageal irritation, observed with daily oral bisphosphonates, may be substantially reduced with once-weekly dosing. This dosing regimen would provide patients with increased convenience and would be likely to enhance patient compliance. We compared the efficacy and safety of treatment with oral once-weekly alendronate 70 mg (N=519), twice-weekly alendronate 35 mg (N=369), and daily alendronate 10 mg (N=370) in a one-year, double-blind, multicenter study of postmenopausal women (ages 42 to 95) with osteoporosis (bone mineral density [BMD] of either lumbar spine or femoral neck at least 2.5 SDs below peak premenopausal mean, or prior vertebral or hip fracture). The primary efficacy endpoint was the comparability of increases in lumbar spine BMD, using strict pre-defined equivalence criteria. Secondary endpoints included changes in BMD at the hip and total body and rate of bone turnover, as assessed by biochemical markers. Both of the new regimens fully satisfied the equivalence criteria relative to daily therapy. Mean increases in lumbar spine BMD at 12 months were: 5.1% (95% CI 4.8, 5.4) in the 70 mg once weekly group, 5.2% (4.9, 5.6) in the 35 mg twice-weekly group, and 5.4% (5.0, 5.8) in the 10 mg daily treatment group. Increases in BMD at the total hip, femoral neck, trochanter, and total body were similar for the three dosing regimens. All three treatment groups similarly reduced biochemical markers of bone resorption (urinary N-telopeptides of type I collagen) and bone formation (serum bone-specific alkaline phosphatase) into the middle of the premenopausal reference range. All treatment regimens were well tolerated with a similar incidence of upper GI adverse experiences. There were fewer serious upper GI adverse experiences and a trend toward a lower incidence of esophageal events in the once-weekly dosing group compared to the daily dosing group. These data are consistent with preclinical animal models, and suggest that once-weekly dosing has the potential for improved upper GI tolerability. Clinical fractures, captured as adverse experiences, were similar among the groups. We conclude that the alendronate 70 mg once-weekly dosing regimen will provide patients with a more convenient, therapeutically equivalent alternative to daily dosing, and may enhance compliance and long-term persistence with therapy. PMID- 10746427 TI - Prevention of recurrent hip fracture. AB - Our objective was to describe the interventions aimed at preventing a recurrent hip fracture, and other injurious falls, which were provided during hospitalization for a first hip fracture and during the two following years. A secondary objective was to study some potential determinants of these preventive interventions. The design of the study was an observational, two-year follow-up of patients hospitalized for a first hip fracture at the University Hospital of Lausanne, Switzerland. The participants were 163 patients (median age 82 years, 83% women) hospitalized in 1991 for a first hip fracture, among 263 consecutively admitted patients (84 did not meet inclusion criteria, e.g., age>50, no cancer, no high energy trauma, and 16 refused to participate). Preventive interventions included: medical investigations performed during the first hospitalization and aimed at revealing modifiable pathologies that raise the risk of injurious falls; use of medications acting on the risk of falls and fractures; preventive recommendations given by medical staff; suppression of environmental hazards; and use of home assistance services. The information was obtained from a baseline questionnaire, the medical record filled during the index hospitalization, and an interview conducted 2 years after the fracture. Potential predictors of the use of preventive interventions were: age; gender; destination after discharge from hospital; comorbidity; cognitive functioning; and activities of daily living. Bi- and multivariate associations between the preventive interventions and the potential predictors were measured. In hospital investigations to rule out medical pathologies raising the risk of fracture were performed in only 20 patients (12%). Drugs raising the risk of falls were reduced in only 17 patients (16%). Preventive procedures not requiring active collaboration by the patient (e.g., modifications of the environment) were applied in 68 patients (42%), and home assistance was provided to 67 patients (85% of the patients living at home). Bivariate analyses indicated that prevention was less often provided to patients in poor general conditions, but no ascertainment of this association was found in multivariate analyses. In conclusion, this study indicates that, in the study setting, measures aimed at preventing recurrent falls and injuries were rarely provided to patients hospitalized for a first hip fracture at the time of the study. Tertiary prevention could be improved if a comprehensive geriatric assessment were systematically provided to the elderly patient hospitalized for a first hip fracture, and passive preventive measures implemented. PMID- 10746428 TI - Inhibitory effects of angiotensin-converting enzyme (ACE) inhibitors on oxygen radicals produced by bronchoalveolar lavage cells in young and aged guinea pigs. AB - We examined the effect of angiotensin-converting enzyme (ACE) inhibitors and age on oxygen radical formation by bronchoalveolar lavage (BAL) cells. Lung-free cells, including pulmonary alveolar macrophages, were harvested from young (4 month-old) and aged (28-month-old) male guinea pigs using BAL. The oxygen radicals produced by BAL cells were measured by a lucigenin-dependent chemiluminescence method using a photon counter. Although spontaneous oxygen radical production by BAL cells from young and aged guinea pigs did not differ, the oxygen radical generation after maximal stimulation with phorbol-myristate acetate (PMA) was greater than that produced without PMA stimulation in both young and aged animals. ACE inhibitors with and without an SH-group (alacepril and lisinopril, respectively) were tested for their effect on oxygen radical formation by BAL cells; both ACE inhibitors inhibited oxygen radical production and generation by BAL cells from both young and aged guinea pigs in a dose dependent manner. However, the alacepril concentration giving 50% inhibition (IC50) of oxygen radical generation by BAL cells was smaller than the IC50 of lisinopril in both young and aged guinea pigs. These results indicate that ACE inhibitors, in particular those with an SH-group, effectively reduce oxygen radical production by BAL cells from young and aged guinea pigs, and suggest that treatment with ACE inhibitors may be useful for ameliorating oxidant-associated pulmonary disorders in young and aged patients. PMID- 10746429 TI - An application of generalizability theory to study a physical performance measure in Parkinson's disease. AB - Clinicians and researchers frequently quantify impairments and functional ability to monitor patient's symptoms and progress. For some patients, such as those with Parkinson's disease (PD), symptoms can fluctuate from day to day, making reliable measurement difficult. Multiple measures then may be required to obtain reliable data. Decisions must be made, balancing the optimum measurement schedule to obtain "good reliability" against burden to the patient. This investigation demonstrates the use of Generalizability Theory in determining the testing schedule when designing an experiment involving patients with known fluctuations of symptoms. In this investigation we use "Functional Axial Rotation" (FAR), a measure of spinal flexibility, to illustrate the use of Generalizability Theory for designing an experiment using participants who have PD. Measurements of FAR were taken on 13 participants, aged 60 or older, who were in early and mid-stages of PD. Three measurements were obtained on each of two consecutive days, and repeated on two consecutive days a week later, giving a total of 12 measures of FAR for each individual. Four sources of variation (subject, week, day and trial) were employed to estimate the reliability of FAR under several designs. Assuming different schedules of measures across weeks, days and trials, the estimated reliability of FAR for four measurements is in the range of 0.75 to 0.83, and for eight measurements in the range of 0.82 to 0.86. We discuss the use of this type of analysis in the determination of the optimum measurement design for experiments involving subjects with known fluctuations. PMID- 10746430 TI - Length of hospitalization in elderly patients with community-acquired pneumonia. AB - Community-acquired pneumonia (CAP) is a serious social and medical problem in the elderly. Mortality, hospitalization and length of stay increase with age. The aim of this study was to determine the risk factors associated with prolonged hospital stay in elderly patients with CAP. Clinical and laboratory data were collected for 115 community-living patients, 65 years old and over, admitted to the geriatric ward of a University Hospital from 1995 to 1998 because of symptoms and signs of pneumonia confirmed by a pulmonary infiltrate on chest x-ray. We divided the patients into two groups, with length of stay more than 13 days (70 patients, cases), and length of stay less than 13 days (45 patients, controls) according to Diagnosis Related Groups criteria for complicated and uncomplicated pneumonia, respectively. A prolonged hospital stay was associated with a higher fever peak and a higher number of days with fever (p<0.005), greater comorbidity (p<0.001), urinary catheterization and secondary urinary infections (p<0.001), higher erythrocyte sedimentation rate (p<0.001), dehydration (p<0.005), and caloric-proteic malnutrition (p=0.01). In conclusion, knowledge of the risk factors for prolonged hospital stay in elderly patients with CAP may be used to identify high-risk patients, prevent the risks with prophylactic measures, and contain the costs of hospitalization. PMID- 10746431 TI - Echocardiographic assessment of left ventricular structure and function in elderly women with congestive heart failure. AB - The aim of this investigation was an echocardiographic assessment of left ventricular structure and function in elderly women who were hospitalized due to congestive heart failure. Sixty-three women with heart failure aged 70-100 years (mean age, 82 years) were studied; medical histories were taken, medical examinations and chest X-ray studies, along with ECG and M-mode, 2D-mode and Doppler echocardiography were performed. Echocardiography revealed that the predominant structural lesions involved the cardiac valves; in the majority of cases, there was thickening and calcification of the mitral and aortic valves, but calcification of the mitral and aortic annulus was also frequent. Mitral regurgitation was noted in 84.1% of patients, and tricuspid regurgitation in 50.8%. More than one type of valvular dysfunction was characteristic of 57.1% of women. Among the evaluated cardiac dimensions, left atrial enlargement was observed in 84.1% of women, and ventricular septal hypertrophy in 60.3%. In the majority of patients (55.6%), the values of left ventricular ejection fraction ranged from 51% to 82%, while in the remaining 44.4% they oscillated between 18% and 50%. The most often detected underlying etiological factor in our elderly females with heart failure was coronary artery disease diagnosed in 88.9% of patients, followed by arterial hypertension (54%), and valvular defects (22.2%). In conclusion, the results indicate the presence of some characteristic lesions revealed by echocardiography in elderly patients treated for heart failure; valvular and annular degenerative lesions are commonly detected, valvular dysfunction is frequent, and in the majority of patients (55.6%) left ventricular systolic function is not impaired. PMID- 10746432 TI - The relation of plasma total homocysteine levels to prevalent cardiovascular disease in older patients with ischemic stroke. AB - The objectives of this study were to describe the distribution of serum levels of total homocysteine (HCys) in a sample of older patients consecutively admitted following acute ischemic cerebral stroke, as compared with healthy controls, and to test for possible relationships of HCys levels to some of the prevalent cardiovascular diseases in these stroke patients. One hundred and thirty-seven stroke patients and 132 healthy controls (age > or =60) participated in this study. HCys levels were determined by HPLC method with fluorescence detection. Correlates of HCys levels and clinical data were examined. The results showed that stroke patients (mean age 74.6+/-9.2) had higher HCys levels as compared with controls (13.8 and 9.8 respectively, p<0.001). Advanced age, male gender, absence of diabetes and a positive history of previous myocardial infarction were the factors associated with HCys levels higher than 10 mmol/L (Odds ratio 2.72, 2.54, 3.12, 3.55, respectively). We conclude that hyperhomocysteinemia is prevalent in older patients with acute ischemic stroke. Few factors associated with increased risk for hyperhomocysteinemia in these stroke patients were identified. The study supports earlier observations regarding elevated HCys levels in stroke patients and increased prevalence of associated cardiovascular disease. PMID- 10746433 TI - Current status and perspectives of gerontology in Russia. AB - The current situation of Russian gerontology and its development after 1991 are analyzed in this paper. The potential of Russian gerontology has been evaluated as rather high, and includes not only leading scientific centers (Moscow, St. Petersburg, Novosibirsk, Ekaterinburg), but also many other regions of the country. The main directions of current research in gerontology, the strategy recommended as a base for future research, and medico-social means for health, care and improving the quality of life of the elderly in all its aspects are also discussed. PMID- 10746434 TI - Heterogeneity in breast cancer and the problem of relevance of findings. AB - Many attempts are made to identify critical genetic events responsible for the development and progression of breast cancer. There is increasing evidence that breast cancer is a heterogeneous disease, both, phenotypically as well as with respect to its molecular biologically. It is, therefore, extremely difficult to establish a diagnostically and prognostically relevant tumourigenesis model. Emerging new techniques such as microarrays, will provide us with a wealth of additional data over the next years. The precise sampling of tumour material in clearly defined histopathological lesions will be a prerequisite for the assignment of specific genetic alterations to defined stages of breast disease. PMID- 10746436 TI - Evaluation of heterogeneity of DNA ploidy in early gastric cancers. AB - DNA ploidy has been shown to be a predictive parameter for prognosis in various solid tumours. The prognostic value of DNA-ploidy in gastric cancers is still a matter of controversy. A possible explanation for the discrepant results reported in the literature could be sampling error in tumours with multiple stemlines differing in DNA-ploidy. In order to determine whether or not such heterogeneity exists in early gastric carcinoma, we have performed DNA cytophotometry on multiple samples of a group of 17 early gastric carcinomas, of which 8 were pure intramucosal and 9 were infiltrating into the submucosa. We found an aneuploid DNA-stemline in 8 (47%) early gastric cancers, more often in tumours invading into the submucosa (5/9) than in purely mucosal tumours (3/8). Multiple DNA stemlines were found more frequently in submucosally infiltrating tumours (4/5). These results confirm the presence of DNA-aneuploid early gastric carcinoma which are frequently heterogeneous and suggest that heterogeneity occurs more frequently in tumours invading the submucosa. This heterogeneity is best detected by analysing multiple samples of tumours for DNA-ploidy. PMID- 10746435 TI - Immunocytochemistry and DNA-image cytometry in diagnostic effusion cytology. II. Diagnostic accuracy in equivocal smears. AB - To determine sensitivity and specificity of different antibodies for the immunocytochemical detection of malignant cells in diagnostically equivocal effusions in comparison with those achieved by DNA-image cytometry. 65 cytologically doubtful effusions of the serous cavities were stained with twelve antibodies. Furthermore, DNA-image cytometry was performed. Data were correlated with patient follow-up. Sensitivity of cellular staining of Ber-EP4 for the identification of malignant cells was 77.8%, specificity of absent staining for benign cells was 100%. Positive predictive value for the identification of malignant cells was 100%, negative value 65.5%. Sensitivity of DNA-aneuploidy for the identification of malignancy was 82.9%, specificity of DNA-non-aneuploidy for benignity 94.7%. The positive predictive value of DNA-aneuploidy for the occurrence of malignant cells was 96.7%. Negative predictive value of DNA-non aneuploidy was 72.0%. Combining immunocytochemistry applying Ber-EP4 only and DNA cytometry in equivocal effusions resulted in a sensitivity of 88.9% for the identification of malignant cells associated with a 95.0% specificity. Positive predictive value was 97.7%, the negative one 79.2%. PMID- 10746437 TI - Ha-ras oncogene effect on DNA content and chromatin supraorganization in benzo[a]pyrene-transformed human breast epithelial cells. AB - When transfected to benzo[a]pyrene (BP)-transformed MCF-10F human breast epithelial cells (BP1 cell line) the c-Ha-ras oncogene has proven to enhance the neoplastic changes initiated by exposure to BP, giving rise to an aggressive tumorigenic cell line, BP1-Tras. We have previously demonstrated by image analysis that BP affects the DNA content and the chromatin supraorganization of MCF-10F cells. Here Feulgen-stained BP1-Tras cells were studied by image analysis in order to evaluate possible additional changes in DNA content and chromatin texture induced by insertion of the ras oncogene. A high variability in DNA content also including polyploidy or near-polyploidy, and an increase in the packing states of the chromatin which became still condensed in BP1 cells were found in BP1-Tras cells. The results differed from those reported for the BP1-E1 cell line which is also an aggressive tumorigenic cell line, but was attained through progressive passages of BP-transformed cells. It was demonstrated that different patterns of changes in DNA content and chromatin organization may be involved in equally aggressive tumorigenic BP-transformed cell lines originated from the same cell line by different mechanisms. PMID- 10746438 TI - Flow cytometric immunophenotyping of mature lymphatic neoplasias using knowledge guided cluster analysis. AB - Flow cytometry is widely used for the immunological characterization of hematopoietic malignancies. Discrimination of normal and malignant cellular immunophenotypes is the most critical step in data analysis, especially if multi color analysis is performed on highly heterogenous cell suspensions. We therefore investigated, whether adaptive, simultaneous multiparameter gating allowed automated, operator independent analysis of data obtained from the immunophenotyping of blood or bone marrow samples with regard to the presence of non-Hodgkin lymphoma cells. The identification of physiological and malignant cells was achieved by predefining population boundaries, based on the expectations of the population's location in two-dimensional dot plots. The prospective application of these predefined region boundaries in 52 blood and bone marrow samples enabled identification of lymphoma cells with regard to their presence and immunophenotype, based on the correlation of markers as defined in multiple tubes. Our data confirm that highly standardized data analysis methods can reduce the variability of analysis and support the expert in establishing a rapid classification of the sample. PMID- 10746439 TI - Energy transfer between fluorescein isothiocyanate and propidium iodide--a problem in the estimation of Tpot with the bromodeoxyuridine-DNA flow cytometry technique? AB - Energy transfer in flow cytometry can occur when two fluorochromes are bound in close proximity (generally within 100 A) and the emission spectrum of one fluorochrome overlaps significantly with the excitation spectrum of the other. The latter criterium is fulfilled for the fluorochromes fluorescein isothiocyanate and propidium iodide and also the former when they, e.g., are used in bromodeoxyuridine - DNA flow cytometry methods. In the present growth kinetic study using this method, we show that energy transfer does take place between fluorescein isothiocyanate and propidium iodide which results in a detected increase in DNA content with 2-3%. Despite the erroneous increase in the obtained DNA content values, this does not seem to have any influence on the calculation of DNA synthesis time and potential doubling time where the DNA content, based on the relative movement principle of the labelled cells, is used. PMID- 10746440 TI - An automated 96-well-plate loader for FACScan. AB - Staining multiple samples for data acquisition with a flow cytometer is done in 96-well plates to save time and make large data assessment in one working day possible. However, the inability of the FACScan to take up the samples from 96 well plates is a major drawback. In order to avoid the individual transfer of samples to tubes, we have developed a system, which allows using the FACScan with 96-well plates. The machine consists of a programmable control module and a loader which moves the 96-well plate in 3 axis well by well along the sample collector. The machine is equipped with a wash buffer tank to avoid cross contamination of samples and a shaking option to avoid sedimentation of cells during acquisition. The machine can be further developed into a full automatic loader if connected to the FACS Station. In 24 hours about 7,000 samples with 10,000 cells each can be acquired. PMID- 10746441 TI - Lamellar corneal patch grafts in the management of corneal melting. AB - PURPOSE: To evaluate the clinical indications and results of reconstructive (tectonic) lamellar keratoplasty in corneal melting. METHODS: A nonrandomized, uncontrolled retrospective case series of 64 consecutive patients (80 eyes) who underwent lamellar keratoplasty for corneal melting at our institution over a 17 year period. We reviewed the (a) clinical indications, (b) visual acuities, (c) postoperative corneal clarity, and (d) post-operative complications. Comparisons in visual acuity were made between central and peripheral corneal melts. The statistical influence of patient age, diagnosis, and corneal graft size on pre- and postoperative visual acuity values also was studied. RESULTS: Although reconstructive lamellar keratoplasty for active corneal melting was effective in saving the integrity of the globes in all but four patients, the postoperative visual acuity remained poor in the majority of cases because of the often devastating nature of the underlying ocular diseases. Only 14 patients had best postoperative visual acuities of 20/100 or better. Repeated lamellar keratoplasties were necessitated by corneal opacification, infection, or progressive postoperative corneal dissolution in 14 cases. Subsequent vision restoring surgeries, consisting of penetrating keratoplasties or cataract extractions, were done in 11 eyes with modest improvement of visual acuity. Postoperative visual acuity was significantly better in peripheral corneal melts than in central melts (p = 0.004). CONCLUSION: Lamellar keratoplasty is an effective method of restoring the integrity of the eye ravaged by corneal melting. It is less invasive and consequently safer than penetrating keratoplasty in actively inflamed and unstable eyes. The primary purpose for this surgery is to salvage the integrity of the globe during the acute phase of disease and not so much to achieve visual improvement per se. It allows time for systemic immunosuppression to take effect and for the eye to quiet down before possible future vision-restoring surgery. PMID- 10746443 TI - Penetrating keratoplasty in children. AB - PURPOSE: To determine the success of penetrating keratoplasty in the presence of various pediatric corneal abnormalities. METHODS: We performed a retrospective study of pediatric corneal grafts at L.V. Prasad Eye Institute, Hyderabad, India. Outcome of pediatric corneal transplantation was evaluated in terms of anatomic and optical success and factors contributing to poor graft survival. On the basis of the corneal pathology, patients were divided into three categories: congenital, acquired nontraumatic, and acquired traumatic. RESULTS: A retrospective analysis of 154 penetrating keratoplasties performed in 140 children, aged 14 years or younger, was done. The average follow-up was 1.3 years (range, 1 week-5 years). Grafts remained clear in 102 (66.2%) of 154 eyes. Clear grafts were achieved in 30 (63.8%) of 47 eyes with congenital opacities, 12 (54.5%) of 22 eyes with opacities from trauma, and 60 (70.6%) of 85 eyes with acquired nontraumatic opacities. Most (26 of 52) of the graft failures occurred during the first 26 weeks after surgery. Survival analysis revealed the probability of a graft remaining clear at the end of 26 weeks as 80% (SE, 3.39%). Poor graft survival could be correlated with those younger than 5 years (p = 0.0341) and performance of anterior vitrectomy (p = 0.0002). Most grafts failed because of allograft rejection (42.3%), infectious keratitis (26.9%), or secondary glaucoma (13.4%). Postoperatively, 53 eyes had > or =20/400 vision, 29 of which had > or =20/50. Vision could not be assessed in 33 eyes because of the young age. CONCLUSION: Whereas anatomic success of pediatric keratoplasty is increasing, optical success continues to remain less than satisfactory. Early surgical intervention and intensive amblyopia therapy may promote visual recovery. PMID- 10746442 TI - Penetrating keratoplasty for varicella-zoster virus keratopathy. AB - PURPOSE: To examine and report the results of penetrating keratoplasty performed in patients with varicella-zoster virus keratopathy. METHODS: The authors retrospectively reviewed the records of 15 patients who had penetrating keratoplasty for varicella-zoster virus keratopathy from January 1989 through December 1998 on the Cornea Service at Wills Eye Hospital. RESULTS: Twelve patients had a preoperative diagnosis of herpes zoster ophthalmicus, and three, of varicella. Four eyes had lateral tarsorrhaphies performed in conjunction with penetrating keratoplasty. Three eyes had endothelial rejection episodes that responded well to treatment with topical steroids. One eye had a regraft 1 month after primary failure, and this second graft also failed because of recurrent neurotrophic keratopathy. Three eyes that had repeated penetrating keratoplasty for graft failure had clear grafts at the last examination. At an average follow up time of 50 months, 13 (86.7%) grafts remained clear, and the best corrected visual acuity was 20/100 or better in eight (53.3%) eyes. Five patients had decreased visual acuity because of retinal diseases. CONCLUSION: Although varicella-zoster virus keratopathy is an uncommon indication for penetrating keratoplasty, effective visual rehabilitation can be achieved in these patients. Careful postoperative management, frequent lubrication, and lateral tarsorrhaphies to protect the corneal surface are major factors in the successful outcome of these cases. PMID- 10746444 TI - Diclofenac sodium, 0.1% (Voltaren Ophtha), versus sodium chloride, 5%, in the treatment of filamentary keratitis. AB - PURPOSE: To compare the efficacy and short-term safety of diclofenac sodium, 0.1% (Voltaren Ophtha; Ciba-Vision) and of sodium chloride, 5% ophthalmic solution, in the treatment of filamentary keratitis (FK) in patients with dry-eye syndrome due to secondary Sjogren's syndrome. METHODS: Thirty-two patients (64 eyes) with dry eye syndrome due to secondary Sjogren' syndrome were enrolled in a randomized study (patients and authors were aware of which medication was being used). All patients had FK. Sixteen patients were treated with sodium chloride, 5% drops, and 16 patients received diclofenac sodium, 0.1% eyedrops. Treatment regimen included instillation of 1 drop, 4 times a day for 28 days, for both groups. Clinical assessment was performed once a week during the study period. Data on the efficacy and safety of the different therapeutic regimens were collected and compared. RESULTS: Both medications achieved disappearance of filaments at the end of the study. Treatment with diclofenac sodium, 0.1%, revealed a significantly more rapid improvement of the clinical symptoms as compared with sodium chloride, 5%. No significant adverse effects were observed in both groups. CONCLUSION: Diclofenac sodium, 0.1%, may be an effective and safe topical therapy in patients with FK caused by secondary Sjogren's disease. PMID- 10746445 TI - Assessment of the reliability of human corneal endothelial cell-density estimates using a noncontact specular microscope. AB - PURPOSE: We sought to determine the variance in endothelial cell density (ECD) estimates for human corneal endothelia. METHODS: Noncontact specular micrographs were obtained from white subjects without any history of contact lens wear, or major eye disease or surgery; subjects were within four age groups (children, young adults, older adults, senior citizens). The endothelial image was scanned, and the areas from > or =75 cells measured from an overlay by planimetry. The cell-area values were used to calculate the ECD repeatedly so that the intra- and intersubject variation in an average ECD estimate could be made by using different numbers of cells (5, 10, 15, etc.). RESULTS: An average ECD of 3,519 cells/mm2 (range, 2,598-5,312 cells/mm2) was obtained of counts of 75 cells/ endothelium from individuals aged 6-83 years. Average ECD estimates in each age group were 4,124, 3,457, 3,360, and 3,113 cells/mm2, respectively. Analysis of intersubject variance revealed that ECD estimates would be expected to be no better than +/-10% if only 25 cells were measured per endothelium, but approach +/-2% if 75 cells are measured. CONCLUSION: In assessing the corneal endothelium by noncontact specular microscopy, cell count should be given, and this should be > or =75/ endothelium for an expected variance to be at a level close to that recommended for monitoring age-, stress-, or surgery-related changes. PMID- 10746446 TI - The relationship between human papillomavirus and p53 gene in conjunctival squamous cell carcinoma. AB - PURPOSE: The p53 tumor-suppressor gene has been documented to exist in mutated forms in many types of squamous cell carcinoma in the body. Also in conjunctival squamous cell carcinoma, human papillomavirus (HPV) is accepted as an oncogenic factor. The objective of our study was to establish a correlation between p53 overexpression and the presence of HPV infection within tumor tissues from patients with conjunctival squamous cell carcinoma. METHODS: Tissue sections obtained from paraffin-embedded conjunctival squamous cell carcinoma specimens from 23 patients were examined with light microscopy, polymerase chain reaction (PCR), and immunohistochemistry. RESULTS: Seventy-eight percent of tumors were positive for p53, whereas 22% were positive for HPV. The proportion of patients positive for both p53 and HPV was 17%, whereas another 17% of the patients were negative for both p53 and HPV. Therefore no significant disproportion was found in the distribution of patients' HPV status and p53 status (p = 1.00). No significant correlation or linear association was found between the HPV status and p53 status (r = 0.022; p = 0.920). CONCLUSION: We could not show any statistical association between abnormal p53 gene-product expression by immunohistochemistry in conjunctival squamous cell carcinomas and HPV infection by PCR detection techniques. PMID- 10746447 TI - Confocal microscopy of a patient with irregular astigmatism after LASIK reoperations and relaxation incisions. AB - PURPOSE: Laser-assisted in situ keratomileusis (LASIK) is widely used for correcting refractive errors. If the predicted refractive result is not achieved after the first operation, a re-operation can be performed by ablating more stromal tissue after reopening the flap. The goal of this study was to analyze, by using in vivo confocal microscopy, the morphologic changes associated with repeated LASIKs. METHODS: Clinical examination, computed corneal topography, and real-time in vivo confocal microscopy were performed on both eyes of a 50-year old patient with induced irregular astigmatism leading to decreased best corrected vision in the left eye after LASIK. The left cornea had been operated on 5 times (LASIK with two reoperations followed by two relaxing incisions), and the right cornea twice (LASIK with one reoperation). RESULTS: Microfolds at the level of the Bowman's layer and highly reflective particles at the flap interface were observed in both corneas. The subbasal nerve plexus was severed in the left eye. In addition, we identified epithelial material in the flap margin and inside one of the two relaxing incisions placed inferotemporally. CONCLUSION: Repeated LASIKs may stretch the flap and result in microfolding at the Bowman's layer. This and deposition of particles in the flap interface may increase with the number of reoperations, challenging the healing response. Microfolding and occurrence of foreign material in the interface may add to the irregular astigmatism and poor visual outcome after LASIK. Clinical in vivo confocal microscopy offers new possibilities for the assessment of ultrastructural changes after corneal refractive surgery. PMID- 10746448 TI - Incidence and prevention of epithelial growth within the interface after laser in situ keratomileusis. AB - PURPOSE: To analyze the incidence of epithelial growth within the interface after laser in situ keratomileusis (LASIK) and describe a technique that may prevent its occurrence. METHODS: Results were reviewed for all LASIK procedures performed by two surgeons using identical surgical technique with either the Automated Corneal Shaper or Hansatome microkeratome. After laser ablation, the bed and posterior surface of the flap were irrigated with 0.2 microM filtered basic salt solution, and the surfaces were swept with lint-free sponges to clear the interface of epithelial and other debris. An aspirating lid speculum connected to suction was used to evacuate irrigation fluid and debris from the field. After the flap was smoothed into position, a bandage contact lens was used for the first day after surgery to prevent lifting of the flap edge during blinking. RESULTS: Seven hundred eighty-three eyes of 419 patients had primary LASIK, and 108 eyes had LASIK reoperation. Three eyes developed epithelial growth within the interface, with a minimum of 3 months of follow-up. The interface opacity appeared within 1 month of surgery in all three eyes. Two epithelial nests were small (1.0 and 1.5 mm in diameter, respectively) and did not increase in size on subsequent visits. Neither peripheral epithelial nest affected best-corrected visual acuity or caused any other sequelae. One ingrowth occurred early postoperatively in a case with a donut-shaped flap and was eliminated by transepithelial photorefractive keratectomy (PRK). CONCLUSION: Irrigating, wiping the stromal interface with sponges. aspirating irrigation fluid and debris with a suctioning lid speculum, and use of a bandage contact lens for the first day after surgery during LASIK and LASIK enhancement may markedly reduce the incidence of epithelial growth within the interface. PMID- 10746449 TI - Surgically induced astigmatism after photorefractive keratectomy with the excimer laser. AB - PURPOSE: To evaluate retrospectively the effect of spherical excimer laser photorefractive keratectomy (PRK) on astigmatism. METHODS: Four hundred seventy consecutive eyes of patients who had PRK for the treatment of myopia without astigmatic keratotomy, PRK reoperation, or other surgical procedures were evaluated in a retrospective clinical study. PRK was performed using the Summit Apex excimer laser with attempted corrections from 1 to 7 diopters (D) of myopia. Preoperative and postoperative astigmatism was determined by manifest refraction refined with a 0.25-D Jackson cross cylinder and evaluated with vector analysis. RESULTS: Eighty-five ( 18%) eyes continued to have a spherical refraction after PRK, 53 (11%) eyes had the same preoperative astigmatism, and 332 (71%) eyes had a change in magnitude of astigmatism > or =0.25 D after spherical PRK. The absolute change in astigmatism magnitude irrespective of axis was +0.4 +/- 0.4 (standard deviation) D at 6 months after PRK. Eyes with change in astigmatism power tended to have higher preoperative myopia and higher preoperative astigmatism. Vector analysis revealed surgically induced astigmatism was 0.68 +/- 0.50 D (range, 0-3.25 D) at 1 month and 0.56 +/- 0.47 D (range, 0-3.1 D) at 12 months after spherical PRK. CONCLUSION: Spherical excimer laser PRK is associated with significant surgically induced astigmatism that is likely related to decentration of the ablation, excimer laser beam irregularities, and variations in wound healing across the ablated zone. Surgically induced astigmatism will complicate attempts to correct astigmatism simultaneously at the time of PRK and suggest that such attempts are likely to be problematic for lower levels of astigmatism. PMID- 10746450 TI - One-year results of PRK in low and moderate myopia: fewer than 0.5% of eyes lose two or more lines of vision. AB - PURPOSE: To retrospectively evaluate the results of myopic photorefractive keratectomy (PRK) for different levels of intended correction, including analysis of loss of best spectacle-corrected visual acuity. METHODS: Four hundred seventy five consecutive eyes with 1 year of follow-up that had PRK for the correction of 1-7 diopters (D) of myopia by using the Summit SVS Apex excimer laser. Three hundred forty-eight eyes were examined at 1 year. This study was confined to the 236 eyes with 1 year of follow-up that had PRK without astigmatic keratotomy. Eyes also were analyzed according to the range of attempted correction (0-3 D, low; 3.1-6D, moderate; and > or =6.1 D, high moderate). Manifest refraction, uncorrected visual acuity (UCVA), best spectacle-corrected visual acuity (BSCVA), surface regularity index (SRI), and surface asymmetry index (SAI) were evaluated for each group. RESULTS: One year after PRK, 91% of all eyes were within 1 diopter and 73% of eyes were within 0.5 diopter of emmetropia. Uncorrected visual acuity was 20/25 or better in 79% and 20/40 or better in 96% of eyes. Two lines of BSCVA were lost in only 0.4% of eyes (one of 236). No eye lost >2 lines of BSCVA, and 30% gained one line. Mean SRI and SAI were increased as compared with preoperative values, but were within the normal range for our patient population (ranges, 0.2-1.0 and 0.1-0.7, respectively). UCVA, BSCVA, and predictability decreased, whereas SAI and SRI increased, with increasing attempted correction. CONCLUSION: PRK effectively reduced myopia in all eyes with 12 months' follow-up. Predictability tended to decrease with increasing attempted correction, even for low to moderate myopia. PRK may induce mild surface asymmetry and irregularity, and these alterations tend to increase with higher attempted correction. Fewer than 0.5% of eyes lost > or =2 lines of best-corrected visual acuity. PMID- 10746451 TI - The keratocyte network of human cornea: a three-dimensional study using confocal laser scanning fluorescence microscopy. AB - PURPOSE: Keratocytes of the living human cornea were examined to compare quantitatively spatial arrangement and cell volume of the stromal layers. This knowledge is required for further studies toward a quantitative understanding of cellular alterations in corneal pathology. METHODS: Three human corneas were stained with calcein AM and ethidium homodimer (Live/Dead Kit) directly after enucleation. The fluorescent cells were examined with confocal laser scanning fluorescence microscopy. High-resolution three-dimensional (3-D) volumes of < or =270 microm in the z-axis were reconstructed. Cell density and volume density were determined by computer-aided morphometry. RESULTS: Three keratocyte subpopulations were distinguished. Their spatial arrangement was visualized by 3 D reconstructions of the scanned volumes. Whereas cell density decreased progressively from the anterior (100%) to posterior (53.7%) stroma, volume density was highest in the posterior stroma (17.03 +/- 5.05%) and lowest in the central stroma (9.31 +/- 1.09%). In the anterior stroma, volume density was found to be 10.19 +/- 4.37%. CONCLUSION: Confocal laser scanning fluorescence microscopy allowed quantitative analysis and the visualization of the spatial arrangement of the keratocyte network in the living human corneal tissue for the first time. The results provide a basis for further studies of alterations of the normal cellular arrangements in corneal disease. PMID- 10746452 TI - CxGELSIX: a novel preparation of type VI collagen with possible use as a biomaterial. AB - PURPOSE: This study was initiated to evaluate tissue acceptance and stability of a novel type VI collagen preparation (CxGelsix) as a biomaterial in the rabbit corneal stroma. We hypothesized that CxGelsix, embedded intrastromally, does not have any adverse affect on surrounding corneal tissues, and remains intact in the presence of an acute inflammatory reaction during corneal wound healing. METHODS: Type VI collagen was extracted and purified from rabbit corneal stroma under nondenaturing conditions. This preparation, Gelsix, was concentrated and cross linked with polyethylene glycol to produce a transparent film (CxGelsix). Discs of CxGelsix, 4.0-mm diameter, 9- to 35-microm thick were implanted intrastromally and clinically examined periodically for 4 months. In another experiment, implantation of CxGelsix, 2.0-mm-diameter, was followed by corneal wounding adjacent to the implant and examined clinically for 30 weeks. At the end of these periods, the tissues from these experiments were processed for light and transmission electron microscopy. RESULTS: An intralamellar 4.0-mm-diameter disc of CxGelsix does not alter the structure of corneal epithelium above the implant, suggesting normal transport of nutrients through CxGelsix. Moreover, no structural abnormalities were seen in the rest of the cornea, and the cornea remains transparent. Although the cornea accepts the presence of CxGelsix disc as judged by clinical criteria, gradual degradation of the implant is seen ultrastructurally. CxGelsix is remarkably stable despite its exposure to endogenous enzymes during inflammation and wound healing. Partial degradation of the implant occurs only after many months, and it is gradually replaced with bundles of fine collagen fibrils reminiscent of normal cornea. CONCLUSION: The results of this study suggest that CxGelsix is potentially useful as a biomaterial. PMID- 10746453 TI - Effect of fortified antibiotic solutions on corneal epithelial wound healing. AB - PURPOSE: To evaluate the influence of fortified antibiotic eyedrops on corneal epithelial wound healing. METHODS: We developed an in vitro epithelial wound healing model to evaluate the toxicity of antibiotics. An excimer laser was used to create an epithelial defect 1.5 mm in diameter, 70 microm in depth on the central area of porcine cornea. The intact animal globes were maintained in the incubator by a perfusion system. Fortified antibiotics: 10% piperacillin, 5% cefazolin, 0.5% chloramphenicol, 5% vancomycin, 1% amikacin, 2% gentamicin, and 0.1% amphotericin B were applied to the wound in three applications. The wounds were evaluated 24 h after setup with fluorescein stain and a scoring system. RESULTS: The 0.1% amphotericin B and 2% gentamicin disturbed the corneal epithelial healing rate significantly. The remaining antibiotics did not interfere with the epithelial healing rate in our study design. CONCLUSION: Fortified antibiotic eyedrops demonstrated varied degrees of influence on corneal epithelial wound healing. When antibiotic eyedrops are used, both the efficacy and toxicity of the antibiotics should be the major concern. If efficacy is equivalent, less-toxic agents should be given preference. PMID- 10746454 TI - Lactoferrin protects against UV-B irradiation-induced corneal epithelial damage in rats. AB - PURPOSE: Lactoferrin supplementation suppresses ultraviolet light B (UV-B) induced oxidation of cultures of human corneal epithelial cells. To investigate the protective effect of lactoferrin containing eyedrops against UV-B-induced corneal damage in vivo, we examined lactoferrin efficacy in a rat UV-B keratitis model. METHODS: Sprague-Dawley rats were irradiated with >10 kJ/m2 after anesthetization, and then corneal epithelial defect was observed at 24 h postirradiation. The pre- or postapplication of vehicle or lactoferrin-containing eyedrops was performed, and then corneal epithelial damage was scored based on fluorescein staining. RESULTS: Posttreatment with lactoferrin did not inhibit the extent of corneal damage and did not affect wound healing. However, pretreatment by topical application of lactoferrin suppressed development of a corneal epithelial defect induced by UV-B irradiation in rats. CONCLUSION: These results suggest that the presence of lactoferrin in human tear fluid may inhibit UV induced corneal epithelial damage. PMID- 10746455 TI - Recombinant kringle 1-3 of plasminogen inhibits rabbit corneal angiogenesis induced by angiogenin. AB - PURPOSE: Angiostatin is a potent angiogenesis inhibitor that has been identified as a cryptic fragment of plasminogen molecule containing the first four kringle domain. Angiogenin, a 14-kDa monomeric protein, a potent blood vessel inducer, is expressed in tumors and present in mammalian plasma. The purpose of this study was to determine whether recombinant kringle 1-3 (rKI-3) of human plasminogen could interfere with angiogenesis induced by angiogenin and to evaluate the role of angiogenin in corneal angiogenesis in rabbit. METHODS: A Hydron polymer pellet containing 2.0 microg of angiogenin was implanted intrastromally into the superior cornea of each of 44 rabbit eyes. All eyes received an intrastromal pellet and were randomized into either one group treated with 12.5 microg of rKI 3 (n = 25) or the other group treated with phosphate-buffered saline (PBS; n = 19). Both pellets were positioned in parallel at the site 1.2 mm from the superior limbus. Two masked observers kept the angiogenesis score daily, based on the number and the length of new vessels. The corneas with induced angiogenesis also were examined histologically. RESULTS: On the third day of the angiogenin pellets implantation, the eye treated with rKI-3 had less angiogenesis (mean score, 4.2 +/- 6.6) than eye treated with PBS (mean score, 16.1 +/- 17.1; p < 0.05, Mann-Whitney U test). The cornea treated with PBS also showed much more leukocyte adhesion than the cornea treated with rKI-3. CONCLUSION: Recombinant kringle 1-3 appears to inhibit angiogenin-induced angiogenesis in a rabbit corneal pocket assay. Recombinant kringle 1-3 may have therapeutic potential as an antiangiogenic agent. PMID- 10746457 TI - The corneal endothelium in the blowfish (Torquigener pleurogramma). AB - PURPOSE: In vertebrates, a corneal endothelium is essential for the maintenance of corneal transparency in a variety of environments. Knowledge of the surface structure of the corneal endothelium may assist our understanding of this unique tissue and its evolutionary development. Although there have been many studies of the corneal endothelium of humans and some mammals, there have been few in other vertebrates. METHODS: The field emission scanning electron microscope was used to study the surface structure of the corneal endothelium in the blowfish, Torquigener pleurogramma (Tetraodontidae, Teleostei), and to examine cell density. Cell areas were measured by using image-analysis software. RESULTS: The endothelium is composed of a sheet of interdigitating hexagonal and pentagonal cells with a mean area of 154 microm2 and a density of 6,486 cells/mm2. Two types of surface features are identified; primary cilia and microvilli. The cilia are cylindrical, protrude from either a pore or circular indentation in the cell center, and possess a knob-like ending. The microvilli are button-like protrusions with a density of -3.5 x 105 microvilli/mm2 or 54 microvilli/cell in central cornea. CONCLUSION: The results show that the surface structure of teleost endothelial cells is similar to those described for other vertebrates and indicate that cell density varies across classes, with the presence of cilia a more widespread occurrence than previously believed. PMID- 10746456 TI - A comparative SEM study of the vertebrate corneal epithelium. AB - PURPOSE: The anterior surface of the cornea of mammals, including humans, has numerous folds in the anterior epithelial cell membranes in the form of microvilli and microplicae. The role of these surface irregularities may be to increase cell-surface area and therefore aid in intra- and extracellular movement of nutritional and waste products across the cell membranes in addition to stabilizing the corneal tear film. The aim of this study was to investigate and compare the nature of these corneal-surface features in various vertebrate classes residing in different environments. METHODS: The anterior corneal surfaces of various vertebrates were investigated by using field emission scanning electron microscopy. Cell areas were analyzed by using image-analysis software. RESULTS: Representative species were examined from all the vertebrate classes, with the exception of the Cephalaspidomorphi. The mean epithelial cell density of aquatic vertebrates (17,602 +/- 9,604 cells/mm2) is greater (p = 0.000018) than that of aerial and terrestrial vertebrate species, including amphibians (3,755 +/- 2,067 cells/ mm2). Similarly, the mean epithelial cell density for the marine vertebrates (22,553 +/- 8,878 cells/mm2) is greater (p = 0.0015) than that of the freshwater and estuarine species (10,529 +/- 5,341 cells/mm2). The anterior corneal surfaces of all species examined were found to show a variety of cell-surface structures. Microvilli are predominant in reptiles, birds, and mammals; microridges appear to be characteristic of the Osteichthyes; and microholes were observed only in the Chondrichthyes. CONCLUSION: The function of these morphologic variations in surface structure appear to be correlated with the range of ecologic environments (marine, aerial, and terrestrial) occupied by each species, corneal phylogeny, and the demands placed on the cornea to ensure clear vision. PMID- 10746458 TI - Fungal keratitis after laser in situ keratomileusis: a case report. AB - PURPOSE: To report a case of fungal keratitis resulting after laser in situ keratomileusis (LASIK). METHODS: A 38-year-old white man in good health developed a corneal infiltrate with laboratory confirmation of fungal keratitis after LASIK. Corneal scrapings were taken. Silver stain was positive for hyphae. Culture was positive for Curvularia sp. The patient was started on intensive natamycin 5% and amphotericin 0.15% topical therapy. RESULTS: The patient's keratitis was successfully treated with intensive antifungal therapy. CONCLUSIONS: Infectious keratitis is a rare but a serious potential complication after LASIK. To our knowledge, no previous case of fungal keratitis after LASIK has been reported. This case emphasizes the importance of surveillance for infection after LASIK. PMID- 10746459 TI - Persistent corneal opacity after oral isotretinoin therapy for acne. PMID- 10746460 TI - Spontaneous corneal graft ulcerative perforation due to mixed Acanthamoeba and herpes simplex keratitis: a clinicopathologic study. PMID- 10746461 TI - Delayed onset and recurrent Alcaligenes xylosoxidans keratitis. PMID- 10746462 TI - Necrotizing nocardial scleritis with intraocular extension: a case report. PMID- 10746463 TI - Ultrasound biomicroscopy of an intracorneal epithelial cyst. PMID- 10746464 TI - Pupillary block glaucoma after tissue adhesive application and anterior chamber reformation with air. PMID- 10746465 TI - Medicine's secrets. PMID- 10746466 TI - Orthopedic surgeons build so children can play safely. PMID- 10746467 TI - Anatomy and physiology of peripheral nerve injury and repair. AB - The management of peripheral nerve injury continues to be a major clinical challenge. Despite advancements in microsurgical technique, results after nerve repair have been unpredictable and dis appointing. The management of these nerve injuries relies on having a thorough understanding of peripheral nerve anatomy. This is the basis of the classification schemes by Seddon and Sunderland, in which the prognosis of nerve injuries varies depending on the degree of injury to their substructures. The most recent advances in the management of peripheral nerve injuries rely on the ability to manipulate the pathophysiologic processes triggered by nerve injuries and regeneration. End-to-end primary repair should be sought whenever a tension-free repair can be attained. If there is a significant nerve gap, use of nerve autograft remains the gold standard. In nerve injuries where a nerve autograft is not possible, the use of nerve allograft, as well as autogenous, biodegradable, and synthetic nerve conduits has shown promising results in experimental studies. PMID- 10746468 TI - Disability outcomes in a worker's compensation population: surgical versus nonsurgical treatment of carpal tunnel syndrome. AB - A retrospective record review of patients with occupational carpal tunnel syndrome, nerve conduction velocity studies, and a closed Workers' Compensation case was undertaken to compare the outcome of surgical versus nonsurgical treatment with respect to disability and return to work status. Between January 1, 1991, and December 31, 1993, 182 patients who met the inclusion criteria were identified. Surgical release of the carpal tunnel was performed in 57% of patients and the other 43% were treated conservatively. Overall, 82% of patients returned to full work status, whereas 18% had duty modifications. Surgical treatment decreased the rate of duty modifications and disability ratings compared with nonsurgical treatment and reduced the odds of incurring disability. Severity of carpal tunnel syndrome was also a significant factor affecting disability. Despite the generally held belief that the outcome of treatment of occupational carpal tunnel syndrome is poor, the present study shows that both surgical and nonsurgical treatment is effective. However, patients treated with surgery had decreased disability when compared with those who were treated conservatively. PMID- 10746469 TI - Core decompression and conservative treatment for avascular necrosis of the femoral head: a meta-analysis. AB - Core decompression for avascular necrosis (AVN) of the femoral head continues to be a controversial procedure. Meta-analysis techniques were employed to identify 22 studies with a single surgical core decompression technique. A similar procedure identified eight studies that treated patients conservatively. The success rates for core decompression were 84%, 63%, and 29% for Steinberg stages I, II, and III, respectively. Conservatively treated patients with stage 0, I, III, and III AVN demonstrated success rates of 86%, 61%, 59%, and 25%, respectively. Chi-square analysis showed the success rate of core decompression to be statistically higher than conservative treatment for stage I hips only. Large multicenter prospective double-blinded studies with patients randomized to either core decompression or conservative treatment, then stratified by stage, cause, and bilaterality, are needed to determine the best treatment for early stage AVN. PMID- 10746470 TI - Cervical venous structure in the inter-transverse and intra-transverse foraminal region: an anatomic study. AB - Vertebral venous bleeding is frequently encountered during anterolateral cervical decompression. The present study was undertaken to identify the pattern and location of the vertebral vein in relationship to the vertebral artery in the inter-transverse and intra-transverse foraminal regions. Twenty-one cadavers were dissected to determine the anatomic features of the vertebral vein in the inter transverse and intra-transverse foramina. The vertebral veins in the inter foraminal and intraforaminal regions from C-3 to C-6 can be classified into three types: single or double veins, venous plexus, and absence of the vein. Of the 21 specimens, vertebral veins were found bilaterally in five specimens (24%) and unilaterally in eight specimens (38%). The veins were situated either anterolateral or anteromedial to the vertebral artery. The venous structures showed in venous plexus in two specimens (9.5%). In six specimens (29%), there were no obvious venous structures related to the vertebral artery in its intra transverse and inter-transverse foraminal course. The veins are contained in a fibrous and osseous tunnel as they descend through the transverse foramina. Subperiosteal dissection of fibrous tissue from the lateral aspect of the uncinate process after removal of the anterior wall of the transverse foramen may minimize hemorrhages from the vertebral vein in the inter-transverse and intra transverse foraminal region during resection of the uncovertebraljoint or neural foraminotomy with retraction of the vertebral artery laterally. PMID- 10746471 TI - A simple effective splinting technique for the mallet finger. AB - We describe an inexpensive, simple, and effective technique for the closed treatment of mallet fingers. This technique splints the mallet finger in 0 degrees of extension. It is simple, readily reproducible, and easy to use, requiring materials that are readily available to any physician. Thirty-seven patients, ages 21 to 65 years, presented with a closed mallet finger injury at day zero to 5 months after injury. Ten of these patients had a fracture. All were treated with this splinting technique. We report excellent results in 35 of 37 patients, who recovered either complete or near-complete active extension. Our results compare favorably with results from other techniques and splints. PMID- 10746472 TI - Isolated skeletal metastasis to the patella. AB - Metastases to the patella are rarely described in the literature. A 51-year-old man with stage IIIB adenocarcinoma presented to our clinic. One year after radiation and chemotherapy treatment, it was found that he had a metastasis in the right patella. Six months after incisional biopsy with curettage of the patellar lesion and placement of methylmethacrylate bone cement into the remaining patellar defect, he has no pain and is ambulatory. This case has been presented as a basis for palliative surgery in a patient with a rare metastatic tumor of the patella. PMID- 10746473 TI - Nutritional rickets: report of four cases diagnosed at orthopaedic evaluation. AB - Nutritional rickets, the classic form of vitamin D-deficiency rickets, is rarely seen in the setting of modern medicine. We report four cases of nutritional rickets diagnosed at the time of orthopedic evaluation at our institutions during a 3-year period. All patients were referred by their pediatricians for evaluation of bowlegs. All children lived in a northern climate. Three were black and one was white. Prolonged breast-feeding and an unusual diet were identified as the underlying cause in our cases. Genu varum, seen in the setting of relative short stature and low weight, with a consistent social history, should raise the suspicion for nutritional rickets. The continued prevalence of this disease entity must be recognized in both the primary care and the orthopedic surgery setting. Vitamin D supplementation and enhanced sunlight exposure serve as effective prophylaxis and treatment of this disease. PMID- 10746474 TI - Pyogenic arthritis of a lumbar facet joint. AB - We herein report the case of a 68-year-old man with diabetes who developed pyogenic arthritis of a lumbar facet joint after spinal injection. We performed magnetic resonance imaging (MRI), computed tomography (CT), technetium 99 methylene diphosphonate scintigraphy, and single photon emission computed tomography (SPECT) for this patient. MRI showed a lesion in the facet joint and no evidence of spondylodiscitis. CT showed a swelling of periarticular soft tissue around the facet joint. Bone scintigraphy showed a characteristic vertical uptake. In particular, SPECT was able to clearly confirm the location of the infection. An infection of the facet joint has only been rarely reported, but we recommended that this area should be carefully evaluated whenever a patient develops an infection of the lumbar spine after a spinal injection. PMID- 10746475 TI - Triple central ray amputation for clear cell sarcoma of the hand. AB - A 16-year-old girl presented for evaluation and management of a recurrent soft tissue mass in her left hand after excision at another facility. She was given a diagnosis of clear cell sarcoma. She chose a limb salvage procedure over amputation. A triple central ray amputation of her index, middle, and ring fingers was performed. After 3 years, she has reasonable grasp function with her remaining fingers and has chosen not to use a prosthesis. Long-term follow-up is necessary for this tumor, as local recurrence and metastatic disease have been reported frequently. PMID- 10746476 TI - Traumatic dislocation of trapezium: case report and review of the literature. AB - A complete dislocation of the trapezium from its fossa is rare. We present a case of a 36-year-old man with a complete ulnar and volar dislocation of the trapezium. The trapezium was reduced with manual manipulation and was held in anatomic position by using multiple Kirschner wires. The patient's clinical course is reviewed and similar cases from the literature are discussed. PMID- 10746477 TI - Avascular necrosis of the hip. PMID- 10746478 TI - Ions, genes and insulin release: from basic science to clinical disease. Based on the 1998 R. D. Lawrence Lecture. AB - In 1968, reports of the first microelectrode recordings of insulin-secreting cells were published. Thirty years later it is now established that electrical responses of beta-cells play a critical role in stimulus-secretion coupling. It is now also clear that defects in ion channel genes compromise the mechanisms which govern secretion and lead to the onset of disease. Here, the physiology of insulin release is reviewed in the context of ion channels, the ionic control of insulin release and the pathophysiology of hyperinsulinism of infancy. PMID- 10746479 TI - 'Numbness of the feet' is a poor indicator for polyneuropathy in Type 2 diabetic patients. AB - AIMS: To identify neuropathic sensory symptoms associated with a clinical neurological examination (CNE) and to investigate whether these symptoms could be used as a diagnostic or screening tool for diabetic polyneuropathy in general practice. METHODS: Five hundred and eighty-eight patients with Type 2 diabetes, recruited from 26 general practices in the Netherlands, underwent a CNE and completed a diabetes symptom checklist that included 10 items on neuropathic sensory symptoms. Linear regression analyses were performed to assess the association between neuropathic symptoms and CNE. Receiver operating characteristic (ROC) curves were created to assess the diagnostic properties of neuropathic symptoms. RESULTS: In this population, with a mean age of 66.8 years, 32% were identified with diabetic polyneuropathy according to the CNE. Variables that showed the strongest association with CNE score were age (beta = 0.41), symptoms of sensory alteration (beta = 0.27), and the item 'numbness of the feet' (beta = 0.35) in particular. ROC curves showed that prediction of diabetic polyneuropathy from these symptoms was unsatisfying. The sensitivity and specificity of daily symptoms of 'numbness of the feet' were 28% and 93%, respectively, in patients <68 years, and 22% and 92%, respectively, in patients > or =68 years. CONCLUSIONS: Identification of neuropathic sensory symptoms is not useful as a diagnostic or even a screening tool in the assessment of diabetic neuropathy in daily practice. Therefore, the results reported in this paper mandate an annual foot examination by the general practitioner. PMID- 10746480 TI - Aldose reductase gene polymorphisms and susceptibility to diabetic nephropathy in Type 1 diabetes mellitus. AB - AIMS: To investigate association and linkage between DNA sequence variants in the aldose reductase (AR) gene on chromosome 7q35 and diabetic nephropathy (DN) in Type 1 diabetes mellitus. METHODS: By sequencing the promoter region and 10 exons in eight DN cases and eight controls, a frequent bi-allelic polymorphism (C-106T) was discovered. This polymorphism and the known 5'ALR2 dinucleotide repeat polymorphism were genotyped in unrelated cases with advanced nephropathy (n = 221) and unrelated controls with normoalbuminuria (n = 193). For a family based study, 166 case-trios (case and both parents) and 83 control-trios (control and both parents) were also genotyped. RESULTS: In the case-control study, carriers of the Z-2 allele of the 5'ALR2 polymorphism had a significantly higher risk of DN than non-carriers (odds ratios: 1.6 for heterozygotes and 2.1 for homozygotes, P<0.05 for each). The same was true for carriers of the T allele of the C-106T polymorphism (odds ratios: 1.6 for heterozygotes and 1.9 for homozygotes, P<0.05 for each). Moreover, the haplotype carrying both risk alleles was in excess in DN cases. In the family study, transmission of risk alleles from heterozygous parents was consistent with the case-control study, excess transmission in case trios and deficient in control-trios. CONCLUSIONS: Association between DN and two DNA sequence variants in the promoter region of the AR gene implicates the polyol pathway in the development of kidney complications in Type 1 diabetes mellitus. Further examination of the molecular mechanisms underlying these findings may provide insight into the pathogenesis of DN. PMID- 10746481 TI - Comparison of questionnaire, breath carbon monoxide and urine cotinine in assessing the smoking habits of Type 2 diabetic patients. AB - AIMS: Previous studies have suggested that the self-reporting of smoking amongst diabetic patients is unreliable, and that objective markers such as breath carbon monoxide (CO) or urinary cotinine are needed. As these studies have been mostly in young Type 1 diabetic patients, the reliability of smoking history has been assessed in a large group of older Type 2 diabetic patients. METHODS: Two hundred and ninety-nine Type 2 diabetic patients were investigated for smoking habits by unstructured questionnaire, breath CO, and urinary ratio. RESULTS: Mean age of the group was 63 (range 30-80) years and 41% were female. Direct questioning suggested 77 (26%) to be smokers, and of these 852 had raised (>1 microg/mg) urinary cotinine-creatinine , and 71% raised (>8 p.p.m.) breath CO. Assuming urinary cotinine-creatinine as the 'gold standard', the sensitivity, specificity and positive predictive values for questionnaire were 97%, 95% and 85%, respectively; and for breath CO they were 74%, 98%, and 93%. CONCLUSION: It is concluded that at least in this population of Type 2 diabetic patients, a structured smoking history appeared to identify current smokers with good accuracy. PMID- 10746482 TI - Correlations between insulin sensitivity and bone mineral density in non-diabetic men. AB - AIMS: To investigate relationships between bone mineral density (BMD), insulin secretion and insulin sensitivity, controlling for body composition, in view of data suggesting that hyperglycaemia [corrected] leads to decreased osteoblast proliferation and a negative calcium balance and that insulin stimulates osteoblast differentiation and collagen synthesis, with no clear evidence if this response in impaired in insulin resistance. METHODS: Femur and whole body (WB) BMD was measured in 55 male patients with ischaemic heart disease and 40 healthy men, using a Hologic QDR-2000 densitometer. Insulin sensitivity (Si) was estimated as the rate of glucose disappearance divided by the area under the insulin curve during an intravenous glucose tolerance test. RESULTS: Insulin and C-peptide levels were not correlated with BMD, but Si was a significant predictor of femur (log, r = 0.35) and WB BMD (log r = 0.29, both P<0.01), even after controlling for weight and age. Fat mass (FM) was a predictor of BMD (femur: r = 0.33 P<0.01, WB: r = 0.43 P<0.001). In the femur the association with FM disappeared when log(Si) was entered in the regression. Lean body mass (LBM) contributed significantly to BMD (r = 0.50 and r = 0.66, both P<0.001). CONCLUSIONS: These results are compatible with a direct influence of lean body mass on bone, while the impact of fat mass may consist of insulin resistance with increased insulin exposure of bone. It is hypothesized that patients with insulin resistance in the metabolic pathway do not exhibit resistance to the skeletal actions of insulin. PMID- 10746483 TI - Microcirculatory response of skin to benzoic acid and methyl nicotinate in patients with diabetes. AB - AIMS: In view of the relationship between microvascular pathology and organ complications in diabetes mellitus, the aim of the present study was to examine the microvascular response of upper arm skin to non-immunological contact irritants in 17 insulin-dependent diabetic patients and 11 non-diabetic controls. METHODS: Non-immunological contact urticaria, an inflammatory reaction mediated in a unique way, not previously studied in diabetic patients, was examined. The test agents were benzoic acid and methyl nicotinate. The intensity of the reactions was measured using laser-Doppler flowmetry and colorimetry. The patients were divided into two groups, depending on whether they had had diabetes for less or more than 10 years. RESULTS: There were no differences in the maximal blood flow responses between the groups, but the diabetic patients showed increased blood flow responses to the lowest irritant concentrations compared to the controls. The reactions in the two groups of diabetic patients were similar. CONCLUSIONS: The present study suggests that the microvascular reactivity of diabetic skin to non-immunological contact irritants is increased. PMID- 10746484 TI - Insulin sensitivity in hypertensive Type 2 diabetic patients after 1 and 19 days' treatment with trandolapril. AB - AIMS: The aims of this study were to examine the effects of trandolapril, a long acting angiotensin converting enzyme (ACE) inhibitor with high tissue uptake, on insulin sensitivity and lipid concentrations in hypertensive patients with Type 2 diabetes mellitus. METHODS: Insulin sensitivity was assessed after an acute dose (day 3) and 19 days continuous treatment (days 3-21) using the isoglycaemic, hyperinsulinaemic glucose clamp with D[3-3H] labelled glucose, a variable D[3-3H] priming dose and a 'hot' glucose infusion. Rates of glucose appearance (Ra) and glucose disappearance (Rd) were isotopically determined during the basal and insulin stimulated periods of the clamp. Twenty-four (5 female) hypertensive (blood pressure >75th centile for age and sex) patients with Type 2 diabetes mellitus were studied. Patients were randomized, in a double-blind manner, to either trandolapril 4 mg daily (T) or placebo (P). RESULTS: Baseline (day 1) systolic (mean +/- SD; P 164+/-14 and T 168+/-13 mm Hg) and diastolic (P 93+/-6, and T 98+/-10 mm Hg) blood pressures were comparable. On days 3 and 21, significant reductions were observed in both groups (P<0.001). In the trandolapril-treated group, serum trandolapril concentrations were >200 pg/ml on days 3 and 21, in all patients apart from one subject at a single visit, while trandolapril was undetectable in the placebo group. Body mass index (BMI) was greater in T compared with P (32.2+/-5.4 v. 28.3+/-4.6, P = 0.07). After correcting for BMI, basal hepatic glucose output (HGO) P 2.6 (95% CI 2.23-3.13) and T 1.91 (1.33-2.51) mg x kg(-1) x min(-1) and clamped HGO P 0.32 (-0.44-1.09) and T 0.87 (0.40-1.34) mg x kg(-1) x min(-1) were similar in both groups. The insulin sensitivity index was comparable in both groups on all days. Total cholesterol concentrations were similar in both groups throughout the study. Triglyceride concentrations were significantly lower in group P 1.38 (1.07-1.68); T 2.14 (1.70-2.58) mmol/l, P<0.01), no significant treatment effect being observed. CONCLUSIONS: An acute dose and 19 days' continuous treatment with trandolapril resulted in no change in insulin sensitivity or plasma lipid profiles in patients with Type 2 diabetes mellitus and hypertension. These data support the metabolic neutrality of trandolapril in patients with Type 2 diabetes mellitus and hypertension. PMID- 10746485 TI - Estimating the prevalence of diagnosed diabetes in a health district of Wales: the importance of using primary and secondary care sources of ascertainment with adjustment for death and migration. AB - AIMS: To determine diagnosed diabetic prevalence within our district (population 434398) in 1996 using data from two sources. METHODS: A general practice audit comprising data on patients with diabetes from 61 (82%) of 74 general practices was linked to a record linkage-derived patient index in which data from secondary care and other sources underwent a process of probability matching to identify records relating to the same patient and to flag those with diabetes. By linking this dataset to a mortality dataset, patients known to have died before 1996 could be excluded. Age and sex-stratified emigration rates were applied to those identified by the hospital dataset for each year from 1991 onwards. RESULTS: A total of 386988 residents (89.1%) were listed with a general practitioner participating in the audit, of whom 6050 patients were identified as having diabetes in 1996; a prevalence rate of 1.56%. From the hospital-based source, 7639 patients were identified who were alive in 1996, a period prevalence of 1.76%. By combining the two sources, and extrapolating the general practice audit to the population as a whole, a total of 10 530 patients were identified of whom 8735 were confirmed as still resident within South Glamorgan during 1996. This represented a period prevalence of between 2.01% to 2.42%. By applying age and sex-stratified migration rates to the diabetic population identified by hospital sources, a diagnosed diabetic population of 10,004 was identified, a prevalence of 2.3%. CONCLUSIONS: This study demonstrates that to calculate the true prevalence of diagnosed diabetes from health sources, it is necessary to use both primary and secondary care sources. PMID- 10746486 TI - The prevalence of multiple diabetes-related complications. AB - AIMS: To determine the prevalence of the complications of diabetes and the interrelationship between them within a United Kingdom district health authority population. METHODS: Data extracted from a general practice diabetes audit were combined with data for patients with diabetes derived from a patient index constructed using record linkage techniques. RESULTS: A total of 10709 patients were identified as having diabetes (prevalence 2.47%). Coronary heart disease was present in 25.2%, cerebrovascular disease in 9.6%, complications of the 'diabetic foot' in 18.1%, retinopathy in 16.5% and nephropathy in 2.0%. Over a half of the patients (52.1%) had none of the studied complications, 30.2% had one, 12.7% had two, 4.1% had three, 0.8% had four and 0.1% had all five. All complications were related to both age and duration of diabetes but duration was particularly apparent for the microvascular complications (retinopathy and nephropathy). Macrovascular complications in the Type 2 diabetic population appear advanced in onset compared with Type 1. CONCLUSIONS: Multiple complications are apparent in almost one fifth of patients with diabetes. Macrovascular morbidity in Type 2 diabetes of early onset indicates that a targeted approach to treatment may prove most beneficial in both patient and health service terms. PMID- 10746487 TI - Insulin resistance alone does not explain the clustering of cardiovascular risk factors in southern India. AB - AIMS: To determine whether insulin resistance (IR calculated using the HOMA model) has a dominant role in the clustering of cardiovascular risk factors in the Asian Indian population. METHODS: A total of 654 non-diabetic subjects aged > or =40 years (male 396: female 258) were selected from a population survey. They had estimates of fasting and 2 h plasma glucose, insulin levels, body mass index (BMI), waist-to-hip ratio (WHR) and blood pressure. Factor analysis was carried out using the principle components analysis (PCA) with varimax orthogonal rotation of continuously distributed variables, considered to represent the components of insulin resistance syndrome including the calculated IR. RESULTS: There were three major clusters of cardiovascular disease (CVD) risk variables in men and four clusters in women. Insulin resistance, 2 h plasma glucose, insulin and obesity aggregated as the major domain. Insulin resistance was not linked with hypertension. BMI was a common link for all the three factors in men, and for three of the four in women. CONCLUSIONS: Insulin resistance is not the only underlying factor for the clustering of CVD risk factors in south Indians. These findings are consistent with the presence of several distinct physiological domains, as shown in other ethnic groups. PMID- 10746488 TI - No association between birth weight and Type 1 diabetes mellitus--a twin-control study. AB - AIMS: To investigate, by means of a twin-control study, whether there is a relationship between birth weight and Type 1 diabetes mellitus. METHODS: In the youngest part (n = 20 888 pairs) of the Danish Twin Register, 95 twin pairs (26 monozygotic and 69 dizygotic) with Type 1 diabetes were identified. Information about birth weight and birth length, prematurity, maternal age and parity was obtained from midwives' records and from the Danish Medical Birth Registry and analysed using Student's two-sided t-test, Chi-square test and logistic regression. RESULTS: Birth data were available for 83 diabetic and 51 healthy twin individuals, altogether 67 twin pairs (20 monozygotic and 47 dizygotic). Mean birth weight was 2537 g and 2565 g (NS) in monozygotic and dizygotic pairs and the corresponding length at birth 47.6 cm and 48.2 cm (NS). In pairs discordant for diabetes, the mean birth weight was 2538 g in twins with diabetes and 2549 g in twins without diabetes (NS). Logistic regression in discordant pairs showed no relationship between diabetes status and birth weight, birth length, birth order, sex or prematurity. Logistic regression in concordant vs. discordant pairs showed no effect of mean birth weight and birth length, maternal age, parity or weeks of prematurity. CONCLUSIONS: There is no association between Type 1 diabetes mellitus and birth weight in this study. Furthermore, variables related to birth weight and length cannot explain why some pairs are concordant while other remain discordant. PMID- 10746489 TI - Clinico-pathological features of postural hypotension in diabetic autonomic neuropathy. AB - We report the clinico-pathological features and management of a 49-year-old male with a 30-year history of Type 1 diabetes mellitus who had nephropathy (proteinuria 1.81 g/24 h, creatinine 136 micromol/l), proliferative retinopathy and severe somatic and autonomic neuropathy. A sural nerve biopsy demonstrated marked myelinated fibre loss with unmyelinated fibre degeneration and regeneration combined with extensive endoneurial microangiopathy. The management of the patient's blood pressure problems (supine hypertension) and symptomatic postural hypotension is discussed. PMID- 10746490 TI - When do environmental factors initiate islet cell damage in children with Type 1 diabetes mellitus? PMID- 10746491 TI - Diabetic foot ulcer management: a perspective of primary and secondary care. PMID- 10746492 TI - Bordetella pertussis infection: pathogenesis, diagnosis, management, and the role of protective immunity. AB - Whooping cough is presently one of the ten most common causes of death from infectious disease worldwide. Despite a high vaccine uptake, resurgences of this disease have been observed in several countries. Virulence factors of Bordetella pertussis include agglutinogens, fimbriae, P.69/pertactin, pertussis toxin, filamentous haemagglutinin, adenylate cyclase, tracheal cytotoxin, dermonecrotic toxin, lipopolysaccharide, tracheal colonisation factor, serum resistance factor, and type III secretion. Virulence factor expression is regulated by the bvgAS locus, a two-component signal transduction system. The pathophysiologic sequence consists of attachment (fimbriae, P.69/pertactin, tracheal colonisation factor, pertussis toxin, filamentous haemagglutinin), evasion of host defence (adenylate cyclase, pertussis toxin, serum resistance factor), local effects (tracheal cytotoxin), and systemic effects (pertussis toxin). Bordetella pertussis is transmitted by respiratory droplets and causes disease only in humans. Various diagnostic methods are available, including culture, serological methods, and the polymerase chain reaction. Serotyping of isolates to detect agglutinogens 2 and 3 is useful because serotype 1,2 may be associated with higher mortality, and antibodies to these antigens (agglutinins) may be protective in both animals and humans. Immunisation using whole-cell vaccine is effective but is reactogenic. Acellular vaccines containing one to five components are being used increasingly in various countries. Protective immunity to pertussis correlates with high levels of antibody to each of pertactin, fimbriae, and pertussis toxin; however, doubt remains as to the relationship between agglutinogen 3 and fimbria 3, making results of trials investigating these virulence factors difficult to interpret. PMID- 10746493 TI - Thrice-weekly sulfadiazine-pyrimethamine for maintenance therapy of toxoplasmic encephalitis in HIV-infected patients. Spanish Toxoplasmosis Study Group. AB - An open, randomised, multicentre trial was conducted to evaluate the efficacy of thrice-weekly versus daily therapy with sulfadiazine-pyrimethamine in the prevention of relapses of toxoplasmic encephalitis in HIV-infected patients. Between February 1994 and July 1997, 124 patients with HIV infection were enrolled after resolution of the first acute episode of toxoplasmic encephalitis treated with sulfadiazine-pyrimethamine. Patients were randomly assigned to receive either a daily regimen consisting of sulfadiazine (1 g) twice a day plus 25 mg pyrimethamine and 15 mg folinic acid daily (n = 58), or a thrice-weekly regimen consisting of the same doses of sulfadiazine and folinic acid plus 50 mg pyrimethamine (n = 66). After a median follow-up period of 11 months (range 1-39 months), no differences were found in the incidence of toxoplasmic encephalitis relapses between the groups, there being 14.9 episodes per 100 patient-years (95% CI: 2.8-20.2) in the daily-regimen group versus 14.1 episodes (95% CI: 2.3-17.2) in the intermittent-regimen group. The estimated cumulative percentages of relapse at 12 months were 17% and 19%, respectively (P = 0.91). In a Cox multivariate analysis, not taking antiretroviral therapy was the only variable independently associated with relapse (adjusted risk ratio: 4.08; 95%CI: 1.32 12.66). Baseline CD4+ cell counts, prior AIDS, mental status, sequelae and allocated maintenance therapy regimen were not independent predictors of relapse. No differences were observed in the survival rate (P = 0.42), or in the incidence of severe adverse effects (P = 0.79). The efficacy of the thrice-weekly regimen was similar to that of the daily regimen in the prevention of relapses of toxoplasmic encephalitis. Administration of antiretroviral therapy was the only factor associated with a lower incidence of relapse. PMID- 10746494 TI - Risk factors for esophageal candidiasis. AB - The role of gastric acid inhibitors as predisposing factors for Candida esophagitis is unknown. A retrospective case-control study of esophageal candidiasis was conducted in human immunodeficiency virus (HIV)-negative patients diagnosed from January 1991 to December 1997. The diagnosis of esophageal candidiasis was always made on the basis of endoscopic and histological criteria. Fifty-one patients were diagnosed with esophageal candidiasis, 15 of whom had esophageal complaints and 48 of whom suffered from another previous chronic disease (17 had cancer). In addition, 20 patients had previously been treated with antibiotics, 13 with steroids and 14 with omeprazole. In the multivariate analysis, neoplasm (odds ratio, 5.50; 95% confidence interval, 1.94-15.56) and therapy with antibiotics (odds ratio, 11.97; 95% confidence interval, 3.82 37.45), steroids (odds ratio, 35.52; 95% confidence interval, 3.90-324.01) or omeprazole (odds ratio, 18.23; 95% confidence interval, 4.67-71.03) were all associated with esophageal candidiasis. These data suggest that Candida esophagitis tends to occur in patients with chronic diseases, most of whom have been previously treated with antibiotics, steroids or omeprazole. The findings support the hypothesis that treatment with omeprazole favors the development of esophageal candidiasis. PMID- 10746495 TI - Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Finland. AB - This study reports the recent trends in the occurrence of methicillin-resistant Staphylococcus aureus in Finland, with special focus on characterization of the strains linked to interhospital epidemics and local outbreaks. Between 1981 and 1997, the annual number of methicillin-resistant Staphylococcus aureus isolations ranged from 89 to 272. Of all blood isolates of Staphylococcus aureus reported to the National Infectious Disease Register during the period 1995-97 (n = 2049), only six were resistant to methicillin. Between 1992 and 1997, typing analysis by various methods (i.e., antibiogram, phage typing, ribotyping, and pulsed-field gel electrophoresis) identified 18 different strains capable of causing intrahospital outbreaks or interhospital epidemics. These 18 strains were separated into 13 different ribotypes and 14 major pulsed-field gel electrophoresis types. Multiresistance was investigated as a possible marker for epidemicity. Eight of the ten interhospitally spread strains were multiresistant compared to only three of the eight intrahospitally spread outbreak strains. More than one-third of the epidemic and local outbreak strains were suspected to be of foreign origin. The majority (6 of 10) of the epidemics were localized in southern and western Finland, and the largest epidemic, which occurred in the Helsinki metropolitan area, involved over 200 persons. Thus far, the epidemics have remained primarily intracity problems, and only two strains have become endemic. PMID- 10746496 TI - Antimicrobial resistance trends in Shigella serogroups isolated in Israel, 1990 1995. AB - From a total of 31319 Shigella strains isolated in Israel between 1990 and 1996, 17574 were sent to the National Shigella Reference Center for typing. Of these, 15287 were identified as Shigella sonnei, 1833 as Shigella flexneri, 327 as Shigella boydii and 127 as Shigella dysenteriae. In all, 4395 strains were tested for sensitivity to ampicillin, trimethoprim-sulfamethoxazole, tetracycline, chloramphenicol, nalidixic acid and ofloxacin. All strains tested were sensitive to ofloxacin, and only three strains were resistant to nalidixic acid. Only 113 of 3240 (3.5%) Shigella sonnei strains, 172 of 970 (17.7%) Shigella flexneri strains and 45 of 185 (24.3%) Shigella boydii strains tested were sensitive to four other antibiotic agents. The rates of resistance of Shigella sonnei, Shigella flexneri and Shigella boydii to trimethoprimsulfamethoxazole were 94.4%, 51.3% and 61.6%, respectively. Rates of resistance to ampicillin among these species were 73.4%, 63.5% and 21.4%, respectively. The proportion of strains exhibiting multiple drug resistance was higher for Shigella sonnei than for the other serotypes studied. These results emphasize the need to reassess the use of antibiotic agents in the treatment of shigellosis. PMID- 10746497 TI - Antibiotic susceptibility of bacterial strains isolated from patients with community-acquired urinary tract infections in France. Multicentre Study Group. AB - The aim of this study was to determine the distribution and antibiotic susceptibility patterns of bacterial strains isolated from adults with community acquired urinary tract infections (UTI) in France. From December 1996 to March 1997, each of 15 private laboratories in France consecutively collected about 80 non-duplicate strains isolated from adult outpatients with UTI, including patients receiving care at home, and tested their susceptibility by the disk diffusion test. A total of 1160 strains were collected: 1031 gram-negative bacilli, including Escherichia coli (n = 865), Proteus mirabilis (n = 68) and Klebsiella spp. (n = 40), and 129 gram-positive cocci, including Staphylococcus aureus (n = 16), other staphylococci (n = 25), group B streptococci (n = 25) and enterococci (n = 63). In the case of 430 bacterial isolates, the patients had either been hospitalised in the last 6 months or received antibiotic treatment in the last 3 months. The antibiotic susceptibility rates for Escherichia coli were: amoxicillin (58.7%), amoxicillin-clavulanic acid (63.3%), ticarcillin (61.4%), cephalothin (66.8%) cefuroxime (77.6%), cefixime (83.6%), cefotaxime (99.8%), ceftazidime (99%), nalidixic acid (91.9%), norfloxacin (96.6%), ofloxacin (96.3%), ciprofloxacin (98.3%), cotrimoxazole (78.2%), fosfomycin (99.1%) and gentamicin (98.4%). Of the Enterobacteriaceae, five strains produced an extended spectrum beta-lactamase. Methicillin resistance was detected in nine Staphylococcus aureus isolates. The most important findings were two extended spectrum, beta-lactamase-producing and three methicillin-resistant Staphylococcus aureus strains isolated from patients who had not been hospitalised in the last 6 months or taken antibiotics in the last 3 months. The findings indicate that these strains can spread within the community; therefore, monitoring antibiotic susceptibility of bacteria isolated in the community appears to be mandatory. PMID- 10746498 TI - Spondylodiscitis associated with bacteraemia due to coagulase-negative staphylococci. AB - Three cases are reported of spondylodiscitis caused by coagulase-negative staphylococci in patients without osteosynthetic material. All three patients had bacteraemia associated with an infected intravascular device left in place. On the basis of this observation, it is concluded that such devices should be removed promptly in cases of prolonged or relapsing bacteraemia. Furthermore, spondylodiscitis should be suspected in patients with back pain after bacteraemia caused by coagulase-negative staphylococci. PMID- 10746499 TI - Survival and growth of Burkholderia cepacia within the free-living amoeba Acanthamoeba polyphaga. AB - The aim of this study was to investigate whether Burkholderia cepacia is capable of survival and growth within the free-living amoeba Acanthamoeba polyphaga using a differential immunofluorescence assay of bacterial-amoebal cocultures and viable counts of bacteria determined after amoebal lysis. The numbers of intra amoebal bacteria and the numbers of infected amoebae increased over time; although, when heat-killed bacteria were used, no intracellular bacteria were observed. These findings should be taken into account in future studies of environmental reservoirs of Burkholderia cepacia. PMID- 10746500 TI - In vitro adherence of Enterococcus faecalis and Enterococcus faecium to urinary catheters. AB - The in vitro adherence of ten strains of Enterococcus faecalis and ten strains of Enterococcus faecium to siliconized latex urinary catheters and to silicone elastomer was evaluated. Bacterial suspensions (2.5x10(5) cfu/ml) in tryptic soy broth containing 0.5 cm segments from each type of catheter were incubated at 37 degrees C. At specified intervals, the segments were washed to remove nonadherent bacteria and sonicated for 1 min, and colony-forming units were quantified. Bacterial adherence occurred rapidly, reaching maximal peaks after 24 h of incubation. Enterococcus faecium adherence to both biomaterials was significantly lower than that of Enterococcus faecalis. No differences were observed between the two elastomers. Bacterial adherence was not related to bacterial surface hydrophobicity, hemolysin or gelatinase production. PMID- 10746501 TI - Comparison of three methods for identification of Streptococcus milleri group isolates to species level. AB - A collection of 180 clinical isolates of Streptococcus milleri group were identified to species level using two phenotypic methods (a commercial system and the reference method based on differential phenotypic reactions) and a genotypic method (hybridisation of the 16S rRNA gene with species-specific probes) in order to evaluate the performance of the respective methods. A high level of agreement (80%) was observed between the results of the reference method and the genotypic method. The highest level of agreement was found for the species Streptococcus anginosus (83%), a high level of agreement (76%) also being achieved for Streptococcus constellatus and Streptococcus intermedius. The sensitivity of the commercial system compared to the genotypic method was 76% overall, but it was low (57.5%) for Streptococcus intermedius. Twenty-five strains belonged to the recently described CI strains. PMID- 10746502 TI - Effect of administering short-course, standardized regimens in individuals infected with drug-resistant Mycobacterium tuberculosis strains. AB - Presented here are the cases of three siblings with multidrug-resistant tuberculosis who demonstrated increased antituberculous-drug resistance during the periods in which they received standard regimens of directly observed, short course chemotherapy that were administered before the susceptibility patterns of their Mycobacterium tuberculosis isolates had been checked. More specifically, they acquired resistance to drugs they received as part of ineffective standard treatment and retreatment regimens. Development of antituberculous-drug resistance through inadvertent, inadequate therapy appears to be the most likely explanation for the increased resistance seen in these three patients. PMID- 10746503 TI - In vitro susceptibility to gemifloxacin and trovafloxacin of Streptococcus pneumoniae strains exhibiting decreased susceptibility to ciprofloxacin. AB - The in vitro susceptibility to trovafloxacin and gemifloxacin of Streptococcus pneumoniae strains exhibiting decreased susceptibility to ciprofloxacin (MIC > or =2 microg/ml; 30 strains with intermediate resistance [MIC 2 microg/ml] and 43 strains with complete resistance [MIC > or =4 microg/ml]) was determined. Seventy three strains collected in a surveillance study carried out from May 1996 to April 1997 in Spain (prior to commercialisation of trovafloxacin and gemifloxacin) from patients with respiratory tract infections were tested. The antibacterial activity of gemifloxacin was affected to a lesser extent than that of trovafloxacin by the increase in the MIC of ciprofloxacin, with gemifloxacin showing significantly (P< or =0.001) better antibacterial activity than trovafloxacin in all ciprofloxacin MIC categories (MIC50/MIC90 values of 0.015/0.03, 0.015/0.06, 0.03/0.06 and 0.12/0.25 microg/ml for gemifloxacin vs. 0.12/0.12, 0.12/1, 0.25/0.5 and 2/4 microg/ml for trovafloxacin in the 2, 4, 8 and > or =16 microg/ml ciprofloxacin MIC categories, respectively). Nine (12.3%) of these 73 strains exhibited decreased susceptibility to trovafloxacin (> or =2 microg/ml), whereas all strains were inhibited by 0.25 microg/ml of gemifloxacin. PMID- 10746504 TI - Evaluation of a fluorescence hybridisation assay using peptide nucleic acid probes for identification and differentiation of tuberculous and non-tuberculous mycobacteria in liquid cultures. AB - The performance was evaluated of a fluorescence in situ hybridisation assay using peptide nucleic acid probes (Dako Probe MTB Culture Confirmation Test; Dako, Denmark) for identification of Mycobacterium tuberculosis complex (MTC) organisms and differentiation between tuberculous and non-tuberculous mycobacteria (NTM) in material taken directly from Bactec 12B (Becton Dickinson, USA) and MB/BacT (Organon Teknika, USA) bottles. The test was applied to 129 smear-positive (Ziehl Neelsen stain) clinical specimens, 48 previously identified clinical strains of mycobacteria (12 MTC and 36 NTM), and 51 reference strains (7 MTC and 44 NTM) which were all previously inoculated into Bactec 12B and MB/BacT bottles. The sensitivity and specificity of the assay for MTC-positive cultures was 87.6% and 100%, respectively, for Bactec 12B, and 100%, respectively, for MB/BacT. The sensitivity and specificity of the assay for NTM-positive cultures was 100% for both media. PMID- 10746505 TI - Evaluation of the E test for antifungal susceptibility testing of Candida glabrata. AB - The E test was compared to the reference NCCLS broth macrodilution method for susceptibility testing of Candida (Torulopsis) glabrata. The MICs of amphotericin B, flucytosine, fluconazole and itraconazole were determined using the appropriate culture media (RPMI 1640 agar with 2% glucose, Casitone agar or Antibiotic Medium 3 agar) according to the drug tested. Agreement between the two methods was within plus/minus two dilutions for 77-100% of test results, according to the drug/medium combination. The study revealed problems in determining the MICs of azoles using the E test, and confirmed the suitability of Casitone agar for susceptibility testing of fluconazole even if results were read within 24 h. PMID- 10746506 TI - Association between the use of anticholinergic agents and asymptomatic bacteriuria. PMID- 10746507 TI - Transmission of Staphylococcus aureus between humans and domestic animals in a household. PMID- 10746508 TI - Antimicrobial susceptibility and serotyping of Pseudomonas aeruginosa strains isolated in Bogota, Columbia. PMID- 10746509 TI - How long should patients with osteoporosis be treated with bisphosphanates? PMID- 10746510 TI - Some things only a woman can do. PMID- 10746512 TI - Big ideas for the 21st century. PMID- 10746511 TI - Toward optimal health: the experts respond to osteoporosis.. Interview by Jodi Godfrey Meisler. PMID- 10746513 TI - Multiple pregnancies and assisted reproductive technologies. PMID- 10746514 TI - Sleep disturbance in menopause. AB - Sleep problems (i.e., insomnia) affect midlife women as they approach and pass through menopause at rates higher than at most other stages of life. The purpose of this article is to critically review what is known about insomnia (perceived poor sleep) and physiologically assessed sleep, as well as sleep-related disordered breathing (SDB), in women according to menopausal status and the role of hypothalamic-pituitary-ovarian (HPO) hormones. Self-report evidence that sleep difficulties are related to the hormonal changes of menopause is mixed. Data from studies in which sleep was physiologically measured reveal that sleep problems appear corequisite with hot flashes and sweats. Results are difficult to compare across studies because of varying methodologies in how sleep quality and patterns were assessed and how age cohorts and menopausal status were defined. The risk of SDB increases with age, although women are less susceptible at any age than men. As with men, snoring, obesity, and high blood pressure are clear risk factors. Some women may be underdiagnosed for SDB, as they have somewhat different symptom manifestations than men. Usually, frank apnea is not as evident. Primary care clinicians should be mindful of the potential for SDB in women who are obese, have high blood pressure, are cognizant of snoring, and report morning headaches and excessive daytime sleepiness. Improved care will result from consistently incorporating sleep insomnia assessments into practice as a basis for referring to sleep centers as necessary or prescribing sleep-enhancing behavioral and pharmacological treatments. PMID- 10746515 TI - Has the impact of hormone replacement therapy on health-related quality of life been undervalued? AB - Previous economic evaluations of hormone replacement therapy (HRT) have restricted positive effects to alleviation of postmenopausal symptoms and negative effects to drug side effects. We studied the association between HRT use and postmenopausal women's valuation of both health-related quality of life and potential treatment side effects. Postmenopausal women with either a documented first vertebral fracture within the past 5 years or no history of osteoporotic fractures were recruited from Olmsted County, Minnesota, and from Dartmouth Hitchcock Medical Center in New Hampshire to participate in a study to assess quality of life and women's attitudes toward osteoporosis prevention. Women's valuations of their current health and potential HRT-related side effects were quantified as quality-adjusted life years (QALYs) assessed by an automated utility assessment instrument (U-Titer) and the time tradeoff technique, by a vertical rating scale, and by estimated quality of well-being (QWB) scores. Health status was measured using the Medical Outcomes Study SF-36. Regression methods were used to assess the impact of current HRT use on health-related quality of life and valuation of side effects. There were 106 women with vertebral fracture and 180 with no history of hip, wrist, or vertebral fractures. Altogether, 116 (40.6%) women were currently taking HRT, 64 (22.2%) had taken HRT in the past, and 106 (37.1%) women had never taken HRT. Current HRT users had higher time tradeoff QALYs than never and past HRT users, with gains ranging from 15.0 to 83.7 days per year for current users relative to the others. Benefits were largest for women with a vertebral fracture and limitations in activities. The secondary QALY measures also showed significantly higher values for current HRT users compared with other women, as did SF-36 subscales for general health, physical function, role-emotional function, and vitality. There was substantial variability in women's perceptions of HRT side effects. Overall, the proportion of women willing to trade time to avoid bleeding was largest, at 95.5%, followed by breast tenderness, weight gain, and endometrial biopsy at 90.4%, 87.4%, and 82.7%, respectively. Current HRT users had higher health-related quality of life than past or never users according to all measures studied. Women's perceptions of potential side effects were highly variable and should be considered by physicians when prescribing an HRT regimen. If, as our results suggest, postmenopausal therapy has positive effects beyond the immediate postmenopausal years, previous economic studies may have underestimated the value of HRT. PMID- 10746516 TI - A synergistic effect of a daily supplement for 1 month of 200 mg magnesium plus 50 mg vitamin B6 for the relief of anxiety-related premenstrual symptoms: a randomized, double-blind, crossover study. AB - To investigate single and combined effects of daily dietary supplementation with 50 mg of vitamin B6 and 200 mg magnesium (as MgO) for one cycle for the relief of mild premenstrual symptoms, a randomized, double-blind, placebo-controlled, crossover design was used. Forty-four women with an average age of 32 years took part in the study. Each woman was randomly assigned, according to a Latin square design, to take consecutively all four of the following treatments daily for one menstrual cycle: (1) 200 mg Mg, (2) 50 mg vitamin B6, (3) 200 mg Mg + 50 mg vitamin B6 and (4) placebo. Throughout the study, each volunteer kept a daily record of symptoms using a 5-point ordinal scale in a menstrual diary of 30 symptoms. Symptoms were grouped into six categories: anxiety, craving, depression, hydration, other, and total. Urinary magnesium output for 24 hours was estimated using the Mg/creatinine concentration ratio. ANOVA showed no overall difference between individual treatments, but predefined treatment comparisons using factorial contrasts in ANOVA showed a significant effect of 200 mg/day Mg + 50 mg/day vitamin B6 on reducing anxiety-related premenstrual symptoms (nervous tension, mood swings, irritability, or anxiety) (p = 0.040). Urinary Mg output was not affected by treatment. A small synergistic effect of a daily dietary supplementation with a combination of Mg + vitamin B6 in the reduction of mild premenstrual anxiety-related symptoms was demonstrated during treatment of 44 women for one menstrual cycle. In view of the modest effect found, further studies are needed before making general recommendations for the treatment of premenstrual symptoms. The study indicated that absorption from MgO was poor and daily supplementation for longer than 1 month is necessary for tissue repletion. PMID- 10746517 TI - Improved iron status parameters may be a benefit of hormone replacement therapy. AB - One of the benefits of hormone replacement therapy (HRT) is to decrease cardiovascular disease. A mechanism whereby HRT may play a role in reducing cardiovascular risk is through improved iron status parameters. High serum ferritin has been related to increased risk of coronary heart disease, whereas low iron-binding capacity has been identified as an important risk factor for myocardial infarction. This study examined iron status parameters in a group of postmenopausal women taking oral HRT (n = 27) and those not taking oral HRT (n = 27) at two times 1 year apart. Women were compared on the following serum measures: estradiol, lipids, iron, total iron-binding capacity, and ferritin. Women taking HRT had higher levels of estradiol (p < 0.001) and improved lipid profiles (p < 0.001) (lower total and low-density lipoprotein [LDL] cholesterol and higher levels of high-density lipoprotein [HDL] cholesterol). In addition, women on HRT had better iron status parameters than those not on HRT (p = 0.002). Total iron-binding capacity was greater for women on HRT compared with women not on HRT, and serum ferritin levels were lower in women on HRT than those not on HRT. The groups were comparable in age, body mass index, and physical activity. Our results confirm previous findings and indicate that women taking HRT have higher serum levels of estradiol and improved lipid profiles compared with those not taking HRT. In addition, we have found that iron status parameters are better in women taking HRT, suggesting the need to further examine this effect as it relates to decreased cardiovascular risk in postmenopausal women. PMID- 10746518 TI - Barriers and solutions to implementing a new curriculum: lessons from the women's health education program at MCP Hahnemann School of Medicine. AB - The integration of new knowledge into the medical school curriculum is a difficult process. This article proposes effective strategies for overcoming obstacles to curricular integration of women's health and sex and gender topics. Some techniques developed to overcome barriers to the integration of new material into an existing curriculum include faculty development, faculty rewards, development of competencies and assessment tools, interdisciplinary team teaching, standardized patients, and reference resources. An interdisciplinary approach to implementing women's health education, as with most new curricular material, is endorsed for integration of the new field into the medical school curriculum. This proposed model results in general institutional participation and support, especially from senior level leadership. The outlined process enables students and faculty to learn techniques for incorporating emerging information in all disciplines, helping them to become "life-long learners." PMID- 10746520 TI - Training internal medicine residents to screen for domestic violence. AB - Internal medicine residency programs will need to train residents about domestic violence to comply with the Residency Review Commission standards. To assess the effectiveness of an educational intervention intended to increase screening of patients for domestic violence by internal medicine resident physicians and identify characteristics associated with increased screening, we used a quasi experimental, pretest/posttest trial. This was conducted in an internal medicine residency program and its affiliated primary care clinic with categorical internal medicine and combined medicine/pediatrics resident physicians. Exit interview surveys of patients were conducted at baseline and following the educational program. Patients were questioned about demographics and if they were asked about domestic violence during the current visit. Physicians were questioned about demographics and attitudes and beliefs related to domestic violence. Prior to the intervention, only 0.8% (1 of 122) of patients reported being asked about domestic violence. After the intervention, the percentage asked rose to 17% (20 of 116). The odds ratio (OR) for being asked about domestic violence after training was 25.2 (6.1-104). Patients who were younger than 50 years were more likely to be asked (OR 2.5, 1.5-4.6). Caucasian physicians were more likely to ask (OR 2.8, 1.1-7.6). Patients reporting they were taught breast self-examination at that day's visit were also more likely to be screened (OR 2.9, 1.1-7.9). We found evidence that moderately intense training and focusing on results can increase resident physician screening rates for domestic violence. To do so effectively, we recommend a training session similar to this one and continued monitoring of outcome in the clinical setting. PMID- 10746519 TI - Locating a health advocate in a private obstetrics/gynecology office increases patient's receipt of preventive recommendations. AB - This study was performed to determine if adding a health advocate (HA) to the care team for postmenopausal women increased the number of women for whom the physician recommended screening tests or prevention strategies: cholesterol level, mammography, depression counseling, smoking cessation, or weight reduction. The study took place in two locations of a private obstetrician gynecologist practice. In one location, an HA reviewed screening forms and counseled women about preventive services recommended by the physician. In the second location, women completed the screening form, but treatment occurred as usual. Women were eligible if they were postmenopausal or age 50 or over and were being seen for preventive care. A total of 210 postmenopausal women were screened. Women who were screened when the HA was present (n = 85) did not differ from women screened at the intervention location when the HA was not present (n = 68) or screened at a second practice location (n = 57) in the prevalence of risk factors. Women were significantly more likely to receive indicated preventive recommendations when the HA was present (24% versus 73%, p < 0.001). For breast cancer screening, nearly all women screened when the HA was present received a referral compared to about one third of women screened when the HA was not present (OR = 3.0, 95% CI 1.8-5.2). Women are more likely to receive recommendations based on screening data when ancillary staff are available to assist in patient education and referral and to encourage physician recommendations. Further work is needed to identify cost-effective methods for supporting physicians' preventive care efforts. PMID- 10746521 TI - Variability in mammography screening legislation across the states. AB - Before passage of the Federal legislation, National Breast and Cervical Cancer Screening Act (NBCCSA) in 1991, over half the states (65%) had preexisting laws requiring health insurers, for example, Blue Cross/Blue Shield and HMOs, to provide services beyond the federal coverage for mammography screening and care following breast cancer. This study examined mammography screening legislation across the states. Data were derived from telephone interviews with six NBCCSA program directors or coordinators from July 1999 to October 1999. A review of existing documents from the Institute for Women's Policy Research, online data from the Centers for Disease Control and Prevention, and state laws provided by the Governmental Affairs Division of the American Cancer Society was undertaken. There was considerable variability in relation to factors potentially related to the extent of state laws. The states with the lowest age-adjusted breast cancer mortality rates among black women had the least comprehensive state legislation. Several states with the least percent of women above the federal poverty threshold also had the least comprehensive legislation. Some states had a wide gap between the provision of health insurance coverage and scope of legislation to ensure care following breast cancer. Some states were more aggressive in their efforts to ensure care following breast malignancy at diagnosis. Lessons could be learned by states that enacted the least comprehensive legislation. With the passage of the federal legislation nearly a decade ago, more women are receiving timely and available mammography screening, resulting in earlier diagnosis of breast cancer. Greater efforts must be undertaken by all states to provide the full array of breast cancer treatment for women in the millennium. PMID- 10746523 TI - Predicting depressive symptoms after miscarriage: a path analysis based on the Lazarus paradigm. AB - Twenty percent of all pregnancies end in miscarriage. Findings are mixed about who is most at risk for a depressive response. The purpose of this study was to develop and test a theory-based path model that would enable prediction of the intensity of women's depressive symptoms at 4 months and at 1 year after miscarriage. The model is based on Lazarus's theory of emotions and adaptation. Model constructs examined included stage I contextual variables (gestational age, number of miscarriages, number of children, maternal age, perceived provider caring at the time of loss, and family income), stage II interceding variables (perceived social support, emotional strength, and subsequent pregnancy/birth), stage III primary appraisal of meaning (personal significance of miscarrying), stage IV secondary appraisal (active or passive coping), and stage V emotional response (depressive symptoms). Path analysis employing a series of stepwise, multiple regression equations was used to test the hypothesized model. The sample consisted of 174 women whose pregnancies ended prior to 20 weeks gestation (mean = 10.51, SD = 3.32). The model accounted for 63% of the variance in women's depressive symptoms at 4 months and 54% at 1 year. Findings support the utility of the Lazarus model and confirm that women most at risk for increased depressive symptoms after miscarriage are those who attribute high personal significance to miscarriage, lack social support, have lower emotional strength, use passive coping strategies, have lower incomes, and do not conceive or give birth by 1 year after loss. PMID- 10746522 TI - Women physicians and stress. AB - Most women physicians enjoy better than average physical health and lead satisfying and productive lives. However, higher than average rates of depression, anxiety, marital problems, and substance abuse have been reported by some, but not all, authors. This quantitative survey of 196 women physicians and qualitative focus groups with 48 other women physicians was conducted to determine perceptions of their health, stress, satisfaction, knowledge, and abuse rates in medical practice. Eight specialties plus family practice physicians participated. The average age was 44.1 years (SD 8.8, range 23-77). Seventy-four percent of women physicians were married, with children. Specialists and family physicians were similar in all demographic characteristics except that family physicians were more significantly likely to be divorced, separated, or widowed (p < or = 0.01). Specialists perceived their personal physical health to be better than that of family doctors (p < or = 0.05), and family physicians rated their medical knowledge better than that of specialists (p < or = 0.0001). Women physicians over age 50 or with children over age 19 reported the best mental health (p < or = 0.0001 and 0.003, respectively). Overall, 49% of women physicians reported usually having high levels of stress, 44% felt mentally tired, and 17% took antidepressant drugs. Seventy-three percent reported verbal abuse at work (71% in the last year), and 33% reported physical assault at work (11% in the last year). Focus groups identified three major sources of stress: high expectations, multiple roles, and work environment. These results are discussed and compared with the literature. Both personal and systemic strategies are required to solve the problems identified. Women physicians can facilitate the adoption of some of these strategies by sharing information about successes, challenges, and solutions. PMID- 10746524 TI - Medical ethics. PMID- 10746525 TI - Pain at the cellular level: the role of the cytokine tumor necrosis factor-alpha. PMID- 10746526 TI - Management of pain associated with metastatic epidural spinal cord compression: use of imaging studies in planning epidural therapy. PMID- 10746527 TI - Long-term spinal analgesic delivery: a review of the preclinical and clinical literature. PMID- 10746528 TI - Combined lumbar and sacral plexus block compared with plain bupivacaine spinal anesthesia for hip fractures in the elderly. AB - BACKGROUND AND OBJECTIVES: This prospective randomized study was designed to determine the hemodynamic effects and quality of combined lumbar and sacral plexus block compared with plain bupivacaine spinal anesthesia in the elderly for repair of proximal femoral fractures. METHODS: Twenty-nine elderly patients ranging in age from 68 to 97 years were randomly assigned to 2 groups: a spinal anesthesia group with single-shot 3 mL 0.5% plain bupivacaine, and a combined block group with 30 mL lidocaine 1.33% with epinephrine for the posterior lumbar plexus block and 10 mL same mixture for the parasacral block and an iliac crest block with 5 mL lidocaine 1%. RESULTS: No need for general anesthesia was encountered in either group. Anesthesia was judged unsatisfactory in 1 of 15 patients in the combined block group. The initial decrease of mean arterial pressure was 38% in the spinal group and 27% in the block group and was not significantly different. A more prolonged hemodynamic effect was found in the spinal group, indicated by the more frequent use of ephedrine to stabilize blood pressure (P<.05). Patients over 85 years had a significantly larger decrease in blood pressure than younger patients (P<.01). CONCLUSIONS: Plain bupivacaine spinal anesthesia and combined lumbar/sacral plexus block provided adequate anesthesia for repair of hip fracture in the elderly. Hypotension was induced by both the combined peripheral nerve block and plain bupivacaine spinal anesthesia in aged patients; hypotension was found to be longer lasting after spinal anesthesia and of a larger magnitude in patients over 85 years of age. PMID- 10746529 TI - Microscopic analysis of three different spinal needle tips after experimental subarachnoid puncture. AB - BACKGROUND AND OBJECTIVES: Previous studies have shown the vulnerability of the tips of cutting type thin spinal needles and the possibility of foreign material passing into the subarachnoid space during the lumbar puncture. We made a microscopic analysis to compare two commonly used noncutting pencil-point spinal needles with different tip designs. Needles with a cutting tip design were included as reference. METHODS: Four fresh cadavers were placed in the lateral position and their backs were scrubbed with disinfectant solution containing 0.1% fluorescein. Thirty-two spinal needles (27 gauge) of each type (modified Quincke, modified Sprotte, and modified Whitacre) were inserted through an introducer at interspaces L2-5 into the subarachnoid space. Under visual control (spinal canal opened ventrally) all the needle tips were cut after successful subarachnoid puncture; 16 needles of each tip design were investigated under a fluorescence microscope, and another 16 needle tips were collected into test tubes and cytocentrifuged smears were prepared. The tips and smears with the most obvious findings were photographed under a microscope. RESULTS: On microscopy, only 2 needle tips were damaged (1 modified Quincke and 1 modified Whitacre). Visible fluorescent tissue particles were more frequently seen on modified Quincke needles (56%) compared with modified Sprotte (37%) and Whitacre (37%) needles (NS). In the cytocentrifugation smears, the largest clusters of epithelioid cells and muscle fibers were observed in the Quincke group. In the Whitacre group many fewer and smaller cell clusters including small muscle particles were seen, and only minor epithelioid cells were found in the Sprotte group. CONCLUSIONS: Tissue coring seems to be a common phenomenon during lumbar puncture. The most prominent attachments appeared with a cutting Quincke-type spinal needle. PMID- 10746530 TI - Spinal anesthesia for postpartum tubal ligation after pregnancy complicated by preeclampsia or gestational hypertension. AB - BACKGROUND AND OBJECTIVES: The American College of Obstetricians & Gynecologists has opposed postpartum tubal ligation (PPTL) in patients whose pregnancies have been complicated by a medical condition, including hypertension. A prospective study was conducted to assess the appropriateness of spinal anesthesia for PPTL in patients with hypertensive disorders of pregnancy. METHODS: After institutional review board approval and written informed consent, women scheduled for PPTL were enrolled. The hypertensive group (n = 25) consisted of women whose pregnancies were complicated by gestational hypertension, preeclampsia, or chronic hypertension with superimposed preeclampsia. For each hypertensive patient, a matched control was designated by selecting the next consenting normotensive patient scheduled for PPTL. After an intravenous bolus of lactated ringers solution 500 mL, spinal anesthesia was induced with hyperbaric lidocaine. Hemodynamic measurements were recorded every 2 minutes for 20 minutes after spinal injection. Ephedrine IV was administered for systolic blood pressure < or =90 mm Hg. RESULTS: The mean preoperative mean blood pressure (MAP) was 107+/-17 mm Hg in the hypertensive group and 85+/-11 mm Hg in the controls, P<.0001. The hypertensive group sustained a more profound percentage decrease in MAP than did their control counterparts during spinal anesthesia (29%+/-12% v. 15%+/-8%, P<.0001). However, the lowest intraoperative MAP was similar in both groups. Thirty-five percent of hypertensive patients and 24% of the controls received ephedrine IV. CONCLUSIONS: PPTL during spinal anesthesia in patients whose pregnancies have been complicated by hypertension had results comparable with patients with uncomplicated pregnancies; however, further studies are needed to ascertain its safety. PMID- 10746531 TI - Lack of secondary hyperalgesia and central sensitization in an acute sheep model. AB - BACKGROUND AND OBJECTIVES: We aimed to determine the following in an experimental acute pain model in sheep: (1) whether multimodal analgesia with intravenous fentanyl and ketorolac was more effective than fentanyl alone; (2) whether secondary hyperalgesia (central sensitization) occurred in adjacent (foreleg) dermatomes after thoracic surgery; (3) whether ketorolac used preemptively influenced the development of secondary hyperalgesia after surgery. METHODS: Changes in primary nociception were measured by increases to tolerated pressure, applied to the foreleg by a blunt pin, before foreleg withdrawal occurred. Changes to breath-to-breath interval and estimated end-tidal CO2 were used as indices of respiratory effects. Study 1 (n = 6) compared the paired responses to acute nociception after ketorolac (90 mg) or saline (control) pretreatment, followed by fentanyl (graded, 0 mg to 1.5 mg). Study 2 (n = 6) used a cross-over of ketorolac (90 mg) or saline (control) 24 hours and 1 hour, respectively, before a standardized thoracotomy incision, followed by antinociceptive testing with ketorolac (90 mg) and fentanyl (0.6 mg) daily over 4 days. RESULTS: In study 1, fentanyl produced naloxone-antagonizable antinociception and respiratory depression. Ketorolac did not affect fentanyl antinociception, except for prolonging antinociception at the highest dose; it did not affect the respiratory effects. In study 2, preemptive ketorolac had no effect on the postoperative antinociceptive or respiratory effects of fentanyl. The pharmacokinetics of fentanyl were unaltered by ketorolac. CONCLUSIONS: The results obtained in this acute pain model found no significant evidence of a fentanyl-ketorolac interaction, of central sensitization as shown by secondary hyperalgesia, or of a preemptive analgesic effect. PMID- 10746532 TI - The effects of sarpogrelate on superoxide production by human neutrophils. AB - BACKGROUND AND OBJECTIVES: Superoxide anion (O2-) released from neutrophils plays an important role in antibacterial host defense system and tissue auto-injury. Sarpogrelate, a serotonin-receptor antagonist, has been successfully used for management of chronic pain caused by arterial occlusive or ischemic vascular diseases, or by microcirculation disturbances. Suppression of O2- generation may be detrimental to infection or contribute to the therapeutic approach to these diseases, the pathogenesis of which probably includes neutrophil activation. No data regarding the effects of sarpogrelate on neutrophil functions are available despite the possible clinical concern. The purpose of this study was to determine whether sarpogrelate reduces O2- production by human neutrophils using an in vitro system. In addition, we examined changes in concentrations of the intracellular calcium ion ([Ca2+]i), which is responsible for one of the mechanisms of the neutrophils' O2- production. METHODS: The O2- production by human neutrophils or the xanthine-xanthine oxidase system and [Ca2+]i were measured in the absence and the presence (at clinically relevant concentrations: 0.1x, 10x, and 100x these concentrations) of sarpogrelate. RESULTS: Sarpogrelate inhibited O2- production of neutrophils in a dose-dependent manner. The drug at a clinically relevant concentration suppressed this neutrophil function. In contrast, sarpogrelate failed to inhibit O2-generation by the cell-free (xanthine xanthine oxidase) system. Elevation of [Ca2+]i in neutrophils stimulated by a chemotactic factor was dose-dependently attenuated with sarpogrelate. CONCLUSIONS: These findings suggest that sarpogrelate (even at clinically relevant concentrations) is able to inhibit O2- production by neutrophils. However, the drug failed to quench an excessive amount of O2- (similar to the level produced by neutrophils). There is a possibility that the inhibitory effect of the drug on [Ca2+]i response in neutrophils may contribute to impairment of the neutrophils' O2- production. Further studies using in vivo systems are required to elucidate the inhibitory effects of sarpogrelate on O2- in clinical settings. PMID- 10746533 TI - Ropivacaine attenuates pulmonary vasoconstriction induced by thromboxane A2 analogue in the isolated perfused rat lung. AB - BACKGROUND AND OBJECTIVES: Thromboxane A2 (TXA2) activation is involved in several pathophysiological states in producing pulmonary hypertension. Local anesthetics (LA) inhibit signaling of TXA2 receptors expressed in cell models. Therefore, we hypothesized that LA may inhibit pulmonary vasoconstriction induced by the TXA2 analogue U 46619 in an isolated lung model. METHODS: Isolated rat lungs were perfused with physiological saline solution and autologous blood with or without the LA lidocaine, bupivacaine, ropivacaine, or the permanently charged lidocaine analogue QX 314 (all 1 microg/mL) as a pretreatment. Subsequently, pulmonary vasoconstriction was induced by 3 concentrations of U 46619 (25, 50, and 100 ng/mL) and the change in pulmonary artery pressure (Pa) was compared with each LA. In a second experiment, Pa responses to angiotensin II (0.1 microg), hypoxic pulmonary vasoconstriction (HPV, 3% O2 for 10 minutes), or phenylephrine (0.1 microg) were assessed to determine the specificity of ropivacaine effects on TXA2 receptors. Finally, reversibility of pulmonary vasoconstriction was determined by adding ropivacaine to the perfusate after pulmonary vasoconstriction was established with U 46619. RESULTS: Ropivacaine, but not bupivacaine, lidocaine, or QX 314 significantly attenuated pulmonary vasoconstriction induced by 50 ng/mL U 46619 (35.9%, P<.003) or 100 ng/mL U 46619 (45.2%, P<.001). This effect of ropivacaine was likely to be specific for the thromboxane receptor because pulmonary vasoconstriction induced by angiotensin II, HPV, or phenylephrine was not altered. Ropivacaine did not reverse vasoconstriction when it was administered after U 46619. CONCLUSIONS: Ropivacaine, but not lidocaine, bupivacaine, or QX 314 at 1 microg/mL, attenuates U 46619-induced pulmonary vasoconstriction in an isolated perfused rat lung model. These results support evidence that the clinically used enantiomer S(-) ropivacaine may inhibit TXA2 signaling. PMID- 10746535 TI - Revision of a spinal column stimulator lead without lead replacement. AB - OBJECTIVE: The objective of this case report is to describe a technique for percutaneous spinal column stimulator (SCS) revision without lead replacement. CASE REPORT: A 35-year-old woman with a single-lead SCS implanted for control for left lower extremity pain underwent revision of her SCS lead because of loss of stimulation paresthesia in the area of pain. Using the method described, the lead was revised without lead replacement. CONCLUSION: SCS leads can be revised without placement of a new lead. PMID- 10746534 TI - A prospective randomized double-blinded controlled study of ropivacaine 0.75% versus bupivacaine 0.5%-mepivacaine 2% for peribulbar anesthesia. AB - BACKGROUND AND OBJECTIVES: Ropivacaine 1% has recently been used in clinical trials for peribulbar anesthesia. This study aims to compare the safety and the efficacy of ropivacaine 0.75% with that of a 1:1 mixture of bupivacaine 0.5% and mepivacaine 2% for peribulbar anesthesia. METHODS: Two thousand patients undergoing peribulbar anesthesia for elective cataract phacoemulsification were prospectively studied over a 1-year period and randomly assigned to 1 of 2 groups according to the local anesthetic used. One thousand patients were administered peribulbar anesthesia with 9 mL of ropivacaine 0.75% plus 1 mL of hyaluronidase (group R), and 1,000 patients received peribulbar anesthesia with 4 mL of bupivacaine 0.5% plus 4 mL of mepivacaine 2% plus 1 mL of hyaluronidase plus 1 mL of sodium bicarbonate (group BM). Peribulbar anesthesia was always accomplished by the same physician by 2 injections of 5 mL each, with a 25-gauge 25-mm needle. Evaluation was performed by another physician blinded to the technique used and included assessment of pain on local anesthetic injection, ocular and eyelid akinesia, need for top-up injections, onset time and duration of anesthesia, intraoperative analgesia, duration of surgery, hemodynamic parameters, and incidence of perioperative complications. RESULTS: A greater incidence of pain on injection was found in group BM (P<.001). No difference between the groups was found regarding the onset time and the duration of anesthesia. Perioperative analgesia was satisfactory in both groups with no significant difference. Patients in group R showed a reduced need for top-up injection and a better ocular akinesia at 8 and 10 minutes (P<.01). The akinesia of the eyelid was comparable in the 2 groups and complete in all cases at 8 minutes. Cardiac arrhythmias were more frequent in group BM (P<.01). Local complications did not differ between the groups. An increase in mean artierial blood pressure and heart rate was observed in both groups 1 minute after injection of local anesthetic. CONCLUSIONS: Peribulbar anesthesia with ropivacaine provided better ocular akinesia 8 to 10 minutes after block insertion than a bupivacaine-mepivacaine mixture, which reduced the need for top-up injections. Ropivacaine also caused less pain on injection. PMID- 10746536 TI - Repeated subarachnoid catheter displacement as a complication of spinal infusion using an internal infusion pump. AB - OBJECTIVE: To present and analyze the case of a woman receiving chronic spinal opioid therapy using an implanted infusion pump who experienced repeated displacement of the subarachnoid catheter despite the use of standard techniques for anchoring the catheter. The solution devised to avoid the problem is described. CASE REPORT: A 53-year-old woman was diagnosed with transverse myelitis 10 years earlier and she developed T7-T10 spinal cord atrophy, and pain below the T7 segment. After unsuccessful noninvasive pharmacological treatment, a spinal opioid infusion protocol was begun. On 3 occasions during the course of therapy, despite the use of standard measures for anchoring the system, catheter displacement into the subcutaneous pouch of the pump occurred. After the last such episode, a specially designed technique was used, anchoring the catheter by means of a silicone piece, and the injection of 2.5 mL of fibrin glue in the epidural space. CONCLUSIONS: The application of fibrin glue (Tissucol; Immuno AG, Vienna, Austria) may be considered as an adjuvant for the fixation of subarachnoid catheters used for intraspinal infusions. PMID- 10746537 TI - Stiffen your catheter--not your resolve! PMID- 10746538 TI - The Mansour's sacral plexus block: an effective technique for continuous block. PMID- 10746539 TI - First experiences with a new spinal needle. PMID- 10746540 TI - Eldor spinal needle versus Pencan spinal needle. PMID- 10746541 TI - Acute aphesia following tourniquet release in intravenous regional anesthesia with 0.75% lidocaine. PMID- 10746542 TI - Bupivacaine versus buprenorphine. PMID- 10746543 TI - The envelope protein of an endogenous murine retrovirus is a tumor-associated T cell antigen for multiple murine tumors. AB - Recently, significant progress has been made in identifying specific tumor associated antigens recognized by T cells and defining the specific peptide epitopes within these proteins that are processed and presented on class I major histocompatibility antigens. Most of these antigens have been identified in human melanoma, where many of them appear to be tissue-specific, nonmutated proteins expressed by melanoma and normal melanocytes but not by other tissues. There has been much less progress in identifying the tumor antigens on murine tumors that are recognized by T cells, and this has restricted the development of preclinical animal models for immunotherapy. The authors previously described a method for generating tumor-reactive T cells from murine tumors (tumor infiltrating lymphocytes) that are CD8+ T cells recognizing autologous tumor and that can inhibit established tumor on adoptive transfer. Here the authors show that the envelope protein of an endogenous murine retrovirus of the AKV family, found in the germline of the C57BL/6 mouse, is recognized by tumor-infiltrating lymphocytes from two histologically different tumors syngeneic to that mouse strain. Furthermore, the authors identify the specific 9-amino acid peptide from the p15E transmembrane component of this envelope protein that is recognized in the context of major histocompatibility complex Kb, show that it is naturally presented and recognized on several other H-2b tumors, and that cytotoxic T lymphocytes specific for this epitope are therapeutic for these antigen expressing tumors on adoptive transfer. PMID- 10746544 TI - Antitumor activity of interleukin-18 on mouse glioma cells. AB - Interleukin-18 (IL-18) exhibits antitumor activity in various laboratory models. In the current study, brain tumors in naive mice regressed after an intratumoral injection of a single dose of recombinant IL-18 (rIL-18). Intraperitoneal rIL-18 substantially delayed the growth of subcutaneously inoculated gliomas but not gliomas located in the brain. Efficacy was reduced when studies were performed in mice depleted of natural killer cells. Although intracerebral administration of rIL-18 increased the serum interferon-gamma concentration, the antitumor effect of IL-18 was not mediated by interferon-gamma. These data suggest the therapeutic potential for control of tumor growth by intratumoral administration of rIL-18 in patients with glioma. PMID- 10746545 TI - Cytokines delivered by biodegradable microspheres promote effective suppression of human tumors by human peripheral blood lymphocytes in the SCID-Winn model. AB - A new technology for the local and sustained delivery of immunostimulatory molecules to the tumor environment for cancer immunotherapy was evaluated. The ability of cytokines delivered by biodegradable microspheres to promote the antitumor activity of human peripheral blood lymphocytes (PBL) was tested in a human PBL, human tumor, and SCID mouse (SCID-Winn) model. Co-engraftment of human recombinant IL-12-loaded microspheres with human PBL and tumors in SCID mice promoted complete tumor suppression in as many as 100% of the mice, whereas microspheres loaded with polyethyleneglycol-interleukin-2 suppressed but did not eliminate the growth of tumor xenografts. Control microspheres (loaded with bovine serum albumin) in the presence of human PBL or cytokine-loaded microspheres in the absence of human PBL had no tumor-suppressive effect. Coincident with the enhancement of the human PBL-mediated antitumor activity in mice treated with IL-12-loaded microspheres was the production and release of human IFN-gamma indicating that IL-12 released from the microspheres results in the activation of the engrafted human PBL. The results establish that biodegradable microspheres represent an effective tool for the local and sustained delivery of cytokines to the tumor environment for cancer immunotherapy. PMID- 10746546 TI - An acidic microenvironment inhibits antitumoral non-major histocompatibility complex-restricted cytotoxicity: implications for cancer immunotherapy. AB - Local immunosuppression may explain the failure of an effective immune response against solid tumors. Although it is well known that the interstitial pH is significantly lower in solid tumors than in normal tissue, only a few studies in the mouse system have investigated the influence of this acidic milieu on the anti-tumoral cytotoxic response. Here the authors report the suppression of human non-major histocompatibility complex (MHC)-restricted cytotoxicity against tumor cells by an acidic extracellular pH (pHe). Unstimulated peripheral blood mononuclear cells, lymphokine-activated killer (LAK) cells, and natural killer cell clones were used as effector cells. According to pH measurements in solid tumors, representative pH values of 7.2 to 5.3 were chosen during the cytotoxic assays. Target cell lysis was measured using two nonradioactive fluorometric methods, namely two-color flow cytometry and a modified calcein-release assay, which allowed cell-mediated cytotoxicity to be measured and compared with that in adherent targets. Using K562, Daudi, or Raji as suspended target cell lines, the cytotoxic activity of unstimulated peripheral blood mononuclear cells and of LAK cells was markedly reduced by a decreasing pHe. An extracellular pH of 5.8 to 5.3 resulted in a nearly complete loss of the cytotoxic response. This pHe-dependent impairment of the killing activity could also be shown for killer cells stimulated with interleukins-7 and -12, phytohemagglutinin, or lipopolysaccharide. The lytic potential of homogeneous natural killer cell clones as effectors was also strictly influenced by the surrounding pH. The pHe dependence of the non-MHC-restricted killer cell functions against tumor cells seems to be a general phenomenon, because the cytolytic activity of LAK cells against six human adherent tumor cell lines (HeLa, HepG2, LS174T, LS174Te, MCF-7, and RT112) was also clearly reduced under acidic conditions. To initiate the killing process, adhesion molecules play an important role in recognition and binding of the target cell. However, flow cytometric analysis revealed that the expression pattern of relevant adhesion molecules was unaffected by acidic pHe. In conclusion, these data clearly indicate an inhibition of non-MHC-restricted cytotoxicity against tumor cells by an acidic pHe, which may contribute to the failure of immunosurveillance against solid tumors. Consequently, efforts to enhance the anti-tumoral cytotoxicity by immunotherapies may have limited success. PMID- 10746547 TI - Essential role of nitric oxide and interferon-gamma for tumor immunotherapy with interleukin-10. AB - Several laboratories have reported anti-tumor activity for high levels of interleukin-10 (IL-10) expressed as a transgene or administered as recombinant protein. The authors have reported a positive correlation for nitric oxide production and anti-tumor activity of IL-10 in a murine model of breast cancer. In the current study, they sought evidence of a mechanistic role for nitric oxide in IL-10-mediated tumor growth inhibition. They wanted to determine whether pharmacologic inhibition of nitric oxide synthase (NOS) activity reverses the therapeutic effect of IL-10. Administration of either of two NOS inhibitors, aminoguanidine (AG) or L-lysine,N6-1-iminoethyl-dihydrochloride, appears to abrogate in part the tumor growth inhibition observed when IL-10 is overexpressed as a transgene in two murine mammary tumor cell lines. Nitric oxide levels were assessed at the tumor site by measuring nitrosylated heme levels by electron spin resonance spectroscopy. Nitric oxide hemoglobin levels were lower in tumors from aminoguanidine-treated mice, indicating that effective inhibition of nitric oxide production occurred at the tumor site. Previous investigations showed that the inducible form of NOS protein (iNOS), but not constitutive NOS, was expressed at higher levels in IL-10-expressing tumors. Because iNOS is regulated at the transcriptional level, the authors compared iNOS mRNA levels in IL-10 and control tumors. Northern analysis revealed strong iNOS message expression in all six IL 10-expressing tumors examined, whereas message was faintly detected in parental or 66-neo tumors. The inducible form of NOS is responsive to induction by interferon-gamma (IFN-gamma). The role of IFN-gamma in IL-10-mediated tumor inhibition and iNOS mRNA induction was determined. When tumors were transplanted to IFN-gamma mutant mice, the tumor-inhibitory activity of IL-10 was lost. Furthermore, iNOS mRNA was no longer induced in the absence of host expression of IFN-gamma. These data indicate that nitric oxide contributes to the anti-tumor activity of IL-10 and that expression of iNOS in this context depends on IFN gamma. PMID- 10746548 TI - Immunoregulating properties of peptides related to tumor rejection antigens: effect on human monocytes and natural killer cells. AB - The authors analyzed the effect of several 15-amino acid peptides with sequences related to tumor-rejection antigens, tyrosinase, and the MAGE family on peripheral blood mononuclear cells from healthy donors cultured for periods of 1 to 7 days. Some of these peptides promoted stimulation of monocytes, manifested by phenotypic changes, release of interleukin (IL)-1a, IL-6, and tumor necrosis factor-alpha, and induction of nitric oxide synthase on differentiated CD14++/+ CD16+ DR++ monocytes. An increase in the percentage of cytotoxic monocytes (CD14+/- CD16+) containing granule-associated DNase activity was also observed. Active peptides induced the release of IL-2 and interferon-gamma. Nonspecific natural killer and lymphokine-activated killer cell-mediated cytotoxicity was also observed against classical target cell lines (K-562 and Daudi) and allogenic melanoma cell lines AC and BB, together with an increase in granule-associated DNase in the natural killer cell-enriched population. Monocytes were needed to enhance this innate response, because peptides failed to induce the release of IL 2 on monocyte-depleted peripheral blood mononuclear cells. Data show an enhancement of the rapid innate immune response by peptides related to tumor rejection antigens and suggest that they could also determine the nature of a slow and more definitive specific immune response against tumor cells. PMID- 10746549 TI - CD95-mediated tumor recognition by CD4+ effector cells in a murine mammary model. AB - The authors examined cellular mechanisms involved in anti-tumor reactivity induced by the murine MT-9G1 mammary tumor line, which was transduced to secrete granulocyte macrophage-colony-stimulating factor (GM-CSF). Compared with the parental MT-901 tumor, MT-9G1 subcutaneous tumors elicited an influx of CD4+ cells and dendritic cells. Secondary in vitro activation of tumor-draining lymph node cells with anti-CD3 and interleukin-2 resulted in effector cells that can mediate regression of established pulmonary metastases after adoptive transfer. In vivo depletion of T-cell subsets showed that tumor regression required CD4+ tumor-draining lymph node cells rather than CD8+ cells. The activated CD4+ cells expressed CD95L and mediated lysis of CD95+ MT-901 tumor cells, which were major histocompatibility complex class II negative. The CD4+ cells also released GM-CSF in response to tumor stimulation. A Fas fusion protein inhibited tumor lysis and GM-CSF release by the CD4+ cells. These studies document an alternate pathway by which CD4+ immune cells may recognize major histocompatibility complex class II deficient tumors in which CD95L-bearing T cells induced an anti-tumor response mediated via CD95L:CD95. PMID- 10746550 TI - Adoptive transfer from interferon-alpha-fed mice is associated with inhibition of active experimental autoimmune encephalomyelitis by decreasing recipient tumor necrosis factor-alpha secretion. AB - Ingested type I interferon (IFN) suppresses clinical relapse in murine chronic experimental autoimmune encephalomyelitis (EAE), inhibits clinical attacks more effectively than subcutaneous doses, and decreases the adoptive transfer of EAE. To determine whether splenocytes from IFN-fed donors were "suppressor-like" populations, donor SJL/J mice were immunized and fed with mock IFN-alpha or with IFN-alpha every other day for at least 4 weeks after initial clinical attack. Recipients of adoptively transferred CD8+ T cells from mock IFN-alpha-fed donors showed no clinical improvement of clinical disease compared with actively immunized controls. In contrast, recipients of adoptively transferred CD8+ T cells from IFN-alpha-fed donors showed decreased clinical disease compared with recipients of mock IFN-alpha-fed CD8+ T cells. To evaluate the mechanism of protection by donor CD8+ T cells and to determine if ingested IFN-alpha activates natural immunomodulatory cell populations, the authors used the acute EAE model and naive-fed donor animals as sources of T cells and CD8+ T cells. Con A activated spleen T cells from naive nonimmunized mock IFN-alpha-fed donors inhibited actively induced disease and showed decreased recipient TNF-alpha secretion compared with recipients of T cells from mock IFN-fed mice. Donor activated spleen CD8+ T cells from naive nonimmunized IFN-alpha-fed animals suppressed actively induced EAE in recipients and showed decreased IFN-gamma and TNF-alpha proinflammatory secretion. Decreased recipient TNF-alpha secretion correlates best with the disease protection from IFN-fed T and CD8+ T cells. PMID- 10746551 TI - Semiallogeneic cell hybrids as therapeutic vaccines for cancer. AB - The authors have engineered a cell line that can be used in human studies as a universal donor cell for the formation of semiallogeneic cell hybrids after fusion with patient-derived tumor cells. These hybrids can be irradiated and injected as a patient-tailored therapeutic vaccine in patients affected by virtually any type of cancer. A crucial step in this research effort has been the derivation of an allogeneic cell line (FO1-12) that expresses both a dominant selectable marker (neomycin resistance) and a recessive selectable marker (sensitivity to hypoxanthine, aminopterin, and thymidine), which allows easy selection of semiallogeneic cell hybrids derived from the fusion of FO1-12 cells with patient-derived tumor cells. Tumor-infiltrating lymphocytes derived from select patients with melanoma and exposed to semiallogeneic cell hybrids from the same patient were better able to specifically lyse autologous tumor cells. Furthermore, FO1-12 cells express carcinoembryonic antigen, which is ubiquitous in adenocarcinomas, and fusion of FO1-12 cells with various patient-derived adenocarcinoma cells showed that the hybrid cells also express carcinoembryonic antigen. Because of the results of these preclinical studies, the authors were given permission to use semiallogeneic cell hybrids for immunotherapy of patients with metastatic melanoma or metastatic adenocarcinoma who had not responded to standard treatment regimens. Treatment with semiallogeneic vaccines is associated with minimal or no toxicity and can induce a specific anti-tumor immune response. PMID- 10746552 TI - Detection of T helper responses, but not of human papillomavirus-specific cytotoxic T lymphocyte responses, after peptide vaccination of patients with cervical carcinoma. AB - Human papillomavirus type 16 (HPV16)-encoded E7 oncoprotein is constitutively expressed in cervical carcinoma cells and is required for cellular transformation to be maintained. The E7 protein, therefore, forms an attractive target for T cell-mediated immune intervention to prevent or treat HPV16+ tumors. The authors performed a peptide-based phase I/II vaccination trial to induce anti-tumor immune responses in patients with recurrent or residual cervical carcinoma. Fifteen HLA-A*0201+ patients with HPV16+ cervical carcinoma received vaccinations with synthetic peptides representing 2 HPV16 E7-encoded, HLA-A*0201-restricted cytotoxic T lymphocyte epitopes and a pan-HLA-DR-binding T-helper epitope, PADRE, in adjuvant. No signs of toxicity were observed. Two patients had stable disease for more than 1 year after vaccination, 3 patients died of the disease during or shortly after the vaccination period, and 10 patients maintained progressive cervical carcinoma. Specific immune responses directed against the vaccine components were analyzed in peripheral blood samples. No cytotoxic T lymphocyte responses against the HPV16 E7 peptides were detectable. After vaccination, strong PADRE helper peptide-specific proliferation was detected in 4 of 12 patients. In conclusion, peptide vaccination with 2 HPV16 E7 cytotoxic T lymphocyte epitopes and a universal T helper epitope is well tolerated by patients with advanced cervical carcinoma. Despite a reduction of in vitro cytolytic or proliferative recall responses to some, but not all, conventional antigens in this patient group, peptide-specific proliferative responses were induced in 4 patients. Based on the current study, it is now feasible to perform peptide vaccination in earlier stages of HPV16-induced cervical disease. PMID- 10746553 TI - Results of a dose-intense phase 1 study of a combination chemotherapy regimen with cisplatin and epidoxorubicin including medroxyprogesterone acetate and recombinant interleukin-2 in patients with inoperable primary lung cancer. AB - Based on the role of cytokines in the pathogenesis of cancer-related anorexia cachexia and the ability of progestins, such as medroxyprogesterone acetate, to reduce cytokine production and relieve cancer-related anorexia-cachexia symptoms, the authors designed an open, dose-finding phase I study of a combined chemotherapy regimen (cisplatin [CDDP], epidoxorubicin [EPI]), including recombinant interleukin-2 (IL-2) and medroxyprogesterone acetate for patients with stage IIIB to IV inoperable primary lung cancer. The end points were clinical response and toxicity with definition of dose-limiting toxicity and maximal tolerable dose; relief of cancer-related anorexia-cachexia symptoms; the assessment of patient serum levels of IL-1beta, IL-6, tumor-necrosing factor alpha (TNF-alpha), and soluble IL-2 receptor (sIL-2R). From March to October 1997, 16 patients (M:F ratio, 14:2; mean age, 60.5 years; age range, 41 to 74 years) were enrolled. All patients were evaluable for toxicity and 14 of them for response. The patients were assigned to increasing dose levels of drugs according to a dose-escalation schedule. The weekly schedule consisted of a combination of CDDP given intravenously on day 1, EPI given intravenously on day 1, 1 g/day medroxyprogesterone acetate given orally on days 1 to 7, and recombinant IL-2 1.8 MIU administered subcutaneously on days 2 to 7 plus 300 microg granulocyte-colony stimulating factor support given subcutaneously on days 2 to 5. Administration of medroxyprogesterone acetate began 1 week before the first cycle. Dose escalation of the drugs was as follows: 30 mg x m2 x week(-1) CDDP and 25 mg x m2 x week(-1) EPI (first level, two patients); 30 mg x m2 x week(-1) CDDP and 33 mg x m2 x week(-1) EPI (second level, 2 patients); 40 mg x m2 x week(-1) CDDP and 33 mg x m2 x week(-1) EPI (third level, 6 patients); and 40 mg x m2 x week(-1) CDDP and 40 mg x m2 x week(-1) EPI (fourth level, 6 patients). Six cycles were planned for each patient. The actual dose intensity delivered was more than 80% of the projected dose intensity of all drugs. After six cycles, clinical response (according to World Health Organization criteria), toxicity (according to World Health Organization criteria), Eastern Cooperative Oncology Group (ECOG) performance status, body weight, appetite, and serum levels of cytokines were evaluated. After six cycles, 9 of 14 patients (64.3%) had partial response, 3 of 14 (21.4%) had stable disease, and 2 of 14 (14.3%) had progressive disease, and the objective response rate was 64.3%. ECOG performance status and body weight did not change significantly after treatment, whereas appetite showed an increase that was of borderline statistical significance. Toxicity was acceptable and only hematologic. Dose-limiting toxicity was established at the fourth dose level; consequently, maximal tolerable dose was assessed at the third dose level. Before treatment, the serum levels of IL-1beta, IL-6, and TNF-alpha were significantly greater in the patients than in healthy persons. The comparison between pretreatment and posttreatment serum values of IL-1beta, IL-6, TNF-alpha, and sIL 2R did not reveal significant differences in the patients. Similar results were obtained when the patients were considered as responders (partial response) or non-responders (stable or progressive disease) to therapy. Only IL-6 serum levels were increased (p = 0.014) after treatment. PMID- 10746554 TI - Phase 2 trial of vaccination with tyrosinase peptides and granulocyte-macrophage colony-stimulating factor in patients with metastatic melanoma. AB - This phase II study was performed to determine the induction of a specific T-cell response, the clinical response rate, and toxicity of vaccination with different HLA class I-binding peptide epitopes derived from the melanocyte differentiation antigen tyrosinase in patients with stage IV melanoma. The study population consisted of 16 patients with metastatic disease and two patients who were macroscopically free of disease at study entry after resection of recurrent skin lesions. Patients received intradermal injections of 200 microgram [corrected] peptide corresponding to their HLA type on day 3, and 75 or 150 microg granulocyte-macrophage colony-stimulating factor on days 1 to 4. Vaccinations were repeated at weeks 2, 4, 6, 10, and 14. Monitoring of peptide-specific T-cell frequencies in the peripheral blood was performed using an interferon gamma ELISPOT assay. Eleven of the 16 patients with metastatic disease went off the protocol within the first 10 weeks because of tumor progression. Of the five patients with metastatic disease who received all six vaccinations, one patient showed a mixed response with regression of some lung metastases; two patients with progressive disease before vaccination had stable disease for 6 and 18+ months; and two patients had progression of their disease. The two patients who had all their metastases resected before vaccination did not have relapses for 6 and 12+ months after vaccination. Induction of tyrosinase-reactive T cells was found in these two patients and in two others with metastatic disease, including the one who achieved a mixed response and one with stable disease. This study shows limited clinical and immunologic activity of HLA class 1-peptide vaccination in combination with granulocyte-macrophage colony-stimulating factor in stage IV melanoma patients. PMID- 10746555 TI - Frequency of human leukocyte antigen-A 24 alleles in patients with melanoma determined by human leukocyte antigen-A sequence-based typing. AB - The analysis of immune responses of patients with melanoma has led to the identification of melanoma-associated antigens targeted by T cells. Cytotoxic T lymphocytes recognize peptides from melanoma-associated antigens presented on the cancer cell surface in the context of HLA class I molecules. Immunodominant melanoma-associated antigen epitopes are being evaluated for their ability to immunize patients with advanced melanoma. However, these vaccination efforts are limited by the extensive polymorphism of the HLA class I heavy chain, which occurs in functional domains of the molecule. Patients with melanoma with the HLA A-24 phenotype were recruited for vaccination with the peptide AFLPWHRLF from the melanoma-associated antigen tyrosinase. This peptide is recognized in association with HLA-A*2402. The HLA-A24 family includes at least 15 alleles whose frequency and ability to present the same peptide are unknown. The distribution of HLA-A24 alleles was studied in a melanoma population for the practical purpose of identifying patients suitable for vaccination with HLA-A*2402 epitopes. An HLA-A locus-specific polymerase chain reaction method followed by sequencing was developed to determine the HLA-A alleles in genomic DNA. HLA-A 24 was also typed in healthy persons of various ethnic backgrounds to further explore the HLA-A24 family. In white persons, the HLA-A*2402 allele was most common (in 85% of white persons and in 97% of the patients with melanoma). Fewer persons carried the HLA A*2403 allele (13% in all samples, 3% in melanoma patients). Finally, two new alleles, HLA-A*2422 and HLA-A*24 null, were identified. These results suggest that vaccination with HLA-A*2402-associated epitopes has the potential for broad use in this patient population. PMID- 10746556 TI - Genetics of molybdenum cofactor deficiency. AB - Molybdenum cofactor (MoCo) deficiency leads to a combined deficiency of the molybdoenzymes sulphite oxidase, xanthine dehydrogenase and aldehyde oxidase. Effective therapy is not available for this rare disease, which results in neonatal seizures and other neurological symptoms identical to those of sulphite oxidase deficiency. It is an autosomal recessive trait and leads to early childhood death. Biosynthesis of MoCo can be divided into the formation of a precursor and its subsequent conversion to the organic moiety of MoCo by molybdopterin synthase. These two steps are the molecular basis of the two observed complementation groups A and B and of two types of MoCo deficiency with an identical phenotype. MOCS1 is defective in the majority of patients (group A) and was shown to encode two enzymes functioning in the formation of a precursor. The corresponding transcript is bicistronic with two consecutive open reading frames (ORFs). MOCS2 encodes the small and large subunits of molybdopterin synthase via a single transcript with two overlapping reading frames. This gene carries lesions in the B complementation group less frequently observed in patients. Both genes, MOCS1 and MOCS2, share the unusual bicistronic architecture, have identical and very low expression profiles and extremely conserved C-terminal ends in their 5'-ORF. These observations point to a novel form of microcompartmentalization and render the MOCS genes ideal candidates for a somatic gene therapy approach. PMID- 10746557 TI - Gene organization and rearrangements at the human Rhesus blood group locus revealed by fiber-FISH analysis. AB - The human Rhesus (Rh) blood group locus is composed of two highly homologous genes, the RHD and RHCE genes on chromosome 1, encoding the D, C/c, and E/e antigens in common Rh-positive phenotypes. In general, the RHD gene is either absent or grossly deleted in Rh-negative individuals. In this study, gene organization at the RH locus of Japanese donors with different serological phenotypes was directly analyzed by two-color fluorescence in situ hybridization on DNA fibers released from their lymphocytes (fiber-FISH) and by using DNA probes of introns 3 and 7 of the RHCE and RHD genes. Six Rh-positive samples (two with the D+C-c+E+e-, two with the D+C+c-E-e+, and two with the D+C+c+E+e+ phenotype) showed the presence of two RH genes within a region of less than 200 kb on chromosome 1p36.1. Of great interest was the finding that the genes were arranged in the antidromic order of the telomere -RHCE (5'--> 3') -RHD (3'-->5') centromere. On the other hand, two typical Rh-negative samples (D-C-c+E+e+) showed the presence of only one RHCE gene, as expected. Moreover, further analysis combined with a locus-specific assay of three Rh-negative samples (D C+c+E+e+, D-C+c+E-e+, and D-C+c-E-e+) showed the possible presence of the RHD gene(s) and complex rearrangements, including partial deletion, duplication, and recombination, in this region; these could be responsible for the Rh-negative phenotype. PMID- 10746558 TI - A new approach for identifying non-pathogenic mutations. An analysis of the cystic fibrosis transmembrane regulator gene in normal individuals. AB - Given q as the global frequency of the alleles causing a disease, any allele with a frequency higher than q minus the cumulative frequency of the previously known disease-causing mutations (threshold) cannot be the cause of that disease. This principle was applied to the analysis of cystic fibrosis transmembrane conductance regulator (CFTR) mutations in order to decide whether they are the cause of cystic fibrosis. A total of 191 DNA samples from random individuals from Italy, France, and Spain were investigated by DGGE (denaturing gradient gel electrophoresis) analysis of all the coding and proximal non-coding regions of the gene. The mutations detected by DGGE were identified by sequencing. The sample size was sufficient to select essentially all mutations with a frequency of at least 0.01. A total of 46 mutations was detected, 20 of which were missense mutations. Four new mutations were identified: 1341+28 C/T, 2082 C/T, L1096R, and I11131V. Thirteen mutations (125 G/C, 875+40 A/G, TTGAn, IVS8-6 5T, IVS8-6 9T, 1525-61 A/G, M470V, 2694 T/G, 3061-65 C/A, 4002 A/G, 4521 G/A, IVS8 TG10, IVS8 TG12) were classified as non-CF-causing alleles on the basis of their frequency. The remaining mutations have a cumulative frequency far exceeding q; therefore, most of them cannot be CF-causing mutations. This is the first random survey capable of detecting all the polymorphisms of the coding sequence of a gene. PMID- 10746559 TI - Molecular analysis of autosomal dominant hereditary ataxias in the Indian population: high frequency of SCA2 and evidence for a common founder mutation. AB - Expansion of CTG/CAG trinucleotide repeats has been shown to cause a number of autosomal dominant cerebellar ataxias (ADCA) such as SCA1, SCA2, SCA3/ MJD, SCA6, SCA7, SCA8 and DRPLA. There is a wide variation in the clinical phenotype and prevalence of these ataxias in different populations. An analysis of ataxias in 42 Indian families indicates that SCA2 is the most frequent amongst all the ADCAs we have studied. In the SCA2 families, together with an intergenerational increase in repeat size, a horizontal increase with the birth order of the offspring was also observed, indicating an important role for parental age in repeat instability. This was strengthened by the detection of a pair of dizygotic twins with expanded alleles showing the same repeat number. Haplotype analysis indicates the presence of a common founder chromosome for the expanded allele in the Indian population. Polymorphism of CAG repeats in 135 normal individuals at the SCA loci studied showed similarity to the Caucasian population but was significantly different from the Japanese population. PMID- 10746560 TI - Meiotic segregation analysis of a 14;21 Robertsonian translocation carrier by fluorescence in situ hybridization. AB - Meiotic segregation of chromosomes 14 and 21 in sperm from a 14;21 Robertsonian translocation carrier was analyzed with dual-color FISH using two locus-specific DNA probes (Tel 14q and LSI 21). The frequency of normal or chromosomally balanced sperm, resulting from alternate segregation, was 88.42%. The frequency of unbalanced sperm, resulting from adjacent segregation, was 11.25%. These observed frequencies deviated significantly from the theoretical frequencies (33.33% and 66.67%, respectively) based on random chromosome segregation, with sperm resulting from alternate segregation being preferentially produced in the translocation carrier. With respect to the chromosomally unbalanced sperm, the frequency of 21q disomic sperm was 2.45%, which is in agreement with the frequencies of unbalanced fetuses or offspring at the time of amniocentesis or at term (0-4.3%) reported by others. Although the frequency of 14 or 21 nullisomic sperm should be theoretically equal to that of 14q or 21q disomic sperm in both the carrier and controls, the frequency of nullisomic sperm was significantly higher than that of disomic sperm in the carrier (P=0.0009 for chromosome 14, P<0.0001 for chromosome 21) but not in the controls (P=0.091 for chromosome 14, P=0.74 for chromosome 21). This evidence suggests the occurrence of maturation arrest during spermatogenesis of the carrier. PMID- 10746561 TI - Missense mutations in SURF1 associated with deficient cytochrome c oxidase assembly in Leigh syndrome patients. AB - We have studied the fibroblasts of three patients suffering from Leigh syndrome associated with cytochrome c oxidase deficiency (LS-COX-). Their mitochondrial DNA was functional and all nuclear COX subunits had a normal sequence. The expression of transcripts encoding mitochondrial and nuclear COX subunits was normal or slightly increased. Similarly, the OXA1 transcript coding for a protein involved in COX assembly was increased. However, several COX-protein subunits were severely depressed, indicating deficient COX assembly. Surf1, a factor involved in COX biogenesis, was recently reported as mutated in LS-COX- patients, all mutations predicting a truncated protein. Sequence analysis of SURF1 gene in our three patients revealed seven heterozygous mutations, six of which were new : an insertion, a nonsense mutation, a splicing mutation of intron 7 in addition to three missense mutations. The mutation G385 A (Gly124-->Glu) changes a Gly that is strictly conserved in Surfl homologs of 12 species. The substitution G618 C (Asp202-->His), changing an Asp that is conserved only in mammals, appears to be a polymorphism. The mutation T751 C changes Ile246 to Thr, a position at which a hydrophobic amino acid is conserved in all eukaryotic and some bacterial species. Replacing Ile246 by Thr disrupts a predicted beta sheet structure present in all higher eukaryotes. COX activity could be restored in fibroblasts of the three patients by complementation with a retroviral vector containing normal SURF1 cDNA. These mutations identify domains essential to Surf1 protein structure and/or function. PMID- 10746563 TI - Chromosome analysis of blastomeres from human embryos by using comparative genomic hybridization. AB - Karyotypic studies of aborted fetuses have been used to draw the inference that the proportion of conceptuses with chromosome abnormalities is very high. Fluorescent in situ hybridization (FISH) studies of blastomeres from early cleavage embryos have provided some support for this inference but they are limited to the study of a few chromosomes. We describe the novel application of comparative genomic hybridization (CGH) to the study of numerical and structural abnormalities of single blastomeres from disaggregated 3-day-old human embryos. CGH results were obtained for 63 blastomeres from 12 embryos. Identification of all chromosomes with the exception of chromosomes 17, 19, 20 and 22 was possible. The embryos divided into four groups: (1) embryos with a normal CGH karyotype seen in all blastomeres; (2) embryos with consistent aneuploidy suggesting meiotic non-disjunction had occurred; (3) embryos that were mosaic generally with one or more cells showing aneuploidy for one or two chromosomes but some with cells showing extensive aneuploidy; and (4) one embryo with extensive aneuploidy in all blastomeres. The extensive aneuploidy in group 4 is interpreted as corresponding to the random aneuploidy seen in "chaotic" embryos reported by using interphase FISH. Partial chromosome loss and gain following chromosome breakage was observed in one embryo. Our analysis provides basic biological information on the occurrence of constitutional and post-zygotic chromosome abnormalities in early human embryos. Used in conjunction with embryo biopsy, diagnostic CGH should allow the exclusion of a proportion of embryos that appear normal but that have a poor probability of survival and, therefore, may improve the implantation rate after in vitro fertilization. PMID- 10746562 TI - Further evidence of autosomal dominant congenital zonular pulverulent cataracts linked to 13q11 (CZP3) and a novel mutation in connexin 46 (GJA3). AB - We describe a four-generation family with fully penetrant, autosomal dominant, congenital cataracts (ADCC), presenting with morphologically homogeneous "zonular pulverulent" cataracts (CZP) and typical early-onset phenotype. Linkage analysis was performed with a panel of polymorphic markers mapped to all genomic regions of ADCC susceptibility. Contiguous significant two-point lod scores were generated at autosomal region 13q11-q13 and further linkage and haplotype studies confined the disease locus to 13q11, supporting a previous linkage of CZP (specifically CZP3) to 13q11. Mutations in a gap-junction protein, connexin 46 (alphaa3 subunit or GJA3), have recently been reported as being linked to the 13q11 region. Mutational analysis of connexin 46 in our family revealed a C-->T at position 560 (P187L) of the cDNA sequence creating a novel MnlI restriction site that segregated with affected members of the pedigree. This family represents a second report of CZP3 linkage to 13q and is associated with a novel mutation in the connexin 46 (GJA3) gene. PMID- 10746564 TI - A simple and accurate method for determination of microsatellite total allele content differences between DNA pools. AB - DNA pooling is a potential tool for the efficient analysis of the large numbers of samples and DNA markers that are necessary for genome-wide association studies. A simple accurate method for measuring total allele differences in comparisons between two pools containing large numbers of DNA samples is presented. This method compares relative peak height differences between electrophoretograms for each allele of a microsatellite. The method was evaluated by the analysis of 11 microsatellite markers and DNA pooled sample sizes of 50, 100, and 200 individual DNA samples from the same number of different subjects. Pools were created from previously individually genotyped subjects and constructed so that the pool comparisons would provide real total allele differences varying from 0% to 55%. Calculated pool differences were then compared with the real total allele differences determined by individual genotyping results. Together over 200 comparisons demonstrated a correlation coefficient of 0.96, which compared favorably with other previous methods of analysis. This method could provide a rapid screen for total allele differences of greater than 10%, a threshold that should be applicable to detecting low relative risk genes in common diseases. Therefore, these studies suggest that DNA pooling could be a useful tool in association studies for the determination of candidate regions for a range of complex genetic diseases. PMID- 10746565 TI - SEL1L, the human homolog of C. elegans sel-1: refined physical mapping, gene structure and identification of polymorphic markers. AB - We have cloned the human full-length cDNA SEL1L, which is highly similar to the C. elegans sel-1 gene, an important negative regulator of the "notch" pathway which acts as a key regulator of the cellular proliferation and specification processes in both vertebrates and invertebrates. The SEL1L gene maps to 14q24.3 31 and here we report its fine localization by HAPPY mapping, which determines its molecular distance to microsatellite markers isolated in the region. We have found two new polymorphic (CA)n microsatellites located in the gene, and have identified the exon-intron boundaries. The gene is composed of 21 exons spanning 70 kb of genomic DNA. Human SEL1L protein exhibits a high degree of similarity compared to the mouse and nematode homologs. PMID- 10746566 TI - Compound heterozygous mutations in the flavoprotein gene of the respiratory chain complex II in a patient with Leigh syndrome. AB - Succinate dehydrogenase (SDH) deficiency represents a minor cause of Leigh syndrome (LS). Noticeably, the first mutation in a nuclear-encoded respiratory chain component, a mutation in the 5p15 copy of the flavoprotein (Fp) subunit gene of the SDH, was reported 4 years ago in two siblings with LS and SDH deficiency. We now report a new patient with LS and SDH deficiency. Because two copies of the Fp gene are present in the human genome, we first determined the complete structure of these two copies. This allowed us to identify a 1 bp deletion creating a frameshift in the 3q29 copy, confirming that this second copy was a pseudogene. We also sequenced the promoter region of the 5p 15 gene and, in addition, screened for mutations in the patient. Sequencing of the Fp SDH cDNA in the patient only allowed us to identify a heterozygous C to T transition, changing an alanine to a valine in one allele. This transition was found to be heterozygous in the patient's father but was absent from 150 controls. Transfection of the corresponding mutant cDNA into human Fp-deficient cells failed to restore normal SDH activity, confirming the deleterious effect of this mutation. The second allele, inherited from the mother, carried an A to C substitution changing the methionine translation initiation codon to a leucine. This mutant transcript represented only 10% of total Fp transcript suggesting instability of this transcript. So far, profound deficiencies in complex II activity resulting from mutations in the Fp gene of the SDH present only as LS, a striking observation in view of the ubiquitous expression of this typical housekeeping gene in humans. PMID- 10746567 TI - Complex inheritance of ABCR mutations in Stargardt disease: linkage disequilibrium, complex alleles, and pseudodominance. AB - Stargardt disease is a recessively transmitted disease caused by mutations in the ABCR gene. Linkage disequilibrium has recently been reported between a polymorphism, 2828 A, and a common Western European founder mutation, 2588 C. Here, we confirm this linkage disequilibrium in a North American population. We also describe two complex alleles involving the 2828 A and 2588 C alterations and suggest a possible order of clinical severity of mutations identified in trans to the complex alleles. Finally, we report pseudodominance of Stargardt disease in a family with the 2588 C mutation, further supporting a high frequency of carriers for ABCR mutations in our population. PMID- 10746568 TI - Molecular analysis of the genotype-phenotype relationship in factor X deficiency. AB - Factor X deficiency is a rare haemorrhagic condition, normally inherited as an autosomal recessive trait, in which a variable clinical presentation correlates poorly with laboratory phenotype. The factor X (F10) genes of 14 unrelated individuals with factor X deficiency (12 familial and two sporadic cases) were sequenced yielding a total of 13 novel mutations. Family studies were performed in order to distinguish the contributions of individual mutant F10 alleles to the clinical and laboratory phenotypes. Missense mutations were studied by means of molecular modelling, whereas single basepair substitutions in splice sites and the 5' flanking region were examined by in vitro splicing assay and luciferase reporter gene assay respectively. The deletion allele of a novel hexanucleotide insertion/deletion polymorphism in the F10 gene promoter region was shown by reporter gene assay, to reduce promoter activity by approximately 20%. One family manifesting an autosomal dominant pattern of inheritance possessed three clinically affected members who were heterozygous for a splice-site mutation that was predicted to lead to the production of a truncated protein product. A model which accounts for the dominant negative effect of this lesion is presented. Variation in the antigen level of heterozygous relatives of probands was found to be significantly higher between families than within families, consistent with the view that the nature of the F10 lesion(s) segregating in a given family is a prime determinant of the laboratory phenotype. By contrast, no such relationship could be discerned between laboratory phenotype and polymorphism genotype. PMID- 10746569 TI - Development and evaluation of a PCR-microplate capture hybridization method for direct detection of verotoxigenic Escherichia coli strains in artificially contaminated food samples. AB - For the purpose of detecting, directly in food, verotoxigenic Escherichia coli, a microplate hybridization method for the detection of PCR products from the SLT I and SLT II genes, was developed and evaluated. Two pairs of primers and two probes, specific for the SLT I gene and for the SLT II gene, were designed and tested. For the strains containing both genes, two PCR products of different molecular weights were obtained, whereas when only one gene was present only one fragment resulted from PCR. The use of the biotin-labeled probes allowed the immobilization of the PCR products in the microtiter plate wells and by this means their detection was possible using an ELISA-based technique. Forty artificially contaminated and fifty naturally contaminated food samples were analyzed by using the PCR-microplate hybridization technique developed in this study. All the artificially contaminated food samples were positive, independently of the number of cells inoculated before the enrichment step, whereas the naturally contaminated food samples were all negative. PMID- 10746570 TI - Occurrence and expression of virulence-related properties of Vibrio species isolated from widely consumed seafood products. AB - In this study, widely consumed fresh seafood products were examined for the presence of Vibrio spp. Thirteen percent of the samples examined were found to be contaminated with halophilic vibrios belonging to the species V. alginolyticus (81.48%), V. parahaemolyticus (14.8%) and V. cholerae non 0:1 (3.7%). A greater isolation frequency (18.9%) was found for mussels. Significant adhesiveness and strong cytotoxicity factors were revealed in a significant number of the Vibrio spp. isolated. These results confirm that the presence of Vibrio spp. in seafood products is common, and suggest that routine examination of such products for these pathogenic agents would be advisable. PMID- 10746571 TI - Quantitative survival of native Salmonella serovars during storage of frozen raw pork. AB - The quantitative survival of material contaminants of Salmonella serovars was studied in raw pork during frozen storage. Raw pork samples were obtained from public markets in Guadalajara, Mexico, and tested for Salmonella. Three positive samples were selected for survival studies in three different trials. Populations of Salmonella were determined by the most probable number (MPN) method, with isolation on bismuth sulfite agar plates. One typical colony was selected from each plate and subjected to serovar identification. Approximately 20 colonies were serotyped for each portion of frozen pork at each sampling time during storage. During frozen storage, numbers of Salmonella were reduced from 7-11 to 1.6 MPN g(-1) over a period of 22 weeks in Trial 1, from 1500 9000 to 2.5 MPN g( 1) over 42 weeks in Trial 2, and from 2000-20,000 to 20 MPN g(-1) over 78 weeks in Trial 3. The number of different Salmonella serovars identified was 10, 14 and 29 for Trials 1, 2 and 3, respectively. In Trial 3, S. agona, S. newbrunswick, S. drypool and S. anatum predominated over the other 25 serovars identified. S. agona was not only the most prevalent, but also the most abundant. At 15 weeks of storage, estimated MPNs of this serovar were 700 g(-1) of pork. Most serovars were detected sporadically; nine were isolated only once, and nine only twice. Serovars such as S. derby and S. newlands appeared only at the first sampling time, while others such as S. schwarzengrund, S. dublin and S. newport appeared only at the last sampling time. Most serovars identified in this study are commonly isolated from human clinical sources and from raw or processed foods in Mexico. PMID- 10746572 TI - On the design of optimal dynamic experiments for parameter estimation of a Ratkowsky-type growth kinetics at suboptimal temperatures. AB - It is generally known that accurate model building, i.e., proper model structure selection and reliable parameter estimation, constitutes an essential matter in the field of predictive microbiology, in particular, when integrating these predictive models in food safety systems. In this context, Versyck et al. (1999) have introduced the methodology of optimal experimental design techniques for parameter estimation within the field. Optimal experimental design focuses on the development of optimal input profiles such that the resulting rich (i.e., highly informative) experimental data enable unique model parameter estimation. As a case study, Versyck et al. (1999) [Versyck, K., Bernaerts, K., Geeraerd, A.H., Van Impe, J.F., 1999. Introducing optimal experimental design in predictive modeling: a motivating example. Int. J. Food Microbiol., 51(1), 39-51] have elaborated the estimation of Bigelow inactivation kinetics parameters (in a numerical way). Opposed to the classic (static) experimental approach in predictive modelling, an optimal dynamic experimental setup is presented. In this paper, the methodology of optimal experimental design or parameter estimation is applied to obtain uncorrelated estimates of the square root model parameters [Ratkowsky, D.A., Olley, J., McMeekin, T.A., Ball, A., 1982. Relationship between temperature and growth rate of bacterial cultures. J. Bacteriol. 149, 1-5] describing the effect of suboptimal growth temperatures on the maximum specific growth rate of microorganisms. These estimates are the direct result of fitting a primary growth model to cell density measurements as a function of time. Apart from the design of an optimal time-varying temperature profile based on a sensitivity study of the model output, an important contribution of this publication is a first experimental validation of this innovative dynamic experimental approach for uncorrelated parameter identification. An optimal step temperature profile, within the range of model validity and practical feasibility, is developed for Escherichia coli K12 and successfully applied in practice. The presented experimental validation result illustrates the large potential of the dynamic experimental approach in the context of uncorrelated parameter estimation. Based on the experimental validation result, additional remarks are formulated related to future research in the field of optimal experimental design. PMID- 10746573 TI - The effect of 100% CO2 on the growth of nonproteolytic Clostridium botulinum at chill temperatures. AB - The growth of a cocktail of spores from six nonproteolytic Clostridium botulinum type B and E isolates at 5 and 10 degrees C was used to assess the combined effect of NaCl (0.5-4.5% w/v), pH (5.5-6.5) and atmosphere (10% H2:90% N2, 5% CO2:10% H2:85% N2, or 100% CO2) in buffered peptone, yeast, glucose, starch broth with an Eh of approximately -350 mV. Under all atmospheres growth tended to be slower as the concentration of NaCl increased and with NaCl combined with pH levels below 6.0. Of the atmospheres tested, growth occurred at a slower rate and over a narrower range of conditions when C. botulinum was exposed to 100% CO2. This effect was enhanced when the incubation temperature was 5 degrees C. The results indicate that while CO2 decreased C. botulinum growth at chill temperatures, prevention of growth also depended on the NaCl concentration and the pH of the medium. PMID- 10746574 TI - Physiology of dairy-associated Bacillus spp. over a wide pH range. AB - Bacillus species isolated from alkaline wash solutions used for cleaning in place in South African dairy factories have been suggested to contaminate product contact surfaces of dairy processing equipment and result in post-pasteurization spoilage of milk and milk products. Growth and attachment of such Bacillus isolates under alkaline and acidic conditions have not been previously described. Therefore, the in vitro growth temperature and pH ranges, attachment abilities and hydrophobicity, and enzyme production capabilities of four Bacillus isolates (tentatively identified as B. subtilis115, B. pumilus122, B. licheniformis137 and B. cereus144) previously isolated from the alkaline wash solutions in a South African dairy were examined. Growth pH ranges were determined in buffered Standard One-like Nutrient Broth and in unbuffered 1% Milk Medium at pH values ranging from 3 to 12. Growth and attachment to stainless steel surfaces and production of protease and lipase enzymes were determined in 1% Milk Medium at pH 4, 7 and 10. Colony hydrophobicity of each isolate by the Direction of Spreading Method (DOS) was also determined at pH 4, 7 and 10. In addition, Arrhenius plots were used to examine the growth temperature ranges of the isolates. All isolates grew at pH values ranging from 4.5 to 9.5 in buffered Standard One-like Nutrient Broth, and from pH 4 to 10 in 1% Milk Medium. All isolates also attached to stainless steel at pH 4, 7 and 10 in 1% Milk Medium. Generally the attachment of B. subtilis115, B. pumilus122 and B. lichenformis137 to stainless steel surfaces was enhanced at pH 4 and 10, compared to pH 7. By contrast, the best attachment of B. cereus144 cells to stainless steel surfaces was at pH 7. Planktonic and attached cells of all isolates produced proteolytic enzymes at pH 7 and 10, but not at pH 4. Similarly, planktonic and attached cells of B. subtilis115, B. pumilus122 and B. licheniformis137 produced lipolytic enzymes at pH 7 and 10, and weak lipolysis was observed at pH 4. The Bacillus cereus144 isolate showed no lipolytic activity at pH 10. All isolates exhibited low hydrophobic properties at all pH values even though attachment to stainless steel at the same pH values occurred. None of the isolates grew below 11 degrees C or above 56 degrees C, and optimum growth temperatures were in the high mesophilic range (36-44 degrees C). PMID- 10746575 TI - Suitability of log-linear models to evaluate the microbiological quality of baby clams (Chamelea gallina L.) harvested in the Adriatic Sea. AB - The presence of fecal coliforms or Escherichia coli in baby clams (Chamelea gallina L.) is considered an indicator related to their safety because they can be correlated with the presence of pathogenic bacteria. For this reason the Italian regulation has defined limits for these microorganisms. The presence of these microbial indicators is dependent on various environmental variables. In this work all the variables considered are categorical and, consequently, the traditional approach of predictive microbiology was not applicable. The data were summarized by means of a cross-tabulation and analyzed using the log-linear model technique. This statistical technique is widely used in social and economic studies but only partially developed in food microbiology. The suitability of the log-linear model to analyse microbiological data in relation to environmental variables was evaluated. In particular, the microbiological quality of baby clams harvested in five different areas of the Adriatic Sea coast in Emilia Romagna (Italy) was considered. The influence of the season and geographical origin on microbiological standards was assessed. A logit model was developed to predict the frequencies, depending on geographical origin and season, of samples with concentrations of the indicator organisms below or above the legal standards provided by Italian regulation. PMID- 10746576 TI - Occurrence of pathogenic Yersinia enterocolitica in Norwegian pork products determined by a PCR method and a traditional culturing method. AB - From October 1997 to April 1998, a survey was conducted to assess the occurrence of pathogenic Yersinia enterocolitica in Norwegian pork products, using a traditional culturing method and a PCR assay. A total of 300 pork samples was examined. Five slaughterhouses in the Norwegian Meat Cooperative were represented with 249 samples and another 51 samples were obtained from retail outlets in the city of Oslo. Using the NMKL method, Y. enterocolitica 0:3 was isolated from six (2%) of the samples, while the PCR method indicated presence of pathogenic Y. enterocolitica in 50 (17%) of the samples. The results indicate that a reduction has occurred in the prevalence of pathogenic Y. enterocolitica in Norwegian pork products, as compared to a previous Norwegian study conducted in 1988-1989. The study also highlights the need for further development and improvement of methods applied for the detection of pathogenic Y. enterocolitica in foods. PMID- 10746577 TI - Strictly anaerobic halophiles isolated from canned Swedish fermented herrings (Surstromming). AB - Strictly anaerobic halophiles were isolated from canned Swedish fermented herrings (Surstromming). All isolates were phenotypically uniform with some exceptions and were identified as the genus Haloanaerobium and assigned to either Haloanaerobium praevalens or Haloanaerobiuim alcaliphilum. A comparative analysis of 16S rDNA sequences revealed that the representative strain S-8 of the isolates was identical to that of Haloanaerobium praevalens DSM 2228T. Furthermore, this strain exhibited high levels (> 80%) of DNA-DNA homology with Haloanaerobium praevalens DSM 2228T. This is a novel report of halophilic anaerobes isolated from a food product. Such anaerobes may contribute to the intense flavor and the swollen can characteristics of Swedish fermented herring. PMID- 10746578 TI - Pulsed electric fields inactivation of attached and free-living Escherichia coli and Listeria innocua under several conditions. AB - The use of pulsed electric fields (PEF) is considered as a mild process in the inactivation of microorganisms present in liquid food products. PEF treatments of Escherichia coli and Listeria innocua suspended in milk and phosphate buffer, with same pH and same conductivities, yielded to similar inactivation. Reduction rates obtained in distilled water indicated that conductivity of the food product is a main parameter in bacterial inactivation. Bacteria attached to polystyrene beads were inactivated by PEF at a greater (E. coli) or equal rate (L. innocua) than free-living bacteria. Base on the use of selective and non-selective enumeration media, no clear indications were obtained for sublethal damage of microorganisms surviving the PEF treatment. E. coli cells subjected to 60 pulses at 41 kV/cm were examined by transmission and scanning electron microscopy. Changes in the cytoplasm were observed and the cell surface appeared rough. The cells outer membranes were partially destroyed allowing leaking of cell cytoplasm. PMID- 10746579 TI - Bacteriological quality of raw oysters in Trinidad and the attitudes, knowledge and perceptions of the public about its consumption. AB - In Trinidad and Tobago, raw oyster cocktails are a delicacy, but they are generally believed to be responsible for illness in the consumer. The microbial loads of raw oysters, condiments/spices and ready-to-consume oyster cocktails were determined in four sampling areas. Questionnaires were also administered to 72 oyster vendors to determine practices that affect the bacteriological quality of the products. Three hundred members of the public were interviewed to determine the attitudes, knowledge and perceptions of the public about raw oyster consumption. The mean total aerobic plate count (TAPC) per g of 50 samples each, of raw oysters, condiments/spices and ready-to-consume raw oyster cocktails averaged from 1.0 x 10(7)+/-4.3 x 10(7) to 1.4 x 10(8)+/-6.4 x 10(8), 2.0 x 10(5)+/-1.0 x 10(6) to 2.0 x 10(7)+/-1.4 x 10(8), and 4.3 x 10(5)+/-1.0 x 10(6) to 2.2 x 10(6)+/-1.0 x 10(7), respectively. The difference for each product among the four areas was statistically significant (P < or = 0.05; chi2). Using a recommended maximum standard of TAPC per g of 5.0 x 10(5), 115 (57.5%), 27 (13.5%) and 51 (25.5%) of 200 samples each, of raw oysters, condiments/spices and oyster cocktails, respectively, were considered unfit for human consumption. Amongst vendor practices, source of oyster harvest and length of time between separation of oyster meat from shell and sale, significantly affected the mean TAPC per g and the prevalence of unfit oyster cocktail samples. Consumption of raw oyster cocktails was more prevalent amongst males (73.6%) than females (26.4%) (P < 0.002), East Indians (63.2%) as compared with other respondents (36.8%) (P < 0.001), individuals < or = 40 years old (82.1%) than in individuals > 40 years of age (17.9%) (P < 0.01), and in individuals who were aware that raw oysters are considered to be a sexual enhancer (86.8%) as compared to those who did not have this perception (11.3%) (P < 0.03). Fear of falling ill prevented 44 (37.9%) of 116 non-consumers from eating oyster cocktails, while 13 of 106 consumers (12.3%) reported having experienced an oyster-borne illness. The rather high prevalence of raw oyster cocktails found to be unfit for human consumption, coupled with the perceptions and attitudes of the consumers about the product, pose a significant health risk to the public. PMID- 10746580 TI - Inhibitory combinations of nisin, sodium chloride, and pH on Listeria monocytogenes ATCC 15313 in broth by an experimental design approach. AB - The influence of pH (5.0-8.2), NaCl concentrations (0-6% w/v), and incubation time (0-24 h) on the inhibitory activity of nisin (0-100 I.U./ml) against Listeria monocytogenes (10(3) cfu/ml) was studied using the Doehlert experimental design and was confirmed by kinetic experiments. Predicted values were in agreement with experimental values. Experiments were carried out at 22 degrees C in reconstituted TSB-YE1 broth with or without NaCl. Nisin had an immediate pH dependent bactericidal effect, which increased with decreasing pH values. In modified TSB-YE1 broth without NaCl, the bactericidal efficacy of nisin (50 I.U./ml) was maximum at pH 6.6, with no L. monocytogenes survivors until 120 h at 22 degrees C. Nisin (50 I.U./ml) action decreased in the presence of NaCl, with a minimal inhibitory effect between 2 and 4%. This partially protective effect was cancelled at higher levels of nisin. PMID- 10746581 TI - Inhibitory action of potassium sorbate degradation products against Staphylococcus aureus growth in laboratory media. AB - The inhibitory action of potassium sorbate (KS) degradation products against Staphylococcus aureus growth was examined in model aqueous systems. KS degradation products formation was induced by the storage at 70 degrees C for 7 days or at 37 degrees C for 90 days of aqueous systems containing basically brain heart infusion, yeast extract and different amounts of KS at pH 5.5. After storage, residual amounts of KS were measured and each system was inoculated with a pool of three S. aureus strains at 10(4) colony forming units/ml. Degradation products from KS generally inhibited S. aureus growth by two to three log cycles. However, the population of S. aureus reached a higher level when the degradation products were formed at 37 degrees C suggesting that the nature of the degradation products seemed to depend on the temperature at which they were produced. The inhibitory activity produced by KS degradation products along with residual amounts of KS was smaller than the one produced by the initial amounts of the preservative. PMID- 10746582 TI - Outbreak of viral gastroenteritis due to sewage-contaminated drinking water. AB - In August 1998, a large outbreak of gastroenteritis occurred in a Swiss village of 3500 inhabitants whereof more than 50% were affected. A high contamination of drinking water with faecal coliforms revealed a defect in the waste water system. The objective of the present study was to investigate the outbreak in respect of the presence of human pathogenic viruses. Drinking water and clinical samples from patients were examined for the presence of 'Norwalk-like viruses' (NLVs) and enteroviruses. NLVs and enteroviruses were detected by reverse transcription polymerase chain reaction (RT-PCR) in one of two drinking water samples. Five of seven stool samples from ill persons were positive for NLVs. Typing of NLV specific RT-PCR products by DNA sequencing revealed the presence of an identical genogroup-1 strain closely related to Southampton virus in both the water and one of the stool samples. A genogroup-2 NLV strain was identified in all positive stool samples. The enteroviral amplicon showed high sequence similarity with swine vesicular disease virus. These results demonstrate that the drinking water was highly contaminated with enteric viruses and that at least two NLV strains were involved in this outbreak of gastroenteritis. PMID- 10746583 TI - Application of thin agar layer method for recovery of injured Salmonella typhimurium. AB - Xylose lysine decarboxylase (XLD) medium, a selective plating medium, can inhibit heat-injured Salmonella typhimurium from growing, whereas tryptic soy agar (TSA), a nonselective medium, does not. To facilitate recovery of heat-injured S. typhimurium cells while providing selectivity of isolation of S. typhimurium from other bacteria in the sample, a thin agar layer (TAL) procedure was developed by overlaying 14 ml of nonselective medium (TSA) onto prepoured and solidified XLD medium in a 8.5 cm diameter Petri dish. During the first few hours of incubating the plate, the injured S. typhimurium repaired and started to grow in the TSA. During the resuscitation of injured cells, the selective agents from XLD were diffused to the TSA top layer part. Once the selective agents diffused to the top part of the TAL, the resuscitated S. typhimurium started to produce a typical reaction (black color) and other microorganisms were inhibited by the selective agents. The recovery rate for heat-injured (55 degrees C for 15 min) S. typhimurium with the TAL method was compared with TSA, XLD, and the traditional overlay method (OV; pouring selective agar on top of resuscitated cells on TSA agar 3-4 h after incubation). No significant difference occurred among TSA, OV, and TAL (P > 0.05) for enumeration of heat-injured S. typhimurium, but they recovered significantly higher numbers than from XLD agar (P < 0.05). PMID- 10746584 TI - MS: the return of the B cell. PMID- 10746585 TI - Specific cognitive deficits in neurologic and psychiatric disease: targets for treatment. PMID- 10746586 TI - Emotional facial paresis of pontine origin. PMID- 10746587 TI - New nomenclature and DNA testing guidelines for myotonic dystrophy type 1 (DM1). The International Myotonic Dystrophy Consortium (IDMC). PMID- 10746588 TI - Cerebral sinus thrombosis diagnosed by MRI and MR venography in cancer patients. AB - OBJECTIVE: To report the characteristics of cerebral sinus thrombosis (CST) in cancer patients diagnosed by MRI and MR venography (MRV). BACKGROUND: CST is a complication of cancer with multiple etiologies and variable symptoms at presentation. Most reports in cancer patients were before the use of MRI and MRV, which has simplified the diagnosis of CST. METHODS: The neurology database at Memorial Sloan-Kettering Cancer Center was used to identify cancer patients with a diagnosis of CST between January 1994 and April 1998. RESULTS: Twenty patients were identified. Nine had hematologic malignancies (HMs) and 11 had solid tumors (STs). The median interval from cancer diagnosis to presentation was 4 months for HMs and 20 months for STs. The most common symptom was headache. MRI and MRV correlated in all but three patients, and MRV was more sensitive in four patients. The most frequently involved cerebral sinus was the superior sagittal sinus. Multiple sinuses were affected in 8 of 19 patients. Five patients had a cerebral or subarachnoid hemorrhage and three had infarction. Disorders of coagulation were the most frequent etiology in patients with HM; compression or invasion of the cerebral sinus from dural/calvarial metastasis was the main cause in those with ST. Treatment was directed at the underlying cause. Ten of 20 patients improved clinically and 3 of 6 patients improved radiologically. CONCLUSION: MRI and MRV can diagnose CST accurately in cancer patients. Causes of CST depend on cancer type, and treatment varies with etiology. Most patients have a good outcome. PMID- 10746589 TI - Comparison of immunoglobulin G heavy-chain sequences in MS and SSPE brains reveals an antigen-driven response. AB - OBJECTIVE: To better understand B-cell activation in MS by analyzing the immunoglobulin (Ig)G heavy chain variable region (VH) repertoire found in MS brains and comparing it with brain VH sequences in individuals with subacute sclerosing panencephalitis (SSPE)--a chronic encephalitis produced by measles virus (MV)-and characterized by an antigen-driven oligoclonal IgG response to MV antigens. BACKGROUND: The specificity of oligoclonal IgG in MS CSF and plaques, and their relevance to the pathogenesis of MS is unknown. METHODS: Nested PCR was used to amplify and sequence the rearranged IgG heavy-chain VH repertoire in plaques of three acute MS brains and in three SSPE brains. A representative population of VH sequences from each tissue was aligned to the known 51 functional VH germline segments. From this the authors determined the closest VH family germline segment, and the degree and location of somatic mutations for each unique IgG. RESULTS: As expected for an antigen-driven response against MV antigens, most VH sequences from the SSPE brains were mutated extensively compared with their closest germline segments. Furthermore, SSPE VH sequences accumulated replacement mutations preferentially in the complementary-determining regions (CDRs) relative to framework regions-features normally observed during antigen-driven selection. A comparison of VH family and germline usage also demonstrated that each SSPE brain had its own unique IgG response. When the authors compared the VH response in MS plaques with SSPE, MS VH sequences were also mutated extensively, displayed a preferential accumulation of replacement mutations in CDRs, and were unique in each MS brain. CONCLUSION: The presence of an antigen-driven response in MS, rather than a nonconventional mechanism of B cell activation, warrants additional analysis of the specificity of IgG in MS brain and CSF. PMID- 10746591 TI - Methanol and the brain. PMID- 10746590 TI - MS functional composite: relation to disease phenotype and disability strata. AB - OBJECTIVE: The MS Functional Composite (MSFC), a recently developed outcome measure for clinical trials, was applied to 240 patients with MS to explore its utility in different subgroups of MS and for comparison with the Expanded Disability Status Scale (EDSS). METHOD: Three clinical dimensions were examined: arm/hand function, leg function/ambulation, and cognition. Predictions of relative scores on the MSFC and its components in three major MS phenotypes (relapsing-remitting, primary progressive, and secondary progressive) and three strata of disability were developed and tested. Also, correlations with EDSS were calculated and the effect of an external reference population was assessed. RESULTS: Mean MSFC score was positive in the relapsing-remitting (0.4) and mildly disabled (0.4) groups and negative in the secondary progressive (-0.3), primary progressive (-0.4), and moderately (-0.07) and severely disabled (-1.0) groups. The correlation between EDSS and MSFC was strong (-0.68). EDSS correlated strongly with ambulation in secondary and primary progressive patients and severely disabled patients, moderately with arm/hand function for all analyzed groups, and not at all with cognition. Comparison with an external reference population showed changes in MSFC- and Z-scores, but did not result in altered differences between the subgroups. CONCLUSION: Our prospective study in subgroups of MS confirmed and extended the construct validity of the MSFC. The MSFC also showed good concurrent validity with the EDSS, and includes information about cognition. PMID- 10746592 TI - Neurologic complications in immune-mediated heparin-induced thrombocytopenia. AB - OBJECTIVE: To evaluate neurologic complications in patients with immune-mediated heparin-induced thrombocytopenia (HIT) with respect to incidence, clinical characteristics, outcome, and therapy. METHODS: One hundred and twenty consecutive patients with immune-mediated HIT were recruited over a period of 11 years and studied retrospectively for the occurrence of neurologic complications. Diagnosis of HIT was based on established clinical criteria and confirmed by detection of heparin-induced antibodies using functional and immunologic tests. RESULTS: Eleven of the 120 patients (9.2%) presented with neurologic complications; 7 suffered from ischemic cerebrovascular events, 3 from cerebral venous thrombosis, and 1 had a transient confusional state during high-dose heparin administration. Primary intracerebral hemorrhage was not observed. The relative mortality was much higher (Chi-square test, p < 0.01) in HIT patients with neurologic complications (55%) as compared to patients without neurologic complications (11%). The mean platelet count nadir in neurologic patients was 38 +/- 25 x 10(9)/l on average, and was lower in patients with fatal outcome compared to those who survived (21 +/- 13 x 10(9)/l versus 58 +/- 21 x 10(9)/l; p < 0.05, Wilcoxon test). In three patients neurologic complications preceded thrombocytopenia. There was a high coincidence of HIT-associated neurologic complications with other HIT-associated arterial or venous thrombotic manifestations. CONCLUSION: Neurologic complications in HIT are relatively rare, but associated with a high comorbidity and mortality. HIT-associated neurologic complications include cerebrovascular ischemia and cerebral venous thrombosis. They may occur at a normal platelet count. PMID- 10746593 TI - Neural and endothelial control of the microcirculation in diabetic peripheral neuropathy. AB - OBJECTIVE: To examine the role played by endothelium-dependent and endothelium independent vasodilation of the cutaneous microcirculation and their relationship to neural microcirculatory control in type 1 and type 2 diabetic patients. METHODS: Acetylcholine and sodium nitroprusside were iontophoresed using a dose response technique. Endothelium-dependent, endothelium-independent, and C-fiber mediated vasodilation were measured with a laser Doppler device. RESULTS: Endothelium-dependent vasodilation of the forearm cutaneous microcirculation was attenuated in diabetic subjects. The response was less in type 2 diabetic subjects than in controls (p < 0.005). In contrast, there was no significant difference between type 1 diabetic subjects and controls. There was no significant abnormality in endothelium-independent vasodilation in either diabetic group. The C-fiber-mediated axon reflex in the forearm was impaired in both type 1 and type 2 diabetics, which is consistent with a small-fiber neuropathy (p < 0.005). The duration of diabetes in type 2 diabetics was a significant predictor of the maximum endothelium-mediated vasodilation. CONCLUSION: Changes in cutaneous blood flow are seen relatively early in the course of diabetic peripheral neuropathy and are characterized by endothelial and neural but not smooth muscle dysfunction. The presence of significant C-fiber impairment in both diabetic groups, together with significantly greater dysfunction in endothelium-dependent vasodilation in type 2 diabetics, suggests that the endothelial function and nitric oxide may play a greater role in the pathogenesis of diabetic peripheral neuropathy in type 2 diabetic patients. PMID- 10746594 TI - Outcome of surgery for thoracic outlet syndrome in Washington state workers' compensation. AB - OBJECTIVE: To determine the predictors of outcome of thoracic outlet syndrome (TOS) surgery in a population-based cohort of injured workers. METHODS: All injured workers in the Washington State Workers' Compensation system who received TOS surgery during 1986 to 1991 were identified by computerized bill payment records and validated by medical record review (n = 158). The main outcome measure was work disability status 1 year after surgery. Additional functional status and quality of life outcomes were determined by telephone survey an average of 4.8 years after operation. A sample of workers with a TOS diagnosis who did not receive surgery during 1987 to 1989 were identified as a comparison group (n = 95). RESULTS: Sixty percent of workers were still work disabled 1 year after surgery. The strongest predictors of remaining disabled were the amount of work disability before surgery (OR = 1.85; 95% CI, 1.51 to 2.28), longer time between injury and TOS diagnosis (OR = 1.34; 95% CI, 1.09 to 1.64), and older age at injury (OR = 1.07; 95% CI, 1.00 to 1.13). There was no relationship between type of surgery, presence of any provocative tests, or experience of surgeon and work disability outcome. In follow-up surveys an average of 4.8 years after surgery, 72.5% of workers still reported they were "limited a lot" in vigorous activities. Compared with a nonsurgical sample of TOS patients, those receiving surgery had 50% greater medical costs and were three to four times more likely to be work disabled. CONCLUSIONS: The outcome of TOS surgery among injured workers is worse than has generally been reported. The nonspecific neurogenic TOS diagnosis, the complexity of workers' compensation cases, and the adverse event profile are likely substantial contributors to the worse outcomes reported here. Well-designed prospective studies and randomized trials are required to elucidate any role of TOS surgery in nonspecific TOS. PMID- 10746595 TI - Testing memory for self-generated items in dementia: method makes a difference. AB - OBJECTIVE: To learn how pAD (probable Alzheimer's disease), PD+ ("Parkinson's Plus" syndrome), and control subjects remember internally generated material under different conditions. BACKGROUND: "Self-discovered," or internally generated knowledge, prized by educators and therapists, can bring about considerable behavioral change. Both parietal-temporal-limbic (pAD) and frontal subcortical dementia (e.g. PD+) cause dysmemory, but may cause different internal external memory bias. pAD subjects, confusing internal and external information (confabulation) and reporting internal information during memory testing (intrusions), may be biased to remember internal material. PD+ subjects, impaired at generative tests, may be externally biased. METHODS: Ten pAD, 5 PD+, and 10 control subjects generated words in a category without instruction to remember (INR), and took a list-learning test of incidental memory for internally and externally generated words. To test how INR influences memory, subjects then generated and attempted to recall four more words. RESULTS: All three subject groups remembered more internally generated than externally provided words without INR. Recall versus recognition of internally generated words differed by group, with PD+ subjects showing greatest improvement with recognition. The pAD subjects performed worse with INR than without INR, had the most intrusion errors, and, rather than demonstrating a release from proactive inhibition, recalled fewer words outside the category. Groups differed in overall recall/recognition improvement (p = 0.015). CONCLUSIONS: Aged subjects preferentially retained internally generated material. However, among demented subjects, memory for internally generated words was influenced by the testing method used. PD+ subjects have poor internal recall, but excellent internal recognition. In pAD, memory for internally generated words may exceed external memory, but only when subjects are not explicitly trying to remember. PMID- 10746596 TI - Association of vitamin E and C supplement use with cognitive function and dementia in elderly men. AB - OBJECTIVE: To determine whether use of vitamin E and C supplements protects against subsequent development of dementia and poor cognitive functioning. METHODS: The Honolulu-Asia Aging Study is a longitudinal study of Japanese American men living in Hawaii. Data for this study were obtained from a subsample of the cohort interviewed in 1982, and from the entire cohort from a mailed questionnaire in 1988 and the dementia prevalence survey in 1991 to 1993. The subjects included 3,385 men, age 71 to 93 years, whose use of vitamin E and C supplements had been ascertained previously. Cognitive performance was assessed with the Cognitive Abilities Screening Instrument, and subjects were stratified into four groups: low, low normal, mid normal, and high normal. For the dementia analyses, subjects were divided into five mutually exclusive groups: AD (n = 47), vascular dementia (n = 35), mixed/other types of dementia (n = 50), low cognitive test scorers without diagnosed dementia (n = 254), and cognitively intact (n = 2,999; reference). RESULTS: In a multivariate model controlling for other factors, a significant protective effect was found for vascular dementia in men who had reported taking both vitamin E and C supplements in 1988 (odds ratio [OR], 0.12; 95% CI, 0.02 to 0.88). They were also protected against mixed/other dementia (OR, 0.31; 95% CI, 0.11 to 0.89). No protective effect was found for Alzheimer's dementia (OR, 1.81; 95% CI, 0.91 to 3.62). Among those without dementia, use of either vitamin E or C supplements alone in 1988 was associated significantly with better cognitive test performance at the 1991 to 1993 examination (OR, 1.25; 95% CI, 1.04 to 1.50), and use of both vitamin E and C together had borderline significance (OR, 1.18; 95% CI, 0.995 to 1.39). CONCLUSIONS: These results suggest that vitamin E and C supplements may protect against vascular dementia and may improve cognitive function in late life. PMID- 10746597 TI - APOE and the risk of PD with or without dementia in a population-based study. AB - OBJECTIVE: To study the association between APOE genotype and PD with or without dementia. METHODS: The study formed part of the Rotterdam Study, a prospective, population-based cohort study on the frequency, etiology, and prognosis of chronic diseases. The cohort examined for PD consisted of 6,969 independently living or institutionalized inhabitants from a suburb of Rotterdam, the Netherlands, aged 55 years or older. All participants were screened at baseline (1990 to 1993) and at follow-up (1993 to 1994) for symptoms of parkinsonism by study physicians; screen positives received a diagnostic workup by a neurologist. RESULTS: APOE genotyping was available for 107 PD patients (26 with and 81 without dementia) and 4,805 non-PD control subjects. The presence of at least one epsilon2 allele significantly increased the risk of PD (OR = 1.7; 95% CI, 1.0 to 2.8). When we looked separately for demented and nondemented PD patients as compared with nonparkinsonian controls, APOE did not appear to be associated with PD without dementia, but both the epsilon2 and the epsilon4 allele increased the risk of PD with dementia (OR = 5.6; 95% CI, 2.0 to 15.2 and OR = 3.6; 95% CI, 1.3 to 9.9). The risk of dementia for epsilon4 allele carriers was not significantly different for persons with or without PD. However, the epsilon2 allele strongly increased the risk of dementia in patients with PD (interaction p < 0.007). CONCLUSIONS: In the elderly the APOE-epsilon2 allele increases the risk of PD and, in particular, the risk of PD with dementia. PMID- 10746598 TI - White matter abnormalities in mobility-impaired older persons. AB - OBJECTIVE: To investigate the relationship between white matter abnormalities and impairment of gait and balance in older persons. METHODS: Quantitative MRI was used to evaluate the brain tissue compartments of 28 older individuals separated into normal and impaired groups on the basis of mobility performance testing using the Short Physical Performance Battery. In addition, individuals were tested on six indices of gait and balance. For imaging data, segmentation of intracranial volume into four tissue classes was performed using template-driven segmentation, in which signal-intensity-based statistical tissue classification is refined using a digital brain atlas as anatomic template. RESULTS: Both decreased white matter volume, which was age-related, and increased white matter signal abnormalities, which were not age-related, were observed in the mobility impaired group compared with the control subjects. The average volume of white matter signal abnormalities for impaired individuals was nearly double that of control subjects. CONCLUSIONS: This cross-sectional study suggests that decreased white matter volume is age-related, whereas increased white matter signal abnormalities are most likely to occur as a result of disease. Both of these changes are independently associated with impaired mobility in older persons and therefore likely to be additive factors of motor disability. PMID- 10746599 TI - Progression of parkinsonian signs in Alzheimer's disease. AB - OBJECTIVE: To describe the progression of parkinsonian signs in persons with AD. BACKGROUND: Parkinsonian signs are common in AD and appear to be related to morbidity and mortality. However, little is known about individual patterns of progression of parkinsonian signs. METHODS: A cohort of 410 people with clinically diagnosed AD underwent annual clinical evaluations over a 4-year period, with over 90% of survivors participating in follow-up. The entire motor portion of the Unified Parkinson's Disease Rating Scale (UPDRS) was administered at each evaluation. Previously established measures of four parkinsonian signs were derived from the UPDRS. Scores ranged from 0 to 100 and represented the percent obtained of the total possible item score. RESULTS: A growth curve approach was used to estimate individual paths of change. Rates of change in bradykinesia (4.5% increase per year), rigidity (6.0% increase per year), and gait disorder/postural reflex impairment (8.9% increase per year) were substantial and positively correlated (median r = 0.69). Change in tremor was minimal, mostly confined to postural tremor, and weakly correlated with change in other signs (median r = 0.16). The rate of progression in each sign was highly variable across individuals and not strongly related to demographic factors or use of neuroleptic medications. CONCLUSIONS: Parkinsonian signs other than tremor progress rapidly in AD but at widely differing rates. PMID- 10746600 TI - Dementia diagnoses from clinical and neuropsychological data compared: the Cache County study. AB - OBJECTIVE: To validate a neuropsychological algorithm for dementia diagnosis. METHODS: We developed a neuropsychological algorithm in a sample of 1,023 elderly residents of Cache County, UT. We compared algorithmic and clinical dementia diagnoses both based on DSM-III-R criteria. The algorithm diagnosed dementia when there was impairment in memory and at least one other cognitive domain. We also tested a variant of the algorithm that incorporated functional measures that were based on structured informant reports. RESULTS: Of 1,023 participants, 87% could be classified by the basic algorithm, 94% when functional measures were considered. There was good concordance between basic psychometric and clinical diagnoses (79% agreement, kappa = 0.57). This improved after incorporating functional measures (90% agreement, kappa = 0.76). CONCLUSIONS: Neuropsychological algorithms may reasonably classify individuals on dementia status across a range of severity levels and ages and may provide a useful adjunct to clinical diagnoses in population studies. PMID- 10746601 TI - Evaluation of heme oxygenase-1 as a systemic biological marker of sporadic AD. AB - BACKGROUND: Heme oxygenase-1 (HO-1) is a 32-kDa stress protein that catalyzes the degradation of heme to biliverdin. HO-1 immunoreactivity is greatly increased in neurons and astrocytes of the hippocampus and cerebral cortex of individuals with AD and colocalizes to senile plaques and neurofibrillary tangles. METHODS: We investigated whether systemic HO-1 regulation is also deranged in AD patients and whether blood HO-1 measurements provide a peripheral biomarker of the disease. Plasma HO-1 protein levels were measured by competitive ELISA and lymphocyte HO-1 mRNA levels were determined by Northern analysis in patients with early probable sporadic AD, normal elderly controls (NEC), normal younger controls, individuals with age-associated cognitive decline (AACD) not meeting AD criteria, and patients with non-Alzheimer dementia, nondementing neurologic illness, and chronic medical disorders. CSF HO-1 protein concentrations were also determined by ELISA in pathologically confirmed AD and control cases. RESULTS: Mean plasma HO-1 protein concentrations were significantly lower in AD patients (0.85 +/- 0.14 microg/mL) compared with NEC (1.77 +/- 0.34 microg/mL; p < 0.05) and control patients. The AACD group exhibited plasma HO-1 concentrations (1.06 +/- 0.33 microg/mL) intermediate between, but not different from, those of the AD patients and NEC. Lymphocyte HO-1 mRNA levels were lower in the AD cohort relative to NEC (p < 0.001) and individuals with AACD, non-Alzheimer dementia, nondementing neurologic illness, and chronic medical conditions. Lymphocyte HO-1 mRNA levels were also lower in the AACD group relative to NEC (p < 0.05). In comparison with all groups excluding AACD, the sensitivity and specificity of lymphocyte HO-1 mRNA measurement for diagnosis of early sporadic AD are 88% and 75%. Mean CSF HO 1 protein concentrations were lower (p < 0.01) in AD cases (19.07 ng/mL) relative to control values (32.48 ng/mL). CONCLUSIONS: Plasma and CSF HO-1 protein and lymphocyte HO-1 mRNA levels are decreased in subjects with sporadic AD. Quantitative assay for lymphocyte HO-1 mRNA expression may serve as a useful biologic marker in early sporadic AD. PMID- 10746602 TI - MRI volumetric study of dementia with Lewy bodies: a comparison with AD and vascular dementia. AB - OBJECTIVE: To compare global and regional atrophy on MRI in subjects with dementia with Lewy bodies (DLB), AD, vascular dementia (VaD), and normal aging. In addition, the relationship between APOE-epsilon4 genotype and volumetric indices was examined. METHOD: MRI-based volume measurements of the whole-brain, ventricles, frontal lobe, temporal lobe, hippocampus, and amygdala were acquired in elderly subjects with DLB (n = 27; mean age = 75.9 years), AD (n = 25; 77.2 years), VaD (n = 24; 76.9 years), and normal control subjects (n = 26; 76.2 years). RESULTS: Subjects with DLB had significantly larger temporal lobe, hippocampal, and amygdala volumes than those with AD. No significant volumetric difference between subjects with DLB and VaD was observed. Compared with control subjects, ventricular volumes were increased in all patients with dementia, though those with DLB showed a relative preservation of whole-brain volume. There were no significant differences in frontal lobe volumes between the four groups. APOE-epsilon4 status was not associated with volumetric indices. CONCLUSION: The findings support the hypothesis that DLB is associated with a relative preservation of temporal lobe structures. In the differentiation of DLB and AD, this may have important implications for diagnosis. PMID- 10746603 TI - Tourette's syndrome improvement with pergolide in a randomized, double-blind, crossover trial. AB - OBJECTIVE: To determine whether pergolide, a mixed D1-D2-D3 dopamine agonist, is efficacious and safe in the treatment of children with Tourette's syndrome. BACKGROUND: Neuroleptics, which block dopamine transmission, are currently used for treatment of children with severe tics, but major side effects and limited efficacy reduce clinical utility. Prior open-label reports of pergolide suggest potential benefit. METHODS: The authors enrolled 24 children age 7 to 17 years with Tourette's disorder, chronic motor tic disorder, or chronic vocal tic disorder by Diagnostic and Statistical Manual of Mental Disorders (4th ed.) criteria, plus severity criteria on the Yale Global Tic Severity Scale (YGTSS) of > or =20, in a double-blind, placebo-controlled, crossover study. Children were randomized to receive either placebo or up to 300 microg/day pergolide for the first 6-week treatment period, with a 2-week placebo washout, followed by crossover to the alternate treatment. The primary outcome measure was tic severity assessed by YGTSS. RESULTS: Compared with placebo treatment, pergolide treatment was associated with significantly lower YGTSS scores (42.0 +/- 20.4 versus 23.5 +/- 18.7; F = 12.0, df = 1, 17, p = 0.0011). No patient had a serious adverse event and pergolide was well tolerated. CONCLUSIONS: In this randomized, placebo-controlled, crossover trial, pergolide appeared to be a safe and efficacious treatment for Tourette's syndrome in children. PMID- 10746604 TI - Head injury and the risk of AD in the MIRAGE study. AB - OBJECTIVES: It has been suggested in some studies that head injury is a risk factor for AD, and that this risk is heightened among carriers of the APOE epsilon4 allele. We examined the effects of head injury and APOE genotype on AD risk in a large family study. SUBJECTS: A total of 2,233 probands who met criteria for probable or definite AD and their 14,668 first-degree family members (4,465 parents, 7,694 siblings, and 2,509 spouses) were ascertained at 13 centers in the United States, Canada, and Germany participating in the MIRAGE (Multi Institutional Research in Alzheimer Genetic Epidemiology) project. Information on head injury was collected by interview of multiple informants and review of medical records. Nondemented relatives and spouses served as control subjects for this study. METHODS: Odds of AD for head trauma with or without loss of consciousness were computed by comparing probands with unaffected spouses using conditional logistic regression analysis. To account for the unique biologic relationship between probands and their parents and siblings, odds of AD were computed using a generalized estimating equation (GEE) Poisson regression approach. GEE logistic regression was used to examine the joint effects of APOE genotype and head injury on the odds of AD in probands and a control group comprised of unaffected siblings and spouses. RESULTS: Comparison of probands with their unaffected spouses yielded odds ratios for AD of 9.9 (95% CI, 6.5 to 15.1) for head injury with loss of consciousness and 3.1 (2.3 to 4.0) for head injury without loss of consciousness. The corresponding odds derived from the comparison of probands with their parents and sibs were 4.0 (2.9 to 5.5) for head injury with loss of consciousness and 2.0 (1.5 to 2.7) for head injury without loss of consciousness. Head injury without loss of consciousness did not significantly increase the risk of AD in spouses (OR = 1.3; 95% CI, 0.4 to 4.1). The joint effects of head injury and APOE genotype were evaluated in a subsample of 942 probands and 327 controls (spouses and siblings). Head injury increased the odds of AD to a greater extent among those lacking epsilon4 (OR = 3.3) than among epsilon4 heterozygotes (OR = 1.8) or homozygotes (OR = 1.3). CONCLUSION: Head injury is a risk factor for AD. The magnitude of the risk is proportional to severity and heightened among first-degree relatives of AD patients. The influence of head injury on the risk of AD appears to be greater among persons lacking APOE-epsilon4 compared with those having one or two epsilon4 alleles, suggesting that these risk factors may have a common biologic underpinning. PMID- 10746605 TI - Line bisection judgments implicate right parietal cortex and cerebellum as assessed by fMRI. AB - OBJECTIVE: To use functional MRI (fMRI) to determine which brain regions are implicated when normal volunteers judge whether pretransected horizontal lines are correctly bisected (the Landmark test). BACKGROUND: Manual line bisection and a variant thereof involving perceptual judgments of pretransected lines (the Landmark test) are widely used to assess unilateral visuospatial neglect in patients with neurologic disease. Although unilateral (left) neglect most often results from lesions to right temporoparietal cortex, the normal functional anatomy of the Landmark test has not been convincingly demonstrated. METHODS: fMRI was carried out in 12 healthy right-handed male volunteers who judged whether horizontal lines were correctly prebisected. In the control task, subjects detected whether the horizontal lines contained a transection mark irrespective of the position of that mark. Response was by two-choice key press: on half the trials, subjects used the right, and on half, the left hand. Statistical analysis of evoked blood oxygenation level-dependent responses, measured with echoplanar imaging, employed statistical parametric mapping. RESULTS: Performing the Landmark task showed neural activity (p < 0.05, corrected) in the right superior posterior and right inferior parietal lobe, early visual processing areas bilaterally, the cerebellar vermis, and the left cerebellar hemisphere. Only the latter area showed a significant interaction with hand used. CONCLUSIONS: The right hemispheric dominance observed in inferior parietal cortex is consistent with the results of lesion studies. Right superior parietal cortex, vermis, and left cerebellar hemisphere have not been implicated in neglect, but all appear to play a cognitive role in the Landmark task. PMID- 10746606 TI - Functional MRI correlates of real and imagined tool-use pantomimes. AB - OBJECTIVE: To study the pattern of cerebral activation related to the performance of tool-use pantomimes with functional MRI (fMRI) using a task-subtraction design. BACKGROUND: Tool use comprises a particular category of transitive actions. Inability to pantomime the use of tools has been classically associated with retrorolandic dominant hemisphere damage. However, where in the left hemisphere these transitive representations are generated is unclear. METHODS: Echoplanar images were acquired in eight alternating task and control periods. Sixteen right-handed normal adults pantomimed the use of common tools and utensils with each hand. The control condition consisted of a sequence of nonsymbolic complex movements of forearm, hand, and fingers at a self-paced rate. Eight individuals also imagined the execution of the real task and control actions. A repeated measures ANOVA compared activations in five regions of interest in each hemisphere. RESULTS: Regardless of which hand was used, the left hemisphere was more active than the right in both real (p < 0.02) and imagined (p < 0.04) tasks. Activations clustered in the left intraparietal cortex and posterior dorsolateral frontal cortex. CONCLUSIONS: Pantomiming the use of tools is associated with activation of the left intraparietal cortex and dorsolateral frontal cortex. The left intraparietal cortex may store the representations of tool-use formulae, whereas the dorsolateral frontal cortex activation may reflect the switching between innervatory motor programs. PMID- 10746607 TI - Prediction of recovery of continuous memory after traumatic brain injury. AB - OBJECTIVE: To evaluate the ability of measures of initial severity, tests of attention, and demographic characteristics to predict recovery of continuous memory for words over a 24-hour period in patients with acute traumatic brain injury. METHODS: Recovery of continuous memory was assessed prospectively in 94 patients with nonpenetrating traumatic brain injury. A classification and regression tree analysis identified a hierarchical subset of variables that may be used as a simple guideline for predicting recovery of continuous memory. Weibull regression models evaluated and compared the predictive ability of multiple variables. RESULTS: Four groups of patients were identified based on measures of severity of injury and demographic characteristics. These four groups had recovery profiles that were more precise than could be obtained by using the Glasgow Coma Scale alone: mild, about 1 week to recovery of continuous memory; moderate, 1 to 4 weeks; severe, 2 to 6 weeks; and extremely severe, 4 to 8 weeks. Regression analysis confirmed that measures of capacity (inherent resources such as indicated by age) and compromise (general functional brain state measured neuropsychologically) improved prediction over models based only on injury severity measures, such as the Glasgow Coma Scale. CONCLUSIONS: Approaches to predicting recovery of continuous memory in the acute period after traumatic brain injury that take into account multiple measures provide a more sensitive predictive index. PMID- 10746608 TI - Evidence for long-term neurotoxicity associated with methamphetamine abuse: A 1H MRS study. AB - OBJECTIVE: To determine whether proton MRS (1H MRS) can detect long-term metabolite abnormalities in abstinent methamphetamine users. BACKGROUND: Methamphetamine is toxic to dopaminergic and serotonergic neurons in rodents; however, little data are available on the toxic effects of methamphetamine on the human brain. METHODS: 1H MRS was performed in 26 abstinent methamphetamine abusers with a history of methamphetamine dependence (median total cumulative lifetime exposure, 3,640 g; median recency of last methamphetamine use, 4.25 months) and 24 healthy subjects without a history of drug abuse. Cerebral metabolite concentrations on 1H MRS were measured in the frontal cortex, frontal white matter, and basal ganglia. RESULTS: The concentration of N-acetylaspartate ([NA]), a neuronal marker, was reduced significantly (-5 to -6%) in the basal ganglia and frontal white matter of methamphetamine users compared with control subjects. The frontal white matter [NA] correlated inversely with the logarithm of the lifetime methamphetamine use. The methamphetamine users also showed significantly reduced total creatine in the basal ganglia (-8%), and increased choline-containing compounds ([CHO], +13%) and myo-inositol ([MI], +11%) in the frontal grey matter. CONCLUSIONS: The reduced [NA] on 1H MRS provides evidence for long-term neuronal damage in abstinent methamphetamine users. PMID- 10746609 TI - Cortical myoclonus and cerebellar pathology. AB - OBJECTIVE: To study the electrophysiologic and pathologic findings in three patients with cortical myoclonus. In two patients the myoclonic ataxic syndrome was associated with proven celiac disease. BACKGROUND: The pathologic findings in conditions associated with cortical myoclonus commonly involve the cerebellar system, but there has only been one report of cerebellar pathology in a patient in whom cortical myoclonus was physiologically characterized antemortem. METHODS: Cortical somatosensory evoked potentials (SEPs) were recorded and EEG activity was averaged preceding myoclonic electromyographic activity. In one patient cortico-cortical inhibition was tested using two paired ipsilateral magnetic stimuli over the motor strip. Neuropathologic examination was carried out, including linear Purkinje cell densities/millimeter calculations for different regions of the cerebellum. RESULTS: The electrophysiology showed evidence of dysfunction of the sensorimotor cortex with enlarged SEPs and a time-locked cortical potential preceding the action myoclonus. In addition, motor cortical inhibition was abnormal in one case. Pathology showed unremarkable primary sensory, motor, and premotor cerebral cortices, except for unilateral gliosis of the motor cortex in one case. The cerebellum showed patchy atrophy and ongoing degeneration. A striking feature was the greater severity of Purkinje cell loss and Bergmann gliosis in the outer aspects than in the depths of the folia. CONCLUSIONS: Pathologic abnormalities are paradoxically mainly located in the cerebellum in some patients with cortical myoclonus, despite clear electrophysiologic evidence of cortical dysfunction. This observation suggests that enhanced excitability of the sensorimotor cortex may arise as a distant effect of cerebellar pathology. PMID- 10746610 TI - Expression and functional activity of heat shock proteins in human glioblastoma multiforme. AB - OBJECTIVE: To assess the expression of heat shock proteins (HSP) in glioma cells in vitro and in vivo and to examine their role in resistance to apoptosis. BACKGROUND: HSP are expressed in response to various forms of stress. Constitutive HSP expression may confer resistance to cytotoxic stimuli in human cancers. METHODS: HSP expression was assessed by immunoblot analysis in glioma cells in vitro and by immunocytochemistry in human glioblastomas in vivo. Modulation of apoptosis by hyperthermia-mediated HSP induction was examined in glioma cell lines in vitro. RESULTS: Immunoblot analysis revealed constitutive expression of HSP27, HSP72, HSP73, and HSP90 in all 12 human glioma cell lines. B crystallin (alphaBC) was expressed in 3 of 12 cell lines. High levels of alphaBC and HSP72 correlated with drug resistance and high p53 levels in vitro. Transient hyperthermia (43 degrees C/2 hours) induced HSP27 and HSP72 expression but had no effect on the levels of alphaBC, HSP73, or HSP90. HSP induction provided no survival advantage against subsequent cytotoxic challenges, including cytotoxic cytokines and radiochemotherapy. Immunohistochemistry showed strong expression of all HSP in vivo. The comparative analysis of HSP27, alphaBC, HSP72, HSP73, and HSP90 expression in 24 paired samples of first resections and recurrences of human glioblastoma multiforme revealed no impact of HSP expression on response to adjuvant radiochemotherapy and no modulation of HSP expression by radiochemotherapy. CONCLUSIONS: High constitutive, as opposed to inducible, expression of HSP may play a role in the primary resistance of human malignant gliomas to cytotoxic radiochemotherapy. Superinduction of HSP levels by hyperthermia in vitro provided no further survival advantage. PMID- 10746611 TI - Prolongation of midazolam half-life after sustained infusion for status epilepticus. AB - Midazolam clearance was examined in two patients with medically refractory convulsive status epilepticus. One patient received a constant infusion of midazolam for 68 hours and the other patient received a constant infusion of midazolam for 148 hours. In both patients the decline in level was, overall, much slower than expected. The half-lives of the terminal phase were longer than typically published (52.9 hours in Patient 1 and 20.1 hours in Patient 2). Thus, the data suggest that midazolam exhibits use-dependent pharmacokinetic changes that may be important clinically in situations that require prolonged midazolam therapy. PMID- 10746612 TI - Glossopharyngeal neuralgia and MS. AB - Glossopharyngeal neuralgia (GPN) is characterized by a severe lancing pain in the posterior pharynx, tonsillar fossa, and base of the tongue. It is induced frequently by swallowing and yawning. GPN has not been described previously in MS patients. The authors report four MS patients with GPN. Three responded to carbamazepine and one resolved during treatment with adrenocorticotrophin hormone (ACTH) and cyclophosphamide. Withdrawal of carbamazepine after 1 week in one patient resulted in recurrence of pain. GPN may be associated with MS and responds to carbamazepine. PMID- 10746613 TI - Treatment of acute disseminated encephalomyelitis with intravenous immunoglobulin. AB - Acute disseminated encephalomyelitis (ADEM) is a presumed immune-mediated, demyelinating disease of the CNS for which the standard treatment is high-dose steroids. We describe two patients with ADEM in whom treatment with IV methylprednisolone coincided with deterioration in their clinical status. They were subsequently treated with IV immunoglobulin and exhibited dramatic clinical improvement, with return to their previous level of functioning. PMID- 10746614 TI - Mutation in the CAV3 gene causes partial caveolin-3 deficiency and hyperCKemia. AB - Mutations in the caveolin-3 (CAV3) gene are associated with autosomal dominant limb-girdle muscular dystrophy (LGMD1C). The authors report a novel sporadic mutation in the CAV3 gene in two unrelated children with persistent elevated levels of serum creatine kinase (hyperCKemia) without muscle weakness. Immunohistochemistry and quantitative immunoblot analysis of caveolin-3 showed reduced expression of the protein in muscle fibers. Our data indicate that a partial caveolin-3 deficiency should be considered in the differential diagnosis of idiopathic hyperCKemia. PMID- 10746615 TI - Rotational vertebral artery occlusion syndrome with vertigo due to "labyrinthine excitation". AB - Leftward head rotations in a patient with a rotational vertebral artery occlusion syndrome elicited recurrent uniform attacks of severe rotatory vertigo and tinnitus in the right ear. These attacks were accompanied by a mixed clockwise torsional downbeat nystagmus with a horizontal component toward the right. A transient ischemia of the right labyrinth probably induced the attacks and led to a combined transient excitation of the right anterior and horizontal semicircular canals as well as the cochlea. PMID- 10746616 TI - Memory deficits after bilateral anterior fornix infarction. AB - The authors report a patient who suddenly developed memory loss without any other focal neurologic deficits. Neuropsychological testing showed severe anterograde verbal and visual memory deficits that improved gradually over several months, but not to the point of the premorbid state. Brain MRI with diffusion-weighted imaging performed 3 days after onset revealed acute infarction involving the bilateral fornices and the right genu of the corpus callosum. The authors suggest that acute fornix infarction may cause anterograde memory deficits. PMID- 10746617 TI - Verapamil in the prophylaxis of episodic cluster headache: a double-blind study versus placebo. AB - The authors performed a double-blind, double-dummy study to compare the efficacy of verapamil with placebo in the prophylaxis of episodic cluster headache. After 5 days' run-in, 15 patients received verapamil (120 mg tid) and 15 received placebo (tid) for 14 days. The authors found a significant reduction in attack frequency and abortive agents consumption in the verapamil group. Side effects were mild. These findings provide objective evidence for the effectiveness of verapamil in episodic cluster headache prophylaxis. PMID- 10746618 TI - Risk factors for lacunar stroke: a case-control transesophageal echocardiographic study. AB - To reassess the independent risk factors for lacunar stroke and to clarify the role of potential embolic sources, we conducted a case-control study using transesophageal echocardiography and duplex ultrasonography. Among 62 consecutive patients with their first lacunar stroke and 202 normal controls, we found that hypertension (p < 0.001), smoking (p = 0.001), and aortic arch atheroma (p = 0.006) were independently associated with an increased risk of lacunar stroke. Whether proximal aortic arch atheroma is mechanistically associated with lacunar stroke or merely coexistent is uncertain. PMID- 10746620 TI - Hypomania from the left frontal AVM resection. PMID- 10746619 TI - Vocal cord adduction during vagus nerve stimulation for treatment of epilepsy. PMID- 10746621 TI - Acquired neuromyotonia after bone marrow transplantation. PMID- 10746622 TI - Steroid-responsive Devic's variant in Sjogren's syndrome. PMID- 10746623 TI - The relationship of MS to physical trauma and psychological stress: report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology. PMID- 10746624 TI - The relationship of MS to physical trauma and psychological stress: report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology. PMID- 10746625 TI - The relationship of MS to physical trauma and psychological stress: report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology. PMID- 10746626 TI - Charcot and the myth of misogyny. PMID- 10746627 TI - The Webster's dictionary: neurologists on the Internet. PMID- 10746628 TI - Outer retinal dysfunction in patients treated with vigabatrin. PMID- 10746629 TI - Physical rehabilitation has a positive effect on disability in multiple sclerosis patients. PMID- 10746630 TI - Insulin-like growth factor I receptor signaling--overlapping or redundant pathways? PMID- 10746632 TI - Characterization of human and rat glucagon-like peptide-1 receptors in the neurointermediate lobe: lack of coupling to either stimulation or inhibition of adenylyl cyclase. AB - Glucagon-like peptide-1 (GLP-1) has been shown to bind to the posterior pituitary in the rat. We examined GLP-1 binding sites in human postmortem and rat pituitaries. Dense [125I]GLP-1 binding was seen in both human and rat posterior pituitary. In rat neurointermediate lobe membranes the binding site showed a Kd of 0.2 +/- 0.01 nM and a binding capacity of 600 +/- 33 fmol/mg protein (n = 3). In human pituitary membranes the binding site showed a Kd of 0.82 +/-0.05 nM and a binding capacity of 680 +/- 93 fmol/mg protein (n = 3). Chemical cross-linking showed a relative mol wt for the receptor-ligand complex of 73,100 +/- 1,400 (n = 3) in man and 59,300 +/- 900 (n = 3) in rat. GLP-1 (1 microM) failed to increase cAMP levels measured in rat neurointermediate lobes, whereas pituitary adenylate cyclase-activating polypeptide (100 nM) increased cAMP from a basal level of 14 +/-1 to 80 +/- 4 pmol/neurointermediate lobe 15 min (n = 5; P < 0.01). GLP-1 (up to 1 microM) did not affect the pituitary adenylate cyclase-activating polypeptide-stimulated cAMP levels. GLP-1 (up to 1 microM) also did not stimulate release of vasopressin or oxytocin from isolated rat neurointermediate lobes. The posterior pituitary shows the highest density of GLP-1-binding sites yet seen, but their function and signal transduction mechanism remain unknown. PMID- 10746631 TI - Domains of the insulin-like growth factor I receptor required for the activation of extracellular signal-regulated kinases. AB - The type 1 insulin-like growth factor receptor (IGF-IR) activates the extracellular signal-regulated kinases (ERK1 and -2). The two major substrates of the IGF-IR, insulin receptor substrate-1 (IRS-1) and the Shc proteins, are known to contribute to this activation. We investigated the domains of the IGF-IR required for the activation of the ERK proteins. To facilitate this study, we used a cell line (32D cells) that lacks IRS-1. In the absence of IRS-1, ERK activation is inhibited if the IGF-IR is mutated at two domains: tyrosine Y950 and a serine quartet at 1280-1283. Expression of IRS-1 in 32D cells expressing the double mutant IGF-IR restores ERK activation. The importance of the C terminus of the IGF-IR in ERK activation (in the absence of IRS-1) is confirmed by the failure of the insulin receptor to give a sustained activation of ERK. In this model system, there is a good, but not exact, correlation between ERK activation and cell survival after withdrawal of growth factors. PMID- 10746633 TI - Effects of insulin on prenylation as a mechanism of potentially detrimental influence of hyperinsulinemia. AB - To investigate the cause and effect relationship between hyperinsulinemia and the increased amounts of farnesylated p21Ras, we performed hyperinsulinemic euglycemic clamps in normal weight volunteers as well as in normal mice and dogs. Insulin infusions significantly raised the amounts of farnesylated p21Ras in the white blood cells of humans, in liver samples of mice and dogs, and in aorta samples of mice. Obese hyperinsulinemic individuals and dogs (made hyperinsulinemic by surgical diversion of the pancreatic outflow from the portal vein into the vena cava) displayed increased amounts of farnesylated p21Ras before the hyperinsulinemic clamps. Infusions of insulin did not alter the already increased levels of farnesylated p21Ras in these experimental models. To further investigate the role of acquired insulin resistance in modulating insulin's effect on p21Ras prenylation, we induced insulin resistance in rats by glucosamine infusion. Insulin-resistant glucosamine-treated animals displayed significantly increased farnesylated p21Ras in response to insulin infusion compared to that in control saline-treated animals. Transgenic models of insulin resistance (heterozygous insulin receptor substrate-1 knockout mice, A-ZIP/F-1 fatless mice, and animals overexpressing glutamine:fructose-6-phosphate amidotransferase) contained increased amounts of farnesylated p21Ras. We conclude that hyperinsulinemia, either endogenous (a prominent feature of insulin resistance) or produced by infusions of insulin, increases the amounts of farnesylated p21Ras in humans, mice, and dogs. This aspect of insulin action may represent one facet of the molecular mechanism of the potentially detrimental influence of hyperinsulinemia. PMID- 10746634 TI - Vitamin D is an important factor in estrogen biosynthesis of both female and male gonads. AB - In the present study, the role of vitamin D in the regulation of estrogen synthesis in gonads was investigated. Vitamin D receptor null mutant mice showed gonadal insufficiencies. Uterine hypoplasia and impaired folliculogenesis were observed in the female, and decreased sperm count and decreased motility with histological abnormality of the testis were observed in the male. The aromatase activities in these mice were low in the ovary, testis, and epididymis at 24%, 58%, and 35% of the wild-type values, respectively. The gene expression of aromatase was also reduced in these organs. Elevated serum levels of LH and FSH revealed hypergonadotropic hypogonadism in these mice. The gene expressions of estrogen receptor alpha and beta were normal in gonads in these mice. Supplementation of estradiol normalized histological abnormality in the male gonads as well as in the female. Calcium supplementation increased aromatase activity and partially corrected the hypogonadism. When the serum calcium concentration was kept in the normal range by supplementation, the aromatase activity in the ovary increased to 60% of the wild-type level, but LH and FSH levels were still elevated. These results indicated that vitamin D is essential for full gonadal function in both sexes. The action of vitamin D on estrogen biosynthesis was partially explained by maintaining calcium homeostasis; however, direct regulation of the expression of the aromatase gene should not be neglected. PMID- 10746635 TI - Alcohol ingestion inhibits the increased secretion of puberty-related hormones in the developing female rhesus monkey. AB - Alcohol (ALC) use and abuse by adolescents has been rising at an alarming rate. Whether ALC consumption during prepubertal years affects specific hormones and the process of sexual maturation is not known. We used immature female rhesus macaques to assess the effects of ALC on circulating levels of hormones known to be critical for the pubertal process. Ten monkeys averaging 20.3 +/- 0.3 months of age were bled by saphenous vein puncture at 0830 and 2030 h each day for 5 consecutive days to determine baseline levels of GH, insulin-like growth factor I, FSH, LH, estradiol (E2), and leptin. For the next 12 months, each day at 1330 h five monkeys were administered ALC (2 g/kg), and five monkeys were administered an isocaloric sucrose solution via a nasogastric approach. Blood was again collected twice daily on 5 consecutive days at 24, 28, and 32 months for hormone analysis. Food consumption and weight gain were similar for ALC-treated and control animals. The expected night-related increase in serum GH occurred during late juvenile development (28-32 months of age) in control animals, but was suppressed (P < 0.05) in ALC-treated animals. This action was paralleled by a decrease (P < 0.01) in serum insulin-like growth factor I. Serum LH and E2 were also depressed by ALC, with their effects most pronounced at 32 months (LH, P < 0.01; E2, P < 0.001). Serum FSH and leptin were not altered. Although ALC did not affect age at menarche, the interval between subsequent menstruations was lengthened (P < 0.05), thereby showing that ALC affected the development of a regular monthly pattern of menstruation. These results demonstrate the detrimental effects of ALC on the activation of hormone secretion that accompanies puberty in female rhesus monkeys. They also suggest that the subsequent growth spurt and normal timing or progression of puberty may be at risk in human adolescents and teenagers consuming even relatively moderate amounts of ALC on a regular basis. PMID- 10746636 TI - The role of the dorsal vagal complex and the vagus nerve in feeding effects of melanocortin-3/4 receptor stimulation. AB - Fourth intracerebroventricular (4th-icv) administration of the melanocortin-3/4 receptor (MC3/4-R) agonist, MTII, reduces food intake; the antagonist, SHU9119, increases feeding. The dorsal motor nucleus of the vagus nerve (DMX) contains the highest density of MC4-R messenger RNA in the brain. To explore the possibility that the DMX contributes to 4th-icv MC4-R effects, we delivered doses of MTII and SHU9119 that are subthreshold for ventricular response unilaterally through a cannula centered above the DMX. MTII markedly suppressed 2-h (50%), 4-h (50%), and 24-h (33%) intake. Feeding was significantly increased 4 h (50%) and 24 h (20%) after SHU9119 injections. These results suggest that receptors in the DMX, or the dorsal vagal complex more generally, underlie effects obtained with 4th icv administration of these ligands. We investigated possible vagal mediation of 4th-icv MTII effects by giving the agonist to rats with subdiaphragmatic vagotomy. MTII suppressed 2-, 4-, and 24-h liquid diet intake (approximately 80%) to the same extent in vagotomized and surgical control rats. We conclude that stimulation or antagonism of MC3/4-Rs in the dorsal vagal complex yields effects on food intake that do not require an intact vagus nerve. PMID- 10746637 TI - Lasofoxifene (CP-336,156), a selective estrogen receptor modulator, prevents bone loss induced by aging and orchidectomy in the adult rat. AB - It has been well documented that selective estrogen receptor modulators (SERMs) can prevent bone loss in ovariectomized rats and postmenopausal women. The purposes of this study were to determine the effects of a potent and orally active SERM, lasofoxifene (CP-336,156), on bone mass, bone strength, total serum cholesterol, prostate weight, and histology in adult male orchidectomized (ORX) rats. Sprague Dawley male rats at 10 months of age were divided into 6 groups, with 10 rats/group. The first group was necropsied on day 0 and served as basal controls. The remaining rats were either sham operated (n = 10) and treated orally with vehicle, or ORX (n = 40) and treated with either vehicle or lasofoxifene at 1, 10, or 100 microg/kg x day for 60 days. Total serum cholesterol, prostate weight and histology, distal femoral bone mineral density (DFBMD) by dual energy x-ray absorptiometry, and static and dynamic bone histomorphometry of the third lumbar vertebral body were determined. Maximal load and stiffness of the fifth lumbar vertebral body were also determined by compression tests. Age-related decreases in DFBMD (-9%) and trabecular bone volume (TBV; -13%) of the third lumbar vertebral body were found in sham-operated rats compared with basal controls. ORX induced significant increases in total serum cholesterol (+31%), eroded surface (+48%), activation frequency of bone turnover (+103%) and significant decreases in prostate weight (-89%), DFBMD ( 14%), TBV (-23%), and maximal load (-17%) compared with basal controls. Compared with sham controls, ORX induced significant increases in eroded perimeter and activation frequency. Lasofoxifene decreased body weight in all dose groups compared with both sham and ORX control values. Compared with ORX controls, ORX rats treated with lasofoxifene at 10 or 100 microg/kg x day had significantly lower percent eroded perimeter activation frequency and significantly higher DFBMD, TBV, and maximal load. Further, lasofoxifene at 10 and 100 microg/kg x day significantly decreased total serum cholesterol by 46% and 68% in ORX rats, whereas no effect was found in prostate weight and histology parameters compared with ORX control values. These data showed that lasofoxifene prevented bone loss by inhibiting bone turnover associated with aging and orchidectomy in 10-month old male rats. Further, lasofoxifene decreased total serum cholesterol and did not affect the prostate in these rats. These results suggest that SERMs such as lasofoxifene may be useful therapeutic agents for preventing bone loss in elderly men with some degree of hypogonadism. PMID- 10746638 TI - Steroidogenic factor-1 influences protein-deoxyribonucleic acid interactions within the cyclic adenosine 3,5-monophosphate-responsive regions of the murine steroidogenic acute regulatory protein gene. AB - De novo synthesis of the steroidogenic acute regulatory protein (StAR) in response to trophic hormonal stimulation of steroidogenic cells is required for the delivery of cholesterol from the mitochondrial outer membrane to the mitochondrial inner membrane and the cytochrome P450 side-chain cleavage enzyme. StAR expression is transcriptionally regulated by cAMP-mediated mechanisms, and we have identified a 45-bp region within the mouse promoter that is important for cAMP responsiveness of the gene. This region, located between -105 and -60 of the start site of transcription, contains a SF-1-binding site, a highly conserved C/EBPbeta-AP-1-nuclear receptor half-site sequences (CAN region), and a GATA-4 binding site. The SF-1 element and CAN region are required for full basal activity, whereas the GATA-4 element may account for 20% of the cAMP response in MA-10 mouse Leydig tumor cells. A cAMP-dependent protein-DNA complex was observed with the CAN region and mutation of a nonconsensus AP-1 site within this region greatly diminished promoter strength. Complex protein-DNA interactions within the cAMP response region (-105/-60) were shown to require the SF-1 element (-95), suggesting that SF-1 is required for protein-DNA interaction at the CAN (-79) region and maximal activity of the promoter. PMID- 10746639 TI - The rabbit sex hormone-binding globulin gene: structural organization and characterization of its 5-flanking region. AB - Sex hormone-binding globulin (SHBG) transports sex steroids in the blood. In humans and rabbits, the gene encoding SHBG (shbg) is expressed primarily in the liver and testis, whereas the testis is the major site of shbg expression in rodents postnatally. Sequence analysis has revealed that rabbit shbg (rbshbg) spans 2.5 kb and comprises eight exons with consensus splice sites at all exon intron junctions. The major transcription start site ofrbshbg is located 52 bp upstream from the translation initiation codon for the rabbit SHBG precursor. Unlike the situation in humans and rats, rbshbg transcripts contain no alternative exon 1 sequences in the liver or testis, and this suggests that the rbshbg 5'-flanking region plays an equally important role in controlling transcription of this gene in these tissues. Like the human and rat shbg promoter sequences, the rbshbg proximal promoter lacks a typical TATA box. It also contains several transcription factor-binding sites, but deoxyribonuclease I footprinting experiments indicated that the human and rabbit shbg proximal promoters interact quite differently with proteins extracted from rabbit liver nuclei. However, the predominant footprint on the rbshbg promoter is conserved at the same position within the human shbg (hshbg) promoter and includes consensus binding sites for the transcription factor nuclear factor- 1. Transient transfection studies of the rbshbg 5'-flanking sequence (893 bp) revealed regions that actively enhance and repress its activity in human hepatoblastoma and mouse Sertoli cells, but not in Chinese hamster ovary cells. Like the rat shbg proximal promoter, the rbshbg 5'-flanking sequence lacks a region that corresponds to a cis-element, designated footprinted region 4 in the hshbg proximal promoter. Furthermore, the hshbg promoter footprinted region 3 sequence is poorly conserved in rbshbg, and when mutated to resemble the corresponding human sequence it increased the transcriptional activity of the rbshbg promoter by 7-fold in hepatoblastoma cells. Thus, the rabbit and hshbg promoters appear to be controlled by a different set of transcriptional regulators. Further comparisons of their functional activities may shed light on species-specific differences in the spatial and temporal expression of this gene, the products of which play important roles in regulating sex steroid access to target cells. PMID- 10746640 TI - Activation of c-fos expression in hypothalamic nuclei by mu- and kappa-receptor agonists: correlation with catecholaminergic activity in the hypothalamic paraventricular nucleus. AB - Administration of the preferential mu-opioid receptor agonist, morphine, and selective K-opioid receptor agonists elicits activation of the hypothalamus pituitary-adrenocortical axis, although the site or the molecular mechanisms for these effects have not been determined. The expression ofFos, the protein product of the c-fos protooncogene, has been widely used as an anatomical marker of monitoring neuronal activity. In the present study we evaluated 1) the effects of the mu-opioid receptor agonist, morphine, and those of the selective K-opioid receptor agonist, trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl ]benzeneacet amide methane sulfonate (U-50,488H), administration on the expression of Fos in hypothalamic nuclei; and 2) the possible modification of the activity of noradrenergic neurons known to send afferent projections to the paraventricular nucleus (PVN), the site of CRF neurons involved in initiating ACTH secretion. Using immunohistochemical staining of Fos, the present results indicate that acute treatment with either morphine or U-50,488H induces marked Fos immunoreactivity within the hypothalamus, including the medial parvicellular PVN and supraoptic and suprachiasmatic nuclei. Pretreatment with naloxone attenuated the effect of morphine, whereas nor-binaltorphimine, a selective kappa opioid receptor antagonist, abolished the effect of U-50,488H on Fos induction. Correspondingly, morphine and U-50,488H injection increased the production of the cerebral noradrenaline metabolite 3-methoxy-4-hydroxyphenylethylene glycol as well as noradrenaline turnover in the PVN. These effects were antagonized by naloxone and nor-bin-altorphimine, respectively. All of these findings are discussed in terms of specific events that couple opioid-induced activation of the hypothalamus-pituitary-adrenocortical axis and noradrenergic activity with changes in gene expression in selective hypothalamic nuclei. PMID- 10746641 TI - Development of a transgenic mouse that overexpresses a novel product of the growth hormone-releasing hormone gene. AB - The GH-releasing hormone (GHRH) precursor molecule contains a 30-amino acid C terminal region that has been designated GHRH-related peptide (GHRH-RP). To begin to understand the physiological role of GHRH-RP, transgenic (Tg) mice that constituitively express this peptide were developed. To generate these mice, a transgene (SS-RP) was constructed by overlap primer extension PCR. This transgene, under the control of the mouse phosphoglycerate kinase gene, selectively expresses GHRH-RP, but not GHRH. Western blot analysis confirmed that the transgene produces GHRH-RP. Animals were evaluated for the effect of excess GHRH-RP on growth, fertility, behavior, stem cell factor (SCF) expression, and hematopoiesis. Northern blot and RT-PCR were used to demonstrate ubiquitous expression of the transgene in tissues from GHRH-RP Tg animals. These tissues also had marked overexpression of SCF messenger RNA compared with controls. Tg animals had significantly increased cell cycling for granulocyte-macrophage, erythroid, and multilineage progenitor cells. Transgenic animals did not differ from control mice in their growth, fertility, or behavior. These findings demonstrate, for the first time, that in vivo the C-terminal peptide of the pro GHRH molecule is a biologically active peptide that is capable of stimulating the expression of SCF and hematopoiesis in vivo and suggests that GHRH-RP may play a role in normal blood cell development. PMID- 10746642 TI - Dexamethasone counteracts the effect of prolactin on islet function: implications for islet regulation in late pregnancy. AB - Islets undergo a number of up-regulatory changes to meet the increased demand for insulin during pregnancy, including increased insulin secretion and beta-cell proliferation. It has been shown that elevated lactogenic hormone is directly responsible for these changes, which occur in a phasic pattern, peaking on day 15 of pregnancy and returning to control levels by day 20 (term). As placental lactogen levels remain elevated through late gestation, it was of interest to determine whether glucocorticoids (which increase during late gestation) could counteract the effects of lactogens on insulin secretion, beta-cell proliferation, and apoptosis. We found that insulin secretion measured over 24 h in culture and acute secretion measured over 1 h in response to high glucose were increased at least 2-fold by PRL treatment after 6 days in culture. Dexamethasone (DEX) treatment had a significant inhibitory effect on secretion in a dose dependent manner at concentrations greater than 1 nM. At 100 nM, a concentration equivalent to the plasma corticosteroid level during late pregnancy, DEX inhibited secretion to below control levels. The addition of DEX (>1 nM) inhibited secretion from PRL-treated islets to levels similar to those produced by DEX treatment alone. Bromodeoxyuridine (10 microM) staining for the final 24 h of a 6-day culture showed that PRL treatment increased cell proliferation 6-fold over the control level. DEX treatment alone (1-1000 nM) did not reduce cell division below the control level, but significantly inhibited the rate of division in PRL-treated islets. YoYo-1, an ultrasensitive fluorescent nucleic acid stain, was added (1 microM; 8 h) to the medium after 1-3 days of culture to examine cell death. Islets examined under confocal microscopy showed that DEX treatment (100 nM) increased the number of cells with apoptotic nuclear morphologies. This was quantified by counting the number of YoYo-labeled nuclei per islet under conventional epifluorescence microscopy. The numbers of YoYo-1 positive nuclei per islet in control and PRL-treated islets were not different after 3 days of culture. However, DEX treatment increased YoYo-1 labeling 7-fold over that in controls. DEX also increased YoYo-1 labeling in PRL-treated islets 3 fold over the control level. These data show that the increased plasma glucocorticoid levels found during the late stages of pregnancy could effectively reverse PRL-induced up-regulation of islet function by inhibiting insulin secretion and cell proliferation while increasing apoptosis. PMID- 10746643 TI - Regulation of inositol trisphosphate receptor isoform expression in glucose desensitized rat pancreatic islets: role of cyclic adenosine 3',5'-monophosphate and calcium. AB - The regulation of inositol 1,4,5-trisphosphate receptor (IP3R) messenger RNA (mRNA) and protein expression was investigated in glucose-desensitized rat isolated pancreatic islets. Islets were cultured for 4 days with glucose (11 mM; G-treated) to induce desensitization; IP3R-I mRNA levels were similar to basal (5.5 mM glucose) values, whereas IP3R-II mRNA levels were increased and IP3R-III levels were reduced compared with basal levels. Somatostatin increased the expression of IP3R-II mRNA and reduced the expression of IP3R-III mRNA compared with basal values, but did not significantly affect G-treated islet IP3R expression. When forskolin (FSK), 8-bromo-cAMP, and glucagon-like peptide 1-(7 36) amide were added to G-treated islets after 4 days of culture, IP3R-II mRNA levels were reduced, whereas IP3R-III mRNA levels increased, to levels observed in control islets, within 3 h. The levels of IP3R-I mRNA were unaffected by either somatostatin or FSK. The protein kinase A inhibitor. H-89, and actinomycin D prevented the effects of FSK. A Ca2+ ionophore mimicked the effects of FSK on IP3R mRNA expression, whereas blockade of voltage-dependent Ca2+ channels or chelation of intracellular Ca2+ inhibited the actions of FSK. cAMP also increased IP3R-III mRNA in insulinoma cells. In G-treated islets, FSK slowed IP3R-III mRNA degradation. FSK, but not glucose, stimulated protein kinase A activation in G treated islets. Thus, cAMP mediates changes in IP3R-II and -III mRNA transcription and stability in glucose-desensitized islets. The regulated expression of IP3R-II and -III mRNA is mediated in part by intracellular Ca2+ availability. PMID- 10746644 TI - Formation of three-dimensional thyroid follicle-like structures by polarized FRT cells made communication competent by transfection and stable expression of the connexin-32 gene. AB - Pig thyrocytes, either in the intact gland or cultured under conditions leading to thyroid follicle reconstitution, coexpress two gap junction proteins, connexin 32 (Cx32) and connexin-43 (Cx43). As thyrocytes cultured in the form of a monolayer only express Cx43, we hypothesized that Cx32 could play a role in thyroid folliculogenesis. In the present work, we analyzed the ability of polarized FRT cells (that are gap junction deficient) to form follicle-like structures after stable transfection with either Cx32 or Cx43 genes. Wild-type and transfected FRT cells, while growing, showed the capacity to form three dimensional structures corresponding to domes that result from the accumulation of fluid underneath limited areas of the cell layer. The number of domes formed by FRT cells expressing Cx32 (FRT-Cx32) was 2- to 3-fold higher than that obtained with either wild-type or Cx43-transfected FRT cells (FRT-Cx43). Domes generated by FRT-Cx32 cells were stable (beyond 3 weeks of culture), whereas those formed from wild-type or FRT-Cx43 cells were transient, disappearing when cells reached confluence. Inspection of the cell organization within domes formed from FRT-Cx32 cells by phase contrast and confocal microscopy revealed a progressive transition from domes toward closed structures with a lumen. The tightness of the lumen was demonstrated by the retention of a fluorescent probe, lucifer yellow, introduced by microinjection. Electron microscope examinations showed that the neoformed follicle-like structures had an inside-out polarity. Analyses of cell motion and division with time, by fluorescence video microscopy, indicated that the transformation of domes into inside-out follicles brings into play the migration of cells and, to a lesser extent, cell multiplication underneath the domes. In conclusion, FRT cells forced to express Cx32 give rise to domes that transform into closed inside-out follicles. This gain of function appears Cx specific, as FRT-Cx43 cells did not form similar structures. Our data suggest that the formation and/or functioning of Cx32 gap junctions might represent a key event in thyroid epithelium morphogenesis, i.e. formation of a lumen from a tight epithelial cell layer. PMID- 10746645 TI - Testicular heat exposure enhances the suppression of spermatogenesis by testosterone in rats: the "two-hit" approach to male contraceptive development. AB - The objectives of the study were to determine stage-specific changes in the kinetics of germ cell apoptosis induced by administration of exogenous testosterone (T) alone and to examine whether addition of a single testicular heat exposure would enhance the induction of germ cell apoptosis and the suppression of spermatogenesis by T. Adult male rats were implanted with 3-cm SILASTIC brand capsules (Dow Corning Corp.) containing T for up to 6 weeks. Intratesticular T levels declined to 2.9% of control values by 1 week and remained suppressed at 2, 3, and 6 weeks after T administration. The incidence of germ cell apoptosis (expressed as numbers per 100 Sertoli cells) was low in control rats (0-9.52). After T treatment, the mean incidence of apoptosis at stages VII-VIII increased significantly by 1 week (21.43 +/-3.33) and showed further increases by 6 weeks (56.30 +/- 7.47); apoptotic rates remained low at early (I-VI) and later (XII-XIV) stages. To test whether the combination of T with a single testicular heat exposure resulted in more complete suppression of spermatogenesis than either treatment alone, four groups of adult rats received one of the following treatments: 1) a subdermal empty polydimethylsilozane implant, 2) exposure to a single testicular heating (43 C for 15 min) applied on day 14, 3) 3-cm T implant, or 4) 3-cm T implant and a single testicular heat exposure (applied on day 14). All animals were killed at the end of 6 weeks. In the heat-treated group, testis weight and testicular sperm counts were decreased to 65.4% and 28.9% of control levels, respectively. The corresponding values in the T-treated group were 49.7% and 24.9% of control levels, respectively. Notably, addition of heat to T further reduced testis weight to 31.1% of control levels and testicular sperm counts to near zero. Histomorphometric analysis showed that all treatments reduced seminiferous tubular diameter and epithelial and luminal volume, with the greatest decrease after combined T and heat treatment. Heat exposure in animals bearing T implants markedly reduced the number of pachytene spermatocytes and round spermatids through apoptosis, resulting in tubules devoid of mature spermatids. Spermatogonia and preleptotene spermatocytes remained unaffected. These results clearly demonstrate that 1) exogenous T reduces intratesticular T and induces apoptosis mainly at stages VII VIII within 1-6 weeks; 2) the combined treatment of T and heat markedly inhibits spermatogenesis, resulting in near azoospermia within 6 weeks; and 3) meiosis and spermiogenesis are the most vulnerable phases of spermatogenesis in response to T plus heat treatment. These findings suggest that a combination of hormonal treatment such as T and a physical agent (heat exposure) is more effective in suppressing spermatogenesis than either treatment alone. We hypothesize that combination of two antispermatogenic agents ("two hit") working at separate stages of the spermatogenic cycle will lead to greater male contraceptive efficacy. PMID- 10746646 TI - Organization and regulation of the gene encoding the sheep acid-labile subunit of the 150-kilodalton insulin-like growth factor-binding protein complex. AB - In adult animals, most circulating insulin-like growth factor I (IGF-I) and IGF II is sequestered in a 150-kDa complex composed of 1 molecule each of IGF, IGF binding protein-3 or -5, and the acid-labile subunit (ALS). Capture of IGF in ALS containing complexes increases their circulating half-lives and concentrations and suppresses their hypoglycemic potential. ALS has been studied almost exclusively in rodents and primates, and no information exists in the sheep despite its extensive use to study the circulating IGF system. To initiate studies in the sheep, we isolated the ovine ALS gene and characterized its spatial and developmental regulation. The ALS gene spans about 3.0 kb of chromosomal DNA and consists of 2 exons interrupted by a 977-bp intron. Exon 1 encodes the first 5 amino acids of the signal peptide; exon 2 encodes the remaining 27 amino acids of the signal peptide and the entire mature protein of 579 amino acids. Transcription initiation occurs at nucleotides -58 and -29 (ATG, + 1), 2 sites that are not preceded by TATA or initiator sequences. A DNA fragment extending from -727 to - 11 of the sheep ALS gene directed basal expression of a luciferase reporter plasmid in the rat liver cell line H4-II-E. GH increased promoter activity by 1.8-fold, consistent with conservation in the sheep promoter of the GH response element previously identified in the mouse gene. A survey of adult tissues by Northern analysis revealed the presence of a 2.2-kb transcript only in liver. Weak expression was first detected in liver on day 130 of fetal life, increased suddenly on day 7 of postnatal age, and changed little thereafter. The sheep is a useful model to understand the regulation and role of ALS, particularly around the time of birth, when final maturation of the circulating IGF system occurs. PMID- 10746647 TI - Differential regulation of leptin transport by the choroid plexus and blood-brain barrier and high affinity transport systems for entry into hypothalamus and across the blood-cerebrospinal fluid barrier. AB - Leptin is a circulating hormone that controls food intake and energy homeostasis. Little is known about leptin entry into the central nervous system (CNS). The blood-cerebrospinal fluid (CSF) barrier at the choroid plexus and the blood-brain barrier (BBB) at the cerebral endothelium are two major controlling sites for entry of circulating proteins into the brain. In the present study, we characterized leptin transport across the blood-CSF barrier and the BBB by using a brain perfusion model in lean rats. Rapid, high-affinity transport systems mediated leptin uptake by the hypothalamus (KM = 0.2 ng/ml) and across the blood CSF barrier (KM = 1.1 ng/ml). High affinity in vivo binding of leptin was also detected in the choroid plexus (KD = 2.6 ng/ml). In contrast, low affinity carriers for leptin (KM = 88 to 345 ng/ml) were found at the BBB in the CNS regions outside the hypothalamus (e.g. cerebral cortex, caudate nucleus, hippocampus). Our findings suggest a key role of high affinity leptin transporters in the hypothalamus and choroid plexus in regulating leptin entry into the CNS and CSF under physiological conditions. Low affinity transporters at the BBB outside the hypothalamus could potentially contribute to overall neuropharmacological effects of exogenous leptin. PMID- 10746649 TI - Effect of leptin on CYP17 enzymatic activities in human adrenal cells: new insight in the onset of adrenarche. AB - CYP17 is a microsomal enzyme embodying two distinct activities, 17alpha hydroxylase and 17,20-lyase, essential for the synthesis of cortisol and sex hormone precursors, respectively. The two activities are differentially regulated in a tissue and developmental stage-dependent fashion. Leptin might play a role in such differential control. Low dose leptin caused a significant increase in 17,20-lyase activity in adrenal NCI-H295R cells expressing leptin (OB) receptor (OB-R), without significant sustained influence on the 17alpha-hydroxylase activity. To analyze the time dependence of this leptin effect, the impact of long and short-term leptin treatment was studied. To assess the relationship with the OB-R signal transduction pathway, the same experiments were performed in intact cells and in a reconstituted system. The long- and short-term studies in intact cells and in microsomes suggest that the 17alpha-hydroxylase activity of CYP17 can be promptly stimulated by leptin, but that the effect is transient. In contrast, physiological doses of leptin steadily enhance 17,20-lyase activity. This influence is direct, OB-R specific and dependent on the integrity of the signal transduction pathway. The 17,20-lyase activity stimulation relies on phosphate incorporation, as demonstrated by the loss of leptin-dependent 17,20 lyase stimulation after phosphate removal, and by the fact that the DHEA production appears to be related exclusively to the presence of phosphorylated CYP17, independently from novel protein synthesis. The mechanism underlying the observed events seems to involve CYP17 phosphorylation, a feature of the OBR signal transduction pathway, and a process already shown to be crucial for 17,20 lyase activity. PMID- 10746648 TI - A1 adenosine receptor activation increases adipocyte leptin secretion. AB - A1 adenosine receptors (A1ARs) are heavily expressed in adipocytes and influence fat cell metabolism. Because increasing evidence suggests a role for leptin in mediating appetite and fat cell metabolism, we tested whether ALARs regulate leptin production. Rats were treated with the A1AR agonist N6 cyclopentyladenosine (CPA), and changes in circulating levels of leptin and leptin gene expression were examined. Serum leptin levels rose 2- to 10-fold, with peak increases seen 8-16 h after injection of CPA (P < 0.05). In contrast, CPA did not alter steady state levels of adipose tissue leptin mRNA. To assess the influence of endogenous adenosine on circulating leptin levels, rats were also injected with dipyridamole (DPY), an adenosine reuptake blocker. DPY induced 80% increases in serum levels at 8 h after injections (P < 0.05). Supporting the idea that stimulation of leptin production is A1AR mediated, pretreatment with the A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine completely blocked increases in leptin levels after DPY treatment. To complement in vivo studies, the effect of A1AR activation on leptin secretion was also studied in epididymal fat pad cultures. In cultures, CPA treatment increased leptin secretion by 37% (P < 0.05). Collectively, these data show that the adenosinergic system can increase leptin secretion by directly activating A1ARs in fat tissue. PMID- 10746650 TI - 17beta-estradiol antiinflammatory activity in carrageenan-induced pleurisy. AB - We have recently demonstrated that 17beta-estradiol (E2) opposes cytokine dependent increase of inducible nitric oxide synthase (iNOS) activity in rat smooth muscle cells and proposed that this effect might be associated to an antiinflammatory activity of this hormone. In the present study, we examine the E2 effects on a well-known in vivo model of inflammation. We show that, in carrageenan treatment of ovariectomized rats, prior exposure to E2 significantly attenuated inflammatory response as measured by histological examination and exudate production. The effect was visible with a single injection of a physiological dose of E2 1 h before the carrageenan treatment and was blocked by coadministration of the estrogen receptor antagonists ICI 182,780 or tamoxifen. This latter observation suggests that the effect is receptor mediated. The mechanisms by which estradiol has beneficial effects in this model of inflammation are unclear: we show that in hormonally treated rats there is a decrease in polymorphonuclear cells migration as shown by cell counting and myeloperoxidase measurement. In addition, E2 pretreatment opposes carrageneen induced high lipid peroxidation maintaining malondialdehyde activity at control levels. E2 treatment decreases NO production and the activity of iNOS with consequent diminished nitrite synthesis and nitrosine accumulation. Finally, immunohistochemical analysis for poly (ADP-ribose) synthase revealed a positive staining in lungs from carrageenan-treated rats that was blocked by estradiol treatment. We conclude that E2 attenuates the degree of inflammation and tissue damage associated with carrageenan-induced pleurisy in the rat. PMID- 10746651 TI - In vitro stimulation of the prepubertal rat gonadotropin-releasing hormone pulse generator by leptin and neuropeptide Y through distinct mechanisms. AB - Leptin may act as a negative feedback signal to the brain in the control of appetite through suppression of neuropeptide Y (NPY) secretion and stimulation of cocaine- and amphetamine-regulated transcript (CART), a new anorectic peptide. We aimed at studying whether leptin, NPY, and CART have related effects on the hypothalamic control of the pituitary-gonadal system and the developmental changes in NPY and CART effects. Using retrochiasmatic hypothalamic explants from prepubertal 15-day-old male rats, the GnRH interpulse interval (mean +/- SD: 62 +/- 5 min) was significantly reduced by 10(-7) M of leptin (46 +/- 3.3 min) as well as 10(-7) M of NPY (47 +/- 4.4 min) and 10(-6) M of CART (46 +/- 2.7 min), whereas the GnRH pulse amplitude was not affected. The stimulatory effects of different NPY receptor agonists [human PYY 3-36, porcine NPY 13-36, human (D-Trp 32) NPY, porcine (Leu 31 Pro 34) NPY, human pancreatic polypeptide (PP)], as well as the absent effects of rat PP were consistent with the involvement of the Y5 receptor subtype in mediation of NPY effects. Incubation with 10(-7) M of a Y5 receptor selective antagonist prevented the effect of NPY (61 +/- 4 vs. 46 +/- 2 min), whereas leptin and CART effects were not (47 +/- 3 vs. 46 +/- 3 min and 46 +/- 3 vs. 46 +/- 2 min, respectively), suggesting that NPY was not involved in leptin and CART effects. Using an anti-CART antiserum (1:1000), the reduction of GnRH interpulse interval caused by leptin was partially prevented (56.2 +/- 4 vs. 47.9 +/- 3.8 min), whereas the reduction of GnRH interval caused by NPY was not affected (45.9 +/-2.5 vs. 47.8 +/- 3.7). The GnRH interpulse interval was decreased by 10(-7) M of NPY at 5 days (72 +/- 3.8 vs. 91.9 +/- 3.5) as well as at 15 days, whereas such an effect was not observed anymore at 25 and 50 days. Similar effects were observed using 10(-6) M of CART-peptide. Using 10(-6) M of the Y5-receptor antagonist, the GnRH interpulse interval was significantly increased at 15 days (66.6 +/- 2.7 min), 25 days (56.5 +/- 39.9 min), and 50 days (52.5 vs. 38.2 min), whereas no change was observed at 5 days. Using the anti CART antiserum, a significant increase of GnRH interpulse interval was observed at 25 days only. In conclusion, the stimulatory effects of leptin and NPY on the frequency of pulsatile GnRH secretion before puberty involve two distinct mechanisms. NPY causes acceleration of GnRH pulsatility via the Y5-receptor subtype, which is not involved in leptin effects while the CART is involved in leptin effects on GnRH secretion but not in NPY effects. The reduction of pulsatility by the Y5 antagonist provides evidence of endogenous NPY involvement in the control of GnRH secretion from the time of onset of puberty. PMID- 10746652 TI - Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the expression of follicle stimulating hormone receptors during cell differentiation in cultured granulosa cells. AB - Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) is a common environmental pollutant causing public concern. Using a cell culture system derived from rat granulosa cells that provides unique advantages for studying the molecular mechanisms underlying the action of TCDD, the influences of TCDD on FSH receptor (FSH-R) induction were examined. The treatment with FSH produced, as expected, a substantial increase in specific FSH-R expression, whereas concurrent treatment with the environmental amount of TCDD (10 pM) resulted in a significant decrease in FSH-R after being cultured from 24-72 h. Cotreatment with FSH (30 ng/ml) and increasing doses of TCDD inhibited the levels of FSH-induced FSH-R messenger RNA (mRNA) in a dose-dependent manner. Treatment with 8-Br-cAMP (1 mM) produced a significant increase in FSH-R mRNA; concurrent treatment with TCDD (10 pM) produced a significant attenuation of 8-Br-cAMP action. These findings suggest that the ability of TCDD to interfere with FSH action, as regards the induction of FSH-Rs, is exerted at sites distal to those involved in cAMP generation. Because a single transcript of 5.2 kb was seen for the Ah receptor in this granulosa cell system, the effects of TCDD may be mediated by this specific receptor. The rates of FSH-R mRNA gene transcription, assessed by nuclear run-on transcription assay, were decreased by the addition of TCDD. The effect of TCDD on FSH-R mRNA stability was determined by measuring the decay of FSH-R mRNA under conditions known to inhibit transcription. The decay curve for the 2.4-kb FSH-R mRNA transcript was not significantly changed after the addition of TCDD. These findings showed that the effect of TCDD on FSH-R mRNA was, at least in part, the result of decreased transcription. PMID- 10746653 TI - Stimulation of gonadotropin-releasing hormone surges by estrogen. I. Role of hypothalamic progesterone receptors. AB - Estrogen (E2) stimulates GnRH surges by coupling a daily neural signal to neuronal circuitries governing GnRH release. We have hypothesized that E2 promotes this coupling process by inducing expression of neuronal transcription factors, which are subsequently activated by neurotransmitter-mediated mechanisms representing the daily neural signal. These experiments tested the specific hypothesis that the progesterone receptor (PR) functions in this manner, viz. as an E2-induced factor whose activation is necessary for the stimulation of GnRH surges. Two complimentary experiments were performed to determine whether activation of hypothalamic PRs is obligatory for the stimulation of GnRH surges by E2. In the first, the effects of a PR antagonist on GnRH and LH surges were assessed in ovariectomized (OVX), E2-primed rats. Rats were OVX on diestrous day 2, treated with 30 microg estradiol benzoate or oil vehicle, sc, and then administered either oil vehicle or the type I antiprogestin, ZK98299 at 0900 h on proestrus. GnRH release rates and plasma LH levels were determined in each animal by microdialysis of median eminence and atrial blood sampling, respectively. Estrogen, but not oil vehicle, treatment evoked robust and contemporaneous GnRH and LH surges in animals that received no PR antagonist on proestrus. Additional treatment with ZK98299, however, completely blocked both GnRH and LH surges. In a second experiment, specific involvement of anteroventral periventricular (AVPV) PRs in E2-induced GnRH surges was assessed. Additional groups of OVX, E2-primed rats were fitted with intracerebroventricular cannulas, and PR antisense oligonucleotides were infused into the third ventricle adjacent to the AVPV to prevent expression of PR in this periventricular region. Animals infused with PR antisense oligos did not exhibit any LH surges, whereas surges were observed in saline-, missense-, and sense oligo-treated controls. Immunohistochemistry confirmed the effectiveness of PR antisense oligonucleotides in blocking PR expression. These findings provide direct support for the hypothesis that activation of PRs, specifically those in hypothalamic regions including the AVPV, is an obligatory event in the stimulation of GnRH surges by E2. PMID- 10746654 TI - Stimulation of gonadotropin-releasing hormone surges by estrogen. II. Role of cyclic adenosine 3'5'-monophosphate. AB - Release of GnRH surges in female rats is directed by a daily neural signal and occurs only after exposure of the hypothalamus to sustained, elevated estrogen (E2) levels in serum. We have proposed that preovulatory E2 couples the daily neural signal to the circuitry governing GnRH release by a two-step process, which includes stimulation of neuronal progesterone receptors (PRs) by E2 and subsequent activation of PRs by the daily neural signal. In the preceding report we documented that PR activation is obligatory for the stimulation of GnRH surges by E2. In these studies we assess the validity of a second essential feature of this model, that neural signals can activate PRs and thereby prompt the release of GnRH and LH surges. Our efforts specifically focused on the role of cAMP in mediating neural PR trans-activation leading to GnRH surges. To assess whether cAMP may function as a daily neural signal, cAMP levels were examined via a competitive binding assay in anteroventral periventricular nucleus (AVPV) homogenates obtained at 0900, 1200, 1500, 1800, and 2100 h on all days of the estrous cycle. A significant rise in cAMP concentrations was observed at 1500 h on all estrous cycle days. A similar rise at the same time was observed in AVPV tissues of ovariectomized (OVX) rats regardless of steroid treatment. No significant increase in cAMP levels was observed at any time point in homogenates of ventromedial nucleus or cerebral cortex. In a second experiment, female rats were OVX on the afternoon of diestrous day 2 and simultaneously administered 30 microg estradiol benzoate or oil vehicle. On the following day of presumptive proestrus, rats received intracerebroventricular infusions of the cAMP analog, 8 bromo-cAMP, or saline vehicle at 0900 h. Rats treated with 8-bromo-cAMP exhibited LH surges that were advanced by 3 h compared with those in saline-treated controls. This advance did not occur in 8-bromo-cAMP-treated rats not primed with E2, or in E2-treated rats given the antiprogestin RU486. In a third experiment, OVX, estradiol benzoate-primed rats received intracerebroventricular infusions of saline vehicle or the adenylyl cyclase inhibitor SQ22536; although saline-treated rats exhibited normal LH surges, no surges were observed in the rats receiving SQ22536. In additional SQ22536-treated animals, however, LH surge release was rescued and greatly augmented by a pharmacological dose of progesterone. These results demonstrate that 1) cAMP levels in the AVPV are significantly elevated at 1500 h on a daily basis; 2) cAMP elevations in the AVPV can prematurely evoke LH surges by a mechanism that requires PR activation; 3) inhibition of adenylyl cyclase activity in the AVPV blocks LH surges, an action that can be reversed by progesterone; and 4) cAMP generation leads to PR transactivation in the AVPV. Our observations thus provide support for the hypothesis that an increase in intracellular cAMP in the AVPV acts as a component of the daily neural signal required to initiate GnRH and subsequent LH surges, and that transmission of this signal is mediated by cAMP-induced PR trans-activation in the AVPV. PMID- 10746655 TI - Human insulin-like growth factor (IGF) binding protein-1 inhibits IGF-I stimulated body growth but stimulates growth of the kidney in snell dwarf mice. AB - The actions of insulin-like growth factor-I (IGF-I) are modulated by IGF binding proteins (IGFBPs). The effects of IGFBP-1 in vivo are insufficiently known, with respect to inhibitory or stimulatory actions on IGF-induced growth of specific organs. Therefore, we studied the effects of IGFBP-1 on IGF-I-induced somatic and organ growth in pituitary-deficient Snell dwarf mice. Human GH, IGF-I, IGFBP-1, and a preequilibrated combination of equimolar amounts of IGF-I and IGFBP-1 were administered sc during 4 weeks. Treatment with IGF-I alone induced a significant increase in body length (108% of control) and weight (112%) as well as an increase in weight of the submandibular salivary glands (135%), kidneys (124%), femoral muscles (111%), testes (129%), and spleen (126%) compared with saline treated controls. IGFBP-1 alone induced a significant increase in weight of the kidneys (152% of control). Coadministration of IGF-I with IGFBP-1 neutralized the stimulating effects of IGF-I on body length and weight as well as on the femoral muscles and testes. In contrast, the weights of the submandibular salivary glands (143%) were not significantly different from those of IGF-I-treated animals, whereas the weights of the kidneys (171%) and spleen (156%) were significantly increased compared with IGF-I-treated mice. The effect of IGFBP-1 plus IGF-I on kidney weight was not significantly greater than the effect of IGFBP-1 alone. Western ligand blotting showed induction of the IGFBP-3 doublet as well as IGFBPs with molecular masses of 24 kDa, most probably IGFBP-4, by human GH, IGF-I alone, and IGF-I in combination with IGFBP-1. Our data show that coadministration of IGFBP-1 inhibits IGF-I-induced body growth of GH-deficient mice but significantly stimulates the growth promoting effects of IGF-I on the kidneys and the spleen. These data warrant further investigation because differences in concentrations of IGFBP-1 occurring in vivo may influence IGF-I-induced anabolic processes. PMID- 10746656 TI - Prevention of spontaneous autoimmune diabetes in NOD mice by transferring in vitro antigen-pulsed syngeneic dendritic cells. AB - To evaluate the effect of antigen-pulsed dendritic cell (DC) transfer on the development of diabetes, 5-week-old female NOD mice received a single iv injection of splenic syngeneic DC from euglycemic NOD mice pulsed in vitro with human y globulin (HGG). Eleven of 12 mice were protected from the development of diabetes up to the age of 25 weeks, and the insulitis score was significantly reduced. In contrast, NOD mice receiving unpulsed splenic DCs showed histological signs of insulitis and course of type 1 diabetes similar to untreated NOD mice. Treatment with HGG-pulsed DC was associated with profound modifications of cytokine secretory capacities within the islets. Thus, supernatants of islets from these mice contained increased levels of interleukin (IL)-4, IL-10, and, to a lesser extent, interferon-gamma and diminished levels of tumor necrosis factor a compared with controls. Because exogenous IL-4 and IL-10 exert antidiabetogenic effect in NOD mice and early blockade of endogenous tumor necrosis factor-alpha prevents NOD mouse diabetes, these phenomena may be causally related to the antidiabetogenic effect of HGG-pulsed DC treatment. PMID- 10746657 TI - Long-term alterations in adiposity affect the expression of melanin-concentrating hormone and enkephalin but not proopiomelanocortin in the hypothalamus of ovariectomized ewes. AB - We have developed a ruminant model to study long-term alterations in adiposity on the expression of appetite-regulating peptides in the hypothalamus. In this model endocrine and metabolic status are fully defined as well as body composition. The current study sought to define the effects of altered adiposity on the expression of genes for neuropeptide Y (NPY), POMC, enkephalin (ENK), and melanin concentrating hormone (MCH). Ovariectomized ewes with high (60 +/- 1 kg) (FAT) or low (37 +/- 3 kg) body weights (THIN) were blood sampled every 10 min for 8 h to determine metabolic and endocrine status. The animals were then killed and the brains perfused for in situ hybridization. Body composition analysis was performed on the carcass using dual energy x-ray absorptiometry; this indicated that the FAT animals were 36 +/- 1% fat, whereas the THIN animals were 15 +/- 2% fat. The LH interpulse interval was lower and mean GH concentrations were higher in the THIN animals; cortisol and TSH levels were not different between the two groups but free T4 and free T3 levels were lower; the FT3:FT4 ratio was higher in THIN ewes. Levels of insulin, lactate, and nonesterified fatty acids were lower in the THIN group, and plasma glucose and urea concentrations were similar in THIN and FAT animals. Levels of gene expression of NPY and MCH were higher in THIN ewes. POMC expression was similar in the two groups. In the THIN animals, ENK expression was lower in the paraventricular and ventromedial nuclei but higher in the periventricular region. In conclusion, we have shown that alterations in adiposity influence the expression of appetite-regulating peptides in the absence of ovarian steroids. The appetite stimulators, NPY and MCH, appear to be involved in the metabolic response to altered adiposity, whereas ENK in the periventricular region may be linked to the secretion of GH and possibly LH. Our results suggest that altered expression of appetite- regulating peptides can be linked with the endocrine and metabolic adaptations that occur with long-term changes in adiposity. PMID- 10746658 TI - Localization of leptin receptor-like immunoreactivity in the corticotropes, somatotropes, and gonadotropes in the ovine anterior pituitary. AB - Leptin is a secreted product of the adipocytes that regulates a variety of functions. The presence of the leptin receptor (LR) has been demonstrated in the endocrine and neuroendocrine tissue, but only limited information is available regarding cell-specific expression in the anterior pituitary gland. We have used double-label immunofluorescence histochemistry to study the distribution of LR like immunoreactivity (LR-ir) in the corticotropes, somatotropes, and gonadotropes of the ovine anterior pituitary. LR-ir was found in 34% of cells in the pars distalis and 94% of the cells in the pars tuberalis. In the pars distalis, LR-ir was present in 27% of corticotropes, 69% of somatotropes, and 29% of gonadotropes. In contrast, 90% of the gonadotropes in the pars tuberalis were immunopositive for LR. There was no alteration in the number of gonadotropes containing LR-ir during the various phases of the estrous cycle (n = 3/group) in the pars distalis (luteal phase, 36%; follicular phase, 32%; and estrous phase, 32%). In conclusion, we show that, in the pars distalis, LR-ir is expressed to a greater extent in the somatotropes than in the gonadotropes or corticotropes. This is in accordance with the documented effects of leptin on pituitary GH secretion. The differential expression of LR-ir between the gonadotropes of the pars distalis and pars tuberalis probably reflects the different phenotypes of the cells in these two regions. Lower levels of LR-ir expression in gonadotropes and corticotropes of the pars distalis may suggest that leptin does not substantially influence these particular cells, at least in this species. PMID- 10746659 TI - Interleukin-1 receptor antagonist is produced by sertoli cells in vitro. AB - The interleukin-1 (IL-1) system has been suggested to be involved in the cell-to cell cross-talk within the testis. To identify a testicular cell source of IL-1 receptor antagonist (IL-1ra), mouse Sertoli cells were isolated, purified, cultured, and examined for IL-1ra. Our investigation revealed that Sertoli cells produce large amounts of immunoreactive IL-1ra under basal culture conditions, as examined by enzyme-linked immunosorbent assays. Its expression can be induced, showing maximum concentrations after 8 h of stimulation. Lipopolysaccharide, as well as IL-1alpha and -beta, were found to stimulate IL-1ra production in Sertoli cells. FSH is capable to induce IL-1ra production in Sertoli cells in a dose dependent manner. Immunocytochemical staining confirmed the presence of IL-1ra in the cytoplasma of Sertoli cells. The presence of IL-1ra messenger RNA was demonstrated by RT-PCR analysis. Our results may help to better evaluate the IL-1 activity in the testis and may indicate the involvement of IL-1ra in the autocrine and paracrine regulation of testicular cell function. PMID- 10746660 TI - Opposing actions of two transforming growth factor-beta isoforms on pituitary lactotropic cell proliferation. AB - Three transforming growth factor-beta protein isoforms (TGFbeta1, TGFbeta2, and TGFbeta3) have been identified in mammals. These isoforms appear to have similar actions on cell growth in various tissues. In rat pituitary tissue, TGFbeta1 is localized in PRL-secreting lactotropes and has been shown to act on lactotropes to inhibit estradiol-induced cell proliferation. The steroid inhibits the production and secretion of TGFbeta1. It is not known whether the other two isoforms are produced in and/or act on lactotropes. Using immunocytochemical detection techniques, we determined that, like TGFbeta1, TGFbeta3 is colocalized with PRL in the anterior pituitary of Fischer-344 female rats. Administration of estradiol increased TGFbeta3-immunoreactive cell numbers, TGFbeta3 protein, and TGFbeta3 messenger RNA levels in the pituitary. Determinations of TGFbeta3 actions in vitro in primary cultures of pituitary cells indicated that TGFbeta3 concentration dependently increases lactotropic cell proliferation. The growth promoting action of TGFbeta3 was potentiated by estradiol. Immunoneutralization studies indicated that although TGFbeta1 antibody failed to prevent estradiol's mitogenic action, it potentiated the mitogenic action of TGFbeta3. In contrast, TGFbeta3-neutralizing antibody inhibited lactotropic cell proliferation by estradiol. These data indicate that unlike many other tissues, TGFbeta1 and TGFbeta3 have opposite actions on lactotropic proliferation in the pituitary. Furthermore, TGFbeta1 and TGFbeta3 may be involved in estradiol's mitogenic action on lactotropes. PMID- 10746661 TI - Impaired growth, delayed ossification, and reduced osteoclastic activity in the growth plate of calcium-supplemented rats with renal failure. AB - Linear growth is reduced in prepubertal children with adynamic renal osteodystrophy, suggesting that the proliferation and/or differentiation of epiphyseal growth plate chondrocytes is abnormal in this disorder. To examine this issue, in situ hybridization and histochemistry were used to measure selected markers of endochondral bone formation and bone resorption in the proximal tibia of subtotally nephrectomized rats fed a high calcium diet to induce biochemical changes consistent with adynamic osteodystrophy. Blood ionized calcium concentrations were higher and serum PTH levels were lower in nephrectomized, calcium-supplemented rats than in either intact or nephrectomized control animals. Linear growth and tibial length were reduced, but messenger RNA levels for type II collagen, type X collagen, and the PTH/PTHrP receptor did not differ from control values in nephrectomized rats given supplemental calcium. In contrast, both the width of epiphyseal cartilage and the height of the zone of hypertrophic chondrocytes were greater in calcium-supplemented nephrectomized rats. These morphological changes were associated with decreases in histochemical staining for tartrate-resistant acid phosphatase and lower levels of messenger RNA expression for the matrix metalloproteinase MMP-9/gelatinase B immediately adjacent to the epiphyseal growth plate. Diminished chondroclastic/osteoclastic activity alters growth plate morphology and adversely affects linear bone growth in calcium-supplemented, nephrectomized rats. PMID- 10746662 TI - Platelet-derived growth factor activates porcine thecal cell phosphatidylinositol 3-kinase-Akt/PKB and ras-extracellular signal-regulated kinase-1/2 kinase signaling pathways via the platelet-derived growth factor-beta receptor. AB - Platelet-derived growth factor (PDGF) is a potent mitogenic factor for ovarian thecal cells cultured in vitro. PDGF binds to and induces homo- or heterodimerization of PDGF receptor-a or -beta (PDGF-Ralpha or PDGF-Rbeta). Despite this, little information is available about which PDGF receptors are expressed in the ovary, what signaling cascades are activated by PDGF, and the effects of PDGF on thecal cell steroidogenesis. The present study demonstrates the expression of immunoreactive PDGF-Rbeta, but not PDGF-Ralpha, in the thecal and stromal compartments of intact porcine ovaries as well as in cultured porcine thecal cells. Treatment of porcine thecal cells in vitro with PDGF resulted in rapid and sustained tyrosine phosphorylation of PDGF-Rbeta, activation of Src tyrosine kinase and phosphatidylinositol-3-kinase (PI3-kinase), and serine 473 phosphorylation of Akt/protein kinase B. In addition, PDGF stimulated an increase in GTP-Ras (activated Ras) and extracellular signal-regulated kinase (ERK) phosphorylation. Both forms of PDGF, AB and BB, stimulated thecal cell growth approximately 3- to 4-fold over controls and inhibited LH-stimulated progesterone and androstenedione secretion. Blockade of PI3-kinase activation with wortmannin had no effect on PDGF-stimulated thecal cell growth or PDGF inhibition ofLH stimulated steroid secretion, indicating that PI3-kinase activation is not necessary for PDGF-stimulated thecal cell growth or inhibition of LH-stimulated steroidogenesis. Conversely, blockade of the MEK-ERK pathway with PD98059 completely blocked PDGF-stimulated cell growth, indicating that activation of the MEK-ERK pathway is required for PDGF-stimulated thecal cell growth. Additionally, the MEK inhibitor PD98059 restored LH-stimulated steroid secretion, demonstrating that activation of the MEK-ERK pathway can lead to inhibition of LH-stimulated steroid secretion. The present study demonstrates that PDGF acts on ovarian thecal cells via activation of the PDGF beta-receptor and stimulates thecal cell growth via activation of a Rasmitogen-activated protein kinase-dependent, PI3 kinase-independent pathway. The strong expression of PDGF-Rbeta and the potent effects of PDGF on thecal cell growth and steroidogenesis suggest an important role for PDGF in thecal cell recruitment and growth during follicular development in vivo. PMID- 10746663 TI - The role of prostaglandin E receptor subtypes (EP1, EP2, EP3, and EP4) in bone resorption: an analysis using specific agonists for the respective EPs. AB - PGE2 functions as a potent stimulator of bone resorption. The action of PGE2 is thought to be mediated by some PGE receptor subtypes present in osteoblastic cells. In this study, we examined the involvement of PGE receptor subtypes, EP1, EP2, EP3, and EP4, in PGE2-induced bone resorption using specific agonists for the respective EPs. In mouse calvaria cultures, EP4 agonist markedly stimulated bone resorption, but its maximal stimulation was less than that induced by PGE2. EP2 agonist also stimulated bone resorption, but only slightly. EP1 and EP3 agonists did not stimulate it at all. RT-PCR showed that osteoblastic cells isolated from newborn mouse calvaria expressed all of the EPs messenger RNA (mRNA). Both EP2 agonist and EP4 agonist induced cAMP production and the expression of osteoclast differentiation factor (ODF) mRNA in osteoblastic cells. Simultaneous addition of EP2 and EP4 agonists cooperatively induced cAMP production and ODF mRNA expression. In mouse bone marrow cultures, EP2 and EP4 agonists moderately induced osteoclast formation, but the simultaneous addition of the two agonists cooperatively induced it, similar to that by PGE2. In calvaria culture from EP4 knockout mice, a marked reduction in bone resorption to PGE2 was found. In EP4 knockout mice, EP4 agonist failed to induce bone resorption, but EP2 agonist slightly, but significantly, induced bone resorption. These findings suggest that PGE2 stimulates bone resorption by a mechanism involving cAMP and ODF, which is mediated mainly by EP4 and partially by EP2. PMID- 10746664 TI - Differential expression of growth hormone messenger ribonucleic acid by somatotropes and gonadotropes in male and cycling female rats. AB - Past studies have reported the appearance of cells sharing phenotypic characteristics of gonadotropes and GH cells. During diestrus and early proestrus, a subset of somatotropes (40-60%) expressed both GH antigens and gonadotropin (LH-beta, LHbeta, or FSH-beta) messenger RNAs (mRNAs) or GnRH receptors. More recently, we reported that subsets of gonadotropes identified by LHbeta or FSHbeta antigens expressed GH- releasing hormone (GHRH) binding sites. The present studies were designed to learn if these putative multipotential cells also expressed GH mRNA. Biotinylated sense and antisense oligonucleotide probes were developed and cytochemical in situ hybridization tests were optimized for the detection of GH mRNA with GH, LHbeta, and FSHbeta antigens. RNase protection assays were developed with a complementary RNA probe that detected a 380-bp region at the 5' end of the GH mRNA. Both the in situ hybridization and RNase protection assays detected changes in expression of GH mRNA during the estrous cycle with the lowest expression occurring during metestrus and peak expression occurring on the morning of proestrus. Cell counts confirmed the results of the RNase protection assays showing that increases in mRNA levels seen from metestrus to proestrus reflected increased percentages of GH mRNA-bearing cells. In addition, densitometric analyses demonstrated that the higher GH mRNA levels assayed from diestrus to proestrus reflected increased area and density of label per cell. Both types of assays showed sex differences in expression of GH mRNA; male rat cell populations had higher values than female rats in metestrus, diestrus, or estrus. However, percentages of GH cells in male rats were equal to those from proestrous female rats and levels of GH mRNA were lower in male rats than proestrous females. Dual labeling experiments showed that, in male rats and diestrous, proestrous, or estrous females, GH mRNA was expressed in over 70% of GH cells. Expression of GH mRNA was also found in 50-57% of cells with LHbeta or FSHbeta antigens in the same groups. The lowest expression was seen in the metestrous groups (30-40% of GH cells or gonadotropes expressed GH mRNA). Expression of GH mRNA was first increased from metestrus to diestrous largely in GH cells, and slightly in cells with LHbeta antigens. Further increases were seen in GH and LH cells by the morning of proestrus. In contrast, FSH gonadotropes did not show an increased expression of GH mRNA until the morning of proestrus (reaching the same peak reached by LH cells). These data confirm the working hypothesis that a multihormonal cell type develops during diestrus to support both the somatotrope and gonadotrope populations. Collectively, our studies suggest that this multihormonal cell may function to help support the regulatory functions of the gonadotrope during the periovulatory period. In addition, the appearance of significant levels of expression of GH mRNA by male rat gonadotropes suggests that this multihormonal cell may play a role in regulation of the male reproductive system as well. PMID- 10746666 TI - Haystacks and needles. PMID- 10746667 TI - Acronymization of the prostate. PMID- 10746665 TI - Autocrine human growth hormone enhancement of human mammary carcinoma cell spreading is Jak2 dependent. AB - We investigated the role of autocrine production of human (h) GH in the attachment and spreading of mammary carcinoma cells in vitro. We used a previously described model system for the study of the autocrine/paracrine role of GH in which the hGH gene (MCF-hGH) or a translation-deficient hGH gene (MCF MUT) was stably transfected into MCF-7 cells. No differences in attachment to a collagen matrix between MCF-hGH and MCF-MUT cells were observed in either serum free medium (SFM) or medium containing exogenous hGH, 5% serum, or 10% serum. In contrast, MCF-hGH cells spread more rapidly on a collagen matrix than did MCF-MUT cells. Exogenous hGH and 10% serum interacted with autocrine production of hGH in an additive manner to increase cell spreading. MCF-hGH cells formed filipodia and stress fibers earlier than MCF-MUT cells during the process of cell spreading and possessed marked differences in morphology after spreading. MCF-MUT cells displayed uniform and symmetrical formation of stress fibers, whereas MCF-hGH cells displayed irregular and elongated stress fiber formation. The level of cytoplasmic phosphotyrosine was increased in MCF-hGH compared with MCF-MUT cells during spreading and displayed colocalization with Janus kinase 2 (JAK2). Basal JAK2 tyrosine phosphorylation was increased, and it increased further on spreading in MCF-hGH cells compared with MCF-MUT cells. Transient transfection of JAK2 complementary DNA resulted in interaction with autocrine hGH to increase the rate of cell spreading in MCF-hGH cells compared with MCF-MUT cells. Treatment with a selective JAK2 tyrosine kinase inhibitor (AG 490) reduced the rate of MCF hGH cell spreading to the rate of MCF-MUT cell spreading. Thus, we conclude that autocrine production of hGH enhances the rate of mammary carcinoma cell spreading in a JAK2-dependent manner. PMID- 10746668 TI - Expressions of cell-cycle regulatory gene products in conventional gastric adenomas: possible immunohistochemical markers of malignant transformation. AB - In 54 lesions of gastric adenomas consisting of 31 low-grade adenomas (LGAs) and 23 high-grade adenomas (HGAs), 28 intramucosal carcinomas (IMCs), and 23 carcinomas invading the submucosa (SMCs), the expression of cell-cycle regulatory gene products (p53, p21/waf1, p27/kip1, and Ki-67) was studied using immunohistochemical techniques. Several lesions were also analyzed by the fluorescence in situ hybridization method. The overexpression of p53 was found in no LGAs and in 9% of HGAs, whereas a considerable number of cases showed an overexpression in IMCs (39%) and SMCs (43%). A reduced expression of p21/waf1 was present in only 4% of HGAs. Superficial eccentric positivity was present in all LGAs and 74% of HGAs, whereas it was present in 46% of IMCs and 4% of SMCs. P53 positive and p21/waf1-negative lesions, which were supposed to have a mutated p53 gene, were observed in no LGAs, in 4% of HGAs, in 11% of IMCs, and in 26% of SMCs. The expression of cyclin E was more frequently present in carcinomas than in adenomas. However, no high expression of cyclin E was observed among the adenomas. A reduced expression of p27/kip1 was encountered more frequently in carcinoma than adenoma. By a semiquantitative evaluation comparing adenoma and carcinoma in the same stomach, the increased degrees of both p21/waf1 and cyclin E were highlighted. A chromosome gain was detected among 7% of the adenomas and 85% of the carcinomas. In conclusion, the expressions of p53, p21/waf1, p27/kip1, and cyclin E were considered to be of great value for estimating the dysplastic progression of gastric adenomas. Especially, various aspects of protein expression, including its distribution and semiquantitative evaluation of positive cells, and a combined analysis with several proteins, may thus be useful as possible markers of dysplastic evolution in gastric adenomas. PMID- 10746670 TI - Chronic deciduitis in the placental basal plate: definition and interobserver reliability. AB - This study tested whether concordance could be achieved for abnormal inflammation in the basal decidua of placental specimens among 6 pathologists experienced in placental pathology. Thirty microscope slides were evaluated by the pathologists for chronic deciduitis. They also scored the severity and extent of inflammation and the presence of plasma cells. No definition of chronic deciduitis was provided. Concordance (5/6 or 6/6 agreement) was achieved in 23 cases (76%). Spearman's rank correlation showed that the diagnosis of chronic deciduitis was almost identical to the assessment of the severity of the inflammation. A regression analysis showed that the perception of severity (and hence chronic deciduitis) was influenced by the other 2 variables, extent and plasma cells. The results were shared with the pathologists, and 25 cases (excluding those with previous 6/6 consensus) were reevaluated. Concordance was now achieved in the 83% of those remaining cases. Using a threshold based on the severity and the extent of lymphocytes, and the presence of plasma cells, pathologists are able to diagnose chronic deciduitis with sufficient concordance to be of value in clinical correlation studies. PMID- 10746669 TI - Distinction between dysplasia-associated lesion or mass (DALM) and adenoma in patients with ulcerative colitis. AB - Polyps with epithelial dysplasia in ulcerative colitis (UC) represent either dysplasia-associated lesions or masses (DALMs) or sporadic adenomas. DALMs are frequently associated with associated carcinoma and are an indication for colectomy. Removal of the polyp is treatment of choice for sporadic adenomas. Differentiating between these 2 lesions is not always easy. The goal of this study was to distinguish DALMs from adenomas in patients with UC on a genetic basis. We evaluated genetic alterations in DALMs and compared them with a previously published set of dysplastic polyps in patients with UC that were considered adenomas for the following reasons: (1) polyps were located outside of current active disease; (2) polyps had histological features of sporadic adenomas; and (3) patients displayed a uneventful follow-up after polypectomy (UC adenomas). In addition, adenomas not associated with UC were studied. Genetic alterations on chromosome 3p were assessed for the markers D3S1766, D3S2409, and D3S2387. LOH with or without microsatellite instability was found in 70%, 37%, and 57% of cases of DALM, respectively. In contrast, UC-adenomas lesions exhibited genetic alterations in 8.3%, 11.7%, and 15.3% for the respective markers. Spontaneous adenomas exhibited genetic alterations in 10.5%, 7.1%, and 0% of cases, which were not significantly different from the UC-adenoma results. These results indicate that UC-adenomas are genetically and biologically similar to sporadic adenomas and that UC-adenomas may biologically represent sporadic adenomas, supporting on a genetic basis the criteria chosen to diagnose adenomas in UC. Genetic markers on chromosome 3p may be useful in the differential diagnosis between DALM and UC-adenomas. PMID- 10746671 TI - Matrix metalloproteinases and tissue inhibitors of metalloproteinases in bronchial squamous preinvasive lesions. AB - Metalloproteinases and their inhibitors are known to play an important role in the extracellular matrix remodeling associated with preinvasive lesions and invasive carcinomas; however, little is known about their role in early lung carcinoma. Immunohistochemical studies were made of the reactivity of bronchial squamous preneoplastic lesions from cigarette smokers, including basal cell hyperplasia, squamous metaplasia, dysplasia, carcinoma in situ, and invasive squamous cell carcinoma for matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and type IV collagen in 13 patients. Staining for type IV collagen disclosed discontinuities in basement membranes from basal cell hyperplasia to dysplasia, progressing to destruction in carcinoma in situ and invasive carcinoma. Reactivity for MMP-9 was mild in basal cell hyperplasia and squamous metaplasia, increasing in carcinoma in situ and invasive carcinoma. In contrast, reactivity for MMP-1 was strong in basal cell hyperplasia and squamous metaplasia, decreasing in carcinoma in situ and invasive carcinoma. Some neoplastic cells in carcinoma in situ and invasive carcinoma were MMP-3 positive. Staining for MMP-2 and TIMP-1 was moderate to strong in all squamous preinvasive lesions. Confocal microscopy showed MMP-9-positive cells passing through fragmented basement membranes in which type IV collagen and MMP-9 were colocalized. Type IV collagen colocalized with MMP-2 in all lesions and with TIMP 1 in basal cell hyperplasia and squamous metaplasia. The inverse relationships between the reactivity for MMP-1 and MMP-9 with progression of bronchial squamous preinvasive lesions suggest important roles for these MMPs in basement membrane remodeling in these lesions. PMID- 10746672 TI - Recurrent anomalies of 6q25 in chondromyxoid fibroma. AB - Chondromyxoid fibroma is a rare benign bone tumor most commonly arising in the metaphysis of long bones in young adults. Histopathologically, chondromyxoid fibroma may be difficult to distinguish from other cartilaginous neoplasms. Recently, a pericentric inversion of chromosome 6 [inv(6)(p25q13)] has been proposed as a specific genetic marker for chondromyxoid fibroma. In this study, cytogenetic and spectral karyotypic analyses of 2 chondromyxoid fibroma cases showed clonal abnormalities of chromosome 6 but at a breakpoint on the long arm (q25) distal to that described in the pericentric inversion. These findings suggest that several distinct breakpoints on chromosome 6 are nonrandomly involved in chondromyxoid fibroma. PMID- 10746673 TI - PTEN mutations in endometrial carcinomas: a molecular and clinicopathologic analysis of 38 cases. AB - PTEN mutations have been reported to be frequent in endometrioid carinomas of the endometrium (EEC). Some correlation has been found between PTEN mutations and the presence of microsatellite instability (MI) in EEC, but no convincing cause effect relationship for such association has been offered. DNA of 38 patients with endometrial carcinoma (EC) was extracted from blood and from fresh-frozen and paraffin-embedded tumor tissue. PTEN mutations were detected by single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. Results were correlated with MI status and clinicopathologic data. PTEN mutations were detected in 17 tumors (44.7%), and they were more frequent in endometrioid (EEC) (17 of 33, 51.5%) than in nonendometrioid carcinomas (NEEC) (0 of 5, 0%). PTEN mutational spectrum differed between MI+ and MItumors. PTEN mutations were detected in 9 of 15 MI+ tumors (60%), but in only 8 of 23 MI- neoplasms (34.8%). In EC with MI, PTEN mutations were detected in short coding mononucleotide repeats (A)s and (A)6 in 4 of 9 carcinomas (44.4%). These results confirm that PTEN is an important target gene in endometrial carcinogenesis. The occurrence of PTEN mutations in short coding mononucleotide repeats in MI-positive tumors suggests that these mutations may be secondary to deficiencies in mismatch repair and gives some explanation for the frequent presence of PTEN mutations in these tumors. PMID- 10746675 TI - Individualized prognosis for melanoma patients. AB - The clinical course of malignant melanoma is notoriously variable. Current approaches to prognostication allow assignment to risk categories but do not permit accurate assessment of prognosis on an individual patient basis. We analyzed a melanoma histology database that comprises 1,042 sequential melanoma patients evaluated by A.J.C. at UCLA between 1980 and 1990 for 30 separate variables according to a standard protocol. After censoring for absent data, a univariate Cox model analysis was performed that showed 20 individual variables that were significantly linked to clinical outcome. A step-up multivariate analysis was then performed. The combined analysis shows 5 variables: gender, site of primary, age relative to 60 years, Breslow thickness, and presence and width of ulceration to be linked to survival. Probability of survival is calculated using a 2-step approach. The survival-linked variables are multiplied to give an individualized risk score. This is converted into probability of survival by the formula .987 (risk score) for 3-year survival, .975 (risk score) for 5-year survival, and .960 (risk score) for 10-year survival. Thus, a 55-year old woman with a 1.8-mm nonulcerated melanoma on the leg would have a risk score of (1 x 1 x 1 x 2 x 1) = 2 and a predicted probability of survival at 5 years of .9752 (95%) and at 10 years of .9602 (92%). We used similar techniques to develop individualized risk scores for likelihood of recurrence. The significant variables in this case are anatomic site of the primary melanoma, melanoma subtype, Breslow thickness, and presence and width of ulceration. The formulae for likelihood of recurrence at different periods after initial surgical removal of the primary melanoma are at 3 years, .979(risk score); at 5 years, .971(risk score); and at 10 years, .957(risk score). This relatively simple approach to prognostication uses readily available demographic information and is likely to be more accurate than single-factor analysis. PMID- 10746674 TI - Epstein-Barr virus (EBV) genes expression in cervical intraepithelial neoplasia and invasive cervical cancer: a comparative study with human papillomavirus (HPV) infection. AB - To elucidate a causative role of Epstein-Barr virus (EBV) for cervical cancer, presence and expression of EBV genes were examined in 31 cervical carcinomas (ICC), 23 cervical intraepithelial neoplasias (CIN), and 35 normal cervices (NCX). In reverse transcription polymerase chain reaction (RT-PCR) analysis, EBER 1 mRNAwas expressed in 74% (23/31) of ICC, 83% (19/23) of CIN, 37% (13/35) of NCX. LMP-1 was expressed in 52% (16/31) of ICC, 70% (16/23) of CIN, and 23% (8/35) of NCX, and EBNA-2 was expressed in 32% (10/31) of ICC, in 48% (11/23) of CIN, and in 11% (4/35) of NCX. Expression rates of these genes were significantly higher in ICC and CIN than in NCX (P < .05). RNA in situ hybridization analysis showed that EBER-1 was expressed in half (7/14) of ICC and 35% (6/17) of CIN, and BamH-W, which is a leader sequence of EBNA genes, was expressed in 86% (12/14) of ICC and 71% (12/17) of CIN. LMP-1 and EBNA-2 proteins also were detected in ICC and CIN cells by inmunofluorescence staining. PCR analysis showed that EBV genome was detected in 55% (17 of 31) of ICC and in 26% (9/35) of NCX. In contrast, human papillomavirus (HPV) DNA was detected in 84% (26/31) of ICC, whereas none of NCX had HPV DNA. Either EBV or HPV was detected in 61% (19/31) of ICC, whereas both EBV and HPV was detected in 39% (12/31) of ICC. EBV infection may be involved in the development of cervical cancer, although further study should be performed to elucidate a causative role of EBV for the cancer. PMID- 10746676 TI - Sarcomatoid carcinoma of the urinary bladder: a clinicopathologic and immunohistochemical analysis of 14 patients. AB - Sarcomatoid carcinoma of the urinary bladder is a rare entity, in which both the histogenesis and biological behavior remain controversial. We herein describe the clinicopathologic and immunohistochemical profiles of sarcomatoid carcinomas and discuss the significance of cell adhesion molecules in the development of this peculiar neoplasm. The authors examined formalin-fixed and paraffin-embedded tissue samples from 14 patients with sarcomatoid carcinoma of the urinary bladder. An immunohistochemical analysis was performed by using antibodies against epithelial and mesenchymal antigens as well as adhesion molecules. Most patients suffered from an advanced stage of the tumor, extending to the muscular layer (7 cases) or to the perivesical tissues (5 cases). Microscopically, all 14 tumors were composed predominantly of a carcomatoid component and an obviously carcinomatous component. The sarcomatoid component was composed of a mixture of spindle cells, round cells, and pleomorphic giant cells. The carcinomatous components consisted of papillary or nonpapillary high-grade transitional cell carcinoma (TCC). The zones of gradual transition between the carcinomatous and the sarcomatous elements were focally apparent in each tumor. The findings of an immunohistochemical examination indicated that both carcinomatous and sarcomatoid components expressed epithelial antigens (pankeratin or EMA), even though the staining pattern varied from case to case. As for cell adhesion molecules, the carcinomatous components were positive for E-cadherin (8 of 12), CD44s (8 of 12), and CD44v6 (6 of 12). Although the sarcomatoid components were also positive for E-cadherin (5 of 12), CD44s (4 of 12), and CD44v6 (3 of 12), these rates were lower than those in the carcinomatous components. Six patients died of their disease between 5 and 36 months after the diagnosis was made. The recognition of sarcomatoid carcinomas has important therapeutic and prognostic implications. It seems appropriate to treat these neoplasms in the same manner as conventional high-grade TCCs with similar degrees of invasion. We consider that sarcomatoid carcinomas should be regarded as a high-grade carcinoma that shows a prominent pseudosarcomatous dedifferentiation. The sarcomatoid component of sarcomatoid carcinomas may result from either anaplastic changes or dedifferentiation related to the process of losing cell adhesion molecules. PMID- 10746677 TI - Microvasculature and VEGF expression in cartilaginous tumors. AB - We examined the microvasculature and VEGF expression in 26 cartilaginous lesions (CL) including 5 enchondromas, 9 grade 1 chondrosarcoma (CS), 6 grade 2 CS, 4 grade 3 CS, 1 mesenchymal, and 1 myxoid chondrosarcoma. The degree of neovascularization was measured by counting microvessels on H&E and factor VIII related antigen immunostained slides. Vessels were divided into pericartilage vessels (PCV) and intracartilage vessels (ICV). PVC comprised vessels around the lobules or invading the lobules but themselves surrounded by noncartilaginous stroma (ie, fibrous stroma); ICV consisted of those vessels present inside the tumoral nodules and in direct apposition with malignant cells or tumoral stroma. A direct correlation was seen between histological type and grade of CS and pericartilage vessels. In contrast, ICV were found only in higher-grade CS. No enchondromas and only 1 of 9 grade 1 CS had ICV. This patient had Ollier's disease. All but 2 of the grade 2 CS showed ICV (average, 20.5). The exceptions were predominantly grade 1 CS with focal grade 2 areas and extensive areas of necrosis. All but 1 grade 3 CS had ICV, the exception being a case of metastatic CS to the lung. Malignant chondrocytes of high-grade lesions stained strongly for vascular endothelial growth factor (VEGF), a potent angiogenic factor. The only high-grade tumors that did not express VEGF did not show ICV either. Enchondromas and grade 1 CS, most without ICV, did not express VEGE In summary, PCV are present in all categories of tumoral cartilage and the number increases with histological grade; ICV are found in high-grade lesions, and the exceptions show extensive necrosis; VEGF expression by malignant chondrocytes is seen in high grade lesions almost exclusively, and among these in those lesions that showed intracartilage vessels. It is possible that PCV are involved in supporting tumor growth, whereas ICV might be involved in the acquisition of metastatic potential by cartilage tumors. VEGF expression is strongly associated with the presence of ICV. PMID- 10746678 TI - Preoperative chemoradiotherapy alters the expression and prognostic significance of adhesion molecules in Barrett's-associated adenocarcinoma. AB - A variety of prognostic markers have been related to decreased patient survival in patients with epithelial malignancies. These include expression of the homotypic adhesion molecule E-cadherin (ECAD) and the hyaluronic acid receptor CD44. Expression of ECAD and CD44 was evaluated in Barrett's-associated adenocarcinoma (BAd) from 67 patients. Expression was determined by immunoperoxidase staining and graded semiquantitatively based on the proportion of positively stained cells. These data were then correlated with clinical and pathological parameters, including the presence or absence of chemoradiotherapy (chemrad) and patient survival. There were 56 men and 11 women (mean age, 62 years). Thirty-nine (58%) patients received preoperative chemrad. ECAD expression was detected in all (100%) tumors. The ECAD staining grade did not correlate with other pathological features of the tumors. However, ECAD staining was significantly increased in BAd of patients who received chemrad (P = .003), in comparison with those who did not, and in individual patients when prechemrad biopsies and postchemrad resection specimens were compared (P = .04). In terms of prognosis, increased ECAD expression was associated with shortened patient survival only in BAd patients who had received chemrad (univariate analysis of chemrad patients with stage I and II BAd, P = .02). ECAD expression was not significantly associated with survival in BAd patients who did not receive chemrad. CD44 expression was detected in 88% of cases. CD44 expression did not correlate with any of the pathological features of the tumors or with chemrad status. Increased expression of CD44 was significantly associated with shortened patient survival in chemrad patients only (univariate analysis P = .03, multivariate analysis P = .04), although a strong trend was observed when all patients were analyzed regardless of chemrad status (P = .07). The results of this study indicate that chemrad alters the expression of ECAD in BAd. Thus, the prognostic utility of ECAD expression must be evaluated in the context of chemrad status. CD44 also may be a valuable prognostic marker in BAd. PMID- 10746679 TI - Altered expression of hMLH1 and hMSH2 protein in endometrial carcinomas with microsatellite instability. AB - Microsatellite instability (MI) has been observed in approximately 20% of presumably sporadic cases of uterine endometrioid carcinoma (UEC). A previous mutational analysis of the 4 known DNA mismatch repair genes (hMSH2, hMLHI, hPMS1, and hPMS2) on a small number of Ml-positive tumors detected mutations in only 2 of 8 cases, both in hMSH2. To further explore the underlying cause of MI in UEC, we analyzed the protein expression of hMSH2 and hMLHI in UEC of known MI status. Formalin-fixed, paraffin-embedded archival tissue from 21 UECs was analyzed by immunoperoxidase staining with monodonal antibodies against hMLH1 and hMSH2 protein. Tumors were evaluated for presence of nuclear staining by 3 investigators. Lack of nuclear hMLHI staining was found in 7 of 13 carcinomas with MI, but in none of 8 carcinomas without MI (Fischer's exact, 0.018). Lack of nuclear hMSH2 staining was found in 3 of the MI-positive cases, but none of the MI-negative cases (not statistically significant). Taken together, lack of nuclear staining of either hMLH1 or hMSH2 was found in 9 of 13 cases with MI and in none of 8 cases without MI (Fischer's exact, 0.005). We conclude that MI in sporadic UEC appears to be associated with lack of expression of either hMLH1 or hMSH2, suggesting that inactivation of these genes may be responsible for MI in most MI-positive sporadic UECs. PMID- 10746680 TI - Type XV collagen in human colonic adenocarcinomas has a different distribution than other basement membrane zone proteins. AB - In situ carcinomas must penetrate their own basement membrane to be classified as invasive, and subsequently infiltrate surrounding connective tissue and cross vascular basement membranes to metastasize hematogenously. Accordingly, in many studies, integral basement membrane components, including type IV collagen, laminin, and heparan sulfate proteoglycan, have been localized in a spectrum of tumors to gain insight into their role in neoplasia. A number of recently identified extracellular matrix molecules and isoforms of the aforementioned proteins have been localized to the basement membrane zone, illustrating another level of biochemical heterogeneity in these structures. As the complexity of these matrices becomes more apparent, their roles in maintaining homeostasis and in tumor biology falls into question. Of the new group of collagens localized to the basement membrane zone, type XV was the first to be characterized (Cell Tissue Res, 286:493-505, 1996). This nonfibrillar collagen has a nearly ubiquitous distribution in normal human tissues via a strong association with basement membrane zones, suggesting that it functions to adhere basement membrane to the underlying stroma. To begin investigation of this protein in malignant tumors, we have localized type XV in human colonic adenocarcinomas and compared its distribution with that of type IV collagen and laminin. Collagens XV and IV and laminin were found in all normal and colonic epithelial, muscle, fat, neural, and vascular basement membrane zones, as shown previously. In moderately differentiated, invasive adenocarcinomas, laminin and type IV collagen were sometimes observed as continuous, linear deposits around some of the malignant glands, but more often they were seen in either discontinuous deposits or were completely absent. In contrast, type XV collagen was characterized as virtually absent from the basement membrane zones of malignant glandular elements in moderately differentiated tumors. Nevertheless there were also similarities; all 3 proteins were usually present in the stroma and adjacent vascular basement membrane zones surrounding invasive glands. The loss of type XV collagen from these malignant epithelial basement membrane zones and its increased interstitial expression suggests a role for this protein in the invasive process and the possibility that it may provide a sensitive indicator of tumor invasion. PMID- 10746681 TI - Identification of a novel 9 cM deletion unit on chromosome 6q23-24 in papillary serous carcinoma of the peritoneum. AB - To define regions of deletion on chromosome 6q in papillary serous carcinoma of the peritoneum (PSCP), we analyzed 103 tumor tissues from 53 patients by using 11 polymorphic microsatellite markers spanning loci from 6q23 to 6q27. Allelic losses on 6q were observed in 42 of 53 (79.2%) cases. We identified 3 distinct regions with a high percentage (>40%) of loss of heterozygosity. The first region is located at 6q23-24 and defined by D6S311 (15 of 35 informative cases, 42.9%). Detailed deletion mapping of chromosome 6q23-24 in these tumor samples identified a novel 9 cM minimal deletion region flanked by D6S250 and ESR. The second one is located at 6q25.1-25.2 and defined by D6S448 (17 of 36 informative cases, 47.2%). A second minimal deletion region of 4 cM was flanked by D6S420 and D6S442. The third region is located at 6q27 and defined by D6S297 (9 of 19 informative cases, 47.4%). Comparing these results with our cases of advanced staged invasive serous epithelial ovarian carcinoma (SEOC), we observed that allelic losses at D6S311 (6q23) and D6S149 (6q27) were significantly higher for PSCP than for SEOC. The pattern of allelic loss at each tumor site within an individual patient was also studied. A total of 36 cases displayed allelic loss for at least 1 of multiple tumor sites, and 35 of these patients exhibited nonidentical patterns of allelic loss at various tumor sites of the same patient. Furthermore, an alternating pattern of allelic loss in the same patient was identified in 3 of 53 patients studied. These results show that allelic losses on 6q are very frequent in PSCP, and we show 2 discrete minimal deletion regions on 6q, suggesting the existence of at least 2 tumor suppressor genes within 6q that may be involved in the pathogenesis of PSCP. In addition, the finding of different patterns of allelic loss at different tumor sites within the same patient indicate a mutifocal origin in some PSCP cases. These results provide strong evidence to support our previous reports that PSCP is a multifocal disease entity. PMID- 10746682 TI - Large artery vasculopathy in HIV-positive patients: another vasculitic enigma. AB - Human immunodeficiency virus (HIV) infection has impacted on all the systems of the body, and the cardiovascular system is no exception, with small to medium sized vessel vasculitis being most frequently described. We present 16 HIV positive patients with large vessel disease consisting of either aneurysms (often multiple) or occlusive disease. Nine men and 7 women ranging in age from 18 to 38 years presented with rupture of aneurysm, transient ischemic attacks, hypertension, ischemia to the lower extremity, or a mass at the site of the aneurysm. Eight patients had 1 aneurysm, 2 had 2 lesions, and the remaining 6 cases had from 3 to 7 aneurysms. Arteries affected included the common carotid, abdominal aorta, common iliac, femoral, and popliteal. Three patients had intercurrent infections, but none had any obvious infective vascular lesion. Only 1 patient had a positive TPHA test for syphilis. Microbiologic culture of both blood and thrombus contents was positive for Staphylococcus aureus in 1 case; no other organisms were cultured. The key histological features were within the adventitia: leukocytoclastic vasculitis of the vasa vasora and periadventitial vessels, proliferation of slit-like vascular channels, chronic inflammation, and fibrosis. There was associated medial fibrosis with loss and fragmentation of muscle and elastic tissue. Intimal changes consisted of duplication and fragmentation of the internal elastic lamina with calcification. Atheroma and marked intimal thickening were not evident We believe that the occurrence of this large vessel vasculopathy (mainly aneurysmal) often with multiple lesions in young HIV-positive patients, is characteristic of possible infective or immune complex origin, with leukocytoclastic vasculitis of vasa vasora and periadventitial vessels being pivotal in many cases. PMID- 10746683 TI - The digestive system manifestations of Rosai-Dorfman disease (sinus histiocytosis with massive lymphadenopathy): review of 11 cases. AB - Rosai-Dorfman disease (RDD), originally described as sinus histiocytosis with massive lymphadenopathy, is a rare histiocytic proliferative disorder with a distinctive microscopic appearance. Formerly thought to be a process limited to lymph nodes, involvement by RDD has now been documented in many organ systems, notably bone, skin and soft tissue, central nervous system, eye and orbit, and upper respiratory tract. The digestive system, however, is affected only exceptionally, as reflected by the existence of only a handful of individual case reports. In this article, we report 11 patients in which the disease involved intestinal tract, liver, or pancreas, and describe the most salient clinicopathologic features. The specific site of involvement within the digestive system was gastrointestinal tract in 5, liver in 5, and pancreas in 1. Most patients also had evidence of disease in other extranodal sites, as well as in 1 or more lymph node groups. PMID- 10746684 TI - Expression of thyroid transcription factor-1 (TTF-1) in human C cells and medullary thyroid carcinomas. AB - Thyroid transcription factor-1 (TTF-1) has been known to regulate the transcriptional activity of thyroid-specific genes in thyroid follicular cells. We recently identified TTF-1 mRNA expression in rat thyroid C cells. The current study was undertaken to elucidate how TTF-1 is expressed in human C cells and medullary thyroid carcinomas (MTCs), and how this expression influences the functions and clinical behavior of these cells. By immunohistochemistry, the nuclei of normal and hyperplastic C cells distinctively reacted with antibody against TTF-1, whereas the immunostaining intensity in C cells was rather weak and heterogeneous in comparison with that in follicular cells. Identical TTF-1 immunoreactivity was observed in all 15 MTC specimens examined. The reaction intensity did not depend on tumor patterns or cell features. In nonisotopic in situ hybridization, an antisense riboprobe clearly hybridized the cytoplasms of C cells and MTC cells, which concurrently showed immunohistochemical positivity for calcitonin. Northern blot analysis indicated a marked hybridization with TTF-1 mRNA of approximately 2.3 kb in an MTC specimen. Furthermore, the presence of TTF 1 mRNA was confirmed by reverse transcription polymerase chain reaction (RT-PCR) in the human MTC cell line, TT. Our results suggest that human thyroid C cells and MTC cells express TTF-1 in connection with their functional ability. Therefore, TTF-1 expression can be a functional marker not only for follicular cells and follicular cell tumors but also for C cells and medullary C cell carcinomas. PMID- 10746685 TI - JC virus in human glial-derived tumors. AB - To investigate the presence and the role of polyomaviruses JC (JCV), BK (BKV), and the simian polyomavirus (SV40) in human brain tumors, samples from 25 glial derived tumors (10 astrocytomas, 5 ependymomas, 5 oligodendrogliomas, and 5 glioblastomas) were examined by means of molecular biology and immunohistochemistry. Nested PCR of the large T (LT) region and its sequence analysis showed JCV in 6 cases (4 astrocytomas, 1 oligodendroglioma, and 1 ependymoma), while the transcriptional control region (TCR) was amplified only in 1 astrocytoma, the oligodendroglioma, and the ependymoma, one of which (astrocytoma) also stained positively by immunohistochemistry (JCV LT). TCR sequence analysis of the oligodendroglioma showed a JCV rearranged structure not related to a known viral strain, while the astrocytoma and the ependymoma disclosed a JCV Mad-4 strain that is known to induce brain tumors in animals. We suggest that JCV could have played a role in the pathogenesis of these brain tumors. PMID- 10746686 TI - Transient myeloproliferative disease of the newborn: case report with placental, cytogenetic, and flow cytometric findings. AB - Transient myeloproliferative disease (TMD) of the newborn is a rare hematologic abnormality associated with trisomy 21. It is frequently difficult to distinguish the disorder from true congenital leukemia (TCL). Unlike leukemia, which has a clinically aggressive course, TMD generally resolves within weeks to months. We present a case of TMD of the newborn diagnosed on the basis of peripheral blood studies and describe the pertinent pathological findings within the placenta. Flow cytometric analysis of the blasts in the peripheral blood showed phenotypic heterogeneity with features consistent with megakaryocytic differentiation. Cytogenetic studies showed trisomy 21 within the blastic cells. The placenta showed villous dysmaturity with associated chorangiosis and prominent intravascular aggregates of primitive-appearing cells with focal, early vascular wall invasion. The neonate recovered fully and shows no evidence of disease at 2 years of age. PMID- 10746687 TI - Uterine myxoid leiomyosarcoma within a leiomyoma. AB - A case of myxoid leiomyosarcoma of the uterus arising in a leiomyoma is reported. Although the tumor showed very low mitotic activity ranging from zero to 2/10 HPF, the presence of infiltrative pattern of growth and a high MIB-1 index (60% of cells positive) established the diagnosis. Myxoid leiomyosarcoma may arise in leiomyoma. PMID- 10746688 TI - Rules of genome access. PMID- 10746689 TI - Eyes, ears and science PMID- 10746691 TI - Space researchers protest at 'disruptive' export controls PMID- 10746690 TI - Researchers caught in dispute over transgenic mice patents. PMID- 10746693 TI - Northern lobby attacks UK synchrotron siting PMID- 10746692 TI - Could AIDS treatments slip through patents loophole? PMID- 10746694 TI - US/UK statement on genome data prompts debate on 'free access'. PMID- 10746696 TI - To create generalists, teach students how to learn by themselves PMID- 10746695 TI - 'The human genome itself must be freely available to all humankind'. PMID- 10746697 TI - We have touched the dust from dying stars PMID- 10746698 TI - Confusion over cash for Indian biotech centre... PMID- 10746699 TI - Though funding states have promised to pay up. PMID- 10746700 TI - The lasting value of Mitchell's mechanisms. PMID- 10746701 TI - People must be judged in the context of their time. PMID- 10746702 TI - Brains, courage and integrity PMID- 10746703 TI - Bordeaux mixture PMID- 10746704 TI - From forelimbs to two legs. PMID- 10746705 TI - Tricks with a single photon PMID- 10746707 TI - Gamma-ray astronomy. A needle in a cosmic haystack PMID- 10746706 TI - Parkinson's pathology in a fly. PMID- 10746708 TI - Making hard light sharper PMID- 10746710 TI - Robert Rathbun Wilson (1914-2000) PMID- 10746709 TI - Membrane fusion. Changing partners. PMID- 10746711 TI - Y-chromosome variation and Irish origins. PMID- 10746712 TI - Fluctuations caught in the act. PMID- 10746713 TI - Apolipoprotein E and cognitive performance. PMID- 10746714 TI - A tetrodotoxin-producing marine pathogen. PMID- 10746716 TI - Discovery of a new population of high-energy gamma-ray sources in the Milky Way AB - One of the great mysteries of the high-energy gamma-ray sky is the group of approximately 170 unidentified point sources found along the Galactic plane. They are more numerous than all other high-energy gamma-ray sources combined and, despite 20 years of effort, no clear counterparts have been found at other wavelengths. Here we report a new population of such objects. A cluster of approximately 20 faint sources appears north of the Galactic Centre, which is part of a broader class of faint objects at mid-latitudes. In addition, we show in a model-independent way that the mid-latitude sources are distinct from the population of bright unidentified sources along the Galactic plane. The distribution on the sky indicates that the faint mid-latitude sources are associated with the Gould belt of massive stars and gas clouds at approximately 600 light years distance, as has been previously suggested. PMID- 10746715 TI - Three-dimensional structure of the neuronal-Sec1-syntaxin 1a complex. AB - Syntaxin 1a and neuronal Sec1 (nSec1) form an evolutionarily conserved heterodimer that is essential for vesicle trafficking and membrane fusion. The crystal structure of the nSec1-syntaxin 1a complex, determined at 2.6 A resolution, reveals that major conformational rearrangements occur in syntaxin relative to both the core SNARE complex and isolated syntaxin. We identify regions of the two proteins that seem to determine the binding specificity of particular Sec1 proteins for syntaxin isoforms, which is likely to be important for the fidelity of membrane trafficking. The structure also indicates mechanisms that might couple the action of upstream effector proteins to conformational changes in syntaxin 1a and nSec1 that lead to core complex formation and membrane fusion. PMID- 10746717 TI - Trapping an atom with single photons AB - The creation of a photon-atom bound state was first envisaged for the case of an atom in a long-lived excited state inside a high-quality microwave cavity. In practice, however, light forces in the microwave domain are insufficient to support an atom against gravity. Although optical photons can provide forces of the required magnitude, atomic decay rates and cavity losses are larger too, and so the atom-cavity system must be continually excited by an external laser. Such an approach also permits continuous observation of the atom's position, by monitoring the light transmitted through the cavity. The dual role of photons in this system distinguishes it from other single-atom experiments such as those using magneto-optical traps, ion traps or a far-off-resonance optical trap. Here we report high-finesse optical cavity experiments in which the change in transmission induced by a single slow atom approaching the cavity triggers an external feedback switch which traps the atom in a light field containing about one photon on average. The oscillatory motion of the trapped atom induces oscillations in the transmitted light intensity; we attribute periodic structure in intensity-correlation-function data to 'long-distance' flights of the atom between different anti-nodes of the standing-wave in the cavity. The system should facilitate investigations of the dynamics of single quantum objects and may find future applications in quantum information processing. PMID- 10746718 TI - An algorithmic benchmark for quantum information processing AB - Quantum information processing offers potentially great advantages over classical information processing, both for efficient algorithms and for secure communication. Therefore, it is important to establish that scalable control of a large number of quantum bits (qubits) can be achieved in practice. There are a rapidly growing number of proposed device technologies for quantum information processing. Of these technologies, those exploiting nuclear magnetic resonance (NMR) have been the first to demonstrate non-trivial quantum algorithms with small numbers of qubits. To compare different physical realizations of quantum information processors, it is necessary to establish benchmark experiments that are independent of the underlying physical system, and that demonstrate reliable and coherent control of a reasonable number of qubits. Here we report an experimental realization of an algorithmic benchmark using an NMR technique that involves coherent manipulation of seven qubits. Moreover, our experimental procedure can be used as a reliable and efficient method for creating a standard pseudopure state, the first step for implementing traditional quantum algorithms in liquid state NMR systems. The benchmark and the techniques can be adapted for use with other proposed quantum devices. PMID- 10746719 TI - Storage of X-ray photons in a crystal resonator AB - The temporal structure and high brilliance of the X-ray beams produced by third generation synchrotrons open up new possibilities in time-dependent diffraction and spectroscopy, where timescales down to the sub-nanosecond regime can now be accessed. These beam properties are such that one can envisage the development of the X-ray equivalent of optical components, such as photon delay lines and resonators, that have proved indispensable in a wide range of experiments--for example, pump-probe and multiple-interaction experiments--and (through shaping the temporal structure and repetition rate of the beams) time-dependent measurements in crystallography, physics, biology and chemistry. Optical resonators, such as those used in lasers, are available at wavelengths from the visible to soft X-rays. Equivalent components for hard X-rays have been discussed for more than thirty years, but have yet to be realized. Here we report the storage of hard X-ray photons (energy 15.817 keV) in a crystal resonator formed by two plates of crystalline silicon. The photons are stored for as many as 14 back-and-forth cycles within the resonator, each cycle separated by one nanosecond. PMID- 10746720 TI - Soft-mode hardening in SrTiO3 thin films AB - Understanding the behaviour of the dielectric constant in ferroelectric thin films remains a challenging problem. These ferroelectric materials have high static dielectric constants, and so are important for their applications in high storage-density capacitor structures such as dynamic random access memory (DRAM). But the dielectric constant tends to be significantly reduced in thin films, thereby limiting the potential benefit of ferroelectrics for memory devices. Extensive studies have shown that this phenomenon could be caused by a 'dead layer' of very low dielectric constant between the ferroeletric film and the electrode. And, although very few direct measurements are in fact available, it has been recognized that the lattice dynamical properties in the thin films should also play a key role in the reduction of the dielectric constant. Here we report far-infrared ellipsometry and low-frequency dielectric measurements in SrTiO3 thin films, which demonstrate that the Lyddane-Sachs-Teller relation between the optical-phonon eigenfrequencies and the dielectric constant is fully maintained, as is the case in the bulk material. This indicates that the dramatic reduction of the dielectric constant is a consequence of a profound change of the lattice dynamical properties, in particular of the reduced softening of its lowest optical-phonon mode. Our results therefore provide a better understanding of the fundamental limitations of the dielectric constant values in ferroelectric thin films. PMID- 10746721 TI - Extended surface chirality from supramolecular assemblies of adsorbed chiral molecules AB - The increasing demand of the chemical and pharmaceutical industries for enantiomerically pure compounds has spurred the development of a range of so called 'chiral technologies' (ref. 1), which aim to exert the ultimate control over a chemical reaction by directing its enantioselectivity. Heterogeneous enantioselective catalysis is particularly attractive because it allows the production and ready separation of large quantities of chiral product while using only small quantities of catalyst. Heterogeneous enantioselectivity is usually induced by adsorbing chiral molecules onto catalytically active surfaces. A mimic of one such catalyst is formed by adsorbing (R,R)-tartaric acid molecules on Cu(110) surfaces: this generates a variety of surface phases, of which only one is potentially catalytically active, and leaves the question of how adsorbed chiral molecules give rise to enantioselectivity. Here we show that the active phase consists of extended supramolecular assemblies of adsorbed (R,R)-tartaric acid, which destroy existing symmetry elements of the underlying metal and directly bestow chirality to the modified surface. The adsorbed assemblies create chiral 'channels' exposing bare metal atoms, and it is these chiral spaces that we believe to be responsible for imparting enantioselectivity, by forcing the orientation of reactant molecules docking onto catalytically active metal sites. Our findings demonstrate that it is possible to sustain a single chiral domain across an extended surface--provided that reflection domains of opposite handedness are removed by a rigid and chiral local adsorption geometry, and that inequivalent rotation domains are removed by successful matching of the rotational symmetry of the adsorbed molecule with that of the underlying metal surface. PMID- 10746722 TI - Bonding and reactivity at oxide mineral surfaces from model aqueous complexes AB - The kinetic stability of oxide surfaces affects a broad range of physical phenomena, including mineral dissolution and sorption reactions, stable-isotope fractionation, and catalyst support degradation. Our knowledge of the rates of these processes derives mostly from the rates of net mass transfer between the bulk solid and fluid phases. But from such data it is difficult to determine rates of elementary steps that are needed to test theoretical models. Here we determine the rates of oxygen exchange between an aqueous fluid and specific sites on the 'Al13' polyoxocation--AlO4Al12(OH)24(H2O)12(7+)--the structure of which closely resembles the surfaces of some Al-(hydr)oxide minerals in soils and catalyst supports. Extrapolation of these data to 298 K (and near pH 5.3) yields half-lives for oxygen on the complex that range from approximately 0.6 milliseconds for bound water to 41 seconds and 13 hours for the two distinct, but structurally similar, bridging hydroxyls. This surprisingly large range of labilities (approximately 10(7)) indicates that reactivity is very sensitive to molecular structure. Moreover, these results indicate that well chosen aqueous complexes provide important information to relate bonding to reactivity at mineral surfaces. PMID- 10746723 TI - Evidence that humans evolved from a knuckle-walking ancestor. AB - Bipedalism has traditionally been regarded as the fundamental adaptation that sets hominids apart from other primates. Fossil evidence demonstrates that by 4.1 million years ago, and perhaps earlier, hominids exhibited adaptations to bipedal walking. At present, however, the fossil record offers little information about the origin of bipedalism, and despite nearly a century of research on existing fossils and comparative anatomy, there is still no consensus concerning the mode of locomotion that preceded bipedalism. Here we present evidence that fossils attributed to Australopithecus anamensis (KNM-ER 20419) and A. afarensis (AL 288 1) retain specialized wrist morphology associated with knuckle-walking. This distal radial morphology differs from that of later hominids and non-knuckle walking anthropoid primates, suggesting that knuckle-walking is a derived feature of the African ape and human clade. This removes key morphological evidence for a Pan-Gorilla clade, and suggests that bipedal hominids evolved from a knuckle walking ancestor that was already partly terrestrial. PMID- 10746724 TI - Predictive accuracy of population viability analysis in conservation biology. AB - Population viability analysis (PVA) is widely applied in conservation biology to predict extinction risks for threatened species and to compare alternative options for their management. It can also be used as a basis for listing species as endangered under World Conservation Union criteria. However, there is considerable scepticism regarding the predictive accuracy of PVA, mainly because of a lack of validation in real systems. Here we conducted a retrospective test of PVA based on 21 long-term ecological studies--the first comprehensive and replicated evaluation of the predictive powers of PVA. Parameters were estimated from the first half of each data set and the second half was used to evaluate the performance of the model. Contrary to recent criticisms, we found that PVA predictions were surprisingly accurate. The risk of population decline closely matched observed outcomes, there was no significant bias, and population size projections did not differ significantly from reality. Furthermore, the predictions of the five PVA software packages were highly concordant. We conclude that PVA is a valid and sufficiently accurate tool for categorizing and managing endangered species. PMID- 10746725 TI - Low variability in a Y-linked plant gene and its implications for Y-chromosome evolution. AB - Sex chromosomes have evolved independently in several different groups of organisms, but they share common features, including genetic degeneration of the Y chromosome. Suppression of recombination between ancestral proto-X and proto-Y chromosomes is thought to have led to their gradual divergence, and to degeneration of the Y chromosome, but the evolutionary forces responsible are unknown. In non-recombining Y chromosomes, deleterious mutations may be carried to fixation by linked advantageous mutations ("selective sweeps"). Occurrence of deleterious mutations may drive "Muller's ratchet" (stochastic loss of chromosomes with the fewest mutations). Selective elimination of deleterious mutations, causing "background selection" may accelerate stochastic fixation of mildly detrimental mutations. All these processes lower effective population sizes, and therefore reduce variability of genes in evolving Y chromosomes. We have studied DNA diversity and divergence in a recently described X- and Y-linked gene pair (SLX-1 and SLY-1) of the plant Silene latifolia to obtain evidence about the early stages of Y degeneration. Here we show that DNA polymorphism in SLY-1 is 20-fold lower than in SLX-1, but the pattern of polymorphism does not suggest a selective sweep. PMID- 10746726 TI - Representation of a perceptual decision in developing oculomotor commands. AB - Behaviour often depends on the ability to make categorical judgements about sensory information acquired over time. Such judgements require a comparison of the evidence favouring the alternatives, but how the brain forms these comparisons is unknown. Here we show that in a visual discrimination task, the accumulating balance of sensory evidence favouring one interpretation over another is evident in the neural circuits that generate the behavioural response. We trained monkeys to make a direction judgement about dynamic random-dot motions and to indicate their judgement with an eye movement to a visual target. We interrupted motion viewing with electrical microstimulation of the frontal eye field and analysed the resulting, evoked eye movements for evidence of ongoing activity associated with the oculomotor response. Evoked eye movements deviated in the direction of the monkey's judgement. The magnitude of the deviation depended on motion strength and viewing time. The oculomotor signals responsible for these deviations reflected the accumulated motion information that informed the monkey's choices on the discrimination task. Thus, for this task, decision formation and motor preparation appear to share a common level of neural organization. PMID- 10746727 TI - A Drosophila model of Parkinson's disease. AB - Parkinson's disease is a common neurodegenerative syndrome characterized by loss of dopaminergic neurons in the substantia nigra, formation of filamentous intraneuronal inclusions (Lewy bodies) and an extrapyramidal movement disorder. Mutations in the alpha-synuclein gene are linked to familial Parkinson's disease and alpha-synuclein accumulates in Lewy bodies and Lewy neurites. Here we express normal and mutant forms of alpha-synuclein in Drosophila and produce adult-onset loss of dopaminergic neurons, filamentous intraneuronal inclusions containing alpha-synuclein and locomotor dysfunction. Our Drosophila model thus recapitulates the essential features of the human disorder, and makes possible a powerful genetic approach to Parkinson's disease. PMID- 10746728 TI - Interleukin-1 polymorphisms associated with increased risk of gastric cancer. AB - Helicobacter pylori infection is associated with a variety of clinical outcomes including gastric cancer and duodenal ulcer disease. The reasons for this variation are not clear, but the gastric physiological response is influenced by the severity and anatomical distribution of gastritis induced by H. pylori. Thus, individuals with gastritis predominantly localized to the antrum retain normal (or even high) acid secretion, whereas individuals with extensive corpus gastritis develop hypochlorhydria and gastric atrophy, which are presumptive precursors of gastric cancer. Here we report that interleukin-1 gene cluster polymorphisms suspected of enhancing production of interleukin-1-beta are associated with an increased risk of both hypochlorhydria induced by H. pylori and gastric cancer. Two of these polymorphism are in near-complete linkage disequilibrium and one is a TATA-box polymorphism that markedly affects DNA protein interactions in vitro. The association with disease may be explained by the biological properties of interleukin-1-beta, which is an important pro inflammatory cytokine and a powerful inhibitor of gastric acid secretion. Host genetic factors that affect interleukin-1-beta may determine why some individuals infected with H. pylori develop gastric cancer while others do not. PMID- 10746729 TI - PKC-theta is required for TCR-induced NF-kappaB activation in mature but not immature T lymphocytes. AB - Productive interaction of a T lymphocyte with an antigen-presenting cell results in the clustering of the T-cell antigen receptor (TCR) and the recruitment of a large signalling complex to the site of cell-cell contact. Subsequent signal transduction resulting in cytokine gene expression requires the activation of one or more of the multiple isoenzymes of serine/threonine-specific protein kinase C (PKC). Among the several PKC isoenzymes expressed in T cells, PKC-theta is unique in being rapidly recruited to the site of TCR clustering. Here we show that PKC theta is essential for TCR-mediated T-cell activation, but is dispensable during TCR-dependent thymocyte development. TCR-initiated NF-kappaB activation was absent from PKC-theta(-/-) mature T lymphocytes, but was intact in thymocytes. Activation of NF-kappaB by tumour-necrosis factor alpha and interleukin-1 was unaffected in the mutant mice. Although studies in T-cell lines had suggested that PKC-theta regulates activation of the JNK signalling pathway, induction of JNK was normal in T cells from mutant mice. These results indicate that PKC-theta functions in a unique pathway that links the TCR signalling complex to the activation of NF-kappaB in mature T lymphocytes. PMID- 10746730 TI - Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1. AB - Activated T lymphocytes differentiate into effector cells tailored to meet disparate challenges to host integrity. For example, type 1 and type 2 helper (T(H)1 and T(H)2) cells secrete cytokines that enhance cell-mediated and humoral immunity, respectively. The chemokine monocyte chemoattractant protein-1 (MCP-1) can stimulate interleukin-4 production and its overexpression is associated with defects in cell-mediated immunity, indicating that it might be involved in T(H)2 polarization. Here we show that MCP-1-deficient mice are unable to mount T(H)2 responses. Lymph node cells from immunized MCP-1(-/-) mice synthesize extremely low levels of interleukin-4, interleukin-5 and interleukin-10, but normal amounts of interferon-gamma and interleukin-2. Consequently, these mice do not accomplish the immunoglobulin subclass switch that is characteristic of T(H)2 responses and are resistant to Leishmania major. These effects are direct rather than due to abnormal cell migration, because the trafficking of naive T cells is undisturbed in MCP-1(-/-) mice despite the presence of MCP-1-expressing cells in secondary lymphoid organs of wild-type mice. Thus, MCP-1 influences both innate immunity, through effects on monocytes, and adaptive immunity, through control of T helper cell polarization. PMID- 10746731 TI - Betaglycan binds inhibin and can mediate functional antagonism of activin signalling. AB - Activins and inhibins, structurally related members of the TGF-beta superfamily of growth and differentiation factors, are mutually antagonistic regulators of reproductive and other functions. Activins bind specific type II receptor serine kinases (ActRII or IIB) to promote the recruitment and phosphorylation of the type I receptor serine kinase, ALK4 (refs 7-9), which then regulates gene expression by activating Smad proteins. Inhibins also bind type II activin receptors but do not recruit ALK4, providing a competitive model for the antagonism of activin by inhibin. Inhibins fail to antagonize activin in some tissues and cells, however, suggesting that additional components are required for inhibin action. Here we show that the type III TGF-beta receptor, betaglycan, can function as an inhibin co-receptor with ActRII. Betaglycan binds inhibin with high affinity and enhances binding in cells co-expressing ActRII and betaglycan. Inhibin also forms crosslinked complexes with both recombinant and endogenously expressed betaglycan and ActRII. Finally, betaglycan confers inhibin sensitivity to cell lines that otherwise respond poorly to this hormone. The ability of betaglycan to facilitate inhibin antagonism of activin provides a variation on the emerging roles of proteoglycans as co-receptors modulating ligand-receptor sensitivity, selectivity and function. PMID- 10746732 TI - The Gcn5 bromodomain co-ordinates nucleosome remodelling. AB - The access of transcription factors to eukaryotic promoters often requires modification of their chromatin structure, which is accomplished by the action of two general classes of multiprotein complexes. One class contains histone acetyltransferases (HATs), such as Gcn5 in the SAGA complex, which acetylate nucleosomal histones. The second class contains ATPases, such as Swi2 in the Swi/Snf complex, which provide the energy for nucleosome remodelling. In several promoters these two complexes cooperate but their functional linkage is unknown. A protein module that is present in all nuclear HATs, the bromodomain, could provide such a link. The recently reported in vitro binding of a HAT bromodomain with acetylated lysines within H3 and H4 amino-terminal peptides indicates that this interaction may constitute a targeting step for events that follow histone acetylation. Here we use a suitable promoter to show that bromodomain residues essential for acetyl-lysine binding are not required in vivo for Gcn5-mediated histone acetylation but are fundamental for the subsequent Swi2-dependent nucleosome remodelling and consequent transcriptional activation. We show that the Gcn5 bromodomain stabilizes the Swi/Snf complex on this promoter. PMID- 10746733 TI - Occupational exposure to ultraviolet radiation: a health risk assessment. AB - This review applies health risk assessment methodology to the subject of occupational exposure to ultraviolet radiation (UVR). Ultraviolet radiation is produced by many sources, and for most people the sun is their main source of exposure. In the occupational environment, there are also many artificial sources, of which welding arcs are the predominant and most intense. Adverse health effects include photokeratitis, erythema, pterygium, some types of cataract, non-melanocytic skin cancer, and malignant melanoma. Outdoor workers and workers who are exposed to certain artificial sources (particularly welding arcs), frequently are exposed to UVR levels that are above the current exposure limits. Such workers may also be at an additional risk of developing nonmelanocytic skin cancer along with other chronic conditions, including malignant melanoma. A range of control measures is available but nevertheless, many workers (particularly those exposed to solar UVR) do not make full use of these measures. Therefore, the implementation of health education campaigns and other initiatives that encourage use are needed. Overall, although exposure to UVR presents a substantial risk for certain occupations, the current exposure standards and currently available control measures (if used) are considered to provide adequate protection for most workers. PMID- 10746734 TI - Important considerations in the development of public health advisories for arsenic and arsenic-containing compounds in drinking water. AB - Drinking water contamination by arsenic remains a major public health problem. Acute and chronic arsenic exposure via drinking water has been reported in many countries of the world; especially in Argentina, Bangladesh, India, Mexico, Thailand, and Taiwan, where a large proportion of drinking water (ground water) is contaminated with a high concentration of arsenic. Research has also pointed out significantly higher standardized mortality ratios and cumulative mortality rates for cancers of the bladder, kidney, skin, liver, and colon in many areas of arsenic pollution. General health effects that are associated with arsenic exposure include cardiovascular and peripheral vascular disease, developmental anomalies, neurologic and neurobehavioral disorders, diabetes, hearing loss, portal fibrosis of the liver, lung fibrosis, hematologic disorders (anemia, leukopenia, and eosinophilia), and carcinoma. Although, the clinical manifestations of arsenic poisoning appear similar, the toxicity of arsenic compounds depends largely u[on the chemical species and the form of arsenic involved. On the basis of its high degree of toxicity to humans, and the non threshold dose-response assumption, a zero level exposure is recommended for arsenic, even though this level is practically non-attainable. In this review, we provide and discuss important information on the physical and chemical properties, production and use, fate and transport, toxicokinetics, systemic and carcinogenic health effects, regulatory and health guidelines, analytical methods, and treatment technologies that are applied to arsenic pollution. Such information is critical in assisting the federal, state and local officials who are responsible for protecting public health in dealing with the problem of drinking water contamination by arsenic and arsenic-containing compounds. PMID- 10746735 TI - The gastrointestinal tract and acute effects of copper in drinking water and beverages. AB - Copper is an essential element for all living beings. Exposure to copper results almost exclusively from the ingestion of food and water. Generally, potable water contains low levels of copper, but high concentrations of this mineral have been found in water from private wells or when water or beverages with low pH have been conducted through copper piping. Some authors have associated acute gastrointestinal symptoms (diarrhea, abdominal pain, nausea, and vomiting) with elevated levels of copper in water or beverages, but without excluding other confounding factors that could produce such symptoms. Recently, various controlled studies have demonstrated that a concentration of 2 mg Cu/L of potable water does not produce an increase in gastrointestinal symptoms in infants, and that in women, only concentrations greater than 3 mg Cu/L increase the number of episodes of nausea, vomiting, and abdominal pain, but not diarrhea. This critical analysis of scientific publications verifies the WHO provisional level for copper in drinking water (2 mg/L) as safe for human health. PMID- 10746736 TI - Social correlates of cigarette smoking cessation: findings from the 1995 microcensus survey in Germany. AB - BACKGROUND: An increasing percentage of smokers are quitting this unhealthy behavior during their life course. The aim of this study is to analyze which social factors play an important role regarding ex-smoking in Germany. METHODS: Data were derived from the 1995 German Microcensus, which is a representative survey for the population in Germany. Included in the analysis were 44,553 current smokers and 23,780 ex-smokers. The independent variables were education, occupational status, family status, unemployment/social welfare, household income, and community size. A two-stage statistical modeling procedure was used, initially to assess the most important effects of the independent variables on smoking cessation and secondly, to analyze the cumulative effects of the independent variables. RESULTS: The most striking effects observed for smoking cessation were family status and education. For example, in males aged 30 to 49 years, the percentages of ex-smokers of all ever smokers were 44.7% for married males with high education compared with only 14.6% for males with low education. The corresponding percentages for females were 44.0% and 17.6%. CONCLUSIONS: Such striking differences in the social polarization of smoking cessation in Germany demonstrate the importance of anti-smoking policies and new strategies that avoid a further increase in the social inequality of smoking behavior. PMID- 10746737 TI - Maganese retention in rat brain. AB - Manganese retention was observed in brains and in several other tissues of female Wistar rats after the intratracheal instillation of an inorganic manganese compound: manganese dioxide. Two categories of rats, younger (180 to 200g) and older (330 to 350g), were divided into a control group, in which animals received vehicle only (0.5 mL physiological saline), and an experimental group, in which rats received a dose of 0.48 mg of Mn/kg body weight (in 0.5 mL saline), twice a week for 3 months, for a total dosage of 11.80 mg of Mn/kg body weight. At the end of the exposure period, manganese retention in selected rat organs, brain, liver, kidney, and lung, was analyzed using atomic absorption spectrophotometry. At the end of the 6-wk or 12-wk manganese dioxide exposure period, analysis of variance of the manganese retention results revealed significant differences between Mn-exposed and unexposed rats in brain, kidney, and lung tissues (p<0.01) for both experimental age categories. Moreover, at the end of the 12-wk exposure period, significant results (p<0.05) between younger and older rats were obtained for both brain and kidneys. In both types of tissue, the manganese retention in the younger group was higher than that in older animals. PMID- 10746739 TI - The twin-arginine translocation system: a novel means of transporting folded proteins in chloroplasts and bacteria. AB - Protein translocases have been characterised in several membrane systems and the translocation mechanisms have been shown to differ in critical respects. Nevertheless, the majority were believed to transport proteins only in a largely unfolded state, and this widespread characteristic was viewed as a likely evolutionary effort to minimise the diameter of translocation pore required. Within the last few years, however, studies on the chloroplast thylakoid membrane have revealed a novel class of protein translocase which possesses the apparently unique ability to transport fully-folded proteins across a tightly sealed energy transducing membrane. A related system, (the twin-arginine translocation, or Tat system) has now been characterised in the Escherichia coli plasma membrane and considerations of its substrate specificity again point to its involvement in the transport of folded proteins. The emerging data suggest a critical involvement in many membranes for the biogenesis of two types of globular protein: those that are obliged to fold prior to translocation, and those that fold too tightly or rapidly for other types of protein translocase to handle. PMID- 10746738 TI - Desert ecosystems: similarities, characteristics, and health benefits. AB - Salty bodies of water in desert zones are known worldwide. The Salton Sea in California, USA, and the Dead Sea between Israel and Jordan are located in arid areas at approximately the same latitude, which might explain some similarities. Both the Salton and Dead Seas have ecosystems consisting of a singular saline sea/hot desert interface. The Salton Sea, the largest inland body of water in California, is a saline lake in the Coachella and Imperial Valleys. The Imperial Valley is one of the 10 top agricultural areas in the United States. Several thermoelectric plants exploiting geothermal wells operate around the Salton Sea, and some areas comprise a National Wildlife Refuge. The Dead Sea (Sea of Salt in Hebrew), the lowest saline lake on earth, contains high concentrations of salts and is a reservoir of minerals with a unique evaporation regime. The Dead Sea salts are the raw materials for the production of several chemical and health products. Magnesium salts and sulfur-containing mud are used for treating human skin disorders, allergies, arthritis, and respiratory diseases. After visiting both zones, we recorded, analyzed, and compared the similarities and differences between the areas. Some differences were found in the geographic, orographic, hydraulic, and climatic properties, but the main difference is in the economic industrial aspect. The characteristics and health aspects are described in this report. PMID- 10746740 TI - De novo DNA methylation at nonrandom founder sites 5' from an unmethylated minimal origin of DNA replication in latent Epstein-Barr virus genomes. AB - Latent episomal genomes of Epstein-Barr virus, a human gammaherpesvirus, represent a suitable model system for studying replication and methylation of chromosomal DNA in mammals. We analyzed the methylation patterns of CpG dinucleotides in the latent origin of DNA replication of Epstein-Barr virus using automated fluorescent genomic sequencing of bisulfite-modified DNA samples. We observed that the minimal origin of DNA replication was unmethylated in 8 well characterized human cell lines or clones carrying latent Epstein-Barr virus genomes as well as in a prototype virus producer marmoset cell line. This observation suggests that unmethylated DNA domains can function as initiation sites or zones of DNA replication in human cells. Furthermore, 5' from this unmethylated region we observed focal points of de novo DNA methylation in nonrandom positions in the majority of Burkitt's lymphoma cell lines and clones studied while the corresponding CpG dinucleotides in viral genomes carried by lymphoblastoid cell lines and marmoset cells were completely unmethylated. Clustering of highly methylated CpG dinucleotides suggests that de novo methylation of unmethylated double-stranded episomal viral genomes starts at discrete founder sites in vivo. This is the first comparative high-resolution methylation analysis of a latent viral origin of DNA replication in human cells. PMID- 10746741 TI - Trichostatin A modulates expression of p21waf1/cip1, Bcl-xL, ID1, ID2, ID3, CRAB2, GATA-2, hsp86 and TFIID/TAFII31 mRNA in human lung adenocarcinoma cells. AB - Lung adenocarcinoma cells treated for 16 h with trichostatin A (TSA), an inhibitor of histone deacetylases, and untreated cells were analyzed with respect to differential gene expression. Complex hybridization of cDNA arrays revealed repression of Bcl-xL, CRAB2 and TFIID/TAFII31 as well as induction of p21waf1/cip1, GATA-2, hsp86, ID1, ID2 and ID3 mRNA expression, which could be verified by Northern blotting. ID2 induction was further confirmed by Taqman realtime quantitative RT-PCR. The described alterations of gene expression due to TSA renders the lung adenocarcinoma cells susceptible to induction of apoptosis. PMID- 10746742 TI - Effects of the active aldehyde group generated by RNA N-glycosidase in the sarcin/ricin domain of rat 28S ribosomal RNA on peptide elongation. AB - Effects of the active aldehyde group of ribose C1' at position 4324 of rat 28S rRNA, in the inactivated ribosome generated by RNA N-glycosidases (trichosanthin, A-chain of cinnamomin and ricin), on peptide elongation have been studied. The aldehyde group inhibits the activities of eEF1A-dependent aminoacyl-tRNA binding to the inactivated ribosome and eEF1A-dependent GTPase, but increases eEF2 dependent activity. At a high concentration of RNA N-glycosidase, the generated aldehyde group also inhibits aminoacyl-tRNA binding to the inactivated ribosome in the absence of elongation factor and translocation activity. When the aldehyde group is reduced into a hydroxyl group by sodium borohydride or blocked with an amino acid through nucleophilic addition, the activities of eEF1A-dependent aminoacyl-tRNA binding and eEF1A-dependent GTPase of the inactivated ribosome are partially restored, but the altered activities of eEF2-dependent GTPase, translocation and aminoacyl-tRNA binding in the absence of elongation factor are not normalized. Thus, reduction or blockage of the aldehyde group with sodium borohydride or amino acids might change the conformation of the S/R domain in rat 28S ribosomal RNA to meet the requirement for eEF1A-dependent reactions, but not eEF2-involved reactions. PMID- 10746743 TI - Responses to peroxynitrite in yeast: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a sensitive intracellular target for nitration and enhancement of chaperone expression and ubiquitination. AB - Peroxynitrite (ONOO-), a potent oxidizing and nitrating species, has been linked to covalent modifications of biomolecules in a number of pathological conditions. In S. cerevisiae, a model eukaryotic cell system, ONOO- was found to be more potent than hydrogen peroxide in oxidizing thiols, inducing heat shock proteins (Hsp70) and enhancing the ubiquitination of proteins. As identified by microsequence analysis following immunoprecipitation with anti-nitrotyrosine antibodies, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was especially susceptible to nitration by ONOO- in yeast cells. The activity of this enzyme was strongly inhibited upon steady-state exposure of the cells to low doses of ONOO- in yeast and in cultured rat astrocytes. Thus, ONOO- is a potent stressor in yeast capable of inducing oxidative damage and protein nitration, with GAPDH being a preferential target protein that is efficiently inactivated. PMID- 10746744 TI - Enhancing the T-->R transition of insulin by helix-promoting sequence modifications at the N-terminal B-chain. AB - Structurally, the T-->R transition of insulin mainly consists of a rearrangement of the N-terminal B-chain (residues B1-B8) from extended to helical in one or both of the trimers of the hexamer. The dependence of the transition on the nature of the ligands inducing it, such as inorganic anions or phenolic compounds, as well as of the metal ions complexing the hexamer, has been the subject of extensive investigations. This study explores the effect of helix enhancing modifications of the N-terminal B-chain sequence where the transition actually occurs, with special emphasis on N-capping. In total 15 different analogues were prepared by semisynthesis. 80% of the hexamers of the most successful analogues with zinc were found to adopt the T3R3 state in the absence of any transforming ligands, as compared to only 4% of wild-type insulin. Transformation with SCN- ions can exceed the T3R3 state where it stops in the case of wild-type insulin. Full transformation to the R6 state can be achieved by only one-tenth the phenol concentration required for wild-type insulin, i.e. almost at the stoichiometric ratio of 6 phenols per hexamer. PMID- 10746745 TI - Structure-dependent effects of glucose-containing analogs of platelet activating factor (PAF) on membrane integrity. AB - Synthetic choline-containing phospholipids comprise a new class of compounds with antineoplastic properties. We have investigated the effect of recently synthesized glucose-containing analogs of lysophosphatidylcholine (glyceroglucophospholipid, Glc-PC) and of lysoplatelet activating factor (Glc PAF) and its C16, C14 and C12 derivatives (ET-16, ET-14, and ET-12) on proliferation of immortalized human keratinocyte (HaCaT) cells. The data were compared to the ability of the compounds to intercalate into phosphatidylserine liposomes and to form lesions in planar bilayer membranes. A correlation between bioactivity and membrane activity was found. The number of molecules that intercalated into phosphatidylserine liposomes depended on the chemical structure of the compounds and was in the order Glc-PAF approximately ET-16 approximately ET-14 > Glc-PC > ET-12. All compounds induced membrane lesions, and the lesion forming activity was in the same order. Similar activity rankings were found for the release of lactate dehydrogenase from HaCaT cells as a measure of lytic activity and for the influence on cell number as a measure of proliferation. In the latter test, however, proliferation was already inhibited at non-toxic concentrations. From these findings, it may be concluded that the intercalation of the compounds at toxic concentrations leads to the formation of membrane lesions and finally results in membrane rupture leading to cell death. PMID- 10746746 TI - Two different mechanisms for activation of cyclic PIP synthase: by a G protein or by protein tyrosine phosphorylation. AB - The biosynthesis of the functional, endogenous cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP) is performed by the plasma membrane-bound enzyme cyclic PIP synthase, which combines prostaglandin E (PGE) and activated inositol phosphate (n-IP) to cyclic PIP. The Km values of the enzyme for the substrates PGE and n-IP are in the micromolar range. The plasma membrane-bound synthase is activated by fluoride, by the stable GTP analog GMP PNP, by protamine or biguanide, by noradrenaline, and by insulin. The activation by protamine or biguanide and fluoride (10 mM) is additive, which may indicate the presence of two different types of enzyme, comparable to phospholipase Cbeta and phospholipase Cgamma. Plasma membrane-bound cyclic PIP synthase is inhibited by the protein tyrosine kinase inhibitor tyrphostin B46 with an IC50 of 1.7 microM. However, the solubilized and gel-filtrated enzyme is no longer inhibited by tyrphostin, indicating that the activity of cyclic PIP synthase is connected with the activity of a membrane-bound protein tyrosine kinase. Cyclic PIP synthase activity of freshly prepared plasma membranes is unstable. Upon freezing and rethawing of liver plasma membranes, this instability is increased about 2 fold. Protein tyrosine phosphatase inhibitors [vanadate, fluoride (50-100 mM)] stabilize the enzyme activity, but protease inhibitors do not, indicating that inactivation of the enzyme is connected with protein tyrosine dephosphorylation. Cyclic PIP synthase is present in all tissues tested, like brain, heart, intestine, kidney, liver, lung, skeletal muscle, spleen, and testis. Apart from liver, cyclic PIP synthase activity in most tissues is rather low, but it can be increased up to 5-fold when protein tyrosine phosphatase inhibitors like vanadate are present in the homogenization buffer. Preincubation of cyclic PIP synthase of liver plasma membranes with the tyrosine kinase src kinase causes a 2-fold increase of cyclic PIP synthase activity, though this is certainly not the physiological role played by src kinase in intact cells. The data indicate that cyclic PIP synthase can be activated by two separate mechanisms: by a G protein or by protein tyrosine phosphorylation. PMID- 10746747 TI - 3-Hydroxybenzoate:coenzyme A ligase from cell cultures of Centaurium erythraea: isolation and characterization. AB - In xanthone biosynthesis, 3-hydroxybenzoate:coenzyme A ligase (3HBL) supplies the starter substrate for the formation of an intermediate benzophenone. 3HBL from cell cultures of the medicinal plant Centaurium erythraea was purified to apparent homogeneity using a seven-step-procedure. The enzyme was an AMP-forming CoA ligase with a Km = 14.7 microM for 3-hydroxybenzoic acid, 8.5 microM for coenzyme A and 229 microM for ATP. The pH and temperature optima were 7.5 and 35 degrees C, respectively. In SDS-PAGE, two polypeptides of Mr 41,500 and 40,500 were detected. Both proteins were structurally related to each other as shown by tryptic digestion. Their N-termini were blocked. The difference in their apparent molecular masses could not be attributed to glycosylation. 3HBL had a native Mr of approx. 50,000 and is thus active as a monomer. PMID- 10746748 TI - Gel chromatographic characterization of the hydrophobic interaction of glycosylphosphatidylinositol-alkaline phosphatase with detergents. AB - The interaction of a glycosylphosphatidylinositol (GPI) protein with different detergents was studied for the first time with a purified protein. Four differently hydrophobic fractions of GPI-alkaline phosphatase (GPI-AP) from calf intestine were used as model proteins. The mode of interaction was determined by investigating (i) the self-aggregation behaviour of the GPI-AP fractions, (ii) the interference of detergents with GPI-AP binding to octyl-Sepharose, and (iii) the elution of GPI-AP bound to octyl-Sepharose. It was shown that polyoxyethylene type detergents surprisingly interact much stronger than n-octylglucoside with GPI-AP, which is in contrast to the known behaviour of GPI-proteins in natural membranes. Gel filtration chromatography of Triton X-100 at concentrations above the critical micellar concentration yields three different micelle species with apparent molecular weights of about 166, 54, and 16 kDa. GPI-AP fraction II, which is shown to bear only one anchor per dimer, does not bind to any of these micelles. We demonstrate that a complex is formed containing about 150 Triton X 100 molecules and about 4700 molecules of water per molecule of GPI-AP dimer. The experimental findings are in accordance with a simple geometrical model based on the physical data of fatty acids and the arrangement, mean size, and shape of Triton X-100 molecules. PMID- 10746749 TI - Immunolabeling of CD3-positive lymphocytes with a recombinant single-chain antibody/alkaline phosphatase conjugate. AB - G3(3) is a novel murine monoclonal antibody directed against the CD3 antigen of human T lymphocytes which could be used to analyze lymphoid malignancies. We have produced and characterized a recombinant colorimetric immunoconjugate with the antigen-binding specificity of antibody G3(3). A gene encoding a single-chain antibody variable fragment (scFv) was assembled using the original hybridoma cells as a source of antibody variable heavy (VH) and variable light (VL) chain genes. The chimeric gene was introduced into a prokaryotic expression vector in order to produce a soluble scFv fused to bacterial alkaline phosphatase. DNA sequencing and Western blotting analyses demonstrated the integrity of the soluble immunoconjugate recovered from induced recombinant bacteria. The scFv/AP protein was bifunctional and similar in immunoreactivity to the parent G3(3) antibody. Flow cytometry and immunostaining experiments confirmed that the activity of the scFv/AP protein compares favourably with that of the parent antibody. The scFv/AP conjugate was bound to CD3 antigen at the surface of T cells and was directly detected by its enzymatic activity. Thus this novel fusion protein has potential applications as an immunodiagnostic reagent. PMID- 10746750 TI - Phage display of RNase A and an improved method for purification of phages displaying RNases. AB - Functional ribonuclease A was presented on the surface of the filamentous phage M13 by fusion to the minor coat protein. RNase activity of the fusion protein was shown by a zymogram assay. In addition, we established a modified method for preparing RNase-displaying phages without contaminating host RNases. PMID- 10746751 TI - Use of social services by pregnant Medicaid eligible women in Baltimore. AB - OBJECTIVES: To use linked health and social service databases to determine differences in the use of social services by pregnant women in different managed care systems. METHODS: Comparison of service use by women enrolled in a fee-for service primary care case management program (Maryland Access to Care or MAC), in a capitated health maintenance organization (HMO), or not assigned to managed care using six state databases. Participants included 5181 women receiving Medical Assistance (MA) and delivering in Baltimore City in 1993. Outcome measures were receipt of WIC, AFDC, and Food Stamps. RESULTS: The overall proportions of women receiving WIC, AFDC, and Food Stamps at delivery were 52.7%, 89.2%, and 62.7%, respectively. Women enrolled in an HMO at delivery were less likely to be receiving WIC (adjusted odds ratios, 0.8, 95% CI, 0.69 to 0.93), AFDC (OR, 0.20; CI, 0.03 to 0.43 for women with prior children and OR 0.13; CI, 0.09 to 0.20 for women without prior children), and Food Stamps (OR 0.77; CI, 0.59 to 0.95 for women with prior children and OR, 0.49; CI, 0.35 to 0.67 for women without prior children) than their MAC counterparts. Women not assigned to managed care also generally were less likely than their MAC counterparts to receive WIC (OR 0.55; CI, 0.46, 0.66), AFDC (OR 1.07; CI 0.83, 1.30 for women with prior children and OR 0.24; CI 0.18, 0.34 for women without prior children), and Food Stamps (OR 0.31; CI 0.08, 0.55 for women with prior children and OR 0.31; CI 0.23, 0.41 for women without prior children). CONCLUSIONS: Although many low-income pregnant women qualify for select social services, receipt of WIC and Food Stamps was low. Increasing efforts are needed by managed care systems and public health agencies to ensure delivery of appropriate services for women. PMID- 10746752 TI - Low prenatal weight gain among adult WIC participants delivering term singleton infants: variation by maternal and program participation characteristics. AB - OBJECTIVE: To determine the association of maternal and prenatal WIC program participation characteristics with low prenatal weight gain among adult women delivering liveborn, singleton infants at term. METHODS: WIC program data for 19,017 Black and White Alabama women delivering in 1994 were linked with birth certificate files to examine the association of anthropometric, demographic, reproductive, hematologic, behavioral and program participation characteristics with low prenatal weight gain. RESULTS: One third (31.0%) had low prenatal weight gain as defined by the Institute of Medicine. The incidence of low weight gain was increased among women who had < 12 years of education, were single, Black, anemic, had low or normal prepregnancy body mass index (BMI), increased parity, interpregnancy intervals < or = 24 months, used tobacco or alcohol, or entered prenatal care or WIC programs after the first trimester. After adjusting for selected maternal characteristics, the adjusted odds ratios (AOR) for low weight gain were increased with short interpregnancy intervals (AOR 1.21 to 2.20); tobacco use (AOR 1.16 to 1.40), anemia (AOR 1.20 to 1.25), and second trimester entry into prenatal care (AOR 1.14 to 1.20); the size of the AORs and 95% confidence intervals varied by BMI and racial subgroup. CONCLUSIONS: The results of this study suggest that WIC interventions targeting low prenatal weight gain be focused on risk factors present not only during pregnancy, but during the pre- and interconceptional periods as well. Interventions should target low BMI, tobacco use, and anemia, and include attention to nutrition screening and risk reduction among women in postpartum and family planning clinic settings. PMID- 10746753 TI - Implementation of Fetal and Infant Mortality Review (FIMR): experience from the national Healthy Start program. AB - OBJECTIVES: The implementation of the Fetal and Infant Mortality Review (FIMR) process was examined as part of the evaluation of the national Healthy Start program, a federal program designed to reduce infant mortality in several communities. The implementation of the FIMR process over the 5-year funding period is described in terms of productivity, barriers and facilitators to implementation, and project expenditures. METHODS: Data were derived from grant continuation applications and personal interviews with program staff to produce a qualitative description. RESULTS: As of the summer of 1996, 14 of the 15 Healthy Start sites in the national evaluation had successfully implemented the FIMR process. Most sites had developed a two-tiered review process for examination of case data in which a review by health and social services professionals was followed by community review. In the period 1993 to 1995, the percentage of fetal and infant deaths reviewed had a median of 34% with a range of 4-79% across the sites at a cost of $600 to $3400 per death reviewed. Recommendations were variably implemented. CONCLUSIONS: The FIMR process provides an important opportunity to contribute to the knowledge base regarding infant mortality in these communities. The process, however, has important logistical requirements and may require substantial financial resources that may affect implementation of confidential inquiries into infant mortality and other health problems. PMID- 10746754 TI - Assessment of Healthy Start Fetal and Infant Mortality Review recommendations. AB - OBJECTIVES: We examine the scope and nature of the recommendations that emerged from the Healthy Start Fetal and Infant Mortality Review (FIMR) projects and explore their use to promote systems change. METHODS: The FIMR process of 16 of the 22 federal Healthy Start projects was reviewed. We analyzed data from a June 1996 survey developed and administered by the MCH Bureau which gathered information about recommendations produced by the FIMRs. We supplemented these data with information gathered through follow-up telephone interviews and by abstracting information from grant documents. RESULTS: The 16 Healthy Start FIMRs reviewed approximately 1300 cases between 1991 and 1996. A total of 303 specific action strategies were recommended, reflecting eighteen specific substantive areas of concern. Overall, 65% of recommendations fell under the rubric of "program" functions, 31% under "practice," and 4% under "policy." Healthy Start itself was most commonly targeted for action. The second most frequent target for action were public and private provider institutions. Public policymaking entities were rarely targeted. CONCLUSIONS: In the first several years of implementation, with few exceptions these FIMRs sought limited change. They worked almost exclusively within their own span of control to effect important, but limited changes in systems serving women and their infants. As public health professionals seek to monitor population health, the field must strengthen any and all vehicles that draw upon collaborative structures at the community level to not only uncover problems, but to address them as well. PMID- 10746755 TI - The effect of implementing a more comprehensive screening for substance use among pregnant women in North Carolina. AB - OBJECTIVES: Substance use during pregnancy causes significant morbidity and mortality. Screening for substance use provides opportunity for intervention and treatment, but is often overlooked in prenatal care with today's fiscal climate and preconceived ideas about who uses substances. The purpose of this study was to determine the effect of implementing a more comprehensive screening procedure for identifying substance-using pregnant women. METHODS: Two hundred randomly selected prenatal patients screened using the old procedure for substance use during pregnancy were compared to 400 randomly selected patients screened after implementation of a new, more detailed screening procedure. The old screening approach included three check-boxes concerning substance use during pregnancy: one for "Smoking/alcohol," one for "Drug use (any)," and one for "Drug addiction/alcoholism." The new screening procedure included more detailed questions about the frequency of cigarette, alcohol, and illegal drug use during pregnancy. RESULTS: Compared with the old approach, the new screening protocol increased reporting of smoking/alcohol use from 21% to 72% (relative risk = 2.63, 95% confidence interval = 2.231, 3.108); reporting of any drug use from 12% to 18% (relative risk = 1.07, 95% confidence interval = 1.002, 1.150); and reporting of alcoholism/drug abuse from 0% to 6% (relative risk = 1.06, 95% confidence interval = 1.038, 1.090). CONCLUSIONS: Our study suggests that more detailed screening using direct questions concerning the amount and frequency of substance use increases reporting of prenatal substance use as compared to screening approaches using only "yes/no" style check boxes. PMID- 10746756 TI - Validation of a food frequency questionnaire in Native American and Caucasian children 1 to 5 years of age. AB - OBJECTIVE: To assess the validity of the Harvard Service Food Frequency Questionnaire (HFFQ) in the diet assessment of Native American and Caucasian children 1 to 5 years of age participating in the North Dakota WIC program. METHODS: The 84-item HFFQ was administered twice to the parent or guardian of 131 Native American and 102 Caucasian children ages 1 to 5 years (total n = 233), first at the child's routine WIC visit and then following the completion of three 24-hr dietary recalls taken over approximately 1 month. Average nutrient intakes from the three 24-hr dietary recalls were compared to average nutrient intakes from the HFFQs by calculating Pearson correlation coefficients and adjusting for energy intake and within person variation. RESULTS: Correlation coefficients ranged from 0.26 for dietary fiber to 0.63 for magnesium. The average correlation was 0.52, similar to that found in validation studies among adolescents and adults. The following nutrients had correlations of 0.50 or greater: carbohydrate, sucrose, total fat, vitamin C, vitamin E, vitamin B1, vitamin B2, niacin, folate, vitamin B6, calcium, magnesium, and iron. CONCLUSIONS: The HFFQ is a simple self-administered questionnaire completed by the child's parent or guardian and is useful in assessing the diets of Native American and Caucasian children. It may also provide important nutritional information about this age group for future program planning, research, education, and intervention purposes. PMID- 10746757 TI - Fetal and Infant Mortality Review: an evolving process. AB - Fetal and Infant Mortality Review (FIMR) has been a dynamic process from its inception in the 1980s. Essentially, it is a local, community-driven process that has grown and been refined as more communities adopt the methodology. The National Fetal and Infant Mortality Review (NFIMR) Program, a partnership between the federal Maternal and Child Health Bureau and the American College of Obstetricians and Gynecologists, serves as a resource for state and local communities implementing FIMR. In this issue of the Maternal and Child Health Journal, two articles by Baltay et al. and Grason and Misra examine the implementation of FIMR in the original federal Healthy Start programs and present findings of interest to the MCH community. PMID- 10746758 TI - Biofilm resistance to antimicrobial agents. PMID- 10746759 TI - Bacterial respiration: a flexible process for a changing environment. PMID- 10746760 TI - Systematic study of gene expression and transcription organization in the gntZ ywaA region of the Bacillus subtilis genome. AB - Within the framework of the international project 'The functional analysis of the Bacillus subtilis genome' in Japan and Europe, the gene expression and transcription organization of the gntZ-ywaA region (160 kb) of the B. subtilis genome has been systematically analysed. First, all unanalysed genes comprising more than 80 amino acids (125 genes) in this region were inactivated through integration of plasmid pMUTIN. No essential gene was found which could not be inactivated. All the integrants grew normally in both nutrient sporulation medium and glucose minimal medium. But an integrant in the yxbG gene exhibited an oligosporogenic phenotype in the nutrient sporulation medium. The synthesis of beta-galactosidase was examined, as a reporter for expression of the inactivated genes, during growth and sporulation in the two media. The results indicated that 36% of the promoters were inactive when cells were grown in at least one of these two media. Furthermore, the transcription of the 119 genes in this region was analysed by Northern blotting, resulting in a transcription map. The results indicate that the gntZ-ywaA region contains at least 24 polycistronic operons, including several published ones. The operons newly found in this work are yxaAB, yxaGH, yxaJKL, yxbBA-yxnB-asnH-yxaM, yxbCD, yxcED, yxdJK, yxeFGH, yxeKLMNOPQ, yxeR-yxxB, hutPHUIGM, bgIPH-yxiE, wapA-yxxG, yxiM-deaD, katB-yxiS, yxjCDEF, yxjJI and yxkF-mmsX. PMID- 10746761 TI - Stability by multimer resolution of pJHCMW1 is due to the Tn1331 resolvase and not to the Escherichia coli Xer system. AB - The plasmid pJHCMW1 encodes resistance to several aminoglycosides and beta lactams and consists of a copy of the transposon Tn1331, a region including the replication functions, and a sequence with homology to ColE1 cer, designated mwr. In this work, the role of this cer-like site in ensuring the stable inheritance of pJHCMW1 by multimer resolution was studied. The Escherichia coli Xer site specific recombination system acts at sites such as ColE1 cer to resolve plasmid multimers formed by homologous recombination, thereby maintaining plasmids in a monomeric state and helping to ensure stable plasmid inheritance. Despite its high similarity to ColE1 cer, the pJHCMW1 mwr was a poor substrate for Xer recombination in E. coli and did not contribute significantly to plasmid stability. Instead, the Tn1331 co-integrate resolution system was highly active at resolving pJHCMW1 multimers and ensured the stable inheritance of pJHCMW1. Although Xer recombination at pJHCMW1 mwr was inefficient in E. coli, the recombination that did occur was dependent on ArgR, PepA, XerC and XerD. A supercoiled circular DNA molecule containing two pJHCMW1 mwr sites in direct repeat yielded Holliday-junction-containing product when incubated with ArgR, PepA, XerC and XerD in vitro, confirming that pJHCMW1 mwr is a functional recombination site. However, unlike cer, some Holliday-junction-containing product could be detected for mwr in the absence of ArgR, although addition of this protein resulted in formation of more Holliday junctions. Binding experiments demonstrated that XerD bound to pJHCMW1 mwr core with a high affinity, but that XerC bound to this site very poorly, even in the presence of XerD. PMID- 10746762 TI - Mu DNA reintegration upon excision: evidence for a possible involvement of nucleoid folding. AB - Mutations induced by the integration of a Mugem2ts prophage can revert at frequencies around 1x10(-6). In these revertant clones, the prophage excised from its original localization is not lost but reintegrated elsewhere in the host genome. One of the most intriguing aspects of this process is that the prophage reintegration is not randomly distributed: there is a strong correlation between the original site of insertion (the donor site) and the target site of the phage DNA migration (the receptor site). In this paper, it is shown that in the excision-reintegration process mediated by Mugem2ts, the position of the initial prophage site strongly influences the location of the reintegration site. In addition, for each donor site, the receptor site is a discrete DNA region within which the excised Mu DNA can reintegrate and the two sites implicated in phage DNA migration must be located on the same DNA molecule. These data suggest the involvement of nucleoid folding in the excision-reintegration process. PMID- 10746763 TI - A bacteriophage-like particle from Bartonella bacilliformis. AB - Bartonella bacilliformis and Bartonella henselae, the respective agents of Oroya fever and cat-scratch disease in humans, are known to produce bacteriophage-like particles (BLPs) that package 14 kbp segments of the host chromosome. Data from this study suggest that other Bartonella species including Bartonella quintana, Bartonella doshiae and Bartonella grahamii also contain similar BLPs, as evidenced by the presence of a 14 kbp extrachromosomal DNA element in their genomes, whereas Bartonella elizabethae and Bartonella clarridgeiae do not. A purification scheme utilizing chloroform, DNase I and centrifugation was devised to isolate BLPs from B. bacilliformis. Intact BLPs were observed by transmission electron microscopy and were round to icosahedral in shape and approximately 80 nm in diameter. RFLP and Southern blot analysis of BLP DNA from B. bacilliformis suggest that packaging, while non-selective, is less than the near-random packaging previously reported for the B. henselae phage. Data also suggest that the linear, double-stranded BLP DNA molecules have blunt ends with noncovalently closed termini. Packaging of the BLP DNA molecules into a protein coat appears to be closely related to nucleic acid synthesis, as unpackaged phage DNA is not detectable within the host cell. SDS-PAGE analysis of purified BLPs from B. bacilliformis showed three major proteins with apparent molecular masses of 32, 34 and 36 kDa; values that closely correspond to proteins found in B. henselae BLPs. Western blot analysis performed with patient convalescent serum showed that BLP proteins are slightly immunogenic in humans. To determine if BLPs contribute to horizontal gene transfer, mutants of B. bacilliformis were generated by allelic exchange with an internal fragment of the 16S-23S rDNA intergenic spacer region and a suicide vector construct, termed pKB1. BLPs from one of the resultant strains were able to package the mutagenized region containing the kanamycin-resistance cassette; however, numerous approaches and attempts at intraspecies transduction using these BLPs were unsuccessful. PMID- 10746764 TI - Identification of a gene cluster for antibacterial polyketide-derived antibiotic biosynthesis in the nystatin producer Streptomyces noursei ATCC 11455. AB - Streptomyces noursei ATCC 11455 produces the antifungal polyene antibiotic nystatin containing the deoxysugar moiety mycosamine. Part of the deoxythymidyl diphosphate (TDP)-glucose dehydratase gene (gdhA) known to be involved in deoxysugar biosynthesis was amplified by PCR from genomic DNA of S. noursei ATCC 11455. A gene library for S. noursei was made and screened with the gdhA probe. Several overlapping phage clones covering about 30 kb of the S. noursei genome were physically mapped. A partial DNA sequencing analysis of this region resulted in the identification of several putative genes typical of macrolide antibiotic biosynthetic gene clusters. A gene-transfer system for 5. noursei has been established, and gene deletion or disruption experiments within the putative biosynthetic gene cluster were performed. All of the knock-out mutants retained the ability to produce nystatin, suggesting that the identified gene cluster is not involved in biosynthesis of this antibiotic. Culture extracts from the wild type strain and three knock-out mutants were analysed by TLC followed by a bioassay against Micrococcus luteus. Two antibacterial compounds were found to be synthesized by the wild-type strain while only one was produced by the mutants. This provided evidence for the involvement of the identified gene cluster in the biosynthesis of a presumably novel antibacterial macrolide antibiotic in S. noursei. PMID- 10746765 TI - Characterization of the genetic locus responsible for production and immunity of carnobacteriocin A: the immunity gene confers cross-protection to enterocin B. AB - Carnobacteriocin A (CbnA) is a regulated bacteriocin produced by Carnobacterium piscicola LV17A that is encoded on a 72 kb plasmid. A 10.0 kb fragment from this plasmid that contained information necessary for bacteriocin production and immunity was cloned and sequenced. Genetic analysis showed the presence of the previously sequenced structural gene for CbnA, as well as genes encoding proteins homologous to dedicated bacteriocin transport proteins and proteins of three component signal transduction systems. The induction factor (CbnX) was chemically synthesized and induced CbnA production at 10(-11) M or higher in a C. piscicola LV17A culture that had lost the ability to produce bacteriocin as a result of dilution. The gene cbiA for the immunity protein is not located in typical close proximity to the structural gene for CbnA and is encoded in the opposite orientation. CbiA has homology with EniB, the immunity protein for enterocin B that is also encoded in the opposite orientation to the bacteriocin gene. CbiA and EniB cross-protected against the corresponding bacteriocins. PMID- 10746766 TI - Polyadenylylation in mycobacteria: evidence for oligo(dT)-primed cDNA synthesis. AB - The ability of mRNA to direct synthesis of cDNA in the presence of oligo(dT) was analysed using a novel application of fluorescein-11-dUTP incorporation into cDNA by reverse transcriptase. Evidence is provided for the first time that a majority of the mycobacterial mRNA pool is polyadenylylated. mRNA transcripts of hsp65 were also amplified with specific primers from the oligo(dT)-primed cDNA preparation in Mycobacterium bovis BCG, M. smegmatis and M. vaccae. Furthermore, PCR amplication of cDNAs for genes entD, entC and trpE2 from M. bovis BCG yielded the expected products when reverse transcription was primed with oligo(dT), suggesting that polyadenylylation is a general phenomenon in mycobacteria. PMID- 10746767 TI - Overproduction of the secretin OutD suppresses the secretion defect of an Erwinia chrysanthemi outB mutant. AB - OutB is a component of the Erwinia chrysanthemi Out secretion machinery. Homologues of OutB have been described in two other bacteria, Klebsiella oxytoca and Aeromonas hydrophila, but their requirement in the secretion process seems to be different. Study of OutB topology with the BlaM topology probe suggests that it is an inner-membrane protein with a large periplasmic domain. However, fractionation experiments indicate that it could be associated with the outer membrane through its C-terminal part. The secretion deficiency of an Erw. chrysanthemi outB mutant can be reversed by the addition of an inducer of the kdgR regulon. It was shown that this effect results from the increased expression of the secretin OutD and that secretion can be restored in an outB mutant by introducing the outD gene on a plasmid. Several experiments suggest an interaction between OutB and OutD. In Erw. chrysanthemi, the presence of OutD stabilizes OutB. OutD expressed in Escherichia coli can be protected from proteolytic degradation by the coexpression of OutB. This effect does not require the N-terminal, transmembrane segment of outB. OutB can be cross-linked with OutD by formaldehyde. These results indicate that OutB could act with OutD in the functioning of the Out secretion machinery. PMID- 10746768 TI - The gene pvaB encodes oxidized polyvinyl alcohol hydrolase of Pseudomonas sp. strain VM15C and forms an operon with the polyvinyl alcohol dehydrogenase gene pvaA. AB - A 5.7 kbp SphI fragment containing the polyvinyl alcohol (PVA) dehydrogenase gene pvaA and its 1.9 kbp 5'-flanking region was cloned from the PVA-degrading bacterium Pseudomonas sp. VM15C. The pvaB gene, encoding oxidized PVA hydrolase, was found in the region upstream of pvaA. Sequence data and expression studies indicated that pvaA and B constitute an operon in the order pvaBA. The pvaB gene encoded a protein of 379 amino acid residues (40610 Da), and a lipoprotein signal sequence and the lipase consensus sequence, Gly-X-Ser-X-Gly, characteristic of the active-site serine region in serine hydrolases, were detected in the deduced amino acid sequence. The pvaB product with the pvaA product constituted an enzyme system for the cleavage of PVA molecules. The pvaA product introduced beta diketone groups into the PVA molecule, and the pvaB product hydrolysed these beta diketone groups in oxidized PVA. The pvaB product also hydrolysed 4,6-nonanedione at a low rate, but not acetylacetone or 5-nonanone. It was completely inhibited by PMSF and was concluded to be a serine hydrolase. There were no proteins showing high similarity to the pvaB product in the databases, but minor similarity to a number of serine hydrolases including polyhydroxyalkanoate depolymerases was apparent. PMID- 10746769 TI - Molecular characterization of the ferric-uptake regulator, fur, from Staphylococcus aureus. AB - Iron is an essential nutrient for the survival and pathogenesis of bacteria, but relatively little is known regarding its transport and regulation in staphylococci. Based on the known sequences of ferric-uptake regulatory (fur) genes from several Gram-positive and Gram-negative bacteria, a fragment containing the fur homologue was cloned from a genomic library of Staphylococcus aureus RN450. Nucleotide sequence analysis of this fragment revealed the presence of a 447 bp ORF that encodes a putative 149 aa polypeptide with an apparent molecular mass of 17 kDa. A putative ferrichrome-uptake (fhu) operon, containing the conserved Fur-binding sequences (Fur box) in the promoter region, was also cloned from the same S. aureus library. To characterize the impact of Fur on the fhu operon, fur was cloned, overexpressed as a His-tagged protein and purified by Ni2+-affinity column chromatography. The recombinant protein was digested with enterokinase to remove the His tag. Electrophoretic mobility-shift assays indicated that Fur binds to the promoter region of the fhu operon in the presence of divalent cations. Fur also interacted with the promoter region of the recently reported sir operon that has been proposed to constitute a siderophore-transport system in S. aureus. The DNase I-protection assay revealed that Fur specifically binds to the Fur box located in the promoter region of the fhu operon. The primer extension reaction indicated that the transcription-start site of the fhu operon was located inside the Fur box. S. aureus fur partially complemented a fur- mutation in Bacillus subtilis. The data suggest that Fur regulates iron-transport processes in S. aureus. PMID- 10746770 TI - Fur-independent regulation of iron metabolism by Irr in Bradyrhizobium japonicum. AB - Bradyrhizobium japonicum expresses both Fur and Irr, proteins that mediate iron dependent regulation of gene expression. Control of irr mRNA accumulation by iron was aberrant in a fur mutant strain, and Fur repressed an irr::lacZ promoter fusion in the presence of iron. Furthermore, metal-dependent binding of Fur to an irr gene promoter was demonstrated in a region with no significant similarity to the Fur-binding consensus DNA element. These data suggest that the modest control of irr transcription by iron is mediated by Fur. However, Irr protein levels were regulated normally by iron in the fur strain, indicating that Fur is not required for post-transcriptional control of the irr gene. Accordingly, regulation of hemB, a haem biosynthesis gene regulated by Irr, was controlled normally by iron in a fur strain. In addition, the hemA gene was shown to be controlled by Fur, but not by Irr. It was concluded that Fur cannot be the only protein by which B. japonicum cells sense and respond to iron, and that Irr may be involved in Fur independent signal transduction. Furthermore, iron-dependent regulation of haem biosynthesis involves both Irr and Fur. PMID- 10746771 TI - The gene encoding IIAB(Man)L in Streptococcus salivarius is part of a tetracistronic operon encoding a phosphoenolpyruvate: mannose/glucose phosphotransferase system. AB - Glucose and mannose are transported in streptococci by the mannose-PTS (phosphoenolpyruvate:mannose phosphotransferase system), which consists of a cytoplasmic IIAB protein, called IIAB(Man), and an uncharacterized membrane permease. This paper reports the characterization of the man operon encoding the specific components of the mannose-PTS of Streptococcus salivarius. The man operon was composed of four genes, manL, manM, manN and manO. These genes were transcribed from a canonical promoter (Pman) into a 3.6 kb polycistronic mRNA that contained a 5'-UTR (untranslated region). The predicted manL gene product encoded a 35.5 kDa protein and contained the amino acid sequences of the IIA and IIB phosphorylation sites already determined from purified S. salivarius IIAB(Man)L. Expression of manL in Escherichia coli generated a 35 kDa protein that reacted with anti-IIAB(Man)L antibodies. The predicted ManM protein had an estimated size of 27.2 kDa. ManM had similarity with IIC domains of the mannose EII family, but did not possess the signature proposed for mannose-IIC proteins from Gram-negative bacteria. From multiple alignment analyses of sequences available in current databases, the following modified IIC(Man) signature is proposed: GX3G[DNH]X3G[LIVM]2XG2[STL][LT][EQ]. The deduced product of manN was a hydrophobic protein with a predicted molecular mass of 33.4 kDa. The ManN protein contained an amino acid sequence similar to the signature sequence of the IID domains of the mannose-EII family. manO encoded a 13.7 kDa protein. This gene was also transcribed as a monocistronic mRNA from a promoter located in the manN-manO intergenic region. A search of current databases revealed the presence of IIAB(Man)L, ManM, ManN and ManO orthologues in Streptococcus mutans, Streptococcus pyogenes, Streptococcus pneumoniae and Enterococcus faecalis. This work has elucidated the molecular structure of the mannose PTS in streptococci and enterococci, and demonstrated the presence of a putative regulatory protein (ManO) within the man operon. PMID- 10746772 TI - Protonmotive force regulates the membrane conductance of Streptococcus bovis in a non-ohmic fashion. AB - Because the DCCD (dicyclohexylcarbodiimide)-sensitive, F-ATPase-mediated, futile ATP hydrolysis of non-growing Streptococcus bovis JB1 cells was not affected by sodium or potassium, ATP hydrolysis appeared to be dependent only upon the rate of proton flux across the cell membrane. However, available estimates of bacterial proton conductance were too low to account for the rate of ATP turnover observed in S. bovis. When de-energized cells were subjected to large pH gradients (2.75 units, or -170 mV), internal pH declined at a rate of 0.15 pH units s(-1). Based on an estimated cellular buffering capacity of 200 nmol H+ (mg protein)(-1) per pH unit, H+ flux across the cell membrane (at -170 mV) was 108 mmol (g protein)(-1) h(-1). When potassium-loaded cells were treated with valinomycin in low-potassium buffers, initial K+ efflux generated membrane potentials in close agreement with values predicted by the Nernst equation. These artificial membrane potentials drove H+ uptake, and H+ influx was counterbalanced by a further loss of cellular K+. Flame photometry indicated that the rate of K+ loss was 215 (+/-26) mmol K+ (g protein)(-1) h(-1) at -170 mV, but the potassium sensitive fluorescent compound CD222 indicated that this rate was only 110 (+/ 44) mmol K+ (g protein)(-1) h(-1). As pH gradients or membrane potentials were reduced, the rate of H+ flux declined in a non-ohmic fashion, and all rates were <25 mmol (g protein)(-1) h(-1) at a driving force of -80 mV. Previous estimates of bacterial proton flux were based on low and unphysiological protonmotive forces, and the assumption that H+ influx rate would be ohmic. Rates of H+ influx into S. bovis cells [as high as 9x10(-11) mol H+ (cm membrane)(-2) s(-1)] were similar to rates reported for respiring mitochondria, but were at least 20-fold greater than any rate previously reported in lactic acid bacteria. PMID- 10746773 TI - L-[U-14C] lactate binding to a 43 kDa protein in plasma membranes of Candida utilis. AB - To identify the putative lactate transporter protein of Candida utilis, plasma membranes from cells grown either on lactic acid (presence of lactate proton symport) or glucose (absence of lactate proton symport) were incubated with L-[U 14C]lactic acid and the membrane proteins were then separated by SDS-PAGE. A well defined peak of radioactivity occurred in the lane of the gel containing plasma membrane proteins from lactic-acid-grown cells but not from glucose-grown cells. Binding was inhibited by unlabelled pyruvate and lactate, whereas succinate and citrate were not inhibitory. The monocarboxylate transporter inhibitor of animal cells, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, competitively inhibited the lactate proton symport in the whole yeast and also inhibited lactate binding to proteins of isolated plasma membranes. The polypeptide pattern of plasma membranes from lactic-acid-grown cells revealed a 43 kDa polypeptide associated with the peak of labelled lactate. Altogether the results suggest that this polypeptide is either the lactate transporter or a component of it. PMID- 10746774 TI - Yeast spores seem to be involved in biological phosphate removal: a microscopic in situ case study. AB - The principal polyphosphate-accumulating organism (PAO) in a biological-phosphate removal activated-sludge process was assessed microscopically. The organism was recognized by its distinct morphotype most easily after polyphosphate staining. The PAO occurred in large, homogeneous clusters. The cells of the PAO were the biggest cells abounding in the sludge--clearly bigger than average sludge bacteria. Typical of the principal PAO was a variation of cell size, even in fresh sludge. In acetate minimal medium containing ampicillin, the original principal PAO clusters were converted to clusters of clearly larger, polyphosphate-containing, vegetative yeast-like cells. Cycloheximide addition inhibited this and caused flock disintegration, disappearance of the principal PAO clusters and growth of free bacteria. The cell wall of the principal PAO was not of the usual bacterial character. It showed anomalous Gram staining, stained for chitin (not found in bacteria) and bound concanavalin A, like cell walls of many yeasts. In addition, the PAO cell wall was resistant to lysozyme, but sensitive to an enzyme mixture that lyses yeast cell walls. It was concluded that the principal PAO cells in the studied sludge were clustered spores of a yeast. PMID- 10746775 TI - Occurrence of natural dixenic associations between the symbiont Photorhabdus luminescens and bacteria related to Ochrobactrum spp. in tropical entomopathogenic Heterorhabditis spp. (Nematoda, Rhabditida). AB - Bacteria naturally associated with the symbiont Photorhabdus luminescens subsp. akhurstii were isolated from the entomopathogenic nematode Heterorhabditis indica. Bacterial isolates distinct from P. luminescens subsp. akhurstii were obtained from 33% of the samples. Fourteen bacterial isolates, from nematodes collected from three different Caribbean islands, were characterized by conventional phenotypic tests, restriction fragment length polymorphism and sequence analyses of PCR-amplified 16S rRNA genes (16S rDNAs). Isolates were grouped into three genotypes, each one being associated with one Caribbean island. Phenotypic characteristics and 16S rDNA analysis showed that the Photorhabdus-associated bacteria were closely related to Ochrobactrum anthropi for the group from Guadeloupe, and to Ochrobactrum intermedium for the two groups from the Dominican Republic and Puerto Rico. No pathogenicity of the Ochrobactrum spp. to the insects Galleria mellonella and Spodoptera littoralis (Lepidoptera) was detected. Since Ochrobactrum spp. are considered as human opportunist pathogens, the mass production of entomopathogenic nematodes for biological control requires strict vigilance. PMID- 10746776 TI - Evidence for specificity in type 4 pilus biogenesis by enteropathogenic Escherichia coli. AB - Type 4 fimbriae (pili) are surface appendages that are expressed by many species of Gram-negative bacteria. Previous studies have demonstrated that Pseudomonas aeruginosa can express and assemble pilin subunits from several unrelated species, indicating a common mechanism for biogenesis of type 4 pili whereby structural subunits from one system may be interchanged with those of another. In this study, an isogenic mutant of enteropathogenic Escherichia coli (EPEC) was constructed containing the entire tcpA gene from Vibrio cholerae 0395, which encodes the major structural subunit of the toxin-coregulated pilus (TCP), in place of bfpA, which encodes the major structural subunit of the bundle-forming pilus (BFP). Surprisingly, expression of type 4 pilin structures and the associated phenotype of bacterial autoaggregation in culture media were not observed for cells of the EPEC strain containing tcpA nor for those containing an additional mutation in bfpF, which otherwise is associated with a hyperfimbriate phenotype. In addition, cells of a bfpA mutant EPEC strain containing plasmids designed to express either of two different chimeric type 4 pilin subunits containing segments of BfpA and TcpA also failed to form bacterial aggregates and express type 4 pilin structures. Collectively, these results indicate that the type 4 pilin assembly system of EPEC exhibits specificity with regard to pilin subunit recognition and assembly. PMID- 10746777 TI - Identification of new loci involved in adhesion of Listeria monocytogenes to eukaryotic cells. European Listeria Genome Consortium. AB - Insertional mutagenesis was performed with Tn1545 in the genetic background of an inIAB deletion mutant to identify new adhesion determinants in Listeria monocytogenes. Four insertion mutants defective in adhesion to eukaryotic cells were identified. Insertion sites were cloned by inverse-PCR and sequenced. The genetic organization of insertion regions was further analysed by screening and sequencing DNA fragments from a HindIII library and by searching databases. Three adhesion-defective mutants each had one copy of Tn1545 inserted into their chromosome. The insertion sites were different in the three mutants: (i) upstream from two ORFs in tandem, similar to dfp and priA of Bacillus subtilis, respectively; (ii) within an ORF encoding a putative 126 amino-acid-polypeptide with no significant similarity to any known protein; (iii) within an ORF similar to a B. subtilis ORF with no known function, just upstream from an operon similar to an ABC (ATP-binding cassette) transporter operon from B. subtilis. The excisants obtained from these mutants using the excision reporter plasmid pTCR9 recovered full adhesion capacity. A fourth mutant was the most severely defective in adhesion. It had five Tn1545 insertions, one of which was upstream from dfp and priA, and another of which was upstream from ami, a gene encoding a surface exposed autolysin with a C terminus similar to that of InIB. Ami was clearly involved because an ami null mutant constructed in an EGDdeltainIA-F background was adhesion-defective. Thus new regions involved in the adhesion of L. monocytogenes to eukaryotic cells were identified. Further study is required to define more accurately the roles of these regions in the adhesion process itself. PMID- 10746779 TI - Altered patterns of cellular growth, morphology, replication and division in conditional-lethal mutants of the thermophilic archaeon Sulfolobus acidocaldarius. AB - As a basis for studing the essential cellular processes of hyperthermophilic archaea, thermosensitive mutants of Sulfolobus acidocaldarius were isolated and characterized. Exponential-phase liquid cultures were shifted to the nonpermissive temperature and growth, viability, and distributions of cell mass and DNA content were measured as a function of time after the shift. The observed phenotypes demonstrate that chromosome replication, nucleoid organization, nucleoid partition and cell division, which normally are tightly co-ordinated during cellular growth, can be inhibited or uncoupled by mutation in this hyperthermophilic archaeon. PMID- 10746778 TI - Proteins complexed to the P1 adhesin of Mycoplasma pneumoniae. AB - Adherence of Mycoplasma pneumoniae to host cells requires several mycoplasmal membrane proteins and cytoskeleton-like proteins in addition to the adhesin P1, a transmembrane protein of 170 kDa. To analyse interactions of the P1 adhesin with other membrane proteins or with cytoskeleton-like proteins, cross-linking studies were performed in vivo using the permeant reagent paraformaldehyde. The cross linked protein complex was isolated by immunoaffinity chromatography, and proteins complexed to the P1 protein were identified by immunoblot analysis followed by high mass accuracy tryptic peptide mapping using matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). In addition to the P1 protein and a truncated form of the same protein, the adhesin-related 30 kDa protein, two membrane proteins of 40 and 90 kDa, the cytoskeleton-associated 65 kDa protein and two cytoskeleton-forming proteins, HMW1 and HMW3, were found to be components of the isolated protein complex. Furthermore, the cross-linked complex contained the chaperone DnaK and the E1alpha subunit of pyruvate dehydrogenase. In summary, it was shown that cytadherence-associated membrane proteins are located in close proximity to cytoskeleton-like proteins, suggesting a functional interaction between membrane and cytoskeleton-like proteins. DnaK might be involved in translocation of proteins from the cytoplasm to the membrane and pyruvate dehydrogenase might be a structural protein of the attachment organelle. PMID- 10746780 TI - Disordered ultrastructure in lignin-peroxidase-secreting hyphae of the white-rot fungus Phanerochaete chrysosporium. AB - The practice of exposing liquid cultures of the white-rot fungus Phanerochaete chrysosporium to a pure oxygen atmosphere under conditions of nutrient starvation has been widely adopted to induce lignin peroxidase (LiP) synthesis. Transmission electron microscopy was used to examine hyphal cells of carbon-limited cultures that had been exposed to an atmosphere of pure oxygen, and revealed evidence of a major loss in organization of cellular ultrastructure, which may be attributed to oxygen toxicity. Under some conditions (continuous agitation in air with cellulose as the carbon source) cultures will produce LiP without needing to be exposed to a pure oxygen atmosphere. A similar major loss of cellular ultrastructure was found in hyphal cells from such cultures upon examination. Investigation of the levels of H2O2, catalase and carbonyl content of intracellular proteins suggests that the latter cultures developed a hyperoxidant state because the rate of supply of carbon from cellulose hydrolysis was insufficient for oxygen homeostasis. The association of LiP with these cultures and with those exposed to an atmosphere of pure oxygen infers that LiP may be triggered in response to oxidant stress. PMID- 10746781 TI - The CMRF-35H gene structure predicts for an independently expressed member of an ITIM/ITAM pair of molecules localized to human chromosome 17. AB - The CMRF-35 monoclonal antibody recognizes an epitope found on at least two cell surface molecules, differentially expressed by many leukocytes. These molecules, the CMRF-35H (9) and CMRF-35A (CMRF-35) antigens are both members of the immunoglobulin (Ig) superfamily with a single V-like Ig domain. The function of these molecules is unknown, however the presence of putative immunoreceptor tyrosine-based inhibitory motifs (ITIM) in the cytoplasmic domain of the CMRF-35H molecule suggests that this molecule may play a regulatory role in leukocyte function. The CMRF-35H and CMRF-35A molecules show several similarities to the family of molecules containing ITIM or immunoreceptor tyrosine-based activatory motifs (ITAM) suggesting that CMRF-35H/CMRF-35A may be new members of this family. This would further indicate that, like other ITIM/ITAM containing molecules, CMRF-35H/CMRF-35A will also play an important role in the immune response. To further characterize these molecules, we have isolated genomic clones for the CMRF-35H gene and determined its intron-exon organization. The gene spans approximately 12 kb and consists of seven exons. Furthermore, this gene has been mapped to chromosome 17 and thus is not linked to the known human ITIM containing genes which map to human chromosome 19 or the recently characterized molecule, NKp44, localized to human chromosome 6. PMID- 10746783 TI - Genomic characterization of CD84 reveals the existence of five isoforms differing in their cytoplasmic domains. AB - CD84, a new member of the immunoglobulin superfamily, shows high homology with several molecules belonging to the CD2 family of differentiation antigens. By screening a peripheral blood leukocyte cDNA library four CD84 isoforms were obtained differing in their 3' sequence. A reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that these isoforms were normally found on leukocytes and a new isoform was identified. To establish the nature of the five isoforms obtained (CD84a, CD84b, CD84c, CD84d and CD84e) the genomic structure of the CD84 gene was determined. Our results show that it is composed of at least eight exons, with an exon coding for the 5' UTR and the leader peptide, two exons coding for each of the two immunoglobulin-like domains, an exon encoding the transmembrane portion and four exons coding for the cytoplasmic domains. The isoforms are generated by several mechanisms: alternative use of exons, reading frame shift, use of a cryptic splice site or absence of splicing. The differential expression of several potentially phosphorylatable residues on the different isoforms could be a way to regulate its possible activity in signal transduction. PMID- 10746782 TI - DR2 antigens are associated with severity of disease in toxic oil syndrome (TOS). AB - Toxic oil syndrome (TOS) was an epidemic which broke out in Spain in 1981, caused by the ingestion of rapeseed oil denatured with 2% aniline and sold illegally as edible oil. More than 20,000 people were affected and mortality rate was 8.4%. Genetic susceptibility appears to be involved in the pathology of this disease. Several reports have described association between the chronic stage of the disease and DR-DQ antigens (DR3, DR4, DR2 and DQ8). In the present work, we have reassessed the HLA class II antigens in a well-designed case-control study. Triplets of subjects (n=265) composed by chronic patients (n=117), non-affected family members (n=71) and non-related controls (n=77) were studied. Also, HLA class II antigens were analyzed in patients who had died from TOS (n= 34) and in TOS control patients who died from other non-TOS related causes (n=13). Regarding surviving patients no significant association was found between HLA and disease. In contrast, an increase in phenotypic frequency of DR2 antigen, was found in patients who had died from TOS (73.5%) compared with the whole study group: TOS affected alive patients (25.6%, corrected P<0.001), non-affected family members (28.5%, corrected P<0.001), non-related controls (23.9%, corrected P<0.001) and dead controls (38.4%, P=0.03). PMID- 10746784 TI - Analysis of HLA genes and haplotypes in Ainu (from Hokkaido, northern Japan) supports the premise that they descent from Upper Paleolithic populations of East Asia. AB - The Ainu people are assumed to be the descendants of pre-agricultural native populations of northern Japan, while the majority of population of present-day Japan (Hondo-Japanese) is considered to have descended mainly from post-neolithic migrants. Sequence-level polymorphisms of the HLA-class I (HLA-A and HLA-B) genes were investigated in DNA samples of 50 Ainu living in Hidaka district, Hokkaido. HLA-A*2402, A*0201, A*0206, A*2601, A*3101, B*1501, B*5101, B*3901, and B*3501 were observed at frequencies of more than 10% and most of these have previously been found in populations of not only Asians but also North and South American Indians. A*68012, which has not so far been detected in Hondo-Japanese, was found in the Ainu (3%). On the other hand, several alleles common in Hondo-Japanese, including HLA-A*3303, A*1101, B*4403, B*5201, B*5401, B*4601, and B*0702 were infrequent in Ainu (0-1%). Correspondence and neighbor-joining analyses of various populations based on HLA-A, -B and -DRB1 gene frequencies enabled distinction between Asian, Native South American, European, and African populations. The Ainu, as well as Tlingit (Na-Dene), were placed midway between other East Asians, including Hondo Japanese, and Native South Americans (Amerindians) in the correspondence analysis. Furthermore, several HLA-A-B and HLA-B-DR-DQ haplotypes common in the Ainu, are shared with some Native American populations. These observations strongly suggest a unique place for the Ainu as descendants of some Upper Paleolithic populations of East Asia, from whom some Native Americans may have descended. PMID- 10746785 TI - Multiple sclerosis: a modifying influence of HLA class I genes in an HLA class II associated autoimmune disease. AB - Multiple sclerosis (MS) is a presumed autoimmune disease of the central nervous system, shown to be associated with the HLA class II haplotype DRB1*15,DQB1*06. Carrying the HLA class II haplotype DRB1*15,DQB1*06 increases the risk of MS by 3.6. By adopting a polymerase chain reaction (PCR)-based typing technique for HLA class I and class II genes, 200 Swedish MS patients and 210 Swedish healthy controls were analysed for their HLA alleles. Additional HLA class I alleles that increase and decrease the genetic susceptibility to MS were identified. The HLA A*0301 allele increases the risk of MS (odds ratio=2.1) independently of DRB1*15,DQB1*06. HLA-A*0201 decreases the overall risk (odds ratio= 0.52) and the presence of A*0201 reduces the risk of MS for DRB1*15,DQB1*06 carriers from 3.6 to 1.5. Our findings are the first to identify a major modulating effect of HLA class I alleles on the susceptibility to a human autoimmune disease; a phenomenon that has previously only been observed in animal models. PMID- 10746786 TI - A MAGE-A1 peptide is recognized on HLA-B7 human tumors by cytolytic T lymphocytes. AB - Antigens encoded by MAGE genes are of particular interest for cancer immunotherapy because of their strict tumoral specificity and because they are shared by many tumors. MAGE antigenic peptides are currently used in therapeutic vaccination trials. The identification of additional antigenic peptides is likely to be important for the future of these clinical trials in order to increase the number of patients eligible for these vaccinations and to analyze in detail the T cell response of vaccinated patients. We describe here the isolation of a cytolytic T lymphocyte (CTL) clone which recognizes a new MAGE-A1 peptide, RVRFFFPSL (MAGE-A1(289-297)), which is presented by HLA-B7. This CTL clone lysed HLA-B7 tumor cells expressing MAGE-A1. HLA-B7 is expressed by approximately 20% of Caucasians PMID- 10746787 TI - An unusual insertion in intron 2 of apparently functional MHC class I alleles in rhesus macaques. AB - Here we describe a nucleotide insertion in intron 2 of two rhesus major histocompatibility complex (MHC) class I alleles, Mamu-A*05 and Mamu-A*07. This resulted in an intron 2 that was nearly twice the length of any other intron 2 of primate MHC class I genes sequenced to date. This insertion was most similar (93% identity) to the beginning of intron 3 of HLA-A alleles. It was also similar to intron 3 of several human MHC class I pseudogenes and MHC class I pseudogenes from cotton top tamarin and cat. The finding of this insertion in two rhesus MHC class I genes is surprising given the uniformity in length and sequence of introns of functional HLA-A, -B and -C genes. PMID- 10746788 TI - Identification of the immunoreactive peptide sequence for AgSK1, an adenocarcinoma-restricted antigen. AB - SK1, a human immunoglobulin M (IgM) monoclonal antibody was derived from regional nodal lymphocytes of a Dukes B colon carcinoma patient. The antigen recognized by the human monoclonal antibody (HuMab) SK1, termed AgSK1, was shown to be a two chain glycoprotein with an apparent molecular weight range of 42-46 kDa and preferentially expressed by human adenocarcinomas, particularly human gastrointestinal malignancies. To identify the gene encoding the AgSK1 antigenic epitope, a cDNA expression library constructed in lambda gt22A using mRNA from the colon carcinoma cell line HT29 was screened and one of the isolated clones encoding a 1.5-kb cDNA, which showed strong immunoreactivity with HuMab SK1, was selected for further analysis. This clone consisted of an amino terminal open reading frame of 54 amino acids and the carboxyl terminal 20 amino acids of this protein coding region contained the antigenic epitope recognized by HuMab SK1. PMID- 10746789 TI - A new MICA allele with ten alanine residues in the exon 5 microsatellite. AB - The MICA and MICB genes code for protein products that have structural similarities to major histocompatibility complex (MHC) class I genes. These genes are upregulated by heat stress. They have been shown to interact with a common receptor (NKG2D/DAP10) on gammadelta T cells, CD8+ T cells and natural killer (NK) cells. The MICA gene has an expressed microsatellite, GCT, within the exon 5 which encodes for alanine. So far, four different repetitions of this short tandem have been reported. Also one non-synonymous, one synonymous substitution and a 1-bp insertion within this region have also been described. An association of Behcet's disease with the microsatellite A9 has been reported. Here we report a novel allele with 10 GCT repetitions (A10) which was detected by reference strand mediated conformation analysis and confirmed by DNA sequencing. PMID- 10746790 TI - Three novel MICB alleles. AB - The two members of the MHC class I chain-related (MIC) gene family, MICA and MICB, have been shown by several investigators to be polymorphic. Most of the research effort so far has focussed on MICA, so less is known about the extent of polymorphism in the MICB gene. Here we report three novel MICB alleles, which had been detected in the course of an SSOP typing study on a large cohort of cell lines. Two of these alleles are formed by a non-synonymous nucleotide variation. Our results confirm previous findings that most of the polymorphisms in the MICB gene, as in MICA, are coding and suggest that the extent of polymorphism in the two genes might be comparable. PMID- 10746791 TI - Molecular analysis of HLA-DRB1, -DQA1 and -DQB1 polymorphism in Turkey. AB - We report the evaluation of MHC class II polymorphism in the population of Turkey. HLA-DRB1, -DQA1 and -DQB1 have been investigated by polymerase chain reaction and sequence-specific oligonucleotide probe hybridisations (PCR/SSO) and sequence-specific priming (SSP) in 250 randomly selected healthy individuals. We also report the allelic distribution of these genes. The most frequent alleles detected were DRB1*1101 (0.104), *0301 (0.092), *0701 (0.090), DQA1*0501 (0.334), *0102 (0.164) and *03 (0.148) and DQB1*0301 (0.256), *02 (0.164), *0302 (0.128). The frequent 'putative' three-locus haplotypes carry the most frequent alleles at these loci. The most frequently detected class II "haplotypes" are DRB1*1101 DQA1*0501 DQB1*0301 (0.100), DRB1*0301 DQA1*0501 DQB1*02 (0.092) and DRB1*0701 DQA1*0201 DQB1*02 (0.072). The distribution of alleles and 'putative' haplotypes has shown common features with other Mediterranean populations. The results extend the HLA map to another Mediterranean country and provide a database for further HLA-disease association studies and transplantation applications. PMID- 10746792 TI - A new HLA-A*02 allele, A*0234, detected by polymerase chain reaction using sequence-specific primers (PCR-SSP). AB - A novel HLA-A*02 allele, A*0234, was identified in a potential unrelated bone marrow donor typed by polymerase chain reaction using sequence-specific primers (PCR-SSP). Positive reactions obtained upon testing with PCR-SSP did not fit any known combination of alleles indicating the possible presence of a novel allele. Sequencing of clones from this individual revealed the presence of a novel allele, HLA-A*0234. The sequence of exons 2, 3 and part of exon 4 showed that A*0234 differed from A*02011 by a single nucleotide in exon 2 at position 282 (C to G). The nucleotide substitution results in an amino acid change at residue 70 (Histidine to Glutamine) in the alpha1 domain. PMID- 10746793 TI - HLA-A*2416: a new allele of the HLA-A19 lineage. AB - We here report on a new HLA-A19 allele (A*2416) found in a German kidney recipient. Serological class I typing revealed HLA-A11,19 without clear definition of the A19 split antigen. As with serology, polymerase chain reaction (PCR)-based typing also revealed inconclusive results. We therefore sequenced the gene from the 5' flanking region through the 3'-end of exon 4 of this allele after haplotype-specific PCR amplification. The sequence analysis revealed a new HLA-A allele which is identical to A*3101 with the exception of the 3' half of exon 2 which is identical to the common A9 alleles. The phylogenetic analysis constructed with the nearest-neighbor algorithm and based on exons 1-4 or introns 1-3 clearly indicated, that A*2416 belongs unequivocally to the A19 lineage. PMID- 10746794 TI - A novel HLA-B*51 allele (B*5116) identified by nucleotide sequencing. AB - We report here an additional HLA-B*51 variant designated HLA-B*5116. Detected by an abnormal serological reactivity pattern, this variant was identified as a B*51 allele by polymerase chain reaction using sequence-specific primers (PCR-SSP) and characterized by nucleotide sequencing. The new variant sequence match closely with the classical HLA-B*5101 excepted two adjacent nucleotide substitutions at positions 216 and 217 of the third exon and the subsequent Leucine to Glutamic acid change at codon 163 of the alpha2 domain (CTG-->GAG). This new variant was not detected in three different ethnic groups (French, Algerian and Lebanese) suggesting a very rare frequency. PMID- 10746795 TI - HLA-B*4413 identified in a UK Caucasoid potential bone marrow donor. AB - We describe a novel allele belonging to the B*44 allele group. HLA-B*4413 was identified in a Caucasoid potential bone marrow donor from the Anthony Nolan Bone Marrow Trust register. Initial HLA typing of this donor revealed unusual serological reactivity. Further investigation was carried out by reference strand conformation analysis (RSCA) and sequence-based typing (SBT) using DNA extracted from an Epstein-Barr virus (EBV)-transformed B-cell line made from this donor (AMI005AN). In this individual, two B*44 alleles were revealed upon initial investigation: B*4409 and a previously unseen allele which has been named B*4413. B*4413 is identical to B*44031 except for a unique nucleotide substitution resulting in an amino acid difference at residue 61 in the alpha1 helix. PMID- 10746796 TI - Identification of a novel DPB1* allele (DPB1*6901) and the occurrence of HLA haplotypes that extend to DPB1. AB - The sequence of a novel DPB1 allele, DPB1*6901, observed in a Caucasian bone marrow donor phenotype HLA A2; Cw*0501,*1601; B*4402, *4403; DRB1*0401; *07; DQB1*02; *0301; DPB1*0401; *6901, is described. The sequence is consistent with that previously described for DPB1*0601 with the exception of codon 69. The sequence at this codon is consistent with that previously observed only in the DPB1*1101 and *1501 alleles. It is suggested that DPB1*6901 may have arisen as a result of a recombination event occurring between codons 58 and 64 between DPB1*0601 and DPB1*1101. The sequence of DPB1*1501 from codon 64 is not consistent with DPB1*6901. A linkage disequilibria analysis that examined 212 potential bone marrow recipients in which HLA-A to DQ haplotypes had been established by family studies showed linkage disequilibrium between HLA-B, DRB1 and DPB1 in some haplotypes and not others. PMID- 10746797 TI - Nomenclature for factors of the HLA system, update October/November 1999. WHO Nomenclature Committee for Factors of the HLA System. PMID- 10746798 TI - Long-term renal replacement therapy. PMID- 10746799 TI - Pathophysiology of renal anemia. AB - Normochromic normocytic anemia regularly develops in chronic renal failure when the glomerular filtration rate drops below 20-30 ml/min. The reasons include: 1) a moderately reduced red cell life span, 2) blood loss, and 3) an inadequate increase in erythropoiesis relative to the fall in hemoglobin (Hb). The life-span of red blood cells may be shortened by their reduced resistance to mechanical, osmotic or oxidative stress, as well as by extracorpuscular factors. Blood loss results from dialysis, diagnostic sampling and, in particular, occult gastrointestinal bleeding. The predominant cause of inadequate erythropoiesis is a failure to increase erythropoietin (EPO) production in response to the developing anemia. Experience with recombinant EPO has shown that relative EPO deficiency is the key cause of the anemia and that the response of hematopoietic progenitor cells is not usually diminished in renal failure. However, reduced iron availability, inadequate dialysis, infection and hyperparathyroidism can all impair the efficacy of EPO. Therapeutic use of EPO has also shown clearly for the first time that anemia is responsible for a significant proportion of morbidity in patients with chronic renal failure and probably also contributes to increased mortality through its cardiovascular complications. PMID- 10746800 TI - Erythrocyte free radical and energy metabolism. AB - The erythrocyte is a highly specialized cell whose main functions are oxygen transport and the mediation of carbon dioxide transport. Energy production in the mature erythrocyte depends on glycolysis, with glucose as the principal substrate. Glycolysis and the oxidative pentose phosphate pathway generate NADH and NADPH to reduce methemoglobin, which is being continuously produced, and the antioxidant glutathione, which is present in high concentrations. Red blood cells are equipped with a highly effective antioxidant defense even without the glutathione system. Compared with other cell types, they possess high activities of the most important antioxidant enzymes. Most of the nonenzymatic antioxidant capacity of whole blood is likewise localized in the erythrocytes. Circulating red cells are mobile free radical scavengers and provide antioxidant protection to other tissues and organs. An imbalance between pro-oxidant reactions and antioxidant defense is described in patients with chronic renal failure. Oxidative stress increases as antioxidant defenses are weakened by pro-oxidant hemodialysis factors; it increases further still in renal anemia with a very low red cell count. Thus in terms of free radical metabolism, the only arguments remaining over the complete correction of renal anemia are those in favor, with none against. PMID- 10746801 TI - Oxidative stress in anemia. AB - The increased formation of reactive oxygen species under hypoxic conditions often appears paradoxical. A prooxidant shift results from changes in cellular metabolism (especially energy metabolism), higher flux rates in catecholamine metabolism and permanent leukocyte activation. These mechanisms of increased free radical production then find themselves opposed by an antioxidant system that is markedly weakened by anemia. The erythrocytes represent an important component of the antioxidant capacity of blood, comprising in particular intracellular enzymes, e.g. superoxide dismutase and catalase, but also the glutathione system. It is thus possible that some complications of uremia are at least partly due to oxidative stress. These include cardiovascular complications, premature biological aging and increased susceptibility to infection. Strategies to strengthen the complex endogenous free radical defenses can thus be predicted to show long-term benefit. In this context the expansion of EPO therapy may well be a major step in stabilizing free radical metabolism in anemic patients. PMID- 10746802 TI - Does treatment of renal anemia with recombinant erythropoietin influence oxidative stress in hemodialysis patients? AB - Patients with chronic renal failure (CRF) undergoing hemodialysis (HD) are exposed to constant oxidative stress, as shown by elevated malondialdehyde (MDA) plasma concentrations in HD patients. The aim of our study was to investigate the role of renal anemia in oxidative stress. To this end, MDA and 4-hydroxynonenal (HNE) were measured in three groups of patients. Group I comprised 8 patients with hemoglobin (Hb) < 10 g/dl (mean Hb 8.1 +/- 1.3 g/dl) and group II 8 patients with Hb > 10 g/dl (mean Hb 12.4 +/- 1.9 g/dl). None of these 16 patients had been previously treated with recombinant erythropoietin (rhEPO). Group III comprised 27 patients with mean Hb 10.5 +/- 1.6 g/dl after long-term treatment with rhEPO. The plasma concentrations of both MDA and HNE in all 43 HD patients were significantly higher (p < 0.0001) than in 20 healthy controls (MDA 2.85 +/- 0.25 vs 0.37 +/- 0.03 microM, HNE 0.32 +/- 0.03 versus 0.10 +/- 0.01 microM). Comparison between the three groups showed that the HD patients with Hb < 10 g/dl had significantly higher plasma concentrations of lipid peroxidation products (MDA 3.81 +/- 0.86 microM, HNE 0.45 +/- 0.07 microM) than either HD patients with Hb > 10 g/dl (MDA 2.77 +/- 0.58 microM, HNE 0.25 +/- 0.05 microM) or HD patients treated with rhEPO (MDA 2.50 +/- 0.12 microM, HNE 0.29 micro 0.03 microM). An inverse correlation was also demonstrated between plasma HNE and Hb (r= 0.62, p < 0.0001). It follows that a substantial part of the oxidative stress is due to renal anemia. Treatment with rhEPO can therefore effectively reduce oxidative stress in HD patients. PMID- 10746803 TI - Oxidation parameters in complete correction of renal anemia. AB - Chronic hemodialysis (HD) patients are more exposed to oxidative stress, with its adverse impact on many cell functions and not least on patient survival. There is evidence that partial correction of renal anemia by erythropoietin (rhEPO) therapy reduces oxidative stress. The aim of this study was to clarify whether complete correction of renal anemia with rhEPO reduces free radical generation in HD patients and increases antioxidant supply. The following parameters: malondialdehyde (MDA), reduced glutathione (GSH) and glutathione disulfide (GSSG) were investigated in patients with a hematocrit (Hct) normalized on rhEPO therapy (Hct > or = 0.4), and compared with those in anemic patients (Hct 0.3 - 0.39 and Hct < 0.3). The groups were similar in age, sex or body weight. Patients with normal Hct were significantly longer in the chronic HD program (74.0 +/- 70.3 vs. 23.0 +/- 30.9 and 30.6 +/- 34.8 months; p < 0.05) and received significantly lower doses of iron (35.7 +/- 19.5 vs. 55.4 +/- 26.0 and 80.0 +/- 47.1 mg/week; p < 0.05) and rhEPO (68.9 +/- 63.6 vs. 106.5 +/- 63.9 and 152.8 +/- 86.0 IU/kg/week; p < 0.05). MDA levels were significantly lower in the group with normal Hct than in the comparison groups (1.54 +/- 0.27 vs. 1.98 +/- 0.52 and 2.23 +/- 0.93 micromol/l; p < 0.01), but did not differ significantly between the anemic groups. GSH and GSSG concentrations corrected for erythrocyte levels showed no significant differences, but whole-blood levels in patients with Hct > or = 0.4 and 0.3 - 0.39 were significantly higher than in patients with Hct < 0.3 (GSH: 0.97 +/- 0.42 vs. 1.03 +/- 0.38 and 0.62 +/- 0.34 micromol/ml; GSSG: 14.57 +/- 6.06 vs 13.07 +/- 5.18 and 7.28 +/- 3.64 micromol/l; p < 0.05). CONCLUSION: After correction of renal anemia, MDA levels are significantly lower - reflecting decreased free radical generation - than in anemic HD patients. Whole-blood antioxidant capacity is significantly increased. Overall, rhEPO therapy has clearly positive effects on free radical metabolism. PMID- 10746804 TI - Long-term effects of rhEPO therapy on erythrocyte rheology in dialysis patients with different target hematocrits. AB - Successful treatment of renal anemia with recombinant erythropoietin (rhEPO) raises the question of whether the renal anemia symptom complex requires complete correction. Current arguments against increasing hemoglobin (Hb) levels above 10 11 g/dl are impaired hemodynamics, increased risk of vascular access occlusion, unmanageable hypertension and dialysis complications. The aim of the study was to determine whether sustained Hb normalization using long-term rhEPO causes hemorheological changes with a potentially negative hemodynamic impact. The study was conducted in 42 rhEPO-treated dialysis patients with stable Hb > 11.0 g/dl for at least 20 weeks. The mean Hb of the total study group was 12.8 1.1 g/dl. To study the effect of Hb as a risk indicator in greater detail, the patients were divided into two groups, with hematocrits above and below 0.40. Hemorheology (erythrocyte deformability and aggregation, plasma viscosity) showed no significant changes, including vs a healthy control group. Throughout the period of increased rhEPO administration, no increase was observed in the incidence of hypertension or vascular thrombosis. CONCLUSION: the marked additional quality-of life benefit achieved by complete correction of renal anemia harbors no substantial increase in treatment risk. PMID- 10746805 TI - Complete correction of renal anemia by recombinant human erythropoietin. AB - The target-hematocrit (Hct) for the correction of renal anemia by recombinant human erythropoeitin (rhEPO) therapy is discussed controversially. A normalization of the Hct that could lead to a further improvement of the patients status, is often rejected, because of possible side effects as a result of an increase in blood viscosity. Hemodialysis (HD) induces an acute hemoconcentration due to ultrafiltration that might influence these risk factors negatively and therefore conflict with the normalization of Hct. The aim of this study was to investigate the changes in rheological and biochemical parameters in chronic HD patients with a normal initial Hct before hemodialysis. Results in 39 patients are given as mean +/- SD before/after HD: Hct 0.42 +/- 0.05/0.45 +/- 0.05 (p < 0.001), hemoglobin (g/dI) 13.3 +/- 1.0/14.4 +/- 1.3 (p < 0.001), MCV (fl) 99.3 +/ 5.7/99.1 +/- 5.5, MCHC (mM/l) 19.9 +/- 0.6/20.1 +/- 0.6 (p < 0.01), red blood cell (RBC) elongation (%) 60.97 +/- 3.67/60.99 +/- 3.73, RBC aggregation index AI0 0.52 +/- 0.12/0.50 +/- 0.12, AI4 0.52 +/- 0.14/0.51 +/- 0.12, plasma viscosity 1.74 +/- 0.14/1.92 +/- 0.20 (p < 0.001), whole blood viscosity (WBV), etaabs.100(mPas) 5.91 +/- 0.78/6.80 +/- 1.2 (p < 0.001), etaabs.0.01(mPas) 75.81 +/- 35.48/167.656 +/- 98.686 (p < 0.05), ultrafiltration (FM) 2.1 +/- 1.1. The biochemical parameters protein, albumin, IgG, IgA, IgM, cholesterol, transferrin and fibrinogen are significantly increased after HD. The hemoconcentration during HD is associated with a significant increase in WBV, mainly associated with the increase in Hct (r = 0.83), but not exceeding the normal range compared to healthy controls. The increase in plasma viscosity is correlated mainly with an increase in protein (r = 0.80), albumin (r = 0.74), and fibrinogen (r = 0.54). No significant changes in RBC aggregation and deformability were observed during the HD session. In conclusion, from the rheological point of view it is unlikely that the normalization of the Hct will contribute to an increased risk in access thrombosis or thromboembolic events in HD patients. PMID- 10746806 TI - Pressure and flow in the extracorporeal circuit. AB - The impact of pressure and flow changes in the extracorporeal circuit on dialysis efficiency in response to altered hematocrit is discussed in the light of the known properties of blood, published studies and the author's as yet unpublished measurements. Increasing the hematocrit from 0.35 to 0.45 must be expected to reduce the efficiency of urea elimination by 3-5%, which can be offset by increasing dialysis time or blood flow. The change in physical parameters thus represents no obstacle to normalizing the hematocrit. However, basing the erythropoietin dose on the predialysis hematocrit should be reconsidered. PMID- 10746808 TI - Erythropoietin and arterial hypertension. AB - Erythropoietin (EPO) has revolutionized the treatment of anemia in renal failure patients, both in the pre- and postdialysis phase. Not only does the treatment improve well being, but also it positively influences cardiac function and permits cardiac hypertrophy to regress. EPO can lead to an increase in blood pressure; the mechanisms of this effect are not entirely clear. By optimizing dialysis treatment, paying close attention to volume regulation, giving EPO subcutaneously and in a fashion to increase hematocrit gradually, the occurrence of blood pressure increases can be minimized. Hypertension has not proved to be a serious general problem in the EPO treated patient. PMID- 10746807 TI - Amino acid and alpha-keto acid metabolism depends on oxygen availability in chronic hemodialysis patients. AB - Amino acid and alpha-keto acid metabolism depends on oxygen availability in chronic hemodialysis patients. Malnutrition in hemodialysis (HD) patients with renal anemia is reflected in an abnormal plasma amino acid profile. We wished to determine the extent to which malnutrition can be improved by correcting anemia with recombinant human erythropoietin (rhEPO) and supplemental iron. As specific parameters we measured and compared the plasma concentrations of the branched chain amino acids valine, leucine and isoleucine and their transamination products - major participants in proteolysis and protein biosynthesis - using HPLC with fluorescence detection in 45 severely anemic HD patients (group A: Hb 7.2 +/- 0.8 g/dl), 34 patients with partially corrected anemia (group B: Hb 10.3 +/- 0.6 g/dl) and 35 HD patients with totally corrected anemia (group C: Hb 13.5 +/- 0.7 g/dl). Sixty healthy subjects (group N: Hb 14.2 +/- 1.1 g/dl) served as controls. Correlating with the degree of correction (A/B/C/N), a significant shift to anabolic metabolism was observed in the plasma levels of valine: 130/146/155/205 micromol/l; leucine: 73/71/80/110 micromol/l and alpha ketoisocaproate (KIC): 11.4/11.8/15.1/ 30.4 micromol/l. It is concluded that energy metabolism becomes increasingly anabolic as hemoglobin levels are normalized. PMID- 10746809 TI - Iron metabolism in rhEPO-treated hemodialysis patients. AB - A number of factors have been shown to limit the response to recombinant human erythropoietin (rhEPO). One major factor appears to be an inadequate iron supply to the bone marrow. Erythropoiesis is dependent upon a continuous supply of iron to the bone marrow. The rate at which iron can be drawn from existing stores may easily limit the rate of delivery for hemoglobin synthesis. This may result in "functional iron deficiency" which is distinct from "absolute iron deficiency" caused by depletion of iron stores. At present there are three main parameters available to clinicians wishing to monitor iron status in their patients: serum ferritin and transferrin saturation (TFS), which are indirect measurements, and the percentage of hypochromic red cells, which directly reflects marrow iron status. Ferritin levels should be measured before starting rhEPO therapy to ensure adequate iron stores (> 200 microg/l), and when patients move from the correction phase to the maintenance phase of therapy (have stores become depleted during the correction phase?). In addition, ferritin levels can give an indication of iron overload following excess parenteral iron administration. The TFS represents a balance between iron supply by the stores and demand by bone marrow. A saturation below 20% probably indicates iron-deficient erythropoiesis. However, this is an indirect measure of marrow iron supply and wide fluctuations have been observed when determined at different time points. The percentage of hypochromic red blood cells is measured by flow cytometry and a hypochromic subpopulation of more than 10% (normal percentage > 2.5%) indicates iron deficient erythropoiesis. However, not all departments have access to the required equipment. The aim of iron supplementation is to provide sufficient iron for the correction phase and replace iron losses (1,500 - 3,000 mg/year in hemodialysis patients) during the maintenance phase of rhEPO therapy. This amounts to a daily iron need in the range of 5-7 mg, which is well above the normal dietary intake and absorptive capacity of the human intestine. Therefore there is a need for intravenous iron, in particular when the patient ha absolute or functional iron deficiency, is in tolerant of oral iron, or is not complying we with the oral regimen. PMID- 10746810 TI - How can hemodialysis-associated hypotension and dialysis-induced symptoms be explained and controlled--particularly in diabetic and arteriosclerotic patients? AB - Patients with diabetes and/or severe arteriosclerosis are often unable to tolerate volume removal during hemodialysis (HD) and develop hemodialysis-induced symptoms. These problems can be omitted by well-balanced correction of the acid/base status. We compared 20 high-risk patients which were either treated with standard HD (dialysate bicarbonate 33 mmol/l, treatment A) or treated with standard HD and additional administration of NaHCO3 (120-160 ml 8.4% NaHCO3 solution over the venous line during HD) to correct the metabolic acidosis to upper normal values (treatment B). The following parameters were compared: 1. Acid/base status; 2. EEG monitoring and clinical observation of dialysis-induced symptoms; 3. Invasive monitoring of circulation by Swan-Ganz thermodilution; 4. Ventilation, oxygen consumption and lactate production. Optimal correction of acid/base values resulted in symptom-free hemodialysis sessions with stable PaCO2 in the normal range, cardiovascular stability assessed by invasive monitoring, normal ventilation and higher oxygen consumption and decreased lactate production. Optimal correction of acid/base balance further led to the absence of EEG alterations and of dialysis-induced symptoms during treatment B as compared to treatment A. The baroreceptor response in these patients is usually disturbed due to sclerosis of the pressosensible vessels, especially the aortic arch and the pulmonary arteries impairing a compensatory increase of heart rate upon volume removal. However, chemoreceptors are able to increase sympathetic tone with preservation of blood pressure in this situation. In addition a decrease of PaO2 during volume removal can only be answered by an early increase of ventilation response due to stimulation of chemoreceptors provided that PaCO2 is maintained normal. Furthermore, normal cerebral blood flow also depends on a normal PaCO2. Based on these pathophysiological mechanisms the therapeutic strategy of additional bicarbonate administration to correct the acid/base status guarantees a stable normal PaCO2 and facilitates a symptom-free HD in high-risk patients. PMID- 10746811 TI - Cardiac mortality prevention in uremic patients. Therapeutic strategies with particular attention to complete correction of renal anemia. AB - BACKGROUND: Left ventricular hypertrophy represents the major risk factor for cardiac mortality and morbidity, with cardiac mortality being the most important determinant for survival in dialysis patients. The prevalence of left ventricular hypertrophy is already high at initiation of dialysis and increases with time. Anemia is considered as the most important factor for the development of left ventricular hypertrophy. Others already demonstrated that with partial correction of renal anemia by erythropoietin a partial regression of the left ventricular mass can be achieved. PATIENTS AND METHODS: We investigated the effect of complete correction of renal anemia to normal hemoglobin values of 14 g/dl (Hct 42%) on left ventricular hypertrophy by echocardiography. Eight Patients entered the study 4-8 weeks after initiation of chronic hemodialysis with a mean hemoglobin of 9.5 +/- 1.3 g/dl). RESULTS: Left ventricular mass index (LVMI) decreased from 155 +/- 45 g/m2 to 123 +/- 18g/m2 (p < 0.05) within the observation period of 12 +/- 5 months. The results showed, that either normal left ventricular dimensions could be preserved or, if left ventricular hypertrophy was already present, complete regression was possible. CONCLUSION: Therefore, we propose that complete correction of renal anemia should be introduced into the therapy of dialysis patients along with strict adherence to established measures for the control of left ventricular hypertrophy: control of fluid overload and arterial hypertension and the use of ACE-inhibitors and betablockers. In addition, optimal correction of metabolic acidosis, control of the calcium-phosphate product and hyperparathyreoidism must be attempted. Thus, it should be possible to reverse left ventricular hypertrophy and its deleterious consequences in the dialysis population in order to improve survival and quality of life. PMID- 10746812 TI - Why correct renal anemia completely? PMID- 10746813 TI - Improvement in left ventricular function in response to carvedilol is accompanied by attenuation of neurohumoral activation in patients with dilated cardiomyopathy. AB - BACKGROUND: We sought to evaluate whether improvement in ejection fraction (EF) with carvedilol therapy is accompanied by improvement in neurohumoral factors. METHODS AND RESULTS: Forty-two patients with dilated cardiomyopathy were given carvedilol for 3 to 5 months. Changes in EF, plasma atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and norepinephrine levels were determined. Iodine-123 metaiodobenzylguanidine (MIBG) images were also obtained before and after carvedilol therapy. Myocardial uptake of MIBG was calculated as the heart to mediastinal activity ratio (H/M). Storage and release of MIBG was calculated as percent myocardial MIBG washout rate (WR). We divided patients into 2 groups: 27 responders whose EF increased by more than 5% and 15 nonresponders whose EF increased by 5% or less. EF of responders increased by 15 +/- 5% and that of nonresponders by 1 +/- 4%. Although MIBG image-derived indexes of nonresponders remained unchanged, the delayed H/M (1.91 +/- 0.34 v 2.24 +/- 0.53, P < .01) and WR (49 +/- 11 v 39 +/- 9%, P < .01) of responders improved, respectively. The plasma ANP (51 +/- 50 v 27 +/- 24 pg/mL, P < .01) and BNP (194 +/- 197 v 49 +/- 62 pg/mL, P < .01) levels of responders decreased. The degree of changes in the plasma BNP level correlated with changes in EF (r = -.698, P < .01). CONCLUSION: The improvement in EF with carvedilol therapy was proved to be accompanied by an improvement in neurohumoral factors. PMID- 10746814 TI - Effects of carvedilol on left ventricular regional wall motion in patients with heart failure caused by ischemic heart disease. Australia-New Zealand Heart Failure Research Collaborative Group. AB - BACKGROUND: Beta-blocker therapy has been shown to improve left ventricular (LV) ejection fraction and reduce LV volumes in patients with heart failure caused by ischemic heart disease. However, the possible mechanisms of this improvement and the effects of such treatment on regional wall motion have not been established. In a substudy of the Australia-New Zealand trial of carvedilol in patients with heart failure caused by ischemic heart disease, the effects of treatment on LV regional wall motion were assessed using 2-dimensional echocardiography. METHODS AND RESULTS: One hundred nineteen patients from 10 centers were included on this substudy. Patients were randomly assigned to treatment with carvedilol or placebo. Echocardiography was performed before randomization and after 6 and 12 months of treatment. LV regional wall motion was assessed using a semiquantitative scoring system. LV wall motion score index (WMSI) was reduced from 2.40 to 2.29 after 6 and 12 months in the carvedilol group and remained unchanged in the placebo group (2-tailed P = .005, carvedilol vs placebo). The percentage of myocardium with normal function also significantly improved with carvedilol treatment. CONCLUSIONS: Carvedilol improved LV regional WMSI in patients with heart failure caused by ischemic heart disease. These results indicate a mechanism by which beta-blocker therapy may benefit patients with heart failure and are consistent with an intrinsic improvement in LV function after treatment with carvedilol. PMID- 10746815 TI - Felodipine improves left ventricular emptying in patients with chronic heart failure: V-HeFT III echocardiographic substudy of multicenter reproducibility and detecting functional change. AB - BACKGROUND: The echocardiographic substudy of the Vasodilator-Heart Failure Trial III (V-HeFT III) aimed to determine if felodipine treatment in patients with heart failure who were taking an angiotensin-converting enzyme inhibitor had a favorable effect on left ventricular (LV) structure and function. Earlier V-HeFT trials showed that hydralazine-isosorbide dinitrate improved ejection fraction (EF) and survival, whereas enalapril achieved greater survival with smaller increases in EF. Would the combination of a potent vasodilator and enalapril produce greater improvements in function and survival? METHODS AND RESULTS: Doppler-echocardiographic data were collected from 260 males with heart failure who were randomized to felodipine or a placebo. Mean intrasubject differences between baseline, at 3 months, and at 12 months were compared. Intersite and intrareader reproducibilities were measured from duplicate recordings and readings. At 3 months, no changes in ultrasound variables from baseline occurred in either group. At 12 months, felodipine patients achieved greater increases in EF, shortening of LV end-systolic length, and increases in stroke volume index. Reproducibility coefficients of variation were 7.4% (EF), 6.0% (end-diastolic length), and 13.0% (stroke volume index). CONCLUSIONS: The echocardiographic substudy showed that felodipine, added to heart failure therapy, increased EF, shortened end-systolic length, and increased stroke volume index. The changes were small and confirmed that reproducibility from multiple laboratories can be coordinated into a useful research tool. PMID- 10746816 TI - Endothelial dysfunction in patients with heart failure: relationship to disease severity. AB - BACKGROUND: Heart failure is associated with abnormal endothelium-dependent vasodilation. However, the relationship of this abnormality to heart failure severity has not been well defined. METHODS AND RESULTS: We used strain-gauge plethysmography to assess forearm blood flow (FBF) responses to endothelium dependent, endothelium-independent, and reactive hyperemic stimuli in normal subjects (n = 29) and in patients with mild (n = 26) and severe (n = 41) heart failure. FBF responses to intra-arterial methacholine (0.3, 1.5, 3.0 microg/min) were significantly (P < .005) and similarly reduced in patients with mild (2.8 +/ 0.4, 5.9 +/- 0.7, and 7.7 +/- 1.1 mL/min/dL) and severe (2.7 +/- 0.4, 5.4 +/- 0.7, and 6.9 +/- 0.9) heart failure compared with normal subjects (4.5 +/- 0.4, 9.4 +/- 1.0, and 12.0 +/- 1.1). FBF responses to nitroprusside (1, 5, 10 microg/min) were significantly reduced in mild (2.4 +/- 0.3, 6.7 +/- 1.1, and 11.9 +/- 2.0, P < .05) and severe (1.9 +/- 0.2, 5.1 +/- 0.5, and 7.3 +/- 0.9, P < .001) heart failure groups compared with normal subjects (3.8 +/- 0.5, 10.8 +/- 1.2, and 14.9 +/- 1.2). However, FBF responses were reduced to a greater extent (P < .001) in mild heart failure compared with severe heart failure. Peak reactive hyperemia was significantly impaired only in severe heart failure. There was no correlation between methacholine responses and ejection fraction, maximum oxygen consumption, wedge pressure, or serum norepinephrine. CONCLUSION: Impaired endothelium-dependent vasodilation is present and near maximum in mild heart failure. Endothelial dysfunction may be an early finding in human heart failure. PMID- 10746817 TI - Is apoptosis present in progression to chronic hypertensive heart failure? AB - BACKGROUND: Cardiomyocyte apoptosis is believed to occur in hypertension. Isolated myocyte data from spontaneously hypertensive heart failure (SHHF) rats, however, suggest that significant myocyte loss does not occur in this model. To investigate this issue further, heart sections from failing and nonfailing SHHF rats were examined by using in situ terminal deoxynucleotidyltransferase-mediated 2'-deoxyuridine 5'-triphosphate nick end-labeling (TUNEL). Additional hearts were optimally fixed by perfusion with glutaraldehyde and histologically examined for evidence of myocyte damage or loss. METHODS AND RESULTS: Five Sprague-Dawley (SD) rats, 8 failing SHHF rats, and 6 nonfailing SHHF rats were perfusion-fixed with formaldehyde and used for TUNEL assay. Heart sections from each group were also treated with DNase for positive controls. There were no significant differences in the number of TUNEL-positive cells in SD, failing SHHF, and nonfailing SHHF rats. Additionally, extensive screening of 1-microm sections of optimally fixed failing hearts revealed little evidence of myocyte loss or nuclear characteristics suggestive of apoptosis. CONCLUSION: Apoptosis does not appear to be an important component of myocardial remodeling in SHHF rats during hypertrophy or end-stage heart failure. Examination of myocyte nuclear structure by high-resolution microscopy of optimally fixed tissues is recommended as an alternative approach to study apoptosis. PMID- 10746818 TI - Apoptosis in heart failure: is there light at the end of the tunnel (TUNEL)? PMID- 10746819 TI - Mitochondrial respiratory chain activity in idiopathic dilated cardiomyopathy. AB - BACKGROUND: Cardiomyopathy is well recognized in mitochondrial diseases in which it has been associated with defects of mitochondrial function, including cytochrome-c oxidase (COX) deficiencies. This study explores the respiratory chain activity, particularly of COX, in patients with cardiomyopathy to determine whether a relationship exists between respiratory enzyme activity and cardiac function. METHODS AND RESULTS: Myocardial specimens from the left ventricular wall of explanted hearts were obtained from subjects with ischemic (n = 6) or nonischemic dilated (n = 8) cardiomyopathy. Assays for citrate synthase (CS) and complexes II/III and IV activity were performed on cardiac mitochondria and homogenate. Enzyme activities were normalized to CS activity and compared with control activities (n = 10). A significant reduction in COX and/or CS activity was identified in mitochondrial preparations from the transplant group and correlated significantly with ejection fraction (P < .05), although this does not prove a causal relationship. Significantly reduced CS activity in homogenate was identified, suggesting decreased mitochondrial volume in addition to decreased COX activity. Measurements in cardiac homogenates failed to show a significant reduction in COX activity (P > .05) in the transplant group, suggesting that the use of prefrozen tissue homogenates may underestimate existing mitochondrial respiratory defects in cardiac tissue. CONCLUSIONS: Mitochondrial function is altered at a number of levels in end-stage cardiomyopathy. Defective COX activity resulting in deficient adenosine triphosphate generation may contribute to impaired ventricular function in heart failure. Agents capable of improving mitochondrial function may find an adjuvant role in the treatment of cardiac failure. PMID- 10746820 TI - Chronic endothelin blockade in dogs with pacing-induced heart failure: possible modulation of sympathoexcitation. AB - BACKGROUND: Endothelin-1 (ET-1) is a potent vasoconstrictor peptide elaborated by many cell types. Plasma ET-1 levels are significantly augmented in patients and experimental animals with heart failure. Enhanced levels of ET-1 may contribute to myocardial depression and alterations in sympathetic nerve activity in the setting of chronic heart failure. The effects of chronic blockade of endothelin A (ET(A)) receptors on the development and severity of experimental heart failure and sympathoexcitation were evaluated in these experiments using the specific ET(A) antagonist, PD156707. METHODS AND RESULTS: Four groups of conscious, chronically instrumented mongrel dogs were administered either PD156707 (750 mg orally thrice daily) or a placebo starting 1 day before ventricular pacing or a sham (nonpaced) period. Before pacing or the sham period, baseline hemodynamic and plasma norepinephrine (NE) measurements were made. Hemodynamic and NE measurements were made every 3 to 4 days for the next 28 days. All parameters were relatively stable in nonpaced dogs administered placebo. Paced placebo dogs showed classic hemodynamic and sympathoexcitatory changes indicative of heart failure. Nonpaced dogs administered PD156707 showed a significant decrease in mean arterial pressure and total peripheral resistance beginning 3 days after drug administration. Myocardial function was not affected by PD156707 in nonpaced dogs. In paced dogs, PD156707 also reduced arterial pressure and peripheral resistance. Changes in myocardial function were small and insignificant. Paced dogs administered PD156707 showed an approximately 50% lower increase in plasma NE level from days 10 to 24 compared with paced dogs administered placebo (941.8 +/- 122.8 vs 501.1 +/- 92.6 pg/mL at 17 days; P < .01). CONCLUSIONS: These data suggest that ET-1 contributes to the maintenance of arterial pressure in both sham dogs and dogs paced into heart failure. ET-1 does not appear to have a potent effect on inotropic state, but the data strongly suggest that ET-1 may contribute to the progressive deterioration of circulatory function in heart failure by mediating sympathoexcitation and enhancing plasma NE concentration. PMID- 10746821 TI - Increased brain angiotensin receptor in rats with chronic high-output heart failure. AB - BACKGROUND: The renin-angiotensin system (RAS) plays a key role in the pathophysiology of chronic heart failure (CHF). In rats, we reported that CHF enhances dipsogenic responses to centrally administered angiotensin I, and central inhibition of the angiotensin-converting enzyme (ACE) prevents cardiac hypertrophy in CHF. This suggests that the brain RAS is activated in CHF. To clarify the mechanism of the central RAS activation in CHF, we examined brain ACE and the angiotensin receptor (AT) among rats with CHF. METHODS AND RESULTS: We created high-output heart failure in 22 male Sprague-Dawley rats by aortocaval shunt. Four weeks after surgery, we examined ACE mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) and AT by binding autoradiography. ACE mRNA levels were not significantly increased in the subfornical organ (SFO), the hypothalamus, or in the lower brainstem of CHF rats (n = 5) compared with sham operated rats (SHM) (n = 6). Binding densities for type 1 AT (AT1) in the SFO (P < .05), paraventricular hypothalamic nuclei (P < .05), and solitary tract nuclei (P < .05) were higher in rats with CHF (n = 5) than in SHM rats (n = 6). Thus, in rats with CHF, AT1 expression is increased in brain regions that are closely related to water intake, vasopressin release, and hemodynamic regulation. CONCLUSIONS: The fact that AT1 expression was upregulated in important brain regions related to body fluid control in CHF rats indicates that the brain is a major site of RAS action in CHF rats and, therefore, a possible target site of ACE-inhibitors in the treatment of CHF. PMID- 10746822 TI - Rising levels of cardiovascular mortality in Mississippi, 1979-1995. AB - BACKGROUND: Cardiovascular disease rates are improving in the United States, but not for certain subgroups, especially some African Americans. The objective of the study is to assess current levels and trends in cardiovascular disease mortality in Mississippi. METHODS: Mortality statistics from the U.S. vital statistics system for the period 1979-95 were used. Comparison of age-adjusted mortality rates in Mississippi with the other states for the year 1995 and with the nation as a whole over the period of 1979-95 was performed. RESULTS: Mississippians had the highest age-adjusted cardiovascular disease morality rates in the nation in 1995. Overall, the cardiovascular rates in Mississippi were 37% higher than for the U.S. African American men and women from Mississippi had especially high cardiovascular mortality rates, approximately 50% and 70% higher than their white counterparts, respectively. The higher burden of cardiovascular disease in African Americans from Mississippi was especially marked in the younger age groups. Since about 1984-85, cardiovascular mortality rates in Mississippi have been increasing for African Americans, whereas nationally they have been decreasing. In contrast, cardiovascular mortality rates for whites in Mississippi have been declining, but at a much slower rate than seen nationally. The wide divergence in trends for African American and white men and women over that period in Mississippi has lead to an estimated 19,400 excess cardiovascular deaths. Virtually identical trends were found for heart disease. CONCLUSIONS: Cardiovascular diseases are a major public health problem in Mississippi that is especially severe in African American residents, and the problem is growing worse each year. It is important to identify the determinants of and solutions for this enormous public health problem in Mississippi. PMID- 10746823 TI - Transcriptional regulation of transforming growth factor beta1 by glucose: investigation into the role of the hexosamine biosynthesis pathway. AB - BACKGROUND: The hexosamine biosynthesis pathway (HBP) is hypothesized to mediate many of the adverse effects of hyperglycemia. We have shown previously that increased flux through this pathway leads to induction of the growth factor transforming growth factor-alpha (TGF-alpha) and to insulin resistance in cultured cells and transgenic mice. TGF-beta is regulated by glucose and is involved in the development of diabetic nephropathy. We therefore hypothesized that the HBP was involved in the regulation of TGF-beta by glucose in rat vascular and kidney cells. METHODS: A plasmid containing the promoter region of TGF-beta1 cloned upstream of the firefly luciferase gene was electroporated into rat aortic smooth muscle, mesangial, and proximal tubule cells. Luciferase activity was measured in cellular extracts from cells cultured in varying concentrations of glucose and glucosamine. RESULTS: Glucose treatment of all cultured cells led to a time- and dose-dependent stimulation in TGF-beta1 transcriptional activity, with high (20 mM) glucose causing a 1.4- to 2.0-fold increase. Glucose stimulation did not occur until after 12 hours and disappeared after 72 hours of treatment. Glucosamine was more potent than glucose, with 3 mM stimulating up to a 4-fold increase in TGFbeta1-transcriptional activity. The stimulatory effect of glucosamine was also dose-dependent but was slower to develop and longer lasting than that of glucose. CONCLUSIONS: The metabolism of glucose through the HBP mediates extracellular matrix production, possibly via the stimulation of TGF-beta in kidney cells. Hexosamine metabolism therefore, may play a role in the development of diabetic nephropathy. PMID- 10746824 TI - Comparative efficacy and potency of long-term therapy with glipizide or glyburide in patients with type 2 diabetes mellitus. AB - BACKGROUND: Long-term studies on the comparative efficacy and relative potency of glipizide and glyburide are sparse and controversial. METHODS: In a randomized prospective trial, we compared the effectiveness and relative potency of glipizide and glyburide over a 15-month period in 18 patients with type 2 diabetes mellitus (DM2) (9 on glyburide and 9 on glipizide) who were unresponsive to diet therapy. Glycemic control was assessed using 4 methods: 1) quarterly fasting plasma glucose (FPG), and 2-hour postprandial plasma glucose after a standard breakfast; 2) insulin and glucose response to Sustacal (test meal) challenge every 3 to 6 months; 3) quarterly hemoglobin A1c; and 4) intravenous glucose tolerance testing every 6 months to measure first and second phase insulin secretion. Patient characteristics were similar in each treatment group. RESULTS: Similar doses of glipizide (11 mg/day) or glyburide (10 mg/day) resulted in comparable reduction of FPG and hemoglobin A1c and increase in first phase insulin response to intravenous glucose tolerance testing. There was greater reduction in FPG and 2-hour postprandial plasma glucose with glipizide than with glyburide in 6 months. Contrary to the Physicians' Desk Reference, but consistent with another short-term study, our long-term study demonstrated that glipizide and glyburide are equipotent at similar doses in controlling hyperglycemia in DM2. CONCLUSIONS: Glipizide and glyburide are effective in controlling hyperglycemia with similar doses in DM2. Glipizide exhibits greater reduction in FPG and 2PPG at 6 months. Additional studies are needed to validate equipotency of these drugs. PMID- 10746825 TI - Biphasic changes in nitric oxide generation in hemodialyzed patients with end stage renal disease treated with recombinant human erythropoietin. AB - BACKGROUND: Use of recombinant human erythropoietin (rHuEpo) in patients with end stage renal disease (ESRD) improves anemia and reduces the need for blood transfusions. However, one third of patients on rHuEpo develop hypertension, aggravation of preexistent hypertension, or other complications. Nitric oxide (NO) plays a role in blood pressure (BP) regulation. Whether rHuEpo treatment in ESRD is accompanied by alterations in NO production was explored in patients undergoing hemodialysis. METHODS: Of 121 consecutive patients in a hemodialysis clinic, 107 were treated with rHuEpo and 14 were untreated. Plasma was collected before and after hemodialysis for quantification of nitrite and nitrate (NOx). Findings were correlated with various routinely monitored parameters. RESULTS: Predialysis NOx levels were lower in the treated than the untreated group; postdialysis NOx levels were virtually the same. Thus, the change was less in the treated group. Urea reduction ratios (URR) and ultrafiltrate volumes were similar. The mean predialysis systolic BP was higher in the treated group than in the untreated group. The dose of rHuEpo did not correlate with the plasma NOx or the predialysis BPs. No correlation was found between NOx levels and Hb or gender. Of the 107 treated patients, 12 had an increased postdialysis NOx without differences in ultrafiltrate volumes or URR. This group had higher total serum calcium levels, faster pulses, and greater BP reductions than other treated patients. No difference was found in the use of calcium-channel blockers and serum phosphorus and intact parathyroid hormone concentrations did not differ significantly among these groups. CONCLUSIONS: Intermittently hemodialyzed ESRD patients treated with rHuEpo accumulate less NOx in the plasma before dialysis but generate more NOx during dialysis than untreated patients. About 11% of treated patients generated excessive amounts of NOx, thereby maintaining plasma concentrations at the predialysis level or higher. This group experienced significant hemodynamic consequences characteristic of the excessive action of NO. PMID- 10746826 TI - Case records of the VA Maryland Health Care System/University of Maryland Medicine. A psychiatric clinicopathological conference. PMID- 10746827 TI - Syndromes of severe asthma. AB - Asthma responds to conventional therapy in the majority of patients. However, attention has recently focused on the 1 to 15% of asthmatics who are thought to manifest severe asthma, which responds poorly to commonly used regimens. In this review, current knowledge about the pathogenesis of severe asthma is reviewed and several distinct clinical syndromes of severe asthma are discussed. PMID- 10746828 TI - Effect of disease states on theophylline serum concentrations: are we still vigilant? AB - The use of theophylline has decreased over the past decade because of concerns over the risks of serious adverse effects as well as availability of more effective, safer drugs. Because of this decline in use, some clinicians may not be alert to the marked effect of some disease states on theophylline serum concentrations. The purpose of this review is to heighten awareness of the effect of decompensated heart failure, cor pulmonale, hepatic dysfunction, thyroid disease, and febrile illness on theophylline serum concentrations. Because many patients receive some benefit from this drug, safe use by clinicians requires closer monitoring of serum concentrations in patients with factors that alter theophylline clearance, including several disease states. PMID- 10746829 TI - Diffuse panbronchiolitis in Latin America. AB - Diffuse panbronchiolitis (DPB) is a unusual form of bronchiolar disease that has been reported almost exclusively in Asians. We describe DPB in a non-Asian Brazilian citizen who has never traveled outside the country. The clinical, radiographic, and histologic features of this case resemble those described in Japanese patients. The present case shows that DPB, although rare in Western countries, is not a disease restricted to Asia. It always should be considered in the differential diagnosis of nodular radiographic opacities associated with airflow limitation, especially in non-smokers with a history of chronic sinusitis. PMID- 10746831 TI - Anaerobic bacteremia in a neutropenic patient with oral mucositis. AB - An increasing number of anaerobic bloodstream infections in neutropenic cancer patients have been reported in the last decade. The type of anaerobes isolated from most of these patients suggests an oral source of infection. We describe a case of anaerobic bacteremia in a neutropenic patient with oral mucositis that highlights the importance of considering these organisms when selecting empiric prophylactic or therapeutic antimicrobial regimens, especially in the setting of periodontal disease or oral mucositis. PMID- 10746832 TI - Primary hyperparathyroidism in a twin pregnancy and review of fetal/maternal calcium homeostasis. AB - BACKGROUND: Hyperparathyroidism occurs rarely in pregnancy; this is the first reported case in a twin gestation. Management of this unusual case is described and an overview of fetal/maternal calcium homeostasis is discussed. METHODS: The patient presented at 33 weeks' gestation with hypertension and premature labor. Serum calcium and phosphorus were 14.6 and 1.7 mg/dL, respectively. An intact parathyroid hormone (PTH) level was 243 pg/mL (normal, 10-65). RESULTS: The patient was treated with parenteral saline hydration and oral phosphate supplementation that was continued through week 37. Although the calcium remained elevated between 12.6 and 13.3 mg/dL, medical therapy was continued because of the risks of surgery in the third trimester. Alternative medical treatments (bisphosphonates, calcitonin) were considered ill advised in pregnancy. The patient remained asymptomatic without further labor, and at week 37, fraternal twins were delivered by cesarean section. The infants were monitored closely and experienced no hypocalcemic symptoms after delivery. Postpartum, the mother's parathyroid scan and ultrasound were negative. She underwent neck exploration and a single 700-mg adenoma was removed. Transient asymptomatic hypocalcemia (7.5 mg/dL) occurred postoperatively, and she was placed on oral calcium (1500 mg/day) and calcitriol (0.25 mg/day). These were stopped at 8 weeks, when both PTH and parathyroid hormone-related peptide levels were normal. CONCLUSION: Mother and infants continue to do well after 18 months. This case provides an interesting setting to consider the interrelationships between elevated maternal PTH and the fetal/placental factors that regulate calcium metabolism in pregnancy. PMID- 10746830 TI - Spiral computerized tomography and video thoracoscopy in catamenial pneumothorax. AB - Catamenial pneumothorax is a rare disease that is usually diagnosed on clinical grounds. Delay in diagnosis can lead to considerable morbidity. We describe a case in which spiral CT scan and videothoracoscopy led to early objective diagnosis and management. PMID- 10746833 TI - Addison disease after appendicitis. AB - A 14-year-old boy manifested acute abdominal pain, vomiting, high temperature and diarrhea. He also underwent increasing hyponatremia and hyperkalemia after appendectomy. Further testing confirmed Addison disease. The serum adrenal antibody test was positive, and other autoimmune diseases were excluded. PMID- 10746834 TI - Acquired hemophilia masked by warfarin therapy. AB - People without hemophilia but with autoantibodies specifically directed against the procoagulant activity of factor VIII are known to have acquired hemophilia. The bleeding diathesis in these patients is often severe and life-threatening. The definite laboratory diagnosis of this disorder includes demonstration of low factor VIII levels in plasma with a high titer of factor VIII inhibitors, but the initial suspicion for its presence should rise in view of a prolonged partial thromboblastin time (PTT) and a normal prothrombin time associated with an acquired bleeding disorder. Oral anticoagulant treatment is known to prolong PTT as well, and the merger of these 2 situations may cause delayed diagnosis of acquired hemophilia with devastating consequences. We describe here the first reported case of acquired hemophilia diagnosed in a patient treated with warfarin. In such patients prolonged PTT may be ascribed to warfarin therapy rather than to acquired hemophilia, thus causing a dangerous delay in diagnosis. PMID- 10746835 TI - Possibilities of fertility preservation in children and young adults undergoing treatment for malignancy. PMID- 10746836 TI - Hormone replacement therapy and the skin: beneficial effects: the case in favor of it. PMID- 10746837 TI - Systemic estrogens have no conclusive beneficial effect on human skin connective tissue. AB - SUBJECT: Aging of the skin is affected by intrinsic and extrinsic factors. Especially important extrinsic factor is ultraviolet radiation, which causes premature aging of the skin. Intrinsic aging is influenced by genetic factors, and changes in hormones. In menopause, changes in hormonal balance have been suggested to enhance aging of the skin. DISCUSSION: Accordingly, several studies have been accomplished, in which hormone replacement therapy (HRT) has been used in order to prevent skin aging at or after the menopause. In this overview the results of previous studies, and a study done recently, are critically reviewed. CONCLUSION: One year systemic replacement therapy has no beneficial effect on the skin thickness, collagen synthesis, or elastin. Topical estrogens might have some beneficial effects. However, since the number of patients treated with topical estrogens is relatively small, further studies are needed. PMID- 10746838 TI - Magnetic resonance imaging compared to ultrasonography in fetal weight and volume estimation in diabetic and normal pregnancy. AB - OBJECTIVE: To estimate fetal volume and weight in diabetic and normal pregnancy using high-resolution magnetic resonance imaging. METHODS: T1-weighted magnetic resonance imaging was combined with semiautomatic segmentation technique. The accuracy of fetal volume estimations thus obtained was compared with conventional ultrasound-based weight estimations in ten pregnant women with insulin-dependent diabetes mellitus and ten women with normal pregnancy. Examinations were made within 48 hours before delivery. RESULTS: Ultrasound-based estimations of fetal weight showed a correlation rate of r=0.77 with the actual birth weights in the whole material, while volume determinations based on magnetic resonance imaging showed a significantly better correlation rate of r=0.95. Diabetic women did not differ from the normal pregnancy group with regard to birth weight or the accuracy of weight estimations. CONCLUSIONS: High-resolution magnetic resonance imaging combined with semiautomatic segmentation software was found to be accurate in determining fetal volume and, consequently, better than conventional ultrasound-based techniques in estimating fetal weight. The use of magnetic resonance imaging in fetal weight estimation may be recommended for clinical situations where an accurate weight estimate is considered essential. PMID- 10746839 TI - Oral guar gum, a gel-forming dietary fiber relieves pruritus in intrahepatic cholestasis of pregnancy. AB - BACKGROUND: Guar gum is a gel-forming fiber, which increases fecal elimination of bile acids. It may therefore be beneficial in the treatment of intrahepatic cholestasis of pregnancy. METHODS: Forty-eight patients with intrahepatic cholestasis of pregnancy were randomized double-blind to receive either guar gum or placebo until delivery. Serum bile acid concentration was measured. Pruritus was assessed by both the investigator and the patient. RESULTS: At baseline, the intensity of pruritus and the serum bile acid concentration were significantly related. Guar gum diminished or prevented worsening of pruritus, while in the placebo group pruritus was enhanced (p<0.05). In the placebo group serum bile acid concentration increased significantly, whereas in the guar gum group it remained unchanged (p<0.05 between the groups). Guar gum treatment-induced changes of the pruritus score and serum bile acid concentrations were significantly related (p<0.01). CONCLUSIONS: Guar gum relieved the intensity of pruritus without any side effects and prevented the rise in serum bile acid concentration in this placebo-controlled and double-blind study of patients with intrahepatic cholestasis of pregnancy. PMID- 10746840 TI - Emergency cerclage in the presence of protruding membranes: is pregnancy outcome predictable? AB - BACKGROUND: The aim of this retrospective study is to verify whether some maternal features are related to pregnancy outcome in cases of emergency mid trimester cerclage when membranes are protruding through the dilated cervix. METHODS: Between 1988 and 1996 twenty-three pregnant patients with dilated cervix and protruding membranes were treated with emergency cerclage. At the time of cerclage, gestational age ranged from 17 to 27 weeks (median 22). RESULTS: Pregnancy was prolonged from 0 to 20 weeks (median 4). Eleven living infants were born (46%); median gestational age at delivery was 25 weeks (range 21-39) and median birth weight 700 g (range 350-3980 g). Obstetric histories, white blood cell count, and vaginal-cervical and urine cultures obtained on admission were analyzed in the two following groups: data from patients with good pregnancy outcome (live births) versus those from patients with poor outcome (stillbirths and neonatal deaths). No significant difference was found between the groups for the above mentioned maternal features. CONCLUSIONS: The possibility of 46% live births is considered a good result for mid-trimester emergency cerclage when the membranes are protruding. Success of the procedure remains unpredictable on the basis of the maternal features investigated. PMID- 10746841 TI - Symphyseal distention in relation to serum relaxin levels and pelvic pain in pregnancy. AB - OBJECTIVE: To elucidate whether there is an association between symphyseal distention, circulating relaxin levels and pelvic pain in pregnancy. METHODS: Serum relaxin and symphyseal width were assessed in 19 consecutive referral cases with severe pelvic pain at 35 weeks of pregnancy and in a cohort of 49 women at 12 and 35 weeks of pregnancy. The referral cases were received over a period of two years and four months and the cohort was recruited over a period of two months. Relaxin was measured with an ELISA test and symphyseal width assessed using ultrasonography. All women with pelvic pain were clinically assessed. The women were divided into three groups; Group A (n= 38), cohort cases with no or mild pain; Group B (n= 11), cohort cases with disabling pain; and Group C (n= 19), referral cases. RESULTS: At 35 weeks of pregnancy, mean symphyseal width was 4.5 mm (s.d. 1.0 mm) in Group A, 5.7 mm (s.d. 2.6 mm) in Group B, and 7.4 mm (s.d. 3.5 mm) in Group C. The difference between Groups A and B is statistically significant (p=0.044) as is that between Groups A and C (p<0.0001). Serum relaxin levels were not associated with symphyseal distention or disabling pain. CONCLUSION: Severe pelvic pain during pregnancy was strongly associated with an increased symphyseal distention. However, the severity of pain did not predict the degree of symphyseal distention in the individual case, indicating that other mechanisms are also involved. Serum relaxin levels were not associated with the degree of symphyseal distention or with pelvic pain in pregnancy. PMID- 10746842 TI - Fetal erythropoietin and endothelin-1: relation to hypoxia and intrauterine growth retardation. AB - BACKGROUND: We have examined whether endothelin-1 (ET-1) and erythropoietin (EPO) in amniotic fluid, and EPO in fetal serum obtained by cordocentesis from fetuses with signs of intrauterine growth retardation (IUGR), were correlated to fetal growth and/or chronic fetal hypoxia. METHODS: Amniotic fluid and fetal serum were obtained by cordocentesis from 28 fetuses suspected to have IUGR and subsequently analyzed for EPO and ET-1 by ELISA. These data were correlated to blood gas results and fetal/maternal parameters at delivery. RESULTS: A novel finding was that ET-1 correlated to PO2 in amniotic fluid. The average level of ET-1 in amniotic fluid was 48.3+/-4.7 pmol/L. The results also showed a correlation between EPO levels in amniotic fluid and EPO in fetal serum. Furthermore, EPO correlated weakly to birth weight at delivery. Children with the lowest birth weights had the highest EPO levels. High EPO values, similarly to ET-1, correlated to low pO2 values. The level of EPO in amniotic fluid was 8.0+/-1.6 mIU/ml and in cord blood 29.5+/-9.6 mIU/ml. CONCLUSIONS: The results indicate that ET-1 levels may be a marker for short-term hypoxia, but not for fetal growth, since ET-1 in amniotic fluid was correlated to PO2 at the time of cordocentesis, but not to birth weight. The results also indicate that EPO levels in amniotic fluid and in fetal cord serum are highly correlated, and thus both can be used as markers for fetal growth and chronic hypoxia before the onset of labor. PMID- 10746843 TI - The effect of vaginal pH on the efficacy of vaginal misoprostol for induction of labor. AB - BACKGROUND: Misoprostol was reported to be an effective agent for cervical ripening and induction of labor. Our purpose was to evaluate whether vaginal pH affected the efficacy of misoprostol for induction of labor. METHODS: The vaginal pH of 103 women admitted for induction of labor were measured. According to the vaginal pH, two groups were generated, those with a vaginal pH <5 (n= 65), and those with a vaginal pH > or =5 (n=38). All women received intravaginal misoprostol tablets, 50 microg every 4 hours up to three doses. Further medication was not given after entry into active labor or spontaneous rupture of membranes. In cases of failed induction or arrest disorders oxytocin augmentation was used. RESULTS: The average interval from start of induction to vaginal delivery was shorter, and oxytocin augmentation was required less commonly in the lower pH group. We did not find any significant difference in cesarean section rates, or incidence of adverse maternal or fetal outcome. CONCLUSION: Vaginal pH may affect the pharmacokinetics of vaginally administered misoprostol, and may cause an alteration in induction to delivery interval. PMID- 10746844 TI - Factors associated with climacteric symptoms and the use of hormone replacement therapy. AB - BACKGROUND: To investigate factors associated with climacteric symptoms and the use of hormone replacement therapy (HRT). METHODS: A random sample of women aged 46, 50, 54, 58 and 62 years resident in Goteborg, Sweden (n=5,990) were invited by letter to complete a postal questionnaire concerning the menopause and HRT use. RESULTS: The response rate was 76% (n=4,504). Women with a higher education, who exercised regularly and who had regular spare-time activities felt better and had fewer climacteric complaints (p<0.001) compared to lower educated women, women who exercised infrequently and women who had no spare-time activities. Climacteric symptoms were associated with dryness symptoms (dry eyes, skin, hair and mouth), bilateral salpingo-oophorectomy (SOE), use of psychotropical drugs and use of alternative remedies, previous hysterectomy and full-time occupation. Stepwise multiple regression analysis showed significant correlations between vasomotor symptoms and various dryness symptoms, no menstrual periods and use of alternative remedies. Current HRT-users (13%) had used estrogens for 4.0 years and past users (14%) for 4.3 years. A correlation (p<0.005) was found between HRT use and previous use of contraceptive pills, use of alternative remedies and hysterectomy. Education, occupation and the frequency of exercise did not influence the prevalence of HRT use. CONCLUSIONS: This study has shown that the experience of climacteric symptoms and well-being is associated with many different factors. Women with a higher education and those who exercised regularly were more often symptom-free. HRT use was associated with previous use of contraceptive pills, use of alternative remedies and hysterectomy. PMID- 10746845 TI - Comparison of usefulness of estradiol vaginal tablets and estriol vagitories for treatment of vaginal atrophy. AB - BACKGROUND: Atrophic vaginitis is a common condition. This study compared the usefulness of estradiol vaginal tablets (EVT) and estriol vagitories (EV) in treatment of atrophic vaginitis. METHODS: Ninety-six postmenopausal women with symptoms of atrophic vaginitis were treated for 24 weeks with either EVT or with EV. Patients used the medication daily for the first 2 weeks of the study, and twice-weekly thereafter. RESULTS: Both EVT and EV were effective in treating vaginal atrophy and patients in both treatment groups experienced a significant improvement in vaginal symptoms such as itching, irritation, dryness, and dyspareunia. At the end of the study three (6%) EVT treated women reported leakage and none needed to use sanitary towels. Among the EV treated women 31 (65%) reported leakage and 14 (29%) required sanitary protection. Furthermore, 90% in the EVT group perceived the medication as hygienic compared to 79% in the EV group, and 49% in the EVT group indicated that the product was easy to use compared to 28% in the EV group. Endometrial thickness was increased (1.1 mm with EVT and 0.5 mm on EV) in both treatment groups during the first 2 weeks of the study, but returned to baseline levels when the frequency of drug application was reduced to twice-weekly. CONCLUSIONS: Estradiol vaginal tablets provides an effective alternative to traditional forms of local estrogen therapy. PMID- 10746846 TI - The prevalence and bothersomeness of lower urinary tract symptoms in women 40-60 years of age. AB - AIM: To assess the prevalence and bothersomeness of lower urinary tract symptoms in women aged 4060 years. Study design. Ongoing longitudinal cohort study. METHODS: Four thousand women recruited on a random basis from the Civil Registration System, in one rural and one urban county in Denmark, were asked to fill in a self-administered, validated questionnaire on lower urinary tract symptoms. Logistic regression analysis was performed to assess the relationship between LUTS, bothersomeness, age, and county residency. Symptom scores and bother scores were compared in order to obtain a valid measure of lower urinary tract symptoms (LUTS). RESULTS: The prevalence of LUTS occurring more than weekly was 27.8% (95% CI: 26.2%-29.4%) and 16.1% (95% CI: 14.8-17.4) had urinary incontinence. The prevalence of stress incontinence increased from at 40 years up to the age of 55 years (OR=1.9 (95% CI: 1.3-2.7)) and declined thereafter. Irritative symptoms such as urge incontinence and urgency steadily escalated in an almost linear fashion with increasing age ((OR=2.7 (95% CI: 1.6-4.5) and OR=2.1 (95% CI: 1.5-2.8), respectively). Incontinence symptoms were the most bothersome. Age was positively associated with most LUTS, but not with bothersomeness. County residency was not associated with LUTS. CONCLUSION: Women aged 40-60 years frequently have bothersome lower urinary tract symptoms. Age, but not county residency, is an important factor associated with the occurrence of lower urinary tract symptoms in perimenopausal women. LUTS occurring more often than weekly seem to be the most appropriate single measure of LUTS. PMID- 10746847 TI - Cervical intraepithelial neoplasia in pregnancy. AB - OBJECTIVE: To determine the progression/regression rate of cervical intraepithelial neoplasia in pregnancy and to describe the number of patients requiring treatment for cervical neoplasia during or following the pregnancy. METHODS: A retrospective analysis of 305 pregnant women with abnormal cervical cytology was performed. The colposcopic, cytologic and histologic findings of repeated examinations during pregnancy and of the subsequent examination eight weeks postpartum were registered and compared. All smears were obtained by cotton bud and Cytobrush. Colposcopy was performed using standard techniques and cervical biopsies were taken in case of colposcopic abnormalities. Endocervical curettage was omitted during pregnancy. At postpartum evaluation colposcopy, directed biopsies and endocervical curettage were performed in all cases. RESULTS: One hundred and two patients (33%) were followed only by cytology and colposcopy. The remaining 203 patients (67%) had one to four colposcopically directed biopsies during the pregnancy. Comparing the initial histology in pregnancy to the postpartum histologic evaluation 25% showed spontaneous regression while 75% of the women exhibited progression (28%) or persistence (47%) in the severity of cervical neoplasia. Two patients were treated by cervical conization in early pregnancy and 143 women (53%) were treated within the first year after the pregnancy. In the postpartum period microinvasive carcinoma was diagnosed in two patients, but no women advanced to more serious stages of cervical cancer. CONCLUSIONS: The high persistence rate of cervical intraepithelial neoplasia in pregnancy leads us to recommend a liberal use of colposcopically directed biopsies during pregnancy and to ensure a high follow-up rate in the postpartum period. PMID- 10746848 TI - Prognostic significance of serous and clear cell adenocarcinoma in surgically staged endometrial carcinoma. AB - BACKGROUND: The serous adenocarcinoma (SA) and clear cell adenocarcinoma (CCA) of endometrium have been shown to be associated with high relapse rate and poor survival. It is not clear whether prognostic significance of these specific cell types of tumor is independent of retroperitoneal lymph node metastasis and other histopathologic prognostic factors in endometrial carcinoma. METHODS: We examined 240 consecutive patients with clinical stage I to stage III endometrial carcinoma who were treated prospectively with radical surgery and/or platinum-based chemotherapy. Surgery included extended hysterectomy, bilateral salpingo oophorectomy, and systematic pelvic and paraaortic lymph node dissection. Prognostic significance of various histopathologic factors was determined by Cox regression analysis. RESULTS: SA/CCA were more frequently associated with deep myometrial invasion, high nuclear grade (G3), lymph-vascular space invasion (LVSI), and pelvic lymph node metastasis when compared to endometrioid adenocarcinoma (EMA). Of 216 clinically staged stage I or II disease, seven of 12 cases of SA/CCA had extrauterine disease. This incidence was much higher than that for EMA (46/204) (p<0.01). A multivariate Cox regression analysis revealed that cell type, grade, LVSI, and paraaortic node metastasis (PANM) were independent prognosticators. CONCLUSIONS: Prognosis of patients with endometrial carcinoma depends on cell type, grade, LVSI, and PANM. Poor prognosis for patients with SA/CCA is independent of lymph node metastasis and other histopathologic prognostic factors. The SA/CCA should be strictly discriminated from EMA. PMID- 10746849 TI - The risk of premalignant and malignant pathology in endometrial polyps. AB - OBJECTIVE: To evaluate the risk of premalignant and malignant pathology among endometrial polyps. DESIGN: Prospective cohort study. SETTING: Minimal Access Surgical Training (MAST) center in a large teaching hospital. METHODS: Among 248 patients seen in outpatient hysteroscopy clinic (1996-97), 62 had endometrial polyps. All patients had endometrial sampling for histological assessment. To determine the magnitude of malignant potential among polyps, we compared the pathological findings in polyps (cases) with non-polypoidal specimens (controls). RESULTS: Out of 62 polyps, histologically 53 (85.5%) were benign, seven (11.3%) had hyperplasia, and two (3.2%) were associated with malignancy. Hyperplasia was more frequent in endometrial specimens with polyps than in those without (11.3% vs 4.3%, p=0.04), but the incidence of carcinoma in the two groups was the same (3.2% vs 3.2%, p= 1.0). CONCLUSION: In abnormal uterine bleeding, hyperplasia was, but cancer was not, more common in women with endometrial polyps compared to those without polyps. PMID- 10746850 TI - Saline infusion sonohysterography in endometrial cancer: assessment of malignant cells dissemination risk. AB - BACKGROUND: To assess the risk of malignant cells dissemination in patients with endometrial cancer undergoing saline infusion sonohysterography (SIS). METHODS: Fourteen consecutive patients (mean age: 58.1 years, range: 41-74 years) diagnosed as having a clinical stage I endometrial carcinoma and scheduled for surgical staging were entered in this study. SIS was performed at the time of laparotomy just when the abdomen was opened and prior to the start of the surgical procedure. 10-20 mL of saline was infused. All fluid spilled from the fimbrial ends of the fallopian tubes was collected and analyzed for the presence or absence of malignant endometrial cells. Samplings were also taken from ascites, if found, or cytological washings, prior to and after SIS. RESULTS: SIS could be satisfactorily performed in all patients. There was no spillage from either tube in nine patients. Mean fluid volume obtained from the other five patients was 4.4 mL. Cytologic analysis revealed the presence of malignant cells in the spilled fluid in one case (7.1%, one out of fourteen). CONCLUSIONS: Our data show that a small but real risk of malignant cell dissemination exists in patients with endometrial carcinoma who undergo SIS. PMID- 10746851 TI - Pregnancy outcome with intrahepatic cholestasis. PMID- 10746852 TI - Modifications to the Misgav Ladach technique for cesarean section. PMID- 10746853 TI - The alloimmune response and effector mechanisms of allograft rejection. AB - Allograft rejection is the consequence of the recipient's alloimmune response to nonself antigens expressed by donor tissues. Allospecific T lymphocytes are activated by donor peptides which are presented in the context of major histocompatibility complex molecules by either donor or recipient antigen presenting cells. Antigen presenting cells also provide essential costimulatory signals which are required for T cell proliferation and differentiation into either helper or effector lymphocytes. Effector mechanisms of allograft rejection include those mediated by cytotoxic T lymphocytes, macrophages, natural killer cells, and B lymphocytes. Importantly, alloimmune responses are controlled by regulatory molecules which include membrane receptors and cytokines. Novel insights into the interactions between antigen presenting cells and T lymphocytes, and further understanding of how alloimmune responses are regulated, will help in developing effective antirejection and tolerance-inducing strategies. PMID- 10746854 TI - Prospects and applicability of molecular diagnosis of allograft rejection. AB - We believe that changes in blood urea nitrogen and plasma creatinine are, at best, surrogate markers for rejection. It seems certain that rejection, an immunologic process must precede deterioration in renal function. We suggest that the efforts to prevent chronic rejection should include an effective means to diagnose rejection before the advent of fixed renal injury. Through an analysis of transcription for a series of genes that are expressed by activated, but not resting, cytotoxic lymphocytes, eg, granzyme B, a remarkably accurate tool for assessing the presence of rejection has emerged. Although our methods first centered on analysis of kidney transplant biopsy specimens, noninvasive surveillance using transcriptional profiling analysis circulating blood or urine sediment cells has proven equally informative. PMID- 10746855 TI - Immunosuppressive agents in organ transplantation: past, present, and future. AB - The development of immunosuppressive agents reflects the progress in understanding the cellular and molecular mechanisms which mediate allograft rejection. Six paradigms represent the evolution of immunosuppressive strategies for organ transplantation. The proliferation paradigm advances agents which interrupt lymphocyte cell division (azathioprine, cyclophosphamide, mycophenolic acid). The depletion paradigm conscripts drugs that bind to lymphocyte cell surface markers, thereby producing cell lysis and/or inactivation (polyclonal ATGAM and thymoglobulin, and monoclonal OKT3 antilymphocyte antibodies). The cytokine paradigm uses agents that interrupt lymphocyte maturational events; eg, synthesis (calcineurin inhibitors: cyclosporine/tacrolimus), binding to surface receptors (anti-CD25 mAbs), or signal transduction phases of cytokine stimulation (sirolimus). The introduction of calcineurin inhibitors markedly reduces the rate of acute rejection episodes and increases short-term graft survival rates; nephrotoxicity and chronic allograft attrition remain as unanswered challenges. The cyclosporine A (CsA) sparing property of sirolimus permits the use of lower exposure to calcineurin agents, allows for early withdrawal of steroid therapy, and may delay allograft senescence. Furthermore, the combination of SRL with anti IL-2R mAbs proffers an induction approach which allows prolonged periods of holiday from calcineurin inhibitors. To address the tissue nonselectivity of the calcineurin and mTOR inhibitors, which presumably causes the drug toxicities, new agents are being developed to selectively inhibit the T cell target Janus Kinase 3. In the costimulation paradigm, the accessory signals generated by antigen presenting cells are interrupted by distinct agents: the receptor conjugate CTLA4 immunoglobulin and anti-B7 or anti-CD40 ligand mAbs. Another set of drugs (selectin blocking agents, anti-ICAM-1 antisense deoxy oligonucleotides, and the lymphocyte homing inhibitor FTY720) seeks to modulate the ischemia-reperfusion injury, which exacerbates cytokine-mediated events in the donor and the subsequent procurement injury and may also accelerate the progression of transplant senescence. Finally, the transplantation tolerance paradigm is based on the development of strategies which distort alloimmune recognition by antigen reactive cells (MHC peptides or proteins), produce anergy (costimulation blockers), functional inactivation, or deletion of antigen-reactive cells (donor bone marrow infusions and gene therapy). Presently, the optimal immunosuppressive strategy uses combinations of agents that act in synergistic fashion to provide the potency, freedom from toxic reactions, convenience of administration, and cost appropriate for the individual patient. PMID- 10746856 TI - Chronic allograft dysfunction: mechanisms and new approaches to therapy. AB - Renal allograft failure is the most common cause of end-stage renal disease beyond the early posttransplantation period, accounting for 25% to 30% of patients awaiting renal transplantation. Despite recent advances in immunosuppressive therapy, improvements in long-term graft survival have not been commensurate with those observed in 1-year graft survival. The most common cause of chronic allograft loss is an incompletely understood clinicopathological entity sometimes called chronic rejection, chronic allograft dysfunction or in the case of kidneys, chronic allograft nephropathy. Although the precise mechanism(s) responsible for the characteristic pathological changes are still unclear, it is generally agreed that both alloantigen-dependent and alloantigen independent factors influence the development of chronic allograft nephropathy. This article will address the potential mechanisms responsible for the pathogenesis of chronic dysfunction in solid organ grafts and the current approaches to management, including newer therapies designed to prevent the progression of the disease. PMID- 10746857 TI - Recent advances in the diagnosis and management of infection in the organ transplant recipient. AB - Infection remains a significant cause of morbidity and mortality in organ transplant patients, with significant infection being found in more than half of these individuals posttransplant. The most important principles of patient treatment are prevention, early diagnosis, and specific therapy. The nature of the antimicrobial therapy required both for infection prevention and treatment is closely linked to the immunosuppressive therapy being administered. A particular challenge in the transplant patient is that the antiinflammatory effects of antirejection therapy tend to obscure the manifestations of infection until relatively late in the disease process, thus putting particular emphasis on more aggressive diagnostic approaches-imaging procedures, biopsy, and new techniques for microbial detection (antigen and DNA detection). Antimicrobial therapy can be administered in three ways: therapeutically, prophylactically, and preemptively. Particularly given the propensity for adverse interactions between antimicrobial agents and cyclosporine and tacrolimus, there is a particular emphasis on prophylactic and preemptive use of antimicrobials. PMID- 10746858 TI - Recurrent and de novo glomerulonephritis in the renal allograft. AB - Glomerulonephritis is a major cause of end-stage renal disease (ESRD) and is the primary disease in over a third of patients undergoing renal transplantation. An understanding of the incidence and clinical significance of recurrent glomerulonephritis posttransplantation is therefore essential. Indeed, all forms of glomerulonephritis have been reported to recur histologically after renal transplantation, but the incidence and severity of clinical recurrence varies greatly according to the type of glomerular disease. Large registries report that between 5% to 10% of allografts fail secondary to recurrence of primary disease. This review attempts to clarify some of the salient clinical features of recurrent glomerulonephritis from a body of literature full of anecdotal reports and retrospective studies. There are several excellent reviews on recurrent glomerulonephritis; this review aims to complement these by focusing on more recent developments in the field. Development of de novo glomerulonephritis in the transplant will also be discussed. The recurrence of metabolic diseases such as diabetes mellitus, cystinosis, Fabry's disease, and primary hyperoxaluria will not be discussed. PMID- 10746859 TI - Cardiovascular disease after renal transplantation. AB - Cardiovascular disease (CVD) is common after renal transplantation. In the absence of controlled intervention trials, the strength of evidence that modifying a risk factor will reduce the incidence of CVD in renal transplant recipients must rest on: (1) evidence from studies in the general population, (2) observational studies linking the risk factor to CVD in renal transplant recipients, and (3) studies showing that the risk factor can be safely and effectively treated in transplant patients. Accordingly, the evidence is strong that hyperlipidemia should be treated after renal transplantation. Evidence is very suggestive that pretransplant screening for CVD, treatment of hypertension, the use of low-dose aspirin, and smoking cessation will also help to reduce the incidence of posttransplant CVD. Less compelling are data suggesting that intensive glucose control in diabetics will safely decrease the incidence of CVD. Although there is little evidence that treatment of erythrocytosis will reduce CVD, hematocrits above 55% to 60% should probably be treated to prevent venous thrombosis. Vitamins for reducing homocysteine, antioxidant vitamins, and prophylaxis for potentially atherogenic infections are therapies that warrant additional study. In summary, the best current approach to reducing the high incidence of posttransplant CVD is to aggressively identify, and then systematically treat modifiable risk factors. PMID- 10746860 TI - Kidney-pancreas transplantation for diabetic nephropathy. AB - Pancreas transplantation is being performed with increasing frequency and increasing technical success. The availability of new immunosuppressant agents has been associated with a reduction in the previously high rates of allograft rejection in recipients of simultaneous pancreas-kidney transplants. These lower rejection rates have, in turn, led to changes in surgical techniques and a resurgence of interest in isolated pancreas transplantation--either in nonuremic patients or, more commonly, in patients who have already received a prior kidney transplant. Pancreas transplantation has emerged as an important option for the management of patients with type I diabetes mellitus and diabetic nephropathy. PMID- 10746861 TI - Challenges after pediatric transplantation. AB - In this review we address the current challenges facing the pediatric transplant caregiver. We focus most of our discussion on recent developments that have resulted in improved graft survival in renal allograft recipients. We discuss the issue of growth failure posttransplant and the realization that it is time to reassess strategies that optimize the immunosuppression and growth after pediatric transplantation. Lastly, we discuss some recent findings suggesting that these issues are also of critical importance for success after pediatric liver transplantation. PMID- 10746862 TI - Transplantation tolerance: mechanisms and strategies? AB - The goal of transplantation is to induce tolerance to the transplanted tissue. We believe that this will only be possible by exploring strategies normally used to endure and maintain self-tolerance. Tolerance can be defined as a state where the immune system does not respond to a specific antigen. This is in sharp contrast with immunosuppression, which decreases the immune response to a myriad of antigens and requires continued medication. T cells play an essential role in the immune response to alloantigens, because animals devoid of T cells do not reject transplanted organs. In this article we will focus on the mechanism of T cell tolerance and how the immune system may be manipulated to achieve tolerance to alloantigens. PMID- 10746863 TI - Xenotransplantation: is this the future? AB - The modern era of xenotransplantation, ie, transplantation across species, involving use of genetically modified pigs as potential organ and tissue donors for human recipients, is now more than 5 years old but remains essentially an experimental and controversial procedure. The status of the field is reviewed with regard to immunologic barriers, especially delayed xenograft rejection, infectious risks, biocompatibility issues, and associated problems. The case is made that robust clinical application is still at least a decade away although in the interim knowledge developing from the experimental approaches under investigation is proving of unexpected use in several areas of medicine. PMID- 10746864 TI - High-affinity sites for beta-D-galactosidase on membrane-bound vesicles isolated from rat epididymal fluid. AB - Glycosidases in rat epididymal fluid are secreted under androgen stimulation and possess receptors on the sperm surface. One of these enzymes, beta-D galactosidase (gal), was found in the epididymal fluid as a soluble enzyme and also in a heterogeneous population of membrane bound vesicles (mbv). beta-D Galactosidase was specifically localized to a subpopulation of larger, electron dense mbv. The aim of this study was to analyze the high-affinity sites for gal on the membrane of mbv using two different methods: classical fluorometric assay (used in previous papers) and colloidal gold (20 nm) conjugated to gal as a marker in ultrastructural studies. beta-D-Galactosidase bound to mbv with high affinity (Kd in a nanomolar range) are in a saturable form. Furthermore, 25 mM fructose-1,6-diphosphate (f-1,6-dip), a sugar that competes for the binding site, showed 50% inhibition of the binding. The gold conjugates were mostly observed on the surface of the large, electron-dense mbv but not on the small, electron lucent mbv. Gold particles were also observed on the larger vesicles, but less frequently in the presence of f-1.6-dip. Larger mbv possesses high-affinity sites for gal on their membrane. PMID- 10746865 TI - Potassium increases intracellular calcium simulating progesterone action in human sperm. AB - Progesterone (P) and zona pellucida are known to induce acrosome reaction in human sperm by increasing cytosolic calcium. High concentrations of potassium ions (K+) improve the rate of acrosome reaction in human sperm in vitro. This article determined whether the effect of K+ on the acrosome in human sperm is mediated by increasing intracellular calcium ([Ca2+]i). The effect of K+ on [Ca2+]i was examined by using Fura 2 as the fluorescent indicator. The effect of K+ and P on [Ca2+]i in sperm and the involvement of ion channels was compared. Motile sperm were collected by the swim-up method from semen of healthy volunteers and capacitated overnight in BWW containing 0.5% BSA. Incubation of capacitated sperm with different concentrations of potassium chloride (1.25-20 mM) resulted in dose-dependent increase in [Ca2+]i similar to that observed with P. The increase in [Ca2+]i by K+ and P was blocked by the addition of EGTA, a Ca2+ chelator. K+-induced change in [Ca2+] was not altered by the addition of dihydropyridine derivatives. The combined treatment of K+ (20 mM) and P (0.75 microg/mL) caused an additive effect on the increase in [Ca2+]i. It would appear that human sperm plasma membrane possess different Ca2+ channels responsive to P and K+. PMID- 10746866 TI - Testicular peritubular myoid cells are a target for adrenomedullin. AB - Adrenomedullin (AM) is a recently cloned vasorelaxing peptide that belongs to the calcitonin gene-related peptide family. AM inhibits the contraction of several types of smooth muscle cells and is present in the testis as well as in many other organs. The authors investigated whether testicular peritubular myoid cells (PMC) possess specific receptors for AM. Binding of AM to PMC was saturable in a time-dependent manner and 125I-AM binding was effectively displaced by cold AM. The study documents that testicular peritubular myoid cells are a target for adrenomedullin and suggests a role for this peptide in the paracrine regulation of the testis. PMID- 10746867 TI - Correlation of the bovine cervical mucus penetration test with human sperm characterisitics in 1,406 ejaculates. AB - The bovine cervical mucus penetration test (BCMPT) was performed to determine its usefulness in screening the ability of sperm to successfully penetrate mucus in vitro. Ejaculates were obtained by masturbation from patients attending an infertility clinic. Routine semen analysis was performed using a microcomputerized multiple-exposure photography system. The BCMPT was performed. Overall, the average penetration of the mucus was 38 +/- 0.46 mm. Of the 1,406 ejaculates analyzed, 244 (17%) displayed a negative result (0-20 mm), 291 (21%) a questionable result (21-30 mm), and 871 (62%) a positive result (>30 mm). A highly significant (p < .001) correlation between mucus penetration distance and sperm MD (r = 0.541), MI (r = 0.484), count (r = 0.475), motility (r = 0.448), velocity (r = 0.400) and morphology (r = 0.369) was observed. Overall, the finding of an abnormal semen parameter resulted in a 34 +/- 5% accurate prediction of a negative or questionable BCMPT (<30 mm), while a normal semen parameter resulted in a 90 +/- 4% accurate prediction of a positive BCMPT (>30 mm). Sperm MD showed the strongest positive predictive value (98%), while morphology showed the greatest negative predictive value (50%). Of the 1,406 samples, 25 +/- 2% of the samples with normal semen parameters displayed a negative BCMPT. Conversely, 6 +/- 2% of samples with abnormal parameters showed a positive BCMPT. The BCMPT successfully identifies a significant subpopulation of patients as having an inadequate penetration of mucus with otherwise normal semen characteristics. PMID- 10746868 TI - Functional activity of mouse sperm was not affected by low doses of aspirin-like drugs. AB - To investigate some possible effects of low doses of nonsteroidal anti inflammatory drugs upon functional activity of mouse sperm, the authors injected lysine acetyl salicylate (im 14.3 mg/kg day(-1), ibuprofen (ip 5.6 mg/kg day( 1)), or piroxicam (ip 0.28 mg/kg day(-1) to pregnant females (the male cohort was sacrificed at adulthood) (A) or to adult males during 35 (B) or 60 (C) days. Parameters measured were motility, viability, acrosomal integrity, responses to hypoosmotic shock, in vitro fertilization index, and testosterone plasma levels. Salicylate evoked a slight reduction in the percentage of swollen gametes in A, and ibuprofen diminished testosterone plasma levels in B. The other parameters remained unchanged in all groups. Results are well supported by the low doses assayed, which are equivalent to the content of one tablet commercially available for each compound. PMID- 10746869 TI - The use of ebastine, a mast cell blocker, for treatment of oligozoospermia. AB - The effect of mast cell blocker (ebastine) on semen quality was evaluated in 15 idiopathic oligozoospermic males. Ten milligrams/day of ebastine was administered orally for 3 months. Nine cases (66.7%) showed definite improvement in the semen quality. The wives of 3 patients (20%) became pregnant within 6 months of the initial treatment. It would appear that mast cell blocker (ebastine) significantly improves semen quality in men with idiopathic oligozoospermia. PMID- 10746870 TI - Localization of the sex-determining region-Y gene in XX males. AB - Localization of the sex-determining region Y (SRY) was investigated in 2 XX males. Metaphase chromosomes from peripheral lymphocytes were stained by fluorescence in situ hybridization using DXZ1 and SRY probes. An identical hybridization signal with the SRY probe was found on an X chromosome in both cases. The karyotype of the 2 cases was 46,XX, t(X;Y)(p22.3;p11.3). It would appear that XX male is the presence of a Y-chromosome fragment transferred to the X-chromosome short arm by unequal interchange between homologous regions in the short arms of sex chromosomes. PMID- 10746871 TI - PCR analysis of Y-chromosome sequences in a 45,X male patient and a review of the literature. AB - The 45,X karyotype is usually associated with Turner syndrome, while male phenotype is exceptional. The authors report a 45,X male patient with normal external genitalia and sex behavior, but who was azoospermic. He had a normally developed musculature and pilose distribution, testicular volume of 15 mL and no gynecomastia but clinical stigmata of Turner syndrome (short stature, short neck and 4th metacarpal bones) and azoospermia. Hormonal plasma levels of testosterone, estradiol, prolactin, and gonadotrophins were within the normal range as was the response of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (30 and 60 min) after 100 microg iv of LH-RH administration. Testicular biopsy could not be performed. Karyotype was 45,X without evidence of mosaicism. Polymerase chain reaction of genomnic DNA studied with 12 different sequences of Y chromosome revealed only the presence of SRY gene (testis determining factor). It is possible that SRY/autosomal translocation had occurred in this patient. The study of 45,X male should be of great value in elucidating the complex mechanisms involved in normal male sex differentiation. PMID- 10746872 TI - Stimulation and scavestrogen-induced inhibition of reactive oxygen species generated by rat sertoli cells. AB - The ability of Sertoli cells harvested from 18-day-old Sprague-Dawley rats to generate reactive oxygen species (ROS) occurs under unstimulated and stimulated conditions. Thus, the generation of ROS and its regulation by stimulating and inhibiting compounds was determined as a lucigenin-dependent chemiluminescence reaction. According to the data, ROS generation was influenced by different cell preparation conditions--stimulating substances such as PMA, FMLP, C5a, A23187, and scavestrogens characterized by antioxidant, radical-scavenging properties. The mechanical homogenization during cell preparation procedures leads to an increase of ROS generation. ROS generation of Sertoli cells was followed by elected substances in the following rank order of efficacy: PMA > FMLP > or = C5a > Ca-ionophore A23187. The registered inhibiting effects of the scavestrogens J811 and J861 were significant. The measured CL counts decreased at 72 and 77%, respectively, of control experiments done without scavestrogens. The generation of reactive oxygen species in Sertoli cells and especially the increase in oxygen free radicals and their effects on cellular structures of spermatids are directly involved in inducing morphological alterations. Sertoli cells play an important role in spermatogenesis. The measurements of ROS may have clinical relevance in the evaluation of infertility. PMID- 10746873 TI - Liquids storage (5 degrees C) of ram semen in different diluents. AB - This study examined the effects of three diluents--skim milk (M), TesT (TE) and Tris-trehalose (TR)--on quality of ram semen stored at 5 degrees C over a long term. Percentage of motile spermatozoa (PM), motility score (MS), percentage of intact acrosomes (PIA), and percentage of swollen spermatozoa under hypoosmotic conditions (PS) were assessed after cooling and every 24 h up to day 16. There was a significant effect (p < .01) of diluent type in the conservation rates after cooling for PM. The TR diluent achieved greater values (94.7 +/- 1.36%) than TE (82.4 +/- 3.08%), while M showed an intermediate value (89.3 +/- 2.17%). In relation to time of storage, TR diluent showed the highest PM values up to day 12, but differed (p < .05) from TE medium only until day 4. The TR extender maintained a PM > 50% up to day 8, while TE and M were greater than 50% up to day 5. Skim milk maintained progressive movement of sperm cells for longer (MS = 3 until day 5) than the other diluents. There were no significant differences in PIA among the 3 media during the first 6 days of storage (except in day 2), and M showed the lowest value afterward. There was no significant extender effect on PS for the first 3 days, but after day 6 diluent M also showed the lowest values of this variable. PMID- 10746874 TI - Chemotaxonomy and geographical distribution of tropane alkaloids. AB - This review illustrates the distribution of tropane alkaloids within the families Solanaceae, Erythroxylaceae, Proteaceae, Euphorbiaceae, Rhizophoraceae, Convolvulaceae and Cruciferae. Whereas tropane alkaloids are characteristic of the genera Datura, Brugmansia (tree datura) and Duboisia of the Solanaceae, the distribution is more widespread with novel tropane derivatives in families not traditionally associated with these bases. The chemical nature of more recently discovered water-soluble calystegines and the di- and trimeric forms from the Convolvulaceae (e.g. schizanthines from Schizanthus spp.), truxillines from Bolivian coca leaves and moonines of Erythroxylum moonii are highlighted. Where possible and appropriate, links between the phytochemistry and taxonomy are discussed. PMID- 10746875 TI - Cucumisin-like protease from the latex of Euphorbia supina. AB - A protease has been purified from the latex of Euphorbia supina Rafin by two steps of chromatography. The Mr was estimated by SDS-PAGE to be 80 kDa. Its activity was inhibited strongly by diisopropyl fluorophosphate, but not by EDTA, pepstatin, or cysteine protease inhibitors, indicating that the enzyme is a serine protease. The specificity of the protease is broad, but the preferential cleavage sites were C-terminal sites of hydrophobic amino acid residues. The N terminal sequence of the first fifteen residues was determined and six of the residues match those in cucumisin [EC 3.4.21.25], a protease from the sarcocarp of melon fruit (Cucumis melo L. var. Prince). The results indicate that the E. supina protease is a cucumisin-like serine protease. PMID- 10746876 TI - Biosynthesis of the gymnomitrane-type sesquiterpenes in liverworts. AB - The biosynthesis of gymnomitranes in the liverworts Reboulia hemisphaerica (L.) Raddi and Bazzania trilobata (L.) Gray was investigated. Labelling experiments with 1-13C-, 2-13C- and 6,6-D2 glucose, 2,2-D2- and 4,4-D2 mevalolactone and tissue cultures of the liverworts were carried out. PMID- 10746877 TI - Caffeic acid oligomers in Lithospermum erythrorhizon cell suspension cultures. AB - Lithospermum erythrorhizon cells cultured in pigment production (M-9) medium produced lithospermic acid B, a dimerized caffeic acid ester derivative, in quantities similar to the production of shikonin. The cells also produced a related dimer, (+)-rabdosiin. In Linsmaier-Skoog liquid medium, which suppresses shikonin production, both lithospermic acid B and (+)-rabdosiin were still formed. Lithospermic acid, a caffeic acid-rosmarinic acid conjugate, was isolated as a main constituent in Lithospermum hairy root cultures. In the aerial parts of L. erythrorhizon, the content of these phenylpropanoid oligomers was relatively low compared to that of rosmarinic acid. PMID- 10746878 TI - Sugar sensing and Ca2+-calmodulin requirement in Vitis vinifera cells producing anthocyanins. AB - We have previously reported that sucrose modulates anthocyanin biosynthesis in cell suspension cultures of Vitis vinifera L. The main role of sugar in this response does not seem to be that of general carbohydrate source for the supply of energy. In the present work, a number of pharmacological agents were used to further investigate the components of the signal transduction pathway involved in the induction of anthocyanin biosynthesis by sugar. We found that the phosphorylation of hexose by hexokinase, but not its transport, has to be taken into account for the sucrose signal transduction leading to anthocyanin accumulation. Indeed, 3-O-methylglucose, a glucose analog transported into cells but not phosphorylated by hexokinase, has no effect on anthocyanin production. Mannose mimics the effect of sucrose in grape cells, and mannoheptulose, a specific inhibitor of hexokinase, reduces the accumulation of anthocyanins in response to sucrose. The results with the two latter analogs are discussed. Ca2+ channel blockers, verapamil and LaCl3, which were used to investigate the role of extracellular Ca2+, all inhibited the sugar response. Ca2+ depletion by pretreatment with ethylene glycol bis (beta-aminoethylether)-N,N,N',N' tetraacetic acid (EGTA) also blocked the sugar response, which was partially recovered when Ca2+ was added exogenously after Ca2+ depletion. The use of two potent calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphtalenesulphonamide (W7) and chlorpromazine, showed that calmodulin is involved in the sugar signal transduction. A protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), and the protein phosphatase inhibitors, endothall and cantharidin, also inhibited the sugar response. The results of the present study suggest the involvement of several components of general signal transduction pathways such as Ca2+, calmodulin, and protein kinases phosphatases in the induction of anthocyanin biosynthesis by sugar. PMID- 10746879 TI - The African yam bean seed lectin affects the development of the cowpea weevil but does not affect the development of larvae of the legume pod borer. AB - Artificial feeding assays were used to study the effect of purified galactose specific lectins from African yam beans (Sphenostylis stenocarpa) on development of larvae of the cowpea weevil, Callosobruchus maculatus (Coleoptera : Bruchidae) and the legume pod-borer, Maruca vitrata (Lepidoptera : Pyrialidae). Inhibition of development of C. maculatus was observed when larvae were fed on artificial cowpea seeds containing 0.2%, 2.0% and 5.0% (wt/wt) of dietary lectin. Larval mortality was between 30% and 88%, while delays in total developmental time ranged between 7 and 13 days. The lectin had no effect on development of larvae of M. vitrala, when tested through topical artificial diet incorporation assays, except at the extremely high dose of 35% dietary level. PMID- 10746880 TI - Microbial transformation of papaveraldine. AB - Preparative-scale fermentation of papaveraldine (1), the known benzylisoquinoline alkaloid, with Mucor ramannianus 1839 (sih) has resulted in a stereoselective reduction of the ketone group and the isolation of S-papaverinol (2) and S papaverinol N-oxide (3). The structure elucidation of both metabolites was based primarily on 1D-, 2D-NMR analyses and chemical transformations. The absolute configuration of 2 was determined using Horeau's method of asymmetric esterification. These metabolism results were consistent with the previous plant cell transformation studies on papaverine and isopapaverine. PMID- 10746881 TI - DNA triplex stabilization property of natural anthocyanins. AB - The DNA triplex stabilization property of seven natural anthocyanins (five monoglucosides and two diglucosides) has been measured by the mean of triplex thermal denaturation experiments. We have noticed a difference between the diglucosides that do not modify this melting temperature and the monoglucosides (namely 3-O-beta-D-glucopyranoside of malvidin, peonidin, delphinidin, petunidin and cyanidin) which present a weak but significant stabilizing effect. It appears clearly that the difference between the two series could be due to the supplementary sugar moiety at the 5 position for the diglucosylated compounds, that would make them too crowded to allow interaction with the triplex. Among the monoglucoside series, the most active compounds are the only ones to embody a catechol B-ring in their structure that could be important for such an interaction. The need to have pure and fully characterized compounds to run these measurements, made it possible for us to unambiguously assign the 1H and 13C NMR spectra with the help of 2D NMR experiments. Thus, missing data of compounds not totally described earlier, are provided herein. PMID- 10746882 TI - Coumarins and bicoumarin from Ferula sumbul: anti-HIV activity and inhibition of cytokine release. AB - The methanol extract of the dried roots of Ferula sumbul afforded two furanocoumarin esters, fesumtuorin A, B, one bicoumarin, fesumtuorin C, five spirobicoumarins, fesumtuorin D, E, F, G and H, along with nineteen known coumarins. Their structures were established on the basis of spectroscopic studies. Some of the isolated compounds showed anti-HIV activity and very weak inhibition of cytokine release. PMID- 10746883 TI - Unusual phenolic glycosides from Cotoneaster orbicularis. AB - The whole plant of Cotoneaster orbicularis contains the novel di-C glycosylflavone, 4",4"'-di-O-beta-glucopyranosyl-vicenin II, or 6,8-di-C-beta Cellobiosylapigenin, as well as the hitherto unknown natural phenolic glucoside, gentisic acid 2-O-beta-glucopyranoside, or orbicularin. Further phenolics are protocatechuic, anisic, caffeic, p-coumaric acids, catechin, epicatechin, 2"-O alpha-rhamnopyranosylvitexin, vitexin, rutin, isoquercetrin, hyperin and naringenin. All structures were determined by routine methods of analysis and confirmed mostly by 1H- and 13C-NMR. PMID- 10746884 TI - Phloroglucinol derivatives from Hypericum japonicum. AB - The isolation and identification of a new phloroglucinol derivative (2), a diterpenol (4), together with the known compounds flavesone (1) and sarothralen B (3), from the aerial parts of Hypericum japonicum are reported. Their structures were established by extensive spectral analysis and the structure of (3) has also been confirmed by a single crystal X-ray determination. PMID- 10746885 TI - Bacopasaponins E and F: two jujubogenin bisdesmosides from Bacopa monniera. AB - Two new dammarane-type jujubogenin bisdesmosides, bacopasaponins E and F of biological interest have been isolated from the reputed Indian medicinal plant Bacopa monniera and defined as 3-O-[beta-D-glucopyranosyl(1 --> 3)[alpha-L arabinofuranosyl(1 --> 2)]alpha-L-arabinopyranosyl]-20-O-(alpha-L arabinopyranosyl) jujubogenin and 3-O-[beta-D-glucopyranosyl(1 --> 3)[alpha-L arabinofuranosyl(1 --> 2)]beta-D-glucopyranosyl]-20-O-alpha-L-arabinopyranosyl) jujubogenin respectively by spectroscopic methods and some chemical transformations. PMID- 10746886 TI - Terpenoids from Tripterigyum hypoglaucum. AB - Six terpenoids have been isolated from the root bark of Tripterygium hypoglaucum, along with 14 known compounds. The structures of the terpenoids were elucidated as 3,11,14-oxo-abieta-8,12-diene, 3beta-hydroxy-12,14-dimethoxyabieta-8,11,13 triene, 3beta-hydroxy-11alpha-ethoxyurs-12-ene, 3beta-hydroxy-11alpha-methoxyurs 12-ene, 3beta-hydroxy-11alpha-methoxyolean-12-ene-28-oic acid, and 1beta-benzoyl 8alpha-cinnamoyl-4alpha,5alpha- dihydroxydihydroagarofuran. PMID- 10746887 TI - Weight gain in midlife: don't blame your hormones! PMID- 10746888 TI - Soy phytoestrogens: what will be their role in postmenopausal hormone replacement therapy? PMID- 10746889 TI - A decision tree for the use of estrogen replacement therapy or hormone replacement therapy in postmenopausal women: consensus opinion of The North American Menopause Society. AB - OBJECTIVE: Menopause is associated with physiologic changes that may have negative effects on quality of life in some women and/or that may increase morbidity and mortality secondary to osteoporosis and/or coronary heart disease. Estrogen replacement therapy (ERT) and combined estrogen/progestogen therapy (hormone replacement therapy [HRT]) play an important role in reducing these negative effects. The North American Menopause Society (NAMS) sought to develop treatment algorithms that could assist the clinician in deciding whether to recommend ERT/HRT to postmenopausal women. DESIGN: NAMS held a closed conference of experts to develop a decision tree that outlined the rational use of ERT/HRT in postmenopausal women on the basis of risks versus benefits. The proceedings of the conference were used to assist the NAMS Board of Trustees in developing this consensus opinion of the Society. RESULTS: On the basis of the conference proceedings, NAMS developed three algorithms for the clinician to use as a tool in deciding whether to recommend ERT/HRT to a woman who is postmenopausal: (1) menopause-related symptoms, (2) cardiovascular risk, and (3) osteoporosis risk. CONCLUSIONS: The goal of ERT/HRT is to enhance women's quality of life as well as to reduce the risks of death and disability associated with osteoporosis and coronary heart disease. The decision to initiate ERT/HRT must be individualized according to each woman's needs. This decision tree for ERT/HRT presents a rational approach to decision making on the basis of the principles of care; details of specific therapeutic interventions will change as data from clinical trials are presented. PMID- 10746890 TI - Effects of menopause and estrogen replacement therapy or hormone replacement therapy in women with diabetes mellitus: consensus opinion of The North American Menopause Society. AB - OBJECTIVE: Given that the prevalence of non-insulin-dependent diabetes mellitus (type 2 DM) is increasing in postmenopausal women and type 2 DM substantially increases the risk for cardiovascular events in postmenopausal women, a population that is at higher risk for coronary heart disease, The North American Menopause Society (NAMS) developed a consensus opinion on appropriate management strategies for postmenopausal women who have or who are at risk for developing type 2 DM. DESIGN: NAMS held a closed conference of experts in the field to evaluate the published clinical data on the effects of menopause, type 2 DM, and estrogen or hormone replacement therapy (ERT/HRT) in women, especially the effects on cardiovascular risk factors, and to discuss therapeutic options. The proceedings of the conference were used to assist the NAMS Board of Trustees in developing this consensus opinion. RESULTS: On the basis of the current knowledge, NAMS established consensus on the following issues: (1) Controlling cardiovascular risk factors through pharmacologic and nonpharmacologic means can significantly decrease the risk for developing cardiovascular events. (2) A broad based recommendation for ERT/HRT cannot be made; rather, the benefits and risks must be weighed in the context of each woman's risk factors. (3) When ERT/HRT is recommended, the greatest benefits may be obtained from the use of transdermal estrogen preparations, low doses of oral estrogens, progesterone instead of progestin, and/or nonandrogen preparations, although more research is needed in this area. (4) Counseling can help maximize the patient's adherence to multiple medication regimens and increase her understanding of the potential benefits and risks of ERT/HRT. CONCLUSIONS: Controlling an individual woman's risk factors for cardiovascular events should be the focus of any management strategy for a postmenopausal woman who has or is at risk for developing type 2 DM. PMID- 10746891 TI - A longitudinal study of weight and the menopause transition: results from the Massachusetts Women's Health Study. AB - OBJECTIVE: Results of past studies of menopause and weight are inconsistent, in part because of problems in study design and analyses, such as retrospective assessment of age at menopause and failure to control for confounding factors. To address such shortcomings, we conducted multivariate analyses on longitudinal data from a large, community-based sample of initially pre- and perimenopausal women who were making the transition through menopause. DESIGN: Data were from the second phase of the Massachusetts Women's Health Study, a cohort of 418 women aged 50-60 years in 1986. We assessed the relationship between menopause transition and weight, after accounting for previous weight; age; and the behavioral factors of smoking, exercise, and annual ethanol consumption. Menopause status was defined in terms of months of amenorrhea. The association of hormone replacement therapy and weight also was examined. RESULTS: Menopause transition was not consistently associated with increased weight, and use of hormone replacement therapy was not significantly related to weight. Behavioral factors--particularly exercise and ethanol consumption--were more strongly related to weight than was menopause transition. CONCLUSIONS: These results are consistent with findings from other studies that suggest that the weight increases experienced by middle-aged women in the United States are not a result of the menopause transition. PMID- 10746893 TI - The effect of 17beta-estradiol and alpha-tocopherol on the oxidation of LDL cholesterol from postmenopausal women and the minor effect of gamma-tocopherol and melatonin. AB - OBJECTIVE: Estrogens have a potent antioxidant effect on low-density lipoprotein (LDL) cholesterol in vitro and in vivo. A variety of compounds with antioxidant properties, such as vitamins and other hormones, also have been recommended in clinical practice to prevent several diseases related to oxidation. The aim of this study was to compare the antioxidant potency of estradiol (E2), the liposoluble vitamin E (both, alpha- and gamma-tocopherol), and melatonin. DESIGN: LDL was isolated by ultracentrifugation from the plasma of 11 healthy, untreated postmenopausal women. Aliquots containing 0.5 mg of LDL protein were incubated for 4 h with 15 microM of CuSO4 to induce oxidative stress and with one of the four compounds studied: E2, alpha-tocopherol, gamma-tocopherol, or melatonin in doses of 0, 5, 15, 50, and 500 microM and 1 and 2 mM. Malondialdehyde (MDA) was measured as a marker of LDL oxidation. RESULTS: E2 induced a dose-dependent decrease in MDA concentration (nmol/mg protein). MDA values were significantly different as compared with baseline at 5 microM of E2 (F = 47.17; p < 0.0001). Alpha-tocopherol, gamma-tocopherol, and melatonin also showed a significant decrease in MDA concentration but to a lesser degree. The reduction of MDA reached statistical significance at 50 microM with alpha-tocopherol, 500 microM with melatonin, and 1 mM with gamma-tocopherol. The antioxidant effect also reached a plateau at concentrations of 50 microM of E2 and 1 mM of alpha tocopherol; gamma-tocopherol and melatonin did not reach a plateau at any dose tested. CONCLUSIONS: The antioxidant potency of E2 in vitro is at least 10-100 times greater than alpha- and gamma-tocopherol and melatonin. Whether this finding implies a better performance of E2 as a protective agent against oxidation-related diseases remains to be determined. PMID- 10746892 TI - Clinical effects of a standardized soy extract in postmenopausal women: a pilot study. AB - OBJECTIVE: This double-blind, randomized study was aimed at evaluating comparatively, in postmenopausal women, the activity of a standardized soy extract (SOYSELECT) and placebo when given alone or in combination with conjugated equine estrogens (CEE) on early climacteric symptoms. Lipid profile, pituitary hormones, osteocalcin and endothelin levels, and vaginal and endometrial parameters were also evaluated. DESIGN: Participants in the control group were given placebo, and participants in the treated group were given 400 mg/day of a standardized soy extract, corresponding to 50 mg/daily of isoflavones. After 6 weeks of treatment, CEE was also then given to each participant at a dose of 0.625 mg/day for 4 weeks. At the end of this period, soy and placebo treatment were suspended, and, until the end of the study (week 12), participants were administered 10 mg/day of medroxyprogesterone acetate in association with CEE (0.625 mg/day). RESULTS: When compared with pretreatment data, on week 6 of the study, a significant (p < 0.01) reduction in the mean number of hot flushes per week was observed in participants who were receiving the standardized soy extract, whereas a more marked relief was observed in both soy and placebo groups during CEE administration. Concurrently, the severity of hot flushes, assessed by means of the Greene climacteric scale, was also reduced in the soy group participants (p < 0.001, by paired t-test). No soy-related changes were observed on vaginal cytology, endometrial thickness, uterine artery pulsatility index, or metabolic and hormonal parameters tested. Finally, CEE related changes on genital tract, uterine vascular compartment, and pituitary hormones were not modified by soy treatment. CONCLUSIONS: SOYSELECT may be a safe and efficacious therapy for relief of hot flushes in women who refuse or have contraindications for hormone replacement therapy. PMID- 10746894 TI - Transdermal administration of estradiol and norethisterone: effect on the uterus and uterine arteries. AB - OBJECTIVE: To evaluate the short- and long-term effect on the uterus, endometrium, and vascular reactivity of uterine arteries of sequential transdermal estradiol (50 microg/day) and norethisterone (0.25 mg/day in the last 14 days of each cycle). DESIGN: An intravaginal ultrasound evaluation was performed in 48 postmenopausal women before and at the 3rd and 12th month of treatment, during the last 3 days of both estradiol alone and estradiol plus norethisterone. An endometrial biopsy was also performed before and at the end of treatment. In 11 participants, intravaginal ultrasound and endometrial biopsy were repeated after 48 months of treatment. RESULTS: Uterine volume (33.7 +/- 3.3 cm3 to 56.8 +/- 3.7 cm3; p = 0.001) and endometrial thickness (3.07 +/- 0.48 mm to 5.74 +/- 0.41 mm; p = 0.001) increased within 3 months, with no further increases. Thickness was similar in the estradiol and estradiol-norethisterone phase. Endometrial hyperplasia was found in one participant at 12 months of treatment. A significant decrease (p = 0.002) in the pulsatility index of uterine arteries was observed only during the estradiol phase. After 48 months of treatment, the pulsatility index of uterine arteries was lower than at baseline (2.78 +/- 0.24 vs. 2.23 +/- 0.33; p = 0.044) even when evaluated in the combined phase. CONCLUSIONS: The transdermal administration of sequential estradiol and norethisterone reduces uterine artery resistance and induces a self-limiting growth of the uterus and endometrium. PMID- 10746895 TI - Use of unopposed estrogen in women with uteri: prevalence, clinical implications, and economic consequence. AB - OBJECTIVE: Hormone replacement therapy with estrogen/progestin is the treatment of choice for relieving postmenopausal vasomotor symptoms and preventing urogenital atrophy and osteoporosis in women with intact uteri. However, despite the known increased incidence of endometrial hyperplasia when unopposed estrogen is used in such women, this progestin regimen has not been universally adopted. DESIGN: This study was conducted in a managed care organization to determine the extent of the use of unopposed estrogen in women with intact uteri. Pharmacy claims data for all women 55 years or older with claims for estrogen only from September 1, 1996, to December 31, 1996, were reviewed. A total of 5,209 records were identified, from which 480 were randomly selected. A survey of the members' physicians was then carried out to determine hysterectomy status and was confirmed by chart audit. RESULTS: Thirty-three (11%) of the members identified had not undergone hysterectomy. Follow-up physician contact revealed that five women did not have a uterus. Use of estrogen without opposing progestin was documented in a substantial percentage of files reviewed. It is of concern that with the documentation of the risks of endometrial hyperplasia and carcinoma in the intact uterus, unopposed therapy still occurs. In addition to the clinical costs, there are economic consequences to this practice. An economic model of unopposed estrogen use was created. A management cost of $1,504 for 3 years was estimated. CONCLUSIONS: Further educational efforts are needed to ensure the use of opposed estrogen in the woman with an intact uterus. PMID- 10746896 TI - Diagnosis of symptomatic postmenopausal women by traditional Chinese medicine practitioners. AB - OBJECTIVE: To learn more about the way that practitioners of traditional Chinese medicine (TCM) diagnose women who have menopausal symptoms. DESIGN: We assembled a cohort of 23 postmenopausal women who had hot flushes and were otherwise healthy. Each woman was examined independently by nine practitioners of TCM on the same day. Examination consisted of medical history and physical examination. Diagnoses were recorded and counted. RESULTS: The most frequent diagnosis made by the practitioners of TCM was kidney yin deficiency, which was the diagnosis made after 168 of 207 visits (81%); 23 women seen by nine TCM practitioners. Practitioners showed good agreement regarding presence of kidney yin deficiency: in 12 women (52%), this diagnosis was made by eight of nine practitioners; in 16 women (70%), seven of nine practitioners made this diagnosis; and in all 23 women (100%), at least five of nine practitioners made this diagnosis. CONCLUSIONS: Practitioners of TCM who diagnose postmenopausal women with vasomotor symptoms are likely to make a diagnosis that includes kidney yin deficiency. PMID- 10746897 TI - Subthreshold disorders in psychiatry: clinical reality, methodological artifact, and the double-threshold problem. AB - The introduction of modern operationalized classification systems for mental disorders has led to the issue of subthreshold disorders. Definitions for illness do not at the same time define health, e.g., in the sense of the World Health Organization (WHO) definition from 1947. The threshold not only to define disorders but also to define health is open to discussion. So-called subthreshold disorders require the definition of 2 thresholds. Empirical research has suggested that these "between-threshold disorders" are associated with increased disability and many other negative consequences. Part of the problem with subthreshold disorders is methodological in nature. Psychopathology and the Gestalt characteristic of psychopathological signs are ignored, and categorical instead of dimensional concepts are used. Thus, the distinction between syndromes and disorders, as well as the hierarchical structure of disorders, is not taken into account, and statistical problems with the prognostic power, which is dependent on the epidemiological distribution, are not solved. Variations in threshold definitions have important consequences for the individual and for society, be it because of the negative effects of "diagnostic labeling" or because of the costs to the health care system. Treatment options are presently rather insufficient, although modern sequential treatment algorithms and newer treatments (e.g., selective serotonin reuptake inhibitors [SSRIs] and Saint John's-wort) promise interesting perspectives. Also in this context, self-help should become an important area of medical treatment research. PMID- 10746898 TI - Plato versus Aristotle: categorical and dimensional models for common mental disorders. AB - There are two quite different ways of conceptualizing common mental disorders: as categorically distributed or as dimensions. In the former case, individuals with "subthreshold" disorders may contain a small proportion of "true cases"; while in the latter, they are seen as having the same basic disorder, but in insufficient degree to warrant a diagnostic label. The categorical school postulates the phenomenon of "comorbidity" to account for those who are both depressed and anxious. The dimensional school regards them as cases of "anxious depression," and observes that the 2 dimensions are highly correlated with one another. This article considers 3 levels of severity of cases satisfying research diagnostic criteria for depression: mild, moderate, and severe. At ascending levels of initial severity, there is increasing disability, and a worse outcome at 12 months. The only positive treatment effect is that favoring antidepressants over sedatives at 3 months, and this effect is related to initial severity-being much greater for the "severe" group of depressions. Initial severity is linearly related to disability, and there is no disjunction at the usual division between "non-cases" and "cases" of depression. Clinicians are obliged to use categorical concepts, as they must decide who is sufficiently ill to justify treatment. But in our efforts to understand the relationships between social and biological variables, dimensional models are far more appropriate. PMID- 10746899 TI - Public health consequences of different thresholds for the diagnosis of mental disorders. AB - The acceptance of subthreshold disorders in psychiatry is a controversial topic with multiple implications for public health policy. Using subthreshold depression as an example, we provide documentation based on data from epidemiologic and clinical studies suggesting that subthreshold conditions are not uncommon, there is significant associated disability (with significant societal costs), and the course of illness often leads to the development of the full-blown disorder. The potential societal benefits of developing and testing low-cost, low-risk, and nontraditional treatments are considered. The recommendations are to match treatment intensity and cost with severity level in a staged approach. PMID- 10746900 TI - Limitations in activities of daily living: towards a better understanding of subthreshold mental disorders in old age. AB - Based on a representative sample of elderly subjects, a description of the limitations in activities of daily living (ADL) and instrumental ADL (IADL) at subthreshold levels of dementia and depression is presented and compared against a sample of psychiatric non-cases and samples with specified levels of the respective illnesses. Additionally, it was analyzed whether these limitations are useful diagnostic markers with regard to subdiagnostic psychiatric disorders. Even at subthreshold levels of depression and dementia, elderly people suffer quite extensively from ADL and IADL limitations. However, multifactorial analyses indicate little evidence that these limitations are specific for psychiatric morbidity, be it at subdiagnostic or specified levels. By and large, ADL and IADL limitations in an elderly sample have to be considered instead as consequences of physical health-related comorbidity. Thus, issues regarding the treatment of ADL and IADL limitations at subdiagnostic as well as specified levels of psychiatric morbidity may not be solved from a psychiatric point of view alone, and a multifactorial, i.e., multiprofessional, perspective is strongly recommended. PMID- 10746901 TI - Does effective lithium prophylaxis result in a symptom-free state of manic depressive illness? Some thoughts on the fine-tuning of mood stabilization. AB - Recovered depressive or manic-depressive patients on long-term lithium medication may show oscillations in their state of well-being, neuropsychological impairment, and subclinical mood disturbances. It remains an open question as to whether such changes should be labeled as "subthreshold" symptoms of depression or mania, as a particular syndromal entity not directly related to the "mood disorder," or as subtle side effects of lithium. Within a psychological theory of the action of lithium, mental effects of lithium must be postulated and should not be simply considered as side effects. Depending on medical common sense and economic restrictions, the optimization and fine-tuning of lithium prophylaxis may be advisable in selected cases, including counseling, family therapy, cognitive therapy, well-being therapy, or interpersonal psychotherapy. Also, a change of medication may be considered in patients with subjective or objective cognitive impairment. PMID- 10746902 TI - Subthreshold depression in the elderly: qualitative or quantitative distinction? AB - Recent studies revealed that subthreshold depression (or "subclinical" or "subsyndromal" depression) can have clinical validity because it is related to dysfunction and disability and is a risk factor for major depression. However, none of these studies focused on old age. Therefore, one aim of the psychiatric part of the multidisciplinary Berlin Aging Study (BASE) was also to detect milder forms of psychopathological syndromes, especially subthreshold depression, compared with specified forms such as major depression and dysthymia according to the DSM-III-R. The present evaluation shows that subthreshold depression can be characterized in 2 ways: firstly, as a quantitatively minor variant of depression or a depression-like state with fewer symptoms or with less continuity; and secondly, as qualitatively different from major depression with fewer suicidal thoughts or feelings of guilt or worthlessness, while worries about health and weariness of living occur with a similar frequency. PMID- 10746903 TI - Depressive spectrum diagnoses. AB - There has been widespread debate about the validity of the contemporary diagnostic classification system of depression. The major goal of this study is to examine the prognostic significance of each of the major subtypes of depression using data from 5 interviews of a 15-year prospective community-based cohort study. The stability of the following diagnostic subtypes across the duration of the study was examined: major depressive disorder (MDD), dysthymia, recurrent brief depression (RBD), and minor depression. The results show that there was little stability for the specific subtypes of depression among those who continued to manifest depression during the follow-up period; 51% of those with MDD and 44% of those with RBD met criteria for another subtype of depression. When stability was observed, the same subtype often occurred in combination with the development of another subtype. Among individuals with a single subtype, severity was greatest among those with dysthymia, whereas individuals with combined subtypes had greater severity than those with a single subtype. The lack of longitudinal stability of the diagnostic subtypes of depression suggests that depression is better expressed as a spectrum rather than a set of discrete subtypes. PMID- 10746904 TI - Anxiety and anxiety disorders in the old and very old--results from the Berlin Aging Study (BASE). AB - Within the context of the Berlin Aging Study, we examined the distribution of anxiety symptoms and disorders in a representative community sample. The participants were beyond the age of 70 years, thereby extending results from other studies not covering this age range. Additionally, we analyzed the distribution of anxiety symptoms and syndromes not fulfilling specified diagnostic criteria. A sample of 258 old (70 to 84 years) and 258 very old (85 to 103 years) subjects were examined. Anxiety disorders as defined in DSM-III-R and according to clinical judgment (diagnoses termed NOS) were assessed. In addition, items from the Geriatric Mental State-A (GMS-A) covering a wide range of symptoms of anxiety were subjected to factor analysis. The raw score distributions of anxiety subscales obtained by this procedure are examined by age, gender, education, personal living situation, and psychiatric comorbidity. The weighted overall prevalence of anxiety in the elderly community is 4.5% (n = 17), including specified (n = 8) anxiety disorders according to the DSM-III-R and unspecified (n = 9) disorders. Prevalence rates in the younger old were 4.3% and in the older old 2.3%. Weighted prevalence rates for males were 2.9% and for females 4.7%. The most common comorbid disorders were affective disorders in both age groups as well as both genders. Independently of the nosological level, 52.3% reported one or more symptoms of anxiety. Factor analysis of anxiety-related symptoms yielded 5 independent subscales, reflecting hypochondriasis, panic, phobia, worries, and vegetative anxiety. There were more phobic symptoms in the younger age group (P < .001). Except for worries and hypochondriac symptoms, females showed significantly higher anxiety in all other anxiety dimensions. There was no relation between anxiety and cognitive status or socioeconomic status (SES). Only for subjects living alone was more phobic-type anxiety found. Anxiety disorders in old and very old persons are less frequent than other psychiatric disorders of old age, and do not increase with age. Gender differences can still be observed. The symptomatic structure of anxiety seems similar to that found in younger cohorts. Thus, anxiety disorders in old age do not seem substantially different from those in younger age. Their relative contribution to the spectrum of mental disorders seems to decrease, rather than increase, with age, while at the same time anxiety symptoms are an almost daily experience. PMID- 10746905 TI - Mixed anxiety-depression and its implications for models of mood and anxiety disorders. AB - Recent findings have suggested that there is a distinct group of patients presenting with subthreshold levels of mixed anxious and depressive symptoms associated with significant functional impairment. Accumulating evidence of this type ultimately led to a multisite field trial which investigated the possibility of developing a new category of mixed anxiety-depression (MAD) for the DSM-IV. The field trial confirmed both the existence and impairment of a sizable group of patients with mixed subclinical anxious and depressive symptoms, and provisional criteria for MAD were proposed. Although the validity of the tentative MAD category has yet to be established, the unique characteristics of patients presenting with MAD symptoms have important implications for models of mood and anxiety disorders. We argue that the particular pattern of impairment associated with MAD provides additional evidence that anxiety and depressive disorders have a shared diathesis best captured by the construct of nonspecific negative affect. PMID- 10746906 TI - Subclinical cognitive impairment: epidemiology and clinical characteristics. AB - Subclinical cognitive impairment is commonly reported in elderly populations, but its clinical significance is largely undetermined. It is commonly perceived as a normal feature of the aging process or a separate nosological entity. The clinical status of this disorder is explored herein by reference to longitudinal observations. It is concluded that persons with subclinical cognitive impairment constitute a highly heterogeneous group. There is an elevated risk in this group for senile dementia, with an estimated 18% incidence rate over 3 years, but in the majority of cases, this problem is more commonly associated with depressive symptomatology and physical illness. Only 13% of persons with recent observable change in cognitive functioning appear to have a totally benign and transient syndrome. Caution should be exercised in applying single unifying nosological entities such as age-associated memory impairment to this group of the elderly. PMID- 10746907 TI - Prediction of deterioration in mild cognitive disorder in old age- neuropsychological and neurochemical parameters of dementia diseases. AB - In normal senescence, an age-related impairment of cognitive function is observed. The difficult clinical question is in which cases of mild cognitive impairment (MCI) will there be a rapid cognitive decline to a dementia syndrome. Two ways to improve prognosis are discussed: neuropsychological tests and analysis of neurochemical markers. First, the question is asked as to whether there are clusters of MCI. Longitudinal neuropsychological data from the Berlin Aging Study (BASE) are presented, a population-based sample of 516 subjects aged 70 to 103 years. There are clusters found that in part match those clusters, which have been identified by a study from Ritchie et al. in 1996. Especially, a cluster of 13.8% of the nondemented participants with a decline in memory performance is observed. The validation of clusters of cognitive performance and decline opens up the possibility of diagnosing distinctive subgroups of MCI to improve prognosis in old age. Second, the existing data concerning the diagnostic laboratory analysis for Alzheimer's disease (AD) are reviewed. Especially, data regarding nerve growth factor (NGF) are reported. In MCI, preliminary data show a correlation between the NGF serum level and cognitive performance. It can be concluded that the combined investigation of neuropsychological functions and cognitive decline, as well as laboratory measurement of neurochemical markers, might allow an improved prognosis for mental health in very old age. PMID- 10746908 TI - Prodromal states in schizophrenia. AB - The vulnerability-stress-coping (VSC) model is the most influential heuristic concept in understanding the course of schizophrenia, whose prodromal status still offers unsolved conceptual and methodological issues. Improved knowledge about the prodromal phase would provide a better understanding of the developing psychopathology and psychophysiology of schizophrenia and could also be of predictive value to attune therapeutic actions to the course of the illness more precisely. To shed more light on the characteristics of prodromal states, data from a German multicenter study on intermittent versus maintenance neuroleptic long-term treatment in schizophrenia (ANI study) were reanalyzed with respect to the prevalence and profile, nature, time course, and predictive value of prodromal symptoms in impending relapse. The results demonstrate that prodromes are a category of symptoms on their own, but they share variance with other symptom domains. Treatment side effects, psychotic symptoms, dysphoric mood, and social dysfunction are all associated with prodromal states--the direction of this association, however, is still to be clarified. Prodromal symptoms are also related to the neuroleptic treatment strategy and its relapse-preventive efficacy -findings that underscore neuroleptic maintenance medication in preventing both overt and subthreshold psychotic morbidity in schizophrenia. PMID- 10746909 TI - Subthreshold symptoms and vulnerability indicators (e.g., eye tracking dysfunction) in schizophrenia. AB - Subthreshold symptoms in schizophrenia can be prodromal signs of a psychotic relapse. In people without schizophrenia, similar symptoms may indicate the presence of disorders termed schizophrenia spectrum disorders. Subthreshold schizophrenia-like symptoms may indicate a genetically transmitted higher proneness to schizophrenia. Such a higher liability to develop schizophrenia is ascertained on a symptom level. In genetic studies, asymptomatic members of a pedigree are therefore classified as unaffected although they may possess the genes in question. On a biological level, eye tracking dysfunction has been shown to fulfill certain criteria for a vulnerability indicator and therefore promises to offer more information on genetically transmitted proneness to schizophrenia even in people without psychopathological symptoms. Subthreshold symptoms may warrant treatment. The database for prophylactic treatment in populations at high risk, especially those without symptoms, is currently very small. PMID- 10746910 TI - Transition to alcohol dependence: clinical and neurobiological considerations. AB - The transition to alcohol dependence is supposed to occur during a critical period that begins with increased drinking and ends with a loss of control. This process may last about 3 to 4 years, and is modified by gender and accelerated by premorbid traits (e.g., novelty-seeking) and comorbid disorders (e.g., dissocial personality disorders according to ICD-10). Genetic disposition, environmental influences (e.g., stress), and sensitization by exposure are factors implicated in dependence that alter brain functions, some possibly in an irreversible way. Underlying neurobiological mechanisms that may have different time patterns are beginning to be characterized on a systemic, cellular, and molecular level. Repeated free choices of the rewarding compound seem to be necessary for the transition to dependence. PMID- 10746912 TI - Sleep-wake cycle, sleep-related disturbances, and sleep disorders: a chronobiological approach. AB - There is convincing evidence that the functions of sleep include restoration of brain energy storage and memory consolidation. The circadian timing system (CTS) is involved in the daily variation of almost any physiological and psychological variable evaluated thus far. Disturbances of the CTS can be clinically observed by their influence on the sleep-wake cycle, hormones, body temperature, and locomotor activity. This article reviews the basic mechanisms of circadian rhythm sleep disturbances, names the applicable diagnostic tools and specific therapeutic strategies, and thereby hints at the impact of circadian rhythm sleep disturbance on psychiatric disorders, especially disorders of affect and cognition. In light of the preventive, diagnostic, and therapeutic tools now available, a new round of chronobiological studies in psychiatry seems justified, promising, and necessary. PMID- 10746911 TI - Hazardous alcohol use: its delineation as a subthreshold disorder, and approaches to its diagnosis and management. AB - The last 20 years have seen a significant paradigm shift in how we view alcohol misuse. The dichotomous model of "alcoholism" and "normal drinking" has now been replaced by the concept of a spectrum of disorders. In this new framework, "hazardous alcohol use" is defined as a repeated pattern of drinking that confers the risk of harmful consequences. It is a typical example of a subthreshold disorder. Where actual physical or psychological harm or social problems have occurred, the terms "harmful alcohol use" and "alcohol abuse," respectively, are applied. These conditions would typically be considered to be above the clinical threshold. The most severe disorder, alcohol dependence, is a psychobiological syndrome with often severe physical, psychological, and social sequelae. This article describes how the concept of hazardous alcohol use originated, and reviews the intervention techniques that have been developed to induce and assist hazardous drinkers to reduce their consumption to nonhazardous levels. The findings from a series of World Health Organization (WHO) collaborative studies on brief interventions for hazardous alcohol use are described. This work has resulted in the development of the Alcohol Use Disorders Identification Test (AUDIT) screening instrument, which can detect over 90% of hazardous drinkers in a range of settings, and the demonstration that 5 minutes' structured advice can reduce hazardous consumption by 30%. The later phases of this program of work have examined strategies to promote the dissemination of brief interventions for hazardous alcohol use throughout primary health care, and the nationwide, systematic, and sustained utilization of these interventions. PMID- 10746913 TI - Suicidal behavior--symptom or disorder? AB - This article examines the diagnostic status of suicidal ideation with and without additional signs of defined or subthreshold mental disorders. Data from the World Health Organization (WHO) study on Psychological Problems in General Health Care (PPGHC) show that 8.8% of all general practice patients report that they recently had a wish to be dead. Among patients with acute depressive episodes, the rate is 34.5%, as compared with 1.3% in persons without any sign of mental disorder. Rates of suicidal ideation in persons with subthreshold disorders (10.4%) are similar to the rate in persons with other, nondepressed forms of mental disorders (12.9%). Still, 30.1% of all persons with suicidal ideation do not have a defined mental disorder, and 21.5% do not even have subthreshold disorders. But in these persons also, suicidal ideation is associated with an increased rate of depressive complaints. Suicidal ideation therefore, in any case, can be seen as an indicator of mental problems. But it seems impossible to lower the thresholds of defined mental disorders so that all forms of suicidal ideation will be covered. Instead, suicidal thoughts must be regarded as a symptom with the status of a disorder itself which should be handled as a comorbid condition if other mental disorders co-occur. PMID- 10746914 TI - The waxing and waning of mental disorders: evaluating the stability of syndromes of mental disorders in the population. AB - This article examines the stability of symptoms, syndromes, and diagnoses of specific anxiety and depressive disorders, as well as diagnostic shifts from one syndrome to another over time. Using retrospective and longitudinal prospective data from the baseline and first follow-up investigation (19.7 months later) of the Early Developmental Stages of Psychopathology Study (EDSP), we focus on establishing stability measures for early stages of mental disorders in a community sample of adolescents aged 14 to 17 years at baseline. The results are as follows: (1) Although only about 30% developed a full-blown DSM-IV disorder, psychopathological syndromes are widespread in adolescents: 15% of the population aged 14 to 17 at baseline were not affected by at least some clinically relevant symptoms of mental disorders either throughout their previous life or throughout the follow-up period. (2) The likelihood of staying free of symptoms and threshold disorders during follow-up was highest among subjects who were completely well at baseline. The probability of a positive outcome decreased as a function of severity of baseline diagnostic status. (3) There was a considerable degree of fluctuation not only in the diagnostic status and severity of specific disorders, but also in terms of complete remissions and shifts from one syndrome and disorder to another. (4) Anxiety disorders, overall, slightly differ with regard to the persistence and stability of the diagnostic status from depressive disorders. (5) However, there were remarkable differences between specific types of anxiety and depressive disorders. Consistent with other longitudinal epidemiological studies in the general population, this study finds that the syndromes and diagnoses of mental disorders have a strong tendency to wax and wane over time in this age group. PMID- 10746915 TI - Potential treatment for subthreshold and mild depression: a comparison of St. John's wort extracts and fluoxetine. AB - Subthreshold depressive disturbances and depressive episodes of mild severity are frequently associated with disability and socioeconomic burden, and often show an increase in symptomatology over time if untreated. Thus, there is an urgent need for antidepressant active compounds that are more readily available than those that must be obtained by prescription. To get an impression of the efficacy of the widely used phytopharmaceutical St. John's wort, the antidepressant efficacy in mild depressive disorders was compared with that of the standard antidepressant fluoxetine. The present overview includes controlled trials of fluoxetine in depression with a mean initial score on the Hamilton Rating Scale for Depression (HAM-D) < or =24, which were compared to the respective studies on St. John's wort. The mean HAM-D reduction of all St. John's wort studies was 10.2 (52.9%), and the respective figures for fluoxetine were 12.5 points and 55.5%. Thus, no relevant efficacy difference between the groups of investigations was found based on the studies included. The most important restrictions of this overview are no meta-analysis was performed, the studies were performed with heterogeneous methodological standards, and the St. John's wort extracts used were very different. However, St. John's wort might be a treatment option to reduce symptoms in patients not suffering from full-blown depressive disorder. PMID- 10746916 TI - The development of the Maze procedure for the treatment of atrial fibrillation. AB - The Maze procedure was developed for the treatment of atrial fibrillation over a period of several years. Extensive experimental and clinical studies of the underlying electrophysiology of the arrhythmia were performed, and numerous surgical techniques and principles were tried before the Maze procedure was conceived. Few cardiac surgical procedures have undergone more extensive research and experimental trials before being applied clinically. This article gives a brief summary of the work leading up to the eventual Maze-III procedure that is now in clinical use. PMID- 10746917 TI - Current status of the Maze procedure for the treatment of atrial fibrillation. AB - Since the first patient underwent the Maze procedure on September 25, 1987, 346 patients have undergone this operation for the treatment of atrial fibrillation. The procedure was designed as an open-heart operation performed through a median sternotomy. It underwent 2 major modifications relatively early in the series, evolving into the so-called Maze-III procedure, which has been used exclusively since April 16, 1992. Since that time, the Maze-III procedure has been adapted to allow it to be done by minimally invasive techniques. In addition, we recently performed the entire procedure in 2 patients without the use of cardiopulmonary bypass. The operative mortality rate has remained at 2% to 3%. This includes patients undergoing concomitant high-risk cardiac surgical procedures and all re do cases. The overall success rate in curing atrial fibrillation has been 99%. The procedure itself has been shown to cause no permanent damage to the sinus node. The left atrium has been documented to function long-term postoperatively in 93% of patients and the right atrium functions in 99% of patients. The Maze III procedure remains the surgical procedure of choice for the treatment of medically refractory atrial fibrillation. PMID- 10746918 TI - The importance of cryoablation of the coronary sinus during the Maze procedure. AB - Although the Maze procedure has proven to be very effective in the treatment of atrial fibrillation, some authors have chosen to delete some of the important steps of the technique. Both our experimental and clinical experiences with the Maze procedure indicate that 1 of the most important principles is to interrupt conduction across the posterior-inferior portion of the left atrium. This is accomplished by creating a transmural lesion in the myocardium and then creating a circumferential lesion at the same site in the coronary sinus. We have used surgical incisions in the atrium and a cryolesion in the coronary sinus to block conduction in this area. If either fails, there is a high rate of arrhythmia recurrence. PMID- 10746919 TI - The Cox-Maze procedure: the Cleveland Clinic experience. AB - The Cox-Maze procedure was designed to address the consequences of atrial fibrillation, tachycardia, hemodynamic impairment, and thromboembolism. From 1991 until June 1999, 100 patients underwent the Maze operation at the Cleveland Clinic Foundation. The group included 72 men with a mean age of 58 +/- 11 years (range, 23 to 78 years). Initially, the Maze-I procedure was performed primarily for patients with lone atrial fibrillation. However, since 1995, the Maze-III procedure has been performed exclusively, and it is typically combined with mitral valve repair. Twenty-three patients had only a Maze procedure, 60 patients had the Maze procedure/mitral valve repair, 10 patients had Maze procedure/coronary artery bypass, 6 had Maze procedure/mitral valve replacement, and 1 had Maze procedure/atrial septal defect repair. Chronic atrial fibrillation was present in 78% of patients for a mean of 8 +/- 9 years. There was a 1% perioperative mortality and 5% late mortality rate. Median hospital stay was 9 +/ 5 days. Six patients required new early permanent pacemaker insertion. With a mean follow-up of 3 years, 90.4% of patients are in sinus rhythm (or atrial pacing). Preoperative symptoms were reduced: 24% had preoperative syncope; none had syncope in follow-up; 14% of patients preoperatively had cerebral or systemic emboli; and there were no perioperative or late embolic events. The Maze procedure effectively addressed the major complications of atrial fibrillation and was associated with low perioperative and late morbidity rates. PMID- 10746920 TI - Cox-Maze procedure for atrial fibrillation: Mayo Clinic experience. AB - The Cox-Maze procedure corrects atrial fibrillation in 90% of patients, and successful operation restores sinus rhythm, thereby reducing risks of thromboembolism and anticoagulant-associated hemorrhage. Symptoms such as palpitation and fatigability also improve with restoration of atrioventricular synchrony. At the Mayo Clinic, 221 Cox-Maze procedures were performed from March 1993 through March 1999. Over 75% of patients had associated cardiac disease and concomitant operations. Overall, early mortality was 1.4%, and the incidence of postoperative pacemaker implantation was 3.2%. Limiting incisions to the right atrium simplifies the operation for patients who primarily have tricuspid valve disease, and in early follow-up, outcome appeared to be as good as that achieved with biatrial incisions. The Cox-Maze procedure has proved particularly useful for patients with preoperative atrial fibrillation who require valvuloplasty for acquired mitral valve regurgitation; 87 patients have had this combined procedure, and there have been no early deaths. Further, our experience indicates that ventricular dysfunction is not a contraindication for operation and that restoration of sinus rhythm after the Cox-Maze procedure improves left ventricular ejection fraction in most patients. PMID- 10746921 TI - The Maze procedure: the LDS Hospital experience. AB - At our center we have encouraged consideration of the Maze procedure over noncurative therapies for atrial fibrillation, particularly in patients who have other indications for cardiac surgical intervention. As a result, 78 of the 99 Maze procedures we have performed since 1993 have involved combined procedures. These combined operations included procedures on 1 or more valves in 69 of 78 patients (88%). The unmodified "cut-and-sew" Maze-III technique was used in all patients. There has been no operative mortality, and the median postoperative stay has been 7 days. Cure of atrial fibrillation has been observed in 97% of patients, and pacemaker implantation was required for sick sinus syndrome in 6%. Our results favor broader use of the Maze procedure for symptomatic atrial fibrillation and support use of the original Maze-III technique as the procedure of choice. PMID- 10746922 TI - Treatment of atrial fibrillation using the Maze procedure: the Japanese experience. AB - The results of a questionnaire survey show that surgical treatment for atrial fibrillation (AF) has been performed in more than 2,500 patients in Japan. The methods of treatment used were the Cox-Maze-II procedure, the Cox-Maze-III procedure, the Kosakai-Maze procedure, the left atrial Maze procedure, the right atrial Maze procedure, compartment operation, radial operation, and other modifications. The success rates of the right atrial Maze procedure and compartment operation for AF associated with congenital heart disease were approximately 50%. These success rates were significantly lower than those of the other procedures (P < .005), which were more than 70%. Among the latter procedures, there was no significant difference in success rate. In the author's experience, atrial tachycardias caused by micro-re-entries or acceleration of automaticities were seen in approximately 50% of unsuccessful cases. Any type of Maze procedure can be used to cure atrial arrhythmia that is caused by macro reentries alone. In the future it will be very important to establish indications for the Maze procedure and methods of simplifying the operation. PMID- 10746923 TI - The Maze-III procedure combined with valve surgery. AB - Previous studies have suggested that the Maze procedure is not as effective in controlling atrial fibrillation when the arrhythmia is associated with significant valvular heart disease. In this study, we evaluate our own results in 83 patients who underwent 96 valve procedures in combination with the Maze-III procedure. Our results indicate that the Maze-III procedure is just as safe and effective in controlling atrial fibrillation associated with valvular heart disease as it is in controlling atrial fibrillation not associated with valvular heart disease. PMID- 10746924 TI - Stroke prevention as an indication for the Maze procedure in the treatment of atrial fibrillation. AB - The Maze procedure has proven to be extremely effective in curing medically refractory atrial fibrillation. This analysis of our surgical results with the Maze procedure indicates that the Maze procedure, with or without associated cardiac surgery, has the lowest perioperative stroke rate of any major cardiac surgical procedure. This is surprising in view of the fact that all of the patients who undergo the Maze procedure have an elevated risk of stroke because of the presence of atrial fibrillation. In addition, many of the patients have already had strokes, further increasing the likelihood of perioperative stroke. Only 1 patient has had a stroke in the 12-year follow-up period following the Maze procedure. This is comparable to the risk of stroke in the general population and indicates that the Maze procedure essentially abolishes the risk of stroke associated with atrial fibrillation. PMID- 10746925 TI - Observations on the perioperative management of patients undergoing the Maze procedure. AB - In addition to the usual measures that constitute optimal perioperative care after cardiac surgery, the Maze procedure demands several other measures because of certain complications that are unique to this particular operation. These complications include preoperative conditions such as amiodarone therapy, thromboembolism, diastolic dysfunction of the left ventricle, and associated valvular heart disease, as well as intraoperative differences that include multiple atriotomies and excision of both atrial appendages. The most common postoperative complications are atrial arrhythmias, excessive fluid retention, and pulmonary complications. In this article, we outline our own approach to the perioperative care of patients undergoing the Maze procedure. PMID- 10746926 TI - New surgical and catheter-based modifications of the Maze procedure. AB - There is currently an intense interest in applying the principles of the Maze procedure in a less invasive manner so that a wider group of patients with atrial fibrillation can be treated safely and effectively. These efforts have centered around surgical attempts to curtail the number of lesions placed in the atria at the time of valve surgery and catheter-based attempts to re-create a part or all of the Maze procedure with radiofrequency ablation. Thus far, these techniques remain highly experimental and largely without merit. Many of the problems that we encountered several years ago in developing the surgical Maze procedure are now being repeated in patients undergoing these highly experimental and inadequately evaluated procedures. Nevertheless, there are occasional flashes of promise with some of these approaches. Moreover, it is clear that only a miniscule percentage of the patients with atrial fibrillation will ever become candidates for the open-heart Maze procedure as it is now performed. Therefore, the continuing struggle to relieve the invasive downside of the Maze procedure is warranted but with the caveat, especially to our cardiologist colleagues, to proceed with caution. PMID- 10746927 TI - The relative bioavailability and metabolism of bisphenol A in rats is dependent upon the route of administration. AB - Bisphenol A (BPA) is used to produce polymers for food contact applications, thus there is potential for oral exposure of humans to trace amounts via the diet. BPA was weakly estrogenic in screening assays measuring uterine weight/response, although much higher oral doses of BPA were required to elicit a uterotropic response as compared to other routes of administration. The objective of this study was to determine if a route dependency exists in the pharmacokinetics and metabolism of 14C-labeled BPA following single oral (po), intraperitoneal (ip), or subcutaneous (sc) doses of either 10 or 100 mg/kg to Fischer 344 rats. Results indicated a marked route dependency in the pharmacokinetics of BPA. The relative bioavailability of BPA and plasma radioactivity was markedly lower following oral administration as compared to sc or ip administration. The major fraction of plasma radioactivity following oral dosing was the monoglucuronide conjugate of BPA (68-100% of plasma radioactivity). BPA was the major component in plasma at Cmax following sc or ip administration exceeded only by BPA-monoglucuronide in females dosed ip. Up to four additional unidentified metabolites were present only in the plasma of animals dosed ip or sc. One of these, found only following ip administration, was tentatively identified as the monosulfate conjugate of BPA. The monoglucuronide conjugate was the major urinary metabolite; unchanged BPA was the principal component excreted in feces. These results demonstrated a route dependency of BPA bioavailability in rats, with oral administration resulting in the lowest bioavailability, and offer an explanation for the apparent route differences in estrogenic potency observed for BPA. PMID- 10746928 TI - Localization of N-acetyltransferases NAT1 and NAT2 in human tissues. AB - Human acetyl coenzyme A-dependent N-acetyltransferase (EC 2.3.1.5) (NAT) catalyzes the biotransformation of a number of arylamine and hydrazine compounds. NAT isozymes are encoded at 2 loci; one encodes NAT1, formerly known as the monomorphic form of the enzyme, while the other encodes the polymorphic NAT2, which is responsible for individual differences in the ability to acetylate certain compounds. Human epidemiological studies have suggested an association between the "acetylator phenotype" and particular cancers such as those of the bladder and colon. In the present study, NAT1- and NAT2-specific riboprobes were used in hybridization histochemistry studies to localize NAT1 and NAT2 mRNA sequences in formalin-fixed, paraffin-embedded human tissue sections. Expression of both NAT1 and NAT2 mRNA was observed in liver, gastrointestinal tract tissues (esophagus, stomach, small intestine, and colon), ureter, bladder, and lung. In extrahepatic tissues, NAT1 and NAT2 mRNA expression was localized to intestinal epithelial cells, urothelial cells, and the epithelial cells of the respiratory bronchioles. The observed heterogeneity of NAT1 and NAT2 mRNA expression between human tissue types may be of significance in assessing their contribution to known organ-specific toxicities of various arylamine drugs and carcinogens. PMID- 10746929 TI - Effects of dietary oltipraz and ethoxyquin on aflatoxin B1 biotransformation in non-human primates. AB - Following aflatoxin B1 (AFB) exposure, rats readily develop liver tumors. However, treatment of rats with a variety of compounds, including the synthetic dithiolthione oltipraz and the antioxidant ethoxyquin, protects these rodents from AFB-induced hepatocarcinogenesis. Several epidemiological studies strongly suggest that AFB is also a causative agent of liver cancer in humans. However, relatively little is known about the efficacy of cancer chemoprevention in human and non-human primates. To this end, we examined the effects of chemopreventive agents on AFB metabolism in non-human primates. Hepatic aflatoxin B1 metabolism profiles of macaque (Macaca nemestrina) and marmoset (Callithrix jacchus) monkeys were determined and compared to humans. Quantitatively, the oxidative metabolism of this mycotoxin was similar in the three primate species. In contrast to macaques, both humans and marmosets lacked AFB-glutathione conjugating activity. It was concluded that marmosets resembled human AFB metabolism more closely than the macaques, and therefore, marmoset monkeys were chosen for this study. Eleven adult male marmosets were randomly assigned to three groups. Animals received the synthetic dithiolthione oltipraz, the antioxidant ethoxyquin, or vehicle only. In addition, two single doses of AFB were also administered orally before and after animals were treated with aforementioned compounds. Both oltipraz and ethoxyquin induced aflatoxin B1-glutathione conjugating activity in the livers of some but not all marmosets. In addition, 10 microM oltipraz inhibited cytochrome P450 mediated activation of AFB to the ultimate carcinogenic metabolite, aflatoxin B1 8,9-epoxide, in vitro, up to 51%. Furthermore, animals treated in vivo with oltipraz, but not ethoxyquin, exhibited a significant reduction (53% average) in AFB-DNA adduct formation relative to the control animals (p < 0.05). Together, our data suggest that chemoprevention is also effective in primates; however, most likely to a lesser degree than in rodents. PMID- 10746930 TI - Utility of real time breath analysis and physiologically based pharmacokinetic modeling to determine the percutaneous absorption of methyl chloroform in rats and humans. AB - Due to the large surface area of the skin, percutaneous absorption has the potential to contribute significantly to the total bioavailability of some compounds. Breath elimination data, acquired in real-time using a novel MS/MS system, was assessed using a PBPK model with a dermal compartment to determine the percutaneous absorption of methyl chloroform (MC) in rats and humans from exposures to MC in non-occluded soil or occluded water matrices. Rats were exposed to MC using a dermal exposure cell attached to a clipper-shaved area on their back. The soil exposure cell was covered with a charcoal patch to capture volatilized MC and prevent contamination of exhaled breath. This technique allowed the determination of MC dermal absorption kinetics under realistic, non occluded conditions. Human exposures were conducted by immersing one hand in 0.1% MC in water, or 0.75% MC in soil. The dermal PBPK model was used to estimate skin permeability (Kp) based on the fit of the exhaled breath data. Rat skin K(p)s were estimated to be 0.25 and 0.15 cm/h for MC in water and soil matrices, respectively. In comparison, human permeability coefficients for water matrix exposures were 40-fold lower at 0.006 cm/h. Due to evaporation and differences in apparent Kp, nearly twice as much MC was absorbed from the occluded water (61.3%) compared to the non-occluded soil (32.5%) system in the rat. The PBPK model was used to simulate dermal exposures to MC-contaminated water and soil in children and adults using worst-case EPA default assumptions. The simulations indicate that neither children nor adults will absorb significant amounts of MC from non occluded exposures, independent of the length of exposure. The results from these simulations reiterate the importance of conducting dermal exposures under realistic conditions. PMID- 10746931 TI - Glutathione S-transferase in mucus of rat small intestine. AB - Glutathione S-transferases in the small intestine function in detoxification of electrophilic compounds ingested in foods, dietary supplements, and orally administered drug preparations. Although the required substrate glutathione (GSH) is synthesized in the intestinal enterocytes, the rate of synthesis is slow compared to both the maximal GST activity and the rate of uptake of luminal GSH. GSH is supplied to the intestinal lumen in the bile, and normal luminal concentrations in the rat are about 250 microM. The present study was designed to test the hypothesis that exogenous GSH is used for intestinal conjugation by glutathione S-transferase. The results show that 250 microM of extracellular GSH stimulated conjugation of 1-chloro-2,4-dinitrobenzene by approximately 300% in rat intestinal enterocyte preparations. However, an unexpected finding was that most of this stimulated activity did not depend upon uptake of GSH by the enterocytes but was due to glutathione S-transferase associated with mucus. Immunohistochemistry of glutathione S-transferase in the intact small intestine confirmed that a portion of the GST is present in the mucus layer. The presence of this detoxication enzyme in the extracellular mucus layer provides a novel mechanism for preventing direct contact of potentially toxic dietary electrophiles with the intestinal enterocytes. PMID- 10746932 TI - Differential and non-uniform tissue and brain distribution of two distinct 14C hexachlorobiphenyls in weanling rats. AB - Excretion and tissue retention of a coplanar and a non-coplanar hexachlorobiphenyl (HxCB) were determined 48 h after a single intraperitoneal (ip) dose of 8 mg/kg radiolabeled [14C]-HxCBs to weanling male and female Long Evans rats. The objective was to understand the involvement of initial target organs of chlorobiphenyl (CB) accumulation following acute exposure in immature animals. During the short interval, both HxCBs remained sequestered predominantly in mesenteric fat (compared to subcutaneous fat) and less than 1% of the doses were excreted. Excretion was 4- to 8-fold lower than adult rats. Coplanar CB 169 (3,3',4,4',5,5'-HxCB) did not accumulate appreciably in the brain, but was retained at 3-fold higher levels in the liver than was non-coplanar CB 153 (2,2',4,4',5,5'-HxCB). Accumulation of 14C-CB 153 in brains was 4- to 9-fold higher than that of 14C-CB 169 and was adequate to detect non-uniform distribution in serial cryostat sections by phosphor imaging autoradiography. The autoradiographs showed a higher CB 153-derived radioactivity associated with fiber tracts throughout the brain. Specifically, the corpus callosum, internal and external capsules, medial lemniscus, tegmentum of the mesencephalon and metencephalon, and cerebellar peduncles showed significantly higher 14C-CB 153 than the other structures. The 14C-CB 153 was not found in the ventricular system and vascular spaces. These results suggest for the first time that an ortho substituted PCB congener accumulated preferentially in brain in a structure specific manner when compared to a non-ortho-substituted PCB congener. PMID- 10746933 TI - Sensitivity analysis of a physiological model for 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD): assessing the impact of specific model parameters on sequestration in liver and fat in the rat. AB - 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has dose-dependent tissue distribution because of induction of CYP1A2, a TCDD-binding protein, in the liver. Induction requires transcriptional activation of the CYP1A2 gene product by TCDD and the Ah receptor. An empirical model for dose-dependent distribution (Carrier et al., 1995, Toxicol. Appl. Pharmacol. 131, 253-266) included two simple descriptors: one for the maximum liver sequestration (Fmax) and the other for body burden leading to half maximum sequestration (Kd). Physiologically based pharmacokinetic (PBPK) models include specific parameters for protein receptors, protein binding, tissue solubility, and protein induction. We have applied a PBPK model to define two macroscopic constants related to these dose-response curves, i.e., the inflection point, and the maximum values of these curves. The dose-response curves generated from the PBPK model were for the proportion sequestered in liver and the liver to fat concentration ratio. Our analysis assessed the specific biological factors in the PBPK model that governed the values of these two macroscopic constants. For the fraction in liver, the Hill coefficient (a shape exponent describing the relationship between the Ah receptor-TCDD complex with the DNA receptor) resulted in the largest shift in inflection when using PBPK model parameters specific for TCDD. For the liver to fat ratio, the inflection point was most affected by the number of available Ah receptors. Conventional normalized sensitivity coefficients for the liver-to-fat ratio at the maximum were highest for the fat-to-blood partition coefficient, CYP1A2 binding affinity, and maximum extent of induction of CYP1A2. A similar pattern was observed for the liver fraction, except that the sensitivity coefficients were much smaller. The behavior of different TCDD congeners was evaluated by altering the value of key parameters. Our results demonstrate that the inflection point is more related to characteristics of DNA binding/induction steps of the Ah receptor-DNA complex than by the CYP1A2 affinity of TCDD or concentrations of CYP1A2. Surprisingly, the maximum is more sensitive to changes in CYP1A2 concentrations and affinity for TCDD. In addition, the analysis showed that the liver-to-fat ratio is a more useful experimental measure than is proportion in liver because the ratio responds with similar sensitivity over a much wider range of input parameters. PMID- 10746934 TI - Sex-dependent regulation of hepatic cytochrome P-450 by DDT. AB - Dichlorodiphenyltrichloroethane (DDT) is a well-known inducer of microsomal monooxygenase systems in rodent liver. However, little information is available on its effects on the sex-dependent regulation of CYPs preferentially affected. Therefore, our objective was to evaluate the effects of DDT on the sexual expression pattern of some hepatic P-450 isozymes. Single doses of technical DDT (0, 0.1, 1, 5, 10, or 100 mg/kg body wt) were administered by gavage to Wistar rats. The effects on CYPs 1A1, 2B11/2B2, 2C11, 2E1, 3A1, and 3A2, were assessed 24 h later by means of CYP protein content determined by Western blotting and/or enzyme activities participating in alkoxyresorufin and pnitrophenol metabolism. The highest dose induced 18-fold the expression of CYP3A2 in female rats without producing significant induction (< 3-fold) in males. The effects on this isozyme, which is not normally expressed in females, suggest that DDT is able to modulate sexual metabolic dimorphism, as 3A2 expression is androgen dependent. DDT did not significantly alter CYP3A1 in males, suggesting that DDT is not a pure phenobarbital (PB)-type inducer. The effects on CYP2B1/2B2 protein (19-fold) and associated enzyme activities indicated that males had a lower response threshold than females, but that the latter were able to reach a higher relative induction. The preferential induction of CYPs 2B and 3A by DDT in a sex-related manner suggest that CYP regulation could play an important role in endocrine disruption. PMID- 10746935 TI - Stability of hemoglobin and albumin adducts of benzene oxide and 1,4-benzoquinone after administration of benzene to F344 rats. AB - The stability of cysteinyl adducts of benzene oxide (BO) and mono-S-substituted cysteinyl adducts of 1,4-benzoquinone (1,4-BQ) was investigated in both hemoglobin (Hb) and albumin (Alb) following administration of a single oral dose of 400 mg [U-14C/13C6]benzene/kg body weight to F344 rats. Total radiobound adducts to Hb were stable, as were adducts formed by the reaction of [13C6]BO with cysteinyl residues on Hb. In both cases adduct stability was indicated by zero-order kinetics with decay rates consistent with the lifetime of rat erythrocytes. Hb adducts of 1,4-BQ were not detected, possibly due to the production of multi-S-substituted adducts within the erythrocyte. Regarding Alb binding, total radiobound adducts decayed more rapidly than expected (half-life of 0.4 days), suggesting that uncharacterized benzene metabolites were noncovalently bound or formed unstable adducts with Alb. Although adducts from reactions of BO and 1,4-BQ with Alb both decayed with rates consistent with those of Alb turnover in the rat, the half-life for 1,4-BQ-Alb (2.5 days) was shorter than that for BO-Alb (3.1 days), suggesting some instability of 1,4-BQ-Alb. Assuming similar rates of adduct instability in humans and rats, the 1,4-BQ-Alb adducts would be eliminated with a half-life of approximately 8 days, compared with BO-Alb, which would be expected to turnover with Alb (half-life of approximately 21 days). PMID- 10746936 TI - Xenobiotics modulate the p53 response to DNA damage in preneoplastic enzyme altered foci in rat liver; effects of diethylnitrosamine and phenobarbital. AB - Enzyme-altered foci (EAF) develop in rat liver in response to carcinogen treatment. Our hypothesis is that EAF adapt to genotoxic stimuli by lowering their expression of p53 and that such decreased p53 expression confers a growth advantage on the hepatocytes present in EAF. After a single neonatal dose of diethylnitrosamine (DEN), rats were treated with either 2 - 12 additional doses of DEN or phenobarbital (PB) for 3 - 14 months. Twenty-four hours prior to sacrifice, all rats also received a challenging dose of DEN. The numbers of p53 positive hepatocytes (demonstrating immunohistological staining in the nucleus) in EAF and surrounding tissue were subsequently determined. In DEN-treated rats, p53 expression was attenuated in EAF compared to surrounding tissue. The longer the period of treatment and the larger the size of the EAF, the fewer the p53 positive hepatocytes/mm2 were observed in these lesions. These data were confirmed by Western blot analysis. PB-treated rats did not demonstrate this effect seen in DEN-treated rats. In this case, the expression of p53 was not related to size of EAF or length of treatment. Many EAF in PB-treated animals contained very large numbers of p53-positive cells. Upon staining for terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling (the TUNEL procedure), many apoptotic hepatocytes were also seen in EAF. These data indicate that the p53 response to DNA damage can be modulated by xenobiotics. This can be explained as an adaptive alteration in the p53 response. PMID- 10746937 TI - The influence of antioxidants on cigarette smoke-induced DNA single-strand breaks in mouse organs: a preliminary study with the alkaline single cell gel electrophoresis assay. AB - According to published information, the lung is the only clear target organ for tumors when mice are exposed to cigarette smoke. Liver, skin, and upper digestive tract are target organs when orally or dermally exposed to cigarette smoke condensate, but not kidney, brain, or bone marrow. We tested the genotoxicity of cigarette smoke in the known target organ (lung), possible target organs (stomach and liver), and non-target organs (kidney, brain, and bone marrow) of the mouse using the alkaline single-cell gel electrophoresis (SCG, or comet) assay, as modified by us. We also tested the effect of free radical scavengers on the genotoxicity of the smoke. Male ICR mice were exposed to cigarette smoke. DNA single-strand breaks (SSB) were measured by the SCG assay 15, 30, 60, 120, and 240 min after the exposure. Fifteen min after the animals were exposed for 1 min to a 6-fold dilution of smoke, SSB appeared in the lungs, stomach, and liver; the damage in the lungs and liver returned to almost control levels by 60 min, and that of the stomach by 120 min. Kidney, brain, and bone marrow DNA were not damaged. Exposure to more dilute smoke (12- or 24-fold dilution) did not cause DNA damage. Single oral pretreatment (100 mg/kg) of either ascorbic acid (VC) or alpha-tocopherol acetate (VE) 1 h before smoke inhalation prevented SSB in the stomach and liver, while VE but not VC significantly reduced SSB in the lung. Five consecutive days of either VC or VE (100 mg/kg/day) pretreatment completely prevented SSB in the lung, stomach, and liver. Thus, the SCG assay detected DNA SSB, induced by cigarette smoke, in the known target organ, two possible target organs, and none of the non-target organs. Antioxidants could prevent those effects, suggesting that free radicals may have been a source of the damage. Our results suggest the importance of the SCG assay as a tool in the study of genotoxicity and carcinogenicity. PMID- 10746938 TI - Repeated cadmium exposures enhance the malignant progression of ensuing tumors in rats. AB - Prior studies show that a single subcutaneous (sc) exposure to cadmium (Cd) will induce injection site sarcomas (ISS) in rats. These tumors, thought clearly malignant, do not often metastasize or invade subdermal muscle layers because of their location. Recent evidence indicates that when tumorigenic cells chronically exposed to Cd in vitro are inoculated into mice, tumor progression and invasiveness in the mice are enhanced. Thus, we studied the effects of repeated Cd exposures on tumor incidence, progression, and metastatic potential in rats. Wistar (WF) and Fischer (F344) rats (30 per group) were injected sc in the dorsal thoracic midline with CdCl2 once weekly for 18 weeks with doses of 0, 10, 20, or 30 micromol Cd/kg. This resulted in total doses of 0, 180, 360, or 540 micromol/kg. One other group of each strain received a low, loading dose of Cd (3 micromol/kg) prior to 17 weekly injections of 30 micromol/kg (total dose 513 micromol/kg). Rats were observed for 2 years. Many F344 rats (57%) died within one week after the first injection of the highest dose, but WF rats were not affected. The low loading dose prevented acute lethality of the high dose in F344 rats. Surprisingly, latency (time to death by tumor) of ISS was the shortest in the groups given the low loading dose in both strains. ISS in these groups also showed the highest rate of metastasis and subdermal muscle layer invasion. Based on ISS incidence in the groups given the lowest total dose of Cd (180 micromoles/kg), F344 rats were more sensitive to tumor induction, showing an incidence of 37% compared to 3% in WF rats. On the other hand, Cd-induced ISS showed a higher overall metastatic rate in WF rats (18 metastatic ISS/68 total tumors in all treated groups; 27%) compared to F344 rats (6%). Immunohistochemically, the primary ISS showed high levels of metallothionein (MT), a cadmium-binding protein, while metastases were essentially devoid of MT. These results indicate that repeated Cd exposures more rapidly induce ISS. An initial low exposure to Cd further accelerates the appearance and enhances the metastatic potential and invasiveness of these tumors. The primary and metastatic ISS appear to have a differing phenotype, at least with regard to MT production. The association between multiple Cd exposures and enhanced metastatic potential of the ensuing tumors may have important implications in chronic exposures to Cd, or in cases of co-exposure of Cd with organic carcinogens, as in tobacco smoking. PMID- 10746939 TI - 2-Chloro-s-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for estrogenicity? AB - There is increasing concern that certain chemicals in the environment can cause endocrine disruption in exposed humans and wildlife. Investigations of potential effects on endocrine function have been limited mainly to interactions with hormone receptors. A need exists for the development of alternate in vitro methods to evaluate chemicals for their potential to disturb various endocrine functions via other mechanisms. Our laboratory is using the human H295R adrenocortical carcinoma cell line to examine chemicals for their potential to interfere with the activity and/or expression of several key cytochrome P450 (CYP) enzymes involved in the biosynthesis of steroid hormones. In this report we demonstrated that the commonly used 2-chloro-s-triazine herbicides atrazine, simazine, and propazine dose-dependently (0-30 microM) induced aromatase (CYP19) activity to an apparent maximum of about 2.5-fold in H295R cells. Basal- and triazine-induced aromatase activity was completely inhibited by the irreversible aromatase inhibitor 4-hydroxyandrostenedione (100 microM). The triazines increased levels of CYP19 messenger ribonucleic acid (mRNA) between 1.5- and 2 fold. The time-response profile of the induction of aromatase activity and CYP19 mRNA by the triazines was similar to that by 8-bromo-cyclic adenosine monophosphate, a known stimulant of the protein kinase-A pathway that mediates the induction of aromatase in these cells. The observed induction of aromatase, the rate-limiting enzyme in the conversion of androgens to estrogens, may be an underlying explanation for some of the reported hormonal disrupting and tumor promoting properties of these herbicides in vivo. PMID- 10746940 TI - The contribution of hepatic inactivation of testosterone to the lowering of serum testosterone levels by ketoconazole. AB - Hepatic biotransformation processes can be modulated by chemical exposure and these alterations can impact the biotransformation of endogenous substrates. Furthermore, chemically mediated alterations in the biotransformation of endogenous steroid hormones have been implicated as a mechanism by which steroid hormone homeostasis can be disrupted. The fungicide ketoconazole has been shown to lower serum testosterone levels and alter both gonadal synthesis and hepatic inactivation of testosterone. The present study examined whether the effects of ketoconazole on the hepatic biotransformation of testosterone contribute to its lowering of serum testosterone levels. Results also were used to validate further the use of the androgen-regulated hepatic testosterone 6alpha/15alpha-hydroxylase ratio as an indicator of androgen status. Male CD-1 mice were fed from 0 to 160 mg/kg ketoconazole in honey. Four h after the initial treatment, serum testosterone levels, gonadal testosterone secretion, and hepatic testosterone hydroxylase activity decreased, and the hepatic testosterone 6alpha/15alpha hydroxylase ratio increased in a dose-dependent manner. Immunoblot analysis indicated that the transient decline in hepatic biotransformation was not due to reduced P450 protein levels. Rather, hepatic testosterone biotransformation activities were found to be differentially susceptible to direct inhibition by ketoconazole. Differential inhibition was also responsible for the increase seen in the 6alpha/15alpha-hydroxylase ratio. The changes in serum testosterone levels could be explained by decreased gonadal synthesis of testosterone and were not impacted by decreased hepatic biotransformation of testosterone. These results demonstrate that changes in the hepatic hydroxylation of testosterone by ketoconazole, and perhaps other chemicals, have little or no influence serum testosterone levels. PMID- 10746941 TI - The estrogen receptor relative binding affinities of 188 natural and xenochemicals: structural diversity of ligands. AB - We have utilized a validated (standardized) estrogen receptor (ER) competitive binding assay to determine the ER affinity for a large, structurally diverse group of chemicals. Uteri from ovariectomized Sprague-Dawley rats were the ER source for the competitive-binding assay. Initially, test chemicals were screened at high concentrations to determine whether a chemical competed with [3H] estradiol for the ER. Test chemicals that exhibited affinity for the ER in the first tier were subsequently assayed using a wide range of concentrations to characterize the binding curve and to determine each chemical's IC50 and relative binding affinity (RBA) values. Overall, we assayed 188 chemicals, covering a 1 x 10(6)-fold range of RBAs from several different chemical or use categories, including steroidal estrogens, synthetic estrogens, antiestrogens, other miscellaneous steroids, alkylphenols, diphenyl derivatives, organochlorines, pesticides, alkylhydroxybenzoate preservatives (parabens), phthalates, benzophenone compounds, and a number of other miscellaneous chemicals. Of the 188 chemicals tested, 100 bound to the ER while 88 were non-binders. Included in the 100 chemicals that bound to the ER were 4-benzyloxyphenol, 2,4 dihydroxybenzophenone, and 2,2'-methylenebis(4-chlorophenol), compounds that have not been shown previously to bind the ER. It was also evident that certain structural features, such as an overall ring structure, were important for ER binding. The current study provides the most structurally diverse ER RBA data set with the widest range of RBA values published to date. PMID- 10746942 TI - Estrogenic activity of octylphenol, nonylphenol, bisphenol A and methoxychlor in rats. AB - Considerable attention has recently been focused on environmental chemicals that disrupt the reproductive system by altering steroid receptor function. Although numerous in vitro and in vivo methods have been shown to be useful approaches for identifying chemicals that can disrupt reproduction through a direct interaction with the estrogen receptor, it is imperative that the protocols selected be capable of detecting chemicals with a broad range of estrogenic activity. Here we evaluate the reliability of the 3-day uterotrophic assay for detecting chemicals with strong or weak estrogenic activity in both prepubertal and ovariectomized adult Long Evans rats. These data were compared to additional measures of estrogenic activity, which included the age of vaginal opening, the induction of cornified vaginal epithelial cells in ovariectomized adult rats, and estrous cyclicity in intact adult rats. Test chemicals selected for these studies included 17-beta-estradiol, ethynyl estradiol, methoxychlor, 4-tert-octylphenol, 4-nonylphenol and bisphenol A. Data from in vitro receptor binding assays compared the ability of the test chemicals to compete with [3H]-estradiol or [3H] promegestone for binding to estrogen or progesterone receptors. As expected, the binding affinities for the estrogen receptor ranged from high to low, as reflected by Ki concentrations of 0.4 nM for 17-beta-estradiol and ethynyl estradiol, and 0.05-65 microM for 4-tert-octyphenol, 4-nonylphenol, and methoxychlor. Although none of the test chemicals demonstrated a high affinity for binding to the progesterone receptor, 4-tert-octylphenol and 4-nonylphenol exhibited a weak affinity, with Ki concentrations ranging from 1.2 to 3.8 microM. In vivo studies indicated that the 3-day uterotrophic assay in prepubertal rats was the best method for detecting estrogenic activity when compared with all other end points, based upon the dose-response data for ethynyl estradiol (0.01 0.1 mg/kg), 4-tert-octylphenol (50-200 mg/kg, oral), and 4-nonylphenol (25-100 mg/kg, oral). Although oral doses of ethynyl estradiol (0.01 mg/kg) and 4 nonylphenol (50 mg/kg) induced a significant increase in uterine weight in the prepubertal rats, these doses were ineffective for stimulating a similar response in ovariectomized adult rats. The age of vaginal opening was advanced following oral exposure from postnatal days 21-35 to ethynyl estradiol (0.01 mg/kg), methoxychlor (50 mg/kg), 4-tert-octylphenol (200 mg/kg), and 4-nonylphenol (50 mg/kg). Although bisphenol A (200 mg/kg, oral) induced a significant uterotrophic response within 3 days in prepubertal rats, doses up to 400 mg/kg failed to advance the age of vaginal opening. Monitoring changes in the vaginal epithelium of ovariectomized adult rats was the least effective method for detecting estrogenic activity for 4-tert-octylphenol and bisphenol A. The number of 4-5 day estrous cycles was reduced during a 25-day exposure to ethynyl estradiol (0.01 mg/kg), methoxychlor (50 mg/ kg), 4-tert-octylphenol (200 mg/kg), 4-nonylphenol (100 mg/kg), and bisphenol A (100 mg/kg) by oral gavage. Although long periods of extended diestrus (7-14 days) were generally correlated with exposure to ethynyl estradiol and 4-tert-octylphenol, the cycling patterns following exposure to methoxychlor, 4-nonylphenol and bisphenol A were not as clearly defined, with shorter periods of extended diestrus (4-7 days) and/or estrus (3-5 days) intermittently observed throughout the exposure period. Together these data provide a comparison of the 3-day uterotrophic assay with alternative measures of estrogenic activity for a group of test chemicals with a broad range of affinities for the estrogen receptor. These data can be useful during the assessment and validation of methods for screening environmental chemicals for endocrine disrupting activity. PMID- 10746943 TI - Alterations in endocrine responses in male Sprague-Dawley rats following oral administration of methyl tert-butyl ether. AB - Methyl tert-butyl ether (MTBE) is an oxygenated fuel additive used to decrease carbon monoxide emissions during combustion. MTBE is a nongenotoxic chemical that induces Leydig cell tumors (LCT) in male rats. The mechanism of MTBE-induced LCT is not known; however, LCT induced by other nongenotoxic chemicals have been associated with the disruption of the hypothalamus-pituitary-testicular (HPT) axis. The objective of this study was to determine whether MTBE functions as an endocrine-active compound by affecting levels of specific hormones involved in the maintenance of the HPT axis. Nine-week-old male Sprague-Dawley rats were administered MTBE by gavage at 0, 250, 500, 1000, or 1500 mg MTBE/kg/day for 15 or 28 consecutive days and sacrificed 1 h following the last dose. Relative testis weights were increased only in high-dose animals treated for 28 days, and no testicular lesions were observed at any dose level. Adrenal gland, liver, and kidney weights were also increased. Histologic changes included protein droplet nephropathy of the kidney and centrilobular hypertrophy of the liver. Interstitial fluid and serum testosterone levels as well as serum prolactin levels were decreased only in animals treated with 1500 mg MTBE/kg/day for 15 days. At 28 days, serum triiodothyronine (T3) was significantly decreased at 1000 and 1500 mg MTBE/kg/day compared to control animals, and a decrease in serum luteinizing hormone and dihydrotestosterone was observed at 1500 mg MTBE/kg/day. These results indicate that MTBE causes mild perturbations in T3 and prolactin; however, the changes in testosterone and LH levels did not fit the pattern caused by known Leydig cell tumorigens. PMID- 10746944 TI - Effects of the oxidant potassium permanganate on the expression of gill metallothionein mRNA and its relationship to sublethal whole animal endpoints in channel catfish. AB - Potassium permanganate is an oxidant heavily used in fish culture. The effects of this compound were examined utilizing molecular (Metallothionein) and whole animal endpoints following an 8-week exposure to nominal concentrations of 0.5 (daily) and 1.0 and 2.0 mg/L (on alternate days) of potassium permanganate (PM). In order to measure MT, a complementary DNA clone of metallothionein (MT) was cloned and sequenced from the liver of channel catfish treated with a single injection of cadmium chloride (10 mg/kg). The cDNA was obtained by reverse transcriptase polymerase chain reaction (RT-PCR), using 3' rapid amplification of cDNA ends (RACE) technique. No significant correlation was observed with gill MT expression or sublethal endpoints indicative of toxicity (weight, length, condition index [CI], or liver somatic index [LSI). MT mRNA expression in gill was significantly reduced only after 8 weeks in the 2.0 mg/L treatment. Decreases in CI were observed in males at all time points after 4 weeks, at the 2.0 mg/L treatment concentration, with a NOEC of 1 mg/L. Reductions in LSI that were not dose dependent were also observed in both males and females throughout the 8-week study and no consistent reduction in weight gain or length was observed. These data demonstrate that minimal changes in sublethal effects occur in fish following 0.5-2.0 mg/L PM treatment after 4 weeks, but recovery from adverse effects is observed by 8 weeks, suggesting that acute (typically less than 1 week) treatment of channel catfish with PM would not significantly affect fish health. PMID- 10746945 TI - Development and modification of a recombinant cell bioassay to directly detect halogenated and polycyclic aromatic hydrocarbons in serum. AB - Polycyclic and halogenated aromatic hydrocarbons (PAHs/HAHs) are a diverse group of widespread and persistent environmental contaminants that can cause a variety of detrimental effects in vertebrates. As most available methods to detect these contaminants are expensive, labor and time intensive, and require large amounts of tissue for extraction and analysis, several rapid mechanistically based bioassay systems have been developed to detect these chemicals. Here we describe application and optimization of a recently developed recombinant mouse cell bioassay system that responds to both PAHs and HAHs with the rapid induction of firefly luciferase for the detection of these chemicals in whole serum samples. This chemically activated luciferase expression (CALUX) bioassay has been modified to allow rapid (4-h) and direct analysis of small volumes (25-50 microl) of whole serum in a 96-well microtiter plate format without the need for solvent extraction. This bioassay can detect as little as 10 parts per trillion of the most potent HAH, 2,3,7,8-TCDD, and is also sensitive to other HAHs and PAHs. The use of simple procedures corrects for interplate and intraplate variability and the Ah receptor dependence of the induction response is accounted for by use of the antagonist 4-amino-3-methoxyflavone. PMID- 10746946 TI - Gliotoxin-induced cytotoxicity proceeds via apoptosis and is mediated by caspases and reactive oxygen species in LLC-PK1 cells. AB - Renal failure associated with aspergillosis is caused by pathogenic fungi. Gliotoxin is a toxic epipolythiodioxopiperazine metabolite produced by the pathogens. The present study investigated the cytotoxicity and underlying mechanisms induced by gliotoxin in LLC-PK1 cells, a porcine renal proximal tubular cell line. Gliotoxin at 100 ng/ml did not show a cytotoxic effect, but unmasked a dose-dependent cell death induced by TNF-alpha. TNF-alpha-induced cell death in the presence of gliotoxin was associated with hypodiploid nuclei and activation of caspase-3-like proteases. Blockade of caspases by boc-aspartyl (OMe)-fluoromethylketone and z-DEVD.fmk inhibited TNF-alpha-induced cell death. As the concentrations of gliotoxin were increased, gliotoxin killed the cells directly in a dose-dependent manner. Further analyses of DNA fragmentation, hypodiploid nuclei, mitochondrial membrane potential, and plasma membrane integrity revealed that cell death proceeded via apoptosis. Gliotoxin-induced apoptosis was associated with dose-dependent and time-dependent activation of caspase-3-like proteases. Boc-aspartyl (OMe)-fluoromethylketone attenuated the killing effect. Gliotoxin also increased the intracellular levels of reactive oxygen species as measured by flow cytometry. N-acetylcysteine, a well-known antioxidant, completely abolished the gliotoxin-induced caspase-3-like activity, cytotoxicity, and reactive oxygen species. In conclusion, (1) gliotoxin at 100 ng/ml unmasks the ability of TNF-alpha-induced apoptosis, and the effect of TNF alpha is mediated by caspase-3-like proteases; and (2) at higher concentrations gliotoxin itself induces cell death, which is via apoptosis and dependent on caspase-3-like activity and reactive oxygen species. PMID- 10746947 TI - Sex differences in diquat-induced hepatic necrosis and DNA fragmentation in Fischer 344 rats. AB - Redox cycling metabolism of diquat catalyzes generation of reactive oxygen species, and diquat-induced acute hepatic necrosis in male Fischer 344 (F344) rats has been studied as a model of oxidant mechanisms of cell killing in vivo. At equal doses of diquat, female F344 rats sustained less hepatic damage than did male rats, as estimated by plasma alanine aminotransferase (ALT) activities after 6 h. Biliary efflux of glutathione disulfide (GSSG) was greater in male than in female rats at each dose of diquat, but even comparable rates of GSSG excretion were associated with less hepatic injury in female rats. Hepatic activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) were similar in the two genders, and activities of glutathione reductase (GR) and glutathione S transferase-alpha (GST-alpha) activities were higher in the male rats. Previous studies in male rats have implicated formation of 2,4-dinitrophenylhydrazine (DNPH)-reactive "protein carbonyls" and related iron chelate-catalyzed redox reactions as mechanisms critical to diquat-induced acute cell death in vivo. However, diquat-treated female rats showed higher levels of DNPH-reactive proteins in livers and in bile than did males, both at identical doses of diquat and at doses that produced similar elevations in plasma ALT activities. In female rats, fragmentation of hepatic deoxyribonucleic acids (DNA) was increased by doses of diquat that did not increase plasma ALT activities, and increased fragmentation was observed prior to elevation of plasma ALT activities. In the present studies, hepatic necrosis was most closely associated with DNA fragmentation, although additional studies are needed to determine the mechanisms responsible for and the pathophysiological consequences of the fragmentation. PMID- 10746948 TI - Protection from cytotoxic effects induced by the nitrogen mustard mechlorethamine on human bronchial epithelial cells in vitro. AB - The present study was undertaken to find potent molecules against the toxicity of nitrogen mustard mechlorethamine (HN2) on respiratory epithelial cells, using a human bronchial epithelial cell line (16HBE14o-) as an in vitro model. The compounds examined included inhibitors of poly(ADP-ribose) polymerase (PARP), sulfhydryl-group donors as nucleophiles, and iron chelators and inhibitors of lipid peroxidation as antioxidants. Their effectiveness was determined upon observance of metabolic dysfunction induced by HN2 following a 4-h exposure, using (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and ATP-level assays as indicators. Moreover, the fluorescent probe, monobromobimane (mBBr), and 2',7'-dichlorofluorescin-diacetate (H2DCF-DA) were used to assess intracellular sulfhydryl and peroxide level modifications by flow cytometry, respectively, following a 3-h exposure. At last, cell death was assessed by flow cytometry using the propidium iodide (PI)-dye-exclusion assay following 24-h exposure. PARP inhibitors (niacinamide, 3-aminobenzamide, 6(5H) phenanthridinone), and two sulfhydryl-group donors (N-acetylcysteine, WR-1065) were found to be effective in preventing HN2-induced metabolic dysfunction when added in immediate or delayed treatment with HN2. Only N-acetylcysteine, however, was found to prevent cell death induced by HN2, though it must be present at the time of the HN2 challenge. Flow cytometric measurements of intracellular sulfhydryl levels strongly suggested that N-acetylcysteine and WR-1065 are preventive in alkylation of cellular compounds, mainly by direct extracellular interaction with HN2. PARP inhibitors prevent secondary deleterious effects induced by HN2, considering metabolism dysfunction as the endpoint. Elsewhere, the oxidative stress appears to be a side effect in HN2 toxicity only upon considering the inefficiency of several antioxidants. PMID- 10746949 TI - Uptake of styrene in the upper respiratory tract of the CD mouse and Sprague Dawley rat. AB - Inspired styrene is an olfactory toxicant in the mouse and rat. To provide nasal dosimetric information, upper respiratory tract (URT) uptake efficiency (UE) of styrene was measured in the surgically isolated URT of the urethane-anesthetized CD mouse and Sprague Dawley rat throughout a 45-min exposure. In the first studies, the effect of inspiratory flow rate on styrene UE was examined. At flows of 12-, 24-, or 70-ml/min average UE of 17, 9.8, and 4.1%, respectively, were observed in the mouse. For the rat, UE averaged 14, 9.1 and 5.7% at flow rates of 70, 150, and 400 ml/min, respectively. In the second study, UE was measured at inspired concentrations of 5, 10, 25, 50, 100, or 200 ppm at a flow rate of 12 ml/min in the mouse and 70 ml/min in the rat in both naive and metyrapone (150 mg/kg sc) pretreated animals. In the rat, steady state UE decreased with increasing exposure concentration, averaging between 24 and 10% efficiency at 5 to 200 ppm (p < 0.0001). Metyrapone pretreatment resulted in statistically significant reductions in UE with steady-state UE averaging 10-14% at 5-200 ppm. Metyrapone pretreatment abolished the concentration dependence. In naive mice, styrene UE did not maintain a steady state, but steadily declined during exposure. The mechanisms of the non-steady state behavior are not known, but they appear to be due to a styrene metabolite, as evidenced by the fact that steady state UE was observed in metyrapone-pretreated mice. In the mouse, UE averaged between 42 and 10% efficiency at 5 to 200 ppm (p < 0.0001). Metyrapone pretreatment resulted in statistically significant reductions in UE, with steady state UE averaging 20-10% at 5-200 ppm. As in the rat, metyrapone pretreatment abolished the concentration dependence. In toto, these data provide strong evidence that inspired styrene is metabolized in nasal tissues in the rat and mouse and that a metabolic basis exists for the observed inspired concentration dependence of UE. PMID- 10746950 TI - The role of dispersion in particle deposition in human airways. AB - Aerosol dispersion and deposition are processes that occur concurrently in human airways. However, dispersion has not been properly accounted for in most deposition models. In this paper we have incorporated the latest understanding of dispersion into a dosimetry model and study the influence of dispersion on particle deposition in the lung. We show that dispersion influences the total deposition of inhaled particles and in particular increases the pulmonary deposition of fine mode particles. We also discuss how dispersion can help elucidate a number of clinical and epidemiologic results associated with particle deposition in the lung. PMID- 10746951 TI - Inhalation tolerance study for p-aramid respirable fiber-shaped particulates (RFP) in rats. AB - This study was designed to assess the lung clearance function in rats after subchronic exposure to p-aramid respirable fiber-shaped particulates (RFP). Male Wistar rats were exposed 6 hrs/day, 5 days/week for 3 months to 50, 200, and 800 RFP/ml measured by scanning electron microscopy (SEM). Recovery effects were followed up through 9 months postexposure. The retention of RFP (length > 5 microm) was about 25 x 10(6) RFPs per lung in the low dose group after 3 months of exposure. The corresponding values in the medium and high dose groups amounted to overproportionally higher values of 122 x 10(6) and 576 x 10(6) RFPs per lung, respectively. A decrease in the length of the retained RFPs over the 9-month recovery period was observed, indicating a breakage of long fibrils. Alveolar clearance half-times measured by gamma tracers indicated a dust overloading of lungs for the high dose group at 0 and 3 months postexposure. Bronchoalveolar lavage parameters revealed that p-aramid RFPs induced pronounced inflammatory effects in the high and medium dose groups. Histopathologically, slight fibrotic and hyperplastic lesions were observed in the medium and high dose groups directly after the end of exposure. The findings at the 3-month postexposure interval resulted in a reduction of inflammatory changes in the medium and high dose groups compared to the sacrifices upon cessation of exposure. No histopathologic effects were detected in the low dose group. In the high dose group the maximum functionally tolerated dose was exceeded. The No Observed Adverse Effect Level (NOAEL) of RFP was 50 RFP/ml as measured by SEM. PMID- 10746952 TI - Family approach for estimating reference concentrations/doses for series of related organic chemicals. AB - The family approach for related compounds can be used to evaluate hazard and estimate reference concentrations/doses using internal dose metrics for a group (family) of metabolically related compounds. This approach is based upon a simple four-step framework for organizing and evaluating toxicity data: 1) exposure, 2) tissue dosimetry, 3) mode of action, and 4) response. Expansion of the traditional exposure-response analysis has been increasingly incorporated into regulatory guidance for chemical risk assessment. The family approach represents an advancement in the planning and use of toxicity testing that is intended to facilitate the maximal use of toxicity data. The result is a methodology that makes toxicity testing and the development of acceptable exposure limits as efficient and effective as possible. An example is provided using butyl acetate and its metabolites (butanol, butyraldehyde, and butyrate), widely used chemicals produced synthetically by the industrial oxo process. A template pharmacokinetic model has been developed that comprises submodels for each compound linked in series. This preliminary model is being used to coordinately plan toxicity studies, pharmacokinetic studies, and analyses to obtain reference concentrations/doses. Implementation of the family approach using pharmacokinetic modeling to obtain tissue dose metrics is described and its applications are evaluated. PMID- 10746953 TI - Acute hepatotoxicant exposure induces TNFR-mediated hepatic injury and cytokine/apoptotic gene expression. AB - Tumor necrosis factor receptor knockout (TNFR KO) mice were used to examine the role of tumor necrosis factor-alpha (TNFalpha) signaling during acute hepatotoxicant exposure. Mice were exposed intraperitoneally (ip) to either vehicle, phosphate-buffered saline (PBS), or dimethylnitrosamine (DMN, 100 mg/kg) for 24 h. Histological evaluation showed that DMN-treated TNFR-2 KO mice had increased liver damage compared to wild type (WT), TNFR-1 KO, or TNFR double KO (DKO) mice. Also, 3 of 8 TNFR-2 KO mice died following DMN treatment, suggesting that hepatic TNFR-2 signaling produces protective responses that counteract TNFR 1-mediated damage. DMN-induced cellular infiltration was absent in TNFR-1 deficient mice, indicating that infiltrating cells do not exacerbate acute hepatotoxic events. In separate experiments, mice were exposed ip to either DMN (5.0 or 100 mg/kg), carbon tetrachloride (CCl4, 0.3 or 1.0 ml/kg), or corresponding PBS/corn oil controls for 6 or 24 h to compare the hepatic mRNA expression of cytokine- and apoptotic-associated genes. Following 24 h of DMN (100 mg/kg) or 6-24 h of CCl4 treatment, hepatic transcripts for TNFalpha, interferon (IFN)-gamma, IL (interleukin)-1RI, and transforming growth factor (TGF)-betaRII were induced. Hepatotoxicant-treated WT and TNFR DKO mice induced liver transcripts for the pro- and anti-apoptotic genes, Bax and Bcl-X(L), respectively, indicating TNF-independent gene activation. The anti-apoptotic gene, Bfl-1, was highly expressed in CCl4-treated, TNFR-positive strains, but minimally expressed in TNFR DKO mice, suggesting that hepatic Bfl-1 is TNF regulated. Taken together, these data show that acute hepatotoxicant exposure is followed by upregulation of liver cytokine, cytokine receptor, and apoptotic transcripts, and that TNFalpha regulates various aspects of liver inflammation and injury in a TNFR-specific fashion. PMID- 10746954 TI - Capillary electrophoresis as a versatile tool for the bioanalysis of drugs--a review. AB - This review article presents an overview of current research on the use of capillary electrophoretic techniques for the analysis of drugs in biological matrices. The principles of capillary electrophoresis and its various separation and detection modes are briefly discussed. Sample pretreatment methods which have been used for clean-up and concentration are discussed. Finally, an extensive overview of bioanalytical applications is presented. The bioanalyses of more than 200 drugs have been summarised, including the applied sample pretreatment methods and the achieved detection limits. PMID- 10746955 TI - Directly-coupled HPLC-NMR spectroscopic studies of metabolism and futile deacetylation of phenacetin in the rat. AB - The metabolism and futile deacetylation of phenacetin has been investigated in the rat via 1H NMR spectroscopic analysis of urine. Animals were dosed with either phenacetin or phenacetin-C2H3 and urine samples were collected for -24-0 (pre-dosing), 0-8. 8-24, and 24-48 h post-dosing. Drug metabolites of the two compounds were concentrated from the urine using solid-phase extraction prior to the use of directly-coupled HPLC-1H NMR spectroscopy for separation and identification. Following dosing of phenacetin, the metabolites identified were paracetamol glucuronide, paracetamol and N-hydroxyparacetamol, whilst paracetamol and N-hydroxyparacetamol sulphate were identified following dosing of phenacetin C2H3. Quantitatively the percentage futile deacetylation of phenacetin-C2H3 metabolites was found to be 32% in both paracetamol and N-hydroxyparacetamol sulphate. This study further indicated the importance of futile deacetylation in simple analgesics and the value of directly-coupled HPLC-NMR spectroscopy for the study of this process. PMID- 10746956 TI - Nested designs in ruggedness testing. AB - Nested designs were performed in order to execute a ruggedness test according to the United States Pharmacopeia definition for ruggedness, in which mainly non procedure related factors are examined. Several nested designs have been executed on a high performance liquid chromatography assay to determine tetracycline and related substances in bulk samples of tetracycline. Factors such as different laboratories, analysts, instruments, columns, days and batches were examined. The interpretation methods described in the literature were found to cause problems. In these methods the variances of the examined factors are estimated from the calculated mean square values and from the equation for the expected mean squares. Very frequently, negative variance estimates were obtained. Their absolute values were found to be dependent on the influence of the factor examined below it in the design, on the examined response. Therefore an alternative interpretation method for nested designs, based on pooled variances, was proposed and found to be appropriate to use for ruggedness testing purposes. Both approaches, the one from the literature and the one proposed here, were tested on simulated data coming from a nested design with four factors and on the experimentally measured data. PMID- 10746957 TI - 'High throughput' solid-phase extraction technology and turbo ionspray LC-MS-MS applied to the determination of haloperidol in human plasma. AB - A quantitative method for the analysis of haloperidol in human plasma is described. Sample clean-up was performed by means of solid-phase extraction using 3M Empore extraction disk plates in the 96-well format, automated with a Canberra Packard pipetting robot. Separation was performed by reversed phase high performance liquid chromatography with turbo ionspray tandem mass spectrometric detection by monitoring the decay of protonated haloperidol of m/z 376 to its fragment at m/z 165, versus the decay of protonated haloperidol-D4 at m/z 380 to its fragment at m/z 169. The validated concentration range was from 0.100 to 50.0 ng ml(-1), with an inaccuracy and overall imprecision below 10% at all concentration levels. Validation results on linearity, specificity, precision, accuracy and stability are shown and are found to be adequate. The average sample preparation time for a batch of 96 samples is approximately 50 min. The chromatographic run time is 3 min. A sample throughput of at least 240 samples per day can be achieved. PMID- 10746958 TI - Capillary zone electrophoresis determination of meropenem in biological media using a high sensitivity cell. AB - A capillary electrophoresis method with a high sensitivity cell (Z-cell) has been developed for the determination of meropenem in aqueous solution and in biological media (urine, plasma). Water samples were analysed using two calibration curves of meropenem with standard capillary and a capillary with a high sensitivity cell. In urine, the samples were only diluted in buffer and were injected without any further sample preparation. For the analysis of plasma samples, a calibration curve was utilized covering the meropenem concentration range of 0.5-200 microg/ml. The detection limit and the relative standard deviation of the migration times and of the peak areas were determined. PMID- 10746959 TI - The use of high-performance size exclusion chromatography (HPSEC) as a molecular weight screening technique for polygalacturonic acid for use in pharmaceutical applications. AB - Polygalacturonic acid is a linear carbohydrate polymer of monomeric galacturonic acid. It is commercially available as apple and citrus pectins comprised of a mixture of partially methoxylated and/or amidated polygalacturonic acids with molecular weights ranging from 25,000 to > 100,000 Da. Pectin can be chemically or enzymatically hydrolyzed to yield polygalacturonic acid fractions of diverse average molecular weight ranges and polydispersities for a variety of uses. Pectin and polygalacturonic acid are used extensively as gelling agents and stabilizers by the food industry, and have applications as therapeutic, and diagnostic pharmaceutical agents such as the magnetic resonance imaging agent LumenHance. A simple high-performance size exclusion chromatography (HPSEC) method, employing commonly available non-specialized HPLC instrumentation, is described for use as a rapid molecular weight screening technique to determine the average molecular weight range and polydispersity of polygalacturonic acid intended for use in pharmaceutical formulations. A TosoHaas G3000PWXL HPLC column, 50 mM phosphate buffer (pH approximately 6.9) mobile phase, and refractive index detection were used. A molecular weight calibration curve was linear for polysaccharide standards of 180-100,000 Da with a coefficient of correlation of 0.999. The method was employed to screen commercially available polygalacturonic acid raw materials for average molecular weight data (Mn, Mw, and Mp) and polydispersity (Mw/Mn). PMID- 10746960 TI - An enzyme immunoassay to determine the levels of specific antibodies toward bacterial surface antigens in human immunoglobulin preparations and blood serum. AB - Human polyvalent intravenous immunoglobulin (IVIG) preparations are used as a complementary aid to the proper antimicrobial treatment of severely septic patients in intensive care units (ICUs) and/or as a prophylactic agent to immunocompromised hosts, particularly prone to bacterial infections. There is skepticism about the usefulness of IVIGs since it is not known whether their administration ensures the enhancement of humoral immune responses by providing a sufficient amount of specific antibodies towards the specified bacterial pathogen to be treated. In this report, a simple and reproducible enzyme-linked immunosorbent assay for determining the content of specific antibodies against bacterial surface antigens in commercially available IVIG preparations is described. The method is also easily applied to determine the amount of bacterial antibodies in blood serum. The levels of specific antibodies toward gram positive and negative pathogenic isolates often encountered in ICUs were estimated in two IVIG (Sandoglobulin and Gamimmune) preparations. Significant differences regarding the content of antibodies to certain clinically bacterial isolates were identified not only between the two IVIG preparations tested, but also among various lots from each IVIG preparation. No significant variation (P < or = 0.001) among the bottles derived from the same lot was determined in both preparations. The variation in the levels of specific antibodies in IVIG preparations may be attributed to differences between the donor pools as well as the manufacturing procedure. Application of the method to patients with primary immune deficiencies showed that infusion of highly reactive IVIG preparations enhanced significantly their humoral response toward various pathogens. The results of this study suggest that the content determination of pathogen-specific antibodies in IVIG preparations before administration may be of great importance for treating bacterial infections. PMID- 10746961 TI - Simple kinetic method for the analysis of m-dope in pharmaceutical preparations. Part II. PMID- 10746962 TI - Clinicopathological study of pineal parenchymal tumors: correlation between histopathological features, proliferative potential, and prognosis. AB - This study describes the clinicopathologic features of 13 cases with pineal parenchymal tumors. Based on the histopathologic findings, especially the extent of atypia and pineocytic differentiation as determined by Bodian's staining, we classified these tumors into pineocytomas (4), pineocytomas with anaplasia (4) and pineoblastomas (5). All the cases with pineocytoma and pineocytoma with anaplasia were adults, and all the cases with pineoblastoma were younger children. One patient with pineocytoma died of other disease 7 months after initial treatment. One patient with pineocytoma with anaplasia died 168 months after initial treatment. The other patients with pineocytoma and pineocytoma with anaplasia survived between 9 and 179 months after surgery. However, all of the five pineoblastoma patients died within 14 months after initial treatment. The mean MIB-1 index in pineoblastomas was significantly higher than that in other types of pineal parenchymal tumors, but there were no differences between pineocytomas and pineocytomas with anaplasia with respect to the MIB-1 index. The mean MIB-1 index in neurofilament protein-immunopositive cases was significantly lower than that in immunonegative cases. With regard to the malignant potential, we emphasize that a clear distinction should be made between pineoblastomas in children and other types of pineal parenchymal tumors in adults. PMID- 10746963 TI - A case of endolymphatic sac tumor with long-term survival. AB - A 72-year-old man developed left facial palsy at age 14 and left-sided hearing loss at age 20. At the age of 59, he presented with gait disturbance, and a large left cerebellopontine angle tumor was detected, which had markedly destroyed the pyramidal bone. The tumor was subtotally resected, but he required two more operations at the ages of 64 and 69 because of tumor regrowth. At the present time, recurrent tumor has destroyed the occipital bone and is invading the scalp. However, even though he has several cranial nerve palsies and cerebellar ataxia, he remains in stable condition and demonstrates long-term survival. The patient's surgical specimens revealed a papillary adenoma, which was recently thought to be of endolymphatic sac origin, although the origin of this kind of tumor, whether arising from the middle ear or from the endolymphatic sac, has not been established with certainty so far. In this paper, we provide further evidence that this tumor originates from the endolymphatic sac, based on anatomical, histopathological, and embryological evidence. PMID- 10746964 TI - Secretory meningioma with lipomatous component: case report. AB - A case of meningioma coexisting with both lipomatous and secretory components, the latter characterized by hyaline inclusion bodies, is reported. The neuroradiological features of lipomeningioma are reevaluated, and the possible pathogenetic mechanisms of this unique combination in the present case are discussed. PMID- 10746965 TI - Malignant pilocytic astrocytoma in the medulla oblongata: case report. AB - A 27-year-old woman visited our hospital with chief complaints of abducens nerve palsy and cerebellar symptoms. On computerized tomographic scanning and magnetic resonance imaging, a tumor with strong enhancement was found on the dorsal side of the medulla oblongata. A tumor was excised by suboccipital craniotomy and C1 laminectomy. Histologically, many Rosenthal fibers together with pilocytic tumor cells were found in some regions, but a very high Ki-67 labeling rate accompanied by cells with nuclei of irregular size and giant cells was observed in other regions. The tumor was diagnosed as malignant pilocytic astrocytoma originating from pilocytic astrocytoma by transformation. The biological behavior of pilocytic astrocytoma is obscure in several respects. We report our experience of a case of malignant pilocytic astrocytoma that developed in the brain stem and progressed extremely rapidly. PMID- 10746966 TI - Intranuclear inclusions of meningioma associated with abnormal cytoskeletal protein expression. AB - We describe a case of meningothelial meningioma with a large number of intranuclear inclusions. Morphologically, these are divided into cytoplasmic inclusions and nuclear vacuoles. The cytoplasmic inclusion has a limiting membrane with cell organelles and filaments. Inclusions of this type are generally eosinophilic, like the cytoplasm. However, there are many inclusions that are more eosinophilic than the cytoplasm or that have a ground-glass appearance. Some of them may contain fine or coarse granules. On the other hand, the nuclear vacuole lacks a limiting membrane and appears empty. In most of the inclusions of this type, there is a faintly basophilic substance in the margin. Generally, the cytoplasmic inclusions are as immunopositive as cytoplasm with vimentin, but some of these cytoplasmic inclusions are more reactive. Under the electron microscope, abnormal aggregation of intermediate filaments is recognized in the cytoplasmic inclusions. It is considered that a strong reaction of cytoplasmic inclusions with vimentin immunostaining is due to abnormal aggregation of intermediate filaments. The present study distinctly demonstrates abnormal localization of intermediate filaments in the cytoplasmic inclusions, and it is suggested that the cytoskeleton participates in the evolution of the cytoplasmic inclusions. PMID- 10746967 TI - Primary CNS lymphoma associated with streptococcal abscess: an autopsy case. AB - This report describes a case of streptococcal abscess in the nodules of a primary central nervous system (CNS) lymphoma. Magnetic resonance imaging (MRI) of the brain revealed multiple lesions with ringlike enhancement over the bilateral frontal, right temporal, and left parietal lobes. On admission, acute brain edema occurred following angiography, which resulted in respiratory arrest. Autopsy findings showed that the ringlike enhanced lesions on MRI were streptococcal abscesses localized in the lymphoma nodules. The lymphoma was classified as non Hodgkin, diffuse large cells of B-cell lineage. No other lymphoma mass was found extracranially. An immunohistochemical study showed that the lymphoma cells were positive for leukocyte common antigen, Epstein-Barr virus, bax. and bcl-XL, and negative for L-26 and bcl-2. This case demonstrated that an opportunistic streptococcal abscess developed in primary CNS lymphoma in a patient without acquired immunodeficiency syndrome (AIDS), though a few similar cases have been reported in patients with AIDS. PMID- 10746968 TI - Cytoreductive surgery for stage IV epithelial ovarian cancer. AB - We tried to determine the role of cytoreductive surgery for stage IV epithelial ovarian cancer and in what conditions this surgical procedure could carry the best benefits. From January 1986 to December 1997, seventy-one of 73 patients with stage IV epithelial ovarian cancer who were treated in Cancer Hospital of Shanghai Medical University were retrospectively reviewed. Clinical information including age, grade, histology, presence of ascites, size of residual disease, site of extra-abdominal metastasis, whether initially presenting as metastatic disease or not, neo-adjuvant chemotherapy, platinum-based chemotherapy and second line chemotherapy was obtained. Survival was calculated by life-table and survival curves were computed using the Kaplan-Meier method with differences in survival estimated by log-rank test. Independent prognostic factors were identified by Cox's proportional hazards regression model. The median age of the patients' population was 54 years (range 22-82), median follow-up time was 12 months (range 3 to 130) and estimated 5-year survival rate 6.1%. Thirty out of 71 (42.3%) patients were successfully debulked (< or = 1 cm) at the time of initial surgery. There was a significant difference in five-year survival rate between patients optimally (14.1%) vs suboptimally (0%) cytoreduced, with an estimated median survival in the optimal group of 23 months vs 9 months in the suboptimal group (P=0.0001, long-rank test). When the variables were factorized, only in patients with malignant pleural effusion or positive supraclavicular lymph nodes, optimal cytoreduction could get the greatest benefits. Multivariate analysis revealed that the size of residual disease and ascites were independent factors of survival. However, only ascites was the prognostic factor of progression-free survival. Optimal cytoreductive surgery is an important determinant of survival in women with stage IV epithelial ovarian cancer, mainly in those with malignant pleural effusion or positive supraclavicular lymph node pathology. PMID- 10746969 TI - D2 lymphectomy in the treatment of gastric cancer: a retrospective view on our experience (1990-1997). AB - From 1990 to 1997 we observed, in our department, 267 patients affected by gastric cancer. In the first four years of our experience (1990-93) we resected 87 patients out of 136 gastric cancers observed (63.9%): 56 pts. (64.4%) were classified as ASA I-II, 21 (24.1%) as ASA III, 10 (11.5%) as ASA IV. In 2 cases (2.3%), operated in emergency, a DO-1 lymphectomy was performed, with a mean of 4 nodes resected; 67 pts. (77.0%) had a D2, with a mean of 36.5 nodes resected; in 18 pts. (20.7%) we performed a D3, with a mean of 64.3 nodes resected. Post operative technical complications were 13 (14.9%). We observed 5 post-operatory deaths (5.7%), 3 due to technical complications. Absolutely and relatively curative resections have been 62 (71.3%). In the second period (1994-97) we resected 89/131 patients (67.9%): in this group 50 pts. (56.2%) were classified as ASA I-II, 24 (26.9%) as ASA III, 15 (16.9%) as ASA IV. With the exception of 3 patients (3.4%) who were operated in emergency (D0-1 procedures, with a mean number of 2.3 nodes resected) we adopted D2 lymphadenectomy plus hepatic peduncle as the procedure of choice, performed in 86 pts. (96.6%), with a mean of 34.1 nodes resected. Post-operative technical complications were 5 (5.6%). We observed 5 post-operatory deaths (5.6%), 1 due to technical problems. Absolutely and relatively curative resections have been 76 (85.4%). Morbidity and mortality due to technical complications in the second period are lower than observed in the first period, without any difference in the curability rate. D2 lymphectomy seems to be an effective procedure, safe even in high anesthesiological risk patients. Increasing experience and standardization of the technique reduced risk of surgical complications and mortality. PMID- 10746971 TI - Endoscopic follow-up in oncological diseases of the gastrointestinal tract: the experience of the Regina Elena Cancer Institute. AB - In oncology, follow-up refers to the medical procedures aimed to control, over time, both patients at risk of developing cancer, or those already submitted to surgical treatments for neoplastic lesions. The usefulness of an endoscopic follow-up in oncological diseases of the gastrointestinal tract is still being debated and, in some cases, a variety of different protocols are often employed for the same disease. At Regina Elena Cancer Institute, after a critical review of our data and literature, we established and followed guidelines of endoscopic follow-up for patients both at risk and submitted to curative surgery for cancer. PMID- 10746970 TI - Obstructive biliary symptomatology as the first sign of HIV-infection. AB - A 41-year-old white homosexual man presented with epigastric pain and jaundice. Physical examination showed enlargement of bilateral axillar and left inguinal lymph node, while ERCP and a CT scan suggested interruption of bile flow in the intrapancreatic tract of the common bile duct. An endoprosthesis was positioned in the common bile duct during the ERCP. Blood tests (both ELISA and Western blot techniques) showed positivity for anti-HIV antibodies and a CD4 count of 780/mmc (normal: 900-1,200/mmc). A few days later, a dramatic increase of the size of a lymph node in this right axilla occurred, rapidly reaching 5 cm of diameter. A biopsy was performed at this level, and histological examination revealed a high grade B-cell Burkitt type lymphoma. Bone marrow biopsy was negative, as well as lumbar puncture. Aggressive chemotherapy with adriamycin, cyclophosphamide, bleomycine, eldesine and prednisone, together with intratechal administration of methotrexate, was attempted. However, after a marginal and transient regression, the NHL rapidly progressed and the patient eventually died seven months after the diagnosis of NHL. A post mortem examination confirmed the diagnosis of Burkitt lymphoma of the peripancreatic and axillar lymph nodes, with diffusion to the leptomeninges, subaracnoideal spaces and encephalus. No signs of lymphoma were detected in other nodal or extra nodal areas. PMID- 10746972 TI - Facial nerve functionality after parotid tumors surgery. AB - The purpose of this study is to show the connection among tumor histology, surgical treatments and facial nerve postoperative functionality. A retrospective review was conducted on 69 patients with benign and malignant parotid tumors: they underwent surgical treatment for benign and malignant parotid tumors at the Maxillofacial Division of Rome University "La Sapienza" from 1988 to 1997. In our series of patients, we performed conservative superficial parotidectomy, conservative total parotidectomy and radical parotidectomy. Tumor enucleation was reserved for those neoplasms with a limited and restricted mass. The surgical approach was based on mass extension and on histopathologic features. Our findings showed that a careful and a timely diagnosis is very important in order to select a conservative surgical treatment for benign and intermediate grade of malignancy tumors with limited dimensions. Most aggressive histological types, and large mass dimensions require a radical treatment with consequent facial nerve postoperative dysfunctions. Our experience demonstrates that this surgical approach provides a very high rate of success in the cure of tumors and a low rate of facial nerve postoperative dysfunctions. PMID- 10746973 TI - Isolation and purification of phosphate dependent glutaminase from sarcoma-180 tumor and its antineoplastic effects on murine model system. AB - High rate of glutamine use is a characteristic of tumor cell both in vivo and in vitro and experimental cancer therapies have developed by depriving tumor cells of glutamine. In several investigations, bacterial glutaminase was found to be a potent therapeutic agent against varieties of tumor, but it showed suppressive effects on haematopoietic systems and inhibitory effects on normal lymphocytic blastogenesis. No antineoplastic study has nevertheless been undertaken with glutaminase enzyme purified from mammalian source. In the present study we report the purification of glutaminase enzyme from mitochondria of highly malignant S 180 cell using ion exchange chromatography and affinity column chromatography of glutamine. Purified enzyme is a kidney type phosphate dependent glutaminase with Mr 64 KD. Effect of enzyme therapy has been investigated in transplantable as well as induced tumor model in both ascites and solid form. It has been observed that the enzyme at the total dose of 10 unit/mouse successfully inhibited the tumor burden both in ascitic and solid tumor and subsequently increases the host's life span. There was no significant toxic effect on the peripheral blood cells. PMID- 10746974 TI - Cisplatin containing chemotherapy influences HLA-DR expression on monocytes from cancer patients. AB - The effect of cisplatin-containing chemotherapy regimen was evaluated on the expression of HLA-DR antigen in peripheral blood monocytes from patients with lung (LCP) and colorectal (CCP) cancer. Chemotherapeutic schedules employed in patients were etoposide and cisplatin for LCP and 5-fluorouracil and cisplatin for CCP. The results obtained showed a diminished percentage of monocytes expressing HLA-DR antigen in LCP (52.4 +/- 2.6, p < 0.004) and CCP (50.1 +/- 2.1, p < 0.001) respectively versus healthy donors (71.0 +/- 1.1%), and that their values increased during chemotherapy, raising them up to control level after the second cycle of treatment, independently of the course of the cancer growth. We conclude that both modalities of treatment allowed an increase of monocytes expressing HLA-DR antigen, suggesting that this effect may be due to cisplatin action. PMID- 10746975 TI - Dendritic cells stimulate the expansion of PML-RAR alpha specific cytotoxic T lymphocytes: its applicability for antileukemia immunotherapy. AB - Dendritic cells (DC), the most potent "professional" antigen-presenting cells, hold promise for improving the immunotherapy of cancer. In this study, we investigated the ability of normal donor DC pulsed ex vivo with 12 mer PML-RAR alpha (A) peptide (SGAGEAAIETQS) to generate peptide specific autologous cytotoxic T-lymphocytes. The peptide pulsed DC-primed peripheral blood lymphocytes (PBL) displayed significantly higher cytotoxic activity compared with that of peptide non-pulsed DC-primed PBL against peptide-pulsed autologous macrophages (P<0.001). Both CD8+ and CD4+ T lymphocytes were involved in the effector cell populations. The PML-RAR alpha peptide-pulsed DC-primed T-cells were significantly superior in their production of GM-CSF and TNF-alpha, compared with peptide non-pulsed DC-primed T-cells. These intriguing preclinical studies, suggest that PML-RAR alpha pulsed-DC could be a promising immunotherapeutic modality for patients with acute promyelocytic leukemia. PMID- 10746976 TI - Prostate cancer induction in autoimmune rats and modulation of T cell apoptosis. AB - In this study we present an experimental model of prostate gland cancer induced by long term hormonal treatment with testosterone in combination with a chemical carcinogenic agent in male Wistar rats with autoimmune prostatitis (AIP). This system is particularly attractive in order to study the factors involved in the transition from a non-invasive to an invasive carcinoma, decisive in the risk of human cancer. At first, autoimmune prostatitis was induced by immunization in 3 months-old male Wistar rats with autologous accessory glands. Then, rats were treated with continuous intradermal implants of testosterone propionate (TP) and with single doses of the chemical carcinogen 7, 12 dimethylbenz (alpha) anthracene (DMBA) by intraperitoneal injection. Histopathological studies of the prostate gland revealed the presence of pre-malignant lesions, particularly the so-called prostatic intraepithelial neoplasm (PIN) in 50% of animals. Moreover, we observed the development of carcinomas in 50% of treated-animals, which could be histologically discriminated in adenocarcinomas, carcinoma of epidermal origin and undifferentiated carcinoma. In autoimmune rats which did not receive any other treatment, exposure to autoantigens gave rise to an atypical hyperplasia of the prostatic gland which could be attributed to the hyperactive state of the gland. Control groups constituted by autoimmune rats treated with TP or DMBA, and normal rats which were exposed to TP and/or DMBA evidenced the presence of PINs at different degrees, but did not develop carcinomas. Moreover, serum acid phosphatase significantly increased as treatment was accomplished, reaching its maximum levels in animals with carcinoma, in which DNA content, determined by image cytometry, showed to be aneuploid. Finally, we provided biochemical and cytofluorometrical evidence of the induction of apoptosis of spleen T cells in carcinoma-bearing hosts, and to a lesser extent in animals with PIN, but not in autoimmune or normal controls, which could represent an alternative molecular mechanism for explaining host immunosuppression triggered by tumors. PMID- 10746977 TI - HLA class I antigens expression in renal cell carcinoma: histopathological and clinical correlation. AB - Immunohistochemical analysis of HLA class I antigens expression in 26 renal cell carcinomas (RCCs)--18 clear cell, 6 granular and 2 chromophobe--was performed with indirect immunoperoxidase method. Results were correlated with extent and immunophenotype of tumor infiltrating mononuclear cells, histopathological (histology, cytology, grade, presence of necrosis) and clinical (tumor diameter, TNM classification) characteristics of RCC. 4 (15%) RCCs showed reduced HLA class I presence and this was associated with greater tumor diameter and more frequent T3, T4 and M1 stage. All tumors with altered HLA class I antigens expression were grade 2 or 3 and strong correlation with presence of necrosis (p=0.006) was noticed, while mononuclear cell infiltrates (especially CD8+ T lymphocytes) were less extensive compared to tumors with normal HLA class I level. Our results suggest more aggressive clinical behavior of RCCs with reduced HLA class I antigens expression, probably due to impaired cellular antitumor immune response. PMID- 10746978 TI - Serum vascular endothelial growth factor is a predictor of invasion and metastasis in hepatocellular carcinoma. AB - Tumor progression is angiogenesis dependent, and vascular endothelial growth factor (VEGF) is a key growth factor in this process. sVEGF concentrations in 44 patients with hepatocellular carcinoma (HCC) and 5 with benign liver lesions were determined with an enzyme-link immunoadsorbent assay system (ELISA). Reverse transcript-polymerase chain reaction (RT-PCR) was carried out on surgical specimens of 51 patients with HCC. The relative levels of VEGF mRNA expression were measured by determining a ratio between PCR products of VEGF and the endogenous internal standard gene b-actin. UEA-1 was histochemically used to count microvascularity in tumor tissue. Elevated sVEGF concentrations were found in patients with HCC (172.84+/-111.75 pg/ml) as compared to individuals with benign liver lesions (95.74+/-36.20 pg/ml, P<.05). Of 44 cases with HCC, sVEGF concentrations in the patients with PV-emboli or with poor-encapsulated tumors were significantly higher than in those without PV-emboli or with well encapsulated tumors (P<0.05). The expression levels of VEGF mRNA in tumors with PV-emboli and in poor-encapsulated tumors were higher than in those without PV emboli and in well-encapsulated tumors (P<0.05). Microvascular density in HCC tissues was significantly correlated with the expression levels of VEGF mRNA (P<0.01; r=0.7). Circulating VEGF was derived from HCC tissue. sVEGF concentrations could be a new marker for predicting the angiogenesis, invasion and metastasis of HCC. PMID- 10746979 TI - Molecular and clinical study of familial adenomatous polyposis for genetic testing and management. AB - Familial adenomatous polyposis (FAP) is an inherited predisposition to colorectal cancer characterized by the development of numerous adenomatous polyps, predominantly in the colorectal region. Germline mutations of the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis. We examined germline mutations of the APC gene and clinical features among eighty seven individuals who consisted of thirty-nine FAP-patients, thirty-seven of their family members with a 1 in 2 risk of predisposition to this disease, and eleven normal persons. We accurately identified nine heterozygotes, among individuals with a 1 in 2 risk by genetic testing, without the uncertainty of the recurrence risk calculated by Bayes' theorem. Six of the nine heterozygotes were confirmed to have colorectal polyps by colonoscopic examination. Since they were diagnosed at 12.7 years-of-age on average, and were no more than 20 years old, they could be treated to prevent colorectal cancer. Based on the genotype phenotype correlation, we concluded that the germline mutations responsible for the sparse polyps phenotype of FAP-patients tend to locate from codon 1055 in the proximal region of the APC gene, while those for the profuse type locate from codon 1102 in the distal region. Among the thirty-nine FAP-patients, we found that those with the germline mutations within codon 1055 and codon 1262 had colorectal carcinomas of an advanced stage, at a high rate (71.4%). Special attention and aggressive intervention is needed in these patients and relatives at risk. With reasonable and appropriate management, it should be possible to prolong and improve the quality of life of those family members both affected and at risk. PMID- 10746980 TI - Transfection of mammalian cells by the methods of receptor mediated gene transfer and particle bombardment. AB - The efficacy of currently developed methods for gene transfer into mammalian cells depends primarily on the transfection technique, and also on the type of targeted cells. Considering the importance of gene transfer in the creation of gene therapies, our study was aimed at the assessment of transfection capacity of receptor mediated gene transfer method (RMGT), and method of particle bombardment (helios gene gun system--HGG) in different normal and malignant mammalian cells ex vivo. In addition, the HGG was also assessed for its ability to transfect tumor cells of subcutaneous (s.c.) tumors in C57Bl/6 mice in vivo. Using RMGT an average ex vivo transfection rate of 35.7%, and 20.4% was achieved in malignant melanoma B-16, and human breast adenocarcinoma MCF7, respectively. However, in normal fibroblast L929 cells the transfection by RMGT succeeded only in 2.1% of the cells. On the other hand, the transfection efficacy of HGG was comparable in both malignant cell lines resulting in an average gene transfer to 9.6% of B-16 and 10.5% of MCF7 cells, while only 3.9% of normal fibroblasts were successfully transfected. Application of HGG for an in vivo gene transfer into s.c. B-16 melanoma tumors in C57Bl/6 mice resulted in a successful but limited transfection of the epithelium as well as of the superficially sited tumor cells. Taking into consideration both methods, RMGT is more appropriate for ex vivo transfection of cells, under the condition that target cells express a specific receptor for the molecule attached to the carrier. On the other hand, HGG is not complicated to use, no requirements for specific structures on target cells are necessary (potentially usable in different cells), and it has applicability in direct in vivo transfection processes. PMID- 10746981 TI - Microvascular endothelium dysfunction during growth of transplanted lymphosarcoma and glioma in rats. AB - The changes of endothelium functional parameters (adhesivity, permeability, thrombogenity and thromboresistance) in mesenteric microvasculature in rats with two transplanted tumors: fast-growing Pliss' lymphosarcoma (PLS) and slow-growing glioma-35 (G-35) were studied, using a complex method, based upon a computer assisted microscopic video image processing. The endothelial dysfunction in mesenteric microvessels is characterized by the increase of leukocyte adhesion to the endothelial cells, microvessels permeability and their thromboresistance properties decrease in tumor-bearing rats. The observed changes are shown to be the nonspecific manifestation of the tumor process development. They are determined by the duration of the process development and are not dependent on the tumor node size. PMID- 10746982 TI - The study on the expression of membrane HSP70 protein in H22 cell and its immunoprotective mechanism against carcinoma. AB - The mRNA and protein expression of HSP70 were studied using RT-PCR and FCM techniques. It's immune protective effects in vivo and the cytotoxicity in vitro were also observed. Results showed that cell viability didn't change under 42-43 degrees C, but declined in 44-45 degrees C; the level of HSP70 mRNA decreased initially (0.5-4.0 hour) but gradually resumed and increased from 8 to 12 hours at 42 degrees C. The positive cells expressed membrane HSP70 were significantly higher in heat shocked group than in a control group (P<0.001); the highest positive rate was 96.8% at 43 degrees C. The C3H mice immunized with heat shocked H22 cells could resist a secondary subcutaneous inoculation of parental H22 cells and their tumorigenic rate was significantly lower than that in mice immunized with parental H22 cells and RPMI 1640 control mice, which were 1/9.7/8 and 8/8, respectively. Their median survival time was also longer than that of parental cell group and RPMI 1640 control group, which were >90 days, 73.5 days, and 39.5 days, respectively. Through heat-treated tumor cell and lymphocyte mixed culture (TLMC), the induced lymphocytes had higher cytotoxic activities than that of splenic cells. The cytotoxicity against H22 cells reached 65.38% (2 hrs, 42 degrees C) and 67.84% (12 hrs, 43 degrees C) and could be blocked by anti-HSP70 McAb. Phenotype analysis revealed that the rate of TCR gammadelta+ cells rose with increasing cytotoxic activity, but no similar changes could be found in the CD4+ CD8+ TCR alphabeta+ subset. These results suggest that proper heat shock conditions can improve the immunogenicity of tumor cells and CD4- CD8- TCR gammadelta+ T carried on cytotoxic function via the HSP70 molecule. PMID- 10746983 TI - Reversal effect of TTD on human multidrug resistant KBV200 cell line. AB - The reversal effect of TTD (a Chinese medicine) on human multidrug- resistant KBV200 cell line was studied and compared with verapamil (VPL). The chemosensitivity of KBV200 was detected by MTT assay in vitro and the level of MDR1 mRNA of KBV200 was investigated by RT-PCR. The cytotoxicity of TTD to KBV200 and the parent sensitive cell line KB is very low and nearly same with a concentration of 10(-6) mol x L(-1). With the concentration TTD increased VCR cytotoxicity on KBV200, 50% inhibitory concentration (IC50) decreased from 1122.5+/-72.36 mol x L(-1) to 31.76+/-5.4 nmol x L(-1) (p<0.001). It was more effective than VPL was (p<0.01). The combination of low concentration of TTD (10( 8) mol x L(-1)) and VCR (100 nmol x L(-1)) has significantly increased VCR cytotoxicity on KBV200, cell Surviving Fraction decreased from 0.91+/-0.056 to 0.74+/-0.07 (p<0.02). TTD did not inhibit the expression of MDR1 mRNA of KBV200 with the concentration of 10(-6) mol x L(-1). These data indicated that TTD could reverse VCR resistance of KBV200 and may be useful in enhancing the clinical effectiveness of VCR. PMID- 10746984 TI - Surface changes and hormone production in pituitary cells during tumorigenesis. AB - Prolactinomas were induced by chronic estrone acetate treatment in female Wistar rats in vivo. After enzymatic dissociation of the tumor tissue, monolayer cell cultures were obtained. In vitro tumor induction was performed by treatment of normal monolayer anterior pituitary cell cultures with a 1:1 mixture of 7,9 dimethylbenz[c]acridine: 8-methylbenz[c]acridine. For immunohistochemistry, the cell cultures were stained by the peroxidase-antiperoxidase method for standardization; the nonspecific hormone release was induced with 30 mM K+. The prolactin, alpha-melanotropin, and adrenocorticotropin levels in the supernatant were measured by specific, sensitive radioimmunoassays. The results indicated surface differences between the in vivo induced prolactinoma cells and normal pituitary cells: the attachment of the prolactinoma cells required 15% collagen treatment, whereas normal cells required only 3-4% collagen. Tumor cells induced in vitro by methylbenz[c]acridine treatment were able to attach only after ammonia activation of the collagen surface. These findings strongly suggest that the mode of tumor induction can result in differences in membrane fluidity; this phenomenon is possibly connected with the levels of prolactin, adrenocorticotropin and alpha-melanotropin hormone production of these endocrine tumor cells. PMID- 10746985 TI - Effect of normal and tumor factors on different phases of cell populations cycle. AB - In the present experiments we studied the effect of extracts from intact liver (LE), ES2 tumor extract (TE), plasmas from intact mice (PI), and from tumor bearing animals (PT) on different phases of hepatocytes and renocytes cell cycles. C3HS 28-day-old male mice, standardized for periodicity analysis, were injected at 16:00 hours and killed every 4 hours during a circadian cycle at 20:00/04; 00:00/08; 04:00/12; 08:00/16; 12:00/20 and 16:00/24 (time of day/hours post treatment). Colchicine (2 microg/g) was injected 4 hours before killing them. Samples of livers and kidneys were processed for histology and mitotic index determinations. The results were expressed as colchicine arrested metaphases per 1000 nuclei. The TE, LE and PI had a promoting effect on the mitotic activity of hepatocytes during the first 12 hours post treatment. During the subsequent 12 hours, not only these treatments but also the PI had an inhibiting effect on the mitotic activity of the same cell population. Also the TE and the PT had a promoting effect on the mitotic activity of the renocytes during the first 12 hours while the effect of all treatments showed a clear inhibition of the mitotic activity studied during the last 12 hours. Taking into account the time elapsed between the injections and the measurements made in these light-dark synchronized animals, we conclude that the increase in mitotic index observed in those tissues stemmed from a reinitiation of cell-cycle traverse in a subpopulation of G2-arrested, noncycling cells. PMID- 10746986 TI - Coexistence of myelodysplastic syndrome and multiple myeloma. AB - We describe the simultaneous presentation of myelodysplastic syndrome (MDS) and multiple myeloma (MM). Our patient had MDS (RAEB type) and bone marrow infiltration (40% plasma cells), as well as biclonal paraprotein. Patients with MM, MDS have been reported after chemotherapy but few cases documenting the coexistence of MDS and MM at diagnosis have been reported in the literature. PMID- 10746988 TI - Hepatoid carcinoma of the stomach: is it still an unusual anatomo-clinical entity? Six cases-report. AB - Hepatoid carcinoma of the stomach is a rare neoplasm (especially in western countries) characterized by high levels of serum alpha-fetoprotein (AFP), the presence of "hepatoid foci" inside the gastric tumor and poor prognosis, due to the earlier onset of liver metastases. We treated six patients for hepatoid carcinoma of the stomach between 1990 and 1997. The female to male ratio was 1:1, the average age was 71 (54-81) and the average AFP-level was 1160 ng/ml (603 1531). We performed 2 total gastrectomies, 2 subtotal gastrectomies and 2 gastro jejunostomies (due to presence of liver metastases): in one case, the patient underwent a splenectomy as well. All the tumors showed the presence of "hepatoid foci" (the morphological feature is close to the hepatocellular carcinoma) and a positive immunoreactivity to AFP. The mean survival was 3 months: only one patient is still alive and disease-free (with a 52 months follow-up). After radical surgery, she underwent a chemotherapic treatment with cisplatin, epirubicin, 5-fluorouracil and l-leucovorin. We conclude that our series (the widest in Italy and one of most impressive in Europe) confirm the poor prognosis of this neoplasm, but we also want to underline that this tumor is not so "unusual" any more and it requires new types of treatment, like postoperative chemotherapy, besides surgery, to be fighted properly. PMID- 10746987 TI - Mixed follicular and parafollicular thyroid carcinoma. AB - A rare case of mixed follicular-parafollicular thyroid carcinoma which occurred in a 50-year-old man, is reported. The ultrastructural aspects of the tumor showed: a biphasic growth pattern with microfolliculi and solid areas; the coexpression of thyroglobulin and calcitonin antigens in the same follicle-like structures; the presence of neuroendocrine granules, microvilli and intracytoplasmic canaliculi bordered by microvilli. These characteristics lead us to a diagnosis of mixed follicular-parafollicular thyroid carcinoma. PMID- 10746989 TI - Solitary metachronous splenic metastases: an evaluation of surgical treatment. AB - Splenic metastases occurring after primary tumor removal and apparently solitary have been documented only recently in Literature. They are, most of the times, clinically asymptomatic and their presence is casually determined by ultrasonographic follow-up in subjects otherwise in good conditions. The belief that splenic metastases occur only in disseminated cancer is today no longer accepted. Some Authors consider solitary splenic metachronous metastases eligible for surgical treatment as well as pulmonary or hepatic metastases. In the case presented, surgery was required due to abscess formation of a splenic metastasis, which was not responding to chemotherapy. Our experience, like others reported in Literature, verified a long-term post-operative survival in spite of limited disease-free time. Surgical treatment by splenectomy can be indicated in selected patients, considering that chemotherapy has been proved to be ineffective in the treatment of splenic metastases. PMID- 10746990 TI - Papillary renal cell carcinoma presenting as nodal metastases to the neck. AB - Renal cell carcinoma, in a high percentage of patients, metastasizes early, sometimes mimicking other lesions. We present a case of an asymptomatic papillary renal cell carcinoma that presented neck metastases as the initial manifestation. The laterocervical and supraclavicular masses were considered consistent with nodal metastases from a thyroid nodule. A hemithyroidectomy was performed before the renal tumor was diagnosed. Then the patient underwent a left-side radical nefrectomy. We discuss the unpredictability of the clinical course of renal cell carcinoma. PMID- 10746991 TI - Non Hodgkin's lymphoma arising from skeletal muscle. PMID- 10746992 TI - Role of mitogen-activated protein kinases in ischemia and reperfusion injury : the good and the bad. PMID- 10746993 TI - Secretory group II phospholipase A(2) : a newly recognized acute-phase reactant with a role in atherogenesis. PMID- 10746994 TI - A tale of two (Calcium) channels. PMID- 10746995 TI - Prospects for gene therapy for heart failure. AB - Heart failure represents an enormous clinical challenge in need of effective therapeutic approaches. The possibility of gene therapy for heart failure merits consideration at this time because of improvements in vector technology; cardiac gene delivery; and, most importantly, our understanding of the molecular pathogenesis of heart failure. We will first review recent advances in cardiac gene delivery in animal models and then examine several targets being considered for therapeutic intervention. In this context, gene transfer provides not only a potential therapeutic modality but also an important tool to help validate specific targets. Several interventions, particularly those enhancing sarcoplasmic calcium transport, show promise in animal models of heart failure and in myopathic cardiomyocytes derived from patients. However, bridging the gap between these basic investigative studies and clinical gene therapy remains a formidable task. Early experiments in rodents will need to be extended to large animal models with clinical-grade vectors and delivery systems to assess both efficacy and safety. On the basis of a foundation of rigorous science and a growing understanding of heart failure pathogenesis, there is reason for cautious optimism for the future. PMID- 10746996 TI - Stimulation of myocardial Na(+)-independent Cl(-)-HCO(3)(-) exchanger by angiotensin II is mediated by endogenous endothelin. AB - Experiments were performed in isolated cat papillary muscles loaded with the pH sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in the esterified form to study the effect of endothelin-1 (ET-1) on the activity of the Na(+)-independent Cl(-)-HCO(3)(-) exchanger. Exposure to ET-1 (10 nmol/L) raised pH(i) by 0.13+/-0.03 U (P<0.05) in papillary muscles superfused with nominally HCO(3)(-)-free solution, whereas no significant change was detected under CO(2)/HCO(3)(-)-buffered medium. However, if ET-1 was applied to muscles pretreated with the anion exchanger inhibitor 4-acetamido-4'-isothiocyanato stilbene-2, 2'-disulfonic acid, pH(i) increased by 0.09+/-0.02 U (P<0.05) in the presence of CO(2)/HCO(3)(-) buffer. The rate of pH(i) recovery from trimethylamine hydrochloride-induced intracellular alkaline load was enhanced so that net HCO(3) efflux increased about three times in the presence of ET-1 (2.74+/-0.25 versus 9.66+/-1.29 mmol. L(-1). min(-1) at pH(i) 7.55, P<0.05). This effect was canceled by previous exposure to either 50 nmol/L PD 142,893 (nonselective endothelin receptor blocker) or 300 nmol/L BQ 123 (selective blocker of ET(A) receptors). BQ 123 also abolished angiotensin II-induced activation of the Na(+) independent Cl(-)-HCO(3)(-) exchanger. These results show that ET-1 increases the activity of the Na(+)-independent Cl(-)-HCO(3)(-) exchanger in cardiac tissue through the ET(A) receptors. Furthermore, our data suggest that the previously described angiotensin II-induced stimulation of the anion exchanger activity is mediated by endogenous ET-1. PMID- 10746997 TI - T-Type and tetrodotoxin-sensitive Ca(2+) currents coexist in guinea pig ventricular myocytes and are both blocked by mibefradil. AB - Under Na(+)-free conditions, low-voltage-activated Ca(2+) currents in cardiomyocytes from various species have been described either as Ni(2+) sensitive T-type Ca(2+) current (I(Ca(T))) or as tetrodotoxin (TTX)-sensitive Ca(2+) current (I(Ca(TTX))). So far, coexistence of the 2 currents within the same type of myocyte has never been reported. We describe experimental conditions under which I(Ca(T)) and I(Ca(TTX)) can be separated and studied in the same cell. Rat and guinea pig ventricular myocytes were investigated with the whole cell voltage-clamp technique in Na(+)-free solutions. Whereas rat myocytes lack I(Ca(T)) and exhibit I(Ca(TTX)) only, guinea pig myocytes possess both of these low-voltage-activated Ca(2+) currents, which are separated pharmacologically by superfusion with TTX or Ni(2+). I(Ca(T)) and I(Ca(TTX)) were of similar amplitude but significantly differed in their electrophysiological properties: I(Ca(TTX)) activated at more negative potentials than did I(Ca(T)), the potential for half maximum steady-state inactivation was more negative, and current deactivation and recovery from inactivation were faster. I(Ca(TTX)) but not I(Ca(T)) increased after membrane rupture ("run-up"). Isolation of I(Ca(TTX)) by application of the bivalent cation Ni(2+) is critical because of possible shifts in voltage dependence. Therefore, we investigated whether the T-type Ca(2+) channel blocker mibefradil (10 micromol/L) is a suitable tool for the study of I(Ca(TTX)). However, mibefradil not only blocked I(Ca(T)) by 85+/-2% but also decreased I(Ca(TTX)) by 48+/-8%. We conclude that under Na(+)-free conditions I(Ca(T)) and I(Ca(TTX)) coexist in guinea pig ventricular myocytes and that both currents are sensitive to mibefradil. Future investigations of I(Ca(T)) will have to consider the TTX-sensitive current component to avoid possible interference. PMID- 10746998 TI - Identification of a T-type Ca(2+) channel isoform in murine atrial myocytes (AT-1 cells) AB - Calcium channels are important targets for therapeutics, but their molecular diversity complicates characterization of these channels in native heart cells. In this study, we identify a new splice variant of a low-voltage activated, or T type Ca(2+), channel in murine atrial myocytes. To date, alpha1G and alpha1H are the only 2 T-type Ca(2+) channel isoforms found in cardiovascular tissue. We compared alpha1G and alpha1H channel current heterologously expressed in HEK 293 cells with T-type current from the murine atrial tumor cell, AT-1. AT-1 cell T type current (I(T)) has the same voltage dependence of activation and inactivation as alpha1G and alpha1H. The cloned T-type channels and AT-1 T-type current share similar kinetics of macroscopic inactivation and deactivation. The kinetics of recovery from inactivation of T-type currents serves as an electrophysiological signature for T-channel isoform. alpha1G and AT-1 I(T) have a similar recovery from inactivation time course that is faster than that for alpha1H. In all cases, T-type current recovers with a biexponential time course, and the relative amplitude of fast and slow time courses explains the slower alpha1H recovery kinetics, rather than differences in the time constants of the individual transitions. Thus, the T-type channels may be an important contributor to automaticity in heart cells, and molecular diversity is reflected in the pathway of recovery from inactivation. PMID- 10746999 TI - Overexpressed A(1) adenosine receptors reduce activation of acetylcholine sensitive K(+) current by native muscarinic M(2) receptors in rat atrial myocytes. AB - In adult rat atrial myocytes, muscarinic acetylcholine (ACh)-sensitive K(+) current activated by a saturating concentration of adenosine (I(K(ACh),(Ado))) via A(1) receptors (A(1)Rs) amounts to only 30% of the current activated by a saturating concentration of ACh (I(K(ACh),(ACh))) via muscarinic M(2) receptors. The half-time of activation of I(K(ACh),(Ado)) on a rapid exposure to agonist was approximately 4-fold longer than that of I(K(ACh),(ACh)). Furthermore, I(K(ACh),(Ado)) never showed fast desensitization. To study the importance of receptor density for A(1)R-I(K(ACh),(Ado)) signaling, adult atrial myocytes in vitro were transfected with cDNA encoding for rat brain A(1)R and enhanced green fluorescent protein (EGFP) as a reporter. Whole-cell current was measured on days 3 and 4 after transfection. Time-matched cells transfected with only the EGFP vector served as controls. In approximately 30% of EGFP-positive cells (group I), the density of I(K(ACh),(Ado)) was increased by 72%, and its half-time of activation was reduced. Density and kinetic properties of I(K(ACh),(ACh)) were not affected in this fraction. In approximately 70% of transfection-positive myocytes (group II), the density of I(K(ACh),(ACh)) was significantly reduced, its activation was slowed, and the fast desensitizing component was lost. Adenosine-induced currents were larger in group II than in group I, their activation rate was further increased, and a fast desensitizing component developed. These data indicate that in native myocytes the amplitude and activation kinetics of I(K(ACh),(Ado)) are limited by the expression of A(1)R. Overexpression of A(1)R negatively interferes with signal transduction via the muscarinic M(2) receptor-linked pathway, which might reflect a competition of receptors with a common pool of G proteins. Negative interference of an overexpressed receptor with physiological regulation of a target protein by a different receptor should be considered in attempts to use receptor overexpression for gene therapy. PMID- 10747000 TI - Impaired conduction of vasodilation along arterioles in connexin40-deficient mice. AB - Connexins have been hypothesized to play an important role in intercellular communication within the vascular wall and may provide a mechanistic explanation for conduction of vasomotor responses. To test this hypothesis, we studied the transmission of vasomotor responses in the intact skeletal muscle microcirculation of connexin40-deficient mice (Cx40(-/-)). Arterioles were locally stimulated with hyperpolarizing dilators (acetylcholine [ACh] as well as bradykinin [Bk]) or depolarizing K(+) solution, and the resulting changes in diameter were measured using a videomicroscopy technique at the site of application and up to 1.32 mm upstream. Arterial pressure was elevated 25% in Cx40(-/-) mice (94+/-5 versus 75+/-4 mm Hg). Vessels selected for study had equivalent basal diameter and vasomotor tone in both genotypes of mice. Vasomotion was present in small arterioles of both genotypes, but its intensity was exaggerated in Cx40(-/-) mice. ACh and Bk induced dilation (33% and 53%, respectively, of maximal response) at the site of application that was of similar magnitude in both genotypes. These dilations were observed to spread upstream within <1 second without significant attenuation in Cx40(+/+) mice. However, spreading was severely attenuated in Cx40(-/-) animals (11+/-4% versus 35+/-7% with ACh and 38+/-5% versus 60+/-7% with Bk in Cx40(-/-) and Cx40(+/+), respectively; P<0.05). In contrast, conducted vasoconstrictions, induced by K(+) solution decreased equally with distance in both genotypes. These results support a significant role for Cx40 in vascular intercellular communication. Our observations indicate that Cx40 is required for normal transmission of endothelium-dependent vasodilator responses and may underlie altered vasomotion patterns. PMID- 10747001 TI - Relation between renal interstitial ATP concentrations and autoregulation mediated changes in renal vascular resistance. AB - The present study was performed to examine the hypothesis that autoregulation related changes in renal vascular resistance (RVR) are mediated by extracellular ATP. By use of a microdialysis method, renal interstitial concentrations of ATP and adenosine were measured at different renal arterial pressures (RAPs) within the autoregulatory range in anesthetized dogs (n=12). RAP was reduced in steps from the ambient pressure (131+/-4 mm Hg) to 105+/-3 mm Hg (step 1) and 80+/-2 mm Hg (step 2). Renal blood flow and glomerular filtration rate exhibited efficient autoregulation in response to these changes in RAP. RVR decreased by 22+/-2% in step 1 (P<0.01) and 38+/-3% in step 2 (P<0.01). The control renal interstitial concentration of ATP was 6.51+/-0.71 nmol/L and decreased to 4. 51+/-0.55 nmol/L in step 1 (P<0.01) and 2.77+/-0.47 nmol/L in step 2 (P<0.01). In contrast, the adenosine concentrations (117+/-6 nmol/L) were not altered significantly. Changes in ATP levels were highly correlated with changes in RVR (r=0.88, P<0.0001). Further studies demonstrated that stimulation of the tubuloglomerular feedback (TGF) mechanism by increasing distal volume delivery elicited with acetazolamide also led to increases in renal interstitial ATP concentrations, whereas furosemide, which is known to block TGF responses, reduced renal interstitial fluid ATP concentrations. The data demonstrate a positive relation between renal interstitial fluid ATP concentrations and both autoregulation- and TGF-dependent changes in RVR and thus support the hypothesis that changes in extracellular ATP contribute to the RVR adjustments responsible for the mechanism of renal autoregulation. PMID- 10747002 TI - Biphasic actions of prostaglandin E(2) on the renal afferent arteriole : role of EP(3) and EP(4) receptors. AB - Prostaglandin (PG) E(2) is an important modulator of the actions of angiotensin (Ang) II. In the present study, we investigated the renal microvascular actions of PGE(2) and the EP receptor subtypes involved. Ibuprofen potentiated Ang II induced vasoconstriction in in vitro perfused normal rat kidneys and augmented afferent arteriolar, but not efferent arteriolar, responses in the hydronephrotic rat kidney model. This preglomerular effect of endogenous prostanoids was mimicked by exogenous PGE(2), which reversed Ang II-induced afferent arteriolar vasoconstriction at concentrations of 0.1 to 10 nmol/L without affecting the efferent arteriole. The PGE(2)-induced vasodilation was potentiated by the phosphodiesterase inhibitor Ro 20-1724 and was mimicked by 11-deoxy-PGE(1) (0.01 to 1 nmol/L). Butaprost, which acts preferentially at EP(2) receptors, was relatively ineffective. Whereas 0.1 to 10 nmol/L PGE(2) elicited vasodilation, higher concentrations (1 to 10 micromol/L) restored Ang II-induced afferent arteriolar vasoconstriction. This response was blocked by pertussis toxin (200 microg/mL) and was mimicked by the EP(1)/EP(3) agonist sulprostone (1 to 300 nmol/L). Reverse transcription-polymerase chain reaction of individually isolated afferent arterioles revealed the presence of message for EP(4) and all 3 EP(3) splice variants (alpha, beta, and gamma) but not EP(1) or EP(2). Our findings thus indicate that PGE(2) elicits both vasodilatory and vasoconstrictor actions on the afferent arteriole. The vasodilation is mediated by EP(4) receptors coupled to cAMP, presumably via G(alphas). The vasoconstriction is mediated by an EP(3) receptor coupled to G(alphai) and appears to reflect a functional antagonism of the EP(4)-induced vasodilation. PMID- 10747004 TI - Local expression of bovine decorin by cell-mediated gene transfer reduces neointimal formation after balloon injury in rats. AB - Decorin is an extracellular matrix (ECM) proteoglycan that may modify vascular smooth muscle cell (SMC) function by altering the response to growth factors and the accumulation of ECM proteins during vascular injury. To investigate these possibilities in vivo, decorin was overexpressed at the site of arterial injury by cell-mediated gene transfer. Fischer rat SMCs were transduced in vitro with a retroviral construct that contained the bovine decorin gene and were subsequently seeded into injured rat carotid arteries. A species-specific antibody to bovine decorin and polymerase chain reaction primers were used to detect bovine decorin and distinguish it from endogenous rat decorin. Immunohistochemical and Northern analyses of rat carotid arteries revealed only low levels of rat decorin expression up to 8 weeks after balloon injury. However, after cell-mediated transfer of bovine decorin, strong expression of bovine decorin was verified by immunohistochemistry and reverse transcriptase-polymerase chain reaction. Four weeks after injury, the intimal area in vessels seeded with bovine decorin overexpressing SMCs was significantly reduced by 35+/-4% (mean+/-SEM, n=9; P<0.01). Decorin overexpression also induced a higher intimal nuclear density and decreased volume of ECM. Specifically, immunostaining for versican and fibronectin was markedly reduced. In contrast, immunostaining for collagen type I was increased, and electron microscopy confirmed that collagen accumulation was altered. Bromodeoxyuridine labeling indicated that intimal SMC proliferation was not affected by the expression of bovine decorin. In summary, we demonstrate that gene transfer of the ECM proteoglycan, decorin, into the injured arterial wall reduces intimal ECM volume and alters the composition of the ECM. PMID- 10747003 TI - Downregulation of endothelial nitric oxide synthase in rat aorta after prolonged hypoxia in vivo. AB - The goal of this study was to determine whether hypoxia alters expression of endothelial nitric oxide synthase (eNOS) in the systemic circulation. Rats breathed either air or 10% oxygen for 12 hours, 48 hours, or 7 days. Thoracic aortas were excised and either mounted in organ bath myographs or frozen in liquid nitrogen for later extraction of protein and RNA. eNOS protein (Western blotting) was decreased (20% of normoxic control) after 12 hours, 48 hours, and 7 days of hypoxia. eNOS mRNA (ribonuclease protection assay) was similarly reduced. Acetylcholine (10(-4) mol/L) reversed phenylephrine (10(-5) mol/L) preconstriction by 53.3+/-5.6% in aortic rings from normoxic rats and 26.1+/-4.8% in rings from rats exposed to hypoxia for 48 hours (P<0.05), with comparable impairment of relaxation by the calcium ionophore A23187 (10(-5) mol/L). Responses to diethylamine nitric oxide and 8-bromo-cGMP were unaffected. Aortic cGMP levels after incubation with acetylcholine (10(-6) mol/L) averaged 14.0+/ 1.8 fmol/mg in rings from normoxic rats compared with 8.7+/-1.0 fmol/mg in rings from hypoxic rats (P<0. 05). Similarly, nitrate concentration (by capillary electrophoresis) in the media in which the rings were incubated was reduced in the hypoxic group (5.6+/-0.23 micromol/L for hypoxic rats and 7.8+/-0.7 micromol/L for normoxic rats). Impaired endothelial NO release may handicap the vascular responses that defend vital organ function during hypoxia. PMID- 10747005 TI - A mechanism of transition from ventricular fibrillation to tachycardia : effect of calcium channel blockade on the dynamics of rotating waves. AB - Abbreviation of the action potential duration and/or effective refractory period (ERP) is thought to decrease the cycle length of reentrant arrhythmias. Verapamil, however, paradoxically converts ventricular fibrillation (VF) to ventricular tachycardia (VT), despite reducing the ERP. This mechanism remains unclear. We hypothesize that the size and the dynamics of the core of rotating waves, in addition to the ERP, influence the arrhythmia manifestation (ie, VF or VT). The objectives of this study were (1) to demonstrate functional reentry as a mechanism of VF and VT in the isolated Langendorff-perfused rabbit heart in the absence of an electromechanical uncoupler and (2) to elucidate the mechanism of verapamil-induced conversion of VF to VT. We used high-resolution video imaging with a fluorescent dye, ECG, frequency and 2-dimensional phase analysis, and computer simulations. Activation patterns in 10 hearts were studied during control, verapamil perfusion (2x10(-6) mol/L), and washout. The dominant frequency of VF decreased from 16.2+/-0.7 to 13.5+/-0.6 Hz at 20 minutes of verapamil perfusion (P<0.007). Concomitantly, phase analysis revealed that wavefront fragmentation was reduced, as demonstrated by a 3-fold reduction in the density of phase singularities (PSs) on the ventricular epicardial surface (PS density: control, 1.04+/-0.12 PSs/cm(2); verapamil, 0.32+/-0.06 PSs/cm(2) [P=0.0008]). On washout, the dominant frequency and the PS density increased, and the arrhythmia reverted to VF. The core area of transiently appearing rotors significantly increased during verapamil perfusion (control, 4.5+/-0.6 mm(2); verapamil, 9.2+/-0.5 mm(2) [P=0.0002]). In computer simulations, blockade of slow inward current also caused an increase in the core size. Rotating waves underlie VF and VT in the isolated rabbit heart. Verapamil-induced VF-to-VT conversion is most likely due to a reduction in the frequency of rotors and a decrease in wavefront fragmentation that lessens fibrillatory propagation away from the rotor. PMID- 10747006 TI - Inhibition of extracellular signal-regulated kinase enhances Ischemia/Reoxygenation-induced apoptosis in cultured cardiac myocytes and exaggerates reperfusion injury in isolated perfused heart. AB - Three major mammalian mitogen-activated protein kinases, extracellular signal regulated kinase (ERK), p38, and c-Jun NH(2)-terminal protein kinase (JNK), have been identified in the cardiomyocyte, but their respective roles in the heart are not well understood. The present study explored their functions and cross talk in ischemia/reoxygenation (I/R)-induced cardiac apoptosis. Exposing rat neonatal cardiomyocytes to ischemia resulted in a rapid and transient activation of ERK, p38, and JNK. On reoxygenation, further activation of all 3 mitogen-activated protein kinases was noted; peak activities increased (fold) by 5.5, 5.2, and 6.2, respectively. Visual inspection of myocytes exposed to I/R identified 18.6% of the cells as showing morphological features of apoptosis, which was further confirmed by DNA ladder and terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL). Myocytes treated with PD98059, a MAPK/ERK kinase (MEK1/MEK2) inhibitor, displayed a suppression of I/R-induced ERK activation, whereas p38 and JNK activities were increased by 70.3% and 55.0%, respectively. In addition, the number of apoptotic cells was increased to 33.4%. With pretreatment of cells with SB242719, a selective p38 inhibitor, or SB203580, a p38 and JNK2 inhibitor, I/R+PD98059-induced apoptotic cells were reduced by 42.8% and 63.3%, respectively. Hearts isolated from rats treated with PD98059 and subjected to global ischemia (30 minutes)/reoxygenation (1 hour) showed a diminished functional recovery compared with the vehicle group. Coadministration of SB203580 attenuated the detrimental effects of PD98059 and significantly improved cardiac functional recovery. The data taken together suggest that ERK plays a protective role, whereas p38 and JNK mediate apoptosis in cardiomyocytes subjected to I/R, and the dynamic balance of their activities is critical in determining cardiomyocyte fate subsequent to reperfusional injury. PMID- 10747007 TI - Cardiac dysfunction caused by myocardium-specific expression of a mutant thyroid hormone receptor. AB - Thyroid hormone deficiency has profound effects on the cardiovascular system, resulting in decreased cardiac contractility, adrenergic responsiveness, and vascular volume and increased peripheral vascular resistance. To determine the importance of direct cardiac effects in the genesis of hypothyroid cardiac dysfunction, the cardiac myocyte was specifically targeted with a mutant thyroid hormone receptor (TR)-beta (Delta337T-TR-beta(1)) driven by the alpha-myosin heavy chain (alpha-MHC) gene promoter. As a control in these experiments, a wild type (Wt) TR-beta(1) was also targeted to the heart by using the same promoter. Transgenic mice expressing the mutant TR displayed an mRNA expression pattern consistent with cardiac hypothyroidism, even though their peripheral thyroid hormone levels were normal. When these animals were rendered hypothyroid or thyrotoxic, mRNA expression of MHC isoforms remained unchanged in the hearts of the Delta337T transgenic animals, in contrast to Wt controls or transgenic animals expressing Wt TR-beta(1), which exhibited the expected changes in steady state MHC mRNA levels. Studies in Langendorff heart preparations from mutant TR beta(1) transgenic animals revealed evidence of heart failure with a significant reduction in +dP/dT, -dP/dT, and force-frequency responses compared with values in Wt controls and transgenic mice overexpressing the Wt TR-beta(1). In contrast, in vivo measures of cardiac performance were similar between Wt and mutant animals, indicating that the diminished performance of the mutant transgenic heart in vitro was compensated for by other mechanisms in vivo. This is the first demonstration indicating that isolated cardiac hypothyroidism causes cardiac dysfunction in the absence of changes in the adrenergic or peripheral vascular system. PMID- 10747008 TI - Molecular basis for the association of group IIA phospholipase A(2) and decorin in human atherosclerotic lesions. AB - Group IIA secretory nonpancreatic phospholipase A(2) (snpPLA(2)) is associated with collagen fibers in the extracellular matrix of human atherosclerotic plaques. Decorin, a small proteoglycan (PG) carrying chondroitin/dermatan sulfate glycosaminoglycans (GAGs), forms part of the collagen network in human arteries. To explore whether snpPLA(2) may be associated with collagen fibers via interaction with decorin, we performed (1) immunohistochemistry to compare the relative in vivo localization of snpPLA(2) and decorin in human atherosclerotic tissue and (2) in vitro experiments to study the interaction between snpPLA(2) and decorin. In atherosclerotic lesions, decorin was detected within the snpPLA(2)-positive part of the intima close to the media. Electrophoretic mobility shift assay showed that snpPLA(2) binds to decorin synthesized by human fibroblasts. Native and GAG-depleted decorin enhanced the association of snpPLA(2) to collagen types I and VI in a solid-phase binding assay. Furthermore, snpPLA(2) bound efficiently to a recombinant decorin core protein fragment B/E (Asp45-Lys359). This binding was competed with soluble decorin and inhibited at NaCl concentrations >150 mmol/L. The decorin core protein fragment B/E competed better than dermatan sulfate for binding of snpPLA(2) to decorin-coated microtiter wells. The enzymatic activity of snpPLA(2) increased 2- to 3-fold in the presence of decorin or GAG-depleted decorin. The results show that snpPLA(2) binds preferentially to the decorin protein core rather than to the GAG chain and that this interaction enhances snpPLA(2) activity. As a consequence, this active extracellular enzyme may contribute to the pathogenesis of atherosclerosis by modifying lipoproteins and releasing inflammatory lipid mediators at places of lipoprotein retention in the arterial wall. PMID- 10747009 TI - The active site architecture of Pisum sativum beta-carbonic anhydrase is a mirror image of that of alpha-carbonic anhydrases. AB - We have determined the structure of the beta-carbonic anhydrase from the dicotyledonous plant Pisum sativum at 1.93 A resolution, using a combination of multiple anomalous scattering off the active site zinc ion and non crystallographic symmetry averaging. The mol- ecule assembles as an octamer with a novel dimer of dimers of dimers arrangement. Two distinct patterns of conservation of active site residues are observed, implying two potentially mechanistically distinct classes of beta-carbonic anhydrases. The active site is located at the interface between two monomers, with Cys160, His220 and Cys223 binding the catalytic zinc ion and residues Asp162 (oriented by Arg164), Gly224, Gln151, Val184, Phe179 and Tyr205 interacting with the substrate analogue, acetic acid. The substrate binding groups have a one to one correspondence with the functional groups in the alpha-carbonic anhydrase active site, with the corresponding residues being closely superimposable by a mirror plane. Therefore, despite differing folds, alpha- and beta-carbonic anhydrase have converged upon a very similar active site design and are likely to share a common mechanism. PMID- 10747010 TI - Structure and mechanism of action of a novel phosphoglycerate mutase from Bacillus stearothermophilus. AB - Bacillus stearothermophilus phosphoglycerate mutase (PGM), which interconverts 2- and 3-phosphoglyceric acid (PGA), does not require 2,3-diphosphoglyceric acid for activity. However, this enzyme does have an absolute and specific requirement for Mn(2+) ions for catalysis. Here we report the crystal structure of this enzyme complexed with 3PGA and manganese ions to 1.9 A resolution; this is the first crystal structure of a diphosphoglycerate-independent PGM to be determined. This information, plus the location of the two bound Mn(2+) ions and the 3PGA have allowed formulation of a possible catalytic mechanism for this PGM. In this mechanism Mn(2+) ions facilitate the transfer of the substrate's phosphate group to Ser62 to form a phosphoserine intermediate. In the subsequent phosphotransferase part of the reaction, the phosphate group is transferred from Ser62 to the O2 or O3 positions of the reoriented glycerate to yield the PGA product. Site-directed mutagenesis studies were used to confirm our mechanism and the involvement of specific enzyme residues in Mn(2+) binding and catalysis. PMID- 10747011 TI - Structure of the C-terminal laminin G-like domain pair of the laminin alpha2 chain harbouring binding sites for alpha-dystroglycan and heparin. AB - The laminins are large heterotrimeric glycoproteins with fundamental roles in basement membrane architecture and function. The C-terminus of the laminin alpha chain contains a tandem of five laminin G-like (LG) domains. We report the 2.0 A crystal structure of the laminin alpha2 LG4-LG5 domain pair, which harbours binding sites for heparin and the cell surface receptor alpha-dystroglycan, and is 41% identical to the laminin alpha1 E3 fragment. LG4 and LG5 are arranged in a V-shaped fashion related by a 110 degrees rotation about an axis passing near the domain termini. An extended N-terminal segment is disulfide bonded to LG5 and stabilizes the domain pair. Two calcium ions, one each in LG4 and LG5, are located 65 A apart at the tips of the domains opposite the polypeptide termini. An extensive basic surface region between the calcium sites is proposed to bind alpha-dystroglycan and heparin. The LG4-LG5 structure was used to construct a model of the laminin LG1-LG5 tandem and interpret missense mutations underlying protein S deficiency. PMID- 10747012 TI - Mutational analysis of the propensity for amyloid formation by a globular protein. AB - Acylphosphatase can be converted in vitro, by addition of trifluoroethanol (TFE), into amyloid fibrils of the type observed in a range of human diseases. The propensity to form fibrils has been investigated for a series of mutants of acylphosphatase by monitoring the range of TFE concentrations that result in aggregation. We have found that the tendency to aggregate correlates inversely with the conformational stability of the native state of the protein in the different mutants. In accord with this, the most strongly destabilized acylphosphatase variant forms amyloid fibrils in aqueous solution in the absence of TFE. These results show that the aggregation process that leads to amyloid deposition takes place from an ensemble of denatured conformations under conditions in which non-covalent interactions are still favoured. These results support the hypothesis that the stability of the native state of globular proteins is a major factor preventing the in vivo conversion of natural proteins into amyloid fibrils under non-pathological conditions. They also suggest that stabilizing the native states of amyloidogenic proteins could aid prevention of amyloidotic diseases. PMID- 10747013 TI - Regulatory cross-talk between adhesin operons in Escherichia coli: inhibition of type 1 fimbriae expression by the PapB protein. AB - Pathogenic Escherichia coli often carry determinants for several different adhesins. We show a direct communication between two adhesin gene clusters in uropathogenic E.coli: type 1 fimbriae (fim) and pyelonephritis-associated pili (pap). A regulator of pap, PapB, is a key factor in this cross-talk. FimB recombinase turns on type 1 fimbrial expression, and PapB inhibited phase transition by FimB in both off-to-on and on-to-off directions. On-to-off switching requiring FimE was increased by PapB. By analysis of FimB- and FimE LacZ translational fusions it was concluded that the increase in on-to-off transition rates was via an increase in FimE expression. Inhibition of FimB promoted switching was via a different mechanism: PapB inhibited FimB-promoted in vitro recombination, indicating that FimB activity was blocked at the fim switch. In vitro analyses showed that PapB bound to several DNA regions of the type 1 fimbrial operon, including the fim switch region. These data show that Pap expression turns off type 1 fimbriae expression in the same cell. Such cross-talk between adhesin gene clusters may bring about appropriate expression at the single cell level. PMID- 10747014 TI - gC1q-R/p32, a C1q-binding protein, is a receptor for the InlB invasion protein of Listeria monocytogenes. AB - InlB is a Listeria monocytogenes protein that promotes entry of the bacterium into mammalian cells by stimulating tyrosine phosphorylation of the adaptor proteins Gab1, Cbl and Shc, and activation of phosphatidyl- inositol (PI) 3 kinase. Using affinity chromatography and enzyme-linked immunosorbent assay, we demonstrate a direct interaction between InlB and the mammalian protein gC1q-R, the receptor of the globular part of the complement component C1q. Soluble C1q or anti-gC1q-R antibodies impair InlB-mediated entry. Transient transfection of GPC16 cells, which are non-permissive to InlB-mediated entry, with a plasmid expressing human gC1q-R promotes entry of InlB-coated beads. Furthermore, several experiments indicate that membrane recruitment and activation of PI 3-kinase involve an InlB-gC1q-R interaction and that gC1q-R associates with Gab1 upon stimulation of Vero cells with InlB. Thus, gC1q-R constitutes a cellular receptor involved in InlB-mediated activation of PI 3-kinase and tyrosine phosphorylation of the adaptor protein Gab1. After E-cadherin, the receptor for internalin, gC1q R is the second identified mammalian receptor promoting entry of L. monocytogenes into mammalian cells. PMID- 10747015 TI - Direct interaction between the cell division protein FtsZ and the cell differentiation protein SpoIIE. AB - SpoIIE is a bifunctional protein with two critical roles in the establishment of cell fate in Bacillus subtilis. First, SpoIIE is needed for the normal formation of the asymmetrically positioned septum that forms early in sporulation and separates the mother cell from the prespore compartment. Secondly, SpoIIE is essential for the activation of the first compartment-specific transcription factor sigma(F) in the prespore. After initiation of sporulation, SpoIIE localizes to the potential asymmetric cell division sites near one or both cell poles. Localization of SpoIIE was shown to be dependent on the essential cell division protein FtsZ. To understand how SpoIIE is targeted to the asymmetric septum we have now analysed its interaction with FtsZ in vitro. Using the yeast two-hybrid system and purified FtsZ, and full-length and truncated SpoIIE proteins, we demonstrate that the two proteins interact directly and that domain II and possibly domain I of SpoIIE are required for the interaction. Moreover, we show that SpoIIE interacts with itself and suggest that this self-interaction plays a role in assembly of SpoIIE into the division machinery. PMID- 10747016 TI - Highly purified glycosylphosphatidylinositols from Trypanosoma cruzi are potent proinflammatory agents. AB - Intracellular protozoan parasites are potent stimulators of cell-mediated immunity. The induction of macrophage proinflammatory cytokines by Trypanosoma cruzi is considered to be important in controlling the infection and the outcome of Chagas' disease. Here we show that the potent tumour necrosis factor-alpha-, interleukin-12- and nitric oxide-inducing activities of T.cruzi trypomastigote mucins were recovered quantitatively in a highly purified and characterized glycosylphosphatidylinositol (GPI) anchor fraction of this material. The bioactive trypomastigote GPI fraction was compared with a relatively inactive GPI fraction prepared from T. cruzi epimastigote mucins. The trypomastigote GPI structures were found to contain additional galactose residues and unsaturated, instead of saturated, fatty acids in the sn-2 position of the alkylacylglycerolipid component. The latter feature is essential for the extreme potency of the trypomastigote GPI fraction, which is at least as active as bacterial endotoxin and Mycoplasma lipopeptide and, therefore, one of the most potent microbial proinflammatory agents known. PMID- 10747017 TI - The alpha-subunit of the mitochondrial F(1) ATPase interacts directly with the assembly factor Atp12p. AB - The Atp12p protein of Saccharomyces cerevisiae is required for the assembly of the F(1) component of the mitochondrial F(1)F(0) ATP synthase. In this report, we show that the F(1) alpha-subunit co-precipitates and co-purifies with a tagged form of Atp12p adsorbed to affinity resins. Moreover, sedimentation analysis indicates that in the presence of the F(1) alpha-subunit, Atp12p behaves as a particle of higher mass than is observed in the absence of the alpha-subunit. Yeast two-hybrid screens confirm the direct association of Atp12p with the alpha subunit and indicate that the binding site for the assembly factor lies in the nucleotide-binding domain of the alpha-subunit, between Asp133 and Leu322. These studies provide the basis for a model of F(1) assembly in which Atp12p is released from the alpha-subunit in exchange for a beta-subunit to form the interface that contains the non-catalytic adenine nucleotide-binding site. PMID- 10747018 TI - GATE-16, a membrane transport modulator, interacts with NSF and the Golgi v-SNARE GOS-28. AB - Membrane proteins located on vesicles (v-SNAREs) and on the target membrane (t SNAREs) mediate specific recognition and, possibly, fusion between a transport vesicle and its target membrane. The activity of SNARE molecules is regulated by several soluble cytosolic proteins. We have cloned a bovine brain cDNA encoding a conserved 117 amino acid polypeptide, denoted Golgi-associated ATPase Enhancer of 16 kDa (GATE-16), that functions as a soluble transport factor. GATE-16 interacts with N-ethylmaleimidesensitive factor (NSF) and significantly stimulates its ATPase activity. It also interacts with the Golgi v-SNARE GOS-28 in an NSF dependent manner. We propose that GATE-16 modulates intra-Golgi transport through coupling between NSF activity and SNAREs activation. PMID- 10747019 TI - Multiple modes of peptide recognition by the PTB domain of the cell fate determinant Numb. AB - The phosphotyrosine-binding (PTB) domain of the cell fate determinant Numb is involved in the formation of multiple protein complexes in vivo and can bind a diverse array of peptide sequences in vitro. To investigate the structural basis for the promiscuous nature of this protein module, we have determined its solution structure by NMR in a complex with a peptide containing an NMSF sequence derived from the Numb-associated kinase (Nak). The Nak peptide was found to adopt a significantly different structure from that of a GPpY sequence-containing peptide previously determined. In contrast to the helical turn adopted by the GPpY peptide, the Nak peptide forms a beta-turn at the NMSF site followed by another turn near the C-terminus. The Numb PTB domain appears to recognize peptides that differ in both primary and secondary structures by engaging various amounts of the binding surface of the protein. Our results suggest a mechanism through which a single PTB domain might interact with multiple distinct target proteins to control a complex biological process such as asymmetric cell division. PMID- 10747020 TI - Hsp90 is required for c-Mos activation and biphasic MAP kinase activation in Xenopus oocytes. AB - During Xenopus oocyte maturation, the Mos protein kinase is synthesized and activates the MAP kinase cascade. In this report, we demonstrate that the synthesis and activation of Mos are two separable processes. We find that Hsp90 function is required for activation and phosphorylation of Mos and full activation of the MAP kinase cascade. Once Mos is activated, Hsp90 function is no longer required. We show that Mos interacts with both Hsp90 and Hsp70, and that there is an inverse relationship between association of Mos with these two chaperones. We propose that Mos protein kinase is activated by a novel mechanism involving sequential association with Hsp70 and Hsp90 as well as phosphorylation. We also present evidence for a two-phase activation of MAP kinase in Xenopus oocytes. PMID- 10747021 TI - Identification of a peptide blocking vascular endothelial growth factor (VEGF) mediated angiogenesis. AB - Vascular endothelial growth factor (VEGF) binding to the kinase domain receptor (KDR/FLK1 or VEGFR-2) mediates vascularization and tumor-induced angiogenesis. Since there is evidence that KDR plays an important role in tumor angiogenesis, we sought to identify peptides able to block the VEGF-KDR interaction. A phage epitope library was screened by affinity for membrane-expressed KDR or for an anti-VEGF neutralizing monoclonal antibody. Both strategies led to the isolation of peptides binding KDR specifically, but those isolated by KDR binding tended to display lower reactivities. Of the synthetic peptides corresponding to selected clones tested to determine their inhibitory activity, ATWLPPR completely abolished VEGF binding to cell-displayed KDR. In vitro, this effect led to the inhibition of the VEGF-mediated proliferation of human vascular endothelial cells, in a dose-dependent and endothelial cell type-specific manner. Moreover, in vivo, ATWLPPR totally abolished VEGF-induced angiogenesis in a rabbit corneal model. Taken together, these data demonstrate that ATWLPPR is an effective antagonist of VEGF binding, and suggest that this peptide may be a potent inhibitor of tumor angiogenesis and metastasis. PMID- 10747022 TI - Bcl-2 is a monomeric protein: prevention of homodimerization by structural constraints. AB - The pro-apoptotic activity of the Bcl-2 family member Bax has been shown to be facilitated by homodimerization. However, it is unknown whether Bcl-2 or Bcl-x(L) have to homodimerize to protect cells from apoptosis. Here we show by co immunoprecipitation and FPLC analyses that while Bax multimerizes and forms heterodimers with Bcl-2, there is no evidence for Bcl-2 homodimerization, even in conditions under which Bcl-2 protects cells from apoptosis. Immunofluorescence studies confirmed that Bax can attract active, soluble Bcl-2 to mitochondrial membranes, but that nuclear/ER membrane-bound Bcl-2 was incapable of dislocating soluble Bcl-2. The failure of Bcl-2 to homodimerize is due to structural constraints as versions of Bcl-2 deleted or mutated in the BH1 and BH2 domains effectively dimerized with wild-type Bcl-2 and were dislocated by Bcl-2 inside cells. These data indicate that naturally occurring Bcl-2 does not expose protein domains that mediate homodimerization and therefore most likely acts as a monomer to protect cells from apoptosis. PMID- 10747023 TI - Arrest of spermatogenesis in mice expressing an active heat shock transcription factor 1. AB - In mammals, testicular temperature is lower than core body temperature, and the vulnerable nature of spermatogenesis to thermal insult has been known for a century. However, the primary target affected by increases in temperature is not yet clear. We report here that male mice expressing an active form of heat shock transcription factor 1 (HSF1) in the testis are infertile due to a block in spermatogenesis. The germ cells entered meiotic prophase and were arrested at pachytene stage, and there was a significant increase in the number of apoptotic germ cells in these mice. In wild-type mice, a single heat exposure caused the activation of HSF1 and similar histological changes such as a stage-specific apoptosis of pachytene spermatocytes. These results suggest that male infertility caused by thermal insult is at least partly due to the activation of HSF1, which induces the primary spermatocytes to undergo apoptosis. PMID- 10747024 TI - The Escherichia coli RNA polymerase alpha subunit linker: length requirements for transcription activation at CRP-dependent promoters. AB - The C-terminal domain of the Escherichia coli RNA polymerase alpha subunit (alphaCTD) plays a key role in transcription initiation at many activator dependent promoters. This domain is connected to the N-terminal domain by an unstructured linker, which is proposed to confer a high degree of mobility on alphaCTD. To investigate the role of this linker in transcription activation we tested the effect of altering the linker length on promoters dependent on the cyclic AMP receptor protein (CRP). Short deletions within the alpha linker decrease CRP-dependent transcription at a Class I promoter while increasing the activity of a Class II promoter. Linker extension impairs CRP-dependent transcription from both promoters, with short extensions exerting a more marked effect on the Class II promoter. Activation at both classes of promoter was shown to remain dependent upon activating region 1 of CRP. These results show that the response to CRP of RNA polymerase containing linker-modified alpha subunits is class specific. These observations have important implications for the architecture of transcription initiation complexes at CRP-dependent promoters. PMID- 10747025 TI - Dominant-negative mutants reveal a role for the Cdk7 kinase at the mid-blastula transition in Drosophila embryos. AB - The metazoan cyclin-dependent kinase Cdk7 was purified originally as part of a biochemical activity called CAK (Cdk-activating kinase) capable of phosphorylating and activating in vitro the Cdks that promote the different cell cycle transitions. Cdk7 is also found in the transcription factor complex TFIIH, suggesting that it participates in vivo in the control of RNA polymerase II. We have examined the physiological role of Cdk7 during the course of Drosophila development. By expressing dominant-negative forms of the kinase, we were able to alter Cdk7 function at given developmental stages. Expression of Cdk7 mutants severely delayed the onset of zygotic transcription in the early embryo, but did not alter the timing of the first 13 embryonic nuclear cycles. These results implicate Cdk7 in the control of transcriptional machinery in vivo. While cell cycle regulation is not sensitive to our manipulations of Cdk7 activity, it suggests that a distinct pool of CAK activity that is unaffected by expression of the cdk7(DN) mutants is present in these embryos. PMID- 10747027 TI - The structure of mouse HP1 suggests a unique mode of single peptide recognition by the shadow chromo domain dimer. AB - The heterochromatin protein 1 (HP1) family of proteins is involved in gene silencing via the formation of heterochromatic structures. They are composed of two related domains: an N-terminal chromo domain and a C-terminal shadow chromo domain. Present results suggest that chromo domains may function as protein interaction motifs, bringing together different proteins in multi-protein complexes and locating them in heterochromatin. We have previously determined the structure of the chromo domain from the mouse HP1beta protein, MOD1. We show here that, in contrast to the chromo domain, the shadow chromo domain is a homodimer. The intact HP1beta protein is also dimeric, where the interaction is mediated by the shadow chromo domain, with the chromo domains moving independently of each other at the end of flexible linkers. Mapping studies, with fragments of the CAF1 and TIF1beta proteins, show that an intact, dimeric, shadow chromo domain structure is required for complex formation. PMID- 10747026 TI - The atypical PKC-interacting protein p62 channels NF-kappaB activation by the IL 1-TRAF6 pathway. AB - The atypical protein kinase C (aPKC)-interacting protein, p62, has previously been shown to interact with RIP, linking these kinases to NF-kappaB activation by tumor necrosis factor alpha (TNFalpha). The aPKCs have been implicated in the activation of IKKbeta in TNFalpha-stimulated cells and have been shown to be activated in response to interleukin-1 (IL-1). Here we demonstrate that the inhibition of the aPKCs or the down-regulation of p62 severely abrogates NF kappaB activation by IL-1 and TRAF6, suggesting that both proteins are critical intermediaries in this pathway. Consistent with this we show that p62 selectively interacts with the TRAF domain of TRAF6 but not that of TRAF5 or TRAF2 in co transfection experiments. The binding of endogenous p62 to TRAF6 is stimulus dependent, reinforcing the notion that this is a physiologically relevant interaction. Furthermore, we demonstrate that the N-terminal domain of TRAF6, which is required for signaling, interacts with zetaPKC in a dimerization dependent manner. Together, these results indicate that p62 is an important intermediary not only in TNFalpha but also in IL-1 signaling to NF-kappaB through the specific adapters RIP and TRAF6. PMID- 10747028 TI - Histone H2A is required for normal centromere function in Saccharomyces cerevisiae. AB - Histones are structural and functional components of the eukaryotic chromosome, and their function is essential for normal cell cycle progression. In this work, we describe the characterization of two Saccharomyces cerevisiae cold-sensitive histone H2A mutants. Both mutants contain single amino acid replacements of residues predicted to be on the surface of the nucleosome and in close contact with DNA. We show that these H2A mutations cause an increase-in-ploidy phenotype, an increased rate of chromosome loss, and a defect in traversing the G(2)-M phase of the cell cycle. Moreover, these H2A mutations show genetic interactions with mutations in genes encoding kinetochore components. Finally, chromatin analysis of these H2A mutants has revealed an altered centromeric chromatin structure. Taken together, these results strongly suggest that histone H2A is required for proper centromere-kinetochore function during chromosome segregation. PMID- 10747029 TI - SCF(Met30)-mediated control of the transcriptional activator Met4 is required for the G(1)-S transition. AB - Progression through the cell cycle requires the coordination of basal metabolism with the cell cycle and growth machinery. Repression of the sulfur gene network is mediated by the ubiquitin ligase SCF(Met30), which targets the transcription factor Met4p for degradation. Met30p is an essential protein in yeast. We have found that a met4Deltamet30Delta double mutant is viable, suggesting that the essential function of Met30p is to control Met4p. In support of this hypothesis, a Met4p mutant unable to activate transcription does not cause inviability in a met30Delta strain. Also, overexpression of an unregulated Met4p mutant is lethal in wild-type cells. Under non-permissive conditions, conditional met30Delta strains arrest as large, unbudded cells with 1N DNA content, at or shortly after the pheromone arrest point. met30Delta conditional mutants fail to accumulate CLN1 and CLN2, but not CLN3 mRNAs, even when CLN1 and CLN2 are expressed from strong heterologous promoters. One or more genes under the regulation of Met4p may delay the progression from G(1) into S phase through specific regulation of critical G(1) phase mRNAs. PMID- 10747030 TI - Functional characterization of SR and SR-related genes in Caenorhabditis elegans. AB - The SR proteins constitute a family of nuclear phosphoproteins, which are required for constitutive splicing and also influence alternative splicing regulation. Initially, it was suggested that SR proteins were functionally redundant in constitutive splicing. However, differences have been observed in alternative splicing regulation, suggesting unique functions for individual SR proteins. Homology searches of the Caenorhabditis elegans genome identified seven genes encoding putative orthologues of the human factors SF2/ASF, SRp20, SC35, SRp40, SRp75 and p54, and also several SR-related genes. To address the issue of functional redundancy, we used dsRNA interference (RNAi) to inhibit specific SR protein function during C.elegans development. RNAi with CeSF2/ASF caused late embryonic lethality, suggesting that this gene has an essential function during C.elegans development. RNAi with other SR genes resulted in no obvious phenotype, which is indicative of gene redundancy. Simultaneous interference of two or more SR proteins in certain combinations caused lethality or other developmental defects. RNAi with CeSRPK, an SR protein kinase, resulted in early embryonic lethality, suggesting an essential role for SR protein phosphorylation during development. PMID- 10747031 TI - UBP1, a novel hnRNP-like protein that functions at multiple steps of higher plant nuclear pre-mRNA maturation. AB - Efficient splicing of higher plant pre-mRNAs depends on AU- or U-rich sequences in introns. Moreover, AU-rich sequences present in 3'-untranslated regions (3' UTRs) may play a role in 3' end processing of plant mRNAs. Here, we describe the cloning and characterization of a Nicotiana plumbaginifolia nuclear protein that can be cross-linked to U-rich intron and 3'-UTR sequences in vitro, and associates with nuclear poly(A)(+) RNA in vivo. The protein, UBP1, strongly enhances the splicing of otherwise inefficiently processed introns when overexpressed in protoplasts. It also increases the accumulation of reporter mRNAs that contain suboptimal introns or are intronless. The enhanced accumulation is apparently due to UBP1 interacting with the 3'-UTR and protecting mRNA from exonucleolytic degradation. The effect on mRNA accumulation but not on mRNA splicing was found to be promoter specific. The fact that these effects of UBP1 can be separated suggests that they represent two independent activities. The properties of UBP1 indicate that it is an hnRNP protein that functions at multiple steps to facilitate the nuclear maturation of plant pre-mRNAs. PMID- 10747032 TI - U snRNP assembly in yeast involves the La protein. AB - In all eukaryotic nuclei, the La autoantigen binds nascent RNA polymerase III transcripts, stabilizing these RNAs against exonucleases. Here we report that the La protein also functions in the assembly of certain RNA polymerase II transcribed RNAs into RNPs. A mutation in a core protein of the spliceosomal snRNPs, Smd1p, causes yeast cells to require the La protein Lhp1p for growth at low temperatures. Precursors to U1, U2, U4 and U5 RNAs are bound by Lhp1p in both wild-type and mutant cells. At the permissive temperature, smd1-1 cells contain higher levels of stable U1 and U5 snRNPs when Lhp1p is present. At low temperatures, Lhp1p becomes essential for the accumulation of U4/U6 snRNPs and for cell viability. When U4 RNA is added to extracts, the pre-U4 RNA, but not the mature RNA, is bound by Smd1p. These results suggest that, by stabilizing a 3' extended form of U4 RNA, Lhp1p facilitates efficient Sm protein binding, thus assisting formation of the U4/U6 snRNP. PMID- 10747033 TI - A Sm-like protein complex that participates in mRNA degradation. AB - In eukaryotes, seven Sm proteins bind to the U1, U2, U4 and U5 spliceosomal snRNAs while seven Smlike proteins (Lsm2p-Lsm8p) are associated with U6 snRNA. Another yeast Sm-like protein, Lsm1p, does not interact with U6 snRNA. Surprisingly, using the tandem affinity purification (TAP) method, we identified Lsm1p among the subunits associated with Lsm3p. Coprecipitation experiments demonstrated that Lsm1p, together with Lsm2p-Lsm7p, forms a new seven-subunit complex. We purified the two related Sm-like protein complexes and identified the proteins recovered in the purified preparations by mass spectrometry. This confirmed the association of the Lsm2p-Lsm8p complex with U6 snRNA. In contrast, the Lsm1p-Lsm7p complex is associated with Pat1p and Xrn1p exoribonuclease, suggesting a role in mRNA degradation. Deletions of LSM1, 6, 7 and PAT1 genes increased the half-life of reporter mRNAs. Interestingly, accumulating mRNAs were capped, suggesting a block in mRNA decay at the decapping step. These results indicate the involvement of a new conserved Sm-like protein complex and a new factor, Pat1p, in mRNA degradation and suggest a physical connection between decapping and exonuclease trimming. PMID- 10747034 TI - Suppression of post-transcriptional gene silencing by a plant viral protein localized in the nucleus. AB - Post-transcriptional gene silencing (PTGS) is a homology-dependent RNA degradation process that may target RNA exclusively in the cytoplasm. In plants, PTGS functions as a natural defense mechanism against viruses. We reported previously that the 2b protein encoded by cucumber mosaic cucumovirus (CMV) is a virulence determinant and a suppressor of PTGS initiation in transgenic Nicotiana benthamiana. By fusion with the green fluorescent protein, we now show that the CMV 2b protein localizes to the nuclei of tobacco suspension cells and whole plants via an arginine-rich nuclear localization signal, (22)KRRRRR(27). We further demonstrate that the nuclear targeting of the 2b protein is required for the efficient suppression of PTGS, indicating that PTGS may be blocked in the nucleus. In addition, our data indicate that the PTGS suppressor activity is important, but not sufficient, for virulence determination by the 2b protein. PMID- 10747035 TI - Chromatin binding of the fission yeast replication factor mcm4 occurs during anaphase and requires ORC and cdc18. AB - We describe an in situ technique for studying the chromatin binding of proteins in the fission yeast Schizosaccharomyces pombe. After tagging the protein of interest with green fluorescent protein (GFP), chromatin-associated protein is detected by GFP fluorescence following cell permeabilization and washing with a non-ionic detergent. Cell morphology and nuclear structure are preserved in this procedure, allowing structures such as the mitotic spindle to be detected by indirect immunofluorescence. Cell cycle changes in the chromatin association of proteins can therefore be determined from individual cells in asynchronous cultures. We have applied this method to the DNA replication factor mcm4/cdc21, and find that chromatin association occurs during anaphase B, significantly earlier than is the case in budding yeast. Binding of mcm4 to chromatin requires orc1 and cdc18 (homologous to Cdc6 in budding yeast). Release of mcm4 from chromatin occurs during S phase and requires DNA replication. Upon overexpressing cdc18, we show that mcm4 is required for re-replication of the genome in the absence of mitosis and is associated with chromatin in cells undergoing re replication. PMID- 10747036 TI - A novel SMC protein complex in Schizosaccharomyces pombe contains the Rad18 DNA repair protein. AB - In Schizosaccharomyces pombe, rad18 is an essential gene involved in the repair of DNA damage produced by ionizing radiation and in tolerance of UV-induced DNA damage. The Rad18 protein is a member of the SMC (structural maintenance of chromosomes) superfamily, and we show that, like the other SMC proteins in condensin and cohesin, Rad18 is a component of a high-molecular-weight complex. This complex contains at least six other proteins, the largest of which is Spr18, a novel SMC family member closely related to Rad18, and likely to be its heterodimeric partner. SMC proteins have ATP-binding domains at the N- and C termini, and two extended coiled-coil domains separated by a hinge in the middle. We show that the N-terminal ATP-binding domain of Rad18 is essential for all functions, and overexpression of an N-terminal mutant has a dominant-negative effect. We have identified an important mutation (S1045A) near the C-terminus of Rad18 that separates its repair and essential roles. Potential models for the role of the Rad18-Spr18 complex during DNA repair are discussed. PMID- 10747038 TI - Efficient repair of A/C mismatches in mouse cells deficient in long-patch mismatch repair. AB - A previously unrecognized mismatch repair activity is described. Extracts of immortalized MSH2-deficient mouse fibroblasts did not correct most single base mispairs. The same extracts carried out efficient repair of A/C mismatches. A/G mispairs were less efficiently corrected and there was no significant repair of A/A. MLH1-defective mouse extracts also repaired an A/C mispair. A/C correction by Msh2(-/-) mouse cell extracts was not affected by antibodies against the PMS2 protein, which inhibited long-patch mismatch repair. A/C repair activity is thus independent of MutSalpha, MutSbeta and MutLalpha. A/C mismatches were corrected 5 fold more efficiently by extracts of Msh2 knockout mouse cells than by comparable extracts prepared from hMSH2- or hMLH1-deficient human cells. MSH2-independent A/C correction by mouse cell extracts did not require a nick in the circular duplex DNA substrate. Repair involved replacement of the A and was associated with the resynthesis of a limited stretch of 0.8 in selfing populations. The estimation may be unbiased under partial selfing with variable and epistatic mutation effects in predominantly outcrossing populations. The estimation is fairly robust in nonequilibrium populations at different stages approaching M-S balance. The dynamics of populations approaching M-S balance under various parameters are also studied. Under mutation and selection, populations approach balance at a rapid pace. Generally, it takes 400-2000 generations to reach M-S balance even when starting from homogeneous individuals free of DGM. Our investigation here provides a basis for characterizing DGM in partial selfing or outcrossing populations and for nonequilibrium populations. PMID- 10747078 TI - Genetic parentage in large half-sib clutches: theoretical estimates and empirical appraisals. AB - Nearly all of the 906 embryos from a male-tended nest of the sand goby (Pomatoschistus minutus) were genotyped at two hypervariable microsatellite loci to document conclusively the number of mothers and their relative genetic contributions to the nest. The true number of mothers determined by this nearly exhaustive genetic appraisal was compared to computer simulation treatments based on allele frequencies in the population, assumptions about reproductive skew, and statistical sampling strategies of progeny subsets. The "ground-truthed" appraisal and the theoretical estimates showed good agreement, indicating that for this nest a random sample of approximately 20 offspring would have sufficed for assessing the true number of biological parents (but not necessarily their relative genetic contributions). Also, a general dilocus matrix procedure is suggested for organizing and interpreting otherwise cumbersome data sets when extremely large numbers of full-sib and half-sib embryos from a nest are genotyped at two or more hypervariable loci. PMID- 10747079 TI - Reversal of fortune. Do Rab GTPases act on the target membrane? PMID- 10747080 TI - Microtubules in orbit. PMID- 10747081 TI - Acting like actin. The dynamics of the nematode major sperm protein (msp) cytoskeleton indicate a push-pull mechanism for amoeboid cell motility. PMID- 10747082 TI - Pores in the wall: claudins constitute tight junction strands containing aqueous pores. PMID- 10747083 TI - Keratin-dependent, epithelial resistance to tumor necrosis factor-induced apoptosis. AB - Tumor necrosis factor (TNF) is a cytokine produced by macrophages and T lymphocytes that acts through two distinct receptors, TNFR1 (60 kD, CD120a) and TNFR2 (80 kD, CD120b), to affect cellular proliferation, differentiation, survival, and cell death. In addition to its proinflammatory actions in mucosal tissue, TNF is important for liver regeneration. Keratin 8 (K8) and keratin 18 (K18) form intermediate filaments characteristic of liver and other single cell layered, internal epithelia and their derivative cancers. K8-deficient (K8(-)) mice, which escape embryonic lethality, develop inflammatory colorectal hyperplasia, mild liver abnormalities, and tolerate hepatectomy poorly. We show that normal and malignant epithelial cells deficient in K8 and K18 are approximately 100 times more sensitive to TNF-induced death. K8 and K18 both bind the cytoplasmic domain of TNFR2 and moderate TNF-induced, Jun NH(2)-terminal kinase (JNK) intracellular signaling and NFkappaB activation. Furthermore, K8(-) and K18(-) mice are much more sensitive to TNF dependent, apoptotic liver damage induced by the injection of concanavalin A. This moderation of the effects of TNF may be the fundamental function of K8 and K18 common to liver regeneration, inflammatory bowel disease, hepatotoxin sensitivity, and the diagnostic, persistent expression of these keratins in many carcinomas. PMID- 10747084 TI - Imaging constitutive exocytosis with total internal reflection fluorescence microscopy. AB - Total internal reflection fluorescence microscopy has been applied to image the final stage of constitutive exocytosis, which is the fusion of single post-Golgi carriers with the plasma membrane. The use of a membrane protein tagged with green fluorescent protein allowed the kinetics of fusion to be followed with a time resolution of 30 frames/s. Quantitative analysis allowed carriers undergoing fusion to be easily distinguished from carriers moving perpendicularly to the plasma membrane. The flattening of the carriers into the plasma membrane is seen as a simultaneous rise in the total, peak, and width of the fluorescence intensity. The duration of this flattening process depends on the size of the carriers, distinguishing small spherical from large tubular carriers. The spread of the membrane protein into the plasma membrane upon fusion is diffusive. Mapping many fusion sites of a single cell reveals that there are no preferred sites for constitutive exocytosis in this system. PMID- 10747085 TI - Fusion of constitutive membrane traffic with the cell surface observed by evanescent wave microscopy. AB - Monitoring the fusion of constitutive traffic with the plasma membrane has remained largely elusive. Ideally, fusion would be monitored with high spatial and temporal resolution. Recently, total internal reflection (TIR) microscopy was used to study regulated exocytosis of fluorescently labeled chromaffin granules. In this technique, only the bottom cellular surface is illuminated by an exponentially decaying evanescent wave of light. We have used a prism type TIR setup with a penetration depth of approximately 50 nm to monitor constitutive fusion of vesicular stomatitis virus glycoprotein tagged with the yellow fluorescent protein. Fusion of single transport containers (TCs) was clearly observed and gave a distinct analytical signature. TCs approached the membrane, appeared to dock, and later rapidly fuse, releasing a bright fluorescent cloud into the membrane. Observation and analysis provided insight about their dynamics, kinetics, and position before and during fusion. Combining TIR and wide field microscopy allowed us to follow constitutive cargo from the Golgi complex to the cell surface. Our observations include the following: (1) local restrained movement of TCs near the membrane before fusion; (2) apparent anchoring near the cell surface; (3) heterogeneously sized TCs fused either completely; or (4) occasionally larger tubular-vesicular TCs partially fused at their tips. PMID- 10747086 TI - Structure and assembly of the Nup84p complex. AB - The Nup84p complex consists of five nucleoporins (Nup84p, Nup85p, Nup120p, Nup145p-C, and Seh1p) and Sec13p, a bona fide subunit of the COPII coat complex. We show that a pool of green fluorescent protein-tagged Sec13p localizes to the nuclear pores in vivo, and identify sec13 mutant alleles that are synthetically lethal with nup85Delta and affect the localization of a green fluorescent protein Nup49p reporter protein. In the electron microscope, sec13 mutants exhibit structural defects in nuclear pore complex (NPC) and nuclear envelope organization. For the assembly of the complex, Nup85p, Nup120p, and Nup145p-C are essential. A highly purified Nup84p complex was isolated from yeast under native conditions and its molecular mass was determined to be 375 kD by quantitative scanning transmission electron microscopy and analytical ultracentrifugation, consistent with a monomeric complex. Furthermore, the Nup84p complex exhibits a Y shaped, triskelion-like morphology 25 nm in diameter in the transmission electron microscope. Thus, the Nup84p complex constitutes a paradigm of an NPC structural module with distinct composition, structure, and a role in nuclear mRNA export and NPC bio- genesis. PMID- 10747087 TI - Asymmetric requirements for a Rab GTPase and SNARE proteins in fusion of COPII vesicles with acceptor membranes. AB - Soluble NSF attachment protein receptor (SNARE) proteins are essential for membrane fusion in transport between the yeast ER and Golgi compartments. Subcellular fractionation experiments demonstrate that the ER/Golgi SNAREs Bos1p, Sec22p, Bet1p, Sed5p, and the Rab protein, Ypt1p, are distributed similarly but localize primarily with Golgi membranes. All of these SNARE proteins are efficiently packaged into COPII vesicles and suggest a dynamic cycling of SNARE machinery between ER and Golgi compartments. Ypt1p is not efficiently packaged into vesicles under these conditions. To determine in which membranes protein function is required, temperature-sensitive alleles of BOS1, BET1, SED5, SLY1, and YPT1 that prevent ER/Golgi transport in vitro at restrictive temperatures were used to selectively inactivate these gene products on vesicles or on Golgi membranes. Vesicles bearing mutations in Bet1p or Bos1p inhibit fusion with wild type acceptor membranes, but acceptor membranes containing these mutations are fully functional. In contrast, vesicles bearing mutations in Sed5p, Sly1p, or Ypt1p are functional, whereas acceptor membranes containing these mutations block fusion. Thus, this set of SNARE proteins is symmetrically distributed between vesicle and acceptor compartments, but they function asymmetrically such that Bet1p and Bos1p are required on vesicles and Sed5p activity is required on acceptor membranes. We propose the asymmetry in SNARE protein function is maintained by an asymmetric distribution and requirement for the Ypt1p GTPase in this fusion event. When a transmembrane-anchored form of Ypt1p is used to restrict this GTPase to the acceptor compartment, vesicles depleted of Ypt1p remain competent for fusion. PMID- 10747088 TI - A family of proteins with gamma-adaptin and VHS domains that facilitate trafficking between the trans-Golgi network and the vacuole/lysosome. AB - We have cloned and characterized members of a novel family of proteins, the GGAs. These proteins contain an NH(2)-terminal VHS domain, one or two coiled-coil domains, and a COOH-terminal domain homologous to the COOH-terminal "ear" domain of gamma-adaptin. However, unlike gamma-adaptin, the GGAs are not associated with clathrin-coated vesicles or with any of the components of the AP-1 complex. GGA1 and GGA2 are also not associated with each other, although they colocalize on perinuclear membranes. Immunogold EM shows that these membranes correspond to trans elements of the Golgi stack and the TGN. GST pulldown experiments indicate that the GGA COOH-terminal domains bind to a subset of the proteins that bind to the gamma-adaptin COOH-terminal domain. In yeast there are two GGA genes. Deleting both of these genes results in missorting of the vacuolar enzyme carboxypeptidase Y, and the cells also have a defective vacuolar morphology phenotype. These results indicate that the function of the GGAs is to facilitate the trafficking of proteins between the TGN and the vacuole, or its mammalian equivalent, the lysosome. PMID- 10747089 TI - GGAs: a family of ADP ribosylation factor-binding proteins related to adaptors and associated with the Golgi complex. AB - Formation of intracellular transport intermediates and selection of cargo molecules are mediated by protein coats associated with the cytosolic face of membranes. Here, we describe a novel family of ubiquitous coat proteins termed GGAs, which includes three members in humans and two in yeast. GGAs have a modular structure consisting of a VHS domain, a region of homology termed GAT, a linker segment, and a region with homology to the ear domain of gamma-adaptins. Immunofluorescence microscopy showed colocalization of GGAs with Golgi markers, whereas immunoelectron microscopy of GGA3 revealed its presence on coated vesicles and buds in the area of the TGN. Treatment with brefeldin A or overexpression of dominant-negative ADP ribosylation factor 1 (ARF1) caused dissociation of GGAs from membranes. The GAT region of GGA3 was found to: target a reporter protein to the Golgi complex; induce dissociation from membranes of ARF-regulated coats such as AP-1, AP-3, AP-4, and COPI upon overexpression; and interact with activated ARF1. Disruption of both GGA genes in yeast resulted in impaired trafficking of carboxypeptidase Y to the vacuole. These observations suggest that GGAs are components of ARF-regulated coats that mediate protein trafficking at the TGN. PMID- 10747090 TI - The role of the COOH terminus of Sec2p in the transport of post-Golgi vesicles. AB - Sec2p is required for the polarized transport of secretory vesicles in S. cerevisiae. The Sec2p NH(2) terminus encodes an exchange factor for the Rab protein Sec4p. Sec2p associates with vesicles and in Sec2p COOH-terminal mutants Sec4p and vesicles no longer accumulate at bud tips. Thus, the Sec2p COOH terminus functions in targeting vesicles, however, the mechanism of function is unknown. We found comparable exchange activity for truncated and full-length Sec2 proteins, implying that the COOH terminus does not alter the exchange rate. Full length Sec2-GFP, similar to Sec4p, concentrates at bud tips. A COOH-terminal 58 amino acid domain is necessary but not sufficient for localization. Sec2p localization depends on actin, Myo2p and Sec1p, Sec6p, and Sec9p function. Full length, but not COOH-terminally truncated Sec2 proteins are enriched on membranes. Membrane association of full-length Sec2p is reduced in sec6-4 and sec9-4 backgrounds at 37 degrees C but unaffected at 25 degrees C. Taken together, these data correlate loss of localization of Sec2 proteins with reduced membrane association. In addition, Sec2p membrane attachment is substantially Sec4p independent, supporting the notion that Sec2p interacts with membranes via an unidentified Sec2p receptor, which would increase the accessibility of Sec2p exchange activity for Sec4p. PMID- 10747091 TI - Compromised cytoarchitecture and polarized trafficking in autosomal dominant polycystic kidney disease cells. AB - Cystogenesis associated with autosomal dominant polycystic kidney disease (ADPKD) is characterized by perturbations in the polarized phenotype and function of cyst lining epithelial cells. The polycystins, the protein products of the genes mutated in the majority of ADPKD cases, have been described recently, but the pathological mechanism by which causal mutations result in the mislocalization of cell membrane proteins has remained unclear. This report documents the dissociation from the ADPKD cell basolateral membrane of three molecules essential for spatial organization and exocytosis. The adherens junction protein E-cadherin, the subcellular disposition of which governs intercellular and intracellular architecture, was discovered sequestered in an internal ADPKD cell compartment. At the same time, sec6 and sec8, components of a complex critical for basolateral cargo delivery normally arrayed at the apico-lateral apex, were depleted from the ADPKD cell plasma membrane. An analysis of membrane transport revealed that basolateral trafficking of proteins and lipids was impaired as a result of delayed cargo exit from the ADPKD cell Golgi apparatus. Apical transport proceeded normally. Taken together with recent documentation of an association between polycystin-1 and E-cadherin (Huan and van Adelsberg 1999), the data suggest that causal mutations disrupt E-cadherin-dependent cytoarchitecture, adversely affecting protein assemblies crucial for basolateral trafficking. PMID- 10747092 TI - Dynamic localization of protein phosphatase type 1 in the mitotic cell cycle of Saccharomyces cerevisiae. AB - Protein phosphatase type I (PP1), encoded by the single essential gene GLC7 in Saccharomyces cerevisiae, functions in diverse cellular processes. To identify in vivo subcellular location(s) where these processes take place, we used a functional green fluorescent protein (GFP)-Glc7p fusion protein. Time-lapse fluorescence microscopy revealed GFP-Glc7p localizes predominantly in the nucleus throughout the mitotic cell cycle, with the highest concentrations in the nucleolus. GFP-Glc7p was also observed in a ring at the bud neck, which was dependent upon functional septins. Supporting a role for Glc7p in bud site selection, a glc7-129 mutant displayed a random budding pattern. In alpha-factor treated cells, GFP-Glc7p was located at the base of mating projections, again in a septin-dependent manner. At the start of anaphase, GFP-Glc7p accumulated at the spindle pole bodies and remained there until cytokinesis. After anaphase, GFP Glc7p became concentrated in a ring that colocalized with the actomyosin ring. A GFP-Glc7-129 fusion was defective in localizing to the bud neck and SPBs. Together, these results identify sites of Glc7p function and suggest Glc7p activity is regulated through dynamic changes in its location. PMID- 10747093 TI - Evidence for the involvement of KIF4 in the anterograde transport of L1 containing vesicles. AB - In this study we present evidence about the cellular functions of KIF4. Using subcellular fractionation techniques and immunoisolation, we have now identified a type of vesicle that associates with KIF4, an NH(2)-terminal globular motor domain kinesin-like protein. This vesicle is highly concentrated in growth cones and contains L1, a cell adhesion molecule implicated in axonal elongation. It lacks synaptic vesicle markers, receptors for neurotrophins, and membrane proteins involved in growth cone guidance. In cultured neurons, KIF4 and L1 predominantly localize to the axonal shaft and its growth cone. Suppression of KIF4 with antisense oligonucleotides results in the accumulation of L1 within the cell body and in its complete disappearance from axonal tips. In addition, KIF4 suppression prevents L1-enhanced axonal elongation. Taken collectively, our results suggest an important role for KIF4 during neuronal development, a phenomenon which may be related to the anterograde transport of L1-containing vesicles. PMID- 10747094 TI - Orbit, a novel microtubule-associated protein essential for mitosis in Drosophila melanogaster. AB - We describe a Drosophila gene, orbit, that encodes a conserved 165-kD microtubule associated protein (MAP) with GTP binding motifs. Hypomorphic mutations in orbit lead to a maternal effect resulting in branched and bent mitotic spindles in the syncytial embryo. In the larval central nervous system, such mutants have an elevated mitotic index with some mitotic cells showing an increase in ploidy. Amorphic alleles show late lethality and greater frequencies of hyperploid mitotic cells. The presence of cells in the hypomorphic mutant in which the chromosomes can be arranged, either in a circular metaphase or an anaphase-like configuration on monopolar spindles, suggests that polyploidy arises through spindle and chromosome segregation defects rather than defects in cytokinesis. A role for the Orbit protein in regulating microtubule behavior in mitosis is suggested by its association with microtubules throughout the spindle at all mitotic stages, by its copurification with microtubules from embryonic extracts, and by the finding that the Orbit protein directly binds to MAP-free microtubules in a GTP-dependent manner. PMID- 10747095 TI - Toxoplasma gondii exploits host low-density lipoprotein receptor-mediated endocytosis for cholesterol acquisition. AB - The obligate intracellular protozoan Toxoplasma gondii resides within a specialized parasitophorous vacuole (PV), isolated from host vesicular traffic. In this study, the origin of parasite cholesterol was investigated. T. gondii cannot synthesize sterols via the mevalonate pathway. Host cholesterol biosynthesis remains unchanged after infection and a blockade in host de novo sterol biosynthesis does not affect parasite growth. However, simultaneous limitation of exogenous and endogenous sources of cholesterol from the host cell strongly reduces parasite replication and parasite growth is stimulated by exogenously supplied cholesterol. Intracellular parasites acquire host cholesterol that is endocytosed by the low-density lipoprotein (LDL) pathway, a process that is specifically increased in infected cells. Interference with LDL endocytosis, with lysosomal degradation of LDL, or with cholesterol translocation from lysosomes blocks cholesterol delivery to the PV and significantly reduces parasite replication. Similarly, incubation of T. gondii in mutant cells defective in mobilization of cholesterol from lysosomes leads to a decrease of parasite cholesterol content and proliferation. This cholesterol trafficking to the PV is independent of the pathways involving the host Golgi or endoplasmic reticulum. Despite being segregated from the endocytic machinery of the host cell, the T. gondii vacuole actively accumulates LDL-derived cholesterol that has transited through host lysosomes. PMID- 10747096 TI - Fyn-binding protein (Fyb)/SLP-76-associated protein (SLAP), Ena/vasodilator stimulated phosphoprotein (VASP) proteins and the Arp2/3 complex link T cell receptor (TCR) signaling to the actin cytoskeleton. AB - T cell receptor (TCR)-driven activation of helper T cells induces a rapid polarization of their cytoskeleton towards bound antigen presenting cells (APCs). We have identified the Fyn- and SLP-76-associated protein Fyb/SLAP as a new ligand for Ena/ vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Upon TCR engagement, Fyb/SLAP localizes at the interface between T cells and anti-CD3-coated beads, where Evl, a member of the Ena/VASP family, Wiskott Aldrich syndrome protein (WASP) and the Arp2/3 complex are also found. In addition, Fyb/SLAP is restricted to lamellipodia of spreading platelets. In activated T cells, Fyb/SLAP associates with Ena/VASP family proteins and is present within biochemical complexes containing WASP, Nck, and SLP-76. Inhibition of binding between Fyb/SLAP and Ena/VASP proteins or WASP and the Arp2/3 complex impairs TCR-dependent actin rearrangement, suggesting that these interactions play a key role in linking T cell signaling to remodeling of the actin cytoskeleton. PMID- 10747097 TI - An epidermal plakin that integrates actin and microtubule networks at cellular junctions. AB - Plakins are cytoskeletal linker proteins initially thought to interact exclusively with intermediate filaments (IFs), but recently were found to associate additionally with actin and microtubule networks. Here, we report on ACF7, a mammalian orthologue of the Drosophila kakapo plakin genetically involved in epidermal-muscle adhesion and neuromuscular junctions. While ACF7/kakapo is divergent from other plakins in its IF-binding domain, it has at least one actin (K(d) = 0.35 microM) and one microtubule (K(d) approximately 6 microM) binding domain. Similar to its fly counterpart, ACF7 is expressed in the epidermis. In well spread epidermal keratinocytes, ACF7 discontinuously decorates the cytoskeleton at the cell periphery, including microtubules (MTs) and actin filaments (AFs) that are aligned in parallel converging at focal contacts. Upon calcium induction of intercellular adhesion, ACF7 and the cytoskeleton reorganize at cell-cell borders but with different kinetics from adherens junctions and desmosomes. Treatments with cytoskeletal depolymerizing drugs reveal that ACF7's cytoskeletal association is dependent upon the microtubule network, but ACF7 also appears to stabilize actin at sites where microtubules and microfilaments meet. We posit that ACF7 may function in microtubule dynamics to facilitate actin microtubule interactions at the cell periphery and to couple the microtubule network to cellular junctions. These attributes provide a clear explanation for the kakapo mutant phenotype in flies. PMID- 10747098 TI - The function of plakophilin 1 in desmosome assembly and actin filament organization. AB - Plakophilin 1, a member of the armadillo multigene family, is a protein with dual localization in the nucleus and in desmosomes. To elucidate its role in desmosome assembly and regulation, we have analyzed its localization and binding partners in vivo. When overexpressed in HaCaT keratinocytes, plakophilin 1 localized to the nucleus and to desmosomes, and dramatically enhanced the recruitment of desmosomal proteins to the plasma membrane. This effect was mediated by plakophilin 1's head domain, which interacted with desmoglein 1, desmoplakin, and keratins in the yeast two-hybrid system. Overexpression of the armadillo repeat domain induced a striking dominant negative phenotype with the formation of filopodia and long cellular protrusions, where plakophilin 1 colocalized with actin filaments. This phenotype was strictly dependent on a conserved motif in the center of the armadillo repeat domain. Our results demonstrate that plakophilin 1 contains two functionally distinct domains: the head domain, which could play a role in organizing the desmosomal plaque in suprabasal cells, and the armadillo repeat domain, which might be involved in regulating the dynamics of the actin cytoskeleton. PMID- 10747099 TI - Extracellular-regulated kinase activation and CAS/Crk coupling regulate cell migration and suppress apoptosis during invasion of the extracellular matrix. AB - Regulation of cell migration/invasion is important for embryonic development, immune function, and angiogenesis. However, migratory cells must also coordinately activate survival mechanisms to invade the extracellular matrix and colonize foreign sites in the body. Although invasive cells activate protective programs to survive under diverse and sometimes hostile conditions, the molecular signals that regulate these processes are poorly understood. Evidence is provided that signals that induce cell invasion also promote cell survival by suppressing apoptosis of migratory cells. Extracellular-regulated kinase (ERK) activation and molecular coupling of the adaptor proteins p130 Crk-associated substrate (CAS) and c-CrkII (Crk) represent two distinct pathways that induce cell invasion and protect cells from apoptosis in a three-dimensional collagen matrix. CAS/Crk mediated cell invasion and survival requires activation of the small GTPase Rac, whereas ERK-induced cell invasion, but not survival requires myosin light chain kinase activation and myosin light chain phosphorylation. Uncoupling CAS from Crk or inhibition of ERK activity prevents migration and induces apoptosis of invasive cells. These findings provide molecular evidence that during invasion of the extracellular matrix, cells coordinately regulate migration and survival mechanisms through ERK activation and CAS/Crk coupling. PMID- 10747100 TI - Newly recognized herpesvirus causing malignant catarrhal fever in white-tailed deer (Odocoileus virginianus). AB - Malignant catarrhal fever (MCF) was diagnosed by clinical signs and lesions in five out of six white-tailed deer (Odocoileus virginianus) in a North American zoo. The clinical signs and histopathological lesions in these deer were typical of MCF. Antibody to an epitope conserved among the MCF viruses was detected in the sera collected from the deer. PCR failed to amplify viral sequences from DNA extracted from peripheral blood leukocytes (PBL) and/or spleens of the deer with primers specific for ovine herpesvirus 2 (OHV-2) or specific for alcelaphine herpesvirus 1 (AHV-1). By using degenerate primers targeting a conserved region of a herpesviral DNA polymerase gene, a DNA fragment was amplified from the PBL or spleens of all six deer and sequenced. Alignment of the sequences demonstrated that the virus in the deer belongs to the Gammaherpesvirinae subfamily, exhibiting 82% identity to OHV-2, 71% to AHV-1, and 60% to a newly identified bovine lymphotropic herpesvirus. This virus, which causes classical MCF in white tailed deer, is a newly recognized agent belonging to the MCF group of gammaherpesviruses. It is the third reported pathogenic MCF virus, genetically distinct but closely related to OHV-2 and AHV-1. The reservoir for the virus has not been identified. PMID- 10747101 TI - Evaluation of serotype prediction by cpsA-cpsB gene polymorphism in Streptococcus pneumoniae. AB - New pneumococcal conjugate vaccines covering a limited number of serotypes are likely to come into widespread use over the next few years. It is unknown what effect this will have on the relative importance of different serotypes as causes of pneumococcal infection. Hence, it will be important to monitor serotype prevalence before, during, and after the introduction of new vaccines. We have investigated the ability of a PCR method based on polymorphisms in two genes common to the different capsule loci to predict the serotype of 93 clinical isolates of Streptococcus pneumoniae submitted to the Central Public Health Laboratory in 1997. Of 70 isolates with vaccine serotypes, 65 were predicted to belong to the correct serotype; this number was improved to 69 with the inclusion of two additional patterns to the database. Of 23 isolates with other serotypes, 19 were correctly predicted as non-vaccine serotypes, the discrepancy lying with four isolates of 6A (non-vaccine serotype) that were indistinguishable from isolates of 6B (vaccine serotype). In situations in which culture of the organism is not feasible, this method could potentially be applicable directly to clinical specimens and could be a valuable aid to the surveillance of pneumococcal serotypes. PMID- 10747102 TI - Genomic analysis of Blastocystis hominis strains isolated from two long-term health care facilities. AB - The genotype Blastocystis hominis is highly polymorphic. Therefore, a genetic marker would be a powerful tool for the identification or classification of B. hominis subtypes and could be used as a means to resolve the transmission route or origin of the parasite. To this end, 32 B. hominis isolates were collected from patients and/or staff members of two long-term health care facilities (facilities A and B), and these organisms were subjected to genotype analysis based on diagnostic PCR primers and restriction fragment length polymorphism (RFLP) of small subunit rRNA gene (rDNA). Based on PCR amplification using diagnostic primers which were developed from randomly amplified polymorphic DNA analysis of known strains of B. hominis, the 32 isolates of B. hominis were classified into three different subtypes. Thirty isolates, including twenty-four that were isolated from patients and a staff member, from facility A and all isolates isolated from six patients from facility B showed the same genotype. Two of six patients of facility B had been transferred from facility A, and these two patients also had the same-genotype B. hominis that corresponded to 24 isolates from facility A. This genotype strain may have been transmitted by these two patients from facility A to facility B, suggesting human-to-human transmission. In contrast, 2 of 26 isolates from facility A showed distinct genotypes, suggesting that the colonization by these two isolates is attributable to another infectious route. These different subtypes were subjected to RFLP analysis, and the RFLP profiles were correlated with the results obtained by diagnostic PCR primers. This study presents the first molecular evidence of possible human-to human B. hominis infection between and/or among two small communities. PMID- 10747103 TI - New Ehrlichia species closely related to Ehrlichia chaffeensis isolated from Ixodes ovatus ticks in Japan. AB - Seven Ehrlichia strains (six HF strains and one Anan strain) that were obtained from laboratory mice by intraperitoneally inoculating homogenates of adult Ixodes ovatus collected in Japan were characterized. 16S rRNA sequences of all six HF strains were identical, and the sequences were 99.7, 98.2, and 97.7% identical to those of Anan strain, Ehrlichia chaffeensis (human monocytic ehrlichiosis agent), and E. muris, respectively. Partial GroEL amino acid sequencing also revealed that the six HF strains had identical sequences, which were 99.0, 98.5, and 97.3% identical to those of E. chaffeensis, the Anan strain, and E. canis, respectively. All HF strains were lethal to mice at higher dosages and intraperitoneal inoculation, whereas the Anan or E. muris strain induced only mild clinical signs. Light and electron microscopy of moribund mice inoculated with one of the HF strains revealed severe liver necrosis and the presence of numerous ehrlichial inclusions (morulae) in various organs. The study revealed that members of E. canis genogroup are naturally present in Ixodes ticks. HF strains that can cause severe illness in immunocompetent laboratory mice would be valuable in studying the pathogenesis and the roles of both cellular and humoral immune responses in ehrlichiosis caused by E. canis genogroup. PMID- 10747104 TI - Evaluation of the Wider system, a new computer-assisted image-processing device for bacterial identification and susceptibility testing. AB - The Wider system is a newly developed computer-assisted image-processing device for both bacterial identification and antimicrobial susceptibility testing. It has been adapted to be able to read and interpret commercial MicroScan panels. Two hundred forty-four fresh consecutive clinical isolates (138 isolates of the family Enterobacteriaceae, 25 nonfermentative gram-negative rods [NFGNRs], and 81 gram-positive cocci) were tested. In addition, 100 enterobacterial strains with known beta-lactam resistance mechanisms (22 strains with chromosomal AmpC beta lactamase, 8 strains with chromosomal class A beta-lactamase, 21 broad-spectrum and IRT beta-lactamase-producing strains, 41 extended-spectrum beta-lactamase producing strains, and 8 permeability mutants) were tested. API galleries and National Committee for Clinical Laboratory Standards (NCCLS) microdilution methods were used as reference methods. The Wider system correctly identified 97.5% of the clinical isolates at the species level. Overall essential agreement (+/-1 log(2) dilution for 3,719 organism-antimicrobial drug combinations) was 95.6% (isolates of the family Enterobacteriaceae, 96.6%; NFGNRs, 88.0%; gram positive cocci, 95.6%). The lowest essential agreement was observed with Enterobacteriaceae versus imipenem (84.0%), NFGNR versus piperacillin (88.0%) and cefepime (88.0%), and gram-positive isolates versus penicillin (80.4%). The category error rate (NCCLS criteria) was 4.2% (2.0% very major errors, 0.6% major errors, and 1. 5% minor errors). Essential agreement and interpretive error rates for eight beta-lactam antibiotics against isolates of the family Enterobacteriaceae with known beta-lactam resistance mechanisms were 94.8 and 5.4%, respectively. Interestingly, the very major error rate was only 0.8%. Minor errors (3.6%) were mainly observed with amoxicillin-clavulanate and cefepime against extended-spectrum beta-lactamase-producing isolates. The Wider system is a new reliable tool which applies the image-processing technology to the reading of commercial trays for both bacterial identification and susceptibility testing. PMID- 10747105 TI - Comparison of protein A gene sequencing with pulsed-field gel electrophoresis and epidemiologic data for molecular typing of methicillin-resistant Staphylococcus aureus. AB - The epidemiologic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) isolates is currently determined by analysis of chromosomal DNA restriction patterns by pulsed-field gel electrophoresis (PFGE). We have evaluated an alternative typing system (MicroSeq StaphTrack Kit; Perkin-Elmer Biosystems) based on the sequence analysis of the chromosomally encoded polymorphic repeat X region of the S. aureus protein A (spa) gene. A total of 69 clinical MRSA isolates were divided into 18 groups according to the number and nucleotide sequences of the spa repeats. Molecular typing results obtained both by spa sequencing and from the PFGE patterns were concordant except for one group, which contained 20 isolates recovered over a 2-year period from hospitalized patients at the Mayo Clinic. Although the spa typing patterns were indistinguishable for those isolates, PFGE analysis yielded seven related but distinguishable patterns. Further coagulase gene sequence analysis subtyped those 20 strains into four groups which followed distinct temporal and geographic distributions. During a 2-year epidemic period there were up to 7 fragment changes in PFGE patterns among epidemiologically related isolates, suggesting that PFGE may be unsuitable for long-term typing of strains involved in epidemics. Although more limited than PFGE in discriminatory power, spa sequencing analysis could be used as a screening method for typing of MRSA strains because of the shorter turnaround time, ease of use, and the inherent advantages of sequence analysis, storage, and sharing of information. PMID- 10747106 TI - Mitochondrial cytochrome b gene analysis of Aspergillus fumigatus and related species. AB - Nucleotide sequences of 426 bp from the mitochondrial (mt) cytochrome b genes of six anamorph species and two species of Neosartorya teleomophs of Aspergillus section Fumigati were determined. These sequences were used to build nucleotide- and amino acid-based trees for phylogenetic analysis. Thirteen strains of A. fumigatus including 10 clinical isolates of A. fumigatus, 1 type culture of A. fumigatus var. fumigatus, 1 type culture of A. fumigatus var. ellipticus, and 1 strain of A. fumigatus var. albus, had the same nucleotide sequences. One strain of A. fumisynnematus, two strains labeled A. neoellipticus, two strains of A. viridinutans, and one strain of A. duricaulis had distinct nucleotide and amino acid sequences. Two strains of A. brevipes were divided into two types. One produced a 1,500-bp fragment that included an intron. The nucleotide sequences of its two exons were similar to those of the A. fumigatus, and the derived amino acid sequence was the same as that for A. fumigatus. The other produced a 426-bp fragment and had the same nucleotide and amino acid sequences as A. unilateralis. Neosartorya fischeri var. fischeri and N. stramenia had nucleotide sequences that differed from that of A. fumigatus. These species possessed their own characteristic nucleotide sequences that differed from each other. In comparisons of homologous sequences from four other pathogenic species of Aspergillus, regions specific to section Fumigati were found. The mt cytochrome b gene analysis was valuable for the identification, classification, and phylogenetic analysis of isolates of section Fumigati. PMID- 10747107 TI - Heterogeneous antimicrobial resistance patterns in polyclonal populations of coagulase-negative staphylococci isolated from catheters. AB - Most cases of nosocomial bacteremia are catheter related, and coagulase-negative staphylococci (CoNS) are the microorganisms most frequently associated with these infections. Subtle morphological differences are frequently found among CoNS colonies cultured from infected catheters. The aim of this study was to analyze the significance of the morphological heterogeneity observed in these CoNS populations. With this purpose in mind, the clonal composition of the CoNS populations obtained from a selection of nine catheters was analyzed by two different molecular techniques, arbitrarily primed-PCR and DNA macrorestriction analysis by pulsed-field gel electrophoresis. Twenty CoNS morphotypes were included for analysis, and four single colonies representative of each morphotype were selected. Morphological differences between colonies were found to correlate in all cases with differences at the molecular level. Unique fingerprints were also obtained for some isolates which were indistinguishable from other representatives of the same morphotypes. Differences in the molecular patterns among the isolates were associated in most of the cases with differences in the antimicrobial susceptibility patterns. The frequent isolation of polyclonal CoNS populations from catheters, with heterogeneous antimicrobial susceptibility patterns, has relevant epidemiologic and therapeutic implications in the context of catheter-related infections. PMID- 10747108 TI - Comparison of the nucleotide sequences of 16S rRNA, 444 Ep-ank, and groESL heat shock operon genes in naturally occurring Ehrlichia equi and human granulocytic ehrlichiosis agent isolates from Northern California. AB - We examined 11 naturally occurring isolates of Ehrlichia equi in horses and two human granulocytic ehrlichiosis agent isolates in California for sequence diversity in three genes. Ehrlichia equi isolates were from Sierra (n = 6), Mendocino (n = 3), Sonoma (n = 1), and Marin (n = 1) counties, and human granulocytic ehrlichiosis (HGE) agent isolates were obtained from Humboldt county. PCR with specific primers for 16S rRNA, 444 Ep-ank and groESL heat shock operon genes successfully produced amplicons for all 13 clinical samples. The 444 Ep-ank gene of the HGE agent and E. equi isolates from northern California is different from the eastern U.S. isolates BDS and USG3. The translated amino acid sequence of the groESL heat shock operon gene fragment is identical among E. equi, the HGE agent, and E. phagocytophila, with the exception of the northern Californian equine CASOLJ isolate. Microheterogeneity was observed in the 16S rRNA gene sequences of HGE agent and E. equi isolates from northern California. These results suggest that E. equi and the HGE agent found in California are similar or identical but may differ from the isolates of equine and human origin found in the eastern United States. PMID- 10747109 TI - Emergence of drug resistance mutations in human immunodeficiency virus type 2 infected subjects undergoing antiretroviral therapy. AB - The reverse transcriptase (RT) and protease genes from 12 human immunodeficiency virus type 2 (HIV-2)-infected individuals who had been exposed to antiretroviral drugs for longer than 6 months were examined for the presence of mutations which could be involved in drug resistance. Four individuals carried virus genotypes with amino acid substitutions potentially associated with resistance to nucleoside analogues: two at codon 70 (K-->R) and two at codon 184 (M-->V). Moreover, the latter two patients harbored a codon Q151M mutation which is associated to multidrug resistance in HIV-1, and one of these subjects carried some of the typically linked mutations at codons 65 and 69. With regard to the protease inhibitors, substitutions associated with resistance to protease inhibitors at codon 46 were observed in all individuals. Moreover, minor resistance mutations, as well as new ones of unknown meaning, were often seen in the protease gene. In conclusion, amino acid changes in the HIV-2 RT and protease genes which could be associated with drug resistance seem to occur at positions identical to those for HIV-1. PMID- 10747110 TI - Molecular evolution in a multidrug-resistant lineage of Streptococcus pneumoniae: emergence of strains belonging to the serotype 6B Icelandic clone that lost antibiotic resistance traits. AB - Since their first detection in 1988, penicillin-resistant Streptococcus pneumoniae isolates have rapidly spread in Iceland to account for close to 20% of all pneumococcal disease in that country by 1993. The major component (70%) of the resistant pneumococci identified from 1989 to 1992 was the progeny of a single multidrug-resistant clone (Icelandic clone) with a homogeneous chromosomal macrorestriction profile and identical multilocus enzyme type expressing serotype 6B and resistance to penicillin, tetracycline, chloramphenicol, erythromycin, and trimethoprim-sulfamethoxazole. The rest of the non-penicillin-susceptible isolates included bacteria with serotype 6A and serogroups 19 and 23. The unique geographic and epidemiological setting and the availability of a complete collection of all non-penicillin-susceptible isolates of S. pneumoniae in Iceland prompted us to carry out a molecular epidemiological study to monitor the fate of the Icelandic clone between 1989 and 1996; in addition, we wished to extend the characterization to representative groups of all non-penicillin-susceptible serotype 6B pneumococci which showed variations in antibiotype and which were recovered in Iceland between late 1989 and the end of 1996. Also included in the study were non-penicillin-susceptible isolates of serogroup 23. Pulsed-field gel electrophoresis of SmaI-restricted chromosomal DNA and Southern hybridization with the lytA DNA probe and probes specific for antibiotic resistance genes were used to characterize pneumococcal isolates. The results show that (i) the Icelandic clone remained the predominant type among penicillin-resistant S. pneumoniae through 1996; (ii) the emergence of variants of the Icelandic clone which had lost one or more of the antibiotic resistance phenotypes and/or resistant genes, singly or in combination, was documented during the surveillance period; and (iii) isolates belonging to the internationally spread multidrug resistant serotype 23F clone were present in the Icelandic collection since late 1989 but did not increase in number during the subsequent years. PMID- 10747111 TI - Clinical use of capillary PCR to diagnose Mycoplasma pneumonia. AB - In the present study, serologic data were compared with data obtained by capillary PCR to establish the efficacy of capillary PCR for the determination of Mycoplasma infection in samples obtained from throat swabs, bronchoalveolar lavage fluids (BALF), and sputum of patients with Mycoplasma pneumonia. We performed PCR analysis for Mycoplasma DNA on a total of 325 samples from 197 patients with community-acquired pneumonia and in whom Mycoplasma pneumonia was suspected. There were 68 PCR-positive specimens. Review of the differences in PCR positivity rates based on the site of specimen collection showed the highest rate of detection (28.6%) from throat swabs. From among the 31 patients with significantly elevated titers of serum Mycoplasma antibodies, the PCR results were positive for 25 patients. Thus, capillary PCR had a sensitivity of 80.6% (25 of 31). Five of the six false-negative results were from throat swab specimens. Moreover, testing (PCR) had been performed only once for these five patients with false-negative results. From among the PCR-positive findings from BALF specimens, there were no false-positive results. BALF specimens were very useful, except for the technical procedures and increased patient burden required to obtain these specimens. We suggest that the use of throat swab specimens in capillary PCR is much more suitable for diagnosing Mycoplasma pneumonia in routine clinical practice; however, careful throat swab specimen collection and an increase in the number of times that the PCR is performed are necessary to reduce the rate of false-negative results. PMID- 10747112 TI - Rapid enzyme immunoassay for determination of toxigenicity among clinical isolates of corynebacteria. AB - A rapid enzyme immunoassay (EIA) was developed for the phenotypic detection of diphtheria toxin among clinical isolates of corynebacteria. The assay uses equine polyclonal antitoxin as the capture antibody and an alkaline phosphatase-labeled monoclonal antibody, specific for fragment A of the toxin molecule, as the detecting antibody. The assay is rapid, sensitive, and specific: a final result is available within 3 h of colony selection, and the limits of detection are 0.1 ng of pure diphtheria toxin/ml. Toxigenicity could be detected with isolates grown on a diverse range of culture media, including selective agars. Toxin detection using the EIA was compared to that with the Elek test and PCR detection of fragment A of the diphtheria toxin (tox) gene, using 245 isolates of corynebacteria. The results for the EIA were in complete concordance with those of the Elek test: 87 toxigenic and 158 nontoxigenic isolates. Ten of the phenotypically nontoxigenic strains were found to contain fragment A of the tox gene but did not express the toxin protein. These isolates were found to be nontoxigenic in the Vero cell tissue culture cytotoxicity assay and were therefore nontoxigenic for diagnostic purposes. The EIA is a simple rapid phenotypic test which provides a definitive result on toxigenicity within one working day. PMID- 10747113 TI - Diagnosis and differentiation of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis infections in pigs by PCR amplification of the p36 and p46 genes. AB - The genome of Mycoplasma hyopneumoniae encodes several immunodominant proteins, including a cytosolic protein (p36), three membranous proteins (p46, p65, and p74), and an adhesin (p97). Cross-reactions with M. flocculare and M. hyorhinis reduce the specificity of conventional serological detection methods. However, certain antigenic determinants of the p36 and p46 proteins have been shown to be specific for M. hyopneumoniae. In the present study, pairs of oligonucleotide primers were designed to permit PCR amplification of entire p36 and p46 genes and of internal fragments of these genes. Specific amplicons could be obtained with as low as 0.5 to 50 pg of extracted chromosomal DNA. No amplification product was obtained when testing p36 and p46 primer pairs with genomic DNA or RNA from other mycoplasma species, bacteria, and viruses commonly associated with respiratory diseases in pigs. By using the single p36-PCR method, a positive reaction was demonstrated in 100% (30 of 30) of lungs from pigs that developed typical lesions associated with an M. hyopneumoniae infection, and no false-positive results were detected when 62 apparently normal lungs were tested. On the other hand, with the single p46-PCR method a sensitivity of 86.6% (26 of 30) and a specificity of 96.7% (60 of 62) were obtained in comparison with the necropsy findings. A mixed infection with M. hyorhinis was diagnosed in 13.3% (4 of 30) of the cases by using species-specific primers for the heterologous p37 gene. The sensitivity of the single p36-PCR method for the detection of M. hyopneumoniae, when tested on tracheobronchial swabs, was 100% (20 positive samples), with a specificity of 93.3% (14 of 15 negative samples), compared to the necropsy findings. Both expected amplicons were obtained with 86.6% (26 of 30) positive lungs when p36 and p46 primers were used simultaneously (multiplex PCR) to further increase the specificity of the PCR assay. PMID- 10747114 TI - A panel of monoclonal antibodies targeting the rabies virus phosphoprotein identifies a highly variable epitope of value for sensitive strain discrimination. AB - A recombinant rabies virus phosphoprotein fusion product (GST-P) was used to generate a series of monoclonal antibodies (MAbs) with anti-P reactivity. Competitive binding assays classified 27 of these MAbs into four groups (I to IV), and 24 of them were deemed to recognize linear epitopes, as judged by their reaction in immunoblots. The linear epitope recognized in each case was mapped by using two series of N- and C-terminally deleted recombinant phosphoproteins. Assessment of the reactivities of representative MAbs to a variety of lyssavirus isolates by an indirect fluorescent antibody test indicated that group I MAbs, which recognized a highly conserved N-terminal epitope, were broadly cross reactive with all lyssaviruses assayed, while group III MAbs, which reacted with a site overlapping that of group I MAbs, exhibited variable reactivities and group IV MAbs reacted with most isolates of genotypes 1, 6, and 7 only. In contrast, group II MAbs, which recognized an epitope located within a highly divergent central portion of the protein, were exquisitely strain specific. These anti-P MAbs are potentially useful tools for lyssavirus identification and discrimination. PMID- 10747115 TI - Quantitative analysis of human herpesvirus 8 viral load using a real-time PCR assay. AB - We have developed a quantitative real-time PCR (TaqMan) assay aimed at measuring the cellular human herpesvirus 8 (HHV-8) DNA load in various clinical samples. Standard curves were obtained by serial dilutions of a control plasmid containing both HHV-8 (ORF73 gene) and the cellular target (human albumin gene). The assay appeared to be very sensitive (100% detection rate for at least 10 copies per well) and specific and was easily reproducible (less than 3% intra-assay variability, 5% interassay variability). This method allowed us to quantify precisely the average HHV-8 copy number per cell in various persistently HHV-8 infected cell lines (BBG-1 cells, n = 200; BC-1 cells, n = 59; BCBL-1 cells, n = 70). A retrospective study was also conducted to assess the HHV-8 DNA load in 12 human immunodeficiency virus-infected patients with either Kaposi's sarcoma (KS; seven patients monitored over a 3-month period) or multicentric Castleman's disease (MCD; five patients). The HHV-8 DNA load ranged from 0 to 9,171 copies/10(6) cells in low-risk KS patients (T0, I0, S0 according to the classification of the AIDS Clinical Trials group). We also measured the viral loads in MCD patients either during symptomatic periods or during remission. The results are in agreement with previously published data, with high viral loads correlating with clinical symptoms (1.3 x 10(6) copies/10(6) cells) and low viral loads correlating with asymptomatic periods (less than 5,000 copies/10(6) cells). PMID- 10747116 TI - Development of a highly specific recombinant Toxocara canis second-stage larva excretory-secretory antigen for immunodiagnosis of human toxocariasis. AB - The specificity of the recombinant Toxocara canis antigen developed for the immunodiagnosis of human toxocariasis was compared with that of the excretory secretory antigen from T. canis second-stage larvae (TES) by enzyme-linked immunosorbent assay. A total of 153 human serum samples from patients infected with 20 different helminths, including 11 cases of toxocariasis, were examined. No false-negative reactions were observed for the toxocariasis cases. When the TES was used at concentrations of 0.5 and 0.125 microg/ml, cross-reactions were observed in 79 (55.6%) and 61 (43.0%) of 142 cases, respectively. In contrast, when the recombinant antigen was tested at a concentration of 0.5 microg/ml, cross-reactions were observed in 19 (13.4%) of 142 cases. At a concentration of 0.125 microg/ml, however, the cross-reaction rate decreased sharply to only 2.1%, corresponding to 3 of 142 cases. The cross-reactions occurred with one case each of gnathostomiasis, paragonimiasis with Paragonimus miyazakii, and spirometriasis, in which high antibody titers were detected. In addition, the recombinant antigen showed negative reactions with serum samples from patients infected with Ascaris and hookworms, which are the most common parasites in the world. These findings are also supported by experiments with animals infected with Ascaris and hookworm. From these results, the recombinant antigen is highly specific for toxocariasis and may provide more reliable diagnostic results than other methods. PMID- 10747118 TI - Characterization of strains of Mycoplasma mycoides subsp. mycoides small colony type isolated from recent outbreaks of contagious bovine pleuropneumonia in Botswana and Tanzania: evidence for a new biotype. AB - Four strains of Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) isolated from recent outbreaks of contagious bovine pleuropneumonia (CBPP) in Africa have been investigated. One Botswanan strain, M375, displayed numerous and significant phenotypic differences from both contemporary field isolates and older field and vaccine strains (African, Australian, and European strains dating back to 1936). Differences include altered morphology, reduced capsular polysaccharide production, high sensitivity to MmmSC rabbit hyperimmune antisera in vitro, and unique polymorphisms following immunoblotting. While insertion sequence analysis using IS1634 clearly indicates a close evolutionary relationship to west African strains, hybridization with IS1296 shows the absence of a band present in all other strains of MmmSC examined. The data suggest that a deletion has occurred in strain M375, which may explain its altered phenotype, including poor growth in vitro and a relative inability to cause septicemia in mice. These characteristics are also exhibited by Mycoplasma capricolum subsp. capripneumoniae (causal agent of contagious caprine pleuropneumonia [CCPP]), against which M375 antiserum exhibited some activity in vitro (unique among the various MmmSC antisera tested). These findings may have evolutionary implications, since CCPP is believed to be lung specific and without a septicemic phase (unlike CBPP). Since M375 was isolated from a clinical case of CBPP, this novel biotype may be fairly widespread but not normally isolated due to difficulty of culture and/or a potentially altered disease syndrome. Bovine convalescent antisera (obtained from contemporary naturally infected cattle in Botswana) were active against strain M375 in an in vitro growth inhibition test but not against any other strains of MmmSC tested. There exists the possibility therefore, that strain M375 may possess a set of protective antigens different from those of other strains of MmmSC (including vaccine strains). These findings have implications for the control of the current CBPP epidemic in Africa. PMID- 10747119 TI - Assessment of morphology for rapid presumptive identification of Mycobacterium tuberculosis and Mycobacterium kansasii. AB - Mycobacterium tuberculosis often exhibits serpentine cording when grown in liquid medium, whereas Mycobacterium kansasii can be larger and cross-barred. We assessed the use of these morphologic characteristics as a cost-effective method for rapid presumptive identification of isolates from BACTEC bottles. Without specific training, using the Kinyoun acid-fast stain, definitive cording was found in 237 of 373 specimens positive for M. tuberculosis (64%) and cross barring was recognized within 63 of 76 (83%) of the specimens positive for M. kansasii, giving sensitivities specificities, positive predictive values, and negative predictive values of 63.5, 96, 92, and 79%, respectively, for M. tuberculosis and 83, 95, 59, and 98%, respectively, for M. kansasii. With training and experience, these results improved to 74.5, 98, 96, and 84% and 93, 98, 79, and 98%, respectively. The major improvements were in distinguishing the pseudocording, or loose aggregation of Mycobacterium avium complex from M. tuberculosis and the long beaded forms of Mycobacterium gordonae from M. kansasii. Mycobacterium asiaticum and Mycobacterium szulgai, which rarely occur, are genetically related to M. kansasii and morphologically difficult to distinguish. In defined circumstances, serpentine cording and cross-barring can be used for rapid presumptive identification of M. tuberculosis and M. kansasii, respectively, and as guides for initial probe selection to reduce costs. PMID- 10747117 TI - Quantitation of human immunodeficiency virus type 1 RNA in different biological compartments. AB - Little information is available describing viral loads in body fluids other than blood. In addition, the suitability of commercially available assays for human immunodeficiency virus type 1 (HIV-1) RNA quantitation has not been evaluated in most nonblood fluids. We compared Organon Teknika's nucleic acid sequence-based amplification method (NASBA) and Roche's Amplicor HIV-1 Monitor (reverse transcriptase PCR [RT-PCR]) for quantitating HIV-1 RNA in cerebrospinal fluid (CSF), saliva, breast milk, seminal plasma, and cervical-vaginal lavage fluid (CVL). Saliva and breast milk frequently demonstrated some inhibition in the RT PCR assay, similar to the inhibition previously described in seminal plasma. Inhibition of the RT-PCR assay was not observed with CSF or CVL, nor in any of the NASBA assays. When fluids from HIV-infected individuals were tested by RT-PCR and NASBA, 73 and 27% of CSF samples and 60 and 40% of breast milk specimens had detectable RNA, respectively. These differences were not statistically significant. In cross-sectional studies using RT-PCR to measure viral RNA in paired blood plasma and CSF samples, 71% of blood plasma samples and 42% of CSF samples were positive. A similar analysis using NASBA with paired blood plasma and CVL, saliva, or seminal plasma samples revealed 91% were blood plasma positive and 55% were CVL positive, 76% were blood plasma positive and 46% were saliva positive, and 83% were blood plasma positive and 63% were seminal plasma positive. NASBA worked fairly well to quantitate HIV-1 RNA from all fluids without apparent inhibition. RT-PCR performed well on CVL and CSF, frequently with greater sensitivity, although its use in other fluids appears limited due to the presence of inhibitors. These studies demonstrate that viral loads in nonblood fluids were generally lower than in blood. PMID- 10747120 TI - A multisite trial comparing two cytomegalovirus (CMV) pp65 antigenemia test kits, biotest CMV brite and Bartels/Argene CMV antigenemia. AB - A total of 513 blood specimens, predominantly from organ transplant recipients, human immunodeficiency virus-positive patients, and bone marrow transplant recipients, were tested for cytomegalovirus (CMV) by culture and pp65 antigenemia across four test sites. Peripheral blood leukocytes were examined by using both the Biotest CMV Brite and the Bartels/Argene CMV Antigenemia kits. A total of 109 specimens were positive for CMV, 106 (97%) were positive by antigenemia, and 34 (31%) were positive by culture. According to the manufacturers' instructions, 150,000 cells were applied per slide for the Biotest kit and 200,000 cells per slide for the Bartels kit. A total of 93 specimens (88%) were positive by the Biotest kit, and 86 (81%) were positive by the Bartels kit. In specimens found to be positive by only one kit, the positive cell counts were low (median, 1; range, 1 to 7). When the data from all four sites were combined and analyzed, there was no statistical difference between the performance of the two kits; the Biotest and Bartels kits were found to be equivalent in sensitivity, specificity, and positive and negative predictive values for the detection of CMV pp65 antigenemia. PMID- 10747121 TI - Quantitative galactomannan detection is superior to PCR in diagnosing and monitoring invasive pulmonary aspergillosis in an experimental rat model. AB - Two diagnostic tests, an Aspergillus-specific PCR and an enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of galactomannan, were compared for diagnosing and monitoring invasive pulmonary aspergillosis. Persistently neutropenic rats with left-sided invasive pulmonary aspergillosis were sacrificed at regular intervals after inoculation. Blood samples and bronchoalveolar lavage (BAL) fluid were cultured and tested by PCR as well as by ELISA. Disseminated fungal infection in extrapulmonary organs was determined. The sensitivity of the ELISA was higher than that of the PCR on all days of measurements, in both blood and BAL fluid. Positive PCR or ELISA results in blood were not significantly associated with disseminated fungal infection. Serial testing in a separate group of rats showed consistently increasing concentrations of circulating galactomannan during the course of disease, while a positive PCR could be followed by negative results. The concentration of galactomannan was highly predictive for the time of survival (P < 0.0001). It was concluded that, in this model, quantitative galactomannan detection is superior to PCR in diagnosing and monitoring invasive pulmonary aspergillosis. PMID- 10747122 TI - A rapid, automated enzymatic fluorometric assay for determination of D-arabinitol in serum. AB - A rapid enzymatic fluorometric assay for measuring D-arabinitol in serum was developed using recombinant D-arabinitol dehydrogenase from Candida albicans (rArDH). rArDH was produced in Escherichia coli and purified by dye-ligand affinity chromatography. rArDH was highly specific for D-arabinitol, cross reacting only with xylitol (4.9%) among all polyols tested. A Cobas Fara II centrifugal autoanalyzer (Roche) was used to measure NADH fluorometrically when rArDH and NAD were added to serum extracts, and D-arabinitol concentrations were calculated from standard curves derived from pooled human serum containing known amounts of D-arabinitol. The method was precise (mean intra-assay coefficients of variation [CVs], 0.8%, and mean interassay CVs, 1.6%) and rapid (3.5 min per assay) and showed excellent recovery of added D-arabinitol in serum (mean recovery rate, 101%). The mean and median D-arabinitol/creatinine ratios were 2.74 and 2.23 microM/mg/dl, respectively, for the 11 patients with candidemia compared to 1.14 and 1.23 microM/mg/dl, respectively, for 10 healthy controls (P < 0.01). These results confirm earlier studies showing that serum D-arabinitol measurement may help to promptly diagnose invasive candidiasis. The technique shows a significant improvement in terms of accuracy, cost, simplicity, specificity, and speed compared with gas chromatography, mass spectrometry, and earlier enzymatic assays. PMID- 10747123 TI - Antipneumococcal activity of telithromycin by agar dilution, microdilution, E test, and disk diffusion methodologies. AB - Agar dilution and microdilution (both in air) and E test and disk diffusion (both in air and CO(2)) were used to test the activity of telithromycin against 110 erythromycin-susceptible and 106 erythromycin-resistant pneumococci. The MICs at which 50 and 90% of strains are inhibited (MIC(50)s and MIC(90)s, respectively) for erythromycin-susceptible strains varied between 0.008 and 0.016 microg/ml and 0.016 and 0.03 microg/ml when the samples were incubated in air. By comparison, telithromycin MIC(50)s and MIC(90)s for erythromycin-resistant strains were in air 0.03 to 0.125 and 0. 125 to 0.5 microg/ml, respectively. When agar dilution was used as the reference method, essential agreement was found for 112 of 216 strains (51.9%) for microdilution, 168 of 216 (77.8%) for E test in air, and 132 of 216 (61.1%) for E test in CO(2). With the exception of four strains tested by E test in CO(2), all organisms were susceptible to a proposed telithromycin susceptibility breakpoint of < or =1 microg/ml. By disk diffusion with 15-microg telithromycin disks, all strains but one had zones of inhibition > or =19 mm in diameter when incubated in CO(2), while all strains had zone diameters of > or = 22 mm when incubated in air. Zone diameters in air were generally 4 to 5 mm larger than in CO(2). By all methods, MICs and zones of all erythromycin resistant strains occurred in clusters separated from those seen with erythromycin-susceptible strains. The results for macrolide-resistant strains with erm and mef resistance determinants were similar. The results show that (i) telithromycin is very active against erythromycin-susceptible and -resistant strains irrespective of macrolide resistance mechanism; (ii) susceptibility to telithromycin can be reliably tested by the agar, microdilution, E test, and disk diffusion methods; and (iii) incubation in CO(2) led to smaller zones by disk diffusion and higher MICs by E test, but at a susceptible MIC breakpoint of < or =1 microg/ml and a susceptible zone diameter cutoff of > or =19 mm in CO(2), 215 of 216 strains were found to be susceptible to telithromycin. PMID- 10747124 TI - Analysis of plasmid and chromosomal DNA of multidrug-resistant Salmonella enterica serovar typhi from Asia. AB - Molecular analysis of chromosomal DNA from 193 multidrug-resistant (MDR) Salmonella enterica serovar Typhi isolates from 1990 to 1995 from Pakistan, Kuwait, Malaysia, Bangladesh, and India produced a total of five major different pulsed-field gel electrophoresis (PFGE) patterns. Even within a particular country MDR S. enterica serovar Typhi DNA was found to be in different PFGE groups. Similar self-transferable 98-MDa plasmids belonging to either incompatibility group incHI1 or incHI1/FIIA were implicated in the MDR phenotype in S. enterica serovar Typhi isolates from all the locations except Quetta, Pakistan, where the majority were of incFIA. A total of five different PFGE genotypes with six different plasmids, based on incompatibility and restriction endonuclease analysis groups, were found among these MDR S. enterica serovar Typhi isolates. PMID- 10747125 TI - Fractionation of membrane components from tachyzoite forms of Toxoplasma gondii: differential recognition by immunoglobulin M (IgM) and IgG present in sera from patients with acute or chronic toxoplasmosis. AB - Tachyzoite forms of Toxoplasma gondii were subjected to a sequential organic solvent extraction, which allows fractionation of membrane components according to their degrees of hydrophobicity, yielding three fractions named F1 (most hydrophobic) to F3 (least hydrophobic). Fractions F2 (80.85% specificity and 86.95% sensitivity) and F3 (89.36% specificity and 93.61% sensitivity) gave the best results, being preferentially recognized by immunoglobulin M (IgM) and IgG in sera from patients with acute and chronic toxoplasmosis, respectively. Improved scores of specificity (100%) and sensitivity (100%) were achieved when a secondary antibody against human IgG1 instead of total IgG was employed to measure the reactivity of IgG antibodies with the F3 fraction. To purify tachyzoite antigens recognized by human IgM or IgG antibodies, the F2 or F3 fraction was loaded onto an octyl-Sepharose column and eluted with a propan-1-ol gradient. The main antigen(s) recognized by IgM or IgG eluted in a single peak from the octyl-Sepharose resin loaded with either F2 (30 to 50% propan-1-ol) or F3 (15 to 35% propan-1-ol), respectively. These semipurified fractions gave improved scores when used to detect T. gondii-specific IgM (95.7% specificity and 81.8% sensitivity) or IgG (100% specificity and 93. 75% sensitivity) in an enzyme linked immunosorbent assay. Further biochemical and immunological analyses of antigens partially purified from F2 and F3 indicate that glycoinositolphospholipids are preferentially recognized by IgM, whereas proteins of approximately 30 to 40 kDa are recognized by IgG, elicited during T. gondii infection in humans. PMID- 10747126 TI - Evaluation of diagnostic value and epidemiological implications of PCR for Pneumocystis carinii in different immunosuppressed and immunocompetent patient groups. AB - To evaluate the value of single and nested PCRs for diagnosis of Pneumocystis carinii pneumonia (PCP) in a variety of respiratorily distressed patient groups, 574 respiratory samples from 334 patients (89 human immunodeficiency virus [HIV] positive patients, 61 transplant recipients, 66 malignancy patients, 34 otherwise immunosuppressed patients, and 84 immunocompetent patients) were prospectively examined by microscopy and single and nested PCRs. The resulting data were correlated with clinical evidence of PCP. Microscopy and single PCR of bronchoalveolar lavage (BAL) specimens from HIV patients were 100% sensitive and specific in detecting PCP, whereas nested PCR, although being 100% sensitive, reached a specificity of only 97.5%. In the three non-HIV immunosuppressed patient groups, both single and nested PCR invariably produced lower positive predictive values than microscopy. Among immunocompetent patients, the positive predictive values of both PCRs were 0%. Therefore, the diagnostic values of the PCR methods tested do not seem to offer any additional advantage compared to that of conventional microscopy for these patient groups. However, nested PCR identified a significant percentage of clinically silent P. carinii colonizations in about 17 to 20% of immunocompetent and immunosuppressed non-HIV patients. PMID- 10747127 TI - Intraspecies diversity of Cryptococcus laurentii as revealed by sequences of internal transcribed spacer regions and 28S rRNA gene and taxonomic position of C. laurentii clinical isolates. AB - The intraspecies diversity of an opportunistic yeast pathogen, Cryptococcus laurentii, was revealed by analysis of the sequences of the internal transcribed spacer regions and the 28S rRNA gene. Ten strains of C. laurentii were grouped into two major phylogenetic groups and were further divided into at least seven species. Four of the strains isolated from patients did not represent a single species but showed heterogeneity. These results suggest that C. laurentii is a genetically heterogeneous species, and this must be taken into consideration when identifying C. laurentii clinical isolates. PMID- 10747128 TI - Comparison of a baculovirus-based VP2 enzyme immunoassay (EIA) to an Escherichia coli-based VP1 EIA for detection of human parvovirus B19 immunoglobulin M and immunoglobulin G in sera of pregnant women. AB - A split-sample study was conducted to evaluate the clinical performance of an enzyme immunoassay that detects the human parvovirus B19 virus (B19V) immunoglobulin M (IgM) or IgG in the sera of pregnant women. The initial study compared a baculovirus-expressed VP2 enzyme immunoassay (BVP2 EIA) (Biotrin International Inc., Dublin, Ireland) with the currently available and commonly used Escherichia coli-expressed VP1 enzyme immunoassay (EVP1 EIA) (MRL Diagnostics, Cypress, Calif.). There was a high degree of agreement between the two assays in the detection of IgM antibodies (283 of 307 [92.2%]) or IgG antibodies (279 of 311 [89. 7%]), with the majority of discrepancies (IgM, 17 of 24 [71%]; IgG, 16 of 31 [50%]) being due to equivocal data obtained with the EVP1 EIA. Specimens with discordant BVP2 EIA and EVP1 EIA results (23 of 24 IgM and 32 of 32 IgG results) were analyzed further by baculovirus-based VP1 immunofluorescence assays (BVP1 IFAs) (Biotrin International). The BVP2 EIA and BVP1 IFA results for 20 of 23 and 28 of 32 specimens for IgM and IgG, respectively, were concordant. In contrast, the EVP1 EIA and BVP1 IFA data for only 3 of 23 and 4 of 32 specimens for IgM and IgG, respectively, were in agreement, despite the fact that the same capsid antigen was used. Both the BVP2 EIAs and BVP1 IFAs utilize a conformational viral capsid antigen, while the EVP1 EIA uses a denatured viral capsid antigen. In conclusion, the BVP2 EIAs produced far fewer equivocal results for IgM and IgG, correlating more closely to the confirmatory BVP IFAs, than did the EVP1 EIAs and proved to be more accurate for detecting B19V antibodies in the sera of pregnant women. PMID- 10747129 TI - Development and clinical evaluation of a recombinant-antigen-based cytomegalovirus immunoglobulin M automated immunoassay using the Abbott AxSYM analyzer. AB - A new microparticle enzyme immunoassay (MEIA), the Cytomegalovirus (CMV) Immunoglobulin M (IgM) test, was developed on the Abbott AxSYM analyzer. This test uses recombinant CMV antigens derived from portions of four structural and nonstructural proteins of CMV: pUL32 (pp150), pUL44 (pp52), pUL83 (pp65), and pUL80a (pp38). A total of 1, 608 specimens from random volunteer blood donors (n = 300), pregnant women (n = 1,118), transplant recipients (n = 6), and patients with various clinical conditions and disease states (n = 184) were tested during development and evaluation of this new assay. In a preliminary clinical evaluation we tested specimens collected prospectively from pregnant women (n = 799) and selected CMV IgM-positive archived specimens from pregnant women (n = 39). The results from the new CMV IgM immunoassay were compared to the results of a consensus interpretation of the results obtained with three commercial CMV IgM immunoassays. The results for specimens with discordant results were resolved by a CMV IgM immunoblot assay. The relative sensitivity, specificity, and agreement for the AxSYM CMV IgM assay were 94.29, 96.28, and 96.19%, respectively, and the resolved sensitivity, specificity, and agreement were 95.83, 97.47, and 97.37%, respectively. We also tested serial specimens from women who experienced seroconversion or a recent CMV infection during gestation (n = 17) and potentially cross-reactive specimens negative for CMV IgM antibody by the consensus tests (n = 184). The AxSYM CMV IgM assay was very sensitive for the detection of CMV IgM during primary CMV infection, as shown by the detection of CMV IgM at the same time as or just prior to the detection of CMV IgG. Specimens from individuals with lupus (n = 16) or parvovirus B19 infection (n = 6) or specimens containing hyper IgM (n = 9), hyper IgG (n = 8), or rheumatoid factor (n = 55) did not cross-react with the AxSYM assay. One specimen each from individuals infected with Epstein-Barr virus (n = 26), measles virus (n = 10), herpes simplex virus (n = 12), or varicella-zoster virus (n = 13) infection, one specimen from an influenza vaccinee (n = 14), and one specimen containing antinuclear antibody cross-reacted with the assay. The overall rate of cross reactivity of the specimens with the assay was 3.3% (6 of 184). The AxSYM CMV IgM assay is a sensitive and specific assay for the detection of CMV-specific IgM. PMID- 10747130 TI - A simple PCR method for rapid genotype analysis of Mycobacterium ulcerans. AB - Two high-copy-number insertion sequences, IS2404 and IS2606, were recently identified in Mycobacterium ulcerans and were shown by Southern hybridization to possess restriction fragment length polymorphism between strains from different geographic origins. We have designed a simple genotyping method that captures these differences by PCR amplification of the region between adjacent copies of IS2404 and IS2606. We have called this system 2426 PCR. The method is rapid, reproducible, sensitive, and specific for M. ulcerans, and it has confirmed previous studies suggesting a clonal population structure of M. ulcerans within a geographic region. M. ulcerans isolates from Australia, Papua New Guinea, Malaysia, Surinam, Mexico, Japan, China, and several countries in Africa were easily differentiated based on an array of 4 to 14 PCR products ranging in size from 200 to 900 bp. Numerical analysis of the banding patterns suggested a close evolutionary link between M. ulcerans isolates from Africa and southeast Asia. The application of 2426 PCR to total DNA, extracted directly from M. ulcerans infected tissue specimens without culture, demonstrated the sensitivity and specificity of this method and confirmed for the first time that both animal and human isolates from areas of endemicity in southeast Australia have the same genotype. PMID- 10747132 TI - Incidence and identification of Klebsiella planticola in clinical isolates with emphasis on newborns. AB - Studies conducted in France and Germany suggest that up to 19% of clinically identified Klebsiella sp. are actually Klebsiella planticola, an environmental species that has been attributed to two cases of septicemia, with a rare isolate of Klebsiella terrigena (0. 4%) being identified. A 1-year survey of newborns on a neonatal ward, also conducted in Germany, reported that 72% of Klebsiella sp. were Klebsiella oxytoca and 8.7% were K. planticola. The tests necessary to identify these species are not found in most clinical identification schemes or in the database matrices of most commercial identification products. To determine the incidence of unrecognized K. planticola among the Klebsiella sp. isolates in our collection, we used the battery of seven supplemental tests amended from the work of Monnet and Freney to test 352 stock isolates and 84 fresh clinical isolates from four local hospitals. After testing 436 strains of Klebsiella, only one strain was identified as a possible K. planticola and none was identified as K. terrigena. We tested an additional 43 stock strains of K. oxytoca isolated from newborns by using eight biochemical tests and found one additional strain of K. planticola. The occurrence of K. planticola in our collection is far less frequent than that observed in other countries. PMID- 10747131 TI - Identification of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, arcobacter butzleri, and A. butzleri-like species based on the glyA gene. AB - Currently, the detection and identification of Campylobacter and Arcobacter species remains arduous, largely due to cross-species phenotypic similarities and a relatively narrow spectrum of biochemical reactivity. We have developed a PCR hybridization strategy, wherein degenerate primers are used to amplify glyA fragments from samples, which are then subjected to species-specific oligodeoxyribonucleotide probe hybridizations, to identify and distinguish between Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, Arcobacter butzleri, and an A. butzleri-like species. Evaluation of this strategy with genomic DNA from different type strains suggests that this approach is both specific and sensitive and thus may be applicable in a diagnostic assay to identify and differentiate these highly related species. PMID- 10747133 TI - New agar medium for testing susceptibility of Mycobacterium tuberculosis to pyrazinamide. AB - A new agar medium to perform pyrazinamide (PZA) susceptibility testing with Mycobacterium tuberculosis has been developed. This medium has an acidic pH of 6.0 instead of the usual for agar media, pH 6.8, to provide optimal conditions for PZA activity, and it also differs from conventional Middlebrook 7H10/7H11 agar in that animal serum (fetal or calf bovine or fetal equine serum) is used instead of oleic acid-albumin-dextrose-catalase to support good growth of M. tuberculosis at the low pH of 6.0. A critical concentration of 900 or 1,200 microg of PZA/ml in this medium made it possible to differentiate between PZA susceptible and PZA-resistant clinical isolates. This agar medium has the following advantages compared to a liquid medium: it allows determination of the actual proportion of PZA-resistant bacteria in the isolate and it is simple and inexpensive. In addition, it has the potential of being used for a direct susceptibility test with PZA, but this approach will require further confirmation. Further studies to develop critical concentrations of other drugs for this low-pH medium, as well as to investigate the possibility of cultivation in regular (non-CO(2)) incubators, are in progress. PMID- 10747134 TI - Epidemiological study of pap genes among diarrheagenic or septicemic Escherichia coli strains producing CS31A and F17 adhesins and characterization of Pap(31A) fimbriae. AB - The association of the pap operon with the CS31A and F17 adhesins was studied with 255 Escherichia coli strains isolated from calves, lambs, or humans with diarrhea. The three classes of PapG adhesin with different receptor binding preferences were also screened. The pap operon was associated with 50 and 36% of human strains that produced CS31A and ovine strains that produced F17, respectively. Among the bovine isolates, the pap operon was detected in 61% of the CS31A-positive isolates and 72% of the strains that produce both CS31A and F17. The class II adhesin gene was present in bovine (20%) and ovine (71%) isolates. Both class II and III adhesins were genetically associated with 36% of the human strains. The highest prevalence of the pap operon was observed among E. coli strains that produce additional adhesins involved in the binding of bacteria to intestinal cells. Among the bovine isolates, the reference strain for CS31A and F17c was found to be positive for the pap operon. Phenotypic and genotypic characterizations were undertaken. Pap(31A) appeared as fine and flexible fimbriae surrounding the bacteria but did not mediate adhesion to calf intestinal villi. Pap(31A) production was optimal with bacteria cultured on minimal growth media and repressed by addition of exogenous leucine. The deduced amino acid sequence of the PapA(31A) structural subunit showed 57 to 97% identity with the different P-related structural subunits produced by E. coli strains isolated from pigs with septicemia or humans with urinary tract infections. None of the three papG allelic variants was detected, but a homologous papG gene was present in the chromosome of strain 31A. PMID- 10747135 TI - Identification of Aspergillus species using internal transcribed spacer regions 1 and 2. AB - Aspergillus species are the most frequent cause of invasive mold infections in immunocompromised patients. Although over 180 species are found within the genus, 3 species, Aspergillus flavus, A. fumigatus, and A. terreus, account for most cases of invasive aspergillosis (IA), with A. nidulans, A. niger, and A. ustus being rare causes of IA. The ability to distinguish between the various clinically relevant Aspergillus species may have diagnostic value, as certain species are associated with higher mortality and increased virulence and vary in their resistance to antifungal therapy. A method to identify Aspergillus at the species level and differentiate it from other true pathogenic and opportunistic molds was developed using the 18S and 28S rRNA genes for primer binding sites. The contiguous internal transcribed spacer (ITS) region, ITS 1-5.8S-ITS 2, from referenced strains and clinical isolates of aspergilli and other fungi were amplified, sequenced, and compared with non-reference strain sequences in GenBank. ITS amplicons from Aspergillus species ranged in size from 565 to 613 bp. Comparison of reference strains and GenBank sequences demonstrated that both ITS 1 and ITS 2 regions were needed for accurate identification of Aspergillus at the species level. Intraspecies variation among clinical isolates and reference strains was minimal. Sixteen other pathogenic molds demonstrated less than 89% similarity with Aspergillus ITS 1 and 2 sequences. A blind study of 11 clinical isolates was performed, and each was correctly identified. Clinical application of this approach may allow for earlier diagnosis and selection of effective antifungal agents for patients with IA. PMID- 10747136 TI - Diversity in a variable-number tandem repeat from Yersinia pestis. AB - We have identified a tetranucleotide repeat sequence, (CAAA)(N), in the genome of Yersinia pestis, the causative agent of plague. This variable-number tandem repeat (VNTR) region has nine alleles and great diversity (calculated as 1 minus the sum of the squared allele frequencies) (diversity value, 0.82) within a set of 35 diverse Y. pestis strains. In contrast, the nucleotide sequence of the lcrV (low-calcium-response) gene differed only slightly among these strains, having a haplotype diversity value of 0.17. Replicated cultures, phenotypic variants of particular strains, and extensively cultured replicates within strains did not differ in VNTR allele type. Thus, while a high mutation rate must contribute to the great diversity of this locus, alleles appear stable under routine laboratory culture conditions. The classic three plague biovars did not have single identifying alleles, although there were allelic biases within biovar categories. The antiqua biovar was the most diverse, with four alleles observed in 5 strains, while the orientalis and mediaevalis biovars exhibited five alleles in 21 strains and three alleles in 8 strains, respectively. The CAAA VNTR is located immediately adjacent to the transcriptional promoters for flanking open reading frames and may affect their activity. This VNTR marker may provide a high resolution tool for epidemiological analyses of plague. PMID- 10747137 TI - Development of a serological test for Haemophilus ducreyi for seroprevalence studies. AB - We developed a new enzyme immunoassay (rpEIA) for use in determining the seroprevalence of chancroid. Three highly conserved outer membrane proteins from Haemophilus ducreyi strain 35000 were cloned, overexpressed, and purified from Escherichia coli for use as antigens in the rpEIA. Serum specimens from patients with and without chancroid were assayed to determine optimum sensitivity and specificity and to establish cutoff values. On the basis of these data, rpEIA was found to be both sensitive and specific when used to test a variety of serum specimens from patients with genital ulcers and urethritis and from healthy blood donors. PMID- 10747139 TI - Transmission of Pneumocystis carinii DNA from a patient with P. carinii pneumonia to immunocompetent contact health care workers. AB - The transmission of Pneumocystis carinii from person to person was studied by detecting P. carinii-specific DNA in prospectively obtained noninvasive deep nasal-swab samples from a child with a documented P. carinii pneumonia (PCP), his mother, two contact health care workers, and 30 hospital staff members who did not enter the patient's room (controls). Nested-DNA amplification was done by using oligonucleotide primers designed for the gene encoding the mitochondrial large subunit rRNA of rat P. carinii (P. carinii f. sp. carinii) that amplifies all forms of P. carinii and internal primers specific for human P. carinii (f. sp. hominis). P. carinii f. sp. hominis DNA was detected in samples from the patient and all of his contacts versus none of the 30 hospital staff members. The results, as previously shown in murine models of P. carinii pneumonia, document that person-to-person transmission of P. carinii is possible. This observation suggests that immunocompromised patients not on PCP prophylaxis should not enter the room of a patient with PCP, and it also raises the question as to whether healthy contacts can transmit the disease to immunocompromised patients at risk. PMID- 10747138 TI - Development of reverse transcription-PCR assays specific for detection of equine encephalitis viruses. AB - Specific and sensitive reverse transcription-PCR (RT-PCR) assays were developed for the detection of eastern, western, and Venezuelan equine encephalitis viruses (EEE, WEE, and VEE, respectively). Tests for specificity included all known alphavirus species. The EEE-specific RT-PCR amplified a 464-bp region of the E2 gene exclusively from 10 different EEE strains from South and North America with a sensitivity of about 3,000 RNA molecules. In a subsequent nested PCR, the specificity was confirmed by the amplification of a 262-bp fragment, increasing the sensitivity of this assay to approximately 30 RNA molecules. The RT-PCR for WEE amplified a fragment of 354 bp from as few as 2,000 RNA molecules. Babanki virus, as well as Mucambo and Pixuna viruses (VEE subtypes IIIA and IV), were also amplified. However, the latter viruses showed slightly smaller fragments of about 290 and 310 bp, respectively. A subsequent seminested PCR amplified a 195 bp fragment only from the 10 tested strains of WEE from North and South America, rendering this assay virus specific and increasing its sensitivity to approximately 20 RNA molecules. Because the 12 VEE subtypes showed too much divergence in their 26S RNA nucleotide sequences to detect all of them by the use of nondegenerate primers, this assay was confined to the medically important and closely related VEE subtypes IAB, IC, ID, IE, and II. The RT-PCR-seminested PCR combination specifically amplified 342- and 194-bp fragments of the region covering the 6K gene in VEE. The sensitivity was 20 RNA molecules for subtype IAB virus and 70 RNA molecules for subtype IE virus. In addition to the subtypes mentioned above, three of the enzootic VEE (subtypes IIIB, IIIC, and IV) showed the specific amplicon in the seminested PCR. The practicability of the latter assay was tested with human sera gathered as part of the febrile illness surveillance in the Amazon River Basin of Peru near the city of Iquitos. All of the nine tested VEE-positive sera showed the expected 194-bp amplicon of the VEE specific RT-PCR-seminested PCR. PMID- 10747140 TI - Detection of the agent of heartwater, Cowdria ruminantium, in Amblyomma ticks by PCR: validation and application of the assay to field ticks. AB - We have previously reported that the pCS20 PCR detection assay for Cowdria ruminantium, the causative agent of heartwater disease of ruminants, is more sensitive than xenodiagnosis and the pCS20 DNA probe for the detection of infection in the vector Amblyomma ticks. Here, we further assessed the reliability of the PCR assay and applied it to field ticks. The assay detected DNA of 37 isolates of C. ruminantium originating from sites throughout the distribution of heartwater and had a specificity of 98% when infected ticks were processed concurrently with uninfected ticks. The assay did not detect DNA of Ehrlichia chaffeensis, which is closely related to C. ruminantium. PCR sensitivity varied with tick infection intensity and was high (97 to 88%) with ticks bearing 10(7) to 10(4) organisms but dropped to 61 and 28%, respectively, with ticks bearing 10(3) and 10(2) organisms. The assay also detected C. ruminantium in collections of Amblyomma hebraeum and Amblyomma variegatum field ticks from 17 heartwater-endemic sites in four southern African countries. Attempts at tick transmission of infection to small ruminants failed with four of these collections. The pCS20 PCR assay is presently the most characterized and reliable test for C. ruminantium in ticks and thus is highly useful for field and laboratory epidemiological investigations of heartwater. PMID- 10747141 TI - High homogeneity of the Yersinia pestis fatty acid composition. AB - The cellular fatty acid compositions of 29 strains of Yersinia pestis representing the global diversity of this species have been analyzed by gas liquid chromatography to investigate the extent of fatty acid polymorphism in this microorganism. After culture standardization, all Y. pestis strains studied displayed some major fatty acids, namely, the 12:0, 14:0, 3-OH-14:0, 16:0, 16:1omega9cis, 17:0-cyc, and 18:1omega9trans compounds. The fatty acid composition of the various isolates studied was extremely homogeneous (average Bousfield's coefficient, 0.94) and the subtle variations observed did not correlate with epidemiological and genetic characteristics of the strains. Y. pestis major fatty acid compounds were analogous to those found in other Yersinia species. However, when the ratios for the 12:0/16:0 and 14:0/16:0 fatty acids were plotted together, the genus Yersinia could be separated into three clusters corresponding to (i) nonpathogenic strains and species of Yersinia, (ii) pathogenic Yersinia enterocolitica isolates, and (iii) Yersinia pseudotuberculosis and Y. pestis strains. The grouping of the two latter species into the same cluster was also demonstrated by their high Bousfield's coefficients (average, 0.89). Therefore, our results indicate that the fatty acid composition of Y. pestis is highly homogeneous and very close to that of Y. pseudotuberculosis. PMID- 10747143 TI - Comparison of enhanced Mycobacterium tuberculosis amplified direct test with COBAS AMPLICOR Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis complex in respiratory and extrapulmonary specimens. AB - The new Roche COBAS AMPLICOR Mycobacterium tuberculosis Assay was compared to the Gen-Probe enhanced Mycobacterium tuberculosis Amplified Direct Test (AMTDII). A total of 486 specimens (296 respiratory and 190 extrapulmonary) collected from 323 patients were tested in parallel with both assays. Results were compared with those of acid-fast staining and culture, setting the combination of culture and clinical diagnosis as the "gold standard." After resolution of discrepant results, the sensitivity, specificity, and positive and negative predictive values for AMTDII were 85.7, 100, 100, and 90.4% for respiratory specimens and 82.9, 100, 100, and 95. 5% for extrapulmonary specimens, respectively. The corresponding values for AMPLICOR were 94.2, 100, 100, and 96.6% for respiratory specimens and 85, 100, 100, and 96.1% for extrapulmonary specimens, respectively. No significant differences were observed between the results of both assays or, within each one, between respiratory and extrapulmonary specimens. The difference between AMTDII and AMPLICOR sensitivities was related to the presence of inhibitory samples, which the former assay, lacking an internal amplification control (IAC), could not detect. The overall inhibition rate for the AMPLICOR assay was 3.9% (19 specimens). It is concluded that, although both amplification assays proved to be rapid and specific for the detection of M. tuberculosis complex in clinical samples, AMPLICOR, by a completely automated amplification and detection procedure, was shown to be particularly feasible for a routine laboratory setting. Finally, AMTDII is potentially an excellent diagnostic technique for both respiratory and extrapulmonary specimens, provided that an IAC is included with the assay. PMID- 10747142 TI - Application of a fluorogenic PCR assay for typing and subtyping of influenza viruses in respiratory samples. AB - A fluorogenic PCR-based method (TaqMan-PCR) was developed for typing and subtyping of influenza virus genomes in clinical specimens. The TaqMan-PCR employs a probe technology that exploits the endogenous 5'-3' nuclease activity of the Taq DNA polymerase to allow direct detection of the amplicon by release of a fluorescent reporter during the PCR. Therefore, post-PCR analysis is avoided since hybridization with the fluorogenic probe and quantification of the amplified product is performed simultaneously during PCR cycling. The specificity of the method was evaluated on 86 influenza A (25 H1N1 and 61 H3N2) and 49 influenza B virus reference strains and isolates. The sensitivity of the technique was found to be at the level of 0.1 50% tissue culture infective dose. This TaqMan-PCR was applied prospectively to surveillance work by community-based sampling in Germany during the last two influenza seasons. Seven hundred five throat swabs were analyzed during the winter of 1997-1998. A total of 195 of 705 samples (28%) were positive by PCR. Influenza viruses could be isolated from 125 specimens (18%). During the 1998-1999 season, 1,840 respiratory samples were received. Influenza viruses were isolated from 281 specimens (15%) out of 525 throat swabs (29%) which were positive for influenza A or B virus by TaqMan-PCR. Further differentiation of influenza A virus-positive swabs revealed an intensive circulation of the subtype H3N2 during both seasons, 1997-1998 and 1998-1999. The TaqMan-PCR was much more sensitive than culture and revealed an excellent correlation for typing and subtyping of influenza viruses when samples were positive by both methods. PMID- 10747144 TI - Emergence of resistance of Candida albicans to clotrimazole in human immunodeficiency virus-infected children: in vitro and clinical correlations. AB - Oropharyngeal candidiasis (OPC) is a common opportunistic infection in human immunodeficiency virus (HIV)-infected patients and other immunocompromised hosts. Clotrimazole troches are widely used in the treatment of mucosal candidiasis. However, little is known about the potential contribution of clotrimazole resistance to the development of refractory mucosal candidiasis. We therefore investigated the potential emergence of resistance to clotrimazole in a prospectively monitored HIV-infected pediatric population receiving this azole. Adapting the National Committee for Clinical Laboratory Standards M27-A reference method for broth antifungal susceptibility testing of yeasts to clotrimazole, we compared MICs in macrodilution and microdilution assays. We further analyzed the correlation between these in vitro findings and the clinical response to antifungal therapy. One isolate from each of 87 HIV-infected children was studied by the macrodilution and microdilution methods. Two inoculum sizes were tested by the macrodilution method (10(3) and 10(4) CFU/ml) in order to assess the effect of inoculum size on clotrimazole MICs. The same isolates also were tested using a noncolorimetric microdilution method. Clotrimazole concentrations ranged from 0.03 to 16 microg/ml. Readings were performed after incubation for 24 and 48 h at 35 degrees C. For 62 (71.2%) of 87 clinical isolates, the MICs were low (< or =0.06 microg/ml). The MIC for 90% of the strains tested was 0.5 microg/ml, and the highest MIC was 8 microg/ml. There was no significant difference between MICs at the two inoculum sizes. There was 89% agreement (+/-1 tube) between the microdilution method at 24 h and the macrodilution method at 48 h. If the MIC of clotrimazole for an isolate of C. albicans was > or =0.5 microg/ml, there was a significant risk (P < 0.001) of cross-resistance to other azoles: fluconazole, > or = 8 microg/ml (relative risk [RR] = 8.9); itraconazole, > or =1 microg/ml (RR = 10). Resistance to clotrimazole was highly associated with clinically overt failure of antifungal azole therapy. Six (40%) of 15 patients for whom the clotrimazole MIC was > or =0.5 microg/ml required amphotericin B for refractory mucosal candidiasis versus 4 (5.5%) of 72 for whom the MIC was <0.5 microg/ml (P = 0.001; 95% confidence interval = 2.3 to 22; RR = 7.2). These findings suggest that an interpretive breakpoint of 0.5 microg/ml may be useful in defining clotrimazole resistance in C. albicans. The clinical laboratory's ability to determine MICs of clotrimazole may help to distinguish microbiologic resistance from the other causes of refractory OPC, possibly reducing the usage of systemic antifungal agents. We conclude that resistance to clotrimazole develops in isolates of C. albicans from HIV-infected children, that cross-resistance to other azoles may develop concomitantly, and that this resistance correlates with refractory mucosal candidiasis. PMID- 10747145 TI - Borrelia burgdorferi B31 Erp proteins that are dominant immunoblot antigens of animals infected with isolate B31 are recognized by only a subset of human lyme disease patient sera. AB - Sera from animals infected with Borrelia burgdorferi isolates yield intense immunoblot signals from the B31 ErpA/I/N and ErpB/J/O proteins, which have apparent molecular masses of 19 and 60 kDa, respectively. Since B. burgdorferi proteins with those molecular masses are of immunodiagnostic importance, Lyme disease patient sera were used in studies of B31 lysates and recombinant B31 ErpA/I/N and ErpB/J/O proteins. Immunoblot analyses indicated that only a minority of the patients produced antibodies that recognized the tested B31 Erp proteins. Southern blot analyses of Lyme disease spirochetes cultured from 16 of the patients indicated that all these bacteria contain genes related to the B31 erpA/I/N and erpB/J/O genes, although signal strengths indicated only weak similarities in many cases, suggestive of genetic variability of erp genes among these bacteria. These data indicate that Erp proteins are generally not the 19- and 60-kDa antigens observed on serodiagnostic immunoblots. PMID- 10747146 TI - A novel multiresistant Streptococcus pneumoniae serogroup 19 clone from Washington State identified by pulsed-field gel electrophoresis and restriction fragment length patterns. AB - In 1997, a cluster of multiresistant invasive serogroup 19 pneumococcus infections, including two fatalities, was reported in Washington State. Further investigation identified other cases. Fourteen Washington Streptococcus pneumoniae isolates, four from Alaska, and eight isolates from eastern Canada with reduced penicillin susceptibility (MIC of > or =1 microg/ml) were included in the study. Pulsed-field gel electrophoresis (PFGE) with ApaI, SacII, and SmaI restriction enzymes and IS1167 and mef restriction fragment length polymorphism (RFLP) pattern analysis were performed. Twenty of the 26 isolates had identical or related PFGE patterns, with two or all three enzymes, and identical or related IS1167 RFLP patterns, indicating that they were genetically related. These 20 isolates contained the mef gene conferring erythromycin resistance and had identical mef RFLP patterns. The PFGE and RFLP patterns were distinct from those of six multiresistant clones previously described and suggest that a new multiresistant clone has appeared in Washington, Alaska, and eastern Canada. This newly characterized clone should be included in the Pneumococcal Molecular Epidemiology Network. PMID- 10747147 TI - Characterization of Salmonella enterica serovar typhimurium DT104 isolated from Denmark and comparison with isolates from Europe and the United States. AB - A total of 136 isolates of Salmonella enterica serovar Typhimurium DT104 from Denmark (n = 93), Germany (n = 10), Italy (n = 4), Spain (n = 5), and the United Kingdom (n = 9) were characterized by antimicrobial resistance analysis, plasmid profiling, pulsed-field gel electrophoresis (PFGE) with the restriction enzymes XbaI and BlnI, and analysis for the presence of integrons and antibiotic resistance genes. The isolates from Denmark were from nine pig herds, while the isolates from other countries were both of animal and of human origin. All but 10 isolates were resistant to ampicillin, chloramphenicol, spectinomycin, streptomycin, sulfonamides, and tetracycline. Five isolates from the United Kingdom and Spain were sensitive to all antibiotics examined, whereas four isolates from the United Kingdom and the United States were also resistant to one or more of the antibiotics, namely, gentamicin, neomycin, and trimethoprim. All but two strains had the same PFGE profiles when the XbaI restriction enzyme was used, while seven different profiles were observed when the BlnI restriction enzyme was used. Different dominating BlnI types were observed among European isolates compared with the types observed among those from the United States. All the isolates harbored common 95-kb plasmids either alone or in combination with smaller plasmids, and a total of 11 different plasmid profiles were observed. Furthermore, all but one of the multidrug-resistant isolates contained two integrons, ant (3")-Ia and pse-1. Sensitive isolates contained no integrons, and isolates that were resistant to spectinomycin, streptomycin, and sulfonamides had only one integron containing ant (3")-Ia. When restriction enzyme BlnI was used, the 14 isolates from one of the nine herds in Denmark showed unique profiles, whereas isolates from the remaining herds were homogeneous. Among isolates from seven of nine herds, the same plasmid profile (95 kb) was observed, but isolates from two herds had different profiles. Thus, either PFGE (with BlnI) or plasmid profiling could distinguish isolates from three of nine pig herds in Denmark. The epidemiological markers (antimicrobial susceptibility testing, plasmid profiling, and PFGE) applied demonstrated high in vivo stability in the Danish herds. This may indicate that some different strains of multidrug-resistant S. enterica serovar Typhimurium DT104 have been introduced into Danish food animal herds. The presence of isolates from six different countries with similar profiles by PFGE with XbaI and highly homogeneous profiles by PFGE with BlnI indicate that multidrug-resistant S. enterica serovar Typhimurium DT104 has probably been spread clonally in these countries. However, some minor variation could be observed by using plasmid profiling and profiling by PFGE with BlnI. Thus, a more sensitive technique for subtyping of strains of DT104 and a broader investigation may help in elucidating the epidemiological spread of DT104 in different parts of the world. PMID- 10747148 TI - Evaluation of CHROMagar Staph. aureus, a new chromogenic medium, for isolation and presumptive identification of Staphylococcus aureus from human clinical specimens. AB - CHROMagar Staph. aureus (CSA) is a new chromogenic medium for presumptive identification of Staphylococcus aureus as mauve colonies after 24 h of incubation. We conducted a preliminary study with 100 S. aureus and 45 coagulase negative Staphylococcus (CoNS) stock isolates plated on CSA. All S. aureus isolates yielded mauve colonies after 24 h of incubation at 37 degrees C, while CoNS isolates grew as blue, white, or beige colonies. Culture on CSA was then prospectively compared to a conventional laboratory method, i.e. , culture on 5% horse blood agar (HBA), catalase test, and latex agglutination test (HBA-catalase latex), for isolation and presumptive identification of S. aureus from 2,000 consecutive clinical samples. Among the 310 S. aureus isolates recovered by at least one of the two methods, 296 grew as typical mauve colonies on CSA, while only 254 yielded catalase-positive, latex-positive colonies on HBA. The sensitivity of CSA was significantly higher than that of the conventional method (95.5 and 81.9%, respectively; P < 0.001) and allowed the recovery of important clinical isolates that were undetected on blood agar. The specificities of the two methods were not significantly different, although that of CSA was slightly higher (99.4% versus 98.9% for HBA-catalase-latex; P = 0. 08). On the basis of its excellent sensitivity and specificity, ease of identification of positive colonies, and absence of complementary testing, CSA can be recommended as a routine plating medium for presumptive identification of S. aureus in clinical specimens. PMID- 10747149 TI - Comparison of polyvinyl alcohol fixative with three less hazardous fixatives for detection and identification of intestinal parasites. AB - Polyvinyl alcohol (PVA) containing the fixative mercuric chloride is considered the "gold standard" for the fixation of ova and parasites in the preparation of permanently stained smears of stool specimens. However, mercuric chloride is potentially hazardous to laboratory personnel and presents disposal problems. We compared three new alternative, nontoxic fixatives with PVA, analyzing ease of sample preparation and quality of smears. Sixty-eight fresh stool specimens were divided into aliquots and placed in each of four different fixatives: PARASAFE (PS) (Scientific Devices Laboratory, Inc., Des Plaines, Ill.), ECOFIX (EC) (Meridian Diagnostics, Inc., Cincinnati, Ohio), Proto-Fix (PF) (Alpha-Tec Systems, Inc., Vancouver, Wash.), and low-viscosity PVA fixative (PVA) (Meridian). Specimens were processed and stained according to each manufacturer's directions. Parasites were found in 31 of 68 slide preparations with PVA, 31 with PF, 30 with EC, and 30 with PS. Blastocystis hominis and Iodamoeba butschlii were preserved in a readily identifiable state by all methods of fixation. However, some parasites were more easily identified with some of the fixatives because of differences in parasite distortion. For example, Entamoeba histolytica (Entamoeba dispar) was detected in 13 stools fixed with PF, 7 with PVA, and 6 with EC but none with PS. Likewise, Chilomastix mesnili was identified in 13 specimens fixed with PF, 8 with EC, and 5 with PVA but only 1 with PS, while Entamoeba coli was seen much less frequently with PS than with the other three fixatives. A dirty background was observed in 41% of specimens prepared with PS, whereas background quality was acceptable with other fixatives. Sample preparation was most rapid with PS, although the EC method involved the fewest steps. In conclusion, PVA and PF produced the least parasite distortion, while PS proved unsatisfactory for the identification of E. histolytica, E. coli, and C. mesnili. Both PF and EC appear to be acceptable, environmentally safe substitutes for PVA. PMID- 10747150 TI - Evaluation of phenotypic markers for selection and identification of Candida dubliniensis. AB - Candida dubliniensis is often associated with C. albicans in cultures. Easy-to perform selective isolation procedures for these closely related species do not exist. Therefore, we evaluated previously described discriminatory phenotypic markers for C. dubliniensis. A total of 150 oral rinses from human immunodeficiency virus (HIV)-infected patients were cultured on CHROMagar Candida. Dark green colonies described as being indicative of C. dubliniensis and other green colonies, 170 in total, were isolated. Chlamydospore formation, intracellular beta-D-glucosidase activity, ability to grow at 42 degrees C, carbohydrate assimilation pattern obtained by the API ID 32C, and Fourier transform infrared (FT-IR) spectroscopy were used for phenotypic characterization. Sequencing of the 5' end of the nuclear large-subunit (26S) ribosomal DNA gene was used for definitive species identification for C. dubliniensis. C. dubliniensis was found in 34% of yeast-colonized HIV-infected patients. The color of the colonies on CHROMagar Candida proved to be insufficient for selecting C. dubliniensis, since only 30 of 53 proven C. dubliniensis isolates showed a dark green color in primary cultures. The described typical chlamydospore formation can give only some indication of C. dubliniensis. The assimilation pattern proved to be insufficient to discriminate C. dubliniensis from C. albicans. All C. dubliniensis strains showed no or highly restricted growth at 42 degrees C and a lack of beta-D-glucosidase activity. Unfortunately, atypical C. albicans strains can also exhibit these phenotypic traits. FT-IR spectroscopy combined with hierarchical clustering proved to be as reliable as genotyping for discriminating the two species. PMID- 10747151 TI - Reverse cross blot hybridization assay for rapid detection of PCR-amplified DNA from candida species, Cryptococcus neoformans, and Saccharomyces cerevisiae in clinical samples. AB - A PCR-based assay was developed to detect and identify medically important yeasts in clinical samples. Using a previously described set of primers (G. Morace et al., J. Clin. Microbiol. 35:667-672, 1997), we amplified a fragment of the ERG11 gene for cytochrome P-450 lanosterol 14alpha-demethylase, a crucial enzyme in the biosynthesis of ergosterol. The PCR product was analyzed in a reverse cross blot hybridization assay with species-specific probes directed to a target region of the ERG11 gene of Candida albicans (pCal), C. guilliermondii (pGui), C. (Torulopsis) glabrata (pGla), C. kefyr (pKef), C. krusei (pKru), C. parapsilosis (pPar), C. tropicalis (pTro), the newly described species C. dubliniensis (pDub), Saccharomyces cerevisiae (pSce), and Cryptococcus neoformans (pCry). The PCR reverse cross blot hybridization assay correctly identified multiple isolates of each species tested. No cross-hybridization was detected with any other fungal, bacteria, or human DNAs tested. The method was tested against conventional identification on 140 different clinical samples, including blood and cerebrospinal fluid, from patients with suspected fungal infections. The results agreed with those of culture and phenotyping for all but six specimens (two of which grew yeasts not included in the PCR panel of probes and four in which PCR positivity-culture negativity was justified by clinical findings). Species identification time was reduced from a mean of 4 days with conventional identification to 7 h with the molecular method. The PCR-reverse cross blot hybridization assay is a rapid method for the direct detection and identification of yeasts in clinical samples. PMID- 10747152 TI - Genetic structure of population of Bacillus cereus and B. thuringiensis isolates associated with periodontitis and other human infections. AB - The genetic diversity and relationships among 35 Bacillus cereus and Bacillus thuringiensis isolates recovered from marginal and apical periodontitis in humans and from various other human infections were investigated using multilocus enzyme electrophoresis. The strains were isolated in Norway, except for three strains isolated from periodontitis patients in Brazil. The genetic diversity of these strains was compared to that of 30 isolates from dairies in Norway and Finland. Allelic variation in 13 structural gene loci encoding metabolic enzymes was analyzed. Twelve of the 13 loci were polymorphic, and 48 unique electrophoretic types (ETs) were identified, representing multilocus genotypes. The mean genetic diversity among the 48 genotypes was 0.508. The genetic diversity of each source group of isolates varied from 0.241 (periodontal infection) to 0.534 (dairy). Cluster analysis revealed two major groups separated at a genetic distance of greater than 0.6. One cluster, ETs 1 to 13, included solely isolates from dairies, while the other cluster, ETs 14 to 49, included all of the human isolates as well as isolates from dairies in Norway and Finland. The isolates were serotyped using antiflagellar antiserum. A total of 14 distinct serotypes were observed. However, little association between serotyping and genotyping was seen. Most of the strains were also analyzed with pulsed-field gel electrophoresis, showing the presence of extrachromosomal DNA in the size range of 15 to 600 kb. Our results indicate a high degree of heterogeneity among dairy strains. In contrast, strains isolated from humans had their genotypes in one cluster. Most strains from patients with periodontitis belonged to a single lineage, suggesting that specific clones of B. cereus and B. thuringiensis are associated with oral infections. PMID- 10747153 TI - Fluorescent amplified-fragment length polymorphism genotyping of Salmonella enterica subsp. enterica serovars and comparison with pulsed-field gel electrophoresis typing. AB - We have performed the fluorescently labeled amplified-fragment length polymorphism (FAFLP) method on 97 strains of the food-borne pathogen Salmonella enterica subsp. enterica comprising seven different serovars using the restriction enzymes EcoRI and MseI. From the total FAFLP fingerprinted strains, 81 were compared with pulsed-field gel electrophoresis (PFGE) typing of the same strains. The FAFLP method showed a discriminatory power equal to that of PFGE. We report a fast, robust, and high-resolution adaptation of the AFLP assay for fingerprinting S. enterica subsp. enterica serovars with capillary electrophoresis that can be scaled to high throughput on automated analysis instruments. PMID- 10747154 TI - Molecular phylogenetic evidence for noninvasive zoonotic transmission of Staphylococcus intermedius from a canine pet to a human. AB - rRNA-based molecular phylogenetic techniques were used to identify the bacterial species present in the ear fluid from a female patient with otitis externa. We report the identification of Staphylococcus intermedius from the patient and a possible route of transmission. Analysis of 16S ribosomal DNA restriction fragment length polymorphisms indicated that the dominant species present was S. intermedius. A pet dog owned by the patient also was tested and found to harbor S. intermedius. In humans, the disease is rare and considered a zoonosis. Previously, S. intermedius has been associated with dog bite wounds, catheter related injuries, and surgery. This study represents the first reported case of a noninvasive infection with S. intermedius. PMID- 10747156 TI - Evaluation of ciprofloxacin as a representative of veterinary fluoroquinolones in susceptibility testing. AB - Currently in veterinary medicine, ciprofloxacin is often used in susceptibility testing to represent the entire class of fluoroquinolone antimicrobials. Using quality control organisms as well as clinical isolates, we compared the MIC of ciprofloxacin to those of three other fluoroquinolones used in animals and found that ciprofloxacin is not an adequate representative of other members of this class. PMID- 10747155 TI - Characterization of a recurrent clonal type of Escherichia coli O157:H7 causing major outbreaks of infection in Scotland. AB - A particular recurrent clonal type of Escherichia coli O157 has been isolated from multiple clinical, veterinary, food, and environmental sources throughout Scotland since 1989. Significant genotypic variation was detected among isolates from distinct outbreaks, with the presence or absence of single fragments being sufficient to delineate outbreak groups within the clonal type. PMID- 10747157 TI - Long-term persistence of a discotheque-associated invasive Neisseria meningitidis group C strain as proven by pulsed-field gel electrophoresis and porA gene sequencing. AB - A cluster of a Neisseria meningitidis serogroup C strain causing invasive disease was investigated. Five out of seven cases were associated with a particular discotheque. The strains were indistinguishable, as revealed by pulsed-field gel electrophoresis and sequencing of variable regions of the porA gene, but caused strikingly different clinical presentations during 5 months. PMID- 10747158 TI - Molecular characterization of multidrug-resistant Streptococcus pneumoniae isolates in Korea. The Asian Network for Surveillance of Resistant Pathogens (ANSORP) Study Group. AB - Pulsed-field gel electrophoresis, ribotyping, and fingerprinting analysis of 22 invasive isolates of multidrug-resistant (MDR) pneumococci from Korea showed that 59 to 82% were genetically related. DNA sequencing of the PBP 2B gene showed relatively uniform alterations in nucleotides (5.4 to 7.8%) and amino acids (3.0 to 4. 3%), while Asn-276-->Lys, Arg-285-->Cys and Ser-305-->Phe substitutions were unique to Korean MDR strains, suggesting the spread of a few epidemic clones of resistant pneumococci within Korea. PMID- 10747159 TI - Comparison of a commercial enzyme-linked immunosorbent assay with immunofluorescence and complement fixation tests for detection of Coxiella burnetii (Q fever) immunoglobulin M. AB - A commercially available enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Q fever (PanBio Coxiella burnetii immunoglobulin M [IgM] ELISA, QFM 200) was compared to the indirect fluorescent antibody test (IFAT) for C. burnetii IgM and the complement fixation test (CFT). The ELISA demonstrated 92% agreement with the reference method (IFAT), and gave a sensitivity of 99% (69 of 70 samples) and a specificity of 88% (106 of 121). Specificity can be increased with confirmation by IFAT. CFT was found to have a specificity of 90% (107 of 119), although it was lacking in sensitivity (73%; 51 of 70). No cross-reactivity was observed in the ELISA with serum samples from patients with mycoplasma (n = 6), chlamydia (n = 5), or legionella (n = 4) infections, although 2 of 5 patients with leptospirosis and 1 of 4 samples containing rheumatoid factor (RF) demonstrated positive results in the ELISA. Results indicate that the performance of the PanBio C. burnetii (Q fever) IgM ELISA (F = 187) is superior to that of CFT (F = 163), and consequently the ELISA should be a useful aid in the diagnosis of acute Q fever. PMID- 10747160 TI - Comparison of the yields of blood cultures using serum or plasma from patients with early Lyme disease. AB - In an initial experiment, culture-grown Borrelia burgdorferi was added to freshly collected uninfected human blood. This in vitro study demonstrated that more spirochetes were distributed into the plasma than into the serum fraction. In a subsequent clinical study, B. burgdorferi was recovered from plasma cultures of approximately 50% of 42 patients with early Lyme disease associated with erythema migrans. The rate of recovery from plasma cultures was significantly greater than that from serum cultures (P < 0.001). PMID- 10747161 TI - Molecular typing and exopolysaccharide biosynthesis of Burkholderia cepacia isolates from a Portuguese cystic fibrosis center. AB - This work describes the first epidemiological survey of Burkholderia cepacia involved in pulmonary infections among the Portuguese population with cystic fibrosis (CF) who attended the major CF treatment Center in Lisbon at Sta. Maria Hospital from 1995 to the end of 1997. The characterization of the genomic relatedness of the isolates was based on the analysis of their ribopatterns (with EcoRI) followed by construction of a ribotype-based phylogenetic tree. This study was complemented with macrorestriction fragment analysis by pulsed-field gel electrophoresis. After optimization of the solid growth medium, we found that exopolysaccharide (EPS) production by B. cepacia CF isolates is not as rare a phenomenon as was thought before; indeed, 70% of the isolates examined were EPS producers. PMID- 10747162 TI - Identification of an epitope common to genogroup 1 "norwalk-like viruses". AB - A panel of 10 monoclonal antibodies (MAbs) to recombinant Norwalk virus (NV) capsid protein were tested in competition enzyme-linked immunosorbent assays. Patterns of competition indicated that these MAbs recognize six to eight epitopes covering five nonoverlapping regions of the capsid protein. A single epitope, recognized by NV MAbs NV3901, NV3912, and NV2461 was found to occur in the majority of genogroup 1 (G1) but not genogroup 2 (G2) "Norwalk-like viruses" (NLVs). This observation supports the subdivision of human NLVs into two genogroups and provides an assay for the rapid identification of G1 NLVs in fecal specimens. PMID- 10747163 TI - Relevance of commercial diagnostic tests to detection of enteric adenovirus infections in South Africa. AB - The prevalence of enteric adenoviruses detected by an in-house enzyme-linked immunosorbent assay (the RIVM-ELISA) ranged from 13 to 38%, and subgroup F adenoviruses comprised 86%. All subgroup F adenoviruses reacted with both RIVM anti-adenovirus type 40 (Ad40) and anti-adenovirus type 41 (Ad41) monoclonal antibodies but were not detected by Adenoclone Type 40/41 enzyme immunoassay (EIA). The correlation between the Biotrin EIA and RIVM-ELISA results was low (26%). Immunospecific tests suggest that a significant proportion of enteric adenoviruses, possibly comprising previously unidentified or emerging types, are not detected by commercial diagnostic tests in South Africa. PMID- 10747164 TI - Restriction fragment length polymorphism analysis using random chromosomal gene probes for epidemiological analysis of Campylobacter jejuni infections. AB - We have evaluated the ability of a new genotyping method for Campylobacter jejuni based on restriction fragment length polymorphisms using random chromosomal gene probes. DNAs from C. jejuni strains digested with each of three restriction enzymes, HhaI, HaeIII, and HpaII, were analyzed by Southern hybridization using each of two unrelated cosmid clones, P14 and P15 (respectively containing 30- and 35-kb genomic DNA fragments of C. jejuni strain OH4384). The method reported provides a stable and discriminating means for identifying C. jejuni strains and should be useful for epidemiological analyses. PMID- 10747165 TI - A field-suitable, semisolid aerobic enrichment medium for isolation of Campylobacter jejuni in small numbers. AB - The objective of this study was to produce an economical, easy to prepare, field suitable enrichment medium for detection of Campylobacter jejuni in small numbers. A semisolid aerobic enrichment medium was developed. Rates of recovery from inoculated medium, sterile swabs, and mixed cultures of C. jejuni and coliform bacteria were tested. PMID- 10747166 TI - Evaluation of a new commercially available immunoglobulin M and immunoglobulin G immunochromatographic test for diagnosis of melioidosis infection. AB - An immunochromatographic test for the rapid determination of immunoglobulin M (IgM) and IgG antibodies to Burkholderia pseudomallei was evaluated by using sera from bacteriologically confirmed melioidosis patients and high-risk and clinically suspected patients, along with disease control groups. The sensitivities were 100 and 93% for the IgG and IgM tests, respectively, while the specificity was 95% for both assays. The test was rapid and simple to perform, with results obtained in 10 min. PMID- 10747167 TI - PCR assay for species-specific identification of Bacteroides thetaiotaomicron. AB - Bacteroides thetaiotaomicron is the second most frequently encountered species of the anaerobes isolated from clinical specimens. We developed a PCR-based assay for the rapid identification of B. thetaiotaomicron. Specific primers were based on shared amplicons of about 1.2 kb generated from B. thetaiotaomicron by randomly amplified polymorphic DNA. This 1.2-kb fragment was sequenced and then used to design a set of PCR amplification primers. This PCR generated an amplification product of 721 bp, which was unique to all 65 isolates of B. thetaiotaomicron tested. There was no amplification with isolates of other bacterial species. Restriction enzyme digestion of the amplification product and dot blot hybridization further verified the specificity of the assay. These results suggest that this PCR assay targets a nucleotide sequence that is strongly conserved in B. thetaiotaomicron. This simple and rapid PCR assay provides a rapid and accurate method for identification of B. thetaiotaomicron and shows promise for the detection of B. thetaiotaomicron in clinical samples. PMID- 10747168 TI - Identification of coryneform bacterial isolates by ribosomal DNA sequence analysis. AB - Identification of coryneform bacteria to the species level is important in certain circumstances for differentiating contamination and/or colonization from infection, which influences decisions regarding clinical intervention. However, methods currently used in clinical microbiology laboratories for the species identification of coryneform bacteria are often inadequate. We evaluated the MicroSeq 500 16S bacterial sequencing kit (Perkin-Elmer Biosystems, Foster City, Calif.), which is designed to sequence the first 527 bp of the 16S rRNA gene for bacterial identification, by using 52 coryneform gram-positive bacilli from clinical specimens isolated from January through June 1993 at the Mayo Clinic. Compared to conventional and supplemented phenotypic methods, MicroSeq provided concordant results for identification to the genus level for all isolates. At the species level, MicroSeq provided concordant results for 27 of 42 (64.3%) Corynebacterium isolates and 5 of 6 (83.3%) Corynebacterium-related isolates, respectively. Within the Corynebacterium genus, MicroSeq gave identical species level identifications for the clinically significant Corynebacterium diphtheriae (4 of 4) and Corynebacterium jeikeium (8 of 8), but it identified only 50.0% (15 of 30) of other species (P < 0.01). Four isolates from the genera Arthrobacter, Brevibacterium, and Microbacterium, which could not be identified to the species level by conventional methods, were assigned a species-level identification by MicroSeq. The total elapsed time for running a MicroSeq identification was 15.5 to 18.5 h. These data demonstrate that the MicroSeq 500 16S bacterial sequencing kit provides a potentially powerful method for the definitive identification of clinical coryneform bacterium isolates. PMID- 10747169 TI - Pooling of urine specimens for detection of asymptomatic Chlamydia trachomatis infections by PCR in a low-prevalence population: cost-saving strategy for epidemiological studies and screening programs. AB - Pooling, in groups of five, of urine specimens from asymptomatically infected men in a population with 4% prevalence, as determined by case finding, is 100% sensitive and specific and results in a 60.5% reduction in the number of tests needed. Pooling of urine specimens in groups of 10 for the estimation of population-based prevalence is 96.1% sensitive and 100% specific and saves 90% of the test costs. PMID- 10747170 TI - G and P genotyping of rotavirus strains circulating in france over a three-year period: detection of G9 and P[6] strains at low frequencies. The AZAY Group. AB - Rotavirus G and P types from 716 children with acute diarrhea in Dijon from 1995 to 1998 and throughout France during the winter of 1997-1998 were analyzed by reverse transcription-PCR. P[8],G1 predominated, followed by P[8],G4, which emerged during the last winter. G9 and P[6] strains were detected at low frequencies. PMID- 10747171 TI - Selective isolation of eae-positive strains of shiga toxin-producing Escherichia coli. AB - Culture on cefixime, tellurite, and sorbitol-MacConkey agar after HCl treatment facilitated the growth of 410 (94%) of 436 eae-positive Shiga toxin-producing Escherichia coli (STEC) strains and 17 (16%) of 107 eae-negative STEC strains. This selectivity was closely related to acid resistance in E. coli and tellurite resistance in eae-positive STEC strains. PMID- 10747172 TI - Evaluation of the Sirscan automated zone reader in a clinical microbiology laboratory. AB - We compared readings of Kirby-Bauer plates by the Sirscan, an automated image analyzer that measures zone diameters, to those of experienced clinical microbiologists measuring zones with a hand-held caliper interfaced to a computer and with a ruler. To read plates of Escherichia coli, Morganella morganii, and Pseudomonas aeruginosa containing 12 antibiotic disks the Sirscan took 11 s; technologists took 28 s by caliper and 39 s by ruler. Reading times of four different technologists ranged from 22 to 44 s with the caliper and 10 to 12 s with Sirscan. Upon repeated testing zone size variation rarely exceeded 3 mm by caliper and 1 mm by Sirscan. Over a 4-month period, 368 clinical isolates were tested prospectively by both methods in the Clinical Microbiology Laboratory of the Miriam Hospital. There was good correlation of zone sizes for most antibiotics, but Sirscan zone diameter measurements tended to be 3 to 5 mm larger than caliper readings for ciprofloxacin, norfloxacin, aztreonam, erythromycin, clindamycin, and trimethoprim-sulfamethoxazole. Very major errors (resistant by caliper and susceptible by Sirscan) occurred with 10 of 3,770 readings (0.3%), mainly where breakpoint criteria lacked an intermediate zone. They occurred in testing staphylococci with amoxicillin-clavulanate (5 of 127 isolates, 3.9%), pseudomonas with piperacillin (1 of 28, 3.6%), coagulase-negative staphylococci with oxacillin (2 of 74, 2.7%), gram-negative bacilli with cefuroxime (1 of 209, 0.5%), and mixed species with trimethoprim-sulfamethoxazole (1 of 366, 0.3%). The Sirscan zone reader facilitates accurate, fully quantitative susceptibility testing in clinical microbiology laboratories. PMID- 10747173 TI - Disseminated infection caused by Scedosporium prolificans in a patient with acute multilineal leukemia. AB - In this report, we describe a case of disseminated infection caused by Scedosporium prolificans (S. inflatum) in a patient affected by chemotherapy induced acute multilineal leukemia and neutropenia. For the fungus isolated in four blood cultures, high MICs of currently available antifungal agents were found. Postmortem examination revealed multiorgan involvement. PMID- 10747174 TI - Pseudomembranous colitis caused by a toxin A(-) B(+) strain of Clostridium difficile. AB - We report a case of severe pseudomembranous colitis due to a toxin A(-) B(+) strain of Clostridium difficile in an immunosuppressed patient and discuss the implications for diagnostic testing in suspected C. difficile-associated diarrhea. PMID- 10747175 TI - Bartonella vinsonii subsp. berkhoffii as an agent of afebrile blood culture negative endocarditis in a human. AB - We report a case of endocarditis in a human infected with Bartonella vinsonii subsp. berkhoffii, which causes bacteremia and endocarditis in dogs. Bacterial identification was established by PCR amplification and sequencing of an intergenic spacer region (ITS1), 16S ribosomal DNA, and a gene encoding citrate synthase (gltA). Bartonella antibodies were detected by immunofluorescence. PMID- 10747176 TI - Desulfovibrio desulfuricans bacteremia in a dog. AB - Desulfovibrio desulfuricans was isolated from the blood of a dog presenting with fever, anorexia, and rear limb stiffness. The isolate was identified by 16S rRNA gene amplification and sequencing. PMID- 10747178 TI - Combining physiology and genetics in the zebrafish retina. AB - The zebrafish has recently joined the ranks of Drosophila and C. elegans as a tractable model for genetic screens (Fishman, 1999). Zebrafish grow fast, can be kept in large numbers in a small space, and are efficiently mutagenized and screened. Genomic resources are made available at an increasing pace. These days, a mutation can be mapped and cloned in a matter of months. Because a mutant hunt is intrinsically unbiased in terms of the classes of genes that will be tagged, it holds the unique potential to discover novel genes or, in our era of genome sequencing, to identify novel functions for known genes. Zebrafish display dozens of innate behaviours in response to light, of which the optomotor and the optokinetic responses are the most widely studied (Brockerhoff et al. 1995; Easter & Nicola, 1996). Their retinae are crisply layered following the typical vertebrate pattern, and the retinal layers are tiled in an almost crystalline fashion by mosaics of different cell types. Electroretinograms are recorded routinely and therefore, not surprisingly, zebrafish are now also being used for a genetic approach to the visual system. PMID- 10747177 TI - Aerococcus urinae in urinary tract infections. AB - Aerococcus urinae is a rarely reported pathogen, possibly due to difficulties in the identification of the organism. A. urinae is a gram-positive coccus that grows in pairs and clusters, produces alpha-hemolysis on blood agar, and is negative for catalase and pyrrolidonyl aminopeptidase. Some of these characteristics and its being absent from the databases of most commercial identification systems could allow A. urinae to be misidentified as a streptococcus, enterococcus, or staphylococcus. We report two cases of urinary tract infection (UTI) caused by A. urinae and characterize these isolates by morphology, biochemical testing, whole-cell fatty acid analysis, 16S rRNA gene sequencing, and antibiotic susceptibilities. Most patients infected with A. urinae are elderly males with predisposing conditions who present initially with UTI. Because A. urinae is resistant to sulfonamides, treatment could be inappropriate, with infections resulting in serious complications, including death. It is important for the clinician and the microbiologist to consider A. urinae a potential pathogen and proceed with thorough microbiological identification. PMID- 10747179 TI - Multiple forms of feedback inhibition by str. oriens inhibitory interneurons? AB - In the CA1 region of the hippocampus pyramidal neuron basilar dendrites extend into the stratum oriens-alveus while the apical dendrites project deep into the stratum lacunosum-moleculare, a distance several hundred micrometres in extent. This extended dendritic arbor provides a large surface area for afferent input. For example, the axons of CA3 pyramidal neurons synapse onto pyramidal cell dendritic spines across approximately 2/3 of the apical dendritic tree. In contrast a diverse population of local-circuit GABAergic inhibitory interneurons selectively innervate specific postsynaptic domains of principal cells (for review see Freund & Buzsaki, 1996). These cells target their axons either to the axon initial segment, somata, or proximal and distal dendrites, with each cell type implicated in a particular operational role. However, rigid classification of the numerous subpopulations has been problematic (see Parra et al. 1998 for further discussion). PMID- 10747180 TI - Multiple pathways responsible for the stretch-induced increase in Ca2+ concentration in toad stomach smooth muscle cells. AB - 1. A digital imaging microscope with fura-2 as the Ca2+ indicator was used to determine the sources for the rise in intracellular calcium concentration ([Ca2+]i) that occurs when the membrane in a cell-attached patch is stretched. Unitary ionic currents from stretch-activated channels and [Ca2+]i images were recorded simultaneously. 2. When suction was applied to the patch pipette to stretch a patch of membrane, Ca2+-permeable cation channels (stretch-activated channels) opened and a global increase in [Ca2+]i occurred, as well as a greater focal increase in the vicinity of the patch pipette. The global changes in [Ca2+]i occurred only when stretch-activated currents were sufficient to cause membrane depolarization, as indicated by the reduction in amplitude of the unitary currents. 3. When Ca2+ was present only in the pipette solution, just the focal change in [Ca2+]i was obtained. This focal change was not seen when the contribution from Ca2+ stores was eliminated using caffeine and ryanodine. 4. These results suggest that the opening of stretch-activated channels allows ions, including Ca2+, to enter the cell. The entry of positive charge triggers the influx of Ca2+ into the cell by causing membrane depolarization, which presumably activates voltage-gated Ca2+ channels. The entry of Ca2+ through stretch activated channels is also amplified by Ca2+ release from internal stores. This amplification appears to be greater than that obtained by activation of whole cell Ca2+ currents. These multiple pathways whereby membrane stretch causes a rise in [Ca2+]i may play a role in stretch-induced contraction, which is a characteristic of many smooth muscle tissues. PMID- 10747181 TI - Permeant ion binding affinity in subconductance states of an L-type Ca2+ channel expressed in Xenopus laevis oocytes. AB - 1. The relationship between single-channel conductance and ion binding affinity in Ca2+ channels was investigated by measuring differences in the apparent binding affinity (K'D) for Ca2+ among naturally occurring conductance states of an L-type (alpha1C) Ca2+ channel heterologously expressed in Xenopus oocytes. Using cell-attached patch recordings, three or more conductance levels were observed when Ca2+, Ba2+ or Li+ was used as the permeating ion. 2. With Li+ as the charge carrier, low concentrations of Ca2+ (0.1-3.0 microM) produced discrete blocking events in all conductance states. Measurements of open and blocked times as a function of Ca2+ concentration were used to calculate rates of block and unblock. 3. K'D was calculated for three of the conductance levels. Binding affinity for Ca2+ increased as conductance decreased (K'D: large = 7.5 microM, medium = 4.0 microM, small = 2.7 microM). The lower K'D values of the smaller conductance states arose from a combination of larger on-rates and smaller off rates. 4. These results imply that permeant ions such as Ca2+ have both easier access to, and longer dwell time in, the Ca2+ binding locus in the pore when the channel opens to a subconductance level as compared to the fully open level. 5. The difference in K'D between the large and small conductance levels corresponds to a small difference in the free energy of binding, DeltaDeltaG approximately 1kBT, where kB is Boltzmann's constant and T is absolute temperature (kelvin). Nonetheless, an Eyring model of Ca2+ channel permeation incorporating the state specific on- and off-rate constants for Ca2+ was able to reproduce the large difference in channel conductance, indicating that small differences in binding energy may be able to account for large differences in amplitude between conductance states. PMID- 10747182 TI - Lidocaine induces a slow inactivated state in rat skeletal muscle sodium channels. AB - 1. Local anaesthetics such as lidocaine (lignocaine) interact with sodium channels in a manner that is exquisitely sensitive to the voltage-dependent conformational state of the ion channel. When depolarized in the presence of lidocaine, sodium channels assume a long-lived quiescent state. Although studies over the last decade have localized the lidocaine receptor to the inner aspect of the aqueous pore, the mechanistic basis of depolarization-induced 'use-dependent' lidocaine block remains uncertain. 2. Recent studies have shown that lowering the extracellular Na+ concentration ([Na+]o) and mutations in the sodium channel outer P-loop modulate occupancy of a quiescent 'slow' inactivated state with intermediate kinetics (termed IM) that involves structural rearrangements in the outer pore. 3. Site-directed mutagenesis and ion-replacement experiments were performed using voltage-clamped Xenopus oocytes and cultured (HEK-293) cells expressing wild-type and mutant rat skeletal muscle (mu1) sodium channels. 4. Our results show that lowering [Na+]o potentiates use-dependent lidocaine block. The effect of [Na+]o is maintained despite a III-IV linker mutation that partially disrupts fast inactivation (F1304Q). In contrast, the effect of lowering [Na+]o on lidocaine block is reduced by a P-loop mutation (W402A) that limits occupancy of IM. 5. Our findings are consistent with a simple allosteric model where lidocaine binding induces channels to occupy a native slow inactivated state that is inhibited by [Na+]o. PMID- 10747183 TI - Properties of the delayed rectifier potassium current in porcine sino-atrial node cells. AB - Whole-cell currents were recorded in single, spontaneously active cells dissociated from porcine sino-atrial node, and the conductance and gating properties of the delayed rectifier K+ current (IK) were investigated. The isolated cells exhibited spontaneous action potentials at a rate of 80.5 +/- 5.4 min-1 (mean +/- s.e.m., n = 11). Under Ca2+ current block, depolarization from 40 mV to various potentials activated a time-dependent outward current (IK). The activation curve of IK showed a half-activation potential (V1/2) of 20.5 +/- 2.1 mV and a slope factor (S) of 16.4 +/- 1.2 mV (n = 8). As the duration of the depolarizing pulse to either +10 or +60 mV was prolonged, the amplitude of the tail current increased in proportion to that of the activated outward current during depolarization. E4031 (2-5 uM), a selective blocker for the rapidly activating component of IK (IK,r), hardly affected IK, but chromanol 293B, a selective blocker for the slowly activating component (IK,s), inhibited IK with an IC50 of 8.79 uM. The reversal potential of IK was -75.2 +/- 2.3 mV with 5.4 mM external and 150 mM internal K+. The time courses of activation and deactivation of IK were fitted by the sum of two exponential functions at various potentials. The relationship between the time constants and membrane potential showed a bell shaped curve with a peak at around -10 mV for both fast and slow components. The results indicate that in porcine sino-atrial node cells IK is largely derived from IK,s and that IK,s plays a functional role in the slow diastolic depolarization. IK,s may, in part, account for the relatively slower heart rate of pigs than that of rabbit in which IK,r is a functionally dominant component of IK. PMID- 10747184 TI - The role of ClC-3 in volume-activated chloride currents and volume regulation in bovine epithelial cells demonstrated by antisense inhibition. AB - 1. A chloride current with mild outward rectification was induced in the native bovine non-pigmented ciliary epithelial (NPCE) cells by a 23 % hypotonic solution. The current showed no or little inactivation at depolarized steps. 2. ATP blocked 88 and 61 % of the outward and inward components of the volume activated chloride current (ICl,vol) with an IC50 of 5.3 and 9.6 mM, respectively. 3. The volume-activated chloride current was decreased and the activation of the current was delayed by inhibiting endogenous ClC-3 expression using a ClC-3 antisense oligonucleotide. The inhibition of the current as a function of antisense concentration was asymptotic with a maximum about 60 %. The remaining current was probably not derived from ClC-3 and was inhibited by ATP. 4. ClC-3 expression in the bovine NPCE cells was verified by immunofluorescence studies. ClC-3 immunofluorescence was distributed throughout the cells but with the predominant location within the nucleus. The expression of ClC-3 protein was diminished by the ClC-3 antisense oligonucleotide with the greatest diminution occurring in the nuclear region. 5. The size of the volume-activated chloride current was positively correlated with the ClC-3 immunofluorescence level. 6. Regulatory volume decrease of the NPCE cells was reduced by ClC-3 antisense oligonucleotide. 7. We conclude that endogenous ClC-3 is associated with the volume-activated chloride current and is involved in cell volume regulation, but that it can only contribute towards a proportion of the current in NPCE cells. 8. The nuclear predominance of ClC-3 immunofluorescence in NPCE cells, the absence of basal activity of chloride current and the marked pharmacological differences between IClC-3 and ICl,vol argue against ClC-3 being the only, or even the main, volume-activated chloride channel in NPCE cells. PMID- 10747185 TI - ATP stimulates Cl- secretion and reduces amiloride-sensitive Na+ absorption in M 1 mouse cortical collecting duct cells. AB - 1. Using equivalent short circuit current (ISC) measurements we examined the effect of extracellular ATP on transepithelial ion transport in M-1 mouse cortical collecting duct cells. Apical addition of ATP produced a rapid transient peak increase in ISC. This was followed by a fall below basal ISC due to a reduction in the amiloride-sensitive ISC component. 2. The ATP-induced ISC increase was preserved in the presence of apical amiloride while it was reduced in the absence of extracellular Cl- and in the presence of the apical Cl- channel blockers diphenylamine-2-carboxylic acid (DPC, 1 mM), DIDS (300 microM) and niflumic acid (100 microM). 3. The stimulatory effect of apical ATP on ISC was concentration dependent with an EC50 of about 0.6 microM. Basolateral ATP elicited a similar ISC response. Experiments using the ATP scavenger hexokinase demonstrated that the ATP effects were elicited via separate apical and basolateral receptors. 4. ATP and UTP applied to either the apical or the basolateral bath equi-potently stimulated ISC while 'purified' ADP and UDP had no effect consistent with P2Y2 purinoceptors, the expression of which was confirmed using RT-PCR. 5. Intracellular calcium concentration ([Ca2+]i) measurements using fura-2 demonstrated that ATP and UTP elicited a rise in [Ca2+]i with EC50 values of 1.1 and 0.6 microM, respectively. The shape and time course of the calcium response were similar to those of the ISC response. The peak ISC response was preserved in the nominal absence of extracellular calcium but was significantly reduced in cells pre-incubated with the calcium chelator BAPTA AM. 6. We conclude that in M-1 cells extracellular ATP reduces amiloride-sensitive Na+ absorption and stimulates Cl- secretion via calcium-activated Cl- channels through activation of P2Y2 purinoreceptors located in the apical and basolateral membrane. PMID- 10747186 TI - Cell surface domain specific postsynaptic currents evoked by identified GABAergic neurones in rat hippocampus in vitro. AB - 1. Inhibitory postsynaptic currents (IPSCs) evoked in CA1 pyramidal cells (n = 46) by identified interneurones (n = 43) located in str. oriens were recorded in order to compare their functional properties and to determine the effect of synapse location on the apparent IPSC kinetics as recorded using somatic voltage clamp at -70 mV and nearly symmetrical [Cl-]. 2. Five types of visualised presynaptic interneurone, oriens-lacunosum moleculare (O-LMC), basket (BC), axo axonic (AAC), bistratified (BiC) and oriens-bistratified (O-BiC) cells, were distinguished by immunocytochemistry and/or synapse location using light and electron microscopy. 3. Somatostatin immunoreactive O-LMCs, innervating the most distal dendritic shafts and spines, evoked the smallest amplitude (26 +/- 10 pA, s.e.m., n = 8) and slowest IPSCs (10-90 % rise time, 6.2 +/- 0.6 ms; decay, 20.8 +/- 1.7 ms, n = 8), with no paired-pulse modulation of the second IPSC (93 +/- 4 %) at 100 ms interspike interval. In contrast, parvalbumin-positive AACs evoked larger amplitude (308 +/- 103 pA, n = 7) and kinetically faster (rise time, 0.8 +/- 0.1 ms; decay 11.2 +/- 0.9 ms, n = 7) IPSCs showing paired-pulse depression (to 68 +/- 5 %, n = 6). Parvalbumin- or CCK-positive BCs (n = 9) terminating on soma/dendrites, BiCs (n = 4) and O-BiCs (n = 7) innervating dendrites evoked IPSCs with intermediate kinetic parameters. The properties of IPSCs and sensitivity to bicuculline indicated that they were mediated by GABAA receptors. 4. In three cases, kinetically complex, multiphasic IPSCs, evoked by an action potential in the recorded basket cells, suggested that coupled interneurones, possibly through electrotonic junctions, converged on the same postsynaptic neurone. 5. The population of O-BiCs (4 of 4 somatostatin positive) characterised in this study had horizontal dendrites restricted to str. oriens/alveus and innervated stratum radiatum and oriens. Other BiCs had radial dendrites as described earlier. The parameters of IPSCs evoked by BiCs and O-BiCs showed the largest cell to cell variation, and a single interneurone could evoke both small and slow as well as large and relatively fast IPSCs. 6. The kinetic properties of the somatically recorded postsynaptic current are correlated with the innervated cell surface domain. A significant correlation of rise and decay times for the overall population of unitary IPSCs suggests that electrotonic filtering of distal responses is a major factor for the location and cell type specific differences of unitary IPSCs, but molecular heterogeneity of postsynaptic GABAA receptors may also contribute to the observed kinetic differences. Furthermore, domain specific differences in the short-term plasticity of the postsynaptic response indicate a differentiation of interneurones in activity-dependent responses. PMID- 10747187 TI - Granule cell hyperexcitability in the early post-traumatic rat dentate gyrus: the 'irritable mossy cell' hypothesis. AB - 1. Cytochemical and in vitro whole-cell patch clamp techniques were used to investigate granule cell hyperexcitability in the dentate gyrus 1 week after fluid percussion head trauma. 2. The percentage decrease in the number of hilar interneurones labelled with either GAD67 or parvalbumin mRNA probes following trauma was not different from the decrease in the total population of hilar cells, indicating no preferential survival of interneurones with respect to the non-GABAergic hilar cells, i.e. the mossy cells. 3. Dentate granule cells following trauma showed enhanced action potential discharges, and longer-lasting depolarizations, in response to perforant path stimulation, in the presence of the GABAA receptor antagonist bicuculline. 4. There was no post-traumatic alteration in the perforant path-evoked monosynaptic excitatory postsynaptic currents (EPSCs), or in the intrinsic properties of granule cells. However, after trauma, the monosynaptic EPSC was followed by late, polysynaptic EPSCs, which were not present in controls. 5. The late EPSCs in granule cells from fluid percussion-injured rats were not blocked by the NMDA receptor antagonist 2-amino 5-phosphonovaleric acid (APV), but were eliminated by both the non-NMDA glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the AMPA receptor antagonist GYKI 53655. 6. In addition, the late EPSCs were not present in low (0.5 mM) extracellular calcium, and they were also eliminated by the removal of the dentate hilus from the slice. 7. Mossy hilar cells in the traumatic dentate gyrus responded with significantly enhanced, prolonged trains of action potential discharges to perforant path stimulation. 8. These data indicate that surviving mossy cells play a crucial role in the hyperexcitable responses of the post-traumatic dentate gyrus. PMID- 10747188 TI - Axonal stratification patterns and glutamate-gated conductance mechanisms in zebrafish retinal bipolar cells. AB - 1. Whole-cell patch recording and puff pipette techniques were used to identify glutamate receptor mechanisms on bipolar cell (BC) dendrites in the zebrafish retinal slice. Recorded neurons were stained with Lucifer Yellow, to correlate glutamate responses with BC morphology. 2. BC axon terminals (ATs) consisted of swellings or varicosities along the axon, as well as at its end. AT stratification patterns identified three regions in the inner plexiform layer (IPL): a thick sublamina a, with three bands of ATs, a narrow terminal-free zone in the mid-IPL, and a thin sublamina b, with two bands of ATs. BCs occurred with ATs restricted to sublamina a(Group a), sublamina b(Group b) or with ATs in both sublaminae (Group a/b). 3. OFF-BCs belonged to Group a or Group a/b. These cells responded to glutamate or kainate with a CNQX-sensitive conductance increase. Reversal potential (Erev) ranged from -0.6 to +18 mV. Bipolar cells stimulated sequentially with both kainate and glutamate revealed a population of glutamate insensitive, kainate-sensitive cells in addition to cells sensitive to both agonists. 4. ON-BCs responded to glutamate via one of three mechanisms: (a) a conductance decrease with Erev approximately 0 mV, mimicked by L-(+)-2-amino-4 phosphonobutyric acid (APB) or trans-1-amino-1, 3-cyclopentanedicarboxylic acid (trans-ACPD), (b) a glutamate-gated chloride conductance increase (IGlu-like) characterized by Erev >= ECl (where ECl is the chloride equilibrium potential) and partial blockade by extracellular Li+/Na+ substitution or (c) the activation of both APB and chloride mechanisms simultaneously to produce a response with outward currents at all holding potentials. APB-like responses were found only among BCs in Group b, with a single AT ramifying deep within sublamina b; whereas, cells expressing IGlu-like currents had one or more ATs, and occurred within Groups b or a/b. 5. Multistratified cells (Group a/b) were common and occurred with either ON- or OFF-BC physiology. OFF-BCs typically had one or more ATs in sublamina a and only one AT in sublamina b. In contrast, multistratified ON-BCs had one or more ATs in sublamina b and a single AT ramifying deep in sublamina a. Multistratified ON-BCs expressed the IGlu-like mechanism only. 6. Visual processing in the zebrafish retina involves at least 13 BC types. Some of these BCs have ATs in both the ON- and OFF-sublaminae, suggesting a significant role for ON- and OFF-inputs throughout the IPL. PMID- 10747189 TI - Identification of subunits contributing to synaptic and extrasynaptic NMDA receptors in Golgi cells of the rat cerebellum. AB - 1. To investigate the properties of N-methyl-D-aspartate receptors (NMDARs) in cerebellar Golgi cells, patch-clamp recordings were made in cerebellar slices from postnatal day 14 (P14) rats. To verify cell identity, cells were filled with Neurobiotin and examined using confocal microscopy. 2. The NR2B subunit-selective NMDAR antagonist ifenprodil (10 microM) reduced whole-cell NMDA-evoked currents by approximately 80 %. The NMDA-evoked currents were unaffected by the Zn2+ chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN; 1 microM) suggesting the absence of NMDARs containing NR2A subunits. 3. Outside-out patches from Golgi cells exhibited a population of 'high-conductance' 50 pS NMDAR openings. These were inhibited by ifenprodil, with an IC50 of 19 nM. 4. Patches from these cells also contained 'low-conductance' NMDAR channels, with features characteristic of NR2D subunit-containing receptors. These exhibited a main conductance of 39 pS, with a sub-conductance level of 19 pS, with clear asymmetry of transitions between the two levels. As expected of NR2D-containing receptors, these events were not affected by ifenprodil. 5. The NMDAR-mediated component of EPSCs, evoked by parallel fibre stimulation or occurring spontaneously, was not affected by 1 microM TPEN. However, it was reduced (by approximately 60 %) in the presence of 10 microM ifenprodil, to leave a residual NMDAR-mediated current that exhibited fast decay kinetics. This is, therefore, unlikely to have arisen from receptors composed of NR1/NR2D subunits. 6. We conclude that in cerebellar Golgi cells, the high- and low-conductance NMDAR channels arise from NR2B- and NR2D containing receptors, respectively. We found no evidence for NR2A-containing receptors in these cells. While NR2B-containing receptors are present in both the synaptic and extrasynaptic membrane, our results indicate that NR1/NR2D receptors do not contribute to the EPSC and appear to be restricted to the extrasynaptic membrane. PMID- 10747190 TI - Changes in the distribution of synaptic potentials from bulbospinal neurones following axotomy in cat thoracic spinal cord. AB - 1. Plasticity in functional connections of expiratory bulbospinal neurones was investigated by measurement of terminal potentials (TPs) and focal synaptic potentials (FSPs), recorded with spike-triggered averaging in the thoracic spinal cord of anaesthetized, paralysed cats. These measurements were made in normal cats and in those which had previously been subjected to spinal cord lesions that transected the axons of the bulbospinal neurones in the segment below that under investigation, either about 2 weeks or about 16 weeks previously. 2. In both groups of operated animals bulbospinal neurones with firing properties and conduction velocities similar to normal were present. The extracellular recordings that were averaged to reveal TPs and FSPs were made on two standard grids, each consisting of eight sites spaced 0.25 mm apart on two electrode tracks. One grid was positioned at a rostral and one at a caudal location within one segment (T7-T9). 3. Tn the normal animals TPs and FSPs were larger and/or more common at rostral sites than at caudal sites, by a factor of about 1.7. In both 2 week and 16 week animals, TPs and FSPs were observed, both showing normal tine courses and latencies. At rostral sites in 2 week and 16 week animals the amplitudes and/or the frequency of occurrence of TPs and FSPs were similar to normal, as was the case fir caudal sites in the 2 week animals. However, at caudal sites in the 16 week animals the FSPs were mole common and/or significantly larger than normal, with the increase particularly marked on the lateral track, being equivalent to a factor of about 2. A corresponding increase in the amplitude and/or frequency of occurrence of TPs at caudal lateral sites was also seen, but was not significant. 4. The results are interpreted as evidence for short-range sprouting of the bulbospinal axons and the formation by them of new connections in the caudal parts of the segments concerned. PMID- 10747191 TI - Single photon responses in Drosophila photoreceptors and their regulation by Ca2+. AB - 1. Discrete events (quantum bumps) elicited by dim light were analysed in whole cell voltage clamp of photoreceptors from dissociated Drosophila ommatidia. Bumps were automatically detected and analysed for amplitude, rise and decay times, and latency. 2. The bump interval and amplitude distributions, and the 'frequency of seeing' curve conformed to Poisson predictions for the absorption of single photons. 3. At resting potential (-70 mV), bumps averaged 10 pA in peak amplitude with a half-width of ca 20 ms, representing simultaneous activation of ca 15 channels. 4. The macroscopic response to flashes containing up to at least 750 photons were predicted by the linear summation of quantum bumps convolved with their latency dispersion. 5. Bump duration was unaffected by lowering the extracellular Ca2+ concentration ([Ca2+]o) from 1.5 to 0.5 mM, but increased >10 fold between 0.5 mM Ca2+ and 0 Ca2+. Bump amplitude was constant over the range 1.5-100 microM, but decreased ca 5- to 10-fold at lower Ca2+ concentrations. Bump latency increased by ca 50 % between 1.5 mM and 100 microM Ca2+o but returned to near control levels in Ca2+-free solutions. At intermediate [Ca2+]o bumps were biphasic with a slow rising phase followed by rapid amplification and inactivation. This behaviour was mimicked in high [Ca2+]o by internal buffering with BAPTA, but not EGTA. This suggests that Ca2+ influx through the light sensitive channels must first raise cytosolic Ca2+ to a threshold level before initiating a cycle of positive and negative feedback mediated by molecular targets within the same microvillus. Quantum bumps in trp mutants lacking the major class of light-sensitive channel were reduced in size (mean 3.5 pA) representing simultaneous activation of only one or two channels; however, a second rarer (10 %) class of large bump had an amplitude similar to wild-type (WT) bumps. Bumps in trpl mutants lacking the second class of light-sensitive channel were very similar to WT bumps, but with slightly slower decay times. In InaDP215 mutants, in which the association of the TRP channels with the INAD scaffolding molecule is disrupted, bumps showed a defect in quantum bump termination, but their amplitudes and latencies were near normal. PMID- 10747192 TI - Brain-derived neurotrophic factor blocks long-term depression in solitary neurones cultured from rat visual cortex. AB - 1. To address questions of whether long-term depression (LTD) in the visual cortex is expressed in pre- or postsynaptic sites, whether brain-derived neurotrophic factor (BDNF) exerts its LTD-blocking action without involvement of GABAergic inhibition, and whether the action of BDNF is pre- or postsynaptic, we observed excitatory postsynaptic currents (EPSCs) from solitary neurones cultured on glial microislands. In this preparation GABAergic inhibition is not involved and a group of synapses (autapses) which generate evoked EPSCs is thought to be the same as those generating spontaneous EPSCs. 2. A short depolarising voltage step to the soma generated Na+ spikes which were followed by autaptic EPSCs. When this somatic activation was paired with prolonged depolarisation for 100 ms to 30 mV and repeated at 1 Hz for 5 min, LTD was induced in all of the nine cells tested. Then, the frequency of spontaneous EPSCs decreased, but the amplitude did not change, suggesting that the site of LTD expression is presynaptic. 3. Application of BDNF at 50 ng ml-1 blocked the depression of evoked EPSCs and the decrease in the frequency of spontaneous EPSCs. An inhibitor for receptor tyrosine kinases, K252a, antagonised the action of BDNF, suggesting an involvement of BDNF receptors, TrkB. 4. These results suggest that BDNF prevents low-frequency inputs from inducing LTD of excitatory synaptic transmission through presynaptic mechanisms in the developing visual cortex. PMID- 10747193 TI - Effects of 1- or -adrenoceptor stimulation on work-loop and isometric contractions of isolated rat cardiac trabeculae. AB - 1. We studied the effects of alpha1- or beta-adrenoceptor stimulation on the contractility of isolated rat ventricular trabeculae at 24 degrees C using the work-loop technique, which simulates the cyclical changes in length and force that occur during the cardiac cycle. Some muscles were injected with fura-2 to monitor the intracellular Ca2+ transient. 2. Comparison of twitch records revealed that peak force was greater and was reached earlier in work-loop contractions than in corresponding isometric contractions. This was attributed to the changes in muscle length and velocity during work-loop contractions, since the Ca2+ transients were largely unaffected by the length changes. 3. Stimulation of alpha1-adrenoceptors (with 100 microM phenylephrine) increased net work, power production, the frequency for maximum work, and the frequency for maximum power production (fopt). The increase in net work was due to the positive inotropic effect of phenylephrine, which was similar at all frequencies investigated (0. 33 4.5 Hz). The increase in fopt was attributed to an abbreviation of twitch duration induced by alpha1-stimulation at higher frequencies (> 1 Hz), even though the twitch became longer at 0.33 Hz. 4. beta-Adrenoceptor stimulation (with 5 microM isoprenaline) produced marked increases in net work, power output, the frequency for net work, and fopt. These effects were attributed both to the positive inotropic effect of beta-stimulation, which was greater at higher frequencies, and to the reduction in twitch duration. beta-stimulation also abolished the frequency-dependent acceleration of twitch duration. 5. The increase in power output and fopt with alpha1- as well as beta-adrenoceptor stimulation suggested that both receptor types may contribute to the effects of catecholamines, released during stress or exercise, although the greater effects of beta-stimulation are likely to predominate. PMID- 10747194 TI - Rate-dependent changes of twitch force duration in rat cardiac trabeculae: a property of the contractile system. AB - 1. We examined the mechanisms for rate-dependent changes in twitch force duration by simultaneously measuring force and [Ca2+]i in rat cardiac trabeculae. 2. Peak force decreased when the rate of stimulation was increased from 0.2 to 0.5 Hz, whilst it increased from 1 to 2 Hz. Over the same range of frequencies, peak [Ca2+]i transients increased monotonically, whilst both force and [Ca2+]i transient duration were abbreviated. 3. Changes in peak force or peak [Ca2+]i transients were not responsible for the changes in force or [Ca2+]i transient duration. 4. The changes in twitch force and [Ca2+]i transient duration were completed roughly within one beat following an abrupt change in the rate of stimulation. 5. Rate-dependent changes resembled those observed with isoproterenol (isoprenaline) application. However, kinase inhibitors (i.e. K252 a, H-89, KN-62 and KN-93) had no effect on the rate-dependent changes of twitch force and [Ca2+]i transient kinetics, suggesting that protein kinase A (PKA), protein kinase PKG) and Ca2+-calmodulin-dependent protein kinase II (CaM/kinase II) were not responsible for these kinetic changes. 6. Despite the changes in twitch force and [Ca2+]i transient kinetics, the rate-limiting step for the rate dependent force relaxation still resides at the level of the contractile proteins. 7. Our results suggest that rate-dependent changes in force and [Ca2+]i transients do not depend on PKA or CaM/kinase II activity but might result from intrinsic features of the contractile and/or Ca2+-handling proteins. PMID- 10747195 TI - Regulation of force and unloaded sliding speed in single thin filaments: effects of regulatory proteins and calcium. AB - 1. Measurements of the unloaded sliding speed of and isometric force exerted on single thin filaments in in vitro motility assays were made to evaluate the role of regulatory proteins in the control of unloaded thin filament sliding speed and isometric force production. 2. Regulated actin filaments were reconstituted from rabbit F-actin, native bovine cardiac tropomyosin (nTm), and either native bovine cardiac troponin (nTn), troponin containing a TnC mutant, CBMII, in which the sole regulatory site in cardiac TnC (site II) is inactivated (CBMII-Tn), or troponin containing a point mutation in TnT (I79N, where isoleucine at position 79 is replaced with asparagine) associated with familial hypertrophic cardiomyopathy (FHC). 3. Addition of regulatory proteins to the thin filament increases both the unloaded sliding speed and the isometric force exerted by myosin heads on the thin filaments. 4. Variation of thin filament activation by varying [Ca2+] or the fraction of CBMII/TnC bound to the thin filament at pCa 5, had little effect on the unloaded filament sliding speed until the fraction of the thin filament containing calcium bound to TnC was less than 0.15. These results suggest that [Ca2+] primarily affects the number of attached and cycling crossbridges. 5. The presence of the FHC TnT mutant increased the thin filament sliding speed but reduced the isometric force that heavy meromyosin exerted on regulated thin filaments. These latter results, together with the increased sliding speed and isometric force seen in the presence of regulatory proteins, suggest that thin filament regulatory proteins exert significant allosteric effects on the interaction of crossbridges with the thin filament. PMID- 10747196 TI - Role of calcium stores and membrane voltage in the generation of slow wave action potentials in guinea-pig gastric pylorus. AB - 1. Intracellular recordings made in single bundle strips of a visceral smooth muscle revealed rhythmic spontaneous membrane depolarizations termed slow waves (SWs). These exhibited 'pacemaker' and 'regenerative' components composed of summations of more elementary events termed spontaneous transient depolarizations (STDs). 2. STDs and SWs persisted in the presence of tetrodotoxin, nifedipine and ryanodine, and upon brief exposure to Ca2+-free Cd2+-containing solutions; they were enhanced by ACh and blocked by BAPTA AM, cyclopiazonic acid and caffeine. 3. SWs were also inhibited in heparin-loaded strips. SWs were observed over a wide range of membrane potentials (e.g. -80 to -45 mV) with increased frequencies at more depolarized potentials. 4. Regular spontaneous SW activity in this preparation began after 1-3 h superfusion of the tissue with physiological saline following the dissection procedure. Membrane depolarization applied before the onset of this activity induced bursts of STD-like events (termed the 'initial' response) which, when larger than threshold levels initiated regenerative responses. The combined initial-regenerative waveform was termed the SW-like action potential. 5. Voltage-induced responses exhibited large variable latencies (typical range 0.3-4 s), refractory periods of approximately 11 s and a pharmacology that was indistinguishable from those of STDs and spontaneous SWs. 6. The data indicate that SWs arise through more elementary inositol 1,4,5 trisphosphate (IP3) receptor-induced Ca2+ release events which rhythmically synchronize to trigger regenerative Ca2+ release and induce inward current across the plasmalemma. The finding that action potentials, which were indistinguishable from SWs, could be evoked by depolarization suggests that membrane potential modulates IP3 production. Voltage feedback on intracellular IP3-sensitive Ca2+ release is likely to have a major influence on the generation and propagation of SWs. PMID- 10747197 TI - Effect of motor unit recruitment on functional vasodilatation in hamster retractor muscle. AB - 1. The effect of motor unit recruitment on functional vasodilatation was investigated in hamster retractor muscle. Recruitment (i.e. peak tension) was controlled with voltage applied to the spinal accessory nerve (high = maximum tension; intermediate = approximately 50% maximum; low = approximately 25% maximum). Vasodilatory responses (diameter times time integral, DTI) to rhythmic contractions (1 per 2s for 65s) were evaluated in first, second and third order arterioles and in feed arteries. Reciprocal changes in duty cycle (range, 2.5 25%) effectively maintained the total active tension (tension times time integral, TTI) constant across recruitment levels. 2. With constant TTI and stimulation frequency (40 Hz), DTI in all vessels increased with motor unit recruitment. DTI increased from distal arterioles up through proximal feed arteries. 3. To determine whether the effect of recruitment on DTI was due to increased peak tension, the latter was controlled with stimulation frequency (15, 20 and 40 Hz) during maximum (high) recruitment. With constant TTI, DTI then decreased as peak tension increased. 4. To explore the interaction between recruitment and duty cycle on DTI, each recruitment level was applied at 2.5, 10 and 20 % duty cycle (at 40 Hz). For a given increase in TTI, recruitment had a greater effect on DTI than did duty cycle. 5. Functional vasodilatation in response to rhythmic contractions is facilitated by motor unit recruitment. Thus, vasodilatory responses are determined not only by the total tension produced, but also by the number of active motor units. PMID- 10747198 TI - Regional blood flow and nociceptive stimuli in rabbits: patterning by medullary raphe, not ventrolateral medulla. AB - 1. Regional blood flow was measured with Doppler ultrasonic probes in anaesthetized rabbits. We used focal microinjections of pharmacological agents to investigate medullary pathways mediating ear pinna vasoconstriction elicited by electrical stimulation of the spinal tract of the trigeminal nerve or by pinching the lip, and pathways mediating mesenteric vasoconstriction elicited by electrical stimulation of the afferent abdominal vagus nerve. 2. Bilateral injection of kynurenate into the rostral ventrolateral medulla reduced arterial pressure and prevented the mesenteric vasoconstriction and the rise in arterial pressure elicited by abdominal vagal stimulation. However, kynurenate did not prevent ear pinna vasoconstriction or the fall in pressure elicited by trigeminal tract stimulation. Similar injections of muscimol also failed to prevent the trigeminally elicited cardiovascular changes. 3. Injections of kynurenate into the raphe-parapyramidal area did not diminish trigeminally elicited ear vasoconstriction or the depressor response. However, injections of muscimol substantially reduced or abolished the trigeminally elicited ear vasoconstriction, without affecting the depressor response. Raphe-parapyramidal muscimol injections also entirely abolished ear vasoconstriction elicited by pinching the rabbit's lip. 4. The trigeminal depressor response does not depend on either the rostral ventrolateral medulla or the raphe-parapyramidal region. 5. Mesenteric vasoconstriction elicited by stimulation of the afferent abdominal vagus nerve is mediated via the rostral ventrolateral medulla, but ear vasoconstriction elicited by lip pinch or by stimulation of the trigeminal tract is mediated by the raphe-parapyramidal region. Our study is the first to suggest a brainstem pathway mediating cutaneous vasoconstriction elicited by nociceptive stimulation. PMID- 10747199 TI - Changes in muscle strength, muscle fibre size and myofibrillar gene expression after immobilization and retraining in humans. AB - 1. Changes in muscle strength, vastus lateralis fibre characteristics and myosin heavy-chain (MyoHC) gene expression were examined in 48 men and women following 3 weeks of knee immobilization and after 12 weeks of retraining with 1866 eccentric, concentric or mixed contractions. 2. Immobilization reduced eccentric, concentric and isometric strength by 47 %. After 2 weeks of spontaneous recovery there still was an average strength deficit of 11 %. With eccentric and mixed compared with concentric retraining the rate of strength recovery was faster and the eccentric and isometric strength gains greater. 3. Immobilization reduced type I, IIa and IIx muscle fibre areas by 13, 10 and 10 %, respectively and after 2 weeks of spontaneous recovery from immobilization these fibres were 5 % smaller than at baseline. Hypertrophy of type I, IIa and IIx fibres relative to baseline was 10, 16 and 16 % after eccentric and 11, 9 and 10 % after mixed training (all P < 0.05), exceeding the 4, 5 and 5 % gains after concentric training. Type IIa and IIx fibre enlargements were greatest after eccentric training. 4. Total RNA/wet muscle weight and ty I, IIa and IIx MyoHC mRNA levels did not change differently after immobilization and retraining. Immobilization downregulated the expression of type I MyoHC mRNA to 0.72-fold of baseline and exercise training upregulated it to 0.95 of baseline. No changes occurred in type IIa MyoHC mRNA. Immobilization and exercise training upregulated type IIx MyoHC mRNA 2.9-fold and 1.2-fold, respectively. For the immobilization segment, type I, IIa and IIx fibre area and type I, IIa and IIx MyoHC mRNA correlated (r = 0.66, r = 0.07 and r = 0.71, respectively). 5. The present data underscore the role muscle lengthening plays in human neuromuscular function and adaptation. PMID- 10747200 TI - Blood flow and oxygenation in peritendinous tissue and calf muscle during dynamic exercise in humans. AB - 1. Circulation around tendons may act as a shunt for muscle during exercise. The perfusion and oxygenation of Achilles' peritendinous tissue was measured in parallel with that of calf muscle during exercise to determine (1) whether blood flow is restricted in peritendinous tissue during exercise, and (2) whether blood flow is coupled to oxidative metabolism. 2. Seven individuals performed dynamic plantar flexion from 1 to 9 W. Radial artery and popliteal venous blood were sampled for O2, peritendinous blood flow was determined by 133Xe-washout, calf blood flow by plethysmography, cardiac output by dye dilution, arterial pressure by an arterial catheter-transducer, and muscle and peritendinous O2 saturation by spatially resolved spectroscopy (SRS). 3. Calf blood flow rose 20-fold with exercise, reaching 44 +/- 7 ml (100 g)-1 min-1 (mean +/- s.e.m. ) at 9 W, while Achilles' peritendinous flow increased (7-fold) to 14 +/- 4 ml (100 g)-1 min-1, which was 18 % of the maximal flow established during reactive hyperaemia. SRS-O2 saturation fell both in muscle (from 66 +/- 2 % at rest to 57 +/- 3 %, P < 0.05) and in peritendinous regions (58 +/- 4 to 52 +/- 4 %, P < 0.05) during exercise along with a rise in leg vascular conductance and microvascular haemoglobin volume, despite elevated systemic vascular resistance. 4. The parallel rise in calf muscle and peritendinous blood flow and fall in O2 saturation during exercise indicate that blood flow is coupled to oxidative metabolism in both tissue regions. Increased leg vascular conductance accompanied by elevated microvascular haemoglobin volume reflect vasodilatation in both muscle and peritendinous regions. However, peak exercise peritendinous blood flow reaches only approximately 20 % of its maximal blood flow capacity. PMID- 10747202 TI - Tissue-specific Bcl-2 protein partners in apoptosis: An ovarian paradigm. AB - Apoptosis is an essential physiological process by which multicellular organisms eliminate superfluous cells. An expanding family of Bcl-2 proteins plays a pivotal role in the decision step of apoptosis, and the differential expression of Bcl-2 members and their binding proteins allows the regulation of apoptosis in a tissue-specific manner mediated by diverse extra- and intracellular signals. The Bcl-2 proteins can be divided into three subgroups: 1) antiapoptotic proteins with multiple Bcl-2 homology (BH) domains and a transmembrane region, 2) proapoptotic proteins with the same structure but missing the BH4 domain, and 3) proapoptotic ligands with only the BH3 domain. In the mammalian ovary, a high rate of follicular cell apoptosis continues during reproductive life. With the use of the yeast two-hybrid system, the characterization of ovarian Bcl-2 genes serves as a paradigm to understand apoptosis regulation in a tissue-specific manner. We identified Mcl-1 as the main ovarian antiapoptotic Bcl-2 protein, the novel Bok (Bcl-2-related ovarian killer) as the proapoptotic protein, as well as BOD (Bcl-2-related ovarian death agonist) and BAD as the proapoptotic ligands. The activity of the proapoptotic ligand BAD is regulated by upstream follicle survival factors through its binding to constitutively expressed 14-3-3 or hormone-induced P11. In contrast, the channel-forming Mcl-1 and Bok regulate cytochrome c release and, together with the recently discovered Diva/Boo, control downstream apoptosis-activating factor (Apaf)-1 homologs and caspases. Elucidation of the role of Bcl-2 members and their interacting proteins in the tissue-specific regulation of apoptosis could facilitate an understanding of normal physiology and allow the development of new therapeutic approaches for pathological states. PMID- 10747201 TI - The voltage sensor in voltage-dependent ion channels. AB - In voltage-dependent Na, K, or Ca channels, the probability of opening is modified by the membrane potential. This is achieved through a voltage sensor that detects the voltage and transfers its energy to the pore to control its gate. We present here the theoretical basis of the energy coupling between the electric field and the voltage, which allows the interpretation of the gating charge that moves in one channel. Movement of the gating charge constitutes the gating current. The properties are described, along with macroscopic data and gating current noise analysis, in relation to the operation of the voltage sensor and the opening of the channel. Structural details of the voltage sensor operation were resolved initially by locating the residues that make up the voltage sensor using mutagenesis experiments and determining the number of charges per channel. The changes in conformation are then analyzed based on the differential exposure of cysteine or histidine-substituted residues. Site directed fluorescence labeling is then analyzed as another powerful indicator of conformational changes that allows time and voltage correlation of local changes seen by the fluorophores with the global change seen by the electrophysiology of gating currents and ionic currents. Finally, we describe the novel results on lanthanide-based resonance energy transfer that show small distance changes between residues in the channel molecule. All of the electrophysiological and the structural information are finally summarized in a physical model of a voltage dependent channel in which a change in membrane potential causes rotation of the S4 segment that changes the exposure of the basic residues from an internally connected aqueous crevice at hyperpolarized potentials to an externally connected aqueous crevice at depolarized potentials. PMID- 10747203 TI - Development of specific connectivity between premotor neurons and motoneurons in the brain stem and spinal cord. AB - Astounding progress has been made during the past decade in understanding the general principles governing the development of the nervous system. An area of prime physiological interest that is being elucidated is how the neural circuitry that governs movement is established. The concerted application of molecular biological, anatomical, and electrophysiological techniques to this problem is yielding gratifying insight into how motoneuron, interneuron, and sensory neuron identities are determined, how these different neuron types establish specific axonal projections, and how they recognize and synapse upon each other in patterns that enable the nervous system to exercise precise control over skeletal musculature. This review is an attempt to convey to the physiologist some of the exciting discoveries that have been made, within a context that is intended to link molecular mechanism to behavioral realization. The focus is restricted to the development of monosynaptic connections onto skeletal motoneurons. Principal topics include the inductive mechanisms that pattern the placement and differentiation of motoneurons, Ia sensory afferents, and premotor interneurons; the molecular guidance mechanisms that pattern the projection of premotor axons in the brain stem and spinal cord; and the precision with which initial synaptic connections onto motoneurons are established, with emphasis on the relative roles played by cellular recognition versus electrical activity. It is hoped that this review will provide a guide to understanding both the existing literature and the advances that await this rapidly developing topic. PMID- 10747204 TI - Human body composition: in vivo methods. AB - In vivo methods used to study human body composition continue to be developed, along with more advanced reference models that utilize the information obtained with these technologies. Some methods are well established, with a strong physiological basis for their measurement, whereas others are much more indirect. This review has been structured from the methodological point of view to help the reader understand what can be examined with each technique. The associations between the various in vivo methods (densitometry, dilution, bioelectrical impedance and conductance, whole body counting, neutron activation, X-ray absorptiometry, computer tomography, and magnetic resonance imaging) and the five level multicompartment model of body composition are described, along with the limitations and advantages of each method. This review also provides an overview of the present status of this field of research in human biology, including examples of reference body composition data for infants, children, adolescents, and adults. PMID- 10747205 TI - Carbon dioxide transport and carbonic anhydrase in blood and muscle. AB - CO(2) produced within skeletal muscle has to leave the body finally via ventilation by the lung. To get there, CO(2) diffuses from the intracellular space into the convective transport medium blood with the two compartments, plasma and erythrocytes. Within the body, CO(2) is transported in three different forms: physically dissolved, as HCO(3)(-), or as carbamate. The relative contribution of these three forms to overall transport is changing along this elimination pathway. Thus the kinetics of the interchange have to be considered. Carbonic anhydrase accelerates the hydration/dehydration reaction between CO(2), HCO(3)(-), and H(+). In skeletal muscle, various isozymes of carbonic anhydrase are localized within erythrocytes but are also bound to the capillary wall, thus accessible to plasma; bound to the sarcolemma, thus producing catalytic activity within the interstitial space; and associated with the sarcoplasmic reticulum. In some fiber types, carbonic anhydrase is also present in the sarcoplasm. In exercising skeletal muscle, lactic acid contributes huge amounts of H(+) and by these affects the relative contribution of the three forms of CO(2). With a theoretical model, the complex interdependence of reactions and transport processes involved in CO(2) exchange was analyzed. PMID- 10747206 TI - Neurotoxins affecting neuroexocytosis. AB - Nerve terminals are specific sites of action of a very large number of toxins produced by many different organisms. The mechanism of action of three groups of presynaptic neurotoxins that interfere directly with the process of neurotransmitter release is reviewed, whereas presynaptic neurotoxins acting on ion channels are not dealt with here. These neurotoxins can be grouped in three large families: 1) the clostridial neurotoxins that act inside nerves and block neurotransmitter release via their metalloproteolytic activity directed specifically on SNARE proteins; 2) the snake presynaptic neurotoxins with phospholipase A(2) activity, whose site of action is still undefined and which induce the release of acethylcholine followed by impairment of synaptic functions; and 3) the excitatory latrotoxin-like neurotoxins that induce a massive release of neurotransmitter at peripheral and central synapses. Their modes of binding, sites of action, and biochemical activities are discussed in relation to the symptoms of the diseases they cause. The use of these toxins in cell biology and neuroscience is considered as well as the therapeutic utilization of the botulinum neurotoxins in human diseases characterized by hyperfunction of cholinergic terminals. PMID- 10747209 TI - You must cross the bridge which spans the river of time. PMID- 10747208 TI - Regulation of contraction in striated muscle. AB - Ca(2+) regulation of contraction in vertebrate striated muscle is exerted primarily through effects on the thin filament, which regulate strong cross bridge binding to actin. Structural and biochemical studies suggest that the position of tropomyosin (Tm) and troponin (Tn) on the thin filament determines the interaction of myosin with the binding sites on actin. These binding sites can be characterized as blocked (unable to bind to cross bridges), closed (able to weakly bind cross bridges), or open (able to bind cross bridges so that they subsequently isomerize to become strongly bound and release ATP hydrolysis products). Flexibility of the Tm may allow variability in actin (A) affinity for myosin along the thin filament other than through a single 7 actin:1 tropomyosin:1 troponin (A(7)TmTn) regulatory unit. Tm position on the actin filament is regulated by the occupancy of NH-terminal Ca(2+) binding sites on TnC, conformational changes resulting from Ca(2+) binding, and changes in the interactions among Tn, Tm, and actin and as well as by strong S1 binding to actin. Ca(2+) binding to TnC enhances TnC-TnI interaction, weakens TnI attachment to its binding sites on 1-2 actins of the regulatory unit, increases Tm movement over the actin surface, and exposes myosin-binding sites on actin previously blocked by Tm. Adjacent Tm are coupled in their overlap regions where Tm movement is also controlled by interactions with TnT. TnT also interacts with TnC-TnI in a Ca(2+)-dependent manner. All these interactions may vary with the different protein isoforms. The movement of Tm over the actin surface increases the "open" probability of myosin binding sites on actins so that some are in the open configuration available for myosin binding and cross-bridge isomerization to strong binding, force-producing states. In skeletal muscle, strong binding of cycling cross bridges promotes additional Tm movement. This movement effectively stabilizes Tm in the open position and allows cooperative activation of additional actins in that and possibly neighboring A(7)TmTn regulatory units. The structural and biochemical findings support the physiological observations of steady-state and transient mechanical behavior. Physiological studies suggest the following. 1) Ca(2+) binding to Tn/Tm exposes sites on actin to which myosin can bind. 2) Ca(2+) regulates the strong binding of M.ADP.P(i) to actin, which precedes the production of force (and/or shortening) and release of hydrolysis products. 3) The initial rate of force development depends mostly on the extent of Ca(2+) activation of the thin filament and myosin kinetic properties but depends little on the initial force level. 4) A small number of strongly attached cross bridges within an A(7)TmTn regulatory unit can activate the actins in one unit and perhaps those in neighboring units. This results in additional myosin binding and isomerization to strongly bound states and force production. 5) The rates of the product release steps per se (as indicated by the unloaded shortening velocity) early in shortening are largely independent of the extent of thin filament activation ([Ca(2+)]) beyond a given baseline level. However, with a greater extent of shortening, the rates depend on the activation level. 6) The cooperativity between neighboring regulatory units contributes to the activation by strong cross bridges of steady-state force but does not affect the rate of force development. 7) Strongly attached, cycling cross bridges can delay relaxation in skeletal muscle in a cooperative manner. 8) Strongly attached and cycling cross bridges can enhance Ca(2+) binding to cardiac TnC, but influence skeletal TnC to a lesser extent. 9) Different Tn subunit isoforms can modulate the cross-bridge detachment rate as shown by studies with mutant regulatory proteins in myotubes and in in vitro motility assays. (ABSTRACT TRUNCATED) PMID- 10747210 TI - Patient-controlled epidural analgesia with morphine or morphine plus ketamine for post-operative pain relief. AB - Sixty patients were randomly assigned to two equal groups. Group I received epidural morphine 1 mg after surgery and used a patient-controlled analgesia device programmed to deliver morphine 0. 2 mg h-1, 0.2 mg per bolus. Group II received an epidural loading dose of morphine 1 mg plus ketamine 5 mg and used a patient-controlled analgesia device programmed to deliver morphine 0. 2 mg+ketamine 0.5 mg h-1, morphine 0.2 mg+ketamine 0.5 mg per bolus with a lockout time of 10 min. The mean morphine consumption was 8. 6+/-0.7 mg for group I and 6.2+/-0.2 mg for group II. Although group II utilized significantly less morphine (P < 0.05), pain relief was significantly better in group II than in group I (P < 0.05) in the first 3 h. Vomiting occurred more frequently in group I (26%) than in group II (13%). The frequency and severity of pruritus and level of sedation were similar in the two groups. These findings suggest that patient-controlled epidural analgesia with morphine plus ketamine may provide effective analgesia with a lesser dose of morphine and fewer subsequent side effects, compared with patient-controlled epidural analgesia with morphine alone after lower abdominal surgery. PMID- 10747211 TI - Factors associated with post-operative myocardial ischaemia in elderly patients undergoing major non-cardiac surgery. AB - Forty patients (> 65 years) undergoing hip arthroplasty or peripheral vascular surgery both associated with high risk for post-operative myocardial ischaemia were randomized to receive either spinal or general anaesthesia. Ambulatory ECG recording (Holter) until the third post-operative morning, a daily 12-lead ECG and serum creatine kinase and troponine concentraItions were obtained. The number of ischaemic episodes, total duration of ischaemia and ischaemic minutes per hour were noted for each patient peri-operatively. Sixteen of the patients (40%) had post-operative myocardial ischaemia. An intra-operative increase in the plasma concentration of norepinephrine but not epinephrine was detected in the patients who later developed post-operative myocardial ischaemia. The increase in plasma norepinIephrine concentrations correlated with the decrease in core temperature. The type of anaesthesia had no effect on the incidence of myocardial ischaemia during or after surgery. Our results suggests that intra-operatively decreased core temperature and the increase in plasma concentration of norepinephrine probably caused peripheral vasoconstriction leading to latent cardiac dysfunction. These events should be avoided in the patients at risk of post operative cardiac ischaemia. PMID- 10747212 TI - Efficacy and side effects of tramadol versus oxycodone for patient-controlled analgesia after maxillofacial surgery. AB - Tramadol, a weak opioid mu-receptor agonist, may have a favourable potency and side effect profile for intravenous patient-controlled analgesia (PCA). In a prospective, double-blind, randomized study involving 54 patients, tramadol was compared with oxycodone in PCA after maxillofacial surgery. All the patients were given diclofenac sodium 1 mg kg-1 intramuscularly and dexamethasone 8 mg twice a day. Post-operatively patients received tramadol or oxycodone by a PCA apparatus (lockout 5 min, tramadol 0.3 mg kg-1 bolus, oxycodone 0.03 mg kg-1 bolus). During the immediate recovery period, opioid was administered i.v. in a double-blind fashion, either tramadol 10 mg or oxycodone 1 mg increments until the pain control was judged to be satisfactory by the patient. Pain was assessed at rest and during activity (mouth opening) before and after loading, at 2 h after commencing the PCA, as well as at 21.00 and at 09.00 hours on the following morning. Side effects were recorded. The potency ratio of tramadol to oxycodone was found to be approximately 8:1. There was no significant difference between the groups in the VAS scores for pain. No respiratory depression was identified. Tramadol was found to provide adequate analgesia after maxillofacial surgery without risk of respiratory depression. However, the incidence of nausea was slightly greater in the tramadol group than in the oxycodone group (44% vs. 28%, NS). PMID- 10747213 TI - In-flight medical emergencies: response of anaesthetists who were passengers on commercial flights. AB - All consultants and trainees in anaesthesia in a large teaching hospital were surveyed. Details of the number of flights per year and details of any medical emergencies in which they had been involved were recorded. The mean number of flights per year was 7.1 domestic and 3.4 international. Of the 45 anaesthetists surveyed, 14 had dealt with emergencies in flight, four had dealt with more than one. The minor emergencies (12) included transient ischaemic attacks, abdominal pain and otitis media. The seven serious events included seizures, angina, hypoglycaemic coma, respiratory arrest and two fatal cardiac arrests. No flights were diverted. On only two occasions were their medical qualifications checked. Requests for documentation were unusual. On several occasions the equipment which was available was inadequate. All doctors that responded were insured in the UK and most stated that they would assist Americans on American airlines. Medical emergencies were more likely on long haul flights. PMID- 10747215 TI - Referees for 1999 PMID- 10747214 TI - Idiopathic prolonged QT interval and QT dispersion: the effects of propofol during implantation of cardioverter-defibrillator. AB - Local anaesthesia combined with conscious sedation is becoming a popular technique for implantation of cardioverter-defibrillator devices. Propofol was given to provide loss of consciousness during defibrillation shock administration, for induced ventricular fibrillation testing. Propofol was found to decrease QT interval and QT dispersion in two patients with idiopathic prolonged QT interval and QT dispersion. The findings of the procedure are reported. PMID- 10747216 TI - Antagonistic effect of Lactobacillus acidophilus, Saccharomyces boulardii and Escherichia coli combinations against experimental infections with Shigella flexneri and Salmonella enteritidis subsp. typhimurium in gnotobiotic mice. AB - Lactobacillus acidophilus, Saccharomyces boulardii and Escherichia coli are probiotic strains used individually to protect against enteropathogenic agents. In order to determine if a synergistic effect of the individual protective mechanisms ordinarily attributed to each of these biotherapeutic agents is possible, we orally administered Lact. acidophilus H2B20, S. boulardii and E. coli EMO (LSE) to germfree mice. Ten days after colonization of the digestive tract, groups of animals associated (experimental) or not (control) with LSE were challenged orally with streptomycin resistant (Sfr) or streptomycin sensitive (Sfs) Shigella flexneri strains or Salmonella enteritidis subsp. typhimurium. Bacterial counts in faeces from experimental mice showed that the Sfr strain was eliminated 11 d after challenge while Sfs and S. enteritidis subsp. typhimurium colonized the digestive tract and continued to be present at high population levels (108 CFU g-1 of faeces), which is similar to that observed in control animals. All possible di- and monoassociations of the three probiotics with gnotobiotic mice were also performed before experimental oral infection with Sfr. The data showed that antagonism was obtained only when E. coli EMO was present. Different sensitivity of Sh. flexneri Sfr and Sfs to E. coli EMO antagonism could be explained by the different generation times between Sfr and Sfs, as shown by colonization kinetic experiments in the digestive tract of gnotobiotic mice. PMID- 10747217 TI - Re-assessment of the influence of yeast strain and environmental factors on glycerol production in wine. AB - Increasing glycerol production is of concern for wine-makers in improving the quality of certain wines. We have compared the impact of strain and relevant environmental factors influencing glycerol production under the same conditions, i.e. standardized conditions simulating enological fermentation. The glycerol production of 19 industrial wine strains ranged from 6.4 to 8.9 g l-1 and varied significantly between strains. The production of acetate and succinate was also found to differ substantially depending on the strain but no significant strain dependent variation was observed for acetaldehyde. Interestingly, high glycerol production was not correlated to high production of acetate or acetaldehyde, which are undesirable in wine. A detailed study with two low or two high glycerol producing strains showed that temperature and the initial concentration of nitrogen had little effect on the amount of glycerol formed, although agitation or a nitrogen source composed mainly of ammoniacal nitrogen slightly enhanced glycerol production. The influence of environmental factors remained minor while the predominant factor for glycerol variability in wine was attributed to the strain. Taking into account wine-making constraints, the results indicate that achieving a high glycerol content in wine requires the selection or improvement of yeast strains rather than the control of growth and cultivation conditions. PMID- 10747218 TI - Screening and production of rhamnolipids by Pseudomonas aeruginosa 47T2 NCIB 40044 from waste frying oils. AB - World production of oils and fats is about 2.5 million tonnes, 75% of which are derived from plants. Most of them are used in the food industry for the manufacture of different products, or directly as salad oil. Great quantities of waste are generated by the oil and fat industries: residual oils, tallow, marine oils, soap stock, frying oils. It is well known that the disposal of wastes is a growing problem and new alternatives for the use of fatty wastes should be studied. Used frying oils, due to their composition, have great potential for microbial growth and transformation. The use of economic substrates such as hydrophobic wastes meets one of the requirements for a competitive process for biosurfactant production. In the Mediterranean countries, the most used vegetable oils are sunflower and olive oil. Here we present a screening process is described for the selection of micro-organism strains with the capacity to grow on these frying oils and accumulate surface-active compounds in the culture media. From the 36 strains screened, nine Pseudomonas strains decreased the surface tension of the medium to 34-36 mN/M; the emulsions with kerosene remained stable for three months. Two Bacillus strains accumulated lipopeptide and decreased the surface tension to 32-34 mN/m. Strain Ps. aeruginosa 47T2 was selected for further studies. The effect of nitrogen and a C/N of 8. 0 gave a final production of rhamnolipid of 2.7 g l-1 as rhamnose, and a production yield of 0.34 g g-1. PMID- 10747219 TI - The effect of combinations of Fusarium mycotoxins (deoxynivalenol, zearalenone and fumonisin B1) on growth of brewing yeasts. AB - The interactive effect of combinations of the Fusarium mycotoxins deoxynivalenol (DON), zearalenone (ZEA) and fumonisin B1 (FB1) on growth of brewing yeasts was examined. Yeast growth was assessed by measurement of dry weight or relative growth, cell number, viability and conductance change of the growth medium using direct and indirect methods. The interactive effect of a combination of these mycotoxins was subject to the ratio of toxins in the mixture and the toxicity of individual toxins on yeast growth. When a combination of mycotoxins at low concentration was added into the growth medium, no significant inhibitory effect on growth was observed compared to controls. However, when a combination of high concentrations of DON and ZEA which individually inhibited yeast growth was examined, the interactive effect was shown to pass from antagonism to synergism depending on the ratio of the toxins in the mixture. As a synergistic interaction between these Fusarium mycotoxins was observed only at high concentrations, which were far higher than would be expected in good quality grain, they are not a concern when related to yeast growth under the brewing conditions studied. PMID- 10747220 TI - Use of enzymatic methods for rapid enumeration of coliforms in freshwaters. AB - Rapid enumeration methods based on the enzymatic hydrolysis of 4 methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl-beta-D-glucuronide were optimized for freshwaters. The enzymes beta-D-galactosidase (GALase) and beta-D-glucuronidase (GLUase) were shown to be already induced in freshwaters when tested, respectively, with the inducers isopropyl-beta-D thiogalactopyranoside and methyl-beta-D-glucuronide. Both enzymatic activities were compared, respectively, with plate counts of total and faecal coliforms in freshwaters. Enzymatic methods and reference plate counts were significantly correlated in log-log plots. Moreover, the GLUase method allowed the detection of viable (presenting a detectable GLUase activity) but nonculturable Escherichia coli. PMID- 10747221 TI - Combination of hydrostatic pressure and lacticin 3147 causes increased killing of Staphylococcus and Listeria. AB - The use of hydrostatic pressure and lacticin 3147 treatments were evaluated in milk and whey with a view to combining both treatments for improving the quality of minimally processed dairy foods. The system was evaluated using two foodborne pathogens: Staphylococcus aureus ATCC6538 and Listeria innocua DPC1770. Trials against Staph. aureus ATCC6538 were performed using concentrated lacticin 3147 prepared from culture supernatant. The results demonstrated a more than additive effect when both treatments were used in combination. For example, the combination of 250 MPa (2.2 log reduction) and lacticin 3147 (1 log reduction) resulted in more than 6 logs of kill. Similar results were obtained when a foodgrade powdered form of lacticin 3147 (developed from a spray dried fermentatation of reconstituted demineralized whey powder) was evaluated for the inactivation of L. innocua DPC1770. Furthermore, it was observed that treatment of lacticin 3147 preparations with pressures greater than 400 MPa yielded an increase in bacteriocin activity (equivalent to a doubling of activity). These results indicate that a combination of high pressure and lacticin 3147 may be suitable for improving the quality of minimally processed foods at lower hydrostatic pressure levels. PMID- 10747222 TI - The effect of a commercial starter culture addition on the ripening of an artisanal goat's cheese (Cameros cheese). AB - The evolution of physicochemical parameters, and the most important microbial groups, were determined for the following three batches of 'Cameros' goat's milk cheese during ripening: Batch R elaborated with raw milk, Batch RS elaborated with raw milk and with the addition of a starter culture, and Batch PS elaborated with pasteurized milk and with the addition of the same culture. No differences in total solids (TS) or in the content of NaCl, fat and total nitrogen (expressed as percentages of TS) were found during the ripening. The pH, fat acidity and non protein nitrogen (NPN, expressed as a percentage of TN) showed significant differences between the batches. The inoculated batches showed the fastest drop in pH at the beginning of the ripening period, but the cheeses of Batch R showed a higher degree of lipolysis and proteolysis. The addition of a starter influenced the microbiological quality of the cheeses. Differences in the counts of Enterobacteriaceae and faecal coliforms were found between Batches R and RS after 15 days. Staphylococcus aureus increased in number during the early period of ripening and attained a population above 6 log cfu g-1 in Batch R in the period from 5 to 10 days. However, enterotoxins were not detected in this Batch. Batch R showed lower values of lactic acid bacteria at the beginning of the ripening period, but no significant differences were found between batches in the period from 5 to 15 days of ripening. At the beginning of the ripening, Lactococcus was the main lactic acid bacteria, with L. lactis lactis being predominant. After 15 days, the lactic acid bacteria counts decreased in the three batches, especially in the cheeses of Batch PS (only 2.2 log cfu g-1 was found at 60 days), as lactococci (the only lactic acid bacteria present in Batch PS) are incapable of growing under the conditions found in cheeses at the end of their ripening period. At this time, Lactobacillus was the predominant genus in Batches R and RS, with L. plantarum predominant. No lactococci were found from day 30 in Batch R and from day 40 in Batch RS. The cheeses of Batch RS received the most favourable scores from the tasting panel for all attributes judged: cut appearance, colour, aroma, taste, texture and general acceptance. PMID- 10747223 TI - Improvement of the detection of Listeria monocytogenes by the application of ALOA, a diagnostic, chromogenic isolation medium. AB - A new selective agar medium, ALOA, for the selective and differential isolation of Listeria monocytogenes has been evaluated. All stressed cultures of L. monocytogenes serovars tested grew on the medium as bluish colonies surrounded by a distinctive opaque halo and gave a productivity ratio of at least 0.95. Non pathogenic Listeria sp. produced bluish colonies without a halo as was also the case for some enterococci and bacilli. Special attention must be paid to some Bacillus cereus strains and L. ivanovii since their colony appearance can be misleading. Only some unidentified listeria-like bacteria gave false-positive results. ALOA detected 4. 3% more positives from naturally contaminated dairy and meat samples compared with the ISO procedure when used with GenprobeTM or VidasTM for confirmation of presumptive colonies; 13.9% false negatives were found compared with 38.9% using PALCAM/Oxford. ALOA was also clearly superior to Oxford and PALCAM when samples containing both L. monocytogenes and L. innocua were examined. The introduction of ALOA in standard isolation procedures as an additional medium would enhance the detection ratio and reduce the time and cost of analysis for L. monocytogenes. PMID- 10747224 TI - Evaluation of plate count methods for determination of maximum specific growth rate in mixed microbial communities, and its possible application for diversity assessment. AB - Two plate count methods are proposed for direct assessment of the maximum specific growth rate (microm) of bacteria in mixed communities. An estimate of microm of individual colonies is obtained by plating samples on an agar medium and determining either the time required to form macroscopically visible colonies, Tv (diameter < or =0.2 mm), or the linear radial growth rate, Kr, of single colonies. In accordance with theoretical models, a linear relationship was found between microm determined in liquid culture and 1/Tv, and between microm and Kr. Empirical relationships were established for these relationships. The time required to form a visible colony was 17 +/- 9 h longer for cells in the stationary growth phase, whereas the linear radial growth rate was not affected by the physiological state of the cells. The proposed plate count methods are simple and applicable for describing the community structure, and for estimating the frequency distribution of maximum specific growth rates in mixed communities. By using this frequency distribution it is possible to calculate diversity indexes and to assign a microbial community a position on an r/K-gradient. PMID- 10747225 TI - Anti-Listeria effect of enterocin A, produced by cheese-isolated Enterococcus faecium EFM01, relative to other bacteriocins from lactic acid bacteria. AB - Enterocin A produced by Enterococcus faecium EFM01 displayed a narrow antimicrobial spectrum, mainly directed against Listeria spp. In particular, the bacteriocin was extremely active against 13 Listeria monocytogenes strains. This high specificity of action of enterocin A for Listeria spp. relative to lactic acid bacteria, together with its broad range of activity from pH 4.0 to pH 9.0, are factors which may be of great interest with respect to the potential antilisterial use of this bacteriocin in fermented foods. Assessment of the effect of enterocin A concentration on the extent and kinetics of bactericidal activity on L. monocytogenes Lm 6 (107 cfu ml-1 in culture broth), suggested that viability losses of higher than 5 log10, and time intervals necessary for maximum loss of viability of less than 2 h, could not be obtained. Moreover, it was shown that both parameters are closely dependent on the Listeria strain used. On the other hand, at concentrations inducing destruction of approximately 2 log10 cycles, maximum loss of viability was achieved within time intervals which varied widely from one lactic acid bacteria bacteriocin to another. PMID- 10747226 TI - Purification and characterization of an extracellular feruloyl esterase from the thermophilic anaerobe Clostridium stercorarium. AB - Feruloyl esterases act as accessory enzymes for the complete saccharification of plant cell wall hemicelluloses. Although many fungal feruloyl esterases have been purified and characterized, few bacterial phenolic acid esterases have been characterized. This study shows the extracellular production of a feruloyl esterase by the thermophilic anaerobe Clostridium stercorarium when grown on birchwood xylan. The feruloyl esterase was purified 500-fold in successive steps involving ultrafiltration, preparative isoelectric focusing and column chromatography by anion exchange, gel filtration and hydrophobic interaction. The purified enzyme released ferulic, rho-coumaric, caffeic and sinapinic acid from the respective methyl esters. The purified enzyme also released ferulic acid from a de-starched wheat bran preparation. At pH 8.0 and 65 degrees C, the Km and Vmax values for the hydrolysis of methyl ferulate were 0.04 mmol l-l and 131 micromol min-1 mg-1, respectively; the respective values for methyl coumarate were 0.86 mmol l-l and 18 micromol min-1 mg-1. The purified feruloyl esterase had an apparent mass of 33 kDa under denaturing conditions and showed optimum activity at pH 8.0 and 65 degrees C. At a concentration of 5 mmol l-l, the ions Ca2+, Cu2+, Co2+ and Mn2+ reduced the activity by 70-80%. PMID- 10747227 TI - Microbiological examination of cold ready-to-eat sliced meats from catering establishments in the United Kingdom. AB - A microbiological study of cold, ready-to-eat sliced meats from 2579 catering establishments (public houses, hotels, cafes, restaurants, residential homes and other catering premises) found that 2587 of 3494 samples (74%) were of acceptable quality, 892 (26%) were of unsatisfactory quality and 15 (<1%) were of unacceptable quality. Unacceptable results were due to high levels of Escherichia coli, Staphylococcus aureus, Listeria species and/or Clostridium perfringens. Unsatisfactory results were mostly due to high Aerobic Plate Counts. The microbiological quality of cold, ready-to-eat meats was associated with meat type, premises type, management training, hygienic practices, meat supplier and length of storage. The relationship between food hygiene training and microbiological quality is discussed. PMID- 10747228 TI - Distribution of Vibrio cholerae virulence genes among different Vibrio species isolated in Sardinia, Italy. AB - The members of the genus Vibrio include harmless aquatic strains as well as strains capable of causing epidemics of cholera. Diarrhoea caused by Vibrio cholerae is attributed to cholerae enterotoxin (CT) codified by the ctx operon and regulated by a number of virulence genes such as toxT, toxR and toxS. Fifty two Vibrio strains were isolated from different aquatic environments in and around Sardinia and searched by PCR for the presence of ctxA, zot, ace, toxR, toxS, toxT, tcpA and vpi virulence genes in the genomes of the isolates. The toxR operon was found in 27 Vibrio alginolyticus strains out of 42 analysed, in three out of four V. cholerae non-O1 strains and in three Vibrio parahaemolyticus isolates. A positive amplification for the virulence pathogenic island (vpi) was produced by five V. alginolyticus strains. Finally, the ace expected amplification fragment was found in two V. alginolyticus isolates whereas the amplification with zot primers produced the expected fragment in one V. alginolyticus isolate. Differentiation of these strains with a PCR fingerprinting technique revealed no association between the presence of virulence genes and a particular fingerprinting pattern. Although most Vibrio species are considered non-pathogenic or only potentially harmful to humans, the finding of V. cholerae virulence genes in other members of the genus Vibrio, and the recent reports of the creation and evolution of pandemic strains of V. cholerae, may give a new perspective to the significance of these results. PMID- 10747229 TI - Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces. AB - A total of 92 enterococci, isolated from the faeces of minipigs subjected to an in vivo feeding trial, were screened for the production of antimicrobial substances. Bacteriocin production was confirmed for seven strains, of which four were identified as Enterococcus faecalis and three as Enterococcus faecium, on the basis of physiological and biochemical characteristics. The bacteriocins produced by the Ent. faecalis strains showed a narrow spectrum of activity, mainly against other Enterococcus spp., compared with those from the Ent. faecium strains showing a broader spectrum of activity, against indicator strains of Enterococcus spp., Listeria spp., Clostridium spp. and Propionibacterium spp. The bacteriocins of all seven Enterococcus strains were inactivated by alpha chymotrypsin, proteinase K, trypsin, pronase, pepsin and papain, but not by lipase, lysozyme and catalase. The bacteriocins were heat stable and displayed highest activity at neutral pH. The molecular weight of the bacteriocins, as determined by tricine SDS-PAGE, was approximately 3.4 kDa. Only the strains of Ent. faecalis were found to contain plasmids. PCR detection revealed that the bacteriocins produced by Ent. faecium BFE 1170 and BFE 1228 were similar to enterocin A, whereas those produced by Ent. faecium BFE 1072 displayed homology with enterocin L50A and B. PMID- 10747230 TI - Cloning and expression of alpha-amylase from the hyperthermophilic archaeon Pyrococcus woesei in the moderately halophilic bacterium Halomonas elongata. AB - An extracellular alpha-amylase gene from the hyperthermophilic archaeon Pyrococcus woesei has been cloned and sequenced. The 1.4-kb protein-coding sequence is identical to that of the corresponding alpha-amylase gene of the closely related species P. furiosus. By using a shuttle cloning vector for halophilic bacteria, the P. woesei alpha-amylase was expressed in the moderate halophile Halomonas elongata, under the control of a native H. elongata promoter. The hyperthermophilic amylase activity expressed in the halophilic host was recovered completely in the crude membrane fraction of cell homogenates, suggesting the formation of inclusion bodies or that the secretion machinery of H. elongata may fail to recognize and release the pyrococcal alpha-amylase to the extracellular medium. However, thermal stability, metal ion interactions, optimal temperature and pH values for the crude and purified recombinant alpha-amylase were comparable with those of the native pyrococcal enzyme. The P. woesei amylase activity expressed in H. elongata was consistently detected in the cells upon growth on a wide range of NaCl concentrations (0.7-2.5 mol l-1). To our knowledge, this is the first report on the expression of an archaeal gene (P. woesei alpha-amylase) in a moderate halophilic host which serves as a cell factory able to grow under extreme salt conditions and with very simple nutritional requirements. PMID- 10747231 TI - A 5-year epidemiological study of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates in a medium- and long-stay neurological unit. AB - Thirty-eight different strains of extended-spectrum beta-lactamase (ESBL) producing Klebsiella pneumoniae (ESBL Kp), isolated from urine and pus samples of 38 patients hospitalized in a medium- and long-stay neurology department between 1 January 1992 and 31 December 1996, were analysed by antibiotic resistance phenotyping, DNA macrorestriction by pulsed-field electrophoresis and isoelectric focusing of beta-lactamases. An epidemiological survey was conducted to identify risk factors for infection by ESBL Kp in this setting. The 38 isolates were distributed into 13 antibiotypes, three of which predominated (13, six and six isolates). The DNA macrorestriction pattern identified 15 genotypes, four of which predominated (11, six, four and four isolates). A combination of the two typing methods revealed several epidemic clones that emerged consecutively. Two main types of ESBL (SHV-2 and CTX-1) were identified by isoelectric focusing, the former predominating. The case-control study showed that the length of hospital stay, degree of malnutrition and dependency, and urinary sphincter status were the main factors significantly associated with ESBL Kp isolation. PMID- 10747232 TI - The growth and resistance to sodium hypochlorite of Listeria monocytogenes in a steady-state multispecies biofilm. AB - A constant-depth film fermenter (CDFF) was used to culture a steady-state multispecies biofilm consisting of one strain each of Listeria monocytogenes, Pseudomonas fragi and Staphylococcus xylosus. These bacteria were initially grown together in a conventional chemostat to achieve a steady state before being inoculated into the CDFF over an 18-h period. A dilute tryptone soya broth (TSB) medium was supplied to the CDFF and the biofilm allowed to develop over a 28-d period. This mature biofilm was then subjected to increasing levels of sodium hypochlorite solution to measure any antimicrobial effect. The three organisms were seen to reach a steady state after 6 d in the chemostat before being transferred to the CDFF where the mature multispecies biofilm reached steady state at 17 d. Listeria monocytogenes in both planktonic and biofilm growth stabilized at 1. 8 and 1.5%, respectively, of the total plate counts, while Ps. fragi and Staph. xylosus were the predominant organisms in the biofilm at 59% and 39.5%, respectively, of the total microbial population. Steady-state biofilms in the CDFF were exposed to increasing strengths of sodium hypochlorite; 200, 500 and 1000 p.p.m. free chlorine, but a substantial two-log cycle drop in bacterial numbers was only achieved at 1000 p.p.m. free chlorine. In planktonic culture all three organisms were completely eliminated when exposed to 10 p.p.m. free chlorine for a 30-s period. PMID- 10747233 TI - Predictive model for the growth of Yersinia enterocolitica under modified atmospheres. AB - A quadratic response surface model is presented to describe the maximum specific growth rate of Yersinia enterocolitica, at refrigeration temperatures, under modified atmospheres. The presence of CO2 affected mainly the lag phase of the organism. The length of the lag phase increased with higher levels of CO2 in the atmosphere, and this effect was more noticeable at low temperatures. The effect of oxygen was similar but less pronounced. The observed growth was slower with higher CO2. Oxygen also decreased the growth rate, but its effect was significant only when its proportion in the atmosphere was greater than about 40%. Model predictions were compared with growth rates obtained in sea food inoculated with Y. enterocolitica and packaged under modified atmospheres. Predictions were also checked to determine whether they were inside the strict interpolation region of the model. PMID- 10747234 TI - Evidence that water transmits the disease caused by the fish pathogen Photobacterium damselae subsp. damselae. AB - The transmission through water of the disease caused by the fish pathogen, Photobacterium damselae subsp. damselae, as well as the role of the skin mucus in the initial steps of the infection, have been studied. All tested strains resisted the bactericidal activity of the mucus and showed an ability to adhere to it, but only those virulent by the intraperitoneal route were infective through water. Moribund fishes showed the typical signs of the disease: haemorrhaged areas on the body surface and ulcerative lesions with mucus degradation. These results suggest that the pathogen can be transmitted to fish through water and use the skin as a portal of entry. PMID- 10747235 TI - Production of sakacin P by Lactobacillus sakei in a completely defined medium. AB - In order to investigate factors influencing the production of the bacteriocin, sakacin P, Lactobacillus sakei CCUG 42687 was grown in a completely defined medium (DML-B) with 33 components. Although the maximum sakacin P concentration obtained was higher on a complex medium due to higher cell mass, the production per cell mass was higher in DML-B. Sakacin P was produced at 4-30 degrees C, with the highest specific production at low temperatures. More sakacin P was produced at uncontrolled pH compared with cultivation at pH 6.3. Tween-80 had a positive effect on sakacin P production, while addition of sodium chloride and trace metals had negative effects. The decrease in sakacin P concentration during the late growth and stationary phases was shown to be cell-independent and promoted at high temperature and pH. Some differences in production levels of sakacin P were found among six strains of Lactobacillus sakei tested. PMID- 10747236 TI - Role of RpoS and AlgT in Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide and monochloramine. AB - The role of two sigma factors, AlgT and RpoS, in mediating Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide and monochloramine was investigated. Two knock out mutant strains, SS24 (rpoS-) and PAO6852 (algT-), were compared with a wild type, PAO1, in their susceptibility to monochloramine and hydrogen peroxide. When grown as biofilms on alginate gel beads (mean untreated areal cell density 3.7 +/- 0.27 log cfu cm-2) or on glass slides (mean untreated areal cell density 7.6 +/- 0.9 log cfu cm-2), wild type bacteria exhibited reduced susceptibility to both antimicrobial agents in comparison with suspended cells. On alginate gel beads, all strains were equally resistant to monochloramine. rpoS- and algT- gel bead biofilms of 24-hour-old were more susceptible to hydrogen peroxide disinfection than were biofilms formed by PAO1. Biofilm disinfection rate coefficients for the two mutant strains were statistically indistinguishable from planktonic disinfection rate coefficients, indicating complete loss of biofilm resistance. While 48-hour-old algT- biofilm cells became resistant to hydrogen peroxide, 48-hour-old rpoS- biofilm cells remained highly susceptible. With the thicker biofilms formed on glass coupons, all strains were equally resistant to both hydrogen peroxide and monochloramine. It is concluded that while RpoS and AlgT may play a transient role in protecting thin biofilms from hydrogen peroxide, these sigma factors do not mediate resistance to monochloramine and do not contribute significantly to the hydrogen peroxide resistance of thick biofilms. PMID- 10747237 TI - Correlation between serum concentrations following continuous intravenous infusion of dexmedetomidine or medetomidine in cats and their sedative and analgesic effects. AB - Dexmedetomidine (DEX) may have some therapeutic advantages over the racemate medetomidine (MED). Here we have examined how serum concentrations of DEX correlate with some of its anaesthetic effects. Cats (n = 6) were administered with a continuous stepwise intravenous (i.v.) infusion of DEX or MED on different occasions in a cross-over design. Maintenance infusion rates (mg/kg/min) used were: DEX = 0.25 (MED = 0.50); DEX = 1 (MED = 2) and DEX = 4 (MED = 8) for infusion steps 1, 2 and 3, respectively. Each maintenance infusion lasted at least 50 min and was preceded with a loading dose. There was no significant difference between serum DEX and 0.5 serum MED concentrations at any dose level nor was there a significant difference between serum DEX and the (entire) serum MED concentrations. There was no significant difference between DEX and MED for sedation, analgesia, muscular relaxation and heart and respiratory rates. For both DEX and MED, serum drug concentration and analgesia were dose-dependent and sedation increased until the end of infusion step 2 (dose level 2) and decreased at the end of step 3 (dose level 3). Muscular relaxation was not dose-dependent. We conclude that increasing the blood concentration of DEX or MED beyond a certain level decreases the level of sedation instead of increasing it even though analgesia increases. The rate at which DEX and MED are metabolized in cats may not be the same. PMID- 10747238 TI - Bioavailability of amprolium in fasting and nonfasting chickens after intravenous and oral administration. AB - The bioavailability of amprolium (APL) was measured after intravenous (i.v.) and oral (p.o.) administration to chickens. Twelve healthy chickens weighing 1.28 1.41 kg received a dose of 13 mg APL/kg intravenously, and 13 or 26 mg APL/kg orally in both a fasted and a nonfasted condition in a Latin square design. Plasma samples were taken from the subwing vein for determination of APL concentration by HPLC method. The data following intravenous and oral administration were best fitted by 2-compartment and 1-compartment models, respectively, using weighted nonlinear least squares regression. The half-life beta t(1/2)beta, volume of distribution (Vd) and total body clearance (Cl) after intravenous administration were 0.21 h, 0.12 L/kg and 1.32 L/h.kg, respectively. The elimination half-life (t(1/2) Kel) after oral administration was 0.292-0.654 h which is 1.5-3.2 times longer than after intravenous administration, suggesting the presence of a 'flip-flop' phenomenon in chickens. The maximum plasma concentration (Cmax) of 13 mg/kg APL administered orally to chickens during fasting was significantly (about four times) higher than that during nonfasting (P < 0.05). Bioavailability during nonfasting was from 2.3 to 2.6%, and 6.4% during fasting. PMID- 10747239 TI - Clinical effects and pharmacokinetics of medetomidine and its enantiomers in dogs. AB - The clinical effects and pharmacokinetics of medetomidine (MED) and its enanti omers, dexmedetomidine (DEX) and levomedetomidine (LEVO) were compared in a group of six beagle dogs. The dogs received intravenously (i.v.) a bolus of MED (40 microg/kg), DEX (20 and 10 microg/kg), LEVO (20 and 10 microg/kg), and saline placebo in a blinded, randomized block study in six separate sessions. Sedation and analgesia were scored subjectively, and the dogs were monitored for heart rate, ECG lead II, direct blood pressure, respiratory rate, arterial blood gases, and rectal body temperature. Blood samples for drug analysis were taken. Peak sedative and analgesic effects were observed at mean (+/- SD) plasma levels of 18.5 +/- 4.7 ng/mL for MED40, 14.0 +/- 4.5 ng/mL for DEX20, and 5.5 +/- 1.3 ng/mL for DEX10. The overall level of sedation and cardiorespiratory effects did not differ between MED40, DEX20 and DEX10 during the first hour, apparently due to a ceiling effect. However, the analgesic effect of DEX20 lasted longer than the effect of the corresponding dose of racemic medetomidine, suggesting greater potency for dexmedetomidine in dogs. Levomedetomidine had no effect on cardio vascular parameters and caused no apparent sedation or analgesia. The pharmacokinetics of dexmedetomidine and racemic medetomidine were similar, but clearance of levomedetomidine was more rapid (4.07 +/- 0.69 L/h/kg for LEVO20 and 3.52 +/- 1.03 for LEVO10) than of the other drugs (1.26 +/- 0.44 L/h/kg for MED40, 1.24 +/- 0.48 for DEX20, and 0.97 +/- 0.33 for DEX10). PMID- 10747240 TI - Response to criticisms of the US FDA parametric approach for withdrawal time estimation: rebuttal and comparison to the nonparametric method proposed by Concordet and Toutain. AB - The benefits and drawbacks of using nonparametric methods for estimating product withdrawal times have been debated for many years. This issue was recently revived by Concordet & Toutain (1997a, b) when they described a nonparametric method for withdrawal time estimation. The authors urged the international adoption of this approach, basing their recommendation on three fundamental concerns: (1) the lack of a consistent official procedure for determining a withdrawal time within the European Union (EU); (2) the need to identify a statistical method for improving the international harmonization of withdrawal times for new chemical entities; and (3) a lack of confidence in the robustness of the US Food and Drug Administration/Center for Veterinary Medicine (US FDA) procedure, particularly with respect to minor violations in the underlying parametric assumptions. Due to the critical nature of these issues, the US FDA considers it vital to respond to these concerns. This paper provides a description of the US FDA parametric procedure. We also examine the statistical concerns expressed by Concordet and Toutain, identifying the reasons for our confidence in the US FDA parametric approach. Finally, using their Monte Carlo simulation models, we generate additional datasets to explore the behaviour of their nonparametric procedure and evaluate its ability to support US FDA regulatory activities. PMID- 10747241 TI - Cytotoxicity in pig hepatocytes induced by 8-quinolinol, chloramine-T and natamycin. AB - The potential cytotoxic effects of the compounds 8-quinolinol, chloramine-T and natamycin have been studied in isolated pig hepatocytes. The relative cytotoxicity of these compounds was evaluated on the basis of the leakage of cytosolic lactate dehydrogenase (LDH), 3-(4,5 dimethyl)thiazol-2-yl,-2,5-diphenyl tetrazolium bromide (MTT) reduction by mitochondrial dehydrogenases, uptake of neutral red (NR) by cytosolic lysosomes, glutathion (GSH) depletion and oxidized glutathion (GSSG) efflux after 24 h exposure. Evaluation of the 20%, 50% and 80% reduced absorbance data obtained from the parameters NR20, NR50, and NR80, and MTT20, MTT50 and MTT80 enabled us to rank these compounds in decreasing order of cytotoxicity: 8-quinolinol > natamycin > chloramine-T. Also for the parameters LDH and GSH, chloramine-T appears to be less cytotoxic than natamycin and 8 quinolinol. Our study demonstrated that pig hepatocytes may be a useful model for examining cytotoxic events of drugs to be used in pigs, therefore avoiding possible extrapolation problems due to species differences. PMID- 10747242 TI - Pharmacokinetics and oral bioavailability of a doxycycline formulation (DOXYCYCLINE 75%) in nonfasted young pigs. PMID- 10747243 TI - Comparative pharmacokinetics of doramectin and ivermectin in sheep. PMID- 10747244 TI - Oral single-dose pharmacokinetics of thiamphenicol in the sea-bass (Dicentrarchus labrax). PMID- 10747245 TI - Selective control of lactobacilli in kimchi with nisin. AB - The use of nisin to control the lactobacilli responsible for the over-ripening of kimchi, traditional Korean fermented vegetables, was studied. Of the 40 strains of lactic acid bacteria studied, most were sensitive to nisin at a concentration of 100 IU ml-1, while two strains appeared to be resistant. In MRS broth containing nisin at concentrations of 100-300 IU ml-1, the growth of sensitive strains of Lactobacillus plantarum was delayed for 2-3 d at 20 degrees C. When nisin was added to kimchi at a concentration of 100 IU ml-1, the growth of Lactobacillus spp. was inhibited more than the growth of Leuconostoc spp. Scanning electron micrograph observations confirmed the results, demonstrating the predominance of cocci in kimchi containing nisin. These results suggest that at recommended levels, nisin can be used to preserve kimchi by inhibiting lactobacilli more effectively than other lactic acid bacteria involved in kimchi fermentation. PMID- 10747246 TI - A simple HPLC method for analysing diaminopimelic acid diastereomers in cell walls of Gram-positive bacteria. AB - A simple and sensitive method for separating and detecting the LL, DD and meso diastereomers of the dibasic amino acid diaminopimelic acid (DAP) in the peptidoglycan of Gram-positive bacteria is described. This method is based on reverse phase HPLC separation of chiral derivatives of DAP followed by fluorescence detection of the o-phthaldehyde derivatives. Its application to the analyses of cell walls of several Gram-positive bacteria is described, where 10 mg or less of dry cells is required. PMID- 10747247 TI - Medium-size droplets of methyl ricinoleate are reduced by cell-surface activity in the gamma-decalactone production by Yarrowia lipolytica. AB - Size of methyl ricinoleate droplets during biotransformation into gamma decalactone by Yarrowia lipolytica was measured in both homogenized and non homogenized media. In non-homogenized but shaken medium, droplets had an average volume surface diameter d32 of 2.5 microm whereas it was 0.7 microm in homogenized and shaken medium. But as soon as yeast cells were inoculated, both diameters became similar at about 0.7 microm and did not vary significantly until the end of the culture. The growth of Y. lipolytica in both media was very similar except for the lag phase which was lowered in homogenized medium conditions. PMID- 10747248 TI - Properties and stability of glycerophosphate oxidase isolated from a mutant strain of Aerococcus viridans. AB - The properties of microbial L-alpha-glycerophosphate oxidase (GPO) isolated from a mutant strain of Aerococcus viridans DBM 1509 were estimated. The stability at different temperatures and pH were detected. At 4 degrees C the enzyme lost activity during 15 d, at 20 degrees C and 30 degrees C GPO activity decreased during 30 and 25 h, respectively. The highest stability was measured at - 20 degrees C and pH 9. At 4 degrees C the stability was enhanced by the addition of 0.1 M EDTA or by lyophilization in the presence of dextrin. These conditions allow the prolongation of the low stability of microbial GPO which limited its use, and give the opportunity to increase the stability of other enzymes PMID- 10747249 TI - Growth of Helicobacter pylori in various liquid and plating media. AB - The objectives of this research were to compare commonly used liquid and plating media to elucidate whether one medium provided superior growth of Helicobacter pylori in vitro. The liquid media compared were Mueller-Hinton broth, brain heart infusion broth and H. pylori special peptone broth, formulated in this laboratory. No significant differences in growth rates were noted and shaking during the incubation of broths was not essential for good growth. The plating media compared included Columbia agar, Mueller-Hinton agar, modified Glupczynski's Brussels campylobacter charcoal agar, Johnson-Murano agar and H. pylori special peptone agar (HPSPA). None of the non-specific plating media that have been used historically to culture H. pylori exhibited any particular advantage. However, HPSPA provided an obvious advantage in colony size. Helicobacter pylori special peptone agar enhances the cultivation of H. pylori and could improve the recovery of the bacterium from clinical samples in vitro. PMID- 10747250 TI - Lactobacillus farciminis MD, a newer strain with potential for bacteriocin and antibiotic assay. AB - A native isolate Lactobacillus farciminis MD isolated from fermenting mushroom exhibited a high degree of sensitivity to the majority of the bacteriocins produced by strains of lactobacilli, leuconostoc and pediococci. Also, the efficacy of Lact. farciminis MD as a sensitive strain for antibiotic assay was established against different antibiotics including ampicillin, cefazoline, chloramphenicol and nitrofurantoin at concentrations of 30 microg each, showing an inhibition zone of 30 mm diameter. The high degree of sensitivity towards bacteriocins and antibiotics provide potential for the exploitation of Lact. farciminis MD in establishing very well-defined bacteriocin producers. PMID- 10747251 TI - Acceleration of hyaluronidase production in the course of batch cultivation of Clostridium perfringens can be achieved with bacteriolytic enzymes. AB - As Clostridium perfringens hyaluronidase has cell-bound enzyme features, an enzymatic approach has been designed to facilitate the release of hyaluronidase into culture through increasing the clostridial cell wall permeability. As a result of the application of lytic peptidase from Actinomyces rutgersensis, beta N-acetylglucosaminidase and beta-N-acetylmuramidase (both from Bacillus subtilis) commercially available preparations at the end of the producer's exponential growth phase, a 5.3-, 4.8- and 4.0-fold acceleration, respectively (but no enhancement), of hyaluronidase production in the course of batch cultivation of Cl. perfringens has been achieved. This also resulted in an approximately 10-fold reduction in undesirable side lecithinase activity irrespective of the bacteriolytic enzyme preparation used. PMID- 10747252 TI - Development of a combined PCR-culture technique for the rapid detection of Arcobacter spp. in chicken meat. AB - A combined PCR-culture technique was developed to detect Arcobacter spp. in fresh chicken meat. Following a short selective enrichment of chicken samples, bacterial DNA was extracted and amplified using primers targeted at the genes encoding 16S rRNA of Arcobacter spp. The selected primers amplify a 181-bp fragment from all Arcobacter spp., whereas no PCR product is generated for other bacteria, including the closely related Campylobacter and Helicobacter species. The assay was used to screen 96 retail-purchased chicken samples for the presence of Arcobacter spp. Fifty-three percent of the samples analysed were positive for this micro-organism. The assay is simple and sensitive and reduces the amount of time required to positively detect Arcobacter spp. in poultry meat. PMID- 10747253 TI - Frequency of Escherichia coli strains producing the cytotoxic necrotizing factor (CNF1) in nosocomial urinary tract infections. AB - The presence of cytotoxic necrotizing factor 1 (CNF1), together with various associated virulence factors (alpha-haemolysin, P-, S- and A-fimbriae), was screened in 175 uropathogenic Escherichia coli strains isolated from hospitalized adult patients. The cnf1 gene was detected in 30% of the selected strains independently of the severity of the clinical urinary infection. A significant association between CNF1, haemolytic activity and the products of the pap/sfa genes was found. However, CNF1 appeared not to play a major role in nosocomial E. coli urinary tract infections. PMID- 10747254 TI - Detection of Escherichia coli O157:H7 in heifers' faecal samples using an automated immunoconcentration system. AB - Pre-treatment of a 5-h enrichment culture with an automated immunoconcentration (ICE) system greatly improved the isolation of Escherichia coli O157:H7 from spiked heifer faecal samples. Enrichment samples plated directly onto sorbitol MacConkey agar (SMAC) and SMAC agar supplemented with cefixime and potassium tellurite (CT-SMAC) showed recovery rates of 8% and 56%, respectively. However, after ICE treatment, E. coli O157:H7 was recovered from 92% of the samples on SMAC and 100% on CT-SMAC. Immunoconcentration analysis of heifers' faecal samples collected from a slaughter-house in France, during March to June 1998, showed that 1% (three of 300) was positive for E. coli O157:H7. Phenotypic and genotypic analysis showed that all three isolates carried both the O157 and H7 antigens, did not ferment sorbitol or had beta-glucuronidase activity and carried trait virulence factors for E. coli O157:H7 (uidA allele, eaeA and pO157 plasmid). However, only one strain was toxigenic and this strain produced a single toxin, namely verotoxin 2. PMID- 10747255 TI - Caged ATP - an internal calibration method for ATP bioluminescence assays. AB - ATP bioluminescence, based on the firefly luciferase system, is used for the rapid determination of hygienic practices in the food industry. This study has demonstrated the use of caged ATP as an internal ATP standard and quantified the effects of industrial cleansing solutions, alcoholic beverages and pH on firefly luciferase activity. The light signal was quenched 6-47% by five cleansing solutions at standard working concentrations. Ethanol at 1% (v/v) inhibited bioluminescence by 15% (w/v) whereas concentrations above 4% enhanced the light output. The light signal was quenched by 20-25% at pH values below pH 4 and above pH 10. PMID- 10747256 TI - Variations in virulence between different electrophoretic types of Listeria monocytogenes. AB - A total of 245 strains of Listeria monocytogenes, representing 33 different electrophoretic types (ETs), were examined quantitatively for haemolytic activity. No significant difference was observed in the mean haemolytic activity between different ETs. Eighty four out of 91 strains examined were found to be virulent for chick embryos. Strains belonging to ET 2 and ET 4 were found to be less virulent than strains of other ETs (P = 0.0447). Furthermore, strains from clinical cases were found to be more virulent (P = 0.0002) than strains from foods (the MTD among clinical strains was 2.46 in mean compared with 3.64 among food isolates). The explanation for this may be that more virulent strains are more prone to cause human infection. It is, however, also possible that strains of L. monocytogenes may become more virulent while multiplying in a living organism compared with multiplying in foods. PMID- 10747257 TI - Activity of hydrolysed lactoferrin against foodborne pathogenic bacteria in growth media: the effect of EDTA. AB - Lactoferrin was hydrolysed with pepsin and the antimicrobial activity of the resulting hydrolysate (HLF) was studied in 1% peptone, 0.05% yeast extract, 1% glucose (PYG) medium and tryptic soy broth (TSB). HLF was effective against Listeria monocytogenes, enterohaemorrhagic Escherichia coli and Salmonella enteritidis in PYG, however, the highest studied concentration (1.6 mg ml-1) did not inhibit growth of any of these organisms in TSB. The addition of EDTA enhanced the activity of HLF in TSB, indicating that the decreased activity of HLF may have been due, in part, to excess cations in the medium. PMID- 10747207 TI - Synaptic control of motoneuronal excitability. AB - Movement, the fundamental component of behavior and the principal extrinsic action of the brain, is produced when skeletal muscles contract and relax in response to patterns of action potentials generated by motoneurons. The processes that determine the firing behavior of motoneurons are therefore important in understanding the transformation of neural activity to motor behavior. Here, we review recent studies on the control of motoneuronal excitability, focusing on synaptic and cellular properties. We first present a background description of motoneurons: their development, anatomical organization, and membrane properties, both passive and active. We then describe the general anatomical organization of synaptic input to motoneurons, followed by a description of the major transmitter systems that affect motoneuronal excitability, including ligands, receptor distribution, pre- and postsynaptic actions, signal transduction, and functional role. Glutamate is the main excitatory, and GABA and glycine are the main inhibitory transmitters acting through ionotropic receptors. These amino acids signal the principal motor commands from peripheral, spinal, and supraspinal structures. Amines, such as serotonin and norepinephrine, and neuropeptides, as well as the glutamate and GABA acting at metabotropic receptors, modulate motoneuronal excitability through pre- and postsynaptic actions. Acting principally via second messenger systems, their actions converge on common effectors, e.g., leak K(+) current, cationic inward current, hyperpolarization activated inward current, Ca(2+) channels, or presynaptic release processes. Together, these numerous inputs mediate and modify incoming motor commands, ultimately generating the coordinated firing patterns that underlie muscle contractions during motor behavior. PMID- 10747259 TI - Effect of extra aeration on extracellular enzyme activities and ATP concentration of dairy Pseudomonas fluorescens. AB - The effect of forced aeration on extracellular enzyme synthesis during batch growth of a Pseudomonas fluorescens strain of dairy origin on pyruvate mineral salts medium at 7 degrees C was studied. Measurement of oxygen tension, electron micrographs to estimate cell volume, luciferase determination of ATP and plate counts were performed in the course of incubation. Cells from the stationary phase of growth had lower energy status (in terms of intracellular ATP concentration) in the cultures receiving surplus aeration. Those cells produced three times more extracellular proteinase and lipase than control cells. Onset time for production of both enzymes coincided with a sharp fall of intracellular ATP levels. PMID- 10747258 TI - Phylogenetic characterization of several para- and meta-PCB dechlorinating Clostridium species: 16s rDNA sequence analyses. AB - The genus Clostridium has more than 127 species, grouped according to their morphology and functions. Nine Clostridium species were identified based on their ability to dechlorinate meta- and para-PCB (polychlorinated biphenyl) contaminated sediments. The phylogenetic relatedness of these PCB-degrading Clostridium species was studied using ribosomal RNA genes. The diversity of small subunit rRNA genes associated with the domain bacteria was examined using defined operational taxonomic units (OTUs) in samples from PCB contaminated sediments from Lake Medinah, New York. The RFLP (restriction fragment length polymorphism) of the OTVs was measured. OTUs B (105 clones), A (33 clones) and C (45 clones) accounted for 75% of all the 16S rDNA clones expressing anaerobic para- and meta PCB dechlorinating activity. In this report we describe complete 16S rDNA sequences of OTU-A and OTU-B, and partial rDNA sequences of OTUs C-J. The OTU-B and OTU-I form a phylogenetically related cluster, closely affiliated with Clostridium hydroxybenzoicum strains. OTUs A, C, D, G, H and J also belong to the genus Clostridium, but they represent separate species. OTU-E, a close affiliate to Bacteroides forsynthus, is a meta-PCB dechlorinator. The Cl. hydroxybenzoicum strains (OTU-B) are primarily para-PCB dechlorinators and are the most common. Some less prevalent OTUs (- E, -G, -H and -I), are also mostly para-PCB dechlorinators. Other Clostridium species such as Cl. beijerinckii (OTU-A), Cl. intestinalis (OTU-D) and Cl. thermolacticum (OTU-J) are primarily meta-PCB dechlorinators. Cl. paraputrificum (OTU-C) and Cl. cellulosi (OTU-F), were less prevalent in the total consortium, but they could dechlorinate both para- and meta-PCB. Although a few less prevalent Clostridium species can degrade both para and meta-PCBs, this study confirms that para- and meta-PCB dechlorinating species are generally phylogenetically different. PMID- 10747260 TI - Sensitivity of nisin-resistant Listeria monocytogenes to heat and the synergistic action of heat and nisin. AB - Nisin, a bacteriocin produced by some strains of Lactococcus lactis, acts against foodborne pathogen Listeria monocytogenes. A single exposure of cells to nisin can generate nisin-resistant (Nisr) mutants, which may compromise the use of nisin in the food industry. The objective of this research was to compare the heat resistance of Nisr and wild type (WT) Listeria monocytogenes. The synergistic effect of heat-treatment (55 degrees C) and nisin (500 IU ml-1) on the Nisr cells and the WT L. monocytogenes Scott A was also studied. When the cells were grown in the absence of nisin, there was no significant (alpha = 0.05) difference in heat resistance between WT and Nisr cells of L. monocytogenes at 55, 60 and 65 degrees C. However, when the Nisr cells were grown in the presence of nisin, they were more sensitive to heat at 55 degrees C than the WT cells. The D-values at 55 degrees C were 2.88 and 2.77 min for Nisr ATCC 700301 and ATCC 700302, respectively, which was significantly (alpha = 0.05) lower than the D value for WT, 3.72 min. When Nisr cells were subjected to a combined treatment of heat and nisin, there was approximately a four log reduction during the first 7 min of treatment. PMID- 10747261 TI - Characteristics of a sporulation stimulating factor from Clostridium perfringens type A. AB - A product in the culture supernatant fluid of Clostridium perfringens NCTC 8239 stimulated the sporulation of a test strain, NCTC 8679, of the same organism. The responsible factor, termed sporulation factor (SF), was present in seven cultures of Cl. perfringens grown in either a defined or complex medium. The SF reversed glucose-mediated catabolite repression of sporulation by this organism. Preliminary characterization of the SF demonstrated a resistance to elevated temperatures and proteases and a molecular weight of less than 500 Da. The known association of Cl. perfringens enterotoxin with sporulation highlights the importance of interactions between strains of this organism as may occur in the human intestine during foodborne illness. PMID- 10747263 TI - Isolation of Bacillus thuringiensis from Northern Victoria Land, Antarctica. AB - Sixty-two samples of Antarctic soils, mosses, penguin guano, algae and lichens were examined for the presence of aerobic endospore-forming bacteria; 36 samples (58%) yielded such organisms, and two samples from Edmonson Point (74 degrees 21'S 165 degrees 08'E) and one sample from Apostrophe Island (73 degrees 32'S 167 degrees 24'E), northern Victoria Land, yielded strains of Bacillus thuringiensis. Further isolations from two of the samples, appreciable variation in biotypes among the strains, failure of the strains to grow on routine B. thuringiensis media, and the fact that one of the sampling sites is very rarely visited by humans, suggest that the organisms were living in these soils rather than being chance contaminants. A representative strain, from Apostrophe Island, was identified as serovar pirenaica (H57). PMID- 10747264 TI - First, do no harm. PMID- 10747265 TI - An open comparative study of dispersible piroxicam versus soluble acetylsalicylic acid for the treatment of osteoarticular painful attack during sickle cell crisis. AB - We compared the efficacy and tolerability of oral piroxicam 1 mg/kg/day with soluble aspirin given at 100 mg/kg/day taken four-hourly in 58 patients with sickle cell anaemia and severe ostcoarticular painful attacks requiring hospitalization in a randomized, paralleled study. Main investigational criteria were pain relief, limitation of movement, fever, and insomnia or agitation. Both groups were well-matched at the commencement of therapy but most patients on piroxicam showed remarkable and significant pain relief and improvement in other parameters within 24 h. Unwanted effects were absent in the piroxicam-treated group whereas those treated with aspirin experienced nausea and vomiting. There were no significant changes in liver function tests with both forms of treatment. Oral piroxicam is an effective and safe treatment in the management of the osteoarticular painful crisis in sickle cell anaemia. It might prevent the use of parenteral analgesics and hospitalization and reduce the loss of school hours in patients who are being treated for bone pain crises that characterize sickle cell anaemia. PMID- 10747262 TI - Cluster analysis of AP-PCR generated DNA fingerprints of Vibrio vulnificus isolates from patients fatally infected after consumption of raw oysters. AB - Arbitrarily-primed-polymerase chain reaction (AP-PCR) DNA fingerprints were generated for 10 Vibrio vulnificus strains isolated from patients who became infected and died between 1993 and 1996 as a result of consuming raw oysters. Analysis of the DNA fingerprints with gel imaging and cluster analysis software revealed significant genetic heterogeneity among these strains, suggesting that V. vulnificus has a high degree of variation in its genomic organization, and that multiple pathogenic strains with greatly diverse genomic arrangements, rather than a single type of infective strain or serogroup, caused these infections. PMID- 10747266 TI - Aggressive Kaposi's sarcoma in a 6-month-old African infant: case report and review of the literature. AB - Kaposi's sarcoma (KS), known to exist in Africa for a century now, was rare in children and unknown in the newborn. With the onset of the HIV/AIDS epidemic, a more aggressive, disseminated type of KS (AKS) was recognized. Recently KS was diagnosed in a 6-month-old infant in Tanzania. Data support the notion that HHSV8 infectivity can be potentiated with HIV infection and thus produce multiple lesions in different anatomical sites early in life. Furthermore, the available evidence would suggest a nonsexual route of HHSV8 infection, possibly from mother to fetus. PMID- 10747267 TI - Morbidity assessment in urinary schistosomiasis infection through ultrasonography and measurement of eosinophil cationic protein (ECP) in urine. AB - In a Schistosoma haematobium-endemic village in western Madagascar we evaluated ultrasonography and Eosinophil Cationic Protein (ECP) in urine as means to detect the associated urinary tract pathology. 192 individuals were matched according to age and sex, and grouped into infected persons with bladder and, if present, kidney pathology (n = 96); infected persons without pathology (n = 48) and noninfected persons without pathology (n = 48). The median urinary egg count was significantly higher in individuals with ultrasonographically detectable urinary tract pathology (115 eggs/10 ml urine) than in infected persons without (45 eggs/10 ml of urine). At 136 ng/ml, the median ECP level was significantly higher in the 144 infected individuals than in the 48 noninfected persons (0.35 ng/ml). Egg excretion correlated positively with ECP level. The median ECP level was significantly higher in the group with ultrasonographically detectable urinary tract pathology than in the group without (183 ng/ml vs. 67 ng/ml). The results suggest that minor degrees of pathology, particularly at an early stage of infection with S. haematobium, might be overlooked by ultrasonography despite the presence of marked inflammation, as indicated by markedly increased urinary ECP levels in infected individuals without ultrasonographically detectable urinary tract pathology. ECP may therefore provide important information on the evolution of S. haematobium-associated urinary tract morbidity. PMID- 10747268 TI - Seroepidemiology of human plague in the Madagascar highlands. AB - We conducted a seroepidemiological survey of human plague in the general population using random sampling in the area of Ambositra, the main focus of plague in the central highlands of Madagascar (520 confirmed and presumptive cases notified during the past 10 years). Sera were tested using an ELISA IgG F1 assay. Considering the internal validity of the assay and the sampling method, the overall corrected prevalence of F1 antibodies was 0.6% (95% CI: 0.2%-1.8%). Being nearly 0 up to the age of 40, the corrected prevalence increased markedly after 45 years to 6.2%. Six of 20 individuals who declared to have been treated for clinical suspicion of bubonic plague in the past had F1 antibodies. The seroprevalence did not differ according to gender except in individuals > 60, where antibodies were significantly more frequent in males. This study suggests that the number of clinically suspected cases of plague provided by the surveillance network was plausible, despite some true cases being missed and a significant number of false positives. We also confirm that Yersinia pestis infections may occur without marked clinical manifestations and patients may recover without treatment, in accordance with old observations of pestis minor. PMID- 10747269 TI - Crimean-congo haemorrhagic fever: a seroepidemiological and tick survey in the Sultanate of Oman. AB - In 1995 and 1996, 4 persons from the Sultanate of Oman were confirmed with clinical Crimean-Congo haemorrhagic fever (CCHF). To assess the prevalence of CCHF virus infection in Oman, a convenience sample of imported and domestic animals from farms, abattoirs and livestock markets was examined by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to CCHF virus. Ticks were collected from selected animals, identified, pooled by species, host and location and tested for evidence of infection with CCHF virus by antigen capture ELISA. Serum samples from individuals working in animal and nonanimal contact-related jobs were also tested for CCHF antibodies. Serological evidence of infection was noted in 108 (22%) of 489 animals. Most of the ticks collected (618 of 912) from all species of sampled livestock were Hyalomma anatolicum anatolicum, a competent vector and reservoir of CCHF virus. 243 tick pools were tested for CCHF antigen, and 19 pools were positive. Of the individuals working in animal contact-related jobs, 73 (30.3%) of 241 non-Omani citizens and only 1 (2.4%) of 41 Omani citizens were CCHF antibody-positive. Butchers were more likely to have CCHF antibody than persons in other job categories. The presence of clinical disease and the serological results for animals and humans and infected Hyalomma ticks provide ample evidence of the presence of CCHF virus in yet another country in the Arabian Peninsula. PMID- 10747270 TI - Decline of mortality in children in rural Gambia: the influence of village-level primary health care. AB - Using data from a longitudinal study conducted in 40 villages by the UK MRC in the North Bank Division of The Gambia beginning in late 1981, we examined infant and child mortality over a 15-year period for a population of about 17 000 people. Comparisons are drawn between villages with and without PHC. The extra facilities in the PHC villages include: a paid Community Health Nurse for about every 5 villages, a Village Health Worker and a trained Traditional Birth Attendant. Maternal and child health services with a vaccination programme are accessible to residents in both PHC and non-PHC villages. The data indicate that there has been a marked improvement in infant and under-five mortality in both sets of villages. Following the establishment of the PHC system in 1983, infant mortality dropped from 134/1000 in 1982-83 to 69/1000 in 1992-94 in the PHC villages and from 155/1000 to 91/1000 in the non-PHC villages over the same period. Between 1982 and 83 and 1992-94, the death rates for children aged 1-4 fell from 42/1000 to 28/1000 in the PHC villages and from 45/1000 to 38/1000 in the non-PHC villages. Since 1994, when supervision of the PHC system has weakened, infant mortality rates in the PHC villages have risen to 89/1000 in 1994-96. The rates in the non-PHC villages fell to 78/1000 for this period. The under-five mortality rates in both sets of villages have converged to 34/1000 for 1994-96. When the PHC programme was well supported in the 1980s, we saw significantly lower mortality rates for the 1-4-year-olds. These differences disappeared when support for PHC was reduced after 1994. The differential effects on infant mortality are less clear cut. PMID- 10747271 TI - Red cell glucose-6-phosphate dehydrogenase status and pyruvate kinase activity in a Nigerian population. AB - Glucose-6-phosphate dehydrogenase A- (G6PD A-) deficiency is a common enzymopathy in Africa that sporadically leads to manifest haemolytic anaemia. It is not exactly known how far the haematological status of individuals with either homozygous or heterozygous G6PD A- deficiency differs from that of individuals with normal G6PD activity. In a field study in Nigeria, we determined G6PD gene variants, the corresponding G6PD and pyruvate kinase (PK) activities, and basic haematological parameters in clinically healthy individuals, who were, in part, asymptomatically infected by malaria parasites. Red blood cell counts and haemoglobin levels were lower in G6PD A- deficient than in G6PD normal subjects. PK activities were higher in G6PD deficients, indicating a younger red cell population in these individuals. These findings suggest that G6PD A- deficiency is accompanied by chronic subclinical haemolysis. As a consequence, the reduced life span of red cells leads to an impaired diagnosis of G6PD heterozygosity when applying routine biochemical methods. PMID- 10747272 TI - International multi-centre evaluation of a dipstick assay for human leptospirosis. AB - A dipstick assay for the detection of Leptospira-specific immunoglobulin M (IgM) antibodies in human sera was evaluated in 27 laboratories in 23 countries. 873 serum samples from 711 patients including 329 laboratory-confirmed leptospirosis case patients, 239 noncase patients and 69 patients with viral infections causing heamorrhagic fever were tested. Relative to the results of the reference leptospirosis test, the sensitivity of the dipstick assay was 84.5% for serum samples collected during the first 10 days of the disease and 92.1% for serum samples collected 10-30 days after the onset of disease. The specificity was 87.5% and 94.4%, respectively. Similar to viral haemorrhagic fevers, leptospirosis may cause bleeding. A small number of serum samples from patients with haemorrhagic viral infections gave a weak (1 +) stain. All other samples were negative. In conclusion, the dipstick assay is sensitive and specific and reacts well with serum samples from patients infected with a range of leptospiral strains. It is also easy to use and does not require special equipment or refrigeration. Therefore the assay is ideal for use in developing countries and rural settings. PMID- 10747273 TI - Field trial of a haemoglobin colour scale: an effective tool to detect anaemia in preschool children. AB - The objective of this study was to evaluate the performance of the Haemoglobin Colour Scale, developed by Stott and Lewis, to diagnose anaemia in a primary health care setting where anaemia was prevalent and severe. Three measures of anaemia were compared in 535 preschool children: haemoglobin based on the Haemoglobin Colour Scale, clinical assessment in three sites (conjunctiva, palm and nail bed) and haemoglobin based on a digital haemoglobinometer (HemoCue method) taken as gold standard. A statistically significant correlation (r = 0.80, coefficient = 0.77 and Y intercept = 2.33) was obtained between the results of the Haemoglobin Colour Scale and the HemoCue. In more than 80% of cases, the difference between the colour scale readings and the results of the HemoCue was within 1 g/dl. Of 415 anaemic children (Hb < 11 g/dl by HemoCue), 85.2% were so identified by the Haemoglobin Colour Scale and 19.7% were classified anaemic by clinical pallor. Of 19 severely anaemic children (Hb < 7 g/dl by HemoCue), 73.6% were identified as severely anaemic and 100% were classified as anaemic by the colour scale, 61.1% were classified as anaemic using clinical pallor. We found the Haemoglobin Colour Scale to be a useful tool in identifying anaemic and severely anaemic children. Efficiencies in term of cost, accuracy and time make it an important resource in primary health care settings in developing countries. Further testing with other staff in other settings is recommended to determine the usefulness of large-scale distribution. PMID- 10747274 TI - Prospective validation of a standardized questionnaire for estimating childhood mortality and morbidity due to pneumonia and diarrhoea. AB - This paper reports the validation of a 'best-judgement' standardised questionnaire using guidelines and algorithms developed by an expert working group conducted in Nicaragua between 1995 and 1997. Prospective hospital data, including standardised medical recording of selected signs and symptoms, laboratory and radiographic test results and physician diagnoses were collected for children < 5 years admitted with any serious life-threatening condition in 3 study hospitals. The mothers or caregivers of the children were later traced and interviewed using the 'best-judgement' questionnaire. Interviews were completed 1 22 months after admission to hospital for 1115 children (400 who died during the stay in hospital and 715 who were discharged alive). The cause of death or admission to hospital was determined by an expert algorithm applied to hospital data. A similar procedure was used to derive the cause using the answers to questions from interviews. Hospital causes were compared with interview causes and sensitivity and specificity calculated, together with the estimated cause specific fraction for diarrhoea and pneumonia. Multiple diagnoses were allowed; 378 children in the sample (104 deaths, 274 survivors) had a reference diagnosis of diarrhoeal illness, and 506 (168 deaths, 338 survivors) a reference diagnosis of pneumonia. When results for deaths and survivors in all age groups were combined, the expert algorithms had sensitivity between 86% and 88% and specificity between 81% and 83% for any diarrhoeal illness; and sensitivity between 74% and 87% and specificity between 37% and 72% for pneumonia. Algorithms tested in previous validation studies were also applied to data obtained in this study, and the results are compared. Despite less than perfect sensitivity and specificity, reasonably accurate estimates of the cause-specific mortality and morbidity fractions for diarrhoea were obtained, although the accuracy of estimates in other settings using the same instrument will depend on the true cause-specific fraction in those settings. The algorithms tested for pneumonia did not produce accurate estimates of the cause-specific fraction, and are not recommended for use in community settings. PMID- 10747276 TI - Vaccination in the 21st century - new funds, new strategies? PMID- 10747275 TI - Health-seeking behaviour for child illness in Guatemala. AB - Relying on data from the 1995 Guatemalan Survey of Family Health (EGSF), we analyse the relationship between child illness and health-seeking behaviour. Information on illness was collected for 3193 children. This analysis is based on 870 of these who became ill with diarrhoeal or respiratory disease during a 13 day period prior to interview. Estimates are derived from logistic models of the probability of seeing any or a specific type of health care provider as a function of characteristics of the illness on a given day and the child. The results indicate that modern medical care plays a major role in the treatment of infectious illness among children in rural Guatemala, with visits to pharmacists, doctors and the staff at government health facilities occurring much more frequently than visits to curers and other traditional practitioners. In general, families are much more likely to seek out a health care provider when a child experiences fever and gastrointestinal symptoms than when suffering from respiratory and other symptoms, and when a mother perceives the illness to be serious. The results also indicate that infants, low parity children, and children assessed as having generally been in good health are more likely to visit health care providers than other children. However, the particular associations often vary by type of health care provider. PMID- 10747277 TI - Viewpoint: immunization against poverty. PMID- 10747278 TI - Submicroscopic Plasmodium falciparum infections in pregnancy in Ghana. AB - Malarial parasitaemia below the threshold of microscopy but detectable by polymerase chain reaction (PCR) assays is common in endemic regions. This study was conducted to examine prevalence, predictors, and effects of submicroscopic Plasmodium falciparum infections in pregnancy. In a cross-sectional study among 530 pregnant women in Ghana, plasmodial infections were assessed by microscopy and PCR assays. Concentrations of haemoglobin and C-reactive protein (CRP) were measured and antimalarial drugs (chloroquine, pyrimethamine) in urine were demonstrated by ELISA dipsticks. By microscopy, 32% of the women were found to harbour malaria parasites. This rate increased to 63% adding the results of the parasite-specific PCR. P. falciparum was present in all but one infection. With increasing gravidity, infection rates and parasite densities decreased and the proportions of submicroscopic parasitaemia among infected women grew. Correspondingly, anaemia, fever and evidence of inflammation (CRP > 0.6 mg/dl) were more frequent in primigravidae than in multigravidae. Antimalarial drugs were detected in 65% of the women and were associated with a reduced prevalence of P. falciparum infections and a raised proportion of submicroscopic parasitaemia. Both gravidity and antimalarial drug use were independent predictors of submicroscopic P. falciparum infections. These infections caused a slight reduction of Hb levels and considerably increased serum concentrations of CRP. Conventional microscopy underestimates the actual extent of malarial infections in pregnancy in endemic regions. Submicroscopic P. falciparum infections are frequent and may contribute to mild anaemia and inflammation in seemingly aparasitaemic pregnant women. PMID- 10747279 TI - Correlation of in vivo-resistance to chloroquine and allelic polymorphisms in Plasmodium falciparum isolates from Uganda. AB - The efficacy of chloroquine in the treatment of uncomplicated falciparum malaria in Africa is heavily compromised by high levels of drug resistance. The occurrence of active site mutations in the Plasmodium falciparum multi drug resistance-gene 1 (pfmdr1) has been associated with development of resistance to chloroquine. This study investigates the occurrence of several mutations at codons 86, 1042 and 1246 of the pfmdr1-gene in infected blood samples taken from Ugandan children before treatment with chloroquine and their relationship to clinical and parasitological resistance. Even though a clear association of CQR to one certain pfmdr1 single point mutation could not be substantiated, the frequency of resistance was consistently higher for samples revealing any of the mutations than among wild type samples, and 90% of the clinically resistant samples did present a mutation. Thus detection of these allelic pfmdr1 polymorphisms is not a decisive factor for prediction of clinical chloroquine resistance, but an interplay of the different mutations with unknown cofactors is to be assumed and the possible role of other genetic alterations remains to be investigated. PMID- 10747280 TI - Mothers' perceptions and knowledge on childhood malaria in the holendemic Kibaha district, Tanzania: implications for malaria control and the IMCI strategy. AB - Prior to an intervention on improving the quality of malaria case management, we assessed mothers' abilities to recognize nonsevere and severe/complicated malaria in children when a child has fever with other physiological and behavioural symptoms associated with malaria. Malaria was mentioned as the commonest febrile illness (94. 1%), convulsions the least (11.4%). Fever and enteric symptoms featured as the most important symptoms of childhood malaria at frequencies of 93.5% and 73.8%, respectively. The need for laboratory diagnosis was very high (98.3%), the reason being to get accurate diagnosis and treatment (89.4%). Poor outcome of treatment was ascribed to incorrect diagnosis and prescription, noncompliance at home and ineffective drugs (62.1%). Most mothers (86.6%) would take antipyretic measures first when a child has fever, and subsequently the majority (92.9%) would seek care at a modern health facility. About 50% of the mothers would give traditional treatments for childhood convulsions and wait till fits cease before the next action. A high proportion of the mothers (75%) held the belief that an injection in a child with high fever would precipitate convulsions or death. The implications of these findings for chemotherapeutic malaria control in holoendemic areas within the context of the Integrated Management of Childhood Illnesses (IMCI) strategy are discussed. PMID- 10747281 TI - The immunofluorescence antibody test (IFAT) for the diagnosis of schistosomiasis used in a non-endemic area. AB - OBJECTIVE: To evaluate an immunofluorescence antibody test (IFAT) for diagnosis of schistosomiasis in nonimmune travellers and immigrants from endemic areas. METHODS: 65 patients (48 Danes and 17 immigrants) with schistosomiasis were included. The diagnosis of schistosomiasis was based on the presence of schistosome eggs in faeces, urine, sperm, rectal or bladder biopsies and/or the presence of specific antibodies determined by the serological immunofluorescence antibody test (IFAT). Egg excretion was detected using conventional methods and the IFAT performed on whole S. mansoni schistosomula worms, harvested after 8 weeks from mice. Two patterns of immunofluorescence were observed: Fluorescence in the gut of the schistosome called 'Gut Associated Antigen, GAA', and fluorescence of the surface of the schistosomula called 'Membrane Bound Antigen, MBA'. RESULTS: Eggs were found in 44% of the Danish patients and in 76% of immigrants. The diagnosis was based on a positive IFAT in 48% of the patients. In patients from nonendemic areas, the finding of antibodies against GAA was diagnostic while optimal sensitivity in the immigrants was reached by measuring antibodies against both GAA and MBA. CONCLUSION: In patients from nonendemic areas GAA is a sensitive marker of acute infection with schistosomiasis. In patients from endemic areas the demonstration of both GAA and MBA is necessary to properly identify long-lasting, nonacute infections. Egg-detection and/or measurement of CAA and CCA remain the methods of choice to monitor treatment as the immunofluorescence assay may remain positive for several years after treatment. PMID- 10747282 TI - A comparative analysis of the cost-effectiveness of treatment based on parasitological and symptomatic screening for Schistosoma mansoni in Burundi. AB - OBJECTIVES: To assess the cost-effectiveness of three alternative screening strategies in delivering treatment to patients presenting with symptoms suggestive of S. mansoni, using data collected from 17 PHCCs in the Rusizi Plain, Burundi. The three strategies were: (1) screening all symptomatic patients using a Kato-Katz smear and treating only the ones found positive; (2) treating all symptomatic patients or (3) treating only those presenting with symptoms of severe diarrhoea (blood in stool). METHODS: The database consisted of 41 051 visits of symptomatic patients to the 17 PHCCs during 1990. Effectiveness was measured as the number of infected patients treated (gold standard: 25-mg Kato Katz smear). Cost-effectiveness ratios (CERs) (cost in US$ per infected patient treated) were used to compare the control strategies. Sensitivity analysis was performed to assess the effect of drug price and prevalence of infection on the CERs. RESULTS: The overall prevalence of S. mansoni infection was 9.5% (95% CI = 9.1%, 9.9%). Treating only those with severe symptoms of infection would have left 92% of infected patients untreated. Performing a Kato-Katz smear to confirm the diagnosis was more cost-effective than treating all symptomatic patients on a presumptive basis, with CERs of 4.2 US$ and 12.43 US$ per infected person treated, respectively. Sensitivity analysis showed that, for a cost of 0.99 US$ per dose of praziquantel, the Kato-Katz option remained the most cost-effective approach for prevalences under 76%. For a drug price of 0.21 US$ per dose, both strategies would have become equivalent. The latter value varied extensively per PHCC (range 0.17 US$ to 0.51 US$), due to the different prevalences of infection (range 0.5% to 34.3%). CONCLUSION: We found that using severe diarrhoea as an indicator for infection was not appropriate, and that screening symptomatic patients with the Kato-Katz method remained the most cost-effective approach in the given setting. The CER of treating all symptomatic patients on a presumptive basis depended very much on the drug price and the endemic level. PMID- 10747283 TI - Glucose-6-phosphate dehydrogenase deficiency in northern Vietnam. AB - Glucose-6-phosphate dehydrogenase (G6PD) deficiency was evaluated in 1676 schoolboys in northern Vietnam. The trait was nearly absent in boys of the Kinh (0.5%) and the Mong (0.7%) ethnic groups that traditionally have lived outside malaria transmission areas. Prevalences among ethnic groups living in the foothills, the breeding area of the main malaria vector Anopheles minimus, ranged from 9.7% to 31%. These findings support the hypothesis of a selective advantage of the trait in Plasmodium falciparum-endemic areas. PMID- 10747284 TI - Outcome of meningitis caused by Streptococcus pneumoniae and Haemophilus influenzae type b in children in The Gambia. AB - In developing countries, endemic childhood meningitis is a severe disease caused most commonly by Streptococcus pneumoniae or Haemophilus influenzae type b (Hib). Although many studies have shown that fatality rates associated with meningitis caused by these organisms are high in developing countries, little is known about the long-term outcome of survivors. The purpose of this study was to assess the importance of disabilities following pneumococcal and Hib meningitis in The Gambia. 257 children aged 0-12 years hospitalized between 1990 and 1995 with culture-proven S. pneumoniae (n = 134) or Hib (n = 123) meningitis were included retrospectively in the study. 48% of children with pneumococcal meningitis and 27% of children with Hib meningitis died whilst in hospital. Of the 160 survivors, 89 (55%) were followed up between September 1996 and October 1997. Of the children with pneumococcal meningitis that were traced, 58% had clinical sequelae; half of them had major disabilities preventing normal adaptation to social life. 38% of survivors of Hib meningitis had clinical sequelae, a quarter of whom had major disabilities. Major handicaps found were hearing loss, mental retardation, motor abnormalities and seizures. These data show that despite treatment with effective antibiotics, pneumococcal and Hib meningitis kill many Gambian children and leave many survivors with severe sequelae. Hib vaccination is now given routinely in The Gambia; an effective pneumococcal vaccine is needed. PMID- 10747285 TI - Training health workers to assess anaemia with the WHO haemoglobin colour scale. AB - WHO recommends that all pregnant women be screened for anaemia. In rural Africa this is often done by clinical examination which is known to have variable reliability. The recently developed WHO Haemoglobin Colour Scale may be the answer to this problem as it is simple and reliable. This study examines the training procedure recommended by WHO for the Haemoglobin Colour Scale when resources are very limited. We trained 7 laboratory technicians from the Medical Research Council Laboratories Hospital, Fajara, The Gambia and 13 Community Health Nurses (CHNs) from North Bank Division East, a rural area in The Gambia, to use the Colour Scale. The CHNs used the Scale to estimate haemoglobins on all new bookings to the antenatal clinics for a period of one month and recorded how they were managed. At the end of the study period they completed a qualitative questionnaire about the scale. Both groups of trainees were successfully trained although the WHO protocol for training was impossible to follow due to resource limitations. Eight of the 13 trained CHNs used the scale in practice and recorded 307 estimations with a mean haemoglobin of 9.1 g/dl. The results were normally distributed. Six of the 9 patients with Hb readings of < 4 g/dl were managed correctly. In response to the questionnaire the CHNs thought the scale was cheap, easy and quick to use and as good as the haemoglobinometer they had used previously. The main criticism was that it was not robust enough. The development of a low-technology, cheap, simple and reliable method for measuring haemoglobin is a welcome development. However, a simpler training procedure and a standard way of measuring observer performance are necessary. PMID- 10747286 TI - A health club for a community school in south-eastern Nigeria: influence on adult perception of onchocerciasis and compliance with community-based ivermectin therapy. AB - The impact of a school health club on adult perception of onchocerciasis and compliance with ivermectin was evaluated in an onchocerciasis-endemic community in southeastern Nigeria. Venous blood was collected from each of 26, 32 and 124 randomly selected subjects during ivermectin distribution programmes in 1995 1996 and 1997 respectively. Ivermectin concentrations were measured in the samples. Data was also collected from 334 and 319 randomly selected household heads or their representatives (aged 24 to 65 years) before and after health talks by schoolchildren, using interviewer-administered questionnaires. There was an increasing number of subjects who participated in control programmes (116 in 1995, 437 in 1996 and 2055 in 1997). Compliance with ivermectin treatment was low (53.9%) in 1995 but increased dramatically (90.1%) in 1997. A significant proportion (chi2 = 108.7, df = 1, P < 0.0001) of respondents knew about onchocerciasis after health education, predominantly from health workers (64.5%) before the tests and children (92.3%) after. Knowledge and beliefs about causative agents (chi2 = 266.4, df = 5, P < 0.0001), diagnostic method (chi2 = 207.4, df = 3, P < 0.0001), prevention (chi2 = 67.0, df = 4, P < 0.0001) of onchocerciasis and the effectiveness of ivermectin (chi2 = 40.4, df = 1, P < 0.0001) also differed significantly between the periods before and after tests. The school health club increased adult knowledge about onchocerciasis and its treatment. Schoolchildren could therefore supplement the information, education and communication (IEC) aspect of health care delivery in a community through such health clubs. PMID- 10747287 TI - Alteration of Egr-1 mRNA during multistage carcinogenesis in mouse skin. AB - Immediate early genes, including fos, jun, and early growth response-1 (Egr-1), are induced during cellular response to changes in extracellular environment. These immediate early genes are believed to mediate processes of cell growth and differentiation. In particular, Egr-1 is induced during mitogenic stimulation of a variety of cell types, including fibroblasts, B cells, and epithelial cells. In the present study, we examined Egr-1 gene expression during multistage carcinogenesis in mouse skin. After a single topical treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to SENCAR mouse skin, Egr-1 mRNA was induced, and maximal induction was observed at 2 h in both epidermis and dermis. Induction of Egr-1 mRNA by TPA was inhibited by fluocinolone acetonide, a potent inhibitor of tumor promotion by TPA. Egr-1 mRNA was present in primary keratinocytes derived from adult SENCAR mice. The keratinocyte cultures were maintained in low Ca(2+) medium, and Egr-1 mRNA levels became significantly elevated after the cultures were switched to high Ca(2+) medium. Additionally, a large proportion of primary papillomas and carcinomas generated from SENCAR mice by standard initiation-promotion regimens exhibited elevated Egr-1 mRNA compared with normal epidermis. Taken together, these data suggest a possible role of Egr 1 during multistage carcinogenesis in mouse skin. PMID- 10747288 TI - Gamma-irradiation induces matrix metalloproteinase II expression in a p53 dependent manner. AB - Matrix metalloproteinases (MMPs) are a family of proteinases that degrade the basement membrane and have been implicated in promoting tumor metastasis. MMP-2, one member of this family, was recently found to be a p53 target and subject to p53 upregulation. In this study, we examined the correlation between the expression of MMP-2 and the increased expression of p53 after gamma-irradiation. Three human p53-positive cell lines that express wild-type p53, including U2-OS (osteosarcoma), RKO (colon carcinoma), MCF-7 (breast carcinoma), one mouse p53 positive cell line and HepG2 (liver carcinoma), and two p53-negative human cell lines, SAOS-2 (osteosarcoma) and RKO-E6 (colon carcinoma), were used in this study. The MMP-2 activity was analyzed by using gelatin zymography. The p53 level was measured by western blot analysis. Our results show that wild-type p53 induced by ionizing radiation caused a subsequent increase of MMP-2 activity in U2-OS and RKO cells but not in MCF-7, HepG2, SAOS-2, or RKO-E6 cells. These results suggest that the gamma-radiation-induced expression of MMP-2 is dependent on the cell type and presence of functional p53. Thus, ionizing radiation could activate MMP-2 activity in a subset of human cancer cells and may lead to an increase in their metastatic potential. PMID- 10747289 TI - Fourteenth Aspen Cancer Conference: mechanisms of toxicity, carcinogenesis and cancer prevention. PMID- 10747290 TI - When the band begins to play: histone acetylation caught in the crossfire of gene control. AB - Increasing evidence from recent research suggests a connection between cancer and a deranged equilibrium of histone acetylation, which is maintained by two competing enzymatic activities, histone acetyltransferases (HATs) and histone deacetylases (HDACs). It is our hypothesis that a significant proportion of leukemias and possibly also solid tumors have abnormalities involving HATs or HDACs at the genomic level through genetic mutations or chromosomal alterations. In these cases, altered levels of HATs or HDACs may derange the tightly regulated equilibrium of histone acetylation, which may affect the expression of a broad spectrum of cellular genes. On the other hand, HATs and HDACs may be carried to defined target promoters as cofactors of transcription factor-bound repressor or enhancer complexes and thereby carry out unwanted enzymatic activities in the wrong place at the wrong time. We therefore propose a model for disease being associated with a deranged equilibrium of acetylation that affects histone proteins and promoter-bound transcription factors. PMID- 10747291 TI - Role of phosphorylated p50-NF-kappaB in the ultraviolet response of mouse skin. AB - The skin constitutes a primary target for stimuli such as ultraviolet (UV) radiation and tumor promoters, leading to both inflammatory and altered proliferative responses. Since the nuclear factor kappaB (NF-kappaB) family of transcription factors plays a major role in these biological processes, we sought to elucidate its expression in newborn mouse skin upon UV and 12-O tetradecanoylphorbol-13-acetate (TPA) exposures. We have identified the nuclear NF-kappaB binding activity in mouse skin as composed of p50/p65 heterodimers and p50 homodimers by supershift assays using different NF-kappaB-containing sequences. After UV exposure, but not TPA treatment, we detected increased NF kappaB binding activity that correlated with a decrease of IkappaBalpha protein levels, although it was not accompanied by p50 or p65 translocation. Immunostaining of newborn mouse sections confirmed that p50 was predominantly localized in the cytoplasm of epidermal basal cells before and after UV treatment. By immunoblotting, we found distinct phosphorylated forms of p50 in cytoplasmic extracts, while only a hyperphosphorylated form was detected in nuclear extracts. In vitro dephosphorylation of skin extracts dramatically reduced the affinity of p50-containing dimers for DNA. Our data suggest that the NF-kappaB response of mouse skin to UV exposure, contrary to most stimuli in other tissues, implies additional mechanisms other than translocation, such as p50 phosphorylation. PMID- 10747292 TI - Transfection of alpha(1,3)fucosyltransferase antisense sequences impairs the proliferative and tumorigenic ability of human colon carcinoma cells. AB - Sialyl Lewis x and sialyl Lewis a are oncodevelopmental antigens involved in the pathogenesis of colon adenocarcinoma. Biosynthesis of these glycans is controlled by alpha(1,3/1,4)fucosyltransferases. We report the disruption of sialyl Lewis x/a biosynthesis and inhibition of colon carcinoma cell proliferation by stable transfection of antisense sequences directed at the human Lewis alpha(1,3/1,4)fucosyltransferase gene, FUT3, and the plasma alpha(1,3)fucosyltransferase gene, FUT6. COLO-205 cells expressed high levels of sialyl Lewis x/a, alpha(1,3)fucosyltransferase activity, and FUT3/6 transcripts, but COLO14 micromol/L) was 25. 8%. After adjustment for major cardiovascular risk factors, serum albumin, and HbA(1c), the odds ratio (95% CI) for 5-year mortality was 1.56 (1.07 to 2.30) for hyperhomocysteinemia and 1.26 (1.02 to 1. 55) per 5-micromol/L increment of tHcy. The odds ratio for 5-year mortality for hyperhomocysteinemia was 1.34 (0.87 to 2.06) in nondiabetic subjects and 2.51 (1.07 to 5.91) in diabetic subjects (P=0.08 for interaction). CONCLUSIONS: Hyperhomocysteinemia is related to 5-year mortality independent of other major risk factors and appears to be a stronger (1.9-fold) risk factor for mortality in type 2 diabetic patients than in nondiabetic subjects. PMID- 10747343 TI - Carvedilol for prevention of restenosis after directional coronary atherectomy : final results of the European carvedilol atherectomy restenosis (EUROCARE) trial. AB - BACKGROUND: In addition to its known properties as a competitive, nonselective beta and alpha-1 receptor blocker, carvedilol directly inhibits vascular myocyte migration and proliferation and exerts antioxidant effects that are considerably greater than those of vitamin E or probucol. This provides the basis for an evaluation of carvedilol for the prevention of coronary restenosis. METHODS AND RESULTS: In a prospective, double-blind, randomized, placebo-controlled trial, 25 mg of carvedilol was given twice daily, starting 24 hours before scheduled directional coronary atherectomy and continuing for 5 months after a successful procedure. The primary end point was the minimal luminal diameter as determined during follow-up angiography 26+/-2 weeks after the procedure. Of 406 randomized patients, 377 underwent attempted atherectomy, and in 324 (88.9%), a 5.0 microg/L). On arrival, LV function and size were comparable between groups A (n=31) and B (n=16). LV ejection fraction, initially depressed (day 1: group A, 47+/-7% versus group B, 47+/-8%; P=NS), increased significantly in group A (day 360: 54+/-8%, P<0.001) but was unchanged in group B (day 360: 43+/-8%, P=NS). LV volumes increased significantly in group B (P<0. 05) but not in group A. Furthermore, patients in group B developed signs of restrictive LV diastolic filling. Multivariate regression analysis identified PIIINP >5.0 microg/L and deceleration 95% were obtained by selecting cells expressing eGFP. Overexpression of insulin receptors in HUVECs resulted in an approximately 3-fold increase in production of NO in response to insulin. In contrast, HUVECs overexpressing a tyrosine kinase-deficient mutant insulin receptor had a dose response curve similar to that of control cells. Overexpression of inhibitory mutants of either phosphatidylinositol 3-kinase (PI3K) or Akt resulted in nearly complete inhibition of insulin-stimulated production of NO. Overexpression of an inhibitory mutant of Ras had a much smaller effect. CONCLUSIONS: Receptor kinase activity is necessary to mediate production of NO through the insulin receptor. Both PI3K and Akt contribute importantly to this process, whereas the contribution of Ras is small. PMID- 10747348 TI - Genetic determinants of hemostasis phenotypes in Spanish families. AB - BACKGROUND: Recent studies have described genetic mutations that affect the risk of thrombosis as a result of abnormal levels of such hemostatic parameters as protein C, protein S, and the activated protein C resistance ratio. Although these mutations suggest that genes play a part in determining variability in some hemostasis-related phenotypes, the relative importance of genetic influences on these traits has not been evaluated. METHODS AND RESULTS: The relative contributions of genetic and environmental influences to a panel of hemostasis related phenotypes were assessed in a sample of 397 individuals in 21 extended pedigrees. The effects of measured covariates (sex, age, smoking, and exogenous sex hormones), genes, and environmental variables shared by members of a household were quantified for 27 hemostasis-related measures. All of these phenotypes showed significant genetic contributions, with the majority of heritabilities ranging between 22% and 55% of the residual phenotypic variance after correction for covariate effects. Activated protein C resistance ratio, activated partial thromboplastin time, and Factor XII showed the strongest heritabilities, with 71.3%, 83.0%, and 67.3%, respectively, of the residual phenotypic variation attributable to genetic effects. CONCLUSIONS: These results clearly demonstrate the importance of genetic factors in determining variation in hemostasis-related phenotypes that are components of the coagulation and fibrinolysis pathways and that have been implicated in risk for thrombosis. The presence of such strong genetic effects suggests that it will be possible to localize previously unknown genes that influence quantitative variation in these hemostasis-related phenotypes that may contribute to risk for thrombosis. PMID- 10747349 TI - Abnormalities of cardiac sympathetic innervation in arrhythmogenic right ventricular cardiomyopathy : quantitative assessment of presynaptic norepinephrine reuptake and postsynaptic beta-adrenergic receptor density with positron emission tomography. AB - BACKGROUND: The frequent provocation of ventricular tachycardia by stress or catecholamines and the efficacy of antiarrhythmic drugs with antiadrenergic properties suggest an involvement of the cardiac adrenergic system in arrhythmogenesis in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC). Previous studies demonstrated abnormalities of the presynaptic uptake-1 assessed by (123)I-MIBG-single-photon emission computed tomography. METHODS AND RESULTS: This study investigated neuronal reuptake of norepinephrine (uptake-1) and beta-adrenergic receptor density in 8 patients with ARVC and 29 age-matched control subjects. All subjects underwent positron emission tomography with the volume of distribution (V(d)) of [(11)C]hydroxyephedrine ((11)C-HED) used to assess presynaptic norepinephrine reuptake, the maximum binding capacity (B(max)) of [(11)C]CGP-12177 ((11)C-CGP-12177) to assess postsynaptic beta-adrenergic receptor density, and [(15)O]H(2)O for quantification of myocardial blood flow. Patients with ARVC demonstrated a highly significant global reduction in postsynaptic beta-adrenergic receptor density compared with that in control subjects (B(max) of (11)C-CGP-12177: 5.9+/-1.3 vs 10.2+/-2.9 pmol/g tissue, P<0.0007), whereas the presynaptic uptake-1 tended toward reduction only (V(d) of (11)C-HED: 59.1+/-25.2 vs 71.0+/-18.8 mL/g tissue, NS). There were no differences in myocardial blood flow between the groups, and plasma norepinephrine was within normal limits in patients and control subjects. CONCLUSIONS: The findings demonstrate a significant reduction of myocardial beta-adrenergic receptor density in patients with ARVC. This may result from a secondary downregulation after increased local synaptic norepinephrine levels caused by increased firing rates of the efferent neurons or as the result of impaired presynaptic catecholamine reuptake. These findings give new insights into the pathophysiology of arrhythmogenesis in ARVC, with potential impact on diagnostic evaluation and therapeutic management. PMID- 10747350 TI - Results of maze surgery for lone paroxysmal atrial fibrillation. AB - BACKGROUND: If drug refractoriness to paroxysmal atrial fibrillation (PAF) occurs, arrhythmia surgery that involves channelling and the exclusion of specific atrial areas can abolish atrial fibrillation. The purpose of this study was to establish the effectiveness and safety of maze III surgery to abolish PAF. METHODS AND RESULTS: Surgery was performed in 41 selected patients who had long standing, symptomatic, drug-refractory, lone PAF. At discharge, 35 patients (85%) were arrhythmia free, and 6 patients (15%) showed PAF and paroxysmal atrial tachycardia. Death or stroke did not occur during a mean follow-up of 31+/-16 months. At the end of follow-up, 39 patients (95%) had no PAF; however, in 2 patients (5%), PAF persisted and eventually required His bundle ablation and pacing. Three months after surgery, nodal escape rhythm was observed in only 1 patient, whereas sick-sinus syndrome emerged late after surgery in 2 patients. Antiarrhythmic drugs were used in 20% of patients during follow-up. The quality of life improved markedly after surgery and remained unchanged afterward. Echocardiographic findings did not alter, but exercise capacity increased. CONCLUSIONS: This pilot study demonstrates the effectiveness and safety of maze III surgery for lone PAF. In patients without sick-sinus syndrome, this intervention offers a sensible alternative to His bundle ablation and lifelong pacemaker dependency. PMID- 10747351 TI - Global right atrial mapping of human atrial flutter: the presence of posteromedial (sinus venosa region) functional block and double potentials : a study in biplane fluoroscopy and intracardiac echocardiography. AB - BACKGROUND: Previous studies of atrial flutter have found linear block at the crista terminalis; this was thought to predispose the patient to the arrhythmia. More recent observations, however, have demonstrated crista conduction. We sought to characterize the posterior boundary of atrial flutter. METHODS AND RESULTS: Patients with counterclockwise flutter (n=20), clockwise flutter (n=3), or both (n=5) were studied using two 20-pole catheters. Biplane fluoroscopy determined catheter positions. During counterclockwise flutter, craniocaudal activation occurred along the entire lateral and posterior right atrial walls. Septal activation proceeded caudocranially. In all patients, a line of block was seen in the posteromedial (sinus venosa) right atrium; this was manifested by the presence of double potentials where the upward and downward activations collided. Anatomic location was confirmed by intracardiac echocardiography in 9 patients. In patients with clockwise flutter, the line of block and double potentials were seen in the same location during counterclockwise flutter, but the activation sequence around the line of block was reversed. Pacing near the site of double potentials during sinus rhythm excluded a fixed line of block, and premature atrial complexes demonstrated functional block with manifest double potentials. In 2 patients, posterior ectopy organized to subsequently initiate isthmus dependent atrial flutter. CONCLUSIONS: (1) A functional line of block is seen at the posteromedial (sinus venosa region) right atrium during counterclockwise and clockwise atrial flutter. (2) All lateral wall right atrial activation can be uniform during flutter, without linear block or double potentials in the region of the crista terminalis. (3) Activation at the site of posteromedial right atrial functional block can organize to subsequently initiate isthmus-dependent atrial flutter. PMID- 10747352 TI - Enhanced cardiac contractility after gene transfer of V2 vasopressin receptors In vivo by ultrasound-guided injection or transcoronary delivery. AB - BACKGROUND: Systemic levels of arginine vasopressin (AVP) are increased in congestive heart failure, resulting in vasoconstriction and reduced cardiac contractility via V(1) vasopressin receptors. V(2) vasopressin receptors (V2Rs), which promote activation of adenylyl cyclase, are physiologically expressed only in the kidney and are absent in the myocardium. Heterologous expression of V2Rs in the myocardium could result in a positive inotropic effect by using the endogenous high concentrations of AVP in heart failure. METHODS AND RESULTS: We tested gene transfer with a recombinant adenovirus for the human V2R (Ad-V2R) to stimulate contractility of rat or rabbit myocardium in vivo. Ultrasound-guided direct injection or transcoronary delivery of adenovirus in vivo resulted in recombinant receptor expression in the myocardial target area, leading to a substantial increase in [(3)H]AVP binding. In 50% of the cardiomyocytes isolated from the directly injected area, single-cell shortening measurements detected a significant increase in contraction amplitude after exposure to AVP or the V2R specific desmopressin (DDAVP). Echocardiography of the target myocardial area documented a marked increase in local fractional shortening after systemic administration of DDAVP in V2R-expressing animals but not in control virus treated hearts. Simultaneous measurement of global contractility (dP/dt(max)) confirmed a positive inotropic effect of DDAVP on left ventricular function in the Ad-V2R-injected animals. CONCLUSIONS: Adenoviral gene transfer of the V2R into the myocardium increases cardiac contractility in vivo. Heterologous expression of cAMP-forming receptors in the myocardium could lead to novel strategies in the therapy of congestive heart failure by bypassing the desensitized beta-adrenergic receptor-signaling cascade. PMID- 10747353 TI - Inhibition of early atherogenesis by losartan in monkeys with diet-induced hypercholesterolemia. AB - BACKGROUND: Angiotensin II may contribute to atherogenesis by facilitating the proliferative and inflammatory response to hypercholesterolemia. This study determined, in a primate model of diet-induced atherosclerosis, the effect of AT(1) blockade on fatty-streak formation, plasma lipids, and surrogate markers of vascular injury. METHODS AND RESULTS: Male cynomolgus monkeys fed a diet containing 0.067 mg cholesterol/kJ for 20 weeks were given losartan (180 mg/d, n=6) or vehicle (n=8) for 6 weeks starting at week 12 of the dietary regimen. Arterial pressure, heart rate, plasma total and lipoprotein cholesterol concentrations, and lipoprotein particle sizes and subclass distributions were unaffected by treatment. Losartan caused significant (P<0.05) increases in plasma angiotensin II and angiotensin-(1-7). Compared with vehicle-treated controls, losartan reduced the extent of fatty streak in the aorta, the coronary arteries, and the carotid arteries by approximately 50% (P<0.05). A significant (P<0.05) reduction in the susceptibility of LDL to in vitro oxidation, serum levels of monocyte chemoattractant protein-1, and circulating monocyte CD11b expression were also associated with losartan treatment. In addition, serum levels of vascular cell adhesion molecule-1 and E-selectin did not change during treatment but increased after discontinuation of losartan. Serum C-reactive protein, platelet aggregability, and white cell counts were not modified by losartan. CONCLUSIONS: This study demonstrates for the first time an antiatherogenic effect of AT(1) receptor blockade in nonhuman primates. Losartan inhibited fatty-streak formation through mechanisms that may include protection of LDL from oxidation and suppression of vascular monocyte activation and recruitment factors. PMID- 10747354 TI - Cell-surface estrogen receptors mediate calcium-dependent nitric oxide release in human endothelia. AB - BACKGROUND: Although estrogen replacement therapy has been associated with reduction of cardiovascular events in postmenopausal women, the mechanism for this benefit remains unclear. Because nitric oxide (NO) is considered an important endothelium-derived relaxing factor and may function to protect blood vessels against atherosclerotic development, we investigated the acute effects of physiological levels of estrogen on NO release from human internal thoracic artery endothelia and human arterial endothelia in culture. METHODS AND RESULTS: We tested the hypothesis that estrogen acutely stimulates constitutive NO synthase activity in human endothelial cells by acting on a cell-surface receptor. NO release was measured in real time with an amperometric probe. 17beta Estradiol exposure to internal thoracic artery endothelia and human arterial endothelia in culture stimulated NO release within seconds in a concentration dependent manner. 17beta-Estradiol conjugated to bovine serum albumin also stimulated NO release, suggesting action through a cell-surface receptor. Tamoxifen, an estrogen receptor inhibitor, antagonized this action. We further showed with the use of dual emission microfluorometry that 17beta-estradiol stimulated release of endothelial NO was dependent on the initial stimulation of intracellular calcium transients. CONCLUSIONS: Physiological doses of estrogen immediately stimulate NO release from human endothelial cells through activation of a cell-surface estrogen receptor that is coupled to increases in intracellular calcium. PMID- 10747357 TI - Mentors for the new millennium : advice to a young cardiologist PMID- 10747356 TI - Alteration of ventricular fibrillation by flecainide, verapamil, and sotalol: an experimental study. AB - BACKGROUND: The purpose of this study was to determine whether the myocardial electrophysiological properties are useful for predicting changes in the ventricular fibrillatory pattern. METHODS AND RESULTS: Thirty-two Langendorff perfused rabbit hearts were used to record ventricular fibrillatory activity with an epicardial multiple electrode. Under control conditions and after flecainide, verapamil, or d,l-sotalol, the dominant frequency (FrD), type of activation maps, conduction velocity, functional refractory period, and wavelength (WL) of excitation were determined during ventricular fibrillation (VF). Flecainide (1.9+/-0.3 versus 2.4+/-0.6 cm, P<0. 05) and sotalol (2.1+/-0.3 versus 2.5+/-0.5 cm, P<0.05) prolonged WL and diminished FrD during VF, whereas verapamil (2.0+/ 0.2 versus 1. 7+/-0.2 cm, P<0.001) shortened WL and increased FrD. Simple linear regression revealed an inverse relation between FrD and the functional refractory period (r=0.66, P<0.0001), a direct relation with respect to conduction velocity (r=0.33, P<0.01), and an inverse relation with respect to WL estimated during VF (r=0.49, P<0.0001). By stepwise multiple regression, the functional refractory periods were the only predictors of FrD. Flecainide and sotalol increased the circuit size of the reentrant activations, whereas verapamil decreased it. The 3 drugs significantly reduced the percentages of more complex activation maps during VF. CONCLUSIONS: The activation frequency is inversely related to WL during VF, although a closer relation is observed with the functional refractory period. Despite the diverging effects of verapamil versus flecainide and sotalol on the activation frequency, WL, and size of the reentrant circuits, all 3 drugs reduce activation pattern complexity during VF. PMID- 10747355 TI - Inhibition of transplant vasculopathy in a rat aortic allograft model after infusion of anti-inflammatory viral serpin. AB - BACKGROUND: Transplant vasculopathy remains a difficult therapeutic problem, resulting in the majority of late cardiac graft losses. This chronic vascular disease is thought to be triggered by alloantigen-dependent and alloantigen independent inflammatory factors. Despite improved 1-year survival, the incidence of transplant vasculopathy has not improved with current immunosuppressive protocols. Highly effective strategies have evolved in the large DNA viruses that shield infecting viruses from host inflammatory responses. Serp-1 is a secreted myxoma virus anti-inflammatory serine proteinase inhibitor. Serp-1 inhibits plasminogen activators in a manner similar to plasminogen activator inhibitor (PAI-1), a vascular protein that plays a pivotal regulatory role in vascular wound healing. In this study, we tested the ability of purified Serp-1 protein to ameliorate posttransplant vasculopathy after rat aortic allograft surgery. METHODS AND RESULTS: Serp-1 protein or controls were infused into 98 rats immediately after segmental aortic allograft transplantation. After either late (28 days, 64 rats) or early (12 to 48 hours, 24 rats) follow-up, transplanted aortic segments were harvested for morphological and immunohistochemical analysis. Significant reductions in intimal plaque growth (P<0.002) and mononuclear cell invasion (P<0.033) were detected after Serp-1 infusion at nanogram doses. Serp-1 reduced early macrophage (P<0.0016) and nonspecific lymphocyte (P<0.0179) invasion into medial and adventitial layers and inhibited associated depletion of medial smooth muscle cells (P<0.0006). CONCLUSIONS: Infusion of a viral anti-inflammatory serpin, Serp-1, significantly reduces early inflammatory responses and later luminal occlusion in a rat aortic allograft model. PMID- 10747358 TI - Thrombi in acute coronary syndromes : revisited and revised. PMID- 10747359 TI - Images in cardiovascular medicine. Giant J waves in hypothermia. PMID- 10747360 TI - Left upper pulmonary vein stenosis 2 months after radiofrequency catheter ablation of atrial fibrillation. PMID- 10747361 TI - Drugs and disease. PMID- 10747362 TI - Safety net in jeopardy. PMID- 10747363 TI - Recommendations for the nutritional management of patients with diabetes mellitus. PMID- 10747364 TI - Fecal incontinence: a clinical approach. AB - "Fecal incontinence" is defined as the involuntary loss of stool at any time of life after toilet training. It is a socially and psychologically devastating condition for patients and their families, and a topic which both patients and physicians are reluctant to approach. Although the true prevalence of fecal incontinence is unknown, studies have reported it to be as high as 2. 2% in the general population, with significantly higher rates among nursing home residents and hospitalized elderly. Risk factors include advancing age, female gender and multiparity. An understanding of pelvic floor anatomy and physiology is required to appreciate how diverse medical conditions can affect mechanisms involved in normal continence. The rectum serves as a storage reservoir until elimination can take place at a socially acceptable time and place. The pelvic floor muscles help to regulate the defecatory process and maintain continence. These muscles include the internal anal sphincter, the external anal sphincter and the puborectalis muscle. Each muscle contributes to normal continence, although the relative importance of each is controversial. Neurologic integrity and sensation are also key factors. Conditions associated with fecal incontinence include diarrheal states, fecal impaction, idiopathic neurologic injury, surgical and obstetric injury, pelvic trauma, collagen vascular disease, and neurologic impairment related to stroke, diabetes, or multiple sclerosis. Evaluation of the patient with fecal incontinence includes a directed history and physical examination, with particular attention paid to integrity of the perineum and rectum, and a complete neurologic evaluation. Diagnostic tools such as stool studies, anorectal manometry, defecography, electromyography, pudendal nerve conduction, and endoanal ultrasound may be employed in an outpatient setting. Fecal incontinence may be treated conservatively by employing such methods as dietary restriction, stool bulking agents, and biofeedback. Surgery may be the best option for cases refractory to medical treatment, or for those patients with rectocele or obstetrical injury. In this article, we review the presentation, epidemiology, pathophysiology, and etiology of fecal incontinence. Evaluation, including key components of directed history and physical examination, and the appropriate use of diagnostic studies and indications for treatment options are also addressed. PMID- 10747365 TI - GERD and its complications. AB - Gastroesophageal reflux disease (GERD) is the condition that results when gastric material that refluxes into the esophagus or oropharynx causes symptoms, tissue injury, or both. Endoscopic examination usually is not required merely to establish a diagnosis of GERD, but endoscopy is the best diagnostic test for Barrett's esophagus, a sequela of GERD that predisposes to esophageal adenocarcinoma. Patients found to have Barrett's esophagus will require regular endoscopic surveillance for early, curable neoplasia. Esophageal pH monitoring is useful for patients who have symptoms or signs suggestive of GERD, but who have little or no response to antisecretory therapy. For patients who have severe, ulcerative reflux esophagitis, the clinician has only two reasonable therapeutic options: (1) lifelong antisecretory therapy with proton pump inhibitors or (2) antireflux surgery. There are no absolute indications for antireflux surgery, but the operation can be considered for patients with severe GERD who are unwilling to accept lifelong medical therapy or for young patients whose GERD symptoms respond only to proton pump inhibitors administered in high dosages. PMID- 10747366 TI - Regulation of urea transporter proteins in kidney and liver. AB - Due to urea's role in producing concentrated urine, its transport is critically important to the conservation of body water. Within the renal inner medulla, urea is transported by both facilitated and active urea transport mechanisms. The vasopressin-regulated, facilitated urea transporter (UT-A1) in the terminal inner medullary collecting duct (IMCD) permits high rates of transepithelial urea transport and results in delivery of large quantities of urea into the deepest portions of the inner medulla where it is needed to maintain a high interstitial osmolality for maximal urine concentration. Four cDNA isoforms of the UT-A urea transporter family have been cloned. In addition, there are three secondary active, sodium-dependent, urea transport mechanisms in IMCD subsegments: (1) active urea secretion in the apical membrane of the terminal IMCD from untreated rats; (2) active urea absorption in the apical membrane of the initial IMCD from low-protein fed or hypercalcemic rats; and (3) active urea absorption in the basolateral membrane of the initial IMCD from furosemide-treated rats. This review will focus on integrative studies of the rapid and long-term regulation of urea transporters in rats with reduced urine concentrating ability. These studies led to the surprising result that the basal-facilitated urea permeability in the terminal IMCD and UT-A1 protein abundance are increased during in vivo conditions associated with an impaired urine concentrating ability. In contrast, there are two response patterns of active urea transporters: (1) hypercalcemia, a low protein diet, and furosemide result in induction of active urea absorption in the initial IMCD, albeit by different mechanisms, and inhibition of active urea secretion in the terminal IMCD; while (2) water diuresis results in up-regulation of active urea secretion in the terminal IMCD without any active urea absorption in the initial IMCD. The first pattern contributes to the urine concentrating defect by increasing urea delivery to the base of the inner medulla, thus decreasing urea delivery distally to the inner medullary tip. The second response pattern will directly decrease urea content in the deep inner medulla. UT-A urea transporters are also expressed outside the kidney. Recent studies show that the liver has phloretin-inhibitable urea transport and that it occurs via a 49 kDa UT A protein. When rats are made uremic, the abundance of this 49 kDa UT-A protein increases in the liver in vivo. This up-regulation of the 49 kDa UT-A protein may allow hepatocytes to increase ureagenesis to reduce the accumulation of ammonium and/or bicarbonate in uremia. PMID- 10747367 TI - Hepatitis C virus, autoimmunity and lymphoproliferation. AB - We summarize clinical, laboratory and pathologic details regarding a patient who presented with extrahepatic disease manifestations of hepatitis C virus (HCV) infection, including cryoglobulinemic leg ulcers due to cutaneous vasculitis, peripheral sensorimotor neuropathy, and recurrent pulmonary infiltrates. The patient had evidence for B-cell lymphoproliferation, diagnosed as extranodal lymphoma on initial (though not subsequent) bone marrow examination, retroperitoneal lymphadenopathy, and the presence of a Type II IgM6 monoclonal rheumatoid factor which became cryoprecipitable on complexing to IgG. Chronic hepatitis was mild on liver biopsy, though fibrotic changes developed over a three-year period of follow-up. She had consistently normal liver function tests, except for a brief rebound effect on discontinuing interferon-alpha, and preterminally. Symptoms were only partially responsive to trials of corticosteroids, cytotoxic agents, plasmapheresis and interferon, and the patient ultimately died at The Mount Sinai Hospital of sepsis. We review current information regarding the spectrum of extrahepatic HCV infection, including pathogenic factors relevant to its overlapping autoimmune, rheumatic and lymphoproliferative disease manifestations. The exact prevalence of these HCV related syndromes among the 1% of the world population estimated to be infected by this virus remains to be delineated. Chronicity of infection, and lack of efficacy of currently available therapy in effecting sustained clearance of the virus from the host, have made this an important public health problem that is likely to increase in significance. Possible relationships to non-Hodgkin's lymphoma may present novel opportunities to delineate the basis for oncogenesis in HCV infection. PMID- 10747368 TI - To be a physician. PMID- 10747369 TI - The Bell Commission: ethical implications for the training of physicians. AB - In 1989, the New York State Legislature enacted New York State Code 405 in response to the death of a patient in a New York City hospital. Code 405 was the culmination of a report (the Bell Commission Report) that implicated the training of residents as part of the problem leading to that tragic death. This paper explores the consequences of the regulatory changes in physician training. The sleep deprivation of house officers was considered a major issue requiring correction. There is little evidence to support the claim that sleep deprivation is a serious cause of medical misadventures. Nevertheless, the changes in house officers' working hours and responsibilities have profound implications. Changes in the time allotted to teaching, the ability to learn from patients admitted after a shift is over, and the increasing loss of continuity, all may have a negative impact on physician training. It is not clear that trainees are being realistically prepared for the actual practice of medicine - physicians often work extended hours. The most serious concern that has been raised is the loss of professionalism by physicians. Residents are now viewing themselves as hourly workers, and the State has intervened in an area of training formerly left to the profession to manage. We are now training doctors in New York State who will be comfortable working in an hourly wage setting, but not in the traditional practice of medicine as it has been in the United States during this century. We are concerned that this may sever the bond between doctor and patient - a bond that has been the bedrock of our conception of a physician. PMID- 10747370 TI - The ethics of placebo prescribing. AB - The prescribing of placebos brings into focus the practice of being truthful to patients. The term "placebo" has a generally pejorative connotation in medicine, although the Latin is "I shall please." More recently, the definition of "placebo" has expanded to include "medication without proven pharmacological activity." Yet it is known that the use of placebos often leads to substantial improvement. The effectiveness of placebos is a challenging and troubling issue for physicians. PMID- 10747371 TI - Genetic testing: a physician's perspective. AB - Progress in DNA diagnostics has been extremely rapid. We sought to determine attitudes, awareness, and knowledge of genetic testing by physicians affiliated with the Mount Sinai Medical Center. We surveyed 363 physicians within whose fields genetic testing for various diseases and disorders exist. Physicians' awareness of and opinions regarding testing, attitudes toward counseling, knowledge of the field, and interest in further education were assessed. Three hundred forty-one (341) physicians were determined to be eligible for the study and, of these, 89 (26%) returned completed surveys. Of the respondents, 71% rated their knowledge of genetics and genetic testing as "fair" to "poor"; only 37% read articles concerning genetic testing on a regular basis. Physician awareness of currently available testing produced a bell-shaped distribution. Knowledge regarding Mendelian genetics yielded a bimodal distribution, and knowledge reflecting an understanding of the mechanics behind genetic testing produced a bell-like curve, skewed to the right. Those who identified themselves as practicing within an "academic" setting scored significantly higher on the Mendelian genetics and testing mechanics sections than those practicing in a "private" setting. Ninety-eight percent (98%) of the physicians said they would refer their patients to a genetic counselor. Although 91% of the respondents were aware of the existence of genetic counseling services, only 71% were aware of the services available at major New York medical centers. Of those aware of counseling services, 53% had referred a patient to them, and 83% of those who referred were "mostly" to "very" satisfied with the counseling. Ninety-five percent (95%) of the physicians believed that the doctor, among others, has the responsibility to counsel patients about genetic testing, yet only 51% felt that they had the time. No statistically significant preference was found concerning the methods for gaining further education or information about genetic testing. Further education for physicians is required in order for them to accurately convey the risks and benefits of genetic testing to their patients. Furthermore, awareness of the counseling services available within the New York area needs to be heightened in order to provide physicians and patients with the specific services they desire. The most efficient and effective methods for providing information and for heightening awareness need to be determined through additional research. PMID- 10747372 TI - Replacement of the pyloric sphincter with the ileocecal valve: an experimental study. AB - BACKGROUND: Several surgical methods have been devised and applied to overcome the complications associated with the loss of the pyloric sphincter after distal gastrectomy. However, none of these methods creates an efficient sphincteric mechanism at the anastomotic site. The purpose of this experimental study in dogs was to replace the pylorus with the ileocecal valve and determine whether its sphincteric function would be preserved in its new location without affecting gastrointestinal motility and the health of the animals. METHODS: Thirteen dogs underwent surgical removal of the pyloric sphincter and a partial distal gastrectomy. The ileocecal valve, with a short segment of ileum, was then relocated so that the ileal segment was anastomosed to the stomach while the cecal segment was anastomosed to the duodenum. Intestinal continuity was reestablished by anastomosing the distal ileum with the ascending colon. Intraileal and intracolic pressures were measured in all animals prior to and following transposition of the ileocecal valve. In 3 of these animals, pre pyloric (intragastric) and post-pyloric (intraduodenal) pressures were also measured before the pylorus was removed. Pressure measurements on both sides of the transposed ileocecal valve were performed again 4-6 months later. All pressure measurements were made directly with a water manometer. Radiographic and fluoroscopic studies were carried out on all animals to assess gastrointestinal motility, gastric emptying times, and the sphincteric competence of the transposed ileocecal valve. Hematological and biochemical studies intended to assess the nutritional status of all animals were carried out. Also, postoperative measurements were made of gastric basic acid output. RESULTS: All animals were alive and well 4-6 months after the initial operative procedure. Hematological studies and biochemical tests and studies of liver function remained normal. There was a slight reduction in serum B12 levels and, as expected, a significant postoperative reduction in gastric basic acid output. The intraluminal pressure measurements and the radiographic and fluoroscopic studies all showed that the sphincteric mechanism of the ileocecal valve was preserved in its new location, that gastrointestinal motility was not impaired, and that the healthy condition of the animals was maintained. Gross and histological examination of the transposed segments of the intestinal tract did not reveal any abnormalities. CONCLUSION: Because the anatomy and physiology of the human alimentary tract are similar to those of the dog, this technique may be applicable clinically, when indicated, to avoid and/or relieve complications resulting from gastrectomy, when those complications do not respond or have not responded to conservative management. PMID- 10747374 TI - An integrated program for evidence-based medicine in medical school. AB - To provide optimal care for their patients, clinicians must be able to locate and interpret the most current literature. Teaching the necessary skills to medical students is essential, if we wish to train clinicians to be able to keep up with the expansion of biomedical knowledge for their entire working lives. In this paper, we describe our school's four-year curriculum in evidence-based medicine and the performance of three senior classes on the summative evaluation exercise devised to measure the program's success. PMID- 10747373 TI - Gallstone ileus and Crohn's disease without biliary-enteric fistula: report of a unique case. AB - Gallstone ileus is an uncommon cause of small bowel obstruction, accounting for fewer than 3% of laparotomies for intestinal obstruction. Patients with long standing Crohn's disease have an increased risk of developing gallstone disease. However, gallstone ileus is not common in these patients. We report the case of a 70-year-old female with Crohn's disease who presented with gallstone ileus, and present a review of the literature. We discuss the association between gallstone ileus and Crohn's disease, and the treatment options for these patients. We emphasize the importance of including gallstone ileus in the differential diagnosis in patients presenting with intestinal obstruction, especially patients with long-standing Crohn's disease. We advocate the early utilization of computerized tomography to confirm the diagnosis, and prompt early surgical intervention. PMID- 10747375 TI - Clinical trials in hypertension: an uncertain impact on physician practice. PMID- 10747376 TI - Long-term confusion. PMID- 10747377 TI - Bonus of contention. PMID- 10747378 TI - House conversions. PMID- 10747379 TI - Bright ideas. PMID- 10747380 TI - Monitoring diabetes. AB - The incidence of type 2 diabetes in older people is high and getting higher. This article forms the second part of a practical guide to the management of diabetes for nurses working in long-term elderly care settings. It can also be used as a resource when teaching junior staff and health care assistants. The first part, published in the June-July edition of NT Nursing Homes, looked at the Issues involved in diagnosis and treatment. PMID- 10747382 TI - Take a swallow. PMID- 10747381 TI - Wound care. Part 3. PMID- 10747384 TI - Templates, "wheeled reagents", and a new route to rotaxanes by anion complexation: the trapping method AB - The topological aspects of rotaxanes are compared with those of the other families of mechanically interlocked molecules: catenanes and molecular knots. The role of the different types of templates in the well-known threading and clipping procedures often used for rotaxane synthesis are discussed. Finally the conceptually new trapping method that is based on the action of supramolecular nucleophiles formed by anionic stopper-wheel complexes is described. PMID- 10747383 TI - Molar maintenance. PMID- 10747385 TI - Advances in homogeneous and heterogeneous catalysis with metal-containing silsesquioxanes AB - Metal-containing silsesquioxane derivatives provide new catalysts with both homogeneous and heterogeneous applicability. The steric and electronic properties of silsesquioxane silanolate ligands render metal centers more Lewis acidic than conventional alkoxide or siloxide ligands do. This concept has been exploited in newly developed catalysts for alkene metathesis, polymerization, epoxidation, and Diels-Alder reactions of enones. Other applications are envisioned in the near future. PMID- 10747386 TI - Energy-gap dependence of photoinduced charge separation and subsequent charge recombination in 1,4-phenylene-bridged zinc--free-base hybrid porphyrins AB - A series of 1,4-phenylene-bridged ZP-HP hybrid porphyrins (ZP = zinc porphyrin, HP = free-base porphyrin) 1-8 ZH have been prepared in which an electron-donating ZP moiety is kept constant and electron-accepting HP moieties are varied by introducing electron-accepting substituents, so that the energy gap for charge separation, ZP-1HP*--> ZP(+)-HP-, covers a range of about 0.9 eV in DMF. Here selective excitation at the HP moiety was employed to avoid complication in the determination of electron transfer rates derived from energy transfer, 1ZP*-HP - > ZP-1HP*. Definitive evidence for the electron transfer has been obtained in three solvents (benzene, THF, and DMF) through picosecond-femtosecond transient absorption studies, which have allowed the determination of the rates of the photoinduced charge separation, ZP-1HP* --> ZP(+)-HP-, and subsequent thermal charge recombination ZP(+)-HP- --> ZP-HP. Dyad 1ZH in THF exhibits a biphasic fluorescence decay that indicates thermal repopulation of the ZP-1HP* from ZP(+) HP-; this has been also supported by the transient absorption spectra. On this ground, the energy levels of the ZP(+)-HP- ion pairs have been estimated. Similar biphasic fluorescence decay has been observed for 5 ZH in benzene; this allows furhter estimation of the energy level of the ZP(+)-HP- ion pairs. The free energy-gap dependence (energy-gap law) has been probed from the normal to the upper limit region for the rate of the charge separation alone, and only the inverted region for the rate of the charge recombination. It was not possible to reproduce both energy-gap dependencies of the charge separation and the charge recombination assuming common parameter values for the reorganization energy and electronic interaction responsible for the electron transfer with the classical Marcus equation. Although both energy-gap dependencies can be approximately reproduced by means of the simplified semiclassical equation, which takes into consideration the effect of the high-frequency vibrations replaced by one mode of averaged frequency, many features, which include the effects of solvent polarity, electron-tunneling matrix element, and so forth on the energy-gap law, are considerably different from those of the previous studied porphyrin-quinone systems with weaker inter-chromophore electronic interactions. PMID- 10747387 TI - Towards synthetic adrenaline receptors. AB - The macrocyclic bisphosphonate 2 forms complexes with amino alcohols, amines, and amino acid esters with high association constants in polar organic solvents. Exertion of solvophobic interactions inside the macrocyclic cavity in DMSO and methanol leads to specificity for guest molecules with hydrophobic moieties. Experimental evidence is presented for the insertion of the guest molecules' nonpolar groups into the macrocycle's hydrophobic cavity. NMR spectra of complexes with 2 in DMSO show a molecular imprint of the guest molecule; this gives information about its location inside the macrocycle. In aqueous solutions strong self-association of 2 occurs, which is explained by distinct structural similarities between 2 and micelle-forming phospholipids. PMID- 10747388 TI - A new organic nanoporous architecture: dumb-bell-shaped molecules with guests in parallel channels AB - A new type of dumb-bell-shaped host molecule (6-8) has been synthesised, of which 1,8-bis((1)-adamantyl)-1,3,5,7-octatetrayne (8 = BAOT) forms an open porous architecture when cocrystallised with a number of typical solvent molecules. Adamantyl substituents attached to a tetraalkyne spacer build up the walls of parallel channels wherein guest molecules are aligned. Surprisingly, the tetraalkyne unit is significantly bent. Desolvation experiments provide evidence for a reversible inclusion of guests. In the case of the inclusion of 2-butanone, a partial substitution by symmetrical and asymmetrical long-chain chromophores during crystallisation was possible. Stained crystals showed optical frequency doubling. The crystal structure analysis revealed a centric space group, although considerable translational and orientational disorder was present. Application of scanning pyroelectric microscopy revealed that the growth of inclusion compounds with 2-butanone produced polar ordering of guest molecules, which were aligned in two macro-domains of opposing polarity. The resulting orientation of the carbonyl dipoles is in agreement with the theoretical prediction of a Markov model of spontaneous polarity formation based on molecular recognition processes on growing crystal faces. The present case represents a new example of a property driven supramolecular synthesis. PMID- 10747389 TI - A comparative repair study of thymine- and uracil-photodimers with model compounds and a photolyase repair enzyme. AB - Cyclobutane uridine and thymidine dimers with cis-syn-structure are DNA lesions, which are efficiently repaired in many species by DNA photolyases. The essential step of the repair reaction is a light driven electron transfer from a reduced FAD cofactor (FADH ) to the dimer lesion, which splits spontaneously into the monomers. Repair studies with UV-light damaged DNA revealed significant rate differences for the various dimer lesions. In particular the effect of the almost eclipsed positioned methyl groups at the thymidine cyclobutane dimer moiety on the splitting rates is unknown. In order to investigate the cleavage vulnerability of thymine and uracil cyclobutane photodimers outside the protein environment, two model compounds, containing a thymine or a uracil dimer and a covalently connected flavin, were prepared and comparatively investigated. Cleavage investigations under internal competition conditions revealed, in contrast to all previous findings, faster repair of the sterically less encumbered uracil dimer. Stereoelectronic effects are offered as a possible explanation. Ab initio calculations and X-ray crystal structure data reveal a different cyclobutane ring pucker of the uracil dimer, which leads to a better overlap of the pi*-C(4)-O(4)-orbital with the sigma*-C(5)-C(5')-orbital. Enzymatic studies with a DNA photolyase (A. nidulans) and oligonucleotides, which contain either a uridine or a thymidine dimer analogue, showed comparable repair efficiencies for both dimer lesions. Under internal competition conditions significantly faster repair of uridine dimers is observed. PMID- 10747390 TI - Dimeric capsules by the self-assembly of triureidocalix AB - A number of calix[6]arenes bearing ureas at the upper rim positions of alternate rings 1, 3 and 5 were prepared and studied in detail by NMR spectroscopy and gel permeation chromatography. N-Unsubstituted ureas were shown to dimerize through a cyclic array of hydrogen bonds to give cylindrical cavities capable of encapsulating small molecules such as dichloromethane, benzene and fluorobenzene. Slow equilibria between dimer and monomer were observed in [D6]DMSO-CDCl3 mixtures. By contrast, N-substituted ureas are monomeric. All urea monomers with bulky O-substituents display a solvent-dependent, slow equilibrium between C3v and Cs cone conformations. PMID- 10747391 TI - Different C-C coupling reactions of permethyltitanocene and permethylzirconocene with disubstituted 1,3-butadiynes AB - Herein we describe different C-C coupling reactions of permethyltitanocene and zirconocene with disubstituted 1,3-butadiynes. The outcomes of these reactions vary depending on the metals and the diyne substituents. The reduction of [Cp2*MCl2] (Cp* = C5Me5; M = Ti, Zr) with Mg in the presence of disubstituted butadiynes RC triple bond C-C triple bond CR' is suitable for the synthesis of different C-C coupling products of the diyne and the permethylmetallocenes, and provides a new method for the generation of functionalized pentamethyl cyclopentadienyl derivatives. For M = Zr and R = R' = tBu, the reaction gives, by a twofold activation of one pentamethylcyclopentadienyl ligand, the complex [Cp*Zr[-C(=C=CHtBu)-CHtBu-CH2-eta5-C5Me3-CH2-]] (3), containing a fulvene ligand that is coupled to the modified substrate (allenic subunit). When using the analogous permethyltitanocene fragment "Cp2*Ti", the reaction depends strongly on the substituents R and R'. The coupling product of the butadiyne with two methyl groups of one of the pentamethylcyclopentadienyl ring systems, [Cp*Ti[eta5-C5Me3 (CH2-CHR-eta2-C2-CHR'-CH2)]], is obtained with R = R' = tBu (4) and R = tBu, R' = SiMe3 (5). In these complexes one pentamethylcyclopentadienyl ligand is annellated to an eight-membered ring with a C-C triple bond, which is coordinated to the titanium center. A different activation of both pentamethylcyclopentadienyl ligands is observed for R = R' = Me, resulting in the complex [[eta5-C5Me4(CH2)-]Ti[-C(=CHMe)-C(=CHMe)-CH2-eta5-C5Me4]] (6), which displays a fulvene as well as a butadienyl-substituted pentamethylcyclopentadienyl ligand. The influence exerted by the size of the metal is illustrated in the reaction of [Cp2*ZrCl2] with MeC triple bond C-C triple bond CMe. Here the five-membered metallacyclocumulene complex [Cp2*Zr(eta4 1,2,3,4-MeC4Me)] (7) is obtained. The reaction paths found for R = R' = Me are identical to those formerly described for R = R' = Ph. PMID- 10747392 TI - A mechanistic study of the FeO+-mediated decomposition pathways of phenol, anisol, and their thio analogues. AB - The gas-phase oxidations of phenol, anisol, thiophenol, and thioanisol by 'bare' FeO+ are examined by using Fourier transform-ion cyclotron resonance (FT-ICR) and tandem mass-spectrometry. Reaction mechanisms are derived on the basis of isotope labeling experiments, MS/MS studies, and comparison with structural isomers, that is ions formed by independent routes. The chemistry of all substrates is determined by the functional groups, whereas reactions typical of unsubstituted benzene with FeO+ are suppressed. For phenol and thiophenol, four-membered metallacycles are obtained concomitant with a regioselective loss of water, which involves the O atom from the FeO+ entity and hydrogen atoms originating from the functional group and from the ortho position of the ring. C-H bond cleavage of the methoxy group (kH/kD = 2.0) is rate-contributing for the degradation of metastable anisol/FeO+, which is featured by highly regioselective losses of H2O, HCO, H2CO, and [C,H2,O2]. In the oxidation of thioanisol, two different C-H bond activation mechanisms are operating, resulting in the elimination of [Fe,H,O,S] concomitant with the formation of the benzyl cation (kH/kD = 4.7), and loss of water (kH/kD = 2.5). The reactions of independently generated, formal S- and C oxidation intermediates of thioanisol indicate the occurrence of extensive structural isomerizations prior to dissociation. For anisol and thioanisol, analogies and differences between oxidation reactions catalyzed by the enzyme cytochrome P-450 in the condensed phase and those observed for the gas-phase model FeO+ are discussed. PMID- 10747393 TI - Bis- and tetrakis(organosilyl) decatungstosilicate, AB - The high propensity of organosilanes towards polycondensation and reaction with nucleophilic moieties has facilitated the formation of new organic-inorganic hybrids based on the lacunary divacant heteropolyanion [gamma-SiW10O36]8-. Depending on the experimental conditions two different types of derivatives were obtained with the general formula [gamma-SiW10O36(RSi)2O]4- (1) (>90% yield) and [gamma-SiW10O36(RSiO)4]4- (2) (>85% yield) (R = H (1a, 2a), vinyl (1b), C3H6OC(O)C-(Me)=CH2 (1c, 2c), phenyl (1d, 2d)). The structures of the hybrid anions have been inferred from spectroscopic data, in particular from multinuclear (29Si and 183W) NMR solution studies and from MALDI-TOF mass spectrometry. Both species correspond to the grafting of an oxo-bridged siloxane unit onto the surface of the lacunary polyoxoanion. PMID- 10747394 TI - Synthesis of the sialyl Lewis X epitope attached to glycolipids with different core structures and their selectin-binding characteristics in a dynamic test system. AB - Sialyl Lewis X (sLeX)/selectin-mediated leukocyte rolling along endothelial cells has recently gained wide interest. In this paper the influence of the spacer length of laterally clustered neoglycolipids 1a-d on cell rolling in a dynamic test system is investigated. The required di-O-hexadecyl glycerols with none, and with three, six, or nine ethylene glycol units as spacer groups (compounds 4a-d) could be readily obtained. The synthesis of 1-O-thexyldimethylsilyl-protected sLeX 24 was based on sialylation of 2,3,4-O-unprotected galactose derivative 11 with sialyl phosphite 8 as donor; this afforded the desired disaccharide 12, which was transformed into trichloroacetimidate 14 as disaccharide donor. Reaction of 3-O-unprotected glucosamine derivative 18 with fucosyl donor 20 afforded disaccharide 21, which was transformed into the 4-O-unprotected derivative 23. Reaction of 14 with 23 furnished the desired tetrasaccharide 24 in good yield. Transformation of 24 into the trichloroacetimidate 26 as donor, followed by the reaction with 4a-d as acceptor gave, after deprotection, the target molecules 1a-d. For comparison, 4d was also connected with a sialyl residue (-->31) and with an N-acetylglucosamine residue (-->34). Compounds 1c and 1d with a hexaethylene glycol and a nonaethylene glycol spacer, respectively, were much more efficient in mediating selectin-dependent cell rolling in the dynamic test system than compounds 1a and 1b, which had no spacer (1a), or only a triethylene glycol spacer (1b). PMID- 10747395 TI - Development of an unusually highly enantioselective hetero-Diels-Alder reaction of benzaldehyde with activated dienes catalyzed by hypercoordinating chiral aluminum complexes AB - The effect of Lewis acid catalysis of the hetero-Diels-Alder reaction between benzaldehyde and activated dienes (e.g. the Danishefsky's diene) has been investigated. In the present work we decided to study a series of chiral aluminum complexes as potential catalysts for the hetero-Diels-Alder reaction in order to gain a better understanding of the effect on the chiral induction of varying the steric and electronic environment of the metal ion. The results of this study prompted us to conclude that steric effects in the ligand coordination sphere and hypercoordination are strongly contributing factors to the optical yield of the reaction. Optimization of the reaction culminated in the synthesis of the hetero Diels-Alder product in 99.4% ee and 97% yield of the isolated product. Based on the experimental results the mechanism for the hetero-Diels-Alder reaction is discussed and it is postulated that hypercoordination to the chiral aluminum Lewis acid center is of importance for the reaction. PMID- 10747396 TI - A new view on chemistry of solids in solution--cryo energy-filtered transmission electron microscopy (cryo-EFTEM) imaging of aggregating palladium colloids in vitreous ice AB - It is shown that by using cryo-transmission electron microscopy (cryo-TEM) it is possible to image the aggregation behaviour of nanoparticles while they are still in solution. This technique has allowed the study of the arrangement of colloidal palladium particles in solution by preparing the specimen by the plunge-freezing technique. This method of rapidly cooling the specimen avoids rearrangement of the particles during specimen preparation. The palladium particles were identified by energy-filtered cryo-TEM. The aggregation of particles in solution was studied as a function of pH and ionic strength. The results can be used as recommendations for colloidal solutions intended for deposition of single particles. PMID- 10747397 TI - Soluble-polymer-supported synthesis of beta-lactams on a modified poly(ethylene glycol) AB - A modified poly(ethylene glycol) (PEG) has been developed for the soluble-polymer supported synthesis of beta-lactams. The monomethylether of PEG (MeOPEG) with an average M(W) of 5000 was used as the support, a 4-(3-propyl)phenyl residue as the spacer, and a 4-oxyphenylamino group as the moiety with the reactive functionality. From this modified PEG representative aromatic, heteroaromatic, unsaturated, and aliphatic imines were obtained in high yields by different procedures. The polymer-supported imines were then employed to prepare several beta-lactams by enolate/imine condensation and ketene/imine cycloaddition. Examples of the control of the absolute stereochemistry during the azetidinone ring formation are also reported. The reactions carried out on the polymer-bound imines showed a remarkable similarity to those performed on nonimmobilized imines, both in terms of yields and stereoselectivities. Removal of the beta lactams from the polymer has also been accomplished to directly deliver the N unsubstituted azetidinones. PMID- 10747398 TI - Probing the mechanisms of enantioselective hydrogenation of simple olefins with chiral rhodium catalysts in the presence of anions AB - The strong influence of various anions upon the hydrogenation of 2-phenyl-1 butene, catalyzed by chiral rhodium catalysts was investigated. Both sulfonates and halides exert large increases in the enantioselectivity when [Rh[(-) bdpp](NBD)]ClO4 (bdpp = 2,4-bis(diphenylphosphino)pentane, NBD = 2,5 norbornadiene) is used as the catalyst precursor at high pressures (70 atm) of dihydrogen in nonpolar solvents. A dihydride mechanism similar to that for Wilkinson's catalyst [RhCl(PPh3)3] was shown to be operating at both high- and low-pressure conditions through a combination of catalytic studies, 31P, 1H and parahydrogen-induced polarization (PHIP) NMR experiments. With sulfonate and in neat methanol, however, a mechanistic switch takes place from a dihydride route (dihydrogen addition before olefin binding) at high pressure to an unsaturate route (olefin binding before dihydrogen addition) at low pressures (<30 atm). Olefin isomerization is inhibited by halide addition, but occurs with sulfonate and in neat methanol through what is most likely a pi-allyl mechanism. A detailed understanding of the effects of addition of these anions is crucial for development of new classes of catalysts capable of efficient enantioselective reduction of prochiral olefins lacking a secondary polar binding group. PMID- 10747400 TI - Interdigitation, interpenetration and intercalation in layered cuprous tricyanomethanide derivatives AB - Reaction of Cu(I), tricyanomethanide (tcm , C(CN)3-) and L = either hexamethylenetetramine (hmt), 4,4'-bipyridine (bipy) or 1,2-bis(4-pyridyl)ethene (bpe) gives crystals of [Cu(tcm)(hmt)] (1), [Cu(tcm)(bipy)] (2) and [Cu(tcm)(bpe)] x 0.25 bpe x 0.5 MeCN (3), respectively. Crystal structure analysis shows 1-3 all contain closely related puckered (4,4) sheets composed of tetrahedral Cu(I) ions bridged by 2-connecting tcm- and L. The crystal packing, however, varies markedly with L. In 1 the sheets interdigitate in pairs. In 2 the sheets participate in parallel interpenetration in pairs. In 3 guest bpe and MeCN molecules are intercalated in channels formed by the stacking of the sheets. PMID- 10747399 TI - Nonenzymatic template-directed reactions on altritol oligomers, preorganized analogues of oligonucleotides. AB - Altritol nucleic acids (ANAs) are RNA analogues with a phosphorylated D-altritol backbone. The nucleobase is attached at the 2-(S)-position of the carbohydrate moiety. We report that ANA oligomers are superior to the corresponding DNA, RNA, and HNA (hexitol nucleic acid) in supporting efficient nonenzymatic template directed synthesis of complementary RNAs from nucleoside-5'-phosphoro-2-methyl imidazolides. Activated ANA and HNA monomers do not oligomerize efficiently on DNA, RNA, HNA, or ANA templates. PMID- 10747401 TI - Chemoenzymatic synthesis and fluorescent visualization of cell-surface selectin bound sialyl Lewis x derivatives. AB - Sialyl Lewis x (sLe(x)) derivatives conjugated to readily visualized molecular labels are useful chemical probes to study selectin-carbohydrate interactions. Localization of the selectins on the surface of leukocytes and activated endothelial cells can be detected through fluorescence of bound selectin ligands. Herein we present a short chemoenzymatic synthesis of a fluorescently labeled bivalent sLe(x) conjugate. The use of an amino-substituted monovalent sLe(x) to obtain fluorescent- and biotin-labeled sLe(x) derivatives is also described. The cell-staining utility of the fluorescent sLe(x) conjugates is demonstrated for a HUVEC cell line expressing E-selectin and for CHO-K1 cells expressing either L- or E-selectin. PMID- 10747402 TI - 1,2-Diacetals in synthesis: total synthesis of a glycosylphosphatidylinositol anchor of Trypanosoma brucei. AB - A full account on a total synthesis of GPI anchor 1 employing butanediacetal (BDA) groups and a chiral bis(dihydropyran) is presented. The reactivity of selenium and thio glycosides was tuned by the use of BDA groups. This allowed the assembly of an appropriately protected GPI anchor precursor 2 in just six steps from the six building blocks 5-10 including only one protecting group manipulation. myo-Inositol was desymmetrised with the bis(dihydropyran) derivative 15 and appropriately protected to give inositol acceptor 21 in nine steps and 17% overall yield. The use of common starting materials and BDA protections give efficient access to building blocks 5, 6, 7 and 8. A new and improved synthesis of the glucosamine donor 28 is included. In summary, a highly convergent and efficient synthesis of GPI anchor 1, which is clearly adaptable to other GPI anchors, has been reported. PMID- 10747404 TI - Enantioselective enzymes for organic synthesis created by directed evolution. AB - A new concept for the creation of enzymes displaying improved enantioselectivity in a given reaction is described; it is based on "evolution in the test tube". Accordingly, proper molecular biological methods for random mutagenesis, gene expression, and high-throughput screening systems for the rapid assay of enantioselectivity are combined. Several rounds of mutagenesis and screening are generally necessary in order to create mutant enzymes that show high degrees of enantioselectivity, as in the case of the lipase-catalyzed hydrolytic kinetic resolution of a chiral ester in which the original enantioselectivity of 2 % ee (E = 1) increases to > 90% ee (E = 25). PMID- 10747403 TI - A self-assembled cylindrical capsule: new supramolecular phenomena through encapsulation. AB - The synthesis and spectroscopic characterization of self-assembled cylindrical capsule 1a x 1a of nanometer dimensions is described. Encapsulation studies of large organic guest molecules were performed by using 1H NMR sprectroscopy in [D12]mesitylene solution. In addition to the computational (MacroModel 5.5, Amber* force field) analysis of the capsule's shape and geometry, an experimental approach towards estimation of the internal cavity dimensions is described. This involves using series of homologous molecular "rulers" (e.g. aromatic amides 5a i). The available space inside the capsule 1a x 1a can be estimated as 5.7 x 14.7 A (error +/- 0.2 A) with this technique. Dibenzoyl peroxide is readily encapsulated in [D12]mesitylene and was shown to be stable to decomposition for at least three days at 70 degrees C inside the capsule. Moreover, 1a x 1a prevents the encapsulated peroxide from oxidizing Ph3P or diphenyl carbazide present in solution. The normal chemical reactivity of the peroxide is restored by release from the capsule by DMF, a solvent that competes for the hydrogen bonds that hold the capsule together. The protection and release of encapsulated species augurs well for the application of capsules in catalysis and delivery. PMID- 10747405 TI - Hollow capsule processing through colloidal templating and self-assembly AB - Hollow capsules of nanometer to micrometer dimensions constitute an important class of materials that are employed in diverse technological applications, ranging from the delivery of encapsulated products for cosmetic and medicinal purposes to their use as light-weight composite materials and as fillers with low dielectric constant in electronic components. Hollow capsules comprising polymer, glass, metal, and ceramic are nowadays routinely produced by using various chemical and physicochemical methods. The current article focuses on a recent novel and versatile technique, based on a combination of colloidal templating and self-assembly processes, developed for synthesizing uniform hollow capsules of a broad range of materials. The strategy outlined readily affords control over the size, shape, composition, and wall thickness of the hollow capsules. PMID- 10747406 TI - Bridging the final gap in stereocontrolled wittig reactions: methoxymethoxy-armed allylic phosphorus ylides affording conjugated dienes with high cis selectivity AB - After treatment with an appropriate base (butyllithium or sodium amide), 2 alkenyltris(2-methoxymethoxyphenyl)phosphonium salts carrying an allyl, crotyl, or prenyl (3-methyl-2-butenyl) side chain condense with saturated or unsaturated aldehydes to give conjugated dienes with Z/E ratios ranging from 90:10 to > 99:1 and averaging 96:4. Owing to steric congestion, yields are only moderate (on average 41%; extremes 10-79%). The nonvolatile tris(2 methoxymethoxyphenyl)phosphine oxide by-product can be readily isolated and reduced to recover the phosphane starting material, or it may be hydrolyzed to the water-soluble tris(2-hydroxyphenyl)phosphine oxide. PMID- 10747407 TI - Assembly of silver(I) polymers with helical and lamellar structures AB - The new versatile multidentate nonchelating ligand 1,2-bis[(2-pyr-imidinyl) sulfanylmethyl]benzene (bpsb) was designed and prepared for supramolecular syntheses. Self-assembly between silver nitrate and the bpsb ligand resulted in the polymer [Ag4(bpsb)2-(NO3)4]n (1) with a single-stranded helical chain structure. Each bpsb ligand in 1 acts as a tetradentate ligand, in which two sulfur atoms and two nitrogen atoms from different pyrimidine groups coordinate to four Ag atoms in four different directions. The nitrate anions serve as a template for the formation of the helix and are either embedded in the interior of the helix or located in the flank of the helix. Self-assembly between silver perchlorate and the bpsb ligand under the same conditions gave rise to the polymer [Ag2(bpsb)3(ClO4)2]n (2) comprising a two-dimensional lamellar network containing crownlike cavities. The silver atoms in two adjacent layers are arranged staggered in 2. The two-dimensional lamellar network comprising isolated cavities of [Ag6(bpsb)6] is very different from that of usual honeycomb structures. PMID- 10747408 TI - Asymmetric allyl-metal bonding in substituted (eta3-allyl) palladium complexes: X ray structures of cis- and trans-4-acetoxy- AB - Enantiomerically pure cis and trans isomers of 4-acetoxy-[eta3(1,2,3) cyclohexenyl]palladium chloride dimers (cis-1 and trans-1) were prepared from enantiomerically pure trans-1-acetoxy-4-chloro-2-cyclohexene. X-ray analyses of these complexes show that in the trans complex (trans-1) the six-membered ring prefers a chair conformation, whereas in the cis complex (cis-1) the cyclohexenyl ring has a boat conformation. According to the X-ray structure of trans-1 the Pd C3 bond is shorter than the other allylic terminal palladium-carbon bond (Pd-C1). On the other hand, in cis-1 the Pd-C3 and Pd-C1 bond lengths are identical within the experimental error. The calculated structures (B3PW91/LANL2DZ + P) of trans-1 and cis-1 also display differences in the allylpalladium bonding. The asymmetric allylpalladium bonding in trans-1 is explained on the basis of pi-sigma electronic interactions between the 4-acetoxy substituent and the allyl-metal moiety. PMID- 10747409 TI - Synthesis, characterization, and isomerization of an iridabenzvalene AB - Metal- halogen exchange of vinylcyclopropene 4 followed by addition of [(Me3P)2Ir(CO)Cl] produced iridabenzvalene 5. The metallabenzene valence isomer was characterized by NMR and IR spectroscopy and by X-ray crystallography. Isomerization of complex 5 to iridabenzene derivatives 6 and 13 was accomplished by thermolysis and by Ag+ ions, respectively. The former transformation was a clean and quantitative process that displayed first-order kinetics. PMID- 10747410 TI - Calix AB - Chiral calix[4]arene derivatives with four O-(N-acetyl-PhgOMe), (1), (Phg denotes R-phenylglycine), or O-(N-acetyl-LeuOMe) (2) strands have been synthesised. Both compounds exist in chloroform in stable cone conformations with a noncovalently organised cavity at the lower rim that is formed by circular interstrand amidic hydrogen bonds. Such organisation affects both the selectivity and extraction/transport properties of 1 and 2 toward metal cations. Calix[4]arene derivatives with one OCH2COPhgOMe strand (3), two OCH2COPhgOMe strands (5) and with 1,3-OMe-2,4-(O-CH2COPhgOMe) substituents (4) at the lower rim have also been prepared. For 3, a conformation stabilised by a circular hydrogen-bond arrangement is found in chloroform, while 4 exists as a time-averaged C2 conformation with two intramolecular NH ...OCH3 hydrogen bonds. Compound 5 has a unique hydrogen-bonding motif in solution and in the solid state with two three centred NH-.. O and two OH...O hydrogen bonds at the lower rim. This motif keeps 5 in the flattened cone conformation in chloroform. The X-ray structure analysis of 1 revealed a molecular structure with C2 symmetry; this structure is organised in infinite chains by intra- and intermolecular H bonds. The solid-state and solution structures of the [1-Na]ClO4 complex are identical, C4 symmetric cone conformations. PMID- 10747411 TI - A novel approach towards intermolecular stabilization of para-quinone methides. First complexation of the elusive, simplest quinone methide, 4-methylene-2,5 cyclohexadien-1-one AB - A novel approach towards the intermolecular stabilization of "simple" (i.e. methylene-unsubstituted) p-quinone methides (QMs) by their coordination to a transition-metal center is described. 4-Bromomethyl phenols, protected by a silyl group, were employed as the QM precursors and cis-chelating diphosphine Pd0 complexes were chosen as the metal precursors, since they have strong back bonding interactions with the electron-poor QM moiety. Removal of the silyl protecting-group from the corresponding [LPd(benzyl)Br] complex (L=bisphosphine) with fluoride results in the spontaneous rearrangement of the unobserved zwitterionic Pd(II) complex into the QM-Pd0 complex. The feasibility of this approach was demonstrated in the synthesis of the structurally characterized Pd0 complex of BHT-QM (4), a biologically relevant metabolite of 2,6-di-tert-butyl-p cresol, and the synthesis of the complex of 4-methylene-2,5-cyclohexadien-1-one (11), the simplest, and so far unobserved QM molecule. These complexes exhibit a remarkable thermal stability and do not react with alcohol or water. In both cases, the use of an appropriate incoming ligand allowed the release of the coordinated QM into the reaction media in which it was effectively trapped by added nucleophiles. PMID- 10747412 TI - Protease catalysis mediated by a substrate mimetic: a novel enzymatic approach to the synthesis of carboxylic acid amides. AB - We present a protease-based method for the coupling of non-coded and non-amino acid-derived amines with carboxy components. The key feature of this approach is the combination of the substrate-mimetic strategy with the ability of the cysteine protease clostripain to accept a wide spectrum of amines. Firstly, we tested the use of the 4-guanidinophenyl ester leaving group to mediate acceptance of non-coded and non-amino-acid-derived acyl residues. This employed beta-amino acid and simple carboxylic acid moieties as acyl donors, and several amino acid and peptide units as acyl acceptors. The study was completed by the use of non amino-acid-derived acyl acceptors comprising simple amines, amino alcohols, and diamines. The results indicate that the approach presented is a useful strategy for the synthesis of peptide isosteres, peptide analogues, and organic amides. These last open a new range of synthetic applications of proteases completely beyond peptide synthesis, achieving efficient and selective acylations of non amino-acid-derived amines under extraordinarily mild reaction conditions. PMID- 10747413 TI - Experimental and theoretical investigations on the synthesis, structure, reactivity, and bonding of the stannylene-iron complex Bis bis(2-tert-butyl-4,5,6 trimethyl-phenyl)SnFe (eta6-toluene) (Sn-Fe-Sn) AB - The pi-(arene)bis(stannylene) complex bis(bis(2-tert-butyl-4,5,6 trimethylphenyl)SnFe(eta6-toluene) (Sn-Fe-Sn, 15) is accessible in high yields by a metal-atom-mediated synthesis between iron atoms, toluene, and the stannylene [bis(2-tert-butyl-4,5,6-trimethylphenyl)Sn](3). Complex 15 has a half-sandwich structure with short Fe -Sn bonds (2.432(1) A) and a trigonal-planar coordination at both the Fe and Sn atoms. The distance between the two Sn centers is 3.56 A. Complex 15 is stable under ambient conditions and displays a pi-arene lability, so far rarely observed for (arene)iron complexes; this leads to an irreversible substitution of the arene and formation of fivefold-coordinated zerovalent iron complexes. The pi-arene lability of the title compound is a result of the Fe-Sn bonding situation, which can be interpreted, on the basis of an extended Huckel molecular orbital calculation, as being solely a donation of the 5sigma lone-pair of Sn into empty or half-filled acceptor d orbitals on Fe. As the calculations reveal, there is little backbonding from the iron to the tin, and the strong sigma donation leads to an increased occupation of the pi-antibonding orbitals of the eta6-arene, which are mainly responsible for the experimentally observed arene lability. Fe and Sn Mossbauer spectra support the polar character of Sn(sigma+)-->Fe(sigma-) with strong sigma donation from tin to iron, but significantly low iron-to-tin pi backdonation. PMID- 10747414 TI - Reaction of HO* with guanine derivatives in aqueous solution: formation of two different redox-active OH-adduct radicals and their unimolecular transformation reactions. Properties of G(-H)*. AB - The reaction of *OH with 2'-deoxyguanosine yields two transient species, both identified as OH adducts (G*-OH), with strongly different reactivity towards O2, or other oxidants, or to reductants. One of these, identified as the OH adduct at the C-8 position (yield 17% relative to *OH), reacts with oxygen with k=4 x 10(9)M(-1)s(-1); in the absence of oxygen it undergoes a rapid ring-opening reaction (k = 2 x 10(5) s(-1) at pH4-9), visible as an increase of absorbance at 300-310 nm. This OH adduct and its ring-opened successor are one-electron reductants towards, for example, methylviologen or [Fe(III)(CN)6]3-. The second adduct, identified as the OH adduct at the 4-position (yield of 60-70% relative to *OH), has oxidizing properties (towards N,N,N',N'-tetra-methyl-p phenylenediamine, promethazine, or [Fe(II)(CN)6]4-). This OH adduct undergoes a slower transformation reaction (k = 6 x 10(3) s(-1) in neutral, unbuffered solution) to produce the even more strongly oxidizing (deprotonated, depending on pH) 2'-deoxyguanosine radical cation, and it practically does not react with oxygen (k< or = 10(6)M(-1)s(-1)). The (deprotonated) radical cation, in dilute aqueous solution, does not give rise to 8-oxoguanosine as a product. However, it is able to react with ribose with k< or =4 x 10(3)M(-1)S(-1). PMID- 10747415 TI - Electron transfer in DNA from guanine and 8-oxoguanine to a radical cation of the carbohydrate backbone. AB - Photolysis of a 4'-pivaloyl-substituted nucleotide in single- and double-stranded DNA (1) generated an enol ether radical cation 4 that was reduced to enol ether 17 by electron transfer from the nearest guanoside (G). Variation of the nucleotide sequence demonstrated a strong distance dependence of this electron transfer rate with beta = 1.0 +/- 0.1 A(-1). When 8-oxoguanosine (G(oxo)) was used as the electron donor, the rate of the electron transfer increased by a factor of 4 but the distance dependence of the transfer remained unchanged within experimental error. In single strands, the number of intervening A, T, and C nucleotides had a much smaller effect; the rate remained nearly constant for two, three, or four intervening nucleotides. This is explained by the flexibility of the single-stranded oligonucleotides. PMID- 10747416 TI - Metal telluride clusters composed of niobocene carbonyl, telluride, and cobalt carbonyl units: syntheses, structures, and reactivity AB - Abstract: The reaction of [Cp#2NbTe2H] (1#; Cp# = Cp* (C5Me5) or Cp(x) (C5Me4Et)) with two equivalents of [Co2(CO)8] gives a series of cobalt carbonyl telluride clusters that contain different types of niobocene carbonyl fragments. At 0 degrees C, [Cp#2NbTe2CO3(CO)7] (2#) and [Co4Te2(CO)10] (3) are formed which disappear at higher temperatures: in boiling toluene a mixture of [cat2][Co9Te6(CO)8] (5#) (cat= [Cp#2Nb(CO)2]+) and [cat2][Co11Te7(CO)10] (6#) is formed along with [cat][Co(CO)4] (4#). Complexes 6# transform into [cat][Co11Te7(CO)10] (7#) upon interaction with HPF6 or wet SiO2. The molecular structures of 2(Cp(x)), 4(Cp(x)), 5(Cp*), 6(Cp*) and 7(Cp*) have been determined by X-ray crystallography. The structure of the neutral 2(Cp(x)) consists of a [Co3(CO)6Te2] bipyramid which is connected to a [(C5Me4Et)2Nb(CO)] fragment through a mu4-Te bridge. The ionic structures of 4(Cp(x)), 5(Cp*), 6(Cp*) and 7(Cp*) each contain one (4, 7) or two (5, 6) [Cp#2Nb(CO)2]+ cations. Apart from 4, the anionic counterparts each contain an interstitial Co atom and are hexacapped cubic cluster anions [Co9Te6(CO)8]2- (5) or heptacapped pentagonal prismatic cluster anions [Co11Te7(CO)10]n- (n=2: [6]2- , n=1: [7]-), respectively. Electrochemical studies established a reversible electron transfer between the anionic clusters [Co11,Te7(CO)10]- and [Co11Te7(CO)10]2in 6# and 7# and provided evidence for the existence of species containing [Co11Te7(CO),0] and [Co11Te7(CO)0]3-. The electronic structures of the new clusters and their relative stabilities are examined by means of DFT calculations. PMID- 10747417 TI - The formation of heterodimers by vancomycin group antibiotics. AB - The formation of heterodimers in mixtures of glycopeptide antibiotics has been detected by electrospray ionization mass spectrometry (ESI-MS), and dimerization constants have been determined. By using NMR spectroscopy, it has been shown that these heterodimers indeed exist in aqueous solution. The dimerization constants obtained by NMR spectroscopy are in good agreement with those determined by ESI MS. Structural information on the heterodimer interface of some of the heterodimers is obtained by using two-dimensional NMR techniques and reveals that these heterodimers are similar in structure to the homodimers. PMID- 10747418 TI - Palladium-catalyzed synthesis of cephalotaxine analogues. AB - The synthesis of cephalotaxine ring analogues 10 was achieved by two successive intramolecular palladium-catalyzed reactions of 12 via 11, namely an allylic amination and a Heck reaction. The substrates 12 were obtained by alkylation of primary amines 13 with tosylates 14. PMID- 10747419 TI - Reversible dioxygen binding and phenol oxygenation in a tyrosinase model system. AB - The complex [Cu2(L-66)]2+ (L-66 = a,a'-bis?bis[2-(1'-methyl-2' benzimidazolyl)ethyl]amino?-m-xylene) undergoes fully reversible oxygenation at low temperature in acetone. The optical [lambda(max) = 362 (epsilon 15000), 455 (epsilon 2000), and 550 nm (epsilon 900M(-1)cm(-1))] and resonance Raman features (760 cm(-1), shifted to 719cm(-1)(-1) with 18O2) of the dioxygen adduct [Cu2(L 66)(O2)]2+ indicate that it is a mu-eta2:eta2-peroxodicopper(II) complex. The kinetics of dioxygen binding, studied at - 78 degrees C, gave the rate constant k1 = 1.1M(-1) 5(-1) for adduct formation, and k(-1) =7.8 x 10(-5)s(-1), for dioxygen release from the Cu2O2 complex. From these values, the O2 binding constant K= 1.4 x 10(4)M(-1) at -78 degrees C could be determined. The [Cu2(L 66)(O2)]2+ complex performs the regiospecific ortho-hydroxylation of 4 carbomethoxyphenolate to the corresponding catecholate and the oxidation of 3,5 di-tert-butylcatechol to the quinone at -60 degrees C. Therefore, [Cu2(L-66)]2+ is the first synthetic complex to form a stable dioxygen adduct and exhibit true tyrosinase-like activity on exogenous phenolic compounds. PMID- 10747420 TI - ZrIV-tetraphenylporphyrinates as nuclease mimics: structural, kinetic and mechanistic studies on phosphate diester transesterification. AB - The Zr(IV)-tetraphenylpor-phyrinates Zr(TPP)(X,X'), (X,X' = -OAc, -OMe, Cl ) 4-6, 8 were prepared and their complexing properties as well as catalytic properties towards solvolysis of the phosphate diesters hpp (2), dmp (3) and pmp (16) characterised. The diesters 2 and 16, representing model phosphates for RNA and DNA, were substrates for the catalyst Zr(TPP)Cl2 (4), and rate accelerations over background by 6-9 orders of magnitude were measured. These accelerations are comparable to those of dinuclear transition metal catalysts and lanthanide ions. Catalytic turnover was observed. Kinetic studies revealed that the catalytically active species of 4 in the solvolysis of 2 and 16 in methanol-containing solvents are dinuclear complexes containing either one or two phosphate esters depending upon the phosphate concentration. Besides the usual solvolysis pathway of the RNA model hpp (2), which proceeds via the cyclophosphate 20, a second, unusual pathway via direct substitution of the hydroxypropyl substituent was found. X-ray analysis of the Zr(TPP)(dmp) complex 19 revealed a dinuclear structure with two bridging dmp ligands and one monomethyl phosphate unit. In 19 one of the two dmp residues occurs in a very unusual high energy ac,ap conformation. Based on this structure and on the kinetic data, mechanistic models for the two solvolysis reaction pathways were developed. From an extensive CSD search on phosphodiester structures no correlation between P-O ester bond lengths and diester conformations could be found. However, P-O ester bonds decrease in length with increasing formal charge of the complexing metal ions. This underlines the higher importance of electrostatic activation relative to stereoelectronic effects in phosphodiester hydrolysis. PMID- 10747421 TI - Gas-phase reactions of nitronium ions with acetylene and ethylene: an experimental and theoretical study AB - A comparative study of the gas-phase reactions of NO2+ with acetylene and ethylene was performed by using FT-ICR, MIKE, CAD, and NfR/ CA mass spectrometric techniques, in conjunction with ab initio calculations at the MP2/6-31+G* level of theory. Both reactions proceed according to the same mechanism, that is, 1,3 dipolar cycloaddition, but yield products of different stability. The C2H2NO2+ adduct from acetylene has an aromatic character and hence is highly stabilized with respect to the C2H4NO2+ adduct from ethylene. Both cycloadducts tend to isomerize into O-nitroso derivatives, that is, nitrosated ketene and nitrosated acetaldehyde, which represent the thermodynamically most stable products from the addition of NO2+ to acetylene and ethylene, respectively. As prototypal examples of the reactivity of free nitronium ions with most simple pi systems, the reactions investigated are useful starting points to model the mechanism of aromatic nitration. PMID- 10747422 TI - Synthesis, structures and theoretical investigation of AB - The silylated derivative of thiophosphoric acid (S)P(SSiMe3)3 is used as a convenient starting compound for the synthesis of multinuclear Cu and Au cluster complexes. (S)P(SSiMe3)3 reacts with CuCI/PPh3 and [AuCClPPh3] to give the following compounds: [Cu4(P2S6)(PPh3)4] (1), [Cu6(P2S6)Cl2-(PPh3)6] (2) and [Au4(P2S6)(PPh3)4](3). According to X-ray structure determination, these compounds contain P2S6(4-) ions, in which S atoms act as ligands for Cu+ and Au+ ions. Although 1 and 3 have the same stoichiometry, bonding of the metal ions to the P2S6 skeleton displays small but remarkable differences. Au is twofold coordinated, whereas Cu shows a threefold coordination. Ab initio calculations have been carried out to rationalise these structural differences. The theoretical treatment of the corresponding Ag compound indicates the latter to be less stable. PMID- 10747423 TI - Hybrid molecular magnets obtained by insertion of decamethyl-metallocenium cations into layered, bimetallic oxalate complexes: AB - A new series of hybrid organometallic - inorganic layered magnets with the formula [Z(III)Cp*2][M(II)M(III)(ox)3] (Z(III) = Co, Fe; M(III) = Cr, Fe; M(II) = Mn, Fe, Co, Cu, Zn; ox = oxalate; Cp* = pentamethylcyclopentadienyl) has been prepared. All of these compounds are isostructural and crystallize in the monoclinic space group C2/m, as found by X-ray structure analysis. Their structure consists of an eclipsed stacking of the bimetallic oxalate-based extended layers separated by layers of organometallic cations. These salts show spontaneous magnetization below To, which corresponds to the presence of ferro-, ferri-, or canted antiferromagnetism. Compounds in which the paramagnetic deca methylferrocenium is used instead of the diamagnetic decamethylcobaltocenium are good examples of chemically constructed magnetic multilayers with alternating ferromagnetic and paramagnetic layers. The physical properties of this series have been thoroughly studied by means of magnetic measurements and ESR and Mossbauer spectroscopy. We have found that the two layers are electronically quasiindependent. As a consequence, the bulk properties of these magnets have not been significantly affected by the insertion of a paramagnetic layer of S = 1/2 spins in between the extended layers. In fact, the critical temperatures remain unchanged even when comparing [MCp*2]+ derivatives with [XR4]+ compounds (X = N, P; R = Ph, nPr, nBu). Nevertheless, the presence of the paramagnetic layer has been shown to have some influence on the hysteresis loops of these compounds. In the same context, the spin polarization of the paramagnetic units (which arises from the internal magnetic field created by the bimetallic layers in the ordered state) has been observed by Mossbauer and ESR spectroscopy. PMID- 10747424 TI - Methodology and clinical applications of blood pressure and heart rate analysis. PMID- 10747425 TI - ICNP and telematic applications for nurses in Europe. The telenurse experience. PMID- 10747426 TI - Recommendations for international action. Report from the project G8-ENABLE. PMID- 10747427 TI - Rising to the challenge: portering services at the Queen Elizabeth II Health Sciences Centre. AB - With the recent formation of the Queen Elizabeth II Health Sciences Centre in Halifax, Nova Scotia, a number of challenges have arisen that must be addressed. One involves the porter service, which faces increased demands since every department has new and often expanded duties for porters. This paper identifies obstacles facing the porter service and develops recommendations, which may also be of interest to other healthcare facilities facing similar challenges. PMID- 10747428 TI - Community support for families: the Huron County approach to a long-term care issue. AB - This article profiles a pilot project in Huron County, Ontario, which merged two separate government-sponsored programs that provide support to families with disabled children into a single program delivered through a community agency. After almost ten years in operation, the program is working very well, but certain adjustments are necessary to keep the program running smoothly. PMID- 10747429 TI - Transforming U.S. health care: the arduous road to value. AB - The solution to the crisis in U.S. health care lies not in longing for the good old days or lobbying for higher reimbursements, but rather in taking a lesson from industry and producing a higher quality product more efficiently. The way to accomplish such a tall order is through collecting better information and applying it to improve care. The tools required are available: computers to gather and analyze data, the Internet to communicate information, research to advance the practice of medicine. The enduring health care organizations will be those that understand and embrace the coming changes, and change themselves. PMID- 10747430 TI - Focus on case management: linking outcomes and accountability. AB - The ability of case managers to rely on the traditional intuitive appreciation for the value of case management is declining. Case managers must accept the challenge of identifying, measuring, and reporting their outcomes, and clearly link outcomes with accountability. Practicing case managers must be as familiar with outcomes measurement and selecting relevant measures as their managers. The Case Management Society of America (CMSA) sponsored the development of the Council for Case Management Accountability (CCMA) to provide leadership in the development of outcomes. The work and mission of CCMA includes the creation of the "Spectrum of Accountability" and sponsoring three "State of the Science" papers addressing key case management outcomes areas. PMID- 10747431 TI - Trials and tribulations of year one: initiating cross-continuum care management in a community-based hospital. AB - To prepare to enter the Last Great Race, the Iditarod, you need a great team with experience, dedication, training, sponsorship, and the ability to work towards the desired goal. Planning a cross-continuum care management system is equally challenging. In this article, we present a real-life example of how a community based hospital system has begun to integrate existing case management and care coordination efforts into a unified approach to care management. This scenario challenges organizations developing, refining, and evaluating care management efforts to think about what is being done, how, and why. PMID- 10747432 TI - Selecting clinical quality improvement projects: getting a bigger return for your investment. AB - An urban medical center developed and implemented a process for selecting clinical quality-improvement projects. The process was designed to select projects that would deliver greater returns than had previous projects. The four step process involved: (1) establishing project selection criteria, (2) identifying potential projects, (3) assigning points and ranking projects, and (4) selecting projects based on rank and available resources. This process won physician commitment and secured administrative support. It identified projects that had unprecedented success in improving clinical outcomes, increasing patient satisfaction, and reducing cost. PMID- 10747434 TI - Defining quality health care with outcomes assessment while achieving economic value. AB - The effectiveness of a procedure is increasingly guided by the evaluation of patient outcomes. Outcomes data is used to develop clinical pathways of care and to define appropriate resource-use levels without sacrificing quality of care. Integration of the economic implications of medical services into an outcome based guideline allows for the development of disease-management strategies. In cardiovascular medicine, risk reduction is associated with high cost due to the "pay-back" of new technologies and therapies. A major challenge is to define a balance between "high tech" care and cost. This paper devises an outpatient evidence-based guideline using clinical and economic outcomes data for the diagnosis of coronary disease. PMID- 10747435 TI - Planning for successful outcomes in the new millennium. AB - The complexity of the health care environment will increase in the next millennium. Organizations must adopt an approach of selecting outcomes management solutions that are focused on data capture, analysis, and comparative reviews and reporting. They must decisively and creatively implement, in a phased approach, integrated solutions from existing robust systems, while considering future systems targeted for implementation. Outcomes management solutions must be integrated with the organization's information systems strategic plan. The successful organization must be able to turn business-critical data into information that supports both business and clinical decision-making activities. In short, health care organizations will have to become information-driven. PMID- 10747433 TI - Speaking the same language: improving evaluation of patient-focused outcomes using standardized terminology. AB - As the practice of medicine grows in complexity and the public calls for demonstrated value at decreased cost, the need for increasing effectiveness of both providing care and evaluating patient outcomes becomes paramount. This paper will demonstrate the success of utilizing a standardized taxonomy within an automated patient record to provide cues to clinicians regarding individual, patient-focused outcomes as well as timely evaluation of aggregated patient outcomes data. A case study will be used to illustrate dynamic evaluation of clinical outcomes following cardiac surgery. PMID- 10747436 TI - Classifying reasons for hospital readmissions. AB - Readmissions are a key measurement tool in today's outcomes-focused health care environment. Monitoring the volume of readmissions is a straightforward process in light of the database resources available to care providers. Examining and reporting on the actual reasons for readmissions provides opportunities for improvement specific to the needs of a patient population. The readmission coding tool used at the Medical University of South Carolina demonstrates both the ability to assess the causes for patients returning to our institution within thirty days of discharge and the opportunity to correct problems in specific service areas with regard to discharge planning. PMID- 10747437 TI - Automation of practice management in a Lebanese University Health Clinic. AB - Interest in technology and the recognition that clinical practice analysis data is crucial for planning have stimulated the development of information management solutions at all levels in developing countries. The spectrum of such endeavors remains restrained due to the limitations in human resources skills. This paper describes an inexpensive operation for automating practice analysis in a Lebanese University Health Clinic. The system was developed in piecemeal fashion to accommodate the needs of the staff and their computer expertise. It demonstrates the impact of such automation on patient care, including missing medical records, appointment scheduling, referral rates, and repeat prescriptions. PMID- 10747438 TI - Preventive services in the primary care practices of the Practice Partner Research Network. AB - Despite the emphasis of primary care on preventive services over the past decade, and the reminder systems that are available to promote the provision of these services, many patients still do not receive needed services. This study describes the preventive services that the primary care practices of the Practice Partner Research Network (PPRNet) monitors, and documents adherence to them. Preventive services monitored in PPRNet practices and the levels of adherence to them vary by practice and service. The lower-than-desired levels of adherence offer opportunities for improvement interventions. PMID- 10747439 TI - Creating an infrastructure for the productive sharing of clinical information. AB - The need for a patient-centered approach to health care services delivery is well recognized. Health care has become more specialized, with increasing numbers of disciplines and subdisciplines. In addition, both providers and community are increasingly mobile. As a consequence, patients see more providers, which has led to increasing fragmentation of patient-centered care and in particular of patients' personal health records. Clinicians and patients alike recognize the need to ensure that care information is patient-centered, continuous, and integrated in order to optimize the effectiveness of proactive and reactive care. Current arrangements, however, including the architecture of medical record and information management systems, are mainly provider- and service-centered and may not readily support the sharing of data to this end. PMID- 10747440 TI - Changing domains in the management process. Radiographers as managers in the NHS. AB - Examines a group of radiographers developing management roles within the backdrop of a changing NHS. A comparative study of 25 Scottish and English radiographer managers were interviewed using semi-structured interviews. Interviews were based on a number of issues associated with moving from a clinical professional to a clinical manager and were analysed using domain theory. The interviews formed a number of emerging themes, which included management, professionalism, management style, conflicts between the role of both manager and professional, and role change. Radiographer managers are forming new "hybrid" manager roles, which have been developing within a changing NHS. A definite tension was seen in this role change. This transition was not easy for this group of radiographer managers. However, they have shown resilience in undertaking both operational and strategic management decisions, while using their clinical background to inform their decision making. PMID- 10747441 TI - Participatory system development. A case study from Urban Health Lahore component DFID second family health project. AB - To improve effectiveness and efficiency of urban health services there is a need clearly to identify policies and the functions of various stakeholders and individuals in the system. System development, which involves, analysis of situations and the developing of solutions to address the identified issues, is necessary and forms a crucial element of system reforms. Developing systems in a participatory manner along with management capacity building can yield better results in terms of applicability of the system, improved confidence and ability of managers and service providers and sustainability of the reforms. PMID- 10747442 TI - Management of demand in the NHS, including the effects of queues and pensioners. AB - Discusses the methods used in the NHS to bring demand into balance with supply. People with minor illnesses try self-treatments and alternative medicine. Systematic programs to identify ill people are applied to only a few illnesses. Waiting lists for elective surgery cause some richer people to take their demand to private hospitals. An analysis of such waiting lists shows that, other than this, queues are not a method of rationing but are just the effect of bad management of the actual methods, which are then discussed. The same methods are used to ration access to specialist physicians. Providing extra resources would eliminate queues only if another condition was satisfied. It is argued that providing fully adequate medical care for patients of working age, although expensive, might produce a net economic gain, whereas all care for pensioners, including medical care, gives a net economic loss. Therefore it may not be sensible for people to have inadequate medical care for the first 65 years of their lives just because it is economically impracticable for them to have fully adequate medical care when they are pensioners. PMID- 10747443 TI - Managing alliances for health. PMID- 10747444 TI - Throw out the mission statement and bring back the epidemiologists. PMID- 10747446 TI - Management and relationships in total purchasing pilots. Relevance for primary care groups. AB - Primary Care Groups (PCGs) represent a natural evolution of the various models- total purchasing, multifunds and locality commissioning--embraced by the term "primary care commissioning". They will involve large numbers of different professionals working together to commission improved health care for their populations. The total purchasing pilots (TPPs) were the subjects of an extensive evaluation which has highlighted the significance of developing and improving relationships between the key players as a prerequisite for the successful implementation of their strategy. Management arrangements were central to this success. Similar considerations will be of crucial importance for the PCGs which are considerably larger and more complex organisations than the TPPs. PMID- 10747447 TI - Perceptions of the General Medical Council: the views of executive directors. AB - The regulation of the medical profession has recently been the subject of considerable debate. This paper describes a survey of the views of Trust medical directors and health authority directors of public health in the Northern and Yorkshire region on the way the GMC operates. A total of 46 out of 50 (92 per cent) gave a substantive response to the survey, of whom 16 (35 per cent) had not had cause to contact the GMC during the previous year, nine (20 per cent) were happy with their contact with the GMC and 21 (46 per cent) expressed some concern. The most commonly cited causes for concern were the length of time taken to process a complaint and lack of communication. While the individual opinions expressed are entirely subjective, it is argued that collectively they identify issues which the GMC and executive directors need to address. PMID- 10747445 TI - Factors affecting the level of success of community information systems. AB - The factors that influence the ultimate level of success or failure of systems development projects have received considerable attention in the academic literature. However, previous research has rarely targeted different instances of a common type of system within a homogeneous organisational sector. This paper presents the results of a survey of IM&T managers within Community Trusts to gain insights into the factors affecting the success of Community Information Systems. The results demonstrate that the most successful operational systems were thoroughly tested prior to implementation and enjoyed high levels of user and senior management commitment. Furthermore, it has been shown that there is a relationship between the level of organisational impact and systems success, with the most successful systems engendering changes to the host organisation's culture, level of empowerment and clinical working practices. In addition to being of academic interest, this research provides many important insights for practising IM&T managers. PMID- 10747448 TI - Focus of doctor-patient communication in follow-up consultations for patients treated surgically for colorectal cancer. AB - A series of consultations between patients treated surgically for colorectal cancer and their hospital consultants were examined to establish the main focus of the consultation at various stages in the post-surgical period. The results showed that follow-up consultations were predominantly doctor driven. Patients interviewed less than 12 months since the time of surgery (short-term group) were more likely to receive a longer consultation with a significantly higher number of verbal interactions (questions, responses) than patients more than 12 months since surgery at the time of interview (long-term group). Furthermore, patients in the short-term group played a greater participatory role within consultations than patients in the long-term group. The predominant focus throughout all consultations was biomedical, with little attention afforded to patients' expressions of post-operative anxiety. At present, it appears that out-patient consultations play a minimal role in either detecting or addressing psychosocial morbidity amongst colorectal cancer patients in the post-surgical period. PMID- 10747449 TI - Knowledge and attitudes of Malaysian private medical practitioners toward guidelines-based medicine. AB - This paper describes a study of the knowledge and attitudes of a sample of private medical practitioners in Malaysia in relation to questions posed on guidelines-based medicine. The study was conducted through a postal questionnaire sent to a sample of 2,000 private practitioners. Placing the research in context, a description of Malaysia and its health-care system is provided. Issues surrounding guidelines-based medicine in the Malaysian context are discussed. An analysis of the sample population's personal and professional characteristics is presented. Key research findings include identifying a substantial knowledge deficit--less than half of the respondents claimed to understand "guidelines". A majority of doctors (64 per cent) think guidelines are useful. Only 29 per cent of respondents knew where to find guidelines, with 22 per cent claiming to have used guidelines. The paper concludes with a discussion of issues in the implementation of guidelines-based medicine in Malaysia based on the study's findings. PMID- 10747450 TI - Making bureaucracy work. AB - What gives bureaucracy such a bad name? Is it bureaucracy in itself, or the ghosts in the system who, in a million minor drifts, contribute to turning efficiency into red-tape? Undesirable side-effects need not be confused with necessary first-level effects. There is nothing wrong with the bureaucratic system as such. Ultimately we could not work without it as it is the only known way of co-ordinating vast numbers of people to treat mass problems. However, like any tool, it is only as good as the people who use it, and its results are largely linked to the very agendas of the users. Understanding what makes bureaucracy work requires a good look at the implicit biases in the bureaucratic model, mostly seeded by its various founders and theoreticians, as well as tackling pragmatic issues of creating and applying rules--and where and when to change them. PMID- 10747451 TI - How many health managers have asked themselves "What does the Lawrence inquiry mean for us?". PMID- 10747452 TI - A model for calculating costs of hospital wards: an Italian experience. AB - Until recently Italian hospitals had no cost accounting or activity data collection systems, being formally required only to do financial book-keeping. The cost analysis method presented here might be used to set up detailed and complete hospital cost accounting, which would permit a better understanding of patterns of resource distribution among departments, better opportunities for cost saving and cost control for hospital managers and health authorities. The study first identified a framework within which to assess the annual cost related to a hospital ward, then calculated the mean bed day cost for each speciality. Cost data were collected over one year in 1996 from manually compiled records, at one local hospital in Northern Italy. Costs were estimated following a step-down allocation method. Wards requiring a major amount of resources per day of stay are intensive cardio-coronary unit (US$650.689), and ophthalmology (US$483.322). The less expensive ward is general medicine (US$148.645). The cost analysis method presented in this study might be used to set a detailed and complete hospital cost database, which is a necessary tool for hospital managers to realise cost control and cost recovery. PMID- 10747453 TI - Empowerment and the performance of health services. AB - Addresses the issue of empowerment and its possible role in promoting the effectiveness of health services. Empowerment represents the ability of people within organisations to use their own initiative to further organisational interests. However, despite its apparent simplicity, the concept turns out to be quite complex and to have unanticipated implications. We explore some of these implications in health service organisations, and their consequences for health policy. Our conclusion is that many health policies may well act to degrade the empowerment of health service workers, and hence the performance of health service organisations. PMID- 10747454 TI - Workplace health concerns: a focus group study. AB - Addresses the workplace health concerns of employees at an Acute Hospital Trust. The research conducted utilised a focus group methodology to investigate the health concerns of multidisciplinary groups of health care workers (n = 27). The findings indicate that the concern for the majority was workplace stress. Stressors that were identified as important to the groups were for example, the nature of the work they were undertaking, staffing levels, volume of the work, management styles and their work environments. Health promotion and prevention topics such as motivation and health status, smoking, alcohol and drug misuse, nutrition and weight control and physical exercise were seen of subsidiary concern. Limitations of the study are given. Future research in the subject area is identified. PMID- 10747455 TI - A pregnant mother's right to refuse treatment beneficial to her fetus: refusing blood transfusions. PMID- 10747456 TI - The American difference in health care costs: is there a problem? Is medical necessity the solution? PMID- 10747457 TI - When angry patients become angry prosecutors: medical necessity determinations quality of care and the qui tam law. PMID- 10747458 TI - The reasonable and necessary criterion and Medicare contractor review of claims: efforts to combat fraud, waste and abuse in the Medicare program. PMID- 10747459 TI - Salary expectations vs. company profits: the corporate tug of war. PMID- 10747460 TI - Environmental management for healthcare providers. PMID- 10747461 TI - A marketing tool for expert witnesses. PMID- 10747462 TI - A decade of change in clinical waste treatment and disposal in Scotland. AB - Ten years ago there were over 150 small incinerators located on hospital premises throughout Scotland. The majority were operated by portering staff, had poor combustion and no gas cleaning equipment. At that time alternatives to incineration were in their infancy and relatively expensive. Funding for major capital projects lay with the Government, and a strategic study suggested the installation of 12 centralised modern incinerators on the mainland and three on the islands: one per Scottish Health Board. Some health boards proceeded with new installations well ahead of dead-lines for closure of old plant. Others adopted a 'wait and see' policy and were overtaken by political changes. These resulted in a cutback in government-funded capital investment and a shift of non-core services to the private sector. Clinical waste disposal was contracted out as a service contract and some private sector companies offered alternative low temperature technologies for clinical waste treatment. As a result there is now the opportunity to compare the advantages and disadvantages of incineration, sterilization, dry heat disinfection and other techniques. Technological change has also required revised waste segregation methods within the hospitals. In parallel with this there has been an overall reduction in waste quantities. The remaining incinerator operators are now faced with more stringent emission limits to be implemented by June 2000. This is resulting in closures of incinerators that have been operating for a few years. The situation continues to change. PMID- 10747464 TI - There are solutions to problems. AB - In these times of health care reform and change there are an increasing number of problems in the health care workplace that health care managers must face. Solving problems successfully is important for survival of the manager and for smooth delivery of care. The challenge of solving problems can be faced by a systematic response that includes having faith in oneself, clearly defining the problem, not jumping to conclusions, considering the overall goal, getting staff involved as soon as possible, reviewing all possible options for obtaining results, developing a plan of action, selecting a strategic time to take action, conveying appropriate urgency about the problem, having faith in colleagues and staff, implementing the plan, using resources available, and evaluating the outcome as well as the process. PMID- 10747463 TI - Staff turnover: occasional friend, frequent foe, and continuing frustration. AB - Turnover appears to be a relatively simple concept. However, considerable confusion results when discussing turnover because of differences in how it is defined--what is counted, how it is counted, and how the rate of turnover is expressed. Turnover is also costly, although not enough attention is paid to turnover's cost because so much of it is indirect and thus not readily visible. There are a variety of causes of turnover, some which can be corrected and some which cannot be avoided. Reducing or otherwise controlling turnover requires continuing management attention to its causes and constant recognition of what can and should be controlled and what cannot be controlled. Ongoing attention to turnover is an essential part of the department manager's role. PMID- 10747465 TI - Fifty-two effective, inexpensive ways to reward and recognize hospital employees. AB - Effective reward and recognition programs are important in order to retain well qualified hospital employees and actively engage them in satisfying patients, managing scarce resources, and improving performance. The rewards and recognition bestowed may be modest in scale but must be symbolic of genuine caring and appreciation by management. This article outlines a number of reward and recognition tactics that can be used singly or in combination to begin demonstrating management's commitment to improving employee satisfaction. PMID- 10747466 TI - The effective team member: avoiding team burnout. AB - This article outlines specific suggestions for team members designed to help ensure that team membership is a satisfying experience. The suggestions offered provide clear guidelines for the responsibilities individual health care providers must assume when working on teams. Your proactive engagement in addressing the suggestions provided is part of an integrated, holistic approach to teams. PMID- 10747467 TI - Literacy, cultural diversity, and client education. AB - Client education offers many challenges to health care professionals. There is a mismatch between the reading level of most clients and the reading level of printed materials used in health education. Social and cultural factors also influence how our clients are able to learn about their health. These problems can lead to noncompliance with treatments, missed appointments, wrong dosages of medication, and undue fear among our clients. There are things that health care professionals can do to make written materials more user-friendly and culturally more acceptable. PMID- 10747468 TI - Bringing together nursing and law: a fitting combination in today's health care environment. AB - Blending careers is an exciting alternative to choosing one profession or another. In this article, the author relates her experiences as a nurse to her current choice of profession, the law. The author used the same principles and interventions developed while nursing in dealing with clients experiencing personal injuries, working on the estate of a client's deceased family member, or helping a client with the many issues that arise with the end of a marriage. The marriage of nursing and law has been a successful partnership. One career builds on the experience and education of the other, allowing the practitioner to offer clients a total package of legal and health care delivery assistance. PMID- 10747469 TI - What every health care professional should know about AIDS. AB - As more and more clients with acquired immunodeficiency syndrome (AIDS) are encountered in health care agencies, it is important that the health care professional be well informed with current facts and information on treatment. Supportive care by the health professional is essential to assist the client in maintaining maximum quality of life and a sense of self-esteem and self-efficacy. It is important for the professional to be aware not only of the supportive care needed by clients but also of the safeguards necessary in such a high-risk profession. PMID- 10747470 TI - I see a change in your future. AB - Like it or not, peoples' careers are continually being affected by circumstances beyond their control. However, there are steps one can take to become more flexible and adaptable, and thus more employable, in a changing job market. Job security may be decreasing, but opportunities are increasing overall. Options are available in the face of change; it remains only to recognize them, prepare accordingly, and take advantage of them. Doing so means learning to recognize the signs of career stagnation and potential obsolescence, becoming aware of alternatives, and recognizing that security, if such exists, now resides in adaptability. PMID- 10747471 TI - Applied business training: the latest continuing education for physicians. AB - Due to rapid changes in the health care industry, physicians are being asked to do much more than just take care of patients. They need an arsenal of additional skills if they want to survive in a shifting health care environment characterized by cost consciousness, competition, and fear of decreasing service quality. Consequently, doctors are searching for opportunities to learn more about the business side of managed care. The University of Tennessee at Chattanooga's School of Business Administration, along with Blue Cross/Blue Shield of Tennessee, the Chattanooga Unit of the University of Tennessee College of Medicine, and Erlanger Medical Center, has developed management education courses for area physicians. This article outlines the implementation of the continuing education programs and provides suggestions for others considering such a program, as well as ideas for future business school and physician education linkages. PMID- 10747472 TI - Vision, hearing problems in the elderly. PMID- 10747473 TI - Ohio CNAs speak out. Enjoyment ranks high, pay low in satisfaction study. PMID- 10747474 TI - Congress poised to approve BBA relief. PMID- 10747475 TI - Pain often goes untreated in nursing facilities, study finds. PMID- 10747476 TI - Companies merge to form largest health care REIT. PMID- 10747477 TI - Volume, price of seniors housing properties increasing. PMID- 10747478 TI - AHCA helps launch SNF nutrition awareness campaign. PMID- 10747479 TI - Medicare, Medicaid force Texas chain into bankruptcy. PMID- 10747480 TI - Seniors housing especially popular with low-income seniors. PMID- 10747481 TI - Bill would expedite residents' return from hospital. PMID- 10747482 TI - Genesis simplifies multicare joint venture. PMID- 10747484 TI - Solomont Bailis walks where competitors haven't thought to tread--with an eye to the future. PMID- 10747483 TI - 'Heart's Desire' program gives residents a lift. PMID- 10747485 TI - Final rule clarifies coverage issue. Medicare PPS covers five to eight days for the top RUG classifications. PMID- 10747486 TI - Understanding the needs of direct care staff. What it takes to attract and keep quality staff. PMID- 10747487 TI - AHCA 1999 Quality Award winners. Innovators earn the honors. PMID- 10747488 TI - The 1999 Lifespace Design Award winners. PMID- 10747489 TI - 1999 Nurse Scholarship Award winners. PMID- 10747490 TI - Labor and staffing in the PPS era. Providers will find that reining in labor costs helps them ride out the PPS challenge. PMID- 10747491 TI - Reducing the risk of violence. Long term care providers that do not take steps to reduce the risk of workplace violence face possible OSHA violations. PMID- 10747492 TI - Mentoring program yields staff satisfaction. Mentoring through the exchange of information across all organizational levels can help administrators retain valuable staff. PMID- 10747493 TI - Proper assessment soothes chronic pain. PMID- 10747494 TI - Designing strategies to prevent falls. PMID- 10747495 TI - Restorative nursing techniques maximize function. PMID- 10747496 TI - Reaching a new level in outcomes management. PMID- 10747497 TI - Nourishing healthy skin. PMID- 10747499 TI - GAO calls for immediate comparative surveys by HCFA. PMID- 10747498 TI - Hydration strategies focus on outcomes. PMID- 10747500 TI - Republicans work on five appropriations bills. PMID- 10747501 TI - Medicare FIs halt nursing allocation audits. PMID- 10747502 TI - Sun works to build reorganization deal. PMID- 10747503 TI - OIG reports find SNF access a growing problem. PMID- 10747504 TI - Expansion strong in 1999 but will be slower next year. PMID- 10747505 TI - HCFA plans a quality check in financially troubled facilities. PMID- 10747506 TI - Legislation thwarts physician-assisted suicide. PMID- 10747507 TI - California governor vetoes nursing facility reform bill. PMID- 10747508 TI - An interview with Chip Roadman--president and CEO of AHCA. PMID- 10747509 TI - Long-term care in the election limelight. PMID- 10747510 TI - The Y2K fix. Planning for business as usual despite potential disruptions, ranging from water to phone to finances. PMID- 10747511 TI - Standardizing SNF-vendor contracts. PMID- 10747513 TI - PPS changes dialysis coverage rules. PMID- 10747512 TI - Tactics for reducing CNA turnover. PMID- 10747514 TI - Infection control procedures. The elderly's susceptibility to infection mandates stopping the spread of germs. PMID- 10747515 TI - Protecting residents' electronic privacy. PMID- 10747516 TI - Congress nears final vote on BBA. PMID- 10747517 TI - Utility and quality of autologous fresh frozen plasma and autologous fibrin glue for surgical patients. AB - The quality of autologous fresh frozen plasma and autologous fibrin glue prepared in our hospital was evaluated. The Auto-FFP was separated from whole blood or collected using a cell separator and the Auto-FG was then prepared from the Auto FFP. The Auto-FFP showed significantly higher levels of fibrinogen, Factor-V and Factor-VIII than commercially available FFP. The Auto-FG contained approximately 10 times the concentration of fibrinogen, Factor-VIII, Factor-XIII, von Willbrand factor and fibronectin and 1.5 times that of alpha 2-Plasminogen inhibitor compared to those of the Auto-FFP. When the Auto-FG was sprayed over diffusely bleeding sites from surgical wounds, hemostasis occurred within 5 s. These results suggest that Auto-FFP and Auto-FG may have sufficient utility and quality to reduce the use of allogeneic blood products. PMID- 10747518 TI - Pre-separation storage of whole blood: the effect of temperature on red cell 2,3 diphosphoglycerate and myeloperoxidase in plasma. AB - BACKGROUND: Although whole blood intended for component preparation is commonly left to cool at ambient temperature, knowledge is insufficient concerning what effects this may have on red blood cell (RBC) quality, in particular after a prolonged hold. STUDY DESIGN AND METHODS: Whole blood collected in CPD was incubated at 20 degrees C and 28 degrees C for 6 h designed as a paired study. Blood components were prepared and the red blood cell concentrates (RBCs) were stored for 28 days at 4 degrees C +/- 2 degrees C. Blood gases, pH, glucose, lactate, adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG) and plasma myeloperoxidase (MPO) were investigated. RESULTS: After 6 h the 2,3-DPG concentrations had lowered to 88% (20 degrees C) and 54% (28 degrees C) of initial levels, respectively. The difference was significant and was maintained for 28 days, although, at low levels from day 7 (28 degrees C) and day 14 (20 degrees C) of storage. ATP was maintained at the initial level in both groups during the first 6 h of storage but after component separation the levels were significantly higher in the 28 degrees C group during the first 5 days. The release of myeloperoxidase (MPO) was significantly higher in the non-cooled group than in the cooled group. CONCLUSIONS: Pre-separation holding for 6 h of whole blood at temperatures of 28 degrees C causes a great and rapid loss of 2,3-DPG and considerable formation of acid metabolites resulting in clearly subnormal 2,3 DPG levels even on day 1. Active pre-separation cooling to 20 degrees C is to be recommended. PMID- 10747519 TI - Automatic volumetric capillary cytometry in the routine of an apheresis unit involved in multicomponent collection. AB - In apheresis, leukodepletion by secondary filtration of the platelet components or by the primary use of special high efficiency apparatuses is widely used to meet current clinical practice. Leukodepletion of RBC is mandatory for hematooncological patients and new filters for plasma are progressively being introduced in the routine of European blood banks. However, since the monitoring of leukodepletion efficiency continues to be carried out manually using the Nageotte or the microdroplet fluorescence assay (MFA), inaccuracy and labour intensity of counting will limit the possibility of satisfying the increasing demand for leukodepletion monitoring. Volumetric capillary cytometry (VCC) is a totally automated system that has been shown to correlate well with Nageotte, MFA and flow-cytometric countings of residual leukocytes in platelet and RBC product. In this article we describe the application of VCC in the quality control program of our hemapheresis unit in which all apheresis donations are of the multicomponent collection type. PMID- 10747520 TI - The flow rate significantly influences the leukocyte depletion rate during prestorage in-line filtration of platelet concentrates. AB - BACKGROUND: White cell reduction of blood products minimizes the risks of alloimmunization against HLA-antigens, the transmission of viral diseases and the incidence of platelet transfusion reactions. One modern strategy is leukocyte depletion with an integrated filter system immediately after preparation and prior to storage. STUDY DESIGN AND METHODS: We evaluated the efficiency of a novel in-line filter system Sepacell PLX-5 BPS for leukocyte reduction of platelet concentrates (PC) from pooled buffy-coats. A total of 44 PCs were investigated with regard to different filtration flow rates (25-110 ml/min) and leukocyte depletion and thrombocyte recovery rates were analysed. Furthermore, we studied the influence of filtration on PCs over a storage period of 6 days (n = 12) by investigation of pH, lactate and glucose. Platelet function was determined by means of hypotonic shock response, external shape change and expression of CD62p. RESULTS: The mean leukocyte depletion rate was > log 5. After filtration the mean leukocyte count was 0.12 +/- 0.21 x 10(6). In 60% of the PCs the leukocyte count lay below the detection level of the Nageotte chamber, which is < 0.3 x 10(5). The flow rate correlates significantly with the leukocyte count in the PCs (r = 0.325; p = 0.033) and therefore with the leukocyte depletion rate (r = -0.422; p = 0.01). Flow rates under 40 ml lead to a significantly lower leukocyte contamination. Only in one PC, at a flow rate of 84 ml/min, was the leukocyte threshold of 1 x 10(6) exceeded. We did not find a significant correlation between filtration flow rate and thrombocyte recovery (r = 0.315; p = 0.069). The mean platelet count in the PC was 2.88 +/- 0.47 x 10(11). Compared with the thrombocyte count in the pooled buffy coat, the recovery was 68.6%. We observed a decrease of pH, glucose, external shape change and hypotonic shock response over the storage period while lactate and the expression of CD62p increased. CONCLUSION: The filter system Sepacell PLX-5 BPS proved to be suitable for in-line filtration of platelet concentrates prior to storage. Filtration flow rates of up to 40 ml/min allowed efficient leukocyte depletion without significant loss in the quality of the platelet concentrates and the platelet function in vitro. PMID- 10747521 TI - Autoapheresis and intraoperative blood salvage in oncologic surgery. AB - Transfusion of predeposit or salvaged autologous blood has continued to grow since the 1980s. Issues such as the indications for use and cost effectiveness as well as the safety of autologous blood salvaged during cancer surgery have emerged and should be addressed. The concern for possible contamination of autologous RBC with cancer cells responsible for metastasis has limited the use of autologous salvaged blood in cancer patients. Nevertheless, clinical experience has been gained on the use of salvaged blood in patients with colorectal, gastric, renal, hepatic, breast, bladder and lung cancer. No evidence has been reported showing an increase in metastasis or a decrease in patient survival, in spite of the obvious demonstration that salvaged blood is contaminated with viable tumor cells which are not washed out of the RBC layer during intraoperative blood salvage (IOBS). However, a number of limitations have hampered the widespread use of IOBS in these patients and the technique is not well established. Increasing knowledge of the deleterious effects of allogeneic blood transfusion both in terms of the increased number of viral or bacterial infections and the down-regulation of the patient's immune system have recalled attention to IOBS and to the techniques such as filtration, which might reduce the risk of reinfusion of cancer cells, or totally eliminate the risks such as irradiation has been proposed by Hansen's group. This paper reviews the topic with some emphasis on our personal experience with gamma and X-ray irradiation of salvaged blood in a large reference hospital, where IOBS and filtration of salvaged blood were established for use in cancer patients in 1993 and 1996. PMID- 10747523 TI - The UK strategy for monitoring universal leucodepletion. AB - This summary manuscript deals with the NBS approach to quality monitoring of universal leucodepletion based on representative sampling. There is also a brief discussion on the failure rate and potential causes for the inter site variations in white cell enumeration by current methods. PMID- 10747522 TI - Cost effectiveness of quality assurance in plateletpheresis. AB - BACKGROUND AND OBJECTIVES: A quality assurance system (QAS) is part of modern blood banking facilities. Quality control of single donor platelet (SDP) concentrates includes the determination of the platelet (PLT) yield and the white blood cell (WBC) contamination. Improvements in modern apheresis technology allow the collection of high PLT yields and leukoreduced products. Double dose SDPs can be split and WBC-reduced products may be labelled as leukodepleted thereby reducing costs. MATERIAL AND METHODS: 3309 SDPs obtained with the Amicus, AS 104, AS.TEC 204 and MCS + blood cell separators were retrospectively analysed for their PLT yield. SDPs with > or = 4.0 x 10(11) PLTs were considered as double dose SDPs and split. WBCs were determined microscopically in 170 SDPs. SDPs with a leukocyte content < 1.0 x 10(6) were labelled as leukodepleted and no further WBC filtration was recommended. RESULTS: PLT yield was statistically higher in SDPs from the Amicus device, 84.8% of these products could be split. Double dose concentrates were collected in 22.7% with the MCS + machine and in 4.8% with the AS 104/AS.TEC 204 blood cell separators. The savings for disposables was $150,041 and for infectious disease testing $75,766. After the subtraction of the costs for PLT determinations in all SDPs $215,880 could be saved. WBC contamination was statistically lowest in in-line filtered SDPs from the MCS + device (median 0.29 x 10(5), range 0.22-9.96 x 10(5)) and all of these products were considered as fulfilling the criterion of leukodepeletion so that we were able to save $17,135 for bedside filters. Median WBC content was 0.75 x 10(5) (range 0.35-22.5 x 10(5)) in SDPs from the Amicus and 0.9 x 10(5) (range 0.27-99.8 x 10(5)) in SDPs from the AS 104/AS.TEC 204 devices, respectively. CONCLUSION: Blood cell separators of the newest generation allow the collection of leukodepleted double dose SDPs. An intensified QAS in plateletpheresis allows the decision whether a product can be split and/or released as leukodepleted. By this means we were able to save a total of $233,015 per year. PMID- 10747524 TI - Plateletapheresis: instrumentation validation. AB - Plateletapheresis instrumentation validation is required to document that a new or modified instrument or technique is capable of consistently producing acceptable products at the production center using their equipment, personnel, and counting techniques even though the instrument or technique may already have FDA or equivalent approval for use. To pursue the process of validation, several questions need to be addressed: when is it required, what products are validated, what parameters are monitored, and how many products are required. Validation is required when a new instrument or technique (process) is used that could affect the quality of the product. According to the FDA, each apheresis system (e.g., Spectra LRS, Amicus) and each type of product (e.g., single, double, triple) need to be validated separately. Parameters to be validated vary, but usually platelet (plt) yield, white blood cell (WBC) content (if products are labeled "leukoreduced"), and 5-day storage pH are monitored. The number of procedures monitored is also quite variable, but we use 20 samples for highly variable parameters such as platelet yield and WBC content and five samples for less variable parameters such as 5-day storage pH. As an example, we validated the Fenwal Amicus (Baxter Biotech) for single apheresis platelet products. With 20 samples, we found that: 85% of the products contained > or = 3 x 10(11) plt (requirement was at least 75% contain > or = 3 x 10(11) plt); platelet concentration of all products was < or = 1.515 x 10(6) plt/microL (requirement was < or = 2.435 x 10(6) plt/microL), and WBC content was < 1 x 10(6) WBC in all products (requirement was all products contain < 5 x 10(6) WBC). In addition, in five samples, the 5-day storage pH was 6.89-7.25 (requirement was all products should be > or = 6.2 pH). Once validation is complete and acceptable, the process should be monitored on a regular basis using some form of process control. Statistical process control programs are available that can assist in documenting validation and ongoing process control. With the use of process validation and ongoing process control, the plateletapheresis center can assure that acceptable products are consistently being produced. PMID- 10747525 TI - Corrective and preventive action. AB - Recognized principles of quality management include a component for process improvement, comprised largely of corrective and preventive action taken in response to identified problems. The importance of identifying and investigating problems has been clearly established in transfusion medicine. Such problems can be identified in the following ways: error, incident, and accident reports; adverse reaction reports; customer complaints; process indicator measurements; results of proficiency testing; and results of internal or external audits, inspections, or assessments. Responses to reported events can be remedial, in which the symptom is addressed, or corrective, in which the underlying cause is addressed with the intent to prevent recurrence. If identified problems or their root causes are trended to look for patterns or problems not yet occurring are anticipated, the action taken is proactive and considered preventive. Methods to trend events, monitor processes, and perform root cause analysis are discussed as well as use of the following process improvement 'tools': control charts, flowcharting, the 'repetitive why', cause-and-effect diagram, and Pareto analysis. PMID- 10747527 TI - Integration of quality programs by telemedicine in surgical services. AB - Surgical procedures can be performed and monitored in operating rooms physically isolated, but electronically linked to a parent medical center, and certainly from distant consultation. Quality of surgical care has been measured in retrospect rather than in real time, and outcomes have generally not had the benefit of immediate consultation and group analysis. However, telemedicine can connect consultants, surgeons, patients, and databases. The Yale Telemedicine Center has developed network schemes, which connect the OR of trainees with the laboratory of the teacher, and has opened a continuum between pedagogy and product. The program has connected regional hospitals, connected components of hospital systems, and has been used in international testbeds. The particular operative procedures studied have been laparoscopic procedures. The consultant is based at Yale and is connected with the distant OR to share the laparoscopic surgical field. Connection includes real time video, audio and the ability to draw instruction on the OR screen. Bandwidths as low as 12kbs(POTS) have been employed with modification with effective intervention. The potential for quality improvement is substantial, and inclusion in quality programs is strongly recommended. PMID- 10747526 TI - The impact of telemedicine on health care management. Proceedings of the G 8 Global Health Care Applications Project (GHAP) Conference. Regensburg, Germany, 21 November 1998. PMID- 10747528 TI - Telemedicine in neurosurgical daily practice. AB - Telemedicine has been in daily practice at our neurosurgical department for five years. In our experience the standard personal computer-based telemedicine systems Medicom and especially RADO Look are reliable, easy to use, and of high quality performance. Telemedicine makes possible better patient management at regional hospitals and the avoidance of patient transports. Therefore treatment costs in neurosurgical patients can be highly reduced. PMID- 10747529 TI - Perfect DiViSy technology for video network in medicine (Moscow Information Network for Teleoncology). AB - A large telemedicine project is now under development in Moscow. The general task of the project is to link oncology research centres and hospitals meeting the project profile, and to provide on-line (using real-time video conferences) and off-line (via distributed database of medical information) information exchange. There was no existing technology available for use at the project's base which was ready to meet all requirements, so we are using specially created technology for video networks (DiViSy V.97). We suppose this technology to be more applicable for use in telemedicine networks than are business video conference technologies and classical Internet/Intranet technologies. We affirm in our conclusion that now is the time for developing special telemedicine standards, recommendations of compatibility, etc., and that we are ready to take part in this process. PMID- 10747530 TI - International concerted action on collaboration in telemedicine: G8 sub-project 4. AB - The main objective of G-7/8 Global Healthcare Applications sub-project 4 is to enable an international concerted action on collaboration in telemedicine. To promote and facilitate the implementation of telemedicine or health telematics networks around the world, it was felt necessary to solve certain key issues. Five thematic solution-seeking FORUMS are each addressing a specific item. The first FORUM held in Montreal, Canada on May 28-30 1998 focussed on Interoperability of telemedicine and telehealth systems. Other FORUMS address other themes such as: Impacts of Telemedicine on health care management (Regensburg, Germany, November 21-23 1998); Evaluation and Cost Effectiveness of Telemedicine (Melbourne, Australia, February 19-20 1999); Clinical and technical quality and standards (Washington, USA, April 29-30 1999); Medico-legal aspects of national and international applications (Oxford, UK, fall 1999). The main objective of these FORUMS is to arrive at best practices through consultation amongst experts who seek together the best solutions to facilitate global international telemedicine networks. Towards this goal, G-8 sub-project-4 will also conduct the IMPACT (International Multipoint Project of Advanced Communication in Telemedicine) feasibility study which will aim at conducting multipoint exchanges between telemedicine units in the academic centers of the participating G-8 and other countries. More detailed information on this project and summaries of the initial FORUMS are found on our Web site at www.g7sp4.org. PMID- 10747531 TI - A controlled pilot study in the use of telemedicine in the community on the management of heart failure--a report of the first three months. AB - This is a controlled pilot study of twenty patients to see if heart failure management can be optimised in the community using telemedicine. The study seeks to examine the feasibility, acceptability and reliability of using telemedicine in this context. Heart failure is a common condition. It is an important cause of mortality and morbidity and has large cost implications for the NHS. Most patients are managed in the UK in General Practice based on clinical assessment by the practitioner. Twenty patients with a mean age of 75.1 years and mean New York Heart Association grade of 1.75 were randomised in to two equal groups (telemonitoring and control) and observed for a period of three months. All twenty patients had a Cardiologist assessment and quality of life measurement at the beginning and end of the study. Patients in the telemedicine group had their blood pressure, pulse and weight data collected daily and undertook a weekly video conference with the nurse. Control patients had their blood pressure, weight and pulse measured at six weekly intervals. The study has been extended for a further six months beyond its initial three-month observation period to see if the initial short term benefit in the telemedicine group is maintained. PMID- 10747532 TI - Telematic-based model of early diagnosis and treatment of acute coronary syndromes. PMID- 10747533 TI - Telemedicine and diabetes. AB - Telecommunication technology has reached a maturity which can effectively support the promotion of health, prevention and management of diseases and rehabilitation. To achieve effective support harmonization and interoperability of technologies are main requirements. This article tries to point out how interoperability is being met and how the requirements are being applied using DIABCARD as an example. DIABCARD has developed a chip card based medical information system for the care of persons with diabetes. PMID- 10747534 TI - Telemedicine based screening of infants at risk for retinopathy of prematurity. AB - Retinopathy of Prematurity (ROP) is a major risk for permanent visual loss in extreme prematures. In stage 3+ (zone I and II), defined as threshold disease, the risk for retinal detachment with subsequent blindness is about 50% without treatment, compared to only a few percent with laser photoablation. Visualisation and interpretation of the critical retinal changes requires a high degree of expertise. Therefore, screening by general ophthalmologists will not identify all cases at threshold necessary to prevent blindness in as many infants as possible. The development of a new digital wide-field camera system (RetCam 120) allows documentation of nearly the entire retina within minutes. Recently, sensitivity, specificity, positive predictive power and negative predictive power have been evaluated to be high. Data collection is also possible by trained staff of the neonatal care units. Data can be transferred electronically, even though it could be transferred through physical transport. Analyzing the data in a central reading center specialized in the treatment of ROP will allow optimal timing of treatment and thus salvage vision in more infants. In addition, unnecessary referrals that are not only expensive but put also stress on the very vulnerable prematures can be avoided. At present, efforts are made to realize a pilot project in Eastern Bavaria that will test the potentials of telemedicine based screening for ROP. In addition, the potential of the system will be evaluated in other neuroophthalmological diseases. PMID- 10747535 TI - ByOPHTEL: a Bavarian project for rapid telemedical exchange of knowledge, files and skills between practitioners and hospitals in eye care. AB - ByOPHTEL, part of the Bavarian initiative Bayern Online, derived its name from the European project OPHTEL, to which it is closely linked and which, in Bavaria, is more or less worked out and performed by identical persons and institutions. It provides a group of 7 practitioners in and around the city of Munich with the possibility of exchanging general ophthalmologic knowledge as well as patient related information with anyone in the group, or with the related ophthalmologic, internal, resp. scientific centers at the TU Munich and the GSF Neuherberg. Connections are routinely performed in the asynchronous or in the synchronous mode, and all partners participate in the scientific projects Knowledge Based Information System, Glaucoma Monitor, and Diabetic Retinopathy Monitor--as well as in the enhancement of a trustful patient-physician relationship ("second opinion") and a closer cooperation in out-patient and in-patient ophthalmical surgery and medical treatment. PMID- 10747536 TI - Collaborative telemedicine between optometry and ophthalmology: an initiative from the University of Houston. AB - The University of Houston College of Optometry (UHCO) has developed a strong Telemedicine Optometry/Ophthalmology program. Patient care, clinical teaching and research benefit from utilizing Telemedicine technology. This initiative between Optometry and Ophthalmology is at the forefront of eyecare! Presented here: clinical teaching opportunities via tele-education including retinal, uveitic, neuro-ophthalmic disorders and glaucoma. Seminars introduce students to this technology and enhance their clinical experience through exposure to eye pathologies often related to systemic diseases. UHCO's affiliated clinics include multidisciplinary centers staffed by optometrists: the Frost Eye Clinic (HIV multidisciplinary care), Good Neighbor Health Clinic and Rusk School Health Promotion Project (indigent multidisciplinary care). These centers can converse on line with eyecare professionals. Large sites interested in low vision/visual rehabilitation include: The Institute for Rehabilitation and Research (TIRR), the Social Security Hospital (Lima, Peru), the Matagorda General Hospital and the Quentin Mease Hospital. These sites, do offer quality care but require visual rehabilitation programs provided by UHCO. These sites also provide exposure to pathology for the students. A residency program trains optometrists in Telemedicine. The research program focuses on optics and imaging necessary to diagnose glaucoma, diabetic retinopathy, and neuro-ophthalmic disorders through Telemedicine. UHCO supports the advancement of standards in tele-eyecare. PMID- 10747537 TI - Web-based teledermatology consult system: preliminary results from the first 100 cases. AB - The TRICARE Region 1 Teledermatology Consortium has developed a secure web-based "store and forward" consult system designed to allow medical treatment facilities throughout the region to submit dermatology consults. Realizing that there is very little objective data on teledermatology, we have begun to evaluate our system through the integration of questionnaires for patients, referring physicians, and consultants in addition to telephonic patient follow up. We report the preliminary data from the first 100 cases received in the first two months since partly deploying the web based teledermatology consult system in the Tricare Region I (Northeast United States). We primarily address system effectiveness (percentage of cases that required follow up to dermatology and/or primary care physicians, adequacy of evaluating pigmented lesions) diagnostic agreement, acceptance, effect on access to care, and educational value to the primary care physicians. PMID- 10747538 TI - Are dermatologists in private practice interested in teledermatological services? AB - Diagnosing dermatologic skin conditions can be difficult, especially in pigmented skin lesions. Therefore, the consultation of an expert via teledermatology could prove vital. For this purpose, a rapid transfer of medical data including high resolution images is essential. This transfer can be performed with a variety of modern telecommunication technologies, including ISDN, highspeed-ISDN, Internet, and Intranet. As the levels of both communication software and camera-systems can be quite different, our survey investigated the equipment of 84 dermatologists in private practice. A questionnaire was distributed on computer equipment, operating system software, and any image documentation systems used, as well as required telecommunications equipment and possible applications of tele dermatology. This survey showed a response rate of 54% and proves that dermatologists in private practice are interested in telemedicine services. Most dermatologists surveyed use Windows 95 operating software and 74% have access to modern ISDN modems or PC-cards. Dermatologists currently prefer applications with low-tech communication hardware and software requirements. Consultation of dermatological centers was the favored application with 59%. Our survey clearly demonstrates that a high percentage of dermatologists in private practice would use tele-dermatology. In our experience, for the excellence of this service an image documentation system is essential to provide the tele-dermatological expert with standardized images with constant illumination. PMID- 10747539 TI - European field tests with HISTKOM telepathology equipment. AB - HISTKOM telemicroscopy equipment for telepathology is designed for the most challenging application in telepathology: intraoperational frozen section diagnosis. Adapted to this application, it is also excellently suited for all other telepathology modes requesting less sophisticated equipment. The technical concept and user interface are oriented to routine daily pathology. HISTKOM underwent heavy field-tests at several locations. The field-tests designs and the results of five of these are reported in this paper. Telepathology will exploit its advantages in networks hosting participants requesting and offering services. The solution of the interoperability problem caused by different equipment from different suppliers within such a network will be a major task, the solution for which is in progress. The new generation of HISTKOM equipment and software is designed in a modularized concept, allowing the integration of various hardware components from different manufacturers; thus special configurations can be realized easily. HISTKOM is offered as complete turnkey system, but can also be installed in yet existing configurations of the customer if they meet specifications. PMID- 10747540 TI - Economic analysis of teleradiology applications with KAMEDIN. AB - The teleradiology system KAMEDIN (German Telekom), installed on HP-Unix- and NT Workstations, was evaluated in different scenarios and a cost-benefit-analysis was performed. CT examinations were transferred from a PACS workstation (GE) to KAMEDIN using DICOM-3 protocol. Teleconferences were realized with an intensive care unit by LAN, with a radiology department at 5 km. distance by ISDN, and with an on duty radiologist 22 km. away by ISDN. On average, 36 CT slices per patient were transferred. Overall costs (costs for hardware, software, support, ISDN fees, and staff) were compared to possible cost reduction, mainly concerning transportation and films. These three scenarios could be realized during daily routine work. Differing in their amount of transportation cost reduction, two applications (intensive care unit, radiologist on duty) showed a break-even at 1817, respectively 528 teleconferences/year. Improvement of cost-effectiveness can be obtained on the conditions that existing hardware will be used and that the automatic data transfer will be improved. Combining all optimisation factors, the break-even decreased to a minimum of 167, respectively 77 teleconferences/year. The optimisation of patient management is an additional- but in this study, not yet counted--advantage of teleradiology. PMID- 10747541 TI - WWW-based access to radiological patient data: two years of experience. AB - This paper describes the theoretical concept behind, the technical implementation of, and the experiences created with a self-developed system to access radiological patient data by WWW-technology. MATERIALS AND METHODS: The WWW system interfaces the RIS via HL7 and the PACS via DICOM. Core components are a secure Web-Server and an underlying database system (DB). The DB contains all relevant information from the RIS and thumbnails of corresponding images archived in the PACS. After authorization, the user receives this information in the form of dynamically generated Web pages. By mouse-clicking a thumbnail, the original DICOM-image is sent. It is displayed with a helper application (DICOM-Viewer), allowing all kinds of image manipulation and post-processing. RESULTS: Determined advantages of the system were: platform independence, security features, a fixed link of image and report, universal availability, and simple usage. The only critical issue was performance. Cost savings could be seen in: a reduction of DICOM workstations, employment of all available hardware, and reduced training and teaching efforts. CONCLUSIONS: The author believes that a WWW-based concept is the only feasible approach which is in the same way technically possible, clinically acceptable, and financially affordable in order to grant a variety of users access to radiological data--although improvements in performance have to made by, e.g., closer implementation of those systems to the RIS and PACS architecture, and by employing high-grade image compression. PMID- 10747542 TI - Towards a methodology for developing and implementing best practices in telehealth and telemedicine. AB - One objective of the growing telehealth and telemedicine literature base is to inform potential adopters and raise their awareness through pilot projects. However, pilot studies often fail to create sustainable solutions because certain conditions are needed for long term success: (1) matching needs to appropriate solutions: while needs assessment can identify needs, adopters must view, test, and experience potential solutions under a number of circumstances; (2) change and new technology: technological implementation brings both foreseen and unforeseen changes, difficult to forecast, and pilot projects can create artificial expectations; (3) awareness of context: there has been a pervasive tendency to treat telemedicine or telehealth as separate from the context of health care delivery, but telemedicine is only a communication and information tool, and should not be considered as separate from other health care delivery activities; (4) availability of long term funding; while loans, government grants and private sector partnering can bridge gaps temporarily, sustainable solutions require that savings from existing systems can be applied to pay for telehealth and telemedicine installations. This paper makes a case for adopting best practices by proposing that a data base of case studies documenting best practices and success factors be developed and made widely available. PMID- 10747543 TI - Southern Health Board--advanced telematic/telemedicine in healthcare services in the south west of Ireland. AB - The Southern Health Board has been involved in Telematic Healthcare services for the past number of years. Our involvement in a number of EU funded projects has given us extensive experience and expertise in this area. The primary focus of our telematic applications is optimising health services delivery with a particular emphasis towards people who are isolated due to social and physical boundaries and limitations. Our main projects are: NIVEMES (Network of Integrated Vertical Medical Services) includes the exchange of medical information between urban centres and isolated communities including those at sea, rural, elderly and disabled persons. RISE (Remote Information Services for the Elderly) focuses on the enhanced healthcare services of the elderly and disabled through the use of telemedical applications. HEALTHLINE To strengthen the successful outcome of Telehealth implementation by specific projects in the EU by providing the expert Training and Information dissemination services to providers, beneficiaries and the general public, using the Telehealth systems and infrastructure applied by those projects. KATE (Killarney & Telecom Eireann) the SHB in conjunction with the Information Age Town Project in Killarney are working towards benefiting the local community through the innovative use of Information and Communication Technologies. TASTE (Technology Assessment in Telemedicine) An EU funded trans national initiative aimed at developing a methodology for assessing medical technology, particularly in the area of Neurosurgery. FECV (Forward Emergency Control Vehicle) An European Space Agency project through which we are developing an advance communications vehicle for major emergence/event management through Satellite linkage to SHB systems. PMID- 10747544 TI - Remote Communities Services Telecentre Project. AB - The delivery of teleservices to rural communities is challenged by the high cost of infrastructure and maintenance. This is compounded if land-based telecommunication links are absent or if bandwidth is limited, as is the case in many areas of the world. The concept of the Remote Communities Services Telecentre (RCST) is a shared-use facility that can be implemented in rural and remote communities to provide telelearning, telehealth, communications, and information access services. This can be through conventional telephony or cable or, as more likely in remote regions, through the use of a shared wireless facility using satellite. This model is being implemented as a wireless network in the Province of Newfoundland through collaboration between the Telemedicine and Educational Technology Resources Agency (TETRA) of Memorial University and a number of partners. Private sector partners in St John's include Collaborative Network Technologies Inc. (CNTI) and FutureWorks Inc. Partners in Ottawa include Telesat Canada, QTECH Hybrid Systems, and the Communications Research Centre. Funding support has been provided by the Canadian Network for the Advancement of Industry, Research and Education (CANARIE); Operation ONLINE of Newfoundland and Labrador; and the European Space Agency. The Newfoundland partners were part of the Team Canada exhibitors group at the G7 Information Minister's Summits in 1995 (Brussels) and 1996 (Johannesburg). The TETRA Centre at Memorial University has previously been involved with international telehealth and telelearning projects in Africa and the Caribbean. This presentation will discuss the RCST project as a model for widespread implementation in the belief that many rural communities world-wide which currently lack adequate wired telecommunications, will opt strategically for a wireless solution given technologic and economic realities. The concept of the Remote Communities Services Telecentre (RCST) is to provide a telecommunications resource facility in small remote communities that have limited communications infrastructure. Such communities are found throughout the world, in lesser-developed and developed countries. In Canada, these communities tend to be in the north. The Telemedicine and Educational Technology Resources Agency (TETRA) of Memorial University has considerable experience in the delivery of services in education and health to such northern Canadian communities. PMID- 10747545 TI - "HausTeleDienst"--a CATV-based interactive video service for elderly people. AB - Since 1991, 17 elderly persons (aged 75 to over 90) in 15 households have been connected via TV-videophones to a service centre. A standard CATV network was modified to support a reverse channel. This has been world-wide the first fully interactive broadband video communications project implemented in a real setting and operating over an extended period of time. The overall aim has been to prove the ability of frail elderly and mobility-impaired persons to live independently, and to demonstrate strategies for reducing the load on social and health care service resources. Service components include remote care; information and assistance; emergency, counselling, training and exercise services. Practical experience, both from the side of the service provider and that of the users, is discussed. Simplicity of technical design and ease of handling contributed to a feeling of being empowered by the technology. In addition, the very personal and close relationship to the staff--which was strongly supported, not inhibited by the video communications--was a major factor in acceptance of the service. It proved a highly successful activity with a great potential for the future: In different contexts with newer technology, market trials are underway to commercialize this service. PMID- 10747546 TI - Fonetix: building virtual speech therapy practicum over the Internet. AB - The purpose of this project is to create an advanced interactive speech therapy site that can be used over the World Wide Web (WWW). This multimedia site, which we have named Fonetix, includes animation of articulation, audio, and video display and capture. The main function of Fonetix is to augment the role a speech language pathologist can play while interacting with patients remotely over the Internet. High quality consultative services via the Internet means that all individuals, regardless of their locale, can have access to best possible service. Fonetix could allow a patient, no matter how remote they are, to have access to the services often only available in major centres. The basic research work into Fonetix has been completed and a functional model has been constructed that will run on any type of personal computer with standard audio and video cards, therefore avoiding expensive equipment requirements. The ultimate goal of the Fonetix system is to create a multi-functional framework that will allow meaningful interactions via the Internet in a variety of circumstances--including clinical, educational and social functions. PMID- 10747547 TI - Evaluation of a Canadian telepsychiatry service. AB - A telepsychiatry pilot project linking five rural general hospitals and Alberta Hospital Ponoka was implemented in June 1996 as a response to both a scarcity of psychiatric resources in the Province of Alberta and the emphasis on community based care. Psychiatrists located at Alberta Hospital Ponoka provided psychiatric consultations using videoconferencing technology in response to referrals from general practitioners. The project was evaluated through the use of questionnaires administered to consumers, service providers, and psychiatric consultants. Questionnaires were supplemented by interviews, site visits, logs, telephone interviews with consumers, and surveys to general practitioners at the rural sites. A total of 109 consultations occurred during the pilot stage. Results strongly suggested acceptance and satisfaction with the consultation by consumers, general practitioners and psychiatrists. The patient's perceived benefits included timeliness of service, service within their own community, decreased work time loss for self or family, and positive outcomes as a result of specialist expertise. Up until September 30, 1998 a total of 535 consultations had been completed, 381 of which were new referrals and 154 of which were follow up referrals. Seventy-two different physicians referred to the service. The practice profile is dramatically different to outpatient psychiatric practice in which approximately 90% of patient contacts are follow-up visits. PMID- 10747548 TI - Functional characteristics of the telemedical network for the medical service of the Bundeswehr for support of operations outside Germany and civil-military co operation. AB - The maxim governing the accomplishment of medical tasks by the Medical Service of the Bundeswehr stipulates that the soldiers will, in case of illness, accident or injury, receive medical treatment to the standard they can expect in the Federal Republic of Germany. In this context the introduction of telemedical applications into the Medical Service will improve the quality of the medical care through better and faster access for the medical officer assigned to a unit or a ship for requesting support to expert knowledge from military facilities or the civilian sector. The telemedical system is based on a high-class commercial PC. This workstation can be extended as needed with medical devices (x-ray film digitizer, dermatoscope, otoscope) and allows linkage to other imaging methods (e.g., videocamera, ultrasound). Currently this system is under evaluation in a multi centre trial. The participants of this telemedical network communicate via EURO ISDN lines with sufficient bandwidth. If terrestrial lines are not available the communication will be based on satellite capacity. The application of internet technology additionally provides access to medical data bases respectively allows civil-military co-operation and the interoperability between medical services of allied armed forces. This future-orientated telemedical approach with its modular upwards-compatibility will provide, especially in its mobile version and by using communication standards, the possibility to support necessary civil-military co operation in the area of disaster relief and missions outside of Germany in the future. PMID- 10747549 TI - Suggested telemedicine websites. PMID- 10747550 TI - Theory of use behind telehealth applications. AB - A variety of telehealth applications have been implemented throughout the industrialized world. Given that telehealth has yet to become routine in the practices of physicians, the impact of such applications on health care management remains difficult to identify. Only by becoming integrated into the 'normal practice' of clinicians can telehealth facilitate smooth communications in health care delivery processes. In this paper we demonstrate the importance of developing a 'theory of use', describing how clinicians are expected to use teleconsultation in their daily activities. By employing Giddens' Structuration Theory, we can develop a clear vision of the utilization of telehealth, and a better framework with which to assess its added value for health care delivery. We illustrate this claim by referring to our research on the Quebec Inter Regional Telemedicine Network. In this study we compare the 'theory of use' behind the telemedicine network with the perceptions of physicians regarding their needs in terms of communications and access to expert advice. The breach between the theory and the perceptions of physicians may help to explain how the utilization of telehealth could be enhanced, and how new and sustainable routines for its integration into the health care system might be created. PMID- 10747552 TI - Assessing the impact of telemedicine on health care management. AB - Telemedicine is undoubtedly one of the most dynamic developing fields of IT technologies in health care. From research and development to routine clinical use of telemedicine applications, decision-makers demand information on the impact that widespread use will have on structures and processes in health care. To date published evaluations have mostly focused on technical core functions. Future evaluation concepts have to consider side-effects the use of telematic systems in health care might have. The report of effects should consider the 4 major interest groups: patients, providers, third-party payers, and society. Learning from experiences in other IT-fields, system developers can avoid pitfalls that might lead to a poor acceptance of their systems or the discontinuation of their R&D project at an immature stage. PMID- 10747551 TI - The value of an evaluation framework for telehealth initiatives. AB - Healthcare managers and policy makers will, in the immediate and near future, make major decisions about the allocation of scarce healthcare resources for telehealth 'solutions'. In our haste to capitalize on what technology can do we may be obscuring discussion and research about what technology should do. For example, currently much attention is being paid to standardization for technological aspects of telehealth. In contrast few efforts have been made to seek standardization in regards to a broad evaluation framework for telehealth. A body of opinion believes that missing in our rush into the on-line world is a systematic approach to research into the human, social, cultural, economic, and political factors associated with healthcare. As a result we lack the tools and experience necessary to assess the true value and implications of telehealth 'solutions'. Developing general guidelines for an evaluation framework, from needs assessment through integrated research to post-study assessment, would greatly enhance the quality of decision making by healthcare managers and policy makers. We propose a model--the Telehealth Integrated Research Model (TIRM)--as the first step in encouraging discussion and development of an internationally accepted standardized telehealth evaluation framework. PMID- 10747553 TI - A review of Japanese telemedicine research and global co-operation. AB - We can trace 229 telemedicine trials in Japan. Of these, 150 were active in 1998, including 73 in radiology, 24 in home care, 22 in medical imaging, 20 in pathology, 5 in ophthalmology, and 3 in dentistry. Some of them have been reformed into practical daily activities. However, activities using the same method work in some local areas but not in others. Typical successful cases will be presented and discussed. We are constructing Japan-Canada cooperation in numerous areas using several communicative methods: ordinary telephone, ordinary Internet, ISDN (2B/6B), and an offline network using optical memory cards (which patients carry). We have introduced successful domestic cases into global cooperation. Some of them will be useful for exchanging medical information among developing countries. PMID- 10747554 TI - Telehealth in British Columbia: from design to delivery. PMID- 10747555 TI - The SIREN legal workshops: list of urgent legal actions for telemedicine. PMID- 10747556 TI - Health care professionals protocol for secure online transmission of patient data. AB - One of the results of the 49th Bavarian Physician's Conference was that in 1996 the Bavarian Statutory Health Care Administration (Kassenarztliche Vereinigung Bayerns, KVB) and the Bavarian Medical Association (Bayerische Landesarztekammer, BLAK) jointly suggested a project for the development and verification of a security infrastructure for the online transmission of medical patient data. This Project, the so-called "Health Care Professionals Protocol" (HCP-Protokoll), was designed to establish the first consensus standard for a secure and probably open system to be used in the health care system operating under the constraints of the diverse and heterogeneous IT-infrastructure in Bavaria, with a view towards utilization in all of Germany. In January 1997, the HCP-Protocol was accepted as the strategic mainline project for telematics applications in medicine by the State of Bavaria and endowed with more than 1.3 million DM in the framework of "Bavaria Online II". In the meantime, various national organizations of the German medical community as well as important industrial partners have expressed support for this initiative. Mention in the Roland-Berger study "Telematics in Health Care, Perspectives of Telemedicine in Germany", designation as an "exemplary scenario" in the final report of the Working Group 7 of the Forum Info 2000 "Telematics Applications in Health Care", as well as integration of the new German health professional card, make the HCP-Protocol the most promising candidate for a de facto standard in the security infrastructure for all participants in health care telecommunication in Germany. Since the middle of 1998, an expert group under the guidance of the joint "Projektburo Telemedizin" of the KVB and BLAK has been working on the definition of this protocol, taking into consideration the current legal framework of the German medical profession (Berufsordnung), the German signature law (SigG), and the national data security laws (BDSG), as well as today's technical possibilities and existing components. The results of this definition will be published at the first opportunity (sometime during the first half of 1999) and then placed into public domain, so that any solution provider can reference this source to create secure and mutually interoperable application programs for health care professionals in Germany. PMID- 10747557 TI - The G8-Global Health Care Applications Project (GHAP) and its role for the development of national telemedicine activities. PMID- 10747558 TI - Results of European projects enabling secure regional, national and international health care networks. AB - Based on the work of the ISHTAR, the TRUSTHEALTH1, the EUROMED-ETS and the MEDSEC project funded by the European Commission, the synergetic results of these different projects in providing a security infrastructure for the test environment of an oncological network will be demonstrated. The ISHTAR project investigated security issues in Health Care Establishments (HCE). It provides guidance for threat and risk analysis, and defines a schema for specification and implementation of appropriate security services and mechanisms to respond to the HCE security requirements of both communication and application security concepts. The TRUSTHEALTH1 project dealt with specification and test implementation of basic security services and mechanisms based on Health Professional Cards (HPC) and Trusted Third Party (TTP) services. Within the EUROMED-ETS project, the security solution has been extended to the Internet environment and WWW tools, successfully creating an Internet-based international TTP structure between the universities of Athens, Calabria, and Magdeburg. Finally, in the MEDSEC project the specification and implementation of a generic EDI security solution has been delivered to provide secure communication between applications and sites. In the ongoing TRUSTHEALTH2 project, the results of all these projects will be implemented in a real environment of cancer care. PMID- 10747559 TI - NOAH. A mobile emergency care system. Notfall-Organisations- und Arbeitshilfe. AB - Experiencing communication problems in our emergency services, we developed an innovative communication network and system (NOAH, which stands for Notfall Organisations- und Arbeitshilfe, or Emergency-Organisation Active Aide System). In contrast with the currently established emergency services communication in Germany by radio wave, data is transferred from the scene in the form of an electronic record on a notepad-computer by means of the wireless data communication network Modacom (Mobile Data Communication), which is provided by German Telekom. It is received at the dispatch-centre in mentioned format and transferred to the admitting hospital without any loss of data. In a prospective study, the technical and conceptual suitability of this system was investigated. It was shown that an admitting hospital was informed more than 20 minutes in advance about the admittance of a patient. To assure the quality of transferred data, information was ranked by different criteria. This further demonstrated that much more precise information about a patient's condition was already available upon admittance to the hospital. PMID- 10747560 TI - MediLink: a wearable telemedicine system for emergency and mobile applications. AB - The practical needs of the medical professional faced with critical care or emergency situations differ from those working in many environments where telemedicine and mobile computing have been introduced and tested. One constructive criticism of the telemedicine initiative has been to question what positive benefits are gained from videoconferencing, paperless transactions, and online access to patient record. With a goal of producing a positive answer to such questions an architecture for multipurpose mobile telemedicine applications has been developed. The core technology is based upon a wearable personal computer with a smart-card interface coupled with speech, pen, video input and wireless intranet connectivity. The TransPAC system with the MedLink software system is designed to provide an integrated solution for a broad range of health care functions where mobile and hands-free or limited-access systems are preferred or necessary and where the capabilities of other mobile devices are insufficient or inappropriate. Structured and noise-resistant speech-to-text interfacing plus the use of a web browser-like display, accessible through either a flatpanel, standard, or headset monitor, gives the beltpack TransPAC computer the functions of a complete desktop including PCMCIA card interfaces for internet connectivity and a secure smartcard with 16-bit microprocessor and upwards of 64K memory. The card acts to provide user access control for security, user custom configuration of applications and display and vocabulary, and memory to diminish the need for PC-server communications while in an active session. TransPAC is being implemented for EMT and ER staff usage. PMID- 10747561 TI - How do regulatory agencies ensure the release of a safe medical device? AB - This article describes the current regulatory activities of the Food and Drug Administration (FDA), in particular the FDA techniques for regulating medical device approvals, medical-device reporting, and medical-device-company inspections. Also discussed are the FDA Modernization Act and the methods the FDA is using to implement the act. PMID- 10747562 TI - Institutional epidemiologists' roles in evaluating medical devices. AB - As the prevalence and variety of medical devices have increased, so too has the need for active involvement by epidemiologists. Traditionally, epidemiologists enter the chain of events after devices are marketed. It is proposed that a more proactive approach should be taken and that epidemiologists should be involved at all stages of product development. PMID- 10747563 TI - Dollars and sense: a practical guide to cost analysis for hospital epidemiology and infection control. AB - This paper explains practical approaches for collecting inpatient cost data for cost-of-illness and cost-effectiveness analyses. The economic definition of cost of an item is the value of the resources that are consumed in its production. Cost analysis should collect the resources hypothesized to be affected by the illness or intervention. The dollar value of these resources can also be estimated. Diagnosis-related group (DRG) reimbursements are not helpful when all study patients have the same DRG or when no DRG exists (e.g., nosocomial infection). Hospital charges are not a good surrogate for costs. Hence, data needed include resources used, charges, and cost-to-charge ratios, so that cost can be estimated. Resources used can be obtained from hospital information systems. For some resource use (e.g., physician services, pharmacy, and intravenous fluids), charges or cost-to-charge ratios may not be available, and an external standard may be needed to estimate the dollar value. For many types of resources, hospital financial systems provide both charges and cost-to-charge ratios. This yields an estimate of average cost (total cost divided by patient days) when marginal cost (change in variable cost per day of patient stay) is a better estimate of the value of the resources consumed. However, cost-to-charge ratios remain the only practical way of estimating cost in many circumstances and are commonly used in economic studies. Cost-of-illness estimates vary among the various nonrandomized study designs used. "Real-world" randomized trials are potentially useful to obtain advantages of randomization but avoid the protocol induced biases of traditional double-blind controlled trials. PMID- 10747564 TI - The indirect effect of managed care: a true story. PMID- 10747565 TI - Implementing preventive health measures: a pilot study. AB - OBJECTIVE: Implementation of preventive services guidelines is performed inconsistently. In an attempt to reduce variation in guideline implementation, we developed a patient questionnaire based on the US Preventive Services Task Force Guide and the Health Plan Employer Data and Information Set 3.0 performance measures of the National Committee on Quality Assurance. SUBJECTS: 100 hospitalized patients of five primary-care physicians. METHODS: In a pilot study, 100 hospitalized patients of five primary-care physicians were questioned about their compliance with evidence-based, preventive healthcare recommendations. Information was requested on blood pressure measurement, cholesterol screening, fecal occult blood testing, smoking-cessation counseling, Pap testing, mammography, postmenopausal hormonal replacement therapy counseling, prostate examination and prostate-specific antigen (PSA) testing, use of aspirin and beta blockers following an acute myocardial infarction, testing of diabetics for hemoglobin A1c and retinal eye examinations, questioning of the elderly for auditory and visual problems, and receipt of influenza and pneumococcal vaccines. Information on variations from the recommended preventive service was fed back to their physicians. Six months after the initial survey, the patients were requestioned to determine if compliance had improved with the recommendations. RESULTS: We found significant improvement in fecal occult blood testing, smoking cessation, Pap smear testing, mammography use, prostate examinations and PSA testing, hemoglobin A1c testing, seeing or hearing loss follow-up, and the administration of influenza and pneumococcal vaccines. CONCLUSIONS: Improving implementation of preventive services recommendations is a challenge. This pilot study suggests that involving the patient more in the process and informing the physician of the results may improve the process. PMID- 10747566 TI - Factors associated with changes in satisfaction with care. AB - OBJECTIVE: Satisfaction with care is an important outcome for evaluating the effectiveness of medical care. Many factors can influence satisfaction, including disease state, healthcare utilization, and health-status changes. However, few studies have investigated the association between these factors and changes in satisfaction. DESIGN: This study examined the influence of personal characteristics, type of health plan, disease states, and healthcare utilization on changes in satisfaction with care in a prospective cohort over a 12-month period through two surveys, baseline and follow-up. PARTICIPANTS: Enrollees in one of three different commercial health plans: point-of-service product, an unrestricted fee-for-service product, and a preferred-provider organization product. MEASUREMENTS AND MAIN RESULTS: Two multivariate logistic regression models were constructed. The first model evaluated factors that predicted increased satisfaction with care between the two surveys. Compared with respondents who reported no change in health status, both those with improved health status (odds ratio [OR], 1.29, 95% confidence interval [CI95], 1.03-1.61) and those with declines in health (OR, 1.29, CI95, 1.03-1.61) were significantly more likely to report an increase in satisfaction with care. Those with a history of hospitalization were also more likely to report an increase in satisfaction with care (OR, 1.27, CI95, 1.01-1.59). The second multivariate logit model evaluated factors that predicted decreases in satisfaction with care from the baseline survey. Those with reported declines in health status were more likely to report decreases in satisfaction with medical care (OR, 1.43, CI95, 1.13 1.79). Neither age, gender, race, type of health plan, disease state, nor doctor's office visits were related to observed changes in satisfaction with medical care. CONCLUSION: Changes in satisfaction with care appear to be related to changes in health status. However, the relation between these two attributes is not intuitively apparent. PMID- 10747567 TI - Outpatient satisfaction: validation of a French-language questionnaire: data quality and identification of associated factors. AB - OBJECTIVES: Following 1996 legislation requiring French hospitals to assess patient satisfaction, this study developed and validated a brief French-language multidimensional questionnaire designed to measure outpatient satisfaction with hospital visits and compared data quality for two patient-satisfaction survey methods. DESIGN: Authors developed a 19-item questionnaire following a strict procedure (identification of dimensions to explore, formulation, and selection of items). SETTING: Validation data were obtained from patients of six outpatient clinics in a teaching hospital. PARTICIPANTS: 586 consenting eligible patients were randomized to receive the questionnaire 2 weeks after their visit with one of two survey methods: a mailed self-administered questionnaire or a telephone interview. RESULTS: The response rate (79%) was not significantly different between the two survey methods. The risk of having one or more missing values was higher in the mail survey group (odds ratio, 1.65; 95% confidence interval, 1.03 2.63), but mail respondents were less likely to use the "extremely positive" response category. Principal component analysis identified four factors that accounted for 56% of the variance: interpersonal skills and information transfer, physical surroundings, convenience, and appointment delay. Patients' comments on open-ended questions validated the semantic content of the factorial construct. The internal consistency coefficient was greater than 0.70 for three of four subscales. Patient background characteristics accounted for less than 10% of the factorial score variance. Patient satisfaction was correlated with age, type of visit, and, to a lesser extent, gender and education level. CONCLUSION: This easily administered, multidimensional out-patient-satisfaction questionnaire provided encouraging preliminary psychometric characteristics. PMID- 10747568 TI - The cost of burn care and implications for efficient care. AB - OBJECTIVE: To clarify the social issues and problems associated with burn care in Japan, based on a cost analysis of acute burn care. METHODS: A retrospective review was undertaken of 71 patients admitted with burns at Nippon Medical School between January 1 and December 31, 1997. A cost analysis was performed for three major burn patients with a burn surface area (BSA) of 70% to 80% and three minor burn patients with a BSA of 20% to 30%. A questionnaire was administered to both burn patients and medical providers in all 127 emergency centers to help improve long-term quality of life (QOL). RESULTS: 80% of burn patients were under age 70. In the major burn cases, the amount of reimbursement according to the government regulated fee schedule was much less than the cost of treatment. The ratio of the cost of both medication and materials to total cost of treatment was higher for patients with major burns. Patients responding to the survey acknowledged being generally happy despite suffering from psychological and financial problems. A total of 413 medical providers from 63 institutions responded to the survey regarding improving long-term QOL. CONCLUSIONS: Acute burn care is a costly service. The goal of burn care should shift from saving lives to allowing for a productive return to society. Quality improvement, outcome measurement, and technology assessment combined with health economics research for burn care should be carried out in Japan. PMID- 10747569 TI - A comprehensive educational approach to improving patient isolation practice. AB - OBJECTIVE: To describe a workable plan for the successful education of a large, diverse group of healthcare workers in a university hospital setting. DESIGN: A prospective, nonrandomized study of compliance with infection control isolation practice following various educational interventions. SETTING: 300-bed tertiary care, academic medical center with out-patient clinics. PARTICIPANTS: Hospital employees and contract workers. INTERVENTIONS: The infection control department introduced a plan to implement the Centers for Disease Control and Prevention's new isolation guidelines. A comprehensive proposal was presented to administration. It included a time line for institution and a comprehensive educational and performance-improvement plan, including standard lectures and a video that explained Standard and Transmission-Based Precautions. Follow-up consisted of customized in-services and one-on-one continued education tailored to the individual units. RESULTS: Compliance with isolation procedure after standardized lectures and video alone was poor. Compliance improved after institution of smaller, more intensive in-services tailored to individual departments and given during all shifts. CONCLUSIONS: Intensive, individualized education is the key to compliance. This requires sufficient infection control staffing and administrative support. PMID- 10747570 TI - The psychological and physiological effects of an intensive-care unit environment on healthy individuals. AB - OBJECTIVE: The ideal inpatient environment would be one in which patient stress and anxiety are alleviated, but current inpatient hospital settings often do not seem to take this fact into consideration. To date, the effects of the actual hospital environment itself on patients is poorly understood. The purpose of the present study was to investigate the types of psychological and physiological changes that people undergo in response to being placed in an inpatient setting. DESIGN AND SETTING: Ten healthy volunteers were admitted to an intensive-care unit (ICU) for 4 days and 3 nights. The psychological and physiological changes before and after admission were examined. A qualitative assessment was also performed. RESULTS: Most healthy individuals at times develop feelings of depression, which can be measured by the Profile of Mood States and the Zung Self Rating Depression Scale (P < .05). In this study, participants' fatigue and confusion increased (P < .05) and vigor decreased (P < .01) as measured by the Profile of Mood States. There was also an increase in the General Health Questionnaire scores (P < .01). However, no significant physiological effects were apparent, as measured by peripheral lymphocyte counts, natural killer cells activity, and urinary 17-kerosteroid and 17-hydroxycorticosteroid levels. Subjects often engaged in introspection during hospitalization and often had a negative attitude towards the hospital environment. CONCLUSIONS: The findings of increased feelings of depression attributable solely to being in an ICU setting are inconsistent with the type of environment generally considered necessary to alleviate patient anxiety and tension in a critical-care-ward environment. The negative perception of this environment strongly suggests room for improvement. Efforts in this regard should focus on improving the five senses, particularly sight, sound, and taste within the ICU. PMID- 10747571 TI - Emergence of highly antibiotic-resistant Pseudomonas aeruginosa in relation to duration of empirical antipseudomonal antibiotic treatment. AB - OBJECTIVE: This study examines antibiotic resistance in Pseudomonas aeruginosa in hospitalized patients in relation to prior empirical antibiotic therapy. DESIGN: Two retrospective case analyses comparing patients who manifested P aeruginosa with differing patterns of antibiotic resistance. SETTING AND PARTICIPANTS: Patients acquiring P aeruginosa in a community hospital. MEASURES: Patients were compared on duration of hospitalization and days and doses of antibiotics prior to recovery of P aeruginosa. Patients were grouped, based on susceptibility patterns of their P aeruginosa isolates classified as follows: (1) fully susceptible (susceptible to all classes of antipseudomonal antibiotics [SPA]), (2) multidrug-resistant (resistant to two classes of antipseudomonal antibiotics [MDRPA]), or (3) highly drug-resistant (resistant to > or = 6 classes of antipseudomonal antibiotics [HRPA]). To control for duration of hospitalization, antibiotic treatments of HRPA and SPA patients were compared during the first 21 days of care. RESULTS: Prior to recovery of HRPA, six HRPA patients received greater amounts of antibiotics, both antipseudomonal and non-antipseudomonal, than did six SPA patients prior to recovery of SPA. For 14 patients with hospital acquired SPA who later manifested MDRPA, duration and dosage of antipseudomonal antibiotics, but not all antibiotics, were significantly higher for the SPA-to MDRPA interval than for the preceding admission-to-SPA interval. The median duration of antipseudomonal antibiotic treatment prior to the recovery of P aeruginosa was 0 days for SPA, 11 days for MDRPA, and 24 days for HRPA. CONCLUSION: Duration of empirical antipseudomonal antibiotic treatment influences selection of resistant strains of P aeruginosa; the longer the duration, the broader the pattern of resistance. PMID- 10747573 TI - Results of a JCAHO accreditation survey in a university student health center. AB - This article describes the results of our initial accreditation survey by the Joint Commission on the Accreditation of Healthcare Organizations (JCAHO). Strengths, as well as supplemental recommendations in our clinic and laboratory programs, were identified during the accreditation survey. Mechanisms for addressing the supplemental recommendations are described in this report. Direct and indirect costs of the accreditation process also are discussed. PMID- 10747572 TI - Underreporting of percutaneous exposure accidents in a teaching hospital in Spain. AB - The study's objectives were to determine the frequency of biological-risk accidents involving percutaneous exposure and to identify factors associated with underreporting. Two hundred fifty healthcare professionals from inpatient services at high risk for exposure at the Puerta del Mar University Hospital of Cadiz, Spain, participated in the study. A questionnaire was used to measure personal and work variables, the number of accidents suffered and reported in the last year, and the circumstances motivating the reporting or nonreporting. Two hundred thirty-two persons (92.8%) completed the questionnaire. The accident rate was 12 per 100,000 hours worked. Physicians were the most frequent accident victims (rate 22/100,000 hours). The general surgery and emergency services had higher rates than other services (rates 19.82 and 14.17, respectively). Sixty-six percent of the accidents were not reported to the register. The main predictors of the underreporting were length of professional service greater than 19 years, working in the surgery service, and the perception that the accidents did not involve health risk. The true accident rate was higher than that reflected in the Accident Register. Underreporting was high. The main variables associated with underreporting were length of professional service, work area or department, and perception of risk from the accident. PMID- 10747574 TI - Handwashing education practices: a descriptive survey. AB - Hand washing is the single most important function in preventing the spread of infection. However, compliance by healthcare workers still remains under 50%. Intensive observational and interventional programs have been shown to increase compliance, but the increase in compliance is short-term. Therefore, in April 1998, we conducted a random survey of 500 members of the Association for Practitioners in Infection Control and the Society for Healthcare Epidemiology of America to determine the didactic educational practices (in-service lectures) of practitioners with regard to hand washing. Response rate was 18% (n = 90). Eighty eight percent (73) conducted new employee orientation, 64% (73) conducted yearly in-service, whereas monthly, quarterly, and semiannual in-service were 11% (9), 11% (9), and 14% (12), respectively. Thirty-nine percent (35) of our respondents rated their compliance to hand washing as > 50%; 31% (19), < 50%; and, 39% (33) did not know. Only 5% (4) of our respondents determined the outcome measures and cost-effectiveness of their present handwashing program. Our survey had identified the lack of ongoing education for the healthcare worker about handwashing compliance. Continuous reinforcement through monthly in-service is labor-intensive and costly. An alternative model using the patient as ongoing reinforcement may be a solution for increasing compliance. PMID- 10747575 TI - Systems are changing: where can they be improved? AB - Wide-ranging changes of unprecedented scale throughout the delivery of healthcare services are producing consequences we cannot ignore. A need to collaborate to improve healthcare systems is evident. The Society for Healthcare Epidemiology of America (SHEA) and the Health Care Division of the American Society for Quality are responding in jointly sponsoring this symposium concerning problems with medical devices. This presentation and those that follow will explore the quality of management of managed-care corporations; the important threat to out primary source of innovation in medical technology posed by behaviors of consolidated corporations; the impact of these changes on roles of hospital epidemiologists; and measures to ensure proper design, production, delivery, and use of safe medical products. Our profession and SHEA have made significant research-based contributions that document their potential value, but we have not always done as well as we could to market epidemiology as an important asset for sound management decisions in this era of change. We should ally ourselves with related disciplines in order to succeed as we undertake new challenges on a broadening field. PMID- 10747576 TI - Consolidation happened; now comes the hard part. Large MBHOs don't have an easy path to success. PMID- 10747577 TI - Taking the 'dual' out of dual diagnosis. Providers say more integrated treatment means better outcomes. PMID- 10747579 TI - Measuring outcomes in outpatient settings. How one company is using clinical pathways for depression. PMID- 10747578 TI - Dissecting the carve-out. States try to learn from one another's experiences. PMID- 10747580 TI - The state of the child: a profile of many needs. PMID- 10747582 TI - Same community, new partnerships. Can integrated mental health and child welfare funding work? PMID- 10747581 TI - More than just a lock-up. Juvenile jails must provide a spectrum of treatment services. PMID- 10747583 TI - Embracing children in the community. Why the field places faith in systems of care. PMID- 10747584 TI - Treatment issues for the disruptive child. Diagnosis and interventions require flexibility. PMID- 10747585 TI - Instead of coercive treatment, build service systems. PMID- 10747586 TI - Some clients will never pursue treatment. PMID- 10747588 TI - Intimidated by the Internet? Browser basics. PMID- 10747587 TI - Avoid the fine print blues. PMID- 10747589 TI - Toward an understanding of burnout. AB - Burnout is a syndrome characterized by three dimensions: exhaustion, cynicism and ineffectiveness. It arises as a result of an interface between personal factors that are common to many professionals in conjunction with specific factors that are part of the fabric of any organization. The organizational factors include workload, control, the workplace as a supportive community, reward systems, fairness in decision-making and conflicts in group values. Improvement in group function comes with attention to each of these factors. PMID- 10747590 TI - The most important question in your practice. AB - What went wrong when you provided the best medical care, but the patient left and never came back? By not asking a key question to each patient you may be passing up the best opportunity to satisfy and retain your patients. A restaurant experience reveals how to determine simply and effectively for every patient if you are really meeting the patient's needs. This is cost-effective, patient focused service at its best--right to the bottom line. PMID- 10747591 TI - Developing alternate revenue sources. AB - During the 1990s, with the proliferation of managed care and aggressive negotiating postures by insurance companies, the reimbursement paid to physicians decreased drastically. In order to compensate, physicians have had to extend their work week, reduce overhead or seek alternative revenue sources. Developing alternative sources of revenue without having to work additional hours is the most palatable option. In our practice, almost 15 percent of revenue is derived from alternate or non-physician revenues collected at 100 percent of posted charges. This article highlights some of these solutions and discusses their applicability and appropriateness within a practice. PMID- 10747592 TI - Healers & dealers. AB - While some physicians aggressively seek opportunities to build lucrative business ventures, others resist the intrusion of business issues and risks into their practices. A key to success is ensuring a good fit between an organization's business model and its physician's professional values and business dispositions. This article describes how to assess physicians' readiness for business risk, and it provides a framework for evaluating different business models in light of the professional values and business risk profiles of the organization's physicians. Finally the article provides a roadmap for actions to close gaps between the demands of the business model and the business dispositions of its physicians. PMID- 10747593 TI - Heroes and servant leaders in group practice. An interview with new MGMA chair Robert Goldstein. Interview by Bob Readling. PMID- 10747594 TI - Selecting new technology for your practice. PMID- 10747595 TI - Annual report awards issue. Six hospitals capture annual report competition honors. AB - The best of the annual reports entered in this year's annual Healthcare Annual Report Contest are included in this issue on pages four through 32. Every entry in this year's competition had something to commend. They were reflective of the talent that assembled them. The editors and panel judges thank you for submitting your entries. The winners are New York Methodist Hospital, Brooklyn, first overall; Children's Hospital of Orange County, Calif., second overall; Woman's Hospital, Baton Rouge, La., first community health; Hackensack University Medical Center, Hackensack, N.J., first year in review; Chesapeake General Hospital, Chesapeake, Va., first community relations; and Osteopathic Health System of Texas, second community relations. The editors thank all the entrants for participating. Remember, the deadline for 1999 entries is July 1, 2000. PMID- 10747596 TI - Yale-New Haven Hospital offers complex Web site. PMID- 10747597 TI - Healthcare Web sites brace for information demand. AB - Millions of Americans are turning to the Internet to find and share health information. This phenomenon became a major topic of conversation at the recent fourth annual conference of the Society for Healthcare Strategy and Market Development in Denver. Three leading providers are profiled. PMID- 10747598 TI - Cook Hospital provides a good example of what works on a healthcare Web site. PMID- 10747599 TI - Television programs help health network get prostate cancer drive off the ground. AB - Sometimes a friendly media outlet can be right in your backyard. University of Utah Health Network utilized the school's PBS affiliate to help promote a prostate cancer blood test drive. PMID- 10747600 TI - Salt Lake hospital survives close brush with twister, power outage. AB - LDS Hospital in Salt Lake City found that dealing with wounded and casualties from a natural disaster is just one problem; dealing with the natural disaster itself can be a whole other problem. A great success story in the midst of this summer's terrible tornado which hit Utah's capitol. PMID- 10747601 TI - Hearing, GAO report focus on multiple G deficiencies. PMID- 10747602 TI - Seniors housing still booming, not overbuilt, study says. PMID- 10747603 TI - IHS takes over NovaCare's contract therapy business. PMID- 10747604 TI - Study shows significant drop in Medicare payments. PMID- 10747605 TI - Court gives a qualified nod to community placements. PMID- 10747606 TI - SNFs battle to get relief from BBA cuts. PMID- 10747607 TI - Quality indicators help focus survey. QIs to pinpoint areas for further investigation. PMID- 10747609 TI - Wound/skin/continence survey. PMID- 10747608 TI - Managing data, documentation, and resident care. PMID- 10747610 TI - Making quality assisted living affordable. PMID- 10747611 TI - Supreme Court rulings favor employers. PMID- 10747612 TI - Building staff communication. PMID- 10747613 TI - Montessori methods enhance function. PMID- 10747614 TI - Surfing over sixty. Making Internet access available helps residents stay connected. PMID- 10747615 TI - Clinton bill includes relief funds. PMID- 10747616 TI - Volunteers and governance forge a new partnership. PMID- 10747617 TI - Livin' long in the Lone Star State. PMID- 10747618 TI - A special kind of neighbor. Volunteers make a difference in home health care. PMID- 10747619 TI - Savage spouses. Blunt questions are a lifeline for victims of domestic violence. PMID- 10747620 TI - Why volunteers: a resource money can't buy. PMID- 10747621 TI - Gazing into health care's crystal ball. PMID- 10747622 TI - A healthy Davidson back with broader perspective, big plans. The captain's back on deck, and he's plotting a shift in course. Interview by Craig Webb. PMID- 10747624 TI - On leadership. PMID- 10747623 TI - Once upon a culture. PMID- 10747625 TI - Waking the giant: creating a future for hospital volunteering. PMID- 10747626 TI - Some volunteer ABCS. PMID- 10747627 TI - Volunteer orientation checklist. How to make the most of a new helper's first few days. PMID- 10747628 TI - Public health funding mechanisms in New Zealand. AB - The funding of population-based public health services (health protection, health promotion and disease prevention) has received little attention in the international literature on health reforms, and yet these services are of fundamental importance to the health of populations and to the economy. This article provides justification for health policy-makers placing more emphasis on the level of public health funding compared with funding on personal health services, and accountability arrangements for its expenditure, when considering options to improve the performance of their health sectors. The New Zealand experience of funding public health services is described within the context of the health reforms. The strengths and weaknesses of the adopted approach are analysed. PMID- 10747629 TI - A future for primary health care in New Zealand. AB - The attempt to implement a health market in New Zealand by separating funders and providers in 1992 has not delivered improved health outcomes. Indeed there is increasing concern that deprived populations are not accessing appropriate health care. This article describes the models of primary care that have evolved in the new environment and suggests that these new structures, given appropriate support, are ideally placed to increase the focus of primary care on population health. A capitation funding model with patient enrolment and low fee-for-service barriers is proposed as the most promising model for delivering improved health outcomes. The model incorporates a needs-based funding formula, locality health needs assessment, an increased role for primary care nurses and improved responsiveness to local communities, especially Maori. PMID- 10747630 TI - Humbug, health and humanity: a patient's tale of care in a public teaching hospital. PMID- 10747631 TI - Managing unstable angina and coronary care beds effectively. AB - This article is an evaluation of a new chest pain admission policy at Western Hospital. Our aim was to determine the clinical outcomes of patients with unstable angina treated according to this new policy. It involved attempting to identify a group at low risk of serious complications and to manage them in a general ward area without cardiac monitoring, to reduce the pressure on coronary care unit beds. We conducted a retrospective case note review and concluded that that selected patients with an admission diagnosis of unstable angina can be safely managed in unmonitored medical beds provided mechanisms are in place for their transfer to the coronary care unit if recurrent pain or a complication occurs. PMID- 10747632 TI - The role of top management in supporting the use of information technology in Australian hospitals. AB - The progressive use of information systems and information technology has the potential to transform the way complex organisations are managed and the way they operate. This article reports the findings of a study undertaken to examine the importance of various factors related to the progressive use of information technology in Australian hospitals. Our analysis of data from 84 hospitals shows that hospital size has a significant positive relationship with the progressive use of information technology, as does the chief executive officer's attitude to information technology; however chief executive officer participation in information technology activities does not. The implications of these findings for the role of top management are discussed. PMID- 10747633 TI - Quality and general practice accreditation: the approach of Australian General Practice Accreditation Limited. AB - The purpose of this article is to outline the underlying assumptions, principles and processes of a continuous quality improvement approach which underpins the accreditation of general practice offered by Australian General Practice Accreditation Limited. PMID- 10747635 TI - Managed competition: the policy context. PMID- 10747634 TI - A comparison of the impact of hospital reform on medical subcultures in some Australian and New Zealand hospitals. AB - This article examines similarities and differences in the way that hospital staff in Australia and New Zealand are evaluating efforts to improve quality, clinical effectiveness and service integration, and to strengthen clinical accountability. We draw on data from a cross-national study of hospital staff in Australia and New Zealand. The results highlight the way in which respondents' views about reform are influenced by the interplay of two factors: the impact of respondents' occupational backgrounds (our findings point to differences in the profession based subcultures of medicine, nursing and general management and the way that these are reflected in respondents' assessments of particular aspects of reform); and the way that the impact of professional subcultures may be mitigated by differences between the systems in which respondents were located, including differences between the programs of reform that have been pursued in each country. The implications of these findings are discussed. PMID- 10747636 TI - Making sense of integrated care in New Zealand. AB - Integrated care is becoming a significant feature of New Zealand's current health system. Initiatives to date focus on service coordination or devolution of purchasing, which may be viewed as complementary approaches aimed at meeting a common goal of improving services. They are, however, likely to yield different benefits and pose different risks. This article outlines the background to current integrated care developments in New Zealand and offers a conceptual framework for distinguishing the approaches adopted. It also discusses a number of practical issues that will need to be addressed as such initiatives evolve, and considers some of the factors that need to be taken into account when considering where health care purchasing decisions are best made. PMID- 10747637 TI - Health policy and management across the Tasman. PMID- 10747638 TI - Lessons from the other side: what can we learn from the private sector? AB - Business has reacted in an impressive manner to increasing globalisation, short term stock market pressure for performance, emerging industries and new technologies. While the private sector has become increasingly competitive, the public sector has not adopted this commercial rigour. Funding pressures on health services will continue, as will increasing consumer and staff demands and the blurring of public and private health care provision. As a result, there are lessons and techniques the public and private health sectors should learn from each other. I have drawn the issues that follow from my experience in the steel and food industries. PMID- 10747639 TI - Booking systems for elective services: the New Zealand experience. AB - This article provides a brief overview of New Zealand's experience in implementing booking systems for elective services in public hospitals. It identifies the basic features of the booking systems policy and explores the rationale and objectives for these policy settings. Progress with implementation of booking systems is explored and some of the challenges and recent developments are also outlined. The authors argue that booking systems represent a major improvement on waiting lists for patients, providers, purchasers and policy makers. PMID- 10747640 TI - Independent life expectancy in New Zealand, 1996-97. AB - The objective of this article is to describe independent life expectancy (ILE) in New Zealand in 1996-97, including variations in this indicator between age, gender and ethnic groups. ILE is defined as the number of years a person can expect to live without any self-reported functional limitation requiring the assistance of another person or a complex assistive device. ILE is a positive measure of health. Its complement, expectation of life with dependency (LED), is also a useful indicator. Together, ILE and LED add up to total life expectancy (LE). The contribution to ILE from disability and mortality at each age is analysed in this article. The elasticity of ILE to changes in mortality and to changes in disability is also investigated. Finally, the burden of injury is estimated by calculating the potential gain in ILE that would result were injury related disability and mortality to be eliminated. PMID- 10747641 TI - Shifting to capitation in primary care: what might the impact be in New Zealand? AB - Primary medical care in New Zealand has traditionally been delivered by general practitioners and funded by a mix of fee-for-service government subsidies, user part-charges and private payments. In 1998, New Zealand's national purchaser of publicly-funded health care, the Health Funding Authority, proposed to pay health service organisations capitation fees per enrolled patient, as well as fees-for service for immunisations and some performance-related payments. This article considers the implications, drawing on theory and research from New Zealand and elsewhere, of different methods for paying general practitioners and other primary care professionals. The main focus is on whether giving a greater emphasis to capitation will lead to a fairer distribution of resources and better access to services for those groups of people who are not well served by the current system. PMID- 10747642 TI - Revised guidelines for preventing poliomyelitis. PMID- 10747643 TI - Preparing for the next influenza outbreak--or (inevitably) pandemic. PMID- 10747644 TI - Do PAs in clinical practice find their work satisfying? AB - Few PAs--17% of those eligible to practice--have left the profession or chosen another career. One hypothesis is that PAs are satisfied with their profession and their work. To test this hypothesis, the AAPA conducted a survey of the professional satisfaction of AAPA members in clinical practice, collecting data using the 1998 AAPA member opinion survey that had been mailed to 17,336 PAs. Usable responses were received from 12,766 (73.6%) members in clinical practice. Measured were satisfaction with work environment, satisfaction with clinical practice, satisfaction with job, impression of the job market, and outlook on the profession. By all measures, PAs are highly satisfied with their choice of career and job. When respondents were asked to rate all aspects of their job taken together, 90% responded excellent (31%) or good (59%). Ninety percent said that they would definitely (47%) or probably (43%) become a PA if they were starting a career today. PMID- 10747645 TI - Sedating pediatric patients safely. PMID- 10747646 TI - The lure of unconventional therapy for rheumatic disease: how powerful is it? AB - Patients who suffer rheumatologic disease are known to try unconventional therapies in addition to, or instead of, medical therapy prescribed by their provider. Reasons for this include the chronic nature of their illness, the adverse effects of conventional medications, and widespread publicity about the purported efficacy of unconventional therapies. We undertook this study to assess the types of unconventional therapies used by patients in one rheumatology practice. A survey comprising nine questions and a cover letter was mailed to 300 patients in the practice, asking them to rank, in order of perceived effectiveness, unconventional therapies they had used and the treatments provided by the rheumatologist. We determined that the prevalence of unconventional therapy use was 44% (response rate, 45% [135 patients]). Patients' faith, attributes of the physician, and medications prescribed by the physician were ranked more highly effective in the healing process than all other unconventional therapies. We conclude that, although unconventional therapies were utilized by patients in this practice, they valued the physician's medical treatment plan and efforts most. PMID- 10747648 TI - Recruiting, interviewing, and hiring the right person. AB - In 1998 and 1999, unprecedented changes occurred in the U.S. economy: unemployment rates dropped below 4.5%, the stock market soared to more than 11,000, and about 3 million new technology-related jobs were created (1). Faced with worldwide competition and new technologies, many industries and businesses have had to relook at and reengineer their operations. Human resource management has played a key role. Significant resources always have been allocated to the recruiting process to hire competent and loyal employees. To not spend the time and effort to recruit good employees can be costly in many ways. This review offers guidelines to managers and emphasizes just how important this process is to the success of an organization. PMID- 10747647 TI - Strategic human resource management: redefining the role of the manager and worker. AB - Health-care managers have long been sensitive to the importance of physical resources (technology, space) and financial resources to the success of service delivery. During the last several decades, the value and potential for development of a third element, the human resource has gained new recognition. The importance of viewing personnel as an expandable resource, addressed in an organization's strategic plan, is crucial to meet challenges faced in a rapidly changing health-care environment. PMID- 10747650 TI - Staff retention: a major key to management's success. AB - The number of qualified and competent clinical laboratory sciences (CLS) professionals is rapidly declining. As competition grows and the clinical laboratory comes under greater scrutiny, the laboratories that survive will be those that recognize that their, employees are their greatest asset. However, retaining expert CLS professionals requires a dramatic change in management's belief system regarding the value of its personnel assets. Management must create a culture of empowered people who have the freedom to exhibit self-direction in achieving the organization's mission and goals. Management must consider its employees valuable enough to invest in their professional growth. A laboratory's long-term sustainability and economic performance will depend on a committed workforce that supports their organization's success. PMID- 10747649 TI - The new employee: proper orientation and training go a long way toward job success. AB - Considerable money is spent to advertise, interview, and hire a new employee. Considerable amounts of time and energy should be used to train, orient, and encourage this employee. The longevity of employees in their jobs is often directly related to the nurturing of the employee through continuing education. PMID- 10747651 TI - Competency assessment: establishing a program. AB - This article describes how a competency assessment program can be implemented in a clinical laboratory. The author reviews applicable standards and regulations and describes a 10-step process for establishing a program. Sample forms are provided for documenting assessments. PMID- 10747652 TI - Managing difficult employees. AB - The author describes the DISC Trait Theory Model as a means of enabling managers to improve interactions and reduce encounters with difficult employees. A basic understanding of each style (director, interactor, control, and service) is provided as well as strategies for different types of interactions. PMID- 10747653 TI - Involuntary termination of employment: legal and practical considerations. AB - Involuntary termination of employment is the final step in the human resource management life cycle. It often is viewed negatively because it may connote failure on the part of the employee. In some circumstances, however, it may indicate management's failure to turn a situation around. Although you may never expect an involuntary termination to be a pleasant experience, management can find some consolation in assuring that the decision is well founded and based on legitimate, nondiscriminatory reasons. PMID- 10747654 TI - Role of medical, technical, and administrative leadership in the human resource management life cycle: a team approach to laboratory management. AB - We believe the team approach to laboratory management achieves the best outcomes. Laboratory management requires the integration of medical, technical, and administrative expertise to achieve optimal service, quality, and cost performance. Usually, a management team of two or more individuals must be assembled to achieve all of these critical leadership functions. The individual members of the management team must possess the requisite expertise in clinical medicine, laboratory science, technology management, and administration. They also must work together in a unified and collaborative manner, regardless of where individual team members appear on the organizational chart. The management team members share in executing the entire human resource management life cycle, creating the proper environment to maximize human performance. Above all, the management team provides visionary and credible leadership. PMID- 10747655 TI - Understanding the demographics of the evolving workforce. AB - The author discusses the four age cohorts currently in the workplace and the fifth that will arrive by the year 2005: the Pre-Boomers, Boomers, Cuspers, Busters, and Netsters. She describes their workplace and lifestyle characteristics, their social values, and their communication styles. Strategies for motivating and communicating when managing a workforce of diverse ages also are included. PMID- 10747656 TI - The mentoring process: beneficial to manager, employee, and organization. AB - Managers seeking to develop, retain, and stabilize their workforces will find that this article has basic mentoring skill information that will support their effort. It describes benefits the manager and the organization can derive from the process as well as the more commonly expected benefits to the employee. PMID- 10747657 TI - Ethics and race-based home care assignments. PMID- 10747659 TI - Big thinking in small spaces. PMID- 10747658 TI - Finding Mr. (or Ms.) Right. PMID- 10747660 TI - Quality assurance, practical management, and outcomes of point-of-care testing: laboratory perspectives, Part I. AB - Pathologists and nurses have only recently cooperated in point-of-care testing (POCT), after accreditation organizations recommended that the laboratories take responsibility for managing the quality of patient-care testing conducted at the bedside. Laboratories are charged with ensuring that patient-care tests generate comparable results, regardless of the location or method. Many home testing devices, when used in hospitals, physicians' office laboratories, and mobile nursing practices, have presented technical and operational issues that were not foreseen from home use. These problems arise from a number of factors: the way the devices are used, the patient population, and even differences in sample type. Thus, to be successful, management of POCT in the health-care environment requires interdisciplinary cooperation of clinical nursing staff and laboratory staff. The article identifies problems in POCT, describes some solutions, and examines how well these solutions have worked from a laboratory perspective. PMID- 10747661 TI - A task-targeted automation system: a case study. AB - Kaiser Permanente's Northern California Regional Laboratory recently achieved an important milestone in its continuous quality improvement project by automating time-consuming and labor-intensive tasks involved in preanalytical and postanalytical sample processing. The new task-targeted automation system, the most sophisticated of its kind in the world, consists of three key components: the Roche Diagnostics PSD 1 primary sample decapper/sorter/archiver, the Roche Diagnostics VS 250 aliquoter, and a data manager/process controller developed by Data Innovations. The system was placed into service in April 1998, culminating more than a year of planning and implementation. Within months after the system went into operation, the laboratory observed significant improvements in workflow and efficiency, as well as a reduction in work-related injuries. The purpose of this article is to provide management teams in other laboratories with a description of the planning and implementation process and offer suggestions for those contemplating a similar program. PMID- 10747662 TI - Nutrition assessment outcomes: a strategy to improve health care. AB - Nutrition assessment performed as part of hospital admission protocol can significantly reduce length of stay, costs of care, and the incidence of readmission by efficiently identifying patients with malnutrition. As reported in more than 150 clinical studies, malnutrition is a health problem of huge magnitude, potentially affecting more than half of hospitalized patients in the United States. The laboratory director can optimize the use of visceral protein testing in nutrition assessment protocols to realize the greatest benefit for both patient and institution. The accurate identification of patients with protein calorie malnutrition allows the use of malnutrition ICD-9-CM codes to obtain higher reimbursement for the increased acuity of illness. PMID- 10747664 TI - Presentation skills for the reluctant speaker. AB - Presentation skills are vital to clinical systems managers. This article covers four steps to successful presentations: 1) tailoring for an audience, 2) organizing a presentation, 3) mastering presentation techniques, and 4) creating effective visual aids. Tailoring for the audience entails learning about the audience and matching the presentation to their knowledge, educational level, and interests. Techniques to curry favor with an audience include: establishing common ground, relating through universal experiences, and pushing "hot buttons." Tasks involved in organizing the presentation for maximum audience interest begin with arranging the key points in a transparent organizational scheme. Audience attention is sustained using "hooks," such as graphics, anecdotes, humor, and quotations. Basic presentation techniques include appropriate rehearsal, effective eye contact with an audience, and anxiety-reducing strategies. Visual aids include flip charts, slides, transparencies, and computer presentations. Criteria for selecting the type of visual aids are delineated based on audience size and type of presentation, along with respective advantages and disadvantages. The golden rule for presentations is "Never show a slide for which you have to apologize." Rules to maximize visibility and effectiveness, including use of standard templates, sans serif fonts, dark backgrounds with light letters, mixed cases, and effective graphics, ensure that slides or projected computer images are clear and professional. Taken together, these strategies will enhance the delivery of the presentation and decrease the speaker's anxiety. PMID- 10747663 TI - Competing in the new health care paradigm: a novel personnel compensation model, Part I. AB - Operational issues in health care ultimately lead to discussions concerning personnel. Adequate staffing, "rightsizing," compensation, scheduling, morale, supervision, and organizational structure were subjects of the questions the Clinical Pathology Laboratories at the University of Virginia Medical Center asked in 1994. The answers were found in the design of a new skill-based personnel organizational model and compensation plan. PMID- 10747665 TI - The use of the Delphi panel for consensus development on indicators of laboratory performance. AB - The objective of this research study is to identify laboratory performance areas and indicators of performance from the perspective of key laboratory constituencies. A specific technique, the Delphi panel, is used to achieve consensus on a system of indicators that can be used by laboratory service managers to build information pathways, increase efficiency, and improve outcomes. Individual panels represented the five highest ranked stakeholder groups: hospital executives, managed care executives, referring physicians, laboratory regulators, and laboratory managers. The instruments used for the Delphi panels were developed and validated in a pilot study. After three rounds, each panel had identified a set of priority performance areas and indicators for each area. Although concurring that the Delphi was valuable and effective in identifying performance areas, participants indicated that it was less useful in developing specific indicators enabling monitoring of laboratory performance over time or cross-laboratory comparisons. PMID- 10747666 TI - Career development strategies. The psycho-barbarian manager. PMID- 10747667 TI - When a health-care plan does not cover an ordered test. PMID- 10747668 TI - Dollar$ & $en$e. Part III: Measuring added value. PMID- 10747669 TI - As we see it. Outreach. PMID- 10747670 TI - What is your return on your skills investment? PMID- 10747671 TI - Performance management--the balanced scorecard: a framework for managing complex and rapid change. PMID- 10747672 TI - Performance management--scoring on a major-league scale: BJC's five step process. PMID- 10747674 TI - Quality improvement--first-ever healthcare quality chair established at UMass. PMID- 10747673 TI - Reengineering--at New York City's Mount Sinai, reengineering is a way to grow, not a special project. PMID- 10747675 TI - Taming acid reflux. PMID- 10747677 TI - Agita at work. PMID- 10747676 TI - The acid test for formulary policy. PMID- 10747678 TI - Action items for employers. Top 10 tips on acid reflux. PMID- 10747679 TI - That burning sensation. PMID- 10747680 TI - Determining the differences among cost savings, cost avoidance, and cost reduction. AB - Cost savings, cost avoidance, and cost reduction are important factors to measure when valuing clinical pharmacy services. Currently, there are no universally accepted definitions for these terms. The authors of this study evaluated 44 articles which claimed to measure cost savings, cost avoidance, or cost reduction associated with clinical pharmacy services, and offered recommendations for when it would be appropriate to use these terms. Based on the results of our analysis, it is evident that controversy exists in the literature when determining if services result in cost avoidance or cost reduction. Therefore, there is a need for universally accepted definitions of these terms so that future research may evaluate such factors uniformly. PMID- 10747681 TI - Influencing physician prescribing. AB - The drug use process suffers from problems related to quality and cost that have not responded well to administrative or educational interventions. In many cases, attempts to improve the quality of physician prescribing have been clumsy, often based on intuition. This article begins by describing the drug use process and the role of prescribing in that process. In the following section, we describe what is known about how physicians make drug choice decisions. The paper concludes with suggestions, based on evidence, about the design of strategies for influencing prescribing. PMID- 10747683 TI - Health care 2000: top 10 trends for the new century. PMID- 10747682 TI - Anticoagulation in pregnancy. AB - The use of anticoagulants during pregnancy for prevention and treatment of venous thromboembolism and prevention of systemic embolism in patients with valvular heart disease presents several problems. This article discusses the complications associated with warfarin, unfractionated heparin, and low-molecular-weight heparin as well as the benefits of each. While a literature review turned up only limited data, the authors extrapolated from existing data recommendations for treatment during pregnancy, finding that oral warfarin should be replaced by heparin during pregnancy, especially from the 6th to the 12th week and near term. In addition, treatment recommendations are provided for different stages of pregnancy, delivery, and postpartum. PMID- 10747684 TI - "Top 10" trends predictions for 1999 were 100 percent accurate. PMID- 10747685 TI - Clinical practice guidelines. Four new Joint Commission standards for 2000. PMID- 10747686 TI - Practice-based disease management. PMID- 10747687 TI - Food safety update. PMID- 10747688 TI - Special needs of Hispanic clients. Similarities and differences among subgroups in this population. PMID- 10747689 TI - Employer-based health insurance: constant erosion for low-income citizens. PMID- 10747690 TI - Developing tools for better medical management of Medicare populations. AB - Medicare enrollment in HMOs got off to a quick start but has ebbed recently, principally because health plans found that the elderly joining their plans were costing the plan more than what the federal government was willing to pay. For the MCOs that want to continue to participate in Medicare + Choice, it is imperative for them to understand, in as much detail as possible, the potential health care costs of their enrollees. PMID- 10747691 TI - Practice guidelines for the diagnosis and treatment of Alzheimer's disease in a managed care setting: Part II--Pharmacologic therapy. Alzheimer's Disease (AD) Managed Care Advisory Council. AB - The progressive loss of social and physical functioning associated with Alzheimer's disease (AD) results in extensive social and economic costs to society. The early diagnosis and treatment of AD may reduce cognitive and behavioral symptoms of this disease and may slow disease progression, thereby alleviating some of these social and economic costs. The Alzheimer's Disease Managed Care Advisory Council, a panel of experts from managed care, academic medicine, and the Los Angeles chapter of the Alzheimer's Association was convened to synthesize current evidence-based recommendations for AD diagnostic and treatment guidelines and to integrate these guidelines for use in MCOs. This paper presents conclusions from this panel and provides an algorithm for the treatment of AD specifically for managed care settings. When combined with other necessary efforts to educate providers, these guidelines should improve the cost effectiveness and quality of care for individuals with dementia in managed care. PMID- 10747692 TI - Staffing issues and evolution in managed care pharmacy. PMID- 10747693 TI - The treatment of seasonal allergic rhinitis: cost implications of pharmacotherapy for managed care. AB - Seasonal allergic rhinitis affects a large proportion of both the general and the managed care populations. Whereas matching treatment to the needs of individual patients remains somewhat of an art, decisions regarding which drugs to add to the formulary of an MCO should incorporate an assessment of a medication's efficacy and safety profile, as well as a review of the overall costs associated with its utilization. Recognizing that the overall costs of drug utilization include more than the actual price of a prescription, a utilization analysis of annual drug therapy was undertaken. The results of this recent study demonstrate that fewer prescriptions and lower costs were associated with certain products. In this study, azelastine nasal spray was associated with an overall lower cost of pharmacotherapy. PMID- 10747694 TI - The emerging role of telemedicine in extended-care risk management. PMID- 10747695 TI - Managed care and the implementation of the State Children's Health Insurance Program. AB - More than two years after its enactment, the Health Care Financing Administration, Baltimore, issued proposed regulations implementing the State Children's Health Insurance Program (SCHIP), enacted under the Balanced Budget Act of 1997. This program was enacted to provide health insurance coverage to the estimated 10 million uninsured children living in the United States. This article provides an overview of SCHIP and how it has been implemented in the various states. PMID- 10747696 TI - Telehealth technology evaluations process. PMID- 10747697 TI - An enhanced healthcare platform via e-medicine. PMID- 10747698 TI - Behavioral telehealth: using telemedicine to expand behavioral medicine services. PMID- 10747699 TI - Nurses' responses to telemedicine in home healthcare. PMID- 10747700 TI - Evaluating telemedicine in a changing technological era. PMID- 10747701 TI - Managing telehealthcare information. PMID- 10747702 TI - Telehealth or telehype? Some observations and thoughts on the current status and future of telehealth. PMID- 10747703 TI - Teledermatology in Department of Defense Health Services Region 10. PMID- 10747704 TI - Telehealth applications in speech-language pathology. PMID- 10747705 TI - Patient health information confidentiality in telehealth applications. PMID- 10747706 TI - The evolution of telepharmacy: a paradigm shift. PMID- 10747707 TI - The hand via telemedicine. PMID- 10747708 TI - A message to America from America's communities. PMID- 10747709 TI - What experience are you selling? PMID- 10747710 TI - The battle to control online purchasing. PMID- 10747711 TI - Apollo meets Minerva: health in 2025. PMID- 10747712 TI - The downside of patient empowerment. PMID- 10747713 TI - Creating hope and opportunity. The Pembroke Township-Hopkins Park partnership. PMID- 10747714 TI - Changing public policy. PMID- 10747715 TI - Co-producing health: professionals and communities build on assets. PMID- 10747716 TI - Nurturing a new mental model. PMID- 10747717 TI - The community as co-producer of health. PMID- 10747718 TI - The Capacity Inventory: the ultimate equalizer. PMID- 10747719 TI - The decisions of the 1990s: the view from a Monday morning quarterback. PMID- 10747720 TI - Bearing witness. PMID- 10747721 TI - Regional hospital improves efficiency with co-generation retrofit. AB - Feasibility analysis of the co-generation retrofit of the Red Deer Regional Hospital pointed to a reasonable payback of capital cost and increased efficiency in operation of the facility. Budget restrictions nearly stopped the project from proceeding. Innovative construction procedures proposed by the Facility Management Group, in particular, Mr Keith Metcalfe, Director of Maintenance, allowed a worthwhile project to reach successful completion. We feel that this model can perhaps be used by similar facilities in the future to achieve their energy efficiency goals. PMID- 10747722 TI - Specialised lighting for hospital premises. PMID- 10747723 TI - Benchmarking engineering. PMID- 10747724 TI - Heat pumps for cold climates: the heat pump in Stokmarknes Hospital, Norway. PMID- 10747726 TI - 23rd annual EMS state and province survey. PMID- 10747725 TI - Key changes in state EMS offices. PMID- 10747727 TI - Gold Book buyer's guide. PMID- 10747728 TI - Directory of government agencies. PMID- 10747729 TI - Directory of EMS organizations. PMID- 10747730 TI - Fire-based EMS. PMID- 10747731 TI - "Silent MI". PMID- 10747732 TI - Fire-based EMS. PMID- 10747733 TI - Fire-based EMS. PMID- 10747734 TI - Fire-based EMS. PMID- 10747735 TI - Fire-based EMS. PMID- 10747736 TI - Look before you leap. PMID- 10747737 TI - The standard of caring. PMID- 10747738 TI - Prehospital management of epiglottitis. PMID- 10747739 TI - Brain attack. New perspectives on stroke. PMID- 10747740 TI - Ambulance service and managed care: the MCO perspective. AB - EMS and managed care can coexist, but it requires communication and flexibility on both sides. If the managed care organizations whose members you service haven't taken steps to contact you regarding these issues, your management can certainly take the initiative in requesting a meeting. In a typical MCO, transportation is a very small percentage of the overall budget, and day-to-day communications with service providers may be overlooked in dealing with larger financial considerations until there's a problem. After a problem has occurred is not the right time to attempt to establish a relationship. Communication, regardless of who takes the first step, can avoid this scenario. Remember, transportation is your area of expertise, not theirs, and most companies will appreciate your efforts to discuss issues before they become problems. PMID- 10747741 TI - Training the trainers. Emergency International provides EMS systems development. PMID- 10747742 TI - Critical thinking in the classroom. PMID- 10747743 TI - Back to basics. PMID- 10747744 TI - The duty to create and maintain health records. PMID- 10747747 TI - Provider 1999 LTC buyer's guide. PMID- 10747745 TI - Why withholding treatment is not assisted suicide. AB - We have shown why it is that withholding medical treatment is not properly considered to be an assisted suicide. We have said nothing about the desirability or need for new legislation to support assisting a patient in a suicide. We have been concerned only to show that any plausible arguments for assisted suicide must stand on their merits and the attempt to justify a practice of assisted suicide by linking it to the withdrawing of medical treatment through an analysis of causation fails. In this closing section we place our discussion in a broader setting and draw out some of the implications of the distinctions we have made. One central point we wish to emphasize is the role of context in discussions of withholding treatment and assisting a suicide. We have noted the difference between the "normative" and "scientific" sense of "cause". The normative sense is used when holding a person responsible, either legally or morally, for what he or she has actually or scientifically caused (the cause-in-fact). When we hold a person responsible for the consequences of his or her actions, we do so in a way that is sensitive to the context of that action. There is no set formula for determining how broad a context must be considered, whether it be a year and a day, or some shorter or longer interval. The determination of context will involve judgments of relevance and reasonableness and will depend on any special relationships that may hold between the parties involved. Finally, we emphasize how essential context is to a determination of causation. If one fails to consider both the scientific and normative dimensions of causation and relies only on the scientific dimension, one ends up with the counter-intuitive judgments that, in the Olson case, the neurosurgeon who withdrew life support for Erickson is the cause of death, and similarly for the example given by Schaffner and the Nancy B. case discussed by Fish and Singer. Our advice is, "Don't go there". PMID- 10747746 TI - Health care reform in Canada: is there room for efficiency? PMID- 10747748 TI - Genesis Health Ventures restructures multicare acquisition. PMID- 10747749 TI - Staffing standard takes center stage at Senate forum. PMID- 10747750 TI - New Mexico to set new staffing levels for nursing facilities. PMID- 10747751 TI - Baltimore facilities battle over 'whites only' bequest. PMID- 10747752 TI - JCAHO, CARF issue draft standards for assisted living. PMID- 10747753 TI - BBA bill shores up struggling providers. PMID- 10747755 TI - Reaching a new level in outcomes management. PMID- 10747754 TI - Meeting the baby boomer challenge. How will a generation of 70 million elders reshape and redirect long-term care? PMID- 10747756 TI - Customer expectations, today and beyond. PMID- 10747757 TI - Agency issues compliance program guidance. PMID- 10747758 TI - Spiritual care for the new millennium. PMID- 10747759 TI - Techno-savvy boomers dictate services. PMID- 10747760 TI - OSHA releases ergonomics standard. PMID- 10747762 TI - Estimating the long-term cost savings from the treatment of Alzheimer's disease: a modelling approach. PMID- 10747761 TI - Formulary management of low molecular weight heparins. AB - Low molecular weight heparins (LMWHs) are increasingly being utilised as anticoagulants in healthcare settings. These agents offer several advantages over standard unfractionated heparin. Indications for LMWHs include deep vein thrombosis and pulmonary embolism prophylaxis, deep vein thrombosis treatment, use in coronary procedures associated with a high risk for bleeding, and in acute coronary syndromes. Prior to being added to formularies, LMWHs should be evaluated for efficacy, safety and economic benefits over other anticoagulants. Institutions should be prepared to conduct their own economic assessments in the absence of readily available studies. There is clear evidence that LMWHs are cost saving or are at least cost effective as thromboprophylactic agents in major orthopaedic surgery. The economic benefits of LMWHs in other surgical situations is less clear. Consistent evidence from several countries indicate that LMWHs are cost saving as anticoagulants for the initial treatment of DVT. Further studies are needed to evaluate the efficacy, safety and economics of LMWHs in other conditions besides hip and knee arthroplasty and general surgery. PMID- 10747763 TI - A comparative review of generic quality-of-life instruments. AB - The assessment of health-related quality of life (HR-QOL) is an essential element of healthcare evaluation. Hundreds of generic and specific HR-QOL instruments have been developed. Generic HR-QOL instruments are designed to be applicable across a wide range of populations and interventions. Specific HR-QOL measures are designed to be relevant to particular interventions or in certain subpopulations (e.g. individuals with rheumatoid arthritis). This review examines 7 generic HR-QOL instruments: (i) the Medical Outcomes Study 36-Item Short Form (SF-36) health survey; (ii) the Nottingham Health Profile (NHP); (iii) the Sickness Impact Profile (SIP); (iv) the Dartmouth Primary care Cooperative Information Project (COOP) Charts; (v) the Quality of Well-Being (QWB) Scale; (vi) the Health Utilities Index (HUI); and (vii) the EuroQol Instrument (EQ-5D). These instruments were selected because they are commonly used and/or cited in the English language literature. The 6 characteristics of an instrument addressed by this review are: (i) conceptual and measurement model; (ii) reliability; (iii) validity; (iv) respondent and administrative burden; (v) alternative forms; and (vi) cultural and language adaptations. Of the instruments reviewed, the SF-36 health survey is the most commonly used HR-QOL measure. It was developed as a short-form measure of functioning and well-being in the Medical Outcomes Study. The Dartmouth COOP Charts were designed to be used in everyday clinical practice to provide immediate feedback to clinicians about the health status of their patients. The NHP was developed to reflect lay rather than professional perceptions of health. The SIP was constructed as a measure of sickness in relation to impact on behaviour. The QWB, HUI and EQ-5D are preference-based measures designed to summarise HR-QOL in a single number ranging from 0 to 1. We found that there are no uniformly 'worst' or 'best' performing instruments. The decision to use one over another, to use a combination of 2 or more, to use a profile and/or a preference-based measure or to use a generic measure along with a targeted measure will be driven by the purpose of the measurement. In addition, the choice will depend on a variety of factors including the characteristics of the population (e.g. age, health status, language/culture) and the environment in which the measurement is undertaken (e.g. clinical trial, routine physician visit). We provide our summary of the level of evidence in the literature regarding each instrument's characteristics based on the review criteria. The potential user of these instruments should base their instrument selection decision on the characteristics that are most relevant to their particular HR-QOL measurement needs. PMID- 10747764 TI - Economic evaluation of specific immunotherapy versus symptomatic treatment of allergic rhinitis in Germany. AB - OBJECTIVE: To use published data to compare the economic consequences of specific immunotherapy (SIT) lasting 3 years with those of continuous symptomatic treatment in patients with either pollen or mite allergy. DESIGN AND SETTING: The evaluation was conducted from the following 3 perspectives in Germany: (i) society; (ii) healthcare system; and (iii) statutory health insurance (SHI) provider. A modelling approach was used which was based on secondary analysis of existing data. The follow-up period was 10 years. The break-even point of cumulated costs, their difference per patient and the additional cost per additional patient free from asthma symptoms [incremental cost-effectiveness ratio (ICER)] were used as target variables, each from the viewpoint of SIT. The types of costs were direct and indirect (society), direct (healthcare system) and those incurred by SHI (i.e. expenses). In the base-case analysis, the average values of the clinical parameters and average case-related costs/expenses were applied. MAIN OUTCOME MEASURES AND RESULTS: The break-even point was reached between year 6 and year 8 after the start of therapy, resulting in net savings of between 650 and 1190 deutschmarks (DM) per patient after 10 years. The ICERs of SIT were between -DM3640 and -DM7410, depending on study perspective and nature of the allergy (1990 values for symptomatic treatment and treatment of asthma, 1995 values for SIT; DM1 approximately $US0.58). The sensitivity analysis demonstrated the robustness of the model and its results. First, all the independent variables of the model were varied. Secondly, the influence of the model variables was quantified using a deterministic model. SIT was more likely to result in net savings than in additional costs. An economic parameter (cost for symptomatic treatment) had the highest influence on the results. CONCLUSIONS: This evaluation showed that SIT for 3 years is economically advantageous in patients who are allergic to pollen or mites and whose symptoms are inadequately controlled by continuous symptomatic treatment. After 10 years, the administration of SIT leads to net savings from the perspectives of society, the healthcare system and SHI (third-party payer) in Germany. PMID- 10747765 TI - Use and cost of hospital and community service provision for children with HIV infection at an English HIV referral centre. AB - OBJECTIVE: To describe the use of hospital and community services for children infected with HIV and estimate the cost per patient-year by stage of HIV infection during the era of antiretroviral monotherapy. DESIGN: Data on the use of hospital services were collected from case notes; the use of statutory and nonstatutory community services was recorded through diaries and interviews. Total cost estimates were calculated from unit costs from relevant hospital departments and community organisations. SETTING: Children managed at St. Mary's Hospital (London, England) between 1 January 1986 and 31 December 1994, some of whom used statutory and nonstatutory community services in South East England between 1 November 1994 and 31 May 1996. PATIENTS AND PARTICIPANTS: 118 children with positive HIV antibody status. MAIN OUTCOME MEASURES AND RESULTS: Mean inpatient days, outpatient visits, tests and procedures performed, drugs prescribed, community services used, associated unit costs and average cost estimates per patient-year by stage of HIV infection (1995/1996 values), and lifetime costs. Service provision during the study period was predominantly hospital-based. The use of services increased for different stages of HIV infection and increased with increasing severity of HIV infection. A shift from an inpatient-based to an outpatient-based service was seen between the periods 1986 to 1991 and 1992 to 1994. As symptoms evolved, children used more hospital inpatient services, with an accompanying shift in the use of community services from general services, such as schooling, to increased use of nurses, social care and home help. The estimated total cost of hospital and community care was 18,600 Pounds per symptomatic non-AIDS patient per year and 46,600 Pounds per AIDS patient per year. Similar estimates for children with indeterminate HIV infection and asymptomatic infection amounted to 8300 Pounds and 4800 Pounds per patient year, respectively. Nondiscounted lifetime costs for hospital care amounted to 152,400 Pounds (44,300 Pounds to 266,800 Pounds) compared with discounted lifetime costs of 122,700 Pounds (42,000 Pounds to 182,200 Pounds); nondiscounted lifetime costs for community care amounted to 24,300 Pounds (7900 Pounds to 41,600 Pounds) compared with discounted lifetime costs of 21,000 Pounds (6800 Pounds to 32,000 Pounds). CONCLUSIONS: The continued emphasis on the use of hospital services may be due to the small number of children infected with HIV, most of whom lived in the London metropolitan area where specialist care was concentrated in a few centres. A shift from an inpatient- to an outpatient-based service was observed over time; the advent of the use of combination antiretroviral therapy in this population may further facilitate a shift in service provision and promote shared care between specialist centres, local hospital and community-based services. PMID- 10747766 TI - The cost of urinary incontinence in Italian women. A cross-sectional study. Gruppo di Studio Incontinenza. AB - OBJECTIVE: To offer cost estimates of urinary incontinence (UI) in the general population based on prospectively collected data. DESIGN: We analyzed individual costs in a sample of women with UI who were identified in the framework of a cross-sectional study on the prevalence of UI in women aged > 40 years. SETTING: Six areas in Italy. INTERVENTION: Home interview. PATIENTS AND PARTICIPANTS: Women were identified among the patients registered with a network of general practitioners operating in each area using computer-generated random number lists. RESULTS: A total of 2767 women were identified. Of these, 408 (14.7%) reported UI during the year before the interview and 229 underwent a detailed interview on UI-related costs. On the basis of this information, we estimated the direct costs associated with UI from the perspective of the Italian National Health Service (INHS). The lifetime cost per patient of diagnosis was 80,131 Italian lire (L) (exchange rate: $US1 = L1618). Consultations accounted for only 20% of the diagnostic cost, diagnostic tests for 36% and hospital admissions for diagnostic procedures accounted for 44%. The diagnosis cost estimate seems low, partly because several women did not request either consultations or diagnostic tests (the overall rate per patient was 0.76 for consultations and 0.39 for diagnostic tests). The only appreciable treatment cost, according to the INHS perspective, was for diapers. The annual cost per patient for diapers was L255,519. The prevalence of UI in women aged > 40 years in Italy is estimated in the study at 9.3%. Thus, combining this information with the cost estimates, the annual treatment cost of UI in Italian women aged > 40 years is L351,800 billion, considering diapers and drugs only. CONCLUSION: This study has estimated the individual cost of UI in the general population. These figures may be useful when designing economic evaluations of UI. PMID- 10747769 TI - Empirically based conversion factors for calculating couple-years of protection. AB - Couple-years of protection (CYP) is one of several commonly used indicators to assess international family planning efforts. It has been the subject of much debate, relating in part to the specific conversion factors used to translate the quantity of the respective contraceptive methods distributed to a single measure of protection. This article outlines a comprehensive effort to revisit those conversion factors based on the best available empirical evidence. In most instances, the analysis supports previously established standard conversion factors. However, there are two notable departures. Fewer condoms and spermicides are recommended for each CYP (120 vs. 150), primarily because coital frequency among condom users is lower than previously assumed. Furthermore, for sterilization, the authors recommend the use of country or region-specific conversion factors. Every program evaluation indicator has strengths and weaknesses, and the best program evaluation efforts use a variety of indicators. If CYP is used to evaluate programs, however, the authors believe that the conversion factors presented reflect the best available evidence. PMID- 10747768 TI - The relative costs and benefits of telephone interviews versus self-administered diaries for daily data collection. AB - This article compares two methods of collecting daily data: self-administered diaries and telephone interviews. Study participants included 44 men and 56 women between the ages of 16 and 35 who participated in a larger study of drinking, drug use, and sexual activity. Participants were randomly assigned to either the written diary or the telephone interview conditions; question wording and format were identical in both conditions. Daily data were collected for a period of 8 weeks. Results indicate that although telephone interviews resulted in slightly more missed days of data collection, they generally yielded less item-level missing data, produced cleaner data and therefore were less costly to process, and were as palatable to participants as self-administered diaries. Except for reports of drinking and vegetable consumption, telephone and diary conditions did not differ in the amount of behavior reported; more drinking and vegetable consumption were reported with telephone interviews, however. Telephone interviews also imposed considerably higher overall personnel costs. PMID- 10747767 TI - Economic cost of male erectile dysfunction using a decision analytic model: for a hypothetical managed-care plan of 100,000 members. AB - OBJECTIVE: This paper examined the economic cost of male erectile dysfunction (ED) for a hypothetical managed-care (MC) model. DESIGN AND SETTING: A prevalence based cost-of-illness approach was used to estimate the direct medical cost for ED treatment. A treatment plan algorithm was developed from a MC perspective to model the initial treatment selection of various patient groups [vacuum erection device, intracavernosal injection (ICI) therapy, transurethral alprostadil suppository, sildenafil, testosterone replacement therapy, penile prosthesis] and their therapy outcomes during a 3-year period. Overall cost was based on 1998 US dollars. Total direct medical cost of ED considered in this model included the cost of initial physician consultation and evaluation, the cost incurred by patients from various treatment groups (pharmacological and surgical options), as well as the cost related to patients' follow-up for treatment within the 3-year period. Consideration for therapy switches made by patients who failed initial therapy was included as part of the clinical assumptions for this model. Treatment response and expected outcomes (dropouts) were considered for the various treatment options. PARTICIPANTS: A total of 100,000 enrolled members were included in the study. MAIN OUTCOME MEASURES AND RESULTS: The total cost of ED was $US3,204,792 for the 3-year period in the hypothetical MC plan. The treatment portion accounted for approximately 80% of the total cost while the cost of medical services and diagnostic tests were minimal in comparison. The 3 year total cost of nonsurgical treatment was $US2,473,045. Costs associated with each treatment alternative were $US81,866 (testosterone transdermal patch), $US51,930 (vacuum erection device), $US384,624 (ICI therapy), $US226,483 (transurethral alprostadil suppository) and $US1,728,142 (sildenafil citrate). Results from the model showed a noticeable trend of decreasing cost patterns over time and reflected the attrition observed for many of the standard medical therapies for ED. CONCLUSIONS: Sildenafil and the vacuum erection device should be considered as first-line management strategies for ED whereas ICI therapy, transurethral alprostadil suppository and penile prosthesis implant should be reserved for second- or third-line therapy. Because costs associated with switches related to successive treatment failures can be high, treatment considerations should, therefore, focus on achieving long term patient satisfaction. The patient's preferred treatment choice, using goal-directed therapy during the initial consultation and evaluation visit, should be used. PMID- 10747770 TI - Mediators and moderators in the evaluation of programs for children. Current practice and agenda for improvement. AB - The author examines the role of mediators and moderators in the evaluation of programs for children. The terms are defined and examples of each are presented. Using bibliometric analysis, the author examines how evaluators use mediators and moderators in treatment studies in education, juvenile justice, health care, child protection, and mental health. The use of mediators and moderators is sporadic and vague at best. An agenda for improvement is outlined that includes greater use of program theory and intensive case studies to find out why researchers in prevention and health promotion incorporate mediators and moderators more effectively in their evaluations. PMID- 10747771 TI - An application of theory-driven evaluation to a drop-in youth center. AB - This article reports on the theory-driven evaluation of a drop-in center for youth that incorporated a literature search, concept mapping with staff, and focus groups with youth. Findings revealed strong agreement among the three sources of data around specific elements identified as critical components of a program theory of global prevention in after-school-hours initiatives, such as drop-in centers. These results are used to illustrate how a theory-driven approach was relevant for the context and objectives of this evaluation, as well as how it was used to develop knowledge useful for action, social intervention theory, and further research. PMID- 10747772 TI - Policy evaluation research. Measuring the independent variables. AB - The purpose of this article is to evaluate the accuracy of three methods used to obtain policy data: (a) government agency surveys, (b) secondary sources, and (c) historical legal research. Changes in laws were identified for all 48 contiguous states for the period 1968 to 1994. Legal research is most accurate for well established laws that have consistent legal descriptions across nearly all states. Laws that are recently enacted, adopted by only a few states, and treated in a legally inconsistent manner across states require a multistage data collection method to identify accurate policy change information. PMID- 10747773 TI - Physical constraints in the condensation of eukaryotic chromosomes. Local concentration of DNA versus linear packing ratio in higher order chromatin structures. AB - The local concentration of DNA in metaphase chromosomes of different organisms has been determined in several laboratories. The average of these measurements is 0.17 g/mL. In the first level of chromosome condensation, DNA is wrapped around histones forming nucleosomes. This organization limits the DNA concentration in nucleosomes to 0. 3-0.4 g/mL. Furthermore, in the structural models suggested in different laboratories for the 30-40 nm chromatin fiber, the estimated DNA concentration is significantly reduced; it ranges from 0.04 to 0.27 g/mL. The DNA concentration is further reduced when the fiber is folded into the successive higher order structures suggested in different models for metaphase chromosomes; the estimated minimum decrease of DNA concentration represents an additional 40%. These observations suggest that most of the models proposed for the 30-40 nm chromatin fiber are not dense enough for the construction of metaphase chromosomes. In contrast, it is well-known that the linear packing ratio increases dramatically in each level of DNA folding in chromosomes. Thus, the consideration of the linear packing ratio is not enough for the study of chromatin condensation; the constraint resulting from the actual DNA concentration in metaphase chromosomes must be considered for the construction of models for condensed chromatin. PMID- 10747774 TI - Interaction of a semirigid agonist with Torpedo acetylcholine receptor. AB - The binding of the semirigid agonist [(3)H]arecolone methiodide to the Torpedo nicotinic acetylcholine receptor has been correlated with its functional properties measured both in flux studies with Torpedo membrane vesicles and by single-channel analysis after reconstitution in giant liposomes. Under both equilibrium and preequilibrium conditions, the binding of arecolone methiodide is similar to that of other agonists such as acetylcholine. At equilibrium, it binds to two sites per receptor with high affinity (K(d) = 99 +/- 12 nM), and studies of its dissociation kinetics suggest that each of these sites is made up of two subsites that are mutually exclusive at equilibrium. The kinetics of arecolone methiodide binding were monitored by the changes in the receptor intrinsic fluorescence, and the data are consistent with a model in which the initial binding event is followed by sequential conformational transitions of the receptor-ligand complex. In flux studies, arecolone methiodide was approximately 3-fold more potent (EC(50) = 31 +/- 5 microM) than acetylcholine but its maximum flux rate was 4-10-fold lower. This phenomenon has been studied further by single channel analysis of Torpedo receptors reconstituted in giant liposomes. Whereas the flexible agonist carbamylcholine (5 microM) was shown to induce channels with conductances of 56 and 34 pS with approximately equal frequency, arecolone methiodide (2 microM) preferentially induced the channel of lower conductance. These results are interpreted in terms of a simple model in which the rigidity of arecolone methiodide restrains the conformation that the receptor-ligand complex can adopt, thus favoring the lower conductance state. PMID- 10747775 TI - NEM tubulin inhibits microtubule minus end assembly by a reversible capping mechanism. AB - Although microtubule (MT) dynamic instability is thought to depend on the guanine nucleotide (GTP vs GDP) bound to the beta-tubulin of the terminal subunit(s), the MT minus end exhibits dynamic instability even though the terminal beta-tubulin is always crowned by GTP-alpha-tubulin. As an approach toward understanding how dynamic instability occurs at the minus end, we investigated the effects of N ethylmaleimide-modified tubulin (NTb) on elongation and rapid shortening of individual MTs. NTb preferentially inhibits minus end assembly when combined with unmodified tubulin (PCTb), but the mechanism of inhibition is unknown. Here, video-enhanced differential interference contrast microscopy was used to observe the effects of NTb on MTs assembled from PCTb onto axoneme fragments. MTs were exposed to mixtures of PCTb (25 microM) and NTb (labeled on approximately 1 Cys per monomer) in which the NTb/PCTb ratio varied from 0.025 to 1. The NTb/PCTb mixture had a slight inhibitory effect on the plus end elongation rate, but significantly inhibited or completely arrested minus end elongation. For the majority of mixtures that were assayed (0.1-1 NTb/PCTb ratio), minus end MT length remained constant until the NTb/PCTb mixture was replaced. Replacement with PCTb allowed elongation to proceed, whereas replacement with buffer or NTb caused minus ends to shorten. Taken together, the results indicate that NTb associates with both plus and minus ends and that NTb acts to reversibly cap minus ends only when PCTb is also present. Low-resolution mapping of labeled Cys residues, along with previous experiments with other Cys-reactive compounds, suggests that modification of beta-tubulin Cys(239) may be associated with the capping action of NTb. PMID- 10747776 TI - Coordination chemistry of co(II)-bleomycin: its investigation through NMR and molecular dynamics. AB - Previous studies on the coordination chemistry of Co-bleomycin have suggested the secondary amine in beta-aminoalanine, the N5 and N1 nitrogens in the pyrimidine and imidazole rings, respectively, and the amide nitrogen in beta hydroxyhistidine as equatorial ligands to the cobalt ion. The primary amine in beta-aminoalanine and the carbamoyl group of the mannose have been proposed alternatively as possible axial ligands. The first coordination sphere of Co(II) in Co(II)BLM has been investigated in the present study through the use of NMR and molecular dynamics calculations. The data collected from the NMR experiments are in agreement with the equatorial ligands previously proposed, and also support the participation of the primary amine as an axial ligand. The paramagnetic shifts of the gulose and mannose protons could suggest the latter as a second axial ligand. This possibility was investigated by way of molecular dynamics, with distance restraints derived from the relaxation times measured through NMR. The molecular dynamics results indicate that the most favorable structure is six-coordinate, with the primary amine and either the carbamoyl oxygen or a solvent molecule occupying the axial sites. The analysis of the structures previously derived for HOO-Co(III)-bleomycin and HOO-Co(III) pepleomycin led us to propose the six-coordinate structure with only endogenous ligands, as the one held in solution by the Co(II) derivative of bleomycin. PMID- 10747777 TI - Purification, characterization, and preliminary crystallographic study of copper containing nitrous oxide reductase from Pseudomonas nautica 617. AB - The aerobic purification of Pseudomonas nautica 617 nitrous oxide reductase yielded two forms of the enzyme exhibiting different chromatographic behaviors. The protein contains six copper atoms per monomer, arranged in two centers named Cu(A) and Cu(Z). Cu(Z) could be neither oxidized nor further reduced under our experimental conditions, and exhibits a 4-line EPR spectrum (g(x)=2.015, A(x)=1.5 mT, g(y)=2.071, A(y)=2 mT, g(z)=2.138, A(z)=7 mT) and a strong absorption at approximately 640 nm. Cu(A) can be stabilized in a reduced EPR-silent state and in an oxidized state with a typical 7-line EPR spectrum (g(x)=g(y)= 2.021, A(x) = A(y)=0 mT, g(z) = 2.178, A(z)= 4 mT) and absorption bands at 480, 540, and approximately 800 nm. The difference between the two purified forms of nitrous oxide reductase is interpreted as a difference in the oxidation state of the Cu(A) center. In form A, Cu(A) is predominantly oxidized (S = (1)/(2), Cu(1.5+) Cu(1.5+)), while in form B it is mostly in the one-electron reduced state (S = 0, Cu(1+)-Cu(1+)). In both forms, Cu(Z) remains reduced (S = 1/2). Complete crystallographic data at 2.4 A indicate that Cu(A) is a binuclear site (similar to the site found in cytochrome c oxidase) and Cu(Z) is a novel tetracopper cluster [Brown, K., et al. (2000) Nat. Struct. Biol. (in press)]. The complete amino acid sequence of the enzyme was determined and comparisons made with sequences of other nitrous oxide reductases, emphasizing the coordination of the centers. A 10.3 kDa peptide copurified with both forms of nitrous oxide reductase shows strong homology with proteins of the heat-shock GroES chaperonin family. PMID- 10747778 TI - Structural and dynamic characterization of omega-conotoxin MVIIA: the binding loop exhibits slow conformational exchange. AB - omega-Conotoxin MVIIA is a 25-residue, disulfide-bridged polypeptide from the venom of the sea snail Conus magus that binds to neuronal N-type calcium channels. It forms a compact folded structure, presenting a loop between Cys8 and Cys15 that contains a set of residues critical for its binding. The loop does not have a unique defined structure, nor is it intrinsically flexible. Broadening of a subset of resonances in the NMR spectrum at low temperature, anomalous temperature dependence of the chemical shifts of some resonances, and exchange contributions to J(0) from (13)C relaxation measurements reveal that conformational exchange affects the residues in this loop. The effects of this exchange on the calculated structure of omega-conotoxin MVIIA are discussed. The exchange appears to be associated with a change in the conformation of the disulfide bridge Cys8-Cys20. The implications for the use of the omega-conotoxins as a scaffold for carrying other functions is discussed. PMID- 10747779 TI - The crystal structure of N(10)-formyltetrahydrofolate synthetase from Moorella thermoacetica. AB - The structure was solved at 2.5 A resolution using multiwavelength anomalous dispersion (MAD) scattering by Se-Met residues. The subunit of N(10) formyltetrahydrofolate synthetase is composed of three domains organized around three mixed beta-sheets. There are two cavities between adjacent domains. One of them was identified as the nucleotide binding site by homology modeling. The large domain contains a seven-stranded beta-sheet surrounded by helices on both sides. The second domain contains a five-stranded beta-sheet with two alpha helices packed on one side while the other two are a wall of the active site cavity. The third domain contains a four-stranded beta-sheet forming a half barrel. The concave side is covered by two helices while the convex side is another wall of the large cavity. Arg 97 is likely involved in formyl phosphate binding. The tetrameric molecule is relatively flat with the shape of the letter X, and the active sites are located at the end of the subunits far from the subunit interface. PMID- 10747780 TI - Lipases provide a new mechanistic model for polyhydroxybutyrate (PHB) synthases: characterization of the functional residues in Chromatium vinosum PHB synthase. AB - Polyhydroxybutyrate (PHB) synthases catalyze the conversion of beta hydroxybutyryl coenzyme A (HBCoA) to PHB. These enzymes require an active site cysteine nucleophile for covalent catalysis. A protein BLASTp search using the Class III Chromatium vinosum synthase sequence reveals high homology to prokaryotic lipases whose crystal structures are known. The homology is very convincing in the alpha-beta-elbow (with the active site nucleophile)-alpha-beta structure, residues 131-175 of the synthase. A conserved histidine of the Class III PHB synthases aligns with the active site histidine of the lipases using the ClustalW algorithm. This is intriguing as this histidine is approximately 200 amino acids removed in sequence space from the catalytic nucleophile. Different threading algorithms suggest that the Class III synthases belong to the alpha/beta hydrolase superfamily which includes prokaryotic lipases. Mutagenesis studies were carried out on C. vinosum synthase C149, H331, H303, D302, and C130 residues. These studies reveal that H331 is the general base catalyst that activates the nucleophile, C149, for covalent catalysis. The model indicates that C130 is not involved in catalysis as previously proposed [Muh, U., Sinskey, A. J., Kirby, D. P., Lane, W. S., and Stubbe, J. (1999) Biochemistry 38, 826-837]. Studies with D302 mutants suggest D302 functions as a general base catalyst in activation of the 3-hydroxyl of HBCoA (or a hydroxybutyrate acyl enzyme) for nucleophilic attack on the covalently linked thiol ester intermediate. The relationship of the lipase model to previous models based on fatty acid synthases is discussed. PMID- 10747781 TI - Coupling between the N- and C-terminal domains influences transducin-alpha intrinsic GDP/GTP exchange. AB - The N-terminal regions of the heterotrimeric G-protein alpha-subunits represent one of the major Gbetagamma contact sites and have been implicated in an interaction with G-protein-coupled receptors. To probe the role of the N-terminal domain of transducin-alpha in G-protein function, a chimeric Gtialpha subunit with the 31 N-terminal Gtalpha residues replaced by the corresponding 42 residues of Gsalpha (Ns-Gtialpha) has been examined for the interaction with light activated rhodopsin (R). Gtialpha displayed a somewhat higher R-stimulated rate of GTPgammaS binding relative to Ns-Gtialpha, suggesting modest involvement of the Gtalpha N-terminal sequence in recognition of the receptor. However, the intrinsic rate of nucleotide exchange in Ns-Gtialpha was significantly faster (k(app) = 0.014 min(-)(1)) than that in Gtialpha (k(app) = 0.0013 min(-1)) as judged by the GTPgammaS binding rates. Substitution of 42 N-terminal residues of Gsalpha by the Gtalpha residues in a reciprocal chimera, Nt-Gsalpha, had an opposite effect-notable reduction in the intrinsic GTPgammaS-binding rate (k(app) = 0.0075 min(-)(1)) in comparison with Gsalpha (k(app) = 0.028 min(-)(1)). Residue Val30 (His41 in Gsalpha) within the N-terminal region of Gtalpha interacts with the C-terminal residue, Ile339. To test the hypothesis that observed changes in the intrinsic nucleotide exchange rate in chimeric Galpha subunits might be attributed to this interaction, GtialphaVal30His, GtialphaIle339Ala, and Ns-GtialphaHis41Val mutants have been made and analyzed for basal GTPgammaS binding. GtialphaVal30His and GtialphaIle339Ala had increased GTPgammaS binding rates (k(app) = 0. 010 and 0.009 min(-)(1), respectively), whereas Ns-GtialphaHis41Val had a decreased GTPgammaS binding rate (k(app) = 0.0011 min(-)(1)) relative to their parent proteins. These results suggest that the coupling between the N-terminal and C-terminal domains of Gtalpha is important for maintaining a low nucleotide exchange rate in unstimulated transducin. PMID- 10747782 TI - Characterization of the DNA-binding domains from the yeast cell-cycle transcription factors Mbp1 and Swi4. AB - The minimal DNA-binding domains of the Saccharomyces cerevisiae transcription factors Mbp1 and Swi4 have been identified and their DNA binding properties have been investigated by a combination of methods. An approximately 100 residue region of sequence homology at the N-termini of Mbp1 and Swi4 is necessary but not sufficient for full DNA binding activity. Unexpectedly, nonconserved residues C-terminal to the core domain are essential for DNA binding. Proteolysis of Mbp1 and Swi4 DNA-protein complexes has revealed the extent of these sequences, and C terminally extended molecules with substantially enhanced DNA binding activity compared to the core domains alone have been produced. The extended Mbp1 and Swi4 proteins bind to their cognate sites with similar affinity [K(A) approximately (1 4) x 10(6) M(-)(1)] and with a 1:1 stoichiometry. However, alanine substitution of two lysine residues (116 and 122) within the C-terminal extension (tail) of Mbp1 considerably reduces the apparent affinity for an MCB (MluI cell-cycle box) containing oligonucleotide. Both Mbp1 and Swi4 are specific for their cognate sites with respect to nonspecific DNA but exhibit similar affinities for the SCB (Swi4/Swi6 cell-cycle box) and MCB consensus elements. Circular dichroism and (1)H NMR spectroscopy reveal that complex formation results in substantial perturbations of base stacking interactions upon DNA binding. These are localized to a central 5'-d(C-A/G-CG)-3' region common to both MCB and SCB sequences consistent with the observed pattern of specificity. Changes in the backbone amide proton and nitrogen chemical shifts upon DNA binding have enabled us to experimentally define a DNA-binding surface on the core N-terminal domain of Mbp1 that is associated with a putative winged helix-turn-helix motif. Furthermore, significant chemical shift differences occur within the C-terminal tail of Mbp1, supporting the notion of two structurally distinct DNA-binding regions within these proteins. PMID- 10747783 TI - New mechanistic insights from structural studies of the oxygen-sensing domain of Bradyrhizobium japonicum FixL. AB - The FixL heme domain serves as the dioxygen switch in the FixL/FixJ two-component system of Rhizobia. Recent structural studies of the Bradyrhizobium japonicum FixL heme domain (BjFixLH) have suggested an allosteric mechanism that is distinct from the classical hemoglobin model. To gain further insight into the FixL sensing mechanism, structures of BjFixLH bound to dioxygen, imidazole, and nitric oxide have been determined. These structures, particularly the structure of BjFixLH bound to its physiological ligand, dioxygen, have helped to address a number of important issues relevant to the BjFixLH sensing mechanism. On the basis of the oxy-BjFixLH structure, a conserved arginine is found to stabilize the dioxygen ligand in a mode reminiscent of the distal histidine in classical myoglobins and hemoglobins. The structure of BjFixLH bound to imidazole elucidates the structural requirements for accommodating sterically bulky ligands. Finally, the structure of BjFixLH bound to nitric oxide provides evidence for a structural intermediate in the heme-driven conformational change. PMID- 10747785 TI - A common pharmacophore for Taxol and the epothilones based on the biological activity of a taxane molecule lacking a C-13 side chain. AB - Extensive structure-activity studies done with Taxol have identified the side chain at C-13 as one of the requirements for biological activity. Baccatin III, an analogue of Taxol lacking the C-13 side chain, has none of the biological characteristics of Taxol. Since 2-m-azido Taxol, a Taxol derivative with a m azido substituent in the C-2 benzoyl ring, has greater activity than Taxol, we questioned whether 2-m-azido baccatin III might be active. 2-m-Azido baccatin III inhibited the proliferation of human cancer cells at nanomolar concentrations, blocked cells at mitosis, and reorganized the interphase microtubules into distinct bundles, a typical morphological change induced by Taxol. In contrast to 2-m-azido baccatin III, 2-p-azido baccatin III was similar to baccatin III, having no Taxol-like activity, further indicating the specificity and significance of the 2-meta position substituent. Molecular modeling studies done with the C-2 benzoyl ring of Taxol indicated that it fits into a pocket formed by His227 and Asp224 on beta-tubulin and that the 2-m-azido, in contrast to the 2-p azido substituent, is capable of enhancing the interaction between the benzoyl group and the side chain of Asp224. The observation that the C-13 side chain is not an absolute requirement for biological activity in a taxane molecule has enabled the development of a new common pharmacophore model between Taxol and the epothilones. PMID- 10747784 TI - Structure of the complex of Cdc42Hs with a peptide derived from P-21 activated kinase. AB - Cdc42Hs is a member of the Ras superfamily of GTPases and initiates a cascade that begins with the activation of several kinases, including p21-activated kinase (PAK). We have previously used a 46 amino acid fragment of PAK (PBD46) to define the binding surface on Cdc42Hs [Guo et al. (1998) Biochemistry 37, 14030 14037]. Here we describe the three-dimensional solution structure of the Cdc42Hs. GMPPCP-PBD46 complex. Heteronuclear NMR methods were used to assign resonances in the complex, and approximately 2400 distance and dihedral restraints were used to calculate a set of 20 structures using a combination of distance geometry, simulated annealing, and chemical shift and Ramachandran refinement. The overall structure of Cdc42Hs in the complex differs from the uncomplexed structure in two major aspects: (1) the first alpha helix is reoriented to accommodate the binding of the peptide and (2) the regions corresponding to switch I and switch II are less disordered. As suggested by our previous work (Guo et al., 1998) and similar to the complex between Cdc42Hs and fACK [Mott et al. (1999) Nature 399, 384-388], PBD46 forms an intermolecular beta-sheet with beta2 of Cdc42Hs and contacts both switch I and switch II. The extensive binding surface between PBD46 and Cdc42Hs can account for both the high affinity of the complex and the inhibition by PBD46 of GTP hydrolysis. PMID- 10747786 TI - Calmodulin remains extended upon binding to smooth muscle caldesmon: a combined small-angle scattering and fourier transform infrared spectroscopy study. AB - We show that calmodulin (CaM) has an extended conformation in its complexes with sequences from the smooth muscle thin filament protein caldesmon (CaD) by using small-angle X-ray and neutron scattering with contrast variation. The CaD sequences used in these experiments were a C-terminal fragment, 22kCaD, and a smaller peptide sequence within this fragment, MG56C. Each of these sequences contains the CaM-binding sites A and B previously shown to interact with the C- and N-terminal lobes of CaM, respectively [Wang et al. (1997) Biochemistry 36, 15026]. By modeling the scattering data, we show that the majority of the MG56C sequence binds to the N-terminal domain of CaM. FTIR data on CaM complexed with 22kCaD or with MG56C peptide show the 22kCaD sequence contains unordered, helix, and extended structures, and that the extended structures reside primarily in the MG56C portion of the sequence. There are small changes in secondary structure, involving approximately 12 residues, induced by CaM binding to CaD. These changes involve a net decrease in extended structures accompanied by an increase in alpha helix, and they occur within the CaM and/or in the MG56C sequence. PMID- 10747787 TI - Evidence that beta-tubulin induces a conformation change in the cytosolic chaperonin which stabilizes binding: implications for the mechanism of action. AB - The class II chaperonin CCT facilitates protein folding by a process that is not well-understood. One striking feature of this chaperonin is its apparent selectivity in vivo, folding only actin, tubulin, and several other proteins. In contrast, the class I chaperonin GroEL is thought to facilitate the folding of many proteins within Escherichia coli. It has been proposed that this apparent selectivity is associated with certain regions of a substrate protein's primary structure. Using limiting amounts of beta-tubulin, beta-tubulin mutants, and beta tubulin/ftsZ chimeras, we assessed the contribution of select regions of beta tubulin to CCT binding. In a complementary study, we investigated inter-ring communication in CCT where we exploited polypeptide binding sensitivity to nucleotide to quantitate nucleotide binding. beta-Tubulin bound with a high apparent affinity to CCT in the absence of nucleotide (apparent K(D) approximately 3 nM; its apparent binding free energy, DeltaG, ca. -11.8 kcal/mol). Despite this, the interactions appear to be weak and distributed throughout much of the sequence, although certain sites ("hot spots") may interact somewhat more strongly with CCT. Globally averaged over the beta-tubulin sequence, these interactions appear to contribute ca. -9 to -11 cal/mol per residue, and to account for no more than 50-60% of the total binding free energy. We propose that a conformation change or deformation induced in CCT by substrate binding provides the missing free energy which stabilizes the binary complex. We suggest that by coupling CCT deformation with polypeptide binding, CCT avoids the need for high "intrinsic" affinities for its substrates. This strategy allows for dynamic interactions between chaperonin and bound substrate, which may facilitate folding on the interior surface of CCT in the absence of nucleotide and/or productive release of bound polypeptide into the central cavity upon subsequent MgATP binding. CCT displayed negative inter-ring cooperativity like GroEL. When ring 1 of CCT bound MgATP or beta-tubulin, the affinity of ring 2 for polypeptide or nucleotide was apparently reduced approximately 100-fold. PMID- 10747788 TI - Intramolecular activation of a Ca(2+)-dependent protein kinase is disrupted by insertions in the tether that connects the calmodulin-like domain to the kinase. AB - Ca(2+)-dependent protein kinases (CDPK) have a calmodulin-like domain (CaM-LD) tethered to the C-terminal end of the kinase. Activation is proposed to involve intramolecular binding of the CaM-LD to a junction sequence that connects the CaM LD to the kinase domain. Consistent with this model, a truncated CDPK (DeltaNC) in which the CaM-LD has been deleted can be activated in a bimolecular interaction with an isolated CaM-LD or calmodulin, similar to the activation of a calmodulin-dependent protein kinase (CaMK) by calmodulin. Here we provide genetic evidence that this bimolecular activation requires a nine-residue binding segment from F436 to I444 (numbers correspond to CPK-1 accession number L14771). Two mutations at either end of this core segment (F436/A and VI444/AA) severely disrupted bimolecular activation, whereas flanking mutations had only minor effects. Intramolecular activation of a full-length kinase was also disrupted by a VI444/AA mutation, but surprisingly not by a F436/A mutation (at the N-terminal end of the binding site). Interestingly, intramolecular but not bimolecular activation was disrupted by insertion mutations placed immediately downstream of I444. To show that mutant enzymes were not misfolded, latent kinase activity was stimulated through binding of an antijunction antibody. Results here support a model of intramolecular activation in which the tether (A445 to G455) that connects the CaM-LD to the kinase provides an important structural constraint and is not just a simple flexible connection. PMID- 10747789 TI - Histidine 295 and histidine 510 are crucial for the enzymatic degradation of heparan sulfate by heparinase III. AB - The heparinases from Flavobacterium heparinum are powerful tools in understanding how heparin-like glycosaminoglycans function biologically. Heparinase III is the unique member of the heparinase family of heparin-degrading lyases that recognizes the ubiquitous cell-surface heparan sulfate proteoglycans as its primary substrate. Given that both heparinase I and heparinase II contain catalytically critical histidines, we examined the role of histidine in heparinase III. Through a series of diethyl pyrocarbonate modification experiments, it was found that surface-exposed histidines are modified in a concentration-dependent fashion and that this modification results in inactivation of the enzyme (k(inact) = 0.20 +/- 0.04 min(-)(1) mM(-)(1)). The DEPC modification was pH dependent and reversible by hydroxylamine, indicating that histidines are the sole residue being modified. As previously observed for heparinases I and II, substrate protection experiments slowed the inactivation kinetics, suggesting that the modified residue(s) was (were) in or proximal to the active site of the enzyme. Proteolytic mapping experiments, taken together with site-directed mutagenesis studies, confirm the chemical modification experiments and point to two histidines, histidine 295 and histidine 510, as being essential for heparinase III enzymatic activity. PMID- 10747790 TI - Magnetic circular dichroism properties of reaction center complexes isolated from the zinc-bacteriochlorophyll a-containing purple bacterium Acidiphilium rubrum. AB - Reaction center (RC) complexes isolated from a Zn-bacteriochlorophyll (BChl) a containing purple bacterium, Acidiphilium rubrum, were characterized by absorption, circular dichroism, and magnetic circular dichroism (MCD) spectroscopy. The oxidized-minus-reduced difference spectra indicated that, in this RC, the Zn-BChl a is the primary electron donor. The molecular structure of the donor was examined by measuring the ratio of the MCD intensity of the Faraday B-term (B) to the dipole strength (D). In the Q(y) region, B/D for the donor was about half those of bacteriopheophytin a and the accessory Zn-BChl a, indicating that the primary electron donor is a dimer. The magnitude of bleach of the Q(x) band was half that observed in Rhodobacter sphaeroides, suggesting the cation is localized on a single Zn-Bchl a. The absorption intensity of the higher-energy Q(y) exciton band was approximately 28% of that of the lower-energy band, and the exciton splitting was approximately 570 cm(-1), smaller than that in Rb. sphaeroides. These results indicate that, in A. rubrum, the primary electron donor is a Zn-BChl a dimer but that the interaction between the two molecules is rather weak. On the basis of these results, an adaptive strategy for changes in BChl a species is discussed from an evolutionary perspective. PMID- 10747791 TI - Oxidase reaction of cytochrome cd(1) from Paracoccus pantotrophus. AB - Cytochrome cd(1) (cd(1)NIR) from Paracoccus pantotrophus, which is both a nitrite reductase and an oxidase, was reduced by ascorbate plus hexaamineruthenium(III) chloride on a relatively slow time scale (hours required for complete reduction). Visible absorption spectroscopy showed that mixing of ascorbate-reduced enzyme with oxygen at pH = 6.0 resulted in the rapid oxidation of both types of heme center in the enzyme with a linear dependence on oxygen concentration. Subsequent changes on a longer time scale reflected the formation and decay of partially reduced oxygen species bound to the d(1) heme iron. Parallel freeze-quench experiments allowed the X-band electron paramagnetic resonance (EPR) spectrum of the enzyme to be recorded at various times after mixing with oxygen. On the same millisecond time scale that simultaneous oxidation of both heme centers was seen in the optical experiments, two new EPR signals were observed. Both of these are assigned to oxidized heme c and resemble signals from the cytochrome c domain of a "semi-apo" form of the enzyme for which histidine/methionine coordination was demonstrated spectroscopically. These observations suggests that structural changes take around the heme c center that lead to either histidine/methionine axial ligation or a different stereochemistry of bis-histidine axial ligation than that found in the as prepared enzyme. At this stage in the reaction no EPR signal could be ascribed to Fe(III) d(1) heme. Rather, a radical species, which is tentatively assigned to an amino acid radical proximal to the d(1) heme iron in the Fe(IV)-oxo state, was seen. The kinetics of decay of this radical species match the generation of a new form of the Fe(III) d(1) heme, probably representing an OH(-)-bound species. This sequence of events is interpreted in terms of a concerted two-electron reduction of oxygen to bound peroxide, which is immediately cleaved to yield water and an Fe(IV)-oxo species plus the radical. Two electrons from ascorbate are subsequently transferred to the d(1) heme active site via heme c to reduce both the radical and the Fe(IV)-oxo species to Fe(III) OH(-) for completion of a catalytic cycle. PMID- 10747792 TI - Proton NMR studies of Co(II) complexes of the peptide antibiotic bacitracin and analogues: insight into structure-activity relationship. AB - Bacitracin is a widely used metal-dependent peptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis with a potent bactericidal activity directed primarily against Gram-positive organisms. This antibiotic requires a divalent metal ion such as Zn(II) for its biological activity, and has been reported to bind several other transition metal ions, including Co(II), Ni(II), and Cu(II). Despite the wide use of bacitracin, a structure-activity relationship for this drug has not been established, and the structure of its metal complexes has not been fully determined. We report here one- and two-dimensional nuclear magnetic resonance (NMR) studies of the structure of the metal complexes of several bacitracin analogues by the use of paramagnetic Co(II) as a probe. The Co(II) complex of this antibiotic exhibits many well-resolved isotropically shifted (1)H NMR signals in a large spectral window ( approximately 200 ppm) due to protons near the metal, resulting from both contact and dipolar shift mechanisms. The assignment of the isotropically shifted (1)H NMR features concludes that bacitracin A(1), the most potent component of the bacitracin mixture, binds to Co(II) via the His-10 imidazole ring N(epsilon), the thiazoline nitrogen, and the monodentate Glu-4 carboxylate to form a labile complex in aqueous solutions. The free amine of Ile-1 does not bind Co(II). Several different analogues of bacitracin have also been isolated or prepared, and the studies of their Co(II) binding properties further indicate that the antimicrobial activity of these derivatives correlates directly to their metal binding mode. For example, the isotropically shifted (1)H NMR spectral features of the high-potent bacitracin analogues, including bacitracins A(1), B(1), and B(2), are virtually identical. However, Glu-4 and/or the thiazoline ring does not bind Co(II) in the bacitracin analogues with low antibiotic activities, including bacitracins A(2) and F. PMID- 10747793 TI - Structure of the molybdenum site of Rhodobacter sphaeroides biotin sulfoxide reductase. AB - Conditions for heterologous expression of Rhodobacter sphaeroides biotin sulfoxide reductase in Escherichia coli were modified, resulting in a significant improvement in the yield of recombinant enzyme and enabling structural studies of the molybdenum center. Quantitation of the guanine and the molybdenum as compared to that found in R. sphaeroides DMSO reductase demonstrated the presence of the bis(MGD)molybdenum cofactor. UV-visible absorption spectra were obtained for the oxidized, NADPH-reduced, and dithionite-reduced enzyme. EPR spectra were obtained for the Mo(V) state of the enzyme. X-ray absorption spectroscopy at the molybdenum K-edge has been used to probe the molybdenum coordination of the enzyme. The molybdenum site of the oxidized protein possesses a Mo(VI) mono-oxo site (Mo=O at 1.70 A) with additional coordination by approximately four thiolate ligands at 2.41 A and probably one oxygen or nitrogen at 1.95 A. The NADPH- and dithionite-reduced Mo(IV) forms of the enzyme are des-oxo molybdenum sites with approximately four thiolates at 2.33 A and two different Mo-O/N ligands at 2.19 and 1.94 A. PMID- 10747794 TI - Structure and stability effects of the mutation of glycine 34 to serine in Rhodobacter capsulatus cytochrome c(2). AB - Gly 34 and the adjacent Pro 35 of Rhodobacter capsulatus cytochrome c(2) (or Gly 29 and Pro 30 in vertebrate cytochrome c) are highly conserved side chains among the class I c-type cytochromes. The mutation of Gly 34 to Ser in Rb. capsulatus cytochrome c(2) has been characterized in terms of physicochemical properties and NMR in both redox states. A comparison of the wild-type cytochrome c(2), the G34S mutation, and the P35A mutation is presented in the context of differences in chemical shifts, the differences in NOE patterns, and structural changes resulting from oxidation of the reduced cytochrome. G34S is substantially destabilized relative to wild-type (2.2 kcal/mol in the oxidized state) but similarly destabilized relative to P35A. Nevertheless, differences in terms of the impact of the mutations on specific structural regions are found when comparing G34S and P35A. Although available data indicates that the overall secondary structure of G34S and wild-type cytochrome c(2) are similar, a number of both perturbations of hydrogen bond networks and interactions with internal waters are found. Thus, the impact of the mutation at position 35 is propagated throughout the cytochrome but with alterations at defined sites within the molecule. Interestingly, we find that the substitution of serine at position 34 results in a perturbation of the heme beta meso and the methyl-5 protons. This suggests that the hydroxyl and beta carbon are positioned away from the solvent and toward the heme. This has the consequence of preferentially stabilizing the oxidized state in G34S, thus, altering hydrogen bond networks which involve the heme propionate, internal waters, and key amino acid side chains. The results presented provide important new insights into the stability and solution structure of the cytochrome c(2). PMID- 10747795 TI - Function of the extra 5'-phosphate carried by histidine tRNA. AB - Among elongator tRNAs, tRNA specific for histidine has the peculiarity to possess one extra nucleotide at position -1. This nucleotide is believed to be responsible for recognition by histidyl-tRNA synthetase. Here, we show that, in fact, it is the phosphate 5' to the extra nucleotide which mainly supports the efficiency of the tRNA aminoacylation reaction catalyzed by Escherichia coli histidyl-tRNA synthetase. In the case of the reaction of E. coli peptidyl-tRNA hydrolase, this atypical phosphate is dispensable. Instead, peptidyl-tRNA hydrolase recognizes the phosphate of the phosphodiester bond between residues -1 and +1 of tRNA(His). Recognition of the +1 phosphate of tRNA(His) by peptidyl tRNA hydrolase resembles, therefore, that of the 5'-terminal phosphate of other elongator tRNAs. PMID- 10747796 TI - Using a targeted chemical nuclease to elucidate conformational changes in the E. coli 30S ribosomal subunit. AB - Determining the detailed tertiary structure of 16S rRNA within 30S ribosomal subunits remains a challenging problem. The particular structure of the RNA which allows tRNA to effectively interact with the associated mRNA during protein synthesis remains particularly ambiguous. This study utilizes a chemical nuclease, 1, 10-o-phenanthroline-copper, to localize regions of 16S rRNA proximal to the decoding region under conditions in which tRNA does not readily associate with the 30S subunit (inactive conformation), and under conditions which optimize tRNA binding (active conformation). By covalently attaching 1,10-phenanthroline copper to a DNA oligomer complementary to nucleotides in the decoding region (1396-1403), we have determined that nucleotides 923-929, 1391-1396, and 1190 1192 are within approximately 15 A of the nucleotide base-paired to nucleotide 1403 in inactive subunits, but in active subunits only cleavages (1404-1405) immediately proximal to the 5' end of the hybridized probe remain. These results provide evidence for dynamic movement in the 30S ribosomal subunit, reported for the first time using a targeted chemical nuclease. PMID- 10747797 TI - Deletion of C-terminal residues of Escherichia coli ribosomal protein L10 causes the loss of binding of one L7/L12 dimer: ribosomes with one L7/L12 dimer are active. AB - Escherichia coli ribosomal protein L10 binds the two L7/L12 dimers and thereby anchors them to the large ribosomal subunit. C-Terminal deletion variants (Delta10, Delta20, and Delta33 amino acids) of ribosomal protein L10 were constructed in order to define the binding sites for the two L7/L12 dimers and then to make and test ribosomal particles that contain only one of the two dimers. None of the deletions interfered with binding of L10 variants to ribosomal core particles. Deletion of 20 or 33 amino acids led to the inability of the proteins to bind both dimers of protein L7/L12. The L10 variant with deletion of 10 amino acids bound one L7/L12 dimer in solution and when reconstituted into ribosomes promoted the binding of only one L7/L12 dimer to the ribosome. The ribosomes that contained a single L7/L12 dimer were homogeneous by gel electrophoresis where they had a mobility between wild-type 50S subunits and cores completely lacking L7/L12. The single-dimer ribosomal particles supported elongation factor G dependent GTP hydrolysis and protein synthesis in vitro with the same activity as that of two-dimer particles. The results suggest that amino acids 145-154 in protein L10 determine the binding site ("internal-site") for one L7/L12 dimer (the one reported here), and residues 155-164 ("C-terminal-site") are involved in the interaction with the second L7/L12 dimer. Homogeneous ribosomal particles containing a single L7/L12 dimer in each of the distinct sites present an ideal system for studying the location, conformation, dynamics, and function of each of the dimers individually. PMID- 10747798 TI - Yeast resolving enzyme CCE1 makes sequential cleavages in DNA junctions within the lifetime of the complex. AB - CCE1 is a DNA junction-resolving enzyme of Saccharomyces cerevisiae. Such enzymes are required to make two symmetrically paired cleavages in order to resolve the four-way junction productively. Using a cruciform assay, we show here that CCE1 introduces two unilateral cleavages in a sequential manner. This requires that the protein remains bound to the junction, preventing branch migration of the point of strand exchange. From a detailed kinetic analysis, we find that the CCE1 cleavage at a given site is accelerated by a factor of 5-10 when it occurs subsequently to the initial cleavage. These properties ensure a productive resolution of the four-way junction and may be general for junction-resolving enzymes. PMID- 10747799 TI - On the role of the carboxyl-terminal helix of RXR in the interactions of the receptor with ligand. AB - The retinoid X receptor (RXR), a ligand-inducible transcription factor that is activated by 9-cis-retinoic acid, is a member of the superfamily of nuclear hormone receptors. The ligand-induced transcriptional activity of nuclear receptors is coordinated by their C-terminal region termed the ligand-binding domain. Structural analyses of several nuclear receptors showed that the most dramatic ligand-induced conformational change in these proteins involves a positional shift in the receptors' C-terminal helix, termed helix 12. Consequently, in the liganded state, helix 12 is folded over the entrance to the ligand-binding pocket where it serves as a lid, and it has been proposed that this region functions to stabilize ligand binding by at least some nuclear receptors. Here, to examine the possible role of helix 12 in contributing to the association of RXR with its ligand, the equilibrium and kinetic parameters of the interactions of 9-cis-retinoic acid with RXR and with a deletion mutant lacking helix 12 were measured. Deletion of the region did not significantly alter the ligand-binding affinity of RXR at equilibrium. However, both the rate of dissociation and the rate of association of the RXR-9-cis-retinoic acid complex were significantly slower in the absence of helix 12. Taken together, these observations suggest that helix 12 of RXR facilitates both the entry and the exit of the ligand from the binding pocket without affecting the equilibrium ligand binding affinity. The results thus point at a previously unsuspected function for this region. PMID- 10747800 TI - Studies with recombinant Saccharomyces cerevisiae CaaX prenyl protease Rce1p. AB - Eukaryotic proteins with carboxyl-terminal CaaX motifs undergo three post translational processing reactions-protein prenylation, endoproteolysis, and carboxymethylation. Two genes in yeast encoding CaaX endoproteases, AFC1 and RCE1, have been identified. Rce1p is solely responsible for proteolysis of yeast Ras proteins. When proteolysis is blocked, plasma membrane localization of Ras2p is impaired. The mislocalization of undermodified Ras in the cell suggests that Rce1p is an attractive target for cancer therapeutics. Homologous expression of plasmid-encoded Saccharomyces cerevisiae RCE1 under the control of the GAL1 promoter gave a 370-fold increase in endoprotease activity over an uninduced control. Yeast Rce1p was detected by Western blotting with a yRce1p antibody or with an anti-myc antibody to Rce1p bearing a C-terminal myc-epitope. Membrane preparations were examined for their sensitivity to a variety of protease inhibitors, metal ion chelators, and heavy metals. The enzyme was sensitive to cysteine protease inhibitors, Zn(2+), and Ni(2+). The substrate selectivity of yRce1p was determined for a variety of prenylated CaaX peptides including farnesylated and geranylgeranylated forms of human Ha-Ras, Ki-Ras, N-Ras, and yeast Ras2p, a-mating factor, and Rho2p. Six site-directed mutants of conserved polar and ionic amino acids in yRce1p were prepared. Four of the mutants, H194A, E156A, C251A, and H248A, were inactive. Results from the protease inhibition studies and the site-directed mutagenesis suggest that Rce1p is a cysteine protease. PMID- 10747801 TI - Conservative mutations of glutamine-125 in herpes simplex virus type 1 thymidine kinase result in a ganciclovir kinase with minimal deoxypyrimidine kinase activities. AB - The herpes simplex virus type 1 thymidine kinase (HSV-1 TK) is the major anti herpes virus pharmacological target, and it is being utilized in combination with the prodrug ganciclovir as a toxin gene therapeutic for cancer. One active-site amino acid, glutamine-125 (Gln-125), has been shown to form hydrogen bonds with bound thymidine, thymidylate, and ganciclovir in multiple X-ray crystal structures. To examine the role of Gln-125 in HSV-1 TK activity, three site specific mutations of this residue to an aspartic acid, an asparagine, or a glutamic acid were introduced. These three mutants and wild-type HSV-1 TK were expressed in E. coli and partially purified and their enzymatic properties compared. In comparison to the Gln-125 HSV-1 TK, thymidylate kinase activity of all three mutants was decreased by over 90%. For thymidine kinase activity relative to Gln-125 enzyme, the K(m) of thymidine increased from 0.9 microM for the parent Gln-125 enzyme to 3 microM for the Glu-125 mutant, to 6000 microM for the Asp-125 mutant, and to 20 microM for the Asn-125 mutant. In contrast, the K(m) of ganciclovir decreased from 69 microM for the parent Gln-125 enzyme to 50 microM for the Asn-125 mutant and increased to 473 microM for the Glu-125 mutant. The Asp-125 enzyme was able to poorly phosphorylate ganciclovir, but with nonlinear kinetics. Molecular simulations of the wild-type and mutant HSV-1 TK active sites predict that the observed activities are due to loss of hydrogen bonding between thymidine and the mutant amino acids, while the potential for hydrogen bonding remains intact for ganciclovir binding. When expressed in two mammalian cell lines, the Glu-125 mutant led to GCV-mediated killing of one cell line, while the Asn-125 mutant was equally as effective as wild-type HSV-1 TK in metabolizing GCV and causing cell death in both cell lines. PMID- 10747802 TI - Electron paramagnetic resonance and electron nuclear double resonance spectroscopic identification and characterization of the tyrosyl radicals in prostaglandin H synthase 1. AB - The tyrosyl radicals generated in reactions of ethyl hydrogen peroxide with both native and indomethacin-pretreated prostaglandin H synthase 1 (PGHS-1) were examined by low-temperature electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies. In the reaction of peroxide with the native enzyme at 0 degrees C, the tyrosyl radical EPR signal underwent a continuous reduction in line width and lost intensity as the incubation time increased, changing from an initial, 35-G wide doublet to a wide singlet of slightly smaller line width and finally to a 25-G narrow singlet. The 25-G narrow singlet produced by self-inactivation was distinctly broader than the 22-G narrow singlet obtained by indomethacin treatment. Analysis of the narrow singlet EPR spectra of self-inactivated and indomethacin-pretreated enzymes suggests that they reflect conformationally distinct tyrosyl radicals. ENDOR spectroscopy allowed more detailed characterization by providing hyperfine couplings for ring and methylene protons. These results establish that the wide doublet and the 22-G narrow singlet EPR signals arise from tyrosyl radicals with different side-chain conformations. The wide-singlet ENDOR spectrum, however, is best accounted for as a mixture of native wide-doublet and self-inactivated 25-G narrow-singlet species, consistent with an earlier EPR study [DeGray et al. (1992) J. Biol. Chem. 267, 23583-23588]. We conclude that a tyrosyl residue other than the catalytically essential Y385 species is most likely responsible for the indomethacin-inhibited, narrow-singlet spectrum. Thus, this inhibitor may function by redirecting radical formation to a catalytically inactive side chain. Either radical migration or conformational relaxation at Y385 produces the 25-G narrow singlet during self-inactivation. Our ENDOR data also indicate that the catalytically active, wide-doublet species is not hydrogen bonded, which may enhance its reactivity toward the fatty-acid substrate bound nearby. PMID- 10747803 TI - Do cysteine 230 and lysine 238 of biotin carboxylase play a role in the activation of biotin? AB - Biotin carboxylase from Escherichia coli catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl CoA carboxylase, which catalyzes the committed step in long-chain fatty acid synthesis. For the carboxylation of biotin to occur, biotin must be deprotonated at its N1' position. Kinetic investigations, including solvent isotope effects and enzyme inactivation by N-ethylmaleimide, suggested a catalytic role for a cysteine residue and led to the proposal of a mechanism for the deprotonation of biotin. The proposed pathway suggests a catalytic base removes a proton from a nearby cysteine residue, forming a thiolate anion, which then abstracts the proton from biotin. Inactivation studies of pyruvate carboxylase, which has an analogous mode of action to biotin carboxylase, suggests the catalytic base in this reaction is a lysine residue. Using the crystal structure of biotin carboxylase, cysteine 230 and lysine 238 were identified as the likely active site residues that act as this acid-base pair. To test the hypothesis that cysteine 230 and lysine 238 act as an acid-base pair to deprotonate biotin, site directed mutagenesis was used to mutate cysteine 230 to alanine (C230A) and lysine 238 to glutamine (K238Q). Mutations at either residue resulted in a 50 fold increase in the K(m) for ATP. The C230A mutation had no effect on the formation of carboxybiotin, indicating that cysteine 230 does not play a role in the deprotonation of biotin. However, the K238Q mutation resulted in no formation of carboxybiotin, which showed that lysine 238 has a role in the carboxylation reaction. N-Ethylmaleimide was found to inactivate the C230A mutant but not the K238Q mutant, suggesting that N-ethylmaleimide is reacting with lysine 238 and not cysteine 230. The pH dependence of N-ethylmaleimide inactivation revealed that the pK value for lysine 238 was 9.4 or higher, suggesting lysine 238 is not a catalytic base. Thus, the results suggest that cysteine 230 and lysine 238 do not act as an acid-base pair in the deprotonation of biotin. PMID- 10747804 TI - Strand cleavage of supercoiled DNA by water-soluble peroxyl radicals. The overlooked importance of peroxyl radical charge. AB - It is well established that the peroxyl radicals formed during the thermal decomposition of 2,2'-azobis(amidinopropane), ABAP, in oxygenated water can cleave double-stranded DNA, from which fact it has been concluded that peroxyl radicals, as a general class, can induce DNA strand scission. However, the ABAP derived radicals are positively charged, and DNA is a negatively charged polyanion. Moreover, the relatively small and, therefore, free to diffuse peroxyl radicals likely to be formed in vivo will generally be negatively charged or neutral. Plasmid supercoiled DNA [pBR 322, 4361 base pairs (bp)] was reacted with known, equal fluxes of two positively charged peroxyl radicals, a negatively charged peroxyl radical, and a neutral peroxyl radical. The two positively charged peroxyl radicals degraded >/=80% of the supercoiled pBR 322 at a flux of 4 radicals/bp, but the negatively charged and neutral peroxyl radicals had no significant effect even at a flux as high as 24 radicals/bp. The same lack of effect on the DNA was also observed with high fluxes of superoxide/hydroperoxyl radicals. Similar results were obtained with another supercoiled DNA, pUC 19, except that pUC 19 is somewhat more sensitive to strand scission by positively charged peroxyl radicals than pBR 322. We conclude that most of the peroxyl radicals likely to be formed in vivo have little or no ability to induce DNA strand scission and that the potential role of electrostatics in radical/DNA reactions should always be considered. PMID- 10747805 TI - Factors determining mutagenic potential for individual cis and trans opened benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts. AB - Four adducts that would result from trans opening at C-1 of benzo[c]phenanthrene 3,4-diol 1,2-epoxide (B[c]PhDE) isomers (i.e., DE-1 enantiomers, where the epoxide oxygen and benzylic hydroxyl group are cis, and DE-2 enantiomers, where they are trans) by the N(6)-amino group of dAdo, together with the two cis opened N(6)-dAdo adducts of B[c]PhDE-1, were incorporated into two oligonucleotides at the underlined site in 5'-TTTAGAGTCTGCTCCC [context I(A)] and 5'-CAGATTTAGAGTCTGC [context II(A)]. After ligation of these, and the corresponding unsubstituted oligonucleotides, into single-stranded M13mp7L2 bacteriophage and transfection into SOS-induced Escherichia coli SMH77, base substitution mutations induced by the different B[c]PhDE-dAdo adducts were determined. These findings were compared with data [Ponten et al. (1999) Biochemistry 38, 1144-1152] for cis opened B[c]PhDE-2-dAdo adducts in the same sequence contexts. In most cases, adducts with S absolute configuration at the site of attachment of the nucleoside to the hydrocarbon had higher mutation frequencies (1.9-56.5%) than the corresponding adducts with R configuration (0.05-5.6%). For adducts derived from B[c]PhDE-1, the predominant mutations were A-->T transversions in context I(A) and A-->G transitions for most of these adducts in context II(A). For adducts derived from B[c]PhDE-2, A-->T base substitutions predominated for most of the trans adducts, but A-->G mutations were favored by the cis adduct with S configuration in either context. Thus, the structural feature that most dramatically affected mutagenic activity was the configuration of the carbon at the attachment point, with S configuration mostly being associated with greater mutagenicity than the R configuration. However, other structural variations and sequence context also affected mutagenicity, indicating that a combination of structure and context effects define mutagenicity. PMID- 10747806 TI - Overexpression, purification, and analysis of complementation behavior of E. coli SuhB protein: comparison with bacterial and archaeal inositol monophosphatases. AB - The E. coli suhB gene product, which has been suggested to participate in posttranscriptional control of gene expression, also possesses inositol-1 phosphatase (I-1-Pase) activity. To test if SuhB I-1-Pase activity is sufficient for its function in cells, we have cloned the genes for three other I-1-Pases (from the archaea Methanococcus jannaschii and Archaeoglobus fulgidus, and from the bacterium Thermotoga maritima) into the E. coli expression vector pET23a(+) and examined if these extragenic I-1-Pases could complement the suhB mutation in E. coli strain CG1307 (which also has a mutation in dnaB and a cold-sensitive phenotype). None of these I-1-Pase genes restored growth at 30 degrees C although they generated active I-1-Pase enzymes (as measured by I-1-Pase specific activities of crude protein extracts from the transformed CG1307 cells). In contrast, the pET23a(+) recombinant plasmid with the wild-type E. coli suhB gene complemented the cold sensitivity of the chromosomal mutant suhB and restored the temperature-sensitive growth of the dnaB mutation in the double mutant strain CG1307. Further evidence that this relief of the suppressor behavior of the suhB mutation is not related to the I-1-Pase activity of the SuhB protein was provided by construction of the E. coli SuhB mutant D87N. This mutant protein is inactive as an I-1-Pase but fully functional in changing the temperature sensitivity of the E. coli double mutant strain. Therefore, I-1-P phosphatase activity is neither sufficient nor required for complementation of suhB mutant suppressor effects. The wild-type E. coli SuhB protein was also overexpressed to very high levels and purified to homogeneity in high yield (1 mg/10 mL of culture). The major differences of the E. coli I-1-Pase from all the other characterized I-1 Pases are that it exists as a monomer (rather than a dimer or tetramer) in solution and is more hydrophobic. These physical differences, rather than the I-1 Pase activity, may be involved in the biological role of wild-type SuhB in E. coli. PMID- 10747807 TI - Mapping the oligomeric interface of diacylglycerol kinase by engineered thiol cross-linking: homologous sites in the transmembrane domain. AB - This work represents the first stage of thiol-based cross-linking studies to map the oligomeric interface of the homotrimeric membrane protein diacylglycerol kinase (DAGK). A total of 53 single-cysteine mutants spanning DAGK's three transmembrane segments and the first part of a cytoplasmic domain were purified and subjected to catalytic oxidation in mixed micelles. Four mutants (A52C, I53C, A74C, and I75C) were observed to undergo intratrimer disulfide bond formation between homologous sites on adjacent subunits. To establish whether the homologous sites are proximal in the ground-state conformation of DAGK or whether the disulfide bonds formed as a result of motions that brought normally distal sites into transient proximity, additional cross-linking experiments were carried out in three different milieus of varying fluidity [mixed micelles, 1-palmitoyl-2 oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles, and Escherichia coli membranes]. Cross-linking experiments included disulfide bond formation under three different catalytic conditions [Cu(II)-phenanthroline oxidation, I(2) oxidation, and thionitrobenzoate-based thiol exchange] and reactions with a set of bifunctional thiol-reactive chemical cross-linkers presenting two different reactive chemistries and several spacer lengths. On the basis of these studies, residues 53 and 75 are judged to be in stable proximity within the DAGK homotrimer, while position 52 appears to be more distal and forms disulfide bonds only as a result of protein motions. Results for position 74 were ambiguous. In lipid vesicles and mixed micelles DAGK appears to execute motions that are not present in native membranes, with mobility also being higher for DAGK in mixed micelles than in POPC vesicles. PMID- 10747808 TI - Iron-sulfur center of biotin synthase and lipoate synthase. AB - Biotin synthase and lipoate synthase are homodimers that are required for the C-S bond formation at nonactivated carbon in the biosynthesis of biotin and lipoic acid, respectively. Aerobically isolated monomers were previously shown to contain a (2Fe-2S) cluster, however, after incubation with dithionite one (4Fe 4S) cluster per dimer was obtained, suggesting that two (2Fe-2S) clusters had combined at the interface of the subunits to form the (4Fe-4S) cluster. Here we report Mossbauer studies of (57)Fe-reconstituted biotin synthase showing that anaerobically prepared enzyme can accommodate two (4Fe-4S) clusters per dimer. The (4Fe-4S) cluster is quantitatively converted into a (2Fe-2S)(2+) cluster upon exposure to air. Reduction of the air-exposed enzyme with dithionite or photoreduced deazaflavin yields again (4Fe-4S) clusters. The (4Fe-4S) cluster is stable in both the 2+ and 1+ oxidation states. The Mossbauer and EPR parameters were DeltaE(q) = 1.13 mm/s and delta = 0.44 mm/s for the diamagnetic (4Fe-4S)(2+) and DeltaE(q) = 0.51 mm/s, delta = 0.85 mm/s, g(par) = 2.035, and g(perp) = 1.93 for the S = (1)/(2) state of (4Fe-4S)(1+). Considering that we find two (4Fe-4S) clusters per dimer, our studies argue against the early proposal that the enzyme contains one (4Fe-4S) cluster bridging the two subunits. Our study of lipoate synthase gave results similar to those obtained for BS: under strict anaerobiosis, lipoate synthase can accommodate a (4Fe-4S) cluster per subunit [DeltaE(q) = 1.20 mm/s and delta = 0.44 mm/s for the diamagnetic (4Fe-4S)(2+) and g(par) = 2.039 and g(perp) = 1.93 for the S = (1)/(2) state of (4Fe-4S)(1+)], which reacts with oxygen to generate a (2Fe-2S)(2+) center. PMID- 10747809 TI - Mutation of serine 395 of tyrosine hydroxylase decouples oxygen-oxygen bond cleavage and tyrosine hydroxylation. AB - Ser395 and Ser396 in the active site of rat tyrosine hydroxylase are conserved in all three members of the family of pterin-dependent hydroxylases, phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase. Ser395 is appropriately positioned to form a hydrogen bond to the imidazole nitrogen of His331, an axial ligand to the active site iron, while Ser396 is located on the wall of the active site cleft. Site-directed mutagenesis has been used to analyze the roles of these two residues in catalysis. The specific activities for formation of dihydroxyphenylalanine by the S395A, S395T, and S396A enzymes are 1.3, 26, and 69% of the wild-type values, respectively. Both the S395A and S396A enzymes bind a stoichiometric amount of iron and exhibit wild-type spectra when complexed with dopamine. The K(M) values for tyrosine, 6-methyltetrahydropterin, and tetrahydrobiopterin are unaffected by replacement of either residue with alanine. Although the V(max) value for tyrosine hydroxylation by the S395A enzyme is decreased by 2 orders of magnitude, the V(max) value for tetrahydropterin oxidation by either the S395A or the S396A enzyme is unchanged from the wild-type value. With both mutant enzymes, there is quantitative formation of 4a hydroxypterin from 6-methyltetrahydropterin. These results establish that Ser395 is required for amino acid hydroxylation but not for cleavage of the oxygen oxygen bond, while Ser396 is not essential. These results also establish that cleavage of the oxygen-oxygen bond occurs in a separate step from amino acid hydroxylation. PMID- 10747810 TI - A partially unfolded structure of the alkaline-denatured state of pepsin and its implication for stability of the zymogen-derived protein. AB - Pepsin, a gastric aspartic proteinase, is a zymogen-derived protein that undergoes irreversible alkaline denaturation at pH 6-7. Detailed knowledge of the structure of the alkaline-denatured state is an important step in understanding the mechanism of the formation of the active enzyme. An extensive analysis of the denatured state at pH 8.0 was performed using a variety of techniques including (1)H nuclear magnetic resonance spectroscopy and solution X-ray scattering. This analysis indicates that the denatured state under these conditions has a compact and globular conformation with a substantial amount of secondary and tertiary structures. The data suggest that this partially structured species has a highly folded region and a flexible region. The NMR measurements suggest that the folded region contains His53 and is located at least partly in the N-terminal lobe of the protein. The alkaline-denatured state experiences a further reversible denaturation step at higher pH or on heating; the midpoints of the unfolding transition are pH 11.5 (at 25 degrees C) and 53.1 degrees C (at pH 8.0), respectively. The present findings suggest that the proteolytic processing of pepsinogen has substantially modified the ability of the protein to fold, such that its folding process cannot progress beyond the partially folded intermediate of pepsin. PMID- 10747811 TI - Interaction of soluble guanylate cyclase with YC-1: kinetic and resonance Raman studies. AB - The enzyme-soluble guanylate cyclase (sGC), which converts GTP to cGMP, is a receptor for the signaling agent nitric oxide (NO). YC-1, a synthetic benzylindazole derivative, has been shown to activate sGC in an NO-independent fashion. In the presence of carbon monoxide (CO), which by itself activates sGC approximately 5-fold, YC-1 activates sGC to a level comparable to stimulation by NO alone. We have used kinetic analyses and resonance Raman spectroscopy (RR) to investigate the interaction of YC-1 and CO with guanylate cyclase. In the presence of CO and 200 microM YC-1, the V(max)/K(m GTP) increases 226-fold. While YC-1 does not perturb the RR spectrum of the ferrous form of baculovirus/Sf9 cell expressed sGC, it induces a shift in the Fe-CO stretching frequency for the CO bound form from 474 to 492 cm(-1). Similarly, YC-1 has no effect on the RR spectrum of ferrous beta1(1-385), the isolated sGC heme-binding domain, but shifts the nu(Fe-CO) of CO-beta1(1-385) from 478 to 491 cm(-1), indicating that YC-1 binds in heme-binding region of sGC. In addition, the CO-bound forms of sGC and beta1(1-385) in the presence of YC-1 lie on the nu(Fe-CO) vs nu(C-O) correlation curve for proximal ligands with imidazole character, which suggests that histidine remains the heme proximal ligand in the presence of YC-1. Interestingly, YC-1 does not shift nu(Fe-CO) for the CO-bound form of H105G(Im), the imidazole-rescued heme ligand mutant of beta1(1-385). The data are consistent with binding of CO and YC-1 to the sGC heme-binding domain leading to conformational changes that give rise to an increase in catalytic turnover and a change in the electrostatic environment of the heme pocket. PMID- 10747812 TI - Environmental study of subunit i, a F(o) component of the yeast ATP synthase. AB - The topology of subunit i, a component of the yeast F(o)F(1)-ATP synthase, was determined by the use of cysteine-substituted mutants. The N(in)-C(out) orientation of this intrinsic subunit was confirmed by chemical modification of unique cysteine residues with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid. Near-neighbor relationships between subunit i and subunits 6, f, g, and d were demonstrated by cross-link formation following sulfhydryl oxidation or reaction with homobifunctional and heterobifunctional reagents. Our data suggest interactions between the unique membrane-spanning segment of subunit i and the first transmembranous alpha-helix of subunit 6 and a stoichiometry of 1 subunit i per complex. Cross-linked products between mutant subunits i and proteins loosely bound to the F(o)F(1)-ATP synthase suggest that subunit i is located at the periphery of the enzyme and interacts with proteins of the inner mitochondrial membrane that are not involved in the structure of the yeast ATP synthase. PMID- 10747813 TI - Roles of aromatic residues in high interfacial activity of naja naja atra phospholipase A(2) PMID- 10747815 TI - High-density genome scan in Crohn disease shows confirmed linkage to chromosome 14q11-12. AB - Epidemiological studies have shown that genetic factors contribute to the pathogenesis of the idiopathic inflammatory bowel diseases (IBD), Crohn disease (CD) and ulcerative colitis (UC). Recent genome scans and replication studies have identified replicated linkage between CD and a locus on chromosome 16 (the IBD1 locus), replicated linkage between IBD (especially UC) and a locus on chromosome 12q (the IBD2 locus), and replicated linkage between IBD (especially CD) and a locus on chromosome 6p (the IBD3 locus). Since the estimated locus specific lambdas values for the regions of replicated linkage do not account for the overall lambdas in CD, and since the published genome scans in IBD show at least nominal evidence for linkage to regions on all but two chromosomes, we performed an independent genome scan using 751 microsatellite loci in 127 CD affected relative pairs from 62 families. Single-point nonparametric linkage analysis using the GENEHUNTER-PLUS program shows evidence for linkage to the adjacent D14S261 and D14S283 loci on chromosome 14q11-12 (LOD = 3.00 and 1.70, respectively), and the maximal multipoint LOD score is observed at D14S261 (LOD = 3.60). In the multipoint analysis, nominal evidence for linkage (P<.05) is observed near D2S117 (LOD = 1.25), near D3S3045 (LOD = 1.31), between D7S40 and D7S648 (LOD = 0.91), and near D18S61 (LOD = 1.15). Our finding of significant linkage to D14S261 and the finding of suggestive linkage to the same locus in an independent study (multipoint LOD = 2.8) satisfies criteria for confirmed linkage, so we propose that the region of interest on chromosome 14q11-12 should be designated the IBD4 locus. PMID- 10747817 TI - NNRTIs-a new class of drugs for HIV. PMID- 10747818 TI - Interplay between the MexA-MexB-OprM multidrug efflux system and the outer membrane barrier in the multiple antibiotic resistance of Pseudomonas aeruginosa. AB - MexAB-OprM is a constitutively expressed multidrug efflux system of Pseudomonas aeruginosa. Using isogenic pump mutants, the contributions of the MexAB-OprM efflux pump and the outer membrane barrier to multiple antibiotic resistance were evaluated by assessing the influence of pump inactivation and outer membrane permeabilization on antibiotic susceptibility. Both pump inactivation and increased outer membrane permeability enhanced antibiotic susceptibility, although maximal susceptibility was achieved when the two were combined. Thus, inhibition of antibiotic efflux pumps and permeabilization of the outer membrane constitute an effective approach to reversing the antibiotic resistance of this organism. PMID- 10747819 TI - In vitro antibacterial spectrum of a new broad-spectrum 8-methoxy fluoroquinolone, gatifloxacin. AB - The in vitro antibacterial spectrum of gatifloxacin was compared with those of ciprofloxacin and ofloxacin. Gatifloxacin was two- to four-fold more potent than comparator quinolones against staphylococci, streptococci, pneumococci and enterococci (gatifloxacin MIC90s, < or =1 mg/L, except 4 mg/L against methicillin resistant Staphylococcus aureus and Enterococcus faecium). Gatifloxacin was two fold less potent than ciprofloxacin, and the same as or two-fold more potent than ofloxacin against Enterobacteriaceae (MIC90s, 0.06-0.5 mg/L against most members of the Enterobacteriaceae and < or =1 mg/L against Proteus/Morganella spp.). Relative to the comparator quinolones, gatifloxacin was two- to four-fold more potent against Providencia spp., and had good potency against Acinetobacter spp. (MIC90s, 0.25-1 mg/L). Gatifloxacin and ofloxacin had similar anti-pseudomonal potency, with corresponding MIC90s of 4, 8 and 0.25 mg/L for Pseudomonas aeruginosa, Pseudomonas fluorescens and Pseudomonas stutzeri, while ciprofloxacin had two- to eight-fold more potency. The three quinolones were equipotent against Burkholderia cepacia (MIC90s, 8 mg/L), but gatifloxacin was two-fold more potent against Stenotrophomonas maltophilia (MIC90, 4 mg/L). Gatifloxacin was highly potent (MIC90s, 0.03-0.06 mg/L) against Haemophilus influenzae, Legionella spp., Helicobacter pylori and had at least eight-fold better anti-chlamydial and anti mycoplasma potency (gatifloxacin MIC90s, 0.13 mg/L). The higher quinolone MICs for ureaplasma (MIC90s, 4-8 mg/L) may be due to the acidic pH of the ureaplasma test medium, which antagonizes quinolones. Like other quinolones, gatifloxacin had poor potency against Mycobacterium avium-intracellulare, though it was eight- to 16-fold more potent against Mycobacterium tuberculosis (MIC90, 0.25 mg/L). Of the three quinolones, only gatifloxacin had activity against Bacteroides fragilis and Clostridium difficile. In summary, gatifloxacin is a broad-spectrum 8-methoxy fluoroquinolone that is more potent than ciprofloxacin and ofloxacin against Gram positive bacteria, chlamydia, mycoplasma, mycobacteria and anaerobes. PMID- 10747820 TI - Ecological effects of triclosan and triclosan monophosphate on defined mixed cultures of oral species grown in continuous culture. AB - The effects of triclosan and its phosphorylated derivative, triclosan monophosphate were studied using a continuous culture microcosm model. Two conditions were simulated, a caries-like state (pH 5.5 with artificial saliva plus glucose as growth medium) and a periodontal disease-like state (pH 7.5 with BHI plus yeast extract, haemin and cysteine as growth medium). Both cultures were maintained anaerobically at 37 degrees C at a growth rate of 0.1/h. Steady-state chemostats were pulsed with triclosan or triclosan monophosphate (initial concentrations between 20 and 40 mg/L) and changes in the ecological composition noted after 6 h. The caries-like microcosm steady state was dominated by streptococci, Lactobacillus and Veillonella sp. with low but detectable levels of Neisseria, Actinomyces and Fusobacterium sp. No significant ecological shifts occurred following pulses of either antimicrobial agent; all species were affected to approximately the same degree. The periodontal disease-like microcosm steady state was dominated by streptococci, Fusobacterium, Veillonella, Actinomyces, Prevotella and Porphyromonas sp. with low numbers of Neisseria and Lactobacillus sp. Significant ecological shifts were apparent following pulses of triclosan. The streptococci became the dominant group followed by Fusobacterium sp. For triclosan monophosphate, the streptococci again became dominant although Lactobacillus and Actinomyces were now the main sub-dominant species and Gram negative anaerobes including Fusobacterium sp. were markedly inhibited. It is concluded that in the periodontal disease state, both triclosan and triclosan monophosphate affected the Gram-negative anaerobes to a greater extent than the Gram-positive groups and that this effect was more marked for triclosan monophosphate. PMID- 10747821 TI - Activity of nitazoxanide alone and in combination with azithromycin and rifabutin against Cryptosporidium parvum in cell culture. AB - The in vitro activity of nitazoxanide alone and in combination with azithromycin and rifabutin was investigated against four clinical isolates of Cryptosporidium parvum. The susceptibility tests were performed by inoculation of the isolates on toe cell monolayers and determination of the parasite count after 48 h incubation at 37 degrees C. The culture medium was supplemented with Dulbecco's modified Eagle's medium containing serial dilutions of each agent. Antibiotic-free plates were used as controls. Experiments were performed in triplicate. Nitazoxanide showed moderate anticryptosporidial activity: it suppressed the growth of parasites by >50% at 8 mg/L. A parasite reduction of 79.8-83.9% was observed when nitazoxanide 8 mg/L was combined with azithromycin 8 mg/L and rifabutin 8 mg/L. The study suggests that nitazoxanide may be active in inhibiting C. parvum growth in vitro upon combination with azithromycin or rifabutin. PMID- 10747822 TI - Resistance surveillance of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis isolated in Asia and Europe, 1997-1998. AB - A multicentre, collaborative study was performed in Asia and Europe during the winter of 1997-1998 to determine the in vitro activity of selected antimicrobial agents against common respiratory pathogens. Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis isolates were collected from 48 sites in China, France, Germany, Italy, Japan, Spain and the UK and tested in a central laboratory in the USA. Broth microdilution MICs were determined for beta lactams (penicillin, amoxycillin/clavulanate, cefuroxime, ceftriaxone), macrolides (azithromycin, clarithromycin), sulphonamides (co-trimoxazole), glycopeptides (vancomycin) and fluoroquinolones (levofloxacin). The percentage of isolates susceptible to each antimicrobial class varied substantially by country. Penicillin susceptibility amongst pneumococci ranged from 34% in France and Spain to 92% in Germany, and macrolide susceptibility varied between 26% in China and 91% in the UK. In most countries beta-lactam, macrolide and cotrimoxazole resistance was more prevalent amongst penicillin-intermediate and -resistant S. pneumoniae isolates. However, little or no resistance was detected to levofloxacin (0.3% intermediate and resistant) or vancomycin (0% intermediate and resistant). For H. influenzae the prevalence of beta-lactamase production varied from 6% in China and Germany to 32% in Spain, and for M. catarrhalis, from 79% in Germany to 98% in Japan. With the exception of ampicillin, beta-lactamase production had a minimal effect on beta-lactam activity against H. influenzae or M. catarrhalis. Our findings demonstrate that antimicrobial resistance profiles of common respiratory isolates differ dramatically between countries in Asia and Europe. PMID- 10747823 TI - Molecular epidemiology of an outbreak due to IRT-2 beta-lactamase-producing strains of Klebsiella pneumoniae in a geriatric department. AB - In February 1998, 195 patients in the geriatric department of a French hospital were screened for the presence of co-amoxiclav-resistant Klebsiella pneumoniae. Eleven co-amoxiclav-resistant isolates obtained all produced an identical IRT-2 beta-lactamase. These K. pneumoniae isolates were clonally related and harboured a c. 55 kb non-conjugative plasmid encoding a non-class-1 integron-located blaIRT 2 gene. This study underlines that geriatric departments may be a reservoir for antibiotic-resistant strains and that IRT beta-lactamase-producing strains may be nosocomial pathogens. PMID- 10747824 TI - Resistance profile of Bacteroides fragilis isolated in Brazil. Do they shelter the cfiA gene? AB - The epidemiology of antimicrobial resistance of clinical isolates and human intestinal strains of Bacteroides fragilis has assumed great importance in the last few years since this microorganism, like other members of the B. fragilis group, can be responsible for the spread of resistance determinants. It is possible that the presence of B. fragilis in polluted aquatic environments might contribute to the spread of resistance. The antimicrobial resistance profile of 44 clinical B. fragilis strains isolated from 1981-1988 and 1991-1998 from the University hospital of Rio de Janeiro, and of 17 faecal and 17 polluted aquatic environmental B. fragilis strains isolated between 1991 and 1998 was determined. The susceptibility tests against penicillin, cefoxitin, imipenem, meropenem, clindamycin, chloramphenicol and metronidazole were performed by Etest in Wilkins Chalgren agar enriched with 5% sheep blood. Motivated by some high MIC values for cefoxitin and meropenem, the cfiA gene, which codes for a metallo-beta-lactamase, was investigated among all strains, using PCR amplification. The resistance to penicillin was high in the samples from 1981 to 1988 (92.9%) and also in those from 1991 to 1998 (100%), although the MIC90 decreased from 256 mg/L to 24 mg/L. An increase in the resistance level to clindamycin and cefoxitin was seen from one decade to the other, the MIC90 values changing from 4 mg/L to 12 mg/L and from 8 mg/L to 32 mg/L, respectively. The susceptibility profile for metronidazole, chloramphenicol, imipenem and meropenem remained stable, although two clinical strains showed MICs of 6 mg/L and 8 mg/L against meropenem. Almost all human intestinal strains were resistant to penicillin and all of them were susceptible to imipenem, meropenem, chloramphenicol and metronidazole. The MICs of meropenem against two strains isolated from a polluted aquatic environment were 6 mg/L and 32 mg/L. The cfiA gene was detected in five strains, two of which were isolated from clinical specimens against which the MIC values of cefoxitin were high and three from an aquatic environment, whose susceptibility to both cefoxitin and meropenem ranged from sensitive to resistant. PMID- 10747825 TI - Levofloxacin penetrates human monocytes and enhances intracellular killing of Staphylococcus aureus and Pseudomonas aeruginosa. AB - Intracellular bacteria often cause relapsing and refractory infections. However, these infections can be treated effectively with antibiotics such as ofloxacin which penetrate into the cells containing bacteria. As levofloxacin, the levorotatory isomer of ofloxacin, has enhanced antibacterial activity, we tested the levofloxacin concentration in human monocytes and the effects of intracellular levofloxacin on monocyte killing of Staphylococcus aureus strain ATCC 29213 and Pseudomonas aeruginosa strain PA1348A. Human monocytes were incubated with levofloxacin at various pH values and temperatures. Following incubation, the monocytes were separated from incubation media, and intracellular (C) and extracellular (E) levofloxacin concentrations were determined. Mean C/E ratios after 15 min of incubation with 6 and 12 mg/L levofloxacin at pH 7.4 were 6.4 and 7.1, respectively. C/E ratios were similar at pH 7.4 and 8.0, but decreased at lower pH values. To study the effects of levofloxacin on intracellular killing of S. aureus and P. aeruginosa, opsonized bacteria were added to monolayers of monocytes. Following phagocytosis, monocytes were incubated with various concentrations of levofloxacin, ciprofloxacin and rifampicin, alone or in combination. Levofloxacin (2.5 and 4 mg/L) significantly reduced the survival of cell-associated S. aureus and was more effective than ciprofloxacin at similar concentrations (P < 0.01). Enhanced killing of cell associated P. aeruginosa by levofloxacin (0.5 and 1.0 mg/L) was also observed. Activities of levofloxacin and ciprofloxacin against cell-associated P. aeruginosa were similar. Addition of rifampicin did not augment the bactericidal activity of levofloxacin. Since levofloxacin is concentrated in human monocytes and increases their bactericidal activity against intracellular bacteria, it should be considered for treatment of infections caused by susceptible intracellular bacteria. PMID- 10747826 TI - Comparative therapeutic efficacy of clinafloxacin in a pneumococcal meningitis mouse model. AB - The therapeutic efficacy of clinafloxacin, a fluoroquinolone in clinical trials, was compared with that of ciprofloxacin and ceftriaxone in a novel pneumococcal meningitis mouse model. Mice were challenged by the intracerebral ventricular route with 50 IL of a lethal bacterial suspension and treated subcutaneously 2 h later. Both penicillin-susceptible and multidrug-resistant pneumococcal strains were used for evaluation. Survival percentages were calculated as the median curative dose (CD50) using log-probit statistical methods. Ceftriaxone was the most active agent against the penicillin-susceptible strain (CD50 = 2 mg/kg), but showed a 30-fold decrease in potency against the resistant strain. Clinafloxacin was equally effective against both strains, and proved to be the most active agent against the penicillin-resistant pneumococcus. PMID- 10747827 TI - Imipenem, doxycycline and amikacin in monotherapy and in combination in Acinetobacter baumannii experimental pneumonia. AB - Acinetobacter baumannii is a common cause of nosocomial pneumonia and other nosocomial infections. Multiresistant A. baumannii has also a high prevalence, which can make effective treatment difficult. We designed a new model of A. baumannii experimental pneumonia using C57BL/6 immunocompetent mice. This model was used to compare the efficacy of imipenem, doxycycline and amikacin in monotherapy, and the combination of imipenem plus amikacin and doxycycline plus amikacin. Doxycycline plus amikacin were synergic in vitro after 24 h incubation, whereas imipenem plus amikacin showed no in vitro synergy. The number of sterile lungs and the lung clearance of A. baumannii were greater in the group treated with imipenem than in those treated with amikacin or doxycycline in monotherapy (P < 0.05). The combination of imipenem plus amikacin and doxycycline plus amikacin was no more effective than imipenem alone in the clearance of organisms from lungs (2.42 +/- 1.46 cfu/g versus 2.7 +/- 1.5 cfu/g versus 1.23 +/- 1.02 cfu/g). These results suggest that the addition of amikacin does not improve the results obtained by imipenem monotherapy. Doxycycline plus amikacin is an alternative to imipenem in the therapy of A. baumannii pneumonia. PMID- 10747828 TI - A comparison of the photosensitizing potential of trovafloxacin with that of other quinolones in healthy subjects. AB - Treatment with some quinolones is associated with an abnormal skin reaction following exposure to sunlight (photosensitivity). The objective of the current study was to compare the photosensitizing potential of a new quinolone, trovafloxacin, with that of ciprofloxacin, lomefloxacin and placebo. Forty-eight healthy males (age range 19-45 years) were randomized to receive a 7 day course of treatment with: (i) trovafloxacin 200 mg od; (ii) ciprofloxacin 500 mg bd; (iii) lomefloxacin 400 mg od; or (iv) placebo bd. Minimal erythema doses (MEDs) were assessed using a monochromator at baseline and on day 5 of treatment, for wavelengths of 305 +/- 5, 335 +/- 30, 365 +/- 30, 400 +/- 30 and 430 +/- 30 nm; 335 +/- 30 and 365 +/- 30 nm are within the UVA range. Immediate reaction MEDs were similar in all treatment groups. However, between baseline and day 5, the mean decreases in delayed-reaction MED (24 h) at 335 +/- 30 nm were only 18.99% for trovafloxacin versus placebo (P = 0.1267), compared with 53.77% (P 0.0001) and 64.13% (P 0.0001) for ciprofloxacin and lomefloxacin, respectively. Similarly, at 365 +/- 30 nm, trovafloxacin produced the smallest reduction in delayed MED versus placebo (43.66%), compared with ciprofloxacin (61.53%) and lomefloxacin (75.81%). These differences between trovafloxacin and ciprofloxacin and lomefloxacin were significant at both 335 +/- 30 and 365 +/- 30 nm (P 0.029). All MED values returned to baseline levels within 2 days of drug cessation. These results show that trovafloxacin has significantly less photosensitizing potential than either ciprofloxacin or lomefloxacin. This photosensitivity appears to be induced only by wavelengths in the UVA region, is maximal at 24 h and is a short term effect. PMID- 10747830 TI - Combinatorial biochemistry and shuffling of TEM, SHV and Streptomyces albus omega loops in PSE-4 class A beta-lactamase. AB - The class A PSE-4 beta-lactamase was used for studying the importance of amino acids in the omega (Omega) loop and its interactions for hydrolysis of beta lactam antibiotics. By cassette mutagenesis, we replaced the amino acids 163-179 Omega loop in PSE-4 with TEM-1, SHV-1 and Streptomyces albus G beta-lactamase Omega loops. Phenotypic analysis of Escherichia coli recombinants expressing the Omega loop PSE-4 mutant enzymes gave MICs and kinetic data similar to those of wild-type PSE-4. PMID- 10747829 TI - Pharmacokinetic assessment of oral ganciclovir in lung transplant recipients with cystic fibrosis. AB - Oral ganciclovir has been used as prophylaxis and therapy against cytomegalovirus in patients with HIV infection and following organ transplantation. Oral ganciclovir has clear practical advantages over intravenous ganciclovir but has a relatively low bioavailability and this may be problematic in at-risk patients with malabsorption. The bioavailability and therefore therapeutic potential of oral ganciclovir in cystic fibrosis (CF) patients post-lung transplant (LT) might be expected to be inadequate given the high incidence of malabsorption in these patients. An 8 h pharmacokinetic study was performed in 12 CF patients 160 +/- 122 days post-transplant who had been taking 1 g oral ganciclovir tds for 3 days with food (plus normal enzyme supplements). Mean (range) serum creatinine was 150 Imol/L (70-280). Blood was sampled at 0.5, 1, 2, 3, 4, 6 and 8 h post-final dose. Plasma was stored at -20 degrees C and later analysed by highperformance liquid chromatography. Mean peak concentration (C(max)) was 4.8 mg/L (0. 96-12.8), mean minimum concentration (C(min)) was 3.6 mg/L (0.78-11. 7) and mean area under the curve (AUC) was 35.4 mg.8 h/L (8-99). C(max), C(min) and AUC correlated significantly with one another (P < 0.001) as well as with serum creatinine and creatinine clearance (P < 0.01). When corrected for alterations in renal function, plasma oral ganciclovir levels are as predicted for other transplant populations. Three days of oral ganciclovir results in therapeutically useful plasma drug levels in the CF LT population, despite a background of general malabsorption. C(max), C(min) and AUC are highly correlated, allowing for the possibility of steady-state drug monitoring to confirm that the recommended dosing algorithm produces appropriate plasma levels. PMID- 10747832 TI - Activity of gemifloxacin, a new broad-spectrum quinolone, against 200 pneumococci by four different susceptibility testing methods. AB - Agar and microdilution (in air), Etest (in air and CO(2)) and disc diffusion (in air and CO(2)) susceptibility testing methods were used to investigate the activity of gemifloxacin against 200 pneumococci. MIC(50)s were 0.016-0.03 mg/L and MIC(90)s 0.125-0.25 mg/L for all methods. With agar dilution as reference, 187/200 strains gave essential agreement with microdilution and 196 with Etest (air and CO(2)). Disc zones were a few millimetres narrower in CO(2) than in air. With discs in CO(2), all ciprofloxacin-susceptible strains yielded zone diameters 26 mm; values in air were 28 mm. Zones for ciprofloxacin-resistant strains in CO(2) were mostly 21-26 mm; zones in air were a few millimetres wider, but mostly <31 mm. PMID- 10747831 TI - In vitro selected fluoroquinolone-resistant mutants of Citrobacter freundii: analysis of the quinolone resistance acquisition. AB - Ten quinolone-resistant mutants of Citrobacter freundii, which were selected in vitro with fluoroquinolones from two clinical isolates, were studied. The parent isolates were susceptible to quinolones in spite of showing a single substitution in the GyrB (His-417 --> Leu). No change was observed in the outer membrane proteins or in the lipopolysaccharide in any of the ten mutants studied with respect to their parent isolates. The development of quinolone resistance in selected mutants was associated with the appearance of a substitution in the GyrA (Thr-83 --> Ile) in nine of the ten mutants plus enhanced active efflux in all of them. PMID- 10747833 TI - Susceptibility testing of Haemophilus influenzae to clarithromycin. AB - Susceptibility testing of Haemophilus influenzae to the macrolide compounds has often been problematic. This is as a result of the inability of many isolates of H. influenzae to grow without the addition of 5% CO2 to the incubation atmosphere and the subsequent detrimental effect that CO2 has on the activity of the macrolide group of compounds. This report describes refinements and recommendations for susceptibility testing of H. influenzae to the macrolide clarithromycin. PMID- 10747834 TI - Endothelial cell compatibility of trovafloxacin and levofloxacin for intravenous use. AB - Levofloxacin and trovafloxacin have excellent activity against a variety of Gram positive and Gram-negative organisms resistant to the established agents. One local side-effect closely related to the use of parenteral fluoroquinolones is phlebitis. To evaluate the effect of trovafloxacin and levofloxacin on endothelial cell viability, intracellular levels of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), guanosine 5'-triphosphate (GTP) and guanosine 5'-diphosphate (GDP) levels were measured using high-performance liquid chromatography. Trovafloxacin at concentrations of 2 and 1 mg/mL reduced the intracellular ATP content from 12.5 +/- 1.7 to 1.9 +/- 0.3 nmol/10(6) cells and 9.3 +/- 0.8 nmol/10(6) cells, respectively, within 60 min. In addition, ADP, GTP and GDP levels were extensively depleted. Levofloxacin at concentrations of 5 and 2.5 mg/mL led to a significant ATP decline from 12.5 +/- 1.7 to 2.3 +/- 0.2 nmol/10(6) cells and 10.3 +/- 0.9 nmol/10(6) cells, respectively, within 60 min. These data indicate that infusions of high doses of trovafloxacin or levofloxacin are not compatible with maintenance of endothelial cell function. Commercial preparations have to be diluted and should be administered into large veins. PMID- 10747835 TI - Clinical relevance of Proteus mirabilis in hospital patients: a two year survey. AB - A retrospective study was performed on 1072 non-duplicate isolates of Proteus mirabilis, taken in the period April 1996 to March 1998, and on 100 patient charts randomly selected during the same period. P. mirabilis isolates accounted for 7.7% of Enterobacteriaceae. The isolates were predominantly from urine (70.2%); of the total, 38.0% were penicillinase-producing isolates, 6.9% were extended-spectrum beta-lactamase (ESBL)-producing isolates and 3.6% produced inhibitor-resistant beta-lactamase (IRB). ESBL-producing isolates were observed in long-stay and intensive care and IRB-producing isolates in paediatric units. Of the 95 patients whose charts were examined, 69 had a confirmed infection, which in 42 cases was nosocomial. PMID- 10747836 TI - Failure of oily chloramphenicol depot injection to treat plague in a murine model. AB - Effective low-cost single-dose therapy would be invaluable in treating human plague. The efficacy of single- or two-dose injections of oily chloramphenicol (OCm) was compared with that of standard multiple injections of reference drugs (streptomycin or chloramphenicol) in a murine plague model. A single injection of OCm was ineffective. Two doses cleared bacteraemia and limited bacterial growth in the mouse spleen but were less effective in reducing mortality than standard therapy. However, because of the marked pharmacokinetic differences between mice and humans, the failure of depot injection of OCm in murine plague treatment is not indicative of its ineffectiveness in human plague. PMID- 10747837 TI - Genetic basis of natural resistance to erythromycin in Mycoplasma hominis. PMID- 10747838 TI - In vitro susceptibility of Neisseria meningitidis. PMID- 10747839 TI - Selection of resistance to the essential oil of Melaleuca alternifolia in Staphylococcus aureus. PMID- 10747840 TI - Use of essential oil therapies in immunocompromised patients. PMID- 10747841 TI - The use of fluconazole and itraconazole in the treatment of candida albicans infections: a review PMID- 10747842 TI - Analysis of the mechanisms of quinolone resistance in clinical isolates of citrobacter freundii PMID- 10747844 TI - Integrin-mediated RON growth factor receptor phosphorylation requires tyrosine kinase activity of both the receptor and c-Src. AB - Cooperation between integrins and growth factor receptors plays an important role in the regulation of cell growth, differentiation, and survival. The function of growth factor receptor tyrosine kinases (RTKs) can be regulated by cell adhesion to extracellular matrix (ECM) even in the absence of ligand. We investigated the pathway involved in integrin-mediated RTK activation, using RON, the receptor for macrophage-stimulating protein. Adhesion of RON-expressing epithelial cells to ECM caused phosphorylation of RON, which depended on the kinase activity of both RON itself and c-Src. This conclusion is based on these observations: 1) ECM induced RON phosphorylation was inhibited in cells expressing kinase-inactive c Src; 2) active c-Src could phosphorylate immunoprecipitated RON from ECM stimulated cells but not from unstimulated cells; and 3) ECM did not cause RON phosphorylation in cells expressing kinase-dead RON, nor could active c-Src phosphorylate RON immunoprecipitated from these cells. The data fit a pathway in which ECM-induced integrin aggregation causes both c-Src activation and RON oligomerization followed by RON kinase-dependent autophosphorylation; this results in RON becoming a target for activated c-Src, which phosphorylates additional tyrosines on RON. Integrin-induced epidermal growth factor receptor (EGFR) phosphorylation also depended on both EGFR and c-Src kinase activities. This sequence appears to be a general pathway for integrin-dependent growth factor RTK activation. PMID- 10747846 TI - Targeted inactivation of the isoprenylcysteine carboxyl methyltransferase gene causes mislocalization of K-Ras in mammalian cells. AB - After isoprenylation and endoproteolytic processing, the Ras proteins are methylated at the carboxyl-terminal isoprenylcysteine. The importance of isoprenylation for targeting of Ras proteins to the plasma membrane is well established, but the importance of carboxyl methylation, which is carried out by isoprenylcysteine carboxyl methyltransferase (Icmt), is less certain. We used gene targeting to produce homozygous Icmt knockout embryonic stem cells (Icmt-/ ). Lysates from Icmt-/- cells lacked the ability to methylate farnesyl-K-Ras4B or small-molecule Icmt substrates such as N-acetyl-S-geranylgeranyl-L-cysteine. To assess the impact of absent Icmt activity on the localization of K-Ras within cells, wild-type and Icmt-/- cells were transfected with a green fluorescent protein (GFP)-K-Ras fusion construct. As expected, virtually all of the GFP-K-Ras fusion in wild-type cells was localized along the plasma membrane. In contrast, a large fraction of the fusion in Icmt-/- cells was trapped within the cytoplasm, and fluorescence at the plasma membrane was reduced. Also, cell fractionation/Western blot studies revealed that a smaller fraction of the K-Ras in Icmt-/- cells was associated with the membranes. We conclude that carboxyl methylation of the isoprenylcysteine is important for proper K-Ras localization in mammalian cells. PMID- 10747847 TI - Identification and characterization of a new family of guanine nucleotide exchange factors for the ras-related GTPase Ral. AB - Guanine nucleotide exchange factors (GEFs) are responsible for coupling cell surface receptors to Ras protein activation. Here we describe the characterization of a novel family of differentially expressed GEFs, identified by database sequence homology searching. These molecules share the core catalytic domain of other Ras family GEFs but lack the catalytic non-conserved (conserved non-catalytic/Ras exchange motif/structurally conserved region 0) domain that is believed to contribute to Sos1 integrity. In vitro binding and in vivo nucleotide exchange assays indicate that these GEFs specifically catalyze the GTP loading of the Ral GTPase when overexpressed in 293T cells. A central proline-rich motif associated with the Src homology (SH)2/SH3-containing adapter proteins Grb2 and Nck in vivo, whereas a pleckstrin homology (PH) domain was located at the GEF C terminus. We refer to these GEFs as RalGPS 1A, 1B, and 2 (Ral GEFs with PH domain and SH3 binding motif). The PH domain was required for in vivo GEF activity and could be functionally replaced by the Ki-Ras C terminus, suggesting a role in membrane targeting. In the absence of the PH domain RalGPS 1B cooperated with Grb2 to promote Ral activation, indicating that SH3 domain interaction also contributes to RalGPS regulation. In contrast to the Ral guanine nucleotide dissociation stimulator family of Ral GEFs, the RalGPS proteins do not possess a Ras-GTP-binding domain, suggesting that they are activated in a Ras-independent manner. PMID- 10747848 TI - Purification and characterization of a human factor that assembles and remodels chromatin. AB - We have previously reported the isolation and characterization of a nucleosome remodeling and spacing factor, RSF. One of the RSF subunits is hSNF2h, a SNF2 homologue. Here we set out to isolate and characterize other hSNF2h-containing complexes. We have identified a novel hSNF2h complex that facilitates ATP dependent chromatin assembly with the histone chaperone NAP-1. The complex possesses ATPase activity that is DNA-dependent and nucleosome-stimulated. This complex is capable of facilitating ATP-dependent nucleosome remodeling and transcription initiation from chromatin templates. In addition to hSNF2h, this complex also contains a 190-kDa protein encoded by the BAZ1A gene. Since both subunits are homologues of the Drosophila ACF complex (ATP-utilizing chromatin assembly and remodeling factor), we have named this factor human ACF or hACF. PMID- 10747849 TI - Physical and functional interaction of rabphilin-11 with mammalian Sec13 protein. Implication in vesicle trafficking. AB - Rab11a small G protein (Rab11p) is implicated in vesicle trafficking, especially vesicle recycling. We have previously isolated a downstream effector of Rab11p, named rabphilin-11. We found here that rabphilin-11 directly bound the mammalian counterpart of yeast Sec13 protein (mSec13p) in cell-free and intact cell systems. Yeast Sec13p is involved as a component of coat proteins II in the Sar1p induced vesicle formation from the endoplasmic reticulum, but the precise role of mSec13p is unknown. The interaction of rabphilin-11 with mSec13p was enhanced by GTP-Rab11p. Rabphilin-11 localized on the vesicles in perinuclear regions and along microtubules oriented toward the plasma membrane, whereas mSec13p partly colocalized with rabphilin-11 in the perinuclear regions, most presumably the Golgi complex. Disruption of the rabphilin-11-mSec13p interaction by overexpression of the mSec13p-binding region of rabphilin-11 impaired vesicle trafficking. These results indicate that the rabphilin-11-mSec13p interaction is implicated in vesicle trafficking. PMID- 10747850 TI - L-929 cells harboring ectopically expressed RelA resist curcumin-induced apoptosis. AB - Curcumin (diferuloyl methane), the yellow pigment in turmeric (Curcuma longa), is a potent chemopreventive agent. Curcumin induces apoptosis of several, but not all, cancer cells. Many cancer cells protect themselves against apoptosis by activating nuclear factor-kappaB (NF-kappaB)/Rel, a transcription factor that helps in cell survival. Signal-induced activation of NF-kappaB is known to be inhibited by curcumin. To understand the role of NF-kappaB in curcumin-induced apoptosis, we stably transfected relA gene encoding the p65/RelA subunit of NF kappaB, into l-929 cells (mouse fibrosarcoma) and the relA-transfected cells were resistant to varying doses of curcumin (10(-6)-10(-4) m), whereas the parental cells underwent apoptosis in a time- and dose-dependent manner. The relA transfected cells showed constitutive NF-kappaB DNA binding activity that could not be inhibited by curcumin and did not show nuclear condensation and DNA fragmentation upon treatment with curcumin. When a super-repressor form of IkappaB-alpha (known to inhibit NF-kappaB) was transfected transiently into relA transfected cells, the cells were no longer resistant to curcumin. Our results highlight a critical anti-apoptotic role for NF-kappaB in curcumin-induced apoptosis. PMID- 10747851 TI - Suppressor of cytokine signaling-1 inhibits VAV function through protein degradation. AB - Suppressor of cytokine signaling-1 (SOCS1) is an inducible Src homology 2 (SH2) containing protein that negatively regulates cytokine and growth factor signaling required during thymic development. Recent evidence indicates that SOCS1 interacts with elongins B and C, which are components of a ubiquitin ligase complex, VCB (VHL/elonginC/B), based on the VHL (von Hippel Lindau) tumor suppressor protein. SOCS1 has previously been shown to operate as an inhibitor of Janus kinases. Here we show that SOCS1 has the distinct function of targeting the hematopoietic specific guanine nucleotide exchange factor, VAV, for ubiquitin mediated protein degradation. VAV and SOCS1 form a protein complex through interactions between the VAV NH(2)-terminal regulatory region and the SH2 domain of SOCS1 in a phosphotyrosine-independent manner. SOCS1 decreases the steady state levels of cotransfected VAV and onco-VAV and reduces the focus forming activity of onco-VAV. SOCS1 stimulates the polyubiquitination of VAV proteins in vivo, which was stabilized by proteasomal inhibitors. These results suggest that SOCS1 programs VAV degradation by acting as a substrate-specific recognition component of a VCB-like ubiquitin ligase complex. PMID- 10747852 TI - The L18 domain of light-harvesting chlorophyll proteins binds to chloroplast signal recognition particle 43. AB - Chloroplast signal recognition particle (cpSRP) is a novel type of SRP that contains a homolog of SRP54 and a 43-kDa subunit absent from all cytoplasmic SRPs but lacks RNA. It is also distinctive in its ability to post-translationally interact with light-harvesting chlorophyll proteins (LHCP), hydrophobic proteins synthesized in the cytoplasm and targeted to the thylakoid via the stroma. LHCP integration into thylakoid membranes requires the two subunits of cpSRP, cpFtsY, GTP, and the membrane protein ALB3. It had previously been shown that the L18 domain, an 18-amino acid peptide between the second and third transmembrane domains, and a hydrophobic domain are required for interaction with cpSRP. In the present study we used a pull-down assay, with cpSRP43 or cpSRP54 fused to glutathione-transferase, to study interactions between cpSRP43, cpSRP54, LHCP, and cpFtsY. cpFtsY was not observed to form significant interactions with any of the proteins even in the presence of nonhydrolyzable GTP analogs. Our data indicate that cpSRP43 binds to the L18 domain, that cpSRP54 binds to the hydrophobic domain, and that LHCP and cpSRP54 independently bind to cpSRP43. These data confirm that the novel post-translational interaction between LHCP and cpSRP is mediated through binding to cpSRP43. PMID- 10747853 TI - Induction of melanocyte-specific microphthalmia-associated transcription factor by Wnt-3a. AB - Microphthalmia-associated transcription factor (Mitf) plays a critical role in the development of neural crest-derived melanocytes. Here, we show that exogenously added Wnt-3a protein, an intercellular signaling molecule, up regulates the expression of endogenous melanocyte-specific Mitf (Mitf-M) mRNA in cultured melanocytes. The melanocyte-specific promoter of the human MITF gene (MITF-M promoter) contains a functional LEF-1-binding site, which is bound in vitro by LEF-1 and confers the preferential expression on a reporter gene in melanocytes and melanoma cells, as judged by the transient transfection assays. Moreover, the LEF-1-binding site is required for the transactivation of a reporter gene by LEF-1, beta-catenin, or their combination. Exogenously added Wnt 3a protein also transactivates the MITF-M promoter via the LEF-1-binding site; this activation was abolished when a dominant-negative form of LEF-1 was coexpressed. These results suggest that Wnt-3a signaling recruits beta-catenin and LEF-1 to the LEF-1-binding site of the MITF-M promoter. Therefore, the present study identifies Mitf-M/MITF-M as a direct target of Wnt signaling. PMID- 10747854 TI - Deletion of PBP/PPARBP, the gene for nuclear receptor coactivator peroxisome proliferator-activated receptor-binding protein, results in embryonic lethality. AB - We previously isolated and identified peroxisome proliferator-activated receptor (PPAR)-binding Protein (PBP) as a coactivator for PPARgamma. PBP has recently been identified as a component of the multiprotein complexes such as TRAP, DRIP, and ARC that appear to play an important role in the transcriptional activation by several transcriptional factors including nuclear receptors. To assess the biological significance of PBP, we disrupted the PBP gene (PBP/PPARBP) in mice by homologous recombination. PBP(+/-) mice are healthy, fertile, and do not differ significantly from PBP(+/+) control littermates. PBP null mutation (PBP(-/-)) is embryonically lethal at embryonic day 11.5, suggesting that PBP is an essential gene for mouse embryogenesis. The embryonic lethality is attributed, in part, to defects in the development of placental vasculature similar to those encountered in PPARgamma mutants. Transient transfection assays using fibroblasts isolated from PBP mutant embryos revealed a decreased capacity for ligand-dependent transcriptional activation of PPARgamma as compared with fibroblasts derived form the wild type embryos. These observations suggest that there is no functional redundancy between PBP and other coactivators such as steroid receptor coactivator-1 and that PBP plays a critical role in the signaling of PPARgamma and other nuclear receptors. PMID- 10747855 TI - RpoS-dependent promoters require guanosine tetraphosphate for induction even in the presence of high levels of sigma(s). AB - RpoS-dependent promoters require ppGpp for induction in the stationary phase. This has been thought to be a simple consequence of final sigma(S) itself requiring ppGpp for its production. By using four model promoters requiring final sigma(S) for normal induction in the stationary phase, we demonstrate that final sigma(S)-dependent promoters require ppGpp even in the presence of high levels of final sigma(S) produced ectopically. Similar to final sigma(70)-dependent promoters under positive control by ppGpp, the requirement of final sigma(S) dependent promoters for this alarmone is bypassed by specific "stringent" mutations in the beta-subunit of RNA polymerase. The results suggest that stationary phase induction of both final sigma(S)- and final sigma(70)-dependent genes requires the stringent control modulon and that stringency confers dual control on the RpoS regulon by affecting promoter activity and the levels of the required final sigma-factor. PMID- 10747856 TI - A gain-of-function mutation in STAT6. AB - Interleukin-4 (IL-4) is a cytokine that plays a crucial role in the pathophysiology of asthma and allergic diseases. IL-4-induced gene expression is largely mediated through the activation of the latent transcription factor STAT6. We identified a STAT6 mutant (STAT6VT)) that is activated independently of IL-4 stimulation. STAT6VT carries two amino acid changes in the SH2 domain that affect the overall structure and stability of the monomeric and dimeric protein. When overexpressed in mammalian cells, STAT6VT undergoes tyrosine phosphorylation, binds DNA, and activates transcription in the absence of IL-4 stimulation. Using the Jak1- and Jak3-deficient fibroblast line U4A, we demonstrate that phosphorylation is mediated by an IL-4-independent tyrosine kinase that is not able to activate the wild-type STAT6 protein. These results suggest that small changes in STAT6 could result in hyperactivation of the protein and constitutive expression of STAT6-dependent genes. Such a mutation, if found in vivo, could cause genetic predisposition for atopic diseases. PMID- 10747858 TI - A lecithin cholesterol acyltransferase-like gene mediates diacylglycerol esterification in yeast. AB - The terminal step in triglyceride biosynthesis is the esterification of diacylglycerol. To study this reaction in the model eukaryote, Saccharomyces cerevisiae, we investigated five candidate genes with sequence conservation to mammalian acyltransferases. Four of these genes are similar to the recently identified acyl-CoA diacylglycerol acyltransferase and, when deleted, resulted in little or no decrease in triglyceride synthesis as measured by incorporation of radiolabeled oleate or glycerol. By contrast, deletion of LRO1, a homolog of human lecithin cholesterol acyltransferase, resulted in a dramatic reduction in triglyceride synthesis, whereas overexpression of LRO1 yielded a significant increase in triglyceride production. In vitro microsomal assays determined that Lro1 mediated the esterification of diacylglycerol using phosphatidylcholine as the acyl donor. The residual triglyceride biosynthesis that persists in the LRO1 deletion strain is mainly acyl-CoA-dependent and mediated by a gene that is structurally distinct from the previously identified mammalian diacylglycerol acyltransferase. These mechanisms may also exist in mammalian cells. PMID- 10747859 TI - Cell vacuolation induced by the VacA cytotoxin of Helicobacter pylori is regulated by the Rac1 GTPase. AB - Chronic gastric infection with the Gram-negative bacterium Helicobacter pylori is a major contributing factor in the development of duodenal ulcers and is believed to be a significant risk factor in the development of gastric tumors. The VacA cytotoxin of H. pylori is a 90-kDa secreted protein that forms trans-membrane ion channels. In epithelial cells, VacA activity is associated with the rapid formation of acidic vacuoles enriched for late endosomal and lysosomal markers. Rac1 is a member of the Rho family of small GTP-binding proteins that regulate reorganization of the actin cytoskeleton and intracellular signal transduction and are being shown increasingly to play a role in membrane trafficking events. In this study we report that: (i) green fluorescent-tagged Rac1 localizes around the perimeter of the vacuoles induced by VacA; (ii) expression of dominant negative Rac1 in epithelial cells inhibits vacuole formation; (iii) expression of constitutively active Rac1 potentiates the activity of VacA. Taken together, these data demonstrate a role for Rac1 in the regulation of VacA activity. PMID- 10747860 TI - Cloning of an amino acid transporter with functional characteristics and tissue expression pattern identical to that of system A. AB - We report here on the cloning and functional characterization of the protein responsible for the system A amino acid transport activity that is known to be expressed in most mammalian tissues. This transporter, designated ATA2 for amino acid transporter A2, was cloned from rat skeletal muscle. It is distinct from the neuron-specific glutamine transporter (GlnT/ATA1). Rat ATA2 consists of 504 amino acids and bears significant homology to GlnT/ATA1 and system N (SN1). ATA2 specific mRNA is ubiquitously expressed in rat tissues. When expressed in mammalian cells, ATA2 mediates Na(+)-dependent transport of alpha (methylamino)isobutyric acid, a specific model substrate for system A. The transporter is specific for neutral amino acids. It is pH-sensitive and Li(+) intolerant. The Na(+):amino acid stoichiometry is 1:1. When expressed in Xenopus laevis oocytes, transport of neutral amino acids via ATA2 is associated with inward currents. The substrate-induced current is Na(+)-dependent and pH sensitive. The amino acid transport system A is particularly known for its adaptive and hormonal regulation, and therefore the successful cloning of the protein responsible for this transport activity represents a significant step toward understanding the function and expression of this transporter in various physiological and pathological states. PMID- 10747861 TI - Peripherin/rds influences membrane vesicle morphology. Implications for retinopathies. AB - Peripherin/rds is an integral membrane glycoprotein found in the rim regions of vertebrate photoreceptor cell discs. Natural mutations of the encoding gene result in degenerative retinal disorders, such as retinitis pigmentosa. The retinal degeneration slow (rds) phenotype, observed in mice, is considered to be an appropriate model for peripherin/rds-mediated retinitis pigmentosa. Associated abnormalities in the outer segment of photoreceptor cells have implicated peripherin/rds in some aspect of disc morphology, yet it remains unclear whether such morphological effects are the cause or the result of this condition. Here we present the first direct evidence to support a role for peripherin/rds in maintaining the flattened vesicle morphology characteristic of photoreceptor outer segments. In vitro expression yields a 36-kDa immunoreactive species, which is inserted into membranes and undergoes N-glycosylation, inter- and intramolecular disulfide bonding, and dimerization. Electron microscopy reveals that peripherin/rds flattens microsomal vesicles. This effect appears to be dependent on disulfide bond formation but not N-glycosylation. The inability of two pathogenic peripherin/rds mutants (P216L and C165Y) to flatten membrane vesicles implicates such mutations as the primary cause of the retinal degeneration observed in retinitis pigmentosa. PMID- 10747862 TI - Involvement of calmodulin in 1alpha,25-dihydroxyvitamin D3 stimulation of store operated Ca2+ influx in skeletal muscle cells. AB - The steroid hormone 1alpha,25-dihydroxyvitamin D(3) (1, 25-(OH)(2)D(3)) rapidly modulates Ca(2+) homeostasis in avian skeletal muscle cells by driving a complex signal transduction mechanism, which promotes Ca(2+) release from inner stores and cation influx from the outside through both L-type and store-operated Ca(2+) (SOC) channels. In the present work, we evaluated the involvement of calmodulin (CAM) in 1,25-(OH)(2)D(3) regulation of SOC influx in chick skeletal muscle cells. Treatment with 10(-9) m 1,25-(OH)(2)D(3) in Ca(2+)-free medium resulted in a rapid but transient Ca(2+) rise correlated with the sterol-induced inositol 1,4,5-trisphosphate (IP(3)) production. The SOC influx stimulated by the hormone was insensitive to both CAM antagonists (fluphenazine, trifluoperazine, chlorpromazine, compound 48/80) and the CAM-dependent protein kinase II (CAMKII) inhibitor KN-62 when added after the sterol-dependent Ca(2+) transient, but it was completely abolished when added prior to the IP(3)-induced mobilization of Ca(2+) from endogenous stores. Moreover, in cells microinjected with antisense oligonucleotides directed against the CAM mRNA the sterol-stimulated SOC influx was reduced up to 60% respect to uninjected cells. The present results suggest that the 1, 25-(OH)(2)D(3)-induced (IP(3)-mediated) cytosolic Ca(2+) transient is required for CAM, activation which in turn activates SOC influx in a mechanism that seems to include CAMKII. PMID- 10747863 TI - Type I phosphatidylinositol 4-phosphate 5-kinase directly interacts with ADP ribosylation factor 1 and is responsible for phosphatidylinositol 4,5 bisphosphate synthesis in the golgi compartment. AB - Phosphatidylinositol (PtdIns) 4,5-bisphosphate is involved in many aspects of membrane traffic, but the regulation of its synthesis is only partially understood. Golgi membranes contain PI 4-kinase activity and a pool of phosphatidylinositol phosphate (PIP), which is further increased by ADP ribosylation factor 1 (ARF1). COS7 cells were transfected with alpha and beta forms of PI 4-kinase, and only membranes from COS7 cells transfected with PI 4 kinase beta increased their content of PIP when incubated with ARF1. PtdIns(4, 5)P(2) content in Golgi membranes was nonexistent but could be increased to a small extent upon adding either cytosol or Type I or Type II PIP kinases. However, when ARF1 was present, PtdIns(4,5)P(2) levels increased dramatically when membranes were incubated in the presence of cytosol or Type I, but not Type II, PIP kinase. To examine whether ARF1 could directly activate Type I PIP 5 kinase, we used an in vitro assay consisting of phosphatidycholine-containing liposomes, ARF1, and PIP 5-kinase. ARF1 increased Type I PIP 5-kinase activity in a guanine nucleotide-dependent manner, identifying this enzyme as a direct effector for ARF1. PMID- 10747864 TI - Atomic force microscopy reveals two conformations of the 20 S proteasome from fission yeast. AB - The proteasome is a major cytosolic proteolytic complex, indispensable in eukaryotic cells. The barrel-shaped core of this enzyme, the 20 S proteasome, is built from 28 subunits forming four stacked rings. The two inner beta-rings harbor active centers, whereas the two outer alpha-rings play a structural role. Crystal structure of the yeast 20 S particle showed that the entrance to the central channel was sealed. Because of this result, the path of substrates into the catalytic chamber has remained enigmatic. We have used tapping mode atomic force microscopy (AFM) in liquid to address the dynamic aspects of the 20 S proteasomes from fission yeast. We present here evidence that, when observed with AFM, the proteasome particles in top view position have either open or closed entrance to the central channel. The preferred conformation depends on the ligands present. Apparently, the addition of a substrate to the uninhibited proteasome shifts the equilibrium toward the open conformation. These results shed new light on the possible path of the substrate into the proteolytic chamber. PMID- 10747865 TI - Insertional mutation of the murine kisimo locus caused a defect in spermatogenesis. AB - Spermatogenesis is a developmental process that occurs in several phases and is regulated by a large number of gene products. An insertional transgenic mouse mutant (termed kisimo mouse) has been isolated that results in abnormal germ-cell development, showing abnormal elongated spermatids in the lumina of seminiferous tubules. We cloned the disrupted locus of kisimo and identified a novel testis specific gene, THEG, which is specifically expressed in spermatids and was disrupted in the transgenic mouse. The yeast two-hybrid screening method revealed that THEG protein strongly interacts with chaperonin containing t-complex polypeptide-1epsilon, suggesting that THEG protein functions as a regulatory factor in protein assembly. Our findings indicate that the kisimo locus is essential for the maintenance of spermiogenesis and that a gene expression disorder may be involved in male infertility. PMID- 10747866 TI - TASK-3, a novel tandem pore domain acid-sensitive K+ channel. An extracellular histiding as pH sensor. AB - Tandem pore domain acid-sensitive K(+) channel 3 (TASK-3) is a new member of the tandem pore domain potassium channel family. A cDNA encoding a 365- amino acid polypeptide with four putative transmembrane segments and two pore regions was isolated from guinea pig brain. An orthologous sequence was cloned from a human genomic library. Although TASK-3 is 62% identical to TASK-1, the cytosolic C terminal sequence is only weakly conserved. Analysis of the gene structure identified an intron within the conserved GYG motif of the first pore region. Reverse transcriptase-polymerase chain reaction analysis showed strong expression in brain but very weak mRNA levels in other tissues. Cell-attached patch-clamp recordings of TASK-3 expressed in HEK293 cells showed that the single channel current-voltage relation was inwardly rectifying, and open probability increased markedly with depolarization. Removal of external divalent cations increased the mean single channel current measured at -100 mV from -2.3 to -5.8 pA. Expression of TASK-3 in Xenopus oocytes revealed an outwardly rectifying K(+) current that was strongly decreased in the presence of lower extracellular pH. Substitution of the histidine residue His-98 by asparagine or tyrosine abolished pH sensitivity. This histidine, which is located at the outer part of the pore adjacent to the selectivity filter, may be an essential component of the extracellular pH sensor. PMID- 10747867 TI - p300 mediates functional synergism between AF-1 and AF-2 of estrogen receptor alpha and beta by interacting directly with the N-terminal A/B domains. AB - Estrogen receptor (ER) alpha and beta mediate estrogen actions in target cells through transcriptional control of target gene expression. For 17beta-estradiol induced transactivation, the N-terminal A/B domain (AF-1) and the C-terminal E/F domain (AF-2) of ERs are required. Ligand binding is considered to induce functional synergism between AF-1 and AF-2, but the molecular mechanism remains unknown. To clarify this synergism, we studied the role of reported AF-2 coactivators, p300/CREB binding protein, steroid receptor coactivator 1/transcriptional intermediary factor-2 (SRC-1/TIF2) family proteins and thyroid hormone receptor-associated protein-220/(vitamin D3 receptor-interacting protein- 205-(TRAP220/DRIP205) on the AF-1 activity in terms of synergism with the AF-2 function. We found that neither any of the SRC-1/TIF2 family coactivators nor TRAP220/DRIP205 is potent, whereas p300 potentiates the AF-1 function of both human ERalpha and human ERbeta. Direct interactions of p300 with the A/B domains of ERalpha and ERbeta were observed in an in vitro glutathione S-transferase pull down assay in accordance with the interactions in yeast and mammalian two-hybrid assays. Furthermore, mutations in the p300 binding sites (56-72 amino acids in ERalpha and 62-72 amino acids in ERbeta) in the A/B domains caused a reduction in ligand-induced transactivation functions of both ERalpha and ERbeta. Thus, these findings indicate that ligand-induced functional synergism between AF-1 and AF-2 is mediated through p300 by its direct binding to the A/B regions of ERalpha and ERbeta. PMID- 10747868 TI - Polyglutamylation of nucleosome assembly proteins. AB - Polyglutamylation is an original posttranslational modification, discovered on tubulin, consisting in side chains composed of several glutamyl units and leading to a very unusual protein structure. A monoclonal antibody directed against glutamylated tubulin (GT335) was found to react with other proteins present in HeLa cells. After immunopurification on a GT335 affinity column, two prominent proteins of approximately 50 kDa were observed. They were identified by microsequencing and mass spectrometry as NAP-1 and NAP-2, two members of the nucleosome assembly protein family that are implicated in the deposition of core histone complexes onto chromatin. Strikingly, NAP-1 and NAP-2 were found to be substrates of an ATP-dependent glutamylation enzyme co-purifying on the same column. We took advantage of this property to specifically label and purify the polyglutamylated peptides. NAP-1 and NAP-2 are modified in their C-terminal domain by the addition of up to 9 and 10 glutamyl units, respectively. Two putative glutamylation sites were localized for NAP-1 at Glu-356 and Glu-357 and, for NAP-2, at Glu-347 and Glu-348. These results demonstrate for the first time that proteins other than tubulin are polyglutamylated and open new perspectives for studying NAP function. PMID- 10747869 TI - Analysis of transcription of the Col6a1 gene in a specific set of tissues suggests a new variant of enhancer region. AB - The region extending from -5.4 to -3.9 kilobase pairs from the transcription start site of the Col6a1 gene has been previously shown to contain sequences activating tissue-specific transcription in articular cartilage, intervertebral disks, subepidermal, and vibrissae mesenchyme and peripheral nervous system (Braghetta, P., Fabbro, C., Piccolo, S., Marvulli, D., Bonaldo, P., Volpin, D., and Bressan, G. M. (1996) J. Cell Biol. 135, 1163-1177). Analysis of expression of deletions of this region in transgenic mice has identified the 383-base pair fragment E-L as the most active sequence of the region. Linker-scanning mutagenesis analysis of segment E-J, which spans the 5' 245 base pairs of E-L and is sufficient for high frequency expression in articular cartilage, showed that all the mutations reduced transcription considerably, suggesting that the integrity of the entire cluster of elements is necessary for enhancer activity. Electrophoretic mobility shift assays with nuclear extracts derived from various sources showed that fragment E-J binds numerous transcription factors (at least 22). These factors are present in most cells, expressing and nonexpressing alpha1(VI) collagen mRNA, but in different relative proportions, and none of them appears to be cell type-specific. Several lines of evidence indicate that sequence elements of the enhancer may have different functional roles in various cells. The data configure the -5.4/-3.9 region of the Col6a1 gene as a new type of tissue-specific enhancer, characterized by a variety of tissues supporting its activation and by the dependence of its function only on ubiquitous transcription factors. This type of enhancer is postulated to be particularly important for genes such as those of the extracellular matrix, which are often expressed with broad tissue specificity. PMID- 10747870 TI - Activation of phospholipase D1 by Cdc42 requires the Rho insert region. AB - Members of the Rho subfamily of GTP-binding proteins are implicated in the regulation of phospholipase D (PLD). In the present study, we demonstrate a physical association between a Rho family member, Cdc42, and PLD1. Binding of Cdc42 to PLD1 and subsequent activation are GTP-dependent. Although binding of Cdc42 to PLD1 does not require geranylgeranylation, activation of PLD1 is dependent on this lipid modification of Cdc42. Specific point mutations in the switch I region of Cdc42 abolish binding to and, therefore, activation of PLD1 by Cdc42. Deletion of the Rho insert region, which consists of residues 120-139, from Cdc42 does not interfere with binding to PLD1 but inhibits Cdc42 stimulated PLD1 activity. Interestingly, deletion of the insert region from Cdc42 also inhibits activation of PLD1 by Arf and protein kinase C. With the lack of specific inhibitors of PLD activity, the insert deletion mutant of Cdc42 (designated (DeltaL8)Cdc42) is a novel reagent for in vitro studies of PLD1 regulation, as well as for in vivo studies of Cdc42-mediated signaling pathways leading to PLD1 activation. Because the insert region is required for the transforming activity of Cdc42, regulation of PLD1 by this region on Cdc42 is of major interest. PMID- 10747872 TI - Mechanism of STAT3 activation by insulin-like growth factor I receptor. AB - Recent evidence indicates that STAT proteins can be activated by a variety of receptor and non-receptor protein-tyrosine kinases. Unlike cytokine-induced activation of STATs, where JAKs are known to play a pivotal role in phosphorylating STATs, the mechanism for receptor protein-tyrosine kinase mediated activation of STATs remains elusive. In this study, we investigated the activation of STAT proteins by the insulin-like growth factor I receptor (IGF-IR) in vitro and in vivo and assessed the role of JAKs in the process of activation. We found that STAT3, but not STAT5, was activated in response to IGF-I in 293T cells cotransfected with IGF-IR and STAT expression vectors. Moreover, tyrosine phosphorylation of STAT3, JAK1, and JAK2 was increased upon IGF-I stimulation of endogenous IGF-IR in 293T cells transfected with the respective STAT or JAK expression vector. Supporting the observation in 293T cells, endogenous STAT3 was tyrosine-phosphorylated upon IGF-I stimulation in the muscle cell line C2C12 as well as in various embryonic and adult mouse organs during different stages of development. Dominant-negative JAK1 or JAK2 was able to block the IGF-IR-mediated tyrosine phosphorylation of STAT3 in 293T cells. A newly identified family of proteins called SOCS (suppressor of cytokine signaling), including SOCS1, SOCS2, SOCS3 and CIS, was able to inhibit the IGF-I-induced STAT3 activation as well with varying degrees of potency, in which SOCS1 and SOCS3 appeared to have the higher inhibitory ability. Inhibition of STAT3 activation by SOCS could be overcome by overexpression of native JAK1 and JAK2. We conclude that IGF-I/IGF-IR is able to mediate activation of STAT3 in vitro and in vivo and that JAKs are essential for the process of activation. PMID- 10747873 TI - The hydrophilic N-terminal domain complements the membrane-anchored C-terminal domain of the sensor kinase KdpD of Escherichia coli. AB - The putative turgor sensor KdpD is characterized by a large, N-terminal domain of about 400 amino acids, which is not found in any other known sensor kinase. Comparison of 12 KdpD sequences from various microorganisms reveals that this part of the kinase is highly conserved and includes two motifs (Walker A and Walker B) that are very similar to the classical ATP-binding sites of ATP requiring enzymes. By means of photoaffinity labeling with 8-azido-[alpha (32)P]ATP, direct evidence was obtained for the existence of an ATP-binding site located in the N-terminal domain of KdpD. The N-terminal domain, KdpD/1-395, was overproduced and purified. Although predicted to be hydrophilic, it was found to be membrane-associated and could be solubilized either by treatment with buffer of low ionic strength or detergent. The membrane-associated form, but not the solubilized one, retained the ability to bind 8-azido-[alpha-(32)P]ATP. Previously, it was shown that the phosphatase activity of a truncated KdpD, KdpD/Delta12-395, is deregulated in vitro (Jung, K., and Altendorf, K. (1998) J. Biol. Chem. 273, 17406-17410). Here, we demonstrated that this effect was reversed in vesicles containing both the truncated KdpD and the N-terminal domain. Furthermore, coexpression of kdpD/Delta12-395 and kdpD/1-395 restored signal transduction in vivo. These results highlight the importance of the N terminal domain for the function of KdpD and provide evidence for an interaction of this domain and the transmitter domain of the sensor kinase. PMID- 10747874 TI - The SCAN domain mediates selective oligomerization. AB - The SCAN domain is described as a highly conserved, leucine-rich motif of approximately 60 amino acids found at the amino-terminal end of zinc finger transcription factors. Although no specific biological function has been attributed to the SCAN domain, its predicted amphipathic secondary structure led to the suggestion that this domain may mediate protein-protein associations. A yeast two-hybrid screen identified members of two SCAN domain protein families that interact with the SCAN domain of the zinc finger protein ZNF202. The interacting ZNF191 protein represents the family of SCAN domain-containing zinc finger proteins, whereas the novel SDP1 protein establishes a new family of genes that encode an isolated SCAN domain. Isolated SCAN domain proteins may form asymmetric homodimers in solution. Biochemical binding studies confirmed the associations of ZNF191 and SDP1 with ZNF202 and established the SCAN domain as a selective hetero- and homotypic oligomerization domain. SCAN mediated protein associations might therefore represent a new regulatory mechanism of transcriptional activity. PMID- 10747875 TI - A zebrafish ftz-F1 (Fushi tarazu factor 1) homologue requires multiple subdomains in the D and E regions for its transcriptional activity. AB - A zebrafish Ftz-F1 homologue, zFF1A (zebrafish Ff1a or Nr5a2, a member of nuclear receptor superfamily) and its C-terminally truncated variant (zFF1B) were previously identified. Due to lack of the identity box (I-box) and activation function 2 (AF-2) domain, zFF1B lacks transactivation function and fails to synergize with estrogen receptor (ER) in regulating promoters. It was speculated that the I-box might be involved in the zFF1A/ER interaction. In the present study, the function of the I-box was examined. In the absence of the I-box or with an altered heptad 9, the AF-2 of zFF1A was not functional, either in the presence or absence of ER. The GST pull-down assay showed that zFF1A and its mutants exerted similar physical contacts with ER-LBD, suggesting that the "dimerization" domain (I-box) is essential for the transcriptional activity of zFF1A. Moreover, nuclear receptor coactivator selectively activated zFF1 with the I-box but exerted no effect on zFF1B, indicating that the I-box is able to interact with the coactivators. By deletion study and analysis of the identified domains in GAL4-DNA binding domain, other regions of zFF1A critical for its AF were also delineated. Consistent with the mutation analysis, AF-2 was active only in the presence of the I-box. We also identified a novel AF domain (AF-3) located in the hinge region (amino acids 155-267), although the activity of AF-3 was inhibited by its flanking region. We suggest that the D and E regions of zFF1A possess both positive and negative transactivation functions, and interdomain "cross-talk" may confer the full transcriptional activity of the protein. PMID- 10747877 TI - Regulation and intracellular trafficking pathways of the endothelin receptors. AB - The effects of endothelin (ET) are mediated via the G protein-coupled receptors ET(A) and ET(B). However, the mechanisms of ET receptor desensitization, internalization, and intracellular trafficking are poorly understood. The aim of the present study was to investigate the molecular mechanisms of ET receptor regulation and to characterize the intracellular pathways of ET-stimulated ET(A) and ET(B) receptors. By analysis of ET(A) and ET(B) receptor internalization in transfected Chinese hamster ovary cells in the presence of overexpressed betaARK, beta-arrestin-1, beta-arrestin-2, or dynamin as well as dominant negative mutants of these regulators, we have demonstrated that both ET receptor subtypes follow an arrestin- and dynamin/clathrin-dependent mechanism of internalization. Fluorescence microscopy of Chinese hamster ovary and COS cells expressing green fluorescent protein (GFP)-tagged ET receptors revealed that the ET(A) and ET(B) subtypes were targeted to different intracellular routes after ET stimulation. While ET(A)-GFP followed a recycling pathway and colocalized with transferrin in the pericentriolar recycling compartment, ET(B)-GFP was targeted to lysosomes after ET-induced internalization. Both receptor subtypes colocalized with Rab5 in classical early endosomes, indicating that this compartment is a common early intermediate for the two ET receptors during intracellular transport. The distinct intracellular routes of ET-stimulated ET(A) and ET(B) receptors may explain the persistent signal response through the ET(A) receptor and the transient response through the ET(B) receptor. Furthermore, lysosomal targeting of the ET(B) receptor could serve as a biochemical mechanism for clearance of plasma endothelin via this subtype. PMID- 10747878 TI - Lipoprotein receptors in extraembryonic tissues of the chicken. AB - Yolk is the major source of nutrients for the developing chicken embryo, but molecular details of the delivery mechanisms are largely unknown. During oogenesis in the chicken, the main yolk components vitellogenin and very low density lipoprotein (VLDL) are taken up into the oocytes via a member of the low density lipoprotein receptor gene family termed LR8 (Bujo, H., Hermann, M., Kaderli, M. O., Jacobsen, L., Sugawara, S., Nimpf, J., Yamamoto, T., and Schneider, W. J. (1994) EMBO J. 13, 5165-5175). This endocytosis is accompanied by partial degradation of the yolk precursor protein moieties; however, fragmentation does not abolish binding of VLDL to LR8. The receptor exists in two isoforms that differ by a so-called O-linked sugar domain; the shorter form (LR8 ) is the major form in oocytes, and the longer protein (LR8+) predominates in somatic cells. Here we show that both LR8 isoforms are expressed at ratios that vary with embryonic age in the extraembryonic yolk sac, which mobilizes yolk for utilization by the embryo, and in the allantois, the embryo's catabolic sink. Stored yolk VLDL interacts with LR8 localized on the surface of the yolk sac endodermal endothelial cells (EEC), is internalized, and degraded, as demonstrated by the catabolism of fluorescently labeled VLDL in cultured EEC. Addition to the incubation medium of the 39-kDa receptor-associated protein, which inhibits all known LR8/ligand interactions, blocks the uptake of VLDL by EEC. The levels of endogenous receptor-associated protein correspond to those of LR8+ but not LR8-, suggesting that it may play a role in the modulation of surface presentation of LR8+. Importantly, EEC express significant levels of microsomal triglyceride transfer protein and protein disulfide isomerase, key components required for lipoprotein synthesis. Because the apolipoprotein pattern of VLDL isolated from the yolk sac-efferent omphalomesenteric vein is very different from that of yolk VLDL, these data strongly suggest that embryo plasma VLDL is resynthesized in the EEC. LR8 is a key mediator of a two-step pathway, which affects the uptake of VLDL from the yolk sac and the subsequent delivery of its components to the growing embryo. PMID- 10747879 TI - Evolution of human and non-human primate CC chemokine receptor 5 gene and mRNA. Potential roles for haplotype and mRNA diversity, differential haplotype-specific transcriptional activity, and altered transcription factor binding to polymorphic nucleotides in the pathogenesis of HIV-1 and simian immunodeficiency virus. AB - Polymorphisms in CC chemokine receptor 5 (CCR5), the major coreceptor of human immunodeficiency virus 1 (HIV-1) and simian immunodeficiency virus (SIV), have a major influence on HIV-1 transmission and disease progression. The effects of these polymorphisms may, in part, account for the differential pathogenesis of HIV-1 (immunosuppression) and SIV (natural resistance) in humans and non-human primates, respectively. Thus, understanding the genetic basis underlying species specific responses to HIV-1 and SIV could reveal new anti-HIV-1 therapeutic strategies for humans. To this end, we compared CCR5 structure/evolution and regulation among humans, apes, Old World Monkeys, and New World Monkeys. The evolution of the CCR5 cis-regulatory region versus the open reading frame as well as among different domains of the open reading frame differed from one another. CCR5 cis-regulatory region sequence variation in humans was substantially higher than anticipated. Based on this variation, CCR5 haplotypes could be organized into seven evolutionarily distinct human haplogroups (HH) that we designated HHA, -B, -C, -D, -E, -F, and -G. HHA haplotypes were defined as ancestral to all other haplotypes by comparison to the CCR5 haplotypes of non-human primates. Different human and non-human primate CCR5 haplotypes were associated with differential transcriptional regulation, and various polymorphisms resulted in modified DNA nuclear protein interactions, including altered binding of members of the NF kappaB family of transcription factors. We identified novel CCR5 untranslated mRNA sequences that were conserved in human and non-human primates. In some primates, mutations at exon-intron boundaries caused loss of expression of selected CCR5 mRNA isoforms or production of novel mRNA isoforms. Collectively, these findings suggest that the response to HIV-1 and SIV infection in primates may have been driven, in part, by evolution of the elements controlling CCR5 transcription and translation. PMID- 10747880 TI - Activation of the AT1 angiotensin receptor is dependent on adjacent apolar residues in the carboxyl terminus of the third cytoplasmic loop. AB - The C-terminal region of the third intracellular loop of the AT(1) angiotensin receptor (AT(1)-R) is an important determinant of G protein coupling. The roles of individual residues in agonist-induced activation of G(q/11)-dependent phosphoinositide hydrolysis were determined by mutational analysis of the amino acids in this region. Functional studies on mutant receptors transiently expressed in COS-7 cells showed that alanine substitutions of the amino acids in positions 232-240 of the third loop had no major effect on signal generation. However, deletion mutations that removed Ile(238) or affected its position relative to transmembrane helix VI significantly impaired angiotensin II-induced inositol phosphate responses. Substitution of Ile(238) with an acidic residue abolished the ability of the receptor to mediate inositol phosphate production, whereas its replacement with basic or polar residues reduced the amplitude of inositol phosphate responses. Substitutions of Phe(239) with polar residues had relatively minor effects on inositol phosphate signal generation, but its replacement by aspartic acid reduced, and by positively charged residues (Lys, Arg) significantly increased, angiotensin II-induced inositol phosphate responses. The internalization kinetics of the Ile(238) and Phe(239) mutant receptors were impaired in parallel with the reduction in their signaling responses. These findings have identified Ile(238) and Phe(239) as the critical residues in the C-terminal region of the third intracellular loop of the AT(1)-R for receptor activation. They also suggest that an apolar amino acid corresponding to Ile(238) of the AT(1)-R is a general requirement for activation of other G protein-coupled receptors by their agonist ligands. PMID- 10747881 TI - Dityrosine cross-linking promotes formation of stable alpha -synuclein polymers. Implication of nitrative and oxidative stress in the pathogenesis of neurodegenerative synucleinopathies. AB - Intracellular proteinaceous aggregates are hallmarks of many common neurodegenerative disorders, and recent studies have shown that alpha-synuclein is a major component of several pathological intracellular inclusions, including Lewy bodies in Parkinson's disease (PD) and glial cell inclusions in multiple system atrophy. However, the molecular mechanisms underlying alpha-synuclein aggregation into filamentous inclusions remain unknown. Since oxidative and nitrative stresses are potential pathogenic mediators of PD and other neurodegenerative diseases, we asked if oxidative and/or nitrative events alter alpha-synuclein and induce it to aggregate. Here we show that exposure of human recombinant alpha-synuclein to nitrating agents (peroxynitrite/CO(2) or myeloperoxidase/H(2)O(2)/nitrite) induces formation of nitrated alpha-synuclein oligomers that are highly stabilized due to covalent cross-linking via the oxidation of tyrosine to form o,o'-dityrosine. We also demonstrate that oxidation and nitration of pre-assembled alpha-synuclein filaments stabilize these filaments to withstand denaturing conditions and enhance formation of SDS insoluble, heat-stable high molecular mass aggregates. Thus, these data suggest that oxidative and nitrative stresses are involved in mechanisms underlying the pathogenesis of Lewy bodies and glial cell inclusions in PD and multiple system atrophy, respectively, as well as alpha-synuclein pathologies in other synucleinopathies. PMID- 10747882 TI - Evidence that the NH2 terminus of vph1p, an integral subunit of the V0 sector of the yeast V-ATPase, interacts directly with the Vma1p and Vma13p subunits of the V1 sector. AB - The vacuolar-type H(+)-ATPase (V-ATPase) is composed of a peripherally bound (V(1)) and a membrane-associated (V(0)) complex. V(1) ATP hydrolysis is thought to rotate a central stalk, which in turn, is hypothesized to drive V(0) proton translocation. Transduction of torque exerted by the rotating stalk on V(0) requires a fixed structural link (stator) between the complexes to prevent energy loss through futile rotation of V(1) relative to V(0); this work sought to identify stator components. The 95-kDa V-ATPase subunit, Vph1p, has a cytosolic NH(2) terminus (Nt-Vph1p) and a membrane-associated COOH terminus. Two-hybrid assays demonstrated that Nt-Vph1p interacts with the catalytic V(1) subunit, Vma1p. Co-immunoprecipitation of Vma1p with Nt-Vph1p confirmed the interaction. Expression of Nt-Vph1p in a Deltavph1 mutant was necessary to recruit Vma13p to V(1). Vma13p bound to Nt-Vph1p in vitro demonstrating direct interaction. Limited trypsin digests cleaves both Nt-Vph1p and Vma13p. The same tryptic treatment results in a loss of proton translocation while not reducing bafilomycin A(1) sensitive ATP hydrolysis. Trypsin cleaved Vph1p at arginine 53. Elimination of the tryptic cleavage site by substitution of arginine 53 to serine partially protected vacuolar acidification from trypsin digestion. These results suggest that Vph1p may function as a component of a fixed structural link, or stator, coupling V(1) ATP hydrolysis to V(0) proton translocation. PMID- 10747883 TI - New beta -lactamase inhibitory protein (BLIP-I) from Streptomyces exfoliatus SMF19 and its roles on the morphological differentiation. AB - A new beta-lactamase inhibitory protein (BLIP-I) from Streptomyces exfoliatus SMF19 was purified and characterized. The molecular mass of BLIP-I was estimated to be 17.5 kDa by gel filtration fast protein liquid chromatography. The N terminal sequence was NH(2)-Asn-Ser-Gly-Phe-Ser-Ala-Glu-Lys-Tyr-Glu-Gln-Ile-Gln Phe-Gly. BLIP-I inhibited Bacto(R) Penase (Difco), and plasmid encoded TEM-1 beta lactamase, whereas it did not inhibit Enterobacter cloacae beta-lactamases. The K(i) value of BLIP-I against TEM-1 beta-lactamase was determined to be 0.047 nm. The gene (bliA) encoding BLIP-I protein was identified by screening a genomic library using an oligonucleotide probe with a sequence based on the N-terminal sequence of BLIP-I. Analysis of the nucleotide sequence revealed that the gene was 558 base pairs in length and encoded a mature protein of 157 amino acid residues preceded by a 29-amino acid signal sequence. Pairwise comparison of the deduced amino acid sequence showed 38% identity with BLIP of Streptomyces clavuligerus. Furthermore, the 49th amino acid residue of BLIP-I was identical to Asp-49 of BLIP that was characterized to be an important residue for the inhibitory activity of BLIP. A modified BLIP-I in which Asp-49 was replaced by alanine (D49A) was obtained by site-directed mutagenesis. The inhibitory activities of recombinant (r) BLIP-I and its D49A mutant derivative, expressed in Escherichia coli, were compared. The K(i) value of rBLIP-I against TEM-1 beta lactamase was similar to that of wild-type BLIP-I, but the D49A mutation increased the K(i) of rBLIP-I inhibition approximately 200-fold. A disruption mutant of the bliA gene in S. exfoliatus SMF19 was obtained by replacing the wild type bliA gene with a copy inactivated by inserting a hygromycin resistance gene. The disruption mutant showed a bald phenotype, indicating that the bliA gene plays a role in morphological differentiation. PMID- 10747884 TI - Functional probing of the human glucocorticoid receptor steroid-interacting surface by site-directed mutagenesis. Gln-642 plays an important role in steroid recognition and binding. AB - To elucidate which amino acids in the glucocorticoid receptor ligand-binding domain might be involved in determining steroid binding specificity by interaction with the D-ring of glucocorticoids, we have performed site-directed mutagenesis of the four amino acids Met-560, Met-639, Gln-642, and Thr-739 based on their proximity to the steroid in a model structure. Mutations of these residues affected steroid binding affinity, specificity, and/or steroid-dependent transactivation. The results indicate that these residues are located in close proximity to the ligand and appear to play a role in steroid recognition and/or transactivating sensitivity, possibly by changes in the steroid-dependent conformational change of this region, resulting in the formation of the AF-2 site. Mutation of Gln-642 resulted in a marked decrease in affinity for steroids containing a 17alpha-OH group. This effect was alleviated by the presence of a 16alpha-CH(3) group to a varying degree. Thr-739 appears to form a hydrogen bond with the 21-OH group of the steroid, as well as possibly forming hydrophobic interactions with the steroid. Met-560 and Met-639 appear to form hydrophobic interactions with the D-ring of the steroid, although the nature of these interactions cannot be characterized in more detail at this point. PMID- 10747885 TI - Stimulation of DNA synthesis and cell proliferation of human mammary myoepithelial-like cells by hepatocyte growth factor/scatter factor depends on heparan sulfate proteoglycans and sustained phosphorylation of mitogen-activated protein kinases p42/44. AB - Hepatocyte growth factor/scatter factor (HGF/SF) is a heparan/dermatan sulfate binding growth factor produced by stromal cells that acts as a paracrine effector on neighboring epithelia. HGF/SF stimulated DNA synthesis in human mammary (Huma) 109 myoepithelial-like cells grown on collagen I and fibronectin substrata but not when grown on plastic. Dual phosphorylation of mitogen-activated protein kinases (p42/44(MAPK)) was required for this stimulation of DNA synthesis. In Huma 109 cells cultured on plastic, HGF/SF stimulated a transient phosphorylation of p42/44(MAPK), which reached a maximum at 10 min after addition of the growth factor and returned to near basal levels after 20 min. In contrast, the phosphorylation of p42/44(MAPK) stimulated by HGF/SF in cells cultured on collagen I or fibronectin was sustained over 45 min. In Huma 109 cells deficient in sulfated glycosaminoglycans, HGF/SF failed to stimulate p42/44(MAPK) phosphorylation or DNA synthesis on any substratum, even when soluble heparan sulfate proteoglycans purified from the cells or from the culture medium were added. However, HGF/SF stimulated DNA synthesis and a sustained phosphorylation of p42/44(MAPK) in sulfated glycosaminoglycan-deficient Huma 109 cells plated on a substratum of medium HSPGs but not cell HSPGs. The HGF/SF-induced proliferation is thus highly dependent on heparan sulfate proteoglycans in myoepithelial-like cells. PMID- 10747886 TI - Sugar transport through maltoporin of Escherichia coli. Role of polar tracks. AB - The three-dimensional structure of the maltooligosaccharide specific outer membrane channel LamB of Escherichia coli complexed with sugar molecules revealed a hypothetical transport pathway. Sugars are supposed to slide over a stretch of aromatic residues facilitated by continuous making/breaking of hydrogen bonds between the hydroxyl groups of the sugars and charged amino acids, the "polar tracks." The effect of nine single and three multiple mutations in the polar track residues was investigated by current fluctuations, liposome swelling assays, and in vivo uptake of radiolabeled substrates. Additionally, sugar transport through wild-type LamB was investigated by current fluctuation analysis in water and deuterium. This way the effects on k(on) and k(off) could be investigated separately. Analyses of the various mutants revealed a strong effect on the k(on) values. Because steering to the binding site requires only a few interactions, consequently the loss of even one bond will have a strong effect. Deuterium experiments, which changed the characteristic of all hydrogen bonds, showed a strong effect on k(off) rates, because at this stage the sugar has numerous interactions with the channel. Furthermore, all the mutations induces a strong decrease of in vivo uptake of sugars. These results clearly demonstrate the importance of the polar track residues on both on and off rates in sugar transport and reveal a strong cooperative effect of hydrogen bond formation. PMID- 10747887 TI - Distinct roles of two intracellular phospholipase A2s in fatty acid release in the cell death pathway. Proteolytic fragment of type IVA cytosolic phospholipase A2alpha inhibits stimulus-induced arachidonate release, whereas that of type VI Ca2+-independent phospholipase A2 augments spontaneous fatty acid release. AB - Cytosolic phospholipase A(2)alpha (cPLA(2)alpha; type IVA), an essential initiator of stimulus-dependent arachidonic acid (AA) metabolism, underwent caspase-mediated cleavage at Asp(522) during apoptosis. Although the resultant catalytically inactive N-terminal fragment, cPLA(2)(1-522), was inessential for cell growth and the apoptotic process, it was constitutively associated with cellular membranes and attenuated both the A23187-elicited immediate and the interleukin-1-dependent delayed phases of AA release by several phospholipase A(2)s (PLA(2)s) involved in eicosanoid generation, without affecting spontaneous AA release by PLA(2)s implicated in phospholipid remodeling. Confocal microscopic analysis revealed that cPLA(2)(1-522) was distributed in the nucleus. Pharmacological and transfection studies revealed that Ca(2+)-independent PLA(2) (iPLA(2); type VI), a phospholipid remodeling PLA(2), contributes to the cell death-associated increase in fatty acid release. iPLA(2) was cleaved at Asp(183) by caspase-3 to a truncated enzyme lacking most of the first ankyrin repeat, and this cleavage resulted in increased iPLA(2) functions. iPLA(2) had a significant influence on cell growth or death, according to cell type. Collectively, the caspase-truncated form of cPLA(2)alpha behaves like a naturally occurring dominant-negative molecule for stimulus-induced AA release, rendering apoptotic cells no longer able to produce lipid mediators, whereas the caspase-truncated form of iPLA(2) accelerates phospholipid turnover that may lead to apoptotic membranous changes. PMID- 10747888 TI - Human Ca2+ receptor cysteine-rich domain. Analysis of function of mutant and chimeric receptors. AB - The 612-residue extracellular domain of the human Ca(2+) receptor (hCaR) has been speculated to consist of a Venus's-flytrap domain (VFT) and a cysteine-rich domain. We studied the function of the hCaR Cys-rich domain by using mutagenesis and chimera approaches. A chimeric hCaR with the sequence from residues 540-601 replaced by the corresponding sequence from the Fugu CaR remained fully functional. Another chimeric hCaR with the same region of sequence replaced by the corresponding sequence from metabotropic glutamate receptor subtype 1 (mGluR1) still was activated by extracellular Ca(2+) ([Ca(2+)](o)), but its function was severely compromised. Chimeric receptors with the hCaR VFT and mGluR1 seven-transmembrane domain plus C-tail domain retained good response to [Ca(2+)](o) whether the Cys-rich domain was from hCaR or from mGluR1. Mutant hCaR with the Cys-rich domain deleted failed to respond to [Ca(2+)](o), although it was expressed at the cell surface and capable of dimerization. Our results indicate that the hCaR Cys-rich domain plays a critical role in signal transmission from VFT to seven-transmembrane domain. This domain tolerates a significant degree of amino acid substitution and may not be directly involved in the binding of [Ca(2+)](o). PMID- 10747889 TI - Binding of alkylurea inhibitors to epoxide hydrolase implicates active site tyrosines in substrate activation. AB - The structures of two alkylurea inhibitors complexed with murine soluble epoxide hydrolase have been determined by x-ray crystallographic methods. The alkyl substituents of each inhibitor make extensive hydrophobic contacts in the soluble epoxide hydrolase active site, and each urea carbonyl oxygen accepts hydrogen bonds from the phenolic hydroxyl groups of Tyr(381) and Tyr(465). These hydrogen bond interactions suggest that Tyr(381) and/or Tyr(465) are general acid catalysts that facilitate epoxide ring opening in the first step of the hydrolysis reaction; Tyr(465) is highly conserved among all epoxide hydrolases, and Tyr(381) is conserved among the soluble epoxide hydrolases. In one enzyme inhibitor complex, the urea carbonyl oxygen additionally interacts with Gln(382). If a comparable interaction occurs in catalysis, then Gln(382) may provide electrostatic stabilization of partial negative charge on the epoxide oxygen. The carboxylate side chain of Asp(333) accepts a hydrogen bond from one of the urea NH groups in each enzyme-inhibitor complex. Because Asp(333) is the catalytic nucleophile, its interaction with the partial positive charge on the urea NH group mimics its approach toward the partial positive charge on the electrophilic carbon of an epoxide substrate. Accordingly, alkylurea inhibitors mimic features encountered in the reaction coordinate of epoxide ring opening, and a structure based mechanism is proposed for leukotoxin epoxide hydrolysis. PMID- 10747890 TI - Vertical-scanning mutagenesis of a critical tryptophan in the "minor groove binding track" of HIV-1 reverse transcriptase. Major groove DNA adducts identify specific protein interactions in the minor groove. AB - Biochemical and molecular modeling studies of human immunodeficiency virus type 1 reverse transcriptase (RT) have revealed that a structural element, the minor groove binding track (MGBT), is important for both replication frameshift fidelity and processivity. The MGBT interactions occur in the DNA minor groove from the second through sixth base pair from the primer 3'-terminus where the DNA undergoes a structural transition from A-like to B-form DNA. Alanine-scanning mutagenesis had previously demonstrated that Gly(262) and Trp(266) of the MGBT contributes important DNA interactions. To probe the molecular interactions occurring in this critical region, eight mutants of RT were studied in which alternate residues were substituted for Trp(266). These enzymes were characterized in primer extension assays in which the template DNA was adducted at a single adenine by either R- or S-enantiomers of styrene oxide. These lesions failed to block DNA polymerization by wild-type RT, yet the Trp(266) mutants and an alanine mutant of Gly(262) terminated synthesis on styrene oxide-adducted templates. Significantly, the sites of termination occurred primarily 1 and 3 bases following adduct bypass, when the lesion was positioned in the major groove of the template-primer stem. These results indicate that residue 266 serves as a "protein sensor" of altered minor groove interactions and identifies which base pair interactions are altered by these lesions. In addition, the major groove lesion must alter important structural transitions in the template-primer stem, such as minor groove widening, that allow RT access to the minor groove. PMID- 10747891 TI - Involvement of sphingosine in mitochondria-dependent Fas-induced apoptosis of type II Jurkat T cells. AB - Exposure to anti-Fas antibody in Jurkat cells (type II cells), which are characterized by a weak caspase-8 activation at the death-inducing signaling complex (DISC), induced a biphasic increase in ceramide levels. The early generation of ceramide preceded transient activation of acidic ceramidase and subsequent production of sphingosine, followed by cytochrome c release, activation of caspases-2, -3, -6, -7, -8, and -9, Bid cleavage, and a later sustained ceramide accumulation. The caspase inhibitor benzyloxycarbonyl-Val-Ala Asp-fluoromethyl ketone inhibited early increases of ceramide and sphingosine, whereas overexpression of Bcl-x(L) had no effect, and both prevented the later sustained ceramide accumulation. Exogenous sphingosine, as well as cell-permeable C(2)-ceramide, induced cytochrome c release from mitochondria in a caspase independent fashion leading to activation of caspase-9 and executioner caspases and, surprisingly, activation of the initiator caspase-8 and processing of its substrate Bid. These effects were also completely abolished by Bcl-x(L) overexpression. Our results suggest that sphingosine might also be involved in the mitochondria-mediated pathway of Fas-induced cell death in type II cells. PMID- 10747892 TI - The GADD45 inhibition of Cdc2 kinase correlates with GADD45-mediated growth suppression. AB - Cell cycle growth arrest is an important cellular response to genotoxic stress. Gadd45, a p53-regulated stress protein, plays an important role in the cell cycle G(2)-M checkpoint following exposure to certain types of DNA-damaging agents such as UV radiation and methylmethane sulfonate. Recent findings indicate that Gadd45 interacts with Cdc2 protein and inhibits Cdc2 kinase activity. In the present study, a series of Myc-tagged Gadd45 deletion mutants and a Gadd45 overlapping peptide library were used to define the Gadd45 domains that are involved in the interaction of Gadd45 with Cdc2. Both in vitro and in vivo studies indicate that the interaction of Gadd45 with Cdc2 involves a central region of the Gadd45 protein (amino acids 65-84). The Cdc2-binding domain of Gadd45 is also required for Gadd45 inhibition of Cdc2 kinase activity. Sequence analysis of the central Gadd45 region reveals no homology to inhibitory motifs of known cyclin-dependent kinase inhibitors, indicating that the Cdc2-binding and -inhibitory domains on Gadd45 are a novel motif. The peptide containing the Cdc2-binding domain (amino acids 65-84) disrupted the Cdc2-cyclin B1 protein complex, suggesting that dissociation of this complex results from a direct interaction between the Gadd45 and Cdc2 proteins. GADD45-induced cell cycle G(2)-M arrest was abolished when its Cdc2 binding motif was disrupted. Importantly, a short term survival assay demonstrated that GADD45-induced cell cycle G(2)-M arrest correlates with GADD45 mediated growth suppression. These findings indicate that the cell cycle G(2)-M growth arrest mediated by GADD45 is one of the major mechanisms by which GADD45 suppresses cell growth. PMID- 10747893 TI - Dynamics of NF kappa B and Ikappa Balpha studied with green fluorescent protein (GFP) fusion proteins. Investigation of GFP-p65 binding to DNa by fluorescence resonance energy transfer. AB - We investigated the dynamics of nuclear transcription factor kappaB (NF-kappaB) by using fusion proteins of the p65 subunit with mutants of green fluorescent protein (GFP). GFP-NF-kappaB chimeras were functional both in vitro and in vivo, as demonstrated by electrophoretic mobility shift assays and reporter gene studies. GFP-p65 was regulated by IkappaBalpha similar to wild type p65 and associated with its inhibitor even if both proteins were linked to a GFP protein. This finding was also verified by fluorescence resonance energy transfer (FRET) microscopy and studies showing mutual regulation of the intracellular localization of both GFP chimerae. Incubation of GFP-p65 with fluorescently labeled NF-kappaB-binding oligonucleotides also resulted in FRET. This effect was DNA sequence-specific and exhibited saturation characteristics. Application of stopped-flow fluorometry to measure the kinetics of FRET between GFP-p65 and oligonucleotides revealed a fast increase of acceptor fluorescence with a plateau after about 10 ms. The observed initial binding rate showed a temperature dependent linear correlation with the oligonucleotide concentration. The association constant calculated according to pre-steady state kinetics was 3 x 10(6) m(-1), although equilibrium binding studies implied significantly higher values. This observation suggests that the binding process involves a rapid association with a rather high off-rate followed by a conformational change resulting in an increase of the association constant. PMID- 10747894 TI - The C-terminal RG dipeptide repeats of the spliceosomal Sm proteins D1 and D3 contain symmetrical dimethylarginines, which form a major B-cell epitope for anti Sm autoantibodies. AB - The Sm proteins B/B', D1, D2, D3, E, F, and G are components of the small nuclear ribonucleoproteins U1, U2, U4/U6, and U5 that are essential for the splicing of pre-mRNAs in eukaryotes. D1 and D3 are among the most common antigens recognized by anti-Sm autoantibodies, an autoantibody population found exclusively in patients afflicted with systemic lupus erythematosus. Here we demonstrate by protein sequencing and mass spectrometry that all arginines in the C-terminal arginine-glycine (RG) dipeptide repeats of the human Sm proteins D1 and D3, isolated from HeLa small nuclear ribonucleoproteins, contain symmetrical dimethylarginines (sDMAs), a posttranslational modification thus far only identified in the myelin basic protein. The further finding that human D1 individually overexpressed in baculovirus-infected insect cells contains asymmetrical dimethylarginines suggests that the symmetrical dimethylation of the RG repeats in D1 and D3 is dependent on the assembly status of D1 and D3. In antibody binding studies, 10 of 11 anti-Sm patient sera tested, as well as the monoclonal antibody Y12, reacted with a chemically synthesized C-terminal peptide of D1 containing sDMA, but not with peptides containing asymmetrically modified or nonmodified arginines. These results thus demonstrate that the sDMA-modified C terminus of D1 forms a major linear epitope for anti-Sm autoantibodies and Y12 and further suggest that posttranslational modifications of Sm proteins play a role in the etiology of systemic lupus erythematosus. PMID- 10747895 TI - Superoxide production and reactive oxygen species signaling by endothelial nitric oxide synthase. AB - Reactive oxygen species can function as intracellular messengers, but linking these signaling events with specific enzymes has been difficult. Purified endothelial nitric-oxide synthase (eNOS) can generate superoxide (O(2)) under special conditions but is only known to participate in cell signaling through NO. Here we show that eNOS regulates tumor necrosis factor alpha (TNFalpha) through a mechanism dependent on the production of O(2) and completely independent of NO. Expression of eNOS in transfected U937 cells increased phorbol 12-myristate 13 acetate-induced TNFalpha promoter activity and TNFalpha production. N(omega) Methyl-l-arginine, an inhibitor of eNOS that blocks NO production but not its NADPH oxidase activity, did not prevent TNFalpha up-regulation. Likewise, Gln(361)eNOS, a competent NADPH oxidase that lacks NOS activity, retained the ability to increase TNFalpha. Similar to the effect of eNOS, a O(2) donor dose dependently increased TNFalpha production in differentiated U937 cells. In contrast, cotransfection of superoxide dismutase with eNOS prevented TNFalpha up regulation, as did partial deletion of the eNOS NADPH binding site, a mutation associated with loss of O(2) production. Thus, eNOS may straddle a bifurcating pathway that can lead to the formation of either NO or O(2), interrelated but often opposing free radical messengers. This arrangement has possible implications for atherosclerosis and septic shock where endothelial dysfunction results from imbalances in NO and O(2) production. PMID- 10747896 TI - Canonical heat shock element in the alpha B-crystallin gene shows tissue-specific and developmentally controlled interactions with heat shock factor. AB - Oligomerization of the heat shock factor (HSF) and its interaction with the heat shock element (HSE) are the hallmark of active transcriptional response to tangible physical or chemical stress. It is unknown if these interactions are subject to control and modulation by developmental cues and thus have tissue or stage specificity. By using promoter sequences containing a canonical HSE from the alphaB-crystallin gene, we demonstrate a tissue-specific transition from monomeric (in fetal and early neonatal stages that lack oligomeric HSF.HSE complexes) to oligomeric HSF-HSE interactions by postnatal day 10-21 in the ocular lens. Developmental control of these interactions is further demonstrated by induction of oligomeric HSF.HSE complexes in neonatal extracts by in vitro manipulations, interestingly, only in the lens and not in the brain, heart, or liver extracts. The exclusive presence of oligomeric HSF.HSE complexes in the postnatal/adult lens corresponds to known highly increased number of alphaB crystallin transcripts in this tissue. PMID- 10747897 TI - Stress-induced activation of protein kinase CK2 by direct interaction with p38 mitogen-activated protein kinase. AB - Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-alpha stimulate the specific enzyme activity of CK2 in the human cervical carcinoma HeLa cells by up to 8-fold, and this could be blocked by the p38 MAP kinase inhibitor SB203580. We show that p38alpha MAP kinase, in a phosphorylation-dependent manner, can directly interact with the alpha and beta subunits of CK2 to activate the holoenzyme through what appears to be an allosteric mechanism. Furthermore, we demonstrate that anisomycin- and tumor necrosis factor-alpha-induced phosphorylation of p53 at Ser-392, which is important for the transcriptional activity of this growth suppressor protein, requires p38 MAP kinase and CK2 activities. PMID- 10747899 TI - The cyclin D1 gene is transcriptionally repressed by caveolin-1. AB - The cyclin D1 gene encodes the regulatory subunit of the holoenzyme that phosphorylates and inactivates the retinoblastoma pRB protein. Cyclin D1 protein levels are elevated by mitogenic and oncogenic signaling pathways, and antisense mRNA to cyclin D1 inhibits transformation by the ras, neu, and src oncogenes, thus linking cyclin D1 regulation to cellular transformation. Caveolins are the principal protein components of caveolae, vesicular plasma membrane invaginations that also function in signal transduction. We show here that caveolin-1 expression levels inversely correlate with cyclin D1 abundance levels in transformed cells. Expression of antisense caveolin-1 increased cyclin D1 levels, whereas caveolin-1 overexpression inhibited expression of the cyclin D1 gene. Cyclin D1 promoter activity was selectively repressed by caveolin-1, but not by caveolin-3, and this repression required the caveolin-1 N terminus. Maximal inhibition of the cyclin D1 gene promoter by caveolin-1 was dependent on the cyclin D1 promoter T-cell factor/lymphoid enhancer factor-1-binding site between 81 to -73. The T-cell factor/lymphoid enhancer factor sequence was sufficient for repression by caveolin-1. We suggest that transcriptional repression of the cyclin D1 gene may contribute to the inhibition of transformation by caveolin-1. PMID- 10747900 TI - Cysteine-scanning mutagenesis around transmembrane segments 1 and 11 and their flanking loop regions of Tn10-encoded metal-Tetracycline/H+ antiporter. AB - Putative transmembrane helices (TM) 1 and 11 in the metal-tetracycline/H(+) antiporter are predicted to be close to each other on the basis of disulfide cross-linking experiments of the double-cysteine mutants in the periplasmic loop regions (Kubo, Y., Konishi, S., Kawabe, T., Nada, S., and Yamaguchi, A. (2000) J. Biol. Chem. 275, 5270-5274). In this study, each amino acid from Asn-2 to Gly-44 in the putative TM1 and loop1-2 regions or that from Ser-328 to Gly-366 in TM11 and its flanking regions was individually replaced with cysteine. With respect to the TM1 region, 10 mutants, from T5C to L14C, were all not reactive with N ethylmaleimide (NEM), and from D15C to I22C, NEM-reactive and non-reactive mutations periodically appeared every two residues. Three mutants, M23C to V25C, were all NEM-reactive, but the degree of the latter two mutants was very low. Seven mutants, from L26C to E32C, were all highly reactive with NEM. Therefore, the region of TM1 is composed of the 21 amino acid residues from Thr-5 to Val-25. It is a partially amphiphilic helix, that is, the N-terminal (cytoplasmic) half is embedded in the hydrophobic interior, and the C-terminal (periplasmic) half faces a water-filled channel. With respect to TM11, nine mutants, from S328C to G336C, and six mutants, from L361C to G366C, were all reactive with NEM. On the other hand, out of the 24 mutants, from L337C to S360C, 17 were not reactive with NEM, and the 7 NEM-reactive mutants were scattered, indicating that this region is a transmembrane segment. The 7 residues from Val-347 to Phe-353 including Pro 350 formed a central hydrophobic core, and the 7 NEM-reactive mutations were periodically distributed in its flanking regions, indicating that both ends of TM11 face a water-filled channel. Ala-354 is located at about 1/3 of the length from the periplasmic end of TM11. Disulfide cross-linking experiments on double cysteine mutants having the combination of A354C and a cysteine-scanning mutation in the loop1-2 region indicated that loop1-2 is very flexible and close to the periplasmic end of TM11. Tetracycline prevented the cross-linking formation between the periplasmic ends of TM1 and TM11; however, it did not affect the cross-linking between loop1-2 and TM11, indicating that the substrate-induced conformational change involves a shift in the relative locations of TM1 and TM11. PMID- 10747901 TI - Modulation of PAX6 homeodomain function by the paired domain. AB - PAX6 is required for proper development of the eye, central nervous system, and nose. PAX6 has two DNA binding domains, a glycine-rich region that links the two DNA binding domains, and a transactivation domain. There is evidence that the different DNA binding domains of PAX6 have different target genes. However, it is not clear if the two DNA binding domains function independently. We have studied the effect of structural changes in the paired domain on the function of PAX6 mediated through its homeodomain. The R26G and I87R mutations have been reported in different human patients with clinically different phenotypes and are in the N and the C-terminal halves of the paired domain, respectively. Surprisingly, we found that the I87R mutant protein not only lost the transactivation function but also failed to bind DNA by either of its DNA binding domains. In contrast, the R26G mutant protein lost DNA binding through its paired domain but had greater DNA binding and transactivation than wild-type PAX6 on homeodomain binding sites. Like R26G, the 5a isoform showed higher DNA binding than wild-type PAX6. This study demonstrates that the two subdomains of the paired domain influence the function of the homeodomain differentially and also provides an explanation for the difference in phenotypes associated with these mutations. PMID- 10747902 TI - Transcriptional regulation of the antioxidant response element. Activation by Nrf2 and repression by MafK. AB - The antioxidant response element (ARE) mediates the transcriptional activation of many genes encoding phase II drug-metabolizing enzymes in response to oxidative stress. Recent studies using knockout mice suggest that NF-E2-related factor 2 (Nrf2), along with small Maf proteins, binds and activates the ARE. In this study, using in vitro binding assays, Nrf2/MafK heterodimers were found to interact with high affinity to the ARE. However, distinct differences were observed when this interaction was compared with that formed with nuclear proteins from H4II EC3 or HepG2 cells. Overexpression of Nrf2 activated ARE mediated transcription in HepG2 cells, and this activation was further increased by tert-butylhydroquinone. In HeLa cells, overexpression of Nrf2 resulted in activation of the ARE, but this activation was no longer induced by tert butylhydroquinone. Using ARE constructs with point mutations in the core sequence, we found that only mutations at the T or G nucleotides within the core (TGAC) render the ARE unresponsive to Nrf2. Overexpression of MafK led to dose dependent repression of ARE activity. Activation of the ARE by Nrf2 was similarly antagonized by MafK. These data suggest that Nrf2 plays an important role mediating basal activity of the ARE but that small Maf proteins are repressors and not activators of ARE-mediated transcription. PMID- 10747903 TI - p53 suppresses the c-Myb-induced activation of heat shock transcription factor 3. AB - Expression of heat shock proteins (HSPs) is controlled by heat shock transcription factors (HSFs). Vertebrates express multiple HSFs whose activities may be regulated by distinct signals. HSF3 is specifically activated in unstressed proliferating cells by direct binding to the c-myb proto-oncogene product (c-Myb), which plays an important role in cellular proliferation. This suggests that the c-Myb-induced HSF3 activation may contribute to the growth regulated expression of HSPs. Here we report that the p53 tumor suppressor protein directly binds to HSF3 and blocks the interaction between c-Myb and HSF3. In addition, p53 stimulates the degradation of c-Myb through a proteasome dependent mechanism, which is, at least partly, mediated by induction of Siah in certain types of cells. Induction of p53 by a genotoxic reagent in DT40 cells disrupts the HSF3-c-Myb interaction and down-regulates the expression of certain HSPs. Mutated forms of p53 found in certain tumors did not inhibit c-Myb-induced HSF3 activation. The regulation of HSF3 activity by c-Myb and p53 sheds light on the molecular events that govern HSP expression during cellular proliferation and apoptosis. PMID- 10747904 TI - Site-directed cross-linking of b to the alpha, beta, and a subunits of the Escherichia coli ATP synthase. AB - The b subunit dimer of the Escherichia coli ATP synthase, along with the delta subunit, is thought to act as a stator to hold the alpha(3)beta(3) hexamer stationary relative to the a subunit as the gammaepsilonc(9-12) complex rotates. Despite their essential nature, the contacts between b and the alpha, beta, and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b(24-156), a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F(1) sector or to complete F(1)F(0) was attempted. Cross-linking in both the full length and truncated forms of b was obtained at positions 92 (to alpha and beta), and 109 and 110 (to alpha only). Mass spectrometric analysis of peptide fragments derived from the b(24-156)A92C cross-link revealed that cross-linking took place within the region of alpha between Ile-464 and Met-483. This result indicates that the b dimer interacts with the alpha subunit near a non-catalytic alpha/beta interface. A cysteine residue introduced in place of the highly conserved arginine at position 36 of the b subunit could be cross-linked to the a subunit of F(0) in membrane-bound ATP synthase, implying that at least 10 residues of the polar domain of b are adjacent to residues of a. Sites of cross-linking between b(24-156)A92C and beta as well as b(24-156)I109C and alpha are proposed based on the mass spectrometric data, and these sites are discussed in terms of the structure of b and its interactions with the rest of the complex. PMID- 10747905 TI - Cooperative influence of genetic polymorphisms on interleukin 6 transcriptional regulation. AB - Interleukin 6 (IL6) plays key roles in hematopoiesis, immune, and acute phase responses. Dysregulated IL6 expression is implicated in diseases such as atherosclerosis and arthritis. We have examined the functional effect of four polymorphisms in the IL6 promoter (-597G-->A, -572G-->C, -373A(n)T(n), -174G-->C) by identifying the naturally occurring haplotypes and comparing their effects on reporter gene expression. The results indicate different transcriptional regulation in the ECV304 cell line compared with the HeLa cell line, suggesting cell type-specific regulation of IL6 expression. The haplotypes showed functional differences in the ECV304 cell line; transcription was higher from the GG9/11G haplotype and lower from the AG8/12G allele. The differences suggest that more than one of the polymorphic sites is functional; the base differences at distinct polymorphic sites do not act independently of one another, and one polymorphism influences the functional effect of variation at other polymorphic sites. These results show that genetic polymorphisms in the promoter influence IL6 transcription not by a simple additive mechanism but rather through complex interactions determined by the haplotype. PMID- 10747906 TI - NADPH oxidase activation increases the sensitivity of intracellular Ca2+ stores to inositol 1,4,5-trisphosphate in human endothelial cells. AB - Many stimuli that activate the vascular NADPH oxidase generate reactive oxygen species and increase intracellular Ca(2+), but whether NADPH oxidase activation directly affects Ca(2+) signaling is unknown. NADPH stimulated the production of superoxide anion and H(2)O(2) in human aortic endothelial cells that was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium and was significantly attenuated in cells transiently expressing a dominant negative allele of the small GTP-binding protein Rac1, which is required for oxidase activity. In permeabilized Mag-indo 1-loaded cells, NADPH and H(2)O(2) each decreased the threshold concentration of inositol 1,4,5-trisphosphate (InsP(3)) required to release intracellularly stored Ca(2+) and shifted the InsP(3)-Ca(2+) release dose-response curve to the left. Concentrations of H(2)O(2) as low as 3 microm increased the sensitivity of intracellular Ca(2+) stores to InsP(3) and decreased the InsP(3) EC(50) from 423.2 +/- 54.9 to 276.9 +/- 14. 4 nm. The effect of NADPH on InsP(3)-stimulated Ca(2+) release was blocked by catalase and by diphenyleneiodonium and was not observed in cells lacking functional Rac1 protein. Thus, NADPH oxidase-derived H(2)O(2) increases the sensitivity of intracellular Ca(2+) stores to InsP(3) in human endothelial cells. Since Ca(2+) dependent signaling pathways are critical to normal endothelial function, this effect may be of great importance in endothelial signal transduction. PMID- 10747907 TI - Interaction of tau with the neural membrane cortex is regulated by phosphorylation at sites that are modified in paired helical filaments. AB - The axonal microtubule-associated phosphoprotein tau interacts with neural plasma membrane (PM) components during neuronal development (Brandt, R., Leger, J., and Lee, G. (1995) J. Cell Biol. 131, 1327-1340). To analyze the mechanism and potential regulation of tau's PM association, a method was developed to isolate PM-associated tau using microsphere separation of surface-biotinylated cells. We show that tau's PM association requires an intact membrane cortex and that PM associated tau and cytosolic tau are differentially phosphorylated at sites detected by several Alzheimer's disease (AD) diagnostic antibodies (Ser(199)/Ser(202), Thr(231), and Ser(396)/Ser(404)). In polar neurons, the association of endogenous tau phosphoisoforms with the membrane cortex correlates with an enrichment in the axonal compartment. To test for a direct effect of AD specific tau modifications in determining tau's interactions, a phosphomutant that simulates an AD-like hyperphosphorylation of tau was produced by site directed mutagenesis of Ser/Thr residues to negatively charged amino acids (Glu). These mutations completely abolish tau's association with the membrane cortex; however, the construct retains its capability to bind to microtubules. The data suggest that a loss of tau's association with the membrane cortex as a result of phosphorylation at sites that are modified during disease contributes to somatodendritic tau accumulation, axonal microtubule disintegration, and neuronal death characteristic for AD. PMID- 10747908 TI - The intrinsic DNA helicase activity of Methanobacterium thermoautotrophicum delta H minichromosome maintenance protein. AB - Minichromosome maintenance proteins (MCMs) form a family of conserved molecules that are essential for initiation of DNA replication. All eukaryotes contain six orthologous MCM proteins that function as heteromultimeric complexes. The sequencing of the complete genomes of several archaebacteria has shown that MCM proteins are also present in archaea. The archaea Methanobacterium thermoautotrophicum contains a single MCM-related sequence. Here we report on the expression and purification of the recombinant M. thermoautotrophicum MCM protein (MtMCM) in both Escherichia coli and baculovirus-infected cells. We show that purified MtMCM protein assembles in large macromolecular complexes consistent in size with being double hexamers. We demonstrate that MtMCM contains helicase activity that preferentially uses dATP and DNA-dependent dATPase and ATPase activities. The intrinsic helicase activity of MtMCM is abolished when a conserved lysine in the helicase domain I/nucleotide binding site is mutated. MtMCM helicase unwinds DNA duplexes in a 3' --> 5' direction and can unwind up to 500 base pairs in vitro. The kinetics, processivity, and directionality of MtMCM support its role as a replicative helicase in M. thermoautotrophicum. This strongly suggests that this function is conserved for MCM proteins in eukaryotes where a replicative helicase has yet to be identified. PMID- 10747909 TI - Galpha 13 requires palmitoylation for plasma membrane localization, Rho-dependent signaling, and promotion of p115-RhoGEF membrane binding. AB - Most heterotrimeric G protein alpha subunits are covalently modified by palmitate attached to one or more N-terminal cysteine residues. Although a wide variety of proteins undergo palmitoylation, the role of this fatty acid modification in G protein signaling is not well understood. Thus, we examined the role of palmitoylation of alpha(13), a G protein alpha subunit that regulates many pathways involved in cell growth. Both N-terminal cysteines at positions 14 and 18 were required for palmitoylation. Mutant alpha(13), in which both cysteines were changed to serines, failed to localize to plasma membranes in transfected cells and failed to activate Rho-dependent serum response factor-mediated transcription and actin stress fiber formation. However, nonpalmitoylated, cysteine to serine mutant alpha(13) retained the ability to co-immunoprecipitate with a direct effector, p115-RhoGEF. Finally, we report the novel observation that activated alpha(13) induces a redistribution of p115-RhoGEF from the cytoplasm to plasma membranes, but non-palmitoylated mutants of alpha(13) fail to cause p115-RhoGEF translocation. These findings identify palmitoylation of alpha(13) as critical for its proper membrane localization and signaling and provide insight into the mechanism of activation of Rho-dependent signaling pathways by alpha(13). PMID- 10747911 TI - TREK-2, a new member of the mechanosensitive tandem-pore K+ channel family. AB - Recently, several mammalian K(+) channel subunits (TWIK, TREK-1, TRAAK, and TASK) possessing four transmembrane segments and two pore-forming domains have been identified. We report the cloning of a new member of this tandem-pore K(+) channel from a rat cerebellum cDNA library. It is a 538-amino acid protein and shares 65% amino acid sequence identity with TREK-1. Therefore, the new clone was named TREK-2. Unlike TREK-1, whose mRNA has been reported to be expressed in many different tissues, TREK-2 mRNA is expressed mainly in the cerebellum, spleen, and testis as judged by reverse transcriptase-polymerase chain reaction and Northern blot analysis. Expression of TREK-2 in COS-7 cells induced a time-independent and non-inactivating K(+)-selective current. TREK-2 was partially blocked (36%) by 2 mm Ba(2+). In symmetrical 150 mm KCl, the single-channel conductances were 110 picosiemens at -40 mV and 68 picosiemens at +40 mV, and the mean open time was 0.9 ms at -40 mV. TREK-2 was activated by membrane stretch or acidic pH. At -40 mm Hg pressure, channel activity increased 10-fold above the basal level. TREK-2 was also activated by arachidonic acid and other naturally occurring unsaturated free fatty acids. These results show that TREK-2 is a new member of the tandem pore K(+) channel family and belongs to the class of mechanosensitive and fatty acid-stimulated K(+) channels. PMID- 10747910 TI - Gamma1- and gamma2-syntrophins, two novel dystrophin-binding proteins localized in neuronal cells. AB - Dystrophin is the scaffold of a protein complex, disrupted in inherited muscular dystrophies. At the last 3' terminus of the gene, a protein domain is encoded, where syntrophins are tightly bound. These are a family of cytoplasmic peripheral membrane proteins. Three genes have been described encoding one acidic (alpha1) and two basic (beta1 and beta2) proteins of approximately 57-60 kDa. Here, we describe the characterization of two novel putative members of the syntrophin family, named gamma1- and gamma2-syntrophins. The human gamma1-syntrophin gene is composed of 19 exons and encodes a brain-specific protein of 517 amino acids. The human gamma2-syntrophin gene is composed of at least 17 exons, and its transcript is expressed in brain and, to a lesser degree, in other tissues. We mapped the gamma1-syntrophin gene to human chromosome 8q11 and the gamma2-syntrophin gene to chromosome 2p25. Yeast two-hybrid experiments and pull-down studies showed that both proteins can bind the C-terminal region of dystrophin and related proteins. We raised antibodies against these proteins and recognized expression in both rat and human central neurons, coincident with RNA in situ hybridization of adjacent sections. Our present findings suggest a differentiated role of a modified dystrophin-associated complex in the central nervous system. PMID- 10747912 TI - Disulfide bonds are required for folding and secretion of apolipoprotein B regardless of its lipidation state. AB - Apolipoprotein (apo) B-100, an essential protein for the assembly and secretion of very low density lipoproteins depends on lipid binding (lipidation) for its secretion. Seven of its 8 disulfides are clustered within the N-terminal 21%. The role of these disulfides in the secretion of lipidated or unlipidated truncated forms of apoB was studied in C127 cells expressing apoB-17, apoB-29, or apoB-41. These cells do not express microsomal triglyceride transfer protein yet secrete apoB-41 on triacylglycerol-rich lipoproteins while apoB-29 and apoB-17 are secreted with little or no lipid, respectively. Dithiothreitol utilized in pulse chase studies prevented the cotranslational formation of disulfides and when added posttranslationally reduced native disulfides. As a result, the secretion of reduced apoB forms was blocked and they were retained in the cells. Reduced apoB polypeptides were rescued following removal of dithiothreitol, as they underwent post-translational disulfide bonding, attained their mature form, and were subsequently secreted. Together the data suggest that in C127 cells the formation of native disulfides is critical for the folding and secretion of apoB independent of its length, its requirement for lipidation or microsomal triglyceride transfer protein expression. Therefore, these cells provide an appropriate model to study the folding of apoB in great detail. PMID- 10747913 TI - Acyclic permutants of naturally occurring cyclic proteins. Characterization of cystine knot and beta-sheet formation in the macrocyclic polypeptide kalata B1. AB - Kalata B1 is a prototypic member of the unique cyclotide family of macrocyclic polypeptides in which the major structural features are a circular peptide backbone, a triple-stranded beta-sheet, and a cystine knot arrangement of three disulfide bonds. The cyclotides are the only naturally occurring family of circular proteins and have prompted us to explore the concept of acyclic permutation, i.e. opening the backbone of a cross-linked circular protein in topologically permuted ways. We have synthesized the complete suite of acyclic permutants of kalata B1 and examined the effect of acyclic permutation on structure and activity. Only two of six topologically distinct backbone loops are critical for folding into the native conformation, and these involve disruption of the embedded ring in the cystine knot. Surprisingly, it is possible to disrupt regions of the beta-sheet and still allow folding into native-like structure, provided the cystine knot is intact. Kalata B1 has mild hemolytic activity, but despite the overall structure of the native peptide being retained in all but two cases, none of the acyclic permutants displayed hemolytic activity. This loss of activity is not localized to one particular region and suggests that cyclization is critical for hemolytic activity. PMID- 10747914 TI - Prenylation-dependent association of protein-tyrosine phosphatases PRL-1, -2, and -3 with the plasma membrane and the early endosome. AB - PRL-1, -2, and -3 represent a novel class of protein-tyrosine phosphatase with a C-terminal prenylation motif. Although PRL-1 has been suggested to be associated with the nucleus, the presence of three highly homologous members and the existence of a prenylation motif call for a more detailed examination of their subcellular localization. In the present study, we first demonstrate that mouse PRL-1, -2, and -3 are indeed prenylated. Examination of N-terminal epitope-tagged PRL-1, -2, and -3 expressed in transiently transfected cells suggests that PRL-1, -2, and -3 are present on the plasma membrane and intracellular punctate structures. Stable Chinese hamster ovary cells expressing PRL-1 and -3 in an inducible manner were established. When cells were treated with brefeldin A, PRL 1 and -3 accumulated in a collapsed compact structure around the microtubule organizing center. Furthermore, PRL-1 and -3 redistributed into swollen vacuole like structures when cells were treated with wortmannin. These characteristics of PRL-1 and -3 are typical for endosomal proteins. Electron microscope immunogold labeling reveals that PRL-1 and -3 are indeed associated with the plasma membrane and the early endosomal compartment. Expression of PRL-3 is detected in the epithelial cells of the small intestine, where PRL-3 is present in punctate structures in the cytoplasm. When cells are treated with FTI-277, a selective farnesyltransferase inhibitor, PRL-1, -2, and -3 shifted into the nucleus. Furthermore, a mutant form of PRL-2 lacking the C-terminal prenylation signal is associated with the nucleus. These results establish that the primary association of PRL-1, -2, and -3 with the membrane of the cell surface and the early endosome is dependent on their prenylation and that nuclear localization of these proteins may be triggered by a regulatory event that inhibits their prenylation. PMID- 10747915 TI - The topogenic contribution of uncharged amino acids on signal sequence orientation in the endoplasmic reticulum. AB - Signal sequences for insertion of proteins into the endoplasmic reticulum induce translocation of either the C- or the N-terminal sequence across the membrane. The end that is translocated is primarily determined by the flanking charges and the hydrophobic domain of the signal. To characterize the hydrophobic contribution to topogenesis, we have challenged the translocation machinery in vivo in transfected COS cells with model proteins differing exclusively in the apolar segment of the signal. Homo-oligomers of hydrophobic amino acids as different in size and shape as Val(19), Trp(19), and Tyr(22) generated functional signal sequences with similar topologies in the membrane. The longer a homo oligomeric sequence of a given residue, the more N-terminal translocation was obtained. To determine the topogenic contribution of all uncharged amino acids in the context of a hydrophobic signal sequence, two residues in a generic oligoleucine signal were exchanged for all uncharged amino acids. The resulting scale resembles a hydrophobicity scale with the more hydrophobic residues promoting N-terminal translocation. In addition, the helix breakers glycine and proline showed a position-dependent effect, which raises the possibility of a conformational contribution to topogenesis. PMID- 10747916 TI - Differential regulation of endogenous cadherin expression in Madin-Darby canine kidney cells by cell-cell adhesion and activation of beta -catenin signaling. AB - Cadherins mediate cell-cell adhesion, but little is known about how their expression is regulated. In Madin-Darby canine kidney (MDCK) cells, the cadherin associated cytoplasmic proteins alpha- and beta-catenin form high molecular weight protein complexes with two glycoproteins (Stewart, D. B., and Nelson, W. J. (1997) J. Biol. Chem. 272, 29652-29662), one of which is E-cadherin and the other we show here is the type II cadherin, cadherin-6 (K-cadherin). In low density, motile MDCK cells, the steady-state level of cadherin-6 is low, but protein is synthesized. However, following cell-cell adhesion, cadherin-6 becomes stabilized and accumulates by >50-fold at cell-cell contacts while the E-cadherin level increases only 5-fold during the same period. To investigate a role of beta catenin in regulation of cadherin expression in MDCK cells, we examined the effects of expressing signaling-active beta-catenin mutants (DeltaGSK, DeltaN90, and DeltaN131). In these cells, while levels of E-cadherin, alpha- and beta catenin are similar to those in control cells, levels of cadherin-6 are significantly reduced due to rapid degradation of newly synthesized protein. Additionally, these cells appeared more motile and less cohesive, as expression of DeltaGSK-beta-catenin delayed the establishment of tight confluent cell monolayers compared with control cells. These results indicate that the level of cadherin-6, but not that of E-cadherin, is strictly regulated post translationally in response to Wnt signaling, and that E-cadherin and cadherin-6 may contribute different properties to cell-cell adhesion and the epithelial phenotype. PMID- 10747917 TI - Interaction of amphipols with sarcoplasmic reticulum Ca2+-ATPase. AB - Amphipols are short-chain amphipathic polymers designed to keep membrane proteins soluble in aqueous solutions. We have evaluated the effects of the interaction of amphipols with sarcoplasmic reticulum Ca(2+)-ATPase either in a membrane-bound or a soluble form. If the addition of amphipols to detergent-solubilized ATPase was followed by removal of detergent, soluble complexes formed, but these complexes retained poor ATPase activity, were not very stable upon long incubation periods, and at high concentrations they experienced aggregation. Nevertheless, adding excess detergent to diluted detergent-free ATPase-amphipol complexes incubated for short periods immediately restored full activity to these complexes, showing that amphipols had protected solubilized ATPase from the rapid and irreversible inactivation that otherwise follows detergent removal. Amphipols also protected solubilized ATPase from the rapid and irreversible inactivation observed in detergent solutions if the ATPase Ca(2+) binding sites remain vacant. Moreover, in the presence of Ca(2+), amphipol/detergent mixtures stabilized concentrated ATPase against inactivation and aggregation, whether in the presence or absence of lipids, for much longer periods of time (days) than detergent alone. Our observations suggest that mixtures of amphipols and detergents are promising media for handling solubilized Ca(2+)-ATPase under conditions that would otherwise lead to its irreversible denaturation and/or aggregation. PMID- 10747919 TI - Mutations that increase the activity of the promoter of the Escherichia coli melibiose operon improve the binding of MelR, a transcription activator triggered by melibiose. AB - MelR is an Escherichia coli transcription factor that activates expression of the melAB operon in response to the presence of melibiose in the environment. MelR stimulates transcription initiation at the melAB promoter by binding to four sites centered at positions -120.5, -100.5, -62.5, and -42.5 upstream of the transcript start point. In a previous study, we described a spontaneous mutant that exhibited increased melAB expression. Sequence analysis showed that this mutant carries five consecutive base changes at positions -49, -50, -51, -52, and -53 upstream of the melAB transcript start. Here we show that these changes improve MelR binding to the target site centered at position -42.5 at the melAB promoter and that this improvement is responsible for increased promoter activity. Thus, the activity of the melAB promoter is fixed by the occupation by MelR of a DNA site that overlaps the -35 hexamer: MelR appears to be a typical class II-type transcription activator. PMID- 10747918 TI - The YXXL motif, but not the two NPXY motifs, serves as the dominant endocytosis signal for low density lipoprotein receptor-related protein. AB - All members of the low density lipoprotein (LDL) receptor family contain at least one copy of the NPXY sequence within their cytoplasmic tails. For the LDL receptor, it has been demonstrated that the NPXY motif serves as a signal for rapid endocytosis through coated pits. Thus, it is generally believed that the NPXY sequences function as endocytosis signals for all the LDL receptor family members. The primary aim of this study is to define the endocytosis signal(s) within the cytoplasmic tail of LDL receptor-related protein (LRP). By using LRP minireceptors, which mimic the function and trafficking of full-length endogenous LRP, we demonstrate that the YXXL motif, but not the two NPXY motifs, serves as the dominant signal for LRP endocytosis. We also found that the distal di-leucine motif within the LRP tail contributes to its endocytosis, and its function is independent of the YXXL motif. Although the proximal NPXY motif and the proximal di-leucine motif each play a limited role in LRP endocytosis in the context of the full-length tail, these motifs were functional within the truncated receptor tail. In addition, we show that LRP minireceptor mutants defective in endocytosis signal(s) accumulate at the cell surface and are less efficient in delivery of ligand for degradation. PMID- 10747920 TI - Factors determining the composition of inositol trisphosphate receptor hetero oligomers expressed in COS cells. AB - COS-7 cells were transiently transfected with type I and type III myo-inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms to study the processes underlying assembly and oligomerization of these tetrameric proteins. A FLAG epitope was engineered on to the N terminus of the type III IP(3)R to distinguish the transfected from the endogenous isoform. This was not necessary for the type I IP(3)R, since the endogenous levels of this isoform were extremely low. Based on sucrose gradient analysis, the transfected type I or FLAG-type III IP(3)Rs assembled into tetramers. Confocal immunofluorescence experiments confirmed that the constructs were primarily targeted to the endoplasmic reticulum. Recombinant type I IP(3)R expressed in COS cells over a 48-h period showed a negligible capacity to form hetero-oligomers with endogenous type III IP(3)Rs, based upon co immunoprecipitation assays. However, substantial formation of hetero-oligomers was observed between recombinant receptors when the cells were simultaneously transfected with type I and FLAG-type III IP(3)Rs. Co-immunoprecipitation experiments using lysates from metabolically labeled cells allowed the quantitation of homo- and hetero-oligomers in cells transfected with different ratios of type I and FLAG-type III IP(3)R DNA. These studies show that the relative expression level of the two isoforms influences the fraction of hetero oligomers formed. However, the proportion of hetero-oligomers formed were less than predicted by a binomial model in which the association of subunits is assumed to be random. In doubly transfected cells, the early kinetics of (35)S label incorporation into homotetramers showed a lag period corresponding to the time taken to synthesize a full-length receptor. However, hetero-oligomers were synthesized with a longer lag period, suggesting that there may be kinetic constraints that favor homo-oligomers over hetero-oligomers. PMID- 10747921 TI - Identification of the minimal functional unit in the low density lipoprotein receptor-related protein for binding the receptor-associated protein (RAP). A conserved acidic residue in the complement-type repeats is important for recognition of RAP. AB - The low density lipoprotein receptor-related protein (LRP), a member of the low density lipoprotein receptor family, mediates the internalization of a diverse set of ligands. The ligand binding sites are located in different regions of clusters consisting of approximately 40 residues, cysteine-rich complement-type repeats (CRs). The 39-40-kDa receptor-associated protein, a folding chaperone/escort protein required for efficient transport of functional LRP to the cell surface, is an antagonist of all identified ligands. To analyze the multisite inhibition by RAP in ligand binding of LRP, we have used an Escherichia coli expression system to produce fragments of the entire second ligand binding cluster of LRP (CR3-10). By ligand affinity chromatography and surface plasmon resonance analysis, we show that RAP binds to all two-repeat modules except CR910. CR10 differs from other repeats in cluster II by not containing a surface exposed conserved acidic residue between Cys(IV) and Cys(V). By site-directed mutagenesis and ligand competition analysis, we provide evidence for a crucial importance of this conserved residue for RAP binding. We provide experimental evidence showing that two adjacent complement-type repeats, both containing a conserved acidic residue, represent a minimal unit required for efficient binding to RAP. PMID- 10747922 TI - Compulsory order of substrate binding to herpes simplex virus type 1 thymidine kinase. A calorimetric study. AB - Isothermal titration calorimetry has been used to investigate the thermodynamic parameters of the binding of thymidine (dT) and ATP to herpes simplex virus type 1 thymidine kinase (HSV1 TK). Binding follows a sequential pathway in which dT binds first and ATP second. The free enzyme does not bind ATP, whose binding site becomes only accessible in the HSV1 TK.dT complex. At pH 7.5 and 25 degrees C, the binding constants are 1.9 x 10(5) m(-1) for dT and 3.9 x 10(6) m(-1) for ATP binding to the binary HSV1 TK.dT complex. Binding of both substrates is enthalpy driven and opposed by a large negative entropy change. The heat capacity change (DeltaCp) obtained from DeltaH in the range of 10-25 degrees C is -360 cal K(-1) mol(-1) for dT binding and -140 cal K(-1) mol(-1) for ATP binding. These large DeltaCp values are incompatible with a rigid body binding model in which the dT and ATP binding sites pre-exist in the free enzyme. Values of DeltaCp and TDeltaS strongly indicate large scale conformational adaptation of the active site in sequential substrate binding. The conformational changes seem to be more pronounced in dT binding than in the subsequent ATP binding. Considering the crystal structure of the ternary HSV1 TK.dT.ATP complex, a large movement in the dT binding domain and a smaller but substantial movement in the LID domain are proposed to take place when the enzyme changes from the substrate-free, presumably more open and less ordered conformation to the closed and compact conformation of the ternary enzyme-substrate complex. PMID- 10747923 TI - Analysis of CMF1 reveals a bone morphogenetic protein-independent component of the cardiomyogenic pathway. AB - Disruption of the CMF1 function in anterior mesoderm inhibits cardiac myogenesis in avian embryos. In the present study, we show that CMF1 is a member of an emerging family of proteins that includes centromeric protein-F, mitosin, and LEK1. These proteins are characterized by their large size (350 kDa), dynamic subcellular distribution, and potential functions in cell division and differentiation. The current data suggest that CMF1 is a unique member of this family by virtue of its restricted protein expression and variant subcellular distribution. Immunochemical analysis demonstrates that CMF1 protein is expressed in cardiogenic cells prior to the activation of cardiac structural gene products. In addition, we show that expression of CMF1 is not dependent on the bone morphogenetic protein (BMP) signaling pathway during development. Still, CMF1 cannot direct cardiomyogenesis in the absence of such factors as NKX-2.5. Taken with our previous data, this study suggests that CMF1 is a BMP-independent component of the cardiomyogenic pathway. PMID- 10747924 TI - Protection of renal inner medullary epithelial cells from apoptosis by hypertonic stress-induced p53 activation. AB - Acute hypertonicity causes cell cycle delay and apoptosis in mouse renal inner medullary collecting duct cells (mIMCD3) and increases GADD45 expression. Because the tumor suppressor protein p53 may be involved in these effects, we have investigated the role of p53 in mIMCD3 response to hyperosmotic stress. Acute elevation of osmolality with NaCl addition from the control level of 320 mosmol/kg to 500-600 mosmol/kg greatly increased the levels of total and Ser(15) phosphorylated p53 within 15 min. However, similar elevation of osmolality with urea did not increase p53 levels. Our studies indicate that induced p53 is transcriptionally active because NaCl addition to 500-600 mosmol/kg stimulated transcription of a luciferase reporter containing a p53 consensus element and appropriately altered mRNA levels of known transcriptional targets of p53, i.e. increased MDM-2 and decreased BCL-2 levels. Elevating NaCl further to 700-800 mosmol/kg rapidly killed most of the cells by apoptosis. At these higher NaCl concentrations, p53 levels were further increased although Ser(15) phosphorylation and transcriptional activity were significantly lower than levels at 500-600 mosmol/kg. At NaCl-induced 500 mosmol/kg, apoptosis was rare in the presence of control, nonspecific oligonucleotide but highly prevalent upon addition of p53 antisense oligonucleotide that substantially reduced p53 levels. We conclude that induction of active p53 in mIMCD3 cells by hypertonic stress contributes to cell survival. PMID- 10747925 TI - Signaling pathways to the assembly of an interferon-beta enhanceosome. Chemical genetic studies with a small molecule. AB - Small molecules that modulate specific protein functions are valuable tools for dissecting complex signaling pathways. Here, we identified a small molecule that induces the assembly of the interferon-beta (IFN-beta) enhanceosome by stimulating all the enhancer-binding activator proteins: ATF2/c-JUN, IRF3, and p50/p65 of NF-kappaB. This compound stimulates mitogen-activated protein kinase kinase kinase 1 (MEKK1), which is a member of a family of proteins involved in stress-mediated signaling pathways. Consistent with this, MEKK1 activates IRF3 in addition to ATF2/c-JUN and NF-kappaB for the assembly of the IFN-beta enhanceosome. MEKK1 activates IRF3 through the c-JUN amino-terminal kinase (JNK) pathway but not the p38 and IkappaB kinase (IKK) pathway. Taken together with previous observations, these results implicate that, for the assembly of an IFN beta enhanceosome, MEKK1 can induce IRF3 and ATF2/c-JUN through the JNK pathway, whereas it can induce NF-kappaB through the IKK pathway. Thus, specific MEKK family proteins may be able to integrate some of multiple signal transduction pathways leading to the specific activation of the IFN-beta enhanceosome. PMID- 10747926 TI - Evidence that retinoic acid receptor beta induction by retinoids is important for tumor cell growth inhibition. AB - Retinoic acid receptor beta (RARbeta) is thought to be involved in suppressing cell growth and tumorigenicity. Many premalignant and malignant cells exhibit a reduced RARbeta expression. However, in some of these cells (e.g. H157 human squamous cell carcinoma cells), RARbeta can be induced by retinoids (e.g. all trans-retinoic acid, ATRA) because its promoter contains a retinoic acid response element. To examine the hypothesis that RARbeta induction is important for inhibition of cell proliferation by retinoids, we blocked ATRA-induced RARbeta expression in H157 cells using a retroviral vector harboring multiple copies of antisense RARbeta2 sequences. Antisense RARbeta-transfected cells showed not only decreased expression of ATRA-induced RARbeta protein but also reduced ATRA induced RARE binding activity and transactivation. Importantly, all antisense RARbeta transfectants of H157 cells were less responsive than vector-transfected cells to the growth inhibitory effects of the retinoids ATRA and Ch55 in vitro. These results demonstrate that RARbeta induction may play an important role in mediating growth inhibitory effects of retinoids in cancer cells. PMID- 10747927 TI - Receptor chimeras indicate that the vascular endothelial growth factor receptor-1 (VEGFR-1) modulates mitogenic activity of VEGFR-2 in endothelial cells. AB - Vascular endothelial growth factor (VEGF) provokes angiogenesis in vivo and stimulates growth and differentiation of endothelial cells in vitro. Although VEGF receptor-1 (VEGFR-1) and VEGFR-2 are known to be high affinity receptors for VEGF, it is not clear which of the VEGFRs are responsible for the transmission of the diverse biological responses of VEGF. For this purpose we have constructed a chimeric receptor for VEGFR-1 (CTR) and VEGFR-2 (CKR) in which the extracellular domain of each receptor was replaced with the extracellular domain of human colony-stimulating factor-1 receptor (CSF-1R), and these receptors were expressed in pig aortic endothelial (PAE) cells. We show that CKR individually expressed in PAE cells is readily tyrosine-phosphorylated in vivo, autophosphorylated in vitro, and stimulates cell proliferation in a CSF-1-dependent manner. In contrast, CTR individually expressed in PAE cells showed no significant in vivo, in vitro tyrosine phosphorylation and cell growth in response to CSF-1 stimulation. The kinase activity of CKR was essential for its biological activity, since mutation of lysine 866 to arginine abolished its in vivo, in vitro tyrosine phosphorylation and mitogenic signals. Remarkably, activation of CTR repressed CKR-mediated mitogen-activate protein kinase activation and cell proliferation. Similar effects were observed for VEGFR-2 co-expressed with VEGFR 1. Collectively, these findings demonstrate that VEGFR-2 activation plays a positive role in angiogenesis by promoting endothelial cell proliferation. In contrast, activation of VEGFR-1 plays a stationary role in angiogenesis by antagonizing VEGFR-2 responses. PMID- 10747928 TI - Functional coupling of oxygen binding and vasoactivity in S-nitrosohemoglobin. AB - S-Nitrosohemoglobin (SNO-Hb) is a vasodilator whose activity is allosterically modulated by oxygen ("thermodyamic linkage"). Blood vessel contractions are favored in the oxygenated structure, and vasorelaxant activity is "linked" to deoxygenation, as illustrated herein. We further show that transnitrosation reactions between SNO-Hb and ambient thiols transduce the NO-related bioactivity, whereas NO itself is inactive. One remaining problem is that the amounts of SNO Hb present in vivo are so large as to be incompatible with life were all the S nitrosothiols transformed into bioactive equivalents during each arterial-venous cycle. Experiments were therefore undertaken to address how SNO-Hb conserves its NO-related activity. Our studies show that 1) increased O(2) affinity of SNO-Hb (which otherwise retains allosteric responsivity) restricts the hypoxia-induced allosteric transition that exchanges NO groups with ambient thiols for vasorelaxation; 2) some NO groups released from Cys(beta93) upon transition to T structure are autocaptured by the hemes, even in the presence of glutathione; and 3) an O(2)-dependent equilibrium between SNO-Hb and iron nitrosylhemoglobin acts to conserve NO. Thus, by sequestering a significant fraction of NO liberated upon transition to T structure, Hb can conserve NO groups that would otherwise be released in an untimely or deleterious manner. PMID- 10747929 TI - Functional role of charged residues in the transmembrane segments of the yeast plasma membrane H+-ATPase. AB - As defined by hydropathy analysis, the membrane-spanning segments of the yeast plasma membrane H(+)-ATPase contain seven negatively charged amino acids (Asp and Glu) and four positively charged amino acids (Arg and His). To explore the functional role of these residues, site-directed mutants at all 11 positions and at Glu-288, located near the cytoplasmic end of M3, have been constructed and expressed in yeast secretory vesicles. Substitutions at four of the positions (Glu-129, Glu-288, Asp-833, and Arg-857) had no significant effect on ATP hydrolysis or ATP-dependent proton pumping, substitutions at five additional positions (Arg-695, His-701, Asp-730, Asp-739, and Arg-811) led to misfolding of the ATPase and blockage at an early stage of biogenesis, and substitutions of Asp 143 allowed measurable biogenesis but nearly abolished ATP hydrolysis and proton transport. Of greatest interest were mutations of Glu-703 in M5 and Glu-803 in M8, which altered the apparent coupling between hydrolysis and transport. Three Glu-703 mutants (E703Q, E703L, E703D) showed significantly reduced pumping over a wide range of hydrolysis values and thus appeared to be partially uncoupled. At Glu-803, by contrast, one mutant (E803N) was almost completely uncoupled, while another (E803Q) pumped protons at an enhanced rate relative to the rate of ATP hydrolysis. Both Glu-703 and Glu-803 occupy positions at which amino acid substitutions have been shown to affect transport by mammalian P-ATPases. Taken together, the results provide growing evidence that residues in membrane segments 5 and 8 of the P-ATPases contribute to the cation transport pathway and that the fundamental mechanism of transport has been conserved throughout the group. PMID- 10747930 TI - An unusual carbohydrate binding site revealed by the structures of two Maackia amurensis lectins complexed with sialic acid-containing oligosaccharides. AB - Seeds from the legume tree Maackia amurensis contain two lectins that can agglutinate different blood cell types. Their specificity toward sialylated oligosaccharides is unique among legume lectins; the leukoagglutinin preferentially binds to sialyllactosamine (alphaNeuAc(2-3)betaGal(1 4)betaGlcNAc), whereas the hemagglutinin displays higher affinity for a disialylated tetrasaccharide (alphaNeuAc(2-3)betaGal(1-3)[alphaNeuAc(2-6)]alphaG alNAc). The three-dimensional structure of the complex between M. amurensis leukoagglutinin and sialyllactose has been determined at 2.75-A resolution using x-ray crystallography. The carbohydrate binding site consists of a deep cleft that accommodates the three carbohydrate residues of the sialyllactose. The central galactose sits in the primary binding site in an orientation that has not been observed previously in other legume lectins. The carboxyl group of sialic acid establishes a salt bridge with a lysine side chain. The glucose residue is very efficiently docked between two tyrosine aromatic rings. The complex between M. amurensis hemagglutinin and a disialylated tetrasaccharide could be modeled from the leukoagglutinin/sialyllactose crystal structure. The substitution of one tyrosine by an alanine residue is responsible for the difference in fine specificity between the two isolectins. Comparison with other legume lectins indicates that oligosaccharide specificity within this family is achieved by the recycling of structural loops in different combinations. PMID- 10747931 TI - Transgenic mice expressing a truncated form of the high mobility group I-C protein develop adiposity and an abnormally high prevalence of lipomas. AB - Chromosomal translocations in human lipomas frequently create fusion transcripts encoding high mobility group (HMG) I-C DNA-binding domains and C-terminal sequences from different presumed transcription factors, suggesting a potential role for HMG I-C in the development of lipomas. To evaluate the role of the HMG I C component, the three DNA-binding domains of HMG I-C have now been expressed in transgenic mice. Despite the ubiquitous expression of the truncated HMG I-C protein, the transgenic mice develop a selective abundance of fat tissue early in life, show marked adipose tissue inflammation, and have an abnormally high incidence of lipomas. These findings demonstrate that the DNA-binding domains of HMG I-C, in the absence of a C-terminal fusion partner, are sufficient to perturb adipogenesis and predispose to lipomas. We provide data supporting the central utility of this animal model as a tool to understand the molecular mechanisms underlying the development of one of the most common kind of human benign tumors. PMID- 10747932 TI - Genetic analysis of a potential zinc-binding domain of the adenovirus E4 34k protein. AB - E4 34k, the product of adenovirus early region 4 (E4) open reading frame 6, modulates viral late gene expression, viral DNA replication, apoptosis, double strand break repair, and transformation through multiple interactions with components in infected and transformed cells. Conservation of several cysteine and histidine residues among E4 34k sequences from a variety of adenovirus serotypes suggests the presence of a zinc binding domain important for function. Consistent with the hypothesis that E4 34k is a zinc metalloprotein, zinc binding by baculovirus-expressed E4 34k protein was demonstrated in a zinc blotting assay. To investigate the relationship between the potential zinc-binding region and E4 34k function, a series of mutant genes containing single amino acid substitutions at each of the conserved cysteine and histidine residues in E4 34k were constructed. The mutant proteins were examined for the ability to complement the late protein synthetic defect of an E4 deletion mutant, to physically interact with the viral E1b 55-kDa protein (E1b 55k) and cellular p53 protein, to relocalize E1b 55k, and to destabilize the p53 protein. These analyses identified a subset of cysteine and histidine residues required for stimulation of late gene expression, physical interaction with E1b 55k, and p53 destabilization. These data suggest that a zinc-binding domain participates in the formation of the E4 34k-E1b 55k physical complex and that the complex is required in late gene expression and for p53 destabilization. PMID- 10747933 TI - Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting KasA and KasB condensing enzymes in Mycobacterium tuberculosis. AB - Prevention efforts and control of tuberculosis are seriously hampered by the appearance of multidrug-resistant strains of Mycobacterium tuberculosis, dictating new approaches to the treatment of the disease. Thiolactomycin (TLM) is a unique thiolactone that has been shown to exhibit anti-mycobacterial activity by specifically inhibiting fatty acid and mycolic acid biosynthesis. In this study, we present evidence that TLM targets two beta-ketoacyl-acyl-carrier protein synthases, KasA and KasB, consistent with the fact that both enzymes belong to the fatty-acid synthase type II system involved in fatty acid and mycolic acid biosynthesis. Overexpression of KasA, KasB, and KasAB in Mycobacterium bovis BCG increased in vivo and in vitro resistance against TLM. In addition, a multidrug-resistant clinical isolate was also found to be highly sensitive to TLM, indicating promise in counteracting multidrug-resistant strains of M. tuberculosis. The design and synthesis of several TLM derivatives have led to compounds more potent both in vitro against fatty acid and mycolic acid biosynthesis and in vivo against M. tuberculosis. Finally, a three-dimensional structural model of KasA has also been generated to improve understanding of the catalytic site of mycobacterial Kas proteins and to provide a more rational approach to the design of new drugs. PMID- 10747934 TI - Stopped-flow reaction kinetics of recombinant components of proton-translocating transhydrogenase with physiological nucleotides. AB - New information on the high resolution structure of the membrane proton pump, transhydrogenase, now provides a framework for understanding kinetic descriptions of the enzyme. Here, we have studied redox reactions catalyzed by mixtures of the recombinant NAD(H)-binding component (dI) of Rhodospirillum rubrum transhydrogenase, and the recombinant NADP(H)-binding component (dIII) of either the R. rubrum enzyme or the human enzyme. By recording changes in the fluorescence emission of native and engineered Trp residues, the rates of the redox reaction with physiological nucleotides have been measured under stopped flow conditions, for the first time. Rate constants for the binding reaction between NAD(+)/NADH and the R. rubrum dI.dIII complex are much greater than those between nucleotide and isolated dI. For the redox step between the physiological nucleotides on the R. rubrum dI. dIII complex, the rate constant in the forward direction, k(f) approximately 2900 s(-1), and that for the reverse reaction, k(r) approximately 110 s(-1). Comparisons with reactions involving an analogue of NAD(H) indicate that the rate constants at this step are strongly affected by the redox driving force. PMID- 10747935 TI - GTP binding and signaling by Gh/transglutaminase II involves distinct residues in a unique GTP-binding pocket. AB - G(h) is a dual function protein. It has receptor signaling activity that requires GTP binding and Ca(2+)-activated transglutaminase (TGase) activity that is inhibited by GTP binding. G(h) shows no homology with other GTP-binding proteins, and its GTP-binding site has not been defined. Based on sequence analysis of [alpha-(32)P]GTP-photolabeled and proteolytically released internal peptide fragments, we report localization of GTP binding to a 15-residue segment ((159)YVLTQQGFIYQGSVK(173)) of the G(h) core domain. This was confirmed by site directed mutagenesis; a G(h)/fXIIIA chimera (in which residues 162-179 of G(h) were substituted with the equivalent but nonhomologous region of the non-GTP binding TGase factor XIIIA) and a G(h) point mutant, S171E, retained TGase activity but failed to bind and hydrolyze GTP and did not support alpha(1B) adrenergic receptor signaling. Slight impairment of GTP binding (1.5-fold) and hydrolysis (10-fold) in the absence of altered TGase activity did not affect signaling by the mutant K173N. However, greater impairment of GTP binding (6 fold) and hydrolysis (50-fold) abolished signaling by the mutant K173L. Mutant S171C exhibited enhanced GTP binding and signaling. Thus, residues Ser(171) and Lys(173) are critical for both GTP binding and signaling but not TGase activity. Mutagenesis of residues N-terminal to Gly(170) impaired both GTP binding and TGase activity. From computer modeling of G(h), it is evident that the GTP binding region identified here is distinct from, but interacts with, the TGase active site. Together with structural considerations of G(h) versus other GTP binding proteins, these findings indicate that G(h) has a unique GTP-binding pocket and provide for the first time a mechanism for GTP-mediated regulation of the TGase activity of G(h). PMID- 10747936 TI - Mechanism of inactivation of ornithine transcarbamoylase by Ndelta -(N' Sulfodiaminophosphinyl)-L-ornithine, a true transition state analogue? Crystal structure and implications for catalytic mechanism. AB - The crystal structure is reported at 1.8 A resolution of Escherichia coli ornithine transcarbamoylase in complex with the active derivative of phaseolotoxin from Pseudomonas syringae pv. phaseolicola, N(delta)-(N' sulfodiaminophosphinyl)-l-ornithine. Electron density reveals that the complex is not a covalent adduct as previously thought. Kinetic data confirm that N(delta) (N'-sulfodiaminophosphinyl)-l-ornithine exhibits reversible inhibition with a half-life in the order of approximately 22 h and a dissociation constant of K(D) = 1.6 x 10(-12) m at 37 degrees C and pH 8.0. Observed hydrogen bonding about the chiral tetrahedral phosphorus of the inhibitor is consistent only with the presence of the R enantiomer. A strong interaction is also observed between Arg(57) Nepsilon and the P-N-S bridging nitrogen indicating that imino tautomers of N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine are present in the bound state. An imino tautomer of N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine is structurally analogous to the proposed reaction transition state. Hence, we propose that N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine, with its three unique N-P bonds, represents a true transition state analogue for ornithine transcarbamoylases, consistent with the tight binding kinetics observed. PMID- 10747937 TI - Multimerization potential of the cytoplasmic domain of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41. AB - We previously demonstrated that an envelope mutant of human immunodeficiency virus type 1 lacking the entire cytoplasmic domain interferes in trans with the production of infectious virus by inclusion of the mutant envelope into the wild type envelope complex. We also showed that the envelope incorporation into virions is not affected when the wild-type envelope is coexpressed with the mutant envelope. These results suggest that an oligomeric structure of the cytoplasmic domain is functionally required for viral infectivity. To understand whether the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein gp41 has the potential to self-assemble as an oligomer, in the present study we fused the coding sequence of the entire cytoplasmic domain at 3' to the Escherichia coli malE gene, which encodes a monomeric maltose binding protein. The expressed fusion protein was examined by chemical cross linking, sucrose gradient centrifugation, and gel filtration. The results showed that the cytoplasmic domain of gp41 assembles into a high-ordered structural complex. The intersubunit interaction of the cytoplasmic domain was also confirmed by a mammalian two-hybrid system that detects protein-protein interactions in eucaryotic cells. A cytoplasmic domain fragment expressed in eucaryotic cells was pulled down by glutathione-Sepharose 4B beads via its association with another cytoplasmic domain fragment fused to the C terminus of the glutathione S-transferase moiety. We also found that sequences encompassing the lentiviral lytic peptide-1 and lentiviral lytic peptide-2, which are located within residues 828-856 and 770-795, respectively, play a critical role in cytoplasmic domain self-assembly. Taken together, the results from the present study indicate that the cytoplasmic domain of gp41 by itself is sufficient to assemble into a multimeric structure. This finding supports the hypothesis that a multimeric form of the gp41 cytoplasmic domain plays a crucial role in virus infectivity. PMID- 10747938 TI - Activation of tumor necrosis factor-alpha-converting enzyme-mediated ectodomain shedding by nitric oxide. AB - Ectodomain shedding of cell surface proteins is an important process in a wide variety of physiological and developmental events. Recently, tumor necrosis factor-alpha-converting enzyme (TACE) has been found to play an essential role in the shedding of several critical surface proteins, which is evidenced by multiple developmental defects exhibited by TACE knockout mice. However, little is known about the physiological activation of TACE. Here, we show that nitric oxide (NO) activates TACE-mediated ectodomain shedding. Using an in vitro model of TACE activation, we show that NO activates TACE by nitrosation of the inhibitory motif of the TACE prodomain. Thus, NO production activates the release of cytokines, cytokine receptors, and adhesion molecules, and NO may be involved in other ectodomain shedding processes. PMID- 10747939 TI - Nucleotide binding activity of SecA homodimer is conformationally regulated by temperature and altered by prlD and azi mutations. AB - SecA ATPase is critical for protein translocation across the Escherichia coli inner membrane. To understand this activity further, the high affinity nucleotide binding activity of SecA was characterized. We found that at 4 degrees C SecA homodimer binds one ADP molecule with high affinity. This nucleotide binding activity was conformationally regulated by temperature: at low temperature SecA affinity for ADP was high with a slow exchange rate, whereas at high temperature the converse was true. Azi- and PrlD-SecA proteins that confer azide-resistant and signal sequence suppressor phenotypes were found to have reduced affinity for ADP and accelerated exchange rates compared with wild type SecA. Consistent with this observation, fluorescence and proteolysis studies indicated that these proteins had a conformationally relaxed state at a reduced temperature compared with SecA. The level of Azi- and PrlD-SecA protein was also elevated in inverted membrane vesicles where it displayed higher membrane ATPase activity. These results provide the first direct evidence for conformational regulation of the SecA-dependent nucleotide cycle, its alteration in azi and prlD mutants, and its relevance to in vivo protein export. PMID- 10747940 TI - Identification of amino acid sequences in fibrinogen gamma -chain and tenascin C C-terminal domains critical for binding to integrin alpha vbeta 3. AB - Integrin alpha(v)beta(3) recognizes fibrinogen gamma and alpha(E) chain C terminal domains (gammaC and alpha(E)C) but does not require the gammaC dodecapeptide sequence HHLGGAKQAGDV(400-411) for binding to gammaC. We have localized the alpha(v)beta(3) binding sites in gammaC using gammaC-derived synthetic peptides. We found that two peptides GWTVFQKRLDGSV(190-202) and GVYYQGGTYSKAS(346-358) block the alpha(v)beta(3) binding to gammaC or alpha(E)C, block the alpha(v)beta(3)-mediated clot retraction, and induce the ligand-induced binding site 2 (LIBS2) epitope in alpha(v)beta(3). Neither peptide affects fibrinogen binding to alpha(IIb)beta(3). Scrambled or inverted peptides were not effective. These results suggest that the two gammaC-derived peptides directly interact with alpha(v)beta(3) and specifically block alpha(v)beta(3)-gammaC or alpha(E)C interaction. The two sequences are located next to each other in the gammaC crystal structure, although they are separate in the primary structure. Asp-199, Ser-201, Gln-350, Thr-353, Lys-356, Ala-357, and Ser-358 residues are exposed to the surface. This suggests that the two sequences are part of alpha(v)beta(3) binding sites in fibrinogen gammaC domain. We also found that tenascin C C-terminal fibrinogen-like domain specifically binds to alpha(v)beta(3). Notably, a peptide WYRNCHRVNLMGRYGDNNHSQGVNWFHWKG from this domain that includes the sequence corresponding to gammaC GVYYQGGTYSKAS(346-358) specifically binds to alpha(v)beta(3), suggesting that fibrinogen and tenascin C C-terminal domains interact with alpha(v)beta(3) in a similar manner. PMID- 10747941 TI - The product of the rice myb7 unspliced mRNA dimerizes with the maize leucine zipper Opaque2 and stimulates its activity in a transient expression assay. AB - myb7 mRNA is present in rice in spliced and unspliced forms, splicing being enhanced by anoxia. The protein (Mybleu) encoded by the unspliced mRNA is composed of an incomplete Myb domain followed by a leucine zipper; however, it lacks canonical sequences for DNA binding, transcriptional activation, and nuclear localization. We show here that in transiently transformed tobacco protoplasts, Mybleu is able to enhance the transcriptional activity of the maize leucine zipper Opaque2 on its target b32 promoter. The Mybleu transactivation effect is strictly dependent on the presence of Opaque2 and is driven by Mybleu Opaque2 heterodimers. Mybleu is located in the nucleus, both in rice and in transformed tobacco protoplasts. In rice, the protein is expressed in regions corresponding to undifferentiated cells of roots and coleoptiles. Therefore, myb7 mRNA encodes, depending on its splicing, two transcription factors belonging to separate classes. One of them, Mybleu, has novel structural characteristics, suggesting the existence of new mechanisms acting in the activation of transcription. PMID- 10747943 TI - Functionally accepted insertions of proteins within protein domains. AB - Experiments were designed to explore the tolerance of protein structure and folding to very large insertions of folded protein within a structural domain. Dihydrofolate reductase and beta-lactamase have been inserted in four different positions of phosphoglycerate kinase. The resultant chimeric proteins are all overexpressed, and the host as well as the inserted partners are functional. Although not explicitly designed, functional coupling between the two fused partners was observed in some of the chimeras. These results show that the tolerance of protein structures to very large structured insertions is more general than previously expected and supports the idea that the natural sequence continuity of a structural domain is not required for the folding process. These results directly suggest a new experimental approach to screen, for example, for folded protein in randomized polypeptide sequences. PMID- 10747942 TI - Mapping the DNA binding domain of the Zap1 zinc-responsive transcriptional activator. AB - The Zap1 transcriptional activator of Saccharomyces cerevisiae plays a major role in zinc homeostasis by inducing the expression of several genes under zinc limited growth conditions. This activation of gene expression is mediated by binding of the protein to one or more zinc-responsive elements present in the promoters of its target genes. To better understand how Zap1 functions, we mapped its DNA binding domain using a combined in vivo and in vitro approach. Our results show that the Zap1 DNA binding domain maps to the carboxyl-terminal 194 amino acids of the protein; this region contains five of its seven potential zinc finger domains. Fusing this region to the Gal4 activation domain complemented a zap1Delta mutation for low zinc growth and also conferred high level expression on a zinc-responsive element-lacZ reporter. In vitro, the purified 194-residue fragment bound to DNA with a high affinity (dissociation constant in the low nanomolar range) similar to that of longer fragments of Zap1. Furthermore, by deletion and site-directed mutagenesis, we demonstrated that each of the five carboxyl-terminal zinc fingers are required for high affinity DNA binding. PMID- 10747944 TI - Thermodynamic binding studies of lectins from the diocleinae subtribe to deoxy analogs of the core trimannoside of asparagine-linked oligosaccharides. AB - Lectins from seven different species of the Diocleinae subtribe have been recently isolated and characterized in terms of their carbohydrate binding specificities (Dam, T. K., Cavada, B. S., Grangeiro, T. B., Santos, C. F., de Sousa, F. A. M., Oscarson, S., and Brewer, C. F. (1998) J. Biol. Chem. 273, 12082 12088). The lectins included those from Canavalia brasiliensis, Cratylia floribunda, Dioclea rostrata, Dioclea virgata, Dioclea violacea, and Dioclea guianensis. All of the lectins exhibited specificity for Man and Glc residues, but much higher affinities for the branched chain trimannoside, 3,6-di-O-(alpha-d mannopyranosyl)-d-mannose, which is found in the core region of all asparagine linked carbohydrates. In the present study, isothermal titration microcalorimetry is used to determine the binding thermodynamics of the above lectins, including a new lectin from Canavalia grandiflora, to a complete series of monodeoxy analogs of the core trimannoside. From losses in the affinity constants and enthalpies of binding of certain deoxy analogs, assignments are made of the hydroxyl epitopes on the trimannoside that are involved in binding to the lectins. The pattern of binding of the deoxy analogs is similar for all seven lectins, and similar to that of concanavalin A which is also a member of the Diocleinae subtribe. However, differences in the magnitude of the thermodynamic binding parameters of the lectins are observed, even though the lectins possess conserved contact residues in many cases, and highly conserved primary sequences. The results indicate that non-contact residues in the lectins, even those distant from the binding sites, modulate their thermodynamic binding parameters. PMID- 10747945 TI - Phosphorylation of synaptic vesicle protein 2 modulates binding to synaptotagmin. AB - Synaptic vesicle protein 2 (SV2) is a component of all synaptic vesicles that is required for normal neurotransmission. Here we report that in intact synaptic terminals SV2 is a phosphoprotein. Phosphopeptide mapping studies indicate that a major site of phosphorylation is located on the cytoplasmic amino terminus. SV2 is phosphorylated on serine and threonine but not on tyrosine residues, indicating that it is a substrate for serine/threonine kinases. Phosphopeptide mapping, in gel kinase assays, and surveys of kinase inhibitors suggest that casein kinase I is a primary SV2 kinase. The amino terminus of SV2 was previously shown to mediate its interaction with synaptotagmin, a calcium-binding protein also required for normal neurotransmission. Comparison of synaptotagmin binding with phosphorylated and unphosphorylated SV2 amino-terminal peptides reveals an increase in binding with phosphorylation. These results suggest that the affinity of SV2 for synaptotagmin is modulated by phosphorylation of SV2. PMID- 10747946 TI - Biochemical characterization and molecular cloning of an alpha-1,2 fucosyltransferase that catalyzes the last step of cell wall xyloglucan biosynthesis in pea. AB - Pea microsomes contain an alpha-fucosyltransferase that incorporates fucose from GDP-fucose into xyloglucan, adding it preferentially to the 2-O-position of the galactosyl residue closest to the reducing end of the repeating subunit. This enzyme was solubilized with detergent and purified by affinity chromatography on GDP-hexanolamine-agarose followed by gel filtration. By utilizing peptide sequences obtained from the purified enzyme, a cDNA clone was isolated that encodes a 565-amino acid protein with a predicted molecular mass of 64 kDa and shows 62.3% identity to its Arabidopsis homolog. The purified transferase migrates at approximately 63 kDa by SDS-polyacrylamide gel electrophoresis but elutes from the gel filtration column as an active protein of higher molecular weight ( approximately 250 kDa), indicating that the active form is an oligomer. The enzyme is specific for xyloglucan and is inhibited by xyloglucan oligosaccharides and by the by-product GDP. The enzyme has a neutral pH optimum and does not require divalent ions. Kinetic analysis indicates that GDP-fucose and xyloglucan associate with the enzyme in a random order. N-Ethylmaleimide, a cysteine-specific modifying reagent, had little effect on activity, although several other amino acid-modifying reagents strongly inhibited activity. PMID- 10747947 TI - Beta-chemokine receptor CCR5 signals through SHP1, SHP2, and Syk. AB - The beta-chemokine receptor CCR5 has been shown to modulate cell migration, proliferation, and immune functions and to serve as a co-receptor for the human immunodeficiency virus. We and others have shown that CCR5 activates related adhesion focal tyrosine kinase (RAFTK)/Pyk2/CAK-beta. In this study, we further characterize the signaling molecules activated by CCR5 upon binding to its cognate ligand, macrophage inflammatory protein-1beta (MIP1beta). We observed enhanced tyrosine phosphorylation of the phosphatases SHP1 and SHP2 upon MIP1beta stimulation of CCR5 L1.2 transfectants and T-cells derived from peripheral blood mononuclear cells. Furthermore, we observed that SHP1 associated with RAFTK. However, using a dominant-negative phosphatase-binding mutant of RAFTK (RAFTK(m906)), we found that RAFTK does not mediate SHP1 or SHP2 phosphorylation. SHP1 and SHP2 also associated with the adaptor protein Grb2 and the Src-related kinase Syk. Pretreatment of CCR5 L1.2 transfectants or T-cells with the phosphatase inhibitor orthovanadate markedly abolished MIP1beta-induced chemotaxis. Syk was also activated upon MIP1beta stimulation of CCR5 L1.2 transfectants or T-cells and associated with RAFTK. Overexpression of a dominant negative Src-binding mutant of RAFTK (RAFTK(m402)) significantly attenuated Syk activation, whereas overexpression of wild-type RAFTK enhanced Syk activity, indicating that RAFTK acts upstream of CCR5-mediated Syk activation. Taken together, these results suggest that MIP1beta stimulation mediated by CCR5 induces the formation of a signaling complex consisting of RAFTK, Syk, SHP1, and Grb2. PMID- 10747948 TI - Direct binding and activation of STAT transcription factors by the herpesvirus saimiri protein tip. AB - The Tip protein from Herpesvirus saimiri specifically binds to and activates the protein tyrosine kinase, p56(lck). It has been demonstrated that the expression of Tip in T cells is capable of inducing the DNA binding of members of the signal transducers and activators of transcription (STAT) family of transcription factors. We have examined the mechanism behind which STATs 1 and 3 are activated by Tip expression. Tip becomes tyrosine phosphorylated by p56(lck) at two sites in the amino-terminal tail region. One site of phosphorylation lies within a consensus YXPQ binding motif for the SH2 domains of STATs 1 and 3. We demonstrate that tyrosine phosphorylation of Tip at this site is required for the binding of STATs, and the induction of STAT dependent transcription. Furthermore, we demonstrate that, similar to STAT activation by v-Src, the optimum induction of STAT-dependent transcription by Tip requires Ras/Rac mediated signaling events. PMID- 10747949 TI - A novel mammalian iron-regulated protein involved in intracellular iron metabolism. AB - We have isolated and characterized a novel iron-regulated gene that is homologous to the divalent metal transporter 1 family of metal transporters. This gene, termed metal transporter protein (mtp1), is expressed in tissues involved in body iron homeostasis including the developing and mature reticuloendothelial system, the duodenum, and the pregnant uterus. MTP1 is also expressed in muscle and central nervous system cells in the embryo. At the subcellular level, MTP1 is localized to the basolateral membrane of the duodenal epithelial cell and a cytoplasmic compartment of reticuloendothelial system cells. Overexpression of MTP1 in tissue culture cells results in intracellular iron depletion. In the adult mouse, MTP1 expression in the liver and duodenum are reciprocally regulated. Iron deficiency induces MTP1 expression in the duodenum but down regulates expression in the liver. These data indicate that MTP1 is an iron regulated membrane-spanning protein that is involved in intracellular iron metabolism. PMID- 10747950 TI - Protein-protein interactions of the primase subunits p58 and p48 with simian virus 40 T antigen are required for efficient primer synthesis in a cell-free system. AB - DNA polymerase alpha-primase (pol-prim, consisting of p180-p68-p58-p48), and primase p58-p48 (prim(2)) synthesize short RNA primers on single-stranded DNA. In the SV40 DNA replication system, only pol-prim is able to start leading strand DNA replication that needs unwinding of double-stranded (ds) DNA prior to primer synthesis. At high concentrations, pol-prim and prim(2) indistinguishably reduce the unwinding of dsDNA by SV40 T antigen (Tag). RNA primer synthesis on ssDNA in the presence of replication protein A (RPA) and Tag has served as a model system to study the initiation of Okazaki fragments on the lagging strand in vitro. On ssDNA, Tag stimulates whereas RPA inhibits the initiation reaction of both enzymes. Tag reverses and even overcompensates the inhibition of primase by RPA. Physical binding of Tag to the primase subunits and RPA, respectively, is required for these activities. Each subunit of the primase complex, p58 and p48, performs physical contacts with Tag and RPA independently of p180 and p68. Using surface plasmon resonance, the dissociation constants of the Tag/pol-prim and Tag/primase interactions were 1.2 x 10(-8) m and 1.3 x 10(-8) m, respectively. PMID- 10747951 TI - Peptide mimics of the M13 coat protein transmembrane segment. Retention of helix helix interaction motifs. AB - Sequence-specific noncovalent helix-helix interactions between transmembrane (TM) segments in proteins are investigated by incorporating selected TM sequences into synthetic peptides using the construct CKKK-TM-KKK. The peptides are of suitable hydrophobicity for spontaneous membrane insertion, whereas formation of an N terminal S-S bond can bring pairs of TM helices into proximity and promote their parallel orientation. Using the propensity of the protein to undergo thermally induced alpha-helix --> beta-sheet transitions as a parameter for helix stability, we compared the wild type and mutant (V29A and V31A) bacteriophage M13 coat proteins with their corresponding TM peptide constructs (M13 residues 24 42). Our results demonstrated that the relevant helix-helix tertiary contacts found in the intact proteins persist in the peptide mimics. Molecular dynamics simulations support the tight "two in-two out" dimerization motif for V31A consistent with mutagenesis data. The overall results reinforce the notion of TM segments as autonomous folding domains and suggest that the generic peptide construct provides a viable reductionist system for membrane protein structural and computational analysis. PMID- 10747952 TI - Cloning and expression of the histo-blood group Pk UDP-galactose: Ga1beta 4G1cbeta1-cer alpha1, 4-galactosyltransferase. Molecular genetic basis of the p phenotype. AB - The molecular genetic basis of the P histo-blood group system has eluded characterization despite extensive studies of the biosynthesis of the P(1), P, and P(k) glycolipids. The main controversy has been whether a single or two distinct UDP-Gal:Galbeta1-R 4-alpha-galactosyltransferases catalyze the syntheses of the structurally related P(1) and P(k) antigens. The P(1) polymorphism is linked to 22q11.3-ter. Data base searches with the coding region of an alpha4GlcNAc-transferase identified a novel homologous gene at 22q13.2 designated alpha4Gal-T1. Expression of full coding constructs of alpha4Gal-T1 in insect cells revealed it encoded P(k) but not P(1) synthase activity. Northern analysis showed expression of the transcript correlating with P(k) synthase activity and antigen expression in human B cell lines. Transfection of P(k)-negative Namalwa cells with alpha4Gal-T1 resulted in strong P(k) expression. A single homozygous missense mutation, M183K, was found in six Swedish individuals of the rare p phenotype, confirming that alpha4Gal-T1 represented the P(k) gene. Sequence analysis of the coding region of alpha4Gal-T1 in P(1)+/- individuals did not reveal polymorphisms correlating with P(1)P(2) typing. PMID- 10747953 TI - Analysis of the role of the hypervariable region of yeast Ras2p and its farnesylation in the interaction with exchange factors and adenylyl cyclase. AB - Ras proteins from Saccharomyces cerevisiae differ from mammalian Ha-Ras in their extended C-terminal hypervariable region. We have analyzed the function of this region and the effect of its farnesylation with respect to the action of the GDP/GTP exchange factors (GEFs) Cdc25p and Sdc25p and the target adenylyl cyclase. Whereas Ras2p farnesylation had no effect on the interaction with purified GEFs from the Cdc25 family, this modification became a strict requirement for stimulation of the nucleotide exchange on Ras using reconstituted cell-free systems with GEFs bound to the cell membrane. Determination of GEF effects showed that in cell membrane the Cdc25p dependent activity on Ras2p was predominant over that of Sdc25p. In contrast to full-length GEFs, a membrane bound C-terminal region containing the catalytic domain of Cdc25p was still able to react productively with unfarnesylated Ras2p. These results indicate that in membrane-bound full-length GEF the N-terminal moiety regulates the interaction between catalytic domain and farnesylated Ras2p.GDP. Differently from GEF, full activation of adenylyl cyclase did not require farnesylation of Ras2p.GTP, even if this step of maturation was found to facilitate the interaction. The use of Ha Ras/Ras2p chimaeras of different length emphasized the key role of the hypervariable region of Ras2p in inducing maximum activation of adenylyl cyclase and for a productive interaction with membrane-bound GEF. PMID- 10747954 TI - Increased insulin receptor substrate-1 and enhanced skeletal muscle insulin sensitivity in mice lacking CCAAT/enhancer-binding protein beta. AB - CCAAT/enhancer-binding protein beta (C/EBPbeta) controls gene transcription and metabolic processes in a variety of insulin-sensitive tissues; however, its role in regulating insulin responsiveness in vivo has not been investigated. We performed hyperinsulinemic-euglycemic clamps in awake, non-stressed, chronically catheterized adult mice homozygous for a deletion in the gene for C/EBPbeta (C/EBPbeta(-/-)). Fasting plasma insulin, glucose, and free fatty acid (FFA) levels were significantly lower in C/EBPbeta(-/-) mice compared with wild-type (WT) controls. Acute hyperinsulinemia (4 h) suppressed hepatic glucose production, phosphoenolpyruvate carboxykinase mRNA, and plasma FFA to a similar extent in WT and C/EBPbeta(-/-) mice, suggesting that C/EBPbeta deletion does not alter the metabolic and gene regulatory response to insulin in liver and adipose tissue. In contrast, using submaximal (5 milliunits/kg/min) and maximal (20 milliunits/kg/min) insulin infusions, whole-body glucose disposal was 77% (p < 0.01) and 33% (p < 0.05) higher in C/EBPbeta(-/-) mice, respectively, compared with WT mice. Maximal insulin-stimulated 3-O-methylglucose uptake in isolated soleus muscle was 54% greater in C/EBPbeta(-/-) mice (p < 0.05). Furthermore, insulin-stimulated insulin receptor and Akt Ser(473) phosphorylation and phosphatidylinositol 3-kinase activity were 1.6-2.5-fold greater in skeletal muscle from C/EBPbeta(-/-) mice compared with WT mice. The level of insulin receptor substrate-1 protein was increased 2-fold in skeletal muscle from C/EBPbeta(-/-) mice. These results demonstrate that C/EBPbeta deletion decreases plasma FFA levels and increases insulin signal transduction specifically in skeletal muscle, and both contribute to increased whole-body insulin sensitivity. PMID- 10747955 TI - Sequence specificity, conformation, and recognition by HMG1 protein of major DNA interstrand cross-links of antitumor dinuclear platinum complexes. AB - Interactions of high mobility group (HMG) domain proteins with DNA modified by cisplatin plays a role in mechanisms underlying its antitumor activity. A structural motif recognized by HMG domain proteins on cisplatin-modified DNA is a stable, directional bend of the helix axis. In the present work, bending induced in DNA by major adducts of a novel class of antitumor compounds, represented by the formula [?trans-PtCl(NH(3))(2)?H(2)N(CH(2))(2-6)NH(2)]Cl(2), was investigated. The oligodeoxyribonucleotide duplexes containing various site specific interstrand cross-links of these bifunctional dinuclear platinum drugs were purified and characterized by Maxam-Gilbert footprinting, chemical probing, and phasing assay. It was demonstrated that the cross-links of the dinuclear compounds bent the helix much less than those of cisplatin. Gel retardation assay revealed very weak recognition of DNA adducts of dinuclear complexes by HMG1 protein. Hence, the mediation of antitumor properties of dinuclear platinum complexes by HMG domain proteins is unlikely so that polynuclear platinum compounds may represent a novel class of platinum anticancer drugs acting by a different mechanism than cisplatin and its analogues. A further understanding of how polynuclear platinum compounds modify DNA and how these modifications are processed in cells should provide a rational basis for the design of new platinum drugs rather than searching for cisplatin analogues. PMID- 10747956 TI - Caspase inhibitor P35 and inhibitor of apoptosis Op-IAP block in vivo proteolytic activation of an effector caspase at different steps. AB - Signal-induced activation of caspases, the critical protease effectors of apoptosis, requires proteolytic processing of their inactive proenzymes. Consequently, regulation of procaspase processing is critical to apoptotic execution. We report here that baculovirus pancaspase inhibitor P35 and inhibitor of apoptosis Op-IAP prevent caspase activation in vivo, but at different steps. By monitoring proteolytic processing of endogenous Sf-caspase-1, an insect group II effector caspase, we show that Op-IAP blocked the first activation cleavage at TETD downward arrowG between the large and small caspase subunits. In contrast, P35 failed to affect this cleavage, but functioned downstream to block maturation cleavages (DXXD downward arrow(G/A)) of the large subunit. Substitution of P35's reactive site residues with TETDG failed to increase its effectiveness for blocking TETD downward arrowG processing of pro-Sf-caspase-1, despite wild-type function for suppressing apoptosis. These data are consistent with the involvement of a novel initiator caspase that is resistant to P35, but directly or indirectly inhibitable by Op-IAP. The conservation of TETD downward arrowG processing sites among insect effector caspases, including Drosophila drICE and DCP-1, suggests that in vivo activation of these group II caspases involves a P35 insensitive caspase and supports a model wherein apical and effector caspases function through a proteolytic cascade to execute apoptosis in insects. PMID- 10747957 TI - Nonequivalent nucleotide trapping in the two nucleotide binding folds of the human multidrug resistance protein MRP1. AB - Multidrug resistance protein 1 (MRP1) and P-glycoprotein, which are ATP-dependent multidrug efflux pumps and involved in multidrug resistance of tumor cells, are members of the ATP binding cassette proteins and contain two nucleotide-binding folds (NBFs). P-glycoprotein hydrolyzes ATP at both NBFs, and vanadate-induced nucleotide trapping occurs at both NBFs. We examined vanadate-induced nucleotide trapping in MRP1 stably expressed in KB cell membrane by using 8-azido-[alpha (32)P]ATP. Vanadate-induced nucleotide trapping in MRP1 was found to be stimulated by reduced glutathione, glutathione disulfide, and etoposide and to be synergistically stimulated by the presence of etoposide and either glutathione. These results suggest that glutathione and etoposide interact with MRP1 at different sites and that those bindings cooperatively stimulate the nucleotide trapping. Mild trypsin digestion of MRP1 revealed that vanadate-induced nucleotide trapping mainly occurs at NBF2. Our results suggest that the two NBFs of MRP1 might be functionally nonequivalent. PMID- 10747958 TI - Controlled protein degradation regulates ribonucleotide reductase activity in proliferating mammalian cells during the normal cell cycle and in response to DNA damage and replication blocks. AB - Ribonucleotide reductase (RNR) plays a central role in the formation and control of the optimal levels of deoxyribonucleoside triphosphates, which are required for DNA replication and DNA repair processes. Mammalian RNRs are composed of two nonidentical subunits, proteins R1 and R2. The levels of the limiting R2 protein control overall RNR activity during the mammalian cell cycle, being undetectable in G(1) phase and increasing in S phase. We show that in proliferating mammalian cells, the transcription of the R2 gene, once activated in the beginning of S phase, reaches its maximum 6-7 h later and then declines. Surprisingly, DNA damage and replication blocks neither increase nor prolong the R2 promoter activity in S phase. Instead, the cell cycle activity of the mammalian enzyme is controlled by an S phase/DNA damage-specific stabilization of the R2 protein, which is effective until cells pass into mitosis. PMID- 10747959 TI - TatD is a cytoplasmic protein with DNase activity. No requirement for TatD family proteins in sec-independent protein export. AB - The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. Genes known to be involved in this process include tatA, tatB, and tatC that form an operon with a fourth gene, tatD. The tatD gene product has two homologues in E. coli coded by the unlinked ycfH and yjjV genes. An E. coli strain with in-frame chromosomal deletions in all three of tatD, ycfH, and yjjV exhibits no significant defect in the cellular location of five cofactor-containing enzymes that are synthesized with twin arginine signal peptides. Neither these mutations nor overproduction of the TatD protein cause any discernible effect on the export kinetics of an additional E. coli Tat pathway substrate. It is concluded that proteins of the TatD family have no obligate involvement in protein export by the Tat system. TatD is shown to be a cytoplasmic protein. TatD binds to immobilized Ni(2+) or Zn(2+) affinity columns and exhibits magnesium-dependent DNase activity. Features of the tatA operon that may control TatD expression are discussed. PMID- 10747960 TI - Molecular basis for hyperactivity in tryptophan 409 mutants of neuronal NO synthase. AB - A ferrous heme-NO complex builds up in rat neuronal NO synthase during catalysis and lowers its activity. Mutation of a tryptophan located directly below the heme (Trp(409)) to Phe or Tyr causes hyperactive NO synthesis and less heme-NO complex buildup in the steady state (Adak, S., Crooks, C., Wang, Q., Crane, B. R., Tainer, J. A., Getzoff, E. D., and Stuehr, D. J. (1999) J. Biol. Chem. 274, 26907 26911). To understand the mechanism, we used conventional and stopped flow spectroscopy to compare kinetics of heme-NO complex formation, enzyme activity prior to and after complex formation, NO binding affinity, NO complex stability, and its reaction with O(2) in mutants and wild type nNOS. During the initial phase of NO synthesis, heme-NO complex formation was 3 and 5 times slower in W409F and W409Y, and their rates of NADPH oxidation were 50 and 30% that of wild type, probably due to slower heme reduction. NO complex formation slowed NADPH oxidation in the wild type by 7-fold but reduced mutant activities less than 2 fold, giving mutants higher final activities. NO binding kinetics were similar among mutants and wild type, although in ferrous W409Y (and to a lesser extent W409F) the 436-nm NO complex converted to a 417-nm NO complex with time. Oxidation of the ferrous heme-NO complex to ferric enzyme was 7 times faster in Trp(409) mutants than in wild type. Thus, mutant hyperactivity derives from slower formation and faster decay of the heme-NO complex. Together, these minimize partitioning into the NO-bound form. PMID- 10747961 TI - B-ind1, a novel mediator of Rac1 signaling cloned from sodium butyrate-treated fibroblasts. AB - Sodium butyrate is a multifunctional agent known to inhibit cell proliferation and to induce differentiation by modulating transcription. We have performed differential display analysis to identify transcriptional targets of sodium butyrate in Balb/c BP-A31 mouse fibroblasts. A novel butyrate-induced transcript B-ind1 has been cloned by this approach. The human homologue of this transcript contains an open reading frame that codes for a protein of 370 amino acids without known functional motifs. In transfected cells, the B-ind1 protein has been found to potentiate different effects of the small GTPase Rac1, such as c Jun N-terminal kinase activation and transcriptional activity of nuclear factor kappaB (NF-kappaB). In addition, we have demonstrated that B-ind1 forms complexes with the constitutively activated Rac1 protein. To investigate the role of B-ind1 in Rac1 signaling, we have constructed several deletion mutants of B-ind1 and tested their ability to affect the activation of NF-kappaB by Rac1. Interestingly, the fragment encoding the median region of human B-ind1 acted as a dominant-negative variant to block Rac1-mediated NF-kappaB activity. These data define B-ind1 as a novel component of Rac1-signaling pathways leading to the modulation of gene expression. PMID- 10747962 TI - A protein containing a serine-rich domain with vesicle fusing properties mediates cell cycle-dependent cytosolic pH regulation. AB - Initial differentiation in Dictyostelium involves both asymmetric cell division and a cell cycle-dependent mechanism. We previously identified a gene, rtoA, which when disrupted randomizes the cell cycle-dependent mechanism without affecting either the underlying cell cycle or asymmetric differentiation. We find that in wild-type cells, RtoA levels vary during the cell cycle. Cytosolic pH, which normally varies with the cell cycle, is randomized in rtoA cells. The middle 60% of the RtoA protein is 10 tandem repeats of an 11 peptide-long serine rich motif, which we find has a random coil structure. This domain catalyzes the fusion of phospholipid vesicles in vitro. Conversely, rtoA cells have a defect in the fusion of endocytic vesicles. They also have a decreased exocytosis rate, a decreased pH of endocytic/exocytic vesicles, and an increased average cytosolic pH. Our data indicate that the serine-rich domain of RtoA can mediate membrane fusion and that RtoA can increase the rate of vesicle fusion during processing of endoctyic vesicles. We hypothesize that RtoA modulates initial cell type choice by linking vegetative cell physiology to the cell cycle. PMID- 10747963 TI - Functional interaction between the mouse notch1 intracellular region and histone acetyltransferases PCAF and GCN5. AB - The Notch receptor that plays an important role in cell fate determination is intracellularly cleaved by interaction with the ligand. The cleaved intracellular region (RAMIC) of Notch is translocated into the nucleus and interacts with a DNA binding protein RBP-J to activate transcription of genes that regulate cell differentiation. Although RAMIC has been shown to facilitate the RBP-J-mediated transactivation by displacing the histone deacetylase corepressor complex from RBP-J, there is no evidence demonstrating the involvement of histone acetyltransferases (HATs) in the transactivation. Here we show that mouse Notch1 RAMIC interacts with two conserved HATs, mouse PCAF and GCN5, and recruits each of the HATs to RBP-J. The ankyrin repeats and the transactivation domain of RAMIC and the N-terminal regions of PCAF and GCN5, respectively, are required for the interaction. We also show that not only mouse Notch1 but also Drosophila Notch RAMIC interacts with mouse PCAF and GCN5 in mammalian cells. Furthermore, the RBP J-mediated transactivation activity of RAMIC is repressed by two HAT inhibitor proteins, E1A and Twist. These results suggest that HATs including PCAF and GCN5 play an important role in the RBP-J-mediated transactivation by RAMIC. PMID- 10747964 TI - Structural rearrangements of HIV-1 Tat-responsive RNA upon binding of neomycin B. AB - Binding of human immunodeficiency virus type 1 (HIV-1) transactivator (Tat) protein to Tat-responsive RNA (TAR) is essential for viral replication and is considered a promising starting point for the design of anti-HIV drugs. NMR spectroscopy indicated that the aminoglycosides neomycin B and ribostamycin bind to TAR and that neomycin is able to inhibit Tat binding to TAR. The solution structure of the neomycin-bound TAR has been determined by NMR spectroscopy. Chemical shift mapping and intermolecular nuclear Overhauser effects define the binding region of the aminoglycosides on TAR and give strong evidence for minor groove binding. Based on 15 nuclear Overhauser effect-derived intermolecular distance restraints, a model structure of the TAR-neomycin complex was calculated. Neomycin is bound in a binding pocket formed by the minor groove of the lower stem and the uridine-rich bulge of TAR, which adopts a conformation different from those known. The neamine core of the aminoglycoside (rings I and II) is covered with the bulge, explaining the inhibition of Tat by an allosteric mechanism. Neomycin reduces the volume of the major groove in which Tat is bound and thus impedes essential protein-RNA contacts. PMID- 10747965 TI - Maf transcriptionally activates the mouse p53 promoter and causes a p53-dependent cell death. AB - An increase in the level of the tumor suppressor protein p53 can induce cell cycle arrest or cell death. Although mechanisms for regulating the life span of p53 have been described, there is growing evidence that transcriptional regulation of the p53 gene contributes significantly to controlling p53 protein levels and therefore the fate of a cell. However, the signal transduction pathways that lead to transcriptional activation of the p53 gene are poorly understood. The oncoprotein v-Maf and its cellular counterparts belong to the large combinatorially complex basic leucine zipper family of transcription factors, which include the AP1 family. To date few cellular targets of c-Maf have been identified. It is demonstrated here that v-Maf can bind as a homodimer to a variant Maf recognition element located between -66 and -54 upstream in the mouse p53 promoter. V-Maf and its cellular counterparts are shown to activate p53 expression through this site. The ability of v-Maf to activate p53 expression is modulated by AP1 family members. In addition, overexpression of v-Maf in primary cells leads to a p53-dependent cell death. Thus, Maf and members of the AP1 family are able to regulate p53 expression through this site in the p53 promoter. PMID- 10747966 TI - A pleckstrin homology domain specific for phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P2) and fused to green fluorescent protein identifies plasma membrane PtdIns-4,5-P2 as being important in exocytosis. AB - Kinetically distinct steps can be distinguished in the secretory response from neuroendocrine cells with slow ATP-dependent priming steps preceding the triggering of exocytosis by Ca(2+). One of these priming steps involves the maintenance of phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P(2)) through lipid kinases and is responsible for at least 70% of the ATP-dependent secretion observed in digitonin-permeabilized chromaffin cells. PtdIns-4,5-P(2) is usually thought to reside on the plasma membrane. However, because phosphatidylinositol 4 kinase is an integral chromaffin granule membrane protein, PtdIns-4,5-P(2) important in exocytosis may reside on the chromaffin granule membrane. In the present study we have investigated the localization of PtdIns-4,5-P(2) that is involved in exocytosis by transiently expressing in chromaffin cells a pleckstrin homology (PH) domain that specifically binds PtdIns-4, 5-P(2) and is fused to green fluorescent protein (GFP). The PH-GFP protein predominantly associated with the plasma membrane in chromaffin cells without any detectable association with chromaffin granules. Rhodamine-neomycin, which also binds to PtdIns-4,5-P(2), showed a similar subcellular localization. The transiently expressed PH-GFP inhibited exocytosis as measured by both biochemical and electrophysiological techniques. The results indicate that the inhibition was at a step after Ca(2+) entry and suggest that plasma membrane PtdIns-4,5-P(2) is important for exocytosis. Expression of PH-GFP also reduced calcium currents, raising the possibility that PtdIns-4,5-P(2) in some manner alters calcium channel function in chromaffin cells. PMID- 10747967 TI - Nitric oxide modulates a late step of exocytosis. AB - The effects of nitric oxide (NO) on the late phase of exocytosis have been studied, by amperometry, on Ba(2+)-stimulated chromaffin cells. Acute incubation with NO or NO donors (sodium nitroprusside, spermine-NO, S-nitrosoglutathione) produced a drastic slowdown of the granule emptying. Conversely, cell treatment with N(omega)-nitro-l-arginine methyl ester (a NO synthase inhibitor) or with NO scavengers (methylene blue, 2-(4-carboxyphenyl)-4,4,5, 5-tetramethyl-imidazoline 1-oxyl-3-oxide potassium) accelerated the extrusion of catecholamines from chromaffin granules, suggesting the presence of a NO modulatory tone. The incubation with phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine or zaprinast) or with the cell-permeant cGMP analog 8-bromo-cGMP, mimicked the effects of NO, suggesting the involvement of the guanylate cyclase cascade. NO effects were not related to changes in intracellular Ba(2+). NO did not modify the duration of feet. Effects were evident even on pre-fusioned granules, observed under hypertonic conditions, suggesting that the fusion pore is not the target for NO, which probably acts by modifying the affinity of catecholamines for the intragranular matrix. NO could modify the synaptic transmitter efficacy through a novel mechanism, which involves the regulation of the emptying of secretory vesicles. PMID- 10747968 TI - Effects of incorporation of immunoglobulin G and complement component C1q on uptake and degradation of Alzheimer's disease amyloid fibrils by microglia. AB - Microglia are macrophage-like immune system cells found in the brain. They are associated with Alzheimer's Disease plaques, which contain fibrillar beta-amyloid (fAbeta) and other components such as complement proteins. We have shown previously that murine microglia bind and internalize fAbeta microaggregates via the type A scavenger receptor, but degradation of internalized fAbeta is significantly slower than normal degradation. In this study, we compared internalization by microglia of fAbeta microaggregates to that of anti-Abeta antibody-coated fAbeta (IgG-fAbeta) microaggregates and found that the uptake of the latter is increased by about 1.5-fold versus unmodified fAbeta. The endocytic trafficking of IgG-fAbeta is similar to that of fAbeta microaggregates, following an endosomal/lysosomal pathway. We also compared the internalization of fAbeta microaggregates to that of complement protein, C1q-coated fAbeta microaggregates, and found that the levels of uptake are also increased by about 1.5-fold. Rates of degradation of both types of modified fAbeta microaggregates are unchanged compared with unmodified fAbeta microaggregates. We demonstrated by blocking studies that internalization of IgG-fAbeta is mediated by Fc receptors. These data suggest that, in vivo, several different microglial receptors may play a part in internalizing fAbeta, but the involvement of other receptors may not increase the degradation of fAbeta. PMID- 10747969 TI - The noncatalytic beta-propeller domain of prolyl oligopeptidase enhances the catalytic capability of the peptidase domain. AB - Prolyl oligopeptidase, which is involved in memory disorders, is a member of a new family of serine peptidases. In addition to the peptidase domain, the enzyme contains a beta-propeller, which excludes large peptides from the active site. The enzyme is inhibited with thiol reagents, possibly by reacting with Cys-255 located close to the substrate binding site. This assumption was tested with the Cys-255 --> Thr, Cys-255 --> Ala, and Cys-255 --> Ser variants of prolyl oligopeptidase. In contrast to the wild type enzyme, the Cys-255 --> Thr variant was not inhibited with N-ethylmaleimide, indicating that Cys-255, of the 16 free cysteine residues, exclusively accounts for the enzyme inhibition. Unlike the wild type enzyme that showed a doubly bell-shaped pH rate profile, the modified enzyme displayed a single bell-shaped pH dependence with benzyloxycarbonyl-Gly Pro-naphthylamide. It was the high pH form of the enzyme that virtually disappeared with all three enzyme variants. A substantial reduction was also observed in k(cat)/K(m) for the aminobenzoyl-Ser-Pro-Phe(NO(2))-Ala-OH substrate. The high pK(a) (9.77) of Cys-255 determined by titration with N-ethylmaleimide excluded the possibility that ionization of the thiol group was responsible for generation of the two active enzyme forms. The impaired activity of the enzyme variants could be rationalized in terms of weaker binding, which manifests itself in high K(m) for substrates and high K(i) for inhibitors, like benzyloxycarbonyl Gly-Pro-OH and aminobenzoyl-Ser-d-Pro-Phe(NO(2))-Ala-OH. It was concluded that, besides selecting substrates by size, the beta-propeller domain containing Cys 255 remarkably contributed to catalysis of the peptidase domain. PMID- 10747970 TI - The acute phase response is associated with retinoid X receptor repression in rodent liver. AB - The acute phase response (APR) is associated with decreased hepatic expression of many proteins involved in lipid metabolism. The nuclear hormone receptors peroxisome proliferator-activated receptor alpha (PPARalpha) and liver X receptor (LXR) play key roles in regulation of hepatic lipid metabolism. Because heterodimerization with RXR is crucial for their action, we hypothesized that a decrease in RXR may be one mechanism to coordinately down-regulate gene expression during APR. We demonstrate that lipopolysaccharide (LPS) induces a rapid, dose-dependent decrease in RXRalpha, RXRbeta, and RXRgamma proteins in hamster liver. Maximum inhibition was observed at 4 h for RXRalpha (62%) and RXRbeta (50%) and at 2 h for RXRgamma (61%). These decreases were associated with a marked reduction in RXRalpha, RXRbeta, and RXRgamma mRNA levels. Increased RNA degradation is likely responsible for the repression of RXR, because LPS did not decrease RXRbeta and RXRgamma transcription and only marginally inhibited (38%) RXRalpha transcription. RXR repression was associated with decreased LXRalpha and PPARalpha mRNA levels and reduced RXR x RXR, RXR x PPAR and RXR x LXR binding activities in nuclear extracts. Furthermore, LPS markedly decreased both basal and Wy-14,643-induced expression of acyl-CoA synthetase, a well characterized PPARalpha target. The reduction in hepatic RXR levels alone or in association with other nuclear hormone receptors could be a mechanism for coordinately inhibiting the expression of multiple genes during the APR. PMID- 10747971 TI - Methylation increases the open probability of the epithelial sodium channel in A6 epithelia. AB - We used single channel methods on A6 renal cells to study the regulation by methylation reactions of epithelial sodium channels. 3-Deazaadenosine (3-DZA), a methyltransferase blocker, produced a 5-fold decrease in sodium transport and a 6 fold decrease in apical sodium channel activity by decreasing channel open probability (P(o)). 3-Deazaadenosine also blocked the increase in channel open probability associated with addition of aldosterone. Sodium channel activity in excised "inside-out" patches usually decreased within 1-2 min; in the presence of S-adenosyl-l-methionine (AdoMet), activity persisted for 5-8 min. Sodium channel mean time open (t(open)) before and after patch excision was higher in the presence of AdoMet than in untreated excised patches but less than t(open) in cell-attached patches. Sodium channel activity in excised patches exposed to both AdoMet and GTP usually remained stable for more than 10 min, and P(o) and the number of active channels per patch were close to values in cell-attached patches from untreated cells. These findings suggest that a methylation reaction contributes to the activity of epithelial sodium channels in A6 cells and is directed to some regulatory element closely connected with the channel, whose activity also depends on the presence of intracellular GTP. PMID- 10747972 TI - Molecular basis for the transport of cytochrome P450 2E1 to the plasma membrane. AB - Endoplasmic reticulum-resident cytochrome P450 enzymes that face the cytosol are present on the plasma membrane of hepatocytes, but the molecular origin for their transport to this compartment has until now remained unknown. The molecular basis for the transport of rat ethanol-inducible cytochrome P450 2E1 (CYP2E1) to the plasma membrane was investigated by transfection of several different mutant cDNAs into mouse H2.35 hepatoma cells. Two NH(2)-terminal CYP2E1 mutants were constructed: N(++)2E1, which carried two positive charges in the NH(2) terminus, and 2C-2E1, in which the transmembrane domain of CYP2E1 was replaced with that of CYP2C1, which was previously described to cause retention of CYP2C1 in the endoplasmic reticulum, as well as CYP2E1 COOH-terminally tagged with the vesicular stomatitis virus G protein (VSV-G) epitope (2E1-VSV-G). Immunofluorescent microscopy and cell surface biotinylation experiments revealed that all CYP2E1 variants were present on the extracellular side of the plasma membrane. The VSV-G epitope on CYP2E1 was detected on the outside of the plasma membrane using VSV-G-specific antibodies, indicating that the large COOH-terminal part of CYP2E1 is indeed exposed on the outside of the plasma membrane. The relative levels of CYP2E1, 2C-2E1, and 2E1-VSV-G on the cell surface were found to be about 2% of total cellular enzyme, whereas twice this amount of N(++)2E1 was recovered at the cell surface. Protease protection experiments performed on microsomes isolated from cDNA transfected cells revealed that a small fraction of CYP2E1 and all variant proteins was found to be located in the lumen of the endoplasmic reticulum (type II orientation), whereas the majority of the proteins were in the expected cytosolic or type I orientation. It is concluded that the NH(2)-terminal transmembrane domain of CYP2E1 plays a critical role in directing the protein to the cell surface and that topological inversion of a small fraction of CYP2E1 in the endoplasmic reticulum directs the protein to the plasma membrane. PMID- 10747973 TI - c-Jun NH2-terminal kinase targeting and phosphorylation of heat shock factor-1 suppress its transcriptional activity. AB - The mammalian heat shock transcription factor HSF-1 regulates the expression of the heat shock proteins, molecular chaperones that are involved in cellular processes from higher order assembly to protein degradation. HSF-1 is a phosphorylated monomer under physiological growth conditions and is located mainly in the cytoplasm. Upon activation by a variety of environmental stresses, HSF-1 is translocated into the nucleus, forms trimers, acquires DNA binding activity, is hyperphosphorylated, appears as punctate granules, and increases transcriptional activity of target genes. As cells recover from stress, the punctate granules gradually disappear, and HSF-1 appears in a diffused staining pattern in the cytoplasm and nucleus. We have previously shown that the mitogen activated protein kinase ERK phosphorylates and suppresses HSF-1-driven transcription. Here, we show that c-Jun NH(2)-terminal kinase (JNK) also phosphorylates and inactivates HSF-1. Overexpression of JNK facilitates the rapid disappearance of HSF-1 punctate granules after heat shock. Similar to ERK, JNK binds to HSF-1 in the conserved mitogen-activated protein kinases binding motifs and phosphorylates HSF-1 in the regulatory domain. The overexpression of an HSF-1 green fluorescent protein fusion construct lacking JNK phosphorylation sites causes this HSF-1 mutant to form nuclear granules that remain longer in the nucleus after heat shock. Taken together, these findings indicate that JNK phosphorylates HSF-1 and suppresses its transcriptional activity by rapidly clearing HSF-1 from the sites of transcription. PMID- 10747974 TI - Helicobacter pylori activates mitogen-activated protein kinase cascades and induces expression of the proto-oncogenes c-fos and c-jun. AB - Helicobacter pylori is an etiological agent in the development of mucosa associated lymphoid tissue lymphoma and gastric adenocarcinoma. Patients infected with H. pylori carry a 3-6-fold increased risk of developing cancer compared with uninfected individuals. H. pylori strains expressing the cytotoxin-associated antigen A (CagA) are more frequently associated with the development of neoplasia than cagA-negative strains. However, the molecular mechanism by which H. pylori causes neoplastic transformation remains unclear. Here we report that exposure of gastric epithelial cells to H. pylori induces activation of the transcription factor activator protein 1. Activation of the proto-oncogenes c-fos and c-jun is strongly induced. We show that H. pylori activates the ERK/MAP kinase cascade, resulting in Elk-1 phosphorylation and increased c-fos transcription. H. pylori strains that do not express CagA or that are mutated in cag genes encoded by the CagI pathogenicity island do not induce activator protein 1, MAP kinase activity, or c-fos or c-jun activation. Proto-oncogene activation may represent a crucial step in the pathomechanism of H. pylori induced neoplasia. PMID- 10747975 TI - Alpha 1-fetoprotein transcription factor is required for the expression of sterol 12alpha -hydroxylase, the specific enzyme for cholic acid synthesis. Potential role in the bile acid-mediated regulation of gene transcription. AB - Cholesterol conversion to bile acids occurs via the "classic" (neutral) or the "alternative" (acidic) bile acid biosynthesis pathways. Sterol 12alpha hydroxylase/CYP8b1 is the specific enzyme required for cholic acid synthesis. The levels of this enzyme determine the ratio of cholic acid to chenodeoxycholic acid and thus the hydrophobicity of the circulating bile acid pool. Expression of the 12alpha-hydroxylase gene is tightly down-regulated by hydrophobic bile acids. In this study, we report the characterization of two DNA elements that are required for both the 12alpha-hydroxylase promoter activity and bile acid-mediated regulation. Mutation of these elements suppresses 12alpha-hydroxylase promoter activity. Mutations of any other part of the promoter do not alter substantially the promoter activity or alter regulation by bile acids relative to the wild type promoter. These two DNA elements bind alpha(1)-fetoprotein transcription factor (FTF), a member of the nuclear receptor family. We also show that overexpression of FTF in a non-liver cell line activates the sterol 12alpha-hydroxylase promoter. These studies demonstrate the crucial role of FTF for the expression and regulation of a critical gene in the bile acid biosynthetic pathways. PMID- 10747976 TI - Distribution of the native strain in human alpha 1-antitrypsin and its association with protease inhibitor function. AB - Serine protease inhibitors (serpins) are metastable in their native state. This strain, which is released upon binding to target proteases, is essential for the inhibitory activity of serpins. To understand the structural basis of the native strain, we previously characterized stabilizing mutations of alpha(1) antitrypsin, a prototypical inhibitory serpin, in regions such as the hydrophobic core. The present study evaluates the effects of single point mutations throughout the molecule on stability and protease inhibitory activity. We identified stabilizing mutations in most secondary structures, suggesting that the native strain is distributed throughout the molecule. Examination of the substitution patterns and the structures of the mutation sites revealed surface hydrophobic pockets as a component of the native strain in alpha(1)-antitrypsin, in addition to the previously identified unusual interactions such as side chain overpacking and cavities. Interestingly, many of the stabilizing substitutions did not affect the inhibitory activity significantly. Those that affected the activity were confined in the regions that are mobilized during the complex formation with a target enzyme. The results of our study should be useful for designing proteins with strain and for regulating the stability and functions of serpins. PMID- 10747977 TI - Architectural principles for the structure and function of the glucocorticoid receptor tau 1 core activation domain. AB - A 58-amino acid region mediates the core transactivation activity of the glucocorticoid receptor tau1 activation domain. This tau1 core domain is unstructured in aqueous buffers, but in the presence of trifluoroethanol three alpha-helical segments are induced. Two of these putative structural modules have been tested in different combinations with regard to transactivation potential in vivo and binding capacity to the coactivators in vitro. The results show that whereas single modules are not transcriptionally active, any combination of two or three modules is sufficient, with trimodular constructs having the highest activity. However, proteins containing one, two, or three segments bind Ada2 and cAMP-response element-binding protein with similar affinity. A single segment is thus able to bind a target factor but cannot transactivate target genes significantly. The results are consistent with models in which activation domains are comprised of short activation modules that allow multiple interactions with coactivators. Our results also suggest that an increased number of modules may not result in correspondingly higher affinity but instead that the concentration of binding sites is increased, which gives rise to a higher association rate. This is consistent with a model where the association rate for activator-target factor interactions rather than the equilibrium constant is the most relevant measure of activator potency. PMID- 10747978 TI - Guanylyl cyclase activity associated with putative bifunctional integral membrane proteins in Plasmodium falciparum. AB - We report here that guanylyl cyclase activity is associated with two large integral membrane proteins (PfGCalpha and PfGCbeta) in the human malaria parasite Plasmodium falciparum. Unusually, the proteins appear to be bifunctional; their amino-terminal regions have strong similarity with P-type ATPases, and the sequence and structure of the carboxyl-terminal regions conform to that of G protein-dependent adenylyl cyclases, with two sets of six transmembrane sequences, each followed by a catalytic domain (C1 and C2). However, amino acids that are enzymatically important and present in the C2 domain of mammalian adenylyl cyclases are located in the C1 domain of the P. falciparum proteins and vice versa. In addition, certain key residues in these domains are more characteristic of guanylyl cyclases. Consistent with this, guanylyl cyclase activity was obtained following expression of the catalytic domains of PfGCbeta in Escherichia coli. In P. falciparum, expression of both genes was detectable in the sexual but not the asexual blood stages of the life cycle, and PfGCalpha was localized to the parasite/parasitophorous vacuole membrane region of gametocytes. The profound structural differences identified between mammalian and parasite guanylyl cyclases suggest that aspects of this signaling pathway may be mechanistically distinct. PMID- 10747979 TI - Structure and regulation of the envoplakin gene. AB - Envoplakin, a member of the plakin family of proteins, is a component of desmosomes and the epidermal cornified envelope. To understand how envoplakin expression is regulated, we have analyzed the structure of the mouse envoplakin gene and characterized the promoters of both the human and mouse genes. The mouse gene consists of 22 exons and maps to chromosome 11E1, syntenic to the location of the human gene on 17q25. The exon-intron structure of the mouse envoplakin gene is common to all members of the plakin family: the N-terminal protein domain is encoded by 21 small exons, and the central rod domain and the C-terminal globular domain are coded by a single large exon. The C terminus shows the highest sequence conservation between mouse and human envoplakins and between envoplakin and the other family members. The N terminus is also conserved, with sequence homology extending to Drosophila Kakapo. A region between nucleotides 101 and 288 was necessary for promoter activity in transiently transfected primary keratinocytes. This region is highly conserved between the human and mouse genes and contains at least two different positively acting elements identified by site-directed mutagenesis and electrophoretic mobility shift assays. Mutation of a GC box binding Sp1 and Sp3 proteins or a combined E box and Kruppel-like element interacting with unidentified nuclear proteins virtually abolished promoter activity. 600 base pairs of the mouse upstream sequence was sufficient to drive expression of a beta-galactosidase reporter gene in the suprabasal layers of epidermis, esophagus, and forestomach of transgenic mice. Thus, we have identified a regulatory region in the envoplakin gene that can account for the expression pattern of the endogenous protein in stratified squamous epithelia. PMID- 10747980 TI - Poly-N-acetyllactosamine extension in N-glycans and core 2- and core 4-branched O glycans is differentially controlled by i-extension enzyme and different members of the beta 1,4-galactosyltransferase gene family. AB - Poly-N-acetyllactosamines are attached to N-glycans, O-glycans, and glycolipids and serve as underlying glycans that provide functional oligosaccharides such as sialyl Lewis(X). Poly-N-acetyllactosaminyl repeats are synthesized by the alternate addition of beta1,3-linked GlcNAc and beta1,4-linked Gal by i-extension enzyme (iGnT) and a member of the beta1,4-galactosyltransferase (beta4Gal-T) gene family. In the present study, we first found that poly-N-acetyllactosamines in N glycans are most efficiently synthesized by beta4Gal-TI and iGnT. We also found that iGnT acts less efficiently on acceptors containing increasing numbers of N acetyllactosamine repeats, in contrast to beta4Gal-TI, which exhibits no significant change. In O-glycan biosynthesis, N-acetyllactosamine extension of core 4 branches was found to be synthesized most efficiently by iGnT and beta4Gal TI, in contrast to core 2 branch synthesis, which requires iGnT and beta4Gal-TIV. Poly-N-acetyllactosamine extension of core 4 branches is, however, less efficient than that of N-glycans or core 2 branches. Such inefficiency is apparently due to competition between a donor substrate and acceptor in both galactosylation and N acetylglucosaminylation, since a core 4-branched acceptor contains both Gal and GlcNAc terminals. These results, taken together, indicate that poly-N acetyllactosamine synthesis in N-glycans and core 2- and core 4-branched O glycans is achieved by iGnT and distinct members of the beta4Gal-T gene family. The results also exemplify intricate interactions between acceptors and specific glycosyltransferases, which play important roles in how poly-N-acetyllactosamines are synthesized in different acceptor molecules. PMID- 10747981 TI - Biophysical characterization of gp41 aggregates suggests a model for the molecular mechanism of HIV-associated neurological damage and dementia. AB - In human immunodeficiency virus (HIV)-infected individuals, the level of the HIV envelope protein gp41 in brain tissue is correlated with neurological damage and dementia. In this paper we show by biochemical methods and electron microscopy that the extracellular ectodomain of purified HIV and simian immunodeficiency virus gp41 (e-gp41) forms a mixture of soluble high molecular weight aggregate and native trimer at physiological pH. The e-gp41 aggregate is shown to be largely alpha-helical and relatively stable to denaturants. The high molecular weight form of e-gp41 is variable in size ranging from 7 to 70 trimers, which associate by interactions at the interior of the aggregate involving the loop that connects the N- and C-terminal helices of the e-gp41 core. The trimers are predominantly arranged with their long axes oriented radially, and the width of the high molecular weight aggregate corresponds to the length of two e-gp41 trimers (approximately 200 A). Using both light and electron microscopy combined with immunohistochemistry we show that HIV gp41 accumulates as an extracellular aggregate in the brains of HIV-infected patients diagnosed with dementia. We postulate that the high molecular weight aggregates of e-gp41 are responsible for HIV-associated neurological damage and dementia, consistent with known mechanisms of encephalopathy. PMID- 10747982 TI - Analysis of domains in the IKKalpha and IKKbeta proteins that regulate their kinase activity. AB - The IkappaB kinases IKKalpha and IKKbeta are critical in activating the NF-kappaB pathway. Although these proteins have a similar structure that includes kinase, leucine zipper, and helix-loop-helix domains, they exhibit marked differences in their kinase activity and functional properties. For example, IKKbeta has a 10-20 fold higher level of kinase activity for IkappaBalpha than does IKKalpha. Furthermore, disruption of the murine IKKbeta gene, but not the IKKalpha gene, results in severe defects in activating the NF-kappaB pathway. Mice lacking IKKbeta succumb to severe hepatic apoptosis because of failure to activate the NF kappaB pathway, whereas mice deficient in IKKalpha exhibit skin and skeletal abnormalities and an embryonic lethal phenotype. To better characterize differences in the functional properties of these kinases, hybrid IKK proteins were constructed by domain swapping, and their kinase activity was assayed. These studies demonstrated that differences in the IKKalpha and IKKbeta helix-loop helix domains are primarily responsible for differences in their kinase activity. In contrast, their kinase and leucine zipper domains exhibited relatively conserved function. These studies further define the properties of IKKalpha and IKKbeta, which are involved in their unique regulatory roles. PMID- 10747983 TI - Functional redundancy in the nonspecific RNA binding domain of a class I tRNA synthetase. AB - The sequence of a 228-amino acid nonspecific RNA binding domain appended to the N terminus of a eukaryote tRNA synthetase is shown here to have two lysine-rich clusters (LRCs) that are functionally significant in vivo and in vitro. These two LRCs have unrelated sequences and are separated by a spacer of over 100 amino acids. By using a sensitive test for function in vivo, each LRC is shown to be sufficient in the absence of the other. This sufficiency requires fusion of the spacer to either of the LRCs. Experiments in vitro confirmed that the LRCs are each important for RNA binding. Thus, this nonspecific RNA binding domain has two dissimilar lysine-rich sequence elements that are functionally redundant. Further experiments suggest that this redundancy is not used to dock two molecules of RNA but rather to enhance the overall affinity for a single RNA molecule. PMID- 10747984 TI - Evidence that fertilization activates starfish eggs by sequential activation of a Src-like kinase and phospholipase cgamma. AB - Recent evidence has indicated a requirement for a Src family kinase in initiating Ca(2+) release at fertilization in starfish eggs (Giusti, A. F., Carroll, D. J., Abassi, Y. A., Terasaki, M., Foltz, K. R., and Jaffe, L. A. (1999) J. Biol. Chem. 274, 29318-29322). We now show that injection of Src protein into starfish eggs initiates Ca(2+) release and DNA synthesis, as occur at fertilization. These responses depend on the phosphorylation state of the Src protein; only the kinase active form is effective. Like Ca(2+) release at fertilization, the Ca(2+) release in response to Src protein injection is inhibited by prior injection of the SH2 domains of phospholipase Cgamma. These findings support the conclusion that in starfish, sperm-egg interaction causes egg activation by sequential activation of a Src-like kinase and phospholipase Cgamma. Injection of the SH2 domain of Src, which inhibits Ca(2+) release at fertilization, does not inhibit Ca(2+) release caused by Src protein injection. This indicates that the requirement for a Src SH2 domain interaction is upstream of Src activation in the pathway leading to Ca(2+) release at fertilization. PMID- 10747985 TI - An activated L-selectin mutant with conserved equilibrium binding properties but enhanced ligand recognition under shear flow. AB - Selectins mediate the initial tethering and rolling of leukocytes on vessel walls. Adhesion by selectins is a function of both ligand recognition at equilibrium and mechanical properties of the selectin-ligand bond under applied force. We describe an EGF domain mutant of L-selectin with profoundly augmented adhesiveness over that of native L-selectin but conserved ligand specificity. This mutant, termed LPL, was derived by a substitution of the EGF-like domain of L-selectin with the homologous domain from P-selectin. The mutant bound soluble carbohydrate L-selectin ligand with affinity comparable with that of native L selectin but interacted with all surface-bound ligands much more readily than native L-selectin, in particular under elevated shear flow. Tethers mediated by both native and mutant L-selectin exhibited similar lifetimes under a range of shear stresses, but the rate of bond formation by the mutant was at least 10-fold higher than that of native L-selectin toward distinct L-selectin ligands. Enhanced rate of bond formation by the mutant was associated with profoundly stronger rolling interactions and reduced dependence of rolling on a threshold of shear stress. This is the first demonstration that the EGF domain can modulate the binding of the lectin domain of a selectin to surface-immobilized ligands under shear flow without affecting the equilibrium properties of the selectin toward soluble ligands. PMID- 10747986 TI - In vivo structure of the cell cycle-regulated human cdc25C promoter. AB - The cdc25C promoter is regulated during the cell cycle by the transcriptional repressor CDF-1 that inhibits the activation function of upstream transcriptional activators, most notably the nuclear factor Y/CAAT box binding factor (NF-Y/CBF). In this report a detailed analysis of the in vivo structure of the cdc25C promoter was made. Micrococcus nuclease and methidiumpropyl-EDTA footprinting strongly suggest that the proximal promoter encompassing the cell cycle-dependent element/cell cycle genes homology region and the upstream NF-Y sites is organized in a positioned nucleosome throughout the cell cycle. Furthermore, structural perturbations were detected by DNase I, phenanthroline copper, and KMnO(4) footprinting at the NF-Y binding sites in vivo, which is in agreement with the reported property of NF-Y to bend DNA in vitro. Similar results were obtained with the structurally and functionally related cyclin A promoter. The structural perturbations seen in DNase I and phenanthroline copper footprints were less pronounced in G(0) cells when compared with cycling cells. This presumably reflects a weakened in vivo interaction of NF-Y with its cognate DNA element in G(0). It is likely that these structural perturbations, together with the reported ability of NF-Y to recruit histone acetyl transferase activity, contribute to an opened chromatin structure as a prerequisite for optimal regulation through activation and repression. PMID- 10747987 TI - Characterization and functional expression of cDNAs encoding methionine-sensitive and -insensitive homocysteine S-methyltransferases from Arabidopsis. AB - Plants synthesize S-methylmethionine (SMM) from S-adenosylmethionine (AdoMet), and methionine (Met) by a unique reaction and, like other organisms, use SMM as a methyl donor for Met synthesis from homocysteine (Hcy). These reactions comprise the SMM cycle. Two Arabidopsis cDNAs specifying enzymes that mediate the SMM --> Met reaction (SMM:Hcy S-methyltransferase, HMT) were identified by homology and authenticated by complementing an Escherichia coli yagD mutant and by detecting HMT activity in complemented cells. Gel blot analyses indicate that these enzymes, AtHMT-1 and -2, are encoded by single copy genes. The deduced polypeptides are similar in size (36 kDa), share a zinc-binding motif, lack obvious targeting sequences, and are 55% identical to each other. The recombinant enzymes exist as monomers. AtHMT-1 and -2 both utilize l-SMM or (S,S)-AdoMet as a methyl donor in vitro and have higher affinities for SMM. Both enzymes also use either methyl donor in vivo because both restore the ability to utilize AdoMet or SMM to a yeast HMT mutant. However, AtHMT-1 is strongly inhibited by Met, whereas AtHMT-2 is not, a difference that could be crucial to the control of flux through the HMT reaction and the SMM cycle. Plant HMT is known to transfer the pro-R methyl group of SMM. This enabled us to use recombinant AtHMT-1 to establish that the other enzyme of the SMM cycle, AdoMet:Met S-methyltransferase, introduces the pro-S methyl group. These opposing stereoselectivities suggest a way to measure in vivo flux through the SMM cycle. PMID- 10747988 TI - Expression of a specific glycosyltransferase enzyme regulates T cell death mediated by galectin-1. AB - Galectin-1 induces apoptosis of immature thymocytes and activated T cells, suggesting that galectin-1 regulates cell death in the thymus during selection and in the periphery following an immune response. Although it is known that galectin-1 recognizes lactosamine (Gal-GlcNAc) as a minimal ligand, this disaccharide is ubiquitously expressed on a variety of cell surface glycoproteins. Thus, susceptibility to galectin-1 may be regulated by the presentation of lactosamine on specific oligosaccharide structures created by specific glycosyltransferase enzymes. The core 2 beta-1, 6-N acetylglucosaminyltransferase (core 2 GnT) creates a branched structure on O glycans that can be elongated to present multiple lactosamine sequences. In the thymus, the core 2 GnT is expressed in galectin-1-sensitive thymocyte subsets. In the periphery, an oligosaccharide epitope created by the core 2 GnT is expressed on galectin-1-sensitive activated T-cells. In this report, we demonstrate that expression of the core 2 GnT was necessary and sufficient for galectin-1-induced death of murine T cell lines. In addition, overexpression of the core 2 GnT in mice increased the susceptibility of double positive thymocytes to galectin-1. These data demonstrate that expression of a specific glycosyltransferase can control susceptibility to galectin-1, suggesting that developmentally regulated glycosyltransferase expression may be a mechanism to modulate cell death during T cell development and function. PMID- 10747989 TI - Factor H binding to bone sialoprotein and osteopontin enables tumor cell evasion of complement-mediated attack. AB - Metastatic cancer cells, like trophoblasts of the developing placenta, are invasive and must escape immune surveillance to survive. Complement has long been thought to play a significant role in the tumor surveillance mechanism. Bone sialoprotein (BSP) and osteopontin (OPN, ETA-1) are expressed by trophoblasts and are strongly up-regulated by many tumors. Indeed, BSP has been shown to be a positive indicator of the invasive potential of some tumors. In this report, we show that BSP and OPN form rapid and tight complexes with complement Factor H. Besides its key role in regulating complement-mediated cell lysis, Factor H also appears to play a role when "hijacked" by invading organisms in enabling cellular evasion of complement. We have investigated whether BSP and OPN may play a similar role in tumor cell complement evasion by testing to see whether these glycoproteins could promote tumor cell survival. Recombinant OPN and BSP can protect murine erythroleukemia cells from attack by human complement as well as human MCF-7 breast cancer cells and U-266 myeloma cells from attack by guinea pig complement. The mechanism of this gain of function by tumor cell expression of BSP or OPN has been defined using specific peptides and antibodies to block BSP and OPN protective activity. The expression of BSP and OPN in tumor cells provides a selective advantage for survival via initial binding to alpha(V)beta(3) integrin (both) or CD44 (OPN) on the cell surface, followed by sequestration of Factor H to the cell surface and inhibition of complement mediated cell lysis. PMID- 10747990 TI - Ca2+/Calmodulin reverses phosphatidylinositol 3,4, 5-trisphosphate-dependent inhibition of regulators of G protein-signaling GTPase-activating protein activity. AB - Regulators of G protein signaling (RGS proteins) are GTPase-activating proteins (GAPs) for G(i) and/or G(q) class G protein alpha subunits. RGS GAP activity is inhibited by phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) but not by other lipid phosphoinositides or diacylglycerol. Both the negatively charged head group and long chain fatty acids (C16) are required for binding and inhibition of GAP activity. Amino acid substitutions in helix 5 within the RGS domain of RGS4 reduce binding affinity and inhibition by PIP(3) but do not affect inhibition of GAP activity by palmitoylation. Conversely, the GAP activity of a palmitoylation resistant mutant RGS4 is inhibited by PIP(3). Calmodulin binds all RGS proteins we tested in a Ca(2+)-dependent manner but does not directly affect GAP activity. Indeed, Ca(2+)/calmodulin binds a complex of RGS4 and a transition state analog of Galpha(i1)-GDP-AlF(4)(-). Ca(2+)/calmodulin reverses PIP(3)-mediated but not palmitoylation-mediated inhibition of GAP activity. Ca(2+)/calmodulin competition with PIP(3) may provide an intracellular mechanism for feedback regulation of Ca(2+) signaling evoked by G protein-coupled agonists. PMID- 10747991 TI - Conformational stability is a determinant of ribonuclease A cytotoxicity. AB - Onconasetrade mark, a homolog of bovine pancreatic ribonuclease A (RNase A) with high conformational stability, is cytotoxic and has efficacy as a cancer chemotherapeutic agent. Unlike wild-type RNase A, the G88R variant is toxic to cancer cells. Here, variants in which disulfide bonds were removed from or added to G88R RNase A were used to probe the relationship between conformational stability and cytotoxicity in a methodical manner. The conformational stability of the C40A/G88R/C95A and C65A/C72A/G88R variants is less than that of G88R RNase A. In contrast, a new disulfide bond that links the N and C termini (residues 4 and 118) increases the conformational stability of G88R RNase A and C65A/C72A/G88R RNase A. These changes have little effect on the ribonucleolytic activity of the enzyme or on its ability to evade the cytosolic ribonuclease inhibitor protein. The changes do, however, have a substantial effect on toxicity toward human erythroleukemia cells. Specifically, conformational stability correlates directly with cytotoxicity as well as with resistance to proteolysis. These data indicate that conformational stability is a key determinant of RNase A cytotoxicity and suggest that cytotoxicity relies on avoiding proteolysis. This finding suggests a means to produce new cancer chemotherapeutic agents based on mammalian ribonucleases. PMID- 10747992 TI - Steady-state regulation of the human DNA mismatch repair system. AB - Steady-state levels of human DNA mismatch repair (MMR) transcripts and proteins were measured in MMR-proficient and -deficient cell lines by the newly developed competitive quantitative reverse transcription- polymerase chain reaction and Western analysis normalized with purified proteins. In MMR-proficient cells, hMSH2 is the most abundant MMR protein and is expressed 3 to 5 times more than hMLH1. The hMLH1 protein was expressed 1.5 to 2.5 times more than hPMS2. Steady state levels of mRNA expression correlated well with protein expression. hMSH2 mutated LoVo cells did not express detectable hMSH3 or hMSH6 proteins. Similarly, hMLH1-mutated HCT116 cells did not express detectable hMLH1 or hPMS2 protein, whereas in hMLH1-restored HCT116+ch3 cells, hPMS2 protein was re-expressed. In hMSH6-mutated HCT15 cells, both hMSH3 protein and mRNA were increased. In SV40 transformed lung fibroblasts, all MMR mRNAs and proteins examined were expressed at levels 1.5-5-fold higher than in their nontransformed counterpart. The steady state levels of MMR proteins indicate that substantially more hMutS proteins, which are involved in DNA mismatch recognition, are present in comparison with the hMutL proteins. Stability of hMSH3 and hMSH6 proteins appears to depend upon the presence of the hMSH2 protein, and, similarly, the stability of the hPMS2 protein depends upon hMLH1. When the hMSH6 is mutationally inactivated, hMSH3 increases by both transcriptional up-regulation and enhanced protein stability. A balanced up-regulation of all of the components was seen after viral transformation in a fibroblast model. Quantitative changes of the MMR components are a potential mechanism to modify the DNA MMR capabilities of a cell. PMID- 10747993 TI - Role of a short open reading frame in ribosome shunt on the cauliflower mosaic virus RNA leader. AB - The pregenomic 35 S RNA of cauliflower mosaic virus (CaMV) belongs to the growing number of mRNAs known to have a complex leader sequence. The 612-nucleotide leader contains several short open reading frames (sORFs) and forms an extended hairpin structure. Downstream translation of 35 S RNA is nevertheless possible due to the ribosome shunt mechanism, by which ribosomes are directly transferred from a take-off site near the capped 5' end of the leader to a landing site near its 3' end. There they resume scanning and reach the first long open reading frame. We investigated in detail how the multiple sORFs influence ribosome migration either via shunting or linear scanning along the CaMV leader. The sORFs together constituted a major barrier for the linear ribosome migration, whereas the most 5'-proximal sORF, sORF A, in combination with sORFs B and C, played a positive role in translation downstream of the leader by diverting scanning ribosomes to the shunt route. A simplified, shunt-competent leader was constructed with the most part of the hairpin including all the sORFs except sORF A replaced by a scanning-inhibiting structure. In this leader as well as in the wild type leader, proper translation and termination of sORF A was required for efficient shunt and also for the level of shunt enhancement by a CaMV-encoded translation transactivator. sORF A could be replaced by heterologous sORFs, but a one-codon (start/stop) sORF was not functional. The results implicate that in CaMV, shunt-mediated translation requires reinitiation. The efficiency of the shunt process is influenced by translational properties of the sORF. PMID- 10747994 TI - Limited proteolysis of yeast elongation factor 3. Sequence and location of the subdomains. AB - Elongation factor 3 (EF-3) is an ATPase essential for polypeptide chain synthesis in a variety of yeasts and fungi. We used limited proteolysis to study the organization of the subdomains of EF-3. Trypsinolysis of EF-3 at 30 degrees C resulted in the formation of three fragments with estimated molecular masses of 90, 70, and 50 kDa. Yeast ribosomes protected EF-3 and the large fragments from further degradation. ATP exposed a new tryptic cleavage site and stabilized the 70- and 50-kDa fragments. The conformation of EF-3 as measured by fluorescence spectroscopy did not change upon ATP binding. Poly(G) stimulated proteolysis and quenched the intrinsic fluorescence of EF-3. Using gel mobility shift, we demonstrated a direct interaction between EF-3 and tRNA. Neither tRNA nor rRNA altered the tryptic cleavage pattern. The proteolytic products were sequenced by mass spectrometric analysis. EF-3 is blocked NH(2)-terminally by an acetylated serine. The 90-, 70-, and 50-kDa fragments are also blocked NH(2)-terminally, confirming their origin. The 50-kDa fragment (Ser(2)-Lys(443)) is the most stable domain in EF-3 with no known function. The 70-kDa fragment (Ser(2)-Lys(668)) containing the first nucleotide-binding sequence motif forms the core ATP binding subdomain within the 90-kDa domain. The primary ribosome binding site is located near the loosely structured carboxyl-terminal end. PMID- 10747995 TI - Expression, purification, and biochemical characterization of WbpP, a new UDP GlcNAc C4 epimerase from Pseudomonas aeruginosa serotype O6. AB - B-band lipopolysaccharide is an important virulence factor of the opportunistic pathogen Pseudomonas aeruginosa. WbpP is an enzyme essential for B-band lipopolysaccharide production in serotype O6. Sequence analysis suggests that it is involved in the formation of N-acetylgalacturonic acid. To test this hypothesis, overexpression and biochemical characterization of WbpP were performed. By using spectrophotometric assays and capillary electrophoresis, we show that WbpP is a UDP-GlcNAc C4 epimerase. The K(m) for UDP-GlcNAc and UDP GalNAc are 197 and 224 micrometer, respectively. At equilibrium, 70% of UDP GalNAc is converted to UDP-GlcNAc, whereas the yield of the reverse reaction is only 30%. The enzyme can also catalyze the inter-conversion of non-acetylated substrates, although the efficiency of catalysis is significantly lower. Only 15 and 40% of UDP-Glc and UDP-Gal, respectively, are converted at equilibrium. WbpP contains tightly bound NAD(H) and does not require additional cofactors for activity. It exists as a dimer in its native state. This paper is the first report of expression and characterization of a C4 UDP-GlcNAc epimerase at the biochemical level. Moreover, the characterization of the enzymatic function of WbpP will help clarify ambiguous surface carbohydrate biosynthetic pathways in P. aeruginosa and other organisms where homologues of WbpP exist. PMID- 10747997 TI - Fibrillarin-associated box C/D small nucleolar RNAs in Trypanosoma brucei. Sequence conservation and implications for 2'-O-ribose methylation of rRNA. AB - We report the identification of 17 box C/D fibrillarin-associated small nucleolar RNAs (snoRNAs) from the ancient eukaryote, Trypanosoma brucei. To systematically isolate and characterize these snoRNAs, the T. brucei cDNA for the box C/D snoRNA common protein, fibrillarin, was cloned and polyclonal antibodies to the recombinant fibrillarin protein were generated in rabbits. Immunoprecipitations from T. brucei extracts with the anti-fibrillarin antibodies indicated that this trypanosomatid has at least 30 fibrillarin-associated snoRNAs. We have sequenced seventeen of them and designated them TBR for T. brucei RNA 1-17. All of them bear conserved box C, D, C', and D' elements, a hallmark of fibrillarin associated snoRNAs in eukaryotes. Fourteen of them are novel T. brucei snoRNAs. Fifteen bear potential guide regions to mature rRNAs suggesting that they are involved in 2'-O-ribose methylation. Indeed, eight ribose methylations have been mapped in the rRNA at sites predicted by the snoRNA sequences. Comparative genomics indicates that six of the seventeen are the first trypanosome homologs of known yeast and vertebrate methylation guide snoRNAs. Our results indicate that T. brucei has many fibrillarin-associated box C/D snoRNAs with roles in 2'-O ribose methylation of rRNA and that the mechanism for targeting the nucleotide to be methylated at the fifth nucleotide upstream of box D or D' originated in early eukaryotes. PMID- 10747996 TI - cAMP-dependent phosphorylation of the nuclear encoded 18-kDa (IP) subunit of respiratory complex I and activation of the complex in serum-starved mouse fibroblast cultures. AB - A study is presented on the in vivo effect of elevated cAMP levels induced by cholera toxin on the phosphorylation of subunits of the mitochondrial respiratory complexes and their activities in Balb/c 3T3 mouse fibroblast cultures. Treatment of serum-starved fibroblasts with cholera toxin promoted serine phosphorylation in the 18-kDa subunit of complex I. Phosphorylation of the 18-kDa subunit, in response to cholera toxin treatment of fibroblasts, was accompanied by a 2-3-fold enhancement of the rotenone-sensitive endogenous respiration of fibroblasts, of the rotenone-sensitive NADH oxidase, and of the NADH:ubiquinone oxidoreductase activity of complex I. Direct exposure of fibroblasts to dibutyryl cAMP resulted in an equally potent stimulation of the NADH:ubiquinone oxidoreductase activity. Stimulation of complex I activity and respiration with NAD-linked substrates were also observed upon short incubation of isolated fibroblast mitoplasts with dibutyryl cAMP and ATP, which also promoted phosphorylation of the 18-kDa subunit. These observations document an extension of cAMP-mediated intracellular signal transduction to the regulation of cellular respiration. PMID- 10747999 TI - Isohemoglobin differentiation in the bimodal-breathing amazon catfish Hoplosternum littorale. AB - The bimodal gill(water)/gut(air)-breathing Amazonian catfish Hoplosternum littorale that frequents hypoxic habitats uses "mammalian" 2,3-diphosphoglycerate (DPG) in addition to "piscine" ATP and GTP as erythrocytic O(2) affinity modulators. Its electrophoretically distinct anodic and cathodic hemoglobins (Hb(An) and Hb(Ca)) were isolated for functional and molecular characterization. In contrast to Hb(An), phosphate-free Hb(Ca) exhibits a pronounced reverse Bohr effect (increased O(2) affinity with decreasing pH) that is obliterated by ATP, and opposite pH dependences of K(T) (O(2) association constant of low affinity, tense state) and the overall heat of oxygenation. Dose-response curves indicate small chloride effects and pronounced and differentiated phosphate effects, DPG < ATP < GTP < IHP. Hb(Ca)-O(2) equilibria analyzed in terms of the Monod-Wyman Changeux model show that small T state bond energy differences underlie the differentiated phosphate effects. Synthetic peptides, corresponding to N-terminal fragment of the cytoplasmic domain of trout band 3 protein, undergo oxygenation linked binding to Hb(Ca), suggesting a metabolic regulatory role for this hemoglobin. The amino acid sequences for the alpha and beta chains of Hb(Ca) obtained by Edman degradation and cDNA sequencing show unusual substitutions at the phosphate-binding site that are discussed in terms of its reverse Bohr effect and anion sensitivities. PMID- 10747998 TI - The phosphorylation state of poly(A)-binding protein specifies its binding to poly(A) RNA and its interaction with eukaryotic initiation factor (eIF) 4F, eIFiso4F, and eIF4B. AB - The poly(A)-binding protein (PABP) interacts with the eukaryotic initiation factor (eIF) 4G (or eIFiso4G), the large subunit of eIF4F (or eIFiso4F) to promote translation initiation. In plants, PABP also interacts with eIF4B, a factor that assists eIF4F function. PABP is a phosphoprotein, although the function of its phosphorylation has not been previously investigated. In this study, we have purified the phosphorylated and hypophosphorylated isoforms of PABP from wheat to examine whether its phosphorylation state affects its binding to poly(A) RNA and its interaction with eIF4G, eIFiso4G, or eIF4B. Phosphorylated PABP exhibited cooperative binding to poly(A) RNA even under non-stoichiometric binding conditions, whereas multiple molecules of hypophosphorylated PABP bound to poly(A) RNA only after free poly(A) RNA was no longer available. Together, phosphorylated and hypophosphorylated PABP exhibited synergistic binding. eIF4B interacted with PABP in a phosphorylation state-specific manner; native eIF4B increased the RNA binding activity specifically of phosphorylated PABP and was greater than 14-fold more effective than was recombinant eIF4B, whereas eIF4F promoted the cooperative binding of hypophosphorylated PABP. These data suggest that the phosphorylation state of PABP specifies the type of binding to poly(A) RNA and its interaction with its partner proteins. PMID- 10748000 TI - Rapid Ca2+ influx and diacylglycerol synthesis in growth hormone-mediated islet beta -cell mitogenesis. AB - Growth hormone (GH) is an important mitogenic stimulus for the insulin-producing beta-cell. We investigated the effects of GH on Ca(2+) handling and diacylglycerol (DAG) and cAMP formation in the beta-cell. GH elicited a rapid increase in the cytoplasmic free [Ca(2+)], which required extracellular Ca(2+) and was also blocked by pertussis toxin or protein kinase C (PKC) inhibition. GH also elevated islet DAG content, which should lead to PKC activation. Pertussis toxin and PKC inhibitors obliterated the mitogenicity of GH, suggesting involvement of GTP-binding proteins. PKC activation stimulated beta-cell proliferation, and it also activated phospholipase D. Islet cAMP content was not elevated by GH. Addition of a specific protein kinase A antagonist failed to influence the mitogenicity of GH, whereas a stimulatory cAMP agonist stimulated beta-cell replication. We conclude that GH rapidly increases the beta-cell cytoplasmic free [Ca(2+)] and also evokes a similar increase in DAG content via a phosphatidylcholine-specific phospholipase C, but does not affect mitogen activated protein kinases, phospholipase D, or the cAMP signaling pathway. This rise in DAG may be of importance in translation of the stimulatory signal of GH into a proliferative response by the beta-cell, which seems to occur through GTP binding proteins and PKC-dependent mechanisms. PMID- 10748001 TI - Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands. AB - Xenobiotics induce the transcription of cytochromes P450 (CYPs) 2B and 3A through the constitutive androstane receptor (CAR; NR1I3) and pregnane X receptor (PXR; NR1I2), respectively. In this report, we have systematically compared a series of xenobiotics and natural steroids for their effects on mouse and human CAR and PXR. Our results demonstrate dual regulation of PXR and CAR by a subset of compounds that affect CYP expression. Moreover, there are marked pharmacological differences between the mouse (m) and human (h) orthologs of both CAR and PXR. For example, the planar hydrocarbon 1, 4-bis[2-(3,5-dichloropyridyl-oxy)]benzene activates mCAR and hPXR but has little or no activity on hCAR and mPXR. In contrast, the CAR deactivator androstanol activates both mouse and human PXR. Similarly, the PXR activator clotrimazole is a potent deactivator of hCAR. Using radioligand binding and fluorescence resonance energy transfer assays, we demonstrate that several of the compounds that regulate mouse and human CAR, including natural steroids, bind directly to the receptors. Our results suggest that CAR, like PXR, is a steroid receptor that is capable of recognizing structurally diverse compounds. Moreover, our findings underscore the complexity in the physiologic response to xenobiotics. PMID- 10748002 TI - Identification and characterization of a potent, selective, and orally active antagonist of the CC chemokine receptor-1. AB - The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2 [2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl ]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471). Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1alpha (MIP 1alpha), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (K(i) ranged from 1 nm to 5.5 nm). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca(2+) mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000 fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases. PMID- 10748003 TI - The binding between the stem regions of human growth hormone (GH) receptor compensates for the weaker site 1 binding of 20-kDa human GH (hGH) than that of 22-kDa hGH. AB - Despite the lower site 1 affinity of the 20-kDa human growth hormone (20K-hGH) for the hGH receptor (hGHR), 20K-hGH has the same hGHR-mediated activity as 22 kDa human GH (22K-hGH) at low hGH concentration and even higher activity at high hGH concentration. This study was performed to elucidate the reason why 20K-hGH can activate hGHR to the same level as 22K-hGH. To answer the question, we hypothesized that the binding between the stem regions of hGHR could compensate for the weaker site 1 binding of 20K-hGH than that of 22K-hGH in the sequential binding with hGHR. To demonstrate it, we prepared 15 types of alanine-substituted hGHR gene at the stem region and stably transfected them into Ba/F3 cells. Using these cells, we measured and compared the cell proliferation activities between 20K- and 22K-hGH. As a result, the activity of 20K-hGH was markedly reduced than that of 22K-hGH in three types of mutant hGHR (T147A, H150A, and Y200A). Regarding these mutants, the dissociation constant of hGH at the first and second step (KD1 and KD2) in the sequential binding with two hGHRs was predicted based on the mathematical cell proliferation model and computational simulation. Consequently, it was revealed that the reduction of the activity in 20K-hGH was attributed to the change of not KD1 but KD2. In conclusion, these findings support our hypothesis, which can account for the same potencies for activating hGHR between 20K- and 22K-hGH, although the site 1 affinity of 20K-hGH is lower than that of 22K-hGH. PMID- 10748004 TI - A phosphatidylinositol 3-kinase/Akt pathway, activated by tumor necrosis factor or interleukin-1, inhibits apoptosis but does not activate NFkappaB in human endothelial cells. AB - Tumor necrosis factor (TNF) and interleukin-1 (IL-1) activate the transcription of both anti-apoptotic and pro-inflammatory gene products in human endothelial cells (EC) via NFkappaB. Here we report that both TNF and IL-1 activate the anti apoptotic protein kinase Akt in growth factor and serum-deprived EC, assessed by Western blotting for phospho-Akt. Phosphorylation of Akt is blocked by LY294002 or wortmannin, inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase). Consistent with these biochemical observations, TNF and IL-1 reduce apoptosis caused by growth factor and serum deprivation, and this action is also blocked by LY294002. Although Akt has been reported to activate NFkappaB, LY294002 does not prevent TNF- or IL-1-induced degradation of IkappaBalpha, beta, or epsilon, transcription of NFkappaB-dependent E-selectin or ICAM-1 promoter-reporter genes, or surface expression of E-selectin or ICAM-1 in human EC. LY294002 potentiates the activation of mitogen-activated protein kinases and stress-activated protein kinases by TNF and IL-1, suggesting Akt inhibits these responses. We conclude that TNF and IL-1 activate a PI 3-kinase/Akt anti-apoptotic pathway and that the anti-apoptotic effects of Akt are independent of NFkappaB. Moreover, the PI 3 kinase/Akt pathway does not play a major role in the pro-inflammatory responses of EC to TNF or IL-1. PMID- 10748005 TI - The fate of the initiator tRNAs is sensitive to the critical balance between interacting proteins. AB - Formylation of the initiator tRNA is essential for normal growth of Escherichia coli. The initiator tRNA containing the U35A36 mutation (CUA anticodon) initiates from UAG codon. However, an additional mutation at position 72 (72A --> G) renders the tRNA (G72/U35A36) inactive in initiation because it is defective in formylation. In this study, we isolated U1G72/U35A36 tRNA containing a wobble base pair at 1-72 positions as an intragenic suppressor of the G72 mutation. The U1G72/U35A36 tRNA is formylated and participates in initiation. More importantly, we show that the mismatch at 1-72 positions of the initiator tRNA, which was thus far thought to be the hallmark of the resistance of this tRNA against peptidyl tRNA hydrolase (PTH), is not sufficient. The amino acid attached to the initiator tRNA is also important in conferring protection against PTH. Further, we show that the relative levels of PTH and IF2 influence the path adopted by the initiator tRNAs in protein synthesis. These findings provide an important clue to understand the dual function of the single tRNA(Met) in initiation and elongation, in the mitochondria of various organisms. PMID- 10748006 TI - The structural features of concanavalin A governing non-proline peptide isomerization. AB - The reversible binding of manganese and calcium to concanavalin A determines the carbohydrate binding of the lectin by inducing large conformational changes. These changes are governed by the isomerization of a non-proline peptide bond, Ala-207-Asp-208, positioned in a beta-strand in between the calcium binding site S2 and the carbohydrate specificity-determining loop. The replacement of calcium by manganese allowed us to investigate the structures of the carbohydrate binding, locked state and the inactive, unlocked state of concanavalin A, both with and without metal ions bound. Crystals of unlocked metal-free concanavalin A convert to the locked form with the binding of two Mn(2+) ions. Removal of these ions from the crystals traps metal-free concanavalin A in its locked state, a minority species in solution. The ligation of a metal ion in S2 to unlocked concanavalin A causes bending of the beta-strand foregoing the S2 ligand residues Asp-10 and Tyr-12. This bending disrupts conventional beta-sheet hydrogen bonding and forces the Thr-11 side chain against the Ala-207-Asp-208 peptide bond. The steric strain exerted by Thr-11 is presumed to drive the trans-to-cis isomerization. Upon isomerization, Asp-208 flips into its carbohydrate binding position, and the conformation of the carbohydrate specificity determining loop changes dramatically. PMID- 10748007 TI - Characterization of proexosite I on prothrombin. AB - Activation of prothrombin by factor Xa is accompanied by expression of regulatory exosites I and II on the blood coagulation proteinase, thrombin. Quantitative affinity chromatography and equilibrium binding studies with a fluorescein labeled derivative of the exosite I-specific peptide ligand, hirudin(54-65) ([5F]Hir(54-65) (SO(3)(-)), were employed to identify and characterize this site on human and bovine prothrombin and its expression on thrombin. [5F]Hir(54 65)(SO(3)(-)) showed distinctive fluorescence excitation spectral differences in complexes with prothrombin and thrombin and bound to human prothrombin and thrombin with dissociation constants of 3.2 +/- 0.3 micrometer and 25 +/- 2 nm, respectively, demonstrating a 130-fold increase in affinity for the active proteinase. The bovine proteins similarly showed a 150-fold higher affinity of [5F]Hir(54-65)(SO(3)(-)) for thrombin compared with prothrombin, despite a 2-5 fold lower affinity of the peptides for the bovine proteins. Unlabeled, Tyr(63) sulfated and nonsulfated hirudin peptides bound competitively with [5F]Hir(54 65)(SO(3)(-)) to human and bovine prothrombin and thrombin, exhibiting similar, 40-70-fold higher affinities for the proteinases, although nonsulfated Hir(54-65) bound with 7-17-fold lower affinity than the sulfated analog. These studies characterize proexosite I for the first time as a specific binding site for hirudin peptides on both human and bovine prothrombin that is present in a conformationally distinct, low affinity state and is activated with a approximately 100-fold increase in affinity when thrombin is formed. PMID- 10748008 TI - Role of proexosite I in factor Va-dependent substrate interactions of prothrombin activation. AB - Regulatory exosite I of thrombin is present on prothrombin in a precursor state (proexosite I) that specifically binds the Tyr(63)-sulfated peptide, hirudin(54 65) (Hir(54-65)(SO(3)(-))) and the nonsulfated analog. The role of proexosite I in the mechanism of factor Va acceleration of prothrombin activation was investigated in kinetic studies of the effects of peptide binding. The initial rate of human prothrombin activation by factor Xa was inhibited by the peptides in the presence of factor Va but not in the absence of the cofactor. Factor Xa and factor Va did not bind the peptide with significant affinity compared with prothrombin. Maximum inhibition reduced the factor Va-accelerated rate to a level indistinguishable from the rate in the absence of the cofactor. The effect of Hir(54-65)(SO(3)(-)) on the kinetics of prothrombin activation obeyed a model in which binding of the peptide to proexosite I prevented productive prothrombin interactions with the factor Xa-factor Va complex. Comparison of human and bovine prothrombin as substrates demonstrated a similar correlation between peptide binding and inhibition of factor Va acceleration. Inhibition of prothrombin activation by hirudin peptides was opposed by assembly on phospholipid vesicles of the membrane-bound factor Xa-factor-Va-prothrombin complex. Factor Va interactions of human and bovine prothrombin activation are concluded to share a common mechanism in which proexosite I participates in productive interactions of prothrombin as the substrate of the factor Xa-factor Va complex, possibly by directly mediating productive prothrombin-factor Va binding. PMID- 10748009 TI - The sorting signals for peroxisomal membrane-bound ascorbate peroxidase are within its C-terminal tail. AB - Peroxisomal ascorbate peroxidase (APX) is a carboxyl tail-anchored, type II (N(cytosol)-C(matrix)) integral membrane protein that functions in the regeneration of NAD(+) in glyoxysomes of germinated oilseeds and protection of peroxisomes in other organisms from toxic H(2)O(2). Recently we showed that cottonseed peroxisomal APX was sorted post-translationally from the cytosol to peroxisomes via a novel reticular/circular membranous network that was interpreted to be a subdomain of the endoplasmic reticulum (ER), named peroxisomal ER (pER). Here we report on the molecular signals responsible for sorting peroxisomal APX. Deletions or site-specific substitutions of certain amino acid residues within the hydrophilic C-terminal-most eight-amino acid residues (includes a positively charged domain found in most peroxisomal integral membrane-destined proteins) abolished sorting of peroxisomal APX to peroxisomes via pER. However, the C-terminal tail was not sufficient for sorting chloramphenicol acetyltransferase to peroxisomes via pER, whereas the peptide plus most of the immediately adjacent 21-amino acid transmembrane domain (TMD) of peroxisomal APX was sufficient for sorting. Replacement of the peroxisomal APX TMD with an artificial TMD (devoid of putative sorting sequences) plus the peroxisomal APX C-terminal tail also sorted chloramphenicol acetyltransferase to peroxisomes via pER, indicating that the peroxisomal APX TMD does not possess essential sorting information. Instead, the TMD appears to confer the proper context required for the conserved positively charged domain to function within peroxisomal APX as an overlapping pER sorting signal and a membrane peroxisome targeting signal type 2. PMID- 10748010 TI - Cysteines involved in radical generation and catalysis of class III anaerobic ribonucleotide reductase. A protein engineering study of bacteriophage T4 NrdD. AB - Class III ribonucleotide reductase (RNR) is an anaerobic glycyl radical enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides. We have investigated the importance in the reaction mechanism of nine conserved cysteine residues in class III RNR from bacteriophage T4. By using site-directed mutagenesis, we show that two of the cysteines, Cys-79 and Cys-290, are directly involved in the reaction mechanism. Based on the positioning of these two residues in the active site region of the known three-dimensional structure of the phage T4 enzyme, and their structural equivalence to two cysteine residues in the active site region of the aerobic class I RNR, we suggest that Cys-290 participates in the reaction mechanism by forming a transient thiyl radical and that Cys-79 participates in the actual reduction of the substrate. Our results provide strong experimental evidence for a similar radical-based reaction mechanism in all classes of RNR but also identify important differences between class III RNR and the other classes of RNR as regards the reduction per se. We also identify a cluster of four cysteines (Cys-543, Cys-546, Cys-561, and Cys 564) in the C-terminal part of the class III enzyme, which are essential for formation of the glycyl radical. These cysteines make up a CX(2)C-CX(2)C motif in the vicinity of the stable radical at Gly-580. We propose that the four cysteines are involved in radical transfer between Gly-580 and the cofactor S adenosylmethionine of the activating NrdG enzyme needed for glycyl radical generation. PMID- 10748011 TI - Design of potent beta-lactamase inhibitors by phage display of beta-lactamase inhibitory protein. AB - Beta-lactamase inhibitory protein (BLIP) binds tightly to several beta-lactamases including TEM-1 beta-lactamase (K(i) 0.1 nm). The TEM-1 beta-lactamase/BLIP co crystal structure indicates that two turn regions in BLIP insert into the active site of beta-lactamase to block the binding of beta-lactam antibiotics. Residues from each turn, Asp(49) and Phe(142), mimic interactions made by penicillin G when bound in the beta-lactamase active site. Phage display was used to determine which residues within the turn regions of BLIP are critical for binding TEM-1 beta-lactamase. The sequences of a set of functional mutants from each library indicated that a few sequence types were predominant. These BLIP mutants exhibited K(i) values for beta-lactamase inhibition ranging from 0.01 to 0.2 nm. The results indicate that even though BLIP is a potent inhibitor of TEM-1 beta lactamase, the wild-type sequence of the active site binding region is not optimal and that derivatives of BLIP that bind beta-lactamase extremely tightly can be obtained. Importantly, all of the tight binding BLIP mutants have sequences that would be predicted theoretically to form turn structures. PMID- 10748012 TI - A leucine residue "Gates" solvent but not O2 access to the binding pocket of phascolopsis gouldii hemerythrin. AB - A leucine residue, Leu-98, lines the O(2)-binding pocket in all known hemerythrins. Leu-98 in recombinant Phascolopsis gouldii hemerythrin, was mutated to several other residues of varying sizes (Ala, Val), polarities (Thr, Asp, Asn), and aromaticities (Phe, Tyr, Trp). UV-visible and resonance Raman spectra showed that the di-iron sites in these L98X Hrs are very similar to those in the wild type protein, and several of the L98X hemerythrins formed stable oxy adducts. Despite the apparently tight packing in the pocket, all of the L98X Hrs except for L98W, had second order O(2) association rate constants within a factor of 3 of the wild type value. Similarly, the O(2) dissociation rate constant was essentially unaffected by substitutions of larger (Phe) or smaller (Val, Thr) residues for Leu-98. L98Y Hr showed a 170-fold decrease in the O(2) dissociation rate constant and a large D(2)O effect on this rate, which are attributed to a hydrogen-bonding interaction between the Tyr-98 hydroxyl and the bound O(2). Significant increases in autoxidation rates were observed for all of the L98X Hrs other than X = Tyr. These increases in autoxidation rates are attributed to increased solvent access to the binding pocket caused by inefficient packing (Phe), smaller size (Val, Ala), or increased polarity (Thr, Asp, Asn) of the residue 98 side chain. A leucine at position 98 appears to have the optimal size, shape, and hydrophobicity for inhibition of solvent access. Thus, "gating" of small molecule access to the binding pocket of Hr by Leu-98 is not evident for O(2), but is evident for solvent. PMID- 10748013 TI - Kinetic analysis of nucleotide incorporation by mammalian DNA polymerase delta. AB - The kinetics of nucleotide incorporation into 24/36-mer primer/template DNA by purified fetal calf thymus DNA polymerase (pol) delta was examined using steady state and pre-steady-state kinetics. The role of the pol delta accessory protein, proliferating cell nuclear antigen (PCNA), on DNA replication by pol delta was also examined by kinetic analysis. The steady-state parameter k(cat) was similar for pol delta in the presence and absence of PCNA (0.36 and 0.30 min(-1), respectively); however, the K(m) for dNTP was 20-fold higher in the absence of PCNA (0.067 versus 1.2 microm), decreasing the efficiency of nucleotide insertion. Pre-steady-state bursts of nucleotide incorporation were observed for pol delta in the presence and absence of PCNA (rates of polymerization (k(pol)) of 1260 and 400 min(-1), respectively). The reduction in polymerization rate in the absence of PCNA was also accompanied by a 2-fold decrease in burst amplitude. The steady-state exonuclease rate of pol delta was 0.56 min(-1) (no burst, 10(3) fold lower than the rate of polymerization). The small phosphorothioate effect of 2 for correct nucleotide incorporation into DNA by pol delta.PCNA indicated that the rate-limiting step in the polymerization cycle occurs prior to phosphodiester bond formation. A K(d)(dNTP) value of 0.93 microm for poldelta.dNTP binding was determined by pre-steady-state kinetics. A 5-fold increase in K(d)(DNA) for the pol delta.DNA complex was measured in the absence of PCNA. We conclude that the major replicative mammalian polymerase, pol delta, exhibits kinetic behavior generally similar to that observed for several prokaryotic model polymerases, particularly a rate-limiting step following product formation in the steady state (dissociation of oligonucleotides) and a rate-limiting step (probably conformational change) preceding phosphodiester bond formation. PCNA appears to affect pol delta replication in this model mainly by decreasing the dissociation of the polymerase from the DNA. PMID- 10748014 TI - Degradation of the peroxisome proliferator-activated receptor gamma is linked to ligand-dependent activation. AB - The nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) gamma is a ligand-activated transcription factor that regulates several crucial biological processes such as adipogenesis, glucose homeostasis, and cell growth. It is also the functional receptor for a new class of insulin-sensitizing drugs, the thiazolidinediones, now widely used in the treatment of type 2 diabetes mellitus. Here we report that PPARgamma protein levels are significantly reduced in adipose cells and fibroblasts in response to specific ligands such as thiazolidinediones. Studies with several doses of different ligands illustrate that degradation of PPARgamma correlates well with the ability of ligands to activate this receptor. However, analyses of PPARgamma mutants show that, although degradation does not strictly depend on the transcriptional activity of the receptor, it is dependent upon the ligand-gated activation function 2 (AF2) domain. Proteasome inhibitors inhibited the down-regulation of PPARgamma and ligand activation enhanced the ubiquitination of this receptor. These data indicate that, although ligand binding and activation of the AF2 domain increase the transcriptional function of PPARgamma, these same processes also induce ubiquitination and subsequent degradation of this receptor by the proteasome. PMID- 10748015 TI - Structure and promoter analysis of the human unc-33-like phosphoprotein gene. E box required for maximal expression in neuroblastoma and myoblasts. AB - The human unc-33-like phosphoprotein (hUlip/CRMP-4) is a member of a family of developmentally regulated genes that are highly expressed in the nervous system. Mutations in the C. elegans unc-33 gene lead to worms with abnormal movements. The hUlip gene encodes a 570-amino acid protein with 98% homology to its murine (Ulip) (Byk, T., Dobransky, T., Cifuentes-Diaz, C., and Sobel, A. (1996) J. Neurosci. 16, 688-701) and rat (CRMP-4) (Wang, L. H., and Strittmatter, S. M. (1996) J. Neurosci. 16, 6197-6207) counterparts (Gaetano, C., Matsuo, T., and Thiele, C. J. (1997) J. Biol. Chem. 272, 12195-12201). The hUlip gene was isolated from a human genomic library. It contains 15 exons, including an exon defined by an anaplastic oligodendroglioma expressed sequence tag, and spans at least 61.7 kilobases. hUlip lacks sequences corresponding to the first six exons found in unc-33. unc-33 exons correspond to homologous hUlip exons as follows: VII to 1 and 2, VIII to 3-9, IX to 10-12, and X to 13 and 14. Using the hUlip clone 1 phage, fluorescence in situ hybridization analysis indicates that the hybridization signal localizes to human chromosome 5q32. Deletion analysis of 5' flanking sequences delineated the sequences sufficient to express a reporter gene in both neuroblastoma cells and myoblasts. A consensus MyoD/myogenin binding site is located in a region of the downstream promoter that is nearly identical to its mouse homologue. Mutagenesis shows that this conserved MyoD/myogenin site is necessary for full promoter activity in both myoblasts and neuroblastoma cells. PMID- 10748016 TI - Regulation of plasma membrane Ca2+-ATPase by small GTPases and phosphoinositides in human platelets. AB - We have investigated the restoration of [Ca(2+)](i) in human platelets following the discharge of the intracellular Ca(2+) stores. We found that the plasma membrane Ca(2+)-ATPase is the main mechanism involved in Ca(2+) extrusion in human platelets. Treatment of platelets with the farnesylcysteine analogs, farnesylthioacetic acid and N-acetyl-S-geranylgeranyl-l-cysteine, inhibitors of activation of Ras proteins, accelerated the rate of decay of [Ca(2+)](i) to basal levels after activation with thapsigargin combined with a low concentration of ionomycin, indicating that Ras proteins are involved in the negative regulation of Ca(2+) extrusion. Rho A, which is involved in actin polymerization, was not responsible for this effect. Consistent with this, the actin polymerization inhibitors, cytochalasin D and latrunculin A, did not alter the recovery of [Ca(2+)](i). Activation of human platelets with thapsigargin and ionomycin stimulated the tyrosine phosphorylation of the plasma membrane Ca(2+)-ATPase, a mechanism that was inhibited by farnesylcysteine analogs, suggesting that Ras proteins could regulate Ca(2+) extrusion by mediating tyrosine phosphorylation of the plasma membrane Ca(2+)-ATPase. Treatment of platelets with LY294002, a specific inhibitor of phosphatidylinositol 3- and phosphatidylinositol 4-kinase, resulted in a reduction in the rate of recovery of [Ca(2+)](i) to basal levels, suggesting that the products of these kinases are involved in stimulating Ca(2+) extrusion in human platelets. PMID- 10748017 TI - Towards structural determination of the water-splitting enzyme. Purification, crystallization, and preliminary crystallographic studies of photosystem II from a thermophilic cyanobacterium. AB - A photosystem II preparation from the thermophilic cyanobacterium Synechococcus elongatus, which is especially suitable for three-dimensional crystallization in a fully active form was developed. The efficient purification method applied here yielded 10 mg of protein of a homogenous dimeric complex of about 500 kDa within 2 days. Detailed characterization of the preparation demonstrated a fully active electron transport chain from the manganese cluster to plastoquinone in the Q(B) binding site. The oxygen-evolving activity, 5000-6000 micromol of O(2)/(h.mg of chlorophyll), was the highest so far reported and is maintained even at temperatures as high as 50 degrees C. The crystals obtained by the vapor diffusion method diffracted to a resolution of 4.3 A. The space group was determined to be P2(1)2(1)2(1) with four photosystem II dimers per unit cell. Analysis of the redissolved crystals revealed that activity, supramolecular organization, and subunit composition were maintained during crystallization. PMID- 10748018 TI - Phosphorylation of myosin light chain kinase by p21-activated kinase PAK2. AB - Phosphorylation of myosin II regulatory light chains (RLC) by Ca(2+)/calmodulin dependent myosin light chain kinase (MLCK) is a critical step in the initiation of smooth muscle and non-muscle cell contraction. Post-translational modifications to MLCK down-regulate enzyme activity, suppressing RLC phosphorylation, myosin II activation, and tension development. Here we report that PAK2, a member of the Rho family of GTPase-dependent kinases, regulates isometric tension development and myosin II RLC phosphorylation in saponin permeabilized endothelial monolayers. PAK2 blunts tension development by 75% while inhibiting diphosphorylation of myosin II RLC. Cdc42-activated placenta and recombinant, constitutively active PAK2 phosphorylate MLCK in vitro with a stoichiometry of 1.71 +/- 0. 21 mol of PO(4)/mol of MLCK. This phosphorylation inhibits MLCK phosphorylation of myosin II RLC. PAK2 catalyzes MLCK phosphorylation on serine residues 439 and 991. Binding calmodulin to MLCK blocks phosphorylation of Ser-991 by PAK2. These results demonstrate that PAK2 can directly phosphorylate MLCK, inhibiting its activity and limiting the development of isometric tension. PMID- 10748019 TI - Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein. AB - Structural information on intracellular fusions of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria with endogenous proteins is required as they are increasingly used in cell biology and biochemistry. We have investigated the dynamic properties of GFP alone and fused to a single chain antibody raised against lipopolysaccharide of the outer cell wall of gram-negative bacteria (abbreviated as scFv-GFP). The scFv moiety was functional as was proven in binding assays, which involved the use of both fluorescence correlation spectroscopy observing the binding of scFv-GFP to gram-negative bacteria and a surface plasmon resonance cell containing adsorbed lipopolysaccharide antigen. The rotational motion of scFv-GFP has been investigated with time-resolved fluorescence anisotropy. However, the rotational correlation time of scFv-GFP is too short to account for globular rotation of the whole protein. This result can only be explained by assuming a fast hinge motion between the two fused proteins. A modeled structure of scFv-GFP supports this observation. PMID- 10748020 TI - Modulation of estrogen receptor-alpha transcriptional activity by the coactivator PGC-1. AB - A transcriptional coactivator of the peroxisome proliferator-activated receptor gamma (PPARgamma), PPARgamma-coactivator-1(PGC-1) interacts in a constitutive manner with the hinge domain of PPARgamma and enhances its transcriptional activity. In this study we demonstrate that PGC-1 is a coactivator of estrogen receptor-alpha (ERalpha)-dependent transcriptional activity. However the mechanism by which PGC-1 interacts with ERalpha is different from that of PPARgamma. Specifically, it was determined that the carboxyl terminus of PGC-1 interacts in a ligand-independent manner with the ERalpha hinge domain. In addition, an LXXLL motif within the amino terminus of PGC-1 was shown to interact in an agonist-dependent manner with the AF2 domain within the carboxyl terminus of ERalpha. The ability of PGC-1 to associate with and potentiate the transcriptional activity of an ERalpha-AF2 mutant that is unable to interact with the p160 class of coactivators suggests that this coactivator may have a unique role in estrogen signaling. It is concluded from these studies that PGC-1 is a bona fide ERalpha coactivator, which may serve as a convergence point between PPARgamma and ERalpha signaling. PMID- 10748021 TI - Clitocypin, a new type of cysteine proteinase inhibitor from fruit bodies of mushroom clitocybe nebularis. AB - A novel inhibitor of cysteine proteinases has been isolated from fruit bodies of a mushroom Clitocybe nebularis. The inhibitor was purified to homogeneity by affinity chromatography and gel filtration, followed by reverse-phase high pressure liquid chromatography. The active inhibitor has an apparent molecular mass of about 34 kDa by gel filtration and by SDS-polyacrylamide gel electrophoresis without prior boiling of the sample. Boiling in 2.5% SDS or incubation in 6 m guanidine hydrochloride resulted in a single band of 17 kDa, indicating homodimer composition with no intersubunit disulfide bonds. The inhibitor in nondenaturing buffer is resistant to boiling in water, retaining its activity and dimer composition. The mushroom protein is a tight binding inhibitor of papain (K(i) = 0.59 nm), cathepsin L (K(i) = 0.41 nm), cathepsin B (K(i) = 0.48 micrometer), and bromelain (K(i) = 0.16 micrometer) but is inactive toward cathepsin H, trypsin, and pepsin. Its isoelectric point is 4.4, and sugar analysis indicates the absence of carbohydrate. A single protein sequence of 150 amino acids, containing no cysteine or methionine residues, was obtained by amino acid sequencing. The calculated molecular mass of 16854 Da corresponds well with the value obtained by mass spectrometry. A major part of this sequence was verified by molecular cloning. The monomer sequence is clearly devoid of typical cystatin structure elements and has no similarity to any other known cysteine proteinase inhibitors but bears some similarity to a lectin-like family of proteins from mushrooms. The inhibitor, which is present in at least two other members of the Clitocybe genus, has been named clitocypin (Clitocybe cysteine proteinase inhibitor). PMID- 10748022 TI - Saxiphilin, a saxitoxin-binding protein with two thyroglobulin type 1 domains, is an inhibitor of papain-like cysteine proteinases. AB - The type 1 domain of thyroglobulin is a protein module (Thyr-1) that occurs in a variety of secreted and membrane proteins. Several examples of Thyr-1 modules have been previously identified as inhibitors of the papain family of cysteine proteinases. Saxiphilin is a neurotoxin-binding protein from bullfrog and a homolog of transferrin with a pair of such Thyr-1 modules located in the N-lobe. Saxiphilin is now characterized as a potent inhibitor of three cysteine proteinases as follows: papain, human cathepsin B, and cathepsin L. The stoichiometry of enzyme inhibition reveals that both Thyr-1 domains of saxiphilin inhibit papain (apparent K(i) = 1. 72 nm), but only one of these domains inhibits cathepsin B (K(i) = 1. 67 nm) and cathepsin L (K(i) = 0.02 nm). Physical association of saxiphilin and papain blocked from turnover at the active-site cysteine residue can be detected by cross-linking with glutaraldehyde. The rate of association of saxiphilin and cathepsin B is strongly pH-dependent with an optimum at pH 5.2, reflecting control by at least two H(+)-titratable groups. These results further demonstrate that various Thyr-1 domains are selective inhibitors of cysteine proteinases with utility in the study of protein interactions and degradation. PMID- 10748024 TI - A new variant of the gamma subunit of renal Na,K-ATPase. Identification by mass spectrometry, antibody binding, and expression in cultured cells. AB - The gamma subunit is a specific regulator of Na,K-ATPase expressed mainly in kidney. On SDS-polyacryylamide gel electrophoresis, gamma runs as a doublet, but the origin and significance of the doublet is obscure. Mass spectrometry of the gamma chains of rat kidney Na, K-ATPase shows that gamma(a) (upper) has a mass of 7184.0 +/- 1 Da (carbamidomethyl cysteine), corresponding closely to that for the published sequence without the initiator methionine, while gamma(b) (lower) has a mass of 7337.9 +/- 1Da. Tryptic peptide mapping and sequencing by mass spectrometry reveals that the seven N-terminal residues of gamma(a), TELSANH, are replaced by Ac-MDRWYL in gamma(b), but otherwise the chains are identical. Antibodies raised against peptides TELSANHC and MDRWYLC recognize either gamma(a) or gamma(b) of the Na,K-ATPase, respectively. gamma(a) or gamma(b) cDNAs have been expressed in human embryonic kidney and HeLa cells. The major bands expressed correspond to gamma(a) or gamma(b) of renal Na, K-ATPase. Additional minor bands seen after transfection, namely gamma(a)' in human embryonic kidney and gamma(b)' in HeLa, are presumably cell-specific modifications. The present work clarifies earlier uncertainty regarding doublets seen in kidney and in transfected cells. In particular, the results show that renal Na, K-ATPase contains two variants of the gamma subunit with different sequences but otherwise are unmodified. We discuss the possible functional significance of the two variants. PMID- 10748023 TI - Regulation of phospholipase C-beta 3 activity by Na+/H+ exchanger regulatory factor 2. AB - Among the phospholipase C that catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate, four mammalian phospholipase C-beta (PLC-beta) isotypes (isotypes 1-4) are activated through G protein-coupled receptors (GPCRs). Although the regulation of the PLC-betas by GPCRs and heterotrimeric G proteins has been extensively studied, little is known about the molecular determinants that regulate their activity. The PLC-beta isozymes carry a putative PSD 95/Dlg/ZO-1 (PDZ) binding motif (X(S/T)X(V/L)COOH) at their carboxyl terminus, which is implicated in specific interactions with anchor proteins. Using the yeast two-hybrid system, we identified Na(+)/H(+) exchanger regulatory factor 2 (NHERF2) as a protein that interacted with a C-terminal heptapeptide of PLC beta3. Immunoprecipitation studies revealed that NHERF2 interacts specifically with PLC-beta3, but not with other PLC-beta isotypes. Furthermore, PLC-beta3 interacted with NHERF2 rather than with other PDZ-containing proteins. This interaction required the COOH-terminal NTQL sequence of PLC-beta3 and the second PDZ domain of NHERF2. Interestingly, NHERF2 potentiated the PLC-beta activation by carbachol in COS7 and HeLa cells, while mutant NHERF2, lacking the second PDZ domain, had no such effect. Taken together, the data suggest that NHERF2 may act as a modulator underlying the process of PLC-beta3-mediated signaling. PMID- 10748025 TI - Siderophore-iron uptake in saccharomyces cerevisiae. Identification of ferrichrome and fusarinine transporters. AB - A family of four putative transporters (Arn1p-4p) in Saccharomyces cerevisiae is expressed under conditions of iron deprivation and is regulated by Aft1p, the major iron-dependent transcription factor in yeast. One of these, Arn3p/Sit1p, facilitates the uptake of ferrioxamine B, a siderophore of the hydroxamate class. Here we report that ARN family members facilitated the uptake of iron from the trihydroxamate siderophores ferrichrome, ferrichrome A, and triacetylfusarinine C. Uptake of siderophore-bound iron was dependent on either the high-affinity ferrous iron transport system or the ARN family of transporters. The specificity of each siderophore for individual transporters was determined. Uptake of ferrichrome and ferrichrome A was facilitated by both Arn1p and Arn3p. Uptake of triacetylfusarinine C was facilitated by Arn2p, although small amounts of uptake also occurred through Arn1p and Arn3p. In contrast to the trihydroxamates, uptake of iron from the dihydroxamate rhodotorulic acid occurred only via the high affinity ferrous iron system. Epitope-tagged Arn1p was expressed in intracellular vesicles in a pattern that was indistinguishable from that of Arn3p, whereas Ftr1p, a component of the high-affinity ferrous system, was expressed on the plasma membrane. These data indicate that S. cerevisiae maintains two systems of siderophore uptake, only one of which is located on the plasma membrane. PMID- 10748026 TI - Apoptosis-induced cleavage of beta-catenin by caspase-3 results in proteolytic fragments with reduced transactivation potential. AB - Beta-catenin is a member of the Armadillo repeat protein family with a dual cellular function as a component of both the adherens junction complex and the Wnt/wingless signaling pathway. Here we show that beta-catenin is proteolytically cleaved during anoikis and staurosporine-induced apoptosis. Cleavage of beta catenin was found to be caspase-dependent. Five cleavage products of beta-catenin were identified in vivo and after in vitro cleavage by caspase-3. Amino acid sequencing and mass spectrometry analysis indicated two caspase-3 cleavage sites at the C terminus and three further sites at the N terminus, whereas the central Armadillo repeat region remained unaffected. All beta-catenin cleavage products were still able to associate with E-cadherin and alpha-catenin and were found to be enriched in the cytoplasm. Functional analysis revealed that beta-catenin deletion constructs resembling the observed proteolytic fragments show a strongly reduced transcription activation potential when analyzed in gene reporter assays. We therefore conclude that an important role of the beta-catenin cleavage during apoptosis is the removal of its transcription activation domains to prevent its transcription activation potential. PMID- 10748027 TI - The expression and localization of plasma platelet-activating factor acetylhydrolase in endotoxemic rats. AB - The production of platelet-activating factor (PAF) and PAF-like phospholipids that also bind the PAF receptor are implicated in numerous pathological situations including bacterial endotoxemia and injury-induced oxidative damage. PAF and PAF-like phospholipids are hydrolyzed and inactivated by the enzyme PAF acetylhydrolase. In the intact rat, infusion of lipopolysaccharide (LPS) into a mesenteric vein served as an acute, liver-focused model of endotoxemia. We determined that the liver responds to LPS exposure with the production of plasma type PAF acetylhydrolase mRNA and protein expression specifically in the resident macrophages of the liver. Liver macrophages, defined immunohistochemically using antibodies against ED1, present in livers from saline-treated animals contained no detectable PAF acetylhydrolase. Twenty-four hours following in vivo LPS administration, immunohistochemistry detected a slight increase in the number of ED1 staining cells and the ED1-positive cells now contained an abundance of PAF acetylhydrolase. The systemic administration of LPS resulted in increased expression of PAF acetylhydrolase in several tissues. Of the tissues examined, the greatest increase in PAF acetylhydrolase expression was observed in lung followed by increases in spleen, liver, kidney, and thymus. Additionally, the expression of PAF acetylhydrolase mRNA increased in circulating leukocytes and in peritoneal macrophages in response to systemic exposure to LPS. We examined the regulation of PAF acetylhydrolase expression and demonstrated the administration of the PAF receptor antagonists, BN 50739 and WEB 2170, inhibited by 50% the increase in PAF acetylhydrolase expression in response to LPS. The up-regulation of the plasma-type PAF acetylhydrolase expression constitutes an important mechanism for elevating the local and systemic ability to inactivate PAF and oxidized phospholipids in order to minimize PAF-mediated pathophysiology consequent from exposure to endotoxin. The abundance of PAF acetylhydrolase production in the liver lobule likely limits endotoxin-mediated tissue damage due to PAF synthesis. PMID- 10748028 TI - A new subunit of cytochrome b6f complex undergoes reversible phosphorylation upon state transition. AB - A 15.2-kDa polypeptide, encoded by the nuclear gene PETO, was identified as a novel cytochrome b(6)f subunit in Chlamydomonas reinhardtii. The PETO gene product is a bona fide subunit, subunit V, of the cytochrome b(6)f complex, because (i) it copurifies with the other cytochrome b(6)f subunits in the early stages of the purification procedure, (ii) it is deficient in cytochrome b(6)f mutants accumulating little of the complex, and (iii) it colocalizes with cytochrome f, which migrates between stacked and unstacked membrane regions upon state transition. Sequence analysis and biochemical characterization of subunit V shows that it has a one transmembrane alpha-helix topology with two large hydrophilic domains extending on the stromal and lumenal side of the thylakoid membranes, with a lumenal location of the N terminus. Subunit V is reversibly phosphorylated upon state transition, a unique feature that, together with its topological organization, points to the possible role of subunit V in signal transduction during redox-controlled short term and long term adaptation of the photosynthetic apparatus in eukaryotes. PMID- 10748029 TI - Allosteric regulation of the class III anaerobic ribonucleotide reductase from bacteriophage T4. AB - Ribonucleotide reductase (RNR) is an essential enzyme in all organisms. It provides precursors for DNA synthesis by reducing all four ribonucleotides to deoxyribonucleotides. The overall activity and the substrate specificity of RNR are allosterically regulated by deoxyribonucleoside triphosphates and ATP, thereby providing balanced dNTP pools. We have characterized the allosteric regulation of the class III RNR from bacteriophage T4. Our results show that the T4 enzyme has a single type of allosteric site to which dGTP, dTTP, dATP, and ATP bind competitively. The dissociation constants are in the micromolar range, except for ATP, which has a dissociation constant in the millimolar range. ATP and dATP are positive effectors for CTP reduction, dGTP is a positive effector for ATP reduction, and dTTP is a positive effector for GTP reduction. dATP is not a general negative allosteric effector. These effects are similar to the allosteric regulation of class Ib and class II RNRs, and to the class Ia RNR of bacteriophage T4, but differ from that of the class III RNRs from the host bacterium Escherichia coli and from Lactococcus lactis. The relative rate of reduction of the four substrates was measured simultaneously in a mixed-substrate assay, which mimics the physiological situation and illustrates the interplay between the different effectors in vivo. Surprisingly, we did not observe any significant UTP reduction under the conditions used. Balancing of the pyrimidine deoxyribonucleotide pools may be achieved via the dCMP deaminase and dCMP hydroxymethylase pathways. PMID- 10748030 TI - Biochemical analysis of the Kruppel-associated box (KRAB) transcriptional repression domain. AB - The Kruppel-associated box (KRAB) domain is a 75-amino acid transcriptional repressor module commonly found in eukaryotic zinc finger proteins. KRAB-mediated gene silencing requires binding to the RING-B box-coiled-coil domain of the corepressor KAP-1. Little is known about the biochemical properties of the KRAB domain or the KRAB.KAP-1 complex. Using purified components, a combination of biochemical and biophysical analyses has revealed that the KRAB domain from the KOX1 protein is predominantly a monomer and that the KAP-1 protein is predominantly a trimer in solution. The analyses of electrophoretic mobility shift assays, GST association assays, and plasmon resonance interaction data have indicated that the KRAB binding to KAP-1 is direct, highly specific, and high affinity. The optical biosensor data for the complex was fitted to a model of a one-binding step interaction with fast association and slow dissociation rates, with a calculated K(d) of 142 nm. The fitted R(max) indicated three molecules of KAP-1 binding to one molecule of the KRAB domain, a stoichiometry that is consistent with quantitative SDS-polyacrylamide gel electrophoresis analysis of the complex. These structural and dynamic parameters of the KRAB/KAP-1 interaction have implications for identifying downstream effectors of KAP-1 silencing and the de novo design of new repression domains. PMID- 10748031 TI - Cation hexaammines are selective and potent inhibitors of the CorA magnesium transport system. AB - Cation hexaammines and related compounds are chemically stable analogs of the hydrated form of cations, particularly Mg(2+). We tested the ability of several of these compounds to inhibit transport by the CorA or MgtB Mg(2+) transport systems or the PhoQ receptor kinase for Mg(2+) in Salmonella typhimurium. Cobalt(III)-, ruthenium(II)-, and ruthenium(III)-hexaammines were potent inhibitors of CorA-mediated influx. Cobalt(III)- and ruthenium(III)chloropentaammines were slightly less potent inhibitors of CorA. The compounds inhibited uptake by the bacterial S. typhimurium CorA and by the archaeal Methanococcus jannaschii CorA, which bear only 12% identity in the extracellular periplasmic domain. Cation hexaammines also inhibited growth of S. typhimurium strains dependent on CorA for Mg(2+) uptake but not of isogenic strains carrying a second Mg(2+) uptake system. In contrast, hexacyano cobaltate(III) and ruthenate(II)- and nickel(II)hexaammine had little effect on uptake. The inhibition by the cation hexaammines was selective for CorA because none of the compounds had any effect on transport by the MgtB P-type ATPase Mg(2+) transporter or the PhoQ Mg(2+) receptor kinase. These results demonstrate that cation hexaammines are potent and highly selective inhibitors of the CorA Mg(2+) transport system and further indicate that the initial interaction of the CorA transporter is with a fully hydrated Mg(2+) cation. PMID- 10748032 TI - Transcriptional regulation of the transforming growth factor-beta -inducible mouse germ line Ig alpha constant region gene by functional cooperation of Smad, CREB, and AML family members. AB - Smads regulate transcription of defined genes in response to transforming growth factor-beta (TGF-beta) receptor activation. This process involves functional cross-talk of Smads with transcription factors at responsive DNA elements to achieve maximal transcription activation and specificity. TGF-beta has been shown to induce transcription of the germ line (GL) Ig alpha constant region gene and to direct class switching to IgA antibodies. It has been shown that acute myeloid leukemia (AML) transcription factors cooperate with Smad3 to stimulate transcription from the GL Ig alpha constant region gene promoter. We report here that the TGF-beta-induced transcription from this promoter requires DNA binding of cAMP-response element-binding protein (CREB) to the nearby ATF/cAMP-response element site and of Smads to a nearby Smad binding sequence. At these sites, Smad3/4 cooperates with CREB to activate transcription in response to TGF-beta, and disruption of either binding sequence abolished TGF-beta-induced transcription. In addition, AML1 or AML2 also binds to the promoter and cooperates with Smad3/4, and in this way further enhances the TGF-beta-induced transcriptional activation of the GL Ig alpha promoter. Thus, whereas Smad3/4, CREB, and AML family members bind independently to the respective DNA sequences in the GL Ig alpha promoter, functional synergy of Smads with CREB and AML proteins results in maximal TGF-beta-induced transcription. PMID- 10748033 TI - Sp family members and nuclear factor-Y cooperatively stimulate transcription from the rat pyruvate kinase M gene distal promoter region via their direct interactions. AB - The three distal transcriptional regulatory elements of the rat pyruvate kinase M gene, referred to as boxes A, B, and C, are located around -270 base pairs upstream from the transcriptional initiation site. Electrophoretic mobility shift assays with specific competitors and antibodies show that both box A and box B bind to Sp1 and Sp3 and that box C binds nuclear factor-Y (NF-Y). Luciferase reporter assays revealed that although box A and box B alone have no independent effect on luciferase activities, box C alone stimulates transcription. However, the inclusion of all three elements lead to maximal activity because of a synergistic effect, mainly between box B and box C, suggesting that functional synergism between Sp1/Sp3 and NF-Y is critical for the pyruvate kinase M (PKM) gene distal promoter activity. In fact, co-transfection of a dominant negative mutant of NF-YA (NF-YA29) resulted in a decrease in reporter activity in a box C dependent manner. In addition, the overexpression of Sp1 or Sp3 and NF-Y in Drosophila SL2 cells synergistically stimulated PKM gene distal promoter activity. Using a mammalian two-hybrid system in HeLa cells, it was shown that both Sp1 and Sp3 interacted with NF-YA but not NF-YB and NF-YC. Moreover, glutathione S-transferase pull-down assays revealed that only in vitro translated (35)S-labeled NF-YA interacted with both Sp1 and Sp3 in vitro. A subunit interaction domain of NF-YA, which forms a heterotrimer with NF-YB and NF-YC, is not required for these interactions with Sp1 or Sp3. Thus, we conclude that Sp1, Sp3, and NF-Y stimulate the transcription of the PKM gene via their interactions. PMID- 10748035 TI - The sarco/endoplasmic reticulum calcium-ATPase 2b is an endoplasmic reticulum stress-inducible protein. AB - The sarco/endoplasmic reticulum calcium-ATPase (SERCA) translocates Ca(2+) from the cytosol to the lumen of the endoplasmic reticulum. This Ca(2+) storage is important for cellular processes such as calcium signaling and endoplasmic reticulum (ER)-associated posttranslational protein modifications. We investigated the expression of the SERCA2 and SERCA3 isozymes in PC12 cells exposed to agents interfering with different aspects of the posttranslational protein processing within the ER, thereby activating the ER stress-induced unfolded protein response (UPR). All agents increased the SERCA2b mRNA level 3-4 fold, in parallel with increasing mRNA levels for the ER stress marker proteins BiP/GRP78 and CHOP/GADD153. In contrast, SERCA3 mRNA levels did not change. SERCA2b mRNA stability was not changed, indicating that the mechanism of its up regulation was transcriptional, in accordance with the presence of ER stress response elements in the promoter region of the SERCA2 gene. SERCA2b was also increased at the protein level upon ER stress treatments. Induction of ER stress by tunicamycin, dithiothreitol, or l-azetidine 2-carboxylic acid did not result in depletion of ER calcium, showing that such depletion was not necessary for up regulation of SERCA2b expression or UPR activation in general. We conclude that the SERCA2b expression can be controlled by the UPR pathway independently of ER Ca(2+) depletion. PMID- 10748034 TI - BSAP (Pax5)-importin alpha 1 (Rch1) interaction identifies a nuclear localization sequence. AB - BSAP (Pax5) is an essential transcription factor for early B cell and central nervous system development. In later B cell development, BSAP has been implicated in the regulation of 3' Ig enhancers and a number of B cell-specific genes. Previous studies have suggested a role for BSAP-interacting proteins in the regulation of the function of BSAP. Using the yeast two-hybrid system, we identified importin alpha1 (Rch1) as a BSAP-interacting protein. Importin alpha proteins have been shown to escort proteins into the nucleus through interaction with a nuclear localization signal (NLS), composed of short stretches of basic amino acids. A predicted NLS in BSAP (NKRKRDE, located at amino acids 195-201 in the central domain) was confirmed to be essential for interaction with importin alpha1 by the yeast two-hybrid assay. Physical interaction between BSAP and importin alpha1 was detected in vitro by a glutathione S-transferase (GST) pulldown assay. The NLS sequence in BSAP conferred nuclear localization to green fluorescent protein (GFP)-BSAP fusion proteins. Although the N-terminal paired (DNA-binding) domain of BSAP also conferred nuclear localization when coupled to green fluorescent protein, this domain did not bind to importin alpha1 in the yeast two-hybrid assay. The NLS sequence in the central domain of BSAP binds to the C-terminal 98-amino acid fragment of importin alpha1. PMID- 10748036 TI - A new secreted insect protein belonging to the immunoglobulin superfamily binds insulin and related peptides and inhibits their activities. AB - Insulin and related peptides are key hormones for the regulation of growth and metabolism. Here we describe a novel high affinity insulin-related peptide binding protein (IBP) secreted from cells of the insect Spodoptera frugiperda. This IBP is composed of two Ig-like C2 domains, has a molecular mass of 27 kDa, binds human insulin with an affinity of 70 pm, and inhibits insulin signaling through the insulin receptor. The binding protein also binds insulin-like growth factors I and II, proinsulin, mini-proinsulin, and an insulin analog lacking the last 8 amino acids of the B-chain (des-octa peptide insulin) with high affinity, whereas an insulin analog with a Asp-B10 mutation bound with only 1% of the affinity of human insulin. This binding profile suggests that IBP recognizes a region that is highly conserved in the insulin superfamily but distinct from the classical insulin receptor binding site. The closest homologue of the Spodoptera frugiperda binding protein is the essential gene product IMP-L2, found in Drosophila, where it is implicated in neural and ectodermal development (Garbe, J. C., Yang, E., and Fristrom, J. W. (1993) Development 119, 1237-1250). Here we show that the IMP-L2 protein also binds insulin and related peptides, offering a possible functional explanation to the IMP-L2 null lethality. PMID- 10748037 TI - Independent regulation of the myotonic dystrophy 1 locus genes postnatally and during adult skeletal muscle regeneration. AB - Myotonic dystrophy is caused by a CTG(n) expansion in the 3'-untranslated region of a serine/threonine protein kinase gene (DMPK), which is flanked by two other genes, DMWD and SIX5. One hypothesis to explain the wide-ranging effects of this expansion is that, as the mutation expands, it alters the expression of one or more of these genes. The effects may vary in different tissues and developmental stages, but it has been difficult to develop these hypotheses as the normal postnatal developmental expression patterns of these genes have not been adequately investigated. We have developed accurate transcript quantification based on fluorescent real-time reverse transcription-polymerase chain reaction (TaqMan) to develop gene expression profiles during postnatal development in C57Bl/10 mice. Our results show extensive independent postnatal regulation of the myotonic dystrophy-locus genes in selected tissues and demonstrate which are the most highly expressed of the genes in each tissue. All three genes at the locus are expressed in the adult lens, questioning a previous model of cataractogenesis mediated solely by effects on Six5 expression. Additionally, using an in vivo model, we have shown that Dmpk levels decrease during the early stages of muscle regeneration. Our data provide a framework for investigation of tissue-specific pathological mechanisms in this disorder. PMID- 10748038 TI - The aglycone specificity-determining sites are different in 2, 4-dihydroxy-7 methoxy-1,4-benzoxazin-3-one (DIMBOA)-glucosidase (Maize beta -glucosidase) and dhurrinase (Sorghum beta -glucosidase). AB - The maize beta-glucosidase isozyme Glu1 hydrolyzes a broad spectrum of substrates in addition to its natural substrate DIMBOAGlc (2-O-beta-d-glucopyranosyl-4 hydroxy-7-methoxy-1,4-benzoxazin-3-on e), whereas the sorghum beta-glucosidase isozyme Dhr1 hydrolyzes exclusively its natural substrate dhurrin (p-hydroxy-(S) mandelonitrile-beta-d-glucose). To study the mechanism of substrate specificity further, eight chimeric beta-glucosidases were constructed by replacing peptide sequences within the C-terminal region of Glu1 with the homologous peptide sequences of Dhr1 or vice versa, where the two enzymes differ by 4 to 22 amino acid substitutions, depending on the length of the swapped regions. Five Glu1/Dhr1 chimeras hydrolyzed substrates that are hydrolyzed by both parental enzymes, including dhurrin, which is not hydrolyzed by Glu1. In contrast, three Dhr1/Glu1 chimeras hydrolyzed only dhurrin but with lower catalytic efficiency than Dhr1. Additional domain-swapping within the C-terminal domain of Glu1 showed that replacing the peptide (466)FAGFTERY(473) of Glu1 with the homologous peptide (462)SSGYTERF(469) of Dhr1 or replacing the peptide (481)NNNCTRYMKE(490) in Glu1 with the homologous peptide (477)ENGCERTMKR(486) of Dhr1 was sufficient to confer to Glu1 the ability to hydrolyze dhurrin. Data from various reciprocal chimeras, sequence comparisons, and homology modeling suggest that the Dhr1-specific Ser 462-Ser-463 and Phe-469 play a key role in dhurrin hydrolysis. Similar data suggest that DIMBOAGlc hydrolysis determinants are not located within the extreme 47-amino acid-long C-terminal domain of Glu1. PMID- 10748039 TI - Recombinant fibrinogen Vlissingen/Frankfurt IV. The deletion of residues 319 and 320 from the gamma chain of firbinogen alters calcium binding, fibrin polymerization, cross-linking, and platelet aggregation. AB - We synthesized a variant, recombinant fibrinogen modeled after the heterozygous dysfibrinogen Vlissingen/Frankfurt IV, a deletion of two residues, gammaAsn-319 and gammaAsp-320, located within the high affinity calcium-binding pocket. Turbidity studies showed no evidence of fibrin polymerization, although size exclusion chromatography, transmission electron microscopy, and dynamic light scattering studies showed small aggregates. These aggregates did not resemble normal protofibrils nor did they clot. Fibrinopeptide A release was normal, whereas fibrinopeptide B release was delayed approximately 3-fold. Plasmin cleavage of this fibrinogen was not changed by the presence of calcium or Gly-Pro Arg-Pro, indicating that both the calcium-binding site and the "a" polymerization site were non-functional. We conclude that the loss of normal polymerization was due to the lack of "A-a" interactions. Moreover, functions associated with the C terminal end of the gamma chain, such as platelet aggregation and factor XIII cross-linking, were also disrupted, suggesting that this deletion of two residues affected the overall structure of the C-terminal domain of the gamma chain. PMID- 10748040 TI - p21-activated protein kinase gamma-PAK is translocated and activated in response to hyperosmolarity. Implication of Cdc42 and phosphoinositide 3-kinase in a two step mechanism for gamma-PAK activation. AB - A member of the family of p21-activated protein kinases, gamma-PAK, has cytostatic properties and is activated during apoptosis and in response to DNA damage. To determine whether gamma-PAK is activated by other types of cell stress and to assess its mechanism of activation, the response of gamma-PAK to hyperosmotic stress was examined. In 3T3-L1 mouse fibroblasts, there are two pools of gamma-PAK: the majority of the protein kinase is soluble and has low specific activity, whereas gamma-PAK associated with the particulate fraction has significantly higher specific activity. Hyperosmolarity promotes translocation of gamma-PAK from the soluble to the particulate fraction; this parallels activation of the protein kinase. Activation but not translocation of gamma-PAK is wortmannin-sensitive, suggesting the involvement of a phosphoinositide 3-kinase related activity. gamma-PAK translocation in response to hyperosmolarity parallels Cdc42 translocation to the particulate fraction in vivo and can be induced in vitro by guanosine 5'-3-O-(thio)triphosphate. Cotransfection of gamma PAK with constitutively active Cdc42 induces gamma-PAK activation and translocation, whereas inactive Cdc42 inhibits both processes in response to hyperosmotic stress, suggesting that Cdc42 has a role in the translocation and activation of gamma-PAK. alpha-PAK is not activated in response to hyperosmolarity in 3T3-L1 cells. A two-step model of gamma-PAK activation is presented. PMID- 10748041 TI - Saccharomyces cerevisiae GATA sequences function as TATA elements during nitrogen catabolite repression and when Gln3p is excluded from the nucleus by overproduction of Ure2p. AB - Saccharomyces cerevisiae selectively uses good nitrogen sources (glutamine) in preference to poor ones (proline) by repressing GATA factor-dependent transcription of the genes needed to transport and catabolize poor nitrogen sources, a physiological process designated nitrogen catabolite repression (NCR). We show that some NCR-sensitive genes (CAN1, DAL5, DUR1,2, and DUR3) produce two transcripts of slightly different sizes. Synthesis of the shorter transcript is NCR-sensitive and that of the longer transcript is not. The longer transcript also predominates in gln3Delta mutants irrespective of the nitrogen source provided. We demonstrate that the longer mRNA species arises through the use of an alternative transcription start site generated by Gln3p-binding sites (GATAAs) being able to act as surrogate TATA elements. The ability of GATAAs to serve as surrogate TATAs, i.e. when synthesis of the shorter, NCR-sensitive transcripts are inhibited, correlates with sequestration of enhanced green fluorescent protein (EGFP)-Gln3p in the cytoplasm in a way that is indistinguishable from that seen with EGFP-Ure2p. However, when the shorter, NCR-sensitive DAL5 transcript predominates, EGFP-Gln3p is nuclear. These data suggest that the mechanism underlying NCR involves the cytoplasmic association of Ure2p with Gln3p, an interaction that prevents Gln3p from reaching it is binding sites upstream of NCR-sensitive genes. PMID- 10748042 TI - Species differences between rat and mouse CCKA receptors determine the divergent acinar cell response to the cholecystokinin analog JMV-180. AB - The cholecystokinin (CCK) analog JMV-180 acts as a partial agonist in rats and a full agonist in mice. Whether this functional variability is due to species differences in CCK receptor structure or to alterations in the cellular environment is unknown. To address this question, an adenoviral construct encoding the rat CCK(A) receptor (AdCCK(A)R) was used to express the rat receptor in acini from CCK(A) receptor-deficient mice (CCK(A)R -/-). Infection of CCK(A)R /- acini in vitro with pAdCCK(A)R led to a time-dependent increase in (125)I CCK(8) binding. The affinity for JMV-180 of the adenovirally transferred rat and the endogenous mouse CCK(A) receptors was not different. In native mouse acini, JMV-180 acted as a full agonist (both stimulation and inhibition of amylase release). In contrast, in mouse acini expressing pAdCCK(A)R JMV-180 acted as a partial agonist (only stimulation of amylase release). In addition, the pattern of protein synthesis induced by JMV-180 in CCK(A)R -/- mouse acini infected with AdCCK(A)R resembled the pattern observed in wild-type rats (lack of inhibition) rather than the respective pattern in wild-type mice (inhibition). These data suggest that species differences in the CCK(A) receptor of rats and mice account for the observed divergence in the acinar cell response to JMV-180. PMID- 10748043 TI - Chimeric peptides of statherin and osteopontin that bind hydroxyapatite and mediate cell adhesion. AB - Extracellular matrix proteins play key roles in controlling the activities of osteoblasts and osteoclasts in bone remodeling. These bone-specific extracellular matrix proteins contain amino acid sequences that mediate cell adhesion, and many of the bone-specific matrix proteins also contain acidic domains that interact with the mineral surface and may orient the signaling domains. Here we report a fusion peptide design that is based on this natural approach for the display of signaling peptide sequences at biomineral surfaces. Salivary statherin contains a 15-amino acid hydroxyapatite binding domain (N15) that is loosely helical in solution. To test whether N15 can serve to orient active peptide sequences on hydroxyapatite, the RGD and flanking residues from osteopontin were fused to the C terminus. The fusion peptides bound tightly to hydroxyapatite, and the N15 PGRGDS peptide mediated the dose-dependent adhesion of Moalpha(v) melanoma cells when immobilized on the hydroxyapatite surface. Experiments with an integrin sorted Moalpha(v) subpopulation demonstrated that the alpha(v)beta(3) integrin was the primary receptor target for the fusion peptide. Solid state NMR experiments showed that the RGD portion of the hydrated fusion peptide is highly dynamic on the hydroxyapatite surface. This fusion peptide framework may thus provide a straightforward design for immobilizing bioactive sequences on hydroxyapatite for biomaterials, tissue engineering, and vaccine applications. PMID- 10748044 TI - alpha - and beta -phosphorylated amines and pyrrolidines, a new class of low toxic highly sensitive 31P NMR pH indicators. Modeling of pKa and chemical shift values as a function of substituents. AB - Fourteen linear and cyclic alpha- and beta-aminophosphonates in which the P-atom is substituted by alkoxy groups have been synthesized and evaluated as (31)P NMR pH markers in Krebs-Henseleit buffer. pK(a) values varied with substitution in the range 1.3-9.1, giving potentially access to a wide range of pH. Temperature had a weak influence on pH and a dramatic increase in ionic strength slightly modified the pK(a) of the pyrrolidine diethyl(2-methylpyrrolidin-2-yl)phosphonate (DEPMPH). All compounds displayed a 4-fold better NMR sensitivity than inorganic phosphate or other commonly used phosphonates, as assessed by differences delta(b)-delta(a) between the chemical shifts of the protonated and the unprotonated forms. In isolated perfused rat hearts, a non-toxic concentration window of 1.5-15 mm was determined for three representative compounds. Using empirical linear relationships, the experimental values of pK(a), delta(a), and delta(b) have been correlated with the two-dimensional structure, i.e. the chemical nature of substituents bonded to the secondary amine and P-atom. The data suggest that DEPMPH and its cyclic and linear variants are ideal versatile (31)P NMR probes for the study of tenuous pH changes in biological processes. PMID- 10748045 TI - Identification of a Drosophila vitamin K-dependent gamma-glutamyl carboxylase. AB - Using reduced vitamin K, oxygen, and carbon dioxide, gamma-glutamyl carboxylase post-translationally modifies certain glutamates by adding carbon dioxide to the gamma position of those amino acids. In vertebrates, the modification of glutamate residues of target proteins is facilitated by an interaction between a propeptide present on target proteins and the gamma-glutamyl carboxylase. Previously, the gastropod Conus was the only known invertebrate with a demonstrated vitamin K-dependent carboxylase. We report here the discovery of a gamma-glutamyl carboxylase in Drosophila. This Drosophila enzyme is remarkably similar in amino acid sequence to the known mammalian carboxylases; it has 33% sequence identity and 45% sequence similarity to human gamma-glutamyl carboxylase. The Drosophila carboxylase is vitamin K-dependent, and it has a K(m) toward a model pentapeptide substrate, FLEEL, of about 4 mm. However, unlike the human gamma-glutamyl carboxylase, it is not stimulated by human blood coagulation factor IX propeptides. We found the mRNA for Drosophila gamma-glutamyl carboxylase in virtually every embryonic and adult stage that we investigated, with the highest concentration evident in the adult head. PMID- 10748046 TI - Molecular and structural basis of the specificity of a neutralizing acetylcholine receptor-mimicking antibody, using combined mutational and molecular modeling analyses. AB - The antagonist activity of short-chain toxins from snake venoms toward the nicotinic acetylcholine receptor (nAChR) is neutralized upon binding to a toxin specific monoclonal antibody called Malpha2-3 (1). To establish the molecular basis of this specificity, we predicted from both mutational analyses and docking procedures the structure of the Malpha2-3-toxin complex. From knowledge of the functional paratope and epitope, and using a double-mutation cycle procedure, we gathered evidence that Asp(31) in complementarity determining region 1H is close to, and perhaps interacts with, Arg(33) in the antigen. The use of this pair of proximate residues during the selection procedure yielded three models based on docking calculations. The selected models predicted the proximity of Tyr(49) and/or Tyr(50) in the antibody to Lys(47) in the toxin. This was experimentally confirmed using another round of double-mutation cycles. The two models finally selected were submitted to energy minimization in a CHARMM22 force field, and were characterized by a root mean square deviation of 7.0 +/- 2.9 A. Both models display most features of antibody-antigen structures. Since Malpha2-3 also partially mimics some binding properties of nAChR, these structural features not only explain its fine specificity of recognition, but may also further clarify how toxins bind to nAChR. PMID- 10748047 TI - Expression cloning of an oxysterol 7alpha-hydroxylase selective for 24 hydroxycholesterol. AB - The synthesis of 7alpha-hydroxylated bile acids from oxysterols requires an oxysterol 7alpha-hydroxylase encoded by the Cyp7b1 locus. As expected, mice deficient in this enzyme have elevated plasma and tissue levels of 25- and 27 hydroxycholesterol; however, levels of another major oxysterol, 24 hydroxycholesterol, are not increased in these mice, suggesting the presence of another oxysterol 7alpha-hydroxylase. Here, we describe the cloning and characterization of murine and human cDNAs and genes that encode a second oxysterol 7alpha-hydroxylase. The genes contain 12 exons and are located on chromosome 6 in the human (CYP39A1 locus) and in a syntenic position on chromosome 17 in the mouse (Cyp39a1 locus). CYP39A1 is a microsomal cytochrome P450 enzyme that has preference for 24-hydroxycholesterol and is expressed in the liver. The levels of hepatic CYP39A1 mRNA do not change in response to dietary cholesterol, bile acids, or a bile acid-binding resin, unlike those encoding other sterol 7alpha-hydroxylases. Hepatic CYP39A1 expression is sexually dimorphic (female > male), which is opposite that of CYP7B1 (male > female). We conclude that oxysterol 7alpha-hydroxylases with different substrate specificities exist in mice and humans and that sexually dimorphic expression patterns of these enzymes in the mouse may underlie differences in bile acid metabolism between the sexes. PMID- 10748048 TI - Disruption of the oxysterol 7alpha-hydroxylase gene in mice. AB - Mice without oxysterol 7alpha-hydroxylase, an enzyme of the alternate bile acid synthesis pathway with a sexually dimorphic expression pattern, were constructed by the introduction of a null mutation at the Cyp7b1 locus. Animals heterozygous (Cyp7b1(+/-)) and homozygous (Cyp7b1(-/-)) for this mutation were grossly indistinguishable from wild-type mice. Plasma and tissue levels of 25- and 27 hydroxycholesterol, two oxysterol substrates of this enzyme with potent regulatory actions in cultured cells, were markedly elevated in Cyp7b1(-/-) knockout animals. Parameters of bile acid metabolism as well as plasma cholesterol and triglyceride levels in male and female Cyp7b1(-/-) mice were normal. The cholesterol contents of major tissues were not altered. In vivo sterol biosynthetic rates were unaffected in multiple tissues with the exception of the male kidney, which showed a approximately 40% decrease in de novo synthesis versus controls. We conclude that the major physiological role of the CYP7B1 oxysterol 7alpha-hydroxylase is to metabolize 25- and 27 hydroxycholesterol and that loss of this enzyme in the liver is compensated for by increases in the synthesis of bile acids by other pathways. A failure to catabolize oxysterols in the male kidney may lead to a decrease in de novo sterol synthesis. PMID- 10748049 TI - Characterization of ABCB9, an ATP binding cassette protein associated with lysosomes. AB - We have cloned full-length human and mouse cDNAs of ABCB9, which encodes a predicted multiple-spanning transmembrane domain and a nucleotide-binding domain with Walker motifs. It is therefore designated as a "half" ATP binding cassette (ABC) transporter. Northern analysis shows that the ABCB9 mRNA is expressed at a high level in testes and moderate levels in brain and spinal cord. A splice variant mRNA deleted in the last pair of predicted transmembrane segments was shown to be expressed in human tissues. Phylogenetic analysis indicates that ABCB9 is closely related to TAP1 and TAP2, two "half" ABC proteins found in endoplasmic reticulum. ABCB9 protein colocalized with the lysosomal markers, LAMP1 and LAMP2, in transfected cells. ABCB9 protein appears to be most highly expressed in the Sertoli cells of the seminiferous tubules in mouse and rat testes. These cells have high levels of phagocytosis and secretory activities. These findings pave the way for further investigation into the potential novel function of ABCB9 in lysosomes. PMID- 10748050 TI - Homeostatic modulation of cell surface KDR and Flt1 expression and expression of the vascular endothelial cell growth factor (VEGF) receptor mRNAs by VEGF. AB - Vascular endothelial cell growth factor (VEGF) is a potent angiogenic factor expressed during embryonic development, during wound healing, and in pathologies dependent on neovascularization, including cancer. Regulation of the receptor tyrosine kinases, KDR and Flt-1, to which VEGF binds on endothelial cells is incompletely understood. Chronic incubation with tumor-conditioned medium or VEGF diminished (125)I-VEGF binding to human umbilical vein endothelial cells, incorporation of (125)I-VEGF into covalent complexes with KDR and Flt1, and immunoreactive KDR in cell lysates. Receptor down-regulation desensitized VEGF activation of mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2) and p38 mitogen-activated protein kinase. Preincubation with VEGF or tumor-conditioned medium down-regulated cell surface receptor expression but up-regulated KDR and Flt-1 mRNAs, an effect abrogated by a neutralizing VEGF antibody. Removal of VEGF from the medium led to recovery of (125)I-VEGF binding and resensitization of human umbilical vein endothelial cells. Recovery of receptor expression was inhibited by cycloheximide, indicating that augmented VEGF receptor mRNAs, and not receptor recycling from a cytoplasmic pool, restored responsiveness. As the VEGF receptors promote endothelial cell survival, proliferation, and other events necessary for angiogenesis, the noncoordinate regulation of VEGF receptor proteins and mRNAs suggests that human umbilical vein endothelial cells are protected against inappropriate or prolonged loss of VEGF receptors by a homeostatic mechanism important to endothelial cell function. PMID- 10748051 TI - The FtsJ/RrmJ heat shock protein of Escherichia coli is a 23 S ribosomal RNA methyltransferase. AB - Ribosomal RNAs undergo several nucleotide modifications including methylation. We identify FtsJ, the first encoded protein of the ftsJ-hflB heat shock operon, as an Escherichia coli methyltransferase of the 23 S rRNA. The methylation reaction requires S-adenosylmethionine as donor of methyl groups, purified FtsJ or a S(150) supernatant from an FtsJ-producing strain, and ribosomes from an FtsJ deficient strain. In vitro, FtsJ does not efficiently methylate ribosomes purified from a strain producing FtsJ, suggesting that these ribosomes are already methylated in vivo by FtsJ. FtsJ is active on ribosomes and on the 50 S ribosomal subunit, but is inactive on free rRNA, suggesting that its natural substrate is ribosomes or a pre-ribosomal ribonucleoprotein particle. We identified the methylated nucleotide as 2'-O-methyluridine 2552, by reverse phase high performance liquid chromatography analysis, boronate affinity chromatography, and hybridization-protection experiments. In view of its newly established function, FtsJ is renamed RrmJ and its encoding gene, rrmJ. PMID- 10748052 TI - Direct binding of the signaling adapter protein Grb2 to the activation loop tyrosines on the nerve growth factor receptor tyrosine kinase, TrkA. AB - We demonstrate that the signaling adapter, Grb2, binds directly to the neurotrophin receptor tyrosine kinase, TrkA. Grb2 binding to TrkA is independent of Shc, FRS-2, phospholipase Cgamma-1, rAPS, and SH2B and is observed in in vitro binding assays, yeast two-hybrid assays, and in co-immunoprecipitation assays. Grb2 binding to TrkA is mediated by the central SH2 domain, requires a kinase active TrkA, and is phosphotyrosine-dependent. By analyzing a series of rat TrkA mutants, we demonstrate that Grb2 binds to the carboxyl-terminal residue, Tyr(794), as well as to the activation loop tyrosines, Tyr(683) and Tyr(684). By using acidic amino acid substitutions of the activation loop tyrosines on TrkA, we can stimulate constitutive kinase activity and TrkA-Shc interactions but, importantly, abolish TrkA/Grb2 binding. Thus, in addition to providing the first evidence of direct Grb2 binding to the neurotrophin receptor, TrkA, these data provide the first direct evidence that the activation loop tyrosines of a receptor tyrosine kinase, in addition to their essential role in kinase activation, also serve a direct role in the recruitment of intracellular signaling molecules. PMID- 10748053 TI - The cysteine-rich protein A from Helicobacter pylori is a beta-lactamase. AB - Among the large number of hypothetical proteins within the genomes of Helicobacter pylori, there is a family of unique and highly disulfide-bridged proteins, designated family 12, for which no function could originally be assigned. Sequence analysis revealed that members of this family possess a modular architecture of alpha/beta-units and a stringent pattern of cysteine residues. The H. pylori cysteine-rich protein A (HcpA), which is a member of this family, was expressed and refolded from inclusion bodies. Six pairs of cysteine residues, which are separated by exactly seven residues, form disulfide bridges. HcpA is a beta-lactamase. It slowly hydrolyzes 6-aminopenicillinic acid and 7 aminocephalosporanic acid (ACA) derivatives. The turnover for 6-aminopenicillinic acid derivatives is 2-3 times greater than for ACA derivatives. The enzyme is efficiently inhibited by cloxacillin and oxacillin but not by ACA derivatives or metal chelators. We suggest that all family 12 members possess similar activities and might be involved in the synthesis of the cell wall peptidoglycan. They might also be responsible for amoxicillin resistance of certain H. pylori strains. PMID- 10748054 TI - CD22 forms a quaternary complex with SHIP, Grb2, and Shc. A pathway for regulation of B lymphocyte antigen receptor-induced calcium flux. AB - CD22 is a cell surface molecule that regulates signal transduction in B lymphocytes. Tyrosine-phosphorylated CD22 recruits numerous cytoplasmic effector molecules including SHP-1, a potent phosphotyrosine phosphatase that down regulates B cell antigen receptor (BCR)- and CD19-generated signals. Paradoxically, B cells from CD22-deficient mice generate augmented intracellular calcium responses following BCR ligation, yet proliferation is decreased. To understand further the mechanisms through which CD22 regulates BCR-dependent calcium flux and proliferation, interactions between CD22 and effector molecules involved in these processes were assessed. The adapter proteins Grb2 and Shc were found to interact with distinct and specific regions of the CD22 cytoplasmic domain. Src homology-2 domain-containing inositol polyphosphate-5'-phosphatase (SHIP) also bound phosphorylated CD22, but binding required an intact CD22 cytoplasmic domain. All three molecules were bound to CD22 when isolated from BCR stimulated splenic B cells, indicating the formation of a CD22.Grb2.Shc.SHIP quaternary complex. Therefore, SHIP associating with CD22 may be important for SHIP recruitment to the cell surface where it negatively regulates calcium influx. Although augmented calcium responses in CD22-deficient mice should facilitate enhanced c-Jun N-terminal kinase (JNK) activation, BCR ligation did not induce JNK activation in CD22-deficient B cells. These data demonstrate that CD22 functions as a molecular "scaffold" that specifically coordinates the docking of multiple effector molecules, in addition to SHP-1, in a context necessary for BCR-dependent SHIP activity and JNK stimulation. PMID- 10748055 TI - Octamers of mitochondrial creatine kinase isoenzymes differ in stability and membrane binding. AB - Octamer stability and membrane binding of mitochondrial creatine kinase (MtCK) are important for proper functioning of the enzyme and were suggested as targets for regulatory mechanisms. A quantitative analysis of these properties, using fluorescence spectroscopy, gel filtration, and surface plasmon resonance, revealed substantial differences between the two types of MtCK isoenzymes, sarcomeric (sMtCK) and ubiquitous (uMtCK). As compared with human and chicken sMtCK, human uMtCK showed a 23-34 times slower octamer dissociation rate, a reduced reoctamerization rate and a superior octamer stability as deduced from the octamer/dimer ratios at thermodynamic equilibrium. Octamer stability of sMtCK increased with temperature up to 30 degrees C, indicating a substantial contribution of hydrophobic interactions, while it decreased in the case of uMtCK, indicating the presence of additional polar dimer/dimer interactions. These conclusions are consistent with the recently solved x-ray structure of the human uMtCK (Eder, M., Fritz-Wolf, K., Kabsch, W., Wallimann, T., and Schlattner, U. (2000) Proteins 39, 216-225). When binding to 16% cardiolipin membranes, sMtCK showed slightly faster on-rates and higher affinities than uMtCK. However, human uMtCK was able to recruit the highest number of binding sites on the vesicle surface. The observed divergence of ubiquitous and sarcomeric MtCK is discussed with respect to their molecular structures and the possible physiological implications. PMID- 10748056 TI - Proton block and voltage gating are potassium-dependent in the cardiac leak channel Kcnk3. AB - Potassium leak conductances were recently revealed to exist as independent molecular entities. Here, the genomic structure, cardiac localization, and biophysical properties of a murine example are considered. Kcnk3 subunits have two pore-forming P domains and unique functional attributes. At steady state, Kcnk3 channels behave like open, potassium-selective, transmembrane holes that are inhibited by physiological levels of proton. With voltage steps, Kcnk3 channels open and close in two phases, one appears to be immediate and one is time-dependent (tau = approximately 5 ms). Both proton block and gating are potassium-sensitive; this produces an anomalous increase in outward flux as external potassium levels rise because of decreased proton block. Single Kcnk3 channels open across the physiological voltage range; hence they are "leak" conductances; however, they open only briefly and rarely even after exposure to agents that activate other potassium channels. PMID- 10748057 TI - Overexpression of epidermal growth factor induced hypospermatogenesis in transgenic mice. AB - The in vivo role of epidermal growth factor (EGF) is not well defined even though its effects on culture cells were well studied. To understand the developmental, physiological, and pathological roles of EGF, we have generated transgenic mice widely expressing human EGF with the use of the beta-actin promoter. EGF and transforming growth factor alpha (TGFalpha) bind with equal affinity to the EGF receptor, a transmembrane tyrosine kinase, to trigger various biological responses. EGF and TGFalpha signaling are implicated in the development of the reproductive system. EGF also plays a physiological role in reproduction. Removal of the salivary gland in rodents, which reduces circulating EGF, reduces spermatogenesis, which can be corrected by EGF replacement. Here we show that in our transgenic males, only few post-meiosis II gametes were found, and the mice were sterile. This resembles a common cause of infertility in humans. Furthermore, the transgenic males had reduced serum testosterone. Our findings contrast the previous report on transgenic mice overexpressing TGFalpha in testis, which showed normal spermatogenesis. These data suggest that EGF is the active ligand for EGF receptor reported in germ cells, and proper EGF expression is important for completion of spermatogenesis. PMID- 10748058 TI - CrkII participation in the cellular effects of growth hormone and insulin-like growth factor-1. Phosphatidylinositol-3 kinase dependent and independent effects. AB - We have examined the role of CrkII in the cellular response to both human growth hormone (hGH) and human insulin-like growth factor-1 (hIGF-1). We have demonstrated that overexpression of the adaptor molecule enhances both basal phosphatidylinositol 3-kinase (PI 3-kinase) activity and also dramatically enhances the ability of both hormones to stimulate PI 3-kinase activity in the cell. Many of the effects of CrkII overexpression on hGH- and hIGF-1-stimulated cellular function can then be attributed to CrkII enhancement of PI 3-kinase stimulation by these hormones. Thus, CrkII-enhanced PI 3-kinase activity is used to enhance actin filament reorganization in response to both hGH and hIGF-1, to enhance stress activated protein kinase (SAPK) activity in response to hGH, and to diminish STAT5-mediated transcription in response to hGH. It is apparent, however, that CrkII also regulates cellular function independent of its ability to stimulate PI 3-kinase activity. This is evidenced by the ability of CrkII, in a PI 3-kinase-independent manner, to diminish the activation of p44/42 mitogen activated protein kinase in response to both hGH and hIGF-1 and to inhibit the activation of SAPK by hIGF-1. Therefore, despite the common use of CrkII to activate PI 3-kinase, CrkII also allows hGH or hIGF-1 to selectively switch the activation of SAPK. Thus, common utilization of CrkII by hGH and hIGF-1 allows the execution of common cellular effects of these hormones, concomitant with the retention of hormonal specificity. PMID- 10748059 TI - Nic1p, a relative of bacterial transition metal permeases in Schizosaccharomyces pombe, provides nickel ion for urease biosynthesis. AB - The Schizosaccharomyces pombe genome sequencing project identified an open reading frame (O74869 and O74912, named Nic1p in the present study) with significant similarity to members of a family of bacterial transition metal permeases. These uptake systems transport Ni(2+) ion with extremely high affinity across the bacterial cytoplasmic membrane, but they differ in selectivity toward divalent transition metal cations. An S. pombe mutant harboring an interrupted nic1 allele (nic1-1) was strongly impaired in (63)Ni(2+) uptake in the presence of a high molar ratio of Mg(2+) relative to Ni(2+), conditions that reflect the natural situation. Under these conditions, the nic1-1 mutant contained only background activities of the nickel-dependent cytoplasmic enzyme urease and could not catabolize urea. Among a series of divalent transition metal cations tested (Cd(2+), Co(2+), Cu(2+), Mn(2+), and Zn(2+)), only Co(2+) caused considerable inhibition of Nic1p-mediated Ni(2+) uptake. On the other hand, experiments with (57)Co(2+) (at nm concentrations) did not show significant differences in Co(2+) uptake between the nic1-1 mutant and the parental strain. Our data suggest that Nic1p acts as a plasma-membrane nickel transporter in fission yeast, a finding that invites searches for isologous counterparts in higher eukaryotes. PMID- 10748060 TI - Phosphorylation of smooth muscle myosin heads regulates the head-induced movement of tropomyosin. AB - It has been shown that skeletal and smooth muscle myosin heads binding to actin results in the movement of smooth muscle tropomyosin, as revealed by a change in fluorescence resonance energy transfer between a fluorescence donor on tropomyosin and an acceptor on actin (Graceffa, P. (1999) Biochemistry 38, 11984 11992). In this work, tropomyosin movement was similarly monitored as a function of unphosphorylated and phosphorylated smooth muscle myosin double-headed fragment smHMM. In the absence of nucleotide and at low myosin head/actin ratios, only phosphorylated heads induced a change in energy transfer. In the presence of ADP, the effect of head phosphorylation was even more dramatic, in that at all levels of myosin head/actin, phosphorylation was necessary to affect energy transfer. It is proposed that the regulation of tropomyosin position on actin by phosphorylation of myosin heads plays a key role in the regulation of smooth muscle contraction. In contrast, actin-bound caldesmon was not moved by myosin heads at low head/actin ratios, as uncovered by fluorescence resonance energy transfer and disulfide cross-linking between caldesmon and actin. At higher head concentration caldesmon was dissociated from actin, consistent with the multiple binding model for the binding of caldesmon and myosin heads to actin (Chen, Y., and Chalovich, J. M. (1992) Biophys. J. 63, 1063-1070). PMID- 10748061 TI - TFIIH interacts with the retinoic acid receptor gamma and phosphorylates its AF-1 activating domain through cdk7. AB - Retinoic acid receptor gamma (RARgamma) is phosphorylated in COS-1 cells at two conserved serine residues located in the N-terminal region (serines 77 and 79 in RARgamma1 and serines 66 and 68 in RARgamma2) that contains the activation function AF-1. These serines are phosphorylated in vitro by cdk7, a cyclin dependent kinase associated to cyclin H and MAT1 in the CAK complex (cdk7.cyclin H. MAT1), that is found either free or as a component of the transcription/DNA repair factor TFIIH. RARgamma is more efficiently phosphorylated by TFIIH than by CAK and interacts not only with cdk7 but also with several additional subunits of TFIIH. RARgamma phosphorylation and interaction with TFIIH occur in a ligand independent manner. Our data demonstrate also that phosphorylation of the AF-1 function modulates RARgamma transcriptional activity in a response gene-dependent manner. PMID- 10748062 TI - Inactivation of the peroxisomal multifunctional protein-2 in mice impedes the degradation of not only 2-methyl-branched fatty acids and bile acid intermediates but also of very long chain fatty acids. AB - According to current views, peroxisomal beta-oxidation is organized as two parallel pathways: the classical pathway that is responsible for the degradation of straight chain fatty acids and a more recently identified pathway that degrades branched chain fatty acids and bile acid intermediates. Multifunctional protein-2 (MFP-2), also called d-bifunctional protein, catalyzes the second (hydration) and third (dehydrogenation) reactions of the latter pathway. In order to further clarify the physiological role of this enzyme in the degradation of fatty carboxylates, MFP-2 knockout mice were generated. MFP-2 deficiency caused a severe growth retardation during the first weeks of life, resulting in the premature death of one-third of the MFP-2(-/-) mice. Furthermore, MFP-2-deficient mice accumulated VLCFA in brain and liver phospholipids, immature C(27) bile acids in bile, and, after supplementation with phytol, pristanic and phytanic acid in liver triacylglycerols. These changes correlated with a severe impairment of peroxisomal beta-oxidation of very long straight chain fatty acids (C(24)), 2 methyl-branched chain fatty acids, and the bile acid intermediate trihydroxycoprostanic acid in fibroblast cultures or liver homogenates derived from the MFP-2 knockout mice. In contrast, peroxisomal beta-oxidation of long straight chain fatty acids (C(16)) was enhanced in liver tissue from MFP-2(-/-) mice, due to the up-regulation of the enzymes of the classical peroxisomal beta oxidation pathway. The present data indicate that MFP-2 is not only essential for the degradation of 2-methyl-branched fatty acids and the bile acid intermediates di- and trihydroxycoprostanic acid but also for the breakdown of very long chain fatty acids. PMID- 10748063 TI - Co-translational folding of an eukaryotic multidomain protein in a prokaryotic translation system. AB - Continuous monitoring of the enzymatic activity of newly synthesized firefly luciferase in Escherichia coli cell-free translation system was performed to record folding kinetics of this multidomain eukaryotic protein in the prokaryotic cytosol. Whereas in vitro refolding of denatured luciferase in prokaryotic cytosol occurred with a low yield of active enzyme and took about an hour, the enzyme acquired its native structure immediately upon release from the ribosome, as seen from the immediate halt of active luciferase accumulation upon blocking of translation with inhibitors. The nascent luciferase was also capable of acquiring the active conformation prior to release from the ribosome, when its C terminus was extended with a polypeptide segment. Specific enzymatic activity of the firefly luciferase was found to be equally high irrespective of whether this protein was synthesized in eukaryotic or prokaryotic translation systems. The data presented demonstrate the fundamental ability of prokaryotic cytosol to support effective co-translational protein folding in general and co translational folding of multidomain proteins in particular. PMID- 10748064 TI - Novel function of phosphoinositide 3-kinase in T cell Ca2+ signaling. A phosphatidylinositol 3,4,5-trisphosphate-mediated Ca2+ entry mechanism. AB - This study presents evidence that phosphoinositide (PI) 3-kinase is involved in T cell Ca(2+) signaling via a phosphatidylinositol 3,4, 5-trisphosphate PI(3,4,5)P(3)-sensitive Ca(2+) entry pathway. First, exogenous PI(3,4,5)P(3) at concentrations close to its physiological levels induces Ca(2+) influx in T cells, whereas PI(3,4)P(2), PI(4, 5)P(2), and PI(3)P have no effect on [Ca(2+)](i). This Ca(2+) entry mechanism is cell type-specific as B cells and a number of cell lines examined do not respond to PI(3,4,5)P(3) stimulation. Second, inhibition of PI 3-kinase by wortmannin and by overexpression of the dominant negative inhibitor Deltap85 suppresses anti-CD3-induced Ca(2+) response, which could be reversed by subsequent exposure to PI(3,4,5)P(3). Third, PI(3,4,5)P(3) is capable of stimulating Ca(2+) efflux from Ca(2+)-loaded plasma membrane vesicles prepared from Jurkat T cells, suggesting that PI(3,4,5)P(3) interacts with a Ca(2+) entry system directly or via a membrane-bound protein. Fourth, although D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4, 5)P(4)) mimics PI(3,4,5)P(3) in many aspects of biochemical functions such as membrane binding and Ca(2+) transport, we raise evidence that Ins(1,3,4,5)P(4) does not play a role in anti-CD3- or PI(3,4,5)P(3)-mediated Ca(2+) entry. This PI(3,4,5)P(3)-stimulated Ca(2+) influx connotes physiological significance, considering the pivotal role of PI 3-kinase in the regulation of T cell function. Given that PI 3-kinase and phospholipase C-gamma form multifunctional complexes downstream of many receptor signaling pathways, we hypothesize that PI(3,4,5)P(3) induced Ca(2+) entry acts concertedly with Ins(1,4,5)P(3)-induced Ca(2+) release in initiating T cell Ca(2+) signaling. By using a biotinylated analog of PI(3,4,5)P(3) as the affinity probe, we have detected several putative PI(3,4,5)P(3)-binding proteins in T cell plasma membranes. PMID- 10748065 TI - Localization and characterization of the calsequestrin-binding domain of triadin 1. Evidence for a charged beta-strand in mediating the protein-protein interaction. AB - Triadin is an integral membrane protein of the junctional sarcoplasmic reticulum that binds to the high capacity Ca(2+)-binding protein calsequestrin and anchors it to the ryanodine receptor. The lumenal domain of triadin contains multiple repeats of alternating lysine and glutamic acid residues, which have been defined as KEKE motifs and have been proposed to promote protein associations. Here we identified the specific residues of triadin responsible for binding to calsequestrin by mutational analysis of triadin 1, the major cardiac isoform. A series of deletional fusion proteins of triadin 1 was generated, and by using metabolically labeled calsequestrin in filter-overlay assays, the calsequestrin binding domain of triadin 1 was localized to a single KEKE motif comprised of 25 amino acids. Alanine mutagenesis within this motif demonstrated that the critical amino acids of triadin binding to calsequestrin are the even-numbered residues Lys(210), Lys(212), Glu(214), Lys(216), Gly(218), Gln(220), Lys(222), and Lys(224). Replacement of the odd-numbered residues within this motif by alanine had no effect on calsequestrin binding to triadin. The results suggest a model in which residues 210-224 of triadin form a beta-strand, with the even-numbered residues in the strand interacting with charged residues of calsequestrin, stabilizing a "polar zipper" that links the two proteins together. This small, highly charged beta-strand of triadin may tether calsequestrin to the junctional face membrane, allowing calsequestrin to sequester Ca(2+) in the vicinity of the ryanodine receptor during Ca(2+) uptake and Ca(2+) release. PMID- 10748066 TI - Interaction of CbbR and RegA* transcription regulators with the Rhodobacter sphaeroides cbbIPromoter-operator region. AB - The form I (cbb(I)) Calvin-Benson-Bassham (CBB) reductive pentose phosphate cycle operon of Rhodobacter sphaeroides is regulated by both the transcriptional activator CbbR and the RegA/PrrA (RegB/PrrB) two-component signal transduction system. DNase I footprint analyses indicated that R. sphaeroides CbbR binds to the cbb(I) promoter between -10 and -70 base pairs (bp) relative to the cbb(I) transcription start. A cosmid carrying the R. capsulatus reg locus was capable of complementing an R. sphaeroides regA-deficient mutant to phototrophic growth with restored regulated synthesis of both photopigments and ribulose-bisphosphate carboxylase/oxygenase (Rubisco). DNase I footprint analyses, using R. capsulatus RegA*, a constitutively active mutant version of RegA, detected four RegA* binding sites within the cbb(I) promoter. Two sites were found within a previously identified cbb(I) promoter proximal regulatory region from -61 to -110 bp. One of these proximal RegA* binding sites overlapped that of CbbR. Two sites were within a previously identified promoter distal positive regulatory region between -301 and -415 bp. Expression from promoter insertion mutants showed that the function of the promoter distal regulatory region was helical phase dependent. These results indicated that RegA exerts its regulatory affect on cbb(I) expression through direct interaction with the cbb(I) promoter. PMID- 10748067 TI - Polymorphic gene regulation and interindividual variation of UDP glucuronosyltransferase activity in human small intestine. AB - UDP-glucuronosyltransferases (UGTs) convert dietary constituents, drugs, and environmental mutagens to inactive hydrophilic glucuronides. Recent studies have shown that the expression of the UGT1 and UGT2 gene families is regulated in a tissue-specific fashion. Human small intestine represents a major site of resorption of dietary constituents and orally administered drugs and plays an important role in extrahepatic UGT directed metabolism. Expression of 13 UGT1A and UGT2B genes coupled with functional and catalytic analyses were studied using 18 small intestinal and 16 hepatic human tissue samples. Hepatic expression of UGT gene transcripts was without interindividual variation. In contrast, a polymorphic expression pattern of all the UGT genes was demonstrated in duodenal, jejunal, and ileal mucosa, with the exception of UGT1A10. To complement these studies, interindividual expression of UGT proteins and catalytic activities were also demonstrated. Hyodeoxycholic acid glucuronidation, catalyzed primarily by UGT2B4 and UGT2B7, showed a 7-fold interindividual variation in small intestinal duodenal samples, in contrast to limited variation in the presence of 4 methylumbelliferone, a substrate glucuronidated by most UGT1A and UGT2B gene products. Linkage of RNA expression patterns to protein abundance were also made with several mono-specific antibodies to the UGTs. These results are in contrast to a total absence of polymorphic variation in gene expression, protein abundance, and catalytic activity in liver. In addition, the small intestine exhibits considerable catalytic activity toward most of the different classes of substrates accepted for glucuronidation by the UGTs, which is supported by immunofluorescence analysis of UGT1A protein in the mucosal cell layer of the small intestine. Thus, tissue-specific and interindividual polymorphic regulation of UGT1A and UGT2B genes in small intestine is identified and implicated as molecular biological determinant contributing to interindividual prehepatic drug and xenobiotic metabolism in humans. PMID- 10748068 TI - Biochemical interactions of the neuronal pentraxins. Neuronal pentraxin (NP) receptor binds to taipoxin and taipoxin-associated calcium-binding protein 49 via NP1 and NP2. AB - Neuronal pentraxin 1 (NP1), neuronal pentraxin 2 (NP2), and neuronal pentraxin receptor (NPR) are members of a new family of proteins identified through interaction with a presynaptic snake venom toxin taipoxin. We have proposed that these three neuronal pentraxins represent a novel neuronal uptake pathway that may function during synapse formation and remodeling. We have investigated the mutual interactions of these proteins by characterizing their enrichment on taipoxin affinity columns; by expressing NP1, NP2, and NPR singly and together in Chinese hamster ovary cells; and by generating mice that fail to express NP1. NP1 and NP2 are secreted, exist as higher order multimers (probably pentamers), and interact with taipoxin and taipoxin-associated calcium-binding protein 49 (TCBP49). NPR is expressed on the cell membrane and does not bind taipoxin or TCBP49 by itself, but it can form heteropentamers with NP1 and NP2 that can be released from cell membranes. This is the first demonstration of heteromultimerization of pentraxins and release of a pentraxin complex by proteolysis. These processes are likely to directly effect the localization and function of neuronal pentraxins in neuronal uptake or synapse formation and remodeling. PMID- 10748069 TI - Phospholipid binding of synthetic talin peptides provides evidence for an intrinsic membrane anchor of talin. AB - Talin, an actin-binding protein, is assumed to anchor at the membrane via an intrinsic amino acid sequence. Three N-terminal talin fragments, 21-39 (S19), 287 304 (H18), and 385-406 (H17) have been proposed as potential membrane anchors. The interaction of the corresponding synthetic peptides with lipid model systems was investigated with CD spectroscopy, isothermal titration calorimetry, and monolayer expansion measurements. The membrane model systems were neutral or negatively charged small unilamellar vesicles or monolayers with a lateral packing density of bilayers (32 mN/m). S19 partitions into charged monolayers/bilayers with a penetration area A(p) = 140 +/- 30 A(2) and a free energy of binding of DeltaG(0) = -5.7 kcal/mol, thereby forming a partially alpha helical structure. H18 does not interact with lipid monolayers or bilayers. H17 penetrates into neutral and charged monolayers/bilayers with A(p) = 148 +/- 23 A(2) and A(p) = 160 +/- 15 A(2), respectively, forming an alpha-helix in the membrane-bound state. Membrane partitioning is mainly entropy-driven. Under physiological conditions the free energy of binding to negatively charged membranes is DeltaG(0) = -9. 4 kcal/mol with a hydrophobic contribution of DeltaG(h) = -7.8 kcal/mol, comparable to that of post-translationally attached membrane anchors, and an electrostatic contribution of DeltaG(h) = -1.6 kcal/mol. The latter becomes more negative with decreasing pH. We show that H17 provides the binding energy required for a membrane anchor. PMID- 10748070 TI - Glycosylation efficiency of Asn-Xaa-Thr sequons depends both on the distance from the C terminus and on the presence of a downstream transmembrane segment. AB - Statistical studies of N-glycosylated proteins have indicated that the frequency of nonglycosylated Asn-Xaa-(Thr/Ser) sequons increases toward the C terminus (Gavel, Y., and von Heijne, G. (1990) Protein Eng. 3, 433-442). Using in vitro transcription/translation of a truncated model protein in the presence of dog pancreas microsomes, we find that glycosylation efficiency of Asn-Xaa-Thr sequons indeed is reduced when the sequon is within approximately 60 residues of the C terminus. Surprisingly, the presence of a hydrophobic stop transfer sequence between the Asn-Xaa-Thr sequon and the C terminus results in a very different dependence of glycosylation efficiency on the distance to the C terminus, where the presence of the stop transfer segment inside the ribosome appears to cause a drastic drop in the level of glycosylation. We speculate that this may reflect a change in the structure of the ribosome/translocon complex induced by the stop transfer segment. PMID- 10748071 TI - Determinants of the pH of the Golgi complex. AB - The factors contributing to the establishment of the steady state Golgi pH (pH(G)) were studied in intact and permeabilized mammalian cells by fluorescence ratio imaging. Retrograde transport of the nontoxic B subunit of verotoxin 1 was used to deliver pH-sensitive probes to the Golgi complex. To evaluate whether counter-ion permeability limited the activity of the electrogenic V-ATPase, we determined the concentration of K(+) in the lumen of the Golgi using a null point titration method. The [K(+)] inside the Golgi was found to be close to that of the cytosol, and increasing its permeability had no effect on pH(G). Moreover, the capacity of the endogenous counter-ion permeability exceeded the rate of H(+) pumping, implying that the potential across the Golgi membrane is negligible and has little influence on pH(G). The V-ATPase does not reach thermodynamic equilibrium nor does it seem to be allosterically inactivated at the steady state pH(G). In fact, active H(+) pumping was detectable even below the resting pH(G). A steady state pH was attained when the rate of pumping was matched by the passive backflux of H(+) (equivalents) or "leak." The nature of this leak pathway was investigated in detail. Neither vesicular traffic nor H(+)/cation antiporters or symporters were found to contribute to the net loss of H(+) from the Golgi. Instead, the leak was sensitive to voltage changes and was inhibited by Zn(2+), resembling the H(+) conductive pathway of the plasma membrane. We conclude that a balance between an endogenous leak, which includes a conductive component, and the H(+) pump determines the pH at which the Golgi lumen attains a steady state. PMID- 10748072 TI - p38 mitogen-activated protein kinase regulates a novel, caspase-independent pathway for the mitochondrial cytochrome c release in ultraviolet B radiation induced apoptosis. AB - The mechanisms of UVB-induced apoptosis and the role of p38 mitogen-activated protein kinase (MAPK) were investigated in HaCaT cells. UVB doses that induced apoptosis also produced a sustained activation of p38 MAPK and mitochondrial cytochrome c release, leading to pro-caspase-3 activation. Late into the apoptotic process, UVB also induced a caspase-mediated cleavage of Bid. Caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone and benzyloxycarbonyl Asp-Glu-Val-Asp-fluoromethylketone substantially blocked the UVB-induced apoptosis without preventing the release of mitochondrial cytochrome c and the p38 MAPK activation. The inhibition of p38 MAPK counteracted both apoptosis and cytochrome c release as well as the DEVD-amino-4-methylcoumarin cleavage activity without affecting the processing of pro-caspase-8. These results indicate that UVB induces multiple and independent apoptotic pathways, which culminate in pro caspase-3 activation, and that the initial cytochrome c release is independent of caspase activity. Importantly, we show that a sustained p38 MAPK activation contributes to the UVB-induced apoptosis by mediating the release of mitochondrial cytochrome c into the cytosol. PMID- 10748073 TI - Phosphorylation of the rat vesicular acetylcholine transporter. AB - Metabolic labeling of a mutant PC12 cell line, A123.7, expressing recombinant rat vesicular acetylcholine transporter (VAChT) with radiolabeled inorganic phosphate was used to demonstrate phosphorylation of the transporter on a serine residue. Mutational analysis was used to demonstrate that serine 480, which is located on the COOH-terminal cytoplasmic tail, is the sole phosphorylation site. Phosphorylation of serine 480 was attributable to the action of protein kinase C. Using a permanently dephosphorylated form of rat VAChT, S480A rVAChT, it was shown that this mutant displays the same kinetics for the transport of acetylcholine and the binding of the inhibitor vesamicol as does the wild type transporter. However, sucrose gradient density centrifugation showed that, unlike wild type VAChT, the S480A mutant did not localize to synaptic vesicles. These results suggest that phosphorylation of serine 480 of VAChT is involved in the trafficking of this transporter. PMID- 10748074 TI - Cysteine-rich fibroblast growth factor receptor alters secretion and intracellular routing of fibroblast growth factor 3. AB - Expression of the cysteine-rich fibroblast growth factor (FGF) receptor (CFR) in COS-1 cells strongly inhibits the secretion of co-expressed FGF3. By using a column retention assay and affinity chromatography, we demonstrate that at physiological salt concentrations FGF3 binds with strong affinity to CFR in vivo and in vitro. Furthermore, to show that FGF3 binds to CFR in vivo, truncation mutants of CFR with changed subcellular distributions were shown to cause a similar redistribution of FGF3. Although CFR is a 150-kDa integral membrane glycoprotein that is primarily located in the Golgi apparatus, we show here that in COS-1 cells a substantial proportion of CFR is secreted. This is due to a carboxyl-terminal proteolytic cleavage that releases the intraluminal portion of the protein for secretion. However, the apparent size of the integral membrane and secreted CFR appears similar, since the loss of protein mass is balanced by a gain of complex carbohydrates. The released CFR is associated with the extracellular matrix through its affinity for glycosaminoglycans. These findings show that CFR can modulate the secretion of FGF3 and may control its biological activity by regulating its secretion. PMID- 10748075 TI - Reactive oxygen species expose cryptic epitopes associated with autoimmune goodpasture syndrome. AB - Goodpasture syndrome is an autoimmune disease of the kidneys and lungs mediated by antibodies and T-cells directed to cryptic epitopes hidden within basement membrane hexamers rich in alpha3 non-collagenous globular (NC1) domains of type IV collagen. These epitopes are normally invisible to the immune system, but this privilege can be obviated by chemical modification. Endogenous drivers of immune activation consequent to the loss of privilege have long been suspected. We have examined the ability of reactive oxygen species (ROS) to expose Goodpasture epitopes buried within NC1 hexamers obtained from renal glomeruli abundant in alpha3(IV) NC1 domains. For some hexameric epitopes, like the Goodpasture epitopes, exposure to ROS specifically enhanced recognition by Goodpasture antibodies in a sequential and time-dependent fashion; control binding of epitopes to alpha3(IV) alloantibodies from renal transplant recipients with Alport syndrome was decreased, whereas epitope binding to heterologous antibodies recognizing all alpha3 NC1 epitopes remained the same. Inhibitors of hydrogen peroxide and hydroxyl radical scavengers were capable of attenuating the effects of ROS in cells and kidney by 30-50%, respectively, thereby keeping the Goodpasture epitopes largely concealed when compared with a 70% maximum inhibition by iron chelators. Hydrogen peroxide administration to rodents was sufficient to expose Goodpasture epitope in vivo and initiate autoantibody production. Our findings collectively suggest that ROS can alter the hexameric structure of type IV collagen to expose or destroy selectively immunologic epitopes embedded in basement membrane. The reasons for autoimmunity in Goodpasture syndrome may lie in an age-dependent deterioration in inhibitor function modulating oxidative damage to structural molecules. ROS therefore may play an important role in shaping post-translational epitope diversity or neoantigen formation in organ tissues. PMID- 10748076 TI - Degradation of human thyroperoxidase in the endoplasmic reticulum involves two different pathways depending on the folding state of the protein. AB - Human thyroperoxidase (hTPO), a type I transmembrane glycoprotein, plays a key role in thyroid hormone synthesis. In a previous paper (Fayadat, L., Niccoli, P., Lanet, J., and Franc, J. L. (1998) Endocrinology 139, 4277-4285) we established that after the synthesis, only 15-20% of the hTPO molecules were recognized by a monoclonal antibody (mAb15) directed against a conformational structure and that only 2% were able to reach the cell surface. In the present study using pulse chase experiments in the presence or absence of protease inhibitors followed by immunoprecipitation procedures with monoclonal antibodies recognizing unfolded or partially folded hTPO forms we show that: (i) unfolded hTPO forms are degraded by the proteasome and (ii) partially folded hTPO forms are degraded by other proteases. It was also established upon incubating endoplasmic reticulum (ER) membranes in vitro that the degradation of the partially folded hTPO was carried out by serine and cysteine integral ER membrane proteases. These data provide valuable insights into the quality control mechanisms whereby the cells get rid of misfolded or unfolded proteins. Moreover, this is the first study describing a protein degradation process involving two distinct degradation pathways (proteasome and ER cysteine/serine proteases) at the ER level, depending on the folding state of the protein. PMID- 10748077 TI - Signaling through Ras is essential for ret oncogene-induced cell differentiation in PC12 cells. AB - Specific germline mutations of the receptor tyrosine kinase, Ret, predispose to multiple endocrine neoplasia types 2A and 2B and familial medullary thyroid carcinoma. The mechanisms by which different Ret isoforms (Ret-2A and Ret-2B) cause distinct neoplastic diseases remain largely unknown. On the other hand, forced expression of these mutated versions of Ret induces the rat pheochromocytoma cell line, PC12, to differentiate. Here we used an inducible vector encoding a dominant-negative Ras (Ras p21(N17)) to investigate the contributions of the Ras pathway to the phenotype induced in PC12 cells by the expression of either Ret-2A or Ret-2B mutants. We show that the Ret-induced molecular and morphological changes are both mediated by Ras-dependent pathways. However, even though inhibition of Ras activity was sufficient to revert Ret induced differentiation, the kinetics of morphological reversion of the Ret-2B- was more rapid than the Ret-2A-transfected cells. Further, we show that in Ret transfected cells the suc1-associated neurotrophic factor-induced tyrosine phosphorylation target, SNT, is chronically phosphorylated in tyrosine residues, and associates with the Sos substrate. These results indicate the activation of the Ras cascade as an essential pathway triggered by the chronic active Ret mutants in PC12 cells. Moreover, our data indicate SNT as a substrate for both Ret mutants, which might mediate the activation of this cascade. PMID- 10748078 TI - L-selectin signaling of neutrophil adhesion and degranulation involves p38 mitogen-activated protein kinase. AB - The adhesion molecules known as selectins mediate the capture of neutrophils from the bloodstream. We have previously reported that ligation and cross-linking of L selectin on the neutrophil surface enhances the adhesive function of beta(2) integrins in a synergistic manner with chemotactic agonists. In this work, we examined degranulation and adhesion of neutrophils in response to cross-linking of L-selectin and addition of interleukin-8. Cross-linking of L-selectin induced priming of degranulation that was similar to that observed with the alkaloid cytochalasin B. Activation mediated by L-selectin of neutrophil shape change and adhesion through CD11b/CD18 were strongly blocked by Merck C, an imidazole-based inhibitor of p38 mitogen-activated protein kinase (MAPK), but not by a structurally similar non-binding regioisomer. Priming by L-selectin of the release of secondary, tertiary, and secretory, but not primary, granules was blocked by inhibition of p38 MAPK. Peak phosphorylation of p38 MAPK was observed within 1 min of cross-linking L-selectin, whereas phosphorylation of ERK1/2 was highest at 10 min. Phosphorylation of p38 MAPK, but not ERK1/2, was inhibited by Merck C. These data suggest that signal transduction as a result of clustering L selectin utilizes p38 MAPK to effect neutrophil shape change, integrin activation, and the release of secondary, tertiary, and secretory granules. PMID- 10748079 TI - Glucocorticoids suppress tumor necrosis factor-alpha expression by human monocytic THP-1 cells by suppressing transactivation through adjacent NF-kappa B and c-Jun-activating transcription factor-2 binding sites in the promoter. AB - Glucocorticoid drugs suppress tumor necrosis factor-alpha (TNF-alpha) synthesis by activated monocyte/macrophages, contributing to an anti-inflammatory action in vivo. In lipopolysaccharide (LPS)-activated human monocytic THP-1 cells, glucocorticoids acted primarily on the TNF-alpha promoter to suppress a burst of transcriptional activity that occurred between 90 min and 3 h after LPS exposure. LPS increased nuclear c-Jun/ATF-2, NF-kappaB(1)/Rel-A, and Rel-A/C-Rel transcription factor complexes, which bound specifically to oligonucleotide sequences from the -106 to -88 base pair (bp) region of the promoter. The glucocorticoid, dexamethasone, suppressed nuclear binding activity of these complexes prior to and during the critical phase of TNF-alpha transcription. Site directed mutagenesis in TNF-alpha promoter-luciferase reporter constructs showed that the adjacent c-Jun/ATF-2 (-106 to -99 bp) and NF-kappaB (-97 to -88 bp) binding sites each contributed to the LPS-stimulated expression. Mutating both sites largely prevented dexamethasone from suppressing TNF-alpha promoter luciferase reporters. LPS exposure also increased nuclear Egr-1 and PU.1 abundance. The Egr-1/Sp1 (-172 to -161 bp) binding sites and the PU.1-binding Ets site (-116 to -110 bp) each contributed to the LPS-stimulated expression but not to glucocorticoid response. Dexamethasone suppressed the abundance of the c-Fos/c Jun complex in THP-1 cell nuclei, but there was no direct evidence for c-Fos/c Jun transactivation through sites in the -172 to -52 bp region. Small contributions to glucocorticoid response were attributable to promoter sequences outside the -172 to -88 bp region and to sequences in the TNF-alpha 3' untranslated region. We conclude that glucocorticoids suppress LPS-stimulated secretion of TNF-alpha from human monocytic cells largely through antagonizing transactivation by c-Jun/ATF-2 and NF-kappaB complexes at binding sites in the 106 to -88 bp region of the TNF-alpha promoter. PMID- 10748080 TI - CYP2E1 overexpression in HepG2 cells induces glutathione synthesis by transcriptional activation of gamma-glutamylcysteine synthetase. AB - Induction of CYP2E1 (cytochrome P450 2E1) by ethanol appears to be one of the central pathways by which ethanol generates a state of oxidative stress. CYP2E1 is a loosely coupled enzyme; formation of reactive oxygen species occurs even in the absence of added substrate. GSH is critical for preserving the proper cellular redox balance and for its role as a cellular protectant. Since cells must maintain optimal GSH levels to cope with a variety of stresses, the goal of this study was to characterize the GSH homeostasis in human hepatocarcinoma cells (HepG2) that overexpress CYP2E1. This study was prompted by the finding that toxicity in CYP2E1-overexpressing cells was markedly enhanced after GSH depletion by buthionine sulfoximine treatment. CYP2E1-overexpressing cells showed a 40-50% increase in intracellular H(2)O(2); a 30% increase in total GSH levels; a 50% increase in the GSH synthesis rate; and a 2-fold increase in gamma glutamylcysteine synthetase heavy subunit (GCS-HS) mRNA, the rate-limiting enzyme in GSH synthesis. This GCS-HS mRNA increase was due to increased synthesis since nuclear run-on assays showed increased transcription in CYP2E1-expressing cells, and the GCS-HS mRNA decay after actinomycin D treatment was similar in CYP2E1 expressing cells and empty vector-transfected cells. The facts that treatment with GSH ethyl ester almost completely prevented the increase in GCS-HS mRNA and decreased H(2)O(2) levels and that transient transfection with catalase (but not manganese-superoxide dismutase) produced a decrease in GCS-HS mRNA only in CYP2E1 expressing cells suggest a possible role for H(2)O(2) in the induction of GCS-HS gene transcription. In contrast to results with HepG2 cells expressing CYP2E1, no increase in GCS-HS mRNA was found with a HepG2 cell line engineered to express human cytochrome P450 3A4. In summary, CYP2E1 overexpression in HepG2 cells up regulates the levels of reduced GSH by transcriptional activation of GCS-HS; this may reflect an adaptive mechanism to remove CYP2E1-derived oxidants such as H(2)O(2). PMID- 10748081 TI - Detection of the mitochondrially encoded cytochrome c oxidase subunit I in the trypanosomatid protozoan Leishmania tarentolae. Evidence for translation of unedited mRNA in the kinetoplast. AB - With the aim of identification of kinetoplast-encoded proteins we investigated the subunit composition of cytochrome c oxidase (respiratory complex IV) from kinetoplast mitochondria of the trypanosomatid protozoan Leishmania tarentolae. Eleven stoichiometric subunits were visible in Coomassie-stained, two-dimensional Blue Native/Tricine-SDS electrophoretic gels. Their partial amino acid sequences indicated that these polypeptides are nuclear-encoded. The mitochondrial subunit I was detected with the polyclonal antibodies against an internal region of this polypeptide. In two-dimensional (9 versus 14%) polyacrylamide glycine-SDS gels this subunit is found as a series of spots located off the main diagonal, a property that can be explained by abnormal electrophoretic migration and aggregation. In gels loaded with high amounts of the purified, enzymatically active oxidase, the subunit I spots could be visualized by staining. The determined N-terminal amino acid sequence of the putative monomeric subunit I (MFXLCLVCLSVS) matched with the predicted sequence, thus indicating that the corresponding kinetoplast unedited mRNA is translated into a functional protein. PMID- 10748082 TI - Control of intramolecular interactions between the pleckstrin homology and Dbl homology domains of Vav and Sos1 regulates Rac binding. AB - Vav and Sos1 are Dbl family guanine nucleotide exchange factors, which activate Rho family GTPases in response to phosphatidylinositol 3-kinase products. A pleckstrin homology domain adjacent to the catalytic Dbl homology domain via an unknown mechanism mediates the effects of phosphoinositides on guanine nucleotide exchange activity. Here we tested the possibility that phosphatidylinositol 3 kinase substrates and products control an interaction between the pleckstrin homology domain and the Dbl homology domain, thereby explaining the inhibitory effects of phosphatidylinositol 3-kinase substrates and stimulatory effects of the products. Binding studies using isolated fragments of Vav and Sos indicate phosphatidylinositol 3-kinase substrate promotes the binding of the pleckstrin homology domain to the Dbl homology domain and blocks Rac binding to the DH domain, whereas phosphatidylinositol 3-kinase products disrupt the Dbl homology/pleckstrin homology interactions and permit Rac binding. Additionally, Lck phosphorylation of Vav, a known activating event, reduces the affinities between the Vav Dbl homology and pleckstrin homology domains and permits Rac binding. We also show Vav activation in cells, as monitored by phosphorylation of Vav, Vav association with phosphatidylinositol 3,4,5-trisphosphate, and Vav guanine nucleotide exchange activity, is blocked by the phosphatidylinositol 3 kinase inhibitor wortmannin. These results suggest the molecular mechanisms for activation of Vav and Sos1 require disruption of inhibitory intramolecular interactions involving the pleckstrin homology and Dbl homology domains. PMID- 10748083 TI - The conserved RING-H2 finger of ROC1 is required for ubiquitin ligation. AB - ROC1 is a common component of a large family of ubiquitin E3 ligases that regulate cell cycle progression and signal transduction pathways. Here we present evidence suggesting that a conserved RING-H2 structure within ROC1 is critical for its ubiquitin ligation function. Mercury-containing sulfhydryl modification agents (rho-hydroxymercuribenzoate and mercuric chloride) irreversibly inhibit the ROC1-CUL1 ubiquitin ligase activity without disrupting the complex. Consistent with this, these reagents also eliminate the ability of the Skp1-CUL1 HOS-ROC1 E3 ligase complex to support the ubiquitination of IkappaBalpha. Site directed mutagenesis analysis identifies RING-H2 finger residues Cys(42), Cys(45), Cys(75), His(77), His(80), Cys(83), Cys(94), and Asp(97) as being essential for the ROC1-dependent ubiquitin ligase activity. Furthermore, C42S/C45S and H80A mutations reduce the ability of ROC1 to interact with CUL1 in transfected cells and diminish the capacity of ROC1-CUL1 to form a stable complex with Cdc34 in vitro. However, C75S, H77A, C94S, and D97A substitutions have no detectable effect on ROC1 binding activities. Thus, the ROC1 RING-H2 finger may possess multiple biochemical properties that include stabilizing an interaction with CUL1 and recruiting Cdc34. A possible role of the RING finger in facilitating the Ub transfer reaction is discussed. PMID- 10748084 TI - Subunit IV of cytochrome bc1 complex from Rhodobacter sphaeroides. Localization of regions essential for interaction with the three-subunit core complex. AB - Recombinant subunit IV mutants which identify the regions essential for restoration of bc(1) activity to the three-subunit core complex of Rhodobacter sphaeroides were generated and characterized. Four C-terminal truncated mutants: IV(1-109), IV(1-85), IV(1-76), and IV(1-40) had 100, 0, 0, and 0% of reconstitutive activity of the wild-type IV, indicating that residues 86-109 are essential. IV(1-109) is associated with the core complex in the same manner as the wild-type IV while mutants IV(1-85), IV(1-76), and IV(1-40) do not associate with the core complex, indicating that subunit IV requires its transmembrane helix region (residues 86-109) for assembly into the bc(1) complex. Since GST IV(86-109) fusion protein has little reconstitutive activity, some region(s) in residues 1-85 are required for bc(1) activity restoration after subunit IV is incorporated into the complex through the transmembrane helix, presumably by interaction with cytochrome b in the core complex. The interacting regions are identified as residues 41-53 and 77-85, since mutants IV(21-109), IV(41-109), IV(54-109), and IV(77-109) had 95, 98, 53, and 53% of the reconstitutive activity of the wild-type IV. These two interacting regions are on the cytoplasmic side of the chromatophore membrane and closed to the DE loop and helix G of cytochrome b, respectively. PMID- 10748085 TI - Soluble E-selectin acts in synergy with platelet-activating factor to activate neutrophil beta 2-integrins. Role of tyrosine kinases and Ca2+ mobilization. AB - Selectins play a critical role in neutrophil recruitment to sites of inflammation, in tethering and rolling of neutrophils on vascular endothelium, as well as triggering beta(2)-integrin-mediated adhesion. We have previously demonstrated potential pro-inflammatory effects of soluble E-selectin upon neutrophil effector functions, using a soluble recombinant molecule (E-zz), which increased beta(2)-integrin-mediated adhesion, decreased beta(2)-integrin dependent migration, and triggered reactive oxygen species generation and release. In this study, we have examined the intracellular signals following neutrophil activation by soluble E-selectin. We show that exposure of neutrophils to E-selectin and platelet-activating factor (PAF) in combination induced a synergistic effect upon beta(2)-integrin-mediated adhesion. Although soluble E selectin did not induce Ca(2+) mobilization in neutrophils by itself, elevation of intracellular Ca(2+) was specifically prolonged in response to PAF but not leukotriene B(4) or N-formyl-Met-Leu-Phe. The prolonged Ca(2+) mobilization observed in the presence of E-selectin was dependent on Ca(2+) influx from intracellular stores rather than influx of extracellular Ca(2+) through SKF 96365 sensitive channels. The specific alteration of Ca(2+) mobilization reported here appears not to have a role in the synergistic effects of E-selectin and PAF upon neutrophil O(2) release but may be involved in augmentation of beta(2)-integrin mediated adhesion. PMID- 10748087 TI - Direct interaction of all-trans-retinoic acid with protein kinase C (PKC). Implications for PKC signaling and cancer therapy. AB - Protein kinase C (PKC) regulates fundamental cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. All-trans-retinoic acid (atRA) modulates PKC activity, but the mechanism of this regulation is unknown. Amino acid alignments and crystal structure analysis of retinoic acid (RA)-binding proteins revealed a putative atRA-binding motif in PKC, suggesting existence of an atRA binding site on the PKC molecule. This was supported by photolabeling studies showing concentration- and UV-dependent photoincorporation of [(3)H]atRA into PKCalpha, which was effectively protected by 4-OH-atRA, 9-cis RA, and atRA glucuronide, but not by retinol. Photoaffinity labeling demonstrated strong competition between atRA and phosphatidylserine (PS) for binding to PKCalpha, a slight competition with phorbol-12-myristate-13-acetate, and none with diacylglycerol, fatty acids, or Ca(2+). At pharmacological concentrations (10 micrometer), atRA decreased PKCalpha activity through the competition with PS but not phorbol-12-myristate-13-acetate, diacylglycerol, or Ca(2+). These results let us hypothesize that in vivo, pharmacological concentrations of atRA may hamper binding of PS to PKCalpha and prevent PKCalpha activation. Thus, this study provides the first evidence for direct binding of atRA to PKC isozymes and suggests the existence of a general mechanism for regulation of PKC activity during exposure to retinoids, as in retinoid-based cancer therapy. PMID- 10748088 TI - Calpain-dependent proteolytic cleavage of the p35 cyclin-dependent kinase 5 activator to p25. AB - Cyclin-dependent kinase 5 (CDK5) is a unique CDK, the activity of which can be detected in postmitotic neurons. To date, CDK5 purified from mammalian brains has always been associated with a truncated form of the 35-kDa major brain specific activator (p35, also known as nck5a) of CDK5, known as p25. In this study, we report that p35 can be cleaved to p25 both in vitro and in vivo by calpain. In a rat brain extract, p35 was cleaved to p25 by incubation with Ca(2+). This cleavage was inhibited by a calpain inhibitor peptide derived from calpastatin and was ablated by separating the p35.CDK5 from calpain by centrifugation. The p35 recovered in the pellet after centrifugation could then be cleaved to p25 by purified calpain. Cleavage of p35 was also induced in primary cultured neurons by treatment with a Ca(2+) ionophore and Ca(2+) and inhibited by calpain inhibitor I. The cleavage changed the solubility of the CDK5 active complex from the particulate fraction to the soluble fraction but did not affect the histone H1 kinase activity. Increased cleavage was detected in cultured neurons undergoing cell death, suggesting a role of the cleavage in neuronal cell death. PMID- 10748089 TI - A single C-terminal peptide segment mediates both membrane association and localization of lysyl hydroxylase in the endoplasmic reticulum. AB - Hydroxylation of lysyl residues is crucial for the unique glycosylation pattern found in collagens and for the mechanical strength of fully assembled extracellular collagen fibers. Hydroxylation is catalyzed in the lumen of the endoplasmic reticulum (ER) by a specific enzyme, lysyl hydroxylase (LH). The absence of the known ER-specific retrieval motifs in its primary structure and its association with the ER membranes in vivo have suggested that the enzyme is localized in the ER via a novel retention/retrieval mechanism. We have identified here a 40-amino acid C-terminal peptide segment of LH that is able to convert cathepsin D, normally a soluble lysosomal protease, into a membrane-associated protein. The same segment also markedly slows down the transport of the reporter protein from the ER into post-ER compartments, as assessed by our pulse-chase experiments. The retardation efficiency mediated by this C-terminal peptide segment is comparable with that of the intact LH but lower than that of the KDEL receptor-based retrieval mechanism. Within this 40-amino acid segment, the first 25 amino acids appear to be the most crucial ones in terms of membrane association and ER localization, because the last 15 C-terminal amino acids did not possess substantial retardation activity alone. Our findings thus define a short peptide segment very close to the extreme C terminus of LH as the only necessary determinant both for its membrane association and localization in the ER. PMID- 10748090 TI - Glucose regulates islet amyloid polypeptide gene transcription in a PDX1- and calcium-dependent manner. AB - Islet amyloid polypeptide (IAPP) and insulin are expressed in the beta-cells of the islets of Langerhans. They are co-secreted in response to changes in glucose concentration, and their mRNA levels are also regulated by glucose. The promoters of both genes share similar cis-acting sequence elements, and both bind the homeodomain transcription factor PDX1, which plays an important role in the regulation of the insulin promoter and insulin mRNA levels by glucose. Here we examine the role of PDX1 in the regulation of the human IAPP promoter by glucose. The experiments were facilitated by the availability of a human beta-cell line (NES2Y) that lacks PDX1. NES2Y cells also lack operational K(ATP) channels, resulting in a loss of control of calcium signaling. We have previously used these cells to show that glucose regulation of the insulin gene is dependent on PDX1, but not calcium. In the mouse beta-cell line Min6, glucose (16 mm) stimulated a 3.5-4-fold increase in the activity of a -222 to +450 IAPP promoter construct compared with values observed in 0.5 mm glucose. In NES2Y cells, glucose failed to stimulate transcriptional activation of the IAPP promoter. Overexpression of PDX1 in NES2Y cells failed to reinstate glucose-responsive control of the IAPP promoter. Glucose effects on the IAPP promoter were observed only in the presence of PDX1 when normal calcium signaling was restored by overexpression of the two K(ATP) channel subunits SUR1 and Kir6.2. The importance of calcium was further emphasized by an experiment in which glucose-stimulated IAPP promoter activity was inhibited by the calcium channel blocker verapamil (50 microm). Verapamil was further shown to inhibit the stimulatory effect of glucose on IAPP mRNA levels. These results demonstrate that like the insulin promoter, glucose regulation of the IAPP promoter is dependent on the activity of PDX1, but unlike the insulin promoter, it additionally requires the activity of another, as yet uncharacterized factor(s), the activity of which is calcium-dependent. PMID- 10748091 TI - Measurement of glucose uptake and intracellular calcium concentration in single, living pancreatic beta-cells. AB - There has been no method previously to measure both glucose transport and its effect on the various intracellular functions in single, living mammalian cells. A fluorescent derivative of d-glucose, 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4 yl)amino]-2-deoxy-d-glucose (2-NBDG), that we have developed has made such measurements possible. COS-1 cells that overexpress the human glucose transporter GLUT2 show significantly greater 2-NBDG uptake than mock transfected cells. Using GLUT2-abundant mouse insulin-secreting clonal MIN6 cells, we found that 2-NBDG was incorporated into the cells in a time- and concentration-dependent manner. The 2-NBDG uptake was inhibited by high concentrations of d-glucose in a dose dependent manner and also was almost completely inhibited by 10 micrometer cytochalasin B. We then measured both glucose uptake and the intracellular calcium concentration ([Ca(2+)](i)) in single, living pancreatic islet cells. 2 NBDG and fura-2 were used as the tracer of glucose and indicator of intracellular calcium, respectively. All of the cells that showed an increase in [Ca(2+)](i) in response to a high concentration of glucose (16.8 mm) rapidly incorporated significant 2-NBDG. Immunocytochemical examination confirmed these cells to be insulin-positive beta-cells. All of the cells that showed no significant, rapid 2 NBDG uptake lacked such glucose responsiveness of [Ca(2+)](i), indicating that these cells were non-beta-cells such as glucagon-positive alpha-cells. These results show the uptake of glucose causing a concomitant increase of [Ca(2+)](i) in beta-cells. Because 2-NBDG is incorporated into mammalian cells through glucose transporters, it should be useful for the measurement of glucose uptake together with concomitant intracellular activities in many types of single, living mammalian cells. PMID- 10748092 TI - N-terminal region, C-terminal region, nuclear export signal, and deacetylation activity of histone deacetylase-3 are essential for the viability of the DT40 chicken B cell line. AB - Histone deacetylases (HDACs) are involved in the deacetylation of core histones, which is related to transcription regulation in eukaryotes through alterations in the chromatin structure. We cloned cDNA and genomic DNA encoding a chicken HDAC, chHDAC-3, which was localized in both the nuclei and cytoplasm in DT40 cells. Although one of the two chHDAC-3 alleles could be disrupted with high efficiency, no homozygous mutants were obtained after a second round of the gene-targeting technique due to its necessity for DT40 cells. We introduced a chHDAC-3 transgene under the control of a tetracycline-responsive promoter into the heterozygous mutant and subsequently disrupted the remaining endogenous chHDAC-3 allele to generate the homozygous chHDAC-3-deficient mutant, DeltachHDAC-3/FHDAC3. Inhibition of the expression of the regulatable chHDAC-3 transgene caused apoptotic cell death of the mutant. Complementation experiments involving truncated and missense chHDAC-3 mutant proteins revealed that the 1-23 N-terminal sequence, the 389-417 C-terminal sequence, the nuclear export signal, and the deacetylation activity of chHDAC-3 were essential for the cell viability. Taken together, these results indicate that chHDAC-3 plays an essential role, probably as a scavenger in the cytoplasm, in the proliferation of DT40 cells. PMID- 10748093 TI - Selective inhibition of trypsin by (2R,4R)-4-phenyl-1-[Nalpha-(7- methoxy-2 naphthalenesulfonyl)-L-arginyl]-2-piperidinecarboxylic acid. AB - Evidence is accumulating indicating that trypsin stimulates divergent cellular reactions through the proteinase-activated receptor, in addition to its role as the digestive enzyme. In this report, we introduce (2R,4R)- 4-phenyl-1-[N(alpha) (7-methoxy-2-naphthalenesulfonyl)-l-arginyl]- 2-p iperidinecarboxylic acid as a potent and selective trypsin inhibitor. The agent inhibited trypsin competitively with the K(i) value of 0. 1 micrometer. It inhibited thrombin weakly (K(i) = 2 micrometer) and did not inhibit plasmin, plasma kallikrein, urokinase, and mast cell tryptase (K(i) values for these enzymes are >60 micrometer). Comparative studies with several established proteinase inhibitors revealed that the compound was the first small molecular weight trypsin inhibitor without tryptase inhibitory activity. A docking study has provided a plausible explanation for the molecular mechanism of the selective inhibition showing that the agent fits into the active site of trypsin without any severe collision but that it comes into clash at the 4-phenyl group of piperidine ring against the "60-insertion loop" of thrombin and at the 7-methoxy-2-naphthalenesulfonyl group against Gln(98) of tryptase. PMID- 10748095 TI - IB1 reduces cytokine-induced apoptosis of insulin-secreting cells. AB - IB1/JIP-1 is a scaffold protein that interacts with upstream components of the c Jun N-terminal kinase (JNK) signaling pathway. IB1 is expressed at high levels in pancreatic beta cells and may therefore exert a tight control on signaling events mediated by JNK in these cells. Activation of JNK by interleukin 1 (IL-1beta) or by the upstream JNK constitutive activator DeltaMEKK1 promoted apoptosis in two pancreatic beta cell lines and decreased IB1 content by 50-60%. To study the functional consequences of the reduced IB1 content in beta cell lines, we used an insulin-secreting cell line expressing an inducible IB1 antisense RNA that lead to a 38% IB1 decrease. Reducing IB1 levels in these cells increased phosphorylation of c-Jun and increased the apoptotic rate in presence of IL 1beta. Nitric oxide production was not stimulated by expression of the IB1 antisense RNA. Complementary experiments indicated that overexpression of IB1 in insulin-producing cells prevented JNK-mediated activation of the transcription factors c-Jun, ATF2, and Elk1 and decreased IL-1beta- and DeltaMEKK1-induced apoptosis. These data indicate that IB1 plays an anti-apoptotic function in insulin-producing cells probably by controlling the activity of the JNK signaling pathway. PMID- 10748094 TI - DNA recognition, strand selectivity, and cleavage mode during integrase family site-specific recombination. AB - We have probed the association of Flp recombinase with its DNA target using protein footprinting assays. The results are consistent with the domain organization of the Flp protein and with the general features of the protein-DNA interactions revealed by the crystal structures of the recombination intermediates formed by Cre, the Flp-related recombinase. The similarity in the organization of the Flp and Cre target sites and in their recognition by the respective recombinases implies that the overall DNA-protein geometry during strand cleavage in the two systems must also be similar. Within the functional recombinase dimer, it is the interaction between two recombinase monomers bound on either side of the strand exchange region (or spacer) that provides the allosteric activation of a single active site. Whereas Cre utilizes the cleavage nucleophile (the active site tyrosine) in cis, Flp utilizes it in trans (one monomer donating the tyrosine to its partner). By using synthetic Cre and Flp DNA substrates that are geometrically restricted in similar ways, we have mapped the positioning of the active and inactive tyrosine residues during cis and trans cleavage events. We find that, for a fixed substrate geometry, Flp and Cre cleave the labile phosphodiester bond at the same spacer end, not at opposite ends. Our results provide a model that accommodates local heterogeneities in peptide orientations in the two systems while preserving the global functional architecture of the reaction complex. PMID- 10748096 TI - Electrospray ionization/mass spectrometric analyses of human promonocytic U937 cell glycerolipids and evidence that differentiation is associated with membrane lipid composition changes that facilitate phospholipase A2 activation. AB - Upon differentiation, U937 promonocytic cells gain the ability to release a large fraction of arachidonate esterified in phospholipids when stimulated, but the mechanism is unclear. U937 cells express group IV phospholipase A(2) (cPLA(2)), but neither its level nor its phosphorylation state increases upon differentiation. A group VI PLA(2) (iPLA(2)) that is sensitive to a bromoenol lactone inhibitor catalyzes arachidonate hydrolysis from phospholipids in some cells and facilitates arachidonate incorporation into glycerophosphocholine (GPC) lipids in others, but it is not known whether U937 cells express iPLA(2). We confirm that ionophore A23187 induces substantial [(3)H]arachidonate release from differentiated but not control U937 cells, and electrospray ionization mass spectrometric (ESI/MS) analyses indicate that differentiated cells contain a higher proportion of arachidonate-containing GPC species than control cells. U937 cells express iPLA(2) mRNA and activity, but iPLA(2) inhibition impairs neither [(3)H]arachidonate incorporation into nor release from U937 cells. Experiments with phosphatidate phosphohydrolase (PAPH) and phospholipase D (PLD) inhibitors coupled with ESI/MS analyses of PLD-PAPH products indicate that differentiated cells gain the ability to produce diacylglycerol (DAG) via PLD-PAPH. DAG promotes arachidonate release by a mechanism that does not require DAG hydrolysis, is largely independent of protein kinase C, and requires cPLA(2) activity. This may reflect DAG effects on cPLA(2) substrate state. PMID- 10748097 TI - Identification of a plasma membrane-associated guanine nucleotide exchange factor for ARF6 in chromaffin cells. Possible role in the regulated exocytotic pathway. AB - ADP-ribosylation factors (ARFs) constitute a family of structurally related proteins that forms a subset of the Ras superfamily of regulatory GTP-binding proteins. Like other GTPases, activation of ARFs is facilitated by specific guanine nucleotide exchange factors (GEFs). In chromaffin cells, ARF6 is associated with the membrane of secretory granules. Stimulation of intact cells or direct elevation of cytosolic calcium in permeabilized cells triggers the rapid translocation of ARF6 to the plasma membrane and the concomitant activation of phospholipase D (PLD) in the plasma membrane. Both calcium-evoked PLD activation and catecholamine secretion in permeabilized cells are strongly inhibited by a synthetic peptide corresponding to the N-terminal domain of ARF6, suggesting that the ARF6-dependent PLD activation near the exocytotic sites represents a key event in the exocytotic reaction in chromaffin cells. In the present study, we demonstrate the occurrence of a brefeldin A-insensitive ARF6 GEF activity in the plasma membrane and in the cytosol of chromaffin cells. Furthermore, reverse transcriptase-polymerase chain reaction and immunoreplica analysis indicate that ARNO, a member of the brefeldin A-insensitive ARF-GEF family, is expressed and predominantly localized in the cytosol and in the plasma membrane of chromaffin cells. Using permeabilized chromaffin cells, we found that the introduction of anti-ARNO antibodies into the cytosol inhibits, in a dose dependent manner, both PLD activation and catecholamine secretion in calcium stimulated cells. Furthermore, co-expression in PC12 cells of a catalytically inactive ARNO mutant with human growth hormone as a marker of secretory granules in transfected cells resulted in a 50% inhibition of growth hormone secretion evoked by depolarization with high K(+). The possibility that the plasma membrane associated ARNO participates in the exocytotic pathway by activating ARF6 and downstream PLD is discussed. PMID- 10748098 TI - mHDA1/HDAC5 histone deacetylase interacts with and represses MEF2A transcriptional activity. AB - Recently we identified a new family of histone deacetylases in higher eukaryotes related to yeast HDA1 and showed their differentiation-dependent expression. Data presented here indicate that HDAC5 (previously named mHDA1), one member of this family, might be a potent regulator of cell differentiation by interacting specifically with determinant transcription factors. We found that HDAC5 was able to interact in vivo and in vitro with MEF2A, a MADS box transcription factor, and to strongly inhibit its transcriptional activity. Surprisingly, this repression was independent of HDAC5 deacetylase domain. The N-terminal non-deacetylase domain of HDAC5 was able to ensure an efficient repression of MEF2A-dependent transcription. We then mapped protein domains involved in the HDAC5-MEF2A interaction and showed that MADS box/MEF2-domain region of MEF2A interacts specifically with a limited region in the N-terminal part of HDAC5 which also possesses a distinct repressor domain. These data show that two independent class II histone deacetylases HDAC4 and HDAC5 are able to interact with members of the MEF2 transcription factor family and regulate their transcriptional activity, thus suggesting a critical role for these deacetylases in the control of cell proliferation/differentiation. PMID- 10748099 TI - Alternative antigen receptor (TCR) signaling in T cells derived from ZAP-70 deficient patients expressing high levels of Syk. AB - ZAP-70-deficient patients present with nonfunctional CD4+ T cells in the periphery. We find that a subset of primary ZAP-70-deficient T cells, expressing high levels of the related protein-tyrosine kinase Syk, can proliferate in vitro. These cells (denoted herein as Syk(hi)/ZAP-70(-) T cells) provide a unique model in which the contribution of Syk to TCR-mediated responses can be explored in a nontransformed background. Importantly, CD3-induced responses, such as tyrosine phosphorylation of cellular substrates (LAT, SLP76, and PLC-gamma1), as well as calcium mobilization, which are defective in T cells expressing neither ZAP-70 nor Syk, are observed in Syk(hi)/ZAP-70(-) T cells. However, Syk(hi)/ZAP-70(-) T cells differ from control T cells with respect to the T cell antigen receptor (TCR)-mediated activation of the MAPK cascades: extracellular signal-regulated kinase activity and recruitment of the JNK and p38 stress-related MAPK pathways are diminished. This distinct phenotype of Syk(hi)/ZAP-70(-) T cells is associated with a profound decrease in CD3-mediated interleukin 2 secretion and proliferation relative to control T cells. Thus, ZAP-70 and Syk appear to play distinct roles in transducing a TCR-mediated signal. PMID- 10748100 TI - BMP2-induced apoptosis is mediated by activation of the TAK1-p38 kinase pathway that is negatively regulated by Smad6. AB - Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor beta (TGF-beta) superfamily, regulates a variety of cell fates and functions. At present, the molecular mechanism by which BMP2 induces apoptosis has not been fully elucidated. Here we propose a BMP2 signaling pathway that mediates apoptosis in mouse hybridoma MH60 cells whose growth is interleukin-6 (IL-6) dependent. BMP2 dose-dependently induces apoptosis in MH60 cells even in the presence of IL-6. BMP2 has no inhibitory effect on the IL-6-induced tyrosine phosphorylation of STAT3, and the bcl-2 gene expression which is known to be regulated by STAT3, suggesting that BMP2-induced apoptosis is not attributed to alteration of the IL-6-mediated bcl-2 pathway. We demonstrate that BMP2 induces activation of TGF-beta-activated kinase (TAK1) and subsequent phosphorylation of p38 stress-activated protein kinase. In addition, forced expression of kinase negative TAK1 in MH60 cells blocks BMP2-induced apoptosis. These results indicate that BMP2-induced apoptosis is mediated through the TAK1-p38 pathway in MH60 cells. We also show that MH60-derived transfectants expressing Smad6 are resistant to the apoptotic signal of BMP2. Interestingly, this ectopic expression of Smad6 blocks BMP2-induced TAK1 activation and p38 phosphorylation. Moreover, Smad6 can directly bind to TAK1. These findings suggest that Smad6 is likely to function as a negative regulator of the TAK1 pathway in the BMP2 signaling, in addition to the previously reported Smad pathway. PMID- 10748101 TI - Significance of nuclear relocalization of ERK1/2 in reactivation of c-fos transcription and DNA synthesis in senescent fibroblasts. AB - Two of mitogen-activated protein kinases (MAPK), p44(mapk)/p42(mapk) extracellular signal-regulated kinases (ERK1/2), translocate into nuclei following activation and play critical roles in connecting the signal to gene expression and allowing cell-cycle entry. Here we found that the nuclear translocation of ERK1/2 in response to growth stimuli was significantly inhibited in senescent cells that were irreversibly growth arrested, compared with presenescent cells. The activation step of these enzymes was not impaired, since ERK1/2 were phosphorylated and activated in senescent cells as efficiently as in presenescent cells. By elaborately localizing ERK2 in the nuclei of senescent cells, we could restore c-fos transcriptional activity upon growth stimuli, which was repressed in senescent cells. Furthermore, the nuclear localization of ERK1/2 has been suggested to potentiate the proliferative activity of the senescent cells in collaboration with adenovirus E1A protein. More importantly, SV40 large T antigen, the strong inducer of DNA synthesis, had the inherent ability to restore nuclear relocalization of active ERK1/2 in senescent cells, which was essentially required for the reinitiation of DNA synthesis. Thus, manipulating the relocalization of ERK1/2 into nuclei was expected to open the way to overcome some of the senescent phenotypes. PMID- 10748102 TI - Dissection of the functional domains of the Leishmania surface membrane 3' nucleotidase/nuclease, a unique member of the class I nuclease family. AB - Class I nucleases are a family of enzymes that specifically hydrolyze single stranded nucleic acids. Recently, we characterized the gene encoding a new member of this family, the 3'-nucleotidase/nuclease (Ld3'NT/NU) of the parasitic protozoan Leishmania donovani. The Ld3'NT/NU is unique as it is the only class I nuclease that is a cell surface membrane-anchored protein. Currently, we used a homologous episomal expression system to dissect the functional domains of the Ld3'NT/NU. Our results showed that its N-terminal signal peptide targeted this protein into the endoplasmic reticulum. Using Ld3'NT/NU-green fluorescent protein chimeras, we showed that the C-terminal domain of the Ld3'NT/NU functioned to anchor this protein into the parasite cell surface membrane. Further, removal of the Ld3'NT/NU C-terminal domain resulted in its release/secretion as a fully active enzyme. Moreover, deletion of its single N-linked glycosylation site showed that such glycosylation was not required for the enzymatic functions of the Ld3'NT/NU. Thus, using the fidelity of a homologous expression system, we have defined some of the functional domains of this unique member of the class I nuclease family. PMID- 10748103 TI - Selective activation of the glucocorticoid receptor by steroid antagonists in human breast cancer and osteosarcoma cells. AB - Steroid hormones regulate the transcription of numerous genes via high affinity receptors that act in concert with chromatin remodeling complexes, coactivators and corepressors. We have compared the activities of a variety of glucocorticoid receptor (GR) antagonists in breast cancer and osteosarcoma cell lines engineered to stably maintain the mouse mammary tumor virus promoter. In both cell types, GR activation by dexamethasone occurs via the disruption of mouse mammary tumor virus chromatin structure and the recruitment of receptor coactivator proteins. However, when challenged with a variety of antagonists the GR displays differential ability to activate transcription within the two cell types. For the breast cancer cells, the antagonists fail to activate the promoter and do not promote the association of the GR with either remodeling or coactivator proteins. In contrast, in osteosarcoma cells, the antiglucocorticoids, RU486 and RU43044, exhibit partial agonist activity. The capacity of these antagonists to stimulate transcription in the osteosarcoma cells is reflected in the ability of the RU486 bound receptor to remodel chromatin and associate with chromatin-remodeling proteins. Similarly, the observation that the RU486-bound receptor does not fully activate transcription is consistent with its inability to recruit receptor coactivator proteins. PMID- 10748104 TI - Electron flow between photosystem II and oxygen in chloroplasts of photosystem I deficient algae is mediated by a quinol oxidase involved in chlororespiration. AB - In Chlamydomonas reinhardtii mutants deficient in photosystem I because of inactivation of the chloroplast genes psaA or psaB, oxygen evolution from photosystem II occurs at significant rates and is coupled to a stimulation of oxygen uptake. Both activities can be simultaneously monitored by continuous mass spectrometry in the presence of (18)O(2). The light-driven O(2) exchange was shown to involve the plastoquinone pool as an electron carrier, but not cytochrome b(6)f. Photosystem II-dependent O(2) production and O(2) uptake were observed in isolated chloroplast fractions. Photosystem II-dependent oxygen exchange was insensitive to a variety of inhibitors (azide, carbon monoxide, cyanide, antimycin A, and salicylhydroxamic acid) and radical scavengers. It was, however, sensitive to propyl gallate. From inhibitors effects and electronic requirements of the O(2) uptake process, we conclude that an oxidase catalyzing oxidation of plastoquinol and reduction of oxygen to water is present in thylakoid membranes. From the sensitivity of flash-induced O(2) exchange to propyl gallate, we conclude that this oxidase is involved in chlororespiration. Clues to the identity of the protein implied in this process are given by pharmacological and immunological similarities with a protein (IMMUTANS) identified in Arabidopsis chloroplasts. PMID- 10748105 TI - Identification of a second MutL DNA mismatch repair complex (hPMS1 and hMLH1) in human epithelial cells. AB - Deficiencies of MutL DNA mismatch repair-complex proteins (hMLH1, hPMS2, and hPMS1) typically result in microsatellite instability in human cancers. We examined the association patterns of MutL proteins in human epithelial cancer cell lines with (HCT-116, N87, SNU-1, and SNU-638) and without microsatellite instability (HeLa, AGS, KATO-III, and SNU-16). The analysis of hMLH1, hPMS2, and hPMS1 was performed using Northern blot, Western blot, and co-immunoprecipitation studies. Our data provide evidence that MutL proteins form two different complexes, MutL-alpha (hPMS2 and hMLH1) and MutL-beta (hPMS1 and hMLH1). Gastric and colorectal cancer cells lines with microsatellite instability lacked detectable hMLH1. Decreased levels of hMLH1 protein were associated with markedly reduced levels of hPMS2 and hPMS1 proteins, but the RNA levels of hPMS1 and hPMS2 were normal. In this study, we describe the association of hPMS1 with hMLH1 as a heterodimer, in human cells. Furthermore, normal levels of hMLH1 protein appear to be important in maintaining normal levels of hPMS1 and hPMS2 proteins. PMID- 10748106 TI - Transferrin receptor 2-alpha supports cell growth both in iron-chelated cultured cells and in vivo. AB - In most cells, transferrin receptor (TfR1)-mediated endocytosis is a major pathway for cellular iron uptake. We recently cloned the human transferrin receptor 2 (TfR2) gene, which encodes a second receptor for transferrin (Kawabata, H., Yang, R., Hirama, T., Vuong, P. T., Kawano, S., Gombart, A. F., and Koeffler, H. P. (1999) J. Biol. Chem. 274, 20826-20832). In the present study, the regulation of TfR2 expression and function was investigated. A select Chinese hamster ovary (CHO)-TRVb cell line that does not express either TfR1 or TfR2 was stably transfected with either TfR1 or TfR2-alpha cDNA. TfR2-alpha expressing cells had considerably lower affinity for holotransferrin when compared with TfR1-expressing CHO cells. Interestingly, in contrast to TfR1, expression of TfR2 mRNA in K562 cells was not up-regulated by desferrioxamine (DFO), a cell membrane-permeable iron chelator. In MG63 cells, expression of TfR2 mRNA was regulated in the cell cycle with the highest expression in late G(1) phase and no expression in G(0)/G(1). DFO reduced cell proliferation and DNA synthesis of CHO-TRVb control cells, whereas it had little effect on TfR2-alpha expressing CHO cells when measured by clonogenic and cell cycle analysis. In addition, CHO cells that express TfR2-alpha developed into tumors in nude mice whereas CHO control cells did not. In conclusion, TfR2 expression may be regulated by the cell cycle rather than cellular iron status and may support cell growth both in vitro and in vivo. PMID- 10748107 TI - The two toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase isozymes form heterotetramers. AB - Two isozymes of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) of the apicomplexan protozoan Toxoplasma gondii are encoded by the single HGPRT gene as a result of differential splicing. Western blotting of total T. gondii protein shows that both isozymes I and II, which differ by 49 amino acids, are expressed. Both form enzymatically active homotetramers when overexpressed in Escherichia coli. The specific activity of HGPRT-I is five times that of HGPRT-II. When both isozymes are co-expressed in E. coli, HGPRT-I.HGPRT-II heterotetramers form. The predominant heterotetramer has enzymatic activity similar to HGPRT-II, and gel filtration chromatography demonstrates that its size is intermediate between the sizes of HGPRT-I and HGPRT II. Mass spectrometric analysis of cross-linked homo- and heterotetramers reveals species of distinct molecular mass for HGPRT-I, HGPRT-II, and HGPRT-I.HGPRT-II and suggests that the predominant heterotetramer consists of one HGPRT-I subunit and three HGPRT-II subunits. The implications of this finding are discussed. PMID- 10748108 TI - Bile acid-induced activation of activator protein-1 requires both extracellular signal-regulated kinase and protein kinase C signaling. AB - Elevated concentrations of fecal bile aids are known to promote colon cancer and increasing evidence suggests that alterations in cellular signaling and gene expression may play an important role in this process. In this study, we examined the molecular mechanisms underlying bile acid-mediated gene regulation using GADD153 as our model gene. Promoter deletion analyses revealed that the activator protein-1 (AP-1) transcription factor was crucial for deoxycholic acid (DCA) mediated GADD153 gene transcription. Electrophoretic mobility shift assays and transient transfection analyses demonstrated that both DNA binding and transactivation activities of AP-1 were induced by DCA in a dose-dependent manner. The AP-1 complex induced by DCA consisted of JunD, Fra-1, and c-Fos. Examination of the signaling pathways stimulated by DCA showed that extracellular signal-regulated kinases (ERKs) were required for AP-1 activation. Inhibition of ERK by the mitogen-activated protein kinase/ERK kinase inhibitor PD 98059 or by expression of a dominant negative mutant ERK suppressed AP-1 activation. Notably, the PKC inhibitor, calphostin C, also abolished DCA-induced AP-1 activation but did not affect DCA-mediated ERK activation, suggesting that ERK and PKC function in separate signaling pathways that cooperatively mediate DCA-induced AP-1 activation. Hence, bile acid-stimulated signaling appears to converge on the AP-1 protooncogene. PMID- 10748109 TI - Jak2 acts as both a STAT1 kinase and as a molecular bridge linking STAT1 to the angiotensin II AT1 receptor. AB - Angiotensin II activates the Jak-STAT pathway via the AT(1) receptor. We studied two mutant AT(1) receptors, termed M5 and M6, that contain Y to F substitutions for the tyrosine residues naturally found in the third intracellular loop and the carboxyl terminus. After binding ligand, both the M5 and M6 AT(1) receptors trigger STAT1 tyrosine phosphorylation equivalent to that observed with the wild type receptor, indicating that angiotensin II-mediated phosphorylation of STAT1 is independent of these receptor tyrosine residues. In response to angiotensin II, Jak2 autophosphorylates on tyrosine, and Jak2 and STAT1 physically associate, a process that depends on the SH2 domain of STAT1 in vitro. Evaluation of the wild type, M5, and M6 AT(1) receptors showed that angiotensin II-dependent AT(1) receptor-Jak2-STAT1 complex formation is dependent on catalytically active Jak2, not on the receptor tyrosine residues in the third intracellular loop and carboxyl tail. Immunodepletion of Jak2 virtually eliminated the ligand-dependent binding of STAT1 to the AT(1) receptor. These data indicate that the association of STAT1 with the AT(1) receptor is not strictly bimolecular; it requires Jak2 as both a STAT1 kinase and as a molecular bridge linking STAT1 to the AT(1) receptor. PMID- 10748110 TI - The autoimmune regulator protein has transcriptional transactivating properties and interacts with the common coactivator CREB-binding protein. AB - Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, caused by mutations in the autoimmune regulator (AIRE) gene, is an autosomal recessive autoimmune disease characterized by the breakdown of tolerance to organ-specific antigens. The 545 amino acid protein encoded by AIRE contains several structural motifs suggestive of a transcriptional regulator and bears similarity to cellular proteins involved in transcriptional control. We show here that AIRE fused to a heterologous DNA binding domain activates transcription from a reporter promoter, and the activation seen requires the full-length protein or more than one activation domain. At the structural level AIRE forms homodimers through the NH(2)-terminal domain, and molecular modeling for this domain suggests a four helix bundle structure. In agreement, we show that the common transcriptional coactivator CREB-binding protein (CBP) interacts with AIRE in vitro and in yeast nuclei through the CH1 and CH3 conserved domains. We suggest that the transcriptional transactivation properties of AIRE together with its interaction with CBP might be important in its function as disease-causing mutations almost totally abolish the activation effect. PMID- 10748111 TI - Oxidative stress induces protein kinase D activation in intact cells. Involvement of Src and dependence on protein kinase C. AB - Protein kinase D (PKD) is a protein serine kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids, and activated by phorbol esters, neuropeptides, and platelet-derived growth factor via protein kinase C (PKC) in intact cells. Recently, oxidative stress was shown to activate transfected PKC isoforms via tyrosine phosphorylation, but PKD activation was not demonstrated. Here, we report that oxidative stress initiated by addition of H(2)O(2) (0.15-10 mm) to quiescent Swiss 3T3 fibroblasts activates PKD in a dose- and time- dependent manner, as measured by autophosphorylation and phosphorylation of an exogenous substrate, syntide-2. Oxidative stress also activated transfected PKD in COS-7 cells but not a kinase-deficient mutant PKD form or a PKD mutant with critical activating serine residues 744 and 748 mutated to alanines. Genistein, or the specific Src inhibitors PP-1 and PP-2 (1-10 micrometer) inhibited H(2)O(2)-mediated PKD activation by 45%, indicating that Src contributes to this signaling pathway. PKD activation by H(2)O(2) was also selectively potentiated by cotransfection of PKD together with an active form of Src (v-Src) in COS-7 cells, as compared with PDB-mediated activation. The specific phospholipase C inhibitor, partly blocked H(2)O(2)-mediated but not PDB mediated PKD activation. In contrast, PKC inhibitors blocked H(2)O(2) or PDB mediated PKD activation essentially completely, suggesting that whereas Src mediates part of its effects via phospholipase C activation, PKC acts more proximally as an upstream activator of PKD. Together, these studies reveal that oxidative stress activates PKD by initiating distinct Src-dependent and independent pathways involving PKC. PMID- 10748112 TI - Cloning and characterization of a novel human class I histone deacetylase that functions as a transcription repressor. AB - Histone acetylation alters chromatin state by modifying lysines on histone and plays an important role in modulating gene transcription. A dynamic balance of histone acetylation/deacetylation is maintained by histone acetyltransferases and histone deacetylases. Emerging evidence suggests that a family of histone deacetylases may exist to regulate diverse cellular functions, including chromatin structure, gene expression, cell cycle progression, and oncogenesis. We describe here a novel human histone deacetylase, named HDAC8, cloned from human kidney. HDAC8 encodes 377 amino acid residues and shares extensive homology to several known HDACs, in particular a histone deacetylase from Arabidopsis thaliana. Northern blot analyses revealed that HDAC8 expression pattern for HDAC8 is distinct from that for HDAC1 and HDAC3, and expression of HDAC8 mRNA occurs in multiple organs including heart, lung, kidney, and pancreas. HDAC8 mRNA was also observed in several cell lines derived from cancerous tissues. When expressed in HEK293 cells, HDAC8 exhibited deacetylase activity toward acetylated histone, indicating that this protein is a bona fide histone deacetylase. Its histone deacetylase activity was inhibited by trichostatin and other known histone deacetylase inhibitors. Furthermore, active recombinant HDAC8 was expressed and purified from Escherichia coli. When ectopically expressed in cells, HDAC8 was found to be localized to the nucleus. Co-transfection experiments demonstrated that expression of HDAC8 repressed a viral SV40 early promoter activity. These results indicate that HDAC8 is a novel member of the histone deacetylase family, which may play a role in the development of a broad range of tissues and potentially in the etiology of cancer. PMID- 10748113 TI - The RIM/NIM family of neuronal C2 domain proteins. Interactions with Rab3 and a new class of Src homology 3 domain proteins. AB - RIM1 is a putative effector protein for Rab3s, synaptic GTP-binding proteins. RIM1 is localized close to the active zone at the synapse, where it interacts in a GTP-dependent manner with Rab3 located on synaptic vesicles. We now describe a second RIM protein, called RIM2, that is highly homologous to RIM1 and also expressed primarily in brain. Like RIM1, RIM2 contains an N-terminal zinc finger domain that binds to Rab3 as a function of GTP, a central PDZ domain, and two C terminal C(2) domains that are separated by long alternatively spliced sequences. Unexpectedly, the 3'-end of the RIM2 gene produces an independent mRNA that encodes a smaller protein referred as NIM2. NIM2 is composed of a unique N terminal sequence followed by the C-terminal part of RIM2. Data bank searches identified a third RIM/NIM-related gene, which encodes a NIM isoform referred to as NIM3; no RIM transcript from this gene was detected. To test if NIMs, like RIMs, may function in secretion, we investigated the effect of NIM3 on calcium triggered exocytosis in PC12 cells. NIM3 induced a dramatic increase in calcium evoked exocytosis (50%), with no significant effect on base-line release, suggesting that NIMs, like RIMs, regulate exocytosis The combination of conserved and variable sequences in RIMs and NIMs indicates that the individual domains of these proteins provide binding sites for interacting molecules during exocytosis, as shown for the zinc finger domain of RIM, which binds to GTP-bound Rab3s. To search for additional interacting proteins for RIMs, we employed yeast two-hybrid screens with the C-terminal half of RIM1. Two members of a new family of homologous brain proteins, referred to as RIM-binding proteins (RIM-BPs), were identified. RIM-BPs bind to RIM in yeast two-hybrid and GST pull-down assays, suggesting a specific interaction. In RIMs, the binding site for RIM-BPs consists of a conserved proline-rich sequence between the two C(2) domains, N-terminal to the beginning of NIMs. RIM-BPs are composed of multiple domains, including three fibronectin type III-domains and three Src homology 3 domains, of which the second Src homology 3 domain binds to RIMs. With the RIM-BPs, we have identified a partner for RIMs that may bind to RIMs at the synapse in addition to Rab3. PMID- 10748114 TI - Inhibition of Mcm4,6,7 helicase activity by phosphorylation with cyclin A/Cdk2. AB - A strong body of evidence indicates that cyclin-dependent protein kinases are required not only for the initiation of DNA replication but also for preventing over-replication in eukaryotic cells. Mcm proteins are one of the components of the replication licensing system that permits only a single round of DNA replication per cell cycle. It has been reported that Mcm proteins are phosphorylated by the cyclin-dependent kinases in vivo, suggesting that these two factors are cooperatively involved in the regulation of DNA replication. Our group has reported that a 600-kDa Mcm4,6,7 complex has a DNA helicase activity that is probably necessary for the initiation of DNA replication. Here, we examined the in vitro phosphorylation of the Mcm complexes with cyclin A/Cdk2 to understand the interplay between Mcm proteins and cyclin-dependent kinases. The cyclin A/Cdk2 mainly phosphorylated the amino-terminal region of Mcm4 in the Mcm4,6,7 complex. The phosphorylation was associated with the inactivation of its DNA helicase activity. These results raise the possibility that the inactivation of Mcm4,6,7 helicase activity by Cdk2 is a part of the system for regulating DNA replication. PMID- 10748115 TI - Substrate recognition by the Lyn protein-tyrosine kinase. NMR structure of the immunoreceptor tyrosine-based activation motif signaling region of the B cell antigen receptor. AB - The immunoreceptor tyrosine-based activation motif (ITAM) plays a central role in transmembrane signal transduction in hematopoietic cells by mediating responses leading to proliferation and differentiation. An initial signaling event following activation of the B cell antigen receptor is phosphorylation of the CD79a (Ig-alpha) ITAM by Lyn, a Src family protein-tyrosine kinase. To elucidate the structural basis for recognition between the ITAM substrate and activated Lyn kinase, the structure of an ITAM-derived peptide bound to Lyn was determined using exchange-transferred nuclear Overhauser NMR spectroscopy. The bound substrate structure has an irregular helix-like character. Docking based on the NMR data into the active site of the closely related Lck kinase strongly favors ITAM binding in an orientation similar to binding of cyclic AMP-dependent protein kinase rather than that of insulin receptor tyrosine kinase. The model of the complex provides a rationale for conserved ITAM residues, substrate specificity, and suggests that substrate binds only the active conformation of the Src family tyrosine kinase, unlike the ATP cofactor, which can bind the inactive form. PMID- 10748116 TI - A molecular basis for functional peptide mimicry of a carbohydrate antigen. AB - Peptides may substitute for carbohydrate antigens in carbohydrate-specific immunological reactions. Using the recognition properties of an anti-Lewis Y (LeY) antibody, BR55-2, as a model system, we establish a molecular perspective for peptide mimicry by comparing the three-dimensional basis of BR55-2 binding to LeY with the binding of the same antibody to peptides. The peptides compete with LeY, as demonstrated by enzyme-linked immunosorbent assay and Biacore analysis. The computer program LUDI was used to epitope map the antibody-combining site, correlating peptide reactivity patterns. This approach identified amino acids interacting with the same BR55-2 functional residue groups that recognize the Fucalpha(1-3) moiety of LeY. Molecular modeling indicates that the peptides adopt an extended turn conformation within the BR55-2 combining site, serving to overlap the peptides with the LeY spatial position. Peptide binding is associated with only minor changes in BR55-2, relative to the BR55-2-LeY complex. Anti peptide serum distinguishes the Fucalpha(1-3) from the Fucalpha(1-4) linkage, therefore differentiating difucosylated neolactoseries antigens. These results further confirm that peptides and carbohydrates can bind to the same antibody binding site and that peptides can structurally and functionally mimic salient features of carbohydrate epitopes. PMID- 10748117 TI - Inhibition of tumor necrosis factor mRNA translation by a rationally designed immunomodulatory peptide. AB - Based on sequences of immunomodulatory peptides derived from the heavy chain of HLA Class I, novel immunomodulatory peptides with increased potency were developed by computer-aided rational design. Allotrap 1258 was characterized in detail and shown to inhibit cell-mediated immune responses in vitro and in vivo. Immunomodulatory activity was associated with the capability of the peptides to modulate heme oxygenase (HO) activity. In this study we analyzed the effect of Allotrap 1258 on cytokine expression. Allotrap 1258 inhibited concanavalin A- and lipopolysaccharide-induced human and mouse tumor necrosis factor (TNF) production in vitro and in vivo but had no effect on interleukin (IL)-1, IL-2, IL-4, IL-6, or IL-10 expression. Experiments with HO-1/KO and iNOS/KO mice showed that Allotrap 1258-mediated inhibition of TNF was independent of HO-1 and iNOS. Quantitation of TNF protein expression and mRNA steady state levels demonstrated that Allotrap 1258-mediated inhibition occurred at the translational level. Deletion of the AU-rich element in the 3'-untranslated region (UTR) of TNF mRNA, a region known to be involved in TNF mRNA translation, had minimal effect on Allotrap 1258-mediated inhibition. However, replacement of the TNF 3'-UTR with the human globin 3'-UTR rendered the peptide inactive. This demonstrates that besides AU-rich elements, other sequences in the 3'-UTR of TNF mRNA are involved in translational control of TNF expression. Such sequences are necessary for Allotrap 1258-mediated inhibition of TNF production. PMID- 10748118 TI - Characterization of the aspartate transcarbamoylase from Methanococcus jannaschii. AB - The genes from the thermophilic archaeabacterium Methanococcus jannaschii that code for the putative catalytic and regulatory chains of aspartate transcarbamoylase were expressed at high levels in Escherichia coli. Only the M. jannaschii PyrB (Mj-PyrB) gene product exhibited catalytic activity. A purification protocol was devised for the Mj-PyrB and M. jannaschii PyrI (Mj PyrI) gene products. Molecular weight measurements of the Mj-PyrB and Mj-PyrI gene products revealed that the Mj-PyrB gene product is a trimer and the Mj-PyrI gene product is a dimer. Preliminary characterization of the aspartate transcarbamoylase from M. jannaschii cell-free extract revealed that the enzyme has a similar molecular weight to that of the E. coli holoenzyme. Kinetic analysis of the M. jannaschii aspartate transcarbamoylase from the cell-free extract indicates that the enzyme exhibited limited homotropic cooperativity and little if any regulatory properties. The purified Mj-catalytic trimer exhibited hyperbolic kinetics, with an activation energy similar to that observed for the E. coli catalytic trimer. Homology models of the Mj-PyrB and Mj-PyrI gene products were constructed based on the three-dimensional structures of the homologous E. coli proteins. The residues known to be critical for catalysis, regulation, and formation of the quaternary structure from the well characterized E. coli aspartate transcarbamoylase were compared. PMID- 10748119 TI - Ascorbate is a potent antioxidant against peroxynitrite-induced oxidation reactions. Evidence that ascorbate acts by re-reducing substrate radicals produced by peroxynitrite. AB - Peroxynitrite (ONOO(((-)))/ONOOH) is expected in vivo to react predominantly with CO(2), thereby yielding NO(2)(.) and CO(3) radicals. We studied the inhibitory effects of ascorbate on both NADH and dihydrorhodamine 123 (DHR) oxidation by peroxynitrite generated in situ from 3-morpholinosydnonimine N-ethylcarbamide (SIN-1). SIN-1 (150 micrometer)-mediated oxidation of NADH (200 micrometer) was half-maximally inhibited by low ascorbate concentrations (61-75 micrometer), both in the absence and presence of CO(2). Control experiments performed with thiols indicated both the very high antioxidative efficiency of ascorbate and that in the presence of CO(2) in situ-generated peroxynitrite exclusively oxidized NADH via the CO(3) radical. This fact is attributed to the formation of peroxynitrate (O(2)NOO(-)/O(2)NOOH) from reaction of NO(2)(.) with O(2), which is formed from reaction of CO(3) with NADH. SIN-1 (25 micrometer)-derived oxidation of DHR was half-maximally inhibited by surprisingly low ascorbate concentrations (6-7 micrometer), irrespective of the presence of CO(2). Control experiments performed with authentic peroxynitrite revealed that ascorbate was in regard to both thiols and selenocompounds much more effective to protect DHR. The present results demonstrate that ascorbate is highly effective to counteract the oxidizing properties of peroxynitrite in the absence and presence of CO(2) by both terminating CO(3)/HO( small middle dot) reactions and by its repair function. Ascorbate is therefore expected to act intracellulary as a major peroxynitrite antagonist. In addition, a novel, ascorbate-independent protection pathway exists: scavenging of NO(2)(.) by O(2) to yield O(2)NOO(-), which further decomposes into NO(2)(-) and O(2). PMID- 10748120 TI - Characterization of the unique C terminus of the Escherichia coli tau DnaX protein. Monomeric C-tau binds alpha AND DnaB and can partially replace tau in reconstituted replication forks. AB - A contact between the dimeric tau subunit within the DNA polymerase III holoenzyme and the DnaB helicase is required for replication fork propagation at physiologically-relevant rates (Kim, S., Dallmann, H. G., McHenry, C. S., and Marians, K. J. (1996) Cell 84, 643-650). In this report, we exploit the OmpT protease to generate C-tau, a protein containing only the unique C-terminal sequences of tau, free of the sequences shared with the alternative gamma frameshifting product of dnaX. We have established that C-tau is a monomer by sedimentation equilibrium and sedimentation velocity ultracentrifugation. Monomeric C-tau binds the alpha catalytic subunit of DNA polymerase III with a 1:1 stoichiometry. C-tau also binds DnaB, revealed by a coupled immunoblotting method. C-tau restores the rapid replication rate of inefficient forks reconstituted with only the gamma dnaX gene product. The acceleration of the DnaB helicase can be observed in the absence of primase, when only leading-strand replication occurs. This indicates that C-tau, bound only to the leading-strand polymerase, can trigger the conformational change necessary for DnaB to assume the fast, physiologically relevant form. PMID- 10748121 TI - Neuropilin-2 is a receptor for the vascular endothelial growth factor (VEGF) forms VEGF-145 and VEGF-165 [corrected]. AB - Neuropilin-1 (np-1) and neuropilin-2 (np-2) are receptors for axon guidance factors belonging to the class 3 semaphorins. np-1 also binds to the 165-amino acid heparin-binding form of VEGF (VEGF(165)) but not to the shorter VEGF(121) form, which lacks a heparin binding ability. We report that human umbilical vein derived endothelial cells express the a17 and a22 splice forms of the np-2 receptor. Both np-2 forms bind VEGF(165) with high affinity in the presence of heparin (K(D) 1.3 x 10(-10) m) but not VEGF(121). np-2 also binds the heparin binding form of placenta growth factor. These binding characteristics resemble those of np-1. VEGF(145) is a secreted heparin binding VEGF form that contains the peptide encoded by exon 6 of VEGF but not the peptide encoded by exon 7, which is present in VEGF(165). VEGF(145) binds to np-2 with high affinity (K(D) 7 x 10(-10) m). Surprisingly, VEGF(145) did not bind to np-1. Indeed, VEGF(145) does not bind to MDA-MB-231 breast cancer cells, which predominantly express np 1. By contrast, VEGF(145) binds to human umbilical vein-derived endothelial cells, which express both np-1 and np-2. The binding of VEGF(165) to porcine aortic endothelial cells expressing recombinant np-2 did not affect the proliferation or migration of the cells. Nevertheless, it is possible that VEGF induced np-2-mediated signaling will take place only in the presence of other VEGF receptors such as VEGF receptor-1 or VEGF receptor-2. PMID- 10748122 TI - A novel type I fibroblast growth factor receptor activates mitogenic signaling in the absence of detectable tyrosine phosphorylation of FRS2. AB - A novel variant of the fibroblast growth factor receptor type 1 (FGFR-1) was identified in human placental RNA. In this receptor (FGFR-1L) portions of the second and third immunoglobulin-like (Ig-like) domains are deleted. To determine whether FGFR-1L was functional, full-length variant (pSV/FGFR-1L) and wild-type (pSV/FGFR-1) receptors were stably transfected into rat L6 myoblasts cells. Transfected L6 clones expressed respective proteins and bound (125)I-labeled FGF 2 with K(d) values of 99 pm (FGFR-1) and 26 pm (FGFR-1L). FGF-1 and FGF-2 competed efficiently with (125)I-FGF-2 for binding to FGFR-1 and FGFR-1L, whereas FGF-4 was less efficient. FGF-1, FGF-2, and FGF-4 enhanced mitogen-activated protein kinase (MAPK) activity, increased steady-state c-fos mRNA levels, and stimulated proliferation through either receptor, whereas KGF was without effect. FGFR-1 expressing clones exhibited ligand-induced tyrosine phosphorylation of fibroblast growth factor receptor substrate 2 (FRS2), a 90-kDa adaptor protein that links FGFR-1 activation to the MAPK cascade. In contrast, tyrosine phosphorylation of FRS2 was not evident with FGFR-1L. In addition, phospholipase C-gamma was not tyrosine phosphorylated via activated FGFR-1L. These findings indicate that FGFR-1L binds FGF-1 and FGF-2 with high affinity and is capable of mitogenic signaling, but may activate MAPK to occur via non-classical signaling intermediates. PMID- 10748123 TI - The protein-tyrosine phosphatase PTPMEG interacts with glutamate receptor delta 2 and epsilon subunits. AB - Glutamate receptor (GluR) delta2 is selectively expressed in cerebellar Purkinje cells and plays a crucial role in cerebellum-dependent motor learning. Although GluRdelta2 belongs to an ionotropic GluR family, little is known about its pharmacological features and downstream signaling cascade. To study molecular mechanisms underlying GluRdelta2-dependent motor learning, we employed yeast two hybrid screening to isolate GluRdelta2-interacting molecules and identified protein-tyrosine phosphatase PTPMEG. PTPMEG is a family member of band 4.1 domain containing protein-tyrosine phosphatases and is expressed prominently in brain. Here, we showed by in situ hybridization analysis that the PTPMEG mRNA was enriched in mouse thalamus and Purkinje cells. We also showed that PTPMEG interacted with GluRdelta2 as well as with N-methyl-d-aspartate receptor GluRepsilon1 in cultured cells and in brain. PTPMEG bound to the putative C terminal PDZ target sequence of GluRdelta2 and GluRepsilon1 via its PDZ domain. Examination of the effect of PTPMEG on tyrosine phosphorylation of GluRepsilon1 unexpectedly revealed that PTPMEG enhanced Fyn-mediated tyrosine phosphorylation of GluRepsilon1 in its PTPase activity-dependent manner. Thus, we conclude that PTPMEG associates directly with GluRdelta2 and GluRepsilon1. Moreover, our data suggest that PTPMEG plays a role in signaling downstream of the GluRs and/or in regulation of their activities through tyrosine dephosphorylation. PMID- 10748124 TI - Insulin-stimulated phosphorylation of the protein phosphatase-1 striated muscle glycogen-targeting subunit and activation of glycogen synthase. AB - Protein phosphatase-1 (PP-1) in heart and skeletal muscle binds to a glycogen targeting subunit (G(M)) in the sarcoplasmic reticulum. Phosphorylation of G(M) has been postulated to govern activity of PP1 in response to adrenaline and insulin. In this study, we used biochemical assays and G(M) expression in living cells to examine the effects of insulin on the phosphorylation of G(M), and the binding of PP-1 to G(M). We also assayed glycogen synthase activation in cells expressing wild type G(M) and G(M) mutated at the phosphorylation sites. In biochemical assays kinase(s) prepared from insulin-stimulated Chinese hamster ovary (CHO-IR) cells and C2C12 myotubes phosphorylated a glutathione S transferase (GST) fusion protein, GST-G(M)(1-240), at both site 1 (Ser(48)) and site 2 (Ser(67)). Phosphorylation of both sites was dependent on activation of the mitogen-activated protein kinase pathway, involving in particular ribosomal protein S6 kinase. Full-length G(M) was expressed in CHO-IR cells and metabolic (32)P labeling at sites 1 and 2 was increased by insulin treatment. The G(M) expressed in CHO-IR cells or in C2C12 myotubes co-immunoprecipitated endogenous PP-1, and association was transiently lost following treatment of the cells with insulin. In contrast PP-1 binding to G(M)(S67T), a version of G(M) not phosphorylated at site 2, was unaffected by insulin treatment. Expression of G(M) increased basal activity of endogenous glycogen synthase in CHO-IR cells. Insulin stimulated glycogen synthase activity the same extent in cells expressing wild type G(M) or G(M) mutated to eliminate phosphorylation site 1 and/or site 2. Phosphorylation of G(M) is stimulated by insulin, but this phosphorylation is not involved in insulin control of glycogen metabolism. We speculate that other functions of G(M) at the sarcoplasmic reticulum membrane might be affected by insulin. PMID- 10748125 TI - Cellular mechanisms regulating protein phosphatase-1. A key functional interaction between inhibitor-2 and the type 1 protein phosphatase catalytic subunit. AB - Inhibitor-1 (I-1) and inhibitor-2 (I-2) selectively inhibit type 1 protein serine/threonine phosphatases (PP1). To define the molecular basis for PP1 inhibition by I-1 and I-2 charged-to-alanine substitutions in the Saccharomyces cerevisiae, PP1 catalytic subunit (GLC7), were analyzed. Two PP1 mutants, E53A/E55A and K165A/E166A/K167A, showed reduced sensitivity to I-2 when compared with wild-type PP1. Both mutants were effectively inhibited by I-1. Two-hybrid analysis and coprecipitation or pull-down assays established that wild-type and mutant PP1 catalytic subunits bound I-2 in an identical manner and suggested a role for the mutated amino acids in enzyme inhibition. Inhibition of wild-type and mutant PP1 enzymes by full-length I-2(1-204), I-2(1-114), and I-2(36-204) indicated that the mutant enzymes were impaired in their interaction with the N terminal 35 amino acids of I-2. Site-directed mutagenesis of amino acids near the N terminus of I-2 and competition for PP1 binding by a synthetic peptide encompassing an I-2 N-terminal sequence suggested that a PP1 domain composed of amino acids Glu-53, Glu-55, Asp-165, Glu-166, and Lys-167 interacts with the N terminus of I-2. This defined a novel regulatory interaction between I-2 and PP1 that determines I-2 potency and perhaps selectivity as a PP1 inhibitor. PMID- 10748126 TI - Cooperative interactions between PBX, PREP, and HOX proteins modulate the activity of the alpha 2(V) collagen (COL5A2) promoter. AB - Cell type-specific expression of the human alpha2(V) collagen (COL5A2) gene depends on a cis-acting element that consists of two contiguous protein binding sites (FPA and FPB) located between nucleotides -149 and -95, relative to the transcription start site. The present study focused on the characterization of the FPB-bound complex. DNA binding assays and cell transfection experiments revealed that the bipartite core sequence of FPB (5'-ATCAATCA-3') binds the PBX1/2, PREP1, and HOXB1 proteins, and this in turn leads to promoter transactivation. In the presence of all three nuclear factors, cooperative interactions between recombinant PBX1 and PREP1 or PBX1 and HOXB1 result in binding of the heterodimers to FPB in vitro. Similarly, overexpression of different combinations of PBX1, PREP1, and HOXB1 transactivates FPB-driven transcription. In contrast to the composition of the FPB complex purified from COL5A2-positive cells, the FPB complex from COL5A2-negative cells contains PBX2 and PREP1 but lacks PBX1. However, PBX1 exogenously introduced into COL5A2 negative cells cannot stimulate FPB-driven transcription unless co-expressed with PREP1. Within the intrinsic limitations of the experimental model, our results indicate that combinatorial interactions among PBX and PREP or HOX proteins are involved in regulating tissue-specific production of collagen V. PMID- 10748128 TI - Characterization of retinoic acid receptor-deficient keratinocytes. AB - Retinoids are essential for normal epidermal growth and differentiation and show potential for the prevention or treatment of various epithelial neoplasms. The retinoic acid receptors (RARalpha, -beta, and -gamma) are transducers of the retinoid signal. The epidermis expresses RARgamma and RARalpha, both of which are potential mediators of the effects of retinoids in the epidermis. To further investigate the role(s) of these receptors, we derived transformed keratinocyte lines from wild-type, RARalpha, RARgamma, and RARalphagamma null mice and investigated their response to retinoids, including growth inhibition, markers of growth and differentiation, and AP-1 activity. Our results indicate that RARgamma is the principle receptor contributing to all-trans-retinoic acid (RA)-mediated growth arrest in this system. This effect partially correlated with inhibition of AP-1 activity. In the absence of RARs, the synthetic retinoid N-(4-hydroxyphenyl) retinamide inhibited growth; this was not observed with RA, 9-cis RA, or the synthetic retinoid (E)-4-[2-(5, 5, 8, 8 tetramethyl-5,6,7,8-tetrahydro-2 naphthalenyl)-1-propenyl] benzoic acid. Finally, both RARalpha and RARgamma differently affected the expression of some genes, suggesting both specific and overlapping roles for the RARs in keratinocytes. PMID- 10748127 TI - Arginine methylation inhibits the binding of proline-rich ligands to Src homology 3, but not WW, domains. AB - Src homology 3 (SH3) and WW domains are known to associate with proline-rich motifs within their respective ligands. Here we demonstrate that the proposed adapter protein for Src kinases, Sam68, is a ligand whose proline-rich motifs interact with the SH3 domains of p59(fyn) and phospholipase Cgamma-1 as well as with the WW domains of FBP30 and FBP21. These proline-rich motifs, in turn, are flanked by RG repeats that represent targets for the type I protein arginine N methyltransferase. The asymmetrical dimethylation of arginine residues within these RG repeats dramatically reduces the binding of the SH3 domains of p59(fyn) and phospholipase Cgamma-1, but has no effect on their binding to the WW domain of FBP30. These results suggest that protein arginine methylation can selectively modulate certain protein-protein interactions and that mechanisms exist for the irreversible regulation of SH3 domain-mediated interactions. PMID- 10748129 TI - DcE2F, a functional plant E2F-like transcriptional activator from Daucus carota. AB - In animal cells the progression of the cell cycle through G(1)/S transition and S phase is under the control of the pRB/E2F regulatory pathway. The E2F transcription factors are key activators of genes coding for several regulatory proteins and for enzymes involved in nucleotide and DNA synthesis. In this report we have detected the presence of E2F-like DNA binding activities in carrot nuclear extracts, and we have isolated a carrot cDNA (DcE2F) encoding a plant E2F homologue. The DcE2F gene is expressed in proliferating cells and is induced during the G(1)/S transition of the cell cycle. Supershift experiments using anti DcE2F antiserum have confirmed that the DcE2F protein is a component of the carrot E2F-like nuclear activities. DNA binding assays have demonstrated that the DcE2F protein can recognize a canonical E2F cis-element in association with a mammalian DP protein. Furthermore, transactivation assays have revealed that DcE2F is a functional transcription factor that can transactivate, together with a DP partner, an E2F-responsive reporter gene in both plant and mammalian cells. PMID- 10748130 TI - Inositol 1,4,5-trisphosphate receptor/Ca2+ channel modulatory role of chromogranin A, a Ca2+ storage protein of secretory granules. AB - The secretory granules of neuroendocrine cells, which contain large amounts of Ca(2+) and chromogranins, have been demonstrated to release Ca(2+) in response to inositol 1,4,5-trisphosphate (IP(3)), indicating the IP(3)-sensitive intracellular Ca(2+) store role of secretory granules. In our previous study, chromogranin A (CGA) was shown to interact with several secretory granule membrane proteins, including the IP(3) receptor (IP(3)R), at the intravesicular pH 5.5 (Yoo, S. H. (1994) J. Biol. Chem. 269, 12001-12006). To examine the functional aspect of this coupling, we measured the IP(3)-mediated Ca(2+) release property of the IP(3)R reconstituted into liposomes in the presence and absence of CGA. Presence of CGA in the IP(3)R-reconstituted liposome significantly enhanced the IP(3)-mediated Ca(2+) release from the liposomes. Moreover, the number of IP(3) bound to the reconstituted IP(3)R increased. The fluorescence energy transfer and IP(3)R Trp fluorescence quenching studies indicated that the structure of reconstituted IP(3)R becomes more ordered and exposed in the presence of CGA, suggesting that the coupled CGA in the liposome caused structural changes of the IP(3)R, changing it to a structure that is better suited to IP(3) binding and subsequent Ca(2+) release. These results appear to underscore the physiological significance of IP(3)R-CGA coupling in the secretory granules. PMID- 10748131 TI - Transforming growth factor-beta 1 suppresses serum deprivation-induced death of A549 cells through differential effects on c-Jun and JNK activities. AB - Transforming growth factor (TGF)-beta1, a pleiotropic cytokine involved in regulating growth and differentiation, can exert both pro-apoptotic and anti apoptotic effects depending on the cell type or circumstances. We observed that TGF-beta1 blocked apoptosis resulting from serum withdrawal in A549 human lung carcinoma cells. This was associated with suppression of JNK activation that occurs concomitant with the onset of apoptosis in the absence of TGF-beta1, suggesting that JNK plays an active role in the death process and that TGF-beta1 exerts its protective influence by altering JNK activity. Overexpression of a dominant negative mutant form of SEK1, an upstream activator of JNK, likewise suppressed JNK activation and inhibited apoptosis. Investigation of early events following TGF-beta1 treatment revealed an early induction and phosphorylation of c-Jun that was absent in cells subjected to serum withdrawal alone. That TGF beta1-induced expression of c-Jun is important for survival was supported by the finding that overexpression of non-phosphosphorylatable dominant negative mutant c-Jun, c-Jun(S73A), attenuated the protective influence of TGF-beta1. Our findings suggest that JNK activation is a late but essential event in serum deprivation-induced apoptosis in A549 cells. TGF-beta1 prevents apoptosis, in part, through the early induction and phosphorylation of c-Jun, which in turn results in attenuated JNK activation. PMID- 10748132 TI - Wheat germ poly(A)-binding protein increases the ATPase and the RNA helicase activity of translation initiation factors eIF4A, eIF4B, and eIF-iso4F. AB - Recent studies demonstrated that wheat germ poly(A)-binding protein (PABP) interacted with translation eukaryotic initiation factor (eIF)-iso4G and eIF4B, and these interactions increased the poly(A) binding activity of PABP (Le, H., Tanguay, R. L., Balasta, M. L., Wei, C. C., Browning, K. S., Metz, A. M., Goss, D. J., and Gallie, D. R. (1997) J. Biol. Chem. 272, 16247-16255) and the cap binding activity of eIF-iso4F (Wei, C. C., Balasta, M. L., Ren, J., and Goss, D. J. (1998) Biochemistry 37, 1910-1916). We report here that the interaction between PABP and eIF-iso4G has a substantial effect on the ATPase activity and RNA helicase activity of (eIF4A + eIF4B + eIF-iso4F) complex. ATPase kinetic assays show, in the presence of poly(U), PABP can increase the parameter (k(cat)/K(m)) by 3.5-fold with a 2-fold decrease of K(m) for the (eIF4A + eIF iso4F) complex. In the presence of globin messenger RNA, the ATPase activity of the complex (eIF4A + eIF-iso4F) was increased 2-fold by the presence of PABP. RNA helicase assays demonstrated that the presence of PABP enhanced the RNA duplex unwinding activity of the initiation factor complex. These results suggest that, in terms of the scanning model of translation initiation, PABP may enhance the mRNA scanning rate of the complex formed by eIF4A, eIF4B, and eIF4F or eIF (iso)4F and increase the rate of translation. PMID- 10748133 TI - Determinants of 4-repeat tau expression. Coordination between enhancing and inhibitory splicing sequences for exon 10 inclusion. AB - Mutations in the tau gene are pathogenic causing autosomal dominant frontotemporal dementia with Parkinsonism-chromosome 17 type (FTDP-17). Some mutations in tau exon 10 (E10) and immediately adjacent sequences cause disease by altering E10 splicing. To determine the mechanism of normal E10 splicing regulation and how FTDP-17 mutations alter splicing, mutational analysis of E10 was performed. The results show that E10 contains a complex array of both enhancer and inhibitor cis-acting elements that modulate usage of a weak 5' splice site. The 5' end of E10 contains a previously unrecognized multipartite exon splicing enhancer (ESE) composed of an SC35-like binding sequence, a purine rich sequence, and an AC-rich element. Downstream of this ESE is a purine-rich exon splicing inhibitor. Intronic sequences immediately downstream of E10 also are inhibitory. The results support an alternative model in which I10 inhibitory sequences appear to function as a linear sequence. The cis-elements described are not redundant, and all appear required for normal E10 splicing. Results with double mutations demonstrate that the ESE and the intronic inhibitory element collaborate to regulate splicing. Thus splicing of tau E10 is regulated by a complex set of cis-acting elements that span nearly the entire exon and also include intronic sequences. PMID- 10748134 TI - Marked differences between two isoforms of human pyruvate dehydrogenase kinase. AB - Pyruvate dehydrogenase kinase (PDK) isoforms 2 and 3 were produced via co expression with the chaperonins GroEL and GroES and purified with high specific activities in affinity tag-free forms. By using human components, we have evaluated how binding to the lipoyl domains of the dihydrolipoyl acetyltransferase (E2) produces the predominant changes in the rates of phosphorylation of the pyruvate dehydrogenase (E1) component by PDK2 and PDK3. E2 assembles as a 60-mer via its C-terminal domain and has mobile connections to an E1-binding domain and then two lipoyl domains, L2 and L1 at the N terminus. PDK3 was activated 17-fold by E2; the majority of this activation was facilitated by the free L2 domain (half-maximal activation at 3.3 microm L2). The direct activation of PDK3 by the L2 domain resulted in a 12.8-fold increase in k(cat) along with about a 2-fold decrease in the K(m) of PDK3 for E1. PDK3 was poorly inhibited by pyruvate or dichloroacetate (DCA). PDK3 activity was stimulated upon reductive acetylation of L1 and L2 when full activation of PDK3 by E2 was avoided (e.g. using free lipoyl domains or ADP-inhibited E2-activated PDK3). In marked contrast, PDK2 was not responsive to free lipoyl domains, but the E2-60-mer enhanced PDK2 activity by 10-fold. E2 activation of PDK2 resulted in a greatly enhanced sensitivity to inhibition by pyruvate or DCA; pyruvate was effective at significantly lower levels than DCA. E2-activated PDK2 activity was stimulated >/=3-fold by reductive acetylation of E2; stimulated PDK2 retained high sensitivity to inhibition by ADP and DCA. Thus, PDK3 is directly activated by the L2 domain, and fully activated PDK3 is relatively insensitive to feed-forward (pyruvate) and feed-back (acetylating) effectors. PDK2 was activated only by assembled E2, and this activated state beget high responsiveness to those effectors. PMID- 10748135 TI - Replacement of the transmembrane anchor in angiotensin I-converting enzyme (ACE) with a glycosylphosphatidylinositol tail affects activation of the B2 bradykinin receptor by ACE inhibitors. AB - To investigate further the relationship of angiotensin I-converting enzyme (ACE) inhibitors to activation of the B(2) bradykinin (BK) receptor, we transfected Chinese hamster ovary cells to stably express the human receptor and either wild type ACE (WT-ACE), an ACE construct with most of the cytosolic portion deleted (Cyt-del-ACE), or ACE with a glycosylphosphatidylinositol (GPI) anchor replacing the transmembrane and cytosolic domains (GPI-ACE). BK or its ACE-resistant analogue were the agonists. All activities (arachidonic acid release and calcium mobilization) were blocked by the B(2) antagonist HOE 140. B(2) was desensitized by repeated administration of BK but resensitized to agonist by ACE inhibitors in the cells expressing both B(2) and either WT-ACE or Cyt-del-ACE. In GPI-ACE expressing cells, the B(2) receptor was still activated by the agonists, but ACE inhibitors did not resensitize. Pretreatment with filipin returned the sensitivity to inhibitors. In immunocytochemistry, GPI-ACE showed patchy, uneven distribution on the plasma membrane that was restored by filipin. Thus, ACE inhibitors were inactive as long as GPI-ACE was sequestered in cholesterol-rich membrane domains. WT-ACE and B(2) receptor in Chinese hamster ovary cells co immunoprecipitated with antibody to receptor, suggesting an interaction on the cell membrane. ACE inhibitors augment BK effects on receptors indirectly only when enzyme and receptor molecules are sterically close, possibly forming a heterodimer. PMID- 10748136 TI - Isa1p is a component of the mitochondrial machinery for maturation of cellular iron-sulfur proteins and requires conserved cysteine residues for function. AB - In eukaryotes, mitochondria execute a central task in the assembly of cellular iron-sulfur (Fe/S) proteins. The organelles synthesize their own set of Fe/S proteins, and they initiate the generation of extramitochondrial Fe/S proteins. In the present study, we identify the mitochondrial matrix protein Isa1p of Saccharomyces cerevisiae as a new member of the Fe/S cluster biosynthesis machinery. Isa1p belongs to a family of homologous proteins present in prokaryotes and eukaryotes. Deletion of the ISA1 gene results in the loss of mitochondrial DNA precluding the use of the Deltaisa1 strain for functional analysis. Cells in which Isa1p was depleted by regulated gene expression maintained the mitochondrial DNA, yet the cells displayed retarded growth on nonfermentable carbon sources. This finding indicates the importance of Isa1p for mitochondrial function. Deficiency of Isa1p caused a defect in mitochondrial Fe/S protein assembly. Moreover, Isa1p was required for maturation of cytosolic Fe/S proteins. Two cysteine residues in a conserved sequence motif characterizing the Isa1p protein family were found to be essential for Isa1p function in the biogenesis of both intra- and extramitochondrial Fe/S proteins. Our findings suggest a function for Isa1p in the binding of iron or an intermediate of Fe/S cluster assembly. PMID- 10748137 TI - N-Methyl-D-aspartate receptor stimulation activates tyrosinase and promotes melanin synthesis in the ink gland of the cuttlefish Sepia officinalis through the nitric Oxide/cGMP signal transduction pathway. A novel possible role for glutamate as physiologic activator of melanogenesis. AB - The tyrosinase-catalyzed conversion of l-tyrosine to melanin represents the most distinctive biochemical pathway in the ink gland of the cuttlefish Sepia officinalis; however, the molecular mechanisms underlying its activation have remained so far largely uncharted. In this paper we demonstrate for the first time that l-glutamate can stimulate tyrosinase activity and promote melanin synthesis in Sepia ink gland via the N-methyl-d-aspartate (NMDA) receptor/NO/cGMP signal transduction pathway. Incubation of intact ink glands with either l glutamate or NMDA resulted in an up to 18-fold increase of tyrosinase activity and a more than 6-fold elevation of cGMP levels. Comparable stimulation of tyrosinase was induced by an NO donor and by 8-bromo-cGMP. An NMDA receptor antagonist, NO synthase (NOS) inhibitors, and a guanylate cyclase blocker suppressed NMDA-induced effects. Immunohistochemical evidence indicated that enhanced cGMP production was localized largely in the mature part of the ink gland. Increased de novo synthesis of melanin was demonstrated in NMDA- and NO stimulated ink glands by a combined microanalytical approach based on spectrophotometric determination of pigment levels and high performance liquid chromatography quantitation of pyrrole-2,3, 5-tricarboxylic acid, a specific melanin marker, in melanosome-containing fractions. These results fill a longstanding gap in the understanding of the complex biochemical mechanisms underlying activation of melanogenesis in the mature ink gland cells of S. officinalis and disclose a novel physiologic role of the excitatory neurotransmitter glutamate mediated by the NMDA receptor/NO/cGMP signaling pathway. PMID- 10748138 TI - The nuclease activity of the yeast DNA2 protein, which is related to the RecB like nucleases, is essential in vivo. AB - Saccharomyces cerevisiae Dna2 protein is required for DNA replication and repair and is associated with multiple biochemical activities: DNA-dependent ATPase, DNA helicase, and DNA nuclease. To investigate which of these activities is important for the cellular functions of Dna2, we have identified separation of function mutations that selectively inactivate the helicase or nuclease. We describe the effect of six such mutations on ATPase, helicase, and nuclease after purification of the mutant proteins from yeast or baculovirus-infected insect cells. A mutation in the Walker A box in the C-terminal third of the protein affects helicase and ATPase but not nuclease; a mutation in the N-terminal domain (amino acid 504) affects ATPase, helicase, and nuclease. Two mutations in the N-terminal domain abolish nuclease but do not reduce helicase activity (amino acids 657 and 675) and identify the putative nuclease active site. Two mutations immediately adjacent to the proposed nuclease active site (amino acids 640 and 693) impair nuclease activity in the absence of ATP but completely abolish nuclease activity in the presence of ATP. These results suggest that, although the Dna2 helicase and nuclease activities can be independently affected by some mutations, the two activities appear to interact, and the nuclease activity is regulated in a complex manner by ATP. Physiological analysis shows that both ATPase and nuclease are important for the essential function of DNA2 in DNA replication and for its role in double-strand break repair. Four of the nuclease mutants are not only loss of function mutations but also exhibit a dominant negative phenotype. PMID- 10748139 TI - Recruitment of CD40 and tumor necrosis factor receptor-associated factors 2 and 3 to membrane microdomains during CD40 signaling. AB - Signals delivered to antigen-presenting cells through CD40 are critical for the activation of immune responses. Intracellular tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are key elements of the signal transduction pathways of many TNF receptor family members, including CD40. We show for the first time that engagement of CD40 in intact B cells induces the rapid translocation of TRAF2 from the cytoplasm to the plasma membrane. We found that CD40 engagement also results in its recruitment, together with TRAF2 and TRAF3, to membrane microdomains, regions of the plasma membrane enriched in signaling molecules such as the Src family kinases. Using a membrane-permeable chelator of zinc or a mutant TRAF2 molecule, we show that the putative zinc-binding domains of TRAFs contribute to their recruitment to microdomains and to the downstream activation of c-Jun N-terminal kinase. We suggest that the zinc RING and zinc finger domains of TRAFs are required for communication between CD40 and microdomain-associated signaling molecules and may serve a similar role in the signal transduction pathways of other TNF receptor family members. PMID- 10748140 TI - Identification of transacting factors responsible for the tissue-specific expression of human glucose transporter type 2 isoform gene. Cooperative role of hepatocyte nuclear factors 1alpha and 3beta. AB - We investigated transacting factors binding to the cis-element important in tissue-specific expression of the human glucose transporter type 2 isoform (GLUT2) gene. By transient transfection assay, we determined that the 227-base pair fragment upstream of the ATG start site contained promoter activity and that the region from +87 to +132 (site C) was responsible for tissue-specific expression. DNase I footprinting and electrophoretic mobility shift assay indicated that site C contained one binding site for hepatocyte nuclear factor 1 (HNF1) and two binding sites for HNF3. The mutations at positions +101 and +103, which are considered to be critical in binding HNF1 and HNF3, resulted in a 53% decrease in promoter activity, whereas the mutation of the proximal HNF3 binding site (+115 and +117) reduced promoter activity by 28%. The mutations of these four sites resulted in marked decrease (70%) in promoter activity as well as diminished bindings of HNF1 and HNF3. A to G mutation, which causes conversion of the HNF1 and HNF3 binding sequence to the NF-Y binding site, resulted in a 22% decrease in promoter activity. We identified that both HNF1 and HNF3 function as transcriptional activators in GLUT2 gene expression. Coexpression of the pGL+74 (+74 to +301) construct with the HNF1alpha and HNF3beta expression vectors in NIH 3T3 cells showed the synergistic effect on GLUT2 promoter activity compared with the expression of HNF1alpha, HNF3beta, or a combination of HNF1beta and HNF3beta. These data suggest that HNF1alpha and HNF3beta may be the most important players in the tissue-specific expression of the human GLUT2 gene. PMID- 10748141 TI - Differential activation of brain-derived neurotrophic factor gene promoters I and III by Ca2+ signals evoked via L-type voltage-dependent and N-methyl-D-aspartate receptor Ca2+ channels. AB - Although the brain-derived neurotrophic factor (BDNF) gene is activated by the intracellular Ca(2+) signals evoked via Ca(2+) influx into neurons, little is known about how the activation of alternative BDNF gene promoters is controlled by the Ca(2+) signals evoked via N-methyl-d-aspartate receptors (NMDA-R) and L type voltage-dependent Ca(2+) channels (L-VDCC). There is a critical range in the membrane depolarization caused by high K(+) concentrations (25-50 mm KCl) for effective BDNF mRNA expression and transcriptional activation of BDNF gene promoters I and III (BDNF-PI and -PIII, respectively) in rat cortical culture. The increase in BDNF mRNA expression induced at high K(+) was repressed not only by nicardipine, an antagonist for L-VDCC, but also by dl-amino-5 phosphonovalerate, an antagonist for NMDA-R, which was supported by the effects of antagonists on the Ca(2+) influx. Although the promoter activations at 25 and 50 mm KCl were different, BDNF-PIII was activated by either the Ca(2+) influx through NMDA-R or L-VDCC, whereas BDNF-PI was predominantly by the Ca(2+) influx through L-VDCC. Direct stimulation of NMDA-R supported the activation of BDNF PIII but not that of BDNF-PI. Thus, the alternative BDNF gene promoters responded differently to the intracellular Ca(2+) signals evoked via NMDA-R and L-VDCC. PMID- 10748142 TI - Angiotensin II induces transactivation of two different populations of the platelet-derived growth factor beta receptor. Key role for the p66 adaptor protein Shc. AB - Several signal transduction events induced by angiotensin II (AngII) binding to the angiotensin II type 1 receptor resemble those evoked by platelet-derived growth factor (PDGF) binding to the PDGF-beta receptor (PDGFbeta-R). We report here, in agreement with previous data, that AngII and PDGF-B-chain homodimer (PDGF-BB) stimulate tyrosine phosphorylation of the PDGFbeta-R. Both AngII and PDGF-BB stimulated the phosphorylation of PDGFbeta-R via the binding of tyrosine phosphorylated Shc to PDGFbeta-R. Both PDGF-BB- and AngII-induced phosphorylation of the Shc.PDGFbeta-R complex was inhibited by antioxidants such as N acetylcysteine and Tiron, but not by calcium chelation. However, transactivation of PDGFbeta-R by AngII (measured by PDGFbeta-R tyrosine phosphorylation) differed significantly from PDGF-BB. Evidence to support different mechanisms of PDGFbeta R phosphorylation includes differences in the time course of PDGFbeta-R phosphorylation, differing effects of inhibitors of the endogenous PDGFbeta-R tyrosine kinase and Src family tyrosine kinases, differing results when the PDGFbeta-R was directly immunoprecipitated (PDGFbeta-R-antibody) versus coimmunoprecipitated (Shc-antibody), and cell fractionation studies that suggested that the Shc.PDGFbeta-R complexes phosphorylated by AngII and PDGF-BB were located in separate subcellular compartments. These studies are the first to suggest that transactivation of tyrosine kinase receptors by G protein-coupled receptors involves a unique pathway that regulates a population of tyrosine kinase receptors different from the endogenous tyrosine kinase ligand. PMID- 10748143 TI - Molecular cloning of globotriaosylceramide/CD77 synthase, a glycosyltransferase that initiates the synthesis of globo series glycosphingolipids. AB - The expression cloning of a cDNA for globotriaosylceramide (Gb3)/CD77 synthase (alpha1,4-galactosyltransferase) was achieved using an anti-Gb3 antibody and mouse L cells as a recipient cell line for the transfection. The isolated cDNA clone designated pVTR1 predicted a type II membrane protein with 19 amino acids of cytoplasmic domain, 26 amino acids of transmembrane region, and a catalytic domain with 308 amino acids. Introduction of the cDNA clone into L cells resulted in the neosynthesis of Gb3/CD77, and the extracts of the transfectant cells showed alpha1, 4-galactosyltransferase activity only on lactosylceramide and galactosylceramide. In Northern blotting, a 2.3-kilobase mRNA was strongly expressed in heart, kidney, spleen, and placenta and weakly in colon, small intestine, and brain. Transfection of the cDNA into L cells resulted in the constitution of sensitivity to the apoptosis with Shiga-like toxins (verotoxins). Since Gb3/CD77 synthase initiates the synthesis of globo series glycolipids, the isolation of this cDNA will make possible further investigations into the function of its important series of glycolipids. PMID- 10748144 TI - The nonconserved hydrophilic loop domain of presenilin (PS) is not required for PS endoproteolysis or enhanced abeta 42 production mediated by familial early onset Alzheimer's disease-linked PS variants. AB - Presenilin 1 (PS1) and presenilin 2 (PS2) are polytopic membrane proteins that are mutated in the majority of early onset familial Alzheimer's disease (FAD) cases. Two lines of evidence establish a critical role for PS in the production of beta-amyloid peptides (Abeta). FAD-linked PS mutations elevate the levels of highly amyloidogenic Abeta ending at residue 42 (Abeta42), and cells with ablated PS1 alleles secrete low levels of Abeta. Several recent reports have shown that the hydrophilic loop (HL) domain, located between transmembrane domains 6 and 7, contains sites for phosphorylation, caspase cleavage, and sequences that bind several PS-interacting proteins. In the present report, we examined the metabolism of PS polypeptides lacking the HL domain and the influence of these molecules on Abeta production. We report that the deletion of the HL domain does not have a deleterious effect on the regulated endoproteolysis of PS, saturable accumulation of PS fragments, or the self-association of PS fragments. Abeta production was not significantly altered in cells expressing HL-deleted PS polypeptides compared with cells expressing full-length PS. Importantly, deletion of the HL domain did not affect FAD mutation-mediated elevation in the production of Abeta42. Furthermore, the deletion of the HL domain did not impair the role of PS1 or PS2 in facilitating Notch processing. Thus, our results argue against a biologically or pathologically relevant role for the HL domain phosphorylation and caspase cleavage and the association of PS HL domain-interacting proteins, in amyloid precursor protein metabolism and Abeta production or Notch cleavage. PMID- 10748145 TI - Inhibition of flap endonuclease 1 by flap secondary structure and relevance to repeat sequence expansion. AB - Recent genetic evidence indicates that null mutants of the 5'-flap endonuclease (FEN1) result in an expansion of repetitive sequences. The substrate for FEN1 is a flap formed by natural 5'-end displacement of the short intermediates of lagging strand replication. FEN1 binds the 5'-end of the flap, tracks to the point of annealing at the base of the flap, and then cleaves. Here we examine mechanisms by which foldback structures within the flap could contribute to repeat expansions. Cleavage by FEN1 was reduced with increased length of the foldback. However, even the longest foldbacks were cleaved at a low rate. Substrates containing the repetitive sequence CTG also were cleaved at a reduced rate. Bubble substrates, likely intermediates in repeat expansions, were inhibitory. Neither replication protein A nor proliferating cell nuclear antigen were able to assist in the removal of secondary structure within a flap. We propose that FEN1 cleaves natural foldbacks at a reduced rate. However, although the cleavage delay is not likely to influence the overall process of chromosomal replication, specific foldbacks could inhibit cleavage sufficiently to result in duplication of the foldback sequence. PMID- 10748146 TI - Pituitary adenylyl cyclase-activating peptide activates multiple intracellular signaling pathways to regulate ion channels in PC12 cells. AB - Pituitary adenylyl cyclase-activating peptide (PACAP) stimulates calcium transients and catecholamine secretion in adrenal chromaffin and PC12 cells. The PACAP type 1 receptor in these cells couples to both adenylyl cyclase and phospolipase C pathways, but although phospolipase C has been implicated in the response to PACAP, the role of adenylyl cyclase is unclear. In this study, we show that PACAP38 stimulates Ca(2+) influx in PC12 cells by activating a cation current that depends upon the dual activation of both the PLC and adenylyl cyclase signaling pathways but does not involve protein kinase C. In activating the current, PACAP38 has to overcome an inhibitory effect of Ras. Thus, in cells expressing a dominant negative form of Ras (PC12asn17-W7), PACAP38 induced larger, more rapidly activating currents. This effect of Ras could be overidden by intracellular guanosine-5'-O-3-(thio)triphosphate (GTPgammaS), suggesting that it was mediated by inhibition of downstream G proteins. Ras may also inhibit the current through a G protein-independent mechanism, because cAMP analogues activated the current in PC12asn17-W7 cells, provided GTPgammaS was present, but not in PC12 cells expressing wild type Ras. We conclude that coupling of PACAP to both adenylyl cyclase and phospholipase C is required to activate Ca(2+) influx in PC12 cells and that tonic inhibition by Ras delays and limits the response. PMID- 10748147 TI - Transcriptional autorepression of the stress-inducible gene ATF3. AB - Previously, we demonstrated that ATF3 (activating transcription factor-3) is a stress-inducible gene, and the protein it encodes is a transcriptional repressor. In this report, we present evidence suggesting that ATF3 represses the transcription of its own gene. Interestingly, efficient repression requires a consensus ATF/cAMP-responsive element site in the promoter and a previously unidentified ATF3-binding site immediately downstream from the TATA box. Although this new site resembles the known ATF/cAMP-responsive element sequences at the flanking sequence, it differs from them at the center key residues. These observations indicate that ATF3 can tolerate variations in the center of the binding sites if the flanking sequences are favorable. The repression of the ATF3 promoter by its own gene product provides a mechanistic explanation, at least in part, for the transient expression pattern of the ATF3 gene upon stress induction. PMID- 10748148 TI - Specific functional interaction of human cytohesin-1 and ADP-ribosylation factor domain protein (ARD1). AB - Activation of ADP-ribosylation factors (ARFs) is mediated by guanine nucleotide exchange proteins, which accelerate conversion of inactive ARF-GDP to active ARF GTP. ARF domain protein (ARD1), a 64-kDa GTPase with a C-terminal ADP ribosylation factor domain, is localized to lysosomes and the Golgi apparatus. When ARD1 was used as bait to screen a human liver cDNA library using the yeast two-hybrid system, a cDNA for cytohesin-1, a approximately 50-kDa protein with ARF guanine nucleotide-exchange protein activity, was isolated. In this system, ARD1-GDP interacted well with cytohesin-1 but very poorly with cytohesin-2. In agreement, cytohesin-1, but not cytohesin-2, markedly accelerated [(35)S]guanosine 5'-3-O-(thio)triphosphate binding to ARD1. The effector region of the ARF domain of ARD1 appeared to be critical for the specific interaction with cytohesin-1. Replacement of single amino acids in the Sec7 domains of cytohesin-1 and -2 showed that residue 30 is critical for specificity. In transfected COS-7 cells, overexpressed ARD1 and cytohesin-1 were partially colocalized, as determined by confocal fluorescence microscopy. It was concluded that cytohesin-1 is likely to be involved in ARD1 activation, consistent with a role for ARD1 in the regulation of vesicular trafficking. PMID- 10748149 TI - Dynamics and hydration of the alpha-helices of apolipophorin III. AB - Apolipophorin III (apoLp-III) is an exchangeable apolipoprotein whose structure is represented as a bundle of five amphipathic alpha-helices. In order to study the properties of the helical domains of apolipophorin III, we designed and obtained five single-tryptophan mutants of Locusta migratoria apoLp-III. The proteins were studied by UV absorption spectroscopy, time-resolved and steady state fluorescence spectroscopy, and circular dichroism. Fluorescence anisotropy, near-UV CD and solute fluorescence quenching studies indicate that the Trp residues in helices 1 (N-terminal) and 5 (C-terminal) have the highest conformational flexibility. These two residues also showed the highest degree of hydration. Trp residues in helices 3 and 4 display the lowest mobility, as assessed by fluorescence anisotropy and near UV CD. The Trp residue in helix 2 is protected from the solvent but shows high mobility. As inferred from the properties of the Trp residues, helices 1 and 5 appear to have the highest conformational flexibility. Helix 2 has an intermediate mobility, whereas helices 3 and 4 appear to constitute a highly ordered domain. From the configuration of the helices in the tertiary structure of the protein, we estimated the relative strength of the five interhelical interactions of apoLp-III. These interactions can be ordered according to their apparent stabilizing strengths as: helix 3 helix 4 > helix 2-helix 3 > helix 4-helix 1 approximately helix 2-helix 5 > helix 1-helix 5. A new model for the conformational change that is expected to occur upon binding of the apolipoprotein to lipid is proposed. This model is significantly different from the currently accepted model (Breiter, D. R., Kanost, M. R., Benning, M. M., Wesemberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, M. (1991) Biochemistry 30, 603-608). The model presented here predicts that the relaxation of the tertiary structure and the concomitant exposure of the hydrophobic core take place through the disruption of the weak interhelical contacts between helices 1 and 5. To some extent, the weakness of the helix 1-helix 5 interaction would be due to the parallel arrangement of these helices. PMID- 10748150 TI - A dimer as a building block in assembling RNA. A hexamer that gears bacterial virus phi29 DNA-translocating machinery. AB - Six RNA (pRNA) molecules form a hexamer, via hand-in-hand interaction, to gear bacterial virus phi29 DNA translocation machinery. Here we report the pathway and the conditions for the hexamer formation. Stable pRNA dimers and trimers were assembled in solution, isolated from native gels, and separated by sedimentation, providing a model system for the study of RNA dimers and trimers in a protein free environment. Cryo-atomic force microscopy revealed that monomers displayed a check mark outline, dimers exhibited an elongated shape, and trimers formed a triangle. Dimerization of pRNA was promoted by a variety of cations including spermidine, whereas procapsid binding and DNA packaging required specific divalent cations, including Mg(2+), Ca(2+), and Mn(2+). Both the tandem and fused pRNA dimers with complementary loops designed to form even-numbered rings were active in DNA packaging, whereas those without complementary loops were inactive. We conclude that dimers are the building blocks of the hexamer, and the pathway of building a hexamer is: dimer --> tetramer --> hexamer. The Hill coefficient of 2.5 suggests that there are three binding sites with cooperative binding on the surface of the procapsid. The two interacting loops played a key role in recruiting the incoming dimer, whereas the procapsid served as the foundation for hexamer assembly. PMID- 10748151 TI - Sphingosine is a novel activator of 3-phosphoinositide-dependent kinase 1. AB - 3-Phosphoinositide-dependent kinase 1 (PDK1) has previously been shown to phosphorylate the activation loop of several AGC kinase family members. In this study, we show that p21-activated kinase 1, the activity of which is regulated by the GTP-bound form of Cdc42 and Rac and by sphingosine, is phosphorylated by PDK1. Phosphorylation of p21-activated kinase 1 by PDK1 occurred only in the presence of sphingosine, which increased PDK1 autophosphorylation 25-fold. Sphingosine increased PDK1 autophosphorylation in a concentration-dependent manner and significantly increased phosphate incorporation into known PDK1 substrates. Studies on the lipid requirement for PDK1 activation found that both sphingosine isoforms and stearylamine also increased PDK1 autophosphorylation. However, C(10)-sphingosine, octylamine, and stearic acid were unable to increase PDK1 autophosphorylation, indicating that both a positive charge and a lipid tail containing at least a C(10)-carbon backbone were required for PDK1 activation. Three PDK1 autophosphorylation sites were identified after stimulation with sphingosine in a serine-rich region located between the kinase domain and the pleckstrin homology domain using two-dimensional phosphopeptide maps and matrix assisted laser desorption/ionization mass spectroscopy. Increased phosphorylation of endogenous Akt at threonine 308 was observed in COS-7 cells expressing wild type PDK1, but not catalytically inactive PDK1, when cellular sphingosine levels were elevated by treatment with sphingomyelinase. Sphingosine thus appears to be a true PDK1 activator. PMID- 10748152 TI - Altered sensitivity to single-strand-specific reagents associated with the genomic vascular smooth muscle alpha-actin promoter during myofibroblast differentiation. AB - Stimulation of quiescent AKR-2B mouse fibroblasts with transforming growth factor beta1 results in uniform conversion to a myofibroblast-like phenotype as judged by a rapid accumulation of smooth muscle alpha-actin mRNA and protein. Because transcriptional regulation of the smooth muscle alpha-actin gene in these cells might be mediated by single-stranded DNA-binding proteins, we have examined the sensitivity of genomic DNA to chemical reagents with specificity for unpaired bases in a region of the promoter previously implicated in Puralpha, Purbeta, and MSY1 binding in vitro (Kelm, R. J., Jr., Cogan, J. G., Elder, P. K., Strauch, A. R., and Getz, M. J. (1999) J. Biol. Chem. 274, 14238-14245). Our data reveal specific differences between purified DNA treated in vitro and nucleoprotein complexes treated in living cells. Although some differences were observed in quiescent cells, treatment with transforming growth factor beta1 resulted in the development of additional sensitivity within 1 h. This enhancement was most pronounced in bases immediately upstream of an MCAT enhancer element-containing polypurine-polypyrimidine tract. A TATA-proximal element of similar base distribution showed no such hyperreactivities. These results suggest that activation of the endogenous smooth muscle alpha-actin gene during myofibroblast conversion is accompanied by specific structural changes in the promoter that are consistent with a decline in single-stranded DNA repressor protein binding. PMID- 10748153 TI - A plant plasma membrane H+-ATPase expressed in yeast is activated by phosphorylation at its penultimate residue and binding of 14-3-3 regulatory proteins in the absence of fusicoccin. AB - The Nicotiana plumbaginifolia plasma membrane H(+)-ATPase isoform PMA2, equipped with a His(6) tag, was expressed in Saccharomyces cerevisiae and purified. Unexpectedly, a fraction of the purified tagged PMA2 associated with the two yeast 14-3-3 regulatory proteins, BMH1 and BMH2. This complex was formed in vivo without treatment with fusicoccin, a fungal toxin known to stabilize the equivalent complex in plants. When gel filtration chromatography was used to separate the free ATPase from the 14-3-3.H(+)-ATPase complex, the complexed ATPase was twice as active as the free form. Trypsin treatment of the complex released a smaller complex, composed of a 14-3-3 dimer and a fragment from the PMA2 C-terminal region. The latter was identified by Edman degradation and mass spectrometry as the PMA2 C-terminal 57 residues, whose penultimate residue (Thr 955) was phosphorylated. In vitro dephosphorylation of this C-terminal fragment prevented binding of 14-3-3 proteins, even in the presence of fusicoccin. Mutation of Thr-955 to alanine, aspartate, or a stop codon prevented PMA2 from complementing the yeast H(+)-ATPase. These mutations were also introduced in an activated PMA2 mutant (Gln-14 --> Asp) characterized by a higher H(+) pumping activity. Each mutation directly modifying Thr-955 prevented 14-3-3 binding, decreased ATPase specific activity, and reduced yeast growth. We conclude that the phosphorylation of Thr-955 is required for 14-3-3 binding and that formation of the complex activates the enzyme. PMID- 10748154 TI - Cysteine 230 is essential for the structure and activity of the cytotoxic ligand TRAIL. AB - Unlike other tumor necrosis factor family members, the cytotoxic ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo-2L contains an unpaired cysteine residue (Cys(230)) in its receptor-binding domain. Here we show that the biological activity of both soluble recombinant TRAIL and cell associated, full-length TRAIL is critically dependent on the presence of Cys(230). Mutation of Cys(230) to alanine or serine strongly affected its ability to kill target cells. Binding to its receptors was decreased by at least 200 fold, and the stability of its trimeric structure was reduced. In recombinant TRAIL, Cys(230) was found engaged either in interchain disulfide bridge formation, resulting in poorly active TRAIL, or in the chelation of one zinc atom per TRAIL trimer in the active, pro-apoptotic form of TRAIL. PMID- 10748155 TI - 3-Hydroxy-3-methylglutaryl-CoA synthase. A role for glutamate 95 in general acid/base catalysis of C-C bond formation. AB - Replacement of 3-hydroxy-3-methylglutaryl-CoA synthase's glutamate 95 with alanine diminishes catalytic activity by over 5 orders of magnitude. The structural integrity of E95A enzyme is suggested by the observation that this protein contains a full complement of acyl-CoA binding sites, as indicated by binding studies using a spin-labeled acyl-CoA. Active site integrity is also demonstrated by (13)C NMR studies, which indicate that E95A forms an acetyl-S enzyme reaction intermediate with the same distinctive spectroscopic characteristics measured using wild type enzyme. The initial reaction steps are not disrupted in E95A, which exhibits normal levels of Michaelis complex and acetyl-S-enzyme intermediate. Likewise, E95A is not impaired in catalysis of the terminal reaction step, as indicated by efficient catalysis of a hydrolysis partial reaction. Single turnover experiments indicate defective C-C bond formation. The mechanism-based inhibitor, 3-chloropropionyl-CoA, efficiently alkylates E95A. This is compatible with the presence of a functional general base, raising the possibility that Glu(95) functions as a general acid. Demonstration of a significant upfield shift for the methyl protons of HMG-CoA synthase's acetyl-S-enzyme reaction intermediate suggests a hydrophobic active site environment that could elevate the pK(a) of Glu(95) as required to support its function as a general acid. PMID- 10748157 TI - Interaction of the tumor suppressor PTEN/MMAC with a PDZ domain of MAGI3, a novel membrane-associated guanylate kinase. AB - PTEN/MMAC is a phosphatase that is mutated in multiple human tumors. PTEN/MMAC dephosphorylates 3-phosphorylated phosphatidylinositol phosphates that activate AKT/protein kinase B (PKB) kinase activity. AKT/PKB is implicated in the inhibition of apoptosis, and cell lines and tumors with mutated PTEN/MMAC show increased AKT/PKB kinase activity and resistance to apoptosis. PTEN/MMAC contains a PDZ domain-binding site, and we show here that the phosphatase binds to a PDZ domain of membrane-associated guanylate kinase with inverted orientation (MAGI) 3, a novel inverted membrane-associated guanylate kinase that localizes to epithelial cell tight junctions. Importantly, MAGI3 and PTEN/MMAC cooperate to modulate the kinase activity of AKT/PKB. These data suggest that MAGI3 allows for the juxtaposition of PTEN/MMAC to phospholipid signaling pathways involved with cell survival. PMID- 10748159 TI - Identification of mismatch repair protein complexes in HeLa nuclear extracts and their interaction with heteroduplex DNA. AB - Deficiencies in DNA mismatch repair (MMR) have been found in hereditary colon cancers (hereditary non-polyposis colon cancer, HNPCC) as well as in sporadic cancers, illustrating the importance of MMR in maintaining genomic integrity. We have examined the interactions of specific mismatch repair proteins in human nuclear extracts. Western blot and co-immunoprecipitation studies indicate two complexes as follows: one consisting of hMSH2, hMSH6, hMLH1, and hPMS2 and the other consisting of hMSH2, hMSH6, hMLH1, and hPMS1. These interactions occur without the addition of ATP. Furthermore, the protein complexes specifically bind to mismatched DNA and not to a similar homoduplex oligonucleotide. The protein complex-DNA interactions occur primarily through hMSH6, although hMSH2 can also become cross-linked to the mismatched substrate when not participating in the MMR protein complex. In the presence of ATP the binding of hMSH6 to mismatched DNA is decreased. In addition, hMLH1, hPMS2, and hPMS1 no longer interact with each other or with the hMutSalpha complex (hMSH2 and hMSH6). However, the ability of hMLH1 to co-immunoprecipitate mismatched DNA increases in the presence of ATP. This interaction is dependent on the presence of the mismatch and does not appear to involve a direct binding of hMLH1 to the DNA. PMID- 10748158 TI - Zinedin, SG2NA, and striatin are calmodulin-binding, WD repeat proteins principally expressed in the brain. AB - Striatin is an intracellular protein characterized by four protein-protein interaction domains, a caveolin-binding motif, a coiled-coil structure, a calmodulin-binding domain, and a WD repeat domain, suggesting that it is a signaling or a scaffold protein. Down-regulation of striatin, which is expressed in a few subsets of neurons, impairs the growth of dendrites as well as rat locomotor activity (Bartoli, M., Ternaux, J. P., Forni, C., Portalier, P., Salin, P., Amalric, M., and Monneron, A. (1999) J. Neurobiol. 40, 234-243). Zinedin, a "novel" protein described here, and SG2NA share with striatin identical protein protein interaction domains and the same overall domain structure. A phylogenetic analysis supports the hypothesis that they constitute a multigenic family deriving from an ancestral gene. DNA probes and antibodies raised against specific domains of each protein showed that zinedin is mainly expressed in the central nervous system, whereas SG2NA, of more widespread occurrence, is mainly expressed in the brain and muscle. All three proteins are both cytosolic and membrane-bound. All three bind calmodulin in the presence of Ca(2+). In rat brain, SG2NA and striatin are generally not found in the same neurons. Both localize to the soma and dendrites, suggesting that they share a similar type of addressing and closely related functions. PMID- 10748160 TI - Mutation of Asn-391 within the conserved NPXXY motif of the cholecystokinin B receptor abolishes Gq protein activation without affecting its association with the receptor. AB - Among the most conserved regions in the G-protein-coupled receptors is the (N/D)PX(2-3)Y motif of the seventh transmembrane domain (X represents any amino acid). The mutation of the Asn/Asp residue of this motif in different G-protein coupled receptors was shown to affect the activation of either adenylyl cyclase or phospholipase C. We have mutated the Asn residue (Asn-391) of the NPXXY motif in the CCKBR to Ala and determined the effects of the mutation on binding, signaling, and G-proteins coupling after expression of the mutated receptor in COS cells. The mutated receptor displayed similar expression levels and high affinity CCK binding compared with the wild type CCKBR. However, unlike the wild type CCKBR, the mutated receptor was completely unable to mediate activation of either phospholipase C and protein kinase C-dependent and -independent mitogen activated protein kinase pathways, indicating an essential role of Asn-391 in CCKBR signaling. Coimmunoprecipitation experiments allowed us to show that the inactive mutant retains an intact capacity to form stable complexes with G(q)alpha subunits in response to CCK. These results indicate that the formation of high affinity CCK-receptor-G(q) protein complexes is not sufficient to activate G(q) and suggest that Asn-391 is specifically involved in G(q) proteins activation. PMID- 10748161 TI - Probing contacts between the ribonuclease H domain of HIV-1 reverse transcriptase and nucleic acid by site-specific photocross-linking. AB - Cys(38) and Cys(280) of p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) can be converted to Ser without affecting enzyme function. We have exploited this feature to construct and purify "monocysteine" RT derivatives for site-specific modification with the photoactivable cross linking agent, p-azidophenacyl bromide. Acylation of a unique cysteine residue introduced at the extreme C terminus of the p66 subunit (C(561)) with an azidophenacyl group allowed us to probe contacts between residues C-terminal to alpha-helix E' of the RNase H domain and structurally divergent nucleic acid duplexes. In a binary complex of RT and template-primer, we demonstrate efficient cross-linking to primer nucleotides -21 to -24/-25, and template nucleotides -18 to -21. Cross-linking specificity was confirmed by an analogous evaluation following limited primer extension, where the profile is displaced by the register of DNA synthesis. Finally, contact with a DNA primer hybridized to an isogenic RNA or DNA template indicates subtle alterations in cross-linking specificity, suggesting differences in nucleic acid geometry between duplex DNA and RNA/DNA hybrids at the RNase H domain. These data exemplify how site-specific acylation of HIV-1 RT can be used to provide high resolution structural data to complement crystallographic studies. PMID- 10748162 TI - The bradykinin type 2 receptor is a target for p53-mediated transcriptional activation. AB - The bradykinin type 2 receptor (BK2) is a developmentally regulated G protein coupled receptor that mediates diverse actions such as vascular reactivity, salt and water excretion, inflammatory responses, and cell growth. However, little is known regarding regulation of the BK2 gene. We report here that the rat BK2 receptor is transcriptionally regulated by the tumor suppressor protein p53. The 5'-flanking region of the rat BK2 gene contains two p53-like binding sites: a sequence at -70 base pairs (P1 site) that is conserved in the murine and human BK2 genes; and a sequence at -707 (P2) that is not. The P1 and P2 motifs bind specifically to p53, as assessed by gel mobility shift assays. Transient transfection into HeLa cells of a CAT reporter construct driven by 1.2-kilobases of the BK2 gene 5'-flanking region demonstrated that the BK2 promoter is dose dependently activated by co-expression of wild-type p53. Co-expression of a dominant negative mutant p53 suppresses the activation of BK2 by wild-type p53. Promoter truncation localized the p53-responsive element to the region between 38 and -94 base pairs encompassing the p53-binding P1 sequence. Furthermore, p53 mediated activation of the BK2 promoter is augmented by the transcriptional co activators, CBP/p300. Interestingly, removal of the P2 site by truncation or site directed deletion amplifies p53-mediated activation of the BK2 promoter. These results demonstrate that the rat BK2 promoter is a target for p53-mediated activation and suggest a new physiological role for p53 in the regulation of G protein-coupled receptor gene expression. PMID- 10748163 TI - Structural model of the Fe-hydrogenase/cytochrome c553 complex combining transverse relaxation-optimized spectroscopy experiments and soft docking calculations. AB - Fe-hydrogenase is a 54-kDa iron-sulfur enzyme essential for hydrogen cycling in sulfate-reducing bacteria. The x-ray structure of Desulfovibrio desulfuricans Fe hydrogenase has recently been solved, but structural information on the recognition of its redox partners is essential to understand the structure function relationships of the enzyme. In the present work, we have obtained a structural model of the complex of Fe-hydrogenase with its redox partner, the cytochrome c(553), combining docking calculations and NMR experiments. The putative models of the complex demonstrate that the small subunit of the hydrogenase has an important role in the complex formation with the redox partner; 50% of the interacting site on the hydrogenase involves the small subunit. The closest contact between the redox centers is observed between Cys 38, a ligand of the distal cluster of the hydrogenase and Cys-10, a ligand of the heme in the cytochrome. The electron pathway from the distal cluster of the Fe hydrogenase to the heme of cytochrome c(553) was investigated using the software Greenpath and indicates that the observed cysteine/cysteine contact has an essential role. The spatial arrangement of the residues on the interface of the complex is very similar to that already described in the ferredoxin-cytochrome c(553) complex, which therefore, is a very good model for the interacting domain of the Fe-hydrogenase-cytochrome c(553). PMID- 10748164 TI - Characterization of elements mediating regulation of phosphoenolpyruvate carboxykinase gene transcription by protein kinase A and insulin. Identification of a distinct complex formed in cells that mediate insulin inhibition. AB - The in vivo pattern of induction of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription by cAMP and its inhibition by insulin is reproduced in H4IIe cells and is mediated by a bipartite cAMP/insulin response unit (C/IRU) consisting of a cAMP response element (-95/-87) and an upstream enhancer, AC ( 271/-225). Studies in HepG2 cells showed that binding of AP-1 and CAAT/enhancer binding protein (C/EBP) to AC is required for induction by cAMP, but insulin did not inhibit cAMP-induced PEPCK expression in HepG2 cells. Binding of H4IIe nuclear proteins to an AC element probe was inhibited by antibodies or a consensus site for C/EBP, but not AP-1. Transfection with dominant negative bZIP factors, which prevent endogenous factors from binding to DNA, showed that elimination of cAMP regulatory element-binding protein CREB or C/EBP activity blocked induction by protein kinase A (PKA), whereas elimination of AP-1 activity had no effect. In addition, promoters with multiple CREB sites, or a single CREB site and multiple C/EBP sites, mediated PKA induction, but this was inhibited to no greater extent than basal activity was by insulin. These results indicate that an AC factor other than C/EBP must mediate insulin inhibition. An A-site probe ( 265/-247) or a probe across the middle of the AC element (-256/-237) competed for complexes formed by factors other than AP-1 or C/EBP. However, analysis of competitor oligonucleotides and antibodies for candidate factors failed to identify other factors. Scanning mutations throughout the AC element interfered with induction but allowed us to define five overlapping sites for regulatory factors in AC and to design probes binding just one or two factors. Comparison of the protein-DNA complexes formed on these smaller probes revealed that a specific complex present in rat liver and H4IIe cell nuclear extracts differed from those formed by HepG2 cell nuclear extracts. Our results suggest that multiple factors binding the AC element of the C/IRU interact with each other and CREB to regulate PEPCK induction by cAMP and inhibition by insulin and that the unique factor expressed in H4IIe cells is a candidate for involvement in insulin regulation of PKA-induced PEPCK gene transcription. PMID- 10748165 TI - The B1 subunit of the H+ATPase is a PDZ domain-binding protein. Colocalization with NHE-RF in renal B-intercalated cells. AB - The 56-kDa B1 subunit of the vacuolar H(+)ATPase has a C-terminal DTAL amino acid motif typical of PDZ-binding proteins that associate with the PDZ protein, NHE-RF (Na(+)/H(+) exchanger regulatory factor). This B1 isoform is amplified in renal intercalated cells, which play a role in distal urinary acid-base transport. In contrast, proximal tubules express the B2 isoform that lacks the C-terminal PDZ binding motif. Both the B1 56-kDa subunit and the 31-kDa (E) subunit of the H(+)ATPase are pulled down by glutathione S-transferase NHE-RF bound to GSH Sepharose beads. These subunits associate in vivo as part of the cytoplasmic V1 portion of the H(+)ATPase, and the E subunit was co-immunoprecipitated from rat kidney cytosol with NHE-RF antibodies. The interaction of H(+)ATPase subunits with NHE-RF was inhibited by a peptide derived from the C terminus of the B1 but not the B2 isoform. NHE-RF colocalized with H(+)ATPase in either the apical or the basolateral region of B-type intercalated cells, whereas NHE-RF staining was undetectable in A-intercalated cells. In proximal tubules, NHE-RF was located in the apical brush border. In contrast, H(+)ATPase was concentrated in a distinct membrane domain at the base of the brush border, from which NHE-RF was absent, consistent with the expression of the truncated B2 subunit isoform in this tubule segment. The colocalization of NHE-RF and H(+)ATPase in B- but not A-intercalated cells suggests a role in generating, maintaining, or modulating the variable H(+)ATPase polarity that characterizes the B-cell phenotype. PMID- 10748166 TI - Identification of selective estrogen receptor modulators by their gene expression fingerprints. AB - Clinical studies have shown that estrogen replacement therapy (ERT) reduces the incidence and severity of osteoporosis and cardiovascular disease in postmenopausal women. However, long term estrogen treatment also increases the risk of endometrial and breast cancer. The selective estrogen receptor (ER) modulators (SERMs) tamoxifen and raloxifene, cause antagonistic and agonistic responses when bound to the ER. Their predominantly antagonistic actions in the mammary gland form the rationale for their therapeutic utility in estrogen responsive breast cancer, while their agonistic estrogen-like effects in bone and the cardiovascular system make them candidates for ERT regimens. Of these two SERMs, raloxifene is preferred because it has markedly less uterine-stimulatory activity than either estrogen or tamoxifen. To identify additional SERMs, a method to classify compounds based on differential gene expression modulation was developed. By analysis of 24 different combinations of genes and cells, a selected set of assays that permitted discrimination between estrogen, tamoxifen, raloxifene, and the pure ER antagonist ICI164384 was generated. This assay panel was employed to measure the activity of 38 compounds, and the gene expression fingerprints (GEFs) obtained for each compound were used to classify all compounds into eight groups. The compound's GEF predicted its uterine-stimulatory activity. One group of compounds was evaluated for activity in attenuating bone loss in ovariectomized rats. Most compounds with similar GEFs had similar in vivo activities, thereby suggesting that GEF-based screens could be useful in predicting a compound's in vivo pharmacological profile. PMID- 10748167 TI - Newly synthesized canalicular ABC transporters are directly targeted from the Golgi to the hepatocyte apical domain in rat liver. AB - Newly synthesized canalicular ectoenzymes and a cell adhesion molecule (cCAM105) have been shown to traffic from the Golgi to the basolateral plasma membrane, from where they transcytose to the apical bile canalicular domain. It has been proposed that all canalicular proteins are targeted via this indirect route in hepatocytes. We studied the membrane targeting of rat canalicular proteins by in vivo [(35)S]methionine metabolic labeling followed by preparation of highly purified Golgi membranes and canalicular (CMVs) and sinusoidal/basolateral (SMVs) membrane vesicles and subsequent immunoprecipitation. In particular, we compared membrane targeting of newly synthesized canalicular ABC (ATP-binding cassette) transporters MDR1, MDR2, and SPGP (sister of P-glycoprotein) with that of cCAM105. Significant differences were observed in metabolic pulse-chase labeling experiments with regard to membrane targeting of these apical proteins. After a chase time of 15 min, cCAM105 appeared exclusively in SMVs, peaked at 1 h, and progressively declined thereafter. In CMVs, cCAM105 was first detected after 1 h and subsequently increased for 3 h. This findings confirm the transcytotic targeting of cCAM105 reported in earlier studies. In contrast, at no time point investigated were MDR1, MDR2, and SPGP detected in SMVs. In CMVs, MDR1 and MDR2 appeared after 30 min, whereas SPGP appeared after 2 h of labeling. In Golgi membranes, each of the ABC transporters peaked at 30 min and was virtually absent thereafter. These data suggest rapid, direct targeting of newly synthesized MDR1 and MDR2 from the Golgi to the bile canaliculus and transient sequestering of SPGP in an intracellular pool en route from the Golgi to the apical plasma membrane. This study provides biochemical evidence for direct targeting of newly synthesized apical ABC transporters from the Golgi to the bile canaliculus in vivo. PMID- 10748168 TI - Novel mechanism of beta-lactam resistance due to bypass of DD-transpeptidation in Enterococcus faecium. AB - The peptidoglycan structure of in vitro selected ampicillin-resistant mutant Enterococcus faecium D344M512 and of the susceptible parental strain D344S was determined by reverse phase high performance liquid chromatography and mass spectrometry. The muropeptide monomers were almost identical in the two strains. The substantial majority (99.3%) of the oligomers from the susceptible strain D344S contained the usual d-alanyl --> d-asparaginyl (or d-aspartyl)-l-lysyl cross-link (d-Ala --> d-Asx-l-Lys) generated by beta-lactam-sensitive DD transpeptidation. The remaining oligomers (0.7%) were produced by beta-lactam insensitive LD-transpeptidation, because they contained l-Lys --> d-Asx-l-Lys cross-links. The muropeptide oligomers of the ampicillin-resistant mutant D344M512 contained only these l-Lys --> d-Asx-l-Lys cross-links indicating that resistance was due to the bypass of the beta-lactam-sensitive DD-transpeptidation reaction. The discovery of this novel resistance mechanism indicates that DD transpeptidases cannot be considered anymore as the sole essential transpeptidase enzymes. PMID- 10748169 TI - Presenilin 2 interacts with sorcin, a modulator of the ryanodine receptor. AB - Perturbed Ca(2+) homeostasis is a common molecular consequence of familial Alzheimer's disease-linked presenilin mutations. We report here the molecular interaction of the large hydrophilic loop region of presenilin 2 (PS2) with sorcin, a penta-EF-hand Ca(2+)-binding protein that serves as a modulator of the ryanodine receptor intracellular Ca(2+) channel. The association of endogenous sorcin and PS2 was demonstrated in cultured cells and human brain tissues. Membrane-associated sorcin and a subset of the functional PS2 complexes were co localized to a novel subcellular fraction that is distinctively positive for calcineurin B. Sorcin was found to interact with PS2 endoproteolytic fragments but not full-length PS2, and the sorcin/PS2 interaction was greatly enhanced by treatment with the Ca(2+) ionophore A23187. Our findings reveal a molecular link between PS2 and intracellular Ca(2+) channels (i.e. ryanodine receptor) and substantiate normal and/or pathological roles of PS2 in intracellular Ca(2+) homeostasis. PMID- 10748170 TI - Agonist-induced isomerization in a glutamate receptor ligand-binding domain. A kinetic and mutagenetic analysis. AB - Agonist binding to glutamate receptor ion channels occurs within an extracellular domain (S1S2) that retains ligand affinity when expressed separately. S1S2 is homologous to periplasmic binding proteins, and it has been proposed that a Venus flytrap-style cleft closure triggers opening of glutamate receptor ion channels. Here we compare the kinetics of S1S2-agonist binding to those of the periplasmic binding proteins and show that the reaction involves an initial rapid association, followed by slower conformational changes that stabilize the complex: "docking" followed by "locking." The motion detected here reflects the mechanism by which the energy of glutamate binding is converted into protein conformational changes within S1S2 alone. In the intact channel, these load-free conformational changes are harnessed and possibly modified as the agonist binding reaction is used to drive channel opening and subsequent desensitization. Using mutagenesis, key residues in each step were identified, and their roles were interpreted in light of a published S1S2 crystal structure. In contrast to the Venus flytrap proposal, which focuses on motion between the two lobes as the readout for agonist binding, we argue that smaller, localized conformational rearrangements allow agonists to bridge the cleft, consistent with published hydrodynamic measurements. PMID- 10748171 TI - A novel shuttle protein binds to RNA helicase A and activates the retroviral constitutive transport element. AB - The constitutive transport element (CTE) of type D retroviruses mediates the nuclear export of unspliced viral transcripts. We previously showed that RNA helicase A functionally interacts with CTE and contains a bidirectional nuclear transport domain at the carboxyl terminus. Here we report the identification of a novel human protein, helicase A-binding protein 95 (HAP95), which specifically binds to the carboxyl terminus of RNA helicase A. HAP95 is partially homologous to AKAP95, a member of the A kinase-anchoring protein family, but lacks the protein kinase A binding domain characteristic of this family. HAP95 is a nuclear protein at steady state but shuttles between the nucleus and cytoplasm. Overexpression of HAP95 significantly increases CTE-dependent gene expression but has no effect on general gene expression or that mediated by the Rev/Rev response element of human immunodeficiency virus type 1. PMID- 10748172 TI - Caveolin-1 inhibits epidermal growth factor-stimulated lamellipod extension and cell migration in metastatic mammary adenocarcinoma cells (MTLn3). Transformation suppressor effects of adenovirus-mediated gene delivery of caveolin-1. AB - Caveolin-1 is a principal component of caveolae membranes that may function as a transformation suppressor. For example, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (D7S522; 7q31.1) that is deleted in human cancers, including mammary carcinomas. However, little is known about the role of caveolins in regulating cell movement, a critical parameter in determining metastatic potential. Here, we examine the role of caveolin-1 in cell movement. For this purpose, we employed an established cellular model, MTLn3, a metastatic rat mammary adenocarcinoma cell line. In this system, epidermal growth factor (EGF) stimulation induces rapid lamellipod extension and cell migration. Interestingly, we find that MTLn3 cells fail to express detectable levels of endogenous caveolin-1. To restore caveolin-1 expression in MTLn3 cells efficiently, we employed an inducible adenoviral gene delivery system to achieve tightly controlled expression of caveolin-1. We show here that caveolin-1 expression in MTLn3 cells inhibits EGF-stimulated lamellipod extension and cell migration and blocks their anchorage-independent growth. Under these conditions, EGF-induced activation of the p42/44 mitogen-activated protein kinase cascade is also blunted. Our results suggest that caveolin-1 expression in motile MTLn3 cells induces a non-motile phenotype. PMID- 10748173 TI - Correlated switch binding and signaling in bacterial chemotaxis. AB - In Escherichia coli, swimming behavior is mediated by the phosphorylation state of the response regulator CheY. In its active, phosphorylated form, CheY exhibits enhanced binding to a switch component, FliM, at the flagellar motor, which induces a change from counterclockwise to clockwise flagellar rotation. When Ile(95) of CheY is replaced by a valine, increased clockwise rotation correlates with enhanced binding to FliM. A possible explanation for the hyperactivity of this mutant is that residue 95 affects the conformation of nearby residues that potentially interact with FliM. In order to assess this possibility directly, the crystal structure of CheY95IV was determined. We found that CheY95IV is structurally almost indistinguishable from wild-type CheY. Several other mutants with substitutions at position 95 were characterized to establish the structural requirements for switch binding and clockwise signaling at this position and to investigate a general relationship between the two properties. The various rotational phenotypes of these mutants can be explained solely by the amount of phosphorylated CheY bound to the switch, which was inferred from the phosphorylation properties of the mutant CheY proteins and their binding affinities to FliM. Combined genetic, biochemical, and crystallographic results suggest that residue 95 itself is critical in mediating the surface complementarity between CheY and FliM. PMID- 10748174 TI - An exochelin of Mycobacterium tuberculosis reversibly arrests growth of human vascular smooth muscle cells in vitro. AB - Proliferation of vascular smooth muscle cells (VSMC) is characteristic of restenosis following balloon angioplasty. We show here that a low concentration of a novel iron chelator, desferri-exochelin 772SM, reversibly arrests the growth of human VSMC in vitro, specifically in G(0)/G(1) and S phases. The lipophilic desferri-exochelin is effective more rapidly and at a 10-fold lower concentration than the nonlipophilic iron chelator deferoxamine. Treatment of growth synchronized VSMC with the desferri-exochelin results in down-regulation of cyclin E/ Cdk2 and cyclin A/Cdk2 activity but does not affect the cyclin D/Cdk4/retinoblastoma phosphorylation pathway. Both DNA replication and RNA transcription are inhibited in exochelin-treated cells, but protein synthesis is not. The ability of desferri-exochelin 772SM to reversibly block the growth of VSMC in vitro with no apparent cytotoxicity suggests that the exochelin may be useful as a therapeutic agent to limit restenosis in injured vessels. PMID- 10748175 TI - Distinct protein domains of the yeast Golgi GDP-mannose transporter mediate oligomer assembly and export from the endoplasmic reticulum. AB - The substrates for glycan synthesis in the lumen of the Golgi are nucleotide sugars that must be transported from the cytosol by specific membrane-bound transporters. The principal nucleotide sugar used for glycosylation in the Golgi of the yeast Saccharomyces cerevisiae is GDP-mannose, whose lumenal transport is mediated by the VRG4 gene product. As the sole provider of lumenal mannose, the Vrg4 protein functions as a key regulator of glycosylation in the yeast Golgi. We have undertaken a functional analysis of Vrg4p as a model for understanding nucleotide sugar transport in the Golgi. Here, we analyzed epitope-tagged alleles of VRG4. Gel filtration chromatography and co-immunoprecipitation experiments demonstrate that the Vrg4 protein forms homodimers with specificity and high affinity. Deletion analyses identified two regions essential for Vrg4p function. Mutant Vrg4 proteins lacking the predicted C-terminal membrane-spanning domain fail to assemble into oligomers (Abe, M., Hashimoto, H., and Yoda, K. (1999) FEBS Lett. 458, 309-312) and are unstable, while proteins lacking the N-terminal cytosolic tail are stable and multimerize efficiently, but are mislocalized to the endoplasmic reticulum (ER). Fusion of the N terminus of Vrg4p to related ER membrane proteins promote their transport to the Golgi, suggesting that sequences in the N terminus supply information for ER export. The dominant negative phenotype resulting from overexpression of truncated Vrg4-DeltaN proteins provides strong genetic evidence for homodimer formation in vivo. These studies are consistent with a model in which Vrg4p oligomerizes in the ER and is subsequently transported to the Golgi via a mechanism that involves positive sorting rather than passive default. PMID- 10748176 TI - Inhibition of insulin-like growth factor-I-mediated cell signaling by the von Hippel-Lindau gene product in renal cancer. AB - Insulin-like growth factor-I (IGF-I)-mediated signaling is thought to be involved in the regulation of multiple cellular functions in different tumors including renal cell carcinoma (RCC). Blocking IGF-I signaling by any of the several strategies abolishes or delays the progression of a variety of tumors in animal models. Herein, we demonstrate that in RCC cell lines, IGF-I-mediated signaling is found to be inhibited in the presence of wild type von Hippel-Lindau (VHL) tumor suppresser gene. Moreover, molecular modeling and biochemical approaches have revealed that beta-domain of the VHL gene product by interacting directly with protein kinase Cdelta inhibits its association with IGF-IR for downstream signaling. We also demonstrated that RCC has IGF-I-mediated invasive activity where protein kinase Cdelta is an important downstream molecule, and this invasiveness can be blocked by wild type VHL. These experiments thus elucidate a novel tumor suppresser function of VHL with its unique kinase inhibitory domain. PMID- 10748177 TI - Hydrophobic residues Phe751 and Leu753 are essential for STAT5 transcriptional activity. AB - One facet of cytokine signaling is relayed to the nucleus by the activation, through tyrosine phosphorylation, of latent cytoplasmic signal transducers and activators of transcription (STAT) family members. It has been demonstrated that the C termini of STATs contain the transactivation domain and are essential for the transactivation of target genes. To better understand the function of the STAT C terminus, we have generated a series of C-terminal mutants in STAT5a and examined their effects on transactivation, tyrosine phosphorylation, and DNA binding. Using GAL4 chimerae with the C terminus of STAT5, we have defined a 12 amino acid region essential for STAT5 transactivation. Surprisingly, deletion of these 12 amino acids in the context of the native STAT5 backbone preserved the overall transcriptional activity of the protein. Further analysis revealed that deletion of this region resulted in hyper-DNA binding activity, thus compensating for the weakened transactivation domain. Using site-directed mutagenesis, we show that within this 12-amino acid region the acidic residues were non-essential for transactivation. In contrast, the non-acidic residues were crucial for transactivation. Mutating either Phe(751) or Leu(753) to alanine abolished transactivation suggesting that these residues were essential for connecting STAT5 to the basal transcriptional machinery. PMID- 10748178 TI - Antagonistic action of a 25-carboxylic ester analogue of 1alpha, 25 dihydroxyvitamin D3 is mediated by a lack of ligand-induced vitamin D receptor interaction with coactivators. AB - A 25-carboxylic ester analogue of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25 (OH)(2)D(3)), ZK159222, was described as a novel type of antagonist of 1alpha,25 (OH)(2)D(3) signaling. The ligand sensitivity of ZK159222, in facilitating complex formation between 1alpha,25-(OH)(2)D(3) receptor (VDR) and the retinoid X receptor (RXR) on a 1alpha,25-(OH)(2)D(3) response element (VDRE), was approximately 7-fold lower when compared with 1alpha,25-(OH)(2)D(3). However, ZK159222 was not able to promote a ligand-dependent interaction of the VDR with the coactivator proteins SRC-1, TIF2, and RAC3, neither in solution nor in a complex with RXR on DNA. Functional analysis in HeLa and COS-7 cells demonstrated a 10-100-fold lower ligand sensitivity for ZK159222 than for 1alpha, 25 (OH)(2)D(3) and, most interestingly, a potency that was drastically reduced compared with 1alpha,25-(OH)(2)D(3). A cotreatment of 1alpha,25-(OH)(2)D(3) with a 100-fold higher concentration of ZK159222 resulted in a prominent antagonistic effect both in functional in vivo and in in vitro assays. These data suggest that the antagonistic action of ZK159222 is due to a lack of ligand-induced interaction of the VDR with coactivators with a parallel ligand sensitivity, which is sufficient for competition with the natural hormone for VDR binding. PMID- 10748179 TI - Inhibition of glycogen-synthase kinase 3 stimulates glycogen synthase and glucose transport by distinct mechanisms in 3T3-L1 adipocytes. AB - The role of glycogen-synthase kinase 3 (GSK3) in insulin-stimulated glucose transport and glycogen synthase activation was investigated in 3T3-L1 adipocytes. GSK3 protein was clearly present in adipocytes and was found to be more abundant than in muscle and liver cell lines. The selective GSK3 inhibitor, LiCl, stimulated glucose transport and glycogen synthase activity (20 and 65%, respectively, of the maximal (1 microm) insulin response) and potentiated the responses to a submaximal concentration (1 nm) of insulin. LiCl- and insulin stimulated glucose transport were abolished by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin; however, LiCl stimulation of glycogen synthase was not. In contrast to the rapid stimulation of glucose transport by insulin, transport stimulated by LiCl increased gradually over 3-5 h reaching 40% of the maximal insulin-stimulated level. Both LiCl- and insulin-stimulated glycogen synthase activity were maximal at 25 min. However, insulin-stimulated glycogen synthase activity returned to basal after 2 h, coincident with reactivation of GSK3. After a 2-h exposure to insulin, glycogen synthase was refractory to restimulation with insulin, indicating selective desensitization of this pathway. However, LiCl could partially stimulate glycogen synthase in desensitized cells. Furthermore, coincubation with LiCl during the 2 h exposure to insulin completely blocked desensitization of glycogen synthase activity. In summary, inhibition of GSK3 by LiCl: 1) stimulated glycogen synthase activity directly and independently of PI3-kinase, 2) stimulated glucose transport at a point upstream of PI3-kinase, 3) stimulated glycogen synthase activity in desensitized cells, and 4) prevented desensitization of glycogen synthase due to chronic insulin treatment. These data are consistent with GSK3 playing a central role in the regulation of glycogen synthase activity and a contributing factor in the regulation of glucose transport in 3T3-L1 adipocytes. PMID- 10748180 TI - Identification and characterization of cell lines with a defect in a post adsorption stage of Sendai virus-mediated membrane fusion. AB - In the early stage of infection, Sendai virus delivers its genome into the cytoplasm by fusing the viral envelope with the cell membrane. Although the adsorption of virus particles to cell surface receptors has been characterized in detail, the ensuing complex process that leads to the fusion between the lipid bilayers remains mostly obscure. In the present study, we identified and characterized cell lines with a defect in the Sendai virus-mediated membrane fusion, using fusion-mediated delivery of fragment A of diphtheria toxin as an index. These cells, persistently infected with the temperature-sensitive variant Sendai virus, had primary viral receptors indistinguishable in number and affinity from those of parental susceptible cells. However, they proved to be thoroughly defective in the Sendai virus-mediated membrane fusion. We also found that viral HN protein expressed in the defective cells was responsible for the interference with membrane fusion. These results suggested the presence of a previously uncharacterized, HN-dependent intermediate stage in the Sendai virus mediated membrane fusion. PMID- 10748181 TI - The transcriptional response of yeast to saline stress. AB - Adaptation to changes in extracellular salinity is a critical event for cell survival. Genome-wide DNA chip analysis has been used to analyze the transcriptional response of yeast cells to saline stress. About 7% of the genes encoded in the yeast genome are induced more than 5-fold after a mild and brief saline shock (0.4 m NaCl, 10 min). Interestingly, most responsive genes showed a very transient expression pattern, as mRNA levels dramatically declined after 20 min in the presence of stress. A quite similar set of genes increased expression in cells subjected to higher saline concentrations (0.8 m NaCl), although in this case the response was delayed. Therefore, our data show that cells respond to saline stress by inducing the expression of a very large number of genes and suggest that stress adaptation requires regulation of many cellular aspects. The transcriptional induction of most genes that are strongly responsive to salt stress was highly or fully dependent on the presence of the stress-activated mitogen-activated protein kinase Hog1, indicating that the Hog1-mediated signaling pathway plays a key role in global gene regulation under saline stress conditions. PMID- 10748182 TI - Synergistic activation of NFAT by HIV-1 nef and the Ras/MAPK pathway. AB - Nef is a lentiviral protein involved in pathogenesis of AIDS, but its molecular mechanisms of action remain incompletely understood. Here we report a novel effect of Nef on lymphocyte signaling, which is mediated via a T cell receptor (TCR)-independent contribution of Nef to induction of nuclear factor of activated T cells (NFAT), a transcription factor that plays a central role in coordinating T cell activation. Expression of Nef did not significantly alter the basal level of NFAT activity in Jurkat cells nor the increased activity following T cell receptor stimulation by anti-CD3 or anti-CD3 + anti-CD28. We also mimicked NFAT induction by TCR triggering by simultaneous activation of the Ras and calcium signaling pathways with phorbol 12-myristate 13-acetate and ionomycin, respectively. Strikingly, whereas activation of either of these pathways individually did not induce NFAT activity in control cells, in Nef-expressing cells phorbol 12-myristate 13-acetate treatment alone resulted in a 100-fold increase in NFAT-directed gene expression. Experiments with different dominant negative mutant signaling proteins, inhibitory chemicals, and Lck-deficient Jurkat cells revealed that this effect was mediated via activation of calcineurin by Nef-induced changes in calcium metabolism, but was independent of TCR associated signaling events. This ability of Nef to substitute for triggering of the calcium pathway in induction of NFAT could promote activation of human immunodeficiency virus (HIV)-infected T cells in response to stimuli mediated via TCR or other cell surface receptors under conditions when activation of Ras rather than calcium signaling would otherwise predominate. PMID- 10748183 TI - Dependence of elevated human leukocyte antigen class I molecule expression on increased heavy chain, light chain (beta 2-microglobulin), transporter associated with antigen processing, tapasin, and peptide. AB - Human leukocyte antigen (HLA) class I molecule expression was investigated by DNA mediated gene transfer. Cell surface expression was increased up to 75% by transfection of HLA-A2 or HLA-B8 heavy chain genes but not genes encoding light chains (beta(2)-microglobulin (beta(2)m)), transporter associated with antigen processing (TAP), or tapasin. Interferon (IFN) treatment further increased expression of transfected heavy chains, suggesting that IFN inducible molecules support heavy chain expression. IFN induces beta(2)m, TAP, and tapasin mRNAs. Transfected heavy chain expression increased upon cotransfection with genes encoding TAP1 and TAP2 but not individual TAP subunits, beta(2)m, or tapasin. Tetracycline inducible heavy chain gene expression was also increased by IFN treatment or TAP cotransfection, suggesting that IFN-induced TAP supports heavy chain maturation. Expression of a mutant that does not interact strongly with TAP, HLA-A2-T134K, was also increased by IFN. Inhibition of TAP-dependent peptide transport by ICP47 reduced heavy chain expression. Expression of HLA-A2, but not HLA-B8, was restored in ICP47 cells by HLA-A2-binding (IP-30) signal peptides. However, these peptides did not further increase transfected HLA-A2 expression, suggesting that peptide availability does not limit heavy chain expression in the absence of ICP47. These results suggest that cytokine-induced TAP supports maturation of HLA class I molecules through combined chaperone and peptide supply functions. PMID- 10748184 TI - Determination of the free-energy change for repair of a DNA phosphodiester bond. AB - The repair of phosphodiester bonds in nicked DNA is catalyzed by DNA ligases. Ligation is coupled to cleavage of a phosphoanhydride bond in a nucleotide cofactor resulting in a thermodynamically favorable process. A free energy value for phosphodiester bond formation was calculated using the reversibility of the T4 DNA ligase reaction. The relative number of DNA nicks to phosphodiester bonds in a circular plasmid DNA, formed during this reaction at fixed concentrations of ATP to AMP and PP(i), was quantified. At 25 degrees C, pH 7, the equilibrium constant (K(eq)) for the ligation reaction is 3.89 x 10(4) m. This value corresponds to a standard free energy (DeltaG degrees ') of -6.3 kcal mol(-1). By subtracting the known energy contribution due to hydrolysis of ATP to AMP and PP(i), DeltaG degrees ' for the hydrolysis of a DNA phosphodiester bond is -5.3 kcal mol(-1). PMID- 10748185 TI - Amino-terminal-derived JNK fragment alters expression and activity of c-Jun, ATF2, and p53 and increases H2O2-induced cell death. AB - The stress-activated protein kinase JNK plays an important role in the stability and activities of key regulatory proteins, including c-Jun, ATF2, and p53. To better understand mechanisms underlying the regulation of JNK activities, we studied the effect of expression of the amino-terminal JNK fragment (N-JNK; amino acids 1-206) on the stability and activities of JNK substrates under nonstressed growth conditions, as well as after exposure to hydrogen peroxide. Mouse fibroblasts that express N-JNK under tetracycline-off (tet-off) inducible promoter exhibited elevated expression of c-Jun, ATF2, and p53 upon tetracycline removal. This increased coincided with elevated transcriptional activities of p53, but not of c-Jun or ATF2, as reflected in luciferase activities of p21(Waf1/Cip1)-Luc, AP1-Luc, and Jun2-Luc, respectively. Expression of N-JNK in cells that were treated with H(2)O(2) impaired transcriptional output as reflected in a delayed and lower level of c-Jun-, limited ATF2-, and reduced p53 transcriptional activities. N-JNK elicited an increase in H(2)O(2)-induced cell death, which is p53-dependent, because it was not seen in p53 null cells yet could be observed upon coexpression of p53 and N-JNK. The ability to alter the activity of ATF2, c-Jun, and p53 and the degree of stress-induced cell death by a JNK-derived fragment identifies new means to elucidate the nature of JNK regulation and to alter the cellular response to stress. PMID- 10748187 TI - ERK1b, a 46-kDa ERK isoform that is differentially regulated by MEK. AB - We identified a 46-kDa ERK, whose kinetics of activation was similar to that of ERK1 and ERK2 in most cell lines and conditions, but showed higher fold activation in response to osmotic shock and epidermal growth factor treatments of Ras-transformed cells. We purified and cloned this novel ERK (ERK1b), which is an alternatively spliced form of ERK1 with a 26-amino acid insertion between residues 340 and 341 of ERK1. When expressed in COS7 cells, ERK1b exhibited kinetics of activation and kinase activity similar to those of ERK1. Unlike the uniform pattern of expression of ERK1 and ERK2, ERK1b was detected only in some of the tissues examined and seems to be abundant in the rat and human heart. Interestingly, in Ras-transformed Rat1 cells, there was a 7-fold higher expression of ERK1b, which was also more responsive than ERK1 and ERK2 to various extracellular treatments. Unlike ERK1 and ERK2, ERK1b failed to interact with MEK1 as judged from its nuclear localization in resting cells overexpressing ERK1b together with MEK1 or by lack of coimmunoprecipitation of the two proteins. Thus, ERK1b is a novel 46-kDa ERK isoform, which seems to be the major ERK isoform that responds to exogenous stimulation in Ras-transformed cells probably due to its differential regulation by MEK. PMID- 10748188 TI - Role of COPI in phagosome maturation. AB - Phagosomes mature by sequentially fusing with endosomes and lysosomes. Vesicle budding is presumed to occur concomitantly, mediating the retrieval of plasmalemmal components and the regulation of phagosomal size. We analyzed whether fission of vesicles from phagosomes requires COPI, a multimeric complex known to be involved in budding from the Golgi and endosomes. The role of COPI was studied using ldlF cells, that harbor a temperature-sensitive mutation in epsilon-COP, a subunit of the coatomer complex. These cells were made phagocytic toward IgG-opsonized particles by heterologous expression of human FcgammaRIIA receptors. Following incubation at the restrictive temperature, epsilon-COP was degraded in these cells and their Golgi complex dispersed. Nevertheless, phagocytosis persisted for hours in cells devoid of epsilon-COP. Retrieval of transferrin receptors from phagosomes became inefficient in the absence of epsilon-COP, while clearance of the FcgammaRIIA receptors was unaffected. This indicates that fission of vesicles from the phagosomal membrane involves at least two mechanisms, one of which requires intact COPI. Traffic of fluid-phase markers and aggregated IgG-receptor complexes along the endocytic pathway was abnormal in epsilon-COP-deficient cells. In contrast, phagosome fusion with endosomes and lysosomes was unimpaired. Moreover, the resulting phagolysosomes were highly acidic. Similar results were obtained in RAW264.7 macrophages treated with brefeldin A, which precludes COPI assembly by interfering with the activation of adenosine ribosylation factor. These data indicate that neither phagosome formation nor maturation are absolutely dependent on COPI. Our findings imply that phagosomal maturation differs from endosomal progression, which appears to be more dependent on COPI-mediated formation of carrier vesicles. PMID- 10748189 TI - Reverse gyrase, the two domains intimately cooperate to promote positive supercoiling. AB - Reverse gyrases are atypical topoisomerases present in hyperthermophiles and are able to positively supercoil a circular DNA. Despite a number of studies, the mechanism by which they perform this peculiar activity is still unclear. Sequence data suggested that reverse gyrases are composed of two putative domains, a helicase-like and a topoisomerase I, usually in a single polypeptide. Based on these predictions, we have separately expressed the putative domains and the full length polypeptide of Sulfolobus acidocaldarius reverse gyrase as recombinant proteins in Escherichia coli. We show the following. (i) The full-length recombinant enzyme sustains ATP-dependent positive supercoiling as efficiently as the wild type reverse gyrase. (ii) The topoisomerase domain exhibits a DNA relaxation activity by itself, although relatively low. (iii) We failed to detect helicase activity for both the N-terminal domain and the full-length reverse gyrase. (iv) Simple mixing of the two domains reconstitutes positive supercoiling activity at 75 degrees C. The cooperation between the domains seems specific, as the topoisomerase domain cannot be replaced by another thermophilic topoisomerase I, and the helicase-like cannot be replaced by a true helicase. (v) The helicase like domain is not capable of promoting stoichiometric DNA unwinding by itself; like the supercoiling activity, unwinding requires the cooperation of both domains, either separately expressed or in a single polypeptide. However, unwinding occurs in the absence of ATP and DNA cleavage, indicating a structural effect upon binding to DNA. These results suggest that the N-terminal domain does not directly unwind DNA but acts more likely by driving ATP-dependent conformational changes within the whole enzyme, reminiscent of a protein motor. PMID- 10748190 TI - Ultraviolet radiation-induced interleukin 6 release in HeLa cells is mediated via membrane events in a DNA damage-independent way. AB - Evidence exists that ultraviolet radiation (UV) affects molecular targets in the nucleus or at the cell membrane. UV-induced apoptosis was found to be mediated via DNA damage and activation of death receptors, suggesting that nuclear and membrane effects are not mutually exclusive. To determine whether participation of nuclear and membrane components is also essential for other UV responses, we studied the induction of interleukin-6 (IL-6) by UV. Exposing HeLa cells to UV at 4 degrees C, which inhibits activation of surface receptors, almost completely prevented IL-6 release. Enhanced repair of UV-mediated DNA damage by addition of the DNA repair enzyme photolyase did not affect UV-induced IL-6 production, suggesting that in this case membrane events predominant over nuclear effects. UV induced IL-6 release is mediated via NFkappaB since the NFkappaB inhibitor MG132 or transfection of cells with a super-repressor form of the NFkappaB inhibitor IkappaB reduced IL-6 release. Transfection with a dominant negative mutant of the signaling protein TRAF-2 reduced IL-6 release upon exposure to UV, indicating that UV-induced IL-6 release is mediated by activation of the tumor necrosis factor receptor-1. These data demonstrate that UV can exert biological effects mainly by affecting cell surface receptors and that this is independent of its ability to induce nuclear DNA damage. PMID- 10748191 TI - Virus maturation targets the protein capsid to concerted disassembly and unfolding. AB - Many animal viruses undergo post-assembly proteolytic cleavage that is required for infectivity. The role of maturation cleavage on Flock House virus was evaluated by comparing wild type (wt) and cleavage-defective mutant (D75N) Flock House virus virus-like particles. A concerted dissociation and unfolding of the mature wt particle was observed under treatment by urea, whereas the cleavage defective mutant dissociated to folded subunits as determined by steady-state and dynamic fluorescence spectroscopy, circular dichroism, and nuclear magnetic resonance. The folded D75N alpha subunit could reassemble into capsids, whereas the yield of reassembly from unfolded cleaved wt subunits was very low. Overall, our results demonstrate that the maturation/cleavage process targets the particle for an "off pathway" disassembly, because dissociation is coupled to unfolding. The increased motions in the cleaved capsid, revealed by fluorescence and NMR, and the concerted nature of dissociation/unfolding may be crucial to make the mature particle infectious. PMID- 10748192 TI - Signaling via the T cell antigen receptor induces phosphorylation of Stat1 on serine 727. AB - The Stat1 transcription factor plays a pivotal role in both, the antiviral and antigrowth actions of interferons. Stat1 acquires the ability to bind DNA by becoming phosphorylated on Tyr(701). However, to effectively stimulate gene transcription, it must also be phosphorylated on Ser(727). We show that engagement of T cell antigen receptor (TCR)/CD3 complex in either Jurkat cells or peripheral blood lymphocytes stimulates phosphorylation of Ser(727) but not Tyr(701) of Stat1. This process does not require the expression of tyrosine kinases Lck and Zap-70. Interestingly, pretreatment of T cells with the Src kinase inhibitor PP1 completely abrogated CD3-mediated serine phosphorylation of Stat1, whereas inhibitors to MEK1 and phosphatidylinositol 3-kinase had no effect. Phosphorylation of Ser(727) of Stat1 in T cells is not restricted to TCR/CD3 but also results when cells are stimulated via the costimulatory molecule CD28. The combination of CD3 and CD28 did not augment phosphorylation of Stat1 Ser(727). Surprisingly, Stat1-mediated transcriptional activity in response to IFN-alpha was enhanced with CD3 stimulation, whereas CD3 alone had little effect. These findings suggest that Stat1 is a signaling molecule in TCR signaling and may play a role in T cell function. PMID- 10748193 TI - A broad role for the zinc finger protein ZNF202 in human lipid metabolism. AB - The ZNF202 gene resides in a chromosomal region linked genetically to low high density lipoprotein cholesterol in Utah families. Here we show that the ZNF202 gene product is a transcriptional repressor that binds to elements found predominantly in genes that participate in lipid metabolism. Among its targets are structural components of lipoprotein particles (apolipoproteins AIV, CIII, and E), enzymes involved in lipid processing (lipoprotein lipase, lecithin cholesteryl ester transferase), and several genes involved in processes related to energy metabolism and vascular disease. Based on the linkage and apparent transcriptional function of ZNF202, we propose that ZNF202 is a candidate susceptibility gene for human dyslipidemia. PMID- 10748194 TI - Synaptic targeting of the postsynaptic density protein PSD-95 mediated by a tyrosine-based trafficking signal. AB - Synaptic function requires proper localization of proteins at synaptic sites. Targeting of the postsynaptic density protein 95 (PSD-95) relies on multiple signals within the protein, including twelve C-terminal amino acids. We now show that this C-terminal targeting domain of PSD-95 mediates postsynaptic localization through a short tyrosine-based motif followed by a pair of hydrophobic amino acids. Consistent with a role in cellular trafficking, the tyrosine motif resembles the canonical motif for interactions with clathrin adaptor proteins. In fact, we find that the C-terminal targeting domain of PSD-95 is sufficient to mediate clathrin-dependent endocytosis when appended to a transmembrane protein. Furthermore, systematic mutagenesis reveals that endocytosis mediated by this domain depends on both the tyrosine motif and the dihydrophobic amino acid pair. Thus, postsynaptic targeting of PSD-95 requires a tyrosine-based signal that can mediate clathrin-coated vesicle formation. PMID- 10748195 TI - Lack of obesity and normal response to fasting and thyroid hormone in mice lacking uncoupling protein-3. AB - Uncoupling protein-3 (UCP3) is a mitochondrial protein that can diminish the mitochondrial membrane potential. Levels of muscle Ucp3 mRNA are increased by thyroid hormone and fasting. Ucp3 has been proposed to influence metabolic efficiency and is a candidate obesity gene. We have produced a Ucp3 knockout mouse to test these hypotheses. The Ucp3 (-/-) mice had no detectable immunoreactive UCP3 by Western blotting. In mitochondria from the knockout mice, proton leak was greatly reduced in muscle, minimally reduced in brown fat, and not reduced at all in liver. These data suggest that UCP3 accounts for much of the proton leak in skeletal muscle. Despite the lack of UCP3, no consistent phenotypic abnormality was observed. The knockout mice were not obese and had normal serum insulin, triglyceride, and leptin levels, with a tendency toward reduced free fatty acids and glucose. Knockout mice showed a normal circadian rhythm in body temperature and motor activity and had normal body temperature responses to fasting, stress, thyroid hormone, and cold exposure. The base-line metabolic rate and respiratory exchange ratio were the same in knockout and control mice, as were the effects of fasting, a beta3-adrenergic agonist (CL316243), and thyroid hormone on these parameters. The phenotype of Ucp1/Ucp3 double knockout mice was indistinguishable from Ucp1 single knockout mice. These data suggest that Ucp3 is not a major determinant of metabolic rate but, rather, has other functions. PMID- 10748196 TI - Energy metabolism in uncoupling protein 3 gene knockout mice. AB - Uncoupling protein 3 (UCP3) is a member of the mitochondrial anion carrier superfamily. Based upon its high homology with UCP1 and its restricted tissue distribution to skeletal muscle and brown adipose tissue, UCP3 has been suggested to play important roles in regulating energy expenditure, body weight, and thermoregulation. Other postulated roles for UCP3 include regulation of fatty acid metabolism, adaptive responses to acute exercise and starvation, and prevention of reactive oxygen species (ROS) formation. To address these questions, we have generated mice lacking UCP3 (UCP3 knockout (KO) mice). Here, we provide evidence that skeletal muscle mitochondria lacking UCP3 are more coupled (i.e. increased state 3/state 4 ratio), indicating that UCP3 has uncoupling activity. In addition, production of ROS is increased in mitochondria lacking UCP3. This study demonstrates that UCP3 has uncoupling activity and that its absence may lead to increased production of ROS. Despite these effects on mitochondrial function, UCP3 does not seem to be required for body weight regulation, exercise tolerance, fatty acid oxidation, or cold-induced thermogenesis. The absence of such phenotypes in UCP3 KO mice could not be attributed to up-regulation of other UCP mRNAs. However, alternative compensatory mechanisms cannot be excluded. The consequence of increased mitochondrial coupling in UCP3 KO mice on metabolism and the possible role of yet unidentified compensatory mechanisms, remains to be determined. PMID- 10748197 TI - Identification of the cystic fibrosis transmembrane conductance regulator domains that are important for interactions with ROMK2. AB - In addition to functioning as a cAMP-activated chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR) plays an important role in conferring regulatory properties on other ion channels. It is known, with respect to CFTR regulation of ROMK2 (renally derived K(ATP) channel), that the first transmembrane domain and the first nucleotide binding fold domain (NBF1) of CFTR are necessary for this interaction to occur. It has been shown that under conditions that promote phosphorylation, the ROMK2-CFTR interaction is attenuated. To elucidate the complex nature of this interaction, CFTR constructs were co-expressed with ROMK2 in Xenopus oocytes, and two microelectrode voltage clamp experiments were performed. Although the second half of CFTR can act as a functional chloride channel, our results suggest that it does not confer glibenclamide sensitivity on ROMK2, as does the first half of CFTR. The attenuation of the ROMK2-CFTR interaction under conditions that promote phosphorylation is dependent on at least the presence of the R domain of CFTR. We conclude that transmembrane domain 1, NBF1, and the R domain are the CFTR domains involved in the ROMK2-CFTR interaction and that NBF2 and transmembrane domain 2 are not essential. Lastly, the R domain of CFTR is necessary for the attenuation of the ROMK2-CFTR interaction under conditions that promote phosphorylation. PMID- 10748198 TI - Transducin-like enhancer of split proteins, the human homologs of Drosophila groucho, interact with hepatic nuclear factor 3beta. AB - Members of the hepatic nuclear factor 3 (HNF3) family, including HNF3alpha, HNF3beta, and HNF3gamma, play important roles in embryonic development, the establishment of tissue-specific gene expression, and the regulation of gene expression in differentiated tissues. We found, using the glutathione S transferase pull-down method, that the transducin-like Enhancer of split (TLE) proteins, which are the human homologs of Drosophila Groucho, directly associate with HNF3beta. Conserved region II of HNF3beta (amino acids 361-388) is responsible for the interaction with TLE1. A mammalian two-hybrid assay was used to confirm that this interaction occurs in vivo. Overexpression of TLE1 in HepG2 and HeLa cells decreases transactivation mediated through the C-terminal domain of HNF3beta, and Grg5, a naturally occurring dominant negative form of Groucho/TLE, also increases the transcriptional activity of this region of HNF3. These results lead us to suggest that TLE proteins could influence the expression of mammalian genes regulated by HNF3. PMID- 10748199 TI - Regulation of membrane-type-1 matrix metalloproteinase activity by its cytoplasmic domain. AB - Membrane-type-1 matrix metalloproteinase (MT1-MMP) has transmembrane and cytoplasmic domains, which target it to invasive fronts. We analyzed the role of the cytoplasmic tail by expressing wild type MT1-MMP and three mutants with progressively truncated C termini in human Bowes melanoma cells. We examined gelatinase A activation and the localization and processing of recombinant proteins in stable cell clones using gelatin zymography, immunoblotting, and immunofluorescence. Cell invasion was analyzed in vitro by Matrigel invasion assays. Gelatinase A was activated in all cell clones. However, the localization of MT1-MMP to the leading edge of migrating cells and cell invasion through Matrigel were strongly enhanced only in cells expressing either wild type or truncated MT1-MMP lacking 6 C-terminal amino acid residues (Delta577). Truncations of 10 or 16 amino acid residues in the cytoplasmic domain (Delta567 and Delta573, respectively) disturbed MT1-MMP localization. The expression of wild type and Delta577 MT1-MMPs induced also their cleavage to 43-kDa cell surface forms and the release of soluble, approximately 20-kDa N-terminal fragments containing the catalytic center. A synthetic MMP inhibitor but not a gelatinase inhibitor prevented the processing, suggesting that autocatalytic cleavage occurs. Purified soluble MT1-MMP was also autoproteolytically processed to 43- and 20-kDa forms in vitro. Our results indicate that the cytoplasmic domain has an important role in cell invasion by controlling both the targeting and degradation/turnover of MT1-MMP. PMID- 10748200 TI - The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA. I. Increased functional expression of antibody fragments with and without cis prolines. AB - The production of recombinant proteins in the periplasm of Escherichia coli can be limited by folding problems, leading to periplasmic aggregates. We used a selection system for periplasmic chaperones based on the coexpression of an E. coli library with a poorly expressing antibody single-chain Fv (scFv) fragment displayed on filamentous phage (Bothmann, H., and Pluckthun, A. (1998) Nature Biotechnol. 16, 376-380). By selection for a functional antibody, the protein Skp had been enriched previously and shown to improve periplasmic expression of a wide range of scFv fragments. This selection strategy was now repeated with a library constructed from the genomic DNA of an skp-deficient strain, leading to enrichment of the periplasmic peptidylprolyl cis,trans-isomerase (PPIase) FkpA. Coexpression of FkpA increased the amount of fusion protein displayed on the phage and dramatically improved functional periplasmic expression even of scFv fragments not containing cis-prolines. In contrast, the coexpression of the periplasmic PPIases PpiA and SurA showed no increase in the functional scFv fragment level in the periplasm or displayed on phage. Together with the in vitro data in the accompanying paper (Ramm, K., and Pluckthun, A. (2000) J. Biol. Chem. 275, 17106-17113), we conclude that the effect of FkpA is independent of its PPIase activity. PMID- 10748201 TI - The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA. II. Isomerase-independent chaperone activity in vitro. AB - We recently identified FkpA by selecting for the increased yield of antibody single-chain Fv (scFv) fragments in phage display, even of those not containing cis-prolines. We have now investigated the properties of FkpA in vitro. The peptidylprolyl cis-trans-isomerase activity of FkpA was found to be among the highest of any such enzyme with a protein substrate, yet FkpA is not able to enhance the proline-limited refolding rate of the disulfide-free hu4D5-8 scFv fragment, probably due to inaccessibility of Pro-L95. Nevertheless, the yield of the soluble and functional scFv fragment was dramatically increased in vitro in the presence of FkpA. Similar effects were observed for an scFv fragment devoid of cis-prolines. We are thus forced to conclude that the observed folding assisting function is independent of the isomerase activity of the protein. The beneficial effect of FkpA was found to be due to two components. First, FkpA interacts with early folding intermediates, thus preventing their aggregation. Additionally, it has the ability to reactivate inactive protein, possibly also by binding to a partially unfolded species that may exist in equilibrium with the aggregated form, which may thus be released on a productive pathway. These in vitro measurements therefore fully reflect the in vivo results from periplasmic overexpression of FkpA. PMID- 10748202 TI - Wnt1 and MEK1 cooperate to promote cyclin D1 accumulation and cellular transformation. AB - Members of the Wnt family of signal transducers regulate cellular differentiation/reorganization and cellular proliferation. However, few pro proliferative targets of Wnt have been identified. We now show that cyclin D1, a critical mediator of cell cycle progression, is a downstream target of Wnt dependent signaling. NIH-3T3 cell lines engineered to overexpress Wnt1 displayed reduced glycogen synthase kinase-3beta activity. Wnt1-dependent glycogen synthase kinase-3beta inhibition corresponded with decreased cyclin D1 proteolysis and, thus, hyperaccumulation of active cyclin D1.CDK4 (cyclin-dependent kinase 4) kinase. However, in the absence of serum-derived growth factors, Wnt-1 was not sufficient to drive cyclin D1 accumulation or S-phase entry. In contrast, cells engineered to co-express Wnt1 and activated MEK1 accumulated high levels of cyclin D1 and entered the DNA synthetic phase in the absence of serum-derived growth factors, a characteristic of neoplastic transformation. The ability of a dominant-negative cyclin D1 mutant, D1-T156A, to inhibit Wnt1/MEK1-dependent S phase entry suggests that cyclin D1 is a critical downstream target for Wnt1- and MEK1-dependent cellular proliferation. PMID- 10748203 TI - Functional interactions among yeast Rad51 recombinase, Rad52 mediator, and replication protein A in DNA strand exchange. AB - Rad51-catalyzed DNA strand exchange is greatly enhanced by the single-stranded (ss) DNA binding factor RPA if the latter is introduced after Rad51 has already nucleated onto the initiating ssDNA substrate. Paradoxically, co-addition of RPA with Rad51 to the ssDNA to mimic the in vivo situation diminishes the level of strand exchange, revealing competition between RPA and Rad51 for binding sites on ssDNA. Rad52 promotes strand exchange but only when there is a need for Rad51 to compete with RPA for loading onto ssDNA. Rad52 is multimeric, binds ssDNA, and targets Rad51 to ssDNA. Maximal restoration of pairing and strand exchange requires amounts of Rad52 substoichiometric to Rad51 and involves a stable, equimolar complex between Rad51 and Rad52. The Rad51-Rad52 complex efficiently utilizes a ssDNA template saturated with RPA for homologous pairing but does not appear to be more active than Rad51 when an RPA-free ssDNA template is used. Rad52 does not substitute for RPA in the pairing and strand exchange reaction nor does it lower the dependence of the reaction on Rad51 or RPA. PMID- 10748204 TI - The MEF2A isoform is required for striated muscle-specific expression of the insulin-responsive GLUT4 glucose transporter. AB - Previously, we have demonstrated that an MEF2 consensus sequence located between 473/-464 in the human GLUT4 gene was essential for both tissue-specific and hormonal/metabolic regulation of GLUT4 expression (Thai, M. V., Guruswamy, S., Cao, K. T., Pessin, J. E., and Olson, A. L. (1998) J. Biol. Chem. 273, 14285 14292). To identify the specific MEF2 isoform(s) responsible for GLUT4 expression, we studied the pattern of expression of the MEF2 isoforms in insulin sensitive tissues. Both heart and skeletal muscle were found to express the MEF2A, MEF2C, and MEF2D isoforms but not MEF2B. However, only the MEF2A protein was selectively down-regulated in insulin-deficient diabetes. Co immunoprecipitation with isoform-specific antibodies revealed that, in the basal state, essentially all of the MEF2A protein was presented as a MEF2A-MEF2D heterodimer without any detectable MEF2A-MEF2A homodimers or MEF2A-MEF2C and MEF2C-MEF2D heterodimers. Electrophoretic mobility shift assays revealed that nuclear extracts from diabetic animals had reduced binding to the MEF2 binding site compared with extracts from control or insulin-treated animals. Furthermore, immunodepletion of the MEF2A-MEF2D complex from control extracts abolished binding to the MEF2 element. However, addition of MEF2A to diabetic nuclear extracts fully restored binding activity to the MEF2 element. These data strongly suggest that the MEF2A-MEF2D heterodimer is selectively decreased in insulin deficient diabetes and is responsible for hormonally regulated expression of the GLUT4 gene. PMID- 10748205 TI - The C/EBP bZIP domain can mediate lipopolysaccharide induction of the proinflammatory cytokines interleukin-6 and monocyte chemoattractant protein-1. AB - C/EBPalpha, beta, and delta are all expressed by bone marrow-derived macrophages. Ectopic expression of any of these transcription factors is sufficient to confer lipopolysaccharide (LPS)-inducible expression of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) to a B lymphoblast cell line, which normally lacks C/EBP factors and does not display LPS induction of proinflammatory cytokines. Thus, the activities of C/EBPalpha, beta, and delta are redundant in regard to expression of IL-6 and MCP-1. Surprisingly, the bZIP region of C/EBPbeta, which lacks any previously described activation domains, can also confer LPS-inducible expression of IL-6 and MCP-1 in stable transfectants. Transient transfections reveal that the bZIP regions of C/EBPbeta, C/EBPdelta, and, to a lesser extent, C/EBPalpha can activate the IL-6 promoter and augment its induction by LPS. Furthermore, the transdominant inhibitor, LIP, can activate expression from the IL-6 promoter. The ability of the C/EBPbeta bZIP region to activate the IL-6 promoter in transient transfections is completely dependent upon an intact NF-kappaB-binding site, supporting a model where the bZIP protein primarily functions to augment the activity of NF-kappaB. Replacement of the leucine zipper of C/EBPbeta with that of GCN4 yields a chimeric protein that can dimerize and specifically bind to a C/EBP consensus sequence, but shows a markedly reduced ability to activate IL-6 and MCP-1 expression. These results implicate the leucine zipper domain in some function other than dimerization with known C/EBP family members, and suggest that C/EBP redundancy in regulating cytokine expression may result from their highly related bZIP regions. PMID- 10748207 TI - An open conformation of switch I revealed by the crystal structure of a Mg2+-free form of RHOA complexed with GDP. Implications for the GDP/GTP exchange mechanism. AB - Mg(2+) ions are essential for guanosine triphosphatase (GTPase) activity and play key roles in guanine nucleotide binding and preserving the structural integrity of GTP-binding proteins. We determined the crystal structure of a small GTPase RHOA complexed with GDP in the absence of Mg(2+) at 2.0-A resolution. Elimination of a Mg(2+) ion induces significant conformational changes in the switch I region that opens up the nucleotide-binding site. Similar structural changes have been observed in the switch regions of Ha-Ras bound to its guanine nucleotide exchange factor, Sos. This RHOA-GDP structure reveals an important regulatory role for Mg(2+) and suggests that guanine nucleotide exchange factor may utilize this feature of switch I to produce an open conformation in GDP/GTP exchange. PMID- 10748206 TI - Residue 259 is a key determinant of substrate specificity of protein-tyrosine phosphatases 1B and alpha. AB - The aim of this study was to define the structural elements that determine the differences in substrate recognition capacity of two protein-tyrosine phosphatases (PTPs), PTP1B and PTPalpha, both suggested to be negative regulators of insulin signaling. Since the Ac-DADE(pY)L-NH(2) peptide is well recognized by PTP1B, but less efficiently by PTPalpha, it was chosen as a tool for these analyses. Calpha regiovariation analyses and primary sequence alignments indicate that residues 47, 48, 258, and 259 (PTP1B numbering) define a selectivity determining region. By analyzing a set of DADE(pY)L analogs with a series of PTP mutants in which these four residues were exchanged between PTP1B and PTPalpha, either in combination or alone, we here demonstrate that the key selectivity determining residue is 259. In PTPalpha, this residue is a glutamine causing steric hindrance and in PTP1B a glycine allowing broad substrate recognition. Significantly, replacing Gln(259) with a glycine almost turns PTPalpha into a PTP1B-like enzyme. By using a novel set of PTP inhibitors and x-ray crystallography, we further provide evidence that Gln(259) in PTPalpha plays a dual role leading to restricted substrate recognition (directly via steric hindrance) and reduced catalytic activity (indirectly via Gln(262)). Both effects may indicate that PTPalpha regulates highly selective signal transduction processes. PMID- 10748208 TI - Fidelity of eucaryotic DNA polymerase delta holoenzyme from Schizosaccharomyces pombe. AB - The fidelity of Schizosaccharomyces pombe DNA polymerase delta was measured in the presence or absence of its processivity subunits, proliferating cell nuclear antigen (PCNA) sliding clamp and replication factor C (RFC) clamp-loading complex, using a synthetic 30-mer primer/100-mer template. Synthesis by pol delta alone was distributive. Processive synthesis occurred in the presence of PCNA, RFC, and Escherichia coli single strand DNA-binding protein (SSB) and required the presence of ATP. "Passive" self-loading of PCNA onto DNA takes place in the absence of RFC, in an ATP-independent reaction, which was strongly inhibited by SSB. The nucleotide substitution error rate for pol delta holoenzyme (HE) (pol delta + PCNA + RFC) was 4.6 x 10(-4) for T.G mispairs, 5.3 x 10(-5) for G.G mispairs, and 4.5 x 10(-6) for A.G mispairs. The T.G misincorporation frequency for pol delta without the accessory proteins was unchanged. The fidelity of pol delta HE was between 1 and 2 orders of magnitude lower than that measured for the E. coli pol III HE at the same template position. This relatively low fidelity was caused by inefficient proofreading by the S. pombe polymerase-associated proofreading exonuclease. The S. pombe 3'-exonuclease activity was also extremely inefficient in excising primer-3'-terminal mismatches in the absence of dNTP substrates and in hydrolyzing single-stranded DNA. A comparison of pol delta HE with E. coli pol IIIalpha HE (lacking the proofreading exonuclease subunit) showed that both holoenzymes exhibit similar error rates for each mispair. PMID- 10748209 TI - Individual subunits of the eukaryotic cytosolic chaperonin mediate interactions with binding sites located on subdomains of beta-actin. AB - The chaperonin containing TCP-1 (CCT) of eukaryotic cytosol is composed of eight different subunit species that are proposed to have independent functions in folding its in vivo substrates, the actins and tubulins. CCT has been loaded with (35)S-beta-actin by in vitro translation in reticulocyte lysate and then subjected to immunoprecipitation with all eight anti-CCT subunit antibodies in mixed micelle buffers, conditions that disrupt CCT into its constituent monomers. Interactions between (35)S-beta-actin and isolated CCTalpha, CCTbeta, CCTepsilon, or CCTtheta subunits are observed, suggesting that polar and electrostatic interactions may mediate actin binding to these four CCT subunits. Additionally, a beta-actin peptide array was screened for CCT-binding sequences. Three regions rich in charged and polar amino acid residues, which map to the surface of native beta-actin, are implicated in interactions between actin and CCT. Several of these biochemical results are consistent with the recent cryo-electron microscopy three-dimensional structure of apo-CCT-alpha-actin, in which alpha-actin is bound by the apical domains of specific CCT subunits. A model is proposed in which actin interacts with several CCT subunits during its CCT-mediated folding cycle. PMID- 10748210 TI - Interaction between actin and the effector peptide of MARCKS-related protein. Identification of functional amino acid segments. AB - It is widely assumed that the members of the MARCKS protein family, MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP), interact with actin via their effector domain, a highly basic segment composed of 24-25 amino acid residues. To clarify the mechanisms by which this interaction takes place, we have examined the effect of a peptide corresponding to the effector domain of MRP, the so-called effector peptide, on both the dynamic and the structural properties of actin. We show that in the absence of cations the effector peptide polymerizes monomeric actin and causes the alignment of the formed filaments into bundle-like structures. Moreover, we document that binding of calmodulin or phosphorylation by protein kinase C both inhibit the actin polymerizing activity of the MRP effector peptide. Finally, several effector peptides were synthesized in which positively charged or hydrophobic segments were deleted or replaced by alanines. Our data suggest that a group of six positively charged amino acid residues at the N-terminus of the peptide is crucial for its interaction with actin. While its actin polymerizing activity critically depends on the presence of all three positively charged segments of the peptide, hydrophobic amino acid residues rather modulate the polymerization velocity. PMID- 10748211 TI - Purification of the novel endonuclease, Hpy188I, and cloning of its restriction modification genes reveal evidence of its horizontal transfer to the Helicobacter pylori genome. AB - We have isolated a novel restriction endonuclease, Hpy188I, from Helicobacter pylori strain J188. Hpy188I recognizes the unique sequence, TCNGA, and cleaves the DNA between nucleotides N and G in its recognition sequence to generate a one base 3' overhang. Cloning and sequence analysis of the Hpy188I modification gene in strain J188 reveal that hpy188IM has a 1299-base pair (bp) open reading frame (ORF) encoding a 432-amino acid product. The predicted protein sequence of M.Hpy188I contains conserved motifs typical of aminomethyltransferases, and Western blotting indicates that it is an N-6 adenine methyltransferase. Downstream of hpy188IM is a 513-bp ORF encoding a 170-amino acid product, that has a 41-bp overlap with hpy188IM. The predicted protein sequence from this ORF matches the amino acid sequence obtained from purified Hpy188I, indicating that it encodes the endonuclease. The Hpy188I R-M genes are not present in either strain of H. pylori that has been completely sequenced but are found in two of 11 H. pylori strains tested. The significantly lower G + C content of the Hpy188I R M genes implies that they have been introduced relatively recently during the evolution of the H. pylori genome. PMID- 10748212 TI - Identification and characterization of the iron regulatory element in the ferritin gene of a plant (soybean). AB - Iron increases ferritin synthesis, targeting plant DNA and animal mRNA. The ferritin promoter in plants has not been identified, in contrast to the ferritin promoter and mRNA iron-responsive element (IRE) in animals. The soybean leaf, a natural tissue for ferritin expression, and DNA, with promoter deletions and luciferase or glucuronidase reporters, delivered with particle bombardment, were used to show that an 86-base pair fragment (iron regulatory element (FRE)) controlled iron-mediated derepression of the ferritin gene. Mutagenesis with linkers of random sequence detected two subdomains separated by 21 base pairs. FRE has no detectable homology to the animal IRE or to known promoters in DNA and bound a trans-acting factor in leaf cell extracts. FRE/factor binding was abrogated by increased tissue iron, in analogy to mRNA (IRE)/iron regulatory protein in animals. Maximum ferritin derepression was obtained with 50 microm iron citrate (1:10) or 500 microm iron citrate (1:1) but Fe-EDTA was ineffective, although the leaf iron concentration was increased; manganese, zinc, and copper had no effect. The basis for different responses in ferritin expression to different iron complexes, as well as the significance of using DNA but not mRNA as an iron regulatory target in plants, remain unknown. PMID- 10748213 TI - Identification of a novel function of the alphavirus capping apparatus. RNA 5' triphosphatase activity of Nsp2. AB - Both genomic and subgenomic RNAs of the Alphavirus have m(7)G(5')ppp(5')N (cap0 structure) at their 5' end. Previously it has been shown that Alphavirus-specific nonstructural protein Nsp1 has guanine-7N-methyltransferase and guanylyltransferase activities needed in the synthesis of the cap structure. During normal cap synthesis the 5' gamma-phosphate of the nascent viral RNA chain is removed by a specific RNA 5'-triphosphatase before condensation with GMP, delivered by the guanylyltransferase. Using a novel RNA triphosphatase assay, we show here that nonstructural protein Nsp2 (799 amino acids) of Semliki Forest virus specifically cleaves the gamma,beta-triphosphate bond at the 5' end of RNA. The same activity was demonstrated for Nsp2 of Sindbis virus, as well as for the amino-terminal fragment of Semliki Forest virus Nsp2-N (residues 1-470). The carboxyl-terminal part of Semliki Forest virus Nsp2-C (residues 471-799) had no RNA triphosphatase activity. Replacement of Lys-192 by Asn in the nucleotide binding site completely abolished RNA triphosphatase and nucleoside triphosphatase activities of Semliki Forest virus Nsp2 and Nsp2-N. Here we provide biochemical characterization of the newly found function of Nsp2 and discuss the unique properties of the entire Alphavirus-capping apparatus. PMID- 10748214 TI - Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein-coupled receptors delineate two major classes of receptors. AB - Visual arrestin, betaarrestin1, and betaarrestin2 comprise a family of intracellular proteins that desensitize G protein-coupled receptors (GPCRs). In addition, betaarrestin1 and betaarrestin2 target desensitized receptors to clathrin-coated pits for endocytosis. Whether arrestins differ in their ability to interact with GPCRs in cells is not known. In this study, we visualize the interaction of arrestin family members with GPCRs in real time and in live cells using green fluorescent protein-tagged arrestins. In the absence of agonist, visual arrestin and betaarrestin1 were found in both the cytoplasm and nucleus of HEK-293 cells, whereas betaarrestin2 was found only in the cytoplasm. Analysis of agonist-mediated arrestin translocation to multiple GPCRs identified two major classes of receptors. Class A receptors (beta2 adrenergic receptor, mu opioid receptor, endothelin type A receptor, dopamine D1A receptor, and alpha1b adrenergic receptor) bound betaarrestin2 with higher affinity than betaarrestin1 and did not interact with visual arrestin. In contrast, class B receptors (angiotensin II type 1A receptor, neurotensin receptor 1, vasopressin V2 receptor, thyrotropin-releasing hormone receptor, and substance P receptor) bound both betaarrestin isoforms with similar high affinities and also interacted with visual arrestin. Switching the carboxyl-terminal tails of class A and class B receptors completely reversed the affinity of each receptor for the visual and non-visual arrestins. In addition, exchanging the betaarrestin1 and betaarrestin2 carboxyl termini reversed their extent of binding to class A receptors as well as their subcellular distribution. These results reveal for the first time marked differences in the ability of arrestin family members to bind GPCRs at the plasma membrane. Moreover, they show that visual arrestin can interact in cells with GPCRs other than rhodopsin. These findings suggest that GPCR signaling may be differentially regulated depending on the cellular complement of arrestin isoforms and the ability of arrestins to interact with other cellular proteins. PMID- 10748215 TI - Coupling ribose selection to fidelity of DNA synthesis. The role of Tyr-115 of human immunodeficiency virus type 1 reverse transcriptase. AB - The catalytic efficiency of incorporation of deoxyribonucleotides by wild-type human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) was around 100-fold higher than for dideoxyribonucleotides, in Mg(2+)-catalyzed reactions, and more than 10,000-fold higher than for nucleotides having a 2'-hydroxyl group in Mg(2+)- and Mn(2+)-catalyzed reactions. Mutant RTs with nonconservative substitutions affecting Tyr-115 (Y115V, Y115A, and Y115G) showed a dramatic reduction in their ability to discriminate against ribonucleotides in the presence of both cations. However, selectivity of deoxyribonucleotides versus ribonucleotides was not affected in mutants Y115W and F160W. The substitution of Tyr-115 with Val or Gly had no effect on discrimination against dideoxyribonucleotides, but these mutants were less efficient than the wild-type RT in discriminating against cordycepin 5'-triphosphate. We also show that Tyr 115 is involved in fidelity of DNA synthesis, but substitutions at this position have different effects depending on the metal cofactor used in the assays. In Mg(2+)-catalyzed reactions, removal of the side chain of Tyr-115 reduced misinsertion and mispair extension fidelity, while opposite effects were observed in Mn(2+)-catalyzed reactions. Our results indicate that the aromatic side chain of Tyr-115 plays a role as a "steric gate" preventing the incorporation of nucleotides with a 2'-hydroxyl group in a cation-independent manner, while its influence on fidelity could be modulated by Mg(2+) or Mn(2+). PMID- 10748216 TI - Recognition of RhoA by Clostridium botulinum C3 exoenzyme. AB - The C3-like ADP-ribosyltransferases exhibit a very confined substrate specificity compared with other Rho-modifying bacterial toxins; they selectively modify the RhoA, -B, and -C isoforms but not other members of the Rho or Ras subfamilies. In this study, the amino acid residues involved in the RhoA substrate recognition by C3 from Clostridium botulinum are identified by applying mutational analyses of the nonsubstrate Rac. First, the minimum domain responsible for the recognition by C3 was identified as the N-terminal 90 residues. Second, the combination of the N-terminal basic amino acids ((Rho)Arg(5)-Lys(6)), the acid residues (Rho)Glu(47) and (Rho)Glu(54) only slightly increases ADP-ribosylation but fully restores the binding of the respective mutant Rac to C3. Third, the residues (Rho)Glu(40) and (Rho)Val(43) also participate in binding to C3 but they are mainly involved in the correct formation of the ternary complex between Rho, C3, and NAD(+). Thus, these six residues (Arg(5), Lys(6), Glu(40), Val(43), Glu(47), and Glu(54)) distributed over the N-terminal part of Rho are involved in the correct binding of Rho to C3. Mutant Rac harboring these residues shows a kinetic property with regard to ADP-ribosylation, which is identical with that of RhoA. Differences in the conformation of Rho given by the nucleotide occupancy have only minor effects on ADP-ribosylation. PMID- 10748217 TI - Combined serum paraoxonase knockout/apolipoprotein E knockout mice exhibit increased lipoprotein oxidation and atherosclerosis. AB - Serum paraoxonase (PON1), present on high density lipoprotein, may inhibit low density lipoprotein (LDL) oxidation and protect against atherosclerosis. We generated combined PON1 knockout (KO)/apolipoprotein E (apoE) KO and apoE KO control mice to compare atherogenesis and lipoprotein oxidation. Early lesions were examined in 3-month-old mice fed a chow diet, and advanced lesions were examined in 6-month-old mice fed a high fat diet. In both cases, the PON1 KO/apoE KO mice exhibited significantly more atherosclerosis (50-71% increase) than controls. We examined LDL oxidation and clearance in vivo by injecting human LDL into the mice and following its turnover. LDL clearance was faster in the double KO mice as compared with controls. There was a greater rate of accumulation of oxidized phospholipid epitopes and a greater accumulation of LDL-immunoglobulin complexes in the double KO mice than in controls. Furthermore, the amounts of three bioactive oxidized phospholipids were elevated in the endogenous intermediate density lipoprotein/LDL of double KO mice as compared with the controls. Finally, the expression of heme oxygenase-1, peroxisome proliferator activated receptor gamma, and oxidized LDL receptors were elevated in the livers of double KO mice as compared with the controls. These data demonstrate that PON1 deficiency promotes LDL oxidation and atherogenesis in apoE KO mice. PMID- 10748218 TI - Structural and functional characterization of interaction between hepatitis B virus X protein and the proteasome complex. AB - Hepatitis B virus (HBV) has a unique fourth open reading frame coding for a 16.5 kDa protein known as hepatitis B virus X protein (HBX). The importance of HBX in the life cycle of HBV has been well established, but the underlying molecular function of HBX remains controversial. We previously identified a proteasome subunit PSMA7 that interacts specifically with HBX in the Saccharomyces cerevisiae two-hybrid system. Here we demonstrate that PSMC1, an ATPase-like subunit of the 19 S proteasome component, also interacts with HBX and PSMA7. Analysis of the interacting domains among PSMA7, PSMC1, and HBX by deletion and site-directed mutagenesis suggested a mutually competitive structural relationship among these polypeptides. The competitive nature of these interactions is further demonstrated using a modified yeast two-hybrid dissociator system. The crucial HBX sequences involved in interaction with PSMA7 and PSMC1 are important for its function as a transcriptional coactivator. HBX, while functioning as a coactivator of AP-1 and acidic activator VP-16 in mammalian cells, had no effect on the transactivation function of their functional orthologs GCN4 and Gal4 in yeast. Overexpression of PSMC1 seemed to suppress the expression of various reporters in mammalian cells; this effect, however, was overcome by coexpression of HBX. In addition, HBX expression inhibited the cellular turnover of c-Jun and ubiquitin-Arg-beta-galactosidase, two well known substrates of the ubiquitin-proteasome pathway. Thus, interaction of HBX with the proteasome complex in metazoan cells may underlie the functional basis of proteasome as a cellular target of HBX. PMID- 10748219 TI - Kinetic investigation of the specificity of porcine brain thyrotropin-releasing hormone-degrading ectoenzyme for thyrotropin-releasing hormone-like peptides. AB - Evidence indicates that neuronally released thyrotropin-releasing hormone (TRH) is selectively inactivated by TRH-degrading ectoenzyme (TRH-DE) (EC ). TRH-DE inhibitors may be used to enhance the therapeutic actions of TRH and to investigate the functions of TRH and TRH-DE in the central nervous system. Although TRH-DE appears to exhibit a high degree of specificity toward TRH, systematic specificity studies, which would facilitate inhibitor design, have not been previously conducted for this enzyme. In this paper we present the first description of TRH-DE specificity across a directed peptide library in which the histidyl (P(1)') residue of TRH was replaced by a series of amino acids. Peptides were synthesized using standard solid phase chemistry. Kinetic parameters were measured either by continuous or discontinuous fluorometric assays or by quantitative high pressure liquid chromatography. The P(1)' residue was found to influence significantly both the ability of the peptides to bind to TRH-DE, as measured by their K(i) values, and the ability of TRH-DE to catalyze their hydrolysis. Moderately bulky, uncharged P(1)' residues were found to bind preferentially to TRH-DE. Results from this screen provide valuable information for the development of TRH-DE inhibitors and have led to the identification of two potent, reversible TRH-DE inhibitors, l-pyroglutamyl-l-asparaginyl-l prolineamide (K(i) = 17.5 micrometer) and Glp-Asn-Pro-7-amido-4-methyl coumarin (K(i) = 0.97 micrometer). PMID- 10748220 TI - p110beta is up-regulated during differentiation of 3T3-L1 cells and contributes to the highly insulin-responsive glucose transport activity. AB - Activation of p85/p110 type phosphatidylinositol kinase is essential for aspects of insulin-induced glucose metabolism, including translocation of GLUT4 to the cell surface and glycogen synthesis. The enzyme exists as a heterodimer containing a regulatory subunit (e.g. p85alpha) and one of two widely distributed isoforms of the p110 catalytic subunit: p110alpha or p110beta. In the present study, we compared the two isoforms in the regulation of insulin action. During differentiation of 3T3-L1 cells into adipocytes, p110beta was up-regulated approximately 10-fold, whereas expression of p110alpha was unaltered. The effects of the increased p110 expression were further assessed by expressing epitope tagged p110beta and p110alpha in 3T3-L1 cells using adenovirus transduction systems, respectively. In vitro, the basal lipid kinase activity of p110beta was lower than that of p110alpha. When p110alpha and p110beta were overexpressed in 3T3-L1 adipocytes, exposing cells to insulin induced each of the subunits to form complexes with p85alpha and tyrosine-phosphorylated IRS-1 with similar efficiency. However, whereas the kinase activity of p110beta, either endogenous or exogeneous, was markedly enhanced by insulin stimulation, only very small increases of the activity of p110alpha were observed. Interestingly, overexpression of p110beta increased insulin-induced glucose uptake by 3T3-L1 cells without significantly affecting basal glucose transport, whereas overexpression of p110alpha increased both basal and insulin-stimulated glucose uptake. Finally, microinjection of anti-p110beta neutralizing antibody into 3T3 L1 adipocytes abolished insulin-induced translocation of GLUT4 to the cell surface almost completely, whereas anti-p110alpha neutralizing antibody did only slightly. Together, these findings suggest that p110beta plays a crucial role in cellular activities evoked acutely by insulin. PMID- 10748221 TI - The transactivation domain within cysteine/histidine-rich region 1 of CBP comprises two novel zinc-binding modules. AB - cAMP-response element-binding protein-binding protein (CBP) is a transcriptional coactivator that interacts with a number of DNA-binding proteins and cofactor proteins involved in the regulation of transcription. Relatively little is known about the structure of CBP, but it has been noted that it contains three domains that are rich in cysteine and histidine (CH1, CH2, and CH3). The sequence of CH2 conforms to that of a leukemia-associated protein domain (PHD finger), and it has been postulated that this and both CH1 and CH3 may be zinc finger domains. This has not, however, been demonstrated experimentally. We have studied CH1 and show that it is composed of two novel zinc-binding modules, which we term "zinc bundles." Each bundle contains the sequence Cys-X(4)-Cys-X(8)-His-X(3)-Cys, and we show that a synthetic peptide comprising one zinc bundle from CH1 can fold in a zinc-dependent manner. CH3 also appears to contain two zinc bundles, one with the variant sequence Cys-X(2)-Cys-X(9)-His-X(3)-Cys, and we demonstrate that this variant motif also undergoes Zn(II)-induced folding. CH1 acts as a transcriptional activation domain in cellular assays. We show that mutations in any of the four zinc-chelating residues in either zinc bundle of CH1 significantly impair this activity and that these mutations also interfere with certain protein-protein interactions mediated by CH1. Our results indicate that CBP is a genuine zinc-binding protein and introduce zinc bundles as novel protein interaction domains. PMID- 10748222 TI - Nerve growth factor protects oligodendrocytes from tumor necrosis factor-alpha induced injury through Akt-mediated signaling mechanisms. AB - Tumor necrosis factor-alpha is thought to be one of the most important inflammatory cytokines associated with the demyelinating disease multiple sclerosis. We determined whether neurotrophins could protect oligodendrocytes from tumor necrosis factor-alpha-mediated cytotoxicity. Among the neurotrophins tested, nerve growth factor was most effective at preventing cell death. Nerve growth factor also prevented the tumor necrosis factor-induced loss of mitochondrial membrane potential. Overexpression of constitutively active Akt, a downstream target of phosphatidylinositol 3-kinase, but not of constitutively active MEK, protected oligodendrocytes from tumor necrosis factor-induced injury. Moreover, overexpression of dominant-negative Akt negated the protective effects of nerve growth factor on tumor necrosis factor-mediated oligodendrocyte cytotoxicity. These findings indicate that the Akt pathway is crucial in nerve growth factor-mediated oligodendrocyte protection. PMID- 10748223 TI - Tandem arrangement of the clathrin and AP-2 binding domains in amphiphysin 1 and disruption of clathrin coat function by amphiphysin fragments comprising these sites. AB - Amphiphysin 1 and 2 are proteins implicated in the recycling of synaptic vesicles in nerve terminals. They interact with dynamin and synaptojanin via their COOH terminal SH3 domain, whereas their central regions contain binding sites for clathrin and for the clathrin adaptor AP-2. We have defined here amino acids of amphiphysin 1 crucial for binding to AP-2 and clathrin. Overexpression in Chinese hamster ovary cells of an amphiphysin 1 fragment that binds both AP-2 and clathrin resulted in a segregation of clathrin, which acquired a diffuse distribution, from AP-2, which accumulated at patches also positive for Eps15. These effects correlated with a block in clathrin-mediated endocytosis. A fragment selectively interacting with clathrin produced a similar effect. These results can be explained by the binding of amphiphysin to the NH(2)-terminal domain of clathrin and by a competition with the binding of this domain to the beta-subunit of AP-2 and AP180. The interaction of amphiphysin 1 with either clathrin or AP-2 did not prevent its interaction with dynamin, supporting the existence of tertiary complexes between these proteins. Together with previous evidence indicating a direct interaction between amphiphysin and membrane lipids, these findings support a model in which amphiphysin acts as a multifunctional adaptor linking the membrane to coat proteins and coat proteins to dynamin and synaptojanin. PMID- 10748224 TI - Functional characterization of yeast mitochondrial release factor 1. AB - The yeast Saccharomyces cerevisiae mitochondrial release factor was expressed from the cloned MRF1 gene, purified from inclusion bodies, and refolded to give functional activity. The gene encoded a factor with release activity that recognized cognate stop codons in a termination assay with mitochondrial ribosomes and in an assay with Escherichia coli ribosomes. The noncognate stop codon, UGA, encoding tryptophan in mitochondria, was recognized weakly in the heterologous assay. The mitochondrial release factor 1 protein bound to bacterial ribosomes and formed a cross-link with the stop codon within a mRNA bound in a termination complex. The affinity was strongly dependent on the identity of stop signal. Two alleles of MRF1 that contained point mutations in a release factor 1 specific region of the primary structure and that in vivo compensated for mutations in the decoding site rRNA of mitochondrial ribosomes were cloned, and the expressed proteins were purified and refolded. The variant proteins showed impaired binding to the ribosome compared with mitochondrial release factor 1. This structural region in release factors is likely to be involved in codon dependent specific ribosomal interactions. PMID- 10748225 TI - The aconitase function of iron regulatory protein 1. Genetic studies in yeast implicate its role in iron-mediated redox regulation. AB - Iron regulatory proteins (IRP) are sequence-specific RNA-binding proteins that mediate iron-responsive gene regulation in animals. IRP1 is also the cytosolic isoform of aconitase (c-aconitase). This latter activity could complement a mitochondrial aconitase mutation (aco1) in Saccharomyces cerevisiae to restore glutamate prototrophy. In yeast, the c-aconitase activity of IRP1 was responsive to iron availability in the growth medium. Although IRP1 expression rescued aco1 yeast from glutamate auxotrophy, cells remained growth-limited by glutamate, displaying a slow-growth phenotype on glutamate-free media. Second site mutations conferring enhanced cytosolic aconitase-dependent (ECA) growth were recovered. Relative c-aconitase activity was increased in extracts of strains harboring these mutations. One of the ECA mutations was found to be in the gene encoding cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDP2). This mutation, an insertion of a Ty delta element into the 5' region of IDP2, markedly elevates expression of Idp2p in glucose media. Our results demonstrate the physiological significance of the aconitase activity of IRP1 and provide insight into the role of c-aconitase with respect to iron and cytoplasmic redox regulation. PMID- 10748226 TI - Choline release and inhibition of phosphatidylcholine synthesis precede excitotoxic neuronal death but not neurotoxicity induced by serum deprivation. AB - N-Methyl-d-aspartate (NMDA) receptor overactivation has been proposed to induce excitotoxic neuronal death by enhancing membrane phospholipid degradation. In previous studies, we have shown that NMDA releases choline and reduces membrane phosphatidylcholine in vivo. We now observed that glutamate and NMDA induce choline release in primary neuronal cortical cell cultures. This effect is Ca(2+) dependent and is blocked by MK-801 ((+)-5-methyl-10, 11-dihydro-5H dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate). In cortical neurons, the NMDA receptor-mediated choline release precedes excitotoxic cell death but not neuronal death induced by either osmotic lysis or serum deprivation. Glutamate, at concentrations that release arachidonic acid, does not release choline in cerebellar granule cells, unless these cells are rendered susceptible to excitotoxic death by energy deprivation. The NMDA-evoked release of choline is not mediated by phospholipases A(2) or C. Moreover, NMDA does not activate phospholipase D in cortical cells. However, NMDA inhibits incorporation of [methyl-(3)H]choline into both membrane phosphatidylcholine and sphingomyelin. These results show that the increase in extracellular choline induced by NMDA receptor activation is directly related with excitotoxic cell death and indicate that choline release is an early event of the excitotoxic process produced by inhibition of phosphatidylcholine synthesis and not by activation of membrane phospholipid degradation. PMID- 10748227 TI - Notch1 deficiency dissociates the intrathymic development of dendritic cells and T cells. AB - Thymic dendritic cells (DCs) form a discrete subset of bone marrow (BM)-derived cells, the function of which is to mediate negative selection of autoreactive thymocytes. The developmental origin of thymic DCs remains controversial. Although cell transfer studies support a model in which T cells and thymic DCs develop from the same intrathymic pluripotential precursor, it remains possible that these two types of cells develop from independent intrathymic precursors. Notch proteins are cell surface receptors involved in the regulation of cell fate specification. We have recently reported that T cell development in inducible Notch1-deficient mice is severely impaired at an early stage, before the expression of T cell lineage markers. To investigate whether development of thymic DCs also depends on Notch1, we have constructed mixed BM chimeric mice. We report here that thymic DC development from Notch1(-/)- BM precursors is absolutely normal (in terms of absolute number and phenotype) in this competitive situation, despite the absence of Notch1(-/)- T cells. Furthermore, we find that peripheral DCs and Langerhans cells are also not affected by Notch1 deficiency. Our results demonstrate that the development of DCs is totally independent of Notch1 function, and strongly suggest a dissociation between intrathymic T cell and DC precursors. PMID- 10748228 TI - Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmune inflammation and cell cycle progression. AB - The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis of tumor cells but not normal cells; its role in normal nontransformed tissues is unknown. We report here that chronic blockade of TRAIL in mice exacerbated autoimmune arthritis, and that intraarticular TRAIL gene transfer ameliorated the disease. In vivo, TRAIL blockade led to profound hyperproliferation of synovial cells and arthritogenic lymphocytes and heightened the production of cytokines and autoantibodies. In vitro, TRAIL inhibited DNA synthesis and prevented cell cycle progression of lymphocytes. Interestingly, TRAIL had no effect on apoptosis of inflammatory cells either in vivo or in vitro. Thus, unlike other members of the tumor necrosis factor superfamily, TRAIL is a prototype inhibitor protein that inhibits autoimmune inflammation by blocking cell cycle progression. PMID- 10748229 TI - Crystal structure of the cysteine-rich domain of mannose receptor complexed with a sulfated carbohydrate ligand. AB - The macrophage and epithelial cell mannose receptor (MR) binds carbohydrates on foreign and host molecules. Two portions of MR recognize carbohydrates: tandemly arranged C-type lectin domains facilitate carbohydrate-dependent macrophage uptake of infectious organisms, and the NH(2)-terminal cysteine-rich domain (Cys MR) binds to sulfated glycoproteins including pituitary hormones. To elucidate the mechanism of sulfated carbohydrate recognition, we determined crystal structures of Cys-MR alone and complexed with 4-sulfated-N-acetylgalactosamine at 1.7 and 2.2 A resolution, respectively. Cys-MR folds into an approximately three fold symmetric beta-trefoil shape resembling fibroblast growth factor. The sulfate portions of 4-sulfated-N-acetylgalactosamine and an unidentified ligand found in the native crystals bind in a neutral pocket in the third lobe. We use the structures to rationalize the carbohydrate binding specificities of Cys-MR and compare the recognition properties of Cys-MR with other beta-trefoil proteins. PMID- 10748230 TI - The cysteine-rich domain of the macrophage mannose receptor is a multispecific lectin that recognizes chondroitin sulfates A and B and sulfated oligosaccharides of blood group Lewis(a) and Lewis(x) types in addition to the sulfated N-glycans of lutropin. AB - The mannose receptor (MR) is an endocytic protein on macrophages and dendritic cells, as well as on hepatic endothelial, kidney mesangial, tracheal smooth muscle, and retinal pigment epithelial cells. The extracellular portion contains two types of carbohydrate-recognition domain (CRD): eight membrane-proximal C type CRDs and a membrane-distal cysteine-rich domain (Cys-MR). The former bind mannose-, N-acetylglucosamine-, and fucose-terminating oligosaccharides, and may be important in innate immunity towards microbial pathogens, and in antigen trapping for processing and presentation in adaptive immunity. Cys-MR binds to the sulfated carbohydrate chains of pituitary hormones and may have a role in hormonal clearance. A second feature of Cys-MR is binding to macrophages in marginal zones of the spleen, and to B cell areas in germinal centers which may help direct MR-bearing cells toward germinal centers during the immune response. Here we describe two novel classes of carbohydrate ligand for Cys-MR: chondroitin 4 sulfate chains of the type found on proteoglycans produced by cells of the immune system, and sulfated blood group chains. We further demonstrate that Cys MR interacts with cells in the spleen via the binding site for sulfated carbohydrates. Our data suggest that the three classes of sulfated carbohydrate ligands may variously regulate the trafficking and function of MR-bearing cells. PMID- 10748231 TI - Modulation of the major histocompatibility complex class II-associated peptide repertoire by human histocompatibility leukocyte antigen (HLA)-DO. AB - Antigen presentation by major histocompatibility complex class II molecules is essential for antibody production and T cell activation. For most class II alleles, peptide binding depends on the catalytic action of human histocompatibility leukocyte antigens (HLA)-DM. HLA-DO is selectively expressed in B cells and impedes the activity of DM, yet its physiological role remains unclear. Cell surface iodination assays and mass spectrometry of major histocompatibility complex class II-eluted peptides show that DO affects the antigenic peptide repertoire of class II. DO generates both quantitative and qualitative differences, and inhibits presentation of large-sized peptides. DO function was investigated under various pH conditions in in vitro peptide exchange assays and in antigen presentation assays using DO(-) and DO(+) transfectant cell lines as antigen-presenting cells, in which effective acidification of the endocytic pathway was prevented with bafilomycin A(1), an inhibitor of vacuolar ATPases. DO effectively inhibits antigen presentation of peptides that are loaded onto class II in endosomal compartments that are not very acidic. Thus, DO appears to be a unique, cell type-specific modulator mastering the class II-mediated immune response induced by B cells. DO may serve to increase the threshold for nonspecific B cell activation, restricting class II peptide binding to late endosomal compartments, thereby affecting the peptide repertoire. PMID- 10748232 TI - T cells can use either T cell receptor or CD28 receptors to absorb and internalize cell surface molecules derived from antigen-presenting cells. AB - At the site of contact between T cells and antigen-presenting cells (APCs), T cell receptor (TCR)-peptide-major histocompatibility complex (MHC) interaction is intensified by interactions between other molecules, notably by CD28 and lymphocyte function-associated antigen 1 (LFA-1) on T cells interacting with B7 (B7-1 and B7-2), and intracellular adhesion molecule 1 (ICAM-1), respectively, on APCs. Here, we show that during T cell-APC interaction, T cells rapidly absorb various molecules from APCs onto the cell membrane and then internalize these molecules. This process is dictated by at least two receptors on T cells, namely CD28 and TCR molecules. The biological significance of T cell uptake of molecules from APCs is unclear. One possibility is that this process may allow activated T cells to move freely from one APC to another and eventually gain entry into the circulation. PMID- 10748233 TI - Antigen-specific B cell memory: expression and replenishment of a novel b220(-) memory b cell compartment. AB - The mechanisms that regulate B cell memory and the rapid recall response to antigen remain poorly defined. This study focuses on the rapid expression of B cell memory upon antigen recall in vivo, and the replenishment of quiescent B cell memory that follows. Based on expression of CD138 and B220, we reveal a unique and major subtype of antigen-specific memory B cells (B220(-)CD138(-)) that are distinct from antibody-secreting B cells (B220(+/)-CD138(+)) and B220(+)CD138(-) memory B cells. These nonsecreting somatically mutated B220(-) memory responders rapidly dominate the splenic response and comprise >95% of antigen-specific memory B cells that migrate to the bone marrow. By day 42 after recall, the predominant quiescent memory B cell population in the spleen (75-85%) and the bone marrow (>95%) expresses the B220(-) phenotype. Upon adoptive transfer, B220(-) memory B cells proliferate to a lesser degree but produce greater amounts of antibody than their B220(+) counterparts. The pattern of cellular differentiation after transfer indicates that B220(-) memory B cells act as stable self-replenishing intermediates that arise from B220(+) memory B cells and produce antibody-secreting cells on rechallenge with antigen. Cell surface phenotype and Ig isotype expression divide the B220(-) compartment into two main subsets with distinct patterns of integrin and coreceptor expression. Thus, we identify new cellular components of B cell memory and propose a model for long term protective immunity that is regulated by a complex balance of committed memory B cells with subspecialized immune function. PMID- 10748234 TI - Extracellular K(+) and opening of voltage-gated potassium channels activate T cell integrin function: physical and functional association between Kv1.3 channels and beta1 integrins. AB - Elevated extracellular K(+) ([K(+)](o)), in the absence of "classical" immunological stimulatory signals, was found to itself be a sufficient stimulus to activate T cell beta1 integrin moieties, and to induce integrin-mediated adhesion and migration. Gating of T cell voltage-gated K(+) channels (Kv1.3) appears to be the crucial "decision-making" step, through which various physiological factors, including elevated [K(+)](o) levels, affect the T cell beta1 integrin function: opening of the channel leads to function, whereas its blockage prevents it. In support of this notion, we found that the proadhesive effects of the chemokine macrophage-inflammatory protein 1beta, the neuropeptide calcitonin gene-related peptide (CGRP), as well as elevated [K(+)](o) levels, are blocked by specific Kv1.3 channel blockers, and that the unique physiological ability of substance P to inhibit T cell adhesion correlates with Kv1.3 inhibition. Interestingly, the Kv1.3 channels and the beta1 integrins coimmunoprecipitate, suggesting that their physical association underlies their functional cooperation on the T cell surface. This study shows that T cells can be activated and driven to integrin function by a pathway that does not involve any of its specific receptors (i.e., by elevated [K(+)](o)). In addition, our results suggest that undesired T cell integrin function in a series of pathological conditions can be arrested by molecules that block the Kv1.3 channels. PMID- 10748235 TI - Role for cathepsin F in invariant chain processing and major histocompatibility complex class II peptide loading by macrophages. AB - The major histocompatibility complex (MHC) class II-associated invariant chain (Ii) regulates intracellular trafficking and peptide loading of MHC class II molecules. Such loading occurs after endosomal degradation of the invariant chain to a approximately 3-kD peptide termed CLIP (class II-associated invariant chain peptide). Cathepsins L and S have both been implicated in degradation of Ii to CLIP in thymus and peripheral lymphoid organs, respectively. However, macrophages from mice deficient in both cathepsins S and L can process Ii and load peptides onto MHC class II dimers normally. Both processes are blocked by a cysteine protease inhibitor, indicating the involvement of an additional Ii-processing enzyme(s). Comparison of cysteine proteases expressed by macrophages with those found in splenocytes and dendritic cells revealed two enzymes expressed exclusively in macrophages, cathepsins Z and F. Recombinant cathepsin Z did not generate CLIP from Ii-MHC class II complexes, whereas cathepsin F was as efficient as cathepsin S in CLIP generation. Inhibition of cathepsin F activity and MHC class II peptide loading by macrophages exhibited similar specificity and activity profiles. These experiments show that cathepsin F, in a subset of antigen presenting cells (APCs), can efficiently degrade Ii. Different APCs can thus use distinct proteases to mediate MHC class II maturation and peptide loading. PMID- 10748236 TI - Disruption of T cell homeostasis in mice expressing a T cell-specific dominant negative transforming growth factor beta II receptor. AB - The immune system, despite its complexity, is maintained at a relative steady state. Mechanisms involved in maintaining lymphocyte homeostasis are poorly understood; however, recent availability of transgenic (Tg) and knockout mouse models with altered balance of lymphocyte cell populations suggest that cytokines play a major role in maintaining lymphocyte homeostasis. We show here that transforming growth factor (TGF)-beta plays a critical role in maintaining CD8(+) T cell homeostasis in a Tg mouse model that specifically overexpresses a dominant negative TGF-beta II receptor (DNRII) on T cells. DNRII T cell Tg mice develop a CD8(+) T cell lymphoproliferative disorder resulting in the massive expansion of the lymphoid organs. These CD8(+) T cells are phenotypically "naive" except for the upregulation of the cell surface molecule CD44, a molecule usually associated with memory T cells. Despite their dominance in the peripheral lymphoid organs, CD8(+) T cells appear to develop normally in the thymus, suggesting that TGF-beta exerts its homeostatic control in the peripheral immune system. PMID- 10748237 TI - Activation of lipoxin A(4) receptors by aspirin-triggered lipoxins and select peptides evokes ligand-specific responses in inflammation. AB - Lipoxin (LX) A(4) and aspirin-triggered LX (ATL) are endogenous lipids that regulate leukocyte trafficking via specific LXA(4) receptors (ALXRs) and mediate antiinflammation and resolution. ATL analogues dramatically inhibited human neutrophil (polymorphonuclear leukocyte [PMN]) responses evoked by a potent necrotactic peptide derived from mitochondria as well as a rogue synthetic chemotactic peptide. These bioactive lipid analogues and small peptides each selectively competed for specific (3)H-LXA(4) binding with recombinant human ALXR, and its N-glycosylation proved essential for peptide but not LXA(4) recognition. Chimeric receptors constructed from receptors with opposing functions, namely ALXR and leukotriene B(4) receptors (BLTs), revealed that the seventh transmembrane segment and adjacent regions of ALXR are essential for LXA(4) recognition, and additional regions of ALXR are required for high affinity binding of the peptide ligands. Together, these findings are the first to indicate that a single seven-transmembrane receptor can switch recognition as well as function with certain chemotactic peptides to inhibitory with ATL and LX (lipid ligands). Moreover, they suggest that ALXR activation by LX or ATL can protect the host from potentially deleterious PMN responses associated with innate immunity as well as direct effector responses in tissue injury by recognition of peptide fragments. PMID- 10748238 TI - Opposing effects of transmembrane and soluble Fas ligand expression on inflammation and tumor cell survival. AB - Fas ligand (FasL) has been shown to mediate both apoptotic and inflammatory reactions. To rigorously assess the physiological role of different forms of the FasL molecule with regard to these two distinct processes, we isolated stably transfected lymphoma cell lines that expressed either murine wild-type FasL, membrane-only FasL, or functionally distinct forms of soluble FasL. First, the ability of these lines to induce an inflammatory response was assessed in vivo by injecting the transfectants intraperitoneally and measuring subsequent neutrophil extravasation into the peritoneal cavity. Second, lines were assessed by injecting the transfectants subcutaneously and monitoring their growth as solid tumors. Our study clearly demonstrated that the extent of inflammation induced by the transfectants directly correlated with their relative cytotoxic activities. A neutrophil response could only be elicited in mice with intact Fas death domains although Fas expression by the neutrophils was not essential. Lymphoma cells expressing the soluble FasL form corresponding to the natural cleavage product could not trigger apoptosis and did not induce a neutrophil response. In contrast to the other FasL transfectants, these cells survived as tumor transplants. However, expression of soluble FasL was not benign, but actually suppressed the inflammatory response and protected other transfectants from the effector mechanisms elicted by membrane-bound FasL. PMID- 10748239 TI - Self-tolerance to the murine homologue of a tyrosinase-derived melanoma antigen: implications for tumor immunotherapy. AB - The human tyrosinase-derived peptide YMDGTMSQV is presented on the surface of human histocompatibility leukocyte antigen (HLA)-A*0201(+) melanomas and has been suggested to be a tumor antigen despite the fact that tyrosinase is also expressed in melanocytes. To gain information about immunoreactivity and self tolerance to this antigen, we established a model using the murine tyrosinase derived homologue of this peptide FMDGTMSQV, together with transgenic mice expressing the HLA-A*0201 recombinant molecule AAD. The murine peptide was processed and presented by AAD similarly to its human counterpart. After immunization with recombinant vaccinia virus encoding murine tyrosinase, we detected a robust AAD-restricted cytotoxic T lymphocyte (CTL) response to FMDGTMSQV in AAD transgenic mice in which the entire tyrosinase gene had been deleted by a radiation-induced mutation. A residual response was observed in the AAD(+)tyrosinase(+) mice after activation under certain conditions. At least some of these residual CTLs in AAD(+)tyrosinase(+) mice were of high avidity and induced vitiligo upon adoptive transfer into AAD(+)tyrosinase(+) hosts. Collectively, these data suggest that FMDGTMSQV is naturally processed and presented in vivo, and that this presentation leads to substantial but incomplete self-tolerance. The relevance of this model to an understanding of the human immune response to tyrosinase is discussed. PMID- 10748241 TI - Differentiating between memory and effector CD8 T cells by altered expression of cell surface O-glycans. AB - Currently there are few reliable cell surface markers that can clearly discriminate effector from memory T cells. To determine if there are changes in O glycosylation between these two cell types, we analyzed virus-specific CD8 T cells at various time points after lymphocytic choriomeningitis virus infection of mice. Antigen-specific CD8 T cells were identified using major histocompatibility complex class I tetramers, and glycosylation changes were monitored with a monoclonal antibody (1B11) that recognizes O-glycans on mucin type glycoproteins. We observed a striking upregulation of a specific cell surface O-glycan epitope on virus-specific CD8 T cells during the effector phase of the primary cytotoxic T lymphocyte (CTL) response. This upregulation showed a strong correlation with the acquisition of effector function and was downregulated on memory CD8 T cells. Upon reinfection, there was again increased expression of this specific O-glycan epitope on secondary CTL effectors, followed once more by decreased expression on memory cells. Thus, this study identifies a new cell surface marker to distinguish between effector and memory CD8 T cells. This marker can be used to isolate pure populations of effector CTLs and also to determine the proportion of memory CD8 T cells that are recruited into the secondary response upon reencounter with antigen. This latter information will be of value in optimizing immunization strategies for boosting CD8 T cell responses. PMID- 10748240 TI - Requirement of tumor necrosis factor receptor-associated factor (TRAF)6 in interleukin 17 signal transduction. AB - Signaling through its widely distributed cell surface receptor, interleukin (IL) 17 enhances the transcription of genes encoding proinflammatory molecules. Although it has been well documented that IL-17 activates the transcription factor nuclear factor (NF)-kappaB and c-Jun NH(2)-terminal kinase (JNK), the upstream signaling events are largely unknown. Here we report the requirement of tumor necrosis factor receptor-associated factor (TRAF)6 in IL-17-induced NF kappaB and JNK activation. In embryonic fibroblasts (EFs) derived from TRAF6 knockout mice, IL-17 failed to activate the IkappaB kinases (IKKs) and JNK. Consequently, IL-17-induced IL-6 and intercellular adhesion molecule 1 expression in the TRAF6-deficient cells was abolished. Lack of TRAF6 appeared to be the sole defect responsible for the observed failure to respond to IL-17, because transient transfection of TRAF6 expression plasmid into the TRAF6-deficient cells restored IL-17-induced NF-kappaB activation in a luciferase reporter assay. Furthermore, the levels of IL-17 receptor (IL-17R) on the TRAF6-deficient EFs were comparable to those on the wild-type control cells. Defect in IL-17 response was not observed in TRAF2-deficient EFs. Moreover, when TRAF6 and IL-17R were coexpressed in 293 cells, TRAF6 coimmunoprecipitated with IL-17R. Together, these results indicate that TRAF6, but not TRAF2, is a crucial component in the IL-17 signaling pathway leading to proinflammatory responses. PMID- 10748242 TI - Nitric oxide inhibits hepatitis B virus replication in the livers of transgenic mice. AB - We have previously identified two antiviral cytokines (interferon [IFN]-gamma and IFN-alpha/beta) that downregulate hepatitis B virus (HBV) replication in the liver of transgenic mice. The cytokine-inducible downstream events that inhibit HBV replication have not been identified. One possible factor is nitric oxide (NO), a pleiotropic free radical with antiviral activity that is produced in the liver by the inducible NO synthase (iNOS). To examine the role of NO in our model, we crossed transgenic mice that replicate HBV with mice that lack a functional iNOS. Importantly, iNOS-deficient mice were almost completely resistant to the noncytopathic inhibitory effect of HBV-specific cytotoxic T lymphocytes on viral replication, an effect that we have shown previously to depend on the intrahepatic induction of IFN-gamma. Conversely, iNOS-deficient mice were not resistant to the antiviral effect of IFN-alpha/beta induced by either polyinosinic-polycytidylic acid complex or by lymphocytic choriomeningitis virus (LCMV) infection. These results indicate that NO mediates the antiviral activity of IFN-gamma, whereas the antiviral activity of IFN-alpha/beta is NO independent. We also compared the relative sensitivity of LCMV to control by NO in these animals. Interestingly, LCMV replicated to higher levels in the liver of iNOS-deficient mice than control mice, indicating that NO controls LCMV replication in the liver, as well as HBV. PMID- 10748243 TI - Deficiency in serum immunoglobulin (Ig)M predisposes to development of IgG autoantibodies. AB - Serum immunoglobulin (Ig)M provides the initial response to foreign antigen and plays a regulatory role in subsequent immune response development, accelerating the production of high-affinity IgG. Here we show that mice deficient in serum IgM have an increased propensity to spontaneous autoimmunity as judged by the development with age of serum IgG anti-DNA antibodies and the renal deposition of IgG and complement. They also exhibit augmented anti-DNA IgG production on exposure to lipopolysaccharide. Thus, deficiency in serum IgM leads to diminished responsiveness to foreign antigens but increased responsiveness to self-a paradoxical association reminiscent of that described in humans deficient in complement or IgA. We wondered whether serum IgM might play an analogous role with regard to the response to self-antigens. However, here-in contrast to the sluggish response to foreign antigens-we find that deficiency in serum IgM actually predisposes to the development of IgG antibodies to autoantigens. PMID- 10748245 TI - Structure, function and regulation of the plant vacuolar H(+)-translocating ATPase. AB - The plant V-ATPase is a primary-active proton pump present at various components of the endomembrane system. It is assembled by different protein subunits which are located in two major domains, the membrane-integral V(o)-domain and the membrane peripheral V(1)-domain. At the plant vacuole the V-ATPase is responsible for energization of transport of ions and metabolites, and thus the V-ATPase is important as a 'house-keeping' and as a stress response enzyme. It has been shown that transcript and protein amount of the V-ATPase are regulated depending on metabolic conditions indicating that the expression of V-ATPase subunit is highly regulated. Moreover, there is increasing evidence that modulation of the holoenzyme structure might influence V-ATPase activity. PMID- 10748244 TI - The plant plasma membrane H(+)-ATPase: structure, function and regulation. AB - The proton-pumping ATPase (H(+)-ATPase) of the plant plasma membrane generates the proton motive force across the plasma membrane that is necessary to activate most of the ion and metabolite transport. In recent years, important progress has been made concerning the identification and organization of H(+)-ATPase genes, their expression, and also the kinetics and regulation of individual H(+)-ATPase isoforms. At the gene level, it is now clear that H(+)-ATPase is encoded by a family of approximately 10 genes. Expression, monitored by in situ techniques, has revealed a specific distribution pattern for each gene; however, this seems to differ between species. In the near future, we can expect regulatory aspects of gene expression to be elucidated. Already the expression of individual plant H(+)-ATPases in yeast has shown them to have distinct enzymatic properties. It has also allowed regulatory aspects of this enzyme to be studied through random and site-directed mutagenesis, notably its carboxy-terminal region. Studies performed with both plant and yeast material have converged towards deciphering the way phosphorylation and binding of regulatory 14-3-3 proteins intervene in the modification of H(+)-ATPase activity. The production of high quantities of individual functional H(+)-ATPases in yeast constitutes an important step towards crystallization studies to derive structural information. Understanding the specific roles of H(+)-ATPase isoforms in whole plant physiology is another challenge that has been approached recently through the phenotypic analysis of the first transgenic plants in which the expression of single H(+)-ATPases has been up- or down-regulated. In conclusion, the progress made recently concerning the H(+)-ATPase family, at both the gene and protein level, has come to a point where we can now expect a more integrated investigation of the expression, function and regulation of individual H(+)-ATPases in the whole plant context. PMID- 10748246 TI - Vacuolar H(+)-pyrophosphatase. AB - The H(+)-translocating inorganic pyrophosphatase (H(+)-PPase) is a unique, electrogenic proton pump distributed among most land plants, but only some alga, protozoa, bacteria, and archaebacteria. This enzyme is a fine model for research on the coupling mechanism between the pyrophosphate hydrolysis and the active proton transport, since the enzyme consists of a single polypeptide with a calculated molecular mass of 71-80 kDa and its substrate is also simple. Cloning of the H(+)-PPase genes from several organisms has revealed the conserved regions that may be the catalytic site and/or participate in the enzymatic function. The primary sequences are reviewed with reference to biochemical properties of the enzyme, such as the requirement of Mg(2)(+) and K(+). In plant cells, H(+)-PPase coexists with H(+)-ATPase in a single vacuolar membrane. The physiological significance and the regulation of the gene expression of H(+)-PPase are also reviewed. PMID- 10748247 TI - Molecular aspects of higher plant P-type Ca(2+)-ATPases. AB - Recent genomic data in the model plant Arabidopsis thaliana reveal the existence of at least 11 Ca(2+)-ATPase genes, and an analysis of expressed sequence tags suggests that the number of calcium pumps in this organism might be even higher. A phylogenetic analysis shows that 11 Ca(2+)-ATPases clearly form distinct groups, type IIA (or ECA for ER-type Ca(2+)-ATPase) and type IIB (ACA for autoinhibited Ca(2+)-ATPase). While plant IIB calcium pumps characterized so far are localized to internal membranes, their animal homologues are exclusively found in the plasma membrane. However, Arabidopsis type IIB calcium pump isoforms ACA8, ACA9 and ACA10 form a separate outgroup and, based on the high molecular masses of the encoded proteins, are good candidates for plasma membrane bound Ca(2+)-ATPases. All known plant type IIB calcium ATPases seem to employ an N terminal calmodulin-binding autoinhibitor. Therefore it appears that the activity of type IIB Ca(2+)-ATPases in plants and animals is controlled by N-terminal and C-terminal autoinhibitory domains, respectively. Possible functions of plant calcium pumps are described and - beside second messenger functions directly linked to calcium homeostasis - new data on a putative involvement in secretory and salt stress functions are discussed. PMID- 10748248 TI - Plant ABC transporters. AB - The ATP binding cassette (ABC) superfamily is a large, ubiquitous and diverse group of proteins, most of which mediate transport across biological membranes. ABC transporters have been shown to function not only as ATP-dependent pumps, but also as ion channels and channel regulators. Whilst members of this gene family have been extensively characterised in mammalian and microbial systems, the study of plant ABC transporters is a relatively new field of investigation. Sequences of over 20 plant ABC proteins have been published and include homologues of P glycoprotein, MRP, PDR5 and organellar transporters. At present, functions have been assigned to a small proportion of these genes and only the MRP subclass has been extensively characterised. This review aims to summarise literature relevant to the study of plant ABC transporters, to review methods of cloning, to discuss the utility of yeast and mammalian systems as models and to speculate on possible roles of uncharacterised ABC transporters in plants. PMID- 10748249 TI - Emerging mechanisms for heavy metal transport in plants. AB - Heavy metal ions such as Cu(2+), Zn(2+), Mn(2+), Fe(2+), Ni(2+) and Co(2+) are essential micronutrients for plant metabolism but when present in excess, these, and non-essential metals such as Cd(2+), Hg(2+) and Pb(2+), can become extremely toxic. Thus mechanisms must exist to satisfy the requirements of cellular metabolism but also to protect cells from toxic effects. The mechanisms deployed in the acquisition of essential heavy metal micronutrients have not been clearly defined although a number of genes have now been identified which encode potential transporters. This review concentrates on three classes of membrane transporters that have been implicated in the transport of heavy metals in a variety of organisms and could serve such a role in plants: the heavy metal (CPx type) ATPases, the natural resistance-associated macrophage protein (Nramp) family and members of the cation diffusion facilitator (CDF) family. We aim to give an overview of the main features of these transporters in plants in terms of structure, function and regulation drawing on information from studies in a wide variety of organisms. PMID- 10748250 TI - Molecular insights into the structure and function of plant K(+) transport mechanisms. AB - Our understanding of plant potassium transport has increased in the past decade through the application of molecular biological techniques. In this review, recent work on inward and outward rectifying K(+) channels as well as high affinity K(+) transporters is described. Through the work on inward rectifying K(+) channels, we now have precise details on how the structure of these proteins determines functional characteristics such as ion conduction, pH sensitivity, selectivity and voltage sensing. The physiological function of inward rectifying K(+) channels in plants has been clarified through the analysis of expression patterns and mutational analysis. Two classes of outward rectifying K(+) channels have now been cloned from plants and their initial characterisation is reviewed. The physiological role of one class of outward rectifying K(+) channel has been demonstrated to be involved in long distance transport of K(+) from roots to shoots. The molecular structure and function of two classes of energised K(+) transporters are also reviewed. The first class is energised by Na(+) and shares structural similarities with K(+) transport mechanisms in bacteria and fungi. Structure-function studies suggest that it should be possible to increase the K(+) and Na(+) selectivity of these transporters, which will enhance the salt tolerance of higher plants. The second class of K(+) transporter is comprised of a large gene family and appears to have a dual affinity for K(+). A suite of molecular techniques, including gene cloning, oocyte expression, RNA localisation and gene inactivation, is now being used to fully characterise the biophysical and physiological function of plants K(+) transport mechanisms. PMID- 10748251 TI - Sodium transport in plant cells. AB - Salinity limits plant growth and impairs agricultural productivity. There is a wide spectrum of plant responses to salinity that are defined by a range of adaptations at the cellular and the whole-plant levels, however, the mechanisms of sodium transport appear to be fundamentally similar. At the cellular level, sodium ions gain entry via several plasma membrane channels. As cytoplasmic sodium is toxic above threshold levels, it is extruded by plasma membrane Na(+)/H(+) antiports that are energized by the proton gradient generated by the plasma membrane ATPase. Cytoplasmic Na(+) may also be compartmentalized by vacuolar Na(+)/H(+) antiports. These transporters are energized by the proton gradient generated by the vacuolar H(+)-ATPase and H(+)-PPiase. Here, the mechanisms of sodium entry, extrusion, and compartmentation are reviewed, with a discussion of recent progress on the cloning and characterization, directly in planta and in yeast, of some of the proteins involved in sodium transport. PMID- 10748252 TI - Structure, function and regulation of ammonium transporters in plants. AB - Ammonium is an important source of nitrogen for plants. It is taken up by plant cells via ammonium transporters in the plasma membrane and distributed to intracellular compartments such as chloroplasts, mitochondria and vacuoles probably via different transporters in each case. Ammonium is generally not used for long-distance transport of nitrogen within the plant. Instead, most of the ammonium transported into plant cells is assimilated locally via glutamine synthetases in the cytoplasm and plastids. Ammonium is also produced by plant cells during normal metabolism, and ammonium transporters enable it to be moved from intracellular sites of production to sites of consumption. Ammonium can be generated de novo from molecular nitrogen (N(2)) by nitrogen-fixing bacteria in some plant cells, such as rhizobia in legume root nodule cells, and at least one ammonium transporter is implicated in the transfer of ammonium from the bacteria to the plant cytoplasm. Plant physiologists have described many of these ammonium transport processes over the last few decades. However, the genes and proteins that underlie these processes have been isolated and studied only recently. In this review, we consider in detail the molecular structure, function and regulation of plant ammonium transporters. We also attempt to reconcile recent discoveries at the molecular level with our knowledge of ammonium transport at the whole plant level. PMID- 10748253 TI - Calcium channels in higher plants. AB - Calcium channels are involved principally in signal transduction. Their opening results in increased cytoplasmic Ca(2+) concentration, and the spatial and temporal variations in this are thought to elicit specific physiological responses to diverse biotic and abiotic stimuli. Calcium-permeable channels have been recorded in the plasma membrane, tonoplast, endoplasmic reticulum, chloroplast and nuclear membranes of plant cells. This article reviews their electrophysiological properties and discusses their physiological roles. Emphasis is placed on the voltage-dependent and elicitor-activated Ca(2+) channels of the plasma membrane and the depolarisation-activated (SV), hyperpolarisation activated, IP(3)- and cADPR-dependent Ca(2+) channels of the tonoplast. The closing of stomatal guard cells in the presence of abscisic acid (ABA) is used to illustrate the co-ordination of Ca(2+) channel activities during a physiological response. PMID- 10748254 TI - The ZIP family of metal transporters. AB - Members of the ZIP gene family, a novel metal transporter family first identified in plants, are capable of transporting a variety of cations, including cadmium, iron, manganese and zinc. Information on where in the plant each of the ZIP transporters functions and how each is controlled in response to nutrient availability may allow the manipulation of plant mineral status with an eye to (1) creating food crops with enhanced mineral content, and (2) developing crops that bioaccumulate or exclude toxic metals. PMID- 10748255 TI - Anion channels in higher plants: functional characterization, molecular structure and physiological role. AB - Anion channels are well documented in various tissues, cell types and membranes of algae and higher plants, and current evidence supports their central role in cell signaling, osmoregulation, plant nutrition and metabolism. It is the aim of this review to illustrate through a few selected examples the variety of anion channels operating in plant cells and some of their regulation properties and unique physiological functions. In contrast, information on the molecular structure of plant anion channels has only recently started to emerge. Only a few genes coding for putative plant anion channels from the large chloride channel (CLC) family have been isolated, and current molecular data on these plant CLCs are presented and discussed. A major challenge remains to identify the genes encoding the various anion channels described so far in plant cells. Future prospects along this line are briefly outlined, as well as recent advances based on the use of knockout mutants in the model plant Arabidopsis thaliana to explore the physiological functions of anion channels in planta. PMID- 10748256 TI - Nitrate transporters in plants: structure, function and regulation. AB - Physiological studies have established that plants acquire their NO(-3) from the soil through the combined activities of a set of high- and low-affinity NO(-3) transport systems, with the influx of NO(-3) being driven by the H(+) gradient across the plasma membrane. Some of these NO(-3) transport systems are constitutively expressed, while others are NO(-3)-inducible and subject to negative feedback regulation by the products of NO(-3) assimilation. Here we review recent progress in the characterisation of the two families of NO(-3) transporters that have so far been identified in plants, their structure and their regulation, and consider the evidence for their roles in NO(-3) acquisition. We also discuss what is currently known about the genetic basis of NO(-3) induction and feedback repression of the NO(-3) transport and assimilatory pathway in higher plants. PMID- 10748257 TI - Molecular mechanisms of phosphate and sulphate transport in plants. AB - The application of molecular techniques in recent years has advanced our understanding of phosphate and sulphate transport processes in plants. Genes encoding phosphate and sulphate transporters have been isolated from a number of plant species. The transporters encoded by these genes are related to the major facilitator superfamily of proteins. They are predicted to contain 12 membrane spanning domains and function as H(+)/H(2)PO(-4) or H(+)/SO(2/-4) cotransporters. Both high-affinity and low-affinity types have been identified. Most research has concentrated on genes that encode transporters expressed in roots. The expression of many of these genes is transcriptionally regulated by signals that respond to the nutrient status of the plant. Nutrient demand and the availability of precursors needed in the assimilatory pathways also regulate transcription of some of these genes. Information on the cell types in which phosphate and sulphate transporters are expressed is becoming available. These data, together with functional characterisation of the transporters, are enabling the roles of various transporters in the overall phosphate and sulphate nutrition of plants to be defined. PMID- 10748258 TI - Sucrose transporters in plants: update on function and structure. AB - In plants, sucrose is the major transport form for photoassimilated carbon and is both a source of carbon skeletons and energy for plant organs unable to perform photosynthesis (sink organs). As a molecule translocated over distance, sucrose has to pass through a number of membranes. Membrane transport of sucrose has therefore been considered for a long time as a major determinant of plant productivity. After several decades of physiological and biochemical experiments measuring the activity of sucrose carriers, unequivocal evidence came from the first identification of a cDNA coding a sucrose carrier (SoSUT1, Riesmeier et al. (1992) EMBO J. 11, 4705-4713). At present 20 different cDNAs encoding sucrose carriers have been identified in different plant species, in both dicots and monocots (one case). The total number is increasing rapidly and most importantly, it can be guessed from the results obtained for Arabidopsis, that in each species, sucrose transporters represent a gene family. The sequences are highly conserved and those carriers display the typical 12 transmembrane alpha-helices of members of the Major Facilitator superfamily. Yeast expression of those carriers indicate that they are all influx carriers, all cotransport sucrose and proton and that their affinity for sucrose is surprisingly similar (0.2-2 mM). All their characteristics are in agreement with those demonstrated at the physiological level in plants. These characteristics are discussed in relation to the function in plants and the few data available on the structure of those transporters in relation to their function are presented. PMID- 10748259 TI - Monosaccharide transporters in plants: structure, function and physiology. AB - Monosaccharide transport across the plant plasma membrane plays an important role both in lower and higher plants. Algae can switch between phototrophic and heterotrophic growth and utilize organic compounds, such as monosaccharides as additional or sole carbon sources. Higher plants represent complex mosaics of phototrophic and heterotrophic cells and tissues and depend on the activity of numerous transporters for the correct partitioning of assimilated carbon between their different organs. The cloning of monosaccharide transporter genes and cDNAs identified closely related integral membrane proteins with 12 transmembrane helices exhibiting significant homology to monosaccharide transporters from yeast, bacteria and mammals. Structural analyses performed with several members of this transporter superfamily identified protein domains or even specific amino acid residues putatively involved in substrate binding and specificity. Expression of plant monosaccharide transporter cDNAs in yeast cells and frog oocytes allowed the characterization of substrate specificities and kinetic parameters. Immunohistochemical studies, in situ hybridization analyses and studies performed with transgenic plants expressing reporter genes under the control of promoters from specific monosaccharide transporter genes allowed the localization of the transport proteins or revealed the sites of gene expression. Higher plants possess large families of monosaccharide transporter genes and each of the encoded proteins seems to have a specific function often confined to a limited number of cells and regulated both developmentally and by environmental stimuli. PMID- 10748260 TI - Amino acid transporters in plants. AB - Amino acid transporters are essential participants in the resource allocation processes that support plant growth and development. Recent results have identified several new transporters that contribute to a wide array of physiological activities, and detailed molecular analysis has provided fundamental insights into the structure, function and regulation of these integral membrane proteins. PMID- 10748261 TI - Regulation of sugar, amino acid and peptide plant membrane transporters. AB - During the past few years, various cDNAs encoding the proton cotransporters which mediate the uptake of sucrose, hexoses, amino acids and peptides across the plant plasma membrane have been cloned. This has made possible some preliminary insight into the regulation of the activity of these transporters at various levels. The paper summarises the present status of knowledge and gaps relative to their transcriptional control (organ, tissue and cell specificity, response to the environment) and post-transcriptional control (targeting and turnover, kinetic and thermodynamic control, lipidic environment, phosphorylation). This outline and the description of a few cases (the sink/source transition of the leaf, the pollen grain, the legume seed) serve as a basis for suggesting some directions for future research. PMID- 10748262 TI - Solute pores, ion channels, and metabolite transporters in the outer and inner envelope membranes of higher plant plastids. AB - All plant cells contain plastids. Various reactions are located exclusively within these unique organelles, requiring the controlled exchange of a wide range of solutes, ions, and metabolites. In recent years, several proteins involved in import and/or export of these compounds have been characterized using biochemical and electrophysiological approaches, and in addition have been identified at the molecular level. Several solute channels have been identified in the outer envelope membrane. These porin-like proteins in the outer envelope membrane were formerly thought to be quite unspecific, but have now been shown to exhibit significant substrate specificity and to be highly regulated. Therefore, the inter-envelope membrane space is not as freely accessible as previously thought. Transport proteins in the inner envelope membrane have been characterized in more detail. It has been proved unequivocally that a family of proteins (including triose phosphate-/phosphoenolpyruvate-, and glucose 6-phosphate-specific transporters) permit the exchange of inorganic phosphate and phosphorylated intermediates. A new type of plastidic 2-oxoglutarate/malate transporter has been identified and represents the first carrier with 12 putative transmembrane domains, to be located in the inner envelope membrane. The plastidic ATP/ADP transporter also contains 12 putative transmembrane domains and possesses striking structural similarity to ATP/ADP transporters found in intracellular, human pathogenic bacteria. PMID- 10748263 TI - The role of aquaporins in cellular and whole plant water balance. AB - Aquaporins are water channel proteins belonging to the major intrinsic protein (MIP) superfamily of membrane proteins. More than 150 MIPs have been identified in organisms ranging from bacteria to animals and plants. In plants, aquaporins are present in the plasma membrane and in the vacuolar membrane where they are abundant constituents. Functional studies of aquaporins have hitherto mainly been performed by heterologous expression in Xenopus oocytes. A main issue is now to understand their role in the plant, where they are likely to be important both at the cellular and at the whole plant level. Plants contain a large number of aquaporin isoforms with distinct cell type- and tissue-specific expression patterns. Some of these are constitutively expressed, whereas the expression of others is regulated in response to environmental factors, such as drought and salinity. At the protein level, regulation of water transport activity by phosphorylation has been reported for some aquaporins. PMID- 10748264 TI - Xenopus oocytes as an expression system for plant transporters. AB - The Xenopus oocyte provides a powerful system for the expression and characterisation of plant membrane proteins. Many different types of plant membrane proteins have been expressed and characterised using this system. As there are already several general reviews on the methodology for oocyte expression of channel proteins, we have summarised the particular advantages and disadvantages of using the system for the characterisation of plant cotransporter proteins. As an example of how the system can be used to identify transporters, we describe evidence for a low affinity nitrate transporter in oocytes injected with poly(A) RNA extracted from nitrate-induced barley roots. Furthermore, we describe evidence that the expression of some transporters in oocytes can modify the properties of endogenous membrane proteins. We conclude that although care must be taken in the interpretation of results and in choosing appropriate controls for experiments, oocyte expression is an excellent tool which will have an important role in characterising plant membrane proteins. PMID- 10748265 TI - Functional characterization of bradykinin analogues on recombinant human bradykinin B(1) and B(2) receptors. AB - We have examined the activity of a range of kinins on recombinant human bradykinin receptors, using a high throughput functional assay which measures intracellular Ca(2+) responses. The most potent agonist for Chinese hamster ovary (CHO) cells stably expressing recombinant human bradykinin B(1) receptors were Des-Arg(9)-bradykinin (EC(50)=7.9 nM) and Des-Arg(10)-kallidin (EC(50)=8.6 nM), while the most potent agonist for CHO cells expressing human bradykinin B(2) receptors was bradykinin (EC(50)=2.0 nM). These findings confirm the validity of the recombinant system and the microtitre plate imaging-based characterization system when compared to known agonist properties of the native receptors. The concentration-response relationship for bradykinin at bradykinin B(2) receptors was potently inhibited by [D-Arg(0),Hyp(3), beta-(2-thienyl)-Ala(5),D Tic(7),Oic(8)]-bradykinin (Hoe140) (IC(50)=71 nM), which was 500-fold more potent against the B(2)-expressing cells than the B(1) cells. Bradykinin B(1) receptor mediated responses activated by Des-Arg(10)-kallidin were fully antagonized by Des-Arg(9)-[Leu(8)]bradykinin (IC(50)=59 nM), Des-Arg(10)-Hoe140 (IC(50)=211 nM) and most potently by Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic (B9858) (IC(50)=14 nM), none of which displayed any activity against the bradykinin B(2) receptor cell line up to 3 microM. None of the antagonists displayed partial agonism activity in these cell lines. All bradykinin B(1) and B(2) receptor antagonists tested acted in an apparently non-competitive manner that is likely to be due in part to their kinetics and to the nature of the functional assay used. PMID- 10748266 TI - Inhibitory effect of novel oral hypoglycemic agent nateglinide (AY4166) on peptide transporters PEPT1 and PEPT2. AB - The novel oral hypoglycemic agent nateglinide (AY4166) is a nonsulfonylurea insulin secretagogue, and its pharmacokinetic features include rapid absorption and elimination. As nateglinide is a dipeptide-like drug, we investigated the interaction of nateglinide with peptide transporters PEPT1 and PEPT2, which mediate the absorption of various peptide-like drugs. Nateglinide exhibited a potent inhibitory effect on [14C]glycylsarcosine uptake by the human colon adenocarcinoma cell line Caco-2 and rat PEPT-transfectants. Kinetic analysis revealed that these inhibitory effects were noncompetitive. Na(+)-coupled alanine or threonine uptake by Caco-2 cells was not inhibited by nateglinide, suggesting that the inhibitory effect of nateglinide on peptide transporters was not due to nonspecific interaction. There was little uptake of [14C]nateglinide by peptide transporters. Various sulfonylureas, such as glibenclamide, also inhibited [14C]glycylsarcosine uptake by rat PEPT-transfectants. In conclusion, nateglinide as well as sulfonylureas inhibit the transport activity of PEPT1 and PEPT2, although nateglinide itself is not transported by these transporters. PMID- 10748267 TI - Connective tissue growth factor induces apoptosis via caspase 3 in cultured human aortic smooth muscle cells. AB - Connective tissue growth factor (CTGF) stimulates proliferation of fibroblasts and endothelial cells, but nothing is known about its role in smooth muscle cells. In this study, the effects of recombinant human CTGF (r-hCTGF, 0.5-10 microgram/ml) on cultured human aortic vascular smooth muscle cells were investigated. r-hCTGF significantly reduced cell viability, increased apoptosis, and augmented caspase 3 activity. Moreover, r-hCTGF-induced apoptosis was significantly inhibited by an antibody to CTGF and a caspase-3 inhibitor, Z Asp(Ome)-Glu-(Ome)Val-Asp(Ome)-FMK. These results suggest that r-hCTGF activates caspase 3 and induces apoptosis. PMID- 10748268 TI - Histamine H(3) receptor-mediated inhibition of endogenous acetylcholine release from the isolated, vascularly perfused rat stomach. AB - We studied the effects of histamine H(3) receptor ligands on the release of endogenous acetylcholine from the isolated, vascularly perfused rat stomach. The stomach was perfused via the celiac artery with modified Krebs-Ringer solution containing physostigmine. Released acetylcholine from the portal vein was electrochemically measured using high-performance liquid chromatography and an enzyme system. Vagus nerves were electrically stimulated twice for 2 min (0.5 or 2.5 Hz). Acetylcholine release evoked at 2.5 Hz was slightly inhibited by histamine and effectively potentiated by thioperamide, a histamine H(3) receptor antagonist. Acetylcholine release evoked at 0.5 Hz in the presence of atropine was not influenced by thioperamide, but effectively inhibited by histamine, R alpha-methylhistamine or imetit, histamine H(3) receptor agonists. These inhibitory effects were abolished by thioperamide or pertussis toxin. These results suggest that histamine attenuates acetylcholine release from vagus nerves through histamine H(3) receptor-mediated and pertussis toxin-sensitive mechanisms in the rat stomach. PMID- 10748269 TI - In vivo evidence that erythropoietin has a neuroprotective effect during subarachnoid hemorrhage. AB - To ascertain in vivo whether recombinant human erythropoietin has a neuroprotective effect on the cortex during subarachnoid hemorrhage, 56 rabbits were divided into the following groups: Group 1 control sham operated plus placebo (n=14; saline solution - NaCl 0.9%); Group 2 control sham operated plus recombinant human erythropoietin (n=14); Group 3 subarachnoid hemorrhage plus placebo (n=14); Group 4 subarachnoid hemorrhage plus recombinant human erythropoietin (n=14; intraperitoneal administration of recombinant human erythropoietin immediately after inducing subarachnoid hemorrhage). In none of the Groups 1 and 2 animals was subarachnoid hemorrhage induced. In Group 3 rabbits, an increase in locomotor activity (open field apparatus) was observed 24, 48 and 72 h after surgery, and the mortality rate was 42.9% within 72 h after surgery, and, no increase in locomotor activity was observed in Group 4 rabbits, which survived for at least 72 h. Our findings suggest that recombinant human erythropoietin may be of benefit in the treatment of subarachnoid hemorrhage. PMID- 10748270 TI - Ontogeny of the motor inhibitory role of dopamine D(3) receptor subtype in rats. AB - We have examined the motor responses to the dopamine D(3) receptor-preferring agonist, S(+)-(4aR,10bR)-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H-1-benzopyranol[4,3 b]-1,4-oxazin+ ++-9-ol ((+)-PD128,907), by non-habituated male rats during postnatal development. (+)-PD128,907 (0.025 and 0.1 mg/kg) increased motor activity (rearing, motility and locomotion) in 14-day-old rats without inducing oral stereotypies. However, in 21-, 28- and 70-day-old rats, (+)-PD128,907 caused a significant reduction in motor activity. This reduction was most pronounced in 70-day-old rats. In addition, the stimulatory effects of (+)-PD128,907 in 14-day old rats were fully blocked by the dopamine D(3) receptor antagonist 5,6 dimethoxy-2-(di-u-propylamino) indan (U99194A). These results suggest that the motor inhibition mediated by the activation of the dopamine D(3) receptors develops between the second and the third postnatal weeks. PMID- 10748271 TI - Hyperphagic effect of novel compounds with high affinity for imidazoline I(2) binding sites. AB - Previous studies have suggested that imidazoline I(2) receptors play a role in feeding control in rats. The effect of subcutaneous (s.c.) injections of four novel imidazoline I(2) ligands, 2-naphthalen-2yl-4,5-dihydro-1H-imidazole hydrochloride (benazoline), 2-styryl-4,5-dihydro-1H-imidazole oxalate (tracizoline), o-nitro-tracizoline and o-methyl-tracizoline (metrazoline) on food intake during the light phase was now evaluated in freely feeding male Wistar rats. Their effect was compared to that of idazoxan, a high-affinity ligand at imidazoline I(2) binding sites, but also a potent alpha(2)-adrenoceptor antagonist. Compared to idazoxan, metrazoline exhibits a higher pK(i) for imidazoline I(2) binding sites in rat liver, while the other compounds have a slightly lower pK(i); on the other hand, the novel compounds have much lower affinity than idazoxan at alpha(2)-adrenoceptors. Idazoxan stimulated drinking at a dose as low as 1 mg/kg, and evoked feeding at a higher dose (30 mg/kg). The selective alpha(2)-adrenoceptor antagonist 2-methoxy-idazoxan (RX821002), with negligible affinity at imidazoline I(2) binding sites, significantly increased drinking but failed to stimulate feeding at doses of 10-50 mg/kg. Metrazoline induced hyperphagia and water drinking at doses of 50 mg/kg or higher. Its dipsogenic effect was secondary to the hyperphagic effect, since it was not observed in rats without access to food. Benazoline significantly increased feeding only in response to 30 mg/kg, but its effect was less pronounced than that of metrazoline. Tracizoline and o-nitro-tracizoline were inactive. Following injection into the lateral cerebroventricle at doses up to 100 microgram/rat, and into the third or fourth brain ventricle at doses up to 50 microgram/rat, neither idazoxan nor metrazoline induced hyperphagia. The present results support the idea that imidazoline I(2) ligands influence feeding in rats, and suggest that their site of action is not in the central nervous system. The finding that idazoxan elicits a more potent hyperphagic effect than metrazoline and benazoline, although its affinity for imidazoline I(2) binding sites is lower than that of metrazoline and similar to that of benazoline, raises the question whether its hyperphagic effect might also be due to interaction with other receptors. PMID- 10748272 TI - Role of endothelium in thapsigargin-induced arterial responses in rat aorta. AB - We assessed the role of endothelium in the arterial response to thapsigargin, the Ca(2+)-ATPase inhibitor of the endoplasmic reticulum, in rat isolated aortic rings. Thapsigargin induced an endothelium-dependent relaxation of phenylephrine contracted aortic rings with an EC(50) of 2.6+/-0.4 nM and a 75% maximum relaxation, while it was less effective against 30 mM K(+)-induced contraction. Pretreatment of aortic rings with N(G)-nitro-L-arginine methyl ester (30 microM) or methylene blue (1 microM) reduced thapsigargin-induced relaxation by approximately 85%. Thapsigargin failed to relax the endothelium-denuded rings. L Arginine (3 mM) partially, but significantly, antagonized the effect of 30 microM N(G)-nitro-L-arginine methyl ester. Pretreatment with indomethacin (3 microM), glibenclamide (1 microM) or iberiotoxin (100 nM) did not alter the thapsigargin induced relaxation. In contrast, pretreatment with tetrapentylammonium ions (TPA(+), 1-3 microM) or with 300 microM Ba(2+) suppressed the relaxant response to thapsigargin. TPA(+) (3 microM) also attenuated acetylcholine-induced relaxation. Thapsigargin-induced endothelium-dependent relaxation was primarily dependent on the presence of extracellular Ca(2+). Interestingly, when the tissues were exposed to very low concentrations of thapsigargin (1-3 nM) the nitric oxide-dependent relaxation induced by acetylcholine or A23187 was markedly reduced. While thapsigargin (3 nM) did not influence the relaxation induced by endothelium-independent dilators, sodium nitroprusside and verapamil. These results indicate that thapsigargin produced complex vascular effects primarily by acting on the endothelial cells. Thapsigargin causes an endothelial nitric oxide dependent relaxation; on the other hand, it inhibits nitric oxide-mediated relaxation at the similar concentrations. Activation of TPA(+)- and Ba(2+) sensitive but not Ca(2+)-activated or ATP-sensitive K(+) channels may be also involved in thapsigargin-induced relaxation of rat isolated aortic rings. PMID- 10748273 TI - Unaltered endothelium-dependent modulation of contraction in the pulmonary artery of hypertensive rats. AB - Involvement of endothelium-derived nitric oxide (EDNO) in alpha-adrenoceptor agonist-induced contractile responses was studied in isolated pulmonary arteries from Wistar Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). In the presence of propranolol, noradrenaline-induced contraction was potentiated by endothelium removal or by N(G)-nitro-L-arginine (L-NOARG). The magnitude of the potentiation was independent of the noradrenaline concentration. L-NOARG also shifted the concentration-response curves for phenylephrine and methoxamine to the left and upward. Contractile responses to 2-amino-5,6,7,8, tetrahydro-6-ethyl-4H-oxazolo-(5,4-d)-azepine-dihydrochloride (BHT-933) and 5 bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK-14304) were augmented by L-NOARG in a concentration-dependent manner. There were no differences in the effects of L-NOARG on the contractile responses to alpha-adrenoceptor agonists between the preparations from WKY and SHRSP. Endothelium-dependent relaxation in response to acetylcholine was not impaired in the preparations from SHRSP when compared with those from WKY. These observations suggest that the contractile responses to the alpha(1)-adrenoceptor agonists were depressed mainly by basally released EDNO, while the responses to the alpha(2)-adrenoceptor agonists were depressed mainly by EDNO released in response to alpha(2)-adrenoceptor stimulation. The comparable influence of the endothelium on the alpha-adrenoceptor agonist-induced contractions in the pulmonary arteries from WKY and SHRSP, which were markedly different from other arteries, could be explained by the unaltered endothelium dependent relaxation in the preparations from SHRSP. PMID- 10748274 TI - Acceleration of gastrointestinal transit by momordin Ic in mice: possible involvement of 5-hydroxytryptamine, 5-HT(2) receptors and prostaglandins. AB - Possible involvement of 5-hydroxytryptamine (5-HT), 5-HT receptors and prostaglandins in the acceleration of gastrointestinal transit by momordin Ic was investigated in mice. Accelerative effect of momordin Ic (25 mg/kg, p.o.) on gastrointestinal transit was attenuated by pretreatment with a bolus of DL-p chlorophenylalanine methyl ester (an inhibitor of 5-HT synthesizing enzyme), but not repeated pretreatment with DL-p-chlorophenylalanine methyl ester. Furthermore, cyproheptadine (a nonselective 5-HT(2) receptor antagonist), ritanserin (a 5-HT(2A/2B/2C) receptor antagonist) and clozapine (a 5-HT(2A/2C) receptor antagonist) also attenuated the effect of momordin Ic, but methiothepin (a 5-HT(1) receptor antagonist), MDL 72222 (3-tropanyl-3,5-dichlorobenzoate) and metoclopramide (5-HT(3) receptor antagonists), tropisetron (a 5-HT(3/4) receptor antagonist), ketanserin and haloperidol (5-HT(2A) receptor antagonists) did not. These results suggested a possible involvement of endogenous 5-HT and 5-HT(2B/2C) over 5-HT(2A) receptors. Attenuation by pretreatment with indomethacin (an inhibitor of prostaglandins synthesis) suggested involvement of prostaglandins. It is postulated that momordin Ic accelerates gastrointestinal transit partially by stimulating synthesis of 5-HT to act through 5-HT(2), possibly 5-HT(2C) and/or 5-HT(2B) receptors, which, in turn, increases synthesis of prostaglandins. PMID- 10748276 TI - Hepatocyte growth factor region specifically activates mucin synthesis in rat stomach. AB - We investigated the effects of hepatocyte growth factor (HGF) on mucin biosynthesis and the expression of its receptor in distinct sites and layers of rat gastric mucosa. HGF stimulated the mucin biosynthesis in the surface and gland mucus cells of corpus, but not in the antrum, without its trophic effects. The HGF-receptor mRNA expression was high in the surface and deep corpus mucosa, but low in the antrum. These results demonstrate that HGF has distinct effects on mucin biosynthesis in a specific region of rat stomach, suggesting different regulatory mechanisms underlying the mucus metabolism of distinct mucus-producing cells. PMID- 10748275 TI - Inhibition of the contractile response of the rat detrusor muscle by the beta(2) adrenoceptor agonist clenbuterol. AB - The action of clenbuterol, beta(2)-adrenoceptor agonist, on the contractile response of isolated rat detrusor muscle strips was investigated in vitro. Clenbuterol (10(-5) M) inhibited the detrusor muscle frequency response (1-40 Hz, p<0.02) with a more pronounced effect at 1 Hz than 40 Hz. Clenbuterol (10(-6) M) significantly inhibited the contractile response to exogenous ATP (10(-4) to 10( 2) M, p<0.05) but not to carbachol (10(-9) to 10(-4) M). The presence of 10(-5) M ICI 118, 551, beta(2)-adrenoceptor antagonist, shifted significantly the clenbuterol dose-response to 1 Hz electrical field stimulation (EC(50) 3.4x10(-6) M (+/-2.2x10(-6) M) for clenbuterol alone, to 4.1x10(-4) M (+/-8.8 x10(-5) M), P<0.05). In conclusion, clenbuterol inhibits electrical field and ATP-stimulated contractions of detrusor muscle. Reversal of the clenbuterol inhibition of detrusor muscle contraction by ICI 118, 551 shows that clenbuterol is probably acting through postsynaptic beta(2)-adrenoceptors, which modulate the response to ATP released from purinergic nerves. PMID- 10748277 TI - Effects of the phosphodiesterase 4 inhibitor RPR 73401 in a model of immunological inflammation. AB - The study was performed to investigate effects of the phosphodiesterase 4 inhibitor RPR 73401 [N-(3, 5-dichloropyrid-4-yl)-3-cyclopentyl-oxy-4 methoxybenzamid] on an allergic skin reaction. To simulate an immunological inflammation, BALB/c mice were sensitized to dinitrochlorobenzene or toluenediisocyanate. At first, the abdominal skin was shaved and 50 microliter Freund's adjuvant were injected intracutaneously once. Then, the horny layer was removed by adhesive tape stripping and 100 microliter 0.5% dinitrochlorobenzene or 5% toluenediisocyanate were administered on the epidermis for 4 days. After repeated local treatment of the ear skin with 20 microliter 3% RPR 73401 or intraperitoneal administration of 1 and 5 mg/kg RPR 73401, 20 microliter 1% dinitrochlorobenzene or 0.5% toluenediisocyanate were given topically as a challenge. The vehicle controls showed a high increase in ear thickness over 48 h after challenge, whereas RPR 73401 administered on either route reduced this increase significantly. Nevertheless after topical administration, RPR 73401 had a longer lasting effect. These and other results may point to an indication for RPR 73401 in immunological dermatitis. PMID- 10748278 TI - Gender-related differences in murine T- and B-lymphocyte proliferative ability in response to in vivo [Met(5)]enkephalin administration. AB - Gender-related differences in response to drugs of abuse, such as cocaine and morphine, have been reported both in humans and in experimental animals. Besides causing analgesia, morphine has recently been shown to exert strong immunosuppressive activity. However, no data on the influence of gender on the immunomodulatory effects of morphine or opioid peptides have been reported yet. The aim of this study was to test the influence of gender on the immunomodulatory ability of the endogenous opioid peptide [Met(5)]enkephalin (MENK) in mice. This was done by comparing the proliferative ability of splenic T- and B-lymphocytes 14 h after systemic (intraperitoneal; i.p.) administration of [Met(5)]enkephalin (2.5, 5 or 10 mg/kg body weight). The proliferative ability of T- and B lymphocytes was assessed by testing their in vitro response to graded concentrations of the T- and B-cell mitogens, concanavalin-A (Con-A) and lipopolysaccharide (LPS), respectively. The results obtained showed that [Met(5)]enkephalin, at a dose of 2.5 mg/kg, enhanced the proliferative ability of T-lymphocytes in male mice, but not in female mice. Similarly, [Met(5)]enkephalin, at doses of 2.5 and 5 mg/kg, enhanced the proliferative ability of splenic B-lymphocytes in male mice, whereas in female mice a decrease was observed ([Met(5)]enkephalin 2.5 mg/kg, LPS 10 microg/ml). [Met(5)]enkephalin, at a dose of 10 mg/kg, did not affect the proliferative ability of either lymphocyte population, regardless of gender. The [Met(5)]enkephalin-induced stimulatory effect on both T- and B-lymphocyte proliferation was reversed by naloxone (10 mg/kg body weight), injected prior to [Met(5)]enkephalin, suggesting an involvement of opioid receptors. Thus, the data presented provide evidence for the gender-related response of murine splenic lymphocytes to immunomodulation by [Met(5)]enkephalin, administered in vivo. This finding may be relevant to the potential use of [Met(5)]enkephalin in adjuvant therapy for immunocompromised states, such as acquired immunodeficiency syndrome (AIDS) or malignancies. PMID- 10748279 TI - The effects of delayed treatment with sialyl Lewis X against lipopolysaccharide induced acute lung injury in rabbits. AB - The therapeutic effects of a selectin inhibitor against lipopolysaccharide induced acute lung injury were studied in rabbits by using sialyl Lewis X oligosaccharide. Lipopolysaccharide-induced acute lung injury, as characterized by an impairment of pulmonary gas exchange, clinically resembles that of the acute respiratory distress syndrome. Delayed treatments with sialyl Lewis X oligosaccharide (55 mg kg(-1) i.v. bolus injection 0.5, 1 or 2 h after lipopolysaccharide administration+36 mg kg(-1) h(-1) i.v. infusion for 5.5, 5 or 4 h, respectively) prevented the lipopolysaccharide-induced impairments in pulmonary gas exchange, as well as the accumulation of polymorphonuclear leukocytes in the lung tissue. In contrast, this agent had no significant effects on lipopolysaccharide-induced systemic hypotension, the decrease in the number of circulating white blood cells and platelets or the decline in blood pH. This is the first demonstration that sialyl Lewis X-oligosaccharide is effective against the impairments in pulmonary gas exchange even if administered 0.5, 1 or 2 h following the lipopolysaccharide injection. PMID- 10748280 TI - Inhibitory actions of ropivacaine on tumor necrosis factor-alpha-induced leukocyte adhesion and tissue accumulation in vivo. AB - We have examined the effect of ropivacaine, a local anesthetic, on leukocyte endothelium interactions induced by tumor necrosis factor-alpha (TNF-alpha) in vivo by the use of intravital microscopy in the mouse cremaster microcirculation. It was found that ropivacaine markedly reduced venular leukocyte adhesion and tissue recruitment in response to TNF-alpha challenge, whereas leukocyte rolling was unchanged. Thus, treatment with ropivacaine may be a useful pharmacological tool to control acute inflammation. PMID- 10748282 TI - Analysis of a mathematical model of the effect of inhibitors on the growth of tumors. AB - In this paper, we study a model of tumor growth in the presence of inhibitors. The tumor is assumed to be spherically symmetric and its boundary is an unknown function r=R(t). Within the tumor the concentration of nutrient and the concentration of inhibitor (drug) satisfy a system of reaction-diffusion equations. The important parameters are Lambda(0) (which depends on the tumor's parameters when no inhibitors are present), gamma which depends only on the specific properties of the inhibitor, and beta; which is the (normalized) external concentration of the inhibitor. In this paper, we give precise conditions under which there exist one dormant tumor, two dormant tumors, or none. We then prove that in the first case, the dormant tumor is globally asymptotically stable, and in the second case, if the radii of the dormant tumors are denoted by R(s)(-),R(s)(+) with R(s)(-)infinity)R(t)=R(s)(-), provided the initial radius R(0) is smaller than R(s)(+); if however R(0)R(s)(+) then the initial tumor in general grows unboundedly in time. The above analysis suggests an effective strategy for treatment of tumors. PMID- 10748281 TI - Nitric oxide-mediated mucus hypersecretion protects the stomach of ovariectomized rats. AB - The actions of ovariectomy on nitric oxide synthase (assessed by the citrullin assay), mucus secretion (assessed by the Alcyan blue technique) and ulcerogenic response (indomethacin (30 mg kg(-1), s.c. , 4 h) or cysteamine (400 mg kg(-1), s.c., 24 h)) were studied in the female rat stomach. Ovariectomy increased nitric oxide synthase and mucus secretion, and decreased the severity of lesions, effects reversed by the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 10 mg kg(-1), s.c., 4 h before measurements). Therefore, estrogen-deficiency protects the gastric mucosa by nitric oxide (NO)-mediated mucus hypersecretion. PMID- 10748283 TI - A new statistic for the analysis of association between trait and polymorphic marker loci. AB - Inference for detecting the existence of an association between a diallelic marker and a trait locus is based on the chi-squared statistic with one degree of freedom. For polymorphic markers with m alleles (2), three approaches are mainly used in practice. First, one may use Pearson's chi-squared statistic with m-1 degrees of freedom (d.f.) but this leads to a loss in test power. Second, one can select an allele to be the most associated and then collapse the other allele categories into a single class. This reduces in a biased way, the locus to a diallelic system. Third, one may use the Terwilliger [J.D. Terwilliger, Am. J. Hum. Genet. 56 (1995) 777] likelihood ratio statistic which has a non-standard unknown limiting probability distribution. In this paper, we propose a new statistic, L(D), based on the second testing approach. We derive the asymptotic probability distribution of L(D) in an easy way. Simulation studies show that L(D) is more powerful than Pearson's chi-squared statistic with m-1 d.f. PMID- 10748284 TI - Dynamic behaviors of the Ricker population model under a set of randomized perturbations. AB - We studied the dynamics of the Ricker population model under perturbations by the discrete random variable epsilon which follows distribution P?epsilon=a(i)?=p(i),i=1,ellipsis,n,0/=1. Under the perturbations, n+1 blurred orbits appeared in the bifurcation diagram. Each of the n+1 blurred orbits consisted of n sub-orbits. The asymptotes of the n sub orbits in one of the n+1 blurred orbits were N(t)=a(i) for i=1,ellipsis,n. For other n blurred orbits, the asymptotes of the n sub-orbits were N(t)=a(i)exp[r(1 a(i))]+a(j),j=1,2,ellipsis,n, for i=1,ellipsis,n, respectively. The effects of variances of the random variable epsilon on the bifurcation diagrams were examined. As the variance value increased, the bifurcation diagram became more blurred. Perturbation effects of the approximate continuous uniform random variable and random error were compared. The effects of the two perturbations on dynamics of the Ricker model were similar, but with differences. Under different perturbations, the attracting equilibrium points and two-cycle periods in the Ricker model were relatively stable. However, some dynamic properties, such as the periodic windows and the n-cycle periods (4), could not be observed even when the variance of a perturbation variable was very small. The process of reversal of the period-doubling, an important feature of the Ricker and other population models observed under constant perturbations, was relatively unstable under random perturbations. PMID- 10748285 TI - Waning immunity and sub-clinical infection in an epidemic model: implications for pertussis in The Netherlands. AB - In The Netherlands, an epidemic outbreak of pertussis took place in 1996-1997. Understanding of the causes of the epidemic is hampered by the fact that many cases of infection with Bordetella pertussis go by unnoticed, and by the fact that immunity against infection does not last lifelong. Motivated by these observations, we develop and analyze an age-structured epidemic model that takes these factors into account. A distinction is made between infection in immunologically naive individuals, and infection in individuals whose immune system has been primed before by infection or vaccination. While the former often lead to severe symptoms and thus are more often diagnosed and notified, the latter are largely sub-clinical. The main questions are: (1) to what extent do sub-clinical infections contribute to the circulation of B. pertussis; and (2) what might be the causes for the recent epidemic? To answer these questions, we first present a new method to estimate the force of infection from notification data. The method is applied to the 1988-1995 case notification data from The Netherlands. Estimates of the force of infection vary greatly, depending on the rate at which immunity is lost, and on the fraction of sub-clinical infections. For the 1988-1995 period, our analysis indicates that if immunity is lost at a small rate and if a majority of infections is sub-clinical, the contribution of infection in adults to the transmission process cannot be neglected. Our results furthermore indicate that a decrease in the duration of protection after vaccination due to a change in the pathogen is the most likely factor to account for the 1996-1997 epidemic. PMID- 10748286 TI - A simple vaccination model with multiple endemic states. AB - A simple two-dimensional SIS model with vaccination exhibits a backward bifurcation for some parameter values. A two-population version of the model leads to the consideration of vaccination policies in paired border towns. The results of our mathematical analysis indicate that a vaccination campaign φ meant to reduce a disease's reproduction number R(φ) below one may fail to control the disease. If the aim is to prevent an epidemic outbreak, a large initial number of infective persons can cause a high endemicity level to arise rather suddenly even if the vaccine-reduced reproduction number is below threshold. If the aim is to eradicate an already established disease, bringing the vaccine-reduced reproduction number below one may not be sufficient to do so. The complete bifurcation analysis of the model in terms of the vaccine-reduced reproduction number is given, and some extensions are considered. PMID- 10748287 TI - Uniqueness of limit cycle in the predator-prey system with symmetric prey isocline. AB - We consider a special form of the Gause model of interactions between predator and prey populations. Using the ideas of Cheng, we prove the uniqueness of the limit cycle for more general systems, satisfying some additional conditions. These include also a condition due to Kuang and Freedman. Moreover, in this paper it is shown that the similar generalization of Cheng's uniqueness proof by Conway and Smoller is not correct. PMID- 10748289 TI - Sequential numerical changes of chromosomes 7 and 18 in diffuse-type stomach cancer cell lines: combined comparative genomic hybridization, fluorescence in situ hybridization, and ploidy analyses. AB - Sequential changes of chromosomal copy number were analyzed retrospectively in five diffuse-type gastric cancer cell lines by comparative genomic hybridization (CGH), DNA cytometry, and fluorescence in situ hybridization (FISH) with centromeric and painting probes. By CGH, we found loss of 18q21 in all of the cell lines and gains of 7p11-q31, 20q, and 22 in four of the five cell lines. Actual copy numbers of chromosomes 7 and 18 were determined by FISH: disomy 18 with (partial) loss of 18q in the two DNA-diploid cell lines (AGS and MKN-45), trisomy 7 in MKN-45, disomy 18 and tetrasomy 7 with one-copy loss of 7p and one copy gain of 7q tip in DNA-triploid HSC-39/40A, and trisomy 18 and hexasomy 7 with one-copy loss of 7q in DNA-tetraploid KATO-III. Because the DNA aneuploidy is thought to result through tetraploidization, and the duplicated chromosomal changes in DNA aneuploid tumors seem to precede tetraploidization, the duplicated gain of chromosome 7 and one-copy loss of 7q in KATO-III were inferred to have occurred before and after tetraploidization, respectively. Similarly, HSC-39/40A were inferred to be preceded by the DNA-diploid stage with disomy 7 and monosomy 18. As the loss of 18q21 and the gain of 7p11-q31 were inferred to have occurred already in the DNA diploid stage in at least four and two of the cell lines, respectively, the 18q21 loss may be more important than the 7q gain as an earlier event in the genesis of diffuse-type stomach cancer. The combined CGH, FISH, and ploidy analyses thus give us a clue to extract important earlier events from the chromosomal changes that were screened by CGH alone. PMID- 10748288 TI - Frequent loss of 9p21 (p16(INK4A)) and other genomic imbalances in human malignant fibrous histiocytoma. AB - To search for new recurrent genetic aberrations in malignant fibrous histiocytoma (MFH), a combination of conventional cytogenetic, comparative genomic hybridization (CGH), and Southern blot analyses was applied to a series of 34 tumors. Cytogenetic analysis revealed the presence of multiple structural and numerical aberrations, including marker chromosomes, telomeric associations, double minutes, and ring chromosomes. The most frequent genomic imbalances in this series of neoplasms as detected by CGH were gains of 1q21-q22 (69%), 17q23 qter (41%), and 20q (66%), and losses of 9p21-pter (55%), 10q (48%), 11q23-qter (55%), and 13q10-q31 (55%). Southern blot analyses with p16(INK4A) (CDKN2A; 9p21) and RB1 (13q14) probes provided clear indications for frequent deletions of these tumor suppressor genes, and as such, substantiated the CGH results. Additionally, examination of the TP53 and MDM2 genes showed frequent loss and amplification, respectively. These data indicate that genes involved in the RB1- and TP53 associated cell cycle regulatory pathways may play prominent roles in the development of human MFH. PMID- 10748290 TI - Enhanced detection of chromosomal abnormalities with the use of RxFISH multicolor banding technique. AB - Hematological disorders often have complex karyotypes with multiple markers. Proper assignment of chromosome number or aberration or both can be difficult. Specific identification of chromosomal abnormalities aids in the diagnosis and selection of treatment of patients. Fluorescence in situ hybridization (FISH) has been applied to the identification of translocations, markers, and other chromosomal abnormalities in clinical cytogenetics. However, the standard FISH technique is unable to detect the entire genome in a single experiment. This report presents the use of a cross-species comparative genomic hybridization color-banding technique (RxFISH) that permits examination of an entire karyotype at one time. Specimens from two patients, one with acute lymphocytic leukemia (ALL) and the other with multiple myeloma (MM), were studied. Metaphases were prepared by standard culture techniques. Conventional cytogenetic analysis (GTG banding) showed multiple clones in each of the cases. These clones were hyperdiploid metaphases with complex chromosomal abnormalities and multiple markers. The slides were then hybridized with FITC-, Cy-3-, and Cy-5-labeled RxFISH probes; the results were analyzed by a digital imaging system. The RxFISH color banding confirmed the hyperdiploid metaphases and identified multiple chromosomal abnormalities. In the specimen from the patient with ALL, several chromosomes, which had been classified as markers by G-banding, were found to be specific chromosomes. This study suggests that RxFISH can provide more accurate and specific identification of complex chromosomal abnormalities. RxFISH is a useful complement to the clinical cytogenetic laboratory armamentarium. PMID- 10748291 TI - Establishment and characterization of HKESC-1, a new cancer cell line from human esophageal squamous cell carcinoma. AB - The establishment of an esophageal cancer cell line can facilitate the search for molecular mechanisms involved in esophageal carcinogenesis. A new human cancer cell line, HKESC-1, was established from a primary moderately-differentiated squamous cell carcinoma of the esophagus from a 47-year-old Hong Kong Chinese man. The pathological characteristics (morphology, immunohistochemical, and electron microscopic studies), the tumorigenecity in nude mice, the cytogenetic features, the DNA ploidy, and telomerase activity of the cell line were investigated. The HKESC-1 cells have been maintained continuously in vitro for more than 16 months and passaged over 96 times. HKESC-1 cells grow as a monolayer, with a doubling time of 46 hours. The HKESC-1 cells are of a squamous epithelial origin, as shown by their immunopositivity with the anti-cytokeratin antibodies and ultrastructural demonstration of tonofilaments and desmosomes. The HKESC-1 cells possess characteristics of malignancy because they are highly tumorigenic in nude mice and have strong telomerase activity. The HKESC-1 cells had an aneuploid DNA content, as demonstrated by flow cytometric analysis. Cytogenetic analysis revealed hyperdiploidy of greater than 50 in 80% of analyzable metaphases. Chromosome gains and losses were common, and loss of the Y chromosome was a consistent numerical aberration. Additionally, many structural chromosomal abnormalities were encountered, with frequent breakpoints at 1p32, 7p22, 7q34, and 20q13. This newly established cell line serves as a useful model for studying the molecular pathogenesis, and testing new therapeutic reagents for esophageal squamous cell carcinoma. PMID- 10748292 TI - A FISH study of variant Philadelphia rearrangements. AB - A total of 39 variant Philadelphia (Ph) translocations were studied by fluorescence in situ hybridization (FISH) using MBCR/ABL, mBCR/ABL, or DBCR/ABL probes. Seven cases did not have a BCR/ABL fusion signal. Of a total of 32 fusion positive cases, 5 were simple variants involving chromosome 22 and another chromosome apart from chromosome 9; 23 were complex variants involving chromosomes 22, 9, and a third chromosome (18 cases), or 22, 9, and two other chromosomes (4 cases). Masked Ph rearrangements were detected in 4 cases. One case was a Ph chromosome mimic. Fluorescence in situ hybridization has become a widely used method for studying Ph rearrangements. The latest probe that is being used is the DBCR/ABL (double reciprocal BCR/ABL signals). The expected pattern for this probe is one green ABL signal (1G) on the normal 9, one red BCR signal (1R) on the normal 22, and two fusion signals, BCR/ABL and ABL/BCR (2F), on a derivative 22 and a derivative 9, respectively. Deviant patterns from 1G1R2F, and sometimes 1G1R2F, were indicative of a variant, as long as there was a fusion signal. However, in interphase analysis, it is not possible to visualize a variant rearrangement, and when a deviant pattern involving at least one fusion signal is observed, the following possibilities should be contemplated. The different patterns observed in fifteen Ph variants are described. The patterns observed in variants studied with the DBCR/ABL probe were 2G2R1F (40%), 1G1R2F (20%), 1G1R1F (20%), 1G2R1F (13.3%), and 2G1R1F (6.66%). A single mechanism is involved in the formation of each of these patterns. A 2G2R1F, FISH pattern in 6 cases appears to involve a single concerted event of simultaneous breaks on the participating chromosomes followed by mismatched joining. The three cases with 1G1R2F most probably arose by two sequential rearrangements. The 1G1R1F pattern suggests that either the BCR and ABL breakpoints are different, or there are deletions at the breakpoints, because residual signals are not observed. Two independent events appear to be involved in 1G2R1F with a reverse cryptic 9,22 rearrangement as the first event. In one case of 2G1R1F, the plausible explanation is an insertion of ABL next to BCR and either a simultaneous or a sequential translocation with another chromosome. PMID- 10748293 TI - Mutation of K-RAS protooncogene and loss of heterozygosity on 6q27 in serous and mucinous ovarian carcinomas. AB - The genetic etiology of serous and mucinous ovarian carcinomas was investigated in 76 affected patients, focusing on the possible loss of heterozygosity (LOH) involving chromosome band 6q27 and K-RAS mutations at codon 12. The incidence of LOH in 6q27 (6q27 LOH) was 41% in 64 informative cases; 53% (20/38) and 23% (6/26) in cases of serous ovarian carcinoma and in those of mucinous ovarian carcinoma, respectively, indicating that the incidence of 6q27 LOH was significantly higher in cases of serous ovarian carcinoma (P < 0.05). The incidence of K-RAS mutations at codon 12 was 23% (15/64); 5% (2/38) and 50% (13/26) in cases of serous ovarian carcinoma and in those of mucinous ovarian carcinoma, respectively, indicating that the incidence of the K-RAS mutations was significantly higher in cases of mucinous ovarian carcinoma (P < 0.0001). Thus, K RAS mutations at codon 12 and 6q27 LOH were suggested to be involved in the development and/or progression of mucinous ovarian carcinoma and serous ovarian carcinoma, respectively. PMID- 10748294 TI - Cytogenetic and interphase FISH analyses of 73 basal cell and three squamous cell carcinomas: different findings in direct preparations and short-term cell cultures. AB - Cytogenetic analysis performed on 73 sporadic basal cell carcinomas (BCCs) and three squamous cell carcinomas (SCCs) showed different findings in direct preparations (24 hours) and in short-term cell cultures. Except for loss of the Y chromosome, not one of the other clonal (+6, +16, add(2)(q37), del(3)(q13), add(1)(p31), and near triploidy) or sporadic changes found in direct preparations was found in cell cultures and vice versa. Clonal trisomy 6 found in two BCC direct preparations and demonstrated by interphase fluorescence in situ hybridization in 8 other cases seems to be a nonrandom change in basal cell carcinoma. Immunohistochemistry showed that the cell type investigated was different in the two methods of analysis used: epithelial in direct preparations and fibroblastic in cell cultures. Thus, the results obtained in direct preparations indicate the BCC or SCC epithelial karyotype, whereas the aberrations found in cell cultures indicate the presence of chromosome instability in the fibroblastic stroma. The apparent lack of correspondence between direct and indirect preparations and the presence of clonal chromosome changes in both epithelial and stromal cells suggest tumor cell heterogeneity of BCC. The fibroblastic stroma seems to be implicated in the neoplastic process. This is not evident in SCC, in which clonal changes are present only in direct preparations. The chromosomal distribution of the breakpoints involved in structural changes in direct and cell culture preparations is random; together with those reported in the literature, the breakpoints found in BCC cultures show, however, a cluster to 1p36, 3q13, 9q22, 14p11, 15p11, and Xp11 bands. We did not find any significant correlations between BCC cytogenetic results and the clinical data (site, age, sex, recurrence). The incidence of cases of BCC (38%) and of SCC (100%) showing clonal chromosome changes agree with their benign and malignant nature, respectively. Finally, a significantly high incidence of constitutional inv(9) and dup(9)(q11q21) was found in the group of patients with BCC. PMID- 10748295 TI - Supernumerary ring chromosomes derived from the long arm of chromosome 12 as the primary cytogenetic anomaly in a rare soft tissue chondroma. AB - Supernumerary ring chromosomes varying with respect to both size and number were found as the primary cytogenetic anomaly in a rare benign soft tissue chondroma resected from the floor of the mouth of a 3-year-old girl. Reverse fluorescence in situ hybridization paint probes prepared by polymerase chain reaction from microdissected rings produced fluorescent signal over two large but discontinuous parts of the chromosome 12 long arm, subdivided into four regions. This case expands the spectrum of mesenchymal neoplasms in which ring chromosomes have been described as the primary genetic anomaly. A review of the literature reporting similar findings in other soft tissue tumors further supports the possibility that low-level amplification of chromosome 12 long-arm regions may contribute to abnormal cellular proliferation in a variety of mesenchymal tumors. Genes implicated in the control of the cell cycle such as sarcoma amplified sequence (SAS), the human homolog of the murine double-minute type 2 gene (MDM-2), proto oncogenes CHOP/GADD153, GLI, A2MR, cyclin-dependent kinase (CDK4), and the high mobility group (HMGIC) gene implicated in mesenchymal tumorigenesis are all located on the long arm of chromosome 12. Chromosomal abnormalities involving the 12q13-q15 region are associated with a wide range of benign soft tissue tumors and sarcomas. PMID- 10748296 TI - Trisomy 10 in acute myeloid leukemia: three new cases. AB - Trisomy 10 is a rare nonrandom cytogenetic abnormality found in association with acute myeloid leukemia (AML). The hematological and clinical features associated with this finding have not yet been clearly defined. A literature review revealed 13 cases of trisomy 10 in AML, some reported as a minority component of a more comprehensive AML study and therefore lacking a full description of both clinical and hematological features. We present a summary of these reports and add three new cases to the literature. PMID- 10748297 TI - Chromosomal aberrations in ovarian malignant brenner tumor cell line using chromosome painting. AB - Cytogenetic changes in an ovarian malignant Brenner tumor cell line, SNU-840, were investigated by chromosome painting and G-banding. All chromosome alterations were confirmed by the use of multiple chromosome paintings, which also demonstrated a number of additional alterations. PMID- 10748298 TI - Two cases of AML (M2) with a t(8;19)(q22;q13): a new cytogenetic variant. AB - "Simple" variants of the t(8;21) translocation involving chromosome 8 and a chromosome other than number 21 are rare. To our knowledge, only t(3;8)(q29;q22), t(8;11)(q22;q13), t(8;16)(q22;q24), t(8;20)(q22;p13), and t(8;22) have been reported in the literature. This paper describes for the first time two patients with acute myelogenous leukemia with a consistent t(8;19)(q22;q13) translocation. Their myelograms were compatible with the FAB-M2 subtype. The blasts from case 2 expressed CD34, CD33, CD13, and CD19. Karyotype analyses were performed on bone marrow cells using R- and G-banding at presentation. A t(8;19)(q22;q13) translocation was found in 28/30 metaphases for case 1 and in 23/25 metaphases for case 2. The latter case also had a deletion of chromosome 9, del(9)(q12q22) as an additional abnormality. Reverse transcriptase-polymerase chain reaction study revealed no AML1/ETO fusion transcript in case 2. Dual-color fluorescence in situ hybridization (FISH) assay using two probes (BAC92 and YAC412A4) convincingly demonstrated that the chromosomal material from 8q was translocated onto 19q rather than 19p in case 2. Thus, we consider t(8;19)(q22;q13) a true "simple" variant of t(8;21), and assume that a fusion gene resulting from the t(8;19) may contain the ETO gene located at 8q22 and an unknown partner gene from 19q13, which probably is a new transcription factor, whose molecular entity warrants further study. PMID- 10748299 TI - 17p- syndrome arising from a novel dicentric translocation in a patient with acute myeloid leukemia. AB - The cytogenetic contribution to the poor prognosis when myelodysplastic syndrome (MDS) progresses to acute myeloid leukemia (AML) is not well understood. We present a 66-year-old male who had thrombocytopenia with dysplastic features in peripheral blood neutrophils (hypogranular, hyposegmented neutrophils) comprising the Pelger-Huet anomaly, increased blasts in the marrow, and markers consistent with AML. Diagnostic marrow cytogenetics showed a complex karyotype including del(5q), a novel unbalanced dicentric translocation, t(17;20), resulting in both del(20q) and del(17p). Fluorescence in situ hybridization (with probe TP53) showed deletion of 17p13 on the dicentric chromosome, completing the criteria for the 17p- syndrome. Fluorescence in situ hybridization with probes for two tumor suppressor genes on chromosome 5q also showed deletion (CSF1R [at 5(q33.2-q33.4) and EGR-1 [5(q31-q32)]). Remission was difficult to achieve and cytogenetic relapse occurred 6 months postdiagnosis, and clinical relapse approximately one month later. Our case provides a novel mechanism for the 17p- syndrome, and highlights the difficulty of attributing prognostic significance to a particular cytogenetic abnormality in AML. PMID- 10748300 TI - Monosomy 16 as the sole abnormality in myeloid malignancies. AB - The majority of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients reported with chromosome 16 abnormalities had the inv(16)(p13q22) or t(16;16)(p13;q22) rearrangements, which were associated with a favorable prognosis. In contrast, del(16)(q22) was reported less commonly but was associated with a less favorable prognosis. We describe an 80-year-old woman who presented with MDS (refractory anemia). Chromosome analysis from bone marrow aspirate cultures showed monosomy 16 as the sole cytogenetic abnormality. Comparison of this patient with previously reported cases of monosomy 16 showed that this uncommon abnormality was associated with myeloid disorders. Monosomy 16 patients, similar to del(16)(q22) patients, tended to be elderly, presented with MDS or AML, and had a poor prognosis. The similarity in clinical course for del(16)(q22) and monosomy 16 patients suggests that the phenotype in both groups resulted from loss of important gene(s) on 16q, as distinct from the fusion gene product identified in the inv(16) and t(16;16) rearrangements. PMID- 10748301 TI - Neurofibromatosis 2 phenotypes and germ-line NF2 mutations determined by an RNA mismatch method and loss of heterozygosity analysis in NF2 schwannomas. AB - We used a novel RNase cleavage assay (NIRCA) to screen for neurofibromatosis 2 (NF2) mutations in NF2 schwannomas. Mutations were found in tumors in 16 of 20 patients. Eleven patients (55%) had loss of heterozygosity or loss of one allele, indicating that the mutation was a germ-line mutation. The phenotypes of these patients were consistent with previous NF2 genotype-phenotype correlation studies: patients with nonsense mutations had severe phenotypes, whereas those with splice-site or missense mutations had milder and variable phenotypes. These results confirm the utility of NIRCA as a rapid and convenient method for screening for germ-line NF2 mutations. PMID- 10748302 TI - Myocardial revascularisation: the search for the holy grail. PMID- 10748305 TI - The importance and complexity of neurohumeral over-activity in chronic heart failure PMID- 10748304 TI - Chronic beta-blocker treatment in patients with advanced heart failure. Effects on neurohormones. AB - BACKGROUND: To date, the use of beta-blockers in treating patients with chronic heart failure gains support, this since several large clinical trials reported reduced mortality after chronic beta-blockade. Part of these beneficial effects may result from inhibition of deleterious neurohormone activation that accompanies progression of chronic heart failure. The present study evaluates whether this neurohormone inhibition is preserved after chronic beta-blockade. METHODS: In a retrospective analysis the neurohormonal profiles of patients with moderate to severe chronic heart failure were studied from three treatment subgroups: (1) Without beta-blockers or ACE-inhibitors (n=15), (2) without beta blockers, with ACE-inhibitors (n=324), (3) with beta-blockers and ACE-inhibitors (n=31). Patients were on beta-blockers for an average period of 3.8 years. Plasma samples were obtained under controlled conditions. RESULTS: Despite uneven group sizes, the groups were well matched for clinical characteristics. Plasma renin levels were significantly lower in patients treated adjunctively with beta blockers. Plasma aldosterone and endothelin-I levels also tended to be lower after chronic beta-blockade, however, this did not reach statistical significance. CONCLUSIONS: Chronic adjunctive beta-blocker treatment shows significantly lower plasma renin levels when compared to single ACE-inhibition. This persistent reduction of plasma neurohormone activation may concomitantly reduce the chance of neurohormones to escape from inhibition. PMID- 10748303 TI - Exercise training in heart failure: effect on morbidity and mortality. PMID- 10748306 TI - Superiority of endothelin-1 over norepinephrine in exercise-induced alterations of the conduit artery tone of the non-exercised arm in patients with chronic heart failure. AB - This study is aimed at examining the relative importance of norepinephrine and endothelin-1 in treadmill exercise-induced changes in brachial arterial tone of the non-exercised arm in patients with chronic heart failure (CHF). Brachial artery diameter and blood flow were measured before and after exercise in eight healthy volunteers and 18 patients with stable chronic heart failure by high resolution ultrasound. Maximal exercise resulted in brachial artery dilatation in controls (4.42+/-0.39 vs. 4.77+/-0.39 mm; P<0. 0001) in contrast to constriction seen in the patients (5.27+/-0.67 vs. 5.12+/-0.66 mm; P=0.07). Both groups demonstrated a significant increase in blood flow after exercise. The pre exercise (2.83+/-0.76 vs. 1.69+/-0.15 pmol/l; P=0.0004), post-exercise (4.15+/ 1.5 vs. 2. 02+/-0.34 pmol/l; P=0.0004) and the percent increase (47.15+/-32.5 vs. 19.0+/-10.5%; P=0.02) in endothelin-1 levels were significantly greater in patients than in controls. In contrast to endothelin-1, the exercise-induced percent increase in norepinephrine was greater in controls than patients (100.7+/ 51.8 vs. 49.8+/-43.4%; P=0.01). The percent change in the diameter of the brachial artery in response to maximal exercise was significantly correlated to pre- (r=0.634; P=0.003) and post-exercise (r=0.467; P=0.05) endothelin-1 levels in patients but not in controls [pre-exercise (r=0.07; P=0. 86), post-exercise (r=0.310; P=0.47)]. The change in the diameter of the brachial artery did not correlate with pre- or post-exercise plasma norepinephrine levels in either group. These findings suggest that endothelin-1 is potentially more important than norepinephrine in contributing exercise-induced brachial artery constriction in patients with chronic heart failure. PMID- 10748307 TI - Indapamide blocks the rapid component of the delayed rectifier current in atrial tumor cells (AT-1 cells). AB - We studied the effects of a well known blocker (indapamide) of the slow component (I(ks)) of the delayed rectifier (I(k)) on K(+) currents in atrial tumor myocytes derived from transgenic mice (AT-1 cells) using one electrode voltage clamp method. These cells have been shown to express mRNAs encoding cardiac K(+) channels and display a cardiac electrophysiological phenotype. The major K(+) current is the rapid component (I(kr)) of the delayed rectifier current (I(k)). The purpose of this study was to show that a diuretic agent, indapamide, which was shown to be a selective blocker of the slow component (I(ks)) of delayed rectifier, also blocks I(kr) in a dose dependent manner. The steady state current at the end of a 1s pulse (I(1s), step to +40 mV from a holding potential of -40 mV) was 1070.4+/-202.2 pA (n=5) and the tail current (I(tail)) was 416.3+/-112.9 pA. Indapamide (750 microM) reduced I(1s) and I(tail) to 254.5+/-62.3 pA and 42.2+/-37.7 pA respectively. Indapamide induced block was partially reversible for higher concentrations (> or =750 microM). PMID- 10748308 TI - Atrial fibrillation and stroke: clinical presentation of cardioembolic versus atherothrombotic infarction. AB - The aim of the study was to compare demographic characteristics, anamnestic findings, cerebrovascular risk factors, and clinical and neuroimaging data of cardioembolic stroke patients with and without atrial fibrillation and of atherothrombotic stroke patients with and without atrial fibrillation. Predictors of early diagnosis of cardioembolic vs. atherothrombotic stroke infarction in atrial fibrillation patients were also determined. Data of cardioembolic stroke patients with (n=266) and without (n=81) atrial fibrillation and of atherothrombotic stroke patients with (n=75) and without (n=377) were obtained from 2000 consecutive patients included in the prospective Sagrat Cor-Alianza Hospital of Barcelona Stroke Registry. Risk factors, clinical characteristics and neuroimaging features in these subgroups were compared. The independent predictive value of each variable on early diagnosis of stroke subtype was assessed with a logistic regression analysis. In-hospital mortality in patients with atrial fibrillation was significantly higher than in non-atrial fibrillation patients both in cardioembolic (32.6% vs. 14.8%, P<0. 005) and atherothrombotic stroke (29.3% vs. 18.8%, P<0.04). Valvular heart disease (odds ratio (OR) 4.6; 95% confidence interval (95% CI) 1.19-17.68) and sudden onset (OR 1.8; 95% CI 0.97-3.63) were predictors of cardioembolic stroke, and subacute onset (OR 8; 95% CI 1.29-49.42), COPD (OR 5.2; 95% CI 1.91-14.21), hypertension (OR 3. 63; 95% CI 1.92-6.85), hypercholesterolemia (OR 2.67; 95% CI 1.13-6. 28), transient ischaemic attack (OR 2.49; 95% CI 1.05-5.90), ischaemic heart disease (OR 2.30; 95% CI 1.15-4.60) and diabetes (OR 2.26; 95% CI 1.14-4.47) of atherothrombotic stroke. In conclusion, some clinical features at stroke onset may help clinicians to differentiate cerebral infarction subtypes in patients with atrial fibrillation. Atrial fibrillation is associated with a higher in-hospital mortality both in cardioembolic and atherothrombotic stroke patients. PMID- 10748309 TI - Ventricular tachyarrhythmias following coronary surgery: predisposing factors. AB - The perioperative factors potentially associated with post-coronary artery bypass grafting (CABG) ventricular tachyarrhythmias (VT) onset have not been deeply investigated. Monomorphic or polymorphic ventricular tachycardia and ventricular fibrillation represent the most dreadful arrhythmic events that can complicate the postoperative course of CABG. As a consequence, the aim of our paper was to identify which perioperative variables might predict post-CABG VT occurrence. One hundred and fifty-two consecutive patients who underwent CABG surgery at our Institution were included in the study. Post-CABG VT occurred in 13 out of 152 patients (8.5%, six cases of monomorphic ventricular tachycardia and seven cases of ventricular fibrillation). At univariate analysis, VT patients were significantly younger (54.8+/-6.6 vs. 60.1+/-8.8, P=0.038), exhibited a more severe coronary artery disease (CAD) (number of diseased vessels 2.92+/-0.3 vs. 2.45+/-0.7, P=0.023, and percentage of patients with three-vessel CAD 91.7% vs. 57.3%, P=0.043) and received a greater number of CABGs than those remaining in sinus rhythm (SR) (percentage of patients receiving three or more CABGs 76. 9% vs. 38.8%, P1000 76.9% vs. 38%, Pnormal range 72.7% vs. 30.7%, P=0.014), electrolyte derangement (84.6% vs. 45.6%, P=0.017) and a severe haemodynamic impairment (need for IABP 23% vs. 2.9%, P1000, postoperative electrolyte imbalance, the need for three or more CABGs and of IABP all were independent correlates for VT. In conclusion, post-CABG VT seem to be related to the preexistence of a severe underlying coronary artery disease along with perioperative triggering factors such as acute ischemia, electrolytic disorders and a sudden haemodynamic impairment. PMID- 10748310 TI - beta-blocker therapy in patients after aortic valve replacement for aortic regurgitation. AB - BACKGROUND: beta-blocker therapy for dilated or ischemic cardiomyopathy is now an accepted and effective treatment. However, little is known about its efficacy in patients with postoperative impaired left ventricular function. This retrospective study was designed to assess the effects of beta-blocker therapy in patients after aortic valve replacement (AVR) for aortic regurgitation (AR). METHODS: A total of 59 patients who underwent AVR for chronic AR were assigned to four groups. Twelve patients were treated with both ACE inhibitors and beta blockers, 12 patients with only ACE inhibitors, eight patients with only beta blockers, and 27 patients without beta-blockers or ACE inhibitors. A postoperative echocardiographic study was performed one year after surgery. RESULTS: The heart rate was significantly reduced in patients with beta-blockers despite the use of ACE inhibitors after surgery. Postoperative left ventricular volume was more significantly decreased in beta patients than in non-beta patients despite the use of ACE inhibitors. There were also significant reductions in left ventricular mass index in ACE+beta patients compared to ACE+non-beta patients. However, there were no significant differences in NYHA functional class and survival rate between beta patients and non-beta patients. CONCLUSIONS: beta-blocker therapy may improve cardiac performance by reducing cardiac volume and mass in patients with impaired LV function after AVR for AR. PMID- 10748311 TI - Effect of mibefradil on heart rate variability in patients with chronic heart failure. AB - BACKGROUND: Mibefradil was recently withdrawn from the market because of an unfavorable clinical profile in patients with chronic heart failure. Although drug interactions appear to play a role, other mechanisms such as proarrhythmia and autonomic deterioration could also be relevant. Chronic heart failure is accompanied by autonomic impairment and analysis of heart rate variability can be used to examine autonomic modulation of heart rate. METHODS: We studied 18 heart failure patients (age 63.2+/-10.1 years (mean+/-S.D. ), ejection fraction 0.21+/ 0.07) treated with mibefradil or placebo, who participated in the MACH-I (Mortality Assessment in Chronic Heart failure) trial in our center, and compared them with 18 healthy matched controls. Heart rate variability analysis was performed at baseline and after 7 months of treatment. RESULTS: At baseline, heart rate variability parameters were impaired in patients with heart failure compared to healthy controls (P<0.05). After 7 months of treatment a reduction in (24-h) heart rate was observed (P=0.02, versus placebo). Apart from the effect on mean NN, no significant differences were observed for the remaining heart rate variability parameters. CONCLUSIONS: Mibefradil does not impair autonomic balance and in fact reduces heart rate in patients with heart failure. These findings suggest that autonomic activation did not contribute to the adverse effects of mibefradil. PMID- 10748312 TI - Exercise endpoints in patients with chronic heart failure. AB - BACKGROUND: Peak oxygen consumption (V(O(2))) is a powerful predictor of outcome in patients with chronic heart failure. This is not a test that is readily clinically available. We therefore sought to establish a method of assessing peak V(O(2)) from non-invasively acquired data. METHODS: We analysed the results from incremental treadmill exercise tests in 60 patients [aged 59.0 (S.D. 12.4) years] with chronic heart failure or left ventricular dysfunction [left ventricular ejection fraction (29.6 (15.2)%)] and 52 control subjects [aged 36.7 (12.3)]. Metabolic gas exchange during exercise was measured with a respiratory mass spectrometer. Heart rate and blood pressure were measured. RESULTS: Peak V(O(2)) was lower in patients than controls [19.9 (7.7) ml/kg/min vs. 38.3 (9.0), P<0. 001]. Exercise time (r=0.84, P<0.001), heart rate at peak exercise (r=0.63, P<0.0001), change in heart rate (r=0.72, P<0.0001), rate pressure product at peak exercise (r=0.64, P<0.0001) and change in systolic blood pressure (r=0.31, P=0.002) all correlated with peak V(O(2)). In a stepwise regression model, exercise time was the most powerful predictor of peak V(O(2)) (r(2)=0.79). The only additional independent variable was change in heart rate from rest to peak exercise, which increased r(2) to 0.80. In a survival analysis, measured peak V(O(2)) and the peak V(O(2)) estimated from exercise time and change in heart rate had similar predictive power. CONCLUSIONS: In this preliminary study, peak V(O(2)) can be estimated from non-invasively acquired parameters. Estimated peak V(O(2)) and measured peak V(O(2)) have similar predictive power for outcome. Further work is necessary to see if estimated peak V(O(2)) is widely applicable in a clinical setting. PMID- 10748313 TI - Angiographic presentation of coronary artery spasm in heart transplant recipients. AB - We describe the angiographic characteristics of coronary artery spasm observed in 12 out of 247 (4.9%) patients who underwent 808 coronary angiographies after heart transplantation. Coronary artery spasm was diagnosed when localized and reversible narrowing of the coronary lumen was identified. After coronary artery spasm identification all patients were followed-up clinically for a mean period of 5.1 years. Coronary artery spasm was documented 1-3 years after heart transplant. Coronary artery spasm affected 1 main coronary artery in 10 patients and 2 in 2 patients; in 3 patients 1 or more secondary branches were also affected. The right coronary artery was affected by coronary artery spasm in 8 patients and the anterior descending coronary artery in 6 patients. In 6 patients coronary artery spasm was mechanically induced by the catheter tip. The degree of luminal narrowing due to coronary artery spasm ranged from mild to almost complete occlusion. Coronary artery spasm appeared as a single tubular smooth and concentric stenosis in 8 patients, was discrete in 2 patients and multiple on the same vessel in 2 patients. In 1 patient coronary artery spasm was erroneously interpreted as an organic lesion and percutaneous transluminal coronary angioplasty was planned. During follow-up 3 patients out of 4 who had shown multiple coronary artery spasm died and 2 patients developed critical organic stenosis. In conclusion coronary artery spasm after heart transplant is less rare than commonly believed. Although it usually has a peculiar appearance, it can be misinterpreted as an organic lesion. Multiple coronary artery spasm appears to carry a poor prognosis. PMID- 10748314 TI - Recurrent pulmonary adenoid cystic carcinoma presenting as a cardiac tumor. AB - A 58-year-old patient is presented who had a pulmonary adenoid cystic carcinoma which recurred 10 years after sleeve left pneumonectomy. The patient developed acute heart failure because the lesion obstructed blood flow by compressing the left atrium. Transesophageal echocardiography and magnetic resonance imaging demonstrated a solid mass arising from the pericardium which displaced the posterior wall of the left atrium. The mass was resected. Postoperative radiation was not performed. PMID- 10748319 TI - Tissue transglutaminase: a possible role in neurodegenerative diseases. AB - Tissue transglutaminase is a multifunctional protein that is likely to play a role in numerous processes in the nervous system. Tissue transglutaminase posttranslationally modifies proteins by transamidation of specific polypeptide bound glutamines. This action results in the formation of protein crosslinks or the incorporation of polyamines into substrate proteins, modifications that likely have significant effects on neural function. Tissue transglutaminase is a unique member of the transglutaminase family as in addition to catalyzing the calcium-dependent transamidation reaction, it also binds and hydrolyzes ATP and Guanosine 5'-triphosphate and may play a role in signal transduction. Tissue transglutaminase is a highly regulated and inducible enzyme that is developmentally regulated in the nervous system. In vitro, numerous substrates of tissue transglutaminase have been identified, and several of these proteins have been shown to be in situ substrates as well. Several specific roles for tissue transglutaminase have been described and there is evidence that tissue transglutaminase may also play a role in apoptosis. Recent findings have provided evidence that dysregulation of tissue transglutaminase may contribute to the pathology of several neurodegenerative conditions including Alzheimer's disease and Huntington's disease. In both of these diseases tissue transglutaminase and transglutaminase activity are elevated compared to age-matched controls. Further, immunohistochemical studies have demonstrated that there is an increase in tissue transglutaminase reactivity in affected neurons in both Alzheimer's and Huntington's disease. Although intriguing, many issues remain to be addressed to definitively establish a role for tissue transglutaminase in these neurodegenerative diseases. PMID- 10748320 TI - The yin and yang of tenascin-R in CNS development and pathology. AB - An important biological consequence of the initial interactions between the cell surface and its extracellular environment is the diversity of cellular responses ranging from overt repulsion or avoidance reaction to stable adhesion or final positioning. It is now evident that positive and negative guiding mechanisms are equally relevant to normal pattern formation during development and decisive for the outcome of a regenerative process. In this context, the present review summarizes the knowledge about the extracellular matrix glycoprotein tenascin-R, a member of the tenascin gene family. In contrast to all other known family members, tenascin-R is exclusively expressed in the central nervous system of vertebrates by oligodendrocytes and neuronal subsets at later developmental stages and in adulthood. We focus on the glycoprotein's structure, tissue distribution and functional implications in the molecular control of axon targeting, neural cell adhesion, migration and differentiation during nervous system morphogenesis and pathology. PMID- 10748321 TI - The somatosensory evoked magnetic fields. AB - Averaged magnetoencephalography (MEG) following somatosensory stimulation, somatosensory evoked magnetic field(s) (SEF), in humans are reviewed. The equivalent current dipole(s) (ECD) of the primary and the following middle latency components of SEF following electrical stimulation within 80-100 ms are estimated in area 3b of the primary somatosensory cortex (SI), the posterior bank of the central sulcus, in the hemisphere contralateral to the stimulated site. Their sites are generally compatible with the homunculus which was reported by Penfield using direct cortical stimulation during surgery. SEF to passive finger movement is generated in area 3a or 2 of SI, unlike with electrical stimulation. Long-latency components with peaks of approximately 80-120 ms are recorded in the bilateral hemispheres and their ECD are estimated in the secondary somatosensory cortex (SII) in the bilateral hemispheres. We also summarized (1) the gating effects on SEF by interference tactile stimulation or movement applied to the stimulus site, (2) clinical applications of SEF in the fields of neurosurgery and neurology and (3) cortical plasticity (reorganization) of the SI. SEF specific to painful stimulation is also recorded following painful stimulation by CO(2) laser beam. Pain-specific components are recorded over 150 ms after the stimulus and their ECD are estimated in the bilateral SII and the limbic system. We introduced a newly-developed multi (12)-channel gradiometer system with the smallest and highest quality superconducting quantum interference device (micro-SQUID) available to non-invasively detect the magnetic fields of a human peripheral nerve. Clear nerve action fields (NAFs) were consistently recorded from all subjects. PMID- 10748322 TI - Neurotoxicity associated with neuroleptic-induced oral dyskinesias in rats. Implications for tardive dyskinesia? AB - Tardive dyskinesia is a serious motor side effect of long-term treatment with neuroleptics, with an unknown pathophysiologic basis. Brain damage and aging are prominent risk-factors, and together with the persistent character of the disorder, it is likely that long-lasting neuronal changes are involved in the pathogenesis. It has been hypothesized that striatal neurodegeneration caused by excitotoxic mechanisms and oxidative stress may play an important role in the development of the disorder, and the scope of the present work is to review the evidence supporting this hypothesis. The rat model of tardive dyskinesia has been used extensively in the field, and the usefulness of this model will be discussed. Neuroleptics are able to induce oxidative stress in vitro and increase striatal glutamatergic activity in rats, which may lead to toxic effects in the striatum. Drugs that block excitotoxicity inhibit the development of persistent oral dyskinesia in the rat model, and impaired energy metabolism leads to increased frequency of oral dyskinesia. There are also signs of altered striatal histology in rats with high frequency of oral dyskinesia. Furthermore, markers of increased oxidative stress and glutamatergic neurotransmission have been found in the cerebrospinal fluid of patients with tardive dyskinesia. In conclusion, several lines of evidence implicate neurotoxic events in the development of neuroleptic induced tardive dyskinesia. PMID- 10748323 TI - An examination of gender differences in DPOAE phase delay measurements in normal hearing human adults. AB - This study examined gender differences in f(1)- and f(2)-sweep distortion product otoacoustic emission (DPOAE) phase delay measures in 60 normal-hearing human adults. Phase delay measures were obtained at six different f(2) frequencies ranging from 1.1 to 6.0 kHz (f(2)/f(1) ratios were 1.1-1.3). Primary levels for f(2) were 45 and 50 dB SPL (f(1)f(2)). Gender differences have been observed in normal-hearing human adults in both auditory brainstem response (ABR) and f(1) sweep DPOAE studies. Gender differences in delay have been attributed to differences in the average length of the cochlea, where female cochleas are 13% shorter than male cochleas. Previously, the authors have proposed that the f(1) sweep phase delay estimate is predominantly composed of a level-independent transport time to the site of DPOAE generation and a small proportion of the level and frequency-dependent filter build-up time. The f(2)-sweep delay also contains the transport time, however, it is predominantly composed of the filter build-up time. Therefore, delay differences between stimulation paradigms are equal to a proportion of the filter build-up time. In this study, mean f(1)- and f(2)-sweep delays were significantly longer in male ears than female ears at 1.1 kHz (45 and 50 dB). At 50 dB, f(1)-sweep phase delay measures were 18% longer in male ears (6.5 ms) than female ears (5.5 ms). Mean f(2)-sweep delays were 23% longer in male ears (10.0 ms) than female ears (8.1 ms). This gender difference was not observed when the isolated filter build-up time was calculated from the DPOAE phase delay difference. These observations may therefore be attributed to a gender-related anatomical difference in cochlear length. PMID- 10748324 TI - Abnormal cochlear connective tissue mineralization in the palmerston north autoimmune mouse. AB - Inner ear fibrosis and osteogenesis are common features of human autoimmune disease, although the cellular mechanisms are unknown. The Palmerston North (PN) autoimmune strain mouse has been shown to develop modiolar sclerotic lesions with progression of its systemic disease. Therefore, lesion development was studied in the cochleas of PN mice to gain insight into potential autoimmune osteogenic processes in the human ear. Cochleas from PN mice were examined with electron microscopy to characterize the cellular and extracellular matrix changes that lead to abnormal mineralization. Initially, activated fibroblasts produced extracellular matrix fibers, ranging in size from fine fibrils to larger collagen like fibers. These proliferating fibers appeared to 'seed' the mineralizing lesions by serving as the framework for mineral deposition. As mineralization continued, the foci grew in size and fused to form large sclerotic masses within the connective tissue. However, the lesions never invaded nor degraded the normal modiolar bone. These observations of abnormal mineralization of cochlear connective tissue fibers show some parallels with human cochlear autoimmune osteogenesis, suggesting similar molecular processes may be involved. PMID- 10748325 TI - Masculinizing effects on otoacoustic emissions and auditory evoked potentials in women using oral contraceptives. AB - The otoacoustic emissions (OAEs) and auditory evoked potentials (AEPs) measured in two separate large scale studies were examined retrospectively for potential differences between those women using, and those not using, oral contraception (OC). Fourteen dependent variables were examined, all of which exhibited substantial sex differences. For 13 of those 14 dependent variables, the means for the users of OC were shifted away from the means of the non-users in the direction of the males. Specifically, for four different measures of OAE strength, for seven of eight measures of AEP latency or amplitude, and for two cognitive tests (mental rotation and water level), the means for the users of OC were located intermediate to those of the non-users of OC and the males. Few of these differences between users and non-users of OC achieved statistical significance, but the near universality of the direction of the difference suggests that oral contraceptives do produce a weak masculinizing effect on some auditory structures. These weak masculinizing effects appear to run contrary to the facts that the levels of both free testosterone and estradiol are lower in women using OC than in normal-cycling women. Past findings on auditory sex differences may have underestimated those sex differences. PMID- 10748326 TI - Antioxidants attenuate gentamicin-induced free radical formation in vitro and ototoxicity in vivo: D-methionine is a potential protectant. AB - We have recently suggested antioxidant therapy against aminoglycoside-induced hearing loss based on the hypothesis of a redox-active aminoglycoside-iron complex causing ototoxicity. The present study compares seven antioxidants and iron chelators for their ability to attenuate gentamicin-induced free radical generation in vitro and ototoxicity in guinea pig in vivo. Free radical formation by gentamicin was measured by chemiluminescence detection both in a non-enzymatic system in vitro and in cell culture. Deferoxamine, 2,3-dihydroxybenzoate, or salicylic acid suppressed gentamicin-induced luminescence in both tests. This indicated the usefulness of the assay as a screen for potential protectants since these agents had previously been shown to attenuate gentamicin-induced ototoxicity in vivo. Histidine and D-methionine, amino acids with chelating and antioxidant properties, also suppressed gentamicin-mediated luminosity both in vitro and in cell culture. In contrast, the metal chelators succimer (2, 3 dimercaptosuccinic acid (DMSA)) and trientine (N, N'-bis[2-aminoethyl]-1,2 ethanediamine) promoted free radical formation and were excluded from further studies. Histidine and D-methionine were then administered to guinea pigs receiving concurrent treatment with gentamicin (120 mg/kgx19 days). Threshold shifts induced by gentamicin were significantly attenuated by twice-daily injections of D-methionine. Once-daily injections of histidine or D-methionine were less effective, pointing to the importance of pharmacokinetics in antioxidant protection in vivo. The study presents a simple screening system for agents with the potential to attenuate gentamicin-induced hearing loss. It also supports the hypothesis of free radical formation as an underlying cause of gentamicin ototoxicity. PMID- 10748327 TI - Glial cell line-derived neurotrophic factor has a dose dependent influence on noise-induced hearing loss in the guinea pig cochlea. AB - We examined the effectiveness of glial cell line-derived neurotrophic factor (GDNF) to attenuate cochlear damage from intense noise stress. Subjects were exposed to 115 dB SPL one octave band noise centered at 4 kHz for 5 h. They received artificial perilymph with or without GDNF into the left scala tympani at 0.5 microliter/h from 4 days before noise exposure through 8 days following noise exposure. Different concentrations of GDNF (1 ng/ml, 10 ng/ml, 100 ng/ml, and 1 microgram/ml) were applied chronically directly into the guinea pig cochlea via a microcannula and osmotic pump. Noise-induced hearing loss was assessed with pure tone auditory brainstem responses (at 2, 4, 8 and 20 kHz), measured prior to surgery, 1 day before noise exposure, and 7 days following noise exposure. Subjects were killed on day 8 following exposure for histological preparation and quantitative assessment of hair cell (HC) damage. A dose-dependent protective effect of GDNF on both sensory cell preservation and hearing function was found in the treated ears. At 1 ng/ml, GDNF showed no significant protection; at 10 ng/ml, GDNF showed significant HC protection; and at 100ng/ml, it was greater and bilateral. At 1 microgram/ml, GDNF appeared to have a toxic effect under noise stress in some cochleae. These findings indicate that GDNF at certain concentrations can effectively protect the inner ear from noise-induced hearing loss. PMID- 10748328 TI - Neither endocochlear potential nor tegmentum vasculosum are affected in hearing impaired belgian waterslager canaries. AB - We previously showed that the Belgian Waterslager canary strain is affected by a hereditary hearing loss that is associated with a reduced number of hair cells and hair cell pathologies in the basilar papilla. Since hair cell pathologies were also present in the sacculus, Weisleder et al. (1994) suggested that these birds are afflicted by Scheibe's like dysplasia, a cochleo-saccular defect. In mammals, cochleo-saccular defects are characterized primarily by the lack of an endocochlear potential and abnormalities in the Stria vascularis which only secondarily lead to hair cell loss (Steel and Bock, 1983; Steel, 1994; 1995). Here we report the endocochlear potential of six ears from three non-Belgian Waterslager canaries and three ears of two Belgian Waterslager canaries to decide if Waterslager canaries are affected by a cochleo-saccular or by a neuroepithelial defect. The mean endocochlear potential was 17.6+/-2. 5 mV in the non-Waterslager canaries and 20.3+/-0.6 mV in Waterslager canaries. In addition, and consistent with the presence of a normal endocochlear potential, light microscopy of the tegmentum vasculosum provided no evidence for pathology. These data show that Belgian Waterslager canaries are affected by a neuroepithelial rather than a cochleo-saccular inner ear defect. PMID- 10748329 TI - Effects of contralateral noise on measurement of the psychophysical tuning curve. AB - The effects of the addition of contralateral noise on the psychophysical tuning curve (PTC) were examined in subjects with normal hearing. The masking threshold of the tail part of the PTC tended to decrease with the addition of contralateral noise, although the threshold reduction was usually less than 5 dB. On the other hand, the effects of contralateral noise were relatively small around the tip of the PTC contour. Focusing on the effects of contralateral noise on the masking threshold at the tail part of the PTC, the effects of changing the time between initiation of masking the tone and the presentation of the masked probe tone on the threshold reduction at the tail part of the PTC were also observed. The results indicate that the reduction of the masking threshold by the addition of contralateral noise tended to be larger when the presentation of the signal tone was delayed after the onset of the masker. Usually, when the signal tone was presented under conditions of the forward masking paradigm, the reduction of the threshold was most remarkable. Results obtained in the present study are discussed based on the known characteristics of the olivocochlear (OC)-efferent fibers activated by contralateral noise. PMID- 10748330 TI - Thermal damage threshold at 633 nm of tympanic membrane of pig. AB - Doppler vibrometers are used by many research groups to monitor the motion of the tympanic membrane (TM) and of middle ear ossicles for in vivo and in vitro studies. Power densities in these applications reach 80 W/cm(2). To determine the safe limit of exposure, a cw dye laser at a wavelength of 633 nm was used to investigate the threshold of thermal damage of TM of pigs under exposure times of 60 s. To determine the applied power density accurately, the spot size of the laser beam was monitored by an objective lens and a CCD camera. Twenty-six laser exposed samples of TM were stained by haematoxylin and eosin stain and the semi thin sections were examined microscopically. In none of the sections was any laser induced damage observed with power densities below 7.1 kW/cm(2), whereas serious damage occurred showing coagulation, carbonisation and perforation in all cases with laser powers above 8.2 kW/cm(2). The threshold for damage and the conical shape of the damage zone is explained by photon propagation and absorption in the tissue especially by the increase of the scattering factor at higher tissue temperature. The thermal damage threshold of 8 kW/cm(2) is compared to the maximum permissible exposure given in laser safety standards for skin. PMID- 10748332 TI - Moderate noise trauma in juvenile cats results in profound cortical topographic map changes in adulthood. AB - Cortical topographic map changes have been reported after profound drug-induced hearing loss in neonates, after progressive high-frequency hearing loss, and after mechanically induced lesions in the cochlea of adult animals. The present study demonstrates that exposure of 5-week-old kittens to a loud 6 kHz tone, producing mild to moderate high-frequency hearing loss, induces a profound reorganization of the frequency map in auditory cortex. In the reorganized cortical region, the frequency-tuning curves were of normal sharpness with near normal thresholds. Inhibitory tuning curve bandwidths were similar to those in control animals. Spontaneous activity in the reorganized part of the cortex was significantly increased. In contrast, the strength of the cross-correlation of the spontaneous activity of units recorded on different electrodes was the same in the normal and reorganized part. Minimum first-spike latency was significantly increased in trauma cats, largely for units at the dorsal side of the sampled region. Because most other neural response properties are normal in the reorganized part of cortex, sub-cortical topographic map changes are likely involved in producing the altered cortical topographic maps. PMID- 10748331 TI - Effects of exposure to an augmented acoustic environment on auditory function in mice: roles of hearing loss and age during treatment. AB - The effects of exposure to an augmented acoustic environment (AAE) on auditory function were evaluated in mouse strains that exhibit various degrees and time courses of progressive hearing loss (BXD-22, BXD-12, BXD-16, BXD-14, BALB/cJ), and in normal-hearing CBA/CaJ mice. Beginning at age 25 days, mice were exposed 12 h every night to a 70 dB SPL broadband noise AAE. The AAE was maintained for at least 30 days in each strain. Same-strain control mice were age-matched and maintained under normal vivarium acoustic conditions. The auditory brainstem response (ABR), acoustic startle response amplitude, and prepulse inhibition (PPI) were used to assess the auditory system. Exposure to the AAE resulted in improved auditory performance (better PPI, lower ABR thresholds) when hearing impairment was present, but not when hearing was normal. The ameliorative effects occurred irrespective of a mouse's age at the onset of hearing loss, as long as initiation of AAE treatment preceded the occurrence of severe hearing loss. If AAE treatment was delayed beyond such a point, loss of threshold sensitivity progressed as usual, although PPI could still benefit. Finally, AAE treatment can slow, but not prevent, the occurrence of severe genetically determined hearing loss. PMID- 10748333 TI - Auditory perception in vestibular neurectomy subjects. AB - The auditory efferent nerve is a feedback pathway that originates in the brainstem and projects to the inner ear. Although the anatomy and physiology of efferents have been rather thoroughly described, their functional roles in auditory perception are still not clear. Here, we report data in six human subjects who had undergone vestibular neurectomy, during which their efferent nerves were also presumably severed. The surgery had alleviated these subjects' vertigo but also resulted in mild to moderate hearing loss. We designed our experiments with a focus on the possible role of efferents in anti-masking. Consistent with previous studies, we found little effects of vestibular neurectomy on pure-tone detection and discrimination in quiet. However, we noted several new findings in all subjects tested. Efferent section increased loudness sensation (one subject), reduced overshoot effect (five subjects), accentuated 'the midlevel hump' in forward masking (two subjects), and worsened intensity discrimination in noise (four subjects). Poorer speech in noise recognition was also observed in the surgery ear than the non-surgery ear in three out of four subjects tested, but this finding was confounded by hearing loss. The present results suggest an active role of efferents in auditory perception in noise. PMID- 10748334 TI - Characteristics of reliable tone-evoked oscillations in the rat thalamo-cortical auditory system. AB - Tone-evoked oscillations were studied from simultaneous recordings collected in the auditory cortex, auditory thalamus and auditory sector of the reticular nucleus in urethane anesthetized rats. These oscillations were precisely time locked to tone onset and were easily observed on peristimulus time histograms (PSTHs). Visual inspection of PSTHs and rasters led us to distinguish between 'reliable' oscillations (which exhibited oscillatory patterns in more than 50% of the trials) and 'labile' oscillations (which exhibited oscillations in less than 50% of the trials). Systematic quantification of oscillations based on several indices derived from power spectra confirmed this distinction. 'Reliable' stimulus-locked oscillations were observed in 51/184 (28%) of the recordings from auditory cortex, 9/55 (17%) of the recordings from auditory thalamus and 11/26 (42%) of the recordings from the auditory sector of the reticular nucleus. The frequency range of these oscillations was the same in the three structures (5-14 Hz). Within the same animal, when one electrode exhibited oscillations, there was a high probability of detecting similar oscillations from electrodes located in the same structure, but not from electrodes located in the other structures. These oscillations were observed for pure tone frequency (or for clicks) whatever the tone duration (1 s, 100 ms, 10 ms). The inter-tone interval (ITI) was found to be the critical factor controlling the occurrence of these oscillations: they were present for ITIs of 2 s or longer, but were absent for ITIs of 1 s or less. In contrast, the occurrence of the oscillations was a function neither of the strength of the 'on' evoked response nor of the animal's temperature. However, lowering the animal's temperature from 37-38 degrees C to 35-36 degrees C systematically led to a decrease in the frequency and an increase in the duration of the tone-evoked oscillations. These results suggest that, even in well defined conditions (temperature, EEG, ITI, level of anesthesia), the oscillations triggered by presentation of the same stimulus can be stable or unstable. This temporal instability of stimulus-evoked oscillations has to be taken into account before stating percentages of oscillations in a given brain structure. They also suggest that some general factors such as the animals temperature or the inter stimulus interval can considerably affect their characteristics and/or their occurrence. PMID- 10748335 TI - Age-related changes in auditory function of rhesus monkeys (Macaca mulatta). AB - The purpose of this study was to investigate the changes in auditory function associated with aging in rhesus monkeys (Macaca mulatta) as a model for age related changes in humans. One advantage of using monkeys from the Wisconsin Regional Primate Research Center was that lifestyle factors such as diet and excessive noise exposure were controlled. Twenty younger (mean: 10 years, 9 months, S.D.=+/-6 months) and 20 older (mean: 25 years, 11 months, S.D.=+/-11 months) monkeys were used in this study. Cochlear function in these monkeys was measured with distortion product otoacoustic emissions (DPOAEs); neural function was measured with auditory brainstem responses (ABRs) and middle latency responses (MLRs). Older monkeys had (1) significantly smaller DPOAEs, (2) significantly smaller ABR peak amplitudes, and (3) significantly longer ABR peak latencies compared to younger monkeys. Overall, these results suggest that older monkeys have decreased cochlear and neural function to the level of the brainstem as compared to younger monkeys. The decrease in DPOAE level and the increase in age accounted for approximately 70% of the increase in peak IV latency. PMID- 10748336 TI - Discharge rate of the auditory nerve during noise revealed by electrocochlear stimulation. AB - The purpose of this study was to evaluate the average discharge rate of all fibres in the whole auditory nerve (R(wn)) when a broad-band noise with steady state effects is applied to the ear. We assessed the R(wn) parameter by detecting the state of refractoriness of the nerve during noise stimulation using an electric stimulus (ES) as a probe. The technique, applied in awake pre-implanted guinea pigs (Charlet de Sauvage et al., 1994), made it possible to obtain electro acoustic responses (EARs), from which an estimate of the R(wn) parameter could be deduced. Negative current pulses of 100 micros duration, each followed by an identical pulse of positive polarity for charge balance, were applied between round window and indifferent vertex electrodes at intervals of 160 ms. The 120 ms wide-band noise masker started 92 ms before every other negative ES. The signal on the stimulating electrodes was averaged over a 5.12 ms window in synchrony with the negative pulse. EARs were obtained by alternately subtracting recordings during noise from those during silence. The R(wn) parameter was determined by comparing experimental and computed EAR patterns. For this purpose, a model of unit response incorporating changes in amplitude and conduction velocity during the relative refractory period was designed. The recovery function of the firing probability in response to ES was evaluated. Fibres were classified in different categories according to their background discharge rates. The probability of response of single fibres to ES in each category was calculated on the basis of their interval histograms during silence and noise. Individual spikes were combined accordingly to obtain the computed EAR waveform. R(wn) was determined by adjusting the EAR amplitude of the model in relation to that of the experimental EAR. R(wn) generally increases in a linear fashion with respect to noise intensity expressed in dB, thus following the increase in loudness perception estimated by Weber's law. At the highest noise levels, R(wn) tends to saturate. The estimated saturation rate was found to be about 380 spikes/s. PMID- 10748337 TI - Evidence for active, nonlinear, negative feedback in the vibration response of the apical region of the in-vivo guinea-pig cochlea. AB - The transverse vibration response of the organ of Corti near the apical end of the guinea-pig cochlea was measured in vivo. For cochleae in good physiological condition, as ascertained with threshold compound action potentials and the endocochlear potential, increasing amounts of attenuation and phase lag were found as the intensity was decreased below 80 dB SPL. These nonlinear phenomena disappeared post mortem. The data suggest that an active, nonlinear damping mechanism exists at low intensities at the apex of the cochlea. The phase nonlinearity, evident at all frequencies except at the best frequency (BF), was limited to a total phase change of 0.25 cycles, implying negative feedback of electromechanical force from the outer hair cells into a compliant organ of Corti. The amplitude nonlinearity was largest above BF, possibly due to interaction with a second vibration mode. The high-frequency flank of the amplitude response curve was shifted to lower frequencies by as much as 0.6 octave (oct) for a 50-dB reduction of sound intensity; the reduction of BF was 0.3 oct, but there was no change of relative bandwidth (Q(10 dB)). Detailed frequency responses measured at 60 dB SPL were consistent with non-dispersive, travelling-wave motion: travel time to the place of BF (400 Hz at 60 dB SPL) was 2.9 ms, Q(10 dB) was 1.0; standing-wave motion occurred above 600 Hz. Based on comparison with neural and mechanical data from the base of the cochlea, amplitudes at the apex appear to be sufficient to yield behavioural thresholds. It is concluded that active negative feedback may be a hallmark of the entire cochlea at low stimulus frequencies and that, in contrast to the base, the apex does not require active amplification. PMID- 10748338 TI - Characteristics of the travelling wave in the low-frequency region of a temporal bone preparation of the guinea-pig cochlea. AB - This study provides a detailed quantitative description of the acoustically evoked vibration responses in the low-frequency region of the in vitro guinea-pig cochlea. Responses of the basilar membrane, the reticular lamina and Hensen cells were measured with a laser Doppler vibrometer, without the need for introducing artificial light reflectors. The apex of the cochlea was opened, leaving the helicotrema intact. Two response components were detected: a 'fast' component, which was probably caused by the hole in the cochlea, and a 'slow' component, which shared the features of a classical travelling wave. The velocity response of the 'slow' component exhibited a relatively flat low-frequency slope (15 dB/oct) and a much steeper high-frequency roll-off (third turn: -47 dB/oct; fourth turn: -35 dB/oct). The group delay was dependent on the characteristic frequency. In the fourth turn, the sharpness of the velocity tuning curves (Q(10 dB): 1.0) was similar to those of in vivo mechanical and neural recordings, whereas in the third turn the tuning (Q(10 dB): 1.1) was much less than for in vivo recordings. The results indicate that cochlear amplification, which is responsible for the high sensitivity and sharp tuning in the basal part of the cochlea, is much less pronounced in the apical turn of the cochlea. PMID- 10748339 TI - Activities of Na(+),K(+)-ATPase and Ca(2+)-ATPase in cochlear lateral wall after acoustic trauma. AB - Na(+),K(+)-ATPase and Ca(2+)-ATPase are well known participants in the active transport of ions in the inner ear. These two enzymes play an important role in maintaining cochlear function. Although changes in these enzymes' activities in the cochlea have been implicated in noise-induced hearing loss, no evidence of quantitative alteration of Na(+),K(+)-ATPase or Ca(2+)-ATPase activities has ever been shown. The present study was undertaken to determine the quantitative alterations of their activities by microcolorimetric assay in the cochlear lateral wall after acoustic trauma. Adult albino guinea pigs were exposed to white noise at 105+/-2 dB A for 10 min or 40 h. The age-matched control animals were not exposed to noise. Noise exposure resulted in a significant threshold shift of the auditory brainstem response (P<0.001). Significant decreases in activities of Na(+),K(+)-ATPase and Ca(2+)-ATPase were found in the cochlear lateral wall after noise exposure (P<0.001). Statistical analysis indicated that a good correlation held not only between the decline of these enzyme activities and noise-induced hearing loss, but also between the gradual partial recovery of these parameters during the first 10-day recovery period. The present findings suggest that metabolic damage and ionic disturbance may contribute, at least partially, to noise-induced hearing threshold shift. PMID- 10748352 TI - Past and present in myocardial protection. AB - Both the certainty of anatomical repair and early complete recovery of cardiac function are essential elements of successful cardiac surgery. Successful operations require adequate visualization of the operative field, arrest and relaxation of the hearts and sufficient operative time. Extracorporeal circulation and aortic cross-clamping are necessary to maintain a bloodless field. However, interruption of coronary perfusion by the aortic clamp produces myocardial ischemic injury, and unclamping induces reperfusion injury. For years, hypothermia was the most commonly used strategy for myocardial protection. However, the practice of warm heart surgery under normothermic extra corporeal circulation has increased recently. Cardiac surgeons now have a variety of myocardial protection strategies from which to choose. We introduce here the history of myocardial protection and our own recent investigations. PMID- 10748353 TI - Conservative management (packing) of hemorrhage complicating mediastinoscopy. AB - Major hemorrhage is an uncommon complication of mediastinoscopy. Although there is a paucity of published data on the management of this problem, sternotomy and arterial repair is usually considered the treatment of choice. Two major hemorrhagic complications (1 definite and 1 possible pulmonary artery tear) occurred in a series of 324 mediastinoscopies. Mediastinoscopic gauze packing successfully controlled the hemorrhage. PMID- 10748354 TI - Lung function following cardiac surgery is not affected by postoperative ventilation time. AB - We investigated the effect of postoperative ventilation time on lung function following cardiac surgery. A prospective observational study of 100 elective patients. Anaesthetic technique and postoperative ventilation times reflected the routine of participating anaesthetists. Group I (n = 29) patients were extubated prior to leaving the operating room, Group II (n = 37) within 8 hours and Group III (n = 28) after 8 hours. Oxygen saturation on air, spirometry and chest x-rays were performed preoperatively and on postoperative days 2, 3 and 4. No demographic differences were detected between groups. There was no mortality, perioperative myocardial infarction or reintubation in any group. Lung function significantly declined following surgery in all groups (p<0.0001) for all endpoints, but was not different between groups. Chest x-ray changes were common in all groups but not significantly different between groups. Immediate extubation does not worsen lung function compared to early or late extubation. PMID- 10748355 TI - The effect of modified ultrafiltration in pediatric open heart surgery. AB - Since 1997 we have performed modified ultrafiltration (MUF) in pediatric open heart operations. To elucidate the clinical effects of MUF, patients under 20 kg in weight who underwent corrective open heart operation since 1997 are divided into 2 cohorts according to the enforcement of MUF (control group versus MUF group) retrospectively. Procedures, age, bodyweight, cardiopulmonary bypass (CPB) time, operation time, amount of donor blood use, postoperative inotrope dose, postoperative intubation time, and postoperative gas exchange capacity of lung expressed with respiratory index (RI) were compared between groups. Furthermore, a multiple linear regression analysis was performed to find independent correlates with postoperative RI. Finally scattergrams of intubation time and RI were drawn against several factors. In univariate analyses, the MUF group had significantly shorter intubation time and better RI. Multivariate analysis revealed that the enforcement of MUF was an independent correlate of postoperative RI. Analyses on the scattergrams revealed that the above mentioned favorable effects of MUF were prominent in the patients younger than 3 years or weighing less than 10 kg. We concluded that MUF had significant effects on pulmonary function preservation in pediatric open heart operations, especially for smaller children. PMID- 10748356 TI - Effects of supplemental L-arginine during warm blood cardioplegia. AB - OBJECTIVES: Effects of supplemental L-arginine, nitric oxide precursor, during warm blood cardioplegia were assessed in the blood perfused isolated rat heart. METHODS: The isolated hearts were perfused with blood at 37 degrees C from a support rat. After 20 minutes of aerobic perfusion, the hearts were arrested for 60 minutes with warm blood cardioplegia given at 20-minute intervals. This was followed by 60 minutes of reperfusion. The hearts were divided into the following three groups according to the supplemental drugs added to the cardioplegic solution. The control group (n = 10) received standard warm blood cardioplegia. The L-ARG group (n = 10) received warm blood cardioplegia supplemented with L arginine (3 mmol/l). The L-NAME group (n = 10) received warm blood cardioplegia supplemented with L-arginine (3 mmol/l) and L-nitro-arginine methyl ester, a competitive inhibitor of nitric oxide synthase (1 mmol/l). After 60 minutes of cardioplegic arrest, cardiac function, myocardial metabolism and myocardial release of circulating adhesion molecules were measured during reperfusion. RESULTS: Left ventricular end-diastolic pressure was significantly lower (p<0.05) in the L-ARG group than in the control group and the L-NAME group during reperfusion. Isovolumic left ventricular developed pressure, dp/dt and coronary blood flow were significantly greater (p< 0.05) in the L-ARG group during reperfusion. The L-ARG group resulted in early recovery of lactate metabolism during reperfusion. Myocardial release of circulating intercellular adhesion molecule-1 (ICAM-1) and E-selectin were significantly less (p<0.05) in the L-ARG group at 15 minutes of reperfusion. CONCLUSIONS: The results suggest that augmented nitric oxide by adding L-arginine to warm blood cardioplegia can preserve left ventricular function and ameliorate endothelial inflammation. The technique can be a novel cardioprotective strategy in patients undergoing cardiac surgery. PMID- 10748357 TI - Mid-term results of ATS open pivot bileaflet mechanical prosthetic heart valve. AB - We investigated mid-term results of the patients with an ATS bileaflet valve in our institution. In the past 6 years, 69 patients received valve replacement with an ATS valve. We assessed the changes of serum lactate dehydrogenase (LDH) level in the hospital, and the valve's closing sound. The serum LDH level had almost normalized one week postoperatively and they have maintained normal levels since then. According to the questionnaire about the valvular sound at a random period after surgery, it was unnoticeable in 61 (88.4%) of the patients with ATS valve. In the 8 patients (12.6%) who recognized the valve sound, 7 of them were reoperation cases. As to the frequency analysis for the valve's closing sound, the sound peak was indicated at around 1.2 kHz in the patients with ATS valves. In patients with St. Jude Medical (SJM) valves, it appeared not only around 1.2 kHz but also around 2 to 7 kHz. Postoperative cerebral infarction was complicated in one patient. Mortality occurred in 4 (5.7%) of the patients with ATS valves. The follow up periods were from one to 66 months. Thromboembolic event free and actuarial survival rate in the patients with ATS valves were 98.6% and 94.2%, respectively. These results indicated that the ATS valve is considered to be a safe valve and mid-term follow-up shows excellent results in terms of the patients quality of life. PMID- 10748358 TI - The benefits and new predictors of early extubation following coronary artery bypass grafting. AB - OBJECTIVE: This study researched cardiorespiratory effects and cost-effectiveness of early extubation in patients following coronary artery bypass grafting. METHODS: We retrospectively examined the character of 86 (38 preoperative, 12 intraoperative, 36 postoperative) variables in 78 patients after coronary artery bypass grafting (CABG) at our hospital. The patients were classified into three groups according to the time of extubation: Group A (less than 8 hours), Group B (8 to 24 hours) and Group C (24 hours or more). RESULTS: There were 43 patients in Group A (55%), 27 patients in Group B (35%), and 8 patients in Group C (10%). Univariate preoperative analysis revealed several differences between Groups A and B: 15 variables were found to reach statistical significance. Stepwise logistic regression analysis implicated decreased forced expiratory volume rate in the first second (FEV1.0%), postoperative cardiac index (CI) after admission to the intensive care unit (ICU) and usage of postoperative temporary pace-makers as predictors of late extubation. Also, there was a statistical significance in usage of diuretics within the first day (15.8+/-11.6 vs 28.8+/-22.6 mg; p = 0.044) and in the ICU days (3.6+/-0.8 vs 4.2+/-1.0; p = 0.004) between early and late extubation groups. CONCLUSIONS: Early extubation within 8 hours into the ICU stay was accomplished in 55% of the patients in this study. We speculate that the benefits of early extubation contain improvement of cardiac function, reduction in respiratory infection and complications, and cost saving as the result of diminished ICU admission. Also, we have shown that decreased FEV1.0%, decreased CI and the usage of a postoperative temporary pacemaker were independent predictors of prolonged intubation in our study. PMID- 10748360 TI - Intraoperative acute occlusion of aortic bifurcation during extracorporeal circulation. AB - A 36-year-old male patient showed a significant decrease of arterial pressure in the lower extremities during coronary artery bypass grafting (CABG) with extracorporeal circulation (ECC). Arterial pressure measured in the femoral artery fell to 10-20 mmHg at the end of ECC, whereas in the upper extremities arterial pressure levels were normal. At the end of the surgery a complete ischemia of both lower extremities was observed. We suspected Leriche's syndrome and performed a successful aortic embolectomy through bilateral femoral arteriotomies immediately. An insufficient anticoagulation could be excluded by prolonged "activated clotting time" (ACT), therefore we presumed that the source of embolus was a small aneurysm of the left ventricle. The shape and superficial structure of the extracted embolus, which was partly covered with endocardium, confirmed our suspicion. No complications occurred throughout the postoperative period. On the 10th postoperative day, the patient left our department for postoperative rehabilitation with a normal perfusion of the lower extremities. PMID- 10748359 TI - Cerebral function after aortic surgery using retrograde cerebral perfusion: report of three cases. AB - INTRODUCTION: The neurological analysis undertaken on three cases of aortic aneurysm treated surgically using retrograde cerebral perfusion (RCP) are reported herein. This is the first detailed analysis of postoperative neurological studies in such cases. METHODS: The oxygenation state of cerebral Hb and cytochrome aa3 levels were monitored by near-infrared spectroscopy (NIR) during RCP. Postoperative neurological scoring, computed tomography (CT), magnetic resonance imaging (MRI), and monitoring of local cerebral blood flow (LCBF) quantatively by N-isopropyl-p-[123I] iodoamphetamine (IMP) single-photon emission computed tomography (SPECT) were performed for an extended follow-up period after surgery. RESULTS: The T2WI of MRI findings which consisted of many spotty high intensity areas in the striatum, cerebellum and hippocampus, may reflect the postoperative severe consciousness and mental disturbances (2-4 weeks). However, in all cases the neurological deficit gradually improved until they returned to their normal preoperative states. The improvement of LCBF correlated well with neurological recovery. CONCLUSION: The conditions of the conventional method of RCP must be improved to prevent transient severe consciousness and mental disturbances which occur after the operation in relationship with the appearance of the high intensity area on the MRI T2WI. PMID- 10748361 TI - Closure of calcified patent ductus arteriosus. AB - Patent ductus arteriosus (PDA) in adults is occasionally associated with calcification and pulmonary hypertension, for which an anterior approach through a median sternotomy with cardiopulmonary bypass is often used. Sutures are placed without circulatory arrest by using a transpulmonary balloon catheter as an occluder. To secure the suture tie, we used Nelaton's catheters as tourniquets. PMID- 10748362 TI - Coronary artery bypass grafting for spontaneous coronary artery dissection: a case report and a review of the literature. AB - A 37 year-old woman underwent coronary angiography because of chest pain at rest. Selective coronary angiography demonstrated dissection and stenosis with a filling delay from the left main trunk to the left anterior descending coronary artery. The patient was successfully managed with urgent coronary artery bypass grafting. Spontaneous coronary artery dissection is relatively rare and threatens both elderly and young patients with acute coronary disturbances. Patients can be divided etiologically into three groups. The first was comprised of those in the postpartum period. The second was those with atherosclerotic coronary artery disease, and the third was those associated with coronary vasospasm. Dissections are frequently fatal and most of the known cases have been diagnosed at autopsy. Only a few cases have been documented by coronary angiography, and operative cases of spontaneous coronary artery dissection have rarely been reported. PMID- 10748363 TI - Combined heart transplantation and resection of dissecting aneurysm of ascending aorta and aortic arch: a case report. AB - A 21-year-old male patient had suffered from palpitation and exertional dyspnea since October, 1997. He was admitted to our hospital, and a series of examinations were performed. Chest computed tomography (CT) revealed marked dilatation of the ascending aorta (about 7.5 cm at the proximal portion) and aortic annulus, an intimal flap in the ascending aorta and aortic arch was also noted. Cardiac catheterization revealed the pulmonary capillary wedge pressure was 33 mmHg, pulmonary artery pressure was 47/38 mmHg with a mean of 35.4. The cardiac index was 1.01 l/min/m2. Poor left ventricular contractility was shown by a left ventricular ejection fraction (LVEF) of 13.8% and a right ventricular ejection fraction (RVEF) of 5.13% by a radionuclide angiogram (RNA) study. Under the diagnosis of dilated cardiomyopathy and dissecting aortic aneurysm of the ascending aorta and aortic arch, he was put on a waiting list for heart transplantation. On November 11, 1997 he received heart transplantation. Resection of the dissecting aneurysm of the ascending aorta and the aortic arch and replacement with a 26 mm Vascutek graft were performed first under deep hypothermia and retrograde cerebral perfusion. Then while he was rewarming up, heart implantation was performed. He was discharged 30 days after surgery and has been doing well since then. As far as we know, no literature regarding combined heart transplantation and resection of a dissecting aneurysm of the ascending aorta and aortic arch has been reported. PMID- 10748364 TI - Surgical treatment of spontaneous dissection of the superior mesenteric artery: a case report. AB - A case of a 46-year-old man with spontaneous dissection of the superior mesenteric artery (SMA) demonstrated by ultrasonography is presented. He was successfully treated by emergency aorto-SMA bypass surgery, but complicated with bilateral internal iliac aneurysm. The patient has remained asymptomatic with full employment. PMID- 10748365 TI - An unusual indication for surgical treatment of iliac artery aneurysm treated with stented graft previously. AB - A 70-year-old man was seen with an iliac artery aneurysm and this was treated by an intraluminal graft-stent device introduced through the femoral artery. After the procedure, patency of the artery was proved to be good with control angiography but the mass effect of the aneurysmal sac on the gastrointestinal system did not disappear and we replaced the aneurysmatic segment with a collagen coated graft. The patient then recovered without any complications and gastrointestinal symptoms. PMID- 10748366 TI - Right-sided pleural effusion in spontaneous esophageal perforation. AB - Spontaneous esophageal perforation (Boerhaave's syndrome) is a rare clinical entity in which overindulgence in a large meal precedes vomiting and chest pain. Early diagnosis and aggressive management are keys to minimizing the morbidity and mortality. We report an unusual presentation of this already uncommon occurrence in a 33-year-old female. She presented to the Emergency Department with severe chest pain following vomiting with hematemesis after a large meal. The initial chest radiograph showed up nothing in particular. Dyspnea developed two days later, and a right-sided pleural effusion was seen on chest x-ray. Panendoscopy was highly suggestive of Boerhaave's syndrome. She underwent emergency operation. After three months of hospital care, she was discharged in relatively good condition. This case of right-sided pleural effusion extends the reported description of Boerhaave's syndrome. PMID- 10748367 TI - Inside washington PMID- 10748368 TI - The power of perception. PMID- 10748369 TI - ENA's 30th anniversary: pearls of wisdom PMID- 10748370 TI - Children need to be taught dangers of discarded syringes PMID- 10748372 TI - Astronaut interview PMID- 10748371 TI - Legal column on patient refusing intubation is appreciated PMID- 10748373 TI - Blending CNS and NP roles increases employment opportunities PMID- 10748375 TI - Response to editorial on air medical crashes PMID- 10748374 TI - The changing face of emergency care with "managed care" PMID- 10748376 TI - Response to editorial on air medical crashes PMID- 10748377 TI - Response to editorial on air medical crashes PMID- 10748378 TI - Sample ED education leaflets sought PMID- 10748379 TI - Education internet sites no longer functioning PMID- 10748381 TI - Management of a 36-year-old man with pit viper envenomation. PMID- 10748382 TI - Near-fatal rattlesnake envenomation. PMID- 10748383 TI - Gender and age bias in triage decisions. AB - INTRODUCTION: Recently it has been recognized that women are less likely than men to be diagnosed with a myocardial infarction (MI) or to receive early or aggressive treatment and are more likely than men to die of an MI. The purpose of this qualitative study was to examine the triage decisions made by ED nurses for persons with symptoms suggestive of MI. The theoretical framework for this investigation was Hammond's lens model for clinical inference and Evan's two stage reasoning model. METHOD: Four focus group sessions were conducted. The participant's oral descriptions were tape-recorded, transcribed verbatim, and analyzed using the Krueger method. RESULTS: Content analysis revealed several important issues influencing triage decisions: patient presentation, nursing knowledge and experience, practice environment, intuition, the fear of liability, and gender-specific behaviors. ED nurses held different perceptions regarding the significance and likelihood of MI for male and female patients seeking evaluation and treatment. In addition, ED nurses admitted that MI is not the first diagnosis considered for middle-aged women. DISCUSSION: The inability of ED nurses to associate middle-aged women's presenting symptoms with MI may contribute to the increased morbidity and mortality experienced by this population. The findings of this study have implications for nursing research, education, and practice. PMID- 10748384 TI - Delay in seeking treatment for acute myocardial infarction: why? PMID- 10748385 TI - Hyponatremia as a consequence of acute adrenal insufficiency and hypothyroidism. PMID- 10748386 TI - A scientific basis for choosing the technique of hair removal used prior to wound closure. PMID- 10748388 TI - Clinical nurses forum PMID- 10748387 TI - CEN review questions PMID- 10748389 TI - Memorial service honors EMS personnel following "line of duty" deaths. PMID- 10748390 TI - Common deficiencies cited during CAMTS flight program accreditation surveys. PMID- 10748391 TI - From the feds PMID- 10748393 TI - Managers forum PMID- 10748392 TI - Infections in pregnant women: the need to assess and possibly treat the fetus and the sexual partner as well. AB - Assessing and treating pregnant women in the emergency department for complaints unrelated to pregnancy are complex processes at best. Obtaining a consultation from an obstetrician is always prudent, even if it is simply by telephone. Careful attention to laboratory and other diagnostic test results is imperative, and communication with the patient's primary care physician and/or obstetrician is a must. Assessment of fetal well-being should be documented, and implications for the fetus of all treatments and/or omitted treatments should be considered. With infections in pregnant women, remember to think about implications for the baby and the woman's sexual partner; both may need to be assessed and treated. PMID- 10748394 TI - Public speaking survival strategies. PMID- 10748395 TI - Our new pediatric emergency department. PMID- 10748396 TI - Health care customer satisfaction surveys still running amok: surveys degrade nurses. PMID- 10748397 TI - Tips on navigating your research proposal through the institutional review board. PMID- 10748398 TI - The Community Awareness Rape Education (CARE) program for high school students. PMID- 10748399 TI - Cardiac contusion: two case vignettes. AB - When patients with blunt chest trauma and suspected cardiac contusion are brought to the emergency department, focus on detecting subtle signs of myocardial dysfunction. Obtain the important first EKG, monitor for arrhythmia development, and assess for signs of failure of the right side of the heart. PMID- 10748400 TI - An obtunded elderly woman with tachycardia and tachypnea. PMID- 10748401 TI - Karen Daley--an emergency nurse advocate for other nurses.. Interview by Judith Surveyer Mitiguy. PMID- 10748402 TI - A promise to Megan's dad. PMID- 10748403 TI - Homogeneity of the age at diagnosis in sibs with Type 2 diabetes: implications for sib-pair analysis. AB - Homogeneity of the age at diagnosis of Type 2 diabetes was studied in 1,228 sibs in 300 unrelated families: 100 consecutive single-affected and 200 consecutive multiple-affected ones. There were 635 diabetic sibs. The mean and median age at diagnosis in all affected individuals was 50 years (range, 19-75 years). The mean age at diagnosis in the multiply affected families was 49 years (median 50); the between-sibs range of age at diagnosis within multiple-affected families was (mean and median) 17 years (range, 0-55), with 42% of these diagnosed within a 5 year age span, 66% within 10 years, and 90% within 13 years. When one parent had diabetes, it was more often the mother (79% P = 0.0023). In order to examine this apparent tendency toward homogeneity of age at diagnosis within families, with full regard for and parsimonious to right-censored data, we employed a Cox proportional-hazards survival analysis, with family as the explanatory variable. Deviance residuals resulting from that model were analyzed in a variance components, random effects model ANOVA which indicated a significant (P << 0.001) effect of family on age of diagnosis, with an intraclass correlation of 0.29. In many families the clustering of age at diagnosis appeared very tight, with single outliers, and in 20 families with the longest history, diabetes was diagnosed in 68 sibs within the span of 8 +/- 7 years, whereas 38 unaffected sibs remain free of diabetes 25 +/- 8 years later. The wide differences of the age at diagnosis between families and its intrafamilial homogeneity should be considered in planning genetic analysis of Type 2 diabetes. PMID- 10748404 TI - Molecular mapping of an idic(Yp) chromosome in an Ullrich-Turner patient. AB - We describe a woman with Ullrich-Turner manifestations and a 45,X/46, X,+mar karyotype. Fluorescence in situ hybridization (FISH) and DNA analysis were carried out in order to determine the origin and structure of the marker. FISH showed that the marker was a Y-derived dicentric chromosome. The breakpoint at Yq11 (interval 6) was mapped using Southern blotting and polymerase chain reaction (PCR). There were no nucleotide alterations in the SRY conserved domain. Histological analysis of the gonads showed an ovarian-like stroma with no signs of testicular tissue. These findings indicate that the patient was a mosaic 45,X/46,X,idic(Yp) whose phenotypic expression, including sex determination, appeared to have had more influence from the 45,X cell line. PMID- 10748405 TI - Isodicentric Y chromosome in an Ullrich-Turner patient without virilization. AB - We report on a 17-year-old young woman with Ullrich-Turner syndrome (UTS), who was found to have a karyotype 45,X/46,X,idic(Y)(q11). She had age-appropriate genitalia without virilization in spite of the presence of the Y-derived marker chromosome and SRY locus in 70% of her lymphocytes. Having reviewed the literature, we conclude that a possible explanation for the lack of virilization in these mosaic patients is most likely an uneven distribution of tissue mosaicism (gonadal mosaicism). PMID- 10748406 TI - Molecular cytogenetic characterization and origin of two de novo duplication 9p cases. AB - We report on two additional cases with duplication of 9p, minor with facial anomalies and developmental delay. Using fluorescence in situ hybridization and single-copy probes, we showed that the first case was a direct duplication, whereas the second case was inverted. The extent of the direct duplication was defined as 9p12 --> p24 by microdissection and microcloning of the aberrant chromosome and subsequent chromosome-specific comparative genomic hybridization. DNA polymorphism analysis with eight microsatellite markers revealed that the origin of the dup(9p) was maternal in the first case, whereas it was paternal in the second. PMID- 10748407 TI - Glycogen storage disease type Ia: molecular diagnosis of 51 Japanese patients and characterization of splicing mutations by analysis of ectopically transcribed mRNA from lymphoblastoid cells. AB - Glycogen storage disease type Ia (GSD-Ia) is an autosomal recessive disorder of glycogen metabolism caused by a deficiency of glucose-6-phosphatase (G6Pase) that is expressed in the liver, kidney, and intestinal mucosa. Clinical manifestations include short stature, hepatomegaly, hypoglycemia, hyperuricemia, and lactic acidemia. To elucidate a spectrum of the G6Pase gene mutations and their frequencies, we analyzed mutations in 51 unrelated Japanese patients with GSD-Ia. The most prevalent mutation was g727t, accounting for 88 of 102 mutant alleles examined, followed by R170X mutation, which accounted for 6 mutant alleles, and R83H mutation which was observed in 3 mutant alleles. In addition, 3 different, novel mutations, IVS1-1gqter) resulting from a maternal pericentric inversion of chromosome 2 (p25. 2q34). The infant had clinical findings similar to the characteristic phenotype associated with a partial duplication of chromosome 2q3. Carriers of pericentric inversions of chromosome 2 have an increased risk of pregnancy loss but have only rarely been reported to have a liveborn offspring with an unbalanced chromosome constitution. This case further confirms the risks associated with a pericentric inversion of chromosome 2 and is the second report with manifestations of the trisomy 2q3 phenotype. PMID- 10748412 TI - Spondylocarpotarsal synostosis with ocular findings. AB - We report on three sibs presenting with spondylocarpotarsal synostosis, short trunk dwarfism of postnatal onset, scoliosis, unsegmented thoracic vertebrae with unilateral bar, and carpal bone fusion. Tarsal bone fusion and dental abnormalities were noted in some of them, indicating pleiotropy and intrafamilial variability. Lens opacities, rarefaction of retinal pigmentation, and narrowing of retinal vessels, detected in two patients, are findings that have not been described to date in this condition. PMID- 10748413 TI - OFD II, OFD VI, and Joubert syndrome manifestations in 2 sibs. AB - We present 2 sibs with manifestations of oral-facial-digital syndromes (OFD) and Joubert syndrome. The index patient was the 5th child of healthy nonconsanguineous Turkish parents. At birth this female patient had large hydrocephalus, hypertelorism, deep-set eyes, nystagmus, broad mouth, thick oral frenula, cleft palate, hamartomas of the tongue, postaxial polydactyly of fingers, normal toes, and hypotonia. Cranial MRI showed hydrocephalus and Dandy Walker malformation. The child had no psychomotor development, was unable to swallow and had severe seizures. She died at 2 months of recurrent apneic episodes. At birth the brother of the index patient showed prominent forehead, broad, deep nasal bridge, cleft palate, multiple hamartomas of the tongue, irregular alveolar ridges, retrognathia, bilateral postaxial polydactyly of the hands and feet, and broad halluces. He had an abnormal breathing pattern with phases of tachypnea and apnea. Cranial MRI showed hydrocephalus, hypoplasia of the cerebellar vermis, Dandy-Walker malformation, and hypomyelination of the corpus callosum. Renal ultrasonography demonstrated multiple small cysts. Ocular fixation was absent and he had a mild nystagmus. PMID- 10748414 TI - Novel 7-DHCR mutation in a child with Smith-Lemli-Opitz syndrome. AB - Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder characterized by minor facial anomalies, mental retardation, and multiple congenital abnormalities. Biochemically, the disorder is caused by deficient activity of 7-dehydrocholesterol reductase, which catalyzes the reduction of the Delta7 double bond of 7-dehydrocholesterol to produce cholesterol. Recently, mutations in the gene encoding 7-dehydrocholesterol reductase (7DHCR) were found to cause SLOS. We report the first molecular characterization of an Italian SLOS patient. Interestingly, his paternal 7DHCR allele, of Arab origin, harbored a novel P329L mutation which in combination with a maternal splice-site (IVS8-1 G>C) mutation resulted in a relatively milder phenotype. PMID- 10748415 TI - Arterial tortuosity syndrome. AB - We describe a patient with arterial tortuosity syndrome (ATS), a rare disorder comprising generalized tortuosity and elongation of all major arteries, soft skin, joint laxity, severe keratoconus, and diffuse tortuosity of the carotids and of intracranial arteries. The patient's probably affected brother and sister died at an early age. Cytochemical studies excluded Ehlers-Danlos type IV and type VII syndromes. We review 11 previously described patients. PMID- 10748416 TI - Clinical involvement and protein expression in individuals with the FMR1 premutation. AB - Most individuals with the fragile X premutation are clinically unaffected; however, some show clinical manifestations, including learning difficulties, emotional problems, or even mental retardation. The basis of clinical involvement in these individuals is unknown. Premutation alleles are reportedly associated with normal levels of mRNA and protein (FMRP). To examine this issue in more detail, we studied six individuals with a premutation. We are reporting these cases to demonstrate a spectrum of phenotypic involvement which can be seen clinically. These cases include one individual with the premutation who has no evidence of FMR1 gene dysfunction but has mental retardation from other causes. Other cases presented here show varying degrees of FMR1 gene dysfunction as assessed by FMRP and FMR1 mRNA levels and various clinical features of fragile X. In two cases we observed a significant reduction in FMRP expression and an elevated FMR1 mRNA expression level associated with moderate cognitive deficit. Thus, the utilization of FMRP measures can be helpful in understanding for which premutation patients clinical involvement is caused by dysfunction of the FMR1 gene. PMID- 10748417 TI - Intrafamilial phenotypic variability in Engelmann disease (ED): are ED and Ribbing disease the same entity? AB - We report on clinical and radiologic manifestations in a 3-generation Japanese family with Engelmann disease (ED) or progressive diaphyseal dysplasia. A large variation of phenotype was remarkable among 12 affected family members. Of the 12 patients, 7 had full manifestations of ED, such as bilateral, symmetrical diaphyseal sclerosis of long bones with myopathy and limb pain, whereas the other 5 exhibited only segmental (rhizomelic and/or mesomelic) involvement and asymmetric diaphyseal sclerosis without any clinical symptoms. The phenotype of the latter group of patients resembled Ribbing disease (RD). We propose that ED and RD represent phenotypic variation of the same disorder. PMID- 10748418 TI - Biliary atresia in Kabuki syndrome. PMID- 10748419 TI - Monozygotic twins with Crouzon syndrome: concordance for craniosynostosis and discordance for thumb duplication. PMID- 10748420 TI - 1q32-q41 microdeletion with reference to Van der Woude syndrome and allied clefting entities. PMID- 10748421 TI - Kabuki syndrome and diaphragmatic defects: a frequent association in non-Asian patients? PMID- 10748422 TI - Hyperpolarized 129Xe NMR as a probe for blood oxygenation. AB - Optically enhanced NMR with (129)Xe and (3)He is emerging as a novel and promising technique for medical imaging of lungs and other tissues. Here it is shown that hyperpolarized (129)Xe NMR provides a powerful means of measuring blood oxygenation quantitatively and noninvasively. The interaction of xenon with hemoglobin is responsible for an oxygen-dependent NMR shift of (129)Xe in red blood cells, in sharp contrast to the current model of xenon-hemoglobin binding. This effect could be exploited in brain functional studies, and in the assessment of conditions and diseases affected by blood oxygenation. PMID- 10748423 TI - Reduced (1)H-NMR visibility of creatine in isolated rat hearts. AB - The aim of this study was to measure the concentration of creatine in Langendorff perfused rat hearts, both by quantitative (1)H-MRS and by high-pressure liquid chromatography (HPLC). First, the relaxation times and other parameters affecting absolute quantification by MRS were determined. At 11.75 T, the relaxation times of myocardial creatine were T(1) = 1.1+/-0.29 sec (mean +/- SD, n = 5) and T(2) = 56.4+/-6.2 ms (n = 9). In phantom experiments the MRS measurements gave accurate values for the known relative concentrations of the detected substances. In glucose-perfused rat hearts, the creatine concentration measured by HPLC was 14.2+/-1.9 mmol/kg wet weight (n = 8), in good agreement with literature values. The (1)H-MRS measurements, however, resulted in creatine concentrations of only approximately 60% of this value. The application of CHESS-pulses for water suppression led to a further 30% reduction of the creatine MRS signal. These results indicate a reduced (1)H-NMR visibility of creatine in the myocardium, which suggests a compartmentation of myocardial creatine into various pools. PMID- 10748424 TI - Phase-contrast with interleaved undersampled projections. AB - MR phase-contrast techniques provide velocity-sensitive angiograms and quantitative flow measurements but require long scan times. Recently it has been shown that undersampled projection reconstruction can acquire higher resolution per unit time than Fourier techniques with acceptable artifacts when used in contrast-enhanced MR angiography. Undersampled projection reconstruction has similar potential for phase-contrast acquisitions. Flow sensitization gradients are used with projection trajectories to acquire velocity-dependent phase information. An acquisition scheme that acquires three flow encoding directions on three sets of angular-interleaved projections is introduced. Depending on the resolution, acquisition times for 3D datasets can decrease by factors of two to four. PMID- 10748425 TI - Surface-coil polarization transfer for monitoring tissue metabolism in vivo. AB - Polarization transfer methods can substantially enhance NMR signals from nuclei of low gamma, which are J-coupled to nuclei of high gamma, but to date have had limited application for in vivo (31)P MRS studies. They require both accurate flip angles and good localization in order to achieve their potential. Described here is an implementation of the insensitive nuclei enhanced by polarization transfer (INEPT) polarization transfer method using segmented adiabatic BIR4 RF pulses. Localization uses ISIS, applied to the coupled (1)H spins. Detailed analysis is performed to evaluate the specific absorption rate power deposition when using surface coils. Polarization transfer, localization capability, and use with surface coils are demonstrated using suitable test objects. Finally, in vivo data are presented from the liver of a normal volunteer in which the signals from the phosphodiester peaks are substantially enhanced. PMID- 10748426 TI - Proton MR spectroscopy of wild-type and creatine kinase deficient mouse skeletal muscle: dipole-dipole coupling effects and post-mortem changes. AB - Localized proton MR spectra of mouse skeletal muscle obtained at 7 T show dipole dipole coupling effects for creatine and putative taurine resonances and for the lactate methine signal. These effects are independent of the presence of creatine kinase. The intensity of the methylene (1)H resonance of creatine is not different between wild-type and creatine kinase deficient mice, which have a lower phosphocreatine content. (1)H-MR spectra acquired post-mortem from wild type mouse skeletal muscle parallel to B(0) show a linewidth decrease for the methyl resonance of creatine and a 20% signal intensity loss for its methylene peak concurrent with the total breakdown of phosphocreatine as observed by (31)P MR spectroscopy. However, with the muscle at the magic angle no changes in the appearance and intensity of creatine (and taurine) resonances are observed. These results indicate that the changes observed for creatine resonances are related to altered dipolar couplings and that the intensity of the methylene peak does not necessarily reflect muscular phosphocreatine content. PMID- 10748427 TI - Human brain glucose metabolism mapping using multislice 2D (1)H-(13)C correlation HSQC spectroscopy. AB - A method for multivolume 2D (1)H-(13)C correlation spectroscopy, multislice heteronuclear single quantum coherence (HSQC), is proposed. This permits human brain metabolism from glucose to amino acids to be followed using a 2-T whole body scanner. The modifications from the conventional HSQC are that the 180 degrees ((13)C) and 180 degrees ((1)H) pulses are separated in time in the preparation period and that the 180 degrees ((13)C) pulse is applied at 1/(4J(CH)) before the 90 degrees ((1)H) polarization transfer (PT) pulse. The preparation (echo) time can be set longer than 1/(2J(CH)) so that, even in a whole-body system, slice-selective pulses and gradients can be applied. Another modification is that the 90 degrees ((1)H) reverse PT pulses after the creation of 2I(z)S(z) are used as multislice pulses. The time-course of glutamate C4 could be followed with 15-min temporal resolution from the HSQC spectra obtained from the brains of volunteers after the oral administration of glucose C1, and the maximum S/N was 3. PMID- 10748428 TI - Electromyography in MRI--first recordings of peripheral nerve activation caused by fast magnetic field gradients. AB - Prior studies on the evaluation of stimulation by MRI were based on the subjective feeling of the volunteers. A wide variety of stimulation thresholds between the subjects was observed. In order to exclude subjective perception levels as a cause of this variation, we developed a method to investigate the activation of peripheral nerves after gradient switching by electromyography (EMG) within the MR-imager. Five healthy volunteers were positioned in the MR scanner with the bridge of the nose at isocenter. The amplitude of sinusoidal pulse trains of the anterior-posterior gradient (rise-times: 200 or 300 micros, various numbers of oscillations) was increased stepwise. Four surface electrodes were placed on the region where a muscle-twitch was reported. Electric activity of the muscle during stimulation experiments was recorded with an MR-compatible electro-physiologic amplifier. Stimulation thresholds were defined by the appearance of an EMG-signal. Thresholds were sharp and consistent with the report of the subjects. PMID- 10748429 TI - Event-related fMRI contrast when using constant interstimulus interval: theory and experiment. AB - Event-related functional magnetic resonance imaging (ER-fMRI) involves the mapping of averaged hemodynamic changes resulting from repeated, brief (<3 sec) brain activation episodes. In this paper, two issues regarding constant interstimulus interval ER-fMRI were addressed. First, the optimal interstimulus interval (ISI), given a stimulus duration (SD), was determined. Second, the statistical power of ER-fMRI relative to that of a blocked-design paradigm was determined. Experimentally, it was found that with a 2-sec SD, the optimal ISI is 12 to 14 sec. Theoretically, the optimal repetition interval (T(opt) = ISI + SD) is 12 to 14 sec for stimuli of 2 sec or less. For longer stimuli, T(opt) is 8 + 2 x SD. At the optimal ISI for SD = 2 sec, the experimentally determined functional contrast of ER-fMRI was only -35% lower than that of blocked-design fMRI. Simulations that assumed a linear system demonstrated an event-related functional contrast that was -65% lower than that of the blocked design. These differences between simulated and experimental contrast suggest that the ER-fMRI amplitude is greater than that predicted by a linear shift-invariant system. PMID- 10748430 TI - A reduced field-of-view method to increase temporal resolution or reduce scan time in cine MRI. AB - In some dynamic applications of MRI, only a part of the field-of-view (FOV) actually undergoes dynamic changes. A class of methods, called reduced-FOV (rFOV) methods, convert the knowledge that some part of the FOV is static or not very dynamic into an increase in temporal resolution for the dynamic part, or into a reduction in the scan time. Although cardiac imaging is an important example of an imaging situation where changes are concentrated in a fraction of the FOV, the rFOV methods developed up to now are not compatible with one of the most common cardiac sequences, the so-called retrospective cine method. The present work is a rFOV method designed to be compatible with cine imaging. An increase by a factor n in temporal resolution or a decrease by n in scan time is obtained in the case where only one nth of the FOV is dynamic (the rest being considered static). Results are presented for both Cartesian and spiral imaging. PMID- 10748431 TI - Whole brain quantitative CBF and CBV measurements using MRI bolus tracking: comparison of methodologies. AB - Three different deconvolution techniques for quantifying cerebral blood flow (CBF) from whole brain T*(2)-weighted bolus tracking images were implemented (parametric Fourier transform P-FT, parametric single value decomposition P-SVD and nonparametric single value decomposition NP-SVD). The techniques were tested on 206 regions from 38 hyperacute stroke patients. In the P-FT and P-SVD techniques, the tissue and arterial concentration time curves were fit to a gamma variate function and the resulting CBF values correlated very well (CBF(P-FT) = 1.02 x CBF(P-SVD), r(2) = 0.96). The NP-SVD CBF values (i.e., original unfitted curves were used) correlated well with the P-FT CBF values only when a sufficient number of time series volumes were acquired to minimize tracer time curve truncation (CBF(P-FT) x 0.92 x CBF(NP-SVD), r(2) = 0.88). The correlation between the fitted CBV and the unfitted CBV values was also maximized in regions with minimal tracer time curve truncation (CBV(fit) = 1.00 x CBV(unfit), r(2) = 0.89). When a sufficient number of time series volumes could not be acquired (due to scanner limitations) to avoid tracer time curve truncation, the P-FT and P-SVD techniques gave more reliable estimates of CBF than the NP-SVD technique. PMID- 10748432 TI - Numerical study of blood flow in an anatomically realistic aorto-iliac bifurcation generated from MRI data. AB - Magnetic resonance imaging and computational fluid dynamics (CFD) have been used in combination to simulate flow patterns at the human aorto-iliac bifurcation. Vascular anatomy was reconstructed from stacked two-dimensional (2D) time-of flight images, and revealed asymmetric, nonplanar geometry with curvature in the abdominal aorta and right iliac artery. The left iliac artery was straight and exhibited a smaller take off angle than the right iliac artery. The anatomical reconstruction was used to generate a computational mesh and obtain CFD predictions of flow and wall shear stress (WSS) within the region of interest. The dynamic boundary conditions necessary were specified by 2D cine phase contrast measurements of velocity profiles in each component vessel. Predicted flow patterns were in good quantitative agreement with experiment and demonstrated major differences in WSS distributions between the iliac arteries. This noninvasive approach has considerable potential to evaluate local geometries and WSS as risk factors for arterial disease in individual subjects. PMID- 10748433 TI - TSE-sequences with spin-echo contrast. AB - The article presents a discussion of the basic signal behavior of contrast modified RARE(TSE,FSE...)-sequences which have been modified such that the echo train used for image encoding is preceded by a long echo interval in order to introduce the T(2)-contrast of conventional spin-echo sequences while maintaining the high imaging speed of TSE. Sequences aimed at breathhold abdominal imaging as well as for the detection of hemorrhages in the CNS have been implemented and optimized. The significant difference in image contrast at identical echo times compared to unmodified TSE is demonstrated for different tissues. PMID- 10748434 TI - Rapid autocorrection using prescan navigator echoes. AB - Autocorrection is an adaptive motion correction algorithm that does not require an in vivo measurement of the motion record. A novel method for ensuring convergence of this algorithm when motion is severe is presented. A limited number of navigator echoes are acquired before the imaging sequence to obtain a "snapshot" of the object. Phase differences between the navigator and image k space data are used as an estimate of motion-induced phase shifts in the image, followed by autocorrection. In phantom data a six-fold reduction in computation time compared to autocorrection alone was realized. These results indicate that this navigator/autocorrection combination may be useful for reducing motion artifacts and computation time for MR exams when motion along the image phase encoding axis is severe. PMID- 10748435 TI - Correction for B(1) and B(0) variations in quantitative T(2) measurements using MRI. AB - A new method is described for compensating for the bias introduced by variations in radiofrequency (RF) field strength and main magnetic field strength when making quantitative T(2) measurements using MRI. Field measurements made during the MRI study are used in combination with a signal model for off-resonance and imperfect RF pulses to correct the estimated T(2) value at every voxel. Applicable to both multicomponent and conventional single-component T(2) studies, the method has been validated experimentally using paramagnetic salt solutions in a multicompartment phantom. Studies of the human head are used to demonstrate the method in practice. PMID- 10748436 TI - Spatial and temporal evolution of hemorrhage in the hyperacute phase of experimental spinal cord injury: in vivo magnetic resonance imaging. AB - To follow the spatial and temporal evolution of hemorrhage, in vivo MRI studies of experimental spinal cord injury (SCI) were performed on 17 rats in the very acute phase (hyperacute), starting as early as 9 min and continued up to 400 min posttrauma. Axial MR images were processed slice by slice over a 21 mm length around the epicenter of the injury. The data were analyzed statistically and fitted to an empirically derived function to characterize the spatial and temporal evolution of hemorrhage. The results indicated that 1) the initial hemorrhage in the very early phase of the injury area covered 12.5% of the total cord area and subsequently increased with a time constant of 700 min, 2) a major portion of the hemorrhage was concentrated spatially within the 4 mm distance from the epicenter, 3) the volume of hemorrhage normalized to its initial value increased linearly at a rate of approximately 0.0015 min(-1), and 4) edema was observed at the gray- and white-matter junction as early as 12 min postinjury. In general, edema appeared to be focal and scattered in this phase of the injury, which made its quantification unreliable on MRI. PMID- 10748437 TI - Time-course of the auditory BOLD response to scanner noise. AB - It is a concern for auditory fMRI studies that acoustic noise generated by the scanner produces an auditory response that can confound stimulus-induced activation. To establish how to minimize this problem, the present study mapped the time-course of the auditory response to a burst of acoustic scanner noise by employing a single-event method. Recorded bursts of scanner noise were interspersed with clustered-volume acquisitions at a range of stimulus-to-imaging delays to map the response with a temporal resolution of 1 sec. There were strong responses (1.5% signal change) to scanner noise in primary and secondary auditory cortex. In both cortical areas, the mean response rose to a peak by 4-5 sec after stimulus onset and decayed after a further 5-8 sec. The time course indicates that noise contamination in auditory fMRI can be substantially reduced by using a 9-12-sec repetition time, thus maximizing the dynamic range available for displaying the response to acoustical stimuli of interest. PMID- 10748438 TI - Limits of detection of polydimethylsiloxane in (29)Si NMR spectroscopy. AB - Limits of detection for neat poly(dimethylsiloxane) (PDMS), as detected via (29)Si NMR spectroscopy, were determined by the IUPAC, graphical, and propagation of errors methods from calibration curves obtained using a 7.05 Tesla superconducting magnet equipped with a magic angle spinning (MAS) NMR probe. Under conditions permitting full relaxation of the nuclear spins (recycle time >150 sec), the limit of detection with 128 transients was 1.7, 2.2, and 3.7 mg PDMS (23, 30, and 50 micromoles elemental Si) for the IUPAC, graphical, and propagation of errors methods, respectively. A higher limit of detection results when additional sources of uncertainty are considered. From the strict dependence of the limit of detection on the inverse of the square root of the number of transients signal-averaged, it is possible to extrapolate to a limit of detection of 183 microg PDMS (2.47 micromoles elemental Si) for a 24-hr signal averaging period. This limit of detection is 3 orders of magnitude higher than silicon levels found in human blood. PMID- 10748439 TI - Temperature dependence of human gastrocnemius pH and high-energy phosphate concentration by noninvasive techniques. AB - It is well established that ADP is an important regulator of the oxidative phosphorylation in the mitochondria. Thus, by means of noninvasive techniques it is demonstrated that the relationship between O(2) consumption of the human gastrocnemius at rest and its temperature is likely determined by at least two factors: 1) the modulation of the rate of the chemical reactions imposed by the "physical" temperature-effect; 2) the influence of temperature-induced ADP concentration changes ( approximately 0.83 microM degrees C(-1)) on oxidative phosphorylation. ADP was assessed by applying the temperature-corrected Lohmann equilibrium equation. PCr and ATP were found to increase, with decreasing temperature (-0.54+/-0.05 and -0.17+/-mM degrees C(-1), respectively), while pH varies following the alpha-stat hypothesis (-0.016+/-0.001 pH degrees C(-1)). These findings should be of value when dealing with muscle physiology in extreme environments or clinical applications of hypothermia. PMID- 10748440 TI - Reduction of resonant RF heating in intravascular catheters using coaxial chokes. AB - The incorporation of RF coils into the tips of intravascular devices has been shown to enable the localization of catheters and guidewires under MR guidance. Furthermore, such coils can be used for endoluminal imaging. The long cable required to connect the coil with the scanner input inadvertently acts as a dipole antenna which picks up RF energy from the body coil during transmit. Currents are induced on the cable which can lead to localized heating of surrounding tissue. Cables of various lengths were measured to determine if a resonance in the heating as a function of cable length could be found. Coaxial chokes with a length of lambda/4 were added to coaxial cables to reduce the amplitude of the currents induced on the cable shield. A 0.7-mm diameter triaxial cable, small enough to fit into a standard intravascular device, was developed and measured both with and without a coaxial choke. It is demonstrated that resonant heating does occur and that it can be significantly reduced by avoiding a resonant length of cable and by including coaxial chokes on the cable. PMID- 10748441 TI - Water diffusion, T(2) and compartmentation in frog sciatic nerve PMID- 10748442 TI - Prognostic significance of the distribution of neck node metastasis from oral carcinoma. AB - BACKGROUND: Carcinoma of the oral cavity presents a high risk for neck metastasis, which reduces the probability of regional control and survival. OBJECTIVES: The main objective of this study is to analyze prognostic implications of the distribution of neck metastasis in 513 patients with squamous cell carcinoma of the oral cavity. PATIENTS AND METHODS: All patients underwent surgery from 1970-1992. Tumor stages were I, 63; II, 120; III, 173; and IV, 157. Neck dissections were performed in 448 patients (115 bilateral). RESULTS: By use of multivariate regression techniques the level of lymph node involvement was the most important prognostic factor (relative risks from 1.8 to 2.5). The following variables were also associated with prognosis: mobility of lymph nodes, sex, T stage, age, and tumor thickness. CONCLUSIONS: The level of ipsilateral lymph node involvement was the most significant prognostic factor patients with in oral cancer who underwent surgical treatment. A significant decrease in survival also was seen with regard to the involvement of multiple contralateral lymph nodes. Our results support the indication of elective neck dissections in high-risk patients because among the cases that had metastases at follow-up, 50% were not candidates for salvage treatment. PMID- 10748443 TI - Nasopharyngectomy in the treatment of recurrent nasopharyngeal carcinoma: a twelve-year experience. AB - BACKGROUND: The incidence of recurrent nasopharyngeal carcinoma (NPC) after primary radiotherapy is considerable. The result of re-irradiation to the recurrent tumor is not satisfactory. METHODS: Thirty-one patients who received nasopharyngectomy for treatment of their recurrent nasopharyngeal carcinoma (NPC) (rT1 to rT3) at the Prince of Wales Hospital during the period 1986 to 1997 were reviewed retrospectively. Eleven patients had nasopharyngectomy performed by the transoropalatal approach, 9 patients by the maxillary swing approach, and 11 patients by the mandibular approach. RESULTS: Most recurrences (29 of 31) were rT1 and rT2 tumors. No hospital mortality occurred. The common complications of nasopharyngectomy were palatal defect (17 of 31), trismus (15 of 31), otitis media with effusion (20 of 31), dysphagia (12 of 31), and nasal regurgitation (8 of 31). Nine surgical specimens had microscopic invasion of the resection margins. The 5-year actuarial overall survival, actuarial disease-free survival, and tumor control were 47%, 42%, and 43%, respectively. The administration of postoperative radiotherapy significantly enhanced survival and tumor control after nasopharyngectomy. CONCLUSIONS: Nasopharyngectomy supplemented by postoperative radiotherapy achieved significant survival and tumor control with acceptable complications in selected recurrent NPC. It appears to be a better salvage treatment than re-irradiation alone for selected recurrent NPC. PMID- 10748444 TI - A probability prediction rule for malignant cervical lymphadenopathy using sonography. AB - BACKGROUND: Our purpose was to weigh various sonographic parameters as predicting malignant cervical lymphadenopathy and build a reliable prediction rule. METHODS: One hundred and eighty-nine cervical lymph node lesions from 125 consecutive patients were used for building the prediction model. Sonographic variables, including 15 morphologic features of B-mode, 5 vascular parameters of color Doppler mode, along with age and sex, were analyzed with multivariate logistic regression to evaluate the joint effect of a set of independent variables. A prediction rule for malignant lymphadenopathy was established, and prospective validation was assessed on a new group consisting of 100 lymph nodes from another 60 consecutive patients. RESULTS: The association of heterogeneous content, long transverse diameter, pathologic vascular pattern, high vascular density, and older age provided the most robust prediction value. Scoring scale was designed as 1x (age) + 2x (vascularity index) + 3x (short axis) + 4x (vascular pattern) + 4x (internal echo) according to the parameter estimates of multivariate logistic regression analysis. Cut-off value of score >==10 as malignancy resulted in 89.2% sensitivity and 85.2% specificity. Prospective validation also showed satisfactory results (sensitivity, 82.9%; specificity, 86.2%). CONCLUSIONS: By measuring only 4 sonographic parameters and age, this prediction rule could provide the physician a nonconfusing and reliable probability reference for managing cervical lymphadenopathy. PMID- 10748445 TI - Correlation between tumor consistency and cervical metastasis in tongue carcinoma. AB - BACKGROUND: The purpose of this study was to assess the correlation between the tumor consistency and cervical lymph node metastasis in tongue carcinoma. METHODS: Fifty-three patients with squamous cell carcinoma of the tongue were examined with an instrument capable of measuring consistency. These cases were compared in relation to TN classification, pathologic status of nodes, and consistency. RESULTS: The mean values of consistency in tongue carcinoma were 19. 7 +/- 2.9 g, 13.6 +/- 3.7 g, 27.4 +/- 4.5 g, and 21.2 +/- 6.7 g, corresponding to pathologically positive node group (PN[+]) in T1, pathologically negative node group (PN[-]) in T1, PN(+) in T2, and PN(-) in T2, respectively. Difference in consistency between PN(+) and PN(-) was significant (T1: p <.01, T2: p <.05). In the N0 patients, difference in consistency between PN(+) and PN(-) was also significant (p <.01). CONCLUSIONS: We suggest that quantitative evaluation of tumor consistency would be useful in determining whether selective neck dissections can safely be omitted in some patients. PMID- 10748446 TI - Cyclin D1 expression is predictive of occult metastases in head and neck cancer patients with clinically negative cervical lymph nodes. AB - BACKGROUND: The aim of this study was to investigate the value of p53 and cyclin D1 gene expression in predicting the risk of occult lymph node metastases in patients with head and neck squamous cell carcinoma (HNSCC). METHODS: The expression of cyclin D1 and p53 was evaluated by means of immunohistochemical analysis in 32 HNSCC patients with clinically and radiologically negative lymph nodes in whom metastatic involvement was subsequently demonstrated at histologic examination (pN+). A group of 64 head and neck cancer patients with histologically negative laterocervical lymph nodes (pN0) was used as a control. RESULTS: Cyclin D1 and p53 expression were observed respectively in 42 (43.7%) and 48 cases (50%). Cyclin D1 expression significantly correlated with tumor extension and advanced clinical stage (p =.002 and p =.001, respectively). At univariate regression analysis, cyclin D1 expression significantly correlated with the presence of occult lymph node metastases (p =. 0007), and it remained an independent predictor at multivariate regression analysis (p =.0059). CONCLUSIONS: Our study indicates that the expression of cyclin D1 correlates with the presence of occult cervical metastases in head and neck carcinoma patients, thus suggesting that its immunohistochemical evaluation in biopsy samples may be used as an additional tool for identifying patients to be treated with elective neck dissection. PMID- 10748447 TI - A feasibility study of salivary gland autograft transplantation for xerostomia. AB - BACKGROUND: Radiation-induced xerostomia is a frequent sequela in patients treated for cancer of the head and neck. One strategy to treat xerostomia would be to relocate portions of salivary tissue to adjacent submucosal sites that lie outside the radiation portals such as the anterior oral vestibule. It is not known whether salivary tissue transplanted as an autogenous free graft can survive, function adequately, and not produce mucoceles. METHODS: Salivary gland tissue from the parotid and submandibular glands of the Syrian hamster were transplanted into the submucosal layer of the cheek pouch. After 3 months of observation, looking at graft size, graft extrusion, ulceration, infection, and mucocele formation, the graft sites were harvested. The specimens then underwent pathologic analysis by hematoxylin and eosin staining, as well as immunohistochemical methods to determine positivity for cytokeratin, smooth muscle actin (SMA), and amylase. RESULTS: Histologic analysis of tissue harvested from Syrian hamsters grafted into the cheek pouch demonstrated intact, viable, organized salivary gland tissue. Eighty percent of the animals in the submandibular group and 63% of the animals in the parotid group had at least 1 graft with viable salivary tissue without undue complications. CONCLUSIONS: Salivary gland tissue can be transplanted successfully as free autogenous grafts in the Syrian hamster model. Further studies are needed to determine whether the grafts will subsequently become functional and whether growth can be biologically stimulated. This approach may be a useful strategy to protect salivary gland tissue in patients undergoing radiotherapy for head and neck cancer. PMID- 10748448 TI - Effects of sodium butyrate on growth, differentiation, and apoptosis in head and neck squamous carcinoma cell lines. AB - BACKGROUND: Biologic agents that reverse early changes in the aerodigestive tract mucosa have potential treatment applications for patients with field cancerization of the upper aerodigestive tract. Sodium butyrate (BA) is a normal dietary constituent that induces differentiation and inhibits growth in several malignant cell types in vitro, but its effect on head and neck squamous cell carcinoma (HNSCC) has not been evaluated. METHODS: Using five HNSCC cell lines, the effects of BA on cell proliferation and apoptosis were examined by colorimetric and fluorescence-labeling methods, and the expression of differentiation markers and apoptosis-related proteins were analyzed using Western and Northern blotting, flow cytometry, and cell cycle analysis. RESULTS: BA-induced growth inhibition and apoptosis in HNSCC cells at millimolar concentrations. Apoptosis induction did not depend on the p53 status of the cell lines or on expression of members of the Bcl-2/Bax family. CONCLUSIONS: These results demonstrate that butyrate has activity against HNSCC in vitro and may have clinical applications for management of HNSCC patients. PMID- 10748449 TI - Prolactin as a local growth promoter in patients with locally advanced tongue cancer: GCRI experience. AB - BACKGROUND: The purpose of this study was to assess the role of prolactin (PRL) in men with locally advanced tongue cancer. METHODS: Circulating PRL was assayed immunoradiometrically in pretherapeutic and sequential blood samples of 99 patients with locally advanced tongue cancer. Patients were followed for 3 years or until their death within a stipulated time. Immunohistochemical localization of PRL was performed on formalin-fixed paraffin-embedded tissue sections. Tumoral prolactin receptors (PRLR) were estimated by ligand binding assay; the expression of PRL mRNA and PRLR mRNA were carried out by reverse transcriptase polymerase chain reaction (RT-PCR). Furthermore, PRL amplimer was sequenced and compared with human pituitary PRL amplimer. RESULTS: Pretherapeutic PRL levels were significantly higher in patients with locally advanced tongue cancer compared with controls (p =.01). Thirty-four percent (34 of 99) of the patients had hyperprolactinemia (PRL >/=15.0 ng/mL). Univariate survival analysis showed that patients with pretherapeutic hyperprolactinemia had a significantly shorter overall survival than patients with pretherapeutic PRL <15.0 ng/mL serum (p =.0009). In multivariate analysis, PRL emerged as the most significant independent prognostic factor influencing overall survival. Furthermore, changes in serial PRL levels showed excellent correlation with response to therapy and progression of disease. Forty-four percent (24 of 54) of the tumors showed positive immunoreactivity with PRL antibody, indicating that PRL or a molecule similar to it is produced by tongue tumors. PRL mRNA expression was seen in 85% (43 of 50) of the tumors and confirmed the de novo synthesis of PRL. Sequence analysis of the 234 bp PRL amplimer revealed that the sequence was homologous to exon 5 of pituitary PRL mRNA. The action of PRL is mediated by PRLR, and it was observed that the PRLR positivity by ligand binding assay was 33%. The expression of PRLR mRNA by RT-PCR showed two forms of PRLR mRNA (ie, intermediate form [500 600 bp] seen in 82% (41 of 50 ) of the tumors and the long form [800-900 bp] seen in 36% (18 of 50) of the tumors. In 82% (41 of 50) of the tumors, either the intermediate or long form was seen. CONCLUSIONS: This multifaceted study of PRL suggests that tongue cancer cells produce PRL, and this ectopically produced PRL might be acting as a major local growth promoter by means of autocrine and paracrine mechanisms. Looking at its prognostic value and correlation with disease activity, it may provide new insights into treatment of tongue cancer. PMID- 10748450 TI - Cancer spread in the larynx: a pathologic basis for conservation surgery. AB - BACKGROUND: Previous pathologic studies of the spread of laryngeal carcinoma have drawn inferences about the site of origin of tumors, their mechanisms of growth, or the role of structures as potential barriers to tumor spread. Most of the information is based on the study of advanced or recurrent tumors and is difficult to apply to conservation surgical technique. We carried out a systematic analysis of a wide range of laryngeal tumors with the aim of providing a basis for conservation surgery. METHODS: We analyzed tumor invasion of the laryngeal spaces and the laryngeal framework in relation to the mucosal tumor extent by axial sectioning of 80 sequential partial and total laryngectomy specimens. RESULTS: Invasion of a particular laryngeal space could be accurately predicted by mucosal tumor extent and vocal cord mobility. Invasion of the laryngeal framework was associated with tumor extension to the ventricle, subglottis, or pyriform fossa. The thyroid cartilage and the cricothyroid space and ligament were the most frequent sites of invasion. CONCLUSIONS: The mucosal distribution of a tumor and observation of vocal cord mobility can be used to determine accurately the extent of tumor invasion of the laryngeal spaces and framework and therefore the extent of resection necessary. PMID- 10748451 TI - Neck node metastases from nasopharyngeal carcinoma: MR imaging of patterns of disease. AB - BACKGROUND: The purpose was to use MR imaging to document the patterns of nodal involvement in the upper neck in nasopharyngeal carcinoma (NPC). METHODS: The MR images of 150 patients with newly diagnosed NPC were reviewed retrospectively. Nodes were considered abnormal on MR criteria of size, necrosis, and extracapsular spread. RESULTS: Retropharyngeal nodes (RN) were more frequently involved than nonretropharyngeal nodes (NRNs) (94% vs 76% in 115 patients with nodal metastases). NRN involvement without RN was seen in only 7 of 115 patients (6%). Involvement of RN at the level of the oropharynx (82%) was as common as at the nasopharynx (83%) level. Internal jugular nodes were the most frequently involved NRN nodes (72%). Spinal accessory nodal involvement was also common (57%) but seldom in isolation (8%). Submandibular (3%) and parotid (2%) nodal metastases were uncommon and were always associated with advanced nodal metastases in the ipsilateral RN, internal jugular, and spinal accessory regions. CONCLUSION: Retropharyngeal nodes are the first echelons of nodal metastases. Direct lymphatic spread to the neck without involvement of the RN nodes is uncommon. RN metastases at the level of the oropharynx are more common than previously suspected, and this should influence radiotherapy planning. NRN outside the internal jugular and spinal accessory chains are rare and only occur when the usual routes of lymphatic spread have already been blocked by tumor. PMID- 10748452 TI - The ability of tumor volume to predict local control in surgically treated squamous cell carcinoma of the supraglottic larynx. AB - PURPOSE: Pretreatment CT volumetric measurement of the primary tumor has been shown to be a predictor of local control in patients with laryngeal carcinoma treated with radiation therapy (RT) alone. A direct association has been demonstrated between tumor volume of supraglottic squamous cell carcinoma (SGSCCA) and local control. However, the association between tumor volume of SGSCCA and local control has not been investigated in patients treated surgically. The purpose of this study was to determine the relationship between SGSCCA tumor volume and local control in patients treated surgically. MATERIALS AND METHODS: Primary site tumor volume was calculated from pretreatment CT studies in 37 laryngeal supraglottic carcinomas treated surgically. All patients had clinical follow-up for evidence of recurrent tumor along the surgical margins at the primary site for a minimum of 2 years after completion of treatment. Statistical analysis consisted of Mantel-Haenszel chi-square tests and Fisher's exact test. RESULTS: Overall local control rate was 92% (33 of 37). Tumor volume was significantly associated with local control (p <. 05). Local control rate for tumors with volumes <16 cc was 94% (32 of 34) (p <.05). CONCLUSIONS: Pretreatment CT volumetric analysis is useful for predicting local control in patients with SGSCCA carcinoma treated surgically. PMID- 10748453 TI - Assessment of quality of life in patients treated with accelerated radiotherapy for laryngeal and hypopharyngeal carcinomas. AB - BACKGROUND: This study was conducted to evaluate quality of life (QOL) and functional outcome in patients with carcinomas of the larynx and hypopharynx treated with accelerated radiotherapy (RT). METHODS: Between January 1991 and September 1996, 21 patients treated with accelerated concomitant boost RT schedule (69.9 Gy in 5. 5 weeks) for laryngeal (n = 10) or hypopharyngeal (n = 11) carcinomas and who remained free of disease at 1-year minimum follow-up were evaluated. The functional outcome was assessed by the subjective Performance Status Scale for Head and Neck cancer (PSSHN) and general QOL by the European Organization for Research and Treatment of Cancer Core QOL questionnaire (EORTC QLQ-C30). The median length of follow-up was 37 months (range, 13 to 75). RESULTS: The PSSHN scores were 89, 84, and 86, respectively, for eating in public, understandability of speech and normalcy of diet (100 = normal function). Significantly lower scores for understandability of speech were observed in patients with advanced and laryngeal carcinomas. Normalcy of diet was affected negatively by the severity of xerostomia. All mean functional scale scores of the EORTC QLQ-C30 module were 20% to 25% below the higher score. Most of these scale scores were significantly affected by the severity of xerostomia. CONCLUSIONS: Patients treated with concomitant boost RT for laryngeal and hypopharyngeal carcinomas appear to have similar QOL and functional outcome to those reported for patients treated with conventional or hyperfractionated RT. As expected, many QOL scales were affected by the severity of xero- stomia. PMID- 10748455 TI - Cancer of the tongue in patients less than forty. AB - INTRODUCTION: It is the opinion of many surgeons that the biologic potential of cancer that develops in young people is different compared with older patients. Prior reports on small series of patients addressing this issue have inadequate statistical power to resolve the question. METHODS: By use of the techniques of meta-analysis, patients less than 40 years old who had undergone treatment of squamous cell carcinoma (SCC) of the oral tongue were examined. Twenty-eight patients who were encountered in the Department of Otolaryngology, University of Pittsburgh School of Medicine, and 94 patients were identified in the literature for a total of 122 patients <40 years old. A control group of 150 patients, aged 40 years and older treated for SCC of the oral tongue between 1982 and 1994 was identified. RESULTS: Three-year disease-free survival in the group of patients aged less than 40 was 53.3% compared with 3-year disease free survivorship of 55.0% in the older cohort of patients. CONCLUSION: These data strongly suggest that the outcomes of treatment for SCC of the oral tongue in young patients are similar compared with patients older than 40 with similar extent of disease. PMID- 10748454 TI - Primary surgery as treatment for early squamous cell carcinoma of the tonsil. AB - BACKGROUND: The management of tonsil carcinoma has gradually evolved such that the literature is replete with outcome summaries of this disease treated with primary RT and chemotherapy. Recently there have been no reports of patient outcomes with primary surgical therapy. Nonsurgical treatment is warranted when tumors are unresectable or if the patient refuses surgery. Our policy has been to treat operable squamous cell carcinoma (SCCA) of the tonsil with surgery. The decision to use adjuvant therapy is based on the surgical and histologic findings. We herein report our results with this treatment protocol. METHODS: A retrospective review of 162 patients with SCCA of the tonsil was performed. Eighty-four patients were treated with surgery, which was followed by RT and/or chemotherapy if histologic signs of aggressive behavior were identified. Patients were followed 2 to 15 years after treatment. RESULTS: Of the 9 patients with stage I disease, 89% are without evidence of recurrent disease and 91% of patients with stage II tonsil cancers are also disease free. The survival rates for stage III and stage IV cancer patients are 79 and 52%, respectively. CONCLUSION: Our data suggest that patients with early tonsil cancer can be effectively treated with surgery. Surgery allows pathologic staging so that patients with advanced tumors can be treated with adjuvant therapy. PMID- 10748456 TI - Surgical treatment of recalcitrant radiation-induced gastric erosions. AB - BACKGROUND: Uncontrolled bleeding as a result of radiation gastritis in patients who have pharyngo-laryngo-esophagectomy and gastric pull-up is seldom reported. Surgical resection in the management of this condition has rarely been described. METHOD: A 66-year-old man with hypopharyngeal cancer was treated by pharyngo laryngo-esophagectomy and gastric transposition. He received postoperative radiotherapy and had recurrent hemorrhagic gastritis, necessitating surgical resection. The manubrium was resected to access the mediastinal part of the gastric conduit. The diseased part of the gastric conduit was removed and a free jejunal graft was interposed to replace the resected stomach. RESULTS: Manubrial resection offered adequate access to the stomach transposed in the mediastinum, and the life-threatening bleeding gastritis was successfully controlled by surgical resection. CONCLUSION: Surgical resection of the radiation-damaged transposed stomach through a manubrial resection approach can safely be performed. Free jejunal graft is the choice of reconstruction of the circumferential defect. PMID- 10748457 TI - Abstract PMID- 10748458 TI - Identification of a prx1 limb enhancer. AB - Mice with a loss of function of prx1, a paired-related homeobox gene formerly called Mhox, showed craniofacial defects, limb shortening, and incompletely penetrant spina bifida. To investigate the mechanisms that regulate prx1 expression, we analyzed a 2.4-kb prx1 genomic flanking region in transgenic mice. This region of the prx1 gene contains an enhancer element that directs expression of a LacZ reporter gene in limb bud mesenchyme and a subset of craniofacial mesenchyme. Deletional analysis in transgenic founders identified a necessary 530 bp core element. Comparison of this core element with human Prx1 sequence showed two highly conserved cassettes that also contained a prx recognition element. Moreover, transgene expression was diminished in posterior handplate of prx1; prx2 double mutant mice. Our data reveal that the prx1 limb enhancer is proximally located within the prx1 gene and suggest that prx1 may have an autoregulatory function in limb mesenchyme. PMID- 10748459 TI - Optimal N-ethyl-N-nitrosourea (ENU) doses for inbred mouse strains. AB - ENU is a powerful germline mutagen in the mouse, providing the opportunity to analyze the functions of large numbers of genes in the mammalian genome. In many mutagenesis experiments, it would be beneficial to exploit the advantages of inbred mouse strains. To perform an effective ENU mutagenesis screen using inbred mice, a dosage regimen is required to determine the optimal dose of ENU for that inbred strain, a time-consuming preliminary process. We have carried out dosage regimens for mutagenizing doses of ENU in ten inbred strains of mouse: 129X1/SvJ, 129S6/SvEv, A/J, BALB/cJ, BTBR/N, C3He/J, C3HeB/FeJ, C57BL/6J, C57BR/cdJ, and CBA/CaJ, and determined an optimal dose for each strain, defined by length of sterile period and number of males to survive treatment. Three strains: A/J, BALB/cJ and C57BL/6J, are able to tolerate high doses, up to 300 mg/kg body weight, and are highly recommended for mutagenesis studies. PMID- 10748460 TI - The sequence and associated null phenotype of a C. elegans neurocalcin-like gene. AB - The neuronal calcium sensor (NCS) proteins belong to a subfamily of the EF-hand calcium binding proteins. These proteins are primarily expressed in the nervous system and currently include more than 20 members across species [Nakayama et al., J Mol Evol 34:416-448, 1992]. Two homologues of the ncs genes, Ce-ncs-1 and Ce-ncs-2, have recently been identified in the nematode C. elegans. Here we report the cDNA sequence of a third C. elegans ncs homologue, Ce-ncs-3. We demonstrate that a null mutation in this gene caused by a large deletion in the locus does not confer a visible phenotype in C. elegans. This, in addition to the strong homology between Ce-NCS-3 and the other C. elegans NCS proteins, may indicate functional redundancy between the three genes. PMID- 10748461 TI - Temperature-dependent gene silencing by an expressed inverted repeat in Drosophila. AB - Posttranscriptional gene silencing (PTGS) induced by double-stranded RNA (dsRNA) is an intriguing phenomenon that has been observed in a variety of organisms, including Drosophila melanogaster. Although PTGS in Drosophila is typically observed following direct injection of the dsRNA into embryos, it is theoretically possible that the in vivo transcription of an inverted repeat transgene might also produce a dsRNA "hairpin" that is capable of triggering PTGS. Here we test this idea, and show that an expressed inverted repeat of a portion of the sex differentiation gene, transformer-2, (tra-2), driven by a GAL4 dependent promoter, does genetically repress the endogenous wild-type tra-2 function, producing a dominant loss-of-function mutant phenotype. Remarkably, this effect is temperature-sensitive, with phenotypic consequences seen at 29 degrees, but not at 22 degrees. Moreover, by altering the dosage of either the transgenes or the endogenous tra2(+) loci, one can vary the effect over a wide range of mutant phenotypes. PMID- 10748462 TI - Gene-trap-based target site for cre-mediated transgenic insertion. AB - There is an increasing need for tissue-specific gene expression regulatory elements to study normal and disease development in the mouse. However, the cloning and characterization of these elements are time-consuming and costly. Thus, there is a particular need to be able to identify gene expression patterns without having to clone the promoter elements. Gene-trap strategies identify expression patterns assigned for endogenous genes using reporters, such as LacZ (Gossler et al., 1989; Skarnes, 1990) or green fluorescent protein (GFP) (Ishida and Leder, 1999; Zheng and Hughes, 1999). The gene-trap vector randomly inserts into the genome and "steals" regulatory elements for the reporter. Here we describe an improved gene-trap strategy, which allows an efficient Cre recombinase-mediated insertion of any transgene into the trapped loci as a post integrational modification and links the expression of the transgene to that of the reporter. PMID- 10748463 TI - The cerberus-related gene, Cerr1, is not essential for mouse head formation. AB - The Xenopus cerberus gene encodes a secreted factor expressed in the Spemann organizer that can cause ectopic head formation when its mRNA is injected into Xenopus embryos. In mouse, the cerberus-related gene, Cerr1, is expressed in the anterior mesendoderm that underlies the presumptive anterior neural plate and its expression is downregulated in Lim1 headless embryos. To determine whether Cerr1 is required for head formation we generated a null mutation in Cerr1 by gene targeting in mouse embryonic stem cells. We found that head formation is normal in Cerr1(-/-) embryos and we detected no obvious phenotypic defects in adult Cerr1(-/-) mice. However, in embryonic tissue layer recombination assays, Cerr1( /-) presomitic/somitic mesoderm, unlike Cerr1-expressing wild-type presomitic/somitic mesoderm, was unable to maintain expression of the anterior neural marker gene Otx2 in ectoderm explants. These findings suggest that establishment of anterior identity in the mouse may involve the action of multiple functionally redundant factors. PMID- 10748464 TI - Targeted insertion of a lacZ reporter gene into the mouse Cer1 locus reveals complex and dynamic expression during embryogenesis. AB - The mouse Cer1 (mCer1, Cer-l, Cerr1) gene encodes one member of a family of cytokines structurally and functionally related to the Xenopus head-inducing factor, Cerberus (xCer). We generated a mouse line in which the Cer1 gene was inactivated by replacing the first coding exon with a lacZ reporter gene. Mice homozygous for this allele (Cer1(lacZ)) showed no apparent perturbation of embryogenesis or later development. However, the lacZ reporter revealed a number of hitherto uncharacterised sites of Cer1 expression in late fetal and adult tissues. Preliminary analysis suggests that Cer1 is not essential for their morphogenesis, differentiation, or homeostasis. PMID- 10748465 TI - Cerberus-like is a secreted BMP and nodal antagonist not essential for mouse development. AB - Mouse cerberus-like (cer-l) is a member of the Cerberus/Dan family of secreted factors. As other members of this family of proteins, Cer-l functions in the extracellular space, inhibiting signaling molecules. Here we show that the neural inducing and mesoderm-inhibiting activities of Cer-l result from specific binding to BMP and Nodal molecules, respectively. These properties resemble the ones from the related factor Xenopus Cerberus. However, Xenopus Cerberus in addition to BMP4 and Nodal also binds to and inhibits Wnt proteins. We show that Cer-l does not directly inhibit Wnt signals. A null allele of the mouse Cer-l gene was generated by targeted inactivation in ES cells. Homozygous embryos show no anterior patterning defects, are born alive, and are fertile. Since mouse Cer-l and Xenopus Cerberus differ in biochemical activities, we propose the existence of additional members of this family of inhibitors, which may compensate for the loss of cer-l. PMID- 10748467 TI - The synthetic multivulval genes of C. elegans: functional redundancy, Ras antagonism, and cell fate determination. AB - Development of the C. elegans vulva requires coordination between a strikingly complex set of molecular regulators and pathways. In particular, the correct specification of vulval cell-fates requires both the activation of RTK/Ras/Map kinase members as well as negative regulation by a set of genes known as the SynMuvs. SynMuvs comprise two functionally redundant sets of genes that appear to antagonize Ras pathway signaling. In this way, SynMuv genes act to limit the number of cells adopting vulval fates. Recently, a number of SynMuv genes have been shown to encode worm homologs of the Rb transcriptional-regulatory complex. These and other results are discussed and we present several models for understanding the role of SynMuv genes in vulval development. PMID- 10748466 TI - Deletion of Dad1 in mice induces an apoptosis-associated embryonic death. AB - Dad1 is a putative anti-apoptosis gene identified in several distantly related organisms. Expression of Dad1 in transfected cells inhibits apoptosis in vitro. To determine whether Dad1 has a similar function in vivo, we used gene targeting to delete Dad1. Heterozygous adult mice (+/-) show no obvious phenotype or abnormalities, but genotype analysis of over 100 offspring from heterozygous matings detected no weanling, homozygous Dad1 null (-/-) mice. Subsequent analysis of embryos from heterozygous matings detected Dad1 null (-/-) embryos at E3.5 but no later, suggesting Dad1 is required for development beyond the late blastocyst stage. Increased levels of apoptosis were observed in cultured embryos lacking a functional copy of the gene, consistent with an anti-apoptotic role for Dad1. PMID- 10748468 TI - Caveat e-mptor PMID- 10748469 TI - Genetic supremacy is so 20th century PMID- 10748470 TI - Equipping scientists for the new biology. PMID- 10748471 TI - The protocol's illusionary principle. PMID- 10748473 TI - Reply to "Hazardous CaMV promoter?" PMID- 10748472 TI - Agbiotech and world food security - threat or boon? PMID- 10748474 TI - Hazardous CaMV promoter? PMID- 10748475 TI - Human genome bombshell PMID- 10748476 TI - Drosophila sequence "done" PMID- 10748479 TI - Japan bans human cloning PMID- 10748477 TI - Pigs cloned for first time PMID- 10748478 TI - GMO roundup PMID- 10748480 TI - Population genetics deal PMID- 10748481 TI - Transatlantic biotech deal PMID- 10748482 TI - Beyond nine lives PMID- 10748483 TI - Stem cells as research tools PMID- 10748484 TI - EPO admits patent mistake PMID- 10748485 TI - Research collaborations PMID- 10748486 TI - Elan buys liposome PMID- 10748488 TI - Edible vaccine success PMID- 10748487 TI - Call for food safety body PMID- 10748489 TI - Reversible immortality PMID- 10748491 TI - Gene therapy hits a nerve PMID- 10748490 TI - Amyloid inhibitor get rational PMID- 10748493 TI - Particles on the nose PMID- 10748494 TI - Microarrays get into print PMID- 10748492 TI - Bionic shocker PMID- 10748496 TI - Artificial antigen presenting cells PMID- 10748495 TI - Excising selectable markers from transgenic plants PMID- 10748497 TI - A "cool" new system for gene expression PMID- 10748498 TI - In and out PMID- 10748500 TI - Accessorizing biomaterials PMID- 10748499 TI - Human stem cells become more established PMID- 10748501 TI - Identifying DNA targets of chromatin proteins PMID- 10748502 TI - Technical reports PMID- 10748503 TI - Cellular magnetism PMID- 10748504 TI - Review PMID- 10748505 TI - Unleashing a killer protein PMID- 10748506 TI - Opinions evolve on Kauffman patent. PMID- 10748507 TI - German Health Minister calls time out for Bt maize. PMID- 10748508 TI - French genomics setup questioned. PMID- 10748509 TI - GM food singled out for labeling in the US. PMID- 10748510 TI - Three-way merger regenerates new company. PMID- 10748511 TI - Scrutiny of gene therapy broadens, intensifies. PMID- 10748512 TI - Validating a business model. PMID- 10748513 TI - Creating molecular clues to uncover gene function. PMID- 10748514 TI - New tools for human developmental biology. PMID- 10748515 TI - Target watching with a beady eye. PMID- 10748516 TI - Injecting new ideas into microarray printing. PMID- 10748517 TI - New paths to pain relief. PMID- 10748518 TI - Large-scale functional analysis using peptide or protein arrays. AB - The array format for analyzing peptide and protein function offers an attractive experimental alternative to traditional library screens. Powerful new approaches have recently been described, ranging from synthetic peptide arrays to whole proteins expressed in living cells. Comprehensive sets of purified peptides and proteins permit high-throughput screening for discrete biochemical properties, whereas formats involving living cells facilitate large-scale genetic screening for novel biological activities. In the past year, three major genome-scale studies using yeast as a model organism have investigated different aspects of protein function, including biochemical activities, gene disruption phenotypes, and protein-protein interactions. Such studies show that protein arrays can be used to examine in parallel the functions of thousands of proteins previously known only by their DNA sequence. PMID- 10748519 TI - Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. AB - We describe the derivation of pluripotent embryonic stem (ES) cells from human blastocysts. Two diploid ES cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent primate cells. Human ES cells express the transcription factor Oct-4, essential for development of pluripotential cells in the mouse. When grafted into SCID mice, both lines give rise to teratomas containing derivatives of all three embryonic germ layers. Both cell lines differentiate in vitro into extraembryonic and somatic cell lineages. Neural progenitor cells may be isolated from differentiating ES cell cultures and induced to form mature neurons. Embryonic stem cells provide a model to study early human embryology, an investigational tool for discovery of novel growth factors and medicines, and a potential source of cells for use in transplantation therapy. PMID- 10748520 TI - Induction of human cytotoxic T lymphocytes by artificial antigen-presenting cells. AB - The adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) is a promising therapeutic approach for a number of diseases. To overcome the difficulty in generating specific CTLs, we established stable artificial antigen presenting cells (AAPCs) that can be used to stimulate T cells of any patient of a given human leukocyte antigen (HLA) type. Mouse fibroblasts were retrovirally transduced with a single HLA-peptide complex along with the human accessory molecules B7.1, ICAM-1, and LFA-3. These AAPCs consistently elicit strong stimulation and expansion of HLA-restricted CTLs. Owing to the high efficiency of retrovirus-mediated gene transfer, stable AAPCs can be readily engineered for any HLA molecule and any specific peptide. PMID- 10748521 TI - Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. AB - The ability to track the distribution and differentiation of progenitor and stem cells by high-resolution in vivo imaging techniques would have significant clinical and research implications. We have developed a cell labeling approach using short HIV-Tat peptides to derivatize superparamagnetic nanoparticles. The particles are efficiently internalized into hematopoietic and neural progenitor cells in quantities up to 10-30 pg of superparamagnetic iron per cell. Iron incorporation did not affect cell viability, differentiation, or proliferation of CD34+ cells. Following intravenous injection into immunodeficient mice, 4% of magnetically CD34+ cells homed to bone marrow per gram of tissue, and single cells could be detected by magnetic resonance (MR) imaging in tissue samples. In addition, magnetically labeled cells that had homed to bone marrow could be recovered by magnetic separation columns. Localization and retrieval of cell populations in vivo enable detailed analysis of specific stem cell and organ interactions critical for advancing the therapeutic use of stem cells. PMID- 10748522 TI - Enzymatic incorporation of bioactive peptides into fibrin matrices enhances neurite extension. AB - Fibrin plays an important role in wound healing and regeneration, and enjoys widespread use in surgery and tissue engineering. The enzymatic activity of Factor XIIIa was employed to covalently incorporate exogenous bioactive peptides within fibrin during coagulation. Fibrin gels were formed with incorporated peptides from laminin and N-cadherin alone and in combination at concentrations up to 8.2 mol peptide per mole of fibrinogen. Neurite extension in vitro was enhanced when gels were augmented with exogenous peptide, with the maximal improvement reaching 75%. When this particular fibrin derivative was evaluated in rats in the repair of the severed dorsal root within polymeric tubes, the number of regenerated axons was enhanced by 85% relative to animals treated with tubes filled with unmodified fibrin. These results demonstrate that it is possible to enhance the biological activity of fibrin by enzymatically incorporating exogenous oligopeptide domains of morphoregulatory proteins. PMID- 10748523 TI - Efficient delivery of transgenes to human hair follicle progenitor cells using topical lipoplex. AB - The topical delivery of transgenes to hair follicles has potential for treating disorders of the skin and hair. Here we show that the topical administration of liposome-DNA mixtures (lipoplex) to mouse skin and to human skin xenografts resulted in efficient in vivo transfection of hair follicle cells. Transfection depended on liposome composition, and occurred only at the onset of a new growing stage of the hair cycle. Manipulating the hair follicle cycle with depilation and retinoic acid treatment resulted in nearly 50% transfection efficiency-defined as the proportion of transfected, newly growing follicles within the xenograft. Transgenes administered in this fashion are selectively expressed in hair progenitor cells and therefore have the potential to affect the characteristics of the follicle. These findings form a foundation for the future use of topical lipoplex applications to alter hair follicle phenotype and treat diseases of the hair and skin. PMID- 10748524 TI - Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. AB - We have developed a novel technique, named DamID, for the identification of DNA loci that interact in vivo with specific nuclear proteins in eukaryotes. By tethering Escherichia coli DNA adenine methyltransferase (Dam) to a chromatin protein, Dam can be targeted in vivo to native binding sites of this protein, resulting in local DNA methylation. Sites of methylation can subsequently be mapped using methylation-specific restriction enzymes or antibodies. We demonstrate the successful application of DamID both in Drosophila cell cultures and in whole flies. When Dam is tethered to the DNA-binding domain of GAL4, targeted methylation is limited to a region of a few kilobases surrounding a GAL4 binding sequence. Using DamID, we identified a number of expected and unexpected target loci for Drosophila heterochromatin protein 1. DamID has potential for genome-wide mapping of in vivo targets of chromatin proteins in various eukaryotes. PMID- 10748525 TI - A temperature-regulated replicon-based DNA expression system. AB - We present a temperature-regulated, alphavirus replicon-based DNA expression system. The system is regulated by a viral temperature-sensitive RNA-dependent RNA replicase, creating a temperature-dependent RNA amplification loop. Because of this positive feedback, the system exhibits both low background and high inducibility. We observed 700-fold induction in transiently transfected cells, and over 104-fold induction in stably transfected cells. The high stringency of inducibility allowed the generation of stable cell lines expressing a highly toxic protein upon temperature shift. These data suggest that the present expression system could simplify bioprocess engineering strategies, especially in situations where the cloned protein has detrimental effects on host cell metabolism. PMID- 10748526 TI - A genetic system for detection of protein nuclear import and export. AB - We have developed a simple genetic assay to detect active nuclear localization (NLS) and export signals (NES) on the basis of their function within yeast cells. The bacterial LexA protein was modified (mLexA) to abolish its intrinsic NLS and fused to the activation domain of the yeast Gal4p (Gal4AD) with or without the SV40 large T-antigen NLS. In the import assay, if a tested protein fused to mLexA Gal4AD contains a functional NLS, it will enter the cell nucleus and activate the reporter gene expression. In the export assay, if a tested protein fused to mLexA SV40 NLS-Gal4AD contains a functional NES, it will exit into the cytoplasm, decreasing the reporter gene expression. We tested this system with known NLS and NES and then used it to demonstrate a NES activity of the capsid protein of a plant geminivirus. This approach may help to identify, analyze, and select for proteins containing functional NLS and NES. PMID- 10748527 TI - Microarray fabrication with covalent attachment of DNA using bubble jet technology. AB - We have developed a method for fabricating DNA microarrays that uses a Bubble Jet ink jet device to eject 5'-terminal-thiolated oligonucleotides to a glass surface. The oligonucleotides are covalently attached to the glass surface by heterobifunctional crosslinkers that react with the amino group on the substrate and a thiol group on the oligonucleotide probe. Using this method, we fabricated DNA microarrays that carried 64 groups of 18-mer oligonucleotides encoding all possible three-base mutations in the mutational "hot spot" of the p53 tumor suppressor gene. These were screened with a fluorescently labeled synthetic 18 mer oligonucleotide derived from the p53 gene, or segments of the p53 gene that had been PCR amplified from genomic DNA of two cell lines of human oral squamous cell carcinoma (SCC). This allowed us to discriminate between matched hybrids and 1 bp-mismatched hybrids. PMID- 10748528 TI - Intrachromosomal recombination between attP regions as a tool to remove selectable marker genes from tobacco transgenes. AB - Recombinant genes conferring resistance to antibiotics or herbicides are widely used as selectable markers in plant transformation. Once transgenic material has been selected, the marker gene is dispensable. We report a novel strategy to remove undesirable parts of a transgene after integration into the tobacco genome. This approach is based on the transfer of a vector containing a NPTII gene flanked by two 352 bp attachment P (attP) regions of bacteriophage lambda, and the identification of somatic tissue with deletion events following intrachromosomal recombination between the attP regions. This system was used to delete a 5.9 kb region from a recombinant vector that had been inserted into two different genomic regions. As the attP system does not require the expression of helper proteins to induce deletion events, or a genetic segregation step to remove recombinase genes, it should provide a useful tool to remove undesirable transgene regions, especially in vegetatively propagated species. PMID- 10748529 TI - Antifungal activity of a virally encoded gene in transgenic wheat. AB - The cDNA encoding the antifungal protein KP4 from Ustilago maydis-infecting virus was inserted behind the ubiquitin promoter of maize and genetically transferred to wheat varieties particularly susceptible to stinking smut (Tilletia tritici) disease. The transgene was integrated and inherited over several generations. Of seven transgenic lines, three showed antifungal activity against U. maydis. The antifungal activity correlated with the presence of the KP4 transgene. KP4 transgenic, soil-grown wheat plants exhibit increased endogenous resistance against stinking smut. PMID- 10748530 TI - Enhanced phosphorus uptake in transgenic tobacco plants that overproduce citrate. AB - Phosphorus (P) is one of the most important nutrients limiting agricultural production worldwide. In acid and alkaline soils, which make up over 70% of the world's arable land, P forms insoluble compounds that are not available for plant use. To reduce P deficiencies and ensure plant productivity, nearly 30 million tons of P fertilizer are applied every year. Up to 80% of the applied P fertilizer is lost because it becomes immobile and unavailable for plant uptake. Therefore, the development of novel plant varieties more efficient in the use of P represents the best alternative to reduce the use of P fertilizers and achieve a more sustainable agriculture. We show here that the ability to use insoluble P compounds can be significantly enhanced by engineering plants to produce more organic acids. Our results show that when compared to the controls, citrate overproducing plants yield more leaf and fruit biomass when grown under P limiting conditions and require less P fertilizer to achieve optimal growth. PMID- 10748531 TI - Targeted screening for induced mutations. AB - With the accumulation of large-scale sequence data, emphasis in genomics has shifted from determining gene structure to testing gene function, and this relies on reverse genetic methodology. Here we explore the feasibility of screening for chemically induced mutations in target sequences in Arabidopsis thaliana. Our TILLING (Targeting Induced Local Lesions IN Genomes) method combines the efficiency of ethyl methanesulfonate (EMS)-induced mutagenesis with the ability of denaturing high-performance liquid chromatography (DHPLC) to detect base pair changes by heteroduplex analysis. Importantly, this method generates a wide range of mutant alleles, is fast and automatable, and is applicable to any organism that can be chemically mutagenized. PMID- 10748532 TI - High-fidelity mRNA amplification for gene profiling. AB - The completion of the Human Genome Project has made possible the comprehensive analysis of gene expression, and cDNA microarrays are now being employed for expression analysis in cancer cell lines or excised surgical specimens. However, broader application of cDNA microarrays is limited by the amount of RNA required: 50-200 microg of total RNA (T-RNA) and 2-5 microg poly(A) RNA. To broaden the use of cDNA microarrays, some methods aiming at intensifying fluorescence signal have resulted in modest improvement. Methods devoted to amplifying starting poly(A) RNA or cDNA show promise, in that detection can be increased by orders of magnitude. However, despite the common use of these amplification procedures, no systematic assessment of their limits and biases has been documented. We devised a procedure that optimizes amplification of low-abundance RNA samples by combining antisense RNA (aRNA) amplification with a template-switching effect (Clonetech, Palo Alto, CA). The fidelity of aRNA amplified from 1:10,000 to 1:100,000 of commonly used input RNA was comparable to expression profiles observed with conventional poly(A) RNA- or T-RNA-based arrays. PMID- 10748534 TI - Recent patents in combinatorial chemistry PMID- 10748533 TI - Analyzing the new written description guidelines. With the USPTO's revised guidelines, the patent landscape has changed yet again. PMID- 10748535 TI - People PMID- 10748536 TI - New products PMID- 10748537 TI - Progress against AIDS. PMID- 10748538 TI - Boosting plant phosphate uptake PMID- 10748539 TI - Transfection of hair progenitor cells PMID- 10748540 TI - [IEA Congress 1999: Epidemiology at the time of globalization]. PMID- 10748541 TI - [Influence of the asbestos industry and controversies in international organizations]. PMID- 10748542 TI - [The case of Coca Cola: a mass sociogenic illness?]. PMID- 10748543 TI - [Oncological screening in postmodern era]. PMID- 10748544 TI - [The hot potato of the Pusztai case]. PMID- 10748545 TI - [Europe bans asbestos. Finally!]. PMID- 10748546 TI - [Barolo bottle label: is it necessary to warn that alcohol does harm?]. PMID- 10748547 TI - [Environment and genetics in the etiology of cleft lip and cleft palate with reference to the role of folic acid]. AB - The combined birth prevalence of cleft palate [CP] and cleft lip with or without cleft palate [CL(P)] in Europe is approximately one in 700 with characteristic regional variations. Orofacial clefting (OC) is therefore now one of the most frequent congenital anomalies, with a higher birth prevalence that Down's Syndrome or Neural Tube defects, but still lower than cardiovascular malformation. Babies with OC require a multidisciplinary medical approach, surgery and rehabilitative treatments over time. This means an important effort in terms of social organization as well as economical costs for the health care system. In Italy, the health care costs for approximately 800 children born with orofacial clefting per year has been estimated at around 150 billion Lire (80 million Euros). The etiology of OC is complex and heterogeneous both for isolated and associated defects; causes linked to environment, genetics and gene environment interaction are known, although there is still a lot to do, especially in clarifying the role of genetics in producing susceptibility to the environment. Four categories of genes for which there are results suggestive of a genetic susceptibility to OCs are: 1) genes expressed in a particular area of the embryo or in a particular period of the palatine arch development, such as the transforming growth factors alpha and beta (TGF alpha, TGF beta 2, TGF beta 3); 2) genes having biological activities linked to the OC's pathogenesis without direct involvement (e.g. the retinoic acid receptor (RARA), the methylenetetrahydrofolate reductase receptor (MTHFR) and the folic acid receptor (FOLR1); 3) genes or locus identified in experimental animals as the homeotic genes MSX-1 and MSX-2; 4) genes involved in the interaction with the xenobiotics metabolism as those in P-450 cytochrome system. Several environmental factors have been implicated in the OC etiology; among those, the folic acid supplementation during the periconceptional period that was found effective in the prevention of neural tube defects. In fact, folic acid deficiency may be responsible for different malformations through a common mechanism that interferes with the embryonic development, depending on the maternal or embryo genotype. Further investigation is required to study in depth how the genotype would modify the role of environmental factors like folic acid. Well-designed and conducted epidemiological studies seem to be able to give worthwhile information. Studies carried out in Europe on these issues are a few, particularly those on gene-environment interaction. Recent results obtained in molecular biology and the availability of wealth of data can allow to perform ad hoc investigations, being important not only for the basic research but also for their public health implications. For this objective a specific scientific network at the European level has been set by the European Science Foundation (ESF), whose first step will be to establish consistent case ascertainment and data collection across Europe and to develop standardized protocols and methods of analysis. It is hoped that in the longer term such multicentre collaborative research will enable combined analysis and lead to the identification of genetic susceptibility to certain environmental factors, including nutrition. Such studies would inform the current debate about the efficacy of folic acid and other nutritional factors in prevention of disease in the developing embryo. Subsequent public health measures targeted according to risk might reduce the prevalence of disorders such as orofacial clefting. PMID- 10748548 TI - [Distribution of causes of death in communities with different urbanization levels]. AB - The aim of the present work was to compare mortality data during 1980-94 (24 causes of death for males and 25 for females) in populations living in municipalities with different urbanization levels of seven Italian regions (Piemonte, Lombardia, Emilia Romagna, Marche, Lazio, Campania and Sicilia). Urbanization levels were established following the classification of the Italian Institute of Statistics (ISTAT, Classificazione dei comuni secondo le caratteristiche urbane e rurali, 1986). A quite uniform configuration of causes of death with characteristic excesses in the urban environment was evident. The only exception was represented by the male population in Lombardia, probably due to the prevalence of occupational exposure. The urban excesses included: all tumors (also for the population < 64 year old), trachea, bronchi and lung tumors (also < 64 year old people), colorectal cancer, bladder cancer, male liver cancer and female breast cancer. Mortality due to stomach cancer and, only in males, to larynx cancer was lower in urban than in the other types areas, except the Southern regions. Concerning some non tumoral pathologies a more dyshomogeneous situation was observed, with a prevalence of urban defects in Piemonte and Lombardia, of urban excesses in the Southern regions and of both excesses and defects in Emilia Romagna and the Central regions. The "semi-rural" and "rural" types municipalities resulted quite similar, with the exception of Campania and Lazio. In fact, in these two regions a greater association with stomach cancer and lower mortality for all other tumors compared to the urban and, at a lesser extent, to the semi-urban municipalities was found. However, the lower tumor mortality was partly balanced by some other causes of death, again with the exception of Campania and at a lesser extent of Sicilia. On the basis of the causes of death comparisons, the semi-urban municipalities seem to represent a transition type between the urban and the rural ones. PMID- 10748549 TI - [Changing role of the family physician in British National Health Service]. AB - Over the last ten years the traditional role of primary care in Great Britain has been undergoing major changes with the major impact being the introduction of what is termed "GP fundholding", a process by which GPs hold budgets to purchase care from hospitals on behalf of their patients. This paper addresses how the system of fundholding operates in practice and points out the difference between the new and the previous system in which the health authority would plan care for the whole of a district and costs by hospitals would be covered by an all inclusive system. The paper studies the rationale of the introduction of the fundholding and examines the evidence on how successful the approach has been. With reference to a systematic literature review of the evidence on fundholding's outcomes undertaken by the author, the paper examines the evidence for and against fundholding under the following headings: efficiency, equity, quality, choice and responsiveness. The paper then addresses more recent changes to the British health care system and shows how these have changed the role of the family doctor irrevocablly. PMID- 10748550 TI - [Reference data on survival of cancer patients in 17 European countries]. PMID- 10748551 TI - Production and usefulness of monoclonal antibodies against B cells. AB - We have described new monoclonal antibodies (FUN-1, -2, FK61, FB1, FB21) which recognize B cells in the peripheral blood and lymphoid tissues. In preliminary reports FB1 and FUN-1 were described previously as anti-CD20 and -CD86 antibodies, respectively. FB1 and FB21 recognize an intracytoplasmic epitope (35, 38 kD) and a sialic acid-dependent carbohydrate epitope, respectively. FB1 reacts with pan B cells and FB21 with a subpopulation of B cells. In addition, FB21 shows relatively specific reaction with papillary or follicular carcinoma of the thyroid gland, but not with normal thyroid follicules and most benign thyroid gland tumors. Since FB1 and FB21 can be used with formalin-fixed paraffin embedded tissue sections, they are useful for diagnosis of B cell lymphoma or thyroid carcinoma. FUN-1 recognizes surface antigen (CD86) on activated B cells, monocytes in peripheral blood and germinal center B cells in lymphoid tissues. CD86 has an important role in T cell activation and the antigen-specific T-cell dependent immune response. PMID- 10748552 TI - Reverse transcriptase (RT)-independent as well as RT-dependent HIV-1 replication exists in syncytia following cell fusion. AB - We studied the role of reverse transcriptase (RT) in human immunodeficiency virus (HIV)-1 replication in syncytia following cell fusion. A chronically HIV-1 infected MOLT-4 (MOLT-4/IIIB) cells allow HIV-1 replication and induce syncytium formation between uninfected MOLT-4 cells. AZT (3'-azido-3'-deoxythymidine, 1 microM) inhibited neither HIV-1 replication in MOLT-4/IIIB cells nor the syncytium formation induced by concultivation of MOLT-4/IIIB cells with uninfected MOLT-4 cells. In the supernatant of the syncytium containing culture a remarkably higher titer of p24 antigen was produced than in that of MOLT/IIIB cell culture. AZT inhibited p24 antigen production by HIV-1 in the syncytia to levels to comparable to that in MOLT-4/IIIB cells which were treated with AZT. In addition, p24 production by HIV-1 in the syncytia formed by cocultivation of CL-2 cells, which are chronically infected with HIV-1 but lack functional RT, with uninfected MOLT-4 cells was not different from that in CL-2 cells alone. The results suggest that HIV-1 RT plays an important role in HIV-1 replication within the syncytia but an RT-independent replication process which is essential for syncytium formation also exists in the syncytia. These results indicate that not only RT inhibitors but also inhibitors of syncytium formation are essential for anti-HIV therapy. PMID- 10748553 TI - Effect of prostaglandin E2 on cytosolic calcium ion and morphology in cultured bovine retinal pigment epithelium. AB - Inflammatory conditions of the eye may cause migration and proliferation of retinal pigment epithelium (RPE) cells. Therefore we evaluated the effects of prostaglandin E2 (PGE2) on cytosolic calcium ion concentration ([Ca2+]i) and on morphological changes in cultured bovine RPE cells. PGE2 caused an increase in [Ca2+]i, and contraction of RPE cells noted on phase-contrast biomicroscopy. The increase of [Ca2+]i was restrained by removing calcium ion from the medium and the addition of verapamil. Contraction of RPE cells was evaluated further by measuring the diffusion of 3H-dextran. The PGE2-induced increase in diffusion of 3H-dextran was suppressed by verapamil. We conclude that the contraction of RPE cells induced by PGE2 is dependent upon calcium ion transient and may be countered by calcium-blocking agents. PMID- 10748554 TI - Non-traumatic aneurysms of the cavernous sinus causing massive epistaxis--report of three cases. AB - Three cases of non-traumatic intracavernous aneurysms that gave rise to massive epistaxis and required operative intervention, are reported. This is a rare but serious cause of epistaxis, because delayed diagnosis and management can lead to repeated bleedings and even to death. It is important, therefore, to diagnose the source of epistaxis and treat it as soon as possible. In our three patients, an emergency angiography enabled us to diagnose an aneurysm in the cavernous sinus. These aneurysms were located in the C3 or C4 portion of the internal carotid artery and were 3-5 mm in diameter. The projections of these aneurysms were infero-medial, antero-inferior, and lateral, respectively. All three patients underwent emergent surgery. Neck clipping of the aneurysm was performed in two patients, and gradual occlusion of the internal carotid artery in the third patient. Emergency surgery successfully prevented further episodes of epistaxis in all three patients. PMID- 10748555 TI - Peripheral T-cell lymphoma other than angioimmunoblastic T-cell lymphoma (AILD), with follicular dendritic cells proliferation and infection of B immunoblasts with Epstein Barr virus. AB - We describe a case of peripheral T-cell lymphoma, unspecified (REAL) with co existence of follicular dendritic cell (FDC) proliferation and EB virus infection. A 55-year-old Japanese man complained of generalized lymphadenopathy and physical examination showed systemic lymphadenopathy, and hepatosplenomegaly. HTLV-1 antibody titers and gamma-globulin level were within normal limits. Histopathologic examination of the right cervical lymph node showed peripheral T cell lymphoma, unspecified (REAL classification). A diffuse infiltration of lymphoma cells with abundant small venules were found throughout the lymph node. There were few arborizing venules. An irregular meshwork of FDCs was found by immunostaining with DRC-1 and CD21 in the area rich in venules, but not in the area of diffuse lymphoma cell-infiltration. EBER-1 RNA-in situ hybridization showed positive signal on the nuclei of mainly non-neoplastic B immunoblasts. The present case, therefore, was regarded as a rare case of peripheral T-cell lymphoma, unspecified with FDC proliferation and EB virus infection. PMID- 10748556 TI - Host immune profiles and genotyping analysis of HIV-1 among Japanese hemophiliac patients. AB - We studied the immune profiles of 21 Japanese hemophiliac patients who had been infected with human immunodeficiency virus type 1 (HIV-1) by the blood preparations during the 1982-84 period, and carried out the genotyping of HIV-1 V3 region for uncultivated peripheral blood mononuclear cells (PBMC) from 8 patients. Ten years after infection, asymptomatic carrier (AC, 14 cases) yet outnumbered those in AIDS related complex (ARC, 4 cases) and AIDS (3 cases), and the CD4+ and CD8+ T-cell numbers were not correlated with clinical stages. Macrophage tropic sequences, as identified according to the known tropism determinants, appeared to be more frequent as the CD4+ T-cell numbers were higher. There was no correlation of the cell tropism with the disease stages, however. It might be due to HIV-1 of CD4+ T-cells being more productive. The mutation in the V3 region appears to differentially influence the escape from antibody attack and clinical stages, and the cell tropism may not be related to the cell immunity of the host. PMID- 10748557 TI - Stage-specific effects of antimalarials on an Indian isolate of Plasmodium falciparum. AB - In this study, effects of timed sequential exposure to various concentrations of chloroquine and its two metabolites (SA100387/050 B, SA 100487/053 B), quinine, hydroquinine and quinidine were monitored by morphological analyses, using synchronous cultures of Plasmodium falciparum. The trophozoites and schizonts were sensitive to chloroquine and its metabolites. However, quinine and two of its derivatives were lethal without particularly being stage-specific. PMID- 10748558 TI - Variations in the response to Bacillus sphaericus toxins in different strains of Anopheles stephensi Liston. AB - Bacillus sphaericus has a potential of use as a larvicide in water storage practices but no such study has been done against Anopheles stephensi. Baseline susceptibility status of eleven wild and three mutant strains of An. stephensi to Spherix, a formulation of B. sphaericus, strain B-101 (serotype H5a, 5b) was determined to find the variations in the degree of their response to B. sphaericus toxins and to study the possibility of development of resistance. The LC50 and LC90 values of different strains of An. stephensi to B. sphaericus formulation varied from 0.088 to 1.42 mg/l and 0.314 to 10.98 mg/l, respectively. Among all the strains tested, Sarojini Nagar, Delhi strain of An. stephensi (a wild type strain) was least susceptible. Laboratory selection of Sarojini Nagar strain of An. stephensi with B. sphaericus at a concentration of 4 mg/l resulted in the development of a high degree of resistance (LC50 > 1600 mg/l) to B. sphaericus within four generations. PMID- 10748559 TI - Impact of Spherix (Bacillus sphaericus B-101, serotype H5a, 5b) spraying on the control of mosquito breeding in rural areas of Farrukhabad District, Uttar Pradesh. AB - Spray impact of Spherix (Bacillus sphaericus B-101, Serotype H5a, 5b) was assessed against larval and adult populations of mosquito species in rural areas of Farrukhabad district, Uttar Pradesh. High mortality (> 95%) was observed against anopheline and culicine larvae within 48 h of spray @ 1 g/sq m. But the biolarvicidal effect declined within a week which indicated nonrecycling capacity and weekly reapplications were required. The encouraging results obtained through weekly sprays were short-lived because of decline in efficacy in some perennial intradomestic Culex breedings after 15 rounds of spray. The two year intensive field trials, however, had no appreciable impact on adult densities of both malaria vector An. culicifacies and bancroftian filariasis vector Culex quinquefasciatus. Environmental disturbances and manmade problems also affected the efficacy of the biolarvicide. PMID- 10748560 TI - Mosquito fauna and breeding habitats of anophelines in Car Nicobar Island, India. AB - A total of 31 species of mosquitoes belonging to 10 genera, i.e. Anopheles, Aedes, Armigeres, Culex, Harpagomyia, Hodgesia, Mansonia, Orthopodomyia, Toxorhynchites and Uranotaenia were collected from Car Nicobar Island. Four Anopheles species, An. barbumbrosus, An. insulaeflorum, An. kochi and An. roperi were recorded for the first time from this Island. An. sundaicus was the most predominant species encountered. The results of the study on anophelines with emphasis on species-specific breeding preference in various aquatic habitats have been recorded. PMID- 10748561 TI - Microbial control of mosquitoes with special emphasis on bacterial control. AB - There are a number of microbial agents including fungus, protozoa, virus and bacteria which act as mosquitocidal agents. However, among these agents, Bacillus thuringiensis var israelensis and B. sphaericus are the most potent mosquitocidal agents. Bacillus thuringiensis var israelensis and B. sphaericus are gram positive sporulating bacteria which produce protoxin crystals during sporulation and are highly toxic to susceptible mosquito larvae when they ingest them. These bacterial agents are environmentally safe due to their host specificity, require in very low dosage, easy to prepare commercially in large-scale and are less costly. Field trials with various formulations of B. sphaericus and B. thuringiensis var israelensis have demonstrated their safety and potential for controlling mosquitoes. Moreover, cloning and expression of the toxin genes of these organisms in other environment friendly bacteria have also made them important for further investigation. Recent advances with novel types of recombinant micro-organisms with new cloning strategies and cloning the toxin genes under strong promoter for over expression together with in vitro gene manipulation and site directed mutagenesis of the active sites for increased toxicity have the potential to provide more effective control of mosquitoes by exploiting these two bacteria. But the toxins of B. sphaericus and B. thuringiensis var israelensis in particular, do not persist long in nature and require frequent application which is a limiting factor for these organisms to be most successful and potent biolarvicide. Nevertheless, they are by far the best choice for controlling mosquitoes. In this review article different mosquitocidal agents with particular emphasis on B. thuringiensis var israelensis and B. sphaericus have been described. PMID- 10748562 TI - Prevalence of malaria and ABO blood groups in a seaport area in Raigad, Maharashtra. PMID- 10748563 TI - Speaking up; speaking out. PMID- 10748564 TI - Professional practice. The practice of leadership. PMID- 10748565 TI - Public policy. Insuring America's children--a call for nurse participation. PMID- 10748566 TI - Research. If you call me names, I'll call you numbers. PMID- 10748567 TI - The nurse executive. Healthy work environments for all nurses. PMID- 10748568 TI - Relationships among student and graduate caring ability and professional school climate. AB - This study represents the second phase in a longitudinal study of the development of caring ability in registered nurses. A nationwide sample of female graduates of US baccalaureate nursing programs was resurveyed 3 years after the phase-one survey taken during nursing school. The purpose of the study was to continue an evaluation of several variables contributing to the development of professional caring ability. Previously validated instruments were used to measure caring ability (Caring Ability Inventory), maternal and paternal care (Parental Bonding Instrument), and the caring climate of the respective nursing schools (Charles F. Kettering Ltd. School Climate Profile). Although parental bonding scores remained unchanged from the initial survey, caring ability scores significantly increased after entry into practice (paired student's t test, P < .001). The strongest predictor of postgraduate caring ability was student caring ability scores (r = .58, P < .001). When student caring ability scores were excluded as a potential predictor during stepwise multiple regression analysis, school climate scores were the strongest predictor of postgraduate caring ability (r = .17, P < .05). This suggests that development of professional caring ability is related to caring school environments. Furthermore, caring ability as a student predicts further development of caring ability after entry into practice. PMID- 10748569 TI - The relationship among medical diagnosis, nursing diagnosis, and nursing intervention and the implications for home health care. AB - The purpose of this study was to identify the most commonly used medical diagnoses, nursing diagnoses, and nursing interventions for home health care based on 244 patient records. Nursing interventions were categorized into three groups: assessment, instruction, and other. The results showed that for the three most commonly used medical diagnosis groups--"infectious and parasitic disease," "disease of the circulatory system," and "neoplasms"--the most related nursing diagnoses were "alteration in mobility," "alteration in cardiac status," and "alteration in comfort: pain." The most used nursing interventions were "instructions." The results indicated that nurses identified patients' physiological problems mostly in relation to medical diagnoses, and teaching was the most frequently used nursing intervention in home health care. PMID- 10748570 TI - The relationship between health care reform and nurses' interest in union representation: the role of workplace climate. AB - Market-based reforms are rapidly changing the landscape of the American health care system. Over the last decade, a great deal of attention has been paid to these reforms, particularly the advent and rapid growth of managed care and the vertical and horizontal integration of provider institutions. The ramifications of these changes on both individual and institutional consumers has also been widely considered by scholars and policymakers. Yet, although these market reforms are driving the massive restructuring of work in hospitals and other health care organizations, comparatively little attention has been paid to the impact these changes have on health care employees and the environment in which they work. This article reports the results of an exploratory study examining the relationship between health care market reforms, nurses' perceptions of the climate for patient care in hospital settings, and nurses' interest in union representation. A model is proposed to help explain nurses' support for union representation in the presence of market-based reforms. PMID- 10748571 TI - Nursing faculty: one generation away from extinction? AB - Concern over an impending shortage of qualified nursing faculty sparked a statewide survey of nurses with graduate degrees in one midwestern state. A return rate of 61 per cent yielded a sample of 288. In general, nursing faculty members were older than nurses with graduate degrees employed in other roles. Three fourths of the sample worked full-time in stable positions in which they intended to remain until retirement. Fewer than one third were nurse educators; one-third were hospital employees. Nearly half the sample had nonnursing graduate degrees despite local availability of two nursing master's programs. Noncompetitive academic salaries, desire for clinical practice, and rising expectations in higher education were factors deterring nurses with graduate degrees from pursuing faculty roles. Improved compensation, greater respect, closer proximity to work, more realistic expectations, and improved opportunity to continue clinical practice were viewed as incentives to entice practicing nurses into faculty roles. Recommendations for addressing these important challenges are summarized. The next generation of nurses depends on strategic planning now to build the pool of qualified faculty for the future. PMID- 10748573 TI - Becoming an academic administrator: is it for you? AB - Faculty members who think they want to advance into educational administration should consider whether they have the needed qualities, whether they understand the role and its advantages and disadvantages, and whether they are educationally prepared to do so. Making the transition into first-line administration (chairperson, director, assistant dean, or dean) can be stressful, but proper preparation can enhance adaptation. Formal coursework in educational administration is recommended, as is the development of a relationship with a mentor. Well-prepared administrators in academic nursing are always in demand. PMID- 10748572 TI - Development of new faculty in higher education. AB - Faculty development programs in nursing, as well as in other disciplines, are needed to respond to changing expectations about the quality of undergraduate education, student populations, decreasing resources, and widespread use of technology in education. Problems facing new and senior faculty center on these changing demands. Preparing faculty to use technology in teaching is especially important. The problem of diminishing resources for faculty development must be balanced with the overwhelming need for faculty education in this changing environment. PMID- 10748574 TI - Distance technology in nursing education: assessing a new frontier. American Association of Colleges of Nursing. PMID- 10748575 TI - The invisible majority: nurses in the media. PMID- 10748576 TI - Differences in the sensitivity to UVB radiation of two cultivars of rice (Oryza sativa L.) based on observation of long-lived radicals. AB - Free radicals with a long lifetime were observed in the leaves of two rice cultivars (Oryza sativa L.), Sasanishiki (UVB resistant) and Norin-1 (UVB sensitive), by electron spin resonance spectroscopy. The leaves of both cultivars grown with visible light show very similar ESR spectra composed of radical 1 (R1) and radical 2 (R2), which may be attributable to P700 cation radicals in the reaction center of photosystem I, and tyrosine cation radicals in the reaction center of photosystem II, respectively. The ESR spectrum composed of R1 and R2 radicals in the leaves of Sasanishiki grown under visible light with supplemental UVB was similar to that in the plant grown without supplemental UVB. On the other hand, the amount of R2 radicals in the leaves of Norin-1 grown under visible light with supplemental UVB was significantly smaller than that in the plant grown without supplemental UVB. It is suggested that the loss of R2 radicals in Norin-1 upon UVB irradiation is related to the instability of the plant. PMID- 10748577 TI - Delayed cell death, giant cell formation and chromosome instability induced by X irradiation in human embryo cells. AB - We studied X-ray-induced delayed cell death, delayed giant cell formation and delayed chromosome aberrations in normal human embryo cells to explore the relationship between initial radiation damage and delayed effect appeared at 14 to 55 population doubling numbers (PDNs) after X-irradiation. The delayed effect was induced in the progeny of X-ray survivors in a dose-dependent manner and recovered with increasing PDNs after X-irradiation. Delayed plating for 24 h post irradiation reduced both acute and delayed lethal damage, suggesting that potentially lethal damage repair (PLDR) can be effective for relieving the delayed cell death. The chromosome analysis revealed that most of the dicentrics (more than 90%) observed in the progeny of X-ray survivors were not accompanied with fragments, in contrast with those observed in the first mitosis after X irradiation. The present results indicate that the potentiality of genetic instability is determined during the repair process of initial radiation damage and suggest that the mechanism for formation of delayed chromosome aberrations by radiation might be different from that of direct radiation-induced chromosome aberrations. PMID- 10748579 TI - External doses of residents near Semipalatinsk nuclear test site. AB - Accumulated external radiation doses of residents near the Semipalatinsk nuclear test site of the former USSR are presented as a results of study by the thermoluminescence technique for bricks sampled at several settlements in 1995 and 1996. The external doses that we evaluated from exposed bricks were up to about 100 cGy for resident. The external doses at several points in the center of Semipalatinsk City ranged from a background level to 60 cGy, which was remarkably high compared with the previously reported values based on military data. PMID- 10748578 TI - Effect of ionizing radiation on the differentiation of ROS 17/2.8 osteoblasts through free radicals. AB - Although the acceleration of bone regeneration by radiation has been reported, the mechanisms of action of radiation on bone are unclear. The present results indicate that ionizing radiation-stimulated differentiation could result from the generation of reactive oxygen species during radiation exposure. The free radical release is considered as the most important mechanism of bone effect by radiation treatment. In addition, we report that radiation induced transient activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activation and the transcription factor, AP-1. The JNK and AP-1 activation is mediated with radiation-released free radicals in ROS 17/2.8 osteoblasts. These results indicate that ionizing radiation at a single dose of up to 5 Gray stimulates differentiation of ROS 17/2.8 osteoblasts via free radial release which may affect JNK/SAPK and AP-1 activities. PMID- 10748580 TI - Creating a culture of safety: a shared responsibility. PMID- 10748581 TI - Is the goal of a BSN as the criteria for entry into professional nursing practice still worthwhile and realistic? PMID- 10748582 TI - Nutritive sucking and neurobehavioral development in preterm infants from 34 weeks PCA to term. AB - PURPOSE: To investigate changes in nutritive sucking patterns, behavioral state, and neurobehavioral development of preterm infants from the 34 weeks postconceptional age (PCA) to term. DESIGN: Nonexperimental descriptive correlational design was used. METHODS: A convenience sample included 66 preterm infants with a gestational age between 24 and 34 weeks at birth. Mean gestational age was 29.7 weeks, with a mean birthweight of 1379 grams. Instruments included the Kron Nutritive Sucking Apparatus, the Anderson Behavioral State Scale (ABSS), and the Brazelton Neonatal Behavioral Assessment Scale (BNBAS). A feeding procedure was administered at 34 and 40 weeks PCA with BNBAS administered at 40 weeks PCA. Data analysis strategies included t-tests, Spearman rho correlations, Wilcoxon matched-pairs signed-ranks test, and a Random Coefficients Model. RESULTS: There were significant differences in number of sucks (p < 0.001), intensity of sucking pressures (p < 0.001), average time between sucks (p < 0.001), and average time between bursts (p < 0.001) from 34 weeks PCA to term. With maturation, it was noted that the preterm infants were significantly more alert during the sucking protocol from 34 weeks to term (p < 0.001). Yet, infant sucking parameters at 34 weeks PCA and term were not significantly related to the BNBAS or to the infant's state. CLINICAL IMPLICATIONS: These results demonstrate that high risk preterm infants mature significantly in their sucking behaviors and in their ability to maintain a more alert behavioral state from 34 weeks PCA to term. However, unlike full term infants--where state is positively correlated to feeding (sucking) success--the preterm infant state of alertness may not enhance sucking skills at 34 weeks PCA. These infants may be too stressed to manage both feeding and stimulation from the environment that is unavoidable when alert. However, this relationship does appear to change by 40 weeks PCA. PMID- 10748583 TI - Mothers, infants, and DHA. Implications for nursing practice. AB - The purpose of this article is to describe the professional literature and current controversies concerning the relationship between essential fatty acids, especially Docohexaenoic Acid (DHA), and neurologic function. Although there is debate in the literature concerning just how much DHA is required for optimal neurologic functioning of infants, it is known that adequate DHA levels are dependent on an adequate dietary intake. However, common dietary practices today may not provide enough DHA. Because pregnancy and lactation are key times of rapid brain growth for the developing fetus and infant, nurses can be instrumental in teaching pregnant and lactating women diet-related information and promoting practices that help increase DHA levels. By understanding the importance of DHA in pregnancy and infancy, the nurse can take a more active role in essential health education. PMID- 10748584 TI - A comparison of four methods of normal newborn temperature measurement. AB - PURPOSE: The purpose of this study was to: (a) compare newborn temperature measurements obtained by digital disposable, electronic, and tympanic thermometers with glass mercury thermometers, and (b) compare financial implications of each method. METHODS: In this correlational study, 12 perinatal and neonatal nurses obtained temperature measurements of 184 newborns between 1 and 168 hours of age. The stratified convenience sample was selected using medical records numbers. Temperature instruments included glass thermometer, tympanic thermometer, electronic thermometer, and a digital thermometer. Data were analyzed by Pearson r coefficients, mean, standard deviation, and range using an SPSS statistical package. RESULTS: The glass thermometer, electronic thermometer, and digital thermometer temperature assessments were highly correlated (0.748-1.0). The tympanic thermometer had a low correlation coefficient (0.35). Use of the glass thermometer had the highest accompanying cost. Tympanic thermometers were the most cost effective. CLINICAL IMPLICATIONS: In healthy newborns, the use of electronic and digital thermometers can be encouraged if there is concern about using glass thermometers. These results cannot be extrapolated to sick infants. While tympanic thermometers had the lowest associated cost, their lack of correlation with the gold standard glass thermometers for accurate temperature assessment makes them a poor choice for healthy newborns. PMID- 10748585 TI - The nurse's role during oxytocin administration. AB - This article addresses the importance of the nursing role in the management of oxytocin during induction/augmentation of labor. It is nurses at the bedside of laboring women who make oxytocin titration decisions based on their nursing assessments. Those decisions must be based on a sound knowledge of the pharmacologic properties of oxytocin, the physiology of uterine contractions, and the response of the woman and fetus to contractions. In addition, nurses must be aware of the standards and guidelines of care that govern their actions during induction/augmentation. PMID- 10748586 TI - Mothers and others. African-American women's preparation for motherhood. AB - PURPOSE: To describe motherhood, maternal role requirements, and family life as perceived by poor, urban, African-American women. DESIGN: Ethnographic qualitative research. METHOD: Using ethnographic methodology, 15 new mothers between the ages of 14 and 44, along with their families and significant others, were observed and interviewed in their homes and communities, using semi structured and open-ended questions. The initial interview occurred within 24 hours of birth of a normal healthy infant. The interviews were analyzed for emergence of themes. Descriptions of select actions, interactions, and activities exemplified themes. RESULTS: Motherhood was found to be a significant demarcation in the women's lives, and they described both positive and negative maternal experiences. The mothers followed organized, culturally grounded steps in their pursuit of the maternal role; these steps were learned in the family of origin. The four steps discovered were preparing, checking, becoming, and evaluating. Role failure involved undesirable consequences for the mother. The results of this study reveal that these women are mothers by design, not by default. CLINICAL IMPLICATIONS: These mothers expended a great deal of energy to maintain their families; they valued successful maternal role fulfillment. If health care providers could become better sensitized to mothers' cultural values and social stresses, it could be possible to better help identify mothering needs. Health care providers could use culturally sensitive information to help support positive healthy mothering techniques for young mothers from diverse cultural groups. PMID- 10748588 TI - A case study of infant massage outcomes. AB - This article describes a case study of infant massage for a neonate in a neonatal intensive care unit (NICU). Infant massage is grounded in the theory of touch. In an NICU, the infant's tactile experiences can be uncomfortable or painful. This can lead to touch aversion. It is possible that providing pleasurable touch experiences through infant massage can help to develop touch acceptance. It is important to first assess the infant's readiness for massage according to his or her need and response. Infant massage as a complementary therapy in the NICU is demonstrated here in a photo-study of "Nicholas." The initial uncertainty and gradual acceptance by Nicholas of the massage experience is clearly depicted. At the end of the massage, Nicholas was relaxed and asleep in a semi-fetal position. The behavioral change in this infant indicates that he made a transition from touch aversion to touch acceptance. PMID- 10748587 TI - Kangaroo skin-to-skin care for premature twins and their adolescent parents. AB - Risk factors associated with prematurity and adolescent parenting greatly increase when combined with multiple birth. Kangaroo care (KC) for preterm infants is well documented, although KC with twins or with adolescent parents is mentioned only briefly. In this case study, adolescent parents experience KC with their 32-week twin sons beginning 19 hours postbirth. These young parents interacted with, responded to, and took responsibility for their newborn sons, behaviors that are sometimes difficult for adolescent parents. Thus, KC appeared to be an effective intervention for these adolescent parents. Additionally, three kinds of KC with twins were identified: separate, sequential, and shared. PMID- 10748590 TI - Informed consent for adult patients--a review of basic principles. PMID- 10748589 TI - Neonatal herpes simplex virus type II. AB - This description of a tragic case of infection with neonatal herpes simplex virus type II is presented to alert professional nurses to the fact that this disease is sometimes difficult to recognize, but must be considered when a newborn has any unusual symptoms. Neonatal herpes simplex type II may have a slow and insidious onset, and be masked by many other symptoms. Even though the virus might be present in a newborn, the symptoms might not be apparent until several days after birth; this is a problem because infants are now leaving hospitals much earlier and can become ill after hospital discharge. Parents, especially first-time parents, may not recognize subtle signs of illness in their child. Therefore, the onus is on the health care provider to evaluate each infant for symptomatology and provide education to parents along with instructions to obtain early follow-up care for all newborns. PMID- 10748591 TI - Educating women about hepatitis. PMID- 10748592 TI - Cyber nurseries. PMID- 10748593 TI - Now is the time. PMID- 10748594 TI - Now is the time. PMID- 10748595 TI - [BAK (Federal Physician Committee) discusses gene check for embryos. Human cultivation in Germany?]. PMID- 10748596 TI - [Online shopping for physicians. Using virtual shopping colleague Zahn saves time and money]. PMID- 10748597 TI - [When caregivers kill patients. Compassion or criminal motive?. Interview by Dr. Beate Schumacher]. PMID- 10748598 TI - [Urticaria frequently disappears spontaneously after a brief period. Expensive diagnosis only in chronic course]. PMID- 10748599 TI - [Long prodromal phase in schizophrenia. By recognizing it, the prognosis of the patient can be significantly improved]. AB - Both Kraepelin and E. Bleuler reported that nonspecific symptoms are frequently present before the first psychotic symptoms manifest. In the ABC schizophrenia study, we were able to show that, in three-quarters of the cases, initial psychotic symptoms are preceded by a prodromal phase of several years standing that begins with non-specific signs such as negative and depressive symptoms. These early symptoms lead to social disabilities that--already before the onset of acute psychotic symptoms--have an adverse effect on the life situation of those who subsequently go on to develop schizophrenia. With the aim of preventing these early social consequences, patients at risk of developing schizophrenia should be identified in good time, and referred to early detection/intervention centers for a diagnostic work-up. In addition to the initial, non-specific symptoms that mark the beginning of the prodromal phase, basic disturbances also need to be considered, since they have considerable prognostic importance for the transition to psychosis. Other factors of importance are retarded development and behavioral anomalies in childhood and familial load, since premorbid risk factors are indicative of a congenital psychotic risk. PMID- 10748600 TI - [Early intervention in increased risk for schizophrenic illnesses. Multimodal psychotherapy--antipsychotic drugs only in treatment failure]. AB - Interventional strategies for the prodromal phase of schizophrenia are based largely on treatment concepts that have already been shown to be effective for the prevention of relapses. Such a multimodal concept for early psychotherapeutic intervention has been developed at the psychiatric department of the University of Cologne. This model emphasizes individual symptom-oriented cognitive/behavioral treatment and psychoeducation directed both to the patient and his/her contact person(s). In addition, stress and symptom management, training of social skills and computer-aided cognitive training tailored to individual needs are also offered. In contrast, pharmacological treatment is considered only when, despite the above-mentioned strategy, the clinical picture worsens appreciably and attenuated psychotic symptoms have appeared. PMID- 10748601 TI - [At risk for schizophrenic psychosis? Case report of a patient with suspected prodromal symptoms]. PMID- 10748602 TI - [Oral antidiabetic agents--2. Individualized stepwise therapy]. PMID- 10748603 TI - [A rare cause of carpal tunnel syndrome: leprosy. Latency period of decades is possible]. AB - In a 63-year-old German woman with no apparent exposure to leprosy, clinical and histological evidence of tuberculoid leprosy was found. A noteworthy feature of this case is either the very long incubation time of 45 years, or the mode of infection involving an only short encounter in an endemic region. In the event of unclear lesions--on white skin not, typically, hypopigmented, but reddish- accompanied by disordered sensation, we in Europe must also give consideration to the possibility of a sporadic case of leprosy. A particular characteristic of this case was leprous neuritis involving the median nerve which was diagnosed clinically as carpal tunnel syndrome, and scheduled for surgical treatment. Under treatment with dapsone and rifampicin, however, the condition cleared up completely. PMID- 10748604 TI - [Differential gout diagnosis. Spectrum of crystalloid arthropathies, 1]. PMID- 10748605 TI - [Diagnostic quiz. A small girl with thick fingers. Enchondromatosis (Ollier disease)]. PMID- 10748606 TI - [In Process Citation] PMID- 10748607 TI - Use of nutritional scores to predict clinical outcomes in chronic diseases. AB - Many hospitalized patients are malnourished, and the relationship between malnutrition and patient outcome is well established. To determine which patients are at nutritional risks, clinical scores are probably more accurate than using a single nutritional parameter. Among the numerous scores published, both the Prognostic Nutritional Index and the Subjective Global Assessment were prospectively validated. One is based on objective measurements, whereas the second is based on medical history and physical examination. The Nutritional Risk Index has been used in many studies including the "Veterans Study." The Mini Nutritional Assessment is a promising score for evaluating malnutrition in the elderly. The development of nutritional scores for use by nurses may facilitate screening of a large number of hospitalized patients. PMID- 10748608 TI - Beta carotene: from biochemistry to clinical trials. AB - Three large-scale clinical trials tested the effects of supplemental beta carotene on the risk for chronic diseases such as cancer. The populations involved were Finnish male heavy smokers (the Alpha Tocopherol Beta Carotene [ATBC] trial), male asbestos workers and male and female heavy smokers (Beta Carotene and Retinol Efficacy Trial [CARET]), and U.S. male physicians, 11% of whom were current smokers (Physician's Health Study). All three trials concluded that beta-carotene provided no protection against lung cancer; however, quite unexpectedly, two of the trials found a higher risk for lung cancer for those subjects given beta-carotene compared with those that were not. Several authors concluded from these beta-carotene trials that the protective effects of antioxidants against chronic disease are not as great as had been hoped. As reviewed here, however, beta-carotene may or may not be an antioxidant; it certainly differs in many respects from the prototypical antioxidant, vitamin E. In any case, the majority of beta-carotene's effects in vivo are probably not derived from any antioxidant properties that it may possess, but rather from its effect on a number of biochemical systems. Whether taking supplemental antioxidants can reduce the risk for chronic diseases remains to be established, although the case for vitamin E and heart disease appears strong. However, the association between eating a diet sufficient in fruits and vegetables and reduced risk for a number of diseases is consistent. There is no evidence at present that consuming small amounts of supplemental beta-carotene, i.e., amounts in foods or in a multivitamin tablet, is unwise for any population. The role of supplementation, however, particularly at high levels, with compounds that may be anti-oxidants but that are less well understood than vitamin E (e.g., carotenoids, plant polyphenols, and other phytochemicals), is less clear. The surprising results of the ATBC and CARET trials are a red flag, signaling the need for further research; a number of areas for future work are suggested here. Future research should lead to a clearer understanding of the effects of beta carotene and other phytochemicals, as well as to more refined strategies for intervention, with important clinical and public health implications. PMID- 10748609 TI - Association between body mass index and adenocarcinoma of the esophagus and gastric cardia. AB - Obesity is an increasing problem for industrialized nations. The incidence of adenocarcinoma of the esophagus and gastric cardia has also risen during the past two decades. A recently published population-based study attempted to relate this rise to increases in obesity or body mass index. Obesity may not only increase the risk for adenocarcinoma of the upper gastrointestinal tract, but it may also increase the risk of colon cancer and overall mortality. More research is needed to understand obesity's health impact and ways to control this epidemic. PMID- 10748610 TI - Omega-3 fatty acids alter soluble markers of endothelial function in coronary heart disease patients. AB - Several studies have indicated that omega-3 poly-unsaturated fatty acids in fish oil have protective effects on cardiovascular disease by reducing vascular endothelial inflammation in atherosclerosis. In a recent study, supplementation of patients with fish oil following coronary angioplasty elevated levels of lipid peroxides and two adhesion molecules in plasma suggesting a proinflammatory action of fish oil. However, decreases in several plasma markers of endothelial hemostatic activity indicated a healthier and better vasculature associated with fish oil supplementation. PMID- 10748611 TI - Nutrition and infection: malnutrition and mortality in public health. PMID- 10748612 TI - Viruses: evolution, propagation, and defense. PMID- 10748613 TI - Coxsackievirus as a model of viral evolution driven by dietary oxidative stress. PMID- 10748614 TI - Immune and other responses to viral infections. PMID- 10748616 TI - Mother-to-child transmission of HIV infection: nutrition/HIV interactions. PMID- 10748615 TI - Nutrition resistance to viral propagation. PMID- 10748617 TI - Vitamin A and measles. PMID- 10748618 TI - The potential impact of nutritional change on the global burden of viral disease. PMID- 10748619 TI - Stability of L-asparaginase: an enzyme used in leukemia treatment. AB - L-asparaginase from Escherichia coli is an important enzyme widely used in leukemia treatment under the trade name Elspar. Up to now, however, the aspects of its stability and storage has not been studied in detail. The aim of this work is to analyze the factors that could interfere in the enzyme's stability. The enzymatic activity was found to be stable in wide pH range (4.5-11.5), showing a slight increase in activity and stability in alkaline pHs, which indicates a more stable conformation of the molecule. The enzyme proved to have a high activity restoration capacity when submitted to temperatures of 65 degrees C, in pH 8.6 buffer and, surprisingly, in physiologic solution. This suggests a positive effect of sodium ions on such restoration capacity. Stability was high in different diluents used as parenteral solutions and in recipients used in medical practice without significant loss of activity for at least 7 days. These results lead us to conclude that the enzyme has a high stability after the lyophilized form has been reconstituted (at least 7 days), since the necessary precautions are taken in terms of sterile manipulation and if it is stored in a suitable parenteral vehicle under low temperature (about 8 degrees C). PMID- 10748620 TI - Synthesis, antibacterial, antifungal and anti-HIV evaluation of Schiff and Mannich bases of isatin derivatives with 3-amino-2-methylmercapto quinazolin 4(3H)-one. AB - Isatin (Indole 2,3-dione), its 5-chloro and 5-bromo derivatives were added to 3 amino-2-methylmercapto quinazolin-4(3H)-one to form Schiff bases and the N Mannich bases of these compounds were synthesized by reacting with formaldehyde and several secondary amines. Their chemical structures have been confirmed by means of their IR, 1H-NMR data and by elemental analysis. Investigation of antimicrobial activity of compounds was done by an agar dilution method against 26 pathogenic bacteria, 8 pathogenic fungi and anti-HIV activity against replication of HIV-1 (III B) in MT-4 cells. Among the compounds tested 5-chloro-3 (3',4'-dihydro-2'-methylmercapto-4'-oxoquinazolin-3'-yl )- l-morpholino methyl imino isatin was the most active antimicrobial agent. PMID- 10748621 TI - Studies on antiplatelet agents from natural safrole. II. Synthesis and pharmacological properties of novel functionalized oxime O-benzylether derivatives. AB - In an ongoing research program aiming at the synthesis and pharmacological evaluation of new possible prototype candidates exploring the molecular hybridation and bioisosterism principles for molecular designing, we describe in this paper the design and synthesis of a series of new functionalized oxime O benzylethers (4a-b) and (14a-b) as antiplatelet agents based on the inhibition of arachidonic acid (AA) cascade enzymes. For the synthesis of these new bioactive derivatives we used safrole (5), a Brazilian abundant natural product, as starting material. The platelet anti-aggregating evaluation of these oxime O benzylether compounds (4a-b) and (14a-b) in model induced by ADP, collagen and AA, has permitted to evidence an antithrombotic profile to these new derivatives, being the most active the derivative methyl [[3,4 methylenedioxyphenyl]methylene]amino]oxy]-4-methylenephenylacet ic acid (14a). PMID- 10748622 TI - Taste masking as a consequence of the organisation of powder mixes. AB - Usually, chemical or technological operations are used to mask the taste of unpleasant-tasting drugs. To reduce the development cost of such drugs, we propose a new approach which does not require the modification of the existing formulation nor the use of additional costly technological operations. Different particle size fractions of two unpleasant-tasting drugs (niflumic acid and ibuprofen) were blended in binary mixes with different particle size fractions of two non-tasting excipients (ethyl cellulose and hydroxypropyl methylcellulose). By selecting the appropriate mixes of identical composition but different organisations, as predicted from surface energy data, it was possible to use the different organisations to modify the taste of the mixes for a panel of 10 healthy volunteers. PMID- 10748623 TI - Studies on controlled release dimenhydrinate from matrix tablet formulations. AB - In this study, controlled release dosage forms of dimenhydrinate were prepared with different polymers as MC, HEC, Carbopol 934, Eudragit RLPM and Eudragit NE 30 D at different concentrations (2.5-10%). Direct compression (DC) and wet granulation (WG) techniques were used to prepare the tablets. Magnesium stearate was the lubricant while starch gel was the binder. For the quality control of tablets prepared according to 11 different formulations, weight deviation, hardness, friability, diameter-height ratio, content uniformity of the active substance and in vitro dissolution techniques were performed. Dissolution rate of these tablets was controlled by USP XXII dissolution method and the profile of each tablet was plotted and only for F 5 was evaluated kinetically. PMID- 10748624 TI - Poly(phthaloyl-L-lysine)-coated multilamellar vesicles for controlled drug delivery: in vitro and in vivo performance evaluation. AB - Nonionic surfactant vesicles were prepared using Span 60, cholesterol and dicetyl phosphate. The prepared multilamellar vesicles (MLVs) were coated by interfacial polymerization technique using p-phthaloyl dichloride and L-lysine. The formation of the polymeric coat was confirmed by optical microscopic and transmission electron microscopic studies. The prepared, plain and polymer-coated MLVs were studied for their size, shape, encapsulation efficiency, in vitro release profile and effect of osmotic shock on vesicle. The results observed showed that the polymer-coated MLVs were stable under various osmotic conditions. In vivo studies were carried out on albino rats. The half-life and area under curve were found to be high in the case of polymer-coated MLVs as compared to plain MLVs and plain drug solution. In vivo studies using inflammed rat model also indicated that the polymer-coated MLVs were more stable and could release the drug in a controlled fashion as compared to plain MLVs. PMID- 10748625 TI - Creative collaborations. Writers and editors. AB - Creativity, which the culture often depicts as a solitary enterprise, is almost always impacted or even dependent upon one or another aspect of a real relationship or an internalized object relationship. This paper utilizes the example of creative collaborations between writers and editors to illustrate aspects of co-creativity, co-construction, and mutual influence. It draws on Howard Gardner's observation, based on a series of biographical studies, that a creator during a time of artistic or intellectual breakthrough requires both an affective support system and a cognitive one. His observation echoes that of child researchers who emphasize that the contour of developmental lines depends not only on an internal dynamic but on interactions with significant others and on cultural and social influences as well. This paper describes both successful and problematic collaborations between writers and editors and demonstrates the strengths of such collaborations and some of their potential faultlines as well. PMID- 10748626 TI - Coordinating the developmental and psychoanalytic processes: three case reports. Introduction. PMID- 10748627 TI - Treatment of a boy with atypical ego development. AB - A multifaceted mode of therapeutic action is delineated as the complex neuropsychological and psychogenic factors in the development and functioning of an unusual four-year-old boy became elucidated. In addition to standard technique, the author developed a variety of psychoanalytically informed ways to facilitate his growth and ameliorate deviational aspects, especially his difficulties in appreciating and responding to the social-emotional world and establishing stable, integrated mental representations of self and other. The evolving treatment process is presented as well as attempts to coordinate and harmonize analytic and developmental goals. PMID- 10748628 TI - Detours in adolescent development. Implications for technique. AB - Dora's six-year analysis began when she was sixteen. In presenting this material, I shall focus on coordinating the technical demands of working analytically during a period of intense developmental change. This affords us an opportunity to examine the adolescent's use of analytic interventions both for work with neurotic conflicts and to facilitate development. A central issue in my work with Dora has been to examine her potential for navigating the tumultuous currents of adolescence while maintaining sufficient neutrality and abstinence so that later in the treatment, as the patient approached young adulthood, the analysis could be conducted in a manner consistent with our usual work with adults. PMID- 10748629 TI - The psychoanalytic treatment of a child with deviational development. AB - This paper presents the analysis of Peter, a five-year-old boy with compromised ego functions including difficulties in maintaining stable internalized object representations. Despite these serious problems, which are often seen as contraindicating analysis, a predominantly traditional psychoanalytic position emphasizing dynamic interpretations was utilized. Detailed clinical material is presented to demonstrate the effectiveness of this approach in facilitating therapeutic changes in Peter. PMID- 10748630 TI - Constant mental change and unknowability in psychoanalysis. AB - Conflict and compromise formation are central aspects of mental life. They dispose to constant mental change. Limitations of knowability in psychoanalysis are inextricably connected with constant mental change--the latter sometimes diminishing unknowability and sometimes exacerbating it. This paper explores three kinds of unknowability in psychoanalysis and their relation to constant mental change. That relation clarifies to some degree the implications of certain controversial proposals for change in psychoanalytic theory, method, and technique. PMID- 10748631 TI - Therapeutic functions of the real relationship in psychoanalysis. AB - This paper attempts to give a comprehensive picture of the concept and therapeutic functions of the real relationship in clinical psychoanalysis. After introducing this theme, the author presents an extensive review of the literature, citing not only the major recent contributors to the concept but also many contributions from the past. A third part summarizes Freud's development of the analytic relationship and then quotes from Freud's patients' written accounts of their analyses in order to illustrate how Freud's actual analytic style contained many elements of a real relationship. The fourth part presents the author's own formulation of the therapeutic contributions of the real relationship to psychoanalysis and offers a clinical vignette as an illustration of its therapeutic role in the termination phase of an analysis. PMID- 10748632 TI - Clocks, engines, and quarks--love, dreams, and genes. What makes development happen? AB - That psychological growth and maturation throughout the lifespan involve progressive linear processes is an implicit assumption of all models of development. Within psychoanalysis, a particular focus has been those processes that hinder forward development and manifest themselves as regressions or fixations or in character structure. However, the implicit assumption of progressive, linear development leaves unexplored the central question of what are the processes that govern developmental progressions. What makes psychological development happen in more or less predictable ways and yet allows for considerable individual variability? And are those developmental progressions inevitably forwardly progressive? Questions regarding what regulates and integrates development are relevant not only for understanding the normal building up of the internal world and of childhood psychopathology but also for those times of dramatic mental reorganization in adulthood surrounding events such as pregnancy and aging and for issues of psychological change during and after an analysis. Clinical material from analyses with a child and an adult and from interviews with four- to five-year-old children is used to explore individual fantasies of how development and change happens. The central role of internalization and object relations in regulating psychological development is emphasized. PMID- 10748633 TI - The interaction between self and others. A different perspective on narcissism. AB - This paper explores a perspective that expands the scope of the "healthy" narcissistic function to include the narcissistic preservation of self-identities during interaction between self and others. After a brief review of the literature from Freud to current views on the subject, the author describes the mutual influence of narcissistic processing and object relation and goes on to theorize that self-preservation is accomplished by the narcissistic envelope through immunization and defensive processing. Narcissistic immune processing provides familiarity, cohesiveness, equilibrium, integrity, and continuity to the self's separateness in an affective state of stability, which may make the libidinal need for object relation and interrelation with the world possible without undermining the constancy and safety of the self. The defensive processing preserves the self from incohesiveness and vulnerability during exposure to the unfamiliar (by creating a false self-identity, withdrawing into a narcissistic state, or preventing new interactions). The theoretical implications of this argument and their practical application are illustrated by several clinical vignettes. PMID- 10748634 TI - Obsessional manifestations in children. AB - Material is presented from the analyses of three children who developed obsessional behavior during the course of their analytic work. The author's intent is to use a careful examination of the emergence of these children's obsessions to try to understand the unconscious determinants that lead to the development of obsessive-compulsive behavior as a way to deal with psychic distress. PMID- 10748635 TI - Narrative performance mode (NPM) of discourse. AB - The nature of narrative is explored as well as why narrative has been so natural and so important, universal to people worldwide through the centuries. The function served by stories is explored. Attempts are made to mine the structure of narrative and to answer the reader's question: how will this help me in my day to-day work as a psychoanalyst? The basic necessities of story are enumerated, and some of the constitutive elements of narrative are contrasted with other modes of discourse. Freud's comments in The Poet and the Daydream are taken quite literally in this essay, which stresses the influence of unconscious process on changing modes of discourse. That moment when the analyst or analysand switches from one mode of discourse to another, here called a juncture, is compared to the pauses, doubts, and changes of venue within the dream. Modes of discourse psychology, dream psychology, and neurosis psychology are studied on parallel or similar tracks. The analysand's recognition of author, protagonist, antagonist, narrator, and listener (audience) is a new way of thinking about therapeutic action. A brief synopsis of the technical similarities and differences of a number of discourse modes is given, followed by a survey of recent experimental work on children's theories of the mind and narrative acquisition. PMID- 10748636 TI - Ego erection. Regressive perceptual phenomena in relation to psychic growth. AB - Regressive phenomena involving alterations of perceptual and other ego functions are exemplified by the Isakower phenomenon. Originally considered to be a defensive process involving regression to the oral phase in the face of conflict, a number of additional dimensions have been clarified in the literature over time. This paper describes analytic work with a patient who experienced distortions in the perception of his body on the couch. It examines the relationship of these experiences to the Isakower phenomenon. With close attention to analytic case material, it corroborates the oral core, the defensive structure, and the transformational processes characteristic of the episodes. Further, this case demonstrates that orally rooted ego phenomena, arising in response to oedipal conflict, can call forth the analysis of attachment. The vicissitudes of these phenomena throughout an analysis can give representation to the developing capacity for emotional attachment. The capacity for physical attachment, achieved in infancy via the mouth and breast, gives rise incrementally to the capacity for psychic attachment, achieved in adulthood by the waning of the Oedipal complex. PMID- 10748637 TI - The dread of integration. Integrative processes in a chronically ill borderline patient. AB - Integration of the patient's mental organization is an important part of all psychotherapeutic experiences. Generally, it is welcomed and thought well worth the effort needed to achieve it. However, there are some patients who feel terrified by this process. They seem to think that integration involves a loss of the self: they feel it is dangerous and even resist it with psychotic-type defenses. This was certainly the case for the patient described in this paper, who was affected from an early age by brittle diabetes. For her, this reaction was always activated by separation, and it also appeared prior to any developmental step she needed to take--e.g., in recognizing self-boundaries, sexual identity, and facing the oedipal conflict. On all these occasions her reaction was to run away from treatment in a state of deep regression, feeling suicidal, and liable to seriously harm herself through the mistreatment of her diabetes. The issue of integration, seen to arise with the concomitant loss of omnipotence, presents itself at different levels of development of the self and of the drives. PMID- 10748638 TI - Adolescents and popular culture. A psychodynamic overview. AB - Adolescents occupy a difficult and seemingly elusive developmental space, which makes them enigmas to most adults, including psychotherapists. Building upon dynamic theory such as that formulated by Winnicott or Erikson, this paper explores the relationship between adolescents and material elements of popular culture within a psychodynamic and developmental framework. Theoretical perspectives are integrated with case material to illustrate some of the roles of popular music and fashion in the lives of teenagers as a means of expression and in potential therapeutic alliance formation, dynamic understanding and working through developmental conflicts in displacement. PMID- 10748639 TI - Use of insight in child analysis. AB - The role of insight in working with a preadolescent girl, who was engaged in a seven-year analysis, is examined from the perspective of therapeutic action. The patient's use of insight, within the context of a transference neurosis, contributed substantively to changes in the depth of object relations, modulation of affect, return of development to its anticipated pathways, marked reduction of obsessive-compulsive symptomatology, and profound flowering of intellectual development. PMID- 10748640 TI - Acquisition of the algorithms of social life: a domain-based approach. AB - Proposing that the algorithms of social life are acquired as a domain-based process, the author offers distinctions between social domains preparing the individual for proximity-maintenance within a protective relationship (attachment domain), use and recognition of social dominance (hierarchical power domain), identification and maintenance of the lines dividing "us" and "them" (coalitional group domain), negotiation of matched benefits with functional equals (reciprocity domain), and selection and protection of access to sexual partners (mating domain). Flexibility in the implementation of domains occurs at 3 different levels: versatility at a bioecological level, variations in the cognitive representation of individual experience, and cultural and individual variations in the explicit management of social life. Empirical evidence for domain specificity was strongest for the attachment domain; supportive evidence was also found for the distinctiveness of the 4 other domains. Implications are considered at theoretical and applied levels. PMID- 10748641 TI - On inhibition/disinhibition in developmental psychopathology: views from cognitive and personality psychology and a working inhibition taxonomy. AB - Disinhibition is a common focus in psychopathology research. However, use of inhibition models often is piecemeal, lacking an overarching taxonomy of inhibitory processes. The author organizes key concepts and models pertaining to different kinds of inhibitory control from the cognitive and temperament/personality literatures. Within the rubrics of executive inhibitory processes, motivational inhibitory processes, and automatic attentional inhibition processes, 8 kinds of inhibition are distinguished. Three basic temperament traits may address key executive and motivational inhibitory processes. Future developmental psychopathology research should be based on a systematic conceptual taxonomy of the kinds of inhibitory function relevant to a given disorder. Such an approach can clarify which inhibition distinctions are correct and which inhibition deficits go with which disorders. PMID- 10748642 TI - Self-regulation and depletion of limited resources: does self-control resemble a muscle? AB - The authors review evidence that self-control may consume a limited resource. Exerting self-control may consume self-control strength, reducing the amount of strength available for subsequent self-control efforts. Coping with stress, regulating negative affect, and resisting temptations require self-control, and after such self-control efforts, subsequent attempts at self-control are more likely to fail. Continuous self-control efforts, such as vigilance, also degrade over time. These decrements in self-control are probably not due to negative moods or learned helplessness produced by the initial self-control attempt. These decrements appear to be specific to behaviors that involve self-control; behaviors that do not require self-control neither consume nor require self control strength. It is concluded that the executive component of the self--in particular, inhibition--relies on a limited, consumable resource. PMID- 10748643 TI - The development of infant intersensory perception: advantages of a comparative convergent-operations approach. AB - Despite impressive demonstrations of human infants' intersensory capabilities over the past several decades, there has been little focus on the contributions of prenatal and postnatal experience or the specific developmental processes underlying the emergence of intersensory functioning. Research with nonhuman animals has, however, provided a number of advances in understanding early intersensory perception. The authors explore the value of a comparative, convergent-operations approach to the study of early intersensory perception and examine how this approach has highlighted the study of (a) prenatal factors, (b) brain-behavior relations, and (c) context and experience variables contributing to infants' intersensory responsiveness. Examples of how human and animal research programs can cross-fertilize one another in their attempts to understand developmental processes underlying intersensory perception are considered. PMID- 10748644 TI - The development of intersensory temporal perception: an epigenetic systems/limitations view. AB - Several theories have stressed the importance of intersensory integration for development but have not identified specific underlying integration mechanisms. The author reviews and synthesizes current knowledge about the development of intersensory temporal perception and offers a theoretical model based on epigenetic systems theory, proposing that responsiveness to 4 basic features of multimodal temporal experience--temporal synchrony, duration, temporal rate, and rhythm--emerges in a sequential, hierarchical fashion. The model postulates that initial developmental limitations make intersensory synchrony the basis for the integration of intersensory temporal relations and that the emergence of responsiveness to the other, increasingly more complex, temporal relations occurs in a hierarchical, sequential fashion by building on the previously acquired intersensory temporal processing skills. PMID- 10748645 TI - The neighborhoods they live in: the effects of neighborhood residence on child and adolescent outcomes. AB - This article provides a comprehensive review of research on the effects of neighborhood residence on child and adolescent well-being. The first section reviews key methodological issues. The following section considers links between neighborhood characteristics and child outcomes and suggests the importance of high socioeconomic status (SES) for achievement and low SES and residential instability for behavioral/emotional outcomes. The third section identifies 3 pathways (institutional resources, relationships, and norms/collective efficacy) through which neighborhoods might influence development, and which represent an extension of models identified by C. Jencks and S. Mayer (1990) and R. J. Sampson (1992). The models provide a theoretical base for studying neighborhood mechanisms and specify different levels (individual, family, school, peer, community) at which processes may operate. Implications for an emerging developmental framework for research on neighborhoods are discussed. PMID- 10748646 TI - [White-coat normal blood pressure]. PMID- 10748647 TI - [The HIV-positive child]. PMID- 10748648 TI - [The probability of assessing the pathogenesis of ischemic stroke. Study of 81 patients]. AB - 81 patients with ischemic stroke were studied in order to assess the pathogenesis, the accuracy of the pathogenetic diagnosis and the relationship between pathogenesis and infarct site. The pathogenetic mechanisms were grouped according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) system. TOAST system settles five pathogenetic diagnosis: 1) Large artery atherosclerosis (LM); 2) Cardioembolism (CE); 3) Small artery occlusion (SAO); 4) Other etiologies (SOE); 5) Undetermined pathogenesis. As regards the site, the patients were divided into the following groups: Posterior Circulation Infarcts (POCI) 8.6%, Total Anterior Circulation Infarcts (TACI) 19.8%, Partial Anterior Circulation Infarcts (PACI) 29.6%, Lacunar Anterior Circulation Infarcts (LACI) 30.9%, Multiple Site Infarcts 11.1%. A probable or certain diagnosis was issued only in 33.3%. The diagnosis was not complete in 22.2%. That was due either to the severe clinical status or the patient and his relatives' refusal. Most of the incomplete diagnosis occurred in TACI patients. The undetermined pathogenesis due to absent clues occurred in 18.5%. CE often brought about TACI, whereas LM was likely to provoke PACI. SOE, like systemic hypotension, brought about Multiple Site Infarcts. Unfortunately assessing the pathogenetic diagnosis is still a controversial issue and TOAST method itself is not satisfactory as an overall classification system. PMID- 10748649 TI - [Imported malaria in Emergency Department]. AB - It occasionally happens that patients don't suspect to have malaria and diagnosis becomes difficult for the Emergency Department physicians. Since September 1995 to February 1999, 8 cases of malaria have been diagnosed in our first aid station; 5 occurred in european and 3 in extraeuropean immigrants. Incidence of imported malaria was greater after holiday period for european, and equally distributed along the year for immigrants. Patients' provenience was sub-Saharian Africa and Asia. Standard blood examination and thick and thin blood smears were performed showing P. falciparum and P. malariae infection. All patients were successfully treated with mefloquine as recommended. The aspecificity of the symptoms and signs makes diagnosis difficult if malaria is not suspected, in presence of fever in people returning from the tropics. Even in presence of symptoms and abnormal laboratory examinations, diagnosis is possible only with microscopic blood examination. PMID- 10748650 TI - [Carotenodermia: an erroneous diagnosis of jaundice]. AB - Carotenoids are essential pigments in vegetal photosynthetic processes, but they also have important biological functions in animal physiology: beta carotene in particular represents provitamin A. The accumulation of carotenoids in a man results in a cutaneous hyperpigmentation very similar to jaundice: usually this happens in vegetarian subjects after an excessive food eating. In the described case, a situation similar to jaundice appeared in a 56 years old man affected by psychic disorders, his diet consisted for several years almost exclusively of eggs (an average of 27 eggs a day). As this type of food is very rich in carotenoids (especially lutein giving to yolk the characteristic colour), it is inevitable that, in addition to hypercholesterolemia, a consistent amount of carotenodermia is present. This recalls the possible antiaterogenal action of carotenoids. PMID- 10748652 TI - [In a while it will be normal]. PMID- 10748651 TI - [Celiac disease and abortion: focusing on a possible relationship]. AB - Celiac disease represents one of the most frequent chronic inflammatory diseases. In Italy the prevalence among school-age population has been calculated in 1:180 subjects. Along with typical forms of the disease characterized by overt symptoms and signs of malabsorption, many cases are undiagnosed because they are subclinical, atypical or even symptomless. In adults, the disease may present with infertility; in particular celiac disease may be responsible of multiple abortions. These manifestations, whose pathogenesis is unknown, are not related to the severity of the disease; the gluten-free diet strongly ameliorates the fertility. In this paper we have focused the connection between abortion and celiac disease. A better knowledge of this relationship may lead to correctly diagnose and consequently to treat the cause of some cases of abortion, previously labelled as cases of unidentified origin. PMID- 10748653 TI - [Dysmetabolic syndrome related to HIV-1 protease inhibitors. Review of the literature and personal data]. AB - HIV-positive patients receiving antiretroviral therapy with HIV-1 protease inhibitors (PI) frequently show insulin-resistance, impaired glucose tolerance, hypertriglyceridaemia and lipodystrophy (LD). LD has often been reported only after the beginning of PI therapy. Some authors link LD to HIV chronic infection, some others suggest that PIs increase pre-existent disturb. Preliminary data of an observational study drawn in IV day-hospital of Spallanzani Institute in Rome showed hypertriglyceridaemia in 36.4% and hyperglycaemia in 11.2% of patients treated with PI. Carr suggests that such drugs should have this lipid-increasing effect because of their inhibition of low density lipoprotein-receptor-related protein, cytoplasmic retinoic-acid binding protein type 1 and P450 3A cytochrome. This theory doesn't explain why both untreated patients and treated with only reverse transcriptase inhibitors show sometimes the same disorders. According to another hypothesis Tumor necrosis factor-alpha, through inhibition of lipoprotein lipase, would determine high fat-storage in the adipose tissue. Cardiovascular risk factors have always to be assessed before starting a therapy with PI. Glycaemia, triglyceridaemia, cholesterolaemia have to be performed every three months during the treatment and, if necessary, C-Peptide and insulinaemia too. A treatment with lipid-lowering drugs is always recommended in patients with hypertriglyceridaemia > 500 mg/dl and/or hypercholesterolaemia LDL > 190 mg/dl in two following checks. Fibrates have proven to be effective in reducing hypertriglyceridaemia, but there is no certainty that such therapies could have good effects on the LD itself too. PMID- 10748654 TI - [Diabetes, coagulation and vascular events]. AB - Excessive oxidative stress due to hyperglycemia and glycoxidation leads to an increased production of F2-isoprostanes, one of which, 8-iso-PGF2 alpha, reaches high concentrations in plasma and urine in both insulin-dependent and non-insulin dependent diabetics. This is associated with an increase in platelet activation, reflected by an increased urinary excretion of platelet-derived TxB2. Improved metabolic control or vitamin E supplementation reduces urinary 8-iso-PGF2 alpha and TxB2, whereas aspirin or indobufen reduces TxB2 but not 8-iso-PGF2 alpha. Since TxB2 in the urine seems to represent the common link between diabetes (as well as other risk factors) and the thrombotic complications of vascular disease, platelet activation due to lipid-glycoxidation is an important aspect in the pathogenesis of vascular complications of diabetes mellitus. Among the various plasma coagulation and fibrinolysis factors that are found to be altered in diabetes, the increased level of plasminogen activator inhibitor (PAI-1) in the plasma and in the vessel wall is of the utmost importance. Indeed, it is suspected that the atherosclerotic plaques formed in the presence of high concentrations of PAI-1 are more prone to rupture and ensuing thrombosis. The thrombosis-oriented modifications of blood platelets, coagulation and fibrinolysis are an important cause behind the high prevalence of vascular events in diabetes. PMID- 10748655 TI - [Shame: anthropological and clinical dimension]. PMID- 10748656 TI - [Activities of the Brazilian Senate in the area of public health, 1995 and 1996]. AB - The objective of this study was to identify and describe the actions of Brazilian senators in the field of public health in 1995 and 1996. We also sought to determine if profession, regional background, or political party influenced the senators' actions. The actions were divided into three types: legislative (proposal and review of bills and petitions); supervisory (information requests to the executive branch and the establishment of inquiry committees and other special committees); and parliamentary (speeches). The data were collected from two databases maintained by the Senate, namely MATE and DISC. Of the 89 senators who were in office during the study period, 76 were involved with public health issues. Of the total of 667 actions studied, there was a predominance of speeches (43% of all actions), most of them responding to news reported by regional or national media. Supervisory activities were limited (5% of all actions were information requests). The subjects dealt with most frequently were health policies (30%), drugs (9%), regulation of health professions (8%), disease control (7%), and worker health (6%). Concerning the professions of the senators, the most frequent categories were physicians, teachers, and journalists. The senators representing the North and Northeast regions performed 62% of the actions and were involved with almost all the health subjects. Although 43% of the actions were carried out by liberal and right-wing senators, the senators from socialist and labor parties had a stronger proportional participation (both in terms of senators involved and actions performed). It is interesting to note that socialist and labor senators showed minimal involvement in the issue of worker health. The predominance of speeches as a prevalent type of action, the limited and disjointed scope of legislative actions, and, especially, the poor monitoring and control show the need for deep changes in the work process of the Senate's technical committees and, therefore, of the Senate's technical support unit. PMID- 10748657 TI - [Consumption of licit and illicit drugs in Chile: results of the 1998 study and comparison with the 1994 and 1996 studies]. AB - The principal results are presented here from the Third National Study of the Consumption of Drugs, which was carried out in Chile by the National Board for Narcotics Control between 1 September 1998 and 15 January 1999. The 1998 study used the same methodological design as the first and second studies, which were done in 1994 and 1996. The 1998 study expanded the sample to 31,665 individuals, who were representative of a population of 6,940,727 people from 12 to 64 years old, both sexes, and five socioeconomic levels and who were residents of urban areas in 62 commune administrative divisions of the 13 regions of the country. Results from the 1998 study are compared with those from 1996 and 1994. The 1998 study shows that 17.5% of Chileans have at some time in their life used one of the three illicit drugs most popular in the country: marijuana (16.8%), coca paste (2.3%), and cocaine hydrochloride (4.0%). The prevalence of use of any of the three drugs, mainly marijuana, during the preceding year was 5.3% and during the preceding month 2.2%. With respect to licit drugs, 28.4% of Chileans have at some time in their lives used antianxiety drugs, 84.4% of them have consumed alcohol, and 71.9% have used tobacco. Most of the people who once used illegal drugs have stopped doing so: 71.6% in the case of marijuana, 64.1% with coca paste, and 66.8% with cocaine hydrochloride. For legal drugs, the percentages were lower: 55.5% for anxiolytics, 16.0% for alcohol, and 34.5% for tobacco. The consumption of licit and illicit drugs was several times greater among men than among women, except for anxiolytics, whose use was three times greater among women. Drug use was more frequent among persons between 19 and 25 years old. Consumption of illegal drugs was more frequent at higher socioeconomic levels, and use of licit drugs was more common in the lower socioeconomic levels. The 50th percentile of the age of initiating drug use was 17 years for alcohol, 15 for tobacco, 30 for antianxiety agents, 17 for marijuana, 20 for coca paste, and 21 for cocaine hydrochloride. Comparing the results of the three studies shows that, after an increase in the use of licit drugs between 1994 and 1996, there was a stabilization in 1998. With illegal drugs there was a modest increase in consumption between 1996 and 1998, following a small reduction between 1994 and 1996. PMID- 10748658 TI - Death and injury from motor vehicle crashes in Colombia. AB - We report data on the distribution and determinants of road deaths and injuries for all victims in Colombia, with the aim of defining targets and priorities for highway death prevention in that country and other rapidly urbanizing nations. Using information from Colombia's Fund for the Prevention of Road Injury and the national death registry, we studied data on deaths and injuries from 1991 to 1995 for the nation as a whole and for the country's two largest cities, Santa Fe de Bogota and Medellin. Deaths and injuries are rising in the nation as a whole. Of the deaths, 75% occur in urban areas, and 80% are in males. Pedestrians aged 15 34 are a peak subgroup. Thirty-four percent of deaths are attributable to speeding and/or alcohol consumption. Death tolls are highest at night and on weekends. Specific priority targets for intervention are indicated by the fact that 75% of road deaths in Colombia occur in urban areas and that 80% of all victims are males. PMID- 10748659 TI - [Cervical cancer screening among indigenous women in the Xingu Indian Reservation, central Brazil]. AB - Although the literature presents worrisome data regarding the incidence of cervical cancer among indigenous populations, in Brazil there is very little information regarding the occurrence of this type of cancer among indigenous peoples. Therefore, the objective of the present descriptive study was to assess the prevalence of cervical cancer and of cervical and vaginal infections among 423 indigenous women living in the Xingu Indian Reservation, in the state of Mato Grosso, Brazil. These women were or had been sexually active. Data were collected between 1989 and 1996. Clinical and gynecological examinations were carried out prior to the collection of cervical specimens and to the performance of cytologic analyses. Upon detection of abnormalities, a colposcopy and a biopsy were also performed. Our results show that 1% of the women studied presented invasive carcinoma and that 3% presented premalignant lesions. In addition, 84% presented inflammatory atypia, resulting from sexually transmitted genital infections. The present findings are in accordance with the results of other international reports regarding the high prevalence of cervical conditions among indigenous populations, and they underscore the need to extend to the indigenous peoples of Brazil programs aiming at the control of sexually transmitted diseases and at the early detection and treatment of cervical cancer. PMID- 10748660 TI - [Prenatal care in the border city of Tijuana, Mexico]. AB - This study was intended to explore the conditions under which prenatal care is delivered in the border city of Tijuana, Baja California, Mexico, and to assess the possible associations between that care and neonatal results in terms of birthweight, health of the neonate, and prematurity. Seven hospitals serving persons from different socioeconomic strata were chosen, and between December 1993 and March 1994 interviews were conducted with 279 women who were in the first 24 to 48 hours of puerperium. During the interviews data were collected on socioeconomic level; the mothers' knowledge, attitudes, and practices concerning obstetric health; the mothers' perceptions of access to prenatal care; the quality of prenatal care visits (evaluated in terms of having blood and urine tested and weight and blood pressure measured); and the gynecological and obstetric and health history of the mother. A database was created using the SPSS statistics software package. Possible associations were explored, with prenatal care as the independent variable and various dependent variables, by means of contingency tables and a two-tailed Fisher's exact test. None of the neonates was premature, ill, or had a birthweight of < or = 2,500 g. For this reason it was decided to divide the variable corresponding to birthweight into two groupings, < or = 3,000 g and > 3,000 g. A significant (P < 0.00038) relationship was found between a lack of prenatal care and low birthweight. In addition, a lack of prenatal care was associated with: low family income; the mother's financial dependence on the father; the mother being in an unmarried relationship; little communication with the partner; having no medical insurance; an unwanted pregnancy; and giving delivery at the General Hospital. Out of the total sample of 279 women, 15 (5.4%) had received no prenatal care. None of these 15 women reported they had encountered difficulties that prevented them from obtaining prenatal care, but only 7 of those women answered that question. The prenatal care was of good quality in 190 of the cases (68%). Both the number and the timing of the prenatal visits were adequate in 142 of the cases (50%). The mother's having worked during pregnancy or before had a positive predictive value in terms of utilization of prenatal care services. Additional studies are needed to evaluate the possible relationship between domestic violence and the lack of prenatal care or a delay in seeking such care. PMID- 10748661 TI - Evaluation of a sexuality education program for young adolescents in Jamaica. AB - Despite their increasing numbers, few of the sexuality education and pregnancy prevention programs in developing countries have been evaluated. This study, conducted in 1995-1997, assesses the impact of a school-based sexuality education program, the Grade 7 Project, on 945 Jamaican seventh graders (aged 11-14) and their initiation of sexual activity and use of contraception at first intercourse, as well as the knowledge and attitudes that influence their behaviors. Using a quasi-experimental design, the study measured the effects of the Grade 7 Project when the nine-month intervention was completed (short term) and one year after that (long term). Multivariate logistic regression analysis indicated that the project had no effect on initiation of sexual activity, but it had a positive short-term impact on use of contraception at first intercourse (P = .08); adolescents in the intervention group were more than twice as likely to use contraception. The project also had a positive short-term influence on several aspects of the adolescents' knowledge of and attitudes about sexuality and pregnancy. The modest impact of the Grade 7 Project is encouraging, as school based sexuality education programs of limited duration rarely have a long-term impact. Moreover, competing socioeconomic and cultural forces in Jamaica encourage early sexuality and parenthood among adolescents. The use of more participatory teaching methods and smaller class sizes might strengthen the Grade 7 Project and enhance its impact. PMID- 10748663 TI - [Correlation between homicide rates and economic indicators in Sao Paulo, Brazil, 1996]. AB - Around 30% of the deaths due to violent causes in Brazil result from homicides. Violence has traditionally been related to larger social problems, such as poverty. Recently, however, a positive correlation has been observed between higher incomes and an increase in the homicide rate, so that some researchers have begun to consider inequality, rather than poverty, as an explanation for the epidemic of violence. The objective of this study was to investigate the correlation between urbanization, poverty, and economic inequality and homicide rates in the state of Sao Paulo, Brazil, in 1996. Information regarding population size, average monthly income of household heads, income distribution, and Gini index was obtained for each municipality, based on the demographic census. Homicide rates were calculated based on official data. Data were analyzed in terms of correlation and relative risk with 95% confidence intervals. Homicide rates rose in direct proportion to city population and ranged from 6.96 (per 100,000 inhabitants) in municipalities with a population smaller than 10,000 inhabitants, to 55.54 in municipalities with more than 1 million inhabitants. Relative risk ranged from 1.35 to 7.98. A significant correlation with population size was found only for incomes above 3.11 times the minimum wage and a Gini index greater than 0.50. There was a strong, direct, and significant correlation between homicide rates and the income ratio between the ninetieth and the twentieth percentiles of the population. It is necessary to probe more deeply into the macrosocial determinants of homicide rates in order to identify indicators of inequality that can generate meaningful data for developing public health strategies. PMID- 10748662 TI - [Antimicrobial resistance of Shigella spp. in Cordoba, Argentina, during the period 1990-1997]. AB - This study analyzed the evolution of antimicrobial resistance in 771 isolates of Shigella spp. obtained from a total of 9,195 feces cultures done between 1990 and 1997 in a children's hospital in Cordoba, Argentina. S. flexneri, which was responsible for 73% of the Shigella infections, was the species with the greatest resistance. The frequency of S. flexneri resistance to the three antibiotics most used (ampicillin, trimethoprim-sulfamethoxazole, and chloramphenicol) increased from 10% in 1990 to 58% in 1997 (P < 0.001). Considering each of the drugs individually, the resistance to ampicillin increased from 60% to 100% (P < 0.001), the resistance to chloramphenicol from 13% to 71% (P < 0.001), and the resistance to trimethoprim-sulfamethoxazole from 79% to 84% (P = 0.22). For S. sonnei, the increase in resistance to ampicillin (from 36% in 1990 to 54% in 1997) was not statistically significant (P = 0.20), nor was the reduction in resistance to trimethoprim-sulfamethoxazole, which went from 82% in 1990 to 55% in 1997 (P = 0.08). Only two S. sonnei isolates were found that were resistant to chloramphenicol, one in 1995 and another in 1997; two S. sonnei isolates were found with resistance to all three antibiotics. We consider it essential to carry out susceptibility tests of each Shigella clinical isolate, to detect changes in the resistance profile and thus modify empiric treatment. PMID- 10748664 TI - [Treatment of gonarthrosis]. PMID- 10748665 TI - [Infection of cattle by bovine respiratory syncytial virus]. PMID- 10748666 TI - [Poisoning by household products]. AB - Caustic products are responsible for the most serious cases of poisoning, which are always emergency cases. Application of current intervention procedures has decreased both morbidity and mortality. Water-diluted bleach, the ingestion of which remains extremely frequent, is a moderate irritant rather than a caustic product. Emission of gas produced when mixing bleach with other agents can be responsible for choking gas poisoning. Anionic and nonionic detergents are mostly dangerous because of their foam-producing properties. Mercury vapours and methanol are other potentially hazardous products. PMID- 10748667 TI - [Pesticide poisoning]. AB - Pesticide intoxications are mainly accidental with a benign course, such as ingestions of diluted fertilizers or low concentration antivitamin K rodenticides, ant-killing products or granules of molluscicides containing 5% metaldehyde. Voluntary intoxications by chloralose, strychnine, organophosphorus or organochlorine insecticides, concentrated anti-vitamin K products, herbicides such as paraquat, chlorophenoxy compounds, glyphosate or chlorates may be severe. Toxicity is due to active substances but also to solvents or surfactants included in the composition. Analysis done in a toxicology laboratory help in establishing diagnosis, prognosis and treatment. Poison centres may be called constantly to help for the evaluation of these intoxications. PMID- 10748668 TI - [Acute poisoning with industrial products]. AB - Poisonings with industrial products represent approximately 7% of the cases reported to the poison centres. Ingestion of petroleum distillates induces irritation of the gastrointestinal tract, central nervous system depression and aspiration pneumonitis which may be severe; treatment is mainly supportive. Ethylene and diethylene glycol poisonings produce central nervous system depression, anion gap metabolic acidosis, osmolar gap and acute tubular necrosis; in severe cases, hypocalcaemia, cerebral oedema and heart failure may be observed; treatment often associates supportive measures, haemodialysis and administration of competitive inhibitors of alcohol dehydrogenase (ethanol or 4 methylpyrazole). Glycol ethers induce central nervous system depression and metabolic acidosis; in addition, ethylene glycol monobutyl ether produces haemolysis; monomethyl and monoethyl ethers are responsible for bone marrow and lymphoid organ toxicity, they adversely affect spermatogenesis and are teratogens. PMID- 10748669 TI - [Recent advances in environmental toxicology]. AB - Among the sanitary effects of environmental chemicals, 3 examples illustrate the complexity of the issues to be solved: 1. endocrine disrupters, xenobiotics which interfere with hormonal systems, could increase the risk of reproductive and developmental disturbances and explain the rising incidence of hormone-dependent cancers; 2. multiple chemical sensitivity due to odorous chemicals has severe social consequences and warrants further research; 3. the reappearance of well known poisoning cases and the emergent new toxic diseases due to substitutive products for solvents, cooling agents and spray propellants banned for ecological purpose, demonstrate the need for surveillance programmes of industrial and household products. PMID- 10748670 TI - [Chemical-induced cancers]. AB - Most chemical carcinogenic agents are industrial. About 4% of all cancers have an occupational origin, but the percentage is higher in exposed populations. Evidence of carcinogenicity is provided by epidemiological studies, animal experiments and other biological sources like experimental mutagenesis. The IARC (International Agency for Research on Cancer) classification and the European Union classification of carcinogenic agents for humans are useful for prevention and regulation. Some cancers are classified as occupational diseases but only a few persons receive compensation given the difficulties of aetiologic diagnosis and the frequent absence of declaration. PMID- 10748671 TI - [Mushroom poisoning]. AB - Time elapsed between absorption of toxic mushrooms and occurrence of first disorders (mainly digestive), lead to distinguish 2 kinds of syndromes. Early syndromes, beginning before the 6th hour, follow a generally favourable course. Incriminated toxins induce digestive, neurosensory or neurovegetative disorders, sometimes severe but reversible. Late syndromes, beginning after the 6th hour, endanger patient's life. Incriminated toxins can lead to irreversible liver and kidneys damage. Medical treatment aims at eliminating toxins and inhibiting cellular penetration of cytolytic toxins. Symptomatic intensive care, directed by clinical and biological indications, ensure correction of plasmatic disorders and compensation for hepatic and renal severe failure. PMID- 10748672 TI - [Envenomations]. AB - Although venomous animals are less frequent in Europe than in tropical countries, some dangerous species can be responsible of envenomations in France. Bites by two species of adders (Vipera aspis and Vipera berus) can lead to extensive swelling with multiorgan failure. Treatment is based on intravenous infusions of specific antivenom. Bites and stings by other animal species encountered in France are characterised by important pain with local signs like necrosis, bleeding or oedema: weevers, scorpion fish, centipedes, bees, wasps.... Contrary to scorpions of North Africa or India, the 5 scorpion species which live in France are completely harmless. Such is not the case with spiders: latrodectism (neurotoxicity) and loxoscelism (extensive necrosis) observed in Provence and in Corsica. PMID- 10748673 TI - [Non-accidental criminal poisonings]. AB - Among the different types of chemical aggressions, murder is very rare. Most cases are less straight forward: "jokes", ill-wills, impulsive acts.... Pesticides, metals, household products and illicit drugs are most often in cause, in addition to self-defence sprays. Intoxication may be suspected by the victim, in which case paranoia should be eliminated. In other cases, unusual circumstances or symptoms are suspected by the physician. The toxicological analysis must be guided by the clinical context. PMID- 10748674 TI - [Taxonomy, attitudes and competence]. PMID- 10748675 TI - [Drugs and the eye]. PMID- 10748676 TI - [Myasthenic syndrome. Diagnostic trends]. PMID- 10748677 TI - [Antibacterial antibiotics. General data on mode of action and mechanisms of resistance]. PMID- 10748678 TI - [Foreign bodies of the respiratory tract: diagnosis and treatment]. PMID- 10748679 TI - [Leg ulcer: etiology, physiopathology, diagnosis, course, treatment principles]. PMID- 10748680 TI - [Acute metabolic complications of diabetes mellitus (ketoacidosis, hypoglycemia, hyperosmolarity, lactic acidosis). Etiology, physiopathology, diagnosis, emergency treatment of ketoacidosis with drug posology]. PMID- 10748682 TI - [The future of neurosciences. The challenges of the 21st century and ethical responsibilities]. PMID- 10748681 TI - [Contraception: methods, contraindications, surveillance]. PMID- 10748683 TI - [Lipids and AIDS]. AB - HIV infection induces an early decrease of cholesterol and a late increase of triglycerides (TG) with a reduction of HDL. These changes are proportional with the lowering of CD4, which reflects the infection's severity. Both the increase of TG synthesis and the decrease of TG catabolism, in relation with a reduction of lipoprotein lipase activity, are responsible of these changes. Moreover, LDL catabolism is enhanced by macrophage scavenger receptors, due to a high proportion of small, dense LDL which are more easily oxidized. Many cytokines (interferon alpha, interleukins, TNF) play probably a pathogenic role in the dyslipidemia. Some HIV patients who received antiproteases may develop lipodystrophy with central obesity, insulino-resistance, glucose intolerance and sometimes diabetes (like in syndrome X). Other patients present a cushingoid, buffalo hump. This complication may be observed also with antiretroviral treatment other than antiproteases. The physiopathology of these findings could be in relation with structural homologies between antiproteases and some important proteins, involved in lipid and adipocyte metabolism. Cardiovascular risk linked to these perturbations is evident. The treatment is not different from the treatment for seronegative, hyperlipidemic patients: struggle against risk factors, diet advices, fibrates or statins. The antiproteases bring huge contribution to the prognosis of AIDS patients but the risk of cardiovascular complications could impair this therapeutic progress. So, it is essential to understand the pathogeny of these complications in order to discover new antiproteases, without these adverse side effects. PMID- 10748684 TI - [The selection of insulin and adjustment of the dosage in diabetic children and adolescents: personal experiences]. AB - The principal aims of therapeutic management of the child, adolescent and adult with type 1 diabetes are to allow good quality of life and to avoid long-term complications (retinopathy, neuropathy, nephropathy, etc.) by maintaining blood glucose concentrations close to the normal range. Repeated determinations of glycated hemoglobin levels (HbA1c) provide a good criterion of overall control. They must be under 7%. The number of daily insulin injections, (2 or 4), by itself does not necessarily give better results, but the 4-injection regimen allows greater freedom, taking into account that the proper insulin adjustment is difficult before adolescence. Successful glycemic control in young patients depends mainly on the quality and intensity of diabetes education. The aim of this paper is to propose some "recipes", concerning the choice of the insulin regimens and the criteria for insulin adjustments, in order obtain good HbA1c levels. Because medical literature shows that treatment of type 1 diabetic patients is inadequate in general and that levels of HbA1c are often too high, diabetes treatment teams should individually explore the reasons for failure, without any prejudice or bias. PMID- 10748685 TI - [Hemoptysis]. AB - An 82 years-old man is admitted for suspicion of bronchial cancer. He has a persistent cough, repeated bronchial infections and haemoptysis. On thoracic CT scan, there is an increased thickness of the bronchial walls situated at the tracheobronchial cross. PMID- 10748686 TI - [Raloxifene (Celvista, Evista)]. AB - The prevention of osteoporotic fractures in post-menopausal women must be viewed in the framework of the treatment of menopause. SERMs ("Selective Estrogen Receptor Modulators") derivative from steroid hormones have estrogenic and antiestrogenic properties according to the substance and the target tissue. Raloxifene is a second generation SERM. It increases bone mass by 1 to 3% according to the measured site and, after 3 years of therapy at the dose of 60 mg per day, it reduces the incidence of vertebral fractures by 30 to 50% if patients have or do not have vertebral fractures before therapy. This drug is approved for the prevention of vertebral fractures in post-menopausal women at increased risk of fractures. A significant reduction in the incidence of hip fractures has not been demonstrated. Raloxifene exerts favorable effects on cardiovascular risk factors but one has to wait for the results of controlled prospective trials before concluding that raloxifene reduces the risk of atherogeniec disease. Preliminary results indicate a substantial reduction of the risk of invasive breast cancer, still to be confirmed. The incidence of vaginal bleeding does not differ from placebo as raloxifene does not stimulate endometrial proliferation. The most serious adverse event, although infrequent, consists in an increase of the relative risk of thromboembolic disease by 3.1 as compared to placebo. Longer term studies are necessary to compare raloxifene with the estrogen replacement therapy and to determine the extra-bone effects. PMID- 10748687 TI - [Bilateral spondylolysis: the value of bone computed scintigraphy]. PMID- 10748688 TI - [Intradermal reaction to tuberculin: application, interpretation and therapeutic implications]. PMID- 10748689 TI - [The epidemiology of gastroesophageal reflux in Belgium and its approach by general practitioners: results of 2 national surveys]. PMID- 10748690 TI - [The physiopathology of gastroesophageal reflux]. PMID- 10748692 TI - [Indications and surgical technics in the treatment of gastroesophageal reflux]. PMID- 10748691 TI - [The extra-digestive manifestations of gastroesophageal reflux]. PMID- 10748693 TI - [Psychopharmacology as the 20th century turns to the 21st]. PMID- 10748694 TI - [Therapeutic monitoring of psychotropic drugs and pharmacogenetic tests in psychiatry]. PMID- 10748695 TI - [Importance of drug surveillance in psychiatry--the AMSP project]. PMID- 10748696 TI - [New psychopharmacologic studies on methadone: implications for the treatment of opiate dependency]. AB - New psychopharmacological studies performed these last few years should allow, in future, to improve methadone prescription. In particular, thanks to the identification of enzymes involved in the metabolism of this molecule, it is now possible to better understand the problems of metabolic interactions, to foresee and to avoid them. Studies showing the influence of genetic and environmental factors on blood concentrations of methadone, as well as a recent clinical study performed with methadone doses largely in excess of 100 mg/day, stress the importance of adapting methadone treatment individually, particularly as regards to the choice of the dose. This choice must not result from an administrative standardization, as it is essential for patients that, with an adequate social and psychological treatment, an adequate pharmacological treatment should be associated. PMID- 10748697 TI - [Psychotropic drug prescription in adolescent]. PMID- 10748698 TI - [Psychopharmacotherapy in the adult psychiatric domain]. PMID- 10748699 TI - [Psychopharmacology in the psychogeriatric domain: current data, problems, perspectives]. PMID- 10748700 TI - [Psychotropic pharmacotherapy, treatment compliance and prescription observance]. PMID- 10748701 TI - [Integration of clinical approaches in psychiatry. The example of obsessive compulsive disorder]. AB - The objective of this paper is to discuss the complex challenge of psychiatry which tries to integrate knowledge and therapeutic strategies from very different fields. Obsessive compulsive disorder is chosen as an example of a pathological condition where that integration of scientific knowledge is essential and where the combined use of pharmacotherapy, psychoeducation and cognitive behavioural therapy is particularly required for optimal treatment. PMID- 10748702 TI - [L-tryptophan plasma levels in treatment resistant depressive states]. AB - 10 to 30% of depressions are resistant to standard treatment. Different therapeutic strategies are used to treat the resistant depressions. Therefore, before initiating an antidepressant treatment, it would be important to know which patients will probably not respond to a standard treatment. Numerous studies have shown that serotonin is involved in depressive illness and its synthesis in the brain is dependent on the availability of tryprophan from plasma. As tryptophan plasma level is decreased in depression, resistant depressions may also be characterized by alterations of tryptophan plasma level. 141 depressed patients were admitted in our psychiatric unit in Geneva between 1984 and 1990. 36 were diagnosed as treatment resistant depression. Although treatment resistant patients group had more women and a more severe score of depression on the AMDP-4 scale, we did not observe a significant difference in tryptophan plasma level compared to patients who respond to treatment. PMID- 10748703 TI - [Ambulatory management of patients after myocardial infarction]. AB - After a myocardial infarction, the physician should evaluate if his patient has a high risk of complications. Main factors for prognosis are left ventricular systolic function, myocardial ischemia, ventricular arrhythmias and coronary anatomy. Treatment of symptoms and complications (angina, heart failure, ventricular arrhythmias) and drugs aimed at improving prognosis (beta-blockers, platelet inhibitors, angiotensin-converting enzyme inhibitors, estrogen) are discussed. Finally the importance of correcting risk factors is stressed with emphasis on the essential role of statins. PMID- 10748704 TI - [Aspects of Fribourg hospital hygiene during the time of Louis Pasteur]. PMID- 10748706 TI - [Research in the medical office. For organized and quality clinical research in the practicing physician's office in Switzerland]. PMID- 10748705 TI - [Is homeopathy a scientific therapy?]. AB - BACKGROUND: Homoeopathy is not considered as a scientific therapy. AIM OF THE STUDY: To systematically re-examine research concerning homoeopathy and to analyse whether these works show homoeopathy as being a scientific therapy. METHOD: The review of international literature on the subject. RESULTS: Scientific research on homoeopathy is divided into four principal directions: the demonstration of the clinical activity of homoeopathic medicines, the demonstration of the biological activity of the high dilutions and of the principle of similarity, the research of the mechanical action and the medical conception of homoeopathy. Homoeopathy cannot rest on a fixed doctrine but must be continually brought up to date by medical discoveries and present day science. More and more research in these four domaines is being published in the best international medical journals, providing publicity for homoeopathy and its activities. Up to now, no research has categorically proven that homoeopathy has a specific pharmacological action, consequently it is not a proven scientific therapy. CONCLUSION: If homoeopathy wants to be recognised by classical medicine, it must continue its academic research. It is only by doing this that homoeopathy will be accepted by the scientific world. PMID- 10748707 TI - [Concerning the article: autopsies: from the past towards the future (Z. Sando, B. Borisch, F.T. Bosman), 119: 763-767, October 1999]. PMID- 10748708 TI - [The occurrence of Toxoplasma gondii and Neospora caninum as regards meat hygiene]. AB - Toxoplasma gondii and Neospora caninum are phenotypically and phylogenetically closely related cyst-forming coccidia, both of which may cause abortion in livestock animals. T. gondii exhibits also zoonotic potential by causing diaplacental infections in the human fetus and harmful infections in immunosuppressed individuals. Humans get infected either by consuming inappropriately prepared cyst-containing meat or by ingesting oocysts originating from cat feces. Therefore, in order to assess infection risk we need to have knowledge on the prevalence of the parasite in consumable meat and thus slaughtered animals. So far, no data indicate any zoonotic potential for N. caninum. Due to its high economic impact in the bovine production in Switzerland, we included this parasite in the present study as well. The prevalence of both parasite species were investigated by PCR in muscle and brain samples of slaughtered bovines, sheep, pigs and horses. Comparatively, a serum sample from each animal was simultaneously tested serologically by a Toxoplasma-P30-ELISA and a Neospora-SA-ELISA. The prevalences determined by the T. gondii-PCR were the followings: adult cows 3%, young bulls 2%, young cows prior to gravidity 6%, calves 1%, sheep 6%, horses and pigs each 0%. For N. caninum, the PCR-prevalence was 2% for adult cows and 0% for all other animal groups. Conversely, the seroprevalences were much higher for both parasite species and all animal groups, with the exception of the fattening pigs. However, as T. gondii was principally detectable in bovine (cows and calves) as well as in sheep meat, the consumption of this meat harbours a potential infection risk for humans. In contrast, the lack of any parasite detectability in fattening pig and horse meat allows to consider this infection source as neglectable when compared to bovine and ovine meat. PMID- 10748709 TI - [Occurrence of verotoxin-producing Escherichia coli (VTEC) in fecal swabs from slaughter cattle and sheep--an observation from a meat hygiene view]. AB - Fecal samples from 544 beef cattle and 140 sheep were investigated by PCR for verotoxin (VT)-producing Escherichia coli (VTEC) without and with an enrichment step. 6.1% (after enrichment 14%) of cattle samples and 10% (after enrichment 29.2%) of sheep samples were VT-PCR-positive. Moreover, a noticeable age depending prevalence in cattle was found. Eleven VTEC strains isolated from fecal samples of 5 cattle and 6 sheep were taken for further characterization. None of the strains belonged to serogroup O157. But, as reported previously, we also found in this study strains with virulence genes that are associated with increased pathogenicity. The importance of slaughter hygiene and of bacteriological monitoring of carcass contamination has to be pointed out. PMID- 10748710 TI - [Distribution of antigen types of canine parvovirus in Switzerland, Austria and Germany]. AB - In this study the distribution of the various antigenic types of canine parvovirus in Switzerland, Austria, and Germany was investigated. From 14 samples from Switzerland, 35 samples from Austria, and 82 samples from Germany exclusively the new antigenic types CPV-2a and CPV-2b were isolated. Most of these isolates had a Ser-Val mutation at amino acid 297, which was shown to be prevalent in recent CPV isolates from various parts of the world. These findings are discussed with regard to the use of modified live virus vaccines. PMID- 10748711 TI - [Characterization of Swiss new world camelid breeds using microsatellite markers]. AB - Molecular markers allow a characterization of animal populations on DNA level. They help to estimate the genetic variability, they are useful in paternity control, to diagnose hereditary diseases, in epidemiology and last but not least support traditional tools to take breeding decisions. Seventy-six new-world camelids in Switzerland were genetically typed for six microsatellite markers and their parentage controlled. Calculation of allele frequencies, heterozygosity, polymorphism information content, deviation from Hardy-Weinberg equilibrium and exclusion probability of markers showed a high genetic variability within the chosen populations. This will allow selection for certain traits. The estimated genetic distance between different camelid species and breeds was in accordance with their expected phylogenetic origins. PMID- 10748713 TI - Preparation and pharmacological evaluation of novel glycoprotein (Gp) IIb/IIIa antagonists. 1. The selection of naphthalene derivatives. AB - The synthesis and design using molecular modeling techniques for non-peptide, low molecular weight novel fibrinogen receptor (glycoprotein IIb/IIIa: Gp IIb/IIIa) antagonists, is reported. We used a highly potent serine protease inhibitor, Nafamostat, having an amidinonaphthyl unit as the starting compound. The compounds 4-(6-amidino-2-naphthylaminocarbonyl)phenoxyacetic acid (5a) and 4-(6 amidino-2-naphthalenecarboxamido)phenoxyacetic acid (5b) inhibited adenosin-5' diphospate (ADP)-induced aggregation of human platelet-rich plasma (PRP) with IC50 values of 0.05 and 0.07 microM, respectively, and had lost their ability to inhibit a variety of serine proteases, including thrombin, factor Xa, plasmin and trypsin. PMID- 10748712 TI - Synthesis and antitumor activity of novel pyrimidinyl pyrazole derivatives. AB - Novel pyrimidinyl pyrazole derivatives were synthesized and examined for cytotoxic and antitumor activity. Mannich reaction was employed to construct this scaffold. Among the compounds synthesized, a series of propene derivatives exhibited a potent cytotoxic activity against some tumor cell lines including multidrug resistant cell lines due to the overexpression of P-glycoprotein. The vinyl bond moiety in the scaffold was believed to be required for the cytotoxic activity. Among them, compound 14 g, when administered intraperitoneally, showed potent antitumor activity against the malignant ascites caused by intraperitoneal inoculation of P388 cells in mice. This compound also showed high activity against a solid tumor Meth A mouse fibrosarcoma when administered both intraperitoneally and orally. PMID- 10748714 TI - Preparation and pharmacological evaluation of novel glycoprotein (Gp) IIb/IIIa antagonists. 2. Condensed heterocyclic derivatives. AB - A novel series of platelet receptor glycoprotein (Gp) IIb/IIIa antagonists with condensed heterocycles as their basic core was synthesized. In an in vitro assay, trans-4-(5-amidinobenzofuran-2-carboxamido)cyclohexyloxyacetic+ ++ acid 17e and trans-3-[4-(5-amidinobenzofuran-2- carboxamido)cyclohexyl]propionic acid 17f produced marked inhibitions with IC50 values of 0.018 and 0.006 microM, respectively in a human platelet adenosin-5'-diphospate (ADP)-induced aggregation assay; they also exhibited a wide spectrum of inhibition toward major aggregation agonists (ADP, collagen, thrombin, PMA (tumor promoter) and arachidonic acid). These compounds were > 2-3 orders of magnitude more effective in inhibiting platelet aggregation than human umbilical vein endothelial cell (HUVEC) binding. The oral administration of 10 mg/kg of either 17e and 17f to guinea pig, resulted in a 60% inhibition of ex vivo platelet aggregation after 5 h. Oral administration of ethyl trans-4-(5-amidinoben-zofuran-2- carboxamido)cyclohexyloxyacetate 18e (10 mg/kg) resulted in 80% inhibition of platelet aggregation in dogs for 6 h after oral administration with a return to baseline by 24 h. Ethyl trans-3-[4-(5-amidinobenzofuran-2 carboxamido)cyclohexyl]propionat e 18f (AR0598) produced 80% inhibition for 5 h after oral administration. Prodrug 18e showed a good profile in dogs with a long duration of action. 18e (AR0510) was selected as suitable clinical candidate for development as an orally active antithrombotic agent. PMID- 10748715 TI - Effect of UV-absorbing agents on photodegradation of tranilast in oily gels. AB - Tranilast (TL) oily gels containing UV-absorbing agents (UV absorber) were prepared, and the effect of the agents against photodegradation of TL was investigated. When 0.1% TL oily gel without UV absorber was exposed to light, TL was photochemically decomposed to the extent of 74.1% of its initial content at the end of the first hour. Although there were differences in the preventive effect on photodegradation of TL depending on the UV absorbers employed, 2-(2 benzotriazolyl)-p-cresol (BTPC) was the most effective absorber. The addition of UV absorbers to the oily gel did not affect the release of TL from the gel, the skin permeation, or the skin concentration of TL following topical application. UV absorbers added to TL oily gel penetrated into skin; however, their concentration in skin was similar to that following application of commercial sunscreen. These results suggest that the addition of UV absorbers to the oily gel of TL may be useful in preventing photodegradation of TL in the gel. PMID- 10748716 TI - Antidiabetic principles of natural medicines. IV. Aldose reductase and qlpha glucosidase inhibitors from the roots of Salacia oblonga Wall. (Celastraceae): structure of a new friedelane-type triterpene, kotalagenin 16-acetate. AB - The aqueous methanolic extract of an Indian natural medicine, the roots of Salacia oblonga Wall. (Celastraceae), was found to show inhibitory activity on the increase in serum glucose level in sucrose- and maltose-loaded rats. The water-soluble and ethyl acetate-soluble portions from the aqueous methanolic extract showed inhibitory activities on alpha-glucosidase and aldose reductase, respectively. From the water-soluble portion, potent alpha-glucosidase inhibitors, salacinol and kotalanol, were isolated, together with nine sugar related components, while a new friedelane-type triterpene, kotalagenin 16 acetate, was isolated from the ethyl acetate-soluble portion along with known diterpenes and triterpenes. The structure of kotalagenin 16-acetate was elucidated on the basis of physicochemical evidence. Principal components from this natural medicine were examined in terms of inhibitory activity on aldose reductase, and the diterpene and triterpene constituents, including the new kotalagenin 16-acetate, were found to be responsible components for the inhibitory activity on aldose reductase. PMID- 10748718 TI - Chemical constituents from the leaves of Hydrangea macrophylla var. thunbergii (III): Absolute stereostructures of hydramacrosides A and B, secoiridoid glucoside complexes with inhibitory activity on histamine release. AB - Following the characterization of dihydroisocoumarin constituents, two secoiridoid glucoside complexes, called hydramacrosides A and B, were isolated from the leaves of Hydrangea macrophylla Seringe var. thunbergii Makino. The absolute stereostructures of hydramacrosides A and B were elucidated on the basis of chemical and physicochemical evidence, which included the application of the 13C-NMR glycosylation shift rule of 1,1'-disaccharides and the modified Mosher's method. Hydramacrosides A and B exhibited an inhibitory effect on histamine release from rat mast cells induced by an antigen-antibody reaction. PMID- 10748717 TI - Bioactive saponins and glycosides. XIV. Structure elucidation and immunological adjuvant activity of novel protojujubogenin type triterpene bisdesmosides, protojujubosides A, B, and B1, from the seeds of Zizyphus jujuba var. spinosa (Zizyphi Spinosi Semen). AB - Following the elucidation of jujubosides A1 and C and acetyljujuboside B, novel protojujubogenin type triterpene bisdesmosides, protojujubosides A, B, and B1, were isolated from Zizyphi Spinosi Semen, the seeds of Zizyphus jujuba Mill. var. spinosa Hu. The structures of protojujubosides A, B, and B1 were determined on the basis of chemical and physicochemical evidence, which included the conversion of protojujubosides to known jujubosides using enzymatic hydrolysis. Protojujubosides A and jujubosides A, B, and C were found to show potent immunological adjuvant activity. PMID- 10748719 TI - Bioactive saponins and glycosides. XV. Saponin constituents with gastroprotective effect from the seeds of tea plant, Camellia sinensis L. var. assamica Pierre, cultivated in Sri Lanka: structures of assamsaponins A, B, C, D, and E. AB - The saponin fraction from the seeds of the tea plant, Camellia sinensis L. var. assamica Pierre cultivated in Sri Lanka, was found to show a potent protective effect on gastric mucosal lesions induced by ethanol in rats. Nine new acylated polyhydroxyoleanene-type triterpene oligoglycosides called assamsaponins A-I were isolated from the active saponin fraction together with three known saponins, theasaponin E1 and E2 and camelliasaponin B1. The structures of assamsaponins A-E were elucidated on the basis of chemical and physicochemical evidence. Theasaponin E1 exhibited potent gastroprotective activity. PMID- 10748720 TI - Synthesis and antibacterial activity of novel 7-substituted-6-fluoro-1- fluoromethyl-4-oxo-4H-[1,3thiazeto[3,2-a]quinoline-3-carboxylic acid derivatives. AB - A series of 7-substituted-6-fluoro-1-fluoromethyl-4-oxo-4H- [1,3]thiazeto[3,2 a]quinoline-3-carboxylic acid derivatives (2a-1) was prepared and evaluated for antibacterial activity. These compounds were obtained by deacylation of 4 benzoyloxy-2-(1-chloro-2-fluoroethyl)thio-6,7- difluoroquinoline-3-carboxylate (10) and subsequent intramolecular cyclization followed by substitution with cyclic amines and then hydrolysis. The intramolecular cyclization reaction of 18, one of the diastereomers (17, 18) revealed that the cyclization reaction proceeded through an inversion to afford (-)-11a in good chemical and optical yield. The enantiomers of 2a were prepared from the enantiomers of 11a, which were obtained by the optical resolution of the racemate using high-performance liquid chromatography (HPLC). Compounds 2a,b showed excellent in vitro and in vivo antibacterial activity against both gram-negative and gram-positive bacteria including quinolone and Methicillin-resistant Staphylococcus aureus. PMID- 10748721 TI - Retinoid X receptor-antagonistic diazepinylbenzoic acids. AB - Several dibenzodiazepine derivatives were identified as novel retinoid X receptor (RXR) antagonists on the basis of inhibitory activity on retinoid-induced cell differentiation of human promyelocytic leukemia cells HL-60 and transactivation assay using retinoic acid receptors (RARs) and RXRs in COS-1 cells. 4-(5H-2,3 (2,5-Dimethyl-2,5-hexano)-5-n- propyldibenzo[b,e][1,4]diazepin-11-yl)benzoic acid (HX603, 6c) is an N-n-propyl derivative of an RXR pan-agonist HX600 (6a), and exhibited RXR-selective antagonistic activity. Similar RXR-antagonistic activities were observed with 4-(5H-2,3-(2,5-dimethyl-2,5-hexano)-5-methyl- 8 nitrodibenzo[b,e][1,4]diazepin-11-yl)benzoic acid (HX531, 7a) and 4-(5H-10,11 dihydro-5,10-dimethyl-2,3-(2,5-dimethyl- 2,5-hexano)-dibenzo[b,e][1,4]diazepin-11 yl)benzoic acid (HX711, 8b), which also inhibited transactivation of RARs induced by an RAR agonist, Am80. These compounds inhibited HL-60 cell differentiation induced by the combination of a low concentration of the retinoid agonist Am80 with an RXR agonist (a retinoid synergist, HX600). These results indicated that HX603 (6c), and the related RXR antagonists inhibit the activation of RAR-RXR heterodimers as well as RXR homodimers, which is a distinct characteristic different from that of the known RXR antagonist, LG100754 (9). PMID- 10748722 TI - Roles of two basic nitrogen atoms in 1-substituted 4-(1,2 diphenylethyl)piperazine derivatives in production of opioid agonist and antagonist activities. AB - To ascertain roles of the two basic nitrogen atoms in 1-substituted 4-[2,(3 hydroxyphenyl)-1-phenylethyl]-piperazine derivatives (1) in the expression of opioid agonist and antagonist activities, a methine group (CH) was isosterically substituted for nitrogen atom at the 1-position (N-1) in compound 1 to obtain 4 substituted 1-[2-(3-hydroxyphenyl)-1-phenylethyl]piperidine derivatives (2). Their analgesic action and ability to produce physical dependence (jump-producing activity) as the mu-opioid receptor specific in vivo actions, and narcotic antagonist action in mice were compared with those of compound 1. Results of this study showed that, in cases of the racemate and the (S)-(+) enantiomer, opioid agonist activities (analgesic and jump-producing activities) were not greatly affected by the methine-substitution for N-1 in compound 1, but that the narcotic antagonist activity of the (R)-(-) enatiomer was abolished by this substitution. It thus appears that N-1 in compound 1 contributes to the expression of narcotic antagonist activity, whereas the nitrogen atom at the 4-position corresponds to the tyramine moiety necessary for the expression of mu-opioid agonist activity. PMID- 10748723 TI - Synthesis of cyclic oligopeptides containing an Arg-Gly-Asp (RGD) sequence. AB - The synthesis of cyclic RGD peptides with 8-10 residues cyclized by an amide bond and the relationship between their structure and activity (i.e. circular dichroism spectrum and inhibition of platelet aggregation) are reported. The linear peptides were synthesized by the solution method and their cyclization was performed in high dilution using DPPA. PMID- 10748724 TI - Synthesis and central nervous system depressant effects of N3-substituted 2',3'-O isopropylideneuridines. AB - N3-Substituted derivatives of 2',3'-O-isopropylideneuridine (1) were synthesized and their pharmacological effects on the central nervous system (CNS) examined using mice. Methyl (2), ethyl (3), propyl (4), butyl (5), allyl (6), benzyl (7), o-, m-, p-xylyls (8, 9, 10), and alpha-phenylethyl (11) derivatives of 1 were administered to mice by intracerebroventricular (i.c.v.) injection for evaluating hypnotic activity, pentobarbital-induced sleep prolongation, and spontaneous activity as indices. Only 3 possessed hypnotic activity by i.c.v. injection at the dose of 2.0 mumol/mouse. Compounds 3, 4, and 10 significantly showed synergism with a barbiturate, indicating that the derivatives have some CNS depressant effects. Moreover, 3 and 4 caused decrease in the spontaneous activity of mice, even at low doses. The present study indicated that substitution by ethyl, propyl, and p-xylyl groups at the N3-position of 2',3'-O isopropylideneuridine imparted the CNS depressant effects. PMID- 10748725 TI - Matrix assisted synthetic transformations: a mosaic of diverse contributions. II. The pattern is completed. PMID- 10748726 TI - Evaluation of gaseous hydrogen fluoride as a convenient reagent for parallel cleavage from the solid support. AB - We have tested the limits of gaseous hydrogen fluoride as an agent for parallel detachment of organic molecules from the solid support. Peptides were chosen as relatively sensitive models for this reaction. Acid-catalyzed amide bond hydrolysis, side chain modification (tryptophan and other unnatural amino acids) by the protecting group residues as well as dehydration of serine and asparagine was followed. The technique of cleavage of side chain protection prior to the resin cleavage has given satisfactory results. Two-step deprotection and cleavage from benzhydrylamine resin by TFA and HF was compared to the deprotection and cleavage by TFA from Knorr resin. PMID- 10748727 TI - Selection of modified oligonucleotides with increased target affinity via MALDI monitored nuclease survival assays. AB - Reported here is how modified oligonucleotides with increased affinity for DNA or RNA target strands can be selected from small combinatorial libraries via spectrometrically monitored selection experiments (SMOSE). The extent to which target strands retard the degradation of 5'-acyl-, 5'-aminoacyl-, and 5' dipeptidyl-oligodeoxyribonucleotides by phosphodiesterase I (EC 3.1.4.1) was measured via quantitative MALDI-TOF mass spectrometry. Oligonucleotide hybrids were prepared on solid support, and nuclease selections were performed with up to 10 modified oligonucleotides in one solution. The mass spectrometrically monitored experiments required between 120 and 300 pmol of each modified oligonucleotide, depending on whether HPLC-purified or crude compounds were employed. Data acquisition and analysis were optimized to proceed in semiautomated fashion, and functions correcting for incomplete degradation during the monitoring time were developed. Integration of the degradation kinetics provided "protection factors" that correlate well with melting points obtained with traditional UV melting curves employing single, pure compounds. Among the components of the five libraries tested, three were found to contain 5' substituents that strongly stabilize Watson--Crick duplexes. Selecting and optimizing modified oligonucleotides via monitored nuclease assays may offer a more efficient way to search for new antisense agents, hybridization probes, and biochemical tools. PMID- 10748728 TI - Fluorescence-quenched solid phase combinatorial libraries in the characterization of cysteine protease substrate specificity. AB - To map the substrate specificity of cysteine proteases, two combinatorial peptide libraries were synthesized and screened using the archetypal protease, papain. The use of PEGA resin as the solid support for library synthesis facilitated the application of an on-resin fluorescence-quenched assay. Results from the screening of library 2 indicated a preference for Pro or Val in the S3 subsite and hydrophobic residues in S2; the most prevalent residue not being Phe but Val. The S1 subsite exhibited a dual specificity for both small, nonpolar residues, Ala or Gly, as well as larger, Gln, and charged residues, Arg. Small residues predominated in the S1'-S4' subsites. Active peptides from the libraries and variations thereof were resynthesized and their kinetics of hydrolysis by papain assessed in solution phase assays. Generally, there was a good correlation between the extent of substrate cleavage on solid phase and the kcat/KM's obtained in solution phase assays. Several good substrates for papain were obtained, the best substrates being Y(NO2)PMPPLCTSMK(Abz) (kcat/KM = 2109 (mM s) 1), Y(NO2)PYAVQSPQK(Abz) (kcat/KM = 1524 (mM s)-1), and Y(NO2)PVLRQQRSK(Abz) (kcat/KM = 1450 (mM s)-1). These results were interpreted in structural terms by the use of molecular dynamics (MD). These MD calculations indicated two different modes for the binding of substrates in the narrow enzyme cleft. PMID- 10748729 TI - Toward designing drug-like libraries: a novel computational approach for prediction of drug feasibility of compounds. AB - Prediction of the degree of drug-like character in small molecules is of great industrial interest. The major barrier, however, is the lack of a definition for drug-like character. We used the concept of the multilevel chemical compatibility (MLCC) between a compound and a drug library as a measure of the drug-like character of a compound. The rationale is that the local chemical environment of each atom or group of atoms in a compound largely contributes to the stability, toxicity, and metabolism in vivo. A systematic comparison of the local environments within a compound and those within the existing drugs provides a basis for determining whether and how much a compound is drug-like. We applied the MLCC calculations to four test sets: top selling drugs, compounds under biological testing prior to the preclinical test, anticancer drugs, and compounds known to have poor drug-like character. The following conclusions were obtained: (1) A convergent number of unique local structure types were found in the analysis of the library of the existing drugs. It suggests that the current drug library contains about 80% of all the viable types; therefore, discovery of a drug with new local structures is only an event of relatively small probability. (2) The method is highly selective in discerning drug-like compounds: most of the top drugs are predicted to be drug-like, about one-quarter of the biological testing compounds are drug-like, and about one-fifth of the anticancer drugs are drug-like. (3) The method also correctly predicted that none of the known problematic compounds are drug-like. (4) The method is fast enough for computational screening of virtual combinatorial chemistry libraries and databases of available compounds. PMID- 10748730 TI - Parallel synthesis of prostaglandin E1 analogues. AB - The first demonstration of the rapid parallel synthesis of diverse prostaglandin derivatives is reported. Upper (alpha-) side chain diversity was introduced to core 1 via the parallel Suzuki coupling of hydroborated alkenes. Conversion to the enones 3 and 9 was followed by the addition of the lower (omega-) side chains as higher-order cuprates 4. Upper side chains incorporating an N-acylsulfonamide protecting group were further transformed into prostaglandin amide analogues. Cleavage from support with HF/pyridine followed by scavenging provided 26 prostaglandin E1 analogues in high purity. PMID- 10748731 TI - Exploring the scope of poly(ethylene glycol) (PEG) as a soluble polymer matrix for the Stille cross-coupling reaction. AB - The optimization and efficient parallel synthesis and purification of a library of biaryl, heterobiaryl, and styryl derivatives, via the first reported poly(ethylene glycol)-supported palladium-catalyzed Stille procedure, are described. Preliminary investigations into the reaction between monomethoxy poly(ethylene glycol)5000-supported iodide 1a with tributylphenyltin 2 revealed that the optimal "liquid-phase" conditions employ PdCl2(PPh3)2 (0.1 equiv) catalysis with LiCl (10 equiv) in DMF at 80 degrees C for either 48 h (at 20 mM concentration of 1a) or 24 h (at 10 mM concentration of 1a). The soluble polymer supported reaction is superior to its solution-phase counterpart because the tributyltin side products and excess reagents are easily separated from the product intermediate 3a by precipitation of 3a into diethyl ether followed by recovery of the polymer by filtration in > 99%. In addition, the homocoupled byproduct 6 is also removed during this precipitation step. Under these conditions the transesterified biaryl adduct 4a can be isolated in 97-98% yield. The scope of this reaction was probed in a parallel format with the PEG-supported electrophiles 1a-b and a range of tributyl stannanes 2 and 7-13 under the optimized conditions vide supra. Subsequent cleavage of the polymer-supported adducts, by transesterification, and short column chromatography yielded a library of substituted methyl benzoates 4a-b and 14a-b to 20a-b in high yield (69 99%) and purity (> 95%). PMID- 10748732 TI - Matrix assisted synthetic transformations: a mosaic of diverse contributions. I. The pattern emerges. PMID- 10748733 TI - Selection of an optimized adsorbent for preparative chromatographic enantioseparation by microscale screening of a second-generation chiral stationary phase library. PMID- 10748734 TI - Solid phase organic synthesis of piperazinone containing enkephalin mimetics: a readily derivatized, traceless scaffold. AB - The solid phase synthesis of a series of piperazinone-derived Leu-enkephalin analogues is presented. The initial step in the synthesis involved the N alkylation of Wang resin bound N-(4-tert-butyloxy-phenethyl)-glycine with D or L Boc-serine-beta-lactone (the Vederas lactone). The resulting carboxylic acid was then coupled to a variety of monosubstituted benzylamine derivatives using benzotriazol-1-yloxy-tris(dimethyl-amino)phosphonium hexafluorophosphate (the BOP reagent) to yield a series of resin bound tertiary amides. Treatment with 5% H2O in TFA resulted in the facile cleavage, deprotection, and cyclization of this linear precursor to yield a series of piperazinones (compounds 1-8). PMID- 10748735 TI - A combinatorial approach toward the discovery of non-peptide, subtype-selective somatostatin receptor ligands. AB - The tetradecapeptide somatostatin is widely distributed throughout the body and is thought to be involved with a variety of regulatory functions. Recently, five human somatostatin receptors (hSSTR1-5) have been cloned and characterized. Several selective peptidal agonists of the hSSTR receptors are known, and we sought to apply this information to the design of novel non-peptide small molecule ligands for each receptor. Initial computational methods identified a 200 nM murine SSTR2 active compound via a database search of our sample collection. A combinatorial library was designed around the structural class of the compound with the goal of rapidly developing this initial lead into the desired subtype-selective small molecules in order to characterize the pharmacology of each of the receptor subtypes. The library was synthesized using the resin-archive, iterative deconvolution format. The total number of unique compounds in the library was expected to be 131,670, present in 79 mixtures of 1330 or 2660 compounds per mixture. Through sequences of screening and mixture deconvolution, the components of selective and highly active (Ki = 50 pM to 200 nM) non-peptide small molecule ligands for somatostatin subtypes 1, 2, 4, and 5 were identified. In addition to discovering compounds with the desired activity and selectivity, useful structure/activity information was generated which can be used in the design of new compounds and second-generation combinatorial libraries. PMID- 10748736 TI - Comprehensive survey of combinatorial library synthesis: 1998. PMID- 10748737 TI - Combinatorial synthesis and modification of functional porphyrin libraries: identification of new, amphipathic motifs for biomolecule binding. PMID- 10748738 TI - Comparison of resin and solution screening methodologies in combinatorial chemistry and the identification of a 100 nM inhibitor of trypanothione reductase. AB - Two identical polyamine peptide conjugate libraries were screened against the parasitic enzyme trypanothione reductase. One of these libraries was in a solution format, while the other was resin-based and was used in two resin-based screens (a diminution assay and a direct bead screening). Potent inhibitors (100 nM) of trypanothione reductase were identified both in the solution screen and in the resin-based screens when using the PEGA resin of Meldal. Resin screening of both types failed to work with TentaGel resin. Importantly there was excellent agreement between the solution and resin-based assays, suggesting both methods are reliable for the screening of combinatorial libraries. PMID- 10748739 TI - [Use of the pharmacophore concept in drug design]. AB - The application of the pharmacophore concept to design new drugs is discussed. The focus is on the application of computer-assisted drug design methods (CADD) in the discovery of new leads. PMID- 10748740 TI - [Chalcones and their heterocyclic analogs as potential antifungal chemotherapeutic agents]. AB - Chalcones and their heterocyclic analogues show various biological effects, e.g. anti-inflammatory, antitumour, antibacterial, antituberculous, antiviral, antiprotozoal, gastroprotective, and others. The present review discusses in greater detail the fungistatic and fungicide properties of these compounds and presents also their chemical structures. The mechanism of antifungal effects of chalcones and their analogues has not been investigated in greater detail. Due to the presence of a reactive ketovinyl moiety in the molecule the compounds of this type are able to react with the thiol groups of enzymes. It cannot be excluded that chalcones interfere with the normal function of the membranes of fungi and moulds. Further investigation of chemical, physical, and biological properties of chalcones and their analogues could lead to the elucidation of the mechanism of their action and finding of effective fungicidal and fungistatic agents in this group of organic substances. PMID- 10748741 TI - [Production of anthracene derivatives in elicited tissue cultures of Rheum palmatum L]. AB - With a few exceptions, the characteristic problem of cultivation of plant explants in in vitro cultures is a low production of secondary metabolites by these cultures. One of the methods, which can achieve an increase in the production of natural substances in in vitro cultures, is elicitation of cell cultures. The effect of the biotic elicitor Pseudomonas aeruginosa in the form of a homogenate and an aqueous suspension of dead cells on the production of anthracene derivatives by the tissue culture of Rheum palmatum L. of different age and origin was examined. The culture was cultivated on a Murashige-Skoog medium with an addition of 10 mg.l-1 of alpha-naphthylacetic acid. The maximal content of anthracene derivatives found by photometric determination according to PhBs 4 was demonstrated in an eight years old culture (1.027%) after six hour elicitation with an aqueous suspension of dead cells of Pseudomonas aeruginosa (1.7 mg of dry matter/1 ml of solution). The culture newly derived from the root of the intact plant Rheum palmatum L. responded to elicitation more sensitively than the culture derived from the seed; nevertheless the content of anthracene derivatives was lower than in the eight years old culture. PMID- 10748742 TI - [The effect of elicitors from Pseudomonas aeruginosa on the production of flavonoids in cultures of Ononis arvensis L]. AB - The effect of the elicitor, killed cells of Pseudomonas aeruginosa, on the production of flavonoids by a callus and a suspension culture of Ononis arvensis L. was examined. The tested elicitor influenced the flavonoid production both in the callus and suspension culture. An increased content of flavonoids in the callus culture was observed with the use of the elicitor in concentrations I and II (5.81 x 10(-2) and 5.81 x 10(-4) g/l). The maximal production took place after a seven-day elicitation with concentration II. This production was higher by 83% as compared with the control. In suspension cultures an increased production was observed in concentrations I and III (5.81 x 10(-2) and 5.81 x 10(6) g/l). The maximal content of flavonoids was measured after 48-hour elicitation with concentration I. In comparison with the control the increase in the flavonoid content was higher by up to 125%. PMID- 10748743 TI - [Determination of selected secondary metabolites and extracts of Philadelphus coronarius L]. AB - The paper deals with the determination of selected secondary metabolites in the branches and leaves of Philadelphus coronarius L. Colorimetric methods were employed to determine the content of flavonoids (0.63% and 0.01%, respectively) and phenolic substances (3.25% and 0.70%, respectively) in leaves or branches. Also the content of extractive substances was determined in ethanolic, aqueous ethanolic (85% and 60% ethanol), and aqueous extracts. This yielded 9.56%, 22.46%, 24.93%, and 33.15% of extractive substances in leaves and 2.20%, 6.04%, 8.84%, and 10.39% of extractive substances in branches. PMID- 10748744 TI - [The effect of biotic elicitors on the production of flavonoids in suspension cultures of Bellis perennis L]. AB - The effect of two biotic elicitors (homogenates of Pseudomonas aeruginosa and Candida utilis) in a suspension culture of Bellis perennis L. cultivated in the dark and day light period were examined. The growth of the culture was not negatively affected by the action of elicitors. Increased production of flavonoids in the culture depended on the sort, concentration and space of time of action of the elicitor, and on the manner of cultivation from the viewpoint of the light regimen. The best results were achieved with the use of the homogenate of Candida utilis after 24 hours in cultivation under the day light period and after 48 hours in cultivation in the dark. PMID- 10748745 TI - [In vitro effect of quinolones on the hydrophobicity of Acinetobacter baumannii]. AB - Effects of subinhibitory concentrations (sub-MICs) (1/4, 1/8, 1/16 or 1/32 of the MICs) of four quinolone antibiotics on surface hydrophobicity of two Acinetobacter baumannii strains (R1 and R2) were tested. Hydrophobicity was evaluated by adherence of bacteria to xylene and their aggregation in ammonium sulphate solutions. Norfloxacin in concentrations of 1/4 or 1/8 of the MIC decreased hydrophobicity of R1 strain and of R2 strain in concentration of 1/16 of the MIC. Ciprofloxacin was efficient for both strains mainly in concentration of 1/4 of their MICs. Enoxacin (1/8 MIC) more effectively reduced hydrophobic properties only in R2 strain. The other concentrations of the above-mentioned antibiotics as well as all tested concentrations of pefloxacin practically did not affect bacterial surface hydrophobicity. PMID- 10748746 TI - [Studies of local anesthetics. CL. Models of the dependence of local anesthetic effectiveness of basic esters of alkoxy-substituted phenylcarbamic acids on capacity factor k']. AB - On the basis of proposed six models of the dependence of local anaesthetic effectiveness of series of alkoxysubstituted derivatives of phenylcarbamic acid, this activity was studies in dependence on the capacity factor k' as the parameter of lipophilicity. The most suitable models proved to be y = (Cx + Ci).k' for surface anaesthesia, and y = Cx + Ci log k' for infiltration anaesthesia, y being the value of the decadic logarithm of surface or infiltration local anaesthetic activity, Cx the coefficient characterizing the nitrogenous base, Ci the coefficient characterizing the individual alkoxygroups, and k' the capacity factor from HPLC. PMID- 10748747 TI - [Substituted benzo[b]thiophenes with anti-leukotriene activity]. AB - The cyclizing reaction of cinnamic acid (Ia) with thionyl chloride was optimized and a series of 3-chloro-6-subst.benzo[b]thiophene-2-carbonyl chlorides Va-Vn was prepared. Chloride Va was transformed into a series of N-aryl-3-subst. (Cl, OCH3, OH) benzo[b]thiophene-2-carboxamides VII, IX, X. The drugs were subjected to an evaluation of selected antileucotriene activities in vitro and of the anti inflammatory effect in vivo. In agreement with the assumed mechanism, inhibition of the ear inflammation is conditioned by the antileucotriene activity, whereas inhibition of the carrageen oedema is not due to this mechanism alone. PMID- 10748749 TI - [Czech pharmaceutical education during the German Occupation from 1939 to 1945]. AB - When Czech universities were closed down in 1939, the already prepared reform of pharmaceutical graduate studies could not be implemented. The education of pharmacists, assistants, and aspirants was taken over by the Central Union of Pharmacists through its departments, in particular the Czech Pharmaceutical Academy and the Czech Society of Pharmacists. Training of pharmaceutical aspirants took place at the schools for aspirants in Prague and Brno. The experience obtained at these pharmaceutical courses and the new knowledge concerning drugs and pharmaceutical preparations provided by industry and research laboratories contributed to the improvement of the reform of pharmaceutical graduate studies in 1948. PMID- 10748748 TI - [Dissolution and signs of supersaturation of nimodipine in aqueous solutions of the cyclodextrins, alpha-CD, HP-alpha-CD, M-beta-CD and HP-gamma-CD]. AB - The dissolution curves of the substance of the calcium antagonist nimodipine in aqueous solutions of four cyclodextrins were determined at ambient temperature in the course of 14 days. The used cyclodextrins were alpha-cyclodextrin (alpha-CD), hydroxypropyl-alpha-cyclodextrin (HP-alpha-CD), methyl-beta-cyclodextrin (M-beta CD, random-methylated), and hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD) and their respective concentrations were always 0.05 mol/l. According to the measured dissolution curves, M-beta-CD in aqueous medium was a highly efficient solubiliser, capable to dissolve otherwise sparingly soluble nimodipine into a time-stable aqueous solution, with the saturated concentration of nimodipine 5.15 x 10(-4) mol/l (21.5 mg/100 ml) under the given conditions. M-beta-CD thus appeared to be a more efficient solubiliser than the previously studied HP-beta CD. The solubilising power of HP-alpha-CD and HP-gamma-CD was much lower and alpha-CD practically did not improve long-term solubility of nimodipine in water. However, in the presence of alpha-CD, HP-alpha-CD, and HP-gamma-CD, respectively, repeated shortterm episodes of formation of supersaturated solutions of nimodipine were observed on the dissolution curves, characterised by peaks of nimodipine concentration. Similar supersaturation episodes were previously observed in the presence of HP-beta-CD. Since the supersaturation caused by cyclodextrins reportedly substantially improved the biological availability of some drugs, the above-mentioned cyclodextrins, and especially natural alpha-CD, could be useful for the enhancement of the low availability of nimodipine from solid oral drug preparations. PMID- 10748750 TI - [Hysteroscopic resection of submucosal myomas in abnormal uterine bleeding: results of a 4-year prospective study]. AB - OBJECTIVE: The evaluation of the effect of transcervical resection of submucous myoma/s in patients with abnormal uterine bleeding. DESIGN: Prospective clinical study. SETTING: Department of Obstetrics and Gynaecology, 1st Medical Faculty, Charles University and General Faculty Hospital in Prague, Czech Republic. METHODS: 45 patients with resected submucous myoma/s within the period 1995-1998 were selected. Patients in whom resection of myoma was combined with endometrial ablation were excluded. The average age of the cohort was 43 years (29-53). In 37 patients (82%) therapeutical curettage was performed for severe bleeding in the past (3 procedures on average, interval 1-7). In 16 patients (36%) transfusion was administered in the treatment of anaemia secondary to abnormal uterine bleeding in the past. Hysteroscopy, ultrasound scan or both techniques were used to diagnose and classify myomas. In 39 patients 1 myoma was resected, in 6 patients 2 myomas. In 12 (27%) patients myoma of 0., in 29 (64%) of I. and in 4 (9%) of II. has been diagnosed according to the ESH classification. In case of several myomas, the classification was based on the one of the highest degree. Surgery was performed in the early proliferative phase of the menstrual cycle. Purisol (sorbitol and mannitol) was used as a distension medium. Myomas were resected using loop high frequency electroendoresection technique. In 3 (7%) patients myomas of ESH II. were resected in more steps--in 2 cases (5%) in 2 steps and in 1 case (2%) in 3 steps. In patients with myomas classified as a 0. and I., only the character of menstrual cycle has been monitored after surgery. Ultrasound investigation was performed in patients with myomas of II. 3 months after surgery. RESULTS: The bleeding was controlled in terms of eumenorrhoea or hypomenorrhoea in all 45 patients (100%). Ultrasound investigation showed no residual myoma in any of 4 patients after resection of II. myoma. One case fluid overload syndrome of minor degree was documented as the only complication. CONCLUSION: Hysteroscopic high frequency endorsection is a safe and effective method and method of choice in the treatment of submucous myomas in patients with abnormal uterine bleeding. The high success rate in our cohort is certainly influenced by the number of patients included and by the length of follow-up. PMID- 10748751 TI - [Laparoscopic hysterectomy using ultrasonic surgical technics: prospective study]. AB - OBJECTIVE: The use of the harmonic scalpel and Laparosonic Coagulating Shears (LCS) for laparoscopic hysterectomy (LH) was evaluated in thirty six women with other gynaecological conditions. DESIGN: Prospective clinical study. SETTING: Department Gynaecology and Obstetrics, Endoscopic Training Centre, Baby Friendly Hospital Kladno, Kladno, Czech Republic. METHODS: Clinical data and cases of hysterectomy performed via ultrasonic operative laparoscopy were summarized and assessed. The primary hemostatic treatment was harmonic scalpel and LCS. Patients were assessed to efficacy, safety, utility and applicability of laparosonic operative technique. RESULTS: From all laparoscopic procedure were 91.6% successfully completed. There were three cases which were finished electrosurgically. The mean order of total operating time was 86.03 min and the mean order of blood loss was 170 ml (range 50-400 ml). There were no major complications, only one case of postoperative bleeding from vaginal cuff. CONCLUSION: Clinical prospective study demonstrates that ultrasonic activated technology in laparoscopic hysterectomy is easy to use, cost effective, and affords the surgeon a greater margin of safety. The harmonic scalpel produces surgical incision with concomitant hemostasis. This is only a preliminary report, and further randomised study is needed. PMID- 10748752 TI - [Chlamydia trachomatis in men with impaired fertility]. AB - OBJECTIVE: The objective of the prospective study was to assess whether there exists a relationship between the results of positive laboratory tests for the presence of Chlamydia infection (assessment of the presence of Chlamydia trachomatis in the ejaculate by the DNA amplification method, IgA and IgG antichlamydia antibodies in the ejaculate and serum), an abnormal spermiogram and total number of sexual partners in men from infertile couples examined at the Gynaecological and Obstetric Clinic of the First Medical Faculty, Charles University Prague. DESIGN: Prospective clinical study. SETTING: Department of Gynaec.-Obstet., the 1st Medical Faculty of the Charles University, Prague, Czech Republic--Institut of Clinical Biochemy, the 1st Medical Faculty of Charles University, Prague, Czech Republic. METHOD: 101 men were examined. In all the spermiogram was evaluated according to WHO criteria. Specific antichlamydial antibodies IgA and IgG is serum and the ejaculate were assessed by the ELISA method. Evidence of the DNA portion of Chlamydia trachomatis was provided by the MEIA method. Amplification of DNA by LCR was made on a Thermocycler. By means of an aimed questionnaire the number of all sexual partners was assessed. The men were divided into three groups with 1-5, 6-9 and with 10 or more partners. Patients where Chlamydia infection was detected were treated with Ig Azitromycin and their spermiogram was checked 6-8 weeks after treatment. The results of the examination were tested by the chi square test. RESULTS: An abnormal spermiogram was found in 44 (44%) of the examined subjects. With the increasing number of sexual partners the number of men with pathospermia increased. In none LCR for the presence of Chlamydia trachomatis was positive in the ejaculate. In 15 examined patients IgA antichlamydia antibodies were found in the ejaculate. At the same time in all an abnormal spermiogram was found and 11 (73%) of them had six or more sexual partners. After treatment in 8 (62%) of 13 (two did not attend the check-up) marked improvement of the spermiogram occurred. CONCLUSION: IgA antichlamydial antibodies in the ejaculate proved in 62% of the examined men with an abnormal spermiogram a useful indicator of infection in the genital tract. With the increasing number of sexual partners the number of pathospermias increased. PMID- 10748753 TI - [Guidelines for malignant gynecologic tumors. II. Standard--primary comprehensive treatment in operable stages of malignant tumors of the uterine cervix. Gynecologic Oncology Section of the Czech Gynecology and Obstetrical Society]. PMID- 10748754 TI - [Assisted reproduction--present status and perspectives]. AB - OBJECTIVE: To review the current status and new trends in assisted reproduction. DESIGN: The literature on intrauterine insemination, IVF, ICSI, MESA/TESE, preimplantation genetic diagnosis, cryopreservation, in vitro maturation, cloning and stem cells. SETTING: Assisted Reproduction Centre, Ist Medical Faculty of Charles University Prague and General Hospital Prague, Apolinarska 18, 128,00, Prague 2, Czech Republic. RESULTS: IUI, IVF, ICSI also in combination with MESA/TESE are widely used relatively high effective procedures. According to Czech Republic Assisted Reproduction Registry, the pregnancy rate per transfer using IVF, ICSI is 25%, 30% respectively. Cryopreservation of sperm and embryos is a basic part of assisted reproduction, the result with oocytes freezing are still not satisfactory. Preimplantation genetic diagnosis is performed in few countries only due to technical barriers and ethical restrictions. Other methods like cloning, nuclear transfer, embryotherapy or gene manipulation using embryonic stem cells are still only subjects of research. CONCLUSION: During the last ten years, assisted reproduction showed rapid development in a number of treated couples and the spectrum of used methods changed radically. PMID- 10748755 TI - [Fixation of the prolapse vagina to the sacrotuberal ligament]. AB - OBJECTIVE: To present personal experience in the fixation of the vaginal stump to the sacrotuberal ligament. METHOD: Description of the surgery including modified surgical instruments. RESULTS: 32 surgical procedures were performed during a 4 year period. Two relapses were recorded. DISCUSSION: Various surgical procedures including abdominal approach to the fixation are mentioned, complications and prevention of relapses are discussed. CONCLUSION: The method is appropriate for sexually active patients. It preserves the length, axis and volume of the vagina. PMID- 10748756 TI - [Present views on treatment of genital prolapse]. AB - The uterine and vaginal descent create multidisciplinary issue. Its consequences interfere in gynaecology, urology, geriatrics, general medicine and psychiatry. Collateral symptomatology, pathology of the anatomy, diagnostics, both conservative therapy and surgery are discussed. The author mentions the abdominal and vaginal approach, being most experienced with the latter. Prevention is seen shortening of the labour stress, reduction of chronic elevation of the intraabdominal pressure and use of hormonal replacement therapy in the menopause. PMID- 10748757 TI - [Response by the authors to the article by K. Klimova and Z. Maly: Indications and risks of invasive methods of prenatal diagnosis--who indicates and who bears the risk. PMID- 10748758 TI - [In Process Citation] PMID- 10748759 TI - [Regulation of diversity of muscle fiber types]. AB - The muscle phenotype is the result of interaction between intrinsic genetic factors and extrinsic factors including innervation and level of thyroid hormones. Although these factors were intensely studied, the direct comparison of the results is difficult because various models, muscles and animal species have been used. We have therefore introduced a model of heterochronous isotransplantation in order to study in a single model the influence of muscle cell lineage, innervation and level of thyroid hormones on the expression of muscle phenotype of the regenerated muscle grafts. The aim of the present article is to compare the current knowledge of this problem. The expected experimental results will be primarily of the cognitional value, as they will provide a deeper insight into regulatory processes controlling expression of muscle genes. Controlled regulation of muscle differentiation may be, however, of clinical importance for muscle transplantations in man. PMID- 10748760 TI - [Methods of verification of aging theories]. AB - The methodology of verification of theories of aging belongs to the most crucial problems in theoretical and experimental gerontology. Extensive number of hypotheses of the nature of the basic mechanisms of aging exists on the field of theoretical gerontology. This situation claims the development of the effective methodology of verification. Traditional ways of the verification were indirect and insufficient. The progress in theoretical gerontology provides the possibility to verify the theories in more exact way. Survey of the contemporary state in the theories of aging and available methods of verification is presented in the paper. The tools of verification derived from survival curves and mortality curves are analyzed and discussed. Further the tools of verification derived from biomarkers of aging and batteries of biological age are presented. PMID- 10748761 TI - [Mechanisms of remodeling of pulmonary blood vessels in chronic hypoxia]. AB - Chronic lung hypoxia results in the hypoxic pulmonary hypertension, which is caused by the remodeling of peripheral pulmonary blood vessels. Vascular smooth muscle cells proliferate into the prealveolar arteries, the turnover and deposition of connective tissue proteins is increased. We observed an enhanced collagenolytic activity in the extracts from isolated peripheral lung arteries of hypoxic rats. SDS electrophoresis of collagenous proteins extracted from these vessels showed presence of the low molecular weight cleavages of collagen type I. We hypothesize that the activation of collagenolytic metalloproteinases is related to the release of reactive oxygen species, NO and products of their interaction (peroxynitrite). Collagen cleavages may stimulate mesenchymal proliferation in the vascular wall. PMID- 10748762 TI - [Neurohypophyseal hormones: the Prague contribution to their chemistry and pharmacology]. PMID- 10748763 TI - Level of left ventricular dysfunction in endomyocardial biopsy-proven myocarditis. AB - The level of myocardial functional impairment in the course of myocarditis still remains associated with controversial data. However, many investigators agree that there is a significant heart failure during myocarditis and a large number of studies suggest development of global chronic or acute heart failure. The objective of this study was to establish haemodynamic parameters as descriptors of the level of myocardial systolic and diastolic impairment. From 131 investigated patients, 95 were assigned to the study group and 36 to the control group. All patients underwent right and left heart catheterization. A group of patients underwent EMB (endomyocardial biopsy) of the right ventricle. According to the Dallas Criteria, patients were divided into three groups: active myocarditis with fibrosis, active myocarditis without fibrosis and borderline myocarditis. The following haemodynamic parameters were evaluated: left ventricle EF (ejection fraction), cardiac index and end diastolic pressure of the left ventricle. EF, which reflects left ventricular systolic impairment, showed a difference related to the control group, while there was no difference in cardiac index between the groups. Diastolic dysfunction (end diastolic pressure of the left ventricle) was noted in the group with active myocarditis, but not in the group with borderline myocarditis. PMID- 10748764 TI - [The Koebner phenomenon, a prognostic sign of PUVA therapy effectiveness in patients with psoriasis vulgaris--yes or no?]. AB - INTRODUCTION: Former investigations of Koebner phenomenon had demonstrated its higher incidence in patients with severe generalized and/or unstable forms of psoriasis which expressed increased resistance to various treatment modalities. The aim of this study was to establish the correlation between the presence of Koebner phenomenon and the PUVA therapy effects, total number of PUVA treatments, total dose of UVA radiation and the duration of remission after PUVA therapy discontinuation. MATERIAL AND METHODS: Sixty patients with severe clinical picture of psoriasis vulgaris, treated with PUVA therapy, were included in this research. According to the presence of Koebner phenomenon they were divided into two groups, 20 patients with positive and 40 patients with negative Koebner reaction, who were the control group at the same time. RESULTS AND DISCUSSION: 95% of patients treated with PUVA, were cleared of psoriatic changes in the Koebner positive, as well as in the Koebner negative group. There were also no differences between the Koebner positive and Koebner negative group in the mean number of PUVA treatments, mean total dose and the last dose of UVA radiation, which led up to the clinical remission of psoriasis. Our results of investigation have demonstrated increased relapse of psoriasis, during the first 6 months after cessation of PUVA therapy, in the Koebner positive group, with a high statistical significance (p < 0.001), comparing with Koebner negative group in the same period. Furthermore, the tendency of relapse of Koebner positive and Koebner negative psoriatic patients was higher in Koebner positive group even in the first 3 months after PUVA therapy. CONCLUSIONS: PUVA therapy effects, total number of PUVA treatments, total dose of UVA radiation didn't depend on presence of Koebner phenomenon. However, Koebner phenomenon was a mark of high relapsing tendency of psoriasis in the first 6 months after PUVA therapy cessation. PMID- 10748766 TI - [Congenital adrenal hyperplasia]. AB - INTRODUCTION: Congenital adrenal hyperplasia is generally considered to be a rare disease; however, the incidence of severe forms of CAH is probably greater than 1 in 10,000 people and the incidence of milder forms is probably 10 times higher. It is a group of autosomal recessive disorders of adrenal steroidogenesis caused by a genetic disorder in one of the steroidogenic enzymes. These disorders impair cortisol synthesis, resulting in stimulation of pituitary proopiomelanocortin and hypersecretion of adrenocorticotropin, which in turn causes adrenal growth. A genetic disorder in one of the steroidogenic enzymes interferes with normal steroid hormone synthesis. The sings and symptoms of the disease derive from a deficiency of the steroidal end product and from the effects of the accumulated steroidal precursors proximal to the disordered step. Because five enzymatic steps are involved in cortisol biosynthesis, there are five distinct CAH syndromes. CAH-SYNDROMES: 21-Hydroxylase deficiency is the most common variant of CAH, accounting for about 90% of all cases. The clinical syndrome is consequence of defective 21-hydroxylation of progesterone and 17-OH progesterone, with resulting deficient production of both cortisol and aldosterone. The disease occurs in a wide spectrum of clinical variants, including a severely affected form with defect in aldosterone biosynthesis ("salt-wasting" type), a form with apparently normal aldosterone biosynthesis ("simple virilizing" type) and a mild "nonclassic" form that may be asymptomatic or may be associated with symptoms of androgen excess developing during childhood or at puberty. In "classic" forms ("salt-wasting" and "simple virilizing") sings of androgen excess often are prominent. The key measurement for the diagnosis of 21-hydroxylase deficiency is basal plasma 17-OH progesterone. It will be elevated and will hyper-respond to ACTH stimulation. 11 beta-hydroxylase deficiency is the second most frequent cause of CAH. The abnormality is an inherited deficiency of 11 beta-hydroxylase enzyme of the adrenal cortex with consequences in impaired conversion of 11 deoxycortisol to cortisol and of deoxycorticosterone to corticosterone and aldosterone. About two thirds of patients with the severe, "classic" form have hypertension, often beginning within the first few years of life. Patients with mild ("late onset" or "nonclassic") form have normal or, at most, mildly elevated blood pressure. Signs of androgen excess are also often prominent. The diagnosis can be confirmed by demonstrating marked accumulation of deoxycorticosterone and 11-deoxycortisol in plasma and their tetrahydrometabolites in urine. 17 alpha hydroxylase/17, 20 lyase deficiency is the second of two hypertensive forms of CAH. Deficient 17 alpha-hydroxylation of pregnenolone and progesterone result in decreased secretion of cortisol and sex steroids, but increased secretion of sodium-retaining corticoids (corticosterone and deoxycorticosterone). Females present with normal external genitalia but failure to develop secondary sex characteristics and primary amenorrhea. Males frequently fail to develop masculine external genitalia fully (male pseudohermaphroditism). Both sexes have hypertension, hypokalemic alkalosis, and failure to progress into puberty. The diagnosis can be established by the demonstration of elevated plasma deoxycorticosterone and corticosterone 3 beta-hydroxysteroid dehydrogenase is rare cause of CAH. Patients with this biosynthetic defect have deficient production of adrenal glucocorticoids, mineralocorticoids, and testicular testosterone. However, they have excessive production of 5-adrenal androgens. In "classic" form, affected newborns present with symptoms and sings of adrenal insufficiency of varying degree and ambiguous genitalia in both sexes. The "late onset" form is a mild type of disorder. It has been described in women with hirsutism and menstrual abnormalities and may be quite common. (ABSTRACT TRUN PMID- 10748765 TI - [Levels of fetal fibronectin in the cervix and vagina during pregnancy]. AB - INTRODUCTION: Fibronectin is a glycoprotein produced by different types of cells. It can be divided into two main groups--soluble fibronectin, found in human plasma and other body fluids (amniotic fluid) and tissue fibronectin, found in basal membrane and connective tissue between endothelial cells (2). There are three subtypes--plasmatic, cellular and onco-fetal fibronectin, the most important during pregnancy, with different concentrations during the course of pregnancy. The aim of the study is to establish normal distribution of fetal fibronectin in cervicovaginal fluid during pregnancy in our population and afterwards establish the relation between concentrations in different stages of pregnancy and the pregnancy outcome. MATERIAL AND METHODS: This prospective, observational study was done at the Department of Obstetrics and Gynaecology, Clinical Centre Novi Sad, during the period June-October 1988. The presented results are preliminary ones. The patients were randomly chosen from the population that came for routine consultations as well as the ones hospitalized at the Department, at the High Risk Pregnancy Unit, for reasons other than threatening miscarriage and preterm delivery. One-hundred and thirty two pregnant women were examined, divided into three groups, according to gestational age. In group I were women between 7-20 weeks, in group II between 21 and 37 weeks and in group III 38 and more weeks. In every case general and obstetric data were collected, and cervico-vaginal fibronectin was taken from the posterior vaginal fornix, using a special kit (Specimen Collection Package, Adeza Biomedical). Fetal fibronectin concentration was tested using enzyme immunoassay (Adeza Biomedical) and positive was considered the concentration of and above 0.05 microgram/ml. RESULTS: The mean cervico-vaginal fibronectin concentration, uncorrected for the outcome of the pregnancy was as follows--in group I 0.045 (0 0.11) microgram/ml, group II 0.037 (0-1.22) microgram/ml, and in group III 0.226 (0.001-1.05) microgram/ml. The concentration trend was from weakly negative during the period 7-20 weeks, over highly negative (21-37 weeks) to very positive, after 38 weeks. The positive/negative relation in group I was 29.2%/70.8%, group II 11.7%/88.3% and group III 48.4%%/51.6%. After the correction of the results for miscarriages/preterm deliveries/failed induction in postterm pregnancies, the mean concentrations were somewhat different--group I 0.029 microgram/ml, II 0.019 microgram/ml and III 0.282 microgram/ml. The relations of positive and negative results were changed as well and in group I the relation was 27.3%/72.7%, group II 6.8%/93.2% and group III 71.4%/28.6%. DISCUSSION: After the correction for duration and outcome of the pregnancy, our results differed from the results in the literature. Thus in the 1st trimester fetal fibronectin was positive in cervico-vaginal fluid of 27.3% pregnant women, which is double the number usually found in the literature, between 21 and 37 weeks, when a positive result (> 0.05 microgram/ml) would be expected in only 3% of cases, it was positive in 6.8%, whereas 2.3 of the women delivered at term had a positive concentration. The discrepancy in group II (21-37 weeks) cannot be explained neither with a higher incidence of late miscarriages, nor preterm deliveries, for the incidence of such complications was only 3%, which is far less than usually found in the non-selected population (10%) (9). It is also possible that the discrepancy in our results and the results found in literature is based on a fact that our population was not preselected, which was the case in other studies' populations that did not include women with heavy, non-treated vaginal discharge, nor the ones that had sexual intercourse within 24 hours from the moment of fibronectin sampling. (ABSTRACT TRUNCATED) PMID- 10748767 TI - [Ultrasonography and computerized tomography in the evaluation of tumor invasion in renal adenocarcinoma]. AB - INTRODUCTION: Renal cell carcinoma, also known as renal adenocarcinoma is the most common primary renal malignancy. Protean and often nonspecific clinical manifestations of renal carcinoma have lead to its description as the "great imitator" in medicine. Because of the importance of early diagnosis and lack of specific clinical features, imaging has a vital role in its diagnosis. Since the only successful curative treatment of renal adenocarcinoma is surgery, preoperative evaluation of the extent of the disease may play an important role for determining surgical approach and tumor resectability. Over the past decade, computerized tomography has become the most widely used technique for staging renal adenocarcinoma, partly due to the very high overall accuracy of up to 90% that has been achieved. Ultrasonography is an effective, noninvasive and safe procedure that can be used as an initial method in patients suspected for renal adenocarcinoma. MATERIAL AND METHODS: We studied 58 patients (24 females and 34 males, mean age 55.7 years) between 1995-1997 with a histologically confirmed diagnosis of renal adenocarcinoma. A total of 58 patients underwent radical nephrectomy for renal adenocarcinoma. Preoperatively. All patients were examined both by ultrasound and computerized tomography for various tumors characteristics, most commonly evaluation of tumor extension into perirenal structures and major veins invasion. RESULTS AND DISCUSSION: The sensitivity of computerized tomography was higher than that of ultrasonography for the demonstration of tumor extension into the perirenal structures (73.3% vs. 20%). CT does not demonstrate microscopic transcapsular spread of tumor. Criteria for perinephric invasion required a soft-tissue mass at least 1 cm in diameter in the perinephric space. False positive diagnoses do occur. This can be attributed to perinephric oedema, fat necrosis or fibrosis from remote inflammation which may simulate perinephric soft tissue nodules. For surgical planning it is important to determine whether the tumor extends into renal veins and inferior vena cava. Computerized tomography is better to study invasion of inferior vena cava than ultrasonography (100% vs. 66.6%). With regard to renal veins invasion both imaging procedures made false negative results, mainly ultrasonography. In relatively high proportion of cases overlying bowel gas precludes adequate visualization of the renal veins by ultrasonography. CONCLUSION: Newer imaging modalities such as ultrasonography and computerized tomography have superseded conventional radiological methods in the diagnosis and evaluation of renal adenocarcinoma. Our results show that ultrasonography alone is unreliable in detecting tumor spread. Computerized tomography appears to be a more sensitive means of identifying tumor thrombus and tumor extension into perirenal structures. Perinephric tumor spread is the most difficult to detect but is of minor importance if radical nephrectomy is planned. PMID- 10748768 TI - [Viral hepatitis C]. AB - INTRODUCTION: Viral hepatitis type C became one of the most dangerous hepatic diseases, bearing high risk of eventually fatal complications. Now a great deal of public health funds has to be used for prevention and treatment of this serious disease. Only very detailed knowledge of the disease could help to a medical practitioner in his everyday confrontation with this serious problem. MATERIALS AND METHODS: Data, diagnostic, therapeutic and preventive suggestions given in this paper are result of a comprehensive review of relevant literature. RESULTS: The causative agent of hepatitis type C is an RNA virus with six different genotypes. It is easily transmitted from one host to the other only by transferring large amounts of body fluids (blood or plasma transfusion, or prolonged, repeated inoculations of small quantities of infected fluids intravenous drug abusers, recipients of clotting factors, accidental needle sticks). The quantification of the disease activity could be done by a numerical scoring system, originally issued by Knodell, which takes into account four categories: periportal necrosis, intralobular necrosis, portal inflammation and fibrosis. The incubation period of hepatitis C varies from 5 to 7 weeks. It starts like a relatively mild acute disease, but eventually it progresses to chronicity. About 10-20% of patients develops cirrhosis, and yet unknown percentage of patients develops hepatocellular carcinoma. On average, it takes about 30 years for chronic hepatitis C to progress to cirrhosis or cancer. DISCUSSION: Serologic testing for anti-HCV proves the existence of specific antibodies against hepatitis C virus. It becomes positive only after 5-6 weeks from clinical onset. Much more sensitive test is PCR, which proves the viral RNA in body fluids. PCR is positive as early as 2 weeks from the onset of hepatitis. Up to now, the only 100% certain way to prove existence of chronic hepatitis is liver biopsy. Interferon alpha is nowadays used for management of this serious disease. The accepted dose is 3,000,000 U three times weekly for 24 weeks. About 46% of treated patients will have both serological and histological improvement. Total liver collagen and iron staining in portal areas are significantly decreased after the treatment course, giving hope for postponing the onset of cirrhosis. However, half of the responders will experience relapse of the disease within 8 months from the end of treatment, and sustained biochemical and virological response could be seen in only 5% of patients. The sustained response rate was increased in some studies to 29% when iron reduction was undertaken along with interferon. CONCLUSION: Since there is no effective treatment for hepatitis C, much of the efforts should be directed to prevention. Since hepatitis C virus is transmitted only by parenteral route or close personal contact (sexual contact mostly), in the family environment general hygienic measures are considered sufficient. Hands should be washed properly, food, clothing, utensils, linen and excreta of the patient should be handled separately. During sexual intercourse, prophylactics should be used. The most important measure for prevention of posttransfusion hepatitis C is regular testing of all blood donors for anti-HCV antibodies. PMID- 10748769 TI - [Specifics of mastication with complete dentures]. AB - INTRODUCTION: Mastication is a process characterized by alternating phases of physiological inactivity, food intake, initial food crumbling, mouthful formation and deglutition. Although there is a so called human type of mastication, this process is individual for variety of reasons. Mastication is not an innate characteristic and it must be learned, but once it is learned, it gets a reflexive function, demanding maximal involvement of the highest nervous system centers. DISCUSSION: Specificities of mastication in complete denture wearers are caused by specificities connected to changes of the oral cavity due to teeth loss, technical features of complete dentures and quality of their manufacture. Loss of teeth causes loss of periodontal receptors consequently damaging the source of information which, as long as teeth are present, travels from the oral cavity towards the central nervous system and regulates the masticatory muscles activity. As one of the consequences of periodontal receptors' loss, interocclusal perception decreases, because the number of receptors within the masticatory mucosa is negligible. Another problem is that loss of teeth causes loss of the occlusal surface. Occlusal surface is considered to be the only reference coordinate of the zero intermaxillary separation with precise spatial representation within the someostatic field cortex. Permanent loss of antagonistic contact causes disappearance of the memory pattern of the occlusal surface. Therefore completely edentulous mouths are characterized by disintegration of the free interocclusal space. Masticatory forces' value directly depends on the: size of muscles creating the forces, their position in the mandibula, shape of the edentulous alveolar ridge, type of chewing, the relationship between the height of the branch, length of the mandibular body, gonial angle, mandibular angle in relation to the anterior cranial base and posterior facial height and the degree of intermaxillary separation. Bite forces in complete denture wearers are significantly decreased in relation to persons with natural teeth. There is a fundamental difference in the distribution of the functional energy in complete denture wearers and subjects with intact teeth. Persons with natural teeth tend to generate energy through bolus, whereas in complete denture wearers most of the chewing energy is distributed outside bolus, directly through the denture base on the supporting tissues. RESULTS: As the result of this specific dynamics stress of supporting tissues often occurs with consequential mucosal inflammation and rapid alveolar bone resorption. Reduction of masticatory efficacy in denture wearers may be caused by irregular activity of masticatory musculature or is the consequence of irregular flow of energy during mastication. In comparison with young examinees with healthy teeth, authors have registered that complete denture wearers need about seven times more masticatory movements to achieve equal number of sample parts. CONCLUSION: It has been documented by experience and also scientifically proved, that the satisfaction of complete denture wearers is not often proportional with objective functional denture values. Comparison of subjective parameters with objective test results reveals that the percentage of complete denture wearers who consider their dentures to be good, although they objectively are not such, is significantly higher than the percentage of those who complain about their dentures, although they are functionally proper. Our study comprised patients (average age of 69) who wore complete dentures longer than five years. PMID- 10748770 TI - [Helicobacter pylori infection and a decrease in the pH of gastric juice as a possible cause of bleeding in erosive gastritis]. AB - INTRODUCTION: Bleeding erosive gastritis and ulcer disease are the most common causes of bleeding from the upper parts of the alimentary tract. Over the period of the last 10 years there has been an increased number of patients where Helicobacter pylori is mentioned as the possible cause of infection. At the same time in this group of patients pH values were decreased. During our research we wanted to examine the influence of local pH value and Helicobacter pylori infection on the occurrence of bleeding erosive gastritis. MATERIAL AND METHODS: During this research we examined two groups of patients. The following parameters were examined within these two groups of patients: 1. presence of Helicobacter pylori infection; 2. histopathologic changes in mucus; 3. there were 57 patients with a bleeding form of gastritis of unknown etiology, whereas the second group consisted of 351 patients with non-bleeding form of gastritis. We examined the localization: 4. local pH. RESULTS: The presence of Helicobacter pylori infection is statistically significantly higher in the group of patients with bleeding gastritis, when compared to the non-bleeding group (72%:57%). Within the Helicobacter positive patients the most common change of mucose (over 90%) is active chronic gastritis. The presence of erosions and histopathological changes characteristic for Helicobacter pylori infection coincides with colonization of Helicobacter in the antrum. The pathogenetic action of bacteria in antrum decreases the local pH. Therefore the lowest value of local pH occurred in the group of bleeding Helicobacter pylori positive gastritis (1.72). DISCUSSION: Results of this research helped us in treatment of helicobacter pylori positive patients making a risk group for development of recurrent bleeding. We accomplished therapeutical effect in 80% of patients by using a three component eradication therapeutic protocol for helicobacter pylori. Within the group of patients who have been cured, regression of histopathological changes occurred and increase of local pH took place. In the group with persistent infection we have come across with high percentage of recurrent bleeding (37.5%). CONCLUSION: In our opinion helicobaceter pylori infection and decrease of local pH, which take place during the reaction of mucose on the presence of infection, are very aggressive factors which can cause bleeding erosions. PMID- 10748771 TI - The frequency, characteristics and outcome of infections in patients with acute nonlymphoblastic leukemias. AB - A retrospective study was conducted in 91 patients treated at the Clinic of Hematology in Novi Sad in the period January 1, 1994,-November 15, 1997. The frequency, types, characteristics and outcome of infections were examined. The causative microorganism was determined in 65% of 133 febrile episodes, in 55% Gram-negative bacteria, 39% Gram-positive bacteria and in 6% fungi. Gram-negative bacteria were causative microorganisms in 80% of pneumonia. 77% of skin infections and 93% of urinary infections. Gram-positive bacteria were causative microorganisms in 53% of sepsis, Gram-negative in 41% of sepsis and Candida in 6%. The significant resistance to antibiotics was present in 47% of Gram-negative sepsis (causative microorganisms were Pseudomonas aeruginosa and Acinetobacter species) and in 18% of Gram-positive sepsis (susceptibility to imipenem only in Gram-negative sepsis and susceptibility to vankomycin in Gram-positive sepsis). Infections were the cause of death in 62.8% of patients. PMID- 10748772 TI - [Preemptive analgesia in cholecystectomy using pethidine]. AB - INTRODUCTION: Preemptive analgesia given before noxious stimulation prevents or reduces subsequent pain. Pain associated with central sensitization is called pathological pain. Preemptive analgesia could be defined as analgesia that prevents the development of pathological pain. The clinical significance of central sensitization lies in prediction that preemptive analgesia may prevent the establishment of central sensitization and reduce pain experienced following peripheral injury. Various pharmacological agents and methods have a potential in prevention of acute postoperative pain by blocking the somatosensory system and abolishing hypersensitivity. But the role of preemptive analgesia in postoperative pain is still controversial. The goal of the present study was to examine whether pethidine administration before skin incision is more effective in reducing postoperative pain than the same dose of pethidine given intraoperatively. MATERIAL AND METHODS: Thirty patients (ASA 1 to 2), aged from 40 to 65 years, admitted for elective laparotomic cholecystectomy entered the study. Group 1 (n = 15) received 1 mg/kg of pethidine i.v. 5 minutes before induction of anaesthesia (before skin incision) and 0.9% NaCl of equal volume intraoperatively (after peritoneal opening). Group 2 (n = 15) received pethidine and 0.9% NaCl in a reverse manner. Premedication was omitted. No other analgesics were administered at induction and intraoperatively. Anaesthesia was induced with midazolam, thiopentone and succinylcholine for tracheal intubation. Pancuronium was administered for muscle relaxation and halothane with O2/N2O for maintenance of anaesthesia. The duration of surgery (time from skin incision to skin closure) and time from skin closure to the first analgesic request were measured and recorded. In the ward, patients were given metamizol (2500 mg) i.m. at request during the first 12 h. If the regimen was not sufficient, piritramide (2 mg) i.m. was given at request. The intensity of pain was estimated before the first analgesic request and 4, 8, 12 and 24 h thereafter. Pain scores were recorded using VAS (0 = no pain; 10 = worst pain). Data are presented as mean values with their standard deviations and as the ranges of each parameter. The differences in data between two groups were evaluated with Student's t-test. P > 0.05 was considered statistically significant. RESULTS: Demographic data, duration of surgery and time from skin closure to the first analgesic request are presented in Table 1. No significant difference was found between the two groups. The postoperative analgesic requirements in two groups were similar and piritramide requirement was omitted. VAS scores at each time (Table 2) did not differ between the two groups. DISCUSSION: The findings of many clinical investigations remain controversial. Some clinical studies comparing the same analgesic intervention before and after the painful stimulus have shown a benefit of preemptive analgesia. The results of our study did not show a significant difference in pain scores neither in analgesic requirements of patients who received systemic pethidine before the painful stimulus compared with the patients treated with the opioid intraoperatively. However, epidural opioid administration may be more effective (17,18). It is important to say that inhalational anaesthetics, including N2O and some i.v. anaesthetic agents may have preemptive effects themselves, significantly reducing spinal sensitization (19,20). In clinical studies when the preemptive effect of analgesics is under investigation, inhalational and i.v. anaesthetics which are administered to induce and maintain anaesthesia are given before surgery. Therefore development of central sensitization may be attenuated or prevented by the anaesthetics overlapping the preemptive effect of analgesics. CONCLUSION: The results of this study did not demonstrate a preemptive effect of pethidine. (ABSTRACT TRUNCATED) PMID- 10748774 TI - [Physical therapy of subjective symptoms of the cervical syndrome]. AB - INTRODUCTION: Cervical syndrome is represented by a group of symptoms and signs mainly localized in the neck region, but also can be present in other regions such as head, humeroscapular, precordium, scapular and interscapular, upper limbs and sometimes lower limbs. Despite the fact that cervical syndrome is primarily a painful syndrome, clinical manifestations show variety of painless symptoms caused by functional disorders of somatic nerves, vegetative nerves, circulation or psychoneurotic disorders. CERVICAL SYNDROME THERAPY: Conservative management includes medicamentous therapy or physical procedures and application of neck collar, neck traction and ergonomic education. Surgical treatment is reserved for cases with massive neurological deficiency and cases with persistent pain which does not respond to conservative therapy. Final effects of physical, medicamentous and surgical therapy in treating cervical syndrome are identical after a long-term monitoring of patients. However, immediate results of therapy favor physical procedures rather than medicamentous therapy which is important regarding the quality of life of these patients. Application of various physical procedures in the therapy of cervical syndrome includes application of low frequency current electrotherapy followed by ultrasound and thermotherapy representing a significant part of physical therapy. Therapy with low frequency pulsating electromagnetic field and laser photobiostimulation have also been proved successful. These procedures eliminate circulatory and neurological disturbances that cause the appearance of signs in cervical syndrome. CONCLUSION: The most common painful symptoms can be successfully treated with various physical procedures. Laser photobiostimulation and application of pulsating electro-magnetic field are very effective in treatment of vascular and neurological cervical syndrome symptoms, but for the treatment of psychoneurotic symptoms classical physical procedures such as diadinamic currents and ultrasound are more effective. PMID- 10748773 TI - [Evaluation of the usefulness of suction drainage in the treatment of nonspecific bone infections monitored with C-reactive protein]. AB - INTRODUCTION: Irrigation suction drainage (ISD) is an additional method in treatment of infected bones. Two liters of saline solution daily with an antibiotic is usually recommended for ISD, although some authors suggest that more fluid should be used without antibiotics, stressing the importance of the mechanical effect. Although the method is nowadays accepted as a routine procedure, the reported results of ISD are based on treatment of a limited number of patients and the only criteria for assessment have been clinical and bacteriologic findings. The aim of this prospective study is to show the importance of the mechanical effect of an increased fluid run and the optimal time necessary for cleaning of the osteomyelitic cavity by follow-up the C reactive protein (CRP) levels as an indicator of an acute inflammation of the affected bone. METHODS: The prospective study was an open, centralized and randomized clinical investigation including over 105 patients with bone infection treated in the period 1986-1996. Two parameters with possible influence on the CRP levels were determined during investigation: 1. patients were divided in three groups depending on the duration of the irrigation suction drainage (3-6 days, 7-10 days and over 10 days) and 2. quantity of the liquid used for the irrigation suction drainage (two groups: 2000 ml per day and 6000 ml per day). Groups were similar in regard to age, sex, bacteriologic agents, duration of infection, localization and previous operations. C-reactive protein (CRP) is a nonspecific, but extremely sensitive indicator of an acute inflammatory disease. The erythrocyte sedimentation rate is not always a reliable indicator of infection. CRP as a protein of an acute phase appears and disappears quickly. Decreased values are a sign of a healing process used in this study for the estimation of the efficacy of the ISD. The CRP level was controlled semiquantitatively before operation and every third day over three weeks. If the values were below 6 mg/l, the results were classified as negative. RESULTS: There were not significant statistical differences in different groups as far as a preoperative average CRP values were concerned neither in the quantity of employed liquid nor in the duration of the ISD and interaction between these two groups. It was the same on the third postoperative day. The differences appear in the significantly lower average values of the CRP in patients of the second group (6000 ml/day) where the ISD lasted for seven or more days. During further follow up, (6th to 21st day) one can observe a significant statistical difference in average CRP values between the first (2000 ml/day) and second group (6000 ml/day) but there is not difference when the duration and sort of employed liquid is concerned. There is not significant difference in average statistical values of the CRP in blood for interaction of fluid quantity and time of ISD as well. DISCUSSION: The main disadvantage in the function of ISD is nonuniform rinsing of the osteomyelitic cavity. Prolonged ISD has no influence on the clinical result as well as on the average values of the CRP. Starting from the third postoperative day results indicate that the quantity of employed liquid has an important influence on the average level of the CRP, while the duration of ISD is not important. Prolonged ISD can not compensate for an inappropriate surgical intervention because it is only an additional method in the treatment of an osteomyelitic process. CONCLUSION: By observing changes in CRP levels during treatment one can conclude that the efficacy of ISD is based mostly on mechanical rinsing and the removal of debris. This points to the importance of the quantity of fluid employed in irrigation suction drainage. Application of 6000 ml liquid per day without antibiotics during 5-7 days gives opportunity of better rinsing increasing the effect of ISD and reducing the incidence of complications. PMID- 10748775 TI - [A case report of pyridoxine-responsive homocystinuria]. AB - INTRODUCTION: Homocystinuria is a rare, congenital, autosomal-recessive, metabolic disease biochemically characterized by homocysteinemia and homocystinuria and by multi-system clinical disorders. It is a biochemical abnormality of methionine metabolism caused either by transulfuration pathway disorders or by disorders of homocysteine remethilation into methionine and as such it can be a result of numerous specific and different genetic lesions. CASE REPORT: Homocystinuria is most commonly caused by deficiency of cystationine beta synthetase enzyme which catalyses the synthesis of cystathionine from homocysteine and serine in the methinione pathway. This results in accumulation of homocysteine and methionine in plasma and leads to excretion of excessive urinary homocysteine. Depending on specific property of the mutant enzyme molecule some patients respond to very high doses of pyridoxine with decreases of methionine and homocystine and some not. Pyridoxine responsiveness is based on the presence of small residual activity of cystathionine beta synthetase which is not present in nonresponsive patients. Homocystinuria due to cystathionine beta synthase (CBS) deficiency is characterized by numerous different clinical abnormalities, but changes in four organ systems are dominant (eye, skeletal, central nervous and vascular system). CASE DESCRIPTION: Six years ago a six year old boy was admitted to the hospital with vision problems. Ophthalmologic examination revealed a lens dislocation and because of many stigmates the child was sent to endocrinologist. The child had a marfanoid stature, with pectus carinatum and genu valgum, ataxic gait with motoric discoordination, muscle tone which ranged from hypotonia to hypertonia of extrapvramidal type and low intellectual abilities. A simple cyanide-nitroprusside test of patient's urine was positive suggesting homocystinuria. The diagnosis was established after plasma and urine amino acid analysis by HPLC. Concentration of homocystine and methionine were 52 mumol/l and 57 mumol/l in plasma and 249 mumol/du and 55 mumol/du in urine, respectively. After four months of treatment with pyridoxine there were not any significant changes in plasma homocysteine and methionine, but at the same time decrease in urine of these two amino acids were more than 2.5 times higher. This confirms that the patient has a pyridoxine-responsive type of homocystinuria and the dose was increased to 60 mg/day and 600 mg/day. This results in further decline of homocysteine and methionine in plasma and urine which persists up to now (for six years). PMID- 10748776 TI - [A quiet clinical course in an otogenic brain abscess]. AB - INTRODUCTION: The occurrence of otogenic intracranial complications has been significantly diminished by introduction of antibiotic therapy, improved diagnostic methods and surgical treatment of chronic otitis. Meningitis is still the most frequent otogenic intracranial complication, otogenic brain abscesses are following, while other complications occur rarely (1,2,3). Otogenic meningitis and brain abscesses are often associated, which makes the on time diagnosis of each complication harder. Mortality from otogenic brain abscesses is still relatively high, despite diagnostic and therapeutic achievements (4.5). CASE DESCRIPTION: The aim of this study is to present a patient with brain abscess, which arose quietly, as a consequence of chronic suppurative otitis and to point at possibilities of diagnostics and treatment of this complication. The diagnosis of brain abscess by clinical methods of examination is not always reliable. It is often accompanied by most severe form of meningitis, so that neurological examination, in most cases, reveals only signs of meningitis. In our patient, the abscess has been detected by computerized tomography, in its late stage, when it already had completely formed a capsule and had shown clinically manifesting focal signs. This case is interesting because of quiet and unperceived evolution of brain abscess. In the active phase of chronic suppurative otitis, the patient was receiving antibiotic therapy, which has most likely hidden the early symptoms of evolution of intracranial infections. The appearance of signs of focal intracranial infection (photophobia) required applying computerized tomography revealing brain abscess, which was clearly bounded, with a developed capsule, pointing at its late stage (about two months). These findings lead to a conclusion that an otologist should always keep in mind and check whether there is an intracranial infection in cases of active phase chronic otitis. PMID- 10748777 TI - [Folic acid in the prevention of neural tube defects]. AB - INTRODUCTION: The term neural tube defects (NTD) stands for anencephaly, iniencephaly, cephalocoele and spina-bifida. The cause of these anomalies is failure of brain spinal cord to properly develop, together with their protective shield of skull and spine, around the 4th gestational week. The prevalence of NTD in continental Europe is 11.2 10,000 live births. THE LEVEL OF FOLATES IN SERUM AND NTD: The level of folates in serum can influence the risk of a child affected with NTD. Studies on women with previous pregnancies with NTD showed that supplementary intake of folic acid, with or without other vitamins, preconceptual period throughout the first trimester has a preventive effect on its recurrence. Inadequate intake of folic acid is also connected with preterm delivery, intrauterine growth retardation and placental abruption and infarction. FOLATES IN NUTRITION: It is not folic acid, but folates, from the vitamin B group, that can naturally be found in food. There are several groups of folates that differ in the quantity by which they can be absorbed from food. Folates are temperature and storage sensitive and cooking can cause a significant fall of their concentration in food. FOLIC ACID SUPPLEMENTATION: The mean daily intake of folates by food is 0.218 mg whereas a reference nutritive intake for a woman of reproductive age is 0.2 mg per day. The currently recommended daily dose for prevention of first NTD occurrence is 0.4 mg, so it is clear that a certain amount of folic acid has to be supplemented preconceptionally and during the first trimester. It can be done in two ways, by telling all women to take it before conceiving, or to fortify food with sufficiently high doses of folic acid in order to achieve adequate serum levels. Neither of the ways is ideal, for not all women would take the supplement, and by aggressively fortifying the food, we create a potential hazard to those that do not need it and may have some problems with the excess of it. The best solution would be a widespread campaign about the need for folic acid and the risks of NTD. CONCLUSION: Recommendations of The Expert Advisory Group on Folic Acid in prevention of neural tube defects has several aspects (1) reducing the risk of the first NTD occurrence by preconceptional vitamin supplementation of folic acid in the dose of 0.4 mg day, which would go on until the end of the 12th week (2) reducing the risk of NTD recurrence in offspring of men and women with spina-bifida or with obstetric history affected with NTD by preconceptional vitamin supplementation of folic acid in the dose of 4 mg daily during the first 12 weeks and (3) organizing educational programmes for medical staff as well as the whole population in order to popularize vitamin supplementation. PMID- 10748778 TI - [An anniversary of the Maxillofacial Surgery Clinic in Novi Sad--a half-century of progress, education, learning and practice]. AB - Maxillofacial Surgery is an independent surgical specialty dealing with diagnostics and therapy of malignant and benign tumors, injuries, deformities and congenital anomalies of head and neck, as well as infections in the same region. It originates in central European states. Origin and development of Maxillofacial surgery in Serbia is connected to the name of dr Atanbas Pulja, in 1912 y. during the First Balkans War. Maxillofacial surgery in Novi Sad represents a scientific, educational and research base and center. The members of the staff include 7 specialists, out of which 2 are professors and 2 assistant professors. Maxillofacial Clinic is a part of the Institute of Surgery, which is a part of the Clinical Centre of Novi Sad. Head of the Clinic is Full Professor dr Jovan Ristic. PMID- 10748780 TI - [Homeopathy]. PMID- 10748779 TI - [In vitro modulation of adriamycin-induced cytotoxicity of fulerol C60(OH)24]. AB - INTRODUCTION: The aim of this paper was to examine the effect of fullerol C60(OH)24 on the growth of tumor cells in culture and its modulating effect on adriamycin (ADR) induced cytotoxicity in three different human breast carcinoma cell lines. MATERIAL AND METHODS: Growth inhibition was evaluated by tetrazolium colorimetric WST1 assay. ESR spin "trapping" method was used to assess OH-radical scavenger activity of fullerol during Fenton's reaction. RESULTS AND DISCUSSION: Fullerol C60(OH)24 inhibited human breast cancer cell lines in different manner depending on the cell type, dose and time of exposure to it. However, none of the fullerol concentrations induced 50% growth inhibition. In the model system of ADR induced cytotoxicity fullerol strongly suppressed ADR cytotoxicity for more than 50% in almost all concentrations, at each time point. ADR cytotoxicity was decreased by 80%, if the fullerol (1.9 micrograms/ml) was added to culture one hour before ADR. The same rate of suppression was achieved with 20 times less concentration in comparison to well defined natural antioxidant proanthocyanidol BPI. The hydroxyl radical scavenger activity of fullerol during Fenton's reaction, was assassed by the electron spin resonance (ESR) spin "trapping" method. It was shown that fullerol was a potent hydroxyl radical scavenger. RI of ESR signals of PBN-OH adduct in the presence of 0.5 g/ml of fullerol was decreased by 88%. CONCLUSION: The obtained results suggest that antiproliferative effect of fullerol and its protective effect on ADR cytotoxicity might be mediated through antioxidative and hydroxyl-radical scavenger activity of fullerol, but additional investigations are necessary for better understanding of these mechanisms. PMID- 10748781 TI - Albumin in meningococcal disease. PMID- 10748782 TI - Variant Creutzfeldt-Jakob disease: an update. PMID- 10748783 TI - Female urinary incontinence: aetiology and pathophysiology. AB - Genuine stress incontinence and detrusor instability are the main causes of female urinary incontinence. Treatments are not always satisfactory and a better understanding of the aetiology and pathophysiology should result in more successful therapies and aid prevention. PMID- 10748784 TI - Modern approaches to management of menorrhagia. AB - Menorrhagia and its management is a common problem in both the primary health care setting and in hospitals. There is an increasing number of options for management of menorrhagia and these will be discussed in this article. PMID- 10748785 TI - Human parvovirus B19 in pregnancy. AB - Parvovirus B19 infection can result in an adverse outcome when acquired during pregnancy. However, in the majority of cases a successful outcome can be anticipated. Public awareness of this condition is essential and obstetricians should be familiar with the options available to them if they are presented with this clinical problem. PMID- 10748786 TI - Other uses of non-vascular stents. AB - New generation metallic stents are making an impact on coronary, peripheral vascular and biliary diseases. However, their use is not limited to these: other areas include the oesophagus and upper gastrointestinal tract, the small bowel, large bowel and dacryocystic duct. PMID- 10748787 TI - Melanoma. AB - Cutaneous melanoma is becoming more common. Although still rarer than some cancers it is important, particularly as it afflicts young adults relatively frequently. Furthermore early detection with resultant better prognosis should be achievable if the public was to become more aware, and if health-care professionals were more familiar with melanoma and the behaviour of normal moles. PMID- 10748788 TI - Recent advances in the sleep apnoea/hypopnoea syndrome. AB - This review highlights some of the advances in sleep apnoea/hypopnoea syndrome made over the last few years, particularly in diagnosis and treatment. Recent evidence of the controversial associations of sleep apnoea/hypopnoea syndrome with increased morbidity and mortality, particularly from cardiovascular disease, is presented. PMID- 10748789 TI - Repaglinide: a novel oral antidiabetic agent. AB - Repaglinide is a novel oral antidiabetic agent, marking the development of a new class of drugs for type 2 diabetes. This article examines repaglinide and its position in the treatment of type 2 diabetes. PMID- 10748790 TI - Burns resuscitation: what place albumin? AB - The Cochrane analysis of the use of albumin in critical illness has highlighted the need for more well-conducted studies on colloid use in burns. The lack of objectivity in the press regarding this material has compromised our ability to deliver those studies. The analysis provides no evidence that albumin is unsafe for the initial resuscitation of uncomplicated burns, and the fall in its use is more likely to be cost-related. PMID- 10748791 TI - Bisoprolol in chronic heart failure. AB - The evidence for the use of beta-blockers in heart failure is substantial. Latest recommendations and guidelines suggest that most heart failure patients should be on a drug regimen of an angiotensin-converting enzyme inhibitor, diuretic and a beta-blocker. However, despite the evidence from trials, the majority of patient are not yet treated appropriately, with only 5% receiving a beta-blocker. PMID- 10748792 TI - Job sharing in medical training: an evaluation of a 3-year project. AB - Job sharing has been introduced on a major scale in one deanery to help accommodate increasing demand for flexible (part-time) training. We arranged 37 job shares for 74 trainees between 1996 and 1999. Job shares lasted from 6 months to 2 years. Trainees in job shares were as satisfied with their training as those in supernumerary posts or in full-time training. PMID- 10748794 TI - Bias in cohort studies. AB - Cohort studies allow an exploration of patient change over time. They can provide information on the incidence of disease, prognosis (including patient satisfaction) and likely health-care resource use. Nonetheless, bias can be present in cohort studies in the way patients are selected and followed-up, the way measures are taken, or the way data are analysed. This short paper explores ways in which such flaws can be uncovered in published studies, so that their findings can be interpreted appropriately. PMID- 10748793 TI - Perceptions of flexible training in medicine. AB - Most flexible trainees believe that their full-time colleagues perceive flexible training as flawed in some way. However, most consultants and full-time trainees actually view flexible trainees and their posts in a positive light. PMID- 10748795 TI - Gastric cancer in patients under the age of 30. PMID- 10748796 TI - Quadruple primary neoplasms in a 27-year-old woman. PMID- 10748797 TI - Coronary artery fistula: a rare cause of a left to right shunt. PMID- 10748798 TI - Politicians never learn: don't believe the hype? PMID- 10748799 TI - Ciprofloxacin-induced oral facial dyskinesia in a patient with normal liver and renal function. PMID- 10748800 TI - Variant CJD: finding a needle in a haystack? PMID- 10748801 TI - Variant CJD: finding a needle in a haystack? PMID- 10748802 TI - Indications for lanreotide. PMID- 10748803 TI - Tuberculous peritonitis: a non-invasive test. PMID- 10748804 TI - Web watch. PMID- 10748805 TI - Jargon busters: sickness protection. PMID- 10748806 TI - Personal title: a message for the millennium. PMID- 10748807 TI - Postero-medio-anterior approach of the ankle for the pilon fracture. AB - A good view of the operative field is important for better reduction and fixation in surgical treatment of fractures. The exposure of the ankle joint for the pilon fracture is commonly through the anterior approach, or combined with the medical approach. But sometimes it is still difficult to have complete viewing of the articular surface and to apply internal fixation by that approach. In recent years, we developed a "postero-medio-anterior" approach of the ankle joint by one incision. This approach provides an excellent exposure of the anterior, medial and posterior aspects of the ankle joint with a clear view of the articular surface. In our 45 cases of pilon fracture during 1991 to 1995, there was no incisional injury to the neurovascular bundle. Superficial wound edge necrosis was noted in two cases which healed later without further procedure. Therefore, we recommend this approach as a simple and reliable incision for open reduction of pilon fractures. PMID- 10748808 TI - Displaced intra-articular fractures of distal radius: a comparative evaluation of results following closed reduction, external fixation and open reduction with internal fixation. AB - Fractures of the distal end of the radius are common injuries and are the commonest bony injury around the wrist. Management of these fractures has remained controversial as far as modality of treatment is concerned. In this study 90 adult cases of acute displaced intra-articular fractures of the lower end of the radius were classified according to Frykman's and AO classifications after obtaining radiographs in antero-posterior and lateral planes. These were randomly treated by one of three methods: (1) closed reduction and plaster immobilisation, (2) external fixation and (3) open reduction and internal fixation, and were followed for an average of 4 yr. In the final functional assessment (Sarmiento) the results were (1) plaster 43% good and excellent, 50% fair and 7% poor, (2) external fixator 80% good and excellent, 20% fair and poor results, (3) open reduction and internal fixation 63% good and excellent, 26% fair, 11% poor. We recommend that displaced severely comminuted intra-articular fractures should be treated with an external fixator. PMID- 10748809 TI - Improved outcome after early fixation of acetabular fractures. AB - This study was done in order to evaluate the effect of the timing of fixation for acetabular and pelvic ring fractures on patient outcome. Demographic, clinical and outcome data for 5821 trauma patients admitted from January 1993 through January 1996 were retrospectively reviewed. Pelvic fractures were classified according to Young and Burgess. Patients who had fixation within 24 h of admission were compared with those who had later operation. Main outcome measures were Multiple Organ Dysfunction Score according to Moore, hospital and intensive care unit length of stay and discharge disposition. Out of 416 patients with pelvic fractures, one hundred patients had fracture fixation [90 open reduction and internal fixation, 10 external fixation]. There were 59 acetabular fractures and 41 pelvic ring fractures. The overall mortality was 4%. Early fixation of acetabular fractures was associated with lower MODS (p < 0.006) and decreased total length of stay (p < 0.026). Length of hospital stay was also less with early fixation of pelvic ring fractures (p < 0.04). Functional outcome was improved in early fixation of acetabular fractures with a greater proportion of patients being discharged home rather than to rehabilitation or skilled care (p = 0.05). Patients who underwent early repair of acetabular and pelvic ring fractures had a shorter length of hospital stay compared to those with late fixation. Patients with early repair of acetabular fractures had significantly less organ dysfunction and exhibited improved functional outcome. PMID- 10748810 TI - Wear and corrosion of sliding counterparts of stainless-steel hip screw-plates. AB - Wear of the lag screw and barrel of stainless-steel sliding hip screw-plates (SHSP) produces particles and corrosion that jam the sliding screw. This alters the mechanics of the SHSP causing failure and difficulty in removal. We examined 15 surgically-removed SHSP for the extent of wear and corrosion of metal, and damage to the surrounding tissue. SHSP implants were in place from 2 months to 11 years (average 38.9 months) and they were removed because of fracture non-union, implant fracture or pain. None was infected. Electron microscopy was used to examine the implant surfaces and to determine the chemistry of corrosion products both on the metal and in the tissue. The distal ends of all the lag screws and inside walls of the barrels showed wear and corrosion. The severity of corrosion increased with increasing duration of implant use. Surfaces of the inside walls of the barrels were rough from the manufacturing process. This had contributed to wear. Better manufacturing practice to improve surface smoothness of the lag screws and inside wall of the barrels is needed. Use of cobalt-chromium alloys would improve hardness and resistance to corrosion compared with the stainless steel presently used, which would be an advantage for sliding hip screw-plates in younger patients. Deliberate texturing of screws is counter productive and should be avoided. PMID- 10748811 TI - The timing of disability measurements following injury. AB - Disability following trauma has profound personal, social and economic costs. Currently, measurement of disability is not standardised and no agreed time interval between injury and disability measurement exists. This study was designed to define the best time at which to measure disability following trauma. The functional independence measurement (FIM) and Glasgow outcome scale (GOS) were assessed at 3, 6, 12 and more than 24 months after injury for 201 trauma patients. The best time to measure was defined as the point at which a steady state was reached, i.e. when further functional improvement ceased. Motor FIM showed significant change between 3 and 6 month assessments (p < 0.002) and 6 and 12 months (p < 0.002). No statistically significant change occurred beyond 12 months (p > 0.02). For Cognitive FIM, there was significant change between 3 and 6 months (p = 0.02), but not beyond 6 months (p > 0.2). For GOS there was significant change between 3 and 6 months (p < 0.002) and 6 and 12 months (p < 0.002) but not beyond 12 months (p > 0.2). Disability measurements should be performed 12 months after injury, when patients have reached a steady state. This time of measurement should be adopted as the standard for trauma databases and outcome studies. PMID- 10748812 TI - Evaluation of disability and handicap following injury. AB - Disability and handicap outcome measures are fundamental components of trauma system evaluation. These outcomes are described for survivors of major trauma, attended by the HEMS, London system. Together with measures of injury severity, three functional instruments (Functional Independence Measure (FIM), Glasgow Outcome Scale (GOS) and return to pre-injury work status (RTW)) were used to measure outcome in 201 trauma patients. By 12 months post injury 84.1% of cases were independent in Motor FIM, 88.1% in Cognitive FIM, 79.1% had good outcomes in GOS (grades 4 and 5) and 69.2% had returned to work. The functional measures showed a statistically significant relationship with minor and major and trauma (ISS < 16 and > or = 16): FIM (motor p < 0.002; cognitive p < 0.0003), GOS (p < 0.002) and RTW (p < 0.002). Division according to severity of principal injury confirmed the greatest disability and handicap resulted from the severest injuries (AIS 4-5): 68.9% achieved independence in Motor FIM, 73% in Cognitive FIM and only 40% returned to work. When grouped according to body region of principal injury, neurological injury, particularly severe injury (AIS 4-5) to head and spinal cord regions showed the poorest outcomes. FIM, GOS and RTW are recommended as standard indicators of disability and handicap for trauma registries and outcome studies. PMID- 10748813 TI - The sequelae of posterior talar fractures. PMID- 10748814 TI - Ilizarov bone transport over a primary tibial nail and free flap: a new technique for treating Gustilo grade 3b fractures with large segmental defects. PMID- 10748815 TI - Simultaneous carpometacarpal joint dislocations of the thumb and all four fingers. PMID- 10748816 TI - Spontaneous reduction of a fractured medial epicondylar fragment. PMID- 10748817 TI - Air bag associated fatal intra-abdominal injury. PMID- 10748818 TI - Magnetic nasal foreign bodies. PMID- 10748819 TI - Tension pneumopericardium following blunt thoracic trauma and lobectomy. PMID- 10748820 TI - Minimal access approach to supracondylar nailing. PMID- 10748821 TI - The future of neuro-otology. PMID- 10748822 TI - Measurement of surface area in human mastoid air cell system. AB - The total surface areas of human mastoid air cells were measured using digital CT scans. Using 24 normal ears, the lining of the mastoid air cells was digitally scanned, traced and analysed. The mean surface area of the mastoid air cell system was 167.0 cm2 and ranged from 74.78 to 330.01 cm2. The volume was also measured, the mean being 10.43 cm3 ranging from 6.25 to 20.52 cm3. The correlation coefficient between surface area and volume was 0.95. These results indicate that well pneumatized mastoid air cells serve to provide an extremely large surface area in the temporal bone which in normal mastoid air cells is proportional to the volume. PMID- 10748823 TI - The complications of chronic otitis media: report of 93 cases. AB - The aim of this study was to investigate the incidence, mortality and morbidity of complications due to chronic otitis media (COM). During the nine-year period 1990-1999, 2890 cases of COM were reviewed, 93 (3.22 per cent) having 57 (1.97 per cent) intracranial complications (IC) and 39 (1.35 per cent) extracranial complications (EC). In three patients more than one complication was observed. Meningitis and brain abscess were common in the IC group. Subperiosteal abscess (mastoid and Bezold's abscess) was a common complication in the EC group. Cholesteatoma and granulation/polyp in the middle ear/mastoid were the major findings in both groups. Fifteen patients died from IC. Overall, the mortality rate was 16.1 per cent in all patients having complications, and in patients with IC it was 26.3 per cent. In all of the patients with complications, the morbidity rate was 11.8 per cent. Our study supports the finding that meningitis and brain abscess are the common complications of COM and the main reason for mortality. PMID- 10748824 TI - Immune response and immunopathology of the inner ear: an update. AB - Immune-mediated inner-ear disease includes clinical conditions associated with unilateral or bilateral rapidly progressive forms of sensorineural hearing loss. A systemic autoimmune disorder can be present in less than one-third of cases. Because of the lack of well defined detection methods to identify immune-mediated processes within the inner ear, and the fact that the human inner ear is not amenable to diagnostic biopsy, there has been great interest in developing animal models. Experimental models of sterile and virus-induced labyrinthitis support the participation of the immune system in the aetiopathogenesis of inner-ear disorders: interleukin-2 emanates from the endolymphatic sac and assists in changing the spiral modiolar vein, as in the expression of intercellular adhesion molecule 1, which allows the egrees of immune cells from the circulation. The formation of a fibro-osseous matrix ultimately results in degeneration of the inner ear. These investigations have allowed us to alter the immune response for the purpose of regulating its intensity and the subsequent damage to patients. PMID- 10748825 TI - Clinically staged T3N0M0 laryngeal cancer: how is it best treated? Definitive radiotherapy with salvage surgery v/s combined surgery and radiotherapy. AB - Controversy surrounds the optimum treatment of T3N0 cancer larynx. Curative radiotherapy with salvage surgery in reserve is an accepted methodology as is also a combined protocol of surgery and radiotherapy. A retrospective analysis of the survival results of 119 cases of clinically staged T3N0 cancer larynx treated over a 14-year period at a single centre with either of the above two modalities has been undertaken. The selection of the treatment modality for an individual patient was decided jointly by the patient and the clinicians at a combined cancer clinic. The combined surgery plus radiotherapy treatment group was comprised of a relatively greater number of transglottic tumours while the curative radiotherapy group had a higher proportion of glottic tumours. Actuarial four-year disease-free survival rates were significantly better with combined treatment (79.3 per cent) than with radical radiotherapy and surgical salvage (65.3 per cent)--p value = 0.024. In the radical radiotherapy group, failure was almost always at the primary site and the probability of surviving with an intact larynx was approximately half of the total survival. As per this study, a policy of radical radiotherapy (with salvage surgery for failure) for unselected clinically staged T3N0 cancer larynx, does not provide for comparable cure rates or for satisfactory laryngeal preservation. PMID- 10748826 TI - Uvulopalatopharyngoplasty changes fundamental frequency of the voice--a prospective study. AB - The aim of the study was to find whether a muscle-sparing uvulopalatopharyngoplasty (UPPP) and tonsillectomy cause a measurable change in the voice. The fundamental frequency and the first two formants of five sustained vowels were measured before and nine (six to 15) months following operation. The operation consisted of tonsillectomy and UPPP with preservation of the musculature of the soft palate. All patients received a pre- and post-operative 12-canal polysomnography (level-I sleep study). No patient showed signs of any post-operative velopharyngeal insufficiency. Acoustic analysis showed a significant raising of the fundamental frequency of up to 10 Hz. There was also a lowering of the second formant in two of the five vowels. This was correlated with the volume of the excised tissue. The minimal changes will probably have no significance for those who place no special reliance on their voice, but the possible post-operative changes should be made clear to singers and those relying on their voice for professional reasons. PMID- 10748827 TI - Head and neck schwannomas--a 10 year review. AB - Schwannomas of the head and neck are uncommon tumours that arise from any peripheral, cranial or autonomic nerve. Twenty-five to 45 per cent of extracranial schwannomas occur in the head and neck region and thus are usually in the domain of the otolaryngologist. They usually present insidiously and thus are often diagnosed incorrectly or after lengthy delays, however, better imaging and cytological techniques have lessened this to some degree more recently. For benign lesions conservative surgical excision is the treatment of choice bearing in mind possible vagal or sympathetic chain injury. Malignant schwannomas are best treated with wide excision where possible. The role of adjuvant therapy remains uncertain and irrespective of treatment modality prognosis is poor with an overall survival of 15 per cent. However, recent advances in ras oncogene inhibitors may hold hope for the future. PMID- 10748828 TI - Vagus nerve stimulation: quality control in thyroid and parathyroid surgery. AB - This paper describes the use of the Neurosign 100 Nerve Monitor and vagus nerve stimulation in the identification and assessment of the integrity of the recurrent laryngeal nerve (RLN) during thyroid and parathyroid surgery. Vocal fold function was assessed pre- and post-operatively in all patients undergoing thyroid and parathyroid surgery. The nerve monitor, used in association with endotracheal electrodes, was used to confirm correct RLN identification and demonstrate its integrity at the completion of surgery. There were 21 unilateral and 19 bilateral neck explorations. In these 40 patients, 57 of 59 RLNs were identified. The nerve monitor demonstrated RLN continuity in all but one case (equipment failure: electrode misplacement) after initial identification. Vagus nerve stimulation was performed in 21 patients without adverse sequelae. Damage to the RLN was identified in one of these patients, in whom direct RLN stimulation close to the larynx had failed to indicate discontinuity. Post operatively this patient had a transient unilateral vocal fold palsy. The use of the Neurosign 100 Nerve Monitor is no substitute for meticulous surgery. Stimulation of the vagus nerve may be a more sensitive means of assessing RLN integrity during thyroid and parathyroid surgery than stimulation of the RLN itself. Confirmation of RLN integrity allows the surgeon to proceed with confidence to the contralateral side of the neck during hazardous bilateral explorations. PMID- 10748829 TI - Thyroglossal duct remnants. AB - Thyroglossal duct remnants presenting as a lump in the neck are usually called thyroglossal cysts. Meticulous dissection of the cyst and duct, along with the body of the hyoid bone (Sistrunk's operation) is necessary to avoid recurrence. The authors have reviewed the histology of 61 consecutive specimens diagnosed preoperatively as thyroglossal cysts and have found that a true cyst exists in only 46 per cent of cases. PMID- 10748830 TI - A traumatic ossicular disruption successfully repaired with n-butyl cyanoacrylate tissue adhesive. AB - Tissue adhesives have been available and in use for more than 40 years. They have, however, never been popular for ossiculoplasty because of the reported excessive inflammatory reaction and ototoxicity. The authors present a case of ossicular disruption successfully repaired with tissue adhesive and review the literature. PMID- 10748831 TI - Idiopathic familial facial nerve paralysis. AB - A 26-year-old man was seen one day after developing a left facial palsy of unknown aetiology. He had previously had a left facial palsy at age 14 and a right facial palsy at 19, both with minimal residual paresis. Both his mother and grandmother have had facial palsies. The role of hereditary influences in idiopathic facial paralysis, as well as the treatment of this condition, is discussed. PMID- 10748832 TI - Oculostapedial synkinesis following Bell's palsy. AB - A case of oculostapedial synkinesis occurring after Bell's palsy is described. This rare phenomenon has not previously been reported following Bell's palsy. The authors discuss the method of objectively proving the diagnosis, which can be difficult. The patient was successfully treated by stapedius tendon section under local anaesthesia. PMID- 10748833 TI - Bilateral cerebrospinal fluid rhinorrhoea. AB - Cerebrospinal fluid (CSF) rhinorrhoea is leakage of fluid from the subarachnoid space to the frontal, sphenoidal or ethmoidal sinuses, and may occur spontaneously. The authors present the first reported case of bilateral spontaneous CSF rhinorrhoea. Bony defects on both sides of the cribriform plate were identified using endoscopic and radiological techniques, and the CSF fistula was closed endoscopically. The aetiology, diagnosis and contemporary surgical treatment of spontaneous CSF leaks is discussed. Endoscopic repair was successful in this case, and in view of the high success and low reported complication rates this surgical approach should be considered for treatment of spontaneous CSF rhinorrhoea. PMID- 10748834 TI - The role of radiation in delayed hearing loss in nasopharyngeal carcinoma. AB - Although radiation damage to the inner ear can be induced experimentally in animals, its incidence in humans as a complication of therapy for nasopharyngeal carcinoma (NPC) remains a matter of conjecture due to inadequate histological validation. A case of advanced NPC with a mixed conductive and neurosensory hearing loss is presented that at necropsy showed tumour invasion of the VIIIth cranial nerve in the internal auditory meatus with associated infection. The architecture of the organ of Corti was well-preserved despite heavy doses of radiation but degeneration, the cause of which could not be substantiated, was present in the auditory nerve pathway. A diagnosis of radiation damage to the end organ of hearing in the absence of supporting histological evidence should be made with caution. PMID- 10748835 TI - Hoarseness due to leech ingestion. AB - This paper presents a case of hoarseness caused by the pharyngolaryngeal localization of a leech. This pathological lesion is extremely rare in western European countries, but is more frequent in endemic areas. Possibly lethal dyspnoea, haemoptysis or haematemesis can be the revealing symptoms. When the diagnosis is suspected simple examination under anaesthesia and removal of the leech will effect a cure. PMID- 10748836 TI - A benign parathyroid cyst presenting with hoarse voice. AB - Parathyroid tumours and cysts are rare and, when presenting as neck masses, can be clinically misdiagnosed as thyroid lesions. Symptoms may be caused by compression of the surrounding structures or hormonal overactivity. This paper describes a patient with recurrent hoarseness owing to the pressure effects of a parathyroid cyst on the recurrent laryngeal nerve. PMID- 10748837 TI - Primary extracranial meningioma of the soft palate. AB - Extracranial meningiomas comprise two per cent of all meningiomas. Primary extracranial meningiomas are even less common. The authors report the first case of a primary extracranial meningiomas of the soft palate, which presented as an intraoral mass. This was treated by surgical excision and there was no evidence of tumour recurrence at four years of follow-up. PMID- 10748838 TI - Acute purulent thyroiditis with retropharyngeal and retrotracheal abscesses. AB - An unusual case of acute purulent thyroiditis in a 45-year-old man forming a large abscess in the left lobe of the thyroid gland is described. It perforated the capsule at the posterior wall of the thyroid, causing extensive retropharyngeal and retrotracheal abscesses. The patient was treated with broad spectrum antibiotics and surgical excision of the infected tissues. He has remained well one year after the operation. PMID- 10748840 TI - Nasal non-Hodgkin's lymphoma causing mucocele of the maxillary antrum. AB - The authors report a case of an antral mucocele secondary to obstruction of the antral ostium by a non-Hodgkin's lymphoma. Antral mucoceles are rare, and this is the first report of one such caused by a nasal lymphoma. PMID- 10748839 TI - The management of dysphasia in skeletal hyperostosis. AB - Diffuse idiopathic skeletal hyperostosis (DISH), or Forestier's disease, is an ossifying condition frequently encountered in otolaryngology as it affects 12-28 per cent of the adult population. This form of hyperostosis can manifest clinically with dysphagia, food impaction, hoarseness, stridor, myelopathies and other neurological problems. Judicious management of severe dysphagia proves challenging. The failure of conservative care often leaves surgery as the only option. In this report an anterolateral transcervical surgical approach to the confluent osteophytes is discussed and the value of videofluoroscopic swallow highlighted. PMID- 10748841 TI - Spindle cell angiosarcoma of the oropharynx. AB - Angiosarcomas are uncommon malignant tumours of vascular endothelium. In the head and neck, the scalp is the commonest site of origin. A rare case of spindle-cell angiosarcoma of the oropharynx, treated by surgery and post-operative radiotherapy, is reported. We discuss the clinical presentation and histopathological diagnosis of this lesion. PMID- 10748842 TI - Three-dimensional reconstruction of the incudostapedial joint with helical computed tomography. AB - Three-dimensional images of the incudostapedial joint (I-S joint) were reconstructed using helical computed tomography (CT). The images of the joint were most often reconstructed when threshold values were selected at -500 to -700 Hounsfield Units. These shapes were changed by choosing various threshold values. Histological examinations of the cadaver specimen indicate that these changes are due to structure of the I-S joints. We conclude that these 3-D images may be useful in assessing pathology of the I-S joint. PMID- 10748843 TI - Radical revision mastoidectomy for chronic otitis media without cholesteatoma: the relevance of excenteration of all rest cells. AB - Patients who, despite treatment, suffer persistent chronic otitis media (COM), a radical cavity and a hearing loss of more than 55 decibels for more than five years, can be classified as having an 'endstage' COM. It is generally agreed that retained infected residual cell tracts or a too small meatus are common causes for failure of surgery for COM. Radical revision mastoidectomy (RRM) aims to remove all residual cell tracts in the mastoid, in the middle-ear cleft, around the labyrinth and around the Eustachian tube. The mastoid cavity is obliterated with a pedicled muscle flap. Thus the mastoid cavity is reduced and self cleansing is improved. The first 16 consecutive patients who underwent a RRM for 'endstage' COM were included in this study. In about 80 per cent the treatment resulted in a dry ear. We, therefore, conclude that RRM is an effective and safe method for treating 'endstage' COM. PMID- 10748844 TI - Useful residual hearing despite radiological findings suggestive of anacusis. AB - A severe malformation of the inner ear, often referred to as severe labyrinthine dysplasia or common cavity deformity, consists of an absent or dilated cochlear basal coil, wide communication with the vestibule and a tapered internal acoustic meatus and can be associated with absent hearing. We discuss two children with severe labyrinthine dysplasia as shown by computed tomography (CT) scans and, in the first case, an absent VIIIth nerve bilaterally shown by magnetic resonance imaging (MRI). In 1995, both cases were precluded from cochlear implantation, on the basis of the absent VIIIth nerve (first case) and increased risk of CSF leak during operation (second case). However, audiometric results and vocalization patterns of both children suggested the presence of some residual hearing function, while recently reported specific surgical techniques have been found to be safe and effective in the cochlear implantation of the common cavity deformity. The management of such cases should be decided on the grounds of a full audiological assessment in conjunction with the radiological features, in the light of current surgical trends shown to be safe and effective. PMID- 10748845 TI - Subtemporal exposure of the intrapetrous internal carotid artery. An anatomical study with surgical application. AB - This work describes the exposure of the intrapetrous internal carotid artery (ICA) through the subtemporal approach. The anatomical details of 25 fresh temporal bones were studied and provided the initial background for this procedure. Bone drilling in the meatal plane anterior to the internal auditory canal could create a four-sided quadrangular area. The exposure of the ICA through this area was applied on three occasions. It proved safe as regards the surrounding structures and also provided an ample working space for the extirpation of tumours surrounding the artery. PMID- 10748846 TI - Septal perforations closure utilizing the backwards extraction-reposition technique of the quadrangular cartilage. AB - Nasal septal perforations are anatomical defects of the nasal septum, causing dynamic alterations in nasal physiology which may lead to variable symptoms and otolaryngological referral. Repair of nasal septal perforations continues to remain a difficult surgical problem, and nowadays there is no definitive solution for their successful surgical closure. Thirty patients with small- or medium sized anterior nasal septal perforations were treated with a simple technique of backwards extraction-reposition of the quadrangular cartilage. Prior nasal septal surgery and repeated cautery were the most common cause of perforation. After a minimum follow-up of two years the success rate for relief of symptoms and closure of the perforation was 87 per cent. This technique showed very good results in small-sized and selected cases with medium-sized perforations, but the mucosal dissection employed is not suitable for medium to large perforations. PMID- 10748848 TI - The one-stage rhinoplasty septal perforation repair. AB - A combined septal perforation repair and rhinoplasty was performed in 20 patients (12 males, eight females; age range 16-36, mean age 29.6) presenting with septal perforations (size 1-4 cm) and external nasal deformities. The external rhinoplasty approach was used for all cases and the perforation was repaired using bilateral intranasal mucosal advancement flaps with a connective tissue interposition graft in between. The perforation was totally closed in 18 cases (90 per cent) with complete resolution of the pre-operative symptoms occurring in 16 (80 per cent). Cosmetically, 19 cases (95 per cent) were very satisfied with their aesthetic result. The exposure provided by the external approach proved to be very helpful in the process of septal perforation repair. Our results show that septal perforation repair could safely be combined with rhinoplasty and that some of the rhinoplasty manoeuvres used could even facilitate the process of septal perforation repair. PMID- 10748847 TI - Nasal splints, revisited. AB - A study was carried out to compare the effectiveness of nasal splints (in preventing intranasal adhesions) with the morbidity associated with their use in nasal surgery. One hundred and ten patients undergoing a routine nasal operation were randomly allocated into two equal groups, one with splints and the other without. Post-operative pain and discomfort was assessed by a visual analogue scale at 48 hours and a week following surgery when either nasal suction (non splinted group) or removal of splints was carried out. All patients were examined after six weeks for development of adhesions. Results showed that there was no significant difference in the incidence of adhesions between the splinted and non splinted patient groups. However, the patients with splints had significantly more pain and nasal discomfort when assessed one week after surgery. It is concluded that the morbidity associated with nasal splints does not justify their use in routine nasal surgery if the aim is to prevent nasal adhesions, but they may still be indicated for enhancing the stability of the septum following septoplasty. PMID- 10748849 TI - Ex juvantibus approach for chronic posterior laryngitis: results of short-term pantoprazole therapy. AB - The aim of this study was to investigate whether patients with chronic posterior laryngitis and symptoms of gastro-pharyngeal reflux benefit from a six-week therapy with pantoprozole. Twenty-nine out-patients with voice disorders (case history of at least two months) and simultaneous symptoms of gastro-pharyngeal reflux were recruited for this study. At the entry to the study a symptom questionnaire and a video-laryngo/stroboscopy were completed. The symptom questionnaire and the video-laryngo/stroboscopy were repeated after the six weeks of therapy with pantoprazole 40 mg once a day and again six weeks and three months after this follow-up, during which time the patient was without therapy. Hoarseness, globus pharyngeus, sore throat, heartburn, and coughing were the symptoms which showed a significant (p < 0.05) recovery at the follow-ups (mean of hoarseness index: 7.28 to 0.92; mean of globus pharyngeus index: 3.14 to 0.58; mean of heartburn index: 2.86 to 0.5; mean of cough index: 1.72 to 0.25; mean of throat soreness index: 1.72 to 0.15). Laryngoscopy scores of the posterior laryngeal region, the glottic and the supraglottic region showed statistically significant improvement (p < 0.05) after the treatment with pantoprazole. The therapeutic effect exceeded the drug administration until the last follow-up (after three months). The medication was tolerated without side-effects in all patients. A primary (ex juvantibus) therapy with proton pump inhibitors seems to be a therapeutic option for patients with long-lasting chronic inflammation of the larynx not responding to common therapy. In this case a six-week course of treatment has been shown to be sufficient. PMID- 10748850 TI - Irrigation during temporal bone surgery. PMID- 10748851 TI - Myxoid chondrosarcoma of the external auditory meatus. AB - Extraskeletal myxoid chondrosarcoma presenting in the head and neck is extremely rare. Histological diagnosis is difficult and requires close co-operation between clinician, radiologist and pathologist. The tumour has a good prognosis in comparison to myxoid chondrosarcoma of the bone but surgical resection may be difficult due to its gelatinous nature. We present a case of extraskeletal myxoid chondrosarcoma originating in the external auditory meatus of a 42-year-old man. This is the first report of this tumour in this site. PMID- 10748853 TI - Nasolabial cyst: a report of eight cases and a review of the literature. AB - Nasolabial cyst is a cystic ectodermal developmental swelling which occurs as a cyst on the lateral half of the floor of the nasal vestibule at the base of the alae of the nose. Although, it is a rare disease, this is a report of eight cases in a population of 500,000 in one year. Seven of the patients were female aged between 25-50 years. Seven cysts were left-sided and one was bilateral. The purpose of this paper is to present an additional example of patients with nasolabial cyst. It may be a more common disease than previously thought. PMID- 10748852 TI - Spontaneous carotico-cavernous fistula presenting as pulsatile tinnitus. AB - A patient with sudden onset pulsatile tinnitus resulting from a spontaneous carotico-cavernous fistula is presented. The case is discussed and illustrated to highlight the clinical features and natural history of a condition rarely seen by ENT surgeons. PMID- 10748854 TI - Reversible hyposmia caused by intracranial tumour. AB - Two patients with hyposmia caused by an intracranial tumour recovered olfactory functions after craniotomy. The first case was a 68-year-old male with a tumour metastasized from the lung to the right frontal lobe. The second case was a 75 year-old male with meningioma of the right frontal lobe. Results of T & T olfactometry and venous olfaction tests also indicated suspected central hyposmia. Magnetic resonance imaging (MRI) indicated compression of the frontal lobe by intracranial tumour. Pressure on the olfactory centre located in the frontal lobe produced hyposmia. Decompression of the frontal lobe by craniotomy improved the sense of smell. Therefore, some cases of olfactory disturbance caused by intracranial tumour may be reversible if they are the result of simple compression of the olfactory centre. PMID- 10748855 TI - Medial orbital protrusion--a potentially hazardous anomaly during endoscopic sinus surgery. AB - We report the case of a 57-year-old patient with a presumed developmental anomaly of the medial orbital wall. The resultant protrusion of orbital contents into the ethmoidal complex was clearly demonstrated on coronal computed tomography (CT) scans of the paranasal sinuses. This anomaly presents a high risk of iatrogenic injury to the medial rectus and orbit during functional endoscopic sinus surgery and has not previously been described. PMID- 10748856 TI - Surgical emphysema following dental treatment. AB - Surgical emphysema is a relatively rare complication of dental surgery. Many cases go unrecognized or are misdiagnosed. Although the majority of cases resolve spontaneously, some can lead to potentially life-threatening complications requiring emergency intervention. A case of surgical emphysema following a routine restorative dental procedure is presented. The differential diagnosis and management of this condition is discussed. PMID- 10748857 TI - Necrotizing fasciitis after peritonsillar abscess in an immunocompetent patient. AB - Cervical necrotizing fasciitis (CNF) is a rapidly progressive, severe bacterial infection of the fascial planes of the head and neck. Group A beta haemolytic Streptococcus spp. (GABHS), Staphylococcus spp., or obligatory anaerobic bacteria are the most common causative pathogens. The disease usually results from a dental source or facial trauma. Extensive fascial necrosis and severe systemic toxicity are common manifestations of CNF. Review of the literature reveals only seven such cases, with four successful outcomes. The authors present the case of a 50-year-old immunocompetent female with CNF arising from a peritonsillar abscess. Intravenous immunoglobulins in conjunction with surgery and antibiotics were used successfully. The authors also suggest the importance of the early diagnosis, aggressive surgical debridement, broad-spectrum antibiotics, and possible usefulness of the intravenous immunoglobulins in the treatment of CNF, especially when the disease is associated with toxic shock syndrome. PMID- 10748858 TI - The use of inhalers in patients with tracheal stomas or tracheostomy tubes. AB - Patients with chronic obstructive airways disease (COAD) or asthma who have a tracheostomy tube or tracheal stoma have difficulty using metered dose inhalers (MDIs) because of a failure to achieve a good seal between the tracheostomy tube or stoma and the MDI or spacer device mouthpiece. Many such patients therefore utilize nebulizers. MDIs in comparison to nebulizers have the advantages of being more compact, portable, easy to use, less time-consuming, and cheaper. We present the case of a 74-year-old man who underwent a laryngectomy with tracheal stoma formation who had a poor response with nebulizers and required oral steroids. He was subsequently, with the help of a number of attached devices, able to use his MDIs to good effect. We describe a number of devices and adaptors to enable patients with laryngectomy stomas or tracheostomy tubes to utilize MDIs and undergo respiratory function tests. We recommend that all such patients should have the benefit of a consultation with a dedicated respiratory nurse who can provide the appropriate MDIs, devices and adaptors to optimize the treatment of their lower respiratory tract condition. PMID- 10748859 TI - An unusual cutaneous presentation of Hodgkin's disease. AB - Hodgkin's disease is a neoplasm of lymphoid tissue defined histopathologically by the presence of Reed-Sternberg cells in an appropriate cellular background. Hodgkin's disease extends only rarely into the skin. Sinus and fistula formation has been reported in very occasional cases. We now report a case of a 34-year-old woman presenting with a cutaneous lesion surrounding a discharging blind-ending sinus in the neck, subsequently diagnosed as Hodgkin's disease. To our knowledge this form of presentation of Hodgkin's disease has not been reported in the English literature before, and at the same time we would like to outline the difficulties in diagnosis encountered with these cutaneous lymphoid lesions. PMID- 10748860 TI - Penetrating oesophageal foreign bodies in the thyroid gland. AB - Oesophageal penetration and migration of oesophageal foreign bodies into the thyroid gland is extremely rare with only occasional case reports appearing in the medical literature over the years. This is a retrospective review of four patients who were managed for penetrating oesophageal foreign bodies in the thyroid gland over an 11-year period. The clinical, radiological and intra operative findings of the four cases are discussed. PMID- 10748861 TI - Monostotic fibrous dysplasia of the temporal bone. AB - Fibrous dysplasia is a slowly progressive bony disorder where normal bone is replaced by abnormal fibro-osseous tissue. Its monostotic variety in the temporal bone is very rare and such a case is presented here. Computed tomography (CT) may be adequate for the diagnosis and follow-up of these patients. Limited surgery should only be considered in cases of symptomatic disease. PMID- 10748862 TI - Skull base osteomyelitis interpreted as malignancy. AB - Lesions in the skull base may present difficulties in diagnosis primarily because the access needs to be invasive and one has to rely on imaging that may still be misleading. The case presented here illustrates this example in that the patient had abnormalities on computed tomography (CT) and magnetic resonance image (MRI) scans quite convincing of malignancy but which with time proved, essentially through clinical judgment, to be skull base osteomyelitis secondary to malignant external otitis (OME). PMID- 10748863 TI - Turban tumour with involvement of the parotid gland. AB - Familial autosomal dominant cylindromatosis (FADC, turban tumour syndrome, Brooke Spiegler-syndrome and many more, MacKusick catalogue numbers 123850, 313100) is a rare hereditary disease usually presenting in the second or third decade. With female preponderance dermal cylindromas predominantly arise in hairy areas of the body with approximately 90 per cent on the head and neck. Transformation to malignancy seems to be scarce. Although cylindromas of the skin resemble basal cell adenomas of the salivary gland, there is usually no salivary gland involvement. On the other hand, patients with basal cell adenomas of a salivary gland usually do not show dermal lesions. We report one of the rare cases of FADC combined with multiple basal cell adenomas of the parotid glands and present a review of the literature. PMID- 10748865 TI - Differential detection of type II collagen N-terminal and C-terminal denaturation epitopes in degrading cartilage. AB - AIMS: To investigate the relative stability of collagen metabolites in degrading cartilage. METHODS: New antipeptide antibodies to denaturation epitopes located in the N-terminal and C-terminal regions of the type II collagen helix have been made and characterized. Type II collagen fragments in the conditioned medium from cultures of degrading bovine nasal cartilage were detected by immunoblotting with the new antisera as well as by N-terminal sequencing. The antibodies were also used in immunohistochemical studies of normal and osteoarthritic human cartilage. RESULTS: Type II collagen fragments with an apparent molecular mass of approximately 30 kDa were detected in cartilage conditioned media using antibody AH12L3, which recognizes N-terminal epitope AH12. The N-terminal sequence of one of these fragments matched exactly a sequence in the N-terminal region of type II collagen. Antibody AH9L2, which recognizes C-terminal epitope AH9, did not bind to any protein bands in the immunoblotted culture medium. In immunohistochemical studies, antibody AH12L3 detected extensive regions of degraded collagen in osteoarthritic cartilage and a more restricted pattern of staining in nonarthritic cartilage. Far less immunostaining was apparent in all cartilage specimens with antibody AH9L2. CONCLUSIONS: These results indicate that the N terminal region of type II collagen is more resistant to proteolysis than the C terminal region, an observation that has important implications for the choice of epitopes that are likely to be good markers of damage to cartilage collagen in patients with arthritis. PMID- 10748866 TI - Decreased metastatic spread in mice homozygous for a null allele of the cystatin C protease inhibitor gene. AB - AIMS: Increased or altered activities of cysteine proteases have been implicated in serious human disorders such as cancer, rheumatoid arthritis, sepsis, and osteoporosis. To improve the current knowledge of the regulatory role of a major mammalian cysteine protease inhibitor, cystatin C, in such disease processes, a cystatin C deficient mouse was generated and characterized. METHODS: The mouse cystatin C gene was inactivated by insertion of a bacterial neo gene through homologous recombination in 129/Sv embryonic stem cells. Embryonic stem cell clones were injected into C57BL/6J blastocysts followed by injection of the blastocysts into pseudopregnant female mice. F1 offspring with agouti coat colour after mating of chimaeric males with C57BL/6J females were examined by DNA analysis, and mice carrying the targeted mutation were intercrossed to obtain homozygous cystatin C deficient (CysC-/-) mice. To study the role of cysteine proteases and their inhibitors in metastasis, the spread of B16-F10 melanoma cells in CysC-/- and wild-type mice was compared. Analysis of the formation of remote metastases was carried out by intravenous injection of beta-galactosidase transfected B16-F10 cells and subsequent determination of cancer cell colonies in the lungs. RESULTS: Cystatin C deficient mice were fertile and showed no gross pathological abnormality up to 6 months of age. Compared with wild-type mice, seven times fewer large metastatic colonies were counted by means of a dissecting microscope in CysC-/- mice two weeks after tail vein injection of B16-F10 cells. At all of eight time points from 15 minutes to two weeks after intravenous injection of tumour cells, the CysC-/- mice had significantly fewer lung metastases. The observed differences were smaller when beta-galactosidase transfected cells were used to allow counting of small colonies. Subcutaneous and intracerebral tumour growth was not different in the CysC-/- mice. CONCLUSIONS: Cystatin C concentrations in vivo might influence metastasis in some tissues. The decreased metastatic spread of B16-F10 cells in CysC-/- mice is the result of both reduced seeding and reduced growth of tumour cells in their lungs. PMID- 10748867 TI - Abl expression, tumour grade, and apoptosis in chondrosarcoma. AB - AIMS: To determine whether Abl immunoreactivity correlates with grade and cell kinetics (apoptosis and mitosis) in chondrosarcoma. METHODS: Sections from 16 chondrosarcomas were stained immunohistochemically using a polyclonal antibody to the c-Abl/Bcr-Abl oncoprotein. Apoptotic indices and mitotic indices were assessed in all tumours. Sections from 24 paraffin wax blocks of human fetal rib (gestational ages, 15-42 weeks) were also stained to determine whether the Abl protein is synthesised consistently throughout endochondral ossification. RESULTS: Abl staining in immature fetal rib chondrocytes at all stages of development was predominantly nuclear, and 70% of cells showed moderate to strong staining. Abl immunoreactivity was minimal or absent in hypertrophic chondrocytes about to undergo apoptosis at the growth plate. There was strong Abl staining in grade 1 and grade 2 chondrosarcomas but staining was greatly reduced or absent in grade 3 chondrosarcomas. There was a very significant linear correlation between apoptotic index (mean, 0.68%; range, 0-3.2%) and mitotic index (mean, 0.23%; range, 0-0.9%), and both indices were significantly lower in grade 1 than in grade 2 and grade 3 chondrosarcomas. CONCLUSIONS: These data suggest that abl gene expression is associated with differentiation and apoptosis inhibition in fetal and neoplastic chondrocytes. However, these putative effects cannot be ascribed solely to the Abl protein, because several additional factors contribute to the regulation of both differentiation and apoptosis. PMID- 10748864 TI - The Epstein-Barr virus and its association with human cancers. AB - The Epstein-Barr virus (EBV) has been linked to the development of a variety of human malignancies, including Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma, some T cell lymphomas, post-transplant lymphoproliferative disease, and more recently, certain cancers of the stomach and smooth muscle. This review summarizes these associations and in particular the role of the viral latent genes in the transformation process. PMID- 10748868 TI - HHV-8 (KSHV) is not associated with bacillary angiomatosis. AB - AIMS: Bacillary angiomatosis is a rare pseudoneoplastic angioproliferative lesion occurring in patients with AIDS. This condition has been associated with Bartonella henselae and Bartonella quintana infections. Human herpesvirus 8 (HHV 8) is thought to be the causative agent of Kaposi's sarcoma, a vasoproliferative neoplasm, also commonly found in patients with AIDS. The presence of HHV-8 in a cohort of patients with bacillary angiomatosis was investigated. METHODS: Eight cutaneous cases of biopsy confirmed bacillary angiomatosis were assessed for HHV 8 using standard solution phase polymerase chain reaction (PCR). RESULTS: No case of bacillary angiomatosis harboured HHV-8 DNA. CONCLUSIONS: HHV-8 was not demonstrated in the lesions of bacillary angiomatosis and therefore does not appear to play a role in the pathogenesis of this pseudoneoplastic angioproliferative disorder. This finding might be useful in the distinction of bacillary angiomatosis from Kaposi's sarcoma, because lesions from patients with Kaposi's sarcoma almost always contains HHV-8 DNA. PMID- 10748869 TI - Parvovirus B19 is associated with benign testes as well as testicular germ cell tumours. AB - AIMS: Parvovirus B19 has been demonstrated in testes of patients with germ cell tumours but not in controls, raising the possibility that the virus has an aetiological role in these tumours. The aims of this study were to investigate the association of the virus with germ cell tumours and to localise the virus histologically. METHODS: DNA was extracted from paraffin wax embedded sections of testes from 10 seminomas, eight teratomas, two mixed seminoma/teratomas, and 10 testes showing benign histology. Polymerase chain reaction (PCR) amplification of three regions within the NS and VP1/2 genes was carried out in duplicate on all samples. One PCR positive case (seminoma/teratoma) was examined by microdissection of histologically defined tissue components followed by PCR amplification of parvoviral sequences. Samples from PCR positive patients were immunostained using a B19 specific monoclonal antibody. RESULTS: Seven cases were PCR positive, these comprised two of 10 seminomas, one of two mixed tumours, none of eight teratomas, and four of 10 benign controls. PCR analysis of the material microdissected from the seminoma/teratoma showed the presence of the virus in regions of seminoma, teratoma, intratubular germ cell neoplasia, normal tubules, and connective tissue. All patient samples studied immunohistochemically were negative. CONCLUSIONS: This confirms the presence of parvovirus B19 in a proportion of germ cell tumours; however, in one patient, the virus was widespread in the tissue components and not confined to tumour cells. In addition, the virus was present in control benign testes. These data suggest that B19 might not be of aetiological importance in germ cell tumours of testis. PMID- 10748871 TI - Amplification of FISH signals using intermittent microwave irradiation for analysis of chromosomal instability in gastric cancer. AB - Gastrointestinal tract tumours are notorious for their difficulty in relation to conventional cytogenetic analysis. In particular, necrosis, the presence of stromal inflammatory and other cells, and poor attachment of tumour cells have led to problems with the quality and reliability of cytogenetic preparations, even with the recently developed fluorescence in situ hybridisation (FISH) technique. Furthermore, background autofluorescence masks the weak hybridisation signals in the nuclei. To overcome this problem, brief microwave treatment was applied for the identification of centromeres by in situ hybridisation in gastric cancer cells. Using this technique, a panel of 17 centromeric specific alpha satellite probes was used to detect chromosomal instability in these cells. Lymphocyte controls and cancer cells subjected to irradiation achieved the hybridisation threshold in 30 minutes, providing a significant difference when compared with the non-irradiated samples (mean (SD) frequency of diploid cells 97% (2.1%) v 76% (4.6%), respectively). Therefore, this protocol of intermittent microwave treatment is recommended as a simple, rapid, and highly reproducible technique for application to various types of probe. It also gives well defined hybridisation signals and reduces background "noise". PMID- 10748870 TI - Mutational analysis of the PTEN/MMAC1 gene in primary oesophageal squamous cell carcinomas. AB - AIM: To investigate whether PTEN/MMAC1 mutations play a role in the carcinogenesis of oesophageal squamous cell carcinoma. METHODS: A panel of 33 primary oesophageal squamous cell carcinoma tumour samples and 20 corresponding morphologically normal tissues was examined for mutations in all nine exons of the PTEN/MMAC1 gene by means of polymerase chain reaction single strand conformational polymorphism analysis (PCR-SSCP) and direct DNA sequencing methods. RESULTS: Only one of 33 oesophageal squamous cell carcinomas showed an aberrant SSCP band. Further sequencing analysis of this sample revealed an 802 29 T-->C substitution in intron 7. PTEN/MMAC1 mutations were not found in the mutational "hot spot" in exon 5, even after direct sequencing of six oesophageal squamous cell carcinoma samples and three normal tissues. However, a deletion of one nucleotide T at position 492 +8 in intron 5 was seen in all samples. CONCLUSION: These results suggest that PTEN/MMAC1 mutations do not play a major role in the carcinogenesis of oesophageal squamous cell carcinoma. PMID- 10748872 TI - Comparative genomic hybridisation. AB - Comparative genomic hybridisation (CGH) is a technique that permits the detection of chromosomal copy number changes without the need for cell culturing. It provides a global overview of chromosomal gains and losses throughout the whole genome of a tumour. Tumour DNA is labelled with a green fluorochrome, which is subsequently mixed (1:1) with red labelled normal DNA and hybridised to normal human metaphase preparations. The green and red labelled DNA fragments compete for hybridisation to their locus of origin on the chromosomes. The green to red fluorescence ratio measured along the chromosomal axis represents loss or gain of genetic material in the tumour at that specific locus. In addition to a fluorescence microscope, the technique requires a computer with dedicated image analysis software to perform the analysis. This review aims to provide a detailed discussion of the CGH technique, and to provide a protocol with an emphasis on crucial steps. PMID- 10748873 TI - ATM protein synthesis patterns in sporadic breast cancer. AB - AIMS: The gene mutated in ataxia-telangiectasia (A-T), designated ATM (for "A-T mutated"), is believed to be associated with an increased risk of developing breast cancer. Most patients with A-T have null mutations of the ATM gene that appear to give rise to a truncated nonfunctional ATM protein. Therefore, the increased risk of breast cancer reported in A-T heterozygotes appears to be the result of haplo-insufficiency of ATM in breast tissues. This study aimed to determine whether reduced synthesis of ATM was also an important factor in sporadic breast cancer. METHODS: Paraffin wax embedded tissues from patients with breast invasive ductal carcinoma (IDC) (n = 42), patients with ductal carcinoma in situ (DCIS) (n = 17), and others with lymph node metastases (n = 14) were studied. A streptavidin-biotin-peroxidase system was used to stain tissue sections for the ATM protein using the ATM-4BA and CT-1 polyclonal and monoclonal antibodies, respectively. The protein truncation test was used to screen for mutations in the ATM gene in those patients who had greatly reduced ATM protein immunoreactivity in the primary carcinoma (n = 3). RESULTS: Most metastatic breast carcinomas in lymph nodes (71%) had greatly reduced or absent ATM protein synthesis, which was significant when compared with that observed in non metastatic invasive breast carcinomas (p = 0.029; chi 2 test). Although not significant (p = 0.045; chi 2 test), some sporadic breast carcinomas (14 of 42) also had reduced or absent ATM protein immunoreactivity. The protein truncation test did not reveal any gross ATM gene abnormality in the cases tested, indicating that the patients were not A-T heterozygotes, who are predisposed to breast cancer. CONCLUSIONS: A reduction in immunohistochemically detectable ATM protein in sporadic breast carcinoma implicates ATM in the progression of the disease. PMID- 10748874 TI - K-ras mutations appear in the premalignant phase of both microsatellite stable and unstable endometrial carcinogenesis. AB - AIMS: Sequential events of endometrial tumorigenesis can be studied by comparison of genetic lesions seen in normal, premalignant, and malignant tissues. The distribution of k-ras mutations in microsatellite stable and unstable premalignant lesions was studied to determine whether this gene is implicated in both tumorigenic pathways. METHODS: K-ras mutations were analysed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and direct sequencing in matched endometrial normal, premalignant (atypical hyperplasias), and adenocarcinoma tissues from individual patients. Identification of precancers solely by their appearance as atypical endometrial hyperplasias is very subjective; therefore, in addition to histopathological assessment, we performed molecular testing (non-random X inactivation or clonal altered microsatellites) for an expected feature of precancers--that is, monoclonality. RESULTS: Equivalent K-ras mutation frequencies were seen in microsatellite stable (six of 33) and unstable (three of 23) cancers. In both types, K-ras mutation in monoclonal precancers usually corresponded to a change from normal to an equivocal (two of 12) or hyperplastic (10 of 12) histology. Divergent K-ras genotypes among multiple neoplastic tissues of individual patients (two of six patients) are exceptions explained either by multicentric premalignant disease, or acquisition of K-ras mutation late in neoplastic progression. CONCLUSIONS: K ras mutation occurs in both premalignant microsatellite stable and unstable endometrial neoplasia, sometimes before acquisition of features readily diagnostic as atypical endometrial hyperplasia. PMID- 10748875 TI - p53 abnormalities in adenocarcinoma of the gastric cardia and antrum. AB - AIM: To compare the frequency and type of p53 alterations (gene mutation and/or protein overexpression) in a consecutive series of surgically resected adenocarcinomas arising in the gastric cardia and gastric antrum, and to evaluate associations with clinicopathological findings (age, sex, and tumour histology, grade, and stage). METHODS: The series comprised 50 patients with adenocarcinoma of the cardia and 20 patients with adenocarcinoma of the antrum. p53 gene mutations (exons 5-8) were detected by denaturing gradient gel analysis and DNA sequencing. Nuclear p53 overexpression was detected by immunohistochemistry with the DO7 antibody. RESULTS: p53 gene mutations were found in 21 of 50 and five of 20 adenocarcinomas of the cardia and the antrum, respectively. Base transitions occurring at CpG dinucleotides were frequent in both types of tumour. p53 protein overexpression was seen in 32 of 50 and seven of 20 adenocarcinomas of the cardia and of the antrum, respectively. p53 gene mutation and/or protein overexpression were significantly more frequent in adenocarcinomas of the cardia (37 of 50) than in adenocarcinomas of the antrum (seven of 20). There were no differences in the clinicopathological characteristics of the tumours between p53 positive and p53 negative cases in both types of cancer. CONCLUSIONS: This study shows that p53 alterations are more frequent in adenocarcinoma of the cardia than in adenocarcinoma of the antrum. This feature is consistent with the clinical and epidemiological characteristics of these cancers, which suggest that adenocarcinoma arising in the gastric cardia might be related to oesophageal adenocarcinoma, and unrelated to adenocarcinomas of the gastric body and antrum. PMID- 10748876 TI - Differential cytokine expression in EBV positive peripheral T cell lymphomas. AB - AIM: To investigate whether specific cytokines are secreted locally at the tumour site in Epstein-Barr virus (EBV) positive peripheral T cell lymphoma (PTCL). METHODS: An RNase protection assay system was used to study the differential expression of 21 cytokines in parallel in eight cases of EBV positive non-nasal PTCL, and compared with 11 EBV negative non-nasal PTCLs and three EBV positive nasal natural killer (NK) cell lymphomas. RESULTS: Among the eight EBV positive cases, interferon gamma (IFN-gamma), lymphotoxin beta (LT beta), interleukin 10 (IL-10), tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta 1 (TGF-beta 1), and IL-1 receptor a (IL-Ra) were frequently detectable. IL 15, IL-6, IL-4, IL-1 beta, TNF-beta, and IL-9 were sporadically detectable. Of the frequently detectable cytokines, IFN-gamma and LT beta were commonly detected in the EBV negative cases. For cases with > 50% EBV encoded small non polyadenylated RNA (EBER) positive cells, IL-10, TNF-alpha, and TGF-beta 1 were detected in three of three cases, and IL-1Ra in two of three cases. For cases with < 20% EBER positive cells, IL-10 was detected in three of five cases, TNF alpha in two of four cases, but TGF-beta 1 and IL-1Ra were not detected. Interestingly, IL-6 was detected in two of three cases with > 50% EBER positive cells, but only in one of five cases with < 20% EBER positive cells. For comparison, in NK cell lymphomas, IL-10, TNF-alpha, IL-1Ra, and IL-6 were all detectable, but TGF-beta 1 was not detected at all. Immunohistochemical staining revealed IL-10 in many cells; in contrast, EBV latent membrane protein 1 (LMP1) was only found to be positive in isolated cells. CONCLUSIONS: Certain cytokines, such as IL-10 and TNF-alpha, might be expressed preferentially in EBV positive peripheral T cell lymphomas. It is likely that such a cytokine environment enhances EBV infection and contributes towards tumorigenesis. PMID- 10748877 TI - The integration of HPV-18 DNA in cervical carcinoma. AB - AIMS: Little information is available on the patterns of integration into the host chromosomal DNA of cervical carcinomas of human papillomavirus type 18 (HPV 18) DNA, which is associated with up to 20% of these carcinomas. Because integration of the viral genome may be extremely important in the pathogenesis of cervical carcinoma, the aim of this study was to investigate which regions of HPV 18 DNA are integrated into the cellular DNA of cervical carcinomas. METHODS: Southern analysis using four subgenomic probes covering the entire HPV-18 genome was used to map viral DNA integrated within cellular DNA. The polymerase chain reaction (PCR) was used to confirm the presence of specific regions of the viral genome. RESULTS: In all 11 carcinomas there was a single major HPV-18 DNA integrant, retaining approximately 4000 bp of HPV-18 DNA, indicating that approximately half of the virus genome had been lost upon integration. Southern analysis suggested strongly that the viral breakpoint was within the E1/E2 gene boundary, with concomitant loss of part or all of the E2 ORF (open reading frame), all of the E4, E5, and L2 ORFs and part of the L1 ORF. These data were supported by the PCR results, which confirmed that the region of integrated HPV 18 DNA from nucleotides 6558 to 162 was present in all the carcinoma samples studied. Assuming that no genomic rearrangements, deletions, or insertions had occurred, 4131 bp of integrated HPV-18 DNA could be accounted for in eight cervical carcinoma samples. The results of Southern analysis also suggested that integration of HPV-18 DNA may have occurred at a specific host chromosomal site. CONCLUSIONS: Broadly, the viral sequences retained upon HPV-18 integration resemble those found when HPV-16 is integrated. However, it appears that the HPV 18 E2 region is more consistently deleted. PMID- 10748878 TI - PCR based detection of mycobacteria in paraffin wax embedded material routinely processed for morphological examination. AB - BACKGROUND: The incidence of mycobacterial infections has increased during the past five years. A prompt diagnosis is indispensable for initiating appropriate treatment. Because culturing of mycobacteria takes three to six weeks and sensitivity of microscopic detection of acid fast bacilli is low, amplification methods provide promising possibilities. Recently, the polymerase chain reaction (PCR) has been shown to be useful for confirming a mycobacterial infection, especially in cases with unexpected histological findings or lack of suitable material for culturing. AIMS: To evaluate the impact of PCR based techniques in the detection of mycobacterial infections in uncultured routine histological specimens as an alternative to surgical pathology. METHODS: Two hundred and twenty nine formalin fixed and paraffin wax embedded samples from 141 patients with clinical or histological suspicion of a mycobacterial infection were investigated using three different PCR assays and Southern blotting. PCR results were compared with histology and culture and the patients' clinical findings. RESULTS: When using culture as the reference method, the sensitivity for the detection of mycobacteria of the tuberculosis complex was 90%, specificity was 92%, the positive predictive value was 81%, and the negative predictive value was 96%. The sensitivity for the detection of nontuberculous mycobacteria was 100% and specificity was 78%, the positive predictive value was 26%, and the negative predictive value was 100%. The patients' clinical findings supported the PCR positive results, indicating a mycobacterial infection in 11 of 18 initially culture negative cases and in 21 of 35 PCR positive cases without culture results. CONCLUSIONS: These results indicate that PCR based techniques are sensitive, specific, and rapid methods for the detection of mycobacteria in routinely processed paraffin wax embedded and formalin fixed histological samples. PMID- 10748879 TI - Simplified preparation of human arterial sections for PCR analysis of Chlamydia pneumoniae and human DNA. AB - AIMS: To investigate multiple techniques for the preparation of solid tissue for polymerase chain reaction (PCR) analysis, and to identify the most simple techniques for routine use in the laboratory. METHODS: Techniques for the preparation of arterial tissue samples including homogenisation, ultrafiltration, and treatments involving proteinase K, Gene Clean, lectin, and Fe3+ specific chelators were evaluated using the PCR to amplify both Chlamydia pneumoniae and human DNA. RESULTS: Treatment with either Gene-Clean or lectin and the Fe3+ specific chelator deferoxamine mesylate removed PCR inhibitors from tissue homogenates. Homogenisation followed by GeneClean treatment resulted in the amplification of C pneumoniae DNA from within a section of atherosclerotic carotid artery, implying that C pneumoniae elementary bodies had been disrupted. In eight further clinical samples from patients not known to have C pneumoniae infection, human DNA was amplified and no cross contamination was observed between samples. These samples contained no evidence of C pneumoniae by PCR. CONCLUSIONS: A simple preparation of solid tissue for PCR analysis, involving homogenisation followed by GeneClean treatment has been developed, and is effective for the amplification of both C pneumoniae and human DNA. PMID- 10748880 TI - High throughput genotyping for the detection of a single nucleotide polymorphism in NAD(P)H quinone oxidoreductase (DT diaphorase) using TaqMan probes. AB - AIMS: The two electron reduction of quinones to hydroquinones by NAD(P)H quinone oxidoreductase (NQO1) plays an important role in both activation and detoxification of quinone and similarly reactive compounds. A single nucleotide polymorphism at exon 6 leads to an amino acid change at codon 187 from proline to serine. The variant allele has been associated with decreased NQO1 enzyme activity and increased cancer risks. The aim of this study was to develop a rapid genotyping procedure for epidemiological and clinical research into the potential biological and toxicological implications associated with this genetic polymorphism. METHODS: A high throughput genotyping method using fluorogenic probes has been developed to screen this single nucleotide polymorphism. This assay utilises the 5' nuclease activity of Taq polymerase in conjunction with fluorogenic TaqMan probes. The TaqMan genotyping procedure was validated by a restriction fragment length polymorphism method and direct sequencing. RESULTS: This method can be used for the rapid screening of known polymorphisms in large populations. In a population of 143 unrelated individuals, Pro/Pro (wildtype), Pro/Ser (heterozygous), and Ser/Ser (mutant) genotypes were 69.2%, 26.6%, and 4.2%, respectively. CONCLUSIONS: This genotyping method is highly accurate and could be applied to automated large scale genotyping studies. PMID- 10748881 TI - HLA-B*27 typing by sequence specific amplification without DNA extraction. AB - HLA-B27 appears to play a direct role in the pathogenesis of ankylosing spondylitis and almost all patients with this disease have HLA-B27. Therefore, a diagnosis of ankylosing spondylitis can virtually be excluded in the absence of HLA-B27. Many techniques have been used for HLA-B*27 typing. Of these, molecular methods are the most sensitive and specific but require extracted DNA as the testing material. A technique where HLA-B*27 is amplified directly from whole blood using sequence specific primers has been developed. This technique uses small sample volumes, is not restricted by choice of anticoagulant or sample age up to at least six weeks, and can be applied to other clinical polymerase chain reaction based procedures. PMID- 10748882 TI - Variability in the interpretation of microsatellite patterns with different electrophoretic conditions. AB - Microsatellite markers permit the analysis of microsatellite instability and loss of heterozygosity. Frequently, the allelotypes of microsatellites are interpreted in the presence of numerous bands in gels. The importance of different gel electrophoresis conditions in the interpretation of microsatellite patterns was tested. Microsatellite markers were used to amplify DNA from gastric cancer samples and adjacent gastric mucosa. Polymerase chain reaction (PCR) products were separated by electrophoresis through 7% polyacrylamide gels containing either 5.6 M urea and 32% formamide or 7 M urea. PCR reactions separated on urea/formamide gels resulted consistently in clear allele definition (one or two bands), whereas 7 M urea gels resulted in allele patterns that comprised multiple bands. Analysis of microsatellite abnormalities using nonformamide gels gave false negative results in just under a third of cases (four of 13). In conclusion, the interpretation of microsatellite alterations in cancer DNA is improved by using electrophoresis conditions that result in complete DNA denaturation, such as urea/formamide/acrylamide gel electrophoresis. PMID- 10748883 TI - Vertical transmission of HIV-1 infection. AB - Vertical transmission is the dominant mode of acquisition of infection for HIV infection in children, and about 1600 infants are newly infected each day worldwide. Without interventions the risk of transmission is between 15% and 35%, and associated with maternal disease progression, prematurity, duration of rupture of membranes, length of labour, and vaginal delivery. Breastfeeding approximately doubles the risk of vertical transmission; the additional risk of transmission through breastfeeding is approximately 15-20%, with about one-third of this accounted for by late postnatal transmission after 3 months of age. Current strategies to reduce the risk of transmission include a short course of anti-retroviral therapy, avoidance of breastfeeding and Caesarean section delivery. However, even if interventions late in pregnancy or around the time of delivery are highly effective in preventing perinatal infection, it is likely that as a public health policy they are of interest only if alternatives to breastfeeding are feasible, affordable, safe and available. PMID- 10748884 TI - HIV infection in children in developing countries. AB - Disease progression in children acquiring HIV infection vertically from their mothers is more rapid in developing countries compared with developed countries. The probability of death by 12 months in sub-Saharan Africa ranges from 0.23 to 0.35, and by 5 years is 0.57-0.68. Data from Europe in the era before highly active anti-retroviral therapy (HAART) yielded probabilities of 0.1 and 0.2, respectively. Confirming the diagnosis can be difficult in resource-limited settings. Existing clinical case definitions are useful epidemiologically, but of low positive-predictive value in individual children. Priorities for research into management issues include nutrition (infant feeding, vitamin A and micronutrient supplementation), prophylaxis against Pneumocystis carinii pneumonia (PCP), and bacterial infections, case management of persistent diarrhoea, diagnosis/prevention/management of tuberculosis in children and prevention of sexual transmission in adolescents. PMID- 10748885 TI - Childhood tuberculosis--problems ahead. AB - Childhood tuberculosis (TB) is on the increase, both in developing countries and in the UK. Children cannot usually be diagnosed as having TB by sputum microscopy and culture alone, so millions of children are destined to die of undiagnosed TB in poor countries. Drug resistance is likely to affect a greater proportion of TB cases in children, because they have been recently infected by adults. Whilst BCG vaccination can protect against miliary TB and TB meningitis, it will not interrupt the chain of transmission. HIV co-infected mothers are capable of passing congenital TB to their children. PMID- 10748886 TI - Dengue: emergence as a global public health problem and prospects for control. AB - Dengue causes more illness and death than any other arboviral infection: there are at least 20 million infections in the world each year and several hundred thousand cases of a severe, life-threatening syndrome known as dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS). In recent years, the geographical range of dengue has extended and DHF/DSS is occurring in new areas and with increased incidence. The reasons for the resurgence are complex, but parallel demographic changes and reduced efforts towards disease control. Control of dengue currently depends on controlling its mosquito vector. However, development of a vaccine offers greater hope in the long-term. The dengue group of flaviviruses is unique in that it comprises 4 distinct serotypes which have the potential to cause sequential infections with increased severity. It is reasoned, therefore, that any vaccine should induce solid immunity to all 4 serotypes. Knowledge regarding dengue immunity and pathogenesis is rapidly advancing and it is reasonable to believe that this information can be used to devise a safe and efficacious vaccine for dengue, but it seems unlikely that this will become available in the near future. PMID- 10748887 TI - Integrated management of childhood illness in resource-poor countries: an initiative from the World Health Organization. AB - It is estimated that each year around 12 million children aged < 5 years die in resource-poor countries and that 70% of these deaths are due to communicable diseases and/or malnutrition. The same conditions are responsible for an even higher percentage of childhood illness. Since the mid-1990s the World Health Organization has been leading the development of an integrated approach to care for ill children at the primary care level, a programme know as Integrated Management of Childhood Illness (IMCI). The approach essentially combines improved management of childhood illness with aspects of nutrition, immunization and maternal health. IMCI replaces or complements a number of 'vertical' child health programmes aimed at specific groups of conditions including control of diarrhoeal diseases (CDD), acute respiratory infections (ARI) and the Expanded Programme on Immunization (EPI). As of late 1998 the programme, at various stages of development, had been introduced to 51 countries: Introduction (19 countries), Early Implementation (29 countries) or Expansion (9). The approach has many advantages not least that it is well accepted by tropical country paediatricians because it conforms to practice in secondary care. In some countries paediatricians are playing a greater leadership role than they did with previous specific programmes. Many problems remain: programmatic issues, probable over diagnosis of malaria, relationships with other specific initiatives ('Roll Back Malaria' and new-born care) and how to integrate HIV infection into the diagnosis and care 'package'. However the initiative deserves support by paediatricians and public health specialists in industrialized countries. PMID- 10748888 TI - Malnutrition: trials and triumphs. AB - Severe malnutrition is uncommon but often fatal, particularly in very young infants or when oedema is present. Another major contributor to mortality is undiagnosed infection. Three pilot studies have recently been performed in severely malnourished patients in therapeutic feeding centres in sub-Saharan Africa. In each, a practical management problem was addressed and a potential solution tested. Three conclusions were reached: young breastfeeding infants were best managed using a supplemented suckling technique; routine antibiotics from admission reduced mortality; and in adults with oedematous malnutrition, therapeutic diets with a lower-than-usual protein:energy ratio were effective in reducing mortality and permitting catch-up weight gain. PMID- 10748889 TI - Open questions in the case management of sick children. AB - Despite much progress in the specific treatment of diseases in children, some basic management questions remain controversial. Examples for this are fluid management of children with infectious diseases and the control of fever in children. Fluid restriction in children with meningitis has been recommended by several authorities in the field; the basis for these recommendations is reviewed, and reasons are outlined why these recommendations may not be appropriate. Similarly, there are few data on which to base recommendations for fluid management in severe pneumonia and cerebral malaria. Some activities supported by WHO are presented. In the management of fever, opinions vary whether fever is a useful protective factor in combating infections, or whether fever might be harmful and should be lowered. Results of a recent survey of experts on this topic are presented, and the research agenda in the field of supportive management of childhood infections is outlined. PMID- 10748890 TI - Household risk factors for malaria among children in the Ethiopian highlands. AB - Malaria transmission varies from village to village and even from family to family in the same village. The current study was conducted in northern Ethiopia to identify risk factors responsible for such variations in a hypoendemic highland malaria setting: 2114 children aged < 10 years living in 6 villages situated close to small dams at altitudes from 1775 to 2175 m were monitored. Monthly malaria incidence was determined 4 times over a 1-year period during 1997. Incidence results were then analysed by 14 individual and household factors using Poisson multivariate regression. Among 14 factors analysed, use of irrigated land (rate ratio[RR] = 2.68, 95% CI 1.64-4.38), earth roof (RR = 2.15, 95% CI 1.31-3.52), animals sleeping in the house (RR = 1.92, 95% CI 1.29-2.85), windows (RR = 1.84, 95% CI 1.30-2.63), open eaves (RR = 1.85, 95% CI 1.19-2.88), no separate kitchen (RR = 1.57, 95% CI 1.10-2.23), and 1 sleeping room (RR = 1.52, 95% CI 1.05-2.20), were significantly associated with malaria. The proportion of infection among children exposed to one or no risk factor was 2.1%, increasing with the number of risk factors and reaching 29.4% with 5 or more. Further studies are needed to confirm the importance of particular risk factors, possibly leading to simple health education and control measures that could become part of routine control programmes, implemented with inter-sectoral collaboration. PMID- 10748891 TI - Hookworms in dogs of Kuching, Sarawak (north Borneo). PMID- 10748892 TI - Natural ecotopes of Triatoma infestans dark morph and other sylvatic triatomines in the Bolivian Chaco. AB - A survey of natural ecotopes of Triatoma infestans dark morph and other triatomine sylvatic species was performed in an uninhabited area of the Bolivian Chaco. Among the 321 triatomines collected by light trapping, only 4 T. infestans dark morph specimens were identified. Predominant flying species were T. guasayana and T. sordida group 2 (51.7% and 37.1% of capture, respectively). The same species prevailed in terrestrial and epiphytic bromeliads where scarce T. infestans dark morph nymphal instars were also detected. In parrot nests T. delpontei prevailed broadly over other species (90.2% of the capture) and only 4 T. infestans dark morph adults were collected. In contrast, T. infestans dark morph was the predominant species captured in hollow trees (46.0% of the total collected). The abundance of immature forms (88.2% of the collection) shows that hollow trees constitute a favourable ecotope for this species. Of the 421 trees investigated, 33.7% were positive for triatomines. T. infestans dark morph, found inside 15.0% of them, also had higher apparent density than other species (average number of T. infestans in positive trees, 2.0 +/- 1.6 vs 1.3 +/- 0.6 for other species). Light trapping seems to be an efficient method to sample the T. sordida-T. guasayana complex in that it shows a similar distribution to that observed in natural ecotopes; however, this method is ineffective for the assessment of the local abundance of T. infestans dark morph. PMID- 10748893 TI - Transmission ecology of the fly Musca sorbens, a putative vector of trachoma. AB - Recent evidence suggests that eye-seeking flies are important trachoma vectors. We conducted a series of investigations to identify which species of synanthropic flies are potential vector(s) of this blinding disease in The Gambia. Several species of fly were caught in fish-baited attractant traps placed in villages throughout the year (1997/98) but only 2 species, Musca sorbens and M. domestica, were caught from the eyes of children. M. sorbens comprised < 10% of the total number of flies caught with attractant traps but was responsible for > 90% of fly eye contacts, the remainder were made by M. domestica. All fly species were more numerous in the wet season than the dry season. Eyes of young children are considered to be the main reservoir of Chlamydia trachomatis, the causative agent of trachoma. Collections of eye-seeking flies from children showed frequent fly eye contacts (median [interquartile range], 3 [1.5-7] every 15 min). Children with potentially infective ocular or nasal discharge had twice as many fly-eye contacts than children with no discharge (P < 0.001). There was no difference in exposure to fly-eye contacts if a child sat inside or outside a house (P = 0.273). Female flies were more commonly caught from eyes than male (P < 0.001). The presence of Chlamydia DNA was demonstrated by PCR on 2 of 395 flies caught from the eyes of children with a current active trachoma infection. Both positive flies were M. sorbens, one male and the other female. Further elucidation of M. sorbens behavioural ecology and the development of sustainable strategies to control these flies should be a priority. It is likely that M. sorbens is the principal insect vector of trachoma in The Gambia. PMID- 10748894 TI - Detection of antibodies to Leishmania donovani in animals in a kala-azar endemic region in eastern Sudan: a preliminary report. AB - The prevalence of antibodies against Leishmania donovani in selected domestic and wild animal species in 2 villages in Sudan with active L. donovani transmission in humans was investigated. Screening of domestic animals (donkeys, cows, sheep, goats, camels and dogs) with the direct agglutination test (DAT) detected reaction rates above the cut-off titres in donkeys (68.7%), cows (21.4%) and goats (8.5%), and which were also found in wild rats (5.5%). Sera of sheep, camels and dogs had a weak agglutination reaction below the cut-off titre. Testing of the same sera by enzyme-linked immunosorbent assay (ELISA), against a lysate of L. donovani promastigotes, showed reaction rates above the cut-off optical density in cows (47.6%), goats (13.6%), and in rats (4.1%). No Leishmania parasite was isolated from spleen, liver, bone-marrow or spleen of Nile rats. PMID- 10748895 TI - Mapping and estimating the population at risk from lymphatic filariasis in Africa. AB - Lymphatic filariasis remains a major public health problem in Africa and is 1 of the World Health Organization's 6 diseases targeted for global eradication. However, no detailed maps of the geographical distribution of this disease exist, making it difficult to target control activities and quantify the population at risk. We hypothesized that the distribution lymphatic filariasis is governed by climate. The climate at sites in Africa where surveys for lymphatic filariasis had taken place was characterized using computerized climate surfaces. Logistic regression analysis of the climate variables predicted with 76% accuracy whether sites had microfilaraemic patients or not. We used the logistic equation in a geographical information system to map risk of lymphatic filariasis infection across Africa, which compared favourably with expert opinion. Further validation with a quasi-independent data set showed that the model predicted correctly 88% of infected sites. A similar procedure was used to map risk of microfilaraemia in Egypt, where the dominant vector species differs from those in sub-Saharan Africa. By overlaying risk maps on a 1990 population grid, and adjusting for recent population increases, we estimate that around 420 million people will be exposed to this infection in Africa in the year 2000. This approach could be used to produce a sampling frame, based on estimated risk of microfilaraemia, for conducting filariasis surveys in countries that lack accurate distribution maps and thus save on costs. PMID- 10748896 TI - Murine typhus in Mexico City. PMID- 10748897 TI - Resurgence of cases of epileptic seizures and burns associated with cysticercosis in Assologaima, Jayawijaya, Irian Jaya, Indonesia, 1991-95. AB - Historically, neurocysticercosis (NCC) caused by the larval stage, cysticercus or cysticerci, of the pork tapeworm Taenia solium was recognized in Paniai District, western Irian Jaya Province, Indonesia, in the early 1970s. In the 1990s, we observed a rapid increase in the number of cases of epileptic seizures and burns in Assologaima Sub-District, Jayawijaya District, eastern Irian Jaya. There were totals of 1120 new cases of burns and 293 new cases of epileptic seizures during 1991-95 in Assologaima where the number of inhabitants was 15,939. Histopathological examination of resected cysts from patients and a pig revealed that they were cysticerci of T. solium. DNA analysis of these cysts revealed that the nucleotide sequences of 391 base-pair fragments of the mitochondrial cytochrome c oxidase subunit 1 gene were exactly the same in those from patients and the pig. Although 3 of 391 base-pair fragments might differ from that of T. solium reported previously, there were no differences in the amino-acid sequences. Approximately 67% and 65% of persons with epileptic seizures and with subcutaneous nodules, respectively, showed antibody responses highly specific to cysticercosis. Therefore, most cases of epileptic seizures and burns were considered to be associated with cysticercosis in Irian Jaya. PMID- 10748898 TI - A new case of cutaneous infection by a presumed monoxenous trypanosomatid in the island of Martinique (French West Indies). PMID- 10748899 TI - Distribution of hepatitis B virus markers among surgical specialties in Lagos, Nigeria. PMID- 10748900 TI - Performance of a commercial immunochromatographic test for the diagnosis of vivax malaria in Turkey. PMID- 10748901 TI - ParaSight-F test results in cerebral malaria patients before and after treatment in Chittagong Medical College Hospital, Bangladesh. PMID- 10748902 TI - Supra-sternal notch tuberculous abscess: a report of three cases. AB - Extra-pulmonary tuberculosis remains a diagnostic and therapeutic challenge; its clinical presentation can mimic a wide range of pathological conditions. Here we report on 3 female patients who presented with supra-sternal masses that were suspected clinically to be of thyroid origin. By use of fine-needle aspiration cytology (FNAC), they were proved to be tuberculous lesions involving the pre tracheal lymph nodes. Serological examination for HIV-I/II was not reactive in the 3 patients. The patients responded well to a regimen of multi-drug therapy. It is concluded that extra-pulmonary tuberculosis should be considered in the differential diagnosis of thyroid or para-thyroid swellings and that FNAC is a simple, quick and reliable procedure in the diagnosis of extra-pulmonary tuberculous lesions involving the neck. PMID- 10748903 TI - Possible association between Taenia solium cysticercosis and cancer: increased frequency of DNA damage in peripheral lymphocytes from neurocysticercosis patients. AB - Helminths, particularly some Schistosoma species, have been associated with cancer in humans. Neurocysticercosis, produced by cysticerci of the helminth Taenia solium, has been associated with the emergence of brain tumours and haematological malignancies. Local tumours, such as glioblastoma, could be explained by the induction of DNA damage in cells surrounding the cysticercus and chronically exposed to an inflammatory host response. However, systemic effects such as haematological malignancies are not easy to understand. The present work was conducted in Mexico to find out whether DNA damage arises in peripheral lymphocytes in patients with neurocysticercosis. We utilized a highly sensitive technique to analyse chromosomal aberrations, in-situ hybridization with probes against chromosomes 1, 2 and 4, and in addition the blocked-cytokinesis technique was used to determine the formation of micronuclei, a peculiar form of DNA damage. The study was made in lymphocytes from 8 patients before and after the administration of praziquantel, 1 of the 2 drugs used for neurocysticercosis treatment. The frequencies of chromosome aberrations and micronuclei in peripheral blood lymphocytes were higher in the infected patients as compared to those observed both in healthy donors and in the group of patients after praziquantel therapy. Our results suggest that chromosome aberrations induced in peripheral cells during neurocysticercosis could be associated with the development of haematological neoplasias. PMID- 10748904 TI - Hyaluronate levels and markers of oxidative stress in the serum of Sudanese subjects at risk of infection with Schistosoma mansoni. AB - We showed previously that infection by Schistosoma mansoni not only triggers the production of reactive oxygen species in mouse liver but also leads to the alteration of antioxidant defences. To determine whether such events occur in humans, we measured the serum markers of oxidative stress, i.e., lipid peroxides and protein carbonyl, as well as hyaluronate levels in subjects in the Managil area of the Sudan. Grades of fibrosis were determined by ultrasonographic examination. Two groups were used as controls, one Sudanese and the other European. We found that Sudanese subjects in the endemic area differed from the control groups, both Sudanese and European, insofar as they had higher levels of the serum metabolites measured. The latter increased with the grade of fibrosis. Moreover, protein carbonyl and hyaluronic acid levels correlated positively with lipid peroxide levels. These findings indicate that oxidative stress might contribute to S. mansoni-associated pathology in man. The serum markers considered in our study, obtained by relatively simple techniques, may provide a useful biochemical index for the identification of almost asymptomatic patients who, however, are at risk of developing severe schistosomiasis. PMID- 10748905 TI - Pulmonary haemorrhage as a predominant cause of death in leptospirosis in Seychelles. AB - We examined the cause of death during a 12-month period (1995/96) in all consecutive patients admitted to hospital with leptospiral infection in Seychelles (Indian Ocean), where the disease is endemic. Leptospirosis was diagnosed by use of the microscopic agglutination test and a specific polymerase chain reaction assay on serum samples. Seventy-five cases were diagnosed and 6 patients died, a case fatality of 8%. All 6 patients died within 9 days of onset of symptoms and within 2 days of admission for 5 of them (5 days for the 6th). On autopsy, diffuse bilateral pulmonary haemorrhage (PH) was found in all fatalities. Renal, cardiac, digestive and cerebral haemorrhages were also found in 5, 3, 3 and 1 case(s), respectively. Incidentally, haemoptysis and lung infiltrate on chest radiographs, which suggest PH, were found in 8 of the 69 non fatal cases. Dengue and hantavirus infections were ruled out. In conclusion, PH appeared to be a main cause of death in leptospirosis in this population, although haemorrhage in other organs may also have contributed to fatal outcomes. This cause of death contrasts with the findings generally reported in endemic settings. PMID- 10748907 TI - The efficacy of pyrimethamine-sulfadoxine (Fansidar) in the treatment of uncomplicated Plasmodium falciparum malaria in Kenyan children. PMID- 10748906 TI - The mosquito transmission of malaria: the effects of atovaquone-proguanil (Malarone) and chloroquine. AB - Despite its recognized importance, the prevention of patients with malaria from continuing to infect mosquitoes after treatment is not always achieved in practice. An inevitable consequence of the prolonged life-span and relative metabolic stasis of the mature gametocytes of Plasmodium falciparum is that they are not cleared by most antimalarials, and few antimalarials block infection in the mosquito vector. Previous research on the constituents of Malarone, a new 'combined antimalarial', suggested that the active components, atovaquone and proguanil, might inhibit infectivity of gametocytes to mosquitoes. We contrast here the impact of atovaquone-proguanil and chloroquine on the transmission of P. falciparum and P. berghei. While chloroquine enhanced infectivity of P. falciparum, atovaquone-proguanil caused a significant reduction. Surprisingly, sporontocidal activity against the rodent parasite persisted long after the levels of the constituent drugs would have been expected to have fallen below effective plasma concentrations on the basis of the established pharmacokinetics of atovaquone and proguanil. The P. berghei model may thus have provided a sensitive bioassay, detecting drug(s) at levels below that normally found with the usual chemical assays. PMID- 10748908 TI - Mass treatment against human taeniasis for the control of cysticercosis: a population-based intervention study. AB - An intervention study with mass treatment against taeniasis to prevent neurocysticercosis due to Taenia solium in a rural community in Mexico was performed in 1991-96. Information and biological samples were obtained at the beginning of the study, at 6 months and at 42 months after mass treatment with praziquantel at a single dose of 5 mg/kg. Prevalence rates of taeniasis were measured by the detection of Taenia coproantigens and Taenia eggs in faeces; neurocysticercosis was suggested by clinical data and by serum antibodies in humans and also in swine. A reduction of 53% after 6 months and of 56% after 42 months for human taeniasis was seen after treatment. Late-onset general seizures decreased 70%. Anti-cysticercus antibodies in the human population were reduced by 75% after 42 months. Antibodies in pigs also showed a significant reduction of 55% after 6 months. In conclusion, an impact of mass chemotherapy against taeniasis to control cysticercosis in the short and long term was demonstrated. Praziquantel for tapeworm treatment should not be given at doses lower than 10 mg/kg. Late-onset convulsive crisis and specific antibodies are good indicators of neurocysticercosis and of exposure to the parasite, respectively. PMID- 10748909 TI - Risk factors for severe malaria: importance of careful study design: a reply. PMID- 10748910 TI - Efficacy of artesunate against Schistosoma mansoni infections in Richard Toll, Senegal. PMID- 10748911 TI - Studies in vitro on the relative efficacy of current acaricides for Sarcoptes scabiei var. hominis. AB - Resistance of Sarcoptes scabiei to various topical therapies has been described, but clinical assessment of treatment failure is problematic and in-vitro assays are generally not available. We describe a simple in-vitro analysis used to evaluate the relative efficacy of a range of topical, oral, and herbal treatments available in Australia for the treatment of scabies. S. scabiei var. hominis mites were collected from skin scrapings obtained from 7 crusted scabies patients over a period of 2 years (1997 and 1998). Larvae, nymphal instars, and adult mites were tested within 3 h of collection and continuously exposed to selected commercially available treatment products until death, with the elapsed time recorded. Neem was the only product to show little acaricidal activity. Survival curves indicated that, of the other agents, 5% permethrin (Lyclear) had the slowest killing time, with 35% of mites still alive after 3 h, and 4% still alive after 18-22 h of constant exposure. In contrast, no mites were alive after 3 h exposure to 25% benzyl benzoate (Ascabiol), 1% lindane (Quellada), 5% tea tree oil and 100-8000 ng/g of ivermectin (Equimec). Despite the slower killing time with 5% permethrin, there was no evidence of any mite tolerance in vivo or treatment failure in any patients or contact cases. PMID- 10748912 TI - Assessment of the immunological surveillance value of humoral and lymphocyte assays in severe human cystic echinococcosis. AB - Thirty cystic echinococcosis (CE) patients in Uruguay with severe bone or secondary disseminated echinococcosis were immunologically assessed using cellular (lymphocyte transformation assay, LTA) and humoral (specific antibody and subclass responses, circulating antigen and circulating immune-complexes) immunological assays during the course of chemotherapy (albendazole and/or praziquantel). CE patients were divided into 4 groups, according to clinical treatment and outcome: (I) surgery and chemotherapy, (II) chemotherapy with outcome unchanged, (III) chemotherapy with outcome improved, and (IV) chemotherapy considered cured. Increased circulating antigen was of prognostic value in some severe CE cases where levels remained high and/or increased. The lymphoproliferative response in vitro to Echinococcus granulosus antigen was statistically greater in all patient groups compared to normal individuals but at lower levels in improved or cured CE patients. Levels of non-specific LTA response were significantly lower than controls for all groups during albendazole treatment (P < 0.001) but returned to normal levels in cured patients, a result consistent with parasite-induced suppression of cellular responses. This study suggests that, at least in severe osseous and secondary CE, immunosurveillance by specific antibodies, especially total specific immunoglobulin, was overall of more practical use than antigen-specific in-vitro lymphocyte transformation assays. PMID- 10748913 TI - Comparison of artificial membrane feeding with direct skin feeding to estimate infectiousness of Plasmodium falciparum gametocyte carriers to mosquitoes. AB - Human infectiousness to mosquitoes can be estimated by 2 tests: direct feeding on the skin and membrane feeding on venous blood. To validate the membrane feeding assay, the infectiousness of Plasmodium falciparum gametocyte carriers to Anopheles gambiae was estimated by these 2 methods in the same individuals in a rural area of Cameroon. Results from 37 experiments showed that direct feeding gave significantly higher infection rates than membrane feeding. We observed an average of 19.4% infected mosquitoes by direct feeding compared with 12.1% by membrane feeding, and a mean oocyst load of 5.63 by direct feeding compared with 2.65 by membrane feeding. However, there was a very good concordance between the 2 tests: 84.3% with the Kappa test on percentages of infected mosquitoes and 98.7% with the interclass correlation coefficient on oocyst loads. In addition, we found a good linear correlation between the 2 methods. PMID- 10748914 TI - Infectivity of malaria vector mosquitoes: correlation of positivity between ELISA and PCR-ELISA tests. PMID- 10748915 TI - Monitoring for the emergence of new foci of onchocerciasis (river blindness) in the Americas. PMID- 10748916 TI - Sickle cell disease and vitamin E deficiency in children in developing countries. PMID- 10748917 TI - L-arginine transport in retinas from streptozotocin diabetic rats: correlation with the level of IL-1 beta and NO synthase activity. AB - Several evidences suggest that the pro-inflammatory cytokines IL-1 beta and the radical NO are implicated as effectors molecules in the pancreatic beta-cells dysfunction; an event preceding the pathogenesis of diabetes. IL-1 beta induces the expression of the inducible isoform of NO synthase (iNOS), which use L arginine as substrate to overproduce NO. However, it is not known whether these events may participate in the development of diabetic retinopathy, which is the main cause of blindness. In this work, we found an increased level of IL-1 beta in retinas from streptozotocin-induced (STZ) diabetic rats. We also observed that the activity of the NO synthase (NOS) and the L-arginine uptake are enhanced in retinas from STZ-induced diabetic rats as compared to retinas from control rats. We found that the uptake of L-arginine in retinas from control and diabetic rats occurs through a transporter resembling the Y + system, i.e. it is saturable, not affected over the pH range 6.5 to 7.4, and is independent of the extracellular Na+. Nevertheless, the L-arginine transport in retinas from diabetic rats occurs through a carrier with lower affinity (K(m) = 25 microM) and higher capacity (Vmax = 295 +/- 22.4 pmol L-arginine/mg protein) than in retinas from control rats (K(m) = 5 microM and Vmax = 158 +/- 12.8 pmol L-arginine/mg protein) which is correlated with the increased NOS activity and consequent depletion of the intracellular pool of L-arginine. PMID- 10748918 TI - Spatial frequency bandwidth used in the recognition of facial images. AB - The purpose of the study was to find out what spatial frequency information human observers use in the recognition of face images. Signal-to-noise ratio thresholds for the recognition of facial images were measured as a function of the centre spatial frequency of narrow-band additive spatial noise. The relative sensitivity of recognition to different spatial frequencies was derived from these results. The maximum sensitivity was found at 8-13 c/face width and the bandwidth was just under two octaves. Qualitatively similar results were obtained with stimuli in which Fourier phase was randomised within a narrow band of different centre spatial frequencies. This resulted in a considerable increase of energy threshold around 8 c/face width and less elsewhere. Further, contrast energy thresholds were measured as a function of the centre spatial frequency of band-pass filtered face images. As a function of object spatial frequency (c/face width), energy threshold first decreased and then increased. The lowest energy thresholds found around 10 c/face width were lower than the energy threshold for unfiltered images. This is what one would expect if face recognition is narrow-band, since band-pass filtered images of optimal centre spatial frequency do not contain unused contrast energy at low and high spatial frequencies. In conclusion, the results suggest that the recognition of facial images is tuned to a relatively narrow band (< 2 octaves) of mid object spatial frequencies. PMID- 10748919 TI - Interaction between the perceived shape of two objects. AB - The difference between the way in which binocular disparity scales with viewing distance and the way in which motion parallax scales with viewing distance introduces a potential indirect cue for viewing distance: the viewing distance is the only distance at which disparity and motion specify the same depth. The present study examines whether this information is used. Two simulated ellipsoids were presented on a computer screen in complete darkness. The two ellipsoids were 6 degrees to the left and right of straight ahead. Subjects set the width and depth of each ellipsoid to match a tennis ball, and set the distance of the one on the right to half that of the one on the left. The distance of the left ellipsoid varied between trials. On half of the trials it was static. On the other half it was rotating up and down around its frontal horizontal axis. Rotating the left ellipsoid influenced its set depth: rotating ellipsoids were set to be much more spherical. There was no influence on the set depth of the other ellipsoid, or on the set width of either. The set distance of the right ellipsoid was also unaffected. We conclude that subjects do not combine binocular disparity and motion parallax to obtain more veridical information about viewing distance. PMID- 10748920 TI - Concurrent measurement of perceived speed and speed discrimination threshold using the method of single stimuli. AB - Velocity matching using the method of Constant Stimuli shows that perceived velocity varies with contrast [Thompson, P. (1982). Perceived rate of movement depends upon contrast. Vision Research, 22, 377-380]. Random contrast jitter would therefore be expected to increase the slopes of psychometric functions, and thus the velocity discrimination threshold. However, McKee, S., Silverman, G., and Nakayama, K. [(1986) Precise velocity discrimination despite random variation in temporal frequency. Vision Research, 26, 609-620] found no effect of contrast jitter on thresholds, using the method of single stimuli. To determine whether this apparent discrepancy is due to the difference in methodology, or to the different ranges of temporal frequencies used in the two studies, we used the method of single stimuli to measure psychometric functions at three different velocities (0.5, 2.0 and 4.0 degrees/s). We found that contrast jitter increased thresholds at low but not at high velocities. Separate analysis of the psychometric functions at each contrast level showed that increases in contrast increased perceived velocity at low standard speeds (0.5 degree/s) but not at high. We conclude that the effect of contrast on perceived speed is real, and not a methodological artefact, but that it is found only at low temporal frequencies. PMID- 10748921 TI - Pattern detection in the presence of maskers that differ in spatial phase and temporal offset: threshold measurements and a model. AB - Four experiments are described in which brief Gabor patterns are detected in the presence of full-field gratings or Gabor patterns that are superimposed in space, but vary in spatial phase and temporal offset (SOA). E1: Threshold versus masker contrast (TvC) functions were determined for relative phases of 0, 90, 180 and 270 degrees at SOA = 0. For 0 degree relative phase, TvC functions decrease (facilitation) and then increase (masking) as contrast increases. For 90 degrees, there is little or no facilitation and thresholds increase with masker contrast. For 180 degrees, the form of the TvC function varies with observer and conditions. E2: Like E1, except that maskers are Gabor patterns. TvC functions are similar in form to those for full-field maskers, but there is less masking. E3: Forward masking. TvC functions were determined for relative phases of 0, 90, and 180 degrees at SOA = -33 ms. The forms of the TvC functions for 0 and 180 degrees are reversed relative to those at SOA = 0. E4: TvP (threshold versus phase) functions were determined for SOA's of -100, -67, -33, 0 and 33 ms at a constant masker contrast of 0.063. Masking occurs at all relative phases. For simultaneous and backward masking, the threshold is minimum for a relative phase of 0 and maximum at 180 degrees. For forward masking, the form of the function is inverted. A model of pattern masking and facilitation (Foley, J. M. (1994a) Journal of the Optical Society of America A, 11, 1710-1719) is extended to account for masker phase and SOA effects. The model assumes four mechanisms tuned to phases 90 degrees apart, and divisively inhibited by stimuli of all phases. Performance depends on the detection strategy of the observer. PMID- 10748922 TI - Bezold-Brucke hue-shift as functions of luminance level, luminance ratio, interstimulus interval and adapting white for aperture and object colors. AB - For spectral and nonspectral aperture color 3 s stimuli, Bezold-Brucke (B-B) hue shift was measured for one to six subjects for four functions: (1) luminance (L) level from 0.1:1.0 through 100:1000 cd/m2; (2) L ratio from 1:2 through 1:1000; (3) illuminant color temperature (for seven illuminants including A and D65) by two adaptation methods; and (4) interstimulus interval from 0 through 40 s; the latter removes contrast effects and inverts hue-shift, so B-B 'invariants' become hue-shift maxima. SUMMARY: four wavelengths are 'invariant' for a given L level or ratio. With higher L level, invariants shift as opposed pairs: to longer wavelength for b and y, shorter for g and r. With higher L ratio, b and y invariants shift some 10 nm longer but g and r remain about 508 nm and 493.5 c. With higher illuminant color temperature, invariants (1:10 cd/m2) shift shorter for b and y, r slightly longer (spectral to nonspectral 494 c) but g seems constant. Hue-shift data for real object color are very similar to aperture color. The full hue cycle is graphed to the recent extended wavelength scale for CIE dominant wavelength. Many effects are newly reported. PMID- 10748923 TI - Unique and binary hues as functions of luminance and illuminant color temperature, and relations with invariant hues. AB - For aperture color 3 s stimuli, the four unique hues, and at times the four (equally-balanced) binary hues, were measured for: (1) adapting white color temperatures 2850, 3400, 5500, 6500 K; and (2) luminance (L) for 5 and 6 log L ranges (about 0.01-3200 cd/m2) in 3.2:1 L steps (viewed singly) for 1 subject per white, 3 log L in 10:1 steps for 6 subjects for white 6500 K, and 1 log L for 2 11 subjects per white. The full hue cycle is graphed to the extended wavelength scale. With higher L, unique hues *b and *y shift longer, *g and *r shorter, and spectral binaries and 460 nm are invariant wavelengths, for Ls viewed singly. But for Ls viewed in successive contrast (Bezold-Brucke effect), unique hues are practically invariants. As interstimuli interval increases from 0-40 s (from successive L contrast to no-contrast), invariants shift away from uniques (which become hue-shift maxima) and coincide with spectral binaries (but not r/b, 565 c) at 495, 546, 600 nm. Successive L contrast switches spectral uniques' hue-shift off (as invariants), and spectral binaries on (as hue-shift maxima); and no contrast switches the reverse. With higher illuminant color temperature 2850-6500 K, wavelength of 10 constant hues shortens 5-10 nm for *b, *y, g/y, y/r, but others are constant +/- 2 nm. PMID- 10748924 TI - Development of the optokinetic system in macaque monkeys. AB - Optokinetic nystagmus in response to horizontal movement of a whole field random dot pattern was measured in infant macaque monkeys from the first week to about 5 months after birth using electrooculography. During monocular and binocular viewing conditions stimulus velocities were varied between 10 and 120 degrees/s. Monocular stimulation in the temporonasal direction yielded slow phase gain of the optokinetic system which was relatively constant for a given stimulus velocity over the whole period of observation. Gain during nasotemporal stimulation was also clearly present but significantly lower at early stages and increased during further development. This asymmetry of monocular horizontal optokinetic nystagmus (OKN) clearly depended on the stimulus velocity. At lower stimulus velocities (10-20 degrees/s) OKN was largely symmetrical at 2-5 weeks of age. At higher stimulus velocities (40 degrees/s) symmetry was reached at about 12 weeks of age or even much later (80-120 degrees/s). PMID- 10748925 TI - The memory template in Drosophila pattern vision at the flight simulator. AB - Pattern recognition is studied in flight orientation of fixed flying Drosophila melanogaster controlling the horizontal rotations of an arena. Earlier experiments had suggested a simple mechanism of pattern recognition in which a memory template and the actual image are retinotopically matched. In contrast, we now show that Drosophila extracts at least two and probably four pattern parameters: size, vertical position of the center of gravity and, presumably horizontal/vertical extent as well as vertical separatedness of pattern elements. Moreover, the fly treats isolated pattern elements as a compound figure. Retinal transfer is possible between training and test if the centers of gravity of the compound figures are retained. PMID- 10748926 TI - The variation of torsion with vergence and elevation. AB - Two recently developed kinematic models of human eye movements predict systematic departures from Listing's law which are associated with changes in vergence. This vergence-dependent torsion t is proportional to elevation e and vergence v, that is t = kev/2. The proposed value for k is either 1 (Van Rijn, L. J., & Van den Berg, A. V. (1993). Vision Research, 33, 691-708) or 1/2 (Minken, A. W. H., Gielen, C. C. A. M., & Van Gisbergen, J. A. M. (1995). Vision Research, 35, 93 102). One implication of both models is that an eye with a constant fixation direction should exhibit systematic torsional variation during movements of the other eye. This paper therefore examines the torsion produced by moving a fixation target inwards and outwards along the line-of-sight of the right eye at five different viewing elevations (0, +/- 15 and +/- 30 degrees). In a monocular analysis, each eye generally showed intorsion during convergence at positive elevation angles, whereas extorsion occurred at negative elevations; the opposite was true during divergence. However, the torsion response was visibly different between the five subjects, and depended on the direction of target motion. In a binocular analysis, cycloversion (mean of left and right eye torsion) varied dramatically both between subjects and between convergence and divergence; however, cyclovergence (torsional difference) was much less variable. Least squares methods were used to estimate the constant k from monocular torsion, yielding values between 0.2 and 1.0; however, corresponding estimates based on cyclovergence were all close to 1/2. These findings support suggestions that a binocular control system couples the three-dimensional movements of the eyes, and that an existing model of monocular torsion should be generalised to the binocular case. PMID- 10748927 TI - Increment-threshold spectral sensitivity during saccadic eye movements in uniform visual field. AB - We measured increment-threshold spectral sensitivity functions during saccades, immediately after saccades and during fixation. A uniform white background field which covered observer's whole visual field prevented any retinal masking effects. Visual sensitivity was degraded during saccades or after saccades. The reduction in sensitivity depended upon the wavelength of the test stimulus. The spectral sensitivity function during fixation produced a broad smooth curve in the middle and long wavelength region, while saccades caused a prominent dip around 570 nm. This finding indicates that saccadic suppression, which cannot be attributed to retinal masking, has more effect on the achromatic channel than the chromatic channel. The role of central and retinal processes dealing with the perceptual clearness and stableness across saccades will be discussed. PMID- 10748928 TI - Visual adaptation as optimal information transmission. AB - We propose that visual adaptation in orientation, spatial frequency, and motion can be understood from the perspective of optimal information transmission. The essence of the proposal is that neural response properties at the system level should be adjusted to the changing statistics of the input so as to maximize information transmission. We show that this principle accounts for several well documented psychophysical phenomena, including the tilt aftereffect, change in contrast sensitivity and post-adaptation changes in orientation discrimination. Adaptation can also be considered on a longer time scale, in the context of tailoring response properties to natural scene statistics. From the anisotropic distribution of power in natural scenes, the proposal also predicts differences in the contrast sensitivity function across spatial frequency and orientation, including the oblique effect. PMID- 10748929 TI - Aging and dark adaptation. AB - Older adults have serious difficulty seeing under low illumination and at night, even in the absence of ocular disease. Optical changes in the aged eye, such as pupillary miosis and increased lens density, cannot account for the severity of this problem, and little is known about its neural basis. Dark adaptation functions were measured on 94 adults ranging in age from the 20s to the 80s to assess the rate of rod-mediated sensitivity recovery after exposure to a 98% bleach. Fundus photography and a grading scale were used to characterize macular health in subjects over age 49 in order to control for macular disease. Thresholds for each subject were corrected for lens density based on individual estimates, and pupil diameter was controlled. Results indicated that during human aging there is a dramatic slowing in rod-mediated dark adaptation that can be attributed to delayed rhodopsin regeneration. During the second component of the rod-mediated phase of dark adaptation, the rate of sensitivity recovery decreased 0.02 log unit/min per decade, and the time constant of rhodopsin regeneration increased 8.4 s/decade. The amount of time to reach within 0.3 log units of baseline scotopic sensitivity increased 2.76 min/decade. These aging-related changes in rod-mediated dark adaptation may contribute to night vision problems commonly experienced by the elderly. PMID- 10748930 TI - Multiple gain control processes in contrast-contrast phenomena. AB - Spatial interactions among orientation-tuned gain control processes are presumed to mediate center-surround contrast-contrast phenomena. In this paper, we assess contributions of gain control processes that pool over orientation. We measured the apparent contrast of a luminance-modulated center disk embedded in various modulated surrounds. In all conditions, observers compared the apparent contrast of the test center to an identically modulated disk with no surround. When center and surround are simple, vertical sinusoids and presented in phase, suppression depends upon surround contrast and is marked at high contrasts. When components are presented 180 degrees out of phase, no suppression occurs at any contrast. When a horizontal component is added to the surround, much less suppression occurs. However, strong suppression is reinstated when both center and surround are plaids. Neither of the latter two effects are phase dependent. We suggest that two different sources of gain control are revealed by the simple sinusoidal and the plaid stimuli. One is orientation tuned and phase-dependent. The other pools over all orientations and includes neurons tuned to multiple phases. PMID- 10748931 TI - Long-term survival of retinal cell cultures on retinal implant materials. AB - The aim of the present study was to evaluate cell adhesion and cell survival of mammalian retinal neurons on different materials used for the production of multi photodiode arrays (MPDAs) intended for implantation in the subretinal space of patients suffering from progressive photoreceptor cell loss. The survival rates of different types of retinal neurons and glia cells were monitored by conventional histochemical techniques and immunocytochemistry up to 4 weeks. Whereas most of the materials tested showed good biocompatibility, cell survival of retinal glia and neurons was markedly reduced on titanium nitride (TiN), especially for culturing periods longer than 2 weeks. The effect was not mediated by diffusible factors released from TiN material. In conclusion, most of the materials tested in this study are suitable for the production of functional MPDAs and no complications are to be expected from long-term implantations of them in the subretinal space. PMID- 10748932 TI - Photoreceptors are preferentially affected in the rat retina following permanent occlusion of the carotid arteries. AB - Carotid artery occlusion (two vessel occlusion; 2-VO) for 3 or 9 months causes a suppression of the electroretinogram. However, after 3 months the retinal morphology appears unaffected judging from the localisation of GABA, ChAT, alpha PKC, Thy-1 and GFAP immunoreactivities. Moreover, no difference in NMDA-R1, opsin or Thy-1 mRNA levels were detected. In contrast, after 9 months 2-VO photoreceptor degeneration occurred as indicated by thinning of the outer nuclear layer and reduced Ret-P1 immunoreactivity. All other immunoreactivities appeared normal. These findings were supported by analysis of retinal mRNA levels. We conclude that the major effect of prolonged 2-VO is photoreceptor degeneration. PMID- 10748933 TI - Lighting direction affects recognition of untextured faces in photographic positive and negative. AB - Face recognition in photographic positive and negative was examined in a same/different matching task in five lighting direction conditions using untextured 3-D laser-scanned faces. The lighting directions were +60, +30, 0, -30 and -60 degrees, where negative values represent bottom lighting and positive values represent top lighting. Recognition performance was better for faces in positive than in negative when lighting directions were at +60 degrees. In one experiment, the same effect was also found at +30 degrees. However, faces in negative were recognized better than positive when the direction was -60 degrees. There was no difference in recognition performance when the lighting direction was 0 and -30 degrees. These results confirm that the effect of lighting direction can be a determinant of the photographic negative effect. Positive faces, which normally appear to be top-lit, may be difficult to recognize in negative partly because of the accompanying change in apparent lighting direction to bottom-lit. PMID- 10748934 TI - Extraction of depth from opposite-contrast stimuli: transient system can, sustained system can't. AB - The ability of observers to extract depth from opposite luminance-contrast polarity stimuli was investigated. The stimuli consisted of two dichoptic-pairs of Gaussians, with one of the Gaussians in each pair having a positive contrast polarity and the other a negative contrast-polarity. Stimulus durations ranging from 0.2 to 4 s were used. This range of durations was employed to reveal stereo mechanisms that were preferentially sensitive to transient or sustained stimuli. Stimuli were presented in a raised-cosine temporal envelope. Performance with stimuli of the same contrast-polarity was also tested. Observers could easily perceive depth with the same-polarity stimuli, at both long and short durations. Depth could be perceived with low-contrast opposite-polarity stimuli only at short durations. However, depth could be perceived with long-duration stimuli presented within a raised cosine temporal-envelope if a high contrast was used. Depth could also be perceived with low-contrast long-duration stimuli if they were presented within a rectangular temporal-envelope. These findings suggest there are separate sustained and transient mechanisms for stereopsis and that the transient-stereoscopic system can extract depth from opposite-contrast stereograms while the sustained system cannot. Further, it is likely that depth perception with opposite-contrast stereograms found in many previous studies was mediated by the transient-stereopsis system. PMID- 10748935 TI - The orientation discrimination deficit in strabismic amblyopia depends upon stimulus bandwidth. AB - We show that the previously reported orientation deficit in amblyopia (Skottun, B. C., Bradley, A., & Freeman, R. D. (1986). Orientation discrimination in amblyopia. Investigative Ophthalmology and Visual Science, 30, 532-537) also occurs for arrays of randomly positioned Gabor micropatterns for which explanations based on either neural disarray or local neural interactions would not hold. Furthermore, when using Gabors, we show that the deficit varies with the spatial frequency and orientational bandwidth of the stimuli used to measure it. We discuss two competing explanations for this, one based on a broader underlying detector bandwidth in amblyopia (both orientation and spatial frequency) and the other based on a selective deficit of first-order, as opposed to second-order orientation processing in strabismic amblyopia. Our results favour the latter interpretation. PMID- 10748936 TI - Object recognition: view-specificity and motion-specificity. AB - This paper describes an experiment to distinguish between two theories of human visual object recognition. According to the view-specificity hypothesis, object recognition is based on particular learned views, whereas the motion-specificity hypothesis states that object recognition depends on particular directed view sequences. Both hypotheses imply a degree of view-bias (i.e. recognition of a given object is associated with a small number of views). Whereas the view specificity hypothesis attributes this view-bias to a preference for particular views, the motion-specificity hypothesis attributes view-bias to a preference for particular directed view-sequences. Results presented here suggest that recognition of 3D rotating objects involves significant view-bias. This view-bias appears to be associated with an underlying bias for particular directed view sequences, and not for particular views. PMID- 10748937 TI - Post-retinal processing of background luminance. AB - It is generally thought that mean luminance and low spatial frequency information in a visual image are sharply attenuated at the retina, due to processes of light adaptation and the spatial filtering effects of lateral inhibition. Our results from interocular luminance masking suggest, however, that cortical masking effects play a primary role in the attenuation of low frequency sensitivity. Results also revealed that interocular luminance masking saturates and that semisaturation occurs where left and right eye luminances are equal, implying that the test luminance limits the effectiveness of the mask through interocular gating. PMID- 10748938 TI - Spatial limitations of temporal segmentation. AB - We investigated the spatial parameters that permit temporal phase segmentation. Subjects identified a stimulus quadrant which was modulated 180 degrees out of phase with the rest of the stimulus at temporal frequencies between 2 and 30 Hz. We determined the modulation sensitivity for regular square lattices of Gaussian spots and a stimulus made from solid quadrants with varying separation. Sensitivity declined rapidly when spatial separation of the modulating areas was approximately 0.4 degree, but was relatively unchanged by further spatial separations. The results suggest that there are two systems that can detect temporal phase differences. The first is a segregation process that operates below 10 Hz, where phase can be consciously followed and compared across large retinal distances. The second system is a segmentation mechanism that operates at higher temporal frequencies but only over a short range. PMID- 10748940 TI - Contrast inconstancy across changes in polarity. AB - In this study, we show that negative polarity noise patterns appear to have a higher contrast than positive polarity noise patterns with identical expected Fourier amplitude spectra. This demonstrates a failure of contrast constancy over changes in pattern polarity. An examination of local contrast measures shows that negative polarity noise has a wider distribution of local contrast values than positive polarity noise. We propose that the difference in apparent contrast between the two patterns may be based upon spatial non-linearities in the combination of local contrast measures. PMID- 10748939 TI - Experience-dependent integration of texture and motion cues to depth. AB - Previous investigators have shown that observers' visual cue combination strategies are remarkably flexible in the sense that these strategies adapt on the basis of the estimated reliabilities of the visual cues. However, these researchers have not addressed how observers' acquire these estimated reliabilities. This article studies observers' abilities to learn cue combination strategies. Subjects made depth judgments about simulated cylinders whose shapes were indicated by motion and texture cues. Because the two cues could indicate different shapes, it was possible to design tasks in which one cue provided useful information for making depth judgments, whereas the other cue was irrelevant. The results of experiment 1 suggest that observers' cue combination strategies are adaptable as a function of training; subjects adjusted their cue combination rules to use a cue more heavily when the cue was informative on a task versus when the cue was irrelevant. Experiment 2 demonstrated that experience-dependent adaptation of cue combination rules is context-sensitive. On trials with presentations of short cylinders, one cue was informative, whereas on trials with presentations of tall cylinders, the other cue was informative. The results suggest that observers can learn multiple cue combination rules, and can learn to apply each rule in the appropriate context. Experiment 3 demonstrated a possible limitation on the context-sensitivity of adaptation of cue combination rules. One cue was informative on trials with presentations of cylinders at a left oblique orientation, whereas the other cue was informative on trials with presentations of cylinders at a right oblique orientation. The results indicate that observers did not learn to use different cue combination rules in different contexts under these circumstances. These results are consistent with the hypothesis that observers' visual systems are biased to learn to perceive in the same way views of bilaterally symmetric objects that differ solely by a symmetry transformation. Taken in conjunction with the results of Experiment 2, this means that the visual learning mechanism underlying cue combination adaptation is biased such that some sets of statistics are more easily learned than others. PMID- 10748941 TI - Perceived visual direction near an occluder. AB - When an opaque object occludes a more distant object, the two eyes often see different parts of the distant object. Hering's laws of visual direction make an interesting prediction for this situation: the part seen by both eyes should be seen in a different direction than the part seen by one eye. We examined whether this prediction holds by asking observers to align a vertical monocular line segment with a nearby vertical binocular segment. We found it necessary to correct the alignment data for vergence errors, which were measured in a control experiment, and for monocular spatial distortions, which were also measured in a control experiment. Settings were reasonably consistent with Hering's laws when the monocular and binocular targets were separated by 30 arcmin or more. Observers aligned the targets as if they were viewing them from one eye only when they were separated by 2 arcmin; this behavior is consistent with an observation reported by Erkelens and colleagues. The same behavior was observed when the segments were horizontal and when no visible occluder was present. Perceived visual direction when the two eyes see different parts of a distant target is assigned in a fashion that minimizes, but does not eliminate, distortions of the shape of the occluded object. PMID- 10748942 TI - Facilitation between the luminance and red-green detection mechanisms: enhancing contrast differences across edges. AB - Previous studies have shown that detection of a red-green test pattern, such as a spot or grating, may be facilitated two to three times by a suprathreshold luminance pedestal of the same shape. We measured facilitation between the red green (RG) and luminance (LUM) detection mechanisms using sine and square-wave gratings. Facilitation of RG by luminance pedestals was 3-fold for in phase sine wave gratings of 0.8 cpd and a remarkable 7-fold for square-wave gratings. The latter facilitation was greatly reduced at intermediate relative phases and was generally reduced at higher spatial frequencies. We show that on a uniform field, the red or green regions of low spatial frequency test patterns are detected approximately independently, but in the presence of the LUM pedestal RG becomes sensitive to the red-green difference across the luminance edges. Under optimal conditions (with the low-frequency, square-wave luminance pedestal) this increased red-green sensitivity corresponds to a wavelength discrimination threshold as small as approximately 0.04 nm. This conversion of RG into an 'edge detector' may explain why facilitation is twice as large for square-wave gratings (bipolar patterns) than spots (unipolar patterns). The reverse facilitation, that of LUM by the red-green pedestal, is weaker and the results suggest that this is because LUM is initially sensitive to the light-dark difference across luminance edges even in the absence of the red-green pedestal. PMID- 10748943 TI - Large repulsion, but not attraction, tilt illusions occur when stimulus parameters selectively favour either transient (M-like) or sustained (P-like) mechanisms. AB - A vertical test grating appears tilted away from a surrounding inducing grating which is 15 degrees from vertical (repulsion effect) but towards an inducer 75 degrees from vertical (attraction effect). This is the tilt illusion (TI) and similar effects occur when inducing and test stimuli are presented successively (tilt after-effect or TAE). When it was reported [Wolfe, J. (1984). Vision Research, 24, 1959-1964] that large repulsion TAEs occurred with short test flashes, Wolfe postulated that either there are distinct mechanisms which process brief and longer duration stimuli; or that there are distinct mechanisms which are not primarily concerned with duration but are differentially responsive to temporal parameters, amongst several others. Other evidence that TI attraction effects are not modulated by test flash duration resulted in an hypothesis that repulsion and attraction effects are mediated by transient and sustained mechanisms, respectively [Wenderoth, P., van der Zwan, R., & Johnstone, S. (1989). Perception, 18, 715-728]. We demonstrate that large repulsion TIs can be induced when parameters other than duration are manipulated, including contrast and spatial frequency but that these parameters fail to modulate attraction TIs. These results are consistent with some previous hypotheses regarding the origin of repulsion and attraction effects and with Wolfe's latter hypothesis but do not support the view that the two effects are processed, respectively, by transient and sustained mechanisms. PMID- 10748944 TI - Visual evoked potentials related to motion-onset are modulated by attention. AB - The effects of attention on visual evoked potentials triggered by motion-onset were examined in four experiments. A set of randomly oriented bars was used as stimuli. The first experiment showed that responses to motion-onset following pattern-onset by less than 300 ms were suppressed. In the other three experiments, the amplitude of N170 was reduced when attention was drawn away from the moving elements and towards spatially interspersed bars that remained static. The superposition of the two sets made spatial selection unlikely. These results support the existence of an attentional 'motion filter' (separating stationary from moving elements) that can operate at early stages of visual processing. PMID- 10748945 TI - Allergic rhinitis: epidemiology and natural history. AB - Establishing a reliable estimate of the prevalence of allergic rhinitis is difficult; prevalence estimates range from as low as 4% to more than 40%. Epidemiology studies suggest the prevalence of allergic rhinitis in the United States and around the world is increasing. The cause of this increase is unknown; however, contributing factors may include higher concentrations of airborne pollution, rising dust mite populations, less ventilation in homes and offices, dietary factors, and the trend toward more sedentary lifestyles. Allergic rhinitis symptoms typically begin in childhood and adolescence and continue into adulthood. In general, allergic rhinitis symptoms slowly improve and skin-test reactivity tends to wane with increasing age. There is a significant trend for symptom improvement with younger age of onset of allergic rhinitis. As the complicated etiology of allergic rhinitis becomes better understood, it may be possible to reverse the trend for increased prevalence. PMID- 10748946 TI - Cognitive, social, and economic costs of allergic rhinitis. AB - Allergic rhinitis is a highly prevalent, chronic condition. In addition to physical discomfort, rhinitis symptoms have been associated with detrimental effects on the psychological and social aspects of patients' lives. In allergy specific questionnaires, subjects with allergic rhinitis consistently report lower quality of life than nonallergic controls. Untreated patients are embarrassed and frustrated by their allergy symptoms. Atopic individuals consistently exhibit significant declines in cognitive processing, psychomotor speed, verbal learning, and memory during allergy season. The discomfort, cognitive impairment, and absenteeism associated with allergic rhinitis exact a significant economic toll on U.S. businesses through decreased productivity. When combined with direct medical expenditures, the economic burden of allergic rhinitis is considerable. The effect of treatment on the economics of allergic rhinitis is highly variable: relatively inexpensive medications (lower direct costs) have central nervous system side effects that can cause somnolence and impair learning, memory, and performance (higher indirect costs). Health outcomes data on the effects of allergic rhinitis and its treatments can help establish, monitor, and improve standards of care; as well as inform priority setting, direct resource allocation, and eliminate unnecessary practices. PMID- 10748947 TI - Safety of second generation antihistamines. AB - The sedation related to first-generation antihistamine use has been shown to compromise performance at school and at work, impair driving, and decrease the ability to handle tasks that require a high degree of alertness or concentration. Second-generation antihistamines are less likely to produce sedation. Loratadine, cetirizine, and fexofenadine are the most commonly prescribed second-generation antihistamines. Many tests have been conducted to assess the central effects of these three drugs. Compared with placebo, at recommended doses loratadine is not associated with performance impairment. Cetirizine, at recommended doses, has been shown to impair performance and cognition in several studies, although to a much lesser degree than older antihistamines. Clinical trials show fexofenadine is nonsedating, even at very high doses; psychomotor and driving tests reinforce these findings. Loratadine, cetrizine, and fexofenadine all have excellent safety records. Their cardiovascular safety has been demonstrated in drug-interaction studies, elevated-dose studies, and clinical trials. These three antihistamines have also been shown safe in special populations, including pediatric and elderly patients. PMID- 10748948 TI - Differential diagnosis of the patient with unexplained flushing/anaphylaxis. AB - In unusual cases of flushing and anaphylaxis, and after the elimination of the more obvious causes of anaphylaxis or those that may be evaluated by readily available techniques, it is possible to confront a limited and difficult differential diagnosis, which includes idiopathic flushing, anaphylaxis, and neoplastic syndromes associated with mastocytosis and carcinoid tumor. Interestingly, there are rather few features that distinguish one of these possibilities from another. However, the presence of allergic signs and symptoms tend to favor the diagnosis of recurrent idiopathic anaphylaxis; and right-sided valvular heart disease, the presence of excessive 5-HIAA in the urine, and a response to somatostatin favor the diagnosis of carcinoid syndrome. The distinguishing features of mastocytosis include the presence of characteristic skin lesions and diagnostic histopathologic findings on bone marrow biopsy. Counts of absolute mast cell numbers in the skin are less helpful. Following such guidelines, it is often possible to focus on the most likely diagnosis, be it idiopathic anaphylaxis, benign cutaneous flushing, mastocytosis, or carcinoid tumor. PMID- 10748949 TI - Approach to the patient with a history of adverse reactions to aspirin or NSAIDs: diagnosis and treatment. AB - Aspirin and nonsteroidal antiinflammatory drugs can induce several different reactions. It is incumbant upon the astute physician to recognize the differences between these reactions, the clinical settings in which they occur, and the management of each type of reaction. In this article, I have attempted to clarify and simplify, as much as possible, the approach I take in addressing the problems of adverse reactions to these drugs. In addition, the problems of cross-reactions are explained in terms of COX-1 and 2 isoenzymes. Aspirin desensitization and treatment with daily ASA are also addressed. PMID- 10748950 TI - Community-acquired pneumonia: an approach to antimicrobial therapy. AB - Community-acquired pneumonia (CAP), the sixth leading cause of death in the United States, has undergone significant changes in the past 30 years. In addition to the fact that it increasingly is a disease affecting the elderly and those patients with underlying comorbidities, the spectrum of microbiological agents causing pneumonia has greatly expanded and includes in addition to Streptococcus pneumoniae many other agents including Mycoplasma, Chlamydia, and respiratory viruses. A major problem encountered by the clinician facing a patient with CAP derives from the imprecise clinical presentation, which in most instances does not permit a precise diagnosis of the etiological agent. As pneumonia, if untreated, is frequently a rapidly progressive illness, the clinician usually chooses antimicrobial agents on an empirical basis. Careful attention to historical, physical, and laboratory findings, as well as age and presence of comorbidities has led to a categorization of CAP into four groupings that assist in deciding whether the patient should be hospitalized and what empirical antimicrobial regimen should be started. Careful follow-up and familiarity with the clinical pneumonic syndromes associated with different microbial agents is essential to assure a successful outcome. PMID- 10748951 TI - Approach to the patient with multiple antibiotic sensitivities. AB - Allergists sometimes are asked to evaluate patients who have experienced multiple adverse drug reactions. By the time the patient reaches the allergist, the referring physician, as well as the patient, often are frustrated. Each seeks a quick and simple answer regarding the particular drugs the patient may safely receive. Unfortunately, however, in most instances, simple answers cannot be given. Currently we have limited knowledge of the mechanisms and of the clinically-relevant drug metabolites responsible for many of the reactions demonstrated and, for these reasons, we have few valid diagnostic tests that are able to provide clear-cut answers for our patients who present with multiple drug "allergies." Despite these limitations, using accurate and complete historical information along with limited diagnostic testing, logical and practical management approaches can be devised. In addition, due to new exciting data that are being generated from basic research studies in drug allergy, the mechanisms underlying the immunopathology of many drug reactions are becoming more clear. Through the acquisition of scientific information of this type, it is hoped that valid diagnostic tools soon will be developed so that definitive answers, desired by both the patient and the referring physician, can be provided. PMID- 10748952 TI - Rhinitis and asthma connection: management of coexisting upper airway allergic diseases and asthma. AB - Asthma is a chronic inflammatory disease of the lower airways. Epidemiologic surveys and clinical reports have documented that allergic rhinitis coexists with asthma in many patients. Provocative bronchial challenge with allergens responsible for allergic rhinitis in susceptible asthma patients can elicit asthma, and these responses have been linked to bronchial airway hyperreactivity. Provocative bronchial methacholine challenge in allergic rhinitis patients will demonstrate increased airway responsiveness to the bronchial challenge in 30% of those allergic rhinitis patients who had no past history of asthma. These data suggest that subclinical asthma may be present in certain patients with allergic rhinitis. The focus of the National Heart, Lung, and Blood Institute (NHLBI) guidelines for the pharmacologic treatment of asthma focuses on medications to relieve the symptoms of asthma, i.e., bronchodilators and anti-inflammatory agents (i.e., inhaled corticosteroids, cromolyn, and leukotriene modifiers) to control asthma. Avoidance of allergens such as house dust mite are also recommended. Although not emphasized in these NHLBI guidelines, recent studies have observed that treatments, including intranasal steroid, cromolyn, antihistamines, and decongestants, which provide relief of nasal symptoms in patients with both allergic rhinitis and asthma, will also improve the pulmonary symptoms of allergic asthma. This article will review the recent literature. PMID- 10748953 TI - Genetics of asthma and allergy. AB - Asthma and allergies are complex conditions involving multiple steps and pathways, which are influenced by both genetic and environmental factors. The genes involved in these processes are just being identified. Most likely asthma is a result of several genes and their interaction with other genes as well as the environment. Management involves the proper diagnosis, modulating the genetic and environmental factors involved as well as interfering with the activated pathways. Using this approach will lead to a more rational method of managing individuals with allergies and asthma. PMID- 10748955 TI - Evidence-based complementary medicine--challenges and future directions. PMID- 10748954 TI - From immunity to autoimmune disease, a historic trail. Part I. PMID- 10748956 TI - Complementary medicine and evidence. PMID- 10748957 TI - The role of alternative therapy in the management of partial thickness burns of the face--experience with the use of moist exposed burn ointment (MEBO) compared with silver sulphadiazine. AB - INTRODUCTION: Conventional management of partial thickness facial burn wounds includes the use of silver sulphadiazine dressings. Silver sulphadiazine forms an overlying slough that makes wound healing assessment difficult. Moist exposed burn ointment (MEBO) has been proposed as the ideal burn wound dressing both for burns of the face and other sites. Proponents of MEBO claim that it accelerates wound healing and results in scarless wound healing and at the same time reduce bacterial colonisation and the need for analgesics. We present here our experience with MEBO in the management of partial thickness burns of the face. MATERIALS AND METHODS: One hundred and fifteen patients with partial thickness burns were randomly assigned to conventional treatment or MEBO. Out of this, 112 were analysed. Thirty-nine patients sustained facial burns; 17 received MEBO and 22 received silver sulphadiazine. Patients were followed up daily until the burn wounds were reduced by 75% of original body surface area (BSA). RESULTS: In patients with facial burns, MEBO was similar to silver sulphadiazine therapy with respect to rate of wound healing. Minimal slough was present over the wounds in MEBO-treated wounds resulting in clearer assessment of healing progression. CONCLUSIONS: Advantages of MEBO as compared to silver sulphadiazine in the management of partial thickness burns of the face include convenient change of dressing and easier assessment of healing progression. This suggests that MEBO is a useful alternative therapy for partial thickness burns of the face. PMID- 10748958 TI - Traditional Chinese medicines as immunosuppressive agents. AB - INTRODUCTION: Traditional Chinese Medicines (TCM) have been used for centuries in China to treat various immune-mediated disorders. METHODS: This review focuses on the clinical and experimental studies that have been performed with TCM as immunosuppressive agents for the treatment of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), atopic eczema and solid organ transplantation. RESULTS: The "thunder god" vine, Tripterygium wilfordii Hook F (TWHf), has been extensively used in China to treat SLE and RA. TWHf not only inhibited mitogen stimulated lymphoproliferation, but its active derivatives have also been shown to inhibit production of proinflammatory cytokines by monocytes and lymphocytes, as well as prostaglandin E2 production via the cyclooxygenase, COX-2, pathway, a potential mechanism of action in patients with RA. Demethylzelasteral (TZ-93), a triterpenoid isolated from the root cortex of TWHf, the plant alkaloid berbamine, and the hydrophobic extract of a Chinese herbal decoction, CMX-13, were all shown to be active in prolonging allograft survival in experimental animal models of heart, skin and single lung transplants, respectively. There are few well designed randomised placebo-controlled clinical trials demonstrating the efficacy of TCM in various diseases. Zemaphyte, a decoction of 10 herbs, has been shown to be efficacious in the treatment of atopic dermatitis in both children and adults in two randomised double-blind placebo-controlled trials. CONCLUSION: There is both laboratory and clinical evidence that the derivatives of many of these herbs may have significant beneficial immunosuppressive effects, however, concerns of toxicity must also be addressed, as exact dosing of the active derivatives is difficult to achieve with the current prescriptions of TCM. PMID- 10748959 TI - Acupuncture and chronic pain management. AB - INTRODUCTION: Although acupuncture has been widely used to treat a variety of pain conditions, convincing scientific evidence for its efficacy is lacking. Earlier randomised controlled trials attempted to follow a double-blind, placebo controlled model. This approach has encountered many problems such as the virtual impossibility of blinding the acupuncturist and the uncertainties inherent in choosing control acupuncture points. The objective of this review is to assess if acupuncture is an effective treatment for chronic pain. In addition, a number of key methodological issues that arise in the controlled evaluation of acupuncture will be discussed. METHODS: This review is based on the result of previous reviews, meta-analyses and consensus conference. The search was performed with MEDLINE (from 1966), EMBASE (from 1980) and Cochrane library (1999, volume 1). Only randomised trials of acupuncture (involved needling) for subjects with chronic pain published in English were included. RESULTS: Basic science research has demonstrated convincingly that at least in the context of acute pain, acupuncture's effects are related to the release of a variety of natural opioids. Acupuncture has been shown to be effective for postoperative dental pain. There are reasonable studies showing relief of pain with acupuncture on diverse pain conditions such as menstrual cramps, tennis elbow, low back pain and fibromyalgia. This suggests that acupuncture may have a more general effect on pain. However, there are also studies which provide equivocal results because of design, sample size and other factors. The issue is further complicated by inherent difficulties in the use of appropriate controls, such as placebo and sham acupuncture groups. CONCLUSION: As most of the studies were of poor methodological quality, there is a need for further high quality randomised controlled trials. Future studies should also have larger sample sizes, use a valid acupuncture treatment, and have both a short-term and long-term follow-up. PMID- 10748960 TI - Proerectile pharmacological effects of Tribulus terrestris extract on the rabbit corpus cavernosum. AB - INTRODUCTION: The objective of the present study was to investigate the effect of oral treatment of Tribulus terrestris (TT) extract on the isolated corpus cavernosal tissue of New Zealand white (NZW) rabbits and to determine the mechanism by which protodioscin (PTN), a constituent of the TT, exerts its pharmacological effects. MATERIALS AND METHODS: Twenty-four NZW rabbits were randomly assigned to 4 experimental groups of 6 each. Group I served as control. Groups II to IV were treated with the extract at different dose levels, i.e. 2.5 mg/kg, 5 mg/kg and 10 mg/kg body weight, respectively. The TT extract was administered orally, once daily, for a period of 8 weeks. The rabbits were then sacrificed and their penile tissue isolated to evaluate the responses to both contracting and relaxing pharmacological agents and electrical field stimulation (EFS). RESULTS: PTN on its own had no effect on the isolated corpus cavernosal strips. The relaxant responses to EFS, acetylcholine and nitroglycerin in noradrenaline precontracted tissues from treated groups showed an increase in relaxation of a concentration dependent nature compared to that of the tissues from control group. However, the contractile, anti-erectile response of corpus cavernosal tissue to noradrenaline and histamine showed no significant change between the treatment and the control groups. CONCLUSIONS: The relaxant responses to acetylcholine, nitroglycerin and EFS by more than 10%, 24% and 10% respectively compared to their control values and the lack of such effect on the contractile response to noradrenaline and histamine indicate that PTN has a proerectile activity. The enhanced relaxant effect observed is probably due to increase in the release of nitric oxide from the endothelium and nitrergic nerve endings, which may account for its claims as an aphrodisiac. However, further study is needed to clarify the precise mechanism of its action. PMID- 10748961 TI - Investigating plant-based medicines for wound healing with the use of cell culture technologies and in vitro models: a review. AB - INTRODUCTION: Cell culture and molecular technologies are basic yet sophisticated research tool used to investigate plant-based medicine for wound healing. METHODS: Cell viability and proliferation assay is used to determine whether there are any positive effects and to discover what is the limiting cytotoxic concentration in vitro. The scratch technique, fibroblast-populated collagen lattices and aortic rings embedded gels are used as the in-vitro models of wound re-epithelialization, contraction and angiogenesis. The immunofluorescence, immunoblotting and organotypic culture can be used to detect expression of specific proteins that are modulated by plant extracts during the wound healing process. MAIN FINDINGS: Given the dynamics of the wound healing process, cell culture and molecular technologies are advantageous in providing us with detailed studies and analysis of each intricate process. CONCLUSION: The scientific approaches for the study of traditional plant-based remedies for wound healing will provide us an important platform for rigorous testing and evaluation of their clinical efficacy based on accepted rules of evidence. PMID- 10748962 TI - Traditional Indian systems of medicine. AB - INTRODUCTION: A number of traditional systems of medicine exist in India of which Ayurveda is the most popular. Despite being in use for more than 3000 years, few properly designed trials have scientifically examined the clinical potential of Ayurvedic and other medications. METHODS: We reviewed the MEDLINE database to identify clinical trials conducted using traditional Indian medicines. Single case reports were excluded. RESULTS: Ayurvedic preparations have been successfully used for the treatment of bronchial asthma, ischaemic heart disease and hyperlipidaemia. Formulations containing curcumin were reported to reduce inflammation and disability in double-blind clinical trials on patients with rheumatoid arthritis. A number of products are reported to be useful in patients with acute viral hepatitis. A multicentric study by the Indian Council of Medical Research showed that a preparation from Pterocarpus marsupium was effective in reducing levels of blood glucose and glycosylated haemoglobin in patients with non-insulin-dependent diabetes mellitus. In another multicentric trial, patients with fistula-in-ano were randomised to surgery or application of medicated seton (Ksharsootra). Surgical treatment led to a faster cure but recurrence rates were lower with medicated seton. Administration of extract from Bacopa monnieri, to children with mental retardation, was reported to significantly improve short term and long-term memory. CONCLUSIONS: Evidence-based studies on the efficacy and safety of traditional Indian medicines are limited. The essential ingredient in most formulations is not precisely defined. High quality studies are necessary to evaluate and compare the value of traditional Indian drugs to modern medicine. PMID- 10748963 TI - Panax (ginseng)--panacea or placebo? Molecular and cellular basis of its pharmacological activity. AB - INTRODUCTION: The use of ethnobotanical drugs amongst Asians as complementary medicine is prevalent and is also gaining increasing popularity in the West. The most well-known herb traditionally used as a drug is the root of the ginseng species. There are many traditional and anecdotal claims to the therapeutic properties of ginseng. In recent years, there have been systematic efforts to analyse the bioactivities of ginseng saponins. METHODS: A comprehensive review of published literature covering molecular and cellular research as well as animal and human studies on ginseng and its derivatives. RESULTS AND CONCLUSION: Current published data would serve as a framework to understand the pharmacology of ginseng in its entirety, from its molecular action to actual therapeutic effects observed in human use. A new paradigm is emerging whereby the pharmacological effects of traditional herbs such as ginseng can be understood in the light of their polyvalent actions as demonstrated by ginseng saponins with their positive anti-mutagenic, anti-cancer, anti-inflammatory, anti-diabetes and neurovascular effects. With increasing understanding, evidence-based incorporation of traditional herbs as complementary medicine into mainstream medical science can be achieved in the near future. PMID- 10748964 TI - Fibre-optic aided bougie (FAB) for simulated difficult tracheal intubation. AB - INTRODUCTION: Difficult intubation remains a key problem and the value of the gum elastic bougie as a first approach is well recognised. MATERIALS AND METHODS: A fine fibre-optic endoscope (Rapiscope, Cook Critical Care) was used in 50 patients to verify placement of a custom-designed hollow plastic bougie prior to "rail-roading" a tracheal tube. Following induction and muscle relaxation, direct laryngoscopy was performed after two-minutes of assisted ventilation. The laryngoscope blade was lowered to simulate difficult intubation and the bougie passed behind the epiglottis. The position of the introducer bougie was then checked using the Rapiscope. Following correct bronchoscopic identification of the tracheobronchial anatomy, a tracheal tube was then "rail-roaded" following withdrawal of the scope. RESULTS: All patients were successfully intubated following identification of the bronchial tree by the Rapiscope: three on the second attempt and the rest on the first. Mean (SD, range) time to successful bronchoscopic confirmation of correct placement of the bougie was 38 s (9.1 s, 19 to 60 s). All bronchoscopic assisted intubation were subsequently confirmed by capnography after tracheal tube insertion. Mean (SD, range) time to successful intubation was 106 s (14 s, 52 to 132 s). CONCLUSION: The fibre-optic assisted bougie (FAB) offers a promising technique in patients who may be difficult to intubate but who can be ventilated. Further developments are required to achieve a faster intubation time but oxygenation may be achieved by jetting down the hollow bougie. PMID- 10748965 TI - End-of-life issues--preferences and choices of a group of elderly Chinese subjects attending a day care centre in Singapore. AB - INTRODUCTION: This was an exploratory study that was intended to provide a descriptive analysis of the choices and preferences of a group of elderly Chinese subjects attending a day care centre in Singapore with regard to end-of-life issues. MATERIALS AND METHODS: A semi-structured one-to-one interview was conducted to collect data from the subjects. Qualitative techniques were used to analyse the data. RESULTS: Forty-three subjects were interviewed. The median age was 71 years. There were more women than men (58.1% vs. 41.9%). The predominant religion was Buddhism/Taoism. 83.7% and 76.7% of the subjects preferred to be told of the diagnosis and prognosis of a terminal illness, respectively. The person most preferred to reveal the diagnosis was the attending doctor (60.5%). About 83.7% of the subjects have never heard of the Advanced Medical Directive Act, while 37.2% agreed that making an advanced directive would be necessary. Twenty-three (53.5%) would choose the doctor, while 15 (34.9%) would choose a family member as a surrogate decision-maker. Twenty-two (51.2%) thought that euthanasia should be allowed, while 15 (34.9%) disagreed. With regard to supportive measures at the end of life, 67.4% wanted cardiopulmonary resuscitation, 62.8% wanted artificial ventilation, 55.8% wanted nasogastric feeding, 65.1% wanted intravenous hydration and 41.9% wanted renal dialysis. CONCLUSION: There is a need for closer communication between older persons and their carers with regard to end-of-life care. The attending doctor appears to have an important role in this respect. PMID- 10748966 TI - Results of surgical resection of oesophageal carcinoma in Singapore. AB - INTRODUCTION: The 5-year survival rate after resection of oesophageal cancer is still poor, reported to be between 5% to 34% in the literatures. We conducted this retrospective study to determine the prognostic factors and long-term survival rate for surgical resection of oesophageal carcinoma in Singapore. MATERIALS AND METHODS: Sixty-seven consecutive patients who underwent surgical resection of oesophageal carcinoma from January 1989 to December 1996 were reviewed. RESULTS: Majority of the patients were Chinese (94%), males (81%) with a history of dysphagia (96%), cigarette smoking (84%) and alcohol ingestion (58%). The median age at surgery was 63.6 (range 29 to 81) years. Sixty-four patients had squamous cell carcinoma of the oesophagus and 3 patients had adenocarcinoma. The lower one-third of the oesophagus was the most common site (48.6%) followed by the middle third (46%). Postoperative complications included pneumonia (43.3%), vocal cord paralysis/paresis (19.4%) and anastomotic leakage (17.9%). Thirty-day postoperative mortality was 7.5%. There was an almost equal number of patients undergoing transhiatal (34 patients) and transthoracic approach (33 patients). There was no significant difference in postoperative complications and survival outcome for both approaches. The median survival was 15.3 months after surgery. Tumour features such as depth of involvement and TNM stage were useful in predicting survival (P < 0.05, log-rank test). CONCLUSIONS: We conclude that the poor outcome of surgical resection (5-year survival rate was 18% by Kaplan-Meier survival plots) was mainly attributed to late presentation (80% patients had T3 or T4 tumour). PMID- 10748967 TI - Evaluation of a rapid screening test for microalbuminuria with a spot measurement of urine albumin-creatinine ratio. AB - INTRODUCTION: Microalbuminuria has been established as a marker of incipient diabetic nephropathy, and a regular screening programme has been advocated in patients with diabetes mellitus. We investigated if urine albumin levels normalised by creatinine give results comparable to the urinary albumin excretion rate (UAER) with a timed 24-h urine for detecting microalbuminuria. MATERIALS AND METHODS: Morning urine specimens and 24-h collections were obtained from 65 diabetic patients. Albumin and creatinine levels in the spot urine specimens were measured in a single rapid (7 min) assay format using the Bayer DCA2000+ desktop system. Results of albumin/creatinine ratio and urine albumin concentration were then compared to the reference laboratory method of measuring UAER with the timed 24-h urine sample. RESULTS: The determination of the albumin/creatinine ratio gave good performance characteristics for diagnosis of microalbuminuria (defined as > 30 mg/g creatinine) with sensitivity of 71.4% and specificity of 98.0%. With urine albumin concentration alone, sensitivity was 64.3% and specificity was 96.1%. Receiver operating characteristic (ROC) curves, however, suggest similar diagnostic usefulness for screening microalbuminuria with albumin levels expressed in concentration units or as a ratio of creatinine compared to the reference method. Analysis also indicated that lowering the established cut-off values in general, improves diagnostic performance. CONCLUSIONS: Measurement of microalbuminuria with a spot morning specimen using the DCA2000+ desktop system that simultaneously measures albumin and creatinine levels, provides a rapid and reliable method for incipient diabetic nephropathy in clinical practice. PMID- 10748968 TI - Endobronchial stenting in patients requiring mechanical ventilation for major airway obstruction. AB - OBJECTIVE: To examine the value of therapeutic rigid bronchoscopy on subsequent ventilator weaning in ventilated patients with major airway obstruction. MATERIALS AND METHODS: Retrospective review of the medical records of patients who were receiving mechanical ventilation up to the time of rigid bronchoscopy over the period from September 1994 to January 1999. The setting is in an acute tertiary hospital. All patients underwent rigid bronchoscopy with endoscopic techniques (forceps removal, balloon dilation Nd:YAG laser resection and stent insertion) aimed at restoration of major airway patency. RESULTS: Seven patients were identified; 2 with benign stenosis and 5 with malignant stenosis. Six out of the 7 patients had presented with acute respiratory distress requiring emergent intubation, whilst 1 patient had extubation difficulties following general anaesthesia with endotracheal intubation. Three out of the 7 patients had previously failed extubation attempts. All 7 patients were successfully extubated within 48 hours following the procedure. CONCLUSIONS: Therapeutic rigid bronchoscopy is effective in the acute management of respiratory distress from central airway obstruction. Dramatic improvements in ventilatory status following therapeutic rigid bronchoscopy allow ventilator liberation in a select group of patients with an otherwise good performance status. PMID- 10748969 TI - Thrombotic and haemorrhagic complications in patients with mechanical heart valve prostheses attending the Singapore General Hospital Anticoagulation Clinic. AB - INTRODUCTION: This study evaluated the intensity of anticoagulation required for prevention of thromboembolic episodes with minimal haemorrhagic complications in patients with mechanical heart valve prostheses. MATERIALS AND METHODS: The incidence of thrombotic and haemorrhagic events was retrospectively assessed in a series of 143 patients attending the anticoagulation clinic over a period of 67 months. Risk factors for haemostatic events were also analysed. RESULTS: All embolic events occurred when the International Normalised Ratio (INR) was less than 2.2, suggesting an INR of 2.5 to 3.0 should be adequate for thromboembolic prophylaxis. Risk factors for embolic events were atrial fibrillation, mitral valve prosthesis and a previous thromboembolic event. Most embolic events involved the central nervous system with resultant permanent residual disability. Most major bleeding episodes occurred in the presence of underlying pathological states such as peptic ulcers or fibroids and were not related to the intensities of anticoagulation. There was no mortality or long-term morbidity associated with bleeding episodes. CONCLUSIONS: In agreement with recent data from other centres, a moderate intensity of anticoagulation of INR 2.5 to 3.0 is now recommended. Hence, with this lowered intensity of anticoagulation and better patient education, we hope that thrombotic as well as haemorrhagic complications will be reduced in future. PMID- 10748970 TI - Contrasting clozapine prescribing patterns in the east and west? AB - INTRODUCTION: Polypharmacy is common in the treatment of schizophrenia and it may be especially common in the East. In this survey, we compared the effect of clozapine, an atypical neuroleptic, on polypharmacy in two groups of patients with schizophrenia in two different settings. MATERIALS AND METHODS: The medical charts of patients on stable doses of clozapine from a Canadian psychiatric centre and that of a Southeast Asian centre were analysed to evaluate the daily dose requirement and the concomitant medications prescribed. The beliefs of the Asian psychiatrists on the concomitant use of another neuroleptic were also examined. RESULTS: The mean daily dose of clozapine was 408 mg/day for the Canadian subjects and 169 mg/day for the Asian sample. Of the Canadian sample, none were prescribed another neuroleptic, 45% were on a benzodiazepine, and 10% were receiving an anticholinergic agent. The Singapore sample indicated 28% of the subjects on another neuroleptic, with 36% taking an anticholinergic agent and 28% on a benzodiazepine. The Asian psychiatrists prescribed a second neuroleptic in the belief that it would reduce cost and enhance the antipsychotic effect. CONCLUSIONS: This study suggests that there are regional differences in the prescription patterns and daily dose requirements of clozapine. PMID- 10748971 TI - Survival of colorectal cancer patients in Singapore by anatomic subsite: a population-based study. AB - INTRODUCTION: Studies of the prognostic value of anatomic subsite in colorectal cancer survival have yielded conflicting results. Two explanations for possible differences in survival patterns between proximal and distal lesions in the colorectum are biological difference between subsites and the presence of more early-stage lesions in distal than in proximal large bowel. MATERIALS AND METHODS: A total of 435 cases with proximal lesions and an equal number with distal lesions diagnosed between 1990 and 1992 were randomly selected from the Singapore Cancer Registry. Information on vital status at 31 December 1996 were obtained by computerised matching with data from the National Registry of Births and Deaths. RESULTS: Persons with proximal cancers in our study population did not present at a later stage than persons with distal cancer, local lesions (Dukes' Stage A + B) being 45.5% and 45.1%, respectively. Our analysis showed no significant differences in survival between subsites on a stage-for-stage basis. The 5-year survival rates were 42% and 44% for proximal and distal lesions, respectively (median survival times 3.98 and 4.27 years). Stage at diagnosis was the strongest predictor of survival. Among proximal lesions, 5-year survival rates were 57%, 36% and 12% for local, regional and metastatic lesions, respectively. The corresponding figures for the distal group were 65%, 37% and 10%. Age at diagnosis had a significant influence on survival. CONCLUSIONS: Our results, based on population-based figures on survival of colorectal cancer patients where the impact of screening has not been large, do not support an independent influence of anatomic subsite in predicting survival of colorectal cancer. PMID- 10748972 TI - Sharps and needlestick injuries: the impact of hepatitis B vaccination as an intervention measure. AB - INTRODUCTION: This paper studies the epidemiology of sharps and needlestick injuries amongst health care workers and the effectiveness of intervention measures implemented at a regional hospital, Singapore. MATERIALS AND METHODS: A retrospective review of sharps and needlestick injuries among healthcare workers of a regional hospital at Singapore between 1992 and 1997. Various interventions namely education, policy changes and a hospital-wide hepatitis B immunisation programme were reviewed for effectiveness of programmes implementation. RESULTS: Of the 347 reported sharps and needlestick injuries, 45.7% occurred in the nursing staff, 25.1% medical staff, 7.5% health attendants, 5.2% hospital cleaners and 3.7% laboratory technicians. A steady rise in reporting was noted amongst the doctors from 1994 and this correlated with the implementation of educational talks given to new medical staff in May 1993. The number of healthcare workers with no previous history of hepatitis B immunisation decreased significantly from 17 in 1996 to 9 in 1997 (P < 0.001, odds ratio = 1.806 with 95% CI 1.443 to 2.261) after the implementation of the hospital-wide hepatitis B immunisation programme. CONCLUSIONS: Levels of awareness may contribute towards changes seen in the number of reporting of injuries. In our experience, we contend that education and appropriate policy changes towards easier reporting help to decrease sharps and needlestick injuries in healthcare workers. The hospital-wide hepatitis B immunisation programme helped to raise the immune status of the staff so as to reduce the costly prophylactic usage of hepatitis B immunoglobulin. PMID- 10748973 TI - Sleep/wake cycle and circadian disturbances in shift work: strategies for their management--a review. AB - INTRODUCTION: There has been a growing concern about the ability of individuals to maintain adequate levels of performance over long work shifts, particularly when those shifts span night-time hours. It has, therefore, become expeditious to understand and apply principles of circadian rhythms in order to establish simple, rational and appropriate strategies to help our shift workers maximise their performance and minimise their health problems under the various shift work regimes. This review sought to outline several principles of circadian rhythms and the sleep/wake cycle and some possible strategies to manage disturbances in the sleep and performance arising from shift works. METHODS: Many studies in this field had been carried out. The present review concerns studies which elucidate the general circadian principles as well as those which may provide helpful information applicable for us in the work environment we are living in. RESULTS: It has been found that shift workers invariably suffer from a constellation of symptoms, which can sometime severely compromise their ability to perform optimally during their shift work. There are many factors that influence the sleep/wake cycle and thus, the performance of shift work. These include 1) circadian factors, 2) type of shift work, 3) how a person adapt to circadian disruption, 4) ageing, 5) sleep factors and 6) social and domestic factors. CONCLUSIONS: Several possible strategies could be adopted to improve sleep and performance. These include 1) appropriate scheduling of shift work, 2) proper consideration of the speed of shift rotation, 3) strategies for sleep and napping, 4) installing appropriate lighting at the workplace, 5) the use of sleeping pills/hypnotics such as melatonin and melatonin agonists. PMID- 10748974 TI - Diagnosis of pacemaker lead infection using transoesophageal echocardiography: a case report. AB - INTRODUCTION: Vegetative lead infection is an uncommon complication of permanent pacemaker implantation. The diagnosis is difficult using conventional imaging methods. CLINICAL PICTURE: An elderly Indian woman with a history of diabetes mellitus developed Staphylococcus aureus infection after implementation of a permanent pacemaker. Following a non-diagnostic transthoracic echocardiogram, transoesophageal echocardiography was performed and showed a large vegetative mass attached to the pacemaker lead within the right atrium. TREATMENT: The pacemaker was removed and intravenous vancomycin administered for six weeks. OUTCOME: She was discharged well but demised two months later from a second episode of septicaemia. CONCLUSIONS: Pacemaker lead infection remains a challenging management problem. Transoesophageal echocardiography can facilitate its diagnosis. PMID- 10748975 TI - Early hepatocellular carcinoma presenting with biliary ductal invasion--a case report. AB - Early and small hepatocellular carcinoma (HCC) rarely presents with biliary ductal invasion. We present a case of early HCC presenting with biliary invasion, in the absence of a mass on computed tomographic scanning. The patient, a hepatitis B carrier, was initially diagnosed to have "liver fluke infection" after a "leaf-like structure" was found within the right hepatic duct on endoscopic retrograde cholangiopancreatography (ERCP). The specimen was retrieved with a Dormia basket. Histology revealed HCC. This report highlights an unusual case of early and small HCC presenting with biliary ductal invasion. A high index of suspicion has to be entertained in the background of hepatitis B regardless of the atypicality of presentation. PMID- 10748976 TI - Lichen amyloidosus: a bullous variant. AB - INTRODUCTION: Lichen amyloidosus is a common skin disease seen among Asian patients. CLINICAL PICTURE: Typical features range from macular hyperpigmentation to pruritic, lichenified, hyperpigmented papules. However, in this rare bullous variant of lichen amyloidosus, bullae and vesicles are present. Histopathologically, deposits of amyloid were seen in the papillary dermis, associated with an intraepidermal or subepidermal blister. TREATMENT AND OUTCOME: No good treatment so far, but pruritus can be relieved by topical steroid. CONCLUSION: It is important to screen for systemic amyloidosis with the relevant investigations as it can present similarly with blistering eruptions, in which the prognosis would be grave. PMID- 10748977 TI - Endobronchial mass in a patient with Burkholderia pseudomallei infection. AB - INTRODUCTION: Burkholderia pseudomallei infection, the great mimicker of infectious diseases, has protean clinical manifestations. CLINICAL PICTURE: A 37 year-old man who presented with community-acquired pneumonia affecting the right upper lobe had unremitting fever. Bronchoscopy showed an endobronchial mass in the right upper lobe bronchus. TREATMENT: Intravenous ceftriaxone and oral erythromycin, with empiric antituberculous treatment added later. This was subsequently switched to intravenous ceftazidime and oral doxycycline after the diagnosis was made. OUTCOME: There was resolution of the endobronchial mass. CONCLUSION: This case illustrates a unique and unreported presentation of melioidosis. PMID- 10748978 TI - A case report of total finger joint replacement arthroplasty after traumatic amputation. AB - INTRODUCTION: Joint reconstruction following trauma at the proximal interphalangeal or metacarpophalangeal levels remain a difficult problem in hand surgery. Function of the injured finger depends on the mobility and stability at these joints. In fingers amputated at these levels with joint destruction, the hand surgeon can perform either an arthrodesis or a replacement arthroplasty (either as an emergency or as a secondary procedure). Arthrodesis will give a stable pain-free joint, but at the cost of sacrificing mobility and even cosmesis. CLINICAL PICTURE: We report a case of traumatic amputation through the proximal interphalangeal joint (PIPJ) of the right middle finger. Arthrodesis of the PIPJ in extension using Kirschner wires was performed following replantation due to extensive periarticular soft tissue loss, so as to enable repair and healing of the extensor mechanism. This results in poor cosmesis and stiffness of finger interfering with function three months after surgery. TREATMENT AND OUTCOME: A prosthetic total joint replacement of PIPJ was performed as a secondary procedure. Satisfactory hand function and finger movement ensued two years after the procedure, with no loosening or infection of the implant. CONCLUSION: Total interphalangeal joint arthroplasty is a useful secondary procedure, after the initial replantation, for finger amputation with periarticular soft tissue loss. PMID- 10748979 TI - Neonatal lupus erythematosus: our local experience. AB - INTRODUCTION: Neonatal lupus erythematosus (NLE) presents clinically with either cutaneous lesions or cardiac involvement and is related to the transplacental passage of anti-Ro antibodies from mother to foetus. We report a series of 10 cases seen at the National Skin Centre between 1990 and 1998 including a pair of siblings. CLINICAL PICTURE: The female to male ratio was 2.3:1 and most presented with rash between 3 to 5 weeks of life while 4 had lesions at birth. Four of the 9 mothers had SLE, one was diagnosed to have a lupus-like illness, 2 had Sjogren's syndrome and the remaining two had very high ANA titres but the final diagnosis was uncertain. OUTCOME: The rash resolved spontaneously by 3 to 6 months of age. PMID- 10748980 TI - Case reports of nocardiosis in patients with human immunodeficiency virus (HIV) infection. AB - INTRODUCTION: We present 4 local cases of nocardiosis in HIV-infected patients and discuss the diagnosis, clinical syndromes and therapy of nocardiosis. CLINICAL PICTURE: Two cases presented with pulmonary nocardiosis, one had a cervical lymph node abscess and one had disseminated nocardiosis with pulmonary, cerebral and soft tissue involvement. TREATMENT: Combination therapy is often employed. Sulphonamides or co-trimoxazole, amikacin, imipenen, minocycline and ceftriaxone are some of the drugs that could be used. OUTCOME: Outcome hinges on the early recognition and optimal treatment of this infection. CONCLUSIONS: Clinical presentations vary and diagnosis is difficult and frequently delayed. Nocardiosis should be suspected in patients who present with pulmonary lesions with soft tissue and/or cerebral abscesses. PMID- 10748981 TI - Active management of a patient with endstage pulmonary emphysema using lung volume reduction surgery and intensive rehabilitation. AB - INTRODUCTION: Lung volume reduction surgery (LVRS) and pulmonary rehabilitation are newer options available in the treatment of advanced emphysema. We describe the progress of our first local patient to have undergone these 2 new treatment modalities. CLINICAL PICTURE: A 65-year-old man with advanced emphysema, limited by crippling dyspnoea despite maximal medical therapy with inhaled bronchodilator therapy, methylxanthines and supplementary oxygen. TREATMENT: Physical reconditioning with a 4-week inpatient pulmonary rehabilitation programme, followed by LVRS and a further 6-week outpatient pulmonary rehabilitation. OUTCOME: Dramatic improvements in spirometric indices, arterial blood gases, exercise capacity and overall functional status. Improvements maintained at one year follow up period. CONCLUSIONS: LVRS with pulmonary rehabilitation is an exciting new treatment option in a select group of patients with advanced emphysema. Their role in improving overall performance status and quality of life should be considered in patients otherwise considered to have reached the limits of medical therapy. PMID- 10748982 TI - Pseudomembranous colitis in a patient treated with paclitaxel for carcinoma of the breast: a case report. AB - INTRODUCTION: The concomitant antimicrobial properties of antineoplastic agents may play a role in causing pseudomembranous colitis, which has been documented for cisplatin, cyclophosphamide and 5-fluorouracil. CLINICAL PICTURE: We describe the first case reported in the English literature of severe pseudomembranous colitis occurring in a patient given paclitaxel as adjuvant chemotherapy for breast cancer. There was no prior antibiotic therapy. Stool culture confirmed Clostridium difficile. TREATMENT: Oral vancomycin, metronidazole and cholestyramine led to prompt improvement of intestinal symptoms, which resolved in two weeks. OUTCOME: Repeat colonoscopy performed 3 months later showed total resolution of the colitis. CONCLUSIONS: Awareness of pseudomembranous colitis after paclitaxel chemotherapy can avert life-threatening complications. PMID- 10748983 TI - Muscle dysmorphia in a young Chinese male. AB - INTRODUCTION: To describe a young Chinese male with muscle dysmorphia, a recently proposed variant of body dysmorphic disorder. CLINICAL PICTURE: A 24-year-old Chinese male with a morbid fear of weight and muscle loss with associated compulsive weight training, avoidance behaviour, forced eating, depressed mood and disturbed body image. TREATMENT: Antidepressants and cognitive-behavioural psychotherapy. OUTCOME: Preoccupation with muscularity was reduced. CONCLUSIONS: Muscle dysmorphia has been reported in Western populations, mostly among body builders and anabolic steroid abusers. To our knowledge, this is the first case report originating from a non-Western population. The patient illustrates the nosological difficulty of this recent entity. It is likely to be a culture-bound phenomena associated with a Western concept of an ideal body shape for males. PMID- 10748984 TI - 10th Chapter of Surgeons' Lecture: the challenges in surgery--past, present and future, and in search of the 4Cs. AB - Challenges facing surgical practice in the immediate future will not only be in the fields of clinical and operative surgical techniques, but will also involve a new complexity of clinical, para-clinical and economical issues. The impact of casemix, clinical carepaths, evidence-based medicine, high-technological advances, minimally invasive surgery, teleconferencing, robotic surgery, etc will transform the scene of surgical advancement in a dynamic way. Whatever the winds of change may be, ultimately patient's good and welfare must be uppermost in our minds. The challenge is how to maintain and achieve the 4Cs in this age of medical and surgical revolutionary changes. Commitment, Continuing medical education, Consistency, Compassion and a Caring spirit are the Cs we must always keep alive in our clinical practice. PMID- 10748985 TI - Socioeconomic background and war mortality during Vietnam's wars. AB - To understand the experience of North Vietnamese soldiers and civilians during the American war, I explore the paths leading Vietnamese men into battle by considering the relationship between socioeconomic status and war mortality. I use data obtained from retrospective information on kin survival and other socioeconomic characteristics given by respondents in the 1995 Vietnam Longitudinal Survey conducted in Vietnam's Red River Delta. My findings are opposite to those often cited to describe the experience of young Americans who fought in the Vietnam war. In Vietnam, sons of better-educated fathers bore the burden of war disproportionately in relation to sons of fathers with less education, both in proportion serving in the military and in diminished survival chances in combat. The Vietnamese experience during the American war testifies to the ability of a nation to reorder society temporarily and to persuade higher status groups to contribute fully to the war effort. An appreciation of the meaning of this social reshuffling during the American war is critical for understanding the war, Vietnam, and that country's political outlook. PMID- 10748986 TI - The residency decision of elderly Indonesians: a nested logit analysis. AB - This paper is the first study of which the author is aware that examines elderly Indonesians' residency decisions. The 1993 Indonesian Family Life Survey provides detailed data on the living children of a sample of elderly individuals. This allows a nested logit to be estimated, which pays due respect to the role of children's characteristics in determining the residency outcome. The estimated earnings potentials of the parents and their children are included as explanatory variables but are not found to be important determinants of coresidency. PMID- 10748987 TI - Educational assortative mating across marriage markets: non-Hispanic whites in the United States. AB - Whether local marriage market conditions shape marriage behavior is a central social demographic question. Most work on this subject, however, focuses on one type of market condition--sex ratios--and on a single outcome--marital timing or sorting. We examine the impact of local marriage markets' educational composition on educational assortative mating and on how sorting varies with age. We estimate a discrete-time competing-risks model of educational sorting outcomes, using individual data from the NLSY and community descriptors aggregated from census microdata. Results show that residents of educationally less favorable marriage markets are more likely to marry down on education, and that (for women) their chance of doing so increases with age more than for residents of more favorable markets. PMID- 10748988 TI - The declining significance of race among American men during the latter half of the twentieth century. AB - The extent to which racial minority groups face discrimination in the labor market is the subject of considerable debate. Using William J. Wilson's thesis of the declining significance of race as our theoretical context, we provide further empirical evidence about labor market discrimination by investigating wages among African American, American Indian, Chinese American, Hispanic white, Japanese American, and non-Hispanic white men. We find, during the period before the civil rights movement, that a substantively significant wage disadvantage is evident for these minority groups with controls for observed labor force characteristics. In recent data, these net disadvantages are reduced substantially for each of these groups except Hispanics. With the exception of Hispanics, the results support Wilson's thesis. PMID- 10748989 TI - Understanding the twentieth-century decline in chronic conditions among older men. AB - I argue that the shift from manual to white-collar jobs and reduced exposure to infectious disease were important determinants of declines in chronic disease rates among older men from the early 1900s to the 1970s and 1980s. The average decline in chronic respiratory problems, valvular heart disease, arteriosclerosis, and joint and back problems was about 66%. Occupational shifts accounted for 29% of the decline; the decreased prevalence of infectious disease accounted for 18%; the remainder are unexplained. The duration of chronic conditions has remained unchanged since the early 1900s, but when disability is measured by difficulty in walking, men with chronic conditions are less disabled now than they were in the past. PMID- 10748990 TI - Obesity, disease, and functional limitation in later life. AB - Little is known about the effects of obesity late in life. Using data from the Longitudinal Study of Aging and the Assets and Health Dynamics of the Oldest Old Survey, this study finds an increased prevalence of obesity, over time, among those 70 and older. Obesity is related most strongly to limitations in activities of daily living (ADLs) for women and to activities related to mobility. One ADL, eating, has a negative association to obesity. Obesity is associated with an increased prevalence of arthritis, diabetes, and hypertension. These results are cross-sectional and are based on self-reports of height and weight; they must be interpreted cautiously. PMID- 10748991 TI - The effect of pregnancy intention on child development. AB - In this paper, we use data from the National Longitudinal Survey of Youth to investigate the empirical link between unintended pregnancy and child health and development. An important contribution of our study is the use of information on siblings to control for unmeasured factors that may confound estimates of the effect of pregnancy intentions on infant and child outcomes. Results from our study indicate that unwanted pregnancy is associated with prenatal and postpartum maternal behaviors that adversely affect infant and child health, but that unwanted pregnancy has little association with birth weight and child cognitive outcomes. Estimates of the association between unwanted pregnancy and maternal behaviors were greatly reduced after controls for unmeasured family background were included in the model. Our results also indicate that there are no significant differences in maternal behaviors or child outcomes between mistimed and wanted pregnancies. PMID- 10748992 TI - Son preference and sex composition of children: evidence from India. AB - Although the effect of son preference on sex composition of children ever born is undetectable in national-level estimates that aggregate across all families, this article provides empirical evidence from India that son preference has two pronounced and predictable family-level effects on the sex composition of children ever born. First, data from India show that smaller families have a significantly higher proportion of sons than larger families. Second, socially and economically disadvantaged couples and couples from the northern region of India not only want but also attain a higher proportion of sons, if the effects of family size are controlled. PMID- 10748993 TI - Are births underreported in rural China? Manipulation of statistical records in response to China's population policies. AB - Under the current family planning policy in China, the criterion for evaluating all parties involved in the birth planning system provides an incentive for everyone to see that the policy is met, either in reality through strict enforcement of family planning regulations, or statistically through manipulation of statistical records. We investigate underreporting of births in four rural counties of northern China, using data from a 1992 sample survey featuring a reproductive history. To clarify the mechanisms of underreporting, we focus on the ways in which reporting errors may affect the distribution of first births by time since marriage. The results of our investigation suggest that in three of the four counties, first-birth intervals are lengthened by underreporting of girl babies and by replacing them with second births reported as first births. PMID- 10748994 TI - The New Immigrant Survey Pilot (NIS-P): overview and new findings about U.S. legal immigrants at admission. AB - This paper provides an overview of the New Immigrant Survey Pilot (NIS-P), a panel survey of a nationally representative sample of new legal immigrants to the United States based on probability samples of administrative records of the U.S. Immigration and Naturalization Service (INS). The NIS-P links survey information about immigrants' pre- and post-immigration labor market, schooling, and migratory experiences with data available from INS administrative records, including the visa type under which the immigrant was admitted. Results indicate that the procedures followed for locating, interviewing, and reinterviewing respondents yielded representative samples of new legal immigrants and high quality data. On the basis of data obtained from the first round of the survey, we present new information never before available on the schooling and language skills of new immigrants and their earnings gains from immigration. PMID- 10748995 TI - Synthetic surgical gloves. AB - Surgical gloves are used by healthcare workers primarily to minimize the transmission of bloodborne pathogens and other potential infectants. Gloves made of synthetic materials are getting more attention with the increasing prevalence of allergies to natural rubber latex (NRL) among patients and medical staff. However, synthetic gloves are perceived by some people as providing less protection and being less comfortable than NRL gloves. In this study, we evaluated eight models of synthetic surgical gloves from six manufacturers. We examined their barrier effectiveness (that is, their resistance to viral penetration, resistance to puncture, and strength) durability, and comfort and compared these characteristics to those of NRL gloves. We found that all the gloves offered good barrier protection, but that their level of comfort varied widely. Three gloves were rated Preferred, three were Acceptable, and the remaining two gloves were Not Recommended. PMID- 10748996 TI - Trocars: new data on safety and selection. AB - In our November 1998 issue, we published a Guidance Article on the issues surrounding trocar selection and use. In the current article, we update that information with the results of a survey conducted in conjunction with the monthly newsletter OR Manager. The survey characterizes trocar practices in more than 60 U.S. healthcare facilities, focusing on the following issues: Do surgeons use shielded or nonshielded trocars for primary insertions? And do surgeons use disposable or reusable models? The survey indicated that most surgeons in the responding healthcare facilities use nonshielded trocars during at least half of their laparoscopic procedures. It also indicated that most surgeons use disposable models. These findings suggest that many surgeons support the views presented in our 1998 Guidance Article. In the supplementary article "Eye on Medical Errors," we discuss the likely prevalence of trocar-related injuries. This discussion includes recent--and rare--data from insurance claims for trocar injuries. PMID- 10748997 TI - After midnight. Applying the lessons learned from Y2K. PMID- 10748998 TI - Needlestick-prevention devices. AB - In the Introduction to our October 1999 Evaluation of needlestick-prevention devices (NPDs), we discussed how changes in the U.S. regulatory landscape were beginning to compel many healthcare facilities to switch from conventional needle using devices to protective alternatives, such as NPDs. Our discussion focused on recent legislation introduced at the state and federal levels. Less than a month after we published that article, the pace of change increased dramatically. Most notably, two U.S. agencies--the Occupational Safety and Health Administration (OSHA) and the National Institute for Occupational Safety and Health (NIOSH)- issued recommendations stressing the importance of using NPDs, and several additional states began considering NPD legislation. In this Update, we discuss the changes that have occurred since our October 1999 article. Also in this Update, we describe recent changes to our NPD ratings rationale and provide revised ratings for three previously evaluated NPDs. We discovered the need for these changes while compiling information for our new Special Report, the Health Devices Needlestick-Prevention Device Selection Guide. (This Special Report, which incorporates information from our three most recent NPD studies, is described on page 80.) We believe that, with these changes, our ratings more clearly convey the technical merits of each product. PMID- 10748999 TI - Possible infection hazard from incomplete sealant on OEC Series9600 C-arm system. PMID- 10749000 TI - Inappropriate Fisher & Paykel heater-wire adapter melts allegiance breathing circuit. PMID- 10749001 TI - Anchors away! Don't use drywall anchors to mount medical equipment. PMID- 10749002 TI - Bird TBird AVS Series ventilator may not handle certain international gas pressures. PMID- 10749003 TI - Lessons from experienced guideline implementers: attend to many factors and use multiple strategies. AB - BACKGROUND: Studies of clinical guideline implementation have focused almost entirely on changing individual clinician behavior with single intervention strategies and without much attention to the situational context. The goal of this project was to learn from clinic leaders, seasoned in the guideline implementation process, what contextual variables they viewed as important and whether implementation success could be expected if only a single implementation strategy was used. METHODS: In 1998, 12 people with extensive experience in leading clinical guideline implementation were identified who were thought to have particularly keen insight into the process. They were interviewed to generate variables they considered important, as well as strategies they considered effective when used appropriately. A modified nominal group/Delphi process was then used for rating these variables and strategies, and the reactions of international experts were obtained to add perspective to this information. RESULTS: Eighty-seven variables and 25 strategies were identified, clustering in 6 categories (ranked in order of importance by the panel): organizational capabilities for change, infrastructure for implementation, implementation strategies, medical group characteristics, guideline characteristics, and external environment. All six categories were considered to be important, key, or essential by the experienced implementers, although variables within a medical group that directly affect its ability to undertake planned change were rated as much more important than either guideline characteristics or the external environment. DISCUSSION: Although the opinions of those experienced in the process of guideline implementation are primarily of value for generating hypotheses, panel members believe that implementation efforts focusing on the individual physician with a single strategy are unlikely to be successful. Rather, implementation efforts must use multiple strategies that take account of multiple characteristics of the guideline, practice organization, and external environment. PMID- 10749004 TI - Developing performance indicators that reflect an expanded view of health: findings from the use of an innovative methodology. AB - BACKGROUND: The increasing presence of managed health care in the United States has been accompanied by the widespread use of performance indicators to assess health plans along various dimensions of quality. Current performance indicator sets virtually ignore psychosocial and behavioral factors in the prevention and management of illness, especially chronic illness, in spite of documented evidence in the medical literature of the importance of these factors. Instead, current indicator sets focus primarily on biomedical interventions to prevent, treat, and manage illness. METHODOLOGY: In a novel method for developing performance indicators--the use of a storytelling methodology--eight interdisciplinary panels, composed of health care experts at the community, state, and national levels, each completed two stories about patients with chronic illnesses. The first story described experiences a patient might have in the health care system as it is today; the second story retold the events that might transpire if attention to psychosocial and behavioral factors were integrated into the health care system. FINDINGS: Differences between the two sets of stories developed by the panels revealed common themes and specific areas where indicator development might prove fruitful. Performance indicators were identified from these themes, and work is underway to operationalize them; to identify barriers and opportunities for their inclusion in indicator sets; and to further document their potential health and cost-effectiveness. DISCUSSION: Although not scientifically rigorous, the storytelling method was found to provide consistent results and may be applied to many aspects of the health care planning process, health education, and quality improvement efforts. PMID- 10749005 TI - Measuring antidepressant prescribing practice in a health care system using administrative data: implications for quality measurement and improvement. AB - BACKGROUND: Up to one in eight Americans experiences an episode of depression that requires treatment in his or her lifetime. The direct and indirect costs associated with major depression are high but may be reduced with appropriate treatment. To decrease the probability of relapse, guidelines specify that treatment with antidepressant medications should continue for at least 4 months after symptom remission and that adequate doses of antidepressants be used. A study was conducted in 1997-1999 to examine how different specifications in the construction of quality of care measures for depression treatment influence conclusions about the adequacy of antidepressant prescribing practices. METHODS: Subjects were all adult members of two United Healthcare plans who each had at least one outpatient or inpatient claim with a diagnosis of depression during the years 1993-1995 and were continuously enrolled for 12 months. Pharmacy claims data were used to construct measures of duration of treatment, dose, and type of antidepressant. The effects of two different definitions of a new episode (4 month versus 9-month clean period) and two different ways of identifying an episode of depression (one visit versus two visits with a code for depression) were examined on conclusions about adequacy of antidepressant prescribing practices (dose and duration). Whether antidepressant type was related to the likelihood that antidepressants were prescribed at therapeutic doses was also examined. RESULTS: Patients with two or more visits with depression diagnosis codes were significantly more likely to receive antidepressants than those with only one visit, and were more likely to receive therapeutic doses at each time period (1-5 months). The duration of the clean period was not related to conclusions about therapeutic dosing. Among persons receiving antidepressants, those receiving selective serotonin reuptake inhibitors (SSRIs) were more likely to receive therapeutic doses and to continue treatment for at least 5 months than were those prescribed other classes of antidepressants. In multivariate analysis, being prescribed an SSRI versus another class of antidepressants was significantly associated with receiving both 1 month (OR = 7.3 [5.7-9.3]) and 5 months (OR = 2.0 [1.6-2.5]) of therapeutic treatment. DISCUSSION: Conclusions regarding the appropriateness of antidepressant prescribing can vary markedly, depending on how the quality measure is specified. Given that administrative data are and will continue to be used for both monitoring and quality improvement purposes in the short run, it is critical that we understand how variations in measurement specifications influence the conclusions that are drawn about treatment of depression in health plans. PMID- 10749006 TI - Developing a patient complaint tracking system to improve performance. AB - BACKGROUND: Assessing patient satisfaction exclusively through close-ended scaled survey questions may not provide a complete picture of patients' concerns. Only recently has the role of complaint data as a management tool received attention. FORMATION OF THE TEAM AND THE DATABASE: The Complaint Management Team was created in January 1997 at Hartford Hospital (Conn) to develop a coding and reporting mechanism for complaints (negative comments) gathered from patient surveys. Developing the codebook was an evolutionary process. A database was designed to collect three separate complaints and the verbatim text associated with the code. REPORTING: Department-specific, location-specific, and organization wide reports are generated. Quarterly department-specific reports are used to trend the incidence of complaint themes, identify specific locations with problems, and initiate improvement efforts. OVERALL FINDINGS: Since March 1997, most complaints have fallen into five major categories--accommodations (environment), quality of care (care and treatment), respect and caring (humaneness or attitudes and behaviors), timeliness, and communication. The hospital's real estate department has completed a project focused on increasing patient satisfaction with parking. Two projects are still in progress; one is focused on increasing patient satisfaction with respect and staff caring attitude/behaviors, and one on improving satisfaction with the level of noise on the units. DISCUSSION: Approximately 4,000 survey complaints are coded every year. One limitation of the database is that all sources of complaints received throughout the organization are not yet captured. Another limitation is that the outcomes measurement section has exclusive access to the database. CONCLUSIONS: The patient complaint tracking system enables staff, managers, teams, and departments to develop improvement efforts based on quantitative and qualitative data. PMID- 10749007 TI - To err is human: an interview with the Institute of Medicine's Linda Kohn. PMID- 10749009 TI - Knowledge about genetic risk for breast cancer and perceptions of genetic testing in a sociodemographically diverse sample. AB - Informed consent for genetic testing for breast-ovarian cancer susceptibility requires that women understand basic concepts about the inheritance of cancer susceptibility and the benefits and risks associated with genetic testing. Women awaiting routine medical services (N = 220) were surveyed about their knowledge of breast cancer and cancer genetics and their perceptions of genetic testing and personal risk. There were no racial differences in median income or mean level of education. Compared to Caucasian women, African American women knew significantly less about breast cancer and about genetic risk for breast cancer. African American women had different psychological, social, and economic concerns as evidenced by how they weighted the benefits and risks of genetic testing. This study is the first to assess several dimensions of informed consent for genetic testing among a sociodemographically diverse group. The findings should enable health professionals to target the African American and lower-income populations with the appropriate education and counseling. PMID- 10749008 TI - Sleep quality and presleep arousal in chronic pain. AB - This study was designed (1) to characterize the extent and nature of sleep complaints of chronic pain patients and (2) to examine the factors that predict sleep quality. A heterogeneous sample of 51 outpatients with benign, chronic pain was recruited from newspaper and pain clinic advertisements. Patients completed a variety of self-report instruments including the Multidimensional Pain Inventory, the Pittsburgh Sleep Quality Index, the Pre-Sleep Arousal Scale, and the Beck Depression Inventory. Sleep complaints were reported by 88% of the sample. Presleep cognitive arousal, rather than pain severity, was found to be the primary predictor of sleep quality. PMID- 10749010 TI - Bulimia interventions via interpersonal influence: the role of threat and efficacy in persuading bulimics to seek help. AB - The causes and medical consequences of bulimia are well established in the literature, yet prevalence rates for bulimia continue to be quite high. Eating disorder books and popular magazines have offered a variety of ways in which individuals can communicatively influence bulimics to seek the help they need to treat their disorder. Unfortunately, many of the recommended influence strategies have been atheoretically derived and sorely lack empirical data affirming their effectiveness. The present study investigates and empirically tests the effectiveness of interpersonal intervention messages aimed at getting bulimic individuals to seek medical help. Several theoretical perspectives, as they relate to messages of threat and efficacy, were used to predict the effects of bulimics' threat and efficacy perceptions on message acceptance and message rejection. A main and positive effect for efficacy was found on message acceptance. A threat by efficacy interaction on message acceptance was partially supported for both cognitive and behavioral message acceptance. The threat by efficacy interaction was not significant for message rejection. Overall, this study confirmed the notion that high levels of both threat and efficacy are necessary to convince bulimic individuals to seek help. These findings can be used to aid concerned individuals in their quest to intervene and to get bulimics to seek out the necessary medical assistance and support. PMID- 10749011 TI - Development and evaluation of an interactive CD-ROM refusal skills program to prevent youth substance use: "refuse to use". AB - An interactive CD-ROM program designed to reduce adolescent substance use was developed and evaluated. The program uses video vignettes to teach refusal skills and socially acceptable responses to substance use situations, specifically offers of marijuana. In a randomized pretest-to-posttest experiment with 74 public school students from six classes in three high schools, significant changes were observed at posttest on (1) the adolescent's personal efficacy to refuse the offer of marijuana, (2) the adolescent's intention to refuse marijuana if offered, and (3) the adolescent's perceptions of the social norms associated with substance use and the importance of respecting another's decision to refuse a drug offer. In addition, adolescents in the treatment condition were able to recall approximately 50% of the portrayed refusal strategies. Findings are discussed with regard to the potential benefits of an interactive multimedia approach for conducting substance use interventions. PMID- 10749012 TI - Life event exposure, physiological reactivity, and psychological strain. AB - The study tested the prediction that the experience of life events would be reflected in greater autonomic reactivity and that this might play a moderating role between reported stress and psychological symptoms. Eighty-seven undergraduates were screened with a life events scale and thirty-nine were allocated to three groups representing high, medium, and low life event scores. The General Health Questionnaire, Profile of Mood States, the Spielberger Trait Anxiety Scale, and the Marlowe-Crowne Scale were administered. Electrodermal activity and heart rate were recorded during an habituation series of tones and a digit-symbol substitution task. The groups differed both on the GHQ and the POMS and, also, in their physiological responses to the tones. Unexpectedly, the high life event group, compared to the other groups, appeared to display smaller physiological responses. The cognitive task resulted in elevated physiological activity but no consistent group differences. Cardiovascular reactivity moderated the relationship between life event scores and reported distress. These findings are discussed in relation to theories of psychophysiological adaptation to negative events. PMID- 10749013 TI - Hostility in marital dyads: associations with depressive symptoms. AB - We examined the relations of hostility (of self and spouse) with self-ratings of depressive symptoms in 898 spouse pairs. Self-ratings of hostility were initially examined as predictors of depression. Next, spouse self-ratings of hostility were added to the model. Finally, the interaction of self x spouse hostility was investigated. These relations were explored for three components of hostility (Cynicism, Aggressive Responding, and Hostile Affect). Age and education were controlled in all models and effects were examined separately for women and men. Self-ratings of Hostile Affect were positively related to depressive symptoms for both women and men. Self-ratings of Cynicism were also significantly related to depression, but only for men. All three components of spouse's hostility were positively related to one's own symptoms of depression for women. For men, however, spouse's hostility was not related to symptoms of depression. These findings highlight the need to study psychosocial risk factors in social units and have potential implications for intervention. PMID- 10749014 TI - Liposomes as vehicles for targeted therapy of cancer. Part 1: preclinical development. PMID- 10749015 TI - Liposomes as vehicles for targeted therapy of cancer. Part 2: clinical development. PMID- 10749016 TI - Iridium-192 implantation for node-negative carcinoma of the penis: the Cookridge Hospital experience. AB - Carcinoma of the penis is a rare tumour of the male urogenital tract, which may be treated by using several modalities. We present a single-centre experience of iridium-192 implantation. From 1980 to 1997, 31 patients with node-negative penile cancer were treated with an iridium-192 implant to the penis. A retrospective analysis of the case notes was made. Survival curves were estimated by the Kaplan-Meier method. The median age at treatment was 61.5 years. Twenty seven patients presented with Jackson Stage I disease and four with Stage II disease. They were treated with an iridium-192 implant to the penis after biopsy (n = 25) or tumour excision (n = 6), with a 'watch and wait' policy for inguinal nodes. Four patients did not complete their implantation treatment and had additional external beam radiotherapy. The median follow-up was 61.5 months. The primary tumour was controlled in 25 of 31 patients (80.6%) by the implant. In all but one patient with primary relapse, surgical salvage was successful, although one patient died of septicaemia 3 weeks after surgery. Nodes were the initial site of relapse in seven patients, with associated relapse in the primary in one. The actuarial 5-year survival rates were as follows: overall survival 69.0 %, disease-specific survival (corrected for intercurrent deaths) 85.4%, relapse-free survival 57.8% and local relapse-free survival 75.6%. One patient underwent amputation for necrosis and 11 of 25 patients (44%) who achieved penile conservation required dilatation for urethral stenosis. In conclusion, iridium 192 implantation is a successful method of treatment for penile cancer in terms of local control, with preservation of function in the majority of patients. In those who do relapse at the primary site, surgical salvage is usually possible. PMID- 10749017 TI - The role of staging CT scans in the treatment of prostate cancer: a retrospective audit. AB - A retrospective audit was performed to review the use of diagnostic and planning computed tomographic (CT) scans in the management of patients treated with radical radiotherapy for prostate cancer at Mount Vernon Hospital. All 97 patients had a planning CT scan. In addition, 85 also underwent a diagnostic scan for staging purposes. Fifty-one (60%) had both pelvic and abdominal imaging. Twenty abnormalities were detected in 19 patients. Although 13 of these were 'malignant' abnormalities considered to represent metastatic disease, only four altered the treatment intent. Overall, only 4% of patients were denied radical treatment on the basis of CT findings. Malignant intra-abdominal disease was not identified in the absence of metastatic disease in the pelvis. This study confirms that abdominal CT scans contribute very little useful prognostic information in men with prostate cancer, and are not necessary for routine staging prior to radiotherapy. We propose that a single CT scan of the pelvis in patients who are suitable for radical radiotherapy can provide adequate information for both staging and planning purposes, resulting in significant reductions in cost, radiation exposure and scanner time. PMID- 10749019 TI - Education and training of senior house officers in clinical oncology departments. AB - A survey of clinical oncology departments has been undertaken to ascertain the educational training of senior house officers (SHOs). Questionnaires were sent to the clinical directors of all UK clinical oncology departments, who were asked to forward questionnaires to individual SHOs. Replies were received from 26 of 54 departments, identifying 88 SHO posts and, from these, 48 SHOs have returned questionnaires. The clinical directors considered that the main function of the SHOs was to supervise the care of ward patients, but that gaining experience in oncology was also important. Forty-four of the 88 posts (50%) were part of a fixed medical rotation. Twenty-three of 26 (88%) departments had a designated consultant to supervise SHO training. In 22 of the 26 (85%) departments there was protected teaching time for SHOs, the majority organized and carried out by consultant clinical oncologists. SHOs in clinical oncology departments are pluripotential; many pursue careers other than clinical oncology and have differing career objectives. Thirty-one of 48 (65%) SHOs considered that the post had met their expectations. Recommendations are made for the maintenance and improvement of the educational opportunities for SHOs in clinical oncology departments. PMID- 10749018 TI - Evaluation of a nurse-led telephone clinic in the follow-up of patients with malignant glioma. AB - The aim of this study was to evaluate nurse-led telephone follow-up (NTF) for patients with high-grade glioma as an alternative to conventional clinic follow up (CCF) and to assess patient satisfaction with this approach. Patients who were completing primary therapy for high-grade glioma and were suitable for CCF were offered the alternative of nurse-led telephone follow-up. NTF was arranged by the nurse at mutually agreed times. Assessment was by open discussion and a semistructured questionnaire, together with the Barthel Activities of Daily Living Index. Formal medical assessment in the clinic was arranged at 4-month intervals or earlier if indicated. Twenty-two patients were asked to complete a satisfaction questionnaire. Between February 1996 and October 1997, 43 patients with high-grade glioma, one with primitive neuroectodermal tumour and one with oligoastrocytoma agreed to be monitored by NTF. Their median survival from diagnosis was 16 months (95% confidence interval 13-23 months). At the time of analysis, the median time of follow-up by the telephone clinic was 6 months (range 2-21), with symptomatic progressive disease the reason for discontinuation of NTF in all patients. Two-hundred and fifty-four telephone calls were made, of which 234 were routine and 20 non-routine, being initiated by the patients or their carers. NTF was considered as a sufficient replacement for CCF during the stable phase of the disease. There were 41 unscheduled clinic visits, of which 31 were at the time of progression and usually initiated at NTF. The majority of unplanned visits were due to a change in symptoms and would not have been avoided with CCF carried out at the same time intervals. Patient satisfaction was high, with a median satisfaction score of 9, (range 3.6-10 ) on a scale of 0-10. NTF provides an alternative approach to conventional hospital attendance and moves the emphasis away from cancer surveillance to a more patient centred supportive model. It can be carried out without apparent detriment to the patient and is associated with high satisfaction rating. PMID- 10749020 TI - Radical radiotherapy for inoperable non-small cell lung cancer: what factors predict prognosis? AB - We set out to determine the factors that predict the outcome of conventional radical radiotherapy for inoperable non-small cell lung cancer. A retrospective casenote review was carried out of all 69 patients treated between 1986 and 1992 at the Northern Ireland Centre for Clinical Oncology, Belfast, with radical radiotherapy for inoperable non-small cell lung cancer. The tumour dose ranged from 45 Gy to 67.5 Gy, delivered in 15-30 fractions, 5 days per week over 3-6 weeks. All patients were followed up for 5 years. The disease was TNM Stage T1 T4N0-N2M0. The majority of tumours (51) were squamous. Overall survival was 63.8% (44-patients; 95% confidence interval (CT) 51.3-75.2) at one year; median survival was 16 months and 5-year survival was 13% (nine patients; 95% CI 6.1 23.3). Five-year survival for the 36 patients with stage T1 or T2 disease was 5.6% (2 patients). Five-year survival for the 33 patients with stage T3 or T4 disease, all with tumours at or near the carina, was 21.2% (seven patients). A WHO performance status of 0 or 1 (P = 0.03, Cox proportional hazards model) was associated with a better chance of survival. PMID- 10749021 TI - Complete pathological response to chemotherapy for non-small cell lung cancer demonstrated by gamma camera positron emission tomography. AB - We report the case history of a 61-year-old female smoker who presented with an inoperable T2N2M0 squamous cell carcinoma of the right upper lobe bronchus. This was staged by computed tomography (CT), positron emission tomography (PET) using a modified dual-headed gamma camera, and mediastinoscopy. She then underwent three cycles of cisplatin-containing chemotherapy. After the chemotherapy, CT demonstrated a residual 10 mm mass in the right upper lobe and a considerable reduction in size of the mediastinal lymphadenopathy. Functional tumour imaging with PET showed no abnormality, suggesting that there was no remaining viable tumour. At right upper lobectomy a complete pathological response was confirmed. We discuss PET, the potential new applications of gamma camera technology, and the use of cisplatin-containing chemotherapy in non-small cell lung cancer. PMID- 10749022 TI - Pulmonary tumour emboli: a difficult ante-mortem diagnosis. AB - A woman with known metastatic breast cancer presented with subacute dyspnoea. Investigations were non-conclusive; provisional diagnoses of pulmonary thromboemboli and carcinomatous lymphangitis were made. Despite treatment the patient died 10 days after admission. Post-mortem examination showed extensive tumour emboli within the pulmonary arteries. The appropriate investigation and management of such patients is discussed. PMID- 10749023 TI - Clear cell carcinoma of the fimbria of the fallopian tube in a BRCA1 carrier undergoing prophylactic surgery. AB - We report the case history of a patient with a family history of breast and ovarian cancer who was subsequently found to be a carrier of the BRCA1 gene, in whom a tiny focus of clear cell carcinoma was found at the fimbrial end of one fallopian tube when she underwent prophylactic hysterectomy and bilateral salpingoophorectomy. The implications of this finding are discussed. PMID- 10749024 TI - Metastatic adenocarcinoma of the uterine cervix to the thyroid gland. AB - Clinically diagnosed metastasis to the thyroid gland is rare. The authors present the first reported case of metastasis to the thyroid gland from a primary adenocarcinoma of the uterine cervix. PMID- 10749025 TI - Avascular necrosis in patients treated with BEP chemotherapy for testicular tumours. PMID- 10749026 TI - Dietary requirements for indispensable amino acids in adult humans: new concepts, methods of estimation, uncertainties and challenges. PMID- 10749027 TI - Effect of body build on the validity of predicted body fat from body mass index and bioelectrical impedance. AB - The objective of this study was to test whether differences between body fat percent (BF%) measured by densitometry and BF% predicted from body mass index (BMI) or bioimpedance (BIA) can be explained by differences in body build. Weight, height, sitting height (leg length), arm length, skeletal widths, BIA, bone mineral content from dual-energy X-ray absorptiometry, and BF% from densitometry were measured in 90 apparently healthy, young adult subjects (age range 18-31 years). The BMI was calculated and BF% predicted from BMI, age, and sex. BF% was also predicted from BIA. BF% measured by densitometry was 29.3 +/- 5.0% for women and 14.6 +/- 5.1% for men. BF% predicted from BIA (27.1 +/- 6.7% in women, 19.0 +/- 6.0% in men) was significantly different from measured values in both sexes. BF% predicted from BMI (25.9 +/- 2.2% for women, 15.6 +/- 2.4% for men) was only significantly different from BF% from densitometry in women, not in men. In both sexes skeletal widths, especially elbow width, were related to the prediction error of BF% from BMI, confirming the hypothesis that BF% predicted from BMI underestimates BF% in persons with a relatively slender body build or frame. The prediction error of BF% from BIA was found to be related to the length of the arms and legs, confirming the hypothesis that BIA overestimates BF% in persons with relatively long limbs. PMID- 10749028 TI - Protective function of alpha-tocopherol against the process of cataractogenesis in humans. AB - In a random trial 50 patients with unilateral/ bilateral idiopathic immature senile cataract (cortical n = 25, nuclear n = 25) requiring surgery at least in one eye were included in the present study. Reduced glutathione (GSH), vitamin E, malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) were studied in these patients receiving either vitamin E (n = 12 in each subgroup) or placebo (n = 13 in each group) for 30 days. A comparable increase of vitamin E in both types of lens homogenates of cataractous patients was observed in the study group. The level of GSH in cortical cataractous lenses in the study group was significantly raised (p < 0.001) whereas it was not increased significantly in nuclear cataractous lenses (p > 0.05) as compared to the placebo group. The percentage decrease in MDA levels was greater in cortical cataracts (38.07%) than in the nuclear type (27.94%). The activity of GSH-Px in cortical lenses was higher than that in the nuclear cataractous lens. The change in the size of lens opacity in cortical cataractous patients receiving vitamin E therapy was significantly decreased as compared to the placebo group. There may be a direct modulating effect of vitamin E on some GSH-related enzymes and the cortex of the lens might be protected more than the nucleus. PMID- 10749029 TI - Effects of dietary fish oil supplementation on the phospholipid composition and fluidity of cell membranes from human volunteers. AB - BACKGROUND: Membrane fluidity is an important aspect of cellular physiology which may be manipulated by diet. METHODS: We studied the effect of dietary fish oil on the membrane composition of erythrocytes and cheek cells, and on membrane fluidity of erythrocytes as assessed by fluorescence recovery after photobleaching (FRAP). Healthy volunteers received a daily supplement of fish oil (930 mg EPA, 630 mg DHA) for 42 days. RESULTS: The intervention reduced the ratio of n-6 to total fatty acid in the phospholipid fraction of erythrocyte membranes but the n-3 fraction remained stable and the ratio of cholesterol to phospholipid increased. The level of EPA and DHA in cheek cells increased significantly during the intervention period. The mean diffusion coefficient of the fluorescent probe in erythrocyte membranes increased from 7.2 +/- 0.7 x 10-9 cm2/s at the start to 9.8 +/- 0.5 x 10-9 cm2/s after 21 days. Membrane fluidity remained higher than the initial value 42 days after withdrawal of the supplement. Fish oil also reduced platelet aggregation in response to ADP but there was no effect on plasma lipid profiles. CONCLUSION: We conclude that n-3 fatty acids influence erythrocyte membrane composition at relatively low levels of supplementation by a mechanism which does not necessarily involve an increase in the level of EPA or DHA present in the membrane. PMID- 10749030 TI - Consumption of oat milk for 5 weeks lowers serum cholesterol and LDL cholesterol in free-living men with moderate hypercholesterolemia. AB - The aim of this study was to investigate whether consumption of a newly developed oat milk deprived of insoluble fiber would result in lower serum cholesterol and low-density lipoprotein (LDL) cholesterol levels in men with moderate hypercholesterolemia. The study had a randomized, controlled double-blind design, and oat milk was compared with an identically flavored control drink. Sixty-six men were recruited from a screening program and were randomly assigned to two groups. Each group took either oat milk or a control drink (rice milk) for 5 weeks (0.75 liters/day) and then switched to the other drink regimen for another 5-week period with a 5-week washout period between the test periods. The oat milk contained more dietary fiber, especially beta-glucan (0.5 g/100 g), than the control drink (<0.02 g/100 g). Both drinks were well appreciated and got similar sensory evaluation, indicating that the double-blind design had been attained. In the final analysis 52 subjects remained. Compared with the control drink, intake of oat milk resulted in significantly lower serum total cholesterol (6%, p = 0.005) and LDL cholesterol (6%, p = 0.036) levels. The decrease in LDL cholesterol was more pronounced if the starting value was higher (r = -0.55, p < 0.001). The concentration of high-density lipoprotein cholesterol was not significantly different after consumption of the two drinks. Serum triglycerides did not change significantly after intake of oat milk, but a significant increase was observed after intake of the control drink (p = 0.003). It is concluded that also oat milk deprived of insoluble fiber has cholesterol-reducing properties. PMID- 10749032 TI - Protective efficacy of trypsin inhibitor on the the gut following rotavirus infection in malnourished infant mice. AB - Epidemiologic data suggest a close relationship between protein energy malnutrition (or protein energy malnourished) (PEM) and development of infectious diarrheal diseases. In the present studies, the impact of trypsin inhibitor (TI) on the gut following rotavirus (RV) infection was investigated in PEM young growing inbred balb/c mice. The animals were divided into four groups: (1) control; (2) PEM; (3) PEM+RV and (4) PEM+ RV+TI. Control and PEM animals were orally inoculated with normal saline, PEM+RV animals were given 50 microl RV (100ID50) and PEM+RV+TI animals were inoculated with 0.6 mg TI/g body weight + 50 micronl RV. The body weight of each animal was recorded. Six animals from each group were sacrificed on days 0, 1, 3, 5, 7 and 10 post inoculation by cervical dislocation. The intestines were removed, everted and homogenized in ice-cold saline. Leucine aminopeptidase (LAP), glutathione (GSH) levels and L-leucine uptake were determined in the jejunum and ileum. Body weight was decreased in PEM and PEM+RV groups compared to the controls. LAP and leucine uptake levels were elevated in the PEM group but decreased in the PEM+RV group. GSH levels were decreased in the PEM+RV group. Enzyme activity, GSH and LAP uptake levels were restored in the PEM+RV+TI group. The study shows the protective role of soybean TI against RV infection in malnourished mice. PMID- 10749031 TI - Effect of dietary hydrogenated fish oil on the plasma lipoprotein profile and on the fatty acid composition of different tissues on the rat. AB - Dietary fatty acids are actively incorporated into membrane lipids, and fat intake can modify the composition and the biochemical activity of cellular membranes and the pattern of plasma lipoproteins. Industrial hydrogenation of polyunsaturated oils leads to the formation of isomeric trans fatty acids which are incorporated into cellular membranes when they are present in the diet. The trans fatty acid amount present in hydrogenated oils depends on the degree of hydrogenation, being high for partially hydrogenated oils and low for highly hydrogenated oils. Hydrogenated fish oil is widely used in some countries for the production of margarine and industrial fats. This study compares the fatty acid composition of plasma, erythrocytes, subcutaneous adipose tissue, and hepatic microsomal membranes and the plasma lipoprotein profile after feeding rats with a synthetic diet containing either fish oil, partially hydrogenated fish oil, or highly hydrogenated fish oil. It is observed that the tissue content of monounsaturated fatty acids increases and that the content of polyunsaturated fatty acids decreases after an increase of the degree of hydrogenation of the dietary fat. Tissues from animals fed partially hydrogenated fish oil show significant amounts of trans fatty acids only. The plasma triacylglyceride composition and the lipoprotein profile are also altered by the degree of hydrogenation of the dietary fat. Triacylglycerides decrease after highly hydrogenated fat feeding only. Total cholesterol and low-density lipoprotein cholesterol are significantly increased after partially hydrogenated fat feeding. Although no direct evidence is presented, this effect may be attributable to the high content of trans isomers of this dietary fat which nutritionally may behave as saturated fatty acids. PMID- 10749033 TI - Evaluating a new mobility device: feedback from women with disabilities in India. AB - PURPOSE: To gather the opinions of potential wheeled mobility device users at an early stage in the design process to ensure the development of technology which would meet their functional needs. METHOD: Eight women with bilateral lower extremity disabilities living in Gujarat state, India, participated in this study. The women were introduced to a working model of a new wheeled ground mobility device (GADI2) for a brief trial and participated in a feedback interview which solicited information on different aspects of the design, specifically the interface between the device and the user, the physical environment and the sociocultural environment. Both qualitative and quantitative data were collected and analysed. RESULT: Although the overall response to the device was positive, there was a lack of consensus in some of the feedback gathered. There were varying opinions across the participants and recommendations were often in opposition to what would typically be recommended in a traditional rehabilitation setting. CONCLUSIONS: This study investigates and discusses the research findings from a rehabilitation perspective with a focus on the functional versus technical design aspects. The importance of involving potential consumers in the design of technology is highlighted. The small sample size and lack of consensus in some of the results indicates the need for further research and field testing of this new mobility device design. PMID- 10749034 TI - Hand performance assessment of ten people with rheumatoid arthritis when using a range of specified saucepans. AB - PURPOSE: The aim of the pilot study was to provide information about the design and use of saucepan handles to enable clinicians and designers to specify and provide products that are more appropriate for use by people with Rheumatoid Arthritis (RA). The objectives were: to evaluate aspects of new handle design for saucepans in terms of their ease of use for people with RA; document hand grip strength and configuration (grip patterns); record relevant anthropometric data to aid the development of new designs and perform an assessment of lifting techniques used in conjunction with perceived optimum handle configuration. METHOD/RESULTS: Observation and video footage show that subjects continued to use familiar, but damaging, ways of lifting the saucepan even after extensive joint protection training by occupational therapists. Grip strengths recorded using a sphygmomanometer were similar to those found by other studies. The anthropometric measurements taken from the sample group were found to be within available anthropometric surveys of able-bodied people. However, hand length within the sample group with RA was longer than the equivalent in surveys of able-bodied subjects. CONCLUSIONS: Subjects preferred the narrower handles with some surface texture to the larger and more rounded tapered handles. Large handled saucepans were found not to be viable due to the constraints of UK cooking hob sizes and existing British Standards relating to saucepan specification. PMID- 10749035 TI - Functional recovery of community stroke patients. AB - PURPOSE: The aims were to describe the functional abilities of and services provided to stroke patients not admitted to hospital a year after stroke and to investigate factors associated with receiving rehabilitation services. METHOD: A cohort of stroke patients who had not been admitted to hospital were notified by general practitioners. Patients were assessed at a month and a year after stroke on measures of impairment and disability. The rehabilitation received was recorded. There were 124 stroke patients notified by GP's who had not been admitted to hospital. Of these 70 were assessed at both one month and one year after stroke. RESULTS: Patients showed persistent impaired levels of disability one year after stroke, as measured by the Extended Activities of Daily Living (EADL) scale. There was no significant improvement in Barthel scores and only a slight improvement on the EADL from one month to one year after stroke. There were no patients with severe mobility or severe arm impairment. Most patients had some cognitive impairment but there was no significant change between a month and a year. The provision of rehabilitation services increased between a month and a year after stroke. Rehabilitation was provided to those patients with impaired motor function. CONCLUSIONS: Stroke patients not admitted to hospital have significant levels of disability which does not change substantially in the year after stroke. Rehabilitation was provided on the basis of physical function, rather than cognitive function. PMID- 10749036 TI - Recovery from stroke: does rehabilitation counselling have a role to play? AB - AIMS AND OBJECTIVES: The aim of this paper is to explore the stroke literature, with particular emphasis on the management of psycho-social issues during and in the aftermath of stroke rehabilitation. Within the literature, there are a number of studies that indicate that the present 'medical/physical' approach to stroke rehabilitation may not be achieving desirable long-term psycho-social outcomes and these are considered. The studies highlight the potential effect of the human emotional and social aspects of having a stroke upon the physical and mental well being of the person. For the person living with stroke, the ability to maintain a positive sense of direction in life as well as hold onto their, or develop a new, identity is crucial in terms of adapting to change. Holding onto one's self-worth and unity with the past and anticipating the future enhances the ability to cope and adapt in the aftermath of stroke rehabilitation. This links with the investigation into rehabilitation counselling as a potential strategy for the management of long-term psycho-social functional status following stroke. CONCLUSION: It is suggested that the medical model of stroke rehabilitation places undue emphasis on clinical diagnosis and treatment. By default, measurement of physical independence or disability that is compressed into standardized scales has arguably led to the neglect of the emotional and social consequences of stroke and a partial or inhibited view of the person. It is proposed that there is a need to develop and evaluate the effectiveness of rehabilitation counselling from short-term stroke rehabilitation through to long term psychosocial adaptation, for this client group to provide clear evidence for service provision. PMID- 10749037 TI - Towards a new ICIDH. International Classification of Impairments, Disabilities and Handicaps. AB - In 1995 a special issue of Disability and Rehabilitation was published on the use of the International Classification of Impairments, Disabilities and Handicaps (ICIDH) which was issued by WHO in 1980. Since 1995 a lot of energy has been spent by WHO and other organizations aiming at the development of a new and improved ICIDH. In 1997, a draft ICIDH-2 (Beta-1 version) was circulated by WHO in order to be tested in the field. This paper informs the reader about the content of the draft ICIDH, the revision process planning and some Dutch comments relating to the actual draft. PMID- 10749038 TI - Why did the patient choose an inappropriate orthosis? AB - PURPOSE/METHOD: A 70-year-old engineer, suffering from drop foot, designed and produced a strange orthosis. Although on ambulation the orthotic device elevated his foot, an abnormally strange walking pattern was required. Of his own volition the patient attended an orthotic clinic requesting that a local manufacturer produce a replica of the orthosis. The patient received an explanation pertaining to the range of regular orthotics available and in what way they would improve his functional ability. RESULTS/CONCLUSION: The patient saw all the options available and physically tried several. The patient was afforded the opportunity of free choice and chose his own, self-designed orthotic. From a professional point of view this was the most inappropriate option. The rehabilitation team erred in its psychological management of the case, for it overlooked the question of the patient's motivation and commitment to change. PMID- 10749039 TI - Veterinary education and students' attitudes towards animal welfare. AB - Veterinary students at two British universities in their first preclinical, first clinical and final years of study, completed questionnaires designed to assess their attitudes towards the welfare of animals. These attitudes were divided into their two constituent components: emotional (emotional empathy with animals) and cognitive (belief in the sentience of animals). Analyses of variance revealed that the year of study was significantly related to the perceived sentience of dogs, cats and cows, with students in their later years of study rating them as having lower levels of sentience. The female students rated themselves as having significantly higher levels of emotional empathy with animals than did the male students. There was also a significant interaction between sex and year of study, the female students maintaining relatively high levels of empathy throughout the three years, whereas the male students showed lower levels of empathy in their later years. PMID- 10749040 TI - Causes of mortality and culling in adult pheasants. AB - The causes of the deaths or culling of 155 adult pheasants in breeding pens on one site between 1995 and 1997 were investigated. Approximately half the deaths were the result of problems associated with the reproductive tract or trauma, including injuries acquired during fighting or mating. Sinusitis was the commonest infectious cause of mortality or culling, despite medication of the flocks for mycoplasmosis. Marble spleen disease and pheasant coronavirus associated nephritis, two viral conditions capable of causing high mortality, were diagnosed in a few birds in 1996 and 1997. Histomoniasis (blackhead) contributed to the mortality in 1996. A lymphomatous condition of uncertain aetiology was detected in a small number of birds. PMID- 10749041 TI - Tearing of the dura mater in three dogs. AB - Tearing of the thoracolumbar dura mater was diagnosed after myelography in three large breed dogs. All three dogs lost the ability to walk suddenly, after a period of vigorous running or struggling. Radiographic changes suggested that the tearing of the dura was associated with intervertebral disc injury in two dogs. These dogs regained the ability to walk after medical treatment. Postmortem findings suggested that the tearing of the dura mater in the third dog was associated with nerve root injury and spinal cord haemorrhage after an episode of violent struggling. PMID- 10749042 TI - Suspected case of stiff-horse syndrome. PMID- 10749043 TI - Availability of medicines--a report by the BVA Medicines Select Committee. British Veterinary Association. AB - In 1998, in view of continuing concerns about the availability of medicines, the BVA set up a Medicines Select Committee to review policy in this area and to report on any steps that could be taken to introduce greater flexibility into controls. The committee's report was finalized in September 1999, and approved by BVA Council in December (see VR, December 18/25, 1999, pp 718-719). The text of the report, which the Association intends should form a framework for action, is given here. PMID- 10749044 TI - Tendering for LVI work. PMID- 10749045 TI - Cydectin 1 per cent injectable solution for sheep. PMID- 10749046 TI - Control of digital dermatitis. PMID- 10749047 TI - Camelids and the Dangerous Wild Animals Act. PMID- 10749048 TI - Plesiomonas shigelloides--an uncommon cause of diarrhoea in cats? PMID- 10749050 TI - The diamond vermilion flap--a new technique for vermilion augmentation in cleft lip repair. AB - OBJECTIVE: In most cleft lips patients, the vermilion on the medial aspect of the repair is deficient in height. Not many surgeons have dealt with ways to augment the height of the vermilion at this location. In the unilateral cleft lip, the repair may include the insertion of a small triangular vermilion flap from the cleft side into an incision made for it in the muco-vermilion junction of the noncleft side. We have developed a new technique to further augment the vermilion in incomplete cleft lip patients. METHOD: Vermilion remnants may be present in these patients on the scar tissue between the margins of the cleft. A diamond shaped vermilion flap based on its attachments to the orbicularis oris muscle may be transposed into the noncleft side to an incision made caudal and parallel to the muco-vermilion border. Then the triangular vermilion flap may be inserted just distal to the island flap. Using this method, the vermilion is augmented and the muco-vermilion line parallels more the white skin roll. PMID- 10749049 TI - Posterior pharyngeal flap and sphincter pharyngoplasty: the state of the art. AB - Surgical management of velopharyngeal insufficiency by attachment of posterior pharyngeal flap or construction of sphincter pharyngoplasty is reviewed. Posterior pharyngeal flap surgery is well established, with a long history dating back to the 19th century. Flaps have been based superiorly, inferiorly, or laterally. There have been reports of airway obstruction and obstructive sleep apnea associated with posterior pharyngeal flap surgery. The concept of surgical creation of a dynamic sphincter pharyngoplasty to provide velopharyngeal closure was first introduced by Hynes in 1950. Hynes and others have proposed several subsequent anatomic modifications. Airway dysfunction has also been reported following sphincter pharyngoplasty, but may not be as frequent or severe as with posterior pharyngeal flap. While several studies have compared posterior pharyngeal flap and sphincter pharyngoplasty in terms of speech outcome or complications, there is not, as yet, a consensus regarding the specific choice of one versus the other for surgical management of velopharyngeal insufficiency. PMID- 10749051 TI - Modification of the Koken nasal splint. AB - Koken nasal splints have been found effective postoperatively in treating cleft lip nose deformity. In our experience, their retention has been poor, so reducing the molding effect. This paper presents a modification designed to overcome the problem of retention. PMID- 10749052 TI - Closure of palatal fistula with a local mucoperiosteal flap lined with buccal mucosal graft. AB - OBJECTIVE: Oro-nasal fistula is a common complication of palatoplasty. Current methods for fistula repair utilize mucoperiosteal flaps or pedicled flaps. These procedures are often cumbersome and leave a raw nasal surface, which may increase the incidence of postoperative risks and problems. In addition, the recurrence rate of the fistula is as high as 34%. We propose a simple two-layer method of fistula repair to avoid recurrences. DESIGN: A standard mucoperiosteal flap is raised on the oral side. A buccal mucosal graft is harvested from the cheeks and sutured to the nasal side of the flap that is then inset into the fistula. SETTING: Patients were either referred to the senior author's private practice (four patients) or were patients who had previously been operated on by the senior author himself (three patients). SUBJECTS: Study subjects consisted of seven patients, four males and three females, ages 14 months to 8 years. All patients had previously undergone cleft palate repair, complicated by subsequent oro-nasal fistula formation. INTERVENTIONS: All patients underwent oro-nasal fistula repair under general anesthesia with a local mucoperiosteal flap lined with buccal mucosal grafts placed on the nasal side of the flap. RESULTS: In all cases, the fistula was completely closed at first attempt without complications. Patients were followed for a minimum of 2 years, without evidence of recurrence. CONCLUSIONS: Our proposed surgical procedure for fistula closure using a standard mucoperiosteal flap lined with a buccal mucosal graft is a suitable alternative for the repair of postpalatoplasty oro-nasal fistulas. Further study and long term follow-up is needed to establish this method as a new standard form of repair. PMID- 10749053 TI - The anchor of the nasal ala in cleft lip-nose patients: a morphological description and a new surgical approach. AB - OBJECTIVE: The purpose of this article is to describe a morphological condition that is readily seen in both primary and secondary deformed noses of cleft patients and to present a new surgical technique to correct this specific deformity that afflicts the alar dome. This anomaly resembles a taut cord-like tissue that runs vertically from the pyriform aperture and is anchored onto the alar cartilage, thus impeding the cartilage's normal growth process. This new surgical technique releases the anchor and corrects the deformity of the nasal rim by using a triangular flap V-Y advancement technique. RESULTS: From 1994 to 1997, 88 cleft lip-nose cases were operated on by the author using the triangular flap V-Y advancement technique for releasing the anchor. Forty-two cases were primary repairs and 46 were secondary repairs. CONCLUSIONS: By studying the normal and pathological anatomy of the nose and its proportions, the surgeon can obtain ideas for new surgical corrective techniques that can be used to restore anatomical balance and harmony between the nose and the face. Only in the last 15 to 20 years has the plastic surgeon begun to be interested in primary rhinoplasty in cleft lip-nose patients. Within this time, many different techniques have been proposed. All primary and secondary cases that have undergone this specific technique for release of the anchor have shown good results for restoring the height of the alar dome's affected side to that of the noninvolved side, thus establishing a more anatomically balanced nose. PMID- 10749054 TI - Three-dimensional analysis of the child cleft face. AB - OBJECTIVE: This study examined the facial surfaces of cleft children and unaffected children aged 8-11 years with the aim of identifying and assessing differences in their facial surface morphology. The investigation was carried out using an Optical Surface Scanner, an instrument that utilizes laser light to construct and archive a three-dimensional image of the face suitable for linear measurement and direct surface comparisons. DESIGN, SETTING, AND PATIENTS: Thirty nine cleft lip and palate (CLP) patients and 25 unaffected subjects were voluntarily recruited from two southeast England hospitals. A range of linear facial measurements was compared. Three-dimensional differences between the cleft subgroups and the control group were visualized by superimposition of averaged cleft scans over the averaged control group images. RESULTS: Statistically significant dimensional differences (p < or = .05) in interocular width, nose base widths, mouth widths, and nose base/mouth width ratios were found between the cleft group and the control group. Qualitative differences over the whole of the face were readily demonstrated between the groups by superimposition. Face width and submandibular area depth differed consistently between the groups, the cleft face appearing narrower with a deeper submandibular area. CONCLUSION: Significant differences exist between the facial surface morphology of CLP patients and control subjects. PMID- 10749055 TI - Three-dimensional Bolton-Brush Growth Study landmark data: ontogeny and sexual dimorphism of the Bolton standards cohort. AB - OBJECTIVE: The treatment of craniofacial reconstructive surgery patients may benefit from comparison to average referent three-dimensional landmark data. These data may be useful for diagnosis, treatment planning, prosthetic design, or outcomes assessment. With regard to subadult patients, we hypothesize that the pattern of ontogenetic shape change of same sex, same ethnicity, referent populations will show gross uniformity. We present a preliminary shape analysis of 50 three-dimensional landmarks derived from 317 Bolton-Brush Growth Study biorthogonal image pairs. We determine which landmarks can be collected from scanned radiographs reliably by four operators for the precisely locatable points, ontogenetic trends in landmark configuration shape change, and patterns of sexual dimorphism. PARTICIPANTS: Participants were Bolton standards individuals (16 male and 16 female) who contributed biplane cephalograms seven or more times with annual or greater spacing between ages 3 and 18 years. DESIGN: After removing outliers, we searched for ontogenetic heterogeneity, including sexual dimorphism and within sex-specific Procrustes coordinate shape spaces. RESULTS: A cut-off of 4.3-mm interoperator error left 32 landmarks in our analysis. Three different approaches (principal component analysis, age-trend analysis, and principal components of age residuals) all found no patterns of individual variation around sex-specific average trends of shape change. Male shape change peaks at age 15, a correlate of the growth spurt. CONCLUSIONS: Simultaneous frontal and lateral anatomic landmark identification improves three dimensional localization reliability. Three-dimensional craniodental shape change from ages 8 to 18 within the Bolton standards presents little heterogeneity. Considerations of ethnicity aside, these may be initial grounds for use of these data as a normative referent. PMID- 10749057 TI - Skull base factors in relation to hearing impairment in cleft palate children. AB - OBJECTIVE: This investigation was performed to determine if an easily measurable, reproducible, bony parameter could be identified that might predict hearing loss in cleft palate children. SUBJECTS: In this prospective study performed at the Sheffield Children's Hospital (U.K.), 34 children with successfully repaired cleft palate who responded to a postal request for volunteers were assessed clinically, audiologically, and by lateral soft tissue neck radiography. Six children were excluded because of previous otologic surgery or poor quality radiographs. Twenty-six children who had the same series of investigations were randomly selected from routine otolaryngological outpatient clinics and acted as controls. METHODS: The sphenopalatine angle (SPA), which relates the facial and cranial components of the skull, was measured on each child's radiograph. RESULTS: The median SPA in the cleft palate group was significantly greater than in the control group (p = 0.01). In those cleft palate children with a hearing loss the sphenopalatine angle was smaller than in their normal hearing counterparts (p = 0.01). No significant difference was found in the SPA between the hearing loss and normal hearing controls. There was no significant difference in age ranges between the hearing and hearing loss subgroups in each of the two primary groups. CONCLUSIONS: In this study, those cleft palate children with a smaller SPA have a greater incidence of hearing loss. PMID- 10749056 TI - Quantitative analysis of the velopharyngeal sphincter function during speech. AB - OBJECTIVE: This study reports a new concept for quantitative analysis of velopharyngeal function using electromagnetic articulography and simultaneous transoral video endoscopy. PARTICIPANTS: The velopharyngeal closure mechanism was studied in 16 healthy German subjects during production of the vowel-consonant vowel sequences /afa/ and /apa/. DESIGN: Velar movements in the sagittal and vertical direction were measured by electromagnetic articulography (EMA) while video endoscopy was used for recording in the sagittal and transverse directions. MAIN OUTCOME MEASURE: A transverse sagittal quotient (TSQ) was defined after the measurement of sagittal and transverse diameters of the velopharyngeal port (VPP) in the video-endoscopic pictures on the basis of a frame-by-frame analysis. RESULTS: The sphincter morphology was objectively determined with the TSQ base value. A terminal sphincter deformation (TSD) for closure phases of articulatory movement was revealed by a qualitative shape analysis of the TSQ curves. CONCLUSIONS: The assessment of the TSQ of velopharyngeal port diameters and the TSD in the terminal closure period lead to a new dynamic interpretation of velopharyngeal closure patterns. PMID- 10749058 TI - A comparative study of prespeech vocalizations in two groups of toddlers with cleft palate and a noncleft group. AB - OBJECTIVE: This study examined the prelinguistic contoid (consonant-like) inventories of 14 children with unilateral cleft lip and palate (C-UCLP) at 13 months of age. The children had received primary veloplasty at 7 months of age and closure of the hard palate was performed at 3-5 years. The results of this investigation were compared to results previously reported for 19 children with cleft palate and 19 noncleft children at the age of 13 months. The children with clefts in that study received a two-stage palatal surgery. This surgical procedure was formerly used at our center and included closure of the lip and hard palate at 3 months of age and soft palate closure at 22 months of age. DESIGN: Retrospective study. SETTING: The participants were videorecorded in their homes during play with their mothers. The videotapes were transcribed independently by three trained speech pathologists. PATIENTS: Fourteen consecutive patients born with C-UCLP and no known mental retardation or associated syndromes served as subjects. RESULTS: The children who received delayed closure of the hard palate demonstrated a significantly richer variety of contoids in their prespeech vocalizations than the cleft children in the comparison group. Both groups of subjects with clefts had significantly fewer plosives in their contoid inventory than the noncleft group, and there was no difference regarding place of articulation between the group that received delayed closure of the hard palate and the noncleft group. PMID- 10749059 TI - The presurgical status of the alveolar cleft and success of secondary bone grafting. AB - OBJECTIVE: The primary purpose of this study was to evaluate presurgical status of the alveolar cleft site and success of secondary alveolar bone grafting. DESIGN: Thirty patient records were retrospectively reviewed. Patients selected for inclusion had isolated cleft of at least the primary palate. Patients with additional anomalies were not selected. The study population consisted of 15 female sites and 16 male cleft sites. There were two bilateral cleft lip and palate (CLP) patients and 28 unilateral CLP patients. The age at the time of the secondary alveolar bone grafting ranged from 7 years to 14 years, 4 months. SETTING: The study was conducted at the Cleft Palate Clinic at the University of Minnesota, School of Dentistry. METHOD: Presurgical radiographs taken at least 1 month prior to the secondary bone grafting and postsurgical radiographs taken at least 6 months after bone surgery were measured. Measurements included size of the cleft defect and bone support for distal and mesial teeth adjacent to the cleft. Evaluation of success was determined on the basis of postsurgical measurements of satisfactory, intermediate, and unsatisfactory outcomes. RESULTS AND CONCLUSION: The size of the cleft defect was not correlated with the success rate of the secondary alveolar bone grafting. If the amount of distal bone support for the mesial tooth was the same as those in a periodontally healthy individual, a satisfactory outcome was 5.8 times more likely. If the amount of mesial bone support for the distal tooth was the same as those in a periodontally healthy individual, the satisfactory outcome was 3.8 times more likely. Although not a primary purpose of the study, it was found that in this study population, if the patient was female, a satisfactory outcome was 3.8 times more likely. PMID- 10749060 TI - Comparison between palatal configurations in UCLP infants with and without a Hotz plate until four years of age. AB - OBJECTIVE: To evaluate the efficacy of presurgical orthopedic treatment three dimensionally, we previously developed a novel measuring system and described the efficacy of the treatment until 18 months of age. The aim of this study was to compare palatal configuration in complete unilateral cleft lip and palate (UCLP) infants fitted with a Hotz plate to those without it until four years of age using the same measuring system. PATIENTS: Twenty complete UCLP infants were divided into two groups. A Hotz plate was used with 12 patients who first visited our clinic from January 1990 to March 1992 and was not used with an additional eight patients who first visited from April 1992 to July 1993. MAIN OUTCOME MEASURES: Palatal casts taken from birth until four years of age were measured three dimensionally and were compared between the two groups. RESULTS AND CONCLUSIONS: The width of the palate was larger in the group with a Hotz plate than in the group without it at four years of age. No difference was observed between the anteroposterior distance of the palate in the two groups at four years of age. Similarly, the alveolar arch form did not differ between the two groups. The degree of curvature of the palatal surface was smaller in the group with a Hotz plate than in the group without it. These data were similar to data obtained at 18 months of age. PMID- 10749061 TI - An epidemiologic study of oral clefts in Iran: analysis of 1,669 cases. AB - OBJECTIVE: The objective of this study was to assess the epidemiology and some genetic aspects of oral clefting in Iran. DESIGN: The study was a 15-year cross sectional (prevalence) study from August 1976 to September 1991. SETTING: The setting for the study was two plastic surgery departments, both mostly referral centers, and a maternity hospital in Iran. PARTICIPANTS: The participants were 1,669 consecutive surgical cases with oral clefts (79% between 1 day and 18 months of age), registered in two centers. In a parallel study, 19,369 livebirths born in a maternity hospital within the same period were surveyed to ascertain prevalence of clefting at birth. Normal children of this population were used as the control group. Samples were analyzed by prevalence of clefting at birth, type of oral cleft, side of clefting, sex ratio, associated malformations, clefts in relatives, and parental consanguinity. RESULTS: The prevalence of clefts was 1.03 per 1,000 births. Cleft lip (without cleft palate) had a higher (34.9%) and cleft palate alone had a significantly lower prevalence (17.4%) than expected. Cleft lip (without cleft palate) was more common in females (53.6%) than in males (46.4%). The rate of associated malformations in cleft patients (7.73%) was higher than in controls (0.093%). Association between clefting and consanguinity was significant (45.8% in cases versus 43.1% in controls). Occurrence of noncleft congenital malformations among first-degree relatives of our cases (2.77%) was nearly two times that of controls (1.55%). CONCLUSION: Our study reveals that the population incidence of oral clefts in Iran is much closer to European than Arab African or South East Asian countries. However, significant variations in other epidemiologic and some genetic features were observed. PMID- 10749062 TI - Role of the superior pharyngeal constrictor muscle in forced breathing in dogs. AB - OBJECTIVE: Respiratory-related electromyographic (EMG) activity of the superior pharyngeal constrictor (SPC) muscle was analyzed during the early stage of forced breathing. DESIGN: Four adult dogs anesthetized with sodium pentobarbital were used. In the first part of the study, oral and nasal breathing tubes were placed into the respective cavities, and a tracheotomy tube was placed in the second part of the study. Two conditions, the presence (oral-nasal tube breathing) and absence (tracheotomy breathing) of airflow in the upper airway, were achieved in each dog. Following quiet breathing, animals were connected to a closed breathing system, first by an oral-nasal tube and then by a tracheotomy tube. We proposed to induce a forced breathing condition mechanically by using this system for 1 minute. We increased resistance to airflow during forced breathing by means of connecting tubes and a bag. Our aim was not to produce chemical drive but to produce a forced respiration by increasing the resistance to airflow. Tidal volume, breathing frequency, minute volume, chest wall movement, and EMG activity of the SPC muscle were measured and analyzed. RESULTS: During quiet breathing through an oral-nasal or tracheotomy tube, low-amplitude EMG activity of the SPC muscle corresponding to the expiratory cycle of the respiration was observed. In both study conditions, phasic expiratory EMG activity increased immediately after the advent of the breathing from the closed system. Tidal volumes and frequencies also increased rapidly during forced breathing. CONCLUSIONS: An increase in the resistance to airflow increased the activity of the SPC muscle. This augmented respiratory activity probably assists the patency of the upper airway. The augmented respiratory activity was independent of the local reflex pathways. Respiratory-related activity of the SPC muscle may help dilate and stiffen the pharyngeal airway, promoting airway patency. PMID- 10749063 TI - Gastroesophageal reflux in Pierre Robin sequence--early surgical treatment. AB - OBJECTIVE: To assess the prevalence of gastroesophageal reflux (GER) in patients with Pierre Robin sequence and to assess whether early palatoplasty with subperiosteal release of the floor of the mouth eliminates upper respiratory tract infections and gastroesophageal reflux. METHODS: In the period from 1993 to 1997, 24 children with Pierre Robin sequence were treated. Because GER is known to occur in patients with this syndrome, we screened for GER in each child for whom an early palatoplasty was to be performed at 3 months of age. In 21 cases, the presence of GER was confirmed. Within the past 2 years, palatoplasty has been combined with subperiosteal release of the floor of the mouth (SRFM). Prompt clinical improvement and shortened hospital stays have resulted. RESULTS AND CONCLUSIONS: Follow-up studies performed several months after the surgery showed no evidence of GER. The authors conclude that an early closure of the impaired palatopharyngeal ring and the elimination of the mechanism that causes the posterior rotation of the tongue can reduce the need for such surgical procedures as glossopexy, gastrostomy, tracheostomy, and Nissen's fundoplication. PMID- 10749064 TI - Intrasphenoidal migration of a premaxillary Kirschner wire. AB - OBJECTIVE: The use of Kirschner wire for the fixation of premaxilla is a well known method in bilateral cleft lip surgery. We report a case in which the Kirschner wire of the premaxillary fixation had migrated intrasphenoidally. RESULTS AND CONCLUSIONS: The foreign body was accidentally discovered during a cephalometric analysis and was taken out surgically through an upper lip sulcus incision. Although the wire remained asymptomatic for 10 years, it constituted a potential danger for intracranial migration. PMID- 10749065 TI - Time-frequency analyses of TEOAE recordings from normals and SNHL patients. AB - This study evaluated the characteristics of transient evoked otoacoustic emission (TEOAE) time-frequency (TF) representations from normal and hearing-impaired ears. Linear and non-linear TEOAE recordings from normally-hearing subjects (40) and non-linear recordings from patients with sensorineural hearing loss (SNHL) (40) were analysed using the short-time-Fourier-transform spectrogram, the Gabor spectrogram, and the adaptive spectrogram. The TF representations of the TEOAE recordings indicated a considerable dispersion of energy across frequencies and TEOAE time segments >4.0 ms. The linear and non-linear recordings from the normal subjects showed common frequency peaks. The TF representations from the patients with SNHL indicated that the significantly reduced energy in the mid-to-high TEOAE frequencies did not correlate closely with the threshold elevation. As in the recordings from the normal subjects, a high percentage of the TEOAE cumulative energy was found within a short TEOAE segment (4-14 ms). PMID- 10749066 TI - Post-operative stapedius reflex tests with simultaneous loudness scaling in patients supplied with cochlear implants. AB - The estimation of the maximum comfort loudness levels (MCL) by measurements of the electrically elicited stapedius reflex was examined in six experienced cochlear implant users supplied with the COMBI 40 implant system. The stapedius reflex was tested and loudness scaling was performed simultaneously using an up/down stimulation protocol close to the reflex threshold with automated recording of both test procedures. The electrical stapedius reflex threshold (ESRT) and loudness scaling were evaluated separately. Scaling at the reflex threshold ranged between normal and loud. The range of stimulus intensities corresponding to ESRT is much smaller than that at a particular loudness category. The overall correlation between ESRT and MCL was high (r=0.92), with a similar dependence of ESRT and MCL on the channel stimulated. Thus, when the stapedius reflex can be detected post-operatively, the ESRT can be applied successfully for the fitting procedure of the speech processor. Simultaneous loudness scaling during the entire reflex test showed that overstimulation via the implant can be avoided effectively. PMID- 10749067 TI - Electrocochleographic study in patients with vestibular schwannomas and U-shaped audiograms. AB - To determine the nature of sensorineural hearing loss in the middle-frequency range (U-shaped audiogram), we compared the differences in electrocochleographic findings for 15 ears with vestibular schwannomas and 10 ears without tumours. Short-tone bursts of 0.5, 1, 2, and 4 kHz were used to evoke cochlear microphonics (CM). Ears with tumours had normal or lower CM detection thresholds than ears without tumours. Input-output curves for 1-kHz frequency were normal in 10 ears with tumours and in 1 ear without tumours. These indicate that tumour ears have no or mild cochlear dysfunction. In addition, CM detection thresholds of ears with tumours were lower than audiometric thresholds, particularly at the 1- and 2-kHz region. These findings suggest that the loss seen by audiometry in ears with vestibular schwannomas was from a retrocochlear component. PMID- 10749068 TI - Cisplatin-induced increases in spontaneous neural activity in the dorsal cochlear nucleus and associated outer hair cell loss. AB - Tinnitus is one of the consequences of cisplatin chemotherapy, but its underlying mechanisms are not well understood. Since it has been shown that cisplatin causes outer hair cell loss, it is possible that loss of these cells might induce tinnitus by increasing spontaneous activity in the central auditory system. To test this possibility, the present study examined the effects of cisplatin treatment on cochlear hair cells and on spontaneous neural activity in the dorsal cochlear nucleus of hamsters. Recordings, carried out approximately 1 month after cisplatin treatment, demonstrated significant increases in spontaneous activity across broad regions of the dorsal cochlear nucleus relative to levels in saline treated controls. Histological results showed that cisplatin-treated animals also displayed dramatic loss of outer hair cells over most of the basal turn of the cochlea. Inner hair cells remained intact, although some evidence of damage to their stereocilia was evident. These findings indicate that cisplatin treatment causes abnormalities in spontaneous activity in the dorsal cochlear nucleus that are associated with widespread damage to outer hair cells. However, since some damage to inner hair cells was also observed, the role of inner hair cell injury in contributing to higher spontaneous activity cannot be ruled out. PMID- 10749069 TI - Cortical centres underlying auditory temporal processing in humans: a PET study. AB - We have used positron emission tomography (PET) to test a specific hypothesis of a neural system subserving auditory temporal processing (acoustical stimulus duration discrimination). Maps of the cerebral blood flow distribution during specific stimulations were obtained from five normally-hearing and otherwise healthy subjects. The auditory stimuli consisted of sounds of varying duration and of auditorily presented words in which the duration of the initial phoneme was manipulated. All stimuli alternated with conditions of silence in a subtraction paradigm. The blood flow distribution was mapped with O-15-labelled water. The results demonstrated that stimuli requiring recognizing, memorizing, or attending to specific target sounds during temporal processing generally resulted in significant activation of both frontal lobes and the parietal lobe in the right hemisphere. Based on these results, we hypothesise that a network consisting of anterior and posterior auditory attention and short-term memory sites subserves acoustical stimulus duration perception and analysis (auditory temporal processing). PMID- 10749070 TI - Measuring the understanding of sentences by hearing-impaired children: comparison with connected discourse ratings. AB - Measuring the understanding of connected speech by hearing-impaired children is desirable if an appropriate rehabilitative or special educational program is to be implemented or monitored. This study was undertaken to explore the potential of a Cantonese version of the Helen sentence test as a measure of understanding connected speech by a group of 54 mildly to profoundly hearing-impaired children, and to compare the results from this sentence test with those achieved by the same children on a connected discourse test. The effects of two different hearing aid frequency responses and two noise conditions on the results from both tests were also examined. Findings from the present study revealed that the Helen sentence test could be successfully adapted for use with Cantonese-speaking hearing-impaired children in Hong Kong. When compared to the connected discourse test, the modified Helen test produced significantly higher scores and was more sensitive to changes in noise level, especially for the severely and profoundly hearing-impaired children. PMID- 10749071 TI - Inherited sensorineural low-frequency hearing impairment: some aspects of phenotype and epidemiology. AB - This contribution forms part of the HEAR project. It describes some phenotypes of inherited low-frequency sensorineural hearing impairment (LFSHI) and estimates the prevalence of this inherited hearing impairment (HI) based on a clinical series. During a 10-year period (1987-1996), 418 subjects (134 males and 284 females), with a median age of 68 years (range 4-98), had been examined with LFSHI, defined as hearing loss most pronounced in the low frequencies (i.e., 250 and 500 Hz > 20 dB HL with better hearing, i.e., > or =15-dB difference at 1 and/or 2 and/or 4 kHz with an air-bone gap <15 dB for the average of 0.5, 1, and 2 kHz). The 418 subjects comprising 0.6 per cent of the total number of subjects examined (N=69,309) were subdivided into four categories: category I positive genetic subjects (N=69); category II, probably genetic (N=339); category III, uncertain genetic (N=6); and category IV, subjects with contradictory audiological findings (N=4). The phenotype in category I demonstrated a symmetrical LFSHI, with a pattern of progression showing a slow deterioration in the high frequencies (i.e., 2, 4, and 8 kHz as a function of age)--the progression comprising 40-45 dB. In the low frequencies (i.e., 250, 500, and 1,000 Hz), a deterioration of 15-25 dB could be demonstrated from the youngest to the oldest age group. In category II, a symmetrical LFSHI was found in 179 subjects, showing the same pattern of progression as in category I. However, in the age group 20-39 years, a significantly poorer hearing was found in the low frequencies compared to category I, implying that several phenotypes may be present in LFSHI. A subgroup (A) in category II exhibited normal hearing in one ear with LFSHI in the opposite ear with the same pattern of progression as in category I. Three other subgroups with LFSHI and flat/sloping audiogram in the opposite ear and asymmetrical LFSHI also showed the same type of progression in the ear with LFSHI as in category I. A prevalence of 0.18/1,000 (95 per cent CI 0.13-0.22) of LFSHI was estimated based on the background population with a fairly constant prevalence throughout life. It is concluded that inherited nonsyndromal LFSHI is a rare disease and that the many different phenotypes of LFSHI probably are associated with pronounced genetic heterogeneity. PMID- 10749072 TI - Variations in patterns of attraction to same- and other-sex peers during early adolescence. AB - Differences in attraction to same- and other-sex peers as a function of sex, age, individual characteristics (i.e., aggression), and context were examined in a longitudinal study of early adolescent boys and girls (N = 217) that covered the transition from elementary school (Time 1) to middle school (Times 2 and 3). Consistent with T. Moffitt's (1993) concept of the "maturity gap," attraction to aggressive peers, especially attraction to aggressive boys among girls, increased with age and upon entry to middle school, as did attraction to peers who stood out in the peer group in easily observable ways. Attraction to peers who presented features associated with good classroom-based behavior decreased. These patterns are discussed in terms of the developmental needs served by associating with particular peers. PMID- 10749073 TI - Infants' association of linguistic labels with causal actions. AB - Six experiments examined infants' ability to associate nonsense words with 2 causal actions: pushing and pulling. Although Experiment 1 found that 14-month olds failed to form word-action associations, 18-month-olds in Experiment 2 provided reliable evidence of doing so. Additional experiments explored why 14 month-olds may not have formed such an association. Experiment 3 examined 14 month-olds' ability to discriminate a change in either the action or the label when the other element was held constant. Infants discriminated the change in label but not the change in action. When the language labels were replaced with music (Experiments 4-6), 14-month-old infants responded in terms of and discriminated between pushing and pulling. These results, in comparison with those from Experiments 1 and 3, suggest that for 14 month-olds, attempting to associate labels with actions may interfere with their discrimination of similar actions. PMID- 10749074 TI - Understanding inference as a source of knowledge: children's ability to evaluate the certainty of deduction, perception, and guessing. AB - Three experiments investigated children's understanding of inference as a source of knowledge. Children observed a puppet make a statement about the color of one of two hidden toys after the puppet (a) looked directly at the toy (looking), (b) looked at the other toy (inference), or (c) looked at neither toy (guessing). Most 4-, 5-, and 6-year-olds did not rate the puppet as being more certain of the toy's color after the puppet looked directly at it or inferred its color than they did after the puppet guessed its color. Most 8 and 9-year-olds distinguished inference and looking from guessing. The tendency to explain the puppet's knowledge by referring to inference increased with age. Children who referred to inference in their explanations were more likely to judge deductive inference as more certain than guessing. PMID- 10749075 TI - Pubertal timing and substance use: associations between and within families across late adolescence. AB - In the present study, between-family analyses of data from adolescent twin girls offer new evidence that early menarche is associated with earlier initiation and greater frequency of smoking and drinking. The role of personality factors and peer relationships in that association was investigated, and little support was found for their involvement. Novel within-family analyses replicating associations of substance use with pubertal timing in contrasts of twin sisters selected for extreme discordance for age at menarche are reported. Within-family replications demonstrated that the association of pubertal timing with substance use cannot be explained solely by between-family confounds. Within-family analyses demonstrated contextual modulation of the influence of pubertal timing: Its impact on drinking frequency is apparent only among girls in urban settings. Sibling comparisons illustrate a promising analytic tool for studying diverse developmental outcomes. PMID- 10749076 TI - Intersensory redundancy guides attentional selectivity and perceptual learning in infancy. AB - This study assessed an intersensory redundancy hypothesis, which holds that in early infancy information presented redundantly and in temporal synchrony across two sense modalities selectively recruits attention and facilitates perceptual differentiation more effectively than does the same information presented unimodally. Five-month-old infants' sensitivity to the amodal property of rhythm was examined in 3 experiments. Results revealed that habituation to a bimodal (auditory and visual) rhythm resulted in discrimination of a novel rhythm, whereas habituation to the same rhythm presented unimodally (auditory or visual) resulted in no evidence of discrimination. Also, temporal synchrony between the bimodal auditory and visual information was necessary for rhythm discrimination. These findings support an intersensory redundancy hypothesis and provide further evidence for the importance of redundancy for guiding and constraining early perceptual learning. PMID- 10749077 TI - In Cinderella's slippers? Story comprehension from the protagonist's point of view. AB - Research on text comprehension shows that readers construct a model of the situation described in a narrative. A major factor in constructing a situational model is the perspective from which the action of the narrative is imagined. J. B. Black, T. J. Turner, and G. H. Bower (1979) found that adults recall a deictic verb of motion more accurately if it is spatially consistent with the point of view of the main protagonist. Recall is more accurate for the verbs come and bring if they describe a movement toward the protagonist; recall is more accurate for go and take if they describe a movement away from the protagonist. Thus, adults interpret movements in a narrative from the perspective of the protagonist. This study indicates that 3- and 4-year-old children show the same pattern of recall. They accurately recall verbs of motion that are consistent with the protagonist's perspective but make substitution errors on verbs inconsistent with that perspective. PMID- 10749078 TI - Do birds of a feather flock together? The variable bases for African American, Asian American, and European American adolescents' selection of similar friends. AB - Variability in adolescent-friend similarity is documented in a diverse sample of African American, Asian American, and European American adolescents. Similarity was greatest for substance use, modest for academic orientations, and low for ethnic identity. Compared with Asian American and European American adolescents, African American adolescents chose friends who were less similar with respect to academic orientation or substance use but more similar with respect to ethnic identity. For all three ethnic groups, personal endorsement of the dimension in question and selection of cross-ethnic-group friends heightened similarity. Similarity was a relative rather than an absolute selection criterion: Adolescents did not choose friends with identical orientations. These findings call for a comprehensive theory of friendship selection sensitive to diversity in adolescents' experiences. Implications for peer influence and self-development are discussed. PMID- 10749079 TI - Effortful control in early childhood: continuity and change, antecedents, and implications for social development. AB - The course, antecedents, and implications for social development of effortful control were examined in this comprehensive longitudinal study. Behavioral multitask batteries and parental ratings assessed effortful control at 22 and 33 months (N = 106). Effortful control functions encompassed delaying, slowing down motor activity, suppressing/initiating activity to signal, effortful attention, and lowering voice. Between 22 and 33 months, effortful control improved considerably, its coherence increased, it was stable, and it was higher for girls. Behavioral and parent-rated measures converged. Children's focused attention at 9 months, mothers' responsiveness at 22 months, and mothers' self reported socialization level all predicted children's greater effortful control. Effortful control had implications for concurrent social development. Greater effortful control at 22 months was linked to more regulated anger, and at 33 months, to more regulated anger and joy and to stronger restraint. PMID- 10749080 TI - The role of proactive and reactive aggression in the formation and development of boys' friendships. AB - This study tested the hypothesis that friends are more similar in proactive aggression than in reactive aggression. Interpersonal processes that may account for this similarity (i.e., selection and mutual influence) were also examined. In the fall and spring of the school year, the friendships of 185 4th-, 5th-, and 6th-grade boys were identified. Proactive and reactive aggressive behavior were assessed with a teacher-rating instrument for each boy. The results support the general hypothesis and suggest that proactively aggressive boys tend to select proactively aggressive peers as friends; however, mutual influence between stable friends does not appear to account for similarity. These findings are discussed within the framework of G. R. Patterson, J. B. Reid, and T. J. Dishion's (1992) theory of antisocial behavior. PMID- 10749081 TI - Sexual identity, attractions, and behavior among young sexual-minority women over a 2-year period. AB - Previous research suggests that the sexual identities, attractions, and behaviors of sexual-minority (i.e., nonheterosexual) women change over time, yet there have been few longitudinal studies addressing this question, and no longitudinal studies of sexual-minority youths. The results of 2-year follow-up interviews with 80 lesbian, bisexual, and "unlabeled" women who were first interviewed at 16 23 years of age are reported. Half of the participants changed sexual-minority identities more than once, and one third changed identities since the first interview. Changes in sexual attractions were generally small but were larger among bisexuals and unlabeled women. Most women pursued sexual behavior consistent with their attractions, but one fourth of lesbians had sexual contact with men between the two interviews. These findings suggest that there is more fluidity in women's sexual identities and behaviors than in their attractions. This fluidity may stem from the prevalence of nonexclusive attractions among sexual-minority women. PMID- 10749082 TI - Sequelae of cesarean and vaginal deliveries: psychosocial outcomes for mothers and infants. AB - Mother-infant dyads grouped according to whether the infants had been delivered vaginally (n = 74) or by planned (n = 37) or unplanned cesarean (n = 56) were compared on psychosocial outcomes at 4 and 12 months postpartum. Hypotheses were that unplanned cesarean delivery would be related to less optimal outcomes and that this relationship would be mediated by mother's appraisal of the delivery and would attenuate over time. No delivery-related differences in mother-infant interactions were found at 4 or 12 months postpartum with one exception: Women low in neuroticism who delivered by unplanned cesarean showed less positive affect toward their infants at 4 months than did women high in neuroticism who delivered by unplanned cesarean or women in any other group. There was some evidence of the mediating role of maternal appraisal of the delivery on these effects. In general, the results indicate little cause for concern about the quality of mother-infant interactions following cesarean deliveries. PMID- 10749083 TI - Attention and joint attention in preschool children with autism. AB - Three experiments examined the role of attention in explaining dyadic (child adult) and triadic (child-adult-object) joint attention difficulties in autism. Experiments 1 and 2 investigated children's ability to orient to an adult's attention bid and to follow the direction of a human or nonhuman cue. Experiment 3 tested ability to disengage and shift attention to objects. Results showed autism-specific difficulties at both dyadic and triadic levels. Children with autism were less responsive than developmentally delayed controls in orienting to attention bids and in following a human head-turn cue yet had no difficulty in shifting attention and were faster overall in orienting to targets. Results suggest a specific developmental delay in which children with autism rely on the presence of objects in the visual field to guide action. The relation between this problem and autistic children's difficulties with human communicative signals is discussed. PMID- 10749084 TI - Q-sort assessment of attachment security during the preschool years: links from home to school. AB - This study investigated relations among preschoolers' secure-base behavior with mother at home, stressful family conditions, and qualities of developing relationships with peers and teachers in preschool. Preschool-age children (N = 94; 51 boys and 43 girls) and their mothers were observed during multiple home visits, and their security of attachment was assessed with the Attachment Q-Set. Children in families with lower levels of stress had more secure attachment relationships with mothers. Both boys and girls who were less securely attached to their mothers expressed significantly more anger-aggression in preschool. For boys, higher levels of family stress were also significantly related to anger aggression and to social competence in preschool. In addition, boys who were more securely attached to their mothers were more securely attached to their preschool teachers and were more popular with peers in preschool. PMID- 10749085 TI - Conceivable mechanisms by which Helicobacter pylori provokes duodenal ulcer disease. AB - A conceivable concept for the development of duodenal ulcers in Helicobacter pylori (H. pylori) infected subjects is presented in this chapter. The concept includes an explanation of the fact that only a minority of all H. pylori infected subjects will develop a duodenal ulcer. Helicobacter pylori infection of the antrum induces a hypersecretion of gastric acid secretion, giving rise to gastric metaplasia in the duodenal bulb. This gastric metaplasia is a prerequisite for H. pylori colonization of the bulb. These events are common to all H. pylori-infected subjects. However, a much higher density of H. pylori bacteria and colonization with virulent organisms has been found in the bulb of duodenal ulcer patients, resulting in a much stronger inflammatory reaction with active duodenitis and an impaired bicarbonate secretion. These characteristics, together with acid hypersecretion, seem to be the important factors in evoking a duodenal ulcer. PMID- 10749086 TI - The role of gastrin in ulcer pathogenesis. AB - Duodenal ulcer patients are characterized by an antrum-predominant, body-sparing, nonatrophic Helicobacter pylori (H. pylori) gastritis, which results in increased gastrin release and increased acid secretion. The increased gastrin release is caused by the infection impairing the acid-mediated inhibitory control of gastrin release. The elevated levels of the gastrin stimulate the healthy uninflamed, non atrophic acid-secreting region of the stomach to secrete excess amounts of acid. The increased gastrin also exerts trophic effects on the oxyntic mucosa, causing hyperplasia of both the enterochromaffin-like cells and the parietal cells. These trophic changes in the mucosa further enhance its ability to secrete acid. The increased acid secretion results in an increased duodenal acid load, causing gastric metaplasia of the duodenal bulb and eventually the development of ulceration. In H. pylori-infected subjects without duodenal ulceration, a different pattern of gastritis is seen. This includes atrophy of the antrum, which reduces the number of G-cells and thus the degree of hypergastrinaemia induced by the antral infection. There are usually also varying degrees of inflammation and atrophy of the acid-secreting mucosa, which impair its ability to secrete acid in response to gastrin stimulation. The combined effects of the atrophy of the antrum and the inflammation of the antrum of the body mucosa therefore prevent H. pylori-induced acid hypersecretion and may result in varying degrees of hypochlorhydria. The particular pattern of gastritis that a subject develops in response to H. pylori infection and their likelihood of developing a duodenal ulcer is likely to be determinded by host genetic factors plus dietary factors. PMID- 10749087 TI - Patterns of inflammation linked to ulcer disease. AB - Peptic ulcers are accompanied by different patterns of chronic gastritis and duodenitis that generally run parallel to the topography of colonization by Helicobacter pylori (H. pylori). Duodenal ulcers arise on a background of a gastroduodenitis; the gastritis is antrum-predominant while the duodenitis requires acid-induced gastric metaplasia in the duodenal mucosa before bacterial colonization can occur. The colonized and inflamed metaplastic areas in the duodenum (and inflamed pre-pyloric antrum) are the initial sites of ulceration. Proximal gastric ulcers arise in a diffuse (pan-) gastritis or a corpus predominant H. pylori gastritis when the weakened gastric mucosa (especially in the antrum-body transitional zone) is susceptible to ulceration even in the presence of subnormal acid production. These distinctive patterns of gastritis are sufficiently consistent for them to be used to predict ulcer risk. PMID- 10749088 TI - Differences in peptic ulcer between East and West. AB - In general, peptic ulcer occurs at equal rates in the East and the West but with marked regional differences in both, even within the same country. In the West, the incidence of peptic ulcer, particularly duodenal ulcer, rose sharply at the turn of the century and has shown a rapid decline in the past three decades. In the East, the rise was equally impressive, but the decline appears to have been delayed, only starting in the past decade. Asians present their ulcer symptoms a decade earlier than Caucasians, and it has been suggested that this early presentation may be attributable to Helicobacter pylori (H. pylori) infection at a younger age. Interestingly, the male-to-female ratio is much higher in the East than in the West, and the duodenal-to-gastric ulcer ratio manifests a much wider variation in Asians than in Caucasians. As in Western countries, peptic ulcer occurrence in the East shows a cyclical trend, with a peak frequency in the winter months. In the West, the placebo healing rate varies widely up to 78%, whereas in the East it is rather consistent at around one-third. These variations in geographical distribution, time trends, sex and ulcer ratios, seasonal rates and behavioral response to placebo treatment indicate that while H. pylori is a major cause of peptic ulceration, other environmental and genetic factors contribute to ulcer formation. The parietal cell mass and acid secretory capacity of Asian patients with duodenal ulcer are only slightly more than half of those of Caucasian patients, which may explain why Asian patients respond equally well to half the standard dose of anti-secretory agents used in Caucasians. H. pylori infection is generally more prevalent in the East than in the West and is more resistant to metronidazole. The response to standard triple therapies for eradication, however, appears to be as effective in the East as in the West. PMID- 10749089 TI - Peptic ulcer disease in children. AB - A peptic ulcer in a child looks the same as it does in an adult, and many of the aetiologies of peptic ulcer disease in children are similar to those in adults. However, there are many differences between children and adults, especially in the areas of clinical presentation, the prevalences of different types of ulcer disease, and the prevalence of complications of ulcer disease. Therefore the approach to diagnosis and management in children is often at variance with that in adults. One important example is the approach to suspected Helicobacter pylori (H. pylori) disease in children, in which consensus groups have advised a considerably different approach in children. While the chapter deals with the full range of peptic ulcer disease in children, the focus is on those aspects in which there are differences between adults and children. PMID- 10749090 TI - Animal models of gastroduodenal ulcer disease. AB - Animal models have played a significant role in research that aims to understand peptic ulceration. Firstly, they have helped define basic mechanisms of gastric mucosal defence and repair. The basis for gastric injury following NSAID administration was facilitated by animal models that correlated well with disease in humans. In early studies, ulceration was induced by grossly damaging insults to the gastric mucosa that were unphysiological. With refinement these models provided a clearer appreciation of stress ulceration. The discovery of Helicobacter pylori (H. pylori), as the cause of most ulcers, resulted in a need to re-evaluate the early literature and to look for new models. To date, these have contributed little to our understanding of the pathogenesis of H. pylori induced ulcer. A major aim of this chapter is to suggest that thorough understanding of the animal models of Helicobacter infection may provide important new insights, in particular the factors controlling gastritis, the essential precursor lesion of ulceration. Available models include primates, cats, guinea pigs, ferrets and pigs. The mouse models provide opportunity for identifying both essential bacterial and host factors. The most severe pathologies are seen in the H. pylori-infected Mongolian gerbil with ulcers being formed in most animals. This is likely to become the standard animal model for investigation of peptic ulcer disease. PMID- 10749091 TI - Pathogenesis of duodenal ulcer disease: the rest of the story. AB - Although several duodenal ulcer disease-specific abnormalities in gastric function have been described (e.g. exaggerated gastrin releasing peptide stimulated acid secretion and an abnormal sensitivity of the parietal cells to gastrin), none has withstood careful examination. We describe here the critical nature of the duodenal acid load in precipitating and washing out bile salts, which inhibit the growth of Helicobacter pylori (H. pylori) in the development of duodenal ulcer disease. The risk of duodenal ulcer is enhanced by infection with proinflammatory H. pylori (e.g. with an intact cag pathogenicity island). Progressive damage to the duodenum promotes gastric metaplasia, resulting in sites for H. pylori growth and more inflammation. This cycle results in an increasing inability of the duodenal bulb to neutralize acid entering from the stomach until changes in duodenal bulb structure and function are sufficient for an ulcer to develop. Cure of the H. pylori infection results in a sustained fall in duodenal acid load as well as a marked (and continuing) reduction in inflammation, which results in the cure of chronic ulcer disease. PMID- 10749092 TI - The problem of Helicobacter pylori-negative idiopathic ulcer disease. AB - Ulcer disease is an infectious disease, but for how much longer? Reports of a large number of non-infectious ulcers are becoming more frequent, paralleling the changing prevalence of Helicobacter pylori (H. pylori) in many parts of the world. This chapter will address factors involved in the increasing proportion of H. pylori-negative ulcers, the probable cause of such ulcers and the clinical implications related to their management. This discussion is currently most relevant to those regions of the world where the prevalence of H. pylori is already low or rapidly decreasing. However, it is possible that, even in other areas of the world, the prevalence of infection will also eventually change and H. pylori-negative ulcer disease will become more important. PMID- 10749093 TI - Cure of Helicobacter pylori-associated ulcer disease through eradication. AB - The eradication of Helicobacter pylori (H. pylori) infection has led to a dramatic benefit for patients with gastroduodenal ulcer disease, as the majority of these patients receive a lifelong cure. Relapses after successful H. pylori cure may be caused by either recrudescence or reinfection, both rare events nowadays, or be attributed to non-steroidal anti-inflammatory drugs or aspirin intake. In certain geographical areas, H. pylori-negative relapses are proposed as a new, pathophysiological and not yet elucidated entity. The cure of H. pylori infection in uncomplicated duodenal ulcer diseases consists of 7 days of proton pump inhibitor (PPI) based triple therapy, containing two antibiotics from clarithromycin, amoxicillin and metronidazole. In gastric ulcer, it is recommended that the PPI is continued for a further 3 weeks as these ulcers have a prolonged healing time. Rescue therapies after failure need to take into consideration the resistance pattern of the micro-organism and are offered in the form of quadruple therapy or a high-dose PPI with amoxicillin. PMID- 10749094 TI - Consequences of Helicobacter pylori cure in ulcer patients. AB - In the complex pathogenesis of genuine ulcer disease Helicobacter pylori (H. pylori) is an essential, although not on its own sufficient, causal factor. Eradication of the infection heals the active ulcer and, in the long term, leads to a drastic reduction in ulcer recurrence and attendant complications. Some patients remain symptomatic even without ulcer recurrence, and in these, pre existing, exacerbated or induced gastro-oesophageal reflux disease is probably of some significance. Possible causes of ulcer relapse are reinfection, the use of ulcerogenic drugs and persistent gastric hypersecretion. In adults, and probably also in children from the age of six years, H. pylori reinfection is rare, provided that a sensitive and specific test for H. pylori is carried out at the earliest 4 weeks after concluding anti-bacterial treatment. The most common cause of the reappearance of H. pylori is recrudescence - true reinfection hardly ever occurs. The healing of H. pylori-associated ulcer disease improves the patient's quality of life and possibly also life expectancy. Computer-aided calculations, together with a randomized controlled study, have shown not only that individual patients benefit, but that also the health system profits financially. The hypothetical negative effects of H. pylori eradication treatment are still being controversially discussed. PMID- 10749095 TI - How do NSAIDs cause ulcer disease? AB - Gastroduodenal ulceration and bleeding are the major limitations to the use of non-steroidal anti-inflammatory drugs (NSAIDs). The development of safer NSAIDs or of effective therapies for the prevention of the adverse effects of existing NSAIDs requires a better understanding of the pathogenesis of NSAID-induced ulcer disease. NSAIDs can cause damage to the gastroduodenal mucosa via several mechanisms, including the topical irritant effect of these drugs on the epithelium, impairment of the barrier properties of the mucosa, suppression of gastric prostaglandin synthesis, reduction of gastric mucosal blood flow and interference with the repair of superficial injury. The presence of acid in the lumen of the stomach also contributes to the pathogenesis of NSAID-induced ulcers and bleeding, by impairing the restitution process, interfering with haemostasis and inactivating several growth factors that are important in mucosal defence and repair. In recent years, a fuller understanding of the pathogenesis of NSAID induced ulcer disease has facilitated some new, very promising approaches to the development of stomach-sparing NSAIDs. PMID- 10749096 TI - How does Helicobacter pylori infection interact with non-steroidal anti inflammatory drugs? AB - There have been conflicting clinical data on whether Helicobacter pylori (H. pylori) contributes to the pathogenesis of ulcers induced by non-steroidal anti inflammatory drugs (NSAIDs). The discrepant findings reflect the complex interaction between H. pylori and NSAIDs, which has generated divergent results under different clinical conditions. This chapter reviews the pathogenetic mechanisms in ulcer formation that are common to H. pylori and NSAIDs, and explains how a better understanding of these factors might resolve some of the controversies. Existing evidence indicates that the interaction between H. pylori and NSAIDs is not an 'all-or-none' relationship. Factors such as previous exposure to NSAIDs, a past history of ulcer complication, gastric acid output, neutrophil infiltration, concurrent acid suppressive therapy and the type of NSAID used (aspirin versus non-aspirin NSAIDs) would influence the role of H. pylori as a risk factor in NSAID users. Recommendations on H. pylori eradication for different subgroups of NSAID users are proposed. PMID- 10749097 TI - Management of gastroduodenal ulcers caused by non-steroidal anti-inflammatory drugs. AB - Non-steroidal anti-inflammatory drugs (NSAIDs) are a major cause of morbidity and mortality, probably resulting in the death of 1200 patients per annum in the UK. The main mechanism of toxicity involves an inhibition of prostaglandin synthesis that results in mucosal erosion as a result of the abrogation of defence mechanisms. However, acid peptic attack can deepen this initial injury. Thus, logical treatments include prostaglandin analogues as 'replacement therapy', acid suppression, enteric coating to avoid topical effects and the use of safer NSAIDs, including those that have little or no effect on gastric mucosal prostaglandin synthesis. There is less logic to the strategy of Helicobacter pylori (H. pylori) eradication, and the status of this approach is controversial. Overall, proton pump inhibitors have the best profile of efficacy and side effects for the healing and prevention of NSAID-associated ulcers. Misoprostol is also effective and appears to be superior to proton pump inhibitors for superficial erosive injury. Early indications are that selective inhibitors of the inducible cyclooxygenase-2 enzyme have little or no effect in causing ulcers. Growing experience with these agents will probably revolutionize the management of patients with arthritic conditions. However, the increasing use of low-dose aspirin for cardiovascular prophylaxis means that gastroenterologists will have to continue to grapple with the problems of NSAID-associated ulcers for some time to come. PMID- 10749098 TI - Adrenomyeloneuropathy: a neuropathologic review featuring its noninflammatory myelopathy. AB - The neuropathologic features of adrenomyeloneuropathy (AMN) are reviewed by supplementing those few previously published cases with 5 additional cases collected over the years. The endocrine involvement in AMN is briefly presented to serve as a pathogenetic backdrop and to emphasize that most of the lesions in AMN, as in adreno-leukodystrophy (ALD), are noninflammatory in the traditional sense of the word. The myeloneuropathy is emphasized, but the dysmyelinative/inflammatory demyelinative lesions also are presented. The preponderance of available data indicates that the myeloneuropathy of AMN is a central-peripheral distal (dying-back) axonopathy, as was originally proposed. The severity of the myeloneuropathy does not appear to correlate with the duration or severity of endocrine dysfunction. Microglia are the dominant participating cells in the noninflammatory myelopathy. Abnormalities in the ALD gene, which encodes a peroxisomal ABC half-transporter, do not correlate with clinical phenotypes. The relationship of the gene product, ALDP, to the peroxisomal very long chain fatty acid (VLCFA) synthetase, the activity of which is deficient in ALD/AMN, is unclear. An ALD-knockout mouse model has developed axonal degeneration, particularly in spinal cord, and is therefore more reminiscent of AMN than ALD. We continue to postulate that the fundamental defect in the myeloneuropathy of AMN is an axonal or neuronal membrane abnormality perhaps due to the incorporation of VLCFA-gangliosides, which perturbs the membrane's microenvironment and leads to dysfunction and atrophy. PMID- 10749099 TI - Selective reduction in the nicotinic acetylcholine receptor and dystroglycan at the postsynaptic apparatus of mdx mouse superior cervical ganglion. AB - Our previous data suggested that in mouse sympathetic superior cervical ganglion (SCG) the dystrophin-dystroglycan complex may be involved in the stabilization of the nicotinic acetylcholine receptor (nAChR) clusters. Here we used SCG of dystrophic mdx mice, which express only the shorter isoforms of dystrophin (Dys), to investigate whether the lack of the full-length dystrophin (Dp427) could affect the localization of the dystroglycan and the alpha3 nAChR subunit (alpha3AChR) at the postsynaptic apparatus. We found a selective reduction in intraganglionic postsynaptic specializations immunopositive for alpha3AChR and for alpha- and beta-dystroglycan compared with the wild-type. Moreover, in mdx mice, unlike the wild-type, the disassembly of intraganglionic synapses induced by postganglionic nerve crush occurred at the slower rate and was not preceded by the loss of immunoreactivity for Dys isoforms, beta-dystroglycan, and alpha3AChR. These data indicate that the absence of Dp427 at the intraganglionic postsynaptic apparatus of mdx mouse SCG interferes with the presence of both dystroglycan and nAChR clusters at these sites and affects the rate of synapse disassembly induced by postganglionic nerve crush. Moreover, they suggest that the decrease in ganglionic nAChR may be one of the factors responsible for autonomic imbalance described in Duchenne muscular dystrophy patients. PMID- 10749100 TI - Bcl-2 immunoreactive cells with immature neuronal phenotype exist in the nonepileptic adult human brain. AB - Bcl-2, a cell death suppressor protein, is expressed during brain development but is largely down-regulated in the adult central nervous system. We previously reported strong expression of bcl-2 in small, "oligodendrocyte-like" cells (OLC) found in glioneuronal hamartias. These hamartias are microscopic cell rests found in temporal lobe resections from patients with intractable epilepsy and are considered a form of cerebral microdysgenesis. However, a causative relationship between these rests and seizures is not clear. We now report the identification, lineage characterization, and postnatal ontogeny of hamartia-like cell rests in temporal lobes of nonepileptic humans. Postmortem temporal lobes from 28 patients without history of neurologic disease (mean age = 53 years; range = 20 to 83 years) were studied. Microscopic cellular aggregates containing immature appearing, bcl-2-immunoreactive cells (BIC) (identical to OLC) were observed in 23 of 28 (82%) temporal lobes from nonepileptic individuals. BIC were strongly immunoreactive for neuronal-specific class III beta tubulin, neuronal nuclear antigen, and MAP-2, but were consistently negative for neurofilament proteins and Ki67. Such cells were localized to subventricular regions of the caudal amygdala and often extended into the adjacent subcortical white matter and periamygdaloid cortex. BIC became less abundant with advancing age. These findings suggest that hamartia-like rests containing immature postmitotic neurons are normally present in the human brain and that glioneuronal hamartias may not always represent a maldevelopmental lesion associated with epilepsy. PMID- 10749101 TI - Kinetics and polarization of the membrane expression of cytokine-induced ICAM-1 on rat brain endothelial cells. AB - ICAM-1 is a major cellular adhesion molecule by which lymphocytes attach to vascular endothelial cells. Rat brain endothelial cells (RBEC) in culture show very low levels of ICAM-1. However, after exposure to IL-1beta and IFN-gamma, the ICAM-1 expression increases up to 20-fold (as judged by FACS analysis). We used immunogold electron microscopy to examine the kinetics in distribution of cytokine-induced ICAM-1 on the surfaces of tight-junction RBEC (grown on matrigel coated transwells) when exposed to cytokines from either the luminal (upper well) or the abluminal (lower well) surface. Luminal stimulation produced an early upregulation of ICAM-1 not only on the luminal surface of the endothelial cells but also on the lateral surface below the tight junctions and on the abluminal surface. Peak expression on the abluminal surface of the monolayer occurred at the time of maximal "trapping" of lymphocytes seen during an in vitro migration assay. This suggests that the in vitro trapping, as well as the in vivo trapping described by others, may have its basis in a receptor-ligand interaction. We also demonstrate that when the monolayer is stimulated with cytokine from the abluminal surface there is a delayed but preferential upregulation of ICAM-1 on the luminal surface. PMID- 10749102 TI - B7 expression and antigen presentation by human brain endothelial cells: requirement for proinflammatory cytokines. AB - Interaction between systemic immune cells with cells of the blood-brain barrier is a central step in development of CNS-directed immune responses. Endothelial cells are the first cells of the blood-brain barrier encountered by migrating lymphocytes. To investigate the antigen-presenting capacity of human adult brain endothelial cells (HBECs), we used HBECs derived from surgically resected temporal lobe tissue, cocultured with allogeneic peripheral blood derived CD4+ T lymphocytes. HBECs in response to IFN-gamma, but not under basal culture conditions, expressed HLA-DR, B7.1 and B7.2 antigens. Despite such up-regulation, these IFN-gamma-treated HBECs, in contrast to human microglia and PB monocytes, did not sustain allogeneic CD4+ cell proliferation, supported only low levels of IL-2 and IFN-gamma production, and did not stimulate IL-2 receptor expression. CD4+ T cell proliferation and increased IL-2 receptor expression could be obtained by addition of IL-2. Our data suggests that, although HBECs cannot alone support T cell proliferation and cytokine production, HBECs acting in concert with cytokines derived from a proinflammatory environment could support such a response. PMID- 10749103 TI - Increase in HLA-DR immunoreactive microglia in frontal and temporal cortex of chronic schizophrenics. AB - Glia play a major role in neuronal migration, synapse formation, and control of neurotransmission in the developing and mature nervous system. This study investigated whether chronic schizophrenia is associated with glial changes in 3 regions of the cerebral cortex: dorsolateral prefrontal cortex (Brodmann's area 9), the superior temporal gyrus (area 22), and the anterior cingulate gyrus (area 24). In a blind study, astroglia and microglia were identified immunocytochemically in frozen sections from postmortem schizophrenic and control brains. Astroglia and microglia were identified using antibodies to glial fibrillary acidic protein (GFAP) and class II human leucocyte antigen (HLA-DR) respectively. They were then quantified for each cortical layer. Significant differences were found in HLA-DR+ microglial numerical density in 2 of the areas. A 28% increase (p < 0.05) was found in area 9 in 8 schizophrenics (115 +/- 9 cells/mm2) compared with 10 controls (89 +/- 5 cells/mm2), when combining all cortical layers and both cerebral hemispheres. For area 22, there was a 57% increase (p < 0.01) in microglia in 7 schizophrenics (139 +/- 6 cells/mm2) compared with 10 controls (88 +/- 5 cells/mm2). In area 24 the same trend was evident, but the results did not reach significance. Microglial number was further analyzed for each cortical layer, which confirmed the overall pattern. For all areas, numerical density of astroglia showed no significant differences between schizophrenics and controls. Cortical thickness was measured in all areas and total neuronal numerical density was estimated for area 22. Again, no significant differences were found between schizophrenics and controls. This study demonstrates a specific increase in the numerical density of HLA-DR+ microglia in temporal and frontal cortex of chronic schizophrenics, not related to aging, which might be implicated in possible changes in cortical neuropil architecture in schizophrenia. PMID- 10749104 TI - STAT3 and NFkappaB activation precedes glial reactivity in the excitotoxically injured young cortex but not in the corresponding distal thalamic nuclei. AB - In this study we evaluated the activation of the cytokine and growth factor responsive transcription factors signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa B (NFkappaB) after different grades of neural damage in the immature rat brain using double immunocytochemical techniques and electron microscopy. Following neocortical N-methyl-D-aspartate induced excitotoxic cell death, both these transcription factors are mainly activated in astrocytes, although microglia, endothelial cells, and neurons show transient activation at specific times and locations. Interestingly, activation of both transcription factors is only observed in cortical areas affected by severe tissue damage, neuronal degeneration, and blood-brain barrier (BBB) disruption. In contrast, the milder glial response occurring in the distal thalamus is not preceded by immunocytochemically detectable STAT3 and NFkappaB activation, although microglial response, astroglial hypertrophy, and glial fibrillary acidic protein (GFAP) overexpression do occur. In the cortex, astrocytes show STAT3 and NFkappaB activation already at 2 to 4 hours post-lesion, preceding cell hypertrophy and GFAP upregulation, and being maintained in the long-term formed glial scar. STAT3 and NFkappaB activation in microglial cells is protracted and observed at 10 to 24 hours post-lesion. The early activation of both transcription factors in astroglial cells could contribute to the changes in gene expression leading to astrogliosis and the release of signalling molecules which may contribute to the subsequent activation of these transcription factors in microglial cells. PMID- 10749105 TI - Increased expression of beta-chemokines in muscle of patients with inflammatory myopathies. AB - Idiopathic inflammatory myopathies (IIM) are muscle diseases of autoimmune pathogenesis characterized by mononuclear cell infiltration within muscle tissue. Since immune cell homing and accumulation at the site of antigenic challenge is usually mediated by chemokines, we evaluated the expression of 2 beta-chemokines- monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein 1alpha (MIP-1alpha)--by immunohistochemistry and polymerase chain reaction in muscles of polymyositis, inclusion body myositis, and dermatomyositis patients, and related their expression to immunopathological alterations in muscle. MCP-1 and MIP-1alpha transcripts were detected by PCR in all IIM muscles, but not in controls. By immunohistochemistry, the chemokines were found in all IIM muscle sections located in infiltrating inflammatory cells and also in neighboring extracellular matrix. The extent to which extracellular matrix was filled by each chemokine differed in each disease. In view of the known ability of chemokines to bind extracellular matrix and their possible synthesis by extracellular matrix components, we suggest that chemokine storage in the extracellular matrix can act as a microenvironmental factor amplifying lymphocyte activation and migration, thereby maintaining the autoimmune attack against unknown muscle antigens. PMID- 10749106 TI - Immunocytochemical, ultrastructural and neurochemical evidences on synaptogenesis and dopamine release of rat chromaffin cells co-cultured with striatal neurons. AB - The results reported herein address the question of synaptogenesis between adrenal chromaffin cells and striatal neurons. The release of dopamine from chromaffin cells in the presence of striatal neurons was also examined. Co culture of newborn rat chromaffin cells and striatal neurons at 1:1 ratio was made. Cultures were examined morphologically using immunocytochemistry and ultrastructural techniques (transmission electron microscopy), while quantitation of dopamine in the culture media by HPLC-ECD was also determined. Neurite outgrowth from chromaffin cells was enhanced in the presence of striatal neurons and numerous synaptic-like contacts between these two cell types were observed. Higher concentration of dopamine was also present in the co-culture medium as compared with those containing only chromaffin cells. The development of synapses between these two types of cells may give support to the functionality of transplants in human cases of Parkinson disease (PD). PMID- 10749107 TI - Genetics and biology of adult human male germ cell tumors. AB - Adult human male germ cell tumors (GCTs) provide a unique opportunity to study the generation of a transformed pluripotential cell from a totipotential GC in lineage differentiation and on the path to gametogenesis. The pluripotentiality of the tumor cells manifests as histological differentiation into GC-like undifferentiated (SE), primitive zygotic (EC), embryonal-like somatically differentiated (TE), and extra-embryonally differentiated (CC, YST) phenotypes. The tumors and cell lines derived from them comprise exceptional model systems for the molecular analysis of human embryonal cell fate and lineage differentiation. The majority of GCTs show exquisite sensitivity to cisplatin based treatment and have served as models for the development of chemotherapy for solid tumors. Until recently, the molecular mechanisms of GC transformation, GCT differentiation, or GCT chemotherapy sensitivity and resistance were understood poorly. Very recent studies of GCTs have suggested that: (a) overexpression of cyclin D2 is a very early, possibly the oncogenic, event in GC tumorigenesis; (b) differentiation in GCTs may be governed by several possibly interacting pathways, such as loss of regulators of GC totipotentiality and of embryonic development, and genomic imprinting; and (c) chemotherapy sensitivity and resistance may be rooted in part in a p53-dependent apoptotic pathway. In this review, these new data are discussed in the context of GC and GCT biology, and several novel testable genetic models are proposed. PMID- 10749108 TI - Nuclear expression of the c-erbB-4/HER-4 growth factor receptor in invasive breast cancers. AB - The prevalence and sites of expression of the c-erbB-4 receptor have been determined by immunocytochemical staining in a series of 178 human breast cancers. Most tumors displayed cytoplasmic staining of variable intensity. When compared with adjacent normal tissue, 32 cases (18%) showed lower than normal expression, and 13 (7%) showed greater than normal expression. Nuclear immunoreactivity, confirmed by two different antibodies, was present in 87 cancers (49%) but was found in normal adjacent breast epithelial cells in <5% of cases. There were no significant associations with cytoplasmic or membrane immunoreactivity, but cases showing nuclear expression in >25% of cells were associated with good histological grade, epidermal growth factor receptor expression, c-erbB-3 positivity, cripto, amphiregulin, and transforming growth factor-alpha overexpression. PMID- 10749109 TI - Monitoring of herpes simplex virus thymidine kinase enzyme activity using positron emission tomography. AB - 9-[(1-[18F]Fluoro-3-hydroxy-2-propoxy)methyl]guanine ([18F]FHPG) was evaluated as a tracer for noninvasive positron emission tomography (PET) imaging of herpes simplex virus type 1 thymidine kinase (HSV-tk) gene expression. C6 rat glioma cells with and without the HSV-tk gene were incubated with [18F]FHPG for 2 h. The in vitro tracer uptake in HSV-tk-containing C6tk cells was 35 +/- 5 times higher than that in control cells. In nude rats carrying both a C6 and a C6tk tumor, the average ratio of tracer accumulation between the tumors was 15 +/- 5 at 2 h postinjection. The tracer is rapidly cleared from nontarget tissue into the urine because only the HSV-tk-expressing tumor, kidneys, and bladder remained visible on the late PET images. HPLC analysis revealed that three metabolites, tentatively assigned as FHPG mono-, di-, and triphosphate, were formed in the C6tk tumors only. In conclusion, we have demonstrated that [18F]FHPG is a promising tracer for monitoring HSV-tk enzyme activity in vivo with PET. PMID- 10749110 TI - Fusogenic membrane glycoproteins as a novel class of genes for the local and immune-mediated control of tumor growth. AB - We report here the use of viral fusogenic membrane glycoproteins (FMGs) as a new class of therapeutic genes for the control of tumor growth. FMGs kill cells by fusing them into large multinucleated syncytia, which die by sequestration of cell nuclei and subsequent nuclear fusion by a mechanism that is nonapoptotic, as assessed by multiple criteria. Direct and bystander killing of three different FMGs were at least one log more potent than that of herpes simplex virus thymidine kinase or cytosine deaminase suicide genes. Transduction of human tumor xenografts with plasmid DNA prevented tumor outgrowth in vivo, and cytotoxicity could be regulated through transcriptional targeting. Syncytial formation is accompanied by the induction of immunostimulatory heat shock proteins, and tumor associated FMG expression in immunocompetent animals generated specific antitumor immunity. PMID- 10749111 TI - Cell permeable Bcl-2 binding peptides: a chemical approach to apoptosis induction in tumor cells. AB - Bcl-2 is a potent suppressor of apoptosis, and its overexpression contributes to tumorigenesis in many types of human cancers. To test the possibility of modulating Bcl-2 function as an anticancer strategy, a cell permeable Bcl-2 binding peptide, cell permeable moiety (cpm)-1285, was designed by chemically attaching a fatty acid to a peptide derived from the proapoptotic protein Bad. cpm-1285 entered HL-60 tumor cells, bound Bcl-2 protein, and induced apoptosis in vitro. In contrast, cpm-1285 had little effect on normal human peripheral blood lymphocytes. Furthermore, cpm-1285 had in vivo activity in slowing human myeloid leukemia growth in severe combined immunodeficient mice. These results demonstrate a novel approach for therapeutic intervention of tumor growth in vivo with small molecule inhibitors of Bcl-2. PMID- 10749112 TI - Antiangiogenic gene therapy of cancer utilizing a recombinant adenovirus to elevate systemic endostatin levels in mice. AB - Gene therapy represents a possible alternative to the chronic delivery of recombinant antiangiogenic proteins to cancer patients. Inducing normal host tissues to produce high circulating levels of these proteins may be more effective than targeting antiangiogenic genes to tumor tissue specifically. Previously reported gene therapy approaches in mice have achieved peak circulating endostatin levels of 8-33 ng/ml. Here we report plasma endostatin levels of 1770 ng/ml after administration of a recombinant adenovirus. Growth of MC38 adenocarcinoma, which is relatively resistant to adenoviral infection, was inhibited by 40%. These findings encourage gene delivery approaches that use the host as a "factory" to produce high circulating levels of antiangiogenic agents. PMID- 10749114 TI - Eradication diminishes enhancing effects of Helicobacter pylori infection on glandular stomach carcinogenesis in Mongolian gerbils. AB - To investigate the nature of the link between Helicobacter pylori (Hp) infection and stomach carcinogenesis, a study of the glandular stomach of Mongolian gerbils (MGs) was performed. MGs were treated with N-methyl-N-nitrosourea (MNU), followed by inoculation with Hp (groups 1 and 2) or without Hp (group 3), or infected with Hp (groups 4 and 5) or inoculation without Hp (group 6) followed by MNU administration. At week 21, the animals in groups 2 and 5 underwent an eradication procedure. At week 50, the incidences of adenocarcinomas in group 1 (15 of 23) and group 4 (9 of 26) were significantly higher than in group 3 (1 of 15) and group 6 (1 of 18), respectively. Moreover, those in group 2 (5 of 24) and group 5 (2 of 22) were lower than in groups 1 and 4, respectively. This study shows that Hp eradication may be useful as a prevention approach against stomach cancer. PMID- 10749113 TI - Deoxyguanosine adducts of t-4-hydroxy-2-nonenal are endogenous DNA lesions in rodents and humans: detection and potential sources. AB - t-4-Hydroxy-2-nonenal (HNE) is a free radical-mediated oxidation product of polyunsaturated fatty acids. As an electrophile, HNE readily binds to proteins and yields diastereomeric cyclic 1,N2-propano adducts with deoxyguanosine (dG). Here, we report the detection and identification of the HNE-derived cyclic 1,N2 propano-dG adducts as endogenous DNA lesions in tissues of untreated rats and humans using a highly sensitive 32P-postlabeling method in conjunction with high performance liquid chromatography. These adducts were first verified by their comigration with the synthetic UV standards of HNE-dG adducts. Subsequently, their identities were unequivocally established by two independent reactions. An approximately 37-fold increase in the levels of HNE-dG adducts was observed in the liver DNA of F344 rats after treatment with CCl4, suggesting that tissue lipid peroxidation is a likely source of their formation. Our studies in vitro further indicate that omega-6 polyunsaturated fatty acids are likely a unique class of fatty acids involved in HNE-dG adduct formation. PMID- 10749115 TI - Eradication of disseminated lymphomas with CpG-DNA activated T helper type 1 cells from nontransgenic mice. AB - Various evidence suggests that adoptive transfer of polyclonal, tumor-specific, IFN-gamma-producing CD4+ T cells [T helper type 1 (Th1) cells] should be highly efficient for tumor immune therapy. However, this approach could not be tested because very few MHC class II-restricted tumor peptides have been defined. Here we show that stimulation of freshly isolated T helper cells with syngeneic tumor cells and antigen-presenting cells in the presence of immunostimulatory CpG DNA allows the generation of large numbers of strongly polarized, tumor-specific Th1 cells within 3 weeks of culture, even when T helper cells were derived from tumor bearing mice. A single injection of 0.5 x 10(6) A20-specific Th1 cells even eradicated disseminated A20 lymphomas and provided lifelong protection without inducing autoimmune disease. The therapy was largely independent of CD8+ cells but required IFN-gamma and CD40-CD40L interactions, suggesting that tumor specific Th1 cells eradicate established tumors by activating proinflammatory macrophages. PMID- 10749117 TI - Layered expression scanning: rapid molecular profiling of tumor samples. AB - Layered expression scanning is a new approach to comprehensive molecular analysis of tumor samples that uses a layered array of capture membranes coupled to antibodies or DNA sequences to perform multiplex protein or mRNA analysis. Cell or tissue samples are transferred through a series of individual capture layers, each linked to a separate antibody or DNA sequence. As the biomolecules traverse the membrane set, each targeted protein or mRNA is specifically captured by the layer containing its antibody or cDNA sequence. The two-dimensional relationship of the cell populations is maintained during the transfer process, thereby producing a molecular profile of each cell type present. Reduction-to-practice of the technique is demonstrated by analysis of prostate-specific antigen (PSA) protein, gelatinase A protein, and POV1 (PB39) cDNA. As layered expression scanning technology progresses, we envision a laboratory method that will have multiple applications for high-throughput molecular profiling of normal and tumor samples. PMID- 10749116 TI - Abnormal RNA expression of 11p15 imprinted genes and kidney developmental genes in Wilms' tumor. AB - Wilms' tumor (WT) is caused by abnormal development of embryonal kidney cells. WT cells are frequently affected by deletions or functional inactivation of maternal alleles at chromosome 11p15, which indicates that the loss of maternally expressed genes in this region plays an important role in WT pathogenesis. Maternally expressed genes indeed exist within an imprinted region at 11p15.5. Among these, BWR1C is highly expressed in fetal but not in adult kidney, which suggests that it may fulfil an important role in kidney development. Here, we demonstrate that the lack of BWR1C expression is common in WT. Its homology with the proapoptotic gene TDAG51 suggests that the loss of BWR1C expression may be relevant in WT development. In addition, the analysis of the expression of other 11p15 imprinted genes and kidney-developmentally regulated genes indicates that IGF2 overexpression, inappropriate coexpression of RET and GDNF and, in some cases, down-regulation of CDKN1C may also play an important role in the pathogenesis of WT. Our results add new elements to the understanding of the biological basis of WT, which may have implications for WT diagnosis and therapy. PMID- 10749118 TI - Potential role of BRCA2 in a mitotic checkpoint after phosphorylation by hBUBR1. AB - BRCA2, a gene responsible for inherited susceptibility to breast cancer in a number of families, is thought to be critical for replication and repair of DNA during S-phase. To elucidate the physiological functions of BRCA2, we used a yeast two-hybrid system to screen for proteins that could associate with BRCA2. Here we report interaction of BRCA2 with a mitotic checkpoint protein, hBUBR1, and its phosphorylation by hBUBR1 in vitro. After cotransfection of BRCA2 and hBUBR1 expression vectors into the COS7 cell line, both proteins were stained together in the nuclei of cells whose spindle fibers were disrupted, but not in undamaged cells. Treatment with vincristine, which disrupts microtubules, significantly increased expression of both hBUBR1 and BRCA2 in the MCF7 cells. The results suggest that BRCA2 protein might be involved in a mitotic checkpoint in vivo after it has been phosphorylated by hBUBR1. PMID- 10749119 TI - EWS-FLI1, EWS-ERG, and EWS-ETV1 oncoproteins of Ewing tumor family all suppress transcription of transforming growth factor beta type II receptor gene. AB - Ewing sarcoma-specific chromosomal translocations fuse the EWS gene to a subset of ets transcription factor family members, most commonly the FLI1 gene and less frequently ERG, ETV1, E1A-F, or FEV. These fusion proteins are thought to act as aberrant transcription factors that bind DNA through their ets DNA binding domain. Recently, we have shown (K-B. Hahm et al., Nat. Genet., 23: 222-227, 1999) that the transforming growth factor beta (TGF-beta) type II receptor (TGF beta RII), a putative tumor suppressor gene, is a target of the EWS-FLI1 fusion protein. Here, we also examined effects of EWS-ETV1 and EWS-ERG on expression of the TGF-beta RII gene. We show that relative to the control, NIH-3T3 cell lines stably transfected with the EWS-FLI1, EWS-ERG, or EWS-ETV1 gene fusion express reduced levels of TGF-beta RII mRNA and protein, and that these cell lines have reduced TGF-beta sensitivity. Cotransfection of these fusion genes and the TGF beta RII promoter suppresses TGF-beta RII promoter activity and also FLI1-, ERG-, or ETV1-induced promoter activity. These results indicate that transcriptional repression of TGF-beta RII is an important target of the EWS-FLI1, EWS-ERG, or EWS-ETV1 oncogene, and that EWS-ets fusion proteins may function as dominant negative forms of ets transcription factors. PMID- 10749121 TI - Coordinate up-regulation of Sp1 DNA-binding activity and urokinase receptor expression in breast carcinoma. AB - The regulatory mechanisms underlying the overexpression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in highly invasive breast carcinomas remain poorly understood. In this study, we have simultaneously determined the level of uPAR and the activity of the transcription factor Sp1 in 14 breast carcinomas and 5 benign lesions. We found that uPAR levels and Sp1 binding activity are coordinately elevated in malignant tumors (r, 0.94; P < 0.001). On the contrary, undetectable or only barely detectable levels of uPAR and Sp1 activity were found in benign breast lesions. Finally, the engagement of uPAR by catalytically inactive uPA in the MDA-MB-231 breast carcinoma cell line results in a rapid up-regulation of Sp1-binding activity followed by an increase of uPAR protein. These results, taken together, suggest the existence of a uPA dependent positive regulatory loop that may progressively enhance malignant breast cell invasiveness. PMID- 10749120 TI - Modulation of hypoxia-inducible factor 1alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. AB - Dysregulated signal transduction from receptor tyrosine kinases to phosphatidylinositol 3-kinase (PI3K), AKT (protein kinase B), and its effector FKBP-rapamycin-associated protein (FRAP) occurs via autocrine stimulation or inactivation of the tumor suppressor PTEN in many cancers. Here we demonstrate that in human prostate cancer cells, basal-, growth factor-, and mitogen-induced expression of hypoxia-inducible factor 1 (HIF-1) alpha, the regulated subunit of the transcription factor HIF-1, is blocked by LY294002 and rapamycin, inhibitors of PI3K and FRAP, respectively. HIF-1-dependent gene transcription is blocked by dominant-negative AKT or PI3K and by wild-type PTEN, whereas transcription is stimulated by constitutively active AKT or dominant-negative PTEN. LY294002 and rapamycin also inhibit growth factor- and mitogen-induced secretion of vascular endothelial growth factor, the product of a known HIF-1 target gene, thus linking the PI3K/PTEN/AKT/FRAP pathway, HIF-1, and tumor angiogenesis. These data indicate that pharmacological agents that target PI3K, AKT, or FRAP in tumor cells inhibit HIF-1alpha expression and that such inhibition may contribute to therapeutic efficacy. PMID- 10749122 TI - Transfection of constitutively active mitogen-activated protein/extracellular signal-regulated kinase kinase confers tumorigenic and metastatic potentials to NIH3T3 cells. AB - Cellular growth and differentiation are controlled by multiple extracellular signals, many of which activate extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinases. Components of the MAP kinase pathways also cause oncogenic transformation in their constitutively active forms. Moreover, expression of activated ras can confer metastatic potential upon some cells. Activation of MAP kinases requires phosphorylation of both Thr and Tyr in the catalytic domain by a family of dual-specificity kinases, called MEKs (MAP kinase/ERK kinase). MEK1 is activated by phosphorylation at Ser218 and Ser222 by Raf. Mutation of these two sites to acidic residues, specifically [Asp218], [Asp218, Asp222], and [Glu218, Glu222], results in constitutively active MEK1. Using these mutant variants of MEK1, we showed previously that transfection of NIH/3T3 or Swiss 3T3 cells causes morphological transformation and increases growth on soft agar, independent of ERK activity. The transformed cell lines show increased expression of matrix metalloproteinases 2 and 9 and cathepsin L, proteinases that have been implicated in the metastatic process. We tested NIH3T3 cells transfected with the [Asp218] or [Asp218, Asp222] for metastatic potential after i.v. injection into athymic mice. Parental 3T3 cells formed no tumors grossly or histologically. However, all MEK1 mutant transformants formed macroscopic metastases. Thus, like activated Ras, MEK1 can confer both tumorigenic and metastatic potential upon NIH3T3 cells. These results refine the mechanism through which ras could confer tumorigenic and metastatic potential (ie., the critical determinants of tumorigenic and metastatic potential are downstream of MEK1). PMID- 10749123 TI - Adenovirus E1A does not induce the Ewing tumor-associated gene fusion EWS-FLI1. AB - Rearrangement of the EWS gene with FLI1 is thought to occur early in the pathogenesis of Ewing's sarcoma family tumors (EFTs) because the chromosomal aberration is pathognomonic for this disease. Recently, adenovirus (Ad) 5 E1A protein has been reported to induce this gene rearrangement in a variety of cell types. This finding, if generally substantiated, not only suggests an etiological role for viral agents in the generation of oncogenic chromosomal aberrations but would also significantly impact the use of adenoviral vectors for gene therapy. In contrast, we now report on the absence of EWS-FLI1 chimeric products from short- and long-term cultures of stably Ad-transformed cells lines and from transiently E1A-expressing cell lines. In addition, we demonstrate the absence of E1A from EFTs. We conclude that there is no role for Ads in EFT pathogenesis. Consequently, evidence for a viral genesis of tumor-specific gene rearrangements is not available. PMID- 10749124 TI - Constitutive expression of insulin-like growth factor-1 in epidermal basal cells of transgenic mice leads to spontaneous tumor promotion. AB - Transgenic mice overexpressing insulin-like growth factor-1 (IGF-1) in the basal layer of skin epidermis were generated using the bovine keratin 5 promoter (BK5). Neonatal transgenic mice were slightly smaller at birth and exhibited early ear unfolding, wrinkled and thickened skin, and slightly enlarged ears compared with nontransgenic littermates. Morphological evaluation of the skin revealed that persistent overexpression of IGF-1 in the basal layer of the epidermis resulted in epidermal hyperplasia, hyperkeratosis, and an increased labeling index that persisted in adult mice. Phenotypic changes observed in skin were associated with transgene expression in the basal layer of the epidermis and activation of the IGF-1 receptor. Squamous papillomas (some of which converted to carcinomas) developed in a significant proportion (approximately 50%) of older BK5.IGF-1 mice. Treatment of BK5.IGF-1 transgenic mice with multiple topical applications of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, in the absence of tumor initiation led to the development of additional skin papillomas. Furthermore, treatment of BK5.IGF-1 transgenic mice with an initiating dose of 7,12-dimethylbenz[a]anthracene only led to the formation of additional papillomas in the absence of promotion. In two-stage carcinogenesis experiments, BK5.IGF-1 transgenic mice developed 7-fold more papillomas than nontransgenic littermates. Phosphatidylinositol-3-kinase and protein kinase B (Akt) activities were elevated (3-4-fold), and mitogen-activated protein kinase activity was elevated approximately 1.7-fold in the epidermis of transgenic mice compared with nontransgenic mice. In addition, UV light-induced epidermal apoptosis was significantly suppressed in BK5.IGF-1 transgenic mice. These data suggest that persistent activation of IGF-1 receptor signaling pathways in basal epithelial cells leads to spontaneous tumor promotion and that up-regulation of both mitogenic and cell survival signaling pathways may play an important role in the action of IGF-1 in this model system. PMID- 10749125 TI - Genotype-specific Trp53 mutational analysis in ultraviolet B radiation-induced skin cancers in Xpc and Xpc Trp53 mutant mice. AB - We have examined the mutational spectrum in the Trp53 gene from UVB radiation induced skin cancers in Trp53+/+ and Trp53+/- mutant mice of all three possible Xpc genotypes. Mutations were detected in exons 2-10 of the Trp53 coding region in approximately 90% of >80 different skin cancers examined. In contrast to Trp53+/+ mice in which most mutations in the Trp53 gene were located in exons 5 8, the majority of the mutations in Trp53+/- mice were at other exons. We observed a high predilection for C-->T transition mutations at a unique CpG site in codon 122 (exon 4) of the Trp53 gene in Xpc-/- Trp53+/- mice. This site is not part of a pyrimidine dinucleotide. Mutations at this codon, as well as in codons 124 and 210, were observed exclusively in Xpc-/- or Xpc+/- mice. Mutations at the corresponding codons (127 and 213) in the human p53 gene have been reported in skin tumors from human patients with xeroderma pigmentosum. Hence, mutations at codons 122 (125), 124 (127), and 210 (213) may constitute signatures for defective or deficient nucleotide excision repair in mice (humans). In Xpc-/- mice, the majority of mutations were located at C residues in CpG sites, in which the C is presumably methylated. A similar bias can be deduced from studies in human XP individuals. PMID- 10749126 TI - Ultraviolet B radiation-induced skin cancer in mice defective in the Xpc, Trp53, and Apex (HAP1) genes: genotype-specific effects on cancer predisposition and pathology of tumors. AB - Mutations in nucleotide excision repair (NER) genes in humans result in the UV induced skin cancer-prone disease xeroderma pigmentosum (XP). Mouse models that mimic XP have provided an informative experimental system with which to study DNA repair, as well as the molecular pathology of UV radiation-induced skin cancer. We reported previously that mice defective in the Xpc gene (Xpc-/-) are highly predisposed to UVB radiation-induced skin cancer and that the appearance of skin cancer is more rapid in Xpc Trp53 double mutants. Extended studies now demonstrate an increased predisposition to UVB radiation-induced skin cancers in Xpc heterozygous mice compared with normal mice. We also show that Xpc Trp53 double heterozygous mutants are more predisposed to skin cancer than Trp53 single heterozygous mice. No mutations were detected in the cDNA of the remaining Xpc allele, suggesting that haploinsufficiency of the Xpc gene may be operating and is a risk factor for UVB radiation-induced skin cancer in mice. Skin tumors from Xpc-/- mice were exclusively well or moderately well-differentiated squamous cell carcinomas. In Xpc+/+ and Xpc+/- mice, many of the squamous cell carcinomas were less well differentiated. We also documented previously increased predisposition to UV radiation-induced skin cancers in Xpc-/- Apex+/- mice. Here we show the absence of mutations in the cDNA of the remaining Apex allele, a further suggestive indication of haploinsufficiency and its resulting predisposition to skin cancer. The Trp53 and Apex heterozygous conditions altered the skin tumor spectrum to more poorly differentiated forms in all Xpc genotypes. PMID- 10749127 TI - Prognostic significance of p53 nuclear accumulation in localized prostate cancer treated with radical prostatectomy. AB - The role of p53 in the pathogenesis of, and as a predictive biomarker for, localized prostate cancer (PCa) is contested. Recent work has suggested that patterns of p53 nuclear accumulation determined by immunohistochemistry are prognostic, whereas studies using other methods question the role of p53 mutations in predicting outcome. We studied 263 men with localized PCa treated with radical prostatectomy to determine whether p53 nuclear accumulation predicts relapse and disease-specific mortality. We combined two p53 immunohistochemistry scoring systems: (a) percentage of p53-positive tumor nuclei in all major foci of cancer within the prostate; and (b) clustering, where the presence of 12 or more p53-positive cells within a x 200 power field was deemed "cluster positive." Analysis was undertaken using chi2, Kruskal-Wallis, and Mann-Whitney tests for clinicopathological variables and the Kaplan-Meier method, log-rank test, and univariate and multivariate Cox regression modeling for evaluation of contribution to relapse and disease-specific survival. At mean follow-up of 55.1 months (range, 4.9-123.0 months), 39% (102 of 263) of patients had relapsed and 2.3% (6 of 253) had died of PCa. Pretreatment serum prostate-specific antigen concentration, pathological tumor stage, lymph node involvement, Gleason score, and p53 nuclear accumulation, as determined by either percentage score or cluster status, were independent predictors of relapse in multivariate analysis. Clustering of p53-positive cells distinguished between favorable and poor prognosis patients within the lowest p53-positive stratum (>0 to <2%) and was the most discriminatory threshold for predicting relapse in the entire cohort. p53 status predicted outcome in patients with a Gleason score of 5 and above but not those with a score of 4 and below. In patients treated with neoadjuvant hormonal therapy, p53 cluster positivity carried a 90% (19 of 21) risk of relapse by 36 months. All six patients who died from PCa in the period of the study exhibited p53 nuclear accumulation in 20% or more tumor nuclei. This study demonstrates strong relationships between p53 nuclear accumulation and relapse and disease specific mortality in a large series of localized PCas. Furthermore, the presence of clusters of p53-positive nuclei delineates a group of patients with poor prognosis not identified by traditional scoring methods and supports the hypothesis that p53 dysfunction within PCa may exist in foci of tumor cells that are clonally expanded in metastases. PMID- 10749128 TI - Prognostic significance of the metastasis-inducing protein S100A4 (p9Ka) in human breast cancer. AB - The calcium-binding protein S100A4 is capable of inducing metastasis in rodent models for breast cancer. We now show that rabbit antibodies to recombinant rat S100A4 recognize specifically human S100A4 using Western blotting techniques and use them to assess the prognostic significance of S100A4 in primary tumors from a group of 349 patients treated between 1976 and 1982 for stage I and stage II breast cancer. The antibody stains normal breast tissue heterogeneously, but stains positively 41% of the carcinomas, leaving the remaining 59% as negatively stained. In addition to the carcinoma cells, some host stromal cells and lymphocytes are also stained, but these have been discounted in subsequent analyses. There is an association of staining of carcinomas for S100A4 with some tumor variables considered to be associated with poor prognosis for patients: tumor present in axillary lymph nodes (borderline P = 0.058), staining for c-erbB 3 (P = 0.002), cathepsin D (P = 0.024), and c-erbB-2 (P = 0.048). The association of staining for S100A4 with patient survival has been evaluated using life tables and analyzed using generalized Wilcoxon statistics. Eighty percent of the S100A4 negative patients but only 11% of the S100A4-positive patients are alive after 19 years of follow-up, and this association is highly significant (P < 0.0001); the former have a median survival of >228 months and the latter 47 months. The other tumor variables that show significant association with survival time are nodal status (P < 0.0001), tumor size (P = 0.0035), histological grade (P = 0.013), staining for c-erbB-2 (P = 0.0015), estrogen receptor (P = 0.028), and p53 (P = 0.032). Analysis of the association of patients with carcinomas staining for S100A4 and their survival in subgroups defined by these other tumor variables shows that in each subgroup, staining for S100A4 is associated with poorer survival. Patients whose tumors stain for S100A4 and possess involved lymph nodes (P < 0.0001), which are fixed to the chest wall (P = 0.015) or which stain for c erbB-2 (P = 0.050), show a significant reduction in survival times over those with only S100A4-staining tumors. Patients with involved lymph nodes, or staining for c-erbB-2 in the S100A4-negative group fail to show any significant reduction in survival times. Multivariate regression analysis for 137 patients shows that staining for S100A4 is most highly correlated with patient deaths (P < 0.0001), but involved lymph nodes (P = 0.001), fixed tumors (P = 0.0002), and high histological grade (P = 0.022) are also significant independent prognostic variables. These results suggest that in this group of patients, the metastasis inducing protein S100A4 is most tightly correlated with patient demise. PMID- 10749129 TI - Peroxisome proliferator-activated receptor gamma ligands inhibit estrogen biosynthesis in human breast adipose tissue: possible implications for breast cancer therapy. AB - Estrogen biosynthesis is catalyzed by aromatase cytochrome P-450 (the product of the CYP19 gene). Adipose tissue is the major site of estrogen biosynthesis in postmenopausal women, with the local production of estrogen in breast adipose tissue implicated in the development of breast cancer. In human adipose tissue, aromatase is primarily expressed in the mesenchymal stromal cells and is a marker of the undifferentiated preadipocyte phenotype. Aromatase expression in adipose tissue is regulated via the distal promoter I.4, under the control of glucocorticoids and class I cytokines such as oncostatin M, interleukin 6, and interleukin 11, as well as tumor necrosis factor alpha. These cytokines, which are expressed in adipose, also inhibit adipocyte differentiation. Therefore, we hypothesized that factors which stimulate adipocyte differentiation should inhibit aromatase expression. These factors include synthetic peroxisome proliferator-activated receptor gamma (PPARgamma) ligands such as thiazolidinediones, e.g., troglitazone and rosiglitazone (BRL49653) and the endogenous PPARgamma ligand 15-deoxy-delta12,14-prostaglandin J2. We have demonstrated by measurement of aromatase activity and by reverse transcription PCR/Southern blotting that these PPARgamma ligands inhibit aromatase expression in cultured breast adipose stromal cells stimulated with oncostatin M or tumor necrosis factor alpha plus dexamethasone in a concentration-dependent manner, whereas a metabolite of troglitazone that does not activate PPARgamma has no effect. We have also shown that troglitazone inhibits luciferase activity of reporter constructs containing various lengths of the upstream region of promoter I.4 transfected into mouse 3T3-L1 preadipocyte mesenchymal cells, whereas the troglitazone metabolite does not. Because local estrogen production in breast fat is implicated in breast cancer development in postmenopausal women, the actions of PPARgamma ligands suggest that they may have potential therapeutic benefit in the treatment and management of breast cancer. PMID- 10749130 TI - Normal bronchial epithelial cell expression of glutathione transferase P1, glutathione transferase M3, and glutathione peroxidase is low in subjects with bronchogenic carcinoma. AB - Normal bronchial epithelial cells (NBECs) are at risk for damage from inhaled and endogenous oxidative species and from epoxide metabolites of inhaled polycyclic aromatic hydrocarbons. Epidemiological and in vitro data suggest that interindividual variation in this risk may result from variation in NBEC expression of enzymes that inactivate reactive species by conjugating them to glutathione. Quantitative competitive reverse transcription-PCR was used to measure mRNA levels of glutathione transferases (GSTs) and glutathione peroxidases (GSHPxs) in primary NBECs from subjects with or without bronchogenic carcinoma. Mean expression levels (mRNA/10(3) beta-actin mRNA) in NBECs from 23 subjects without bronchogenic carcinoma compared to those from 11 subjects with bronchogenic carcinoma respectively (in parentheses) were: mGST (26.0, 6.11), GSTM3 (0.29, 0.09), combined GSTM1,2,4,5 (0.98, 0.60), GSTT1 (0.84, 0.76), GSTP1 (287, 110), GSHPx (140, 62.1), and GSHPxA (0.43, 0.34). Levels of GSTP1, GSTM3, and GSHPx were significantly (P < 0.05) lower in NBECs from subjects with bronchogenic carcinoma. Further, the gene expression index formed by multiplying the values for mGST x GSTM3 x GSHPx x GSHPxA x GSTP1 had a sensitivity (90%) and specificity (76%) for detecting NBECs from bronchogenic carcinoma subjects that was better than any individual gene. In cultured NBECs derived from eight individuals without bronchogenic carcinoma and incubated under identical conditions such that environmental effects were minimized, the mean level of expression and degree of interindividual variation for each gene evaluated was less than that observed in primary NBECs. Data from these studies support the hypotheses that (a) interindividual variation in risk for bronchogenic carcinoma results in part from interindividual variation in NBEC expression of antioxidant genes; (b) gene expression indices will better identify individuals at risk for bronchogenic carcinoma than individual gene expression values; and (c) both hereditary and environmental exposures contribute to the level of and interindividual variation in gene expression observed in primary NBECs. Many epidemiological studies have been designed to evaluate risk associated with polymorphisms or gene expression levels of putative susceptibility genes based on measurements in surrogate tissues, such as peripheral blood lymphocytes. Based on data presented here, it will be important to include the assessment of NBECs in future studies. Measurement of antioxidant gene expression in NBECs may identify the 5-10% of individuals at risk for bronchogenic carcinoma. Bronchoscopic sampling of NBECs from smokers and ex-smokers then will allow susceptible individuals to be entered into surveillance and/or chemoprevention studies. PMID- 10749131 TI - Chromosomal aberrations in lymphocytes predict human cancer independently of exposure to carcinogens. European Study Group on Cytogenetic Biomarkers and Health. AB - An increased risk of cancer in healthy individuals with high levels of chromosomal aberrations (CAs) in peripheral blood lymphocytes has been described in recent epidemiological studies. This association did not appear to be modified by sex, age, country, or time since CA test, whereas the role played by exposure to carcinogens is still uncertain because of the requisite information concerning occupation and lifestyle was lacking. We evaluated in the present study whether CAs predicted cancer because they were the result of past exposure to carcinogens or because they were an intermediate end point in the pathway leading to disease. A nested case-control study was performed on 93 incident cancer cases and 62 deceased cancer cases coming from two prospective cohort studies performed in Nordic countries (Denmark, Finland, Norway, and Sweden) and Italy. For each case, four controls matched by country, sex, year of birth, and year of CA test were randomly selected. Occupational exposure and smoking habit were assessed by a collaborative group of occupational hygienists. Logistic regression models indicated a statistically significant increase in risk for subjects with a high level of CAs compared to those with a low level in the Nordic cohort (odds ratio, 2.35; 95% confidence interval, 1.31-4.23) and in the Italian cohort (odds ratio, 2.66; 95% confidence interval, 1.26-5.62). These estimates were not affected by the inclusion of occupational exposure level and smoking habit in the regression model. The risk for high versus low levels of CAs was similar in subjects heavily exposed to carcinogens and in those who had never, to their knowledge, been exposed to any major carcinogenic agent during their lifetime, supporting the idea that chromosome damage itself is involved in the pathway to cancer. The results have important ramifications for the understanding of the role played by sporadic chromosome damage for the origin of neoplasia-associated CAs. PMID- 10749132 TI - Association of SRD5A2 genotype and pathological characteristics of prostate tumors. AB - The enzyme product of SRD5A2, 5alpha-reductase type II, is responsible for converting testosterone to the more metabolically active dihydrotestosterone. Therefore, SRDSA2 may be involved in the development or growth of prostate tumors. To examine the effects of allelic variants in the gene SRDSA2 on the presentation of prostate tumors, we studied a sample, primarily Caucasian, of 265 men with incident prostate cancer who were treated by radical prostatectomy. We assessed the relationship of the A49T and V89L polymorphisms at SRD5A2 with clinical and pathological tumor characteristics of these patients. We found no association of V89L genotypes with any of the characteristics studied. The presence of the A49T variant was associated with a greater frequency of extracapsular disease [odds ratio (OR), 3.16; 95% confidence interval (CI), 1.03 9.68] and a higher pathological tumor-lymph node-metastasis (pTNM) stage (OR, 3.11; 95% CI, 1.01-9.65). In addition, the A49T variant was overrepresented in two poor prognostic groups, which have been correlated with reduced rates of biochemical disease-free survival. One group included men with at least two of the following poor prognostic variables: (a) stage T3 tumor; (b) PSA level >10; and/or (c) Gleason score, 7-10 (OR, 3.46; 95% CI, 1.04-11.49). The second group included men with positive margins and high Gleason score (OR, 6.28; 95% CI, 1.05 37.73). Our results suggest that the A49T mutation may influence the pathological characteristics of prostate cancers and, thus, may affect the prognosis of these patients. PMID- 10749133 TI - Synergistic enhancement of herpes simplex virus thymidine kinase/ganciclovir mediated cytoxicity by hydroxyurea. AB - We have previously demonstrated (L. Z. Rubsam et al., Cancer Res., 59: 669-675, 1999) that low ganciclovir (GCV) triphosphate (TP) levels similar to cellular deoxynucleotide concentrations can induce multilog killing in cells stably expressing herpes simplex virus thymidine kinase (HSV-TK). In this study, we evaluated whether reducing the endogenous competitor of GCV-TP, dGTP, enhanced GCV-mediated cytotoxicity. In SW620 human colon carcinoma cells stably expressing HSV-TK, the addition of the ribonucleotide reductase inhibitor, hydroxyurea (HU), decreased cellular dGTP pools and simultaneously increased the accumulation of GCV-TP levels. The amount of GCV nucleotide transfer from HSV-TK-expressing to nonexpressing (bystander) cells was quantitated in physically separated pHook expressing bystander cells. Elevation of the GCV-TP:dGTP ratio by HU resulted in increased levels of GCV nucleotides transferred from HSV-TK-expressing to bystander cells during a 24 h drug incubation and enhanced GCV monophosphate incorporation into DNA after drug removal. Isobologram analysis demonstrated that the combination of GCV and HU was additive in 100% HSV-TK cultures and synergistic in HSV-TK/bystander mixtures. IC50 values for GCV in 1:1 cocultures of HSV-TK-expressing and nonexpressing SW620 cells were reduced from 1.5 microM to 0.07 microM with 2 mM HU. A similar reduction was also observed with HT-29 cells and U251 cells. With 2 mM HU, IC50 values for GCV in 10:90, 5:95, and 1:99 SW620 HSV-TK-expressing and nonexpressing cocultures were reduced from 55 microM to 0.3 microM, 71 microM to 0.8 microM, and 118 microM to 7 microM, respectively. These results demonstrate the ability to pharmacologically enhance HSV-TK/GCV mediated bystander killing and may have an important therapeutic impact. PMID- 10749134 TI - Photodynamic therapy-mediated oxidative stress as a molecular switch for the temporal expression of genes ligated to the human heat shock promoter. AB - Oxidative stress associated with photodynamic therapy (PDT) is a transcriptional inducer of genes encoding stress proteins, including those belonging to the heat shock protein (hsp) family. The efficiency of PDT to function as a molecular switch by initiating expression of heterologous genes ligated to the human hsp promoter was examined in the present study. Selective and temporal reporter gene expression was documented after PDT in mouse radiation-induced fibrosarcoma cells stably transfected with recombinant vectors containing an hsp promoter ligated to either the lac-z or CAT reporter genes and in transfected radiation-induced fibrosarcoma tumors grown in C3H mice. Hyperthermia treatments were included as a positive control for all experiments. Expression vectors containing either human p53 or tumor necrosis factor (TNF)-alpha cDNA under the control of an hsp promoter were also constructed and evaluated. A p53 null and TNF-alpha-resistant human ovarian carcinoma (SKOV-3) cell line was stably transfected with either the p53 or TNF-alpha constructs. Inducible expression and function of p53 as well as inducible expression, secretion, and biological activity of TNF-alpha were documented after PDT or hyperthermia in transfected SKOV cells. These results demonstrate that PDT-mediated oxidative stress can function as a molecular switch for the selective and temporal expression of heterologous genes in tumor cells containing expression vectors under the control of an hsp promoter. PMID- 10749135 TI - The role of Apaf-1, caspase-9, and bid proteins in etoposide- or paclitaxel induced mitochondrial events during apoptosis. AB - Ectopic overexpression of Apaf-1 (2.5-fold) in human acute myelogenous leukemia HL-60 cells (HL-60/Apaf-1 cells) induced apoptosis and sensitized HL-60/Apaf-1 cells to etoposide- and paclitaxel-induced apoptosis (C. Perkins et al., Cancer Res., 58: 4561-4566, 1998). In this report, we demonstrate that in HL-60/Apaf-1 cells, the activity of caspase-9 and -3 induced by Apaf-1 overexpression was associated with a significant increase (5-fold) in the cytosolic accumulation of cytochrome c (cyt c), loss of mitochondrial membrane potential (deltapsim), and an increase in the reactive oxygen species. These were also associated with the processing of procaspase-8 and Bid (cytosolic, proapoptotic BH3 domain containing protein). Transient transfection of Apaf-1 into the Apaf-1-containing mouse embryogenic fibroblasts (MEFs; Apaf-1+/- MEFs) or Apaf-1-/- MEFs also induced the processing of procaspase-9 and procaspase-8, Bid cleavage, and apoptosis. These events were secondary to the activity of the downstream caspases induced by Apaf 1. This conclusion is supported by the observation that in HL-60/Apaf-1 cells, ectopic expression of dominant negative caspase-9, its inhibitory short isoform caspase-9b, or XIAP or treatment with the caspase inhibitor zVAD (50 microM) inhibited Apaf-1-induced caspase-8 and Bid cleavage, mitochondrial deltapsim, release of cyt c, and apoptosis. In contrast, a transient transfection of dominant negative caspase-8 or CrmA or exposure to caspase-8 inhibitor zIETD-fmk inhibited the processing of procaspase-8 and Bid but did not inhibit the cytosolic accumulation of cyt c in either the untreated HL-60/Apaf-1 cells or the etoposide-treated HL-60/Apaf-1 and HL-60/neo cells. These results indicate that Apaf-1 overexpression lowers the apoptotic threshold by activating caspase-9 and caspase-3. This triggers the mitochondrial deltapsim and cyt c release into the cytosol through a predominant mechanism other than cleavage of caspase-8 and/or Bid. This mechanism may involve a cytosolic mitochondrial permeability transition factor, which may be processed and activated by the downstream effector caspases, thereby completing an amplifying feedback loop, which triggers the mitochondrial events during apoptosis. PMID- 10749136 TI - Heterogeneous expression of the SSX cancer/testis antigens in human melanoma lesions and cell lines. AB - The SSX genes, located on the X chromosome, encode a family of highly homologous nuclear proteins. The SSX1 and SSX2 genes were initially identified as fusion partners of the SYT gene in t(X;18)-positive synovial sarcomas. Recently, however, it was found that these two genes, as well as the highly homologous SSX4 and SSX5 genes, are aberrantly expressed in different types of cancers, including melanomas. Because normal SSX expression has been detected only in the testis and, at very low levels, the thyroid, these proteins are considered as new members of the still growing family of cancer/testis antigens. These antigens are presently considered as targets for the development of cancer immunotherapy protocols. In the present study, we developed a monoclonal antibody found to recognize SSX2, SSX3, and SSX4 proteins expressed in formaldehyde-fixed and paraffin-embedded tissues. This antibody was used to investigate SSX expression in normal testis and thyroid, benign melanocytic lesions, melanoma lesions, and melanoma cell lines. SSX nuclear expression in the testis was found to be restricted to spermatogenic cells, mainly spermatogonia. Of 18 melanoma cell lines analyzed, 9 showed SSX RNA and protein expression, although heterogeneously and at variable levels. Treatment of an SSX-negative cell line with 5-aza-2' deoxycytidine, a demethylating agent, led to SSX RNA and protein expression, indicating a role for methylation in transcription regulation. Thirty-four of 101 primary and metastatic melanoma cases and 2 of 24 common nevocellular and atypical nevus cases showed SSX nuclear staining. Again, SSX expression was heterogeneous, ranging from widespread to scarce. Our findings stress the importance of assessing the a priori SSX expression status of melanoma cases that may be selected for immunotherapeutic trials. PMID- 10749137 TI - Preclinical evaluation of "whole" cell vaccines for prophylaxis and therapy using a disabled infectious single cycle-herpes simplex virus vector to transduce cytokine genes. AB - The development of genetically modified "whole" tumor cell vaccines for cancer therapy relies on the efficient transduction and expression of genes by vectors. In the present study, we have used a disabled infectious single cycle-herpes simplex virus 2 (DISC-HSV-2) vector constructed to express cytokine or marker genes upon infection. DISC-HSV-2 is able to infect a wide range of tumor cells and efficiently express the beta-galactosidase reporter gene, granulocyte macrophage colony-stimulating factor (GM-CSF), or IL-2 genes. Gene expression occurred rapidly after infection of tumor cells, and the level of production of the gene product (beta-galactosidase, GM-CSF, or IL-2) was shown to be both time and dose-dependent. Vaccination with irradiated DISC-mGM-CSF or DISC-hIL-2 infected murine tumor cells resulted in greatly enhanced immunity to tumor challenge with live parental tumor cells compared with control vaccines. When used therapeutically to treat existing tumors, vaccination with irradiated DISC mGM-CSF-infected tumor cells significantly reduced the incidence and growth rates of tumors when administered locally adjacent to the tumor site, providing up to 90% protection. The prophylactic and therapeutic efficacy of DISC-mGM-CSF infected cells was shown initially using a murine renal cell carcinoma model (RENCA), and the results were confirmed in two additional murine tumor models: the M3 melanoma and 302R sarcoma. Therapy with DISC-infected RENCA "whole" cell vaccines failed to reduce the incidence or growth of tumor in congenitally T-cell deficient (Nu+/Nu+) mice or mice depleted of CD4+ and/or CD8+ T-lymphocytes, confirming that both T-helper and T-cytotoxic effector arms of the immune response are required to promote tumor rejection. These preclinical results suggest that this "novel" DISC-HSV vector may prove to be efficacious in developing genetically modified whole-cell vaccines for clinical use. PMID- 10749138 TI - The beta-catenin binding domain of adenomatous polyposis coli is sufficient for tumor suppression. AB - Inactivation of the adenomatous polyposis coli (APC) gene is a critical event in the development of human colorectal cancers. At the biochemical level, several functions have been assigned to the multidomain APC protein, but the cellular effects of APC expression and how they relate to its biochemical functions are less well defined. To address these issues, we generated a recombinant adenovirus (Ad-CBR) that constitutively expresses the central third of APC, which includes all of the known beta-catenin binding repeats. When expressed in colon cancer cells, Ad-CBR blocked the nuclear translocation of beta-catenin and inhibited beta-catenin/Tcf-4-mediated transactivation. Accordingly, expression of endogenous targets of the APC/beta-catenin/Tcf-4 pathway was down-regulated. Ad CBR infection of colorectal cancer cell lines with mutant APC but wild-type beta catenin resulted in substantial growth arrest followed by apoptosis. These effects were attenuated in lines with wild-type APC but with mutated beta catenin. These findings suggest that the beta-catenin-binding domain in the central third of APC is sufficient for its tumor suppressor activity. PMID- 10749139 TI - Identification of differentially expressed genes in human prostate cancer using subtraction and microarray. AB - We have identified human prostate cancer- and tissue-specific genes using cDNA library subtraction in conjunction with high throughput microarray screening. Subtracted cDNA libraries of prostate tumors and normal prostate tissue were generated. Characterization of subtracted libraries showed enrichment of both cancer- and tissue-specific genes. Highly redundant clones were eliminated by colony hybridization. The remaining clones were selected for microarray to determine gene expression levels in a variety of tumor and normal tissues. Clones showing overexpression in prostate tumors and/or normal prostate tissues were selected and sequenced. Here we report the identification of two genes, P503S and P504S, from subtracted libraries and a third gene, P510S, by subtraction followed by microarray screening. Their expression profiles were further confirmed by Northern blot, real-time PCR (TaqMan), and immunohistochemistry to be overexpressed in prostate tissues and/or prostate tumors. Full-length cDNA sequences were cloned, and their subcellular locations were predicted by a bioinformatic algorithm, PSORT, to be plasma membrane proteins. The genes identified through these approaches are potential candidates for cancer diagnosis and therapy. PMID- 10749140 TI - Chromosomal fragile site FRA16D and DNA instability in cancer. AB - It has been proposed that common aphidicolin-inducible fragile sites, in general, predispose to specific chromosomal breakage associated with deletion, amplification, and/or translocation in certain forms of cancer. Although this appears to be the case for the fragile site FRA3B and may be the case for FRA7G, it is not yet clear whether this association is a general property of this class of fragile site. The major aim of the present study was to determine whether the FRA16D chromosomal fragile site locus has a role to play in predisposing DNA sequences within and adjacent to the fragile site to DNA instability (such as deletion or translocation), which could lead to or be associated with neoplasia. We report the localization of FRA16D within a contig of cloned DNA and demonstrate that this fragile site coincides with a region of homozygous deletion in a gastric adenocarcinoma cell line and is bracketed by translocation breakpoints in multiple myeloma, as reported previously (Chesi, M., et al., Blood, 91: 4457-4463, 1998). Therefore, given similar findings at the FRA3B and FRA7G fragile sites, it is likely that common aphidicolin-inducible fragile sites exhibit the general property of localized DNA instability in cancer cells. PMID- 10749141 TI - A 700-kb physical map of a region of 16q23.2 homozygously deleted in multiple cancers and spanning the common fragile site FRA16D. AB - We have identified a >600-kb region at 16q23.2 that is homozygously deleted from malignant ovarian ascites using representational difference analysis. Overlapping homozygous deletions were also observed in the colon carcinoma cell line HCT116 and a xenograft established from the small cell lung cancer cell line WX330. This region coincides with that described previously by others as showing loss of heterozygosity in prostate and breast cancers (C. Li et al., Genes Chromosomes Cancer, 24: 175-182, 1999; A. Latil et al., Cancer Res., 57: 1058-1062, 1997; K. Driouch et al., Genes Chromosomes Cancer, 19: 185-191, 1997; A. Iida et al., Br. J. Cancer, 75: 264-267, 1997). In addition, the minimally deleted region spans the common fragile site FRA16D. We have constructed a 700-kb physical map encompassing the deleted region. By fluorescence in situ hybridization of aphidicolin-induced metaphase chromosomes, we have preliminary data to suggest that P1-derived bacterial artificial chromosome clones from the contig lie on both sides of FRA16D. This is confirmed by extensive fluorescence in situ hybridization analysis of the region reported in the accompanying article (M. Mangelsdorf et al., Cancer Res., 60: 1683-1689, 2000) and is consistent with an involvement of this common fragile site in the loss of 16q23.2 material in various cancer types. The minimally deleted region of approximately 210 kb has been characterized using our own markers and public domain markers. Eleven distinct expressed sequences mapped to the region, providing a basis for identifying the predicted tumor suppressor gene in this region. PMID- 10749142 TI - Somatic mutation rates and specificities at TC/AG and GT/CA microsatellite sequences in nontumorigenic human lymphoblastoid cells. AB - We have examined mutational events at TC/AG microsatellites, the second most abundant dinucleotide repetitive motif in the human genome. Mutational targets were constructed containing TC/AG alleles up to 20 units in-frame within the coding region of the herpes simplex virus thymidine kinase (HSV-tk) gene. These targets were incorporated into oriP shuttle vectors, which replicate episomally in human lymphoblastoid cells. The overall HSV-tk mutant frequencies measured after 10 population doublings in cells derived from a clinically normal donor were slightly increased over the background of mutations recovered in Escherichia coli. DNA sequence analyses revealed that replication of TC/AG vectors in human cells increased the mutation frequencies at the microsatellite motif up to 3 fold, relative to background. Additionally, the median HSV-tk mutation rate of single-cell clones carrying the [TC/AG]17 vector was significantly different from that of clones harboring the control vector. The median rate of allele length alterations within the [TC/AG]11 tract was 2 x 10(-6) mutations/cell generation, with an equivalent rate of deletion and expansion mutations. In contrast, a [GT/CA]10 vector showed no increase in microsatellite mutation frequency after replication in human cells, and mutation rates of clones carrying a [GT/CA]16 vector were not significantly different from controls. Intriguingly, replication in human cells of all microsatellite-containing vectors resulted in elevated mutation frequencies at the downstream HSV-tk coding sequence of up to 20-fold, an effect not observed for the control vector. These results demonstrate that the frequency of mutational events at TC/AG motifs is slightly greater than at GT/CA motifs of similar allele length. This is the first report to our knowledge of the mutation rates at TC/AG microsatellite alleles in eukaryotic or prokaryotic cells. PMID- 10749143 TI - DMBT1 encodes a protein involved in the immune defense and in epithelial differentiation and is highly unstable in cancer. AB - The gene deleted in malignant brain tumors 1 (DMBT1) has been proposed as a candidate tumor suppressor for brain, gastrointestinal, and lung cancer. It codes for a protein of unknown function belonging to the superfamily of scavenger receptor cysteine-rich proteins. We aimed at getting insights into the functions of DMBT1 by expression analyses and studies with a monoclonal antibody against the protein. The DMBT1 mRNA is expressed throughout the immune system, and Western blot studies demonstrated that isoforms of DMBT1 are identical to the collectin-binding protein gp-340, a glycoprotein that is involved in the respiratory immune defense. Immunohistochemical analyses revealed that DMBT1 is produced by both tumor-associated macrophages and tumor cells and that it is deregulated in glioblastoma multiforme in comparison to normal brain tissue. Our data further suggest that the proteins CRP-ductin and hensin, both of which have been implicated in epithelial differentiation, are the DMBT1 orthologs in mice and rabbits, respectively. These findings and the spatial and temporal distribution of DMBT1 in fetal and adult epithelia suggest that DMBT1 further plays a role in epithelial development. Rearrangements of DMBT1 were found in 16 of 18 tumor cell lines, and hemizygous deletions were observed in a subset of normal individuals, indicating that the alterations in tumors may be a result of both pre-existing deletions uncovered by a loss of heterozygosity and secondary changes acquired during tumorigenesis. Thus, DMBT1 is a gene that is highly unstable in cancer and encodes for a protein with at least two different functions, one in the immune defense and a second one in epithelial differentiation. PMID- 10749144 TI - A DNA damage signal is required for p53 to activate gadd45. AB - We provide direct evidence that overexpression of p53 is not sufficient for robust p53-dependent activation of the endogenous gadd45 gene. When p53 was induced in TR9-7 cells in the absence of DNA damage, waf1/p21 and mdm2 mRNA levels were increased, but a change in gadd45 mRNA was barely detectable. Activation of the gadd45 gene was observed when camptothecin was added to cells containing p53 in the absence of a further increase in the p53 level. Phosphorylation of p53 at serine 15 and acetylation at lysine 382 were detected after drug treatment. It has been suggested that p53 posttranslational modification is critical during activation. However, inhibition of these modifications by wortmannin was not sufficient to block the transactivation of gadd45. Interestingly, after camptothecin treatment, increased DNase I sensitivity was detected at the gadd45 promoter, suggesting that an undetermined DNA damage signal is involved in inducing chromatin remodeling at the gadd45 promoter while cooperating with p53 to activate gadd45 transcription. PMID- 10749145 TI - Both normal and transforming PCPH proteins have guanosine diphosphatase activity but only the oncoprotein cooperates with Ras in activating extracellular signal regulated kinase ERK1. AB - Previous reports from our laboratory described the activation of the PCPH gene into the PCPH oncogene (mt-PCPH, reported previously as Cph) by a single point mutational deletion. As a consequence, the mt-PCPH oncoprotein is a truncated form of the normal PCPH protein. Although both proteins have ribonucleotide diphosphate-binding activity, only mt-PCPH acted synergistically with a human H Ras oncoprotein to transform murine NIH3T3 fibroblasts. We report here the expression of the PCPH and mt-PCPH proteins in Escherichia coli and the finding that the purified bacterial recombinant proteins have intrinsic guanosine diphosphatase (GDPase) activity. However, expression of the Syrian hamster PCPH and mt-PCPH proteins in haploid yeast strains engineered to be GDPase deficient by targeted disruption of the single GDA1 allele did not complement their glycosylation-disabled phenotype, suggesting the existence of significant functional differences between the mammalian and yeast enzymes. Results from transient cotransfections into NIH3T3, COS-7, or 293T cells indicated that, in mammalian cells, both PCPH and mt-PCPH cause an overall down-regulation of the stimulatory effect of epidermal growth factor or the activated ras or raf oncogenes on the Ras/mitogen-activated protein kinase/extracellular signal regulated kinase (ERK) signaling pathway. However, despite this overall negative regulatory role on Ras signaling, mt-PCPH, but not PCPH, cooperated with the Ras oncoprotein to produce a prolonged stimulation of the phosphorylation of ERK1 but had no effect on the phosphorylation levels of ERK2. These results represent a clear difference between the mechanisms of action of PCPH and mt-PCPH and suggest that the ability to cause a sustained activation of ERK1 may be an important determinant of the transforming activity of mt-PCPH. PMID- 10749147 TI - TSG101 protein steady-state level is regulated posttranslationally by an evolutionarily conserved COOH-terminal sequence. AB - Antisense inactivation of the tsg101 tumor susceptibility gene in murine NIH3T3 fibroblasts leads to neoplastic transformation and tumorigenesis, which are reversed by restoration of tsg101 activity. tsg101 deficiency is associated with a series of mitosis-related abnormalities, whereas overexpression of TSG101 can also result in neoplastic transformation and the perturbation of cell cycling. Together, these observations imply that TSG101 production outside of a narrow range can lead to abnormal cell growth. We report here that the TSG101 protein is maintained at an almost constant steady-state level in cultured murine and human cells and that this occurs through a posttranslational process involving TSG101 protein degradation. Sustained overproduction of TSG101 from chromosomally inserted adventitious constructs resulted in compensatory down-regulation of endogenous TSG101 and replacement of the native protein by the adventitious one. Using deletion mutants of TSG101, we mapped the region responsible for autoregulation of the TSG101 steady-state level to an evolutionarily conserved sequence, here termed the "steadiness box," located near TSG101's COOH-terminal end. Our results suggest a model in which the biological effects of TSG101 are modulated either by self-promoted proteolysis or participation with other cellular protein(s) in a proteolytic feedback-control loop. PMID- 10749146 TI - Respective roles of cyclobutane pyrimidine dimers, (6-4)photoproducts, and minor photoproducts in ultraviolet mutagenesis of repair-deficient xeroderma pigmentosum A cells. AB - The role of UV light-induced photoproducts in initiating base substitution mutation in human cells was examined by determining the frequency and spectrum of mutation in a supF tRNA gene in a shuttle vector plasmid transfected into DNA repair deficient cells (xeroderma pigmentosum complementation group A). To compare the role of two major UV-induced photoproducts, cis-syn cyclobutane-type pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), each photoproduct was removed from UV-irradiated plasmid by photoreactivation before transfection. Removal of either CPDs or 6-4PPs by in vitro photoreactivation reduced the mutation frequency while keeping the mutation distribution and the predominance of G:C-A:T transitions as UV-irradiated plasmid without photoreactivation, indicating that both cytosine-containing CPDs and 6 4PPs were premutagenic lesions for G:C-A:T transitions. On the other hand, A:T G:C transitions were not recovered from plasmids after the removal of 6-4PPs, whereas this type of mutation occurred at a significant level (11%) after the removal of CPDs. Thus, the premutagenic lesions for the A:T-G:C transition are 6 4PPs. Removal of both CPDs and 6-4PPs resulted in the disappearance of mutational hot spots and random distribution of mutation as observed in unirradiated control plasmids. However, the mutational spectrum of photoreactivated plasmids differed significantly from that of unirradiated plasmids. A characteristic feature is a high portion of A:T-T:A transversions (11%) in the photoreactivated plasmid. This mutation is due to nondipyrimidinic "minor" photoproducts, and the mutation spectrum suggests that TA*, the major photoproduct of thymidylyl-(3'-5') deoxyadenosine, is the premutagenic lesion for this mutation. This is the first report revealing the distinct mutagenic roles of the major UV photoproducts and "minor" photoproducts by the use of (6-4)photolyase. PMID- 10749148 TI - Effect of cyclooxygenase and nitric oxide synthase inhibitors on tumor growth in mouse tumor models with and without cancer cachexia related to prostanoids. AB - The potential interaction between cyclooxygenase (Cox) and NO metabolic pathways in the control of local tumor growth was evaluated. Mice bearing either a sarcoma derived tumor (C57B1; MCG 101) or a malignant melanoma (C3H/HeN; K1735-M2) were used. These models were principally different because they demonstrate, in tumor hosts, conditions with and without cancer cachexia, seemingly related to high and low production of prostanoids, respectively. Cox inhibitors (Cox-1 and Cox-2) decreased tumor growth by 35-40% in MCG 101-bearing mice but had no such effect on melanoma-bearing mice, despite the expression of the Cox-2 protein in melanoma cells. Indomethacin reduced prostanoid production in both tumor (MCG 101) and host tissues and reduced tumor cell proliferation, mainly in vivo. Nitric oxide synthase (NOS) inhibitors (N(omega)-nitro-L-arginine methyl ester and N(omega) nitro-L-arginine) reduced tumor growth in vivo by approximately 50% in both tumor models. Tumor growth reduction, related to NOS inhibition, was unrelated to prostanoid production and was an in vivo phenomenon in both tumor models. Specific inhibitors of inducible NOS activity, unexpectedly, had no effect in any tumor model, although inducible NOS protein was present in tumor tissues in large amounts. A combination of Cox and NOS inhibitors had no additive effect on tumor growth (MCG 101). Cox inhibition increased tumor tissue (MCG 101) expression of cNOS mRNA but had no significant effect on tumor tissue expression of the transferrin receptor, vascular endothelial growth factor, or basic fibroblast growth factor. NOS inhibition increased tumor tissue content of cNOS mRNA but showed as well a trend to increase mRNA content of the transferrin receptor and vascular endothelial growth factor. Our results suggest that NOS inhibitors can decrease the local growth of tumors that are either responsive or unresponsive to Cox inhibition. This effect may reflect cross-talk between Cox and NOS pathways within or among tumor cells, or it may represent unrelated effects on tumor and host cells. Whether NO inhibition may be used therapeutically in clinical tumors that are unresponsive to eicosanoid intervention remains to be evaluated. PMID- 10749149 TI - Frequency and prognostic impact of microsatellite instability in a large population-based study of endometrial carcinomas. AB - The replication error repair (RER) phenotype has been reported in 9-43% of sporadic endometrial carcinomas, but there are conflicting data about its effect on prognosis in this disease. This study was performed to establish the frequency of the RER phenotype and to determine its effect on prognosis in a population based series of 259 endometrial carcinomas with long-term follow-up. Five mononucleotide and dinucleotide microsatellite markers on different chromosomes were analyzed, and tumors exhibiting microsatellite instability at two or more loci were classified as RER+. A total of 116 of 259 tumors (45%) were RER+. The 5 year survival rate for the RER- group was 76.2% compared with 79.6% for RER+ cases (P = 0.6). The 5-year recurrence-free survival rate among the 228 patients surgically treated for cure was 80.6% in the RER- group compared with 83.6% in the RER+ group (P = 0.6). The analysis indicates that the RER phenotype is common in endometrial carcinomas, but there is no association with prognosis in this large population-based series of endometrial carcinomas. PMID- 10749150 TI - Role of mitogen-activated protein kinases in the induction of parathyroid hormone related peptide. AB - Tumor production of parathyroid hormone-related protein (PTHRP) is responsible for most cases of hypercalcemia of malignancy. The transplantable rat Leydig tumor H-500 is known to cause hypercalcemia in rats by the release of abundant PTHRP and to closely reproduce the human syndrome. We have demonstrated recently that Ras oncogene can stimulate PTHRP gene expression in Fr3T3 fibroblasts in vitro and cause hypercalcemia in vivo. Using rat Leydig tumor H-500 cells, we have investigated the role of effector pathways downstream of Ras in serum induced PTHRP expression. The Ras inhibitors B-1086 and Lovastatin decreased PTHRP mRNA expression. i.p. administration of B-1086 (50-100 mg/kg/day) into H 500 tumor-bearing male Fischer rats resulted in a dose-dependent reduction in tumor volume, serum calcium, plasma PTHRP, and tumoral PTHRP mRNA expression. Transient transfection of dominant-negative Ras (Ras N17) and Raf (Raf C4B) reduced, whereas activated Raf-1 (Raf BXB) increased, basal expression of PTHRP in H-500 cells. A similar decrease in PTHRP production was seen with a mitogen activated protein kinase kinase (MEK) inhibitor (PD 098059), implicating the involvement of Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway. In addition, stimulation with UV light, which can activate c-Jun NH2-terminal kinase (JNK), or expression of an activated form of Rac (Rac V12) was sufficient to increase PTHRP mRNA. Moreover, a dominant-negative Rac (Rac N17) blocked serum induced PTHRP gene expression. Collectively, these results demonstrate that PTHRP is induced via both Raf-ERK and Rac-JNK mediated pathways, effects which can be blocked by chemical inhibitors and dominant-negative mutants of these pathways in vitro and in vivo. Availability of selective inhibitors of Ras signaling molecules may therefore add to our existing armamentarium to control hypercalcemia of malignancy. PMID- 10749151 TI - Continuous administration of angiostatin inhibits accelerated growth of colorectal liver metastases after partial hepatectomy. AB - Human plasminogen-derived angiostatin is one of the most potent antiangiogenic agents currently known. However, it is unclear whether angiostatin is also effective against accelerated tumor growth induced by local up-regulation of growth factors, including angiogenesis stimulators, such as in regenerating liver. Prior to addressing this question, we tested, in mice, whether continuous administration of angiostatin could improve its biological effects. This assumption was based on the relatively short half-life of angiostatin in mice, as well as on the theoretical necessity to suppress tumor-induced angiogenesis continually. The findings presented here clearly indicate continuous administration to be superior to the conventional twice-daily bolus injections. Using the maximally effective regimen of 100 mg/kg/day via s.c. pump infusion, we found angiostatin to not only suppress s.c. primary tumors but also to significantly inhibit the outgrowth of colorectal hepatic metastases in resting liver and even to inhibit accelerated tumor growth in regenerating liver after 70% partial hepatectomy. In conclusion, angiostatin could play an important role in patients subjected to partial hepatectomy to prevent outgrowth of residual micrometastases, provided it is administered continuously to obtain maximal biological effects. PMID- 10749152 TI - Fas drives cell cycle progression in glioma cells via extracellular signal regulated kinase activation. AB - Recent studies have revealed that a variety of malignant tumors express Fas and/or its ligand FasL. However, tumor cells expressing Fas are not always susceptible to Fas-mediated cell death, and the biological significance of simultaneous expression of Fas and FasL in the same tumor is not known. In the present study, we addressed this question in three glioma cells lines, A-172, T98G, and YKG-1, which express both Fas and FasL endogenously and their Fas transfectants. We report here that: (a) in gliomas, [3H]TdR incorporation was enhanced by anti-Fas IgM monoclonal antibody CH-11 and conversely inhibited by anti-FasL monoclonal antibody NOK-2; (b) cross-linking of Fas with CH-11 drove both cell cycle progression and apoptosis as demonstrated by the induction of the S-G2 phase of DNA and RNA and fragmented nuclei; (c) phosphorylation of extracellular signal-regulated kinase (ERK), but not of c-Jun NH2-terminal kinase or p38, was induced by cross-linking of Fas; (d) a mitogen-activated protein kinase/ERK kinase 1 (MEK1) inhibitor PD98059 completely blocked CH-11-induced ERK phosphorylation as well as cell cycle progression without affecting induction of apoptosis; and (e) a broad-spectrum caspase inhibitor Z-Asp-CH2-DCB inhibited CH 11-induced ERK phosphorylation, cell cycle progression, and apoptosis. These results indicate that Fas-mediated caspase activation elicits two independent cellular responses; one is to induce apoptosis and another is to promote cell cycle progression; the latter is closely linked to the MEK-ERK pathway. Together, our data strongly suggest that FasL may play a role as an autocrine growth factor in gliomas. PMID- 10749153 TI - The surgical treatment of unilateral severe congenital blepharoptosis: the controversy continues. PMID- 10749155 TI - The effect of upper blepharoplasty on eyelid position when performed concomitantly with Muller muscle-conjunctival resection. AB - PURPOSE: To determine the effect on eyelid elevation of excising excess skin, orbicularis oculi muscle, and herniated orbital fat and reconstructing the upper eyelid crease (blepharoplasty) concomitant with a Muller muscle-conjunctival resection. METHODS: The charts of 202 patients who had undergone Muller muscle conjunctival resection during an 8-year interval were reviewed. Three hundred forty-five eyelids were divided into two groups. Group 1 (n = 162) underwent a Muller muscle-conjunctival resection only, and group 2 (n = 183) had this procedure combined with excision of skin, orbicularis muscle, and herniated orbital fat with upper eyelid crease reconstruction. Each group was divided into three subgroups based on the amount of Muller muscle-conjunctival resection. Subgroup A had resection less than 7.75 mm; subgroup B, resection of 7.75 to 8.75 mm; and subgroup C, resection greater than 8.75 mm. The change in margin reflex distance-1 (MRD1) measurements of the upper eyelid levels (postoperative MRD1 minus preoperative MRD1) were calculated and compared between groups. RESULTS: The mean (+/- standard deviation) change in MRD1 was, respectively, 2.3 +/-1.0 mm and 1.9+/-1.0 mm for groups 1A and 2A; 3.1+/-1.3 mm and 2.1+/-1.2 mm for groups 1B and 2B; and 3.4+/-1.2 mm and 2.8+/-1.3 for groups 1C and 2C. CONCLUSIONS: Blepharoplasty performed concomitant with a Muller muscle-conjunctival resection reduced the anticipated postoperative eyelid elevation by as much as 1 mm. Surgeons who perform these procedures together should be aware that a larger Muller muscle-conjunctival resection may be required to obtain the desired increase in eyelid height postoperatively. PMID- 10749154 TI - Frontalis muscle flap advancement for correction of blepharoptosis. AB - PURPOSE: To describe a technique of frontalis muscle flap advancement to repair myogenic ptosis in lieu of a graft or suture material. METHODS: Ten ptotic eyelids in eight patients were repaired using the frontalis flap technique. Patients were selected at random by two separate surgeons; all patients had eyelid excursion measured as poor (or less than 6 mm). RESULTS: Nine of 10 ptotic eyelids were adequately corrected by the frontalis flap technique, with follow-up intervals ranging from 18 to 42 months. Adequate correction was defined as ptosis corrected within 1 mm of the fellow eyelid. Complications of frontalis advancement were few and primarily transient. CONCLUSIONS: Frontalis flap advancement is a technically simple, safe, and effective technique for the repair of myogenic ptosis. The primary advantage of frontalis muscle flap advancement over a graft or suture material that it elevates the eyelid directly by moving the insertion of the frontalis muscle into the eyelid, rather than by graft or suture material. PMID- 10749156 TI - The effects of tetanus toxin on the orbicularis oculi muscle. AB - PURPOSE: Tetanus toxin can cause localized neuromuscular weakness, but it also can produce systemic tetany. The action of tetanus toxin on the orbicularis muscle has not been studied in animals immunized to prevent systemic tetany. Our objective was to determine whether tetanus toxin could be used to treat orbicularis oculi muscle spasms. METHODS: We analyzed the clinical, electrophysiologic, and histopathologic effects of tetanus toxin injected into the orbicularis oculi muscle of rabbits with passive immunity to tetanus toxin. In six rabbits, the orbicularis oculi function in both eyes was assessed clinically, and the baseline orbicularis oculi muscle action potential was measured physiologically with electromyography (EMG). The rabbits then were immunized against tetanus toxin with tetanus immunoglobulin for immediate and definitive immunity. Tetanus toxin was injected into the left orbicularis oculi muscles, leaving the right eyes as controls. Ten days later, the rabbits were again assessed by clinical examination and with EMGs on both the injected side and the noninjected side. The animals were killed at 14 days, and the orbicularis muscle was removed from both sides. The injected and control tissues were examined microscopically for signs of neuromuscular denervation. RESULTS: All six rabbits showed weakness in eye closure on the side injected with tetanus toxin. In addition, four rabbits developed complete ear ptosis on the tetanus toxin injected side because of spread of the toxin to adjacent ear muscles. EMGs showed both a denervation of the orbicularis oculi muscle and a poor blink potential on the side injected with tetanus toxin. Histopathologic studies of the orbicularis oculi muscle injected with tetanus toxin showed angulation of both slow and fast types of muscle fibers compatible with neuromuscular denervation. CONCLUSIONS: Tetanus toxin can cause localized orbicularis oculi weakness, as documented clinically, physiologically, and microscopically, without producing systemic tetany in immunized rabbits. Tetanus toxin may have a potential application in the treatment of blepharospasm and hemifacial spasm. PMID- 10749157 TI - The safety and efficacy of mitomycin C in endonasal endoscopic laser-assisted dacryocystorhinostomy. AB - PURPOSE: The adjunctive use of mitomycin C (MMC) in glaucoma and pterygium surgery has been widely published, but there has not been any large long-term study of its use in endonasal endoscopic laser-assisted dacryocystorhinostomy (ELA-DCR). The purpose of this study was to examine the safety and efficacy of the adjunctive use of MMC in this procedure. METHODS: A nonrandomized, retrospective, single-masked, interventional case-controlled study was performed wherein 123 consecutive procedures using MMC in ELA-DCR procedures, using the Holmium:YAG laser, were compared with a historical control group consisting of 48 consecutive procedures in which MMC was not used. One surgeon performed all procedures. The two groups were compared with regard to complications as well as success rate. The main outcome measures for success were the resolution of epiphora and patency with lacrimal irrigation. Success rates were analyzed using the Fisher's exact test. The main outcome determinants for complications were the presence or absence of delayed wound healing, wound necrosis, infection, or excessive bleeding. Postoperative follow-up interval ranged from 30 months to 72 months. RESULTS: There were no complications in the group receiving MMC. One case of turbino-septal synechia formation occurred in the non-MMC group. The success rate of the MMC group was 99.2% compared with 89.6% in the control group. This difference (9.6%) was statistically significant using Fisher's exact test (p = 0.007). CONCLUSION: This study supports the safety and efficacy of the intraoperative use of MMC in endonasal ELA-DCR. PMID- 10749158 TI - Effect of brief exposure to mitomycin C on cultured human nasal mucosa fibroblasts. AB - PURPOSE: To observe the effect of mitomycin C (MMC) on cultured human nasal mucosa fibroblasts. METHODS: Cultured human nasal mucosa fibroblasts were exposed to MMC (0.1-0.4 mg/ml) for 1 to 5 minutes. The viability of the fibroblasts was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay; DNA fragmentation (apoptosis) by terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL); apoptotic percentage by flow cytometry; and morphology by light microscopy. RESULTS: A portion of the fibroblasts survived the mitomycin treatment and showed evidence of regrowth within 2 to 3 days. These cells reached confluence in 5 to 7 days. The inhibition rates by MTT assay of 0.4 mg/ml MMC for 5-minute exposures was 31.3%. Dose-response effect was noted with the lower concentrations and shorter exposure times where the inhibition rates were lower (but not significantly so). DNA fragmentation was observed in fibroblasts 24 hours after MMC exposure (0.4 mg/ml) for 5 minutes compared with normal control. The apoptotic rate of fibroblasts treated by 0.4 mg/ml MMC was significantly higher than the control (p < 0.05). CONCLUSIONS: Short MMC exposure times have a variable cytotoxic effect and inhibit proliferation of cultured nasal mucosa fibroblasts. MMC also can induce apoptosis with a 5-minute exposure time. Therefore, it is possible that MMC has a complex effect in dacryocystorhinostomy. PMID- 10749159 TI - Anterior tarsal V-wedge resection for cicatricial entropion. AB - PURPOSE: To determine the effectiveness of using a radiofrequency instrument to resect a V-shaped tarsal wedge for the correction of cicatricial upper eyelid entropion. METHODS: Prospective evaluation of 16 upper eyelids of 11 consecutive patients. RESULTS: The operation was successful in all 16 eyelids, with excellent cosmetic and functional outcomes. CONCLUSIONS: Tarsal V-wedge resection is an effective alternative to other complicated techniques in the management of upper eyelid cicatricial entropion. PMID- 10749160 TI - Causes of involutional ectropion and entropion--age-related tarsal changes are the key. AB - PURPOSE: To measure tarsal plates across various age-groups, to determine whether tarsal size changes with increasing age and whether size correlates with involutional ectropion and entropion. METHODS: Comparative, observational, case control study design. Data were obtained for length and height of tarsus in each of the four eyelids. The data were constructed to determine: (I) right-to-left side comparison data, (II) sex difference data, (III) age normal data, (IV) involutional entropion data, (V) involutional ectropion data. RESULTS: (I) There is no difference in tarsal dimensions between right and left sides; (II) males have larger tarsal dimensions than females; (III) tarsal plates are on average smaller in older age ranges; (IV) patients with entropion have smaller than average age-normal tarsal plates; (V) patients with ectropion have larger than average age-normal tarsal plates. CONCLUSIONS: (I) Right and left tarsal plates have equal dimensions, and involutional changes likely occur on both right and left sides equally frequently; (II) males have larger tarsal plates than females and entropion is more frequent in females and ectropion in males; (III) tarsal plates may have a general tendency to atrophy or shrink with age; this may explain why some eyelids develop entropion and others ectropion; (IV) entropion results from the mechanical effect of an atrophied or smaller than age-normal, partially or fully disinserted, tarsal plate being overcome by the normal or increased tone of the preseptal/pretarsal orbicularis muscle; (V) ectropion results from an age-normal or larger than normal tarsal plate mechanically overcoming the normal or decreased tone of the preseptal/pretarsal orbicularis muscle in combination with medial/lateral canthal tendon laxity. PMID- 10749161 TI - Direct internal eyelash bulb extirpation for trichiasis. AB - PURPOSE: To evaluate the success of internal eyelash bulb extirpation in the management of trichiasis. METHODS: The procedure was performed on 26 consecutive eyelids. RESULTS: Our technique was successful in all cases, with no recurrence of signs or symptoms. CONCLUSIONS: Internal eyelash bulb extirpation is a safe and effective technique to remove trichiatic lashes, while avoiding injury to the conjunctiva. PMID- 10749162 TI - Porous polyethylene as a spacer graft in the treatment of lower eyelid retraction. AB - PURPOSE: An experimental study was performed to: 1) assess the tolerance and incorporation of porous polyethylene (Medpor) in the posterior lamella of the rabbit lower eyelid; 2) analyze the effect of implant thickness on incorporation; 3) investigate the ability of conjunctiva to grow over vascularized Medpor and; 4) determine the effects of Medpor surface modification on biocompatibility and fibrovascularization. METHODS: In phase I, 10 rabbit eyelids were operated on to analyze the effects of implant thickness and to develop the surgical technique used in phase II of the study. In phase II, 20 lower eyelids of 10 rabbits received 0.85-mm-thick Medpor grafts, each rabbit receiving both an uncoated implant and one coated with an immobilized collagen. RESULTS: There were no extrusions in phase II. with a postoperative follow-up from 14 to 17 weeks. Fourteen of 20 eyelids had full-thickness conjunctival incisions or excisions placed over the Medpor implant to determine the growth potential of conjunctiva over a vascularized implant. All but one eyelid showed complete defect coverage, occurring in as little as 3 days. Histopathology indicated complete Medpor fibrovascularization as early as 4 weeks after implantation. Because neither coated nor uncoated implants extruded in phase II, no conclusions can be drawn regarding the efficacy of Medpor surface modification. CONCLUSION: Medpor was well tolerated in this soft tissue application, and it offers advantages over other graft materials. PMID- 10749163 TI - A comparative study of bovine pericardium (periguard) and homologous sclera as lower eyelid spacer graft analogs in New Zealand white rabbits. AB - PURPOSE: To compare bovine pericardium (Periguard; Bio-Vascular, Inc., St. Paul, MN, U.S.A.) and homologous sclera as spacer graft analogs in a rabbit model. METHODS: A nonrandomized experimental animal study was conducted. Elliptical subconjunctival implants were placed in one lower eyelid each of 10 rabbits. Five rabbits received homologous sclera implants, and five received bovine pericardium implants. The rabbits were killed 7 weeks later, and the lower eyelids were examined clinically and histopathologically. RESULTS: Rabbit eyelids implanted with Periguard had a more marked inflammatory reaction and more significant collagen lamellar disruption than eyelids implanted with homologous sclera, although clinically they appeared quite similar. CONCLUSIONS: Although bovine pericardium elicited a more intense inflammatory response at the histopathologic level, both implants were similarly tolerated at the clinical level. These findings are consistent with animal and human investigations of bovine pericardium in other parts of the body, and warrant further studies into its potential use in the human eyelid. PMID- 10749165 TI - Orbital decompression. PMID- 10749164 TI - Mucoepidermoid carcinoma of an accessory lacrimal gland with orbital invasion. AB - PURPOSE: To report a case of mucoepidermoid carcinoma of an accessory lacrimal gland with orbital invasion. METHODS: The clinical history and pathologic findings of a patient with a left upper eyelid lesion were reviewed. RESULTS: The patient was evaluated and found to have an epithelial tumor arising in an accessory lacrimal gland. Special stains showed mucin production by individual tumor cells. The tumor was classified as mucoepidermoid carcinoma. CONCLUSIONS: Mucoepidermoid carcinoma may arise in accessory lacrimal glands and invade the orbit. PMID- 10749166 TI - Cloning, sequencing, and expression of three Bartonella henselae genes homologous to the Agrobacterium tumefaciens VirB region. AB - A 17-kDa, immunodominant antigen of Bartonella henselae Houston-1 has previously been cloned, sequenced, and characterized. This clone (H13) contains the 17-kDa antigen gene plus a partial open reading frame, designated ORF1, which is 459 nucleotides long and is directly upstream of the 17-kDa gene. Comparison of the deduced partial amino acid sequence of ORF1 with that of other known genes in GenBank revealed significant identity with several other bacterial virulence genes, including VirB4 of the Agrobacterium tumefaciens virB operon (56/149 amino acids). An overlapping clone, pGB3, was recovered and shown to contain a 3.0-kb region upstream of the 17-kDa gene. Sequence analysis revealed three ORFs upstream of the gene. The deduced amino acid sequence of each ORF was compared with sequences in GenBank, and identity was found with VirB2, VirB3, and VirB4 of A. tumefaciens. In vitro transcription/translation and SDS-PAGE demonstrated that three proteins of 9 kDa, 10 kDa, and 92 kDa, corresponding to the predicted molecular weight of 10.9 kDa, 11.7 kDa, and 89.9 kDa of VirB2, VirB3, and VirB4, respectively, could be expressed from these coding regions. These results indicate that virulence-associated genes and their overall chromosomal arrangement are relatively well conserved between B. henselae and other gram negative bacteria such as A. tumefaciens. PMID- 10749167 TI - Genomic structure and characterization of the Drosophila S3 ribosomal/DNA repair gene and mutant alleles. AB - The Drosophila S3 protein is known to be associated with ribosomes, where it is thought to play a role in the initiation of protein translation. The S3 protein also contains a DNA repair activity, efficiently processing 8-oxoguanine residues in DNA via an N-glycosylase/apurinic-apyrimidinic (AP) lyase activity. The gene that encodes S3 has previously been localized to one of the Minute loci on chromosome 3 in Drosophila. This study focused on the genomic organization of S3 at M(3)95A, initial promoter characterization, and analysis of three mutant alleles at this locus. The S3 gene was found to be a single-copy gene 2 to 3 kb in length and containing a single intron. The upstream 1.6-kb region was analyzed for promoter activity, identifying a presumptive regulatory domain containing potential enhancer and suppressor elements. This finding is of interest, as the S3 gene is constitutively expressed throughout development and mRNA is most likely maternally inherited. Lastly, three Minute alleles from the same locus were sequenced and two alleles found to contain a 22-bp deletion in exon 2, resulting in a truncated S3 protein, although wildtype levels of S3 mRNA and protein were detected in the viable heterozygous Minute alleles, possibly reflecting dosage compensation. PMID- 10749168 TI - Basic mechanisms of DNA-raised antibody responses to intramuscular and gene gun immunizations. AB - DNA-raised antibody (Ab) responses have been compared for the dependence on CD4+ and CD8+ cells, the longevity of functional antigen (Ag) expression, and the nature of the Ag-presenting cell after intramuscular (i.m.) and gene gun inoculations. A plasmid expressing the hemagglutinin (HA) glycoprotein of influenza virus was used for immunizations of BALB/c mice. Intramuscular and gene gun-raised Abs had similar dependencies on CD4+ and CD8+ cells but different temporal patterns of functional Ag expression. The two methods of DNA immunization also appeared to have different frequencies or types of Ag presenting cells in the draining lymph nodes and spleen. For both methods of DNA delivery, Ab was independent of CD8+ cells but dependent on CD4+ cells. The CD4 dependence occurred at priming but not booster immunizations and resulted in a 1 month delay in the Ab response. Temporal T-cell transfers from TCR+/+ mice into immunized TCR-/- mice revealed the persistence of DNA-expressed Ag for up to 1 month after both i.m. and gene gun inoculations. For gene gun, but not i.m. immunizations, approximately 90% of the functional Ag expression was lost by 1 week, consistent with the sloughing of the epidermal target site. Despite similar titers of raised Ab, Ag-presenting dendritic cells could be detected in the draining lymph nodes and spleen of gene gun- but not i.m. DNA-immunized mice. In the gene gun-immunized mice, Ag-presenting dendritic cells appeared in the draining lymph nodes before the spleen. PMID- 10749169 TI - Sp family of transcription factors is involved in iron-induced collagen alpha1(I) gene expression. AB - The purpose of this study was to identify the cis-acting elements and the trans acting factors involved in the iron-induced expression of the collagen alpha1(I) (COL1aI) gene. Rat hepatic stellate cells were cultured in the presence of 50 microM ferric chloride, 50 microM ascorbic acid, and 250 microM citric acid (Fe/AA/CA), and the effects on collagen gene expression and the binding of nuclear proteins to the COL1aI promoter were measured. The Fe/AA/CA treatment induced a time- and dose-dependent increase in the cellular levels of COL1aI mRNA that was abrogate by pretreating cells with cycloheximide, antioxidants, and inhibitors of aldehyde-protein adduct formation. Transient transfection experiments showed that Fe/AA/CA exerted its effect through regulatory elements located between -220 and -110 bp of the COL1aI promoter. Gel retardation assays showed that Fe/AA/CA increased the binding of nuclear proteins to two elements located between -161 and -110 bp of the COL1aI promoter. These bindings were blocked by unlabeled consensus Sp1 oligonucleotide and supershifted with Sp1 and Sp3 antibodies. Finally, Fe/AA/CA increased cellular levels of the Sp1 and Sp3 proteins and Sp1 mRNA. Treatment with Fe/AA/CA stimulates COL1aI gene expression by inducing the synthesis of Sp1 and Sp3 and their binding to two regulatory elements located between -161 and -110 bp of the COL1aI promoter. PMID- 10749170 TI - Vpr-GFP virion particle identifies HIV-infected targets and preserves HIV-1Vpr function in macrophages and T-cells. AB - Human immunodeficiency virus type 1 (HIV-1) is known for its ability to infect immune cells, including T-cells and macrophages. The 96-amino acid Vpr, a virion associated protein, is essential for viral replication in monocytes/macrophages and increases viral replication in primary and established T-cell lines. The Vpr protein regulates a number of host cellular events, including proliferation, differentiation, apoptosis, cytokine production, and NF-kappaB-mediated transcription. Most of these functions have been analyzed using either endogenous Vpr protein or cells transfected with a Vpr expression plasmid. We developed a lentiviral vector complemented with a Vpr expression plasmid that results in viral particles packaged with Vpr protein. To facilitate identification of the target cells infected with the particles containing Vpr, we fused green fluorescent protein (GFP) with the Vpr open reading frame and analyzed the biology of this novel particle. Vpr itself is expressed as a 14-kDa protein; however, in vitro translation of the pVpr-GFP plasmid resulted in the expression of 39-kDa fusion protein. The fusion molecule exhibited the same activity in arresting the cell cycle in G2 as does the wildtype Vpr molecule. Subcellular localization of Vpr and Vpr-GFP by immunofluoresence in human and murine cell lines indicated that Vpr by itself or with the reporter GFP showed a perinuclear staining pattern. Replication kinetics showed no significant difference between Vpr-GFP and native complemented pseudovirus replication in a single-round infectivity assay. A flow cytometry analysis of peripheral blood lymphocytes and macrophages infected with Vpr-GFP-packaged virions and selected by GFP showed 56.7% infectivity for lymphocytes and 84.6% infectivity for macrophages. Additional analysis of CD24 (HSA)-positive cells showed infection of CD4+ cells, macrophages, and, importantly, dendritic cells. This system will allow us to identify specific cell populations including antigen-presenting cells, and allow quantitative analysis of the precise effect of Vpr on both target and bystander cells in vitro as well as in vivo. PMID- 10749173 TI - A job half done PMID- 10749172 TI - Is there a spin doctor in the house? PMID- 10749171 TI - Differential expression of four human dynamin-like protein variants in brain tumors. AB - Dynamin-like protein, a large GTP-binding protein, has recently been cloned, and studies have suggested that it is involved in the formation of coated vesicles. In this report, the differential expression of four human dynamin-like protein splice variants (HdynIV-wildtype [WT], -11, -26, and -37) from various brain tumors was identified by reverse transcription/polymerase chain reaction (RT PCR). One novel variant (HdynIV-11), not described previously, was identified. The four alternatively spliced variants exhibited tissue specificity in normal tissues. The HdynIV-WT was strongly expressed in the brain, whereas HdynIV-37 was expressed in all tissues examined. Moreover, HdynIV-26 was dominant in the liver and apparently overexpressed in all astrocytomas and most meningiomas and adenomas. This report suggests that HdynIV-26 may cause aberrant protein trafficking and alter vesicle formation in brain tumors. Our results also suggest that dynamin-like protein is associated with various brain tumors and, more importantly, that aberrant expression of the HdynIV-26 variant may play a role in brain tumorigenesis. PMID- 10749174 TI - Europe joins race to turn the Internet into one vast computer. PMID- 10749175 TI - Homegrown computer roots out phylogenetic networks. PMID- 10749176 TI - Irish researchers divided over how to spend huge new fund. PMID- 10749177 TI - NEON to shed light on environment research. National Ecological Observatory Network. PMID- 10749178 TI - Are AIDS dissidents advising South Africa? PMID- 10749179 TI - German research agency stifles creativity. PMID- 10749181 TI - New form of hydrogen power provokes scepticism PMID- 10749180 TI - EU centres saved from 'catastrophe'. European Union. PMID- 10749183 TI - Older women scientists fight USGS over layoffs. US Geological Survey PMID- 10749182 TI - Art imitating high-energy physics PMID- 10749184 TI - There's enough food for everyone, but the poor can't afford to buy it. PMID- 10749185 TI - Spanish university study ignores research PMID- 10749186 TI - Not too late to apologize. PMID- 10749188 TI - A 1,000-year chain of thinkers PMID- 10749187 TI - DoE still involved in the Human Genome Project. Department of Energy. PMID- 10749189 TI - Reality check PMID- 10749190 TI - Push-button entanglement PMID- 10749191 TI - Pathogen-driven forest diversity. PMID- 10749192 TI - Hydrocarbon fuels. Hopes for a flame-free future PMID- 10749194 TI - Laser light splits atom PMID- 10749193 TI - In search of the whales' sisters. PMID- 10749195 TI - The element of uncertainty PMID- 10749196 TI - Cell biology. Reeling CASK into the nucleus. PMID- 10749197 TI - Growth of carbon micro-trees PMID- 10749198 TI - Scaling in athletic world records. PMID- 10749199 TI - Gene silencing in worms and fungi. PMID- 10749200 TI - Quantum information and computation AB - In information processing, as in physics, our classical world view provides an incomplete approximation to an underlying quantum reality. Quantum effects like interference and entanglement play no direct role in conventional information processing, but they can--in principle now, but probably eventually in practice- be harnessed to break codes, create unbreakable codes, and speed up otherwise intractable computations. PMID- 10749201 TI - Experimental entanglement of four particles AB - Quantum mechanics allows for many-particle wavefunctions that cannot be factorized into a product of single-particle wavefunctions, even when the constituent particles are entirely distinct. Such 'entangled' states explicitly demonstrate the non-local character of quantum theory, having potential applications in high-precision spectroscopy, quantum communication, cryptography and computation. In general, the more particles that can be entangled, the more clearly nonclassical effects are exhibited--and the more useful the states are for quantum applications. Here we implement a recently proposed entanglement technique to generate entangled states of two and four trapped ions. Coupling between the ions is provided through their collective motional degrees of freedom, but actual motional excitation is minimized. Entanglement is achieved using a single laser pulse, and the method can in principle be applied to any number of ions. PMID- 10749202 TI - Quantum distribution of protons in solid molecular hydrogen at megabar pressures AB - Solid hydrogen, a simple system consisting only of protons and electrons, exhibits a variety of structural phase transitions at high pressures. Experimental studies based on static compression up to about 230 GPa revealed three relevant phases of solid molecular hydrogen: phase I (high-temperature, low pressure phase), phase II (low-temperature phase) and phase III (high-pressure phase). Spectroscopic data suggest that symmetry breaking, possibly related to orientational ordering, accompanies the transition into phases II and III. The boundaries dividing the three phases exhibit a strong isotope effect, indicating that the quantum-mechanical properties of hydrogen nuclei are important. Here we report the quantum distributions of protons in the three phases of solid hydrogen, obtained by a first-principles path-integral molecular dynamics method. We show that quantum fluctuations of protons effectively hinder molecular rotation--that is, a quantum localization occurs. The obtained crystal structures have entirely different symmetries from those predicted by the conventional simulations which treat protons classically. PMID- 10749203 TI - Towards the clarity limit in optical fibre AB - An important scientific and technological goal in the field of optical communications is the achievement of the clarity limit in optical fibres--that is, ensuring that the SiO2 glass from which fibres are made is as transparent as possible. The clarity of the wavelength transmission window (and the width of that window) in existing fibres is already sufficient to form the basis of a world-wide optical communication system, yet it is still limited by contamination of the fibre by water. Here we measure the spatial distribution of water in the glass rods from which optical fibres are drawn and explain the distribution quantitatively with a mathematical model of diffusion in a medium with essentially perfect cylindrical symmetry. Our analysis shows that the water enters from the outside of the rod and diffuses into the molten, flowing glass much faster than is expected from extrapolation of low-temperature measurements. Our elucidation of the physics underlying the contamination process has already led to the fabrication of dry fibres, which have a clarified and broadened communications window. The improved operational range of wavelengths should yield applications for new lasers, optical amplifiers and detectors, and should substantially increase the information-carrying capacity of optical communications systems. PMID- 10749204 TI - Direct oxidation of hydrocarbons in a solid-oxide fuel cell AB - The direct electrochemical oxidation of dry hydrocarbon fuels to generate electrical power has the potential to accelerate substantially the use of fuel cells in transportation and distributed-power applications. Most fuel-cell research has involved the use of hydrogen as the fuel, although the practical generation and storage of hydrogen remains an important technological hurdle. Methane has been successfully oxidized electrochemically, but the susceptibility to carbon formation from other hydrocarbons that may be present or poor power densities have prevented the application of this simple fuel in practical applications. Here we report the direct, electrochemical oxidation of various hydrocarbons (methane, ethane, 1-butene, n-butane and toluene) using a solid oxide fuel cell at 973 and 1,073 K with a composite anode of copper and ceria (or samaria-doped ceria). We demonstrate that the final products of the oxidation are CO2 and water, and that reasonable power densities can be achieved. The observation that a solid-oxide fuel cell can be operated on dry hydrocarbons, including liquid fuels, without reforming, suggests that this type of fuel cell could provide an alternative to hydrogen-based fuel-cell technologies. PMID- 10749205 TI - Single crystals of an ionic anthracene aggregate with a triplet ground state AB - Crystalline supramolecular aggregates consisting of charged organic molecules, held together through metal-cluster-mediated Coulomb interactions, have attracted interest owing to their unusual structural, chemical and electronic properties. Aggregates containing metal cation clusters 'wrapped' by lipophilic molecular anions have, for example, been shown to be kinetically stable and soluble in nonpolar liquids such as saturated hydrocarbons. The formation of supramolecular aggregates can even be exploited to generate aromatic hydrocarbons that carry four negative charges and crystallize in the form of organic poly(metal cation) clusters or helical polymers. Here we report the anaerobic crystallization of an ionic organic aggregate--a contact ion septuple consisting of a fourfold negatively charged 'tripledecker' of three anthracene molecules bridged by four solvated potassium cations. Its electronic ground state is shown experimentally, using temperature-dependent electron paramagnetic resonance spectroscopy, to be a triplet. Although the spins in this biradical ionic solid are separated by a considerable distance, density functional theory calculations indicate that the triplet ground state is 84 kJ mol(-1) more stable than the first excited singlet state. We expect that the successful crystallization of the ionic solid we report here, and that of a covalent organic compound with a triplet ground state at room temperature, will stimulate further attempts to develop new triplet-ground-state materials for practical use. PMID- 10749206 TI - Seismic hazard in the Marmara Sea region following the 17 August 1999 Izmit earthquake AB - On 17 August 1999, a destructive magnitude 7.4 earthquake occurred 100 km east of Istanbul, near the city of Izmit, on the North Anatolian fault. This 1,600-km long plate boundary slips at an average rate of 2-3 cm yr(-1), and historically has been the site of many devastating earthquakes. This century alone it has ruptured over 900 km of its length. Models of earthquake-induced stress change combined with active fault maps had been used to forecast that the epicentral area of the 1999 Izmit event was indeed a likely location for the occurrence of a large earthquake. Here we show that the 1999 event itself significantly modifies the stress distribution resulting from previous fault interactions. Our new stress models take into account all events in the region with magnitudes greater than 6 having occurred since 1700 as well as secular interseismic stress change, constrained by GPS data. These models provide a consistent picture of the long term spatio-temporal behaviour of the North Anatolian fault and indicate that two events of magnitude equal to, or greater than, the Izmit earthquake are likely to occur within the next decades beneath the Marmara Sea, south of Istanbul. PMID- 10749207 TI - Fine-scale heterogeneity in the Earth's inner core AB - The seismological properties of the Earth's inner core have become of particular interest as we understand more about its composition and thermal state. Observations of anisotropy and velocity heterogeneity in the inner core are beginning to reveal how it has grown and whether it convects. The attenuation of seismic waves in the inner core is strong, and studies of seismic body waves have found that this high attenuation is consistent with either scattering or intrinsic attenuation. The outermost portion of the inner core has been inferred to possess layering and to be less anisotropic than at greater depths. Here we present observations of seismic waves scattered in the inner core which follow the expected arrival time of the body-wave reflection from the inner-core boundary. The amplitude of these scattered waves can be explained by stiffness variations of 1.2% with a scale length of 2 kilometres across the outermost 300 km of the inner core. These variations might be caused by variations in composition, by pods of partial melt in a mostly solid matrix or by variations in the orientation or strength of seismic anisotropy. PMID- 10749208 TI - The oldest known anthropoid postcranial fossils and the early evolution of higher primates. AB - The middle Eocene primate family Eosimiidae, which is known from sites in central and eastern China and Myanmar, is central to efforts to reconstruct the origin and early evolution of anthropoid or 'higher' primates (monkeys, apes and humans). Previous knowledge of eosimiid anatomy has been restricted to the dentition and an isolated petrosal bone, and this limited anatomical information has led to conflicting interpretations of early anthropoid phylogeny. Here we describe foot bones of Eosimias from the same middle Eocene sites in China that yield abundant dental remains of this primate. Tarsals of Eosimias show derived anatomical traits that are otherwise restricted to living and fossil anthropoids. These new fossils substantiate the anthropoid status of Eosimias and clarify the phylogenetic position of anthropoids with respect to other major primate clades. Early anthropoids possessed a mosaic of primitive and derived traits in their postcranial skeletons, reflecting their derivation from haplorhine ancestors that retained many prosimian-like features. PMID- 10749209 TI - Soil pathogens and spatial patterns of seedling mortality in a temperate tree. AB - The Janzen-Connell hypothesis proposes that host-specific, distance- and/or density-dependent predators and herbivores maintain high tree diversity in tropical forests. Negative feedback between plant and soil communities could be a more effective mechanism promoting species coexistence because soil pathogens can increase rapidly in the presence of their host, causing conditions unfavourable for local conspecific recruitment. Here we show that a soil pathogen leads to patterns of seedling mortality in a temperate tree (Prunus serotina) as predicted by the Janzen-Connell hypothesis. In the field, the mean distance to parent of seedling cohorts shifted away from maternal trees over a period of 3 years. Seedlings were grown in soil collected 0-5 m or 25-30 m from Prunus trees. Sterilization of soil collected beneath trees improved seedling survival relative to unsterilized soil, whereas sterilization of distant soil did not affect survival. Pythium spp., isolated from roots of dying seedlings and used to inoculate healthy seedlings, decreased survival by 65% relative to controls. Our results provide the most complete evidence that native pathogens influence tree distributions, as predicted by the Janzen-Connell hypothesis, and suggest that similar ecological mechanisms operate in tropical and temperate forests. PMID- 10749210 TI - The ecological cost of sex. AB - Why sex prevails in nature remains one of the great puzzles of evolution. Sexual reproduction has an immediate cost relative to asexual reproduction, as males only express their contribution to population growth through females. With no males to sustain, an asexual mutant can double its relative representation in the population in successive generations. This is the widely accepted 'twofold cost of males'. Many studies have attempted to explain how sex can recoup this cost from fitness benefits associated with the recombination of parental genotypes, but these require complex biological environments that cycle over evolutionary timescales. In contrast, we have considered the ecological dynamics that govern asexual invasion. Here we show the existence of a threshold growth rate for the sexual population, above which the invasion is halted by intraspecific competition. The asexual population then exerts a weaker inhibitory effect on the carrying capacity of the sexual population than on its own carrying capacity. The stable outcome of this is coexistence on a depleted resource base. Under these ecological circumstances, longer-term benefits of sex may eventually drive out the asexual competitor. PMID- 10749211 TI - NMDA spikes in basal dendrites of cortical pyramidal neurons. AB - Basal dendrites are a major target for synaptic inputs innervating cortical pyramidal neurons. At present little is known about signal processing in these fine dendrites. Here we show that coactivation of clustered neighbouring basal inputs initiated local dendritic spikes, which resulted in a 5.9 +/- 1.5 mV (peak) and 64.4 +/- 19.8 ms (half-width) cable-filtered voltage change at the soma that amplified the somatic voltage response by 226 +/- 46%. These spikes were accompanied by large calcium transients restricted to the activated dendritic segment. In contrast to conventional sodium or calcium spikes, these spikes were mediated mostly by NMDA (N-methyl-D-aspartate) receptor channels, which contributed at least 80% of the total charge. The ionic mechanism of these NMDA spikes may allow 'dynamic spike-initiation zones', set by the spatial distribution of glutamate pre-bound to NMDA receptors, which in turn would depend on recent and ongoing activity in the cortical network. In addition, NMDA spikes may serve as a powerful mechanism for modification of the cortical network by inducing long-term strengthening of co-activated neighbouring inputs. PMID- 10749212 TI - The Ras-MAPK pathway is important for olfaction in Caenorhabditis elegans. AB - The Ras-MAPK (mitogen-activated protein kinase) signal transduction pathway is well known to control cellular proliferation and differentiation in response to extracellular signals, but its other functions are less understood. In Caenorhabditis elegans this pathway regulates several developmental events, such as vulval induction and progression of meiosis, but its function in the nervous system is unknown. Here we report that the Ras-MAPK pathway is involved in olfaction in this organism. Mutational inactivation and hyperactivation of this pathway impairs efficiency of chemotaxis to a set of odorants. Experiments in which let-60 ras was expressed using a heat-shock promoter and a cell-specific promoter show that a normal activity of LET-60 Ras is required in mature olfactory neurons. Application of the odorant isoamylalcohol to wild-type animals leads to the activation of MAP kinase in olfactory neurons within 10 seconds. This induction is dependent on the function of the nucleotide-gated channel TAX 2/TAX-4 and the voltage-activated calcium channel subunit UNC-2. These results suggest a dynamic regulatory role for the Ras-MAPK pathway in perception and transmission of sensory signals in olfactory neurons. PMID- 10749213 TI - An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. AB - In a diverse group of organisms that includes Caenorhabditis elegans, Drosophila, planaria, hydra, trypanosomes, fungi and plants, the introduction of double stranded RNAs inhibits gene expression in a sequence-specific manner. These responses, called RNA interference or post-transcriptional gene silencing, may provide anti-viral defence, modulate transposition or regulate gene expression. We have taken a biochemical approach towards elucidating the mechanisms underlying this genetic phenomenon. Here we show that 'loss-of-function' phenotypes can be created in cultured Drosophila cells by transfection with specific double-stranded RNAs. This coincides with a marked reduction in the level of cognate cellular messenger RNAs. Extracts of transfected cells contain a nuclease activity that specifically degrades exogenous transcripts homologous to transfected double-stranded RNA. This enzyme contains an essential RNA component. After partial purification, the sequence-specific nuclease co-fractionates with a discrete, approximately 25-nucleotide RNA species which may confer specificity to the enzyme through homology to the substrate mRNAs. PMID- 10749214 TI - A genetic link between co-suppression and RNA interference in C. elegans. AB - Originally discovered in plants, the phenomenon of co-suppression by transgenic DNA has since been observed in many organisms from fungi to animals: introduction of transgenic copies of a gene results in reduced expression of the transgene as well as the endogenous gene. The effect depends on sequence identity between transgene and endogenous gene. Some cases of co-suppression resemble RNA interference (the experimental silencing of genes by the introduction of double stranded RNA), as RNA seems to be both an important initiator and a target in these processes. Here we show that co-suppression in Caenorhabditis elegans is also probably mediated by RNA molecules. Both RNA interference and co-suppression have been implicated in the silencing of transposons. We now report that mutants of C. elegans that are defective in transposon silencing and RNA interference (mut-2, mut-7, mut-8 and mut-9) are in addition resistant to co-suppression. This indicates that RNA interference and co-suppression in C. elegans may be mediated at least in part by the same molecular machinery, possibly through RNA-guided degradation of messenger RNA molecules. PMID- 10749215 TI - Nuclear translocation and transcription regulation by the membrane-associated guanylate kinase CASK/LIN-2. AB - Membrane-associated guanylate kinases (MAGUKs) contain multiple protein-binding domains that allow them to assemble specific multiprotein complexes in particular regions of the cell. CASK/LIN-2, a MAGUK required for EGF receptor localization and signalling in Caenorhabditis elegans, contains a calmodulin-dependent protein kinase-like domain followed by PDZ, SH3 and guanylate kinase-like domains. In adult rat brain, CASK is concentrated at neuronal synapses and binds to the cell surface proteins neurexin and syndecan and the cytoplasmic proteins Mint/LIN-10 and Veli/LIN-7. Here we report that, through its guanylate kinase domain, CASK interacts with Tbr-1, a T-box transcription factor that is involved in forebrain development. CASK enters the nucleus and binds to a specific DNA sequence (the T element) in a complex with Tbr-1. CASK acts as a coactivator of Tbr-1 to induce transcription of T-element containing genes, including reelin, a gene that is essential for cerebrocortical development. Our findings show that a MAGUK which is usually associated with cell junctions has a transcription regulation function. PMID- 10749216 TI - Phosphorylation of CPE binding factor by Eg2 regulates translation of c-mos mRNA. AB - Full-grown Xenopus oocytes arrest at the G2/M border of meiosis I. Progesterone breaks this arrest, leading to the resumption of the meiotic cell cycles and maturation of the oocyte into a fertilizable egg. In these oocytes, progesterone interacts with an unidentified surface-associated receptor, which induces a non transcriptional signalling pathway that stimulates the translation of dormant c mos messenger RNA. Mos, a mitogen-activated protein (MAP) kinase kinase kinase, indirectly activates MAP kinase, which in turn leads to oocyte maturation. The translational recruitment of c-mos and several other mRNAs is regulated by cytoplasmic polyadenylation, a process that requires two 3' untranslated regions, the cytoplasmic polyadenylation element (CPE) and the polyadenylation hexanucleotide AAUAAA. Although the signalling events that trigger c-mos mRNA polyadenylation and translation are unclear, they probably involve the activation of CPEB, the CPE binding factor. Here we show that an early site-specific phosphorylation of CPEB is essential for the polyadenylation of c-mos mRNA and its subsequent translation, and for oocyte maturation. In addition, we show that this selective, early phosphorylation of CPEB is catalysed by Eg2, a member of the Aurora family of serine/threonine protein kinases. PMID- 10749217 TI - The structure of malaria pigment beta-haematin. AB - Despite the worldwide public health impact of malaria, neither the mechanism by which the Plasmodium parasite detoxifies and sequesters haem, nor the action of current antimalarial drugs is well understood. The haem groups released from the digestion of the haemoglobin of infected red blood cells are aggregated into an insoluble material called haemozoin or malaria pigment. Synthetic beta-haematin (FeIII-protoporphyrin-IX)2 is chemically, spectroscopically and crystallographically identical to haemozoin and is believed to consist of strands of FeIII-porphyrin units, linked into a polymer by propionate oxygen-iron bonds. Here we report the crystal structure of beta-haematin determined using simulated annealing techniques to analyse powder diffraction data obtained with synchrotron radiation. The molecules are linked into dimers through reciprocal iron carboxylate bonds to one of the propionic side chains of each porphyrin, and the dimers form chains linked by hydrogen bonds in the crystal. This result has implications for understanding the action of current antimalarial drugs and possibly for the design of new therapeutic agents. PMID- 10749219 TI - Changing the face of training for science at the interface. PMID- 10749218 TI - Pushing the frontiers of interdisciplinary research: an idea whose time has come. PMID- 10749220 TI - Crossing the divide between theory and practice. PMID- 10749221 TI - Helical CT in the evaluation of the acute abdomen. PMID- 10749222 TI - Evaluation of subsecond gated helical CT for quantification of coronary artery calcium and comparison with electron beam CT. AB - OBJECTIVE: Since its introduction early in the 1990s, helical CT has become the predominant technology for obtaining CT images for medical applications. Recent improvements in the temporal resolution of helical CT (subsecond) and the addition of retrospective cardiac gating are combined in this report evaluating cardiac-gated helical CT for quantifying coronary artery calcium. We compare total calcium scores determined on subsecond gated helical CT with the current reference for coronary calcium evaluation, electron beam CT. MATERIALS AND METHODS: We compared total calcium scores obtained using a general purpose, unmodified helical CT scanner with scores obtained using electron beam CT in 36 individuals who were 68+/-11 years old (age range, 41-85 years). RESULTS: Correlation coefficients ranged from 0.97 to 0.98 (Pearson's product moment) and from 0.95 to 0.96 (Spearman's rank order), depending on the coronary calcium scoring method used. Agreement in the classification of participants as "healthy" or "diseased" at threshold total calcium scores of 10, 100, 160, 200, 400, and 680 was, respectively, 94%, 97%, 89%, 92%, 94%, and 100% using the conventional electron beam CT scoring method and an equivalent method with helical CT. CONCLUSION: A general purpose, current generation helical CT scanner equipped for retrospective cardiac gating can accurately quantify coronary calcium, and the results are highly correlated to scores obtained with electron beam CT. As an alternative method for measuring coronary calcium, gated subsecond cardiac helical CT offers greater availability and lower cost, thereby making population based screening for coronary artery calcium more feasible. PMID- 10749223 TI - What today's department says about the future. PMID- 10749224 TI - Malpractice issues in radiology. Alliterative errors. PMID- 10749225 TI - Changes in the market for diagnostic radiologists as measured through a help wanted index. AB - OBJECTIVE: We sought to create and validate a help wanted index for tracking changes in the radiology job market. SUBJECTS AND METHODS: All jobs advertised in Radiology and the American Journal of Roentgenology from January 1991 through December 1998 were tracked according to three separate parameters: academic versus private practice, subspecialty, and region. Statistical comparison was made between the first and second 48-month subperiods to identify changes. RESULTS: Thirteen thousand seven hundred one advertised positions were coded. A dramatic decrease in job advertisements was noted after December 1991, with advertisements falling to one eighth of their late 1991 peak. A recovery has occurred, with advertising now approaching peak levels. Shifts were seen toward more private practice, midwestern location, vascular and interventional, and mammography positions. Declines occurred in the share of positions in California, the Southwest, and several radiology subspecialties. Other trends were noted but were statistically less significant. A strong correlation (R = 0.98) was found between the annual number of positions advertised and radiologists' median incomes relative to those of all physicians. CONCLUSION: The job market in radiology, much as in other fields, can be tracked in a coincident manner with the use of a help wanted index. Changes in the makeup of radiology practice are important and are identified in a well-constructed index. These findings have validity and can be useful as an adjunct to other information for policy and planning purposes. PMID- 10749226 TI - Comparison of sonographic and CT guidance techniques: does CT fluoroscopy decrease procedure time? AB - OBJECTIVE: Procedure times for percutaneous biopsies were compared for various guidance techniques including helical CT, CT fluoroscopy, sonography with an attached needle guide, and freehand sonography with computer guidance. MATERIALS AND METHODS: Three interventional radiologists experienced in CT- and sonographically guided procedures performed biopsies on a phantom model. The phantom simulated hepatic metastases of various sizes and depths with subcostal or intercostal locations. Lesion sizes were 7, 10, and 20 mm, at 3- and 7-cm depths. Using self-aspirating needles, two passes were performed in each lesion. Mean procedure time per biopsy pass was calculated. A two-tailed Student's t test was used to compare guidance techniques. RESULTS: Mean procedure time per biopsy pass for the four guidance techniques was sonography with a needle guide, 36+/-9 sec; sonography with computer guidance, 43+/-10 sec; helical CT, 146+/-42 sec; and CT fluoroscopy, 50+/-18 sec. CT fluoroscopy required 2.6+/-1.0 sec per biopsy. Helical CT required more procedure time than sonography with a needle guide, CT with computer guidance, and CT fluoroscopy (p < 0.0001). Sonography with a needle guide required less procedure time than sonography with computer guidance (p < 0.002) and CT fluoroscopy (p = 0.0003). Procedure times for CT fluoroscopy and sonography with computer guidance were not statistically different (p = 0.06). CT and sonographic guidance were equally effective regardless of lesion size, depth, or location. CONCLUSION: Traditional sonographic biopsy techniques are faster and more cost-effective than traditional CT techniques; however, CT fluoroscopy offers the localization advantages of CT with improved procedure times. PMID- 10749227 TI - Noninterpretive skills for radiology residents. Critical thinking: deciding whether to incorporate the recommendations of radiology publications and presentations into practice. PMID- 10749228 TI - Preoperative detection of malignant hepatic tumors: comparison of combined methods of MR imaging with combined methods of CT. AB - OBJECTIVE: We compared radiologists' performance on combined unenhanced, gadolinium-enhanced, and ferumoxides-enhanced MR imaging with their performance on helical CT during arterial portography (CTAP) and biphasic CT during hepatic arteriography (CTHA) for the preoperative detection of malignant hepatic tumors. SUBJECTS AND METHODS: MR images and CT scans obtained in 33 patients were retrospectively analyzed. Images of the liver were reviewed on a segment-by segment basis; a total of 261 segments with 39 hepatocellular carcinomas and 21 metastases were independently reviewed by three radiologists who were invited from outside institutions. Unenhanced and gadolinium-enhanced MR images were reviewed first, then ferumoxides-enhanced MR images were added for combined review. CTAP images and biphasic CTHA images were reviewed together. RESULTS: Sensitivity for the detection of hepatic tumors was analogous for combined unenhanced, gadolinium-enhanced, and ferumoxides-enhanced MR images (86%) and for combined CTAP images and biphasic CTHA images (87%). Specificity was higher with MR images (95%, p < 0.01) than with CT images (91%). Radiologists' performances were improved (Az = 0.962, p = 0.0502) by combining ferumoxides-enhanced MR images with unenhanced and gadolinium-enhanced MR images (Az = 0.950), and were analogous for combined unenhanced, gadolinium-enhanced, and ferumoxides-enhanced MR images and for combined CTAP images and biphasic CTHA images (Az = 0.959). CONCLUSION: Radiologists' performances on combined unenhanced, gadolinium enhanced, and ferumoxides-enhanced MR imaging compared with their performances on combined helical CTAP and biphasic CTHA are analogous for the preoperative detection of malignant hepatic tumors. Such a dedicated combination of MR imaging may obviate the need for more invasive angiographically assisted helical CT for the preoperative detection of malignant hepatic tumors. PMID- 10749229 TI - Revealing hepatic metastases from colorectal cancer: value of combined helical CT during arterial portography and CT hepatic arteriography with a unified CT and angiography system. AB - OBJECTIVE: The purpose of our study was to evaluate the use of combined helical CT during arterial portography and CT hepatic arteriography in the preoperative assessment of hepatic metastases from colorectal cancer using a unified CT and angiography system. MATERIALS AND METHODS: Fifty-four patients with hepatic metastases from colorectal cancer preoperatively underwent combined CT during arterial portography and CT hepatic arteriography using the unified CT and angiography system. Three radiologists independently and retrospectively reviewed the images of CT during arterial portography alone, CT hepatic arteriography alone, and combined CT during arterial portography and CT hepatic arteriography. Image review was conducted on a segment-by-segment basis; a total of 432 hepatic segments with (n = 103) 118 metastatic tumors ranging in size from 2 to 160 mm (mean, 25.8 mm) and without (n = 329) tumor were reviewed. RESULTS: Relative sensitivity of combined CT during arterial portography and CT hepatic arteriography (87%) was higher than that of CT during arterial portography alone (80%, p < 0.0005) and CT hepatic arteriography alone (83%, p < 0.005). Relative specificity of CT hepatic arteriography alone (95%, p < 0.0005) and combined CT during arterial portography and CT hepatic arteriography (96%, p < 0.0001) was higher than that of CT during arterial portography alone (91%). Diagnostic accuracy, determined by a receiver operating characteristic curve analysis, was greater with combined CT during arterial portography and CT hepatic arteriography than with CT during arterial portography alone (p < 0.05) or CT hepatic arteriography alone (p < 0.01). CONCLUSION: Using a unified CT and angiography system, we found that combined CT during arterial portography and CT hepatic arteriography significantly raised the detectability of hepatic metastases from colorectal cancer. PMID- 10749230 TI - Fulminant Clostridium septicum infection of hepatic metastases presenting as pneumoperitoneum. PMID- 10749231 TI - MR imaging of diffuse liver disease. PMID- 10749232 TI - Prevalence and significance of gallbladder abnormalities seen on sonography in intensive care unit patients. AB - OBJECTIVE: We evaluated sonographic abnormalities of the gallbladder other than acalculous cholecystitis across a broad range of intensive care unit (ICU) patients. SUBJECTS AND METHODS: Fifty-five consecutive patients (age range, 18-94 years old; mean age, 56 years; 33 men, 22 women), who were admitted to the ICU with a variety of diagnoses, underwent sonography of the gallbladder twice a week. Patients with gallbladder calculi were excluded from the study. The gallbladder was examined for the recognized sonographic features of acalculous cholecystitis: gallbladder wall thickening, gallbladder distention, intramural gallbladder lucencies (striated gallbladder wall), pericholecystic fluid, gallbladder sludge, and Murphy's sign. These findings were correlated with clinical and laboratory parameters that are associated with acalculous cholecystitis: fever, WBC, liver function tests, levels of serum bilirubin, mechanical ventilation status, and administration of parenteral nutrition, narcotic analgesics, antibiotics, and pressor agents. RESULTS: Eleven of the 55 patients were found to have gallbladder calculi and were excluded from the study. Thirty-seven (84%) of the remaining 44 patients had at least one sonographic abnormality while in the ICU. Twenty-five (57%) of the 44 patients had as many as three abnormalities found on sonography, and six (14%) of 44 patients had four or five sonographic findings of gallbladder abnormalities while in the ICU. No statistically significant correlation was found among any of these sonographic abnormalities and the clinical and laboratory parameters. CONCLUSION: Gallbladder abnormalities are frequently seen on sonography in ICU patients, even if these patients are not suspected of having acalculous cholecystitis; therefore, sonography appears to be of limited value in diagnosing acalculous cholecystitis in ICU patients. PMID- 10749233 TI - Xanthogranulomatous cholecystitis: clinical, sonographic, and CT findings in 26 patients. AB - OBJECTIVE: The purpose of our study was to evaluate the sonographic and CT features of xanthogranulomatous cholecystitis, correlating the pathologic and surgical findings. MATERIALS AND METHODS: Xanthogranulomatous cholecystitis was pathologically diagnosed in 26 patients from January 1996 to August 1998. The patients were 15 women and 11 men with a mean age of 63 years. All patients had preoperative sonography and nine also underwent CT In five patients, sonography was performed on the surgical specimen. Clinical indications for imaging included cholecystitis (14 patients), biliary colic (six patients), stone-induced pancreatitis (three patients), tumor (two patients), and gallstone ileus (one patient). RESULTS: The most characteristic sonographic finding, confirmed by sonographic study of the surgical specimens, was the presence of hypoechoic nodules or bands in the gallbladder wall, which were seen in 35% of the patients. Cholelithiasis and a thickened gallbladder wall were frequent findings. The most characteristic (specific) CT finding was a hypodense band in the gallbladder wall, seen in 33% of the patients. Two of twelve patients who underwent laparoscopic cholecystectomy required conversion to open surgery. CONCLUSION: Although the preoperative imaging diagnosis of xanthoganulomatous cholecystitis is difficult, the presence of hypoechoic nodules or bands in the gallbladder wall on sonography or of a hypodense band around the gallbladder on CT, is highly suggestive of this disease. PMID- 10749234 TI - Choledochojejunostomy: possible risk factor for septic complications after percutaneous hepatic tumor ablation. PMID- 10749236 TI - CT of small-bowel obstruction: another perspective using multiplanar reformations. PMID- 10749235 TI - Complications of continuous ambulatory peritoneal dialysis: findings on MR peritoneography. AB - OBJECTIVE: The purpose of this study was to assess the diagnostic value of MR peritoneography in complications of continuous ambulatory peritoneal dialysis. SUBJECTS AND METHODS: Twenty consecutive patients treated with continuous ambulatory peritoneal dialysis who were clinically suspected of dialysis-related complications were prospectively studied with MR peritoneography. For MR peritoneography, 20 ml of gadodiamide was added to 2000-ml dialysate solution (1.36% glucose) that was instilled into the peritoneal cavity. MR peritoneography was performed with the peritoneal cavity filled (n = 12) and after complete drainage of the contrast material-dialysate mixture (n = 20) on a 1.5-T MR unit with a phased array coil. Imaging included axial T1-weighted fast low-angle shot (TR/TE, 174/4.2) with and without fat saturation and axial and coronal T2 weighted fat-saturated turbo spin-echo (3000/138) sequences. All studies were performed without IV contrast material. Images were reviewed for evidence of peritoneal leaks, hernias, loculated fluid collections, and adhesions. RESULTS: Abnormal findings were detected in 13 (65%) of 20 patients and included retroperitoneal leaks (n = 6), diaphragmatic leaks (n = 2), catheter exit-site leaks (n = 2), inguinal hernias (n = 2), and peritoneal adhesions (n = 1). CONCLUSION: MR peritoneography is useful for the evaluation of complications related to continuous ambulatory peritoneal dialysis, and it offers excellent tissue contrast and multiplanar imaging for assessment of complications. PMID- 10749237 TI - The "foamy" esophagus: a radiographic sign of Candida esophagitis. AB - OBJECTIVE: The purpose of this study was to describe the radiographic features of the "foamy" esophagus, a new sign of Candida esophagitis seen on double-contrast esophagography, in six patients. CONCLUSION: The foamy esophagus was characterized by innumerable tiny (1-3 mm), round lucencies (bubbles) that intermingled with the barium suspension along the top of the barium column, producing a layer of foam. Candida esophagitis should be strongly suspected when a foamy esophagus is detected on double-contrast esophagography, particularly in patients with underlying esophageal involvement by scleroderma. PMID- 10749238 TI - Esophageal pseudomass: extrinsic compression of the esophagus due to a narrow thoracic inlet. PMID- 10749239 TI - FDG PET evaluation of mucinous neoplasms: correlation of FDG uptake with histopathologic features. AB - OBJECTIVE: Our goal was to assess the sensitivity of positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) for the detection of mucinous carcinoma and to determine the histologic features of these tumors that may affect lesion detectability. MATERIALS AND METHODS: A retrospective review of all patients with mucinous carcinoma who had undergone FDG PET at our institution from 1995 through 1998 identified 25 patients with new or recurrent mucinous carcinoma at the time of PET. In 22 of these patients, tissue specimens available from either core biopsy or surgical resection allowed detailed histologic analysis. RESULTS: FDG PET revealed mucinous carcinoma in only 13 (59%) of 22 patients, resulting in an unusually high percentage (41%) of false-negative results. Two histologic features were found to be predictive of FDG PET results: tumor cellularity (p = 0.011) and the amount of mucin within the tumor mass (p = 0.009). There was a positive correlation between tumor FDG uptake and cellularity but a negative correlation with the amount of mucin. CONCLUSION: FDG PET is limited in the evaluation of mucinous tumors, particularly in hypocellular lesions with abundant mucin. PMID- 10749240 TI - Esophageal atresia and tracheal stenosis: use of three-dimensional CT and virtual bronchoscopy in neonates, infants, and children. AB - OBJECTIVE: The objective of this study was to evaluate the use of three dimensional CT and virtual bronchoscopy in the treatment of neonates, infants, and children with esophageal atresia and tracheal stenosis. CONCLUSION: Long-gap (n = 1) and short-gap (n = 5) esophageal atresia, long-segment stenosis (n = 2), patent poststenting trachea (n = 1), normal trachea without fistula (n = 1), and tracheal bronchus (n = 1) were studied. Fistulas between the lower esophagus and carina were noted in all six cases of esophageal atresia. All CT findings correlated with operative or bronchoscopy findings. Sensitivity and specificity were 100%. Three-dimensional CT and virtual bronchoscopy are accurate and useful techniques in the preoperative assessment of esophageal atresia and tracheal stenosis in neonates, infants, and children. PMID- 10749241 TI - "Daughter cyst" sign: a sonographic finding of ovarian cyst in neonates, infants, and young children. AB - OBJECTIVE: The purpose of this study was to assess the value of the "daughter cyst" sign, a sonographic finding of neonatal ovarian cysts, in differentiating ovarian cysts from other cystic masses in neonates, infants, and young children. SUBJECTS AND METHODS: In a prospective study, 23 neonates, infants, and young children (age range, 1 day to 36 months) with a lower abdominal cystic mass underwent sonography. We defined the daughter cyst sign as the presence of a small cyst along the wall of a cystic mass. The diagnosis of ovarian cyst was made when this sign was present. Detailed pathologic correlation was available in four ovarian cysts. The size, wall thickness, and contents of the cysts were also evaluated. RESULTS: The 23 cystic lesions included ovarian cyst (n = 11), lymphangioma (n = 3), enteric duplication cyst (n = 3), enteric cyst (n = 1), meconium pseudocyst (n = 2), hydrometrocolpos (n = 2), and urachal cyst (n = 1). The daughter cyst sign was seen in nine (82%) of 11 ovarian cysts but in none of the other cystic lesions. Sensitivity, specificity, and positive predictive value of the daughter cyst sign for differentiating ovarian cysts from other cystic lesions were 82%, 100%, and 100%, respectively. The daughter cyst corresponded to an ovarian follicle on pathologic examination. CONCLUSION: The daughter cyst sign is a specific sonographic finding for an ovarian cyst and may be useful in differentiating uncomplicated ovarian cysts from other cystic masses in neonates, infants, and young children. PMID- 10749242 TI - MR imaging of renal masses interpreted on CT to be suspicious. AB - OBJECTIVE: Prior studies have shown that renal MR contrast enhancement improves the efficacy of mass and proximal vascular evaluation. This study assessed the usefulness of different sequences for characterization of masses that appeared suspicious on CT and for prediction of their potential for malignancy. SUBJECTS AND METHODS: In a prospective manner 32 patients (age range, 26-78 years: average age, 54 years), each with at least one suspicious mass on CT, were examined with MR imaging. The following sequences were performed: conventional spin-echo with and without fat saturation, fast spin-echo, and dynamic gadopentetate dimeglumine enhanced infusion using a 1.5-T superconducting magnet. Results were analyzed and compared with pathologic results after resection. RESULTS: A total of 65 renal masses of average size 2.6 cm (range, 1-10 cm) were detected with dynamic MR imaging. Seventeen of the 65 masses were malignant. Of the 17 malignant masses, three did not enhance on dynamic MR imaging (because of hemorrhage). Sixteen of the 17 malignant masses were heterogeneous on T2-weighted images. Three enhancing masses contained fat and all were angiomyolipomas. Thirty-five of the 65 masses (four with hemorrhage) did not show enhancement, all of which were homogeneous on T2-weighted images and were proven to be cysts. Five masses resulted from infections and had heterogeneous T2 appearance. The remaining masses were three hematomas with hemorrhage, one column of Bertin, and one aneurysm. CONCLUSION: Renal masses that are interpreted as suspicious on CT may lack MR enhancement because of hemorrhage effects; heterogeneity of their T2 appearance is thus critical in differentiating malignancy from benign disease. Odds-ratio calculations give an adjusted estimate of a 3.36-fold increase (95% confidence interval, 1.8-6.27) in the likelihood of malignancy when masses are heterogeneous on T2-weighted images and a 29-fold increase (95% confidence interval, 3.67 241.8) for predicting malignancy when enhancement is present. PMID- 10749243 TI - Diagnosis of renal pelvis subepithelial hemorrhage using unenhanced helical CT. PMID- 10749244 TI - CT of recurrent retroperitoneal sarcomas. AB - OBJECTIVE: We reviewed the medical records and CT scans of 33 patients with recurrent retroperitoneal sarcomas to determine the patterns of recurrent disease. MATERIALS AND METHODS: We reviewed the medical records and CT examinations obtained at the time the recurrence was diagnosed and tabulated data for all patients. Data for patients with high-grade malignancies were compared with those of patients with low-grade malignancies to determine whether there were differences in the interval between initial tumor resection and recurrence. We also compared CT appearances to determine patterns of recurrent disease. RESULTS: Twenty-five of 33 recurrences were detected within 2 years of initial surgery. Only 16 patients had symptoms, and when present, most symptoms were nonspecific. In 28 (85%) patients, recurrent tumor was in the abdomen at the time of diagnosis. In nine patients, the largest detectable abdominal tumor was less than 5 cm in diameter. Interval to recurrence was similar for patients with low- and high-grade tumors. Although the CT appearance was similar for both grades, distant metastases were identified only in patients with high-grade malignancies. CONCLUSION: Primary retroperitoneal malignancies frequently recur within 2 years of initial surgical resection. For asymptomatic patients, diagnosis is typically made during routine follow-up CT. Most patients have abdominal recurrences that may be small when first detected. PMID- 10749245 TI - Percutaneous ethanol ablation of an adrenal tumor. PMID- 10749246 TI - Adrenal mass evaluation in patients with lung carcinoma: a cost-effectiveness analysis. AB - OBJECTIVE: This study evaluates the cost-effectiveness of various imaging and biopsy strategies for characterizing adrenal masses in patients with newly diagnosed non-small cell carcinoma of the lung. MATERIALS AND METHODS: A decision analysis model was used to compare the cost-effectiveness of nine strategies. Initial imaging included unenhanced CT using an adenoma or nonadenoma threshold of 0 or 10 H or in- and opposed-phase MR imaging. When initial imaging did not confirm an adenoma, CT-guided biopsy or subsequent imaging was performed. Medicare reimbursement was used as a surrogate of cost. Net costs were calculated as the difference in costs between two limbs of the decision tree. Net benefits were calculated as the difference between strategies and were calculated for life expectancy in years. MR imaging, CT, and biopsy accuracy, average life expectancy, and surgical mortality rates were based on the literature. RESULTS: The base case analysis determined that the most cost-effective strategy was CT with an adenoma or nonadenoma threshold of 10 H followed by MR imaging, if necessary. CT with a threshold of 0 H followed by biopsy, if necessary, was the least costly. The incremental cost-effectiveness ratio between these two strategies was $16,370 per year of life gained. CONCLUSION: Unenhanced CT using a 10 H threshold followed by MR imaging, if needed, was the most cost-effective strategy for evaluating an adrenal mass in a patient with newly diagnosed non small cell lung cancer. PMID- 10749247 TI - Pulmonary embolism revealed on helical CT angiography: comparison with ventilation-perfusion radionuclide lung scanning. AB - OBJECTIVE: We compared helical CT angiography and ventilation-perfusion radionuclide lung scanning as initial tests in the diagnosis of acute pulmonary embolism. SUBJECTS AND METHODS: Two hundred sixteen consecutive patients who were clinically suspected of having acute pulmonary embolism underwent helical CT angiography, ventilation-perfusion radionuclide lung scanning, and Doppler sonography of the veins of the legs. On the basis of concordance of the results for ventilation-perfusion radionuclide lung scanning and helical CT angiography and on the degree of clinical suspicion, certain patients underwent pulmonary angiography. Patients without pulmonary embolism at initial evaluation in whom no treatment was instituted were followed up for at least 3 months to determine the potential recurrence of thromboembolic disease. RESULTS: Of the 216 patients, 37 (17%) were excluded because of insufficient data to assess the initial event. Final diagnosis for the 179 remaining patients was pulmonary embolism in 68 (37.9%) and no pulmonary embolism in 111 (62.0%), based on pulmonary angiography in 23 patients (12.8%) and concordant imaging findings and outcome in the remaining patients. Statistically significant differences (p < 0.05) were found between sensitivity, specificity, positive predictive value, and negative predictive value for helical CT angiography and ventilation-perfusion radionuclide lung scanning (94.1% versus 80.8%; 93.6% versus 73.8%; 95.5% versus 82%; and 96.2% versus 75.9%, respectively). Interobserver agreement was excellent for helical CT angiography (kappa = 0.72) and moderate for ventilation-perfusion radionuclide lung scanning (kappa = 0.22). CONCLUSION: Helical CT angiography could replace ventilation-perfusion radionuclide lung scanning as the initial test for screening patients who are clinically suspected of having pulmonary embolism. PMID- 10749248 TI - Reduction of contrast material dose and artifacts by a saline flush using a double power injector in helical CT of the thorax. AB - OBJECTIVE: Our objective was to evaluate a combined method of contrast material bolus followed by saline solution flush for thoracic helical CT and statistical comparison with a uniphasic injection protocol. MATERIALS AND METHODS: Fifty patients underwent helical CT of the thorax using 60 ml of contrast material (370 mg I/ml) followed by flushing with 30 ml of physiologic saline solution. These 50 patients had been examined before using our previous protocol, 75 ml of the same contrast material without a subsequent saline solution. Mean attenuation values for both protocols were measured in the superior vena cava, the pulmonary trunk, and the ascending aorta. Image artifacts and mediastinal and hilar depiction were graded and compared. RESULTS: Mean attenuation values in the superior vena cava were considerably higher in the regimen without saline solution flush (459 H versus 352 H) and in the pulmonary trunk and the ascending aorta were almost identical for both protocols. Injection of saline solution diminished surrounding artifacts (p = 0.001). Grading results for the evaluation of mediastinal and hilar structures were not significantly different in the two protocols (p = 0.564). CONCLUSION: Injection of contrast material followed by a saline solution bolus using a double power injector when performing thoracic helical CT allows a 20% reduction of contrast material volume to 60 ml with a similar degree of enhancement. In addition, perivenous artifacts in the superior vena cava are significantly reduced. PMID- 10749249 TI - Fractal analysis of nuclear medicine images for the diagnosis of pulmonary emphysema: interpretations, implications, and limitations. AB - OBJECTIVE: The purpose of this study was to investigate, on images obtained in nuclear medicine examinations, the physical meanings and consequent implications of fractal analysis developed in a recent study that was reported to be effective in quantifying the heterogeneous distribution of carbon particle radioaerosol in the lungs. MATERIALS AND METHODS: Fractal dimensions were computed for 108 sets of radionuclide imaging data from 28 patients according to the methods in a previous report, and were then correlated with the ratio of tissue areas segmented at two thresholds (15% and 35% of maximal radioactivity). RESULTS: Fractal dimension was found to linearly correlate with the ratio natural logarithm of tissue areas segmented at two different threshold levels (n = 108, r = 0.999), with regression slope accurately predicted (error = 0.06%). Bland Altman analysis showed that fractal dimensions ranging from 0.2 to 1.9 can be explained by this area ratio with disagreement of only 5.13% at two standard deviations; thus, fractal dimension seems to be an over-simplified parameter unrelated to spatial heterogeneity of radioaerosol distribution. CONCLUSION: The analysis of this study suggested that the fractal dimension defined in a previous report was limited to the indication of the percentage area of low-radioactivity regions with respect to total tissue area in the image. Because the fractal dimension partially reflects, but is not specific to, a certain degree of focal spots of low radioactivity, we suggest using fractal analysis in clinical practice only with careful control and thorough understanding of the physical meanings. PMID- 10749250 TI - Angiofollicular lymphoid hyperplasia (Castleman disease) of the axilla. PMID- 10749251 TI - Hypersensitivity pneumonitis. PMID- 10749252 TI - 1K versus 2K monitor: a clinical alternative free-response receiver operating characteristic study of observer performance using pulmonary nodules. AB - OBJECTIVE: The aim of this study was to investigate whether and how observer performance in detecting pulmonary nodules is influenced by the use of 1K and 2K monitors with and without voluntary postprocessing. MATERIALS AND METHODS: The study was conducted with clinical digital chest radiographs of 48 patients. CT images of the same patient group served as the gold standard. Data on four different monitor conditions (1K overview, 2K overview, 1K with postprocessing, and 2K with postprocessing) were collected using a 6-point confidence-rating scale and interpreted with an alternative free-response receiver operating characteristic. RESULTS: When magnification and window settings were applied on the 1K monitor at the expense of an increased interpretation time, observer performance with the 1K monitor was not significantly different from that with the 2K monitor. A significant difference only occurred between the 1K monitor postprocessing condition and the 1K monitor overview condition. CONCLUSION: Considering diagnostic accuracy, the 1K monitor is sufficient for the detection of pulmonary nodules, provided that postprocessing options--especially magnification--are applied. Further comparative monitor studies on the detectability of other abnormalities (e.g., fine interstitial structures) need to be performed. PMID- 10749253 TI - Role of sonography in evaluation of radial scars of the breast. AB - OBJECTIVE: We investigated the usefulness of sonography in revealing radial scars suspected on mammography. CONCLUSION: Many radial scars are visible on sonography and, when visible, may present features virtually identical to those of carcinoma of the breast. Findings indicative of a radial scar are often more conspicuous on sonography than on mammography; thus, sonography may have a definitive role when evaluating subtle findings suggestive of a radial scar or when features of a radial scar are evident on only one mammographic view and cannot be localized with certainty. PMID- 10749254 TI - MR imaging-guided sonography followed by fine-needle aspiration cytology in occult carcinoma of the breast. AB - OBJECTIVE: In patients with axillary metastases as clinical evidence of possible occult breast cancer, a combined approach of MR imaging, sonography, and aspiration biopsy cytology was evaluated. SUBJECTS AND METHODS: Thirty-one women with metastatic adenocarcinoma in their axillary lymph nodes originating from an unknown primary site underwent MR imaging of the breast because physical examination and mammography findings were normal. Twenty of the 31 women had no history of malignancy, 10 had been previously treated for contralateral breast cancer, and one patient had nodal metastases in the contralateral axilla at the time breast cancer was detected. When a contrast-enhancing lesion was revealed on MR imaging of the breast, sonography and fine-needle aspiration cytology were also performed. RESULTS: MR imaging revealed the primary breast cancer in eight (40%) of the 20 patients without a history of malignancy. MR imaging of the breast revealed a second primary cancer in three (27%) of the 11 patients with previous or simultaneous breast cancer. All lesions were identified with sonography and verified by cytology and histology. CONCLUSION: In women with axillary lymph node metastases from adenocarcinoma, MR imaging of the breast should be added to clinical examination and mammography before defining the breast cancer as occult. The combined approach of MR imaging, sonography, and aspiration fine-needle cytology is a good alternative to the MR imaging-guided biopsy. PMID- 10749255 TI - Increasing breast tissue depth during stereotactic needle biopsy. PMID- 10749256 TI - Herpes simplex virus mastitis: clinical and imaging findings. PMID- 10749257 TI - Has the mammography quality standards act affected the mammography quality in North Carolina? AB - OBJECTIVE: The United States Food and Drug Administration implemented federal regulations governing mammography under the Mammography Quality Standards Act (MQSA) of 1992. During 1995, its first year in implementation, we examined the impact of the MQSA on the quality of mammography in North Carolina. MATERIALS AND METHODS: All mammography facilities were inspected during 1993-1994, and again in 1995. Both inspections evaluated mean glandular radiation dose, phantom image evaluation, darkroom fog, and developer temperature. Two mammography health specialists employed by the North Carolina Division of Radiation Protection performed all inspections and collected and codified data. RESULTS: The percentage of facilities that met quality standards increased from the first inspection to the second inspection. Phantom scores passing rate was 31.6% versus 78.2%; darkroom fog passing rate was 74.3% versus 88.5%; and temperature difference passing rate was 62.4% versus 86.9%. CONCLUSION: In 1995, the first year that the MQSA was in effect, there was a significant improvement in the quality of mammography in North Carolina. This improvement probably resulted from facilities' compliance with federal regulations. PMID- 10749258 TI - Thermography and the venous diameter ratio in the detection of the non-palpable breast carcinoma. 1968. PMID- 10749259 TI - MR imaging of the knee at 0.2 and 1.5 T: correlation with surgery. AB - OBJECTIVE: The purpose of this study was to compare the diagnostic efficacy of low- and high-field-strength MR imagers in the diagnosis of anterior cruciate ligament tears and meniscus tears. SUBJECTS AND METHODS: In 219 patients with suspected internal derangement of the knee, MR imaging at 0.2 and 1.5 T was performed with similar sequences. Only patients with surgically confirmed diagnosis (n = 90) were included in the statistical analysis. Radiologists were unaware of diagnosis and field strength. Sensitivity, specificity, diagnostic accuracy, and inter- and intraobserver variability were determined. RESULTS: There was excellent correlation between the field strengths in accuracy, sensitivity, and specificity for anterior cruciate ligament and meniscus tears. Accuracy for medial meniscus, lateral meniscus, and anterior cruciate ligament tears was 91-93%, 88-90%, and 93-96%, respectively, at 0.2 T and 91-94%, 91-93%, and 97-98%, respectively, at 1.5 T. Inter- and intraobserver variability values showed excellent correlation (kappa > 0.8). CONCLUSION: The level of diagnostic accuracy in anterior cruciate ligament tears and meniscus tears is comparable for low- and high-field-strength MR imagers. PMID- 10749260 TI - Treatable chondral injuries in the knee: frequency of associated focal subchondral edema. AB - OBJECTIVE: In the knee, chondral flaps and fractures are radiographically occult articular cartilage injuries that can mimic meniscal tears clinically; once correctly diagnosed, these injuries can be treated surgically. We investigated an associated MR imaging finding--focal subchondral bone edema--in a series of surgically proven lesions. MATERIALS AND METHODS: Two musculoskeletal radiologists retrospectively reviewed the MR studies of 18 knees with arthroscopically proven treatable cartilage infractions, noting articular surface defects and associated subchondral bone edema; subchondral edema was defined as focal regions of high signal intensity in the bone immediately underlying an articular surface defect on a T2-weighted or short inversion time inversion recovery (STIR) image. RESULTS: The first observer saw focal subchondral edema deep relative to a cartilage surface defect in 15 (83%) of the 18 cases; in two additional cases a surface defect was seen without underlying edema. The second observer identified 13 knees (72%) with surface defects and associated subchondral edema and three with chondral surface defects and no associated edema. Subchondral edema was seen more frequently on fat-suppressed images and on STIR images than non-fat-suppressed images. CONCLUSION: Focal subchondral edema is commonly visible on MR images of treatable, traumatic cartilage defects in the knee; this MR finding may prove to be an important clue to assist in the detection of these traumatic chondral lesions. PMID- 10749261 TI - High-field and low-field MR imaging of superior glenoid labral tears and associated tendon injuries. AB - OBJECTIVE: The purpose of this study was to determine the performance characteristics of high-field and low-field MR imaging for the diagnosis of a glenoid superior labral anteroposterior (SLAP) tear. MATERIALS AND METHODS: High field (n = 46) or low-field (n = 21) MR imaging was performed on 41 patients with SLAP tears and 26 patients with normal superior labra. The superior labrum was classified into one of four types on the basis of patterns of intralabral signal intensity. The relative frequency of rotator cuff tears and long head of the biceps tendinopathy was also assessed. RESULTS: For the diagnosis of SLAP tear, the sensitivity of high-field MR imaging was 90% (95% confidence interval = 74%, 98%), specificity was 63% (35%, 85%), and accuracy was 80% (66%, 91%). The sensitivity of low-field MR imaging was 64% (31%, 89%), specificity was 70% (35%, 93%), and accuracy was 67% (43%, 85%). A branched linear or stellate focus of abnormal intralabral signal intensity was associated with a SLAP tear in 86% of patients. Conversely, two other labral patterns correlated with a normal superior labrum in 71% of patients. Abnormal signal intensity in the biceps tendon was seen in 15% of patients with a SLAP tear. Full-thickness (37%) and partial thickness (31%) rotator cuff tears were often seen. CONCLUSION: The performance characteristics of high-field MR imaging are superior to those of low-field MR imaging for the diagnosis of a superior labral tear. Rotator cuff tears can be seen in many patients with superior labral tears, but abnormal signal intensity in the biceps tendon is uncommon. PMID- 10749262 TI - Using sonography to diagnose an unossified medial epicondyle avulsion in a child. PMID- 10749263 TI - Diagnostic imaging of thoracic aortic atherosclerosis. PMID- 10749264 TI - Pelvic and lower extremity arterial imaging: diagnostic performance of three dimensional contrast-enhanced MR angiography. AB - OBJECTIVE: The diagnostic performance of a three-dimensional MR angiography-based strategy was assessed with regard to its ability to characterize the arterial vasculature from the aortic bifurcation to the lower extremity runoff vessels. A single-injection, two-station protocol in combination with a lower-extremity vascular coil was used. SUBJECTS AND METHODS: Both conventional digital subtraction angiography and three-dimensional contrast-enhanced MR angiography with a dedicated peripheral vascular coil were performed in 61 patients with suspected peripheral vascular disease. In a prospective analysis, one reviewer evaluated the digital subtraction angiographic images and a second reviewer evaluated the MR angiographic images; both were unaware of the results of the other imaging technique. Each vascular segment (29 segments per patient) was evaluated for the presence of occlusive vessel disease. The following grading system was applied: 0, normal; 1, vessel irregularity with a luminal reduction of less than 10%; 2, mild stenosis (lumen reduction, 10-49%); 3, severe stenosis (lumen reduction, 50-99%); and 4, occlusion (lumen reduction, 100%). In 11 patients surgical graft patency was assessed. RESULTS: MR angiography provided an image quality comparable with that of digital subtraction angiography. Overall sensitivity and specificity for MR angiography were 92% and 96.6%, respectively, for the detection of hemodynamically significant disease and 92.3% and 99.4%, respectively, for the detection of occlusions. CONCLUSION: Two-station contrast enhanced three-dimensional MR angiography with a dedicated lower-extremity vascular coil proved effective enough to consider it as a noninvasive alternative to digital subtraction angiography in the assessment of the pelvic and lower extremity arterial vasculature. PMID- 10749265 TI - Intratesticular pseudoaneurysm after blunt trauma. PMID- 10749266 TI - MR angiography for the long-term follow-up of dissecting aneurysms of the extracranial internal carotid artery. AB - OBJECTIVE: We used MR angiography to examine and follow up the changes of dissecting aneurysms of the extracranial internal carotid artery (ICA). MATERIALS AND METHODS: We retrospectively reviewed the records of 101 consecutive patients with dissecting aneurysms of the extracranial ICA. Twenty patients with 26 spontaneous dissecting aneurysms were followed up with MR angiography every 1-2 years (men, 16; women, four; age range, 28-67 years; mean age, 51 years). RESULTS: The mean duration of follow-up was 41 months (range, 10-93 months). At MR angiography follow-up, 20 aneurysms did not change, four decreased from their original size by 33-53% (mean, 43%), and two resolved. One patient had an asymptomatic recurrent dissecting aneurysm of the extracranial ICA. Clinically, no patient had a thromboembolic stroke or transient ischemic attack during the follow-up period. CONCLUSION: MR angiography revealed that dissecting aneurysms of the extracranial ICA remain stable, decrease in size, or resolve--but they do not increase in size. PMID- 10749267 TI - Nasal T-cell/natural killer cell lymphoma: CT and MR imaging features of a new clinicopathologic entity. AB - OBJECTIVE: Nasal T-cell/natural killer cell lymphoma is a new clinicopathologic entity with a characteristic immunophenotypic profile and distinct clinical features. This study describes the radiologic features of nine cases of proven nasal T-cell/natural killer cell lymphoma. CONCLUSION: Nasal T-cell/natural killer cell lymphoma is often a locally destructive (T stages 3 and 4) disease, typically presenting with obliteration of the nasal passages and maxillary sinuses. Involvement of the adjacent alveolar bone, hard palate, orbits, and nasopharynx is found in more than 50% of cases and is associated with extensive soft-tissue masses. Presence of bone erosion is suggestive but not diagnostic of the disease. PMID- 10749268 TI - Perfusion MR imaging of brain neoplasms. PMID- 10749269 TI - Quantitative measurements of medical images for pharmaceutical clinical trials: comparison between on-site and off-site assessments. AB - OBJECTIVE: In pharmaceutical clinical trials, quantitative measurements on medical images are often conducted to confirm drug efficacy. This study aims to compare the quantitative image analysis performance of an off-site core laboratory with the performance of investigators from multiple clinical sites. MATERIALS AND METHODS: In a phase I clinical trial, 25 healthy subjects underwent dynamic brain single-photon emission computed tomography (SPECT) scintigraphy with 123I-Altropane, a cocaine analogue with high affinity and selectivity for dopamine transporter sites in the striatum. In 20 patients examined on-site and off-site, a total of 80 measurements were made to calculate the drug's binding potential. A trained technologist off-site at a central core laboratory and on site investigators at different clinical sites performed the image analysis. These results were compared with measurements made by a subspecialty radiologist whose assessments were the reference standard. Statistical analysis was performed using multiple regression analysis. RESULTS: Measurements from the central core laboratory (off-site) highly correlated (r = 0.95) with measurements of the reference standard. Measurements from the clinical sites (on-site) grouped together had lower correlation (r = 0.84) with the reference standard. This difference was statistically significant (p < 0.05). CONCLUSION: Training and experience in the specific type of image analysis are critical in obtaining consistent data. Quantitative analysis by dedicated personnel at a core laboratory provides highly reproducible results. The findings support off-site assessment of medical images in pharmaceutical clinical trials. PMID- 10749270 TI - Small-bowel stricture in a woman with oculocutaneous albinism (Hermansky-Pudlak syndrome). PMID- 10749271 TI - Cystic adrenal lymphangioma: incidental diagnosis on abdominal sonography. PMID- 10749272 TI - Granular cell tumor of the presacral space. PMID- 10749273 TI - Nasal chondroma. PMID- 10749274 TI - Tuberculous abscesses of the liver. PMID- 10749275 TI - MR imaging of small cell carcinoma of the uterus with associated inappropriate secretion of antidiuretic hormone. PMID- 10749276 TI - The predicted and observed course of psychotherapy for anxiety and mood disorders. AB - By applying hierarchical linear modeling (HLM) techniques, patient clinical characteristics at the beginning of treatment were used to predict individual patient responses (N = 160) to psychotherapy. Four diagnostic groups (mood, anxiety, other, and no diagnosis) were formed among the patients based on intake administered Structured Diagnostic Interview for the Diagnosis of DSM-III-R axis I Disorders. Patients with mood and anxiety disorders had predicted courses of response to psychotherapy that were similar but different from patients with other disorders and no diagnosis. Predicted and observed courses of response to psychotherapy in a subsample (N = 75) who had provided enough data to model the actual course of treatment showed high levels of congruence, thus supporting the validity of predicting course of response. HLM predictive profiling offers a new approach for assessing treatment effectiveness of psychotherapy with patients having axis I diagnostic conditions by considering an individual patient's clinical characteristics. PMID- 10749277 TI - A critical time intervention with mentally ill homeless men: impact on psychiatric symptoms. AB - We describe the impact of a psychosocial intervention, critical time intervention (CTI), on the cardinal symptom dimensions of schizophrenia, namely negative, positive, and general psychopathology. Ninety-six men with schizophrenia and other psychotic disorders who were discharged from a homeless shelter were randomly assigned to receive either CTI or usual services only. CTI is a time limited intervention designed to enhance continuity of care during the transition from institution to community. Symptom severity at baseline and at 6 months was assessed using the Positive and Negative Syndrome Scale. Using data on 76 subjects for whom we have complete symptom data, we assessed the impact of CTI on change in symptoms. The results suggest that CTI was associated with a statistically significant decrease in negative symptoms at the 6-month follow-up, reflecting modest clinical improvement. There was no significant effect on positive or general psychopathology symptoms. PMID- 10749278 TI - Mortality among homeless and nonhomeless mentally ill veterans. AB - This study directly compared mortality risk in homeless and nonhomeless mentally ill veterans and compared mortality rates in these groups with the general U.S. population. The study used a retrospective cohort design to assess mortality over a 9-year period in homeless (N = 6,714) and nonhomeless (N = 1,715) male veterans who were treated by Department of Veterans Affairs specialized mental health programs. The study showed that mortality rates in all homeless members of the cohort were significantly higher than the general U.S. population. Relative to nonhomeless cohort members, significant increases in mortality risk were observed in cohort members who at baseline were age 45 to 54 and had been homeless 1 year or less (RR = 1.55, 95% CI = 1.02, 2.36) and those age 55 and older who had been homeless 1 year or less (RR = 1.83, 95% CI = 1.33, 2.52). Similar, but nonsignificant trends were observed in cohort members who had been homeless more than 1 year at baseline. Additionally, medical problems at baseline and history of prior hospitalization for alcohol problems elevated mortality risk. Employment at baseline and minority group membership reduced mortality risk. The study suggests that mentally ill veterans served by specialized VA mental health programs are at elevated risk of mortality, relative to the general population. Homelessness increases this risk, particularly in older veterans, and this difference does not abate after entry into a health care system. PMID- 10749279 TI - Alcohol use, abuse, and related problems among children of problem drinkers: findings from a national survey of college alcohol use. AB - The purpose of this study was to estimate the prevalence of parental problem drinking reported by a national sample of college students and to examine the relations between such reports and respondents' patterns of drinking, alcohol abuse, abstinence, and utilization of treatment/counseling. Responses to mailed questionnaires completed by a random sample of 17,592 students at a representative national sample of 140 four-year colleges were analyzed using logistic regression. About 10% of college students reported problem-drinking parents. These children of problem drinkers (COPDs) exhibited a bimodal pattern of drinking behavior, showing higher than normal odds of past year abstinence or heavy episodic drinking. Males were more likely than females to report heavy episodic drinking, and children of affected mothers were at greatest risk. Approximately 23% of COPDs met proxy DSM-IV alcohol abuse criteria COPDs in college are a heterogeneous group showing both vulnerability and resistance. Those reporting treatment are more likely to be abstainers. PMID- 10749280 TI - Compulsivity and obsessionality in opioid addiction. AB - Seventy-one individuals with opioid dependence undergoing ambulatory treatment participated in a personal interview assessing the importance of their opioid-use rituals, the severity of compulsivity and obsessionality in relation to their drug use, and the number of obsessive compulsive disorder (OCD) symptoms. The level of compulsivity and obsessionality in opioid dependence was comparable to that found in OCD and alcohol addiction. The importance of rituals was inversely related to the number of opioid lapses during rehabilitation treatment and positively correlated with the number of non-drug-related OCD symptoms. Based on established norms for OCD symptoms, we estimated that 11.4% of our sample would meet diagnostic criteria for OCD, a rate which is over 4 times higher than the rate of OCD in the general population. PMID- 10749281 TI - Impact of combat and sexual harassment on the severity of posttraumatic stress disorder among men and women peacekeepers in Somalia. AB - The impact of combat and sexual harassment on the severity of posttraumatic stress disorder (PTSD) is compared for 1,307 men and 197 women peacekeepers who served in the same military units. A theoretical model was proposed to express the nature of the impact. Structural equation modeling was used to evaluate the model separately for men and women. Good-fitting, parsimonious models were developed that showed substantial similarity for men and women. For men, severity of PTSD symptoms was impacted by exposure to combat directly and indirectly through fear and sexual harassment. For women, severity of PTSD symptoms was impacted by combat indirectly through the same two influences, although the mechanisms involving fear and sexual harassment were somewhat different. For both genders, moreover, PTSD severity was impacted directly by exposure to the dying of the Somali people. These similarities suggest that in modern stressful overseas military missions, both genders may be susceptible to the same types of risk for the development of PTSD. The incidence and impact of sexual harassment is particularly noteworthy in the case of men and calls for more detailed investigation in future studies. PMID- 10749282 TI - Quality of life for patients with body dysmorphic disorder. AB - Body dysmorphic disorder (BDD) is a relatively common, distressing, and impairing disorder. Quality of life in BDD, however, has not been investigated. In this study, 62 consecutive outpatients with BDD were evaluated with the self-report Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36) and other scales. SF-36 scores were descriptively compared to published norms for several populations. Physical health-related quality of life scores were generally worse than general U.S. population norms and better than norms for outpatients with a medical illness or depression. However, in all mental health domains, BDD subjects' scores were notably worse than norms for the general U.S. population and for patients with depression, diabetes, or a recent myocardial infarction. More severe BDD symptoms and greater delusionality were associated with poorer mental health-related quality of life. These results indicate that patients with BDD have notably poor mental health status and mental health-related quality of life. PMID- 10749283 TI - Development and validation of a mania scale for the symptom checklist 90. PMID- 10749284 TI - Expression of the endocytosis regulatory proteins Rab5 and Rabaptin-5 in glial cytoplasmic inclusions from brains with multiple system atrophy. AB - BACKGROUND: Glial cytoplasmic inclusions (GCIs) occur specifically in oligodendrocytes in brains with multiple system atrophy (MSA). Oligodendrocytes in MSA appear to be functionally altered in their nature in terms of the occurrence of GCIs and aberrant expression of various proteins such as neuron specific protein, MAP2 or pre-synaptic protein, ct-synuclein. The present study examined whether or not aberrant expression of the endocytosis regulatory proteins Rab5 and Rabaptin-5 occcur in oligodendrocytes of brains with MSA. MATERIALS AND METHODS: We examined immunohistochemically the post-mortem brain tissues from 5 patients with MSA and 5 controls. Immunohistochemistry was done using monoclonal anti-Rab5 and anti-Rabaptin-5 antibodies based on ABC method. RESULTS: We have observed Rab5 and Rabaptin-5 immunoreactivity in the neuronal somata and axons of the controls, suggesting that Rab5 and Rabaptin-5 are involved in the regulation of the endocytosis in neurons of the human central nervous system. In the brain tissues from patients with MSA, we have found Rab5 and Rabaptin-5 immunoreactivity in GCIs. CONCLUSION: Rab5, in association with Rabaptin-5, has been demonstrated in the early endosome and regulates the endocytosis. Since Rab5 and Rabaptin-5 have been immunolocalized to neurons in the human brains, we propose that oligodendrocytes may ectopically express Rab5 and Rabaptin-5 in MSA. Thus, the oligodendrocytes in MSA brains appear to be functionally significantly altered, which may be associated with the formation of GCIs in the oligodendrocytes. PMID- 10749285 TI - Bcl-2, bcl-x, and bax expression in dysembryoplastic neuroepithelial tumors. AB - BACKGROUND: Bcl-2, bcl-x and bax are regulatory proteins which are variably expressed in brain tissue and are known to be involved in the regulation of apoptosis; bcl-2 and bcl-x inhibit apoptosis and bax generally promotes apoptosis. This study is a retrospective clinicopathologic and immunohistochemical review of 18 dysembryoplastic neuroepithelial tumors (DNTs), specifically looking for evidence of aberrant expression of apoptosis regulatory proteins which may promote the survival of one or more of the cellular constituents of the tumor. MATERIALS AND METHODS: Eighteen patients (11 males) with DNTs comprise the study group. Patients at the time of surgery ranged in age from 2.1-52 years (mean 16.1 years). Mean seizure duration prior to surgery was 6.4 years (n = 16 patients). Sixteen patients were alive with markedly reduced or no seizures at a mean postsurgical follow-up interval of 63 months; 2 patients were lost to follow-up. RESULTS: All tumors were characterized by an admixture of oligodendroglial cells, neurons and astrocytic cells, focally arranged against a microcystic background. Coexistent cortical dysplasia was noted in 14 evaluable cases. Mitotic figures were rarely noted in 2 tumors. MIB-labeling indices ranged from 0-0.6 (mean 0.2). Astrocytes which were part of the tumor stained with all three antibodies in all cases. The oligodendroglial-like cells of DNT stained positively for bcl-2 in 2/17 tumors, bcl-x in 10/17 tumors, and bax in 12/17 tumors. The neuronal cell component of the DNT stained positively with bcl-2 in 15/17 tumors, bcl-x in 5/17 tumors, and bax in 8/17 tumors. CONCLUSION: Aberrant expression of apoptosis-associated proteins, similar to what has been previously described in gangliogliomas (another epilepsy-related, dysplasia-associated tumor), may play a role in the pathogenesis of DNT. PMID- 10749286 TI - Sinonasal teratocarcinosarcoma ("mixed olfactory neuroblastoma craniopharyngioma") presenting with syndrome of inappropriate secretion of antidiuretic hormone. AB - Sinonasal teratocarcinosarcoma (SNTC) is a rare, aggressive, histologically heterogeneous neoplasm of the paranasal sinuses and nasopharnyx of adults that is composed of variably benign or malignant neuroepithelial, epithelial, and mesenchymal elements. Occasional cases show intracranial extension and may be operated on by neurosurgeons and encountered by neuropathologists who may not be familiar with the entity. STNCs have not previously been associated with functional hypersecretory status. We report a 59-year-old male who presented with headache and syndrome of inappropriate secretion of antidiuretic hormone (SIADH) and was subsequently found to have a bulky tumor of the frontal and ethmoid sinuses with focal dural invasion. The tumor was predominantly composed of olfactory neuroblastoma areas (90% of tumor) admixed with unusually well developed craniopharyngioma-like mature squamous epithelium and ghost cells ( 10% of tumor). Scattered neuroblastoma tumor cells showed strong immunoreactivity with antibodies to arginine vasopressin, supporting ectopic hormone secretion by the tumor. While the coexistence of neuroectodermal and oral ectodermal-like differentiation in SNTCs is characteristic, in our case it was developed to an extreme functional and morphologic degree and was unassociated with other mesenchymal or epithelial elements often found in these complex tumors. SNTCs with limited differentiation have prompted controversy in classification. PMID- 10749287 TI - Endoneurial vasculitis and tubuloreticular inclusions in peripheral nerve biopsy. AB - We describe 3 patients in whom nerve biopsy revealed endothelial tubuloreticular inclusions in association with peripheral nerve endoneurial vasculitis. Two of the patients had systemic lupus erythematosus (SLE), while the third was HIV positive. Review of our biopsy material featuring the much more common finding of epineurial vasculitis failed to disclose any instances in which endothelial tubuloreticular inclusions (TRIs) were present. We conclude that TRIs and endoneurial vasculitis are closely associated. Moreover, if detected on a nerve biopsy specimen, TRIs are very suggestive of SLE or HIV infection. Finally, literature evidence is cited to suggest that an "acid-labile" alpha-interferon may be pathogenically related to the vasculitic process in these patients, perhaps through a process mediated by tumor necrosis factor. PMID- 10749288 TI - Neuron death and glial response in pontosubicular necrosis. The role of the growth inhibition factor. AB - AIM AND METHODS: Fluorochrome and immunohistochemical studies were performed on neonates with pontosubicular necrosis (PSN), aged 26 - 42 weeks of gestation (GW), compared with preterm and term controls aged from 10 GW to 3 months of age. RESULTS: A fluorochrome study using a confocal microscope revealed that nuclear DNA changes occurred earlier than cytoplasm degeneration with diminished RNA orange-red fluorescence. These changes were restricted to the small immature neurons in the pons and subiculum with PSN. On the other hand, although glial fibrillary acidic protein-positive reactive astrocytes were not increased in number, growth inhibitory factor- (GIF) immunoreactive glia with vesicular large nuclei were increased in number within the gray matter of the pons, subiculum, and cerebral cortex in the PSN group. The nuclei of GIF-containing astrocytes became round and vesicular, nearly twice in size and increased in number. Thus, the neuronal death began at the nuclei of selective neurons in specific areas in PSN, although GIFcontaining astrocytes were increased in widespread areas. CONCLUSION: These facts suggest that immature neurons in the pontine nuclei and subiculum are selectively vulnerable to some insults such as hypocarbia and hyperoxygenation, and PSN involves a possible apoptotic neuron death mechanism and a characteristic glial response. PMID- 10749289 TI - A case of Joubert's syndrome with extensive cerebral malformations. AB - Joubert's syndrome is a relatively rare, autosomal-recessive syndrome defined by vermis hypoplasia, hypotonia, developmental delay and at least one of two additional manifestations: abnormal breathing pattern or abnormal eye movements. Detailed descriptions of the neuropathological findings in this syndrome are scarce. We present a radiological and pathological correlation of a case of Joubert's syndrome in which, apart from the classic vermis aplasia and some malformations of the brain stem and the spinal cord, extensive malformations of the cerebrum were found. The dentate nuclei were broken into islands and showed a few heterotopias within the superior cerebellar pedunculi, the inferior olives were plump and dysplastic, and an almost complete absence of the pyramidal decussation was found. In the spinal cord, the dorsal columns were located in a dorsal position within the spinal grey matter. In the cerebrum, absence of the corpus callosum was found. Many nodular heterotopias of the cerebral cortex and of the basal ganglia, the amygdala and the diencephalon were observed. The present case is compared to previous pathological descriptions of the Joubert syndrome and to other syndromes with comparable malformations of the posterior fossa. PMID- 10749290 TI - An autopsy case with clinically and molecular genetically diagnosed Huntington's disease with only minimal non-specific neuropathological findings. AB - An autopsy case with clinically and molecular genetically diagnosed Huntington's disease (HD) accompanied with minimal non-specific neuropathological features was reported. When the patient was 45 years old, he had faulty memory, mood swing, personality change and agitation. Neurological and psychiatric examinations revealed choreoathetoid movements in limbs and trunk, generalized hyperreflexia and mental deterioration. However, cerebellar ataxia and muscle rigidity were not disclosed. Neuroimaging study did not show a definite atrophy of heads of caudate nuclei. Neuroacanthocytosis and Wilson's disease were ruled out by the peripheral blood examination and serum Cu and ceruloplasmin examination. At the age of 55 he died of pneumonia. Post-mortem examination revealed minimal non-specific neuropathological features for HD (Vonsattel's grade 0), that is, no visible fibrillary gliosis in the striatum, and few neuronal loss and only proliferation of astrocytes (astrocytosis) in the striatum. Molecular-genetic study the patient's brain tissues and his youngest son's blood was performed. These studies revealed 40 CAG repeats in the patient, 56 CAG repeats in his youngest son. These results suggest they may be HD. Vonsattel et al. [ 1998] insist that grade 0 comprises 1% of all HD brains, and grade 1 comprises 4% of all HD brains. But we could not find any reports in which the clinical and neuropathological features were described in detail on the cases with clinically and molecular genetically diagnosed HD without specific pathological findings. Therefore, we present in detail the clinical and neuropathological features of such case. PMID- 10749291 TI - Benign glandular peripheral nerve sheath tumor of the seventh and eighth cranial nerve complex. AB - A unique, deep-seated, benign, glandular peripheral nerve sheath tumor (PNST) of the cerebellopontine angle is described. The tumor arose from the seventh and eighth cranial nerve complex in a 15-year-old boy without neurofibromatosis. Histologically, benign glands were embedded in a bland spindle cell stroma. The epithelial cells were immunoreactive for CAM 5.2, and focally for chromogranin. The spindle cells were positive for S100P. The benign glandular PNST unassociated with neurofibromatosis is a controversial entity. The superficial location of most reported cases has made it difficult to exclude entrapped adnexae as a source for the glands. This tumor was separate from the internal auditory meatus on MRI scan, the most likely source of entrapped glands at this site. This case is the first report of a deep-seated, benign, glandular peripheral nerve sheath tumor. It suggests that glandular differentiation in PNSTs, while unusual, is not synonymous with neurofibromatosis or malignancy. PMID- 10749292 TI - Salt intake and hypertension in renal transplant patients. AB - BACKGROUND: Dietary salt restriction is currently widely recommended as an important non-pharmacological measure for the treatment of hypertension. However, the relationship between dietary salt intake and post-transplant hypertension has not been extensively investigated. PATIENTS AND METHODS: We examined the relationship between dietary salt intake and the prevalence of hypertension in 129 renal transplant patients with stable allograft function (serum creatinine < 400 micromol/l, variation in serum creatinine during the preceding two months < 20%). Salt intake was assessed by measuring 24-hour urinary excretion of sodium on an unrestricted diet. Hypertension was defined based on the prescription of antihypertensive medication, and the number of antihypertensive drugs was considered a surrogate marker for severity of hypertension. Patients were divided into tertiles based on urinary sodium excretion and analyzed by chi2-testing. RESULTS: The prevalence of hypertension was 74% and the mean sodium excretion was 178 mmol/d (range: 56 to 603). There was no statistical difference in the frequency of antihypertensive medication between patients with low (76%, UNa = 107 mmol/d), medium (73%, UNa = 178 mmol/d), or high sodium (73%, UNa = 272 mmol/d) excretion. Furthermore, the number of antihypertensive drugs (in treated patients) was similar between the tertiles. There was also no correlation between urinary sodium excretion and systolic (r = -0.05) or diastolic (r = 0.08) blood pressure levels. CONCLUSION: We conclude that dietary salt intake in transplant patients with stable allograft function is higher than currently recommended. There is, however, no relationship between salt intake and the prevalence of hypertension in these patients. These data do not support the hypothesis that the prevalence or severity of post-transplant hypertension is markedly affected by dietary salt intake. PMID- 10749293 TI - Hypertension in chronic hemodialysis patients: current view on pathophysiology and treatment. AB - BACKGROUND: Hypertension accounts for 65 - 85% of patients beginning dialysis, and dialysis alone controls hypertension in over 50% of patients. PATIENT AND METHODS: We have surveyed the status of BP control in 113 hemodialysis patients, 66 men and 47 women, aged 59 +/- 13 years old, with a mean duration on hemodialysis 42 +/- 44 months. The following measurements were recorded: predialysis mean arterial pressure (pre-MAP), post-dialysis MAP (post-MAP), percentage of change in MAP, pre-dialysis weight, post-dialysis weight, fluid removed by ultrafiltration during each dialysis session, interdialytic weight gain and excess weight over the desirable dry weight. RESULTS: Our results showed a hypertension prevalence of 59% (hypertension defined as pre-MAP +/- 110 mmHg). MAP was not different between men and women, and only 4.5% of patients had isolated systolic hypertension. All hypertensive patients were on treatment with antihypertensives. Reduction in post-MAP by > or = 5% (controlled by ultrafiltration) was found in 68.5% of hypertensive and in 87.5% of normotensive patients. Age, primary renal disease, time on dialysis and adequacy of dialysis were not correlated with pre-MAP. Excess volume and interdialytic weight gain were found to correlate with pre-MAP (p = 0.03). Also, the weekly dosage of EPO had a significant correlation with pre-MAP (p = 0.03). No differences were found among four classes of antihypertensive drugs regarding the BP control. Patients with hypertension requiring one drug achieved a significantly (p < 0.05) lower pre-MAP than the group of patients receiving three or more drugs. In conclusion, hemodialysis population shows high prevalence of hypertension, resistant to antihypertensive treatment. CONCLUSION: Current methods of hemodialysis are not effective in controlling BP. This implies that more insight into the role of excess volume and vasomotor systems in the pathogenesis of dialysis hypertension is warranted. PMID- 10749294 TI - Oxidative stress status in adults with nephrotic syndrome. AB - BACKGROUND: Excessive generation of reactive oxygen species is one of the incriminated mechanisms in the pathogenesis of progressive renal injury. The role of oxidant stress in acute and chronic glomerular diseases has been investigated through experimental and clinical studies. SUBJECTS, MATERIALS AND METHODS: In the present study, oxidative stress status in adult nephrotic patients was studied by determining plasma selenium levels, erythrocyte and plasma glutathione peroxidase (GSH-Px) activities, erythrocyte superoxide dismutase (Cu-Zn-SOD) activity, erythrocyte and plasma levels of malondialdehyde (MDA). RESULTS: Twenty adult nephrotic syndrome patients included into the study had lower activities of erythrocyte (17.17 +/- 2.29 U/gHb) and plasma (153.76 +/- 20.12 U/l) GSH-Px activities when compared the controls ( 27.05 +/- 7.30 U/gHb and 308.89 +/- 55.04 U/l for erythrocyte and plasma GSH-Px activities, respectively). They also had lower erythrocyte Cu-Zn-SOD activity (1896.30 +/-94.31 U/gHb) than that of the controls (2506.17 +/- 461.08 U/gHb). Erythrocyte (483.40 +/- 37.45 nmol/gHb in patients vs 210.35 +/- 55.55 nmol/gHb in controls) and plasma (4.84 +/- 0.65 nmol/ml in patients vs 2.03 +/- 0.41 nmol/ml in controls) levels of MDA were higher in patients. Plasma selenium levels of the patients (48.0 +/- 7.28 ng/ml) were lower than that of the controls (69.25 +/-5.80 ng/ml). CONCLUSION: In conclusion, these results obtained in adult nephrotic syndrome patients support the previous data indicating an abnormality in antioxidative system of nephrotic patients. PMID- 10749295 TI - Does severe nutcracker phenomenon cause pediatric chronic fatigue? AB - BACKGROUND: In the past five years we experienced 9 fatigued disabled children who were intermittently or persistently absent from school. PATIENTS: They had been suspected to be burdened with psychosomatic disorders, having orthostatic hypotension, postural tachycardia, or other autonomic dysfunction symptoms. RESULTS: Investigating the cause of moderate orthostatic proteinuria in some of them, we found by chance severe typical nutcracker phenomenon (NC), which was present in all 9 children complaining of chronic fatigue. CONCLUSION: Their symptoms filled the criteria of chronic fatigue syndrome or idiopathic chronic fatigue (CFS/CF). An association between severe NC and autonomic dysfunction symptoms in children with CFS/CF has been presented. PMID- 10749296 TI - A 10-year follow-up study of renal transplant recipients treated with cyclosporine. Japanese Cyclosporine Kidney Transplant Study Group. AB - AIM: In this report a 10-year follow-up of Japanese multicentre trial of renal transplantation using cyclosporine is described. MATERIAL AND METHODS: A total of 1,323 recipients, including 1,055 (79.7%) living donor (LD) transplantations and 268 (20. 3%) non-heart-beating cadaveric donor (CD) transplantations, were enrolled in the study. RESULTS: Favorable results; graft survival at 1, 5 and 10 years were 95.8%, 80.9%, 56.8%, respectively, for LD, and 88.7%, 74.2%, 58.8%, respectively, even for CD, were obtained. No serious adverse reactions or complications were observed in this group of patients excepting 28 cases (2.0%) of malignant tumors. There were no cases of abnormality in 49 babies delivered during the term. CONCLUSION: We demonstrate the interesting findings that graft survival of non-heart-beating CD are surprisingly as good as that of LD and the safety issues are almost equal to other available data in other countries. PMID- 10749297 TI - Twice versus thrice weekly administration of intravenous calcitriol in dialysis patients: a randomized prospective trial. Gruppo Italiano di Studio dell'Osteodistrofia Renale. AB - BACKGROUND: Administration of intravenous (i.v.) calcitriol three times weekly effectively controls the synthesis and secretion of PTH in most uremic patients. Administration of a single dose of 1.25(OH)2D3 reduces synthesis of PTH-mRNA for 6 days in rats. Moreover, it can lower PTH levels for up to 4 days in chronic hemodialysis patients. Therefore, a good response to the administration of i.v. calcitriol two times weekly can be expected. We studied - in a multicenter randomized study in patients with moderate to severe secondary hyperparathyroidism - the effects of the same doses of intravenous calcitriol, administered two or three times weekly. METHODS: Twenty-two hemodialysis patients were randomized into two frequencies of treatment groups: two times (G-2/w) and three times weekly (G-3/w). Both groups were treated with increasing doses of intravenous calcitriol for 3 months (first month 3 microg, second month 4 microg, third month 6 microg weekly). RESULTS: After 12 weeks of therapy with intravenous calcitriol the G-2/w group showed a significant reduction in serum PTH levels (from 821 +/- 392 to 350 +/- 246 pg/ml; mean reduction = 57.4%) comparable to the decrease observed in the G-3/w group (from 632 +/- 116 to 246 +/- 190 pg/ml; mean reduction = 61.2%). Ionized calcium (G-2/w from 1.13 +/-0.10 to 1.14 +/- 0.08 and G-3/w 1.21 +/- 0.13 to 1.26 +/- 0.18 mmol/l) and phosphate levels (G-2/w from 4.99 +/- 1.01 to 5.99 +/- 1.78 and G-3/w 5.31 +/- 0.73 to 5.81 +/- 1.18 mg/dl) did not change significantly and phosphate binders were not modified during the study. CONCLUSION: This study confirms that intravenous calcitriol is an effective therapy for moderate to severe secondary hyperparathyroidism. The administration of two doses per week of intravenous calcitriol is as efficacious as three doses per week in suppressing PTH secretion. PMID- 10749298 TI - Serum bile acids and pruritus in hemodialysis patients. AB - BACKGROUND: Chronic renal failure (CRF) patients usually suffer from pruritus. The pathophysiology of pruritus is still incompletely understood. SUBJECTS, MATERIALS AND METHODS: In this paper we determined serum total bile acids (STBA) in hemodialysis patients with advanced CRF (ACRF) in order to obtain STBA concentration in predialysis, to assess their probable relation among patients with pruritus and in postdialysis using a polysulfone membrane for dialysis. STBA were determined in 49 ACRF patients with chronic hemodialysis and values were compared to 20 control subjects. Hemodialysis patients were divided in two groups: with and without pruritus. In all these patients, month of renal replacement therapy, diabetic patients, dose of dialysis (Kt/V), viral markers, serum creatinine, serum glucose, aspartate and alanine aminotransferase, alkaline phosphatase, hematocrits and albumin were determined. The intensity of itching among pruritic patients was measured by a score system: mild (M), moderate (MO) and severe (S). RESULTS: No significant differences were found in patients with and without pruritus in months of renal replacement therapy, duration of dialysis or dose of dialysis (Kt/V). STBA were determined in all ACRF patients in predialysis and they showed significant differences compared to controls (p < 0.05), however, no differences were observed in the results obtained when control subjects were compared to ACRF patients without pruritus. Also in predialysis, pruritic patients showed significant differences in STBA compared to patients without pruritus (p < 0.001). STBA concentration showed a significant decrease in postdialysis using a polysulfone membrane in ACRF patients with and without pruritus. Finally, correlation with STBA and itch score of pruritus was significant (p < 0.02). CONCLUSION: Hemodialysis patients with ACRF and pruritus showed an increase of STBA in predialysis and a decrease in postdialysis. PMID- 10749299 TI - Factors influencing function of temporary dialysis catheters. AB - AIM: To determine risk factors for failure of temporary dialysis catheters, we prospectively studied the outcome of 178 non-tunneled dual lumen catheters placed in 126 consecutive patients requiring treatment of acute renal failure (ARF) or end-stage renal disease (ESRD). METHODS: Internal jugular (IJ) or subclavian (SC) catheters were used in 122 instances and femoral catheters were employed in 56. RESULTS: IJ or SC catheters with tips in the right atrium or superior vena cava (n = 112) failed (defined as a blood flow < 250 ml/min) 17% of the time, compared with a 40% failure rate for catheters with more peripherally located tips (n = 10), p < 0.05, chi2 testing. In a multivariate analysis, use in ESRD and location peripheral to the SVC were risk factors for catheter failure. Use of one of three catheter brands was associated with a lower failure rate. Although mean venous pressures at 200 ml/min blood flow were higher in IJ or SC catheters that failed, the presence of a high venous pressure, number of catheter uses, IJ vs. SC placement, inpatient vs. outpatient status, and fresh venipuncture vs. placement over a guidewire passed through a previous catheter did not predict catheter malfunction. With femoral catheters, the only risk factor for failure was use in ESRD. CONCLUSION: Of the factors that can be influenced by placement technique, catheter tip location is most important. Whether one catheter brand is superior awaits further confirmation. PMID- 10749300 TI - Membranoproliferative glomerulonephritis in congenital portosystemic shunt without liver cirrhosis. AB - We report two cases with congenital portosystemic shunt who developed renal lesions without liver cirrhosis. Clinically, both cases showed proteinuria and mild hematuria at 9 and 6 years of age, respectively. In one case, the renal lesion was associated with normal renal function, but nephrotic syndrome followed by chronic renal failure were noted in the other. Renal biopsies showed characteristic histological features of membranoproliferative glomerulonephritis (MPGN) with IgA deposits along the glomerular capillary wall. Our cases strongly suggest the association between congenital portosystemic shunt and renal region. The shunt ratio may be an important predisposing factor for this type of nephropathy since a high shunt ratio (> 90%) was noted in both cases. PMID- 10749301 TI - Focal segmental glomerulosclerosis in a patient with Prader-Willi syndrome. AB - The authors describe a girl with Prader-Willi syndrome associated with focal segmental glomerulosclerosis. Severe obesity and unilateral renal agenesis, taken together, may have played an important role in the development of her specific renal disease. PMID- 10749302 TI - Von Hippel-Lindau disease with multiple malignant renal tumors: the importance of genetic testing. AB - We present an adult man who, while being evaluated for gross hematuria, was found to have polycystic kidneys and multiple bilateral renal cell carcinomas. Further evaluation and the presence of a significant family history of cancer suggested the diagnosis of von Hippel-Lindau disease. Through the aid of genetic testing, this unusual diagnosis was confirmed and led to the identification of other family members with the von Hippel-Lindau gene. Patients with von Hippel-Lindau disease have an increased incidence of malignant carcinomas and the syndrome can mimic the presentation of other cystic diseases of the kidney. Early diagnosis and genetic screening of family members is essential to improve the prognosis and survival of those affected. PMID- 10749303 TI - A case of transient blindness in a postoperative hyponatremic child. AB - Hyponatremia represents a common electrolyte disorder in postoperative patients. Headache, nausea, emesis, weakness and lethargy are all consistent symptoms of hyponatremia in children. Among the neurologic symptoms that are infrequently seen in severe hyponatremic pediatric patients seizure is included while visual disorders are not. We report a case of an 8-year-old boy who underwent abdominal surgery and developed severe hyponatremia. Five days after surgery the child suffered a seizure and successively experienced bilateral visual loss. However, after prompt correction of serum sodium concentration, a complete resolution of the blindness was obtained. Thus, we discuss the present case speculating on the pathogenesis of hyponatremic blindness and on its possible therapeutic approach. In conclusion, we suggest that blindness is to be considered a rare symptom that can occur in the clinical scenario of hyponatremia and we report its complete reversibility after timely treatment of hyponatremia. PMID- 10749304 TI - DDAVP to prevent rapid correction in hyponatremia. AB - Correction of hyponatremia can be complicated by brisk free water diuresis with a rise in the serum sodium (s-Na) in excess of the generally accepted rate of 10-15 mmol/l/24 hours. We describe this complication and its treatment with desmopressin (dD-AVP), in a 56-year-old female with severe hyponatremia secondary to polydipsia and antidiuretic (ADH) activity. The patient developed a large free water diuresis with a markedly dilute urine (urine osmolality 61 mmol/kg) and a rise in the serum sodium of 19 mmol/l in 19 hours despite the addition of large volumes of free water intravenously and orally. To reduce the free water excretion, desmopressin (dD-AVP) 8 microg was given intravenously. This resulted in a rise in the urinary osmolality, a reduction in the urine volume, and a 2 mmol/l reduction in the serum sodium. Thereafter, the serum sodium rose 4 mmol/l in 24 hours. There were no neurological sequellae. In cases of appropriate but rapid correction of hyponatremia secondary to rapid free water diuresis, dD-AVP can safely reduce the free water excretion, slow the rate of correction of the serum sodium and simplify the fluid therapy of the patient. PMID- 10749305 TI - Remission of a recurrent carpal tunnel syndrome by a new device of the hemodialysis method in a long-term hemodialysis patient. AB - This report concerns a case in which remission was achieved from the recurrent carpal tunnel syndrome employing new methods of hemodialysis. These being the maintenance of low endotoxin in dialysate, a highly permeable membrane and a 32 microglobulin-adsorbent column. A 78-year-old female patient with a 19-year history of hemodialysis was diagnosed as being a suitable recipient of a third operation. The concentration of endotoxin was maintained at under 10 EU/l and the highly permeable dialyzer with a larger sieving coefficient of beta2 microglobulin was introduced. A Lixelle adsorption column for beta2-microglobulin removal was also introduced and the serum concentration of the beta-microglobulin was maintained at under 20 mg/dl. Consequently, within 6 months the symptoms in the right hand had completely disappeared, the motor nerve latency had almost normalized at 5.0 msec and no recurrence was observed. PMID- 10749306 TI - Glucose-free standard hemodialysis and occult hypoglycemia. PMID- 10749307 TI - Glomerulonephritis and Henoch-Schoenlein purpura associated with acute parvovirus B19 infection. PMID- 10749308 TI - A framework describing components of renal physician's accountability. PMID- 10749309 TI - Retina. Introduction. PMID- 10749310 TI - Intraocular gas tamponades. AB - Intraocular gas tamponades are an important part of vitreoretinal surgery and have become a standard of care. The use of intraocular gas began in 1911. Modem use of intraocular gases as a surgical adjunct is continuously broadening. Today, sulfur hexafluoride and perfluoropropane are increasingly being used as intraocular gas tamponades for a wide range of complicated vitreoretinal diseases. This article reviews the types and function of commonly used gases, their indications and efficacy in certain types of vitreoretinal diseases, and their complications. PMID- 10749311 TI - Silicone oil and fluorosilicone. AB - Silicone oil has been used to fill the vitreous cavity for long-term or permanent internal tamponade in eyes with proliferative vitreoretinopathy or complicated retinal detachment due to ocular trauma, giant retinal tears, proliferative diabetic retinopathy, and cytomegalovirus retinitis. Reports from the Silicone Study confirmed its efficacy in the treatment of proliferative vitreoretinopathy and addressed outcome differences in vitrectomized and nonvitrectomized eyes, combined retinotomy, silicone oil removal, and complications associated with silicone oil tamponade, such as intraocular pressure abnormalities and corneal abnormalities. Because silicone oil is lighter than water and not adequate in supporting the inferior quadrants, several heavier-than-water materials have been introduced for intraocular tamponade. Silicone oil can be a potential vehicle for delivering antiproliferative agents to treat proliferative vitreoretinopathy. PMID- 10749312 TI - Perfluorocarbons and semifluorinated alkanes. AB - The introduction of perfluorocarbons (PFCs) and, more recently, semifluorinated alkanes (SFAs) has greatly facilitated vitreoretinal surgery. A distinction is made between the use of these substances as intraoperative tools and internal tamponade agents. This article reviews the physical and chemical properties of PFCs and SFAs and discusses the indications, results, and complications. The effectiveness of these substances as internal tamponade agents is discussed with reference to the specific gravity, contact angle, viscosity and ability to fill model eye chambers and the vitreous cavity. The evidence for the toxicity in animal and human is examined. PMID- 10749313 TI - Intraoperative adjunctive agents in vitrectomy: serum, cytokines, and glue. AB - The surgical goal in the treatment of retinal breaks is to seal the edges of the break, and traditionally, photocoagulation and cryocoagulation have been used to accomplish this. However, it is sometimes difficult in complicated retinal detachments to maintain the seal against tractional forces. Adhesion is achieved through a process of cell necrosis, inflammation and subsequent fibrovascular proliferation. This strategy, however, may not be appropriate in vision-sensitive areas such as macular holes. To improve the success rate of macular hole surgery, a number of authors have advocated the use of biological modifiers, such as transforming growth factor beta, human autologous serum, tissue glue, or platelet concentrates. These materials may enhance the adhesion of the detached retina and therefore lead to a better anatomical and functional success. We have reviewed the advances of intraoperative application of synthetic or biological adhesives. However, through the improvement of surgical techniques and surgeons' skills in recent years, the anatomical success rate of macular hole surgery has increased in most institutions without adjunctive additives. Thus, many surgeons believe that adjunctive additives may not be necessary for most idiopathic macular holes. PMID- 10749314 TI - Tissue plasminogen activator in the treatment of vitreoretinal diseases. AB - Tissue plasminogen activator (tPA) is a thrombolytic agent that activates plasminogen into plasmin almost exclusively in the presence of fibrin. Intraocular injection of tPA has been proposed for the treatment of vitreoretinal diseases, such as vitreous hemorrhage, postvitrectomy fibrin formation, submacular hemorrhage, retinal vascular occlusive disorders, suprachoroidal hemorrhage and endophthalmitis. Currently, intraocular tPA is only used in the treatment of postvitrectomy fibrin formation and submacular hemorrhage. For other indications, tPA has not been shown to be safe or effective. This article reviews the use of tPA in the treatment of vitreoretinal disorders. PMID- 10749315 TI - Pharmacological vitrectomy. AB - Pharmacological vitrectomy refers to the use of enzymes in an effort to liquefy vitreous and to weaken the adhesion of vitreous cortex to the internal limiting membrane during or before performing vitreous surgery. It is well known that the vitreoretinal interface plays important roles in developing many blinding diseases. To make the vitreous surgery easier for better outcome or to avoid vitrectomy, plasmin, dispase, and chondroinase have been used to promote the disinsersion of vitreous cortex to the internal limiting membrane, a basement membrane of Muller cells. On the other hand, hyaluronidase has been used clinically to facilitate the clearance of vitreous hemorrhage liquefying vitreous body and developing posterior vitreous detachment. This article reviews enzymes as an intraoperative adjunctive agent in vitrectomy. PMID- 10749316 TI - Antibacterial therapy for Crohn's disease: a review emphasizing therapy directed against mycobacteria. AB - The most commonly used antibiotics in Crohn's disease are nitroimidazoles and macrolides often combined with corticosteroids or sulfasalazine. There has been interest in a mycobacterial involvement in Crohn's disease since its earliest description. It is not recognized that Mycobacterium avium subspecies paratuberculosis, a proven but uncommon cause of human disease, is widespread in the human food chain especially in dairy products and beef. M. paratuberculosis has been identified in tissues from a higher proportion of Crohn's disease patients than controls, suggesting that it may be one of the causes of Crohn's disease. We review the large number of antibiotic trials in Crohn's disease. Although studies have been performed with many different protocols and variations in the definition of success, preliminary reports of multiple drug therapies are encouraging. Nevertheless, large-well designed preferably placebo-controlled studies are needed before one could recommend such therapy. PMID- 10749317 TI - Discrepancies between effects of recombinant human growth hormone on absorption and secretion of water and electrolytes on the human jejunum compared to results reported on rat jejunum. AB - Previous studies in rats showed that the administration of recombinant human growth hormone markedly increased intestinal absorption of electrolytes and water and suggested that growth hormone would be a useful antidiarrheal agent. We therefore examined the effect of recombinant human growth hormone on the human jejunum in vivo, using a triple lumen nonabsorbable marker technique. Healthy subjects were studied on two different test days, one as a control and a second where recombinant human growth hormone was injected subcutaneously in a dose of 100 microg/kg. With this dose we achieved equal or higher growth hormone serum levels than in previous rat studies. However the administration of recombinant human growth hormone did not stimulate absorption or inhibit secretion of water and electrolytes in the human jejunum in vivo. We believe that the discrepancy between humans and rats is most likely due to the species difference rather than to differences in methods that were used. Therefore recombinant human growth hormone cannot be considered a useful proabsorptive antidiarrheal agent in humans. PMID- 10749318 TI - Age-related clinical severity at diagnosis in 1705 patients with ulcerative colitis: a study by GISC (Italian Colon-Rectum Study Group). AB - Clinical-endoscopic parameters of UC presentation were studied in 1705 out patients, observed consecutively in 17 Italian gastroenterology centers (males 60.2%; average age at diagnosis 38.5 +/- 16.4 years), and were subdivided arbitrarily into quartile age groups at diagnosis (0-25, 26-35, 36-50, >50). A significantly greater prevalence in males, increasing with age, was shown at diagnosis (P = 0.0002), which seems to correlate with the condition of being an ex-smoker, most frequently found in males. The greater frequency of exsmokers could also, in part, justify the second peak of incidence in old age. Greater colitis extent, greater clinical activity, and greater use of steroids as the first therapeutic step are shown to prevail among younger patients and among women (P = 0.02 and P = 0.019, respectively). The same is observed for symptoms mainly representing clinical severity such as diarrhea, fever, and weight loss (P = 0.004; P = 0.006; P = 0.009, respectively). This study confirms the UC risk factor represented by the condition of being an ex-smoker and shows a greater severity of illness on diagnosis in younger patients. PMID- 10749319 TI - Therapeutic role for bismuth compounds in TNBS-induced colitis in the rat. AB - The 2,4,6-trinitrobenzene sulfonic acid (TNBS) -induced model of chronic inflammation of the rat colon was used to determine the efficacy of bismuth subsalicylate (BSS), bismuth subcitrate (CBS), and 5-aminosalicylic acid (5-ASA) administered in enema form. A novel bismuth compound 1, 2-bis[2-(1,3 dithiobismolane)thio]ethane [Bi2(EDT)3] was also tested. On day 1 colitis was induced with 50 mg TNBS/50% ethanol in female Sprague-Dawley rats, while controls received a saline enema. On day 3, twice-daily treatment with enemas of either saline, BSS, CBS, Bi2(EDT)3, or 5-ASA were initiated in the colitis and control rats. All rats were killed on day 14, and the colons excised, weighed, rated macroscopically, and then fixed for hematoxylin and eosin staining. Blinded microscopic scoring was used to determine injury and healing in all groups. Colon mass and macroscopic scores were increased (P < 0.05) in the group of rats treated with TNBS (N = 16) compared to saline controls (N = 12). Colon mass and macroscopic scores in controls treated with BSS (N = 4), CBS (N = 4), Bi2(EDT)3 (N = 4), and 5-ASA (N = 4) alone did not differ from saline control animals. Macroscopic scoring showed a decrease (P < 0.05) in the degree of damage in the group of rats treated with TNBS plus BSS (N = 15), TNBS plus Bi2(EDT)3 (N = 10) and TNBS plus CBS (N = 4) compared to the group of rats treated with TNBS plus saline (N = 16). A decrease (P < 0.05) in injury and an increase (P < 0.05, Kruskal-Wallis) in healing was observed in the groups of rats treated with TNBS plus BSS, TNBS plus CBS, and TNBS plus 5-ASA compared to the group of rats treated with TNBS plus saline. It appeared that Bi2(EDT)3 was not protective against injury at the microscopic level but that the novel Bi2(EDT)3 has an effective healing capacity at the macroscopic level. We conclude that BSS and CBS decrease injury and/or promote healing as effectively as 5-ASA in this model. PMID- 10749320 TI - Urinary choloyl-PABA excretion in diagnosing small intestinal bacterial overgrowth: evaluation of a new noninvasive method. AB - The synthetic substrate cholyl-PABA, developed by conjugating cholic acid with paraaminobenzoic acid, is hydrolyzed by the bacterial enzyme cholyl hydrolase to release free PABA. This study aimed to evaluate whether quantitating urinary excretion of PABA after oral administration of cholyl-PABA can detect small intestinal bacterial overgrowth. In the first phase, investigations were performed on 10 healthy volunteers to study the dynamics of urinary excretion of PABA and any adverse reactions after oral administration of 1.2 g of cholyl-PABA. Another 10 healthy volunteers and 25 adult patients with various gastrointestinal disorders participated in the second phase, where the urinary cholyl-PABA test was compared to the [14C]xylose breath test (XBT). The upper limit of normal levels of urinary PABA excretion at the end of 4 h was 1.1% of the administered dose of cholyl-PABA. The urinary PABA excretion after 4 hr [median (range), in percentage] in the XBT-positive group was 1.6 (0.6-35.0), which was significantly higher than those in the XBT-negative group [0.7 (0.4-1.8)] and the healthy controls [0.7 (0.2-1.1)]. The agreement between the XBT and the urinary cholyl PABA test was 85.7% (P < 0.01). No adverse effect was noted. In conclusion, the urinary cholyl-PABA test offers a simple, safe, noninvasive, and rapid method for diagnosing small intestinal bacterial overgrowth and warrants further clinical evaluation. PMID- 10749322 TI - Alcohol and cigarette smoking and the risk of colorectal adenomas. AB - Whether alcohol and tobacco can be considered as risk factors for the occurrence of adenomas remains inconclusive. A case-control study was carried out to examine these factors while taking into account possible confounding factors. One hundred eighty-two patients with colorectal adenomas and similar numbers of hospital and population controls were compared as to intake of alcohol and various nutrients including smoking and drug intake. There was a positive association between cigarette smoking and adenoma risk compared with hospital controls, the RR being 2.3 (1.1-4.6). Overall alcohol intake was no risk factor in hospital controls, but drinking liquor was associated with an increased risk, the RR being 4.1 (1.3 13.4) and was especially marked in males [RR 10.2 (2.3-46.2)]. Compared with population controls, there was no increased RR associated with smoking or alcohol intake. None of the risk factors was positively associated with disease risk in those with small or large adenomas. These findings suggest that alcohol and tobacco play no major role in the formation or growth of adenomas. PMID- 10749321 TI - Effect of Shiga toxin 2 on water and ion transport in human colon in vitro. AB - Shiga toxin-producing Escherichia coli (STEC) colonize the lower segments of the human gastrointestinal tract, causing gastrointestinal and systemic diseases. In this study, the effects of Shiga toxin 2 (Stx2) on fluid absorption and ion transport in the human colon were examined. Net water movement (Jw) and short circuit current (Isc) were simultaneously measured across the colonic mucosa incubated with crude or purified Stx2. Stx2 significantly inhibited the absorptive J(w) with no effect on the basal I(sc) after 60 min of exposure. These effects may be due to the inhibition of a nonelectrogenic transport system present in the surface colonic villus cells. Morphological studies of the colonic mucosa treated with crude or purified Stx2 demonstrated a selective damage in the absorptive villus epithelial cells. These findings suggest that Stx2 inhibits water absorption across the human colon by acting on a specific cell population: the mature, differentiated absorptive villus epithelium. PMID- 10749323 TI - Immunodiscrimination of colorectal neoplasia using MUC1 antibodies: discrepant findings in tissue versus stool. AB - Colorectal tumor-associated antigens are attractive targets for novel stool screening assays. MUC1, a glycoprotein antigen, is aberrantly expressed in transformed colorectal mucosa and represents a candidate fecal biomarker. In this study, tissue staining and stool testing were performed to further clarify the discriminant potential of MUC1 in markedly different biologic media. One anti MUC1 monoclonal antibody (MA5) was used for immunohistochemistry and two commercially available MUC1 assay kits (ELSA-CA 15-3 and Truquant BR) were used for stool detection. On tissue staining, MUC1 expression was strong in 40/40 (100%) adenocarcinomas, moderate in 42/55 (76%) adenomas, faint in 8/28 (29%) juxtatumoral mucosa specimens, and absent in 15/15 (0%) nonadjacent mucosa specimens. Conversely MUC1 levels in stool testing did not differ between colorectal cancer cases (N = 14) and controls (N = 14). Based on these results, MUC1 appears to be a functional tumor biomarker in colorectal tissue but not in stool. Bacterial metabolism within stool may unmask the core antigen of MUC1 and account for this discordance in immunoreactivity. PMID- 10749324 TI - Discrimination between malignant and nonmalignant ascites using serum and ascitic fluid proteins in a multivariate analysis model. AB - Our objectives were to study the value of different proteins in the serum and ascitic fluid and assess their potential in discriminating between malignant and nonmalignant ascites in a model that could be developed to aid clinical diagnosis. In all, 57 different measurements (30 in serum and 27 in ascitic fluid) including erythrocyte sedimentation rate, number of white blood cells, cytokines, interleukin-1a (IL-1a), IL-1b, IL-2, IL-6, IL-8, tumor necrosis factor alpha, immunoglobulins (IgG, IgA, IgM), complement factors C3 and C4, acute-phase proteins such as alpha1-acid glycoprotein, alpha2-macroglobulin, alpha1 antitrypsin, haptoglobin, C-reactive protein, ferritin, ceruloplasmin and transferin, were performed in 61 patients with ascites (25 with malignant exudates, 13 with nonmalignant exudates, and 23 with transudates). Patients with sepsis were excluded. Correlation tests and one-way ANOVAs were used for comparisons between different groups. Discriminant analyses were used to assess the significance of each parameter in the differentiation process. Correct classification of 100% of cases required the use of all 57 ascitic fluid measurements in the model, which was not considered practical in clinical diagnosis. Discriminant analysis showed that five ascitic fluid measurements total protein, LDH, TNF-alpha, C4, and haptoglobin-were sufficient for a model to correctly classify 89% of cases. Cross-validation showed that 70% of unknown cases were correctly classified using this model. In conclusion, we have shown that five easily taken protein measurements in the ascitic fluid can differentiate to a large extent between cases with ascites and have proposed a relatively simple statistical model with these parameters that could be developed to be extremely useful in the clinical setting. PMID- 10749325 TI - Saturated saline enhances the effect of electrochemical therapy. AB - We conducted this experiment to assess the effect of saline injection in electrochemical therapy. Platinum electrodes using direct current were inserted into egg white or liver parenchyma. Pure water or 0.9%, 3%, or 26% sodium chloride were injected into various objects to compare with the control group (no injection). Power was set at 10 V. In the egg-white experiment, gas bubbles and coagulated protein developed around the electrodes. In ex vivo liver, frothy reddish debris developed around the cathodes, while a hardening and shrunken surface occurred around the anodes. The pH was 14 around the cathodes, 0 around the anodes. The electric current, the amount of coagulated protein, and the severity of tissue damage were all in proportion to the concentrations of the injected saline. The volume destroyed in the 26% saline group was 8.1 times larger than that of the control group. Therefore, injected saline, especially saturated saline, can enhance the effect of electrochemical therapy. PMID- 10749326 TI - Inflammatory pseudotumor of the colon. PMID- 10749327 TI - Comparison of gastric electrical activity and gastric emptying in healthy and dyspeptic children. AB - To assess and compare gastric electrical activity and gastric emptying recorded from dyspeptic and healthy children, cutaneous electrogastrography and ultrasound examination of the gastric emptying were simultaneously performed in 52 children with nonulcer dyspepsia and 114 healthy children. Symptoms were scored from 0 (none) to 6 (severe). A higher percentage of tachygastria, a higher instability of gastric power, and a lower post/preprandial ratio were present in dyspeptic children than healthy children. As regards the ultrasound parameters, the fasting antral area and T1/2 were similar in dyspeptic children and controls. Only 32% of dyspeptic children had a normal gastric emptying time vs 66% of healthy children. Marked postprandial antral dilatation was found in the dyspeptic children, which correlated with the total symptom score. Electrogastrographic and gastric emptying parameters show specific differences in dyspeptic children with respect to controls, both fasting and after a meal. The postprandial antral distension correlates with the severity of the symptoms. PMID- 10749329 TI - Electrophysiological and mechanical activity of the upper gastrointestinal tract after duodenoplasty or segmental resection of benign gastric outlet stenosis. AB - In an animal experimental study we examined the postoperative recovery of the motility of the upper gastrointestinal tract after operative treatment of a benign gastric outlet obstruction. At 45 Days after induction, a duodenal stenosis was resected in six dogs, and resolved by Finney's duodenoplasty in another six dogs. Fourteen days after segmental resection, the gastric emptying was faster [half evacuation time (T1/2) for semisolid food = 44.4 +/- 16.8 min] than following duodenoplasty [T1/2 = 56.8 +/- 25.3). Here motor migrating complexes (MMCs) started in the antrum and could be traced down to the jejunum. After segmental resection we recognized MMC only distal to the anastomosis. The duration of the whole MMC cycle (69.0 +/- 18.6 min) as well as of the single phases was significantly shorter in the resection group than after duodenoplasty (108.0 +/- 15.1 min). At 28 days after operation the differences in the electromyographic findings were smaller (82.0 +/- 15.1 min vs. 111.4 +/- 11.2 min), but still significant. Obviously humoral transmitters and the extrinsic neural system lead to good propagation of the MMC across the anastomosis, even before the intramural pathways are reestablished. Concerning the fast recovery of the motility of the upper gastrointestinal tract, duodenoplasty is superior to segmental duodenal resection. PMID- 10749328 TI - Effect of meal and intravenous erythromycin on manometric and electrogastrographic measurements of gastric motor and electrical activity. AB - Electrogastrography (EGG) measures, on the skin surface, the myoelectrical activity attributable to gastric smooth muscle cells. The physiological significance of signal amplitude and variation has not been clearly established. The increased signal amplitude after eating a meal may be related to increased contractile activity or to gastric distension. This study investigates the effect of increased gastric motor activity, unaccompanied by gastric distension, on the EGG recording and compares it to the effect of a meal. Nine children (3 months to 15 years old), were assessed by antroduodenal manometry for chronic intestinal pseudoobstruction (N = 5), chronic vomiting (N = 2), and abdominal distension (N = 2). Synchronized EGG recording was performed simultaneously. During the study, four children were given a meal and five were given intravenous erythromycin 3 mg/kg over 1 hr. The ratio of the antral motor index (MI = number of waves x sum of amplitudes) determined 1 hr before a meal (or erythromycin) to that determined 1 hr after a meal (or during intravenous erythromycin) was calculated. The ratio of the running total spectrum power of the electrical signal at the same times was also calculated. Antral MI increased after a meal [MI ratio (mean +/- SE) 5.33 +/- 2.2] and after intravenous erythromycin (MI ratio: 9.36 +/- 2.6). The amplitude of the electrical activity also increased after the meal [power ratio (mean +/- SE) 3.01 +/- 0.65] and after intravenous erythromycin (power ratio: 1.23 +/- 0.39), but the increase was greater after the meal (P < 0.05 vs intravenous erythromycin). No correlation was found between antral MI ratio and running total spectrum power ratio. In conclusion, the increased amplitude of the gastric electrical activity recorded by the EGG after a meal seems to be only partly due to the increase in antral motor activity. The increase in power is also related to gastric distension. PMID- 10749330 TI - Human cholecystokinin-A receptor is not an oncofetal protein. AB - The CCK-A (cholecystokinin-A) receptor is selectively expressed by human pancreatic adenocarcinomas, suggesting a possible role in pancreatic tumorigenesis. In animals, pancreatic CCK receptor expression varies during ontogeny and neoplastic transformation. This study examined the temporal expression of CCK receptors in human fetal, postnatal, and adult pancreas to determine whether the appearance of CCK-A receptors in pancreatic adenocarcinomas reflected oncofetal antigen or pancreatic neoantigen expression. Messenger ribonucleic acid (mRNA) was isolated from six paraffin-embedded normal pancreatic autopsy specimens ranging in age from 17 weeks postfertilization through 26 days following full-term delivery, and samples of adult human tissues, including pancreas and pancreatic adenocarcinoma. Using reverse transcription-polymerase chain reactions, CCK-B receptor mRNA was expressed in all specimens of normal fetal and postnatal human pancreas, adult pancreas, and pancreatic adenocarcinomas. CCK-A receptor mRNA was selectively expressed only in pancreatic adenocarcinomas. These data suggest that selective CCK-A receptor expression in pancreatic adenocarcinomas reflects neoantigen expression in humans. PMID- 10749331 TI - Choledochal cyst spontaneously rupturing the hepatic artery. PMID- 10749332 TI - Troglitazone-induced fulminant hepatic failure. Acute Liver Failure Study Group. AB - The three reported cases demonstrate that troglitazone is an idiosyncratic hepatotoxin that can lead to irreversible liver injury. Thus, troglitazone should be prescribed with caution and should not be used as a first-line agent in the treatment of type II DM when potentially less toxic alternatives are available. It remains to be seen whether the hepatotoxicity associated with troglitazone is a drug-class effect or specific to troglitazone. Other thiazolidinediones currently in clinical trials may be able to provide the therapeutic benefits of troglitazone without significant hepatotoxicity. If troglitazone is used, frequent monitoring of serum aminotransferases and symptoms is mandatory. However, as illustrated by these and other cases reported to date, the onset of troglitazone-induced liver injury is insidious and temporally variable. Thus, the value of close monitoring and when, if ever, it is safe to stop such monitoring are currently unclear. PMID- 10749333 TI - Proposed abdominal sonographic staging to predict severity of liver diseases: analysis with peritoneoscopy and histology. AB - Abdominal sonography is a routinely used noninvasive modality for screening and treatment of liver diseases. We attempted to establish a morphological sonographic staging to predict the severity of liver diseases with their consequent analysis with morphological staging of peritoneoscopy and histology. In all, 136 patients were enrolled for the final confirmation of disease state by peritoneoscopy and histology preceded by abdominal sonography. All patients were categorized from stage 0 to stage 5, depending on a proposed criterion of sonographic features based on surface pattern of liver and the appearance of internal echogenic bands relating to the irregularity of the liver texture. A digitized computer quantitation of histogram-based standard deviation (SD) values from different stages of sonographic images was analyzed, and their values were justified by correlation with the definite appearance of an internal echogenic band. The association of different sonographic stages with disease progression was also demonstrated by their relation with peritoneoscopy and histology. In all patients, the different sonographic staging results were significantly correlated with hepatic surface features of peritoneoscopic staging (r = 0.939, P < 0.0001) graded from stage 0 to stage 5 and were also correlated with biopsy-proven staging of fibrosis (r = 0.739, P < 0.0001). The greater SD values of histogram based echo levels, as analyzed from digitized sonographic images of 90 patients, were associated with the appearance of internal echogenic bands (P < 0.0001). Furthermore, the corresponding SDS were significantly correlated with the qualitative staging of sonographic features (r = 0.781, P < 0.0001), peritoneoscopy (r = 0.786, P < 0.0001) and histology (r = 0.779, P < 0.0001). We concluded that our proposed sonographic staging is well correlated with peritoneoscopic and histological staging of liver diseases, with only a small discrepancy, and can be used clinically to demonstrate the ongoing severity of liver diseases. PMID- 10749334 TI - Sustained viral response is rarely achieved in patients with high viral load of HCV RNA by excessive interferon therapy. AB - Adequate dosing of interferon (IFN) and its cost-effectiveness for sustained virological response were evaluated in relation to viral load and subtype. Prospective analysis of IFN therapy on 326 patients with chronic hepatitis C free from cirrhosis was performed using 9 or 6 million unit (MU) of IFN for six months daily and/or three times a week. Sustained virological response was achieved in 50-94% of patients with < or =2 x 10(4) copies/ml (competitive RT-PCR) or <100 x 10(3) copies/ml (Amplicor monitor) of HCV RNA by 468-1206 MU of IFN, but response was only 0-25% of the patients with > or =2 x 10(5.5) copies/ml (competitive RT PCR) or >200 x 10(3) copies/ml (Amplicor monitor), even with 468-1206 MU of IFN. A high sustained rate was demonstrated in patients with 100-200 x 10(3) copies/ml of HCV RNA by 901-1206 MU of IFN, in comparison to that with < or =900 MU of IFN. Multivariate analysis showed that IFN dose had a significant value for the efficacy of IFN therapy in patients presenting 100-200 x 10(3) copies/ml of HCV RNA. Cost efficacy analysis indicated that it cost approximately $10,000, $26,000, and $50,000-227,000 for one person-viral eradication in the patients with <100, 100-200, and >200 x 10(3) copies/ml, respectively. High-dose IFN is only cost effective in patients with intermediate viral loads, and IFN therapy could be recommended in patients with <200 x 10(3) copies/ml of HCV RNA. PMID- 10749335 TI - Analysis of biochemical and virological efficacy of human lymphoblastoid interferon (IFN) in patients with compensated type C liver cirrhosis: comparative study between increase in individual IFN dose and prolonging of treatment period, using a multicenter randomized controlled trial. AB - A controlled trial was conducted to compare the efficacy of interferon (IFN) between two groups of patients with type C liver. Thirty-five patients were randomly assigned to group A (17 patients) or group B (18 patients). The former received 3 megaunits (MU) of human lymphoblastoid IFN six days per week for two weeks, followed by three days per week for 50 weeks; the latter group received 6 MU six days per week for two weeks followed by three days per week for 24 weeks. The percentages of biological sustained responders (B-SR) and virological sustained responders (V-SR) were 29.4 and 23.5%, respectively, in group B, and 17.6% for both in group A. The therapeutic effects were not different between two groups. HCV genotype 2 accounted for significantly higher percentage of B-SR and V-SR (both 57.1%, respectively). These findings indicate that IFN is effective in type C cirrhosis with genotype 2. PMID- 10749337 TI - Progressive focal nodular hyperplasia of the liver in a patient with genetic hemochromatosis--growth promotion by iron overload? PMID- 10749336 TI - Effects of collateral vessel occlusion on oral glucose tolerance test in liver cirrhosis. AB - Alterations in carbohydrate metabolism associated with liver cirrhosis are characterized by a high serum insulin level and prolonged hyperglycemia on oral glucose tolerance test (OGTT). We measured plasma glucose, immunoreactive insulin (IRI), and C-peptide immunoreactivity (CPR) levels during a 75-g OGTT before and after varices obliteration in 10 cirrhotic patients with gastric varices. After obliteration, the indocyanine green retention rate was decreased and the portal flow velocity was increased. A significant decline in plasma glucose and IRI levels was also noted on OGTT. Moreover, the plasma glucose and IRI levels declined at 90 and 120 min in OGTT while they increased progressively by 120 min before obliteration. The levels of CPR were similar before and after treatment. These results indicate that decreased portal flow due to extrahepatic shunt and consequent impairment of insulin metabolism play a role in glucose intolerance observed in cirrhotic patients and that shunt occlusion improves glucose metabolism. PMID- 10749338 TI - Sequential expression of adrenomedullin and its receptor during gastric ulcer healing in rats. AB - Adrenomedullin (AM) is a potent vasodilatory peptide. While its growth-regulating action in some cultured cells has been recognized, expression of AM and its receptor during gastric ulcer healing has not been explored. Specimens of gastric walls from control rats or gastric ulcers were obtained at 1, 3, 7, and 14 days after gastric ulcer induction. AM and its receptor mRNAs expression were determined by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. By RT-PCR, AM mRNA was increased by 167% at three days, while AM receptor mRNA was increased by 234% at seven days (both P < 0.05). By in situ hybridization, AM and AM receptor mRNAs were increased at ulcer margin from three days after ulcer induction. By immunohistochemistry, AM was increased in the ulcer margin at three and seven days. In separate in vitro studies using a rat gastric epithelial (RGM1) cell line, AM treatment significantly increased transforming growth factor-alpha mRNA expression and cell proliferation. PMID- 10749339 TI - Effect of vitamin E on aspirin-induced gastric mucosal injury in rats. AB - We investigated the effect of vitamin E on aspirin-induced gastric mucosal injury in rats. Twenty-eight male Sprague-Dawley rats were divided into four groups and were fed for 20 weeks with a diet containing <0.1 mg/100 g of alpha-tocopherol (vitamin E-deficient), 2 mg/100 g of alpha-tocopherol (normal and vitamin E sufficient), or 50 mg/100 g of alpha-tocopherol (vitamin E-supplemented). In vitamin E-deficient rats, oral administration of aspirin (200 mg/kg) plus HCI created more severe hemorrhagic erosions than in other rats. Vitamin E-deficient rats had higher levels of thiobarbituric acid reactive substances, myeloperoxidase activity, and cytokine-induced neutrophil chemoattractant in the gastric mucosa. Flow cytometry showed that CD18 expression on stimulated neutrophils was higher in vitamin E-deficient rats than in vitamin E-supplemented rats. These results suggest that vitamin E protects against aspirin-induced gastric mucosal injury by inhibiting lipid peroxidation and accumulation of activated neutrophils. PMID- 10749341 TI - Aging enhances susceptibility of diclofenac-treated rats to gastric ulceration, while attenuating enteropathy. AB - Although clinical reports note aging and gender as risk factors for NSAID therapy associated gastroenteropathy, neither variable has been examined in an animal model. We addressed this unknown by comparing the responses of young (4 months) and old (22 months) rats of both genders to oral treatment with diclofenac (10 or 50 mg/kg). Diclofenac produced gastric ulcers only in old rats, with markedly larger lesions in females. In contrast, the small intestines in old rats of both genders given the 50 mg/kg dosage had >30% fewer ulcers and a fourfold decrease in area of ulceration compared to young rats. The small intestine was the only site of lesions after the 10 mg/kg dosage and showed one gender influence, namely, a transiently faster time course of ulcer development in females. Old and young rats given 50 mg/kg showed similar declines in serum levels of the vascular permeability indices-total protein and albumin-despite reduced intestinal damage in the old animals, which suggests additive vascular leakage across the gastric lesions that were evident only in old animals. Serum biochemistry showed no evidence of hepatotoxicity or dysfunction, consonant with small intestine as the primary target for diclofenac toxicity in rats. We provide the first experimental evidence for an aging influence on the gastrointestinal target site of a nonaspirin NSAID. PMID- 10749340 TI - Effects of ecabet sodium, a novel gastroprotective agent, on mucin metabolism in rat gastric mucosa. AB - The effects of ecabet sodium (ecabet), 12-sulfodehydroabietic acid monosodium salt, on gastric mucin biosynthesis in rat antrum were compared with those in the corpus. Intragastric administration of ecabet significantly increased [3H]glucosamine incorporation into antral mucin as well as into corpus mucin during five successive hours of organ culture. In contrast, mucin biosynthesis in either antrum or corpus was not susceptible to the addition of ecabet to the culture medium. Ecabet-induced stimulation of prostaglandin E2 production in the antrum was essentially the same as that seen in the corpus. In antrum treated with 100 mg/kg ecabet, immunoreactivity with three distinct anti-mucin monoclonal antibodies was found not only in the specific mucus-producing cells, but also in the secreted mucus present at the surface gel layer. These results suggest that ecabet enhances the mucin metabolism, and this stimulation occurs in both the corpus and antrum, suggesting that ecabet might be a useful tool for the further clarification of the regulatory mechanism of antral mucin synthesis. PMID- 10749342 TI - Inhibition of lipid peroxidation, NF-kappaB activation and IL-8 production by rebamipide in Helicobacter pylori-stimulated gastric epithelial cells. AB - The present study aimed to investigate whether rebamipide, a novel antiulcer agent that has an oxygen radical scavenging activity, would inhibit lipid peroxidation, NF-kappaB activation, and IL-8 production by H. pylori. Human gastric epithelial cells (AGS and KATO III), treated with rebamipide or not were incubated in the absence or the presence of H. pylori. As a result, H. pylori significantly stimulated IL-8 production, which was similar to time course stimulation of lipid peroxidation. Other cytokines (IL-1alpha, IL-1beta, IL-6, TNF-alpha) were not stimulated by H. pylori. Treatment with H. pylori resulted in the activation of two species of NF-kappaB dimers (a p50/p65 heterodimer and a p50 homodimer). Rebamipide significantly inhibited lipid peroxidation as an indicative of oxidative damage, NF-kappaB complex formation, and IL-8 production by H. pylori. In conclusion, rebamipide may attenuate H. pylori-induced gastric inflammation by inhibiting lipid peroxidation and oxidant-mediated activation of NF-kappaB and thereby decreasing IL-8 production. PMID- 10749343 TI - Antral glutathione concentration and glutathione S-transferase activity in patients with and without Helicobacter pylori. AB - Previously we demonstrated an inverse relation between cancer of the gastrointestinal tract and glutathione S-transferase activity of the gastrointestinal mucosa. Chronic infection with H. pylori has been associated with an increased risk of gastric cancer. The aim of this study was to investigate the levels of glutathione and glutathione S-transferase activity in H. pylori-infected and noninfected antral mucosa. Glutathione and glutathione S transferases were measured in antral biopsies of patients with nonulcer dyspepsia without H. pylori infection (A), with prior H. pylori infection who became H. pylori negative after eradication therapy (B) and with proven H. pylori infection (C). Glutathione concentration and glutathione S-transferase activity in group A were 31.0 (range 6.0-59.6) nmol/mg protein and 810 (range 165-1312) nmol/min/mg protein, in group B 27.0 (range 5.0-53.8) nmol/mg protein and 745 (range 403 1199) nmol/min/mg protein, and in group C 18.5 (range 1.6-55.8) nmol/mg protein and 572 (range 144-1047) nmol/min/mg protein, respectively. The glutathione and glutathione S-transferase values were significantly lower in patients infected with H. pylori than in patients who were H. pylori negative. PMID- 10749344 TI - Severe and relapsing upper gastrointestinal bleeding in a patient with Glanzmann's thrombasthenia. AB - Glanzmann's thrombasthenia (GT) is a rare familial thrombocytic disease inherited as an autosomal recessive disorder that can induce hemorrhages due to a defect of platelet aggregation, resulting from the absence or reduced concentration of the membrane glycoproteic receptor binding the fibrinogen (integrin alpha(IIb)beta3). The gastrointestinal tract is the site of bleeding in only about 10% of cases but the related mortality is high (12.8%). Among the deaths due to hemorrhage, digestive bleeding causes 57.1%. According to reported data, the source of bleeding may be from preexisting gastroduodenal chronic and acute lesions. We report a case of severe and relapsing upper digestive bleeding in a woman with GT and coexisting thrombocytopenia (from HCV-related liver cirrhosis) and H. pylori positive duodenal ulcer. PMID- 10749345 TI - Immunological aspects of oral desensitization in food allergy. PMID- 10749346 TI - Nonfluoroscopic three-dimensional mapping for arrhythmia ablation: tool or toy? AB - INTRODUCTION: Conventional mapping and ablation rely on fluoroscopy, which can result in imprecise positioning of the ablation catheter and long fluoroscopic exposure times. We evaluated a nonfluoroscopic three-dimensional mapping system, termed CARTO, and compared the results of ablation using this technique with those of conventional mapping. METHODS AND RESULTS: We compared the results of 88 arrhythmia ablations (79 patients) using CARTO with 100 ablations (94 patients) using the conventional technique. The ablations were separated into four groups: (1) AV nodal reentrant tachycardia (AVNRT); (2) atrial tachycardia/flutter; (3) ventricular tachycardia (VT); and (4) bypass tract tachycardia. We compared the success rate, complications, and fluoroscopy and procedure times. The ablation outcomes were excellent and comparable in all four types of the arrhythmias between the two techniques. Major complications included one cardiac tamponade in each group and one second-degree AV block in the conventional group. Fluoroscopy time was shorter using the CARTO technique: 10+/-7 versus 27+/-15 minutes for AVNRT (P < 0.01), 18+/-17 versus 44+/-23 minutes for atrial tachycardia and flutter (P < 0.01), 15+/-12 versus 34+/-31 minutes for VT (P < 0.05), and 21+/-14 versus 53+/-32 minutes for bypass tract tachycardia (P < 0.01). Procedure times were similar except for the bypass tract patients, which was shorter in the CARTO group, 4+/-1.3 versus 5.5+/-2.5 hours (P < 0.01). CONCLUSION: The electroanatomic three-dimensional mapping technique reduced fluoroscopy time and resulted in excellent outcome without increasing the procedure time. PMID- 10749347 TI - Patterns of accelerated junctional rhythm during slow pathway catheter ablation for atrioventricular nodal reentrant tachycardia: temperature dependence, prognostic value, and insights into the nature of the slow pathway. AB - INTRODUCTION: Although accelerated junctional rhythm (AJR) is a known marker for successful slow pathway (SP) ablation sites, AJR may just be a regional effect of the anisotropic conduction properties of this area of the heart. We believe that detailed assessment of the AJR might provide insight into the SP specificity of this AJR and perhaps the nature of the SP itself. METHODS AND RESULTS: Our ablation protocol consisted of 30-second, 70 degrees C temperature-controlled ablation pulses with assessment after each pulse. Serial booster ablations were performed at the original successful site and at least 2 to 3 nearby sites to assess for residual AJR after the procedure in 50 consecutive SP ablations. We defined three distinct patterns of AJR: continuous AJR that persisted until the end of energy delivery (group I, 25 patients); alternating or "stuttering" AJR that persisted throughout energy delivery (group II, 9 patients); and AJR that ended abruptly during energy delivery (group III, 16 patients). Mean ablation temperatures in the three groups was 57 degrees+/-5 degrees C, 54 degrees+/-5 degrees C, and 63 degrees+/-5 degrees C, respectively (P = 0.0002 for groups I and II vs group III). Ten of 34 (29%) patients in groups I and II ("low temperature ablation") exhibited residual SP (jump and/or single echo beats) despite tachycardia noninducibility, and 25 of 34 (73%) patients had residual AJR during the booster ablations, but neither of these was seen in any group III patients. CONCLUSION: Ablation temperature correlates with the pattern of AJR produced during SP ablation. That higher temperature lesions simultaneously abolish all SP activity as well as the focus of AJR suggests that this AJR is specific for the SP and is not a nonspecific regional effect. PMID- 10749348 TI - Simplified "ATP test" for noninvasive diagnosis of dual AV nodal physiology and assessment of results of slow pathway ablation in patients with AV nodal reentrant tachycardia. AB - INTRODUCTION: We recently reported that administration of adenosine triphosphate (ATP) during sinus rhythm identifies dual AV nodal physiology (DAVNP) in 76% of patients with inducible sustained AV nodal reentrant tachycardia (AVNRT) at electrophysiologic (EP) study. In that report, however, the ATP test was considered positive for DAVNP only when the results were reproducible at a given dose of ATP. The aim of the present study was to assess the value of a simplified ATP test for noninvasive diagnosis of DAVNP and abolition or modification of the slow pathway (SP) after radiofrequency ablation (RFA) in patients with inducible sustained AVNRT. METHODS AND RESULTS: The value of a single dose of ATP was studied in 105 patients with inducible sustained AVNRT and in 31 control patients before placement of EP catheters in the cardiac chambers. ATP (10 to 60 mg, in 10 mg increments) was injected during sinus rhythm until ECG signs of DAVNP (> or = 50 msec increase or decrease in PR interval in two consecutive beats, or occurrence of > or = 1 AV nodal echo beat) or > or = second-degree AV block was observed. DAVNP was observed in only 1 (3.2%) control patient. The test could be completed in 96 study patients. DAVNP was found by ATP test in 72 (75%) patients, whereas it was diagnosed by EP criteria in 82 (85%) patients. DAVNP by ATP test disappeared in 27 (96%) of 28 patients who underwent SP abolition and in 18 (60%) of 30 patients who underwent SP modification. In the 12 patients with persistent DAVNP determined by ATP test after SP modification, the number of beats conducted over the SP was significantly reduced (from 6.3+/-3.3 to 2.5+/-2.2 beats; P = 0.002). CONCLUSION: A single administration of ATP during sinus rhythm (at a given dose) enables noninvasive diagnosis of DAVNP in a high percentage of patients with inducible AVNRT and reliably confirms the results of RFA of the SP. PMID- 10749349 TI - Preferential depression of conduction around a pivot point in rabbit ventricular myocardium by potassium and flecainide. AB - INTRODUCTION: During reentrant arrhythmias, the circulating wavefront often makes a sharp turn around a functional or anatomic barrier. We tested the hypothesis that lowering the safety factor for conduction by high K+ or flecainide preferentially depresses conduction of sharply turning wavefronts. METHODS AND RESULTS: In 16 Langendorff-perfused rabbit hearts, a thin layer of anisotropic ventricular myocardium was made using a cryoprocedure. In this layer, a linear radiofrequency lesion was made parallel to the fiber orientation. The tip of the lesion was extended by a short incision. U-turning wavefronts were initiated by pacing at one side of the lesion. A mapping electrode (240 electrodes, resolution 350 to 700 microm) was used to measure conduction times and velocity of planar waves (longitudinal and transverse) and U-turning wavefronts. The safety factor for conduction was lowered by high potassium (8, 10, and 12 mmol/L) and flecainide (1 and 2 mg/L). On average, high potassium and flecainide increased the conduction times of U-turning wavefronts 1.6 times more than longitudinal or transverse planar wavefronts (P < 0.01). At a critical lowering of the excitatory current, functional conduction block occurred at the pivot point, which forced the wavefront to make a longer U-turn. In these cases, the total U-turn conduction time increased from 27+/-9 msec to 75+/-37 msec. About 40% of this delay was caused by a shift of the pivot point and consequent lengthening of the returning pathway. CONCLUSION: Lowering the amount of excitatory current by potassium or flecainide preferentially impairs U-turn conduction. The occurrence of long delays and conduction block at pivot points may explain the mode of action of Class I drugs. PMID- 10749350 TI - Role of virtual electrodes in arrhythmogenesis: pinwheel experiment revisited. AB - INTRODUCTION: Recent experimental evidence demonstrates that a point stimulus generates a nonuniform distribution of transmembrane potential (virtual electrode pattern) consisting of large adjacent areas of depolarization and hyperpolarization. This simulation study focuses on the role of virtual electrodes in reentry induction. METHODS AND RESULTS: We simulated the electrical behavior of a sheet of myocardium using a two-dimensional bidomain model with straight fibers. Membrane kinetics were represented by the Beeler-Reuter Drouhard Roberge model. Simulations were conducted for equal and unequal anisotropy ratios. S1 wavefront was planar and propagated parallel or perpendicular to the fibers. S2 unipolar stimulus was cathodal or anodal. With regard to unequal anisotropy, for both cathodal and anodal stimuli, the S2 stimulus negatively polarizes some portion of membrane, deexciting it and opening an excitable pathway in a region of otherwise unexcitable tissue. Reentry is generated by break excitation of this tissue and subsequent propagation through deexcited and recovered areas of myocardium. Figure-of-eight and quatrefoil reentry are observed, with figure-of-eight most common. Figure-of-eight rotation is seen in the direction predicted by the critical point hypothesis. With regard to equal anisotropy, reentry was observed for cathodal stimuli only at strengths > -95 A/m. CONCLUSION: The key to reentry induction is the close proximity of S2 induced excited and deexcited areas, with adjacent nonexcited areas available for propagation. PMID- 10749351 TI - Various ways to make phase singularities by electric shock. PMID- 10749352 TI - Transmural heterogeneity of ventricular repolarization under baseline and long QT conditions in the canine heart in vivo: torsades de pointes develops with halothane but not pentobarbital anesthesia. AB - INTRODUCTION: In vitro studies have provided evidence for the existence of M cells. The present study examines the contribution of the M cell to transmural dispersion of repolarization (TDR) and to the development of torsades de pointes (TdP) in the canine heart in vivo in animals anesthetized with either pentobarbital or halothane. METHODS AND RESULTS: Monophasic action potentials (MAPs) were recorded from 4 to 7 transmural sites, before and after d-sotalol. Cells displaying the longest MAP duration (MAPD) generally were localized to the deep subendocardium to mid-myocardium (M region) in the anterior wall of the left ventricle. d-Sotalol preferentially prolonged the MAPD of the M region, increasing TDR significantly more (P < 0.05) in animals anesthetized with halothane (31+/-5 to 88+/-17 msec) than in those receiving pentobarbital (24+/-9 to 53+/-7 msec; basic cycle length 1,500 msec). In halothane-anesthetized dogs, a remarkable transient increase in M cell MAPD followed interpolation of one or more extrasystole(s), leading to a transient increase in TDR and TdP. TdP was never observed with pentobarbital anesthesia. CONCLUSION: Our results demonstrate that transmural heterogeneity of repolarization is amplified under acquired long QT conditions and that the increase in TDR underlies the development of TdP in halothane- but not pentobarbital-anesthetized dogs. The data support an important contribution of M cells to TDR and to the development of TdP in the canine heart in vivo. Our data also highlight the importance of acceleration-induced prolongation of MAPD (a phenomena observed principally in M cells) in the development of TdP. PMID- 10749353 TI - Safety and efficacy of a steerable temperature monitoring microwave catheter system for ventricular myocardial ablation. AB - INTRODUCTION: Radiofrequency current delivered during cardiac ablation is limited by a rise in impedance secondary to coagulum formation on the ablation electrode. Microwave antennas continue to deliver energy despite the presence of coagulum; thus, temperature control of the ablation electrode may be even more important for microwave than for radiofrequency ablations to avoid thromboembolic risks. The purpose of this study was to test the safety and efficacy of an ablation system utilizing a feedback control system to maintain a fixed target temperature for creating lesions with multiple applications of microwave energy. METHODS AND RESULTS: Microwave ablation was assessed using an 8.5-French catheter at 2 to 4 sites in 11 dogs. Microwave energy delivery was performed for 60 seconds three times at the same site. Power was regulated using a feedback control mechanism to maintain a target temperature of 75 degrees C. Ambulatory ECG monitoring was performed before and after ablation to assess arrhythmia occurrence. After follow up, the dogs were euthanized, and lesion dimensions measured after fixation. The mean power applied to achieve the target temperature of 75 degrees C was 9.3+/-44 W. The mean depth of the lesions was 8.8+/-4.2 mm. The mean volume of the lesions was 304+/-240 mm3. Forty-four percent of the lesions were transmural. No endocardial thrombus was found. Ventricular tachycardia was observed acutely but resolved after 1 week. CONCLUSION: Temperature feedback control systems for microwave ablation using a temperature-controlled system is feasible for myocardial ablation and creates uniform and large lesions; however, such large lesions can be acutely proarrhythmic. PMID- 10749354 TI - Factors determining clockwise and counterclockwise conduction patterns in atrial reentrant tachycardias: a rabbit model of atrial flutter. AB - INTRODUCTION: In the development of atrial flutter due to reentry, the crista terminalis is supposed to pose a conduction barrier, but the role of its longitudinal conduction in determining the propagation pattern of the reentrant impulse is not known. In rabbit right atrial preparations, we induced reentrant atrial tachycardias and examined the effects of transverse section of the crista terminalis on the development and conduction patterns of arrhythmias. METHODS AND RESULTS: Right atrial preparations from 12 albino rabbits were placed endocardial surface down in a chamber with an array of 48 bipolar electrodes to draw activation maps. A single premature stimulus was delivered to induce tachycardias at the free wall. In the control, five instances of tachycardia per preparation were induced and another five were induced after cutting the crista terminalis. In the control, the mean duration of tachycardia was 127.1+/-25.2 seconds. The tachycardia was counterclockwise in 39 of 60 instances, clockwise in 12, and undetermined in 4 defined as "atypical." After transverse section of the crista terminalis, the duration was prolonged to 372.6+/-30.4 seconds, but the conduction patterns were not changed. In the free wall, counterclockwise reentry had a broader wavefront and faster conduction than clockwise reentry. CONCLUSION: Longitudinal conduction block at the crista terminalis contributed to maintenance of reentrant atrial tachycardias, but had no influence on their propagation patterns. Clockwise and counterclockwise rotation of impulses in reentrant tachycardias had different paths and velocities of the wavefront in the free wall of the right atrium. PMID- 10749355 TI - Temperature-sensitive focal atrial tachycardia in the left atrium. AB - Temperature sensitivity has not been reported in focal atrial tachycardia. We describe a patient with a left atrial tachycardia whose tachycardia rate was affected by hot and cold drinks. The effects were still evident after autonomic blockade. The arrhythmia focus was located at the entrance of the left upper pulmonary vein. Radiofrequency ablation was carried out, which proved to be difficult, but it was successful after several applications of energy, suggesting an epicardial location of the arrhythmia focus. Sensitivity of atrial tachycardia rate to the temperature of food or drink ingested suggests a left atrial focus with a posterior and possibly epicardial location. PMID- 10749356 TI - First evidence of premature ventricular complex-induced cardiomyopathy: a potentially reversible cause of heart failure. AB - Tachycardia-induced cardiomyopathy is a well-recognized and reversible condition, but left ventricular dysfunction due to frequent isolated premature ventricular complexes (PVCs) has not been reported. We observed resolution of dilated cardiomyopathy in a patient after a focal source of PVCs was eliminated by radiofrequency ablation. In a subset of patients with heart failure, PVC-induced cardiomyopathy may be a potentially reversible cause of left ventricular dysfunction. PMID- 10749357 TI - Right pulmonary vein potentials recorded from the posterior right atrial endocardium: human case report and validation in a porcine model. AB - A 33-year-old woman underwent successful catheter ablation of an atrial tachycardia emanating from deep within a large right superior pulmonary vein (RSPV). A previous ablation attempt in this patient had failed, during which radiofrequency energy applications were made to the posterior right atrium only. The mistaken impression of a right atrial source for this tachycardia was due to RSPV potentials that were recorded during mapping in a region of the posterior right atrium contiguous to the vein ("contiguity region"). To further evaluate this, we performed activation mapping and radiofrequency ablation in atria of healthy pigs. Similar to the reported case, "biatrial" potentials were recorded from both left and right aspects of the contiguity region. Radiofrequency energy application altered only the potential emanating from the atrium in which the lesion was applied. Histologic analysis confirmed that the lesion was limited to this atrium. It is concluded that, due to their proximity, electrical activity in the RSPV may be recorded from certain areas of the posterior right atrium, and vice versa. In the reported case of left atrial tachycardia, this led to the mistaken impression of right atrial tachycardia. PMID- 10749358 TI - Electrophysiologic characteristics and ablation of an atypical atrial flutter in the right atrium. AB - Subeustachian isthmus-dependent typical atrial flutter has been well studied. We demonstrate a case with atypical atrial flutter involving only the base of the right atrium around the inferior vena cava. Entrainment pacing and mapping studies documented a distinct circuit traversing the subeustachian isthmus, propagating through the posterobasal right atrium, and skirting the inferior vena cava. Successful radiofrequency ablation of the arrhythmia was accomplished by creating a linear lesion at the subeustachian isthmus. Mapping of the inferior vena cava region and the demonstration of concealed entrainment are essential steps in establishing the mechanism of the atypical atrial flutter. PMID- 10749360 TI - Cardiac electrophysiology in genetically engineered mice. AB - The mouse has become the principal animal model for studying biologic processes in mammals. Major advances in transgene and gene targeting technology enabled manipulation of the mouse genome in a predictable fashion. Mutant mouse strains provide important insights into the molecular mechanisms underlying normal and disordered cardiac conduction and sudden cardiac death. A variety of mouse strains harboring gene mutations leading to inherited developmental disorders have been designed. Structural protein abnormalities, connexin protein defects, and ion channelopathies associated with human clinical phenotypes, including congenital heart disease, cardiomyopathies, long QT syndrome, and muscular dystrophy, have been engineered into the mouse genome, creating models of human electrophysiologic disease. Functional analyses of the underlying molecular mechanisms of resultant phenotypes require appropriate and sophisticated experimental methodology. In this review, genetic mouse models pertinent to human arrhythmogenic disorders and their application to present-day ex vivo and in vivo murine electrophysiologic technology at the whole organ and animal levels are discussed. PMID- 10749359 TI - Virtual electrodes and deexcitation: new insights into fibrillation induction and defibrillation. AB - Previous models of fibrillation induction and defibrillation stressed the contribution of depolarization during the response of the heart to a shock. This article reviews recent evidence suggesting that comprehending the role of negative polarization (hyperpolarization) also is crucial for understanding the response to a shock. Negative polarization can "deexcite" cardiac cells, creating regions of excitable tissue through which wavefronts can propagate. These wavefronts can result in new reentrant circuits, inducing fibrillation or causing defibrillation to fail. In addition, deexcitation can lead to rapid propagation through newly excitable regions, resulting in the elimination of excitable gaps soon after the shock and causing defibrillation to succeed. PMID- 10749361 TI - Channel-specific therapy of cardiac arrhythmias in our time? PMID- 10749362 TI - Apparent ventricular bigeminy in the congenital long QT syndrome: what is the mechanism? PMID- 10749363 TI - Identical atrial activation patterns during spontaneous initiations of atrial fibrillation. PMID- 10749365 TI - C-terminal region of VP1 of selected foot-and-mouth disease virus serotypes: expression in E. coli and affinity purification. AB - Foot-and-mouth disease (FMD), one of the most contagious and economically important diseases of farm animals, is caused by a FMD virus (FMDV) which belongs to the family of Picornaviridae. The virus occurs as seven serotypes of which four (A, O, C and Asia 1) are prevalent in India. Immunoprophylaxis supported by precise diagnosis is the prime requirement for achieving the success in controlling the disease. Recently, recombinant DNA technology is gaining importance for the production of cost-effective and safer diagnostics and immunogens. Based on this approach, cDNA of a part of gene for major variable immunogenic region, VP1, of FMDV of four serotypes (A22, O, C and Asia 1) was amplified by PCR and cloned into expression vector. The expression of the 16 K protein gene from the clones was induced with IPTG and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) and [35S]-methionine labeling. The immunoreactivity of the labeled proteins was assayed by immunoprecipitation with anti-FMDV type-specific sera. Since the proteins contain 6 His residues at the N-terminal end, their affinity purification was carried out using nickel nitrilo-tri-acetic acid (Ni-NTA) agarose matrix. The proteins were found to be immunoreactive and the useful in the FMD diagnosis. PMID- 10749364 TI - Characterization of conduction in the ventricles of normal and heterozygous Cx43 knockout mice using optical mapping. PMID- 10749366 TI - Production of viral antigens in culture fluid of C6/36 mosquito cell line infected with dengue type 4 virus strains isolated from patients with different clinical severities. AB - Viral antigen production was examined in the culture fluid of Aedes albopictus clone C6/36 cell line incubated at 28 degrees C and 37 degrees C after infection with four strains of dengue type 4 (DEN-4) virus which were isolated from patients with different clinical severities. During the observation period from day 1 to day 18, the number of infected cells at each day for all four strains did not show any significant difference (P >0.05). Antigen production as determined by the hemagglutination (HA) test and sandwich enzyme linked immunosorbent assay (ELISA) was higher at 28 degrees C than at 37 degrees C for all four DEN-4 virus strains examined. The amount of viral antigen produced was highest for CT93-74 strain (dengue hemorrhagic fever syndrome (DHF) grade II) and was significantly different in comparison to other strains (P <0.001). This strain was followed by CT93-158 and CT93-129 strains (both of DHF grade I), and CT93-77 strain (dengue fever (DF)). The viral antigen production was apparently proportional to the clinical severity of the patient from whom the virus was isolated. These results show that CT93-74 strain could be used to produce DEN-4 virus antigen of sufficiently high titer in the C6/36 cell culture instead of classical extraction of this antigen from suckling mouse brains. PMID- 10749367 TI - Monoclonal antibodies to an Indian strain of type A foot-and-mouth disease virus. AB - A set of five neutralizing monoclonal antibodies (MAbs) to an Indian strain (IND17/77) of type A (subtype A22) foot-and-mouth disease (FMD) virus (FMDV) was used in the study. Four of the MAbs (27S, 37S, 85S, and 143S) identified a trypsin-sensitive (TS) epitope(s) and were specific for VP1, while the remaining MAb (145S) reacted with a trypsin-resistant (TR) epitope and was specific for VP3 in Western blot analysis. Both the epitopes (TS and TR) were conformation independent in nature. Results obtained in MAb-competition enzyme-linked immunosorbent assay (ELISA), and profiling of the (MAb) neutralization-escape mutants in ELISA and cross-neutralization test revealed two overlapping TS epitopes (27S/37S and 85S/143S) on the virus. Variation at both these epitopes was observed in some field isolates of serotype A. Comparison of deduced amino acid sequence in the VP1 region (aa 140-213) between the parent virus and the mutants identified Gly148 and Arg153 as critical for the formation of both the TS epitopes. Substitution of R153 by Gly or Ser was observed in mutants with no reactivity for the MAbs 85S/143S. However, these mutants maintained partial reactivity with MAbs 27S/37S, and substitution of Gly148 by Glu eliminated both the epitopes. No amino acid substitution was observed in the VP1 region of aa 200 213. Efficient neutralization of the MAb neutralization escape mutants (MAb resistant (MAR) mutants) by bovine vaccinate serum (BVS) indicated involvement of other epitopes on the virion surface in eliciting neutralizing antibodies following vaccination. PMID- 10749368 TI - Induction and distribution of amylolytic activity in Cucumis sativus L. in response to virus infection. AB - Inoculation of cucumber (Cucumis sativus L. cv. Laura) cotyledons with tobacco necrosis virus (TNV) causes both qualitative and quantitative changes in the total and fractionated protein extracts as well as in amylolytic activity. Using a specific test it was demonstrated that the virus infection strongly enhances a major band (Rf 0.0645) of amylolytic activity, predominantly located in apoplast space. The accumulation of this extracellular amylolytic activity is regulated by a time-dependent manner and is correlated with the development of necrotic lesions. The amylolytic activity may be related to degradation of starch shown to be accumulated in the immediate vicinity of necrotic lesions associated with the hypersensitive response (HR). The possible biological function of the identified amylolytic activity in the term of ,,pathosmosis" is also discussed. PMID- 10749369 TI - An epitope shared by the hemagglutinins of H1, H2, H5, and H6 subtypes of influenza A virus. AB - The membrane-inserted hemagglutinin (HA) is the most variable protein of influenza viruses. Here we describe the characterization of a shared epitope in the HA of influenza A virus H1, H2, and H5 subtypes which were completely neutralized by a monoclonal antibody (MAb), directed against this epitope. This MAb (C179) also efficiently precipitated the HAs of these viruses. In addition, MAb C179 did not neutralize H6 subtype strains despite complete amino acid homology of the epitope regions. Furthermore, only the non-glycosylated form of the HA of one of the H6 subtype strains could be precipitated by the MAb. The conformational epitope may be masked by glycosylation, although it could not be excluded that differences in the primary amino acid sequence may cause the decreased accessibility of the epitope in H6 subtype strains. PMID- 10749370 TI - Differentiation of infectious bursal disease virus strains by restriction analysis of RT-PCR-amplified VP2 gene sequences. AB - The techniques of reverse transcription-polymerase chain reaction (RT-PCR) and restriction analysis were used to differentiate highly virulent Indian field isolates of infectious bursal disease virus (IBDV) from vaccine strains. Primers were designed to amplify the variable region of VP2 gene coding for major virus neutralizing epitopes. The 552 bp PCR products generated from four vaccine strains and five field isolates were digested with restriction enzymes DraI, HhaI, MvaI, StuI, StyI, and TaqI, which could differentiate field isolates from vaccine strains. Based on restriction enzyme profiles derived from published sequences, Indian field isolates seem to be closely related to highly virulent Japanese, European, and Chinese strains of the virus. PMID- 10749371 TI - Promotion of vesicular stomatitis virus nucleocapsid protein production by arthopod saliva. AB - In a previous study (Hajnicka, V. et al., Parasitology 116, 533-538, 1998), the infectivity titer of vesicular stomatitis virus (VSV) was shown to increase up to 10,000-fold when mouse L cells were treated with tick salivary gland extract (SGE) prior to infection. To examine this effect at the level of viral protein production, radiolabeled VSV-infected cells were analyzed by double-dimensional gel electrophoresis. A pre-treatment of cells with SGE from partially fed ticks in amounts corresponding to 1 or 3 salivary glands increased the level of both viral nucleocapsid (N) protein and phosphoprotein (P) in a dose-dependent manner. The effect was more pronounced for N protein and could account for the dramatic increase in infectious virus yield. Promotion of viral infectivity by arthropod saliva may support the arthropode-borne transmission cycle of VSV. PMID- 10749372 TI - Preparation of diagnostic monoclonal antibodies against two potyviruses. AB - Diagnostic monoclonal and polyclonal antibodies against bean yellow mosaic virus (BYMV) and plum pox virus (PPV) were prepared, characterized and used for detection of these viruses in infected plants by enzyme-linked immunosorbent assay (ELISA), immunoblot analysis and tissue print immunoblot assay (TPIBA). PMID- 10749373 TI - Plum pox virus capsid protein mobility in SDS-polyacrylamide gel electrophoresis. AB - Slovak plum pox virus (PPV) isolates BOR-3 and KR-4 behaved atypically in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS PAGE) of their capsid proteins (CPs). Whereas other tested PPV isolates could be sorted by SDS-PAGE into groups corresponding to the M or D strains, as controlled by restriction fragment length polymorphism (RFLP) analysis, the two above mentioned isolates behaved as the M strain according to RFLP analysis but not SDS PAGE. Slight mobility differences were observed also among the isolates belonging to the D strain. SDS-PAGE of the CP thus cannot clearly distinguish between these two main PPV strains. BOR-3 isolate has been shown atypical also in its biological properties, and it reacted very weakly with a monoclonal antibody (MAb) recognizing well both M and D strains in immunoblot analysis. PMID- 10749374 TI - Cytotoxicity of the synergistic antienteroviral combination of enviroxime and disoxaril. AB - When combined, enviroxime and disoxaril, two selective picornavirus inhibitors, exert a marked synergistic inhibitory effect on poliovirus type 1 replication in FL cells. The cytotoxicity of the compounds applied individually and in combination to the same cells was examined. The quantitative assay of the cytotoxic effect was made by determination of the growth curve of uninfected FL cells in the presence of increasing concentrations of the compounds applied alone and in combination. The obtained results indicate lack of a synergic cytotoxic effect of the combination of enviroxime and disoxaril. The previously established synergistic antiviral effect, and the lack of cross-resistance and synergic cytotoxic effect classify the combination of enviroxime and disoxaril as a very promising chemotherapeutic. PMID- 10749376 TI - The discovery of rhabdovirus-like particles in strawberry in the Czech Republic. PMID- 10749375 TI - Respiratory syncytial virus infection of human respiratory epithelial cells enhances both muscarinic and beta2-adrenergic receptor gene expression. AB - The possible changes in transcriptional activities of the M1 muscarinic acetylcholine receptor (mAChR) and beta2-adrenergic receptor (AR) genes in respiratory syncytial virus (RSV)-infected human type 2 alveolar epithelial cells (A549 cells) were analyzed semiquantitatively by reverse transcription-polymerase chain reaction (RT-PCR). RSV enhanced M1 mAChR gene expression significantly at 4 hrs post infection (p.i.), and this enhancement persisted until 10 hrs, after peaking at 7 hrs. Beta2-AR gene expression also increased significantly as early as at 1 hr p.i. and persisted for more than 10 hrs. These transcriptional enhancements were observed in cells treated with live but not with inactivated virus. Our observations suggest that RSV infection of human respiratory epithelial cells may enhance the expression of both parasympathetic and sympathetic receptors. The upregulated M1 mAChR gene in virus-infected cells may correlate with temporal airway hyperresponsiveness in subjects with RSV or other respiratory virus infection. The enhanced beta2-AR gene expression in peripheral lungs might explain the excessive mucus secretion observed during viral pneumonitis. PMID- 10749377 TI - Expression of cyclin-dependent kinase inhibitors during corneal wound repair. AB - During corneal epithelial wound repair, cells migrating to cover the wound area exhibit a drastic reduction in proliferative activity. In contrast, cells distal to the original wound exhibit a greatly enhanced level of proliferative activity. At least 90% of the basal cells in limbal and peripheral corneal epithelia synchronously progress through the cell cycle. The question addressed in this article is whether cyclin-dependent kinase inhibitors play a role in the alterations in proliferative activity seen during corneal wound repair. These inhibitors specifically block cells in the G1-phase of the cell cycle. Two families of cyclin-dependent kinase inhibitors have been identified. The CIP/KIP family includes p21, p27, and p57, while the INK4 family consists of p16. p15. p18. and pI9. At least five of these inhibitors are present in the corneal epithelium. The expression of two of these, p15 and p27. is dramatically altered during wound repair, suggesting that they may be involved in the changes in cell proliferation observed during corneal wound healing. PMID- 10749378 TI - The regulation of trabecular meshwork and ciliary muscle contractility. AB - Current models of aqueous humor outflow no longer treat trabecular meshwork (TM) as an inert tissue passively distended by the ciliary muscle (CM). Instead, ample evidence supports the theory that trabecular meshwork possess smooth muscle-like properties and is actively involved in the regulation of aqueous humor outflow and intraocular pressure. In this model, trabecular meshwork and ciliary muscle appear as functional antagonists, with ciliary muscle contraction leading to a distension of trabecular meshwork with subsequent reduction in outflow. and with trabecular meshwork contraction leading to the opposite effect. Smooth-muscle relaxing substances would therefore appear to be ideal candidates for glaucoma therapy with the dual goal of reducing intraocular pressure via the trabecular meshwork and of improving vascular perfusion of the optic nerve head. However, for such substances to effectively lower intraocular pressure, the effect on the ciliary muscle would have to he minimal. For this reason, more information is needed on the signalling processes involved in regulating trabecular meshwork and ciliary muscle contractility. This review attempts to outline current knowledge of signal transduction pathways leading to relaxation and contraction of ciliary muscle and trabecular meshwork. Pathways can be classified as involving or not involving changes of membrane voltage and of requiring or not requiring external calcium: possibly, other pathways exist. These different pathways involve different ion channels and isoforms of PKC and are expressed to a differing degree in ciliary muscle and trabecular meshwork, leading to differential responses when exposed to relaxing or contracting pharmacological agents. Some of these agents. like tyrosine kinase inhibitors and inhibitors of PKC. have been shown to relax trabecular meshwork while leaving ciliary muscle comparatively unaffected. This profile makes these substances appear as ideal drugs for simultaneously improving ocular outflow and retinal circulation, parameters that determine the time course of visual deterioration in glaucoma. PMID- 10749379 TI - The optic nerve head in glaucoma: role of astrocytes in tissue remodeling. AB - Primary open angle glaucoma is a common eye disease characterized by loss of the axons of the retinal ganglion cells leading to progressive loss of vision. The site of damage to the axons is at the level of the lamina cribrosa in the optic nerve head. The mechanism of axonal loss is unknown but elevated intraocular pressure and age are the most common factors associated with the disease. Previous studies in human glaucoma and in experimental glaucoma in monkeys have established a relationship between chronic elevation of intraocular pressure and remodeling of the optic nerve head tissues known clinically as cupping of the optic disc. This review focuses on the astrocytes, the major cell type in the optic nerve head. Astrocytes participate actively in the remodeling of neural tissues during development and in disease. In glaucomatous optic neuropathy, astrocytes play a major role in the remodeling of the extracellular matrix of the optic nerve head, synthesize growth factors and other cellular mediators that may affect directly, or indirectly, the axons of the retinal ganglion cells. Due to the architecture of the lamina cribrosa, formed by the cells and the fibroelastic extracellular matrix, astrocytes may respond to changes in intraocular pressure in glaucoma, leading to some of the detrimental events that underlie axonal loss and retinal ganglion cell degeneration. PMID- 10749380 TI - Structural macromolecules and supramolecular organisation of the vitreous gel. AB - The vitreous gel is a transparent extracellular matrix that fills the cavity behind the lens of the eye and is surrounded by and attached to the retina. This gel liquefies during ageing and in 25-30% of the oppulation the residual gel structure eventually collapses away from the posterior retina in a process called posterior retina in a process called posterior vitreous detachment. This process plays a pivotal role in a number of common blinding conditions including rhegmatogenous retinal detachment, proliferative diabetic retinopathy and macular hole formation. In order to understand the molecular events underlying vitreous liquefaction and posterior vitreous detachment and to develop new therapies it is important to understand the molecular basis of normal vitreous gel structure and how this is altered during ageing. It has previously been established that a dilute dispersion of thin (heterotypic) collagen fibrils is essential to the gel structure and that age-related vitreous liquefaction is intimately related to a process whereby these collagen fibrils aggregate. Collagen fibrils have a natural tendency to aggregate so a key question that has to be addressed is: what normally maintains the spacing of the collagen fibrils? In mammalian vitreous a network of hyaluronan normally fills the spaces between these collagen fibrils. This hyaluronan network can be removed without destroying the gel structure, so the hyaluronan is not essential for maintaining the spacing of the collagen fibrils although it probably does increase the mechanical resilience of the gel. The thin heterotypic collagen fibrils have a coating of non-covalently bound macromolecules which, along with the surface features of the collagen fibrils themselves, probably play a fundamental role in maintaining gel stability. They are likely to both maintain the short-range spacing of vitreous collagen fibrils and to link the fibrils together to form a contiguous network. A collagen fibril associated macromolecule that may contribute to the maintenance of short-range spacing is opticin, a newly discovered extracellular matrix leucine-rich repeat protein. In addition, surface features of the collagen fibrils such as the chondroitin sulphate glycosaminoglycan chains of type IX collagen proteoglycan may also play an important role in maintaining fibril spacing. Furthering our knowledge of these and other components related to the surface of the heterotypic collagen fibrils will allow us to make important strides in understanding the macromolecular organisation of this unique and fascinating tissue. In addition, it will open up new therapeutic opportunities as it will allow the development of therapeutic reagents that can be used to modulate vitreous gel structure and thus treat a number of common, potentially blinding, ocular conditions. PMID- 10749381 TI - Exfoliation syndrome and exfoliation glaucoma. AB - Exfoliation syndrome abnormal deposition in the anterior segment of the eye of an unknown substance thought to be related to elastic fibres and basement membrane components is associated with accelerated cataract progression. increased frequency of intraoperative and postoperative complications and increased risk for glaucoma and. therefore, is a clinically important finding. A clear association has been shown with age. The syndrome occurs worldwide but its prevalence seems to vary from country to country. The best-known sign of exfoliation syndrome is deposits of greyish-white material on the anterior lens surface. Sometimes exfoliation material can also be seen at the pupillary border, on the anterior iris surface, corneal endothelium, and on the anterior vitreous face. When clinically detected, exfoliation syndrome is somewhat more often unilateral than bilateral. According to recent investigations clinically unilateral exfoliation syndrome is probably never truly unilateral but rather asymmetric, because exfoliation material has been detected ultrastructurally and immunohistochemically around iris blood vessels of the nonexfoliative fellow eyes. Indeed, electron microscopy identifies in various organs of patients with exfoliation syndrome fibrils similar to those seen in intraocular exfoliation deposits. Other clinical signs associated with exfoliation syndrome are pigment dispersion, transillumination defects of the iris and reduced response to mydriatics. In unilateral exfoliation syndrome, intraocular pressure (IOP) of the exfoliative eye is approximately 2 mmHg higher than IOP of the nonexfoliative fellow eye. Whether elevated IOP, vascular changes or exfoliation syndrome itself is the main factor causing optic nerve head damage and conversion of an exfoliative eye to glaucomatous, is not known. Glaucoma in the exfoliation syndrome has been shown to have a more serious clinical course than in primary open-angle glaucoma (POAG). At the time of diagnosis, IOP and its diurnal variation are generally higher and visual field defects tend to be greater in exfoliation glaucoma than in POAG. Because the decrease in lOP variation and lowering of the mean IOP level has been shown to improve visual field prognosis more in exfoliation glaucoma than in POAG, the glaucomatous process is considered to be more pressure-related in exfoliation glaucoma. Furthermore, progression of optic disc damage has been shown to be similar in exfoliation glaucoma and POAG when lOPs are lowered to a comparable level by the treatment. However, vascular disturbances in the posterior segment of the eye might after all be of equal importance in these two types of glaucoma; optic disc haemorrhages and venous occlusions have been reported to be as frequent in exfoliation glaucoma as in POAG. Perhaps in exfoliation glaucoma circullatory disturbances combined with high IOP lead to a particularly relentlessly progressing form of the disease. PMID- 10749382 TI - Making space for safer sex. AB - There is much concern about the second wave of HIV infections among gay male youth. Yet qualitative research showing how to produce effective HIV prevention programs for this population are scarce. Traditional models for education are not sufficient. This study uses ethnographic data to illustrate a community empowerment HIV prevention project found to significantly reduce rates of unprotected anal sex among young gay males between the ages of 18 and 29. It seeks to show how safer sex norms among gay youth are produced. Analyses of these data reveal that a sense of ownership in a youth space is a critical component of this model. This research should facilitate AIDS educators in their efforts to end the second wave of infections among gay youth by providing a detailed map of how a successful HIV prevention project operates and by stressing the need to make spaces for safer sex education. PMID- 10749383 TI - Changing sources of care for HIV infection in California. AB - A telephone survey was conducted of random samples of primary care physicians in Los Angeles County and in the 26 rural counties of California to assess changes in medical practices that may have occurred with the development of new pharmacological agents and recent changes in the management of infection with the HIV. Seventy-two to seventy-three percent of the physicians selected participated in the survey in both areas. The results indicate a reduction by almost two thirds of the number of primary care physicians who plan to continue to provide care to HIV-infected patients, both in Los Angeles County and the nonmetropolitan counties of California. Although some were no longer caring for HIV patients because of deaths or patient relocation, the overwhelming majority had referred their patients to infectious disease specialists and HIV clinics because of the growing complexity of the management of these patients. PMID- 10749384 TI - HIV prevention among male clients of female sex workers in Kaolack, Senegal: results of a peer education program. AB - This article reports the results of a peer-led HIV prevention education and condom promotion program among transport workers in Kaolack, Senegal. As part of a 2-year longitudinal follow-up study, changes in men's AIDS-related knowledge, sexual behavior, condom use, and perceived barriers to condom use were evaluated by self-reports obtained from a systematic sample of transport workers interviewed before and after intervention. In addition to men's self-reports, preintervention and postintervention data on men's sexual and condom use behavior were gathered from a sample of licensed, commercial sex workers, who cited transport workers as their primary source of clients. Significant increases in men's HIV-related knowledge, previous use of condoms (from 30.4% to 53.5%), and consistent condom use with regular sex partners were documented over the study period, as were significant declines in perceived barriers to condom use. Though men reported significantly fewer sexual encounters with casual and commercial partners at follow-up compared to baseline, these data were unreliable. Women's postintervention reports indicate that a greater proportion of clients (including, but not limited to transport workers) "always" agree to use condoms (p < .01) compared with baseline and that fewer men offer more money for unprotected sex (p < .01). However, women also report taking greater initiative in the mechanics of condom use (supplying the condom, putting it on, and taking it off) than they did prior to the intervention, and significantly (p < .05) fewer women think that most of their clients know how to use a condom. The findings indicate that the peer-mediated intervention had a positive impact on several important outcomes measured and suggest that HIV prevention efforts need to focus on male client groups despite the logistical and methodological challenges. PMID- 10749385 TI - Substance use and HIV risk profile of gay/bisexual males who drop out of substance abuse treatment. AB - This analysis was undertaken to identify (a) the level of HIV sexual risk behaviors of men who drop out of treatment and (b) baseline variables associated with later treatment dropout. A cross-sectional sample of 340 gay/bisexual men were recruited from an outpatient substance abuse treatment facility in San Francisco. We compared participants who completed less than 15 visits with participants who graduated from the program. Men who dropped out were more likely than treatment graduates to report injection drug use, social problems related to substance use, self-blaming coping strategies, and more recent substance use prior to entering treatment and less likely to have a college degree, report using sex for tension relief, and have previously attended Alcoholics Anonymous or Narcotics Anonymous. Given the strong link between the substance abuse and HIV epidemics, substance abuse treatment agencies have been forced into addressing the issues of HIV sexual risk taking with their clients. Strategies toward reducing substance use relapse and HIV risk reduction are offered. PMID- 10749386 TI - Students together against negative decisions (STAND): evaluation of a school based sexual risk reduction intervention in the rural south. AB - Twenty-one 10th graders selected as opinion leaders by their peers in a rural county in a southern state participated in a 36-hour peer-educator training program Students Together Against Negative Decisions (STAND) based on diffusion of innovations theory and the transtheoretical model. Comparison subjects received either a 22-hour leadership training course (n = 20) or no intervention (n = 45). STAND and comparison subjects completed a 154-item written knowledge, attitude, and behavior survey at the beginning of the training (Time 1), at the end of the training (Time 2), and again 8 months later (Time 3). One hundred and sixty-seven other 9th and 10th graders in the intervention county and 74 in the comparison county completed an abbreviated telephone interview at Time 1 and Time 3. At Time 3 STAND-trained peer educators reported significantly greater increases in AIDS Risk Behavior Knowledge (more than 4 times comparison groups), frequency of conversations with peers about birth control/condoms (+180% vs. +12%) and sexually transmitted diseases (STDs; +282% vs. -33%), condom use self efficacy (+16% vs. -1%), and consistent condom use (+28% vs. +15%). STAND teens also reported substantial favorable trends at Time 3, including increased condom use (+213% vs. +31%) and decreased unprotected intercourse (-30% vs. +29%). At Time 3 teens in the intervention county reported significantly greater increases in the number of people who talked with friends in the preceding 3 months about STDs (+39% vs. -19%) or with a parent/adult about sex (+6% vs. -37%). Intervention county teens also reported a substantial but nonsignificant 2.6-fold greater increase in condom use at last intercourse (+64% vs. +25%) but unfavorable changes in other risk behaviors. The STAND peer-educator training program appears to be an effective method for improving selected sexual knowledge, attitudes, and behaviors among participant teenagers in the rural South. PMID- 10749387 TI - Feasibility of chemoprophylaxis studies in high risk HIV-seronegative populations. AB - Administration of antiretroviral medications-recommended to prevent HIV infection after occupational exposure-has not been evaluated for safety or efficacy following nonoccupational exposure. HIV-seronegative persons at increased risk for HIV exposure completed a self-administered questionnaire assessing their willingness to join studies of this approach. Of 4,572 respondents, 60% were willing to join a study of a "morning-after" pill; dosing three times a day and mild side effects reduced willingness to 30%. Men who have sex with men (MSM) who reported unprotected anal intercourse in the prior 6 months were significantly more likely to be willing to join a morning-after study than MSM who did not (p = 0.006). MSM favored a preventive HIV vaccine over oral chemoprophylaxis; other populations preferred oral chemoprophylaxis. Interest in studies declined as the hypothetical regimen became more demanding. Studies must emphasize the unknown efficacy of this approach, given increased interest among MSM at greater risk of exposure. PMID- 10749388 TI - Relationship of psychosocial factors to HIV risk among Haitian women. AB - This study describes the prevalence of HIV risk behaviors among low-income, Haitian women, identifies theoretically relevant mediating psychosocial HIV risk predictors, and provides formative data for developing culturally and gender sensitive interventions for this distinctive, high risk, and understudied population. Confidential interview surveys were administered to 101 women of Haitian descent while they awaited their medical appointments at a local low income, community medical clinic. Moderately high levels of sexual risk behavior (i.e., unprotected sex with nonmonogamous partners; multiple lifetime partners) were reported. On average, these women reported a belief in their HIV susceptibility, relatively little HIV-related anxiety, somewhat inadequate levels of communication regarding safer sex practices, and lack of adequate confidence in their ability to negotiate safer behaviors in sexual encounters. Both personal and partner condom attitudes were unfavorable and these attitudes predicted condom use levels. It was concluded that interventions need to be developed for Haitian women to improve their attitudes toward condom use and their confidence in negotiating safer sexual practices. However, these interventions cannot be developed in a vacuum. Although it is crucial to consider the woman's individual attitudes and behaviors, it is also important to consider the male partner's attitudes toward sex and the woman's relationship with her male partner within the context of Haitian culture. Only by determining and targeting important potential motivations for safe sex within the cultural context can we most effectively reduce HIV sex risk behavior in Haitian women. PMID- 10749389 TI - Geriatric psychopharmacology: why does age matter? AB - Older adults respond less predictably than younger adults to most medications, typically requiring lower daily doses to achieve desired therapeutic effects and minimize adverse effects and toxicity. This unpredictability is particularly evident among the frail elderly, who are at the upper extreme of the life cycle and often suffer from central neurodegenerative disorders and/or a significant burden of comorbid medical problems. Yet this population has a burgeoning need for clinical services and in recent years has become an increasingly important focus of attention among practitioners. The goal of this review is to provide clinicians with a conceptual framework for understanding and responding to aging and age-related events that influence pharmacotherapeutics in older patients with behavioral disorders. Limitations and gaps in our knowledge base are also highlighted. The article includes a phenomenological overview of the aging process, a consideration of age-related factors that influence the pharmacokinetic and pharmacodynamic properties of psychotropic drugs, and suggested methods of enhancing medication compliance. PMID- 10749390 TI - A name, what's in a name? The medicalization of hyperactivity, revisited. AB - Although attention-deficit hyperactivity disorder has in recent years become one of the most common psychiatric problems diagnosed in children, its status as a medical disorder remains controversial, especially in Britain. This study examines the experience of parents and doctors dealing with hyperactive children, focusing in particular on the process of medicalization. It alms at understanding what is at stake for families and doctors and asks about the role of a medical label in the therapeutic process. It uses an anthropologically informed methodology, in which qualitative semistructured interviews of 1-2 hours duration were held with 29 parents of hyperactive children and 10 general practitioners. Interviews were audiotaped; the content was transcribed and analyzed according to grounded hermeneutic theory. It was found that raising a hyperactive child can provoke a profound sense of alienation in parents. Family and social roles are affected, as are parents' views of themselves in their parental and social roles. Parents tended to experience medicalization and labeling as important aspects of validation and legitimation of their experience, which gave them a sense of control and led to improved parent-child relationships. Doctors felt more reluctant about such medicalization, fearing that it could lead to scape-goating the child and to self-fulfilling prophecies. It may be concluded that the medical framework, especially the medical diagnosis, can provide a powerful tool that has both advantages and disadvantages in the therapeutic process. In trying to avoid stigmatization, doctors can delegitimate parents' experience, thus increasing their suffering. PMID- 10749391 TI - The role of identity in the development of relationships and career. PMID- 10749392 TI - Human rights and psychiatry: report on a teaching model. PMID- 10749393 TI - Does the law interfere with ethical patient care? How it can and why it need not. PMID- 10749394 TI - After managed care, what? PMID- 10749395 TI - Role of nutrients and bacterial colonization in the development of intestinal host defense. AB - In this introduction to the supplement on the use of pre- and probiotics in the health and disease of pediatric patients, I have summarized factors affecting the initial colonization of the neonatal intestine. The term bacterial-epithelial cross-talk was defined, and examples of the enterocyte response to both pathologic and indigenous flora stimulation illustrated. Immaturities in the human neonatal intestinal response to bacteria and their toxins were reviewed in the context of the pathogenesis of age-specific, bacterial gastrointestinal infectious diseases. Finally, the importance of pre- and probiotics as measures to strengthen the neonate's intestinal host defenses in the prevention and treatment of specific age-related disease were considered. PMID- 10749396 TI - Oligosaccharides in human milk and bacterial colonization. PMID- 10749397 TI - Neonatal necrotizing enterocolitis: possible role of probiotic supplementation. PMID- 10749398 TI - Role of oligosaccharides and glycoconjugates in intestinal host defense. PMID- 10749399 TI - Probiotics and intestinal inflammatory disorders in infants and children. PMID- 10749400 TI - Eotaxin and eosinophilic homing to the gut. PMID- 10749401 TI - Emerging role of the enteric nervous system in mediating host responses to microbial infections. PMID- 10749402 TI - The efficacy of cisapride; time to review not regurgitate. PMID- 10749403 TI - The efficacy of cisapride: what is needed for digestion? PMID- 10749404 TI - Role of tissue transglutaminase in celiac disease. PMID- 10749405 TI - Gastrointestinal infections in children in the Southeast Asia region: emerging issues. PMID- 10749406 TI - Acid secretion and response to pentagastrin or omeprazole in human fetal stomach xenografts. AB - BACKGROUND: The dual capacity of stomach tissue to secrete acid and to respond to secretagogues is indicative of the terminal stages of gastric functional maturation. In this study 6- to 10-week-old human fetal stomachs xenografted into nude mice were used to study parietal cells' functional maturation. METHODS: Thirty-four transplants were microsurgically grafted either inside a pouch created on the nude peritoneum (n = 15) or on the host stomach and esophagus (n = 19). The mucosa of transplanted tissues was analyzed by immunohistochemical techniques to detect gastric cells. Gastric cell secretions were collected before and after pentagastrin or omeprazole treatment. RESULTS: Parietal, G, and D cells were detected immunohistochemically only after 1 month of grafting. All xenografts actively secreted acid after 1 or 2 months' transplantation at each graft site. Acid secretion was significantly stimulated by intraperitoneally injected pentagastrin (mean pH +/- SD, 3.2 +/- 0.7 vs. 2.0 +/- 0.5; n = 10, P = 0.005) and was dramatically inhibited by intragastrically administered omeprazole (2.3 +/- 0.6 vs. 6.5 +/- 0.7; n = 15, P = 0.0007) after 5 hours. CONCLUSION: Stomach xenografts were able to develop normally. Parietal cells were physiologically mature with functional proton pumps and active gastrin receptors, as demonstrated after omeprazole and pentagastrin treatment, respectively. Because stomach xenografts matured very rapidly, it is possible that a stomach xenograft model can be used for further studies on the functional maturation of human gastric epithelial cells, as well as the factors that influence this maturation in humans. PMID- 10749408 TI - Inflammatory bowel disease in children and adolescents in Sweden, 1984-1995. AB - BACKGROUND: A prospective study of inflammatory bowel disease (IBD) in Sweden was performed to investigate whether the incidence and morbidity have changed from 1984 through 1995. METHODS: Children 15 years of age or less with IBD were included--i.e., those with a definite diagnosis of ulcerative colitis (UC) and Crohn's disease (CD) and those classified as having indeterminate colitis (IC) and probable Crohn's disease (PCD). The study covered 56.5% of the pediatric population of Sweden. RESULTS: The diagnosis of IBD was made in 639 children, which corresponds to a mean annual incidence of 5.8 per 100,000. The incidence increased from 4.6 per 100,000 per year from 1984 through 1986 to 7.0 from 1993 through 1995. It reflected an increase in UC from 1.4 to 3.2 per 100,000 per year, which is a significant yearly percentage of increase (8%; confidence interval, 2-14%; P < 0.05). In contrast, no change occurred in the incidence of CD (1.2-1.3 per 100,000). The incidence of IC and PCD also remained fairly stable. The percentages of children who underwent surgery decreased from 17.3% in the first 6 years to 4.6% in the last 6 years (P < 0.001). Surgery was performed in 27.7% of CD and 5.3% of UC cases. The median age at diagnosis was 12.2 years for UC, 13.0 years for CD, 11.2 for IC, and 11.2 for PCD. At diagnosis, 48 children (7.5%) were 5 years of age or less, whereas most of the patients were 11 years of age or more (398 children, 62.3%). CONCLUSIONS: In Sweden, the incidence of UC has increased, whereas that of CD remains the same. A significant number of children were classified with IC and PCD. In most children, IBD was diagnosed when they were 11 years old or more, but some cases were detected even in those below 6 years of age. A decrease in the frequency of surgery occurred during the study. PMID- 10749407 TI - Difference in dietary intake and activity level between normal-weight and overweight or obese adolescents. AB - BACKGROUND: To investigate the prevalence of overweight and obesity in an adolescent group representative of a rural Mediterranean area and to determine possible associations with energy and nutrient intakes and levels of physical exercise. METHODS: A representative sample of adolescents was drawn from the secondary school of Torre Pacheco (Murcia), a rural Mediterranean area located in the southeast of Spain. The population selected (331 adolescents aged 14-18 years), was divided into two groups: normal-weight subjects with a body mass index less than 23 kg/m2 and overweight or obese subjects with a body mass index of 23 kg/m2 or more. Weight, height, abdominal and hip perimeters, triceps skinfold, and upper arm circumference were measured. A prospective 7-consecutive days food record and physical activity questionnaire were completed. RESULTS: Overweight boys and girls had an apparently lower energy intake (P = 0.001 and P = 0.042, respectively), and carbohydrate intake (P = 0.000, P = 0.032) than their normal-weight counterparts, but they tended to underreport more often. Overweight boys derived a greater percentage of their energy from fat (P = 0.049) and less from carbohydrate (P = 0.016) than their normal-weight counterparts. Among girls, the percentage of energy derived from fat increased with body mass index (r = 0.210, P = 0.008), whereas fiber intake decreased (r = -0.145; P = 0.041). Overweight and obesity were negatively related to physical activity level only among boys (P = 0.033). CONCLUSION: There is a high prevalence of overweight and obesity in the adolescent population studied (48.2% in boys and 30.7% in girls). The study shows an association between overweight and obesity and nutrient intake and activity level. PMID- 10749409 TI - Parietal cell antibodies and Helicobacter pylori in children. AB - BACKGROUND: Gastric autoantibodies are common in Helicobacter pylori-infected adults, and the presence of these antibodies is associated with atrophic gastritis. The role of H. pylori in the autoimmune type of atrophic gastritis is unresolved, and it is not known at what stage the autoantibodies appear in serum during H. pylori infection. Therefore, we screened children with and without H. pylori infection for gastric parietal cell antibodies. METHODS: Seventy-one children with H. pylori infection verified by examination of gastric biopsy specimens (mean age, 9.4 years), 8 children with positive serology but negative histology for H. pylori (mean age, 11.6 years), and 130 children with negative serology for H. pylori (mean age, 7.7 years) were screened for the presence of gastric parietal cell antibodies in serum by indirect immunofluorescence. In addition, 61 children with celiac disease (mean age, 7.1 years) were screened for gastric parietal cell antibodies and H. pylori antibodies. RESULTS: None of the children with H. pylori infection had gastric parietal cell antibodies in serum. Only three positive parietal cell antibody reactions were found: a 14-year-old boy with positive serology for H. pylori but no other signs of infection (titer 5000), a 14-year-old girl with tuberculosis (titer 1250, seronegative for H. pylori) and a 10-year-old girl with insulin-dependent diabetes mellitus (titer 6250, seronegative for H. pylori). CONCLUSIONS: Although gastric autoantibodies are often found in adults with chronic H. pylori gastritis, it seems that H. pylori-infected children are not positive for gastric parietal cell antibodies. It remains to be studied in which H. pylori infections and at what stages gastric autoantibodies appear. PMID- 10749410 TI - Long-term nutritional and neurodevelopmental outcome of liver transplantation in infants aged less than 12 months. AB - BACKGROUND: Liver transplantation is established treatment for children with end stage liver disease and has a 5-year survival rate of 80% to 85%, even in infants under 12 months. Long-term outcome in nutritional rehabilitation and normal development is unknown. This study aimed to prospectively evaluate growth and psychoneurologic performance of children who undergo liver transplantation in infancy. METHODS: Twenty-five infants (18 girls, 7 boys) who underwent liver transplantation at less than 12 months of age (median age, 9 months) were evaluated for 4 years. Growth measurements were expressed as standard deviation scores (SDSs; mean +/- SEM), and psychoneurologic performance was assessed with the unrevised Griffiths Mental Ability Scales (normal range, 80-120). RESULTS: Four children died during the study (4-year survival, 84%). The children were malnourished before transplantation (SDSs: weight, -1.9 +/- 0.2; midarm muscle area, -0.93 +/- 0.3; midarm fat area, -1.52 +/- 0.3; and height, -0.95 +/- 0.3). Nutritional rehabilitation for all parameters occurred within 12 to 24 months after transplantation, which was most significant for weight (-1.1 +/- 0.2, P = 0.001), midarm muscle area (0.74 +/- 0.3, P = 0.001), and midarm fat area (-0.44 +/- 0.3, P = 0.01). There was some improvement in height (-0.72 +/- 0.3, P = 0.14), which was not significant, although infants who were severely stunted before transplantation (mean height standard deviation score [SDS] -2.46) showed significant catch-up at 1 year after transplantation (mean height SDS -1.2, P = 0.003). Psychoneurologic scores were within normal limits before transplantation and were maintained for the 4-year follow-up period, although individual scores varied during this period. Improved nutritional status was associated with increased muscle bulk and subsequent improvement in motor scores from 90.6 at initial assessment to 97.3 at 4 years (P = 0.28). There was a temporary reduction in social skills and eye-hand coordination in the first year, which may have been an effect of the hospital environment or cyclosporine immunosuppression. Language abilities also regressed during the first year, possibly related to the effect of nasogastric tube feeding in delaying normal speech development. CONCLUSIONS: Liver transplantation in infancy has not only a successful outcome but is also associated with long-term catch-up growth and nutrition and maintenance of normal development. PMID- 10749411 TI - A prospective trial of lansoprazole triple therapy for pediatric Helicobacter pylori infection. AB - BACKGROUND: Triple therapy with a proton-pump inhibitor and two antibiotics is widely used in the treatment of Helicobacter pylori infection in adults. Experience with such therapy in the pediatric population is limited. This was a prospective, nonrandomized, open-label trial to evaluate safety and efficacy of a combination of lansoprazole, clarithromycin, and amoxicillin in symptomatic children with H. pylori infection. METHODS: Children with H. pylori gastritis diagnosed by endoscopy performed for persistent nausea, vomiting, recurrent abdominal pain, and diarrhea with consistent histology were treated with the regimen of 0.45 mg/kg per day lansoprazole divided into two doses (maximum dose, 15 mg twice daily), amoxicillin 40 mg/kg per day in two doses (maximum dose, 1.0 g twice daily), and 250 mg clarithromycin twice daily (<10 years old) or 500 mg twice daily (>10 years old) for 2 weeks. Pre- and posttreatment endoscopic biopsy specimens were graded for the severity of gastritis and H. pylori density by a blinded pathologist. A questionnaire for assessing the severity of symptoms at the time of initial and second endoscopy were completed by patient and/or parent. RESULTS: Thirty-two children (age range, 1-25 years; mean age, 11 years; 19 females, 13 males) were treated with this regimen during an 18-month period. H. pylori organisms with varying grades of gastritis were present in tissue specimens of all patients. Only 28 children had follow-up endoscopy, which showed eradication of H. pylori in 15 (54%) children. Histologic symptoms of gastritis improved after therapy in the whole group. Overall, symptoms of vomiting, abdominal pain, diarrhea, anorexia, and halitosis significantly improved (P < 0.05). Minor adverse effects of therapy occurred in 25% of patients. CONCLUSIONS: Symptoms, histologic, and endoscopic findings improved after triple therapy in children with H. pylori gastritis; however, eradication of bacteria was achieved in only 56% of children. PMID- 10749412 TI - Ethnic differences in intestinal disaccharidase values in children in Finland. AB - BACKGROUND: Intestinal disaccharidase activities tend to be low in villous atrophy, but there are only a few reports of enzyme activities in children with normal villous architecture. METHODS: In the current study the data were reviewed on disaccharidase activities in duodenal biopsy specimens of normal villous structure in 223 children undergoing upper gastrointestinal endoscopy in 1997 and 1998. The ancestry was Finnish in 188 children (median age 8.0 years; range, 0.2 18 years), African in 27 children (median age 5.0 years; range, 1-13 years), and other in eight children. RESULTS: The mean activities of lactase, sucrase, and maltase were significantly higher in Finnish children than in children of African origin (P < 0.0001, P < 0.002, and P < 0.02, respectively). Lactase activity decreased with increasing age (P < 0.001), but age had no significant effect on maltase and sucrase activities. Among Finnish children, 31% (59/188) had lactase activity below the established reference range of 20 units (units are micromoles of substrate hydrolyzed per minute at 37 degrees C per gram of protein) and one child had a probable sucrase-isomaltase deficiency. When these 60 children with low enzyme activities were excluded, the geometric means were lactase, 35.7 units (95% confidence interval [CI], 32.8-38.6 units); maltase, 241 units (95% CI, 225 258 units); and sucrase, 57.5 units (95% CI, 53.5-61.6 units). Among the children of African origin, lactase activity was decreased in 67% (18/27). All three enzyme activities were decreased in parallel more often among the African children (8/27) than among the Finnish children (9/188; P < 0.002). CONCLUSIONS: Ethnicity has a strong effect on disaccharidase values in children with normal villous structure. African children have lower activities of lactase, sucrase, and maltase in duodenal specimens than do children of Finnish origin. PMID- 10749413 TI - Lactobacilli and acidosis in children with short small bowel. AB - BACKGROUND: In patients with a short small bowel, D-lactic acidemia and D-lactic aciduria are caused by intestinal lactobacilli. The purpose of this study was to obtain a detailed picture of the metabolic acidosis in young children with short small bowel. METHODS: Feces, blood, and urine of children with short small bowel and acidosis were studied microbiologically and/or biochemically. RESULTS: Previous findings were confirmed that more than 60% of the fecal flora of patients with small short bowel, who are not receiving antibiotics, consists of lactic acid-producing lactobacilli. In blood, D-lactic acid was the most prominent metabolite: the highest serum D-lactate (15.5 mmol/l) was observed in a sample taken immediately after the onset of hyperventilation. The highest D lactate excretion was in urine collected some hours after the onset of hyperventilation, and amounted to 59 mol/mol creatinine. Acidosis in the patients with short small bowel was related to strongly increased serum D-lactate and anion gap and to strongly decreased serum bicarbonate and pH. CONCLUSION: In children with small short bowel and acidosis, the common intestinal flora of mainly lactobacilli abundantly produces D-lactic acid from easily fermentable carbohydrates. Thus, these bacteria directly cause shifts of bicarbonate, pH, and base excess and indirectly cause shifts of the anion gap, as well as hyperventilation. These kinetic parameters are strongly associated. PMID- 10749414 TI - Intestinal colonization leading to fecal urobilinoid excretion may play a role in the pathogenesis of neonatal jaundice. AB - BACKGROUND: Neonatal hyperbilirubinemia remains of concern because of the potential danger for the central nervous system. Because urobilinogen is a nontoxic derivative of bilirubin, the current study was conducted to examine the fecal excretion of urobilinoids and bilirubin in healthy newborns and infants, as well as their intestinal bacteria capable of reducing bilirubin, to assess a possible relation to serum bilirubin levels during the first weeks of life. METHODS: Bilirubin pigments, urobilinoids, and porphyrins were measured in stools of infants during the first week (group A, n = 60) and between the second week and the first 6 months of life (group B, n = 64). Microbiologic analysis of stools was performed in selected cases and bilirubin-converting activity of isolated bacteria was determined in vitro. RESULTS: Urobilinoids were detectable in stools of 57% of the neonates at day 5, but not before. However, fecal urobilinoid production on that day was only a fraction of that observed in adults (0.07 vs. 0.7-3.6 mg/kg per day), whereas at week 6 it increased significantly to an average of 0.9 mg/kg per day. Microbiologic analysis of neonatal stools revealed two novel bacterial strains of Clostridium perfringens and Clostridium difficile capable of reducing bilirubin to urobilinoids. CONCLUSIONS: Urobilinoids can be detected in stools of 57% of newborns at day 5 after delivery. However, the urobilinoid production during the first week of life is quantitatively insufficient to contribute significantly to the removal of bilirubin. Enhancement of the microbial conversion of bilirubin could decrease the intestinal concentration of bilirubin and may decrease the degree or enhance the removal of neonatal hyperbilirubinemia. PMID- 10749415 TI - Stability of insulin-like growth factor I in the gastrointestinal lumen in neonatal pigs. AB - BACKGROUND: Insulin-like growth factor (IGF)-I is present in the milk of various species. A prerequisite for any biological activity of milk-borne IGF-I in the suckling young is to survive the gastrointestinal luminal digestion. In the present study, the stability of IGF-I was examined in the gastrointestinal lumen in neonatal pigs. METHODS: Iodine-labeled IGF-I was incubated in the gastrointestinal luminal fluids of 3-day-old suckling and 45-day-old weaned pigs at 37 degrees C for 20 minutes. Degradation of the peptide was analyzed by trichloroacetic acid (TCA) precipitation, liquid chromatography, and receptor binding assay. RESULTS: IGF-I remained unchanged in the gastric fluids of suckling and weaned pigs when determined by TCA precipitation. IGF-I degraded 3%, 18%, and 37% in the luminal fluids of the proximal, mid and distal small intestine in suckling piglets compared with 53%, 62%, and 54% in weaned pigs. The results were supported by the chromatography and receptor binding analysis. Porcine colostrum had a capacity to protect IGF-I from gastrointestinal luminal digestion in weaned pigs. CONCLUSION: Milk-borne IGF-I is stable in the gastrointestinal lumen in suckling pigs and may play a role in regulating postnatal development in the suckling young. PMID- 10749416 TI - Assessment of nutritional status and body composition in children using physical anthropometry and bioelectrical impedance: influence of diurnal variations. AB - BACKGROUND: To observe whether there are diurnal variations when assessing children's body composition, using physical anthropometric and bioelectrical impedance (BI) methods. METHODS: In 32 children (18 females and 14 males) aged between 7.1 and 14.9 years, weight, height, arm circumference (AC), four skinfolds, and total body impedance (Z) were measured at 8 A.M., 12 noon, 4 P.M., and 8 P.M. on the same day. Body mass index (BMI), skinfolds sum (SS), total body water (TBW), fat-free mass (FFM), and fat mass (FM) were also calculated at these times. RESULTS: Height at 8 P.M. was 1.156 +/- 0.54 cm less than at 8 A.M. (P < 0.001) and BMI increased 0.434 +/- 0.29 kg/m2 at 8 P.M. (P < 0.001). Weight, SS and AC underwent no significant changes in the course of the day. Z was 5.83% less at 8 P.M. than at 8 A.M. (P < 0.001), yielding a TBW and FFM increase at 8 P.M. of 0.855 +/- 1.061 and 1.173 +/- 1.47 kg, respectively (P < 0.001). CONCLUSIONS: In children there is a diurnal decrease in height, impedance and FM, and an increase in BMI, TBW, and FFM. These factors must be taken into account when using such methods for assessing nutritional status in childhood. PMID- 10749417 TI - Risk of inadequate bone mineralization in diseases involving long-term suppression of dairy products. AB - BACKGROUND: Eighty percent of peak bone mass should be achieved from birth through adolescence. An adequate calcium intake is essential, and it is advisable that 60% of the recommended calcium allowance be dairy calcium. This study was conducted to examine bone mineral content (BMC) in patients with diseases that usually involve long-term suppression of dairy products. METHODS: Thirty patients, aged 2 to 14 years (mean, 7 years), 10 with late-onset, genetically induced lactose intolerance, 7 with cow's milk protein allergy, 3 with short bowel syndrome, and 10 with hypercholesterolemia were involved in the study. They were receiving various dietary regimens for periods longer than 2 years: 14 patients received special formulas for children (lactose-free cow's milk formula, highly hydrolyzed cow's milk protein formula, soy protein isolate formula), 4 patients received liquid soy beverages, 6 patients received skim milk (1% fat), and 6 patients had exclusion of dairy products. Bone mineral density (BMD) was assessed by dual-energy x-ray absorptiometry. RESULTS: Nine patients had osteoporosis, 6 had osteopenia, and 15 had results within normal ranges. Overall, the group had a standard deviation score of -1.3 (osteopenia). The statistical correlation between the BMD value and the percentage intake of recommended daily allowance (RDA) of dairy (or substitute) calcium (in milligrams per day) was highly significant (P < 0.0001, r = 0.89). CONCLUSIONS: All patients with diseases involving total or partial withdrawal from milk products for a prolonged period are a group at potential risk of defective bone mineralization and should be monitored through BMD assessment. PMID- 10749418 TI - Monitoring of 25-OH vitamin D levels in children with cystic fibrosis. AB - BACKGROUND: Patients with cystic fibrosis are at risk for malabsorption of fat soluble vitamins, and those with low 25-OH vitamin D levels have a higher risk of low bone mineral density and long-term skeletal complications. It is currently recommended that vitamins A and E be monitored yearly; however, no recommendations exist for 25-OH vitamin D. Because all three vitamins are fat soluble, the hypothesis in the current study was that low levels of vitamins A and E could identify patients at risk for low 25-OH vitamin D, so that 25-OH vitamin D measurements could be obtained in only selected circumstances. METHODS: Forty (21 girls) patients with CF, age 10.5 +/- 3.9 (SD) years, were assessed in a cross-sectional survey for ideal weight for height (percentage of predicted), spirometry (percentage of predicted FEV1, 33/40 patients), and serum levels of vitamins A, E, 25-OH vitamin D, and cholesterol (37/40 patients). RESULTS: Nine (22.5%) of 40 patients were malnourished (percentage of predicted ideal weight for height <85%), 7 (21.2%) of 33 had moderate to severe lung disease (FEV1 <60%), 4 (10%) of 40 had low levels of vitamin A, 3 (7.5%) of 40 had low vitamin E levels, 4 (10.8%) of 37 low vitamin E/cholesterol levels, and 4 (10%) of 40 had marginal or low levels of 25-OH vitamin D (<40 mmol/l). The patients with low 25 OH vitamin D were older, with no child < 12 years of age having a 25-OH vitamin D level less than 40 mmol/l. They also had lower vitamin E and vitamin E/cholesterol levels than those with normal 25-OH vitamin D levels. The groups did not differ in percentage of predicted ideal weight for height, lung function, or vitamin A levels. The best positive predictor for 25-OH vitamin D less than 40 mmol/l was low vitamin E (66.7%), with a negative predictive value of 94.6%. 25 OH vitamin D levels correlated with vitamin E/cholesterol levels (r = 0.41, P < 0.01) and weakly with vitamin E levels (r = 0.28, P < 0.08), but not with vitamin A levels. CONCLUSIONS: These results suggest that children aged less than 12 years and older children with normal vitamin E levels are especially unlikely to have low 25-OH vitamin D levels, and this measure can therefore be omitted. In contrast, those children with low vitamin E levels may warrant monitoring. PMID- 10749419 TI - Clinical quiz. Gastric heterotopia. PMID- 10749420 TI - Intussusception of the appendix: another poorly recognized cause of rectal bleeding. PMID- 10749421 TI - Simultaneous occurrence of autoimmune enteropathy and recurrent deep venous thrombosis. PMID- 10749422 TI - Hemangioma of the cecum: an overlooked cause of rectal bleeding. PMID- 10749423 TI - Neonatal sclerosing cholangitis associated with autoimmune phenomena. PMID- 10749424 TI - Endoscopic retrograde cholangiopancreatography in children. Subcommittee on Endoscopy and Procedures of the Patient Care Committee of the North American Society for Pediatric Gastroenterology and Nutrition. PMID- 10749426 TI - Breast-feeding and childhood obesity. PMID- 10749425 TI - Taming the RAGE of inflammation. PMID- 10749427 TI - Management of severe malnutrition and diarrhea. PMID- 10749428 TI - Oligoclonal gammopathy in phenotypic diarrhea. PMID- 10749429 TI - Chronic hepatitis C virus infection in childhood and early cirrhosis: it is possible? PMID- 10749430 TI - "Lymphonodular hyperplasia as a sign of food allergy in children". PMID- 10749431 TI - Detection of elevated levels of circulating antigen 85 by dot immunobinding assay in captive wild animals with tuberculosis. AB - Antemortem diagnosis of tuberculosis in captive wild animals is often difficult. In addition to the variability of host cellular immune response, which does not always indicate current active infection, reactivity to saprophytic or other mycobacteria is common and may interfere with the interpretation of the intradermal tuberculin skin test. Furthermore, the immobilization required for administering the test and evaluating skin reactions in these animals may result in unacceptable levels of morbidity and mortality, of particular concern in individuals of rare or endangered species. Proteins of the antigen 85 (Ag85) complex are major secretory products of actively metabolizing mycobacteria in vitro. Production of these proteins by mycobacteria during growth in vivo could result in increases in circulating levels of Ag85 in hosts with active tuberculosis. A dot blot immunoassay has been used to detect and quantify circulating Ag85 in captive wild animals with tuberculosis. Elevated levels of Ag85 were observed in animals with active tuberculosis as compared with uninfected animals. Study populations included a herd of nyala (Tragelaphus angasi) (n = 9) with no history of exposure to Mycobacterium bovis. Serum Ag85 levels ranged from <5 to 15 microU/ ml (median, 5 microU/ml). The other group included 11 animals from a mixed collection with a documented history of an M. bovis outbreak. Animals with pulmonary granulomatous lesions (n = 3) had serum Ag85 levels ranging from 320 to 1,280 microU/ml (median, 320 microU/ml). Animals with only chronic mediastinal or mesenteric lymphadenitis (n = 4) had serum Ag85 levels ranging from <5 to 320 microU/ml (median, 52.5 microU/ml). Animals with no lesions present on necropsy (n = 4) had serum Ag85 levels ranging from <5 to 80 microU/ml (median, <5 microU/ml). This assay could provide an important adjunct to intradermal skin testing for antemortem diagnosis of tuberculosis in nondomestic species. PMID- 10749432 TI - Fecal cortisol metabolite analysis for noninvasive monitoring of adrenocortical function in the cheetah (Acinonyx jubatus). AB - A radioimmunoassay was validated for quantifying excreted cortisol metabolites in cheetah (Acinonyx jubatus) feces. High-performance liquid chromatography analysis indicated that immunoreactivity was associated with a water-soluble metabolite in fecal extracts from males and females. None of the immunoreactivity corresponded with free cortisol or corticosterone but rather was associated with a more polar, unidentified metabolite. To determine the biologic relevance of excreted immunoreactive cortisol metabolites, cheetahs were exposed to a variety of situations anticipated to increase cortisol secretion. First, to assess acute changes in adrenal activity, adrenocorticotropic hormone (ACTH; 400 IU i.m.) was administered to two adult males and two adult females. Pre-ACTH baseline serum cortisol and fecal cortisol metabolite concentrations varied among individuals. Serum cortisol concentrations were elevated above baseline within 10 min of ACTH injection, followed by corresponding increases in fecal cortisol metabolite concentrations (690-4,194% above baseline) 48 hr later in three of four cheetahs. In the fourth cheetah, a smaller increase (334% above baseline) in fecal cortisol metabolite excretion was observed 96 hr after ACTH injection. Seven cheetah females also were subjected to a variety of potentially stressful manipulations, including immobilization, translocation, and introduction to a male to assess the ability of this technique to detect physiologic changes in adrenal activity. Increased fecal corticoid metabolite excretion was observed 24-72 hr after exposure to these exogenous stressors. Results indicate that adrenocortical activity can be monitored noninvasively in the cheetah through analysis of these metabolites. This technique could be valuable for evaluating, and thus optimizing, environmental and management conditions and for investigating the role of stress in disease pathogenesis and the usually poor reproductive performance of this species in captivity. PMID- 10749433 TI - Prevalence of helicobacteriosis and gastritis in semicaptive cheetahs (Acinonyx jubatus). AB - Endoscopic biopsy specimens were obtained from the cardiac, fundic, and pyloric stomach regions of 28 semicaptive cheetahs (Acinonyx jubatus) to look for signs of helicobacteriosis and gastritis. Impression smear cytology and urease tests were positive in 23 and 26 of the cheetahs, respectively. Histopathology showed mild or no lesions in 27 cheetahs; only one cheetah had moderate gastritis. Gastritis was uncommon in these semicaptive cheetahs, and stress could play a major role in the development of helicobacteriosis-associated gastritis in the captive cheetah. PMID- 10749434 TI - Noninvasive reproductive monitoring in the okapi (Okapia johnstoni). AB - Fecal progestagen analysis in okapis (Okapia johnstoni) was used for diagnosis of pregnancy and reproductive disorders, including a comparison of urinary and fecal progestagen analysis and endocrine data on the postpartum period. Data were generated on reliability of fecal progestagen analysis in early pregnancy diagnosis, and case reports were compiled involving single animals with missing luteal activity, abortion after twin pregnancy, and abortions due to deficient placental progestagen production. There was approximately 100-200-fold higher progestagen concentration in feces than in urine, thus explaining the high reliability of fecal progestagen evaluations in diagnosing luteal function and pregnancy. The postpartum period was characterized by lactational anestrus of several months duration, and a postpartum estrous cycle about 2-3 wk after parturition was observed in two of eight animals. An animal with five abortions due to deficient placental progestagen production was treated with altrenogest in a subsequent pregnancy and carried the fetus to term. PMID- 10749435 TI - Comparative physiologic effects of telazol, medetomidine-ketamine, and medetomidine-telazol in captive polar bears (Ursus maritimus). AB - The cardiopulmonary effects of three drug combinations in polar bears (Ursus maritimus) were studied. In 1995, five adult polar bears received i.m. injections of either 8.2 +/- 1.3 mg/kg of Telazol or a combination of 159 +/- 34 microg/kg of medetomidine with 4 +/- 0.8 mg/kg of ketamine in a crossover design. Significantly higher mean arterial pressure, lower heart rate, and lower partial pressure of arterial oxygen (Pao2) occurred with medetomidine-ketamine. In 1996, six adult polar bears were immobilized with i.m. injections of either 8.2 +/- 2 mg/kg of zolazepam-tiletamine or a combination of 74.8 +/- 11.8 microg/kg of medetomidine plus 2.2 +/- 0.3 mg/kg of zolazepam-tiletamine in a crossover design. Significantly higher mean arterial pressure and lower heart rate, base excess, and Pao2 occurred with medetomidine-zolazepam-tiletamine compared with zolazepam-tiletamine alone. Hypertension, bradycardia, and decreased Pao2 were observed with both medetomidine-ketamine and medetomidine-zolazepam-tiletamine. Both combinations should be well tolerated by healthy bears, but both have the potential to produce adverse effects in animals with cardiopulmonary disease. Zolazepam-tiletamine produced minimal adverse cardiopulmonary effects, consistent with the wide margin of safety of this combination in bears. The analgesic effect of zolazepam-tiletamine was apparently poor on the basis of the marked increases in pulse rate and mean arterial pressure after noxious stimuli. PMID- 10749436 TI - Subcutaneous implantation of satellite transmitters with percutaneous antennae into male polar bears (Ursus maritimus). AB - Male polar bears (Ursus maritimus) have not been successfully instrumented with satellite transmitters because they readily shed collar-mounted transmitters. Seven male polar bears were captured on the pack ice off the northern coast of Alaska and surgically implanted with satellite transmitters with percutaneous antennae into the subcutaneous space of the dorsal cervical region. Transmitters failed prematurely with lifetimes of 30-161 days (mean = 97 days). Efforts to relocate implanted bears after transmitters failed were not successful. The mean number of location solutions per transmitter was 204 (range 118-369). An average of 10% and 19% of the locations were accurate to <150 m and to 150-350 m, respectively. Our successful tracking of male polar bears, the high quality of locations obtained from transmitters with percutaneous antennae implanted in the subcutaneous space, and the low visibility of such units make further technical development worthwhile if the reason for premature failure of the transmitters can be determined. PMID- 10749437 TI - Reference hematologic and plasma chemistry values of brown tree snakes (Boiga irregularis). AB - Reference hematologic and plasma chemistry values were determined from 103 blood samples collected from 53 clinically healthy brown tree snakes (Boiga irregularis). Female snakes had significantly higher mean plasma values for total solids, total protein, calcium (Ca), phosphorus (P), uric acid, and blood monocyte percentage than did males, whereas males had significantly higher mean plasma fibrinogen values. The variances for hematocrit, monocyte percentage, azurophil percentage, plasma total solids, plasma total protein, albumin, Ca, and P also differed significantly between sexes. The higher mean values and greater variances for plasma total protein, plasma total solids, Ca, and P in the female snakes were probably associated with yolk synthesis and accumulation. PMID- 10749439 TI - Electrocardiography of southern elephant seal (Mirounga leonina) weanlings. AB - The mean values for amplitude and duration of electrocardiogram intervals, cardiac rhythm, and mean frontal plane QRS-axis were measured in 18 free-living weanling southern elephant seals (Mirounga leonina) anesthetized with tiletamine zolazepam. Animals were 30-50 days old and had been weaned about 3 wk earlier. All animals had a normal sinus rhythm. The mean P-wave duration per animal had a range of 0.06-0.09 sec, and mean PR interval and QT interval ranges were 0.08 0.12 sec and 0.25-0.26 sec, respectively. The amplitude of the R wave was 0.22 mV in a VR and 0.5 mV in DI. The electrical axis was between -60 and +30 in 13 of the weanlings. Electrocardiograms were also obtained from five anesthetized juveniles and one adult female. The electrical axis of juveniles was between 0 and 120 , whereas the QRS vector for the adult female had an orientation of -150 . This report is the first detailed description of the southern elephant seal electrocardiogram. PMID- 10749438 TI - Immobilization of babirusa (Babyrousa babyrussa) with xylazine and tiletamine/zolazepam and reversal with yohimbine and flumazenil. AB - Twelve babirusa (Babyrousa babyrussa) (four females/eight males) were immobilized 30 times during a 4-yr interval. Significantly higher premedication and immobilizing doses were needed for females than for males (P < 0.05). An i.m. preanesthetic xylazine dose of 1.88 +/- 0.37 mg/kg (range = 1.20-2.12 mg/kg) was used for females and 1.22 +/- 0.16 mg/kg (range = 0.82-1.43 mg/kg) for males. After xylazine, the animals were induced with i.m. tiletamine/zolazepam; females received 2.20 +/- 0.47 mg/kg (range = 1.78-3.33 mg/kg) and males received 1.71 +/ 0.34 mg/kg (range = 1.08-2.05 mg/kg). Anesthesia was reversed with yohimbine (0.14 +/- 0.03 mg/kg; range = 0.07-0.20 mg/kg) and flumazenil (1 mg flumazenil/20 mg zolazepam) either i.m. or i.v. This anesthetic combination produced smooth induction, good relaxation, and sufficient immobilization to perform routine diagnostic and therapeutic procedures (venipuncture, hoof and tusk trims, transportation, radiographs, ultrasound examination, weight determinations, and skin biopsies). Supplemental ketamine HCl or isoflurane was administered to two animals to effectively deepen or prolong the anesthetic plane, with no resultant adverse effects. PMID- 10749440 TI - Pseudotuberculosis in marmosets, tamarins, and Goeldi's monkeys (Callithrichidae/Callimiconidae) housed at a European zoo. AB - Yersinia pseudotuberculosis is an insidious bacterial infectious agent distributed worldwide and endemic to European countries. It has caused several animal deaths and may threaten the effectiveness of breeding projects for endangered species. In this retrospective study, we examine the prevalence of pseudotuberculosis in Jersey Zoo (Channel Islands, U.K.) over a period of 16 yr to obtain information that can be applied to prevent the infection. The efforts made to control the disease through vaccination are also explored. Our results show that pseudotuberculosis has been endemic to Jersey Zoo since 1979 and is responsible for significant animal loss in the Callithrichidae/Callimiconidae group. Mortality due to Y. pseudotuberculosis was seasonal; a high percentage of deaths occurred during wet and cold seasons. No significant difference was found in mortality rates of vaccinated versus nonvaccinated animals. Although the efficacy of vaccination has not been confirmed, we believe that an improved vaccination program could be an important tool in controlling outbreaks of infection in marmosets and tamarins. PMID- 10749441 TI - Diagnosis of hypothyroidism in a western lowland gorilla (Gorilla gorilla gorilla) using human thyroid-stimulating hormone assay. AB - Primary hypothyroidism was diagnosed in a 26-yr-old female western lowland gorilla (Gorilla gorilla gorilla) on the basis of serum levels of thyroxine (T4), free T4, and thyroid-stimulating hormone (TSH) measured by human immunoassays. Compared with clinically normal gorillas, the TSH level (107 mlIU/L) was markedly elevated, and T4 (<14.0 nmol/L) and free T4 (5.0 pmol/L) levels were decreased. Thyroid hypofunction could explain the weight gain, unsettled appetite, anxious behavior, lethargy, and poor intraspecies interactions shown by this gorilla. The antibodies in the commercial immunoassay used in this study apparently cross reacted with gorilla TSH. Supplementation with levothyroxine sodium was initiated and was followed by a marked decrease in circulating TSH and a noticeable improvement in the animal's physiologic status and activity level. PMID- 10749442 TI - Calcium balance in Drakensberg crag lizards (Pseudocordylus melanotus melanotus; Cordylidae). AB - Eight nonreproductive female Drakensberg crag lizards (Pseudocordylus melanotus melanotus) were each fed diets of mealworms and calcium capsules with various calcium levels. Excreta were collected and analyzed for calcium and uric acid content. The amount of calcium in the feces was calculated. The lizards appeared to be able to maintain calcium balance at calcium intakes equivalent to 1.4-5.6% calcium in the dry matter of feed. Calcium balance was maintained by adapting intestinal calcium absorption. PMID- 10749444 TI - Hematologic parameters of the Spanish ibex (Capra pyrenaica). AB - Thirteen hematologic parameters were measured in 52 Spanish ibexes (Capra pyrenaica) from Sierra Nevada Natural Park, southern Spain. The animals were captured, maintained in captivity, and physically restrained for blood collection. We compared hematologic values for healthy ibexes with animals infected with sarcoptic mites and animals treated for scabies and recovered. Ibexes with scabies showed a decreased number of erythrocytes, a higher mean corpuscular volume and mean corpuscular hemoglobin, and increased numbers of band neutrophils. The number of lymphocytes was significantly higher in animals that had recovered from scabies as compared with infested ones. The only difference observed between healthy and recovered animals was in the number of monocytes, which remained significantly higher in animals that had recovered from scabies. No hemoparasites were found in this study. PMID- 10749443 TI - Listeria monocytogenes subtypes associated with mortality among fallow deer (Dama dama). AB - Different subtypes of Listeria monocytogenes were isolated from various animal and environmental samples during an episode of increased mortality on a fallow deer (Dama dama) farm. During a 4-wk period, six fallow deer died, including four does, one fawn, and one adult buck. Prior to death, one of the does had exhibited central nervous system signs characteristic of listeriosis. Postmortem examination of the six deer showed no histologic changes typical of listeriosis, although inflammatory changes were present in several organs. Different subtypes of L. monocytogenes were isolated from brain samples from six deer, from fodder and soil from the deer feeding area, and from faces of some healthy animals on the farm. Listeria monocytogenes, which was frequently isolated in the environment of the farm, was considered the probable major cause of mortality in these fallow deer. PMID- 10749445 TI - Feline asthma syndrome in African lions (Panthera leo). AB - Feline asthma syndrome, previously recognized only in domestic cats, was diagnosed in three captive African lions (Panthera leo), one of which died as a result of the condition. Two of the lions displayed progressive signs for 7 yr, including severe bouts of coughing, wheezing, dyspnea, rhonchi, and tachypnea that were most severe during the spring and summer, and the third lion displayed acute signs only once. Scattered to diffuse increased interstitial markings, peribronchial cuffing, and focal atelectasis were visible in radiographs. At necropsy, multiple subpleural bullae, 2-3 cm in diameter, were scattered throughout the lung tissue. There were thick-walled bronchi and bronchioles filled with thick grayish mucus, and alveolar spaces were enlarged with severe, diffuse, banded multifocal areas of alveolar wall fibrosis. The lions had significantly elevated IgE type I immediate hypersensitivity responses to recognized aeroallergens. The captive management of lions should address the design and maintenance of allergen-free air supplies. Ventilation systems should be examined routinely and thoroughly cleaned of any residue. The frequency of examination should increase during the summer. Lions and other large cats should be routinely screened for IgE aeroallergen-specific titers, asthma cases should be treated promptly with prednisolone, and investigations of etiology should be initiated. PMID- 10749446 TI - Dermatophytosis in red pandas (Ailurus fulgens fulgens): a review of 14 cases. AB - Fourteen cases of dermatophytosis were identified from medical records of red pandas (Ailurus fulgens fulgens) housed at the Knoxville Zoo between 1980 and 1996. The median age of affected animals on initial presentation was 8.5 wk (3 wk 11 mo). Clinical signs included crusting, purulent exudate, alopecia, thickening of affected skin, ulceration, and necrosis. Seven animals had mild lesions with signs restricted to crusting and/or alopecia, and six animals had more severe infections, with ulceration, skin necrosis, and purulent exudate. Five of the severely affected pandas had tail involvement. The severity of disease affecting one individual was not recorded. Dermatophytosis was confirmed by culture, cytology, histopathology, or culture followed by histopathology. Microsporum gypseum was the only fungal organism cultured. Six animals were treated for mild disease, and all clinical signs resolved. Partial tail amputation was required as part of the treatment regimen for two of the six severely affected animals, and two others had ulcerated tail lesions that left circumferential scarring after resolution of infection. Itraconazole (5 mg/kg p.o. q 12-24 hr) was the most frequently used systemic antifungal agent in animals with severe lesions. All fungal infections resolved, although one panda died from unrelated causes early in the treatment period. PMID- 10749447 TI - Pulmonary silicosis in three North American river otters (Lutra canadensis). AB - Three adult female North American river otters (Lutra canadensis) demonstrated severe anesthetic complications shortly after being immobilized for dental procedures. Two of the animals died shortly after anesthesia, and the third otter died 2 mo after immobilization. All three animals were diagnosed with pulmonary silicosis on the basis of histopathology, polarized light microscopy, and mass spectrometry. One animal also had primary pulmonary bronchoalveolar carcinoma. Significant concurrent disease was not found in other organs. Analysis of the lung tissue by laser microbe mass spectrometry confirmed the presence of an aluminum silicate in the lung tissue associated with insulation material used in nest box construction. PMID- 10749448 TI - Sublingual squamous cell carcinoma in an Atlantic bottlenose dolphin (Tursiops truncatus). AB - A 22-yr-old captive-born Atlantic bottlenose dolphin (Tursiops truncatus) presented with a nonhealing sublingual mucosal ulcer that was diagnosed histologically as a squamous cell carcinoma, the first such report in a dolphin. The lesion was excised completely and has not recurred. PMID- 10749449 TI - Fibrous osteodystrophy in dromedary camels (Camelus dromedarius). AB - Fibrous osteodystrophy of the facial and long bones was diagnosed in four dromedary camels (Camelus dromedarius). None of the animals responded to treatment with antiinflammatory medications or calcium supplements. The lesions were probably caused by multiple factors, including inappropriate diet and gastrointestinal parasitism. A critical factor in lesion formation may have been vitamin D deficiency secondary to gastrointestinal malabsorption and inadequate winter exposure to ultraviolet light. PMID- 10749450 TI - Pansteatitis in a free-ranging red-tailed hawk (Buteo jamaicensis). AB - A free-ranging juvenile female red-tailed hawk (Buteo jamaicensis) that was unable to fly was admitted to a rehabilitation center and died 1 day later. Hematology and serum chemistry abnormalities included moderate regenerative anemia, elevated creatine kinase, and hyperphosphatemia. Necropsy revealed a generalized steatitis, and histology showed a necrotizing and granulomatous pansteatitis with intralesional pigment compatible with ceroid. There was also moderate diffuse myodegeneration, mild multifocal cardiomyopathy, and mild multifocal hepatic necrosis. These changes and lesions resemble those caused by vitamin E deficiency in mammals and fish-eating birds. PMID- 10749451 TI - Vegetative endocarditis in a scimitar-horned oryx (Oryx dammah). AB - Streptococcus uberis was cultured from vegetative endocarditis lesions in a scimitar-horned oryx (Oryx dammah) from the Parc de la Haute Touche, France. This is the first reported single isolation of S. uberis from an oryx with vegetative endocarditis leading to fatal congestive heart failure. PMID- 10749452 TI - Hematology values from clinically healthy Peromyscus leucopus. AB - Reported herein are hematocrit and total and differential WBCs obtained from 132 clinically healthy male and female white-footed mice (Peromyscus leucopus) of four different age groups: 4, 5-8, 9-20, and >32 wk old. Minimal differences were identified between the age groups. The number and percentage of neutrophils in the 4-wk-old mice were significantly higher than those in the other groups; the relative percentage of lymphocytes in the 4-wk-old mice was significantly lower than in the 9-20-wk-old mice. The only significant gender effects identified were higher numbers of WBCs and lymphocytes in females of the 4-wk-old group and higher hematocrits in males of the 5-8- and >32-wk-old groups. PMID- 10749453 TI - Clinical challenge. Osteolysis and cellulitis secondary to Cryptococcus neoformans in a barasingha (Cervus duvaucelii). PMID- 10749454 TI - Early prostate cancer. PMID- 10749455 TI - The pneumococcal conjugate vaccine arrives: a big win for kids. PMID- 10749456 TI - Pneumococcal conjugate vaccination for adults: why it's important for children. PMID- 10749457 TI - Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Northern California Kaiser Permanente Vaccine Study Center Group. AB - OBJECTIVE: To determine the efficacy, safety and immunogenicity of the heptavalent CRM197 pneumococcal conjugate vaccine against invasive disease caused by vaccine serotypes and to determine the effectiveness of this vaccine against clinical episodes of otitis media. METHODS: The Wyeth Lederle Heptavalent CRM197 (PCV) was given to infants at 2, 4, 6 and 12 to 15 months of age in a double blind trial; 37,868 children were randomly assigned 1:1 to receive either the pneumococcal conjugate vaccine or meningococcus type C CRM197 conjugate. The primary study outcome was invasive disease caused by vaccine serotype. Other outcomes included overall impact on invasive disease regardless of serotype, effectiveness against clinical otitis media visits and episodes, impact against frequent and severe otitis media and ventilatory tube placement. In addition the serotype-specific efficacy against otitis media was estimated in an analysis of spontaneously draining ears. RESULTS: In the interim analysis in August, 1998, 17 of the 17 cases of invasive disease caused by vaccine serotype in fully vaccinated children and 5 of 5 of partially vaccinated cases occurred in the control group for a vaccine efficacy of 100%. Blinded case ascertainment was continued until April, 1999. As of that time 40 fully vaccinated cases of invasive disease caused by vaccine serotype had been identified, all but 1 in controls for an efficacy of 97.4% (95% confidence interval, 82.7 to 99.9%), and 52 cases, all but 3 in controls in the intent-to-treat analysis for an efficacy of 93.9% (95% confidence interval, 79.6 to 98.5%). There was no evidence of any increase of disease caused by nonvaccine serotypes. Efficacy for otitis media against visits, episodes, frequent otitis and ventilatory tube placement was 8.9, 7.0, 9.3 and 20.1% with P < 0.04 for all. In the analysis of spontaneously draining ears, serotype-specific effectiveness was 66.7%. CONCLUSION: This heptavalent pneumococcal conjugate appears to be highly effective in preventing invasive disease in young children and to have a significant impact on otitis media. PMID- 10749458 TI - Antimicrobial resistance of Streptococcus pneumoniae isolated from healthy children in day-care centers: results of a multicenter study in Russia. AB - BACKGROUND: It has been previously shown that study of susceptibility of nasopharyngeal isolates in healthy carriers can predict resistance in clinical isolates. The purpose of this multicenter study was to determine the carriage rate of Streptococcus pneumoniae in healthy children attending day-care centers in Moscow, Smolensk and Yartsevo, Russia, and in vitro activity of penicillin G, amoxicillin/clavulanate, cefaclor, erythromycin, roxithromycin, clarithromycin and trimethoprim-sulfamethoxazole (TMP-SMX) against representative isolates. METHODS: Included in this study were 305 pneumococcal isolates from 733 children attending 9 day-care centers in Moscow, Smolensk and Yartsevo. All children enrolled in this study were <7 years of age. MICs of selected antimicrobials were determined by Etest. Serotyping of selected pneumococcal isolates was done with pool and type antisera. RESULTS: The carriage rate of S. pneumoniae in the 3 centers varied from 44.9% to 66.0% (mean, 55.9%). Susceptibility testing was performed with 305 (74.4%) of 410 isolates. Only 23 (7.5%) of 305 pneumococcal isolates were penicillin-intermediate (range, 2.8 to 12.8%) with no penicillin resistant strains. All tested pneumococci were susceptible to amoxicillin/clavulanate. Macrolides possessed comparable activity against S. pneumoniae, at 4.6% resistant strains for both erythromycin (range, 1.1 to 17.1%) and clarithromycin (range, 1.7 to 17.1%). The highest level of resistance was observed with TMP-SMX, 53.4% (range, 43.8 to 70.9%). Of 23 strains 20 (87.0%) with intermediate resistance to penicillin were serotyped. The most prevalent serotype was 14 (5 isolates), followed by serogroups 19 (4) and 23 (4). CONCLUSIONS: Resistance to penicillin, other beta-lactams and macrolides does not seem to be a problem for Russia now. The high level of resistance to TMP-SMX considerably restricts its usage for the treatment of pneumococcal infections. PMID- 10749459 TI - Impact of an antibiotic restriction policy on hospital expenditures and bacterial susceptibilities: a lesson from a pediatric institution in a developing country. AB - BACKGROUND: In an era of growing concern about bacterial resistance and hospital costs, limiting the use of broad spectrum antibiotics is important. OBJECTIVES: To evaluate the effects of an antibiotic restriction policy on expenditures, antimicrobial resistance rates and clinical outcomes of hospitalized children. DESIGN: Starting in January, 1997, a prior consultation with an infectious disease specialist for using restricted antibiotics was required in all hospital areas. A retrospective assessment of study objectives obtained 2 years before (1995, 1996) and 2 years after (1997, 1998) initiation of the restriction policy was performed. SETTING: The present study was conducted in a 500-bed university hospital serving children nationwide of a developing country, Panama. RESULTS: Total expenditures for antimicrobial agents decreased by 50%, from $699,543 (US dollars) during 1995 and 1996 to $347,261 during 1997 and 1998. Susceptibility rates of many nosocomial isolates (especially staphylococci and Gram-negative enteric bacilli) usually improved for restricted antibiotics with >35% reduction in utilization (notably for gentamicin, third generation cephalosporins, piperacillin and vancomycin). Major improvements in bacterial susceptibilities were observed in the nursery, a place harboring microorganisms exhibiting the higher initial resistance rates of the hospital. No differences in days of hospital stay and mortality rates of all patients and of children with nosocomial infections were detected during the study period. CONCLUSIONS: Requirement for prior approval of selected antimicrobial drugs in a pediatric institution decreases hospital expenditures and improves susceptibilities to antibiotics without compromising patient outcomes or length of hospital stays. PMID- 10749460 TI - Autoantibodies directed against bactericidal/permeability-increasing protein in patients with cystic fibrosis: association with microbial respiratory tract colonization. AB - BACKGROUND: Cystic fibrosis (CF) is associated with the appearance of serum autoantibodies directed against bactericidal/permeability-increasing protein (BPI). OBJECTIVES: To determine the age-specific seroprevalence rates of anti-BPI IgG and IgA in a population of patients with CF and to correlate anti-BPI antibody concentrations with microbial respiratory tract colonization and pulmonary function variables at the time of serum sampling and 6 years thereafter. METHODS: Determination of BPI antibodies of the IgG and IgA isotypes using a commercial enzyme-linked immunosorbent assay in sera of a CF serum bank of 1992; correlation of anti-BPI antibody concentrations with age, clinical score, pulmonary function variables in 1992 and 1998, total serum immunoglobulin isotype concentrations and respiratory tract colonization with Pseudomonas aeruginosa and Aspergillus spp. RESULTS: Seventy-one patients (age in 1992, 14.1 +/- 7.5 years) were studied. Reactivities for anti-BPI-IgG and IgA were found in 28 (39%) and 26 (37%) patients, respectively. The seroprevalence of anti-BPI-IgA, but not IgG, increased significantly with age. P. aeruginosa colonization was associated with elevated concentrations of anti-BPI-IgG (P = 0.003) and IgA (P = 0.037). There were significant negative correlations between pulmonary function variables (vital capacity, forced expiratory volume in 1 s) in 1992 and 1998, respectively, and concentrations of anti-BPI-IgG or IgA in a multiple regression analysis. Anti-BPI-IgG, but not IgA, remained significantly associated with P. aeruginosa colonization (P = 0.006) and with reduced vital capacity (P = 0.01) in 1998 after correction for total serum isotype concentration. CONCLUSIONS: Anti BPI-IgG are strongly associated with concurrent P. aeruginosa colonization and with long term restrictive pulmonary function abnormalities. PMID- 10749461 TI - Acute mastoiditis in children: an increase in frequency in Northern Virginia. AB - BACKGROUND: Acute mastoiditis is reported to occur 2 or 3 times annually in the largest children's hospitals. We encountered an average of 1 case annually at our hospital from 1986 to 1991. During an 8-year period ending October 31, 1999, 22 patients were diagnosed and treated at our hospital. Of these, 17 presented during the last 34 months. METHODS: Retrospective chart review from office and hospital records of children from infancy to age 12 years with a discharge diagnosis of acute mastoiditis who were treated from 1992 through 1999. RESULTS: All children were referred to one of the two pediatric otolaryngologists in our community because of forward protrusion of the auricle and retroauricular cellulitis. Eleven (50%) were <14 months old. Ninety-five percent had a concomitant ipsilateral, inflamed, bulging, immobile eardrum. Computerized tomographic imaging, performed on all patients, revealed universal cortical destruction, subperiosteal abscess or bone destruction in four and dural venous thrombosis in two. Mastoidectomy was necessary for eight children (36%) because of complications of mastoiditis (n = 4) or for failure to improve with antibiotics and myringotomy drainage (n = 4). Streptococcus pneumoniae or Streptococcus pyogenes was recovered from 10 of 17 children (59%) from whom cultures were obtained. CONCLUSION: Cases of acute mastoiditis have markedly increased in our suburban children's hospital. The disease was most common during infancy. Serious complications of mastoiditis occurred in four (18%) of the children in this series. PMID- 10749462 TI - Inhaled corticosteroid therapy is safe in tuberculin-positive asthmatic children. AB - BACKGROUND: Although treatment with oral corticosteroids can cause reactivation of latent Mycobacterium tuberculosis (TB) infection in purified protein derivative (PPD)-positive individuals with no evidence of clinical disease, little is known about the effects of inhaled corticosteroids in this respect. OBJECTIVE: This study was undertaken to assess whether inhaled corticosteroid (CS) therapy reactivates latent TB infection in PPD-positive asthmatic children. METHOD: We studied 32 PPD skin test-positive (> or =10 mm) children [age (mean +/ SD), 7.9 +/- 4.1 years] with no family history and no evidence of TB infection on chest radiograms who were receiving inhaled budesonide for the treatment of asthma. They were further evaluated with thorax computed tomography (CT) and erythrocyte sedimentation rate and closely observed for an additional 9 months. RESULTS: At enrollment the mean diameter of PPD reaction was 12.8 +/- 2.7 mm. The mean duration of inhaled CS treatment and the mean cumulative CS dose were 9.8 +/ 7.6 months and 275 +/-199 mg, respectively. Thorax CT studies revealed mediastinal lymph nodes in 7 of the 32 patients. There was no significant difference between children with and without mediastinal lymph nodes according to age, gender, size of PPD skin testing, erythrocyte sedimentation rate and duration and cumulative CS dose of inhaled budesonide therapy before study. A second thorax CT was obtained 9 months later in those 7 patients with lymphadenopathy (additional mean cumulative CS dose, 222.57 mg). There was no change in the size of their lymph nodes. CONCLUSION: Long term inhaled budesonide therapy appears to be safe in PPD-positive asthmatic children. PMID- 10749463 TI - Randomized trial of four vs. seven days of ceftriaxone treatment for bacterial meningitis in children with rapid initial recovery. AB - BACKGROUND: Seven days or more of antimicrobial treatment is the standard for bacterial meningitis, although third generation cephalosporins are usually able to sterilize cerebrospinal fluid within 24 h. The limited experience from shorter regimens in children is encouraging, and we hypothesized that in rapidly recovering patients older than 3 months of age it would pose no risk for adverse outcome. METHODS: Strict clinical and laboratory criteria were used to define rapid initial recovery, in which case ceftriaxone therapy was either stopped after 4 days (4 injections) in children born on even dates (N = 53) or continued for 7 days in patients born on odd dates (N = 47). Outcomes were compared on Day 7 of hospitalization and at 1 to 3 months after discharge. RESULTS: On Day 7 no differences (P > 0.05 for each criteria) were observed between the 4-day and the 7-day groups regarding fever, clinical signs or serum C-reactive protein concentration. At the follow-up visit 1 to 3 months after discharge the 4-day group had fewer sequelae than the 7-day group (0% vs. 5% neurologic sequelae, P = 0.39 and 3% vs. 9% hearing loss, P = 0.49, respectively). One child in the 4-day group who had fully recovered was subsequently readmitted 53 days after the first hospitalization with recurrent Haemophilus influenzae meningitis. CONCLUSIONS: Four days of ceftriaxone therapy proved to be a safe alternative in patients with rapid initial recovery from bacterial meningitis. A 4-day course of treatment is particularly beneficial for countries with limited resources. PMID- 10749464 TI - Comparison of hemocytometer leukocyte counts and standard urinalyses for predicting urinary tract infections in febrile infants. AB - OBJECTIVES: To compare the accuracy of standard and hemocytometer white blood cell (WBC) counts and urinalyses for predicting urinary tract infection (UTI) in febrile infants. METHODS: Enrolled were 230 febrile infants < 12 months of age. All urine specimens were obtained by suprapubic bladder aspiration and microscopically analyzed by the standard urinalysis (UA) and by hemocytometer WBC counts simultaneously, and quantitative urine cultures were performed. Receiver operating characteristic (ROC) curves were constructed for each method of UA. The optimal cutoff point of the UA test in predicting UTI was determined by ROC analysis. RESULTS: There were 37 positive urine cultures of at least 1,000 CFU/ml. Of these 37 patients, 9 females and 28 males, 1 had a positive blood culture (Escherichia coli). Thirty (81%) of the positive urine cultures had a bacterial colony count > or = 100,000 colony-forming units/ml, whereas the remaining had between 1,000 and 50,000 colony-forming units/ml. The area under the ROC curve for standard UA was 0.790 +/- 0.053, compared with 0.900 +/- 0.039 for hemocytometer WBC counts (P < 0.05). For hemocytometer WBC counts, the presence of < or =10 WBC/microl appeared to be the most useful cutoff point, yielding a high sensitivity (83.8%) and specificity (89.6%). Standard UA, with a cutoff point of 5 WBC/high power field, had a lower sensitivity (64.9%) and similar specificity (88.1%). The hemocytometer WBC counts showed significantly greater sensitivity and positive predictive value (83.8 and 60.8%, respectively) than the standard urinalysis (64.9 and 51.1%, respectively) (P < 0.05). The accuracy, specificity and likelihood ratio of hemocytometer WBC counts were also greater than that of standard UA (88.7, 89.6 and 8.08% vs. 84.3, 88.1 and 5.44%). CONCLUSION: Hemocytometer WBC counts provide more valid and precise prediction of UTI in febrile infants than standard UA. The presence of > or =10 WBC/microl in suprapubic aspiration specimens is the optimum cutoff value for identifying febrile infants for whom urine culture is warranted. PMID- 10749465 TI - Nosocomial Legionnaire's disease in a children's hospital. AB - BACKGROUND: Only a few cases of nosocomial Legionella sp. infection have been reported in children. We report the clinical and epidemiologic data of five nosocomial legionellosis cases that occurred in the Pediatric Nephrology Service between August, 1994, and December, 1998, and the control measures adopted. METHODS: The Hospital Materno-Infantil Vall d'Hebron, Barcelona, is a 407-bed tertiary care hospital. The pediatric kidney transplant unit has three isolated beds in the same ward within the Pediatric Nephrology Service. Diagnostic workup to establish Legionella pneumophila infection included culture, fluorescent antibody and serologic studies. Macrorestriction analysis of genomic DNA was used as epidemiologic markers of the isolated strains. RESULTS: In May, 1996, a case of L. pneumophila serogroup 6 pneumonia was identified in a 19-year-old youth who had received a kidney transplant 16 days earlier. Retrospective and prospective analysis of legionellosis cases diagnosed at our center up to August, 1994, yielded four additional cases. Four patients had had a kidney transplant and were receiving immunosuppressive therapy, and the fifth had been diagnosed with systemic lupus erythematosus with renal involvement. L. pneumophila serogroup 6 was isolated in bronchial secretions in four cases; in the fifth patient the diagnosis was made by serology. L. pneumophila serogroup 6 was isolated from potable water of the hospital. Molecular epidemiologic methods revealed the identity of the environmental and clinical isolates. Showering was implicated as the most feasible means of exposure to contaminated water. CONCLUSIONS: Nosocomial legionellosis, albeit rare in children, should be considered in the differential diagnosis of pneumonias, particularly in immunosuppressed children, because the fatality rate may be high without early diagnosis and treatment. PMID- 10749466 TI - Experience with quinupristin/dalfopristin in treating infections with vancomycin resistant Enterococcus faecium in children. AB - BACKGROUND: The emergence and spread of vancomycin-resistant Enterococcus faecium (VREF) has presented serious therapeutic difficulties because of the lack of reliably active antibiotics. Quinupristin/dalfopristin is a new injectable streptogramin antibiotic that is active against most strains of VREF. Experience with this agent in adults with VREF infections is well-documented; however, there are few reports of its use in children. We report on eight children with VREF infections who received quinupristin/dalfopristin under a compassionate use protocol. METHODS: Quinupristin/dalfopristin was administered according to the manufacturer's recommendations. Clinical and laboratory data were recorded for each patient. RESULTS: The infections treated comprised six cases of bacteremia and two of peritonitis. All patients had serious underlying conditions. Seven patients recovered fully. One patient died, having experienced a relapse of his infection after quinupristin/dalfopristin was discontinued. None of the patients experienced side effects or other adverse events. CONCLUSION: Quinupristin/dalfopristin was well-tolerated and generally effective in children with infections caused by VREF. There is increasing evidence that it may be more effective than other currently available antibiotics in some such patients. PMID- 10749468 TI - Neonatal pertussis. PMID- 10749467 TI - Umbilical vein interleukin 6 and tumor necrosis factor alpha plasma concentrations in the very preterm infant. AB - OBJECTIVE: To examine the relationship between umbilical vein plasma concentrations of interleukin 6 (IL-6) and tumor necrosis factor (TNF)-alpha and early neonatal sepsis in the very preterm infant, and the histopathologic findings of chorioamnionitis in the placentas from these pregnancies. METHODS: A prospective study was conducted in 43 very preterm, singleton infants delivered at or before 32 weeks of gestation. IL-6 and TNF-alpha were measured by enzyme linked immunoassay. Placentas from these pregnancies were histologically examined for the presence of chorioamnionitis. Infants were prospectively classified as confirmed sepsis group, clinical sepsis group or control group. IL-6 and TNF alpha plasma concentrations were not normally distributed, so they were transformed to their natural log values for statistical analysis. RESULTS: The enrolled infants had a mean gestational age of 27.2 +/- 2.7 weeks and a mean birth weight of 956 +/- 325 g. Three (7%) infants had confirmed sepsis, 18 (42%) were in the clinical sepsis group and 22 (51%) were in the control group. IL-6 concentrations but not TNF-alpha were significantly higher (P < 0.05) in the confirmed (8.9 +/- 1.7) and clinical sepsis (5.5 +/- 2.4) groups in comparison with the control group (2.1 +/- 1.6). We examined 42 placentas. Twenty-three (55%) had no evidence of chorioamnionitis, 1 (2%) had mild grade, 8 (19%) had a moderate grade and 10 (24%) had a severe grade of chorioamnionitis. IL-6 was significantly elevated in the moderate (5.9 +/- 1.6 vs. 1.9 +/- 1.6) and severe grade (7.2 +/- 2.3 vs. 1.9 +/- 1.6) of chorioamnionitis, in the presence of acute deciduitis (6.0 +/- 2.7 vs. 2.1 +/-1.8), chorionic vasculitis (6.8 +/- 2.1 vs. 2.2 +/- 1.9) and funisitis (7.3 +/- 1.9 vs. 2.7 +/- 2.3) (P < 0.05) TNF-alpha plasma concentrations were not significantly different. CONCLUSION: An elevated umbilical vein IL-6 concentration is a good indicator of sepsis syndrome in the very preterm infant and also correlates with histologic chorioamnionitis in these pregnancies. PMID- 10749469 TI - Sin nombre hantavirus disease in a ten-year-old boy and his mother. PMID- 10749470 TI - Community-acquired pneumonia in children: guidelines for treatment. PMID- 10749471 TI - Management of respiratory syncytial virus infections in the immunocompromised child. PMID- 10749472 TI - Confirmation of mother-to-child transmission of TT virus. PMID- 10749474 TI - Detection of respiratory viruses in the middle ear fluids of children with acute otitis media by multiplex reverse transcription:polymerase chain reaction assay. PMID- 10749473 TI - Otitis media: can clinical findings predict bacterial or viral etiology? PMID- 10749475 TI - Colonization of the female urogenital tract with Streptococcus pneumoniae and implications for neonatal disease. PMID- 10749476 TI - Chylothorax associated with histoplasmosis in a child. PMID- 10749477 TI - Importance of Kingella kingae as a pediatric pathogen in the United States. PMID- 10749478 TI - Listeria monocytogenes meningitis during the incubation period of hepatitis A disease. PMID- 10749479 TI - Cutaneous neonatal herpes simplex infection associated with ritual circumcision. PMID- 10749480 TI - Cefprozil concentrations in middle ear fluid of children with acute otitis media. PMID- 10749481 TI - RSVIG and palivizumab: not the same. PMID- 10749482 TI - Nosocomial Acinetobacter meningitis. PMID- 10749483 TI - Study on the accuracy of the elution by characteristic point method for the determination of single component isotherms. AB - The accuracy of the method of elution by characteristic point (ECP) used to determine single component isotherms was studied numerically. Overloaded elution peaks were calculated using the equilibrium-dispersive model of nonlinear chromatography while varying the four parameters, i.e., the number of theoretical plates, the dimensionless Langmuir equilibrium constant, the retention and the loading factor, involved in the fundamental equations of the problem. Single component isotherms were estimated by analyzing the diffuse profile of the elution peaks by the ECP method. Similar results were obtained with the transport dispersive model. The comparison of these calculated isotherms with the initial Langmuir isotherms provided detailed information on the influence of the mass transfer resistances on the accuracy of the isotherms afforded by the ECP method. The systematic error made on the isotherms depends on the experimental conditions, described by the four parameters. It is expected that this error could be eliminated by correcting the influence of nonequilibrium in the column on the basis of results of the nondimensional calculations. The concentration range of the objective isotherm which can be determined by the ECP method can also be predicted by numerical calculations. The usefulness of the correction strategy and the prediction of the concentration range were experimentally demonstrated. PMID- 10749484 TI - Design of a simulated moving bed unit for sucrose-betaine separations. AB - The results of a series of pulse experiments carried out in a fixed bed column packed with a strongly acid ion-exchange resin and with reference to the separation of an aqueous sucrose-betaine mixture are presented. These data were used to evaluate equilibrium and diffusion parameters for both key components in the frame of linear chromatography theory. The estimated values were used to design a simulated moving bed plant aimed to separate betaine from sucrose in beet molasses. PMID- 10749485 TI - Microcalorimetric studies of interactions between proteins and hydrophobic ligands in hydrophobic interaction chromatography: effects of ligand chain length, density and the amount of bound protein. AB - Using isothermal titration calorimetry (ITC), this investigation directly measured the adsorption enthalpies of proteins on various hydrophobic adsorbents. Various amounts of butyl and octyl groups were attached onto CM-Sepharose to form C4 and C8, two types of hydrophobic adsorbents. The adsorption enthalpies of both trypsinogen and alpha-chymotrypsinogen A were measured at 4.0 M NaCl and pH 10.0, in which most ionic interaction was suppressed. The adsorption isotherms of both proteins on various adsorbents were also measured, thus allowing us to calculate the Gibbs free energy and entropy of adsorption. Experimental results indicated that the adsorption of both proteins on butyl-containing adsorbents was exothermic, while their adsorption on octyl ones was endothermic. In addition, binding of both proteins with the butyl ligand is basically an adsorption process, while binding with the octyl ligand is adsorption and partition processes. Moreover, on both butyl or octyl, the adsorption enthalpy became increasingly positive as the ligand density increased, while the adsorption entropy became more positive as the alkyl chain length or density of the adsorbent increased. In addition, ITC was used to measure protein-protein interaction. The adsorption enthalpy of both proteins increased as the amount of bound protein increased, and the enthalpy increase of trypsinogen appeared to be higher than that of alpha-chymotrypsinogen A. This observation implies that protein-protein repulsion was stronger among trypsinogen molecules in the experiments. PMID- 10749486 TI - Low-energy interactions in high-performance liquid chromatography. AB - Liquid chromatographic systems with very weak excessive analyte-adsorbent interactions have been studied. These systems consisted of a homologous series of n-alkanes as both analytes and mobile phases with a C18 reversed-phase adsorbent. A linear decrease of the analyte retention volume with an increase of the number of analyte carbon atoms was found. Corresponding increases of analyte retention with an increase in the number of eluent carbon atoms was also discovered. An explanation of these two effects on the basis of adsorption theory is proposed. A good correlation of column hold-up volume calculated by interpolation of the retention dependencies for above mentioned systems with that measured by the minor disturbance method has been shown. A study of the temperature dependencies of these alkane systems has shown entropy-governed retention dependencies. PMID- 10749487 TI - High-performance liquid chromatography with on-line coupled UV, mass spectrometric and biochemical detection for identification of acetylcholinesterase inhibitors from natural products. AB - A high-performance liquid chromatography (HPLC) method with on-line coupled ultraviolet (UV), mass spectrometry (MS) and biochemical detection for acetylcholinesterase (AChE) inhibitory activity has been developed. By combining the separation power of HPLC, the high selectivity of biochemical detection, and the ability to provide molecular mass and structural information of MS, AChE inhibitors can be rapidly identified. The biochemical detection was based on a colorimetric method using Ellman's reagent. The detection limit of galanthamine, an AChE inhibitor, in the HPLC-biochemical detection is 0.3 nmol. The three detector lines used, i.e., UV, MS and Vis for the biochemical detection were recorded simultaneously and the delay times of the peaks obtained were found to be consistent. This on-line post-column detection technique can be used for the identification of AChE inhibitors in plant extracts and other complex mixtures such as combinatorial libraries. PMID- 10749488 TI - Development of analytical and preparative chromatographic separations of novel growth hormone secretagogue compounds. AB - Chromatographic separations of new growth hormone secretagogue compounds were developed to support structure-activity relationship (SAR) studies in conjunction with lead optimization. These new compounds differed from Merck's MK-677 by having two chiral centers and thus diastereomeric mixtures were generated. Separation of initial compounds in the SAR was achieved on a Kromasil C18 column using an ammonium acetate buffer and acetonitrile. However, additional candidates were not separable on C18 columns and a chiral Kromasil CHI-DMB column was used to resolve the diastereomeric compounds. The Kromasil CHI-DMB packing was also used in a preparative chromatographic system to resolve multigram quantities of secretagogue candidates for testing. Chiral separations of different intermediates were also developed in support of evolution of an asymmetric synthetic route. This report summarizes development of the preparative chromatographic system used to purify diastereomeric mixtures and chiral separations of intermediates in the synthesis. PMID- 10749489 TI - Liquid chromatography-electrospray time-of-flight mass spectrometry for on-line accurate mass determination and identification of cyclodepsipeptides in a crude extract of the fungus Metarrhizium anisopliae. AB - Electrospray ionisation time-of-flight mass spectrometry (ESI-TOF-MS) has been used for the detection and identification of destruxins (cyclodepsipeptides) in a crude extract of the fungus Metarrhizium anisopliae. HPLC-MS analyses were performed with a post-column addition of erythromycin as a reference compound (lock mass procedure). Seven destruxin derivatives could be identified on-line from their accurate masses (deviation from calculated values <5.5 ppm) through elemental composition calculations. As a highly sensitive and accurate method, ESI-TOF-MS proved to be very powerful for the analysis and dereplication of natural products in complex mixtures. PMID- 10749490 TI - Ion-exclusion controlled size-exclusion chromatography of methacrylic acid-methyl methacrylate copolymers. AB - Controlled ion-exclusion allows compensation of hydrophobic adsorption in size exclusion chromatography of negatively charged methacrylic acid-methyl methacrylate (Eudragit) polymers using methanol as a mobile phase. Properly selected low-ionic-strength conditions below 5 mM LiCl provide correct separation in the size-exclusion mode. Possible disturbing effects, mainly related to light scattering, under low-salt conditions are discussed and shown to be negligible if on-line concentration-light scattering detection is used. The absence of these disturbances is checked by a comparison of experiments performed in methanol containing 1.25 mM and 2.5 mM LiCl. Molecular mass averages and distributions identical within the experimental error are obtained. PMID- 10749492 TI - Use of equations for the description of experimental dependence of the height equivalent to a theoretical plate on carrier gas velocity in capillary gas-liquid chromatography. AB - Optimal equation for fitting the experimental data on the height equivalent to a theoretical plate (HETP) versus carrier gas velocity in GLC was determined. The data obtained by authors and the literary data by other investigators were used for the comparative study of Van Deemter, Golay and the little known Golay Guiochon equations. The Golay-Guiochon equation takes into account instrumental contribution and other sources of additional band broadening. Correlation coefficient R and Pirson's criterion chi2 were used as a criterion of the data correspondence to the equations. The Golay-Guiochon equation is the best for fitting of the experimental data in 71% considered examples, 19% experimental data may be fitted very precisely by the Van Deemter equation, the Golay equation is preferable for 7% experimental data only. Three percent experimental data may be fitted with the same precision by the Golay and the Van Deemter equations. The results obtained are of theoretical and practical interest. The Golay-Guiochon equation must be used more widely in analytical practice and physicochemical measurements. PMID- 10749491 TI - High-performance liquid chromatographic determination of delta9 tetrahydrocannabinol and the corresponding acid in hemp containing foods with special regard to the fluorescence properties of delta9-tetrahydrocannabinol. AB - A solvent programmed reversed-phase HPLC method with UV detection for the determination of delta9-tetrahydrocannabinol (THC) and delta9 tetrahydrocannabinolic acid A (THCA-A) in foods containing parts of hemp such as edible oil, herb-teas (infusion), herbal hemp or hempseed is presented. The THC peak is also detected by fluorescence. The detection limits with UV detection are 0.01 ng for THC and 0.05 ng for THCA-A and with fluorescence detection 0.1 ng for THC. The relative standard deviation under repeatability conditions of the chromatographic procedure is about 0.5% and that of the over-all analytical procedure for THC in vegetable oils 2% (concentration range of 10-100 mg/kg). PMID- 10749493 TI - Pulsed discharge emission detector: an element-selective detector for gas chromatography. AB - An element-specific pulsed discharge emission detector (PDED) has been coupled directly with a vacuum UV monochromator so that vacuum UV atomic emissions from Cl, Br, I and S can be observed. The observed sensitivities for the elements are in the range of mid to high pg/s, but can be lowered by direct absorption of the radiation using a vacuum UV radiation photomultiplier tube. A helium pulsed discharge photoionization detector (He-PDPID) was run simultaneously in parallel with the PDED. The chromatograms recorded with the two detectors had similar peak shapes, suggesting that there is no peak tailing in the PDED. The ratio of the detector responses PDED/He-PDPID can be used for qualitative identification of the Cl-, Br-, I- or S-containing compounds. PMID- 10749494 TI - Development and characterization of chlorine-selective pulsed discharge emission detector for gas chromatography. AB - A novel chlorine-selective pulsed discharge emission detector (Cl-PDED) for gas chromatography has been developed based on a reaction of krypton with chlorine and a unique design of the detector. A krypton ion produced in the krypton-doped helium pulsed discharge reacts with chlorinated compounds within the pulsed discharge to produce an excited species of KrCl* which emits at 221-222 nm. The reaction has the following advantages in respect to the detection of chlorinated compounds: (1) the reaction is an ion-molecule reaction that is 100-1000 times faster than a reaction of neutrals, which greatly enhances the sensitivity; (2) the KrCl* emission wavelength is far separated from interfering C emissions at 193. and 247.3 nm; (3) the KrCl* emission is transparent to air and can be recorded without a helium purge of the monochromator. The detector itself has been designed to have the following features: (1) the detector has a microvolume of the pulsed discharge region, ca. 0.35 microl, which increases the discharge power density to enhance the sensitivity; (2) this microvolume detector allows the use of a low flow-rate of approximately 5 ml/min, which enhances the sensitivity by the lower dilution of the column effluent; (3) the pulsed discharge is sufficiently narrow to replace the monochromator entrance slit, which gives much greater light gathering power; (4) the discharge electrodes are protected with a helium purge to prevent carbon deposition on the electrodes. This new Cl-PDED is the most sensitive chlorine-selective detector with a minimum detectability of approximately 50 fg Cl/s. The selectivity to carbon is 1000. There are no significant carbon emission lines in the KrCl* emission wavelength region, but the carbon continuum interference (stray light) limits the selectivity. The selectivity could be increased if a double monochromator were used to diminish the stray light. The detector linear range is over three orders of magnitude from 40 fg Cl to approximately 130 pg Cl, and the dynamic range is approximately 4 orders of magnitude. The relative standard deviation of the elemental response to chlorinated compounds is about 5%. PMID- 10749495 TI - Applications using the chlorine-selective pulsed discharge emission detector. AB - The use of the chlorine-selective pulsed discharge emission detector (Cl-PDED) for the GC analyses of EPA mixtures 502, 612, 624, organochlorine pesticides, and polychlorinated biphenyls has been demonstrated. The Cl-PDED is the most sensitive chlorine-selective detector with a minimum detectability of 50 fg Cl/s. A constant response/pg Cl was observed for these mixtures regardless of the number of Cl atoms/molecule and structure of the compound to which the Cl atoms are attached. The analysis of standard samples of polychlorinated biphenyls using the Cl-PDED have sensitivities comparable to those of the electron-capture detector; however, the predictable response/pg Cl from the Cl-PDED is preferred over the extremely variable response from the electron capture detector. PMID- 10749496 TI - Characterization of chlorinated compounds using a dual chlorine-selective pulsed discharge emission detector-helium-pulsed discharge photoionization detector system. AB - The Cl-selective pulsed discharge emission detector (Cl-PDED) response is dependent only upon the Cl content, irrespective of the molecular structures of the compounds. This provides a simple, fast quantitative method of analysis for chlorinated compounds. The response of the helium-pulsed discharge photoionization detector (He-PDPID) is a function of the molecular structure and the number of photoionizable electrons using the He2 band at 13.5-17.5 eV. The ratio of the responses of the two detectors is independent of concentration and can be used to characterize the Cl-containing compounds along with the retention time, or the ratio can be used as evidence for coelution. The dual Cl-PDED-He PDPID detector system is a useful tool for peak identification. The effect of coeluting hydrocarbons on the Cl-PDED response was evaluated by spiking a gasoline sample with US Environmental Protection Agency mixture 502. All Cl-PDED responses were greater than 90% of the response in the absence of the hydrocarbons. PMID- 10749497 TI - Determination of phthalate esters in water samples by solid-phase microextraction and gas chromatography with mass spectrometric detection. AB - Solid-phase microextraction (SPME) with an 85 microm polyacrylate fiber, coupled to gas chromatography-mass spectrometry was used to determine six phthalate esters and bis(2-ethylhexyl) adipate in water samples. The variables affecting the SPME absorption process were optimized and the method developed was applied to analyze both tap and commercial mineral water samples as well as water from the Ebro river and fishing and industrial ports. For real samples, the linear range in full scan acquisition mode was between 0.02 and 10 microg l(-1) for most compounds, and the limits of detection of the method were between 0.006 and 0.17 microg l(-1). Commercial water samples contained in recipients which were made from different materials were analyzed, and the influence of the material of the recipients on the concentration of phthalates was evaluated. PMID- 10749498 TI - Determination of sulphur compounds in beer using headspace solid-phase microextraction and gas chromatographic analysis with pulsed flame photometric detection. AB - A simple and sensitive method for the analysis of volatile and semi-volatile sulphur compounds in beer at trace levels was developed using headspace solid phase microextraction (SPME) and gas chromatography with pulsed flame photometric detection. Different SPME fibres were tested and a Carboxen-polydimethylsiloxane coated fibre was found to be the most appropriate. The adsorption and desorption conditions were optimised. The effect of ethanol concentration in the sample on the extraction of analytes was examined. A 60 m non-polar capillary column preceded by a 10 m length of a polar column was found to be capable of separating a wide range of C1-C6 sulphur compounds. The pulsed flame photometric detector enabled increased sensitivity to be obtained over previous methods, such as dynamic headspace followed by conventional flame photometric detection or sulphur chemiluminescent detection, with high sulphur selectivity. Two sulphur compounds, 2-methyl-1-butanethiol and 3-methylthiophene, were identified in beer for the first time. PMID- 10749499 TI - Development of a method for the simultaneous determination of phosphoric and amino acid group containing pesticides by gas chromatography with mass-selective detection optimization of the derivatization procedure using an experimental design approach. AB - An integrated gas chromatographic-mass-selective detection method for the analysis of the phosphoric and amino acid group containing pesticides is presented. The analytes are derivatized using a single-step procedure for the simultaneous esterification and acetylation of the active groups of analytes ( OH, -COOH, -NH2) by means of acetic acid and trimethyl orthoacetate. An experimental design approach based on the central composite design is used to investigate the dependence of the derivatization variables with the total yield of derivatization of pesticides. The variables selected for study were: the amount of reagents, the temperature and the reaction time. When considering the total pesticide derivatization yield, the amount of acetic acid, the reaction temperature and the reaction time are found to be statistically significant. The electron impact ionization mass spectra of the resulting derivatives are acquired and properly interpreted. Under the chromatographic conditions employed, acceptable peak separation is attained. When the selective ion monitoring mode is used for quantitation purposes, low detection limits in the range 0.05 to 14 microg/l are achieved. Recoveries of spiked water samples range from 96 to 103% and the mean RSD of the method do not exceed 3.5%. PMID- 10749500 TI - Investigation of Cr(III) hydrolytic polymerisation products by capillary electrophoresis-inductively coupled plasma-mass spectrometry. AB - The development of a new method for the determination of Cr(III) hydrolytic polymerisation products using capillary electrophoresis-inductively coupled plasma mass spectrometry (CE-ICP-MS) is described. The results indicate that CE ICP-MS can be used to separate and detect monomeric and polymeric Cr(III) species. The various species migrate through the capillary at a rate proportional to their equilibrium distribution, which is dictated by the solution pH, metal ion concentration and ageing period. In general, the data suggest that the relative mobility follows the order trimer>dimer>monomer. The experimentally determined speciation shows a good qualitative agreement with that described in the literature. Independent confirmation of the presence of polymeric Cr(III) species was performed by ionspray mass spectrometry. PMID- 10749501 TI - Utilization of fluorescein sodium salt for the indirect fluorimetric detection in micellar electrokinetic chromatography. AB - The potential of fluorescein sodium salt to generate a background signal in indirect fluorimetric detection in micellar electrokinetic chromatography (MEKC) has been studied by using a sample test containing various alcohols (methanol, ethanol, propan-1-ol, propan-2-ol, 2-methylpropan-1-ol, pentan-3-ol, 2 methylbutan-1-ol, 3-methylbutan-1-ol), acetaldehyde, acetone and ethyl acetate. In order to optimize sample test resolution and detection sensitivity, we have studied different parameters: sodium dodecyl sulfate concentration, electrolyte ionic strength, applied voltage as well as that of the addition of an organic cosolvent within the running electrolyte. Although the micellar pseudo-phase was not able to be saturated with the fluorophore in order to obtain the maximal detection sensitivity, the detection thresholds were satisfactory. By way of example, the detection threshold reached for 3-methylbutan-1-ol is 0.03% (v/v). The quantitative aspect was approached with ethanol titration in a commercial white wine. Finally the repeatability relating to, on the one hand, the effective electrophoretic mobilities and, on the other hand, the corrected areas has also been studied. PMID- 10749502 TI - On-column amperometric detection in capillary electrophoresis with an improved high-voltage electric field decoupler. AB - An improved fabrication method for a decoupler for on-column amperometric detection in capillary electrophoresis (CE) is described. The decoupler is fabricated by etching one side-wall of the capillary with hydrofluoric acid after the polymer coating had been etched by laser, then the etched hole is sealed with adhesive. The steady time, electric conductivity efficiency and performance are investigated. On-column amperometric detection by CE of para-substituted phenols was carried out by coupling with a carbon-fiber microelectrode (10-microm diameter) and a practical small electrochemical detection cell. PMID- 10749503 TI - Investigation of the effects of cyclodextrins and organic solvents on the separation of cationic surfactants in capillary electrophoresis. AB - The use of alpha- and beta-cyclodextrin (CD) to understand and to improve the separation of a series of cationic surfactants, homologues of alkylbenzyldimethyl ammonium compounds (ABDACs) with an alkyl chain of varying length (C10-C18), in capillary electrophoresis (CE) is reported for the first time. Similar to the effects of organic solvents, the presence of alpha- or beta-CD in the running buffer was found to reduce peak tailing/loss for the longer-chain ABDACs. Based on fluorescence measurements, it was found that formation of host-guest complexes occurred between alpha- or beta-CD and various ABDACs, with the likelihood that the hydrophobic alkyl chain including into the CD cavity and the positively charged ammonium group remaining outside the cavity. The effects of alpha- or beta-CD can be interpreted in terms of a shift away from the formation of (1) micelles in the buffer system and (2) surfactant aggregates at the fused-silica capillary walls, as a result of the formation of inclusion complexes between alpha- or beta-CD and ABDACs. PMID- 10749505 TI - Analysis of bisphosphonates by capillary electrophoresis-electrospray ionization mass spectrometry. AB - Capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS) was applied to the direct identification and quantitation of clodronate and its four common impurities. The coaxial interface technique and negative ion mode were used in the detection. Ion source parameters and sheath liquid composition were optimized to produce maximum abundance of singly charged deprotonated molecules used in monitoring. In addition, the effects of electrolyte composition and instrumental parameters of CE on separation were studied. The developed method provides high separation power and specificity to bisphosphonate analysis. In quantitative analysis, the method showed good linearity (r=0.9946-0.9989), satisfactory repeatability (migration time variation: RSD=0.43-1.0%, peak area variation: RSD=2.2-9.4%) and sufficient sensitivity (detection limits: 0.08-0.22 mg ml(-1)) for identification of bisphosphonates from bulk material. PMID- 10749504 TI - Enantiomeric separation of enzymatic hydrolysis products of dihydropyrimidinone methyl ester with cationic cyclodextrin by capillary electrophoresis. AB - The achiral separation of dihydropyrimidinone (DHP) methyl ester and its corresponding carboxylic acid and the chiral separation of their respective enantiomers were achieved in a single analysis using capillary electrophoresis (CE) with quaternary ammonium-beta-cyclodextrin (QA-beta-CD) as a chiral buffer additive. Separation of the DHP methyl ester from the corresponding carboxylic acid was achieved because the acid was negatively charged at pH 8.3 of the running buffer and the ester is neutral. Upon the addition of QA-beta-CD, the enantiomers of the acid and ester were well resolved before and after the electroosmotic flow, respectively. In addition, the minor DHP methyl ester enantiomer (R isomer) was well separated from several impurities. This CE system was used to monitor the progress of a bioresolution reaction that utilizes an enzyme to convert the R isomer of the ester to its corresponding acid. The quantities of all four enantiomers can be determined using a single set of CE conditions. In addition, it is demonstrated that samples can be directly injected into the capillary without sample pretreatment due to the fact that the coating of the cationic CD on the capillary surface prevents adsorption of the positively charged enzyme. The effects of other experimental parameters such as type of CDs, concentration of CDs, pH, temperature, and the preconditioning of capillary were also studied. PMID- 10749506 TI - Trace-level determination of triazines and several degradation products in environmental waters by disk solid-phase extraction and micellar electrokinetic chromatography. AB - An analytical method combining disk solid-phase extraction with micellar electrokinetic chromatography has been developed for the determination of atrazine, simazine, hydroxyatrazine, deisopropylatrazine, deethylatrazine, propazine and prometryn in water samples. The influence of the buffer and sodium dodecyl sulfate (SDS) concentration, pH and organic modifier on the separation has been studied. Baseline separation of the seven triazines was achieved under the following conditions: 10 mM borate buffer, 60 mM SDS, 20% methanol and pH 9.2. C18-bonded silica and poly(styrene-divinylbenzene) (PS-DVB) disks were evaluated for solid-phase extraction of the selected pesticides (11 of water sample). Using two PS-DVB disks, quantitative recoveries were obtained for all pesticides tested. The method was successfully applied for the determination of the seven triazines in drinking and well water at the 0.1 microg l(-1) and 0.5 microg l(-1) concentration levels, respectively. The detection limits for these analytes using the proposed analytical method were within the 0.02-0.06 microg l( 1) range in drinking water and the 0.06-0.30 microg l(-1) range in well water. PMID- 10749508 TI - Capillary electrochromatography of basic compounds using octadecyl-silica stationary phases with an amine-containing mobile phase. AB - The capillary electrochromatographic (CEC) analysis of basic compounds on octadecyl-silica stationary phases (Hypersil ODS and Spherisorb ODS I) was studied. A basic drug (fluvoxamine) and one of its possible impurities were used as test compounds. With an eluent of acetonitrile-phosphate buffer (pH 7.0), the compounds could be baseline-separated; however, broad and tailing peaks were obtained. To minimise detrimental interactions with residual silanol groups, the pH of the mobile phase was lowered to 2.5, but the plate numbers were still quite low (<2.6x10(4) plates/m). Addition of a masking agent (hexylamine or triethylamine) to the mobile phase resulted in much better peak efficiencies (ca. 1x10(5) plates/m). Therefore, the influence of the amine concentration and pH of the mobile phase on the CEC performance (peak width, peak tailing, electroosmotic flow, selectivity) was investigated in detail. Highest efficiencies (2.8x10(5) plates/m) could be obtained with the Spherisorb column, while the Hypersil column offered a better selectivity. Furthermore, the results show that the residual silanol groups are (at least partly) responsible for the separation of the basic compounds and that the amount of injected sample has an unusually large effect on the peak efficiency. The usefulness of the system for impurity profiling was demonstrated with a mixture containing fluvoxamine and its stereoisomer (a possible impurity) at the 0.1% level. The general effectiveness of amine additives in CEC was illustrated by the separation of a mixture of five structurally different basic drugs yielding plate numbers in the 1x10(5)-3x10(5) plates/m range. Comparison with capillary electrophoretic analysis revealed a unique selectivity of the CEC system which is based on both electrophoretic mobility and chromatographic partitioning. PMID- 10749507 TI - Analysis of steroids in environmental water samples using solid-phase extraction and ion-trap gas chromatography-mass spectrometry and gas chromatography-tandem mass spectrometry. AB - This paper describes an improved method for the extraction and determination of three steroids, oestrone, 17beta-oestradiol, and the synthetic contraceptive steroid 17alpha-ethinyloestradiol in aqueous matrices. Samples of wastewater and environmental water were spiked with internal standards, comprising isotopically labelled analogues of the steroids to be determined. The samples were extracted using solid-phase extraction disks and the extracts were then derivatized to form tert.-butyldimethylsilyl derivatives. The derivatised steroids were determined in the final extracts by GC-MS or GC-MS-MS allowing an operational detection limit for each steroid in effluent samples of 1 ng l(-1). PMID- 10749509 TI - Focal upper extremity neuropathy in neonates. AB - Focal upper extremity neuropathies are common in neonates. The brachial plexus is the most common site involved. Brachial plexus injuries may involve different structures, thus producing different clinical presentations: complete brachial plexus palsy, Duchenne-Erb palsy, upper-middle trunk brachial plexus palsy, Klumpke palsy, fascicular brachial plexus palsy, and bilateral brachial plexus palsy. The causes of brachial plexus palsy are obstetric injury, intrauterine compression, humeral osteomyelitis, hemangioma, exostosis of the first rib, neck compression, and neoplasm. The differential diagnosis of brachial plexus palsy includes pseudoparesis, amyoplasia congenita, congenita varicella syndrome, and neurological lesions at other neuroanatomical levels. The cause and the degree of injury dictate the prognosis. The prognosis of obstetric brachial plexus injury is usually good. PMID- 10749510 TI - Imaging the brachial plexus and peripheral nerves in infants and children. AB - Imaging of the brachial plexus and peripheral nerves is challenging in the pediatric patient. Magnetic resonance imaging is the modality of choice as it is not invasive and demonstrates proximal and distal lesions. This may be used to detect nerve root avulsions, nerve ruptures, pseudo-meningoceles, brachial plexus scarring, post-traumatic neuromas, brachial plexus edema, spinal cord damage, abnormalities of the shoulder joint, trauma, neoplasms, and infection. Imaging allows diagnosis and careful preoperative evaluation of children suffering from brachial plexus injuries and peripheral nerve disorders. PMID- 10749511 TI - Neurophysiological evaluation of children with traumatic radiculopathy, plexopathy, and peripheral neuropathy. AB - Neurophysiological evaluation of children with traumatic peripheral nervous system injury is accomplished with electromyography, motor and sensory nerve conduction studies, and somatosensory and motor-evoked potentials. Preoperative neurophysiological tests are performed if motor deficits persist for more than 3 months. Evidence of reinnervation on these neurophysiological tests predates clinical recovery by weeks, hence they help determine the site of the lesion and provide objective measures for selecting candidates for surgical exploration. Intraoperative neurophysiological tests help to identify and confirm the integrity of nerves and to develop the optimal surgical strategy. Postoperative evaluations every 3 to 6 months may determine efficacy of treatment. PMID- 10749512 TI - Early operative intervention for birth injuries to the brachial plexus. AB - Early surgical repair is indicated for selected infants who sustain birth trauma to the brachial plexus. In children with global or total paralysis, surgery should be performed by 3 to 4 months of age to maximize ultimate extremity function. Although the timing of surgery for children with Erb's palsy remains controversial, in properly selected cases, nerve reconstruction leads to an improvement in shoulder function and overall limb function. PMID- 10749513 TI - Improving shoulder and elbow function in children with Erb's palsy. AB - Many children who sustain birth injuries to the brachial plexus suffer significant functional limitations due to various sequelae affecting the shoulder and elbow or forearm. The maintenance of full passive mobility during the period of neurological recovery is essential for normal joint development. Early surgical correction of shoulder contractures and subluxations reduces permanent deformity. Reconstruction of forearm rotation contractures significantly improves the appearance and use of the extremity for many basic activities. Each child must be carefully evaluated, therapy maximized, and the surgical approach individualized to obtain the best result. PMID- 10749514 TI - Rehabilitation program for children with brachial plexus and peripheral nerve injury. AB - An aggressive and integrated physical and occupational therapy program is essential in the treatment of congenital brachial plexus injuries and other severe upper extremity nerve injuries. This article addresses the evaluation, identification of needs, establishment of goals, and the approaches to rehabilitation treatment for patients with brachial plexus palsy and other peripheral nerve injuries. Rehabilitative therapy can preserve and build on gains made possible by medical or surgical interventions; however, therapy is vital to these children regardless of whether surgery is indicated. The therapist uses a problem-solving approach to evaluate the patient and select appropriate occupational and physical therapy treatment modalities. Therapy is continually adjusted based on each child's unique needs. An understanding of the therapy principles aids in making appropriate referrals and prescriptions, and helps to coordinate care between the therapist, pediatrician, neurologist, and surgeon. PMID- 10749515 TI - Congenital brachial plexus palsy: where have we been and where are we now? PMID- 10749516 TI - Is there a role for therapeutic drug monitoring of new anticonvulsants? AB - Despite the fact that all new anticonvulsants have undergone extensive pharmacokinetic scrutiny prior to their introduction to the market, the opportunity to perform good prospective studies on their concentration-effect relationship has been largely missed, in some cases deliberately because therapeutic drug monitoring (TDM) is considered unfavourable for the marketing of a new drug. However, there are reasons to believe that TDM may play a useful role in maximising the therapeutic potential of new anticonvulsants. In fact, these drugs have a narrow therapeutic index, careful individualisation of dosage to optimise response is required, and inter- and intra-individual pharmacokinetic variability may translate into differences in dosage requirements. The wide interindividual variability in the serum concentrations at which therapeutic and toxic effects of these drugs are observed does not necessarily imply that TDM cannot be useful: indeed, a marked pharmacodynamic variability has also been reported for all the currently monitored older anticonvulsants. The new anticonvulsants which, based on their properties, are particularly attractive candidates for TDM include lamotrigine, topiramate, tiagabine, zonisamide and felbamate. However, in the absence on sound information on the target concentration ranges of these drugs, the routine concentration monitoring of these drugs cannot be recommended. Despite this, serial measurements of serum drug concentrations may be useful in selected patients, especially those suspected of poor compliance and those in whom pharmacokinetic changes caused by disease or administration of concomitant medication are anticipated. Even in the presence of marked interindividual pharmacodynamic variability, it is often possible to empirically determine the concentration at which each patient exhibits the best response, and apply that information in subsequent management. Prospective studies, using preferably a randomised concentration-controlled design, are necessary to better characterise concentration-effect relationships for these agents. PMID- 10749517 TI - Clinical pharmacokinetics and pharmacodynamics of isepamicin. AB - Isepamicin is an aminoglycoside antibacterial with properties similar to those of amikacin, but with better activity against strains producing type I 6' acetyltransferase. The antibacterial spectrum includes Enterobacteriaceae and staphylococci. Anaerobes, Neisseriaceae and streptococci are resistant. The lower and upper break-points are 8 and 16 mg/L. Like other aminoglycosides, isepamicin exhibits a strong concentration-dependent bactericidal effect, a long post antibiotic effect (several hours) and induces adaptive resistance. Isepamicin is administered intravenously or intramuscularly at a dosage of 15 mg/kg once daily or 7.5 mg/kg twice daily. Isepamicin is not bound to plasma proteins, and it distributes in extracellular fluids and into some cells (outer hair cells, kidney cortex) by active transport. Isepamicin is not metabolised and is eliminated solely via the renal route with an elimination half-life (t 1/2 beta) of 2 to 3 hours in adults with normal renal function. The clearance of isepamicin is reduced in neonates, and 7.5 mg/kg once daily is recommended in children <16 days old. Clearance is also reduced in the elderly, but no dosage adjustment is required. In patients with chronic renal impairment, isepamicin clearance is proportional to creatinine clearance (CLCR); the recommended regimen is 8 mg/kg with an administration interval of 24 hours in moderate impairment, 48 hours in severe impairment, 72 hours for CL(CR) 0.6 to 1.14 L/h (10 to 19 ml/min) and 96 hours for CL(CR) 0.36 to 0.54 L/h (6 to 9 ml/min). In end-stage renal failure, isepamicin is eliminated by haemodialysis, but the administration interval should be determined by monitoring the plasma concentration. Compared with healthy volunteers, patients in the intensive care unit or with neutropenic cancer have an increased volume of distribution and a lower clearance, but the 15 mg/kg once daily regimen remains adequate. Isepamicin kinetics are linear in the range 7.5 to 25 mg/kg, so that dosage adjustments, if necessary, are straightforward. Isepamicin can induce nephro-, vestibulo- and oto-toxicity. However, animal and clinical studies show that isepamicin is one of the less toxic aminoglycosides. The usefulness of maintaining serum aminoglycoside concentrations within a therapeutic range remains controversial. With isepamicin, it is proposed to achieve a 1-hour concentration (30 minutes after a 30-minute infusion) >40 mg/L to maximise bactericidal efficacy, and a 'trough' concentration (at the end of the administration interval) <5 mg/L to minimise toxicity. These thresholds should be modified on an individual basis, considering covariates such as concomitant treatment, underlying disease, nature of bacterial strain and site of infection. PMID- 10749518 TI - Clinical pharmacokinetics and pharmacodynamics of celecoxib: a selective cyclo oxygenase-2 inhibitor. AB - Celecoxib, a nonsteroidal anti-inflammatory drug (NSAID), is the first specific inhibitor of cyclo-oxygenase-2 (COX-2) approved to treat patients with rheumatism and osteoarthritis. Preliminary data suggest that celecoxib also has analgesic and anticancer properties. The selective inhibition of COX-2 is thought to lead to a reduction in the unwanted effects of NSAIDs. Upper gastrointestinal complication rates in clinical trials are significantly lower for celecoxib than for traditional nonselective NSAIDs (e.g. naproxen, ibuprofen and diclofenac). The rate of absorption of celexocib is moderate when given orally (peak plasma drug concentration occurs after 2 to 4 hours), although the extent of absorption is not known. Celexocib is extensively protein bound, primarily to plasma albumin, and has an apparent volume of distribution of 455+/-166L in humans. The area under the plasma concentration-time curve (AUC) of celecoxib increases in proportion to increasing oral doses between 100 and 800mg. Celecoxib is eliminated following biotransformation to carboxylic acid and glucuronide metabolites that are excreted in urine and faeces, with little drug (2%) being eliminated unchanged in the urine. Celecoxib is metabolised primarily by the cytochrome P450 (CYP) 2C9 isoenzyme and has an elimination half-life of about 11 hours in healthy individuals. Racial differences in drug disposition and pharmacokinetic changes in the elderly have been reported for celecoxib. Plasma concentrations (AUC) of celecoxib appear to be 43% lower in patients with chronic renal insufficiency [glomerular filtration rate 2.1 to 3.6 L/h (35 to 60 ml/min)] compared with individuals with healthy renal function, with a 47% increase in apparent clearance. Compared with healthy controls, it has been reported that the steady-state AUC is increased by approximately 40% and 180% in patients with mild and moderate hepatic impairment, respectively. Celecoxib does not appear to interact with warfarin, ketoconazole or methotrexate; however, clinically significant drug interactions with fluconazole and lithium have been documented. As celecoxib is metabolised by CYP2C9, increased clinical vigilance is required during the coadministration of other substrates or inhibitors of this enzyme. PMID- 10749521 TI - Environmental dosimetry performance criteria: pilot test of proposed ANSI draft standard N13.29. American National Standards Institute. AB - American National Standards Institute (ANSI) standards are a source of technical guidance on the application of passive radiation detectors in personnel and environmental monitoring programs. The proposed new ANSI Draft N13.29 describes performance tests for environmental dosimetry providers that are analogous to those now required in personnel dosimetry. The objective of this study was to pilot test the procedural specifications before Draft N13.29 is submitted for final balloting. The results of the performance tests are relevant to environmental surveillance programs, which may be affected if Draft N13.29 is approved. Seven environmental dosimetry providers submitted dosimeters for the pilot test, which included two phases. The first phase involved exposing dosimeters to laboratory gamma, beta, and x-ray sources at routine and accident dose levels. In the second phase, dosimeters were subjected to 90 d of simulated environmental conditions in a chamber that cycled through prescribed temperature and humidity parameters and lighting conditions. Two participants passed all categories of the laboratory test phase, and all seven passed the environmental test phase. We report here on the results of the performance tests; additional results relevant to finalizing Draft N13.29 are detailed elsewhere. PMID- 10749522 TI - Modeling energy deposition and cellular radiation effects in human bronchial epithelium by radon progeny alpha particles. AB - Energy deposition and cellular radiation effects arising from the interaction of single 218Po and 214Po alpha particles with basal and secretory cell nuclei were simulated for different target cell depths in the bronchial epithelium of human airway generations 2, 4, 6, and 10. To relate the random chord lengths of alpha particle tracks through spherical cell nuclei to the resulting biological endpoints, probabilities per unit track length for different cellular radiation effects as functions of LET were derived from in vitro experiments. The radiobiological data employed in the present study were inactivation and mutation (mutant frequency at the HPRT gene) in V79 Chinese hamster cells and inactivation and transformation in C3H 10T1/2 cells. Based on computed LET spectra and relative frequencies of target cells, probabilities for transformation, mutation, and cell killing in basal and secretory cells were computed for a lifetime exposure of 20 WLM. While predicted transformation probabilities were about two orders of magnitude higher than mutation probabilities, they were still about two orders of magnitude lower than inactivation probabilities. Furthermore transformation probabilities for basal cells are generally higher than those for secretory cells, and 214Po alpha particles are primarily responsible for transformations in bronchial target cells. PMID- 10749523 TI - A radioiodine speciation, deposition, and dispersion model with uncertainty propagation for the Oak Ridge dose reconstruction. AB - Between 1944 and 1956, radioactive 131I was released into the atmosphere from operations at the Oak Ridge National Laboratory in Oak Ridge, TN. The releases occurred from stacks and from building vents and openings in three different chemical forms: elemental, organic, and particulate. During their transport in the atmosphere, different forms of iodine react differently with other atmospheric chemicals and moisture, and are removed from the plume at different rates by the processes of dry and wet deposition. A modified Gaussian plume model was developed to address the processes of radioiodine speciation, deposition, depletion, and dispersion in the atmosphere, and to propagate uncertainties in input parameter values through to the ground-level concentrations of 131I in air. A unique approach was used to develop an implicitly correlated set of hourly meteorological parameters for any day of a month for each month of the year from ten years of available data between 1987 and 1996. The model was validated for both annual average and short-term releases. For the annual average releases, the predictions of ground-level concentrations of 131I from the model were within a factor of 2 of measured field data. For two of the three sets of available weekly data, the measurements fell within the 95% subjective confidence interval of model predictions. Predictions of ground-level air concentrations were made on an annual average basis for the entire period of release and on a short-term, episodic basis for a 1954 accident. PMID- 10749519 TI - Pharmacokinetic considerations in the eradication of Helicobacter pylori. AB - As Helicobacter pylori plays an important role in the aetiopathogenesis of peptic ulcer, therapeutic strategies aimed at maintaining long term remission have shifted from the control of intragastric pH to targeting H. pylori. According to recent international guidelines the clinical goals--rapid ulcer healing and prevention of relapse--can be best accomplished by combination therapy consisting of an antisecretory drug (proton pump inhibitor or ranitidine) and 2 antimicrobial agents (preferable amoxicillin, clarithromycin or metronidazole). When applying such multidrug regimens, possible synergy between the agents suggests that pharmacokinetic considerations might help to improve H. pylori eradication rates, which should be above 85 to 90% on an intention-to-treat basis. The present review summarises the pharmacokinetic properties and interaction potential of all drugs presently used in the various H. pylori eradication regimens, with emphasis on particular patient populations such as the elderly and those with renal impairment. The drugs considered are omeprazole, lansoprazole, pantoprazole, rabeprazole, ranitidine and ranitidine bismutrex, bismuth salts, amoxicillin, clarithromycin, azithromycin, roxithromycin, metronidazole, tinidazole and tetracycline. When addressing the clinically important questions of the efficacy, safety and costs of the recommended regimens, the impact of drug disposition on H. pylori eradication should not be neglected. PMID- 10749524 TI - Monte Carlo calculations of LR115 detector response to 222Rn in the presence of 220Rn. AB - The sensitivities (in m) of bare LR115 detectors and detectors in diffusion chambers to 222Rn and 220Rn chains are calculated by the Monte Carlo method. The partial sensitivities of bare detectors to the 222Rn chain are larger than those to the 220Rn chain, which is due to the higher energies of alpha particles in the 220Rn chain and the upper energy limit for detection for the LR115 detector. However, the total sensitivities are approximately equal because 220Rn is always in equilibrium with its first progeny, which is not the case for the 222Rn chain. The total sensitivity of bare LR115 detectors to 222Rn chain depends linearly on the equilibrium factor. The overestimation in 222Rn measurements with bare detectors caused by 220Rn in air can reach 10% in normal environmental conditions. An analytical relationship between the equilibrium factor and the ratio between track densities on the bare detector and the detector enclosed in chamber is given in the last part of the paper. This ratio is also affected by 220Rn, which can disturb the determination of the equilibrium factor. PMID- 10749525 TI - Long-term changes in body potassium concentration and dose from 40K for a Japanese adult male group. AB - Total body potassium has been measured for a Japanese adult male group using two whole-body counters. The total body potassium data were analyzed to present the longitudinal data on body potassium concentration in aging individuals. The data for each individual showed that potassium concentration decreased linearly with age from 30 y to 60 y of age. The average decrease rate was -0.0154 +/- 0.0049 g K kg body wt(-1) y(-1). On the other hand, a subject group for each of the 23 years (1975-1998) of the study was divided into three age groups. The same age groups were followed with the course of time to analyze changes in potassium concentration due to other factors besides age. The follow-up study of the same age groups indicated that the average potassium concentration decreased with the course of the 23-y study and that the decrease was due to other factors besides age. The decrease in the average potassium concentration for study subjects is the major cause of the decrease in the average annual dose from 40K. The average annual dose for subjects aged 30-59 y was 194 muGy in 1975, changing to 166 muGy in 1998. The decrease in the average annual dose was about 14% over the 23-y study period. PMID- 10749520 TI - Age-related changes in protein binding of drugs: implications for therapy. AB - The plasma protein binding of drugs, particularly those that are highly bound, may have significant clinical implications. Although protein binding is a major determinant of drug action, it is only one of a myriad of factors that influence drug disposition. The extent of protein binding is a function of drug and protein concentrations, the affinity constant for the drug-protein interaction and the number of protein binding sites per class of binding site. Age-related changes in protein binding are usually not clinically important in drug therapy. Albumin levels are generally decreased in the elderly, whereas alpha1-acid glycoprotein levels are not altered by age per se. Alterations in plasma protein binding that occur in the elderly are generally not attributed to age, but rather to physiological and pathophysiological changes or disease states that may occur more frequently in the elderly and most often account for altered protein binding. Age-related physiological changes, such as decreased renal function, decreased hepatic function and decreased cardiac output, generally produce more clinically significant alterations in drug disposition than that seen with alterations in drug plasma protein binding. An understanding of the inter relationships between drug concentrations, protein binding, the physiology of aging, disease, pharmacokinetics and pharmacodynamics is necessary for effective therapeutic monitoring. Monitoring of unbound drug concentrations simplifies these relationships and provides the fundamental information needed for dosage regimen development and adjustment. Drug therapy in the elderly should be individualised taking into account all of these factors. PMID- 10749526 TI - Percutaneous toxicity of uranyl nitrate: its effect in terms of exposure area and time. AB - Different groups have undertaken research work focusing their attention on the biological effects of uranium and have described kidney and bone to be the main target organs in uranium poisoning. In this study we used the skin as the route of entry of uranium. We carried out two sets of experiments in adult rats: in one of them topical applications with uranyl nitrate (UN) over different areas were performed; in the other topical applications with UN on a given area over different times were carried out. In the latter experiment the exposure to UN was stopped by removing it from skin with soap and water. Kidney and bone samples were removed for histological studies. This work is based on the determination of the survival rate of the exposed animals and on the effects elicited in kidney and bone. There is a relation between the area of the surface exposed to uranium and the time of exposure and the subsequent percutaneous toxicity. There were no surviving animals following topical application of UN to an 8 cm2 area nor when the time of exposure was 24 h. The survival rate of the animals increased when either the topical area or the time of exposure to UN was reduced. Although the inhibition of bone formation in metaphysical bone has been previously described by our group as a result of UN poisoning, this is the first time that such an effect is found after percutaneous exposure for such short periods of time. The general toxic effects of UN, evidenced as kidney histological alterations, increased in severity as either one of the two variables studied increased. This is a condition that could be considered as hazardous for those workers engaged in uranium processing and purification. It is noteworthy that a simple method such as washing with soap and water is an effective method to reduce the lethality of UN percutaneous intoxication. PMID- 10749527 TI - Radon and thoron in cave dwellings (Yan'an, China). AB - 222Rn and 220Rn concentrations were measured in cave dwellings and brick houses in the region of Yan'an (China) during summer 1997. The underground dwellings are built into Quaternary loess, and all investigated houses are founded on it. The median values of indoor 222Rn and 220Rn concentrations are 42 (n = 18) and 77 Bq m(-3) (n = 15) for brick houses and 92 (n = 23) and 215 (n = 17) Bq m(-3) for cave dwellings. To classify the dwellings in respect to their "cave-character," the fraction of walls having a direct contact to the loses is calculated for each dwelling. While the 222Rn concentrations are increasing with higher fractions, the 220Rn concentrations are not correlated with this fraction. On the other hand, due to the short half-life of 220Rn the distance from the measuring point to the walls is negatively correlated with the 220Rn concentration, while there is no correlation with the 222Rn concentration. Therefore, concentric isolines of 220Rn concentrations showing a strong gradient were detected in cave dwellings. An influence of the ventilation rate is distinct for 222Rn but weak for 220Rn. The effective dose rates for 222Rn and 220Rn and their progenies are calculated for brick houses (2.7 mSv y(-1)), cave dwellings (7.1 mSv y(-1)), and for traditional cave dwellings with a bed foundation built with loess (16.7 mSv y( 1)). These calculations are based on summer measurements only. It is expected that the true effective dose rates will be significantly higher. PMID- 10749528 TI - Comments on the use of 137Cs/239+240Pu activity ratio to resolve binary mixtures of Pu. PMID- 10749530 TI - Y2K presents little problem for nuclear power plants. PMID- 10749529 TI - Comment on article by Bursik, Meisenhelder and Spahn. PMID- 10749531 TI - New IAEA guides on occupational protection. International Atomic Energy Agency. PMID- 10749532 TI - Purification, partial characterization, and reactivity with aromatic compounds of two laccases from Marasmius quercophilus strain 17. AB - Two isozymes of laccase were obtained from an induced liquid culture of Marasmius quercophilus with p-hydroxybenzoic acid as the inducer. Both the constitutive and the induced isozyme have a molecular mass of 60 kDa as determined by polyacrylamide gel electrophoresis. Using isoelectric focusing, we found three isozymes with the constitutive enzyme (pI 4, 4.2, 4.4) and four of the induced form (pI 4.75, 4.85, 4.95, 5.1). We observed certain differences between these two isozymes; the specific activity of the induced isozyme was twice as high, and two optimum pH levels (5 and 6) were observed with the induced isozyme (only one, pH 5, for the constitutive isozyme). However, both of these enzymes have the same thermal stability and the same temperature for their highest activity (80 degrees C). Furthermore, the reactivity of both these enzymes with aromatic compounds was similar. The use of mediators extended the oxidized substrate range of the laccases studied. Various products of degradation were observed, depending on the mediator used. When laccase was used alone, the decrease of the signal corresponding to the aromatic cycle, without any formations of other peaks at different wavelengths, suggested polymerisation of aromatic compounds. PMID- 10749533 TI - Ribosomal DNA, species structure, and biogeography of the cactophilic yeast Clavispora opuntiae. AB - The ribosomal DNA of the cactophilic yeast species Clavispora opuntiae was studied in order to clarify the global distribution of the yeast. Over 500 strains, including isolates from several new localities worldwide, were characterized by rDNA restriction mapping. An unusual restriction pattern previously encountered only in one strain, from Conception Island in the Bahamas, was found in several Brazilian isolates. Sequences of the D1/D2 and D7/D8 divergent domains of the large subunit (LSU) and of the intergenic spacers (IGS) confirmed that these strains represent a genetically distinct variety of Clavispora opuntiae. This divergence had previously been hypothesized on the basis of reduced genetic recombination in inter-varietal crosses and the presence of a polymorphic ApaI restriction site located in the LSU. The exact position of the ApaI site in the D8 divergent domain and the nature of the variation that it reveals were determined. The complete sequences of 12 intergenic spacers clarified the significance of the species-wide variation uncovered by restriction mapping. Most of the polymorphic sites occur in the IGS1 and IGS2 regions, on either side of the 5S gene, and the variation is largely due to differences in the numbers and the sequences of internal repeats. Two other polymorphic sites are located in the external transcribed spacer (ETS) region. The reliability of various sites as indicators of overall spacer sequence divergence differed from one case to another. Variety-specific probes were devised and used to screen 120 strains for the presence of recombinant rDNA spacers. Three strains gave ambiguous results, but these did not constitute evidence that inter-varietal recombination has taken place in nature. The hypothesis that the global movement of Clavispora opuntiae has been influenced by the worldwide biological control of prickly pear with Cactoblastis cactorum, a moth of Argentinian origin, has received additional support from the demonstration that Argentinian strains have rDNAs similar to those found where the moth has been introduced. A dramatic founder effect was identified in a yeast population collected in cacti (Maui, Hawaii) in a site where the moth had been recently introduced. PMID- 10749534 TI - Isolation and characterization of a Pseudomonas sp. strain PH1 utilizing meta aminophenol. AB - Pseudomonas sp. strain PH1 was isolated from soil contaminated with pharmaceutical and dye industry waste. The isolate PH1 could use m-aminophenol as a sole source of carbon, nitrogen, and energy to support the growth. PH1 could degrade up to 0.32 mM m-aminophenol in 120 h, when provided as nitrogen source at 0.4 mM concentration with citrate (0.5 mM) as a carbon source in the growth medium. The presence of ammonium chloride as an additional nitrogen source repressed the degradation of m-aminophenol by PH1. To identify strain PH1, the 16S rDNA sequence was amplified by PCR using conserved eubacterial primers. The FASTA program was used to analyze the 16S rDNA sequence and the resulting homology patterns suggested that PH1 is a Pseudomonas. PMID- 10749535 TI - Studies on the interactions of immunostimulated macrophages and Yersinia enterocolitica O:8. AB - Immunological and electron microscopy investigations of the phagocytic and killing activities of peritoneal macrophages from rats and mice against Yersinia enterocolitica serotype O:8 cells were performed. The effect of in vivo application of cytoplasmic membranes (CM) from the stable Escherichia coli WF+ L form on macrophage activity was also studied. It was established that rat macrophages more actively phagocytosed the plasmidless pYV(-) Y. enterocolitica cells, compared to the plasmid-bearing pYV(+) Y. enterocolitica cells. The killing ability against both variants of the Y. enterocolitica strain was significantly enhanced in macrophages from CM-treated rats after 2 h, 4 h, and 24 h incubation. The CM treatment enhanced the phagocytic activity of the macrophages. The in vitro interaction of normal and immunostimulated rat macrophages with both pYV(+) and pYV(-) variants of Y. enterocolitica did not lead to any additional apoptotic and necrotic changes in macrophages compared to control macrophages, which were cultivated without Y. enterocolitica. Electron microscopic investigation showed that mouse macrophages eliminated Y. enterocolitica pYV(+) cells in vivo after 24 h. No engulfed or digested bacterial cells were observed. Activation of cell surfaces and vacuolization of macrophage cytoplasm, both of CM-treated non-infected and infected mice, were observed. The experimental results showed that Y. enterocolitica pYV(+) cells could be eliminated by peritoneal macrophages. PMID- 10749536 TI - Stimulation of the ionic transport system in Brassica napus by a plant growth promoting rhizobacterium (Achromobacter sp.). AB - A plant growth-promoting rhizobacterium belonging to the genus Achromobacter was isolated from the oil-seed-rape (Brassica napus) root. Growth promotion bioassays were performed with oilseed rape seedlings in a growth chamber in test tubes containing attapulgite and mineral nutrient solution, containing NO3- as N source. The presence of this Achromobacter strain increased shoot and root dry weight by 22-33% and 6-21%, respectively. Inoculation of young seedlings with the Achromobacter bacteria induced a 100% improvement in NO3- uptake by the whole root system. Observations on the seminal root of seedlings 20 h after inoculation showed that there was an enhancement of both the number and the length of root hairs, compared to non-inoculated seedlings. Electrophysiological measurements of NO3- net flux with ion-selective microelectrodes showed that inoculation resulted in a specific increase of net nitrate flux in a root zone morphologically similar in inoculated and non-inoculated plants. The root area increased due to root hair stimulation by the Achromobacter bacteria, which might have contributed to the improvement of NO3- uptake by the whole root system, together with the enhancement of specific NO3- uptake rate. Moreover, inoculated plants showed increased potassium net influx and proton net efflux. Overall, the data presented suggest that the inoculation of oilseed-rape with the bacteria Achromobacter affects the mineral uptake. PMID- 10749537 TI - Plant growth-promoting bacteria that decrease heavy metal toxicity in plants. AB - Kluyvera ascorbata SUD165 and a siderophore-overproducing mutant of this bacterium, K. ascorbata SUD165/26, were used to inoculate tomato, canola, and Indian mustard seeds which were then grown in soil for 25-42 days in the presence of either nickel, lead, or zinc. The parameters that were monitored included plant wet and dry weight, protein and chlorophyll content in the plant leaves, and concentration of heavy metal in the plant roots and shoots. As indicated by a decrease in the measured values of these parameters, in all instances, plant growth was inhibited by the presence of the added metal. Both bacterial strains were effective, although not always to a statistically significant extent, at relieving a portion of the growth inhibition caused by the metals. In most cases, the siderophore overproducing mutant K. ascorbata 165/26 exerted a more pronounced effect on plant growth than did the wild-type bacterium K. ascorbata SUD165. The data suggest that the ability of these bacteria to protect plants against the inhibitory effects of high concentrations of nickel, lead, and zinc is related to the bacteria providing the plants with sufficient iron. PMID- 10749539 TI - Adhesion to hyphal matrix and antifungal activity of Pseudomonas strains isolated from Tuber borchii ascocarps. AB - Pseudomonas spp. isolates from Tuber borchii ascocarps, known to be able to produce phytoregulatory and biocontrol substances in pure culture, were used to perform studies on their possible physiological role in nature. Antimycotic activity was confirmed against fungal contaminants isolated from the ascocarps, suggesting that populations associated with Tuber borchii fruit bodies may play a role in the maintenance of ascocarp health. Fifty-five percent of strains tested were also able to release metabolites which affected T. borchii mycelial growth and morphogenesis in culture. On the contrary, growth of the arbuscular mycorrhizal fungus Glomus mosseae and the ectomycorrhizal fungus Laccaria bicolor, putative competitors of Tuber for mycorrhizal infection sites on roots, was not influenced by the presence of any bacterial strain. The possibility that these bacteria, which show antifungal activity and fungal growth modulation activities, might be incorporated in the developing ascocarp by means of their preferential adhesion to Tuber mycelium is discussed. PMID- 10749538 TI - Aspects of the pathology and etiology of 'drippy gill' disease of the cultivated mushroom Agaricus bisporus. AB - Agaricus bisporus sporocarps exhibiting characteristic 'drippy gill' symptoms from a natural outbreak were examined. Discrete bacterial droplets on the hymenial lamellae often coalesced to form ribbons of bacterial ooze. Longitudinal splits on the stipe were lined with a similar bacterial ooze. Bacteria isolated from both the hymenium and stipe were identified as Pseudomonas agarici, and were confirmed to be the causal organism by satisfying Koch's postulates. By light and transmission electron microscopy, the causal bacteria were found to colonize the extrahyphal spaces and degrade the extracellular matrix within affected sporocarps. Degradation of the extracellular matrix was shown to reduce the integrity of the sporocarp, and result in stipe splitting and hymenium disruption. In artificial inoculations of the pileus, bacteria were shown to exist predominantly in sporocarp tissue below the point of inoculation and above affected areas of the hymenium, indicating an approximately vertical passage through the sporocarp via the extracellular spaces. The dissolution of the extracellular matrix, and the observed failure of the bacterium to produce a toxin active against A. bisporus, allow the bacteria to pass through protective membranes unnoticed, and infect the stipe and hymenium prior to veil break. These observations dispel previous assumptions of intrahyphal existence and transmission. In the few instances in which the bacteria were observed to be intrahyphal, the host fungal cell wall was often broken, suggesting intrahyphal existence was opportunistic rather than obligatory. The taxonomic position of a bacterium isolated previously from sporocarps exhibiting symptoms similar to those of drippy gill was determined by examining the biochemical and nutritional profiles of the bacterium, and comparing them with other Pseudomonas agarici isolates. PMID- 10749540 TI - Biodegradation of carbazole by Ralstonia sp. RJGII.123 isolated from a hydrocarbon contaminated soil. AB - The use of microorganisms for bioremediation of contaminated soils may be enhanced with an understanding of the pathways involved in their degradation of hazardous compounds. Ralstonia sp. strain RJGII.123 was isolated from soil located at a former coal gasification plant, based on its ability to mineralize carbazole, a three-ring N-heterocyclic pollutant. Experiments were carried out with strain RJGHII.123 and 14C-carbazole (2 mg/L and 500 mg/L) as the sole organic carbon source. At 15 days, 80% of the 2 mg/L carbazole was recovered as CO2, and <1% remained as undegraded carbazole, while 24% of the 500 mg/L carbazole was recovered as CO2 and approximately 70% remained as undegraded carbazole. Several stable intermediates were formed during this time. These intermediates were separated by high performance liquid chromatography (HPLC) and were characterized using high resolution mass spectroscopy (HR-MS) and gas chromatography - mass spectroscopy (GC-MS). At least 10 ring cleavage products of carbazole degradation were identified; four of these were confirmed as anthranilic acid, indole-2-carboxylic acid, indole-3-carboxylic acid, and (1H)-4 quinolinone by comparison with standards. These data indicate that strain RJGII.123 shares aspects of carbazole degradation with previously described Pseudomonas spp., and may be useful in facilitating the bioremediation of NHA from contaminated soils. PMID- 10749541 TI - Type I nitroreductases in soil enterobacteria reduce TNT (2,4,6,-trinitrotoluene) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine). AB - Many enteric bacteria express a type I oxygen-insensitive nitroreductase, which reduces nitro groups on many different nitroaromatic compounds under aerobic conditions. Enzymatic reduction of nitramines was also documented in enteric bacteria under anaerobic conditions. This study indicates that nitramine reduction in enteric bacteria is carried out by the type I, or oxygen-insensitive nitroreductase, rather than a type II enzyme. The enteric bacterium Morganella morganii strain B2 with documented hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) nitroreductase activity, and Enterobacter cloacae strain 96-3 with documented 2,4,6-trinitrotoluene (TNT) nitroreductase activity, were used here to show that the explosives TNT and RDX were both reduced by a type I nitroreductase. Morganella morganii and E. cloacae exhibited RDX and TNT nitroreductase activities in whole cell assays. Type I nitroreductase, purified from E. cloacae, oxidized NADPH with TNT or RDX as substrate. When expression of the E. cloacae type I nitroreductase gene was induced in an Escherichia coli strain carrying a plasmid, a simultaneous increase in TNT and RDX nitroreductase activities was observed. In addition, neither TNT nor RDX nitroreductase activity was detected in nitrofurazone-resistant mutants of M. morganii. We conclude that a type I nitroreductase present in these two enteric bacteria was responsible for the nitroreduction of both types of explosive. PMID- 10749543 TI - Nutritional requirements of Xylella fastidiosa, which causes Pierce's disease in grapes. AB - A defined medium (XF-26) containing 3 inorganic salts, 2 tricarboxylic acids, 17 amino acids, potato starch, phenol red, and agar was used as the starting point for the study. Deletions of one or more ingredients were performed to prepare various media. A medium was considered able to support growth of Xylella fastidiosa strains responsible for Pierce's disease in grapes, only after 10 serial passages had been completed. Of 3 inorganic salts, K2HPO4 and MgSO4 x 7H2O were essential, and (NH4)2HPO4 was nonessential for growth. Of the Krebs cycle intermediates, all (citrate, alpha-ketoglutarate, succinate, fumarate, malate, and oxaloacetate) but isocitrate supported growth of cultivated strains, whereas only citrate alone or citrate plus succinate supported the primary isolation of PD bacterium. Of 17 amino acids, 6 uncharged polar R groups (asparagine, cysteine, glutamine, glycine, serine, and threonine) supported growth, whereas 8 nonpolar R groups (alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine) or 3 positively charged polar groups (arginine, histidine, and lysine) did not. Starch proved to be nonessential. PMID- 10749542 TI - Oral immunization of mice with a glycoconjugate vaccine containing the O157 antigen of Escherichia coli O157:H7 admixed with cholera toxin fails to elicit protection against subsequent colonization by the pathogen. AB - It has been postulated that a humoral immune response directed against the O157 antigen of Escherichia coli O157:H7, and expressed in the intestine, might afford protection from colonization and consequent infection by this enteric pathogen. The present study was conducted to determine whether such an immune response can be experimentally generated in mice. To this end, mice were orally immunized with a glycoconjugate vaccine consisting of horse serum albumin and the O157 polysaccharide admixed with the mucosal adjuvant, cholera toxin. Mice consistently developed robust local and systemic immune responses to the cholera toxin adjuvant, but were far from uniformly reactive to the test vaccine. Moreover, vaccinated mice were as susceptible to transient intestinal colonization following challenge with an isolate of E. coli O157:H7 as unvaccinated control mice. These results indicate that this vaccination approach is unlikely to be straightforward in target bovine or human hosts. PMID- 10749544 TI - Introduction: the role of bacterial infection in chronic bronchitis. AB - Acute exacerbations of chronic bronchitis reflect increased airway inflammation and are characterised by one or more symptoms of increased sputum production, sputum purulence, and breathlessness. The causes are multifactorial, and bacterial infection is involved in about half of cases. A proportion of patients also have chronic colonization of the bronchial tree between exacerbations, and this may act as a stimulant of airway inflammation. Colonization represents a balance in which compromised host defences limit bacterial numbers but do not eradicate them. The balance is upset during an exacerbation, often due to extraneous factors such as a viral infection or air pollution, leading to increased bacterial numbers and consequently more inflammation. In patients with severe airway damage, infective exacerbations are more likely to occur, and serious consequences may result if baseline lung function is impaired or there are comorbid conditions. In these circumstances, the exacerbation is less likely to resolve spontaneously. Antibiotic treatment benefits patients by achieving bacterial eradication and resolution of the inflammatory response. However, since superficial mucosal infections may resolve spontaneously, there are serious concerns about widespread antibiotic use in patients with more trivial illness. Future studies should include better definition of the type of patients enrolled, improved techniques to determine bacteriological response, and better outcome measures. PMID- 10749545 TI - Host defenses and pathogenesis. AB - The lung is protected by an array of interacting defense mechanisms.These include mechanical defenses that filter and remove particulates from the inhaled air as well as mechanisms to capture and remove particulates that penetrate into the lung as well as chemical and cellular defenses that can kill invading microorganisms. Optimal lung defense requires coordinated action of these various defenses. However, whereas failure of any defense mechanism increases the risk of infection, the various mechanisms provide, at least to a certain extent, redundant protection. As a result, many individuals with compromise of a single defense mechanism may not become clinically ill. Syndromes characterized by chronic infection of the lower respiratory tract are therefore associated with several defects in respiratory tract defense. PMID- 10749546 TI - Inflammation--role of the neutrophil and the eosinophil. AB - Neutrophils are regularly identified in the airway secretions of patients with smoking-related chronic bronchitis and in those with chronic obstructive pulmonary disease. The migration and activation of these cells plays a role that is central to the secondary bronchial defences against potential bacterial pathogens. Where these processes persist or are excessive, potent mediators derived from the neutrophil may cause a perpetuation of bronchial inflammation and result in bronchial epithelial damage and impairment of endobronchial defences. Recent evidence has suggested that the eosinophil may also be present in the airways of some patients with chronic bronchitis, although its role in bronchial inflammation is not yet clear. In this article we have reviewed the evidence relating to the roles of both the neutrophil and of the eosinophil in the airway inflammation of chronic bronchitis. PMID- 10749547 TI - The role of the inflammatory response in chronic bronchitis: therapeutic implications. AB - Chronic bronchitis is diagnosed clinically by a chronic productive cough. As implied by the term "bronchitis," chronic airway inflammation is typically found in the central airways in patients with persistent cough and mucous hypersecretion. Although the exact pathogenesis of chronic bronchitis remains unclear, bacterial colonization and the resulting inflammatory response are thought to be of central importance. The generation of pro-inflammatory cytokines and chemotactic stimuli by the airway epithelium likely play central roles in propagating the inflammatory response in patients with chronic bronchitis. Further insights into the initiating events and underlying mechanisms that result in the clinical syndrome of chronic bronchitis will likely provide novel opportunities for therapeutic interventions. PMID- 10749548 TI - Chronic bronchitis: the role of viruses. AB - Mucus is produced by the epithelial cells in the glands, gland ducts, and the cells lining the airway lumen in the lower airways. The chronic cough and sputum production that defines chronic bronchitis is associated with an inflammatory reaction involving this mucus-secreting apparatus. Respiratory viral infections target the epithelial cells of the lung producing desquamation, microvascular dilatation, edema, and an inflammatory cell infiltrate. These changes predispose the lower airways to bacterial infection by interfering with mucociliary clearance and reducing bacterial killing by macrophages. The exact role of those infections in the pathogenesis of chronic bronchitis has not been clearly determined but they probably play a critical role in inducing bacterial colonization and initiating acute exacerbations of COPD. This article reviews the classification of the viral agents responsible for respiratory tract infection and the nature of the changes they produce in the normal airways and in the airways of patients with chronic bronchitis during acute infections. PMID- 10749549 TI - Haemophilus influenzae in chronic bronchitis. AB - Colonization of the adult respiratory tract with nontypable Haemophilus influenzae is a dynamic process with new strains being acquired and replacing old strains periodically. The organism is a common cause of exacerbations of chronic bronchitis based on 3 lines of evidence: quantitative culture of the lower airways obtained by protected specimen brush, antibiotic trials, and serological studies. Nontypable H. influenzae expresses multiple adhesin molecules that mediate adherence to the respiratory tract mucosa. Recent studies have established that the bacterium penetrates the mucosal surface and survives intracellularly and in the interstitium of the submucosa. The organism shows a remarkable degree of antigenic diversity on its surface, including phase variation of lipooligosaccharide, antigenic heterogeneity of surface proteins, point mutations in genes encoding surface proteins and horizontal transfer of genes. These strategies facilitate evasion of the human immune response. Substantial progress has been made in identifying vaccine antigens to prevent infections caused by nontypable H. influenzae. PMID- 10749550 TI - Nonantimicrobial aspects of therapy. AB - Current therapy for chronic obstructive pulmonary disease is symptomatic, and no treatments prevent the progression of the disease. Stopping smoking is the only effective approach, and this may be facilitated by nicotine replacement and bupropion. Treatment with short- and long-acting inhaled beta2-agonists and anticholinergics are the mainstay of therapy. Inhaled corticosteroids have no impact on disease progression, and the small reduction in exacerbations may not justify the potential systemic side effects. Supplementary oxygen therapy is indicated for acute exacerbations and chronic hypoxia. Other treatments, including mucolytics, vaccines, and respiratory stimulants are of little value. Nonpharmacological measures that are useful include pulmonary rehabilitation, exercise, good nutrition, and, in selected cases, surgery. Several new classes of drug are now in development, including mediator antagonists (leukotriene B4, interleukin-8 antagonists, and anti-oxidants) and nonsteroid anti-inflammatory drugs, of which phosphodiesterase-4 inhibitors look the most promising. There is a pressing need for a better understanding of the underlying disease process to develop more logical therapies. PMID- 10749551 TI - Antibiotic therapy of exacerbations of chronic bronchitis. AB - The role of antibiotics in acute exacerbations of chronic bronchitis (AECB) remains controversial because patients commonly harbor the same bacteria in their sputum at times of stability and at times of acute illness. However, prospective randomized controlled trials do show a benefit for the use of antibiotics, compared with placebo, in AECB, particularly if patients have at least 2 of the following 3 symptoms: increased dyspnea, increased sputum volume, increased sputum purulence. In this setting, antibiotics have value, leading to a more rapid resolution of symptoms and a more rapid return of peak flow rate, compared with placebo. In addition, antibiotics may prevent some patients from developing secondary pneumonia and may prolong the time between exacerbations. When antibiotics are used, a variety of factors must be considered in choosing an agent. These include the likelihood of antibiotic-resistant bacteria, a factor that relates to defining subsets of patients. Patients can fall into 1 of 3 categories, each with a different suggested therapy. These categories include simple AECB, complicated AECB, and AECB at risk for infection with P. aeruginosa. In addition, an antibiotic should be chosen with pharmacokinetics and pharmacodynamic behavior in mind. In the future, research will need to confirm that careful selection of specific agents for specific patients can lead to improved patient outcomes, but already some preliminary data are supporting this concept. PMID- 10749552 TI - Cost-effective therapy for acute exacerbations of chronic bronchitis. AB - Pharmacoeconomic analysis involves the measurement of a ratio determining the extra costs required to achieve an additional unit of clinical benefit. Various techniques including modeling studies, retrospective analysis of databases, "piggy-back" economic analysis of prospective randomized clinical trials, and prospective randomized pharmacoeconomic trials have been developed to aid in economic and health decisions. In acute exacerbations of chronic obstructive pulmonary disease, it is possible to identify a group of patients that are at high risk of treatment failure from routine antimicrobial therapy, hospitalization, respiratory failure, and death. The cost of therapy for this relatively small group of patients is extraordinarily high. Data from a variety of approaches have suggested that aggressive antimicrobial therapy may lead to improved outcomes in these patients. The corollary is that aggressive therapy directed toward patients with either acute bronchitis (mainly a viral infection) or exacerbations of trivial chronic obstructive lung disease leads to emergence of resistance and increased costs. PMID- 10749553 TI - Future antibiotic trials. AB - The true value of antibiotics in acute exacerbations of chronic bronchitis remains to be defined. Although short-term benefits are generally accepted, if prescription volume is an indicator, there is little evidence to support their use in mild exacerbations.Thus, most therapeutic episodes are probably unnecessary and certainly encourage bacterial resistance. In more severe exacerbations, trials must be designed to validate the potential benefits-if they exist-of pharmacologically-improved antibiotics by demonstration of both short- and longer-term benefits. The relevance of standard bacteriological and short term clinical outcome assessments must be questioned. Alternative outcome measures, e.g., quality of life, may allow differentiation of antibiotic classes. Long-term assessment of symptom-free intervals, related to baseline status, might open pathways to the prevention of progressive, bacteria-initiated inflammatory damage to small airways. There is little excuse for continued ignorance of the nature of bacterial involvement in chronic bronchitis and the potential for more effective therapy. PMID- 10749554 TI - A 77-year-old man with bilateral pulmonary infiltrates and shortness of breath. PMID- 10749555 TI - Heterogeneity of dermal deposition of eosinophil granule major basic protein in acute lesions of atopic dermatitis. AB - Deposition of eosinophil granule major basic protein (MBP) often occurs in acute and chronic lesions of atopic dermatitis, but it is not clear what the factors may be that are related to the MBP deposition in some skin lesions of the disease. The purpose of this study was to determine whether a personal or family history of respiratory atopy is related to the intensity of MBP deposition in acute lesions. We immunohistochemically stained biopsy specimens from acute, non oozing indurated erythematous lesions of atopic dermatitis with BMK-13, a monoclonal antibody which recognizes MBP. The subjects were 40 adult patients with atopic dermatitis. Of the 40 patients, 22 had a personal history of respiratory atopy, 8 had a family history of respiratory atopy, and 10 had neither a personal nor a family history of respiratory atopy. Deposition of MBP was observed in the specimens from 24 (60%) of the 40 patients examined. Furthermore, there were great individual differences in the intensity of MBP deposition. A strong MBP deposition was often seen in specimens from patients with atopic dermatitis who had a personal or family history of respiratory atopy, but was absent in specimens from those patients with atopic dermatitis who had neither a personal nor a family history of respiratory atopy. We conclude that a strong MBP deposition seems to occur in acute lesions of those patients with atopic dermatitis who have a predisposition to respiratory atopy. PMID- 10749556 TI - Clinical significance of antinuclear matrix antibody in serum from patients with anti-U1RNP antibody. AB - Serum containing anti-U1RNP antibodies reacts with the nuclear matrix, the relatively insoluble component of the cell nucleus, in addition to U1RNP. In this study, we determine the serum titer and clinical correlations of antinuclear matrix antibodies in samples from patients with anti-U1RNP antibodies. The patients with anti-U1RNP antibodies were classified as having mixed connective tissue disease (MCTD, 15 patients), systemic sclerosis (SSc, 12 patients), systemic lupus erythematosus (SLE, 7 patients), and undifferentiated CTD (UCTD, 9 patients). Antinuclear matrix antibodies were detected using indirect immunofluorescence staining on HCl-treated HEp-2 cells. The antinuclear matrix antibody titer was significantly higher in serum from patients with MCTD or SSc than in serum from patients with SLE or UCTD. The antinuclear matrix antibody titer was significantly increased in serum from patients with sclerodactyly, pitting scars, contracture of the phalanges, and decreased carbon monoxide diffusion capacity. Thus, a higher titer of antinuclear matrix antibodies in serum from patients with anti-U1RNP antibodies may be associated with a clinical diagnosis of MCTD or SSc rather than a diagnosis of SLE or UCTD. PMID- 10749557 TI - Reduction of regrowing hair shaft size and pigmentation after ruby and diode laser treatment. AB - Laser pulses which selectively damage pigmented hair follicles are a useful treatment for hypertrichosis. Clinically, regrowing hairs are often thinner and lighter after treatment. In this study, hair shaft diameter and optical transmission (700 nm) were measured before and after ruby (694 nm) and diode (800 nm) laser irradiation. Hair was collected from 47 and 41 subjects treated with ruby (0.3 ms and 3 ms) and diode (10-20 ms) lasers, respectively. "Responders" were defined as subjects with significant long-term hair loss as determined by hair counts at 9 and/or 12 months after treatment. In ruby laser responders (34/47), regrowing hairs were significantly both thinner (decreased diameter) and lighter (increased transmission). In "nonresponders" (13/47), regrowing hairs were lighter, but not thinner. The regrowing hair shaft absorption coefficient (as calculated assuming Beer's law) was significantly decreased by 0.3 ms ruby laser treatment, but was not changed by 3 ms ruby laser or diode laser treatment. After diode laser treatment, 38 of the 41 subjects were responders and regrowing hairs were both thinner and lighter. These results show that laser treatments can affect structural recovery (size of hair), follicular pigmentation (hair absorption coefficient), or both. Regrowth of thinner hair (decreased shaft diameter) occurs in conjunction with actual loss of hair. After long pulses (3 ms ruby; diode), regrowing hair was thinner and also lighter to an extent related to the decrease in hair diameter. In contrast, short ruby laser pulses (0.3 ms) appeared to be capable of inhibiting follicular pigmentation per se, in addition to affecting the hair diameter. This may account for the complete regrowth of lighter hair in "nonresponders" treated with 0.3 ms pulses. Laser-induced reduction in hair diameter and/or pigmentation are both long-term responses which confer cosmetic benefits in addition to actual hair loss. PMID- 10749558 TI - Cathepsin A activity in primary and metastatic human melanocytic tumors. AB - Several lysosomal proteases including cathepsins B, D, H and L have been found to play a role in the metastasis of tumor cells. However, up to now no information on the role of cathepsin A, a lysosomal multifunctional peptidase, in the proliferative, invasive, and metastatic potential of malignant tumors has been available. In the present study we compared the activity of cathepsin A in lysates of 34 human melanocytic tumors: primary (n = 12) and metastatic (n = 5) malignant melanoma, dysplastic pigmented nevi (n = 6) and pigmented nevi without evidence of dysplastic melanocytes (n = 11). The carboxypeptidase activity of cathepsin A was assayed at pH 5.0 with its specific substrate Cbz-Phe-Ala. The amount of released C-terminal alanine was measured by the ninhydrin method. We found that lysates of primary malignant melanoma lesions exhibited significantly higher cathepsin A activity than lysates of dysplastic and normal pigmented nevi. The cathepsin A activity in lysates of metastatic lesions of malignant melanoma was significantly higher than in primary focus lysates. It seems that cathepsin A may play a role in malignant transformation and metastatic dissemination of malignant melanoma. PMID- 10749559 TI - Replication-selective microbiological agents: fighting cancer with targeted germ warfare. PMID- 10749561 TI - Replication-selective adenoviruses as oncolytic agents. PMID- 10749560 TI - Conditionally replicating herpes vectors for cancer therapy. PMID- 10749562 TI - Antagonists to the rescue. PMID- 10749563 TI - Umbilical cord blood: an expandable resource. PMID- 10749564 TI - Playing "telephone": bioactive lipids as mediators of intercompartmental communication in the alveolus. PMID- 10749565 TI - Leptin, nutrition, and the thyroid: the why, the wherefore, and the wiring. PMID- 10749566 TI - Bone morphogenetic protein 4 regulates the budding site and elongation of the mouse ureter. AB - In the normal mouse embryo, Bmp4 is expressed in mesenchymal cells surrounding the Wolffian duct (WD) and ureter stalk, whereas bone morphogenetic protein (BMP) type I receptor genes are transcribed either ubiquitously (Alk3) or exclusively in the WD and ureter epithelium (Alk6). Bmp4 heterozygous null mutant mice display, with high penetrance, abnormalities that mimic human congenital anomalies of the kidney and urinary tract (CAKUT), including hypo/dysplastic kidneys, hydroureter, ectopic ureterovesical (UV) junction, and double collecting system. Analysis of mutant embryos suggests that the kidney hypo/dysplasia results from reduced branching of the ureter, whereas the ectopic UV junction and double collecting system are due to ectopic ureteral budding from the WD and accessory budding from the main ureter, respectively. In the cultured metanephros deprived of sulfated glycosaminoglycans (S-GAGs), BMP4-loaded beads partially rescue growth and elongation of the ureter. By contrast, when S-GAGs synthesis is not inhibited, BMP4 beads inhibit ureter branching and expression of Wnt 11, a target of glial cell-derived neurotrophic factor signaling. Thus, Bmp4 has 2 functions in the early morphogenesis of the kidney and urinary tract. One is to inhibit ectopic budding from the WD or the ureter stalk by antagonizing inductive signals from the metanephric mesenchyme to the illegitimate sites on the WD. The other is to promote the elongation of the branching ureter within the metanephros, thereby promoting kidney morphogenesis. PMID- 10749567 TI - Altered focal adhesion regulation correlates with cardiomyopathy in mice expressing constitutively active rac1. AB - The ras family of small GTP-binding proteins exerts powerful effects upon cell structure and function. One member of this family, rac, induces actin cytoskeletal reorganization in nonmuscle cells and hypertrophic changes in cultured cardiomyocytes. To examine the effect of rac1 activation upon cardiac structure and function, transgenic mice were created that express constitutively activated rac1 specifically in the myocardium. Transgenic rac1 protein was expressed at levels comparable to endogenous rac levels, with activation of the rac1 signaling pathway resulting in two distinct cardiomyopathic phenotypes: a lethal dilated phenotype associated with neonatal activation of the transgene and a transient cardiac hypertrophy seen among juvenile mice that resolved with age. Neither phenotype showed myofibril disarray and hypertrophic hearts were hypercontractilein working heart analyses. The rac1 target p21-activated kinase translocated from a cytosolic to a cytoskeletal distribution, suggesting that rac1 activation was inducing focal adhesion reorganization. Corroborating results showed altered localizations of src in dilated cardiomyopathy and paxillin in both cardiomyopathic phenotypes. This study, the first examination of rac1 mediated cardiac effects in vivo, demonstrates that dilation and hypertrophy can share a common molecular origin and presents evidence that both timing and concurrent signaling from multiple pathways can influence cardiac remodeling. PMID- 10749568 TI - Pharmacological chaperones rescue cell-surface expression and function of misfolded V2 vasopressin receptor mutants. AB - Over 150 mutations within the coding sequence of the V2 vasopressin receptor (V2R) gene are known to cause nephrogenic diabetes insipidus (NDI). A large number of these mutant receptors fail to fold properly and therefore are not routed to the cell surface. Here we show that selective, nonpeptidic V2R antagonists dramatically increase cell-surface expression and rescue the function of 8 mutant NDI-V2Rs by promoting their proper folding and maturation. A cell impermeant V2R antagonist could not mimic these effects and was unable to block the rescue mediated by a permeant agent, indicating that the nonpeptidic antagonists act intracellularly, presumably by binding to and stabilizing partially folded mutants. In addition to opening new therapeutic avenues for NDI patients, these data demonstrate that by binding to newly synthesized mutant receptors, small ligands can act as pharmacological chaperones, promoting the proper folding and maturation of receptors and their targeting to the cell surface. PMID- 10749569 TI - Combined factor VII/protein C deficiency results in intrauterine coagulopathy in mice. AB - To determine whether an additional loss of the coagulation factor VII (FVII) gene influenced the coagulopathy observed in protein C gene-deficient (PC(-/-)) embryos and neonates, we crossed mice doubly heterozygous for the factor VII (FVII(+/-)) and protein C (PC(+/-)) genes to produce offspring possessing the 9 predicted genotypic combinations. FVII(-/-)/PC(-/-) embryos, although present at their expected Mendelian frequency, displayed a phenotype that had not been observed in either the FVII or PC singly deficient embryos. At E12.5 days postcoitum (dpc), FVII(-/-)/PC(-/-) embryos demonstrated an intra- and extravascular coagulopathy that progressed with substantial concomitant hemorrhage and peripheral edema by E17.5dpc, resulting in mortality immediately after birth. FVII(+/-)/PC(-/-) embryos showed a less severe phenotype, suggesting a gene dosage effect. The lack of rescue of PC(-/-) embryos and neonates and augmented coagulopathy resulting from an additional heterozygous or homozygous FVII deficiency are probably due to increased factor Xa and thrombin generation, resulting from loss of FVIIa-dependent tissue factor pathway inhibitor function and the absence of control at the levels of factors Va and VIIIa. The presence of fibrin in embryos in the absence of fetal FVII suggests that significant clot generating potential exists outside of the embryonic factor VII-dependent pathway. PMID- 10749570 TI - A novel signaling mechanism between gas and blood compartments of the lung. AB - Propagation of inflammatory signals from the airspace to the vascular space is pivotal in lung inflammation, but mechanisms of intercompartmental signaling are not understood. To define signaling mechanisms, we microinfused single alveoli of blood-perfused rat lung with TNF-alpha, and determined in situ cytosolic Ca(2+) concentration ([Ca(2+)](i)) by the fura-2 ratio method, cytosolic phospholipase A(2) (cPLA(2)) activation and P-selectin expression by indirect immunofluorescence. Alveolar TNF-alpha increased [Ca(2+)](i) and activated cPLA(2) in alveolar epithelial cells, and increased both endothelial [Ca(2+)](i) and P-selectin expression in adjoining perialveolar capillaries. All responses were blocked by pretreating alveoli with a mAb against TNF receptor 1 (TNFR1). Crosslinking alveolar TNFR1 also increased endothelial [Ca(2+)](i). However, the endothelial responses to alveolar TNF-alpha were blocked by alveolar preinjection of the intracellular Ca(2+) chelator BAPTA-AM, or the cPLA(2) blockers AACOCF(3) and MAFP. The gap-junction uncoupler heptanol had no effect. We conclude that TNF alpha induces signaling between the alveolar and vascular compartments of the lung. The signaling is attributable to ligation of alveolar TNFR1 followed by receptor-mediated [Ca(2+)](i) increases and cPLA(2) activation in alveolar epithelium. These novel mechanisms may be relevant in the alveolar recruitment of leukocytes. PMID- 10749572 TI - Salt-sensitive hypertension in endothelin-B receptor-deficient rats. AB - The role of the endothelin-B receptor (ET(B)) in vascular homeostasis is controversial because the receptor has both pressor and depressor effects in vivo. Spotting lethal (sl) rats carry a naturally occurring deletion in the ET(B) gene that completely abrogates functional receptor expression. Rats homozygous for this mutation die shortly after birth due to congenital distal intestinal aganglionosis. Genetic rescue of ET(B)(sl/sl) rats from this developmental defect using a dopamine--hydroxylase (DBH)-ET(B) transgene results in ET(B)-deficient adult rats. On a sodium-deficient diet, DBH-ET(B);ET(B)(sl/sl) and DBH ET(B);ET(B)(+/+) rats both exhibit a normal arterial blood pressure, but on a high-sodium diet, the former are severely hypertensive. We find no difference in plasma renin activity or plasma aldosterone concentration between salt-fed wild type, DBH-ET(B);ET(B)(+/+) or DBH-ET(B);ET(B)(sl/sl) rats, and acute responses to intravenous L-NAME and indomethacin are similar between DBH-ET(B);ET(B)(sl/sl) and DBH-ET(B);ET(B)(+/+) rats. Irrespective of diet, DBH-ET(B);ET(B)(sl/sl) rats exhibit increased circulating ET-1, and, on a high-sodium diet, they show increased but incomplete hypotensive responses to acute treatment an ET(A) antagonist. Normal pressure is restored in salt-fed DBH-ET(B);ET(B)(sl/sl) rats when the epithelial sodium channel is blocked with amiloride. We conclude that DBH-ET(B);ET(B)(sl/sl) rats are a novel single-locus genetic model of severe salt sensitive hypertension. Our results suggest that DBH-ET(B);ET(B)(sl/sl) rats are hypertensive because they lack the normal tonic inhibition of the renal epithelial sodium channel. PMID- 10749571 TI - Osteopenia and decreased bone formation in osteonectin-deficient mice. AB - Bone continuously remodels in response to mechanical and physiological stresses, allowing vertebrates to renew bone as adults. Bone remodeling consists of the cycled synthesis and resorption of collagenous and noncollagenous extracellular matrix proteins, and an imbalance in this process can lead to disease states such as osteoporosis, or more rarely, osteopetrosis. There is evidence that the extracellular matrix glycoprotein osteonectin or secreted protein acidic and rich in cysteine (BM-40) may be important in bone remodeling. Osteonectin is abundant in bone and is expressed in areas of active remodeling outside the skeleton. In vitro studies indicate that osteonectin can bind collagen and regulate angiogenesis, metalloproteinase expression, cell proliferation, and cell-matrix interactions. In some osteopenic states, such as osteogenesis imperfecta and selected animal models for bone fragility, osteonectin expression is decreased. To determine the function of osteonectin in bone, we used contact x-ray, histomorphometry, and Northern blot analysis to characterize the skeletal phenotype of osteonectin-null mice. We found that osteonectin-null mice have decreased bone formation and decreased osteoblast and osteoclast surface and number, leading to decreased bone remodeling with a negative bone balance and causing profound osteopenia. These data indicate that osteonectin supports bone remodeling and the maintenance of bone mass in vertebrates. PMID- 10749573 TI - Insulin receptor substrate-1 in osteoblast is indispensable for maintaining bone turnover. AB - Insulin receptor substrates (IRS-1 and -2) are essential for intracellular signaling by insulin and IGF-I, anabolic regulators of bone metabolism. Mice lacking the IRS-1 gene IRS-1(-/-) showed severe osteopenia with low bone turnover. IRS-1 was expressed in osteoblasts, but not in osteoclasts, of wild type (WT) mice. IRS-1(-/-) osteoblasts treated with insulin or IGF-I failed to induce tyrosine phosphorylation of cellular proteins, and they showed reduced proliferation and differentiation. Osteoclastogenesis in the coculture of hemopoietic cells and osteoblasts depended on IRS-1 expression in osteoblasts and could not be rescued by IRS-1 expression in hemopoietic cells in the presence of not only IGF-I but also 1,25(OH)(2)D(3). In addition, osteoclast differentiation factor (RANKL/ODF) was not induced by these factors in IRS-1(-/-) osteoblasts. We conclude that IRS-1 deficiency in osteoblasts impairs osteoblast proliferation, differentiation, and support of osteoclastogenesis, resulting in low-turnover osteopenia. Osteoblastic IRS-1 is essential for maintaining bone turnover, because it mediates signaling by IGF-I and insulin and, we propose, also by other factors, such as 1,25(OH)(2)D(3). PMID- 10749574 TI - Lymph node trafficking and antigen presentation by endobronchial eosinophils. AB - Because eosinophils recruited into the airways in allergic diseases are exposed to inhaled allergens, we evaluated whether eosinophils within the endobronchial lumen can function in vivo as antigen-presenting cells for inhaled antigens. We recovered eosinophils from the airways after aerosol antigen challenge in sensitized mice or from the peritoneal cavities of IL-5 transgenic mice and fluorescently labeled these cells ex vivo. These labeled cells, instilled intratracheally into normal mice, migrated into draining paratracheal lymph nodes and localized to T cell-rich paracortical areas. The homing of airway eosinophils to lymph nodes was not governed by eotaxin, because CCR3(-/-) and CCR3(+/+) eosinophils migrated identically. Airway eosinophils, recovered after inhalational antigen challenge in sensitized mice, expressed MHC class II and costimulatory CD80 and CD86 proteins and functioned in vitro as CD80- and CD86 dependent, antigen-specific, antigen-presenting cells. Moreover, when instilled into the airways of antigen-sensitized recipient mice, airway eosinophils recovered after inhalational antigen challenge stimulated antigen-specific CD4(+) T cell proliferation within paratracheal lymph nodes. Thus, eosinophils within the lumina of airways can process inhaled antigens, traffic to regional lymph nodes, and function in vivo as antigen-presenting cells to stimulate responses of CD4(+) T cells. PMID- 10749575 TI - Regulation of pancreatic PC1 and PC2 associated with increased glucagon-like peptide 1 in diabetic rats. AB - The pancreatic processing enzymes, PC1 and PC2, convert proinsulin to insulin and convert proglucagon to glucagon and glucagon-like peptide 1 (GLP-1). We examined the effect of streptozotocin (STZ) treatment on the regulation of these enzymes and the production of insulin, glucagon, and GLP-1 in the rat. Pancreatic PC1 and PC2 mRNA increased >2-fold and >4-fold, respectively, in rats receiving intraperitoneal STZ (50 mg/kg) daily for 5 days. Immunocytochemistry revealed that, although pancreatic islet cells in the STZ-treated rats were sparse and atrophic PC1, PC2, glucagon, and GLP-1 immunoreactivity increased dramatically in the remaining islet cells. Heightened PC1 and PC2 expression was seen in cells expressing glucagon but not in insulin-expressing cells. Furthermore, in STZ treated rats, bioactive GLP-1(7-36 amide) accumulated in pancreatic extracts and serum 3- and 2.5-fold, respectively, over control animals. This treatment also caused a 2-fold increase in the ratio of amidated forms of GLP-1 immunoreactivity to total glucagon immunoreactivity in the pancreas but did not affect the ratio of proinsulin to insulin. We conclude that hyperglycemic rats have an increased expression of prohormone converting enzymes in islet alpha cells, leading to an increase in amidated GLP-1, which can then exert an insulinotropic effect on the remaining beta cells. PMID- 10749576 TI - Glatiramer acetate (Copaxone) induces degenerate, Th2-polarized immune responses in patients with multiple sclerosis. AB - We examined the effect of glatiramer acetate, a random copolymer of alanine, lysine, glutamic acid, and tyrosine, on antigen-specific T-cell responses in patients with multiple sclerosis (MS). Glatiramer acetate (Copaxone) functioned as a universal antigen, inducing proliferation, independent of any prior exposure to the polymer, in T-cell lines prepared from MS or healthy subjects. However, for most patients, daily injections of glatiramer acetate abolished this T-cell response and promoted the secretion of IL-5 and IL-13, which are characteristic of Th2 cells. The surviving glatiramer acetate-reactive T cells exhibited a greater degree of degeneracy as measured by cross-reactive responses to combinatorial peptide libraries. Thus, it appears that, in some individuals, in vivo administration of glatiramer acetate induces highly cross-reactive T cells that secrete Th2 cytokines. To our knowledge, glatiramer acetate is the first agent that suppresses human autoimmune disease and alters immune function by engaging the T-cell receptor. This compound may be useful in a variety of autoimmune disorders in which immune deviation to a Th2 type of response is desirable. PMID- 10749577 TI - A chemokine-to-cytokine-to-chemokine cascade critical in antiviral defense. AB - Macrophage inflammatory protein 1alpha (MIP-1alpha) promotes natural killer (NK) cell inflammation in livers during murine cytomegalovirus (MCMV) infections, and NK cell-produced interferon gamma (IFN-gamma) contributes to defense against MCMV infections. A specific role for local NK cell IFN-gamma production, however, has not been established. The importance of MIP-1alpha and NK cell-produced IFN-gamma in shaping endogenous immune responses and defense in different compartments was examined. MIP-1alpha deficiency profoundly decreased resistance to MCMV and was associated with dramatically reduced NK cell accumulation and IFN-gamma production in liver. MIP-1alpha-independent IFN-gamma responses were observed in serum and spleen, and infection-induced elevations in blood NK cell populations occurred in absence of the factor, but peak liver expression of another chemokine, the monokine induced by IFN-gamma (Mig), depended upon presence of MIP 1alpha, NK cells, and IFN-gamma. The Mig response was also important for viral resistance. Thus, serum cytokine responses are insufficient; MIP-1alpha is critical for NK cell migration and IFN-gamma delivery to mediate protection; and Mig induction in tissues is a downstream protective response resulting from the process. These results define a critical chemokine-to-cytokine-to-chemokine cascade required for defense during a viral infection establishing itself in tissues. PMID- 10749578 TI - A stable latent reservoir for HIV-1 in resting CD4(+) T lymphocytes in infected children. AB - HIV-1 persists in a latent state in resting CD4(+) T lymphocytes of infected adults despite prolonged highly active antiretroviral therapy (HAART). To determine whether a latent reservoir for HIV-1 exists in infected children, we performed a quantitative viral culture assay on highly purified resting CD4(+) T cells from 21 children with perinatally acquired infection. Replication-competent HIV-1 was recovered from all 18 children from whom sufficient cells were obtained. The frequency of latently infected resting CD4(+) T cells directly correlated with plasma virus levels, suggesting that in children with ongoing viral replication, most latently infected cells are in the labile preintegration state of latency. However, in each of 7 children who had suppression of viral replication to undetectable levels for 1-3 years on HAART, latent replication competent HIV-1 persisted with little decay, owing to a stable reservoir of infected cells in the postintegration stage of latency. Drug-resistance mutations generated by previous nonsuppressive regimens persisted in this compartment despite more than 1 year of fully suppressive HAART, rendering untenable the idea of recycling drugs that were part of failed regimens. Thus the latent reservoir for HIV-1 in resting CD4(+) T cells will be a major obstacle to HIV-1 eradication in children. PMID- 10749579 TI - The central melanocortin system affects the hypothalamo-pituitary thyroid axis and may mediate the effect of leptin. AB - Prolonged fasting is associated with a downregulation of the hypothalamo pituitary thyroid (H-P-T) axis, which is reversed by administration of leptin. The hypothalamic melanocortin system regulates energy balance and mediates a number of central effects of leptin. In this study, we show that hypothalamic melanocortins can stimulate the thyroid axis and that their antagonist, agouti related peptide (Agrp), can inhibit it. Intracerebroventricular (ICV) administration of Agrp (83-132) decreased plasma thyroid stimulating hormone (TSH) in fed male rats. Intraparaventricular nuclear administration of Agrp (83 132) produced a long-lasting suppression of plasma TSH, and plasma T4. ICV administration of a stable alpha-MSH analogue increased plasma TSH in 24-hour fasted rats. In vitro, alpha-MSH increased thyrotropin releasing hormone (TRH) release from hypothalamic explants. Agrp (83-132) alone caused no change in TRH release but antagonized the effect of alpha-MSH on TRH release. Leptin increased TRH release from hypothalami harvested from 48-hour-fasted rats. Agrp (83-132) blocked this effect. These data suggest a role for the hypothalamic melanocortin system in the fasting-induced suppression of the H-P-T axis. PMID- 10749580 TI - Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor. AB - Here, we demonstrate a significant ex vivo expansion of human hematopoietic stem cells capable of repopulating in NOD/SCID mice. Using a combination of stem cell factor (SCF), Flk2/Flt3 ligand (FL), thrombopoietin (TPO), and a complex of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R), we cultured cord blood CD34(+) cells for 7 days and transplanted these cells into NOD/SCID mice. Bone marrow engraftment was judged successful when recipient animals contained measurable numbers of human CD45(+) cells 10-12 weeks after transplantation. When cells were cultured with SCF+FL+TPO+IL-6/sIL-6R, 13 of 16 recipients were successfully engrafted, and CD45(+) cells represented 11.5% of bone marrow cells in engrafted recipients. Cells cultured with a subset of these factors were less efficiently engrafted, both as measured by frequency of successful transplantations and prevalence of CD45(+) cells. In animals receiving cells cultured with all 4 factors, human CD45(+) cells represented various lineages, including a large number of CD34(+) cells. The proportion of CD45(+) cells in recipient marrow was 10 times higher in animals receiving these cultured cells than in those receiving comparable numbers of fresh CD34(+) cells, and the expansion rate was estimated at 4.2-fold by a limiting dilution method. Addition of IL-3 to the cytokine combination abrogated the repopulating ability of the expanded cells. The present study may provide a novel culture method for the expansion of human transplantable hematopoietic stem cells suitable for clinical applications. PMID- 10749581 TI - Cellular and molecular mechanisms of aging and age related diseases. AB - This review on aging is focused on those cellular and molecular mechanisms which concern age related pathologies. The central question addressed is the relationship between normal aging and age-related pathologies such as osteoarthritis, cardiovascular diseases, emphysema, malignant tumors and cognitive decline, dementias. The mechanisms recognized as most important in cell and tissue aging are briefly outlined. Emphasis is laid on the importance of post synthetic modifications of the macromolecules of the extracellular matrix and on cell matrix interactions. Loss of intercellular communication and cell-matrix interactions as a result of receptor decay and receptor uncoupling were recently recognized as key events. Unavoidable poly-pathology at advanced age may be the answer to the above question. PMID- 10749582 TI - Phenotypic alterations in Kaposi's sarcoma cells by antisense reduction of perlecan. AB - Metastasis is a sequence of events including proliferation, migration, adhesion, invasion and subsequent metastatic growth of tumour cells in distant organs. We previously showed that highly metastatic variants of murine melanoma cells express higher levels of the basement membrane proteoglycan perlecan than low or non metastatic variants and expression of an antisense perlecan can reduce metastatic potential. In contrast, antisense expression of perlecan in fibrosarcoma cells was reported to enhance tumorigenesis. To better understand the role of perlecan in angiogenesis we have transfected KS-IMM, an immortalized cell line derived from a human Kaposi s sarcoma, with an antisense perlecan construct and investigated the positive/negative role of perlecan in KS. KS-IMM cells were transfected with either empty vector (neo) or the antisense perlecan construct and clones were isolated. Immuno-blot analysis showed a reduction of perlecan levels in two (AP3 and AP4) isolated clones, in Northern blot analysis endogenous perlecan was undetectable in the AP3 and AP4 clones, while it was present in the neo control clones. AP clones had a reduced migration to HGF in Boyden chambers as compared to neo clones. Proliferation in low serum or serum free conditions was strongly reduced in the AP clones as compared to the neo control cells. The neotransfected cells showed rapid proliferation in low serum supplemented with HGF and VEGF, while antisense transfected clones showed little response. Finally, AP-trasfected KS-IMM cells had significantly reduced migration to VEGF and HGF with respect to controls. In contrast, when the AP transfected cells were injected in nude mice they paradoxically showed enhanced tumor growth as compared to controls. Our preliminary data indicate that perlecan reduction plays a crucial role on Kaposi s sarcoma cell migration and proliferation in vitro. However, in vivo KS-IMM depleted of perlecan had a growth advantage. A possible hypothesis is that perlecan is necessary for growth of KS-IMM cells in vitro, however its down-regulation might promote angiogenesis through increased angiogenic growth factor diffusion, resulting in enhanced tumor growth in vivo. PMID- 10749583 TI - Detection of TNFalpha expression in the bone marrow and determination of TNFalpha production of peripheral blood mononuclear cells in myelodysplastic syndrome. AB - TNFalpha is a highly active cytokine which plays an important role in the regulation of apoptotic cell death, a mechanism involved in the pathophysiology of myelodysplastic syndrome (MDS). In this study we investigated the expression of TNFalpha of the bone marrow trephine biopsies by immunohistochemical method and the TNFalpha production of peripheral blood mononuclear cells by ELISA method in 15 patients affected by MDS. Five of seven patients without excess of blasts showed high or intermediate TNFalpha expression in the bone marrow biopsies, whereas two patients with excess of blasts were negative and one had low expression. The five CMML patients revealed low or intermediate expression. The production of TNFalpha by the PBMC was analysed in 10 patients, four patients with RA and two with CMML produced higher level of TNFalpha which increased after stimulation with phorbol myristic acetate, but none of the RAEB patients revealed increase in TNFalpha production. In conclusion we suppose that increased TNFalpha expression and production by PBMC may be an indirect evidence of the role of increased apoptosis in low risk MDS patients. PMID- 10749584 TI - High production of SPARC/osteonectin/BM-40 in mouse metastatic B16 melanoma cell lines. AB - Production of SPARC/osteonectin/BM-40 was determined in mouse B16 melanoma clones BL6 and F10 (high metastatic) and F1 (low metastatic). SPARC was produced greater amount in BL6 and F10 than in F1 cells, showing a good agreement with their metastatic potentials. Moreover, SPARC production was not influenced by culture pH, even in the acidic conditions (= pH 5.9). Although tumor tissues show often low pH due to excessive amount of acidic metabolites such as lactate, most studies have been done in neutral pH. High SPARC production in the acidic medium, therefore, is thought to be an important potential for tumor invasive behaviour. PMID- 10749585 TI - Unique morphological alterations of the HTLV-I transformed C8166 cells by infection with HIV-1. AB - C8166 cells express T lymphocyte markers, a monocyte-specific esterase, taxpolypeptide of HTLV-I. In spite of this transactivator, their HIV-1 yield is low. Their culture conditions were modified, and infected cells were immobilized on a poly-L-lysine sheet under semisolid overlays to study their phenotypic alterations and HIV-1 production by microscopy and electron microscopy. Another lymphoid cultures (MT-4, CEM, CEM-ss, AdCEM) similarly treated were infected with either HIV-1/RF or IIIB. Specificity of HIV-1 was compared to the effects of vesicular stomatitis virus (VSV). Unlike other cultures, HIV-1/RF infected C8166 cells in Eagle s MEM exhibited surface projections resembling hairy leukemia cells, which was followed by balloon degeneration and apoptosis. Immobilized HIV 1 infected cultures formed flat syncytia with several interdigitating dendritic projections. Syncytia shrunk with condensed nuclear material and axon-like filaments characteristic for infected macrophages. VSV induced enlargement and necrotic lysis of all cell types. Early postinfection with HIV-1, electron microscopy revealed irreversible membrane fusion above cell nuclei, and transient fusion between filaments. Transient presence of coated vesicles containing intact HIV-1 particles, Birbeck granule-like structures of Langerhans cells, fibrillar lamellar structures resembling hairy leukemia or Sezary cells were detected. Late postinfection, high proportion of HIV-1 bud from polarized cytoplasm was empty particle, while that bud and entrapped in cytoplasmic vacuoles contained two or multiple cores in a fused envelope. The effect of early gene products of HIV-1 on HTLV-I and C8166 cells might elicit their latent potentials for monocyte or interdigitating dendritic cells, while in the later phase HTLV-I products might alter HIV-1 virion assembly. PMID- 10749586 TI - CRP, TNF-alpha, IL-1ra, IL-6, IL-8 and IL-10 in blood serum of colorectal cancer patients. AB - Blood serum cytokines: TNFalpha, IL-1ra, IL-6, IL-8, IL-10 as well as CRP were investigated in patients with colorectal cancer, prior treatment and 1, 10 and 42 days after surgery. There was an increase of the levels of CRP, IL-6 and IL-10 in most patients 24 hours after surgery. The levels of IL-1ra were elevated in patients in stage C and in several patients in stage B of the disease and there was a decrease of circulating TNFalpha in stage B patients. On day 10 and 42 after surgery, the levels of cytokines followed various patterns. PMID- 10749587 TI - Serum levels of the soluble adhesion molecules in patients with malignant melanoma. AB - The incidence of malignant melanoma has been steadily increasing over the past decades. CD 44 is a transmembrane glycoprotein which is implicated in a number of adhesive and migratory events. Downregulation of CD 44 is implicated in the metastatic process. P-Selectin is a member of the selectin family of cell surface molecules. The levels of P-Selectin in biological fluids may be elevated in subjects with a variety of pathological conditions. In malignant melanoma, elevation of the plasma level of soluble intercellular adhesion molecule-1 (sICAM 1) has been associated with a reduction in disease-free survival. This study was performed to investigate the differences in the serum concentrations of the adhesion molecules in patients with malignant melanoma. The study group consisted of 52 patients with malignant melanoma and 20 healthy subjects. No meaningful difference was observed for P-selectin and sICAM 1 levels. A statistically significant decrease was observed in the cancer patients for serum CD 44 levels. PMID- 10749588 TI - A comparative survival evaluation and assessment of interclassification concordance in adult supratentorial astrocytic tumors. AB - Classification and grading of astrocytic tumors has been the subject of several controversies and no universally accepted classification system is yet available. Nevertheless, acceptance of a common system is important for assessing prognosis as well as easy comparative evaluation and interpretation of the results of multi center therapeutic trials. We report the results of a single center study on comparative survival evaluation along with assessment of inter-classification concordance in 102 cases of supratentorial astrocytic tumors in adults ((3) (3)16 years of age). Hematoxylin and eosin (H&E) stained slides of these 102 cases were reviewed independently by two pathologists and each case classified or graded according to four different classification systems viz. Kernohan, Daumas-Duport (SAM-A), TESTAST-268 and WHO. The histological grading was then correlated with the survival curves as estimated by the Kaplan-Meier method. The most important observation was that similar survival curves were obtained for any one grade of tumor by all the four classification systems. Fifty three of the 102 cases (51.9%) showed absolute grading concordance using all 4 classifications with maximum concordant cases belonging to grades 2 and 4. Intra-classification grade wise survival analysis revealed a statistically significant difference between grade 2 and grades 3 or 4, but no difference between grades 3 and 4 in any of the classification systems. It is apparent from the results of this study that if specified criteria related to any of the classification systems is rigorously adhered to, it will produce comparable results. Hence, preferential adoption of any one classification system in practice will be guided by the relative ease of histologic feature value evaluation with maximum possible objectivity and reproducibility. We recommend the Daumas-Duport (SAM-A) system since it appears to be the simplest, most objectivized for practical application and highly reproducible with relative ease. PMID- 10749589 TI - Possibilities of preventing osteoradionecrosis during complex therapy of tumors of the oral cavity. AB - In recent years, there has been a dramatic increase in the number of tumors of the head and neck. Their successful treatment is one of the greatest challenges for physicians dealing with oncotherapy. An organic part of the complex therapy is preoperative or postoperative irradiation. Application of this is accompanied by a lower risk of recurrences, and by a higher proportion of cured patients. Unfortunately, irradiation also has a disadvantage: the development of osteoradionecrosis, a special form of osteomyelitis, in some patients (mainly in those cases where irradiation occurs after bone resection or after partial removal of the periosteum). Once the clinical picture of this irradiation complication has developed, its treatment is very difficult. A significant result or complete freedom from complaints can be attained only rarely. Attention must therefore be focussed primarily on prevention, and the oral surgeon, the oncoradiologist and the patient too can all do much to help prevent the occurrence of osteoradionecrosis. Through coupling of an up-to-date, functional surgical attitude with knowledge relating to modern radiology and radiation physics, the way may be opened to forestall this complication that is so difficult to cure. PMID- 10749590 TI - Microvessel count, proliferating cell nuclear antigen and Ki-67 indices in gastric adenocarcinoma. AB - The aim of the present study was to immunohistochemically investigate the prognostic value of neovascularization (expressed as microvessel count-MVC) and tumor cell proliferation (expressed as PCNA labeling index PLI and Ki-67 labeling index KLI) in gastric adenocarcinoma. Correlations with clinicopathologic features were also evaluated. Tumor specimens from 74 patients diagnosed as gastric adenocarcinoma were included in this study. Formalin fixed, paraffin embedded tissue sections stained immunohistochemically with F-VIII, PC10 and MIB 1 monoclonal antibodies. By ocular grid subdivided into 100 areas, number of microvessels and PC10, MIB-1 positive and negative cells were counted at x400 magnification. Chi-square test, Kaplan-Meier method and cox regression analysis were used for statistical analysis. The results showed that, MVC and PLI had a significant correlation with invasion and lymph node metastasis. The prognosis was significantly worse in patients with high MVC (>14 ) and with high PLI (>49%). However any relationship was not observed between KLI (38%) and clinicopathologic parameters, so KLI failed to predict the prognosis. Cox model showed that, MVC and PLI were independent prognostic variables. Ki-67 labeling index in gastric carcinomas has no prognostic relevance. However, the evaluation of microvessel count and proliferating cell nuclear antigen index in gastric carcinomas could be reliable indicators of prognosis. PMID- 10749591 TI - Overexpression of cyclin D1 mRNA in colorectal carcinomas and relationship to clinicopathological features: an in situ hybridization analysis. AB - Increased expression of a key cell cycle regulator, cyclin D1, may have relevance to carcinogenesis and clinicopathological characteristics of some cancers. This study represents the first application of in situ hybridization, ISH, to detect cyclin D1 mRNA in tissue sections from colorectal carcinomas. This approach was selected because of its unique potential to clarify whether increased expression of cyclin D1 mRNA correlates with clinical and pathological parameters. The ISH ofa non-radioactive oligonucleotide probe (Biogenex) was immunocytochemically detected in paraffin embedded sections from biopsy or resection specimens. Tumors ranged from well to poorly differentiated, and from stages A, B, C, and D. Ten year survival data were available on the majority of patients. Intensity of tumor and background (smooth muscle) signals were independently scored from 0 to 3. Overexpressed cyclin D1 mRNA was seen in 86% of cases compared to background. This frequency is similar to that reported for pancreatic carcinoma. The average signal intensity score in tumor foci was 1.9 with a background score of 0.05 (p<001). All cases showed specific staining judged by the cytoplasmic localization and a tumor signal:background ratio >1. Expression did not differentiate cancers based on grade, stage or survival (p>1), but did differentiate carcinoma and severe dysplasia from mild dysplasia. We conclude that ISH of cyclin D1 mRNA is an effective and relatively specific means of detecting activity of this gene in colonic neoplasms. The high frequency of overexpression implies that gene activity by itself is not likely to predict a tumor s biological or clinical behavior. On the other hand, these data suggest that increased cyclin D1 gene activity may be an early event in colorectal carcinogenesis. They also are consistent with findings showing cyclin D1 is inducible by a variety of oncogene products. PMID- 10749593 TI - Is 1-week treatment for peptic ulcer healing sufficient and safe? AB - OBJECTIVE: To confirm whether 1-week anti-Helicobacter therapy to achieve ulcer healing is sufficient and safe. METHODS: We retrospectively analyzed patients with peptic ulcer who were infected with Helicobacter pylori and treated with 3 different 7-day regimens, according to predefined protocols in 3 different centers in the same geographical area (Aragon, Spain). Three combinations commonly described in the literature were used: a) omeprazole (40 mg/24 h), tetracycline hydrochloride (2 g/24 h), colloidal bismuth subcitrate (480 mg/24 h) and metronidazole (750 mg/24 h) (OBTM, n = 105); b) omeprazole (40 mg/24 h), clarithromycin (1.5 g/24 h) and amoxicillin (3 g/24 h) (O40C1.5A3, n = 13); and c) omeprazole (40 mg/24 h), clarithromycin (1 g/24 h) and amoxicillin (2 g/24 h) (O40C1A2, n = 4). In all patients the diagnosis of peptic ulcer disease was confirmed endoscopically, and H. pylori infection was verified with urease testing and histological analysis. After treatment ended, no other antacids were allowed until after endoscopic examination to check eradication and ulcer healing. RESULTS: 122 patients were included (107 with duodenal ulcer, 12 with gastric ulcer and 3 with both). Compliance was good and side effects infrequent and mild. Eradication rates were 88.5% (93/105) in the OBTM group, 100% (13/13) with O40C1.5A3, and 75% (3/4) with O40C1A2. Healing was achieved in 98.16% (107/109) of the patients in whom the bacterial infection was eradicated, and in 23.07% (3/13) of those in whom it was not (p < 0.0001). No patient had any complications during the period without treatment. CONCLUSIONS: 1-week eradication therapy with previously described combinations commonly used in clinical practice achieves high ulcer healing rates with no complications in the period without antacid treatment. We consider that it is not necessary, at least in most patients, to prolong antacid therapy. PMID- 10749594 TI - Stromal tumors of the stomach. Review of our experience and reclassification of a series of patients. AB - OBJECTIVE: To retrospectively review a series of 12 patients operated on in our department for stromal tumor of the stomach. Clinical and morphological data, and the patients' postoperative course, were analyzed. METHODS: Medical records for 12 patients (mean age 63.3 years) were retrospectively reviewed to obtain data on clinical presentation, diagnosis and treatment. Surgical morbidity and mortality were analyzed. A pathologist reviewed the resected specimens to determine the morphological factors of prognostic value. The biological nature of the tumor was reclassified based exclusively on mitotic index, and all tumors were staged according to the TGM system. Recurrence and survival rates were also calculated. RESULTS: The most frequent clinical presentation was abdominal pain and gastrointestinal bleeding. The most sensitive diagnostic methods were computerized tomography and echographic endoscopy. Operability and resectability rates were 100% and 91.6% respectively. Local resection was done in 5 patients, partial gastrectomy in 5, and extended total gastrectomy in 1. Histologically, 6 cases were muscular tumors (2 leiomyomas, 3 low-grade leiomyosarcomas and 1 high grade leiomyosarcoma), 2 were gastrointestinal autonomic nerve (GAN) tumors, and 4 were pure stomal tumors. The morbidity rate was 33.3% and the mortality rate was 8.3% (1 patient). All patients were followed up: 1 patient each died after 9 months and 4 years, 1 developed liver metastases after a disease-free interval of 14 months, and the other 9 patients were still alive and free of disease after intervals ranging from 4 months to 7 years. CONCLUSIONS: Stromal tumors include a group of tumors which may present muscular differentiation (the most frequent type), neural differentiation (GAN tumors) or no differentiation at all (pure stromal tumors). The mitotic index is the most valid parameter to determine biological nature, considering that classification as a benign tumor requires the total absence of mitoses. Treatment was mostly surgical, and local resection with adequate safety margins was effective. Prognosis was relatively good, but long term follow-up is needed to assess the malignant potential of these tumors. PMID- 10749592 TI - Papillary microcarcinoma of the thyroid gland in renal transplant patients. AB - Among organ transplant recipients there is a world wide increase in the number of de novo tumors as well as a decrease in the time of the first appearance after the transplantation. Between 1973 and the 31st of August 1999 1709 cadaver renal allograft transplantations were performed in our Department. Four thyroid cancers were detected among the renal transplanted patients. Two of them proved to be papillary microcarcinomas. Although the elevated risk of thyroid cancers is well established in the literature papillary microcarcinomas have never been reported before in an immunosuppressed patient. Authors highlight that the thyroid gland should always be carefully checked in organ transplant recipients, since better survival might be achieved even in the immunosuppressed population. Metastatic tumor is relatively benign which is in correlation with the literature, but there has been little experience in organ transplanted patients so far. PMID- 10749595 TI - Laboratory evaluation of the effect of prophylaxis for thromboembolism with fractionated heparin in patients undergoing laparoscopic cholecystectomy. AB - OBJECTIVE: We studied the effect of prophylaxis for thromboembolism with low molecular-weight heparin (LMWH) during hospitalization on the biological hemostasis system in patients who had undergone laparoscopic cholecystectomy. METHODS: This was a prospective paired cohort study without a control group (i.e., a before-after study). The subjects were 20 patients operated on laparoscopically for uncomplicated cholelithiasis. All patients received LMWH 2 h before the operation and 24 h after the first dose. Mean duration of surgery was 70 min. Pneumoperitoneum was accomplished at 14 mmHg, and all patients were operated on in the inverted Trendelenberg position (30 degrees). Patients were mobilized within 24 h, and were discharged within 48 h after surgery. As parameters of hemostasis we studied anti-Xa factor activity (anti-Xa), antithrombin III (AT III), partial active thromboplastin time (PTT) and fibrinogen. Samples were taken for laboratory analyses under basal conditions the day before the operation (first determination), 1 h after the first preoperative dose of LMWH was given (second), at the end of the operation (third), 24 h after surgery (fourth), and on postoperative day 7 (fifth). RESULTS: Mean basal values of all parameters were within the normal range. Mean anti-Xa activity was significantly higher in the second and third determinations than in the first and fifth measurements (p < 0.05). Mean PTT was significantly elevated on the second determination and decreased thereafter; however, none of the results differed significantly from the normal value. Mean AT III was significantly lower in the third determination in comparison with the first and fifth measurements. Fibrinogen was significantly higher in the fourth and fifth determinations than in the second and third measurements. Among all parameters and sampling times, the only values outside the normal range were anti-Xa activity on the second, third and fourth determinations. CONCLUSIONS: Plasma anti-Xa factor activity was increased preoperatively, and remained elevated for 24 h after surgery, returning to basal values on postoperative day 7. Partial thromboplastin time was slightly prolonged after the first dose of LMWH, indicating good antithrombotic action. PMID- 10749596 TI - Perianastomotic colonic tumors after inclusion of titanium or Lactomer in the anastomotic suture line. An experimental study in rats. AB - OBJECTIVE: The aim of the study was to find out if there are differences in the incidence of colonic tumors at the anastomosis after the inclusion of titanium or the absorbable material Lactomer in the anastomotic suture line. METHODS: Experimental study with 30 male Sprague-Dawley rats, assigned to 1 of 3 study groups: control (colonic anastomosis with interrupted suture); inclusion of titanium (8 mg) in the anastomotic suture line; and inclusion of Lactomer (8 mg) in the anastomotic line. After pharmacological carcinogenesis with 1-2 dimethylhydrazine, perianastomotic tumors were studied in the 20th postoperative week. RESULTS: The inclusion of titanium in the anastomotic line led to more tumors, a larger anastomotic tumoral area and a larger percentage of tumoral area than Lactomer (p < 0.05). The inclusion of Lactomer may have a protective effect against the induction of cancer in the anastomotic area. CONCLUSIONS: Titanium, a material used in mechanical instruments for digestive tract anastomoses, is not an innocuous material. Its presence in the anastomotic line can promote colonic carcinogenesis after induction. The use of mechanical staplers for colonic anastomoses should be relegated to difficult anastomoses that cannot be sewn manually. PMID- 10749597 TI - [Colonic involvement in the Klippel-Trenaunay-Weber syndrome]. AB - We report the case of a 12-year-old boy, diagnosed of Klippel Trenaunay Weber syndrome, with hemangiomas and venous varicosities in the right leg, who complaint about incidental hematochezia. In the colonoscopy some violet and plain angiodysplastic lesions like in the skin, were observed in rectum and distal sigmoid colon. We think this case is interesting, because of the unusual affectation of the gut in this disease and the importance of considering the endoscopic procedures in the evaluation of these patients. PMID- 10749598 TI - [An aneurysm of the gastroduodenal artery: its treatment via selective embolization]. PMID- 10749599 TI - [A symptomatic splenic hamartoma]. PMID- 10749600 TI - [Intrahepatic cholestasis in untreated hyperthyroidism]. PMID- 10749601 TI - [Chronic intestinal pseudo-obstruction and malnutrition due to intestinal endometriosis]. PMID- 10749602 TI - [Peptic pyloric stenosis treated by an endoprosthesis]. PMID- 10749604 TI - Intraperitoneal chemotherapy: biologic implications for clinical outcome. PMID- 10749603 TI - Adjuvant intraperitoneal 5-fluorouracil in high-risk colon cancer: A multicenter phase III trial. AB - OBJECTIVE: To evaluate the results of a prospective multicenter randomized study of adjuvant intraperitoneal 5-fluorouracil (5-FU) administered during 6 days shortly after resection of stages II and III colon cancers. SUMMARY BACKGROUND DATA: Systemic adjuvant chemotherapy improves the survival of patients with stage III colon cancer receiving treatment for 6 months. Intraperitoneal chemotherapy theoretically combines peritoneal and hepatic effects. METHODS: After resection, 267 patients were randomized into two groups. Patients in group 1 (n = 133) underwent resection followed by intraperitoneal administration of 5-FU (0.6 g/m2/day) for 6 days (day 4 to day 10). These patients also received intravenous 5-FU (1 g) during surgery. Patients in group 2 underwent resection alone (n = 134). RESULTS: In group 1, 103 patients received the total dose, 18 received a partial dose as a result of technical or tolerance problems, and 12 did not receive the chemotherapy. Rates of surgical death and complications were similar in both groups. Tolerance to treatment was excellent or fair in 97% of the patients and poor in 3%. After a median follow-up of 58 months, 5-year overall survival rates were 74% in group 1 and 69% in group 2; disease-free survival rates were 68% and 62%, respectively. Survival curves were superimposed until 3 years after treatment and began diverging thereafter. Among patients receiving the full treatment, the 5-year disease-free survival rate was improved in the treatment group in patients with stage II cancers but was unchanged in patients with stage III cancers. CONCLUSIONS: Chemotherapy with intraperitoneal 5-FU administered during a short period after surgery was well tolerated but was not sufficient to reduce the risk of death significantly. However, it reduced the risk of recurrence in stage II cancers. These results suggest that it should be associated with systemic chemotherapy to reduce both local and distant recurrences. PMID- 10749605 TI - Extracorporeal perfusion for the treatment of acute liver failure. AB - OBJECTIVE AND SUMMARY BACKGROUND DATA: Because of the shortage of available donor organs, death rates from liver failure remain high. Therefore, several temporary liver-assisting therapies have been developed. This article reviews various approaches to temporary liver support as well as immunologic and metabolic developments toward a solution for this problem. METHODS: A literature review was performed using Medline and additional library searches to obtain further references. Only articles with a well-defined aim of study and methodology and a clear description of the outcome of the experiments were included. CONCLUSIONS: Renewed interest has developed in old and new methods for an extracorporeal approach to the treatment of acute liver failure. Although temporary clinical improvement has been established, further research is needed to achieve a successful long-term clinical outcome. New developments in the field of genetic modification and tissue engineering await clinical application in the near future. PMID- 10749606 TI - Ex vivo and in situ resection of inferior vena cava with hepatectomy for colorectal metastases. AB - OBJECTIVE: To describe the surgical techniques and early results of inferior vena cava (IVC) resection in patients with advanced liver tumors. SUMMARY BACKGROUND DATA: Involvement of the IVC by hepatic tumors, although rare, is considered inoperable by standard resection techniques. Concomitant hepatic and IVC resection is required to achieve adequate tumor clearance. METHODS: Between February 1995 and February 1999, 158 patients underwent hepatic resection for colorectal metastases in the authors' unit. Eight patients, aged 42 to 80 years (mean 62 years), with hepatic metastases from colorectal cancer underwent concomitant resection of the IVC and four to six hepatic segments. Resections were carried out under total hepatic vascular exclusion in four patients and ex vivo in four patients. Between 30 degrees and 360 degrees of the retrohepatic IVC was resected and replaced with an autogenous vein patch (n = 1), a ringed Gore Tex tube graft (n = 2), a Dacron tube graft (n = 1), or a patch (n = 3) or was repaired by primary suturing (n = 1). RESULTS: There were two early deaths from multiple organ failure. One patient survived 30 months after ex vivo resection but died of renal cell carcinoma, and another died with recurrent disease at 9 months. The remaining four patients remained alive 5 to 12 months after surgery, with no hepatic failure or venous obstruction; tumor recurrence was present in two. Nonthrombotic occlusion of the neocava occurred in one patient and was stented successfully. CONCLUSIONS: Although concomitant hepatic and IVC resection is associated with a considerable surgical risk, this aggressive surgical approach offers hope for patients with hepatic tumors involving the IVC, who would otherwise have a dismal prognosis. This procedure can be performed under total hepatic vascular exclusion, with or without venovenous bypass, and by ex vivo bench resection. PMID- 10749607 TI - Resection of nonresectable liver metastases from colorectal cancer after percutaneous portal vein embolization. AB - OBJECTIVE: To assess the influence of preoperative portal vein embolization (PVE) on the long-term outcome of liver resection for colorectal metastases. SUMMARY BACKGROUND DATA: Preoperative PVE of the liver induces hypertrophy of the remnant liver and increases the safety of hepatectomy. METHODS: Thirty patients underwent preoperative PVE and 88 patients did not before resection of four or more liver segments. PVE was performed when the estimated rate of remnant functional liver parenchyma (ERRFLP) assessed by CT scan volumetry was less than 40%. RESULTS: PVE was feasible in all patients. There were no deaths. The complication rate was 3%. The post-PVE ERRFLP was significantly increased compared with the pre-PVE value. Liver resection was performed after PVE in 19 patients (63%), with surgical death and complication rates of 4% and 7% respectively. PVE increased the number of resections of more than four segments by 19% (17/88). Actuarial survival rates after hepatectomy with or without previous PVE were comparable: 81%, 67%, and 40% versus 88%, 61%, and 38% at 1, 3, and 5 years respectively. CONCLUSIONS: PVE allows more patients with previously unresectable liver tumors to benefit from resection. Long-term survival is comparable to that after resection without PVE. PMID- 10749608 TI - Extension of the frontiers of surgical indications in the treatment of liver metastases from colorectal cancer: long-term results. AB - OBJECTIVE: To evaluate retrospectively the long-term results of an approach consisting of performing surgery in every patient in whom radical removal of all metastatic disease was technically feasible. SUMMARY BACKGROUND DATA: The indications for surgical resection for liver metastases from colorectal cancer remain controversial. Several clinical risk factors have been reported to influence survival. METHODS: Between March 1980 and December 1997, 235 patients underwent hepatic resection for metastatic colorectal cancer. Survival rates and disease-free survival as a function of clinical and pathologic determinants were examined retrospectively with univariate and multivariate analyses. RESULTS: The overall 3-, 5-, 10-, and 15-year survival rates were 51%, 38%, 26%, and 24%, respectively. The stage of the primary tumor, lymph node metastasis, and multiple nodules were significantly associated with a poor prognosis in both univariate and multivariate analyses. Disease-free survival was significantly influenced by lymph node metastasis, a short interval between treatment of the primary and metastatic tumors, and a high preoperative level of carcinoembryonic antigen. The 10-year survival rate of patients with four or more nodules (29%) was better than that of patients with two or three nodules (16%), and similar to that of patients with a solitary lesion (32%). CONCLUSIONS: Surgical resection is useful for treating liver metastases from colorectal cancer. Although multiple metastases significantly impaired the prognosis, the life expectancy of patients with four or more nodules mandates removal. PMID- 10749609 TI - Hepatic metastases from leiomyosarcoma: A single-center experience with 34 liver resections during a 15-year period. AB - OBJECTIVE: To describe a large single-center experience with hepatic resection for metastatic leiomyosarcoma. SUMMARY BACKGROUND DATA: Liver resection is the treatment of choice for hepatic metastases from colorectal carcinoma. In contrast, the role of liver resection for hepatic metastases from leiomyosarcoma has not been defined. METHODS: The records of 26 patients who between 1982 and 1996 underwent a total of 34 liver resections for hepatic metastases from leiomyosarcoma were reviewed. There were 23 first, 9 second, and 2 third liver resections. The records were analyzed with regard to survival and predictive factors. RESULTS: In the 23 first liver resections, there were 15 R0, 3 R1, and 5 R2 resections. Median survival was 32 months after R0 resection and 20.5 months after R1/2 resection. The 5-year survival rate was 13% for all patients and 20% after R0 resection. In 10 patients with extrahepatic tumor at the time of the first liver resection, 6 R0 and 4 R2 resections were achieved. After R0 resection, the median survival was 40 months (range 5-84 months), with a 5-year survival rate of 33%. After repeat liver resection, the median survival was 31 months (range 5-51 months); after R0 resection, median survival was 31 months and after R1/2 resection it was 28 months. There was no 5-year survivor in the overall group after repeat liver resection. CONCLUSIONS: Despite frequent tumor recurrence, the long-term outcome after liver resection for hepatic metastases from leiomyosarcoma is superior to that after chemotherapy and chemoembolization. Although survival after tumor debulking also seems to be more favorable than after nonoperative therapy, these data indicate that only an R0 resection offers the chance of long-term survival. The presence of extrahepatic tumor should not be considered a contraindication to liver resection if complete removal of all tumorous masses appears possible. In selected cases of intrahepatic tumor recurrence, even repeated liver resection might be worthwhile. In view of the poor results of chemoembolization and chemotherapy in hepatic metastases from leiomyosarcoma, liver resection should be attempted whenever possible. PMID- 10749610 TI - Systemic cytokine response after laparoscopic-assisted resection of rectosigmoid carcinoma: A prospective randomized trial. AB - OBJECTIVE: To compare the systemic cytokine response in patients after laparoscopic-assisted resection with those after open resection of rectosigmoid carcinoma. SUMMARY BACKGROUND DATA: Laparoscopic resection of colorectal carcinoma is technically feasible, but objective evidence of its benefit is scarce. Systemic cytokines are accepted as markers of postoperative tissue trauma and mediators of the host immune response. METHODS: Thirty-four patients with rectosigmoid carcinoma, without evidence of metastatic disease and suitable for laparoscopic resection, were randomized to undergo either laparoscopic (n = 17) or conventional open (n = 17) resection of the tumor. Clinical parameters were recorded. Sera were collected before surgery and at appropriate time points afterward and assayed for interleukin-1beta, tumor necrosis factor-alpha, interleukin-6, and C-reactive protein. The primary end points were the cytokine and C-reactive protein levels. Data were analyzed by intention to treat. RESULTS: The demographic data of the two groups were comparable. The clinical outcome of both groups was satisfactory, with no surgical deaths and a reasonable complication rate. Both interleukin-1beta and interleukin-6 levels peaked 2 hours after surgery, with the responses in the laparoscopic group significantly less than those in the open group. C-reactive protein levels peaked at 48 hours, and the difference was also statistically significant. Levels of tumor necrosis factor-alpha were not elevated after surgery, and there was no difference between the groups. CONCLUSIONS: Tissue trauma, as reflected by systemic cytokine response, was less after laparoscopic resection than after open resection of rectosigmoid carcinoma. The difference in the systemic cytokine response may have implications on the long-term survival. PMID- 10749611 TI - Physiologic responses to laparoscopic aortofemoral bypass grafting in an animal model. AB - OBJECTIVE: To measure and compare the physiologic, metabolic, and hemodynamic responses to aortofemoral bypass grafting by three techniques: open or conventional laparotomy, laparoscopic-assisted (minilaparotomy), and totally laparoscopic grafting. METHODS: Twenty-four laboratory-bred hounds were randomized to one of three groups (open, laparoscopic-assisted, or totally laparoscopic). Four sets of parameters were measured: hemodynamic (intraoperative continuous cardiac output monitoring), inflammatory or hematologic (serial leukocyte and platelet levels), metabolic responses (serial blood glucose, serum cortisol and insulin, plasma epinephrine, plasma norepinephrine, and dopamine levels), and catabolic (24-hour urinary nitrogen excretion). RESULTS: Cardiac output increased transiently with aortic cross-clamping, more in the laparoscopic assisted and total laparoscopic groups than in the open group, but the differences were not significant. White blood counts nearly doubled within 12 hours of surgery but were similar in all three groups. Platelet counts decreased significantly in all three groups, but no significant intergroup effects were observed. Metabolic parameters (e.g., blood glucose, cortisol, and catecholamine) rose significantly during surgery but fell to normal within 24 hours, with no important difference between groups. For the first 24 hours, urinary urea excretion fell by 50% but returned to normal by 7 days in all three groups. CONCLUSIONS: In the experimental animal model, the hemodynamic, hematologic, and metabolic responses to laparoscopic and laparoscopic-assisted aortofemoral bypass grafting are similar to those produced by conventional laparotomy graft placement. These data call into question whether laparoscopic techniques for aortic surgery have a significant physiologic advantage in humans. PMID- 10749612 TI - Extensive abdominal surgery after caustic ingestion. AB - OBJECTIVE: To report the authors' experience in extensive abdominal surgery after caustic ingestion, and to clarify its indications. SUMMARY BACKGROUND DATA: After caustic ingestion, extension of corrosive injuries beyond the esophagus and stomach to the duodenum, jejunum, or adjacent abdominal organs is an uncommon but severe complication. The limit to which resection of the damaged organs can be reasonably performed is not clearly defined. METHODS: From 1988 to 1997, nine patients underwent esophagogastrectomy extended to the colon (n = 2), the small bowel (n = 2), the duodenopancreas (n = 4), the tail of the pancreas (n = 1), or the spleen (n = 1). Outcome was evaluated in terms of complications, death, and function after esophageal reconstruction. RESULTS: Five patients required reintervention in the postoperative period for extension of the caustic lesions. There were two postoperative deaths. Seven patients had secondary esophageal reconstruction 4 to 8 months (median 6 months) after initial resection. Three additional patients died 8, 24, and 32 months after the initial resection. Three survivors eat normally, and one has unexplained dysphagia. CONCLUSIONS: An aggressive surgical approach allows successful initial treatment of extended caustic injuries. Early surgical treatment is essential to improve the prognosis in these patients. PMID- 10749613 TI - Late outcome of isolated gastric bypass. AB - OBJECTIVE: To complete a long-term (>5 years) follow-up of patients undergoing isolated gastric bypass for severe obesity. SUMMARY BACKGROUND DATA: Previous experience as well as randomized trials suggested that the ideal operation for obesity should rely on manipulation of satiety rather than the production of malabsorption. Such an operation should incorporate a small gastric pouch of less than 30 mL placed in a dependent position on the lesser curvature of the stomach, not dependent on staples, and separated from the remaining stomach with a retrocolic, retrogastric Roux-en-Y gastrojejunostomy without external support. METHODS: The authors established an obesity clinic where patients were seen six times during the first year and semiannually thereafter. Emphasis was placed on defining success in terms of approximation to normal body-mass index. RESULTS: Of 274 patients, 243 (89%) were followed up for 5.5 +/- 1.5 years. Before surgery, the patients were obese (n = 13), morbidly obese (n = 134), or super-obese (n = 96). The obese and morbidly obese group achieved an excellent result, and the super-obese a good result. Individual results showed considerable variation from the mean. CONCLUSIONS: This study of isolated gastric bypass with a 5.5-year follow-up rate of 88.6% revealed a success rate of 93% in obese or morbidly obese patients and 57% in super-obese patients. Isolated gastric bypass compares favorably with biliopancreatic diversion in terms of weight loss, maximum weight loss, weight regain, current body-mass index, and percentage of patients with a body-mass index less than 35 kg/m2. PMID- 10749614 TI - Complications and death after surgical treatment of small bowel obstruction: A 35 year institutional experience. AB - OBJECTIVE: To study factors influencing complications and death after operations for small bowel obstruction (SBO) using multifactorial statistical methods. SUMMARY BACKGROUND DATA: Death after surgery for SBO is believed to be influenced by factors such as old age, comorbidities, bowel gangrene, and delay in treatment. No studies have been reported in which adverse factors related to death and complications have been systematically investigated with modern statistical methods. METHODS: The authors studied retrospectively 877 patients who underwent 1,007 operations for SBO from 1961 to 1995. Patients with paralytic ileus, intussusception, and abdominal cancer were excluded. Odds ratios for death, complications, postoperative hospital stay, and strangulation were calculated by means of logistic regression analyses. RESULTS: Death and complication rates decreased during the study period. Old age, comorbidity, nonviable strangulation, and a treatment delay of more than 24 hours were significantly associated with an increased death rate. The rate of nonviable strangulation increased markedly with patient age. Major factors increasing the complication rate were old age, comorbidity, a treatment delay of more than 24 hours, and the need for repeat surgery. CONCLUSION: Death and complication rates after SBO decreased from 1961 to 1995. Major factors influencing the rates were age, comorbidity, nonviable strangulation, and treatment delay. Nonviable strangulation was more common in old patients. PMID- 10749615 TI - Genotype and phenotype factors as determinants for rectal stump cancer in patients with familial adenomatous polyposis. Hereditary Colorectal Tumors Registry. AB - OBJECTIVE: To identify factors influencing the occurrence of cancer in the rectal remnant in patients with familial adenomatous polyposis (FAP) after colectomy and ileorectal anastomosis (IRA). SUMMARY BACKGROUND DATA: The risk for rectal cancer in patients with FAP after colectomy and IRA remains a major concern. METHODS: Between 1955 and 1997, 371 patients (206 men, 165 women) from the Registry of Hereditary Colorectal Tumors underwent colectomy and IRA as a primary surgical procedure. Survival was estimated using the Kaplan-Meier method. Cox proportional hazard models were fitted to assess the relative excess risk of rectal cancer and to control for confounding factors. A multivariate analysis was performed to assess the relation between cancer risk in the rectum and sex, age, number of rectal polyps, colon cancer, and APC germline mutation. RESULTS: Median follow-up was 81 months. Eighty-nine patients (24%) had colon cancer at the time of surgery. The APC mutation was found in 200 patients. In 27 patients, cancer developed in the retained rectum 1 to 26 years after surgery. The incidence of rectal carcinoma appears to increase with time: at 10, 15, and 20 years after surgery, the cumulative risk was 7.7%, 13.1%, and 23.0%, respectively. Multivariate analysis identified as independent predictors the presence of colon cancer at IRA and a mutation occurring between codons 1250 and 1464; both factors increased the risk nine times. CONCLUSIONS: The presence of cancer at IRA and APC mutation type are the most important risk factors for the future development of cancer in the rectal remnant in patients with FAP. PMID- 10749616 TI - Significance of resection margin in hepatectomy for hepatocellular carcinoma: A critical reappraisal. AB - OBJECTIVE: To evaluate the influence of the width and histologic involvement of the resection margin on postoperative recurrence after resection of hepatocellular carcinoma (HCC). SUMMARY BACKGROUND DATA: The significance of the resection margin in hepatectomy for HCC remains controversial. A precise evaluation of the effects of the width and histologic involvement of the resection margin on postoperative recurrence is required to clarify the issue. METHODS: Two hundred eighty-eight patients with macroscopically complete resection of HCC were divided into groups with narrow (<1 cm) or wide (>/=1 cm) resection margins. The two groups were compared for postoperative recurrence rate and pattern of recurrence. A further analysis was performed to investigate the effects of histologic involvement of the resection margin on postoperative recurrence. RESULTS: Recurrence rates were similar between 150 patients with a narrow margin and 138 patients with a wide margin; the groups were comparable in other clinicopathologic variables. Most recurrent tumors occurred in the liver remnant at a segment distant from the resection margin or at multiple segments. Thirty-four patients had margin involved histologically by microscopic invasion from the main tumor (n = 13), venous tumor thrombi (n = 13), or microsatellites separate from the main tumor (n = 8). These patients had significantly higher recurrence rates than those with a histologically clear margin. However, a positive histologic margin was not a significant risk factor for recurrence by multivariate analysis. Tumor stage and perioperative transfusion were the only independent risk factors. CONCLUSIONS: The width of the resection margin did not influence the postoperative recurrence rates after hepatectomy for HCC. A positive histologic margin was associated with a higher incidence of postoperative recurrence, but in most patients this was related to the underlying venous invasion or microsatellites. Most intrahepatic recurrences were considered to arise from intrahepatic metastasis by means of venous dissemination, which a wide resection margin could not prevent. PMID- 10749617 TI - Serum interleukin-10 but not interleukin-6 is related to clinical outcome in patients with resectable hepatocellular carcinoma. AB - OBJECTIVE: To evaluate the clinical significance of preoperative serum levels of interleukin-10 (IL-10) and interleukin-6 (IL-6) in patients with resectable hepatocellular carcinoma (HCC). SUMMARY BACKGROUND DATA: IL-10 is an immunosuppressive factor and IL-6 is a multifunctional cytokine that plays a role in host defense mechanisms. Both have been reported to be related to the disease prognosis in some human solid tumors. Their role in human HCC has not been investigated. METHODS: Preoperative serum samples of 67 patients with HCC who underwent potentially curative resection and 27 normal healthy donors were assayed. Levels of IL-10 and IL-6 were determined by enzyme-linked immunosorbent assay. The clinical significance of serum IL-10 and IL-6 was evaluated and compared with conventional clinicopathologic factors. RESULTS: Levels of IL-10 and IL-6 were significantly higher in patients with HCC than in healthy subjects. There was no correlation between IL-10 and IL-6 levels. Tumor resection resulted in a decrease in IL-10 and IL-6 levels. On univariate analysis, patients with high IL-10 levels had a worse disease-free survival, but IL-6 levels had no correlation with the disease-free survival. Multivariate analysis identified IL 10 levels as a predictor of postresectional outcome, in addition to the well established clinical risk factors. CONCLUSIONS: In patients with HCC, the preoperative serum IL-10 level is related to the clinical outcome. IL-10 may play an important role in the progression of HCC. PMID- 10749618 TI - Direct, minimally invasive adenomectomy for primary hyperparathyroidism: An alternative to conventional neck exploration? AB - OBJECTIVE: To evaluate the feasibility and efficacy of a direct, minimally invasive adenomectomy (MIA) as an alternative to conventional neck exploration (CNE) in patients with primary hyperparathyroidism. SUMMARY BACKGROUND DATA: Because primary hyperparathyroidism is caused by a solitary adenoma in 85% to 90% of patients, a direct adenomectomy through a mini-incision would theoretically suffice whenever an adenoma is correctly localized on preoperative imaging. If effective, a less invasive method could spare the patient an unnecessary bilateral neck exploration, thus saving time and rendering future surgical procedures in the neck less problematic. METHODS: Between October 1994 and October 1998, 110 consecutive patients with biochemically proven primary hyperparathyroidism who were to undergo surgery were enrolled in this study. Ultrasound and spiral CT were routinely performed as standard preoperative imaging modalities in the first series of 65 patients. In the second series of 45 patients, ultrasound was performed as the sole initial modality; it was supplemented by CT only in case of inconclusive test results. If test results were unequivocal (one adenoma), the patient was offered MIA. CNE was performed if the results were equivocal or if multiglandular disease was suspected. RESULTS: Overall, 84 patients were selected for MIA and 26 for CNE. In the first series, 2 MIA procedures (2/51) were converted to CNE because of negative perioperative findings. All 65 procedures resulted in normocalcemia. In the second series, all but five (4/33 MIAs, 1/12 CNEs) resulted in normocalcemia. A reexploration (CNE) was performed in three patients, resulting in normocalcemia after resection of a second or third adenoma. Two patients are still awaiting reexploration. In both series together, 78 of the 110 patients were successfully treated with MIA and spared CNE. CONCLUSION: MIA is a safe and effective alternative to CNE that may replace CNE in approximately two thirds of all patients. PMID- 10749620 TI - Protein-sparing effect in skeletal muscle of growth hormone treatment in critically ill patients. AB - OBJECTIVE: To investigate the effect of growth hormone (GH) treatment on skeletal muscle protein catabolism in patients with multiple organ failure in the intensive care unit (ICU). SUMMARY BACKGROUND DATA: Skeletal muscle depletion affects the incidence of complications and the length of hospital stay. A protein sparing effect of GH treatment in skeletal muscle of long-term ICU patients was hypothesized. METHODS: Twenty critically ill ICU patients were randomized to treatment with GH (0.3 U/kg/day) or as controls. Percutaneous muscle biopsy samples were taken before and after a 5-day treatment period starting on day 3 to 42 of the patient's ICU stay. Protein content, protein synthesis, water, nucleic acids, and free amino acids in muscle were analyzed. RESULTS: The protein content decreased by 8% +/- 11% in the control patients, with no significant change in the GH group. The fractional synthesis rate of muscle proteins increased in the GH group by 33% +/- 48%, and muscle free glutamine increased by 207% +/- 327% in the GH group. Total intramuscular water increased by 12% +/- 14% in the control group as a result of an increase in extracellular water of 67% +/- 86%; these increases were not seen in the GH group. In contrast, the intracellular water increased by 6% +/- 8% in the GH group. CONCLUSION: Treatment with GH for 5 days in patients with multiple organ failure stimulated muscle protein synthesis, increased muscle free glutamine, and increased intracellular muscle water. PMID- 10749619 TI - Angiotensin II inhibitor DuP753 attenuates burn- and endotoxin-induced gut ischemia, lipid peroxidation, mucosal permeability, and bacterial translocation. AB - OBJECTIVE: To investigate the role of angiotensin II as a mediator of burn- and sepsis-induced gut ischemia and reperfusion injury and to determine whether treatment with the angiotensin II inhibitor DuP753 can attenuate mucosal injury and bacterial translocation in a burn/endotoxemia porcine model. SUMMARY BACKGROUND DATA: Thermal injuries and endotoxemia have been shown to induce ischemia and reperfusion injury to the intestine, leading to increased mucosal permeability and bacterial translocation. Angiotensin II, the production of which has been reported to increase after burn, is thought to be one of the primary mediators of postburn mesenteric vasoconstriction. METHODS: An ultrasonic flow probe was inserted into the superior mesenteric artery and a catheter into the superior mesenteric vein in 21 female pigs. After 5 days, all animals were anesthetized, and 14 received 40% total body surface area third-degree burn. DuP753 was administered intravenously at 1 microg/kg to seven pigs immediately after burn. Eighteen hours after burn, 100 microg/kg Escherichia coli lipopolysaccharide (LPS) was intravenously administered. Systemic and splanchnic hemodynamics were measured and blood samples were drawn for blood gas analysis. Plasma conjugated dienes (PCDs), an index of lipid peroxidation, were measured every 6 hours. Intestinal permeability was assessed every 6 hours by measuring the lactulose/mannitol excretion ratio. At the end of the study (42 hours), tissue samples were harvested for bacteriologic cultures. RESULTS: Burn caused a significant decrease in mesenteric blood flow, to approximately 58% of baseline. Postburn endotoxemia significantly reduced the blood flow in the superior mesenteric artery to 53% of baseline. Treatment with DuP753 prevented postburn vasoconstriction and subsequently abrogated the impact of postburn endotoxemia on blood flow in the superior mesenteric artery. Mesenteric oxygen supply was significantly reduced after burn and endotoxin to 60% and 51% of baseline levels, respectively. DuP753 administration significantly improved mesenteric oxygen supply after both insults. Burn- and LPS-induced mesenteric hypoxia, as indicated by decreased mesenteric oxygen consumption, was also ameliorated by DuP753 treatment. PCD levels were significantly elevated 8 hours after burn. LPS caused a higher and prolonged increase in PCD levels. Treatment with DuP753 significantly reduced PCD levels after burn and after LPS. Intestinal permeability, as assessed by the lactulose/mannitol ratio, showed 6-fold and 12 fold increases after thermal injury and LPS, respectively. In contrast, the lactulose/mannitol ratio was only doubled in DuP753-treated animals. Bacterial translocation was significantly increased after burn and endotoxin. The incidence of bacterial translocation in the DuP753-treated animals was similar to that in the sham group. CONCLUSIONS: Angiotensin II appears to play a pivotal role in the burn- and endotoxin-induced intestinal ischemia and reperfusion injury, with subsequent increases in permeability and bacterial translocation. Postburn administration of the angiotensin II receptor antagonist DuP753 significantly reduces the extent of these events. PMID- 10749621 TI - Neutrophil adhesion to vascular prosthetic surfaces triggers nonapoptotic cell death. AB - OBJECTIVE: To test the hypothesis that neutrophil adhesion to expanded polytetrafluoroethylene (ePTFE) and Dacron triggers cell death. SUMMARY BACKGROUND DATA: Vascular prosthetic infections are intransigent clinical dilemmas associated with excessive rates of death and complications. Impaired neutrophil function has been implicated in the infection of implanted cardiovascular devices. ePTFE and Dacron are potent neutrophil stimuli able to elicit activation responses such as reactive oxygen species production independent of exogenous/soluble agonists. Reactive oxygen species that are released into the medium when neutrophils are challenged by soluble agonists are known to cause self-destruction. The authors therefore sought to examine whether neutrophil adhesion to prosthetic graft materials decreases neutrophil viability by means of reactive oxygen species production. METHODS: Neutrophils were adhered to surfaces for up to 6 hours. Cell viability was monitored with propidium iodide staining and lactate dehydrogenase release. RESULTS: Within 6 hours of adhesion to ePTFE and Dacron, respectively, 59% +/- 11% and 44% +/- 5% (n = 7) of the neutrophils were stained by propidium iodide. Indistinguishable results were obtained with plasma-coated ePTFE and Dacron. In contrast, less than 2% of the neutrophils adherent to fibrinogen-, immunoglobin-, or fetal bovine serum-coated polystyrene surfaces for 6 hours were positive for propidium iodide. The increase in membrane permeability to propidium iodide was accompanied by a two- to threefold increase in lactate dehydrogenase release. Pretreatment of neutrophils with N-acetyl-L-cysteine, cytochalasin D, or cyclosporin A significantly reduced the number of propidium iodide-positive ePTFE and Dacron adherent neutrophils. CONCLUSIONS: Neutrophil adhesion to ePTFE and Dacron triggers a rapid nonapoptotic cell death. The effect of ePTFE and Dacron on neutrophil viability appears to be caused by reactive oxygen species production. The premature death of graft-adherent neutrophils provides a novel explanation of the defect in neutrophil bacterial killing associated with vascular prosthetic grafts. PMID- 10749622 TI - Sequential cytokine therapy for pressure ulcers: clinical and mechanistic response. AB - OBJECTIVE: To compare the healing response of sequential topically applied cytokines to that of each cytokine alone and to a placebo in pressure ulcers, and to evaluate the molecular and cellular responses. SUMMARY BACKGROUND DATA: Because of a deficiency of cytokine growth factors in chronic wounds and the reversal of impaired healing in animal models, pressure ulcer trials have been performed with several exogenously applied growth factors. Because single-factor therapy has not been uniformly successful, combination or sequential cytokine therapy has been proposed. Laboratory data have suggested that sequential treatment with granulocyte-macrophage/colony-stimulating factor (GM-CSF)/basic fibroblast growth factor (bFGF) might augment the previously reported effect of bFGF alone. METHODS: A masked, randomized pressure ulcer trial was performed comparing sequential GM-CSF/bFGF therapy with that of each cytokine alone and with placebo during a 35-day period. The primary measure was wound volume decrease over time. Cytokine wound levels and mRNA levels were serially determined. Fibroblast-populated collagen lattices (FPCLs) were constructed from serial fibroblast biopsies. Cellular ultrastructure was evaluated by electron microscopy. Changes in ease of surgical closure and its relative cost were determined. RESULTS: Ulcers treated with cytokines had greater closure than those in placebo-treated patients. Patients treated with bFGF alone did the best, followed by the GM-CSF/bFGF group. Patients treated with GM-CSF or bFGF had higher levels of their respective cytokine after treatment. Patients with the greatest amount of healing showed higher levels of platelet-derived growth factor (PDGF) on day 10 and transforming growth factor beta (TGFbeta1) on day 36. Message for the bFGF gene was upregulated after treatment with exogenous bFGF, suggesting autoinduction of the cytokine. FPCLs did not mimic the wound responses. Ultrastructure of wound biopsies showed response to bFGF. Treatment with any of the cytokines improved the wound by allowing easier wound closure. This was most marked for the bFGF-alone treatment, with a cost savings of $9,000 to $9,200. CONCLUSIONS: Treatment with bFGF resulted in significantly greater healing than the other treatments in this trial. The clinical response appeared to be related to upregulation of the bFGF message and to increased levels of PDGF AB, bFGF, and TGFbeta1 in the wounds and changes in ultrastructure. The resultant improvements could be correlated with cost savings. PMID- 10749623 TI - Neither pelvic nor abdominal drainage is needed after anastomosis in elective, uncomplicated, colorectal surgery. PMID- 10749624 TI - Transanal endoscopic microsurgery (TEM) PMID- 10749625 TI - Subareolar versus peritumoral injection of location of sentinel lymph node. PMID- 10749626 TI - Letter to the Editor. PMID- 10749627 TI - Re: Chu et al "Do all patients with sentinel node metastasis from breast carcinoma need complete axillary node dissection?". PMID- 10749629 TI - Effects of specimen length on the monosegmental motion behavior of the lumbar spine. AB - STUDY DESIGN: An in vitro biomechanical analysis of the segmental motion behavior of the same segments in polysegmental (five segments), bisegmental, and monosegmental specimens using sheep lumbosacral spines. OBJECTIVES: To investigate the effect of specimen length on monosegmental motion behavior. These data may be helpful in planning in vitro tests and in comparing results of studies using specimens of different lengths. SUMMARY OF BACKGROUND DATA: The length of spinal specimens used for in vitro stability tests varies greatly, depending on the purpose of the study. Some investigators prefer testing specimens with one adjacent segment on either end of the region of interest. Others favor specimens as short as possible. METHODS: In a first step, seven sheep spine specimens, L3-S1 (note that sheep spines normally have seven lumbar vertebrae), each were tested without preload in a spine-loading apparatus. Alternating sequences of pure lateral bending, flexion/extension, and axial rotation moments (+/-3.75 Nm) were applied continuously. The motion in each single segment was measured simultaneously. Then, these polysegmental specimens were cut into two bisegmental specimens, L3-L5 and L6-S1, and tested in the same way. Finally, another vertebra was removed to obtain two monosegmental specimens, L3-L4 and L7-S1, and to test them as described. RESULTS: In general, the range of motion at L3-L4 and L7-S1 was smaller when tested in polysegmental than in monosegmental specimens. In polysegmental specimens (five segments), the range of motion at L3-L4 and L7-S1 was approximately 80% (range, 70.6-92.5%) and in bisegmental specimens approximately 95% (range, 66.7-100%) of their range of motion measured in monosegmental specimens. Neutral zone and coupled motions showed the inverse behavior. Significant differences were found. However, they were not consistent with either the loading direction or with the specimen length. CONCLUSIONS: For comparison of results, the specimen length should be kept constant within one experiment. Segmental motion behavior of specimens with different lengths should be compared only qualitatively. PMID- 10749628 TI - Injury to dorsal root ganglia alters innervation of spinal cord dorsal horn lamina involved in nociception. AB - STUDY DESIGN: A study of the relation between the development of mechanical allodynia and the reorganization of primary afferent terminals in the sensory lamina of the rat spinal cord dorsal horn after partial dorsal root ganglion injury in rats. OBJECTIVES: To investigate the pathologic mechanisms of mechanical allodynia after partial dorsal root ganglion injury. SUMMARY OF BACKGROUND DATA: After experimental peripheral nerve injury causing neuropathic pain, myelinated afferent fibers sprout into lamina II of the dorsal horn. This lamina is associated with nociceptive-specific neurons that generally are not stimulated by myelinated fiber input from mechanical receptors. These morphologic changes are suggested to have significance in the pathogenesis of chronic mechanical allodynia, although it is not known whether this kind of morphologic change occurs after dorsal root ganglion injury. METHODS: After partial dorsal root ganglion crush injury, the mechanical force causing footpad withdrawal was measured with von Frey hairs, and myelinated primary afferents were labeled with cholera toxin B subunit horseradish peroxidase, a selective myelinated fiber tracer that identifies transganglionic synapses. RESULTS: After partial dorsal root ganglion injury, mechanical allodynia developed in the corresponding footpad within 3 days and persisted throughout the experimental period. At 2 and 4 weeks after the injury, B subunit horseradish peroxidase-positive fibers, presumably myelinated afferents, were observed to be sprouting into lamina II of the dorsal horn on the injured side, but not on the contralateral control side. CONCLUSIONS: Morphologic change in spinal cord dorsal horn lamina II occurs after partial dorsal root ganglion injury. This change may have significance in the pathogenesis of chronic mechanical allodynia after partial dorsal root ganglion injury. PMID- 10749630 TI - Surgical outcome of cervical expansive laminoplasty in patients with diabetes mellitus. AB - STUDY DESIGN: The results from cervical laminoplasty in 18 patients with diabetes mellitus were compared with results from the same procedure in 34 nondiabetic patients matched for age, gender, and disease. OBJECTIVE: To analyze the effects of diabetes mellitus on the surgical outcome after cervical laminoplasty. SUMMARY OF BACKGROUND DATA: There have been no reports on the results of cervical laminoplasty patients with diabetes. METHODS: A retrospective analysis of 18 patients with diabetes mellitus who underwent cervical laminoplasty and 34 nondiabetic patients who underwent the same surgical procedure was undertaken. The postoperative score, intra- and postoperative findings, complications, and radiologic factors were compared between the two groups. In the group with diabetes, the correlation between the recovery rate of the Japanese Orthopedic Association score and the factors indicating the severity of diabetes was assessed. RESULTS: There was no statistical difference between the total Japanese Orthopedic Association scores of the two groups. However, the group with diabetes mellitus showed a poor recovery of sensory function of the lower extremities. Three patients in the group with diabetes had superficial wound complication after surgery. In contrast, none of the patients in the control group had a wound problem. Furthermore, a negative correlation was observed between the recovery rate and the preoperative HbA1 level in the group with diabetes. CONCLUSIONS: Although patients with diabetes mellitus who had cervical myelopathy experienced benefits from cervical laminoplasty similar to those of nondiabetic patients, the patients with diabetes were more likely to have wound complication. Furthermore, the negative correlation between the recovery rate and the preoperative HbA1 value might suggest that long-term diabetes control of more than 2 to 3 months before surgery at least is recommended for a favorable surgical outcome. PMID- 10749631 TI - Surgical and nonsurgical management of lumbar spinal stenosis: four-year outcomes from the maine lumbar spine study. AB - STUDY DESIGN: A prospective cohort study of patients with lumbar spinal stenosis recruited from the practices of orthopedic surgeons and neurosurgeons throughout Maine. OBJECTIVE: To assess 4-year outcomes for patients with lumbar spinal stenosis treated surgically or nonsurgically. SUMMARY OF BACKGROUND DATA: Surgery for lumbar spinal stenosis has increased dramatically despite the lack of randomized trials comparing surgical with nonsurgical treatments. Long-term evaluation of surgical series has documented deterioration in initial symptomatic improvement, but few studies have compared long-term outcomes of surgical and nonsurgical treatment. METHODS: Eligible, consenting patients had baseline interviews with mailed follow-up questionnaires at 3, 6, and 12 months, then annually thereafter. Clinical data were obtained at baseline from a physician questionnaire. Outcomes included patient-reported symptoms of leg and back pain, functional status, and satisfaction. RESULTS: Of 148 patients with lumbar spinal stenosis initially enrolled, 4-year outcomes were available on 119 patients (80.4%): 67 of 81 (83%) treated surgically and 52 of 67 (78%) treated nonsurgically. The surgically treated patients had more severe symptoms and worse functional status at baseline and better outcomes at 4-year evaluation than the nonsurgically treated patients. After 4 years, 70% of the surgically treated and 52% of the nonsurgically treated patients reported that their predominant symptom, either leg or back pain, was better (P = 0.05). Satisfaction of patients with their current state at 4 years was reported by 63% of the surgically treated and 42% of the nonsurgically treated patients (P = 0.04). Surgical treatment remained a significant determinant of 4-year satisfaction, even after adjustment for other independent predictors (P = 0.001). For the nonsurgically treated patients, there was no significant change in outcomes over 4 years, whereas the initial improvement seen in the surgically treated patients modestly decreased over the subsequent 4 years. CONCLUSIONS: For the patients with severe lumbar spinal stenosis, surgical treatment was associated with greater improvement in patient-reported outcomes than nonsurgical treatment at 4-year evaluation, even after adjustment for differences in baseline characteristics among treatment groups. The relative benefit of surgery declined over time but remained superior to nonsurgical treatment. Outcomes for the nonsurgically treated patients improved modestly and remained stable over 4 years. Determining whether outcomes continue to converge will require longer-term evaluation. PMID- 10749632 TI - A synthetic porous ceramic as a bone graft substitute in the surgical management of scoliosis: a prospective, randomized study. AB - STUDY DESIGN: A prospective randomized study. OBJECTIVES: To assess the clinical and radiologic performances of a synthetic ceramic as a bone graft substitute in scoliosis surgery. SUMMARY OF BACKGROUND DATA: Surgery on the skeleton frequently requires harvesting of autogenous bone grafts from the pelvis, but this procedure often is complicated by problems. METHODS: Fifty-eight patients with idiopathic scoliosis, ages 13 to 25 years, were treated by posterior correction and arthrodesis using Cotrel-Dubousset instrumentation. Posterior spinal fusion was performed using local bone grafts combined with autogenous iliac bone grafts in 30 patients, and combined with porous biphasic calcium phosphate ceramic blocks comprising hydroxyapatite and tricalcium phosphate in another 28 patients. The patients were observed for a minimum of 24 months after surgery, with a mean postoperative observation time of 48 months. The results were assessed clinically and radiographically. RESULTS: Patients in the ceramic group had a lower average blood loss than those in the iliac graft group. They also were free from postoperative local complications in the iliac region, which were experienced by a significantly high proportion of patients belonging to the iliac graft group. Radiography demonstrated successful incorporation of the ceramic blocks within 12 months. The correction of the deformity was maintained similarly and satisfactorily in both groups. CONCLUSIONS: These results justify and favor the use of calcium phosphate ceramics as bone graft substitutes for instrumented posterior spinal fusion in teenagers and young adults. Potentially hazardous harvesting of pelvic bone is no longer necessary for such operations. PMID- 10749633 TI - Development of trunk asymmetry in a cohort of children ages 11 to 22 years. AB - STUDY DESIGN: A cohort study with a follow-up period of 11 years. OBJECTIVES: To study the growth of the spine with a focus on the development of trunk asymmetry and scoliosis. SUMMARY OF BACKGROUND DATA: Trunk asymmetry, a common phenomenon at adolescence, can be considered the clinical expression of scoliosis. The importance of the pubertal growth spurt has been stressed in the natural history of scoliosis. However, no cohort studies have focused on the ascending and descending phase of the spine's peak growth and the development of trunk asymmetry. METHODS: The cohort consisted of all the fourth-grade school children in the Western school district of Helsinki, Finland, in the spring of 1986. These 1060 children (515 girls and 545 boys), from the average age of 11 to 14 years, were invited to undergo annual examinations. The 855 children (80.7%) who had participated in the study at the age of 14 years were invited to a reexamination at the age of 22 years. This invitation was accepted by 430 (208 women and 222 men; 54%) of those invited. The forward bending test, the spinal pantography, and the anthropometric measurements were carried out by the same author (M.N.) throughout this study. RESULTS: At 22 years of age, 30% of the adults were found to be symmetric, with a hump less than 4 mm in the forward bending test, whereas 51% had a hump of 4 to 9 mm, and 19% had a hump 10 mm or larger (major asymmetry). The directional asymmetry of trunk surface, a skew to the right at the thoracic level and to the left at the lumbar level at puberty, remained constant at adult age. The prevalence of major trunk asymmetry at adult age was the same in both women and men, in contrast to the female predominance at puberty in this cohort. There were close correlations in the degrees of thoracic and lumbar asymmetry between puberty and adult ages. CONCLUSIONS: The shape of the back develops mainly during the pubertal growth spurt at ages 12 to 14 years in girls and boys. Trunk asymmetry (and mild scoliosis) seems as prevalent in young adult women as in men, although at puberty idiopathic scoliosis was twice as prevalent among girls as among boys in this cohort. PMID- 10749634 TI - Lumbopelvic lordosis and pelvic balance on repeated standing lateral radiographs of adult volunteers and untreated patients with constant low back pain. AB - STUDY DESIGN: Twenty volunteers and 20 patients with no prior spine surgery had two standing lateral radiographs taken, on the average, 66 months apart and 2 weeks apart, respectively. OBJECTIVES: To first determine the reliability of the measurement techniques used, and then the longitudinal variation between radiographs for the sagittal spinopelvic alignments measured in two stable populations, the one manifesting no back symptoms (volunteers) and the other showing no changes in symptoms (patients). Pelvic morphology also was assessed quantitatively, and significant correlations for the measurements were studied. SUMMARY OF BACKGROUND DATA: There are no published studies on longitudinal variation for measurements of sagittal spinal alignments in asymptomatic control subjects or untreated patients with stable back problems. It may be helpful to know not only how much variation in alignments can be expected between radiographs of the same individual, but also which measurements and measurement techniques offer the greatest clinical reliability and application. METHODS: Each patient in this study reported mechanical type low back pain that was constant in location and character as well as clinically consistent with symptomatic degenerative lumbar disc disease. Each patient and volunteer had 36-inch-long lateral radiographs taken of the entire thoracic and lumbar spine, which included the pelvis. After intervening periods of 1 to 4 weeks (patients) and 5 to 6 years (volunteers), a second radiograph was taken for comparison. Two observers made 24 different measurements on the radiographs including determinations for lumbopelvic lordosis, pelvic balance, and pelvic morphology using the pelvic radius technique. Reliabilities, longitudinal variations, and correlations for the measurements were compared. RESULTS: The most reliable measurements were for pelvic morphology, pelvic balance, and regional lumbopelvic lordosis by the pelvic radius technique. Pelvic morphology was the most constant measurement between individual radiographs. Pelvic morphology and total lumbosacral lordosis were dependent measurements that were complementary in determining total lumbopelvic lordosis. Lumbopelvic lordosis and pelvic balance also had strong correlation, whereas lumbosacral lordosis and pelvic balance were independent measurements. CONCLUSIONS: The pelvic radius technique is recommended for evaluating lordosis to the pelvis because this approach provided not only good measurement reliability on standing radiographs for lumbopelvic lordosis, but also determination of pelvic balance over the hips and the option to assess pelvic morphology quantitatively. Lumbopelvic lordosis and pelvic balance were strongly correlative. This finding, along with higher reliability and lower longitudinal variation on repeated radiographs, indicated greater clinical application for these specific measurements. PMID- 10749635 TI - Impairment magnification during dynamic trunk motions. AB - STUDY DESIGN: An examination of a group of patients with low back disorder and a group of healthy (asymptomatic) individuals asked to produce trunk motions under sincere and insincere experimental conditions. Trunk motion components were examined to determine which combination of motion components could best distinguish between sincere and insincere efforts. OBJECTIVE: To determine whether examination of trunk motion components could be used to identify impairment magnification during unresisted repeated bending tasks. SUMMARY OF BACKGROUND DATA: Trunk motion measures can be used to assess and "benchmark" the status of the low back. However, these measures typically are clinically useful only if the individual is producing an effort that does not magnify the impairment during the functional evaluation. This study addressed the issue of impairment magnification during the production of free dynamic trunk motion. METHODS: The trunk motion characteristics of 100 healthy individuals and 100 patients with chronic low back disorders were documented. All participants were asked to produce the trunk motions in two different types of conditions. In the one experimental condition, they were asked to produce sincere trunk motions. In the other experimental condition they were asked to pretend either that they were experiencing low back pain (the asymptomatic group) or that their pain was worse than it actually was (group with low back pain). RESULTS: A combination of trunk motion measures was able to distinguish well between the conditions. Sensitivity and specificity for the asymptomatic group were 92%, whereas they were 75% for the group with low back pain. Overall, sensitivity and specificity were 81.5% for all the participants combined. CONCLUSIONS: These results indicate that motion measures can be used to help assess impairment magnification during functional trunk motion testing. These measures can provide a means by which to scrutinize the quality of quantitative measures indicating the extent of a low back disorder. These objective motion measures also can be used to complement other subjective observational methods for the assessment of impairment. PMID- 10749636 TI - Predictors of blood transfusions in spinal instrumentation and fusion surgery. AB - STUDY DESIGN: A retrospective review of 244 adult spine instrumentation and fusion surgery cases (1994-1995) from one institution. OBJECTIVES: To ascertain the predictors of blood transfusions for adult patients undergoing different types of multilevel spine surgery. SUMMARY OF BACKGROUND DATA: Blood loss and transfusion requirements during and after multilevel spine surgeries have always been perceived as great. Identifying the predictors of blood transfusion with this type of surgery may aid in reducing the amount of blood loss and the transfusion requirements. METHODS: The charts of 244 adult patients who underwent multilevel spine surgery from January 1994 to July 1995 were retrospectively reviewed. RESULTS: A large percentage of patients required blood transfusion. The significant determinants for increased amounts of allogeneic red blood cell units transfused on the day of surgery using linear multiple regression modeling were low preoperative hemoglobin concentration, tumor surgery, increased number of posterior levels surgically fused, history of pulmonary disease, decreased amount of autologous blood available, and no use of the Jackson table (R2 = 0. 63). The significant determinants for an increased amount of autologous red blood cell units transfused on the day of surgery using linear multiple regression modeling were increased autologous red blood cells available, low preoperative hemoglobin concentration, and increased number of posterior levels surgically fused (R2 = 0. 60). CONCLUSION: The need for transfusion is associated with multiple factors, suggesting that a multifaceted, integrated approach may be necessary to reduce this risk. PMID- 10749637 TI - Use of the surgical blood order equation in spinal instrumentation and fusion surgery. AB - STUDY DESIGN: A retrospective review of 182 cases of adult spine instrumentation and fusion surgery (1994-1995) from one institution. OBJECTIVES: To develop and retrospectively evaluate the use of the surgical blood ordering equation for patients undergoing spinal instrumentation and fusion surgery. SUMMARY OF BACKGROUND DATA: The provision of effective and safe blood and blood products is the primary function of the hospital transfusion service. A quantification of blood bank efficiency is the crossmatch-to-transfusion ratio. The maximal surgical blood order schedule system has been used to improve the efficiency of surgical ordering practices. The current authors have developed a theoretically more efficient system, the surgical blood ordering equation, which incorporates patient factors for ordering red blood cell units for surgical patients. METHODS: The charts of 63 patients with autologous red blood cells available and 119 adult patients with none available, who underwent multilevel spine surgery from January 1994 to July 1995, were reviewed. RESULTS: The surgical blood ordering equation was exactly correct in the ordering for 37 (20.3%) of 182 patients. The maximal surgical blood order schedule was exactly correct in ordering blood for 14 patients (7.6%). Use of the new surgical blood ordering equation to order red blood cells for surgery would result in a lower crossmatch-to-transfusion ratio than with the current system, the maximal surgical blood order schedule, for patients with autologous red blood cells available (1. 0 vs. 1.3) and patients with none available (0.9 vs. 1.2). CONCLUSION: Incorporating patient factors resulted in increased efficiency of blood ordering practices. PMID- 10749638 TI - Comparative results between conventional and computer-assisted pedicle screw installation in the thoracic, lumbar, and sacral spine. AB - STUDY DESIGN: A comparative study on the position of pedicle screws in patients treated surgically with and without computer assistance. OBJECTIVES: To evaluate the accuracy of computer-assisted pedicle screw installation, and to evaluate its clinical benefit as compared with conventional pedicle screw installation techniques. SUMMARY OF BACKGROUND DATA: In vitro and clinical studies have documented a significant rate of misplaced screws in the thoracolumbar area. Neurologic complications are recognized problems caused by screw misplacement. METHODS: Patients treated surgically with computer assistance were compared with a historical control group of patients treated surgically with conventional techniques in the same hospital and by the same surgical team. All screw positions were measured with a postoperative magnetic resonance tomography, and cortical effractions were categorized in 2-mm increments. Patients' charts also were reviewed to assess individual neurologic outcomes. RESULTS: The control cohort was composed of 100 patients, with 544 screws from T5 to S1. The computer assisted cohort was composed of 50 patients, with 294 screws from T2 to S1. In the control cohort, 461 of 544 screws (85%) were found completely within their pedicles as compared with 278 of 294 screws (95%) correctly placed in the computer-assisted group (P < 0.0001). All 16 screws incorrectly placed with computer assistance were found 0.1 mm to 2 mm from the pedicle cortex. In the control cohort, 68 screws were found 0.1 mm to 2 mm, 10 screws 2.1 mm to 4 mm, and 5 screws more than 4 mm from the pedicle cortex. Seven patients in the control cohort were surgically retreated because of postoperative neurologic deficits, whereas no patients in the computer-assisted group were surgically retreated. CONCLUSIONS: Computer assistance can decrease the incidence of incorrectly positioned pedicle screws. PMID- 10749639 TI - Clinical evaluation and computed tomography scan analysis of screw tracts after percutaneous insertion of pedicle screws in the lumbar spine. AB - STUDY DESIGN: An examination of the accuracy of percutaneous pedicle screw placement in the lumbar spine. Using computed tomography scan analysis after implant removal, the screw tracts could be analyzed regarding the degree and direction of screw dislocation. OBJECTIVES: To investigate the misplacement rate and related clinical complications of percutaneous pedicle screw insertion in the lumbar spine. SUMMARY OF BACKGROUND DATA: The feasibility of the external fixation test has been investigated in several studies. Although pedicle screw misplacement has been reported as one of the main complications, there are no reliable data on the misplacement rate for this difficult surgical procedure. METHODS: In this study, 51 consecutive patients with suspected segmental instability were investigated after external transpedicular screw insertion for the external fixation test. Computed tomography scans of all instrumented pedicles from L2 to S1 were performed after screw removal. The screw tracts were analyzed, and the direction and degree of the pedicle violations were noted. In addition, the screw and pedicle angles were measured. RESULTS: Of 408 percutaneously inserted pedicle screws, only 27 screws (6.6%) were misplaced. There were 19 medial pedicle violations, 6 lateral cortical defects, and only 1 cranial and 1 caudal displacement. With respect to the spinal level, S1 showed the highest misplacement rate, with 11 screw dislocations (12%). After surgery, found two nerve root injuries were found. Only one of the injuries (L4) was related to the malposition of a screw. CONCLUSIONS: This study has shown that percutaneous insertion of pedicle screws in the lumbar spine is a safe and reliable technique. Despite the low misplacement rate of only 6.6%, it should be kept in mind that the surgical procedure is technically demanding and should be performed only by experienced spine surgeons. PMID- 10749640 TI - Progression of idiopathic thoracolumbar scoliosis after breast reconstruction with a latissimus dorsi flap: a case report. AB - STUDY DESIGN: A report of a patient in whom progressive symptomatic thoracolumbar scoliosis developed after breast reconstruction with a latissimus dorsi myocutaneous flap. OBJECTIVES: To present the first reported case of progressive symptomatic scoliosis after breast reconstruction with a latissimus dorsi myocutaneous flap and to suggest that latissimus flap harvest may be contraindicated in patients with preexisting scoliosis. SUMMARY OF BACKGROUND DATA: Latissimus dorsi myocutaneous flap harvest incorporated into several surgical operations including breast reconstruction has been presented as a relatively benign procedure without significant biomechanical consequence. Nevertheless, various anatomic and animal studies have suggested an important role for balanced latissimus function in terms of proper spinal alignment. Long term follow-up evaluation of patients after latissimus flap harvest is insufficient and fails to address the specific issue of spinal deformity. METHODS: Postoperative radiographs demonstrated significant progression of the patient's thoracolumbar scoliosis as compared with radiographs taken before her latissimus harvest. Curve progression accompanied by development of severe and disabling back pain were considered indications for surgical curve correction and stabilization. RESULTS: At the time of 1-year follow-up assessment after posterolateral spinal fusion and instrumentation, the patient had experienced complete relief from her back pain and satisfactory spinal fusion. CONCLUSIONS: Although a cause and effect relation cannot be established, this case study suggests that latissimus harvest may have a destabilizing effect on the thoracolumbar spine in the long term, especially in patients with preexisting scoliosis. Alternative procedures should be considered in these patients. PMID- 10749641 TI - Mycobacterium xenopi infection of the spine: a case report and literature review. AB - STUDY DESIGN: A case report of Mycobacterium xenopi. OBJECTIVES: To present a case report of a vertebral osteomyelitis caused by M. xenopi and to review the world literature on the subject. SUMMARY OF BACKGROUND DATA: M. xenopi is most commonly a pulmonary pathogen in immunosuppressed patients or those with underlying lung disease. Infection of the spine is very rare, with only four cases reported. Three of these cases occurred in immunosuppressed patients, and the fourth in a patient with previous tuberculosis osteomyelitis of the spine. METHODS: The information was obtained from a review of the patient's clinical notes and follow-up appointments. RESULTS: The patient was a 73-year-old woman with an M. xenopi osteomyelitis of T6-T7, confirmed by magnetic resonance imaging and a computed tomography-guided biopsy. She was treated with surgical decompression and stabilization before a 2-year antibiotic regimen was begun. At follow-up assessment 1 year after the antibiotics were finished, she still had some ongoing back pain, but no evidence of relapse. CONCLUSIONS: This is the first reported case of an M. xenopi infection of the spine in a patient with no predisposing factors. This type of infection can be difficult to treat, with a high relapse rate reported despite prolonged courses of antibiotics. PMID- 10749642 TI - Foraminal epidural extra osseous cavernous hemangioma of the cervical spine: a case report. AB - STUDY DESIGN: A case report of purely epidural foraminal cervical cavernous angioma assessed by magnetic resonance imaging and diagnosed at pathologic study. OBJECTIVE: To illustrate a rare cause of cervical foraminal mass mimicking a schwannoma. SUMMARY OF BACKGROUND DATA: Several cases of epidural cavernous angioma have been reported. A purely epidural cavernous angioma in a cervical foramen has never been reported in the literature. METHOD: A 36-year-old man sought treatment for acute weakness in his right upper limb with radicular distribution. On magnetic resonance images, the lesion appeared as a foraminal mass with no bone invasion or hematic components. It enhanced after intravenous administration of Gadolinium. Pathologic study after surgery showed a typical cavernous angioma. RESULTS: The patient improved slightly after surgery. CONCLUSION: Clinical and radiologic presentation could be confusing in a purely foraminal epidural cervical cavernous hemangioma. Cavernous hemangioma must be known as a differential diagnosis of a foraminal schwannoma. Diagnosis of cavernous angioma is made easily on pathologic examination. PMID- 10749643 TI - Cervical vertigo after hair shampoo treatment at a hairdressing salon: a case report. AB - STUDY DESIGN: A case report of cervical vertigo that occurred after shampoo treatment in a hairdressing salon. Abnormalities were detected on magnetic resonance images and magnetic resonance angiography scans. OBJECTIVES: To describe the diagnosis of cervical vertigo caused by neck hyperextension. SUMMARY OF BACKGROUND DATA: Neck hyperextension may induce vertigo and dizziness; the pathophysiology remains unclear, however, because subjective findings are usually difficult to document. METHODS: The diagnosis, treatment, and outcome of a patient with cervical vertigo that occurred after hair shampoo treatment in a hair dressing salon were assessed. RESULTS: Magnetic resonance angiography demonstrated narrowing of the left vertebral artery, whereas magnetic resonance imaging showed cerebellar infarction. Treatment included rest and drugs that activate cerebral circulation and prevent platelet aggregation. Improvement was noted within few days. CONCLUSIONS: The authors suggest that the hyperextended neck position during hair shampoo treatment in a beauty parlor may be a risk factor for back lifting or cerebellum vascular insufficiency. Public education should lead to avoidance of this position during hair shampoo treatment at hair dressing salons. PMID- 10749644 TI - Synovial chondromatosis in a facet joint of a cervical vertebra. AB - STUDY DESIGN: A case report of a cervical facet joint synovial chondromatosis. OBJECTIVES: To correlate the radiologic and histologic features of vertebral synovial chondromatosis with review of the literature. SUMMARY OF BACKGROUND DATA: Only two previous cases of vertebral facet joint synovial chondromatosis were found in a review of the English language medical literature. METHODS: A 39 year-old woman had severe cervical pain associated with neurologic signs and symptoms in the left upper extremity. Computed tomographic and magnetic resonance imaging studies were performed. RESULTS: Imaging studies showed lytic defects in the laminae of C3 and C4, with intermediate T1 and high T2 signal intensities. The diagnostic impression was that of a lymphangioma or synovial cyst. A laminectomy showed synovial tissue in both the C3-C4 facet joint and the lamina bone. Histologic examination disclosed synovial chondromatosis. CONCLUSIONS: Synovial chondromatosis of the vertebral spine is quite rare, this being only the third reported example. Direct invasion of the cancellous bone, as in this case, also is a very uncommon feature of chondromatosis. It is emphasized that when radiologic studies demonstrate a lesion with cartilaginous characteristics within or juxtaposed to a joint, synovial chondromatosis, despite its rarity, should be included in the differential diagnosis, regardless of the anatomic site. PMID- 10749645 TI - A solitary bone cyst in the spinous process of the cervical spine: a case report. AB - STUDY DESIGN: A case report. OBJECTIVES: To illustrate a rare case of histologically confirmed solitary bone cyst involving the spinous process of C7. SUMMARY OF BACKGROUND DATA: A solitary bone cyst involving the spine is very unusual. Although four cases of a solitary bone cyst in the spine have been reported in the literature, the current authors have been able to find only one case of solitary bone cyst in the spinous process. All four patients reported in the literature were over 30 years of age. The patient in the current case was a 13-year-old girl with no history of trauma. METHODS: Radiographs and a computed tomography scan of the cervical spine were performed before the operation, as was a histologic examination to make a diagnosis of the lesion. RESULTS: The intraoperative findings from examination of the stagnant fluid within the lesion and the histologic examination indicated the diagnosis of a solitary bone cyst. CONCLUSIONS: A solitary bone cyst in the spine is rare, especially in the young. An osteolytic lesion in the spinous process of the spine tends to be diagnosed as an osteoblastoma or as a giant cell tumor of the bone. A solitary bone cyst of the spine, although rare, must be considered as a differential diagnosis. PMID- 10749646 TI - Spine update: cervical spine internal fixation using screw and screw-plate constructs. AB - Screw and screw-plate constructs have been used successfully in fixation of the cervical spine. This update focuses on the indications, complications, and nuances in the technique used for odontoid screws, transarticular C1-C2 screws, occipitocervical plating, posterior lateral mass screws, pedicle screws, and anterior plating. PMID- 10749647 TI - The management of malignant spinal cord compression compression. PMID- 10749648 TI - Letter to the editor PMID- 10749649 TI - Imagery PMID- 10749650 TI - Response to editorial on air medical crashes PMID- 10749657 TI - Mutation analysis and embryonic expression of the HLXB9 Currarino syndrome gene. AB - The HLXB9 homeobox gene was recently identified as a locus for autosomal dominant Currarino syndrome, also known as hereditary sacral agenesis (HSA). This gene specifies a 403-amino acid protein containing a homeodomain preceded by a very highly conserved 82-amino acid domain of unknown function; the remainder of the protein is not well conserved. Here we report an extensive mutation survey that has identified mutations in the HLXB9 gene in 20 of 21 patients tested with familial Currarino syndrome. Mutations were also detected in two of seven sporadic Currarino syndrome patients; the remainder could be explained by undetected mosaicism for an HLXB9 mutation or by genetic heterogeneity in the sporadic patients. Of the mutations identified in the 22 index patients, 19 were intragenic and included 11 mutations that could lead to the introduction of a premature termination codon. The other eight mutations were missense mutations that were significantly clustered in the homeodomain, resulting, in each patient, in nonconservative substitution of a highly conserved amino acid. All of the intragenic mutations were associated with comparable phenotypes. The only genotype-phenotype correlation appeared to be the occurrence of developmental delay in the case of three patients with microdeletions. HLXB9 expression was analyzed during early human development in a period spanning Carnegie stages 12 21. Signal was detected in the basal plate of the spinal cord and hindbrain and in the pharynx, esophagus, stomach, and pancreas. Significant spatial and temporal expression differences were evident when compared with expression of the mouse Hlxb9 gene, which may partly explain the significant human-mouse differences in mutant phenotype. PMID- 10749661 TI - Increased incidence of unsulphated and 4-sulphated residues in the chondroitin sulphate linkage region observed by high-pH anion-exchange chromatography. AB - We report the isolation, characterization and quantification of five octasaccharides, four hexasaccharides and two tetrasaccharides, derived from the chondroitin sulphate (CS) linkage region of 6-8-year-old bovine articular cartilage aggrecan, following digestion with chondroitin ABC endolyase. Using a novel high-pH anion-exchange chromatography (HPAEC) method, in conjunction with one- and two-dimensional (1)H-NMR spectroscopy, we have identified the following basic structure for the CS linkage region of aggrecan: DeltaUA(beta1 3)GalNAc[0S/4S/6S](beta1-4)GlcA(beta1-3)GalNAc[0S/4S/6S](beta1-4)GlcA(beta1 3)Gal[0S/6S](beta1-3)Gal(beta1-4)Xyl, where DeltaUA represents 4,5-unsaturated hexuronic acid, and 4S and 6S represent an O-ester sulphate group on C-4 and C-6 respectively. The octa-, hexa- and tetra-saccharide linkage region fragments were used to develop a HPAEC fingerprinting method, with detection at A(232 nm), and a linear response to approx. 0.1 nmol of substance. The sulphation patterns of CS linkage regions, of up to octasaccharide in size, from articular and tracheal cartilage aggrecan were examined. The results show that in articular cartilage, for the majority (53%) of octasaccharides the 2-deoxy-2-N-acetyl amino-D galactose (GalNAc) residues closest to the linkage region are both 6-sulphated; however, in a significant portion (34%), one or more of these GalNAc residues are unsulphated, and in 8% both are unsulphated. Approximately 10-18% of the chains have a 4-sulphated GalNAc in the first disaccharide, and 12% have a sulphated linkage region Gal residue. No evidence was found for uronic acid sulphation. These data show that there is a significant increase in the incidence of unsulphated and 4-sulphated GalNAc residues adjacent to the linkage region compared with the rest of the chain. Bovine tracheal cartilage linkage regions displayed very similar sulphation profiles to those from articular cartilage, despite the presence of a higher level of GalNAc 4-sulphation within the repeat region of the main CS chain. PMID- 10749662 TI - 27-Oxygenation of C27-sterols and 25-hydroxylation of vitamin D3 in kidney: cloning, structure and expression of pig kidney CYP27A. AB - This paper describes the molecular cloning of a cytochrome P450 enzyme in pig kidney that catalyses the hydroxylations of vitamin D(3) (cholecalciferol) and C(27)-sterols. DNA sequence analysis of the cDNA revealed that the enzyme belongs to the CYP27 family. The first 36 amino acids have many hallmarks of a mitochondrial signal sequence. The mature pig kidney CYP27 protein contains 498 amino acids. The M(r) of the mature protein was calculated to be 56607. The structure of pig kidney CYP27, as deduced by DNA sequence analysis, shows 77-83% identity with CYP27A in rat, rabbit and human liver. Transfection of the renal CYP27A cDNA into simian COS cells resulted in the synthesis of an enzyme that catalysed the 25-hydroxylation of vitamin D(3) and the 27-hydroxylation of 5beta cholestane-3alpha,7alpha,12alpha-triol, and the further oxidation of the product into the corresponding C(27)-acid 3alpha,7alpha,12alpha-trihydroxy-5beta cholestanoic acid. As part of these studies, the enzymic activities of cultured human embryonic kidney cells were examined using vitamin D(3) and C(27)-sterols as substrates. The cells were found to express CYP27A mRNA and to convert the respective substrates into the same products as recombinantly expressed CYP27A, i.e. 25-hydroxyvitamin D(3) and 27-oxygenated C(27)-sterols. The results of the present study describing the structure and expression of CYP27A in kidney suggest that this enzyme is involved in the renal metabolism of vitamin D(3) and that the kidney plays a role in the metabolism of cholesterol and other C(27)-sterols. PMID- 10749663 TI - Very-low-density lipoprotein binding to the apolipoprotein E receptor 2 is enhanced by lipoprotein lipase, and does not require apolipoprotein E. AB - The apolipoprotein (apo)E receptor 2 (apoER2) is a recently cloned member of the low-density lipoprotein (LDL) receptor (LDLR) family, showing a high homology with both the LDLR and the very-low-density lipoprotein (VLDL) receptor (VLDLR). In the present study, the binding characteristics of the apoER2 with respect to apoE and lipoprotein lipase (LPL) were investigated. VLDL was isolated from both apoE-deficient mice and mice expressing the human APOE2 (Arg(158)-->Cys) and APOE3-Leiden isoforms on an Apoe(-/-),Ldlr(-/-) double knock-out background. apoE rich rabbit beta-VLDL was used as a positive control for binding. Binding experiments performed with Chinese hamster ovary cells expressing the human apoER2 showed that the receptor was able to bind VLDL containing either of the apoE isoforms, as well as the apoE-deficient VLDL. Hence, in contrast with the VLDLR, the apoER2 is not strictly dependent on apoE for VLDL binding. Since LPL has been shown to enhance the binding of lipoproteins to several members of the LDLR family, including the LDLR-related protein, VLDL receptor, gp330 and the LDLR itself, VLDL binding experiments were performed in the presence of LPL. Addition of LPL resulted in a significant increase in apoER2 binding for all VLDL fractions used in this study. In conclusion, lipoprotein binding of VLDL to the apoER2 is enhanced in the presence of LPL, and is not restricted to apoE containing lipoproteins. PMID- 10749664 TI - Yeast cell-free system that catalyses joint-molecule formation in a Rad51p- and Rad52p-dependent fashion. AB - One of the central reactions of homologous recombination is the invasion of a single strand of DNA into a homologous duplex to form a joint molecule. Here we describe the isolation of a cell-free system from meiotic yeast cells that catalyses joint-molecule formation in vitro. The active components in the system required ATP and homologous DNA and operated in both 0.5 and 13 mM MgCl(2). When the cell-free system was prepared from rad51/rad51 and rad52/rad52 mutants and joint-molecule formation was assayed at 0.5 mM MgCl(2), the specific activity decreased to 6% and 13.8% respectively of the wild-type level. However, when the same mutant extracts were premixed, joint-molecule formation increased 4-8-fold, i.e. the mutant extracts exhibited complementation in vitro. These results demonstrated that Rad51p and Rad52p were required for optimal joint-molecule formation at 0.5 mM MgCl(2). Intriguingly, however, Rad51p and Rad52p seemed to be more dispensable at higher concentrations of MgCl(2) (13 mM). Further purification of the responsible activity has proven problematical, but it did flow through a sizing column as a single peak (molecular mass 1.2 MDa) that was co-eluted with Rad51p and RFA, the eukaryotic single-stranded DNA-binding protein. All of these characteristics are consistent with the known properties of the reaction in vivo and suggest that the new cell-free system will be suitable for purifying enzymes involved in homologous recombination. PMID- 10749660 TI - Regulation of cytochrome P450 (CYP) genes by nuclear receptors. AB - Members of the nuclear-receptor superfamily mediate crucial physiological functions by regulating the synthesis of their target genes. Nuclear receptors are usually activated by ligand binding. Cytochrome P450 (CYP) isoforms often catalyse both formation and degradation of these ligands. CYPs also metabolize many exogenous compounds, some of which may act as activators of nuclear receptors and disruptors of endocrine and cellular homoeostasis. This review summarizes recent findings that indicate that major classes of CYP genes are selectively regulated by certain ligand-activated nuclear receptors, thus creating tightly controlled networks. PMID- 10749665 TI - The CB1 cannabinoid receptor is coupled to the activation of protein kinase B/Akt. AB - Cannabinoids exert most of their effects in the central nervous system through the CB(1) cannabinoid receptor. This G-protein-coupled receptor has been shown to be functionally coupled to inhibition of adenylate cyclase, modulation of ion channels and activation of extracellular-signal-regulated kinase. Using Chinese hamster ovary cells stably transfected with the CB(1) receptor cDNA we show here that Delta(9)-tetrahydrocannabinol (THC), the major active component of marijuana, induces the activation of protein kinase B/Akt (PKB). This effect of THC was also exerted by the endogenous cannabinoid anandamide and the synthetic cannabinoids CP-55940 and HU-210, and was prevented by the selective CB(1) antagonist SR141716. Pertussis toxin and wortmannin blocked the CB(1) receptor evoked activation of PKB, pointing to the sequential involvement of a G(i)/G(o) protein and phosphoinositide 3'-kinase. The functionality of the cannabinoid induced stimulation of PKB was proved by the increased phosphorylation of glycogen synthase kinase-3 serine 21 observed in cannabinoid-treated cells and its prevention by SR141716 and wortmannin. Cannabinoids activated PKB in the human astrocytoma cell line U373 MG, which expresses the CB(1) receptor, but not in the human promyelocytic cell line HL-60, which expresses the CB(2) receptor. Data indicate that activation of PKB may be responsible for some of the effects of cannabinoids in cells expressing the CB(1) receptor. PMID- 10749666 TI - Post-translational modification of the myxoma-virus anti-inflammatory serpin SERP 1 by a virally encoded sialyltransferase. AB - SERP-1 is a secreted serpin (serine-proteinase inhibitor) encoded by myxoma virus, a poxvirus pathogen of rabbits. SERP-1 is required for myxoma-virus virulence, and the purified protein has been shown to possess independent anti inflammatory activity in animal models of restenosis and antigen-induced arthritis. As an inhibitor of serine proteinases, SERP-1 acts against tissue-type plasminogen activator, urokinase-type plasminogen activator, plasmin, thrombin and Factor Xa. In the present study, examination of SERP-1 glycosylation-site mutants showed that the N-linked glycosylation of Asn(172) was essential for SERP 1 secretion, whereas mutation of Asn(99) decreased secretion efficiency, indicating that N-linked glycosylation plays an essential role in the processing and trafficking of SERP-1. Furthermore, comparison of SERP-1 from wild-type myxoma virus and a virus containing a targeted disruption of the MST3N sialyltransferase locus demonstrated that SERP-1 is specifically modified by this myxoma-virus-encoded sialyltransferase, and is thus the first reported viral protein shown to by modified by a virally encoded glycosyltransferase. Sialylation of SERP-1 by the MST3N gene product creates a uniquely charged species of secreted SERP-1 that is distinct from SERP-1 produced from other eukaryotic expression systems, though this has no apparent effect upon the kinetics of in vitro proteinase inhibition. Rather, the role of viral sialylation of SERP-1 likely relates to masking antigenicity or targeting SERP-1 to specific sites of action in vivo. PMID- 10749667 TI - Expression and characterization of a recombinant cysteine proteinase of Leishmania mexicana. AB - A major cysteine proteinase (CPB) of Leishmania mexicana, that is predominantly expressed in the form of the parasite that causes disease in mammals, has been overexpressed in Escherichia coli and purified from inclusion bodies to apparent homogeneity. The CPB enzyme, CPB2.8, was expressed as an inactive pro-form lacking the characteristic C-terminal extension (CPB2.8DeltaCTE). Pro-region processing was initiated during protein refolding and proceeded through several intermediate stages. Maximum enzyme activity accompanied removal of the entire pro-region. This was facilitated by acidification. Purified mature enzyme gave a single band on SDS/PAGE and gelatin SDS/PAGE gels, co-migrated with native enzyme in L. mexicana lysates, and had the same N-terminal sequence as the native enzyme. The procedure yielded >3.5 mg of active enzyme per litre of E. coli culture. PMID- 10749668 TI - Among translational effectors, p70S6k is uniquely sensitive to inhibition by glucocorticoids. AB - Fundamental cellular processes such as cell differentiation and growth, apoptosis and cellular metabolism are regulated differentially by glucocorticoid hormones in a cell-context-related fashion. However, these basic processes are not governed by isolated signals but are influenced by the integration of both synergistic and antagonistic extracellular and intracellular stimuli. Because glucocorticoids and insulin-like growth factor I (IGF-I) reciprocally modulate growth-regulated processes such as translation initiation, especially in skeletal muscle, a study was undertaken to address the nature of this counter-regulation. Quiescent L6 skeletal myoblasts pretreated for 4 h with the synthetic glucocorticoid dexamethasone exhibited a marked attenuation of IGF-I-induced activation of the ribosomal protein S6 kinase (p70(S6k)). The adverse effects of glucocorticoids on the activity of the endogenous enzyme were due to differential dephosphorylation at discrete residues, suggesting that, physiologically, some but not all phosphorylation sites are subject to mitogenic regulation. Furthermore, the translational repressor eIF4E-binding protein 1 (4E-BP1), which in many circumstances is co-ordinately regulated with p70(S6k), was dephosphorylated in response to glucocorticoids; however, hyperphosphorylation of the protein after stimulation with IGF-I was refractory to inhibition by glucocorticoids, as was its dissociation from its binding partner, eIF4E. Although both basal and IGF-I-stimulated rates of protein synthesis were modestly affected by glucocorticoids, the synthesis of EF1A, whose mRNA precursor is a prototype for the terminal oligopyrimidine ('TOP') transcript family and whose expression is controlled by the activity of p70(S6k), was markedly affected. Therefore in this cell system it seems that, despite the mutual control of p70(S6k) and 4E-BP1 that is often observed, p70(S6k) is more sensitive to down regulation by glucocorticoids under growth-promoting conditions than is 4E-BP1. PMID- 10749669 TI - Glucose and amino acids modulate translation factor activation by growth factors in PC12 cells. AB - In PC12 phaeochromocytoma cells, protein synthesis is activated by epidermal and nerve growth factors (EGF and NGF). EGF and NGF also regulate a number of components of the translational machinery in these cells. Here we show that the ability of EGF and NGF to induce the phosphorylation of the 70 kDa ribosomal protein, S6 kinase, and the eukaryotic initiation factor (eIF), 4E-binding protein 1, is dependent upon the presence of amino acids (but not glucose) in the medium. This resembles the regulation of these proteins by insulin, which also requires amino acids. Glucose, but not amino acids, is required for the activation of eIF2B by EGF and NGF. In contrast, EGF and NGF can still activate protein synthesis in the absence of nutrients, suggesting that other regulatory events are important in this. In nutrient-deprived cells, an increase in the phosphorylation of eIF4E, and the assembly of the eIF4F complex by EGF and NGF, coincided with the activation of protein synthesis. In serum-starved cells, activation of protein synthesis, phosphorylation of eIF4E, and formation of the eIF4F complex, were blocked by inhibition of MEK, a component of the extracellular regulated kinase (ERK) signalling pathway. Thus the ERK pathway plays a key role in the regulation of protein synthesis in PC12 cells. PMID- 10749670 TI - Involvement of activator protein 1 complexes in the epithelium-specific activation of the laminin gamma2-chain gene promoter by hepatocyte growth factor (scatter factor). AB - Laminin-5 is a trimer of laminin alpha3, beta3 and gamma2 chains that is found in the intestinal basement membrane. Deposition of the laminin gamma2 chain at the basement membrane is of great interest because it undergoes a developmental shift in its cellular expression. Here we study the regulatory elements that control basal and cytokine-activated transcriptional expression of the LAMC2 gene, which encodes the laminin gamma2 chain. By using transient transfection experiments we demonstrated the presence of constitutive and cytokine-responsive cis-elements. Comparison of the transcriptional activity of the LAMC2 promoter in the epithelial HT29mtx cells with that in small-intestinal fibroblastic cells (C20 cells) led us to conclude that two regions with constitutive epithelium-specific activity are present between positions -1.2 and -0.12 kb. This was further validated by transfections of primary foetal intestinal endoderm and mesenchyme. A 2.5 kb portion of the LAMC2 5' flanking region was equally responsive to PMA and hepatocyte growth factor (HGF), whereas it was less responsive to transforming growth factor beta1. A minimal promoter limited to the initial 120 bp upstream of the transcriptional start site maintained inducibility by PMA and HGF. This short promoter fragment contains two activator protein 1 (AP-1) elements and the 5'-most of these is a composite AP-1/Sp1 element. The 5'AP-1 element is crucial to the HGF-mediated activity of the promoter; analysis of interacting nuclear proteins demonstrated that AP-1 proteins containing JunD mediate the response to HGF. PMID- 10749671 TI - Molecular cloning and biochemical characterization of a new mouse testis soluble zinc-metallopeptidase of the neprilysin family. AB - Because of their roles in controlling the activity of several bio-active peptides, members of the neprilysin family of zinc metallopeptidases have been identified as putative targets for the design of therapeutic agents. Presently, six members have been reported, these are: neprilysin, endothelin-converting enzyme (ECE)-1 and ECE-2, the Kell blood group protein, PHEX (product of the phosphate-regulating gene with homologies to endopeptidase on the X chromosome) and X-converting enzyme (XCE). In order to identify new members of this important family of peptidases, we designed a reverse transcriptase-PCR strategy based on conserved amino acid sequences of neprilysin, ECE-1 and PHEX. We now report the cloning from mouse testis of a novel neprilysin-like peptidase that we called NL1. NL1 is a glycoprotein that, among the members of the family, shows the strongest sequence identity with neprilysin. However, in contrast with neprilysin and other members of the family which are type II integral membrane proteins, NL1 was secreted when expressed in cultured mammalian cells, likely due to cleavage by a subtilisin-like convertase at a furin-like site located 22 amino acid residues in the C-terminus of the transmembrane domain. The recombinant enzyme exhibited neprilysin-like peptidase activity and was efficiently inhibited by phosphoramidon and thiorphan, two inhibitors of neprilysin. Northern blot analysis and in situ hybridization showed that NL1 mRNA was found predominantly in testis, specifically in round and elongated spermatids. This distribution of NL1 mRNA suggests that it could be involved in sperm formation or other processes related to fertility. PMID- 10749672 TI - Purification and characterization of secretory phospholipase B, lysophospholipase and lysophospholipase/transacylase from a virulent strain of the pathogenic fungus Cryptococcus neoformans. AB - Infection caused by the fungus Cryptococcus neoformans is potentially fatal. A highly active extracellular phospholipase, demonstrating phospholipase B (PLB), lysophospholipase (LPL) and lysophospholipase/transacylase (LPTA) activities, was purified to homogeneity from C. neoformans using (NH(4))(2)SO(4) fractionation, and hydrophobic-interaction, anion-exchange and gel-filtration chromatography. All three enzyme activities co-purified as a single protein with an apparent molecular mass of 70-90 kDa by SDS/PAGE and 160-180 kDa by gel filtration. The ratio of the three activities remained constant after each purification step. The amino acid composition, as well as the sequences of the N-terminus and of five internal peptide fragments were novel. The protein was an acidic glycoprotein containing N-linked carbohydrate moieties, with pI values of 5.5 and 3.5. The apparent V(max) values for PLB and LPL activities were 12.3 and 870 micromol/min per mg of protein respectively; the corresponding K(m) values were approx. 185.3 and 92.2 microM. The enzyme was active only at acidic pH (pH optimum of 4.0 for PLB and 4.0-5.0 for LPL and LPTA). Enzyme activity did not require added cations, but was inhibited by Fe(3+). LPL and LPTA activities were decreased by 0.1% (v/v) Triton X-100 to 50% of the control value. Palmitoylcarnitine (0.5 mM) inhibited PLB (97% inhibition) and LPL and LPTA activities (35% inhibition) competitively. All phospholipids except phosphatidic acid were degraded by PLB, but dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylcholine were the preferred substrates. This is the first complete description of the purification and properties of a phospholipase, which may be involved in virulence, from a pathogenic fungus. PMID- 10749674 TI - Glucocorticoid regulation of branched-chain alpha-ketoacid dehydrogenase E2 subunit gene expression. AB - Regulation of the mammalian branched-chain alpha-ketoacid dehydrogenase complex (BCKAD) occurs under a variety of stressful conditions associated with changes in circulating glucocorticoids. Multiple levels of regulation in hepatocytes, including alteration of the levels of the structural subunits available for assembly (E1, alpha-ketoacid decarboxylase; E2, dihydrolipoamide acyltransferase; and E3, dihydrolipoamide dehydrogenase), as well as BCKAD kinase, which serves to phosphorylate the E1alpha subunit and inactivate complex activity, have been proposed. The direct role of glucocorticoids in regulating the expression of the murine gene encoding the major BCKAD subunit E2, upon which the other BCKAD subunits assemble, was therefore examined. Deletion analysis of the 5' proximal 7.0 kb of the murine E2 promoter sequence, using E2 promoter/luciferase expression minigene plasmids introduced into the hepatic H4IIEC3 cell line, suggested a promoter proximal region responsive to glucocorticoid regulation. Linker-scanning mutagenesis combined with deletion analysis established this functional glucocorticoid-responsive unit (GRU) to be located near the murine E2 proximal promoter site at -140 to -70 bp upstream from the transcription initiation site. The presence of this region in plasmid minigenes, containing varying amounts of the murine genomic sequence 5' upstream from proximal E2 promoter sequences, conferred 2-10 fold increases in luciferase reporter gene expression in H4IIEC3 cells, whether introduced by transient transfection or following co-selection for stable transfectants. The GRU region itself appeared to contain multiple interacting elements that combine to regulate overall E2 promoter activity in response to changing physiological conditions associated with varying concentrations of glucocorticoids and likely other hormonal effectors. PMID- 10749673 TI - Protein-tyrosine-phosphatase-mediated epidermal growth factor (EGF) receptor transinactivation and EGF receptor-independent stimulation of mitogen-activated protein kinase by bradykinin in A431 cells. AB - Transactivation of the epidermal growth factor (EGF) receptor (EGFR) has been proposed to represent an essential link between G-protein-coupled receptors and the mitogen-activated protein kinase (MAPK) pathway in various cell types. In the present work we report, in contrast, that in A431 cells bradykinin transinactivates the EGFR and stimulates MAPK activity independently of EGFR tyrosine phosphorylation. Both effects of bradykinin are mediated by a pertussis toxin-insensitive G-protein. Three lines of evidence suggest the activation of a protein tyrosine phosphatase (PTP) by bradykinin: (i) treatment of A431 cells with bradykinin decreases both basal and EGF-induced EGFR tyrosine phosphorylation, (ii) this effect of bradykinin can be blocked by two different PTP inhibitors, and (iii) bradykinin significantly increased the PTP activity in total A431 cell lysates when measured in vitro. The transmembrane receptor PTP sigma was identified as a putative mediator of bradykinin-induced downregulation of EGFR autophosphorylation. Activation of MAPK in response to bradykinin was insensitive towards AG 1478, a specific inhibitor of EGFR tyrosine kinase, but was blocked by wortmannin or bisindolylmaleimide, inhibitors of phosphatidylinositol 3-kinase (PI3-K) and protein kinase C (PKC) respectively. These results also suggest that the bradykinin-induced activation of MAPK is independent of EGFR and indicate a pathway involving PI3-K and PKC. In addition, bradykinin evokes a rapid and transient increase in Src kinase activity. Although Src does not participate in bradykinin-induced stimulation of PTP activity, inhibition of Src by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4 d)pyrimidine leads to an increase in MAPK activation by bradykinin. Our results suggest that in A431 cells the G(q/11)-protein-coupled bradykinin B(2) receptor may stimulate PTP activity and thereby transinactivate the EGFR, and may simultaneously activate MAPK by an alternative signalling pathway which can bypass EGFR. PMID- 10749675 TI - N- and C-termini modulate the effects of pH and phosphorylation on hepatic 6 phosphofructo-2-kinase/fructose-2,6-bisphosphatase. AB - Liver and skeletal muscle isoforms of 6-phosphofructo-2-kinase/fructose-2,6 bisphosphatase (6PF2K/Fru-2,6-P(2)ase) isoenzymes are products of alternatively spliced first exons of the same gene, with common kinase and bisphosphatase domains. The muscle-specific exon-1 encodes nine unique amino acids, that lack the cAMP-dependent protein kinase (PK-A) phosphorylation site, and differ in sequence from those encoded by the liver-specific exon-1 (32 amino acids), contributing to its much lower affinity for fructose 6-phosphate (Fru-6-P). PK-A phosphorylation of the liver isoform at Ser(32) reduces the affinity of the kinase for Fru-6-P, and stimulates the bisphosphatase V(max). In the present study, we have defined the locus of interaction of the N-terminal residues with the N-terminal kinase and C-terminal domains by successive N- and C-terminal deletions. This study shows that: (1) residues Gly(5)-Glu(6)-Leu(7) of the liver isoform are responsible for increasing the affinity of 6PF2K for Fru-6-P, maintaining the inhibition of Fru-2,6-P(2)ase activity, and mediating the effects of PK-A phosphorylation on the two activities; (2) the loss of Fru-6-P inhibition of the bisphosphatase and the enhancement of its V(max), rather than the inhibition of the kinase, may be responsible for the behaviour of the muscle isoform primarily as a bisphosphatase; (3) the composition of residues 24-32 of the liver form appears to confer the enhanced kinase catalytic rate of this form over that of the muscle isoform. It is concluded that specific regions of the N terminus of liver and skeletal muscle 6PF2K/Fru-2,6-P(2)ase have a role in adapting the two activities to work in the physiological range of pH and substrate concentrations found in each particular tissue. PMID- 10749676 TI - Thrombospondin-1 binds to polyhistidine with high affinity and specificity. AB - Thrombospondin-1 (TSP1) is a secreted trimeric glycoprotein of 450 kDa with demonstrated effects on cell growth, adhesion and migration. Its complex biological activity is attributed to its ability to bind to cell-surface receptors, growth factors and extracellular-matrix proteins. In this study, we used a (125)I solid-phase binding assay to demonstrate that TSP1 binds specifically to proteins containing polyhistidine stretches. Based on studies with three different six-histidine-containing recombinant proteins, we derived an average dissociation constant of 5 nM. The binding of (125)I-labelled TSP1 to these proteins was inhibited by peptides containing histidine residues, with the degree of competition being a function of the number of histidines within the peptide. Binding was not inhibited by excess histidine or imidazole, indicating that the imidazole ring is not sufficient for recognition by TSP1. Heparin was a potent inhibitor of binding with a K(i) of 50 nM, suggesting that the heparin binding domain of TSP1 may be involved in this interaction. This was confirmed by the ability of a recombinant heparin-binding domain of TSP1 to directly compete for TSP1 binding to polyhistidine-containing proteins. Affinity chromatography with a polyhistidine-containing peptide immobilized on agarose revealed that TSP1 in platelet releasates is the major polypeptide retained on the six-histidine peptide column. We conclude that TSP1 contains a high-affinity binding site for polyhistidine and this is likely to be the molecular basis for the observed binding of TSP1 to histidine-rich glycoprotein. The possibility that other polyhistidine-containing proteins also interact with TSP1 warrants further study. PMID- 10749678 TI - Characterization of a chicken retinoid X receptor-gamma gene promoter and identification of sequences that direct expression in retinal cells. AB - Development of the cellular complexity of the vertebrate neural retina relies on an intricate interplay between extracellular signals and intracellular factors. In particular, transcription factors play a key role in determining the competence of cells to respond to extracellular signals. We have previously shown that, in the developing chick neural retina, expression of the retinoid X receptor-gamma (RXR-gamma2) nuclear receptor gene is restricted to photoreceptors. To characterize the mechanisms that regulate expression of this gene in the neural retina, we isolated a chicken RXR-gamma genomic clone containing the RXR-gamma2 promoter and mapped the transcription initiation site by means of ribonuclease protection. We analysed promoter activity by transient transfection of luciferase reporter gene constructs into cultured cells isolated from embryonic-chick neural retina or facial mesenchyme, which does not normally express detectable RXR-gamma2 transcripts. The DNA fragment lying between nucleotides -657 and +37 with respect to the transcription initiation site had basal promoter activity in both cell types. The fragment lying between nucleotides -1198 and -991 directed 10-20-fold higher levels of luciferase activity in neural retina cells, but only basal levels in facial mesenchyme cells. This 208 bp fragment also enhanced the activity of the simian-virus-40 promoter, when placed upstream in either orientation. Electrophoretic-mobility shift assays using this 208 bp fragment demonstrated the formation of four neural retina-specific protein-DNA complexes. These results indicate that regulation of RXR-gamma2 transcription in the developing chick neural retina involves the binding of one or more neural retina-specific protein factors to an enhancer element located approx. 1 kbp upstream of the transcription initiation site. PMID- 10749677 TI - Nitric oxide-induced autoinhibition of neuronal nitric oxide synthase in the presence of the autoxidation-resistant pteridine 5-methyltetrahydrobiopterin. AB - Nitric oxide synthase (NOS) catalysis results in formation of NO or superoxide (O(2)(-.)) depending on the presence or absence of the cofactor tetrahydrobiopterin (BH4). In the absence of O(2)(-.) scavengers, net NO formation cannot be detected even at saturating BH4 concentrations, which is thought to be due to O(2)(-.) production by BH4 autoxidation. Because the N-5 methylated analogue of BH4 (5-Me-BH4) sustains NOS catalysis and is autoxidation resistant, net NO formation by the neuronal isoform of NOS (nNOS) can be observed at saturating 5-Me-BH4 concentrations. Here we compare the effects of 5-Me-BH4 on L-citrulline formation, NADPH oxidation, H(2)O(2) production and soluble guanylate cyclase (sGC) stimulation. All activities were stimulated biphasically (EC(50) approx. 0.2 microM and more than 1 mM), with an intermediate inhibitory phase at the same pterin concentration as that required for net NO generation and sGC stimulation (4 microM). Concomitantly with inhibition, the NADP(+)/L citrulline stoichiometry decreased from 2.0 to 1.6. Inhibition occurred only at high enzyme concentrations (IC(50) approx. 10 nM nNOS) and was antagonized by oxyhaemoglobin and by BH4. We ascribe the first stimulatory phase to high affinity binding of 5-Me-BH4. The inhibitory phase is due to low-affinity binding, resulting in fully coupled catalysis, complete inhibition of O(2)(-.) production and net NO formation. At high enzyme concentrations and thus high NO levels, this causes autoinhibition. NO scavenging by 5-Me-BH4 at concentrations above 1 mM, resulting in the antagonization of inhibition of NOS, explains the second stimulatory phase. In agreement with these assignments 5-Me-BH4 was found to stimulate formation of a haem-NO complex during NOS catalysis. The observation of inhibition with 5-Me-BH4 but not with BH4 implies that, unless O(2)(-.) scavengers are present, a physiological role for NO-induced autoinhibition is unlikely. PMID- 10749680 TI - The Shc-related adaptor protein, Sck, forms a complex with the vascular endothelial-growth-factor receptor KDR in transfected cells. AB - Despite much progress in recent years, the precise signalling events triggered by the vascular-endothelial-growth-factor (VEGF) receptors, fms-like tyrosine kinase (Flt1) and kinase insert domain-containing receptor (KDR), are incompletely defined. Results obtained when Flt1 and KDR are individually expressed in fibroblasts or porcine aortic endothelial cells have not been entirely consistent with those observed in other endothelial cells expressing both receptors endogenously. It has also been difficult to demonstrate VEGF-induced phosphorylation of Flt1, which has led to speculation that KDR may be the more important receptor for the mitogenic action of VEGF on endothelial cells. In an attempt to identify physiologically important effectors which bind to KDR, we have screened a yeast two-hybrid mouse embryo library with the cytoplasmic domain of KDR. Here we describe the identification of the adaptor protein, Shc-like protein (Sck), as a binding partner for KDR. We demonstrate that this interaction requires phosphorylation of KDR, and identify the binding site for the Src homology 2 (SH2) domain as tyrosine-1175 of KDR. We have also shown that the SH2 domain of Sck, but not that of Src-homology collagen protein (Shc), can precipitate phosphorylated KDR from VEGF-stimulated porcine aortic endothelial cells expressing KDR, and that an N-terminally truncated Sck protein can associate with KDR, in a phosphorylation-dependent fashion, when co-expressed in human embryonic kidney 293 cells. Furthermore, we demonstrate that in the two hybrid assay, both Shc and Sck SH2 domains can associate with the related receptor Flt1. PMID- 10749679 TI - Regional evolution of venom-gland phospholipase A2 isoenzymes of Trimeresurus flavoviridis snakes in the southwestern islands of Japan. AB - Conventional chromatographic analysis showed that phospholipase A(2) (PLA(2)) isoenzymes of the venom of Trimeresurus flavoviridis (Habu snake) of Okinawa island are profoundly different in composition from those of T. flavoviridis of Amami-Oshima and Tokunoshima islands. The most striking feature was that myotoxic [Lys(49)]PLA(2) isoenzymes, called BPI and BPII, which are expressed abundantly in the venoms of Amami-Oshima and Tokunoshima T. flavoviridis, are missing from the venom of Okinawa T. flavoviridis. Northern blot analysis of Okinawa T. flavoviridis venom-gland mRNA species showed the absence of BPI and BPII mRNA species. Analysis by single-stranded conformational polymorphism-PCR of venom gland mRNA species of T. flavoviridis from three islands, with reference to five DNA species each encoding different PLA(2) isoenzymes from Tokunoshima T. flavoviridis venom gland, also suggested that BPI and BPII mRNA species are not expressed in Okinawa T. flavoviridis venom gland. In contrast, genomic Southern blot analysis with a variety of probes showed that only the bands corresponding to the upstream and downstream regions of the genes for BPI and/or BPII can be detected in Okinawa T. flavoviridis. These results suggested that the genes for BPI and BPII in Okinawa T. flavoviridis genome had been inactivated to form pseudogenes. Differently from Amami-Oshima and Tokunoshima T. flavovirdis genomic DNAs, PCR amplification of the segments of BPI and BPII genes between the 5' moiety of second exon and the middle portion of second intron failed for Okinawa T. flavoviridis genomic DNAs. In sequence analysis of the two segments involving polymorphism between BPI and BPII genes, which are located in first exon and third exon, respectively, only one base was detected at the polymorphic positions for pseudogene in Okinawa T. flavoviridis genome. Based on these facts, it became evident for pseudogene that the upstream region of BPI gene down to the 5' moiety of second exon and the downstream region of BPII gene starting from the middle portion of second intron are in a linked form with a possible insertion. Such observations suggest that venom-gland genes for PLA(2) isoenzymes in T. flavoviridis snakes isolated for one to two million years have evolved independently. Their evolution is regional and seems, from several lines of consideration and observation, to be adaptive to the environment. PMID- 10749681 TI - Regulation of expression of the human beta-1,2-N-acetylglucosaminyltransferase II gene (MGAT2) by Ets transcription factors. AB - Oncogenic transformation of fibroblasts by the src oncogene has long been known to cause an increase in the size of cell-surface protein-bound oligosaccharides, owing primarily to increased N-glycan branching mediated by increased beta-1,6-N acetylglucosaminyltransferase V (GnT V) activity. The src-responsive element of the GnT V promoter was localized to Ets-binding sites and the promoter was transcriptionally stimulated by both ets-1 and ets-2 expression [Buckhaults, Chen, Fregien and Pierce (1997) J. Biol. Chem. 272, 19575-19581; Kang, Saito, Ihara, Miyoshi, Koyama, Sheng and Taniguchi (1996) J. Biol. Chem. 271, 26706 26712]. Because GnT V action requires the prior action of beta-1,2-N acetylglucosaminyltransferase II (GnT II) and the human GnT II promoter contains four putative Ets-binding sites [Chen, Zhou, Tan and Schachter (1998) Glycoconj. J. 15, 301-308], GnT II might also be under oncogenic control via Ets transcription factors. We now report that co-transfection into HepG2 or COS-1 cells of either ets-1 or ets-2 expression plasmids together with chimaeric GnT II promoter-chloramphenicol acetyltransferase plasmids results in a 2-4-fold stimulation of promoter activity. Mobility-shift assays and South-Western blots localized the functional Ets-binding site to one of the four putative sites on the GnT II promoter. The GnT II promoter, unlike the GnT V promoter, is not activated by either src or neu. Therefore although both promoters are stimulated by a member of the Ets family of transcription factors, the functional role of this Ets transcriptional control seems to be different for the two genes. PMID- 10749682 TI - Point mutations at multiple sites including highly conserved amino acids maintain activity, but render O6-alkylguanine-DNA alkyltransferase insensitive to O6 benzylguanine. AB - The DNA repair protein, O(6)-alkylguanine-DNA alkyltransferase (AGT), is inactivated by reaction with the pseudosubstrate, O(6)-benzylguanine (BG). This inactivation sensitizes tumour cells to chemotherapeutic alkylating agents, and BG is aimed at enhancing cancer treatment in clinical trials. Point mutations in a 24 amino acid sequence likely to form the BG-binding pocket were identified using a screening method designed to identify BG-resistant mutants. It was found that alterations in 21 of these residues were able to render AGT resistant to BG. These included mutations at the highly conserved residues Lys(165), Leu(168) and Leu(169). The two positions at which changes led to the largest increase in resistance to BG were Gly(156) and Lys(165). Eleven mutants at Gly(156) were identified, with increases in resistance ranging from 190-fold (G156V) to 4400 fold (G156P). Two mutants at Lys(165) found in the screen (K165S and K165A) showed 620-fold and 100-fold increases in resistance to BG. Two mutants at the Ser(159) position (S159I and S159V) were >80-fold more resistant than wild-type AGT. Eleven active mutants at Leu(169) were also resistant to BG, but with lower increases (5-86-fold). Fourteen BG-resistant mutants were found for position Cys(150), with 3-26-fold increases in the amount of inhibitor needed to produce a 50% loss of activity in a 30 min incubation. Six BG-resistant mutants at Asn(157) were found with increases of 4-13-fold. These results show that many changes can render human AGT resistant to BG without preventing the ability to protect tumour cells from therapeutic alkylating agents. PMID- 10749683 TI - Conserved residue lysine165 is essential for the ability of O6-alkylguanine-DNA alkyltransferase to react with O6-benzylguanine. AB - The role of lysine(165) in the activity of the DNA repair protein, O(6) alkylguanine-DNA alkyltransferase (AGT), and the ability of AGT to react with the pseudosubstrate inhibitor, O(6)-benzylguanine (BG), was investigated by changing this lysine to all other 19 possibilities. All of these mutants (except for K165T, which could not be tested as it was too poorly active for assay in crude cell extracts) gave BG-resistant AGTs with increases in the amount of inhibitor needed to produce a 50% loss of activity in a 30 min incubation (ED(50)) from 100 fold (K165A) to 2400-fold (K165F). Lys(165) is a completely conserved residue in AGTs from many species, and all of the mutations at this site also reduced the ability to repair methylated DNA. The least deleterious change was that to arginine, which reduced the rate constant for DNA repair by approx. 2.5-fold. Mutant K165R resembled all of the other mutants in being highly resistant to BG, with an ED(50) value for inactivation by BG>200-fold greater than wild-type. Detailed studies of purified K165A AGT showed that the rate constant for repair and the binding to methylated DNA substrates were reduced by 10-20-fold. Despite this, the K165A mutant AGT was able to protect cells from alkylating agents and this protection was not abolished by BG. These results show that, firstly, lysine at position 165 is needed for optimal activity of AGT towards methylated DNA substrates and is essential for efficient reaction with BG; and second, even if the AGT activity towards methylated DNA substrates is impaired by mutations at codon 165, such mutants can protect tumour cells from therapeutic alkylating agents. These results raise the possibility that the conservation of Lys(165) is due to the need for AGT activity towards substrates containing more bulky adducts than O(6)-methylguanine. They also suggest that alterations at Lys(165) may occur during chemotherapy with BG and alkylating agents and could limit the effectiveness of this therapy. PMID- 10749684 TI - Order and maximum incorporation of N-acetyl-D-galactosamine into threonine residues of MUC2 core peptide with microsome fraction of human-colon-carcinoma LS174T cells. AB - Mucin 2 (MUC2) is the major intestinal mucin. O-glycans are attached to MUC2 in a potentially diverse arrangement, which is crucial for their interaction with endogeneous and exogeneous lectins. In the present report, five oligopeptides [PTTTPITTTT(K), ITTTTTVTPT(K), TVTPTPTPTG(K), PTPTGTQTPT(K) and TQTPTTTPIT(K)] corresponding to portions of the MUC2 tandem repeat domain were synthesized, and incubated with UDP-N-acetyl-D-galactosamine (UDP-GalNAc) and detergent-soluble microsomes, prepared from the human colon carcinoma cell line LS174T. The products were fractionated by reverse-phase HPLC and characterized by matrix assisted laser-desorption ionization-time-of-flight mass spectrometry. Oligopeptides with GalNAc residues derived from PTTTPITTTT(K), containing consecutive threonine residues, were found to be glycosylated with 1-7 GalNAc residues per single peptide. The sequences of all glycopeptides were determined. The results indicated that the predominant sites of the first through to the sixth GalNAc incorporation were Thr(3), Thr(6), Thr(5), Thr(2), Thr(4) and Thr(1), respectively. An exception was the presence of a glycopeptide with three GalNAc residues at Thr(1), Thr(4) and Thr(5). Oligopeptides containing alternating threonine residues [TVTPTPTPTG(K) and PTPTGTQTPT(K)] were not fully glycosylated under the same conditions or even after prolonged incubations. Thus, the preferential order and maximum number of GalNAc incorporation into threonine residues of MUC2 core peptides depends on the peptide sequence, when the microsome fraction of LS174T cells is used as a source of N-acetyl-D galactosaminyltransferases. PMID- 10749685 TI - Apoptosis or senescence-like growth arrest: influence of cell-cycle position, p53, p21 and bax in H2O2 response of normal human fibroblasts. AB - Early-passage human diploid fibroblasts (HDFs) undergo senescence-like growth arrest in response to sublethal concentrations of H(2)O(2) [Chen and Ames (1994) Proc. Natl. Acad. Sci. USA. 95, 4130-4134]. We determine here whether H(2)O(2) can cause apoptosis in HDFs and the molecular changes that differ between apoptosis and senescence-like growth arrest. When exponentially growing early passage IMR-90 cells were treated for 2 h with 50-200 microM (or 0.25-1 pmol/cell) H(2)O(2), a fraction of cells detached at 16-32 h after the treatment. The cells remaining attached were growth-arrested and developed features of senescence in 1 week. The detached cells showed caspase-3 activation and typical morphological changes associated with apoptosis. Caspase-3 activation was H(2)O(2) dose-dependent and preceded nuclear condensation or plasma membrane leakage. Apoptotic cells were mainly distributed in the S-phase of the cell cycle, while growth-arrested cells exhibited predominantly G1- and G2/M-phase distributions. H(2)O(2) pretreatment induced G1 arrest and prohibited induction of apoptosis by a subsequent H(2)O(2) challenge. The p53 protein showed an average 6.1-fold elevation in apoptotic cells and a 3.5-fold elevation in growth arrested cells. Reduction of p53 levels with human papillomavirus E6 protein prohibited the activation of caspase-3 and decreased the proportion of apoptotic cells. Growth-arrested cells had elevated p21, while p21 was absent in apoptotic cells. Bcl-2 was elevated in both growth-arrested and apoptotic cells. Finally, although the overall level of bax did not change in growth-arrested or apoptotic cells, the solubility of bax protein increased in apoptotic cells. Our data suggest that in contrast with growth-arrested cells, apoptotic cells show an S phase cell cycle distribution, a higher degree of p53 elevation, an absence of p21 protein and increased solubility of bax protein. PMID- 10749686 TI - Conserved arginine-516 of Penicillium amagasakiense glucose oxidase is essential for the efficient binding of beta-D-glucose. AB - The effects of mutation of key conserved active-site residues (Tyr-73, Phe-418, Trp-430, Arg-516, Asn-518, His-520 and His-563) of glucose oxidase from Penicillium amagasakiense on substrate binding were investigated. Kinetic studies on the oxidation of beta-D-glucose combined with molecular modelling showed the side chain of Arg-516, which forms two hydrogen bonds with the 3-OH group of beta D-glucose, to be absolutely essential for the efficient binding of beta-D glucose. The R516K variant, whose side chain forms only one hydrogen bond with the 3-OH group of beta-D-glucose, exhibits an 80-fold higher apparent K(m) (513 mM) but a V(max) only 70% lower (280 units/mg) than the wild type. The complete elimination of a hydrogen-bond interaction between residue 516 and the 3-OH group of beta-D-glucose through the substitution R516Q effected a 120-fold increase in the apparent K(m) for glucose (to 733 mM) and a decrease in the V(max) to 1/30 (33 units/mg). None of the other substitutions, with the exception of variant F418A, affected the apparent K(m) more than 6-fold. In contrast, the removal of aromatic or bulky residues at positions 73, 418 or 430 resulted in decreases in the maximum rates of glucose oxidation to less than 1/90. Variants of the potentially catalytically active His-520 and His-563 were completely, or almost completely, inactive. Thus, of the residues forming the active site of glucose oxidase, Arg-516 is the most critical amino acid for the efficient binding of beta-D-glucose by the enzyme, whereas aromatic residues at positions 73, 418 and 430 are important for the correct orientation and maximal velocity of glucose oxidation. PMID- 10749687 TI - Involvement of proline-rich tyrosine kinase 2 in platelet activation: tyrosine phosphorylation mostly dependent on alphaIIbbeta3 integrin and protein kinase C, translocation to the cytoskeleton and association with Shc through Grb2. AB - Proline-rich tyrosine kinase 2 (Pyk2) (also known as RAFTK, CAKbeta or CADTK) has been identified as a member of the focal adhesion kinase (FAK) family of protein tyrosine kinases and it has been suggested that the mode of Pyk2 activation is distinct from that of FAK. In the present study we investigated the mode of Pyk2 activation in human platelets. When platelets were stimulated with thrombin, Pyk2, as well as FAK, was markedly tyrosine-phosphorylated, in a manner mostly dependent on alphaIIbbeta3 integrin-mediated aggregation. The residual Pyk2 tyrosine phosphorylation observed in the absence of platelet aggregation was completely abolished by pretreatment with BAPTA/AM [bis-(o-aminophenoxy)ethane N,N,N',N'-tetra-acetic acid acetoxymethyl ester]. The Pyk2 phosphorylation was inhibited by protein kinase C (PKC) inhibitors at concentrations that inhibited platelet aggregation. In contrast, direct activation of PKC with the active phorbol ester PMA induced the tyrosine phosphorylation of Pyk2 and FAK but only when platelets were fully aggregated with the exogenous addition of fibrinogen (the ligand for alphaIIbbeta3 integrin). Furthermore, PMA-induced Pyk2 (and FAK) tyrosine phosphorylation was also observed when platelets adhered to immobilized fibrinogen. The activation of the von Willebrand factor (vWF)--glycoprotein Ib pathway with botrocetin together with vWF failed to induce Pyk2 (and FAK) tyrosine phosphorylation. Most Pyk2 and FAK was present in the cytosol and membrane skeleton fractions in unstimulated platelets. When platelets were stimulated with thrombin, both Pyk2 and FAK were translocated to the cytoskeleton in an aggregation-dependent manner. In immunoprecipitation studies, Pyk2, as well as FAK, seemed to associate with Shc through Grb2. With the use of glutathione S transferase fusion proteins containing Shc-SH2, Grb2-SH2, and Grb2 N-terminal and C-terminal SH3 domains, it was implied that the proline-rich region of Pyk2 (and FAK) binds to the N-terminal SH3 domain of Grb2 and that the phosphotyrosine residue of Shc binds to the SH2 domain of Grb2. Although Pyk2 and FAK have been reported to be differentially regulated in many cell types, our results suggest that, in human platelets, the mode of Pyk2 activation is mostly similar to that of FAK, in terms of alphaIIbbeta3 integrin-dependent and PKC-dependent tyrosine phosphorylation. Furthermore, Pyk2, as well as FAK, might have one or more important roles in post-aggregation tyrosine phosphorylation events, in association with the cytoskeleton and through interaction with adapter proteins including Grb2 and Shc. PMID- 10749689 TI - The poly(A) tail length of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of milk. AB - The length of casein mRNA from the lactating mouse mammary gland, as assessed on Northern blots, is shorter after weaning, but is elongated following the removal of milk. In order to investigate this phenomenon, the molecular structures of beta- and gamma-casein mRNAs were analysed. The coding and non-coding regions of the two forms were the same length, but the long form of casein mRNA had a longer poly(A) tail than the short form (P<0.05). In order to examine the stability of casein mRNA under identical conditions, casein mRNAs with the long and short poly(A) tails were incubated in the rabbit reticulocyte lysate (RRL) cell-free translation system. Casein mRNA with the long poly(A) tail had a longer half-life than that with the short tail (P<0.05). The beta- and gamma-casein mRNAs were first degraded into 0.92 and 0.81 kb fragments respectively. With undegraded mRNA, the poly(A) tail shortening by exoribonuclease was not observed until the end of the incubation. Northern blot analysis showed that casein mRNA with the long poly(A) tail was protected efficiently from endoribonucleases. We conclude that the length of the poly(A) tail of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of the gland's milk, and we show that the longer poly(A) tail potentially protects the mRNA from degradation by endoribonucleases. PMID- 10749688 TI - Action of rolipram on specific PDE4 cAMP phosphodiesterase isoforms and on the phosphorylation of cAMP-response-element-binding protein (CREB) and p38 mitogen activated protein (MAP) kinase in U937 monocytic cells. AB - U937 monocytic cells are shown here to express a range of PDE4, cAMP-specific phosphodiesterase (PDE) isoenzymes: the long isoenzymes, PDE4A4, PDE4D5 and PDE4D3, plus the short isoenzyme, PDE4B2. These isoenzymes provide around 76% of the total cAMP PDE activity of U937 cells. The specific activities of the total PDE4A, PDE4B and PDE4D activities were 0.63+/-0.09, 8.8+/-0.2 and 34.4+/-2.9 pmol/min per mg of protein respectively. The PDE4 selective inhibitor, rolipram, inhibited immunopurified PDE4B and PDE4D activities similarly, with IC(50) values of approx. 130 nM and 240 nM respectively. In contrast, rolipram inhibited immunopurified PDE4A activity with a dramatically lower IC(50) value of around 3 nM. Rolipram increased phosphorylation of cAMP-response-element-binding protein (CREB) in U937 cells in a dose-dependent fashion, which implied the presence of both high affinity (IC(50) value approx. 1 nM) and low affinity (IC(50) value approx. 120 nM) components. Rolipram dose-dependently inhibited the interferon gamma (IFN-gamma)-stimulated phosphorylation of p38 mitogen-activated protein (MAP) kinase in a simple monotonic fashion with an IC(50) value of approx. 290 nM. On this basis, it is suggested that rolipram inhibition of PDE4A4 is involved in regulating CREB phosphorylation but not IFN-gamma-stimulated p38 MAP kinase phosphorylation. PDE4A4 was also selectively activated by challenge of U937 cells with either bacterial lipopolysaccharide (LPS) or IFN-gamma through a process which was attenuated by both wortmannin and rapamycin. It is proposed that the PDE4A4 isoform is involved in compartmentalized cAMP signalling responses in U937 monocytes. PMID- 10749690 TI - Lignin peroxidase initiates O2-dependent self-propagating chemical reactions which accelerate the consumption of 1-(3',4'-dimethoxyphenyl)propene. AB - Lignin peroxidase (LiP) has been used to study the C(alpha)-C(beta) cleavage of the propylene side chain in 1-(3',4'-dimethoxyphenyl)propene (DMPP) to 3,4 dimethoxybenzaldehyde (veratraldehyde, VAD). Under an air atmosphere, LiP oxidized DMPP to VAD (27.8%) and 1-(3',4'-dimethoxyphenyl)propan-2-one (DMPA, 8.7%), after 10 min of incubation. Dissolved O(2) was rapidly consumed during DMPP conversion, of which one-third was converted into superoxide. The remaining two-thirds of the consumed O(2) was involved in C(alpha)-C(beta) cleavage of DMPP to VAD and in self-propagating chemical reactions stimulating the consumption of DMPP. The involvement of peroxyl radicals, in the chemical consumption of DMPP, was confirmed by using the well-known peroxyl radical reductant Mn(2+). This metal ion severely inhibited the DMPP consumption rate under air, but did not affect the lower enzymic DMPP consumption rate under N(2). The substoichiometric requirement of LiP for H(2)O(2) during DMPP oxidation could be explained in part by dismutation of superoxide, but more importantly by direct chemical reactions of DMPP-derived peroxyl radicals with fresh DMPP. Another VAD-producing route was found by incubating the DMPP oxidation product, DMPA, with LiP. Under air the molar yield of VAD was 29.7%. In the absence of O(2), the C(alpha)-C(beta) cleavage of DMPA to VAD was strongly inhibited and side-chain coupling products (dimers) were formed instead. As a whole, the results suggest two new roles of O(2) in LiP-mediated oxidation of aromatic substrates. First, O(2) is responsible for the formation of reactive peroxyl intermediates, which can directly react with other substrate molecules and thereby accelerate consumption rates. Secondly, O(2) can prevent coupling reactions by lowering the pool of carbon centred radicals accumulating during LiP catalysis. PMID- 10749692 TI - For want of a few good shams. PMID- 10749691 TI - Inositol 1,4,5-trisphosphate-induced Ca2+ release is inhibited by mitochondrial depolarization. AB - We investigated the consequences of depolarizing the mitochondrial membrane potential (Deltapsi(mit)) on Ca(2+) signals arising via inositol 1,4,5 trisphosphate receptors (InsP(3)R) in hormone-stimulated HeLa cells. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or a mixture of antimycin A+oligomycin were found to rapidly depolarize Deltapsi(mit). Mitochondrial depolarization enhanced the number of cells responding to a brief application of a Ca(2+)-mobilizing hormone and prolonged the recovery of cytosolic Ca(2+) after washout of the hormone; effects consistent with the removal of a passive Ca(2+) buffer. However, with repeated application of the same hormone concentration both the number of responsive cells and peak Ca(2+) changes were observed to progressively decline. The inhibition of Ca(2+) signalling was observed using different Ca(2+)-mobilizing hormones and also with a membrane-permeant Ins(1,4,5)P(3) ester. Upon washout of FCCP, the Ca(2+) signals recovered with a time course similar to the re-establishment of Deltapsi(mit). Global measurements indicated that none of the obvious factors such as changes in pH, ATP concentration, cellular redox state, permeability transition pore activation or reduction in Ca(2+)-store loading appeared to underlie the inhibition of Ca(2+) signalling. We therefore suggest that local changes in one or more of these factors, as a consequence of depolarizing Deltapsi(mit), prevents InsP(3)R activation. PMID- 10749693 TI - Action potential propagation in inhomogeneous cardiac tissue: safety factor considerations and ionic mechanism. AB - Heterogeneity of myocardial structure and membrane excitability is accentuated by pathology and remodeling. In this study, a detailed model of the ventricular myocyte in a multicellular fiber was used to compute a location-dependent quantitative measure of conduction (safety factor, SF) and to determine the kinetics and contribution of sodium current (I(Na)) and L-type calcium current [I(Ca(L))] during conduction. We obtained the following results. 1) SF decreases sharply for propagation into regions of increased electrical load (tissue expansion, increased gap junction coupling, reduced excitability, hyperkalemia); it can be <1 locally (a value indicating conduction failure) and can recover beyond the transition region to resume propagation. 2) SF and propagation across inhomogeneities involve major contribution from I(Ca(L)). 3) Modulating I(Na) or I(Ca(L)) (by blocking agents or calcium overload) can cause unidirectional block in the inhomogeneous region. 4) Structural inhomogeneity causes local augmentation of I(Ca(L)) and suppression of I(Na) in a feedback fashion. 5) Propagation across regions of suppressed I(Na) is achieved via a I(Ca(L)) dependent mechanism. 6) Reduced intercellular coupling can effectively compensate for reduced SF caused by tissue expansion but not by reduced membrane excitability. PMID- 10749694 TI - Effect of 6-wk estrogen withdrawal or replacement on myocardial ischemic tolerance in rats. AB - Menopausal status is a risk factor for coronary artery disease death, but the mechanism underlying this association is uncertain. To test whether estrogen ameliorates the effects of acute myocardial ischemia in ways likely to translate into a mortality difference, we compared the response to brief (6-min) and prolonged (45-min) coronary occlusion in vivo in five groups (each n = 16) of rats: ovariectomized females; ovariectomized females after 6 wk 17beta-estradiol replacement; male rats supplemented with estradiol for 6 wk; normal males; and normal females. Coronary occlusion produced a uniform ischemic risk area averaging 53 +/- 3% of left ventricular volume. After a brief occlusion, reperfusion ventricular tachycardia/fibrillation occurred with >85% frequency in all groups. During a prolonged occlusion, ischemic ventricular tachycardia occurred in 100% and sustained tachycardia requiring cardioversion in >75% of rats in all groups. Myocardial infarct size averaged 52 +/- 4% of the ischemic risk area and was similarly unaffected by gender or estrogen status. We conclude that neither short-term estrogen withdrawal, replacement, nor supplementation significantly affects the potentially lethal outcomes from acute coronary occlusion in this species. PMID- 10749695 TI - Temporal contribution of body movement to very long-term heart rate variability in humans. AB - A newly developed, very long-term ( approximately 7 days) ambulatory monitoring system for assessing beat-to-beat heart rate variability (HRV) and body movements (BM) was used to study the mechanism(s) responsible for the long-period oscillation in human HRV. Data continuously collected from five healthy subjects were analyzed by 1) standard auto- and cross-spectral techniques, 2) a cross Wigner distribution (WD; a time-frequency analysis) between BM and HRV for 10-s averaged data, and 3) coarse-graining spectral analysis for 600 successive cardiac cycles. The results showed 1) a clear circadian rhythm in HRV and BM, 2) a 1/f (beta)-type spectrum in HRV and BM at ultradian frequencies, and 3) coherent relationships between BM and HRV only at specific ultradian as well as circadian frequencies, indicated by significant (P < 0.05) levels of the squared coherence and temporal localizations of the covariance between BM and HRV in the cross-WD. In a single subject, an instance in which the behavioral (mean BM) and autonomic [HRV power >0.15 Hz and mean heart rate (HR)] rhythmicities were dissociated occurred when the individual had an irregular daily life. It was concluded that the long-term HRV in normal humans contained persistent oscillations synchronized with those of BM at ultradian frequencies but could not be explained exclusively by activity levels of the subjects. PMID- 10749696 TI - TGF-beta1 downregulates CD36 and scavenger receptor A but upregulates LOX-1 in human macrophages. AB - Transforming growth factor-beta1 (TGF-beta1), a key cytokine for control of cell growth, extracellular matrix formation, and inflammation control, is secreted by many cells present in the arteriosclerotic plaque. Lipid accumulation in the vessel wall is regarded as an early step in atherogenesis and depends on uptake of modified low-density lipoprotein (LDL) by macrophages through scavenger receptors and their transformation into foam cells. Prominent members of the scavenger receptor family are the class A type I and II receptors (ScR-A), the class B receptor CD36, and the recently detected lectin-like oxidized LDL receptor-1 (LOX-1), which, unlike the native LDL receptor (LDL-R), are not feedback controlled. CD36 is responsible for >50% of modified LDL uptake into human monocyte-derived macrophages. We therefore studied whether TGF-beta1 influences expression and function of ScR-A, CD36, and LOX-1 in monocytes using RT-PCR and flow cytometry. Total uptake of oxidized LDL by monocytoid cells, reflecting the combined function of all scavenger receptors, was significantly reduced by TGF-beta1. At initially low picomolar concentrations, TGF-beta1 decreased CD36 mRNA and protein surface expression and ScR-A mRNA levels in the human monocytic cell line THP-1 and in freshly isolated and cultivated human monocytes, whereas LOX-1 mRNA was increased. Expression of LDL-R and beta-actin was not affected by TGF-beta1. In conclusion, depression of scavenger receptor function in monocytes by TGF-beta1 in low concentrations reduces foam cell formation. Together with matrix control by TGF-beta1, this may be important for atherogenesis and plaque stabilization. PMID- 10749697 TI - A chronic mouse model of myocardial ischemia-reperfusion: essential in cytokine studies. AB - Reperfusion of the ischemic myocardium is associated with a cytokine cascade that reflects a cellular response to injury. We studied this cascade in the mouse and found that acute surgical trauma in sham-operated animals obscured early changes in cytokine induction that occur during myocardial ischemia-reperfusion (MI/R). Therefore, we utilized a new implantable device that allows occlusion and reperfusion of the left anterior descending coronary artery in a closed-chest mouse at any time after instrumentation. Induction of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha mRNA in the whole heart was examined by RNase protection assay and quantitated by Phosphor- Imager. At 3 h after instrumentation, levels of IL-6 mRNA in sham-operated animals increased above those of control naive hearts, whereas this increase did not occur until after 1 day for TNF-alpha mRNA. The surgical trauma led to exaggeration of I/R cytokine induction with greater variance in response. At 3 days and 1 wk after instrumentation, levels of both IL-6 and TNF-alpha mRNA in sham-operated animals were comparable to those of naive hearts and induction responses in I/R were much less variant. We also found that 1 h of ischemia and 2 h of reperfusion at all time points of recovery (i.e., 3 h and 1, 3, and 7 days after instrumentation) led to a significant increase in IL-6 and TNF-alpha mRNA levels. In addition, 3 h of permanent occlusion, which did not induce any mRNA increase after 1 wk postinstrumentation, caused marked upregulation of IL-6 mRNA in an acutely prepared animal. This study of early cytokine responses evoked by MI/R highlights the need for dissipation of acute surgical trauma by using a chronic, closed chest mouse preparation. PMID- 10749698 TI - Activation of mRNA translation in rat cardiac myocytes by insulin involves multiple rapamycin-sensitive steps. AB - Insulin acutely activates protein synthesis in ventricular cardiomyocytes from adult rats. In this study, we have established the methodology for studying the regulation of the signaling pathways and translation factors that may be involved in this response and have examined the effects of acute insulin treatment on them. Insulin rapidly activated the 70-kDa ribosomal S6 kinase (p70 S6k), and this effect was inhibited both by rapamycin and by inhibitors of phosphatidylinositol 3-kinase. The activation of p70 S6k is mediated by a signaling pathway involving the mammalian target of rapamycin (mTOR), which also modulates other translation factors. These include the eukaryotic initiation factor (eIF) 4E binding proteins (4E-BPs) and eukaryotic elongation factor 2 (eEF2). Insulin caused phosphorylation of 4E-BP1 and induced its dissociation from eIF4E, and these effects were also blocked by rapamycin. Concomitant with this, insulin increased the binding of eIF4E to eIF4G. Insulin also activated protein kinase B (PKB), which may lie upstream of p70 S6k and 4E-BP1, with the activation of the different isoforms being in the order alpha>beta>gamma. Insulin also caused inhibition of glycogen synthase kinase 3, which lies downstream of PKB, and of eEF2 kinase. The phosphorylation of eEF2 itself was also decreased by insulin, and this effect and the inactivation of eEF2 kinase were attenuated by rapamycin. The activation of overall protein synthesis by insulin in cardiomyocytes was substantially inhibited by rapamycin (but not by inhibitors of other specific signaling pathways, e.g., mitogen-activated protein kinase), showing that signaling events linked to mTOR play a major role in the control of translation by insulin in this cell type. PMID- 10749699 TI - Inactivation of bradykinin by angiotensin-converting enzyme and by carboxypeptidase N in human plasma. AB - Because bradykinin (BK) appears to have cardioprotective effects ranging from improved hemodynamics to antiproliferative effects, inhibition of BK-degrading enzymes should potentiate such actions. The purpose of this study was to find out which enzymes are responsible for the degradation of BK in human plasma. Human plasma from healthy donors (n = 10) was incubated with BK in the presence or absence of specific enzyme inhibitors. At high (micromolar) concentrations, BK was mostly (>90%) degraded by carboxypeptidase N (CPN)-like activity. In contrast, at low (nanomolar) substrate concentrations, at which the velocity of the catalytic reaction is equivalent to that under physiological conditions, BK was mostly (>90%) converted into an inactive metabolite, BK-(1-7), by angiotensin converting enzyme (ACE). BK-(1-7) was further converted by ACE into BK-(1-5), with accumulation of this active peptide. A minor fraction (<10%) of the BK was converted into another active metabolite, BK-(1-8), by CPN-like activity. The present study shows that the most critical step in plasma kinin metabolism, i.e., inactivation of BK, is mediated by ACE. Thus inhibition of plasma ACE activity would be cardioprotective by elevating the concentration of BK in the circulation. PMID- 10749700 TI - Silent alpha(2C)-adrenergic receptors enable cold-induced vasoconstriction in cutaneous arteries. AB - Cold constricts cutaneous blood vessels by increasing the reactivity of smooth muscle alpha(2)-adrenergic receptors (alpha(2)-ARs). Experiments were performed to determine the role of alpha(2)-AR subtypes (alpha(2A)-, alpha(2B)-, alpha(2C) ARs) in this response. Stimulation of alpha(1)-ARs by phenylephrine or alpha(2) ARs by UK-14,304 caused constriction of isolated mouse tail arteries mounted in a pressurized myograph system. Compared with proximal arteries, distal arteries were more responsive to alpha(2)-AR activation but less responsive to activation of alpha(1)-ARs. Cold augmented constriction to alpha(2)-AR activation in distal arteries but did not affect the response to alpha(1)-AR stimulation or the level of myogenic tone. Western blot analysis demonstrated expression of alpha(2A)- and alpha(2C)-ARs in tail arteries: expression of alpha(2C)-ARs decreased in distal compared with proximal arteries, whereas expression of the glycosylated form of the alpha(2A)-AR increased in distal arteries. At 37 degrees C, alpha(2)-AR induced vasoconstriction in distal arteries was inhibited by selective blockade of alpha(2A)-ARs (BRL-44408) but not by selective inhibition of alpha(2B)-ARs (ARC-239) or alpha(2C)-ARs (MK-912). In contrast, during cold exposure (28 degrees C), the augmented response to UK-14,304 was inhibited by the alpha(2C)-AR antagonist MK-912, which selectively abolished cold-induced amplification of the response. These experiments indicate that cold-induced amplification of alpha(2) ARs is mediated by alpha(2C)-ARs that are normally silent in these cutaneous arteries. Blockade of alpha(2C)-ARs may prove an effective treatment for Raynaud's Phenomenon. PMID- 10749701 TI - Dietary coenzyme Q(10) supplement renders swine hearts resistant to ischemia reperfusion injury. AB - To examine whether nutritional supplementation of coenzyme Q(10) (CoQ(10)) can reduce myocardial ischemia-reperfusion injury, a group of swine was fed a regular diet supplemented with CoQ(10) (5 mg x kg(-1) x day(-1)) for 30 days. Another group of pigs that were fed a regular diet supplemented with placebo served as a control. After 30 days, isolated in situ pig hearts were prepared and hearts were perfused with a cardiopulmonary pump system. Each heart was subjected to 15 min of regional ischemia by snaring of the left anterior descending coronary artery, followed by 60 min of hypothermic cardioplegic global ischemia and 120 min of reperfusion. After the experiments were completed, myocardial infarct size was measured by triphenyltrazolium chloride staining methods. Postischemic left ventricular contractile function was better recovered in the CoQ(10) group than in the control group of pigs. CoQ(10)-fed pigs revealed less myocardial infarction and less creatine kinase release from the coronary effluent compared with control pigs. The experimental group also demonstrated a smaller amount of malonaldehyde in the coronary effluent and a higher content of the endogenous antioxidants ascorbate and thiol. Significant induction of the expression of ubiquitin mRNA was also found in the hearts of the CoQ(10)-fed group. The results of this study demonstrate that nutritional supplementation of CoQ(10) renders the hearts resistant to ischemia-reperfusion injury, probably by reducing the oxidative stress. PMID- 10749702 TI - Activation of HSF and selective increase in heat-shock proteins by acute dexamethasone treatment. AB - Heat-shock proteins (HSPs) are an important family of endogenous protective proteins, which increase in response to myocardial ischemia and other stresses. Overexpression of HSP72 is cardioprotective. We were interested in the regulation of heat-shock factor (HSF), the transcription factor for HSP genes. Previously we have observed that the inflammatory cytokine tumor necrosis factor-alpha increases HSP72 levels and postulated that dexamethasone might effect the heat shock response. In the adult rat cardiac myocyte we found that treatment with either low (10 microM)- or high (100 microM)-dose dexamethasone activated HSF by 2-6 h as determined by gel shift assay without evidence of cytotoxicity. Although HSF activation is a key step in expression of HSP72, this may not result in an increase in HSP72. We found that 10 microM dexamethasone increased HSP72 38%, and 100 microM dexamethasone increased HSP72 62% (P < 0.05). HSP27 and HSP60 were unchanged. The selective increase in HSP72 was associated with protection of the cardiac myocytes from hypoxia and reoxygenation. We conclude that dexamethasone is a novel inducer of the heat shock response. PMID- 10749703 TI - Pulsatile flow enhances endothelium-derived nitric oxide release in the peripheral vasculature. AB - The effects of pulsatility in blood flow on endothelium-derived nitric oxide (EDNO) release in the peripheral vasculature were investigated. The basal and flow-stimulated EDNO release were compared between pulsatile and nonpulsatile systemic flows before and after the administration of NO synthase inhibitor N(G) monomethyl-L-arginine (L-NMMA). Peripheral vascular resistance (PVR) was significantly lower in pulsatile flow than in nonpulsatile flow, but this difference disappeared after L-NMMA. The percent increase in PVR by L-NMMA was significantly larger in pulsatile flow. In reactive hyperemia in the hindlimb, the peak flow did not differ; however, both the repayment flow and the duration were significantly larger in pulsatile flow. Percent changes of these parameters by L-NMMA were significantly larger in pulsatile flow. These data indicated that pulsatility significantly enhances the basal and flow-stimulated EDNO release in the peripheral vasculature under in vivo conditions. We also studied the involvement of the Ca(2+)-dependent and Ca(2+)-independent pathways in flow induced vasodilation using calmodulin inhibitor calmidazolium and tyrosine kinase inhibitor erbstatin A. PVR was significantly elevated by erbstatin A but not by calmidazolium, suggesting that flow-induced vasodilation was largely caused by tyrosine kinase inhibitor-sensitive activation of NO synthase. PMID- 10749704 TI - The thyroid hormone analog DITPA restores I(to) in rats after myocardial infarction. AB - Previous studies have established that reductions in repolarizing currents occur in heart disease and can contribute to life-threatening arrhythmias in myocardium. In this study, we investigated whether the thyroid hormone analog 3, 5-diiodothyropropionic acid (DITPA) could restore repolarizing transient outward K(+) current (I(to)) density and gene expression in rat myocardium after myocardial infarction (MI). Our findings show that I(to) density was reduced after MI (14.0 +/- 1.0 vs. 10.2 +/- 0.9 pA/pF, sham vs. post-MI at +40 mV). mRNA levels of Kv4.2 and Kv4.3 genes were decreased but Kv1.4 mRNA levels were increased post-MI. Corresponding changes in Kv4.2 and Kv1.4 protein were also observed. Chronic treatment of post-MI rats with 10 mg/kg DITPA restored I(to) density (to 15.2 +/- 1.1 pA/pF at +40 mV) as well as Kv4.2 and Kv1.4 expression to levels observed in sham-operated controls. Other membrane currents (Na(+), L type Ca(2+), sustained, and inward rectifier K(+) currents) were unaffected by DITPA treatment. Associated with the changes in I(to) expression, action potential durations (current-clamp recordings in isolated single right ventricular myocytes and monophasic action potential recordings from the right free wall in situ) were prolonged after MI and restored with DITPA treatment. Our results demonstrate that DITPA restores I(to) density in the setting of MI, which may be useful in preventing complications associated with I(to) downregulation. PMID- 10749705 TI - Left ventricular torsion is equal in mice and humans. AB - Global cardiac function has been studied in small animals with methods such as echocardiography, cine-magnetic resonance imaging (MRI), and cardiac catheterization. However, these modalities make little impact on delineation of pathophysiology at the tissue level. The advantage of tagged cine-MRI technique is that the twisting motion of the ventricle, referred to as torsion, can be measured noninvasively, reflecting the underlying shearing motion of individual planes of myofibrils that generate wall thickening and ventricular ejection. Thus we sought to determine whether the mechanism of ventricular ejection, as measured by torsion, was the same in both humans and mice. Nine mice and ten healthy humans were studied with tagged cine-MRI. The magnitude and systolic time course of ventricular torsion were equivalent in mouse and humans, when normalized for heart rate and ventricular length. The end-systolic torsion angle was 12.7 +/- 1.7 degrees in humans vs. 2.0 +/- 1.5 degrees in mice unnormalized and 1.9 +/- 0.3 degrees /cm vs. 2.7 +/- 2.3 degrees /cm when normalized for ventricular length). These results support the premise that ventricular torsion may be a uniform measure of normal ventricular ejection across mammalian species and heart sizes. PMID- 10749706 TI - Dominant role of cAMP in regulation of microvessel permeability. AB - We reported previously that increasing cAMP levels in endothelial cells attenuated ATP-induced increases in hydraulic conductivity (L(p)), and that the activation of cGMP-dependent pathways was a necessary step to increase L(p) in response to inflammatory mediators. The aim of the present study was to evaluate the role of basal levels of cAMP in microvessel permeability under resting conditions and to evaluate the cross talk between cAMP- and cGMP-dependent signaling mechanisms in regulation of microvessel permeability under stimulated conditions, using individually perfused microvessels from frog and rat mesenteries. We found that reducing cAMP levels by inhibition of adenylate cyclase or inhibiting cAMP-dependent protein kinase through the use of H-89 increased basal L(p) in both frog and rat mesenteric venular microvessels. We also found that 8-bromocAMP (8-BrcAMP, 0.2 and 2 mM) was sufficient to attenuate or abolish the increases in L(p) due to exposure of frog mesenteric venular microvessels to 8-BrcGMP (2 mM) and ATP (10 microM). Similarly, in rat mesenteric venular microvessels, application of 8-BrcAMP (2 mM) abolished the increases in L(p) due to exposure to 8-BrcGMP alone (2 mM) or with the combination of bradykinin (1 nM). In addition, application of erythro-9-(2-hydroxy-3 nonyl)adenine, an inhibitor of cGMP-stimulated phosphodiesterase, significantly attenuated both 8-BrcGMP- and bradykinin-induced increases in L(p). These results demonstrate that basal levels of cAMP are critical to maintaining normal permeability under resting conditions, and that increased levels of cAMP are capable of overcoming the activation of cGMP-dependent pathways, therefore preventing increases in microvessel permeability. The balance between endothelial concentrations of these two opposing cyclic nucleotides controls microvessel permeability, and cAMP levels play a dominant role. PMID- 10749707 TI - Heterogeneous cardiac sympathetic innervation in heart failure after myocardial infarction of rats. AB - We examined cardiac neuronal function and beta-receptor with a dual-tracer method of [(131)I]meta-iodobenzylguanidine (MIBG) and [(125)I]iodocyanopindolol (ICYP) in rat heart failure after myocardial infarction (MI). In rats with MI, left ventricular (LV) systolic function decreased, and LV dimension and right ventricular (RV) mass increased gradually. MIBG accumulations of the noninfarcted LV (remote region) and RV decreased by 15% at 1 wk compared with sham-operated rats, and these accumulations were restored by 71% and 56%, respectively, at 24 wk compared with age-matched sham rats despite sustained depletion of myocardial norepinephrine contents in these regions. ICYP accumulation of the remote region and of the RV did not decrease at any stages. Myocardial MIBG distribution was heterogeneous at 1 wk when it was lower in the peri-infarcted region than in the remote region, associated with reduced ICYP accumulation in the peri-infarcted region. The heterogeneous distribution of both isotopes disappeared at 12 wk. Thus cardiac sympathetic neuronal alteration was coupled with downregulation of beta-receptors in rat heart failure after MI. The abnormal adrenergic signaling occurred heterogeneously in terms of ventricular distribution and time course after MI. PMID- 10749708 TI - Determination of microvascular flow pattern formation in vivo. AB - Blood flow in microvessels differs significantly from that of red blood cells (RBC) flowing through long, straight glass tubes in vitro. The in vivo situation is characterized by the presence of plasma favoring aggregation, by the irregular geometry of vessel segments, and by frequent branching points. Here, a method is presented to characterize flow patterns in microvascular blood flow during intravital microscopy based on Fourier analysis of recorded light intensity patterns. The interpretation of the resulting power spectra in terms of pattern size distribution was validated by model experiments employing artificial textures and by reverse transformation of idealized spectra. The determined size of RBC flow patterns in microvessels ranged from approximately 8 microm in capillaries to approximately 14 microm in vessels of >30 microm. With increasing shear rate above approximately 100 s(-1) pattern size increased, possibly reflecting formation of short-lived flow clusters. Below approximately 100 s(-1) an increase of pattern size with decreasing shear rate was found in experiments using local occlusion and treatment with high-molecular-weight dextran, suggesting the formation of aggregates. The dynamic process of generation and destruction of RBC flow patterns could well contribute to flow resistance in vivo in peripheral vascular beds. PMID- 10749709 TI - Regulation of DHP receptor expression by elements in the 5'-flanking sequence. AB - The alpha(1)-subunit of the cardiac/vascular Ca(2+) channel, which is the dihydropyridine (DHP)-binding site (the DHP receptor), provides the pore structure for Ca(2+) entry. It contains the binding sites for multiple classes of drugs collectively known as Ca(2+) antagonists. As an initial step toward understanding the mechanisms controlling transcription of the rat cardiac alpha(1C)-subunit gene, we have cloned a 2.3-kb fragment containing the 5' flanking sequences and identified the alpha(1C)-subunit gene transcription start site. The rat alpha(1C)-subunit gene promoter belongs to the TATA-less class of such basal elements. Using deletion analysis of alpha(1C)-subunit promoter luciferase reporter gene constructs, we have characterized the transcriptional modulating activity of the 5'-flanking region and conducted transient transfections in cultured neonatal rat cardiac ventricular myocytes and vascular smooth muscle cells. Sequence scanning identified several potential regulatory elements, including five consensus sequences for the cardiac-specific transcription factor Nkx2.5, an AP-1 site, a cAMP response element, and a hormone response element. Transient transfection experiments with the promoter-luciferase reporter fusion gene demonstrate that the 2-kb 5'-flanking region confers tissue specificity and hormone responsiveness to expression of the Ca(2+) channel alpha(1C)-subunit gene. Electrophoretic mobility shift assays identified a region of the alpha(1C)-subunit gene promoter that can bind transcription factors and appears to be important for gene expression. PMID- 10749710 TI - Cerebral microvascular endothelial cell tube formation: role of astrocytic epoxyeicosatrienoic acid release. AB - Cerebral microvascular endothelial cells (CMVEC) form tubes when cocultured with astrocytes (AS). Therefore, it appears that AS may be important in mediating angiogenesis in the brain. We hypothesized that AS modulate CMVEC tube formation by releasing a soluble factor. Thymidine incorporation in cultured CMVEC increased 305% when incubated with 50% conditioned AS medium for 24 h [control: 52,755 +/- 4,838 counts per minute (cpm) per well, conditioned 161,082 +/- 12,099 cpm/well, n = 8]. Because our laboratory has previously shown that AS can produce epoxyeicosatrienoic acids (EETs), which are known mitogens, we investigated whether release of EETs by AS is responsible for tube formation in the CMVEC-AS coculture. AS were seeded on Lab-Tek slides, CMVEC were seeded on the AS the next day, and cultures were allowed to progress for another 5 days with and without cytochrome P-450 epoxygenase blockade by 17-octadecynoic acid (17-ODYA). Tube formation in cocultures receiving 17-ODYA was significantly inhibited compared with control (93.8%). These data suggest that tube formation requires the release of EETs by AS. PMID- 10749711 TI - Platelet-activating factor receptors in lamb lungs are downregulated immediately after birth. AB - Platelet-activating factor (PAF) is a phospholipid with diverse biological functions mediated by a G protein-coupled receptor. We determined PAF receptor binding in lung membranes of four groups of perinatal lambs. Membrane protein (100 microg/ml) was incubated for 60 min at 30 degrees C with 0.5-24 nM of acetyl [(3)H]PAF in 30 mM Tris buffer, pH 7.2, containing 0.25% BSA, 10 mM MgCl(2), and 125 mM choline chloride. PAF bound to membrane was isolated and quantified by scintillation spectrometry, followed with Scatchard analysis for receptor density (B(max)). The B(max) (means +/- SE, fmol/mg protein) were 445.8 +/- 12.3, 244.2 +/- 3.3, 250.6 +/- 3.6, and 419. 9 +/- 8.6 for the fetal, 90-min-old, <1-day-old, and 6- to 12-day-old lambs, respectively. The B(max) for the 90-min-old and <1 day-old lambs were not different but were significantly lower than those of either the term fetal or 6- to 12-day-old lambs. These data show a significant decrease in PAF binding to its receptor and in PAF B(max) in lung membranes of immediate newborn lambs. The dissociation constants (K(D), nM) were 7.7 +/- 0.52, 11.5 +/- 0.34, 6.9 +/- 0.48, and 5.0 +/- 0.53 for fetal, 90-min-old, <1-day-old, and 6- to 12-day-old newborn lamb lungs, respectively. The K(D) of the 90-min-old lamb was the highest of all. PAF receptor gene measured by RT-PCR showed a significant downregulation of PAF receptor gene mRNA in lungs of lambs <1 day old, suggesting a transcriptional regulation of PAF receptor gene expression in the immediate newborn period. We speculate that decreased PAF receptor binding immediately after birth will facilitate the fall in pulmonary vascular resistance in the immediate newborn period. PMID- 10749713 TI - Increased flow precedes remote arteriolar dilations for some microapplied agonists. AB - This study asks which occurs first in time for remote responses: a dilation or a remote change in flow. Arteriolar diameter (approximately 20 microm) and fluorescently labeled red blood cell (RBC) velocity were measured in the cremaster muscle of anesthetized (pentobarbital sodium, 70 mg/kg) hamsters (n = 51). Arterioles were locally stimulated for 60 s with micropipette-applied 10 microg/ml LM-609 (alpha(v)beta(3)-integrin agonist), 10(-3) M adenosine, or 10( 3) M 3-morpholinosydnonimine (SIN-1, nitric oxide donor) as remote response agonists or with 10(-3) M papaverine, which dilates only locally. Observations were made at a remote site 1,200 microm upstream. With LM-609 or adenosine, the RBC velocity increased first (within 5 s), and the remote dilation followed 5-7 s later. N-nitro-L-arginine (100 microM) blocked the LM-609 (100%) and adenosine (60%) remote dilations. SIN-1 induced a concurrent remote dilation and decrease in RBC velocity (approximately 10 s), suggesting the primary signal was to dilate. Papaverine had no remote effects. This study suggests that, although remote responses to some agonists are induced by primary signals to dilate, additionally, network changes in flow can stimulate extensive remote changes in diameter. PMID- 10749712 TI - Role of a glycocalyx on coronary arteriole permeability to proteins: evidence from enzyme treatments. AB - Whereas the glycocalyx of endothelial cells has been shown to influence solute flux from capillary microvessels, little is known about its contribution to the movement of macromolecules across the walls of other microvessels. We evaluated the hypothesis that a glycocalyx contributes resistance to protein flux measured in coronary arterioles. Apparent solute permeability (P(s)) to two proteins of different size and similar charge, alpha-lactalbumin (alpha-lactalb) and porcine serum albumin (PSA), was determined in arterioles isolated from the hearts of 43 female Yucatan miniature swine. P(s) was assessed in arterioles with an "intact" glycocalyx under control conditions and again after suffusion with adenosine (Ado, 10(-5) M, n = 42 arterioles, N = 29 pigs). In a second set of experiments (n = 21 arterioles, N = 21 pigs) arteriolar P(s) was determined before and after perfusion with enzyme (pronase or heparinase), which was used to digest the glycocalyx. P(s) was assessed a third time on those microvessels after exposure to Ado. Consistent with the hypothesis, P(s) for PSA (P(PSA)(s)) and P(s) for alpha-lactalb (P(alpha-lactalb)(s)) increased from basal levels following enzyme treatment. Subsequent suffusion with Ado, a significant metabolite known to alter coronary vascular smooth muscle tone and permeability, resulted in a significant reduction of basal P(alpha-lactalb)(s) in both untreated and enzyme-treated arterioles. Furthermore, in untreated arterioles, P(PSA)(s) was unchanged by Ado suffusion, whereas Ado induced a pronounced reduction in P(PSA)(s) of enzyme treated vessels. These data demonstrate that in intact coronary arterioles an enzyme-sensitive layer, most likely at the endothelial cell surface, contributes significantly to net barrier resistance to solute flux. PMID- 10749714 TI - Contribution of malonyl-CoA decarboxylase to the high fatty acid oxidation rates seen in the diabetic heart. AB - Myocardial glucose oxidation is markedly reduced in the uncontrolled diabetic. We determined whether this was due to direct biochemical changes in the heart or whether this was due to altered circulating levels of insulin and substrates that can be seen in the diabetic. Isolated working hearts from control or diabetic rats (streptozotocin, 55 mg/kg iv administered 6 wk before study) were aerobically perfused with either 5 mM [(14)C]glucose and 0.4 mM [(3)H]palmitate (low-fat/low-glucose buffer) or 20 mM [(14)C]glucose and 1.2 mM [(3)H]palmitate (high-fat/high-glucose buffer) +/-100 microU/ml insulin. The presence of insulin increased glucose oxidation in control hearts perfused with low-fat/low-glucose buffer from 553 +/- 85 to 1,150 +/- 147 nmol x g dry wt(-1) x min(-1) (P < 0. 05). If control hearts were perfused with high-fat/high-glucose buffer, palmitate oxidation was significantly increased by 112% (P < 0.05), but glucose oxidation decreased to 55% of values seen in the low-fat/low-glucose group (P < 0.05). In diabetic hearts, glucose oxidation was very low in hearts perfused with low fat/low-glucose buffer (9 +/- 1 nmol x g dry wt(-1) x min(-1)) and was not altered by insulin or high-fat/high-glucose buffer. These results suggest that neither circulating levels of substrates nor insulin was responsible for the reduced glucose oxidation in diabetic hearts. To determine if subcellular changes in the control of fatty acid oxidation contribute to these changes, we measured the activity of three enzymes involved in the control of fatty acid oxidation; AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), and malonyl CoA decarboxylase (MCD). Although AMPK and ACC activity in control and diabetic hearts was not different, MCD activity and expression in all diabetic rat heart perfusion groups were significantly higher than that seen in corresponding control hearts. These results suggest that an increased MCD activity contributes to the high fatty acid oxidation rates and reduced glucose oxidation rates seen in diabetic rat hearts. PMID- 10749715 TI - Age-associated arterial wall thickening is related to elevations in sympathetic activity in healthy humans. AB - Arterial wall hypertrophy occurs with age in humans and is a strong predictor of cardiovascular disease risk. The responsible mechanism is unknown, but data from studies in experimental animals suggest that elevated sympathetic-adrenergic tone may be involved. To test this hypothesis in humans we studied 11 young (29 +/- 1 yr; means +/- SE) and 13 older (63 +/- 1) healthy normotensive men under supine resting conditions. Muscle sympathetic nerve activity (MSNA) burst frequency (peroneal microneurography) was 70% higher in the older men (39 +/- 1 vs. 23 +/- 2 bursts/min; P < 0.001). Femoral artery intima media thickness (IMT; B-mode ultrasound) and the femoral IMT-to-lumen diameter ratio (IMT/lumen) were approximately 75% greater in the older men (both P < 0.001). Femoral IMT (r = 0. 82) and the femoral IMT/lumen (r = 0.85) were strongly and positively related to MSNA (both P < 0.001). The significant age group differences in femoral IMT and the IMT/lumen were abolished when the influence of MSNA was removed. In contrast, the relationship between MSNA and femoral wall thickness remained significant after removing the influence of age. We conclude that 1) primary aging is associated with femoral artery hypertrophy in humans and 2) this is strongly related to elevations in sympathetic nerve activity to the vasculature. These results support the hypothesis that tonic elevations in sympathetic nerve activity may be an important mechanism in the arterial remodeling that occurs with human aging. PMID- 10749717 TI - Diabetes abolishes ischemic preconditioning: role of glucose, insulin, and osmolality. AB - Recent evidence indicates that hyperglycemia is an important risk factor for the development of cardiovascular disease. We tested the hypothesis that myocardial infarct size is related to blood glucose concentration in the presence or absence of ischemic preconditioning (PC) stimuli in canine models of diabetes mellitus and acute hyperglycemia. Barbiturate-anesthetized dogs were subjected to a 60-min period of coronary artery occlusion and 3-h reperfusion. Infarct size was 24 +/- 2% of the area at risk (AAR) for infarction in control dogs. PC significantly (P < 0.05) decreased the extent of infarction in normal (8 +/- 2% of AAR), but not diabetic (22 +/- 4% of AAR), dogs. Infarct size was linearly related to blood glucose concentration during acute hyperglycemia (r = 0.96; P < 0.001) and during diabetes (r = 0.74; P < 0.002) in the presence or absence of PC stimuli. Increases in serum osmolality caused by administration of raffinose (300 g) did not increase infarct size (11 +/- 3% of AAR) or interfere with the ability of PC to protect against infarction (2 +/- 1% of AAR). The results indicate that hyperglycemia is a major determinant of the extent of myocardial infarction in the dog. PMID- 10749716 TI - Nitric oxide-induced cardioprotection in cultured rat ventricular myocytes. AB - The aim of this study was to investigate the role of nitric oxide (NO) in a cellular model of early preconditioning (PC) in cultured neonatal rat ventricular myocytes. Cardiomyocytes "preconditioned" with 90 min of stimulated ischemia (SI) followed by 30 min reoxygenation in normal culture conditions were protected against subsequent 6 h of SI. PC was blocked by N(G)-monomethyl-L-arginine monoacetate but not by dexamethasone pretreatment. Inducible nitric oxide synthase (NOS) protein expression was not detected during PC ischemia. Pretreatment (90 min) with the NO donor S-nitroso-N-acetyl-L,L-penicillamine (SNAP) mimicked PC, resulting in significant protection. SNAP-triggered protection was completely abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1 one (ODQ) but was unaffected by chelerythrine or the presence of glibenclamide and 5-hydroxydecanoate. With the use of RIA, SNAP treatment increased cGMP levels, which were blocked by ODQ. Hence, NO is implicated as a trigger in this model of early PC via activation of a constitutive NOS isoform. After exposure to SNAP, the mechanism of cardioprotection is cGMP dependent but independent of protein kinase C or ATP-sensitive K(+) channels. This differs from the proposed mechanism of NO-induced cardioprotection in late PC. PMID- 10749718 TI - Translational inhibition of E-selectin expression stimulates P-selectin-dependent neutrophil recruitment. AB - Although known for its role in hemostasis, there is a growing body of evidence that thrombin can induce leukocyte recruitment and contribute to the inflammatory response. An in vitro parallel-plate flow chamber was used to systematically examine thrombin-induced neutrophil interactions with human endothelium. Stimulation of endothelial cells with thrombin (1 U/ml) resulted in an immediate, P-selectin-dependent increase in neutrophil rolling and adhesion that was comparable in magnitude to optimal levels of histamine (the classical inducer of P-selectin). However, thrombin, but not histamine, induced a delayed (4 h) E selectin-dependent rolling similar to that of tumor necrosis factor-alpha, suggesting that thrombin has the unique ability to recruit neutrophils by an early P-selectin and a delayed E-selectin pathway. Surprisingly, inhibition of E selectin expression with the general protein synthesis inhibitor cycloheximide induced P-selectin expression 4 h after thrombin stimulation. Cycloheximide and thrombin (4 h) induced sufficient P-selectin-dependent rolling to recruit as many neutrophils as were recruited with 4 h of stimulation with thrombin alone. Histamine in the presence of cycloheximide or cycloheximide alone did not evoke the P-selectin response at 4 h, suggesting that this was not due to direct cycloheximide induction of P-selectin. Treatment of endothelium with tumor necrosis factor-alpha (an E-selectin inducer) and cycloheximide also eliminated E selectin expression but, much like thrombin, induced P-selectin expression and neutrophil recruitment. In conclusion, inhibition of E-selectin via protein synthesis inhibition activates the protein synthesis-independent pathway of P selectin expression to support adequate leukocyte recruitment. PMID- 10749719 TI - Organoid culture of cannulated rat resistance arteries: effect of serum factors on vasoactivity and remodeling. AB - We developed an organoid culture technique to study the mechanisms involved in arterial remodeling. Resistance arteries were isolated from rat cremaster muscle and mounted in a pressure myograph at 75 mmHg. Vessels were studied during a 4 day culture period in DMEM with either 2% albumin, 10% heat-inactivated FCS (HI FCS) or 10% dialyzed HI-FCS (12 kDa cut off) added to the perfusate. The albumin group showed a gradual loss of endothelial function and integrity, whereas smooth muscle agonist and myogenic responses were retained. No remodeling was observed. Vessels cultured in the presence of serum showed a progressive constriction. Smooth muscle responses and substance P-induced endothelium-dependent dilation were maintained. An inward remodeling of 17 +/- 4% in the HI-FCS group and 26 +/- 3% in the dialyzed HI-FCS group was found, while media cross-sectional areas were unchanged. These data show that pressurized resistance arteries can be maintained in culture for several days and undergo eutrophic remodeling in vitro in the presence of high molecular weight serum factors. PMID- 10749720 TI - In spontaneously hypertensive rats alterations in aortic wall properties precede development of hypertension. AB - In hypertension arterial wall properties do not necessarily depend on increased blood pressure alone. The present study investigates the relationship between the development of hypertension and thoracic aortic wall properties in 1.5-, 3-, and 6-mo-old spontaneously hypertensive rats (SHR); Wistar-Kyoto rats (WKY) served as controls. During ketamine-xylazine anesthesia, compliance and distensibility were assessed by means of a noninvasive ultrasound technique combined with invasive blood pressure measurements. Morphometric measurements provided in vivo media cross-sectional area and thickness, allowing the calculation of the incremental elastic modulus. Extracellular matrix protein contents were determined as well. Blood pressure was not significantly different in 1.5-mo-old SHR and WKY, but compliance and distensibility were significantly lower in SHR. Incremental elastic modulus was not significantly different between SHR and WKY at this age. Media thickness and media cross-sectional area were significantly larger in SHR than in WKY, but there was no consistent difference in collagen density and content between the strains. Blood pressure was significantly higher in 3- and 6 mo-old SHR than in WKY, and compliance was significantly lower in SHR. The findings in this study show that in SHR, in which hypertension develops over weeks, alterations in functional aortic wall properties precede the development of hypertension. The decrease in compliance and distensibility at a young age most likely results from media hypertrophy rather than a change in intrinsic elastic properties. PMID- 10749721 TI - Inhibition of calcium signaling in descending vasa recta endothelia by ANG II. AB - The intracellular calcium ([Ca(2+)](i)) response of outer medullary descending vasa recta (OMDVR) endothelia to ANG II was examined in fura 2-loaded vessels. Abluminal ANG II (10(-8) M) caused [Ca(2+)](i) to fall in proportion to the resting [Ca(2+)](i) (r = 0. 82) of the endothelium. ANG II (10(-8) M) also inhibited both phases of the [Ca(2+)](i) response generated by bradykinin (BK, 10(-7) M), 835 +/- 201 versus 159 +/- 30 nM (peak phase) and 169 +/- 26 versus 103 +/- 14 nM (plateau phase) (means +/- SE). Luminal ANG II reduced BK (10(-7) M)-stimulated plateau [Ca(2+)](i) from 180 +/- 40 to 134 +/- 22 nM without causing vasoconstriction. Abluminal ANG II added to the bath after luminal application further reduced [Ca(2+)](i) to 113 +/- 9 nM and constricted the vessels. After thapsigargin (TG) pretreatment, ANG II (10(-8) M) caused [Ca(2+)](i) to fall from 352 +/- 149 to 105 +/- 37 nM. This effect occurred at a threshold ANG II concentration of 10(-10) M and was maximal at 10(-8) M. ANG II inhibited both the rate of Ca(2+) entry into [Ca(2+)](i)-depleted endothelia and the rate of Mn(2+) entry into [Ca(2+)](i)-replete endothelia. In contrast, ANG II raised [Ca(2+)](i) in the medullary thick ascending limb and outer medullary collecting duct, increasing [Ca(2+)](i) from baselines of 99 +/- 33 and 53 +/- 11 to peaks of 200 +/- 47 and 65 +/- 11 nM, respectively. We conclude that OMDVR endothelia are unlikely to be the source of ANG II-stimulated NO production in the medulla but that interbundle nephrons might release Ca(2+)-dependent vasodilators to modulate vasomotor tone in vascular bundles. PMID- 10749722 TI - Detection of low- and high-frequency rhythms in the variability of skin sympathetic nerve activity. AB - Spectral analysis of skin blood flow has demonstrated low-frequency (LF, 0.03 0.15 Hz) and high-frequency (HF, 0.15-0.40 Hz) oscillations, similar to oscillations in R-R interval, systolic pressure, and muscle sympathetic nerve activity (MSNA). It is not known whether the oscillatory profile of skin blood flow is secondary to oscillations in arterial pressure or to oscillations in skin sympathetic nerve activity (SSNA). MSNA and SSNA differ markedly with regard to control mechanisms and morphology. MSNA contains vasoconstrictor fibers directed to muscle vasculature, closely regulated by baroreceptors. SSNA contains both vasomotor and sudomotor fibers, differentially responding to arousals and thermal stimuli. Nevertheless, MSNA and SSNA share certain common characteristics. We tested the hypothesis that LF and HF oscillatory components are evident in SSNA, similar to the oscillatory components present in MSNA. We studied 18 healthy normal subjects and obtained sequential measurements of MSNA and SSNA from the peroneal nerve during supine rest. Measurements were also obtained of the electrocardiogram, beat-by-beat blood pressure (Finapres), and respiration. Spectral analysis showed LF and HF oscillations in MSNA, coherent with similar oscillations in both R-R interval and systolic pressure. The HF oscillation of MSNA was coherent with respiration. Similarly, LF and HF spectral components were evident in SSNA variability, coherent with corresponding variability components of R-R interval and systolic pressure. HF oscillations of SSNA were coherent with respiration. Thus our data suggest that these oscillations may be fundamental characteristics shared by MSNA and SSNA, possibly reflecting common central mechanisms regulating sympathetic outflows subserving different regions and functions. PMID- 10749723 TI - Contractile and relaxing reactivity in carotid and femoral arteries of chicken embryos. AB - In the embryo, hypoxemia causes redistribution of cardiac output from the periphery toward the heart and the brain. In view of this, we investigated developmental changes in the contractile and relaxing properties of the peripheral femoral artery (Fem) and the more central carotid artery (Car) at 0.7, 0.8, and 0.9 of the chicken embryo incubation time. Isolated arteries were studied in myographs and were exposed to norepinephrine or phenylephrine. High K(+) (125 mM) and electrical field stimulation (0.25-16 Hz) were used to induce receptor-independent and neurogenic contractions. Relaxing responses to ACh were evaluated in the absence and presence of the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and before and after endothelium removal. alpha(1)-Adrenergic contractile responses increased in a time-dependent manner and were significantly larger in Fem than in Car. Neurogenic contractions and adrenergic nerves could only be demonstrated in Fem at 0.9 incubation. ACh caused relaxation in both Fem and Car at 0.7, 0.8, and 0.9 incubation. The NO independent part of the relaxation was more pronounced in Car than in Fem at all developmental stages. We conclude that the chicken embryo is a useful model to investigate the development of vasomotor control and vascular heterogeneity. The observed regional vascular differences may contribute to cardiac output redistribution during hypoxia in the embryo and might result from endothelial and neurogenic influences on vascular smooth muscle differentiation. PMID- 10749724 TI - Preeclamptic pregnancy is associated with increased sympathetic and decreased parasympathetic control of HR. AB - Previous work from our laboratory using heart rate variability (HRV) has demonstrated that women before menopause have a more dominant parasympathetic and less effective sympathetic regulations of heart rate compared with men. Because it is still not clear whether normal or preeclamptic pregnancy coincides with alternations in the autonomic functions, we evaluated the changes of HRV in 17 nonpregnant, 17 normotensive pregnant, and 11 preeclamptic women who were clinically diagnosed without history of diabetic neuropathy, cardiac arrhythmia, and other cardiovascular diseases. Frequency-domain analysis of short-term, stationary R-R intervals was performed to evaluate the total variance, low frequency power (LF; 0.04-0.15 Hz), high-frequency power (HF; 0.15-0.40 Hz), ratio of LF to HF (LF/HF), and LF in normalized units (LF%). Natural logarithm transformation was applied to variance, LF, HF, and LF/HF for the adjustment of the skewness of distribution. We found that the normal pregnant group had a lower R-R value and HF but had a higher LF/HF and LF% compared with the nonpregnant group. The preeclamptic group had lower HF but higher LF/HF compared with either the normal pregnant or nonpregnant group. Our results suggest that normal pregnancy is associated with a facilitation of sympathetic regulation and an attenuation of parasympathetic influence of heart rate, and such alterations are enhanced in preeclamptic pregnancy. PMID- 10749725 TI - Force-velocity relationship and biochemical-to-mechanical energy conversion by the sarcomere. AB - The intracellular control mechanism leading to the well-known linear relationship between energy consumption by the sarcomere and the generated mechanical energy is analyzed here by coupling calcium kinetics with cross-bridge cycling. A key element in the control of the biochemical-to-mechanical energy conversion is the effect of filament sliding velocity on cross-bridge cycling. Our earlier studies have established the existence of a negative mechanical feedback mechanism whereby the rate of cross-bridge turnover from the strong, force-generating conformation to the weak, non-force-generating conformation is a linear function of the filament sliding velocity. This feedback allows the analytic derivation of the experimentally established Hill's equation for the force-velocity relationship. Moreover, it allows us to derive the transient length response to load clamps and the transient force response to sarcomere shortening at constant velocity. The results are in agreement with experimental studies. The mechanical feedback regulates the generated power, maintains the linear relationship between energy liberated by the actomyosin-ATPase and the generated mechanical energy, and determines the efficiency of biochemical-to-mechanical energy conversion. The mechanical feedback defines three elements of the mechanical energy: 1) external work done; 2) pseudopotential energy, required for cross-bridge recruitment; and 3) energy dissipation caused by the viscoelastic property of the cross bridge. The last two elements dissipate as heat. PMID- 10749726 TI - Caveolin-1 regulates shear stress-dependent activation of extracellular signal regulated kinase. AB - Fluid shear stress activates a member of the mitogen-activated protein (MAP) kinase family, extracellular signal-regulated kinase (ERK), by mechanisms dependent on cholesterol in the plasma membrane in bovine aortic endothelial cells (BAEC). Caveolae are microdomains of the plasma membrane that are enriched with cholesterol, caveolin, and signaling molecules. We hypothesized that caveolin-1 regulates shear activation of ERK. Because caveolin-1 is not exposed to the outside, cells were minimally permeabilized by Triton X-100 (0.01%) to deliver a neutralizing, polyclonal caveolin-1 antibody (pCav-1) inside the cells. pCav-1 then bound to caveolin-1 and inhibited shear activation of ERK but not c Jun NH(2)-terminal kinase. Epitope mapping studies showed that pCav-1 binds to caveolin-1 at two regions (residues 1-21 and 61-101). When the recombinant proteins containing the epitopes fused to glutathione-S-transferase (GST-Cav(1 21) or GST-Cav(61-101)) were preincubated with pCav-1, only GST-Cav(61-101) reversed the inhibitory effect of the antibody on shear activation of ERK. Other antibodies, including m2234, which binds to caveolin-1 residues 1-21, had no effect on shear activation of ERK. Caveolin-1 residues 61-101 contain the scaffolding and oligomerization domains, suggesting that binding of pCav-1 to these regions likely disrupts the clustering of caveolin-1 or its interaction with signaling molecules involved in the shear-sensitive ERK pathway. We suggest that caveolae-like domains play a critical role in the mechanosensing and/or mechanosignal transduction of the ERK pathway. PMID- 10749727 TI - Red blood cell regulation of microvascular tone through adenosine triphosphate. AB - The matching of blood flow with metabolic need requires a mechanism for sensing the needs of the tissue and communicating that need to the arterioles, the ultimate controllers of tissue perfusion. Despite significant strides in our understanding of blood flow regulation, the identity of the O(2) sensor has remained elusive. Recently, the red blood cell, the Hb-containing O(2) carrier, has been implicated as a potential O(2) sensor and contributor to this vascular control by virtue of its concomitant carriage of millimolar amounts of ATP, which it is able to release when exposed to a low-O(2) environment. To evaluate this possibility, we exposed perfused cerebral arterioles to low extraluminal O(2) in the absence and presence of red blood cells or 6% dextran and determined both vessel diameter and ATP in the vessel effluent. Only when the vessels were perfused with red blood cells did the vessels dilate in response to low extraluminal O(2). In addition, this response was accompanied by a significant increase in vessel effluent ATP. These findings support the hypothesis that the red blood cell itself serves a role in determining O(2) supply to tissue. PMID- 10749728 TI - Regulation of ET: pulmonary release of ET contributes to increased plasma ET levels and vasoconstriction in CHF. AB - Endothelin (ET) contributes to the increased systemic vascular resistance and elevated cardiac filling pressures seen in congestive heart failure (CHF). We investigated to what extent ET-mediated vasoconstriction in CHF occurs through an endocrine action of elevated plasma ET or by an autocrine/paracrine mechanism related to induction of vascular ET gene expression. Three weeks of pacing (225 beats/min) induced a marked release of ET-1 from the pulmonary circulation with a sixfold elevation of arterial plasma ET in CHF pigs compared with sham-operated pigs. Arterial plasma ET was the strongest and only independent predictor of systemic vascular resistance. In contrast, vascular preproET-1 and ET-receptor mRNA expression were unaltered or decreased in CHF pigs and did not correlate with indexes of vascular tone. However, myocardial preproET-1 mRNA expression increased twofold in CHF pigs. PreproET-2 and preproET-3 mRNAs were not detectable in cardiovascular tissues. In conclusion, plasma ET was markedly increased because of an augmented release from the pulmonary circulation during CHF, and arterial plasma ET correlated with systemic vascular resistance. The absence of ET induction in the peripheral vasculature suggests that ET increases vascular tone during CHF by an endocrine, not an autocrine/paracrine, mechanism. PMID- 10749729 TI - Role of peroxynitrite in altered fetal-placental vascular reactivity in diabetes or preeclampsia. AB - Oxidative stress may increase production of superoxide and nitric oxide, leading to formation of prooxidant peroxynitrite to cause vascular dysfunction. Having found nitrotyrosine residues, a marker of peroxynitrite action, in placental vessels of preeclamptic and diabetic pregnancies, we determined whether vasoreactivity is altered in these placentas and treatment with peroxynitrite produces vascular dysfunction. The responses of diabetic, preeclamptic, and normal placentas to increasing concentrations of the vasoconstrictors U-46619 (10(-9)-10(-7) M) and ANG II (10(-9)-10(-7) M) and the vasodilators glyceryl trinitrate (10(-9)-10(-7) M) and prostacyclin (PGI(2); 10(-8)-10(-6) M) were compared as were responses to these agents in normal placentas before and after treatment with 3.16 x 10(-4) M peroxynitrite for 30 min. Responses to both vasoconstrictors and vasodilators were significantly attenuated in diabetic and preeclamptic placentas compared with controls. Similarly, responses to U-46619, nitroglycerin, and PGI(2), but not ANG II, were significantly attenuated following peroxynitrite treatment. The presence of nitrotyrosine residues confirmed peroxynitrite interaction with placental vessels. Overall, our data suggest that peroxynitrite formation is capable of attenuating vascular responses in the human placenta. PMID- 10749730 TI - Myofibrillar disruption in hypocontractile myocardium showing perfusion contraction matches and mismatches. AB - Chronically instrumented dogs underwent 2- or 5-h regional reductions in coronary flow that were followed, respectively, by balanced reductions in myocardial contraction and O(2) consumption ("hibernation") and persistently reduced contraction despite normal myocardial O(2) consumption ("stunning"). Previously unidentified myofibrillar disruption developed during flow reduction in both experimental models and persisted throughout the duration of reperfusion (2-24 h). Aberrant perinuclear aggregates that resembled thick filaments and stained positively with a monoclonal myosin antibody were present in 34 +/- 3.8% (SE) and 68 +/- 5.9% of "hibernating" and "stunned" subendocardial myocytes in areas subjected to flow reduction and in 16 +/- 2.5% and 44 +/- 7.4% of subendocardial myocytes in remote areas of the same ventricles. Areas of myofibrillar disruption also showed glycogen accretion and unusual heterochromatin clumping adjacent to the inner nuclear envelope. The degrees of flow reduction employed were sufficient to reduce regional myofibrillar creatine kinase activity by 25-35%, but troponin I degradation was not evident. The observed changes may reflect an early, possibly reversible, phase of the myofibrillar loss characteristic of hypocontractile myocardium in patients undergoing revascularization. PMID- 10749731 TI - Immunochemical evidence for a unique GPI-anchored carbonic anhydrase isozyme in human cardiomyocytes. AB - To clarify the controversial question of cell-specific distribution of carbonic anhydrase (CA) in the heart, endothelial cells and cardiomyocytes were isolated from porcine and human hearts and were characterized with cell-specific markers. CA activity was found in the microsomal fraction of both cell types. It was shown by Triton X-114 phase separation that both cell types possess a membrane-bound form of CA. These CAs share the same mechanism of membrane-anchoring via glycosylphosphatidylinositol (GPI), which excludes identity with transmembrane isoforms CA IX or CA XII. Western blotting analysis of human microsomes with anti human CA IV antibodies revealed a marked difference in immunoreactivity. Endothelial CA activity resulted in 11-fold stronger CA IV bands compared with identical amounts of myocytic CA activity, indicating that cardiac endothelium and cardiomyocytes possess immunologically distinct forms of CA. We conclude that in human hearts CA IV is associated with the endothelium, whereas most of the CA in myocytes is not identical with one of the known CA isozymes. This suggests that cardiomyocytic CA is a novel isozyme. PMID- 10749732 TI - Myocardial substrate metabolism influences left ventricular energetics in vivo. AB - The myocardial oxygen consumption (MVO(2)) to left ventricular pressure-volume area (PVA) relationship is assumed unaltered by substrates, despite varying phosphate-to-oxygen ratios and possible excess MVO(2) associated with fatty acid consumption. The validity of this assumption was tested in vivo. Left ventricular volumes and pressures were assessed with a combined conductance-pressure catheter in eight anesthetized pigs. MVO(2) was calculated from coronary flow and arterial coronary sinus O(2) differences. Metabolism was altered by glucose-insulin potassium (GIK) or Intralipid-heparin (IH) infusions in random order and monitored with [(14)C]glucose and [(3)H]oleate tracers. Profound shifts in glucose and fatty acid oxidation were observed. Contractility, coronary flow, and slope of the MVO(2)-PVA relationship were unchanged during GIK and IH infusions. MVO(2) at zero PVA (unloaded MVO(2)) was 0.16 +/- 0.13 J x beat(-1) x 100 g(-1) higher during IH compared with GIK infusion (P = 0.001), a 48% increase. The study demonstrates a marked energetic advantage of glucose oxidation in the myocardium, profoundly affecting the MVO(2)-PVA relationship. This may in part explain the "oxygen-wasting" effect of lipid-enhancing interventions such as adrenergic drugs and ischemia. PMID- 10749733 TI - In vivo expression profile of an endothelial nitric oxide synthase promoter reporter transgene. AB - Endothelium-derived nitric oxide (NO) is primarily attributable to constitutive expression of the endothelial nitric oxide synthase (eNOS) gene. Although a more comprehensive understanding of transcriptional regulation of eNOS is emerging with respect to in vitro regulatory pathways, their relevance in vivo warrants assessment. In this regard, promoter-reporter insertional transgenic murine lines were created containing 5,200 bp of the native murine eNOS promoter directing transcription of nuclear-localized beta-galactosidase. Examination of beta galactosidase expression in heart, lung, kidney, liver, spleen, and brain of adult mice demonstrated robust signal in large and medium-sized blood vessels. Small arterioles, capillaries, and venules of the microvasculature were notably negative, with the exception of the vasa recta of the medullary circulation of the kidney, which was strongly positive. Only in the brain was the reporter expressed in non-endothelial cell types, such as the CA1 region of the hippocampus. Epithelial cells of the bronchi, bronchioles, and alveoli were scored as negative, as was renal tubular epithelium. Cardiac myocytes, skeletal muscle, and smooth muscle of both vascular and nonvascular sources failed to demonstrate beta-galactosidase staining. Expression was uniform across multiple founders and was not significantly affected by genomic integration site. These transgenic eNOS promoter-reporter lines will be a valuable resource for ongoing studies addressing the regulated expression of eNOS in vivo in both health and disease. PMID- 10749734 TI - Altered membrane proteins and permeability correlate with cardiac dysfunction in cardiomyopathic hamsters. AB - A mutation in the delta-sarcoglycan (SG) gene with absence of delta-SG protein in the heart has been identified in the BIO14.6 cardiomyopathic (CM) hamster, but how the defective gene leads to myocardial degeneration and dysfunction is unknown. We correlated left ventricular (LV) function with increased sarcolemmal membrane permeability and investigated the LV distribution of the dystrophin dystroglycan complex in BIO14.6 CM hamsters. On echocardiography at 5 wk of age, the CM hamsters showed a mildly enlarged diastolic dimension (LVDD) with decreased LV percent fractional shortening (%FS), and at 9 wk further enlargement of LVDD with reduction of %FS was observed. The percent area of myocardium exhibiting increased membrane permeability or membrane rupture, assessed by Evans blue dye (EBD) staining and wheat germ agglutinin, was greater at 9 than at 5 wk. In areas not stained by EBD, immunostaining of dystrophin was detected in CM hamsters at sarcolemma and T tubules, as expected, but it was also abnormally expressed at the intercalated discs; in addition, the expression of beta dystroglycan was significantly reduced compared with control hearts. As previously described, alpha-SG was completely deficient in CM hearts compared with control hearts. In myocardial areas showing increased sarcolemmal permeability, neither dystrophin nor beta-dystroglycan could be identified by immunolabeling. Thus, together with the known loss of delta-SG and other SGs, abnormal distribution of dystrophin and reduction of beta-dystroglycan are associated with increased sarcolemmal permeability followed by cell rupture, which correlates with early progressive cardiac dysfunction in the BIO14.6 CM hamster. PMID- 10749735 TI - Generation of intracellular pH gradients in single cardiac myocytes with a microperfusion system. AB - This study describes the use of a microperfusion system to create rapid, large regional changes in intracellular pH (pH(i)) within single ventricular myocytes. The spatial distribution of pH(i) in single myocytes was measured with seminaphthorhodafluor-1 fluorescence using confocal imaging. Changes in pH(i) were induced by local external application of NH(4)Cl, CO(2), or sodium propionate. Local application was achieved by simultaneously directing two parallel square microstreams, each 275 microm wide, over a single myocyte oriented perpendicular to the direction of flow. One stream contained the control solution, and the other contained a weak acid or base. End-to-end, stable pH(i) gradients as large as 1 pH unit were readily created with this technique. This result indicates that pH within a single cardiac cell may not always be spatially uniform, particularly when weak acid or base gradients are present, which can occur, for example, in regional myocardial ischemia. The microperfusion method should be useful for studying the effects of localized acidosis on myocyte function, estimating intracellular ion diffusion rates, and, possibly, inducing regional changes in other important intracellular ions. PMID- 10749736 TI - Focal extracellular potential: a means to monitor electrical activity in single cardiac myocytes. AB - The focal extracellular potential (FEP) described in this study is an electrophysiological signal related to the transmembrane potential (V(m)) of cardiac myocytes that avoids the mechanical fragility, interference with contraction, and intracellular contact associated with conventional whole cell recording. One end of a frog ventricular myocyte was secured into a glass holding pipette. The FEP was measured differentially between this pipette and a bath pipette while the cell was voltage- or current-clamped by a third whole cell pipette. The FEP appeared as an amplitude-truncated action potential, while FEP duration accurately reflected the action potential duration (APD) at 90% repolarization (APD(90)). FEP magnitude increased as the holding pipette K(+) concentration ([K(+)]) was increased. The FEP-voltage relation was quasi-linear at negative V(m) with a slope that increased with elevated holding pipette [K(+)]. Increasing the membrane conductance inside the holding pipette by adding amphotericin B or cromakalim linearized the FEP-voltage relation across all V(m). The FEP accurately reported electrical activation and APD(90) during changes of stimulation frequency and episodes of cellular stretch. PMID- 10749737 TI - Resistance to myocardial ischemia in five rat strains: is there a genetic component of cardioprotection? AB - There is a need to develop new and more consistent animal models of cardioprotection. Traditionally, outbred dogs, rabbits, and rats have been studied. We determined resistance to ischemia in isolated hearts from inbred strains of rats. Hearts from inbred rats: SS/Mcw (Dahl S, Dahl salt-sensitive), DA/Hsd (Dark Agouti), LEW/Hsd (Lewis), and BN/SsN/Mcw (Brown Norway); and from an outbred rat: Hsd:WIST (Wistar) were subjected to 27 min of global, no-flow ischemia, followed by 3 h of reperfusion. Infarct size in the Brown Norway rat was 2.5 times less than that observed in the Dahl S rat, with the Dark Agouti, Lewis, and Wistar rats intermediate in response. Hearts from Brown Norway rats were also most resistant to ischemia in terms of postischemic enzyme leakage and contractile and vascular function compared with other strains. The average polymorphism rate between strains revealed that such strains were genetically diverse. This study demonstrates strain differences in resistance to myocardial ischemia, suggesting these rats could be used to study a genetic and/or environmental basis for these differences and to provide new animal models for the physiological study of cardioprotection. PMID- 10749738 TI - Fluid shear stress increases membrane fluidity in endothelial cells: a study with DCVJ fluorescence. AB - Fluid shear stress (FSS) has been shown to be an ubiquitous stimulator of mammalian cell metabolism. Although many of the intracellular signal transduction pathways have been characterized, the primary mechanoreceptor for FSS remains unknown. One hypothesis is that the cytoplasmic membrane acts as the receptor for FSS, leading to increased membrane fluidity, which in turn leads to the activation of heterotrimetric G proteins (13). 9-(Dicyanovinyl)-julolidine (DCVJ) is a fluorescent probe that integrates into the cell membrane and changes its quantum yield with the viscosity of the environment. In a parallel-plate flow chamber, confluent layers of DCVJ-labeled human endothelial cells were exposed to different levels of FSS. With increased FSS, a reduced fluorescence intensity was observed, indicating an increase of membrane fluidity. Step changes of FSS caused an approximately linear drop of fluorescence within 5 s, showing fast and almost full recovery after shear cessation. A linear dose-response relationship between shear stress and membrane fluidity changes was observed. The average fluidity increase over the entire cell monolayer was 22% at 26 dyn/cm(2). This study provides evidence for a link between FSS and membrane fluidity, and suggests that the membrane is an important flow mechanosensor of the cell. PMID- 10749739 TI - Adenoviral E3-14.7K protein in LPS-induced lung inflammation. AB - The adenoviral E3-14.7K protein is a cytoplasmic protein synthesized after adenoviral infection. To assess the contribution of E3-14. 7K-sensitive pathways in the modulation of inflammation by the respiratory epithelium, inflammatory responses to intratracheal lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-alpha were assessed in transgenic mice bearing the adenoviral E3-14.7K gene under the direction of the surfactant protein (SP) C promoter. When E3-14.7K transgenic mice were administered LPS intratracheally, lung inflammation as indicated by macrophage and neutrophil accumulation in bronchoalveolar lavage fluid was decreased compared with wild-type control mice. Lung inflammation and epithelial cell injury were decreased in E3-14.7K mice 24 and 48 h after LPS administration. Intracellular staining for surfactant proprotein (proSP) B, proSP C, and SP-B was decreased and extracellular staining was markedly increased in wild-type mice after LPS administration, consistent with LPS-induced lung injury. In contrast, intense intracellular staining of proSP-B, proSP-C, and SP-B persisted in type II cells of E3-14.7K mice, whereas extracellular staining of proSP-B and proSP-C was absent. Inhibitory effects of intratracheal LPS on SP-C mRNA were ameliorated by expression of the E3-14.7K gene. Similar to the response to LPS, lung inflammation after intratracheal administration of TNF-alpha was decreased in E3-14.7K transgenic mice. Levels of TNF-alpha after LPS administration were similar in wild-type and E3-14.7K-bearing mice. Cell selective expression of E3-14.7K in the respiratory epithelium inhibited LPS- and TNF-alpha-mediated lung inflammation, demonstrating the critical role of respiratory epithelial cells in LPS- and TNF-alpha-induced lung inflammation. PMID- 10749740 TI - Possible role of ROS as mediators of hypoxia-induced ion transport inhibition of alveolar epithelial cells. AB - In oxygen-sensitive excitable cells, responses to hypoxia are initiated by membrane depolarization due to closing of the K channels that is thought to be mediated by a decrease in reactive oxygen species (ROS). Because the mechanisms of hypoxic inhibition of ion transport of alveolar epithelial cells (Planes C, Friedlander G, Loiseau A, Amiel C, and Clerici C. Am J Physiol Lung Cell Mol Physiol 271: L70-L78, 1996; Mairbaurl H, Wodopia R, Eckes S, Schulz S, and Bartsch P. Am J Physiol Lung Cell Mol Physiol 273: L797-L806, 1997) are not yet understood, we tested the possible involvement of a hypoxia-induced change in ROS that might control transport activity. Transport was measured as (86)Rb and (22)Na uptake in A549 cells exposed to normoxia, hyperoxia, or hypoxia together with ROS donors and scavengers. H(2)O(2) < 1 mM did not affect transport, whereas 1 mM H(2)O(2) activated (22)Na uptake (+200%) but inhibited (86)Rb uptake (-30%). Also hyperoxia, aminotriazole plus menadione, and diethyldithiocarbamate inhibited (86)Rb uptake. N-acetyl-L-cysteine, diphenyleneiodonium, and tetramethylpiperidine-N-oxyl, used to reduce ROS, inhibited (86)Rb uptake, thus mimicking the hypoxic effects, whereas deferoxamine, superoxide dismutase, and catalase were ineffective. Also, hypoxic effects on ion transport were not prevented in the presence of H(2)O(2), diethyldithiocarbamate, and N-acetyl-L cysteine. These results indicate that ion transport of A549 cells is significantly affected by decreasing or increasing cellular ROS levels and that it is possible that certain species of ROS might mediate the hypoxic effects on ion transport of alveolar epithelial cells. PMID- 10749741 TI - Transcriptional regulation and structural organization of the human cytosolic phospholipase A(2) gene. AB - Cytokines are established regulators of the arachidonic acid cascade in lung cells. The levels of various arachidonic metabolites distinguish the normal and pathogenic states of the human lung. Arachidonyl-selective cytosolic phospholipase A(2) (cPLA(2)) is ubiquitously present in human lung and is most likely the rate-limiting step in eicosanoid generation. We therefore studied the regulation of this pivotal gene in human lung fibroblasts and epithelial cells by proinflammatory cytokines. We demonstrate a dose- and time-dependent induction of human cPLA(2) mRNA by interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma as well as the abrogation of this induction by glucocorticoids. Nuclear runoff studies demonstrate that de novo transcription of the cPLA(2) gene is required for cytokine induction. We have characterized the human cPLA(2) gene, which is encoded by 18 exons and spans in excess of 137 kb. Deletion analysis of a 3.4-kb fragment of the human promoter identified two regions responsible for basal expression of the cPLA(2) gene. Conversely, a CA-dinucleotide repeat in the proximal promoter appears to repress overall promoter activity. Understanding the molecular mechanisms associated with cytokine-dependent expression of the cPLA(2) gene should provide further insight into regulating the level of proinflammatory mediators in pulmonary diseases. PMID- 10749742 TI - The potential of various lipopolysaccharides to release IL-8 and G-CSF. AB - Lipopolysaccharide (LPS) derived from Pseudomonas aeruginosa is less cytotoxic than that from Escherichia coli. But P. aeruginosa induces a prominent sustained lung inflammation as in cystic fibrosis and diffuse panbronchiolotis. The present study examined the potential for several LPSs obtained from E. coli and P. aeruginosa to release neutrophil chemotactic activity (NCA) from lung cells. LPSs differently stimulated A549 cells, BEAS-2B cells, and lung fibroblasts to release NCA [P. aeruginosa > E. coli 0127:B8 (Difco) > E. coli 055:B5 (Sigma) > E. coli 026:B6 (Sigma)]. E. coli 0127:B8 (Sigma) and 0111:B4 (Sigma) did not stimulate these cells. NCA was chemotactic by checkerboard analysis. Molecular-sieve column chromatography revealed three chemotactic peaks. The release of NCA was inhibited by cycloheximide and lipoxygenase inhibitors. Experiments with blocking antibodies suggested that much of the NCA was secondary to the release of interleukin (IL)-8 and granulocyte colony-stimulating factor (G-CSF). Thus we examined the concentrations of IL-8 and G-CSF and found that the potency of the various LPSs to stimulate NCA closely paralleled the potency in releasing IL-8 and G-CSF. But a difference among LPSs to stimulate A549 cells was observed. Finally, the release of IL-6 showed similar results. These data suggest that P. aeruginosa LPS may stimulate lung cells to release more NCA than E. coli LPSs, leading to sustained lung inflammation. PMID- 10749743 TI - IL-10 reduces Th2 cytokine production and eosinophilia but augments airway reactivity in allergic mice. AB - We investigated the effects of interleukin (IL)-10 administration on allergen induced Th2 cytokine production, eosinophilic inflammation, and airway reactivity. Mice were sensitized by intraperitoneal injection of ragweed (RW) adsorbed to Alum and challenged by intratracheal instillation of the allergen. Sensitization and challenge with RW increased concentrations of IL-10 in bronchoalveolar lavage (BAL) fluid from undetectable levels to 60 pg/ml over 72 h. Intratracheal instillation of 25 ng of recombinant murine IL-10 at the time of RW challenge further elevated BAL fluid IL-10 concentration to 440 pg/ml but decreased BAL fluid IL-4, IL-5, and interferon-gamma levels by 40-85% and eosinophil numbers by 70% (P < 0.0001). Unexpectedly, the same IL-10 treatment increased airway reactivity to methacholine in spontaneously breathing mice that had been sensitized and challenged with RW (P < 0.001). IL-10 treatment in naive animals or RW-sensitized mice challenged with PBS failed to increase airway reactivity, demonstrating that IL-10 induces an increase in airway reactivity only when it is administered in conjunction with allergic sensitization and challenge. The results demonstrate that IL-10 reduces Th2 cytokine levels and eosinophilic inflammation but augments airway hyperreactivity. Thus, despite its potent anti-inflammatory activity, IL-10 could contribute to the decline in pulmonary function observed in asthma. PMID- 10749744 TI - Human neutrophil elastase releases two pools of mucinlike glycoconjugate from tracheal submucosal gland cells. AB - Neutrophil elastase can contribute to the pathogenesis of increased airway reactivity and excess mucus secretion in many pulmonary diseases. Ten nanomolar human neutrophil elastase (HNE) effectively empties airway serous cells, raising the question of why HNE is not equally effective at emptying mucous cells of their stored mucin because total release of mucin granules is not seen in postmortem examination of even the most severe disease. To better resolve the mucus secretagogue action of HNE, we measured secretion of mucinlike glycoconjugates (MGCs) released from freshly isolated swine tracheal submucosal gland cells in fractions of the superfusate acquired every 2 min. Six to fifty nanomolar HNE released a fixed quantity of MGCs at an increasing rate with increasing concentrations of enzyme, an action consistent with the release of cell surface mucinlike molecules. The polycation poly-L-lysine (1 microg/ml) released a similar transient of MGCs. A steady-state doubling of MGC rate of release was seen as long as 100 nM HNE was present, but this stimulus represented less than a 1% release of stored MGCs/min and was consistent with release of mucin vesicles from cell stores. Both actions of HNE were inhibited by the specific inhibitors L-680833 and DMP-777 but not by 30 microM erythromycin. Therefore, HNE release of MGCs from tracheal submucosal glands is limited by both the fixed quantity of the MGCs in the transient pool and by the small steady state response to the higher concentrations of enzyme. PMID- 10749745 TI - Residual oil fly ash and charged polymers activate epithelial cells and nociceptive sensory neurons. AB - Residual oil fly ash (ROFA) is an industrial pollutant that contains metals, acids, and unknown materials complexed to a particulate core. The heterogeneous composition of ROFA hampers finding the mechanism(s) by which it and other particulate pollutants cause airway toxicity. To distinguish culpable factors contributing to the effects of ROFA, synthetic polymer microsphere (SPM) analogs were synthesized that resembled ROFA in particle size (2 and 6 microm in diameter) and zeta potential (-29 mV). BEAS-2B human bronchial epithelial cells and dorsal root ganglion neurons responded to both ROFA and charged SPMs with an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the release of the proinflammatory cytokine interleukin-6, whereas neutral SPMs bound with polyethylene glycol (0-mV zeta potential) were relatively ineffective. In dorsal root ganglion neurons, the SPM-induced increases in [Ca(2+)](i) were correlated with the presence of acid- and/or capsaicin-sensitive pathways. We hypothesized that the acidic microenvironment associated with negatively charged colloids like ROFA and SPMs activate irritant receptors in airway target cells. This causes subsequent cytokine release, which mediates the pathophysiology of neurogenic airway inflammation. PMID- 10749746 TI - Antioxidant defense mechanisms of human mesothelioma and lung adenocarcinoma cells. AB - The development of drug resistance of tumors is multifactorial and still poorly understood. Some cytotoxic drugs generate free radicals, and, therefore, antioxidant enzymes may contribute to drug resistance. We investigated the levels of manganese superoxide dismutase (Mn SOD), its inducibility, and its protective role against tumor necrosis factor-alpha and cytotoxic drugs (cisplatin, epirubicin, methotrexate, and vindesin) in human pleural mesothelioma (M14K) and pulmonary adenocarcinoma (A549) cells. We also studied other major antioxidant mechanisms in relation to oxidant and drug resistance of these cells. A549 cells were more resistant than M14K cells toward both oxidants (hydrogen peroxide and menadione) and all the cytotoxic drugs tested. M14K cells contained higher basal Mn SOD activity than A549 cells (28.3 +/- 3.4 vs. 1.8 +/- 0.3 U/mg protein), and Mn SOD activity was significantly induced by tumor necrosis factor-alpha only in A549 cells (+524%), but the induction did not offer any protection during subsequent oxidant or drug exposure. Mn SOD was not induced significantly in either of these cell lines by any of the cytotoxic drugs (0.007-2 microM, 48 h) tested when assessed by Northern blotting, Western blotting, or specific activity. A549 cells contained higher catalase activity than M14K cells (7.6 +/- 1.3 vs. 3.6 +/- 0.5 nmol O(2). min(-1). mg protein(-1)). They also contained twofold higher levels of glutathione and higher immunoreactivity of the heavy subunit of gamma-glutamylcysteine synthetase than M14K cells. Experiments with inhibitors of gamma-glutamylcysteine synthetase and catalase supported our conclusion that mechanisms associated with glutathione contribute to the drug resistance of these cells. PMID- 10749747 TI - Phorbol ester-induced U-937 differentiation: effects on integrin alpha(5) gene transcription. AB - Lung injury is accompanied by increased deposition of fibronectin (FN) matrices. Activated monocytic cells recruited to sites of lung injury express integrin receptors for FN that mediate their interaction with this matrix. One such integrin, alpha(5)beta(1), mediates many of the biological effects of FN, and its expression may be important for immune cell function at sites of lung injury. Herein, we examine the expression of alpha(5)beta(1) in response to the tumor promoter phorbol 12-myristate 13-acetate (PMA) in the human promonocytic cell line U-937. We demonstrate that PMA enhanced the adherence of U-937 cells to FN by increasing the expression of both the alpha(5)- and beta(1)-subunit mRNAs and the surface expression of the protein. In U-937 cells transfected with an alpha(5) promoter-reporter gene, we found that PMA induced the transcription of the alpha(5) gene by acting on very specific promoter sequences other than activator protein-1 in a protein kinase C-dependent manner. Lipopolysaccharide had a similar effect. Modulation of alpha(5)beta(1) expression may be important for regulation of monocytic cell function in lung inflammation after injury. PMID- 10749748 TI - Surfactant protein A prevents silica-mediated toxicity to rat alveolar macrophages. AB - Silicosis is a serious occupational lung disease associated with irreversible pulmonary fibrosis. The interaction between inhaled crystalline silica and the alveolar macrophage (AM) is thought to be a key event in the development of silicosis and fibrosis. Silica can cause direct injury to AMs and can induce AMs to release various inflammatory mediators. Acute silicosis is also characterized by a marked elevation in surfactant apoprotein A (SP-A); however, the role of SP A in silicosis is unknown. We investigated whether SP-A directly affects the response of AMs to silica. In this study, the degree of silica toxicity to cultured rat AMs as assessed by a (51)Cr cytotoxicity assay was shown to be dependent on the time of exposure and the concentration and size of the silica particles. Silica directly injured rat AMs as evidenced by a cytotoxic index of 32.9 +/- 2.5, whereas the addition of rat SP-A (5 microg/ml) significantly reduced the cytotoxic index to 16.6 +/- 1.2 (P < 0. 001). This effect was reversed when SP-A was incubated with either polyclonal rabbit anti-rat SP-A antibody or D-mannose. These data indicate that SP-A mitigates the effect of silica on AM viability, and this effect may involve the carbohydrate recognition domain of SP-A. The elevation of SP-A in acute silicosis may serve as a normal host response to prevent lung cell injury after exposure to silica. PMID- 10749749 TI - Increased production of nitrotyrosine in lung tissue of rats with radiation induced acute lung injury. AB - The purposes of this study were 1) to identify the nitric oxide (NO) synthase (NOS) isoform responsible for NO-mediated radiation-induced lung injury, 2) to examine the formation of nitrotyrosine, and 3) to see whether nitrotyrosine formation and lung injury are reduced by an inducible NOS (iNOS) inhibitor, aminoguanidine. The left hemithorax of rats was irradiated (20 Gy), and the degree of lung injury, the expression of NOS isoforms, and the formation of nitrotyrosine and superoxide were examined after 2 wk. iNOS mRNA was induced, and endothelial NOS mRNA was markedly increased in the irradiated lung. Nitrotyrosine was detected biochemically and immunohistochemically. Aminoguanidine prevented acute lung injury as indicated by decreased protein concentration and lactate dehydrogenase activity in bronchoalveolar lavage fluid and improved NMR parameters and histology. Furthermore, the formation of nitrotyrosine was significantly reduced in the aminoguanidine group. We conclude that iNOS induction is a major factor in radiation-induced lung injury and that nitrotyrosine formation may participate in the NO-induced pathogenesis. PMID- 10749750 TI - Effect of erythromycin on ATP-induced intracellular calcium response in A549 cells. AB - ATP induced a biphasic increase in the intracellular Ca(2+)concentration ([Ca(2+)](i)), an initial spike, and a subsequent plateau in A549 cells. Erythromycin (EM) suppressed the ATP-induced [Ca(2+)](i) spike but only in the presence of extracellular calcium (Ca(2+)(o)). It was ineffective against ATP- and UTP-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] formation and UTP induced [Ca(2+)](i) spike, implying that EM perturbs Ca(2+) influx from the extracellular space rather than Ca(2+)release from intracellular Ca(2+) stores via the G protein-phospholipase C-Ins(1,4,5)P(3) pathway. A verapamil-sensitive, KCl-induced increase in [Ca(2+)](i) and the Ca(2+) influx activated by Ca(2+) store depletion were insensitive to EM. 3'-O-(4-benzoylbenzoyl)-ATP evoked an Ca(2+)(o)-dependent [Ca(2+)](i) response even in the presence of verapamil or the absence of extracellular Na(+), and this response was almost completely abolished by EM pretreatment. RT-PCR analyses revealed that P2X(4) as well as P2Y(2), P2Y(4), and P2Y(6) are coexpressed in this cell line. These results suggest that in A549 cells 1) the coexpressed P2X(4) and P2Y(2)/P2Y(4) subtypes contribute to the ATP-induced [Ca(2+)](i) spike and 2) EM selectively inhibits Ca(2+) influx through the P2X channel. This action of EM may underlie its clinical efficacy in the treatment of airway inflammation. PMID- 10749752 TI - In situ localization and regulation of thromboxane A(2) synthase in normal and LPS-primed lungs. AB - Thromboxane (Tx) A(2) synthase catalyzes the conversion of prostaglandin H(2) to the unstable metabolite TxA(2), which is a potent mediator of vasoconstriction and bronchoconstriction. The cellular localization of TxA(2) synthase was examined by immunohistochemistry and in situ hybridization in human and rat lung tissues. Bronchial epithelial cells, bronchial smooth muscle cells, peribronchial nerve fibers, single cells of bronchus-associated lymphoid tissue, single cells located in the alveolar septum, and alveolar macrophages exhibited positive immunostaining for TxA(2) synthase protein in lung tissue of both species. In addition, vascular smooth muscle cells of muscular and partially muscular vessels displayed strong (rat) and moderate (human) immunostaining for TxA(2) synthase. In situ hybridization performed in the rat lungs demonstrated TxA(2) synthase mRNA localization in accordance with the immunostaining pattern. Perfusing isolated rat lungs with endotoxin for 1 and 2 h resulted in a marked increase in TxA(2) synthase protein staining intensity in most cell types as measured by quantitative image analysis, whereas the in situ hybridization signal was unchanged. We conclude that the pulmonary distribution of TxA(2) synthase displays close similarity between rat and human lung tissues and matches well with the previously described immunolocalization of cyclooxygenase-1 and cyclooxygenase-2 in this tissue. Endotoxin challenge is suggested to cause a rapid upregulation of TxA(2) synthase at the posttranscriptional level. These data provide a morphological basis for the understanding of the role of TxA(2) in the regulation of lung bronchial and vascular tone and in immunologic events. PMID- 10749751 TI - Cloning and expression of guinea pig TIMP-2. Expression in normal and hyperoxic lung injury. AB - Tissue inhibitors of metalloproteinases (TIMPs) play a key regulatory role in extracellular matrix remodeling. By screening a lung library with a human TIMP-2 cDNA probe, we have isolated the cDNA corresponding to guinea pig TIMP-2. The 3.5 kb cDNA presents an open reading frame that predicts a protein of 220 amino acids showing 97.2, 96.8, 97.2, and 77.3% overall identity with human, mouse, rat, and chicken TIMP-2, respectively. Guinea pig TIMP-2 cDNA was expressed in CHO-K1 cells, showing a protein with the expected molecular weight and activity. Northern blot analysis revealed TIMP-2 expression in brain, kidney, intestine, spleen, heart, and lung. Transforming growth factor-beta downregulated TIMP-2 mRNA in guinea pig lung fibroblasts, whereas a variety of other stimuli showed no effect. In normal and hyperoxia-exposed lungs, TIMP-2 mRNA was mainly localized in alveolar macrophages and epithelial cells. No quantitative differences were found by Northern blot. These results confirm that TIMP-2 is highly conserved in mammals and largely expressed in lungs. PMID- 10749753 TI - Ovine surfactant protein cDNAs: use in studies on fetal lung growth and maturation after prolonged hypoxemia. AB - cDNAs for ovine surfactant-associated protein (SP) A, SP-B, and SP-C have been cloned and shown to possess strong similarity to cDNAs for surfactant apoproteins in other species. These reagents were employed to examine the effect of fetal hypoxia on the induction of surfactant apoprotein expression in the fetal lamb. Postnatal lung function is dependent on adequate growth and maturation during fetal development. Insulin-like growth factor (IGF) I and IGF-II, which are present in all fetal tissues studied, possess potent mitogenic and proliferative actions, and their effects can be modulated by IGF-specific binding proteins (IGFBPs). Hypoxia can lead to increases in circulating cortisol and catecholamines that can influence lung maturation. Therefore, the effects of mild hypoxia in chronically catheterized fetal lambs at gestational days 126-130 and 134-136 (term 145 days) on the expression of pulmonary surfactant apoproteins and IGFBPs were examined. Mild hypoxia for 48 h resulted in an increase in plasma cortisol that was more pronounced at later gestation, and in these animals, there was a twofold increase in SP-A mRNA. SP-B mRNA levels also increased twofold, but this was not significant. SP-C mRNA was not altered. No significant changes in apoprotein mRNA were observed with the younger fetuses. However, these younger animals selectively exhibited reduced IGFBP-5 mRNA levels. IGF-I mRNA was also reduced at 126-130 days, although this conclusion is tentative due to low abundance. IGF-II levels were not affected at either gestational age. We conclude that these data suggest that mild prolonged fetal hypoxia produces alterations that could affect fetal cellular differentiation early in gestation and can induce changes consistent with lung maturation closer to term. PMID- 10749754 TI - cDNA cloning of ovine pulmonary SP-A, SP-B, and SP-C: isolation of two different sequences for SP-B. AB - Pulmonary surfactant promotes alveolar stability by lowering the surface tension at the air-liquid interface in the peripheral air spaces. The three surfactant proteins SP-A, SP-B, and SP-C contribute to dynamic surface properties involved during respiration. We have cloned and sequenced the complete cDNAs for ovine SP A and SP-C and two distinct forms of ovine SP-B cDNAs. The nucleotide sequence of ovine SP-A cDNA consists of 1,901 bp and encodes a protein of 248 amino acids. Ovine SP-C cDNA contains 809 bp, predicting a protein of 190 amino acids. Ovine SP-B is encoded by two mRNA species, which differ by a 69-bp in-frame deletion in the region coding for the active airway protein. The larger SP-B cDNA comprises 1,660 bp, encoding a putative protein of 374 amino acids. With the sequences reported, a more complete analysis of surfactant regulation and the determination of their physiological function in vivo will be enabled. PMID- 10749755 TI - Inhaled carbon monoxide does not cause pulmonary vasodilation in the late gestation fetal lamb. AB - As observed with nitric oxide (NO), carbon monoxide (CO) binds and may activate soluble guanylate cyclase and increase cGMP levels in smooth muscle cells in vitro. Because inhaled NO (I(NO)) causes potent and sustained pulmonary vasodilation, we hypothesized that inhaled CO (I(CO)) may have similar effects on the perinatal lung. To determine whether I(CO) can lower pulmonary vascular resistance (PVR) during the perinatal period, we studied the effects of I(CO) on late-gestation fetal lambs. Catheters were placed in the main pulmonary artery, left pulmonary artery (LPA), aorta, and left atrium to measure pressure. An ultrasonic flow transducer was placed on the LPA to measure blood flow to the left lung. After baseline measurements, fetal lambs were mechanically ventilated with a hypoxic gas mixture (inspired O(2) fraction < 0.10) to maintain a constant fetal arterial PO(2). After 60 min (baseline), the lambs were treated with I(CO) [5-2,500 parts/million (ppm)]. Comparisons were made with I(NO) (5 and 20 ppm) and combined I(NO) (5 ppm) and I(CO) (100 and 2,500 ppm). We found that I(CO) did not alter left lung blood flow or PVR at any of the study doses. In contrast, low dose I(NO) decreased PVR by 47% (P < 0.005). The combination of I(NO) and I(CO) did not enhance the vasodilator response to I(NO). To determine whether endogenous CO contributes to vascular tone in the fetal lung, zinc protoporphyrin IX, an inhibitor of heme oxygenase, was infused into the LPA in three lambs. Zinc protoporphyrin IX had no effect on baseline PVR, aortic pressure, or the pressure gradient across the ductus arteriosus. We conclude that I(CO) does not cause vasodilation in the near-term ovine transitional circulation, and endogenous CO does not contribute significantly to baseline pulmonary vascular tone or ductus arteriosus tone in the late-gestation ovine fetus. PMID- 10749756 TI - Developmental changes in endothelin expression and activity in the ovine fetal lung. AB - Mechanisms that regulate endothelin (ET) in the perinatal lung are complex and poorly understood, especially with regard to the role of ET before and after birth. We hypothesized that the ET system is developmentally regulated and that the balance of ET(A) and ET(B) receptor activity favors vasoconstriction. To test this hypothesis, we performed a series of molecular and physiological studies in the fetal lamb, newborn lamb, and adult sheep. Lung preproET-1 mRNA levels, tissue ET peptide levels, and cellular localization of ET-1 expression were determined by Northern blot analysis, peptide assay, and immunohistochemistry in distal lung tissue from fetal lambs between 70 and 140 days (term = 145 days), newborn lambs, and ewes. Lung mRNA expression for the ET(A) and ET(B) receptors was also measured at these ages. We found that preproET-1 mRNA expression increased from 113 to 130 days gestation. Whole lung ET protein content was highest at 130 days gestation but decreased before birth in the fetal lamb lung. Immunolocalization of ET-1 protein showed expression of ET-1 in the vasculature and bronchial epithelium at all gestational ages. ET(A) receptor mRNA expression and ET(B) receptor mRNA increased from 90 to 125 and 135 days gestation. To determine changes in activity of the ET(A) and ET(B) receptors, we studied the effect of selective antagonists to the ET(A) or ET(B) receptors at 120, 130, and 140 days of fetal gestation. ET(A) receptor-mediated vasoconstriction increased from 120 to 140 days, whereas blockade of the ET(B) receptor did not change basal fetal pulmonary vascular tone at any age examined. We conclude that the ET system is developmentally regulated and that the increase in ET(A) receptor gene expression correlates with the onset of the vasodilator response to ET(A) receptor blockade. Although ET(B) receptor gene expression increases during late gestation, the balance of ET receptor activity favors vasoconstriction under basal conditions. We speculate that changes in ET receptor activity play important roles in regulation of pulmonary vascular tone in the ovine fetus. PMID- 10749757 TI - Immunotargeting of glucose oxidase to endothelium in vivo causes oxidative vascular injury in the lungs. AB - Vascular immunotargeting is a novel approach for site-selective drug delivery to endothelium. To validate the strategy, we conjugated glucose oxidase (GOX) via streptavidin with antibodies to the endothelial cell surface antigen platelet endothelial cell adhesion molecule (PECAM). Previous work documented that 1) anti PECAM-streptavidin carrier accumulates in the lungs after intravenous injection in animals and 2) anti-PECAM-GOX binds to, enters, and kills endothelium via intracellular H(2)O(2) generation in cell culture. In the present work, we studied the targeting and effect of anti-PECAM-GOX in animals. Anti-PECAM-GOX, but not IgG-GOX, accumulated in the isolated rat lungs, produced H(2)O(2,) and caused endothelial injury manifested by a fourfold elevation of angiotensin converting enzyme activity in the perfusate. In intact mice, anti-PECAM-GOX accumulated in the lungs (27 +/- 9 vs. 2.4 +/- 0.3% injected dose/g for IgG-GOX) and caused severe lung injury and 95% lethality within hours after intravenous injection. Endothelial disruption and blebbing, elevated lung wet-to-dry ratio, and interstitial and alveolar edema indicated that anti-PECAM-GOX damaged pulmonary endothelium. The vascular injury in the lungs was associated with positive immunostaining for iPF(2alpha)-III isoprostane, a marker for oxidative stress. In contrast, IgG-GOX caused a minor lung injury and little (5%) lethality. Anti-PECAM conjugated with inert proteins induced no death or lung injury. None of the conjugates caused major injury to other internal organs. These results indicate that an immunotargeting strategy can deliver an active enzyme to selected target cells in intact animals. Anti-PECAM-GOX provides a novel model of oxidative injury to the pulmonary endothelium in vivo. PMID- 10749758 TI - Expression of heme oxygenase-1 in the lung in chronic hypoxia. AB - Heme oxygenase (HO)-1 is an oxygen-dependent enzyme that may regulate vascular tone and cell proliferation through the production of carbon monoxide (CO). We tested the hypothesis that HO-1 is upregulated in the lung in chronic hypoxia by exposing male Sprague-Dawley rats to 17,000 feet (395 Torr) for 0, 1, 3, 7, 14, or 21 days. After exposure, blood gases, carboxyhemoglobin (COHb) levels, and hematocrit were measured, and the lungs were either inflation fixed for immunohistochemistry or frozen for later measurement of HO enzyme activity, Western blot for HO-1 protein, and RT-PCR for HO-1 mRNA. The heart was excised and weighed, and the right-to-left heart weight ratio was determined. During hypoxia, the hematocrit increased progressively, reaching significantly higher values than the control value after 3 days. COHb levels increased above the control value after 1 day of hypoxia and increased progressively between 14 and 21 days, whereas arterial PO(2) and arterial PCO(2) did not vary significantly. HO-1 protein determined by Western blot increased for the first 7 days and declined thereafter; however, enzyme activity was elevated only after 1 day. Changes in HO-1 during hypoxia were localized by immunohistochemistry to inflammatory cells (early) and newly muscularized arterioles (later). Lung HO-1 mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase was increased after 1 and 21 days. The data indicate that lung HO-1 protein and activity are upregulated only during early chronic hypoxia, whereas persistent COHb elevations indicate high endogenous CO production rates at nonpulmonary sites. If CO has antiproliferative properties, the lack of HO enzyme activity in the lung may be permissive for pulmonary vascular proliferation in hypoxia. PMID- 10749759 TI - Cell-specific differences in ET-1 system in adjacent layers of main pulmonary artery. A new source of ET-1. AB - Endothelin-1 (ET-1) is a potent vasoconstrictor that causes sustained constriction of the pulmonary artery and modulates normal vascular tone. Endothelial cells were thought to be the major source of ET-1, but recent studies show that vascular smooth muscle cells (SMCs) are also capable of its synthesis. We examined the ET-1 and endothelin-converting enzyme-1 (ECE-1) system in cells cultured from two adjacent layers, subendothelial (L1) and inner medial (L2), of normal sheep main pulmonary artery and the response of this system to exogenous ET-1 and transforming growth factor-beta1 (TGF-beta1). End points include assessment of preproET-1 (ppET-1) and ECE-1 gene coexpression, measurement of intracellular and released ET-1, and ECE-1 activity. RT-PCR analysis revealed that ppET-1 and ECE-1 transcripts were greater in L1 than in L2 cells. The L1 cells also synthesized (L1, 3.2 +/- 0.1; L2, 1.2 +/- 0.1 fmol/10(6) cells) and released (L1, 9.2 +/- 0.5; L2, 2.3 +/-0.1 fmol/ml) greater amounts of ET-1 than L2 cells. The L2 cells internalized exogenous ET-1 in a dose-dependent manner (EC(50) 8 nmol/l) and were more responsive to exogenous ET-1 than L1 cells, showing upregulation of both the ppET-1 and ECE genes. TGF-beta1 downregulated ET 1-stimulated ppET-1 and ECE-1 transcripts but only in L2 cells. In addition, L1 cells showed greater ECE-1 activity than L2 cells, and in both, the activity was sensitive to the metalloprotease inhibitor phosphoramidon. We conclude that the ET-1 system in L1 and L2 cells is distinct. The data suggest that the two cell types have diverse functions in the arterial wall; the L1 cells, like endothelial cells, provide a local source of ET-1; and since the L2 cells are more responsive to exogenous ET-1, they are likely to affect normal pulmonary vascular tone. PMID- 10749760 TI - Neonatal dexamethasone treatment increases the risk for pulmonary hypertension in adult rats. AB - Dexamethasone (Dex) treatment during a critical period of lung development causes lung hypoplasia in infant rats. However, the effects of Dex on the pulmonary circulation are unknown. To determine whether Dex increases the risk for development of pulmonary hypertension, we treated newborn Sprague-Dawley rats with Dex (0.25 microg/day, days 3-13). Litters were divided equally between Dex treated and vehicle control (ethanol) rats. Rats were raised in either room air until 10 wk of age (normoxic groups) or room air until 7 wk of age and then in a hypoxia chamber (inspired O(2) fraction = 0.10; hypoxic groups) for 3 wk to induce pulmonary hypertension. Compared with vehicle control rats, Dex treatment of neonatal rats reduced alveolarization (by 42%; P < 0.05) and barium-filled pulmonary artery counts (by 37%; P < 0.05) in 10-wk-old adults. Pulmonary arterial pressure and the ratio of right ventricle to left ventricle plus septum weights (RV/LV+S) were higher in 10-wk-old Dex-treated normoxic rats compared with those in normoxic control rats (by 16 and 16% respectively; P < 0.05). Small pulmonary arteries of adult normoxic Dex-treated rats showed increased vessel wall thickness compared with that in control rats (by 15%; P < 0.05). After 3 wk of hypoxia, RV/LV+S values were 36% higher in rats treated with Dex in the neonatal period compared with those in hypoxic control rats (P < 0.05). RV/LV+S was 42% higher in hypoxic control rats compared with those in normoxic control rats (P < 0.05). We conclude that Dex treatment of neonatal rats caused sustained lung hypoplasia and increased pulmonary arterial pressures and augmented the severity of hypoxia-induced pulmonary hypertension in adult rats. PMID- 10749761 TI - Binding and uptake of surfactant protein D by freshly isolated rat alveolar type II cells. AB - Alveolar type II cells secrete, internalize, and recycle pulmonary surfactant, a lipid and protein complex that increases alveolar compliance and participates in pulmonary host defense. Surfactant protein (SP) D, a collagenous C-type lectin, has recently been described as a modulator of surfactant homeostasis. Mice lacking SP-D accumulate surfactant in their alveoli and type II cell lamellar bodies, organelles adapted for recycling and secretion of surfactant. The goal of current study was to characterize the interaction of SP-D with rat type II cells. Type II cells bound SP-D in a concentration-, time-, temperature-, and calcium dependent manner. However, SP-D binding did not alter type II cell surfactant lipid uptake. Type II cells internalized SP-D into lamellar bodies and degraded a fraction of the SP-D pool. Our results also indicated that SP-D binding sites on type II cells may differ from those on alveolar macrophages. We conclude that, in vitro, type II cells bind and recycle SP-D to lamellar bodies, but SP-D may not directly modulate surfactant uptake by type II cells. PMID- 10749762 TI - Surfactant-associated protein A inhibits LPS-induced cytokine and nitric oxide production in vivo. AB - The role of surfactant-associated protein (SP) A in the mediation of pulmonary responses to bacterial lipopolysaccharide (LPS) was assessed in vivo with SP-A gene-targeted [SP-deficient; SP-A(-/-)] and wild-type [SP-A(+/+)] mice. Concentrations of tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein-2, and nitric oxide were determined in recovered bronchoalveolar lavage fluid after intratracheal administration of LPS. SP-A(-/-) mice produced significantly more TNF-alpha and nitric oxide than SP-A(+/+) mice after LPS treatment. Intratracheal administration of human SP-A (1 mg/kg) to SP-A(-/-) mice restored regulation of TNF-alpha, macrophage inflammatory protein-2, and nitric oxide production to that of SP-A(+/+) mice. Other markers of lung injury including bronchoalveolar fluid protein, phospholipid content, and neutrophil numbers were not influenced by SP-A. Data from experiments designed to test possible mechanisms of SP-A-mediated suppression suggest that neither binding of LPS by SP-A nor enhanced LPS clearance are the primary means of inhibition. Our data and others suggest that SP-A acts directly on immune cells to suppress LPS induced inflammation. These results demonstrate that endogenous or exogenous SP-A inhibits pulmonary LPS-induced cytokine and nitric oxide production in vivo. PMID- 10749763 TI - Biophysical properties and molecular characterization of amiloride-sensitive sodium channels in A549 cells. AB - Amiloride-sensitive Na(+) channels, present in fetal and adult alveolar epithelial type II (ATII) cells, play a critical role in the reabsorption of fetal fluid shortly after birth and in limiting the extent of alveolar edema across the adult lung. Because of the difficulty in isolating and culturing ATII cells, there is considerable interest in characterizing the properties of ion channels and their response to injury of ATII cell-like cell lines such as A549 that derive from a human alveolar cell carcinoma. A549 cells were shown to contain alpha-, beta-, and gamma-epithelial Na(+) channel mRNAs. In the whole cell mode of the patch-clamp technique (bath, 145 mM Na(+); pipette, 145 mM K(+)), A549 cells exhibited inward Na(+) currents reversibly inhibited by amiloride, with an inhibition constant of 0.83 microM. Ion substitution studies showed that these channels were moderately selective for Na(+) (Na(+)-to-K(+) permeability ratio = 6:1). Inward Na(+) currents were activated by forskolin (10 microM) and inhibited by nitric oxide (300 nM) and cGMP. Recordings in cell attached mode revealed the presence of an amiloride-sensitive Na(+) channel with a unitary conductance of 8.6 +/- 0.04 (SE) pS. Channel activity was increased by forskolin and decreased by nitric oxide and the cGMP analog 8-bromo-cGMP. These data demonstrate that A549 cells contain amiloride-sensitive Na(+) channels with biophysical properties similar to those of ATII cells. PMID- 10749764 TI - A method for measuring the oxygen consumption of intact cell monolayers. AB - This report describes an open-air method for measuring the O(2) consumption (QO(2)) of intact monolayers of cultured cells. This method is based on Fick's second law of diffusion. It requires only a micromanipulator and a miniature O(2) electrode to measure the PO(2) gradient in the culture medium in the well. It was compared with the conventional oxygraph chamber method. Both methods gave the same value for QO(2) in freshly isolated rat type II cells: 166 +/- 15.3 nmol. h( 1). 10(6) cells(-1) for the open-air method and 151 +/- 11.6 nmol. h(-1). 10(6) cells(-1) for the oxygraph chamber method (n = 11 experiments). But the open-air method gave significantly larger values for QO(2) in cells cultured for 2 days (236 +/- 8.8 nmol. h(-1). 10(6) cells(-1)) than the oxygraph method (71 +/- 15.2 nmol. h(-1). 10(6) cells(-1); P < 0.001; n = 12 experiments). This suggests that the way cells are detached from their substratum to be placed in the oxygraph chamber affects their QO(2). The open-air method may be useful for studies on the metabolic properties of monolayers because the cells do not risk being damaged. PMID- 10749765 TI - Macronutrient diet selection in thirteen mouse strains. AB - The strain distribution for macronutrient diet selection was described in 13 mouse strains (AKR/J, NZB/B1NJ, C57BL/6J, C57BL/6ByJ, DBA/2J, SPRET/Ei, CD-1, SJL/J, SWR/J, 129/J, BALB/cByJ, CAST/Ei, and A/J) with the use of a self selection protocol in which separate carbohydrate, fat, and protein diets were simultaneously available for 26-30 days. Relative to carbohydrate, nine strains consumed significantly more calories from the fat diet; two strains consumed more calories from carbohydrate than from fat (BALB/cByJ, CAST/Ei). Diet selection by SWR/J mice was variable over time, resulting in a lack of preference. One strain (A/J) failed to adapt to the diet paradigm due to inadequate protein intake. Comparisons of proportional fat intake across strains revealed that fat selection/consumption ranged from 26 to 83% of total energy. AKR/J, NZB/B1NJ, and C67BL/6J mice self-selected the highest proportion of dietary fat, whereas the CAST/Ei and BALB/cByJ strains chose the lowest. Finally, epididymal fat depot weight was correlated with fat consumption. There were significant positive correlations in AKR/J and C57BL/6J mice, which are highly sensitive to dietary obesity. However, absolute fat intake was inversely correlated with epididymal fat in two of the lean strains: SWR/J and CAST/Ei. We hypothesize that the SWR/J and CAST/Ei strains are highly sensitive to a negative feedback signal generated by increasing body fat, but the AKR/J and C67BL/6J mice are not. The variation in dietary fat selection across inbred strains provides a tool for dissecting the complex genetics of this trait. PMID- 10749766 TI - Gender differentiation of the chemoreflex during growth at high altitude: functional and neurochemical studies. AB - The effect of chronic hypoxia on gender differences in physiology and neurochemistry of chemosensory pathways was studied in prepubertal and adult rats living at sea level (SL; Lyon, France) or at high altitude (HA; La Paz, Bolivia, 3,600 m). HA adult rats had higher hematocrit (Ht%), Hb concentration, resting ventilatory rate (Ve(100)), and higher tyrosine hydroxylase (TH) activity in carotid bodies (CB) than SL animals. At HA and SL, adult females had lower Ht% (46.0 +/- 0.8 vs. 50.4 +/- 0.6% at HA, P < 0.05 and 43.8 +/- 0.9 vs. 47.1 +/- 0.8% at SL, P < 0.05) and Hb (16.1 +/- 0.3 vs. 17.7 +/- 0.2 g/dl at HA, P < 0.05 and 14.5 +/- 0.3 vs. 15.6 +/- 0.1 g/dl at SL, P < 0.05) than males. Females had higher Ve(100) [170 +/- 19 vs. 109 +/- 7 ml. min(-1). 100 g(-1) at HA, P < 0.05 and 50 +/- 3 vs. 40 +/- 2 ml. min(-1). 100 g(-1) at SL, not significant (NS)] and lower CB-TH activity (1.40 +/- 0.2 vs. 3.87 +/- 0.6 pmol/20 min at HA, P < 0.05 and 0.52 +/- 0.1 vs. 0.68 +/- 0.1 pmol/20 min at SL; NS) than males at HA only. The onset of hypoxic ventilatory response during development was delayed at HA. Prepubertal HA females had higher Ve(100) than males (2 wk old, +47%) and higher CB-TH activity (3 wk old, +51%). Medullary noradrenergic groups were sex dimorphic during development at SL. Rats raised at HA had a drop of TH activity between the second and the third postnatal week in all medullary groups. In conclusion, our data support the hypothesis that the CB is the major site for sexual differentiation of the ventilatory control. Ventilatory differences appeared before puberty, and the animals bred at HA had profound alterations in the developmental process of the chemoreflex and its neural pathways. Some of these alterations are under dependence of the sex of the animal, and there is an important interaction between gender and the hypoxic environmental condition during the developmental period. PMID- 10749767 TI - Disorder of salivary secretion in inbred polydipsic mouse. AB - To find mechanisms of an extreme polydipsia in an inbred strain of mice, STR/N, this study was undertaken using Institute of Cancer Research (ICR) mice as a control. During food deprivation, daily water intake of both strains decreased. The decrement in the STR/N mice was larger than that in the ICR mice. During dehydration, daily food intake of the STR/N mice was smaller than that of the ICR mice. These data indicate that prandial drinking was more severely affected for the STR/N mice. Under anesthesia, the stimulated salivary secretion by pilocarpine of the STR/N mice was significantly smaller than that of the ICR mice. The submandibular gland of the STR/N mice was lighter and harder than that of the ICR mice. After desalivation from the major three salivary glands, the ICR mice drank as much as the STR/N mice. Young STR/N mice with undeveloped polydipsia did not show different salivary secretion stimulated by pilocarpine from the young ICR mice. These findings indicate a dysfunction with age in the salivary glands of the STR/N mice, and they suggest that the decreased saliva induces thirst and triggers extraordinary drinking in the polydipsic mice. PMID- 10749768 TI - Modulation of the acute phase response by altered expression of the IL-1 type 1 receptor or IL-1ra. AB - A complete understanding of the role for endogenously produced interleukin-1 (IL 1), tumor necrosis factor-alpha (TNF-alpha), and IL-1 receptor antagonist (IL 1ra) in the acute phase response to inflammation remains unknown. In the present studies, knockout mice lacking either a functional IL-1 type I receptor (IL-1RI( /-)), a TNF type I receptor (TNFR-I(-/-)), or both IL-1 type I and TNF type I receptors (IL-1RI(-/-)/TNFR-I(-/-)) received a turpentine abscess. Additional mice deficient in IL-1ra protein (IL-1ra(-/-)) or overexpressing IL-1ra protein (IL-1ra(tg)) were similarly treated. After a turpentine abscess, IL-1 receptor knockout mice exhibited an attenuated inflammatory response compared with wild type or animals lacking a functional TNFR-I. Mice overexpressing IL-1ra also had an attenuated hepatic acute phase protein response, whereas IL-1ra knockout mice had a significantly greater hepatic acute phase response. We conclude that the inflammatory response to a turpentine abscess is the result of a balance between IL-1ra expression and IL-1 binding to its type I receptor. Endogenously produced IL-1ra plays a central role in mitigating the magnitude of the IL-1-mediated inflammatory response and, ultimately, the outcome to a turpentine abscess. PMID- 10749769 TI - Thalamic lesions dissociate breathing inhibition by hypoxia and adenosine in fetal sheep. AB - The effects of diencephalic lesions on respiratory responses to intra-arterially infused adenosine (ADO) were determined in chronically catheterized fetal sheep (>0.8 term). These studies were designed to test the hypothesis that the inhibitory effects of ADO on fetal breathing, like those of hypoxia, are mediated by the parafascicular nuclear complex (Pf) of the posteromedial thalamus. ADO inhibited breathing [control (C): 26 +/- 2.6, ADO: 4 +/- 1 min/h] in normal fetuses and in a fetus with a lesion that virtually destroyed the thalamus but left intact most of Pf. Neuronal lesions in the diencephalon, produced by injecting ibotenic acid, abolished the inhibitory effects of ADO on breathing (C: 31 +/- 5.1, ADO: 30 +/- 4.5 min/h) when the lesions encompassed Pf or the sector immediately rostral to Pf that retained the capacity to regulate hypoxic inhibition. Smaller lesions created by the insertion of needles also eliminated the depressant effects of ADO when disruptions were within Pf or a rostral component of the thalamic cortical activating system. It is concluded that 1) a medial thalamic sector is critically involved in ADO-induced apnea and 2) ADO dependent and ADO-independent mechanisms mediate hypoxic inhibition. PMID- 10749770 TI - Exogenous growth hormone induces somatotrophic gene expression in neonatal liver and skeletal muscle. AB - The extent to which the local somatotrophic axis is functional in extrahepatic tissues in the neonate is unclear. We therefore determined the expression of growth hormone (GH) receptor (GHR), and insulin-like growth factors I and II (IGF I and IGF-II) mRNA in liver and skeletal muscle (longissimus) of neonatal pigs given daily intramuscular injections of either recombinant porcine GH (1 mg/kg body wt; n = 6) or saline (n = 5) for 7 days. Exogenous GH increased plasma concentrations of GH 30-fold and IGF-I threefold. Abundances of specific mRNA in liver and muscle were measured by RNase protection assays (values are arbitrary density units). In liver, GH treatment increased GHR (6.0 vs. 9.7; P < 0.01) and IGF-I (5.2 vs. 49.0; P < 0.001) but not IGF-II (19.5 vs. 17.2) mRNA. In muscle, GH treatment increased IGF-I mRNA (13.3 vs. 22.8; P < 0.05) but not GHR (8.3 vs. 9.5) or IGF-II (16.1 vs. 16.9). These results demonstrate that exogenous GH can induce local somatotrophic function predominantly in liver but also in muscle of newborn pigs. Our novel finding on the selective increase in muscle IGF-I but not GHR gene expression suggests differences in posttranscriptional regulation and/or intracellular signaling mechanisms. PMID- 10749771 TI - Regulation of myofibrillar protein turnover during maturation in normal and undernourished rat pups. AB - The study tested the hypothesis that a higher rate of myofibrillar than sarcoplasmic protein synthesis is responsible for the rapid postdifferentiation accumulation of myofibrils and that an inadequate nutrient intake will compromise primarily myofibrillar protein synthesis. Myofibrillar (total and individual) and sarcoplasmic protein synthesis, accretion, and degradation rates were measured in vivo in well-nourished (C) rat pups at 6, 15, and 28 days of age and compared at 6 and 15 days of age with pups undernourished (UN) from birth. In 6-day-old C pups, a higher myofibrillar than sarcoplasmic protein synthesis rate accounted for the greater deposition of myofibrillar than sarcoplasmic proteins. The fractional synthesis rates of both protein compartments decreased with age, but to a greater degree for myofibrillar proteins (-54 vs. -42%). These decreases in synthesis rates were partially offset by reductions in degradation rates, and from 15 days, myofibrillar and sarcoplasmic proteins were deposited in constant proportion to one another. Undernutrition reduced both myofibrillar and sarcoplasmic protein synthesis rates, and the effect was greater at 6 (-25%) than 15 days (-15%). Decreases in their respective degradation rates minimized the effect of undernutrition on sarcoplasmic protein accretion from 4 to 8 days and on myofibrillar proteins from 13 to 17 days. Although these adaptations in protein turnover reduced overall growth of muscle mass, they mitigated the effects of undernutrition on the normal maturational changes in myofibrillar protein concentration. PMID- 10749772 TI - Glucocorticoid sensitivity of interleukin-1 agonist and antagonist secretion: the effects of age and gender. AB - Interleukin-1 (IL-1) is a primary mediator of inflammation that is regulated, in part, by the hypothalamic-pituitary-adrenal axis. The purpose of this study was to determine if gender- or age-related differences exist in the sensitivity of IL 1-producing cells to hydrocortisone. Peripheral blood mononuclear cells (PBMC) isolated from men and women (21-77 yr old) were incubated with hydrocortisone (0, 50, 100, 500, or 1,000 ng/ml) with or without lipopolysaccharide (LPS). Secretion of IL-1beta and IL-1 receptor antagonist was inhibited in a dose-dependent manner (P = 0.001) without age- or gender-related differences. Hydrocortisone decreased soluble IL-1 receptor type II (sIL-1RII) secretion by unstimulated cells (P = 0. 0001), but it increased secretion by LPS-stimulated cells (P = 0. 0001) in all groups. Unstimulated cell supernatants from men contained greater concentrations of sIL-1RII than the supernatants from women (P = 0.011). Compared with men, PBMCs from women were less responsive to hydrocortisone inhibition of sIL-1RII secretion, regardless of age (P = 0.001), and compared with the follicular phase, sIL-1RII secretion was lower in the luteal phase of the menstrual cycle (P < 0.05). These data indicate that basal secretion and glucocorticoid modulation of sIL-1RII secretion by cultured PBMCs are gender dependent. Moreover, glucocorticoid influences on sIL-1RII secretion depend on the presence or absence of gram-negative bacterial toxins. PMID- 10749773 TI - Elevation of tail skin temperature in ovariectomized rats in relation to menopausal hot flushes. AB - Menopausal hot flushes (HFs), which manifest as an increase in skin temperature, most frequently occur after menopause and cease with the passage of time. We designed this study to elucidate the characteristics of the elevation of tail skin temperature (TST) in ovariectomized (OVX) rats, which is relevant to human symptoms of HFs. First, we measured TST and rectal temperature (RT) and investigated the time course of their changes up to 20 wk after ovariectomy. The TST in OVX rats (28.4 +/- 0.3 degrees C) was significantly (P = 0.0035) elevated from 2 to 7 wk after the ovariectomy compared with that in sham-operated (Sham) rats (27.0 +/- 0.2 degrees C), whereas the RT in OVX rats was elevated from 8 to 20 wk. We next examined the therapeutic effects of estradiol (E(2)) on the elevation of the TST by continuous subcutaneous infusion. E(2) treatment (1.0 microg/day) completely (P = 0.0232) inhibited the elevation of the TST (28.4 +/- 0.3 degrees C for Sham rats, 29.3 +/- 0.3 degrees C for OVX rats, 28.2 +/- 0.4 degrees C for OVX + E(2) 1.0 microg/day rats). These results demonstrated that the elevation of TST in OVX rats was exhibited soon after the estrogen removal and diminished with time and that it was normalized with continuous E(2) replacement. These characteristics are similar to the symptoms of menopausal HFs in women. PMID- 10749774 TI - Involvement of H(+)-ATPase and carbonic anhydrase in inorganic carbon uptake for endosymbiont photosynthesis. AB - Symbiotic cnidarians absorb inorganic carbon from seawater to supply intracellular dinoflagellates with CO(2) for their photosynthesis. To determine the mechanism of inorganic carbon transport by animal cells, we used plasma membrane vesicles prepared from ectodermal cells isolated from tentacles of the sea anemone, Anemonia viridis. H(14)CO(-)(3) uptake in the presence of an outward NaCl gradient or inward H(+) gradient, showed no evidence for a Cl(-)- or H(+)- driven HCO(-)(3) transport. H(14)CO(-)(3) and (36)Cl(-) uptakes were stimulated by a positive inside-membrane diffusion potential, suggesting the presence of HCO(-)(3) and Cl(-) conductances. A carbonic anhydrase (CA) activity was measured on plasma membrane (4%) and in the cytoplasm of the ectodermal cells (96%) and was sensitive to acetazolamide (IC(50) = 20 nM) and ethoxyzolamide (IC(50) = 2.5 nM). A strong DIDS-sensitive H(+)-ATPase activity was observed (IC(50) = 14 microM). This activity was also highly sensitive to vanadate and allyl isothiocyanate, two inhibitors of P-type H(+)-ATPases. Present data suggest that HCO(-)(3) absorption by ectodermal cells is carried out by H(+) secretion by H(+) ATPase, resulting in the formation of carbonic acid in the surrounding seawater, which is quickly dehydrated into CO(2) by a membrane-bound CA. CO(2) then diffuses passively into the cell where it is hydrated in HCO(-)(3) by a cytosolic CA. PMID- 10749775 TI - Cholecystokinin and leptin act synergistically to reduce body weight. AB - Leptin, the product of the obese gene, reduces food intake and body weight in rats and mice, whereas administration of the gut-peptide CCK reduces meal size but not body weight. In the current experiments, we report that repeated daily combination of intracerebroventricular leptin and intraperitoneal CCK results in significantly greater loss of body weight than does leptin alone. However, leptin plus CCK treatment does not synergistically reduce the size of individual 30-min sucrose meals during this period, and the effect of leptin-CCK combination on daily chow intake, while significant, is small compared with the robust effects on body weight loss. This synergistic effect on body weight loss depends on a peripheral action of CCK and a central action of leptin. These data suggest a previously unsuspected role for CCK in body weight regulation that may not depend entirely on reduction of feeding behavior and suggest a strategy for enhancing the effects of leptin in leptin-resistant obese individuals. PMID- 10749776 TI - Endotoxin administration alters the force vs. pCa relationship of skeletal muscle fibers. AB - Recent work indicates that endotoxemia elicits severe reductions in skeletal muscle force-generating capacity. The subcellular alterations responsible for these decrements have not, however, been fully characterized. One possibility is that the contractile proteins per se are altered in endotoxemia and another is that the mechanism by which these proteins are activated is affected. The purpose of the present study was to assess the effects of endotoxin administration on the contractile proteins by examining the maximum calcium-activated force (F(max)) and calcium sensitivity of single Triton-skinned fibers of diaphragm, soleus, and extensor digitorum longus (EDL) muscles taken from control and endotoxin-treated (8 mg/kg) rats. Fibers were mounted on a force transducer and sequentially activated by serial immersion in solutions of increasing Ca(2+) concentration (i.e., pCa 6.0 to pCa 5.0); force vs. pCa data were fit to the Hill equation. All fibers were typed at the conclusion of studies using gel electrophoresis. F(max), the calcium concentration required for half-maximal activation (Ca(50)), and the Hill coefficient were compared as a function of muscle and fiber type for the control and endotoxin-treated animals. Control group F(max) was similar for diaphragm, soleus, and EDL fibers, i.e., 112.34 +/- 2.64, 111.55 +/- 3.66, and 104.05 +/- 4.33 kPa, respectively. Endotoxin administration reduced the average F(max) for fibers from all three muscles to 80.25 +/- 2.30, 72.47 +/- 2.97, and 78.32 +/- 2.43 kPa, respectively (P < 0.001 for comparison of each to control). All fiber types in diaphragm, soleus, and EDL muscles manifested similar endotoxin-related reductions in F(max). The Ca(50) and the Hill coefficient for all fiber types and all muscles were unaffected by endotoxin administration. We speculate that these alterations in the intrinsic properties of the contractile proteins represent a major mechanism by which endotoxemia reduces muscle force generating capacity. PMID- 10749777 TI - Kinins in humans. AB - The kinin peptide system in humans is complex. Whereas plasma kallikrein generates bradykinin peptides, glandular kallikrein generates kallidin peptides. Moreover, a proportion of kinin peptides is hydroxylated on proline(3) of the bradykinin sequence. We established HPLC-based radioimmunoassays for nonhydroxylated and hydroxylated bradykinin and kallidin peptides and their metabolites in blood and urine. Both nonhydroxylated and hydroxylated bradykinin and kallidin peptides were identified in human blood and urine, although the levels in blood were often below the assay detection limit. Whereas kallidin peptides were more abundant than bradykinin peptides in urine, bradykinin peptides were more abundant in blood. Bradykinin and kallidin peptide levels were higher in venous than arterial blood. Angiotensin-converting enzyme inhibition increased blood levels of bradykinin, but not kallidin, peptides. Reactive hyperemia had no effect on antecubital venous levels of bradykinin or kallidin peptide levels. These studies demonstrate differential regulation of the bradykinin and kallidin peptide systems, and indicate that blood levels of bradykinin peptides are more responsive to angiotensin-converting enzyme inhibition than blood levels of kallidin peptides. PMID- 10749778 TI - Systemic bacterial invasion induced by sleep deprivation. AB - Profound sleep disruption in humans is generally believed to cause health impairments. Through comparative research, specific physical effects and underlying mechanisms altered by sleep deprivation are being elucidated. Studies of sleep-deprived animals previously have shown a progressive, chronic negative energy balance and gradual deterioration of health, which culminate in fatal bloodstream infection without an infectious focus. The present study investigated the conditions antecedent to advanced morbidity in sleep-deprived rats by determining the time course and distribution of live microorganisms in body tissues that are normally sterile. The tissues cultured for microbial growth included the blood, four major organs, six regional lymph nodes, the intestine, and the skin. The principal finding was early infection of the mesenteric lymph nodes by bacteria presumably translocated from the intestine and bacterial migration to and transient infection of extraintestinal sites. Presence of pathogenic microorganisms and their toxins in tissues constitutes a septic burden and chronic antigenic challenge for the host. Bacterial translocation and pathogenic sequelae provide mechanisms by which sleep deprivation appears to adversely affect health. PMID- 10749779 TI - Effect of hyperosmotic solutions on salt excretion and thirst in rats. AB - We investigated urinary changes and thirst induced by infusion of hyperosmotic solutions in freely moving rats. Intracarotid infusions of 0.3 M NaCl (4 ml/20 min, split between both internal carotid arteries) caused a larger increase in excretion of Na(+) and K(+) than intravenous infusions, indicating that cephalic sensors were involved in the response to intracarotid infusions. Intravenous and intracarotid infusions of hyperosmotic glycerol or urea (300 mM in 150 mM NaCl) had little or no effect, suggesting the sensors were outside the blood-brain barrier (BBB). Intracarotid infusion of hypertonic mannitol (300 mM in 150 mM NaCl) was more effective than intravenous infusion, suggesting that cell volume rather than Na(+) concentration of the blood was critical. Similarly, intracarotid infusion (2 ml/20 min, split between both sides), but not intravenous infusion of hypertonic NaCl or mannitol caused thirst. Hyperosmotic glycerol, infused intravenously or into the carotid arteries, did not cause thirst. We conclude that both thirst and electrolyte excretion depend on a cell volume sensor that is located in the head, but outside the BBB. PMID- 10749780 TI - Relationship of atypical melatonin rhythm with two circadian clock outputs in B6D2F(1) mice. AB - Circadian rhythms in body temperature, locomotor activity, and the circadian changes of plasma and pineal melatonin content were investigated in B6D2F(1) mice synchronized by 12 h of light and 12 h of darkness. During 8 wk continuous recording, activity and temperature displayed a marked stable and reproducible circadian rhythm, with both peaks occurring near the middle of darkness. Both 24- and 12-h rhythmic components were also significantly detected. Mean plasma melatonin concentration rose steadily during the light span and reached a maximum (30.6 +/- 10.0 pg/ml) at 11 h after light onset (HALO), then gradually decreased after the onset of darkness to a nadir (4.7 +/- 0.4 pg/ml) at 20 HALO. Mean pineal content followed a pattern parallel to that of plasma concentration (peak at 11 HALO: 17.7 +/- 1.0 pg/gland; trough at 17 HALO: 4.7 +/- 1.0 pg/gland). In addition, a second sharp peak was observed at 21 HALO (20.2 +/- 3.5 pg/gland). Plasma and pineal contents displayed large and statistically significant circadian changes, with a composite rhythm of period (24 + 12 h). This mouse model has predominant production and secretion of melatonin during the day. This possibly contributes to a similar coupling between chronopharmacology mechanisms and the rest-activity cycle in these mice and in human subjects. PMID- 10749781 TI - Gbeta regulation of Na/H exchanger-3 activity in rat renal proximal tubules during development. AB - The decreased natriuretic action of dopamine in the young has been attributed to decreased generation of cAMP by the activated renal D(1)-like receptor. However, sodium/hydrogen exchanger (NHE) 3 activity in renal brush-border membrane vesicles (BBMV) can be modulated independent of cytoplasmic second messengers. We therefore studied D(1)-like receptor regulation of NHE activity in BBMVs in 2-, 4 , and 12-wk-old (adult) rats. Basal NHE activity was least in 2-wk-old compared with 4- and 12-wk-old rats. D(1)-like agonist (SKF-81297) inhibition of NHE activity was also least in 2-wk-old (-1 +/- 9%, n = 3) compared with 4 (-15 +/- 5%, n = 6)- and 12 (-65 +/- 4%, n = 6)-wk-old rats. The decreased response to the D(1)-like agonist in BBMV was not caused by decreased D(1) receptors or NHE3 expression in the young. G(s)alpha, which inhibits NHE3 activity by itself, coimmunoprecipitated with NHE3 to the same extent in 2-wk-old and adult rats. G(s)alpha function was also not impaired in the young because guanosine 5'-O-(3 thiotriphosphate) decreased NHE activity to a similar extent in 4-wk-old and adult rats. Galpha(i-3) protein expression in BBMV also did not change with age. In contrast, Gbeta expression and the amount of Gbeta that coimmunoprecipitated with NHE3 in BBMV was greatest in 2-wk-old rats and decreased with age. Gbeta common antibodies did not affect D(1)-like agonist inhibition of NHE activity in adult rats (8%) but markedly increased it (48%)in 4-wk-old rats. We conclude that the decreased inhibitory effect of D(1)-like receptors on NHE activity in BBMV in young rats is caused, in part, by the increased expression and activity of the G protein subunit Gbeta/gamma. The direct regulation of NHE activity by G protein subunits may be an important step in the maturation of renal tubular ion transport. PMID- 10749782 TI - Activation of renal mechanosensitive neurons involves bradykinin, protein kinase C, PGE(2), and substance P. AB - Increased renal pelvic pressure or bradykinin increases afferent renal nerve activity (ARNA) via PGE(2)-induced release of substance P. Protein kinase C (PKC) activation increases ARNA, and PKC inhibition blocks the ARNA response to bradykinin. We now examined whether bradykinin mediates the ARNA response to increased renal pelvic pressure by activating PKC. In anesthetized rats, the ARNA responses to increased renal pelvic pressure were blocked by renal pelvic perfusion with the bradykinin B(2)-receptor antagonist HOE 140 and the PKC inhibitor calphostin C by 76 +/- 8% (P < 0.02) and 81 +/- 5% (P < 0.01), respectively. Renal pelvic perfusion with 4beta-phorbol 12,13-dibutyrate (PDBu) to activate PKC increased ARNA 27 +/- 4% and renal pelvic release of PGE(2) from 500 +/- 59 to 1, 113 +/- 183 pg/min and substance P from 10 +/- 2 to 30 +/- 2 pg/min (all P < 0.01). Indomethacin abolished the increases in substance P release and ARNA. The PDBu-mediated increase in ARNA was also abolished by the substance P-receptor antagonist RP 67580. We conclude that bradykinin contributes to the activation of renal pelvic mechanosensitive neurons by activating PKC. PKC increases ARNA via a PGE(2)-induced release of substance P. PMID- 10749783 TI - Dose-dependent effects of endotoxin on human sleep. AB - The role of the central nervous system in the host response to infection and inflammation and modulation of these responses by the hypothalamic-pituitary adrenal system are well established. In animals, activation of host defense mechanisms increases non-rapid eye movement (NREM) sleep amount and intensity, which, in turn, are thought to support host defense, or the body's ability to defend itself against challenges to its immune system. In humans, the evidence is conflicting. Therefore, we investigated the effects of three placebo-controlled doses of endotoxin on host response, including nocturnal sleep in healthy volunteers. Administered before nocturnal sleep onset, endotoxin dose dependently increased rectal temperature, heart rate, and the plasma levels of tumor necrosis factor (TNF)-alpha, soluble TNF receptors, interleukin (IL)-1 receptor antagonist, IL-6, and cortisol. The lowest dose reliably increased circulating levels of cytokines and soluble cytokine receptors, but it did not affect rectal temperature, heart rate, or cortisol. This subtle host defense activation increased deep NREM sleep amount, often referred to as slow-wave sleep (stages 3 and 4), and intensity (delta power). Conversely, the highest dose of endotoxin disrupted sleep. Whereas it is well established that the endocrine and thermoregulatory systems are very sensitive to endotoxin, this study shows that human sleep-wake behavior is even more sensitive to activation of host defense mechanisms. PMID- 10749784 TI - Regulation of glucose production in rainbow trout: role of epinephrine in vivo and in isolated hepatocytes. AB - The rate of hepatic glucose production (R(a) glucose) of rainbow trout (Oncorhynchus mykiss) was measured in vivo by continuous infusion of [6 (3)H]glucose and in vitro on isolated hepatocytes to examine the role of epinephrine (Epi) in its regulation. By elevating Epi concentration and/or blocking beta-adrenoreceptors with propranolol (Prop), our goals were to investigate the mechanism for Epi-induced hyperglycemia to determine the possible role played by basal Epi concentration in maintaining resting R(a) glucose and to assess indirect effects of Epi in the intact animal. In vivo infusion of Epi caused hyperglycemia (3.75 +/- 0.16 to 8.75 +/- 0.54 mM) and a twofold increase in R(a) glucose (6.57 +/- 0.79 to 13.30 +/- 1.78 micromol. kg(-1). min(-1), n = 7), whereas Prop infusion decreased R(a) from 7.65 +/- 0.92 to 4.10 +/- 0.56 micromol. kg(-1). min(-1) (n = 10). Isolated hepatocytes increased glucose production when treated with Epi, and this response was abolished in the presence of Prop. We conclude that Epi-induced trout hyperglycemia is entirely caused by an increase in R(a) glucose, because the decrease in the rate of glucose disappearance normally seen in mammals does not occur in trout. Basal circulating levels of Epi are involved in maintaining resting R(a) glucose. Epi stimulates in vitro glucose production in a dose-dependent manner, and its effects are mainly mediated by beta-adrenoreceptors. Isolated trout hepatocytes produce glucose at one-half the basal rate measured in vivo, even when diet, temperature, and body size are standardized, and basal circulating Epi is responsible for part of this discrepancy. The relative increase in R(a) glucose after Epi stimulation is similar in vivo and in vitro, suggesting that indirect in vivo effects of Epi, such as changes in hepatic blood flow or in other circulating hormones, do not play an important role in the regulation of glucose production in trout. PMID- 10749785 TI - Functional and morphological modifications of the urinary bladder in aging female rats. AB - In female Wistar/Rij rats, 10 and 30 mo old, the micturition profiles in conscious animals, the contractile responses of the isolated urinary bladder, and the histology of the vesical tissue have been investigated. During cystomanometry, 60% of conscious senescent rats, but only 25% of young adult rats, showed spontaneous contractions during the bladder-filling phase. In aging rats, micturition pressure and duration of micturition were significantly higher by approximately 40-50%. In contrast, bladder capacity, bladder compliance, micturition volume, and residual volume were not modified with age. In vitro, the contractile responses of the bladder body to KCl, carbachol, arecoline, and alpha,beta-MeATP were similar in tissues from young adult and senescent rats. In contrast, maximum responses to noradrenaline, but not phenylephrine, were two times greater in the older rats. Isoprenaline exhibited the same potency in relaxing KCl-precontracted bladder body of 10- and 30-mo-old animals. Morphometric analysis showed a significant increase in the mean thickness of the muscularis layer with age, whereas the collagen density significantly decreased in the muscularis and in the lamina propria layers. The fact that the majority of senescent rats displayed bladder instability and increased response to alpha adrenergic agonists suggests that this strain of rat seems a good model for the aged human. However, other characteristics of the aging human urinary tract (urinary frequency, decreased cystometric capacity, and decreased detrusor contractility associated with fibrosis) are not present. PMID- 10749786 TI - Carotid and cardiopulmonary chemoreceptor activity increases hippocampal theta rhythm in conscious rabbits. AB - We have examined whether activation of carotid artery chemoreceptors causes alerting in conscious rabbits. Injection of phenylbiguanide (a 5 hydroxytryptamine(3)-receptor agonist) into the common carotid artery of conscious rabbits increased the proportion of theta rhythm in the hippocampal EEG, commencing in the first 5-s epoch after the injection. Intravenous injection of phenylbiguanide also increased the proportion of theta rhythm in the hippocampal electroencephalogram (EEG), but the onset of the change was not until the second 5-s epoch following injection. Injection of Ringer solution, either into the common carotid artery or into the marginal ear vein, did not affect the hippocampal EEG. Results suggest that phenylbiguanide-mediated activation of carotid and cardiopulmonary chemoreceptor afferents alerts the animal, as assessed by induction of theta rhythm in the hippocampal EEG. This alerting response presumably reflects the action of neural inputs that enter the brain with the carotid sinus nerve at the level of the medulla oblongata. PMID- 10749787 TI - Control of blood pressure mediated by baroreflex changes of heart rate in the chicken embryo (Gallus gallus). AB - Pharmacological manipulation of peripheral resistance via sodium nitroprusside and phenylephrine was used to study baroreflex function over the latter two thirds of incubation in embryonic chickens. From day 9 to day 19 of incubation, there is a positive linear relation between heart rate and blood pressure, indicating the feedforward action of arterial pressure on heart rate. A reciprocal relationship between blood pressure and heart rate became pronounced during the last 3 days of incubation. For the purpose of the study, gain of the baroreflex was calculated as maximal gain (only those embryos that demonstrated the response) or average gain (all embryos). Maximal gain increased progressively from 13 +/- 7 beats. min(-1). kPa(-1) at 18 days to 105 +/- 83 beats. min(-1). kPa(-1) in 2-day-old hatchlings. The percentage of embryos older than 18 days with baroreflex responses increased from 33% on day 19 to 56% on day 21, indicating that baroreflex regulation begins late in incubation ( approximately 90% incubation time), and the gain of this reflex exhibits a maturation over the final 3 days of incubation. PMID- 10749788 TI - Rhythmic multiunit neural activity in slices of hamster suprachiasmatic nucleus reflect prior photoperiod. AB - The suprachiasmatic nucleus (SCN) is an endogenous circadian pacemaker, and SCN neurons exhibit circadian rhythms of electrophysiological activity in vitro. In vivo, the functional state of the pacemaker depends on changes in day length (photoperiod), but it is not known if this property persists in SCN tissue isolated in vitro. To address this issue, we prepared brain slices from hamsters previously entrained to light-dark (LD) cycles of different photoperiods and analyzed rhythms of SCN multiunit neuronal activity using single electrodes. Rhythms in SCN slices from hamsters entrained to 8:16-, 12:12-, and 14:10-h LD cycles were characterized by peak discharge rates relatively higher during subjective day than subjective night. The mean duration of high neuronal activity was photoperiod dependent, compressed in slices from the short (8:16 and 12:12 LD) photoperiods, and decompressed (approximately doubled) in slices from the long (14:10 LD) photoperiod. In slices from all photoperiods, the mean phase of onset of high neuronal activity appeared to be anchored to subjective dawn. Our results show that the electrophysiological activity of the SCN pacemaker depends on day length, extending previous in vivo data, and demonstrate that this capacity is sustained in vitro. PMID- 10749789 TI - Medullary lateral tegmental field: an important source of basal sympathetic nerve discharge in the cat. AB - We used blockade of excitatory amino acid (EAA) neurotransmission in the medullary lateral tegmental field (LTF) and rostral ventrolateral medulla (RVLM) to assess the roles of these regions in the control of inferior cardiac sympathetic nerve discharge (SND) and mean arterial pressure (MAP) in urethan anesthetized, baroreceptor-denervated cats. Bilateral microinjection of a non-N methyl-D-aspartate (NMDA)-receptor antagonist [1,2,3, 4-tetrahydro-6-nitro-2,3 dioxobenzo-[f]quinoxaline-7-sulfonamide (NBQX)] into the LTF significantly decreased SND to 46 +/- 4% of control (as demonstrated with power-density spectral analysis) and MAP by 16 +/- 6 mmHg. In contrast, bilateral microinjection of an NMDA-receptor antagonist [D(-)-2-amino-5-phosphonopentanoic acid (D-AP5)] into the LTF did not decrease SND or MAP. These results demonstrate that the LTF is an important synaptic relay in the pathway responsible for basal SND in the cat. Bilateral microinjection of NBQX or D-AP5 into the RVLM significantly decreased power in SND to 48 +/- 5 or 61 +/- 5% of control, respectively, and reduced MAP by 15 +/- 2 or 8 +/- 4 mmHg, respectively. These data indicate that EAA-mediated synaptic drive to RVLM-spinal sympathoexcitatory neurons accounts for a significant component of their basal activity. PMID- 10749791 TI - Cardiac adrenomedullin gene expression and peptide accumulation after acute myocardial infarction in rats. AB - Plasma adrenomedullin (AM) has been shown to increase in the early phase of acute myocardial infarction (MI). However, little information is available regarding cardiac AM synthesis after MI. Accordingly, we examined the time course of ventricular AM production and potential stimulation of AM in the infarcted and noninfarcted regions in MI rats produced by coronary artery ligation. Compared with sham-operated rats, the ventricular AM peptide level 6 h after MI increased 1.5-fold in the infarcted region and 1.7-fold in the noninfarcted region in association with increased left ventricular end-diastolic pressure (EDP). Northern blot analysis also showed marked induction of AM gene expression in the infarcted region (11-fold) and the noninfarcted region (6-fold) 6 h after MI. The AM peptide level in the infarcted region reached its peak (2. 6-fold) 1 wk postinfarction and thereafter decreased to normal. In the noninfarcted region, however, the AM level remained elevated for at least 4 wk. Immunohistochemical studies demonstrated that intense immunostaining for AM was limited to myocytes in both the infarcted and noninfarcted regions. Interestingly, the AM level in the noninfarcted region correlated positively with infarct size (r = 0. 40, P < 0.01) and EDP (r = 0.52, P < 0.001). An oral angiotensin-converting enzyme inhibitor suppressed the overproduction of AM 1 wk postinfarction in association with decreases in EDP and mean arterial pressure. In summary, cardiac AM synthesis was rapidly induced in both the infarcted and noninfarcted regions after MI. The subsequent ventricular AM in the two regions demonstrated different time-concentration curves during 4 wk after MI. AM may be synthesized predominantly by cardiac myocytes, but not by fibroblasts, at least in part, in association with increased ventricular load after MI. PMID- 10749790 TI - Glucocorticoid-induced, caspase-dependent organ apoptosis early after burn injury. AB - Immune suppression and increased apoptotic loss of circulating lymphocytes have been reported after burn injury. However, little is known about the underlying mechanisms responsible for the increased apoptosis of lymphoid and parenchymal cells in solid organs and the role played by inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha) and Fas ligand (FasL), as well as by glucocorticoids. To evaluate the role of endogenously produced glucocorticoids and FasL, mice subjected to a 20% steam burn were pretreated with a glucocorticoid receptor antagonist (mifepristone) or a neutralizing murine Fas fusion protein. Three and twenty-four hours after burn injury, histological analysis, caspase-3 activity, and in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and phenotyping of lymphocyte populations for apoptosis were evaluated. Burn injury increased the number of apoptotic cells and caspase-3 activity in thymus and spleen, but not in other solid organs. Increased apoptosis was seen in several T and B cell populations from both thymus and spleen. Mifepristone pretreatment significantly reduced the apoptosis and caspase 3 activity after burn injury, whereas blocking FasL activity had only minimal effects. We conclude that corticosteroids, and not FasL, are primarily responsible for the increased caspase-3 activity and apoptosis in thymus and spleen cell populations early after burn injury. PMID- 10749793 TI - Effects of hetastarch and mannitol on prolonging survival in stable hypothermia in rats. AB - In rats, prolonged stable hypothermia ( approximately 24 h at body temperature of 19 degrees C) is characterized by a time-dependent increase in hematocrit, plasma osmolality, and red blood cell fragility and a decrease in plasma volume. These changes impede tissue microcirculation and could limit survival. As a countermeasure, we used plasma volume expanders of both long (hetastarch)- and short-lasting (mannitol) characteristics to improve microcirculation and hopefully hypothermia survival. Infusion of 6% hetastarch at hour 3 in hypothermia significantly (P < 0.05) enhanced survival over saline control (33.5 vs. 23.8 h); a significant delay in the increases of hematocrit and cell fragility was also observed compared with those in saline controls. Treating the animal with 6% hetastarch at hour 20 during hypothermia caused a similar but less effective improvement in survival. In contrast, treating the rats with 6% mannitol at hour 3 or 20 during hypothermia failed to enhance survival over saline control, although transient improvement in plasma volume was observed. Our results indicate that by using a long-lasting volume expander, which tends to better maintain plasma volume and rheological parameters governing microcirculation than does saline or a short-lasting volume expander, hypothermia survival can be significantly improved. PMID- 10749792 TI - Alterations in spinal cord Fos protein expression induced by bladder stimulation following cystitis. AB - These studies examined Fos protein expression in spinal cord neurons synaptically activated by stimulation of bladder afferent pathways after cyclophosphamide (CYP)-induced bladder inflammation. In urethan-anesthetized Wistar rats with cystitis, intravesical saline distension significantly (P internal oblique > rectus abdominis congruent with external oblique). In contrast, both laryngeal and tracheobronchial cough resulted in simultaneous activation of all four anterolateral abdominal muscles with peak EMG amplitudes 3- to 10-fold greater than those observed during the largest ETL. Gastric pressures during laryngeal and tracheobronchial cough were at least eightfold greater than those produced by the largest ETL. These results suggest that, unlike their behavior during expiratory loading, the anterolateral abdominal muscles act as a unit during cough. PMID- 10749808 TI - Rat small mesenteric artery function after hindlimb suspension. AB - To determine whether simulated microgravity in rats is associated with vascular dysfunction, we measured responses of isolated, pressurized mesenteric resistance artery segments (157- to 388-microm ID) to vasoconstrictors, pressure, and shear stress after 28-day hindlimb suspension (HS). Results indicated no differences between HS and control (C) groups in 1) sensitivity or maximal responses to vasoconstrictors (norepinephrine, phenylephrine, serotonin, KCl); 2) ID, external diameter, or ratio of wall thickness to ID; 3) distensibility; or 4) vasodilatory responses to shear stress. Myogenic tone was attenuated (P < 0.05) in HS arteries vs. C, as evidenced by 1) decreased magnitude of tone in larger vessels (second order branch off superior mesenteric artery, 261- to 388-microm ID) at pressures >/=40 mmHg in the presence of phenylephrine (10(-7) M) and 2) decreased magnitude of tone in smaller vessels (third-order branch off superior mesenteric artery, 157- to 277-microm ID), which exhibited spontaneous tone, at pressures > or =70 mmHg. This attenuation of myogenic tone after HS could contribute to orthostatic intolerance because myogenic tone contributes to the overall tone of resistance arteries. PMID- 10749810 TI - Passive energy absorption by human muscle-tendon unit is unaffected by increase in intramuscular temperature. AB - The present study measured hamstring intramuscular temperature and muscle-tendon unit viscoelastic properties in healthy young men before and after 10 and 30 min of running with (day S) or without stretch (day NS). On day NS, passive energy absorption and intramuscular temperature were measured before running (Preex), after 10 min of running at 70% of maximum O(2) uptake (Postex10), and after 30 min of running at 75% of maximum O(2) uptake (Postex30). On day S, the protocol was repeated with three stretches (stretches 1-3) added after Postex10. Intramuscular temperature was elevated Postex10 (P < 0.01) and further Postex30 (P < 0.05). On day NS, the total energy absorbed Preex (14.3 +/- 2.3 J), Postex10 (14.5 +/- 3.2 J), and Postex30 (13.5 +/- 2.4 J) was not different. On day S, the total energy absorbed in stretch 3 (10.8 +/- 1.8 J) was lower than that Preex (14.5 +/- 1.7 J, P < 0.01) and Postex10 (13.5 +/- 1.9 J, P < 0.05) but not Postex30 (13.3 +/- 1.8 J). The total energy absorbed Postex30 did not differ from Preex. In conclusion, warm-up and continuous running elevated intramuscular temperature but did not affect the passive energy absorption. Repeated passive stretching reduced the energy absorption immediately; however, the effect did not remain after 30 min of running. These data suggest that passive energy absorption of the human skeletal muscle is insensitive to physiological increases in intramuscular temperature. PMID- 10749811 TI - Cardiovascular response to hypoxia after endurance training at altitude and sea level and after detraining. AB - The purpose of this study was to elucidate 1) the effects of endurance exercise training during hypoxia or normoxia and of detraining on ventilatory and cardiovascular responses to progressive isocapnic hypoxia and 2) whether the change in the cardiovascular response to hypoxia is correlated to changes in the hypoxic ventilatory response (HVR) after training and detraining. Seven men (altitude group) performed endurance training using a cycle ergometer in a hypobaric chamber of simulated 4,500 m, whereas the other seven men (sea-level group) trained at sea level (K. Katayama, Y. Sato, Y. Morotome, N. Shima, K. Ishida, S. Mori, and M. Miyamura. J. Appl. Physiol. 86: 1805-1811, 1999). The HVR, systolic and diastolic blood pressure responses (DeltaSBP/DeltaSa(O(2)), DeltaDBP/DeltaSa(O(2))), and heart rate response (DeltaHR/DeltaSa(O(2)); Sa(O(2)) is arterial oxygen saturation) to progressive isocapnic hypoxia were measured before and after training and during detraining. DeltaSBP/DeltaSa(O(2)) increased significantly in the altitude group and decreased significantly in the sea-level group after training. The changed DeltaSBP/DeltaSa(O(2)) in both groups was restored during 2 wk of detraining, as were the changes in HVR, whereas there were no changes in the DeltaDBP/DeltaSa(O(2)) and DeltaHR/DeltaSa(O(2)) throughout the experimental period. The changes in DeltaSBP/DeltaSa(O(2)) after training and detraining were significantly correlated with those in HVR. These results suggest that DeltaSBP/DeltaSa(O(2)) to progressive isocapnic hypoxia is variable after endurance training during hypoxia and normoxia and after detraining, as is HVR, but DeltaDBP/DeltaSa(O(2)) and DeltaHR/DeltaSa(O(2)) are not. It also suggests that there is an interaction between the changes in DeltaSBP/DeltaSa(O(2)) and HVR after endurance training or detraining. PMID- 10749812 TI - VO(2) kinetics reveal a central limitation at the onset of subthreshold exercise in heart transplant recipients. AB - Because the cardiocirculatory response of heart transplant recipients (HTR) to exercise is delayed, we hypothesized that their O(2) uptake (VO(2)) kinetics at the onset of subthreshold exercise are slowed because of an impaired early "cardiodynamic" phase 1, rather than an abnormal subsequent "metabolic" phase 2. Thus we compared the VO(2) kinetics in 10 HTR submitted to six identical 10-min square-wave exercises set at 75% (36 +/- 5 W) of the load at their ventilatory threshold (VT) to those of 10 controls (C) similarly exercising at the same absolute (40 W; C40W group) and relative load (67 +/- 14 W; C67W group). Time averaged heart rate, breath-by-breath VO(2), and O(2) pulse (O(2)p) data yielded monoexponential time constants of the VO(2) (s) and O(2)p increase. Separating phase 1 and 2 data permitted assessment of the phase 1 duration and phase 2 VO(2) time constant (). The VO(2) time constant was higher in HTR (38.4 +/- 7.5) than in C40W (22.9 +/- 9.6; P < or = 0. 002) or C67W (30.8 +/- 8.2; P < or = 0.05), as was the O(2)p time constant, resulting from a lower phase 1 VO(2) increase (287 +/- 59 vs. 349 +/- 66 ml/min; P < or = 0.05), O(2)p increase (2.8 +/- 0.6 vs. 3.6 +/- 1.0 ml/beat; P < or = 0.0001), and a longer phase 1 duration (36.7 +/- 12.3 vs. 26.8 +/- 6.0 s; P < or = 0.05), whereas the was similar in HTR and C (31.4 +/ 9.6 vs. 29.9 +/- 5.6 s; P = 0.85). Thus the HTR have slower subthreshold VO(2) kinetics due to an abnormal phase 1, suggesting that the heart is unable to increase its output abruptly when exercise begins. We expected a faster in HTR because of their prolonged phase 1 duration. Because this was not the case, their muscular metabolism may also be impaired at the onset of subthreshold exercise. PMID- 10749813 TI - Glucose ingestion and substrate utilization during exercise in boys with IDDM. AB - This study was intended to compare exogenous [(13)C]glucose (Glu(exo)) oxidation in boys with insulin-dependent diabetes mellitus (IDDM) and healthy boys of similar age, weight, and maximal O(2) uptake. In a control trial with water intake (CT) and in a (13)C-enriched glucose trial (GT), subjects cycled for 60 min (58.8 +/- 0.9% maximal O(2) uptake) while the utilization of total glucose, total fat, and Glu(exo) was assessed. In CT, total glucose was 84.7 +/- 9.2 vs. 91.3 +/- 6.6 g/60 min (not significantly different) and total fat was 13.3 +/- 2.2 vs. 11.1 +/- 1.7 g/60 min (not significantly different) in IDDM vs. healthy boys, respectively. In GT, Glu(exo) was 10.4 +/- 1.7 vs. 14.8 +/- 1.1 g/60 min, corresponding to 9.0 +/- 1.0 vs. 12.4 +/- 0.5% of the total energy supply in IDDM and healthy boys, respectively (P < 0.05). Endogenous glucose was spared in both groups by 12.6 +/- 3.5% (P < 0.05). Blood glucose and plasma insulin concentrations were two- to threefold higher in IDDM vs. healthy boys in both trials. In conclusion, Glu(exo) is impaired in exercising boys with IDDM, even when plasma insulin levels are elevated. PMID- 10749814 TI - Vagal and mediator mechanisms underlying the tachypnea caused by pulmonary air embolism in dogs. AB - We investigated the vagal and mediator mechanisms underlying the tachypnea caused by pulmonary air embolism (PAE) in anesthetized and spontaneously breathing dogs. PAE was induced by infusion of air into the right atrium (0.2 ml. kg(-1). min(-1) for 10 min). The first PAE induction caused an increase in respiratory frequency accompanied by a decrease in tidal volume in each of the 30 animals studied. Subsequently, animals were evenly divided into five groups, and a second PAE induction was repeated after various experimental interventions. The tachypneic response to PAE was not significantly altered by pretreatment with a saline vehicle but was largely attenuated by either perivagal capsaicin treatment (a technique that selectively blocks the conduction of unmyelinated C fibers), pretreatment with ibuprofen (a cyclooxygenase inhibitor), or pretreatment with dimethylthiourea (a hydroxyl radical scavenger). Ultimately, the tachypneic response was nearly abolished by a bilateral cervical vagotomy. These results suggest that 1) lung vagal unmyelinated C-fiber afferents play a predominant role in evoking the reflex tachypneic response to PAE and 2) both cyclooxygenase products and hydroxyl radical are important in eliciting this vagally mediated response. PMID- 10749815 TI - Attenuation of force deficit after lengthening contractions in soleus muscle from trained rats. AB - The purposes of this study were 1) to determine the extent to which endurance training reduces the functional deficit induced by lengthening contractions in the soleus (Sol) muscle and 2) to determine whether young and old rats training at a comparable relative exercise intensity would demonstrate a similar protective effect from lengthening-contraction-induced injury. Young (3-mo-old) and old (23-mo-old) male Fischer 344 rats were randomly assigned to either a control or exercise training group [young control (YC), old control (OC), young trained (YT), old trained (OT)]. Exercise training consisted of 10 wk of treadmill running (15% grade, 45 min/day, and 5 days/wk) such that by the end of training the young and old rats were exercising at 27 and 15 m/min, respectively. After training, contractile properties of the Sol muscle were measured in vitro at 26 degrees C. The percent decrease in maximal isometric specific force (P(o)) was determined after a series of 20 lengthening contractions (20% strain from optimal muscle length, 1 contraction every 5 s). After the lengthening contraction protocol, Sol muscle P(o) was decreased by approximately 26% (19.6 vs. 14.6 N/cm(2)) and 28% (14.8 vs. 9.6 N/cm(2)) in the YC and OC rats, respectively. After exercise training, the reduction in P(o) was significantly (P < 0.05) attenuated to a similar degree ( approximately 13%) in both YT rats (18.7 vs. 16.2 N/cm(2)) and OT rats (15.8 vs. 13.7 N/cm(2)). It is concluded that exercise training attenuates the force deficit after repeated lengthening contractions to a comparable extent in young and old rats training at a similar exercise intensity. PMID- 10749816 TI - Modulation of airway caliber by deep inhalation in children. AB - To elucidate whether deep inhalation (DI) modulates changes in airway caliber in childhood, we measured the effect of DI on respiratory impedance before and after inhaled methacholine or salbutamol in 4- to 7-yr-old children (n = 15) suffering from recurrent wheezing. In all children, the real part of impedance between 12 and 16 Hz (Re[Z](12-16)) increased after methacholine from 5.6 +/- 1.2 to 8.2 +/- 1.6 cmH(2)O. l(-1). s (P < 0.001) and resonance frequency from 18 +/- 3 to 25 +/- 5 Hz (P < 0.001). These changes were partially reversed by DI: Re[Z](12-16) decreased to 7.2 +/- 1.2 cmH(2)O. l(-1). s (P < 0.01) and resonance frequency to 19 +/- 5 Hz (P < 0.001). In nine children, on a separate occasion, Re[Z](12-16) decreased after salbutamol from 8.3 +/- 1.9 to 5.1 +/- 0.9 cmH(2)O. l(-1). s (P < 0.001) and resonance frequency from 21 +/- 6 to 15 +/- 3 Hz (P < 0.05). The decrease of Re[Z](12-16) was partially reversed by DI (to 6.2 +/- 1.4 cmH(2)O. l( 1). s, P < 0. 01), but resonance frequency did not change significantly (P = 0.75). We conclude that in 4- to 7-yr-old children pharmacologically induced changes in airway caliber are modulated by DI. These findings suggest that airway to-parenchyma interdependence is operative in this age range. PMID- 10749817 TI - Lack of skeletal muscle hypertrophy in very aged male Fischer 344 x Brown Norway rats. AB - To examine the effect of extreme old age on muscle plasticity, 6- (adult) and 36 mo-old (old) male Fischer 344 x Brown Norway hybrid rats underwent bilateral surgical ablation of the gastrocnemius muscle to functionally overload (OV) the fast-twitch plantaris muscle for 8 wk. Plantaris muscle wet weight, muscle cross sectional area (CSA), and average fiber CSA decreased by 44, 42, and 40%, respectively, in old compared with adult rats, and peak isometric tetanic tension decreased by 83%. Compared with muscles in age-matched controls, plantaris muscle mass increased by 53% and type I, IIA, and IIX/IIB CSA increased by 91, 76, and 103%, respectively, in adult-OV rats, but neither wet mass nor fiber CSA increased in old-OV rats. OV decreased type I, IIA, and IIX/IIB mean fiber CSA by 31, 35, and 30%, respectively, in old-OV rats. Collectively, our data indicate that in extreme old age the plantaris muscle undergoes significant loss of mass, fiber CSA, and contractile function, as well as its capacity to undergo hypertrophy in response to a chronic increase in mechanical load. PMID- 10749818 TI - Interaction of the effects between vitamin D receptor polymorphism and exercise training on bone metabolism. AB - Bone metabolism is strongly influenced by heredity and environmental factors. To investigate interaction of the effects between vitamin D receptor polymorphism by Fok I and resistance exercise training on bone metabolism, young male subjects with FF genotype (F, n = 10) and Ff or ff genotypes (f, n = 10) followed 1 mo of weight training, and changes in bone metabolism were compared. An additional 14 subjects served as a sedentary control. Biomarkers of bone formation, bone specific alkaline phosphatase, and osteocalcin were significantly increased by training in both F and f groups. 1, 25-Dihydroxyvitamin D(3), known to upregulate bone formation, was also increased by the training in the f but not in the F group. Bone resorption assessed by cross-linked NH(2)-terminal telopeptide of type I collagen was significantly suppressed by the training, and the decrease in F was greater and longer lasting than that in f group. In conclusion, stimulation of bone formation and suppression of bone resorption occurred within 1 mo in young men. Despite a significant increase in 1,25-dihydroxyvitamin D(3) in the f group but not in the F group, the response of bone metabolism to the training in the F was similar to or greater than that in f group, suggesting a functional difference between vitamin D receptor genotypes f and F. PMID- 10749819 TI - Adipose tissue lipolysis is increased during a repeated bout of aerobic exercise. AB - The goal of the study was to examine whether lipid mobilization from adipose tissue undergoes changes during repeated bouts of prolonged aerobic exercise. Microdialysis of the subcutaneous adipose tissue was used for the assessment of lipolysis; glycerol concentration was measured in the dialysate leaving the adipose tissue. Seven male subjects performed two repeated bouts of 60-min exercise at 50% of their maximal aerobic power, separated by a 60-min recovery period. The exercise-induced increases in extracellular glycerol concentrations in adipose tissue and in plasma glycerol concentrations were significantly higher during the second exercise bout compared with the first (P < 0.05). The responses of plasma nonesterified fatty acids and plasma epinephrine were higher during the second exercise bout, whereas the response of norepinephrine was unchanged and that of growth hormone lower. Plasma insulin levels were lower during the second exercise bout. The results suggest that adipose tissue lipolysis during aerobic exercise of moderate intensity is enhanced when an exercise bout is preceded by exercise of the same intensity and duration performed 1 h before. This response pattern is associated with an increase in the exercise-induced rise of epinephrine and with lower plasma insulin values during the repeated exercise bout. PMID- 10749820 TI - Carbohydrate loading failed to improve 100-km cycling performance in a placebo controlled trial. AB - We evaluated the effect of carbohydrate (CHO) loading on cycling performance that was designed to be similar to the demands of competitive road racing. Seven well trained cyclists performed two 100-km time trials (TTs) on separate occasions, 3 days after either a CHO-loading (9 g CHO. kg body mass(-1). day(-1)) or placebo controlled moderate-CHO diet (6 g CHO. kg body mass(-1). day(-1)). A CHO breakfast (2 g CHO/kg body mass) was consumed 2 h before each TT, and a CHO drink (1 g CHO. kg(.)body mass(-1). h(-1)) was consumed during the TTs to optimize CHO availability. The 100-km TT was interspersed with four 4-km and five 1-km sprints. CHO loading significantly increased muscle glycogen concentrations (572 +/- 107 vs. 485 +/- 128 mmol/kg dry wt for CHO loading and placebo, respectively; P < 0.05). Total muscle glycogen utilization did not differ between trials, nor did time to complete the TTs (147.5 +/- 10.0 and 149.1 +/- 11.0 min; P = 0.4) or the mean power output during the TTs (259 +/- 40 and 253 +/- 40 W, P = 0.4). This placebo-controlled study shows that CHO loading did not improve performance of a 100-km cycling TT during which CHO was consumed. By preventing any fall in blood glucose concentration, CHO ingestion during exercise may offset any detrimental effects on performance of lower preexercise muscle and liver glycogen concentrations. Alternatively, part of the reported benefit of CHO loading on subsequent athletic performance could have resulted from a placebo effect. PMID- 10749821 TI - Fabrication of elastomer arterial models with specified compliance. AB - A simple way of making elastic tubes using a mechanical lathe for precise control of the wall thickness is proposed in this study. These tubes are particularly useful for modeling properties of large arteries. Tubes with different geometric parameters and hence different elastic behavior have been made with a silicon elastomer (Rhodorsil RTV 1556). They have been created to be used for compliance measurements in hemodynamics research. Within a limited range of pressures, depending on the wall thickness, such tubes can be used to study models in which the compliance value is assumed to be constant. PMID- 10749822 TI - Measurement of thoracoabdominal asynchrony: importance of sensor sensitivity to cross section deformations. AB - Discrepancies in the assessment of thoracoabdominal asynchrony are observed depending on the choice of respiratory movement sensors. We test the hypothesis that these discrepancies are due to a different dependence of the sensors on cross-sectional perimeter and area variations of the chest wall. First, we study the phase shift between perimeter and area (Phi(PA)) for an elliptical model, which is deformed by sinusoidal changes of its principal axes. We show that perimeter and area vary sinusoidally in the physiological range of deformations, and we discuss how Phi(PA) depends on the ellipticity of the cross section, on the ratio of transverse and dorsoventral movement amplitudes, and on their phase difference. Second, we compute the relationship between perimeter, area, and the output of the inductive sensor, and we proceed by comparing inductive plethysmography with strain gauges for several cross section deformations. We demonstrate that both sensors can provide different phase information for identical cross section deformations and, hence, can estimate thoracoabdominal asynchrony differently. Furthermore, the complex dependence of the inductive sensor on perimeter and area warns against this sensor for the evaluation of thoracoabdominal asynchrony. PMID- 10749823 TI - Animal housing influences the response of bone to spaceflight in juvenile rats. AB - The rat has been used extensively as an animal model to study the effects of spaceflight on bone metabolism. The results of these studies have been inconsistent. On some missions, bone formation at the periosteal bone surface of weight-bearing bones is impaired and on others it is not, suggesting that experimental conditions may be an important determinant of bone responsiveness to spaceflight. To determine whether animal housing can affect the response of bone to spaceflight, we studied young growing (juvenile) rats group housed in the animal enclosure module and singly housed in the research animal holding facility under otherwise identical flight conditions (Spacelab Life Science 1). Spaceflight reduced periosteal bone formation by 30% (P < 0.001) and bone mass by 7% in single-housed animals but had little or no effect on formation (-6%) or mass (-3%) in group-housed animals. Group housing reduced the response of bone to spaceflight by as much as 80%. The data suggest that housing can dramatically affect the skeletal response of juvenile rats to spaceflight. These observations explain many of the discrepancies in previous flight studies and emphasize the need to study more closely the effects of housing (physical-social interaction) on the response of bone to the weightlessness of spaceflight. PMID- 10749824 TI - Soft tissue body composition differences in monozygotic twins discordant for spinal cord injury. AB - To determine the effect of paralysis on body composition, eight pairs of male monozygotic twins, one twin in each pair with paraplegia, were studied by dual energy X-ray absorptiometry. Significant loss of total body lean tissue mass was found in the paralyzed twins compared with their able-bodied co-twins: 47.5 +/- 6. 7 vs. 60.1 +/- 7.8 (SD) kg (P < 0.005). Regionally, arm lean tissue mass was not different between the twin pairs, whereas trunk and leg lean tissue masses were significantly lower in the paralyzed twins: -3.0 +/- 3.3 kg (P < 0.05) and 10.1 +/- 4.0 kg (P < 0.0005), respectively. Bone mineral content of the total body and legs was significantly related to lean tissue mass in the able-bodied twins (R = 0.88 and 0.98, respectively) but not in the paralyzed twins. However, the intrapair difference scores for bone and lean tissue mass were significantly related (R = 0.80 and 0.81, respectively). The paralyzed twins had significantly more total body fat mass and percent fat per unit body mass index than the able bodied twins: 4.8 kg (P < 0.05) and 7 +/- 2% (P < 0.01). In the paralyzed twins, total body lean tissue was significantly lost (mostly from the trunk and legs), independent of age, at a rate of 3.9 +/- 0.2 kg per 5-yr period of paralysis (R = 0.87, P < 0.005). Extreme disuse from paralysis appears to contribute to a parallel loss of bone with loss of lean tissue in the legs. The continuous lean tissue loss may represent a form of sarcopenia that is progressive and accelerated compared with that in ambulatory individuals. PMID- 10749825 TI - Renal vasopressin receptor expression and function in rats following spaceflight. AB - It has been suggested there is a decreased renal responsiveness to vasopressin following spaceflight and that this may be the mechanism for the increased urine flow that is observed following return to normal gravity. In the present study, we have therefore measured vasopressin receptor expression and activity in kidneys taken from rats 1 and 14 days following spaceflight of 15 days duration. Measurements of renal vasopressin V(2) and V(1a) receptor mRNA expression by quantitative RT-PCR demonstrated little difference at either 1 day or at 14 days following return from space. Evaluation of (3)H-labeled arginine vasopressin binding to membranes prepared from kidneys indicated that the majority of the vasopressin receptors were V(2) receptors. Furthermore, the data suggested that binding to vasopressin V(2) or V(1a) receptors was unaltered at 1 day and 14 days following spaceflight. Similarly, the ability of vasopressin to stimulate adenylate cyclase suggested no change in vasopressin V(2) receptor activity in these animals. These data suggest that, whatever changes in fluid and electrolyte metabolism are observed following spaceflight, they are not mediated by changes in vasopressin receptor number or vasopressin-induced stimulation of adenylate cyclase. PMID- 10749826 TI - Aging of skeletal muscle: a 12-yr longitudinal study. AB - The present study examines age-related changes in skeletal muscle size and function after 12 yr. Twelve healthy sedentary men were studied in 1985-86 (T1) and nine (initial mean age 65.4 +/- 4.2 yr) were reevaluated in 1997-98 (T2). Isokinetic muscle strength of the knee and elbow extensors and flexors showed losses (P < 0.05) ranging from 20 to 30% at slow and fast angular velocities. Computerized tomography (n = 7) showed reductions (P < 0.05) in the cross sectional area (CSA) of the thigh (12.5%), all thigh muscles (14.7%), quadriceps femoris muscle (16.1%), and flexor muscles (14. 9%). Analysis of covariance showed that strength at T1 and changes in CSA were independent predictors of strength at T2. Muscle biopsies taken from vastus lateralis muscles (n = 6) showed a reduction in percentage of type I fibers (T1 = 60% vs. T2 = 42%) with no change in mean area in either fiber type. The capillary-to-fiber ratio was significantly lower at T2 (1.39 vs. 1. 08; P = 0.043). Our observations suggest that a quantitative loss in muscle CSA is a major contributor to the decrease in muscle strength seen with advancing age and, together with muscle strength at T1, accounts for 90% of the variability in strength at T2. PMID- 10749827 TI - Functional, cellular, and biochemical adaptations to elastase-induced emphysema in hamster medial scalene. AB - The scalene has been reported to be an accessory inspiratory muscle in the hamster. We hypothesize that with the chronic loads and/or dynamic hyperinflation associated with emphysema (Emp), the scalene will be actively recruited, resulting in functional, cellular, and biochemical adaptations. Emp was induced in adult hamsters. Inspiratory electromyogram (EMG) activity was recorded from the medial scalene and costal diaphragm. Isometric contractile and fatigue properties were evaluated in vitro. Muscle fibers were classified histochemically and immunohistochemically. Individual fiber cross-sectional areas (CSA) and succinate dehydrogenase (SDH) activities were determined quantitatively. Myosin heavy chain (MHC) isoforms were identified by SDS-PAGE, and their proportions were determined by scanning densitometry. All Emp animals exhibited spontaneous scalene inspiratory EMG activity during quiet breathing, whereas the scalene muscles of controls (Ctl) were silent. There were no differences in contractile and fatigue properties of the scalene between Ctl and Emp. In Emp, the relative amount of MHC(2A) was 15% higher whereas that of MHC(2X) was 14% lower compared with Ctl. Similarly, the proportion of type IIa fibers increased significantly in Emp animals with a concomitant decrease in IIx fibers. CSA of type IIx fibers were significantly smaller in Emp compared with Ctl. SDH activities of all fiber types were significantly increased by 53 to 63% in Emp. We conclude that with Emp the actively recruited scalene exhibits primary-like inspiratory activity in the hamster. Adaptations of the scalene with Emp likely relate both to increased loads and to factors intrinsic to muscle architecture and chest mechanics. PMID- 10749828 TI - Different ontogeny of rate of force generation and shortening velocity in guinea pig trachealis. AB - Juveniles of many species, including humans, display greater airway responsiveness than do adults. This may involve changes in airway smooth muscle function. In the present work we studied force production and shortening velocity in trachealis from 1-wk-old (1 wk), 3-wk-old (3 wk), and 3-mo-old (adult) guinea pigs. Strips were electrically stimulated (60 Hz, 18 V) at their optimal length (l(o)) to obtain maximum active stress (P(o)) and rate of stress generation. Then, force-velocity curves were elicited at 2.5 s from the onset of the stimulus. By applying a recently developed modification of Hill's equation for airway smooth muscle, the maximum shortening velocity at zero load (V(o)) and the value alpha. gamma/beta, an index of internal resistance to shortening (Rsi), were calculated (alpha, beta, and gamma are the constants of the equation). P(o) increased little with maturation, whereas the rate of stress generation increased significantly (0.40 +/- 0.03, 0.45 +/- 0.03, 0. 51 +/- 0.03 P(o)/s for 1 wk, 3 wk, and adult animals). V(o) slightly increased early with maturation to decrease significantly later (1. 79 +/- 0.67, 2.45 +/- 0.92, and 0.55 +/- 0.09 l(o)/s for 1 wk, 3 wk, and adult animals), whereas the Rsi showed an opposite trend (14.98 +/- 5.19, 8.99 +/- 3.01, and 32.07 +/- 5.54 mN. mm(-2). l(o)(-1). s for 1 wk, 3 wk, and adult animals). This early increase of force generation in combination with late increase of Rsi may explain the changes of V(o) with age. An elevated V(o) may contribute to the incidence of airway hyperresponsiveness in healthy juveniles. PMID- 10749829 TI - Reduced genioglossal activity with upper airway anesthesia in awake patients with OSA. AB - We examined whether topical upper airway anesthesia leads to a reduction in genioglossal (GG) electromyogram (EMG) in patients with obstructive sleep apnea (OSA). Airway mechanics were also evaluated. In 13 patients with OSA, we monitored GG EMG during tidal breathing and during the application of pulses of negative airway pressure (-10 to -12 cmH(2)O). Airflow resistance and airway collapsibility were determined. All measurements were performed with and without topical anesthesia (lidocaine). Anesthesia led to a significant fall in the peak GG EMG response to negative pressure from 36.1 +/- 4.7 to 24.8 +/- 5.3% (SE) of maximum (P < 0.01). This was associated with a fall in phasic and tonic EMG during tidal breathing (phasic from 24.4 +/- 4.1 to 16.4 +/- 3.4% of maximum and tonic from 10.9 +/- 1.6 to 8.0 +/- 1.3% of maximum, P < 0.01). A significant rise in pharyngeal airflow resistance was also observed. Our results demonstrate that topical receptor mechanisms in the nasopharynx importantly influence dilator muscle activity and are likely important in driving the augmented dilator muscle activity seen in the apnea patient. PMID- 10749830 TI - Fiber-type-specific alphaB-crystallin distribution and its shifts with T(3) and PTU treatments in rat hindlimb muscles. AB - Changes in alphaB-crystallin content in adult rat soleus and extensor digitorum longus (EDL) were examined after 8 wk of 3,5, 3'-triiodothyronine (T(3)) and propylthiouracil (PTU) treatments. Cellular distributions of alphaB-crystallin expression related to fiber type, and distribution shifts with these treatments were also examined in detail from the gray level of reactivity to specific anti alphaB-crystallin antibody. alphaB-crystallin content in both soleus and EDL muscles was significantly decreased after T(3), and that in EDL was significantly increased over twofold after PTU treatment. In both control soleus and EDL muscles, the gray level of type I fibers was higher than that of type II fibers. alphaB-crystallin expression among type II subtypes was muscle specific; the order was type I > IIa > IIx > IIb in control EDL muscle and type IIx > or = IIa in soleus muscle. The relation was basically unchanged in both muscles after T(3) treatment and was, in particular, well maintained in EDL muscle. Under hypothyroidism conditions with PTU, the mean alphaB-crystallin levels of type IIa and IIx fibers were significantly lower than levels under control conditions. Thus the relation between fiber type and the expression manner of stress protein alphaB-crystallin is muscle specific and also is well regulated under thyroid hormone, especially in fast EDL muscle. PMID- 10749831 TI - Low-pass filtering, a new method of fractal analysis: application to PET images of pulmonary blood flow. AB - The pattern of a spatial structure that repeats itself independently of the scale of magnification or resolution is often characterized by a fractal dimension (D). Two-dimensional low-pass filtering, which may serve as a method to assess D, was applied to functional images of pulmonary perfusion measured by positron emission tomography. The corner frequency of a low-pass filter is inversely proportional to the resolution scale. The method was applied to three types of images: random noise images, synthetic fractal images, and positron emission tomographic images of pulmonary perfusion. Images were processed with two-dimensional low-pass filters of decreasing corner frequencies, and a spatial heterogeneity index, the coefficient of variation, was calculated for each low-pass-filtered image. The natural logarithm of the coefficient of variation scaled linearly with the natural logarithm of the resolution scale for the PET images studied (average R(2) = 0.99). D ranged from 1.25 to 1.36 for the residual distribution of pulmonary perfusion after vertical gradients were removed by linear regression. D of the same data without removal of vertical gradients ranged from 1.11 to 1.14, but the fractal plots had systematic deviations from linearity and a lower linear correlation coefficient (R(2) = 0. 96). The method includes all data in the lung field and is insensitive to the effects of misregistration. We conclude that low pass filtering offers new insights into the interpretation of D of two dimensional functional images as a measure of the frequency content of spatial heterogeneity. PMID- 10749832 TI - Impact of hetastarch on the intestinal microvascular barrier during ECLS. AB - The effects of hetastarch on microvascular fluid flux were determined in anesthetized dogs undergoing extracorporeal life support (ECLS) with a roller pump and membrane oxygenator. ECLS with a lactated Ringer priming solution resulted in a decrease in microvascular protein reflection coefficient and an increase in transvascular protein clearance. Use of a 6% hetastarch priming solution attenuated the decrease in microvascular protein reflection coefficient and blunted the increase in transvascular protein clearance. Ileal tissue water increased in the group treated with the lactated Ringer priming solution compared with the group treated with 6% hetastarch. The effective plasma-to-interstitial colloid osmotic pressure gradient was greater in the group treated with hetastarch than in the group treated with lactated Ringer solution. Hetastarch decreases the edema associated with ECLS. The reduction in edema is due to the maintenance of the plasma-to-interstitial colloid osmotic pressure gradient and the reduction in the microvascular permeability to protein. PMID- 10749833 TI - Nitric oxide and cerebral blood flow responses to hyperbaric oxygen. AB - We have tested the hypothesis that cerebral nitric oxide (NO) production is involved in hyperbaric O(2) (HBO(2)) neurotoxicity. Regional cerebral blood flow (rCBF) and electroencephalogram (EEG) were measured in anesthetized rats during O(2) exposure to 1, 3, 4, and 5 ATA with or without administration of the NO synthase inhibitor (N(omega)-nitro-L-arginine methyl ester), L-arginine, NO donors, or the N-methyl-D-aspartate receptor inhibitor MK-801. After 30 min of O(2) exposure at 3 and 4 ATA, rCBF decreased by 26-39% and by 37-43%, respectively, and was sustained for 75 min. At 5 ATA, rCBF decreased over 30 min in the substantia nigra by one-third but, thereafter, gradually returned to preexposure levels, preceding the onset of EEG spiking activity. Rats pretreated with N(omega)-nitro-L-arginine methyl ester and exposed to HBO(2) at 5 ATA maintained a low rCBF. MK-801 did not alter the cerebrovascular responses to HBO(2) at 5 ATA but prevented the EEG spikes. NO donors increased rCBF in control rats but were ineffective during HBO(2) exposures. The data provide evidence that relative lack of NO activity contributes to decreased rCBF under HBO(2), but, as exposure time is prolonged, NO production increases and augments rCBF in anticipation of neuronal excitation. PMID- 10749834 TI - Effects of systemic arterial blood pressure on the contractile force of a human hand muscle. AB - The effect of physiological changes in systemic blood pressure on the force output of working abductor pollicis (AP) muscle was studied in six normal subjects. Supramaximal tetanic stimulation at the ulnar nerve produced repeated isometric contractions at 1-s intervals. Force output declined gradually with time. During the train of contractions, subjects voluntarily contracted the knee extensors for 1 min; this raised systemic blood pressure by 29%. Force output from AP rose in parallel with blood pressure so that 18% of the contraction force lost through fatigue was recovered for each 10% increase in blood pressure. When blood pressure in the hand was kept constant despite the increased systemic pressure, force output did not rise. The results show that muscle performance is strongly affected by physiological changes in central blood pressure and suggest that sensory input concerning the adequacy of muscle performance exerts a feedback control over the increase in systemic blood pressure during muscular activity. PMID- 10749835 TI - Adaptive responses during anemia and its correction in lambs. AB - There is limited information available on which to base decisions regarding red blood cell (RBC) transfusion treatment in anemic newborn infants. Using a conscious newborn lamb model of progressive anemia, we sought to identify accessible metabolic and cardiovascular measures of hypoxia that might provide guidance in the management of anemic infants. We hypothesized that severe phlebotomy-induced isovolemic anemia and its reversal after RBC transfusion result in a defined pattern of adaptive responses. Anemia was produced over 2 days by serial phlebotomy (with plasma replacement) to Hb levels of 30-40 g/l. During the ensuing 2 days, Hb was restored to pretransfusion baseline levels by repeated RBC transfusion. Area-under-the-curve methodology was utilized for defining the Hb level at which individual study variables demonstrated significant change. Significant reciprocal changes (P < 0.05) of equivalent magnitude were observed during the phlebotomy and transfusion phases for cardiac output, plasma erythropoietin (Epo) concentration, oxygen extraction ratio, oxygen delivery, venous oxygen saturation, and blood lactate concentration. No significant change was observed in resting oxygen consumption. Cardiac output and plasma Epo concentration increased at Hb levels <75 g/l, oxygen delivery and oxygen extraction ratio decreased at Hb levels <60 g/l, and venous oxygen saturation decreased and blood lactate concentration increased at Hb levels <55 g/l. We speculate that plasma Epo and blood lactate concentrations may be useful measures of clinically significant anemia in infants and may indicate when an infant might benefit from a RBC transfusion. PMID- 10749836 TI - VO(2) kinetics and the O(2) deficit in heavy exercise. AB - The purpose of this study was to examine a new method for calculating the O(2) deficit that considered the O(2) uptake (VO(2)) kinetics during exercise as two separate phases in light of previous research in which it was shown that the traditional O(2) deficit calculation overestimated the recovery O(2) consumption (ROC). Eight subjects completed exercise transitions between unloaded cycling and 25% (heavy, H) or 50% (very heavy, VH) of the difference between the lactic acid threshold (LAT) and peak VO(2) for 8 min. The O(2) deficit, calculated in the traditional manner, was significantly greater than the measured ROC for both above-LAT exercises: 4.03 +/- 1.01 vs. 2.63 +/- 0.80 (SD) liters for VH and 2.36 +/- 0.91 vs. 1.74 +/- 0.63 liters for H for the O(2) deficit vs. ROC (P < 0.05). When the kinetics were viewed as two separate components with independent onsets, the calculated O(2) deficit (2.89 +/- 0.79 and 1.71 +/- 0.70 liters for VH and H, respectively) was not different from the measured ROC (P < 0.05). Subjects also performed the same work rate for only 3 min. These data, from bouts terminated before the slow component could contribute appreciably to the overall VO(2) response, show that the O(2) requirement during the transition is less than the final steady state for the work rate, as evidenced by symmetry between the O(2) deficit and ROC. This new method of calculating the O(2) deficit more closely reflects the expected O(2) deficit-ROC relationship (i.e., ROC >/= O(2) deficit). Therefore, estimation of the O(2) deficit during heavy exercise transitions should consider the slow component of VO(2) as an additional deficit component with delayed onset. PMID- 10749837 TI - Airway distensibility in healthy and asthmatic subjects: effect of lung volume history. AB - Anatomic dead space (VD) is known to increase with end-inspiratory lung volume (EILV), and the gradient of the relationship has been proposed as an index of airway distensibility (DeltaVD). The aims of this study were to apply a rapid method for measuring DeltaVD and to determine whether it was affected by lung volume history. VD of 16 healthy and 16 mildly asthmatic subjects was measured at a number of known EILVs by using a tidal breathing, CO(2)-washout method. The effect of lung volume history was assessed by using three tidal breathing regimens: 1) three discrete EILVs (low/medium/high; LMH); 2) progressively decreasing EILVs from total lung capacity (TLC; TLC-RV); and 3) progressively increasing EILVs from residual volume (RV; RV-TLC). DeltaVD was lower in the asthmatic group for the LMH (25.3 +/- 2.24 vs. 21.2 +/- 1.66 ml/l, means +/- SE) and TLC-RV (24. 3 +/- 1.69 vs. 18.7 +/- 1.16 ml/l) regimens. There was a trend for a lower DeltaVD in the asthmatic group for the RV-TLC regimen (23.3 +/- 2.19 vs. 18.8 +/- 1.68 ml/l). There was no difference in DeltaVD between groups. In conclusion, mild asthmatic subjects have stiffer airways than normal subjects, and this is not obviously affected by lung volume history. PMID- 10749838 TI - Sustained microgravity reduces the human ventilatory response to hypoxia but not to hypercapnia. AB - We measured the isocapnic hypoxic ventilatory response and the hypercapnic ventilatory response by using rebreathing techniques in five normal subjects (ages 37-47 yr) before, during, and after 16 days of exposure to microgravity (microG). Control measurements were performed with the subjects in the standing and supine postures. In both microG and in the supine position, the hypoxic ventilatory response, as measured from the slope of ventilation against arterial O(2) saturation, was greatly reduced, being only 46 +/- 10% (microG) and 52 +/- 11% (supine) of that measured standing (P < 0.01). During the hypercapnic ventilatory response test, the ventilation at a PCO(2) of 60 Torr was not significantly different in microG (101 +/- 5%) and the supine position (89 +/- 3%) from that measured standing. Inspiratory occlusion pressures agreed with these results. The findings can be explained by inhibition of the hypoxic but not hypercapnic drive, possibly as a result of an increase in blood pressure in carotid baroreceptors in microG and the supine position. PMID- 10749840 TI - A new model of chronic cardiac ischemia in rabbits. AB - Chronic cardiac ischemia has mainly been studied in large species such as pigs or dogs. Little research has been performed using small species such as rabbits. In the present study, 1-3 wk after implantation of a novel device (ameroid) on the circumflex coronary artery of New Zealand White rabbits, vessel patency was evaluated by coronary angiography, corrosion cast, and radiolabeled microspheres. Coronary angiograms showed, after 21 days, either total occlusion or severe stenosis in seven of eight arteries, which was confirmed by corrosion casts. The ameroid group had less blood flow in the epicardial (-62%) and endocardial (-54%) layers of the ischemic area compared with sham-operated rabbits (P < 0.05). Blood flow increased in the ischemic area compared with day 0 during acute occlusion, suggesting that progressive coronary occlusion initiated the growth of de novo collateral vessels. Thus we have developed a new model of chronic cardiac ischemia in rabbits with documented progressive coronary stenosis and occlusion that is suitable to test various therapeutic angiogenesis strategies. PMID- 10749839 TI - Revealing deterministic structures in click-evoked otoacoustic emissions. AB - Click-evoked otoacoustic emissions (CEOAEs) were studied by means of recurrence quantification analysis (RQA) and were found to be endowed with a relevant amount of deterministic structuring. Such a structure showed highly significant correlation with the clinical evaluation of the signal over a data set including 56 signals. Moreover, 1) one of the RQA variables, Trend, was very sensitive to phase transitions in the dynamical regime of CEOAEs, and 2) appropriate use of principal component analysis proved able to isolate the individual character of the studied signals. These results are of general interest for the study of auditory signal transduction and generation mechanisms. PMID- 10749841 TI - Developmental myosin heavy chains in the adult human diaphragm: coexpression patterns and effect of COPD. AB - In preliminary experiments we noted developmental (i.e., embryonic and neonatal) myosin heavy chains (MHCs) in the diaphragms of patients with severe chronic obstructive pulmonary disease (COPD). We hypothesized that this finding represented new fiber formation secondary to injury associated with the mechanical stress of COPD or previously undescribed MHCs in the human diaphragm. To distinguish between these possibilities, we analyzed diaphragmatic biopsies obtained from 9 patients with severe COPD (forced expiratory volume in 1 s = 21 +/- 2% predicted, residual volume = 283 +/- 22% predicted) and 10 age-matched controls. First, using immunocytochemistry with specific monoclonal antibodies, we noted that control diaphragms had greater proportions of fibers expressing embryonic (50 +/- 2 vs. 28 +/- 3%, P < 0.0001) and neonatal (52 +/- 2 vs. 32 +/- 3%, P < 0.001) MHCs than COPD diaphragms. Second, SDS-PAGE demonstrated that these developmental MHCs represented only a very small fraction of the diaphragmatic MHC content. Third, the RT-PCR demonstrated mRNA coding for embryonic and neonatal MHCs in COPD and control diaphragms. Last, COPD and control diaphragms exhibited normal histology on light microscopy. We conclude that the presence of developmental MHC isoforms does not indicate new fiber formation in diaphragms of patients with severe COPD. Although these results represent the first systematic description of embryonic and neonatal MHCs in normal adult human diaphragms, their function remains to be elucidated. PMID- 10749842 TI - A new nasal acoustic reflection technique to estimate pharyngeal cross-sectional area during sleep. AB - The conventional acoustic reflection technique in which acoustic waves are launched through the mouth cannot be applied during sleep, nor can it be applied to the nasopharynx, which is the major site of occlusion in patients with obstructive sleep apnea syndrome. We propose a new technique of nasal acoustic reflection to measure pharyngeal cross-sectional areas including the nasopharynx. The acoustic waves are introduced simultaneously to both nostrils during spontaneous nasal breathing. A new algorithm takes into account the nasal septum with asymmetric nasal cavities on both sides and assumes prior knowledge of the cross-sectional area of the nasal cavities and the position of the nasal septum. This method was tested on an airway model with a septum and on healthy human subjects. The conventional technique gave inaccurate measurements for pharyngeal cross-sectional areas for an airway model with asymmetric branching, whereas the new technique measured them almost perfectly. The oro- and hypopharyngeal cross sectional area measurements acquired by the new method were not different from those obtained by the conventional method in normal subjects. This new method can be used as a monitor of upper airway dimensions in nocturnal polysomnography. PMID- 10749843 TI - Historical perspectives of cellular oxygen sensing and responses to hypoxia. AB - The responses to acute and chronic hypoxia begin with oxygen sensing, and this historical perspective is written in line with this concept. The earliest pertinent work started with studies on fermentation in yeast in the 17th century, before the discovery of oxygen. It required 200 yr to localize the oxygen sensing within the cells and another 100 yr to discover the cellular oxidation reactions. Today, the consensus is that the mitochondrial respiratory chain is in part the site of oxygen sensing. In addition, membrane-bound NAD(P)H oxidase possibly takes part in oxygen sensing. Oxygen-sensing mechanisms occur in a tissue specific fashion. For example, the carotid body responds to hypoxia promptly by eliciting a ventilatory response, whereas erythropoietin production in response to hypoxia requires more time, involving new expression of genes. The mechanism has therefore moved from the cells to genes. PMID- 10749844 TI - HIF-1: mediator of physiological and pathophysiological responses to hypoxia. AB - All organisms can sense O(2) concentration and respond to hypoxia with adaptive changes in gene expression. The large body size of mammals necessitates the development of multiple complex physiological systems to ensure adequate O(2) delivery to all cells under normal conditions. The transcriptional regulator hypoxia-inducible factor 1 (HIF-1) is an essential mediator of O(2) homeostasis. HIF-1 is required for the establishment of key physiological systems during development and their subsequent utilization in fetal and postnatal life. HIF-1 also appears to play a key role in the pathophysiology of cancer, cardiovascular disease, and chronic lung disease, which represent the major causes of mortality among industrialized societies. Genetic or pharmacological modulation of HIF-1 activity in vivo may represent a novel therapeutic approach to these disorders. PMID- 10749845 TI - Enhancing our understanding of the molecular responses to hypoxia in mammals using Drosophila melanogaster. AB - Drosophila melanogaster has been used as a genetic model, especially in the past decade, to examine normative biological processes and disease conditions very effectively. These span a wide range of major issues such as aging, cancer, embryogenesis, neural development, apoptosis, and alcohol intoxication. Here, we detail how the Drosophila melanogaster can be used as a genetic model to study the molecular and genetic underpinnings of the response to hypoxia. In our study of the basis of anoxia tolerance, one of the potent approaches that we use is a mutagenesis screen to identify loss-of-function mutants that are anoxia sensitive. The major advantage of this approach is that it is not biased for any particular gene or gene product. Although our screen is in progress, we already have evidence that this approach is useful. PMID- 10749846 TI - Single-unit recordings of arterial chemoreceptors from mouse petrosal ganglia in vitro. AB - A preparation was developed that allows for the recording of single-unit chemoreceptor activity from mouse carotid body in vitro. An anesthetized mouse was decapitated, and each carotid body was harvested, along with the sinus nerve, glossopharyngeal nerve, and petrosal ganglia. After exposure to collagenase/trypsin, the cleaned complex was transferred to a recording chamber where it was superfused with oxygenated saline. The ganglia was searched for evoked or spontaneous unit activity by using a glass suction electrode. Single unit action potentials were 57 +/- 10 (SE) (n = 16) standard deviations above the recording noise, and spontaneous spikes were generated as a random process. Decreasing superfusate PO(2) to near 20 Torr caused an increase in spiking activity from 1. 3 +/- 0.4 to 14.1 +/- 1.9 Hz (n = 16). The use of mice for chemoreceptor studies may be advantageous because targeted gene deletions are well developed in the mouse model and may be useful in addressing unresolved questions regarding the mechanism of chemotransduction. PMID- 10749847 TI - Blunted respiratory responses to hypoxia in mutant mice deficient in nitric oxide synthase-3. AB - In the present study, the role of nitric oxide (NO) generated by endothelial nitric oxide synthase (NOS-3) in the control of respiration during hypoxia and hypercapnia was assessed using mutant mice deficient in NOS-3. Experiments were performed on awake and anesthetized mutant and wild-type (WT) control mice. Respiratory responses to 100, 21, and 12% O(2) and 3 and 5% CO(2)-balance O(2) were analyzed. In awake animals, respiration was monitored by body plethysmography along with O(2) consumption (VO(2)) and CO(2) production (VCO(2)). In anesthetized, spontaneously breathing mice, integrated efferent phrenic nerve activity was monitored as an index of neural respiration along with arterial blood pressure and blood gases. Under both experimental conditions, WT mice responded with greater increases in respiration during 12% O(2) than mutant mice. Respiratory responses to hyperoxic hypercapnia were comparable between both groups of mice. Arterial blood gases, changes in blood pressure, VO(2), and VCO(2) during hypoxia were comparable between both groups of mice. Respiratory responses to cyanide and brief hyperoxia were attenuated in mutant compared with WT mice, indicating reduced peripheral chemoreceptor sensitivity. cGMP levels in the brain stem during 12% O(2), taken as an index of NO production, were greater in mutant compared with WT mice. These observations demonstrate that NOS-3 mutant mice exhibit selective blunting of the respiratory responses to hypoxia but not to hypercapnia, which in part is due to reduced peripheral chemosensitivity. These results support the idea that NO generated by NOS-3 is an important physiological modulator of respiration during hypoxia. PMID- 10749848 TI - The human liver-specific homolog of very long-chain acyl-CoA synthetase is cholate:CoA ligase. AB - Unconjugated bile acids must be activated to their CoA thioesters before conjugation to taurine or glycine can occur. A human homolog of very long-chain acyl-CoA synthetase, hVLCS-H2, has two requisite properties of a bile acid:CoA ligase, liver specificity and an endoplasmic reticulum subcellular localization. We investigated the ability of this enzyme to activate the primary bile acid, cholic acid, to its CoA derivative. When expressed in COS-1 cells, hVLCS-H2 exhibited cholate:CoA ligase (choloyl-CoA synthetase) activity with both non isotopic and radioactive assays. Other long- and very long-chain acyl-CoA synthetases were incapable of activating cholate. Endogenous choloyl-CoA synthetase activity was also detected in liver-derived HepG2 cells but not in kidney-derived COS-1 cells. Our results are consistent with a role for hVLCS-H2 in the re-activation and re-conjugation of bile acids entering liver from the enterohepatic circulation rather than in de novo bile acid synthesis. PMID- 10749850 TI - Specific properties of T-type calcium channels generated by the human alpha 1I subunit. AB - We have cloned and expressed a human alpha(1I) subunit that encodes a subtype of T-type calcium channels. The predicted protein is 95% homologous to its rat counterpart but has a distinct COOH-terminal region. Its mRNA is detected almost exclusively in the human brain, as well as in adrenal and thyroid glands. Calcium currents generated by the functional expression of human alpha(1I) and alpha(1G) subunits in HEK-293 cells were compared. The alpha(1I) current activated and inactivated approximately 10 mV more positively. Activation and inactivation kinetics were up to six times slower, while deactivation kinetics was faster and showed little voltage dependence. A slower recovery from inactivation, a lower sensitivity to Ni(2+) ions (IC(50) approximately 180 micrometer), and a larger channel conductance (approximately 11 picosiemens) were the other discriminative features of the alpha(1I) current. These data demonstrate that the alpha(1I) subunit encodes T-type Ca(2+) channels functionally distinct from those generated by the human alpha(1G) or alpha(1H) subunits and point out that human and rat alpha(1I) subunits have species-specific properties not only in their primary sequence, but also in their expression profile and electrophysiological behavior. PMID- 10749849 TI - The gut-enriched Kruppel-like factor (Kruppel-like factor 4) mediates the transactivating effect of p53 on the p21WAF1/Cip1 promoter. AB - An important mechanism by which the tumor suppressor p53 maintains genomic stability is to induce cell cycle arrest through activation of the cyclin dependent kinase inhibitor p21(WAF1/Cip1) gene. We show that the gene encoding the gut-enriched Kruppel-like factor (GKLF, KLF4) is concurrently induced with p21(WAF1/Cip1) during serum deprivation and DNA damage elicited by methyl methanesulfonate. The increases in expression of both Gklf and p21(WAF1/Cip1) due to DNA damage are dependent on p53. Moreover, during the first 30 min of methyl methanesulfonate treatment, the rise in Gklf mRNA level precedes that in p21(WAF1/Cip1), suggesting that GKLF may be involved in the induction of p21(WAF1/Cip1). Indeed, GKLF activates p21(WAF1/Cip1) through a specific Sp1-like cis-element in the p21(WAF1/Cip1) proximal promoter. The same element is also required by p53 to activate the p21(WAF1/Cip1) promoter, although p53 does not bind to it. Potential mechanisms by which p53 activates the p21(WAF1/Cip1) promoter include a physical interaction between p53 and GKLF and the transcriptional induction of Gklf by p53. Consequently, the two transactivators cause a synergistic induction of the p21(WAF1/Cip1) promoter activity. The physiological relevance of GKLF in mediating p53-dependent induction of p21(WAF1/Cip1) is demonstrated by the ability of antisense Gklf oligonucleotides to block the production of p21(WAF1/Cip1) in response to p53 activation. These findings suggest that GKLF is an essential mediator of p53 in the transcriptional induction of p21(WAF1/Cip1) and may be part of a novel pathway by which cellular responses to stress are modulated. PMID- 10749851 TI - Protein-arginine methyltransferase I, the predominant protein-arginine methyltransferase in cells, interacts with and is regulated by interleukin enhancer-binding factor 3. AB - Arginine methylation is a common post-translation modification found in many proteins. Protein-arginine methyltransferase I (PRMT1) contributes >90% of type I protein-arginine methyltransferase activity in cells and tissues. To expand our knowledge on the regulation and role of PRMT1 in cells, we used the yeast two hybrid system to identify proteins that interact with PRMT1. One of the interacting proteins we cloned is interleukin enhancer-binding factor 3 (ILF3), also known as M phase phosphoprotein 4. ILF3 is closely related to nuclear factor 90 (NF90). Using an immunofluorescence analysis, we determined that ILF3 and PRMT1 co-localize in the nucleus. Moreover, PRMT1 and ILF3 co-precipitate in immunoprecipitation assays and can be isolated together in "pull-down" experiments using recombinant fusion proteins. ILF3 is a robust substrate for methylation by PRMT1 and can modulate PRMT1 activity in in vitro methylation assays. Deletion studies demonstrated that the COOH-terminal region of ILF3, which is rich in arginine, glycine, and serine, is responsible for the strong interaction between PRMT1 and ILF3 and is the site of ILF3 methylation by PRMT1. Although ILF3 and NF90 are highly similar, they differ in their carboxyl-terminal regions. Because of this difference, NF90 does not interact with PRMT1, is a much poorer substrate than ILF3 for PRMT1-dependent methylation, and does not modulate PRMT1 enzyme activity. PMID- 10749852 TI - Interaction of Drosophila melanogaster prohormone convertase 2 and 7B2. Insect cell-specific processing and secretion. AB - The prohormone convertases (PCs) are an evolutionarily ancient group of proteases required for the maturation of neuropeptide and peptide hormone precursors. In Drosophila melanogaster, the homolog of prohormone convertase 2, dPC2 (amontillado), is required for normal hatching behavior, and immunoblotting data indicate that flies express 80- and 75-kDa forms of this protein. Because mouse PC2 (mPC2) requires 7B2, a helper protein for productive maturation, we searched the fly data base for the 7B2 signature motif PPNPCP and identified an expressed sequence tag clone encoding the entire open reading frame for this protein. dPC2 and d7B2 cDNAs were subcloned into expression vectors for transfection into HEK 293 cells; mPC2 and rat 7B2 were used as controls. Although active mPC2 was detected in medium in the presence of either d7B2 or r7B2, dPC2 showed no proteolytic activity upon coexpression of either d7B2 or r7B2. Labeling experiments showed that dPC2 was synthesized but not secreted from HEK-293 cells. However, when dPC2 and either d7B2 or r7B2 were coexpressed in Drosophila S2 cells, abundant immunoreactive dPC2 was secreted into the medium, coincident with the appearance of PC2 activity. Expression and secretion of dPC2 enzyme activity thus appears to require insect cell-specific posttranslational processing events. The significant differences in the cell biology of the insect and mammalian enzymes, with 7B2 absolutely required for secretion of dPC2 and zymogen conversion occurring intracellularly in the case of dPC2 but not mPC2, support the idea that the Drosophila enzyme has specific requirements for maturation and secretion that can be met only in insect cells. PMID- 10749853 TI - Electron transfer, oxygen binding, and nitric oxide feedback inhibition in endothelial nitric-oxide synthase. AB - We studied steps that make up the initial and steady-state phases of nitric oxide (NO) synthesis to understand how activity of bovine endothelial NO synthase (eNOS) is regulated. Stopped-flow analysis of NADPH-dependent flavin reduction showed the rate increased from 0. 13 to 86 s(-1) upon calmodulin binding, but this supported slow heme reduction in the presence of either Arg or N(omega) hydroxy-l-arginine (0.005 and 0.014 s(-1), respectively, at 10 degrees C). O(2) binding to ferrous eNOS generated a transient ferrous dioxy species (Soret peak at 427 nm) whose formation and decay kinetics indicate it can participate in NO synthesis. The kinetics of heme-NO complex formation were characterized under anaerobic conditions and during the initial phase of NO synthesis. During catalysis heme-NO complex formation required buildup of relatively high solution NO concentrations (>50 nm), which were easily achieved with N(omega)-hydroxy-l arginine but not with Arg as substrate. Heme-NO complex formation caused eNOS NADPH oxidation and citrulline synthesis to decrease 3-fold and the apparent K(m) for O(2) to increase 6-fold. Our main conclusions are: 1) The slow steady-state rate of NO synthesis by eNOS is primarily because of slow electron transfer from its reductase domain to the heme, rather than heme-NO complex formation or other aspects of catalysis. 2) eNOS forms relatively little heme-NO complex during NO synthesis from Arg, implying NO feedback inhibition has a minimal role. These properties distinguish eNOS from the other NOS isoforms and provide a foundation to better understand its role in physiology and pathology. PMID- 10749854 TI - Evidence for a tandem two-site model of ligand binding to muscarinic acetylcholine receptors. AB - After short preincubations with N-[(3)H]methylscopolamine ([(3)H]NMS) or R(-) [(3)H]quinuclidinyl benzilate ([(3)H]QNB), radioligand dissociation from muscarinic M(1) receptors in Chinese hamster ovary cell membranes was fast, monoexponential, and independent of the concentration of unlabeled NMS or QNB added to reveal dissociation. After long preincubations, the dissociation was slow, not monoexponential, and inversely related to the concentration of the unlabeled ligand. Apparently, the unlabeled ligand becomes able to associate with the receptor simultaneously with the already bound radioligand if the preincubation lasts for a long period, and to hinder radioligand dissociation. When the membranes were preincubated with [(3)H]NMS and then exposed to benzilylcholine mustard (covalently binding specific ligand), [(3)H]NMS dissociation was blocked in wild-type receptors, but not in mutated (D99N) M(1) receptors. Covalently binding [(3)H]propylbenzilylcholine mustard detected substantially more binding sites than [(3)H]NMS. The observations support a model in which the receptor binding domain has two tandemly arranged subsites for classical ligands, a peripheral one and a central one. Ligands bind to the peripheral subsite first (binding with lower affinity) and translocate to the central subsite (binding with higher affinity). The peripheral subsite of M(1) receptors may include Asp-99. Experimental data on [(3)H]NMS and [(3)H]QNB association and dissociation perfectly agree with the predictions of the tandem two-site model. PMID- 10749856 TI - Cleavage of bovine collagen I by neutrophil collagenase MMP-8. Effect of pH on the catalytic properties as compared to synthetic substrates. AB - The enzymatic processing of bovine collagen I by neutrophil collagenase (MMP-8) has been monitored at 37 degrees C, envisaging the occurrence of multiple intermediate steps, following the initial cleavage, which leads to the formation of (1/4) and (3/4) fragments. Further, the first cleavage event has been investigated at 37 degrees C as a function of pH, and catalytic parameters have been obtained through a global analysis of steady-state kinetic data, such as to get an overall consistent picture of k(cat)/K(m), k(cat), and K(m). These data have been compared with those obtained from the catalysis by MMP-8 of two synthetic fluorogenic substrates under the same experimental conditions. The overall behavior can be accounted for by the existence of five protonating groups, which vary to a different extent their pK(a) values for the three substrates investigated. The main observation concerns the fact the two of these residues, which play a relevant role in the enzymatic activity of MMP-8, are relatively far from the primary recognition site, and they are coming into action only for large macromolecular substrates, such as bovine collagen I. This finding opens the question of appropriate testing for inhibitors of the enzymatic action of MMP-8, which must take into account, and also of these relevant interactions occurring only with natural substrates. PMID- 10749855 TI - Cloning and expression of the human N-acetylneuraminic acid phosphate synthase gene with 2-keto-3-deoxy-D-glycero- D-galacto-nononic acid biosynthetic ability. AB - Sialic acids participate in many important biological recognition events, yet eukaryotic sialic acid biosynthetic genes are not well characterized. In this study, we have identified a novel human gene based on homology to the Escherichia coli sialic acid synthase gene (neuB). The human gene is ubiquitously expressed and encodes a 40-kDa enzyme. The gene partially restores sialic acid synthase activity in a neuB-negative mutant of E. coli and results in N-acetylneuraminic acid (Neu5Ac) and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) production in insect cells upon recombinant baculovirus infection. In vitro the human enzyme uses N-acetylmannosamine 6-phosphate and mannose 6-phosphate as substrates to generate phosphorylated forms of Neu5Ac and KDN, respectively, but exhibits much higher activity toward the Neu5Ac phosphate product. PMID- 10749857 TI - Thiazolidinedione treatment enhances insulin effects on protein kinase C-zeta /lambda activation and glucose transport in adipocytes of nondiabetic and Goto Kakizaki type II diabetic rats. AB - We evaluated effects of the thiazolidinedione, rosiglitazone, on insulin-induced activation of protein kinase C (PKC)-zeta/lambda and glucose transport in adipocytes of Goto-Kakizaki (GK)-diabetic and nondiabetic rats. Insulin effects on PKC-zeta/lambda and 2-deoxyglucose uptake were diminished by approximately 50% in GK adipocytes, as compared with control adipocytes. This defect in insulin induced PKC-zeta/lambda activation was associated with diminished activation of IRS-1-dependent phosphatidylinositol (PI) 3-kinase, and was accompanied by diminished phosphorylation of threonine 410 in the activation loop of PKC-zeta; in contrast, protein kinase B (PKB) activation and phosphorylation were not significantly altered. Rosiglitazone completely reversed defects in insulin stimulated 2-deoxyglucose uptake, PKCzeta/lambda enzyme activity and PKC-zeta threonine 410 phosphorylation, but had no effect on PI 3-kinase activation or PKB activation/phosphorylation in GK adipocytes. Similarly, in adipocytes of nondiabetic rats, rosiglitazone provoked increases in insulin-stimulated 2 deoxyglucose uptake, PKC-zeta/lambda enzyme activity and phosphorylation of both threonine 410 activation loop and threonine 560 autophosphorylation sites in PKC zeta, but had no effect on PI 3-kinase activation or PKB activation/phosphorylation. Our findings suggest that (a) decreased effects of insulin on glucose transport in adipocytes of GK-diabetic rats are due at least in part to diminished phosphorylation/activation of PKC-zeta/lambda, and (b) thiazolidinediones enhance glucose transport responses to insulin in adipocytes of both diabetic and nondiabetic rats through increases in phosphorylation/activation of PKC-zeta/lambda. PMID- 10749858 TI - Interactions of DnaA proteins from distantly related bacteria with the replication origin of the broad host range plasmid RK2. AB - Replication initiation of the broad host range plasmid RK2 requires binding of the host-encoded DnaA protein to specific sequences (DnaA boxes) at its replication origin (oriV). In contrast to a chromosomal replication origin, which functionally interacts only with the native DnaA protein of the organism, the ability of RK2 to replicate in a wide range of Gram-negative bacterial hosts requires the interaction of oriV with many different DnaA proteins. In this study we compared the interactions of oriV with five different DnaA proteins. DNase I footprint, gel mobility shift, and surface plasmon resonance analyses showed that the DnaA proteins from Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa bind to the DnaA boxes at oriV and are capable of inducing open complex formation, the first step in the replication initiation process. However, DnaA proteins from two Gram-positive bacteria, Bacillus subtilis and Streptomyces lividans, while capable of specifically interacting with the DnaA box sequences at oriV, do not bind stably and fail to induce open complex formation. These results suggest that the inability of the DnaA protein of a host bacterium to form a stable and functional complex with the DnaA boxes at oriV is a limiting step for plasmid host range. PMID- 10749859 TI - Cellular uptake of Clostridium botulinum C2 toxin requires oligomerization and acidification. AB - The actin-ADP-ribosylating binary Clostridium botulinum C2 toxin consists of two individual proteins, the binding/translocation component C2II and the enzyme component C2I. To elicit its cytotoxic action, C2II binds to a receptor on the cell surface and mediates cell entry of C2I via receptor-mediated endocytosis. Here we report that binding of C2II to the surface of target cells requires cleavage of C2II by trypsin. Trypsin cleavage causes oligomerization of the activated C2II (C2IIa) to give SDS-stable heptameric structures, which exhibit a characteristic annular or horseshoe shape and form channels in lipid bilayer membranes. Cytosolic delivery of the enzyme component C2I is blocked by bafilomycin but not by brefeldin A or nocodazole, indicating uptake from an endosomal compartment and requirement of endosomal acidification for cell entry. In the presence of C2IIa and C2I, short term acidification of the extracellular medium (pH 5.4) allows C2I to enter the cytosol directly. Our data indicate that entry of C2 toxin into cells involves (i) activation of C2II by trypsin-cleavage, (ii) oligomerization of cleaved C2IIa to heptamers, (iii) binding of the C2IIa oligomers to the carbohydrate receptor on the cell surface and assembly with C2I, (iv) receptor-mediated endocytosis of both C2 components into endosomes, and finally (v) translocation and release of C2I into the cytosol after acidification of the endosomal compartment. PMID- 10749860 TI - Sfb2p, a yeast protein related to Sec24p, can function as a constituent of COPII coats required for vesicle budding from the endoplasmic reticulum. AB - The COPII coat is required for vesicle budding from the endoplasmic reticulum (ER), and consists of two heterodimeric subcomplexes, Sec23p/Sec24p, Sec13p/Sec31p, and a small GTPase, Sar1p. We characterized a yeast mutant, anu1 (abnormal nuclear morphology) exhibiting proliferated ER as well as abnormal nuclear morphology at the restrictive temperature. Based on the finding that ANU1 is identical to SEC24, we confirmed a temperature-sensitive protein transport from the ER to the Golgi in anu1-1/sec24-20 cells. Overexpression of SFB2, a SEC24 homologue with 56% identity, partially suppressed not only the mutant phenotype of sec24-20 cells but also rescued the SEC24-disrupted cells. Moreover, the yeast two-hybrid assay revealed that Sfb2p, similarly to Sec24p, interacted with Sec23p. In SEC24-disrupted cells rescued by overexpression of SFB2, some cargo proteins were still retained in the ER, while most of the protein transport was restored. Together, these findings strongly suggest that Sfb2p functions as the component of COPII coats in place of Sec24p, and raise the possibility that each member of the SEC24 family of proteins participates directly and/or indirectly in cargo-recognition events with its own cargo specificity at forming ER-derived vesicles. PMID- 10749861 TI - Interaction of neuronal Cdc2-like protein kinase with microtubule-associated protein tau. AB - Neuronal Cdc2-like protein kinase (NCLK), a approximately 58-kDa heterodimer, was isolated from neuronal microtubules (Ishiguro, K., Takamatsu, M., Tomizawa, K., Omori, A., Takahashi, M., Arioka, M., Uchida, T. and Imahori, K. (1992) J. Biol. Chem. 267, 10897-10901). The biochemical nature of NCLK-microtubule association is not known. In this study we found that NCLK is released from microtubules upon microtubule disassembly as a 450-kDa species. The 450-kDa species is an NCLK.tau complex, and NCLK-bound tau is in a nonphosphorylated state. Tau phosphorylation causes NCLK.tau complex dissociation, and phosphorylated tau does not bind to NCLK. In vitro, the Cdk5 subunit of NCLK binds to the microtubule-binding region of tau and NCLK associates with microtubules only in the presence of tau. Our data indicate that in brain extract NCLK is complexed with tau in a tau phosphorylation-dependent manner and that tau anchors NCLK to microtubules. Recently NCLK has been suggested to be aberrantly activated and to hyperphosphorylate tau in Alzheimer's disease brain (Patrick, G. N., Zukerberg, L., Nikolic, M., de la Monte, S., Dikkes, P, and Tsai, L.-H. (1999) Nature 402, 615-622). Our findings may explain why in Alzheimer's disease NCLK specifically hyperphosphorylates tau, although this kinase has a number of protein substrates in the brain. PMID- 10749862 TI - Role of the adenomatous polyposis coli gene product in human cardiac development and disease. AB - Expressed sequence tag (EST) and digital Northern analyses of human fetal, adult, and hypertrophic heart cDNA libraries revealed ESTs with high homology to adenomatosis polyposis coli (APC) and its associated protein, beta-catenin, as well as their differential expression. Thus, we hypothesize that the APC/beta catenin pathway may play a role in cardiac development and disease. Reverse transcriptase-polymerase chain reaction analysis exhibited a higher APC expression in adult compared with fetal and hypertrophic heart but no significant difference in beta-catenin mRNA level. However, beta-catenin protein level was higher in fetal and hypertrophic heart compared with adult heart, suggesting the post-translational regulation of beta-catenin by APC in the cardiovascular system. In vitro antisense inhibition of APC resulted a higher beta-catenin protein expression leading to an incomplete myotube formation, suggesting APC/beta-catenin pathway involvement in myotube development. Western blot analysis further reveals three novel isoforms, APC-F, APC-A, and APC-D, ubiquitously expressed in fetal, adult, and hypertrophic heart, respectively. Isoform switching during development and disease pathogenesis suggests functionally distinct roles for each isoform. These data (i) demonstrate the usefulness of genome-based expression analysis for rapid discovery of differentially expressed genes, (ii) implicate the APC/beta-catenin pathway in the cardiovascular development, and (iii) demonstrate APC isoform switching during cardiac development and disease. PMID- 10749863 TI - Expression of oncogenic epidermal growth factor receptor family kinases induces paclitaxel resistance and alters beta-tubulin isotype expression. AB - Oncogenic transformation confers resistance to chemotherapy through a variety of mechanisms, including suppression of apoptosis, increased drug metabolism, and modification of target proteins. Oncogenic epidermal growth factor receptor family members, including EGFRvIII and HER2, are expressed in a broad spectrum of human malignancies. Cell lines transfected with EGFRvIII and HER2 are more resistant to paclitaxel-mediated cytotoxicity, and tubulin polymerization induced by paclitaxel is suppressed compared with cells expressing wild type epidermal growth factor receptor. Because differential expression of beta-tubulin isotypes has been proposed to modulate paclitaxel resistance, we analyzed beta-tubulin isotypes expressed in cell lines transfected with different oncogenes. EGFRvIII- and HER2-expressing cells demonstrated equivalent total beta-tubulin protein compared with cells transfected with wild type receptor or untransfected controls. EGFRvIII-expressing cells demonstrated increases in class IVa (2.5 fold) and IVb (3.1-fold) mRNA, and HER2-expressing cells showed increases in class IVa (2. 95-fold) mRNA. Expression of oncogenic Ha-Ras did not change class IV RNA levels significantly. Inhibition of EGFRvIII kinase activity using a mutant allele with an inactivating mutation in the kinase domain decreased expression of class IVa by 50% and partially reversed resistance to paclitaxel. Expression of oncogenic epidermal growth factor receptor family members is associated with modulation of both beta-tubulin isotype expression and paclitaxel resistance in cells transformed by expression of the receptor. This effect on tubulin expression may modulate drug resistance in human malignancies that express these oncogenes. PMID- 10749864 TI - Cloning, heterologous expression, and distinct substrate specificity of protein farnesyltransferase from Trypanosoma brucei. AB - Protein prenylation occurs in the protozoan that causes African sleeping sickness (Trypanosoma brucei), and the protein farnesyltransferase appears to be a good target for developing drugs. We have cloned the alpha- and beta-subunits of T. brucei protein farnesyltransferase (TB-PFT) using nucleic acid probes designed from partial amino acid sequences obtained from the enzyme purified from insect stage parasites. TB-PFT is expressed in both bloodstream and insect stage parasites. Enzymatically active TB-PFT was produced by heterologous expression in Escherichia coli. Compared with mammalian protein farnesyltransferases, TB-PFT contains a number of inserts of >25 residues in both subunits that reside on the surface of the enzyme in turns linking adjacent alpha-helices. Substrate specificity studies with a series of 20 peptides SSCALX (where X indicates a naturally occurring amino acid) show that the recombinant enzyme behaves identically to the native enzyme and displays distinct specificity compared with mammalian protein farnesyltransferase. TB-PFT prefers Gln and Met at the X position but not Ser, Thr, or Cys, which are good substrates for mammalian protein farnesyltransferase. A structural homology model of the active site of TB PFT provides a basis for understanding structure-activity relations among substrates and CAAX mimetic inhibitors. PMID- 10749865 TI - Inhibition of calcium release-activated calcium current by Rac/Cdc42-inactivating clostridial cytotoxins in RBL cells. AB - Using large clostridial cytotoxins as tools, the role of Rho GTPases in activation of RBL 2H3 hm1 cells was studied. Clostridium difficile toxin B, which glucosylates Rho, Rac, and Cdc42 and Clostridium sordellii lethal toxin, which glucosylates Rac and Cdc42 but not Rho, inhibited the release of hexosaminidase from RBL cells mediated by the high affinity antigen receptor (FcepsilonRI). Additionally, toxin B and lethal toxin inhibited the intracellular Ca(2+) mobilization induced by FcepsilonRI-stimulation and thapsigargin, mainly by reducing the influx of extracellular Ca(2+). In patch clamp recordings, toxin B and lethal toxin inhibited the calcium release-activated calcium current by about 45%. Calcium release-activated calcium current, the receptor-stimulated Ca(2+) influx, and secretion were inhibited neither by the Rho-ADP-ribosylating C3 fusion toxin C2IN-C3 nor by the actin-ADP-ribosylating Clostridium botulinum C2 toxin. The data indicate that Rac and Cdc42 but not Rho are not only involved in late exocytosis events but are also involved in Ca(2+) mobilization most likely by regulating the Ca(2+) influx through calcium release-activated calcium channels activated via FcepsilonRI receptor in RBL cells. PMID- 10749866 TI - Phosphorylation of the nuclear transport machinery down-regulates nuclear protein import in vitro. AB - We have examined whether signal-mediated nucleocytoplasmic transport can be regulated by phosphorylation of the nuclear transport machinery. Using digitonin permeabilized cell assays to measure nuclear import and export, we found that the phosphatase inhibitors okadaic acid and microcystin inhibit transport mediated by the import receptors importin beta and transportin, but not by the export receptor CRM1. Several lines of evidence, including the finding that transport inhibition is partially reversed by the broad specificity protein kinase inhibitor staurosporine, indicate that transport inhibition is due to elevated phosphorylation of a component of the nuclear transport machinery. The kinases and phosphatases involved in this regulation are present in the permeabilized cells. A phosphorylation-sensitive component of the nuclear transport machinery also is present in permeabilized cells and is most likely a component of the nuclear pore complex. Substrate binding by the importin alpha.beta complex and the association of the complex with the nucleoporins Nup358/RanBP2 and Nup153 are not affected by phosphatase inhibitors, suggesting that transport inhibition by protein phosphorylation does not involve these steps. These results suggest that cells have mechanisms to negatively regulate entire nuclear transport pathways, thus providing a means to globally control cellular activity through effects on nucleocytoplasmic trafficking. PMID- 10749867 TI - Evidence for simultaneous protein interactions between human Rad51 paralogs. AB - In yeast, the Rad51-related proteins include Rad55 and Rad57, which form a heterodimer that interacts with Rad51. Five human Rad51 paralogs have been identified (XRCC2, XRCC3, Rad51B/Rad51L1, Rad51C/Rad51L2, and Rad51D/Rad51L3), and each interacts with one or more of the others. Previously we reported that HsRad51 interacts with XRCC3, and Rad51C interacts with XRCC3, Rad51B, and HsRad51. Here we report that in the yeast two-hybrid system, Rad51D interacts with XRCC2 and Rad51C. No other interactions, including self-interactions, were found, indicating that the observed interactions are specific. The yeast Rad51 interacts with human Rad51 and XRCC3, suggesting Rad51 conservation since the human yeast divergence. Data from yeast three-hybrid experiments indicate that a number of the pairs of interactions between human Rad51 paralogs can occur simultaneously. For example, Rad51B expression enhances the binding of Rad51C to XRCC3 and to HsRad51D, and Rad51C expression allows the indirect interaction of Rad51B with Rad51D. Experiments using 6xHis-tagged proteins in the baculovirus system confirm several of our yeast results, including Rad51B interaction with Rad51D only when Rad51C is simultaneously expressed and Rad51C interaction with XRCC2 only when Rad51D is present. These results suggest that these proteins may participate in one complex or multiple smaller ones. PMID- 10749868 TI - Purification, cloning, and expression of a novel salivary anticomplement protein from the tick, Ixodes scapularis. AB - The alternative pathway of complement is an important defense against pathogens and in tick rejection reactions. The tick Ixodes scapularis is able to feed repeatedly on its natural host and has a salivary anticomplement activity that presumably facilitates feeding. In this study, we purified and then obtained the amino-terminal sequence of the I. scapularis salivary anticomplement (Isac). We found a full-length clone coding for Isac by random screening of a salivary gland cDNA library. Expressing Isac cDNA in COS cells reproduced the activity found in tick saliva, namely, inhibition of rabbit erythrocyte lysis by human serum in the presence of Mg(2+) and EGTA, inhibition of C3b binding to agarose in the presence of Mg(2+) and EGTA, and acceleration of factor Bb uncoupling from the C3 convertase generated by the alternative pathway. Recombinant Isac had no effect on the recalcification time of human platelet-poor plasma or in the classical complement pathway, indicating that it is a specific inhibitor similar to the regulators of complement activation of the alternative pathway such as factor H. Isac, however, has no similarity to any protein in the GenBank(TM) data base, indicating that it is a novel and relatively small (18.5 kDa) anticomplement molecule. PMID- 10749869 TI - CaCo-2 cells treated with Clostridium perfringens enterotoxin form multiple large complex species, one of which contains the tight junction protein occludin. AB - The previous model for the action of Clostridium perfringens enterotoxin (CPE) proposed that (i) CPE binds to host cell receptor(s), forming a small ( approximately 90 kDa) complex, (ii) the small complex interacts with other eucaryotic protein(s), forming a large ( approximately 160 kDa) complex, and (iii) the large complex triggers massive permeability changes, thereby inducing enterocyte death. In the current study, Western immunoblot analysis demonstrated that CPE bound to CaCo-2 human intestinal cells at 37 degrees C forms multiple large complex species, with apparent sizes of approximately 200, approximately 155, and approximately 135 kDa. These immunoblot experiments also revealed that occludin, an approximately 65-kDa tight junction protein, is present in the approximately 200-kDa large complex but absent from the other large complex species. Immunoprecipitation studies confirmed that occludin physically associates with CPE in large complex material and also indicated that occludin is absent from small complex. These results strongly suggest that occludin becomes associated with CPE during formation of the approximately 200-kDa large complex. A postbinding association between CPE and occludin is consistent with the failure of rat fibroblast transfectants expressing occludin to bind CPE in the current study. Those occludin transfectants were also insensitive to CPE, strongly suggesting that occludin expression is not sufficient to confer CPE sensitivity. However, the occludin-containing, approximately 200-kDa large complex may contribute to CPE-induced cytotoxicity, because nontoxic CPE point mutants did not form any large complex species. By showing that large complex material is comprised of several species (one containing occludin), the current studies indicate that CPE action is more complicated than previously appreciated and also provide additional evidence for CPE interactions with tight junction proteins, which could be important for CPE-induced pathophysiology. PMID- 10749870 TI - Altered dermatan sulfate structure and reduced heparin cofactor II-stimulating activity of biglycan and decorin from human atherosclerotic plaque. AB - Biglycan and decorin are small dermatan sulfate-containing proteoglycans in the extracellular matrix of the artery wall. The dermatan sulfate chains are known to stimulate thrombin inhibition by heparin cofactor II (HCII), a plasma proteinase inhibitor that has been detected within the artery wall. The purpose of this study was to analyze the HCII-stimulatory activity of biglycan and decorin isolated from normal human aorta and atherosclerotic lesions type II through VI and to correlate activity with dermatan sulfate chain composition and structure. Biglycan and decorin from plaque exhibited a 24-75% and 38-79% loss of activity, respectively, in thrombin-HCII inhibition assays relative to proteoglycan from normal aorta. A significant negative linear relationship was observed between lesion severity and HCII stimulatory activity (r = 0.79, biglycan; r = 0.63, decorin; p < 0.05). Biglycan, but not decorin, from atherosclerotic plaque contained significantly reduced amounts of iduronic acid and disulfated disaccharides DeltaDi-2,4S and DeltaDi-4,6S relative to proteoglycan from normal artery. Affinity coelectrophoresis analysis of a subset of samples demonstrated that increased interaction of proteoglycan with HCII in agarose gels paralleled increased activity in thrombin-HCII inhibition assays. In conclusion, both biglycan and decorin from atherosclerotic plaque possessed reduced activity with HCII, but only biglycan demonstrated a correlation between activity and specific glycosaminoglycan structural features. Loss of the ability of biglycan and decorin in atherosclerotic lesions to regulate thrombin activity through HCII may be critical in the progression of the disease. PMID- 10749872 TI - Characterization of the human B cell RAG-associated gene, hBRAG, as a B cell receptor signal-enhancing glycoprotein dimer that associates with phosphorylated proteins in resting B cells. AB - Affinity-purified polyclonal antibodies against the hBRAG (human B cell RAG associated gene) protein were generated to characterize hBRAG at the biochemical level. Immunoblotting and immunoprecipitation experiments with these antibody reagents demonstrate that this protein can be expressed in B cells as a membrane integrated glycoprotein disulfide-linked dimer. However, both glycosylated and unglycosylated isoforms of hBRAG are detectable with these reagents. Additionally, their use in cell surface biotinylation and flow cytometry reveals subcellular hBRAG pools both at cell surface and intracellular locations. Co immunoprecipitation experiments with hBRAG antisera detected the association of hBRAG with phosphorylated proteins in resting B cells, including the protein tyrosine kinase Hck, which is subsequently dephosphorylated upon B cell receptor (BCR) ligation. Consistent with its cell surface expression and possible link to BCR signaling, experiments in which alpha-hBRAG antibodies were used to generate early activation signals suggest a modest but specific element of tyrosine phosphorylation occurring through a putative hBRAG receptor. Additional experiments also suggest that hBRAG may be involved in positively enhancing BCR ligation-mediated early activation events. Collectively, these results are consistent with a function for hBRAG as a B cell surface signaling receptor molecule. Coupled with the earlier observation that hBRAG expression correlates with early and late B cell-specific RAG expression, we submit that hBRAG may mediate regulatory signals key to B cell development and/or regulation of B cell specific RAG expression. PMID- 10749871 TI - Crystal structure of a conformation-selective casein kinase-1 inhibitor. AB - Members of the casein kinase-1 family of protein kinases play an essential role in cell regulation and disease pathogenesis. Unlike most protein kinases, they appear to function as constitutively active enzymes. As a result, selective pharmacological inhibitors can play an important role in dissection of casein kinase-1-dependent processes. To address this need, new small molecule inhibitors of casein kinase-1 acting through ATP-competitive and ATP-noncompetitive mechanisms were isolated on the basis of in vitro screening. Here we report the crystal structure of 3-[(2,4,6-trimethoxyphenyl) methylidenyl]-indolin-2-one (IC261), an ATP-competitive inhibitor with differential activity among casein kinase-1 isoforms, in complex with the catalytic domain of fission yeast casein kinase-1 refined to a crystallographic R-factor of 22.4% at 2.8 A resolution. The structure reveals that IC261 stabilizes casein kinase-1 in a conformation midway between nucleotide substrate liganded and nonliganded conformations. We propose that adoption of this conformation by casein kinase-1 family members stabilizes a delocalized network of side chain interactions and results in a decreased dissociation rate of inhibitor. PMID- 10749873 TI - Impaired bone resorption to prostaglandin E2 in prostaglandin E receptor EP4 knockout mice. AB - Prostaglandin E(2) (PGE(2)) acts as a potent stimulator of bone resorption. In this study, we first clarified in normal ddy mice the involvement of protein kinase A and induction of matrix metalloproteinases (MMPs) in PGE(2)-induced bone resorption, and then identified PGE receptor subtype(s) mediating this PGE(2) action using mice lacking each subtype (EP1, EP2, EP3, and EP4) of PGE receptor. In calvarial culture obtained from normal ddy mice, both PGE(2) and dibutyryl cyclic AMP (Bt(2)cAMP) stimulated bone resorption and induced MMPs including MMP 2 and MMP-13. Addition of an inhibitor of protein kinase A, H89, or an inhibitor of MMPs, BB94, significantly suppressed bone-resorbing activity induced by PGE(2.) In calvarial culture from EP1-, EP2-, and EP3-knockout mice, PGE(2) stimulated bone resorption to an extent similar to that found in calvaria from the wild-type mice. On the other hand, a marked reduction in bone resorption to PGE(2) was found in the calvarial culture from EP4-knockout mice. The impaired bone resorption to PGE(2) was also detected in long bone cultures from EP4 knockout mice. Bt(2)cAMP greatly stimulated bone resorption similarly in both wild-type and EP4-knockout mice. Induction of MMP-2 and MMP-13 by PGE(2) was greatly impaired in calvarial culture from EP4-knockout mice, but Bt(2)cAMP stimulated MMPs induction similarly in the wild-type and EP4-knockout mice. These findings suggest that PGE(2) stimulates bone resorption by a cAMP-dependent mechanism via the EP4 receptor. PMID- 10749874 TI - Solvent organization in an oligonucleotide crystal. The structure of d(GCGAATTCG)2 at atomic resolution. AB - We describe the crystal structure of d(GCGAATTCG) determined by x-ray diffraction at atomic resolution level (0.89 A). The duplex structure is practically identical to that described at 2.05 A resolution (Van Meervelt, L., Vlieghe, D., Dautant, A., Gallois, B., Precigoux, G., and Kennard, O. (1995) Nature 374, 742 744), however about half of the phosphate groups show multiple conformations. The crystal has three regions with different solvent structure. One of them contains several ordered Mg(+2) ions and can be considered as an ionic crystal. A second region is formed by a network of ordered water molecules with a polygonal organization that binds three duplexes. The third region is formed by channels of solvent in which very few ordered solvent molecules are visible. The less ordered phosphates are found facing this channel. The latter region provides a view of DNA with highly movable charges, both negative phosphates and counterions, without a precise location. PMID- 10749875 TI - Functional analysis of Plp1 and Plp2, two homologues of phosducin in yeast. AB - Mammalian phosducins are known to bind G protein betagamma subunits in vitro, and are postulated to regulate their signaling function in vivo. Here we describe two homologues of phosducin in yeast, called PLP1 and PLP2. Both gene products were cloned, expressed, and purified as glutathione S-transferase fusions. Of the two isoforms, Plp1 bound most preferentially to Gbetagamma. Binding was enhanced by pheromone stimulation and by the addition of GTPgammaS, conditions that favor dissociation of Gbetagamma from Galpha. Gene disruption mutants and gene overexpression plasmids were prepared and analyzed for changes in signaling and nonsignaling phenotypes. Haploid spore products bearing the plp2Delta mutant failed to grow, suggesting that PLP2 is an essential gene. Cell viability was not restored by a mutation in STE7 that blocks signaling downstream of the G protein. Haploid products bearing the plp1Delta mutant were viable and exhibited a 6-7% increase in pheromone-mediated gene induction. Cells overexpressing PLP1 or PLP2 exhibited a 70-80% decrease in gene induction but no change in pheromone-mediated growth arrest. These data indicate that phosducin can selectively regulate early signaling events following pheromone stimulation and has an essential role in cell growth independent of its regulatory role in cell signaling. PMID- 10749876 TI - Ascorbic acid enhances endothelial nitric-oxide synthase activity by increasing intracellular tetrahydrobiopterin. AB - Ascorbic acid enhances NO bioactivity in patients with vascular disease through unclear mechanism(s). We investigated the role of intracellular ascorbic acid in endothelium-derived NO bioactivity. Incubation of porcine aortic endothelial cells (PAECs) with ascorbic acid produced time- and dose-dependent intracellular ascorbic acid accumulation that enhanced NO bioactivity by 70% measured as A23187 induced cGMP accumulation. This effect was due to enhanced NO production because ascorbate stimulated both PAEC nitrogen oxide (NO(2)(-) + NO(3)(-)) production and l-arginine to l-citrulline conversion by 59 and 72%, respectively, without altering the cGMP response to authentic NO. Ascorbic acid also stimulated the catalytic activity of eNOS derived from either PAEC membrane fractions or baculovirus-infected Sf9 cells. Ascorbic acid enhanced bovine eNOS V(max) by approximately 50% without altering the K(m) for l-arginine. The effect of ascorbate was tetrahydrobiopterin (BH(4))-dependent, because ascorbate was ineffective with BH(4) concentrations >10 microm or in PAECs treated with sepiapterin to increase intracellular BH(4). The effect of ascorbic acid was also specific because A23187-stimulated cGMP accumulation in PAECs was insensitive to intracellular glutathione manipulation and only ascorbic acid, not glutathione, increased the intracellular concentration of BH(4). These data suggest that ascorbic acid enhances NO bioactivity in a BH(4)-dependent manner by increasing intracellular BH(4) content. PMID- 10749877 TI - Expression analysis of BACE2 in brain and peripheral tissues. AB - Beta-site amyloid precursor protein cleaving enzyme (BACE) is a novel transmembrane aspartic protease that possesses all the known characteristics of the beta-secretase involved in Alzheimer's disease (Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., Luo, Y., Fisher, S., Fuller, J., Edenson, S., Lile, J., Jarosinski, M. A., Biere, A. L., Curran, E., Burgess, T., Louis, J. -C., Collins, F., Treanor, J., Rogers, G., and Citron, M. (1999) Science 286, 735 741). We have analyzed the sequence and expression pattern of a BACE homolog termed BACE2. BACE and BACE2 are unique among aspartic proteases in that they possess a carboxyl-terminal extension with a predicted transmembrane region and together they define a new family. Northern analysis reveals that BACE2 mRNA is expressed at low levels in most human peripheral tissues and at higher levels in colon, kidney, pancreas, placenta, prostate, stomach, and trachea. Human adult and fetal whole brain and most adult brain subregions express very low or undetectable levels of BACE2 mRNA. In addition, in situ hybridization of adult rat brain shows that BACE2 mRNA is expressed at very low levels in most brain regions. The very low or undetectable levels of BACE2 mRNA in the brain are not consistent with the expression pattern predicted for beta-secretase. PMID- 10749878 TI - Wnt signaling to beta-catenin involves two interactive components. Glycogen synthase kinase-3beta inhibition and activation of protein kinase C. AB - Wnt signaling involves inhibition of glycogen synthase kinase-3beta (GSK-3beta) and elevation of cytoplasmic beta-catenin. This pathway is essential during embryonic development and oncogenesis. Previous studies on both Xenopus and mammalian cells indicate that lithium mimics Wnt signaling by inactivating GSK 3beta. Here we show that serum enhances accumulation of cytoplasmic beta-catenin induced by lithium in both 293 and C57MG cell lines and that growth factors are responsible for this enhancing activity. Growth factors mediate this effect through activation of protein kinase C (PKC), not through Ras or phosphatidylinositol 3-kinase. In addition, Wnt-induced accumulation of cytoplasmic beta-catenin is partially inhibited by PKC inhibitors and by chronic treatment of cells with phorbol ester. Both calphostin C, a PKC inhibitor, and a dominant negative PKC exhibit partial inhibition on Wnt-mediated transcriptional activation. We therefore propose that Wnt signaling to beta-catenin consists of two interactive components: one involves inhibition of GSK-3beta and is mimicked by lithium, and the other involves PKC and serves to augment the effects of GSK 3beta inhibition. PMID- 10749879 TI - Importance of the major extracellular domain of CD9 and the epidermal growth factor (EGF)-like domain of heparin-binding EGF-like growth factor for up regulation of binding and activity. AB - Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family of growth factors. The membrane-anchored form of HB-EGF (proHB-EGF) is mitogenically active to neighboring cells as well as being a precursor of the soluble form. In addition to its mitogenic activity, proHB-EGF has the property of binding to diphtheria toxin (DT), serving as the specific receptor for DT. Tetramembrane-spanning protein CD9, a member of the TM4 superfamily, is physically associated with proHB-EGF at the cell surface and up regulates both mitogenic and DT binding activities of proHB-EGF. To understand this up-regulation mechanism, we studied essential regions of both CD9 and proHB EGF for up-regulation. Immunoprecipitation experiments revealed that not only CD9 but also other TM4 proteins including CD63, CD81, and CD82 associate with proHB EGF on the cell surface. However, these TM4 proteins did not up-regulate DT binding activity of proHB-EGF. Transfection of a series of chimeric constructs comprising CD9 and CD81 showed that the major extracellular domain of CD9 is essential for up-regulation. Assays of DT binding activity and juxtacrine mitogenic activity of the deletion mutants of proHB-EGF and chimeric molecules, derived from proHB-EGF and TGF-alpha, showed that the essential domain of proHB EGF for up-regulation is the EGF-like domain. These results indicate that the interaction of the extracellular domains of both molecules is important for up regulation. PMID- 10749880 TI - Template requirement and initiation site selection by hepatitis C virus polymerase on a minimal viral RNA template. AB - RNA-dependent RNA polymerase, NS5B protein, catalyzes replication of viral genomic RNA, which presumably initiates from the 3'-end. We have previously shown that NS5B can utilize the 3'-end 98-nucleotide (nt) X region of the hepatitis C virus (HCV) genome as a minimal authentic template. In this study, we used this RNA to characterize the mechanism of RNA synthesis by the recombinant NS5B. We first showed that NS5B formed a complex with the 3'-end of HCV RNA by binding to both the poly(U-U/C)-rich and X regions of the 3'-untranslated region as well as part of the NS5B-coding sequences. Within the X region, NS5B bound stem II and the single-stranded region connecting stem-loops I and II. Truncation of 40 nt or more from the 3'-end of the X region abolished its template activity, whereas X RNA lacking 35 nt or less from the 3'-end retained template activity, consistent with the NS5B-binding site mapped. Furthermore, NS5B initiated RNA synthesis from a specific site within the single-stranded loop I. All of the RNA templates that have a double-stranded stem at the 3'-end had the same RNA initiation site. However, the addition of single-stranded nucleotides to the 3'-end of X RNA or removal of double-stranded structure in stem I generated RNA products of template size. These results indicate that HCV NS5B initiates RNA synthesis from a single stranded region closest to the 3'-end of the X region. These results have implications for the mechanism of HCV RNA replication and the nature of HCV RNA templates in the infected cells. PMID- 10749881 TI - The chemotactic action of urokinase on smooth muscle cells is dependent on its kringle domain. Characterization of interactions and contribution to chemotaxis. AB - Urokinase plasminogen activator (uPA) is thought to exert its effects on cell growth, adhesion, and migration by mechanisms involving proteolysis and interaction with its cell surface receptor (uPAR). The functional properties of uPA and the significance of its various domains for chemotactic activity were analyzed using human airway smooth muscle cells (hAWSMC). The wild-type uPA (r uPAwt), inactive urokinase with single mutation (His(204) to Gln) (r-uPA(H/Q)), urokinase with mutation of His(204) to Gln together with a deletion of growth factor-like domain (r-uPA(H/Q)-GFD), the catalytic domain of urokinase (r uPA(LMW)), and its kringle domain (r-KD) were expressed in Escherichia coli. We demonstrate that glycosylated uPA, r-uPAwt, r-uPA(H/Q), and r-uPA(H/Q)-GFD elicited similar chemotactic effects. Half-maximal chemotaxis (EC(50)) were apparent at approximately 2 nm with all the uPA variants. The kringle domain induced cell migration with an EC(50) of about 6 nm, whereas the denaturated r-KD and r-uPA(LMW) were without effect. R-uPAwt-induced chemotaxis was dependent on an association with uPAR and a uPA-kringle domain-binding site, determined using a monoclonal uPAR antibody to prevent the uPA-uPAR interaction, and a monoclonal antibody to the uPA-kringle domain. The binding of iodinated r-uPAwt with hAWSMC was due to interaction with a high affinity binding site on the uPAR, and a lower affinity binding site on an unidentified cell surface target, which was mediated exclusively through the kringle domain of urokinase. Specific binding of r uPA(H/Q)-GFD to hAWSMC involved an interaction with a single site whose characteristics were similar to those of the low affinity site of r-uPAwt binding to hAWSMC. uPAR-deficient HEK 293 cells specifically bound r-uPAwt and r-uPA(H/Q) GFD via a single, similar type of binding site. These cells migrated when stimulated by r-uPA(H/Q)-GFD and uPAwt, but not r-uPA(LMW). HEK 293 cells transfected with the uPAR cDNA expressed two classes of sites that bound r-uPAwt; however, only a single site was responsible for the binding of r-uPA(H/Q)-GFD. Together, these findings indicate that uPA-induced chemotaxis is dependent on the binding of the uPA-kringle to the membrane surface of cells and the association of uPA with uPAR. PMID- 10749882 TI - A role for focal adhesion kinase in phenylephrine-induced hypertrophy of rat ventricular cardiomyocytes. AB - A variety of agonists including phenylephrine (PE) induce hypertrophy in neonatal ventricular cardiomyocytes. Here we report that signals provided by extracellular matrix proteins (ECM) augment the PE-induced hypertrophic response of cardiomyocytes and provide evidence that ECM-dependent signaling is mediated in part by the protein tyrosine kinase, focal adhesion kinase (FAK). Addition of PE to cultured neonatal cardiomyocytes stimulated sarcomeric organization, increased cell size, and induced atrial natriuretic factor in cardiomyocytes plated on the ECM protein laminin or fibronectin. In contrast, cardiomyocytes plated on the non adhesive substrate gelatin exhibited a reduced capacity to undergo these PE stimulated hypertrophic changes. In cardiomyocytes cultured on ECM, PE stimulated a rapid increase in tyrosine phosphorylation of focal adhesion proteins including FAK, paxillin, and p130 Crk-associated substrate and subsequent formation of peripheral focal complexes. Inhibition of the PE-induced hypertrophic response by genistein and herbimycin-A indicated a requirement for protein tyrosine kinases in PE signaling. To determine whether activation of FAK is required for PE induced hypertrophy, a dominant-interfering mutant form of FAK, termed FRNK (FAK related non-kinase), was ectopically expressed in cardiomyocytes using a replication-defective adenovirus expression system. FRNK expression attenuated PE stimulated hypertrophy as assessed by cell size, sarcomeric organization, and induction of atrial natriuretic factor. These data indicate that the signal transduction pathways leading to cardiomyocyte hypertrophy are strongly influenced by and/or dependent upon an integrin-mediated signaling process requiring FAK. PMID- 10749883 TI - Phosphatidylinositol 3-kinase activity regulates alpha -thrombin-stimulated G1 progression by its effect on cyclin D1 expression and cyclin-dependent kinase 4 activity. AB - In this study, we present evidence that PI 3-kinase is required for alpha thrombin-stimulated DNA synthesis in Chinese hamster embryonic fibroblasts (IIC9 cells). Previous results from our laboratory demonstrate that the mitogen activated protein kinase (extracellular signal-regulated kinase (ERK)) pathway controls transit through G(1) phase of the cell cycle by regulating the induction of cyclin D1 mRNA levels and cyclin dependent kinase 4 (CDK4)-cyclin D1 activity. In IIC9 cells, PI 3-kinase activation also is an important controller of the expression of cyclin D1 protein and CDK4-cyclin D1 activity. Pretreatment of IIC9 cells with the selective PI 3-kinase inhibitor, LY294002 blocks the alpha thrombin-stimulated increase in cyclin D1 protein and CDK4 activity. However, LY294002 does not affect alpha-thrombin-induced cyclin D1 steady state message levels, indicating that PI 3-kinase acts independent of the ERK pathway. Interestingly, expression of a dominant-negative Ras significantly decreased both alpha-thrombin-stimulated ERK and PI 3-kinase activities. These data clearly demonstrate that the alpha-thrombin-induced Ras activation coordinately regulates ERK and PI 3-kinase activities, both of which are required for expression of cyclin D1 protein and progression through G(1). PMID- 10749884 TI - Regulation of FGF-3 gene expression in tumorigenic and non-tumorigenic clones of a human colon carcinoma cell line. AB - The FGF-3 gene is constitutively expressed in tumorigenic clones from the SW613-S human colon carcinoma cell line but is silent in non-tumorigenic clones. We have investigated the transcriptional mechanisms responsible for this differential expression. Mapping of DNase I-hypersensitive sites throughout the FGF-3 gene and the region extending 15 kilobases upstream disclosed differences in the patterns obtained between tumorigenic and non-tumorigenic cells. Transient expression assays carried out with a reporter gene driven by FGF-3 promoter fragments of various lengths (0.143 to 11 kilobases) did not reproduce the differential regulation of the resident gene between the two cell types. The same constructs did exhibit a differential activity in stable transfectants, suggesting the involvement of a chromatin-based mechanism in this regulation. Under these conditions, even the 143-base pair minimal promoter fragment was able to drive the differential expression of the reporter gene. During the course of these analyses, several transcriptional modulatory elements (mainly activators) were identified in the FGF-3 upstream region and were found to colocalize with DNase I hypersensitive sites. Moreover, a putative new promoter was discovered 6 kilobases upstream of FGF-3. Altogether, these data provide a basis for the elucidation of the complex regulation of the human FGF-3 gene. PMID- 10749885 TI - Gene 33/Mig-6, a transcriptionally inducible adapter protein that binds GTP-Cdc42 and activates SAPK/JNK. A potential marker transcript for chronic pathologic conditions, such as diabetic nephropathy. Possible role in the response to persistent stress. AB - Chronic stresses, including the mechanical strain caused by hypertension or excess pulmonary ventilation pressure, lead to important clinical consequences, including hypertrophy and acute respiratory distress syndrome. Pathologic hypertrophy contributes to decreased organ function and, ultimately, organ failure; and cardiac and diabetic renal hypertrophy are major causes of morbidity and morality in the developed world. Likewise, acute respiratory distress syndrome is a serious potential side effect of mechanical pulmonary ventilation. Whereas the deleterious effects of chronic stress are well established, the molecular mechanisms by which these stresses affect cell function are still poorly characterized. gene 33 (also called mitogen-inducible gene-6, mig-6) is an immediate early gene that is transcriptionally induced by a divergent array of extracellular stimuli. The physiologic function of Gene 33 is unknown. Here we show that gene 33 mRNA levels increase sharply in response to a set of commonly occurring chronic stress stimuli: mechanical strain, vasoactive peptides, and diabetic nephropathy. Induction of gene 33 requires the stress-activated protein kinases (SAPKs)/c-Jun NH(2)-terminal kinases. This expression pattern suggests that gene 33 is a potential marker for diabetic nephropathy and other pathologic responses to persistent sublethal stress. The structure of Gene 33 indicates an adapter protein capable of binding monomeric GTPases of the Rho subfamily. Consistent with this, Gene 33 interacts in vivo and, in a GTP-dependent manner, in vitro with Cdc42Hs; and transient expression of Gene 33 results in the selective activation of the SAPKs. These results imply a reciprocal, positive feedback relationship between Gene 33 expression and SAPK activation. Expression of Gene 33 at sufficient levels may enable a compensatory reprogramming of cellular function in response to chronic stress, which may have pathophysiological consequences. PMID- 10749886 TI - Regulator of G protein signaling RGS3T is localized to the nucleus and induces apoptosis. AB - RGS3 belongs to a family of the regulators of G protein signaling (RGS). We previously demonstrated that cytosolic RGS3 translocates to the membrane to inhibit G(q/11) signaling (Dulin, N. O., Sorokin, A., Reed, E., Elliott, S., Kehrl, J., and Dunn, M. J. (1999) Mol. Cell. Biol. 19, 714-723). This study examines the properties of a recently identified truncated variant termed RGS3T. Both RGS3 and RGS3T bound to endogenous Galpha(q/11) and inhibited endothelin-1 stimulated calcium mobilization and mitogen-activated protein kinase activity to a similar extent. However, unlike cytosolically localized RGS3, RGS3T was found predominantly in the nucleus and partially in the plasma membrane. Furthermore, RGS3T, but not RGS3, caused cell rounding and membrane blebbing. Finally, 44% of RGS3T-transfected cells underwent apoptosis after serum withdrawal, which was significantly higher than that of RGS3-transfected cells (7%). Peptide sequence analysis revealed two potential nuclear localization signal (NLS) sequences in RGS3T. Further truncation of the RGS3T N terminus containing putative NLSs resulted in a significant reduction of nuclear versus cytoplasmic staining of the protein. Moreover, this truncated RGS3T no longer induced apoptosis. In summary, RGS3 and its truncated variant RGS3T are similar in their ability to inhibit G(q/11) signaling but are different in their intracellular distribution. These data suggest that, in addition to being a GTPase-activating protein, RGS3T has other distinct functions in the nucleus of the cell. PMID- 10749887 TI - A novel cytokine receptor-ligand pair. Identification, molecular characterization, and in vivo immunomodulatory activity. AB - As part of a large scale effort to discover novel secreted proteins, a cDNA encoding a novel cytokine was identified. Alignments of the sequence of the new protein, designated IL-17B, suggest it to be a homolog of the recently described T cell-derived cytokine, IL-17. By Northern analysis, EST distribution and real time quantitative polymerase chain reaction analysis, mRNA was detected in many cell types. A novel type I transmembrane protein, identified in an EST data base by homology to IL-17R, was found to bind specifically IL-17B, as determined by surface plasmon resonance analysis, flow cytometry, and co-immunoprecipitation experiments. Readily detectable transcription of IL-17BR was restricted to human kidney, pancreas, liver, brain, and intestines and only a few of the many cell lines tested. By using a rodent ortholog of IL-17BR as a probe, IL-17BR message was found to be drastically up-regulated during intestinal inflammation elicited by indomethacin treatment in rats. In addition, intraperitoneal injection of IL 17B purified from Chinese hamster ovary cells caused marked neutrophil migration in normal mice, in a specific and dose-dependent manner. Together these results suggest that IL-17B may be a novel proinflammatory cytokine acting on a restricted set of target cell types. They also demonstrate the strength of genomic approaches in the unraveling of novel biological pathways. PMID- 10749889 TI - Estrogen receptor alpha rapidly activates the IGF-1 receptor pathway. AB - Estrogen and insulin-like-growth factor 1 (IGF-1) are potent mitogenic stimuli that share important properties in the control of cellular proliferation. However, the coupling between the signaling cascades of estrogen receptors alpha and beta and the IGF-1 receptor (IGF-1R) is poorly understood. Therefore, we selectively transfected estrogen receptor alpha or beta in COS7 and HEK293 cells, which contain IGF-1R. In presence of estrogen receptor alpha but not beta, 17beta estradiol (E2) rapidly induces phosphorylation of the IGF-1R and the extracellular signal-regulated kinases 1/2. Furthermore, upon stimulation with E2, estrogen receptor alpha but not beta bound rapidly to the IGF-1R in COS7 as well as L6 cells, which express all investigated receptors endogenously. Control experiments in the IGF-1R-deficient fibroblast cell line R(-) showed that after stimulation with E2 only estrogen receptor alpha bound to the transfected IGF-1R. Overexpression of dominant negative mitogen-activated protein kinases kinase inhibited this effect. Finally, estrogen receptor alpha but not beta is required to induce the activation of the estrogen receptor-responsive reporter ERE-LUC in IGF-1-stimulated cells. Taken together, these data demonstrate that ligand bound estrogen receptor alpha is required for rapid activation of the IGF-1R signaling cascade. PMID- 10749890 TI - The high resolution crystal structure of yeast hexokinase PII with the correct primary sequence provides new insights into its mechanism of action. AB - Hexokinase is the first enzyme in the glycolytic pathway, catalyzing the transfer of a phosphoryl group from ATP to glucose to form glucose 6-phosphate and ADP. Two yeast hexokinase isozymes are known, namely PI and PII. The crystal structure of yeast hexokinase PII from Saccharomyces cerevisiae without substrate or competitive inhibitor is determined and refined in a tetragonal crystal form at 2.2-A resolution. The folding of the peptide chain is very similar to that of Schistosoma mansoni and previous yeast hexokinase models despite only 30% sequence identity between them. Distinct differences in conformation are found that account for the absence of glucose in the binding site. Comparison of the current model with S. mansoni and yeast hexokinase PI structures both complexed with glucose shows in atomic detail the rigid body domain closure and specific loop movements as glucose binds. A hydrophobic channel formed by strictly conserved hydrophobic residues in the small domain of the hexokinase is identified. The channel's mouth is close to the active site and passes through the small domain to its surface. The possible role of the observed channel in proton transfer is discussed. PMID- 10749891 TI - Mitotic clonal expansion during preadipocyte differentiation: calpain-mediated turnover of p27. AB - Evidence is presented that calpain, a calcium-activated protease, degrades the cyclin-dependent kinase inhibitor, p27, during the mitotic clonal expansion phase of 3T3-L1 preadipocyte differentiation. Calpain activity is required during an early stage of the adipocyte differentiation program. Thus, inhibition of calpain with N-acetyl-Leu-Leu-norleucinal (ALLN) blocks clonal expansion and acquisition of the adipocyte phenotype only when added between 12 and 24 h after the induction of differentiation. Likewise, inhibition of calpain by overexpression of calpastatin, the specific endogenous inhibitor of calpain, prevents 2-day post confluent preadipocytes from reentering the cell cycle triggered by the differentiation inducers. Inhibition of calpain with ALLN causes preadipocytes to arrest just prior to S phase and prevents phosphorylation of the retinoblastoma gene product, DNA replication, clonal expansion, and subsequent adipocyte differentiation but does not affect the expression of immediate early genes (i.e. fos, jun, C/EBPbeta, and C/EBPdelta). Inhibition of calpain by either ALLN or by overexpression of calpastatin blocks the degradation of p27. p27 is degraded in vitro by cell-free extracts from clonally expanding preadipocytes that contain "active" calpain but not by extracts from pre-mitotic preadipocytes that do not. This action is inhibited by calpastatin or ALLN. Likewise, p27 in preadipocyte extracts is a substrate for purified calpain; this proteolytic action was inhibited by heat inactivation, EGTA, or ALLN. Thus, extracellular signals from the differentiation inducers appear to activate calpain, which degrades p27 allowing density-dependent inhibited preadipocytes to reenter the cell cycle and undergo mitotic clonal expansion. PMID- 10749895 TI - Microsatellite instability, clinical implications, and new methodologies. PMID- 10749896 TI - Improving the cost-effectiveness of colorectal cancer screening. PMID- 10749897 TI - BRCA1 and E-cadherin promoter hypermethylation and gene inactivation in cancer association or mechanism? PMID- 10749898 TI - Tinker with our genetic future? Not yet, say experts. PMID- 10749899 TI - Antiangiogenesis drugs target specific cancers, mechanisms. PMID- 10749901 TI - Several angiogenesis inhibitors show potential in Lab PMID- 10749902 TI - Anti-smoking effort seeks global implementation. PMID- 10749903 TI - From crayons to computers: mapping cancer moves on. PMID- 10749904 TI - Multinational clinical trials click forward. PMID- 10749900 TI - Noncancer uses for angiogenesis inhibitors PMID- 10749906 TI - Correction PMID- 10749905 TI - Stat bite: All-cancer incidence in selected European countries, 1995. PMID- 10749907 TI - National Cancer Institute Workshop Report: the phakomatoses revisited. PMID- 10749908 TI - Alternate ranging methods for cancer mortality maps. AB - BACKGROUND: Mapping techniques can highlight the spatial or temporal variations in rates of cancer mortality. In mapping geographic patterns of cancer mortality, spatial units are grouped into categories defined by specified rate ranges, and then the units in each category are assigned a particular color in the map. We examined the consequences of using different ranging methods when comparing maps over several time intervals. METHODS: Maps of mortality rates for cancers of the breast, lung (including the lung, trachea, bronchus, and pleura), and cervix uteri in the United States by county or state economic area are created for different time intervals between 1950 and 1994. Two ranging methods are employed: 1) Ranges are defined for individual time interval by the deciles of rates in that interval (ranging within intervals), and 2) constant ranges for all time intervals are defined by the deciles of rates for the entire 45-year period from 1950 through 1994 (ranging across intervals). The time intervals from 1950 through 1969 and from 1970 through 1994 were chosen to accommodate the availability of detailed county-level population estimates specifically for blacks starting in 1970. RESULTS: The ranging method has little impact on maps for breast cancer mortality, which changed little over time. For lung cancer, which increased over time, and cervix uteri cancer, which decreased over time, ranging within time intervals shows the geographic variability but does not convey the temporal trends. Trends are evident when ranging across time intervals is employed; however, geographic variability is partially obscured by the predominance of spatial units in the highest rate categories in the recent time intervals for lung cancer and in the early time intervals for cervix uteri cancer. CONCLUSIONS: Ranging within time intervals displays geographic patterns and changes in geographic patterns, regardless of time trends in rates. Ranging across time intervals shows temporal changes in rates but with some loss of information about geographic variability. PMID- 10749909 TI - Standardized approach for microsatellite instability detection in colorectal carcinomas. AB - BACKGROUND: Ubiquitous mutations in microsatellite DNA sequences define a specific type of genetic instability, termed microsatellite instability (MSI). Various approaches have been used to identify the presence and degree of MSI. To define standard diagnostic criteria for MSI, we developed and tested a mathematical model. METHODS: We designed an algorithm for the efficient characterization of MSI and used it to analyze data on six microsatellite markers in colorectal carcinoma and normal tissues from 415 patients. Theoretical models considering one, two, or three populations were tested against the data collected. RESULTS: The observed frequencies of MSI in our series of samples best fit a two-population model, stable and unstable, defined by instability in two or more of four to six markers analyzed. MSI was observed in 7.5% of the tumors. The misclassification rate was less than 5% when any four loci were analyzed and less than 1% when the six markers were used. A stepwise strategy, consisting first of a bulk screening of two loci and then a second screening of two to four additional markers, provided excellent sensitivity (>/=97%) and specificity (100%). Tumors with MSI had distinctive genetic and clinicopathologic features, including better patient survival. CONCLUSION: To assess the presence of MSI in colorectal cancer, we have developed a simple, sensitive, and specific approach based on the apparent good fit of the data to a two-population model. Its application to a prospective series of patients with colorectal carcinomas demonstrates that the presence of MSI characterizes a subset of less aggressive tumors. PMID- 10749910 TI - Tumor characteristics and clinical outcome of elderly women with breast cancer. AB - BACKGROUND: The number of elderly patients with breast cancer is increasing. Limited age-related information available about this disease prompted this study. PATIENTS AND METHODS: The study population was derived from 50828 and 256287 patients with invasive breast cancer in San Antonio breast cancer databases and the Surveillance, Epidemiology, and End Results (SEER) registry, respectively. Tumor biologic and clinical characteristics, local and systemic therapies, and survival according to the patient's age were analyzed. Survival was also compared with that of age-matched women from the general population. RESULTS: In patients 55 years old or older, there was an association between increasing age at diagnosis and the presence of more favorable biologic characteristics of the tumor, including more tumors that express steroid receptors, lower proliferative rates, diploidy, normal p53, and absence of the expression of epidermal growth factor receptor and c-erbB2. In older patients with lymph node-negative disease and/or small tumors, the observed and expected survivals were almost identical. In the SEER registry, the 8-year survival of lymph node-negative patients relative to the expected survival of age-matched women from the general population was 1.01 (95% confidence interval [CI] = 0.98-1. 04) for patients 70 74 years old, 1.06 (95% CI = 1.01-1.11) for patients 75-79 years old, and 1.09 (95% CI = 0.98-1.20) for patients 80-84 years old. CONCLUSION: In women 55 years old or older, advancing age is associated with more favorable tumor biology, and breast cancer survival in older women is similar to survival in the general population irrespective of disease status. This favorable outcome should be considered when making clinical decisions in older patients. PMID- 10749911 TI - Endoscopic colorectal cancer screening: a cost-saving analysis. AB - BACKGROUND: Comprehensive analyses have shown that screening for cancer usually induces net costs. In this study, the possible costs and savings of endoscopic colorectal cancer screening are explored to investigate whether the induced savings may compensate for the costs of screening. METHODS: A simulation model for evaluation of colorectal cancer screening, MISCAN-COLON, is used to predict costs and savings for the U.S. population, assuming that screening is performed during a period of 30 years. Plausible baseline parameter values of epidemiology, natural history, screening test characteristics, and unit costs are based on available data and expert opinion. Important parameters are varied to extreme but plausible values. RESULTS: Given the expert opinion-based assumptions, a program based on every 5-year sigmoidoscopy screenings could result in a net savings of direct health care costs due to prevention of cancer treatment costs that compensate for the costs of screening, diagnostic follow-up, and surveillance. This result persists when costs and health effects are discounted at 3%. The "break-even" point, the time required before savings exceed costs, is 35 years for a screening program that terminates after 30 years and 44 years for a screening program that continues on indefinitely. However, net savings increase or turn into net costs when alternative assumptions about natural history of colorectal cancer, costs of screening, surveillance, and diagnostics are considered. CONCLUSIONS: Given the present, limited knowledge of the disease process of colorectal cancer, test characteristics, and costs, it may well be that the induced savings by endoscopic colorectal cancer screening completely compensate for the costs. PMID- 10749912 TI - Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors. AB - BACKGROUND: Inherited mutations in the BRCA1 gene may be responsible for almost half of inherited breast carcinomas. However, somatic (acquired) mutations in BRCA1 have not been reported, despite frequent loss of heterozygosity (LOH or loss of one copy of the gene) at the BRCA1 locus and loss of BRCA1 protein in tumors. To address whether BRCA1 may be inactivated by pathways other than mutations in sporadic tumors, we analyzed the role of hypermethylation of the gene's promoter region. METHODS: Methylation patterns in the BRCA1 promoter were assessed in breast cancer cell lines, xenografts, and 215 primary breast and ovarian carcinomas by methylation-specific polymerase chain reaction (PCR). BRCA1 RNA expression was determined in cell lines and seven xenografts by reverse transcription-PCR. P values are two-sided. RESULTS: The BRCA1 promoter was found to be unmethylated in all normal tissues and cancer cell lines tested. However, BRCA1 promoter hypermethylation was present in two breast cancer xenografts, both of which had loss of the BRCA1 transcript. BRCA1 promoter hypermethylation was present in 11 (13%) of 84 unselected primary breast carcinomas. BRCA1 methylation was strikingly associated with the medullary (67% methylated; P =.0002 versus ductal) and mucinous (55% methylated; P =.0033 versus ductal) subtypes, which are overrepresented in BRCA1 families. In a second series of 66 ductal breast tumors informative for LOH, nine (20%) of 45 tumors with LOH had BRCA1 hypermethylation, while one (5%) of 21 without LOH was methylated (P =.15). In ovarian neoplasms, BRCA1 methylation was found only in tumors with LOH, four (31%) of 13 versus none of 18 without LOH (P =.02). The BRCA1 promoter was unmethylated in other tumor types. CONCLUSION: Silencing of the BRCA1 gene by promoter hypermethylation occurs in primary breast and ovarian carcinomas, especially in the presence of LOH and in specific histopathologic subgroups. These findings support a role for this tumor suppressor gene in sporadic breast and ovarian tumorigenesis. PMID- 10749913 TI - E-Cadherin gene promoter hypermethylation in primary human gastric carcinomas. AB - BACKGROUND: E (epithelial)-cadherin, the cell adhesion molecule also considered a potential invasion/metastasis suppressor, is mutationally inactivated in nearly half of all undifferentiated-scattered (diffuse-type) gastric carcinomas. In addition, silencing of E-cadherin by CpG methylation within its promoter region has been reported in several gastric carcinoma cell lines. We investigated the methylation status of the E-cadherin promoter region in 53 primary human gastric carcinomas. METHODS: Hypermethylation of the E-cadherin promoter was determined by utilizing methylation-specific polymerase chain reaction (PCR)-single-strand conformation polymorphism (MSP-SSCP) analysis followed by direct sequencing of PCR products. Expression of E-cadherin was studied by western blot analysis. All statistical tests were two-sided. RESULTS: Hypermethylation of the E-cadherin promoter was evident in 27 (51%) of 53 primary gastric carcinomas examined by MSP SSCP. It occurred more frequently in carcinomas of the undifferentiated-scattered type (in 15 [83%] of 18) than in other histologic subtypes (in 12 [34%] of 35) (P =.0011, Fisher's exact test), and it was present at similar rates in early (in six [60%] of 10) versus advanced (in 21 [49%] of 43) carcinomas (P =.73, Fisher's exact test). Methylation occurring at all cytosine-guanosine sequences (CpGs) near the transcriptional start site was confirmed in six of six tumors examined by bisulfite-DNA sequencing, including two early gastric carcinomas. In addition, loss or diminished expression of E-cadherin was confirmed by western blotting in four of the six tumor tissues demonstrating hypermethylation. CONCLUSIONS: The E cadherin promoter frequently undergoes hypermethylation in human gastric cancers, particularly those of the undifferentiated-scattered histologic subtype. E cadherin promoter hypermethylation is associated with decreased expression and may occur early in gastric carcinogenesis. PMID- 10749914 TI - Response PMID- 10749915 TI - Re: Risk/benefit assessment of tamoxifen to prevent breast cancer-still a work in progress? PMID- 10749916 TI - RESPONSE: re: Risk/Benefit assessment of tamoxifen to prevent breast cancer-still a work in progress? PMID- 10749917 TI - Salvage therapy with temozolomide in an immunocompetent patient with primary brain lymphoma. PMID- 10749918 TI - Functional interactions between the p35 subunit of the Arp2/3 complex and calmodulin in yeast. AB - The end9-1 (arc35-1) mutant was identified as an endocytosis mutant and is a mutant allele of ARC35 that encodes a subunit of the Arp2/3 complex. As for other mutants in the Arp2/3 complex, arc35-1 is defective for endocytosis and organization of the actin cytoskeleton. Both defects can be suppressed by overexpression of calmodulin. Analysis of a collection of temperature-sensitive cmd1 mutants for their ability to suppress either the endocytic defect and/or the actin defect indicates that the two defects are tightly coupled. We demonstrate that Arc35p and Cmd1p interact and that Arc35p is required for cortical localization of calmodulin. This is the first report linking Arp2/3 complex function with calmodulin through which it exercises at least one of its endocytic functions. PMID- 10749919 TI - Activation of B-Raf and regulation of the mitogen-activated protein kinase pathway by the G(o) alpha chain. AB - Many receptors coupled to the pertussis toxin-sensitive G(i/o) proteins stimulate the mitogen-activated protein kinase (MAPK) pathway. The role of the alpha chains of these G proteins in MAPK activation is poorly understood. We investigated the ability of Galpha(o) to regulate MAPK activity by transient expression of the activated mutant Galpha(o)-Q205L in Chinese hamster ovary cells. Galpha(o)-Q205L was not sufficient to activate MAPK but greatly enhanced the response to the epidermal growth factor (EGF) receptor. This effect was not associated with changes in the state of tyrosine phosphorylation of the EGF receptor. Galpha(o) Q205L also potentiated MAPK stimulation by activated Ras. In Chinese hamster ovary cells, EGF receptors activate B-Raf but not Raf-1 or A-Raf. We found that expression of activated Galpha(o) stimulated B-Raf activity independently of the activation of the EGF receptor or Ras. Inactivation of protein kinase C and inhibition of phosphatidylinositol-3 kinase abolished both B-Raf activation and EGF receptor-dependent MAPK stimulation by Galpha(o). Moreover, Galpha(o)-Q205L failed to affect MAPK activation by fibroblast growth factor receptors, which stimulate Raf-1 and A-Raf but not B-Raf activity. These results suggest that Galpha(o) can regulate the MAPK pathway by activating B-Raf through a mechanism that requires a concomitant signal from tyrosine kinase receptors or Ras to efficiently stimulate MAPK activity. Further experiments showed that receptor mediated activation of Galpha(o) caused a B-Raf response similar to that observed after expression of the mutant subunit. The finding that Galpha(o) induces Ras independent and protein kinase C- and phosphatidylinositol-3 kinase-dependent activation of B-Raf and conditionally stimulates MAPK activity provides direct evidence for intracellular signals connecting this G protein subunit to the MAPK pathway. PMID- 10749920 TI - The lipid-anchored ectodomain of influenza virus hemagglutinin (GPI-HA) is capable of inducing nonenlarging fusion pores. AB - GPI-linked hemagglutinin (GPI-HA) of influenza virus was thought to induce hemifusion without pore formation. Cells expressing either HA or GPI-HA were bound to red blood cells, and their fusion was compared by patch-clamp capacitance measurements and fluorescence microscopy. It is now shown that under more optimal fusion conditions than have been used previously, GPI-HA is also able to induce fusion pore formation before lipid dye spread, although with fewer pores formed than those induced by HA. The GPI-HA pores did not enlarge substantially, as determined by the inability of a small aqueous dye to pass through them. The presence of 1,1'-dioctadecyl-3, 3,3',3' tetramethylindocarbocyanine perchlorate or octadecylrhodamine B in red blood cells significantly increased the probability of pore formation by GPI-HA; the dyes affected pore formation to a much lesser degree for HA. This greater sensitivity of pore formation to lipid composition suggests that lipids are a more abundant component of a GPI-HA fusion pore than of an HA pore. The finding that GPI-HA can induce pores indicates that the ectodomain of HA is responsible for all steps up to the initial membrane merger and that the transmembrane domain, although not absolutely required, ensures reliable pore formation and is essential for pore growth. GPI-HA is the minimal unit identified to date that supports fusion to the point of pore formation. PMID- 10749921 TI - A novel helicase-type protein in the nucleolus: protein NOH61. AB - We report the identification, cDNA cloning, and molecular characterization of a novel, constitutive nucleolar protein. The cDNA-deduced amino acid sequence of the human protein defines a polypeptide of a calculated mass of 61.5 kDa and an isoelectric point of 9.9. Inspection of the primary sequence disclosed that the protein is a member of the family of "DEAD-box" proteins, representing a subgroup of putative ATP-dependent RNA helicases. ATPase activity of the recombinant protein is evident and stimulated by a variety of polynucleotides tested. Immunolocalization studies revealed that protein NOH61 (nucleolar helicase of 61 kDa) is highly conserved during evolution and shows a strong accumulation in nucleoli. Biochemical experiments have shown that protein NOH61 synthesized in vitro sediments with approximately 11.5 S, i.e., apparently as homo-oligomeric structures. By contrast, sucrose gradient centrifugation analysis of cellular extracts obtained with buffers of elevated ionic strength (600 mM NaCl) revealed that the solubilized native protein sediments with approximately 4 S, suggestive of the monomeric form. Interestingly, protein NOH61 has also been identified as a specific constituent of free nucleoplasmic 65S preribosomal particles but is absent from cytoplasmic ribosomes. Treatment of cultured cells with 1) the transcription inhibitor actinomycin D and 2) RNase A results in a complete dissociation of NOH61 from nucleolar structures. The specific intracellular localization and its striking sequence homology to other known RNA helicases lead to the hypothesis that protein NOH61 might be involved in ribosome synthesis, most likely during the assembly process of the large (60S) ribosomal subunit. PMID- 10749922 TI - Multistep phosphorelay proteins transmit oxidative stress signals to the fission yeast stress-activated protein kinase. AB - In response to oxidative stress, eukaryotic cells induce transcription of genes required for detoxification of oxidants. Here we present evidence that oxidative stress stimuli are transmitted by a multistep phosphorelay system to the Spc1/Sty1 stress-activated protein kinase in the fission yeast Schizosaccharomyces pombe. The fission yeast mpr1(+) gene encodes a novel protein with a histidine-containing phosphotransfer domain homologous to the budding yeast Ypd1. Spc1 activation upon oxidative stress is severely impaired in the Deltampr1 mutant as well as in the mpr1HQ strain, in which the putative phosphorylation site Mpr1-His221 is substituted with glutamine. In response to oxidative stress, Mpr1 binds to the Mcs4 response regulator that functions upstream of the Spc1 cascade, suggesting that Mcs4 is a cognate response regulator for Mpr1. Unexpectedly, when exposed to hydrogen peroxide, Deltampr1 cells can induce the catalase gene ctt1(+), one of the transcriptional targets of the Spc1 pathway, and survive oxidative stress in the absence of significant Spc1 activation. We have found that Pap1, a bZIP transcription factor homologous to human c-Jun, can mediate induction of ctt1(+) expression upon oxidative stress independently of the Spc1 stress-activated protein kinase. These studies show that oxidative stress stimuli are transmitted by multiple pathways to induce specific gene expression. PMID- 10749923 TI - Leishmania mexicana mutants lacking glycosylphosphatidylinositol (GPI):protein transamidase provide insights into the biosynthesis and functions of GPI-anchored proteins. AB - The major surface proteins of the parasitic protozoon Leishmania mexicana are anchored to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. We have cloned the L. mexicana GPI8 gene that encodes the catalytic component of the GPI:protein transamidase complex that adds GPI anchors to nascent cell surface proteins in the endoplasmic reticulum. Mutants lacking GPI8 (DeltaGPI8) do not express detectable levels of GPI-anchored proteins and accumulate two putative protein-anchor precursors. However, the synthesis and cellular levels of other non-protein-linked GPIs, including lipophosphoglycan and a major class of free GPIs, are not affected in the DeltaGPI8 mutant. Significantly, the DeltaGPI8 mutant displays normal growth in liquid culture, is capable of differentiating into replicating amastigotes within macrophages in vitro, and is infective to mice. These data suggest that GPI-anchored surface proteins are not essential to L. mexicana for its entry into and survival within mammalian host cells in vitro or in vivo and provide further support for the notion that free GPIs are essential for parasite growth. PMID- 10749924 TI - Time-lapse video microscopy analysis reveals astral microtubule detachment in the yeast spindle pole mutant cnm67. AB - Saccharomyces cerevisiae cnm67Delta cells lack the spindle pole body (SPB) outer plaque, the main attachment site for astral (cytoplasmic) microtubules, leading to frequent nuclear segregation failure. We monitored dynamics of green fluorescent protein-labeled nuclei and microtubules over several cell cycles. Early nuclear migration steps such as nuclear positioning and spindle orientation were slightly affected, but late phases such as rapid oscillations and insertion of the anaphase nucleus into the bud neck were mostly absent. Analyzes of microtubule dynamics revealed normal behavior of the nuclear spindle but frequent detachment of astral microtubules after SPB separation. Concomitantly, Spc72 protein, the cytoplasmic anchor for the gamma-tubulin complex, was partially lost from the SPB region with dynamics similar to those observed for microtubules. We postulate that in cnm67Delta cells Spc72-gamma-tubulin complex-capped astral microtubules are released from the half-bridge upon SPB separation but fail to be anchored to the cytoplasmic side of the SPB because of the absence of an outer plaque. However, successful nuclear segregation in cnm67Delta cells can still be achieved by elongation forces of spindles that were correctly oriented before astral microtubule detachment by action of Kip3/Kar3 motors. Interestingly, the first nuclear segregation in newborn diploid cells never fails, even though astral microtubule detachment occurs. PMID- 10749925 TI - Axonal membrane proteins are transported in distinct carriers: a two-color video microscopy study in cultured hippocampal neurons. AB - Neurons transport newly synthesized membrane proteins along axons by microtubule mediated fast axonal transport. Membrane proteins destined for different axonal subdomains are thought to be transported in different transport carriers. To analyze this differential transport in living neurons, we tagged the amyloid precursor protein (APP) and synaptophysin (p38) with green fluorescent protein (GFP) variants. The resulting fusion proteins, APP-yellow fluorescent protein (YFP), p38-enhanced GFP, and p38-enhanced cyan fluorescent protein, were expressed in hippocampal neurons, and the cells were imaged by video microscopy. APP-YFP was transported in elongated tubules that moved extremely fast (on average 4.5 micrometer/s) and over long distances. In contrast, p38-enhanced GFP transporting structures were more vesicular and moved four times slower (0.9 micrometer/s) and over shorter distances only. Two-color video microscopy showed that the two proteins were sorted to different carriers that moved with different characteristics along axons of doubly transfected neurons. Antisense treatment using oligonucleotides against the kinesin heavy chain slowed down the long, continuous movement of APP-YFP tubules and increased frequency of directional changes. These results demonstrate for the first time directly the sorting and transport of two axonal membrane proteins into different carriers. Moreover, the extremely fast-moving tubules represent a previously unidentified type of axonal carrier. PMID- 10749927 TI - A family of ADP-ribosylation factor effectors that can alter membrane transport through the trans-Golgi. AB - A family of three structurally related proteins were cloned from human cDNA libraries by their ability to interact preferentially with the activated form of human ADP-ribosylation factor 3 (ARF3) in two-hybrid assays. The specific and GTP dependent binding was later confirmed through direct protein binding of recombinant proteins. The three proteins share large ( approximately 300 residues) domains at their N termini that are 60-70% identical to each other and a shorter (73 residues) domain at their C termini with 70% homology to the C terminal "ear" domain of gamma-adaptin. Although GGA1 is found predominantly as a soluble protein by cell fractionation, all three proteins were found to localize to the trans-Golgi network (TGN) by indirect immunofluorescence. The binding of GGAs to TGN was sensitive to brefeldin A, consistent with this being an ARF dependent event. Thus, these proteins have been named Golgi-localizing, gamma adaptin ear homology domain, ARF-binding proteins, or GGAs. The finding that overexpression of GGAs was sufficient to alter the distribution of markers of the TGN (TGN38 and mannose 6-phosphate receptors) led us to propose that GGAs are effectors for ARFs that function in the regulation of membrane traffic through the TGN. PMID- 10749926 TI - A mutation in gamma-tubulin alters microtubule dynamics and organization and is synthetically lethal with the kinesin-like protein pkl1p. AB - Mitotic segregation of chromosomes requires spindle pole functions for microtubule nucleation, minus end organization, and regulation of dynamics. gamma Tubulin is essential for nucleation, and we now extend its role to these latter processes. We have characterized a mutation in gamma-tubulin that results in cold sensitive mitotic arrest with an elongated bipolar spindle but impaired anaphase A. At 30 degrees C cytoplasmic microtubule arrays are abnormal and bundle into single larger arrays. Three-dimensional time-lapse video microscopy reveals that microtubule dynamics are altered. Localization of the mutant gamma-tubulin is like the wild-type protein. Prediction of gamma-tubulin structure indicates that non-alpha/beta-tubulin protein-protein interactions could be affected. The kinesin-like protein (klp) Pkl1p localizes to the spindle poles and spindle and is essential for viability of the gamma-tubulin mutant and in multicopy for normal cell morphology at 30 degrees C. Localization and function of Pkl1p in the mutant appear unaltered, consistent with a redundant function for this protein in wild type. Our data indicate a broader role for gamma-tubulin at spindle poles in regulating aspects of microtubule dynamics and organization. We propose that Pkl1p rescues an impaired function of gamma-tubulin that involves non-tubulin protein-protein interactions, presumably with a second motor, MAP, or MTOC component. PMID- 10749928 TI - The PC6B cytoplasmic domain contains two acidic clusters that direct sorting to distinct trans-Golgi network/endosomal compartments. AB - The mammalian proprotein convertases (PCs) are a family of secretory pathway enzymes that catalyze the endoproteolytic maturation of peptide hormones and many bioactive proteins. Two PCs, furin and PC6B, are broadly expressed and share very similar cleavage site specificities, suggesting that they may be functionally redundant. However, germline knockout studies show that they are not. Here we report the distinct subcellular localization of PC6B and identify the sorting information within its cytoplasmic domain (cd). We show that in neuroendocrine cells, PC6B is localized to a paranuclear, brefeldin A-dispersible, BaCl(2) responsive post-Golgi network (TGN) compartment distinct from furin and TGN38. The 88-amino acid PC6B-cd contains sorting information sufficient to direct reporter proteins to the same compartment as full-length PC6B. Mutational analysis indicates that endocytosis is predominantly directed by a canonical tyrosine-based motif (Tyr(1802)GluLysLeu). Truncation and sufficiency studies reveal that two clusters of acidic amino acids (ACs) within the PC6B-cd contain differential sorting information. The membrane-proximal AC (AC1) directs TGN localization and interacts with the TGN sorting protein PACS-1. The membrane distal AC (AC2) promotes a localization characteristic of the full-length PC6B cd. Our results demonstrate that AC motifs can target proteins to distinct TGN/endosomal compartments and indicate that the AC-mediated localization of PC6B and furin contribute to their distinct roles in vivo. PMID- 10749929 TI - The Dictyostelium LIM domain-containing protein LIM2 is essential for proper chemotaxis and morphogenesis. AB - We have identified limB, a gene encoding a novel LIM domain-containing protein, LIM2, in a screen for genes required for morphogenesis. limB null cells aggregate, although poorly, but they are unable to undergo morphogenesis, and the aggregates arrest at the mound stage. limB null cells exhibit an aberrant actin cytoskeleton and have numerous F-actin-enriched microspikes. The cells exhibit poor adhesion to a substratum and do not form tight cell-cell agglomerates in suspension. Furthermore, limB null cells are unable to properly polarize in chemoattractant gradients and move very poorly. Expression of limB from a prestalk-specific but not a prespore-specific promoter complements the morphogenetic defects of the limB null strain, suggesting that the limB null cell developmental defect results from an inability to properly sort prestalk cells. LIM2 protein is enriched in the cortex of wild-type cells, although it does not colocalize with the actin cytoskeleton. Our analysis indicates that LIM2 is a new regulatory protein that functions to control rearrangements of the actin cytoskeleton and is required for cell motility and chemotaxis. Our findings may be generally applicable to understanding pathways that control cell movement and morphogenesis in all multicellular organisms. Structure function studies on the LIM domains are presented. PMID- 10749930 TI - Mitotic chromosome condensation requires Brn1p, the yeast homologue of Barren. AB - In vitro studies suggest that the Barren protein may function as an activator of DNA topoisomerase II and/or as a component of the Xenopus condensin complex. To better understand the role of Barren in vivo, we generated conditional alleles of the structural gene for Barren (BRN1) in Saccharomyces cerevisiae. We show that Barren is an essential protein required for chromosome condensation in vivo and that it is likely to function as an intrinsic component of the yeast condensation machinery. Consistent with this view, we show that Barren performs an essential function during a period of the cell cycle when chromosome condensation is established and maintained. In contrast, Barren does not serve as an essential activator of DNA topoisomerase II in vivo. Finally, brn1 mutants display additional phenotypes such as stretched chromosomes, aberrant anaphase spindles, and the accumulation of cells with >2C DNA content, suggesting that Barren function influences multiple aspects of chromosome transmission and dynamics. PMID- 10749931 TI - Chromosome condensation factor Brn1p is required for chromatid separation in mitosis. AB - This work describes BRN1, the budding yeast homologue of Drosophila Barren and Xenopus condensin subunit XCAP-H. The Drosophila protein is required for proper chromosome segregation in mitosis, and Xenopus protein functions in mitotic chromosome condensation. Mutant brn1 cells show a defect in mitotic chromosome condensation and sister chromatid separation and segregation in anaphase. Chromatid cohesion before anaphase is properly maintained in the mutants. Some brn1 mutant cells apparently arrest in S-phase, pointing to a possible function for Brn1p at this stage of the cell cycle. Brn1p is a nuclear protein with a nonuniform distribution pattern, and its level is up-regulated at mitosis. Temperature-sensitive mutations of BRN1 can be suppressed by overexpression of a novel gene YCG1, which is homologous to another Xenopus condensin subunit, XCAP G. Overexpression of SMC2, a gene necessary for chromosome condensation, and a homologue of the XCAP-E condensin, does not suppress brn1, pointing to functional specialization of components of the condensin complex. PMID- 10749932 TI - A new paxillin-binding protein, PAG3/Papalpha/KIAA0400, bearing an ADP ribosylation factor GTPase-activating protein activity, is involved in paxillin recruitment to focal adhesions and cell migration. AB - Paxillin acts as an adaptor molecule in integrin signaling. Paxillin is localized to focal contacts but seems to also exist in a relatively large cytoplasmic pool. Here, we report the identification of a new paxillin-binding protein, PAG3 (paxillin-associated protein with ADP-ribosylation factor [ARF] GTPase-activating protein [GAP] activity, number 3), which is involved in regulation of the subcellular localization of paxillin. PAG3 bound to all paxillin isoforms and was induced during monocyte maturation, at which time paxillin expression is also increased and integrins are activated. PAG3 was diffusely distributed in the cytoplasm in premature monocytes but became localized at cell periphery in mature monocytes, a fraction of which then colocalized with paxillin. PAG3, on the other hand, did not accumulate at focal adhesion plaques, suggesting that PAG3 is not an integrin assembly protein. PAG3 was identical to KIAA0400/Papalpha, which was previously identified as a Pyk2-binding protein bearing a GAP activity toward several ARFs in vitro. Mammalian ARFs fall into three classes, and we showed that all classes could affect subcellular localization of paxillin. We also examined possible interaction of PAG3 with ARFs and showed evidence that at least one of them, ARF6, seems to be an intracellular substrate for GAP activity of PAG3. Moreover, overexpression of PAG3, but not its GAP-inactive mutant, inhibited paxillin recruitment to focal contacts and hampered cell migratory activities, whereas cell adhesion activities were almost unaffected. Therefore, our results demonstrate that paxillin recruitment to focal adhesions is not mediated by simple cytoplasmic diffusion; rather, PAG3 appears to be involved in this process, possibly through its GAP activity toward ARF proteins. Our result thus delineates a new aspect of regulation of cell migratory activities. PMID- 10749933 TI - Clonal tests of conventional kinesin function during cell proliferation and differentiation. AB - Null mutations in the Drosophila Kinesin heavy chain gene (Khc), which are lethal during the second larval instar, have shown that conventional kinesin is critical for fast axonal transport in neurons, but its functions elsewhere are uncertain. To test other tissues, single imaginal cells in young larvae were rendered null for Khc by mitotic recombination. Surprisingly, the null cells produced large clones of adult tissue. The rates of cell proliferation were not reduced, indicating that conventional kinesin is not essential for cell growth or division. This suggests that in undifferentiated cells vesicle transport from the Golgi to either the endoplasmic reticulum or the plasma membrane can proceed at normal rates without conventional kinesin. In adult eye clones produced by null founder cells, there were some defects in differentiation that caused mild ultrastructural changes, but they were not consistent with serious problems in the positioning or transport of endoplasmic reticulum, mitochondria, or vesicles. In contrast, defective cuticle deposition by highly elongated Khc null bristle shafts suggests that conventional kinesin is critical for proper secretory vesicle transport in some cell types, particularly ones that must build and maintain long cytoplasmic extensions. The ubiquity and evolutionary conservation of kinesin heavy chain argue for functions in all cells. We suggest interphase organelle movements away from the cell center are driven by multilayered transport mechanisms; that is, individual organelles can use kinesin-related proteins and myosins, as well as conventional kinesin, to move toward the cell periphery. In this case, other motors can compensate for the loss of conventional kinesin except in cells that have extremely long transport tracks. PMID- 10749934 TI - A screen for dominant negative mutants of SEC18 reveals a role for the AAA protein consensus sequence in ATP hydrolysis. AB - An evolutionarily ancient mechanism is used for intracellular membrane fusion events ranging from endoplasmic reticulum-Golgi traffic in yeast to synaptic vesicle exocytosis in the human brain. At the heart of this mechanism is the core complex of N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). Although these proteins are accepted as key players in vesicular traffic, their molecular mechanisms of action remain unclear. To illuminate important structure-function relationships in NSF, a screen for dominant negative mutants of yeast NSF (Sec18p) was undertaken. This involved random mutagenesis of a GAL1-regulated SEC18 yeast expression plasmid. Several dominant negative alleles were identified on the basis of galactose-inducible growth arrest, of which one, sec18-109, was characterized in detail. The sec18-109 phenotype (abnormal membrane trafficking through the biosynthetic pathway, accumulation of a membranous tubular network, growth suppression, increased cell density) is due to a single A-G substitution in SEC18 resulting in a missense mutation in Sec18p (Thr(394)-->Pro). Thr(394) is conserved in most AAA proteins and indeed forms part of the minimal AAA consensus sequence that serves as a signature of this large protein family. Analysis of recombinant Sec18-109p indicates that the mutation does not prevent hexamerization or interaction with yeast alpha-SNAP (Sec17p), but instead results in undetectable ATPase activity that cannot be stimulated by Sec17p. This suggests a role for the AAA protein consensus sequence in regulating ATP hydrolysis. Furthermore, this approach of screening for dominant negative mutants in yeast can be applied to other conserved proteins so as to highlight important functional domains in their mammalian counterparts. PMID- 10749935 TI - AZU-1: a candidate breast tumor suppressor and biomarker for tumor progression. AB - To identify genes misregulated in the final stages of breast carcinogenesis, we performed differential display to compare the gene expression patterns of the human tumorigenic mammary epithelial cells, HMT-3522-T4-2, with those of their immediate premalignant progenitors, HMT-3522-S2. We identified a novel gene, called anti-zuai-1 (AZU-1), that was abundantly expressed in non- and premalignant cells and tissues but was appreciably reduced in breast tumor cell types and in primary tumors. The AZU-1 gene encodes an acidic 571-amino-acid protein containing at least two structurally distinct domains with potential protein-binding functions: an N-terminal serine and proline-rich domain with a predicted immunoglobulin-like fold and a C-terminal coiled-coil domain. In HMT 3522 cells, the bulk of AZU-1 protein resided in a detergent-extractable cytoplasmic pool and was present at much lower levels in tumorigenic T4-2 cells than in their nonmalignant counterparts. Reversion of the tumorigenic phenotype of T4-2 cells, by means described previously, was accompanied by the up regulation of AZU-1. In addition, reexpression of AZU-1 in T4-2 cells, using viral vectors, was sufficient to reduce their malignant phenotype substantially, both in culture and in vivo. These results indicate that AZU-1 is a candidate breast tumor suppressor that may exert its effects by promoting correct tissue morphogenesis. PMID- 10749936 TI - Intracellular and extracellular leukemia inhibitory factor proteins have different cellular activities that are mediated by distinct protein motifs. AB - Although many growth factors and cytokines have been shown to be localized within the cell and nucleus, the mechanism by which these molecules elicit a biological response is not well understood. The cytokine leukemia inhibitory factor (LIF) provides a tractable experimental system to investigate this problem, because translation of alternatively spliced transcripts results in the production of differentially localized LIF proteins, one secreted from the cell and acting via cell surface receptors and the other localized within the cell. We have used overexpression analysis to demonstrate that extracellular and intracellular LIF proteins can have distinct cellular activities. Intracellular LIF protein is localized to both nucleus and cytoplasm and when overexpressed induces apoptosis that is inhibited by CrmA but not Bcl-2 expression. Mutational analysis revealed that the intracellular activity was independent of receptor interaction and activation and reliant on a conserved leucine-rich motif that was not required for activation of cell surface receptors by extracellular protein. This provides the first report of alternate intracellular and extracellular cytokine activities that result from differential cellular localization of the protein and are mediated by spatially distinct motifs. PMID- 10749937 TI - A dibasic motif in the tail of a class XIV apicomplexan myosin is an essential determinant of plasma membrane localization. AB - Obligate intracellular parasites of the phylum Apicomplexa exhibit gliding motility, a unique form of substrate-dependent locomotion essential for host cell invasion and shown to involve the parasite actin cytoskeleton and myosin motor(s). Toxoplasma gondii has been shown to express three class XIV myosins, TgM-A, -B, and -C. We identified an additional such myosin, TgM-D, and completed the sequences of a related Plasmodium falciparum myosin, PfM-A. Despite divergent structural features, TgM-A purified from parasites bound actin in an ATP dependent manner. Isoform-specific antibodies revealed that TgM-A and recombinant mycTgM-A were localized right beneath the plasma membrane, and subcellular fractionation indicated a tight membrane association. Recombinant TgM-D also had a peripheral although not as sharply defined localization. Truncation of their respective tail domains abolished peripheral localization and tight membrane association. Conversely, fusion of the tails to green fluorescent protein (GFP) was sufficient to confer plasma membrane localization and sedimentability. The peripheral localization of TgM-A and of the GFP-tail fusion did not depend on an intact F-actin cytoskeleton, and the GFP chimera did not localize to the plasma membrane of HeLa cells. Finally, we showed that the specific localization determinants were in the very C terminus of the TgM-A tail, and site-directed mutagenesis revealed two essential arginine residues. We discuss the evidence for a proteinaceous plasma membrane receptor and the implications for the invasion process. PMID- 10749938 TI - EMB-30: an APC4 homologue required for metaphase-to-anaphase transitions during meiosis and mitosis in Caenorhabditis elegans. AB - Here we show that emb-30 is required for metaphase-to-anaphase transitions during meiosis and mitosis in Caenorhabditis elegans. Germline-specific emb-30 mutant alleles block the meiotic divisions. Mutant oocytes, fertilized by wild-type sperm, set up a meiotic spindle but do not progress to anaphase I. As a result, polar bodies are not produced, pronuclei fail to form, and cytokinesis does not occur. Severe-reduction-of-function emb-30 alleles (class I alleles) result in zygotic sterility and lead to germline and somatic defects that are consistent with an essential role in promoting the metaphase-to-anaphase transition during mitosis. Analysis of the vulval cell lineages in these emb-30(class I) mutant animals suggests that mitosis is lengthened and eventually arrested when maternally contributed emb-30 becomes limiting. By further reducing maternal emb 30 function contributed to class I mutant animals, we show that emb-30 is required for the metaphase-to-anaphase transition in many, if not all, cells. Metaphase arrest in emb-30 mutants is not due to activation of the spindle assembly checkpoint but rather reflects an essential emb-30 requirement for M phase progression. A reduction in emb-30 activity can suppress the lethality and sterility caused by a null mutation in mdf-1, a component of the spindle assembly checkpoint machinery. This result suggests that delaying anaphase onset can bypass the spindle checkpoint requirement for normal development. Positional cloning established that emb-30 encodes the likely C. elegans orthologue of APC4/Lid1, a component of the anaphase-promoting complex/cyclosome, required for the metaphase-to-anaphase transition. Thus, the anaphase-promoting complex/cyclosome is likely to be required for all metaphase-to-anaphase transitions in a multicellular organism. PMID- 10749939 TI - A proline-rich region and nearby cysteine residues target XLalphas to the Golgi complex region. AB - XLalphas is a splice variant of the heterotrimeric G protein, Galpha(s), found on Golgi membranes in cells with regulated and constitutive secretion. We examined the role of the alternatively spliced amino terminus of XLalphas for Golgi targeting with the use of subcellular fractionation and fluorescence microscopy. XLalphas incorporated [(3)H]palmitate, and mutation of cysteines in a cysteine rich region inhibited this incorporation and lessened membrane attachment. Deletion of a proline-rich region abolished Golgi localization of XLalphas without changing its membrane attachment. The proline-rich and cysteine-rich regions together were sufficient to target the green fluorescent protein, a cytosolic protein, to Golgi membranes. The membrane attachment and Golgi targeting of the fusion protein required the putative palmitoylation sites, the cysteine residues in the cysteine-rich region. Several peripheral membrane proteins found at the Golgi have proline-rich regions, including a Galpha(i2) splice variant, dynamin II, betaIII spectrin, comitin, and a Golgi SNARE, GS32. Our results suggest that proline-rich regions can be a Golgi-targeting signal for G protein alpha subunits and possibly for other peripheral membrane proteins as well. PMID- 10749940 TI - Calreticulin couples calcium release and calcium influx in integrin-mediated calcium signaling. AB - The engagement of integrin alpha7 in E63 skeletal muscle cells by laminin or anti alpha7 antibodies triggered transient elevations in the intracellular free Ca(2+) concentration that resulted from both inositol triphosphate-evoked Ca(2+) release from intracellular stores and extracellular Ca(2+) influx through voltage-gated, L-type Ca(2+) channels. The extracellular domain of integrin alpha7 was found to associate with both ectocalreticulin and dihydropyridine receptor on the cell surface. Calreticulin appears to also associate with cytoplasmic domain of integrin alpha7 in a manner highly dependent on the cytosolic Ca(2+) concentration. It appeared that intracellular Ca(2+) release was a prerequisite for Ca(2+) influx and that calreticulin associated with the integrin cytoplasmic domain mediated the coupling of between the Ca(2+) release and Ca(2+) influx. These findings suggest that calreticulin serves as a cytosolic activator of integrin and a signal transducer between integrins and Ca(2+) channels on the cell surface. PMID- 10749941 TI - The autocrine mitogenic loop of the ciliate Euplotes raikovi: the pheromone membrane-bound forms are the cell binding sites and potential signaling receptors of soluble pheromones. AB - Homologous proteins, denoted pheromones, promote cell mitotic proliferation and mating pair formation in the ciliate Euplotes raikovi, according to whether they bind to cells in an autocrine- or paracrine-like manner. The primary transcripts of the genes encoding these proteins undergo alternate splicing, which generates at least two distinct mRNAs. One is specific for the soluble pheromone, the other for a pheromone isoform that remains anchored to the cell surface as a type II protein, whose extracellular C-terminal region is structurally equivalent to the secreted form. The 15-kDa membrane-bound isoform of pheromone Er-1, denoted Er 1mem and synthesized by the same E. raikovi cells that secrete Er-1, has been purified from cell membranes by affinity chromatography prepared with matrix bound Er-1, and its extracellular and cytoplasmic regions have been expressed as recombinant proteins. Using the purified material and these recombinant proteins, it has been shown that Er-1mem has the property of binding pheromones competitively through its extracellular pheromone-like domain and associating reversibly and specifically with a guanine nucleotide-binding protein through its intracellular domain. It has been concluded that the membrane-bound pheromone isoforms of E. raikovi represent the cell effective pheromone binding sites and are functionally equipped for transducing the signal generated by this binding. PMID- 10749942 TI - ADAM 23/MDC3, a human disintegrin that promotes cell adhesion via interaction with the alphavbeta3 integrin through an RGD-independent mechanism. AB - ADAM 23 (a disintegrin and metalloproteinase domain)/MDC3 (metalloprotease, disintegrin, and cysteine-rich domain) is a member of the disintegrin family of proteins expressed in fetal and adult brain. In this work we show that the disintegrin-like domain of ADAM 23 produced in Escherichia coli and immobilized on culture dishes promotes attachment of different human cells of neural origin, such as neuroblastoma cells (NB100 and SH-S(y)5(y)) or astrocytoma cells (U373 and U87 MG). Analysis of ADAM 23 binding to integrins revealed a specific interaction with alphavbeta3, mediated by a short amino acid sequence present in its putative disintegrin loop. This sequence lacks any RGD motif, which is a common structural determinant supporting alphavbeta3-mediated interactions of diverse proteins, including other disintegrins. alphavbeta3 also supported adhesion of HeLa cells transfected with a full-length cDNA for ADAM 23, extending the results obtained with the recombinant protein containing the disintegrin domain of ADAM 23. On the basis of these results, we propose that ADAM 23, through its disintegrin-like domain, may function as an adhesion molecule involved in alphavbeta3-mediated cell interactions occurring in normal and pathological processes, including progression of malignant tumors from neural origin. PMID- 10749943 TI - Rearrangements of human mitochondrial DNA (mtDNA): new insights into the regulation of mtDNA copy number and gene expression. AB - Mitochondria from patients with Kearns-Sayre syndrome harboring large-scale rearrangements of human mitochondrial DNA (mtDNA; both partial deletions and a partial duplication) were introduced into human cells lacking endogenous mtDNA. Cytoplasmic hybrids containing 100% wild-type mtDNA, 100% mtDNA with partial duplications, and 100% mtDNA with partial deletions were isolated and characterized. The cell lines with 100% deleted mtDNAs exhibited a complete impairment of respiratory chain function and oxidative phosphorylation. In contrast, there were no detectable respiratory chain or protein synthesis defects in the cell lines with 100% duplicated mtDNAs. Unexpectedly, the mass of mtDNA was identical in all cell lines, despite the fact that different lines contained mtDNAs of vastly different sizes and with different numbers of replication origins, suggesting that mtDNA copy number may be regulated by tightly controlled mitochondrial dNTP pools. In addition, quantitation of mtDNA-encoded RNAs and polypeptides in these lines provided evidence that mtDNA gene copy number affects gene expression, which, in turn, is regulated at both the post-transcriptional and translational levels. PMID- 10749945 TI - Biopsychosocial aspects of prostate cancer. AB - Prostate cancer early detection choices and treatment options are fraught with controversy. To update the consultation-liaison psychiatrist who works with at risk men, the authors reviewed all pertinent citations in the medicine database from 1966 to 1998 and in other relevant publications. Though watchful waiting for early-stage prostate cancer has no side effects, men must cope psychologically with issues of long-term cancer survivorship. Men can choose between different treatment options (e.g., radiation vs. radical prostatectomy) with early detection. Urinary incontinence, sexual dysfunction, and fatigue are major emotional and physical stressors for this population. Consultation-liaison psychiatrists and physicians need to be aware of the psychosocial sequelae of both prostate cancer and treatment-related side effects. PMID- 10749944 TI - A trypanosomal protein synergizes with the cytokines ciliary neurotrophic factor and leukemia inhibitory factor to prevent apoptosis of neuronal cells. AB - Despite the neuronal degeneration in the chronic stage of Chagas' disease, neuron counts actually increase in the preceding, asymptomatic stage, in contrast to the age-related decrease in neuron counts in age-matched normal individuals. Relevant to this observation, we found that the trans-sialidase (TS) of Trypanosoma cruzi, the etiologic agent of Chagas' disease, induces neurite outgrowth and rescues PC12 cells from apoptotic death caused by growth factor deprivation. These properties, novel for a parasite protein, were independent of catalytic activity and were mapped to the C terminus of the catalytic domain of TS. TS activated protein kinase Akt in a phosphoinositide-3 kinase-inhibitable manner, suggesting a molecular mechanism for the TS-induced neuroprotection. TS also triggered bcl-2 gene expression in growth factor-deprived cells, an effect consistent with TS protecting against apoptosis. Ciliary neurotrophic factor and leukemia inhibitory factor, two cytokines critical to the repair of injured motor neurons, specifically potentiated the TS action. The results suggest that TS acts in synergy with host ciliary neurotrophic factor or leukemia inhibitory factor to promote neuronal survival in T. cruzi-infected individuals. PMID- 10749946 TI - Neuropsychiatric sequelae of traumatic brain injury. AB - The authors review the psychiatric disturbances associated with traumatic brain injury. They highlight the close link between traumatic brain injury and psychiatry and provide an overview of the epidemiology, risk factors, classification, and mechanisms of traumatic brain injury. They describe various neuropsychiatric sequelae, and the respective treatments are outlined with emphasis on a multidisciplinary approach. PMID- 10749947 TI - Antidepressant treatment of fibromyalgia. A meta-analysis and review. AB - Fibromyalgia is a common musculoskeletal pain disorder associated with mood disorders. Antidepressants, particularly tricyclics, are commonly recommended treatments. Randomized, controlled trials of antidepressants for treatment of fibromyalgia were reviewed by methodology, results, and potential predictors of response. Twenty-one controlled trials, 16 involving tricyclic agents, were identified; 9 of these 16 studies were suitable for meta-analysis. Effect sizes were calculated for measurements of physician and patient overall assessment, pain, stiffness, tenderness, fatigue, and sleep quality. Compared with placebo, tricyclic agents were associated with effect sizes that were substantially larger than zero for all measurements. The largest improvement was associated with measures of sleep quality; the most modest improvement was found in measures of stiffness and tenderness. Further studies are needed utilizing randomized, double blind, placebo-controlled, parallel designs with antidepressants administered at therapeutic dose ranges, using standardized criteria for fibromyalgia and systematically assessed for co-occurring psychiatric illness. PMID- 10749948 TI - Psychiatric symptom severity and length of stay on an intensive rehabilitation unit. AB - The purpose of this study was to evaluate the role of psychiatric symptoms in the medical and surgical rehabilitation of patients on an intensive rehabilitation unit and to examine whether psychiatric symptom severity contributes to length of hospital stay (LOS). Forty-four patients (21 men and 23 women) were assessed at admission and before discharge with the Functional Independence Measure (FIM). Subjects were evaluated with the Mini-Mental State Exam (MMSE), the Hamilton Rating Scale for Depression (Ham-D), Positive and Negative Symptom Scale, (PANSS), and Scale for the Assessment of Negative Symptoms (SANS). Thirty-six percent of subjects were cognitively impaired (MMSE < 25), 14% had significant depressive symptoms (Ham-D > 12), and 52% had significant negative symptomatology (PANSS-N > 15). A regression analysis yielded a model of three predictive factors. Gender, the PANSS General subscale, and the SANS Attention subscale accounted for 42.4% of LOS variance. These results indicate that psychiatric symptoms are common in medical rehabilitation inpatients and, together with demographic factors, are associated with increased LOS. PMID- 10749949 TI - Factor analysis of the Zung Self-Rating Depression Scale in a large ambulatory oncology sample. AB - Screening cancer patients for depression with self-report inventories presents clinical and methodological challenges. Many investigators separate "somatic" from "cognitive" symptoms when adapting such measures to oncology settings. However, this practice has rarely been empirically validated through factor analytic studies. The following study describes a factor analysis of the Zung Self-Rating Depression Scale (ZSDS) from a large ambulatory sample of cancer patients (N = 1,109). A four-factor solution emerged, consisting of a cognitive symptom factor, a manifest depressed mood factor, and two somatic factors (eating and non-eating related). These factors accounted for 20% (cognitive), 13% (mood), 8% (non-eating), and 7% (eating) of the variance on the Zung, respectively. The authors discuss the implications of these results as they pertain to screening cancer patients for depression. PMID- 10749950 TI - Child and adolescent psychiatry in general children's hospitals. A survey of chairs of psychiatry. AB - This article characterizes the academic, administrative, clinical service, and fiscal characteristics of departments of psychiatry in traditional children's hospitals to determine the characteristics of fiscally successful programs. A survey of chairs of psychiatry from short-term general children's hospitals was conducted based on 38 questions addressing the descriptive characteristics of their respective departments. The characteristics of psychiatry programs identified as fiscally successful were compared to those of programs that required subsidy. Nine of 45 eligible children's hospitals (20%) did not have a department or section of psychiatry, and surveys were returned by 35 of 36 department chairs (97% response). Considerable variation exists in the academic, administrative, clinical services, and fiscal characteristics of programs, although over half are operating at a deficit. Fiscal success was associated with availability of inpatient and intermediate levels of psychiatric care, better integration of the psychiatry program within the children's hospital, and adequate fiscal information being provided to the psychiatry chair. Additional research regarding the potential of psychiatric services to generate clinical success and cost savings is warranted. Pediatric health care professionals and third-party payers should be educated regarding the relevance of psychiatric services within children's hospitals and in physically ill children. PMID- 10749951 TI - Psychological functioning in children and adolescents undergoing radiofrequency catheter ablation. AB - This study examined 38 patients (age 6-18 years) with recurrent cardiac arrhythmias who underwent radiofrequency catheter ablation of ectopic myocardial foci. Psychological functioning was assessed by the Pediatric Symptom Checklist, Short Mood and Feeling Questionnaire, Revised Children's Manifest Anxiety Scale, and Arrhythmia Anxiety Queries prior to ablation and at 3-month follow-up. The patients resembled a normal population without elevations in anxiety or depression. After ablation, the patients showed reductions in the "fear of their heart problem" and increases in "the things that they enjoy." The patients who underwent a curative ablation had better functioning than those who did not show improvement. Children appear to have the opportunity for an improved quality of life after ablation. PMID- 10749952 TI - Resilience factors associated with adaptation to HIV disease. AB - This study examines the hardiness dimensions of commitment, challenge, and control as resilience factors in adaptation among persons with symptomatic HIV disease and AIDS. Two hundred participants completed self-report questionnaires measuring hardiness, psychological distress, quality of life, and core personal beliefs. A series of standard multiple regression analyses revealed that high hardiness was significantly related to 1) lower psychological distress levels; 2) higher perceived quality of life in physical health, mental health, and overall functioning domains; 3) more positive personal beliefs regarding the benevolence of the world and people, self-worth, and randomness of life events; and 4) lowered belief in controllability of life events. Commitment was the hardiness factor that most frequently made a unique contribution to predicting adaptation in the regression models. Implications of these findings for understanding HIV related adaptation and for clinical mental health intervention are considered. Future directions in HIV-related adaptation research are suggested. PMID- 10749953 TI - Psychogenic seizures--why women? AB - The only consistent finding in studies of psychogenic seizures is the approximately threefold higher incidence in women. Therefore, why women? Charcot and Freud emphasized the sexual aspects of the seizure as has the current interest in childhood sexual abuse. From case studies and review of the literature the author believes that psychogenic seizures in women express rage, fear, and helplessness against the dominant and abusive male rather than sexual conflicts. Emphasizing the aggressive component of seizures does not minimize the traumatic effects of sexual abuse but rather includes it as leading to rage and helplessness. PMID- 10749954 TI - Olanzapine-induced neutropenia. PMID- 10749955 TI - Reevaluation of serum cortisol in conversion disorder with seizure (pseudoseizure) PMID- 10749956 TI - Use of mirtazapine as prophylactic treatment for migraine headache. PMID- 10749958 TI - Academy of Psychosomatic Medicine: Proceedings of the 46th Annual Meeting : November 18-21, 1999, New Orleans, LA. PMID- 10749957 TI - Consultation-liaison psychiatry drug-drug interactions update. PMID- 10749959 TI - Patterns and predictors of risk for depressive and anxiety-related disorders during the first three years after heart transplantation. PMID- 10749960 TI - The effect of previous coronary-artery bypass surgery on the prognosis of patients with diabetes who have acute myocardial infarction. Bypass Angioplasty Revascularization Investigation Investigators. AB - BACKGROUND: Acute myocardial infarction in patients with diabetes is associated with high mortality. We studied whether previous revascularization by coronary artery bypass grafting (CABG), as compared with percutaneous transluminal coronary angioplasty (PTCA), influences the prognosis in such patients. METHODS: We classified all patients eligible for the Bypass Angioplasty Revascularization Investigation who underwent coronary revascularization within three months after entry into the study according to whether they had diabetes and whether they had undergone CABG, either initially or after PTCA. The protective effect of CABG with regard to mortality in the presence and in the absence of subsequent spontaneous Q-wave myocardial infarction was estimated with the use of Cox regression models. RESULTS: Among the 641 patients with diabetes and the 2962 without diabetes, the cumulative five-year rates of death were 20 percent and 8 percent, respectively (P<0.001), and the five-year rates of spontaneous Q-wave myocardial infarction were 8 percent and 4 percent (P<0.001). CABG greatly reduced the risk of death after spontaneous Q-wave myocardial infarction in the patients with diabetes (relative risk, 0.09; 95 percent confidence interval, 0.03 to 0.29). Among patients with diabetes who had undergone CABG but did not have spontaneous Q-wave myocardial infarctions, the corresponding relative risk of death was 0.65 (95 percent confidence interval, 0.45 to 0.94). Among the patients without diabetes, no protective effect of CABG was evident. CONCLUSIONS: Among patients with diabetes, previous coronary bypass surgery, as compared with coronary angioplasty, has a highly favorable influence on prognosis after acute myocardial infarction and a smaller beneficial effect among patients who do not have infarction. These findings should influence the type of coronary revascularization procedure selected for patients with diabetes who have multivessel coronary artery disease. PMID- 10749961 TI - Outcome of treatment in children with Philadelphia chromosome-positive acute lymphoblastic leukemia. AB - BACKGROUND: Children with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-positive ALL) have a poor prognosis, and there is no consensus on the optimal treatment for this variant of ALL. METHODS: We reviewed the medical records of patients with Ph-positive ALL who were treated with intensive chemotherapy, with or without bone marrow transplantation, by 10 study groups or large single institutions from 1986 to 1996. Data on 326 children and young adults, who ranged in age from 0.4 to 19.9 years (median, 8.1), were analyzed to determine the rate of complete remission and the probability of event-free, disease-free and overall survival according to standard prognostic factors and type of treatment. RESULTS: The 267 patients who achieved a complete remission after induction chemotherapy (82 percent) were stratified into three subgroups according to the age and leukocyte count at the time of diagnosis: those with the best prognosis (a leukocyte count of less than 50,000 per cubic millimeter and an age of less than 10 years; 95 patients); those with an intermediate prognosis (intermediate-risk features; 92 patients); and those with the worst prognosis (a leukocyte count of more than 100,000 per cubic millimeter; 80 patients). The estimates of disease-free survival at five years (+/-SE) were 49+/-5 percent) for patients with the best prognosis), 30+/-5 percent (for those with an intermediate prognosis), and 20+/-5 percent (for those with the worst prognosis) (P<0.001 for the overall comparison). We also found that transplantation of bone marrow from an HLA-matched related donor offered significantly greater benefit than intensive chemotherapy alone in terms of protecting patients from relapse or other adverse events (relative risk, 0.3; 95 percent confidence interval, 0.2 to 0.5; P<0.001). This finding was consistent in all three groups. CONCLUSIONS: Unlike the usual type of all, Ph-positive ALL is associated with a poor prognosis. Nevertheless, in some patients with favorable prognosis features, the disease can be be controlled by intensive chemotherapy. Transplantation of bone marrow from an HLA matched related donor is superior to other types of transplantation and to intensive chemotherapy alone in prolonging initial complete remissions. PMID- 10749962 TI - The effect of balloon angioplasty on hypertension in atherosclerotic renal-artery stenosis. Dutch Renal Artery Stenosis Intervention Cooperative Study Group. AB - BACKGROUND: Patients with hypertension and renal-artery stenosis are often treated with percutaneous transluminal renal angioplasty. However, the long-term effects of this procedure on blood pressure are not well understood. METHODS: We randomly assigned 106 patients with hypertension who had atherosclerotic renal artery stenosis (defined as a decrease in luminal diameter of 50 percent or more) and a serum creatinine concentration of 2.3 mg per deciliter (200 micromol per liter) or less to undergo percutaneous transluminal renal angioplasty or to receive drug therapy. To be included, patients also had to have a diastolic blood pressure of 95 mm Hg or higher despite treatment with two antihypertensive drugs or an increase of at least 0.2 mg per deciliter (20 micromol per liter) in the serum creatinine concentration during treatment with an angiotensin-converting enzyme inhibitor. Blood pressure, doses of antihypertensive drugs, and renal function were assessed at 3 and 12 months, and patency of the renal artery was assessed at 12 months. RESULTS: At base line, the mean (+/-SD) systolic and diastolic blood pressures were 179+/-25 and 104+/-10 mm Hg, respectively, in the angioplasty group and 180+/-23 and 103+/-8 mm Hg, respectively, in the drug therapy group. At three months, the blood pressures were similar in the two groups (169+/-28 and 99+/-12 mm Hg, respectively, in the 56 patients in the angioplasty group and 176+/-31 and 101+/-14 mm Hg, respectively, in the 50 patients in the drug-therapy group; P=0.25 for the comparison of systolic pressure and P=0.36 for the comparison of diastolic pressure between the two groups); at the time, patients in the angioplasty group were taking 2.1+/-1.3 defined daily doses of medication and those in the drug-therapy group were taking 3.2+/-1.5 daily doses (P<0.001). In the drug-therapy group, 22 patients underwent balloon angioplasty after three months because of persistent hypertension despite treatment with three or more drugs or because of a deterioration in renal function. According to intention-to-treat analysis, at 12 months, there were no significant differences between the angioplasty and drug-therapy groups in systolic and diastolic blood pressures, daily drug doses, or renal function. CONCLUSIONS: In the treatment of patients with hypertension and renal-artery stenosis, angioplasty has little advantage over antihypertensive-drug therapy. PMID- 10749963 TI - Images in clinical medicine. Septic (Aspergillus) embolus. PMID- 10749964 TI - Differences between men and women in the rate of use of hip and knee arthroplasty. AB - BACKGROUND: Previous studies suggest that, for some conditions, women receive fewer health care interventions than men. We estimated the potential need for arthroplasty and the willingness to undergo the procedure in both men and women and examined whether there were differences between the sexes. METHODS: All 48,218 persons 55 years of age or older in two areas of Ontario, Canada, were surveyed by mail and telephone to identify those with hip or knee problems. In these subjects, we assessed the severity of arthritis and the presence of coexisting conditions by questionnaire, documented arthritis by examination and radiography, and conducted interviews to evaluate the subjects' willingness to undergo arthroplasty. The potential need for arthroplasty was defined by the presence of severe symptoms and disability, the absence of any absolute contraindications to surgery, and clinical and radiographic evidence of arthritis. The estimates of need were then adjusted for the subjects' willingness to undergo arthroplasty. RESULTS: The overall response rates were at least 72 percent for the questionnaires and interviews. As compared with men, women had a higher prevalence of arthritis of the hip or knee (age-adjusted odds ratio, 1.76; P<0.001) and had worse symptoms and greater disability, but women were less likely to have undergone arthroplasty (adjusted odds ratio, 0.78; P<0.001). Despite their equal willingness to have the surgery, fewer women than men had discussed the possibility of arthroplasty with a physician (adjusted odds ratio, 0.63). The numbers of people with a potential need for hip or knee arthroplasty were 44.9 per 1000 among women and 20.8 per 1000 among men. After adjustment for willingness to undergo the procedure, the numbers were 5.3 per 1000 for women and 1.6 per 1000 for men. CONCLUSIONS: There is underuse of arthroplasty for severe arthritis in both sexes, but the degree of underuse is more than three times as great in women as in men. PMID- 10749965 TI - "We don't carry that"--failure of pharmacies in predominantly nonwhite neighborhoods to stock opioid analgesics. AB - BACKGROUND: We have observed that many black and Hispanic patients receiving palliative care at a major urban teaching hospital are unable to obtain prescribed opioids from their neighborhood pharmacies. In this study, we investigated the availability of commonly prescribed opioids in New York City pharmacies. METHODS: We surveyed a randomly selected sample of 30 percent of New York City pharmacies to obtain information about their stock of opioids. For each pharmacy, U.S. Census estimates for 1997 were used to determine the racial and ethnic composition of the neighborhood (defined as the area within a 0.4-km [0.25 mile] radius of the pharmacy) and the proportion of residents who were more than 65 years old. Data on robberies, burglaries, and arrests involving illicit drugs in 1997 were obtained for the precinct in which each pharmacy was located. We used a generalized linear model to examine the relation between the racial or ethnic composition of neighborhoods and the opioid supplies of pharmacies, while controlling for the proportion of elderly persons at the census-block level and for crime rates at the precinct level. RESULTS: Pharmacists representing 347 of 431 eligible pharmacies (81 percent) responded to the survey. A total of 176 pharmacies (51 percent) did not have sufficient supplies of opioids to treat patients with severe pain. Only 25 percent of pharmacies in predominantly nonwhite neighborhoods (those in which less than 40 percent of residents were white) had opioid supplies that were sufficient to treat patients in severe pain, as compared with 72 percent of pharmacies in predominantly white neighborhoods (those in which at least 80 percent of residents were white) (P<0.001). CONCLUSIONS: Pharmacies in predominantly nonwhite neighborhoods of New York City do not stock sufficient medications to treat patients with severe pain adequately. PMID- 10749966 TI - Kaposi's sarcoma. PMID- 10749967 TI - Coronary heart disease in patients with diabetes. PMID- 10749968 TI - Renal angioplasty for lowering blood pressure. PMID- 10749969 TI - Disparities in the use of total joint arthroplasty. PMID- 10749971 TI - Correction: Neurologic Complications of the Reactivation of Varicella-Zoster Virus. PMID- 10749970 TI - Racial injustice in health care. PMID- 10749972 TI - California's beleaguered physician groups--will they survive? PMID- 10749973 TI - The Schizosaccharomyces pombe protein Yab8p and a novel factor, Yip1p, share structural and functional similarity with the spinal muscular atrophy-associated proteins SMN and SIP1. AB - The motor neuron disease spinal muscular atrophy (SMA) is caused by reduced levels of functional survival of motor neurons (SMN) protein. Previous studies have shown that SMN binds to the SMN-interacting protein SIP1 and mediates the assembly of spliceosomal U snRNPs in the cytoplasm. In addition, a nuclear function for SMN in pre-mRNA splicing has recently been proposed. Here, we describe the analysis of the Schizo-saccharomyces pombe protein Yab8p and provide evidence that it is structurally and functionally related to SMN found in higher eukaryotes. We show that Yab8p interacts via its N-terminus with a novel protein termed Yip1p. Importantly, Yip1p exhibits homology to SIP1, and the mode of binding to Yab8p is remarkably similar to the SMN-SIP1 interaction. Hence, Yip1p is likely to be the homologue of SIP1 in S.pombe. Yab8p and Yip1p localize predominantly in the nucleus. Genetic studies demonstrate that Yab8p is essential for viability. Strikingly, suppression of YAB8 expression in a conditional knock out strain causes nuclear accumulation of poly(A) mRNA and inhibition of splicing. These data identify Yab8p as a novel factor involved in splicing and suggest that Yab8p exerts a function similar or identical to the nuclear pool of SMN. Our studies provide a model system to study the cellular function of SMN in yeast, and should help in understanding the molecular events leading to SMA. PMID- 10749974 TI - Characterization of the Schizosaccharomyces pombe orthologue of the human survival motor neuron (SMN) protein. AB - Childhood onset spinal muscular atrophy (SMA) is a common autosomal recessive disorder primarily characterized by the loss of lower alpha motor neurons. The underlying chromosomal defects causing SMA have been found in the survival motor neuron (SMN) gene. SMN has been shown previously to play a role in both snRNP biogenesis and mRNA processing, although direct evidence for the relationship between SMN and disease pathology has not been elucidated. SMN orthologues have been isolated in many species including Caenorhabditis elegans and Danio rerio. To study the function of SMN, we have identified and characterized the Schizosaccharomyces pombe orthologue of human SMN, smn1 (+). We have demonstrated that smn1 (+) is essential for viability in S.pombe and yeast expressing missense mutations in Smn1p, which mimic mutations in patients with Type I SMA, show significant mislocalization of the protein and a decrease in cell viability. Wild type Smn1p is localized predominantly in the nucleus whereas yeast expressing Smn1p with missense mutations or deletions of specific domains of the protein accumulate cytoplasmic aggregates. Overexpression of Smn1p results in an increase in the growth rate of cells. Furthermore, mutations within two highly conserved protein interaction domains have a dominant-negative effect on growth, indicating that each domain is of functional significance in S.pombe. These dominant phenotypes can be suppressed by overexpression of murine Smn in the same cell. Given the structural and functional similarities between the protein in fission yeast and higher eukaryotes, S.pombe will be an ideal organism to study the role of SMN in RNA processing. PMID- 10749975 TI - RBMY, a probable human spermatogenesis factor, and other hnRNP G proteins interact with Tra2beta and affect splicing. AB - The RBMY gene family is found on the Y chromosome of all mammals, and microdeletions are strongly associated with infertility in men. RBMY expresses RBM only in the nuclei of germ cells, whereas its X chromosome homologue, RBMX, expresses hnRNP G ubiquitously. We show here that RBM, hnRNP G and a novel testis specific relative, termed hnRNP G-T, interact with Tra2beta, an activator of pre mRNA splicing that is ubiquitous but highly expressed in testis. Endogenous hnRNP G and Tra2beta proteins are associated in HeLa nuclear extracts. RBM and Tra2beta co-localize in two major domains in human spermatocyte nuclei. Phosphorylation enhanced the interaction and reduced competing RNA binding to the interaction domains. Incubation with the protein interaction domain of RBM inhibited splicing in vitro of a specific pre-mRNA substrate containing an essential enhancer bound by Tra2beta. The RNA-binding domain of RBM affected 5' splice site selection. We conclude that the hnRNP G family of proteins is involved in pre-mRNA splicing and infer that RBM may be involved in Tra2beta-dependent splicing in spermatocytes. PMID- 10749976 TI - The short stature homeobox gene SHOX is involved in skeletal abnormalities in Turner syndrome. AB - Turner syndrome is characterized by short stature and is frequently associated with a variable spectrum of somatic features including ovarian failure, heart and renal abnormalities, micrognathia, cubitus valgus, high-arched palate, short metacarpals and Madelung deformity. Madelung deformity is also a key feature of Leri-Weill syndrome. Defects of the pseudoautosomal homeobox gene SHOX were previously shown to lead to short stature and Leri-Weill syndrome, and haploinsufficiency of SHOX was implicated to cause the short stature phenotype in Turner syndrome. Despite exhaustive searches, no direct murine orthologue of SHOX is evident. SHOX is, however, closely related to the SHOX2 homeobox gene on 3q, which has a murine counterpart, Og12x. We analysed SHOX and SHOX2 expression during human embryonic development, and referenced the expression patterns against those of Og12x. The SHOX expression pattern in the limb and first and second pharyngeal arches not only explains SHOX -related short stature phenotypes, but also for the first time provides evidence for the involvement of this gene in the development of additional Turner stigmata. This is strongly supported by the presence of Turner-characteristic dysmorphic skeletal features in patients with SHOX nonsense mutations. PMID- 10749977 TI - Doublecortin mutations cluster in evolutionarily conserved functional domains. AB - Mutations in the X-linked gene doublecortin ( DCX ) result in lissencephaly in males or subcortical laminar heterotopia ('double cortex') in females. Various types of mutation were identified and the sequence differences included nonsense, splice site and missense mutations throughout the gene. Recently, we and others have demonstrated that DCX interacts and stabilizes microtubules. Here, we performed a detailed sequence analysis of DCX and DCX-like proteins from various organisms and defined an evolutionarily conserved Doublecortin (DC) domain. The domain typically appears in the N-terminus of proteins and consists of two tandemly repeated 80 amino acid regions. In the large majority of patients, missense mutations in DCX fall within the conserved regions. We hypothesized that these repeats may be important for microtubule binding. We expressed DCX or DCLK (KIAA0369) repeats in vitro and in vivo. Our results suggest that the first repeat binds tubulin but not microtubules and enhances microtubule polymerization. To study the functional consequences of DCX mutations, we overexpressed seven of the reported mutations in COS7 cells and examined their effect on the microtubule cytoskeleton. The results demonstrate that some of the mutations disrupt microtubules. The most severe effect was observed with a tyrosine to histidine mutation at amino acid 125 (Y125H). Produced as a recombinant protein, this mutation disrupts microtubules in vitro at high molar concentration. The positions of the different mutations are discussed according to the evolutionarily defined DC-repeat motif. The results from this study emphasize the importance of DCX-microtubule interaction during normal and abnormal brain development. PMID- 10749978 TI - Allele diversity and germline mutation at the insulin minisatellite. AB - Previous analysis of germline mutation at highly unstable GC-rich minisatellites with continuous allele size distributions revealed similar meiotic recombinational mechanisms operating at all loci investigated. The insulin minisatellite has been studied intensively due to its associations with diabetes, polycystic ovary syndrome, obesity and birth size. Its bimodal allele size distribution in Caucasians suggests a much lower mutation rate and possible differences in the mutation process compared with highly unstable minisatellites. Mutation at the insulin minisatellite therefore was studied both indirectly from allele diversity surveys and directly by recovering de novo mutants from sperm DNA. Structural analysis of variant repeat distributions in 876 alleles identified 189 different alleles, almost all of which could be assigned to one of three very distinct lineages. Variation within a lineage was minor and due mainly to the gain or loss of one or a few repeat units. These events most probably arise by mitotic replication slippage at a frequency of perhaps 10(-3)per gamete. Sperm DNA analysis revealed a second class of mutation occurring at a frequency of approximately 2 x 10(-5)that involved highly complex intra- and inter-allelic rearrangements very similar to those seen at unstable minisatellites. These complex rearrangements were not seen in somatic DNA and are probably meiotic in origin. Minisatellite homozygosity did not reduce the frequency of these mutants in sperm. The insulin minisatellite therefore appears to evolve by two distinct processes: one involving slippage-like events and the second resulting in complex recombinational turnover of allele structure. PMID- 10749979 TI - High resolution analysis of haplotype diversity and meiotic crossover in the human TAP2 recombination hotspot. AB - Little is known about the nature of recombination hotspots in the human genome and the relationship between crossover activity and patterns of linkage disequilibrium. We have therefore used both haplotype analysis and direct detection of crossovers in sperm to characterize a putative recombination hotspot in the TAP2 gene within the class II region of the MHC. Haplotype diversity provided evidence for a localized hotspot within intron 2 of this gene. Sperm DNA typing using allele-specific PCR primers to selectively amplify recombinant TAP2 molecules revealed a highly localized meiotic crossover hotspot approximately 1.2 kb long, unusually abundant in sequence polymorphisms and flanked by DNA much less active in recombination. Sperm crossover appeared to be fully reciprocal, and almost all crossover products were simple, involving a single exchange between adjacent heterozygous markers. This hotspot appears to be much more active in female than male meiosis. No primary sequence similarities could be found between any of the very few well defined crossover hotspots in the human genome, all of which show recombinationally active domains 1-2 kb long. Direct comparison of recombination frequency and haplotype diversity in TAP2 showed that linkage disequilibrium measures were a poor predictor of crossover frequency in this region, with non-recombining markers sometimes in free association and with examples of pairs of markers spanning the recombination hotspot showing substantial or even absolute linkage disequilibrium. PMID- 10749981 TI - Expression analysis of endoglin missense and truncation mutations: insights into protein structure and disease mechanisms. AB - Hereditary hemorrhagic telangiectasia (HHT) is an inherited autosomal dominant vascular dysplasia caused by mutations in either endoglin (HHT1) or activin-like kinase receptor-1 (ALK-1) (HHT2). The majority of the mutations in endoglin cause frameshifts and premature stop codons. Although initial reports suggested a dominant-negative model for HHT1, more recent reports have suggested that mutations in endoglin lead to haploinsufficiency. In this study, we investigated six different missense mutations and two truncation mutations in the endoglin gene to examine whether mechanisms other than haploinsufficiency might be involved in HHT1. Expression of the missense mutants alone revealed that they are misfolded and that most show no cell surface expression. When co-expressed with wild-type endoglin, the missense mutants are able to dimerize with the normal endoglin protein and are trafficked to the cell surface. We also show that although one truncation mutation acts through haploinsufficiency, the other acts in a dominant-negative way. This implies that either dominant-negative protein interactions or haploinsufficiency can cause HHT1. The biochemical analyses for the different mutations suggest that the endoglin N-terminus is important for correct protein folding and that cysteine residues in the first 350 amino acids are involved in intramolecular disulfide bonds, whereas cysteines located closer to the C-terminus of the extracellular domain are responsible for inter-molecular disulfide bond dimerization. PMID- 10749980 TI - Batten disease: evaluation of CLN3 mutations on protein localization and function. AB - Juvenile neuronal ceroid lipofuscinosis (JNCL), Batten disease, is an autosomal recessive lysosomal storage disease associated with mutations in CLN3. CLN3 has no known homology to other proteins and a function has not yet been described. The predominant mutation in CLN3 is a 1.02 kb genomic deletion that accounts for nearly 85% of the disease alleles. In this mutation, truncation of the protein by a premature stop codon results in the classical phenotype. Additional missense and nonsense mutations have been described. Some missense substitutions result in a protracted phenotype, with delays in the onset of classical clinical features, whereas others lead to classical JNCL. In this study, we examined the effect of naturally occurring point mutations on the intracellular localization of CLN3 and their ability to complement the CLN3-deficient yeast, btn1-Delta. We also examined a putative farnesylation motif thought to be involved in CLN3 trafficking. All of the point mutations, like wild-type CLN3, were highly associated with lysosome-associated membrane protein II in non-neuronal cells and with synaptophysin in neuronal cell lines. In the yeast functional assay, point mutations correlating with a mild phenotype also demonstrated CLN3 activity, whereas the mutations associated with severe disease failed to restore CLN3 function completely. CLN3 with a mutation in the farnesylation motif trafficked normally but was functionally impaired. These data suggest that these clinically relevant point mutations, causative of Batten disease, do not affect protein trafficking but rather exert their effects by impairing protein function. PMID- 10749982 TI - Retention of imprinting of the human apoptosis-related gene TSSC3 in human brain tumors. AB - Genomic imprinting is the result of a gamete-specific modification leading to parental origin-specific gene expression in somatic cells of the offspring. Several embryonal tumors show loss of imprinting of genes clustered in human chromosome 11p15.5, an important tumor suppressor gene region, harboring several normally imprinted genes. TSSC3, a gene homologous to mouse TDAG51, implicated in Fas-mediated apoptosis, is also located in this region between hNAP2 and p57 (KIP2). TSSC3 is the first apoptosis-related gene found to be imprinted in placenta, liver and fetal tissues where it is expressed from the maternal allele in normal human development. This study investigated the imprinting status of TSSC3 in human normal, adult brain and in human neuroblastomas, medulloblastomas and glioblastomas. A polymorphism in exon 1 at position 54 was used to analyze the allelic expression of the TSSC3 gene by a primer oligo base extension (PROBE) assay using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We found that the TSSC3 gene is not imprinted in human normal, adult brain and blood. In contrast, strong allelic bias resembling imprinting could be detected in most examined tumor specimens. The results demonstrate for the first time that the tumors under investigation are associated with a retention of imprinting of a potential growth inhibitory gene. PMID- 10749983 TI - Germline and germline mosaic PTEN mutations associated with a Proteus-like syndrome of hemihypertrophy, lower limb asymmetry, arteriovenous malformations and lipomatosis. AB - Germline PTEN mutations cause Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome (BRR), two hamartoma-tumour syndromes, and somatic PTEN alterations have been shown to participate, to a greater or lesser extent, in a wide variety of sporadic neoplasia. PTEN is a tumour suppressor and dual-specificity phosphatase which affects apoptosis via its lipid phosphatase activity in the phosphoinositol 3-kinase and AKT pathway as well as inhibiting cell spreading via the focal adhesion kinase pathway. CS and BRR share some features, such as hamartomas and lipomatosis. To determine whether other syndromes characterized by overgrowth and lipomas are part of the PTEN syndrome spectrum, we ascertained six individuals with overgrowth and lipomas but who did not meet the diagnostic criteria for CS or BRR. Five had Proteus syndrome and one, a Proteus-like syndrome. When germline DNA and DNA from at least one involved tissue per case were examined for PTEN mutations, only the Proteus-like patient was found to harbour a germline R335X mutation. Interestingly, a lipomatous mass, an epidermoid naevus and arteriovenous malformation tissue, all of which were sampled from physically distinct sites, were all found to carry a second hit R130X mutation on the allele opposite the germline R335X. Both mutations have been described in CS and BRR. We postulate that the second hit, R130X, occurred early in embryonic development and may even represent germline mosaicism. Thus, PTEN may be involved in Proteus-like syndrome with its implications for cancer development in the future. PMID- 10749984 TI - Germline and somatic loss of function of the mouse cpk gene causes biliary ductal pathology that is genetically modulated. AB - The mouse cpk mutation is the most extensively characterized murine model of polycystic kidney disease (PKD) and closely resembles human autosomal recessive PKD (ARPKD), with the exception that B6-cpk/cpk homozygotes do not express the biliary ductal plate malformation (DPM) lesion. However, homozygous mutants from outcrosses to other strains, e.g. DBA/2J (D2), CD-1, BALB/c and Mus mus castaneus (CAST), express the DPM. The current study was designed: (i) to characterize the cpk-associated biliary disease in affected F(2) homozygotes from intercrosses with either CAST or D2; and (ii) to evaluate focal biliary cysts identified in heterozygotes from a D2-cpk congenic strain. We found that all F(2) cpk/cpk pups expressed both the typical renal cystic disease and the DPM. The DPM severity, assessed using semi-quantitative histopathological analysis, was markedly variable in these F(2) progeny. We found no correlation between the severity of the DPM and the renal cystic disease in either F(2) cohort. In addition, we identified focal cysts, apparently of biliary origin, in the livers of both aged D2-+/cpk and F(1) heterozygotes. Genetic analysis demonstrated loss of heterozygosity at the cpk interval and supports a loss-of-function model for biliary cysts. We conclude that the cpk allele contains an inactivating mutation which disrupts tubulo-epithelial differentiation in the kidney and biliary tract. Expression of the biliary lesion is modulated by genetic background, and the specific biliary phenotype is determined by whether loss of function of the cpk gene occurs as a germline or a somatic event. PMID- 10749985 TI - Repeat instability and motor incoordination in mice with a targeted expanded CAG repeat in the Sca1 locus. AB - To elucidate the pathophysiology of spinocerebellar ataxia type 1 (SCA1) and to evaluate repeat length instability in the context of the mouse Sca1 gene, we generated knock-in mice by inserting an expanded tract of 78 CAG repeats into the mouse Sca1 locus. Mice heterozygous for the CAG expansion show intergenerational repeat instability (+2 to -6) at a much higher frequency in maternal transmission than in paternal transmission. The majority of changes transmitted through the female germline were small contractions, as in humans, whereas small expansions occurred more frequently in paternal transmission. The frequency of intergenerational changes was age dependent for both paternal and maternal transmissions. Mice homozygous for mutant ataxin-1 on a C57BL/6J-129/SvEv mixed background performed significantly less well on the rotating rod than did wild type littermates at 9 months of age, although they were not ataxic by cage behavior. Histological examination of brain tissue from mutant mice up to 18 months of age revealed none of the neuropathological changes observed in other transgenic models overexpressing expanded polyglutamine tracts. These data suggest that, even with 78 glutamines, prolonged exposure to mutant ataxin-1 at endogenous levels is necessary to produce a neurological phenotype reminiscent of human SCA1. Pathogenesis is thus a function of polyglutamine length, protein levels and duration of neuronal exposure to the mutant protein. PMID- 10749986 TI - Genetic localization of an autosomal dominant leukodystrophy mimicking chronic progressive multiple sclerosis to chromosome 5q31. AB - The hereditary leukodystrophies represent a group of neurological disorders, in which complete or partial dysmyelination occurs in either the central nervous system (CNS) and/or the peripheral nervous system. Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive, neurological disorder characterized by symmetrical widespread myelin loss in the CNS, and the phenotype is similar to that of chronic progressive multiple sclerosis. We report clinical, neuroradiological and neuropathological data from the originally reported ADLD family. Furthermore, we have localized the gene that causes ADLD to a 4 cM region on chromosome 5q31. Linkage analysis of this family yielded a LOD score of 5.72 at theta = 0.0 with the microsatellite marker D5S804. Genetic localization will lead to cloning and characterization of the ADLD gene and may yield new insights into myelin biology and demyelinating diseases. PMID- 10749987 TI - Mutations in SCO2 are associated with a distinct form of hypertrophic cardiomyopathy and cytochrome c oxidase deficiency. AB - Mutations in SCO2, a cytochrome c oxidase (COX) assembly gene located on chromosome 22, have recently been reported in patients with fatal infantile cardio-encephalomyopathy and severe COX deficiency in heart and skeletal muscle. The Sco2 protein is thought to function as a copper chaperone. To investigate the extent to which mutations in SCO2 are responsible for this phenotype, a complete sequence analysis of the gene was performed on ten patients in nine families. Mutations in SCO2 were found in three patients in two unrelated families. We detected two missense mutations, one of which (G1541A) results in an E140K substitution adjacent to the highly conserved CxxxC metal-binding site. The other (C1634T) results in an R171W substitution more distant from the copper-binding site. A nonsense codon was found on one allele in two siblings presenting with a rapidly progressive fatal cardio-encephalomyopathy. Interestingly, all patients so far reported are compound heterozygotes for the G1541A mutation, suggesting that this is either an ancient allele or a mutational hotspot. The COX deficiency in patient fibroblasts (approximately 50%) did not result in a measurable decrease in the steady-state levels of COX complex polypeptide subunits and could be rescued by transferring chromosome 22, but not other chromosomes. These data indicate that mutations in SCO2 cause a fatal infantile mitochondrial disorder characterized by hypertrophic cardiomyopathy and encephalopathy, and point to the presence of one or more other genes, perhaps in the copper delivery pathway, in this clinical phenotype. PMID- 10749988 TI - Increased motoneuron survival and improved neuromuscular function in transgenic ALS mice after intraspinal injection of an adeno-associated virus encoding Bcl-2. AB - Mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) underlie some familial cases of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by loss of cortical, brainstem and spinal motoneurons. Transgenic mice over- expressing a mutated form of human SOD1 containing a Gly- >Ala substitution at position 93 (SOD1(G93A)) develop a severe, progressive motoneuron disease. We investigated the potential of recombinant adeno-associated virus (rAAV) to transfer neuroprotective molecules in this animal ALS model. Initial experiments showed that injection of an rAAV vector encoding green fluorescent protein unilaterally into the lumbar spinal cord of wild-type mice leads to expression of the reporter gene in 34.7 +/- 5.2% of the motoneurons surrounding the injection site. Intraspinal injection of an rAAV encoding the anti-apoptotic protein bcl-2 in SOD1 (G93A) mice resulted in sustained bcl-2 expression in motoneurons and significantly increased the number of surviving motoneurons at the end-stage of disease. Moreover, the compound muscle action potential amplitude elicited by nerve stimulation and recorded by electromyographic measurements was higher in the rAAV-bcl-2-treated group than in controls. Local bcl-2 expression in spinal motoneurons delayed the appearance of signs of motor deficiency but was not sufficient to prolong the survival of SOD1 (G93A) mice. To our know-ledge, this study describes the first successful transduction and protection of spinal motoneurons by direct gene transfer in a model of progressive motoneuron disease. Our results support the use of AAVs for the delivery of protective genes to spinal cord moto-neurons as a possible way to enhance motoneuron survival and repair. PMID- 10749989 TI - Saethre-Chotzen mutations cause TWIST protein degradation or impaired nuclear location. AB - H-TWIST belongs to the family of basic helix-loop-helix (bHLH) transcription factors known to exert their activity through dimer formation. We have demonstrated recently that mutations in H-TWIST account for Saethre-Chotzen syndrome (SCS), an autosomal dominant craniosynostosis syndrome characterized by premature fusion of coronal sutures and limb abnormalities of variable severity. Although insertions, deletions, nonsense and missense mutations have been identified, no genotype-phenotype correlation could be found, suggesting that the gene alterations lead to a loss of protein function irrespective of the mutation. To assess this hypothesis, we studied stability, dimerization capacities and subcellular distribution of three types of TWIST mutant. Here, we show that: (i) nonsense mutations resulted in truncated protein instability; (ii) missense mutations involving the helical domains led to a complete loss of H-TWIST heterodimerization with the E12 bHLH protein in the two-hybrid system and dramatically altered the ability of the TWIST protein to localize in the nucleus of COS-transfected cells; and (iii) in-frame insertion or missense mutations within the loop significantly altered dimer formation but not the nuclear location of the protein. We conclude that at least two distinct mechanisms account for loss of TWIST protein function in SCS patients, namely protein degradation and subcellular mislocalization. PMID- 10749991 TI - Longer polyglutamine tracts in the androgen receptor are associated with moderate to severe undermasculinized genitalia in XY males. AB - The androgen receptor (AR) is essential to the normal development of the male internal and external genitalia. Consequently, impairment of AR function can result in undermasculinized genitalia that vary from a completely female appearance to isolated hypospadias. Since in vitro studies demonstrate that AR function is reduced by expansion of the polyglutamine tract within the receptor [AR(Gln)(n)]; this study examined whether longer AR(Gln)(n) repeats are associated with moderate to severe undermasculinization. The average AR(Gln)(n) length of the undermasculinized group (n = 78, median 25, interquartile range 23 26) was significantly greater than that of the control population (n = 850, median 23, inter-quartile range 22-26, P = 0.002). The odds ratio of having >/=23 repeats (as opposed to EcR A>>EcR-B2. Upon co-expression with ultraspiracle (Usp), transcriptional activation was further increased with EcR-B1 or EcR-A, but was unchanged with EcR B2 or a truncated EcR lacking the A/B N-terminal domain (EcRDeltaA/B). Thus, the enhanced activity from Usp may depend on the presence of an N-terminal domain of EcR. Co-expression with Usp of several chimeric receptors of the EcR and the mouse androgen receptor (mAR) identified one chimera, composed of the mAR N terminus and the remainder from EcR (mAR?EcR-CDEF) that was transcriptionally silent and inducible by Usp. In contrast, the vertebrate homologue, human retinoic acid receptor (RXRalpha), showed ligand-independent transcription when co-expressed with EcRDeltaA/B but not mAR? EcR-CDEF. Therefore, RXRalpha does not require its partner to possess an N-terminal domain, yet is intolerant of a heterologous N-terminus. Similarly, the human vitamin D receptor, which has a short N-terminal region, showed greater ligand-independent transcription in the presence of RXRalpha than in the presence of Usp. These results reveal a mechanistic basis for the differential activities among the EcR isoforms, and between Usp and RXRalpha. Furthermore, they provided the foundation for a genetic screen to identify potential insecticides as well as accessory proteins for Usp and EcR. PMID- 10750020 TI - Transcriptional activation of cathepsin D gene expression by growth factors. AB - Insulin-like growth factor-I (IGF-I), transforming growth factor alpha (TGFalpha) and epidermal growth factor (EGF) induced cathepsin D gene expression and reporter gene activity in MCF-7 human breast cancer cells transiently transfected with a construct (pCD1) containing a -2576 to -124 cathepsin D gene promoter insert. In contrast, IGF-I, but not TGFalpha or EGF, induced reporter gene activity in cells cotransfected with wild-type estrogen receptor (ER) expression plasmid and a construct (pCD2) containing estrogen-responsive downstream elements from -208 to -101. Promoter deletion and mutational analysis experiments identified four GC-rich sites and an imperfect palindromic estrogen responsive element required for IGF-I activation of the ER (ligand-independent). Subsequent studies with the mitogen-activated protein kinase (MAPK) inhibitor, PD98059, and a serine(118(-ER mutant confirmed the role of the MAPK pathway for IGF-I activation of the ER in MCF-7 cells. Thus, growth factor activation of ER can mediate transactivation vs ER/Sp1 binding to GC-rich sites and represents a novel pathway for ligand-independent ER action. The divergent pathways for IGF-I and TGFalpha/EGF activation of the ER observed in MCF-7 cells contrast with previous data indicating that pathways for growth factor activation of the ER are dependent on the gene and/or gene promoter and on cell context. PMID- 10750021 TI - Identification of Sp1 as the transcription factor for the alternative promoter P2 of the bovine growth hormone receptor gene. AB - Growth hormone receptor (GHR) mRNA variants that differ in the 5'-untranslated regions (5'-UTR) have been isolated in various species. These 5'-UTR variants are generated from the use of alternative promoters and/or alternative splicing. The 5'-UTR 1B is one of the GHR 5'-UTR variants isolated in the bovine but its homologues are also present in other species. The 5'-UTR 1B is a predominant GHR 5'-UTR expressed in many tissues. In the present study, we screened a bovine genomic library and isolated a 1.7 kb bovine GHR genomic sequence including exon 1B and its 5' flanking region from which the GHR 5'-UTR 1B is generated. Using primer extension, two major transcription start sites were mapped in the bovine exon 1B. Transient transfection analysis of the 5' flanking region of exon 1B confirmed its promoter activity (termed P2) in both Hep G2 and BHK-21 cells. Furthermore, analysis of deletion promoter-reporter constructs found that the basal activity of P2 resided in the proximal region of P2. DNase I footprinting analysis and electromobility shift assay (EMSA) identified the ubiquitous transcription factor Sp1 as the binding protein to a GC box-containing DNA element within the proximal P2. Deletion of the GC box greatly reduced the activity of P2 in cell lines. The GC box-containing site also appeared to bind Sp1 in the nuclear extracts from diverse bovine tissues. This suggests that interactions of Sp1 with the GC box-containing element in the proximal region of P2 may be part of the mechanism for the expression of the bovine GHR 5'-UTR 1B in diverse tissues. PMID- 10750022 TI - Expression of the prolactin receptor (tiPRL-R) gene in tilapia Oreochromis niloticus: tissue distribution and cellular localization in osmoregulatory organs. AB - The expression of the prolactin receptor (PRL-R) gene has been investigated in various tissues of tilapia (Oreochromis niloticus) reared in fresh or brackish water. Using a cDNA probe spanning the extracellular domain of the tilapia PRL-R and Northern blot analysis, the presence of tilapia PRL-R mRNA has been confirmed in the osmoregulatory organs and has been detected in other tissues, including the skin, the brain, the reproductive organs, and the two major hematopoietic organs (spleen and head kidney), as well as circulating lymphocytes. These findings suggest a conservation of the physiological processes regulated by prolactin throughout the vertebrates, including immunity and central nervous activity. A non-radioactive in situ hybridization procedure has allowed us to detect the expression of the tilapia PRL-R in the branchial chloride cells and the intestinal mucosal layer of fresh water animals, confirming the direct control exerted by prolactin on the water and ionic exchanges in tilapia. In all the tissues examined one unique PRL-R transcript has been detected with a similar size (3.2 kb) whatever the salinity conditions. Thus, the transcriptional expression of the tilapia PRL-R strongly differs from the complex RNA pattern reported for the higher vertebrates PRL-R and provides an additional argument for the existence of a single PRL-R for both prolactin isoforms in this fish species. PMID- 10750023 TI - Analysis of the 5'-upstream region of mouse P/Q-type Ca2+ channel alpha1A subunit gene for expression in pancreatic islet beta cells using transgenic mice and HIT T15 cells. AB - The omega-agatoxin-IVA-sensitive P/Q-type Ca(2+) channel plays a role in insulin release from the pancreatic islets of beta cells. To dissect the molecular mechanisms underlying beta cell expression of the P/Q-type channel, we characterized the 5'-upstream region of the mouse alpha(1A) subunit gene using transgenic mice and HIT insulinoma cells. The E. coli lacZ reporter gene was expressed in pancreatic acini and islets in transgenic mice carrying the 6.3 kb or 3.0 kb of the 5'-upstream region, although those with 1.5 kb or 0. 5 kb of the 5'-upstream region failed to show reporter expression on histological examination. As the expression of alpha(1A)subunit gene could not be detected in acini using RT-PCR analysis, the reporter expression in acini might have been ectopic expression. When linked to the placental alkaline phosphatase reporter gene to examine promoter activity for beta cell expression, the 6.3 kb and 3.0 kb fragment of the 5'-upstream region, but not the smaller 1.5 kb fragment, were able to drive reporter gene expression in HIT cells. The sequence between 3.0 and 1.5 kb upstream of the start codon enhanced thymidine kinase promoter activity in HIT cells, but not in fibroblast NIH3T3 cells. These results suggested that the beta cell-specific elements of the alpha(1A) subunit gene are likely to be located in the distal upstream region (-3021 to-1563) of the 5'-upstream sequence and that the 6.3 kb fragment of the 5'-upstream region alone might be a lack of a negative cis-regulatory element(s) to suppress the alpha(1A) subunit gene expression in acini. PMID- 10750024 TI - Cyclic AMP-induced expression of steroidogenic acute regulatory protein is dependent upon phosphoprotein phosphatase activities. AB - In addition to the well-documented role of protein kinases in the regulation of steroid production, phosphoprotein phosphatase (PP) activity is required for steroidogenesis. In the present study, we have used the mouse Y1 adrenocortical cell line to identify the site of action of PPs on steroid production by measuring the effects of PP inhibition on the expression of the steroidogenic acute regulatory (StAR) protein and on steroid production. Forskolin-induced activation of cyclic AMP-dependent protein kinase (PKA) enhanced steroidogenesis and this was accompanied by an increased expression of StAR protein. Both steroidogenesis and StAR protein expression were inhibited by two structurally dissimilar inhibitors of PP1 and PP2A activities, okadaic acid and calyculin A. These results suggest that inhibition of PP1 and PP2A inhibits steroid production by preventing the expression of the StAR protein, implicating PP1/2A dephosphorylation reactions as important regulators of stimulus-dependent StAR protein expression, and thus of steroidogenesis. PMID- 10750025 TI - Isolation and analysis of the 3'-untranslated regions of the human relaxin H1 and H2 genes. AB - The human has two relaxins, termed H1 and H2, both of which are biologically active and co-expressed in the decidua, placenta and prostate; in the corpus luteum, the main source of circulating relaxin, only the H2 form is expressed. The reasons for this differential expression of the relaxin genes are unknown. The possibility that their 3'-untranslated regions (UTRs) contribute to this differential expression by affecting their mRNA stabilities was investigated. Thus the 3'-UTRs of both relaxin genes were isolated through a combined 3'-rapid amplification of cDNA ends-PCR (RACE-PCR) using poly (A)(+)RNA from human decidua, placenta, prostate and corpus luteum. The sequences obtained for each 3' UTR were identical in the tissues examined, were AT-rich (72%) and showed 91% homology between relaxin H1 and H2 when maximally aligned to include several gaps, the significance of which is unknown. Relaxin H1 has two, and relaxin H2 has one, poly (A)(+) signal, in addition to one cytoplasmic polyadenylation element 30 nucleotides upstream of this. The mRNA levels of relaxin H1 and H2 in the prostate adenocarcinoma LNCaP.FGC cell line were determined by quantitative competitive RT-PCR. Relaxin H1 had a 10-fold greater number of molecules (approximately 2.5x10(7)) per microgram of total RNA than relaxin H2 (approximately 2.5x10(6)). The stability of relaxin H1 and H2 mRNAs were compared in LNCaP cells treated with the transcription inhibitor actinomycin D (10 mM) for 0, 1, 2, 4, 8, 10, 14, or 24 h. Half-lives of 3.17 days for relaxin H1 mRNA and 11. 4 h for relaxin H2 mRNA were obtained from semi-logarithmic plots. Thus both mRNAs are relatively stable; however, relaxin H1 mRNA is considerably more stable than relaxin H2, at least in LNCaP cells. This difference in their mRNA stability may partly explain the greater level of expression of relaxin H1 in these cells. PMID- 10750026 TI - Androgen ablation results in differential regulation of transforming growth factor-beta isoforms in rat male accessory sex organs and epididymis. AB - The male accessory sex organs and epididymis regress following androgen depletion, although the onset of apoptosis varies temporally depending upon the tissue type. Transforming growth factor-beta1 (TGF-beta1) is an androgen repressed gene and believed to be an apoptotic agent in the regressing rat ventral prostate (VP). Hence, in order to investigate the status of TGF-beta isoforms following castration in androgen-dependent tissues other than VP, this study was undertaken. Northern blot analysis using total RNA from these tissues of intact animals showed higher levels of TGF-beta1 expression as compared with VP, indicating a function other than that of an apoptotic agent for this isoform. Following orchiectomy, TGF-beta1 was induced in all organs studied and the levels were highest at day 3 following castration in seminal vesicle (SV) and the epididymis and decreased by day 5 despite the absence of androgens. This observation implies that TGF-beta1 might not be a truly androgen-repressed gene in these tissues. TGF-beta2 was up-regulated in VP, SV, caput and corpus epididymis but was undetectable in the dorsolateral prostate and cauda epididymis. On the other hand, TGF-beta3 expression was refractory to the androgen status in corpus epididymis and SV but was up-regulated in the remaining tissues. The castration-induced induction of mRNAs was attenuated after exogenous androgen administration. Most importantly, all the isoforms differed significantly in the time and magnitude of induction following castration, suggesting that a single hormone, testosterone, modulates the expression of TGF betas in an isoform- and tissue-specific manner. PMID- 10750027 TI - Molecular cloning of equine transforming growth factor-beta1 reveals equine specific amino acid substitutions in the mature peptide sequence. AB - This study cloned and sequenced equine transforming growth factor (TGF)-beta1, yielding a unique nucleotide structure which predicted amino acid substitutions not seen in other mammalian species. The nucleotide sequence homology was 89% to bovine, 91% to man, 90% to ovine, and 86% to rat. Derived amino acid sequence comparison showed that the equine protein was unique, differing by two residues from man, cow, sheep, pig, and dog, and by three residues in the rat. Subsequent use of the cDNA clones to examine the expression of the TGF-beta1 gene in various tissues indicated predominant expression in adult spleen and kidney, with an age related peak in cartilage expression at 12 months, followed by a decline as the animals matured. Northern blots showed that the predominant transcript sizes were 2.5 and 1.9 kb. More sensitive mRNA detection using PCR reaction showed peak cartilage TGF-beta mRNA levels in horses 0.7 and 1 year of age, with declining expression in older animals (2.5 and 5.5 years of age). In conclusion, although the primary nucleotide sequence of equine TGF-beta was relatively homologous to that of other species, the resulting amino acid sequence was unique to the horse, differing by two residues from the majority of mammalian sequences, where the peptide structure is identical. Expression of TGF-beta was particularly evident in spleen and kidney, and showed an age-related increase in expression in cartilage as the animals approached maturity and then a decline with progressive aging. PMID- 10750029 TI - Structural characterization and effects on corticosteroid secretion of endothelin 1 and endothelin-3 from the frog Rana ridibunda. AB - ABSTRACT Despite the intensive study of endothelin (ET) in mammals, the primary structure and biological activity of the peptide is not known for any species of non-mammalian tetrapod. Extracts of the stomach and the liver of the European green frog Rana ridibunda contained ET-like immunoreactivity measured by RIA using an antiserum raised against human ET-1. The amino acid sequence of the peptide that was isolated in pure form from the stomach extract was identical to that of human ET-1 and the peptide purified from the liver extract was identical to human ET-3 except for a single amino acid substitution (Phe(4)-->Tyr). These observations demonstrate that the amino acid sequences of ET family peptides have been very strongly conserved during evolution of tetrapods and suggest that the pathway of post-translational processing of preproendothelin in the frog is similar to that in mammals. Both frog/human ET-1, frog ET-3 and human ET-3 produced a concentration-dependent increase in the production of corticosteroids from perifused slices of the frog interrenal gland. The maximum responses produced by the peptides (approximately 2-fold increase over basal levels for both corticosterone and aldosterone production) were not significantly different. The potency of ET-1 (-log EC(50)=9.81+/-0.01 (s.e.m.) for corticosterone and 9.52+/-0.29 for aldosterone production) was significantly (P<0.01) greater than that of frog ET-3 (-log EC(50)=8.13+/-1.6 for corticosterone and 8.15+/-0.33 for aldosterone production) but the potencies of frog ET-3 and human ET-3 (-log EC(50)=8.29+/-0.34 and 7.87+/-0.18) were not significantly different. PMID- 10750028 TI - Endothelin-2 synthesis is stimulated by the type-1 tumour necrosis factor receptor and cAMP: comparison with endothelin-converting enzyme-1 expression. AB - ABSTRACT The synthesis of the vasoconstrictor peptide endothelin-2 (ET-2) is dependent on hydrolysis of the biologically inactive intermediate big ET-2 by an endothelin-converting enzyme (ECE). Here, mechanisms inducing ET-2 synthesis have been investigated using the human renal adenocarcinoma cell line (ACHN). Synthesis of ET-2 by ACHN cells was inhibited by phosphoramidon (IC(50( congruent with11 microM). To determine whether ET-2 synthesis occurs in parallel with the metallopeptidase ECE-1, a putative processing peptidase for big ET-2, changes in the levels of their mRNAs were compared by semi-quantitative RT-PCR under conditions causing the upregulation of ET-2 synthesis. Tumour necrosis factor alpha (TNFalpha), forskolin and a cell-permeable cAMP analogue (dibutyryl cAMP) caused concentration-dependent increases in ET-2 synthesis. Combination of forskolin or dibutyryl cAMP with TNFalpha produced a significantly greater increase in ET-2 production than these agents alone, indicating that adenylate cyclase and TNFalpha induce ET-2 synthesis by separate signalling pathways. Studies using receptor selective TNFalpha mutants, (125(I-TNFalpha binding and TNF receptor mRNA showed that type-1 TNF receptors mediate the ET-2 response to TNFalpha. PreproET-2 mRNA levels were increased by TNFalpha at 1 h and 2 h, but returned to control levels at 4 h. Treatment with forskolin significantly increased preproET-2 mRNA levels after 1 h and 4 h. ACHN cells expressed ECE-1b and ECE-1c, but not the ECE-1a isoform of this peptidase. RT-PCR for the combined isoforms ECE-1b/c/d showed TNFalpha to increase mRNA levels at 2 h and 4 h. Forskolin had no effect on ECE-1b/c/d mRNA levels. Thus, expression of ET-2 and ECE-1b/c/d mRNAs in ACHN cells do not display the co-ordinated regulation observed with typical peptide prohormone processing enzymes and their substrates. PMID- 10750030 TI - Thyroid hormone and central nervous system development. PMID- 10750031 TI - Effect of thyroid hormone on mtHsp70 expression, mitochondrial import and processing in cardiac muscle. AB - Mitochondrial heat shock protein 70 (mtHsp70), an important mitochondrial chaperone, is increased in cardiac muscle mitochondria of hyperthyroid rats. To determine the mechanism(s) underlying this increase, we used variations in thyroid status. In Series I, rats were made hyperthyroid by injecting them with 3,3', 5-triiodo-l-thyronine (T(3)) for 5 days, or by treating them with vehicle. In Series II, animals were given 6-n-propyl-2-thiouracil in their drinking water (0.05% w/v) for a period of 32-42 days to make them hypothyroid. During the last 5 days of treatment these animals received injections of either T(3) or vehicle. T(3) treatment resulted in parallel increases in mtHsp70 protein and mRNA levels in a variety of tissues, suggesting transcriptional regulation. However, evidence of tissue-specific post-transcriptional regulation was also apparent. In isolated heart mitochondria, T(3) treatment resulted in a 1.8-fold increase in mtHsp70. This was due to the 1. 6-fold greater import of mtHsp70 into mitochondria in T(3), compared with hypothyroid animals, and it could not be attributed to an altered rate of intramitochondrial mtHsp70 degradation. The rate of processing of mtHsp70 to its mature form, reflecting mitochondrial processing peptidase activity, was unaffected by T(3), but was more rapid than mtHsp70 import. These data indicate a novel mechanism by which T(3) modifies the mitochondrial phenotype via the adaptations in the protein import pathway. PMID- 10750032 TI - Contribution of endogenous oxytocin to sodium excretion in anaesthetized, surgically operated rats. AB - In order to determine the possible role of endogenous oxytocin in controlling electrolyte and water excretion in animals whose renal function is being assessed by invasive techniques, rats were anaesthetized and subjected to micropuncture surgery. Clearance measurements were made in the presence and absence of the potent oxytocin receptor antagonist d(CH(2))(5)[Tyr(Me)(2), Thr(4), Orn(8), Tyr(NH(2))(9)]-vasotocin. In rats infused with vehicle alone, glomerular filtration rate (GFR), sodium excretion and urine flow rate remained stable. In contrast, in antagonist-treated rats GFR was modestly reduced (P<0.05), and there were large falls in both absolute and fractional sodium excretion (P<0.01 in each case) and absolute and fractional water excretion (P<0.05 in each case), indicating effects on both filtered load and fractional tubular reabsorption. The antinatriuresis was not accompanied by a change in the fractional excretion of lithium, suggesting that proximal tubular function is unaffected by oxytocin receptor antagonism; nor was it accompanied by a change in the fractional excretion of potassium, suggesting that the tubular effect is located beyond the potassium secretory site, i.e. downstream of the cortical collecting tubule. We conclude that circulating plasma concentrations of oxytocin during anaesthesia and moderate surgery are sufficient to enhance GFR and reduce fractional tubular sodium and water reabsorption. This has important implications for the interpretation of invasive studies such as micropuncture. PMID- 10750033 TI - Corticosteroid regulation of amiloride-sensitive sodium-channel subunit mRNA expression in mouse kidney. AB - Corticosteroid control of distal nephron sodium handling, particularly through the amiloride-sensitive sodium channel (ENaC), has a key role in blood pressure regulation. The mechanisms regulating ENaC activity remain unclear. Despite the generation of useful mouse models of disorders of electrolyte balance and blood pressure, there has been little study of distal nephron sodium handling in this species. To investigate how corticosteroids regulate ENaC activity we isolated cDNA for the three mouse ENaC subunits (alpha, beta and gamma), enabling their quantitation by competitive PCR and in situ hybridisation. Kidneys were analysed from mice 6 days after adrenalectomy or placement of osmotic mini-pumps delivering aldosterone (50 microg/kg per day), dexamethasone (100 microg/kg per day), spironolactone (20 mg/kg per day) or vehicle alone (controls). In controls, renal ENaCalpha mRNA exceeded beta or gamma by approximately 1.75- to 2.8-fold. All subunit mRNAs were expressed in renal cortex and outer medulla, where the pattern of expression was fully consistent with localisation in collecting duct, whereas the distribution in cortex suggested expression extended beyond the collecting duct into adjacent distal tubule. Subunit mRNA expression decreased from cortex to outer medulla, with a gradual reduction in beta and gamma, and ENaCalpha decreased sharply ( approximately 50%) across the outer medulla. Expression of ENaCbeta and gamma (but not alpha) extended into inner medulla, suggesting the potential for inner medulla collecting duct cation channels in which at least ENaCbetagamma participate. Aldosterone significantly increased ENaC subunit expression; the other treatments had little effect. Aldosterone caused a 1.9- to 3.5-fold increase in ENaCalpha (particularly marked in outer medullary collecting duct), but changes for beta and gamma were minor and limited to the cortex. The results raise the possibility that medullary ENaCalpha upregulation by aldosterone will create more favourable subunit stoichiometry leading to a more substantial increase in ENaC activity. In cortex, such a mechanism is unlikely to have a major role. PMID- 10750034 TI - Estrogen treatment in vivo increases keratinocyte growth factor expression in the mammary gland. AB - Proliferation and differentiation of mammary epithelia are regulated by the combined action of systemic hormones and locally derived paracrine growth factors. Keratinocyte growth factor (KGF) is a potential candidate stromal factor that may participate in the hormonal control of stromal/epithelial interactions. In this study, we have examined the in vivo effect of 17beta-estradiol (E) treatment on KGF expression in mammary glands of peripubertal (5-week-old) and mature (11-week-old) mice. Mice received subcutaneous injections of hormone after which KGF mRNA levels were assayed by ribonuclease protection analysis of mammary gland RNA. E treatment caused a dose- and time-dependent increase in KGF mRNA levels in intact mice from both age groups. Neither 17alpha-estradiol nor progesterone injection affected KGF mRNA levels. Comparison of the relative expression of KGF in parenchyma-free fat pads and in intact glands demonstrated that the basal and E-dependent KGF mRNA levels did not require the presence of mammary epithelium. ELISA assay of KGF tissue content demonstrated that concomitantly with an up-regulation of mRNA, E treatment also increased KGF protein in mammary glands from peripubertal and mature mice. These data show that E treatment stimulates both KGF mRNA and protein expression in mammary stroma in vivo and raises the possibility that KGF has a role in E-regulated mammary gland development. PMID- 10750035 TI - Localization of prostaglandin synthase type-1 (PGHS-1) mRNA and prostaglandin synthase type-2 (PGHS-2) mRNA in ovine myometrium and endometrium throughout gestation. AB - Increased prostaglandin production by tissues in the sheep uterus and placenta are thought to be important for the onset of parturition. In the sheep placenta, this is most likely due to increased expression of prostaglandin synthase type-2 (PGHS-2) rather than prostaglandin synthase type-1 (PGHS-1). However, there is no information concerning expression of PGHS isoenzymes in maternal uterine tissues during pregnancy. Therefore, the purpose of the present study was to examine the expression of PGHS-1 and PGHS-2 in the sheep myometrium and endometrium during late gestation using in situ hybridization and immunohistochemistry. Using (35)S labelled oligonucleotide probes, which give specific hybridization signals in other tissues, we localized PGHS-2 mRNA to endometrial epithelium, and apparently to other cells in both endometrium and myometrium. This artefactual signal was still present with 100-fold excess unlabelled oligonucleotide probe and with sense probes, but was resolved with the use of (33)P-oligonucleotides. Using (33)P-labelled oligonucleotide probes we could not detect either PGHS-1 or PGHS-2 mRNA in myometrium, and found expression only of PGHS-2 mRNA in endometrium. PGHS 2 mRNA localized to the endometrial epithelium and was undetectable in glandular epithelium. The level of PGHS-2 expression rose significantly between days 80 and 85 of pregnancy and term, and this corresponded to the appearance of immunoreactive PGHS-2 protein, measured by immunohistochemistry, in the endometrial epithelium. Therefore we conclude that (33)P-labelled probes are preferred for detection of mRNAs encoding PGHS-2 in ovine uterine tissues. Expression of PGHS-2 mRNA is greater than that of PGHS-1, increases during gestation, and predominates in the endometrial epithelium, consistent with the site of PGHS-2 protein localization. PMID- 10750036 TI - Differential distribution of oestrogen receptor-alpha and -beta mRNAs in the female reproductive organ of rats as revealed by in situ hybridization. AB - The cellular distribution of two oestrogen receptor (ER) subtypes, ERalpha and ERbeta mRNAs, was studied in the entire female reproductive organ of the rat using in situ hybridization. Expression of ERalpha and ERbeta mRNAs was predominant in the reproductive tract and ovary respectively. ERalpha mRNA had the most pronounced expression in epithelial cells and subepithelial stromal cells from the oviduct to the vagina, while in the ovary it was moderately detected in only the theca folliculi and interstitial glands. The oviduct showed a region-dependent expression of ERalpha mRNA: the isthmus had the most intense signals while the infundibulum revealed a low intensity of expression. Signals for ERbeta mRNA in the ovary were most intense in the granulosa cells of healthy follicles, whereas degenerating follicles lacked any significant expression. Less intense signals for ERbeta mRNA were localized in the theca folliculi and corpus luteum. Detectable levels of ERbeta mRNA were observed in the subepithelial stromal cells from the oviduct to the vagina. This study shows that the two ER subtypes are differentially expressed in cells and compartments of the reproductive organ, suggesting that the mediation of oestrogen action in these tissues may be accomplished through the respective predominant receptor. PMID- 10750037 TI - Expression of ErbB receptors during pancreatic islet development and regrowth. AB - We have characterized expression of the ErbB receptor family and one of its ligands, heregulin, in an effort to identify molecules associated with pancreatic development and regeneration. In addition to studying expression during fetal pancreatic development, we have also studied expression during pancreatic regeneration in the interferon-gamma (IFNgamma)-transgenic mouse, which exhibits significant duct cell proliferation and new islet formation. These studies demonstrate significant expression of the ErbB2, ErbB3, and ErbB4 receptors, in addition to heregulin isoforms, in the developing murine fetal pancreas. We also report significant ductal expression of these proteins during IFNgamma-mediated pancreatic regeneration. This striking expression was absent in 1-week-old neonates, but was clearly visible in pups by 5 weeks of age. These data therefore indicate that ErbB receptor and ligand expression decline by birth in both the IFNbeta-transgenic and non-transgenic mice, and that expression resumes early in postnatal life in the IFNbeta-transgenic mice. The expression of ErbB receptor family members at sites of islet development and regrowth suggests that these molecules might be relevant to these processes. PMID- 10750038 TI - Effects of repeated maternal betamethasone administration on growth and hypothalamic-pituitary-adrenal function of the ovine fetus at term. AB - Synthetic glucocorticoids have become an important clinical tool with which to advance fetal lung maturation in women at risk of early preterm birth, and this has succeeded in reducing neonatal mortality and morbidity from respiratory distress syndrome. Although previous studies have shown that glucocorticoids have deleterious consequences on fetal development, there is little information regarding the effects of clinically relevant repeated maternal doses of glucocorticoids on fetal growth and hypothalamic-pituitary-adrenal (HPA) function. We hypothesised that repeated prenatal exposure to increased concentrations of glucocorticoids would alter fetal growth and HPA axis development. Pregnant ewes were injected with betamethasone (0.5 mg/kg) or vehicle at 104, 111 and 118 days of gestation (term 150 days). Animals were sacrificed at 125 and 146 days of gestation, at which time fetal weights were recorded. Maternal and fetal blood samples were gathered and fetal tissue collected. Maternal oestradiol concentrations were significantly greater than those in controls at 125 days of gestation, but were not different at 146 days. Maternal plasma progesterone concentrations were similar between groups at both 125 and 146 days of gestation. Weight at birth was significantly reduced by 23% at 125 days and 19% at 146 days of gestation (P<0.05) after exposure to glucocorticoid. Cord plasma ACTH concentrations were not significantly different between groups at day 125, but were significantly increased in day 146 fetuses of ewes that had received betamethasone (P<0.05). Cord plasma cortisol concentrations followed the same trend, although differences were not statistically significant. Cord plasma corticosteroid binding capacity (CBC) was significantly increased at 125 days of gestation in fetuses of betamethasone treated animals (P<0.05), but not at 146 days of gestation. To examine the mechanisms regulating the increase in cord plasma ACTH of 146-day fetuses, we used in situ hybridisation to determine the distribution and levels of mRNA encoding key pituitary and hypothalamic neuropeptides of the HPA axis. In pituitaries of 146-day fetuses, there were no significant differences in the regional pattern of distribution or amounts of pro-opiomelanocortin (POMC) mRNA between betamethasone-treated animals and controls, in either the pars intermedia or the inferior and superior regions of the pars distalis. Neither prohormone convertase (PC)-1 nor PC-2 mRNA levels in pituitaries of 146-day fetuses were significantly different between treatment groups. After maternal betamethasone, immunoreactive ACTH peptide content in the fetal pars distalis was not different but glucocorticoid receptor (GR) mRNA levels in the pars distalis were increased significantly (P<0.05). No significant difference in distribution pattern or concentrations of corticotrophin-releasing hormone (CRH) mRNA, GR mRNA, oxytocin mRNA and pre-proenkephalin mRNA were found in hypothalami from fetuses at 146 days of gestation after betamethasone treatment. We conclude that antenatal betamethasone given to pregnant sheep in a manner similar to that used in human obstetric practice results in reduced weight at birth at 125 and 146 days, and altered basal cord levels of plasma ACTH and corticosteroid binding capacity, but these changes are not reflective of changes in steady state concentrations of POMC and CRH mRNA in the fetal pituitary or hypothalamus. PMID- 10750039 TI - Islet loss and alpha cell expansion in type 1 diabetes induced by multiple low dose streptozotocin administration in mice. AB - The aim of this study was to investigate the alpha cell population during the development of type 1 diabetes following multiple low-dose streptozotocin administration in mice. For this purpose C57BL/Ks male mice were injected with streptozotocin (40 mg/kg body weight for 5 days). Development of hyperglycemia was monitored over 28 days and a morphometric analysis of islet endocrine cells was performed. A reduction of islet cell area was observed after two injections of streptozotocin. The subsequent decrease of the area throughout the study period averaged 35%. Insulin-positive beta cells gradually disappeared from the identified islets. Hyperglycemia was present from day 7 onwards and in parallel with hyperglycemia, insulitis developed. An analysis of the alpha cell number per islet area revealed a 2- to 3-fold increase in this cell population, with the highest value on day 21. Confocal microscopy analysis of the ICA 512 protein tyrosine phosphatase revealed strong expression in the alpha cells at day 21, suggesting high secretory activity in the diabetic state. It is concluded that multiple low-dose streptozotocin treatment of C57BL/Ks male mice causes the disappearance of a fraction of the islets of Langerhans. In the remaining islet tissue an expansion of alpha cells occurs, reflecting a loss of intraislet beta cells as well as a regeneration of alpha cells. PMID- 10750041 TI - The effect of epidermal growth factor and IGF-I infusion on hepatic and renal expression of the IGF-system in adult female rats. AB - Systemic administration of epidermal growth factor (EGF) in neonatal rats results in reduced body weight gain and decreased circulating levels of IGF-I, suggesting its involvement in EGF-induced growth retardation. We investigated the effect of EGF and/or IGF-I administration for 7 days on circulating IGF-I and IGFBP levels and hepatic and renal IGF-system mRNA expression profiles in adult female rats. EGF administration (30 microg/rat/day) did not influence body weight, liver or kidney weight. In contrast, IGF-I (400 microg/rat/day) and EGF/IGF-I administration increased both body weight and kidney weight. Also, serum IGF-I and the 30 kDa IGFBPs (IGFBP-1 and -2) were significantly increased in these groups. Serum IGFBP-3 levels increased in the IGF-I group along with increased hepatic IGFBP-1 and -3 mRNA levels. In contrast, in the EGF administration group serum IGFBP-3 levels were significantly decreased; however, the mRNA levels remained unchanged. In the EGF/IGF-I administration group, serum IGF-I and IGFBP 3 levels were significantly lowered when compared with the IGF-I administration group. This was in contrast to the effect on kidney weight increase that was identical for the IGF-I and EGF/IGF-I groups. The decrease in serum IGFBP-3 was not reflected at the hepatic IGFBP-3 mRNA level. IGFBP-3 expression might be regulated at a post-transcriptional level although EGF induced IGFBP-3 proteolysis could not be demonstrated in vitro. We conclude that EGF administration reduced serum IGFBP-3 whereas IGF-I administration increased the level of IGFBP-3 and IGF-I and resulted in an increased body and kidney weight in adult female rats. PMID- 10750040 TI - Expression of mRNA encoding IGF-I, IGF-II and type 1 IGF receptor in bovine ovarian follicles. AB - IGFs regulate gonadotrophin-stimulated proliferation and differentiation of granulosa and theca cells in vitro. However, the detailed pattern of mRNA expression of IGFs in bovine follicles remains controversial. The objectives of this study were therefore to describe the temporal and spatial pattern of expression of mRNA encoding IGF-I, IGF-II and the type 1 IGF receptor in bovine follicles in vivo. The expression of mRNA encoding IGF-II was detected in theca tissue from around the time of antrum formation up to and during the development of dominance. No IGF-II mRNA expression was detected in granulosa cells. In the majority of follicles we were unable to detect mRNA encoding IGF-I in either granulosa or theca tissue from follicles at any stage of development. Occasionally low amounts of mRNA encoding IGF-I were detected in the theca externa and connective tissue surrounding some follicles. Type 1 IGF receptor mRNA was detected in both granulosa and theca cells of preantral and antral follicles. Expression was greater in granulosa tissue compared with theca tissue. We also measured IGF-I and -II mRNA in total RNA isolated from cultured granulosa and theca cells using reverse transcriptase PCR. In contrast to the in vivo results, IGF-II mRNA was detected in both granulosa and theca tissue. IGF-I mRNA was detected in theca tissue and in very low amounts in granulosa cells. Using a specific IGF-I RIA we were unable to detect IGF-I immunoreactivity in granulosa conditioned cell culture media. Using immunohistochemistry we detected IGF-I immunoreactivity in some blood vessels within the ovarian stroma. We conclude from these results that IGF-II is the principal intrafollicular IGF ligand regulating the growth of bovine antral follicles. In preantral follicles the expression of mRNA encoding type 1 IGF receptor but absence of endogenous IGF-I or -II mRNA expression, highlights a probable endocrine mechanism for the IGF regulation of preantral follicle growth. PMID- 10750042 TI - IGF-I stimulates chemotaxis of human neuroblasts. Involvement of type 1 IGF receptor, IGF binding proteins, phosphatidylinositol-3 kinase pathway and plasmin system. AB - SH-SY5Y human neuroblastoma cells express IGF receptors, IGFs and IGF binding proteins (IGFBPs), and provide a model for studying the role of the IGF system in human neuronal development. We investigated the effect of IGF-I and des(1-3)IGF-I on the motility of SH-SY5Y cells by a cell migration assay based on the assessment of the number of cells which migrated across 8 microm pore size membranes and around an agarose drop. IGF-I and des(1-3)IGF-I stimulated neuroblast chemotaxis in a dose-dependent manner. Treatment of cells with these agents for 24 h resulted in a significant increase (IGF-I by 70% and des(1-3)IGF I by 90%; P<0. 0001) in cell motility relative to control conditions. Addition of monoclonal antibody against type 1 IGF receptor (alpha-IR3), significantly (P<0.05) reduced the cell motility induced by IGF-I (by 30%) and des(1-3)IGF-I (by 70%). Wortmannin, a specific inhibitor of phosphatidylinositol (PI)-3 kinase intracellular signalling, also reduced the IGF-stimulated cell migration (by over 40%, P<0.01), indicating a key role of the PI-3 kinase pathway in mediating the IGF effect on neuroblast migration. Finally, cell treatment with plasminogen (PLG) markedly enhanced neuroblast migration (by over 200%, P<0.01), whereas incubation with the PLG inhibitor 4-(2-aminoethyl)-benzenesulphonyl fluoride reduced cell motility (by 80%, P<0.01), thus suggesting an involvement of PLG dependent IGFBP proteolysis in the regulation of neuroblast motility. In conclusion, IGF-I is a potent stimulator of neuroblast migration through the activation of type 1 IGF receptor and the PI-3 kinase intracellular pathway. IGFBPs and the plasmin system seem to play a role in cell motility, although the nature and the extent of their involvement has yet to be elucidated. PMID- 10750043 TI - Desensitisation of calcitonin gene-related peptide responsiveness but not adrenomedullin responsiveness in vascular smooth muscle cells. AB - Adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP) are distantly related peptides. Both act through G protein-coupled receptors on vascular smooth muscle cells to increase intracellular cAMP concentrations, causing vasorelaxation. Recent evidence suggests that both peptides bind to a common heptahelical receptor, with specificity for each peptide being determined by a receptor activity modifying protein (RAMP). This hypothesis predicts that each peptide should desensitise the cellular response to subsequent stimulation by the other. We have studied the patterns of desensitisation of ADM/CGRP receptors in rat aortic vascular smooth muscle cells. Cells were incubated for 20 min in either serum free medium (SFM), alone (control) or in SFM containing vasoactive agonist (e.g. ADM 10(-8) M, CGRP 10(-7) M, angiotensin II 10(-9) M or isoproterenol 10(-6) M). Cells were then washed and incubated for a further 20 min in SFM containing a second agonist and 1 mM isobutyryl methyl xanthine. Cells were harvested and assayed for cAMP. Pre-exposure of cells to CGRP, isoproterenol, angiotensin II or ADM, decreased cAMP generation in response to subsequent stimulation with CGRP by 84% (+/-5), 66% (+/-18), 45% (+/-5) and 60% (+/-10) respectively (mean+/-s.d.). Pre-incubation of cells with 100 nM H-89, a protein kinase A (PKA) inhibitor, abolished the desensitisation of CGRP by itself, implying that this desensitisation was mediated through PKA. In contrast, there was no attenuation of the cAMP response to stimulation with ADM by pre exposure to ADM and all other agonists tested. Identical results were seen with or without PKA inhibition by H-89. These results indicate that the ADM receptor does not desensitise over this time period in RAVSMCs, in contrast to the CGRP receptor, which is desensitised by pre-exposure to CGRP and other vaso-active agonists. These data also suggest that ADM and CGRP act through separate receptors in these cells. PMID- 10750044 TI - Specific binding sites for synthetic growth hormone secretagogues in non-tumoral and neoplastic human thyroid tissue. AB - The presence of specific receptors for synthetic growth hormone secretagogues (GHSs) has been investigated in non-tumoral and neoplastic human thyroid tissue using a radio-iodinated peptidyl GHS ((125)I-labelled Tyr-Ala-hexarelin) as ligand. Specific binding sites for Tyr-Ala-hexarelin were detected in membranes from non-tumoral and follicular-derived neoplastic thyroid tissue, but not in thyroid tumours (medullary carcinomas) of parafollicular (C cell) origin. The binding activity was greatest in well differentiated neoplasms (papillary and follicular carcinomas), followed by poorly differentiated carcinomas, non-tumoral thyroid parenchyma, follicular adenomas and anaplastic carcinomas. Both peptidyl (Tyr-Ala-hexarelin, hexarelin, growth hormone releasing peptide (GHRP6) and non peptidyl (MK 0677) GHSs completely displaced the radioligand from binding sites of non-tumoral thyroid gland, but MK 0677 was significantly less potent. The IC(50) values were (1. 9+/-0.3)x10(-8) mol/l for Tyr-Ala-hexarelin, (2.1+/ 0.2)x10(-8) mol/l for hexarelin, (2.4+/- 0.3)x10(-8) mol/l for GHRP6 and only (1. 5+/-0.4)x 10(-7) mol/l for MK 0677. Similar IC(50) values were found in neoplastic thyroid tissue. Scatchard analysis of the binding revealed a finite number of binding sites in non-tumoral (B(max): 1232+/-32 fmol/mg protein, n=3) and neoplastic (papillary carcinomas) thyroid tissue (B(max): 2483+/-380 fmol/mg protein, n=5), with dissociation constants (K(d)) of (3.8+/-0.3)x10(-9) and (4. 4+/-0.6)x 10(-9) mol/l, respectively. On the basis of this evidence, we investigated the effects of some GHS on the proliferation of three different human follicular thyroid carcinoma cell lines (NPA, WRO and ARO) in which the presence of specific GHS receptors was also demonstrated. Tyr-Ala-hexarelin, GHRP6 and MK 0677 were able to inhibit serum-stimulated [(3)H]thymidine incorporation in NPA cells at concentrations close to their binding affinity. These substances also caused a significant inhibition of cell proliferation, which was evident at the earliest time of treatment (24 h) in all the cell lines, and at the latest time (96 h) in NPA cells only. In conclusion, this paper confirms the existence of specific binding sites for GHS in normal thyroid tissue and demonstrates, for the first time, that these binding sites are present in papillary and follicular carcinomas, low in anaplastic carcinomas and absent in medullary carcinomas of the thyroid. This work also provides evidence of a growth inhibitory effect of GHS on cell lines derived from follicular thyroid cancers. PMID- 10750045 TI - Angiotensin II modulates the activity of the Na+/K+ATPase in eel kidney. AB - We have previously shown that angiotensin II (Ang II) has a role at the level of the eel gill chloride cell regulating sodium balance, and therefore osmoregulation; the purpose of the present study was to extend these findings to another important osmoregulatory organ, the kidney. By catalytic histochemistry Na(+)/K(+)ATPase activity was found in both sea water (SW)- and freshwater (FW) adapted eel kidney, particularly at the level of both proximal and distal tubules. Quantitation of tubular cell Na(+)/K(+)ATPase activity, by imaging, gave values in SW-adapted eels which were double those found in FW-adapted eels (Student's t-test: P<0.0001). This was due to a reduced number of positive tubules present in FW-adapted eels compared with SW-adapted eels. By conventional enzymatic assay, the Na(+)/K(+)ATPase activity in isolated tubular cells from SW adapted eels showed values 1.85-fold higher those found in FW-adapted eels (Student's ttest: P<0.0001). Perfusion of kidney for 20 min with 100 nM Ang II provoked a significant increase (1.8-fold) in Na(+)/K(+)ATPase activity in FW, due to up-regulation of Na(+)/K(+)ATPase activity in a significantly larger number of tubules (Student's t-test: P<0.0001). The effect of 100 nM Ang II in SW adapted kidneys was not significant. Stimulation with increasing Ang II concentrations was performed on isolated kidney tubule cells: Ang II provoked a dose-dependent stimulation of the Na(+)/K(+)ATPase activity in FW-adapted eels, reaching a maximum at 100 nM (1.82-fold stimulation), but no significant effect was found in SW-adapted eels (ANOVA: P<0.001 and P>0.05 respectively). Isolated tubule cells stimulated with 100 nM Ang II showed a significant generation of inositol trisphosphate (InsP(3)) and an increment in calcium release from intracellular stores. In conclusion, our results suggest that tubular Na(+)/K(+)ATPase is modulated by environmental salinity, and that Ang II has a role in regulating its activity in FW-adapted eels, probably through an InsP(3) dependent mechanism. PMID- 10750046 TI - Increased maternal serum activin A but not follistatin levels in pregnant women with hypertensive disorders. AB - Activin A levels are elevated in maternal serum of pregnant women with hypertensive disturbances. Because follistatin is a circulating binding protein for activin A, the present study was designed to evaluate whether serum follistatin and activin A levels also change in patients with hypertensive disorders in the last gestational trimester. The study design was a controlled survey performed in the setting of an academic prenatal care unit. Healthy pregnant women (controls, n=38) were compared with patients suffering from pregnancy-induced hypertension (PIH, n=18) or pre-eclampsia (n=16). In addition, the study included a subset of patients with pre-eclampsia associated with intrauterine growth restriction (IUGR, n=5). Maternal blood samples were withdrawn at the time of diagnosis (patients) or in a random prenatal visit (controls), and serum was assayed for follistatin and activin A levels using specific enzyme immunoassays. Hormone concentrations were corrected for gestational age by conversion to multiples of median (MoM) of the healthy controls of the same gestational age. Follistatin levels were not different between controls and patients, while activin A levels were significantly increased in patients with PIH (1.8 MoM), pre-eclampsia (4.6 MoM), and pre eclampsia+IUGR (3.2 MoM, P<0.01, ANOVA). The ratio between activin A and follistatin was significantly increased in patients with PIH (1.5 MoM) and was further increased in patients with pre-eclampsia (4.5 MoM) and in the group with pre-eclampsia+IUGR (2.6 MoM). Follistatin levels were positively correlated with gestational age in control subjects (r=0. 36, P<0.05) and in patients with PIH (r=0.46, P<0.05) or pre-eclampsia (r=0.61, P<0.01), while activin A correlated with gestational age only in the healthy control group (r=0.69, P<0.0001). The finding of apparently normal follistatin and high activin A levels in patients with PIH and pre-eclampsia suggests that unbound, biologically active, activin A is increased in women with these gestational diseases. PMID- 10750047 TI - Effects of long-term administration of vitamin D3 analogs to mice. AB - This study explores the effects of chronic administration of vitamin D(3) compounds on several biological functions in mice. Knowledge of long-term tolerability of vitamin D(3) analogs may be of interest in view of their potential clinical utility in the management of various pathologies such as malignancies, immunological disorders and bone diseases. Four unique vitamin D(3) analogs (code names, compounds V, EO, LH and LA) and 1,25-dihydroxyvitamin D(3) (1, 25(OH)(2)D(3)) were administered i.p. for 55 weeks to Balb/c mice. Each analog had previously been shown to have potent in vitro activities. After 55 weeks of administration, the mice had a profound decrease in their serum levels of interleukin-2 (IL-2). Likewise, several analogs depressed serum immunoglobulin G concentrations (compounds LH and LA), but levels of blood lymphocytes and splenic lymphocyte subsets (CD4, CD8 and CD19) were not remarkably depressed. The percent of committed myeloid hematopoietic stem cells was 4- to 5-fold elevated in the bone marrow of the mice that received analogs LH and V; nevertheless, their peripheral blood white and red cell counts and platelets were not significantly different in any of the groups. The mice that received 1,25(OH)(2)D(3) had a decrease in bone quantity and quality with a decrease in cross-sectional area and cortical thickness, and a 50% reduction in both stiffness and failure load compared with the control group. In contrast, the cohort that received a fluorinated analog (compound EO) developed bones with significantly larger cross-sectional area and cortical thickness as well as stronger mechanical properties compared with the control group. At the conclusion of the study, body weights were significantly decreased in all experimental mice. Their blood chemistries were normal. Extensive gross and microscopic autopsy analyses of the mice at the conclusion of the study were normal, including those of their kidneys. In conclusion, the vitamin D(3) analogs were fairly well tolerated. They did suppress immunity as measured by serum IL-2 and may provide a means to depress the immune response after organ transplantation and for autoimmune diseases. Use of these analogs prevented the detrimental effects of vitamin D(3) administration on mechanical and geometric properties of bone, while one analog (compound EO) actually enhanced bone properties. These results suggest that long-term clinical trials with the analogs are feasible. PMID- 10750048 TI - Plasticity and constraints in development and evolution. AB - Morphological similarities between organisms may be due to either homology or homoplasy. Homologous structures arise by common descent from an ancestral form, whereas homoplasious structures are independently derived in the respective lineages. The finding that similar ontogenetic mechanisms underlie the production of the similar structures in both lineages is not sufficient evidence of homology, as such similarities may also be due to parallel evolution. Parallelisms are a class of homoplasy in which the two lineages have come up with the same solution independently using the same ontogenetic mechanism. The other main class of homoplasy, convergence, is superficial similarity in morphological structures in which the underlying ontogenetic mechanisms are distinct. I argue that instances of convergence and parallelism are more common than is generally realized. Convergence suggests flexibility in underlying ontogenetic mechanisms and may be indicative of developmental processes subject to phenotypic plasticity. Parallelisms, on the other hand, may characterize developmental processes subject to constraints. Distinguishing between homology, parallelisms and convergence may clarify broader taxonomic patterns in morphological evolution. PMID- 10750049 TI - "Paleogenomics": looking in the past to the future. AB - The complete sequence of the human and other vertebrate and nonvertebrate genomes provide a wealth of information on the organization, relationships and evolution of the metazoans. Soon the fine structure of our innermost biological identity will be unveiled and what has so far remained deep and secret will shine like an unearthed treasure and shape and fuel our future quests. A key treasure, for many molecular scientists interested in molecular evolution and development would be the knowledge of the genome of the ancestral precursor of all metazoans. In the absence of fossil DNA, this knowledge will forever remain a yearning for dreamy molecular biologists. And yet, will not the power of deduction and reconstitution of information gained through man's sophisticated technologies one day recreate a "virtual" metazoan ancestor? PMID- 10750050 TI - T-Brain expression in the apical organ of hemichordate tornaria larvae suggests its evolutionary link to the vertebrate forebrain. AB - T-box genes encode a novel family of sequence-specific activators that appear to play crucial roles in various processes of animal development. Although most of the T-box genes are involved in the mesoderm formation of chordate embryos, mammalian T-Brain is expressed in the developing central nervous system, and defines molecularly distinct domains within the cerebral cortex. Here we report the first invertebrate T-Brain homologue from the hemichordate acorn worm, Ptychodera flava, which we designate Pf-Tbrain. Developmental expression of Pf Tbrain was examined by whole mount in situ hybridization to various stages of P. flava embryos. A weak, broad in situ hybridization signal of the Pf-Tbrain transcript is first detected during gastrulation in cells around the archenteron, but this signal disappears as gastrulation proceeds. At mid-gastrula an intense signal appears in several apical ectoderm cells of the gastrula. This signal becomes restricted to the apical region, where the eyespots or the light-sensory organ of the tornaria larva form. Expression of Pf-Tbrain in the apical sensory organ of the tornaria and vertebrate T-Brain in the forebrain suggests an evolutionary relationship between the non-chordate deuterostome larval apical sensory organ and the chordate forebrain. PMID- 10750051 TI - Evolution of exon 1 of the dopamine D4 receptor (DRD4) gene in primates. AB - The dopamine D4 receptor (DRD4) gene exhibits a large amount of expressed polymorphism in humans. To understand the evolutionary history of the first exon of DRD4-which in humans contains a polymorphic 12bp tandem duplication, a polymorphic 13bp deletion, and other rare variants-we examined the homologous exon in thirteen other primate species. The great apes possess a variable number of tandem repeats in the same region as humans, both within and among species. In this sense, the 12bp tandem repeat of exon 1 is similar to the 48bp VNTR of exon 3 of DRD4, previously shown to be polymorphic in all primate species examined. The Old World monkeys show no variation in length, and a much higher conservation of amino acid sequence than great apes and humans. The New World monkeys show interspecific differences in length in the region of the 12bp polymorphism, but otherwise show the higher conservation seen in Old World monkeys. The different patterns of variation in monkeys compared to apes suggest strong purifying selective pressure on the exon in these monkeys, and somewhat different selection, possibly relaxed selection, in the apes. PMID- 10750052 TI - Divergent decapentaplegic expression patterns in compound eye development and the evolution of insect metamorphosis. AB - In the fruit fly Drosophila, the patterning genes decapentaplegic and wingless contribute to the spatial control of retina development in an antagonistic manner. We examined the expression patterns of these genes in the developing visual system of the hemimetabolous grasshopper Schistocerca americana and the primitive holometabolous beetle species Tribolium castaneum. The pattern of wingless expression was strongly conserved as a pair of lateral domains at the anterior margins of both the developing retina and the developing optic lobes. The expression of decapentaplegic, on the other hand, is different. Unlike in Drosophila, no decapentaplegic expression was detected before the onset of photoreceptor differentiation in the retinal precursor tissue of either grasshopper or beetle. Moreover, the subsequent expression of decapentaplegic in the latter species was not concentrated in the moving front of retina differentiation, as in Drosophila, but observed in anterior and posterior regions. Our results indicate that Drosophila eye development contains elements of both ancestral and derived regulatory gene functions. The requirement for decapentaplegic as an antagonist of wingless during the early development of the Drosophila retina might have originated during the evolution of insect metamorphosis. PMID- 10750053 TI - Mitochondrial DNA from Myotragus balearicus, an extinct bovid from the Balearic Islands. AB - DNA was extracted from teeth and bones of Myotragus balearicus, a bovid that evolved in isolation on the Balearic Islands (Western Mediterranean) from the end of the Miocene, becoming extinct 4,000 years BP, after the arrival of humans in the islands. The numerous morphological apomorphies of Myotragus, most strikingly its dwarfism, frontal eyes, and ever-growing incisors, obscure its phylogenetic relationships with extant bovids. Therefore, the recovery of genetic information from Myotragus is of significant interest to help clarify the taxonomic position of this species. In this study we amplified and sequenced a 95 bp (base pair) fragment of the mtDNA cytochrome b gene from 6,000-year-old specimens of Myotragus. Several experimental controls, such as amino acid analysis, independent reproduction in two different laboratories, and cloning of the PCR product, support the authenticity of the ancient DNA sequence recovered. Phylogenetic comparison with orthologous sequences from supposedly related extant genera (serow, goral, mountain goat, chamois, takin, sheep, goat, Himalayan tahr, arctic musk ox, barbary sheep, blue sheep, and saiga) from the Caprinae subfamily suggests that Myotragus is related to some of these species. However, the real phylogenetic position of Myotragus is difficult to assess, due to the lack of resolution of the present molecular study, which can be partially attributed to the short length of the genetic fragment recovered. PMID- 10750054 TI - Haplotype dimorphism in a SNP collection from Drosophila melanogaster. AB - A moderate resolution single nucleotide polymorphism (SNP) map of the genome of Drosophila melanogaster that is designed for use in quantitative genetic mapping is described. Seventeen approximately 500 nucleotide gene sequences spaced at 10 to 20 centimorgan intervals were combined with 49 shorter sequence tag sites (STSs) at 5 to 10 centimorgan intervals to generate a map that should not leave any gaps greater than one half of a chromosome arm when any two wild type lines are compared. Of 20 markers with sufficient polymorphism to construct haplotype cladograms, 13 showed evidence for two divergent classes of haplotype. The possible mechanisms for and implications of the unexpected finding that two thirds of all short gene sequences in D. melanogaster may be dimorphic are discussed, including the suggestion that admixture between two separate lineages may have been a major event in the history of the species. PMID- 10750055 TI - Total evidence: molecules, morphology, and the phylogenetics of cichlid fishes. AB - We present a most comprehensive phylogenetic analysis of the family Cichlidae. New data analyzed include mitochondrial 16S rRNA sequences and two nuclear loci (Tmo-M27 and Tmo-4C4) for a large taxonomic sampling with emphasis on South American species. We also incorporate a published morphological data set for a total evidence analysis. Character congruence among mitochondrial (74 taxa) and nuclear data (50 taxa) was high. However, partition-homogeneity tests suggest significant heterogeneity among molecular and morphological data. In agreement with results obtained from molecular data alone, total evidence analysis (1,460 characters for 34 taxa) supports a robust phylogenetic hypothesis for the family Cichlidae that is congruent with drift-vicariance events associated with the fragmentation of Gondwana. Our analyses confirm the placement of Malagasy/Indian cichlids as the most basal lineages, with a sister-group relationship to the monophyletic African and Neotropical clades. Total evidence suggests that the controversial African genus Heterochromis is at the base of the African radiation. Among more than 50 Neotropical genera analyzed, Retroculus is identified as the basal taxon, with successive branching of Cichla, Astronotus, geophagines (including crenicichlines) + chaetobranchines, and cichlasomines + heroines. Relative rate tests applied to mitochondrial DNA suggest significantly higher rates of genetic variation in Neotropical than in African taxa, and both mitochondrial and nuclear sequences show that rate heterogeneity among Neotropical lineages is confined to the geophagine cichlids. PMID- 10750056 TI - Groger H, callaerts P, gehring walter Jurgen, schmid volker. 1999. Gene duplication and recruitment of a specific tropomyosin into striated muscle cells in the jellyfish podocoryne carnea. J exp zool (Mol dev evol) 285:378-386 AB - The third author of the article was listed as Walter Jurgen Gehring. His correct name is Walter Jakob Gehring. The authors regret the error. PMID- 10750057 TI - Generalized treatment effects for clinical trials. AB - Practice in the analysis of clinical trials with continuously measured endpoints is to focus on the difference or percentage change in mean or median response. However, treatments may have effects on the distribution of responses other than on the average response. We sought an approach to such generalized treatment effects that: (i) targets a parameter that is easily understood by our clinical colleagues; and (ii) employs confidence intervals as the basis for inference. We consider one such approach based on work in reliability theory, namely setting Pr[Y>X] as the target parameter, and compare this approach to an earlier one due to O'Brien. The two approaches have similar properties when they both seek to reject the null hypothesis of no effect due to different variances but differ when the larger variance corresponds to the larger mean. In that case, our approach views that the larger variance attenuates the effect of the larger mean. Out suggested approach applies easily to positive control (clinical) equivalence trials. PMID- 10750058 TI - Re-calculating the sample size in internal pilot study designs with control of the type I error rate. AB - When designing a clinical trial, there is usually some uncertainty about the variability of the primary outcome variable. This may lead to an unnecessarily high or inadequately low sample size. The internal pilot study approach uses data from patients recruited up to an interim stage to re-estimate the variance and to re-calculate the final sample size accordingly. Previously, simulation studies have shown that this methodology may highly improve the chance to obtain a well powered trial. However, it also turned out that the type I error rate may be inflated by this procedure. We quantify the maximum excess of the type I error rate for normally distributed outcomes. If strict control of the alpha-level is considered to be an important issue, a method is proposed to achieve this when re calculating the sample size in internal pilot studies. The characteristics of the power distributions are investigated for various sample size adaptation rules and implications are discussed. PMID- 10750059 TI - Detecting dose response with contrasts. AB - Analyses of dose response studies should separate the question of the existence of a dose response relationship from questions of functional form and finding the optimal dose. A well-chosen contrast among the estimated effects of the studied doses can make a powerful test for detecting the existence of a dose response relationship. A contrast-based test attains its greatest power when the pattern of the coefficients has the same shape as the true dose response relationship. However, it loses power when the contrast shape and the true dose response shape are not similar. Thus, a primary test based on a single contrast is often risky. Two (or more) appropriately chosen contrasts can assure sufficient power to justify the cost of a multiplicity adjustment. An example shows the success of a two-contrast procedure in detecting dose response, which had frustrated several standard procedures. PMID- 10750060 TI - Sample size to test for interaction between a specific exposure and a second risk factor in a pair-matched case-control study. AB - We discuss a sample size calculation for a pair-matched case-control study to test for interaction between a specific exposure and a second risk factor. The second risk factor could be either binary or continuous. An algorithm for the calculation of sample size is suggested which is based on a logistic regression model that relates the logarithm of the disease-exposure odds ratio to the second risk factor. This problem is motivated by a study comparing the prevalence of GP IIIa Pl(A2) polymorphism (the exposure) in individuals with and without myocardial infarction (case-control). One of the hypotheses in this study is whether or not there is an interaction between the prevalence of GP-IIIa Pl(A2) polymorphism and a second risk factor such as smoking status and homocysteine level. We introduce the algorithm in detail with several numerical examples. PMID- 10750061 TI - Bayesian inference for a generalized population attributable fraction: the impact of early vitamin A levels on chronic lung disease in very low birthweight infants. AB - In this paper, the population attributable fraction is studied using the potential responses framework of Rubin's causal model. This framework facilitates definition of a general measure of population attributable effect which can accommodate many-valued and multivariate exposures as well as many-valued responses. Inferential issues are considered from the Bayesian perspective. Finite population inference is emphasized with inference in the case of a fully observed population given particular attention. The key inferential issue concerns computation of the posterior distribution of unobserved potential responses, given observed responses, exposures and covariates. A dependency on model parameters about which observed data are uninformative is highlighted and this reflects the unobservable nature of causal effects. In an application to a small cohort study of respiratory problems in very low birthweight infants, posterior inferences were found to be insensitive to assumptions concerning the joint distribution of potential response variables but sensitive to the assumption of weak ignorability, a weaker form of the more familiar assumption of no confounding by omitted covariates. In a model-based set-up, the weak ignorability assumption is identified with setting a model parameter to zero, and consequently uncertainty concerning this assumption can, in principle, be handled via the prior distribution for the model parameters. PMID- 10750062 TI - A bayesian analysis for spatial processes with application to disease mapping. AB - In epidemiology, maps of disease rates and disease risk provide a spatial perspective for researching disease aetiology. For rare diseases or when the population base is small, the rate and risk estimates may be unstable. We propose using a Bayesian analysis based on the conditional autoregressive (CAR) process that will spatially smooth disease rates or risk estimates by allowing each site to 'borrow strength' from its neighbours. Covariates may be included in the model in such a way as to establish a possible association between risk factors and disease incidence. Bayesian inferences are implemented from a direct resampling scheme where large samples are generated from the various posterior distributions. The methodology is demonstrated with a simulation that assesses the effect of sample size and the model parameters on inferences for the parameters. Our approach is also used to spatially smooth district lip cancer rates in Scotland using the CAR model with a covariate that allows for exposure to sunlight. PMID- 10750063 TI - A robust mixed linear model analysis for longitudinal data. AB - This paper describes robust procedures for estimating parameters of a mixed effects linear model as applied to longitudinal data. In addition to fixed regression parameters, the model incorporates random subject effects to accommodate between-subjects variability and autocorrelation for within-subject variability. Robust empirical Bayesian estimation of subject effects is briefly discussed. As an illustration, the procedures are applied to data from a multiple sclerosis clinical trial. PMID- 10750065 TI - Immunoprophylaxis of respiratory syncytial virus infection. PMID- 10750064 TI - Functions of oral candidiasis episodes that are highly prognostic for AIDS. AB - Although oral candidiasis lesions are widely recognized as markers for AIDS, the relative prognostic significance of functions of these episodes has not been examined. We compare the associations with time to AIDS of one fixed and six time dependent metrics of oral candidiasis lesions, including proximity of a lesion to seroconversion, any candidiasis history, and recency of a lesion. We show in Cox regression models that two metrics are clinically and statistically far more significant than the others, alone or in combination: any history of candidiasis since HIV seroconversion, and recency of a candidiasis episode. The latter metric indicates that the risk of an AIDS diagnosis is high during a candidiasis episode. The results hold for two cohorts of male seroconverters, 627 haemophiliacs and 196 men who have sex with men. Identification of highly prognostic functions of a patient's oral candidiasis history allows clinicians and researchers to focus on these aspects of the history and to omit extraneous information from data collection. Our method extends well beyond candidiasis and AIDS, and may shed light on associations of covariates with outcomes in a variety of settings. PMID- 10750066 TI - President's address. XXXVII National Conference of the Indian Academy of Pediatrics, January 27,2000. PMID- 10750067 TI - Identification of an appropriate strategy to control anemia in adolescent girls of poor communities. AB - OBJECTIVES: To obtain baseline data on hemoglobin (Hb) levels of adolescent girls belonging to the low-socio-economic groups; investigate the comparative efficacy of once 'weekly' and 'daily' administration of iron-folate tablets with respect to impact on the Hb levels; and find out the effect of added ascorbic acid supplementation on the efficacy of iron-folate administration with respect to increment in Hb levels. DESIGN: Randomized experimental. SETTING: Adolescent girls of poor communities in urban areas of Delhi and rural parts of Bharatpur (Rajasthan). METHODS: The baseline investigations included measurements of height, weight, and Hb levels. The Hb levels of the participating subjects were measured again after 3 months and 6 months of supplementation. RESULTS: 61.9% of the subjects in the urban and 85.4% in the rural area were anemic. The response of Hb levels to daily iron/folate supplementation was better in comparison to once-weekly supplementation. The increment in Hb levels of subjects due to addition of vitamin C to iron/folate supplementation was more than that with supplementation of iron/folate alone. CONCLUSIONS: Considering compliance, feasibility and cost-factors, a public-health approach consisting of once-weekly distribution of iron/folate supplementation through schools and welfare centers is better and can be recommended as an appropriate strategy for combating anemia in adolescent girls of poor communities in developing countries like India. PMID- 10750069 TI - Helicobacter pylori infection in children. PMID- 10750068 TI - Sustained hypertension in children. AB - OBJECTIVE: To study the etiology and clinical profile of children with sustained hypertension. DESIGN: Retrospective hospital-based study. SETTING: Tertiary care, referral center. SUBJECTS: 246 children with sustained hypertension presenting between January 1983 and December 1996. RESULTS: The mean age at presentation was 8.2+/-3.9 yr; range 2 months-16 yr. There were 180 boys. An underlying cause for hypertension was identified in 242 (98.4%); 4 patients were considered to have essential hypertension. The chief causes included chronic glomerulonephritis (GN) in 121 (49.2%), obstructive uropathy in 39 (15.8%), reflux nephropathy in 30 (12.2%), thrombotic microangiopathy in 15 (6.1%) and renovascular disease in 14 (5.7%). Takayasu's disease was the most common cause of renovascular hypertension. Coarctation of aorta was the commonest cause of hypertension in infancy, being present in 53.3% of cases. In 198 subjects (80.5%) hypertension was detected as a feature of a known underlying disease. Thirty-five patients however, presented for the first time with complications of severe hypertension, including congestive cardiac failure in 21 and encephalopathy in 23. Thirteen patients presented with nonspecific symptoms and hypertension was detected on clinical examination. CONCLUSIONS: Most patients with sustained hypertension have an underlying etiology. A significant proportion of patients with renovascular and endocrine conditions may present, for the first time, with complications of hypertension. PMID- 10750070 TI - Reference values in medicine and validity of diagnostic test. PMID- 10750071 TI - National consultation on benefits and safety of iodized salt. PMID- 10750072 TI - Profile of complaints and clinical syndromes of children under 5 in Rio de Janeiro, Brazil: implications for the implementation of the Integrated Management of Childhood Illness strategy. PMID- 10750073 TI - Clinical response to two days of oral amoxycillin in children with non-severe pneumonia. PMID- 10750074 TI - Seasonality of births and possible factors influencing it in a rural area of Haryana, India. PMID- 10750075 TI - Feeding practices in under-fives during diarrhea before and after educational intervention. PMID- 10750076 TI - Variable clinical phenotypes of velocardiofacial syndrome in a pair of brothers. PMID- 10750077 TI - Aortic aneurysm in a child with tuberous sclerosis. PMID- 10750078 TI - Uric acid nephropathy as an unusual cause of actual renal failure in a neonate. PMID- 10750080 TI - Systemic toxicity with cyclopentolate eye drops. PMID- 10750079 TI - Anticonvulsant induced osteomalacia. PMID- 10750081 TI - Shelf life of vaccines in multidose vials. PMID- 10750083 TI - Perinatal infections. PMID- 10750082 TI - BCG vaccination. PMID- 10750084 TI - Intertriginous xanthomas--a marker of homozygous familial hypercholesterolemia. PMID- 10750085 TI - Cooling arm to the solar warming system for newborn babies. PMID- 10750086 TI - Trends in antimicrobial resistance of Shigella species isolated from children with acute diarrhea. PMID- 10750087 TI - Spontaneous rupture of umbilical hernia in an infant. PMID- 10750088 TI - Investigation of an epidemic of Reye's syndrome in northern region of India. PMID- 10750089 TI - Reye's syndrome--an interesting epidemiological correlation. PMID- 10750090 TI - Interesterification (acidolysis) of butterfat with conjugated linoleic acid in a batch reactor. AB - Six commercial lipases, in free or immobilized form, were tested for their ability to catalyze acyl exchange between conjugated linoleic acid and anhydrous butterfat under solvent-free conditions. Immobilized Candida antarctica lipase exhibited the best activity. Experiments were conducted for this lipase in butterfat to conjugated linoleic acid ratios of 10:1 (vol/vol), temperatures from 30 to 70 degrees C, enzyme concentrations of 50 to 200 mg/g of reaction mixture, and water contents of 0.15 to 2% (wt/wt). At the maximum enzyme concentration used, equilibrium was reached within the first 24 h of reaction. The optimum temperature was 50 degrees C. The triacylglycerol profile of the product butterfat reflected changes in the relative proportions of fatty acid residues as the reaction proceeded, with increases in those triacylglycerols containing 46 to 54 carbon atoms and concomitant decreases in those triacylglycerols containing 34 to 42 carbon atoms. PMID- 10750092 TI - Plasmin levels in fresh milk whey and commercial whey protein products. AB - Growth of psychrotrophic bacteria in nonfat dry milk at refrigeration temperatures was shown previously in our laboratory to cause a shift in plasmin (a native milk protease) from the casein to the whey fraction. The whey fraction from cheesemaking is commonly used to make whey protein concentrates and isolates, which then are used as functional ingredients in various food systems. Plasmin activity in whey protein products may cause breakdown of food proteins to have desirable or undesirable effects on food quality. This raised questions about the level of plasmin in commercial whey protein products and factors that affect this plasmin level. Therefore, the objectives of this study were to determine: 1) plasmin concentrations in sweet and acid whey protein products as influenced by Pseudomonas growth during storage of fresh milk, and 2) plasmin concentrations in commercial whey protein products. Whey type (sweet or acid) had a significantly (P < 0.05) greater effect on whey-associated plasmin activity than did Pseudomonas fluorescens M 3/6 growth. Acid whey protein products had significantly (P < 0.05) higher plasmin concentrations than sweet whey. Plasmin activities associated with acid and sweet whey protein products were both significantly (P < 0.0001) affected by the growth of Pseudomonas fluorescens M 3/6. The interaction effect between bacterial growth and whey type on plasmin activity was not significant (P = 0.2457). Plasmin activity in the reconstituted commercial whey protein concentrates (i.e., sweet and acid) varied considerably (16.3 to 330 micrograms/g of protein), but was significantly lower (2.1 to 4.4 micrograms/g of protein, P < 0.05) in whey isolates. These quantitative data were supported by plasmin activity visualized by casein SDS-PAGE. PMID- 10750091 TI - The two-stage coagulation of milk proteins in the minimum of the heat coagulation time-pH profile of milk: effect of casein micelle size. AB - Milks with casein micelles larger or smaller than control milk were prepared by differential centrifugation. The heat stability of these modified milks increased markedly throughout the pH range 6.4 to 7.1 with decreasing casein micelle size. Within the region of the minimum in the heat coagulation time-pH profile, the control milk coagulated by a two-stage process, but the modified milks, because of their narrower casein micelle size distribution, coagulated by a single-stage process at the pH of minimum stability. The content of kappa-CN and protein hydration increased as the size of the casein micelles decreased, and the level of glycosylation of kappa-CN and protein surface hydrophobicity increased as a function of micelle size. The effect of casein micelle size on the heat stability of milk is likely to be related to changes in the above physico-chemical properties. PMID- 10750093 TI - Technological properties of milks fermented with thermophilic lactic acid bacteria at suboptimal temperature. AB - In the present work the synergistic relationship between different strains of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus was studied at optimal (44 degrees C) and suboptimal temperatures (30 degrees C). Acidification, viscosity, whey syneresis, and bacterial concentration of the final product were evaluated on single-strain and mixed cultures after 24 h at 30 degrees C and 6 h at 44 degrees C. Three pairs of strains (LBB + CP2, LBP + CP2, and LBR + CP2) showed synergistic effect, which was reflected by the viscosity and syneresis of the coagulum. These results were more significant when cultures were incubated at 30 degrees C, reaching apparent viscosity values of 19 to 28 mPa x s. On the other hand, lactobacilli cultures enhanced the growth of two streptococci strains (CP2 and CP4). These results were confirmed by cultures of streptococci supplemented with supernatants of culture of lactobacilli. Those supernatants stimulate the viscosity produced by CP2 and CP4 strains and reduce the syneresis of all cultures of streptococci. Neither the increase of viscosity nor reduction of syneresis could be attributed to a decrease of pH. PMID- 10750094 TI - Effect of Lactobacillus reuteri on the prevention of hypercholesterolemia in mice. AB - Administration of Lactobacillus reuteri CRL 1098 (10(4) cells/d) to mice for 7 d before inducing hypercholesterolemia (by feeding mice with a fat-enriched diet for the subsequent 7 d) was evaluated. At this low dose, L. reuteri was effective in preventing hypercholesterolemia in mice, producing a 17% increase in the ratio of high-density lipoprotein to low-density lipoprotein. Total cholesterol and triglycerides decreased by 22 and 33%, respectively, in the group that was not fed the lactobacilli. The hypocholesterolemic effect produced by L. reuteri CRL 1098 might be considered as indirect evidence of the permanency of the lactobacilli in the gut. PMID- 10750095 TI - Composition of Ragusano cheese during aging. AB - Ragusano cheese is a brine-salted pasta filata cheese. Composition changes during 12 mo of aging were determined. Historically, Ragusano cheese has been aged in caves at 14 to 16 degrees C with about 80 to 90% relative humidity. Cheeses (n = 132) included in our study of block-to-block variation were produced by 20 farmhouse cheese makers in the Hyblean plain region of the Province of Ragusa in Sicily. Mean initial cheese block weight was about 14 kg. The freshly formed blocks of cheese before brine salting contained about 45.35% moisture, 25.3% protein, and 25.4% fat, with a pH of 5.25. As result of the brining and aging process, a natural rind forms. After 12 mo of aging, the cheese contained about 33.6% moisture, 29.2% protein, 30.0% fat, and 4.4% salt with a pH of 5.54, but block-to-block variation was large. Both soluble nitrogen content and free fatty acid (FFA) content increased with age. The pH 4.6 acetate buffer and 12% TCA soluble nitrogen as a percentage of total nitrogen were 16 and 10.7%, respectively, whereas the FFA content was about 643 mg/100 g of cheese at 180 d. Five blocks of cheese were selected at 180 d for a study of variation within block. Composition variation within block was large; the center had higher moisture and lower salt in moisture content than did the outside. Composition variation within blocks favored more proteolysis and softer texture in the center. PMID- 10750096 TI - A viscoelasticity index for cheese meltability evaluation. AB - A device especially designed for uniaxial creep test was used in this study. Cheddar cheeses of different fat content were used. To study the linear viscoelastic response of the cheese, temperature of 40 degrees C and stress of 1119.5 Pa were chosen. Tests were carried out at cheese ages of 1, 3, 6, and 12 wk after production date. A six-element Kelvin model was used to model the creep data. Instantaneous slope of the creep curve was defined as the viscoelasticity index. The results showed that the viscoelasticity index based on viscoelastic parameters could be used for predicting cheese meltability. From the analysis of variance test, it was evident that that the viscoelasticity index can be used to distinguish the meltability of Cheddar cheeses of different ages and fat levels. PMID- 10750097 TI - Use of bovine primary mammary epithelial cells for the comparison of adherence and invasion ability of Staphylococcus aureus strains. AB - Adherence and invasion of epithelial cells are thought to play a role in the pathogenesis of Staphylococcus aureus mastitis. A cell culture model with primary mammary epithelial cells originating from the secretory tissue from the bovine udder was used to study adherence and invasion of S. aureus. The cells were characterized with antibodies against several cell markers that had been validated on histologic cryostat sections of bovine mammary tissue. All cells stained positively with the anticytokeratin antibodies, which are restricted to epithelial cells. The cell cultures contained a small number of alpha-smooth muscle-actin positive cells (< 1%), probably myoepithelial cells. The use of bovine primary mammary epithelial cells and bovine S. aureus isolates, which were cultured in milk serum, results in a system similar to in vivo. Strain differences for adherence and invasion of S. aureus strains cultured in milk serum were studied. In addition, the correlation between adherence and invasion was evaluated. The number of adhered and invaded bacteria was strain dependent. The percentage of adherence after 5 min of incubation was correlated to the percentage of adherence after 3 h of incubation (r = 0.94; Pearson's correlation test). Fourteen of the 20 strains were able to invade epithelial cells. The percentage of invasion was correlated to the percentage of adherence after 5 min and to the percentage adherence after 3 h (r = 0.95 and 0.90, respectively; Pearson's correlation test). Results indicate that strain differences of adherence and invasion exist for S. aureus and that the invasion is a post adherence event. PMID- 10750098 TI - Effects of bovine somatotropin on milk yield and composition in Holstein cows in advanced lactation fed low- or high-energy diets. AB - The objective of this study was to evaluate the response of Holstein cows to bovine somatotropin (bST) during advanced lactation and its relationship to energy intake. Twenty-four lactating Holstein cows averaging 21 kg of milk/d, and 292 d in milk were assigned to one of three treatment groups in a randomized block design. Blocks were based on the 14 d of pretreatment milk production, and treatment groups were balanced for days in milk. Treatment 1 was a low-energy diet (1.49 Mcal/kg of dry matter) without bST injection; treatment 2 was the low energy diet plus injection of 500 mg of bST every 14 d; and treatment 3 was a high-energy diet (1.71 Mcal/kg of dry matter) with bST injections as in treatment 2. Treatment was divided into two periods (1 to 49 and 50 to 98 d) to determine if response to bST and energy changed with time on treatment. Results showed that bST significantly (P < 0.05) increased milk, fat-corrected milk, and fat and protein yields; and feed efficiency (fat-corrected milk per dry matter intake) for both periods. Milk yield responses to bST were greater for cows fed the low than the high-energy diet in both periods. These data show that bST injections for cows in advanced lactation increased performance, but excessive energy diminished the bST response. PMID- 10750099 TI - Analytic validation of an infrared milk urea assay and effects of sample acquisition factors on milk urea results. AB - The objective of this study was to determine if milk samples, as they are routinely collected by Ontario Dairy Herd Improvement, would yield accurate milk urea results with an infrared assay. This investigation involved analytic validation of the infrared assay and assessment of the effect of DHI routine sample acquisition factors on milk urea results. Analytic validation of an automated milk urea assay was performed by assessing the relative accuracy and precision of milk urea results produced by the Fossomatic 4000 Milk Analyzer, an infrared method of analysis, compared with the Eurochem test, an accepted reference method. Results indicated that, when interpreted at the group level, milk urea results between the infrared method and the reference test were in good agreement. The two tests shared a similar and high level of precision. Milk urea concentrations obtained from composite (metered) milk samples, and not quarter stripping samples, were most representative of concurrent serum urea concentrations. The addition of bronopol preservative did not result in a numerically important change in milk urea concentrations. Storage of preserved metered milk samples for up to 4 d at either room temperature or by refrigeration, or for up to 3 d by freezing, did not result in changes in milk urea concentrations. We concluded that milk samples, as they are routinely collected and handled by DHI, are suitable for measurement of milk urea concentrations with the infrared method of analysis if data are interpreted at the group level. PMID- 10750100 TI - Least-cost ration formulations for Holstein dairy heifers by using linear and stochastic programming. AB - Four mathematical programming models were developed to formulate rations for large breed replacement dairy heifers in each of 11 different weight classes from 50 to 550 kg and daily growth rates of 600, 700, and 800 g, with the objective of achieving a final calving weight of 600 kg. First, a base linear programming model was developed; then, to account for variability in the crude protein content of ration ingredients three other methods were used: right-hand side adjustment, incorporation of a safety margin, and stochastic programming. The average daily cost to calving, given a daily gain of 600, 700, and 800 g, was $0.62, $0.64, and $0.68, respectively. The total feed cost to 600 kg was $89.87 more for a growth rate of 600 over 800 g/d. The stochastic programming model performed better, on the bases of cost and protein-feeding, than did the right hand side adjustment or the safety margin methods. The stochastic programming model over-adjusted crude protein by 5% and cost an average of 3.5% more than the linear programming solution for a dairy heifer growing at 800 g/d with a desired probability of 80% of crude protein intake achieving the NRC minimum. The other two methods over-adjusted crude protein by 10 and 13% and cost an extra 5.5 and 7.6%, respectively, for the right-hand side adjustment and the safety margin methods. PMID- 10750101 TI - A homologous radioimmunoassay for quantification of insulin-like growth factor binding protein-2 in blood from cattle. AB - Insulin-like growth factor-I and -II (IGF-I, IGF-II) circulate in biological fluids bound to six different IGF-binding proteins that regulate IGF bioactivity. The IGF-binding protein-2 is regulated by growth hormones, and its concentration depends on nutrition and physiological state. Specific antibodies directed against bovine IGF-binding protein-2 were produced, and IGF-binding protein-2 levels in bovine blood samples were quantified by radioimmunoassay. Parallel displacement curves showed strong cross-reactivity with bovine and ovine plasma, were low with porcine plasma, and no cross-reactivity with rat or chicken plasma. Addition of IGF-I or -II to a control pool of bovine plasma did not significantly alter control IGF-binding protein-2 values in a radioimmunoassay. Six nycthemeral periods, determined for three young bulls bled on two occasions, showed that IGF binding protein-2 plasma levels were stable throughout the day; two or three samples were sufficient to characterize the animal. Cows treated with recombinant bovine somatotropin (bST) had significantly lower serum levels of IGF-binding protein-2 than did control cows. Furthermore, IGF-binding protein-2 levels were dramatically increased at the onset of lactation. This radioimmunoassay for bovine IGF-binding protein-2, which enables quantitative assessment of IGF binding protein-2 concentration in cattle, confirmed that IGF-binding protein-2 concentrations are depressed by administration of bST, enhanced after calving, and showed absence of diurnal variation. PMID- 10750102 TI - Milk urea nitrogen and infertility in Florida Holstein cows. AB - The objective of this study was to evaluate the association between milk urea nitrogen (MUN) and risk of nonpregnancy after first breeding in a commercial dairy herd in Florida. A total of 515 and 558 cows were classified as having high (17 to 25 mg/dl) or low MUN (6 to 16 mg/dl) within 30 d before first breeding; a total of 158 (30.6%) and 189 (33.8%) cows were diagnosed as pregnant, respectively. Logistic regression was used to evaluate the association between MUN and risk of nonpregnancy controlling for other variables associated with fertility (parity, calving season, breeding season). An interaction was found showing that cows with high MUN that were bred during the summer were 18 times (OR = 17.9; 95% CI = 10.0 to 31.7) at higher risk of nonpregnancy compared to cows with low MUN that were bred during the winter. PMID- 10750103 TI - Treatment of noncyclic lactating dairy cows with progesterone and estradiol or with progesterone, GnRH, prostaglandin F2 alpha, and estradiol. AB - The efficacy of two programs for treating noncyclic cows was compared. In trial 1, 478 cows in five herds were randomly divided into two groups. Cows in one group (C group) were treated with an intravaginal progesterone device for 8 d followed in 48 h by 1 mg of estradiol benzoate to cows that had not been detected in estrus since device removal. Those in the other group (CGP group) were treated with progesterone and estradiol as for the C group plus 10 micrograms of a GnRH agonist (buserelin) at device insertion and 25 mg of PGF2 alpha 7 d after device insertion. In trial 2 with 729 cows in nine herds, the treatments were similar to those in trial 1 except that the duration of progesterone treatment was 7 d. No significant difference was found between trials and results from both trials were combined. Compared with C group cows, CGP group cows had a greater estrous response rate (93.2 vs. 89.1%), a greater conception rate to first artificial insemination (AI, 47.1 vs. 29.4%), marginally lower conception rate to second AI (52.9 vs. 59.7%), lower nonpregnancy rate (8.3 vs. 11.1%), and shorter intervals from the start of breeding to conception by AI (9.8 vs. 15.3 d) or by AI or natural mating (21.6 vs. 26.3 d). The treatment protocol used for the CGP group achieved better reproductive performance than that used for the C group. PMID- 10750104 TI - Estrus synchronization of lactating dairy cows with GnRH, progesterone, and prostaglandin F2 alpha. AB - The reproductive performance of synchronized cows was compared with that of nonsynchronized cows. In trial 1, cyclic cows in five seasonal herds were randomly divided into two groups. Cows in one group (n = 515) were treated with a GnRH agonist and an intravaginal progesterone device, followed in 7 d by a PGF2 alpha injection, and the device was removed 1 d after PGF2 alpha. Cows in the other group (n = 512) did not receive any treatment and acted as control. In trial 2, the treatments were similar to those used in trial 1 except that the progesterone device was removed at the time of PGF2 alpha injection (synchronized: n = 516; control: n = 512). The estrus synchronization rate was 92.8% in trial 1 and 92.2% in trial 2. Conception rate to first artificial insemination (AI) was lower for synchronized cows than for control cows in trial 1 (56.5 vs. 62.7%), but similar in trial 2 (64.6 vs. 63.3%). Across both trials, the pregnancy rate during the AI breeding period was greater for the synchronized cows (85.6%) than for the control cows (81.2%). The synchronization treatment reduced the interval from start of the breeding season to conception for cows conceiving by AI (8.9 vs. 14.8 d) or by AI or natural mating (14.1 vs. 21.6 d). The synchronization protocol used in trial 2 achieved better conception rate than that used in trial 1, but the precision of estrus was less in trial 2 than in trial 1. PMID- 10750105 TI - Effect of superovulation prior to mating on milk production performance during lactation in ewes. AB - Thirty lactating ewes were used to evaluate the effect of superovulation on milk production. Twelve ewes had been injected, prior to mating, with 700 IU of pregnant mare serum gonadotropin; 18 ewes were injected with saline as a control. Thirteen ewes (nine control and four superovulated ewes) were fed at low plane of nutrition; the other ewes (nine control and eight superovulated ewes) were fed at high plane of nutrition. Superovulated ewes, fed at both low and high planes of nutrition, had dramatically higher milk yields (59%), and their milk composition was not changed. Plane of nutrition increased milk lactose and P contents without significant effect on milk production. The increased milk yields in the superovulated ewes were accompanied by increases in dry matter, gross energy intakes, and gross efficiency of milk synthesis. At the end of lactation, superovulated ewes had higher mammary dry fat-free tissue, total DNA, and total RNA. The results demonstrated that superovulation prior to mating dramatically increased milk production and efficiency regardless of plane of nutrition. Increased milk production and efficiency in the superovulated ewes were due to the increased mammary secretory cell numbers and their synthetic activities presumably through the increased endogenous hormonal stimulation of mammary growth and development during pregnancy. PMID- 10750106 TI - Comparing follicle stimulating hormone from two commercial sources for oocyte production from out-of-season dairy goats. AB - Until recently, two sources of follicle stimulating hormone (FSH-P; Schering Plough; Kenilworth, NJ and Super-Ov; FSH-SOV; AUSA International, Tyler, TX) have been commercially available in the United States and routinely used for superovulation of ruminants. Because there have been no comparative follicle stimulating hormone studies on small ruminants, we determined the difference between the number of follicles induced and the number of oocytes that can subsequently be harvested from goats stimulated with either of these two follicle stimulating hormone products. Anestrous Saanen does were fitted with a progestin implant then randomly assigned to one of two ovarian stimulation groups. Starting 4 d after introducing the progestin implant, donors in treatment 1 were administered daily injections of FSH-P for 4 d. Does in treatment 2 were similarly treated but were administered FSH-SOV for 4 d. Follicle aspirations were performed by laparotomy in the morning of treatment d 8. In summary, no difference was detected between the two stimulatory agents for the number of follicles and quality of oocytes harvested from stimulated does, indicating that these two commercial FSH products could be used successfully for ovarian stimulation of anestrous dairy goats. PMID- 10750107 TI - A quantitative approach to classifying Holstein cows based on antibody responsiveness and its relationship to peripartum mastitis occurrence. AB - A quantitative approach was developed to classify Holstein cows and heifers based on phenotypic variation of serum antibody response and to determine associations with peripartum mastitis. Using an index, 136 cows and heifers were classified into high (Group 1), average (Group 2), or low (Group 3) antibody groups following immunization with ovalbumin at wk -8, -3, and 0 relative to parturition. The ranking of groups based on the quantitative index of serum antibody response to ovalbumin were similar for sera and whey antibody such that Group 1 > Group 2 > Group 3. Animals were also vaccinated with Escherichia coli J5 (Rhone Merieux, Lenexa, KS) at wk -8 and -3 relative to parturition. The ranking of groups for E. coli J5 was similar to that observed for serum and whey antibody to ovalbumin. Serum and whey IgG1 and IgG2 concentrations were measured at wk 0, 3, and 6 but differences between groups were not significant. There was no occurrence of mastitis for Group 1 animals in two of the herds. In contrast, Group 1 animals from the third herd had the highest occurrence of mastitis; however, these cases all occurred in first-parity heifers. According to pooled data across all herds, Group 3 animals had the highest occurrence of mastitis. Heritability estimates of serum antibody response to ovalbumin varied between 0.32 to 0.64 depending on week relative to parturition. Heritability estimates of serum antibody response to E. coli J5 also varied between 0.13 to 0.88 depending upon week relative to parturition. These results indicate that high peripartum antibody may be beneficial in some herds. PMID- 10750108 TI - Cysteines involved in the interconversion between dehydrogenase and oxidase forms of bovine xanthine oxidoreductase. AB - Mammalian xanthine oxidoreductase exists intracellularly in its dehydrogenase form. However, outside of this reducing milieu the enzyme quickly transforms into an oxidase form. Interconversion can be controlled by sulfhydryl reactive reagents, suggesting that disulfide bridging is linked to this phenomenon. The present work identified cysteines involved in the interconversion process. Purified enzyme was subjected to mild reduction with 1,4-dithioerythriol to regain dehydrogenase activity, and the accessible cysteines were labeled with specific radioactive alkylation reagents, iodoacetic acid. This partial alkylation stabilizes the dehydrogenase form, presumable by hindering formation of disulfide bond(s). Six of 38 cysteines were found to be labeled (residues 169, 170, 535, 992, 1317, and 1325). The significance of this labeling of bovine xanthine oxidoreductase is discussed in relation to structural knowledge about the enzyme, and especially by comparison with the AA sequences of avian and invertebrate enzymes, which do not undergo conversion. Cysteines 535 and 992 are the most likely marked residues to be involved in the interconversion, whereas the other cysteines are located too far from the cofactorbinding areas in xanthine oxidoreductase. PMID- 10750110 TI - Metabolic alterations associated with an attempt to induce laminitis in dairy calves. AB - The objective of this study was to investigate metabolic alterations in young ruminating calves associated with the sudden introduction of readily fermentable diets in an attempt to induce laminitis. Sixteen dairy bull calves, at 17 wk of age were fed equal amounts of one of four diets that contained either 71 or 81% total digestible nutrients (TDN) and 15 or 20% crude protein in a 2 x 2 factorial with time as a factor. Jugular blood and ruminal fluid were sampled, and hoof temperature was measured postfeeding at frequent intervals over a subsequent 2-d period. Hooves were examined for abnormalities and the orientation of the pedal bone radiographed prior to the experiment, 48 to 72 h into the experiment and at 3 and 7 mo later. Calves responded acutely to the 81% TDN diets by inappetence, stiffness, and diarrhea. Ruminal pH was lower and both D- and L-lactate concentrations were greater in the rumens of calves fed the 81% TDN diets. Total ruminal volatile fatty acid concentration decreased as pH declined. Whole blood L lactate did not differ across treatments, but blood D-lactate increased in calves fed the 81% TDN diets, peaking at 32 h (7.2 mM). Hoof temperature responses could not be explained by dietary treatments. Laminitis was not detected despite the reduction of ruminal pH and a manyfold increase in blood D-lactate. PMID- 10750109 TI - Calf intestinal mucin: isolation, partial characterization, and measurement in ileal digesta with an enzyme-linked immunosorbent assay. AB - The aim of this study was to develop a specific ELISA for calf intestinal mucin to quantify its contribution to ileal endogenous losses. Mucin was isolated from intestinal scrapings by cesium chloride density gradient ultracentrifugation. The isolated mucin had a high concentration of glutamic and aspartic acids, threonine, and serine (13.2, 11.2, 9.6, and 9.2 mol % of total amino acids assayed, respectively). The carbohydrates present were (mol % of total hexoses): galactose 42.1, N-acetylglucosamine 24.1, N-acetylgalactosamine 23.6, fucose 4.7, mannose 3.1, and sialic acids 2.4. Amino acids and carbohydrates represented 52.6 and 47.4% of the mucin by weight, respectively. A rabbit hyperimmune plasma was raised against purified mucin and used to set up an ELISA. The linear range of this assay was 20 to 640 ng/ml. The plasma cross-reacted with calf abomasal and colonic mucin. It showed no cross-reactivity with nonmucin components and no reactivity with intestinal mucin from other animal species except for the rat. Mucin accounted for approximately 3.5% of the dry matter output at the ileum of calves fed a substitute milk based on skim milk powder. This represented a flow of 3.4 g of mucin/kg of dry matter intake. Mucin flow increased when dietary protein was provided by cow's colostrum. Finally, the developed assay is a suitable tool to investigate the impact of dietary factors on the flow of mucin along the gut of preruminant calves. PMID- 10750111 TI - The effect of Lactobacillus buchneri, Lactobacillus plantarum, or a chemical preservative on the fermentation and aerobic stability of corn silage. AB - Several microorganisms and one chemical preservative were tested for their effects on the fermentation and aerobic stability of corn silage. Whole-plant corn (one-half milk line, 31.3% dry matter) was ensiled in quadruplicate 20-L laboratory silos untreated or after the following treatments: Lactobacillus buchneri at 1 x 10(5) and 1 x 10(6) cfu/g of fresh forage; two different strains of L. plantarum, each at 1 x 10(6) cfu/g; and a buffered propionic acid-based product at 0.1% of fresh forage weight. After 100 d of ensiling, silage treated with L. buchneri (1 x 10(6) cfu/g) had a lower concentration of lactic acid compared with the untreated silage, but was similar to other treated silages. The silage treated with the high (1 x 10(6) cfu/g), but not the moderate rate (1 x 10(5) cfu/g) of L. buchneri also had a greater concentration of acetic acid (3.60%) and less yeasts (2.01 log cfu/g) when compared with other treatments (average of 1.88% acetic acid and 5.85 log cfu of yeasts/g). Silages treated with L. plantarums, the moderate rate of L. buchneri, and the chemical preservative took longer to heat than untreated silage when exposed to air, but improvements were numerically small (6.3 to 10.5 h). In contrast, silage treated with the high rate of L. buchneri never heated throughout a 900-h period of monitoring. Inoculating corn silage with 1 x 10(6) cfu/g of L. buchneri resulted in a more heterolactic fermentation and dramatically improved the aerobic stability of corn silage. PMID- 10750112 TI - Effects of Tween 60 and Tween 80 on protease activity, thiol group reactivity, protein adsorption, and cellulose degradation by rumen microbial enzymes. AB - Microbial enzymes extracted from mixed ruminal microorganisms were incubated for 2 h with casein and Tween 60 or Tween 80 at 10 concentrations ranging from 0 to 2.0% (vol/vol) to determine the effects of these nonionic surfactants on protease activation and thiol reactivity (unmasking of thiol groups). Rate and extent of protein adsorption to cellulosic substrate (barley straw) was measured in the presence of 0, 0.05, 0.10, 0.25, and 0.50% (vol/vol) Tween 80. Degradation of cellulose by a rumen bacterial fraction was measured over 48 h of incubation with and without Tween 60 or Tween 80 at 0.25% (vol/vol). Maximum accelerations of protease activity achievable with Tween 60 and Tween 80 (calculated from a Michaelis-Menten kinetics model) were 99.2 and 166.8%, respectively. Concentrations of Tween 60 and Tween 80 at which half the maximal velocities were attained were 0.28 and 0.20% (vol/vol), respectively. Tween 80 increased (P < 0.05) the rate and extent of adsorption of microbial protein to barley straw, and the effect was related to concentration of Tween 80 up to 0.10% (vol/vol). Initial rates of cellulose degradation with no surfactant, 0.25% Tween 60, or 0.25% Tween 80 were 0.60, 0.87, and 1.04 micrograms/ml per h, respectively. These nonionic surfactants were effective for enhancing rumen microbial protease and cellulase activities. Thus, further study is warranted to determine their potential for improving ruminant feeding. PMID- 10750113 TI - Evaluation of a nonstarch polysaccharidase feed enzyme in dairy cow diets. AB - The objective of this study was to evaluate a commercial feed enzyme product (Natugrain 33-L; BASF Corporation, Ludwigshafen, Germany) used mainly in poultry diets for use in ruminant diets. The product contained mainly beta-glucanase, xylanase, and endocellulase activities. The study was conducted as a double 3 x 3 Latin square design with six lactating dairy cows (84 +/- 12 DIM) to measure intake, chewing activities, total tract digestion, and milk production. An additional three cows fitted with ruminal cannulae were used to measure dietary effects on ruminal fermentation and in sacco digestion characteristics. Cows received a diet consisting of 45% forage containing 0, 1.22, or 3.67 L of enzyme product/tonne of total mixed ration (DM basis). Using a low or high concentration of enzyme supplementation increased feed intake, but total tract digestibility only increased with the low concentration of enzyme. As a result, intake of digestible nutrients was increased to a greater extent for cows fed the low concentration than for cows fed the high concentration. Because the cows used in this experiment were in positive energy balance, increased intake of digestible energy due to enzyme supplementation did not increase milk yield or milk component yield. Further research is necessary to determine the mechanism by which adding a fibrolytic enzyme mixture enhanced intake, but only increased feed digestion when used at a low level. PMID- 10750114 TI - Effects of barley grain processing on extent of digestion and milk production of lactating cows. AB - Effects of barley processing on site and extent of digestion and milk production in dairy cows were evaluated in a 4 x 4 Latin square design with four lactating cows with ruminal and duodenal cannulas. Barley grain was steam-rolled to four thicknesses: coarse, medium, medium-flat, and flat. The processing index (PI), measured as volume weight of barley after processing expressed as a percentage of its volume weight before processing, was 81.0, 72.5, 64.0, and 55.5% for the four treatments, respectively. Diets consisted of 53% concentrate (dry matter basis) containing one of the four processed barleys. Cows were offered ad libitum access to a total mixed ration three times daily. Dry matter intake was quadratically increased with decreasing PI, with maximum intake for cows fed medium-flat barley. Although ruminal digestibilities of organic matter, starch, and crude protein were not affected by grain processing, intestinal and total tract digestibilities were linearly increased as PI of barley was reduced. Milk yield was quadratically increased (25.6, 28.1, 30.8, and 29.0 kg/d) with decreasing PI, and maximum milk yield was for cows fed medium-flat barley. Milk fat and lactose contents were similar, but milk protein content was increased with decreasing PI. These results indicate that the optimal extent of barley processing for dairy cows fed diets supplying adequate fiber was medium-flat, corresponding to a processing index of about 64%. Coarsely or flatly rolled barley is not recommended, because extensive processing did not further improve intake of digestible nutrients, and coarsely processed barley resulted in the lowest intake of digestible organic matter; hence, lowest milk production. Processing index is a reliable and practical method to quantitatively measure extent of steam rolling. PMID- 10750115 TI - Feeding oleamide to lactating Jersey cows. 2. Effects on nutrient digestibility, plasma fatty acids, and hormones. AB - Six lactating Jersey cows were used in a 6 x 6 Latin square with 14-d periods to evaluate different ratios of canola oil and oleamide on nutrient digestibility, plasma fatty acids, and plasma hormones. The control diet contained no added fat. All other diets contained 3.5% added fat consisting of 0, 25, 50, 75, and 100% as oleamide and the remainder as canola oil. Data were collected during the final 4 d of each period. Dry matter intake was reduced by the addition of canola oil to the diet, and further reduced by replacing canola oil with oleamide. Milk yield was not affected by diet but increasing oleamide proportion in the fat supplement caused linear increases in cis-C18:1 and linear decreases in C4 to C16 fatty acids in milk. Adding canola oil reduced total tract digestibilities of fiber and fatty acids, but had no effect on the digestibilities of dry matter or protein. Replacing canola oil with oleamide increased protein digestibility linearly, and increased digestibility of fiber (quartic relationship) and fatty acids (quadratic relationship). Oleic acid concentration in plasma increased by adding canola oil to the diet, and was further increased by replacing canola oil with oleamide. Diet had no effect on plasma concentrations of insulin or IGF-I. Oleamide fed to Jersey cows in this study was highly digestible and had no deleterious effects on total tract digestility of fiber or protein. Increasing oleic acid concentration in plasma lipids while maintaining a constant level of added fat in the ration had no effect on circulating concentrations of insulin or IGF-I in Jerseys. PMID- 10750116 TI - Genetic correlation between days until start of luteal activity and milk yield, energy balance, and live weights. AB - The objective of this study was to estimate genetic correlations among fertility and measures of energy balance, dry matter intake, and live weight (change). Data from 622 first lactation cows (fed ad libitum a complete ration) included milk, fat, and protein yields; energy balance; and live weights for the first 15 wk of lactation. For a subset of the heifers (n = 329) and 91 contemporaries, progesterone values were measured in the milk twice a week and were used to determine the interval between calving and first luteal activity (CLA). To obtain correlations, a series of bivariate animal model analyses was performed. The heritabilities for CLA and energy balance were 0.16 and 0.33, respectively. Heritability for live weights were 0.48 to 0.61 and for live weight changes were 0.17 and 0.24. Genetic correlations of CLA with milk, fat, and protein yields were high and unfavorable (0.51, 0.65, and 0.48, respectively), whereas energy balance, live weight during lactation, and weight gain all had a favorable genetic correlation with CLA (range -0.40 to -0.80). When genetic variation in CLA was adjusted genetically for milk, fat, and protein yields, the correlation with feed intake became -0.49 and explained approximately half of the genetic variance in CLA. This supports that increasing genetic merit for feed intake, at a common genetic merit for yield, improved CLA. Furthermore, selection on an index including protein yield and energy balance or live weight (change) allowed 0.71 to 0.80 of the maximum response in protein yield without a negative selection effect on CLA. PMID- 10750117 TI - Microbiology and biochemistry of cheeses with Appelation d'Origine Protegee and manufactured in the Iberian Peninsula from ovine and caprine milks. AB - To support legal protection with objective technical data and to promote enforcement of high quality standards a few European countries have created Appelation d'Origine Protegees. This paper reviews and updates fundamental and applied aspects encompassing microbiological and biochemical characteristics of traditional cheeses with Appelation d'Origine Protegee manufactured in the Iberian Peninsula from ovine, caprine, or both milks. Ovine and caprine cheeses with Appelation d'Origine Protegee from Portugal and Spain can be divided into four distinct groups based on milk source and rennet type: 1) Azeitao, Castelo Branco, Evora, Nisa, Serpa, Serra da Estrela, and La Serena cheeses are manufactured with raw ovine milk and coagulated via plant rennet; 2) Terrincho, Idiazabal, Manchego, Roncal, and Zamorano cheeses are manufactured with raw ovine milk and coagulated via animal rennet; 3) Cabra Transmontano and Majorero are manufactured with raw caprine milk and coagulated via animal rennet; and 4) Amarelo da Beira Baixa, Picante da Beira Baixa, and Rabacal are manufactured with mixtures of raw ovine and caprine milks and coagulated via animal rennet. PMID- 10750118 TI - Effect of age at first calving on production traits and on difference between milk yield returns and rearing costs in Italian Holsteins. AB - The 305-d yield records of 1,048,942 Italian Holstein-Friesian heifers were used to determine the effect of age at first calving on milk yield and fat and protein concentration. Research showed that there is a positive effect of age at first calving on milk yield and fat percentage and a negative effect on protein percentage. In six scenarios of milk price and rearing costs (2 x 3, respectively), the effect of age at first calving on the difference between milk yield returns and rearing costs was calculated. We concluded that the most positive difference was achieved with age at first calving between 23 and 24 mo. PMID- 10750119 TI - Discovering asthma in the older adult. AB - New-onset asthma may occur at any time in the life cycle, but it is often difficult to diagnose in older adults. The symptoms of asthma are strikingly similar to the symptoms of many cardiac and pulmonary diseases prevalent in this age group. This article discusses the presentation of undiagnosed asthma in the older adult, key management issues for this population, and techniques for assisting clinicians in identifying triggers that precipitate or worsen symptoms. PMID- 10750120 TI - Exercise and pregnancy in primary care. AB - An increasing number of women exercise throughout pregnancy. New evidence illustrates that moderate intensity exercise in healthy pregnant women, with certain considerations, does not increase adverse pregnancy risks. Exercise during pregnancy may actually decrease pregnancy-associated discomforts and improve maternal fitness and well-being. By following the American College of Obstetricians and Gynecologists recommendations, clinicians can confidently prescribe exercise to women during preconception, pregnancy, and the postpartum period. PMID- 10750121 TI - The effects of an advanced practice nurse-directed heart failure program. AB - A study was conducted to determine if an inpatient heart failure program directed by an advanced practice nurse (APN) affects the following patient outcomes: length of hospital stay, mortality, readmission rates, and adherence to the recommended clinical regimen. Evaluating APN-directed heart failure programs may assist hospitals and clinics to determine whether such programs should be replaced, modified, continued, or replicated. For those facilities that are considering beginning a heart failure program, the data provided in this study may help establish the necessity of developing such a program or provide ideas for program design. The APN has an integral role in program design and can assist in achieving positive patient outcomes. PMID- 10750122 TI - Advanced nursing practice in the United Kingdom. PMID- 10750123 TI - Diagnosing and treating hypothyroidism. AB - Hypothyroidism is a common endocrine disorder affecting 1.4% to 2.0% of women and 0.1% to 0.2% of men. The prevalence of both overt and subclinical hypothyroidism increases with age, affecting 5% to 10% of women over age 50 and 1.25% of men over age 60, with an increasing incidence in women ages 40 to 50. Typical symptoms are consistent with declining metabolic functions and range from vague complaints of fatigue in subclinical deficiency to overt clinical symptoms involving changes in mentation and memory, lethargy, weight gain, cold intolerance, constipation, and goitrous enlargement of the thyroid gland. Atypical presentations such as weight loss, hearing impairment, tinnitus, and carpal tunnel syndrome may occur, especially in the elderly. This case report reviews the presenting symptomatology of an otherwise healthy 43-year-old woman who exhibited typical and atypical symptoms of underlying thyroid deficiency. PMID- 10750124 TI - Risedronate: a new bisphosphonate for the treatment of osteoporosis. PMID- 10750125 TI - Hypertension, chronotherapy, and patient management. AB - This article highlights information from a symposium held at the 1999 National Conference for Nurse Practitioners, Washington, D.C., November 11, 1999, sponsored by The Nursing Institute and supported by an unrestricted educational grant from Schwarz Pharma Inc. "Hypertension" was the first topic and was discussed by Elijah Saunders, MD, FACC, FACP. "Medical Chronobiology and Chronotherapy" was the second topic and was discussed by Michael H. Smolensky, PhD. The third topic, "The Nurse Practitioner and the Hypertensive Patient," was discussed by Helen C. Noel, ANP, PhD. Dr. Noel also served as chair of the symposium. PMID- 10750126 TI - Arrhythmia induction by antiarrhythmic drugs. PMID- 10750127 TI - Steady-state versus non-steady-state QT-RR relationships in 24-hour Holter recordings. AB - The aim of the present study was to investigate the QT-RR interval relationship in ambulatory ECG recordings with special emphasis on the physiological circumstances under which the QT-RR intervals follow a linear relation. Continuous ECG recordings make it possible to automatically measure QT duration in individual subjects under various physiological circumstances. However, identification of QT prolongation in Holter recordings is hampered by the rate dependence of QT duration. Comparison of QT duration and QT interval rate dependence between different individuals implies that the nature of the QT-RR relationship is defined in ambulatory ECG. Holter recordings were performed in healthy volunteers at baseline and after administration of dofetilide, a Class III antiarrhythmic drug. After dofetilide, beat-to-beat automated QT measurements on Holter tapes were compared with manually measured QT intervals on standard ECGs matched by time. The QT-RR relationship was analyzed at baseline in individual and group data during three different periods: 24-hour, daytime, and nighttime. Data were collected under steady-state or non-steady-state conditions of cycle length and fitted with various correction formulae. Our study demonstrated an excellent agreement between manually and automated measurements. The classic Bazett correction formula did not fit the QT-RR data points in individual or group data. When heart beats were selected for a steady rhythm during the preceding minute, QT-RR intervals fit a linear relationship during the day and night periods, but not during the 24-hour period in both individual and group data. In contrast, in the absence of beat selection, data fit a more complex curvilinear relationship irrespective of the period. Our study provides the basis for comparison of QT interval durations and QT-RR relationships between individuals and between groups of subjects. PMID- 10750128 TI - Electrophysiological characteristics of the atrium in sinus node dysfunction with and without postpacing atrial fibrillation. AB - In patients with sinus node dysfunction (SND) with or without associated paroxysmal atrial fibrillation (AF), the effectiveness of atrial pacing in reducing the incidence of AF is not definitive. In addition, despite several studies involving large populations of implanted patients, little attention has been paid to the electrophysiological (EP) atrial substrate and the effect of permanent atrial pacing. The aim of this study is to correlate EP data and the risk of AF after DDD device implantation. We reviewed EP data of 38 consecutive patients with SND, mean age 70 +/- 8 years, who were investigated free of antiarrhythmic treatment, for the evaluation of the atrial substrate. We also considered as control group 25 subjects, mean age 63 +/- 14 years, referred to our EP laboratory for unexplained syncope or various atrioventricular disturbances. Following pharmacological washout and at a drive cycle length of 600 ms, effective and functional refractory periods (ERP, FRP), S1-A1 and S2-A2 latency, A1 and A2 conduction duration, and latent vulnerability index (ERP/A2) were measured. AF induction was tested with up to three extrastimuli at paced cycle lengths of 600 and 400 ms in 20 patients. Induction of sustained AF (> 30 seconds) was considered as the endpoint. P wave duration on the surface ECG in lead II/V1 was also measured. DDD pacing mode was chosen in all patients with the minimal atrial rate programmed between 60 and 75 beats/min (mean 64 +/- 4 beats/min). After implantation, the patients were followed-up for 29 +/- 17 months and clinically documented occurrence of AF was determined. When comparing patients with SND and subjects of the control group, we did not find any significant statistical differences in terms of ERP (237 +/- 33 vs 250 +/- 29 ms), FRP (276 +/- 30 vs 280 +/- 32 ms) and S1-A1 (39 +/- 16 vs 33 +/- 11 ms) and S2-A2 latency (69 +/- 24 vs 63 +/- 25 ms). In contrast, we observed significant differences regarding A1 (55 +/- 19 vs 39 +/- 13 ms; P < 0.001), A2 (95 +/- 34 vs 57 +/- 18 ms; P < 0.001) and P wave duration (104 +/- 18 vs 94 +/- 15 ms; P < 0.05), and ERP/A2 (2.8 +/- 1.2 vs 4.8 +/- 1.6; P < 0.001). When comparing patients with (n = 11) or without (n = 27) postpacing AF occurrence, we did not find any difference with reference to ERP, FRP, S1-A1, S2-A2, A1 duration, or follow-up duration. In patients with postpacing AF occurrence, A2 was longer (116 +/- 41 vs 87 +/- 27 ms; P < 0.01), ERP/A2 lower (2.1 +/- 0.4 vs 3.1 +/- 1.4; P < 0.05), P wave more prolonged (116 +/- 22 vs 99 +/- 14 ms; P < 0.01), and preexisting AF history predominant (6/11 vs 5/27 patients; P < 0.05). No difference was observed between patients with (n = 8) and without (n = 12) AF induction during the EP study. In patients with SND, the atrial refractoriness appears normal and the most important abnormality concerns conduction slowing disturbances. Persistence of AF despite pacing stresses the importance of mechanisms responsible for AF not entirely brady-dependent. In this setting, more prolonged atrial conduction disturbances, responsible for a low vulnerability index, and a preexisting history of AF enable us to identify a high risk patient group for AF in the follow-up. PMID- 10750130 TI - Far-field QRS complex sensing: prevalence and timing with bipolar atrial leads. AB - Sensing of far-field QRS complex through the atrial pacemaker lead may cause a number of pacemaker function disturbances, most of which are rarely seen with modern pulse generators. However, certain pulse generator algorithms will still be jeopardized by far-field QRS complex sensing. Intracardiac electrograms with markers were obtained by telemetry in 30 patients following implantation of a permanent bipolar atrial lead and a DDDR pulse generator. The occurrence and timing of far-field QRS complex sensing was studied at different atrial amplifier sensitivity settings. With paced ventricular complexes, QRS sensing was documented in all 30 cases at the maximum atrial sensitivity (0.1 mV). The median QRS complex sensing threshold was 0.3 mV, and the sensing window at high atrial sensitivities was 67-202 ms following the ventricular pacing impulse. In one case, QRS complex sensing was seen up to an atrial sensitivity of 1.5 mV. In 12 of 13 patients with 1:1 AV conduction, atrial sensing of spontaneously conducted ventricular complexes was seen (median sensing threshold 0.2 mV; the sensing window was -23 to 114 ms relative to the ventricular amplifier sensing event). Far-field QRS complex sensing was also found in all 12 patients in whom ventricular fusion complexes were obtained (median sensing threshold 0.2 mV; the window of sensing was 64-187 ms after the ventricular pacing impulse). Constant or intermittent QRS complex sensing via the atrial bipolar lead was thus universally demonstrable. It occurred in only a minority (20%) of patients at a sensitivity of 0.5 mV or less. Knowledge regarding the timing of the oversensing as related to the atrial sensitivity setting may aid in the design of algorithms of future pacemakers and cardioverter defibrillators. PMID- 10750129 TI - Soluble L-selectin and neutrophil derived oxidative stress after pacing induced myocardial ischemia in chronic stable coronary artery disease. AB - We studied the effect of atrial pacing induced myocardial ischemia on levels of soluble L-selectin (sL-selectin) and generation of neutrophil derived reactive oxygen species (ROS) in 10 patients with coronary artery disease (CAD) and stable angina and in six individuals without CAD. Myocardial ischemia was measured metabolically by lactate sampling from the coronary sinus (CS) and arterial blood at each pacing step. Before each pacing step, at peak pacing and shortly after cessation, plasma concentrations of sL-selectin and generation of ROS using the chemiluminescence method were measured in CS and femoral artery blood. Baseline sL-selectin levels in CS samples were significantly lower in the CAD compared to the control group (547 +/- 80 vs 836 +/- 82 ng/mL, P = 0.03). At peak pacing, nine of ten patients with CAD developed myocardial ischemia (lactate extraction ratio at rest 28% +/- 7%, at peak pacing -16% +/- 6%). In these patients, luminol enhanced chemiluminescence (CL, 0.88 +/- 0.45 vs 1.9 +/- 0.9 cpm x 10(5), P = 0.09) and levels of sL-selectin (547 +/- 80 vs 764 +/- 86 ng/mL, P = 0.03) from naive neutrophils increased significantly in CS blood suggesting a potent in vivo activation of neutrophils. In control patients, incremental pacing caused neither myocardial ischemia nor a significant change of chemiluminescence or of sL selectin levels. In conclusion, myocardial ischemia induced by pacing tachycardia is able to activate neutrophils in patients with chronic stable coronary artery disease leading to increased generation of ROS and shedding of L-selectin into the coronary circulation. PMID- 10750131 TI - Automatic threshold tracking activation without the intraoperative evaluation of the evoked response amplitude. AUTOCAP Investigators. AB - Automatic threshold tracking (Autocapture) controls the amplitude of the pacing pulse and adjusts it to the actual pacing threshold. The algorithm is based on the proper detection of the evoked response (ER) amplitude after the pacing pulse. For this reason an intraoperative evaluation of ER and polarization is recommended. The aims of the study were to evaluate the ER signal and polarization and the performance of automatic threshold tracking without any intraoperative testing of the ER signal. In addition, the ER amplitude was correlated with the pacing threshold, pacing impedance, spontaneous R wave amplitude, and with the clinical data. The study included 60 patients who received the VVIR pacemaker Regency connected to the Membrane E 1450/1452 pacing lead (St. Jude-Pacesetter). At implantation, a pacing threshold < 0.7 V at 0.5 ms was achieved in all patients. ER and polarization were assessed for the first time at hospital predischarge testing. Follow-up measurements were conducted at month 1, 3, and 6. The ER amplitude at hospital discharge was 8.4 +/- 4.2 mV and increased to 9.4 +/- 4.8 mV at the 6-month follow-up. The pacemaker recommended not to program automatic threshold tracking on in one patient permanently and in three patients intermittently. The ER amplitudes were not differently distributed in men compared with women or in right-sided compared to left-sided implants. The correlation between age and the evoked response was r = 0.15. The correlation between ER amplitude and pacing threshold was r = -0.08, with pacing impedance r = 0.02, and with R wave amplitude r = 0.44. In conclusion, despite no operative evaluation of the ER amplitude being performed, the mean ER amplitude was about 9 mV at 6-month follow-up. Automatic threshold tracking could be programmed on in 93% of the patients throughout the time. Neither the clinical data nor the conventional electrical parameters help to predict patients who will have low ER amplitude or to optimize the ER signal at implantation. PMID- 10750132 TI - Frequency-domain analysis of heart rate variability during positive and negative head-up tilt test: importance of age. AB - The study of autonomic behavior during a head-up tilt test (HUT) has been deemed important to understand the loss of consciousness mechanism. Though HRV in patients with HUT(+) and HUT(-) has been compared, few trials emphasized the importance of age. HRV in frequency domain was analyzed based on 5-minute samples in the supine position, and between 5 and 10 minutes during early tilt test (R1) in 102 patients with one or more episodes of syncope (mean age 44.3 +/- 20.8, range 15-85 years, 55 women). Two subgroups were selected afterwards: (1) young patients between 15 and 35 years of age (41 patients) and (2) elderly patients aged 60 or more (36 patients). The following parameters were taken into account: the sum of low (LF) and high frequency (HF) (LF and HF in absolute values and in normalized units), the LF/HF ratio (L/H ratio), and the percentage of change between baseline and R1 values. The HRV behavior in young and elderly patients with positive and negative HUT was established. We then analyzed the correlation between HRV and age and HUT outcome. A multiple regression analysis encompassing age, HUT outcome, gender, and number of syncope episodes was performed. In young patients, the LF and HF areas and the L/H ratio changed significantly between baseline and R1. The L/H ratio increases from baseline to R1. Conversely, these differences were not significant in the elderly. No differences between HUT(+) and HUT(-) within the same age group were observed. Age related significantly to practically all HRV parameters analyzed, whereas the tilt test outcome correlates poorly with HF normalized units and LF normalized units during R1, and the L/H ratio changes between baseline and R1. By means of a multivariate analysis, only age shows a significant correlation with the HRV values. Despite an all age triggering of vasovagal syncope during HUT, the young and elderly patients' autonomic behavior differs. The young considerably increase their sympathovagal balance during HUT, whereas the elderly have a mitigated autonomic response. No significant differences were observed during the first minutes of the test between those with a HUT(+) and those with a HUT(-) within the same age group. Age, and not the HUT response, is the major determinant of the autonomic behavior during early HUT. PMID- 10750133 TI - Study of interactions between permanent pacemakers and electronic antitheft surveillance systems. AB - Interference of electronic antitheft systems (EASs) with pacemakers has been an object of controversy. This study was performed in 204 patients followed by our pacemaker surveillance center. The data from a total of 408-patient exposures to the EAS were analyzed. The device tested consisted of 129 DDD, 71 VVI, and 4 VDD pacemakers from seven manufacturers. The EAS studied consisted of an "acoustomagnetic" system that emits an intermittent 58-kHz signal, and a magnetic audio frequency system that emits a continuous 73-Hz signal. Complete interrogation of the pacemakers was performed before and after the consecutive exposure of the patients to both EASs. Electrocardiograms were recorded while the patients were exposed to the magnetic fields of each EAS for up to 30 seconds. One or more EAS interferences occurred in 17% of patients. EAS was observed in 26 (20%) of 129 patients with DDD, 7 (10%) of 71 patients with VVI, and 2 (50%) of 4 patients with VDD pacemakers. Over twice as many instances of EAS interference were observed with the "acoustomagnetic" system as were with the magnetic audio frequency system. Among pacemakers programmed in the DDD mode, a considerably greater prevalence of interference was observed at the atrial versus ventricular level, despite the same programmed sensing polarity in both chambers in all but one case. Sensing anomalies were the most common EAS induced disturbance, and typically lasted for the duration of exposure. In a few instances of pacing inhibition, the phenomenon was limited to 1 cycle at the onset of EAS exposure. No changes occurred in the programming of the pacemakers, and a single patient experienced palpitation during EAS induced rapid pacing. During exposure to EAS mimicking the normal use of the systems, interference with a variety of pacemakers was relatively common. However, the anomalies observed were transient and the cause of no symptom or device reprogramming. Patients should be advised to not stand unnecessarily in the close proximity of EASs. PMID- 10750135 TI - The postural orthostatic tachycardia syndrome: a potentially treatable cause of chronic fatigue, exercise intolerance, and cognitive impairment in adolescents. AB - Head upright tilt table testing has become an accepted method to measure an individual's predisposition to autonomically mediated periods of hypotension and bradycardia severe enough to cause frank syncope. At the same time it has become increasingly apparent that less profound falls in blood pressure, while not sufficient to result in loss of consciousness, may cause symptoms such as near syncope, vertigo, and dizziness. We describe a subgroup of adolescents that have a mild form of autonomic dysfunction that exhibit disabling symptoms such as postural tachycardia and palpitations, extreme fatigue, lightheadedness, exercise intolerance, and cognitive impairment. During baseline tilt table testing at a 70 degrees angle, these patients demonstrated a heart rate increase of > or = 30 beats/min (or a maximum heart rate of > or = 120 beats/min) within the first 10 minutes upright (not associated with profound hypotension), which reproduced their clinical symptom complex. Similar observations have been made in the adult population and has been termed the postural orthostatic tachycardia syndrome (POTS). We report that POTS may also occur in adolescents and represents a mild, potentially treatable form of autonomic dysfunction that can be readily identified during head upright tilt table testing. PMID- 10750134 TI - Defibrillators in nonischemic cardiomyopathy treatment evaluation. AB - The Defibrillators in Nonischemic Cardiomyopathy Treatment Evaluation (DEFINITE) is a multicenter randomized trial. Patients will have nonischemic cardiomyopathy (LVEF < or = 35%), a history of symptomatic heart failure and spontaneous arrhythmia (> 10 PVCs/hour or nonsustained ventricular tachycardia defined as 3 15 beats at a rate of > 120 beats/min) on Holter monitor or telemetry within the past 6 months. Patients will be randomized to an implantable cardioverter defibrillator (ICD) versus no ICD. All patients will receive standard oral medical therapy for heart failure including angiotensin converting enzyme inhibitors and beta-blockers (if tolerated). Patients will be followed for 2-3 years. The primary endpoint will be total mortality. Quality-of-life and pharmacoeconomics analyses will also be performed. A registry will track patients who meet basic inclusion criteria but are not randomized. We estimate an annual total mortality of 15% at 2 years in the treatment arm that does not receive an ICD. The ICD is expected to reduce mortality by 50%. Approximately 204 patients will be required in each treatment group. Twenty-five centers will be included in a trial designed to last an estimated 4 years. PMID- 10750136 TI - Clinical and electrocardiographic predictors of recurrent atrial fibrillation. AB - Patients with frequent episodes of paroxysmal atrial fibrillation (AF) are prone to develop permanent AF and have an increased thromboembolic risk. We have previously shown that P wave dispersion (P dispersion), defined as the difference between the maximum and the minimum P wave duration, and maximum P wave duration (P maximum) can distinguish patients with paroxysmal lone AF. The ability of those ECG markers and of other clinical and ECG variables to detect patients at risk for recurrent AF was tested in 88 patients, aged 64 +/- 12 years. All patients had a history of symptomatic episodes of AF during the last 2 years and had not previously received any antiarrhythmic prophylaxis. P maximum and P dispersion were calculated from a 12-lead surface ECG recorded in all patients during sinus rhythm. A computerized ECG system was used and P maximum and P dispersion were calculated on screen from the averaged complexes of all 12 leads. Age (P = 0.01), history of organic heart disease (P = 0.03), P maximum (P < 0.001), minimum P wave duration (P = 0.05), and P dispersion (P < 0.001) were found to be significant univariate predictors of recurrent AF, whereas only P maximum (P < 0.001) and age (P = 0.037) remained significant independent predictors of frequent AF paroxysms in the multivariate analysis. It is concluded that advanced age and prolonged P wave duration may be used as predictors of frequently relapsing AF. Therefore, simple AF predictors exist that could possibly distinguish the patients in whom prophylaxis with antiarrhythmic medicines should be instituted. PMID- 10750137 TI - QT dispersion from body surface ECG does not reflect the spatial dispersion of ventricular repolarization in sheep. AB - The correlation between the QT dispersion on body surface ECG and the dispersion in ventricular repolarization from the cardiac surface was studied in six sheep anesthetized with pentobarbital. The standard 12-lead body surface ECG and multiple ventricular epicardial ECGs were simultaneously recorded. The activation recovery interval (ARI) was measured from the unipolar epicardial ECGs. The pooled QT dispersion from the six animals was significantly smaller than the pooled ARI dispersion (22.7 +/- 2.6 vs 33.0 +/- 6.9 ms, P < 0.01). There was no correlation between the QT and ARI dispersion. The unipolar epicardial ECGs were then converted into bipolar ECGs and epicardial QT intervals were subsequently acquired from these ECGs. The average value of epicardial QT dispersion from the six animals was similar to that of body surface ECG, but was less than the ARI dispersion (27.5 +/- 6.8 vs 33.0 +/- 6.9, P < 0.01). A good correlation between the epicardial QT dispersion and ARI dispersion was identified (r = 0.84, P < 0.05). In addition, a prolongation in ventricular repolarization, induced by an increase in coronary flow, elicited a pooled ARI dispersion of 62.3 +/- 6.2 ms (n = 6), which was larger than the simultaneously recorded body surface QT dispersion (28.3 +/- 9.8 ms, n = 6, P < 0.01). No correlation between the ARI and QT dispersion was found in the presence of the prolonged ventricular repolarization. In conclusion, QT dispersion from a 12-lead body surface ECG seems to underestimate the spatial dispersion of ventricular repolarization acquired from sheep epicardium. PMID- 10750138 TI - Preliminary results with the simultaneous use of implantable cardioverter defibrillators and permanent biventricular pacemakers: implications for device interaction and development. AB - We report our preliminary experience with the combined use of implantable cardioverter defibrillators (ICD) and biventricular pacemakers in six patients with heart failure and malignant ventricular arrhythmia. Two patients underwent ICD implantation for malignant ventricular arrhythmia after previous biventricular pacemaker implantation. One patient underwent biventricular pacemaker insertion for NYHA Class III heart failure after previous ICD implantation. Two patients underwent single device implantation. In the sixth patient, a combined implantation failed due to an inability to obtain a satisfactory left ventricular pacemaker lead position. The potential for device interaction was explored during implantation. In two patients a potentially serious interaction was discovered. Subsequent alterations in device configuration and programming prevented these interactions with long-term use. No complication of combined device use has been demonstrated during a mean follow-up of 2 months (range 1-4 months). Satisfactory ICD and pacemaker function has also been demonstrated. We conclude that combined device implantation may be feasible with currently available pacing technology and that further prospective studies are required in this area. PMID- 10750139 TI - Prevention of the initiation of atrial fibrillation: mechanism and efficacy of different atrial pacing modes. AB - Several atrial pacing modes have been reported to be effective in the prevention of atrial fibrillation (AF); they included biatrial pacing, dual site right atrial pacing, Bachmann's bundle (BB) pacing, and coronary sinus pacing. However, the relative efficacy and electrophysiological mechanisms of these pacing modes in the prevention of AF are not clear. In 15 patients (age 54 +/- 14 years) with paroxysmal AF, P wave duration, effective refractory period, and atrial conduction time were determined with six different atrial drive pacings, that were right atrial appendage (RAA), BB, right posterior interatrial septum (RPS), distal coronary sinus (DCS), RAA plus RPS simultaneously (DSA), and RAA plus DCS simultaneously (BiA). All these patients consistently had AF induced with early RAA extrastimulation coupling to RAA drive pacing. No patient had AF induced with RAA extrastimulation coupled to BB, RPS, or DCS drive pacing, but seven and eight patients had AF induced with RAA extrastimulation coupled to DSA and BiA drive pacing, respectively. The P wave duration was longest during RAA pacing, and became shorter during other atrial pacing modes. Analysis of electrophysiological change showed that early RAA extrastimulation coupled to RAA drive pacing caused the longest atrial conduction delay among these atrial pacing modes; BB, RPS, and DCS drive pacing caused a greater reduction of this conduction delay than DSA and BiA drive pacing. In addition, the effective refractory periods of RAA determined with BB, RPS, and DCS drive pacing were similar and longer than that determined with DSA and BiA drive pacing. In patients with paroxysmal AF, this arrhythmia was readily induced with RAA extrastimuli coupled to RAA drive pacing. BB, RPS, and DCS pacing were similar and more effective than DSA and BiA pacing in preventing AF. PMID- 10750140 TI - Reduced frequency of retention wire fractures suggests that elective explantation of affected atrial leads is no longer indicated. AB - Since 1994, 611 patients with atrial Accufix leads have been followed. Consideration was given to explanting these leads based on the presence or absence of fractures of the retention wire. All leads were followed according to a protocol proposed by the manufacturer, chiefly by regular 6-month high quality X rays and fluoroscopic evaluation. The impact of various conditions on the incidence of fracture was evaluated, such as the shape and location of the lead, prior open heart surgery, and the implantation route. The only factor related to the frequency of fractures was the shape of the lead, fractures occurring statistically more frequently if the leads were pulled from their normal J shape into an L or straightened configuration. The frequency of fractures has plateaued at 6 through 9 years with no further occurrence of Class III or IV fractures (protrusion of the fractured segment). Altogether there were nine (1.5%) Class III and Class IV fractures at the 9-year follow-up. Actuarial survival, in Class I or II, was 97%. We concluded, that the frequency of retention wire fractures has plateaued. This information coupled with the knowledge that the Accufix lead extraction can be difficult and dangerous, suggests that the remaining leads are best left in place. PMID- 10750141 TI - De duobos malis minus est semper eligendum (of two evils the lesser should always be chosen). PMID- 10750142 TI - In vivo potentiostatic studies at the electrode tissue interface: filter properties of the monophasic action potential (Ag/AgCl) electrode in living rat heart. AB - The monophasic action potential (Franz) catheter is regarded as the criterion standard for high fidelity recording of a class of physiological signals. However, its signal modulation characteristics have never been reported. Broadband impedance spectroscopy was performed in perfused living rat heart in a three-electrode potentiostatic configuration to determine the filtering characteristics of the MAP and model Ag/AgCl electrode-tissue interfaces. The filter transfer function H(f) (attenuation [dB] vs log(f) [log(Hz)]) was derived for the frequency range 10 Hz-10(6) Hz. As a filter, the MAP interface is characterized by two ranges of filtering behavior. At high frequency the MAP interface is a high-pass filter with passband frequency 54 kHz-549 kHz (median 321 kHz) and with -3 dB cutoff points ranging from 10 kHz to 302 kHz. In this high frequency range the transfer function is characterized by decreasing attenuation per decade. However, in the lower frequency range relevant to physiological signals (the monophasic action potential, 0.1-40 Hz), it is a severely attenuating nondiodic high-pass filter element with an average attenuation of 16.87 dB relative to passband. In this physiological range, rolloff is nonlinear with increasing attenuation per decade. While the MAP electrode and model Ag/AgCl electrodes are high-pass filters with robust transfer functions for high frequency signals in the living heart, the attenuation of signals in a frequency range relevant to in vivo physiological recording imparts extreme attenuation that may distort physiological signals unpredictably. This disadvantage may be mitigated by amplitude scaling to a calibrated pure tone signal within the physiological frequency band to recover a reproducible signal. PMID- 10750144 TI - Loss of atrial tracking following dual chamber ICD implantation. PMID- 10750143 TI - Nonpharmacological treatment of atrial fibrillation: a heretic's appraisal. PMID- 10750145 TI - Brugada syndrome characterized by the appearance of J waves. AB - We describe a patient with Brugada syndrome. The ST-segment elevation in precordial leads was revealed during admission, but the appearance of J waves was characteristic before ventricular fibrillation (VF), rather than ST-segment elevation. J waves have been reported to be associated with the presence of an Ito-mediated prominent action potential notch in the epicardium. It is considered that one of the mechanisms of this VF is due to heterogeneous distribution of the refractory period according to changes in K+ channels including Ito. PMID- 10750146 TI - Spontaneous sustained monomorphic ventricular tachycardia after administration of ajmaline in a patient with Brugada syndrome. AB - We present the case of a 13-year-old boy with an episode of aborted sudden death, absence of structural heart disease, and a characteristic ECG pattern of right bundle branch block with persistent ST-segment elevation in the right precordial leads, in whom a monomorphic sustained ventricular tachycardia developed spontaneously after the administration of ajmaline. This effect may be related to an increased inhomogeneity of repolarization mediated by the drug and demonstrates the arrhythmogenic potential of Class I antiarrhythmic drugs in patients with Brugada syndrome. PMID- 10750147 TI - Near fatal electrical storm in a patient equipped with an implantable cardioverter defibrillator for Brugada syndrome. AB - A patient with Brugada syndrome experienced incessant ventricular fibrillation 7 years after implantation of an ICD. General anesthesia, fast ventricular pacing, bretylium, and atenolol infusion were ineffective; amiodarone infusion was started. After 190 defibrillation shocks over 36 hours, the electrical storm stopped. A multiorgan failure occurred as a complication of the electrical storm and necessitated prolonged reanimation. Thorough cardiac evaluation revealed no structural abnormality and the patient, now on chronic oral amiodarone therapy, remains free of arrhythmia 1 year after the event. PMID- 10750148 TI - ST segment and T wave alternans in a patient with Brugada syndrome. AB - We describe a patient with Brugada syndrome in whom J point and ST-segment elevation in leads V1 and V2 were augmented by atrial pacing and intravenous administration of propranolol or cibenzoline. Significant T wave alternans with a 2:1 appearance of terminal negative T wave was observed in the absence and presence of atrial pacing after the administration of cibenzoline. The cellular mechanism responsible for T wave alternans, beat-to-beat appearance of terminal negative T wave and augmented J point and ST-segment elevation is discussed. PMID- 10750149 TI - Unmasking effect of propafenone on the concealed form of the Brugada phenomenon. AB - A case report of a patient with frequent ventricular premature beats but with an otherwise normal ECG and no structural heart disease. Propafenone in therapeutical doses unmasked the ECG picture of the Brugada phenomenon. PMID- 10750150 TI - [The taxonomic rank and place of Colpodellida in the system of the Protista]. AB - The analysis of ultrastructure organisation and divergent processes in Colpodellida, Perkinsida, Gregarinea and Coccidea has confirmed the presence of unique basic structures in all of these organisms and the necessity to combine them into the single phylum Sporozoa. A taxonomic rank and place of Colpodellida in the system of living organisms is represented as follows: phylum Sporozoa Leuckart, 1879; em. Krylov, Mylnikov, 1986. (Syn.: Apicomplexa Levine, 1970). Predators or parasites. Common basic structure: pellicular membranes, subpellicular microtubules, micropores, conoid, rhoptries and micronemes, tubular mitochondrial cristae. Class Perkinsea Levine, 1978. Predators or parasites, vegetative stages with two heterodynamic flagella. Subclass 1. Colpodellia nom. nov. (Syn.: Spiromonadia Krylov, Mylnikov, 1986). Predators, two heterodynamic flagella with string-like mastigonemes (if present), division is exclusively within a cyst, with 2-4 daughter cells being produced, extrusomes are trichocyst like. Subclass 2. Perkinsia Levine, 1978. Parasites, zoospores with two heterodynamic flagella, mastigonemes (if present) bristle-like or string-like. PMID- 10750151 TI - [The organizational characteristics of the generative material and the the proliferative dynamics of trematode mother sporocysts]. AB - Analysis of miracidia germinal material organization and proliferation dynamics of mother sporocysts enabled us to divide them into three well-defined groups. The first one includes species whose miracidia possess only differentiated (mature) generative cells and embryos of earlier stages of cleavage. In this case during parasite phase of development of maternal sporocysts the generative function is not performed. To the first group therefore trematode species with pedogenetic larvae could be attributed also. The next group embraces species whose miracidia as well as mature generated cells have some undifferentiated cells; thus the parasitic phase of mother sporocyst development acquires restricted proliferative capacity. The third group consists of species with higher trematodes dominating. They perform generative function exclusively at parasitic phase of mother sporocyst development. Representatives of more archaic and ancient species are the bases of the first two groups on the contrary. Such type of distribution can not occur occasionally and apparently reflects first steps of emergenes of parthenogenetic generations of trematodes. PMID- 10750152 TI - [The parasitic fauna of the Anadyr chum salmon (Oncorhynchys keta Walbaum)]. AB - Data on a parasite fauna of the chum salmon from the Anadyr' River basin are given. 9 parasite species have been found including 3 species of medical importance. PMID- 10750153 TI - [The carbohydrate metabolic end products of trematodes parasitic in cattle]. AB - The trematodes Eurytrema pancreaticum and Calicophoron ijimai during the incubation in vitro assimilated glucose from the incubation medium and utilized the endogenous glycogen. Final products of the carbohydrate metabolism in the calicophorones were lactic, acatic, propionic, isobutyric and alpha-methylbutyric acids; in the eurytremes they were lactic, acetic, propionic, isobutyric, alpha methylbutyric, valerianic and capronic acids. The effect of anthelminthic preparations on the carbohydrate metabolism and its final products was investigated. PMID- 10750154 TI - [The concept of the parasitological typing of lakes]. AB - The parasitological typology of lakes is proposed (with example of the Karelia Kola limnological region). The characteristics of the main types of lakes with allowance of the parasitological data are given. The parasitological typology of lakes is based on studies of the parasite fauna diversity and differs from the trophical classification for lakes proposed by S. V. Gerd (1949). PMID- 10750155 TI - [The adaptive reactions of the gill ectoparasites of the bream (Abramis brama) and of the white bream (Blicca bjoerkna) to exposure to an anthropogenic factor in the Ivan'kovo Reservoir]. AB - Adaptive responses of some gill ectoparasites of the common bream Abramus brama and the white bream Blicca bjoerkna have been studied in five zones of the Ivan'kovo Reservoir: in the region of favorable ecological conditions, in two eutrophic zones and in two areas under the toxic loading. It is found out, that at the response to the pollution the infestation of fishes with most parasite species decreases. The replacement of dominant species is also can be considered as the adaptive response of parasites. It is recorded, that each examined zone with a specific ecological conditions obtains a specific parasite species grouping with a certain composition and abundance of parasite species. The monogeneans of the genus Dactylogyrus express their adaptation to increased temperature by decrease of chitinoid structure size in attachment disc, while the copepods Ergasilus sieboldi react to this factor by faster maturation of egg sacks. PMID- 10750157 TI - [The circadian dynamics of the attacks of the synanthropic population of Culex pipiens molestus mosquitoes at the Staraia Russa Health Resort]. AB - The diurnal biting activity of Culex pipiens molestus in rooms of populated houses was studied in August 1998. The highest activity was recorded at 3.00-4.00 a. m., when 25-30 females attacked one volunteer rer 10 minutes. PMID- 10750156 TI - [Differences in the blocking of the proventriculus in male and female Xenopsylla cheopis (Siphonaptera: Pulicidae)]. AB - Frequency of block formation was studied in Xenopsylla cheopis infected with the triptophan-dependent and the typical strains of Yersinia pestis from the Mountain Altai natural plague focus. It was shown that the rate of blocked males in comparison with that of females was statistically higher. In experiments with the triptophan-dependent strain the frequency of blocking in males was 40.9% and exceeded that in females at four times. The highest rate of blocked males was marked at 7-10th and that of females at 21st days. The tryptophan-dependent strain did not concede to the typical strain in the ability to form a proventricular block in fleas. PMID- 10750158 TI - [The life cycle of the trematode Pneumonoeces nanchangensis major (Plagiorchidae) -a parasite of the lungs in frogs in the Maritime Territory]. AB - The life cycle of the trematode Pneumonoeces nanchangensis major includes three hosts: primary intermediate host (molluscs Helicorbis sujfunensis, Polypylis semiglobosa), secondary intermediate host (larvae of the dragonfly genus Lestes), and final host (frogs Rana nigrimaculata, R. semiplicata). Descriptions and measurements of cercariae and metacercariae are proposed. PMID- 10750159 TI - [The distribution of Phyllodistomum umblae and Phyllodistomum folium (Trematoda: Gorgoderidae) in the excretory system of fishes]. AB - It is shown, that the distribution of Phyllodistomum umblae in the excretory system of all investigated species of salmonids has a principal similarity. The distribution of P. umblae in the excretory system of the Siberian dace and the Siberian roach is distinguished from that of P. folium. PMID- 10750160 TI - [Entobdella hippoglossi (Monogenoidea: Capsalidae) from a perciform fish from the Pacific Ocean and new information on Sessilorbis limopharynx]. AB - A monogenean Entobdella hippoglossi (Capsalidae: Entobdellinae), a common parasite of the flat fishes (Pleuronectidae), is recorded for the first time from an ocean perch Sebastes glaucus (Scorpaenidae) in the north-east part of Pacific ocean. New data on hosts and location of Sessilorbis limopharynx (Capsalidae: Trichopodinae) are also given. PMID- 10750161 TI - [The first information on Schistocephalus nemachili (Cestoda: Ligulidae) from the Siberian groundling in the Lake Baikal region]. AB - Morphological data on Schistocephalus nemachili plerocercoid (a parasite of Nemachilis babatulus) and a nucleotide sequence of its 18S rRNA are presented. Samples were collected from the groundling inhabiting the Isumrudnoe lake (karst lake, belonging to the Lena River basin, 1200 m altitude). Length of plerocercoids 37-70 mm (mean 52.9 mm), width 1.6-7.3 (3.8), number of segments in srobile--229-348 (290). The sequence data on the 5'-terminal major domain of small rRNA of S. nemachili were obtained by mean of polymerase chain reaction and direct sequencing, they displayed 574 base positions. The nucleotide sequence was compared with known Plathelminthes sequences from the EMBL data base to compute data matrix under Juke and Cantor's model (1969). Distance matrix was used for obtaining a similarity dendrogram by Saitou and Nei's method (1987). Comparative analysis showed S. nemachili nucleotide sequence clustering with the cestode sequence (E. granulosus) within the dendrogram containing four trematodes, one cestode and one turbellarian sequences. PMID- 10750162 TI - Locus of control and the spontaneous use of mnemonic strategies in a motor memory task. AB - A test was developed which enabled isolating the effects of locus of control over specific parts of the learning process, while studying its effect on the spontaneous use of mnemonic strategies. Fifty-six adults with mild or moderate mental retardation were randomly assigned to four groups that differed by both internal versus external control, and empty versus filled interval. Recall after an empty interval was significantly better than that following a filled one, suggesting the spontaneous use of mnemonic strategies. No effect was found for the locus of control variable. A longer "warm up decrement" in this population is suggested. PMID- 10750164 TI - Reliability of the Child Behavior Checklist for the assessment of behavioral problems of children and youth with mild mental retardation. AB - The assessment of psychopathology in persons with mental retardation requires reliable and valid instruments. In the present study, the reliability of the Child Behavior Checklist was determined, using data of 42 children and youth with mild mental retardation, with ages from 10 to 18 years. Kappa coefficients and intra-class correlations were computed to determine the reliability at item level and syndrome level. At item level, mean kappa's for inter-rater and test-retest reliability were 0.267 and 0.52, respectively. At syndrome level, mean intra class correlations for inter-rater and test-retest reliability were 0.493 and 0.775, respectively. These results suggest that the Child Behavior Checklist may not always represent a reliable checklist for the assessment of psychopathology among children and youth with mild mental retardation. PMID- 10750163 TI - Body-rocking in college students and persons with mental retardation: characteristics, stability, and collateral behaviors. AB - Body-rocking was analyzed in four studies. The first showed that rocking chairs are not necessarily preferred by people with mental retardation, and that a rocking chair produces a higher amplitude of body-rocking. A second study demonstrated the stability of various measures of body-rocking over a 21-month period. Stable idiosyncratic behaviors accompanying body-rocking also were described. The third study showed that, when compared with college students who engage in habitual body-rocking, persons with mental retardation engage in more body-rocking than college students on various dimensions, and that they also show different collateral behaviors. Finally, in the fourth study, a reanalysis of the data from Study I showed that collateral behaviors do not necessarily occur as part of a body-rocking complex. PMID- 10750166 TI - Further evaluation of the multiple-stimulus preference assessment. AB - Previously researchers have shown that multiple-stimulus preference assessments can produce results comparable to those achieved using the paired-stimulus presentation format. However, extensive experimental validation of this procedure has not yet been accomplished. The purpose of this study was to provide a systematic extension of a study reported by DeLeon and Iwata (1996). We conducted brief stimulus preference assessments with nine participants diagnosed with severe or profound mental retardation. The highest ranked stimuli were then delivered contingent on a target behavior in a multielement format. The stimulus identified as most highly preferred functioned as a reinforcer in six of the nine participants. The results of this study are discussed in the context of current stimulus preference assessment findings. PMID- 10750165 TI - Aggression and the termination of "rituals": a new variant of the escape function for challenging behavior? AB - Aggression and stereotyped behaviors are not uncommon among people with intellectual disabilities and they are often treated separately as operant behaviors. In this single case study, it is argued that the function of a young woman's aggressive behavior appeared to be that of avoiding or escaping the termination of a chain of complex stereotyped behavior (or "ritual"). She became aggressive only when this chain of stereotyped behavior was terminated and the aggression appeared to extinguish when it no longer led to escape from the termination of the "ritual". It is suggested that this is an example of a complex interaction between two behaviors and that it illustrates the need for very careful analysis of the functions of challenging behavior. Furthermore, it is proposed that the lengthening list of variables already documented as determinants of challenging behavior (provision of attention (verbal and physical), mechanical restraint, sensory or tangible events, escape from demands or from social attention, denials, escape from intrusive medical procedures, escape from task difficulty) be lengthened to include the possibility of escape from (or avoidance of) the interruption of a chain of complex stereotyped behavior or "ritual". PMID- 10750167 TI - Side effect profiles of atypical antipsychotics, typical antipsychotics, or no psychotropic medications in persons with mental retardation. AB - Antipsychotic medications have been used to treat a variety of behavioral and psychiatric disturbances in persons with mental retardation. Given the well documented side effects of traditional antipsychotics, newer "atypical" antipsychotics have been well received in this population due to initial reports of a more favorable side effect profile. We compared the side effect profiles of both the typical and atypical antipsychotics using a comprehensive instrument, the Matson Evaluation of Drug Side Effects (MEDS) scale. Participants taking atypical antipsychotics did not differ in overall side effects from a matched control group taking no psychotropic medication, and both groups showed significantly fewer overall side effects than participants taking typical antipsychotics. Subscales designed to measure involuntary movements (e.g., akathisia, tardive dyskinesia) detected differences between participants taking either atypical or typical antipsychotics with respect to akathisia only. Implications of these findings and directions for future research are discussed. PMID- 10750168 TI - Interactions of pyridostigmine bromide, DEET and permethrin alter locomotor behavior of rats. AB - Drug interactions have been suggested as a cause of Gulf War Syndrome. Pyridostigmine bromide (PB), a prophylactic treatment against potential nerve gas attack, the insect repellent DEET, and permethrin (PERM) impregnated in soldiers' uniforms may have interacted and caused greater than expected toxicity. We tested those 3 drugs singly and in combinations on male and female Sprague-Dawley rats in open field arenas to find the effects on rate of locomotion and thigmotaxis. Administration rates were 10 mg PB/kg; 50, 200, or 500 mg DEET/kg; 15, 30, or 60 mg PERM/kg; 5 mg PB/kg + 100 mg DEET/kg; 5 mg PB/kg + 15 mg PERM/kg; 100 mg DEET/kg + 15 mg PERM/kg; or vehicle by gavage and i.p. injection. Locomotor behavior was quantified by video-computer analysis for 2 h post-treatment. Female rats were tested in either pro- or metestrus. Drug interactions were determined by the isobolographic method. Blood serum drug concentrations were estimated by high performance liquid chromatography or gas chromatography-mass spectrometry. Single drug effects were very limited within the ranges tested. Double-drug administrations at half the single-drug rates resulted in statistically significant interactions in male rats for both locomotion rate and thigmotaxis. Combination of PB + PERM and DEET + PERM significantly affected speed, whereas only the combination of DEET + PERM significantly affected thigmotaxis. Female rats did not show significant interactions. Our data suggest that serum concentrations of PB and DEET may have been higher in females than males. Administration of PB + DEET may have reduced the serum concentration of DEET, and administration of PB + PERM may have increased the serum concentration of PERM. PMID- 10750169 TI - Repeated coadministrations of pyridostigmine bromide, DEET, and permethrin alter locomotor behavior of rats. AB - Interactions of pyridostigmine bromide (PB), permethrin (PERM), and the insect repellent DEET (DEET) have been suggested as possible causes of Gulf War Syndrome (GWS) in humans. Open field locomotor studies have long been used in behavioral toxicology. Using male and female Sprague-Dawley rats, video-computer analyses, and the isobolographic method we have determined the effects on locomotor speed and thigmotaxis following repeated administration of single-, double-, or triple drug or vehicle controls in an open field. The effects were measured 24 hours after 7 daily drug administrations. Single-drug administrations caused no significant effects. Double-drug administrations resulted in significant effects in the following cases: males given PB + DEET had a significantly slower locomotion rate; males given DEET + PERM had a significantly faster locomotion rate; females given PB + DEET had a significantly slower locomotion rate; and females given PB + PERM spent significantly more time in the center zone (less thigmotaxis). Triple-drug administration caused no significant effect. These results in comparison with behavioral studies in chickens and insects show certain similarities. The implications of the lasting effects on animal models are relevant to GWS in humans. PMID- 10750170 TI - Acute toxicity, primary irritancy, and genetic toxicity studies with 3 (methylthio)propionaldehyde. AB - Basic acute toxicity, primary irritancy, and genetic toxicity studies were conducted with 3-(methylthio)propionaldehyde (3-MTP). The acute rat peroral LD50 (with 95% confidence limits) for 3-MTP as a 25% (v/v) dilution in corn oil was 1.00 (0.59-1.70) ml/kg (males) and 1.68 (0.95-2.99) ml/kg (females); most deaths occurred 1.5 to 4 h postdosing. By 24-h occluded contact with undiluted 3-MTP, the rabbit acute percutaneous LD50 was 0.71 (0.43-1.15) ml/kg (males) and 0.79 (0.49-1.30) ml/kg (females): times to death ranged from 2 h to 2 d after the start of dosing. Exposure of rats to a statically generated saturated atmosphere killed all 5 males with a 40 min exposure and all 5 females with a 24 min exposure. In contrast, a 4-h exposure of rats to a dynamically generated saturated vapor atmosphere of 3-MTP did not produce any mortalities or signs of toxicity. A 4-hr occluded contact with 0.5 ml undiluted 3-MTP caused moderate to severe erythema and severe edema resolving by 7 to 17 d. Five/6 animals had necrosis apparent on removal of the occlusive dressing and persisting 10 to 17 d. On the rabbit eye, 0.1 ml undiluted 3-MTP produced moderate to severe corneal injury with iritis and moderate conjunctival inflammation which persisted 21 d in 3/6 animals; 0.01 ml caused moderate diffuse corneal injury and moderate conjunctival inflammation with healing by 7 d. 3-MTP did not produce mutagenic activity either in the absence or presence of metabolic activation with a Salmonella typhimurium reverse mutation assay using strains TA98, TA100, TA1535, TA1537 and TA1538. In a mouse lymphoma cell (L5178Y/tk +/-) assay, 3-MTP produced concentration-related increases in mutant colonies, both in the absence and presence of metabolic activation. Increases were mainly in the sigma (chromosomal damaging) colonies. In a mouse bone marrow micronucleus study, with vapor exposures to 37.4, 88.5 and 155.6 ppm for 1 h/d for 2 consecutive d, there were exposure concentration-related increases in micronucleated erythrocytes which were statically significant for male mice. PMID- 10750171 TI - Proliferation of periodontal squamous epithelium in mink fed 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). AB - The maxilla and mandible from 2 adult female mink fed 5.0 ppb 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) for 6 mo were grossly unremarkable, but histologically had nests of squamous epithelium within the periodontal ligament. There was osteolysis of the adjacent alveolar bone. PMID- 10750172 TI - Ammonia toxicity from urea in a Brazilian dairy goat flock. AB - A flock of goats received a diet with 1% urea for at least 1 y. A new batch of concentrate was offered increasing the level of urea to 4.2%. Eighteen of 54 goats showed acute signs of ammonia toxicosis. Ten goats died within 60 min; 4 goats and a buck with convulsions recovered when treated by administration of vinegar and infusion of saline solution, diuretics, and atropine. Three goats with mild signs recovered within 1 h without treatment. The mean ammonia concentration and rumen pH content were 820 mg/L and 7.7, respectively. Generalized congestion, intense pulmonary edema, and slight tubular nephrosis were found in 3 goats on necropsy. The outbreak was self-limiting and no more cases occurred when the diet was removed. PMID- 10750173 TI - The futility of hemoperfusion and hemodialysis in Amanita phalloides poisoning. AB - Amanita phalloides mushrooms are extremely toxic. A variety of treatments have been proposed based as often on anecdotal experience as on firm evidence. General consensus exists regarding some treatments, such as the use of silibinin, penicillin, and activated charcoal. The most polarized debate concerns the value of extracorporeal elimination. We describe a case of 2 adults with confirmed Amanita phalloides poisoning treated with hemodialysis (HD) immediately after arrival at our tertiary care hospital (23 h after ingestion) and later with hemoperfusion (HP); a series blood samples were taken to determine the clearance of the toxin by each method. No amatoxin was detected before treatment, after treatment, or in the HD/HP circuits. Neither HD nor HP contributed to the clearance of amatoxin. PMID- 10750174 TI - Outbreak of copper poisoning in cattle fed poultry litter. AB - In a feedlot of about 1,000 head of cattle, 146 animals died within a period of a few months affected by a disease characterized by anorexia, icterus, hemoglobinuria, constipation, or diarrhea. The clinical course of the disease lasted a few days. Postmortem findings were generalized icterus and a yellow discolored liver. The kidneys were dark brown, and the urinary bladder was filled with urine of the same dark-brown color. The main histopathological findings were centrolobular coagulative necrosis, apoptosis, bilestasis, and proliferation of bile ducts in the portal space. Changes in the kidneys included nephrosis and the presence of bile and precipitates, and cylinders of albumin and of hemoglobin in the uriniferous tubules. Liver samples, collected from 3 animals on which postmortem examinations were performed, had 2,008, 2,783 and 4,906 ppm copper in their dry matter. Two samples of poultry litter fed to the cattle contained 362 and 323 ppm copper. The green forage that formed the rest of their feed only had 4.7 ppm copper. Copper poisoning was diagnosed, most probably caused by feeding litter from poultry that had been fed a ration treated with copper sulfate to avoid aspergillosis. PMID- 10750175 TI - A fatal case of gun blue ingestion in a toddler. AB - Acute selenium poisoning occurs infrequently. The form of selenium encountered plays a great role in toxicity. Several fatalities have been reported and all but I involved ingestion of selenious acid or selenium dioxide. A healthy 22-mo-old male ingested up to 15 ml of Gun Blue solution (selenious acid). Initially he was pink, alert, and combative in the ambulance but his condition rapidly deteriorated. There was no measurable blood pressure, his oxygen saturation was 84% by pulse oximetry, and his mental status deteriorated to require hand ventilation. The child was cyanotic, unresponsive, and without palpable pulses upon presentation. Cardiopulmonary resuscitation was initiated unsuccessfully and was terminated after 35 minutes. PMID- 10750176 TI - A suicide attempt with an oral calcium channel blocker. AB - Calcium channel blockers are widely used in all-aged populations. The drugs are generally safe in therapeutic dosage, but severe side effects with elevated intake are increasingly described, mainly in adult patients. We report an adolescent girl who intentionally ingested an overdose of nifedipine. PMID- 10750177 TI - Pediatric pesticide poisoning in the Carolinas: an evaluation of the trends and proposal to reduce the incidence. AB - Studies from North and South Carolina on hospitalizations following pesticide exposure have shown that about 30% involve children. During 1990-1993, 29% of North Carolinians hospitalized for pesticide poisoning were children. Between 1971 and 1996, 28% to 37% of the patients hospitalized following pesticide exposure in South Carolina were children. Data from a South Carolina study, from the Association of American Poison Control Centers and the Vital Statistics of the US, suggest that pesticide poisonings in children are part of the overall problem of children being poisoned by household chemicals. Most poisonings occur in toddlers about 1-y-of-age. Pesticide-related fatalities in children have steadily decreased for the last 20-y while poisonings from other household chemicals have not decreased dramatically. The data suggest that increased public awareness of the risks of household chemicals could decrease that poisoning incidence. PMID- 10750178 TI - Are poisonings inflicted upon others always criminal? AB - While self-poisoning is a common cause for hospitalization, in the experience of our toxicological intensive care unit and emergency department, poisoning of other persons appears to be quite rare. We identified 32 patients in a non exhaustive review of patients presenting over a 28-y period who had been unequivocally poisoned by others. We examine here the circumstances leading up to these poisonings, along with their clinical outcomes, and propose a classification scheme based on underlying intent. PMID- 10750179 TI - Urginea maritima L (Squill): a poisonous plant of North Africa. AB - Poisoning of livestock by ingestion of Urginea maritima L (squill), a common plant of coastal Mediterranean regions of North Africa is reviewed. PMID- 10750180 TI - The roots of toxicology: an etymology approach. AB - We investigated the roots of toxicology and showed the Greek origin of the word. A number of selected ancient Greek and Byzantine works show the etymology and meaning of the adjective "toxic" or "toxical" as the first composition of "toxicology". Even from early antiquity "toxic" or "toxical" have been connected with substances which cause death, a meaning which has prevailed through the ages. PMID- 10750181 TI - Waste management: how to deal with outdated drugs, chemicals, and pet foods. PMID- 10750182 TI - Waste management in food and animal practice: agriculture chemicals and feed additives. PMID- 10750183 TI - Fatal privet toxicosis in Tennessee cows. PMID- 10750184 TI - A needed technical fix? PMID- 10750185 TI - [A journey the long way to the half-century]. PMID- 10750186 TI - [The genetics of behavior]. AB - A review of the papers published in the Journal of Higher Nervous Activity dedicated to behavioral and neurophysiological studies carried out using the genetic approach is presented. The review is based on classification, which subdivides the diversity of behavioral phenomena into three large categories: instinctive, acquired behavior, and elementary reasoning phenomena. The main contribution to these publications was made by researches at the Institute of Cytology and Genetics, Russian Academy of Sciences, Siberian Branch (Novosibirsk), Institute of Physiology, Russian Academy of Sciences (St. Petersburg), and Department of Physiology of Higher Nervous Activity, Biological Faculty, Lomonosov State University (Moscow). The traditional lines of investigations are: behavioral and neurochemical consequences of domestication, numerous physiological correlations in rat strains selected for high and low excitability levels, and genetic studies of animal reasoning. PMID- 10750187 TI - [The emotional characteristics of the sounding word]. AB - The four-dimensional spherical emotional space has been obtained by multi dimensional scaling of subjective differences between the emotional expressions in sound samples (the words "Yes" and "No" pronounced in different emotional conditions). Euclidean space axes are interpreted as the following neural mechanisms. The first two dimensions are related with the estimation of a sign of emotional condition: the dimension 1--pleasant/unpleasant, useful or not, the dimension 2--an extent of information certainty. The third and the fourth axes are associated with the incentive. The dimension 3 encodes active (anger) or passive (fear) defensive reaction, and the dimension 4 corresponds to achievement. Three angles of four-dimensional hypersphere: the one between the axes 1 and 2, the second between the axes 3 and 4, the third between these two planes determine subjectively experienced emotion characteristics such as described by Vundt emotion modality (pleasure-unpleaure), excitation-quietness suppression, and tension-relaxation, respectively. Thus, the first and the second angles regulate the modality of ten basic emotions: five emotions determined by a situation and five emotions determined by personal activity. In case of another system of angular parameters (three angles between the axes 4 and 1, 3 and 2, and the angle between the respective planes), another system of emotion classification, which is usually described in the studies of facial expressions (Shlosberg's and Izmailov's circular system) and semantics (Osgood) can be realized: emotion modality or sign (regulates 6 basic emotions), emotion activity or brightness (excitation-rest) and emotion saturation (strength of emotion expression). PMID- 10750188 TI - [The cortical interactions in short time intervals during the search for verbal associations]. AB - Cortical connectivity was studied in tasks of generating the use of words in comparison with reading aloud the same words. These tasks were used earlier in PET and high-density ERP recording studies, which described both the functional anatomy and time course of involvement of cortical areas in word processing. We developed a new method for studying the synchrony of EEG spectral components within the short time intervals compatible with the duration of particular cognitive operations. The wavelet transform of the ERP records and calculation of correlations between the wavelet curves were used to reveal connections between cortical areas. Three stages of intracortical communications developing over the course of task performance were discovered: between the right and left frontal areas (0-200 ms after the stimulus presentation), between the left frontal and left posterior temporo-parietal areas (250-500 ms), and, finally, between the left temporal and right fronto-centro-temporal areas. These findings are in good agreement with the results of the previous PET and ERP studies and supplement them with the circuitry of cortical information transfer. Also, they suggest some differences in information processing during automated reading and performance of more complicated use-generation task. PMID- 10750189 TI - [Spectral analysis of the human EEG while listening to music]. AB - EEG was recorded from 16 electrodes (International 10-20 System) in 14 healthy volunteers aged from 18 to 45 in the state of rest and during listening to music (popular classical symphonic pieces). The EEG spectral analysis was carried out. Presentation of musical pieces increased the EEG spectral power in the alpha range in the parietal and occipital areas of both hemispheres. During repeated listening of the same musical fragment there was a tendency for attenuation of these changes. A significant decrease in the peak frequency of the alpha-band was found in the process of listening. The spectral values did not significantly differ between the left and right hemispheres. The findings suggest a decrease in the level of CNS activation under the influence of music. PMID- 10750190 TI - [Postradiation changes in the brain asymmetry and higher mental functions of right- and left-handed subjects (the sequelae of the accident at the Chernobyl Atomic Electric Power Station)]. AB - The present study was aimed at comparative electrophysiological, clinical, and neurophysiological assessment of the brain functional state in dextrals (50 men) and sinistrals (5 men), who participated in the Chernobyl clean. The patients were observed over the course of 10 years (from 1990 to 1999). The results of examination of healthy persons (20 dextrals and 10 sinistrals) were used as a control. The clinical examination revealed the earlier manifestation and more severe development of paroxysmal and epileptic seizures in sinistrals than in dextrals. Electrophysiological study showed a progressive decrease in interhemispheric asymmetry of the EEG coherence characteristic for a healthy brain to inversion of its sign. These changes were more pronounced in sinistrals. In the remote terms after radiation, the interhemispheric EEG coherence decreased over the whole cortex, especially, in the frontal and central areas in both groups of patients. Neurophysiological study revealed a progressive impairment of voluntary motor activity and tactile sensibility, especially, in the left hand. These defects were more expressed in sinistrals than in dextrals. The results of complex and longitudinal examination suggest that the observed changes in brain asymmetry and interhemispheric interaction can be not only a result of a dysfunction of subcortical limbic-reticular and mediobasal brain structures but also a result of the white matter damage including corpus callosum. More expressed impairments in sinistrals than in dextrals can be explained by specific morphofunctional organization of the brain in persons with different sensor and motor asymmetries. PMID- 10750191 TI - Vector representation of associative learning. AB - I. P. Pavlov [12] has shown that conditioned reflexes are selective both with respect to conditioned stimuli and to conditioned reflexes elicited by those conditioned stimuli. At the neuronal level selective aspects of conditioned stimuli are based on detectors selectively tuned to respective stimuli. The selective aspects of conditioned reflexes are due to command neurons representing specific unconditioned reflexes. It can be assumed that conditioned reflexes result from association between selective detectors and specific command neurons. The detectors activated by a conditioned stimulus constitute a combination of excitations--a detector excitation vector. The detector excitation vector acts on a command neuron via a set of plastic synapses--a synaptic weight vector. Plastic synapses are modified in the process of learning making command neuron selectively tuned to a specific conditioned stimulus. The selective tuning of a particular command neuron to a specific excitation vector referred to a conditioned stimulus is a basis of associative learning. The probabilities of conditioned reflexes elicited by conditioned and differential stimuli implicitly contain information concerning excitation vectors that encode respective stimuli. Contribution of the vector code to associative learning was explored combining differential color conditioning with intracellular recording from color-coding neurons. It was shown that colors in carps and monkeys are represented on a hypersphere in the four-dimensional space similar to human color space. The basis of the color space is constituted by red-green, blue-yellow, brightness and darkness neurons. PMID- 10750192 TI - [The expression of the c-fos gene in the brain of mice in the dynamic acquisition of defensive behavioral habits]. AB - Levels of c-fos mRNA expression in mouse cerebral cortex and hippocampus at different stages of footshock escape and avoidance learning were studied by Northern hydridization. In the first series of experiments a mouse was presented with 30 electric footshock daily in a chamber where it could escape from the floor by jumping on the safe platform attached to the wall. A large increase in c fos mRNA level in the cerebral cortex and hippocampus was observed during the first day of training. Mice that were trained for 9 consecutive days and acquired a footshock escape reaction showed no elevation of c-fos expression in the brain as compared to the quiet control group. In the second series of experiments the levels of c-fos expression were compared in individual mice trained to avoid the footshock by jumping on the platform in response to an auditory conditioned stimulus. Mice which acquired avoidance behavior more rapidly had lower c-fos mRNA levels than slow learners. There was no such to difference between the corresponding yoked control groups which consisted of animals matched the rapid and slow learners by the number of footshocks received. It is concluded that achievement of adaptive results in the course of learning leads to a suppression of further c-fos induction by motivational excitation. PMID- 10750193 TI - [The determination of the parameters of anxiety in C57BL/6J, CBA/Lac and BALB/c mice under the influence of a serotonin C1A receptor agonist [correction of antagonist]. AB - Three strains of inbred mice, C57BL/6J (C57), CBA/Lac (CBA), and BALB/c (BALB) were examined in the elevated plus-maze after the injection of an anxiotropic drug, a 5-HT1A agonist ipsapirone (3 mg/kg; i.p.; 30 min). Treatment with ipsapirone had different anxiogenic effects on the behavior of mice in accordance with their genotype. In C57 mice the drug produced a significant decrease in the percentage of the open-arm time and the number of open-arm entries as well as in the number of full entries (when an animal was between the half and the end of an open-arm) and in the number of head dippings. Besides; the number of C57 mice which performed full entries after the ipsapirone injection decreased. In CBA mice ipsapirone reduced the number of enclosed-arm entries, the number of the passages from one enclosed arm to another and the number of head dippings. Only the number of passages dropped in BALB mice after the drug injection. Probably, just these parameters reflect anxiety in mice of the genotypes under study. It was suggested that the sensitivity of 5-HT1A receptors in C57 mice is the highest. PMID- 10750194 TI - [The participation of the cholinergic system of the rat sensorimotor cortex in regulating different types of movements]. AB - To study the role of the cholinergic system of the sensorimotor cortex in regulation of different manipulatory movements and locomotion of Wistar rats, the effects of injections of cholinergic drugs (a cholinergic agonist carbachol and an antagonist scopolamine) into the area of forepaw representation in the sensorimotor cortex on motor activity and performance of manipulatory movements (with prolonged and short pushing) were analyzed. The drugs were injected via special cannulae stereotaxically implanted into the cortex during surgery carried out under Nembutal anesthesia. Carbachol injections (0.03-3 micrograms in 1 microliter of physiologic solution) into the cortex resulted in a significant slowing down of both types of movements as well as an increase in locomotion in the open-field test. Injections of scopolamine (0.3-3 micrograms) into the same cortical area were accompanied by an increase in the number of fast manipulatory movements without significant changes in locomotor activity. The obtained evidence suggests that the cholinergic system of the sensorimotor cortex indifferent manners regulates the innate (locomotion) and acquired movements which require different periods of maintaining the muscle tone of the forepaw (short-time periods for the usual movements necessary for food taking from the narrow horizontal tube and prolonged periods for the learned slow movements with additional tactile and tonic components). PMID- 10750195 TI - [The participation of GFAD-synthesizing neurons in behavior regulation in the snail (Helix lucorum L.)]. AB - It was shown earlier that some neurons in Helix CNS express the mRNA of the precursor of neuropeptide GFAD. Using the data obtained with the help of the whole-mount in situ hybridization, we tried to identify a group of such neurons, namely, the pedal caudo-ventral group and to determine their possible functions. The local extracellular stimulation of the pedal caudo-ventral group resulted in movements of reproductive organs in the semi-intact preparation and suppressed the activity of the modulatory neurons controlling feeding and defensive behavior. Application of synthetic peptide GFAD (10(-8) mol/l) also activated movements of the reproductive organs and suppressed the activity of the modulatory neuron controlling feeding behavior. Stimulation of the labial nerves resulted in suppression of caudo-ventral neurons with simultaneous activation of the modulatory neuron controlling feeding behavior. The obtained evidence suggests that the caudo-ventral neurons can regulate movements of the reproductive organs and also coordinate different functions in realization of the integral sexual behavior. This group of neurons inhibits the modulatory neurons controlling the forms of behavior incompatible with courtship, i.e., feeding and defensive forms. PMID- 10750196 TI - [Changes in glutamate release in the rat nucleus accumbens during food and pain reinforcement]. AB - In vivo microdialysis combined with HPLC-EC analysis was used to monitor extracellular glutamate in the n. accumbens of Sprague-Dawley rats during footshock and food delivery. The footshock presentation resulted in a delayed increase in extracellular glutamate level, whereas the food intake caused its decrease. The intra-accumbens infusion of glutamate reuptake blocker D,L-threo beta-hydroxiaspartate (1 mM) completely prevented the food-induced decrease in glutamate level. The intra-accumbens infusion of sodium channel blocker tetrodotoxin (1 microM) led to an increase in glutamate extracellular level in the n. accumbens in response to food intake. The results suggest that the food induced decrease in glutamate extracellular level in the n. accumbens occurs due to an enhancement of high-affinity glutamate uptake that is probably under the neuronal control during feeding. PMID- 10750197 TI - [A comparison of the efficiency of stimulation of the right and left lateral hypothalamus during a self-stimulation reaction]. AB - Higher rate of the right-side self-stimulation of the lateral hypothalamus than of the left-side was found in freely behaving (30 +/- 8 versus 16 +/- 5 pressings per minute) and fixed rabbits (15 +/- 3 versus 10 +/- 2 pressings per minute, accordingly) under conditions of optimal current (current strength was leveled about the thresholds of food motivational reactions). PMID- 10750198 TI - [Vibrissectomy in rats in early ontogeny leads to disordered functional properties of the cortical projection neurons]. AB - Vibrissae trimming during the first 20 days of postnatal life caused alternations of the properties of the receptive fields of single neurons in the barrel-field cortex in rats. The following changes were found in the deafferented cortex: (i) an extension of the receptive fields of single neurons as judged from an increase in the number of vibrissae with short-latency excitatory responses to stimulation and (ii) a depression of the inhibitory receptive field tuning mechanisms. PMID- 10750200 TI - [The pre-Pavlov period in the development of neurophysiology in the Academy of Sciences]. AB - Over the course of the XVIIIth-XIXth centuries, in the Russian Academy of Sciences investigations were actively pursued into the physiology of the nervous system. These studies were published and widely discussed. A valuable contribution was made by D. Bernulli, P. A. Zagorskii, G. F. Bidder, F. V. Ovsiannikov, I. M. Sechenov, I. R. Tarkhanov, N. E. Vvedenskii, I. G. Orshanskii and others. PMID- 10750199 TI - [A trial of the use of aekol in preventing psychoautonomic disorders]. AB - Autonomic dysfunction in chronic emotional stress is well documented. The aim of this study was to analyze the effects of natural antioxidant vitamin E (aekol). Twenty persons (16 women and 4 men, mean age 38 +/- 4 years) who reported recent occurrence of emotional stress were examined before and after a 4-week treatment with aekol (5 ml twice a day). Heart rate variability (taking into account very low-frequency (VLF, 0.003-0.04 Hz), low-frequency (LF, 0.04-0.15 Hz), and high frequency (HF, 0.15-0.40 Hz) components) was computed from the power spectra (5 min epochs) of the EKG recorded in the patients in supine position. After the treatment, the HF power of the heart rate variability (an index of cardiac parasympathetic activity) increased (p < 0.05), whereas the VLF power (an index of the cerebral sympathetic activity) decreased (p < 0.01). The decrease in the VLF was accompanied by a reduction of anxiety level (p < 0.01). According to our hypothesis, the absolute and relative power of the VLF can be used as an index of anxiety or cerebral sympathetic activity, which significantly decreases after the aekol treatment. PMID- 10750201 TI - [I. P. Pavlov and K. Lorenz]. AB - In the thirtieth, the founder of ethology Austrian zoologist Konrad Lorenz put forward the new theory of behavior, which was met with considerable resistance of the dominant views on the mechanisms of behavior, including Pavlov's concept. From his first theoretical works and later on Lorenz debated with Pavlov. However, these debates were not reduced to a disagreement. He appreciated greatly the scientific contribution of Pavlov, and the ideas of the Russian physiologist were often the starting point of his own speculations. His polemics with Pavlov differed very much from his uncompromising controversies with behaviorists. When Lorenz compared Pavlov's views with behaviorism, he often preferred Pavlov's ideas. Lorenz also draw some parallels between the Pavlov's understanding of behavior and the ethological approach. Lorenz's discussion with Pavlov about the nature of conditioned reflex is of particular interest, since it stimulated Lorenz to develop the theory of this phenomenon. PMID- 10750202 TI - Primary care groups and research networks: opportunities for R&D in context. PMID- 10750203 TI - A cautious welcome for the new guidelines on management of drug dependence. PMID- 10750204 TI - A pharmaceutical needs assessment in a primary care setting. AB - BACKGROUND: As part of a reconfiguration of its general medical services, Ardach Health Centre has integrated a community pharmacist into the centre to provide pharmaceutical care. In order to systematically identify areas of 'pharmaceutical need', a needs assessment was carried out during October 1997. AIM: To prioritize and assist the planning of pharmaceutical care provision within the centre, such that maximum gain could be achieved from inevitable limited resources. METHOD: A four-stage pharmaceutical needs assessment method was created around a selection of techniques: gap analysis, the nominal group technique, and rapid participatory appraisal. This was then applied to a random sample of people drawn from the patient register of Ardach Health Centre and all the health care professionals associated with their care. RESULTS: Through the four-stage process, a pharmaceutical service priority league table was constructed to reflect the unmet pharmaceutical needs of patients and their primary health care providers. The table provided a structured framework around which pharmaceutical service provision within the health centre could be planned. CONCLUSION: We have developed a pragmatic, systematic method of identifying the prevalence of unmet pharmaceutical needs of a community. The assessment assisted service selection, balancing what should be done with what could be done and what could be afforded. PMID- 10750205 TI - Do GPs working in practice with high or low prescribing costs have different views on prescribing cost issues? AB - BACKGROUND: In a previous study we found that a minority of general practitioners (GPs) had different views to health authority advisers on a number of prescribing cost issues. However, there were few differences between subgroups of GPs. We hypothesised that subgroups that might show differences were GPs from practices with either high or low prescribing costs. AIM: To assess differences in views on prescribing cost issues between GPs working in practices with either high or low prescribing costs. METHOD: Using PACTLINE data, prescribing costs were obtained for general practices within the Trent Region for the financial year 1996 to 1997. A questionnaire was sent anonymously to 340 GPs working in those practices with high prescribing costs, and to 322 GPs working in practices with the lowest prescribing costs. RESULTS: A total of 216 (63.5%) GPs from high-cost practices and 194 (60.2%) from low-cost practices responded. There were statistically significant differences between the two groups on seven out of 22 statements. However, when the confounding effect of fundholding was taken into account, significant differences were found for just three statements and each of these related to substitution with comparable but cheaper drugs. CONCLUSIONS: GPs working in practices with either high or low prescribing costs had different views on a number of statements concerning substitution with comparable but cheaper drugs. When encouraging GPs to control their prescribing costs, a different approach may be required for doctors in some high-cost practices. PMID- 10750206 TI - Patients with cancer holding their own records: a randomised controlled trial. AB - BACKGROUND: The burden of cancer care in general practice is increasing. Patient held records may facilitate effective, coordinated care, but no randomised controlled trials of their use in cancer care have been conducted, and concerns about possible negative effects remain. AIM: To evaluate the use of a supplementary patient-held record in cancer care. METHOD: Six hundred and fifty radiotherapy outpatients with any form of cancer were randomised either to hold a supplementary record or to receive normal care. It was explained to record holders that the supplementary record was intended to improve communication with health professionals and act as an aide memoire. After three months, patients' satisfaction with communication and with participation in their own care were assessed. Global health status, emotional functioning, and cognitive functioning were measured using the European Organization for Research and Treatment of Cancer QLQ-C30 questionnaire. RESULTS: There were no significant differences between groups in any of the outcome measures. Patients in both groups expressed a high level of satisfaction with communication and participation in their care. Mean (SD) scores in the intervention and control groups were: global health status, 66.8 (24.2) and 65.3 (23.7); emotional functioning, 75.0 (24.6) and 77.4 (22.8); cognitive functioning, 84.5 (21.0) and 84.0 (21.3). CONCLUSION: A supplementary patient-held record for radiotherapy outpatients appears to have no effect on satisfaction with communication, participation in care, or quality of life. PMID- 10750208 TI - The process of outpatient referral and care: the experiences and views of patients, their general practitioners, and specialists. AB - BACKGROUND: The primary care system in the United Kingdom, involving the general practitioner (GP) as gatekeeper to further services, has helped to keep health care costs down. Despite this, unexplained variation in referral rates and increasing health care costs have led to the search for methods of improving efficiency. There is relatively little recent descriptive data on the processes of care at the primary-secondary care interface. The study reported here provides information about this. AIM: To analyse the patterns and process of care for the referral of outpatients, together with the views of patients, their GPs, and specialists. METHOD: A questionnaire survey of outpatients, their hospital specialists, and GPs in randomly sampled district health authorities in the North Thames Region. The measures included items and scales measuring satisfaction and processes. RESULTS: Almost all of the outpatients thought that their consultation with the specialist was 'necessary' and 'worthwhile'. Most of the GPs felt that they could not have given the study patients the care, treatment, and investigations they received in hospital, and most of the sampled patients' attendances were rated by the specialists as 'appropriate'. However, for just over one-fifth of new patients, the specialists reported that the GP could have done more tests and examinations prior to referring the study patient. Large proportions of GPs in this survey also reported having technical equipment in their practices, as well as direct access to a range of services and hospital based facilities. CONCLUSION: A large amount of work is carried out in general practice prior to the hospital referral of patients, and GPs have direct access to some technologies and services that can act to reduce the burden on hospitals. The discrepancy between GPs' and specialists, perceptions about the potential for further investigative work prior to patient referral merits further investigation. PMID- 10750207 TI - Changes in receptionists' attitudes towards involvement in a general practice based trial of screening and brief alcohol intervention. AB - BACKGROUND: Primary health care receptionists are increasingly expected to be involved in research. However, little is known about receptionists' attitudes to research or health programmes. AIM: To examine changes in receptionists' attitudes, with different levels of training and support, towards involvement in a general practice-based trial of screening and brief alcohol intervention. METHOD: Subjects were 84 receptionists, one per practice, who assisted in the implementation of a screening and brief alcohol intervention programme. Receptionists were randomly assigned to one of three conditions: control (no training or support), training alone, and training plus ongoing telephone support. Baseline and follow-up questionnaires were used to assess changes in receptionists' attitudes. RESULTS: Of 40 items that measured receptionists' attitudes to involvement in the programme, 70% had deteriorated after three months, 20% significantly so. There was no effect of training and support condition. Receptionists' and GPs' attitudes to research and health programmes conflicted. CONCLUSION: Receptionists developed more negative views about involvement in research and health programmes over the three-month study period, regardless of level of training and support. PMID- 10750209 TI - Are specialist mental health services being targeted on the most needy patients? The effects of setting up special services in general practice. AB - BACKGROUND: Around 25% of patients with psychoses lose contact with specialist psychiatric services, despite the government's policy to focus the efforts of community teams on this group. AIM: To identify patient and practice factors associated with continuing contact and loss of contact with specialist services. METHOD: Cross-sectional comparison was made of patients in and out of specialist contact, through detailed interviews with 102 patients among 26 south west London practices. Associations were sought between contact with specialist services and patient factors (illness severity, social functioning, quality of life, needs for care, and satisfaction with general practitioner [GP] services) and practice factors (size, location, fundholding status, training status, and the presence of mental health professionals on site). RESULTS: Thirty-one (30%) patients were currently out of specialist contact. No significant differences were found between those in and out of contact on any measures of diagnosis or psychiatric history. Those in contact had significantly more symptoms, poorer social functioning, poorer quality of life, and more needs for care. The proportion out of contact was significantly higher in two practices that had employed their own mental health professionals to provides services on site for severe mental illnesses. Two factors remained significant predictors of contact in a logistic regression model: whether or not the patient's practice offered a special service on site, and greater patient needs for care. CONCLUSIONS: Secondary mental health services are being targeted towards the more needy patients. The provision of special services in practices can shift care further away from secondary care while still meeting patients' needs. PMID- 10750210 TI - Estimation of cardiovascular risk in hypertensive patients in primary care. AB - Assessment of absolute cardiovascular risk is a rational method of managing hypertension. General practitioners and practice nurses were asked to estimate absolute risk in a group of elderly hypertensive patients during clinical practice. Risk was correctly estimated in 21% of patients, underestimated in 63% of patients, and overestimated in 16% of patients. Unless primary health care professionals use cardiovascular risk charts or tables, treatment decisions in primary care may not be made against realistic estimates of patients' susceptibility to cardiovascular disease. PMID- 10750211 TI - Survey of the use of selective culture for Neisseria gonorrhoeae in specimens from the female genital tract sent by general practitioners to a microbiology laboratory. AB - A retrospective survey of the number of cultures found to be positive for Neisseria gonorrhoeae in genital specimens from female patients sent by general practitioners (GPs) over a three-year period was carried out. The organism could be detected in only four specimens out of over 28,000 specimens sent. Specifically, additional selective culture for N gonorrhoeae had been carried out in 8529 of these specimens. An estimate of the cost savings achievable if this laboratory was no longer to culture routinely for N gonorrhoeae was made. GPs should be aware of their local laboratory's normal practice when processing such specimen and should request specific culture if appropriate. The low number of specimens from which N gonorrhoeae could be cultured might suggest that GPs are referring 'at-risk' patients to genitourinary medicine clinics already. PMID- 10750212 TI - Factors influencing general practitioner referrals to a tertiary paediatric neurodisability service. AB - This study investigated factors influencing general practitioner (GP) referrals to a tertiary paediatric neurodisability service, including the reasons for referral, the children's characteristics, and the involvement of secondary specialist services in the referral. Fifty general practitioners (GPs) referring consecutively to the Neurodisability Service, Great Ormond Street Hospital for Children, London, were interviewed in a cross-sectional study using a postal questionnaire. The main findings were that two-thirds of the GPs did not contact secondary specialist providers prior to referring. The majority (78%) of GPs were responding to parental concerns in the context of a lack of medical diagnosis in 74% of the children. The main (90%) reason given for referral was for parents seeking a second opinion outside their district network. The findings highlighted the influence of parents over GPs' referrals to tertiary neurodisability, and, in light of this, raised issues for consideration for effective organisation of child disability health services. PMID- 10750213 TI - Antimicrobial agents in lower respiratory tract infections in Dutch general practice. AB - This study describes the prescription of antimicrobial agents in cases of lower respiratory tract infections in Dutch general practice. A secondary analysis of data from the National Study of Illness and Procedures of The Netherlands Institute of Primary Health Care (a nationwide group of 161 general practitioners with data from 334,449 patients) had been carried out. Antimicrobial agents were prescribed in 30% of all contacts: in about half of the first contacts and contacts for recurrences, and in one out of six repeat contacts. The prescription rates were associated with diagnosis and reason for encounter but rarely with older age or comorbidity. Amoxycillin and doxycycline were most frequently prescribed. While most lower respiratory tract infections are virus-induced and antibiotics are not effective in most cases, antimicrobial agents might still be overprescribed. PMID- 10750214 TI - Nebulised steroid in the treatment of croup: a systematic review of randomised controlled trials. AB - BACKGROUND: Croup is one of the commonest respiratory complaints among children. There is growing evidence that steroids may be an effective treatment. AIM: To assess the effectiveness of treatment with nebulised steroid for children with croup. METHOD: Systematic review of randomised controlled trials comparing administration of nebulised steroid with placebo. Trials were identified from searches of three bibliographic databases, the Cochrane Controlled Trials Register, correspondence with the manufacturers of nebulised steroid, and one round of manual citation searching. RESULTS: Eight randomised controlled trials were identified including 574 children with mild to severe croup. Overall, the mean age was 25.2 months and 72% of children were male. All trials were hospital based and of good methodological quality, with adequate concealment of treatment allocation and blind outcome assessment. Children treated with nebulised steroid were significantly more likely to show an improvement in croup score by five hours (combined relative risk = 1.48, 95% confidence interval [CI] = 1.27 to 1.74) and significantly less likely to need hospital admission after attending the emergency department (combined relative risk = 0.56, 95% CI = 0.42 to 0.75) than the placebo group. The funnel plot indicated the presence of publication bias, with smaller studies showing the larger effects, but this could also be owing to less pronounced effects in studies of older children with milder croup. CONCLUSIONS: Nebulised steroids are effective in the treatment of children attending hospital departments with croup. A meta-analysis based on individual patient data could clarify to what extent the effect depends on age and severity of disease. New trials are needed to define the indications for, and effectiveness of, steroid treatment of croup in the community. PMID- 10750216 TI - QT lengthening and life-threatening arrhythmias associated with fexofenadine. PMID- 10750215 TI - Not so simple cystitis: how should prescribers be supported to make informed decisions about the increasing prevalence of infections caused by drug-resistant bacteria? AB - Trimethoprim is a safe, effective, and inexpensive treatment for cystitis. However, at least 25% of bacteria isolated from urine samples in general practice are now resistant to trimethoprim in the laboratory. The relationship between laboratory resistance and clinical outcome is complex. Cephalexin appears to be more active than trimethoprim in the laboratory but has been consistently less effective in clinical trials. There is little point in collecting data about the prevalence of drug resistance in urinary bacteria unless it is linked to evidence about the impact of resistance on clinical outcomes. Pragmatic clinical trials are required to provide practices with clear thresholds for managing their antibiotic policies; for example, 'Change from trimethoprim to drug X when the probability of trimethoprim resistance reaches Y%.' Prescribers should be aware that trimethoprim resistance is most likely to occur in patients who have been exposed to trimethoprim or other antibiotics in the previous six months, and that the risk increases with age. This information could be used to stratify women according to risk of infection by trimethoprim-resistant bacteria. Health education leaflets are an effective method for reducing the frequency of recurrent cystitis. Symptomatic treatment can control symptoms and allow time for microbiological investigation. Both of these strategies may help to reduce unnecessary prescribing of antibiotics in general and quinolones in particular. PMID- 10750217 TI - Sex differences in incidence rates and referral ratios for first attack angina pectoris. PMID- 10750218 TI - Patient-centredness and outcomes in primary care. PMID- 10750219 TI - Randomised controlled trials in general practice. PMID- 10750220 TI - Colorectal cancer. PMID- 10750221 TI - Prescribing and French trainees. PMID- 10750222 TI - Non-attendance at psychiatric outpatient clinics. PMID- 10750223 TI - Waiting for the great leap forward. PMID- 10750224 TI - A scandal of inaction: how to help GPs implement evidence-based health promotion. PMID- 10750225 TI - Lessons from the London Initiative Zone Educational Incentives funding: associations between practice characteristics, funding, and courses undertaken. AB - BACKGROUND: Following the Tomlinson report of 1992, London Initiative Zone Educational Incentives (LIZEI) funding was introduced for a three-year period to improve recruitment, retention, and educational opportunities for general practitioners working within inner London. AIM: To test the hypothesis that general practices that show evidence of good organisation achieved better access to LIZEI funding than less organised practices. METHOD: Observational practice based study involving all 164 general practices in EAst London and the City Health Authority during the first two years of the scheme, April 1995 to March 1997. RESULTS: Univariate analysis showed that higher levels of LIZEI funding were associated with practices where there was evidence of good organisation, including higher targets for cervical cytology screening and immunisation rates for under two-year-olds, better asthma prescribing, and training status. Using ten practice and population explanatory variables, multiple regression models were developed for fundholding and non-fundholding practices. Among non fundholding practices, the asthma prescribing ratio was the variable with the greatest predictive value, explaining 14.7% of the variation in LIZEI funding between practices. Strong positive associations existed between taking further degrees and diplomas, practice size, training, and non-fundholding status. CONCLUSION: Larger practices, training practices, and those that demonstrated aspects of good practice organisation gained more LIZEI funding: an example of the 'inverse funding law'. Practices within a multifund, based in the Newham locality, gained LIZEI funding regardless of practice organisation. Networks of practices, and, potentially, primary care groups, have a role in equalising the opportunities for education and development between practices in east London. PMID- 10750226 TI - A competency model for general practice: implications for selection, training, and development. AB - BACKGROUND: The role of the general practitioner (GP) has changed significantly over the past decade. This problem is compounded by growing concern over postgraduate attrition rates from medicine, with current estimates as high as 19%. AIM: To define a comprehensive model of the competencies required for the job role of GP. METHOD: Three independent studies were conducted to define GP competencies including (1) critical incidents focus groups with GPs, (2) behavioural coding of GP-patient consultations, and (3) critical incidents interviews with patients. Study 1 was conducted with GPs (n = 35) from the Trent region. Study 2 involved observation of GP-patient consultations (n = 33 consultations), and Study 3 was conducted with patients (n = 21), all from a Midlands-based medical practice. RESULTS: The data collected from the three studies provided strong evidence for a competency model comprising 11 categories with a summary of the associated behavioural descriptions. Example competencies included empathy and sensitivity, communication skills, clinical knowledge and expertise, conceptual thinking, and coping with pressure. CONCLUSIONS: Triangulation of results was achieved from three independent studies. The competencies derived imply that a greater account of personal attributes needs to be considered in recruitment and training, rather than focusing on academic and clinical competency alone. The model could be employed for future research in design of selection techniques for the role of GP. PMID- 10750227 TI - Prioritising referrals to a community mental health team. AB - BACKGROUND: Current national policies encourage prioritisation of people with severe mental illness (SMI) as well as the development of a primary care-led National Health Service. Where resources for mental health are limited, there is a potential conflict between the needs of people with SMI and the much more common depressive and anxiety disorders that form the bulk of the mental health workload in primary care. AIM: To describe the re-organisation of a community mental health team in order to prioritise people with SMI. METHOD: The number and type of referrals received in the 12 months before and after re-organisation were compared, and general practitioners' (GP) views on the changes sought. RESULTS: There was a significant reduction in GP referrals of patients with less severe disorders in the second year. In both years the proportion of patients with a possible psychotic diagnosis or risk of self-harm was much higher among referrals from within the psychiatry department (92% of referrals) than among GP referrals (20% of referrals). Using data from a postal survey, 46% of referring GPs reported a significant improvement in the service provided to patients with SMI, but 34% reported a deterioration in services for other patient groups. GPs were more likely to be satisfied with the service for people with SMI than with the service for other patient groups. CONCLUSIONS: Improvements in the service provided for those with SMI can be achieved, but this may be at the expense of services for other patient groups. Primary care groups will need to consider this potential conflict in setting priorities for mental health. PMID- 10750228 TI - Parents and GPs at cross-purposes over hyperactivity: a qualitative study of possible barriers to treatment. AB - BACKGROUND: Although childhood hyperactivity is a common, serious, and treatable disorder, most affected children in Britain do not receive effective treatment. AIM: To investigate the views that parents and GPs hold about hyperactivity, and to explore how far these views, and clashes between these views, influence access to services. METHOD: Qualitative study making use of semi-structured interviews with 10 general practitioners (GPs) and 29 parents of hyperactive children drawn from parents' groups, community services, and specialist clinics. RESULTS: The views of parents and GPs differed markedly. Parents generally saw severe hyperactivity as a long-lasting, biologically-based problem that needed treatment in its own right and that benefited from diagnosis. Most of the GPs were unsure whether hyperactivity was a medical disorder warranting a label and specific treatment, and often saw it as a passing phase related to family stresses. Parents worried that professionals would blame them for their child's problem, whereas many GPs saw the parent's tendency to medicalise as a way to avoid thinking about their own shortcomings in parenting. CONCLUSION: Access to treatment was influenced by the views of parents and GPs, by the clashes between these views, and by each group's perceptions of the other group's beliefs. Clashes between the views of parents and GPs were particularly likely to lead to misunderstandings, dissatisfaction, and lack of access to effective help. PMID- 10750229 TI - Psychological morbidity in general practice managers: a descriptive and explanatory study. AB - BACKGROUND: Proposals to establish an occupational health service for primary care should be informed by knowledge of the health needs of general practice employees. AIM: To determine the prevalence and occupational correlates of stress, anxiety, and depression among practice managers in two contrasting health authorities in England. METHOD: A postal questionnaire, soliciting information about stress induced by work-related activities, which contained the General Health Questionnaire (GHQ) and Hospital Anxiety and Depression Scale (HADS), was sent to all 149 practice managers in two health authorities areas of south-east England. RESULTS: Completed questionnaires were returned by 111 (75%) managers; 41/111 (37%) achieved GHQ case status with scores on HADS indicating that 49/111 (44%) classified themselves as anxious and 19/111 (17%) as depressed. The likelihood of being a case was found to be higher in managers from practices with larger numbers of GP partners (P = 0.02) and in managers from practices not in receipt of deprivation payments (P = 0.03). Multiple logistic regression showed that managers' perceived difficulties with general practice administration duties (relative ratio [RR] = 3.27, 95% confidence interval [CI] = 1.22-8.75) and dealings with GPs (RR = 1.86, 95% CI = 1.03-3.34) were the most powerful predictors of case status. CONCLUSION: The questionnaire uncovered high prevalences of self-reported stress, anxiety, and depression in general practice managers. Although the vast majority of National Health Service (NHS) employees have access to an occupational health service, no such source of support exists for those working in general practice. The NHS needs to establish an occupational health service that caters to the needs of clinical and non-clinical members of primary health care teams. PMID- 10750230 TI - Factors influencing discussion of smoking between general practitioners and patients who smoke: a qualitative study. AB - BACKGROUND: Anti-smoking advice from general practitioners (GPs) is effective and recent evidence-based guidelines urge GPs to advise all patients against smoking at every opportunity. GPs do not exploit many opportunities to discuss smoking with patients and the reasons for this are unclear. AIM: To elicit, relate, and interpret GPs' accounts of why they discuss smoking with some patients and not others. METHOD: Thirty-nine Leicestershire GPs were purposively selected so as to have a range of attitudes towards discussing smoking with patients. Each GP had one surgery session video-recorded and afterwards participated in a qualitative, semi-structured interview. Prior to each interview, GPs were shown a video recording of one of their consultations with a smoker to enhance their recall of events. RESULTS: Being aware of patients' smoking status did not necessarily result in GPs discussing smoking with patients. GPs were keen to preserve good doctor-patient relationships and avoid negative responses from patients once the topic of smoking had been raised, and this was felt to be best achieved by restricting most discussions about smoking to situations where patients presented with smoking-related problems and in circumstances where the doctors perceived the doctor-patient relationship was strong. Doctors also thought it important to address patients' agendas relating to the current consultation before discussing smoking. CONCLUSIONS: General practitioners have strong reasons for preferring to discuss smoking when patients present with smoking-related problems. Those wishing to increase the amount of advice-giving by GPs might be more successful if they encouraged GPs to make greater use of problem-orientated opportunities to discuss smoking. PMID- 10750231 TI - General practitioners' views of working with team midwifery. AB - This report presents the results of a survey of general practitioners (GPs) working alongside a midwifery team in south-east England. Sixty-nine per cent of the GPs thought team midwifery was a good idea in theory. However, just 37% thought it was working well locally and 56% reported that they would like to go back to working in the way they did before. Of greatest concern was the decline in interprofessional communications and the loss of continuity for patients. Therefore, team midwifery, as implemented in this locality, may not attain the goals aimed at by the organisation of care in this way. PMID- 10750232 TI - Chlamydia trachomatis in primary care: knowledge and practice in Glasgow. AB - A recent Department of Health report recommended the establishment of a selective screening programme for Chlamydia trachomatis infection. We report a survey which suggests that primary care clinicians may not yet be prepared for this task. PMID- 10750233 TI - Consultation rates with a doctor in 1996. AB - Data from 23 Sheffield practices showed general practitioner in-surgery consultation rates running at an average of 3395 per 1000 patients in 1996-1997. This is 30% above the equivalent contact rate found in the Fourth National Morbidity Survey of England and Wales in 1991-1992. PMID- 10750234 TI - Quality of information on hospice referral. AB - Good quality referral information provides hospice staff with essential information at a time when patients are particularly vulnerable. An Ideal Referral Criteria tool for measuring the quality of general practitioners' information was piloted at one hospice site. Overall inter-rater reliability was 90%, with individual categories ranging from 19% to 34%. Cronbach's alpha was 0.35. Further psychometric testing is recommended. PMID- 10750235 TI - General practitioners' habits and knowledge in relation to the management of H. pylori-associated dyspepsia and their views about a locally available 13-carbon urea breath test. AB - We report the results of general practitioners' views on Helicobacter pylori associated dyspepsia and use of screening tests in the community. The use of office serology tests in screening is of concern as independent validation in specialist units has been disappointing. PMID- 10750236 TI - Sex and relationships following childbirth: a first report from general practice of 131 couples. AB - Changes in a couple's sexual relationship following childbirth may be more significant than previous studies have suggested. Around 50% of first-time parents in this study described their sex life as 'poor' or 'not very good' eight months after the birth of their baby, and one in five said that they would like help for this. First-time parents rating their general relationship as 'poor' or 'not very good' rose from 1% before pregnancy to 20% eight months after childbirth. Changes in the general relationship and the quality of sex life were associated in these couples. PMID- 10750237 TI - A prospective study of primary care patients with musculoskeletal pain: the identification of predictive factors for chronicity. AB - Primary care faces the challenge of reducing the proportion of patients continuing with musculoskeletal pain beyond the acute phase. This study assessed patients presenting in general practice with a four- to 12-week history of pain and re-assessed them 12 weeks later. Patients whose pain was described as 'none' or 'slight' were allocated to the 'acute group', and those whose pain continued to be 'moderate' or 'severe' were allocated to the 'chronic group'. Comparative analysis of the two groups' responses at initial assessment identified pain intensity, active coping score, and previous pain episode to be factors independently predictive of chronicity. PMID- 10750238 TI - Can district-wide audits improve primary care epilepsy management? An audit of seizure frequency recording. AB - A district-wide epilepsy audit in general practice showed that levels of seizure frequency recording were too low to evaluate the quality of control of epilepsy. A repeat audit after multi-faceted interventions showed an improvement in seizure frequency recording of 13.2% (CI = 8.9 to 17.6) from 54.7% to 68%. This illustrates the difficulties of evaluating quality of care using routine records and the problems of implementing changes in general practice. PMID- 10750239 TI - Membership by Assessment of Performance: developing a method for assessing established general practitioners. AB - Over 200 general practitioners (GPs) and others have contributed to the development of Membership by Assessment of Performance (MAP): a new scheme for assessing established GPs. By means of a Delphi consultation with a broad cross section of the profession, a working conference, piloting with potential candidates, and repeated checking with reference panels, the assessment was developed within two years. We report the development of MAP, including the results of the Delphi consultation, which asked 'Which aspects of a general practitioners performance are important to assess?'. PMID- 10750240 TI - Medical cannibalism, strangury, and the spread of erratic hyphenitis. PMID- 10750241 TI - Is there enough clinical time available in primary care? PMID- 10750242 TI - Are postal urine specimens a feasible method for genital chlamydial infection screening? PMID- 10750243 TI - The colorectal cancer jigsaw puzzle. PMID- 10750244 TI - Health technology assessment in primary and community care. PMID- 10750245 TI - Supporting practice-based audit. PMID- 10750246 TI - Needs assessment in primary care. PMID- 10750247 TI - Adult learning theory. PMID- 10750248 TI - Dermaclinic: preliminary triage by GP specialoids? PMID- 10750249 TI - 2000--a time to reflect, look forward, look back and worry about funding. PMID- 10750250 TI - Anticoagulation and the cardioversion of atrial flutter. PMID- 10750251 TI - The future of NSAID therapy: selective COX-2 inhibitors. PMID- 10750253 TI - Traumatic rupture of the diaphragm. AB - Diaphragmatic rupture following trauma is often an associated and missed injury. The diagnosis is difficult, so is usually made intraoperatively. Twenty-one patients with traumatic rupture of the diaphragm (TRD) who presented between 1995 and 1998 were retrospectively analysed: 12 had penetrating injuries and nine had blunt injuries. Right-sided defects exceeded left (12 vs 9). Only seven patients had signs and symptoms directly referrable to rupture of the diaphragm. All patients were operated on through a midline laparotomy. Diaphragmatic hernia was seen in six patients (28.5%); 20 (95%) patients had concomitant injuries. The liver was the most commonly injured organ (10 patients). The aim of this study was to report our experiences with TRD and review the literature. We conclude that correct preoperative diagnosis of TRD needs a high index of suspicion. It can be diagnosed intraoperatively by explorative laparotomy. Most ruptures can be repaired by the abdominal approach. PMID- 10750254 TI - Imaging assessment of uncomplicated bladder outflow obstruction. AB - To assess the impact of the use of plain X-ray films and ultrasound examinations on clinical decision-making in patients referred with symptoms of bladder outflow obstruction. Patients with symptoms of bladder outflow obstruction were recruited prospectively. After clinical assessment they underwent standard investigations of plain X-ray of the kidneys, ureters and bladder, and upper and lower urinary tract ultrasound. A management decision was made on the basis of all results. These decisions were then reviewed with specific reference to radiological findings to assess the role of imaging in the decision-making process. A total of 320 men were recruited for the study. X-ray of the kidneys, ureters and bladder detected 10 upper tract calculi, none of which has required active intervention to date. Four bladder calculi were detected. Two of these were also seen on ultrasound scan: one was small and insignificant, the other had caused microscopic haematuria, necessitating cystoscopy regardless of the X-ray findings. Upper tract ultrasound revealed abnormalities and anatomical variants which did not affect management. Four patients had upper tract dilatation but these had already had surgery planned on the basis of other investigations, including residual urine volume determination by lower tract ultrasound. Lower urinary tract ultrasound should be the only routine imaging used in the initial assessment of patients with bladder outflow obstruction, with consequent implications of reduced patient stress and reduced investigation cost. PMID- 10750252 TI - A study of the efficacy and safety of irbesartan in combination with conventional therapy, including ACE inhibitors, in heart failure. Irbesartan Heart Failure Group. AB - Because heart failure therapy with angiotensin-converting enzyme (ACE) inhibitors may not be optimal, owing to persistent levels of angiotensin II occurring through incomplete blockade and alternate pathways, the benefit of adding irbesartan, an angiotensin receptor antagonist, to conventional therapy, including ACE inhibitors, was examined. In this multicentre, randomised, double blind, placebo-controlled study, 109 patients with heart failure (New York Heart Association functional class II and III) and left ventricular ejection fraction (LVEF) < or = 40% received stable doses of ACE inhibitors and diuretics before and throughout the study. Irbesartan was titrated as tolerated to 150 mg once daily in all patients. Exercise tolerance time (ETT), LVEF and clinical status were assessed at baseline and after 12 weeks. Compared with placebo, irbesartan in combination with conventional therapy, including ACE inhibitors, produced favourable trends in ETT and LVEF and was well tolerated in patients with mild to moderate heart failure. PMID- 10750256 TI - Social drift in forensic psychiatric inpatients with schizophrenia. AB - The highest social class before admission of 27 forensic psychiatric inpatients in a regional secure unit who suffered from schizophrenia was found to be significantly lower than that of their fathers. This is the first report of social drift in this group of patients. PMID- 10750255 TI - Outcome in children under 5 years of age with constipation: a prospective follow up study. AB - Constipation in the pre-school child is common and causes considerable distress to children and their parents. There is a lack of information regarding prognosis in this group of patients although some are clearly at risk of developing long term difficulties with defaecation. We have previously reported characteristics of children under 5 years of age referred to hospital over a one-year period with idiopathic constipation. We now report outcome data after 18 months of follow-up in 41 of the original cohort of 42 children. We have also summarised what we regard as good practice in managing constipation in this age group. Eighteen months after initial outpatient assessment, the symptoms of constipation were reported to have resolved in 36 (88%), although seven of these were still needing regular laxative treatment. It was our subjective impression that the five children who apparently did not improve came from families that had a greater degree of psychosocial problems and where compliance with treatment was suspect. This study shows that with simple treatment measures and ongoing support, an optimistic prognosis can be given to families from the outset. The minority of pre-school children who do not improve may benefit from a more intensive psychological approach to management. PMID- 10750257 TI - Treatment of status epilepticus with midazolam in the critical care setting. AB - Status epilepticus (SE) is a potentially life-threatening condition that requires prompt and aggressive treatment. Prolonged status seizures are associated with significant physiological sequelae and neurological deficits. Although systemic events such as hyperthermia and anoxia contribute to neuronal damage, SE in and of itself can induce cell death. In general, the sooner it is brought under control, the more favourable is the prognosis. Benzodiazepines, as a group, are the most frequently used anticonvulsants in the management of status seizures. Midazolam, a water-soluble benzodiazepine, is a potent anticonvulsant that offers many advantages over typical benzodiazepines. Because of its stability in aqueous media, midazolam dissolves in common diluents such as normal saline or dextrose water. Consequently, midazolam both intravenously (i.v.) and intramuscularly (i.m.) is well tolerated locally and is associated with less venoirritation than benzodiazepines or antiepileptics that require organic solvents. The water solubility of midazolam also allows rapid and reliable absorption of the drug from the i.m. injection site. Because it is rapidly metabolised and its metabolites are pharmacologically inactive, midazolam has a short duration of action. Most patients regain full conscious state and can be evaluated soon after the cessation of treatment. Midazolam by continuous i.v. infusion and by the i.m. route has been successfully used in the treatment of SE. Although some respiratory and haemodynamic side-effects have been associated with midazolam, no clinically significant side-effects were observed with its use for the indication of SE. It is suggested that midazolam is a safe and rapidly effective treatment option in the management of SE in the critical care setting. PMID- 10750258 TI - The role of alpha 1-blockers in combination therapy for hypertension. AB - Antihypertensive monotherapy provides adequate blood pressure control in less than 50% of patients with hypertension (BP > 140/90 mmHg), especially those with stage 2-3 disease. This article reviews clinical studies that demonstrate that add-on therapy with an alpha1-blocker (doxazosin, terazosin or prazosin) is an effective and well-tolerated regimen for improving blood pressure control in patients with inadequately controlled hypertension. Furthermore, alpha1-blockers have therapeutic benefits that go beyond blood pressure management. They have a small but positive effect on the serum lipid profile and they have favourable or benign effects on conditions that frequently coexist with hypertension, such as type 2 diabetes mellitus and benign prostatic hyperplasia. PMID- 10750259 TI - Small bowel carcinoid tumour presenting with intestinal ischaemia. AB - A rare cause of intermittent intestinal ischaemia is presented to highlight the importance of thorough investigation, including the usefulness of laparoscopy, in the assessment of patients with persistent postprandial abdominal pain and hyperactive bowel sounds. PMID- 10750260 TI - The stigma of anxiety disorders. AB - People with genuine anxiety disorders can be misdiagnosed and mismanaged, because of negative public and professional attitudes to anxiety and the 'worried well'. Misdiagnosis includes not taking symptoms seriously and giving patients pejorative labels such as 'personality disorder' or 'inadequate'. This can lead to mismanagement such as reluctance to investigate symptoms fully, with regard to both physical and psychological aetiologies. Also, poor understanding of appropriate treatments can lead to mismanagement of these disorders. This paper reviews sources of stigma in dealing with anxiety disorders and ways in which this stigma might be avoided. PMID- 10750261 TI - Safe and effective management of hypertension with fixed-dose combination therapy: focus on losartan plus hydrochlorothiazide. AB - Treatment of hypertensive patients with fixed-dose combination therapy consisting of losartan and hydrochlorothiazide (HCTZ) has several potential benefits over monotherapy with each of the individual components: more effective blood pressure control, a reduction in the likelihood of adverse effects, and facilitation of patients staying on therapy due to a simple once-daily regimen. Losartan plus HCTZ fixed-dose combination therapy lowers blood pressure in mild to moderate or severe hypertensive patients to a level comparable with other classes of antihypertensive drugs in combination with HCTZ. Fixed-dose combination therapy with losartan plus HCTZ is therefore an excellent choice for hypertensive patients in whom combination therapy is necessary to achieve additional blood pressure reductions. PMID- 10750262 TI - Giant pilomatricoma simulating carcinoma of the male breast. AB - We present the case of a 65-year-old man with a large calcifying epithelioma in the right breast. The case is unusual not only for the size of the tumour but also for its location in a male breast. Despite its large size (12 x 10 x 7 cm), the tumour did not show any clinical, radiological or histological evidence of malignancy or aggressive behaviour. One year after surgery there was no recurrence or metastatic disease. The possibility of malignancy, recurrence or metastasis of these large tumours, as well as the necessity of follow-up and investigations, are emphasised. PMID- 10750263 TI - Eicosapentaenoic acid treatment in schizophrenia associated with symptom remission, normalisation of blood fatty acids, reduced neuronal membrane phospholipid turnover and structural brain changes. AB - The administration of the omega-3 fatty acid eicosapentaenoic acid (EPA) to a drug-naive patient with schizophrenia, untreated with conventional antipsychotic medication, led to a dramatic and sustained clinical improvement in both positive and negative symptoms. This was accompanied by a correction in erythrocyte membranes of abnormalities in both n-3 and n-6 highly unsaturated fatty acids and with reduced neuronal membrane phospholipid turnover, as evidenced by serial 31 phosphorus cerebral magnetic resonance spectroscopy. Using recently developed techniques of image segmentation, subvoxel registration and quantitation, analysis of serial high-resolution 3D cerebral MRI scans showed that, in the year before EPA treatment, cerebral atrophy was taking place and that this atrophy was reversed by six months of EPA treatment. These results demonstrate that EPA can reverse both the phospholipid abnormalities previously described in schizophrenia and cerebral atrophy. They provide strong further evidence in support of the membrane phospholipid model of schizophrenia. PMID- 10750264 TI - Interpretation of the direct antiglobulin test in neonates with haemolytic disease of the newborn. AB - Neonatal blood grouping and direct antiglobulin test results need to be interpreted with caution following multiple intrauterine blood transfusions for haemolytic disease of the newborn (HDN). Except for a few reports published about 20 years ago, this fact is not well documented. We report a case emphasising the problem and its consequences. PMID- 10750265 TI - The impact of community diabetic eye screening. PMID- 10750266 TI - Forensic apartheid? PMID- 10750267 TI - The influence of manufacturing processes on the identification of bullets and cartridge cases--a review of the literature. AB - A basic hypothesis of firearms identification is the uniqueness of the striations and marks left on fired bullets and cartridge cases respectively. This paper reviews the studies so far reported in the literature on this issue, applied to consecutively (or closely) manufactured components of a firearm. A correct firearms identification starting from the striae on a bullet can be performed if one takes into account a few basic rules which are independent of the specific rifling technique applied to the barrel. However, the identification starting from a cartridge case has to be performed with care, exploiting as much as possible all the marks left during the firing process. PMID- 10750268 TI - Novel technique for the combined recovery, extraction and clean-up of forensic organic and inorganic trace explosives samples. AB - This paper describes a simple processing and analysis scheme for explosives trace swab samples which deals both with organic and inorganic materials. Swabs, wetted with ethanol or ethanol/water mixture, were extracted with ethanol/water mixture. The extract was passed directly through a simple column containing an acrylonitrile/styrene copolymer adsorbent. The adsorbent retained common organic explosives, which were recovered with an efficiency of 30-50% as a relatively clean ethyl acetate solution. The concentrated ethyl acetate eluate was analysed using gas chromatography with chemiluminescence or mass spectrometric detection. The unretained inorganic ions and sugars, which were recovered with generally high efficiency as an ethanol/water solution, could be directly analysed using ion chromatography and/or capillary electrophoresis. Minor difficulties encountered in the analysis of sugars, fluoride and phosphate were examined. PMID- 10750269 TI - The use of the Foster & Freeman Fx5 Fibre Finder in forensic textile examinations. AB - The Fx5 Fibre Finder has been developed by Foster & Freeman Ltd to automatically search tape lifts for coloured fibres specified by the operator. Experiments and casework studies have been conducted to assess primarily the performance of the instrument but also, where possible, to compare it with manual searching. Tape lifts have always been manually searched at this laboratory and it is a laborious task. The use of a machine such as the Fx5 would release a scientist from many hours of low power microscopy to be free for other duties and therefore could be a valuable labour saving device in forensic textile examination. PMID- 10750270 TI - Combustion of animal fat and its implications for the consumption of human bodies in fires. AB - This paper describes experiments in which the combustion of animal tissue (pork) was measured under a variety of conditions that may be encountered in fire scenes. Combustion depends on substantial preheating of the tissue by an external heat source and the availability of a porous wick (such as charred cellulosic material). Combustion of moderate-size samples can proceed at a moderate rate of 1-3 g/s (3.6-10.8 kg/hr) if provided with an adequate wick and results in only a small fire of 30-50 kW. In the final test, combustion of 26 kg of fat and skin created a fire of 120-130 kW. Such a fire is more likely to cause fire spread to other combustibles nearby. The presence of other, less efficient fuels (like skin and muscle) and the absence of large fuel masses (such as in the very lean pig carcasses used here) results in significantly smaller fires of 40-50 kW. Such fires are more typical of burning human remains when there are minimal contributions from other fuels. PMID- 10750272 TI - The persistence of gunshot residue on shooters' hands. PMID- 10750271 TI - Cryptic chemical identification as a crime intelligence aid. AB - A dead body was found near the sea and a commercial port in north-east Italy. The man had been shot and then burnt, by using a large volume of fire accelerant. The chemical composition of the flammable mixture had to be determined in order to aid police investigations. GC-MS analysis of residual cloth and soil identified a common gasoline, together with some unrelated compounds deriving from the container used to carry the inflammable liquid. A reconstruction of the event, an examination of the surroundings where the crime took place and the cryptic chemicals found, enabled the investigators to restrict and intensify their enquiries within a specific area. PMID- 10750274 TI - Assessment of a sheriff's office evidence drying room. PMID- 10750273 TI - Feature extraction of optical projectiles images. PMID- 10750275 TI - Control of wood dust from table saws, shapers, and automated routers. PMID- 10750276 TI - Internet offers exciting new alternative for chemical hazard information management. PMID- 10750277 TI - Hard metal exposures. Part 1: Observed performance of three local exhaust ventilation systems. AB - Not every ventilation system performs as intended; much can be learned when they do not. The purpose of this study was to compare observed initial performance to expected levels for three saw-reconditioning shop ventilation systems and to characterize the changes in performance of the systems over a one-year period. These three local exhaust ventilation systems were intended to control worker exposures to cobalt, cadmium, and chromium during wet grinding, dry grinding, and welding/brazing activities. Prior to installation the authors provided some design guidance based on Industrial Ventilation, a Manual of Recommended Practice. However, the authors had limited influence on the actual installation and operation and no line authority for the systems. In apparent efforts to cut costs and to respond to other perceived needs, the installed systems deviated from the specifications used in pressure calculations in many important aspects, including adding branch ducts, use of flexible ducts, the choice of fans, and the construction of some hoods. After installation of the three systems, ventilation measurements were taken to determine if the systems met design specifications, and worker exposures were measured to determine effectiveness. The results of the latter will be published as a companion article. The deviations from design and maintenance failures may have adversely affected performance. From the beginning to the end of the study period the distribution of air flow never matched the design specifications for the systems. The observed air flows measured within the first month of installation did not match the predicated design air flows for any of the systems, probably because of the differences between the design and the installed system. Over the first year of operation, hood air flow variability was high due to inadequate cleaning of the sticky process materials which rapidly accumulated in the branch ducts. Poor distribution of air flows among branch ducts frequently produced individual hood air flows that were far below specified design levels even when the total air flow through that system was more than adequate. To experienced practitioners, it is not surprising that deviations from design recommendations and poor maintenance would be associated with poor system performance. Although commonplace, such experiences have not been documented in peer-reviewed publications to date. This publication is a first step in providing that documentation. PMID- 10750279 TI - Personal fluoride and solvent exposures, and their determinants, in semiconductor manufacturing. AB - Personal air sampling for fluorides and solvents was done at 35 semiconductor fabrication facilities in the United States. Fluoride compounds were used in etching and cleaning operations, and solvents were used in photoresist and developing operations. All personal solvent and fluoride levels were less than 2 percent of current Occupational Safety and Health Administration (OSHA) standards. Statistical models of the exposure determinants for the target agents found production level, as indicated by number of semiconductor wafer cassettes loaded/unloaded from the target machines or baths, was predictive of fluoride, xylene and 1-methoxy-2-propyl acetate exposures. The percent of fresh air ventilation and the percent of xylene in the photoresist were also significant determinants in the statistical model predicting personal xylene exposure levels. PMID- 10750278 TI - Hard metal exposures. Part 2: Prospective exposure assessment. AB - Hard metal exposures may precipitate lung disease in exposed workers. This article reports on a project investigating the relationship between local exhaust hood air flow levels and workplace hard metal exposures. Airborne cobalt, chromium, and cadmium exposure concentrations, and ventilation system function were monitored for three consecutive days prior to installation of three new ventilation systems, and then were followed monthly for one year. Work activities included wet and dry grinding of saw blades, brazing, welding, and setup. Work task exposures were highly variable over the period of the study. Ventilation air flows failed to meet design goals due to low total air volume and poor distribution; however, worker exposures to metals were controlled in most cases. Hood design, worker acceptance, and use of the hoods were as important in controlling exposures as were exhaust hood air flow levels. PMID- 10750280 TI - Skin cleaning with kerosene facilitates passage of carcinogens to the lungs of animals treated with used gasoline engine oil. AB - Solvents such as kerosene or gasoline may be used by workers to clean their skin following contact with oily materials. This practice is not recommended, as it is well known that the solvent will defat the skin. Many also suspect that solvent washing may increase exposure by carrying materials through the skin; however, there is little documentation of this. Auto mechanics may be exposed to used gasoline engine oil (UGEO), an animal carcinogen which forms carcinogen-DNA adducts in skin and lung following topical application. This study was designed to determine if cleaning with kerosene following exposure to UGEO altered absorption of carcinogens from this material. UGEO or new oil (NO) was applied to the shaved skins of groups of HSD-ICR mice for five days. At 1 or 8 hours after application, the treated skins were cleaned with either kerosene or a commercial cleaner, or were not cleaned. Animals were sacrificed 24 hours after the last application, skins and lungs harvested, and DNA analyzed for carcinogen-DNA adducts by 32P-postlabeling. Five applications of UGEO significantly increased carcinogen-DNA adduct levels in both lungs and skin compared to animals treated with NO. DNA adduct levels in the skin were reduced significantly in groups washed with kerosene or commercial cleaner. Washing at one as opposed to eight hours after UGEO application resulted in lower adduct levels regardless of cleaner. DNA adduct levels in the lung were reduced when the commercial cleaner was used, again in a time-related fashion. However, cleaning with kerosene resulted in mean carcinogen-DNA adduct levels in the lung which were significantly higher than even the positive controls, regardless of cleaning time. This is the first demonstration that kerosene cleaning facilitates passage of carcinogens through the skin, resulting in higher levels of genetic damage in a critical internal organ. PMID- 10750281 TI - Mist concentration measurements. II: Laboratory and field evaluations. AB - Sampling methods to determine occupational exposures to metalworking fluid mists are subject to bias. Light-scattering devices may respond differently to variations in particle size, shape, and refractive index. Gravimetric samplers are prone to evaporative losses of semi-volatile components. The performance of two light scattering devices, an electrostatic precipitator, and filters followed by gravimetric analysis was investigated when measuring metalworking fluid mist in laboratory and field settings. Laboratory tests with soluble oil and field tests with soluble oil, straight oil, and semi-synthetic fluid showed significant evaporative losses from filters. Light-scattering devices tended to overestimate mist concentrations when mass median diameters were less than about 2 microns and to underestimate mist concentrations when mass median diameters were larger. Filters will underestimate occupational exposures to metalworking fluid mists when semi-volatile components are present. PMID- 10750282 TI - The relationship between TLV-TWA compliance and TLV-STEL compliance. AB - The purpose of this study was to investigate the relationship between the American Conference of Governmental Industrial Hygienists' (ACGIH) time-weighted average (TLV-TWA) and short-term exposure limit (TLV-STEL) Threshold Limit Values (TLVs). It is of value to determine if one or the other of these exposure limits is inherently more stringent so that exposure monitoring strategies may be devised which efficiently use available resources and effectively control exposures to meet both exposure limits. The ACGIH short-term exposure limit (TLV STEL) imposes three conditions on short-term (15-minute) exposures. These conditions involve exceeding the TLV-TWA and TLV-STEL levels and the time separation of those short-term exposures that exceed the TLV-TWA level. These conditions were analyzed to produce eight unique component probabilities for TLV STEL non-compliance. The sum of these eight components is the total probability of TLV-STEL non-compliance. Mathematical expressions for the eight probability components are derived in terms of the probability that a single 15-minute exposure exceeds the time-weighted average threshold limit value (TLV-TWA) and the geometric standard deviation of these 15-minute exposures. These expressions were applied to various hypothetical workplaces, and the relationship between TLV TWA and TLV-STEL compliance is presented. The results showed that non-compliance of 15-minute exposures with the TLV-STEL level is only one part of overall TLV STEL non-compliance. The additional ACGIH provisions for TLV-STEL compliance--the number of 15-minute exposures exceeding the TLV-TWA level and the number of clean periods separating such exposures--can be important factors in determining TLV STEL compliance. It is concluded that compliance with all provisions of the ACGIH TLV-STEL adds a degree of stringency that greatly enhances the likelihood of TLV TWA compliance for most workplace environments. PMID- 10750283 TI - [The J.E. Purkinje Czech Medical Society and leading journals. The journals Casopis Lekaru Ceskych and Prakticky lekar at the turn of the century. PMID- 10750284 TI - [Development of the maternal bond during pregnancy]. AB - Formation of the maternal bond during pregnancy is an area of scientific interest explored very little so far. The interaction between mother and child during pregnancy is conceived with increasing frequency as a peculiar process of learning where the infant starts to develop his genetic predispositions. In this way it is possible to influence the further development of the infant during the first few months after delivery. The presented paper summarizes selected results of an empirical study implemented in 1996 to 1998. The group comprised 481 pregnant women with a permanent domicile in Brno. Within the framework of the project it proved possible to apply a previously elaborated scale for the assessment of the maternal bond during pregnancy. From the results ensues that a strong maternal bond is present during the seventh month of pregnancy in some two thirds of women. The investigation was also concerned with cultural and situational factors which influence the formation of a strong bond to the child during pregnancy. It proved possible to test the effect of the style of the pregnant womans' mother during her childhood, the amount of experienced stress, the level of social support and the influence of the partner on the maternal bond of the pregnant women. PMID- 10750285 TI - [Dyspnea remains a complicated medical (clinical) problem]. AB - Dyspnoea is a subjective sensation which probably develops as a result of integration of signals from the central nervous system and some peripheral receptors. Known dyspnoegenic stimuli include the sensation of an increased effort of the respiratory muscles, information from chemoreceptors on the inadequacy of ventilation, from pulmonary receptors on the compression of the airways and disharmony between information on the tension of the respiratory muscles and the corresponding shortening of the muscle. Pathological dyspnoea, incl. that of cardiac origin is usually associated with impaired breathing. Dyspnoea can be measured. Possible influencing of dyspnoea depends on knowledge of the mechanism of genesis of this sensation. Treatment of dyspnoea can be focused on increasing the efficiency of respiratory muscles, reduction of afferentation from receptors or suppression of the central integration of the mentioned stimuli. PMID- 10750286 TI - [Myocardial revascularization in malignant ventricular tachyarrhythmia- prognostic significance]. AB - BACKGROUND: The survival of patients with chronic ischaemic heart disease and malignant ventricular tachyarrhythmia is influenced positively in some instances by revascularization of the heart muscle and implantation of a cardioverter defibrillator. The objective of the submitted work was to evaluate by perspective follow-up of subjects with chronic ischaemic heart disease and malignant ventricular tachyarrhythmia: a) the effect of revascularization of the heart muscle on the prognosis, making use of programmed stimulation of the ventricles and testing the effectiveness of antiarrhythmic treatment; b) the importance of implantation of a cardioverter-defibrillator in revascularized and non revascularized subjects for the prevention of sudden "arrhythmic" deaths. METHODS AND RESULTS: The authors examined 37 patients (32 men and 5 women), age bracket 34 to 78 years (mean age 61 +/- 11) with IHD and spontaneous ventricular tachyarrhythmia after ruling out acute myocardial infarction. The group was divided into sub-groups without revascularization (21 subjects) and with revascularization (16 subjects). In both sub-groups programmed stimulation of the ventricles was implemented. During the diagnostic finding of programmed stimulation they tested antiarrhythmic drugs, most frequently amiodarone administered orally. A cardioverter-defibrillator was implanted to 10 patients. All patients were followed-up to death, the longest period being 24 months. They evaluated the frequency of cardiac deaths (death on cardiac grounds incl. sudden "arrhythmic" death) and sudden "arrhythmic" deaths (death within on hour after onset of symptoms or first recorded malignant ventricular tachyarrhythmia). In the sub-group without revascularization with diagnostic inducibility of the heart muscle in 85.7% of patients the authors described 9 cardiac deaths (42.9%) and 8 sden "arrhythmic" deaths (38.1%). Conversely in the sub-group with revascularization and with diagnostic programmed stimulation of the ventricles in half the subjects 5 clinical deaths were found (31.3%) and 3 sudden "arrhythmic" deaths (18.8%). Analysis of 11 sudden "arrhythmic" deaths revealed that no subjects with an implanted cardioverter-defibrillator (5) died (documented malignant ventricular tachyarrhythmia). Five of the six patients who died (all without a cardioverter-defibrillator) were not revascularized. CONCLUSIONS: Revascularization of the heart muscle in patients with ischaemic heart disease (after elimination of acute cardiac infarction) and malignant ventricular tachyarrhythmia reduces the risk of relapse of this arrhythmia. The benefit of implantation of a cardioverter-defibrillator was recorded in all subjects regardless of the revascularization of the heart muscle. PMID- 10750287 TI - [Fatty acid composition and parameters of VLDL and LDL in persons with dyslipidemia]. AB - BACKGROUND: Oxidatively modified LDL play an important role in the pathogenesis of endothelial dysfunction, initiation and development of atherosclerosis and stability of the atheromatous plaque. The increased oxidative stress is apparent from a number of deviations, which are part of the insulin resistance syndrome (hypertension, hypoalphacholesterolaemia, diabetes and hyperlipoproteinaemia). The objective of the work was to examine the degree of oxidation and oxidability of LDL and VDL in subjects with dyslipidaemia. METHODS AND RESULTS: In 40 subjects with dyslipidaemia, defined as a triglyceride concentration above 2.30 mmol/l and a drop of HDL cholesterol below 0.90 mmol/l, the authors assessed the fatty acid profile in plasma lipid classes and LDL by capillary gas chromatography. Lipoperoxidation in VLDL and LDL was examined by the method of kinetics of conjugated dienes according to Esterbauser. The results were compared with a group of healthy controls. The group of dyslipidaemic subjects had higher concentrations of NEFA, IRI, blood sugar and uric acid. In these subjects the concentration of conjugated dienes in VLDL was significantly higher and the lag stage in VLDL and LDL was reduced. Both groups differed as to the composition of VLDD and LDL. The group of dyslipidaemic subjects had a higher concentration of cholesterol, phospholipids, triglycerides and apolipoprotein B. A constant finding in the fatty acid profile of all lipid classes was a raised concentration of palmitoleic acid and reduced linoleic acid concentration. CONCLUSIONS: Dyslipidaemic subjects have, as compared with a control group, higher NEFA, IRI and uric acid concentrations. Furthermore they differed not only by the composition of VLDL and LDL but also by a higher degree of VLDL oxidation and reduced resistance to lipoperoxidation of VLDL and LDL particles. A consistent finding in the fatty acid profile was an increased level of palmitoleic acid in all plasma lipid classes and LDL and a drop of linoleic acid in phosphatidylcholine LDL and plasma cholesterolesters. PMID- 10750288 TI - [Czech gynecology on the threshold of the millenium]. PMID- 10750289 TI - [Suspension of the lower third of the urethra in ambulatory practice--minimally invasive treatment of urinary stress incontinence--technique and initial experience]. AB - OBJECTIVE: The aim of the article is the presentation of a new, mini-invasive method for the treatment of urethral incompetence in women--TVT (tension-free vaginal tape). DESIGN AND SETTING: The design was a prospective comparison of the first 10 patients at the Department of Obstetrics and Gynaecology of the 1st Medical Faculty and General Faculty Hospital in Prague where urethral incompetence = genuine stress incontinence regardless of their history was diagnosed consecutively. METHODS: We present the technique of the operation, examination procedure including the application of dynamic magnetic resonance before and after operation at rest and under Valsalva, the subjective and objective outcome in the analyzed group. We compare also the parameters of urethrovesical junction mobility in the MR image and discuss their importance for the new continence mechanism in the TVT method. RESULTS: All patients are 10-18 months after the operation continent. The functional morphology of the lower urinary tract and of the pelvic floor on the MR image after TVT did not reveal any relevant changes in bladder neck dynamic mobility. CONCLUSION: The experience of other authors concerning possible complications, postoperative care and results are evaluated; they are without exception positive. PMID- 10750290 TI - [The effect of bladder filling on changes in ultrasonography parameters of the lower urinary tract in women with urinary stress incontinence]. AB - OBJECTIVE OF THE STUDY: Was to evaluate the effect of filling the bladder on position and mobility of UV junction, and to determine other possible changes of measured ultrasound parameters of the lower urinary tract in relation to filling of the bladder. TYPE OF STUDY: Prospective randomized clinical study. NAME AND ADDRESS OF THE WORKPLACE: Department of Gynaecology--Obstetrics, 1st Faculty of Medicine of Charles University and VFN in Prague. METHODOLOGY: 20 women were included in our study with proved stress incontinence of urine ("GSI"). Their average age was 46 years, average weight 70 kg, average parity 1.57. Perineal and introital ultrasound examination was performed in a supine position, using Acuson 128 XP 10 equipment, a convex probe of 5 MHz frequency and a vaginal probe of 7.0 MHz. Position and mobility of UVJ was monitored based on the following parameters: angle gamma, abscissas p, h, x, y. Bladder filling was 300 ml, 200 ml and empty bladder. RESULTS: Statistically significant differences were observed in UVJ mobility during contractions of pelvic floor muscles between measurements, when the bladder contained 300 ml of liquid, or was empty (p = 0.00983). UV junction of and empty bladder is higher during a contraction, and the mobility is greater (p = 0.006). Distances x, y, p in case of an empty bladder are shorter during contraction and the gamma angle is smaller (p = 0.01). The thickness of the bladder wall was greater in an empty bladder (p = 0.01). Changes in area and thickness of the urethral sphincter were not significant. CONCLUSIONS: Based on our preliminary results we can state that a bladder filling of 300 ml is not significant for evaluation of UV-junction mobility. Rather the opposite; a higher mobility of UV-junction was observed with an empty bladder. Monitoring of the inner orifice of the urethra with an empty bladder presents a slight problem. To evaluate vesicalisation of the proximal urethra and to confirm insufficiency of the inner sphincter (ISD), a filling in the bladder is necessary. The thickness of the bladder wall is measured with an empty bladder. PMID- 10750291 TI - [Laparoscopic excision of endometriosis of the rectovaginal septum using the Diomed laser]. AB - OBJECTIVE: Laparoscopic excision of deep endometriosis of rectovaginal septum and evaluation of technical capabilities of Diomed laser. DESIGN: Prospective pilot study. SETTING: Department of Gynaecology and Minimally Invasive Surgery, Na Homolce Hospital, Prague. METHODS: Eight patients with clinically and laparoscopically diagnosed endometriosis of the rectovaginal septum were selected for laparoscopic surgery. The extent of the lesion was assessed by vaginal palpations and laparoscopic visualisation of the Douglas space with a sponge forceps inserted into the posterior vaginal fornix and using rectal probe simultaneously. To exclude rectal wall infiltration baryum radiography was performed. RESULTS: 7 out of 8 patients experienced a complete disappearance of symptoms such as dyspareunia, dysmenorrhea and pelvic pain. Follow-up is in the range of 1-12 months. SUMMARY: Deep infiltrating rectovaginal endometriosis is a specific disease very different from peritoneal or ovarian endometriosis. A more precise term is rectovaginal adenomyosis. Radical laparoscopic excision is needed. Although simple this is demanding pelvic surgery, the results are gratifying. The use of a laser seems superior to dissection technique. PMID- 10750292 TI - [Hormone replacement therapy in women with surgical treatment of endometriosis and adenomyosis: prospective and follow-up study. Part I]. AB - OBJECTIVE: To determine whether the initiation of estrogen replacement therapy (ERT) in the postoperative period increases the incidence of symptom recurrence following laparoscopic hysterectomy (LH) and/without bilateral salpingo oophorectomy (BSO) for the treatment of endometriosis and adenomyosis. DESIGN: Prospective partially randomized patient preference follow up trial (PRPPT). SETTING: Department of Gynaecology, Endoscopic Training Centre, Kladno Hospital. METHODS: Chart review of 286 consecutive patients who underwent LH and/or without BSO for endometriosis or adenomyosis between April 1994 and June 1999 and who subsequently received ERT. The methods of second phase of trial was prepared. The symptoms of recurrence in patients who started ERT after surgery and in those who did not start ERT (control group) will be compared and adjusted. RESULTS: Seventy three percent had preoperative pelvic pain. One hundred thirty two women (46.2%) had only endometriosis. Of the cases of endometriosis, 35.4% was stage I and 64.4% was stage II.-IV. One hundred fifty four women had adenomyosis, 60 with endometriosis. CONCLUSION: In the first phase of follow study the collection of clinical data was performed. Preliminary results from the PRPPT demonstrate that laparoscopic approach to surgery for endometriosis increased significantly the number of this disease. PMID- 10750293 TI - [Pharmacology of progestins used in oral hormonal contraceptive preparations]. AB - OBJECTIVE: Review of the pharmacology of progestins used in combined oral contraceptives. DESIGN: Review article. SETTING: Department of Obstetrics and Gynaecology, General Teaching Hospital, Charles University, Prague. CONCLUSION: The author presents a review of pharmacology of progestins used in combined oral contraceptives. He compares important pharmacokinetic and pharmacodynamic properties of different progestins. The paper includes comparison of the influence of progestins on lipids, carbohydrates, steroid transport capacity and coagulation. The different pharmacology of progestins is an important factor in the process of pill selection. PMID- 10750294 TI - [Use of human sperm protein monoclonal antibodies in the diagnosis of sperm pathology and selection of a suitable assisted reproduction method for fertilization]. AB - OBJECTIVE: Use of monoclonal antibodies against sperm proteins in human medicine. DESIGN: Experimental and clinical studies. SETTING: Dep. Biology and Biochemistry of Fertilization, Institute of Molecular Genetics, Prague, Laboratory IVF, Iscare IVF, Prague, Dep. of Immunobiology, Institute for the Care of Mother and Child, Prague. METHODS: Monoclonal antibodies against human sperm intra-acrosomal and cell surface proteins were used for quantitative analysis of these proteins by the immunofluorescence test in samples of human sperm of good and poor qualities. RESULTS: The detection of intra-acrosomal proteins was decreased and, on the other hand, detection of surface proteins was the same or higher in pathological spermatozoa. CONCLUSIONS: Monoclonal antibodies can be used for diagnostics of sperm pathology (quantitative detection of proteins) and for evaluation of the physiological state of sperm cells (state of acrosome before or after acrosome reaction). Finally, monoclonal antibodies could be useful for selection of a suitable method of fertilization (IUI, standard IVF, ICSI) in the laboratories of assisted reproduction. PMID- 10750295 TI - [Levels of peripheral circulating nucleated erythrocytes in pregnant women for noninvasive prenatal diagnosis]. AB - OBJECTIVE: Enrichment of nucleated red blood cells (NRBCs) from maternal blood for non-invasive prenatal diagnosis. DESIGN: Pilot study. SETTING: 2nd Clinic of Paediatrics, University Hospital Motol, Prague, Czech Republic. METHODS: Mononuclear cells were isolated from 13-28 ml of peripheral maternal blood between 13 and 37 weeks of gestation. Leukocytes from maternal peripheral blood were depleted from mononuclear cells by treatment with anti-CD14 and anti-CD45 microbeads and high-gradient magnetic cell separation (MACS) on VarioMACS. NRBCs were sorted from CD14-/CD45- fraction by positive selection using anti-CD71 microbeads on MiniMACS. All sorting steps were analysed by three-colour cytometric analysis with FACScan flow cytometer. RESULTS: In 68 out of 78 pregnant woman (87%) NRBCs were found in range 2 x 10(5) - 1.02 x 10(6). NRBC were enriched with an average enrichment rate of 138-fold ranging from 4-526 fold. In our cohort of pregnant woman the number of isolated NRBCs was individual. We identified NRBCs from the 13th week of gestation. CONCLUSION: The aim of the study is to establish and standardise the method of enrichment of NRBCs from maternal blood samples and verify the applicability of this alternative source for non-invasive prenatal diagnosis. PMID- 10750296 TI - [Prenatal diagnosis in families with cytochrome C oxidase disorder]. AB - OBJECTIVE: Cytochrome c oxidase (COX) deficiency presents with severe impairment of brain, muscle or heart. Prenatal diagnosis in affected families is difficult because the disease may be caused by mutations in nuclear or mtDNA. This study shows the results of prenatal diagnosis in two families where the first child died because of a generalised COX defect. In both cases the low activity of COX was accompanied by a low content of the enzyme. SUBJECTS: In the first family the amniocentesis was performed during the second pregnancy and cultured amniocytes showed a marked decrease of COX activity and ATP production. Based on decision of the parents the pregnancy was terminated. Analysis of the foetal tissues confirmed a generalised COX defect. In the second family the nuclear origin of the COX defect was found using transmitochondrial cybrids derived from COX deficient fibroblasts of the affected child. In the successive pregnancy with dizygotic twins a combined amniocentesis and chorionic villi biopsy has been performed. Prenatal diagnosis was based in both foetuses on three independent approaches. COX activity, the ATP production and protein content of COX complex was measured in cultivated foetal cells. The results of all investigations excluded a putative COX defect and both children are healthy at the age of 2 and half years. CONCLUSION: Prenatal diagnosis of COX disorders is available in families with the generalised form of the disease based on a nuclear origin of COX deficiency. Three independent approaches to characterise COX at a functional, enzymatic and protein level may be used. PMID- 10750297 TI - [Paracervical anesthesia in hysteroscopy and transcervical surgery]. AB - OBJECTIVE: To evaluate the efficacity of local-paracervical anaesthesia in hysteroscopy and transcervical surgery. DESIGN: Prospective study. SETTING: Department of Obstetrics and Gynaecology, 1st medical Faculty, Charles University, Prague. METHODS: In 1998 we performed 144 hysteroscopic procedures under local anaesthesia--paracervical block. In 47 (32.6%) cases we performed transcervical surgery. As anaesthetic agent we used bupivacain (0.25%, Marcain, Astra) combined with an intravenous sedative--midazolam (Dormicum, Hoffman LaRoche). RESULTS: In 130 (90.2%) cases patients evaluated the procedure as comfortable, in 7 (4.86%) cases the procedure caused discomfort and in 7 (4.86%) cases we used general anaesthesia. CONCLUSION: Paracervical anaesthesia is a safe method for hysteroscopy and transcervical surgery due to its minimal invasivity. PMID- 10750299 TI - [Personal experience with laparoscopic treatment of sterility]. AB - OBJECTIVE: Evaluation of laparoscopy in treatment of sterility. DESIGN: Retrospective clinical study. SETTING: Department of Gynaecology and Obstetrics, Hospital in Bruntal, Czech Republic. METHODS: In 1997 and 1998 we performed 36 laparoscopic operations in patients with primary or secondary sterility at our department. In women with anovulatory cycles we performed drilling of the ovaries. Adhesions both in and beyond the pelvis were treated by lysis of adhesions, in women under 35 with mild or moderate hydrosalpinx we performed terminal salpingostomy while large hydrosalpinges were treated with salpingectomy and the patients were recommended for assisted reproduction. Success of the treatment was evaluated at least half a year after laparoscopy. RESULTS: In the anovulatory group, 60% conceived on average 2 months after operation. 36.4% conceived in the group with pelvic adhesions, on average in 5 months and 50% in the salpingostomy group conceived in 8 months. CONCLUSIONS: Although the number of patients is small, the obtained results prove the usefulness of operative laparoscopy in the treatment algorithm of sterile patients. PMID- 10750298 TI - [Congenital vaginal aplasia: laparoscopic reconstruction of a neovagina using the Vecchietti technique in the Mayer-von Rokitansky-Kuester-Hauser syndrome]. AB - OBJECTIVE: Creation of a neovagina in patients with congenital vaginal aplasia and evaluation of laparoscopic approach. DESIGN: Pilot prospective. SETTING: Department of Gynaecology and Minimally Invasive Surgery Hospital Na Homolce. METHODS: Four patients with the above mentioned syndrome were included and the laparoscopic modification of the open Vecchietti's technique was used. All patients had a complete clinical examination and ultrasound evaluation. RESULTS: No complications were found. The mean duration of surgery was 96 min. After two weeks a satisfactory vaginal length was achieved in all patients. SUMMARY: The laparoscopic approach has all advantages of minimally invasive surgery. At the same time the advantages of Vecchietti's open technique are maintained. PMID- 10750300 TI - [Cervical carcinoma during pregnancy]. AB - OBJECTIVE: To evaluate management and outcome of pregnant women with cervical carcinoma. DESIGN: Retrospective clinical study. SETTING: Institute for Mother and Child Care, Prague, Czech Republic. METHODS: Clinical data of pregnant women suffering from cervical carcinoma were summarized and assessed. The primary treatment was radical hysterectomy and pelvic lymphadenectomy including the foetus in utero in the first and early second trimester and caesarean section and radical surgery after viability of the foetus has been reached. Patients were assessed with regard to the stage of the disease and the stage of pregnancy. RESULTS: In this retrospective study we discuss 8 cases of cervical cancer diagnosed during pregnancy. According to the TNM classification 3 patients were in stage Ib1, 3 patients in stage Ib2, one patient in stage IIa and one patient in stage IIb. With the exception of one patient in stage IIb all cases were operable with no complication after radical surgery. After a follow-up of at least 12 months we are convinced that pregnancy has no negative effect on the prognosis of cervical cancer. PMID- 10750301 TI - [Disorders of thyroid function and fertility disorders]. AB - The treatment of young infertile women and women with menstrual cycle dysfunction by thyroid gland extracts and later by pure thyroid hormones led to improvement of their menstrual cycle and frequently also to desired conception. Therefore a functional connection between thyroid and ovary, i.e. an effect of thyroid hormones on ovarian reproductive function was assumed. However, till recently exact scientific evidence did not exist, which could explain the functional connection between these two endocrine glands. The scientific discoveries of the last years in the area of human reproduction (which continues in experimental work on the superfamily of steroid/thyroid receptors, which have been coded by protoonkogen c-erb-A variants), presented proof of the presence of thyroid stimulating hormone (TSH) as well as of T3-receptors in ovary and thus way of direct TSH- and T3-effect on steroidogenesis and oocyte maturation. The authors present scientific discoveries from recent years, which seem to be crucial for the explanation of the thyroid-ovary relation and present the treatment algorithm for practical clinical application of experimental knowledge of human reproduction. PMID- 10750302 TI - [Malignant gynecologic neoplasms in the Czech Republic 1986-1995]. AB - OBJECTIVE: The incidence and mortality of malignant tumours of the uterine cervix, uterine body and ovary are analysed. DESIGN: Retrospective study. SETTING: Department of Obstetrics and Gynecology, Medical Faculty and Faculty Hospital, Charles University, Plzen. METHODS: Analysis of relevant dates from the National Oncological Registry 1986-1995. RESULTS: The incidence of the cervical carcinoma oscilated around 22 (22.7-21.6), the mortality slightly decreased (9.6 8.4). The incidence of the corpus carcinoma rose again (25.9-28.9), its mortality declined (7.4-6.9). The incidence of malignant ovarian tumours significantly increased (16.3-23.4), the mortality became slightly higher (14.8-16.2). The incidence of the ovarian carcinoma got ahead the incidence of the cervical carcinoma (23.4:21.6) and it occupied the second place after the corpus carcinoma. CONCLUSIONS: It is necessary to screen systematically gynecological malignant tumours and concentrate its treatment to specialized centers. Correct reports of these tumours and their precise staging is needed, too. PMID- 10750303 TI - [Statistics for physicians--part I]. AB - Authors present a series of papers describing basic statistical methods used in biomedical research and publications. Part one deals with parameters of description statistics. PMID- 10750304 TI - Long-term survivors of heart transplantation: the Hartford Hospital experience. AB - Heart transplantation has been a clinical program at Hartford Hospital for the past 15 years, resulting in 206 transplants. The five-year survival rate is 69% and is 43% at 10 years. The first recipient is surviving and has had full rehabilitation. Thirteen patients have survived 10 years or more. Advances in immunosuppression are ongoing and will result in further long-term survivors. Graft vasculopathy and lack of organ donation are current problems. PMID- 10750305 TI - An uncommon presentation of a common disease: the importance of the history in medicine. AB - A 32-year-old recent Russian immigrant, mother of a 20-month-old son, presented with right hip pain. She had a history of peptic ulcer disease and a positive Helicobacter pylori serology. Her pain was not relieved by analgesics. Spine and pelvic films were unremarkable. A bone scan was consistent with metastatic disease. She underwent several diagnostic tests including computed tomography of the chest and abdomen, magnetic resonance imaging of the spine, mammogram, and breast ultrasound. A bone marrow biopsy revealed adenocarcinoma, primary site unknown. An upper endoscopy performed eight weeks after her initial presentation showed an ulcerating gastric carcinoma. She was treated with chemotherapy but died two months after diagnosis. Our patient had an uncommon presentation of a common disease. Recognizing her country of origin, and other risk factors, may have facilitated an earlier diagnosis. PMID- 10750306 TI - Recurrent upper gastrointestinal bleeding due to the Dieulafoy's lesion. AB - Dieulafoy's lesion of the gastrointestinal tract, an abnormally dilated artery that penetrates through the mucosa, has been diagnosed more frequently in recent years. Bleeding occurs when the vessel ruptures. Despite widespread awareness of this entity, it remains a diagnostic challenge for gastroenterologists because of its small size and hidden location. Several different diagnostic methods and treatments have been introduced throughout the years. Endoscopy plays a major role in diagnosis and therapy. In this case report, the patient was successfully treated endoscopically using epinephrine injection and heater probe thermoco agulation. The characteristics of the Dieulafoy's lesion, its current diagnosis, treatment, and complications are discussed. PMID- 10750307 TI - A patient's good humored views. PMID- 10750308 TI - Re: "The relationship of the general practitioner to the hospital staff". PMID- 10750309 TI - Experts recommend minimum nurse staffing standards for nursing facilities in the United States. AB - The experts concluded that current data show that the average nurse staffing levels (for RNs, LVN/LPNs, and NAs) in nursing homes are too low in some facilities to provide high quality of care. Caregiving, the central feature of a nursing home, needs to be improved to ensure high quality of care to residents. Because detailed time studies have not been conducted on the amount of time that is required to provide high quality of care to residents, expert opinion is currently the best approach to addressing the problem of inadequate staffing. Increases in the education level and training of nursing staff are also strongly recommended as a step to improving quality of care and reducing turnover rates in nursing homes. These recommendations are designed for consideration by Congress, HCFA regulators, policymakers, nursing home administrators, and nurses. Ideally, Congress would pass legislation establishing these recommendations as minimum standards for all nursing homes or direct HCFA to establish detailed minimum nurse staffing standards to ensure that staffing levels take into account the number and the case-mix of the residents. Alternatively, HCFA could introduce minimum staffing standards through the regulatory process. In 1999 there were a number of efforts at the state level to increase minimum staffing levels. Mohler (1999) surveyed selected states and found that 21 states had either proposed new legislation or were considering proposals for new legislation or new regulations. In California, for example, in 1999 the state budget approved $31 million in new state funds (to be matched with $31 million in federal Medicaid dollars) to increase nursing home staffing minimum requirements from 2.8 to 3.2 hr per resident day and to increase wage rates. Overall, nursing facilities need to be held accountable by HCFA for providing adequate levels and types of staffing to meet the needs of their residents, especially because government is paying for 61% of the expenditures. Adopting these minimum standards will have an important impact on improving the quality of the nation's nursing home care. Additional research is needed to determine appropriate levels and types of staff to provide high quality of care to residents. These studies could test the proposed staffing standards against existing staffing levels to examine the impacts on quality. As new data become available on staffing levels, revisions of staffing standards should be made if necessary to ensure that high standards are maintained. PMID- 10750310 TI - Older people and voting participation: past and future. AB - Contemporary politicians and their advisors focus on older voters as a pivotal segment of the American electorate. Some analysts predict that this preoccupation will intensify in the years ahead and the demands of older persons will dominate American politics. One reason for this focus on older voters is that they constitute a substantial proportion of voters today, largely because of age-group differences in voting turnout rates, and they will be a considerably larger proportion in the future because of the aging of the baby boom cohort. This article examines the voting participation of age groups in past presidential elections and explores what the voting participation of older persons could be like when the baby boom cohort reaches old age. The political significance of older persons being a large percentage of voters is considered with respect to both the past and the future. PMID- 10750311 TI - Specialized dementia programs in residential care settings. AB - The authors conducted a telephone survey in 7 states to determine the prevalence of residential care specialized dementia programs (RC-SDPs) and to identify a sample of homes (n = 56) for more detailed study. The 56 homes were site visited, and data were gathered on facility administration, therapeutic environment, and characteristics of 259 randomly selected residents. Comparison data from 138 nursing home Special Care Units (NH-SCUs) and 1,340 of their residents were obtained from 4 studies conducted in the same 7 states. RC-SDPs were smaller, provided a more homelike environment, and had a higher proportion of residents paying privately, compared with NH-SCUs. Mean levels of cognitive and physical impairment among residents were higher in NH-SCUs; prevalences of psychotropic medication use and problem behaviors were similar. Among RC facilities, small homes were more homelike, provided fewer structured activities, and charged less than larger facilities. RC-SDPs include 5 types: small, independently operated homes; multiple small homes with joint administration; larger, all-dementia facilities; SDPs operated within larger, exclusively RC facilities; and RC-SDPs in multilevel facilities. PMID- 10750312 TI - Anticipating response to predictive genetic testing for Alzheimer's disease: a survey of first-degree relatives. AB - Two hundred and three children and siblings of patients with Alzheimer's disease (AD) (age range: 30-92 years, 75% female) were surveyed regarding potential predictive testing options for the disorder. A mailed questionnaire posed various hypothetical scenarios and assessed the following variables: interest in testing, perceptions of its pros and cons, and psychological and demographic predictors of test intentions. In 5 of 6 scenarios, a majority of participants expressed intentions to pursue testing, with perceived pros outweighing cons. The most important reasons for seeking testing were informing later-life decisions and planning future AD care. Predictors of test intentions were male gender, information-seeking style, higher perceived AD threat, and appraisal of test pros versus cons. Situational factors such as available treatment options and certainty of test information also affected responses. Results suggest a positive view of predictive testing, with its limitations and risks underrated. Study findings may inform AD genetic counseling and health education efforts. PMID- 10750313 TI - Coping with disease-related stressors in Parkinson's disease. AB - This study examines three categories of disease-related stressors (i.e., physical, cognitive, psychosocial), differential coping, and mental and physical health outcomes in individuals with Parkinson's disease (PD). Findings show that tremors, lack of mental energy, and being dependent on others were the most stressful symptoms in each category. Although there was evidence that PD patients match coping efforts to types of stressors, emotional regulation was the most common coping strategy. Hierarchical regression analyses show that disease related variables influence every domain of quality of life, whereas the effects of coping are more selective. Specifically, the use of distancing was related to poorer mental and physical health outcomes. Findings focus attention on the disease-related stressors that create the experience of being chronically ill. PMID- 10750314 TI - Unbalanced social exchanges and living arrangement transitions among older adults. AB - Using an elaborated person-environment perspective, this research clarifies the role unbalanced social exchanges play in older adults' living arrangement transitions. Data from the National Survey of Families and Households (NSFH) are used to estimate destination-specific hazard models that include measures of baseline living arrangements, demographic characteristics, financial resources, family structure, and social exchanges. Distinctions are made between transitions that occur within the community and transitions into an institution or death. The results indicate that unbalanced exchange relationships are an integral part of the living arrangement transition process in later life. PMID- 10750315 TI - Young and middle-aged adults' perceptions of elder abuse. AB - Middle-aged and young adults (ns = 201 and 422, respectively) completed an adaptation of the Severity of Violence Against Women Scale and the Elder Abuse Attitudes and Behavioral Intentions Scale--Revised to examine the impact of (a) respondent age, (b) age and gender of perpetrator and victim, and (c) history of experienced violence on perceptions of elder abuse. Results suggested that middle aged respondents viewed psychological behaviors more harshly than did younger respondents and that both middle-aged women and young men were less tolerant of middle-aged perpetrators. Although history of participatory violence toward older persons was predictive of perceptions of elder abuse as it interacted with respondent age, history of experienced abuse was not predictive. These data support a view of elder abuse that emphasizes its relativistic nature, wherein perceptions of elder abuse depend on both the characteristics of the perceiver and the victim and perpetrator variables. PMID- 10750317 TI - Gerontology education in transition: considering disciplinary and paradigmatic evolution. AB - The disciplinary status of gerontology has long been the subject of much debate, but recent changes in higher education suggest the early development of a more integrated, interdisciplinary approach to gerontological education and research. We assert that gerontology is currently a multidisciplinary field of inquiry, but at selected schools it is moving toward an interdisciplinary field and may ultimately evolve into a new discipline. The risks and benefits attendant with this disciplinary evolution are reviewed in light of the historical development of other disciplines. Factors that may accelerate the development of interdisciplinary gerontology education include paradigm development in gerontology and the success of gerontology PhD programs, especially the placement of their graduates. PMID- 10750316 TI - Nutritional self-management of elderly widows in rural communities. AB - Studies of the nutritional status of older adults (by marital status) and of older women recently widowed suggest that widows are nutritionally vulnerable. Yet few studies have examined nutrition-related behaviors among widows to see why this is true. We conceptualize these behaviors as nutritional self-management strategies, encompassing behaviors related to obtaining food, consuming it, and maintaining food security. Data come from in-depth interviews conducted with 64 widowed women age 70+ in rural North Carolina (23 African American, 24 European American, 17 Native American). Transcripts were coded and analyzed using a systematic text-analysis procedure. Length of widowhood ranged from less than 1 year to 39 years. Themes identified in recent widows' interviews and corroborated in those widowed longer indicate that there are varied responses to widowhood. Some may have a positive impact on nutritional strategies (e.g., following own dietary needs), but most are likely to be negative (e.g., meal skipping, reduced home food production, less dietary variety). Rural communities need to develop ways to identify such widows and assist them in finding acceptable ways to meet nutritional needs. PMID- 10750318 TI - Montessori-based activities for long-term care residents with advanced dementia: effects on engagement and affect. AB - Sixteen residents in long-term care with advanced dementia (14 women; average age = 88) showed significantly more constructive engagement (defined as motor or verbal behaviors in response to an activity), less passive engagement (defined as passively observing an activity), and more pleasure while participating in Montessori-based programming than in regularly scheduled activities programming. Principles of Montessori-based programming, along with examples of such programming, are presented. Implications of the study and methods for expanding the use of Montessori-based dementia programming are discussed. PMID- 10750319 TI - The role of adversity and stress in psychopathology: some evidence and its implications for theory and research. AB - Three lines of research--studies of extreme situations, epidemiological investigations of relations between socioeconomic status (SES) and psychiatric disorders, and a quasi-experimental test of the social causation-social selection issue raised by the epidemiological findings--provide strong evidence that environmental adversity is important in the occurrence not only of post-traumatic stress disorder (PTSD) but also of other types of psychopathology, including major depression, alcoholism, substance use disorders, antisocial personality disorder, and nonspecific distress. Leads from this triad of studies are developed into a basic proposition about the nature of the role of adversity and stress. The core of this proposition is that the likelihood of onset of the above types of disorder increases with two factors: (1) the proportion of the individual's usual activities in which uncontrollable negative changes take place following a major negative event; and (2) how central the uncontrollable changes are to the individual's important goals and values. The role of environmental adversity in bringing about these uncontrollable changes varies with gender, ethnic/racial status, and SES in our own and other modern, urban societies. The types of psychopathology that develop and their course vary with both the types of adversity (e.g., whether life threat is involved) and with the personal predispositions of the individuals who experience the adversity and stress (e.g., family history of particular types of psychopathology). PMID- 10750320 TI - Education and the activation, course, and management of anger. AB - Using data from the 1996 General Social Survey, I examine education's association with the activation, course, and management of anger. I argue that education--as a source of stratification (status) and as a personal resource (human capital)- organizes the conditions that influence anger-related processes. In analyses of anger activation, education is associated with lower odds of family-related anger. The well educated have fewer children and more income--factors associated with a lower risk of family anger. Conversely, education is associated with higher odds of work-related anger, but income and personal control account for that association. In analyses of the course of anger, I document a nonlinear association between education and anger duration. Adjustment for the sense of control--which is negatively associated with anger duration--sharpens that parabolic association. Education is positively associated with perceived appropriateness of anger and negatively associated with the display of anger. In both cases, adjustment for control accounts for education's effect. The sense of control also suppresses education's significant positive effect on anger processing. In analyses of anger management, education increases the odds of cognitive flexibility and problem solving, but its effect on communication depends on the sense of control. In sum, education organizes personal and social circumstances that influence anger-related processes. PMID- 10750321 TI - Marijuana use and depression. AB - The primary goal of this study is to examine the association between marijuana use and adult depressive symptomatology. The key independent variables examined are age of marijuana initiation, frequency of current marijuana use, the use of other licit and illicit drugs, and whether marijuana was used to cope with problems. The relationships among these variables are assessed using data from the Young Men and Drugs Survey (n = 1,941), a nationally representative sample of men from the 1944-1954 birth cohort. Results show that early marijuana initiation appears to be weakly associated with increased depression in adulthood. This effect, however, is mediated by educational attainment, employment status, marital status, and other drug use, notably alcohol and tobacco use. Adult frequency of marijuana use is not significantly associated with increased depression in adulthood. Finally, marijuana users who use the drug to cope with problems are more depressed than those who do not use to cope with problems. PMID- 10750322 TI - The dimensionality of stigma: a comparison of its impact on the self of persons with HIV/AIDS and cancer. AB - Does the impact of stigma on the self differ by illness type? This study focuses on a comparison of the effects of the stigma associated with HIV/AIDS and cancer on self-esteem, body image, and personal control. We test the hypothesis that individuals' perceptions of stigma account for significant differences in the impact of an illness on the self. We examine four dimensions of perceived stigma: social rejection, internalized shame, social isolation, and financial insecurity. In turn, we consider how these dimensions medicate the effects of HIV/AIDS and cancer. Our sample includes 130 persons with HIV/AIDS and 76 persons with cancer. We control for illness severity by including a measure of functional health status that is based on participants' subjective perspectives of the severity of their symptomatology. Our findings provide additional support for modified labeling theory; however, our findings also point to the dimensionality of stigma and its differential negative impact on particular elements of the self, regardless of illness type. PMID- 10750323 TI - Deinstitutionalization, social rejection, and the self-esteem of former mental patients. AB - Modified labeling theorists have long argued that the stigma of mental illness has important consequences for the lives of people with mental illness. We propose that social rejection is an enduring force in the lives of people with mental illness and that these experiences are central to understanding the poor self-concepts described by many former psychiatric patients. We explore changes in a cohort of recently deinstitutionalized mental patients' (N = 88) self-esteem and experiences with social rejection using data from a three wave panel survey conducted while institutionalized and over a two-year period following the patients' discharge from a long-term state hospital. Our results indicate that social rejection is a persistent source of social stress for the discharged patients. Moreover, these experiences increase feelings of self-deprecation that, in turn, weaken their sense of mastery. Where the patients' received their follow up care--whether in a community setting or in another state hospital--had little impact on their self-related feelings or on their experiences of social rejection. Our results provide further support for modified labeling theory and underscore the need to consider the dynamic relationship between stigmatizing experiences and self-related changes. PMID- 10750324 TI - Social roles as process: caregiving careers and women's health. AB - Is involvement in multiple roles beneficial for women's health or do the often noted health benefits of multiple roles reflect an ongoing process of role management? We address this question by looking at two roles, caregiving and employment, and by investigating changes in women's health as they move into and out of both roles. We examine changes in physical health limitations and psychological distress over a two-year period with data from a nationally representative sample of 2,929 late-midlife women. Looking first at health changes associated with caregiving, we find that psychological distress increases as women move into and continue caring for an ill or disabled person in their household. Caregiving has a weaker effect on physical health, but increases in physical limitations prompt exists from caregiving. Increases in physical limitations also appear to be greater for non-employed women, but some or all of this difference reflects selection out of the labor force for women having difficulty combining both roles. Our findings provide further evidence that care work has implications for women's health, while also suggesting a need for further attention to the ways that women actively manage problematic role combinations. PMID- 10750325 TI - Converging health inequalities in later life--an artifact of mortality selection. AB - An emergent issue in the health inequalities debate is how socioeconomic status (SES) and physical health relate over the life course. Many studies indicate that the SES-health relationship diminishes in later life. The present research tests the hypothesis that this convergence in health inequalities is an artifact of mortality selection, which biases downwards the "true" association between SES and health in later life. By including respondents who had subsequently died or were loss-to-followup into the analysis, I assess the sensitivity of the age specific association between education and health to sample selection processes. I study U.S. adults followed for approximately ten years using the NHANES I Epidemiologic Followup Study. Results based on the surviving sample are robust to the inclusion of people selected out of the sample due to mortality or attrition. Sample selection biases do not appear to explain the convergence in health inequalities in late life. PMID- 10750326 TI - The history and development of CBF measurements. PMID- 10750327 TI - Clinical application of cerebrovascular reserve assessment as a strategy for stroke prevention. AB - Patients diagnosed as having symptomatic carotid occlusion, who are at increased risk for stroke, can be readily identified by methods designed to measure cerebrovascular reserves. This paper reviews the use of xenon-enhanced computed tomography (Xe/CT) cerebral blood flow (CBF) methods for quantitatively assessing cerebrovascular reserves. PMID- 10750328 TI - The influence of the input function on quantitative rCBF by the Xe/CT method. AB - Measurements of rCBF by the Xe/CT method are based on the assumption of identity between the end-tidal xenon curve which is applied as input function, and the arterial xenon curve being the true input function to the brain. In this study corresponding end-tidal and arterial xenon curves were measured in an experimental animal model (part 1) and in 5 patients with traumatic brain injury (part 2) and used for rCBF calculation. In both studies rCBF was underestimated by using the end-tidal xenon concentration curve as brain input function. In part 1 rCBF underestimation was depended on pulmonary gas exchange; high or low levels of rCBF; tissue type; and xenon inhalation protocols. In part 2 the mean rCBF underestimation was 18.8 +/- 8.3%. In conclusion, non-invasive estimate of the input function should be considered as a source of error when defining quantitative blood flow values e.g. the flow thresholds of ischaemic infarction. PMID- 10750329 TI - Current status and future challenges in cerebral blood flow mapping in intracranial tumors. AB - CBF studies have been applied in patients with cranial tumors using XenonCT in order to evaluate the blood flow of both the tumor and the peritumoral brain tissue. The histology included mostly meningiomas or gliomas, less frequently neurinomas or metastases. Blood flow was determined in tumor tissue (TBF), peritumoral areas (PTBF) and cerebral hemispheres (hCBF). The CBF was compared to normative CBF values. In supratentorial tumors characteristical blood flow patterns were found according to the histology. PTBF was usually lower in edematous tissue than in normal brain tissue. TBF in malignant gliomas revealed a heterogeneous pattern with a central core of low flow and a peripheral zone with high flow. Global CBF values in patients with malignant gliomas were lower than in controls. Meningiomas showed significantly higher blood flow compared to gliomas or neurinomas. In some cases with large cerebellopontine angle tumors a reduction in brain stem blood flow was detected. In cavernous sinus tumors with carotid artery compression CBF and cerebrovascular reserve capacity was not impaired. These findings indicate that tumor induced alterations in cerebral circulation can be detected by CBF mapping. This diagnostic tool offers a further insight into perfusion related tumor pathophysiology and may thereby optimize individual neurooncological treatment strategies. PMID- 10750331 TI - Reliability and error analysis on xenon/CT CBF. AB - This article provides a quantitative error analysis of a simulation model of xenon/CT CBF in order to investigate the behavior and effect of different types of errors such as CT noise, motion artifacts, lower percentage of xenon supply, lower tissue enhancements, etc. A mathematical model is built to simulate these errors. By adjusting the initial parameters of the simulation model, we can scale the Gaussian noise, control the percentage of xenon supply, and change the tissue enhancement with different kVp settings. The motion artifact will be treated separately by geometrically shifting the sequential CT images. The input function is chosen from an end-tidal xenon curve of a practical study. Four kinds of cerebral blood flow, 10, 20, 50, and 80 cc/100 g/min, are examined under different error environments and the corresponding CT images are generated following the currently popular timing protocol. The simulated studies will be fed to a regular xenon/CT CBF system for calculation and evaluation. A quantitative comparison is given to reveal the behavior and effect of individual error resources. Mixed error testing is also provided to inspect the combination effect of errors. The experiment shows that CT noise is still a major error resource. The motion artifact affects the CBF results more geometrically than quantitatively. Lower xenon supply has a lesser effect on the results, but will reduce the signal/noise ratio. The lower xenon enhancement will lower the flow values in all areas of brain. PMID- 10750330 TI - Why emergency XeCT-CBF should become routine in acute ischemic stroke before thrombolytic therapy. AB - Intravenous thrombolytic therapy using recombinant tissue plasminogen activator (rtpa) has been approved for the treatment of acute ischemic stroke in the USA, if treatment is initiated within 3-hours (NINDS tpa Stroke Study Group) but not 6 hours (ECASS II) after time of onset. Favorable outcome in the placebo arm was much higher than expected possibly because patients with TIA's are likely to be included as progressive ischemic stroke subjects when a brief 3-6 hours duration of stroke is defined as the therapeutic window. Yonas' group at the University of Pittsburg demonstrated that adding stable xenon inhalation to routine CT scanning performed during emergency screening of acute stroke, predicted which cases became irreversibly infarcted if thrombolytic therapy was not administered within a few hours of stroke onset, since non-contrasted CT scans are usually normal this early. Adding a few minutes for inhalation of 26% xenon is justified in order to measure LCBF values which predict size, severity and volumes of impending cerebral infarctions and rule out TIA's which have relatively normal CT CBF values. CT-CBF measures provide positive indications for thrombolytic therapy. This is not possible by MRI and SPECT methods which are not sufficiently quantitative to discern LCBF values persistently below ischemic thresholds of 16 mls/100 gm/min, thereby predicting impending infarction. PMID- 10750332 TI - Analysis of xenon CT data with a personal computer. AB - We transferred stable xenon cerebral blood flow (CBF) data to a personal computer by a software of the digital imaging and communications in medicine (DICOM) output, and handled raw data with the NIH image software. CBF values were able to calculate with the NIH image accurately. This system made a precise color image and correct grey scale image submit to a magazine. DICOM output liberates us from a limit of time and place to analyze xenon CT data. PMID- 10750333 TI - Effect of hematocrit on calculation of cerebral blood flow and lambda in xenon CT. AB - Knowledge of the patient's hematocrit is necessary for calculation of cerebral blood flow (CBF) with xenon CT, and is a potential source of error. This study quantifies the effect of hematocrit on the calculation of CBF and lambda, and determines if the magnitude of the effect is dependent on actual CBF or lambda. The effect of hematocrit was measured empirically using software employing the numerical calculation method. CBF and lambda were found to be inversely related to hematocrit. The percent error produced by an inaccurate hematocrit is greater for lower actual CBF values. PMID- 10750334 TI - Effect of the end-tidal xenon correction method on cerebral blood flow determination. AB - The objective of this work is to show how the end-tidal xenon correction method contributes to determining the cerebral blood flow (CBF) values. Respiratory xenon data from 443 patients (mean age, 59.1 +/- 13.5) were analyzed using two methods: the conventional fitting method and the end-tidal correction method. For both methods, xenon saturation rate constant (Ka1) and desaturation rate constant (Ka2) were calculated for washin and washout phases respectively. By applying the correction method, both the histograms for Ka1 and Ka2 change significantly; they become to have narrower distribution with smaller mean value. As the average effect based on computer simulation, if calculated CBF values using corrected Ka1 and Ka2 are 80 cc/100 g-tissue/min for the gray matter and 20 for the white matter, those using conventional Ka1 and Ka2 become 62.8 for the gray matter and 17.9 for the white matter. By applying the end-tidal correction method, obtained CBF values increase, and the extent of increase is larger when xenon inhalation speed is slower. PMID- 10750335 TI - Effects of premedication with oral hydroxyzine on patient motion during inhalation of 32% xenon for regional cerebral blood flow mapping. AB - Because of its anesthetic properties, inhalation of 30-35% Xenon is associated with uncontrolled patient motion in 3-15% of the cases. This constitutes a major setback to regional cerebral blood flow studies with Xenon-enhanced computed tomography (Xe-CT CBF). The present study attempted to determine the effects of oral premedication with hydroxyzine (H) in the control of motion. Patients scheduled for Xe-CT CBF, aged 20-55 years, were randomly allocated to 3 groups: H 50 mg (n = 41), H 100 mg (n = 36) or Placebo (n = 43). The drugs were administered orally 90 minutes before Xenon inhalation. This consisted a gas mixture of 32% Xe and 25% oxygen. Motion was classified as controlled or uncontrolled depending on whether CBF data acquisition was possible or not. Anxiolysis and sedation were evaluated by a visual analogue scale. Motion was significantly reduced in the H 50 mg (0.8% vs 2.5% in the H 100 mg and 6.7% in the Placebo group). An anxiolytic effect of hydroxyzine was suggested. PMID- 10750336 TI - CBF determination in brain stem and cerebellar hemispheres. AB - CBF of brain stem and cerebellar hemispheres is of particular significance in different cranial diseases and can be determined by PET, SPECT and XenonCT. We have used XenonCT CBF method to study 78 patients with posterior fossa tumors or cerebrovascular disorders. A retrospective analysis of the first 56 CBF studies showed that bone artifacts were the most common cause of study failure. Based on these experiences we have developed a protocol to study brain stem and cerebellar blood flow. Because bone artifacts significantly degrade flow information, we obtained images over the petrous bone with a CT gantry angle of approximately 25 degrees to the orbitomeatal line. The failure rate could be reduced from 41% to 13% with this study protocol. Brain stem blood flow determination may help to clarify the prognosis in head trauma patients and show more physiological details in tumors of the posterior fossa. The phenomenon of crossed cerebellar diaschisis in cerebral insults or neoplasms can be investigated by XenonCT easily. PMID- 10750337 TI - Diagnostic value of perfusion MRI in classifying stroke. AB - Our study was designed to determine whether supplementary information obtained with perfusion MRI can enhance accuracy. We used delayed perfusion, as represented by time to peak map on perfusion MRI, to classify strokes in 39 patients. Strokes were classified as hemodynamic if delayed perfusion extended to a whole territory of the occluded arterial trunk; as embolic if delayed perfusion was absent or restricted to infarcts; as arteriosclerotic if infarcts were small, multiple, and located mainly in the basal ganglias; or as unclassified if the pathophysiology was unclear. We compared these findings with vascular lesions on cerebral angiography, neurological signs, infarction on MRI, ischemia on xenon enhanced CT (Xe/CT) and collateral pathway development. Delayed perfusion clearly indicated the area of arterial occlusion. Strokes were classified as hemodynamic in 13 patients, embolic in 14 patients, arteriosclerotic in 6 patients and unclassified in 6 patients. Hemodynamic infarcts were seen only in deep white matter areas such as the centrum semiovale or corona radiata, whereas embolic infarcts were in the cortex, cortex and subjacent white matter, and lenticulo striatum. Embolic and arteriosclerotic infarcts occurred even in hemo-dynamically compromised hemispheres. Our findings indicate that perfusion MRI, in association with a detailed analysis of T2-weighted MRI of cerebral infarcts in the axial and coronal planes, can accurately classify stroke as hemodynamic, embolic or arteriosclerotic. PMID- 10750338 TI - Cerebral blood flow in volunteers measured by PET and Xe CT/CBF. A comparison. AB - Aim of this study was to compare two quantitative CBF methods. Seven young, healthy volunteers were studied with PET (15-0 labelled water) and afterwards with Xe CT/CBF (30% xenon in oxygen, 3 minutes wash-in, 5 minutes washout protocol). Xe CT/CBF showed greater differences between high and low flow areas than PET CBF. Correlation was found within subjects between ROI's, but no agreement or correlation between the methods could be demonstrated. The disagreement in this study could be due to changes in PCO2. PMID- 10750339 TI - Application of laser Doppler flowmetry to measure cerebral microvascular perfusion in the fetal sheep. AB - Laser Doppler flowmetry (LDF) has been used to measure flow in various organs of the adult, but has not been applied to the mammalian fetus. The purpose of this study was to apply LDF to measure cerebral blood flow of the fetal sheep and to assess the possible errors and artifacts of the method caused by myometrial, fetal, and maternal movements. By three days after probe placement, the flow signal had decreased 55% from initial post surgical readings and thereafter it became stable. During fetal hypoxia, the signal increased 48% and during hypercarbia it increased 59%. After fetal death, the signal decreased to 48% of control level. After maternal death, it decreased to 9% and electrical zero could not be reached. LDF is useful to measure changes of fetal cerebral microvascular perfusion because it can provide continuous signals but care is required in data handling and probe fixation when used for the mammalian fetus. PMID- 10750340 TI - Changes in cerebral blood oxygenation induced by deep brain stimulation: study by near-infrared spectroscopy (NIRS). AB - Previous studies have demonstrated that neural activation causes changes in cerebral blood oxygenation (CBO), i.e. increases in tissue levels of oxy-Hb and total-Hb with a decrease in deoxy-Hb concentration. It is unclear, however, whether neural activation always induces the same pattern of CBO changes or not. In the present study, employing a near-infrared spectroscopy (NIRS), we investigated the CBO changes in the frontal lobe induced by direct stimulation of the thalamus (Vim) or globus pallidus (GPi) in patients with Parkinson's disease or essential tremor. The results indicated that under conditions of neural activation in the frontal lobe, oxy-Hb and total-Hb increased in all 6 cases. Deoxy-Hb decreased in 2 cases during GPi stimulation, and increased in 4 cases during low frequency stimulation of the Vim. The above findings suggest that neural activation induces various patterns of CBO change, especially in deoxy-Hb. This implies that functional MRI based on the BOLD contrast may not consistently detect the area of neural activation. PMID- 10750341 TI - Cerebral blood flow on xenon CT: correlation with the blood flow detected at the common carotid artery on ultrasonography. AB - To correlate cerebral blood flow (CBF) on xenon CT with the flow at common carotid artery (CCA) detected by color doppler ultrasonography, 82 patients (29 men, 53 women; 20-90 yrs) were examined. They included normal volunteers (n = 33), patients with cerebral infarction (n = 8), multiple lacunar infarcts (n = 12), dementia (n = 14), and parkinson disease (n = 15). Flow at the CCA was graded as extremely low (< 0.3 l/min), low (0.3-0.4), and normal (> 0.4). CBF was measured in the following distribution: anterior, middle, posterior cerebral arteries (ACA, MCA, PCA); white matter border zones (BZ); basal ganglia (BA), thalamus in two slices. CBF may be reduced in the BZ, cortical and deep gray matter with extremely low flow at CCA. We suggest that color doppler ultrasonography may aid in triage of patients for further CBF evaluation. As some overlap in CBF exists between normal and diseased groups with respect to low flow at CCA, color doppler ultrasonography must be evaluated in combination with xenon CT to reflect cerebral blood flow. PMID- 10750342 TI - Cerebral blood flow, vascular response and metabolism in patients with MELAS syndrome--xenon CT and PET study. AB - Two patients with MELAS syndrome underwent serial measurement of cerebral blood flow (CBF) with xenon CT while they were presenting stroke like episodes accompanying cerebral lesion detectable with CT. One of them underwent PET measurement of regional cerebral metabolic rate of oxygen (CMRO2) and glucose (CMRGlu) after his symptoms and lesion disappeared. METHODS: The xenon CT CBF study was performed by 4 min wash-in and 3 min wash-out protocol with serial measurement of endexpiratory concentration of xenon gas. The CBF after acetazolamide loading was also quantified in one of them. The PET study was performed to quantify CBF, CMRO2, oxygen extraction fraction (OEF) and cerebral blood volume (CBV) by continuous inhalation of O-15 labeled gases and arterial blood sampling. The PET measurement of CMRGlu was performed by i.v. injection of F-18 FDG and arterial blood sampling. RESULTS: 1) During the symptomatic period, Xe-CBF was normal or slightly increased both in and outside the low density lesion. 2) The CBF response to acetazolamide loading was well preserved both in and outside the low density lesions. 3) After the neurological symptoms and low density lesions disappeared, Xe-CBF pattern and vascular response was the same as during the symptomatic period. 4) In the PET study, normal or slightly increased PET-CBF, increased CMRGlu and markedly decreased CMRO2 in comparison to normal control was noted resulting in a marked decrease in OEF and CMRO2/CMRGlu ratio, a characteristic metabolic pattern for MELAS. CONCLUSION: In the present cases, resting CBF and vasomotor reactivity was well preserved both in symptomatic and remission period. On the contrary, abnormal metabolic pattern was noted. The stroke like episode of the present patients is more likely attributed to metabolic failure than vascular accident. PMID- 10750343 TI - Recovery of decreased local cerebral blood flow detected by the xenon/CT CBF method in a patient with eclampsia. AB - A 23-year-old woman presented in our hospital with toxemia, underwent cesarean section at about 36 weeks gestation, and became eclamptic in the immediate postpartum period. Following a complex partial seizure a few hours after the cesarean section, the patient experienced drowsiness, then cortical blindness. Cranial computed tomography (CT) performed at about 24 hours after the onset of the seizure showed low density areas in the bilateral occipital lobes. Intravenous magnesium sulfate was given, and the neurological symptoms disappeared within three weeks. Xenon/CT cerebral blood flow (CBF) was measured during the acute and chronic stages of the patient's eclampsia and compared with cranial magnetic resonance imaging (MRI) performed at about the same time. In the acute stage, MRI showed abnormal T2-hyperintensity signals in the head of the left caudate nucleus and in the bilateral occipital lobes, predominantly in the white matter. Xenon/CT CBF measurement showed decreased local cerebral blood flow (LCBF) in the area of the left anterior cerebral artery (ACA), the bilateral posterior cerebral arteries (PCAs), and the watershed areas of the left hemisphere. In the chronic stage, abnormal T2-hyperintensity signals remained in that part of the left occipital lobe where, in the acute stage, a marked decrease in LCBF had been detected. The main mechanism of eclampsia in this patient is thought to be a reactive vasoconstriction against hypertension rather than a vasodilatation. PMID- 10750344 TI - Regional cerebral blood flow after status epilepticus. AB - Two patients with status epilepticus due to specific conditions were examined using MRI and stable Xe/CT CBF. [Case 1] A 30-year-old woman developed a grand mal seizure during delivery. She was comatose, and MRI revealed abnormal high intensity areas bilateral basal ganglia, compatible with eclampsia. Regional CBF was decreased in bilateral occipital lobes and right basal ganglia. Six days after onset. Regional gray matter flow was increased, especially in the thalami and basal ganglia. [Case 2] The patient is a 31-year-old male diagnosed with temporal lobe epilepsy since 10 years. At the onset, he had a prolonged right hemiconvulsion followed by generalized tonic-clonic convulsion. MRI 13 days after onset showed left hemispheric edematous swelling of gray matter. Stable Xe/CT 3 weeks after onset demonstrated increased cortical CBF corresponding to edematous area. The results suggested that regional CBF decreased immediately after status epilepticus and then increased for 1-3 weeks in the interictal period. We speculate that the energy debt incurred during prolonged seizure causes relative ischemic condition in the neurons, with the increase in CBF resulting from accelerated energy production for a long period. PMID- 10750345 TI - A profound hemispheric hypoperfusion with relatively small infarcts indicates a progressing stroke. AB - We determined clinical features of patients who had a profound hemispheric hypoperfusion with relatively small, acute cerebral infarcts. One hundred and thirty-five patients with acute cerebral infarction underwent both magnetic resonance imaging (MRI) and cerebral blood flow (CBF) measurement in the acute phase of stroke. Eleven (8.1%) had a profound hemispheric hypoperfusion with relatively small infarcts. In these patients, magnetic resonance angiography or conventional angiography was performed, demonstrating the internal carotid artery (ICA) stenosis or occlusion on the ipsilateral side of the infarcts. MRI and CBF measurement were repeated one month later. All of 11 patients suffered from a mild consciousness disturbance, and showed a progress of neurological symptoms during a few days, indicative of a progressing stroke. Five patients of 11 concomitantly had atherosclerotic lesions in the arteries distal to the circle of Willis. The remaining six patients had only the ICA lesions. The prognosis of the former was poor and the hemispheric hypoperfusion pattern did not improved. On the other hand, the prognosis of the latter was good and the hemispheric hypoperfusion was recovered soon. In conclusion, if the collateral flow through the circle of Willis could compensate the misery perfusion, the prognosis of low flow infarctions with ipsilateral ICA lesions might be good. PMID- 10750346 TI - Changes in cerebral blood flow after carotid endarterectomy. AB - We evaluated changes in cerebral blood flow (CBF) after carotid endarterectomy (CEA) in patients with internal carotid (ICA) stenosis. We studied 46 patients with ICA stenosis who underwent CEA. The mean age of the patients was 63 years, and their mean ICA stenosis was 73%. CBF in the middle cerebral artery territory was measured with xenon-enhanced CT tomography (Xe-CT) before and 3 weeks after CEA. In addition, cerebrovascular reactivity (CVR) was measured after intravenous administration of acetazolamide (ACZ) in 16 patients. There was no significant relationship between the degree of stenosis and CBF. Ten patients had decreased CBF before CEA, and CBF improved in nine of these after CEA. The CVR in 6 of 7 patients with impaired CVR before CEA improved to varying degrees after CEA. The CBF in patients with ICA stenosis varied according to the degree of collateral circulation. In conclusion, CEA can increase CBF and improve CVR in patients with low CBF or low CVR by restoring blood flow through the ICA. PMID- 10750347 TI - Usefulness of hemodynamic evaluation in patients with major cerebral arterial occlusive disease before cardiac surgery. AB - Patients with ischemic heart disease are often complicated with cerebrovascular disease. The purpose of this study is to examine the usefulness of Xe-CT CBF study in patients with cerebral arterial occlusive disease before cardiac surgery with cardiopulmonary bypass. This study was carried out in 11 patients suffered from ischemic heart disease with cerebrovascular diseases. They had severe stenoses or occlusions of cerebral arteries. Cerebral hemodynamics was measured by Xe-CT. There were no ischemic complications in the brain or heart during the study. Hemispheric CBF in the occlusive side is lower than that in the non occlusive side. Cerebral ischemic events occurred in one patient after the cardiac surgery. Xe-CT CBF study can be performed safely in patients with ischemic heart disease. The patients with low CBF and low cerebrovascular reserve, had a greater risk of cerebral complication after cardiac surgery with cardiopulmonary bypass. PMID- 10750348 TI - The preoperative and postoperative cerebral blood flow and vasoreactivity in childhood moyamoya disease. AB - PURPOSE: To report the hemodynamic pattern in childhood moyamoya disease before and after vascular reconstruction. METHODS: We performed the xenon cerebral blood flow (CBF) study in 10 cases of moyamoya disease in last 3 year. In 6 of them, study before and after operation was obtained for comparison. Preoperative study was obtained in 2 cases and postoperative study was obtained in 2 cases. They were 5 males and 5 females with age ranging from 1 to 12 years old. We studied their regional CBF before and after acetazolamide (CCBF) using stable xenon computed tomography. The angiography study was obtained for comparison. RESULT: In the routine study, the CBF and CCBF were poor in the ACA and MCA territory and well correlated to the angiography finding. However, in case of basal ganglion and posterior cerebral artery involvement, xenon CBF study was more sensitive than the angiography. On the postoperative cases, the CBF could increase to a level beyond the level of CCBF shown on the preoperative study. CONCLUSION: Hemodynamic study using stable xenon computed tomography can provide an objective reference for the treatment of moyamoya disease especially in case that surgical management is considered. It is a better modality for the evaluation of surgical outcome than the conventional contrast angiography study. PMID- 10750349 TI - Local cerebral hemodynamic changes through the angiographic stages of moyamoya disease. AB - In order to elucidate the cerebral hemodynamic changes that occur in Suzuki's six angiographic stages of moyamoya disease, local cerebral blood flow (LCBF) during the stable state and CO2 responsiveness of LCBF (L-CO2R: delta %LCBF/delta PaCO2) were measured by the Xenon CT-CBF method. Nineteen patients with moyamoya disease (mean age: 36.8 +/- 11.6 years) and 11 age-matched normal volunteers were studied. The LCBF during the steady state at all stages was not significantly different from that in normal volunteers. At stage 6, however, the LCBF was slightly decreased in the anterior part of the brain, resulting in loss of "hyperfrontality." On the other hand, the L-CO2R in the anterior part of the brain tended to diminish with progression through the stages. Especially in the frontal cortex, the L-CO2R at stage 5 was significantly less than that in normal volunteers (p < 0.01) or at stage 3 (p < 0.05). In conclusion, the cerebrovascular reserve in the anterior circulation became insufficient after stage 4, although the posterior circulation was well maintained. Revascularization surgery involving the anterior circulation may be crucial to prevent ischemic events. PMID- 10750351 TI - Global reduction of cerebral glucose metabolism in persons with symptomatic as well as asymptomatic lacunar infarction. AB - To clarify the hemodynamic changes in lacunar infarction, we evaluated cerebral glucose metabolism by using positron emission tomography with 18F fluorodeoxyglucose (FDG-PET) in patients with asymptomatic and symptomatic lacunar infarction and in persons without cerebral infarction on MRI. Subjects in this study were 27 patients with symptomatic lacunar infarction (SCI group), 73 subjects with asymptomatic lacunar infarction (ACI group), and 134 persons without infarction (NC group). CMRgl in the ACI group was significantly lower than that in the NC group in the cerebral cortex (P < 0.05) and thalamus (P < 0.05). CMRgl in the SCI group was significantly reduced from that in the NC group in the cerebral cortex (P < 0.005), basal ganglia (P < 0.001), thalamus (P < 0.05) and white matter (P < 0.005). The reduction in CMRgl in the SCI group was more severe than that in the ACI group in basal ganglia (P < 0.05) and thalamus (P < 0.05). Our results indicated that glucose metabolism in patients with asymptomatic lacunar infarction is reduced throughout the whole brain as compared with non-infarcted elderly persons. Follow-up and treatment of risk factors if present, may be necessary in such patients. PMID- 10750350 TI - XE-CT CBF changes during normative aging, cognitive decline and dementia. AB - Factors accelerating cerebral degenerative changes represent potentially modifiable risks for cognitive decline. Putative risk factors accelerating mild cognitive decline and dementia were correlated with repeated measures of cerebral atrophy, CT densitometry, perfusions and cognitive testing among neurologically and cognitively normative volunteers. TIAs, hypertension, smoking and male gender accelerate cerebral degenerative changes, mild cognitive decline and dementia. Intervention by control of risk factors and cholinesterase inhibitors should prevent cerebral atropho-degenerative changes so that optimal cognitive performance is maintained. PMID- 10750352 TI - Distinguishing patients with senile dementia of Alzheimer type and normal elderly subjects utilizing xenon CT-CBF and multivariate analysis. AB - Local cerebral blood flow (LCBF) was measured in 17 patients with senile dementia of Alzheimer type (SDAT) and 17 normal controls, utilizing stable xenon computed tomography (Xe CT-CBF). In patients with SDAT, LCBF values were decreased in the cerebral cortex and subcortical structures including the thalamus, basal ganglia and white matter of both hemispheres. Linear discriminant function analysis of LCBF values separated patients with SDAT from normal elderly subjects, with an error of 8.8%. Variables helpful in distinguishing SDAT patients from normal subjects were LCBF values for the frontal and temporal cortex. Multiple regression equation for predicting cognitive performance scores from LCBF values showed the best correlations with LCBF values for the frontal and occipital cortex and thalamus. Xe CT-CBF measurements provide useful information concerning diagnosis and brain function in patients with SDAT. PMID- 10750353 TI - Semi-automatic ROI placement system for analysis of brain PET images based on elastic model: application to diagnosis of Alzheimer's disease. AB - PET with 18F-fluorodeoxyglucose (FDG) is a useful technique to image cerebral glucose metabolism and to detect patients with Alzheimer's disease in the early stage, in which characteristic temporoparietal hypometabolism is visualized. We have developed a new system, in which the standard brain ROI atlas made of networks of segments is elastically transformed to match the subject brain images, so that standard ROIs defined on the segments are placed on the individual brain images and are used to measure radioactivity over each brain region. We applied this methods to Alzheimer's disease. METHODS: This method was applied to the images of 10 normal subjects (ages 55 +/- 12) and 21 patient clinically diagnosed as Alzheimer's disease (age 61 +/- 10). The FDG uptake reflecting glucose metabolism was evaluated with SUV, i.e. decay corrected radioactivity divided by injected dose per body weight in (Bq/ml)/(Bq/g). RESULTS: The system worked all right in every subject including those with extensive hypometabolism. Alzheimer patients showed markedly lower in the parietal cortex (4.0-4.1). When the threshold value of FDG uptake in the parietal lobe was set as 5(Bq/ml)/(Bq/g), we could discriminate the patients with Alzheimer's disease from the normal subjects. The sensitivity was 86% and the specificity was 90%. CONCLUSIONS: This system can assist diagnosis of FDG images and may be useful for treating data of a large number of subjects; e.g. when PET is applied to health screening. PMID- 10750354 TI - Abstinence from drink ameliorated cerebral blood flow and vasoreactivity in patients with chronic alcoholism. AB - High dose ethanol consumption is a risk factor for both ishemic and hemorrhagic cerebrovasucular disease. This link between heavy drinkers and the risk factor of stroke has been considered as hypertension, liver dysfunction, abnormality of platelet function or other unknown mechanisms. Recently some of the experimental study suggest that direct action of ethanol on the inhibition of the synthesis/release of nitric oxide from endothelium and neurons may contribute to this link. Few studies in this field, however, were performed clinically. We examined cerebral blood flow (CBF) and vaso-reactivity in the patients with chronic alcoholism on abstinence from drink. CBF of nine male patients were measured by use of stable Xe-CT method before and after acetazolamide load. Regional CBF increased in second measurement after abstinence, but there were no significant changed statistically. However, %vaso-reactivity in right ACA and MCA significantly improved. We considered that large brain vessels dilated then small vessels could response to acetazolamide. PMID- 10750355 TI - Cerebral circulation and prognosis of the patients with hypoxic encephalopathy. AB - Recent progress in cardiopulmonary resuscitation techniques improved the survival rate of patients with acute cardiopulmonary disturbances. However, severe cerebral complications remained frequently in patients who survived the acute stage. Early prediction of cerebral prognosis is important to optimize the management of these patients. We examined the relations between radiological findings (Xe-CT and MRI) and cerebral prognosis. Patients included in this study were selected from all patients with hypoxic encephalopathy admitted to our hospital. There were 11 men and 10 women. Causes of hypoxic encephalopathy were heart disease (11 cases), suffocation (4 cases), CO intoxication (2 cases), asthma (1 case), pneumothorax (1 case), anaphyraxy shock (1 case) and electric shock (1 case). Xe-CT and MRI were carried out 3 weeks after the onset. Cerebral blood flow (CBF) of the patients was measured at rest and 15 minutes after intravenous administration of acetazolamide (1 g). The prognosis was evaluated 3 months after the onset in accordance with Glasgow Outcome Scale (GOS). Low hemispheric CBF (30 ml/100 g/min), poor reactivity of acetazolamide challenge test (10 ml/100 g/min), presence of hyperintensity areas in the basal ganglia in T1 weighted images (T1WI) and T2 weighted images (T2WI) are the factors associated with poor outcome in hypoxic encephalopathy. PMID- 10750356 TI - Regional cerebral blood flow in the patients with depressive disorders. AB - Regional Cerebral blood flow (rCBF) and metabolism in major depression have been studied intently. Those studies are expected to reveal the mechanisms of the mood disorders, and also to give us valuable informations that the specific brain regions participating mood regulation. However, the data still have been inconsistent. In the present study we measured rCBF in patients with major depression and depressive state using xenon-CT CBF method. There is a tendencies that the rCBF values of normal control is the highest, and those of major depression is the lowest in most of the regions. The rCBF values in the patients with major depression are significantly lower than those in normal control in the right anterior frontal cortex, temporal cortex, putamen and thalamus. We also measured rCBF before and 90 minutes after giving calcium antagonist nilvadipine (4 mg, p.o.) in those subject. There is a tendency that nilvadipine increased in rCBF in normal control, however, it decreased in rCBF in major depression. PMID- 10750357 TI - Benzodiazepine receptor distribution and cerebral blood flow in early blindness- a PET study. AB - We studied benzodiazepine receptor (BZR) distribution, which is thought to be affected by neuronal density in the cerebral cortex, and CBF using [11C]flumazenil and [15O]water PET in early blind (EB) and in blindfold sighted control (SC) subjects. PET images were co-registered to the subject's MRI. Using SPM96, MRI images were normalized in the Talairach and Tournoux coordinate system, and accordingly MRI-registered PET images were spatially normalized. Statistical parametric maps were computed on a voxel-by-voxel basis, using the general linear model. CBF for EB was significantly larger in the Brodmann area 17 and 18, especially anterior area, than that for SC, while there was no significant difference in BZR distribution. Our BZR data suggest that the amount of neurons do not change due to early visual deprivation in the visual cortex, in spite of high CBF in visual cortex of EB subjects. PMID- 10750358 TI - Acetazolamide reactivity in atherothrombotic, cardioembolic and lacunar infarctions. AB - Ten cases of atherothrombotic brain infarction, 10 cases of cardioembolic brain infarction, 10 cases of lacunar brain infarction, 10 cases of transient ischemic attack (TIA) and 10 age-matched controls were studied. The cerebral blood flows in the cerebral cortex and cerebral white matter were significantly lower in the atherothrombotic, cardioembolic and lacunar infarction groups than in the TIA and control groups. The acetazolamide reactivity in the cerebral cortex was significantly lower in the atherothrombotic and lacunar infarction groups than in the cardioembolic, TIA and control groups. The rate of association of hypertension was significantly higher in the atherothrombotic, lacunar and TIA groups than in the cardioembolic and control groups. Plasma fibrinopeptide A, platelet factor 4 and beta-thromboglobulin concentrations were higher in the atherothrombotic, cardioembolic and lacunar groups than in the TIA and control groups. The present study suggests that the degree of thrombolysis and platelet activation is less in TIA than in cerebral infarction and that underlying cerebral arteriosclerosis is more severe in atherothrombotic and lacunar infarction than in cardioembolic infarction. PMID- 10750359 TI - The value of acetazolamide challenge test in the evaluation of acute stroke. AB - The acetazolamide (ACZ) challenge test provides a useful information about compromised hemodynamic state in chronic stroke. However, there is no consensus whether this test is of any value in the evaluation of acute ischemic stroke. The purpose of this study is to examine the value of ACZ challenge test in the management of acute ischemic stroke. Study 1: Nineteen patients with acute embolic stroke were subjected to the Xe CT with and without ACZ (17 mg/kg, i.v.) within 6 hours from the onset. The cases included 12 middle cerebral artery (MCA) occlusions and 7 internal carotid artery (ICA) occlusions. The baseline cerebral blood flow (CBF) values and cerebrovascular reserve (CVR) (% increase in CBF after ACZ) were analyzed in 53 affected regions of interest (ROI). The study indicated that the CBF threshold of subsequent permanent infarction was 15 ml/100 g/min and the ROI with negative CVR had a higher incidence of hemorrhagic infarction. Study 2: Xe-CT with and without ACZ was performed in 32 patients with acute occlusion of the main trunks of cerebral arteries within 6 hours after the onset. Occluded arteries were MCA in 20 patients, ICA in 7, both ICA and MCA in 4 and anterior cerebral artery (ACA) in one. The abnormal hemispheric CBF (< 20 ml/100 g/min) and CVR (< 10%) were correlated with the Glasgow outcome scales of the patients. The predictability of Good Recovery, Moderately Disabled, Severely Disabled, Vegetative Survival and Dead were 80%, 50%, 50%, 100% and 100% by CBF criteria, and 80%, 60%, 80%, 100% and 100% by CVR criteria, respectively. There was no significant increase in the predictability of final outcome of the patients by adding the CVR information of the acute stage. The ACZ challenge test has a potential value in the prediction of hemorrhagic transformation of the ischemic regions. It does not increase the predictability of the long-term outcome. We do not recommend performing ACZ challenge test on routine basis in the evaluation of acute ischemic stroke. PMID- 10750360 TI - Activation of fronto-limbic system in the human brain by cigarette smoking: evaluated by a CBF measurement. AB - Nicotine produces profound behavioral effects in humans, but little is known about the sites of its action. There is a hypothesis that frontal lobe and limbic/cingulate cortical structures might be the sites. In this study, we examined the effects of cigarette smoking on feeling and cerebral blood flow (CBF) in human subjects. Young and healthy 9 cigarette smokers (all males, 24-33 years, average, 26.4) were included. After prohibiting them from smoking for 15 hours, CBF was measured using a Xenon CT-CBF system. Fifteen minutes later after allowing them to smoke two pieces of cigarette, the second CBF measurement was performed. Subtraction CBF map was created to display the changes after smoking. CT images were taken at three levels so as to include the cerebral lobes, basal ganglia, limbic system, brainstem and cerebellum. Arterial nicotine increased up to the levels 8 times higher than before smoking. The increases of blood pressure and pulse rate were minimal. Arterial carbon dioxide level and hematocrit did not change. Feeling after smoking was variable in individual subject. In 8 subjects with a relatively high feeling, CBF increased mainly in the frontal lobe, hippocampus, uncus, thalamus and caudate nucleus. CBF did not change in the parietal, temporal and occipital lobes, and in the putamen, insula, brainstem and cerebellum. In two subjects with uncomfortable feeling, CBF did reduce in the whole brain. The CBF increase in frontal lobe and limbic structures seems to be secondary to nicotine-induced neuronal activation in each structure. Mesocorticolimbic dopamine system, which is believed to influence learning, memory or emotional performance, appears to be a target for nicotine. The CBF reduction in the whole brain might be due to cerebral vasoconstriction or be secondary to a systemic hypotension. PMID- 10750361 TI - Assessment of regional cerebral blood flow by xenon-enhanced computed tomography during mastication in humans. AB - It is suggested that mastication stimulates the brain and accelerates its energy consuming metabolism. This study was designed to determine its effects on regional cerebral blood flow (rCBF) using xenon-enhanced computed tomography (Xe CT). Seven male volunteers, aged 24-57 years, inhaled 30% xenon in a 4 minutes wash-in and 4 minutes wash-out protocol. CT was scanned every 54.5 seconds. The subjects were instructed to chew a gum continuously at a rate of 1 bite per second except at the time of CT scanning (5.5 seconds). A second CBF was done 20 minutes later. Subtraction (mastication-baseline) maps were created. CT images were taken at three levels so as to include the cortex, basal ganglia, limbic system, brainstem and cerebellum. The results demonstrated a significant rCBF increase in the fronto-temporal cortex, caudate nucleus, thalamus and minor increase in the rolandic areas, insula, cingulate and cerebellum. Further studies are needed to validate the clinical significance of these findings. PMID- 10750362 TI - The utility of XeCT cerebral blood flow in the management of acute stroke. AB - The fundamental pathophysiologic event underlying acute ischemic stroke is a reduction in cerebral blood flow (CBF). Over 100 patients with acute stroke have been studied with xenon-enhanced computed tomography (XeCT) during the past three years. A retrospective analysis of these patients has revealed several important observations. XeCT is very sensitive in detecting acute ischemia. The pattern and depth of ischemia can be correlated with the physical examination, CT findings, and angiographic results. Patients with large volumes of severely ischemic tissue are more likely to suffer herniation, hemorrhagic conversion, and death than patients with small volumes of less ischemic tissue. Patients with normal CBF studies are more likely to have their deficits resolved within 24 hours that patients with abnormal CBF maps. It is likely that future prospective studies will better define the precise utility of XeCT in the acute management of cerebral ischemia. PMID- 10750363 TI - Cerebral blood flows in the acute and chronic stages of cerebral infarction. AB - Regional cerebral blood flows (rCBFs) were studied in the acute and the chronic stages of 8 patients with supratentorial cerebral infarction. In 2 patients with cardioembolic infarction, the rCBF increased in one patient and decreased in the other during the chronic stage. This decrease was explained by the risk factors of the latter patient because the patient had more risk factors than the former patient with increased rCBF. In 6 patients with lacunar infarction, the rCBF increased in 2 patients and decreased in 4 during the chronic stage. In all the patients with decreased rCBF, the number of lacunar infarction and/or the degree of leuko-araiosis on MRI increased during the chronic stage. Therefore, the decrease in rCBF during the chronic stage in lacunar infarction is considered to be due to the increase in the number of lacunar infarction, the increased degree of leuko-araiosis or both. PMID- 10750364 TI - Diaschisis and acetazolamide reactivity in brainstem infarction. AB - Regional cerebral blood flows (rCBF) were studied in 6 patients with lateral medullary infarction and 4 patients with pontine infarction, using stable xenon computed tomography method. In lateral medullary infarction, the rCBF and acetazolamide reactivity were decreased in the ipsilateral cerebellum and the rCBF was decreased with normal acetazolamide reactivity in the contralateral frontal lobe in one patient accompanying ipsilateral cerebellar infarction. rCBF was decreased with normal acetazolamide reactivity in the cerebellum in 3 patients, and both rCBF and acetazolamide reactivity were decreased in the whole brain in 2 patients. In pontine infarction, rCBF was decreased in ipsilateral frontal lobe and contralateral cerebellum in one patient, rCBF was decreased in the cerebellum with normal acetazolamide reactivity in the other patients. The hypoperfusion with normal acetazolamide reactivity was considered to be due to diaschisis. The rCBF decrease in the remote areas in patients with brainstem infarction was considered to be due to diaschisis or underlying arteriosclerosis. PMID- 10750365 TI - A specific thrombin inhibitor (argatroban) ameliorated cerebral blood flow in the patients with acute cerebral infarction. AB - Argatroban, one of the arginine derivatives, has been reported to have a safe and potent antithrombin action. This compound is active in several animal models of thrombosis and also has been shown to improve general neurological symptomatology, general subjective symptomatology and general daily behavior in the patients with acute thrombosis. This was considered to reflect remarkable improvement of microcirculation. No published clinical data, however, exist on the effect of argatroban on cerebral blood flow (CBF) change during acute stroke. Three patients with acute cerebral infarction were subjected to this study. Intravenous argatroban injection (2.5 mg/hr) was continued in 48 hours. Regional CBF (rCBF) was measured before and after injection of argatroban using Xe-CT method. Argatroban increased CBF not only in the injured side hemisphere or penumbra, but also contralateral side of lesion in the patients with acute cerebral infarction. PMID- 10750366 TI - The regional cerebral blood flow amelioration of argatroban in the acute stage of cerebral thrombosis. AB - Cerebral blood flow changes by argatroban in the acute stage of cerebral thrombosis have been investigated with the use of stable xenon computed tomography (Xe/CT). The study group consisted of 14 cases (7 males, 7 females) with the average age of 59 years old (ranging from 21 to 79 years of age). We evaluated the cerebral blood flow change after an intravenous drip infusion of 10 mg of argatroban. After the administration of argatroban, we recognized a significant increase in the blood flow (24.4 +/- 5.0 ml/100 g/min to 28.4 +/- 2.7 ml/100 g/min, p < 0.05) in the blood flow area of lower than 30 ml/100 g/min in the affected vascular territory. As a conclusion, we found that argatroban improves ischemic symptoms through the amelioration of blood flow in the penumbra area. PMID- 10750367 TI - Effect of cilostazol on cerebral blood flows in chronic stage of cerebral circulation. AB - In order to find out the difference between cilostazol and ticlopidine hydrochloride in the cerebral vasodilating effect in the chronic stage of cerebral infarction, cerebral blood flows were measured while the patients were on ticlopidine hydrochloride and after ticlopidine hydrochloride was switched to cilostazol. Single photon emission computed tomography (SPECT) was performed using Prism 2000XP gamma camera system. Ultrasound examinations of the carotid artery was performed using Ultramark 9. The blood flows in the frontal white matter, temporal cortex and occipital cortex after cilostazol were significantly higher than those before cilostazol. The peak systolic velocity, time-averaged peak velocity and volume flow after cilostazol were significantly higher than those before cilostazol. The total cholesterol, triglyceride and apolipoprotein B concentrations after cilostazol were significantly lower than those before cilostazol. The present study suggests that cilostazol has better influence on cerebral circulation than ticlopidine hydrochloride in the chronic stage of cerebral infarction. PMID- 10750368 TI - Xenon-enhanced computed tomography in the management of cerebral vasospasm following aneurysmal subarachnoid hemorrhage. AB - At the University of Pittsburgh, xenon-enhanced computed tomography (XeCT) serves numerous critical roles in the management of patients following aneurysmal subarachnoid hemorrhage (SAH). Routine baseline XeCT studies are obtained within 24 hours of surgery and are used for comparison to later studies. In the setting of a delayed neurological deficit, XeCT is used emergently to distinguish vasospasm from other possible causes and to triage patients to appropriate non invasive or invasive therapies. In cases of a delayed neurologic deficit without CBF changes, regardless of transcranial doppler results which can be unreliable, XeCT can prevent unnecessary angiography and potentially harmful invasive treatments. For patients demonstrating territorial ischemia diagnostic of symptomatic vasospasm, XeCT is used to monitor the efficacy of both invasive and non-invasive interventions. This paper summarizes the results and significance of clinical studies of XeCT in the management of patients after SAH at our institution. PMID- 10750369 TI - Correlation between cardiac output and cerebral blood flow following subarachnoid hemorrhage. AB - The authors examined the correlations between cerebral blood flow (CBF) during the stage of vasospasm following subarachnoid hemorrhage and four parameters, namely, cardiac output (cardiac index), mean arterial blood pressure, age, and the Glasgow coma scale score. Forty-two patients who were diagnosed to have subarachnoid hemorrhage were included in this study, and 50 measurements were performed between day 5 and 12 following the subarachnoid hemorrhage. The CBF was measured by stable xenon-enhanced CT and the mean values of four CBF maps were corrected for a PaCO2 of 34 mm Hg (CBF34). The cardiac output and cardiac index were continuously monitored during the CBF measurement. The correlation coefficients of cardiac index, mean arterial blood pressure, age, and GCS against CBF34 were, respectively, 0.436, 0.227, 0.037, and 0.225, and the p values were, respectively, 0.002, 0.074, 0.789, and 0.087. The CBF34 was positively correlated with only the cardiac index and not with any of the other three parameters. Therefore, an increase in the cardiac output is apparently associated with an increase in the CBF during the stage of vasospasm following subarachnoid hemorrhage. Furthermore, we measured the CBF and cerebral perfusion pressure before and after increasing cardiac output in three patients during the stage of vasospasm. The CBF increased by 22.5% +/- 2.9 (SD), with a 42.0% +/- 16.4 increase in the cardiac index, however, no significant change in cerebral perfusion pressure was observed. Therefore, the increase in CBF associated with the increase in cardiac output seems to be attributable to a reduction in the cerebrovascular resistance. PMID- 10750370 TI - A case of subarachnoid hemorrhage presenting with temporary blindness disclosed by Xe CT-CBF study. AB - A 69-year-old male with right oculomotor palsy caused by non-ruptured right internal carotid-posterior communicating artery aneurysm suddenly became blind after cerebral angiography. He lost consensual response of left eye, in spite of no preretinal hemorrhage. Xenon computed tomography(Xe-CT) showed moderate cerebral blood flow(CBF) decrease of bilateral posterior cerebral artery territories at the values of 26.9 ml/100 g/min(left) and 28.3 ml/100 g/min(right), which were about 30% under our control value. However, MR T2 weighted imaging demonstrated no cerebral infarction. Two weeks later, regional CBF values were improved and 6 months later his visual acuity were almost cured. We conclude that Xe-CT CBF study supports the hypothesis of the temporary blindness due to the optic chiasm and nerves injuries by subarachnoid hemorrhage. PMID- 10750371 TI - Evaluation of regional cerebral blood flow in chronic subdural hematoma. AB - To clarify the mechanism responsible for neurological impairment associated with chronic subdural hematoma (CSDH), we performed quantitative measurements of cerebral blood flow (CBF) with xenon-enhanced computed tomographic scans in eight patients with unilateral CSDH. Vascular reserve capacity was also evaluated with acetazolamide challenge. CBF was depressed in all regions examined except the corona radiata. There was no statistical difference in hemispheric and regional CBF between the lesion and non-lesion sides. A significant increase in CBF values ranging from 32% to 69% was observed after acetazolamide administration in the whole brain. Postoperatively CBF remained depressed in all regions we analyzed except for the frontal and temporal lobes, despite the fact that all patients had improved clinical symptoms. Amplitude of N20 and central conduction time (N13-20) in SSEP showed no significant change in CSDH patients compared to normal control. So we conclude that preoperative neurological signs in CSDH are related to a reduction of CBF in the whole brain. However, other mechanisms must be involved to explain preoperative focal signs and good postoperative recovery. PMID- 10750372 TI - Cerebral blood flow measurement of severely head-injured patients during mild hypothermia. AB - In 14 patients with severe head injury, the cerebral blood flow (CBF) during mild hypothermia therapy was measured. Their Glasgow Coma Scale scores on admission were 8 or less and the intracranial pressures were greater than 20 mmHg despite conventional therapy. The CBF was measured with two-level stable xenon CT techniques. And in 11 patients, the cerebral metabolic rates for oxygen (CMRO2) was also calculated. All cases were divided into two groups according to the outcome at discharge by using the Glasgow Outcome Scale, good outcome group in 6 and poor outcome one in 8. The values of mean CBF and CMRO2 of each group were 25.6 +/- 6.6 vs 24.4 +/- 6.4 ml/100 g/min and 1.26 +/- 0.45 vs 0.79 +/- 0.31 ml/100 g/ml, respectively. There were no statistically significant differences between both groups. Single CBF measurement during this therapy may not be helpful as a factor of prognosis evaluation in patients with severe head injury. PMID- 10750373 TI - Effects of 30% stable xenon on regional cerebral blood flow in patients with intracranial pathology. AB - Regional cerebral blood flow was assessed continuously in 22 patients with severe intracranial pathology undergoing xenon-enhanced computed tomography by means of an intraparenchymal thermodiffusion based microprobe. Thirty-four blood flow studies were analysed revealing an overall xenon-induced flow activation from about 12%. Regional CBF rose from 25 +/- 17 ml/100 g/min (mean +/- sd; range: 5.2 41.8 ml/100 g/min) before xenon administration to 28 +/- 21 ml/100 g/min (p = 0.012; range: 6.5-46.4 ml/100 g/min) when "steady-state" during xenon "wash-in" was reached. Flow activation curve demonstrated a logarithmic shape with an increase in rCBF between 3% and 7% within the first 90 seconds of xenon "wash in", 12% after 160 seconds, and showed no further augmentation until the end of the blood flow study after 310 seconds. It is concluded that xenon inhalation leads to flow augmentation in patients with cerebral insult, which does not exceed flow activation obtained in normal subjects. The impact of the results on xenon-enhanced computed tomography cerebral blood flow calculations remains to be established. PMID- 10750374 TI - Blood flow study of meningothelial and fibrous meningiomas by xenon-CT. AB - Cerebral blood flow was studied by xenon-enhanced computed tomography in six patients with incidentally detected intracranial meningiomas. All of the tumors were small (< 32 mm) and there was little or no peritumoral edema. Three patients had meningothelial meningioma and three patients had fibrous meningioma. The tumor blood flow(TBF) and the contralateral tissue blood flow(CLBF) were determined. The ratio of these parameters(TBF/CLBF) was 1.753 +/- 0.467 for meningothelial meningiomas and 0.809 +/- 0.105 for fibrous meningiomas, with a significant difference between the two tumor subtypes (p = 0.0185). There was no correlation between the signal intensity on magnetic resonance imaging and tumor subtype, and the findings on cerebral angiography also did not indicate the subtype. The small meningothelial and fibrous meningiomas with little effect on the surrounding brain tissue could be distinguished from each other by xenon enhanced computed tomography. PMID- 10750375 TI - CD19 and CD22 regulate a B lymphocyte signal transduction pathway that contributes to autoimmunity. AB - The fate of B lymphocytes is dependent on intrinsic and B cell antigen receptor (BCR)-induced signals. These signals are modified and interpreted by other cell surface molecules such as CD19 and CD22 that govern mature B cell activation. This review assesses our current understanding of how CD19 and CD22 regulate B lymphocyte signaling and how alterations in these response-regulators contribute to autoimmunity in mice and humans. We propose that CD19 functions as a specialized adapter protein that regulates B lymphocyte signaling and autoantibody production. Overexpression of CD19 by B cells in systemic sclerosis patients correlates with autoantibody production and transgenic mice that overexpress CD19 produce similar autoantibodies. CD19 establishes a novel Src family kinase activation loop that regulates basal signal transduction thresholds in resting B cells and amplifies Src-family kinase activation following BCR ligation. Reciprocally, CD22 is a potent regulator of CD19 function. These observations provide insight into how CD19 and CD22 govern the molecular ordering and intensity of signals transduced in B cells that may contribute to autoimmunity. PMID- 10750376 TI - Medial trigonal arteriovenous malformations. AB - A retrospective analysis of 48 patients of medial paratrigonal arteriovenous malformations (AVMs) which constituted 18% of the total 258. AVMs surgically managed in our institute for 16 years was carried out to study their clinical presentation, radiological features, operative approaches and functional outcome. While hemorrhage was the initial presenting symptom in 35, it was longstanding headache in 10, focal seizures in two and progressive weakness in one. However, 41 of these presented with bleed at the time of admission to our hospital and in 15 there was history of multiple bleeds. Twelve and eleven patients had field cut and hemiparesis respectively. Arterial supply to the malformation was quite uniform with posterior (43 patients) and anterior cerebral (25 patients) being the most frequent source. Venous drainage was predominantly into the galenic system (70%). Preoperative embolization was carried out in six patients. While the AVM was excised through a parasagittal interhemispheric approach in 34 patients, the nidus was approached through a direct transcortical (superior parietal lobule) approach in fourteen patients. There was only one operative mortality. Twelve patients whose preoperative fields were normal developed field cut postoperatively. An improvement of the preoperative field deficit was noted only in one patient. While eight of the ten patients with preoperative weakness improved and remained independent the remaining two were moderately disabled. Only two of the 10 patients who developed postoperative weakness remained severely disabled. Our operative results suggest that these AVMs which are prone to recurrent bleeds can be resected with acceptable morbidity. Hemianopia resulting from bleed or surgery is unlikely to improve. PMID- 10750377 TI - Update on psoriasis therapy: a perspective from the USA. AB - Because physicians from different nations frequently acquire the use of a new medication at different times, the international exchange of experiences with the new medication is valuable in maximizing its efficacy worldwide. In recent years, many new therapeutic agents have been approved for treating psoriasis in the United States. These include the topical agent calcipotriol and the systemic agents acitretin and cyclosporine. In addition to new agents, a new therapeutic paradigm, sequential therapy, has been introduced recently. It is the hope of the authors that by sharing this paradigm and experiences with these agents in the United States, dermatologists in Japan may gain further insight into optimizing the use of these agents in the treatment of psoriasis. PMID- 10750378 TI - Regulation of cerebral microcirculation--update. AB - The present symposium during Brain 99 was convened to explore the current aspects of the neural (extrinsic and intrinsic) and chemical control of the microvasculature in the brain with specific relevance to stimuli and rapid flow responses. N. Suzuki demonstrated the presence of neurokinin-1 receptors along the axons of vasoactive intestinal polypeptide-containing cerebrovascular parasympathetic nerves. Since the receptors were activated by substance P, calcitonin gene-related peptide and neurokinin released from coexisting sensory nerve fibers, the parasympathetic (vasodilating) fibers could effect rapid local flow increases. N. Suzuki, however, considered this as part of an elaborate defensive network protecting the brain from invasions by noxious substances. E. Hamel discussed the responses of the microvessels to neurotransmitters and suggested that nitric oxide (NO) released from intrinsic neurons may serve as a relay in the flow activation responses by intracerebral cholinergic fibers originating in the basal forebrain nuclei. D. Busija summarized a vasodilating system of activated N-methyl-D-asparate receptors located on neurons involving Ca influx-NO production, and activated ATP-sensitive potassium channels located in the vascular system. According to Busija, such interactions were disrupted during hypoxia and ischemia due to cyclooxygenase-derived superoxide anion. M. Lauritzen observed a 10 times larger increase in blood flow on stimulation of the climbing nerve as compared with that following the parallel nerve stimulation. The former transmitters are considered by him to be NO and K, and the latter NO and adenosine. Each speaker singled out NO as a common mediator for the microvasculature in the rapid local flow increases. PMID- 10750379 TI - A case of aortoduodenal fistula occurring after surgery and radiation for pancreatic cancer. AB - The patient was a 58-year-old woman given curative treatment (pancreatectomy (body and tail) + intraoperative irradiation (25 Gy)) on the basis of a diagnosis of pancreatic carcinoma. Having a favorable postoperative course, she was discharged 24 days after surgery. A week after discharge, she was readmitted for a hemorrhagic gastric ulcer. She was later discharged again on conservative treatment, and followed up at the outpatient clinic, but nine months postoperatively, was readmitted complaining of loss of appetite and abdominal pain. Subsequent tests revealed stricture of the horizontal portion of the duodenum with distension oral to the stricture. Around the celiac artery, the paraaortic lymph nodes were swollen, and a diagnosis of stricture due to recurrent pancreatic carcinoma was made. On the day before bypass surgery was scheduled, the patient vomited blood, so the operation was postponed, conservative treatment such as blood transfusion was administered, and emergency angiography was performed simultaneously. The findings were an aortic pseudoaneurym 1 cm in diameter immediately below the origin of the superior mesenteric artery and between the left and right renal arteries, and a hemorrhage, caused by an aortoduodenal fistula, issuing from the horizontal portion of the duodenum. Hemostasis via a laparotomy was judged difficult, and so an indwelling stent-graft in the aorta was tried to stanch the blood, but without success. Another stent then had to be inserted within the first, thus stopping the flow, but the blood supply to the celiac artery, the superior mesenteric arteries and the renal arteries was impaired, and the patient died about six hours later. Postmortem examination revealed aortoduodenal fistula without recurrence of the carcinoma. The duodenal wall around the fistulous tract showed delayed radiation changes with deep ulceration. The intraoperative radiation may have played an important part in the formation of the fistula. PMID- 10750380 TI - [The inner ear--a "white" spot on the therapeutic map. It's difficult to repair hearing damages so far]. PMID- 10750381 TI - [Risk of hearing damage in connection with pop and rock concerts. The maximum permissible sound level should be legally confirmed]. PMID- 10750382 TI - [Future cure of hearing disorders? Gene therapy and stem cell implantation are possible new therapeutic alternatives]. AB - Hearing loss is a very common disorder; nearly 10 per cent of the population is affected. Recently, a few findings such as the roles of neurotrophins, nitric oxide, reactive oxygen species and glutamate receptors in the peripheral hearing system have been highlighted. In this review, focus is set on possible mechanisms of peripheral hearing disorders, and on recent advances to prevent and treat hearing loss. Clinically useful treatment strategies, especially gene therapy and the use of embryonic stem cells, are particularly stressed. PMID- 10750383 TI - [Diagnosis of Ramsey Hunt syndrome is both simple and difficult. The viral attack is more extensive than expected earlier]. AB - In Ramsay Hunt's syndrome (herpes zoster of the head and neck in combination with facial palsy), the vesicles often appear on the external ear (herpes zoster oticus) but they can also be found on the exterior of the neck. Serologically verified cases without vesicles occur (zoster sine herpeticum). Complications from the eighth cranial nerve (hearing loss and vertigo) are common. MR and PCR studies show a more extensive viral attack than was earlier believed to be the case. Due to the risk of remaining cranial nerve dysfunctions, as exemplified in a case report, antiviral treatment is indicated, in severe cases combined with corticosteroids. The potential value of varicella vaccination to reduce zoster complications is discussed. PMID- 10750384 TI - [Respiratory syncytial virus infections are the most common cause of hospitalization among children. Prophylaxis of high-risk individuals is red but the local epidemiology should be decisive]. PMID- 10750385 TI - [Ophthalmology during the 20th century and in the future. Enormous technical development till now, demands of the population when it comes to good vision will be increasing in the future]. PMID- 10750387 TI - [Quality of life is the most important goal of nutritional support of the dying]. AB - In early palliative stages effective nutrition can improve well-being. In late stages and in dying patients excessive amounts of proteins and lipids may induce nausea and vomiting, due to cachexia and subsequent changes in the metabolism. Excessive hydration may give rise to oedema and dyspnoea. In these late stages the patient rarely feels hungry or thirsty. The goal should therefore be to do good, not to harm and to respect the autonomy of the patient. Thus, the well being of the patient should be in focus: to avoid hunger, thirst, nausea, vomiting, oedemas and dyspnoea. The consequences are that small amounts of carbohydrates and water often constitute the optimum for these patients. PMID- 10750386 TI - [Risky alcohol drinking surveyed at a GP unit. Secondary prevention of alcohol problems in primary care patients]. AB - This article describes an implementation of secondary prevention of alcohol abuse at a GP unit in southern Gothenburg, Sweden. During several periods between 1994 and 1996, screening for alcohol problems was performed using either AUDIT or a 4 item instrument called SWAG. In one part of the study, screening was simultaneously carried out using gamma-GT and MCV. The main object of screening efforts was to stimulate interest for alcohol-related conditions, and this goal was reached. The staff was trained in treatment techniques such as motivational interviewing (MI), bio-feedback using gamma-GT and delivery of concise information. Simple methods to determine level of motivation were used for treatment stratification. Some doctors reported that they had insufficient time for adequate MI treatment, and therefore a condensed model was sometimes used. A nurse-staffed treatment unit was started and successfully promoted work with alcohol problem. Attempts were made to spread these methods to other GP units in the region and this was partially successful, although support from the central primary care administration was not secured. PMID- 10750388 TI - [Pathologists--in short supply in the year of 2000! 200 new specialists must be trained during the next 15 years]. PMID- 10750390 TI - [Can tests like Rorschach be used for more than severe psychopathologic assessment?]. PMID- 10750389 TI - [Projective tests are reliable]. PMID- 10750391 TI - [Reflections on consolation--physicians, too, have the right to grief]. PMID- 10750392 TI - [Two contributions on chronic fatigue syndrome: biological causes are involved in most cases]. PMID- 10750393 TI - [Hazardous to psychiatrize something one can not handle]. PMID- 10750394 TI - [STAR--a method for therapeutic anamnestic reporting]. PMID- 10750395 TI - [The Medical Society must defend the free right of prescription]. PMID- 10750396 TI - For better or worse. PMID- 10750397 TI - Dental economics. Doing better into the second half of the 1990s. PMID- 10750398 TI - Treating chronic pain how do we measure success? AB - In this article, the practitioner is provided with information on common techniques to determine whether a particular treatment for a chronic temporomandibular disorder (TMD) is successful. Although most clinicians rely on patient reports of relief as the primary evidence for success, we have found that these reports are poorly correlated with changes in pain. Reports of relief may reflect other aspects of therapy, such as the patient's relationship with the health care provider or improved ability to cope. Therefore, in order to know if a treatment is truly efficacious, we suggest that the clinician measure the patient's pain before, during and after treatment. Questionnaires designed to measure pain and relief in the clinic, as well as instructions for their use, are included. These can be used to document progress and for medico-legal purposes. PMID- 10750399 TI - A thousand years of dentistry. AB - As we contemplate what the future holds for dentistry, it is helpful to look back at where it's been and how far it has come. PMID- 10750400 TI - [Current pharmacotherapy of Parkinson's disease]. AB - Parkinson's disease (PD) is one of the most common neurodegenerative disorders. The morbidity is about 1-2 of 1000 under 65 years, but it increases to 1-2 of 100 in the population over 65 years. PD became the model of degenerative disorders either concerning the etiopathogenesis or the therapeutic possibilities. Recently the therapy is focused mostly on pharmaceutics, although the treatment of PD is complex. The new drugs improve the quality of life, and there is some evidence that some of them may slow down the progression of the disease. These drugs may provide a neuroprotective therapy, and so a better life expectancy. PMID- 10750401 TI - [Inflammatory markers after surgical treatment of esophageal tumors]. AB - Oesophagectomies carry the risk of postoperative sepsis and mortality. The aim of this study was to evaluate the course of microalbuminuria, serum procalcitonin and C-reactive protein levels following oesophagectomies. Twenty one patients undergoing elective oesophagectomy were studied. Serum procalcitonin and C reactive protein levels were determined on arrival on the intensive care unit (t0) and then daily (t24, t48, t72). Microalbuminuria (expressed as urine albumin:creatinine ratio, mg/mmol) was measured before (tpre), and after surgery (t0, t6, t24, t48, t72). For statistical analysis Wilcoxon test was used. The clinical course of the patients studied was uneventful during the first 72 hours as monitored by daily Multiple Organ Dysfunction Scores. Preoperative microalbuminuria levels were normal (< 10 mg/mmol). Levels at t0 increased significantly but then (t6-24) they returned to normal. Serum procalcitonin (normal: < 0.5 ng/ml) at t0 was slightly elevated and by t24 it increased significantly (median: 2.7 ng/ml, p < 0.05) and remained high for the rest of the study: t48-72. C-reactive protein was normal at t0 (< 10 mg/l) and by t24 it increased dramatically (up to 10-20 times to the normal value) until t48. At t72 it decreased, but still remained in the abnormal range. This study found, that the surgical insult resulted a significant increase in microalbuminuria, serum procalcitonin and C-reactive protein levels. However, the changes were not accompanied by the clinical signs of sepsis or multiple organ dysfunction in the early postoperative period following oesophagectomies. PMID- 10750402 TI - [Adult respiratory distress syndrome after open heart surgery]. AB - Cardiopulmonary bypass used in open heart surgery is responsible for nearly 15% of adult respiratory distress syndrome. The condition has a high mortality rate and still today we do not have a specific therapy for it. The aim of this study was to reveal the factors that are responsible for the adult respiratory distress syndrome developing after open heart operations. A retrospective statistical analysis of the database of 837 consecutive open heart operations was performed. Those patients in whom the adult respiratory distress syndrome has not developed have served as control group. chi 2 probe, Student t test and the Mann-Whitney test were used for the analysis. The authors applied logistic regression analysis for the multivariate investigation. Adult respiratory distress syndrome has developed in 10 patients (1.2%) in the postoperative period. One patient in whom the adult respiratory distress syndrome was accompanied by multiorgan failure has been lost. Between the two subgroups the authors found significant difference in the amount of blood and fresh frozen plasma transfusion, in the duration of ischaemic period and cardiopulmonary bypass, in anaesthesia time, and also in the occurrence of postoperative low cardiac output syndrome and acute myocardial infarction. Fresh frozen plasma can be a risk factor in the development of postoperative adult respiratory distress syndrome. In order to avoid the occurrence of this syndrome a great emphasis must be put on the pulmonary prevention. PMID- 10750404 TI - [Professor Dr.Ern"o Emil Moravcsik--at the 75th anniversary of his death]. PMID- 10750403 TI - [Simultaneous leptospirosis and hantavirus infection in the same patient]. AB - The leptospirosis and the hantaviral infections are worldwide distributed zoonoses having the similar epidemiology and clinical symptoms. Both in Europe and Asia those hantaviral serotypes are common which are responsible for the haemorrhagic fever with renal syndrome, while on the American continent the hantaviral pulmonary syndrome has also been diagnosed. Authors describe a patient who had simultaneous leptospira and hantaviral infections with haemorrhagic fever as well as with mild, transient renal insufficiency and liver damage. The dual infection was proved by serology. PMID- 10750406 TI - [The Rockefeller Foundation for the public health in Hungary. 1924]. PMID- 10750405 TI - [Nikolai Sergeevich Korotkov (1874-1920)]. PMID- 10750407 TI - Research and development. PMID- 10750408 TI - A case-control study of primary caesarean section at the Port Moresby General Hospital, Papua New Guinea, to identify epidemiological predictors of abdominal delivery. AB - A retrospective study of 274 consecutive primary caesarean sections and 274 unmatched controls was carried out at Port Moresby General Hospital from January to December 1992. The primary caesarean section rate was 3.5%. Stepwise logistic regression analysis showed that primary caesarean section was significantly associated with maternal height of less than 150 cm; nulliparity; symphysis fundal height of more than 38 cm at admission in labour; cervical dilatation of less than 4 cm at admission in labour; and the level of fetal head at admission in labour of 3/5 or higher. PMID- 10750409 TI - A retrospective survey of patients with one previous caesarean section delivered at the Port Moresby General Hospital: a comparative study of those delivered vaginally and those delivered by repeat caesarean section. AB - We studied 510 patients in a retrospective, nonrandomized, comparative survey of vaginal births and repeat caesarean section after one primary caesarean section at the Port Moresby General Hospital. 478 (94%) were allowed a trial of scar (TOS). The most common indications for elective caesarean section in the other 32 patients were cephalopelvic disproportion (CPD) 31%, contracted pelvis 19% and preeclampsia 12.5%. In 41% of patients TOS was terminated by emergency caesarean section. Logistic regression analysis showed that the following were significantly associated with repeat caesarean section after TOS: parity of one, no vaginal birth after the primary caesarean section, narrow obstetric conjugate, birthweight of 2500 g or greater, short stature, high level of the head at admission to the labour ward and region of origin. PMID- 10750411 TI - Familial ovarian cancer: report of ovarian carcinoma in three sisters. AB - This is a report of ovarian carcinoma occurring in two sisters diagnosed almost at the same time, prompting prophylactic oophorectomy in a third sister. Histology of the overtly normal ovary in the third sister showed a focus of ovarian cancer. Discussion and a review of the literature suggest that any program designed to reduce the incidence of late-stage ovarian carcinoma should include the surveillance of family members of the index case, including the performance of prophylactic oophorectomy in the unaffected members of the family after they have completed their families. PMID- 10750410 TI - A case-control study of singleton low birthweight babies at the Port Moresby General Hospital. AB - A retrospective study of 432 consecutive singleton low birthweight babies and 432 unmatched controls was carried out at the Port Moresby General Hospital from January to December 1988. Of the 432 low birthweight babies 65% were preterm, 27% were light for gestational age, 6% were both preterm and light for gestational age and 2.5% could not be classified. The results of the analysis showed low birthweight to be significantly associated with the past delivery of a low birthweight infant, very young and elderly mothers, lack of antenatal care, poor family planning, hypertensive disease in pregnancy and intrauterine death. This study reveals that maternal education and improved antenatal care and family planning would ultimately reduce the incidence of low birthweight babies and perinatal mortality in Papua New Guinea. PMID- 10750412 TI - Oral health status of students at the age of 12-15 years in southern Highlands Province of Papua New Guinea: results of a descriptive epidemiological study. AB - A sample of 555 students from three geographically and socioculturally different areas within the Southern Highlands Province, between the ages of 12 and 15 years, was examined in June 1995 to assess the oral health status for planning, monitoring and evaluation purposes. The prevalence of dental caries was found to be 57%, with a DMFT (decayed, missing and filled teeth) score of 1.70 (+/- 2.25) with DT, MT and FT scores of 1.47, 0.12 and 0.10 respectively. 54% of students had active caries and 27% had a DMFT score more than 3. Gingivitis was prevalent among students and visible calculus was present in 35% of them. Only 29% were dentally fit and did not need treatment. About 3% needed some form of orthodontic treatment, and endodontic and cosmetic treatment were respectively needed in 3.5% and 2% of the children. PMID- 10750413 TI - Operation open heart 1995: lessons learned and thoughts for the future. PMID- 10750414 TI - Kawasaki disease in a Papua New Guinean child: the first recorded case. PMID- 10750415 TI - Hypokalaemic paralysis associated with renal tubular acidosis. PMID- 10750416 TI - [Physician-patient relations in tuberculosis]. AB - The "rating design" procedure was first used to examine 168 patients with tuberculosis. The image of an ideal doctor as perceived by patients was studied and it was compared with the traits of a practitioner by sex, age, residence, marital status, social class, disease duration, hospital stay, etc. It is shown that there is a great discrepancy of these images, which requires psychohygienic work with phthisiologists in order to create partner relations during treatment. PMID- 10750417 TI - [Experience in organizing ambulatory control of patients with sarcoidosis in Moscow]. AB - Dispensary record group VIII for patients with sarcoidosis was introduced in Moscow in 1994 and a center based on the Moscow Research Production Center?? was set up to diagnose and treat such patients. In addition to advisory work, the sarcoidosis center performs organizational and methodological functions. A scheme of subgroup (A, B, and C) distribution of Group III patients by the activity and pattern of sarcoidosis has been developed to have unique follow-up methods. The diagnosis made in the patients and their dispensary registration, the efficiency of treatment, and their movement by subgroups are checked up. This system of a center-dispensary interaction revealed a dynamics in sarcoidosis incidence and prevalence in the city in 1994-1997. Evidence is provided for that it is expedient and timely to organize a follow-up of patients with sarcoidosis particularly when the share of paid medical services is increasing. PMID- 10750418 TI - [Concept of rendering antituberculosis care to the population of Sverdlovsk Region under present socioeconomic conditions]. AB - The regional concept of rendering antituberculosis care to the population includes: the creation of a legal basis by passing a law on tuberculosis, special planning of tuberculosis control measures by adopting a special programme, an interdepartmental approach to rendering antituberculosis care to the population by setting up interdepartmental commission on tuberculosis control, computer aided tuberculosis monitoring, activities in the prevention and detection of tuberculosis mainly in high-risk groups, the activities of phthisiological facilities by the developed standards and regulations, and the centralization of the antituberculosis drug supply system. PMID- 10750419 TI - [Characteristics of patient contingents at G.A.Zakhar'in tuberculosis Moscow clinical hospital No.3]. AB - The paper analyzes patient contingents at one of the largest tuberculosis hospitals in Russia in 1995 to 1998. Ti has been ascertained that there has been an increase in the number of patients transferred from general hospitals from 9.7 to 27.4%. Patients with caseous pneumonia and intrathoracic lymph node tuberculosis have doubled. PMID- 10750420 TI - [Efficiency of tuberculosis control measures and their impact on epidemiological situation in Armenia in 1987 and 1997]. AB - Examining epidemiological and qualitative data on the prevention, detection, and treatment tuberculosis in Armenia in 1987 and 1997 revealed 58.6 and 24.0%, rises in tuberculosis incidence and prevalence, respectively; its mortality has increased by 2.5 times. The qualitative indices have become worse: the proportion of destructive pulmonary tuberculosis has risen from 58.0 to 62.4%, cavernous closure has decreased from 78.0 to 57.8%, and cessation of bacterial isolation lowered from 85.0 to 74.5%. Social and organizational reasons of the above worse indices are presented. A package of tuberculosis control measures, which has been adjusted to the present conditions, is recommended. PMID- 10750421 TI - [Intravenous laser radiation treatment of acute and progressive forms of tuberculosis in teenagers]. AB - In 25 of 44 teenagers suffering from acute and progressive tuberculosis, intravenous blood laser radiation was included into its multimodality treatment following 2-4 weeks of the initiation of chemotherapy. The use of laser enhanced the efficiency of treatment, accelerated positive changes by 1.5-2 months by major clinical and laboratory indices, made the disease run smoothly and caused less pronounced residual changes in the lung. PMID- 10750422 TI - [Use of perfluorane in the transfusion therapy of tuberculosis and other bronchopulmonary diseases]. AB - The paper presents the first experience with the Russian blood substitute Perfluorane at pulmonectomy and pleuropulmonectomy in patients with pulmonary tuberculosis. In the patients operated on, the use of PF during simultaneous administration of red blood cells ensures stable hemodynamics, better blood rheological properties and red blood cell metabolic characteristics. Hemostatic events developed in none of 5 cases. PF used in a patient with severe chronic respiratory failures provided a beneficial clinical effect, improved the rheological properties and bioenergy of red blood cells; however, the latter showed an increase in lipid peroxidation. PMID- 10750423 TI - [Stepwise biopsy in diagnosis of bronchial tuberculosis]. AB - Stepwise forceps biopsy was used in 29 patients with different forms of pulmonary tuberculosis, which may ascertain the pattern of bronchial inflammation and the pathogenetic mechanisms of extension of an abnormal process to the bronchial wall, which is important in determining treatment policy. A specific process was found to spread to the bronchial wall from the focus of lesion in the lung tissue itself in a bronchogenic fashion in most (62%) cases and from the bronchopulmonary lymph nodes by contact (38%). There are also data on the impact of controlled short-term chemotherapy in 24 patients with tuberculosis and specific bronchial lesion. The controlled short-term chemotherapy has been found to be effective in slower abacillation rates. PMID- 10750424 TI - [Use of antibiotics in chronic bronchitis]. AB - Whether it is advisable to use tarivide, a wide-spectrum acting antibiotic, in 193 patients aged 23 to 50 years who had chronic catarrhal bronchitis. The experimental group included 43 and 57 patients with chronic nonobstructive and obstructive bronchitis, respectively. For comparison, a matched group receiving penicillin comprised 40 and 53 patients, respectively. Tarivide eliminated most strains of pathogens and improves cellular immunity. Cure and improvement were achieved in 147 (77%) and 13 (12%) patients, respectively. The favourable pharmacokinetics of tarivide, its easy-to-use regimen make it the drug of choice for oral treatment. PMID- 10750425 TI - [Pulmonary and cardiac functions in patients with pulmonary tuberculosis]. AB - Four hundred and nineteen elderly and old patients with pulmonary tuberculosis were examined. One hundred and ninety six patients were found to have an active tuberculous process and contaminant diseases: essential hypertension in 20.7%, functional classes II-III angina pectoris in 55.6%, postinfarct cardiosclerosis in 23.7%. Hypertrophy of the right cardiac cavities was frequently accompanied with that of the left ones (38.1%). Respiratory diseases caused by comorbidity were prevalent among acid-alkali balance disorders. Metabolic disturbances were detected in 90 (21%) patients. Evaluation of pulmonary and cardiac functions by the analysis of acid-alkali balance, gases, blood, and ECG in geriatric patients allows pathogenetic therapy to be timely and soundly. PMID- 10750426 TI - [Pulmonary function changes induced by bronchial asthma in children, teenagers and adults]. AB - The study has established that pulmonary dysfunctions in bronchial asthma are less marked in children and teenagers than in adults. The magnitude of pulmonary dysfunctions are chiefly determined by the severity of bronchial asthma. There was a less close relationship of pulmonary function to the duration of bronchial asthma. There was no association of the severity of pulmonary dysfunctions with the duration of bronchial asthma. PMID- 10750427 TI - [Specific features of immunity in patients with different forms of pulmonary tuberculosis]. AB - The cellular immunity was studied in 59 patients with pulmonary tuberculosis. The development of tuberculous infection was ascertained to be accompanied by decreases in the relative counts of CD9, CD8, and CD72 lymphocytes, as well as monocytes, expressing class II histocompatibility antigens (DR). The patients with tuberculosis were found to have suppressed proliferative T-cell activity and IL-2 production, moderately decreased IL-1 production and increased TNF alpha secretion. PMID- 10750429 TI - [Mechanisms of endotoxicosis in pulmonary tuberculosis]. AB - The intensity of a specific inflammatory process in pulmonary tuberculosis causes an increase the body's tissue proteolytic activity, which is followed by an elevation the blood levels of the toxic products of incomplete proteolysis (medium molecular-weight peptides) and induces the endogenous intoxication syndrome. The severity of endotoxicosis is also characterized by less life of paramecia and higher leukocytic intoxication index. PMID- 10750428 TI - [Molecular mechanisms of mycobacterium tuberculosis strains resistance to rifampicin and isoniazid]. AB - The levels and spectra of the drug resistance of clinical M. tuberculosis strains were defined. There was a relationship of treatment regimens to the drug resistance of mycobacteria isolated from the strains. The fragments of genes rpoB, inhA, and katG were analyzed by polymerase chain reaction and sequencing. In addition to earlier identified substitutions, new mutations were found in rpoB: A-->T/233, G-->A/395, C-->T/232, G-->T/202, C-->T/221, C-->T/260, GA- >TT/202-203, delta 199-207 ATGGACCAG. Strain 12/7 was found to have 30 point mutations leading to substitution of only 3 amino acids and to have GGG(Gly)354 deletion as well. Most mutations in this strain are "silent". Substitutions at 944 and 463 positions were revealed in katG. PMID- 10750430 TI - [Case of chronic eosinophilic pneumonia taken as infiltrative pulmonary tuberculosis]. PMID- 10750431 TI - [Total blood analysis in assessing status of patient with caseous pneumonia]. AB - The relative and absolute parameters of blood cells were examined in patients with caseous pneumonia before and 2 and 4 months after treatment. The absolute count of stab neutrophils and monocytes proved to be most informative. The long term presence of stab neutrophils indicated that these cells are of great importance in the antituberculosis immunity of patients with caseous pneumonia. PMID- 10750432 TI - [Goodpasture's syndrome as a cause of pulmonary hemorrhages]. AB - Analyzing the case histories of 5 patients with Goodpasture's syndrome who have admitted to an emergency clinic for suspected tuberculosis leads to the conclusion that the onset of the disease appeared as intoxication and lung damage, and evolving general weakness, fever, cough. Hemopoiesis appeared just when overall clinical manifestations appeared, it varied from single sputum blood filaments to more frequent mows of pure red blood sputum for several weeks, but there was never an increasing hourly progressively and this failed to cause a rapid drop of hemoglobin. Anemia is attributable by pulmonary blood imbibition, intoxication, and suppressed hemopoiesis in renal failure rather than by external blood loss as hemoptysis. PMID- 10750433 TI - [Extrapulmonary tuberculosis (lecture)]. PMID- 10750434 TI - The past, present and future of nuclear cardiology. PMID- 10750435 TI - Current state of radiotracers for nuclear cardiology. PMID- 10750436 TI - The role of pharmacologic stress testing for myocardial perfusion imaging. PMID- 10750437 TI - The clinical value of assessing left ventricular function from gated SPECT perfusion studies. AB - Gated myocardial perfusion SPECT is a technique that is rapidly becoming widespread in the nuclear cardiology arena, and it currently accounts for a majority of all perfusion SPECT acquisitions performed in the USA. Its main advantage is that of providing important incremental clinical information over SPECT perfusion assessment alone, at essentially no extra cost. With respect to the diagnosis of cardiac disease, gated SPECT has been demonstrated to improve specificity by helping identify attenuation artifacts in patients with suspected coronary artery disease, and is also key in identifying patients with non ischemic cardiomyopathies. In prognostic assessment, gated SPECT-derived measurements of ejection fraction achieve substantial incremental stratification of patients at risk for cardiac death or myocardial infarction, compared to perfusion scores, and volume measurements further improve risk stratification. New quantitative algorithms allow the fast, accurate and reproducible measurement of parameters of global cardiac function (ejection fraction, end-systolic and end diastolic volume) as well as regional cardiac function (myocardial wall motion and thickening), all of which have been validated against a wide variety of gold standards. PMID- 10750438 TI - Comparison of global and regional left ventricular function assessed by gated SPECT and 2-D echocardiography. AB - Left ventricular (LV) function and volumes have major diagnostic and prognostic importance in patients with heart diseases. Those measurements are most commonly obtained with echocardiographic techniques. Recently, with the use of electrocardiographic gating during the acquisition of myocardial perfusion scintigraphy, it has become possible to simultaneously assess LV perfusion, function and volumes. Both technetium-99m labeled agents and thallium-201, the most commonly used tracers for perfusion scintigraphy, can be used for gated perfusion purposes. Many authors compared gated perfusion images to echocardiography, in regard to LV global and segmental wall motion as well as volumes. We performed gated single photon emission computed tomography (SPECT) and echocardiography in 109 consecutive patients (53 male, 56 female, mean age 63 +/- 14 years) within 15 days of each other. Gated tomographic data, including LV volumes, LV ejection fraction and segmental wall motion, were processed using an automatic algorithm whereas echocardiography used standard techniques. To obtain interobserver and intraobserver variability of regional wall motion, we randomly re-analyzed 34 of the 109 gated SPECT studies. The correlations between gated tomography and echocardiography with respect to end-diastolic volume, end systolic volume and left ventricular ejection fraction were good to excellent (all p < 0.001, r values > or = 0.68) regardless of the use of post-stress or rest/redistribution images, thallium-201 or technetium-99m tracers. The agreement between both techniques, regarding segmental wall motion analysis were also good. Intraobserver and interobserver variability for regional wall motion were good to excellent, with an agreement of 90% and 88%, respectively. Other authors also had similar results in different studies with different populations, tracers, imaging acquisition and processing protocols. Thus, quantitative gated SPECT, using a variety of protocols and with either Tl-201 or Tc-99m tracers, has a good correlation with echocardiography for the measurements of absolute LV volumes and LV global and regional function. This technique is highly reproducible and can be used clinically for those measurements, with the additional advantage that the ventricular performance parameters are obtained from the perfusion images. Furthermore, contrary to echocardiographic techniques used to assess LV volumes and LV function, which are quite labor intensive and more observer-dependent, the gated SPECT technique is nearly totally automatic, and highly reproducible. PMID- 10750439 TI - Can nuclear cardiology really help in the emergency departments of the 21st century? AB - The cardiac troponins are a highly sensitive and specific marker for myocardial damage and have been shown to be useful in the management of patients with acute coronary syndromes. However, some patients with significant ischaemia have no myocardial damage, and therefore negative troponins, despite high risk of subsequent events. Furthermore as a result of the time delay before the troponins can be measured in the peripheral blood, serial sampling is required to rule out myocardial damage. Nuclear perfusion imaging provides an instantaneous assessment of perfusion at the time of injection which is not dependent of on myocardial necrosis, but the presence of flow heterogeneity. Thus these two techniques provide complementary information for the assessment of patients with chest pain and non-diagnostic electrocardiograms, and early perfusion imaging offers the possibility of more rapid assessment and decision making than is possible with troponins. PMID- 10750440 TI - The role of radionuclide imaging in the triage of patients with chest pain in the emergency department. AB - The triage of patients presenting to the emergency department with chest pain and a normal or nondiagnostic ECG poses a significant diagnostic challenge to emergency physicians and cardiologists, leading to unnecessary hospital admissions and substantial associated costs. Radionuclide myocardial perfusion imaging can potentially play an important role in this setting, by providing both a safe and efficient means to risk stratify patients with a low-to-moderate likelihood of unstable angina. The proposed algorithm may serve as a strategy to improve utilization of hospital resources while safely identifying the subgroup of patients with acute chest discomfort who do not need to be admitted to the hospital. PMID- 10750441 TI - The role of nuclear cardiology for preoperative risk assessment prior to noncardiac surgery. AB - Preoperative risk assessment, before noncardiac surgery, aims to reduce mortality and morbidity, during the perioperative period. Cardiac risk could be minimised through treatment and stabilisation of the underlying disease, careful monitoring and prophylactic medications. However, cardiac complications, especially, ischemic events are still frequent causes of death during surgery and hospital recovery. A teamwork approach that involves cardiologist, anaesthesiologist and surgeon is required for optimal risk assessment and monitoring in the perioperative period. The incidence of coronary artery disease increases with age and with the presence of risk factors such as diabetes or hypertension. There is also a strong relation between coronary and vascular disease as they are part of the same pathophysiology and have common risk factors. Careful cardiac evaluation is essential when vascular surgery is planned. Clinical evaluation is the first step of preoperative risk assessment and allows the identification of low and high-risk patients. If there is absent or low cardiac risk, surgery may be carried out. In the presence of high-risk markers and if the surgery is elective, coronary arteriography should be considered before non-cardiac surgery. The information provided by non-invasive diagnostic testing is essential in those patients with an intermediate clinical risk for cardiac events. Concerning coronary artery disease, myocardial function and ischemic burden are strong prognostic markers and in this particular setting, nuclear cardiology can play an important role. The extent and localisation of ischemia is well defined by myocardial perfusion scintigraphy and ventricular function can be evaluated by radionuclide ventriculography or by gated perfusion studies. PMID- 10750442 TI - The prognostic value of myocardial perfusion imaging in patients with coronary artery disease. AB - Myocardial perfusion imaging provides important prognostic information in patients with known or suspected coronary artery disease (CAD) and sustained myocardial infarction. The combination of several scintigraphic variables allows separation of patients at low and high risk for future events. In patients with known or suspected CAD, myocardial perfusion imaging has been demonstrated to provide incremental information over clinical, exercise ECG and angiographic data. Also, an increase in referral-rate for cardiac catheterization and revascularization, paralleling the increase in severity of abnormalities on myocardial perfusion imaging, was observed. Perfusion imaging results appear to be interpreted and implemented appropriately in the diagnostic work-up and subsequent treatment of patients with known or suspected CAD. Therefore, myocardial perfusion imaging may serve as a gatekeeper for more costly and invasive procedures. With the introduction of gated SPECT imaging, it is now possible to assess both perfusion and left ventricular function simultaneously. It is anticipated that gated SPECT imaging will provide very strong prognostic data. PMID- 10750443 TI - Clinical relevance of the evaluation of myocardial viability in patients with left ventricular dysfunction. PMID- 10750444 TI - The clinical role of positron emission tomography in management of the cardiac patient. AB - Positron emission tomography (PET) is the most advanced scintigraphic imaging technique developed for in-vivo assessment of cardiac physiology and biochemistry. The currently available PET technology allows the measurement of regional tracer activity with high spatial and temporal resolution. Several radiopharmaceuticals have been introduced to study myocardial perfusion enabling accurate diagnosis and localization of coronary artery disease (CAD). Assessing myocardial blood flow at rest and under stress conditions and calculating (regional) coronary flow reserve by N-13 ammonia, Rb-82 or 0-15 water PET is the most sensitive means to detect any abnormal vasoreactivity which is already found at very early stages of the atherosclerotic process before any angiographic or clinical evidence of CAD. Flow tracers provide also quantitative information on the hemodynamic effects of any local, invasive (angioplasty) or systemic (risk factor modification) intervention. Metabolic imaging with F-18 deoxyglucose (FDG) PET represents currently the gold-standard for tissue viability assessment with well-validated information about the presence and extent of viable myocardium. The prognostic information of metabolic imaging extends to (preoperative) risk stratification in patients with congestive heart failure and facilitates the decision making between revascularization or conservative management. FDG has been also used in combination with SPECT providing comparable physiologic and pathophysiologic information but without need for the expensive and rarely available imaging technology of PET. More recently, newer tracers such as radiolabeled catecholamine analogs allowed the evaluation of cardiac autonomic innervation in a variety of cardiac diseases with involvement of neuronal innervation. C-11 hydroxyephedrine (HED) enabled imaging of alterations in neuronal innervation in diabetes, congestive heart failure and after heart transplantation. The unique functional and prognostic potential provided by PET imaging together with an expected easier access to this technology with better availability of lower cost instrumentation should raise its clinical acceptance in the near future. PMID- 10750446 TI - Assessing allocation concealment and blinding in randomized controlled trials: why bother? PMID- 10750445 TI - Evolving techniques for nuclear cardiology. PMID- 10750447 TI - Review: antihypertensive drugs improve maternal outcomes in mild chronic and pregnancy-induced hypertension. PMID- 10750448 TI - The only truly important problem. PMID- 10750449 TI - Ritalin use in BC. PMID- 10750450 TI - Heat and light in the childhood spanking debate. PMID- 10750451 TI - Heat and light in the childhood spanking debate. PMID- 10750452 TI - Heat and light in the childhood spanking debate. PMID- 10750453 TI - Heat and light in the childhood spanking debate. PMID- 10750454 TI - Heat and light in the childhood spanking debate. PMID- 10750455 TI - Heat and light in the childhood spanking debate. PMID- 10750456 TI - Heat and light in the childhood spanking debate. PMID- 10750457 TI - Heat and light in the childhood spanking debate. PMID- 10750458 TI - Heat and light in the childhood spanking debate. PMID- 10750459 TI - Heat and light in the childhood spanking debate. PMID- 10750460 TI - Heat and light in the childhood spanking debate. PMID- 10750461 TI - Incidence and outcomes of diabetes mellitus in elderly people: report from the Canadian Study of Health and Aging. AB - BACKGROUND: The epidemiology of diabetes in elderly people is not well understood. The purpose of this study was to estimate the incidence of diabetes mellitus among elderly people in Canada and the relative risks of death and admission to an institution among elderly diabetic patients. METHODS: The study was a secondary analysis of data for a community-dwelling sample from the Canadian Study of Health and Aging, a nationwide representative cohort study of 9008 elderly people (65 years of age or older at baseline) in Canada. Diabetes was identified primarily by self-reporting, and a clinician's diagnosis and the presence of treatments for diabetes were used to identify diabetic patients who did not report that they had the condition. RESULTS: The reliability of self reported diabetes (the kappa statistic) was 0.85. The estimated annual incidence of diabetes was 8.6 cases per thousand for elderly Canadians. Incidence decreased with age, from 9.5 for subjects 65-74 years of age, to 7.9 for those 75-84 years of age and then to 3.1 for those 85 years of age and older. Diabetes was associated with death (relative risk 1.87, 95% confidence interval 1.59-2.19) and admission to an institution (relative risk 1.58, 95% confidence interval 1.28 1.94). INTERPRETATION: Diabetes mellitus is common among elderly people, but the incidence declines among the very old. PMID- 10750462 TI - Benchmarking the vital risk of waiting for coronary artery bypass surgery in Ontario. AB - BACKGROUND: Deaths among patients awaiting coronary artery bypass grafting (CABG) are a source of private grief and public concern in Canada. However, some deaths are expected over time among patients with coronary artery disease. Methods of benchmarking the burden of delayed care may be useful in understanding and managing waiting lists for CABG and other health services. The authors therefore determined the vital risk among people waiting for CABG in Ontario and compared it with the risk in the general population and among people living with coronary artery disease. METHODS: Patients registered to undergo CABG in Ontario between 1991 and 1995 were followed to ascertain numbers and dates of preoperative deaths or completed operations. Linking hospital discharge abstract data to vital statistics for 1991 to 1994, the authors defined a cohort of people who had survived 6 months after an acute myocardial infarction (AMI) and followed them for an additional 6 months to determine numbers and dates of deaths. They matched patients by age and sex and then calculated the standardized mortality ratio for each cohort (i.e., the ratio of observed deaths to those expected based on age- and sex-specific daily probabilities of death for the provincial population). RESULTS: Among 21,220 patients awaiting CABG, there were 82 preoperative deaths over a median follow-up of 18 days; the standardized mortality ratio was 2.92 (95% confidence limit [CL] 2.29-3.55). Among 21,220 matched 6-month survivors of an AMI, there were 663 deaths over a median follow up of 185 days; the standardized mortality ratio was 3.84 (95% CI 3.54-4.14). INTERPRETATION: Patients awaiting CABG in Ontario are at a much greater risk of death than the general population. However, when compared with thousands of other patients living with coronary artery disease, they are at similar or decreased vital risk. PMID- 10750463 TI - Determinants of hospital admission among HIV-positive people in British Columbia. AB - BACKGROUND: This study was initiated to evaluate the demographic and clinical determinants of admission to hospital among HIV-positive men and women receiving antiretroviral therapy in British Columbia. METHODS: The analysis was restricted to participants enrolled in the HIV/AIDS Drug Treatment Program between September 1992 and March 1997 who had completed an annual participant survey, had a viral load determination and had signed a consent form allowing electronic access to their inpatient hospital records. A record linkage was conducted with the BC Ministry of Health to obtain all records of hospital admissions from April 1991 to March 1997. Statistical analyses were carried out using parametric and nonparametric methods and multivariate logistic analyses. RESULTS: The study sample comprised 947 participants (859 men, 88 women). Of these, 165 (17%) were admitted to hospital during the study period from May 1, 1996, to Mar. 31, 1997. The median number of admissions was 1 (interquartile range [IQR] 1-2 admissions), and the median length of stay per admission was 3 days (IQR 1-8 days). Admission to hospital was associated with being unemployed (82% of those admitted v. 58% of those not admitted), being an injection drug user (24% v. 17%), reporting a fair or poor health status (46% v. 29%) and having a physician experienced in the management of HIV/AIDS (31% v. 24%). Examination of clinical determinants demonstrated that hospital admission was associated with a previous admission (72% v. 46%), a high viral load (median 74,000 v. 14,000 HIV-1 RNA copies/mL), a low CD4 count (median 0.16 v. 0.27 x 10(9)/L) and an AIDS diagnosis (44% v. 24%). Multivariate logistic regression analysis revealed that being admitted to hospital was independently associated with being unemployed (odds ratio [OR] 2.64, 95% confidence interval [CI] 1.66-4.20), having been previously admitted to hospital (OR 2.30, 95% CI 1.53-3.46), having a high viral load at baseline (OR 1.45, 95% CI 1.16-1.80), being an injection drug user (OR 1.63, 95% CI 1.02-2.62) and having an experienced physician (OR 1.98, 95% CI 1.29-3.03). INTERPRETATION: Hospital admission among participants in this study was found to be associated with marginalization and poor health status. PMID- 10750464 TI - Hockey injuries of the spine in Canada, 1966-1996. PMID- 10750465 TI - PSA screening: a view from the front lines. North York General-Branson Practice Based Small Group. PMID- 10750466 TI - PSA screening: the bottom line. PMID- 10750467 TI - Preventing spinal cord injuries: is this the best we can do? PMID- 10750468 TI - What can comparisons of mortality rates tell us about waiting lists? PMID- 10750469 TI - Licence requirements for international medical graduates: should national standards be adopted? PMID- 10750470 TI - Program for licensure for international medical graduates in British Columbia: 7 years' experience. AB - British Columbia has funded a program for licensure for international medical graduates since 1992, providing 2 entry positions per year for postgraduate training. Each year 25-35 candidates are eligible for the program, 13-16 enter the evaluation process, 4 go on to a clinical evaluation and 2 are offered funding by the Ministry of Health. Other candidates may access community funding if they meet the requirements of the program. Twenty of 26 candidates have successfully completed the postgraduate training and achieved full licensure; 6 are still in training. In this article we describe the development of the program, the evaluation and selection process, characteristics of the candidates and the outcomes of the program. PMID- 10750471 TI - Recognition and management of Kawasaki disease. AB - Kawasaki disease is the leading cause of acquired heart disease in children in the developed world, with coronary artery aneurysms occurring in up to 25% of untreated cases. The mean annual incidence of Kawasaki disease across Canada is about 13 per 100,000 children less than 5 years of age, based on hospital discharge data from 1990 to 1995. The cause remains unknown, and the diagnosis is based on the same clinical criteria used to describe the disease over 30 years ago. However, nonspecific clinical features, evolving presentations and atypical or incomplete presentations make early diagnosis and timely treatment difficult. Delays in diagnosis and treatment, which occur more frequently in older children, are associated with an increased risk of coronary artery aneurysms. Hence, high diagnostic suspicion and prompt referral are required to reduce the rate of cardiac complications. PMID- 10750472 TI - Cost analysis of a provincial drug program to guide the treatment of upper gastrointestinal disorders. AB - BACKGROUND: Concerned with the rising costs of its drug programs for seniors and social-assistance recipients, the government of Newfoundland and Labrador requested physicians and pharmacists at the Memorial University of Newfoundland, and members of the Newfoundland and Labrador Medical Association and the Newfoundland Pharmaceutical Association to provide guidance to the health care community for the use of drugs to treat upper gastrointestinal disorders. METHODS: Algorithms for the management of dyspepsia and gastrointestinal reflux disease were created and distributed to all physicians and pharmacists in the province in June 1996. On July 1, 1996, the provincial government implemented a program to restrict payment for proton-pump inhibitors through its drug programs to situations defined by the algorithms. Restrictions were not applied to the prescribing of cimetidine, ranitidine and prokinetic agents. The status of famotidine and nizatidine was changed from "open benefit" to "special consideration," which requires prescribers to request authorization of their use on a case-by-case basis. RESULTS: Between July 1 and Dec. 31, 1996, 973 of 1078 requests for a proton-pump inhibitor were approved (679 for gastroesophageal reflux, 186 for Helicobacter pylori eradication, 55 for ulcer treatment and 53 for other reasons). The program resulted in a sustained reduction in drug expenditures. Total drug expenditures, which had risen from $39.0 million in 1992/93 to $50.8 million in 1995/96, fell after implementation of the program to $46.4 million in 1996/97 because of a decrease of more than 80% in the use of proton-pump inhibitors. Expenditures on proton-pump inhibitors, which had increased from $0.7 million for the 6 months ending March 1993 to $1.6 million for the 6 months ending March 1996, decreased to $0.3 million for the 6 months ending March 1997. The use of H2-antagonists, but not prokinetic agents, increased concomitantly with the decline in proton-pump inhibitor use. Compared with the year preceding implementation of the program, annual combined expenditures in the subsequent 3 years for H2-antagonists, prokinetic drugs and proton-pump inhibitors were reduced by $1.6 million, $1.7 million and $1.0 million, respectively. Feedback from physicians and pharmacists was supportive for the clinical information and prescribing guidelines. Concerns were mostly limited to process issues. INTERPRETATION: The program, designed by health care professionals, approved by health care associations and implemented by the province of Newfoundland and Labrador to guide the treatment of upper gastrointestinal disorders, has achieved a substantial reduction in drug expenditures. PMID- 10750474 TI - Abuse against women a public health issue: MD. PMID- 10750473 TI - Chronic viral hepatitis C: management update. AB - The management of chronic viral hepatitis C is evolving rapidly. Monotherapy with interferon, the accepted standard of treatment until recently, achieves only a modest sustained virological response rate of 15%. Combination treatment with alpha-2b interferon and ribavirin has been shown to increase sustained response rates to 40% in patients who have never been treated with interferon and to 50% in those who have relapsed following monotherapy with interferon. However, side effects, which have led to the discontinuation of combination treatment in a significant proportion of patients, must be carefully monitored. Treatment with interferon alpha-2b and ribavirin has now been approved in Canada, but the selection and monitoring of patients suitable for combination treatment requires special expertise. Although improvements in current therapeutic options may be possible with more frequent, higher doses or long-acting forms of interferon together with ribavirin, low sustained response rates (i.e., below 30%) for patients with hepatitis C virus genotype 1 emphasize the need for novel antiviral medications that will target the functional sites of the HCV genome. PMID- 10750475 TI - US pharmacy chain tracks customer's use of herbals. PMID- 10750476 TI - College to appeal discrimination ruling. PMID- 10750477 TI - Drug testing a growth industry in Salt Lake City. PMID- 10750478 TI - Popularity growing rapidly as CMA Online turns 5. PMID- 10750479 TI - Coping with flu season. PMID- 10750480 TI - Precautionary principle leads to "may contain" clause for genetically modified foods. PMID- 10750481 TI - Despite some opposition, BC pharmacists to dispense morning-after pill without prescription. PMID- 10750482 TI - Is scarcity of resources a valid legal defence? PMID- 10750483 TI - Solid phase asymmetric synthesis of isoxazolines. PMID- 10750484 TI - Efficient split synthesis for targeted libraries. AB - We propose a new approach for fabricating more sophisticated combinatorial chemistry libraries via split synthesis and evaluate its potential through extensive simulation. Our algorithmically intensive method promises to reduce the time and materials costs of synthesizing libraries which are (1) too large to synthesize economically by sequential or parallel synthesis, (2) too long or irregular for conventional split synthesis generation techniques, and (3) not used in sufficient quantity to justify the setup costs of array makers. It also encourages the design of more focused and interesting libraries than are typically constructed using split synthesis. Our algorithms automate the design of efficient synthesis procedures for motif-based libraries which are too complex to design by hand. Our software allows the user to select the most desirable tradeoff between minimizing the number of steps in the synthesis process and containing the combinatorial explosion of the number of compounds synthesized. PMID- 10750485 TI - Preparation of trifluoromethylpyridine libraries. AB - S-Alkylation followed by heterocyclization of trifluoromethyl-3-cyano-2(1H) pyridinethiones was used for preparation of libraries of S-alkyl trifluoromethylpyridines and thieno[2,3-b]pyridines. The S-alkylation (in water- DMF mixtures) was successful for all 18 alkylating agents employed (yields typically > 50%). S-Alkyl derivatives were further converted to corresponding thieno[2,3-b]pyridines via heterocyclization in base conditions (yields > 65%). Structures of new compounds were elucidated by a combination of IR and 1H NMR spectroscopy and elemental analysis and were confirmed by means of single-crystal X-ray diffraction analysis. PMID- 10750486 TI - Solid-phase synthesis of phenolic steroids: from optimization studies to a convenient procedure for combinatorial synthesis of biologically relevant estradiol derivatives. AB - During the course of our studies on therapeutic agents for the treatment of breast cancer, we became interested in the solid-phase combinatorial synthesis of estradiol derivatives that contain a functionalized side chain at either position 16 beta or 7 alpha. Both types of compounds have already demonstrated inhibitory activity toward both biosynthesis and action of estradiol. As a first step, two versatile precursors bearing an azidoalkyl side chain at either position 16 beta or 7 alpha of estradiol were synthesized using standard solution-phase methods. Afterward, the effectiveness of five linkers to attach the phenolic function of these estradiol derivatives to a polystyrene resin was investigated; they were benzylic ether (Merrifield), 4-alkoxy-benzylic ethers (Wang, Sheppard), tetrahydropyranyl ether (Ellman), benzoic ester, and o-nitrobenzyl ether. To test the linker in a synthetic context, a short sequence of reactions, including reduction of the azide and acylation of the corresponding amine, was performed on the polymer-bound estradiol derivative. While all of the tested linkers proved effective in attaching the phenol functionality of the precursor, only the o nitrobenzyl ether photolabile linker enabled the release of the final products in acceptable purities. Consequently, this linker was used to perform successfully the solid-phase synthesis of four different classes of estradiol derivatives in acceptable yields and excellent purities. This study was preliminary to the combinatorial synthesis of larger libraries of biologically relevant estradiol derivatives. PMID- 10750487 TI - An efficient procedure for traceless solid-phase synthesis of N,N'-substituted thioureas by thermolytic cleavage of resin-bound dithiocarbamates. AB - A novel and efficient procedure which is compatible with high-throughput process for the traceless solid-phase synthesis of thioureas is described. In the presence of carbon disulfide, Merrifield resin reacts with an amine to give a resin-bound dithiocarbamate moiety. Heating this supported dithiocarbamate in the presence of a second amine at 60 degrees C for 12 h led to the formation of the thiourea with the release of benzylthiol bound to the resin. This process allows the preparation of N,N'-di- and trisubstituted thioureas in good yields and with satisfactory purity. Furthermore, the mild reaction conditions involved are compatible with many functional groups. PMID- 10750488 TI - (1S)-1-[(4R)-2,2-dimethyl-1,3-dioxolan+-4-yl][-2-hydroxyethylamm onium benzoate, a versatile building block for chiral 2-aminoalkanols: concise synthesis and application to nelfinavir, a potent HIV-protease inhibitor. AB - A concise synthesis of a versatile chiral C4 building block for 2-aminoalkanols, (1S)-1-[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]-2-hydroxyethylammo nium benzoate (1a), was described. 1 (1a and its enantiomer 1b) acted as four stereoisomers of optically active 2-amino-1,3,4-butanetriol. The versatility of 1 was demonstrated by its application to the practical synthesis of nelfinavir (2), a potent HIV protease inhibitor, as well as by the stereospecific synthesis of three diastereomers of 2. PMID- 10750489 TI - 3-Cyclobutenyl-1,2-dione-substituted porphyrins. A general and efficient entry to porphyrin-quinone and quinone-porphyrin-quinone architectures. AB - A new and efficient synthesis of meso-linked porphyrin-quinone dyads and quinone porphyrin-quinone triads has been developed via the intermediacy of porphyrins bearing 3-cyclobutenyl-1,2-dione and 3-(1-ethenyl)cyclobutenyl-1,2-dione substituents at one or two nonadjacent meso-positions. The free-base porphyrins 5 bromo-10,20-diphenylporphyrin and 5,15-dibromo-10,20-diphenylporphyrin undergo facile palladium-catalyzed Stille coupling with 3-isopropoxy-2-tri-n-butylstannyl cyclobutene-1,2-dione to produce the corresponding mono- and bis(3-cyclobutenyl 1,2-dione)-substituted porphyrins in good yields. In contrast, the zinc bromoporphyrins reacted with the same tin reagent only slowly and with the formation of side products. The free-base bromoporphyrins also were coupled with tri-n-butylvinyltin to afford vinylporphyrins in very good yields. 5,15-Diphenyl 10-vinylporphyrin was converted into trans-bromovinylporphyrin, which underwent facile Stille coupling with 3-isopropoxy-2-tri-n-butylstannylcyclobutene-1,2 dione to afford the vinylogous 3-cyclobutenyl-1,2-dione-substituted porphyrin. The molecular structure of 5,15-bis(3-cyclobutenyl-1,2-dione)-10,20 diphenylporphyrin(Z n) was determined by X-ray crystallography. Although the data revealed a fairly large dihedral angle between the cyclobutenedione and the porphyrin rings (57 degrees), the UV-vis spectra of both the mono- and bis(3 cyclobutenyl-1,2-dione)-substituted porphyrins showed B- and Q-band red shifts indicative of strong electronic coupling between the porphyrin and cyclobutenedione chromophores in solution. Introduction of a double bond between the cyclobutenedione and porphyrin rings resulted in a significant red shift of both the B- and Q-bands compared to those of the nonvinylogous system. All porphyrinic cyclobutenediones were metalated with zinc and then, using established cyclobutenedione chemistry, converted into a variety of porphyrin quinones in excellent yields with aryllithium and vinylic Grignard reagents. From the mono(3-cyclobutenyl-1,2-dione)-substituted porphyrin, 7, a variety of directly linked monoquinone-porphyrin dyads were easily synthesized. Substituents could also be introduced at the free meso-position of 7 by bromination followed by palladium-catalyzed cross-coupling reactions, and additional porphyrinic monoquinones were then prepared from these starting materials. The vinylogous squarylporphyrin was converted into a double bond linked porphyrin-quinone via reaction with phenyllithium followed by thermal rearrangement and oxidation. As a result of the hindered rotation around the C-C bond between the porphyrin and the quinone, pairs of stable, separable, and thermally interconvertable atropisomers of porphyrin-quinones were obtained from 5,15-bis(3-cyclobutenyl-1,2-dione)-10,20 diphenylporphyrin(Z n). The structure of one of the atropisomers was determined by X-ray crystallography. PMID- 10750490 TI - 3-Cyclobutenyl-1,2-dione-substituted porphyrins. 2. A simple and general entry to quinone-porphyrin-porphyrin-quinone tetrads and related molecules. AB - The synthesis of meso-linked quinone-porphyrin-porphyrin-quinone tetrads has been accomplished by the simple treatment of a CH2Cl2 solution of zincated 5 substituted-10,20-diphenylporphyrins (where the substituent = 3-cyclobutenyl-1,2 dione, various quinonyl derivatives, and p-anisyl) with 2,3-dichloro-5,6 dicyanoquinone (DDQ). The zincated porphyrinic quinones were synthesized from 5 (3-cyclobutenyl-1,2-dione)-10,20-diphenylporphyrin(Zn). The meso-linked dimer of 5-(3-cyclobutenyl-1,2-dione)-10,20-diphenylporphyrin(Zn) was also shown to be a useful precursor to meso-linked quinone-porphyrin-porphyrin-quinone tetrads. This DDQ-based oxidative dimerization appears to be general and effective for various zincated 5-substituted-10,20-diphenylporphyrins bearing both electron-withdrawing and -donating substituents. The oxidative dimerization was very sensitive to the reaction solvent (dimerization occurred in CH2Cl2, not in THF) and required the zincated porphyrins (the corresponding free base porphyrins did not undergo dimerization). When this solvent effect was applied to the reaction of I2/AgO2CCF3 with zincated porphyrins, either the dimeric porphyrins or iodoporphyrins could be selectively prepared simply by selecting methylene chloride or THF as the reaction solvent, respectively. PMID- 10750491 TI - Investigations on the enzyme specificity of clostripain: a new efficient biocatalyst for the synthesis of peptide isosteres. AB - To explore the ability of the cysteine protease clostripain as a biocatalyst for the synthesis of peptide isosteres, the S'-subsite specificity of this enzyme toward unnatural substrates was investigated. First, the function of clostripain for acylating aliphatic noncyclic and cyclic amines varying in chain length and ring size was analyzed using a standard acyl donor. Additionally, this series was expanded by use of aromatic amines, amino alcohols, derivatives of non-alpha amino carboxylic acids, and symmetric and asymmetric diamines, respectively. The results obtained give a detailed picture of the unique reactivity of clostripain toward synthetic substrates, allowing insights into the basic enzyme-substrate interactions. Furthermore, the data provide a guideline for the use of clostripain as a biocatalyst for synthesis of peptide isosteres. The study was completed by the utilization of a model substrate mimetic enabling clostripain to react with noncoded and non-amino acid-derived amines as well as nonspecific acyl moieties. The results of this study indicate that this approach may extend the application range of clostripain as a biocatalyst outside of peptide synthesis. PMID- 10750492 TI - Enzymatic desymmetrization of a meso polyol corresponding to the C(19)-C(27) segment of rifamycin S. AB - The stereoselective acylation of meso polyol 2 by vinyl acetate (solvent and acyl donor) in the presence of porcine pancreas lipase gave the corresponding monoester 5 in good yield (76%) and high enantiomeric purity (ee > 98%). The enzymatic reaction was also highly regioselective for a primary alcohol end group, and the two unprotected secondary alcohols were not involved. Compound 5 corresponds to the C(19)-C(27) fragment of rifamycin S. PMID- 10750493 TI - Formal total synthesis of (-)-balanol: concise approach to the hexahydroazepine segment based on RCM. AB - A concise synthesis of the hexahydroazepine moiety 13 of (-)-balanol 1 is described that comprises only eight steps and is distinctly shorter than all previous reported approaches to this particular compound. Sharpless epoxidation of divinylcarbinol 4 and ring closing alkene metathesis (RCM) reaction for the formation of the heterocyclic scaffold 9 constitute the key transformations of this sequence. The latter reaction is best achieved with catalytic amounts of the ruthenium indenylidene complex 18 recently reported. Furthermore, it is demonstrated that RCM can be successfully carried out even in the presence of an azido function provided that Schrock's molybdenum alkylidene complex Mo(=NAr)(=CHCMe2Ph)[OC(Me)(CF3)2]2 (Ar = 2,6-diisopropylphenyl) is used as precatalyst. PMID- 10750494 TI - A new route toward 4-substituted pyrazino[2,1-b]quinazoline-3,6-dione systems. Total synthesis of glyantrypine. AB - Treatment of sodium N-(o-azidobenzoyl)aminoacylglycinates 8 with acetic anhydride afforded 1-acetyl-4-(o-azidobenzoyl)-2,5-piperazinediones 7, with complete retention of the stereochemistry. The intramolecular aza Wittig reactions of compounds 7 in the presence of tributylphosphine followed by deacetylation gave 1,2-unsubstituted pyrazino[2,1-b]quinazoline-3,6-diones 1. This route was adapted to the synthesis of both enantiomers of the alkaloid glyantrypine. PMID- 10750495 TI - Synthetic studies on nogarol anthracyclines. Enantioselective total synthesis of an aminohydroxy epoxybenzoxocin. AB - A chiral synthesis of the aminohydroxy expoxybenzoxocin 6 is described. Enantioselective Friedel-Crafts coupling using a chiral titanium catalyst was employed to produce the optically active atrolactic ester 16a from the phenol 11 and l-menthyl pyruvate (12). The phenolic group in 16a was protected as the benzyl ether and the t-alcohol functionality as the MEM ether to give 20, which after sequential reduction/oxidation provided the aldehyde 22. Addition of the acetylide anion of propargyl aldehyde diethyl acetal (23) to aldehyde 22, followed by oxidation of the resultant diastereoisomeric carbinols, gave the acetylenic ketone 24. Lindlar reduction of 24 afforded the trans-enone 26. Reaction of 26 with thiophenylate anion furnished 27, which was then cyclized to the alpha-methyl pyranoside 29. Oxidation of 29 to the sulfoxide and subsequent thermolysis afforded the hexenulose 30. Sequential epoxidation of 30, reduction of the keto epoxide 31, and reaction of the resultant epoxycarbinol 32 with dimethylamine produced the aminohydroxy pyranose 33a. Debenzylation of 33a to the phenol 33b, followed by intramolecular cyclization, completed the fabrication of the optically active aminohydroxy epoxybenzoxocin 6. The 17-step sequence from the phenol 11 to 6 was achieved in 22% overall yield. PMID- 10750496 TI - Synthesis, electrochemistry, and interactions with beta-cyclodextrin of dendrimers containing a single ferrocene subunit located "off-center". AB - Two series of dendrimers containing a single ferrocene unit located in the focal point of these macromolecules have been synthesized and characterized. The first series of dendrimers has considerable lipophilic character, with tert-butyl ester groups located in their peripheral regions. In contrast, the second series of dendrimers was obtained by the hydrolysis of these peripheral ester groups, yielding water-soluble dendrimers with carboxylic acid groups in their surfaces. The electrochemical properties of these macromolecules were strongly affected by the dendritic groups attached to the ferrocene subunits. Host-guest interactions between the water-soluble dendrimers and the well-known receptor beta cyclodextrin were also investigated. The dendritic groups were found to hamper markedly the formation of inclusion complexes between the cyclodextrin receptor and the dendrimer's ferrocene unit. PMID- 10750497 TI - A novel, one-pot ring expansion of cyclobutanones. Syntheses of carbovir and aristeromycin. AB - A novel, one-pot ring-expansion procedure was developed using Me3S(O)I, NaH, and Sc(OTf)3. The scope and limitations were briefly examined, and a tentative mechanism was proposed. Application of the methodology to known cyclobutanone 1 provided the corresponding cyclopentanone, which was successfully advanced to (+) carbovir and (+)-aristeromycin. PMID- 10750498 TI - A practical preparation of terminal alkynes from aldehydes. PMID- 10750499 TI - Role of laboratory services in health care: the present status in eastern Africa and recommendations for the future. PMID- 10750500 TI - Laboratory services in health centres within Amhara region, north Ethiopia. AB - OBJECTIVE: To identify current problems in laboratory services and elicit suggestions from the technicians aimed at improving the services in health centres within Amhara region, north Ethiopia. DESIGN: Cross-sectional study using a pre-tested questionnaire. SETTINGS: Twenty seven health centres in Amhara region, north Ethiopia. PARTICIPANTS: Twenty seven laboratory technicians. RESULTS: Physical facilities were seriously lacking, consisting of only one room in 85.2% of the health centres. Supply of electricity was never continuous. Whereas virtually all the health centres had up to five medical doctors each, laboratory manpower was grossly below the ideal requirement. Over half of the health centres had only one technician each, resulting into heavy workload. Laboratory reagents were insufficient, equipment lacking, and maintenance and repair were not accorded adequate attention. Eighty different tests were offered by the health centres in various combinations. Twenty categories of suggestions were advanced by the respondents. CONCLUSION: There is a great shortage of manpower, equipment, chemicals and other supplies to provide adequate laboratory services in the health centres within the region. Consequently, the laboratories are functioning below capacity. PMID- 10750501 TI - In vitro susceptibility of gram-negative bacterial isolates to chlorhexidine gluconate. AB - OBJECTIVE: To investigate the susceptibility of clinical isolates of gram negative bacteria to chlorhexidine gluconate. DESIGN: Prospective laboratory study. SETTING: Tikur Anbessa Hospital, Addis Ababa, Ethiopia. SUBJECTS: Clinical specimens from 443 hospital patients. MAIN OUTCOME MEASURES: Significant number of gram negative bacteria were not inhibited by chlorhexidine gluconate (0.02 0.05%) used for antisepsis. RESULTS: Four hundred and forty three strains of gram negative bacteria were isolated from Tikur Anbessa Hospital patients. Escherichia coli (31.6%) and Klebsiella pneumoniae (23%) were the most frequently isolated bacteria followed by Proteus species (13.3%), Pseudomonas species (9.2%), and Citrobacter species (6.1%). Each organism was tested to chlorhexidine gluconate (CHG), minimum inhibitory concentration (MIC) ranging from 0.0001% to 1%w/v. All Salmonella species and E. coli were inhibited by CHG, MIC < or = 0.01%. Twenty nine per cent of Acinetobacter, 28% of K. pneumoniae and Enterobacter species and 19-25% of Pseudomonas, Proteus and Providencia species were only inhibited at high concentrations of CHG (> or = 0.1%). CONCLUSION: Our results showed that a significant number of the gram-negative bacterial isolates were not inhibited by CHG at the concentration used for disinfection of wounds or instruments (MIC 0.02 0.05% w/v). It is therefore important to select appropriate concentration of this disinfectant and rationally use it for disinfection and hospital hygiene. Continuing follow up and surveillance is also needed to detect resistant bacteria to chlorhexidine or other disinfectants in time. PMID- 10750502 TI - Galactosaemia in black South African children. AB - OBJECTIVE: To evaluate the clinical and biochemical features of all black children confirmed to have galactosaemia from the KwaZulu Natal Province of South Africa. DESIGN: Prospective laboratory study. SUBJECTS: These included all black children with the presenting clinical features suggestive of the diagnosis of galactosaemia. SETTING: Department of Chemical Pathology, King Edward VIII Hospital, Durban, South Africa. METHOD: In each case, urine was screened for the presence of a reducing substance using urinary dipstick followed by thin layer chromatography to establish the presence of galactosaemia. The diagnosis of galacotosaemia was then confirmed by analysis of galactose-1 phosphate uridyl transferase (GALT) activity in the erythrocytes using the established Beutler enzyme assay procedure. Age and sex-matched samples were used as controls for GALT activity. The presenting clinical features of each patient on admission were also recorded. INTERVENTIONS: Patients confirmed to have galactosaemia were immediately placed on a galactose free diet. RESULTS: The age distribution of affected individuals varied from six weeks to 27 months with 60% of the children being males. The most common presenting clinical features were jaundice in 77% of the patients, failure to thrive 62%, and cataracts 54%. Four patients had complete absence of GALT activity. Two infants who displayed acute toxicity symptoms and positive urine galactose, exhibited normal GALT activity. CONCLUSION: GALT deficiency is the most common form of galactosaemia in black children in the KwaZulu Natal region. Cases of galactokinase or epimerase enzyme deficiency appear to be present. Further investigation is required to establish the occurrence and prevalence of the latter in affected individuals in this region. PMID- 10750503 TI - Prevalence of anaemia in patients attending an outpatient clinic in western Rift Valley in Kenya during a low malaria season. AB - OBJECTIVE: To assess the prevalence of anaemia in outpatients attending a rural health clinic in an area of seasonal malaria, during the low transmission season. METHODS: Haemoglobin estimation and blood slide examination for malaria parasites were performed on 280 consecutive patients attending outpatient curative services at Entasopia Health Centre, Kajiado District, Kenya, between April-May 1996. Anaemia was defined according to World Health Organisation guidelines for age, sex and pregnancy status. RESULTS: In all groups except adult males, more than half of the patients tested had haemoglobin values below the lower reference limits, suggesting that anaemia is widely present in this population even during the low malaria season. Only 5% of patients were positive for Plasmodium falciparum malaria. Peripheral blood film examination suggested iron deficiency as the major cause of anaemia. CONCLUSIONS: Further studies to define the underlying causes of anaemia and to develop community strategies to prevent anaemia are required. The association between fever and anaemia and the use of pallor to diagnose anaemia, are discussed. PMID- 10750504 TI - Intrafamilial person-to-person spread of bacillary dysentery due to Shigella dysenteriae in southwestern Saudi Arabia. AB - OBJECTIVE: To identify the factors that influence transmission of bacillary dysentry (BD) within families during a propagated outbreak of bacillary dysentery. DESIGN: A retrospective cohort study. SETTING: Eighteen neighbouring villages in rural Gizan, southwestern Saudi Arabia. SUBJECTS: Two hundred and thirty three cases of BD were identified among seventy nine families. RESULTS: Secondary cases of BD occurred in 57 of 79 families with a primary case of BD. The secondary attack rate per cent (AR%) within families ranged between 7.7% and 80%. Age of primary cases did not correlate with degree of secondary AR% in exposed families (p > 0.04; p > 0.05); however, within households, the age of the first secondary cases (median = two years) was usually less than the age of the primary case (median = six years). Children under five years of age constituted 43% of secondary cases. The median interval between successive cases within a house ranged from three and seven days. Two hundred and twenty cases (94.4%) gave history of close contact within another case of BD. Cases of BD were exposed to close relatives with BD (79.1%), neighbours (11.4%), and friends (9.5%). Risk factors influencing the spread of BD within families included two rooms or fewer per house (OR = 4.3, 9.5% CI 1.3-14.3), family size of five or more (p = 0.012, two-tailed Fisher's exact test), and presence of more than two persons per room (OR = 11.2, 95% CI 3.1-42.4). CONCLUSION: Person-to-person secondary transmission can amplify the spread of bacillary dysentery within households and neighbouring villages. Crowding was a risk factor that amplified transmission of BD within families. PMID- 10750505 TI - Is fine needle aspiration cytology a useful tool for the diagnosis of tuberculous lymphadenitis? AB - OBJECTIVE: To study the role of fine needle aspiration cytology (FNAC) in the diagnosis of tuberculous lymphadenitis and find a place for FNAC as laboratory diagnostic method in tuberculosis (TB) control programmes. DESIGN: Prospective laboratory study. METHOD: Duplicate smears from 127 lymph node aspiration were prepared. Both slides were air-dried. Giemsa stain for cytological examination and Ziehl-Neelsen stain for demonstration of acid fast bacillus(AFB) were used and examined by a pathologist and laboratory technologist respectively. SETTING: Tigrai Regional Health Research and Laboratory Centre which is the only unit with microbiological and cytopathological service in the region. SUBJECTS: Patients with one or more enlarged lymph nodes who were sent for FNAC were included. RESULTS: The AFB positivity among the cytologically diagnosed cases of TB lymphadenitis was 56.77%. If we had used culture media for Mycobacterium spp, the positivity would have probably been higher. The positivity rate varied depending on the type of the aspirate. Caseous aspirate showed a higher positivity rate of 60.47% whereas no AFBs were detected in haemorrhagic aspirates. CONCLUSION: This study has demonstrated the usefulness of FNAC in the diagnosis of TB lymphadenitis and the national TB and leprosy control programmes should encompass FNAC as a diagnostic means instead of biopsy which is more invasive and costly. PMID- 10750506 TI - Comparative study of conservative resection and radical operation for thyroid carcinomas. AB - OBJECTIVE: To compare mortality and morbidity in management of thyroid cancers by total lobectomy (C-R) and near-total thyroidectomy (R-O) and to relate pathological subtype to prognosis. DESIGN: A retrospective study of patients with thyroid cancers that were managed during an eleven-year period. SETTING: Jos University Teaching Hospital, Nigeria. SUBJECTS: A total of 44 patients who underwent C-R (n = 26) and R-O (n = 18) were studied. INTERVENTION: A total of 509 goitrous specimens including the excised thyroid cancers were histopathologically studied. MAIN OUTCOME MEASURES: Cases were analysed for mortality and morbidity data in the two groups during a partial follow-up period of two years. RESULTS: Mortality figures for C-R was 4% versus 11 for R-O. Postoperative haemorrhage occurred in 14% for C-R versus six for R-O. Similarly, bilateral vocal cord paralysis occurred in 11% versus 22; voice changes in 11% versus 33, transitory hypoparathyroidism 8% versus 50, local recurrence in 18% versus eleven. Hypothyroidism occurred in all patients undergoing R-O and stitch granuloma in 11% of patients in C-R group versus six for R-O. Follicular carcinoma constituted 59% of the total number thyroid cancers with papillary cancer constituting 35%. CONCLUSION: C-R is recommended as the operation of choice for thyroid cancers. PMID- 10750507 TI - Pain control after major surgery. AB - OBJECTIVE: To assess the adequacy and efficacy of postoperative pain management. DESIGN: A prospective clinical study. SETTING: The Georgetown and New Amsterdam Public Hospitals, Guyana. SUBJECTS: Two hundred consecutive patients undergoing major abdominal surgery. MAIN OUTCOME MEASURES: Presence or absence of significant postoperative pain during the first 24 hours. RESULTS: All the patients experienced pain postoperatively. Sixty one per cent of patients considered their pain severe, 30% rated it moderate and only 9% mild. Reasons for this deficiency of care are partly attributable to the patients themselves and also the health care staff. CONCLUSIONS: Postoperative pain is poorly managed in our general hospitals. PMID- 10750508 TI - Cost saving with ultrasonography in a developing country district hospital. AB - OBJECTIVE: To estimate the potential monetary reduction induced by the introduction of an ultrasound unit in a major district hospital in a developing country. DESIGN: Propective study. SUBJECTS: Patients referred with abnominal diseases and pregnancy. SETTING: Wad Medani Teaching Hospital, Central Sudan. RESULTS: Local specialists referring the patients stated that an estimated 792 radiologic procedures would have been carried out to obtain the same amount of information as achieved by ultrasound. Such procedures budgeted approximately 8100 US dollars, in terms of equipment, material and personnel by 1987 rates. Such savings have benefited all departments of the hospital. CONCLUSION: The authors consider this as evidence for the fact that despite its initial high investment (15,000 US dollars), availability of ultrasound virtually reduced expenditure on other radiological diagnostic procedures. This is of special benefit for the limited budgets of hospitals in non-industrialised countries. PMID- 10750509 TI - CT findings in head scans at Moi Teaching and Referral hospital. AB - BACKGROUND: The first CT scanner in western Kenya was launched on 20th February, 1998. OBJECTIVE: To determine the pattern of CT findings in head scans. DESIGN: Prospective study over a one year period. SETTING: Moi Teaching and Referral Hospital, Eldoret, Kenya. SUBJECTS: Four hundred and ninety five consecutive patients who underwent CT head scans. RESULTS: Intracranial haemorrhage was the leading disorder with 17.8%, followed by brain infarcts at 10.5%, hydrocephalus at 6.3% and brain tumours at 5.9%. CONCLUSION: Intracranial disorders secondary to trauma, stroke and their sequelae are major causes of head disease in western Kenya. PMID- 10750510 TI - Pseudoexfoliation syndrome in Ethiopian glaucoma patients. AB - BACKGROUND: Pseudoexfoliation syndrome (PXS) has variable prevalence rates in the glaucoma population depending on geographic location and racial composition of the population studied. There is no data available on this subject in Ethiopia. OBJECTIVE: To determine the prevalence of PXS among Ethiopian glaucoma clinic population. DESIGN: A prospective study. SETTING: The Glaucoma Clinic of Menelik II-Hospital, which is a tertiary eye care centre in Addis Ababa, Ethiopia. PARTICIPANTS: All new patients with glaucoma or ocular hypertension (ocular HPN) seen during the study period from 1st April to 30th September 1996 were included in the study. RESULTS: Our of the 432 new patients of glaucoma or ocular HPN seen during the study, 108 patients (25%) had PXS. The age of PXS cases ranged from 32 years to 86 years (mean 62.3 years). There were 80 males and 28 females. Of the PXS patients, 104 (96.3%) were cases of chronic open angle glaucoma, while angle closure glaucoma and ocular HPN were rare comprising only two patients (1.85%) each. CONCLUSION: The relatively high prevalence rate of PXS among Ethiopian glaucoma clinic population found in this study stresses the need for further community-based research in order to prevent blindness due to glaucoma associated with PXS. PMID- 10750511 TI - Galactosaemia in an infant: case report. AB - Galactosaemia is a disorder of galactose metabolism in which raised levels of galactose and galactose-l-phosphate damage various organs. It is a very rare disease (incidence 1 in 60,000) and the diagnosis is often missed, leading to poor prognosis. A case of clinical galactosaemia that was diagnosed at the age of 11 months is reported. It is important to be aware of this condition as early treatment may prevent some of the complications. PMID- 10750512 TI - Peutz-Jeghers syndrome: case report. AB - A case of Peutz-Jeghers syndrome (PJS) in an African girl is presented to document the clinical presentation, management and follow up of this condition. The patient who presented with black buccal mucosal hyperpigmentation and clinical features of jejuno-jejunal intussusception was successfully managed by operative reduction of the intussusception and polypectomy. She is being followed up for evidence of malignant transformation in associated intestinal polyps and development of extra-intestinal malignancies at other sites. The management of PJS in light of recent trends is discussed, especially with reference to suggested protocols for screening and surveillance of sites at supposed risk of tumour development. PMID- 10750513 TI - Performing percutaneous transphepatic cholangiography using simple x-ray equipment. AB - Percutaneous transhepatic cholangiography (PTC) is a valuable procedure used for diagnosing diseases of bile duct. It is simple to perform, safe, and inexpensive. The only equipment needed to perform PTC is a simple x-ray machine and puncture needle 14 cm long and of 0.1 cm core diameter. As long as the puncture point is selected accurately and operation is performed correctly, an excellent image can be achieved. This procedure can therefore be used in developing countries where expensive, modern technology is not available. We report a case of a 50 year old female patient in whom the procedure was successfully used. PMID- 10750514 TI - Drug use and abuse. PMID- 10750515 TI - Prevalence of substance use among students in a Kenyan University: a preliminary report. AB - OBJECTIVE: To investigate the prevalence and pattern of substance use among an undergraduate population in a Kenyan university. DESIGN: Cross-sectional survey, cluster sampling of classrooms, self-administered questionnaires. SETTING: Private international university in Nairobi province, Kenya. PARTICIPANTS: Five hundred fifty eight undergraduate students of both sexes, age range 16-50 and mean age (S.E) of 21.1 +/- 0.2. MAIN OUTCOME MEASURES: Self-reported use of psychoactive substances. RESULTS: Percentages of lifetime prevalence rates of commonly used substances were tobacco, 54.7%; alcohol, 84.2%; cannabis, 19.7% and inhalants, 7.2%. The corresponding "past use" and "current use" rates were relatively lower but followed the same pattern. The percentage rates were significantly higher in males than females (p < 0.005). Rates for regular use (> 20 days/month) were higher for tobacco (24.7%) than alcohol (11.5%). The lifetime prevalence rates of other drugs (heroin, cocaine, mandrax, amphetamines and LSD) were low (< 5%), while modest rates were recorded for tranquilizers (10.8%), local brews (13.6%) and cough mixtures (35.1%). Less than 20% of respondents initiated substance use in lower primary school, while more than 50% started using in upper primary and secondary school and 11% to 25% started using substances at university. CONCLUSION: Substances most commonly used by respondents studied were of the licit variety (alcohol and tobacco). The rate of use of the two substances is rather high. The use of illicit drugs seems to be growing and may soon escalate to alarming levels. The findings suggest an urgent need to gather more data, which can be used to guide formulation of health promotion and prevention programmes. PMID- 10750516 TI - Tuberculosis chemotherapy and sputum conversion among HIV-seropositive and HIV seronegative patients in south-eastern Uganda. AB - OBJECTIVE: To investigate if there is a difference in response to tuberculosis treatment between HIV seronegative and HIV seropositive patients following two months of intensive phase tuberculosis treatment. DESIGN: Prospective cohort study. SETTING: St. Francis Leprosy Centre, south-east Uganda. SUBJECTS: Four hundred fifty seven patients with never previously treated sputum smear-positive tuberculosis admitted during a two-year period in 1991/1993. INTERVENTION: Intensive phase treatment with streptomycin, isoniazid, rifampicin and pyrazinamide. MAIN OUTCOME MEASURES: Sputum conversion from a positive to a negative smear at eight weeks of treatment. RESULTS: HIV seropositivity prevalence was 28%. Among HIV seronegative patients, conversion to a negative smear status occurred in 76% persons compared to 78% in HIV seropositive patients. This difference was not statistically significant (OR = 0.9; 95% CI, 0.6-1.5). HIV seropositive patients, however, were more likely to die (p = 0.017). A high prevalence of resistance to isoniazid and streptomycin was found. Isoniazid resistance was more likely in HIV seronegative patients with M. tuberculosis strains compared to HIV seropositive persons (p < 0.005). Initial resistance to antituberculosis drugs did not have a significant effect on smear conversion. CONCLUSION: This study demonstrates that HIV-seropositive status is not a principal factor in delaying sputum conversion among patients receiving intensive phase tuberculosis treatment. PMID- 10750518 TI - Influence of health education on community participation in rapid assessment of onchocerciasis prior to distribution of ivermectin. AB - OBJECTIVE: To investigate the impact of health education on community participation in the rapid assessment of onchocerciasis prior to distribution of ivermectin in Nigeria. DESIGN: There was health education with use of pictorial monographs to an adult population and school children in Umulumgbe and Okpatu communities, respectively. The school children in turn transferred the knowledge acquired to their parents through a health club, and a third community (Awhum) had no health education. Randomly selected subjects in each community were then assessed for their ability to recognise clinical manifestations of disease. SETTING: The study took place in three onchocerciasis-endemic, autonomous communities in Udi local government area of Enugu state in eastern Nigeria. SUBJECTS: Fifty, thirty seven, and thirty three male subjects, aged 20 years and above in Umulumgbe, Okpatu and Awhum respectively were involved in the study. RESULTS: 89.3%, 100% and 25.6% of the total number of onchocercal nodules were rightly indicated by the subjects in Umulumgbe, Okpatu and Awhum respectively. 100% of skin depigmentation was also reported in Umulumgbe and Okpatu each, and 50% in Awhum. Although some of the clinical manifestations (onchocercal nodules and skin depigmentation) were wrongly indicated, others (hanging groin and enlarged scrotum) were not reported by the subjects at all. CONCLUSION: This study clearly shows that health education is necessary for control programmes that are meant to be sustainable, especially the WHO-supported community-directed treatment with ivermectin (CDTI). PMID- 10750517 TI - Comparative efficacy and safety of chloroquine and alternative antimalarial drugs: a meta-analysis from six African countries. AB - OBJECTIVE: To evaluate the safety and efficacy of the currently used antimalarial drugs in six African countries. DESIGN: A meta-analysis. MAIN OUTCOME MEASURES: The role of efficacy, safety and cost on the selection of antimalarial drugs. RESULTS: The comparative efficacy study showed that amodiaquine (with > 90% cure rate) was superior to chloroquine and sulphadoxine-pyrimethamine at seven days schedule. The efficacy of amodiaquine was also observed to be comparable to that of mefloquine and halofantrine. The parasite clearance time (PCT) of these drugs ranged between two days and a week and the fever clearance time (FCT) was within 48 hours. The recrudescence rate at D14-D21 was found to be 12-17% in chloroquine and amodiaquine, while sulphadoxine-pyrimethamine showed a trend similar to halofantrine and mefloquine (0-12% recrudescence rate). Similarly, a big difference was also noted in the cost of the different antimalarial drugs. The pharmacokinetic data, however, showed that they are of similar profile, except in adverse features and contraindications, and values like their half-life (t1/2) where the long (t1/2) in drugs like sulphadoxine-pyrimethamine endows them with suppressive-cure feature, especially against recrudescent strains. Nevertheless, as these data are obtained from resident population in Africa, who however naive are exposed to few malaria challenges in their life, the results should not be directly extrapolated to total non immunes such as visitors from Europe. CONCLUSION: The choice of alternative antimalarial drugs should be mainly based on their relative efficacy, safety and cost. PMID- 10750519 TI - Health education and community participation in the control of urinary schistosomiasis in Ghana. AB - OBJECTIVE: To study the role of health education and community participation for the provision of facilities necessary for the control of urinary schistosomiasis in southern Ghana. HYPOTHESIS: Health education facilitates community participation in the provision of facilities for the control of bilharzia. STUDY AREA: Three rural communities drained by the Densu river in southern Ghana. PARTICIPANTS: Individuals aged 14 years and above formed groups of 10-12 persons by age, sex, ethnic and educational background; 15-16 groups were formed. INTERVENTIONS: Based on existing structures, one community received active, another passive health education and the third had no education. All three communities received chemotherapy. DESIGN: Study was carried out in three phases: pre-intervention phase--during which baseline data on residents' knowledge, attitude, beliefs and perception about bilharzia were collected using focus group discussions (FGD) prior to the second phase, intervention. Another FGD was held after 18 months to evaluate the intervention--third phase. RESULTS: This study suggests that most community members were aware of schistosomiasis but not as a disease. Before the health education, some residents believed bilharzia was a sign of manhood while others attributed the red colour of the urine to the red colour of a variety of sugar cane eaten in the area. After the health education, residents in the three areas constructed hand-dug wells. In addition, those who received active health education constructed two toilets for the schools and weeded the banks of the rivers. Residents also associated the disease with the water snail. CONCLUSION: Health education was useful in changing community perception on bilharzia. PMID- 10750520 TI - Susceptibility pattern of Neisseria gonorrhoeae to antimicrobial agents in Dar es Salaam. AB - OBJECTIVE: To determine the susceptibility pattern of local strains of Neisseria gonorrhoeae from Dar es salaam, Tanzania to locally used antibiotics. METHOD: Out of 429 Neisseria gonorrhoeae strains isolated between 1993 and 1995, one hundred and ninety nine were recovered and tested. Minimum inhibitory concentrations (MIC) of penicillin, doxycycline, erythromycin, cefuroxime and ciprofloxacin were determined by the E-test method while that of spectinomycin was measured by the agar dilution method. Penicillinase producing N. gonorrhoeae were identified by the chromogenic cephalosporin method. RESULTS: Of the 199 strains tested 128 (64%) were found to be penicillinase producing Neisseria gonorrhoeae (PPNG). Only 19 (10%) were penicillin sensitive while all penicillin resistant strains were found to be PPNG. One hundred and seventy five (88%), 11(5%) and 13 (7%) of the tested isolates were resistant, less susceptible and fully susceptible to doxycycline respectively. Resistance to cotrimoxazole, cefuroxime and ciprofloxacin was 36 (18%), 11 (6%), and 3 (2%) respectively. The trend of antibiotic susceptibility rates over the three year period of study showed a significant increase in the proportion of susceptible strains to cotrimoxazole. All of the 75 strains tested against spectinomycin were susceptible. There was a statistically significant difference between the susceptibility patterns of non PPNG and PPNG. Non-PPNG isolates were more susceptible to doxycycline (chi 2 = 78.2, df 2, p = < 0.0001). CONCLUSION: These findings have shown that spectinomycin, ciprofloxacin and cefuroxime could continue to be used to treat gonorrhoea in our settings. Continuous surveillance of susceptibility to the commonly used antibiotics is important in order to detect emergence of resistance early and control the possible wide spread of resistant strains. PMID- 10750521 TI - Antifertility activity of Ricinus communis seed in female guinea pigs. AB - OBJECTIVE: To investigate the anti-fertility effect of Ricinus communis seed extract. DESIGN: Laboratory-based experiment. SETTING: Laboratory of the Department of Pharmacology, Faculty of Medicine, Addis Ababa University, Addis Ababa, Ethiopia in 1996. RESULTS: The seed extract was found to possess anti implantation and abortifacient effects. It was also observed that the seed extract prolonged the oestrus cycle of guinea pigs. The dioestrus phase was significantly prolonged as well. After stopping administering the extract, however, the normal dioestrus phase and oestrus cycle started to resume. The seed extract also reduced the weight of the uterus without affecting that of the ovaries significantly. CONCLUSION: Ricinus communis possesses an anti-fertility effect in female guinea pigs, which might be extrapolated in human beings. These findings might support the accredited claim of its traditional use to avoid unwanted pregnancies. Further studies, however, should be pursued. PMID- 10750522 TI - Anomalies of the vermiform appendix and prevalence of acute appendicitis in Khartoum. AB - OBJECTIVE: To investigate certain anatomical features of the vermiform appendix and the prevalence of acute appendicitis among Sudanese citizens in Khartoum province. DESIGN: A prospective descriptive study of consecutive patients operated by a particular doctor. SETTING: Omdurman and Khartoum Teaching Hospitals from November 1995 to February 1996. INTERVENTION: Appendicectomy and recording particular characteristics of the appendix. MAIN OUTCOME MEASURES: (i) appendix position, (ii) variations of mesopharynx and (iii) length of appendix. RESULTS: Acute appendicitis was found to be more common in males than in females and among adults than children. The average length was 8.9 cm in males and 9.4 cms in females. The appendix was commonly found to be retrocaecal (58.3%) on pelvic (21.7%) or paracaecal (11.7%). Anomalies of the appendix were more common in children than adults and occurred in 47% of cases. CONCLUSION: Average length of the vermiform appendix was longer in females than in males. Ectopic positions such as left-sided appendix were not detected in this study. PMID- 10750523 TI - Serum heat-stable alkaline phosphatase activity in normal pregnancy. AB - OBJECTIVE: To determine the reference values for heat-stable alkaline phosphatase in apparently healthy non-pregnant and in normal pregnant females and to assess the suitability of this parameter as a measure of placental function and indirectly as a measure of foetal survival. DESIGN: A prospective study. SETTING: Ante-natal clinic, University of Nigeria Teaching Hospital, Enugu, Nigeria. PARTICIPANTS: Ninety eight apparently normal pregnant females in various gestational ages and 47 apparently healthy non-pregnant females. MAIN OUTCOME MEASURES: The serum heat-stable alkaline phosphatase activity. RESULTS: Variability in values exists between individuals in the same gestational age. The mean and standard deviation of serum heat-stable alkaline phosphatase was found to be 0.79 +/- 1.54 iu/L in the control group. No significant difference was found (p < 0.05) between this and the mean and standard deviation of serum alkaline phosphatase of 0.98 +/- 1.28 iu/L found in the first eight weeks of gestation. However the means and standard deviations of first trimester, second and third trimester were significantly higher than the controls. CONCLUSION: In the face of worsening economic situation in third world countries this simple method could be used in urban and some rural hospitals in assessing placenta insufficiency. PMID- 10750524 TI - Relationship between hypertension and hearing disorders in the elderly. AB - OBJECTIVE: To investigate the relationship between hypertension and hearing disorders of the elderly. DESIGN: Prospective case-control study. SETTING: Lin Yi People's Hospital, Shandong Province, the People's Republic of China, from October 1995 to June 1997. PARTICIPANTS: Fifty five hypertensive patients and fifty five normal elderly subjects (control group) between the ages of fifty five and eighty nine years old were enrolled in the study. All patients were male and were divided into three groups according to age as follows: Group A: 55-64; Group B: 65-74; Group C: 75-89. INTERVENTIONS AND MAIN OUTCOME MEASURES: SIMENS DA-64 audiometer and MEDSON 2250 auditory brainstem response audiometry (ABR) were utilised to determine the degree of hearing disorders. Serum triglycerides and cholesterol were also tested in our central laboratory. RESULTS: Auditory thresholds collected during audiometric tests increased gradually in proportion with age, especially in the hypertensive group (p < 0.05). The results of the ABR demonstrated that the latencies of wave V, I-VIPL and III-VIPL were also prolonged compared with normal elderly subjects. The patient's duration of illness and the complications of hypertension had an influence on hearing disorders in relation to the ageing process. CONCLUSION: Hypertension, with a high level of blood triglycerides and cholesterol, worsens the hearing of the elderly. The hearing disorder in elderly people is a result of long duration of illness and the complications of the hypertension. PMID- 10750526 TI - Metabolic response to injury. AB - OBJECTIVE: To outline the modern understanding of the aetiology of the metabolic response to injury. DATA RESOURCES: Review of the relevant literature of the twentieth century. STUDY SELECTION: Papers were selected for their relevance to the topic. Preference was for relevant animal and human studies. DATA EXTRACTION AND SYNTHESIS: Relevant papers were obtained through MEDLINE search strategies. Papers were read and analysed for accuracy of methodology, statistics, and application to the review in hand. The review also relied heavily on the author's own work. CONCLUSION: Cytokines are central to the metabolic response to injury and that understanding of their compartmentalisation in critical illness could lead to new therapeutic strategies. The complexity of the system is recognised and suggestions are made for the practising surgeon. PMID- 10750527 TI - Review of oesophageal foreign bodies in Harare Central Hospital. AB - OBJECTIVE: To review our experience in the management of patients with oesophageal foreign bodies. DESIGN: Retrospective analysis. SETTING: Harare Central Hospital, Harare, Zimbabwe. SUBJECTS: Sixty eight patients who presented with foreign body ingestion from October 1990 to December 1991. INTERVENTION: All but two patients underwent rigid oesophagoscopic examination. RESULTS: Sixty five of the cases with foreign bodies in the oesophagus had their foreign bodies retrieved by rigid oesophagoscopic procedure. CONCLUSION: A high index of suspicion will help in early diagnosis especially in children who present with recent and inexplicable onset of respiratory symptoms. PMID- 10750528 TI - A simple and inexpensive medium for culture of splenic aspirates in visceral leishmaniasis. PMID- 10750525 TI - Amniotic fluid index for screening late pregnancies. AB - OBJECTIVE: To find out whether amniotic fluid index (AFI) could be used to screen late pregnancies which presented at the clinic for the first time for any adverse perinatal outcome. DESIGN: Retrospective study that compared patients with AFI > or = 5 with those with AFI > or = 5. SETTING: Korle Bu Teaching Hospital, Accra, Ghana. PATIENTS: One thousand one hundred and eleven consecutive cases referred to the ultrasound unit for the first time after 36 weeks of pregnancy between 1st January 1997 and 31st December 1997. MAIN OUTCOME MEASURES: AFI and rate of induction, Caesarean Section; and incidence of foetal distress. RESULTS: Out of the 1111 patients, 350 had AFI < 5 and 761 had AFI > or = 5. The relative risk for induction and Caesarean section were 6.08 and 3.38 respectively. The relative risk for developing foetal distress was 8.48. The sensitivity was 79.6% and the specificity 89.5% in using AFI < 5 for predicting foetal distress. CONCLUSION: The results indicate that AFI < 5 could be used to screen late pregnancies for foetal distress. PMID- 10750529 TI - Socialization for survival: developmental issues among working street children in India. PMID- 10750530 TI - Daily reality on the streets of Campinas, Brazil. PMID- 10750531 TI - Street children in Nairobi: gender differences in mental health. PMID- 10750532 TI - Homeless youth in the United States: description and developmental issues. PMID- 10750533 TI - Methodological and ethical issues in research with street children. PMID- 10750534 TI - Children at the margins of society: research and practice. PMID- 10750535 TI - Keeping the peace or peace of mind? Maternal cognitions about sibling conflict and aggression. PMID- 10750536 TI - Predicting mother's use of physical punishment during mother-child conflicts in Sweden and Canada. PMID- 10750537 TI - Conceptual and empirical links between children's social spheres: relating maternal beliefs and preschoolers' behaviors with peers. PMID- 10750538 TI - Exploring the effects of low power schemas in mothers. PMID- 10750539 TI - Parenting cognitions and parent-child conflict: current issues and future directions. AB - The chapters in this volume explore a number of important issues in parenting cognitions. It is clear that they have opened up several new areas for consideration. We have suggested yet another new direction for researchers interested in cognitions and conflict having to do with the identification of automatic styles of information processing and their separation from more considered approaches. We have come some way in the study of parenting cognitions since the early 1980s; we have some distance yet to go. PMID- 10750540 TI - Institutional care: risk from family background or pattern of rearing? AB - Previous research has shown that children receiving substitute parental care tend to have high rates of emotional/behavioural disturbance, but uncertainty remains on the extent to which this derives from genetic risk, adverse experiences before receiving substitute care, or from risks associated with substitute care experiences. In order to examine the effects of institutional rearing (as a specific form of substitute care), two groups of primary school children reared in substitute care from before the age of 12 months were compared: 19 children in residential group (institutional) care and 19 in continuous stable foster family care (matched for age and gender). The two groups were similar in coming from biological families with high rates of psychopathology and social malfunctioning, but differed with respect to pattern of rearing. Both groups were compared with classroom controls, using teacher questionnaires, systematic classroom observations, and standardised cognitive testing. Parental questionnaires were also obtained for the two substitute care groups. As found previously, the combined substitute care groups differed from controls in showing a high level of hyperactivity/inattention. The observational measures showed a similar effect, indicating that the elevated rate was not attributable to rater bias. The teacher questionnaire and observational measures showed, however, that the increased level of hyperactivity/ inattention was substantially higher in the institutional group than the foster family group. Parental questionnaire ratings showed the same contrast between the groups, except that the main difference was on unsociability and emotional disturbance rather than hyperactivity/ inattention. It is concluded that, against a background of genetic and early environmental risk, institutional rearing predisposes to a pattern of hyperactivity/inattention. PMID- 10750541 TI - Emotional disclosure in school children. AB - Recent research with adults by Pennebaker and his colleagues has found that emotional disclosure through writing about stressful events appears to have significant benefits in terms of psychological and physical health outcomes. This report describes a controlled trial of emotional disclosure, adapted for school children, with the major hypothesis that the repeated description of negative events will have beneficial effects on measures of mental health, attendance, and school performance. The sample consisted of children aged 8-13 years from four schools, a primary and a secondary school both from a suburban and an inner-city area. Children were randomly assigned to one of three conditions: writing about negative events, writing about nonemotional events, and a non-writing control group. Children in all groups were seen four times during a single week and were then followed up after 2 months with measures of health and school performance. The intervention was well received by both schools and children, and the scripts written by the emotional and nonemotional writing groups differed in content in the predicted ways. Contrary to expectation, there was little evidence of a specific effect of emotional disclosure, and several possible reasons for this are discussed. Nevertheless, there was a general reduction in symptom measures, indicating that children may have benefited from their involvement in the study. Although there are several possible explanations for our findings, they indicate that it is both feasible and potentially valuable to give children opportunities to engage in discussion about sources of stress and their reactions to them. PMID- 10750542 TI - Children's understanding of martial conflict and the marital relationship. AB - Children's understanding of marital conflict and the marital relationship was investigated in 60 children of 5, 7, and 9 years old. Age differences were hypothesized in four areas of children's understanding of marital conflict and the marital relationship, based on the development between 5 and 9 years of age of children's abilities to (a) analyse complex sequences of behavior in terms of the mental states of protagonists and (b) integrate multiple social roles. The four areas investigated were: conceptions of marital conflict, conflict resolution, family triangulation, and integration of the spousal and parental roles. Children were read stories involving family interactions. Open-ended questions were asked about their understanding of these interactions and a coding scheme was devised that reflected theoretical concepts. Nine-year-olds differed from 5-year-olds on all aspects of their understanding of marital conflict and the marital relationship, and 7-year-olds showed levels of understanding between 5- and 9-year-olds. Younger children were less likely than older children to explain marital conflict in terms of the divergent goals of each parent, to understand conflict resolution as dependent on one person changing their beliefs or goals, to understand that parents were simultaneously spouses and parents and to understand the nature of triangulation. Marital conflict is interpreted very differently by children of different ages. PMID- 10750543 TI - Antisocial, angry, and unsympathetic: "hard-to-manage" preschoolers' peer problems and possible cognitive influences. AB - This study is the first to provide direct observations of dyadic interactions with friends for preschool-aged disruptive children. Forty preschoolers (mean age 52 months) rated by parents as "hard to manage" on Goodman's (1997) Strengths and Difficulties Questionnaire (SDQ), as well as 40 control children (matched for age, gender, school, and ethnic background) were filmed for 20 minutes on two occasions playing with a teacher-nominated best friend. The videos were transcribed and coded for antisocial behaviour, displays of negative emotion, and empathic/prosocial responses to friend's distress. Individual differences in social behaviour were considered in relation to false-belief performance, affective perspective taking, and executive function skills (planning and inhibitory control). Compared with controls, the hard-to-manage group showed significantly higher rates of both antisocial behaviour and displays of negative emotion, as well as significantly lower rates of emphatic/prosocial responses. Across both groups combined, frequencies of angry and antisocial behaviours were related to poor executive control. Mental-state understanding was not significantly correlated with antisocial behaviour, emotion display, or empathy, suggesting that the interpersonal problems of young disruptive children owe more to failure of behavioural regulation than to problems in social understanding per se. However, given the relatively low power of the study, these findings require replication with a larger sample. PMID- 10750544 TI - Predictive power of peer behavioral assessment for subsequent maladjustment in community samples of disruptive and nondisruptive children. AB - In a prospective, longitudinal study with community samples of disruptive and nondisruptive children, the predictive relations between peer-assessed dimensions of behavioral reputation in elementary school and teacher-rated behavioral adjustment and test scores of academic achievement were examined over a 4-year period. The Revised Class Play (RCP), a social role matching instrument completed by students about their classmates, was used to assess behavioral dimensions of children's peer reputation at baseline. Regression analyses showed that the RCP dimensions of aggressive-disruptive, sensitive-isolated, and social etiquette were the best predictors of later teacher-rated externalizing problems, internalizing problems, and subsequent adaptive skills respectively. Only IQ predicted later academic achievement. When IQ and disruptive/nondisruptive group status were added to each regression equation they further contributed as significant predictors. When parent and teacher ratings of behavior problems at baseline were entered jointly with RCP dimensions into regression equations, the peer dimensions further explained outcome variance. Additive effects of the RCP dimensions were found only for the externalizing problems outcome. Disruptive children with mild levels of aggressive behavior and high levels of sensitive isolated behavior had less externalizing problems at outcome. A comparison of the difference between disruptive and nondisruptive groups in the relationship of baseline RCP scores to teacher outcomes showed no difference between groups, suggesting that the groups did not depart appreciably from their initial differences relative to each other over time. PMID- 10750545 TI - Childhood peer relationship problems and later risks of educational under achievement and unemployment. AB - This paper examines relations between the extent of children's peer relationship problems at age 9 and their later risks of educational under-achievement and unemployment by the age of 18 years. Results showed that children with high rates of early peer relationship problems were at increased risk of under-achievement and unemployment when compared with children with low rates of early peer relationship problems. These elevated educational and occupational risks were explained by two processes. First, associations between early peer difficulties and later disadvantage were in part noncausal, and arose because of the personal characteristics (IQ and attentional problems) and social backgrounds (socioeconomic adversity, exposure to parental change, and punitive parent-child interaction) of children with early peer problems. Second, problematic peer relations during childhood appeared to place young people at increased risk of a range of adolescent interpersonal and school-related difficulties, including weaker peer attachments, interpersonal problems with teachers, school truancy, suspension, and early school leaving. In turn, these experiences and behaviours served to reduce the educational and employment opportunities of children with early peer problems. Results highlight the importance of childhood peer relationships for academic and occupational success. PMID- 10750546 TI - Precursors of literacy delay among children at genetic risk of dyslexia. AB - This paper reports the literacy skills of 63 children selected as being at genetic risk of dyslexia compared with 34 children from families reporting no history of reading impairment. Fifty-seven per cent of the at-risk group were delayed in literacy development at 6 years compared with only 12% of controls. The "unimpaired" at-risk group were not statistically different from controls on most cognitive and language measures at 45 months, whereas the literacy-delayed group showed significantly slower speech and language development, although they did not differ from controls in nonverbal ability. Letter knowledge at 45 months was the strongest predictor of literacy level at 6 years. In addition, early speech and language skills predicted individual differences in literacy outcome and genetic risk accounted for unique variance over and above these other factors. The results are discussed in terms of an interactive developmental model in which semantic and phonological skills support early reading acquisition. PMID- 10750547 TI - Social and emotional adjustment in children affected with Gilles de la Tourette's syndrome: associations with ADHD and family functioning. Attention Deficit Hyperactivity Disorder. AB - This study examined social-emotional functioning in children with Gilles de la Tourette's syndrome (TS) alone and children with TS and Attention Deficit Hyperactivity Disorder (ADHD). In addition, the contribution of family functioning to social competence was examined. Children with a clinical diagnosis of TS were recruited from the Yale Child Study Center TS specialty clinic. Unaffected control children were recruited through newspaper advertisements and announcements within the university and at area schools. The final sample consisted of 72 children (45 boys and 27 girls) between the ages of 8 and 14. Sixteen children met DMS-III-R criteria for TS, 33 children met criteria for TS and ADHD, and 23 children had no psychiatric diagnoses. Children with TS and ADHD evidenced more externalizing and internalizing behavior problems and poorer social adaptation than children with TS only or unaffected controls. Children with TS only were not significantly different from unaffected controls on most measures of externalizing behaviors and social adaptation but did exhibit more internalizing symptoms. Tic symptom severity was not associated with social, behavioral, or emotional functioning among children with TS, even after stratifying by medication status. However, ADHD diagnosis, obsessional symptom severity, and family functioning were significantly associated with social and emotional adjustment among TS children. Moreover, family functioning was associated with social and emotional adjustment even after controlling for TS and ADHD diagnostic status. These findings demonstrate that much of the social and behavioral dysfunction in children with TS is ADHD-specific and children with TS alone have a very different social-emotional profile than do those with TS plus ADHD. Finally, social-emotional adjustment in children with TS is best understood within the family context. PMID- 10750548 TI - Stimulation seeking and hyperactivity in children with ADHD. Attention Deficit Hyperactivity Disorder. AB - Thirty hyperactive and 30 non-hyperactive children were confronted with a delay, consisting of a waiting situation of 15 minutes, either with or without extra stimulation provided by the presentation of a videotape. The behaviour of the child during the waiting period was videotaped and later coded by two naive observers. In line with theories that emphasise the stimulation-seeking function of hyperactive behaviours, such as the optimal stimulation account and the delay aversion theory, a group by stimulation effect was hypothesised. For two categories of activity this was found, with ADHD children displaying more activity than non-ADHD children in the no-stimulation but not in the stimulation condition. These data provide support for the stimulation-seeking function of certain features of ADHD hyperactivity. PMID- 10750549 TI - Verbal short-term memory deficits in Down syndrome: a consequence of problems in rehearsal? AB - Individuals with Down syndrome suffer from relatively poor verbal short-term memory. Previous explanations of this deficit have been framed in terms of inefficient or absent rehearsal of verbal material in Down syndrome within the phonological loop component of Baddeley and Hitch's (1974) working memory model. Two experiments are presented which test this explanation by looking for the markers of rehearsal in children with Down syndrome and verbal mental age matched controls. Both experiments confirm that individuals with Down syndrome show poorer verbal short-term memory performance than controls. However, they rule out rehearsal as an explanation of these deficits because the evidence suggests that neither individuals with Down syndrome nor matched controls are engaging in spontaneous subvocal rehearsal. Other explanations of poor verbal short-term memory performance in Down syndrome, in terms of impairments both within and outside of the phonological loop system, are discussed. Practical implications for intervention strategies aimed at improving verbal short-term memory skills in Down syndrome are also outlined. PMID- 10750550 TI - Latina mothers' attributions, emotions, and reactions to the problem behaviors of their children with developmental disabilities. AB - We examined the applicability of attribution theory to mothers' perceptions and reactions to their child's problem behavior. Participants were 149 Latina mothers of children with developmental disabilities who were interviewed regarding specific incidents in which their child exhibited a behavior problem. The findings indicate that most mothers viewed their child as not being responsible for the behavior problem. Furthermore, as predicted by attribution theory, mothers who ascribed relatively high responsibility to the child were significantly more likely to report negative emotions (anger and frustration) and aggressive/harsh behavioral reactions than mothers who ascribed low responsibility. Also. mothers were more likely to ascribe high responsibility to the child when the problem was characterized as a behavioral excess than as a behavioral deficit. The results provide support for the applicability of an attributional framework and may have important implications for helping parents in addressing the problem behaviors of their children with developmental disabilities. PMID- 10750551 TI - Behavioral and emotional problems in Chinese children: teacher reports for ages 6 to 11. AB - This study examined the applicability of the Chinese Version of Teacher's Report Form (TRF-CV) and estimated the prevalence of behavioral problems in a general population sample of 2,936 children aged 6 through 11 years in the Shandong Province of China. Teachers completed the TRF-CV and the Conners Hyperkinesis Index (CHI). The TRF-CV total scale showed satisfactory 2-week test-retest reliability (r = .83) and internal consistency (Cronbach's alpha = .94). The TRF CV Total Problems, Attention Problems, Delinquent Behavior, and Aggressive Behavior had acceptable concurrent validity with the CHI (mean r = .62). With the TRF-CV Total Problems score of 26 as a cutoff, an overall correct classification rate of 90% for clinical sample and nonreferral required children was obtained. Exploratory factor analysis yielded six syndromes: Aggressive/Delinquent Behavior, Withdrawn/Depressed, Somatic Complaints, Attention Problems, Social Problems, and Thought Problems, with significant correlations with corresponding American cross-informant syndromes (mean r = .84). The overall prevalence rate of behavioral problems was 15.5% (95% CI = 14.2-16.8%), with a boy-to-girl ratio of 2.0:1 (chi2 = 59.70, p < .001). Younger boys exhibited more externalizing problems. These findings indicate that the TRF-CV is applicable for Chinese children, and the prevalence of behavioral problems shown by it among Chinese children seems comparable to that found in other countries. Although most of the American syndromes were well replicated, the differences in the present subjects, when submitted to principal components analysis, from American samples from whom the original syndromes were derived, could have prevented the study from replicating distinctions between aggressive vs. delinquent and depressed vs. withdrawn syndromes. PMID- 10750552 TI - Bilingualism, school achievement, and mental wellbeing: a follow-up study of return migrant children. AB - In the first phase of this follow-up study we investigated how the use of more than one language affects mental wellbeing and school achievement among 320 school-aged Finnish-Swedish re-migrant children. Now, in the second phase, we screened the same series of children 6 years after migration for psychiatric and psychosomatic symptoms. Out of five groups distinguished in terms of patterns of language use, two had fared well and three showed evident vulnerability. Both successful groups were marked by consistent use of the two languages, Finnish and Swedish, whereas the risk groups were characterised by mixed use of languages before re-migration or substantial language shift after re-migration. PMID- 10750553 TI - Genetic epidemiological approaches to the search for osteoporosis genes. AB - Important progress has been made in the identification of specific environmental factors and estimation of hereditary components in bone density, quantitative ultrasound (QUS), and bone turnover indices. By contrast, the search for specific genes that regulate bone mass has progressed rather slowly, and the results are more difficult to interpret and reproduce. This article reviews the genetics of osteoporosis and problems plaguing genetic research. It is argued that the search for genes involved in the expression of osteoporotic phenotypes should be based on linkage studies in relatively homogeneous populations. Strategies for increasing the power of studies, such as making use of information from extended pedigrees and multivariate analysis, are discussed. With the advent of a comprehensive human genetic linkage map, a complete identification of genes for osteoporosis appears feasible. Understanding the genetic mechanisms and their interactions with environmental factors should allow more focused and cost effective osteoporosis prevention and treatment strategies. PMID- 10750554 TI - Osteoporotic fractures are associated with an 86-base pair repeat polymorphism in the interleukin-1--receptor antagonist gene but not with polymorphisms in the interleukin-1beta gene. AB - Interleukin-1beta (IL-1beta) is a potent stimulator of bone resorption, and has been implicated in the pathogenesis of high bone turnover and osteoporosis. IL-1 receptor antagonist (IL-1ra) is a competitive inhibitor of IL-1beta effects and the biological effects of IL-1beta are therefore proportional to the ratio IL 1beta/IL-1ra. The coding regions of IL-1beta were examined for sequence variations by SSCP and sequencing after polymerase chain reaction (PCR) of genomic DNA. Three previously described polymorphisms (C(-511)-T, G(3877)-A and C(3954)-T) in the IL-1beta gene were determined by restriction fragment length polymorphism (RFLP) using Ava I, Aci I, and Taq I after PCR. The 86-base pair repeat polymorphism in IL-1ra was examined by PCR and electrophoresis and the T11100-C polymorphism in the IL-1ra gene was examined by RFLP using MspA1I after PCR. All polymorphisms were related to bone mass, biochemical markers of bone turnover, and presence of fracture in a study including 389 osteoporotic patients with vertebral fractures and normal controls. Two normal women were heterozygous for a shift from cytosine to thymine (C3263-T) in exon 4 of the IL-1beta gene. This substitution did not affect the amino acid sequence. We did not find other sequence variations in the IL-1beta gene apart from the already known polymorphisms. The distribution of C(-511)-T, G(3877)-A, and C(3954)-T genotypes was similar in the osteoporotic and the normal controls. No significant differences could be shown in bone mass or bone turnover. In the IL-1ra gene almost complete linkage was confirmed between the already known polymorphisms: G(1731)-A, G(1821)-A, A(1868)-G, G(1887)-C, T(8006)-C, C(8061)-T, 86 base pair variable number tandem repeat (VNTR), A(9589)-T, and a new polymorphism: T(1934) C. The A1A1/A3 genotypes of the IL-1ra VNTR polymorphism were significantly more frequent in osteoporotic patients (56.2%) compared with age-matched normal controls (43.3%) (chi2 = 4.09; p = 0.043). The relative risk of osteoporotic fractures was increased to 1.68 (95% CI, 1.01-2.77) in individuals with A1A1/A3 genotypes. Bone mineral density (BMD) of the lumbar spine was reduced in individuals with A1A1/A3 genotypes (p = 0.014, analysis of variance [ANOVA]). The difference in bone mass between A1A1/A3 and A2A1/A2 tended to increase with increasing age. T1100-C genotypes were distributed similarly in osteoporotic patients and normal controls and the polymorphism was without effect on bone mass and biochemical markers of bone turnover. In conclusion, an 86-base pair repeat polymorphism in the IL-lra gene is associated with increased risk of osteoporotic fractures. Other polymorphisms in the IL-1ra and the IL-1beta genes are not associated with osteoporotic fractures or alterations in bone mass or bone turnover. PMID- 10750555 TI - Association of transforming growth factor beta1 genotype with therapeutic response to active vitamin D for postmenopausal osteoporosis. AB - Transforming growth factor beta (TGF-beta) is an important regulator of bone metabolism, its effects being intertwined with those of estrogen and vitamin D. A T-->C polymorphism in exon 1 of the TGF-beta1 gene, which results in the substitution of proline for leucine, is associated with bone mineral density (BMD). However, it is not known whether this polymorphism affects the response to treatment with active vitamin D or to hormone replacement therapy (HRT) in individuals with osteoporosis. Changes in BMD at the lumbar spine (L2-L4 BMD) were compared among TGF-beta1 genotypes in 363 postmenopausal Japanese women who were divided into three groups: an untreated, control group (n = 130), an active vitamin D treatment group (n = 117), and an HRT group (n = 116). TGF-beta1 genotype was determined with an allele-specific polymerase chain reaction assay. In the control group, the rate of bone loss decreased according to the rank order of genotypes TT (homozygous for the T allele) > TC (heterozygous) > CC (homozygous for the C allele), with a significant difference detected between the CC and TT genotypes. The positive response of L2-L4 BMD to HRT increased according to the rank order of genotypes TT < TC < CC, although the differences among genotypes were not statistically significant. Individuals with the CC genotype responded to active vitamin D treatment with an annual increase in L2-L4 BMD of 1.6%, whereas those with the TT or TC genotypes similarly treated lost bone to a similar extent as did untreated subjects of the corresponding genotype. These results suggest that TGF-beta1 genotype is associated with both the rate of bone loss and the response to active vitamin D treatment. PMID- 10750556 TI - Genomic organization of the human chondromodulin-1 gene containing a promoter region that confers the expression of reporter gene in chondrogenic ATDC5 cells. AB - Chondromodulin-1 (ChM-1) is a cartilage-specific glycoprotein that stimulates the growth of chondrocytes and inhibits the tube formation of endothelial cells. To clarify the tissue-specific expression and the role of ChM-1 in pathophysiological conditions, we analyzed the structure of the human ChM-1 gene and its promoter. On the screening of a human genomic cosmid library using the human ChM-1 complimentary DNA (cDNA) as a probe, two clones were obtained that contained ChM-1 cDNA. The restriction enzyme map and nucleotide sequence revealed the human ChM-1 gene consisting of seven exons and exon-intron boundaries. The human ChM-1 gene was assigned to chromosome 13q14-21 by fluorescence in situ hybridization (FISH) using the clone as a probe. A primer extension analysis using total RNA extracted from human cartilage revealed a major transcription start site with the sequence CGCT+1GG. The region approximately 3-kilobase (kb) nucleotides upstream of the translation start site was then sequenced and analyzed in terms of promoter activity. We found that a region 446 base pairs (bp) upstream of the start site had promoter activity in COS7, HeLa, and ATDC5 cells. In structure the promoter is a TATA-less type without a GC-rich region. The transcription factors Sox9, Og12, and Cart-1 did not affect the promoter activity. The transcription factor Ying-Yang1 suppressed the promoter activity but GABP protein did not change the promoter activity. The construct containing 446/+87 fused to the SV40 enhancer and green fluorescent protein (GFP) exhibited expression of GFP corresponding to the differentiation of ATDC5 cells to mature chondrocytes. These results suggest that the element -446/+87 confers the cartilage-specific expression of this gene by some factor(s) other than Sox9, Og12, and Cart-1. PMID- 10750558 TI - Cytoskeletal abnormalities in chondrocytes with EXT1 and EXT2 mutations. AB - The EXT genes are a group of putative tumor suppressor genes that previously have been shown to participate in the development of hereditary multiple exostoses (HME), HME-associated and isolated chondrosarcomas. Two HME disease genes, EXT1 and EXT2, have been identified and are expressed ubiquitously. However, the only known effect of mutations in the EXT genes is on chondrocyte function as evidenced by aberrant proliferation of chondrocytes leading to formation of bony, cartilage-capped projections (exostoses). In this study, we have characterized exostosis chondrocytes from three patients with HME (one with EXT1 and two with EXT2 germline mutations) and from one individual with a non-HME, isolated exostosis. At the light microscopic level, exostosis chondrocytes have a stellate appearance with elongated inclusions in the cytoplasm. Confocal and immunofluorescence of in vitro and in vivo chondrocytes showed that these massive accumulations are composed of actin bundled by 1.5-microm repeat cross-bridges of alpha-actinin. Western blot analysis shows that exostosis chondrocytes from two out of three patients aberrantly produce high levels of muscle-specific alpha actin, whereas beta-actin levels are similar to normal chondrocytes. These findings suggest that mutations in the EXT genes cause abnormal processing of cytoskeleton proteins in chondrocytes. PMID- 10750557 TI - Biomineralization, life-time of odontogenic cells and differential expression of the two homeobox genes MSX-1 and DLX-2 in transgenic mice. AB - Msx and Dlx homeobox genes encode for transcription factors that control early morphogenesis. More specifically, Msx-1, Msx-2, and Dlx-2 homeobox genes contribute to the initial patterning of the dentition. The present study is devoted to the potential role of those homeobox genes during the late formation of mineralized tissues, using the rodent incisor as an experimental system. The continuously erupting mandibular incisor allows (1) the coinvestigation of the whole sequences of amelogenesis and dentinogenesis, aligned along the main dental axis in a single sample in situ and (2) the differential characterization of transcripts generated by epithelial and ectomesenchymal odontogenic cells. Northern blot experiments on microdissected cells showed the continuing expression of Msx-2 and Dlx-2 in the later stages of dental biomineralization, differentially in epithelial and ectomesenchymal compartments. Transgenic mice produced with LacZ reporter constructs for Dlx-2 and Msx-1 were used to detect different components of the gene expression patterns with the sensitive beta galactosidase histoenzymology. The results show a prominent epithelial involvement of Dlx-2, with stage-specific variations in the cells involved in enamel formation. Quantitative analyses identified specific modulations of Dlx-2 expression in ameloblasts depending on the anatomical sites of the incisor, showing more specifically an inverse linear relationship between the Dlx-2 promoter activity level and enamel thickness. This investigation extends the role of homeoproteins to postmitotic stages, which would control secretory cell activity, in a site-specific manner as shown here for Dlx-2. PMID- 10750560 TI - Prevalence of pelvic Paget's disease of bone in the United States. AB - The objective of this article was to estimate the prevalence of Paget's disease of bone in the United States from a statistically derived sample of the general population. Pelvic radiographs obtained in the First National Health and Nutrition Examination Survey (NHANES-I) were reviewed for the presence of Paget's disease. Age, sex, and geographic distribution of Paget's disease of the pelvic region were determined. The overall prevalence of Paget's disease in the United States was estimated. Pelvic Paget's disease is estimated to be present in 0.71 + 0.18% of the radiographs of the general population. The disease was higher in frequency in people who were in the older decades of life with the highest prevalence of 2.32 + 0.54% in the 65- to 74-year-old people. There is a slight male predominance in the 45- to 74-year age group. The regional distribution suggests the highest prevalence in the Northeast (1.48 + 0.52%) with the lowest prevalence in the South (0.26+0.25%). The prevalence was equal in white people and black people. An estimate of the overall prevalence of Paget's disease in the United States was at least 1% and perhaps as much as 2 % of the general population with near equal sex distribution and the highest prevalence in the northeastern United States. PMID- 10750559 TI - The microphthalmia transcription factor regulates expression of the tartrate resistant acid phosphatase gene during terminal differentiation of osteoclasts. AB - The defective terminal differentiation of osteoclasts in mice homozygous for the mi allele of the microphthalmia transcription factor (MITF) gene implies that MITF plays a critical role in regulating gene expression during osteoclast ontogeny. To begin addressing the role of this transcription factor in the osteoclast, target genes need to be identified. In the present work, several lines of evidence show that the gene encoding the enzyme tartrate-resistant acid phosphatase (TRAP) is a target of MITF. Analysis of osteoclasts in vivo in the embryonic forelimb showed that MITF and TRAP RNA were coexpressed in a dynamic pattern during the process of endochondral ossification of long bone. Primary osteoclast-like cells (OCLs) produced from mi/mi mutant mice expressed TRAP messenger RNA (mRNA) at 8-fold lower levels than in OCLs derived from normal mice, indicating a direct link between MITF function and TRAP expression. The activity of mouse TRAP promoter-reporter genes was assayed in the primary OCLs by DNA-mediated transfection, and this activity was shown to depend on a conserved sequence (GGTCATGTGAG) located in the proximal promoter. Recombinant MITF protein recognized specifically this conserved sequence element. Expression of a TRAP promoter-green fluorescent protein (GFP) transgene mimicked the expression of the endogenous TRAP gene during differentiation of osteoclast-like cells, and the expression of the transgene was decreased 8-fold when placed into the mutant mi/mi background. These results are consistent with a role for MITF in gene expression during terminal differentiation of the osteoclast and will allow osteoclast-specific mechanisms of gene regulation to be studied in greater detail. PMID- 10750561 TI - Direct and indirect actions of fibroblast growth factor 2 on osteoclastic bone resorption in cultures. AB - Fibroblast growth factor 2 (FGF-2 or basic FGF) is known to show variable actions on bone formation and bone resorption. This study was undertaken to elucidate the mechanisms whereby FGF-2 affects bone metabolism, especially bone resorption, using three different culture systems. FGF-2 at 10(-9) M and higher concentrations induced osteoclastic cell formation in the coculture system of mouse osteoblastic cells and bone marrow cells, and this induction was abrogated by nonsteroidal anti-inflammatory drugs (NSAIDs). 45Ca release from prelabeled cultured mouse calvariae stimulated by FGF-2 (10(-8) M) was also inhibited by NSAIDs, and the inhibition was stronger by NSAIDs, which are more selective for inhibition of cyclooxygenase 2 (COX-2) than COX-1, suggesting the mediation of COX-2 induction. COX-2 was highly expressed and its messenger RNA (mRNA) level was stimulated by FGF-2 in osteoblastic cells whereas it was undetectable or not stimulated by FGF-2 in cells of osteoclast lineage. To further investigate the direct actions of FGF-2 on osteoclasts, resorbed pit formation was compared between cultures of purified osteoclasts and unfractionated bone cells from rabbit long bones. FGF-2 (> or = 10(-12) M) stimulated resorbed pit formation by purified osteoclasts with a maximum effect of 2.0-fold at 10(-11) M, and no further stimulation was observed at higher concentrations. However, FGF-2 at 10( 9) M - 10(-8) M stimulated resorbed pit formation by unfractionated bone cells up to 9.7-fold. NS-398, a specific COX-2 inhibitor, did not affect the FGF-2 stimulation on purified osteoclasts but inhibited that on unfractionated bone cells. We conclude that FGF-2 at low concentrations (> or =10(-12) M) acts directly on mature osteoclasts to resorb bone moderately, whereas at high concentrations (> or = 10(-9) M) it acts on osteoblastic cells to induce COX-2 and stimulates bone resorption potently. PMID- 10750562 TI - Decreased nitric oxide levels stimulate osteoclastogenesis and bone resorption both in vitro and in vivo on the chick chorioallantoic membrane in association with neoangiogenesis. AB - High nitric oxide (NO) levels inhibit osteoclast (OC)-mediated bone resorption in vivo and in vitro, and nitrate donors protect against estrogen-deficient bone loss in postmenopausal women. Conversely, decreased NO production potentiates OC bone resorption in vitro and is associated with in vivo bone loss in rats and humans. Previously, we reported that bone sections from rats administered aminoguanidine (AG), a selective inhibitor of NO production via inducible NO synthase, exhibited both increased OC resorptive activity as well as greater numbers of OC. Here, we investigated further whether AG promoted osteoclastogenesis, in addition to stimulating mature OC function, using a modified in vivo chick chorioallantoic membrane (CAM) system and an in vitro chick bone marrow OC-like cell developmental model. AG, focally administered in small agarose plugs placed directly adjacent to a bone chip implanted on the CAM, dose-dependently elicited neoangiogenesis while stimulating the number, size, and bone pit resorptive activity of individual OC ectopically formed in vivo. In addition to enhancing OC precursor recruitment via neoangiogenesis, AG also exerted other vascular-independent effects on osteoclastogenesis. Thus, AG promoted the in vitro fusion and formation from bone marrow precursor cells of larger OC-like cells that contained more nuclei per cell and exhibited multiple OC differentiation markers. AG stimulated development was inversely correlated with declining medium nitrite levels. In contrast, three different NO donors each dose-dependently inhibited in vitro OC-like cell development while raising medium nitrite levels. Therefore, NO sensitively regulates OC-mediated bone resorption through affecting OC recruitment (angiogenesis), formation (fusion and differentiation), and bone resorptive activity in vitro and in vivo. Possibly, the stimulation of neoangiogenesis and OC-mediated bone remodeling via AG or other pro-angiogenic agents may find clinical applications in reconstructive surgery, fracture repair, or the treatment of avascular necrosis. PMID- 10750563 TI - Culture in vector-averaged gravity under clinostat rotation results in apoptosis of osteoblastic ROS 17/2.8 cells. AB - Space flight experiments and studies carried out in altered gravity environments have revealed that exposure to altered gravity conditions results in (mal)adaptation of cellular function. In the present study, we used a clinostat to generate a vector-averaged gravity environment. We then evaluated the responses of osteoblast-like ROS 17/2.8 cells subsequent to rotation at 50 revolutions per minute (rpm) for 6-24 h. We found that the cells started to detach from the substrate between 12 h and 24 h of rotation in clinostat but not in stationary cultures or after horizontal rotation (the latter serving as a motion control for turbulence, shear forces, and vibrations). At 24 h, 35% of clinorotated cells had detached and the cells underwent apoptotic death as evidenced by DNA fragmentation analysis, terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining, and flow cytometry with Annexin V staining. The apoptotic death was associated with perinuclear distribution of cell-surface integrin beta1 and disorganization of actin cytoskeleton. These results suggest that vector-averaged gravity causes apoptosis of osteoblasts by altering the organization of the cytoskeleton. We hypothesize that apoptotic death of osteoblasts might play an important role in the pathogenesis of osteoporotic bone loss as observed in actual space flights. PMID- 10750564 TI - Direct action of naturally occurring estrogen metabolites on human osteoblastic cells. AB - This article describes experiments that were performed to examine the direct action of estrogen metabolites on cultured human osteoblast cells. The human fetal osteoblastic cell line, hFOB/ER9, which expresses high levels of the estrogen receptor (ER) alpha, was used to examine the direct effects of 16alpha hydroxyestrone (16alpha-OHE1) and 2-hydroxyestrone (2-OHE1) on osteoblast differentiation. The 16alpha-OHE1 caused a decrease in osteocalcin (OC) secretion to a maximum of 40% of control values (vehicle-treated cells) at 10(-7) M. Alkaline phosphatase (AP) activity was significantly induced at 10(-7) M 16alpha OHE1 with greater than 500% of control at 10(-6) M 16alpha-OHE1. Finally, AP steady-state messenger RNA (mRNA) levels were increased within 24 h of 16alpha OHE1 treatment. In contrast to 16alpha-OHE1, 2-OHE1 had no effects on the secretion of OC, AP activity, or AP gene expression. The 2-OHE1 also did not display any antiestrogen activity because treatment in combination with 17beta estradiol (E2) and 16alpha-OHE1 had no significant effect on the reduction in OC secretion or induction of AP activity. Similar to E2, 16alpha-OHE1 stimulated the expression of an early response gene, a TGF-beta inducible early gene, designated TIEG, as early as 60 minutes after treatment, whereas treatment with 2-OHE1 displayed no effect. Support that the 16alpha-OHE1 regulation of these osteoblasts (OB) markers was mediated through the ER is shown by the fact that the estrogen antagonist ICI 182,780 abrogated these effects. These data suggest that is a potent estrogen agonist on human osteoblastic hOB/ER9 cells. In contrast, 2-OHE1 displayed no estrogenic or antiestrogenic activity in this human osteoblast cell model. PMID- 10750565 TI - Bone has a sexually dimorphic response to aromatase deficiency. AB - Aromatase synthesizes estrogen from androgen precursors. To better understand the role of estrogen in skeletal metabolism and growth, we have assessed long bone growth and histomorphometry in aromatase-deficient (ArKO) mice. The age range for the animals was 5-7 months. At this age mice have already achieved peak bone density but continue slow bone growth. Femur length, an index of long bone growth, showed decreased growth in ArKO males compared with wild-type (wt) littermates but no significant difference in females. Radiographically, compared with age- and sex- matched littermates both ArKO males and females showed osteopenia in the lumbar spine. Histologically, both ArKO males and females showed an osteoporotic-type picture, characterized by significant decreases in trabecular bone volume and trabecular thickness. However, compared with wt littermates female ArKO animals showed a bone remodeling picture consistent with increased bone turnover, much like early postmenopausal osteoporosis in humans. On the other hand, male ArKO animals showed decreases in both osteoblastic and osteoclastic surfaces compared with wt littermates, similar to age-related osteopenia. These findings suggest that osteoporosis seen in aromatase-deficient mice may arise from different bone remodeling activities between males and females. These results also show that the ArKO model exhibits the expected results of estrogen deficiency and may be a good model for investigating sex specific responses to estrogen deficiency. Furthermore, they imply that estrogen is important for attaining peak bone mass in male as well as in female mice. PMID- 10750566 TI - Vitamin K2 (menatetrenone) effectively prevents fractures and sustains lumbar bone mineral density in osteoporosis. AB - We attempted to investigate whether vitamin K2 (menatetrenone) treatment effectively prevents the incidence of new fractures in osteoporosis. A total of 241 osteoporotic patients were enrolled in a 24-month randomized open label study. The control group (without treatment; n = 121) and the vitamin K2-treated group (n = 120), which received 45 mg/day orally vitamin K2, were followed for lumbar bone mineral density (LBMD; measured by dual-energy X-ray absorptiometry [DXA]) and occurrence of new clinical fractures. Serum level of Glu-osteocalcin (Glu-OC) and menaquinone-4 levels were measured at the end of the follow-up period. Serum level of OC and urinary excretion of deoxypyridinoline (DPD) were measured before and after the treatment. The background data of these two groups were identical. The incidence of clinical fractures during the 2 years of treatment in the control was higher than the vitamin K2-treated group (chi2 = 10.935; p = 0.0273). The percentages of change from the initial value of LBMD at 6, 12, and 24 months after the initiation of the study were -1.8 +/- 0.6%, -2.4 +/- 0.7%, and -3.3 +/- 0.8% for the control group, and 1.4 +/- 0.7%, -0.1 +/- 0.6%, and -0.5 +/- 1.0% for the vitamin K2-treated group, respectively. The changes in LBMD at each time point were significantly different between the control and the treated group (p = 0.0010 for 6 months, p = 0.0153 for 12 months, and p = 0.0339 for 24 months). The serum levels of Glu-OC at the end of the observation period in the control and the treated group were 3.0 +/- 0.3 ng/ml and 1.6 +/- 0.1 ng/ml, respectively (p < 0.0001), while the serum level of OC measured by the conventional radioimmunoassay (RIA) showed a significant rise (42.4 +/-6.9% from the basal value) in the treated group at 24 months (18.2 +/- 6.1% for the controls;p = 0.0081). There was no significant change in urinary DPD excretion in the treated group. These findings suggest that vitamin K2 treatment effectively prevents the occurrence of new fractures, although the vitamin K2 treated group failed to increase in LBMD. Furthermore, vitamin K2 treatment enhances gamma-carboxylation of the OC molecule. PMID- 10750567 TI - Transient chondrogenic phase in the intramembranous pathway during normal skeletal development. AB - Calvarial and facial bones form by intramembranous ossification, in which bone cells arise directly from mesenchyme without an intermediate cartilage anlage. However, a number of studies have reported the emergence of chondrocytes from in vitro calvarial cell or organ cultures and the expression of type II collagen, a cartilage-characteristic marker, in developing calvarial bones. Based on these findings we hypothesized that a covert chondrogenic phase may be an integral part of the normal intramembranous pathway. To test this hypothesis, we analyzed the temporal and spatial expression patterns of cartilage characteristic genes in normal membranous bones from chick embryos at various developmental stages (days 12, 15 and 19). Northern and RNAse protection analyses revealed that embryonic frontal bones expressed not only the type I collagen gene but also a subset of cartilage characteristic genes, types IIA and XI collagen and aggrecan, thus resembling a phenotype of prechondrogenic-condensing mesenchyme. The expression of cartilage-characteristic genes decreased with the progression of bone maturation. Immunohistochemical analyses of developing embryonic chick heads indicated that type II collagen and aggrecan were produced by alkaline phosphatase activity positive cells engaged in early stages of osteogenic differentiation, such as cells in preosteogenic-condensing mesenchyme, the cambium layer of periosteum, the advancing osteogenic front, and osteoid bone. Type IIB and X collagen messenger RNAs (mRNA), markers for mature chondrocytes, were also detected at low levels in calvarial bone but not until late embryonic stages (day 19), indicating that some calvarial cells may undergo overt chondrogenesis. On the basis of our findings, we propose that the normal intramembranous pathway in chicks includes a previously unrecognized transient chondrogenic phase similar to prechondrogenic mesenchyme, and that the cells in this phase retain chondrogenic potential that can be expressed in specific in vitro and in vivo microenvironments. PMID- 10750568 TI - Vascular endothelial growth factor is expressed in human fetal growth cartilage. AB - Angiogenesis is a crucial event in endochondral ossification. Chemoattractants and mitogens for endothelial cells (such as basic fibroblast growth factor [bFGF] and transforming growth factor beta [TGF-beta]), which act as local regulators of the process, are synthesized by chondrocytes under several stimuli and in relation to the differentiation stage of the cartilage. Vascular endothelial growth factor (VEGF) is a 44-kDa protein well known as a potent angiogenic molecule owing to its mitogenic and permeability-causing properties. In this work, VEGF was located by immunohistochemistry in growth plate cartilage of human fetuses (20-22 weeks old) and its expression was demonstrated by reverse transcription polymerase chain reaction (RT-PCR). Primary culture of human fetal epiphyseal chondrocytes (HFEC) maintained VEGF expression at protein and messenger RNA (mRNA) levels and this expression was stimulated by cartilage promoting growth factors incorporated into the culture media (rFGF-b, rTGF-beta1, and insulin-like growth factor [rFGF-b] at 50 ng/ml). The conditioned medium (CM) of HFEC stimulated the proliferation of endothelial cells, and this was partially blocked by anti-VEGF antibody. These studies showed VEGF production by chondrocytes of the epiphyseal growth cartilage and suggested a role of this factor in cartilage physiology and the angiogenic process. PMID- 10750569 TI - Comparative effects of estrogen and raloxifene on B lymphopoiesis and bone loss induced by sex steroid deficiency in mice. AB - Estrogen deficiency caused by ovariectomy (OVX) results in a marked bone loss because of stimulated bone resorption. We have reported that OVX selectively stimulates B lymphopoiesis in mouse bone marrow, which is somehow related to bone resorption. Estrogen prevents both the increased B lymphopoiesis and the bone resorption caused by estrogen deficiency. Raloxifene also has a potent estrogenic activity for bone with minimal estrogenic activity for the uterus. To examine the effects of raloxifene on B lymphopoiesis and bone resorption, OVX mice were given either estrogen or raloxifene subcutaneously for 2-4 weeks using a miniosmotic pump. Reduced uterine weight in OVX mice was restored completely by 17beta estradiol (E2). Some 300-fold higher doses of raloxifene increased uterine weight of OVX mice, but only slightly. The number of B220- positive pre-B cells was increased markedly in bone marrow after OVX. The increased B lymphopoiesis was prevented not only by E2 but by raloxifene. In OVX mice, the trabecular bone volume (BV) of the femoral distal metaphysis was reduced markedly, when measured by microcomputed tomography (microCT) scanning and dual-energy X-ray absorptiometry. Both E2 and raloxifene similarly restored it. Like estrogen deficiency, androgen deficiency induced by orchidectomy (ORX) also resulted in a marked bone loss and increased B lymphopoiesis. Both E2 and raloxifene prevented the changes in ORX mice. These results indicate that both estrogen deficiency and androgen deficiency similarly stimulate B lymphopoiesis in mouse bone marrow, which accompany bone loss. Raloxifene exhibits estrogenic actions in bone and bone marrow to prevent bone loss and regulate B lymphopoiesis without inducing estrogenic action in the uterus. PMID- 10750570 TI - Direct measurement of hormone-induced acidification in intact bone. AB - Previous findings have shown that osteoblasts respond to parathyroid hormone (PTH) with an increase in extracellular acidification rate (ECAR) in addition to the known effect of PTH to increase local acidification by osteoclasts. We, therefore, investigated use of the Cytosensor to measure the ECAR response of whole intact bone to PTH employing microphysiometry. The Cytosensor measures a generic metabolic increase of cells to various agents. Using neonatal mouse calvaria, we found that the area surrounding the sagittal suture was particularly responsive to PTH. In this bone, the increase in ECAR was slower to develop (6 minutes) and more persistent than in cultured human osteoblast-like SaOS-2 cells and was preceded by a brief decrease in ECAR. Salmon calcitonin also produced an increase in ECAR in this tissue but with a different pattern than that elicited by PTH. Because PTH stimulates osteoclastic bone resorption in mouse calvaria via a cyclic adenosine monophosphate (cAMP)-mediated mechanism, we showed that the adenylyl cyclase activator forskolin also stimulated ECAR in this tissue. When the protein kinase A (PKA) pathway was activated by maintaining a high intracellular concentration of cAMP using N6-2'-0-dibutyryladenosine-cAMP (db cAMP), there was a reduction of PTH-induced acidification, while isobutylmethylxanthine pretreatment potentiated the PTH-induced acidification, consistent with a PKA-mediated pathway. Thapsigargin and the protein kinase C (PKC) activator phorbol myristate acetate had no effect on the PTH-induced increase in ECAR in calvaria, indicating that PKC does not play a major role in the ECAR response in intact bone. These results indicate the utility of using microphysiometry to study ECAR responses in intact tissue and should enable elucidation of the relative importance of extracellular acidification by osteoblasts and osteoclasts to the anabolic and catabolic activities of PTH, respectively. PMID- 10750571 TI - A detailed assessment of alterations in bone turnover, calcium homeostasis, and bone density in normal pregnancy. AB - The effects of pregnancy on bone turnover and the potential risk of developing an osteoporotic fracture in pregnancy are controversial. Utilizing biochemical markers of bone formation and resorption and dual-energy X-ray absorptiometry (DEXA), bone turnover before, during, and after pregnancy was studied in detail. Ten women (mean age 30 years; range 23-40) were recruited. Prepregnancy data were obtained and then a review was performed at 2-week intervals , once pregnancy was confirmed, until 14 weeks of gestation and thereafter monthly until term. Bone mineral density (BMD) was estimated by DEXA scanning of hip, spine, and forearm preconception and postpartum. In addition, BMD of the forearm at 14 weeks and 28 weeks gestation was obtained. All pregnancies had a successful outcome. Urinary free pyridinium cross-links, free pyridinoline (fPyr) and free deoxypyridinoline (fDPyr), were normal prepregnancy (mean [+/-SD]) 14.6 nmol/mmol (1.8) and 5.0 nmol/mmol (1.0) creat, respectively. By 14 weeks, they had increased to 20.8 nmol/mmol (4.3) and 6.1 nmol mmol (1.4) (both p < 0.02) and by 28 weeks to 26.3 nmol/mmol (5.6) and 7.4 nmol/mmol (1.6) (both p < 0.01). The ratio of fPyr to fDPyr remained constant. A similar significant increase was observed in N telopeptide (NTx). Bone formation was assessed by measurement of carboxyterminal propeptide of type 1 collagen (P1CP) and bone-specific alkaline phosphatase (BSAP). Neither were altered significantly before 28 weeks, but subsequently mean P1CP increased from 110 microg/liter (23) to 235 microg/liter (84) at 38 weeks and mean BSAP increased from 11.1 U/liter (5.0) to 28.6 U/liter (11.1) (p < 0.01 for both variables). Lumbar spine (L1-L4) BMD decreased from a prepregnancy mean of 1.075 g/cm (0.115) to 1.054 g/cm2 (0.150) postpartum (p < 0.05). Total hip BMD decreased from a prepregnancy mean of 0.976 g/cm2 (0.089) to 0.941 g/cm2 (0.097) (p < 0.05). Forearm BMD at midradius, one-third distal and ultradistal decreased but did not reach statistical significance. As assessed by these bone markers, in the first 2 trimesters of pregnancy, bone remodeling is uncoupled with a marked increase in bone resorption. A corresponding increase in formation markers is not observed until the third trimester. Spinal BMD exhibits a significant decrease from prepregnancy to the immediate postpartum period with a mean reduction in BMD of 3.5 % in 9 months. PMID- 10750572 TI - Morphometric X-ray absorptiometry and morphometric radiography of the spine: a comparison of prevalent vertebral deformity identification. AB - Prevalent vertebral deformities are associated with a substantially increased risk of subsequent vertebral and nonvertebral fractures. Knowledge of vertebral fracture status is an important component in the prediction of further fractures in patients with osteoporosis. This study reports a comparison of the quantitative identification of vertebral deformities on morphometric X-ray absorptiometry (MXA) scans and conventional radiographs (MRX) in 161 postmenopausal women (mean age +/- SD, 64 +/- 7.1 years) recruited from patients referred by their family doctor for bone density measurement (n = 119) and osteoporotic subjects with known vertebral deformities attending an osteoporosis clinic (n = 42). Each subject had MXA scans and MRXs of the thoracolumbar spine, to image the vertebrae from T4-L4, at a single visit. The scans and radiographs were analyzed by two trained observers using six points to quantify the shape of each vertebral body. From these points, three vertebral heights were measured: anterior, middle, and posterior. Vertebral deformities were identified using the algorithms proposed by Eastell and by McCloskey. Generally good to excellent agreement (per vertebra, kappa = 0.87-0.93; per subject, kappa = 0.81-0.91) was observed between the two algorithms used for quantitative vertebral deformity identification using MXA or MRX. More moderate agreement (per vertebra, kappa = 0.70-0.79; per subject, kappa = 0.67-0.75) was seen when comparing the same algorithm between MXA and MRX. Agreement between MXA and MRX for the McCloskey algorithm was better than for the Eastell algorithm, largely because of the lower number of false positives produced by the McCloskey methodology. Deformity identification by MXA was limited because of poor image quality, primarily in the upper thoracic spine. One in six MRX deformities were missed by MXA as they occurred in vertebrae not visualized sufficiently for analysis on the MXA scans. Deformity identification was poorer in the upper thoracic spine in analyzable vertebrae with a sensitivity of 50.0% for MXA in terms of MRX using the Eastell algorithm for the vertebral levels T4-T7, compared with 80.6% for L1-L4A. MXA proved to be more effective at identifying moderate to severe MRX deformities producing a sensitivity of 22.0% for MXA in terms of identifying MRX grade 1 deformities using the Eastell algorithm, compared with 81.6% for grade 2 deformities. Although MXA image quality is inferior to that of conventional radiographs, MXA has distinct advantages such as a substantially reduced effective dose to the patient and acquisition of a single image of the spine. MXA is a potentially useful, relatively fast, low-radiation technique to identify prevalent vertebral deformities, particularly moderate to severe deformities in the middle/lower thoracic and lumbar spine, in conjunction with morphometric radiography in some patients. PMID- 10750573 TI - Identification of vertebral deformities in women: comparison of radiological assessment and quantitative morphometry using morphometric radiography and morphometric X-ray absorptiometry. AB - Our aim was to compare normal vertebral reference values for morphometric radiography (MRX) and morphometric X-ray absorptiometry (MXA) and to compare these methods for the identification of vertebral deformities. We calculated MXA reference values (Hologic QDR 4500 A) for 327 women (ages 22-88 years) randomly selected from local General Practice lists in Sheffield, U.K. MRX reference values were calculated from spinal radiographs for 123 of these subjects (ages 56 88 years). We used these reference values to identify deformities in the MRX and MXA reference populations and in 83 women with osteoporosis (ages 49-87 years). We observed differences in mean deformity of vertebral height ratios measured by MRX and MXA, especially for the mid-to-posterior ratio. We compared agreement between quantitative methods (MRX and MXA) and qualitative radiological assessment. Severity of deformity was defined by semiquantitative (SQ) assessment. Agreement was moderate for MRX (k = 0.59; 95% CI = 0.43-0.77) and for MXA (k = 0.47; 95% CI = 0.29-0.66) in the reference population. Agreement was good for MRX (k = 0.86; 95% CI = 0.82-0.89) and MXA (k = 0.71; 95% CI = 0.66 0.75) in the osteoporotic population. MRX and MXA correctly identified a greater proportion of moderate or severe deformities compared with mild deformities. Sensitivity, specificity, predictive values, and accuracy were slightly better for MRX than for MXA. Although MXA agrees well with qualitative radiological assessment, the large proportion of vertebrae excluded from analysis because of poor image quality limits the diagnostic value of the technique. Reference intervals should be technique specific. PMID- 10750575 TI - Biological variability of serum and urinary N-telopeptides of type I collagen in postmenopausal women. AB - Measurement of N-telopeptides of type I bone collagen (NTX) provides a specific indicator of the current level of bone resorption. The biological intrasubject variability of NTX in urine and serum was studied in 277 postmenopausal women, mean age, 63.6 years +/- 10.2 (+/-SD) years. Second-morning void urine and serum specimens were collected at baseline and for two consecutive days to determine short-term variability (% CV). Long-term variability was determined by comparing NTX results at baseline and two consecutive months. Subjects were instructed to maintain current diet, lifestyle, and medications during the study. The median short-term %CV was 13.1% for urine NTX. This compared with 6.3% for serum NTX. Calculation of long-term %CV showed similar trends, with the %CV for NTX measured in serum (7.5%) lower than when measured in urine (15.6%). Using the least significant change (LSC) calculation, our data show that to be 90% confident that a decrease in NTX after initiation of antiresorptive therapy in an individual patient is not caused by variability alone, a 31 % decrease in urine NTX and a 14% decrease in serum NTX is required. As reported changes in NTX caused by antiresorptive therapy are greater than these calculations; our results support the use of either specimen to measure NTX to monitor the effect of therapy. PMID- 10750574 TI - The effect of alendronate on bone mass after distal forearm fracture. AB - Fracture and immobilization of an extremity lead to bone loss at the fracture and at adjacent sites. We conducted a 1-year, single-center, prospective, randomized, double-blind study to determine whether bone loss would occur in the distal radius after a Colles' fracture and whether this loss could be prevented using an antiresorptive drug (alendronate). Thirty-seven women with a recent fracture of the distal forearm and low bone mineral density (BMD) of the lumbar spine were randomized to receive either 10 mg alendronate daily or placebo. BMD of both forearms was measured at baseline and after 3, 6, and 12 months. The results of four women who developed reflex sympathetic dystrophy were not included in the analysis. In the placebo group, there was a significant reduction at 3 months and 6 months in BMD of total radius (p < 0.01), one-third distal radius (p < 0.01), middistal radius (p < 0.05), and ultradistal radius (p < 0.01) on the fractured side. The loss in BMD at one-third distal radius remained significant at month 12 (p < or = 0.001). In the alendronate group BMD of total distal radius, one-third distal radius, and middistal radius at the fractured side remained unchanged. BMD of ultradistal radius increased significantly at months 3, 6, and 12, compared with baseline (p < 0.05). The difference between the two treatment groups was significant at 3 months and 6 months and borderline significant (p = 0.054) after 1 year in total distal radius. In ultradistal radius the differences were significant at all time points. We conclude that BMD of the distal radius of a recently fractured forearm decreases significantly in the 6 months after fracture and the resulting deficit remains evident at least 1 year after fracture. This bone loss can be prevented by alendronate. PMID- 10750576 TI - Chronic intravenous aminobisphosphonate therapy increases high-density lipoprotein cholesterol and decreases low-density lipoprotein cholesterol. AB - Nowadays, bisphosphonates are considered the drugs of choice for the treatment of several bone disorders. Their exact mechanism of action is not clear but recently it has been reported that the aminobisphosphonates inhibit cholesterol biosynthesis and that this might be relevant for their actions on bone osteoclasts. The study includes 87 postmenopausal women with moderate to severe osteoporosis. The patients were randomly assigned to intravenous (iv) infusion of 50 mg of the aminobisphosphonate Neridronate dissolved in 100 ml of saline solution every 2 months for a year (44 patients). The remaining 43 served as controls. At the time of each infusion blood samples were obtained for the evaluation of total cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C), apolipoprotein A-I (Apo A-I), apolipoprotein B (Apo B), and total and bone alkaline phosphatase (AP). Free deoxypyridinoline (f-DPD) was measured in fasting urine specimens. In the control group no significant changes were observed throughout the study period for any of the biochemical variables. In the Neridronate-treated patients both bone AP and f-DPD excretion fell significantly by 15-20%. In these patients serum total cholesterol and serum triglycerides showed marginal decreases, which were occasionally significant. LDL-C and Apo B fell by 5-6% and these changes were statistically significant at most time points. Apo A-I and HDL-C rose progressively with time. At the 12th month, HDL-C rose 17-18% (p < 0.0001) above the baseline values. Similar findings were obtained in four postmenopausal women given high iv doses of Pamidronate or Alendronate. In conclusion aminobisphophonates, at least when given iv, induce remarkable and unexpected effects on lipid metabolism with a final profile that might be clinically relevant. PMID- 10750577 TI - The discrepancy between the binding affinity of PTH (1-34) and RS 66271 is explained by interaction of the PTH/PTHrP receptor with G-protein. PMID- 10750578 TI - Perspective: from increased energy metabolism to cardiac hypertrophy and failure: mediators and molecular mechanisms. PMID- 10750579 TI - The E.K. Frey - E. Werle Foundation award ceremony for the 1998 commemorative gold medalist. PMID- 10750581 TI - Familial hypertrophic cardiomyopathy and atrial fibrillation caused by Arg663His beta-cardiac myosin heavy chain mutation. AB - More than 40 different beta-cardiac myosin heavy chain (beta-MHC) missense mutations have been identified that cause familial hypertrophic cardiomyopathy (FHC). Some of these are recognized to have important clinical manifestations, such as an increased incidence of sudden death. We report that the beta-MHC missense mutation Arg663His causes predominant cardiac morphology and atrial fibrillation. Longitudinal clinical evaluations were performed in a kindred with FHC. The nucleotide sequence of the beta-MHC gene was analyzed to define the causal mutation. A missense mutation in the beta-MHC gene, Arg663His, was identified in 24 individuals. Clinical studies demonstrated modest left ventricular hypertrophy in affected individuals, predominantly localized in the proximal segment of the interventricular septum, which increased (average = 40 +/ 8%) during 7 years of follow-up. Results showed that 47% of Arg663His adults (age > 16 years) with ventricular hypertrophy developed atrial fibrillation, significantly more (p <0.001) than observed in ungenotyped FHC populations. Survival of affected individuals remained near normal. The beta-MHC missense mutation Arg663His causes a characteristic pattern of ventricular hypertrophy. Arg663His individuals have a markedly higher prevalence of atrial fibrillation, compared with a population with ungenotyped hypertrophic cardiomyopathy. The demonstration of phenotype as a direct consequence of genotype further extends the utility of molecular data in clinical medicine. Early identification of Arg663His individuals has the potential to minimize the serious sequelae of this arrhythmia in this FHC group. PMID- 10750580 TI - Heterogeneities in myocardial flow and metabolism: exacerbation with abnormal excitation. AB - Because regional myocardial blood flows are remarkably heterogeneous-with a 6- to 10-fold range of flows in normal hearts-and because the spatial profiles of the flows are stable over long periods and over a range of conditions, the relation between flows and other physiologic functions has been explored. Local fatty acid uptake and oxygen consumption are almost linearly related to the flows. Coronary network structure and hydrodynamic resistances give suitable flow heterogeneity but are thought to be a response to local needs rather than being causative. Presumably the cause is the need for adenosine triphosphate (ATP) synthesis locally, and therefore flows, substrate delivery, and oxygen utilization are driven primarily by local rates of ATP hydrolysis, mainly by contractile proteins. This hypothesis is by no means fully tested. Data on pacing dog hearts from different sites, on patients with left bundle branch block, and on unloading transplanted rat hearts, all point in the same direction: unloading ventricular muscle leads to diminished flow and exaggeratedly diminished glucose uptake. The mechanism is likely to be that discovered by Taegtmeyer and colleagues, namely, the expression of fetal genes in regions where the muscle is unloaded and particular metabolic enzymes and transporters are downregulated. PMID- 10750582 TI - 31P nuclear magnetic resonance spectroscopy: a noninvasive tool to monitor metabolic abnormalities in left ventricular hypertrophy in human. AB - 31p nuclear magnetic resonance (NMR) spectroscopy represents a unique instrument to noninvasively monitor myocardial metabolism in humans. The technique has been used to study the metabolism in myocardial hypertrophy in humans with hypertension, aortic stenosis, aortic incompetence, mitral regurgitation, and hypertrophic cardiomyopathy, as well as after maintenance dialysis or long-term physical exercise in elite cyclists. A primary aim is the determination of the phosphocreatine (PCr)/adenosine triphosphate (ATP) ratio, which reflects the energetic state of the myocardium. Recent investigations take advantage of proton decoupling in 31p NMR spectroscopy, which, besides the PCr/ATP ratio, also allows the determination of the inorganic phosphate/ PCr and the phosphomonoester/PCr ratios as additional indicators for alterations in myocardial metabolism. Abnormal myocardial metabolism was found in humans with aortic stenosis, mitral regurgitation, hypertrophic cardiomyopathy, and in patients who undergo maintenance dialysis. A trend toward a lower PCr/ATP ratio was reported in hypertension and aortic incompetence patients. Several studies have revealed a dependence of the metabolic abnormalities on the degree of heart failure, and one study claimed that a correlation with the extent of hypertrophy exists. No metabolic abnormalities were found in elite cyclists. PMID- 10750583 TI - Regulation of myocardial glucose uptake and transport during ischemia and energetic stress. AB - Myocardial glucose utilization increases in response to the energetic stress imposed on the heart by exercise, pressure overload, and myocardial ischemia. Recruitment of glucose transport proteins is the cellular mechanism by which the heart increases glucose transport for subsequent metabolism. Moderate regional ischemia leads to the translocation of both glucose transporters, GLUT4 and GLUT1, to the sarcolemma in vivo. Myocardial ischemia also stimulates 5' adenosine monophosphate-activated protein kinase, which may be a fuel gauge in the heart and other tissues signaling the need to turn on energy-generating metabolic pathways. Pharmacologic stimulation of this kinase increases cardiac glucose uptake and transporter translocation, suggesting that it may play an important role in augmenting glucose entry in the setting of ischemic or energetic stress. Thus, recent work has provided insight into the cellular and molecular mechanisms responsible for glucose uptake during energetic stress, which may lead to new approaches to the treatment of patients with coronary artery disease. PMID- 10750584 TI - Transcriptional modulators targeted at fuel metabolism of hypertrophied heart. AB - The transition of nonfailing to failing cardiac hypertrophy cannot be prevented by current drug regimens. This investigation examined whether possible drug targets have remained unexplored because they do not result in acute improvement of heart function. Of major importance, in this respect, is an inadequate performance of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2). In the present approach, binding sequences within the proximal promoter of SERCA2 are described which may be useful in the development of drugs (i.e., transcriptional modulators) that interfere selectively with the transcription of genes of the cardiomyocyte. The proximal promoter region of the SERCA2 genes has a thyroid response element, 9 potential Sp1-binding sites (5'-GGGCGG-3', 5'-CCGCCC-3' and 5'-GGGAGG-3'), and an E-box motif (5'-CACATG-3'), which may function as glucose response elements. This region also has 2 putative fatty-acid response elements (5'-GGGGGA-3'). It is proposed that the beneficial effects of the camitine palmitoyltransferase-1 inhibitor etomoxir arise from a shift in fuel metabolism involving glucose response elements and/or peroxisomal proliferator-activated receptors. Although the relative contribution of these DNA regulatory elements remains to be defined, it appears that they provide the driving force that prevents the decrease in transcriptional activity of the SERCA2 gene in the hypertrophic heart. It is further concluded that etomoxir represents a member of a novel class of transcriptional modulators that improve function of hypertrophied hearts with unimpeded blood flow by modulating gene expression of the cardiomyocyte. PMID- 10750585 TI - Beta3-integrin-mediated focal adhesion complex formation: adult cardiocytes embedded in three-dimensional polymer matrices. AB - In vivo studies show that beta3-integrin-mediated focal adhesion formation (FAF) causes recruitment of nonreceptor tyrosine kinases to the cytoskeleton in pressure-overloaded myocardium. To define the mechanism of beta3-integrin mediated signaling, we developed a cell culture model (adult feline cardiocytes embedded in a 3-dimensional matrix of native type 1 collagen, fibronectin, and vitronectin) wherein beta3-integrin-mediated focal adhesion kinase occurs. Focal adhesion kinase was analyzed immunocytochemically using confocal microscopy. Initial studies suggested that cardiocytes cultured in a 3-dimensional matrix formed focal adhesions consisting of both beta3-integrin and the muscle-specific isoform, beta1-integrin (beta1D). The focal adhesions were associated with focal adhesion kinase on both costameres and intercalated disks. To determine the cause of beta1D-integrin-mediated focal adhesion kinase in this model, time course studies were done. Beta3-integrin-mediated focal adhesion kinase occurred within 30 minutes after embedding cardiocytes and persisted for >24 hours, whereas beta1D-integrin-mediated focal adhesion kinase was present from the outset. Because confocal microscopy showed that laminin was present on the surface of freshly isolated cardiocytes, we hypothesized that this was causative of beta1D integrin-mediated focal adhesion kinase. Freshly isolated cardiocytes washed with acidic medium (2 minutes, pH 3.0) to remove laminin and then embedded in a 3 dimensional matrix showed complete absence of beta1D-integrin-mediated focal adhesion kinase, but beta3-integrin-mediated focal adhesion kinase occurred with a time course similar to that seen in cultured, unwashed cardiocytes. Acid washing did not alter the binding ability of beta1D-integrin, because acid-washed cardiocytes in the presence of laminin showed beta1D-integrin-mediated focal adhesion kinase. Thus, cardiocytes embedded in a 3-dimensional matrix show beta3 integrin-mediated focal adhesion kinase and provide an in vitro model to study beta3-integrin-mediated signaling in response to hemodynamic cardiac loading. PMID- 10750586 TI - Paracrine actions of cardiac fibroblasts on cardiomyocytes: implications for the cardiac renin-angiotensin system. AB - Conditioned medium of cardiac fibroblasts was found to induce protein synthesis and signal transduction events rapidly, and to increase angiotensinogen messenger RNA (mRNA) levels in neonatal rat ventricular myocytes. Within 4 hours, fibroblast-conditioned medium (FCM) stimulated protein synthesis in cardiac myocytes, independent of the contractile state, and induced marked increases within 24 hours in total protein content. Endothelin- released by cardiac fibroblasts was not responsible for the stimulation of protein synthesis. FCM rapidly activated signal transduction events in cardiac myocytes associated with hypertrophic stimuli, including: (1) increased tyrosine phosphorylation of several prominent protein bands; (2) mitogen-activated protein kinases (ERK 1 and ERK 2); and (3) protein kinase C. Finally, FCM caused an increase at 8 hours in angiotensinogen mRNA levels of cardiac myocytes, whereas no effect was observed on mRNA levels for renin or the type 1 angiotensin II receptor (AT1). Our results suggest that cardiac fibroblasts produce a factor that rapidly activates cardiac myocyte growth through a membrane receptor that couples to conventional signal transduction pathways. PMID- 10750587 TI - Role of angiotensin AT1, and AT2 receptors in cardiac hypertrophy and disease. AB - Angiotensin II modulates beat-to-beat cardiac performance as a potent vasocontrictor, inotrope, and regulator of water and electrolyte balance. It is also a growth factor that can stimulate the early molecular growth responses of proto-oncogene activation and new protein synthesis, and the later event of cardiocyte hypertrophy independent from load. Its effects are mediated through the angiotensin II type 1 (AT1) receptor, which exists as the AT1a and AT1b isoforms, and the angiotensin II type 2 (AT2) receptor. There is still controversy regarding the role of activation of the AT1 receptor subtype(s) as a mandatory signal versus modulatory regulator of the transduction of mechanical load in pressure-overload hypertrophy due to hypertension or aortic stenosis. The role of the AT2 receptor subtype in the heart is even less well understood, although this receptor appears to serve as an antigrowth signal in proliferating cells. Here we review current data on these controversies, including new data that support the notion that angiotensin II activation of the cardiac AT2 receptor subtype inhibits the effects of angiotensin II on the immediate growth response in the adult heart. PMID- 10750588 TI - Role of the renin-angiotensin system in cardiac hypertrophy. AB - Hypertrophy is an adaptive response of the heart to hemodynamic overload such as hypertension. However, it is generally accepted that cardiac hypertrophy is one of the most critical risk factors of heart disease. Therefore, for the treatment of hypertension it is important to understand the mechanism of cardiac hypertrophy and to establish effective pharmaceutical interventions. Mechanical stretch induced by hypertension is an initial factor leading to cardiac hypertrophy. In an in vivo study using spontaneously hypertensive rats, an angiotensin II type 1 receptor antagonist, TCV116, decreased left ventricular weight, left ventricular wall thickness, transverse myocyte diameter, relative amount of V3 myosin heavy chain, and interstitial fibrosis, whereas treatment with hydrolazine did not. In an in vitro study using cultured cardiomyocytes of neonatal rats, mechanical stretch activated second messengers, such as extracellular signal-regulated protein kinase (ERK), followed by increased protein synthesis. Additionally, in the stretch-conditioned medium, the levels of angiotensin II and endothelin-1 concentrations were increased. Moreover, the Na+/H+ exchanger activated by mechanical stretch modulated the hypertrophic responses of cardiomyocytes. To further elucidate whether angiotensin II is indispensable for mechanical stress-induced cardiac hypertrophy, mechanical stretch-induced ERK activation was examined in angiotensin II type 1a receptor knock-out mice. Although the addition of angiotensin II had no effects on the ERK activity in cardiomyocytes of angiotensin II type 1a receptor knockout mice, mechanical stretch induced a larger increase in the ERK activity in cardiac myocytes from these mice compared with cardiac myocytes of wild-type mice. These results suggest that mechanical stretch could induce hypertrophic responses in cardiac myocytes even in the absence of angiotensin II. The pathways leading to ERK activation differed between cell types. In cardiac fibroblasts, angiotensin II activated ERK via the G(beta)gamma subunit of Gi, Src, Shc, Grb2, and Ras, whereas Gq and protein kinase C were critical in cardiomyocytes. PMID- 10750589 TI - Transcriptional adaptation of the heart to mechanical unloading. AB - Novel strategies in the treatment of heart failure include mechanical unloading with a left ventricular assist device. Although first considered as a bridge to cardiac transplantation, this surgical treatment may improve cardiac function in patients with heart failure, even after removal of the device. The molecular adaptation of the heart to unloading remains largely unknown. Most of the enzymes involved in the regulation of myocardial energetics (including contractile proteins, ion pumps, and metabolic enzymes)exist in "fetal" and "adult" isoforms. It is known that cardiac hypertrophy due to increased work load in vivo involves a switching from the normally expressed adult isoform to the fetal isoform. Our work has now shown that the same pattern occurs in the unloaded heart. In both conditions, this switching is accompanied by the reexpression of growth factors and proto-oncogenes. The functional improvement of the failing heart after mechanical unloading may in part be the result of a reexpression of fetal genes. PMID- 10750590 TI - Activation of protein kinase cascades in the heart by hypertrophic G protein coupled receptor agonists. AB - Cardiac myocyte hypertrophy involves changes in cell structure and alterations in protein expression regulated at both the transcriptional and translational levels. Hypertrophic G protein-coupled receptor (GPCR) agonists such as endothelin-(ET-1) and phenylephrine stimulate a number of protein kinase cascades in the heart. Mitogen-activated protein kinase (MAPK) cascades stimulated include the extracellularly regulated kinase cascade, the stress-activated protein kinase/c-Jun N-terminal kinase cascade, and the p38 MAPK cascade. All 3 pathways have been implicated in hypertrophy, but recent ex vivo evidence also suggests that there may be additional effects on cell survival. ET-1 and phenylephrine also stimulate the protein kinase B pathway, and this may be involved in the regulation of protein synthesis by these agonists. Thus, protein kinase-mediated signaling may be important in the regulation of the development of myocyte hypertrophy. PMID- 10750591 TI - Catecholamines in cardiac hypertrophy. AB - There has been intense interest in the roles catecholamines may play in compensatory myocardial hypertrophy. This article reviews the following: (1) chronic infusions of catecholamines in experimental animals result in cardiac hypertrophy, but in many of the studies mechanical factors have played a role; (2) experiments using isolated papillary muscles and isolated hearts, stretched isolated myocytes, and denervated hearts in vivo demonstrate that mechanical activity is sufficient to cause increased protein synthesis and cell growth; (3) in neonatal myocyte cell cultures, alpha-adrenergic agonists are powerful stimulants for protein synthesis and cell growth. Beta-adrenergic stimulation of nonmyocyte myocardial cells causes release of a factor that promotes protein synthesis in neonatal myocytes. Either alpha or beta stimulation, probably through different mechanisms, appears to have growth-promoting effects on isolated adult myocytes in culture; (4) alpha stimulation is transduced through the Gq pathway and its activation of phospholipase C, cleavage of phosphatidylinositol (4,5)-bisphosphate, and then further through the ras/raf, mitogen-activated protein (MAP) kinase system; (5) transgenic mice with upregulation of catecholamine-related systems have not clarified the independent role of either the alpha- or beta-adrenergic pathway; and (6) observations in humans suggest that mechanical factors predominate in the development and regression of cardiac hypertrophy. Humoral mechanisms, including catecholamines, may play a role, but their quantitative importance has not been determined. It is hypothesized that catecholamines may play a role in transition from the adaptive to the maladaptive state. PMID- 10750592 TI - Agonist-like beta-adrenoceptor antibodies in heart failure. AB - Anti-beta1-adrenoceptor antibodies may play a harmful role, and the elimination of these antibodies could have beneficial effects for some patients with dilated cardiomyopathy. In vitro experiments showed that the antibody was able to influence the function of cultured cardiomyocytes. In these experiments, the antibody prevented the down-regulation of the beta-adrenoceptor-mediated chronotropic response. This lack of desensitization, which resulted in permanent stimulation, could also influence the Ca2+ homeostasis of cardiomyocytes. However, in longer-term (72 hours)-treated cells, the antibodies were able to decrease subtype-specific expression of the beta1 adrenoceptor. In animal experiments, it was shown that long-term immunization with a peptide corresponding to the second extracellular loop of the beta1 adrenoceptor induced a failing heart similar to that in dilated cardiomyopathy. In humans, we observed a remarkable correlation between disappearance of the antibodies and improvement of heart function. Furthermore, in anti-beta1-adrenoceptor-positive patients with dilated cardiomyopathy treated with the immunoadsorption technique, removal of the antibodies also led to improvement of cardiac function and quality of life. This finding indicates that autoimmune processes may be involved in some patients with dilated cardiomyopathy. PMID- 10750593 TI - Beta-adrenergic receptor-G protein-adenylyl cyclase signal transduction in the failing heart. AB - The beta-adrenergic receptor signal transduction pathway is critical for rapid adjustments to increased cardiovascular demand (e.g., during exercise). In the face of chronic stimulation of this pathway, as occurs in the pathogenesis of heart failure, beta-adrenergic receptor stimulation may become maladaptive. Under these conditions, elevation of circulating catecholamines and depletion of cardiac tissue stores of norepinephrine occur in the failing heart, resulting in desensitization. Whether or not stimulation or inhibition of the beta-adrenergic receptor signaling pathway is beneficial in heart failure is controversial. One approach to address this question is to specifically overexpress a component of the beta-adrenergic receptor signaling pathway in a transgenic mouse heart. We have characterized young and old adult mice with overexpressed cardiac G(s alpha) which couples the beta-adrenergic receptor to adenylyl cyclase. In younger animals, beta-adrenergic receptor stimulation results in an augmented heart rate and cardiac contractility. Over the life of the animal, however, a picture of cardiomyopathy develops. The result is a dilated heart with a large amount of fibrosis and myocyte hypertrophy, degeneration atrophy, and apoptosis. Conversely, chronic beta-adrenergic receptor blockade prevents the development of cardiomyopathy. These experiments support the point of view that chronic beta adrenergic stimulation during the development of heart failure is deleterious and that protecting the heart with chronic beta-adrenergic receptor blockade is salutary, conceptually consistent with results of recent clinical trials examining the effects of beta-adrenergic receptor blockers in patients with heart failure. PMID- 10750594 TI - Inotropy, calcium, and heart failure: a personal perspective. AB - The medical treatment of heart failure has improved significantly in the last decade, yet despite the reduction of morbidity and mortality, heart attacks and stroke cause 15 million deaths each year. Cardiovascular disease (CVD) constitutes 50% of all deaths worldwide. Indeed, heart disease represents a continuing global burden, despite all the advances. When considering the major objective of medical treatment of heart failure, the paradigm has shifted from increasing cardiac output of the failing heart by augmenting contractility to a decrease in afterload and inhibition of neurohumors affecting heart muscle. Nevertheless, a direct increase in contractility is still appropriate in some conditions of heart failure for a short-term beneficial effect. This article reviews certain aspects of cellular and molecular mechanisms to provide a framework for problem solving in this vital field. PMID- 10750595 TI - Calcium regulatory proteins and their alteration by transgenic approaches. AB - Abnormalities in calcium flux have been linked to abnormal contractile behavior of the heart in patients with congestive heart failure as well as in animal models. Decreased activity or levels of the calcium adenosine triphosphatase of the sarco(endo)plasmic reticulum (SERCA2) particularly have been known to cause a delay in calcium transients. The SERCA2 protein pumps 2 moles of calcium per mole of adenosine triphosphate (ATP) split from the cytoplasm into the sarcoplasmic reticulum, thus lowering the free cytoplasmic calcium concentration. It therefore is of interest to identify mechanisms by which SERCA activity could be increased in the heart. To determine influences of increased expression of the SERCA2 gene on calcium transient and contractile behavior, we constructed transgenic mice and rats expressing a SERCA2 transgene in their heart. In these animals, a 20% increase in SERCA levels occurs due to additional expression of the SERCA transgene. This leads to a corresponding increase in contractile activity as determined by the increase in left ventricular pressure measured as dP/dt(max) and decrease in diastolic ventricular pressure determined as dP/dt(min). Similarly, isolated cardiac myocytes obtained from the heart of transgenic mice showed an accelerated calcium transient and increased speed of shortening and relengthening as determined by edge detection. To determine if SERCA2 transgene expression could have a compensatory effect on the contractile behavior of the heart in transgenic mice expressing SERCA2, these mice were made hypothyroid, and papillary muscle function was determined. Contractile behavior of the papillary muscle of wild-type hypothyroid mice showed a significant increase in muscle relaxation time (RT50). In contrast, SERCA2 transgenic hypothyroid mice showed normal contractile behavior of papillary muscle. A compensatory effect of SERCA transgene expression was therefore demonstrated. In addition, we constructed transgenic rats expressing a SERCA2 transgene in which constriction of the ascending aorta induced cardiac hypertrophy and a delayed contraction of papillary muscle. In preliminary results, we found that SERCA2 transgenic rats submitted to ascending aortic constriction did not show the delayed relaxation of papillary muscle as was found in wild-type rats submitted to aortic constriction. In addition, adenoviral vectors expressing transgenes for calcium-handling proteins can be used to improve cardiac myocyte contraction. Adenoviruses expressing a SERCA transgene or a mutant phospholamban transgene exhibiting dominant negative action were used to infect isolated myocytes treated with a phorbol ester (phorbol 12-myristate 13-acetate), which delays the calcium transients. The calcium transients and contractile behavior of the isolated myocytes indicated that increased SERCA expression or increased expression of mutant phospholamban transgene led to increased SERCA2 activity, resulting in an increased contractile phenotype. Recent findings by other investigators also indicate that decreased SERCA2 activity can be increased under in vivo conditions using adenoviral vector-based SERCA2 expression. A gene therapy type of approach delivering increased amounts of SERCA or phospholamban mutants leading to increased SERCA activity should therefore be considered in the future. PMID- 10750596 TI - Synergy of amlodipine and angiotensin-converting enzyme inhibitors in regulating myocardial oxygen consumption in normal canine and failing human hearts. AB - The production of endogenous nitric oxide, which regulates myocardial oxygen consumption, is decreased in heart failure. As with angiotensin-converting enzyme (ACE) inhibitors, amlodipine, a calcium antagonist, increases kinin-mediated nitric oxide production in coronary microvessels. We investigated the possibility of synergy between ACE inhibitors and amlodipine in regulating myocardial oxygen consumption. Left ventricular myocardium was isolated from 6 healthy dog hearts and 5 human hearts with end-stage heart failure at the time of orthotopic heart transplantation. Myocardial oxygen consumption was measured before and after administration of bradykinin, S-nitroso N-acetyl penicillamine (SNAP, a nitric oxide donor), ramiprilat (an ACE inhibitor), amlodipine, and the combination of a sub-threshold dose of ramiprilat (10(-8) md/L) + amlodipine. These experiments were repeated with L-nitro-arginine methyl ester (L-NAME, an inhibitor of nitric oxide synthesis), dichloroisocoumarin (an inhibitor of kinin synthesis), and HOE 140 (a B2 kinin-receptor antagonist). Baseline myocardial oxygen consumption in canine hearts was 182 +/- 21 nmol/g/min. Bradykinin and SNAP caused dose dependent reductions in myocardial oxygen consumption (p <0.05). Ramiprilat and amlodipine caused a 10 +/- 3.2% and 11 +/- 0.8% reduction in myocardial oxygen consumption, respectively, when used alone (p <0.05). In the presence of a subthreshold dose of ramiprilat, amlodipine caused a larger (15 +/- 1.7%) reduction in myocardial oxygen consumption compared with either drug used alone (p <0.05). In human hearts, baseline myocardial oxygen consumption was 248 +/- 57 nmol/g/min. Amlodipine caused a larger reduction in myocardial oxygen consumption when used with ramiprilat (22 +/- 3.2%) as compared with amlodipine alone (15 +/- 2.6%). The effect of both drugs was attenuated by L-NAME, dichloroisocoumarin, and HOE 140 (p <0.05). In conclusion, ACE inhibitors and amlodipine act synergistically to regulate myocardial oxygen consumption by modulating kinin mediated nitric oxide release, and this combination of drugs may be useful in the treatment of heart failure. PMID- 10750597 TI - The public health applications of unlinked anonymous seroprevalence monitoring for HIV in the United Kingdom. AB - BACKGROUND: In order to monitor the epidemiology of human immunodeficiency virus (HIV), integrated national programmes of unlinked anonymous (blinded) HIV sero surveys have taken place in the UK since 1990. METHODS: The programmes comprise multi-centre surveys primarily using specimens gathered routinely for screening groups of patients. All specimens are irreversibly unlinked from patient identifiers before being tested. RESULTS: The surveys have met their prime aim of providing at low cost minimally biased estimates of current HIV prevalence and trends in sentinel populations. The surveys have remained acceptable to professionals and the public, being successfully implemented without breech of their founding principles. The findings have had major public health applications, have influenced HIV policy and funding, been used for monitoring the spread of HIV, for targeting and evaluating health promotion and improving projections of severe HIV disease. The surveys have detected substantial prevalence rises and under-diagnosis of HIV which would otherwise have been unrecognised. The programmes' value is being increased by sub-typing HIV-1 isolates, capturing additional demographic information to detect spread among minority groups. The same specimens are used for monitoring other infections (initially hepatitis A, B and C). CONCLUSIONS: Monitoring HIV and other infections through unlinked anonymous HIV surveillance has become an integral essential part of national HIV and AIDS surveillance. Although it has unique applications the value of unlinked anonymous surveillance is maximized when used in conjunction with behavioural data, information from HIV and AIDS reporting, and behavioural data and surveillance for other sexually transmitted infections. PMID- 10750598 TI - Mammographic parenchymal patterns and risk of breast cancer at and after a prevalence screen in Singaporean women. AB - BACKGROUND: The objective of this study was to assess the effect of mammographic parenchymal patterns on risk of breast cancer detected at first screen or in the period following a negative screen. METHODS: The study utilizes a nested case control design with 132 breast cancer patients detected at first screen (from a total of 29 193 screened) and 42 breast cancer patients detected in the period following the first screen. These patients were matched to 348 screened-negative controls. The mammograms were classified according to Tabar's classification for parenchymal pattern and statistical analysis was done by conditional logistic regression. RESULTS: The risk of breast cancer for women with Tabar pattern IV was significantly high when compared to the remaining patterns (odds ratio 2.59). Risk factors for Tabar pattern IV coincided largely with established risk factors for breast cancer. CONCLUSION: The study confirms the increased risk of breast cancer associated with Tabar pattern IV (approximately Wolfe pattern P2), in an Asian population. This pattern is associated with nulliparity and high educational status and is strongly associated with grade 3 cancers. PMID- 10750599 TI - Fat intake and breast cancer risk in an area where fat intake is low: a case control study in Indonesia. AB - BACKGROUND: Associations of fat and other macronutrients with breast cancer risk are not clear in areas where fat intake is low. METHODS: We conducted a hospital based case-control study from 1992 to 1995 in Jakarta, Indonesia. RESULTS: The study, based on 226 cases and 452 age and socioeconomic status matched controls, provided the following findings. (a) In the pre-marriage period, the greater the fat or protein consumption, the larger the risk, whereas decreasing risk with increasing carbohydrate intake was detected. The odds ratio (OR) for the highest quartile of intake relative to the lowest was 8.47 (95% CI: 4.03-17.8) for fat, 2.19 (95% CI: 1.30-3.69) for protein, and 0.16 (95% CI: 0.08-0.31) for carbohydrate. A positive association with fat and a negative one with carbohydrate were also observed for the post-marriage period, but of weaker magnitude compared to the pre-marriage period. (b) The effects of macronutrient intakes were stronger among premenopausal than among postmenopausal women. (c) Most of the associations of protein and carbohydrate were insignificant after adjustment for fat intake. CONCLUSIONS: These findings suggest that fat intake might be an important determinant of breast cancer among populations with a low fat diet in Indonesia. PMID- 10750600 TI - A prospective study of physical activity and risk of prostate cancer in US physicians. AB - BACKGROUND: Exercise can suppress androgen production and may thus decrease the risk of prostate cancer. However, findings from epidemiological studies assessing physical activity and risk of prostate cancer are inconsistent. METHODS: We prospectively examined the association between physical activity and prostate cancer risk in the Physicians' Health Study (PHS), a randomized trial of low-dose aspirin and beta-carotene among 22,071 men aged 40-84 without self-reported myocardial infarction, stroke and cancer. At baseline in 1982, men were asked about the frequency of exercise vigorous enough to work up a sweat. Physical activity was assessed in a similar fashion again at 36 months of follow-up. RESULTS: During 11.1 years of follow-up (258 779 person-years), 982 cases of prostate cancer occurred and were confirmed by medical record review. After adjustment for potential confounding factors (including age, height, randomized treatment assignment, smoking status, alcohol intake, use of multivitamins, history of diabetes, history of hypertension and history of high cholesterol), the relative risks for prostate cancer associated with exercise vigorous enough to work up a sweat were 1.0 (referent) for frequency less than once per week, 1.02 (95% CI: 0.82-1.26) for once per week, 1.07 (95% CI: 0.90-1.27) for 2-4 times per week, and 1.11 (95% CI: 0.90-1.36) for 5+ times per week. Across all subgroups of men categorized by age, body mass index, smoking status, alcohol intake, use of multivitamins, history of diabetes, history of hypertension and history of high cholesterol, there were no significant associations between frequency of exercise vigorous enough to work up a sweat and prostate cancer risk. After excluding cases of prostate cancer that occurred during the first 36 months of follow-up, again, there was no significant association. Combining physical activity assessments at baseline and at 36 months also yielded no significant association with prostate cancer risk. CONCLUSIONS: These observational data from the Physicians' Health Study do not support the hypothesis that increased physical activity reduces the risk of prostate cancer. PMID- 10750601 TI - Tobacco type and risk of squamous cell cancer of the oesophagus in males: a French multicentre case-control study. AB - BACKGROUND: The association between tobacco smoking and oesophageal cancer is well established. However, some major questions remain unanswered such as the importance of kind of tobacco and smoke inhalation. The aim of this study was to investigate the effect of each kind of tobacco on the risk of squamous cell cancer of the oesophagus in men and to test whether the effect of kind of tobacco is similar whatever the sub-site of cancer. Tobacco consumption was assessed by the number of years of consumption and time since quitting. METHODS: We conducted a multicentre case-control study in three university hospitals in France (Caen, Dijon, and Toulouse). From 1991 to 1994, 208 cases and 399 controls, all male, were selected. During the interview, the subject's entire tobacco history was recalled, noting each type of tobacco consumed throughout life. RESULTS: Strong tobacco, dark tobacco and non-filter-tipped cigarettes were associated with an increase in risk whatever the adjustments, whilst light, filter-tipped cigarettes and mild tobacco were not. Hand-rolled cigarettes were more strongly associated with risk than manufactured cigarettes. The effect of hand-rolled cigarettes appeared stronger for the lower third whilst those of strong cigarettes and dark tobacco appeared stronger for the upper third of the oesophagus. The effect of inhaling was confined to the upper third. CONCLUSIONS: Our results, emphasizing the role of dark tobacco, hand-rolled cigarettes, strong cigarettes and non filter-tipped cigarettes are in line with previous publications. Moreover, they suggest that the mechanism underlying the tobacco effect could be different according to the sub-site of cancer. PMID- 10750602 TI - Snowball sampling by mail: application to a survey of smokers in the general population. AB - BACKGROUND: In a series of surveys intended for current and former smokers but sent to a random sample of the general population, we asked never smokers and smokers who did not wish to participate to transmit the questionnaire to any ever smoker they knew. We compared participants who received the questionnaire directly from us (original participants) to participants who received it from an addressee (secondary participants). METHODS: Questionnaires on smoking were mailed to 3300 residents of Geneva (Switzerland) in 1997, and returned by 1167 people (35%). RESULTS: The final sample consisted of similar numbers of original participants (n = 578, primary response rate = 18% of total sample, or about 46% of ever smokers) and secondary participants (n = 566). Original participants were 1.7 years older than secondary participants (P = 0.03) and were more likely to be men (50% versus 43%, P = 0.009). Proportions of current smokers, stages of change, confidence in ability to quit smoking, cigarettes per day and attempts to quit smoking were similar in the two groups. Secondary participants had lower self-efficacy scores (-0.30 standard deviation (SD) units, P < 0.03), and they derived more pleasure from smoking (+0.25 SD units, P = 0.04). Among ex-smokers, direct participants were less active than secondary participants in coping with the temptation to smoke (-0.58 SD units, P = 0.002). Associations between smoking related variables were similar in original and secondary participants. CONCLUSION: Allowing non-eligible addressees to transmit the questionnaire to someone else doubled the response rate, produced moderate bias on some variables only and had no detectable impact on associations between smoking-related variables. PMID- 10750603 TI - Cardiovascular risk factor changes in Finland, 1972-1997. AB - BACKGROUND: The cardiovascular risk factor levels of the population have been assessed in Finland since 1972. In the beginning the surveys were done to evaluate the North Karelia Project, which was a community-based preventive programme. A national cardiovascular disease (CVD) prevention strategy was developed and implemented during the late 1970s. Subsequently, a national cardiovascular risk factors monitoring system was developed to assess the effectiveness of the national strategy. METHODS: Cross-sectional population samples were studied in 1972 and 1977 in North Karelia and Kuopio provinces in eastern Finland. An area in southwestern Finland was included in 1982, followed by the Helsinki metropolitan area in 1992 and Oulu province in northern Finland in 1997. A total of 19 761 men and 20 761 women aged 30-59 participated in the six surveys (1972, 1977, 1982, 1987, 1992, 1997). RESULTS: The serum cholesterol levels of both genders have continuously declined. Systolic blood pressure levels have declined since 1972, but no further decline in diastolic blood pressure was found in 1997. Smoking prevalence among men continued to decline mainly due to an increase in the percentage of never-smokers. For the first time the increase in smoking prevalence among women levelled off and started to decline, mainly because the number of female quitters had increased. CONCLUSIONS: These data suggest that the cardiovascular health programme in Finland has succeeded in decreasing the general risk factor level of the population. PMID- 10750604 TI - Alcohol consumption, metabolic cardiovascular risk factors and hypertension in women. AB - BACKGROUND: Low to moderate alcohol consumption is associated with reduced mortality, primarily due to a reduction in coronary heart disease (CHD). Conversely, heavy drinking increases mortality, mainly due to haemorrhagic stroke and non-cardiovascular diseases. It is important to identify the threshold of alcohol consumption above which the balance of risk and benefit becomes adverse. We examine the relationship between reported alcohol consumption, cardiovascular disease (CVD) risk factors, a 10-year CHD risk score and hypertension in women. METHODS: In all, 14 077 female employees aged 30-64 years, underwent screening for CVD risk factors. Information was available on a range of personal and lifestyle factors, including height, weight, blood pressure, lipids, lipoproteins, apolipoproteins and blood glucose. Age-adjusted means were computed for the risk factors in each of five groups of reported alcohol intake: <1 (non drinkers), 1-7, 8-14, 15-21, > or = 22 units/week. The relationships between alcohol and a derived coronary risk score and hypertension were also examined. RESULTS: Increasing consumption was associated with an age-adjusted increase in high density lipoprotein cholesterol (HDL-C) and apolipoprotein A1 (both P < 0.001), a decline in body mass index, total cholesterol (TC), TC/HDL-C ratio, low density lipoprotein cholesterol (LDL-C) and apolipoprotein B (all P < 0.001), and no trend in triglycerides (P = 0.06), lipoprotein (a) (P = 0.09) or fasting glucose (P = 0.14). Except for LDL-C (P = 0.06) the relationships remained statistically significant after adjustment for possible confounders. Compared to non-drinkers, there was a decrease in 10-year CHD risk with increasing consumption, with the greatest reduction in risk in women consuming 1-7 units/week, odds ratio (OR) = 0.79, (95% CI: 0.72-0.87), and an increase in the prevalence of hypertension among those consuming 15-21 units/week, OR = 1.68, (95% CI: 1.14-2.46). CONCLUSIONS: This study provides biological support for an inverse association between alcohol intake and CHD in women, associated with favourable changes in lipid and lipoprotein risk factors. Women consuming 1-14 units/week had a reduction in CHD risk, but there was an increased prevalence of hypertension among those consuming > or = 15 units/week. These data suggest that, in terms of the reduced risk of CVD, women should be advised to restrict their alcohol consumption to < or = 14 units/week. PMID- 10750605 TI - High density lipoprotein cholesterol increases with living altitude. AB - BACKGROUND: The relationship between high density lipoprotein cholesterol (HDL) serum level and the altitude at which people live is controversial. METHODS: A cross-sectional study was carried out in the adult population (30-64 years) of the Island of El Hierro (Canary Islands, Spain). In all, 594 individuals representative of the El Hierro population for gender, age, district and the altitude at which they lived were included. The factors measured included HDL, living altitude, body mass index (BMI), smoking habits, alcohol consumption, diabetes, menopause in women, and physical activity and dietary habits. RESULTS: The HDL showed a correlation with living altitude (r = 0.14, P < 0.01) and with BMI (r = -0.19, P < 0.01). Smokers had lower HDL levels than ex-smokers and non smokers (P < 0.05). Men who were moderate drinkers had higher HDL levels than heavy or mild drinkers and non-drinkers (P < 0.01). Physical activity was only related to HDL in men with levels >1.52 mmol/l, who walked on the average more than the rest (P < 0.05). Variables not showing the expected relationship with HDL were diabetes and the menopause in women (probably due to a low statistical power of their subsamples). Regression analysis, with HDL as dependent variable showed that the association between HDL and altitude persists when taking altitude as a categorical or a continuous variable. CONCLUSIONS: High density lipoprotein cholesterol levels are linearly and significantly increased when living at a higher altitude. This fact should be taken into account when comparing cardiovascular risk in populations living at different altitudes. PMID- 10750606 TI - Relation of high blood pressure to glucose intolerance, plasma lipids and educational status in an Arabian Gulf population. AB - BACKGROUND: In Bahrain and other populations of the Arabian Peninsula, glucose intolerance is associated with raised plasma total cholesterol, postmenopausal status and low educational status. These associations are not generally seen in other populations with high diabetes prevalence. A study was undertaken in order to determine if hypertension in Bahrainis is associated with the same factors as those related to glucose intolerance. METHODS: A cross-sectional survey of 2120 Bahrainis aged 40-69 years. RESULTS: The age-adjusted prevalence of hypertension (defined as current treatment for hypertension, systolic blood pressure > or = 160 mmHg or diastolic blood pressure > or = 95 mmHg) rose with increasing degrees of glucose intolerance. Age- and sex-standardized prevalence of hypertension was 21% (95% CI: 19-24%) in those with normal glucose tolerance, 31% (95% CI: 27-36%) in those with impaired glucose tolerance, and 38% (95% CI: 34-42%) in those with diabetes. In a multivariate analysis adjusting for age and sex, raised blood pressure was independently associated with waist girth, plasma cholesterol, glucose intolerance, family history of hypertension and (in women) postmenopausal status. There was an inverse relationship between blood pressure and educational status that was independent of other variables. This association parallels the inverse relationship of diabetes to educational level and is consistent with low educational level being a marker for socioeconomic deprivation in early life in this population. CONCLUSION: The high prevalence rates of hypertension and diabetes in Bahrainis are manifestations of a pattern of metabolic disturbances that includes raised plasma cholesterol levels. Both hypertension and diabetes are associated with low educational status, which in this population is a marker for socioeconomic deprivation in early life. This suggests that the risk of hypertension may be set by environmental factors in early life. PMID- 10750607 TI - Comparison of electrocardiographic findings between Northern and Southern Chinese population samples. AB - BACKGROUND: Cardiovascular disease is rare in China, but there are few data on the prevalence of electrocardiographic (ECG) abnormalities in Chinese populations. METHODS: The ECG surveys were carried out in four Chinese population samples, in a total of 9,666 adults aged 35-54 in Beijing and Guangzhou, China from 1981 to 1984. Twelve-lead resting ECG tracings were coded by the Minnesota Code. RESULTS: Prevalence per 1,000 of abnormal ECG ranged from 77.4 to 209.8, and was higher for men than women and higher for Guangzhou than Beijing. Prevalence per 1,000 of major abnormalities in Guangzhou was 29.8 for men and 78.4 for women, higher than the 18.4 and 29.6 for counterparts in Beijing. The ECG changes attributed in 'Western' populations to coronary heart disease (CHD), such as large Q waves (Minnesota Code 1-1, 1-2) and ST-T abnormalities, were similar between Beijing and Guangzhou men, but Guangzhou women had much higher prevalence of ST-T abnormalities than Beijing women. Other ECG abnormalities such as A-V block, left branch bundle block, and left ventricular hypertrophy were rare in people of both sites. CONCLUSIONS: Compared with similar data from the US, these Chinese populations had a relatively low prevalence of ECG abnormalities putatively related to CHD. This corresponds with the low incidence of CHD in the Chinese population. However, within the Chinese populations of this study, a high abnormality rate appeared in a population with low incidence of CHD and hypertension (Guangzhou women). Reasons why ECG abnormalities do not parallel prevalence levels of CHD and hypertension remain to be elucidated. PMID- 10750608 TI - Diet, smoking and anthropometric indices and postmenopausal bone fractures: a prospective study. AB - OBJECTIVE: Bone fractures are an important cause of morbidity and mortality among the elderly in the US. The present study assesses the possible role of a number of risk factors for postmenopausal bone fractures. METHODS: We analysed the relationships of anthropometric, demographic and lifestyle factors with the risk of bone fracture among 6250 postmenopausal women in a prospective cohort study, the New York University Women's Health Study. RESULTS: After an average of 7.6 years of follow-up, 1025 new incident bone fractures were reported, including 34 hip and 159 wrist fractures (incidence rates; 71.6 and 334.7 per 105 woman-years, respectively). The risk of fracture increased with increasing age, body height and total fat intake, while it was significantly lower among obese and African American women. The relative risk among African Americans was 0.45 (95% CI: 0.32 0.63) compared with non-African Americans. Women taller than 170 cm had a 64% increase in risk of fractures, as compared with those under 155 cm. These associations were generally more pronounced when fractures were limited to those at the hip and wrist. CONCLUSIONS: The present study provides an indication for a potential role of dietary fat in the development of postmenopausal fractures and further evidence to support protective effects of obesity, short stature and African American ethnicity. PMID- 10750609 TI - Marital status and mortality in British women: a longitudinal study. AB - BACKGROUND: Most previous studies on marital status and mortality did not adjust for the effect of 'marital selection'. Little research has been done about the relation between marital status and mortality in British women, with the exception of research on bereavement. METHODS: Subjects consisted of women aged > or = 35 in a longitudinal study of a nationally representative sample. Marital status and covariates were enumerated at a baseline interview in 1984/85 and a follow-up interview in 1991/92. Death data up to May 1997 were obtained from the National Health Service Central Register. Cox regression was used to estimate hazard ratios (HR) for the single, divorced and widowed states in relation to the married state. RESULTS: Having adjusted for age and martial selection factors, being single (HR = 1.45) was significantly associated with higher all-cause mortality. Being divorced and being widowed showed no excess mortality risk (each HR = 1.09). CONCLUSIONS: Being single was associated with higher mortality. A causal interpretation is plausible. Being divorced and being widowed were not associated with higher mortality. PMID- 10750610 TI - Induced abortion and low birthweight in the following pregnancy. AB - BACKGROUND: To examine whether induced abortion increases the risk of low birthweight in subsequent singleton live births. METHODS: Cohort study using the Danish Medical Birth Registry (MBR), the Hospital Discharge Registry (HDR), and the Induced Abortion Registry (IAR). All women who had their first pregnancy during 1980-1982 were identified in the MBR, the HDR, and the IAR. We included all 15,727 women whose pregnancy was terminated by a first trimester induced abortion in the induced abortion cohort and 46,026 women whose pregnancy was not terminated by an induced abortion were selected for the control cohort. All subsequent pregnancies until 1994 were identified by register record linkage. RESULTS: Low birthweight (<2500 g) in singleton term live births occurred more frequently in women with one, two, three or more previous induced abortions, compared with women without any previous induced abortion of similar gravidity, 2.2% versus 1.5%, 2.4% versus 1.7%, and 1.8% versus 1.6%, respectively. Adjusting for maternal age and residence at time of pregnancy, interpregnancy interval, gender of newborn, number of previous spontaneous abortions and number of previous low birthweight infants (control cohort only), the odds ratios (OR) of low birthweight in singleton term live births in women with one, two or more previous first trimester induced abortions were 1.9 (95% CI: 1.6, 2.3), and 1.9 (95% CI: 1.3, 2.7), respectively, compared with the control cohort of similar gravidity. High risks were mainly seen in women with an interpregnancy interval of more than 6 months. CONCLUSIONS: The findings suggest a positive association between one or more first trimester induced abortions and the risk of low birthweight in subsequent singleton term live births when the interpregnancy interval is longer than 6 months. This result was unexpected and confounding cannot be ruled out. PMID- 10750611 TI - Maternal mortality in a rural district of southeastern Tanzania: an application of the sisterhood method. AB - BACKGROUND: Deaths from maternal causes represent the leading cause of death among women of reproductive age in most developing countries. It is estimated that the highest risk occurs in Africa, with 20% of world births but 40% of the world maternal deaths. The level of maternal mortality is difficult to assess especially in countries without an adequate vital registration system. Indirect techniques are an attractive cost-effective tool to provide estimates of orders of magnitude for maternal mortality. METHOD: The level of maternal mortality estimated by the sisterhood method is presented for a rural district in the Morogoro Region of Southeastern Tanzania and the main causes of maternal death are studied. Information from region-specific data using the sisterhood method is compared to data from other sources. RESULTS: The maternal mortality ratio (MMR) was 448 maternal deaths per 100,000 live births (95%CI : 363-534 deaths per 100,000 live births). Maternal causes accounted for 19% of total mortality in this age group. One in 39 women who survive until reproductive age will die before age 50 due to maternal causes. The main cause of death provided by hospital data was puerperal sepsis (35%) and postpartum haemorrhage (17%); this is compatible with the main causes reported for maternal death in settings with high levels of maternal mortality, and similar to data for other regions in Tanzania. The sisterhood method provides data comparable with others, together with a cost-effective and reliable estimate for the determination of the magnitude of maternal mortality in the rural Kilombero District. PMID- 10750612 TI - Factors that determine the effectiveness of screening for congenital heart malformations at child health centres. AB - BACKGROUND: The actual yield from current screening for clinically significant congenital heart malformations in Dutch child health care is far from optimal. In this study factors that determine the effectiveness of this screening are identified and recommendations for the optimization of the screening programme are formulated. METHODS: Eighty-two patients with a clinically significant congenital heart malformation were consecutively included in this study. Parents and child health centre physicians were interviewed in order to establish the screening, detection and referral history. Paediatric cardiologists established whether these patients were diagnosed 'in time' or 'too late'. RESULTS: Incomplete performance of the screening examination has more influence on the occurrence of delayed diagnoses than failure by parents to adhere to the complete visit schedule. Adequate screening advances detection of congenital heart malformations. Severity, however, is the most predominant determinant of the age at referral and diagnosis, as well as of the risk of complications. In only 7 out of 39 patients diagnosed 'too late', could no avoidable cause for an adverse outcome be found. In 10 cases (25%) there was a prolonged interval between first referral and diagnosis. CONCLUSION: To optimize the yield of the screening programme, improvement in the performance of the child health centre physicians and the co-operation of other physicians involved in reducing the interval between referral and diagnosis are required. Thus a considerable improvement in the prevention of complications of congenital heart malformations can be obtained. PMID- 10750613 TI - Social determinants of birthweight and length of gestation in Estonia during the transition to democracy. AB - BACKGROUND: To investigate social variation in birthweight and length of gestation in Estonia in the period of transition to a democracy and market economy. METHODS: All live births resulting from singleton pregnancies reported to the Estonian Medical Birth Registry in 1992-1997 (n = 84, 629) were studied with respect to social variation in birthweight and preterm delivery (<37 weeks gestation). The results were adjusted for maternal age, parity, education, nationality, marital status, smoking in pregnancy, sex of the infant (and gestational age). RESULTS: Between 1992 and 1997, mean birthweight increased from 3,465g to 3,497g (P < 0.001) and the preterm rate fell from 5.8% to 5.1% (P = 0.001). Maternal education, marital status and nationality were all independently related to the mean birthweight and the risk of preterm birth. The mean difference in birthweight between children of mothers with basic and university education was 87 g (95% CI : 74-100). Children born to mothers of non-Estonian compared to Estonian nationality were on average 77 g lighter (95% CI: 70-84). While the effect of nationality and marital status on birthweight was relatively stable during the study period, differences in birth outcome by maternal education became stronger. CONCLUSIONS: The mean birthweight increased and the preterm rate decreased in Estonia as a whole during the transition. However, the improvements were not shared equally by all social groups. An increase in variation in birthweight by maternal education was particularly notable. PMID- 10750614 TI - Sensitization to individual allergens as risk factors for lower FEV1 in young adults. European Community Respiratory Health Survey. AB - BACKGROUND: Atopy may impair ventilatory function, but results are controversial. We assess the association between individual reactivity to allergens and the level of baseline maximal one-second forced expiratory volume (FEV1), by smoking and respiratory symptoms. METHODS: The 1472 participants (response 44.5%) of the five Spanish areas of the European Community Respiratory Health Survey (ECRHS) who performed respiratory function tests, skin prick tests and/or specific IgE against common aeroallergens (e.g. mites, pets, mould, pollens) are included. Bronchial hyperreactivity (BHR) was measured with a methacholine challenge. RESULTS: After adjusting for BHR and smoking, in addition to the other allergens, skin reactivity to Alternaria (-208 ml; 95% CI :-451, 35) and IgE antibodies against cat (-124 ml; 95% CI:-269, 21) and Timothy grass (-115 ml, 95% CI:-190, 40) were associated with a decrease in FEV1 in females. Among males, skin reactivity to olive showed the strongest association (-111 ml; 95% CI: -261, 38). The associations were stronger in females. Smoking modifies the association for Alternaria and cat (P for interaction < 0.05). While cat is associated with a decrease in FEV1 in current smokers (-190 ml), Alternaria (-336 ml) was associated among never smokers. The exclusion of subjects with asthma symptoms, or adjustment for respiratory symptoms, led to similar results. CONCLUSIONS: We conclude that immunoresponse to individual allergens (particularly outdoor) is associated with the level of FEV1, and this association occurred independently of asthma, and in smokers and non-smokers, which may be of interest in natural history of chronic obstructive pulmonary disease (COPD). PMID- 10750615 TI - A prospective study of limiting longstanding illness in early adulthood. AB - BACKGROUND: Chronic illness and disability are of increasing public health importance but little is known about the lifetime influences involved in their onset and progression. We aim to (i) establish whether an individual's rating of limiting illness is stable over a 10-year period from age 23 to 33; (ii) assess the relationship between childhood and adult disability; and (iii) identify lifecourse influences on limiting illness in early adulthood. METHODS: Data were from the 1958 British birth cohort, including the original birth survey and follow-ups at ages 7, 11, 16, 23 and 33 years. Limiting longstanding illness was the outcome at both ages 23 and 33. Potential predictors included childhood health and physical development, socioeconomic conditions in early life and adulthood, and behavioural factors. We estimated the effect of potential explanatory factors using logistic regression, in both univariate and multivariate analyses, separately for limiting illness at 23 and 33 years. RESULTS: Prevalence of limiting illness increased from 5.1% (men) and 4.1% (women) at age 23 to 6% for both sexes at age 33. Risk of limiting illness at age 33 was greater for those reporting an illness at age 23 (29.4%, compared with 4.7% of those without illness), though the majority (66%) of 33-year limiting illnesses had no previous record at age 23 or for childhood. Multivariate analysis of limiting illness at age 23 confirmed the high risk for those with childhood disability and also established two further major predictors, namely, injury (adjusted odds ratio [OR] = 1.42, 95% CI: 1.09-1.86) and intermediate socio-emotional status (adjusted OR = 1.73, 95% CI: 1.29-2.31). Additional risks were identified for limiting illness at age 33, including: (i) injury in the preceding 10 years (adjusted OR = 1.55, 95% CI : 1.18-2.04); (ii) body mass index (BMI), for which the relationship was non-linear, with elevated risks for the underweight (adjusted OR = 1.53, 95% CI: 1.03-2.26) and overweight (OR = 1.28, 95% CI: 0.87-1.89); (iii) childhood disadvantage at either or both ages 7 and 11 (adjusted OR = 1.53, 95% CI : 1.07-2.17); and (iv) height at age 7, with a significant non-linear relationship (the adjusted OR for height less than 15th percentile was 1.43 and for height more than the 85th percentile, 1.30). CONCLUSIONS: Both childhood and adult factors predict limiting illness in early adulthood. Childhood is important because some adult illnesses originate in early life, and also because childhood environment influences the risk of adult limiting illness several years later. Our findings suggest that studies seeking to understand the causes of limiting illness, that currently tend to focus exclusively on contemporary factors, need also to consider the contribution of environment in early life. PMID- 10750616 TI - Lack of improvement of life expectancy at advanced ages in The Netherlands. AB - BACKGROUND: Several countries have reported an increase in life expectancy at advanced ages. This paper analyses recent changes in life expectancy at age 60 and 85 in The Netherlands, a low mortality country with reliable mortality data. METHODS: We used data on the population and the number of deaths by age, sex and underlying cause of death for 1970-1994. Life expectancy at age 60 and 85 was estimated using standard life-table techniques. The contribution of different ages and causes of death to the change in life expectancy during the 1970s (1970/74-1980/84) and the 1980s (1980/84-1990/94) were estimated with a decomposition technique developed by Arriaga. RESULTS: Life expectancy at age 60 increased in the 1970s and 1980s, whereas life expectancy at age 85 decreased (men) and stagnated (women) in the 1980s, and has decreased in both sexes since 1985/89. The decomposition by age showed that constant mortality rates in women aged 85-89, and increasing mortality rates at ages 85+ (men) and 90+ (women) have caused this lack of increase in life expectancy. The decomposition by cause of death showed that smaller mortality reductions from other cardiovascular and cerebrovascular diseases, which contributed most to the increase in life expectancy at age 85 in the 1970s, and mortality increases from, amongst others, chronic obstructive pulmonary disease (COPD), mental disorders and diabetes mellitus produced the decrease (men) and plateau (women) in life expectancy at age 85. CONCLUSIONS: Life expectancy at advanced ages stopped increasing during the 1980s in The Netherlands due to mortality increases at ages 85+ (men) and 90+ (women). Cause-specific trends suggest that, in addition to (past) smoking behaviour in men, changes in the distribution of morbidity and frailty in the population might have contributed to this stagnation. PMID- 10750617 TI - Dichotomous or categorical response? Analysing self-rated health and lifetime social class. AB - BACKGROUND: Self-rated health is a commonly used measure of health status, usually having three to five categories. The measure is often collapsed into a dichotomous variable of good versus less than good health. This categorization has not yet been justified. METHODS: Using data from the 1958 British birth cohort, we examined the relationship between socioeconomic conditions, indicated by occupational class at four ages, and self-rated health. Results obtained for a dichotomous variable using logistic regression were compared with alternative methods for ordered categorical variables including polytomous regression, cumulative odds, continuation ratio and adjacent categories models. RESULTS AND CONCLUSIONS: Findings concerning the relationship between socioeconomic position and self-rated health yielded by a logistic regression model were confirmed by alternative statistical methods which incorporate the ordered nature of self rated health. Similarity of results was found regarding size and significance of main effects, type of association and interactive effects. PMID- 10750618 TI - Principles of multilevel modelling. AB - BACKGROUND: Multilevel modelling, also known as hierarchical regression, generalizes ordinary regression modelling to distinguish multiple levels of information in a model. Use of multiple levels gives rise to an enormous range of statistical benefits. To aid in understanding these benefits, this article provides an elementary introduction to the conceptual basis for multilevel modelling, beginning with classical frequentist, Bayes, and empirical-Bayes techniques as special cases. The article focuses on the role of multilevel averaging ('shrinkage') in the reduction of estimation error, and the role of prior information in finding good averages. PMID- 10750619 TI - Record-linkage between two anonymous databases for a capture-recapture estimation of underreporting of AIDS cases: France 1990-1993. The Clinical Epidemiology Group from Centres d'Information et de Soins de l'Immunodeficience Humaine. AB - OBJECTIVE: To estimate the completeness of the French mandatory AIDS surveillance system (Declaration Obligatoire DO) over the 1990-1993 period using a capture recapture approach, by matching the mandatory reports with the AIDS cases present in the French Hospital Database on HIV infection (FHDH). METHODS: An anonymous record-linkage algorithm was developed to identify those cases common to both anonymous surveillance systems. The linkage was based on sex, date of birth, and infection risk group, all strictly matched, and on the dates of AIDS diagnosis and of death, the places of diagnosis and residence, and the AIDS-defining diseases at diagnosis. The total number of AIDS cases and completeness of both surveillance systems were estimated using a capture-recapture approach, assuming independence of the ascertainment sources. RESULTS: The completeness of the mandatory reporting was estimated at 83.6% (95% CI: 82.9-84.3), and that of the FHDH at 47.6% (95% CI: 46.9-48.3) for the surveillance of AIDS cases diagnosed among adults in France between 1990 and 1993. The completeness of the system based on FHDH increased over the study period as more hospitals joined the project, while the completeness of the DO surveillance system remained stable. CONCLUSION: This approach was useful in estimating the underreporting of AIDS cases in France. Regularly performed, it will allow the impact of underreporting to be monitored over time. PMID- 10750620 TI - Antibodies to human herpes virus type 8 (HHV8) in general population and in individuals at risk for sexually transmitted diseases in Western Sicily. AB - BACKGROUND: Human herpes virus 8 (HHV8) appears to be the primary aetiologic agent of Kaposi sarcoma (KS). To study the distribution of HHV8, a seroepidemiological study was carried out in western Sicily, where a high incidence rate of classical KS is well documented. METHODS: A total of 970 sera of healthy human immunodeficiency virus (HIV) negative individuals of general population (1-70 years old) and 742 sera of individuals in different risk groups for HIV infection were evaluated by means of an indirect immunofluorescence assay able to detect antibodies to lytic and latent HHV8 antigens. RESULTS: Crude seroprevalence to HHV8 antigens was 11.5% in the general population, and it increased significantly with age from 6% under age 16 to 22% after age 50. Significantly higher HHV8 seroprevalence rates were detected among HIV positive and negative homosexual men (62% and 22%, respectively), men who had sex with prostitutes (40% and 29%, respectively); female prostitutes (42% and 30%, respectively), and clients at a sexually transmitted disease clinic (male: 60% and 33%, respectively, female: 63% and 43%, respectively). In contrast, heterosexual intravenous drug users had seroprevalence rates comparable to those found in the general population. CONCLUSIONS: The results suggest that HHV8 infection is widespread in Western Sicily. The high seroprevalence in individuals with high risk sexual activity point to the role of sexual behaviour in the transmission of the infection in adults, whereas the detection of antibodies in younger population (under 16 years old) is suggestive of a non-sexual route of transmission, probably occurring during childhood by close personal contact. PMID- 10750621 TI - Mathematical modelling of infection and disease due to Neisseria meningitidis and Neisseria lactamica. AB - BACKGROUND: Invasive meningococcal disease, due to Neisseria meningitidis, is an important cause of morbidity and mortality in young children and adolescents. Nasopharyngeal carriage of meningococci (MC), is most prevalent in young adults whereas carriage of Neisseria lactamica (LC), a related non-pathogenic organism, is most prevalent in young children. The objective of this study was to use modelling techniques to test hypotheses on the processes that govern the incidence of meningococcal disease (MD). METHODS: Deterministic compartmental models were fitted to age structured data sets of MC, LC and MD. RESULTS: The model most consistent with the available data sets is one where LC inhibits MC, an inhibition that lasts for a mean of 4.7 years. The hypothesis that LC also acts as a natural immunogen against MD was consistent with this model. The second peak of MD observed among adolescents could be due to the peak in the acquisition of MC in this age group. CONCLUSIONS: The role of LC as a natural immunogen against asymptomatic and symptomatic meningococcal infection was consistent with available field data. If the introduction of novel meningococcal vaccines into a population changes the prevalence of MC or LC, this could have a substantial impact on the effectiveness of immunization programmes. This paper demonstrates the potential utility of modelling to estimate these effects. PMID- 10750622 TI - Can children's health be predicted by perinatal health? PMID- 10750623 TI - The 'healthy migrant effect'--not merely a fallacy of inaccurate denominator figures. PMID- 10750624 TI - Managing physical symptoms: the clinical assessment as treatment. AB - Physical symptoms are a common cause of attendance at general hospital out patient clinics. There is good evidence that cognitive therapy is effective in the management of such physical symptoms. This narrative review suggests that the assessment itself, without formal psychological therapy, may be used as a treatment, regardless of whether relevant pathology is absent or present. Changing patients' beliefs about their symptoms may improve a broad range of outcomes, including symptoms, disability, distress, and health-care resource use. The evidence for investigations as treatment is reviewed, along with potential for further development and possible pitfalls. A rationale is presented for a brief psychoeducational intervention that can be delivered in the clinic. This would be a logical extension of the kind of simple explanation and reassurance that occurs routinely today, but which is not explicitly used as, or regarded as, treatment. The dearth of relevant evidence is emphasized, and recommendations are made for future research. PMID- 10750625 TI - Psychological functioning and quality of life following bone marrow transplantation: a 3-year follow-up study. AB - OBJECTIVE: To assess changes in quality of life and psychological distress following bone marrow transplantation (BMT) and variables related to this change. METHODS: One hundred twenty-five consecutive patients who underwent bone marrow transplantation (BMT) at the Leiden University Medical Centre between 1987 and 1992 filled in questionnaires measuring quality of life, functional limitations, psychological distress, anxiety, depression, self-esteem, and health locus of control. Measurements were taken before the BMT; 1 month after discharge; and 6 months, 1 year, and 3 years after the BMT. RESULTS: Three years after BMT, a quarter of the patients continued to experience serious functional limitations. Thirteen percent of the patients scored >4 on the General Health Questionnaire (GHQ-12), a percentage comparable to general population prevalence. Quality of life was reported to be good to excellent by almost 90% of the patients at three years. Changes in quality of life could be explained entirely by changes in functional limitations and somatic symptoms. Changes in psychological distress were also related to these measures, and furthermore to baseline psychological functioning. CONCLUSION: Although patients were doing well three years after BMT, there was a group of patients needing help. In interventions special attention should be given to patients with ongoing psychological problems. Emphasis should be on coping with physical limitations. PMID- 10750626 TI - Psychological response to exercise training and/or energy restriction in obese women. AB - OBJECTIVE: The effect of 12 weeks of exercise training (five 45-minute walking sessions/week at 60% to 75% maximum heart rate) and/or moderate energy restriction (4.19 to 5.44 MJ or 1200 to 1300 kcal/day) on psychological general well-being (GWB) and profile of mood states (POMS) was studied in obese women (n = 91, age 45.6 +/- 1.1 years, body mass index 33.1 +/- 0.6 kg/m2). METHODS: Subjects were randomized to one of four groups: control (C); exercise (E); diet (D); and exercise and diet (ED). Psychological variables were measured in all subjects at three timepoints--prestudy, 3 weeks, and poststudy--and analyzed using a 4 x 3 repeated measures design. RESULTS: GWB but not POMS was improved in ED (but not E or D) relative to C poststudy, especially in four of six subscales: freedom from health concern or worry, life satisfaction; cheerful versus depressed mood; and relaxed versus tense feelings. CONCLUSION: These data demonstrate that the combination of moderate energy restriction and exercise training during a 12-week period improves psychological general well-being scores but not mood states in obese subjects. PMID- 10750627 TI - Rationality/antiemotionality personality and selected chronic diseases in a community population in Japan. AB - OBJECTIVE: To clarify the relationships of the rationality/antiemotionality (R/A) personality with past histories of seven chronic diseases and current smoking habits in a community population in Japan. METHODS: A questionnaire survey was conducted of all residents aged 35 years old or over (n = 37,287) in Takayama city, Gifu prefecture, Japan; the response rate was 90.3%. Data from 13,091 males and 14,061 females who completed all relevant questions were analyzed. The subjects were asked to complete an 11-item R/A personality scale developed by Grossarth-Maticek et al. (1985). Past histories of seven major chronic diseases and current smoking habit were also asked in the questionnaire. RESULTS: Among males, the R/A scale score was significantly lower in those who had stroke, diabetes or allergy than in those who did not. Among females, the R/A scale score was significantly lower in those who had stroke, allergy or stomach cancer than in those who did not. Among females, current smokers had significantly lower R/A scores than non-smokers. After controlling for age, smoking and drinking, the R/A score was significantly and negatively associated with stroke among males and females; it was significantly and negatively associated with diabetes and allergy among males. CONCLUSION: Our findings are contrary to those observed in Yugoslavia and West Germany, and suggest a cultural difference in the relationship between the R/A personality and disease occurrence. PMID- 10750628 TI - Empowering diabetes out-patients with structured education: short-term and long term effects of functional insulin treatment on perceived control over diabetes. AB - OBJECTIVE: To investigate short-term and long-term effects of structured outpatient education for Functional Insulin Treatment (FIT: selective insulin dosages for eating, fasting or correcting hyperglycaemia) on perceived control over diabetes and related health beliefs. FIT was thought to influence the perception of self-efficacy in diabetes, in contrast to conventional treatment, based on scheduled, rigid food intake and insulin delivery--it allows flexible eating, provided independent control of glycemia. METHODS: Structured, comprehensive, outpatient group training in FIT for selective use of insulin either for eating, fasting or correction included practical "insulin games." The FIT program focused on everyday criteria for choices of insulin dosages and thus on the patient's ability to execute his/her newly gained flexible treatment to his/her everyday life while preventing acute and late complications. To evaluate effects of FIT on "Perceived Control over Diabetes" and related "Health Beliefs Concerning Diabetes" (Bradley's questionnaires, 1984), a fully randomized short term controlled Study 1 (four weeks, 32 patients), and long-term uncontrolled pilot Study 2 (three years, 68 patients) were performed. RESULTS: The short-term study revealed that FIT induced the feeling of independence from situational control while self-managing diabetes. In the long-term study, the patients were increasingly freed from the feeling of being under the control of physician and treatment-related restrictions, which--together with higher perceived self efficacy--contributed to the feeling of "empowerment." This was associated with high treatment satisfaction and significant improvement of glycemic control. CONCLUSION: Structured out-patient group training for FIT results in measurable improvement of patients' perceived control over diabetes and self-efficacy. PMID- 10750629 TI - Effects of melatonin on the quality of life in patients with delayed sleep phase syndrome. AB - OBJECTIVE: The purpose of this study was to compare health-related quality of life of delayed sleep phase syndrome (DSPS) patients with a random Dutch sample and four samples of patients with other chronic conditions. We also investigated the effectiveness of treatment with 5 mg of melatonin on the quality of life of DSPS patients. METHODS: Forty-three DSPS patients completed a quality-of-life questionnaire (Medical Outcome Study Short Form-36 [MOS SF-36] health survey) just before and 2-9 months after participation in a clinical trial involving the administration of melatonin. Scores were compared with responses to the same survey by a random Dutch sample and by patients with sleep apnea, clinical depression, migraine, and osteoarthritis. RESULTS: MOS SF-36 scales scores were significantly lower in DSPS patients relative to age- and gender-adjusted norms for the Dutch sample. Some health dimensions were more affected, and others less affected, by DSPS compared with the other chronic conditions. Melatonin treatment improved all scales except the scale "role due to emotional problems." CONCLUSION: DSPS has a unique significant quality-of-life burden that seems to be improved by treatment with melatonin. PMID- 10750630 TI - Psychiatric symptoms and distress differ between patients with postherpetic neuralgia and peripheral vestibular disease. AB - OBJECTIVE: No previous studies have investigated the psychiatric characteristics of patients with postherpetic neuralgia (PHN). Similarly, no studies have been performed on patients with different chronic somatic symptoms due to a defined medical disease to compare the characteristics of psychiatric morbidity associated with each etiology. METHODS: After completing the subscales of the Symptom Checklist 90-R, a psychiatrist administered the Diagnostic Interview Schedule to all subjects. The psychiatric comorbidity in 35 patients with pain due to PHN was compared with a control group of 34 patients with the nonpainful aversive symptom of vertigo due to a peripheral vestibular disorder that caused unilateral hypofunction. RESULTS: PHN patients had significantly more symptoms of major depression and somatization disorder. No significant differences were found between groups for psychiatric diagnoses. Patients with PHN reported significantly less acutely distressing somatic symptoms. CONCLUSION: These results suggest that the psychiatric symptoms of patients with PHN are distinct from nonspecific acute distress and may be related to the experience of suffering from chronic neuropathic pain. Patients with PHN may not meet criteria for a psychiatric diagnosis, but their psychiatric comorbidity places them at substantial risk for increased pain, suicidal ideation, sustained disability, and the numerous complications of excessive medical evaluation and treatment. Patients with PHN should be evaluated specifically for psychiatric symptoms to reduce potential negative consequences through appropriate treatment. PMID- 10750631 TI - Symptom patterns in long-duration chronic fatigue syndrome. AB - OBJECTIVE: Our objective was to evaluate symptom patterns in patients with chronic fatigue syndrome (CFS) who were ill for 10 or more years. METHODS: This cross-sectional self-report study compared patient groups with long-duration (median = 18 years; n = 258) and short-duration (median = 3 years; n = 28) CFS to a group of healthy significant others (n = 79) on symptomatic, neurocognitive, and psychological variables. Data were gathered from a 574-item postal questionnaire. RESULTS: A principal-components analysis of CFS symptom data yielded a three-factor solution: cognitive problems; flu-like symptoms; and neurologic symptoms. Compared with the short-duration CFS group, the long duration group had significantly higher CFS symptom severity scores (p < 0.04), largely attributable to increased cognitive difficulties. A subgroup comparison of subjects ill for < 3 years versus those ill 4-7 years suggested that denial coping strategies were more likely in those participants with the shorter illness duration. Significant differences between both CFS groups and healthy controls were found in a number of comorbid disorders. Participants with CFS most often endorsed immune/viral abnormalities and persistent stress as important perceived causes of their illness. CONCLUSION: Participants with long-duration CFS reported a large number of specific cognitive difficulties that were greater in severity than those reported by participants with short-duration CFS. The pattern of comorbid disorders in the CFS groups was consistent with hypersensitivity and viral reactivation hypotheses. PMID- 10750632 TI - Ejaculatory latency and control in men with premature ejaculation: an analysis across sexual activities using multiple sources of information. AB - OBJECTIVE: Men with premature ejaculation (PE) exhibit diminished control over and short latency to ejaculation. The present study attempted to delineate further characteristics of men with PE and to address a number of presumed hypotheses regarding the etiology of this disorder. METHODS: Twenty-six men with PE were compared with an age-matched group of 13 sexually functional men on multiple indices of erectile and ejaculatory response during coital and masturbatory activities. These data were collected through retrospective, prospective, and laboratory methods. RESULTS: Psychophysiological testing indicated greater ejaculatory vulnerability to penile stimulation, although not visual erotic stimulation, in PE men than functional controls. PE men also showed subtle anomalies in the way they perceived their somatic response. The correlation between measures of ejaculatory latency and control was positive and high for intercourse, but low or even negative for masturbation. Whereas functional men showed consistency in ejaculatory latency over coital and masturbatory activities, PE men exhibited much shorter latencies during coitus than masturbation. Data collected under various methodologies (retrospective, prospective, and laboratory) showed greater consistency among sexually functional subjects; and preliminary analysis of laboratory data suggests psychophysiological methodology is as effective in differentiating dysfunctional from functional men as prospective and retrospective methodologies. CONCLUSION: Although ejaculatory latency and control tend to be related, these measures are not always stable over different kinds of sexual activity or using different methods of data collection. Psychophysiological methodology is effective in differentiating group membership (PE vs. control), but does not predict individual ejaculatory responses measured prospectively. PMID- 10750633 TI - Relationship between left ventricular mass and hemodynamic responses to physical and mental stress. AB - OBJECTIVE: Increased left ventricular mass (LVM) is predictive of future cardiac morbidity and mortality. Although casual and ambulatory blood pressure (BP) predict LVM, other hemodynamic determinants of LVM are incompletely understood. The present study examines laboratory-induced hemodynamic responses (to exercise, cold, and mental stress) and 24-hour ambulatory measures as predictors of LVM. METHODS: Thirty-six healthy non-hypertensive subjects (mean age 33.9 +/- 9.4 years; 23 women, 13 men) were tested with mental stress, cold pressor, and treadmill exercise in the laboratory and 24-hour ambulatory BP monitoring. LVM was measured using two-dimensional targeted M-mode echocardiography and indexed for body surface area (LVMI). RESULTS: All laboratory tasks produced significant hemodynamic responses (p's < 0.01). Systolic blood pressure responses to mental stress (r = 0.42, p < 0.01) and cold pressor (r = 0.34, p < 0.05) were significantly related to LVM. After adjusting for body size, the mental stress induced SBP responses was the only significant predictor of LVMI (r = 0.32, p < 0.05). Exercise SBP responses were associated to LVMI in men (r = 0.63, p = 0.02), but not in women (r = 0.02, p = n.s.). Multivariate regression analyses revealed that SBP during mental stress was significantly predictive of LVMI (beta = 0.65, p = 0.05), independent of baseline SBP, 24-hour ambulatory SBP, and other control variables. CONCLUSION: The present results indicate that SBP responses to mental stress are significantly related to LVM among healthy individuals, independently of baseline SBP, 24-hour ambulatory BP, age, body size, and sex. Blood pressure responses to exercise show a robust association with LVM in men but not in women. Hemodynamic responses elicited during laboratory tasks may therefore reveal important information about the pathophysiological processes involved in the development of cardiac end-organ damage. PMID- 10750634 TI - Hostility and ill health: role of psychosocial resources in two contexts of working life. AB - OBJECTIVE: The purpose of this study was to examine the extent to which increased risk to health problems in hostile employees is associated with psychosocial resources and life context. METHODS: Social relationships, job control, and sickness absence were assessed among 757 hostile and nonhostile municipal employees in two life contexts: during a stressful organizational downsizing; and during a period after the downsizing. The follow-up time was 550 person-years for men and 1677 person-years for women during the period of downsizing, and 519 person-years for men and 1568 person-years for women during the period after downsizing. RESULTS: The risk of sick leave was 1.2-1.4-fold higher in the hostile individuals than in the others. Small network size in hostile employees related to a 1.4-2.5-fold higher risk of sick leave compared to large network size. In nonhostile employees, network size did not associate with sick leave. Poor job control effected a 50% higher risk of sick leave in hostile than in nonhostile individuals during downsizing. During the less stressful period, both hostility and poorjob control increased absence rates independently of one another. Strong spouse support protected only nonhostile employees from sickness. No differences between hostile and nonhostile employees were found in the levels of psychosocial resources or changes in them. CONCLUSION: The risk of health problems in hostile persons could be linked with heightened vulnerability in poor psychosocial resource conditions and with the inability to benefit from existing psychosocial resources. Such personal deficits seem to be resource-specific and vary somewhat according to an individual's life context. PMID- 10750635 TI - Depression is strongly associated with alexithymia in the general population. AB - OBJECTIVE: This study examines how alexithymia and depression are related to each other in men and women in a sample of Finnish general population (n = 2018). METHODS: Alexithymia was screened using the 20-item version of the Toronto Alexithymia Scale. Level of depression was assessed using the 21-item Beck Depression Inventory (BDI). Life satisfaction was estimated with a structured scale. RESULTS: The prevalence of alexithymia was 12.8% in men and 8.2% in women. However, the prevalence of alexithymia was 32.1% among those having BDI scores of > or = 9, but only 4.3% among the nondepressed subjects (p < 0.001). The BDI scores explained 29.2% of the variation in TAS-20 scores. Alexithymia was associated with several sociodemographic factors if depression was not taken into account. However, after including depression in the logistic regression models, only depression and low life satisfaction were associated with alexithymia, both in men and women. CONCLUSION: These results suggest that alexithymia has a close relationship to depression in the general population. The impact of social factors on alexithymia may be primarily explained by depression. Depression must be taken into account as a confounding factor when studying alexithymia in general populations due to the strong association between alexithymia and depression. PMID- 10750636 TI - Mechanisms of blood pressure alterations in response to the Valsalva maneuver in postural tachycardia syndrome. AB - The postural tachycardia syndrome (POTS) is characterized clinically by orthostatic lightheadedness and tachycardia. When these patients perform a Valsalva maneuver, there is an excessive blood pressure increment after cessation of the maneuver (phase IV) that is sometimes associated with headaches. It is not known whether excessive phase IV is due to excessive peripheral vascular tone (an alpha-adrenergic mechanism) or is a manifestation of increased beta-adrenergic tone (hyperadrenergic state). The authors undertook a pharmacologic study evaluating the effect of intravenous phentolamine (alpha-adrenergic antagonist) and propranolol (beta-adrenergic antagonist) on the different phases of the Valsalva maneuver in a group of patients with POTS and age-matched normal control subjects. Patients with POTS had mean phases, when compared with controls, that were characterized by more negative II_E (p = 0.07), smaller II_L (p = 0.04), and significantly larger phase IV (p = 0.001). The effect of phentolamine was qualitatively and quantitatively different in POTS when compared with controls. Ten mg phentolamine in controls resulted in a significant accentuation of phase II_E (p = 0.001), attenuation of phase II_L (p = 0.002), and increase of phase IV (57.6 vs 30.7 mm Hg; p = 0.025). These changes resembled those of patients with POTS at baseline. In patients with POTS, the phase II abnormalities, already present, were further accentuated (p <0.001), and phase IV became smaller (50.6 vs 73.8 mm Hg; p = 0.09). Propranolol had no significant effect on phases II_E and II_L, but significantly reduced phase IV in both controls (p <0.05) and in patients with POTS (p <0.001) and improved the headache symptoms, when present, during and after phase IV. The authors conclude that phase IV is mainly under beta-adrenergic regulation and that the exaggerated phase IV in POTS is a result of a hyperadrenergic state. PMID- 10750637 TI - Intravenous cannulation of adolescents does not affect the modulation of autonomic tone assessed by heart rate and blood pressure variability. AB - Invasive arterial monitoring alters autonomic tone. The effects of intravenous (i.v.) insertion are less clear. The author assessed the effects of i.v. insertion on autonomic activity in patients aged 11 to 19 years prior to head-up tilt by measuring heart rate, blood pressure, heart rate variability, blood pressure variability, and baroreceptor gain before and after i.v. insertion with continuous electrocardiography and arterial tonometry in patients with orthostatic tachycardia syndrome (OTS, N = 21), in patients who experienced simple fainting (N = 14), and in normal control subjects (N = 6). Five-minute samples were collected after 30 minutes supine. Fifteen minutes after i.v. insertion, data were collected again. These 5-minute samples were also collected in a separate control population without i.v. insertion after 30 minutes supine and again 30 minutes later. This population included 12 patients with OTS, 13 patients who experienced simple fainting, and 6 normal control subjects. Heart rate variability included the mean RR, the standard deviation of the RR interval (SDNN), and the root mean square of successive RR differences (RMSSD). Autoregressive spectral modeling was used. Low-frequency power (LFP, 0.04-0.15 Hz), high-frequency power (HFP, 0.15-0.40 Hz), and total power (TP, 0.01-0.40 Hz) were compared. Blood pressure variability included standard deviation of systolic blood pressure, LFP, and HFP. Baroreceptor gain at low frequency and high frequency was calculated from cross-spectral transfer function magnitudes when coherence was greater than 0.5. In patients with OTS, RR (790 +/- 50 msec), SDNN (54 +/- 6 msec), RMSSD (55 +/- 5 msec), LFP (422 +/- 200 ms2/Hz), HFP (846 +/- 400 ms2/Hz), and TP (1550 +/- 320 ms2/Hz) were less than in patients who experienced simple fainting (RR, 940 +/- 50 msec; SDNN, 84 +/- 10 msec; RMSSD, 91 +/- 7 msec; LFP, 880 +/- 342 ms2/Hz; HFP, 1720 +/- 210 ms2/Hz; and TP, 3228 +/- 490 ms2/Hz) or normal control subjects (RR, 920 +/- 30 msec; SDNN, 110 +/- 29 msec; RMSSD, 120 +/- 16 msec; LFP, 1600 +/- 331 ms2/Hz; HFP, 2700 +/- 526 ms2/Hz; and TP, 5400 +/- 1017 ms2/Hz). Blood pressure and blood pressure variability were not different in any group. Standard deviation, LFP, and HFP were, respectively, 5.24 +/- 0.8 mm Hg, 1.2 +/- 0.2, and 1.5 +/- 0.3 for patients with OTS; 4.6 +/- 0.4 mm Hg, 1.2 +/- 0.2, and 1.4 +/- 0.3 for patients who experienced simple fainting; and 5.55 +/- 1.0 mm Hg, 1.4 +/- 0.2, and 1.6 +/- 0.3 for normal control subjects. Baroreceptor gain at low frequency and high frequency in patients with OTS (16 +/- 4 msec/mm Hg, 17 +/- 5) was comparable to that in patients who experienced simple fainting (33 +/- 4, 32 +/- 3) and that in normal control subjects (31 +/- 8, 37 +/- 9). Heart rate variability differed between patients with OTS and patients who experienced simple fainting or normal control subjects, and blood pressure and blood pressure variability were not different, but no parameter changed after i.v. insertion. There were no differences from the groups that did not receive i.v. insertions. Data suggest, at most, a limited effect of i.v. insertion on autonomic function in adolescents. PMID- 10750638 TI - Long-term effect of acetyl-L-carnitine on myocardial 123I-MIBG uptake in patients with diabetes. AB - Carnitine derivatives may have beneficial effects on cardiac and nerve function in patients with diabetes. The aim of this study was to investigate the effect of acetyl-L-carnitine (ALC) on myocardial sympathetic nervous function as measured with 123I-meta-iodobenzyl guanidine (MIBG) and single-photon emission tomography (SPET) in 19 patients with diabetes (placebo group, n = 6; ALC group, n = 13) at the beginning and at the end of a 1-year randomized, placebo-controlled, double blind trial. The coefficient of variation for the MIBG analysis was 4%. In patients who were given a placebo, global myocardial MIBG uptake deteriorated during the study (MIBG uptake 1-year follow-up/baseline, 0.86 +/- 0.05, mean +/- standard error of mean), whereas in patients treated with ALC, MIBG uptake did not change significantly (1-year follow-up/baseline, 1.07 +/- 0.08; p = 0.03 between the groups). On the basis of these preliminary data, we conclude that long-term treatment with ALC may be of potential value in preventing the progressive loss of myocardial sympathetic nervous function in patients with diabetes. MIBG-SPET is a sensitive and thus valuable method in assessing the development of myocardial sympathetic nervous dysfunction. PMID- 10750639 TI - Autonomic neuropathy in patients with HIV: course, impact of disease stage, and medication. AB - The purpose of this article is to examine the prevalence, degree, and natural course of pupillary neuropathy (PANP), cardiovascular autonomic neuropathy (CANP), and sensorimotor neuropathy (SNP) and to study the impact of disease stage and medication on neuropathy in 61 consecutive patients with HIV. PANP, CANP, and SNP were assessed by standardized test procedures. Overall prevalence of PANP, CANP, and SNP were 66%, 15%, and 15%, respectively. The maximal pupillary area (pupillary measure, p <0.0001) and the lying-to-standing ratio (cardiovascular measure, p <0.0001) were abnormal as compared with control subjects. The changes in CD4+ T-lymphocytes and respiratory sinus arrhythmia percentile during 2 years of follow-up correlated significantly (r = 0.758, p = 0.007). Patients with CANP were more often in an advanced disease stage than patients without CANP (p = 0.004). SNP, but not PANP or CANP, was associated with the intake of the neuropathogenic drugs dideoxycytidine, dideoxyinosine, and 2',3' didehydro-2',3' dideoxythymidine (p <0.05). Autonomic and sensorimotor neuropathy are frequent in patients with HIV, and progression of CANP may put patients at risk for unexpected cardiorespiratory arrest. PMID- 10750640 TI - Autonomic response to real versus illusory motion (vection). AB - This study explored the cardiovascular responses to illusions of self-motion (vection) induced in normal subjects according to the hypothesis that vection may be a model for vertigo in vestibular disease. Responses were obtained from 10 men who were exposed to rapid tilts of 20 degrees and 30 degrees rolling from the upright position down to the right or left shoulder. These responses were compared with those evoked during the illusion of roll-tilt vection provoked by a torsionally rotating visual field. Comparisons were made between 10-second data epochs before and after stimulus onset. In response to vection, blood pressure (BP) in the radial artery rose consistently in six subjects, and in all of these, a pressor response to real tilt was also observed. The remaining four subjects consistently had decreased BP in response to vection, and their BPs were affected little by tilt. Subjects whose BP increased with vection and tilt may have been dominated by tendency to arousal, whereas those whose BP decreased may reveal the more appropriate response to tilt from the upright position, which is a decrease in BP. This may reflect individual stereotypes and differences in the relative contributions of somatosensory and vestibular control of autonomic regulation. PMID- 10750641 TI - Hemodynamic and symptomatic effects of acute interventions on tilt in patients with postural tachycardia syndrome. AB - A variety of approaches have been used to alleviate symptoms in postural tachycardia syndrome (POTS). Drugs reported to be of benefit include midodrine, propranolol, clonidine, and phenobarbital. Other measures used include volume expansion and physical countermaneuvers. These treatments may influence pathophysiologic mechanisms of POTS such as alpha-receptor dysfunction, beta receptor supersensitivity, venous pooling, and brainstem center dysfunction. The authors prospectively studied hemodynamic indices and symptom scores in patients with POTS who were acutely treated with a variety of interventions. Twenty-one subjects who met the criteria for POTS were studied (20 women, 1 man; mean age, 28.7 +/- 6.8 y; age range, 14-39 y). Patients were studied with a 5-minute head up tilt protocol, ECG monitoring, and noninvasive beat-to-beat blood pressure monitoring, all before and after the administration of an intervention (intravenous saline, midodrine, propranolol, clonidine, or phenobarbital). The hemodynamic indices studied were heart rate (ECG) and systolic, mean, and diastolic blood pressure. Patients used a balanced verbal scale to record any change in their symptoms between the tilts. Symptom scores improved significantly after the patients received midodrine and saline. Midodrine and propranolol reduced the resting heart rate response to tilt (p <0.005) and the immediate and 5-minute heart rate responses to tilt (p <0.002). Clonidine accentuated the immediate decrease in blood pressure on tilt up (p <0.05). It was concluded that midodrine and intravenous saline are effective in decreasing symptoms on tilt in patients with POTS when given acutely. Effects of treatments on heart rate and blood pressure responses generally reflected the known pharmacologic mechanisms of the agents. PMID- 10750643 TI - Familial vasovagal syncope and pseudosyncope: observations in a case with both natural and adopted siblings. AB - This report describes an 11-year-old girl with recurrent syncope beginning at the age of 2 1/2 years. Her paternal grandmother, father, and three of her five natural siblings had similar symptoms, often linked to emotional upsets. There were three adopted children from a single family, and none of these had syncope. Prior to referral there was an increase in attacks, some with convulsions, but with no other features of epilepsy. Vasovagal syncope was confirmed. However, continuous electroencephalogram, blood pressure, and heart rate recordings during attacks indicated that in some episodes there was neither cardiovascular change nor epileptiform activity, implying feigned syncope (pseudosyncope) with pseudoseizures. A psychological origin was sought, found, and in part rectified. The separation of vasovagal syncope from pseudosyncope, in the context of the family history, is discussed. PMID- 10750642 TI - Fludrocortisone and sleeping in the head-up position limit the postural decrease in cardiac output in autonomic failure. AB - Treatment with head-up tilt sleeping and low-dose fludrocortisone effectively minimizes orthostatic symptoms and increases orthostatic blood pressure in patients with neurogenic orthostatic hypotension. The aim of the present study was to examine whether the improvement in orthostatic blood pressure during combined treatment with low-dose fludrocortisone and nocturnal head-up tilt in patients with neurogenic orthostatic hypotension can be attributed to expansion of plasma volume or to increased total peripheral resistance. The effects of a 3 week treatment with fludrocortisone and nocturnal head-up tilting on the postural changes in arterial pressure, heart rate, and cardiac output (pulse contour) were evaluated in eight consecutive patients with orthostatic hypotension. The period during which the patients were able to remain in the standing position without orthostatic complaints increased minimally from 3 to 10 minutes. The decrease in arterial pressure after 1 minute of standing--(means with standard deviations in parentheses) systolic, 49 (20) mm Hg; diastolic, 18 (11) mm Hg--before treatment was produced by a greater than normal decrease in cardiac output: 37% (10%) in patients with neurogenic orthostatic hypotension versus -14% (8%) in control subjects. Treatment increased upright arterial pressure from 83 (19) mm Hg systolic and 55 (13) mm Hg diastolic to 114 (22) mm Hg systolic and 60 (16) mm Hg diastolic by limiting the decrease in cardiac output. Body weight increased but hematocrit did not change. Leg pressure-volume relationship decreased in the two patients studied. The responses of plasma renin activity and aldosterone to orthostatic stress prior to treatment were subnormal and became even lower after treatment. The improvement in upright blood pressure in orthostatic hypotension during treatment with fludrocortisone and nocturnal head-up sleeping is the result of a reduction in the orthostatic decrease in cardiac output. Preliminary data suggest that the expanded body fluid volume is allocated to the perivascular space rather than to the intravascular space. PMID- 10750644 TI - Therapeutic effects of the endothelin receptor antagonist Ro 48-5695 in the TNBS/DNBS rat model of colitis. AB - OBJECTIVE: Endothelins can act as polyfunctional cytokines. It is therefore possible that endothelins could play an active role in gut inflammation. Elevated levels of endothelin-1 have been reported in ulcerative colitis and Crohn's disease. The aim of this study was to establish the therapeutic effect of a 'new' endothelin receptor antagonist Ro 48-5695 in an animal model of inflammatory bowel disease. This study compares the effect of Ro 48-5695 on colonic damage induced by two haptens: trinitrobenzenesulphonic (TNBS) or dinitrobenzenesulphonic acid (DNBS). METHODS: Colitis was induced by intra-rectal administration of TNBS or DNBS. After TNBS/DNBS injury, rats were treated with 10.0, 3.0, 1.0 or 0.3 mg/kg of Ro 48-5695 orally, daily for 5 days. On day 6 post hapten treatment, colonic tissues were removed and examined in a blinded fashion for macroscopic damage (damage score) and myeloperoxidase (MPO) activity. Stool consistency and adhesions were also measured. RESULTS: Oral administration of Ro 48-5695 almost completely prevented TNBS-induced damage at a dose of 10 mg/kg. The same dose in this model also had a therapeutic effect as measured by MPO and incidence of diarrhoea and adhesions. In DNBS-induced colonic damage, Ro 48-5695 was more potent and at 1.0 and 3.0 mg/kg decreased the damage score by 50 and 60% respectively; also the incidence of adhesions and diarrhoea was significantly reduced. However, MPO activity in this model was affected only by the highest dose of Ro 48-5695 tested (3.0 mg/kg) where it was reduced by 48%. CONCLUSIONS: These data provide evidence for the involvement of endothelins in the pathophysiology of inflammatory bowel disease and support the possibility of exploring a new therapeutic approach. PMID- 10750645 TI - The role of the resident intestinal flora in acute and chronic dextran sulfate sodium-induced colitis in mice. AB - OBJECTIVE: There is increasing evidence that the intestinal microflora plays an important role in the pathogenesis of inflammatory bowel disease. In the present study, we examined the role of the resident intestinal flora in our model of dextran sulfate sodium (DSS)-induced acute and chronic colitis in mice. METHODS: Acute colitis was induced in BALB/c mice with 5% DSS in their drinking water for 7 days. Chronic colitis was established after four cycles of feeding 5% DSS for 7 days and water for 10 days. For eliminating intestinal bacteria, mice were injected intraperitoneally with metronidazole and ciprofloxacin. We analysed four parameters: (1) body weight, (2) length of the colon, (3) histological score, and (4) myeloperoxidase activity. RESULTS: In acute DSS colitis treatment with antibiotics led to an improvement of the histological parameters (epithelial damage, P< 0.05; inflammatory infiltrate, P< 0.05) and colon length (P < 0.0028). A significant reduction in granulocyte infiltration was indicated by a 52.6% reduced myeloperoxidase activity in colonic biopsies. By contrast, in chronic colitis, treatment of mice with antibiotics failed to show significant effects. CONCLUSION: In acute DSS-induced colitis bacteria and/or bacterial products play a major role in initiation of inflammation but not in chronic DSS colitis. PMID- 10750646 TI - Pre-operative kinetic parameter determination of colorectal adenocarcinomas. Prognostic significance. AB - BACKGROUND: The aim of this study was to test the prognostic value of pre operative assessment of tumour kinetics in colorectal adenocarcinoma. METHODS: The study of tumour kinetics was performed using an in vivo injection of bromodeoxyuridine. Endoscopic biopsies were obtained from the tumour and analysed using flow cytometry. This procedure enables calculation of the in vivo S-phase fraction labelling index (LI), the duration of S-phase (Ts) and the potential tumour doubling time (Tpot). Disease-free survival curves were calculated by a Kaplan-Meier method. The statistical significance between curves was tested by the log rank test. A multivariate analysis was performed using the Cox's proportional hazards model to determine the effect of pathological staging (lymph node involvement), ploidy and kinetic parameters. RESULTS: Thirty-eight colorectal carcinomas were studied without prior chemotherapy or radiation therapy. In univariate analysis, lymph node involvement, labelling index > 10% and Tpot < 5 days were associated with poor prognosis, with P= 0.0006, 0.049 and 0.029 respectively; no significant differences were found in Ts (P = 0.214), and ploidy (P= 0.095). In multivariate analysis, lymph node involvement, ploidy and Tpot were found to be independent factors of colorectal cancer prognosis (P= 0.028, 0.032 and 0.035 respectively) in all tumours. Tpot was considered a independent prognostic factor in diploid tumours (P= 0.047) but not in aneuploid tumours (P= 0.345). CONCLUSIONS: These results suggest that kinetic parameters determined by pre-operative biopsies of colorectal adenocarcinoma represent a prognosis factor, independent of pathological staging, particularly in diploid tumours. PMID- 10750647 TI - Controlled clinical trial of doxorubicin and tamoxifen versus tamoxifen monotherapy in hepatocellular carcinoma. AB - OBJECTIVES: Treatment results of advanced hepatocellular carcinoma have remained unsatisfactory; the response rates to intravenous doxorubicin are no better than 20%. Oral tamoxifen has been proposed on the basis of beneficial results in some trials. The aim of this study was to evaluate whether the addition of doxorubicin to oral tamoxifen improves survival compared to oral tamoxifen alone. METHODS: Thirty-two consecutive patients with a priori defined contra-indications against surgery (transplantation, resection) or chemo-embolization were evaluated to receive chemotherapy. All patients received oral tamoxifen 30 mg bid; 16 also received intravenous doxorubicin 50 mg/m2 every 4 weeks. The control group consisted of the remaining 16 patients who either were considered unfit for doxorubicin because of a Karnofsky index < 50% (n = 5), cardiac disease (n = 6) or who refused to have cytotoxic drug therapy (n = 5). RESULTS: Median survival time was 148 days (95% CI 89.2-206.8) in the doxorubicin group and 96 days (95% CI 49.0-143.0) in the control group, and this was not significantly different (P= 0.408), regardless of the presence or absence of cirrhosis. CONCLUSIONS: In conclusion, the results of our study indicate that combination therapy using doxorubicin and tamoxifen is unlikely to considerably improve survival compared to tamoxifen alone in patients with advanced hepatocellular carcinoma. PMID- 10750648 TI - Early detection of haemobilia associated with percutaneous ethanol injection for hepatocellular carcinoma. AB - OBJECTIVE: Haemobilia often results from iatrogenic injury caused by therapeutic procedures. The objective of this study was to evaluate the efficacy of early diagnosis of haemobilia based on ultrasonography in patients with hepatocellular carcinoma undergoing percutaneous ethanol injection. PATIENTS AND METHODS: A combination retrospective and prospective study on the early detection of haemobilia caused by percutaneous ethanol injection was conducted on 365 patients in 1995-1996. The retrospective study reviewed the clinical, laboratory and imaging data of 172 patients who had undergone ethanol injection therapy in 1995. The results showed that ultrasonographic changes in the gallbladder, namely the rapid appearance of echogenic material in the gallbladder lumen, are a useful early sign of haemobilia. Based on the results of the retrospective study, a prospective study on the early detection of haemobilia was carried out in 1996. In the prospective study, percutaneous ethanol injection was halted as soon as haemobilia was detected. RESULTS: The incidence of haemobilia in the prospective group (3.6%) was not different from that in the retrospective group (4.7%). However, the mean duration between percutaneous ethanol injection and diagnosis of haemobilia was only 0.3 +/- 0.2 days in the prospective group, compared with 2.8 +/- 2.1 days in the retrospective group (P < 0.001), and the mean duration of jaundice in the prospective group (4.3 days) was significantly shorter than in the retrospective group (40.0 days) (P< 0.05). CONCLUSION: Early diagnosis of haemobilia based on ultrasonographic findings of the gallbladder lumen effectively reduces the severity of haemobilia-related complications due to immediate interruption of the interventional procedure. PMID- 10750649 TI - Calcium concentration in hepatocytes during liver ischaemia-reperfusion injury and the effects of diltiazem and citrate on perfused rat liver. AB - OBJECTIVE: To clarify the role of the calcium concentration in hepatocytes in liver ischaemia-reperfusion injury in relation to the protective effect of calcium-related agents. DESIGN: Serial calcium concentrations of extramitochondrial cytosolic sites (EMCa) and in mitochondria (MCa) in hepatocytes of isolated perfused rat liver were measured by an electron probe X ray microanalyser, and the effects of calcium-related agents were evaluated. METHODS: Ischaemia was induced for 2 h, followed by 2 h of reperfusion in group I (without drug), group II (with diltiazem, 9.3 microM) and group III (with citrate, 5 mM). RESULTS: In group I, EMCa increased rapidly after reperfusion (before ischaemia, 2.0 mmol/kg wet weight; 1 min after reperfusion, 4.4 mmol/kg), and MCa increased (before ischaemia, 2.6 mmol/kg; 1 min after reperfusion, 5.07 mmol/kg). Thereafter the levels decreased, but remained high at 120 min after reperfusion in group I (EMCa, 3.2 mmol/kg, MCa 4.1 mmol/kg). At 1 min after reperfusion, EMCa in group III (2.9 mmol/kg) and MCa in groups II (3.4 mmol/kg) and III (4.0 mmol/kg) were significantly lower than in group I. At 120 min after reperfusion, the EMCa and MCa in groups II (2.5 and 3.1 mmol/kg, respectively) and III (2.4 and 3.0 mmol/kg, respectively) remained at significantly lower levels. Mitochondrial function in groups II and III was better preserved than in group I. The levels of hepatic enzymes in the perfused fluid of group III were lower than in group I. CONCLUSIONS: Calcium concentration in hepatocytes may play an important role in ischaemia-reperfusion injury. Diltiazem and citrate demonstrated a protective effect by maintaining a low calcium level in hepatocytes. PMID- 10750650 TI - Relationship between genotypes of hepatitis C virus and histopathological manifestations in chronic hepatitis C patients. AB - OBJECTIVE: The aim of this study was to assess the relationship between HCV genotype and histological liver injury. DESIGN: Prospective study on a cohort of patients with biopsy proven chronic hepatitis C. SETTING: University medical centre. PARTICIPANTS: Enrolled were 324 consecutive patients (male 197, median age 52 years, range 19-68; chronic hepatitis, 224; cirrhosis, 100). METHODS: HCV genotype was determined by the INNO LiPA assay and HCV RNA levels by the bDNA assay. The histological features were scored according to the histology activity index. RESULTS: The distribution of HCV genotypes was 1a, 4.6%; 1b, 52.4%; 2a/c, 27%; 3a, 8%; 4, 2%; mixed, 6%. Serum HCV RNA levels were similar for all genotypes. There was no difference in the distribution of HCV genotypes between patients with chronic hepatitis and those with cirrhosis. Patients with genotype 1b and those with type 2a/c showed a similar prevalence of cases of cirrhosis (33% versus 31%, respectively). In addition, in a subgroup of 102 patients with an established date of infection, the progression to cirrhosis occurred with a similar length of time for HCV type 1b and 2a/c (median 16 versus 15 years, respectively). Patients with HCV genotype 2a/c or mixed genotype showed a higher histology activity index than those with type 1b (P< 0.01), whereas there was no difference in the fibrosis score for the different genotypes. Patients with genotype 3a showed a significantly higher prevalence of steatosis compared to those infected with other genotypes. Alanine aminotransferase (ALT) values were higher in patients with HCV type 2a/c, 3a and mixed genotype than those with type 1 (P < 0.002). CONCLUSIONS: The data indicate that there is no association between a particular HCV genotype and the progression to cirrhosis, and that specific genotypes are associated with distinct histopathological and biochemical manifestations although none of them is correlated with an increase of the fibrosis stage. PMID- 10750652 TI - Prevention and healing of experimental indomethacin-induced gastric lesions: effects of ebrotidine, omeprazole and ranitidine. AB - BACKGROUND: Ebrotidine is a new H2 receptor antagonist that potentiates the gastric mucosal barrier. AIM: To compare ebrotidine with other anti-secretory drugs in the prevention and healing of indomethacin-induced gastric lesions. METHODS: Three different models of indomethacin-induced gastric lesions were used. (1) Fasted rat model: indomethacin was intra-gastrically administered in rats pre-treated with different doses of the anti-secretory drugs. (2) Re-fed rat model: rats orally treated with different doses of anti-secretory drugs had free access to chow pellets and were then treated with parenteral indomethacin. (3) Healing model: either oral or parenteral anti-secretory drugs were given after indomethacin administration. Computer-assisted analysis of the area of damage was expressed as ulcer index. Gastric secretion was evaluated in the pylorus-ligated rat model. RESULTS: Inhibition of acid secretion was in the order omeprazole > ebrotidine = ranitidine. Ebrotidine at the highest dose used (100 mg/kg) and omeprazole, but not ranitidine, significantly prevented indomethacin-induced corpus (fasted rat) and antrum (re-fed rat) gastric lesions. In the ulcer healing model, oral administration of omeprazole and both ranitidine and ebrotidine at the highest dose used improved the ulcer index. The parenteral administration of these drugs had a lesser effect than the oral route and was in the order ebrotidine > omeprazole > ranitidine. CONCLUSIONS: Ebrotidine is effective in both the prevention and healing of indomethacin-induced experimental gastric lesions. In these models, the effect of ebrotidine is comparable to omeprazole and more effective than ranitidine. PMID- 10750651 TI - Extracorporeal shock wave lithotripsy: elimination of densely calcified gallstones and gallstones with calcified rims. AB - OBJECTIVE: Until now, radiopaque gallstones have been excluded from extracorporeal shock wave lithotripsy (ESWL), because these stones in vivo are less sensitive to the forces that cause disintegration. In Japan there is a higher percentage of patients with radiopaque gallstones than in Western countries. Our purpose in working with patients in Japan was to warrant extensive indication of ESWL to radiopaque gallstones, especially densely calcified stones. DESIGN/METHODS: Retrospective analysis of clinical data. Patients were classified by computed tomography (CT) of stones. Group A consisted of 78 patients whose gallstones were densely calcified (CT attenuation values in Hounsfield units (HU), 473 +/- 323). Group B consisted of 22 patients whose stones had a calcified rim (CT attenuation values, 357 +/- 244). Ninety-eight patients received adjuvant dissolution therapy with ursodeoxycholic acid. Other recommendations, such as a glass of milk at night, were not given to the patients. SETTING: One university hospital and one general hospital. PATIENTS: One hundred consecutive Japanese patients with radiopaque stones in contractile gallbladder (CT attenuation values, > 150 HU, 447 +/- 310, mean +/- SD) were the subjects. With respect to the efficacy of ESWL, a degree of calcification for stones and its relationship with the rates of stone fragmentation and disappearance were assessed. Fragmentation to less than 3 mm in stone diameter was the aim, without limit of shock wave discharges and sessions. RESULTS: After ESWL sessions stones were fragmented successfully in 74 of the 100 patients (57 of the 78 patients in Group A, and 17 of the 22 patients in Group B). The mean number of discharges per patient was 10,435 +/- 8,726. The mean number of discharges for successful stone fragmentation of Group A (9,839 +/- 8,187) was not significantly different from that of Group B (11,376 +/- 6,344). One year after lithotripsy, 60 of the 100 patients were free of stones (45 in Group A, and 15 in Group B). CONCLUSION: It appears that patients with either densely calcified gallstones, or those in whom the stones have a calcified rim, are both suitable candidates for lithotripsy. PMID- 10750653 TI - Costs and benefits of a test-and-treat strategy in Helicobacter pylori-infected subjects: a prospective intervention study in general practice. AB - OBJECTIVE: To identify health outcomes and costs/savings of a Helicobacter pylori test-and-treat strategy in patients using acid suppressants chronically. DESIGN: Prospective intervention study. Patients were tested for H. pylori infection and treated with 14 days of ranitidine bismuth citrate (RBC) 400 mg (b.i.d.) and clarithromycin 500 mg if infected. Cure was determined after six months. SETTING: General practice. PARTICIPANTS: Patients using acid suppressants chronically were identified by a computer search; 184 patients gave written consent and were included. MAIN OUTCOME MEASURES: Serology, symptom questionnaire, medication history, quality of life determination, costs/savings. RESULTS: Out of 184 patients, 85 (46%) had positive serology. A cure rate of 61/80 (76%) was achieved. The intervention group showed significant symptom relief. Benefits were evident in patients with ulcer disease but also in patients with uninvestigated dyspepsia. Quality of life improved for cured patients in the intervention group. No improvements for dyspeptic symptoms or quality of life occurred in the H. pylori-negative group. After six months, significant savings for medication use had occurred in treated patients diagnosed as ulcer disease or non-ulcer dyspepsia. Savings on drug use and doctor visits equalize with costs for tests and antibiotics after nine months. Although less, costs for drugs also decreased significantly in the H. pylori-negative group. Therefore, for the study population, costs and savings are even after 6.5 months. CONCLUSIONS: A test-and treat strategy for H. pylori, systematically applied at the population level in patients using acid suppressants chronically, results in significant health benefits and economic savings within 1 year of follow-up. PMID- 10750654 TI - A twenty-year history of alveolar echinococcosis: analysis of a series of 117 patients from eastern France. AB - OBJECTIVES: Alveolar echinococcosis of the liver is a very rare and severe parasitic disease due to the growth of the larva of Echinococcus multilocularis. The aim of this paper was to describe a 20-year study of the epidemiological, clinical and therapeutic aspects of alveolar echinococcosis in eastern France. DESIGN: One hundred and seventeen consecutive cases, diagnosed and followed in our liver unit, were studied from 1972 to 1993. METHODS: Data from 85 patients followed since 1983 (period B) were compared to data from a first series of 32 patients (period A) collected from 1972 to 1982; 1983 was chosen as the cut-off year because of the numerous changes that occurred in the diagnosis, follow-up and treatment of the disease at this time, in particular the introduction of parasitostatic benzimidazoles. RESULTS: The results of patient follow-up were evaluated in December 1997. The cumulative prevalence was 2.5 per 100,000 persons in period A whereas it reached 6.6 per 100,000 in period B. The annual incidence in period B was 7.3 on average, compared with 2.7 in period A. Twenty-nine per cent of patients from period B were asymptomatic at the time of diagnosis compared with 10% in period A. This change was correlated with less advanced liver lesions, and was related to the extensive use of abdominal ultrasound, and from 1987, serological screening. Curative resections were performed in 24% of the cases in period B versus only 3% in period A. From 1986, liver transplantations were performed in eight patients from period A and 13 patients from period B. In period B, palliative surgery was frequently replaced by radiological non-operative procedures to treat abscesses and jaundice. From 1982, 73 patients received benzimidazoles for a period of time ranging from 4 to 138 months. Stabilization of the lesions was observed in two-thirds of the patients. Episodes of jaundice or digestive haemorrhage due to portal hypertension were 31.5 and 11 times less frequent respectively in patients from period B compared with period A. Actuarial survival at 5 years improved from 67% in period A to 88% in period B in patients of similar age. CONCLUSIONS: Radical changes in the diagnosis and the management of alveolar echinococcosis have occurred during the last decade. Together they have contributed to an improvement in the status of the patients affected by this very severe parasitic disease. PMID- 10750655 TI - Digestive haemorrhage in patients with anti-Anisakis antibodies. AB - OBJECTIVE: Anisakis simplex parasite causes anisakidosis, a disease that often mimics other gastrointestinal diseases such as peritonitis, appendicitis, Crohn's disease, ulcer, etc. Patients with digestive haemorrhage caused by ulcers, varices or Mallory syndrome were analysed for antibodies to the worm A. simplex. METHODS: Antibody detection was carried out by enzyme-linked immunosorbent assay (ELISA) and immunoblot using crude extracts of antigen and excretory/secretory products. Total immunoglobulin (Ig), IgG, IgM, IgA and IgE were studied. RESULTS: Eighty-seven patients were studied. The following prevalence rates were found with crude antigen: total Ig 30% (95% confidence interval 21-40), with values for IgG, IgM, IgA and IgE of 22 (CI 14-31), 17 (CI 10-26), 37 (CI 27-47) and 12% (CI 6-20), respectively. Twenty-four positive sera for total Ig response and crude products were selected for determination of specific antibodies with excretory/secretory antigens. We obtained 8, 13, 3 and 16 positive cases for total Ig, IgG, IgM and IgA respectively. The percentages of positivity within the varices and Mallory groups of patients were higher although differences were not significant (35 and 50% respectively). In a healthy population, the prevalence for total Ig is much lower (6%). Twenty-five positive sera for total Ig response were tested by means of immunoblot analysis against crude larval antigen. Concerning total Ig antibody response, 12 of the sera showed an immuno recognition pattern concordant with the human anisakidosis reference serum (E17). Specific IgG bands were visualized in 30 sera; specific IgM and IgA in 6 and 12, respectively. Different clinical variables of these patients were studied: leucocytes, eosinophils, haemoglobin, prothrombin activity, thromboplastin time, fibrinogen, platelets and erythrocyte sedimentation rate. There were a few significant differences: for total Ig in prothrombin activity and platelets, and for IgM in eosinophils. CONCLUSIONS: The prevalence of detectable antibodies against A. simplex is higher in patients with digestive haemorrhage than in the healthy population. PMID- 10750657 TI - Pseudogastroparesis as a presentation of small-bowel malignancy. AB - Establishing the diagnosis of small-bowel malignancy is sometimes an extremely difficult challenge owing to its non-specific symptoms. The mainstay of treatment is early recognition, diagnosis and surgical resection. The prognosis depends primarily on the degree of spread and stage at presentation. We present two cases with initially obscure presentations of a small-bowel tumour. One was a jejunal adenocarcinoma, but an initial upper gastrointestinal and small-bowel series did not disclose the lesion; the other was a primary ileal lymphoma, first thought to be diabetes mellitus gastroparesis. Therefore, a negative small-bowel series or presentation of a systemic disease-associated intestinal pseudo-obstruction or gastroparesis does not exclude the possibility of a small-bowel malignancy, if the clinical symptoms are not alleviated after prokinetic medications. The clinicians should further pursue the possibility of an obstructing lesion. PMID- 10750656 TI - Incidence and presentation of coeliac disease in South Glamorgan. AB - OBJECTIVE: To determine the incidence and presenting features of coeliac disease and dermatitis herpetiformis in the population of South Glamorgan between 1981 and 1995. DESIGN: Retrospective case-finding study using pathology, dietetic and clinical records, data from hospital activity analysis, general practice records and a Coeliac Society questionnaire. Incidence rates were calculated using the Registrar General's mid-year estimates. SETTING: Regional hospitals, South Glamorgan, Wales. PARTICIPANTS: All new cases of coeliac disease or dermatitis herpetiformis. MAIN OUTCOME MEASURES: Crude incidence rates (per quinquennia) for both children and adults. Age, sex, family history, symptoms at the time of diagnosis and time to diagnosis from symptom onset. RESULTS: In total, 137 cases of coeliac disease (27 children, 110 adults) and 19 cases of dermatitis herpetiformis were detected. In adults with coeliac disease, incidence rates have risen from 1.32 to 3.08 per 100,000 with a 3:1 female predominance. Almost 50% of adults were over fifty years old when diagnosed and 25% had no abdominal symptoms. In children, the disease incidence has remained stable but with a rising trend in mean age at diagnosis and higher likelihood of atypical symptoms in older children. There has been no change in the incidence of dermatitis herpetiformis. Only 8.3% of all patients had an affected first-degree relative. CONCLUSIONS: In contrast to other reports of declining incidence, coeliac disease in children has remained constant in South Glamorgan, but has markedly increased in adults, particularly women. Presentation may be at any age, often with atypical symptoms, which may delay diagnosis. PMID- 10750658 TI - Primary gastric lymphoma associated with human T-cell leukaemia virus I. AB - We herein report a case of primary gastric lymphoma with reference to human T cell leukaemia virus type I. A 65-year-old male was admitted to our hospital for emergency treatment of abdominal pain and haematemesis. Upper gastrointestinal endoscopy on admission revealed a disintegrated tumour with bleeding in the cardia. Total gastrectomy with distal pancreatomy and splenectomy was performed. The patient soon fell into cachexic condition due to early recurrence of abdominal lymph nodes and a rapid increase of the tumour, and died 80 days after operation. The pathological diagnosis was non-Hodgkin's lymphoma with diffuse large cell type and T-cell lineage. The patient was serologically positive for human T-cell leukaemia virus type I (HTLV-I) antibody. Southern blot analysis on the resected tumour tissue showed a monoclonal integration of HTLV-I pro-viral DNA. These results confirmed the current case to be primary adult T-cell leukaemia/lymphoma of the stomach. PMID- 10750659 TI - Hepatic hyper-vitaminosis A: importance of retinyl ester level determination. AB - We report the case of a 32-year-old man with portal hypertension without cirrhosis due to chronic vitamin A intoxication. Portal hypertension revealed by oesophageal varice rupture progressively worsened and ascites occurred 5 years after the patient stopped vitamin A intake. Initially, serum retinyl palmitate concentration was increased whereas serum retinol concentration was normal. There was no hepatic fibrosis on light microscopic examination of liver biopsy specimens. Five years after the patient stopped excessive vitamin A intake, serum retinol and retinol-binding protein concentrations were below the normal range even though there was an increased hepatic retinyl ester content. This was attributed to the late development of peri-sinusoidal fibrosis. This case mainly shows the importance of retinyl ester level determination: serum retinyl palmitate should be measured immediately after intoxication and hepatic retinyl esters should be measured initially and particularly later. Indeed, later serum and hepatic retinol levels in chronic hyper-vitaminosis A may be normal and lead to under-estimation of liver vitamin A overload. PMID- 10750660 TI - Ursodeoxycholic acid (UDCA) for the treatment of amoxycillin-clavulanate potassium (Augmentin)-induced intra-hepatic cholestasis: report of two cases. AB - This report describes two patients who developed jaundice within two weeks of receiving an amoxycillin-clavulanate potassium combination. Causes of jaundice, other than drug administration, were excluded. The patients' jaundice and clinical symptoms did not respond to stopping the drug. Ursodeoxycholic acid (750 mg/day) led to a prompt and sustained improvement in their hyperbilirubinaemia and symptoms such as pruritus and fatigue. These cases suggest that ursodeoxycholic acid may be an effective treatment for drug-associated cholestasis. PMID- 10750661 TI - The Fourth DeWitt S. Goodman lecture. Novel approaches to the prevention of colon cancer by nutritional manipulation and chemoprevention. AB - Large bowel cancer is one of the most common human malignancies in Western countries including North America. This report details the preventive strategies aimed at reducing the incidence and mortality of large bowel cancer by nutritional manipulation and chemopreventive agents. During recent decades, multidisciplinary research in epidemiology and laboratory animal model studies have contributed much to our understanding of the etiology of this cancer; more importantly, it has enabled us to approach cancer prevention. An impressive body of data thus far accumulated has provided important concepts about dietary factors such as fat and fiber as key modulators of large bowel cancer. Compelling experimental evidence indicates that certain dietary lipids and fibers influence tumorigenesis in the colon. Data obtained in metabolic epidemiological and laboratory animal model studies are sufficiently convincing in showing the enhancement of colon cancer by certain types of fat and protection against it by certain dietary fibers. Our approach to the primary prevention of large bowel cancer is to translate the findings from clinical epidemiological and laboratory studies into sound advice for patients and for the public at large to reduce fat intake and increase fiber intake, specifically cereals and grains. Preclinical efficacy studies have provided scientifically sound evidence as to how several phytochemicals and their synthetic analogues act to retard, block, or reverse carcinogenesis. Equally exciting are opportunities for effective chemoprevention with nonsteroidal anti-inflammatory agents, both synthetic and naturally occurring, or selective cyclooxygenase-2 inhibitors. Our exploration of the multistep process of carcinogenesis has provided substantial insights into the mechanisms by which chemopreventive agents modulate these events. Growing knowledge in this area has brought about an innovative combination of agents with different modes of action as a means of increasing efficacy and minimizing toxicity. There is growing optimism for the view that realization of preventive concepts in large bowel cancer will also serve as a model for preventing malignancies such as cancer of the prostate and breast. PMID- 10750662 TI - Oral field cancerization: carcinogen-induced independent events or micrometastatic deposits? AB - Patients with a head and neck squamous cell carcinoma (HNSCC) often develop multiple (pre)malignant lesions. This finding led to the field cancerization theory, which hypothesizes that the entire epithelial surface of the upper aerodigestive tract has an increased risk for the development of (pre)malignant lesions because of multiple genetic abnormalities in the whole tissue region. Demonstration of alterations in histologically normal tumor-adjacent mucosa from HNSCC patients supported this hypothesis. Currently, the question has been raised whether multiple lesions develop independently from each other or from migrated malignant or progenitor cells. The majority of the mucosal alterations appear to be related to the exposure to alcohol and/or tobacco. Moreover, almost all primary remote tumors from HNSCC patients appear to be clonally unrelated. Therefore, there is more evidence that field cancerization is due to multiple independent events than to migration of genetically altered cells. PMID- 10750663 TI - Differential regulation of apoptosis in normal versus transformed mammary epithelium by lutein and retinoic acid. AB - We examined the effects of all-trans retinoic acid (ATRA) and lutein (a nonprovitamin A carotenoid), on apoptosis and chemosensitivity in primary normal human mammary epithelial cells, SV40 transformed mammary cells, and MCF-7 human mammary carcinoma cells. ATRA and lutein selectively induced apoptosis in transformed but not normal human mammary cells. In addition, both compounds protected normal cells, but not transformed cells, from apoptosis induced by the chemotherapy agents etoposide and cisplatin. Furthermore, lutein and ATRA selectively increased the ratio of Bcl-xL:Bax protein expression in normal cells but not transformed mammary cells, suggesting a possible mechanism for selective modulation of apoptosis. The differential effects of lutein and ATRA on apoptotic pathways in normal versus transformed mammary epithelial cells may have important implications for chemoprevention and therapy. PMID- 10750664 TI - 25-Hydroxyvitamin D3, the prohormone of 1,25-dihydroxyvitamin D3, inhibits the proliferation of primary prostatic epithelial cells. AB - The hormonal metabolite of vitamin D, 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] is known to inhibit the proliferation of prostatic epithelial cells. This has stimulated interest in vitamin D compounds as therapeutic agents for prostate cancer. However, the therapeutic use of 1,25(OH)2D3 is limited because elevations in serum 1,25(OH)2D3 can cause dangerous elevations in serum calcium levels. We wondered whether the prohormone of 1,25(OH)2D3, 25-hydroxyvitamin D3 (25-OH-D3), which is much less calcemic, could also achieve antiproliferative effects in prostatic cells. 25-OH-D3 is converted to 1,25(OH)2D3 by the mitochondrial enzyme 1-alpha-hydroxylase. We have recently shown that human prostatic cells also possess significant 1-alpha-hydroxylase activity (Schwartz et al., Cancer Epidemiol. Biomark. Prev., 7: 391-395, 1998). We studied 1-alpha-hydroxylase gene expression in four strains of primary human prostatic epithelial cells by reverse transcription PCR amplification (RT-PCR) of 1-alpha-hydroxylase. Human prostatic stromal cells were negative for 1-alpha-hydroxylase by RT-PCR. This led us to hypothesize that 25-OH-D3 would inhibit the proliferation of prostatic epithelial cells because 25-OH-D3 would be converted to 1,25(OH)2D3 intracellularly. We studied the effects of 25-OH-D3 and 1,25(OH)2D3 on the proliferation of prostatic epithelial cells using high density growth and clonal growth assays on two different primary cell strains derived from normal human prostatic peripheral zone. 25-OH-D3 and 1,25(OH)2D3 each inhibited growth in a dose- and time dependent manner. Growth inhibition was evident at 1 nM, and maximal inhibition was observed at 100 nM within 10-12 days of exposure. The potencies of 25-OH-D3 and 1,25(OH)2D3 were not significantly different. These data demonstrate that 25 OH-D3, which previously was thought to have little biological activity, can become a potent antiproliferative hormone for prostatic cells that express 1 alpha-hydroxylase. Because 25-OH-D3 exhibits similar potency to 1,25(OH)2D3 but is less calcemic, 25-OH-D3 may offer a safer option than 1,25(OH)2D3 for prostate cancer therapy. Moreover, because 25-OH-D3 is produced endogenously from vitamin D, these findings support a potential role for vitamin D in the chemoprevention of prostate cancer. PMID- 10750665 TI - Risk of breast cancer and organochlorine exposure. AB - A prospective investigation of breast cancer and organochlorine (OC) exposures was undertaken in the New York University Women's Health Study. Cases (n = 148) and individually matched controls (n = 295) were identified among women whose blood had been obtained 6 months or more prior to breast cancer diagnosis. In addition, among 84 cases and 196 controls, two or more consecutive annual blood samples were available to estimate half-lives of 1,1-dichloro-2,2-bis(p chlorophenyl) ethene (DDE) and polychlorinated biphenyls (PCBs). Cases and controls had similar levels of DDE (geometric mean, 6.95 versus 7.27 ng/ml; lipid adjusted geometric mean, 977 versus 1100 ng/g) and PCBs (5.04 versus 4.97 ng/ml; lipid-adjusted geometric mean, 683 versus 663 ng/g). These differences remained nonsignificant when estrogen receptor status of tumors was considered. DDE and PCB half-lives did not differ in case versus control patients. In control patients, DDE and PCB half-lives were strongly correlated (r(s) = 0.71), and the half-life of DDE (but not that of PCB) was inversely correlated with body mass index (BMI), yet the blood serum levels of PCB (but not those of DDE) were correlated with BMI. We conclude that there is no evidence for an association of breast cancer risk with DDE or PCB levels in blood (based on samples collected during the period 1987-1992) nor with their elimination half-lives. However, changes in DDE and PCBs over time are influenced by metabolism, BMI, and current OC exposures, and each may affect interpretation of OC levels in risk assessment models. PMID- 10750666 TI - Ki-ras point mutation and p53 expression in human pancreatic cancer: a comparative study among Chinese, Japanese, and Western patients. AB - The aim of this study was to clarify features of Ki-ras point mutation (PM) and p53 expression in Chinese pancreatic cancer and to compare those with that in other countries. Dot blot hybridization and immunohistochemical methods were performed in 59 Chinese patients. The results showed that Ki-ras PMs at codon 12 and p53 expression were frequent in this group. No relationships were found between Ki-ras PM alone and p53 expression alone, and clinicopathological parameters, including age, gender, clinical stage, and histological grade and classification in Chinese patients. However, their cooperation was significantly associated with a poor prognosis in this group. Comparison showed that there were significant differences in the overall frequency and substitution of Ki-ras PM and in the ratio of transition:transversion in pancreatic cancer among various countries. In addition, the effect of Ki-ras PM and p53 expression on a poor prognosis of pancreatic cancer may be different among various countries. These findings suggested that not only Ki-ras PM and p53 expression are frequent in Chinese pancreatic cancer, but also a gene component to pancreatic cancer may be different between Asian and Western pancreatic cancer. In addition, it seems that cooperation of Ki-ras PM and p53 expression may predict a poor prognosis in Chinese patients with pancreatic cancer. PMID- 10750667 TI - Definition of tumor-associated antigens in hepatocellular carcinoma. AB - With an estimated annual incidence of about one million cases, hepatocellular carcinoma (HCC) is one of the most common neoplasms worldwide. Of all malignant diseases, it is the major cause of death in some regions of Africa and Asia. The pathogenic mechanisms responsible for HCC are not well defined, and therapeutic means, especially in inoperable HCCs, are still unsatisfactory and await improvement. In the quest for tumor antigens exploitable for gene therapy, we studied immune responses in the context of HCC. A cDNA library derived from a human HCC sample was screened using the SEREX approach. Nineteen distinct antigens reactive with autologous IgG were identified. Sequence analysis revealed three of the cDNA clones to code for hitherto unknown proteins and 16 known genes products. Proteins as diverse in function as LDH, albumin, and kinectin were found. Furthermore, proteins involved in the transcription/translation machinery had elicited an immune response in the autologous host. A panel of allogenic sera including sera from patients with hepatitis, liver cirrhosis, HCC, and other tumor entities, as well as sera from normal individuals, was used for frequency analysis of antibody responses. Whereas allogenic sera of HCC patients detected most antigens at a high percentage, control sera were rarely antibody-positive. The nature of the major fraction of antigens described here are linked to liver. Thus, our findings demonstrate not only the complexity of the humoral immune response against HCC, but may also offer new insight into mechanisms underlying transformation of the liver cell. PMID- 10750668 TI - Association of surface ultraviolet B radiation levels with melanoma and nonmelanoma skin cancer in United States blacks. AB - Ultraviolet B (UVB) radiation exposure increases the risk of skin cancer in whites. Motivated by indications that United States geographic variation of relative skin cancer risk in blacks approaches that in whites, we used Poisson regression to estimate the risk of skin cancer in blacks as a function of average annual surface-levels of UVB radiation, measured by Robertson-Berger meters. United States data were used on deaths in 506 state economic areas, 1970-1994, and on incident cases in the nine areas of the Surveillance, Epidemiology, and End Results Program, 1973-1994. For black males, the age-adjusted relative risk of mortality for a 50% increase in UVB radiation was significantly above one for malignant melanoma, 1970-1994 (1.16; 95% confidence interval, 1.02-1.32) and nearly so for nonmelanoma skin cancer, 1970-1981 (1.18; 95% confidence interval, 1.00-1.39), for which the time period was chosen to avoid AIDS-related deaths from Kaposi's sarcoma. However, for black females, the relative risk of mortality was not significantly elevated for either skin cancer, and, for both black males and females, the relative risk of incidence was not significantly elevated for melanoma in the period 1973-1994. Incidence data on nonmelanoma skin cancer were not available. Although the public health implication is uncertain because of the much lower absolute risk of skin cancer in blacks compared with whites, the findings suggest that sunlight exposure increases skin cancer risk in blacks. PMID- 10750669 TI - Ubiquitous presence of O6-methylguanine in human peripheral and cord blood DNA. AB - O6-Methylguanine (O6-meG) is a powerful premutagenic lesion that can arise from exposure to methylating agents. Although it has been reported to occur in human DNA, no systematic epidemiological analysis of its occurrence in populations suffering general environmental exposure is available. We report here results from a study of the presence of O6-meG in maternal and cord blood leukocyte DNA of women not knowingly exposed to methylating agents. Using a modification of an already existing method capable of detecting the lesion at levels as low as 16 nmol/molG, the adduct was detected in 31 of 36 maternal and 30 of 36 cord samples, at levels ranging up to 192 nmol/molG. Adduct levels in maternal blood DNA were significantly higher than those in cord blood DNA (P < 0.05), and there was a strong correlation between adduct levels in the two tissues (P < 0.001). In bivariate analysis, no significant association of adduct levels in either tissue and residence air pollution, active and passive smoking status, or eating habits was found. However, intake of fruits/vegetables and of vitamin supplements showed nonstatistically significant trends toward being associated with lower adduct levels in both maternal and cord blood DNA. The same trend was observed after multivariate analysis where all the above variables were controlled for. These findings indicate that premutagenic methylation DNA damage is commonplace in individuals not known to have suffered excessive exposure to environmental methylating agents or their precursors and are compatible with an endogenous origin of this damage, possibly associated with endogenous nitrosation processes. PMID- 10750671 TI - Cimetidine use and risk of prostate and breast cancer. AB - Histamine (H2) receptor antagonists, such as cimetidine and ranitidine, became available in the late 1970s and presently number among the most commonly used drugs. Cimetidine has been hypothesized to exert a cancer preventive effect on the prostate due to its ability to inhibit the binding of dihydrotestosterone to androgen receptors. Other hormonal effects of this drug include increases in serum prolactin levels and inhibition of 2-hydroxylation of estradiol. We assessed risk of prostate and breast cancers in a cohort of 48,512 members of the Group Health Cooperative of Puget Sound prescribed cimetidine or another H2 blocker between 1977 and 1995. Standardized incidence ratios were calculated comparing the observed numbers of cancers to those expected based on population rates in western Washington State. Because cimetidine, but not other H2 blockers, influences hormonal activity and metabolism, we conducted nested case-control studies comparing cancer risk among individuals treated with cimetidine to individuals who used other H2 blockers. Risks of breast and prostate cancers were identical among users of cimetidine and users of other H2 blockers (relative risk, 1.0 for both cancers). We observed no trend in risk of breast cancer according to time since first or last cimetidine prescription or number of cimetidine prescriptions filled. For prostate cancer, our findings were similar save for a modest increase in risk among men who had filled > or =21 cimetidine prescriptions (relative risk, 1.4; 95% confidence interval, 1.0-1.9). Our results suggest that use of cimetidine does not influence risk of female breast cancer. Further, these data provide little evidence to support the previously hypothesized preventive effect of cimetidine on risk of prostate cancer. PMID- 10750670 TI - Increasing vegetable and fruit intake: randomized intervention and monitoring in an at-risk population. AB - High vegetable and fruit (V&F) consumption has been associated with a lower risk of several cancers. However, little is known about the ability of individuals to increase their intakes markedly. In this 1-year randomized, controlled diet intervention study of men and women with a recent history of adenomas, the intervention group (n = 100) was asked to increase V&F intake to at least eight servings per day; the control group (n = 101) continued eating their usual diet. End-point measures included V&F intake assessed by 3-day diet records, plasma carotenoids, serum lipids, urinary sodium and potassium, and body weight. The intervention group increased their daily V&F intake an average of 5.5 servings over 1 year; the control group had an average decrease of 0.5 servings per day (P < 0.001). Plasma total carotenoids, alpha-carotene, beta-carotene, beta cryptoxanthin, and lutein/zeaxanthin were each statistically significantly elevated over baseline (11-54%) in the intervention group compared with the control group over the duration of follow-up (P < 0.001). Urinary potassium excretion was elevated 14% over baseline in the intervention group compared with no change in the control group (P < 0.001). Modest decreases in the intervention but not the control group were observed for total and low-density lipoprotein cholesterol. Plasma lycopene, triglycerides, high-density lipoprotein cholesterol, body weight, and urinary sodium were not affected by the intervention. V&F intake was significantly increased in this motivated population at higher risk of colon cancer and maintained for at least 12 months, as assessed using diet records and an ensemble of biomarkers. PMID- 10750672 TI - Early onset baldness and prostate cancer risk. AB - Prostatic carcinoma is the leading cancer among American men, yet few risk factors have been established. Although increased androgen levels have long been associated with both prostatic carcinoma and baldness, to date no studies have shown an association between hair patterning and prostate cancer risk. A lack of standardized instruments to assess baldness or the assessment of hair patterning during uninformative periods of time may have precluded the ability of previous studies to detect an association. We hypothesized that baldness, specifically vertex baldness, should be assessed using standardized instruments and during early adulthood if an association with prostate cancer risk is to be found. To test this hypothesis, we included identical items related to hair patterning in surveys that were administered in two distinct prostate cancer case-control studies (Duke-based study, n = 149; 78 cases; 71 controls and community-based study, n = 130; 56 cases; 74 controls). In each, participants were provided with an illustration of the Hamilton Scale of Baldness and asked to select the diagrams that best represented their hair patterning at age 30 and again at age 40. From these data, the following five categories were created and compared: not bald (referent group); vertex bald early onset (by age 30); vertex bald later onset (by age 40); frontal bald early onset (by age 30); frontal bald later onset (by age 40); and frontal (at age 30) to vertex bald (at age 40). Separate analyses of the two studies are consistent and suggest an association between vertex baldness and prostate cancer [vertex bald early onset odds ratios, 2.44 [confidence interval (CI), 0.57-10.46)] and 2.11 (CI, 0.66-6.73), respectively; vertex bald later onset odds ratios, 2.10 (CI, 0.63-7.00) and 1.37 (CI, 0.47 4.06), respectively]. Although statistical significance was not achieved in either one of these studies, the concordance between the data suggests a need for future studies to determine whether early onset vertex baldness serves as a novel biomarker for prostate cancer and whether androgen production, metabolism, or receptor status differs among these men when compared to those who exhibit other types of hair patterning. PMID- 10750673 TI - Prevalence of polymorphisms in the human UDP-glucuronosyltransferase 2B family: UGT2B4(D458E), UGT2B7(H268Y), and UGT2B15(D85Y). AB - UDP-glucuronosyltransferases (UGTs) of the UGT2B family conjugate steroid hormones as well as bile acids and xenobiotics. UGT2Bs are expressed in numerous human tissues, such as skin, breast, prostate, adipose, and intestine and are hypothesized to modulate steroid metabolism and excretion. Polymorphisms have been identified that may modify substrate specificities or enzyme activities of UGT2B family isozymes. We determined the prevalence of the UGT2B4(D458E), UGT2B7(H268Y), and UGT2B15(D85Y) polymorphisms in a sample of 233 individuals. The allele frequencies were significantly different (P < 0.02) between individuals of Caucasian and Asian descent for all three polymorphisms. In Asians (n = 32), the frequencies of the UGT2B4(D458), UGT2B7(H268), and UGT2B15(D85) alleles were 1.00, 0.73, and 0.64, respectively, whereas, in Caucasians (n = 202), the frequencies of UGT2B4(D458), UGT2B7(H268), and UGT2B15(D85) were 0.75, 0.46, and 0.45, respectively. The distribution of the UGT2B4(D458E), UGT2B7(H268Y), and UGT2B15(D85Y) genotypes also differed by ethnic group (P < 0.0001, P = 0.002, and P = 0.02, respectively). All Asians were homozygous for UGT2B4(D458) and had a greater than 2-fold higher prevalence of the UGT2B7(H268) and UGT2B15(D85) homozygous genotypes compared with Caucasians: 56.2% versus 21.8%, and 46.9% versus 22.3%, respectively. Concomitantly, only 9.4% of Asians were UGT2B7(Y268) homozygous and 18.7% were UGT2B15(Y85) homozygous compared with 29.2% and 32.2%, respectively, of Caucasians. The data suggest that there may be large differences in UGT2B polymorphisms between Asians and Caucasians. This warrants evaluation both in larger, multiethnic cohorts and in relation to known ecological differences in risk of sex hormone-dependent cancers. PMID- 10750674 TI - Alpha-linolenic acid and risk of prostate cancer: a case-control study in Uruguay. AB - In the time period of 1994-1998, a case-control study on diet and prostate cancer was carried out in Uruguay to examine the risk associated with fat intake. Two hundred and seventeen (217) incident cases afflicted with advanced prostate cancer were frequency-matched with 431 controls on age, residence, and urban/rural status. The analysis was carried out using unconditional multiple logistic regression. Alpha-linolenic acid was associated with a strong positive association (fourth quartile of intake odds ratio, 3.91; 95% confidence interval, 1.50-10.1) after controlling for total calorie intake and for the other types of fat. The effect was similar when alpha-linolenic acid was analyzed by its sources of origin (odds ratio for vegetable linolenic acid, 2.03; 95% confidence interval, 1.01-4.07). Including this report, five of six studies that have examined the relationship between alpha-linolenic acid and prostate cancer yielded a positive association, which was significant in four studies. Thus, there appears to be evidence of a role of alpha-linolenic acid in prostate carcinogenesis. PMID- 10750675 TI - Role of polymorphisms in codons 143 and 160 of the O6-alkylguanine DNA alkyltransferase gene in lung cancer risk. AB - O6-Alkylguanine DNA alkyltransferase (AGT) plays an important role in the repair of alkylating agent-induced DNA damage and protection from the carcinogenic effects of environmental agents. To examine the importance of the AGT codon 143 and codon 160 polymorphisms in risk for lung cancer and to assess the prevalence of these polymorphisms in different racial groups, we performed genotype analysis of lung cancer patients and matched controls. The prevalence of the AGT143Val allele in controls was 0.07 in Caucasians and 0.03 in African Americans. The AGT143Val allele was not detected in an unmatched Asian control cohort. The prevalence of the AGT160Arg variant allele was 0.01 in Caucasians, 0.02 in African Americans, and 0.03 in Asians. A marginally significant association was observed between the AGT codon 143 (isoleucine/valine) genotype and risk for lung cancer (odds ratio = 2.1; 95% confidence interval = 1.01-4.7). The prevalence of the AGT160Arg variant allele was similar in lung cancer cases versus matched controls. These results suggest that the AGT codon 143 polymorphism may play an important role in risk for lung cancer. PMID- 10750676 TI - Guidelines for statistical reporting. PMID- 10750677 TI - Cochrane pregnancy and childbirth database: resource for evidence-based practice. AB - The Cochrane Pregnancy and Childbirth database is an ongoing meta-analysis of evidence documenting effective health care practices for childbearing women and their neonates. It is proving invaluable to nurse educators, researchers, clinicians, and administrators working in a variety of health care delivery settings. Evidence-based nursing practice that is safe and effective can enhance rather than overpower pivotal and celebratory life events such as childbirth. PMID- 10750678 TI - Two prenatal alcohol use screening instruments compared with a physiologic measure. AB - OBJECTIVE: To compare two prenatal alcohol use screening instruments with a physiologic measure of prenatal alcohol use. DESIGN: Retrospective comparison of the Prenatal Alcohol Use Interview (PAUI) and the ACOG Antepartum Record with CDTect. SETTING: An inner-city, high-volume, prenatal clinic. PARTICIPANTS: Fifty six women selected and enrolled at their first prenatal visits. MAIN OUTCOME MEASURE: An assessment of relative sensitivity and specificity of two prenatal alcohol use screening instruments. RESULTS: Women identified as Drinkers by the CDTect were more likely to be identified as Drinkers by the PAUI (59%) than by the ACOG Antepartum Record (19%). Also, the PAUI had a lower false negative rate (41%) than the ACOG record (80%). That is, the PAUI was less likely to identify as Quitters women the CDTect identified as Drinkers than was the ACOG record. CONCLUSION: The PAUI is a more sensitive screen than the ACOG record and should be the instrument preferred for screening prenatal alcohol use. PMID- 10750679 TI - Employment patterns and timing of birth in women with high-risk pregnancies. AB - OBJECTIVE: To describe the characteristics of employed women with high-risk pregnancies, their pattern of employment prenatally and postpartum, and the relationship of prenatal employment to preterm or full-term birth. DESIGN: Secondary analysis with a sample of 171 women with high-risk pregnancies. SETTING: Women's homes and a tertiary care hospital. PARTICIPANTS: Women who were primarily single, African American, and poor; 33% worked or attended school during their pregnancies. MAIN OUTCOME MEASURES: Gestational age at birth, employment, and school attendance. RESULTS: Preterm delivery was not related to when the women stopped working or attending school or were prescribed bed rest. Women employed prenatally were older, had higher incomes, and were more likely to be white or of ethnicity other than African American. Fifty-seven percent of women with a history of prenatal employment and 85% of the women who intended to work after delivery returned to work during the first postpartum year. CONCLUSIONS: Women employed during high-risk pregnancies are similar demographically to women with low-risk pregnancies in other studies. Most of the women stopped working or attending school because of prescribed bed rest. Bed rest, however, was not related to preterm delivery. Most women who planned to return to work did so. Factors other than the women's high-risk pregnancies, such as attitudes toward employment, employability, and family circumstances, most likely influenced their employment status. Current welfare reform initiatives will increase the number of women working while pregnant. This article provides pre-welfare-reform baseline data concerning patterns and effects of employment for women with high-risk pregnancies. These data will enable nurses to examine the effects of welfare reform on employment during pregnancy and preterm birth. PMID- 10750680 TI - Differential effects of breast- and formula-feeding on preterm infants' sleep wake patterns. AB - OBJECTIVE: To compare sleep-wake patterns of breastfed and formula-fed preterm infants. DESIGN: Data were taken from an exploratory study of infant biorhythm maturation. Parents completed a 24-hour diary of infant Sleep, Awake, and Cry states and feedings, recorded at 30-minute intervals. Infant health data were collected from medical records and parents' reports. SETTING: Infants were studied in the home after discharge from a neonatal intensive-care unit. PARTICIPANTS: The convenience sample included 12 breastfed and 25 formula-fed preterm infants (gestational age, 26-33 weeks; corrected postnatal age, 4-6 weeks). Groups were comparable in terms of gestational age, postnatal age, Apgar scores, maternal age, and home environment. MAIN OUTCOME MEASURE: The 24-hour recording period was divided into day (0600-1800) and night (1800-0600). Study variables were Day, Night, and 24-hour Sleep, Awake, and Cry. RESULTS: Breastfed preterm infants exhibited more Day Cry and 24-hour Cry than did formula-fed infants. Infants demonstrated a diurnal pattern in Cry, Awake, and Sleep. Breastfed preterm infants cried approximately 1 hour per day more than formula fed infants. CONCLUSION: Preterm breastfed infants experienced more cry than did formula-fed infants. The relationship between feeding method and sleep-wake pattern has implications for supporting lactation as well as for research design. PMID- 10750681 TI - Caring behaviors by nurses: women's perceptions during childbirth. AB - OBJECTIVE: To identify nursing behaviors perceived as caring by women during childbirth. DESIGN: Descriptive research design using a structured questionnaire. SETTING: Interviews were conducted after vaginal birth and before hospital discharge. PARTICIPANTS: A convenience sample of 31 childbearing women. RESULTS: Women's perceptions of caring behaviors used by nurses were measured via the Caring Behavior Assessment, an instrument that lists 63 nursing behaviors in seven subscales and is congruent with Watson's (1988) caring factors. Mothers who had uncomplicated vaginal births were asked to rate behaviors of their nurses. Behaviors in the human needs assistance subscale, which included items such as "help me with my care until I'm able to do it for myself," "give my treatments and medication on time," and "check my condition closely," were perceived as the most caring. CONCLUSION: Identification of nursing behaviors that are perceived as caring will help nurses working in managed care environments where time spent with patients is decreased. PMID- 10750682 TI - Procedures used to prepare and administer intramuscular injections: a study of infertility nurses. AB - OBJECTIVE: To describe the procedures infertility nurses use to prepare and administer intramuscular injections of fertility medications. DESIGN: Descriptive survey. PARTICIPANTS: Nurses listed as members of the Nurses Professional Group of the American Society for Reproductive Medicine (N = 645) were surveyed. Completed questionnaires were returned by 219 of the nurses. MAIN OUTCOME MEASURES: Volume of diluent, needle selection, site selection, internal rotation of the extremity distal to the injection site, and use of the z-track technique. RESULTS: There was wide variation in the gauge and length of needles used to administer the medications, with most nurses using a 22 g, 1-1/2-in needle for all medications. Most nurses changed the needle between preparing and administering medications; however, filter needles were seldom used. There was wide variation in the volume of diluent used to reconstitute medications. Most of the nurses used the dorsogluteal site for injections. Although almost all of the nurses indicated that they routinely rotated injection sites, they infrequently used sites other that the dorsogluteal site. Most nurses did not rotate the extremity distal to the injection site when administering injections and even fewer used the z-track technique. CONCLUSIONS: This study demonstrated wide variation in the procedures used by infertility nurses to prepare and administer intramuscular injections of fertility medications. Many nurses did not use procedures that can reduce the pain and tissue trauma associated with intramuscular injections. PMID- 10750683 TI - Intrapartum pain: pharmacologic management. AB - Even with the tremendous therapeutic benefit of nonpharmaceutical pain relief measures for laboring women, pharmaceutical therapies often are needed. Nurses and other health care providers need to be familiar with the differing pharmaceutical properties of commonly prescribed pain-relieving drugs. The pharmaceutical properties of sedatives and hypnotics, opioids, and local anesthetic agents used to relieve pain during labor and delivery are reviewed. Individualization of drug therapy and maximal therapeutic effects result when the health care provider is informed about the pharmaceutical properties of analgesic and anesthetic agents so that a wise choice can be made. PMID- 10750684 TI - Common antiviral agents used in women's and children's care, part 1. AB - Antiviral medications interfere with one or more of the six parts of the viral reproductive cycle. The five mechanisms of action of antiviral agents are used to group pharmaceuticals into categories: uncoating inhibitors, nucleic acid synthesis inhibitors, nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors. The pharmacokinetics and nursing implications of specific uncoating inhibitors for respiratory viruses and nucleic acid synthesis inhibitors for respiratory syncytial virus, herpes simplex, and varicella zoster viruses are described in detail. PMID- 10750685 TI - Common antiviral agents used in women's and children's care, part 2. AB - Antiviral agents that act in the later phases of the viral reproductive cycle are used primarily against human immunodeficiency virus (HIV). Combination therapy for HIV with drugs from different categories is more effective than monodrug therapy in slowing disease progression and improving survival. Antiviral treatment for all pregnant women with HIV is recommended for prevention of vertical transmission. Procedures are discussed for postexposure prophylaxis for health care providers exposed to HIV. PMID- 10750686 TI - Fertility drug therapies: past, present, and future. AB - Throughout the last 30 years there has been an evolution of drug therapies aimed at the treatment of infertility. These agents primarily address the induction of ovulation or enhancing ovulation by allowing more oocytes to mature simultaneously. As this evolution has progressed, drugs have moved away from human products to the advent of the recombinant or genetically engineered technology. The drugs have not cured infertility, but they have affected positively the quest of families confronted by infertility. PMID- 10750688 TI - Omega-3 polyunsaturated fatty acid regulation of gene expression. AB - This review describes the mechanisms by which polyunsaturated fatty acids regulate the activity of the nuclear transcription factors, peroxisome proliferator-activated receptor and sterol regulatory element binding protein-1, and it describes the role that the peroxisome proliferator-activated receptor and sterol regulatory element binding protein-1 play in coordinating the regulation of lipid synthesis, lipid oxidation, and thermogenesis. Finally, the requirement for dietary polyunsaturated fatty acids, particularly n-3 fatty acids, is defined in terms of the effects polyunsaturated fatty acids exert on gene expression and the role that these effects play in overall energy balance. PMID- 10750687 TI - Effects of nutrients on vascular function. PMID- 10750689 TI - Endothelial dysfunctions: common denominators in vascular disease. AB - The past 20 years have witnessed enormous progress in our understanding of the biology of vascular endothelium and its role in cardiovascular disease. Stemming from the seminal observations of Furchgott, the concept of a continuous regulation of vascular tone by normal endothelium and alterations of such control in disease states has become one of the most enlightening concepts of cardiovascular research. This review covers a few updates on the topic, illustrating selective areas of recent progress in our understanding of endothelial function in the control of leucocyte adhesion, atherogenesis and vascular tone, as well as the alterations that cause and accompany vascular disease. PMID- 10750690 TI - The value of apolipoprotein E knockout mice for studying the effects of dietary fat and cholesterol on atherogenesis. AB - The ability of the apolipoprotein E-deficient mouse to develop spontaneous atherosclerosis, which resembles the human process, is an excellent model in which to assess the impact of dietary factors. This review discusses the role of several nutrients in the development of atherosclerosis and the mechanisms through which they act. PMID- 10750691 TI - In-vivo and in-vitro nutrient-gene interactions. AB - Association studies of gene variants and response to dietary challenges represent one way of investigating gene-nutrient interactions. Several studies reported in the present review concentrate on evaluating variation at the apolipoprotein AI CIII-AIV and apolipoprotein E gene loci, as well as the fatty acid binding protein gene. In addition, the effect of nutrients can be directly evaluated at the level of gene expression, and reports of in-vitro studies of control of fatty acid and triglycerides synthesis are discussed in the present review. PMID- 10750692 TI - Trans fatty acids and cardiovascular disease risk. AB - Recent studies continue to confirm previous observations that trans fatty acids elevate low density lipoprotein cholesterol levels, and at relatively high intakes decrease high density lipoprotein cholesterol levels. Considerable interest is focused on the potential benefits of trans-free margarines. Both adipose and plasma trans fatty acid levels reflect dietary intake. Current estimates of trans fatty acid intake in developed countries range from 0.5 to 2.6% of energy, contributed to primarily by differences in food availability and preference, and partly by the methodological differences used to calculate the data. PMID- 10750693 TI - Plant-derived phenolic antioxidants. AB - Antioxidant nutrients are important for limiting damaging oxidative reactions in cells, which may predispose to the development of major clinical conditions such as heart disease and cancer. There is great interest in the possibility that the antioxidant potential of plant-derived phenolic compounds, such as flavonoids, may reduce the risk of developing these conditions. Antioxidant effectiveness in vivo depends on the bioavailability of these compounds, which was assumed to be low. However, recent studies with improved methodology indicate that some plant phenolics appear in plasma and body tissues and, thus, may be important nutritional antioxidants. However, this cannot be established with certainty until their effects on biomarkers of oxidative stress are established. PMID- 10750694 TI - Viscous and nonviscous fibres, nonabsorbable and low glycaemic index carbohydrates, blood lipids and coronary heart disease. AB - Viscous fibres such as guar, glucomannans, pectins, oat betaglucan and psyllium continue to be seen as hypocholesterolaemic. Nevertheless, in large cohort studies, ironically it is the insoluble cereal fibre that has been demonstrated to relate negatively to cardiovascular disease and diabetes, despite an absence of effect on fasting lipids or postprandial glycaemia. In general, resistant or nonabsorbable starch is lipid neutral, whereas some nonabsorbable sugars or oligosaccharides may raise serum cholesterol, possibly through providing more acetate after colonic fermentation by colonic microflora. On the other hand, fructo-oligosaccharides appear to reduce serum triglycerides for reasons that are not entirely clear. Of possibly greater recent interest have been the carbohydrates that are not so much resistant to absorption, but rather are slowly absorbed. They possess some of the features of dietary fibre in providing a substrate for colonic bacterial fermentation. In the small intestine, however, they form lente or sustained release carbohydrate. In the form of low glycaemic index foods, lente carbohydrate consumption has been shown to relate to improved blood lipid profiles in hyperlipidaemic individuals and improved glycaemic control in diabetes. In larger cohort studies, low glycaemic index foods or low glycaemic load diets have been associated with higher HDL-cholesterol levels and reduced incidence of diabetes and cardiovascular disease. PMID- 10750695 TI - n-3 polyunsaturated fatty acids and the cardiovascular system. AB - n-3 Polyunsaturated fatty acids, mainly those contained in fish oils, are candidates for inclusion in secondary prevention programmes for coronary heart disease, based on the results of recent randomized trials in humans. Marine n-3 polyunsaturated fatty acids retard coronary atherosclerosis and appear to prevent fatal arrhythmias; and they decrease mortality subsequent to myocardial infarction. PMID- 10750697 TI - Bibliography. Current world literature. Nutrition and metabolism. PMID- 10750696 TI - Weight loss and plasma lipids. AB - Although weight loss is associated with improvements in the plasma lipid profile, factors other than weight loss per se are involved. Energy restriction resulting in even modest weight loss suppresses endogenous cholesterol synthesis, which contributes to observed decline in circulating lipid concentrations. Whether individuals have stabilized weight or are still actively losing weight affects the magnitude of LDL cholesterol reduction as well as the direction of HDL cholesterol change. Hence, it is important to consider the timing of lipid measurements in the interpretation of the plasma lipid response to weight loss. Another important factor is the dietary composition of the weight loss strategy, with evidence that dietary fatty acid profile and amount can differentially influence the lipid response similar to that observed in energy balance studies. Other issues such as gender, and exercise during weight loss are also relevant. However, whether the lipid changes that are observed in the short term are sustained in the long term and whether the manner of weight loss has any impact on long-term outcomes remains to be determined. PMID- 10750698 TI - Nutrition and metabolism. PMID- 10750699 TI - Genetics and molecular biology. PMID- 10750700 TI - Hyperlipidaemia and cardiovascular disease. PMID- 10750701 TI - Therapy and clinical trials. PMID- 10750702 TI - Involvement of ethylene and gibberellin signalings in chromosaponin I-induced cell division and cell elongation in the roots of Arabidopsis seedlings. AB - Chromosaponin I (CSI), a triterpenoid saponin isolated from pea, stimulates the growth of roots in Arabidopsis thaliana seedlings on wetted filter paper in the light for 14 d. The growth rates of roots in Columbia (Col) and Landsberg erecta (Ler) wild-types were 0.92 and 0.26 mm d(-1), respectively, and they were accelerated to 3.46 (Col) and 2.20 (Ler) mm d(-1) by treating with 300 microM CSI. The length of mature epidermal cells was increased by 1.8-fold (Col) and 2.81-fold (Ler) compared with control and the number of epidermal cells was increased by a factor of 1.65 (Col) and 2.12 (Ler). Treatment with 2 aminoethoxyvinylglycine (AVG), an inhibitor of ethylene biosynthesis, also increased cell length but not cell number. The effects of CSI on root growth were not detected in the ethylene-insensitive mutant ein2-1. CSI did not inhibit ethylene production but stimulated the growth of roots in ctr1-1, the constitutive triple response mutant for ethylene, indicating that CSI inhibits ethylene signaling, especially downstream of CTR1. In the GA-insensitive mutant gai and the mutant spy-3, in which the basal level of GA signaling is activated, CSI did not increase cell number, although both CSI and AVG stimulated cell elongation in these mutants. These results suggest that the inhibition of ethylene signaling is the cause of CSI-induced cell elongation. A possible involvement of both GA and ethylene signalings is discussed for the CSI-induced cell division. PMID- 10750703 TI - Role of malate synthesis mediated by phosphoenolpyruvate carboxylase in guard cells in the regulation of stomatal movement. AB - To clarify the pathway and role of malate synthesis in guard cells, epidermal strips isolated from Vicia faba L. leaflets were treated with 3,3-dichloro-2 dihydroxyphosphinoylmethyl-2-propenoate (DCDP), a specific inhibitor of phosphoenolpyruvate carboxylase (PEPC). When dark-closed stomata were illuminated, malate accumulated in guard cells and stomata opened; these were inhibited by 60% and 30%, respectively, by 5 mM DCDP treatment. When light-opened stomata were treated with DCDP, both malate level in guard cells and stomatal aperture decreased. Treatment with 5 mM DCDP partially inhibited CO2 incorporation into malate in guard cells. Treatment with mannitol at 0.4 M (osmotic stress) in the light increased malate level in guard cells and closed stomata. DCDP treatment decreased both malate level and stomatal aperture under stressed condition. These results show that malate synthesis in the light under both non-stressed and stressed conditions is dependent on PEPC activity. The extent of the decrease in malate level by DCDP treatment was larger under stressed condition than under nonstressed condition, suggesting that osmotic stress may enhance the activity of this pathway of malate synthesis which is induced by light. Role of malate synthesis in guard cells is discussed. PMID- 10750704 TI - Male flower-specific expression of genes for polygalacturonase, pectin methylesterase and beta-1,3-glucanase in a dioecious willow (Salix gilgiana Seemen). AB - We isolated cDNA clones (pSgPG1 through pSgPG4, pSgPME1 and pSgGN1) for the polygalacturonases (PGs), pectin methylesterase (PME) and beta-1,3-glucanase (GN) that are expressed specifically in male flowers of the dioecious willow (Salix gilgiana Seemen). The structural characteristics of the deduced proteins, designated SgPGs, SgPME1 and SgGN1, respectively, suggest that these enzymes function in pollens or anthers. The four SgPGs were more than 91.9% homologous to one another at the amino acid level, indicating that their genes are members of a single family. Although the expression of the SgPGs, SgPME1 and SgGN1 was specific to male catkins (inflorescences), these genes were found in the genomes of both male and female plants. The expression of the transcripts of SgPGs, SgPME1 and SgGN1 was regulated developmentally in male reproductive organs. Maximal expression of SgPGs and SgPME1 was detected when male flowers were fully open and mature, while maximal expression of SgGN1 occurred at an earlier time. In situ hybridization revealed that the expression of SgPGs and SgPME1 was restricted to mature pollen grains after microspore mitosis. These results suggest that the pollen-specific or anther-specific expression of genes for PGs, PME and GN occurs in a dioecious plant, willow, just as it does in monoecious plants, and that the expression of these genes is related to the developmental stage of pollen grains during male gametogenesis. PMID- 10750705 TI - A secreted peptide growth factor, phytosulfokine, acting as a stimulatory factor of carrot somatic embryo formation. AB - Somatic embryogenesis of the carrot (Daucus carota L.) depends on a set of factors, some of which accumulate in culture medium (conditioned medium, CM). When embryogenic cell clusters were transferred to an embryo-inducing medium, addition of CM derived from somatic embryo culture markedly stimulated somatic embryo formation. The active principles were purified using a simple bioassay system and identified to be phytosulfokines (PSKs), sulfated oligopeptide growth factors originally isolated from a CM derived from asparagus (Asparagus officinalis L.) mesophyll culture. Quantification studies using a competition ELISA system employing an anti-PSK-alpha polyclonal antibody showed that PSK production might be related to growth of cells, rather than development of somatic embryos. Thus the stimulatory effect of PSK on somatic embryo formation might be due to promotion of cell proliferation. PMID- 10750706 TI - Accumulation of plastocyanin mRNA lacking 5' region in the green alga Pediastrum boryanum grown under copper-deficient conditions. AB - In the green alga Pediastrum boryanum NIES-301, plastocyanin accumulates under copper-sufficient conditions and cytochrome c6 accumulates under copper-deficient conditions. We cloned the cDNA which encodes pre-apoplastocyanin from P. boryanum cultured under the copper-sufficient condition. The deduced amino acid sequence of the pre-apoplastocyanin protein consists of 151 amino acid residues including a putative bipartite presequence of 53 amino acid residues. Southern blot analysis of P. boryanum genomic DNA indicated that pre-apoplastocyanin is encoded by a single nuclear gene. Northern blot analysis showed that copper-deficient cells accumulated a shorter form of the mRNA of pre-apoplastocyanin, which did not generate pre-apoplastocyanin in the wheat-germ translation system. The difference in size was ascribed to the absence of the 5' region in the mRNA of pre-apoplastocyanin obtained from the copper-deficient cells, which accounts for the absence of plastocyanin under these conditions. This phenomenon represents a novel regulatory mechanism, although details of the mechanism are not yet known. PMID- 10750707 TI - The promoter for the maize C4 pyruvate, orthophosphate dikinase gene directs cell and tissue-specific transcription in transgenic maize plants. AB - The pyruvate,orthophosphate dikinase (PPDK) gene coding the chloroplast enzyme involved in C4 photosynthesis has a dual promoter system. The first promoter is responsible for the transcription of a larger transcript and its product is targeted to the chloroplast (hence, it is designated as C4Pdk promoter) while the second promoter is responsible for the transcription of a smaller transcript and its product remains in the cytosol. In this study, chimeric maize C4Pdk promoter (0.9 or 1.5 kb)-beta-glucuronidase or luciferase fusion genes were introduced into maize plants by Agrobacterium-mediated transformation. The cell- and tissue specificities of the maize C4Pdk promoter in the transgenic maize plants were examined by histochemical and enzymic activity analyses of the reporters in different photosynthetic cells and tissues. The results showed that the reporter proteins are almost exclusively localized in leaf mesophyll cells. Among the tissues tested, leaf blade had the highest reporter activities with sheath exhibiting about 10% of the activities in blade. Husk, stem, tassel and root had no or very little reporter activities. Taken together, these results suggest that the maize C4Pdk promoter is specifically transcribed in the mesophyll cells of leaf blade and to a much less extent in the mesophyll cells of sheath, but not in leaf bundle sheath cells or other tissues. Furthermore, the 0.9 kb maize C4Pdk promoter sequences appear to contain the necessary cis-acting elements for its cell- and organ-specific expression. PMID- 10750708 TI - Identification of the Ndh (NAD(P)H-plastoquinone-oxidoreductase) complex in etioplast membranes of barley: changes during photomorphogenesis of chloroplasts. AB - In the last few years the presence in thylakoid membranes of chloroplasts of a NAD(P)H-plastoquinone oxidoreductase complex (Ndh complex) homologous to mitochondrial complex I has been well established. Herein, we report the identification of the Ndh complex in barley etioplast membranes. Two plastid DNA encoded polypeptides of the Ndh complex (NDH-A and NDH-F) were relatively more abundant in etioplast membranes than in thylakoids from greening chloroplasts. Conversion of etioplast into chloroplast, after light exposure of barley seedlings grown in the dark, was accompanied by a decrease in the NADH dehydrogenase activity associated to plastid membranes. Using native-PAGE and immunolabelling techniques we have determined that a NADH specific dehydrogenase activity associated with plastid membranes, which was more active in etioplasts than in greening chloroplasts, contained the NDH-A and NDH-F polypeptides. These results complemented by those obtained through blue-native-PAGE indicated that NDH-A and NDH-F polypeptides are part of a 580 kDa NADH dependent dehydrogenase complex present in etioplast membranes. This finding proves that accumulation of the Ndh complex is independent of light. The decrease in the relative levels and specific activity of this complex during the transition from etioplast to chloroplasts was accompanied by a parallel decrease in the specific activity of peroxidase associated to plastid membranes. Based on the mentioned observations it is proposed that an electron transport chain from NADH to H2O2 could be active in barley etioplasts. PMID- 10750709 TI - Involvement of CUP-SHAPED COTYLEDON genes in gynoecium and ovule development in Arabidopsis thaliana. AB - When mutations in CUP-SHAPED COTYLEDON1 (CUC1) and CUC2 are combined, severe defects involving fusion of sepals and of stamens occur in Arabidopsis flowers. In addition, septa of gynoecia do not fuse along the length of the ovaries and many ovules have their growth arrested. CUC2 is expressed at the tips of septal primordia during gynoecium development and at the boundary between nucellus and chalaza during ovule development. These expression patterns are partially consistent with the phenotype of the mutant gynoecium. CUC2 mRNA is also shown to be expressed at the boundaries between meristems and organ primordia during both the vegetative and reproductive phases. This expression pattern indicates that CUC2 is generally involved in organ separation in shoot and floral meristems. PMID- 10750710 TI - An Arabidopsis cotyledon-specific albino locus: a possible role in 16S rRNA maturation. AB - We report here the isolation and characterization of a cotyledon-specific albino locus of Arabidopsis, WHITE COTYLEDONS (WCO). This recessive mutation in the WCO locus, located on the top of Chromosome 1, results in albino cotyledons but green true leaves. An accumulation profile of chlorophylls and ultrastructure of chloroplasts indicate that WCO is necessary for development of functional chloroplasts in cotyledons but is dispensable in true leaves. This was further supported by the fact that the mutants request feeding of sucrose for their survival at the early seedling stage where true leaves have not emerged, but the mutants which have developed true leaves are able to grow autotrophically without sucrose supplementation. The wco mutants accumulate low levels of chloroplast mRNA encoding photosynthesis-related proteins and have a specific defect in 16S rRNA maturation in a cotyledon-specific manner. Although wco mutants exhibited abnormal chloroplasts and chloroplast gene expression in cotyledons, nuclear genes for photosynthetic components are expressed at similar levels to those found in wild-type siblings. This lack of suppression of the nuclear genes is not due to a defect in the signaling of the so-called "plastid factor" to the nucleus since normal suppression of the nuclear genes was observed in response to the photo-oxidative damage due to norflurazon application. PMID- 10750711 TI - Isolation and characterization of arsenate-sensitive and resistant mutants of Chlamydomonas reinhardtii. AB - Arsenate-sensitive and resistant mutants of Chlamydomonas reinhardtii were obtained by screening mutants generated by random insertional mutagenesis for growth in the presence of various concentrations of arsenate. The intracellular concentrations of arsenic in the mutants kept in the arsenate-containing medium were determined with an atomic absorption spectrophotometer. The intracellular levels of arsenic in the arsenate-resistant mutants were all lower than that of the parent strain CC425. Some of the arsenate-sensitive mutants, AS1 and AS3, showed obviously higher levels of arsenic than that of CC425, while other sensitive mutant, AS2, did not accumulate arsenic so much. Analysis of the chemical species of arsenic suggested that inorganic arsenic was converted to dimethylarsinic acid (DMAA) in CC425. However, DMAA was hardly detected in AS2. The mechanisms of the resistance to arsenate are discussed on its uptake and detoxification. PMID- 10750712 TI - Blue light-induced chloroplast relocation in Arabidopsis thaliana as analyzed by microbeam irradiation. AB - Chloroplast relocation in mesophyll cells of Arabidopsis thaliana was observed microscopically and analyzed by microbeam irradiation. Chloroplasts located along the anticlinal walls in dark-adapted cells. When part of a cell was irradiated with a microbeam of high fluence rate blue light (B) simultaneously with background red light (R) on the whole cell, the chloroplasts moved towards the B irradiated area, but did not enter the beam. The background R illumination activated cytoplasmic motility as well as chloroplast movement. Without R illumination, there was little chloroplast relocation. In light-adapted cells in which the chloroplasts were spread over the cell surface perpendicular to the incident light, R-illumination had the same effect. Under background R, the chloroplasts moved out of the area irradiated with a B microbeam of 8 or 30 W m( 2) (avoidance response), but chloroplasts outside the beam moved towards the area irradiated with the B microbeam (accumulation response). These results suggest that the signals for accumulation and avoidance responses were generated in a single cell by high fluence rate B. cry1cry2, npq1 and nph1 mutants showed B induced chloroplast relocation. Both the accumulation and avoidance responses were observed in all the mutants, although in the nph1 mutant, the sensitivity of accumulation movement was slightly lower than that of the wild type. We discuss the possible photoreceptor for B-induced chloroplast relocation. PMID- 10750713 TI - Isolation and characterization of an Arabidopsis mutant, fireworks (fiw), which exhibits premature cessation of inflorescence growth and early leaf senescence. AB - To examine the mechanism underlying the reproductive development in monocarpic plants, we screened for mutants that exhibit premature cessation of inflorescence growth in Arabidopsis. We identified a novel mutant line that exhibited earlier cessation of flower formation and inflorescence stem elongation. This mutant also exhibited accelerated rosette leaf senescence after the cessation of the inflorescence growth. We designated the mutant fireworks (fiw) because flowers and siliques were clustered at the top of the fiw inflorescence. The fiw mutation was a single, recessive mutation and mapped on the lower part of chromosome 4. The fiw phenotype was not observable during vegetative growth, but the inflorescence growth was arrested more than 7 d earlier than the wild type (WT). Microscopic observation revealed that the fiw apical meristem was structurally preserved. The premature arrest of growth was observed not only in the primary inflorescence but also in the lateral inflorescence, which is consistent with the global proliferative arrest observed later in WT. Regardless of such dramatic phenotypic features, the fiw plants bore normal flowers and set fully matured siliques. PMID- 10750714 TI - Expression of two uricase (Nodulin-35 genes in a non-ureide type legume, Medicago sativa. AB - Two cDNA clones, MsU2 and MsU9 encoding uricase (EC 1.7.3.3, Nodulin-35) were isolated from a cDNA library prepared from nodule tissues of alfalfa, Medicago sativa, plants. Both MsU2 and MsU9 encoded 308 amino acid polypeptides with a difference of 5 amino acids, and the deduced amino acid sequences shared 98% homology. Between these two cDNA clones and uricase genes of soybean which were designated as Nod-35s, more than 80% identity was observed in nucleotides and deduced amino acid sequences, suggesting that these MsU2 and MsU9 are homologs of Nod-35. Using the reverse transcription-PCR technique, we detected the transcripts of these two genes in almost all tissues of alfalfa. The operation of uricase genes was confirmed by the presence of ureide in the xylem sap and uricase activity in the nodules. In situ hybridization analysis revealed that MsU2 and MsU9 were expressed only in uninfected cells of the infected zone of the nodule tissue. The cell specific-expression of the two uricase genes was observed in an identical manner to that of Nod-35 in soybean nodules. PMID- 10750715 TI - Stress-induced factor involved in flower formation of Lemna is an alpha-ketol derivative of linolenic acid. AB - A stress-induced substance(s) (factor C) incubated with norepinephrine (NE) has strong flower-inducing activity in Lemna paucicostata. We isolated an essential component (FIF) of factor C, and clarified its chemical structure as 9-hydroxy-10 oxo-12(Z),15(Z)-octadecadienoic acid, an alpha-ketol derivative of linolenic acid, which is formed via 9-hydroperoxy linolenic acid. Synthesized FIF showed flower-inducing activity after incubation with NE (factor C activity) equivalent to that formed in the stressed Lemna. Jasmonic acid and 13-hydroxy-12-oxo 9(Z),15(Z)-octadecadienoic acid (12,13-alpha-ketol linolenic acid), both of which are formed via 13-hydroperoxide of linolenic acid and all other derivatives of FIF synthesized by chemical and enzymatic processes failed to show the factor C activity. These results suggest that the molecular structure of FIF is very specific for the factor C activity. PMID- 10750716 TI - Water permeability and revolving movement in Phaseolus vulgaris L. twining shoots. AB - Osmotic water permeability (Pos) was measured in protoplasts isolated from different tissues of Phaseolus vulgaris twining shoot. Parenchyma protoplasts exhibited more frequently high Pos values than epidermis protoplasts did. Water channels could facilitate water movement between parenchyma cells whereas cell-to cell water transport mostly occurs through plasmodesmata in epidermis. PMID- 10750717 TI - Identification of multi-gene families encoding isopentenyl diphosphate isomerase in plants by heterologous complementation in Escherichia coli. AB - Two cDNAs encoding isopentenyl diphosphate isomerase (IPI) in Adonis aestivalis, Arabidopsis thaliana, and Lactuca sativa, and single examples from Oryza sativa and Tagetes erecta were identified. An analysis of these and other ipi leads us to suggest a separate origin for green algal and plant genes and propose that a single gene encodes plastid and cytosolic IPI in plants. PMID- 10750719 TI - Veno-occlusive disease of the liver. AB - The success of high-dose cytoreductive strategies depends not only on antitumor activity but also on the tolerability of treatment. Although advances in supportive care have significantly reduced mortality due to infection and hemorrhage, regimen-related toxicities remain problematic. Hepatic veno-occlusive disease (VOD) is the most serious regimen-related toxicity after high-dose cytoreductive therapy. Risk factors for VOD are well established, but the biology of the syndrome remains poorly understood. Unfortunately, no pharmacologic approaches that clearly prevent or treat VOD have been identified. A better understanding of the pathogenesis of VOD will lead to more effective prevention and treatment strategies. PMID- 10750718 TI - A new method for direct introduction of chemicals into a single sieve tube of intact rice plants. AB - We present a novel method for direct introduction of chemicals into a sieve tube of intact rice plants-namely, the method of micro-introduction using stylet of insect (the MUSI method). Fluorescent dyes were successfully introduced into a sieve tube through a severed stylet of planthopper and the distribution of the dyes was observed. PMID- 10750720 TI - High dose cytarabine or transplantation for consolidation of younger patients with acute myeloid leukemia. AB - Collaborative study groups have invested considerable effort in the last decade in defining the role of allogeneic and autologous bone marrow transplantation as consolidation treatment for first remission of acute myeloid leukemia in younger patients. These efforts have been able, more precisely, to quantitate the degree to which patients who receive transplantation are a selected group. There has been a trend in recent years to increase the intensity of chemotherapy, which has improved treatment results, and the factors which determine the risk of relapse have become more widely acknowledged. These developments have made the appropriate choice of consolidation treatment less clear. Transplantation significantly reduces the risk of relapse and in some trials has improved the disease-free survival. However, a clear benefit in overall survival has been less clear and more difficult to demonstrate, partly because some patients who fail first-line chemotherapy can be salvaged in second remission. The trials that included high-dose cytarabine (ara-C) in the chemotherapy schedule were the ones in which no survival benefit was seen. PMID- 10750722 TI - Improving availability and safety of unrelated donor transplants. AB - Transplants of hematopoietic stem cells from unrelated donors have become feasible for a growing population of patients with hematologic malignancy. More than 2,000 patients with acute and chronic leukemia, lymphoma, myeloma, and myelodysplasia are transplanted each year worldwide using marrow or blood stem cells from unrelated volunteers. Many patients have achieved complete immunologic tolerance and have become long-term survivors. The probability of finding a suitable donor has increased, because of the expansion of the network of registries containing more than 5 million HLA-typed donors worldwide. The selection of compatible donors has become more effective, thanks to the discovery of new HLA alleles and the development of precise and efficient HLA typing methods using DNA technology. Prophylaxis of viral and fungal infections has decreased morbidity and improved survival. The availability of more selective immunosuppressive agents provides the opportunity to decrease treatment-related toxicity and graft-versus-host disease. PMID- 10750721 TI - Hemopoietic stem cell transplantation for myelodysplastic syndrome. AB - The term myelodysplastic syndrome (MDS) describes a spectrum of disorders that are characterized by dysplastic marrow cell morphology, the development of peripheral blood cytopenias, and a tendency to evolve into acute myeloid leukemia. MDS has been recognized as a stem-cell disease, and hemopoietic stem cell transplantation is currently the only potentially curative therapy. In patients with less advanced MDS (<5% blasts in the marrow), 3-year survival rates of 70% and 65% can be achieved with HLA-identical related and HLA-matched unrelated donors, respectively. The overall probability of disease recurrence in these patients is less than 5%. Of patients with advanced disease (5% marrow blasts or more), about 40% to 45% and 25% to 30% are surviving in remission after transplantation from a related or an unrelated donor, respectively. This inferior outcome is largely due to a higher incidence of post-transplantation relapse (20% to 30%). Inclusion of the International Prognostic Scoring System criteria into outcome analyses shows an inverse correlation between overall risk category and relapse-free survival after transplantation. Future trials should explore the usefulness of different transplantation regimens for different risk categories. Among patients with less advanced disease, use of a conditioning regimen that combines cyclophosphamide and busulfan, dose adjusted to reach target plasma levels, has been associated with improved survival in recipients of transplants from related and unrelated donors. It has also permitted successful hemopoietic stem-cell transplantation in patients as old as 66 years of age. Improved survival with transplants from unrelated volunteer donors has been achieved with selection of donors based on high-resolution HLA typing. Autologous stem-cell transplantation may provide excellent consolidation for selected patients who have obtained complete remission with conventional chemotherapy. High treatment related morbidity and mortality rates, particularly after allogeneic transplantation, remain challenges that must be addressed with innovative approaches. PMID- 10750723 TI - Medical complications of combined surgical and nonsurgical therapy. AB - The poor prognosis of patients with preoperatively identified stage IIIa N2 non small-cell lung cancer has led to the use of various combinations of chemotherapy, radiation, and surgery in phase II clinical trials and, more recently, as standard therapy. The survival benefits of these combination approaches have been noted, but the morbidity associated with these approaches has received less attention. Compared with surgery alone, combination treatments almost always lead to a higher percentage of patients requiring pneumonectomy and greater numbers of complex resections and technical problems. The risks for postoperative complications and death can also be expected to be higher. It is well documented, for example, that pulmonary morbidity related to the adult respiratory distress syndrome and bronchopleural fistulae is increased when pneumonectomy is done after chemoradiation therapy. Other toxicities that can affect the fate of the surgical patient include myelosuppression, cardiomyopathy, and renal disorders. Fortunately, the proper performance of anesthesia and surgery can minimize the incidence of these toxicities and reduce their effect on patients. PMID- 10750724 TI - Positron emission tomography in initial staging and diagnosis of persistent or recurrent disease. AB - Positron emission tomography (PET) constitutes a major advance in the diagnosis, staging, prognostic assessment, and follow-up of lung cancer. However, it is not a magic bullet that can solve all of the uncertainties that beguile the imaging of this disease. Small lesions, particularly those in the brain, may often be missed with PET, and three-dimensional localization of suspected sites may also be unreliable. We are still learning how best to apply this new technology in an environment that demands the efficient use of medical resources. PET will probably be used most enthusiastically in the prethoracotomy staging of patients who are considered operable or probably operable on the basis of computed tomography and in the assessment of treatment response and disease recurrence when clinical management will be determined by prompt recognition of these events. PMID- 10750725 TI - Gene therapy for lung cancer. AB - The advances that have been made over the past decade in the field of gene transfer as well as in the fields of immunology and the molecular biology of tumorigenesis have brought to reality the possibility of using gene transfer as an anti-cancer treatment modality. The published results of clinical trials using this approach to date have been very limited, and a considerable amount of work still needs to be done in order to make this an effective treatment modality. However the developments that have occurred in the past several years indicate that this modality will become efficacious in the foreseeable future. PMID- 10750726 TI - Early detection and prevention of lung cancer. AB - Lung cancer remains the leading cause of cancer death in the United States and is one of the world's leading causes of preventable death. Technologic advances have brought new modalities that may be useful for the early detection of lung cancer. However, because of the large number of persons at increased risk for lung cancer, screening is a formidable task. There are several risk factors that can be identified, including potential susceptibility factors, which may aid in pinpointing individuals who need to participate in regular screening programs. Aside from recognized environmental exposures including cigarette smoking, there are a number of genetic and metabolic susceptibility factors that have been examined. These include polymorphisms in the cytochrome p450 enzymes and the metabolizing capability of glutathione s-transferase or acetylation. Additionally, defects in DNA repair and in bleomycin sensitivity assays may also aid in identifying individuals who are at an increased risk for lung cancer. Additional work has been done in the area of characterizing the molecular alterations in the bronchial epithelium in high-risk smokers. This manuscript addresses only selected molecular alterations that have been examined in preneoplastic bronchial epithelium. In addition to mutations in the k-ras oncogene and the p53 gene, which are frequently seen in malignancy, alterations in the p16 gene, microsatellite instability and loss of heterozygocity are also promising potential markers of preneoplasia. The hnRNP A2/B1 gene also shows some promising increased expression in preneoplasia. Lung cancer prevention has made some strides. A number of trials with molecular and morphologic intermediate endpoints have been conducted and have suggested that some of the molecular alterations and morphologic alterations are reversible. However, the rate of spontaneous regression of these lesions is, as yet, uncharacterized. Two recent large studies, the beta-carotene and retinol efficacy trial (CARET) trial conducted in the United States and the Alpha-Tocopherol Beta Carotene (ATBC) trial conducted in Finland, both demonstrated an unexpected increased risk for lung cancer associated with beta-carotene supplementation. The EUROSCAN trial evaluation of vitamin A and N-acetylcystine also showed no benefit to supplementation in reducing risk for lung cancer. Results from the Intergroup study of 1 3-cis-retinoic acid are pending, and plans are underway for an Intergroup trial studying high selenium yeast to reduce lung cancer risk. Hopefully, the combination of identifying markers of increased risk among the numerous current and former smokers will identify high-risk populations to participate in future trials of promising agents that may lead to reduction in incidence and mortality of the leading cause of cancer death. PMID- 10750727 TI - Adjuvant therapy of resected non-small-cell lung cancer. AB - The administration of adjuvant therapy after complete resection of non-small-cell lung cancer is controversial. Radiation therapy and chemotherapy have been used individually and concomitantly in efforts to prevent local recurrence and improve survival. However, recent phase II and III trials and a meta-analysis have produced conflicting results. Postoperative adjuvant therapy remains a subject of active investigation. PMID- 10750728 TI - Sunscreen. AB - Skin cancer prevention is increasingly a focus of public health campaigns. The most important avoidable cause of skin cancer is ultraviolet radiation from the sun, but sunscreens are often used in a suboptimal manner, compromising their effectiveness. People may "compensate" for the use of sunscreen by increasing their time in the sun, which can have adverse effects on health. Epidemiologic studies of sunscreen use have suffered from important methodologic limitations. A clearer picture of the impact of sunscreens is emerging from randomized trials that have recently been published or presented. The available evidence supports the current recommendation to "Slip! Slop! Slap!" when in the sun. PMID- 10750729 TI - Melanoma vaccines. AB - Remarkable advances in tumor vaccination have been made since Coley first deliberately infected cancer patients with both live and heat-killed bacteria. Melanoma is the most immunogenic solid tumor and, as such, has served as the major model for tumor vaccine investigation in both the laboratory and the clinic. Many advances in the field of melanoma vaccination have been based on an improved understanding of the cellular interaction required to induce a specific antitumor immune response. As a result of this new knowledge, many clinical trials of melanoma vaccines are now under way, and vaccines for metastatic melanoma have shown evidence of clinical effectiveness. This paper outlines the current status of melanoma vaccination. PMID- 10750730 TI - Update on the pathogenesis and treatment of Kaposi sarcoma. AB - Kaposi sarcoma (KS), an unusual tumor of vascular origin, was one of the first recognized manifestations of AIDS. In the past few years, it has become clear that human herpesvirus-8 (HHV-8) is critical to the development of KS in the setting of immunosuppression, such as that seen with HIV infection. Other genetic and environmental factors may also play a role in the pathogenesis of KS. Of note, several endogenous substances elaborated by HIV-infected cells may promote angiogenesis and the growth of KS. With advances in our understanding of the pathogenesis of this tumor have come novel treatments for KS. Suppression of HIV replication has substantially decreased the incidence of KS in the western world, and treatment directed at angiogenesis or virus-induced tumorigenesis may ultimately lead to more effective control of KS. For most patients, however, standard chemotherapy, radiation therapy, topical therapies, and interferon-alpha remain the mainstays of treatment. In this review, recent advances in our understanding of the pathogenesis of KS are highlighted and a brief overview of current approaches to the treatment of this tumor is provided. PMID- 10750732 TI - Bibliography. Current world literature. Transplantation. PMID- 10750731 TI - Primary and metastatic diseases in malignant melanoma of the gastrointestinal tract. AB - In this review, the gastrointestinal (GI) manifestations of malignant melanoma including primary mucosal melanoma of the GI tract and metastatic melanoma to the GI tract are discussed. Although malignant melanoma most commonly arises in the skin, primary melanomas can also arise from the mucosal epithelial lining of the gastrointestinal tract. The vast majority of gastrointestinal melanoma is metastatic from a cutaneous primary; however, there is evidence that melanoma can arise de novo from within certain areas of the gastrointestinal system. The sporadic nature and small numbers of patients reported in the literature with mucosal melanomas have prevented a good understanding of the pathogenesis, natural history, and optimal treatment of this uncommon presentation of melanoma. PMID- 10750733 TI - Bibliography. Current world literature. Lung and mediastinum. PMID- 10750734 TI - Bibliography. Current world literature. Melanoma and other skin neoplasms. PMID- 10750736 TI - Biphasic regulation of Fc-receptor mediated phagocytosis of rabbit alveolar macrophages by surfactant phospholipids. AB - Dipalmitoyl phosphatidylcholine (DPPC) is a major phospholipid constituent in the pulmonary surfactant, whereas lysophosphatidylcholine (Lyso-PC) is a minor constituent, this membrane phospholipid being produced at inflammatory sites in association with activation of phospholipase A2. To determine the role of these two different forms of phospholipids in the phagocytic function of alveolar macrophages (AM), we examined the effects of DPPC or Lyso-PC on Fc-mediated phagocytosis. We demonstrated a significant decrease of the ingestion activity of AM for anti-sheep erythrocyte immunoglobulin G-coated sheep erythrocytes (EA: IgG) by DPPC. On the other hand, Lyso-PC caused significantly increased ingestion of EA: IgG by AM. These data indicate that increase of Lyso-PC due to the hydrolysis of DPPC through activation of phospholipase A, may up-regulate AM mediated phagocytic functions in the alveolar milieu associated with infections and inflammation. DPPC may suppress and stabilize the AM-mediated phagocytosis in the normal alveolar environment. PMID- 10750735 TI - Demographic and reproductive factors for high seroprevalence of Chlamydia trachomatis among pregnant women in Japan. AB - In order to elucidate demographic and reproductive factors associated with Chlamydia trachomatis seropositivity, serological screening and questionnaire survey were conducted on pregnant women in Nagasaki Prefecture, Japan. Serum samples were taken from 1718 pregnant women between September and December, 1996, at the cooperative obstetric hospitals and clinics, and tested for the presence of antibodies to C. trachomatis using the enzyme immunoassay. A questionnaire was administered on a sub-sample (n -409), among whom 85 (20.8%) were seropositive. A multiple logistic analysis revealed that four characteristics showed a significant association with the seropositivity: (i) experience of premarital pregnancy, (ii) non use of condoms, (iii) short duration of education, and (iv) more frequent induced abortion. The unsafe sexual behavior of young people lacking proper knowledge of how to prevent STD is the most important intervention target for control of the C. trachomatis epidemic in Japan. PMID- 10750737 TI - Screening for pyrimidine metabolism disorders using dried filter-paper urine samples: method development and a pilot study in Nagoya City, Japan. AB - A screening system for pyrimidine metabolism disorders by measurement with high performance liquid chromatography using dried filter-paper urine samples is presented. This system permits the simultaneous determination of dihydrouracil, uracil, orotic acid and pseudouridine. The coefficient of variations for the four compounds on the filter-paper urine samples were 0.010 approximately 0.069 and the recoveries were 98.5 approximately 107.1%. The detection limits of the four compounds were 2 approximately 20 micromol/liter. The correlation between the filter-paper urine samples and liquid urine samples was excellent (0.938-0.988). We supeculated that this method could be used to detect pyrimidine metabolism disorders, such as dihydropyrimidinuria, dihydropyrimidine dehydrogenase deficiency and hereditary orotic aciduria. As a pilot study, we have analyzed dried filter-paper urine samples from 34, 200 healthy Japanese, and found three cases of dihydropyrimidinuria without clinical symptoms. PMID- 10750738 TI - Total spondylectomy for solitary spinal metastasis of the thoracolumbar spine: a preliminary report. AB - Eight cases of solitary spinal metastasis with neurological deficit in thoracolumbar spine in which total en bloc spondylectomy (TS) was performed by bisecting the affected vertebra through both pedicles using fine thread wire saws were reviewed. Patient age ranged from 40 to 78 (mean, 62) years. Primary lesions were in the lung (2), kidney (2), thyroid (3) and prostate (1). TS was performed through a posterior approach in 5 cases (T6-7, T12, L2, L2 and L2-3) and through a one-stage anterior and posterior combined approach in the others (L2, L3 and L4). The spine was reconstructed with a ceramic vertebral prosthesis and a pedicle screw fixation system in 5 cases, and with augmented anterior spinal instruments in 3 cases. There were neither surgical complications nor surgical mortality. All patients became ambulatory and pain-free after surgery. Histologically, a marginal surgical margin was achieved in only one case. The other 7 cases had intralesional margin at the osteotomized pedicles. Four patients died from causes unrelated to local recurrence 4 to 44 (mean, 19) months after surgery. Effectiveness of surgery was maintained until death in all 4 of these patients. Asymptomatic local recurrence occurred in 2 patients with renal cancer at 10 and 33 months after surgery. These preliminary results suggest that TS for solitary spinal metastasis can achieve good control of local recurrence without major complications and is clinically acceptable surgery. PMID- 10750739 TI - A case report of sinonasal teratocarcinosarcoma. AB - A sinonasal teratocarcinosarcoma (SNTCS) is a rare and aggressive malignant neoplasm histologically characterized by the combination of one or more epithelial elements and mesenchymal components. We report a case of a 78-year-old man with SNTCS involving the nasal cavity and paranasal sinuses. He complained of epiphora and exophthalmos with weight loss. Physical and diagnostic images resulted T4N0M0. The tumor was completely and widely resected via a trans-facial approach to perform total maxillectomy with orbital exenteration. The clinical presentation, pathologic features, and clinical course are described with a review of the literature. PMID- 10750740 TI - Prenatal confirmation of periventricular leukomalacia in a surviving monochorionic-diamniotic twin after death of the other fetus: a case report. AB - A 30-year-old woman was found to be carrying monochorionic-diamniotic twins at 7 weeks of gestation. The growth-retarded fetus died at 21 weeks of gestation. At 28 weeks of gestation, periventricular leukomalacia was detected in the brain of the surviving fetus by transvaginal ultrasonography. A female baby presenting with microcephaly was born at 39 weeks of gestation, and CT of the brain showed microcephaly and marked hydrocephalus. At 12 months of age, the surviving infant presented with severe physical growth retardation, and frequent episodes of clonic convulsions. PMID- 10750741 TI - Lessons to be learnt from the Bristol affair. PMID- 10750743 TI - Age does not influence early and late tumor-related outcome after surgery for bronchogenic carcinoma. PMID- 10750742 TI - Ethics of rapid surgical technological advancement. PMID- 10750744 TI - The STS National Database: current changes and challenges for the new millennium. Committee to Establish a National Database in Cardiothoracic Surgery, The Society of Thoracic Surgeons. AB - BACKGROUND: The Society of Thoracic Surgeons (STS) established the National Database (NDB) for Cardiac Surgery in 1989. Since then it has grown to be the largest database of its kind in medicine. The NDB has been one of the pioneers in the analysis and reporting of risk-adjusted outcomes in cardiothoracic surgery. METHODS AND RESULTS: This report explains the numerous changes in the NDB and its structure that have occurred over the past 2 years. It highlights the benefits of these changes, both to the individual member participants and to the STS overall. Additionally, the vision changes to the NDB and reporting structure are identified. The individuals who have participated in this effort since 1989 are acknowledged, and the STS owes an enormous debt of gratitude to each of them. CONCLUSIONS: Because of their collective efforts, the goal to establish the STS NDB as a "gold standard" worldwide for process and outcomes analysis related to cardiothoracic surgery is becoming a reality. PMID- 10750745 TI - Outcome of thoracoabdominal aortic operations using deep hypothermia and distal exsanguination. AB - BACKGROUND: Operation of the descending and thoracoabdominal aorta may be affected by a significant perioperative morbidity, mainly because of ischemic damage of the spinal cord and malperfusion of the abdominal organs. METHODS: A comparative analysis was performed on two consecutive series of patients operated between 1982 and 1998. Group 1 consisted of 90 patients operated with moderate hypothermic left heart bypass. Group 2 included 38 patients operated using deep hypothermic cardiopulmonary bypass and a period of circulatory arrest while performing the proximal anastomosis and distal exsanguination during confection of the distal anastomosis. RESULTS: Main demographic factors and causes of the aortic disease were similar in both groups. Early mortality was significantly higher in the group of patients with aortic cross-clamping (15 of 90, 16%) than in those operated with circulatory arrest (2 of 38, 5.2%), p < 0.001. Paraplegia occurred in 8 patients in the group operated with mild hypothermia (8.8%) but in only 1 patient (2.6%) when deep hypothermia had been used. CONCLUSIONS: In our experience, deep hypothermia combined with distal exsanguination significantly improved the early postoperative outcome after operation of the descending and thoracoabdominal aorta. This technique allowed easy confection of proximal and distal anastomoses, and the duration of the operation was not prolonged significantly through this approach. PMID- 10750747 TI - Safety and efficacy of off-pump coronary artery bypass grafting. AB - BACKGROUND: We evaluated the application of the off-pump coronary artery bypass (OPCAB) procedure relative to safety and efficiency as measured by operative mortality postoperative complications and longitudinal outcome. METHODS: Three hundred and fifty OPCAB patients were compared to 3,171 on-pump or conventional coronary artery bypass (CCAB) patients between January 1, 1997 and December 31, 1998. The groups were divided into three preoperative predicted risk categories: low-risk (0 to 2.59%), medium-risk (2.6 to 9.9%), and high-risk (> or =10%). Society of Thoracic Surgeons National Cardiac Surgery Database definitions and predicted risk group models were utilized to compare all preoperative, intraoperative, and postoperative variables using univariate analysis. RESULTS: Overall comparison of the immediate outcome of CCAB and OPCAB shows little statistical significance in the variables analyzed. The operative mortality was 3.4% in both groups. When the immediate outcome was compared between groups (CCAB vs OPCAB), as well as individual risk groups (low, medium, and high), similar patterns of operative variables and postoperative complications were observed. The operative mortality in the low-risk group was 1.1% for CCAB and 1.4% for OPCAB; 7% for CCAB and 6% for OPCAB in the medium-risk group; and in the high risk group 28.5% for CCAB compared to 7.7% for OPCAB group (p = 0.008). Short term follow-up shows a trend of increased recurring angina and reinterventional procedures in the OPCAB patients. CONCLUSIONS: Safety for OPCAB is assessed through retrospective data review. Longitudinal follow-up for survival, reintervention, and quality of postoperative document efficacy and patency rates, compared to on-pump procedures, is mandatory. This study documented the immediate safety of the OPCAB procedure. Preliminary findings at 1-year follow-up is an important finding in this study, but it is not conclusive at this time. Long-term longitudinal follow-up is required to assess the future effectiveness of OPCAB. PMID- 10750746 TI - Hypothermic circulatory arrest causes multisystem vascular endothelial dysfunction and apoptosis. AB - BACKGROUND: Multiple organ failure after deep hypothermic circulatory arrest (DHCA) may occur secondary to endothelial dysfunction and apoptosis. We sought to determine if DHCA causes endothelial dysfunction and apoptosis in brain, kidney, lungs, and other tissues. METHODS: Anesthetized pigs on cardiopulmonary bypass were: (1) cooled to 18 degrees C, and had their circulation arrested (60 minutes) and reperfused at 37 degrees C for 90 minutes (DHCA, n = 8); or (2) time-matched normothermic controls on bypass (CPB, n = 6). Endothelial function in cerebral, pulmonary, and renal vessels was assessed by vasorelaxation responses to endothelial-specific bradykinin (BK) or acetylcholine (ACh), and smooth muscle specific nitroprusside. RESULTS: In vivo transcranial vasorelaxation responses to ACh were similar between the two groups. In small-caliber cerebral arteries, endothelial relaxation (BK) was impaired in CPB vs DHCA (maximal 55% +/- 2% [p < 0.05] vs 100% +/- 6%). Pulmonary artery ACh responses were comparable between CPB (110% +/- 10%) and DHCA (83% +/- 6%), but responses in pulmonary vein were impaired in DHCA (109% +/- 3%, p < 0.05) relative to CPB (137% +/- 6%). In renal arteries, endothelial (ACh) responses were impaired in DHCA (71% +/- 13%) relative to CPB (129% +/- 14%). Apoptosis (DNA laddering) occurred primarily in duodenal tissue, with a greater frequency in DHCA (56%, p < 0.05) compared with normothermic CPB (17%) and nonbypass controls (0%). CONCLUSIONS: DHCA is associated with endothelial dysfunction in cerebral microvessels but not in the in vivo transcranial vasculature; in addition, endothelial dysfunction was noted in large-caliber renal arteries and pulmonary veins. DHCA is also associated with duodenal apoptosis. Vascular endothelial dysfunction and apoptosis may be involved in the pathophysiology of multisystem organ failure after DHCA. PMID- 10750748 TI - Endothelin receptor pathway in human left ventricular myocytes: relation to contractility. AB - BACKGROUND: Increased synthesis and release of the potent bioactive peptide endothelin-1 (ET-1) occurs during and after cardiac surgery. However, the cellular and molecular basis for the effects of ET-1 on human left ventricular (LV) myocyte contractility remains unknown. METHODS: LV myocyte contractility was examined from myocardial biopsies taken from patients (n = 30) undergoing elective coronary artery bypass. LV myocytes (n = 997, > 30/patient) were isolated using microtrituration and contractility examined by videomicroscopy at baseline and after ET-1 exposure (200 pmol/L). In additional studies, myocytes were pretreated to inhibit either protein kinase C (PKC) (chelerythrine, 1 micromol/L), the sodium/hydrogen (Na/H) exchanger (EIPA, 1 micromol/L), both PKC and the Na/H exchanger, or the ET(A) receptor (BQ-123, 1 micromol/L), followed with ET-1 exposure. RESULTS: Basal myocyte shortening increased 37.8 +/- 6.3% with ET-1 (p < 0.05). Na/H exchanger, PKC, and dual inhibition all eliminated the effects of ET-1. Furthermore, ET(A) inhibition demonstrated that ET-1 effects on myocyte contractility were mediated through the ET(A) receptor subtype. CONCLUSIONS: ET-1 directly influences human LV myocyte contractility, which is mediated through the ET(A) receptor and requires intracellular activation of PKC and stimulation of the Na/H exchanger. PMID- 10750749 TI - Cosgrove-Edwards Annuloplasty System: midterm results. AB - BACKGROUND: The Cosgrove-Edwards Annuloplasty System includes a universally flexible band that corrects mitral annular dilatation via measured plication of the posterior annulus. The purpose of this study was to evaluate midterm clinical and functional results in the first 197 patients receiving this flexible annuloplasty band at mitral valve repair. METHODS: From February 1993 to July 1994, 197 consecutive patients with mitral regurgitation had mitral valve repair using this system. Valve disease was degenerative in 73%, rheumatic in 15%, ischemic in 5%, infectious in 2.5%, and other in 4%. RESULTS: Immediately after repair, echocardiographic mitral regurgitation was none or trivial in 92%, 1+ in 5%, and 2+ in 3%. There were no hospital deaths. Late follow-up was available in 195 patients (99%), with 661 patient-years of follow-up available for analysis. Four-year actuarial survival was 93%, freedom from thromboembolism 94%, from endocarditis 98%, and from reoperation 95%. At a mean interval of 18 months, echocardiography in 157 patients demonstrated no or trace mitral regurgitation in 56%, 1+ in 24%, 2+ in 9%, 3+ in 6%, and 4+ in 3%. At a mean of 61 +/- 5 months, reconstruction of the mitral annulus from real-time three-dimensional echocardiographic images in 10 patients confirmed preserved nonplanar shape and sphincter mechanism of the mitral annulus. Annular orifice area decreased 28% +/- 11% during the cardiac cycle from a mean of 10.1 +/- 3.9 cm2 in diastole to 7.2 +/- 2.8 cm2 in systole. CONCLUSIONS: This annuloplasty system is effective for repair of mitral regurgitation secondary to all causes and preserves mitral annular flexibility and function at 5-year follow-up. PMID- 10750750 TI - Preoperative quality of life as a predictive factor of 3-year survival after open heart operations. AB - BACKGROUND: The aim of this prospective study was twofold: to determine the evolution of quality of life in heart surgery patients through the first 3 postoperative years using the Nottingham Health Profile questionnaire and to determine whether preoperative quality of life influences 3-year survival. METHODS: From January to July 1994, 215 patients underwent elective open heart operation in our department. Patients filled in the Nottingham Health Profile questionnaire five times: preoperatively, postoperatively at month 3, and at each anniversary of their operation for 3 years. The evolution of quality of life scores through time were compared using analysis of covariance with repeated measures. Analysis of 3-year survival prognostic factors was achieved using the Cox proportional hazards model. RESULTS: Quality of life scores varied through time, but not significantly. Multivariate analysis showed two independent risk factors to influence 3-year survival: dyspnea class (III-IV versus I-II, relative risk = 2.80, 95% confidence interval = 1.2 to 6.5) and the energy section of the Nottingham Health Profile questionnaire (relative risk = 1.02 by unit, 95% confidence interval = 1.01 to 1.03). CONCLUSIONS: Our study shows quality of life scores to be stable for the first 3 years after operation and the preoperative energy score to be predictive of 3-year survival. PMID- 10750751 TI - Theoretical analysis of right gastroepiploic artery grafting to right coronary artery. AB - BACKGROUND: The right gastroepiploic artery (GEA) has been used as the second reliable arterial graft for coronary artery bypass grafting (CABG). However, concern regarding the flow competition with the recipient coronary artery has remained. METHODS: An application of in situ GEA grafting to the right coronary artery (RCA) was studied by using a theoretical model. The theoretical model of CABG was given variables; ie, the diameters and the lengths of both in situ GEA and proximal segment of the RCA, and the degree of proximal stenosis in the RCA. According to the range of these variables obtained from clinical data, the ratio of the GEA flow to the flow of the RCA distal to the anastomosis was calculated. RESULTS: Main factors to determine the flows in the two parallel paths were the inner diameters of both vessels, and the degree of the proximal stenosis. When the inner diameters of the GEA were 0.5 mm larger than that of the RCA, the GEA carried more than 50% of the total flow of the RCA distal to the anastomosis despite a moderate stenosis in the RCA. When the inner diameter of the GEA was equal to, or 0.5 mm smaller than, that of the RCA, the GEA flow was dominated by the native RCA flow unless the proximal stenosis was critical. CONCLUSIONS: If the inner diameter of the GEA is 0.5 mm larger than that of the RCA, CABG with the GEA can be applied more widely. If not, the application would basically be limited. PMID- 10750752 TI - Low-dose continuous infusion of human atrial natriuretic peptide during and after cardiac surgery. AB - BACKGROUND: We evaluated the effects of human atrial natriuretic peptide (hANP) during cardiopulmonary bypass (CPB). METHODS: Forty patients undergoing coronary artery bypass grafting were investigated. A group of patients given hANP for 24 hours from the start of CPB (hANP group) was compared with a non-hANP group. Parameters examined were hemodynamics, urine volume, dosage of furosemide, respiratory index, pleural effusion, ANP, cyclic guanosine monophosphate, renin activity (renin), angiotensin-II, aldosterone, and glomerular filtration rate. RESULTS: Central venous pressure, systemic vascular resistance index, and pulmonary vascular resistance index were significantly lower in the hANP group than in the non-hANP group. The hANP group showed significantly higher levels of ANP, cyclic guanosine monophosphate, glomerular filtration rate, and respiratory index, and significantly lower levels of renin, angiotensin-II, aldosterone, and pleural effusion, as compared with the non-hANP group. The dosage of furosemide was significantly lower and the urine volume was significantly larger in the hANP group. CONCLUSIONS: hANP can satisfactorily compensate for the shortcomings of CPB by decreasing the peripheral vascular resistance, suppressing the renin angiotensin-aldosterone system, and exerting a strong diuretic effect. PMID- 10750753 TI - Mitral valve replacement with homograft and Maze III procedure. AB - BACKGROUND: Mitral valve disease is often accompanied by atrial fibrillation, which may compromise the patient even after the valvular lesion has been repaired. METHODS: Three patients with rheumatic type mitral valve disease and chronic atrial fibrillation were treated by mitral valve replacement with cryopreserved mitral valve homograft and Maze III procedure, as a method to relieve both the valvular pathology and the rhythm disorder. The patients' clinical courses have been followed for up to 1 year after operation. RESULTS: All patients survived operation, and all have normal sinus rhythm. None are taking cardiac medications. CONCLUSIONS: Long-term treatment with warfarin should not be required, and other cardiac medicines may be eliminated following mitral valve replacement with homograft combined with Maze III procedure. PMID- 10750754 TI - No benefit of reduced heparinization in thoracic aortic operation with heparin coated bypass circuits. AB - BACKGROUND: Heparin coating of the cardiopulmonary bypass circuit attenuates inflammatory response and confer clinical benefits in cardiac operations. The positive effects may be amplified with reduced systemic heparin dosage. We studied markers of inflammation and coagulation in thoracic aortic operations with heparin-coated circuits and standard vs reduced systemic heparinization. METHODS: Thirty patients were randomized to standard (group S; 300 IU/kg initially; activated clotting times [ACT] > 480 seconds; 5,000 IU in prime; n = 16) or reduced (group R; 100 IU/kg initially; ACT > 250 seconds; 2,500 IU in prime; n = 14) dose systemic heparin. The following markers were analyzed perioperatively: (a) inflammatory response; acute phase cytokine interleukin-6, and granulocytic proteins myeloperoxidase and lactoferrin; (b) complement activation; factor C3a and the C5a-9 terminal complement complex [TCC]; and (c) coagulation; thrombin-antithrombin III complex. RESULTS: The clinical outcome did not differ between groups. Four (29%) patients in group R had a perioperative thromboembolic event. All studied markers were significantly elevated during and throughout cardiopulmonary bypass in both groups. Maximal values were higher in group R for all variables except for TCC. There were no statistically significant intergroup differences regarding markers of inflammation, complement activation, or coagulation activation. CONCLUSIONS: The blood trauma in thoracic aortic operation is extensive, as reflected by the elevation of the studied biochemical markers, even when heparin-coated cardiopulmonary bypass circuits are used. In this study, we did not detect any benefits, either biochemical or clinical, of reducing the dose of systemic heparin. PMID- 10750755 TI - Neuron-specific enolase increases in plasma during and immediately after extracorporeal circulation. AB - BACKGROUND: Minor cerebral complications are common after cardiac surgery. Several biochemical markers for brain injury are under research; one of these is neuron-specific enolase (NSE). The purpose of this study was to investigate the release of this enzyme into the blood during and immediately after extracorporeal circulation and to evaluate the effect of hemolysis on this release. METHODS: Sixteen patients scheduled for elective heart surgery were included in the study. Blood samples for analysis of NSE and free hemoglobin in plasma were drawn before, during, and up to 48 hours after the end of extracorporeal circulation. The release of NSE from erythrocytes and its correlation to the release of free hemoglobin was studied by serial dilution and hemolysis in vitro. RESULTS: The peri- and postoperative course was uneventful in all patients. Extracorporeal circulation initiated a release of NSE that reached a maximum 6 hours after the end of perfusion. Thereafter, the levels declined with an estimated t1/2 of 30 hours. The concentration of free hemoglobin increased during the perfusion, with maximum levels at the end of perfusion, after which they fell rapidly to normal values. The in vitro study showed a strong linearity between the release of NSE and free hemoglobin after induced hemolysis. CONCLUSIONS: The increased levels of enolase at the end of cardiopulmonary bypass can, to a major part, be explained by the release from hemolysed erythrocytes. The value of NSE as a marker for brain injury in these situations is therefore doubtful. PMID- 10750756 TI - Anterior mitral leaflet prolapse as a primary cause of pure rheumatic mitral insufficiency. AB - BACKGROUND: This study was designed to revise the mechanisms and repair techniques of anterior mitral leaflet prolapse observed during the correction of pure rheumatic mitral regurgitation in children. METHODS: From March 1993 to May 1998, 36 children suffering from pure rheumatic mitral regurgitation due to anterior leaflet prolapse underwent mitral valve repair. The mean age was 12.5 years (range, 6 to 16 years). Anterior leaflet prolapse was due to chordal elongation in 25 patients (group A), chordal rupture in 6 patients (group B), and retraction of anterior secondary chordae tendineae, creating a V-shaped deformity in the middle of the anterior leaflet, thus moving the free edge of the anterior leaflet away from the coaptation plane, in 5 patients (group C). Chordal shortening, transposition, and resection of anterior secondary chordae tendineae were used to correct anterior leaflet prolapse according to the predominantly responsible mechanism. RESULTS: All patients were available for clinical follow up, which ranged from 6 months to 5 years (mean follow-up, 3 years). Echocardiographic studies were obtained until the 3rd postoperative month, and all patients showed significant improvement in their left ventricular and atrial dimensions. There was one late death related to endocarditis. Two patients in group C who had mitral valve repair underwent mitral valve replacement on the 19th and 24th postoperative months, respectively, because of failure of mitral valve repair. CONCLUSIONS: Mitral valve repair for pure mitral regurgitation due to rheumatic anterior leaflet prolapse can be performed safely for all types of mechanisms. Although the techniques we used provide stable short-term results in each of these groups, midterm results are better in groups A and B, where tissue thickening is less important, recurrences of rheumatic carditis are lower, and the interval between the first rheumatic attack and the surgical procedure is shorter than in group C. PMID- 10750757 TI - Effect of low molecular weight heparin (fragmin) on bleeding after cardiac surgery. AB - BACKGROUND: Fragmin (Dalteparin, Pharmacia Ltd, Milton Keynes, UK), a low molecular weight heparin, is now recommended in the treatment of unstable angina. Due to the greater bioavailability and longer half-life of Fragmin compared with conventional heparin we postulated that this may influence postoperative bleeding after cardiac surgery for unstable angina. METHODS: We investigated the influence of the agent on postoperative bleeding after cardiac surgery. Patients undergoing first-time coronary artery bypass grafting were prospectively studied in four groups: group A (n = 100) were elective patients; group B (n = 60) had unstable angina and received conventional heparin intravenously until operation; group C (n = 115) received Fragmin with the last dose administered more than 12 hours before surgery; and group D (n = 115) received Fragmin within 12 hours of operation. RESULTS: Patients in group D had significantly greater blood loss (p < 0.001) and increased blood transfusion than groups A, B, and C (p = 0.047). Patients receiving Fragmin more than 12 hours before surgery (group C) had similar rates of blood loss and transfusion to group B (p > 0.05) but greater than in group A (p = 0.021). There were no differences in reopening rate. CONCLUSIONS: The risks of bleeding and transfusion must be weighed against the risks of acute ischemic events if Fragmin is stopped more than 12 hours before operation. PMID- 10750758 TI - Conservative surgical treatment of valvular injury after blunt chest trauma. AB - BACKGROUND: Blunt injury to the cardiac valves leads to progressive ventricular failure often requiring surgical management. Most frequently, prosthetic replacement is the chosen management. METHODS: Three consecutive patients presenting to one surgeon with blunt traumatic valve lesions formed the study group. RESULTS: At operation, the valvular pathology was assessed, and reparative techniques were used to correct the defects. All the patients had an excellent outcome at follow-up periods of 2 to 3 years. CONCLUSIONS: Conservative operation to repair traumatic valve lesions is feasible and has potential advantages over replacement. PMID- 10750759 TI - Autopsy findings in early and late postoperative death after partial left ventriculectomy. AB - BACKGROUND: Partial left ventriculectomy (PLV) is an alternative to heart transplantation for patients with severe heart failure. However, this procedure is accompanied by high morbidity and mortality. Therefore, we studied the hearts of 12 patients who underwent this procedure to increase our understanding of the causes of bad outcome. METHODS: We analyzed the autopsy hearts of 11 of 16 patients who died after PLV, and one heart from a patient who underwent heart transplantation. RESULTS: Six patients died less than 30 days postoperatively, 4 of cardiogenic shock, 1 of arrhythmia, and 1 of coagulopathy. Five patients died from 36 to 120 days after the procedure, 4 of cardiogenic shock and 1 of arrhythmia. The patient who underwent heart transplantation had a cardiogenic shock 230 days after PLV. Ten hearts weighed more than 500 g and nine had myocardial infarction that extended to the papillary muscles. Four patients had infarction of both papillary muscles and 3 of them had episodes of arrhythmia, suggesting some relation between these events. CONCLUSIONS: We found several important morphologic clues for bad outcome: infarction of both papillary muscles, which may be associated with the development of arrhythmia, and myocardial infarction and pericardial hemorrhage, which may contribute to the outcome of heart failure. PMID- 10750760 TI - Anticoagulative management of patients requiring left ventricular assist device implantation and suffering from heparin-induced thrombocytopenia type II. AB - BACKGROUND: Heparin-induced thrombocytopenia type II (HIT II) is a rare but life threatening side effect of heparin therapy. We describe the perioperative anticoagulative management of patients tested positive for HIT II and requiring implantation of a left ventricular assist device (LVAD). METHODS: We report on 3 patients with a different perioperative anticoagulative management (preoperative, intraoperative, and postoperative anticoagulation with danaparoid-sodium; preoperative anticoagulation with recombinant hirudin, anticoagulation with danaparoid-sodium intraoperatively and postoperatively; preoperative anticoagulation with recombinant hirudin, intraoperative anticoagulation with heparin, and postoperative anticoagulation with danaparoid-sodium) and discuss the difficulties of the treatment. RESULTS: Anticoagulation with alternative drugs such as recombinant hirudin and danaparoid-sodium led to serious and life threatening bleeding complications as well as to thromboembolic events in the first 2 patients. Therefore the third patient underwent LVAD implantation using heparin for intraoperative anticoagulation to avoid administration of high doses of recombinant hirudin or danaparoid-sodium. Despite very low anti-factor Xa activities, when using danaparoid-sodium postoperatively, the patient suffered from a bleeding complication on the 4th day after LVAD implantation requiring reexploration. CONCLUSIONS: In selected cases (negative heparin-induced platelet aggregation (HIPA) test at the time of LVAD implantation and continuation of postoperative anticoagulation with recombinant hirudin or danaparoid-sodium), heparin may be used for LVAD implantation in HIT II patients to reduce bleeding complications. PMID- 10750761 TI - Management of aortic valve disease during aortic surgery. AB - BACKGROUND: Alternative management strategies for aortic valve disease and aortic operation include valve preservation and aortic repair (VPR), composite valve graft (CVG), or separate valve and aortic repair (SVR). We evaluated these approaches. METHODS: Of 250 ascending/arch operations, 151 patients had aortic valvular disease and dissection (n = 56, 37%) or aneurysms operated between November 1990 and January 1998. Sixty-seven patients underwent CVG insertion, 50 SVR, 13 VPR, and 21 only aortic repair alone (RA). Sixty (40%) patients also had aortic arch repairs and 53 (35%) coronary artery bypasses. RESULTS: The early 30 day survival and stroke rates were 99% (150 of 151) and 0% (0 of 151), respectively: CVG 100% (67 of 67), 0%; VPR 100% (13 of 13), 0%; SVR 98% (49 of 50), 0%; RA 100% (21 of 21), 0% (p = not significant [NS]). On late follow-up of all patients (5 to 92 months; 96% complete 1998), 3 CVG, 2 VPR, 6 SVR, and 0 RA patients died with respective 5-year Kaplan-Meier survival rates of 88.4%, 70%, 69%, and 100% (p = 0.07, log-rank test). The respective linear rates for stroke were 0%, 5.5% (n = 1), 0%, and 0%; for hemorrhage were 0%, 0%, 0%, and 0%; and for endocarditis were 2.2% (n = 3), 0%, 0%, and 0% (p = NS). There were 11 late deaths and no patient required reoperation or ruptured the ascending aorta or the aortic arch. CONCLUSIONS: With careful selection of the appropriate method excellent early and late results can be achieved. PMID- 10750762 TI - Off-pump bypass graft operation significantly reduces oxidative stress and inflammation. AB - BACKGROUND: This study investigated whether off-pump coronary bypass graft operations on the beating heart under normothermic conditions reduces the systemic oxidative stress and inflammatory reaction seen in patients operated under cardiopulmonary bypass (CPB). METHODS: A cardiac stabilizer (Octopus Tissue Stabilizer; Medtronic Inc, Minneapolis, MN) was used to perform the coronary anastomoses on the normothermic beating heart with or without CPB. Serial blood samples were taken at various intervals. Plasma was analyzed for several oxidative stress and inflammatory markers. RESULTS: Significant increases from prior anesthesia values of lipid hydroperoxides (190% at 4 hours), protein carbonyls (250% at 0.5 hours) and nitrotyrosine (510% at 0.5 hours) were seen in the CPB group, but they were abolished or significantly reduced in the off-pump group. Complement C3a and elastase levels were rapidly increased upon the institution of CPB, and this was followed by increases in IL-8, TNF-alpha, and sE selectin. In contrast, the rise of these factors was blunted in patients operated without CPB. CONCLUSIONS: Off-pump coronary bypass graft operation on a beating heart significantly reduces oxidative stress and suppresses the inflammatory reaction associated with the use of CPB. PMID- 10750763 TI - Lazaroid reduces production of IL-8 and IL-1 receptor antagonist in ischemic spinal cord injury. AB - BACKGROUND: 21-aminosteroids (lazaroids) have demonstrated the protective effect against cerebral ischemic injury through the inhibition of lipid peroxidation. We examined whether lazaroids affected the production of proinflammatory and antiinflammatory cytokines in ischemic spinal cord injury model. MATERIALS: Anesthetized New Zealand white rabbits underwent a 20-minute infrarenal aortic cross-clamping (AXC) with pretreatment of either an intravenous 3 mg/kg lazaroid U74389G (group L; n = 10) or the same volume saline (group P; n = 10). Sham operation group (group S; n = 6) underwent only exposure of the aorta. Plasma concentrations of interleukin (IL)-8, -1beta, -1 receptor antagonist (IL-1ra) and tumor necrosis factor (TNF)-alpha were measured at four time points. Functional assessment with Tarlov score at 24 and 48 hours after pretreatment, pathologic assessment of the spinal cord, and measurements of cytokine levels in the spinal cord were performed. RESULTS: The maximum elevation of plasma IL-8 and -1ra levels occurred at 1 hour after declamping in four measurement points. Plasma IL 8 and -1ra levels in group L were significantly lower than those in group P (*p < 0.05). Plasma TNFalpha peaked at 5 minutes after declamping, but decreased afterwards. Plasma TNFalpha levels were not different among three groups. Spinal IL-8 levels in group L (0.98 +/- 0.34 ng/g tissue) were lower than those in group P (7.26 +/- 2.26 ng/g tissue)(*p < 0.05). Spinal IL-1ra and TNFalpha were not significantly different. Tarlov score and pathologic assessment were better in group L. CONCLUSIONS: Lazaroid U-74389G reduced the production of systemic IL-8 and -1ra and spinal IL-8 when AXC caused spinal cord injury. These results indicate that lazaroids may attenuate ischemic endothelial cell injury or activation of leukocytes. PMID- 10750764 TI - Mesenteric dysfunction after cardiopulmonary bypass: role of complement C5a. AB - BACKGROUND: We investigated the effects of cardiopulmonary bypass (CPB) on ileal homeostasis, and the influence of functional inhibition of complement C5a on CPB induced mesenteric injury. METHODS: Pigs were perfused on CPB for 1 hour and then perfused off CPB for an additional 2 hours. Antiporcine C5a monoclonal antibody (C5a MAb) was administered 20 minutes before onset of CPB to 6 pigs; 6 controls received saline vehicle. Total complement activity, ileal myeloperoxidase, and indices of ileal integrity were examined. RESULTS: Treatment with C5a MAb ameliorated CPB-induced abnormalities in endothelium-dependent relaxation to ADP and substance P, and the hypercontractile response to phenylephrine of ileal microvessels (88 to 168 microm). Ileal myeloperoxidase activity [units/g protein] was 41 +/- 11 in the C5a MAb group, compared to 83 +/- 13 in the saline group (19 +/- 10 base line). Total hemolytic complement activity was similar in the C5a MAb and saline groups (0.6 +/- 0.2 and 0.7 +/- 0.2 CH50 units). During CPB, ileal mucosal blood flow and mucosal pH, edema formation, and epithelial permeability deteriorated similarly in saline and C5a MAb groups. Inducible nitric oxide synthase (iNOS) mRNA expression was similar before and after CPB. CONCLUSIONS: CPB is associated with significant physiologic alterations in mucosal perfusion, epithelial permeability, edema formation, and blood flow regulation. Inhibition of C5a limits neutrophil-mediated impairment of ileal microvascular regulation after bypass, but does not improve extravascular mesenteric dysfunction after CPB. PMID- 10750765 TI - Aprotinin and tranexamic acid for high transfusion risk cardiac surgery. AB - BACKGROUND: Studies have shown that aprotinin and tranexamic acid can reduce postoperative blood loss after cardiac operation. However, which drug is more efficacious in a higher risk surgical group of patients, has yet to be defined in a randomized study. METHODS: With informed consent, 80 patients undergoing elective high transfusion risk cardiac procedures (repeat sternotomy, multiple valve, combined procedures, or aortic arch operation) were randomized in a double blind fashion, to receive either high dose aprotinin or tranexamic acid. Patient and operative characteristics, chest tube drainage and transfusion requirements were recorded. RESULTS: There was no significant difference between the 2 treatment groups with respect to age, cardiopulmonary bypass time, complications (myocardial infarction, stroke, death), chest tube drainage (6, 12, or 24 hours), blood transfusions up to 24 hours postoperatively, total allogeneic blood transfusions for entire hospital stay, or induction/postoperative hemoglobin levels. However, multiple regression analysis revealed a positive relationship between cardiopulmonary bypass time and 24 hour blood loss in the tranexamic acid group (p = 0.001), unlike the aprotinin group where 24 hour blood loss is independent of cardiopulmonary bypass time (p = 0.423). CONCLUSIONS: Overall, there was no significant difference in blood loss, or transfusion requirements, when patients received either aprotinin or tranexamic acid for high transfusion risk cardiac operation. Aprotinin, when given as an infusion in a high-dose regimen, was able to negate the usual positive effect of cardiopulmonary bypass time on chest tube blood loss. PMID- 10750766 TI - Comparative rest and exercise hemodynamics of 23-mm stentless versus 23-mm stented aortic bioprostheses. AB - BACKGROUND: The hemodynamic superiority of stentless valves at rest has been generally accepted, but there is a lack of studies on exercise hemodynamics. METHODS: We assessed aortic valve hemodynamics at rest and during exercise in 10 patients with a 23-mm stentless aortic bioprosthesis (Medtronic Freestyle; Medtronic Europe SA/NV, St. Stevens Woluwe, Belgium), in 10 patients with a 23-mm stented aortic bioprosthesis (Carpentier-Edwards, SAV, model 2650; Baxter Edwards AG, Horw, Switzerland), and in 10 healthy volunteers (control group) by means of Doppler echocardiography. RESULTS: Gradients at rest and gradients on comparable maximum exercise levels were significantly lower in patients with stentless valves compared to those with stented valves (rest: 6 +/- 2/11 +/- 4 mm Hg [mean/peak] versus 12 +/- 3/21 +/- 10 mm Hg; exercise: 9 +/- 3/18 +/- 6 mm Hg [mean/peak] versus 22 +/- 8/40 +/- 11 mm Hg). Patients with stentless valves revealed, in comparison to healthy young men, significantly higher gradients, but the small gradient difference of 3/7 mm Hg (mean/peak) at rest remained nearly unchanged throughout the exercise protocol (4/8 mm Hg [mean/peak] at 25 W, 4/9 mm Hg at 50 W and 4/9 mm Hg at 75 W). In contrast, the gradient difference between patients with stented and stentless valves increased significantly from one exercise level to the next (6/12 mm Hg [mean/peak] at rest, 8/14 mm Hg at 25 W, 12/17 mm Hg at 50 W, and 15/25 mm Hg at 75 W). CONCLUSIONS: A stentless aortic bioprosthesis seems to be an appropriate aortic valve substitute, especially in patients who perform regular physical exercise. PMID- 10750767 TI - Bedside estimation of risk as an aid for decision-making in cardiac surgery. AB - BACKGROUND: Evaluations of the cardiac-surgery mortality rates of hospitals and surgeons can be fair and realistic only when the observed mortality rates are compared with expected rates with preoperative risk factors taken into account. Risk-approximation calculations also can assist patients and physicians in discussing the risk of cardiac surgery, especially if the estimation of surgical mortality takes all of the important risk factors into account. METHODS: A logistic regression model was developed in which 47 potential risk factors were considered, and a method requiring only simple addition and graphic interpretation was designed for approximating the estimated risk easily and quickly, with paper and pencil alone. RESULTS: The estimates provided by the simplified model correlated well with the observed mortality rates. CONCLUSIONS: A simple approximation of a logistic regression model has been found to be helpful in discussions between physicians and patients contemplating aortocoronary bypass or valve-related surgery. PMID- 10750769 TI - Craniocervical and aortic atherosclerosis as neurologic risk factors in coronary surgery. AB - BACKGROUND: Advanced age is associated with increased systemic atherosclerosis and is a consistent neurologic risk factor after coronary artery bypass grafting (CABG). METHODS: We studied prospectively whether varying degrees of a total atherosclerotic score derived from the brain, carotid arteries, and ascending aorta predicted postoperative neuropsychologic (NP) dysfunction and stroke in 177 elderly patients (> or = 60 years) undergoing CABG. RESULTS: Group L (low total atherosclerotic score) had rates of NP dysfunction of 25% and 4%, group I (intermediate) had rates of 33% and 22%, and group H (high) had rates of 79% and 43% on postoperative days 1 and 7, respectively (p < 0.001). The incidence of stroke was higher in group H (14.3%) than in groups I and L (7.8% and 0.9%; p = 0.013). Stepwise logistic regression analysis demonstrated the significant predictors of NP dysfunction on postoperative day 7 to be total atherosclerotic score, peripheral vascular disease, and diabetes mellitus, and those of stroke to be total atherosclerotic score, peripheral vascular disease, and hyperlipidemia. CONCLUSIONS: Perioperative evaluation of craniocervical and aortic atherosclerosis is useful to identify a high-risk patient at postoperative NP dysfunction and stroke after CABG. PMID- 10750768 TI - Trends during 25 years of coronary artery bypass operation at St. Luke's Medical Center in Milwaukee, Wisconsin. AB - BACKGROUND: Most studies of changes in coronary artery bypass graft (CABG) operations are from major academic institutions. The present study evaluated changes in CABG operations since 1968 in a community hospital. METHODS: The data were from the St. Luke's Medical Center Cardiovascular Data Registry in Milwaukee, Wisconsin. Mortality rates, risk factors, overall patient risk, and surgical procedures were compared from 1968 to 1994. RESULTS: There was a dramatic decrease in 30-day mortality rates from 1968 to 1972. After 1976, mortality rates increased because of higher risk patients, but the mortality rate, adjusted for patient risk, continued to decline. Both internal mammary arteries and sequential grafts were widely used by 1972, followed by a decline in use until 1980, and then a steep increase in use from 1980 to the present. CONCLUSIONS: This study provided evidence from a community hospital that the skills of the surgical teams improved first dramatically then gradually. The pattern of adapting new surgical techniques suggested that these techniques were critically evaluated for several years after they were introduced. PMID- 10750770 TI - Technical aspects of double-skeletonized internal mammary artery grafting. AB - BACKGROUND: Bilateral internal mammary artery (IMA) grafting is performed to provide complete arterial myocardial revascularization with the intention of decreasing postoperative return of angina and the need for reoperation. We present here technical views of double-skeletonized IMA grafting, and evaluate its clinical outcome. METHODS: Skeletonized IMA is harvested gently with scissors and silver clips, without use of cauterization, and embedded in a small syringe filled with papaverine. Three strategies for arterial revascularization were employed in 762 consecutive patients: (1) the cross arrangement (242 patients, 32%), where the in situ right internal mammary artery (RIMA) is used for the left anterior descending artery (LAD), in situ left internal mammary artery (LIMA) to circumflex marginal branches and the gastroepiploic artery for the right coronary artery (RCA); (2) the composite arrangement (476 patients, 62%), where free IMA is attached end-to-side to the other in situ IMA; and (3) the natural arrangement (44 patients, 6%), where the in situ RIMA is connected to the RCA and in situ LIMA to LAD. Mean age was 66 years (range 30 to 92). Two hundred ninety-two patients (38%) were older than 70, and 229 (30%) were diabetic. RESULTS: Operative mortality was 2.5% (n = 19). The mortality of urgent and elective cases was 1.2% (8 of 663), and that of emergency operation was 11% (11 of 99). There were 9 (1.2%) perioperative myocardial infarctions, and 10 patients (1.3%) sustained strokes. Sternal wound infection occurred in 14 (1.8%). CONCLUSIONS: The three strategies described here provide the surgeon with the versatility required for arterial revascularization with bilateral IMAs in most patients referred for coronary artery bypass grafting. PMID- 10750771 TI - The effect of cardiotomy suction on the brain injury marker S100beta after cardiopulmonary bypass. AB - BACKGROUND: An increase of S100beta in serum during cardiopulmonary bypass (CPB) has been interpreted as a sign of brain injury. Cardiotomy suction may cause fat embolization, and its role in the S100beta increase was examined. METHODS: Twenty coronary artery operation patients were randomly assigned to two groups, 10 with suction during CPB to cardiotomy reservoir (CR), 10 to cell saving device (CS). S100beta was measured (immunoassay) in blood from the patients and from cell saving device after processing. In 7 additional patients S100beta was measured in the cell saving device before processing and directly from the wound at sternotomy. RESULTS: Before anesthesia, serum S100beta was 0.03+/-0.06 microg/L. At the end of CPB it was 2.47+/-1.31 microg/L and 0.44+/-0.27 microg/L (CR vs CS; p < 0.001). S100beta was 33+/-12 microg/L in CS reservoir and 42+/-18 microg/L in blood from the wound. CONCLUSIONS: Most serum S100beta after CPB with cardiotomy suction may be of extracerebral origin. S100beta after CPB with cell saving device was the same as after off-pump operation. The interpretation that an increase in S100beta during CPB in patients reflects cerebral injury must be questioned. PMID- 10750772 TI - Arterial switch after failed atrial baffle procedures for transposition of the great arteries. AB - BACKGROUND: Late failure of the systemic right ventricle after atrial baffle procedures in patients with transposition of the great arteries poses significant management problems. We reviewed our experience with staged conversion to arterial switch operation (ASO) in these patients. METHODS: Between 1984 and 1999, 11 patients underwent pulmonary artery band (PAB) to prepare the left ventricle for ASO conversion. One additional patient had subpulmonic stenosis and was naturally prepared. Mean age at the initial PAB was 12.2+/-7 years (range, 1.9 to 23 years). Four patients underwent reoperation to tighten the PAB before ASO. Mean interval from PAB to ASO was 1.3+/-0.9 years. RESULTS: There was no mortality from PAB. Six patients had ASO conversion and 2 died. Recent surgical modifications at the time of ASO were used to prevent neoaortic valve insufficiency and to cryoablate atrial reentry tachycardia. Four patients developed biventricular failure after PAB and had orthotopic cardiac transplantation (OCT) 14+/-10 months after PAB. The other 2 patients are still with PAB: 1 is awaiting ASO conversion and the other has insufficient left ventricular hypertrophy necessary for ASO conversion despite two preparatory PABs. CONCLUSIONS: A select group of patients with right ventricular failure after atrial baffle operations can undergo staged conversion to ASO with the opportunity for excellent long-term outcome. Surgical modifications at the time of ASO can address the problems of neoaortic insufficiency and persistent atrial arrhythmias. PAB may be a therapeutic endpoint in some patients not responding with adequate left ventricular hypertrophy. Those patients who develop biventricular failure after PAB will require cardiac transplantation. PMID- 10750773 TI - Surgical intervention for complications of transcatheter dilation procedures in congenital heart disease. AB - BACKGROUND: Transcatheter interventions have assumed an important role in the management of many forms of congenital heart disease. While complications of transcatheter interventions are uncommon and usually minor, significant complications requiring operation do occur on occasion. The purpose of this report is to present our experiences with seven such complications, and to review the literature on this topic. METHODS: Seven patients who required operation after a transcatheter dilation procedure between 1992 and 1998 are described. Three patients required retrieval of retained foreign bodies (stents or balloons), and repair of the underlying abnormality. Two patients underwent repair of fistulas between 2 great vessels, or a great vessel and a cardiac chamber. One patient required operation for a postdilation aneurysm. One patient underwent urgent repair of severe aortic regurgitation after balloon aortic valvuloplasty. RESULTS: All patients survived and are doing well, with no further need for catheter or operative intervention, from 8 months to 6 years after operation. Additional reported complications requiring operation are discussed as well. CONCLUSIONS: Operation for complications of catheter interventions in congenital heart disease is seldom necessary. Though uncommon, a variety of such complications may occur, including vascular, valvar, intracardiac, and foreign body complications. When operation is required, results are typically very good. PMID- 10750774 TI - Ultra fast track in elective congenital cardiac surgery. AB - BACKGROUND: Changes in healthcare delivery have affected the practice of congenital cardiac surgery. We recently developed a strategy of limited sternotomy, early extubation, and very early discharge, and reviewed the perioperative course of 198 pediatric patients undergoing elective cardiovascular surgical procedures, to assess the efficacy and safety of this approach. METHODS: One hundred ninety-eight patients aged 0 to 18 years (median 3.2 years) underwent 201 elective cardiovascular surgical procedures over a 1-year period. All patients were admitted on the day of surgery. Patients were divided into six diagnostic groups: group 1, complex left-to-right shunts (n = 14, 7.0%); group 2, simple left-to-right shunts (n = 83, 41.3%); group 3, right-to-left shunts with pulmonary obstruction (n = 33, 16.4%); group 4, isolated, nonvalvular obstructive lesions (n = 30, 14.9%); group 5, isolated valvular anomalies (n = 20, 10.0%); and group 6, miscellaneous (n = 21, 10.4%). RESULTS: After 201 procedures, 175 patients (87.1%) were extubated in the operating room and 188 (93.6%) within 4 hours from operation. Four patients (2.0%) were extubated more than 24 hours from completion of the procedure, and 2 (1.0%) died while on respiratory support (never weaned). Five patients (2.6%) failed early extubation (<4 hours). Early discharge was achieved for the vast majority of patients. Overall median length of stay (LOS, including day of surgery as day 1) was 2.0 days, with a median LOS of 3.0 days for those patients requiring circulatory arrest duration exceeding 20 minutes. Of 195 patients, 43 (24.6%), 121 (74.0%), and 159 (81.5%) were discharged, respectively, at <24, <48, <72 hours from admission. Longest and shortest mean postoperative LOS were in group 6 (9.9+/-14.5 days) and group 2 (1.6 = 0.7 days), respectively. Six patients (2.9%) died, and 11 (5.5%) suffered in-hospital complications. Thirty patients (15.4%) were either treated as outpatients (n = 11, 5.7%) or readmitted (n = 19, 9.7%) within 30 days from the time of surgery. Only 8 of 195 patients (4.1%) were readmitted with true surgical complications requiring invasive therapeutic procedures. CONCLUSIONS: Selected patients with a broad spectrum of congenital heart disease may enjoy same-day admission, limited sternotomy, immediate extubation, and very early discharge with excellent outcomes and acceptable morbidity. PMID- 10750775 TI - Fast-track congenital heart operations: a less invasive technique and early extubation. AB - BACKGROUND: Many novel techniques have been described for "minimally invasive" congenital cardiac operations to achieve an improved cosmetic result. There is little information on incorporation of such techniques into fast-track congenital heart operations. METHODS: We have developed an approach to fast-track congenital heart operations, which includes a cosmetic approach for repair of congenital heart defects without sacrificing adequate exposure or requiring specialized equipment, along with a simple approach to intraoperative anesthetic management that allows extubation in the operating room. The heart is exposed through a short midline skin incision and a full median sternotomy. The conventional technique of cannulation is performed. Between October 1997 and January 1999, 88 patients were operated on with this method. Cardiac anomalies included simple and complex ostium secundum atrial septal defect, sinus venous atrial septal defect, partial atrioventricular septal defect, simple and complex ventricular septal defect, and bicuspid aortic valve stenosis. RESULTS: There were no operative or late deaths. The majority of patients were extubated in the operating room or within 2 hours of operation. No patient underwent reoperation and the mean length of hospital stay was 3.9 days. Sternal instability or wound infection were not observed. CONCLUSIONS: We believe that our approach to fast-track congenital heart operation is safe and effective. The surgical technique provides good exposure and has excellent cosmetic results. Moreover, it is easy to learn and, if necessary, the surgeon can quickly gain direct access to the heart. The anesthetic management facilitates early tracheal extubation and a shorter duration of postoperative stay. PMID- 10750776 TI - Modification of the subclavian patch aortoplasty for repair of aortic coarctation in neonates and infants. AB - BACKGROUND: Coarctation repair in neonates or small infants, using a subclavian patch, has a relatively high risk of restenosis, especially if complicated by the presence of a short subclavian artery or long coarctation segment. We introduce a technical modification that facilitates the use of a subclavian flap, and decreases the restenosis rate in this subgroup of patients. It consists of a side to-side transverse aortic anastomosis at the level of the coarctation, which widens the coarctation segment, shortens the isthmus, and pulls the distal end of the aortotomy proximally, allowing a tension-free subclavian flap aortoplasty. METHODS: Fifty-three consecutive neonates or infants less than 18 weeks old, with complex coarctation, underwent repair using this technique. Mean age was 26+/-3 days and 36 patients (68%) were less than 28 days old. Weights ranged from 1.4 to 6.4 kg (mean 3.4+/-0.2 kg), and 26 patients had other cardiac anomalies. Preoperative gradient by Doppler measurement ranged from 25 to 90 mm Hg (mean 49+/-2 mm Hg). RESULTS: Mean aortic cross-clamp time was 27+/-1 minutes (range 19 to 34 minutes). There were no deaths or surgical complications. Follow-up echocardiogram 4 to 52 months postoperatively (mean 25+/-2 months) demonstrated no significant pressure gradient (less than 20 mm Hg) in 51 of 53 patients (96%), and a significant gradient in 2 patients (4%), which was subsequently corrected with balloon angioplasty. CONCLUSIONS: The technical modification described shortens the isthmus, and thus allows for a longer aortotomy distal to the area of coarctation resulting in a tension-free repair especially in patients with a short subclavian artery. It also widens the area of coarctation, and as a result leads to a lower early recoarctation rate in this high-risk group. With increasing emphasis on the need for a longer aortotomy to prevent restenosis, this modification will have increasing application, especially in the neonatal population. PMID- 10750777 TI - Molecular biologic substaging of stage I lung cancer according to gender and histology. AB - BACKGROUND: This study is designed to assess molecular biologic substaging according to gender and histology in patients with stage I non-small cell lung cancer (NSCLC). METHODS: Pathologic specimens were collected from 408 consecutive patients after complete resection for stage I NSCLC, with follow-up of at least 5 years. A panel of nine molecular markers was chosen for immunohistochemical analysis of the tumor: recessive oncogenes p53 and bcl-2, the protooncogene erbB 2, KI-67 proliferation index, retinoblastoma oncogene (Rb), epidermal growth factor receptor (EGFr), angiogenesis factor viii, sialyl-Tn antigen (STN), and CD 44. Cox proportional hazards regression analysis was used to construct a risk model for cancer-specific survival according to marker status, gender, and histologic subtype. RESULTS: Among men, the only molecular marker associated with decreased cancer-specific survival is erbB-2; among women, there are four markers: p53, Rb, CD-44, and factor viii. Among patients with squamous cell carcinoma, the only molecular marker associated with decreased cancer-specific survival is erbB-2; among patients with adenocarcinoma (AC), there are three markers: p53, CD-44, and factor viii. Multivariable analysis of interactions among molecular markers, gender, and histology demonstrates two important relationships (hazard ratio): p53+/women (2.269) and CD-44+/AC (2.266). CONCLUSIONS: Molecular biologic substaging of patients with stage I NSCLC demonstrates differential cancer-specific survival according to marker expression, gender, and histologic subtype. PMID- 10750778 TI - Significance of cyclic adenosine monophosphate and nitroglycerin in ET-Kyoto solution for lung preservation. AB - BACKGROUND: We previously demonstrated that the supplement of both dibutyryl cyclic adenosine monophosphate (db-cAMP) and nitroglycerin to the conventional ET Kyoto solution improved lung preservation significantly. However, the significance of each component in lung preservation remained unclear. We examined the efficacy of the two components on lung preservation in the current study. METHODS: Rat lung grafts (eight per group) were studied in an isolated lung perfusion model. Group 1 grafts were flushed and preserved with ET-Kyoto solution containing 2 mmol/L of db-cAMP. Group 2 grafts were flushed and preserved with ET Kyoto solution containing 100 mg/L of nitroglycerin. In group 3, the grafts were flushed and preserved with ET-Kyoto solution containing neither db-cAMP nor nitroglycerin as control group. After 4-hour cold storage, the lung grafts were reperfused for 50 minutes. RESULTS: The lung grafts in groups 1 and 2 showed significantly better lung function after reperfusion than those in group 3 with regard to arterial oxygen tension, shunt fraction, peak inspiratory airway pressure, mean pulmonary arterial pressure, and pulmonary vascular resistance. The supplementation of db-cAMP improved especially the pulmonary arterial pressure and pulmonary vascular resistance, while the supplementation of nitroglycerin improved especially the oxygenation and airway pressure of the grafts. CONCLUSIONS: Both of db-cAMP and nitroglycerin had beneficial effects on lung preservation and are essential to the ET-Kyoto solution. There was a difference between the two components in the effects on preserved lungs. PMID- 10750779 TI - Prognostic significance of the size of central fibrosis in peripheral adenocarcinoma of the lung. AB - BACKGROUND: The prognostic significance of the characteristics of central fibrosis in peripheral adenocarcinoma of the lung has been reported. However, the prognostic significance of the size of central fibrosis has never been evaluated. METHODS: A total of 100 consecutive surgically resected peripheral adenocarcinomas of the lung measuring 3.0 cm or less in maximum dimension were reviewed histologically, and the maximum dimension of central fibrosis was measured on conventional hematoxylin and eosin stain. RESULTS: Median follow-up for patients alive was 54 months. The overall 5-year survival rate was 75%. Twenty-one patients with adenocarcinoma having central fibrosis 5 mm or smaller in maximum dimension had a 5-year survival rate of 100%, whereas the other 79 patients had a 5-year survival less than 70%. Multivariate analysis showed the size of central fibrosis to be an independent prognostic factor as significant as vascular invasion and locoregional lymph node metastasis (p = 0.010, 0.024, and 0.024, respectively). CONCLUSIONS: The size of central fibrosis is an independent prognostic factor in peripheral lung adenocarcinoma, as significant as the well established prognostic factors vascular invasion and lymph node metastasis. PMID- 10750780 TI - Aspergilloma: a series of 89 surgical cases. AB - BACKGROUND: Surgery for pleuropulmonary aspergilloma is reputed to be risky. We reviewed our results, focusing attention on the postoperative complications. METHODS: During a 20-year period, 87 patients were operated on for pulmonary (86) or pleural (3) aspergillomas. Seventy-two percent of patients were complaining of hemoptysis. Eighty-nine resections were performed because there were two bilateral cases. Seventy percent of aspergillomas had developed in cavitation sequelaes from tuberculosis disease. Thirty-four patients had severe respiratory insufficiency that allowed us to perform only lobectomy (18), segmentectomy (2), or cavernostomy (14). RESULTS: Thirty-seven lobectomies (five with associated segmentectomies), two bilobectomies, 21 segmentectomies, 10 pneumonectomies, and 17 cavernostomies were performed. Total blood loss exceeded 1,500 mL in 14 cases, and 71% of patients required blood transfusion. There were five postoperative deaths (5.7%), related to respiratory failure (2), infectious complication (1), pulmonary embolus (1), and cardiorythmic disorder (1). Incomplete reexpansions were frequently seen in patients undergoing lobectomies or segmentectomies. No death or major complications occurred in asymptomatic patients. During follow-up, none of the patients had recurrent hemoptysis. CONCLUSIONS: Surgical resection of aspergilloma is effective in preventing recurrence of hemoptysis. It has low risk in asymptomatic patients and in the absence of underlying pulmonary disease. Incomplete reexpansion is frequent after lobectomy and segmentectomy, especially when there is underlying lung disease. Cavernostomy is an effective treatment in high-risk patients. Long-term prognosis is mainly dependent on the general condition of patients. PMID- 10750781 TI - Successful treatment of acute, ongoing rat lung allograft rejection with the novel immunosuppressant SDZ-RAD. AB - BACKGROUND: Recent experimental data have shown that coadministration of microemulsion cyclosporine and the novel immunosuppressant SDZ-RAD potentiates the immunosuppressive efficacies of both drugs to suppress allograft rejection. Our study was designed to assess the potential of delayed SDZ-RAD administration, in addition to cyclosporine maintenance therapy, to reverse acute rejection in an allogeneic rat lung transplant model. METHODS: Unilateral left lung transplantation was performed using Brown-Norway donors implanted into Lewis recipients. An untreated control group and a cyclosporine monotherapy group (7.5 mg/kg) were followed for 7 days. An additional cyclosporine monotherapy group (7.5 mg/kg), and a combined therapy group treated with cyclosporine (7.5 mg/kg) plus SDZ-RAD (2.5 mg/kg), were followed for 21 days. For treatment of ongoing rejection, 7.5 mg/kg cyclosporine was given as maintenance therapy, and SDZ-RAD (2.5 mg/kg) was added on postoperative day 7. Drugs were given orally, and in the combined therapy regimens, administered 6 hours apart. Outcome variables included daily weight, radiographs, and histology. RESULTS: Radiographs on postoperative day 7 showed mild and moderate opacification of the left chest in the cyclosporine monotherapy groups and the untreated control group. Addition of SDZ RAD to cyclosporine treatment on postoperative day 7 reversed opacification by postoperative days 14 and 21. Monotherapy with microemulsion CsA resulted in mild histological rejection by day 7, which progressed to moderate rejection by day 21. Addition of SDZ-RAD on postoperative day 7 reversed acute rejection, resulting in none or minimal rejection at day 21. CONCLUSIONS: SDZ RAD reverses acute rejection under cyclosporine maintenance therapy in a stringent lung allotransplant model. PMID- 10750782 TI - Technique of controlled reperfusion of the transplanted lung in humans. AB - BACKGROUND: Reperfusion injury remains a significant and sometimes fatal problem in clinical lung transplantation. Controlled reperfusion of the transplanted lung using white cell-filtered, nutrient-enriched blood has been shown recently to significantly ameliorate reperfusion damage in a porcine model. We modified this experimental technique and applied it to human lung transplantation. METHODS: Approximately 1,500 mL of arterial blood was slowly collected in a cardiotomy reservoir during the lung implant, and mixed to make a 4:1 solution of blood:modified Buckberg perfusate. This solution was passed through a leukocyte filter and into the transplant pulmonary artery for 10 minutes, at a controlled rate (200 mL/min) and pressure (less than 20 mm Hg), immediately before removal of the vascular clamp. RESULTS: Five patients underwent lung transplantation (1 bilateral, 4 single lung) using this technique. All patients were ventilated on a 40% fraction of inspired oxygen within a few hours and extubated on or before the first postoperative day. CONCLUSIONS: Controlled reperfusion of the transplanted lung with white cell-filtered, nutrient-enriched blood has given excellent functional results in our small initial clinical series. PMID- 10750783 TI - Age does not influence early and late tumor-related outcome for bronchogenic carcinoma. AB - BACKGROUND: The influence of age on early and late outcome after surgical resection of bronchogenic carcinoma is unknown. In an attempt to clarify this issue, we reviewed the outcome of 212 consecutive patients with primary lung cancer who had surgical treatment for bronchogenic carcinoma. METHODS: Ninety-two patients were younger than 50 years (group 1), and 120 patients were older than 70 years of age (group 2). Squamous cell carcinoma and adenocarcinoma were the most common histologic types in both groups. According to the new international staging classification, a similar proportion of stage I, II, and III were observed in both groups. RESULTS: Only the rate of pneumonectomy was significantly higher in younger patients (41% versus 22%, p = 0.002). The overall operative mortality rate in group 1 was 2.2% and 2.6% after pneumonectomy. In group 2 the overall mortality rate was 2.5% and 3.8% after pneumonectomy. Advanced age did not affect operative mortality. The adjusted (tumor-related) survival rate at 5 years was 56% in group 1 and 53% in group 2 (p = 0.93). The adjusted survival rate for patients with stage I was 61% in group 1 and 65% in group 2 (p = 0.21), and for stage IIIa 39% in group 1 and 48% in group 2 (p = 0.43). The adjusted 5-year survival rate was 56% in group 1 and 59% in group 2 for squamous cell carcinoma (p = 0.53) and 49% in group 1 and 42% in group 2 for adenocarcinoma (p = 0.76). CONCLUSIONS: Perioperative risk and midterm survival were similar in younger and older patients after surgical resection of bronchogenic carcinoma. We believe that this result is because surgical candidates constitute already a highly selected group of patients. From these data it is not possible to conclude that biologic behavior of lung cancer is more aggressive in younger patients. PMID- 10750784 TI - Functional assessment of chest wall integrity after methylmethacrylate reconstruction. AB - BACKGROUND: All patients with extensive resection of the anterolateral chest wall and the sternum followed by reconstruction with methylmethacrylate substitutes were assessed prospectively 6 months after the operation to delineate chest wall integrity with pulmonary function and cine-magnetic resonance imaging. METHODS: Twenty-six patients underwent chest wall reconstruction by use of methylmethacrylate between 1994 and 1998 due to primary tumors in 35%, metastases in 27%, T3 lung cancer in 19%, and debridement for radionecrosis and osteomyelitis in 19% of patients. Three to eight ribs were resected and additional sternum resection was performed in 39% of patients. RESULTS: There was no 30-day mortality. All patients were extubated after the operation without need for reintubation. Prosthesis dislocation occurred in 1 patient and infection in 2 patients during follow-up. Nineteen patients (73%) suffered no restrictions of daily activities. Clinical examination revealed normal shoulder girdle function in 77% of patients. There was no significant difference between preoperative and postoperative FEV1 (forced expiratory volume in 1 second) measurements in patients with lobectomy or wedge resections. Cinemagnetic resonance imaging revealed concordant chest wall movements during respiration in 92% of patients without paradoxical movements or implant dislocations being observed. CONCLUSIONS: Large defects of the anterolateral chest wall and sternum can be reconstructed efficiently with methylmethacrylate substitutes with minimal morbidity and excellent cosmetic and functional outcome. PMID- 10750785 TI - Percutaneous vertebral angioplasty before coronary artery bypass grafting. AB - We report a case of a 63-year-old male with three-vessel coronary heart disease complicated by stenosis of the bilateral vertebral arteries. Triple coronary bypass grafting, using arterial conduits, was successfully performed after percutaneous balloon angioplasty of the left vertebral artery. Precedent angioplasty of a stenotic vertebral artery is safe and protects the brain from ischemia during extracorporeal circulation. PMID- 10750786 TI - Prolonged extracorporeal membrane oxygenation as a bridge to cardiac transplantation. AB - Cardiac transplantation provides the best option for neonates with congenital heart disease that is not amenable to surgical repair or palliation. The scarcity of suitable organs for this group has resulted in prolonged waiting times; many infants die awaiting transplantation. We present the case of a newborn with severe Ebstein's anomaly and low cardiac output who was supported with extracorporeal membrane oxygenation for 1,126 hours, until an appropriate organ became available. PMID- 10750787 TI - Uncommon complication of arterial switch operation: tracheobronchial compression. AB - We describe 2 patients in whom symptoms of airway compression developed after arterial switch operation for correction of the transposition of the great arteries. The clinical features, diagnosis, management, and proposed mechanisms of this complication are described. PMID- 10750788 TI - Lemierre's syndrome with bilateral empyema thoracis. AB - A 31-year-old patient is described with thrombophlebitis of the right jugular vein, and anerobic septicemia (Lemierre's syndrome). Multiple pulmonary abscesses and bilateral fibrinopurulent empyema were also present. Treatment included intravenous antibiotics, heparin, and video-assisted thoracoscopic debridement of pleural cavities. A favorable outcome was observed. PMID- 10750789 TI - Successful treatment of massive arterial air embolism during open heart surgery. AB - We report a case of a 5-year-old girl who suffered a massive arterial air embolism during surgical closure of an atrial septal defect. The risk of permanent neurologic deficits or even fatal outcome is significant (mortality rate, 31%). We successfully treated a proven arterial air embolism with intraoperative (retrograde cerebral perfusion) combined with postoperative procedures (deep barbiturate anesthesia and hyperbaric oxygenation). At discharge the girl had fully recovered from the initial neurologic defects. PMID- 10750790 TI - Bronchioloalveolar carcinoma arising in a bronchogenic cyst. AB - We report the case of a 37-year-old woman with a radiographically cystic lung lesion. Lobectomy was performed. Histopathologic examination showed a bronchioloalveolar carcinoma arising in a bronchogenic cyst. This suggests that epithelial cells of bronchogenic cysts can undergo malignant transformation. It may be prudent to recommend complete resection of any bronchogenic cyst. PMID- 10750792 TI - Preoperative embolization in surgical treatment of mediastinal hemangiopericytoma. AB - The case of a 47-year-old man with a tumor of the posterosuperior mediastinum is reported. Surgical biopsy sample revealed a hemangiopericytoma, but radical excision was impossible because of massive bleeding. Percutaneous embolization of mediastinal tumor was performed to reduce peroperative blood loss. It allowed uneventful complete removal of the lesion. We recommend preoperative embolization in cases of hypervascular mediastinal tumors. PMID- 10750791 TI - The Ross operation in a Jehovah's Witness: a paradigm for heart surgery in children without transfusion. AB - A 3-year-old 18 kg male child of the Jehovah's Witness faith presented with severe aortic regurgitation. A successful Ross procedure was performed using a pulmonary autograft, without the use of blood or blood product transfusion. Blood conservation strategy included: (1) preoperative treatment with recombinant human erythropoietin; (2) intraoperative strategies, including technical modifications to the Ross procedure, and the prophylactic use of fibrin glue; (3) utilization of a heparin-bonded cardiopulmonary bypass circuit and assisted venous drainage; and 4) the use of prebypass phlebotomy, cell-saving device and autotransfusion. The patient was discharged home on postoperative day 7 with a hemoglobin level of 11.9. PMID- 10750793 TI - Salmonella infection of a ventricular aneurysm with mural thrombus. AB - We describe a case of salmonella infection of a left ventricular aneurysm with a mural thrombus and review 12 cases described in the literature. This entity should be looked for in any patient with persistent or relapsing salmonella bacteremia in whom an intracardiac thrombus is demonstrated. Nuclear imaging may help in the diagnosis. A combined medical and surgical approach should be aggressively pursued because patients who do not undergo an aneurysmectomy are unlikely to survive. PMID- 10750794 TI - Systemic-to-pulmonary shunt in a patient with isolation of the subclavian artery. AB - A surgical procedure is described that was carried out to treat an 18-month-old boy with tetralogy of Fallot, right aortic arch, and isolation of the left subclavian artery. The patient underwent a descending aorta-right pulmonary artery shunt using a polytetrafluoroethylene graft, as a systemic-pulmonary shunt. The effectiveness of this procedure in patients with some special conditions is discussed. PMID- 10750795 TI - Leaflet escape in a TEKNA and an original duromedics bileaflet valve. AB - We report a case of leaflet escape in an Edwards-TEKNA bileaflet valve, in the mitral position. The examination findings of the explanted valve are compared with a similar case of leaflet escape in an original Edward-Duromedics prosthesis. Based on our findings alone, it is not certain whether the TEKNA valve continues to have a higher risk for fracture. PMID- 10750796 TI - Mediastinal amyloidosis. AB - Systemic amyloidosis is an unusual cause of mediastinal lymphadenopathy. Thoracic surgeons are often called upon to establish a diagnosis in patients with mediastinal lymphadenopathy, so familiarity with mediastinal amyloidosis is valuable. We report our experience with 2 patients, and discuss the diagnostic role of mediastinoscopy and other less invasive biopsy techniques. General anesthesia may pose significant risks in this disease; nonoperative biopsy techniques should be considered if the diagnosis of amyloidosis is suspected. PMID- 10750797 TI - Septic paradoxical embolus through a patent foramen ovale after pacemaker implantation. AB - A case of a septic paradoxic embolus due to an infected pacemaker lead associated with a patent foramen ovale (PFO) is described. Treatment consisted of immediate intracardiac embolectomy, pericardial patch closure of the PFO, total removal of the infected pacemaker lead and generator, and placement of a new permanent epicardial lead pacemaker system. PMID- 10750798 TI - Pulmonary artery compression by a giant aortocoronary vein graft aneurysm. AB - Late failure of saphenous vein aortocoronary bypass grafts is predominantly due to vein graft atherosclerotic disease. Rarely, saphenous vein aortocoronary bypass grafts undergo aneurysmal degeneration. We report a case of a giant true aneurysm of a saphenous vein aortocoronary bypass graft producing right heart failure from main pulmonary artery compression. PMID- 10750799 TI - Arterial switch operation: successful bilateral internal thoracic artery grafting. AB - Internal thoracic artery grafting in arterial switch operations for transposition of great arteries has been reported for salvage of myocardial ischemia after initial coronary transfer. We report a situation where we opted for primary coronary bypass grafting to avoid an obviously difficult coronary transfer, with successful outcome. PMID- 10750800 TI - Constrictive pericarditis after bypass leading to internal mammary graft failure. AB - Constrictive pericarditis is an infrequent consequence of cardiac operation. The etiology remains unknown, but the consequences include a restrictive process that leads to congestive failure and diminished cardiac output. In addition, the desmoplastic reaction, associated with fusion of the visceral and parietal pericardial layers, has been implicated in failure of saphenous vein grafts. We report a case of constrictive pericarditis, after coronary bypass operation, with occlusion of an internal mammary graft. PMID- 10750801 TI - Diagnosis of a lung mass with a somatostatin analog scan. PMID- 10750802 TI - Veno-venous bypass to prevent myocardial ischemia during right heart bypass operation in PA, IVS, and RV dependent coronary circulation. AB - There is a risk of myocardial ischemia in patients with pulmonary atresia and intact ventricular septum associated with right ventricle dependent coronary circulation, especially during open heart operation. Cardiopulmonary bypass unloads the right ventricle, and thereby reduces the coronary perfusion pressure in an area that is wholly or partly dependent on the right ventricle. We present a veno-venous bypass technique to keep the right ventricle beating and ejecting to supply the oxygenated blood into the right ventricle dependent myocardium and consequently to prevent myocardial ischemia during right heart bypass operation. PMID- 10750803 TI - Arterial cannulation of the innominate artery. AB - Arterial cannulation of the innominate artery for cardiopulmonary bypass offers the advantage of central cannulation with standard cannulating techniques when the ascending and arch aorta are unavailable (eg, redo, aortic dissection, aneurysms). It avoids the difficulties associated with a second incision (axillary artery cannulation) and retrograde perfusion (femoral artery cannulation). PMID- 10750804 TI - Control of perigraft bleeding during ventricular assist device implantation. AB - This report describes a surgical technique devised to reduce perigraft bleeding in left ventricular assist devices, which has yielded excellent results in our institution. The technique may be applied during implantation of the Novacor N100, HeartMate 1000 VE, and Thoratec systems. PMID- 10750805 TI - Management of closed space infections associated with endoscopic vein harvest. AB - Wound complications are uncommon following endoscopic saphenous vein harvest. However, closed space infections within the endoscopic tunnel may occur and are difficult to manage. We describe the management of closed space infection in 3 patients and a method that allows drainage without unroofing the endoscopic tunnel. PMID- 10750806 TI - Sergei S. Brukhonenko: the development of the first heart-lung machine for total body perfusion. AB - Sergei S. Brukhonenko designed and constructed one of the earliest heart-lung machines. He was the first to experimentally perform a total body perfusion with the heart of the animal isolated from the circulation. His work paved the way to the first experimental operations on heart valves. Although Brukhoneko's pioneering contributions have not received the recognition they deserve, his work represents an important landmark in cardiac surgery. PMID- 10750807 TI - Surgical management of primary aortoesophageal fistula secondary to thoracic aneurysm. AB - Aortoesophageal fistula, secondary to thoracic aortic aneurysm, is an uncommon cause of gastrointestinal bleeding that is uniformly fatal without surgical intervention. These may be primary fistulas, in cases of thoracic aortic aneurysm without previous repair, or secondary fistulas occurring after surgical repair of thoracic aortic aneurysm. Surgical treatment has been successful in a small number of cases of primary aortoesophageal fistula, secondary to thoracic aortic aneurysm, but techniques used have varied. We report a successful repair of primary aortoesophageal fistula, secondary to descending thoracic aortic aneurysm, and review the evolution of management since the three previously reported successful repairs at our institution. PMID- 10750808 TI - Combined resection of T4 lung cancer with invasion of the descending thoracic aorta. PMID- 10750809 TI - Thoracotomy approach in reoperations. PMID- 10750810 TI - Successful treatment of massive pulmonary tumor embolism from renal cell carcinoma. PMID- 10750811 TI - Is it justified to disregard the Bohr effect during alpha-stat hypothermia? PMID- 10750812 TI - The hybrid procedure for myocardial revascularization: intermediate results. PMID- 10750813 TI - Extrapleural intercostal nerve block. PMID- 10750814 TI - Flexible input cannula in ventricular assist device. PMID- 10750815 TI - Outcome of coronary endarterectomy. PMID- 10750816 TI - Minimally invasive, but too many infections. PMID- 10750817 TI - Assessing renal function in "off-pump" versus "on-pump" coronary revascularization. PMID- 10750818 TI - Necessity of postoperative angiography after minimally invasive coronary bypass grafting. PMID- 10750819 TI - Perfectly imperfect. PMID- 10750820 TI - Stroke after repair of atrial septal defect. PMID- 10750821 TI - Less invasive correction of atrial septal defects with transthoracic cannulation. PMID- 10750822 TI - Expression of glycine and the glycine transporter glyt-1 in the developing rat retina. AB - Previous studies show that glycine transporter-1 (glyt-1) is a consistent membrane marker of adult retinal neurons that are likely to release glycine at their synaptic terminals (Pow, 1998; Vaney et al., 1998; Pow & Hendrickson, 1999). The current study investigated when glyt-1 immunoreactivity appeared in the postnatal rat retina, and whether all glycine-containing neurons also labelled for glyt-1. Ganglion cells, horizontal cells, and photoreceptors showed transient labelling. Many cells in the ganglion cell layer are immunoreactive for both glycine and glyt-1 at postnatal day (Pd) 1 but both are minimal by Pd5. Transient immunoreactivity for both glyt-1 and glycine was observed in presumptive horizontal cells between Pd5 and Pd10. At Pd1 many cells in the outer part of the retina which resembled immature photoreceptors were heavily labelled for glycine, but did not express glyt-1; these disappeared at older ages. These findings suggest diverse mechanisms and transient roles for glycine in the developing rat retina. In the adult rat retina, a subpopulation of amacrine cells are prominently immunoreactive for both glycine and glyt-1. These cells labelled for glycine at Pd1, but did not express significant levels of glyt-1 until Pd5. Processes from these amacrine cells did not reach the inner half of the inner plexiform layer until Pd10-14. Bipolar cells became glycine-IR between Pd10 and Pd14, but consistently lacked any glyt-1 immunoreactivity. This temporal pattern of labelling strongly indicates that bipolar cells label for glycine when gap junctions become functional between glycine/glyt-1 immunoreactive amacrine cells and cone bipolar cells. PMID- 10750823 TI - GABA(A) receptor binding and localization in the tiger salamander retina. AB - Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the retina and also appears to act as a trophic factor regulating photoreceptor development and regeneration. Although the tiger salamander is a major model system for the study of retinal circuitry and regeneration, our understanding of GABA receptors in this species is almost exclusively based on the results of physiological studies. Therefore, we have examined the pharmacological binding properties of GABA(A) receptors and their anatomical localization in the tiger salamander retina. Radioligand-binding studies showed that specific 3H-GABA binding to GABA(A) receptors was dominated by a single high-affinity binding site (Kd = 15.6+/-6.9 nM). Specific binding of 3H-GABA was almost completely eliminated by muscimol (Ki = 105+/-62 nM) and bicuculline (Ki = 14.3+/-2.2 microM); however, SR-95531 only displaced about 40% of specific 3H-GABA binding (Ki = 35.0+/-3.8 nM). These data indicate that there are at least two subtypes of GABA(A) receptors present in the salamander retina that can be distinguished by their antagonist binding properties: one sensitive to both bicuculline and SR 95531, and one sensitive to bicuculline but insensitive to SR-95531. Because localization of GABA receptors in the salamander retina by immunocytochemistry is problematic, GABA(A) receptors were localized by fluorescent ligand binding combined with immunocytochemical labeling for cell specific markers. Binding of fluorescently labeled muscimol to GABA(A) receptors was present in both plexiform layers and on photoreceptor cell bodies. GABA(A) receptors in the outer plexiform layer were localized to both photoreceptor terminals and horizontal cell processes. PMID- 10750824 TI - Modeling cat retinal beta-cell arrays. AB - There were three objectives to the work undertaken for this paper: (1) to provide a comprehensive characterization of the statistical properties of arrays of beta cell somata; (2) to develop a model that simulates cellular arrays with the same properties; and (3) to use this model to examine whether the array of beta-cells should be viewed as one array or as two arrays, one each for its OFF- and ON center cells. Beta-cells are morphological correlates of the electrophysiological X-cells and those beta-cells whose dendrites stratify within the outer and inner sublamina of the retina's inner plexiform layer correspond, respectively, to OFF- and ON-center X-cells. Arrays of peripheral beta-cell somata from two retinas were studied. A Delaunay triangulation and a Voronoi tessellation were generated for each array and measures derived from these constructs used to analyze the arrays' spatial organization. As others have shown previously with a less complete statistical characterization, we found that the arrays of OFF- and ON center beta-cells have similar spatial properties and are more regular than the array of all beta-cells. We developed a model to simulate cellular arrays with spatial properties like those of arrays of beta-cells. A good fit between model and real arrays was found when the model assumed an explicit spatial dependence between the placement of OFF- and ON-center cells. We propose therefore that a single array of beta-cells formed of both OFF- and ON-center cells is consistent with the data currently available for beta-cell somatic arrays. PMID- 10750826 TI - Burst and tonic firing in thalamic cells of unanesthetized, behaving monkeys. AB - Thalamic relay cells fire in two distinct modes, burst or tonic, and the operative mode is dictated by the inactivation state of low-threshold, voltage gated, transient (or T-type) Ca2+ channels. Tonic firing is seen when the T channels are inactivated via membrane depolarization, and burst firing is seen when the T channels are activated from a hyperpolarized state. These response modes have very different effects on the relay of information to the cortex. It had been thought that only tonic firing is seen in the awake, alert animal, but recent evidence from several species suggests that bursting may also occur. We have begun to explore this issue in macaque monkeys by recording from thalamic relay cells of unanesthetized, behaving animals. In the lateral geniculate nucleus, the thalamic relay for retinal information, we found that tonic mode dominated responses both during alert behavior as well as during sleep. We nonetheless found burst firing present during the vigilant, waking state. There was, however, considerably more burst mode firing during sleep than wakefulness. Surprisingly, we did not find the bursting during sleep to be rhythmic. We also recorded from relay cells of the somatosensory thalamus. Interestingly, not only did these somatosensory neurons exhibit much more burst mode activity than did geniculate cells, but bursting during sleep was highly rhythmic. It thus appears that the level and nature of relay cell bursting may not be constant across all thalamic nuclei. PMID- 10750825 TI - The NMDAR1 subunit of the N-methyl-D-aspartate receptor is localized at postsynaptic sites opposite both retinal and cortical terminals in the cat superior colliculus. AB - The N-methyl-D-aspartate receptor (NMDAR) is an ionotropic glutamate receptor that is important in neurotransmission as well as in processes of synaptic plasticity in the mammalian superior colliculus (SC). Despite the importance of this receptor in synaptic transmission, there is as yet no evidence that demonstrates directly the synaptic localization of the NMDAR receptor in SC. We have used electron-microscope (EM) immunocytochemistry to localize the NMDAR1 subunit of this receptor protein and its association with sensory afferents in the cat SC. Retinal synaptic terminals were identified by normal morphology and cortical synaptic terminals by degeneration after lesions of areas 17-18 of the visual cortex. At the light-microscope level, label was densest within the superficial gray and upper optic layers, but also present in all other layers. Label was contained within cell bodies, dendrites, and a few putative axons. At the EM level, antibody labeling was found along postsynaptic densifications and internalized within the cytoplasm of a variety of dendrites and some cell bodies. Postsynaptic profiles labeled by NMDAR1 included conventional dendrites and presynaptic dendrites which contained pleomorphic synaptic vesicles and are known to be GABAergic. Many of the labeled postsynaptic densifications of both of these profile types received synaptic inputs from retinal or cortical terminals. Virtually no NMDAR1 immunoreactivity was found on thin dendritic thorns or putative spines, even when these were postsynaptic to retinal or cortical terminals. In summary, these results show that the NMDAR1 subunit is postsynaptic to both retinal and cortical afferents, which are known to be glutamatergic, and are consistent with physiological evidence showing that stimulation of either pathway can activate the NMDA receptor. PMID- 10750827 TI - Dopaminergic and GABAergic amacrine cells are direct targets of melatonin: immunocytochemical study of mt1 melatonin receptor in guinea pig retina. AB - Distribution of the mt1 melatonin receptor in the guinea pig retina was immunocytochemically investigated using peptide-specific anti-mt1 receptor antibody. Western blots of the guinea pig retina showed a single band at approximately 37 kilodalton (kD) immunoreactive to the anti-mt1 antibody. The most intense immunoreactivity for the mt1 receptor was detected in the cell bodies of ganglion cells. Their dendrites and axons were also immunolabeled. Subpopulations of amacrine cells, the inner plexiform layer, and the outer plexiform layer also exhibited moderate to weak immunolabeling. The mt1-positive amacrine cells were located either at the vitreal border of the inner nuclear layer or displaced in the ganglion cell layer. Double immunolabeling using antibodies to the mt1 receptor and tyrosine hydroxylase revealed that the majority of dopaminergic amacrine cells showed mt1 immunoreactivity. Almost all the ICA type dopaminergic cells were mt1 positive while the 2CA type cells less frequently exhibited mt1 immunoreaction. By double immunolabeling for the mt1 receptor and GABA, more than 50% of the mt1-immunoreactive amacrine cells were shown to be GABAergic neurons. Approximately one-third of the GABAergic amacrine cells were immunolabeled for the mt1 receptor. The present results demonstrate expression of the mt1 receptor in diverse neuronal cell types in the guinea pig retina and provide the first evidence for the direct effect of melatonin on dopaminergic and GABAergic amacrine cells via the mt1 receptor. PMID- 10750828 TI - Differential contributions of magnocellular and parvocellular pathways to the contrast response of neurons in bush baby primary visual cortex (V1). AB - How neurons in the primary visual cortex (V1) of primates process parallel inputs from the magnocellular (M) and parvocellular (P) layers of the lateral geniculate nucleus (LGN) is not completely understood. To investigate whether signals from the two pathways are integrated in the cortex, we recorded contrast-response functions (CRFs) from 20 bush baby V1 neurons before, during, and after pharmacologically inactivating neural activity in either the contralateral LGN M or P layers. Inactivating the M layer reduced the responses of V1 neurons (n = 10) to all stimulus contrasts and significantly elevated (t = 8.15, P < 0.01) their average contrast threshold from 8.04 (+/- 4.1)% contrast to 22.46 (+/- 6.28)% contrast. M layer inactivation also significantly reduced (t = 4.06, P < 0.01) the average peak response amplitude. Inactivating the P layer did not elevate the average contrast threshold of V1 neurons (n = 10), but significantly reduced (t = 4.34, P < 0.01) their average peak response amplitude. These data demonstrate that input from the M pathway can account for the responses of V1 neurons to low stimulus contrasts and also contributes to responses to high stimulus contrasts. The P pathway appears to influence mainly the responses of V1 neurons to high stimulus contrasts. None of the cells in our sample, which included cells in all output layers of V1, appeared to receive input from only one pathway. These findings support the view that many V1 neurons integrate information about stimulus contrast carried by the LGN M and P pathways. PMID- 10750829 TI - The role of early retinal lateral inhibition: more than maximizing luminance information. AB - Lateral inhibition is one of the first and most important stages of visual processing. There are at least four theories related to information theory in the literature for the role of early retinal lateral inhibition. They are based on the spatial redundancy in natural images and the advantage of removing this redundancy from the visual code. Here, we contrast these theories with data from the retina's outer plexiform layer. The horizontal cells' lateral-inhibition extent displays a bell-shape behavior as function of background luminance, whereas all the theories show a fall as luminance increases. It is remarkable that different theories predict the same luminance behavior, explaining "half" of the biological data. We argue that the main reason is how these theories deal with photon-absorption noise. At dim light levels, for which this noise is relatively large, large receptive fields would increase the signal-to-noise ratio through averaging. Unfortunately, such an increase at low luminance levels may smooth out basic visual information of natural images. To explain the biological behavior, we describe an alternate hypothesis, which proposes that the role of early visual lateral inhibition is to deal with noise without missing relevant clues from the visual world, most prominently, the occlusion boundaries between objects. PMID- 10750830 TI - Expression of CB2 cannabinoid receptor mRNA in adult rat retina. AB - To date, two cannabinoid receptors, CB1 and CB2, have been cloned. The CB1 receptor has been found in a variety of tissues, particularly in the brain. CB2 receptor mRNA is mainly expressed in the immune system, though one group has found it in mouse cerebellum. Previous immunostaining studies in our lab demonstrated the presence of CB1 receptors in the retina though little evidence exists for the presence of CB2. The putative endogenous ligand for CB2 has been found in retina, however, suggesting that further study of CB2 in retina is warranted. Because glutamate is toxic to retinal ganglion cells in glaucoma and activation of CB2 receptors may be able to protect neurons from glutamate-induced death, we examined the expression of CB2 mRNA in adult rat retina in order to better understand possible neuroprotective mechanisms relevant to glaucoma. Using in situ hybridization, we demonstrated that CB2 cannabinoid receptor messenger RNA was clearly expressed in the adult rat retina, including the somas of retinal ganglion cells. Antisense cRNA probe detected strong signals in the retinal ganglion cell layer, the inner nuclear layer, and the inner segments of photoreceptor cells. Using reverse transcription polymerase chain reaction (RT PCR) in both rat and mouse tissue, we obtained an RT-PCR product with the same sequence as that reported for CB2 in the GenBank database, thus confirming the presence of CB2 mRNA in retina. The presence of CB2 in retina provides new evidence for the presence of CB2 in the central nervous system (CNS) and an excellent model for its study. PMID- 10750831 TI - The rhodopsin cycle is preserved in IRBP "knockout" mice despite abnormalities in retinal structure and function. AB - In the vertebrate retina, vision is initiated and maintained by the photolysis and regeneration, respectively, of light-sensitive pigments in the disk membranes of the photoreceptor outer segments. This cyclical process depends on an exchange of retinoids between the photoreceptors and the retinal pigment epithelium (RPE). There is a great deal of indirect evidence that the transport of retinoids between these cellular compartments is mediated by the interphotoreceptor retinoid-binding protein (IRBP), a large glycoprotein synthesized in the photoreceptors and extruded into the interphotoreceptor matrix (IPM) that fills the subretinal space. Nevertheless, a number of in vitro experiments have demonstrated that an intermembranous transfer of retinoids can occur through an aqueous medium independent of any retinoid-binding protein. This led to the suggestion that IRBP may play the more passive role of an extracellular buffer, serving to prevent the degradation and potentially cytotoxic effects of free retinoids when large amounts are released into the IPM. We have studied the structural and functional properties of transgenic mice in which homologous recombination was used to delete the IRBP gene. Light- and electron-microscopic examination of the retinas of "knockout" (IRBP-/-) mice revealed a significant loss of photoreceptor nuclei, and profound changes in the structure and organization of the receptor outer segments. Consistent with these observations, electroretinographic recordings showed a marked reduction in response amplitude for both rod- and cone-mediated potentials. However, despite the histological and electrophysiological changes, there was no evidence of gross abnormalities in the visual cycle. After bleaching a significant fraction of the available rhodopsin, electroretinogram amplitude and rhodopsin density gradually increased toward their pre-bleach levels, and the rates of recovery were even more rapid than those seen in wild-type (IRBP+/+) mice. PMID- 10750832 TI - Spatiotemporal adaptation through corticothalamic loops: a hypothesis. AB - The thalamus is the major gate to the cortex and its control over cortical responses is well established. Cortical feedback to the thalamus is, in turn, the anatomically dominant input to relay cells, yet its influence on thalamic processing has been difficult to interpret. For an understanding of complex sensory processing, detailed concepts of the corticothalamic interplay need yet to be established. Drawing on various physiological and anatomical data, we elaborate the novel hypothesis that the visual cortex controls the spatiotemporal structure of cortical receptive fields via feedback to the lateral geniculate nucleus. Furthermore, we present and analyze a model of corticogeniculate loops that implements this control, and exhibit its ability of object segmentation by statistical motion analysis in the visual field. PMID- 10750833 TI - High color-vision sensitivity in macaque and humans. AB - Psychophysical (behavioral) detection thresholds and color-discrimination thresholds were determined in a macaque using a two-alternative forced-choice procedure. On a white background, detection thresholds were determined for a white increment and three spectral increments: 618, 516, and 456 nm. Intermixed with detection threshold determinations, color-discrimination thresholds were determined by presenting the white increment, and one of the spectral increments, at 1.0 log units above their respective detection thresholds and dimming both until discrimination performance fell to threshold. The monkey could discriminate the color of the increments at detection threshold because the average color discrimination threshold was 0.98+/-0.14 log attenuation. Because the monkey was much more sensitive to the spectral increments than the white increment, we performed an unconventional experiment. We determined the monkey's detection threshold for the white increment alone, and with broadband color filters in the white light path without adjusting the light's intensity. Insertion of several color filters in the light path lowered detection thresholds of both the macaque and six human trichromats. We believe that this improvement in detection thresholds produced by simply inserting color filters in a white light path is a threshold manifestation of the Helmholtz-Kohlrausch effect and suggests that one of color vision's important evolutionary advantages may be improved detection sensitivity. PMID- 10750834 TI - Distribution of tubulin, kinesin, and dynein in light- and dark-adapted octopus retinas. AB - Cephalopod retinas exhibit several responses to light and dark adaptation, including rhabdom size changes, photopigment movements, and pigment granule migration. Light- and dark-directed rearrangements of microfilament and microtubule cytoskeletal transport pathways could drive these changes. Recently, we localized actin-binding proteins in light-/dark-adapted octopus rhabdoms and suggested that actin cytoskeletal rearrangements bring about the formation and degradation of rhabdomere microvilli subsets. To determine if the microtubule cytoskeleton and associated motor proteins control the other light/dark changes, we used immunoblotting and immunocytochemical procedures to map the distribution of tubulin, kinesin, and dynein in dorsal and ventral halves of light- and dark adapted octopus retinas. Immunoblots detected alpha- and beta-tubulin, dynein intermediate chain, and kinesin heavy chain in extracts of whole retinas. Epifluorescence and confocal microscopy showed that the tubulin proteins were distributed throughout the retina with more immunoreactivity in retinas exposed to light. Kinesin localization was heavy in the pigment layer of light- and dark adapted ventral retinas but was less prominent in the dorsal region. Dynein distribution also varied in dorsal and ventral retinas with more immunoreactivity in light- and dark-adapted ventral retinas and confocal microscopy emphasized the granular nature of this labeling. We suggest that light may regulate the distribution of microtubule cytoskeletal proteins in the octopus retina and that position, dorsal versus ventral, also influences the distribution of motor proteins. The microtubule cytoskeleton is most likely involved in pigment granule migration in the light and dark and with the movement of transport vesicles from the photoreceptor inner segments to the rhabdoms. PMID- 10750835 TI - Motion coherence affects human perception and pursuit similarly. AB - Pursuit and perception both require accurate information about the motion of objects. Recovering the motion of objects by integrating the motion of their components is a difficult visual task. Successful integration produces coherent global object motion, while a failure to integrate leaves the incoherent local motions of the components unlinked. We compared the ability of perception and pursuit to perform motion integration by measuring direction judgments and the concomitant eye-movement responses to line-figure parallelograms moving behind stationary rectangular apertures. The apertures were constructed such that only the line segments corresponding to the parallelogram's sides were visible; thus, recovering global motion required the integration of the local segment motion. We investigated several potential motion-integration rules by using stimuli with different object, vector-average, and line-segment terminator-motion directions. We used an oculometric decision rule to directly compare direction discrimination for pursuit and perception. For visible apertures, the percept was a coherent object, and both the pursuit and perceptual performance were close to the object motion prediction. For invisible apertures, the percept was incoherently moving segments, and both the pursuit and perceptual performance were close to the terminator-motion prediction. Furthermore, both psychometric and oculometric direction thresholds were much higher for invisible apertures than for visible apertures. We constructed a model in which both perception and pursuit are driven by a shared motion-processing stage, with perception having an additional input from an independent static-processing stage. Model simulations were consistent with our perceptual and oculomotor data. Based on these results, we propose the use of pursuit as an objective and continuous measure of perceptual coherence. Our results support the view that pursuit and perception share a common motion integration stage, perhaps within areas MT or MST. PMID- 10750836 TI - The fountain amacrine cells of the rabbit retina. AB - We have characterized a distinctive type of bistratified amacrine cell in the rabbit retina at both the single cell and population levels. These cells correspond to the "fountain" amacrine cells recently identified by MacNeil and Masland (1998). The fountain cells can be distinguished in superfused retinal wholemounts labeled with nuclear dyes, thus enabling them to be targeted for intracellular injection with Neurobiotin. This revealed that the primary dendrites ascend steeply to sublamina b of the inner plexiform layer, where they form an irregular arbor at the border of strata 4 and 5. These dendrites then give rise to multiple varicose processes that descend obliquely to sublamina a, where they form a more extensive arbor in stratum 1. The fountain amacrine cells show strong homologous tracer coupling when injected with Neurobiotin, and this has enabled us to map their density distribution across the retina and to examine the dendritic relationships between neighboring cells. The fountain amacrine cells range in density from 90 to 360 cells/mm2 and they account for 1.5% of the amacrine cells in the rabbit retina. The thick tapering dendrites in sublamina b form highly territorial arbors that tile the retina with minimal overlap, whereas the thin varicose processes intermingle in sublamina a. The fountain cells are immunopositive for y-aminobutyric acid and immunonegative for glycine. We further propose that these cells are homologous to the substance P-immunoreactive (SP-IR) amacrine cells in the cat retina and that they may account for a subset of the SP IR amacrine cells in the rabbit retina. PMID- 10750837 TI - conZA8 encodes an abundant protein targeted to the developing macronucleus in Euplotes crassus. AB - During macronuclear development in the ciliate Euplotes crassus, micronuclear derived chromosomes undergo a series of rearrangements that include polytenization, DNA splicing, chromosome fragmentation, and telomere addition and processing. Although cis-acting signals that may function in the regulation of these events have been characterized, the proteins that mediate these events have not yet been identified. To identify development-specific factors that may be involved in DNA rearrangement, we previously isolated clones of a number of genes that are expressed only during early macronuclear development. Here, we report the genomic and cDNA sequences of one of these genes, conZA8. The analysis indicates that the conZA8 gene encodes a novel, 468-amino acid, proline-rich protein. Antibodies were raised against both a recombinant form of the conZA8 protein and an internal peptide. Immunoblotting and immunofluorescence analyses indicated that the conZA8 protein is highly abundant, expressed only during the polytene chromosome stage of macronuclear development, and localized to the developing macronucleus. Possible functions of the conZA8 protein are discussed. PMID- 10750838 TI - Analysis of the 5.8S rRNA gene and the internal transcribed spacers in Naegleria spp. and in N. fowleri. AB - Internal transcribed spacers (ITS) and the 5.8S ribosomal gene of 21 Naegleria fowleri strains and eight other species including Naegleria gruberi were sequenced. The results showed that this region can help differentiate between and within species. The phylogeny of Naegleria spp. deduced from the ITS and the 5.8S gene produced four major lineages, fowleri-lovaniensis, galeacystis-italica clarki-gruberi-australiensis, andersoni-jamiesoni, and pussardi, that fit perfectly with those inferred from the 18S rRNA gene analysis. The N. gruberi isolate, NG260, was closely related to Naegleria pussardi. The other N. gruberi isolates branched together with Naegleria australiensis in another lineage. The ITS and 5.8S results for N. fowleri were congruent with those previously deduced by RAPD analysis. The phylogenetic analysis inferred from ITS and RAPD data revealed two major groups. The French Cattenom and Chooz and South Pacific strains constituted the first group. The second group encompassed the strains corresponding to the Euro-American and Widespread RAPD variants and shared the same substitution in the 5.8S gene. In addition, it was possible to define species specific primers in ITS regions to rapidly identify N. fowleri. PMID- 10750839 TI - 1-O-alkyl-2-acetyl-sn-glycero-3-phosphate:phosphohydrolase activity in Tetrahymena pyriformis. AB - Within the frame of the de novo formation of Platelet-Activating Factor in Tetrahymena, the occurrence as well as the properties of a lipid phosphate phosphohydrolase activity catalyzing the dephosphorylation of 1-O-alkyl-2-acetyl sn-glycero-3-phosphate was investigated. The activity was distributed in all the membrane fractions of the cell and in the cytosol. It showed preference for acyl acetyl-sn-glycero-phosphate as well, and at a much lower level, for dipalmitoyl glycero-phosphate. Mg2+ and Ca2+ caused a dose-dependent inhibition, while F-, EDTA and EGTA had no effect. The enzymic activity was linear for at least up to 60 min incubation time and up to 150 microg protein. Microsomal activity exhibited two optimal pH areas, around 7.0 and 9.0, while mitochondrial activity showed one peak, at pH 7.0. Acyl-GP, acyl-acetyl-GP and alkyl-GP could replace alkyl-acetyl-GP in significant rates, while dipalmitoyl-GP, beta-GP, fructose-6 GP, p-nitrophenylphosphate, creatine phosphate or ATP had no effect. Side phospholipase A2 and C activities were also detected. Taking into account the presence of PAF and alkylacetylglycerol in the protozoan as well as the presence of a dithiothrcitol-insensitive CDP-choline:cholinephosphotransferase activity that converts alkylacetylglycerol to PAF, we suggest that the present phosphohydrolase activity may be involved in the de novo production of PAF within Tetrahymena. PMID- 10750840 TI - Centrin protein and genes in Trichomonas vaginalis and close relatives. AB - Anti-centrin monoclonal antibodies 20H5 and 11B2 produced against Clamydomononas centrin decorated the group of basal bodies as well as very closely attached structures in all trichomonads studied and in the devescovinids Foaina and Devescovina. Moreover, these antibodies decorated the undulating membrane in Trichomonas vaginalis, Trichomitus batrachorum, and Tritrichomonas foetus, and the cresta in Foaina. Centrin was not demonstrated in the dividing spindle and paradesmosis. Immunogold labeling, both in pre- and post-embedding, confirmed that centrin is associated with the basal body cylinder and is a component of the nine anchoring arms between the terminal plate of flagellar bases and the plasma membrane. Centrin is also associated with the hook-shaped fibers attached to basal bodies (F1, F3), the X-fiber, and along sigmoid fibers (F2) at the pelta axostyle junction, which is the microtubule organizing center for pelta-axostyle microtubules. There was no labeling on the striated costa and parabasal fibers nor on microtubular pelta-axostyle, but the fibrous structure inside the undulating membrane was labeled in T. vaginalis. Two proteins of 22-20 kDa corresponding to the centrin molecular mass were recognized by immunoblotting using these antibodies in the three trichomonad species examined. By screening a T. vaginalis cDNA library with 20H5 antibody, two genes encoding identical protein sequences were found. The sequence comprises the 4 typical EF-hand Ca++ binding domains present in every known centrin. Trichomonad centrin is closer to the green algal cluster (70% identity) than to the yeast Cdc31 cluster (55% identity) or the Alveolata cluster (46% identity). PMID- 10750841 TI - Macronuclear development in conjugants of Tetrahymena thermophila, which were artificially separated at meiotic prophase. AB - Conjugant pairs of Tetrahymena thermophila were mechanically separated by vigorous pipetting at the early stages of meiotic prophase. The complete sequence of conjugational nuclear events including the appearance of pronuclei, development of the new macronuclei (postzygotic development), and resorption of the old macronuclei was observed in the separated cells, without pronuclear exchange. The pronuclei in the separated cells were recognised by the presence of components of the extranuclear cytoskeleton, which were labelled with anti tubulin and anti-fenestrin antibodies in the same way as in undisturbed conjugants. The apical region of the separated conjugants (the post-junction area), corresponding to the junction area of conjugants was labelled with anti fenestrin antibody and maintained the properties required for the nuclear development. The results of the genetic study were consistent with a hypothesis that cytogamy (pronuclear fusion) was induced in the separated conjugants. Therefore, the lasting cell contact is not necessary for the successful completion of conjugational nuclear events. PMID- 10750842 TI - Abundance of terrestrial gymnamoebae at a northeastern U. S. site: a four-year study, including the El Nino winter of 1997-1998. AB - The abundance, sizes, and when appropriate, diversity of gymnamoebae were documented at approximately monthly intervals for four years (1995-1998) at a grassy, terrestrial site slightly upslope from a freshwater pond. Soil samples were analyzed for viable gymnamoebae using a standard laboratory culturing protocol. The mean density of gymnamoebae based on the total data set was ca. 1,600/g (s.e.+/-190). Minimum densities of gymnamoebae (156/g) occurred in January 1995, and a maximum for the sampling period (5,838/g) occurred in July 1997, when a rainy period followed an extended period of drought. Among the environmental variables monitored (precipitation, soil moisture, organic content, and temperature) only precipitation correlated significantly with abundance of gymnamoebae (r = 0.34, p = 0.02). During the mild, moist El Nino winter of 1997 1998, a larger than usual number of gymnamoebae was recorded at the site (approximately 3,800/g) compared to a mean density of approximately 900/g for comparable periods in preceding years. The mean sizes were also larger. Since gymnamoebae are increasingly recognized as major members of soil microbial communities enhancing soil fertility through nutrient mineralization, it is important to document environmental variables that influence their abundance and activity in terrestrial ecosystems. PMID- 10750843 TI - The behavior of the doublet of Oxytricha bifaria (Ciliata, Hypotrichida): a contribution to the understanding of this enigmatic form. AB - The tracks of normal organisms of Oxytricha bifaria and of stage IA, IB, II, III, IV and V doublets were studied to test the hypothesis that the doublet might function as a dispersal form. Stage IA, the only stage to swim, swims straight with only rare interruptions; its rate of mobility (Rmo = 443 micro/s) is roughly twice that of singlets (Rmo = 218 micro/s). Stage IA doublets swim in three dimensional movement which enables them to be carried away by water currents. The other stages seem to represent passage back towards the normal singlet form. The ethological evidence reported here together with other results already published supports the working hypothesis that the doublet of O. bifaria is a dispersal form suggests that the doublet might well represent a special fourth differentiation state of this species in addition to pairs, giants, and cysts. PMID- 10750844 TI - Rotifers ingest oocysts of Cryptosporidium parvum. AB - Six genera of rotifers including Philodina, Monostyla, Epiphanes, Euchlanis, Brachionus, and Asplanchna were exposed to oocysts of Cryptosporidium parvum cleaned of fecal debris. Unstained oocysts and those stained with fluorescein conjugated monoclonal antibody were added to suspensions of viable rotifers and were examined by phase-contrast, differential interference contrast, and fluorescence microscopy. Rotifers of all six genera were observed ingesting oocysts. A maximum of 25 oocysts was observed in the stomachs of Eauchlanis and Brachionus. Euchlanis and Epiphanes were observed excreting boluses containing up to eight oocysts. It was not determined whether rotifers digested or otherwise rendered oocysts nonviable. PMID- 10750845 TI - Use of micromanipulation and "feeder layers" to clone the oyster pathogen Perkinsus marinus. AB - Genetic and biochemical characterization of microbes often requires the use of clonal cultures. A method to clone the oyster parasite Perkinsus marinus is described. Individual cells are isolated via micromanipulation and maintained above an actively proliferating "feeder layer" of P. marinus on a 0.45-microm membrane. Extracellular products released from the proliferating feeder layer can diffuse across the membrane and bathe the isolated cell, stimulating it to proliferate. The method is relatively simple and should be applicable to most protists that can be cultured in the laboratory. PMID- 10750846 TI - Long branch attraction effects and the status of "basal eukaryotes": phylogeny and structural analysis of the ribosomal RNA gene cluster of the free-living diplomonad Trepomonas agilis. AB - The three taxa emerging at the base of the eukaryotic ribosomal RNA phylogenetic tree (Diplomonadida, Microspora, and Parabasalia) include a wide array of parasitic species. and some free-living organisms that appear to be derived from a parasitic ancestry. The basal position of these taxa, which lack mitochondria, has recently been questioned. I sequenced most of the ribosomal RNA gene cluster of the free-living diplomonad Trepomonas agilis and a secondary structure model was reconstructed for the SSU rRNA. I conducted a RASA matrix analysis to identify, independently from tree reconstruction, putative long branch attraction effects in the data matrix. The results show that each of the basal clades and the euglenozoan clade act, indeed, as long branches and may have been engaged in a process of accelerated rate of evolution. A nucleotide signature analysis was conducted in the conserved regions for positions defining the three great domains of life (Eubacteria, Archea, and Eukaryota). For the three basal taxa, this analysis showed the presence of a significant number of different non-eukaryotic nucleotides. A precise study of the nature and location of these nucleotides led to conclusions supporting the results of the RASA analysis. Altogether, these findings suggest that the basal placement of these taxa in the SSU ribosomal RNA phylogenetic tree is artifactual, and flawed by long branch attraction effects. PMID- 10750847 TI - Rumen ciliated protozoan fauna of the yak (Bos grunniens) in China with the description of Entodinium monuo n. sp. AB - Rumen ciliate species composition was surveyed in domestic yaks kept in Tibet, Sichuan, and Inner Mongolia, China. Twelve genera including 36 species with 18 formae were identified. The species compositions were slightly different among the three areas: yaks in Tibet had the simplest fauna, in contrast, the fauna of yaks in Inner Mongolia were the most abundant and similar to those found in the cattle kept in the same area. This suggests that the rumen ciliate composition of yaks is affected by that of cattle kept together or in proximity. A new species belonging to the genus Entodinium, Entodinium monuo n. sp., was recognized from the yaks in all areas. This new species was common in the yaks but was not detected in the cattle fed near yaks in Inner Mongolia. There was a similar generic ciliate composition in yaks kept in respective areas. Entodinium was the most predominate ciliate (51.9-61.0%) with total ciliate densities estimated as 10(5)/ml per yak. PMID- 10750848 TI - Textile dye allergic contact dermatitis prevalence. AB - This article summarizes textile dye prevalence studies and makes recommendations for advancing knowledge about textile-dye sensitization. Prevalence data is provided by study and by dye. Dermatology teams are encouraged to conduct textile dye prevalence studies in countries other than Italy, include dyes for which the least prevalence data has been collected, to standardize method of application and reading, and to verify purity and identity of dyes used for patch testing. Testing with pure dyes and other chemicals in dye formulations should provide insights in choosing dye systems that will decrease sensitization. PMID- 10750849 TI - Airborne contact dermatitis from methylchloroisothiazolinone in wall paint. Abolition of symptoms by chemical allergen inactivation. AB - Preservatives such as isothiazolinones in paints have been reported to cause airborne contact dermatitis. The patients whom we report experienced acute dermatitis on air-exposed skin and respiratory symptoms after staying in recently painted rooms. Kathon (methylchloroisothiazolinone/methylisothiazolinone) added as preservative to the wall paint was identified as causative agent. In one individual symptoms rapidly disappeared after treatment of the painted walls with inorganic sulfur salt, which leads to inactivation of the allergenic properties of methylchloroisothiazolinone/methylisothiazolinone. We describe the patients, the clinical course and review the literature pertinent to such cases. In addition we report on the chemical analyses of the decorating paints used, and on experiments on emission and air concentration of methylchloroisothiazolinone/methylisothiazolinone from a painted surface before and after inactivation by sodium bisulfite. PMID- 10750851 TI - An open assay model to induce subclincal non-erythematous irritation. AB - To avoid the acute irritation and dryness that a single occlusive surfactant application (24-h patch test) may cause, and to approximate clinical use, an open application model was chosen to define subclinical non-erythematous irritation in the stratum corneum. This human test combined a supervised washing at the laboratory with at-home use of the test products by the subjects. Effects of washing with the surfactants on the dorsal hand and volar forearm were compared. The results demonstrated that this situational use model permits the exploration of subclinical surfactant-induced irritation. The forearm appeared to be more discriminative as compared to the dorsal hand. Squamometry proved to be a sensitive, complementary assessment method for detecting surfactant-induced subclinical skin surface alterations and for differentiating surfactant effects in this open assay. PMID- 10750850 TI - Ascertainment of hand dermatitis using a symptom-based questionnaire; applicability in an industrial population. AB - In this study, the applicability of a symptom-based questionnaire on hand dermatitis was assessed in a population of rubber workers. The questionnaire was previously validated in a study among nurses. 224 subjects employed in 9 different companies completed a questionnaire on skin complaints. Subsequently, 202 workers attended the physical examination of the skin by a dermatologist. The ascertainment of skin complaints according to the questionnaire was compared to the medical evaluation. The 2 different diagnostic tools used for assessing dermatitis resulted in dissimilar estimates of the prevalence of active hand dermatitis, ranging from 6.9% to 38.1% of all workers. Using the medical evaluation as 'gold standard' we observed a moderate sensitivity and specificity (respectively 71.4%; 95% CI: 47.7-95.1 and 76.1%; 95% CI: 70.0-82.2), a low positive predictive value (18.2%; 95% CI: 8.0-28.4) and a high negative predictive value (97.3%; 95% CI: 94.7-99.9) for the classification based on the self-administered questionnaire. When evaluated against 'first symptoms of dermatitis' the sensitivity decreased, while the specificity remained almost the same. The deviant findings between the present and the original validation study of the same questionnaire among nurses hamper its applicability in populations with different occupations. Therefore, if questionnaires are to be used, validity studies have to be carried out to evaluate differences in perception of skin diseases between different (occupational) populations. PMID- 10750852 TI - Severe dermographism after topical therapy with diphenylcyclopropenone for alopecia universalis. AB - We describe here a 19-year-old Japanese man with an 11-year history of alopecia universalis, who, after the 1st application of a 0.003% diphenylcyclopropenone (DPCP) solution to the whole scalp, developed acute contact dermatitis at the test site, together with widespread severe dermographism. Every 3 weeks, persistence of the severe urticarial reaction and efficacy of treatment were monitored by constant pressure stimuli in a series of pressure tests, and subsequently evaluated by laser Doppler flowmetry (LDF). Although, on pressure tests, the urticarial response was found to significantly improve after starting treatment, erythematous responses continued to appear for nearly 3 months. The persistent course of these side-effects in our patient strongly suggests that precautions must currently be taken in the therapeutic use of potent sensitizers such as DPCP. PMID- 10750853 TI - The spectrum of inflammatory cell response to dimethyl sulfoxide. AB - Dimethyl sulfoxide (DMSO), depending upon the concentration and mode of application to the skin, can induce either a non-immunological immediate contact urticaria or an irritant reaction. The dermal cellular infiltrate after open application of varying concentrations of DMSO has been studied in an experimental guinea pig model. The composition of the dermal cellular infiltrate showed a spectrum dependent on the concentration and number of applications of DMSO. The immediate reaction infiltrate 3 h after application of 100% DMSO consisted of 50% granulocytes, basophils being predominant. On the other hand, 12% DMSO applied 3 x daily for 3 days (cumulative insult) caused histologically a cellular reaction in which 80% of the infiltrate consisted of mononuclear cells. The present findings are compared to the microscopic findings in 3 other cutaneous reactions previously studied in this animal model, namely, the Type I immediate hypersensitivity reaction, the Type IV delayed hypersensitivity reaction, and the irritant reaction. Differing cellular infiltrate patterns are discernible at the same time points. The study illustrates the spectrum of inflammatory reactions seen in the skin and provides background information for future clinical studies, for instance, on the role of the basophil granulocyte in immediate contact reactions. PMID- 10750854 TI - Rash related to use of scented products. A questionnaire study in the Danish population. Is the problem increasing? AB - Fragrances are used in many types of cosmetic and household products, which are an important part of everyday life in modern society. The aim of the current investigation was to describe the frequency of self-reported rash due to scented products in a random sample of the adult Danish population. Further, it was determined whether risk of self-reported 1st-time rash from scented products had increased during the past 15 years compared to the preceding period. The sample consisted of 1537 persons, 801 female and 736 male, above the age of 15 years. The participants were interviewed person-to-person to obtain a general health profile, and in this connection, questions were asked concerning rash related to the use of scented products. 28.6% (440/1537) had on some occasion experienced rash from scented products, 10.6% had experienced rash within the year prior to interview. A multivariate analysis showed that women had a significantly increased risk of reporting rash from scented products compared to men (odds ratio: 1.56, p<0.0001). Furthermore, it was shown that individuals below the age of 40 years had a significantly increased risk of reporting rash from scented products compared to older age groups. The risk of reporting 1st-time rash occurring after 1978 was significantly increased (odds ratio: 2.34, p<0.0001), as compared to the preceding period. This may be taken as indicative of an increasing problem with scented products, involving potentially severe public health implications. PMID- 10750855 TI - Claim substantiation and efficiency of hydrating body lotions and protective creams. AB - In the present work a practical claim substantiation study is shown by the example of 5 commercially available body lotions. Their efficacy with respect to effects on transepidermal water loss (TEWL) and stratum corneum (SC) hydration of ageing skin has been examined. Results were obtained after single and repeated application (14 days, 2 x a day). The best performing product was then selected and further tested for its potential effects on sodium lauryl sulfate (SLS) damaged skin. This was done in a younger population and the recovery of the impaired barrier function was followed by TEWL measurements. The selected body lotion had a high efficacy, improving both the TEWL and SC hydration of ageing skin by more than 30%. When applied to SLS-damaged skin, the product was able to improve skin barrier repair in comparison with physiological barrier repair. The results of this study show that a combination of non-invasive objective measurements can be used to substantiate product claims. Claims can be made with respect to protective and preventive properties of products, but also as to effectiveness of topical skin treatment in the case of abnormal barrier function or barrier restoration. PMID- 10750856 TI - Prurigo-nodularis-like lesion in parthenium dermatitis. PMID- 10750857 TI - Allergic contact dermatitis from carrot. PMID- 10750858 TI - The azo dye solvent yellow 3 produces depigmentation. PMID- 10750859 TI - Occupational allergic contact dermatitis from Compositae in agricultural work. PMID- 10750860 TI - Allergic contact dermatitis from natural rubber latex. PMID- 10750861 TI - Alternative formulation for patients with contact reactions to topical 2% and 5% minoxidil vehicle ingredients. PMID- 10750862 TI - Ring dermatitis as a clinical presentation of fragrance sensitization. PMID- 10750863 TI - Double active sensitization in an atopic patient. PMID- 10750864 TI - Allergic contact dermatitis from alkylammonium amidobenzoate (Osmaron B). PMID- 10750865 TI - Allergic contact dermatitis from carteolol and timolol in eyedrops. PMID- 10750866 TI - Allergic contact dermatitis from glyceryl trinitrate ointment. PMID- 10750867 TI - Allergic contact dermatitis from camomile used in phytotherapy. PMID- 10750868 TI - Vesicular palmar eczema from the neoprene tongue of an ankle support. PMID- 10750869 TI - Fragrance allergen substitution. PMID- 10750870 TI - HHS awards $527 million for HIV/AIDS care in high-incidence areas. PMID- 10750871 TI - Overcoming the barriers to change: screening for colorectal cancer. PMID- 10750872 TI - Avoiding drug interactions. PMID- 10750873 TI - Rapid 'dipstick' assays for the detection of malaria. PMID- 10750874 TI - The true incidence of GERD-induced globus. PMID- 10750875 TI - Management of gallstones and their complications. AB - The accurate differentiation of gallstone-induced biliary colic from other abdominal disease processes is the most crucial step in the successful management of gallstone disease. Despite the availability of many imaging techniques to demonstrate the presence of gallstones, clinical judgment ultimately determines the association of symptoms with cholelithiasis and its complications. Adult patients with silent or incidental gallstones should be observed and managed expectantly, with few exceptions. In symptomatic patients, the intervention varies with the type of gallstone-induced complication. In this article, we review the salient clinical features, diagnostic tests and therapeutic options employed in the management of gallstones and their complications. PMID- 10750876 TI - Acute and post-traumatic stress disorder after spontaneous abortion. AB - When a spontaneous abortion is followed by complicated bereavement, the primary care physician may not consider the diagnosis of acute stress disorder or post traumatic stress disorder. The major difference between these two conditions is that, in acute stress disorder, symptoms such as dissociation, reliving the trauma, avoiding stimuli associated with the trauma and increased arousal are present for at least two days but not longer than four weeks. When the symptoms persist beyond four weeks, the patient may have post-traumatic stress disorder. The symptoms of distress response after spontaneous abortion include psychologic, physical, cognitive and behavioral effects; however, patients with distress response after spontaneous abortion often do not meet the criteria for acute or post-traumatic stress disorder. After spontaneous abortion, as many as 10 percent of women may have acute stress disorder and up to 1 percent may have post traumatic stress disorder. Critical incident stress debriefing, which may be administered by trained family physicians or mental health practitioners, may help patients who are having a stress disorder after a spontaneous abortion. PMID- 10750877 TI - Treatment of common cutaneous herpes simplex virus infections. AB - Herpes simplex virus infection is increasingly common in the United States. New antiviral medications have expanded treatment options for the two most common cutaneous manifestations, orolabial and genital herpes. Acyclovir therapy remains an effective and often less expensive option. Famciclovir and valacyclovir offer improved oral bioavailability and convenient oral dosing schedules but are more expensive than acyclovir. Patients who have six or more recurrences of genital herpes per year can be treated with one of the following regimens: acyclovir, 400 mg twice daily; valacyclovir, 1 g daily; or famciclovir, 250 mg twice daily. These regimens are effective in suppressing 70 to 80 percent of symptomatic recurrences. Episodic treatment of recurrent genital herpes is of questionable benefit, but it may be helpful in appropriately selected patients. There is little evidence indicating benefit from treatment of recurrent orolabial herpes, which tends to be mild and infrequent. PMID- 10750878 TI - Alcoholism in the elderly. AB - Alcohol abuse and alcoholism are common but underrecognized problems among older adults. One third of older alcoholic persons develop a problem with alcohol in later life, while the other two thirds grow older with the medical and psychosocial sequelae of early-onset alcoholism. The common definitions of alcohol abuse and dependence may not apply as readily to older persons who have retired or have few social contacts. Screening instruments can be used by family physicians to identify older patients who have problems related to alcohol. The effects of alcohol may be increased in elderly patients because of pharmacologic changes associated with aging. Interactions between alcohol and drugs, prescription and over-the-counter, may also be more serious in elderly persons. Physiologic changes related to aging can alter the presentation of medical complications of alcoholism. Management of alcohol withdrawal in elderly persons should be closely supervised by a health care professional. Alcohol treatment programs with an elder-specific focus may improve outcomes in some patients. PMID- 10750879 TI - Management of bacterial endocarditis. AB - Most cases of bacterial endocarditis involve infection with viridans streptococci, enterococci, coagulase-positive staphylococci or coagulase-negative staphylococci. The choice of antibiotic therapy for bacterial endocarditis is determined by the identity and antibiotic susceptibility of the infecting organism, the type of cardiac valve involved (native or prosthetic) and characteristics of the patient, such as drug allergies. Antibiotic therapies discussed in this report are based on recommendations of the American Heart Association. Treatment with aqueous penicillin or ceftriaxone is effective for most infections caused by streptococci. A combination of penicillin or ampicillin with gentamicin is appropriate for endocarditis caused by enterococci that are not highly resistant to penicillin. Vancomycin should be substituted for penicillin when high-level resistance is present. Resistance of enterococci to multiple antibiotics including vancomycin is becoming an increasing problem. Native valve infection by methicillin-susceptible staphylococci is treated with nafcillin, oxacillin or cefazolin. The addition of gentamicin for the first three to five days may accelerate clearing of bacteremia. Infection of a prosthetic valve by a staphylococcal organism should be treated with three antibiotics: oral rifampin and gentamicin and either nafcillin, oxacillin, cefazolin or vancomycin, depending on susceptibility to methicillin. Vancomycin is substituted for penicillin in patients with a history of immediate-type hypersensitivity to penicillin. PMID- 10750880 TI - Clinically significant drug interactions. AB - A large number of drugs are introduced every year, and new interactions between medications are increasingly reported. Consequently, it is no longer practical for physicians to rely on memory alone to avoid potential drug interactions. Multiple drug regimens carry the risk of adverse interactions. Precipitant drugs modify the object drug's absorption, distribution, metabolism, excretion or actual clinical effect. Nonsteroidal anti-inflammatory drugs, antibiotics and, in particular, rifampin are common precipitant drugs prescribed in primary care practice. Drugs with a narrow therapeutic range or low therapeutic index are more likely to be the objects for serious drug interactions. Object drugs in common use include warfarin, fluoroquinolones, antiepileptic drugs, oral contraceptives, cisapride and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. Many other drugs, act as precipitants or objects, and a number of drugs act as both. Regularly updated manuals of drug interactions and CD-ROM-formatted programs are useful office references. PMID- 10750881 TI - Update on colorectal cancer. AB - An estimated 129,400 new cases of colorectal cancer occurred in the United States during 1999. The lifetime risk of developing this cancer is 2.5 to 5 percent in the general population but two to three times higher in persons who have a first degree relative with colon cancer or an adenomatous polyp. Between 70 and 90 percent of colorectal cancers arise from adenomatous polyps, whereas only 10 to 30 percent arise from sessile adenomas. Tumors or polyps that develop proximal to the splenic flexure carry a poorer prognosis than those that arise more distally, in part because of delayed diagnosis secondary to later development of symptoms. The Dukes system is the classic staging method for colorectal cancer; the TNM staging system is more detailed and therefore more useful for surgical purposes. Although screening guidelines vary, most agree that colorectal cancer screening should begin at 50 years of age in patients without a personal or family history of colorectal cancer. PMID- 10750882 TI - Diagnosis and management of acute low back pain. AB - Acute low back pain is commonly encountered in primary care practice but the specific cause often cannot be identified. This ailment has a benign course in 90 percent of patients. Recurrences and functional limitations can be minimized with appropriate conservative management, including medications, physical therapy modalities, exercise and patient education. Radiographs and laboratory tests are generally unnecessary, except in the few patients in whom a serious cause is suspected based on a comprehensive history and physical examination. Serious causes that need to be considered include infection, malignancy, rheumatologic diseases and neurologic disorders. Patients with suspected cauda equina lesions should undergo immediate surgical investigation. Surgical evaluation is also indicated in patients with worsening neurologic deficits or intractable pain that is resistant to conservative treatment. The current recommendation is two or three days of bed rest for patients with acute radiculopathy. The treatment plan should be reassessed in patients who do not return to normal activity within four to six weeks. PMID- 10750883 TI - Osteoarthritis: current concepts in diagnosis and management. AB - Most patients with osteoarthritis seek medical attention because of pain. The safest initial approach is to use a simple oral analgesic such as acetaminophen (perhaps in conjunction with topical therapy). If pain relief is inadequate, oral nonsteroidal anti-inflammatory drugs or intra-articular injections of hyaluronic acid-like products should be considered. Intra-articular corticosteroid injections may provide short-term pain relief in disease flares. Alleviation of pain does not alter the underlying disease. Attention must also be given to nonpharmacologic measures such as patient education, weight loss and exercise. Relief of pain and restoration of function can be achieved in some patients with early osteoarthritis, particularly if an integrated approach is used. Patients with advanced disease may eventually require surgery, which generally provides excellent results. PMID- 10750884 TI - ACC/AHA guidelines on the management of acute myocardial infarction. American College of Cardiology and the American Heart Association. PMID- 10750886 TI - Change in the ratio of free-to-total prostate-specific antigen during progression of advanced prostate cancer. AB - BACKGROUND: The ratio of free-to-total prostate-specific antigen (PSA) is different in benign prostatic hyperplasia and in the early stage of prostate cancer. The present study was undertaken to examine the ratio of free-to-total PSA in the advanced stage of this cancer and its subsequent change during course of the disease. METHODS: Free and total PSA were measured in sera collected from the following patients with benign and cancerous prostatic diseases: 47 cases of benign prostatic hypertrophy, nine in TIC with less than 10 ng/mL of total PSA, 11 in stage C, 16 in D2, 22 in remission under endocrine therapy, and 12 in relapse. In addition, PSA was measured sequentially in four other patients who were also in relapse. RESULTS: The ratio of free-to-total PSA was similar in early and advanced stages of untreated prostate cancer and was lower than that in benign prostatic hyperplasia. The ratio increased to the level of benign prostatic hyperplasia during remission from stages C and D2 under endocrine therapy. There was no correlation with the intervals from the start of the therapy to examination. Following relapse, the ratio came down gradually to the level obtained in untreated prostate cancer. CONCLUSION: The ratio of free-to total PSA was similar in all stages of untreated prostate cancer. Response and relapse to endocrine therapy were associated with increase and decrease in ratio, respectively. PMID- 10750885 TI - Collection of peripheral blood stem cells with granulocyte-colony-stimulating factor alone in testicular cancer patients. AB - BACKGROUND: High-dose chemotherapy with the transplantation of peripheral blood stem cells (PBSC) has been performed for the treatment of advanced testicular cancer patients. Recently, it has been reported that, in healthy donors, a large quantity of stem cells can be transferred to peripheral blood using granulocyte colony-stimulating factor (G-CSF) alone. Therefore, it was decided to try to harvest PBSC from three patients having testicular cancers with G-CSF alone. METHODS: The three patients with testicular cancer were 26, 56 and 62-years-old. They had undergone five, two and three cycles of chemotherapy, respectively, but no radiation therapy. Granulocyte colony-stimulating factor was subcutaneously injected (250 microg) into each patient twice per day for 6 days. Peripheral blood stem cells were harvested for 3 days (days 4-6) and mononuclear cells (MNC), CD34-positive cells and colony-forming units of granulocyte-macrophage (CFU-GM) in PBSC collected by apheresis were measured. RESULTS: Apheresis showed that the total MNC count was 20.2 x 10(8)/kg (range, 10.6-25.9 x 10(8)/kg), the CD34-positive cell count was 0.98 x 10(6)/kg (range, 0.75-1.4 x 10(6)/kg) and the total CFU-GM count was 1.36 x 10(5)/kg (range, 0.25-3.0 x 10(5)/kg). CONCLUSION: After mobilization of peripheral blood stem cells with G-CSF alone, sufficient amounts of MNC were obtained from testicular cancer patients who had undergone chemotherapy several times. However, sufficient amounts of CD34-positive cells and CFU-GM could not be obtained. These results suggested that the G-CSF dose was not adequate for harvesting sufficient amounts of CD34-positive cells and CFU-GM. PMID- 10750887 TI - Ejaculated spermatozoa in patients with non-mosaic Klinefelter's syndrome. AB - BACKGROUND: Non-mosaic Klinefelter patients are generally azoospermic and there is no therapy to improve the spermatogenesis. Some patients have a few spermatozoa in their ejaculates, which can be used for intracytoplasmic sperm injection (ICSI), but only a few cases resulting in a successful birth have been reported. METHODS: Out of 52 non-mosaic 47,XXY Klinefelter patients, four cases having spermatozoa in their ejaculates were retrospectively studied. RESULTS: Intracytoplasmic sperm injection was performed in three cases using their ejaculates and resulted in one chemical abortion and one death in utero (8 weeks). Using testicular sperm, one patient had a healthy baby with a normal karyotype. CONCLUSION: Some non-mosaic Klinefelter patients have ejaculated sperm that can result in a successful birth following ICSI. However, the birth rate is very low compared with the fertilization rate, suggesting increased risk of chromosomal abnormalities. PMID- 10750888 TI - Urinary incontinence among community-dwelling people aged 40 years or older in Japan: prevalence, risk factors, knowledge and self-perception. AB - BACKGROUND: Urinary incontinence (UI) is a common problem in adults, especially among the elderly. We examined the prevalence and risk factors of UI and potential factors hindering individuals from seeking treatment for UI among a community-dwelling population aged over 40 years. METHODS: Data were collected by mailing a 23-item urinary incontinence questionnaire to a random sample of community-dwelling individuals aged 40-75 years (n=3500) in seven towns of Shiga Prefecture, Japan. Collected data were then used to estimate the prevalence of UI and to provide information regarding subtypes of UI, knowledge and self perception about UI. RESULTS: The overall response rate was 52.5%. Prevalence of UI for male and female respondents were 10.5% and 53.7%, respectively. The incidence of urge incontinence increased as age increased in the male group. In women, stress incontinence was prevalent at all ages and the incidence of urge incontinence increased over 70 years of age. Urinary incontinence was more likely as activities of daily living limitations and cystitis increased. Women with a history of hysterectomy or diabetes mellitus and men who had stroke were at increased risk for UI. Of those who reported UI, only 3% had ever consulted doctors or other health care professionals concerning it, 25% recognized their condition as a disease and 38% considered it curable by appropriate treatments. In addition, 63% regarded UI as an unavoidable consequence of aging, 63% considered their condition was embarrassing and 54% were reluctant to seek treatment from a health professional. CONCLUSIONS: Although UI is common among community-dwelling individuals over 40 years of age, the majority of affected individuals remained untreated due to lack of knowledge and/or a negative perception of UI. Thus, community education on UI may be needed to increase the number of UI patients who receive treatment. PMID- 10750889 TI - Trans-mesosigmoid cutaneous ureterostomy. AB - BACKGROUND: A new method was developed in order to create a single stoma cutaneous ureterostomy in which both ureters traverse the abdominal cavity and yet are buttressed by the mesosigmoid and covered by its visceral peritoneum. METHODS: The long mesenterium which is attached to the most mobile part of the sigmoid colon was used for the bilateral ureteral pathway. Tunnels for the ureteral path were made just underneath the visceral peritoneum on the bilateral side of the mesosigmoid. Through the tunnels both ureteral ends were brought from the retroperitoneal space to the mesenterocolonic junction (MCJ) and the MCJ is then approximated and sutured to the inside of the ureteral tract through the abdominal wall. The ureters brought outside the skin, are conjoined and sutured to the V skin flap. Eight patients who carried a high risk for operation and/or had a bladder tumor judged to be incurable underwent this cutaneous ureterostomy. RESULTS: All cases except one with low urinary output could be managed without catheter indwelling during the follow-up period. Three patients suffered from paralytic ileus and one required laparotomy for mechanical ileus during the short postoperative period. Postoperative excretory urography evaluated 14 kidneys during the follow-up period from 2 to 61 months and showed normal upper urinary tract in 11 and a mildly hydronephrotic tract in three. CONCLUSIONS: Transmesosigmoid cutaneous ureterostomy provides a single catheterless stoma even when the available ureters are relatively short. It appears to be a good method for supravesical urinary diversion when indicated. PMID- 10750890 TI - Ectopic pheochromocytoma arising in the spermatic cord 5 years after removal of bilateral carotid body tumors and adrenal pheochromocytomas. AB - A patient with ectopic pheochromocytoma that developed in the spermatic cord about 5 years after successive resections of bilateral carotid body tumors and bilateral adrenal pheochromocytomas is reported. This is thought to be the first case of pheochromocytoma of the spermatic cord in a setting of multiplicity. PMID- 10750891 TI - Gasless retroperitoneoscopic partial nephrectomy for a case with renal cell carcinoma. AB - A 53-year-old man with incidental renal cell carcinoma underwent gasless retroperitoneoscopic partial nephrectomy. Convalescence was uneventful. There remained no evidence of disease during the 58-month follow up. PMID- 10750892 TI - Glomus tumor of the penis. AB - Glomus tumors of the penis are extremely rare. A patient with multiple regional glomus tumors involving the penis is reported. A 16-year-old boy presented with the complaint of painless penile masses and resection of the lesions was performed. The pathologic diagnosis was glomus tumor of the penis. This is the ninth case of glomus tumor of the penis to be reported in the literature. PMID- 10750893 TI - Ion mapping in plant cells--methods and applications in signal transduction research. AB - This review covers both methodical aspects and actual applications of ion imaging techniques in plant cell signal research. The methodological section explains the basic principles of fluorescence ion imaging, the impact of modern developments in fluorescence microscopy and introduces the most important fluorescence probes including aequorin and other photoproteins. It critically comments on loading strategies, intracellular compartmentation of probes and calibration procedures. The second part compiles actual research areas where the application of ion imaging procedures has gained substantial achievements and helped to establish new concepts of calcium- and pH-dependent signalling. Examples comprise the hormonal control of stomatal movements, effects of gibberellic and abscisic acids in aleurone cells, elicitation of phytoalexin production, cytosolic pH and cell development, and signatures of Ca2+ as a universal signal in plant cells. PMID- 10750895 TI - Control of carbon partitioning and photosynthesis by the triose phosphate/phosphate translocator in transgenic tobacco plants (Nicotiana tabacum). II. Assessment of control coefficients of the triose phosphate/phosphate translocator. AB - Transgenic tobacco (Nicotiana tabacum L.) plants with decreased and increased transport capacities of the chloroplast triose phosphate/phosphate translocator (TPT) were used to study the control the TPT exerts on the flux of starch and sucrose biosynthesis, as well as CO2 assimilation, respiration and photosynthetic electron transport. For this purpose, tobacco lines with an antisense repression of the endogenous TPT (alphaTPT) and tobacco lines overexpressing a TPT gene from Flaveria trinervia (FtTPT) were used. In ambient CO2, there was no or little effect of altered TPT transport activities on either rates of photosynthetic electron transport and/or CO2 assimilation. However, in elevated CO2 (1500 microl x 1(-1)) and low O2 (2%) the TPT exerted strong control on the rate of CO2 assimilation (control coefficient for the wild type; C(J(A))(TPT) = 0.30) in saturating light. Similarly, the incorporation of 14C into starch in high CO2 was increased in tobacco plants with decreased TPT activity, but was reduced in plants overexpressing the TPT from F. trinervia. Thus, the TPT exerted negative control on the rate of starch biosynthesis with a C(J(Starch))(TPT) = -0.19 in the wild type estimated from a hyperbolic curve fitted to the data points. This was less than the positive control strength on the rate of sucrose biosynthesis (C(J(Suc))(TPT) = 0.35 in the wild type). Theoretically, the positive control exerted on sucrose biosynthesis should be numerically identical to the negative control on starch biosynthesis unless additional metabolic pathways are affected. The rate of dark respiration showed some correlation with the TPT activity in that it increased in FtTPT overexpressors, but decreased in alphaTPT plants with an apparent control coefficient of C(J(Res))(TPT) = 0.24. If the control on sucrose biosynthesis is referred to as "gain of carbon" (positive control) and the control on starch biosynthesis as well as dark respiration as a "loss of carbon" (negative control) for sucrose biosynthesis and subsequent export, the sum of the control coefficients on dark respiration and starch biosynthesis would be numerically similar to the control coefficient on the rate of sucrose biosynthesis. There was also some control on the rate of photosynthetic electron transport, but only at high light and in elevated CO2 combined with low O2. The control coefficient for the rate of photosynthetic electron transport was C(J(ETR))(TPT) = 0.16 in the wild type. Control coefficients were also calculated for plants with elevated and lowered TPT activity. Furthermore, the extent to which starch degradation/glucose utilisation compensates for the lack of triose phosphate export was assessed. The TPT also exerted control on metabolite contents in air. PMID- 10750896 TI - Expression of a Petunia inflata pectin methyl esterase in Solanum tuberosum L. enhances stem elongation and modifies cation distribution. AB - Transgenic potato (Solanum tuberosum L.) plants were constructed with a Petunia inflata-derived cDNA encoding a pectin methyl esterase (PME; EC 3.1.1.11) in sense orientation under the control of the cauliflower mosaic virus 35S promoter. The PME activity was elevated in leaves and tubers of the transgenic lines but slightly reduced in apical segments of stems from mature plants. Stem segments from the base of juvenile PME-overexpressing plants did not differ in PME activity from the control, whereas in apical parts PME was less active than in the wild-type. During the early stages of development stems of these transgenic plants elongated more rapidly than those of the wild-type. Further evidence that overexpression of a plant-derived PME has an impact on plant development is based on modifications of tuber yield, which was reduced in the transgenic lines. Cell walls from transgenic tubers showed significant differences in their cation binding properties in comparison with the wild-type. In particular, cell walls displayed increased affinity for sodium and calcium, while potassium binding was constant. Furthermore, the total ion content of transgenic potatoes was modified. Indications of PME-mediated differences in the distribution of ions in transgenic plants were also obtained by monitoring relaxations of the membrane potential of roots subsequent to changes in the ionic composition of the bathing solution. However, no effects on the chemical structure of pectin from tuber cell walls could be detected. PMID- 10750894 TI - Control of carbon partitioning and photosynthesis by the triose phosphate/phosphate translocator in transgenic tobacco plants (Nicotiana tabacum L.). I. Comparative physiological analysis of tobacco plants with antisense repression and overexpression of the triose phosphate/phosphate translocator. AB - The physiological properties of transgenic tobacco plants (Nicotiana tabacum L.) with decreased or increased transport capacities of the chloroplast triose phosphate/phosphate translocator (TPT) were compared in order to investigate the extent to which the TPT controls metabolic fluxes in wild-type tobacco. For this purpose, tobacco lines with an antisense repression of the endogenous TPT (alphaTPT) and tobacco lines overexpressing the TPT gene isolated from the C4 plant Flaveria trinervia (FtTPT) were used. The F. trinervia TPT expressed in yeast cells exhibited transport characteristics identical to the TPT from C3 plants. Neither antisense TPT plants nor FtTPT overexpressors showed a phenotype when grown in a greenhouse in air. Contents of starch and soluble sugars in upper source leaves were similar in TPT underexpressors and FtTPT overexpressors compared to the wild type at the end of the photoperiod. The FtTPT overexpressors incorporated more 14CO2 in sucrose than the wild type, indicating that the TPT limits sucrose biosynthesis in the wild type. There were only small effects on labelling of amino acids and organic acids. The mobilisation of starch was enhanced in alphaTPT lines but decreased in FtTPT overexpressors compared to the wild type. Enzymes involved in starch mobilisation or utilisation, such as alpha amylase or hexokinase were increased in alphaTPT plants and, in the case of amylases, decreased in FtTPT overexpressors. Moreover, alpha-amylase activity exhibited a pronounced diurnal variation in alphaTPT lines with a maximum activity after 8 h in the light. These changes in starch hydrolytic activities were confirmed by activity staining of native gels. Activities of glucan phosphorylases were unaffected by either a decrease or an increase in TPT activity. There were also effects of TPT activities on steady-state levels of phosphorylated intermediates as well as total amino acids and malate. In air, there was no or little effect of altered TPT transport activity on either rates of photosynthetic electron transport and/or CO2 assimilation. However, in elevated CO2 (1500 microl x l(-1)) and low O2 (2%) the rate of CO2 assimilation was decreased in the alphaTPT lines and was slightly higher in FtTPT lines. This shows that the TPT limits maximum rates of photosynthesis in the wild type. PMID- 10750897 TI - Gravity independence of seed-to-seed cycling in Brassica rapa. AB - Growth of higher plants in the microgravity environment of orbital platforms has been problematic. Plants typically developed more slowly in space and often failed at the reproductive phase. Short-duration experiments on the Space Shuttle showed that early stages in the reproductive process could occur normally in microgravity, so we sought a long-duration opportunity to test gravity's role throughout the complete life cycle. During a 122-d opportunity on the Mir space station, full life cycles were completed in microgravity with Brassica rapa L. in a series of three experiments in the Svet greenhouse. Plant material was preserved in space by chemical fixation, freezing, and drying, and then compared to material preserved in the same way during a high-fidelity ground control. At sampling times 13 d after planting, plants on Mir were the same size and had the same number of flower buds as ground control plants. Following hand-pollination of the flowers by the astronaut, siliques formed. In microgravity, siliques ripened basipetally and contained smaller seeds with less than 20% of the cotyledon cells found in the seeds harvested from the ground control. Cytochemical localization of storage reserves in the mature embryos showed that starch was retained in the spaceflight material, whereas protein and lipid were the primary storage reserves in the ground control seeds. While these successful seed-to-seed cycles show that gravity is not absolutely required for any step in the plant life cycle, seed quality in Brassica is compromised by development in microgravity. PMID- 10750898 TI - Developmental regulation of the maize Zm-p60.1 gene encoding a beta-glucosidase located to plastids. AB - A beta-glucosidase that cleaves the biologically inactive hormone conjugates cytokinin-O- and kinetin-N3-glucosides is encoded by the maize Zm-p60.1 gene. The expression of the Zm-p60.1 gene was analyzed by Northern blot analysis and in situ hybridization. It was found that the expression levels of the Zm-p60.1 specific mRNA changed after pollination of carpellate inflorescences. The Zm p60.1 cDNA was expressed in E. coli and antibodies were raised against this protein. An antibody was used to determine the tissue-specific localization of this protein. By in situ immunolocalization experiments, this protein was found to be located in cell layers below the epidermis and around the vascular bundles of the coleoptile. In the primary leaf, the Zm-p60.1 protein was detected in cells of the outermost cell layer and around the vascular tissue. In floral tissue, Zm-p60.1 was present in the glumes, the carpels and in the outer cell layer of the style. In coleoptiles, as determined by immuno-electronmicroscopy, the Zmp60.1 protein was located exclusively in the plastids. PMID- 10750899 TI - Alfin1 transcription factor overexpression enhances plant root growth under normal and saline conditions and improves salt tolerance in alfalfa. AB - Plant root development is an essential determinant of plant growth and crop yield that could be enhanced by induced changes in the expression of root-specific regulatory factors. We reported previously that Alfin1 binds DNA in a sequence specific manner and that Alfin1 overexpression in transgenic alfalfa (Medicago sativa L.) enhances expression of the salt-inducible MsPRP2 gene in roots, suggesting that Alfin1 functions to regulate gene expression in roots. Here we show that Alfin1 is an essential gene for root growth and that its overexpression in transgenic plants confers a many-fold increase in root growth under normal and saline conditions. Alfin1-binding sites occur in promoters of genes expressed in roots of a wide variety of plant species and we propose that it is a general root growth regulator. Even though Alfin1 overexpression was under the control of the CaMV 35S promoter, plant shoot growth was not adversely affected. We show further that introduction of the Alfin1 transgene in plants confers a dominant characteristic that significantly increases plant growth and salt tolerance. PMID- 10750900 TI - Osmotically evoked shrinking of guard-cell protoplasts causes vesicular retrieval of plasma membrane into the cytoplasm. AB - The dye FM1-43 was used alone or in combination with measurements of the membrane capacitance (Cm) to monitor membrane changes in protoplasts from Vicia faba L. guard cells. Confocal images of protoplasts incubated with FM1-43 (10 microM) at constant ambient osmotic pressure (pi omicron) revealed in confocal images a slow internalisation of FM1-43-labelled membrane into the cytoplasm. As a result of this process the relative fluorescence intensity of the cell interior (fFM,i) increased with reference to the total fluorescence (fFM,t) by 7.4 x 10(-4) min( 1). This steady internalisation of dye suggests the occurrence of constitutive endocytosis under constant osmotic pressure. Steady internalisation of FM1-43 labelled membrane caused a prominent staining of a ring-like structure located beneath the plasma membrane. Abrupt elevation of pi omicron by 200 mosmol kg(-1) caused, over the first minutes of incubation, a rapid internalisation of FM1-43 fluorescence into the cytoplasm concomitant with a decrease in cell perimeter. Within the first 5 min the cell perimeter decreased by 7.9%. Over the same time fFM,i/fFM,t increased by 0.13, reflecting internalisation of fluorescent label into the cytoplasm. Combined measurements of Cm and total fluorescence of a protoplast (fFM,p) showed that an increase in pi omicron evoked a decrease in Cm but no change in fFM,p. This means that surface contraction of the protoplast is due to retrieval of excess membrane from the plasma membrane and internalisation into the cytoplasm. Further inspection of confocal images revealed that protoplast shrinking was only occasionally associated with internalisation of giant vesicles (median diameter 2.7 microm) with FM 1-43-labelled membrane. But, in all cases, osmotic contraction was correlated with a diffuse distribution of FM1-43 label throughout the cytoplasm. From this, we conclude that endocytosis of small vesicles into the cytoplasm is the obligatory process by which cells accommodate an osmotically driven decrease in membrane surface area. PMID- 10750901 TI - Purification and characterization of a hydroxamic acid glucoside beta-glucosidase from wheat (Triticum aestivum L.) seedlings. AB - A beta-glucosidase (EC 3.2.1.21) with a high affinity for cyclic hydroxamic acid beta-D-glucosides was purified from 48-h-old wheat (Triticum aestivum L.) seedlings. The activity occurred transiently at a high level during the non autotrophic stage of growth, and the nature of the transient occurrence was correlated with that of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc). The glucosidase had maximum activity at an acidic pH (pH 5.5) and the purified enzyme showed a high affinity for DIMBOA-Glc, Vmax and Km being 4100 nkat/mg protein and 0.27 mM, respectively. It also hydrolyzed p-nitrophenol beta glycosides, as well as flavone and isoflavone glucosides, but to a lesser extent. The results indicated that the primary natural substrate for the glucosidase is DIMBOA-Glc and that the enzyme is involved in defense against pathogens and herbivores in non-autotrophic wheat. The glucosidase was found to be present as oligomeric forms with a molecular mass of 260-300 kDa comprising 60- and 58-kDa monomers. The N-terminal 12-amino-acid sequences of the two monomers were identical (Gly-Thr-Pro-(Ser?)-Lys-Pro-Ala-Glu-Pro-Ile-Gly-Pro), and showed no similarity to those of other plant glucosidases. Polyacrylamide gel electrophoresis under nondenaturing condition indicated the existence of at least eight isozymes. Three cultivars of Triticum aestivum had the same zone of glucosidase activity on zymograms, but the activity zones of the Triticum species, T. aestivum L., T. spelta L. and T. turgidum L., had different mobilities. PMID- 10750902 TI - The targeting and accumulation of ectopically expressed oleosin in non-seed tissues of Arabidopsis thaliana. AB - Full-length and N-terminal deletions of a sunflower (Helianthus annuus L.) oleosin protein were expressed ectopically in transgenic Arabidopsis thaliana (L.) Heynh. Immunological detection of the sunflower protein revealed that it accumulated in a range of non-oil-storing tissues, including leaves, roots and petals. This accumulation was shown to result from deposition in the microsomal membrane fraction. Expression in oil-storing tissues (such as seeds) of oleosin N terminal deletions revealed impaired transfer from the endoplasmic reticulum to the oil body. In non-oil-storing tissues, accumulation in the microsomal membrane fraction was progressively reduced by N-terminal deletion. These data confirm the role of the endomembrane system in the targeting of the oleosin and its intimate relationship with oil-body biogenesis. PMID- 10750903 TI - Use of 15N reverse gradient two-dimensional nuclear magnetic resonance spectroscopy to follow metabolic activity in Nicotiana plumbaginifolia cell suspension cultures. AB - Nitrogen metabolism was monitored in suspension cultured cells of Nicotiana plumbaginifolia Viv. using nuclear magnetic resonance (NMR) spectroscopy following the feeding of (15NH4)2SO4 and K15NO3. By using two-dimensional 15N-1H NMR with heteronuclear single-quantum-coherence spectroscopy and heteronuclear multiple-bond-coherence spectroscopy sequences, an enhanced resolution of the incorporation of 15N label into a range of compounds could be detected. Thus, in addition to the amino acids normally observed in one-dimensional 15N NMR (glutamine, aspartate, alanine), several other amino acids could be resolved, notably serine, glycine and proline. Furthermore, it was found that the peak normally assigned to the non-protein amino-acid gamma-aminobutyric acid in the one-dimensional 15N NMR spectrum was resolved into a several components. A peak of N-acetylated compounds was resolved, probably composed of the intermediates in arginine biosynthesis, N-acetylglutamate and N-acetylornithine and, possibly, the intermediate of putrescine degradation into gamma-aminobutyric acid, N acetylputrescine. The occurrence of 15N-label in agmatine and the low detection of labelled putrescine indicate that crucial intermediates of the pathway from glutamate to polyamines and/or the tobacco alkaloids could be monitored. For the first time, labelling of the peptide glutathione and of the nucleotide uridine could be seen. PMID- 10750904 TI - Simulating ozone detoxification in the leaf apoplast through the direct reaction with ascorbate. AB - This paper presents a mathematical model which enables the semi-quantification of ozone (O3) detoxification, based upon the direct reaction of the pollutant with ascorbate (ASC) located in the aqueous matrix associated with the cell wall (i.e. the apoplast). The model describes the uptake of ozone into the leaf and its direct reaction with ASC, taking into consideration the regeneration of dehydroascorbic acid in the cytosol, the rate of replenishment of cell wall ASC and the distribution of ASC between sub-cellular compartments based upon the permeability of biomembranes to the neutral species, ascorbic acid and the pH of various sub-cellular compartments. The importance of various physico-chemical characteristics (e.g. stomatal conductance, mesophyll cell wall thickness and tortuosity, chloroplast volume, apoplast pH, ASC:O3 reaction stoichiometry) in mediating the flux of ozone to the plasmalemma is analysed. Model simulations, supported by experimental observations, suggest that the ASC concentration in the leaf apoplast is high enough to scavenge a significant proportion of the O3 taken up into the leaf interior, under environmentally relevant conditions. However, there is considerable variation between taxa in the potential degree of protection afforded by apoplastic ASC, emphasizing the need for an improved understanding of the reaction chemistry of O3 in the cell wall. PMID- 10750905 TI - Flexible coupling between light-dependent electron and vectorial proton transport in illuminated leaves of C3 plants. Role of photosystem I-dependent proton pumping. AB - The role of cyclic electron transport has been re-examined in leaves of C3 plants because the bioenergetics of chloroplasts (H +/e = 3 in the presence of a Q cycle; H+/ATP = 4 of ATP synthesis) had suggested that cyclic electron flow has no function in C3 photosynthesis. After light activation of pea leaves, the dark reduction of P700 (the donor pigment of PSI) following far-red oxidation was much accelerated. This corresponded to loss of sensitivity of P700 to oxidation by farred light and a large increase in the number of electrons available to reduce P700+ in the dark. At low CO2 and O2 molar ratios, far-red light was capable of decreasing the activity of photosystem II (measured as the ratio of variable to maximal chlorophyll fluorescence, Fv/Fm) and of increasing light scattering at 535 nm and zeaxanthin synthesis, indicating formation of a trans-thylakoid pH gradient. Both the light-induced increase in the number of electrons capable of reducing far-redoxidised P700 and the decline in Fv/Fm brought about by far-red in leaves were prevented by methyl viologen. Antimycin A inhibited CO2-dependent O2 evolution of pea leaves at saturating but not under limiting light; in its presence, far-red light failed to decrease Fv/Fm. The results indicate that cyclic electron flow regulates the quantum yield of photosystem II by decreasing the intrathylakoid pH when there is a reduction in the availability of electron acceptors at the PSI level (e.g. during drought or cold stresses). It also provides ATP for the carbon-reduction cycle under high light. Under these conditions, the Q-cycle is not able to maintain a H+/e ratio of 3 for ATP synthesis: we suggest that the ratio is flexible, not obligatory. PMID- 10750906 TI - Growth, ageing and death of a photoautotrophic plant cell culture. AB - Batch cultures of photoautotrophic cell suspensions of Chenopodium rubrum L., growing in an inorganic medium on CO2 under a daily balanced light-dark regime of 16: 8 h could be maintained for approximately 100 d without subcultivation. The long-lived cultures showed an initial cell division phase of 4 weeks, followed by a stationary phase of another 4 weeks, after which ageing and progressive cell death reduced the number of living cells and the cultures usually expired after another 3-4 weeks. These developmental phases of the cell culture were characterised with respect to photosynthetic performance, dark respiration, content of phytohormones and capacity of cell division. Cell division of the majority of the cells finished in the G1- or G0-phase of the cell cycle, caused by a pronounced decline in the endogenous levels of auxin and cytokinins. Supply of these growth factors to resting cells resulted in resumption of cytokinesis, at least by some of the cells. However, responsiveness to the phytohormones declined during the stationary phase, and subcultivation was no longer possible beyond day 60 when the phases of ageing and death commenced. Ageing was characterised by a further decline in the photosynthetic capacity of the cells, by a climacteric enhancement of dark respiration, but also by a slight increase in the level of IAA and cytokinins concomitant with a decrease in ethylene. Similarities and differences between the development of batch-cultured photoautotrophic cells of C. rubrum and that of a leaf are discussed with respect to using the cell culture as a model for a leaf. PMID- 10750907 TI - The kinetics of calcium and magnesium entry into mycorrhizal spruce roots. AB - The entry of calcium and magnesium from external sources into mycorrhizal roots of 3-year-old Norway spruce trees (Picea abies [L.] Karst.) was monitored. Roots of intact plants were exposed for various periods of time, ranging from 2 min to 48 h, to nutrient solutions which contained the stable-isotope tracers 25Mg and 44Ca. After labelling, samples of roots were excised from the plants, shock frozen, cryosubstituted and embedded. The resulting isotope composition in this material was analysed by a laser-microprobe-mass-analyser (LAMMA) at relevant positions within cross-sections of the roots. For both elements, we determined (i) the fractions of the isotopes originating from the plant prior to labelling, and (ii) the fraction of isotopes originating from the corresponding tracer that penetrated into the root. Both divalent cations rapidly penetrated across the cortical apoplast and reached the endodermis. After 2 min of exposure to the labelling solution, an initial transient gradient of the tracers could be observed within the root cortex. Subsequently, calcium as well as magnesium equilibrated between the apoplast of the entire cortex and the external tracer with a half-time, t1/2, of about 3 min. In contrast, the kinetics of radial movement into the vascular stele showed a delay with a t1/2 of 100-120 min. We take this as strong evidence that there exists a free apoplastic path for divalent cations in the cortex and that the endodermis is a major barrier to the further passage of Mg and Ca into the xylem. While 25Mg in the labelling solution exchanged rapidly with Mg in the cortical apoplast, the exchange across the plasma membrane with Mg present in the protoplasm of the same cortical cells was almost 2 orders of magnitude slower. The kinetics of Ca and Mg entry at + 6 degrees C were similar to those obtained at a root temperature of +22 degrees C. PMID- 10750908 TI - Temperature-dependent internode elongation in vegetative plants of Arabidopsis thaliana lacking phytochrome B and cryptochrome 1. AB - Vegetative plants of Arabidopsis thaliana (L.) Heynh. form a compact rosette of leaves in which internode growth is virtually arrested. Rapid extension of the internodes occurs after flower buds are present in the reproductive apex. Under natural radiation, continuous light from fluorescent lamps, or short photoperiods of light from fluorescent lamps, plants of the phyB cry1 double mutant (lacking both phytochrome B and cryptochrome 1) did not form normal rosettes because all the internodes showed some degree of elongation. Internode elongation was weak in thephyB single mutant and absent in the cry1 mutant, indicating redundancy between phytochrome B and cryptochrome 1. The absence of phytochrome A caused no effects. The failure to form normal rosettes was conditional because internode elongation was arrested at low temperatures in all the mutant combinations. In contrast, the temperature dependence of phytochrome B and cryptochrome 1 effects on hypocotyl growth was weak. The elongation of the internodes in phyB cry1 was not accompanied by early flowering as showed by the lack of effects on the final number of leaves. Apex dissection indicated that in phyB cry1 double mutants internode elongation anticipated the transition from the vegetative to the reproductive stage. Thus, stem growth in Arabidopsis thaliana is not fully dependent on the program of reproductive development. PMID- 10750909 TI - Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD. AB - Microtubule organization plays an important role in plant morphogenesis; however, little is known about how microtubule arrays transit from one organized state to another. The use of a genetically incorporated fluorescent marker would allow long-term observation of microtubule behavior in living cells. Here, we have characterized a Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line that had been stably transformed with a gfp-mbd construct previously demonstrated to label microtubules (J. Marc et al., 1998, Plant Cell 10: 1927-1939). Fluorescence levels were low, but interphase and mitotic microtubule arrays, as well as the transitions between these arrays, could be observed in individual gfp-mbd transformed cells. By comparing several attributes of transformed and untransformed cells it was concluded that the transgenic cells are not adversely affected by low-level expression of the transgene and that these cells will serve as a useful and accurate model system for observing microtubule reorganization in vivo. Indeed, some initial observations were made that are consistent with the involvement of motor proteins in the transition between the spindle and phragmoplast arrays. Our observations also support the role of the perinuclear region in nucleating microtubules at the end of cell division with a progressive shift of these microtubules and/or nucleating activity to the cortex to form the interphase cortical array. PMID- 10750910 TI - Fast track to the trichome: induction of N-acyl nornicotines precedes nicotine induction in Nicotiana repanda. AB - Nicotiana repanda Wildenow ex Lehmann acylates nornicotine in its trichomes to produce N-acyl-nornicotine (NacNN) alkaloids which are dramatically more toxic than nicotine is to the nicotine-adapted herbivore, Manduca sexta. These NacNNs, like nicotine, were induced by methyl jasmonate (MeJA) and wounding, but the 2 fold increase in NacNN pools was much faster (within 6 h) than the MeJA-induced increase in nornicotine pools (24 h to 4 d), its parent substrate. When 15NO(-)3 pulse-chase experiments with intact and induced plants were used to follow the incorporation of 15N into alkaloids in different plant parts over the plant's lifetime, it was found that the root nicotine pool was most rapidly labeled, followed by the shoot nornicotine and NacNN pools. After 3 d, 3.12% of 15N acquired was in nicotine (0.93%), nornicotine (0.32%) and NacNNs (1.73%) while only 0.14% was in anabasine. Once NacNNs are externalized to the leaf surface, they are not readily re-distributed within the plant and are lost with senescing leaves. The wound- and MeJA-induced N-acylation of nornicotine is independent of induced changes in nornicotine pools and the rapidity of the response suggests its importance in defense against herbivores. PMID- 10750911 TI - Morphogenesis in cucumber seedlings is negatively controlled by gravity. AB - Seedlings of most cucurbitaceous plants develop a peg (protuberance caused by cell outgrowth) on the transition zone between the hypocotyl and root. The peg is necessary for removing the seed coat after germination. In our spaceflight experiments on the STS-95 space shuttle, Discovery, we found that cucumber (Cucumis sativus L.) seedlings grown under microgravity conditions developed two pegs symmetrically at the transition zone. Thus, cucumber seedlings potentially develop two pegs and do not require gravity for peg formation itself, but on the ground the development of one peg is suppressed in response to gravity. This may be considered as negative control of morphogenesis by gravity. PMID- 10750912 TI - The influence of an Urgent Care Center on the frequency of ED visits in an urban hospital setting. AB - We examined the effect of a visit to an Urgent Care Center (UCC) on emergency department (ED) use by patients with nonemergent complaints. A study population of 1,629 patients with no previous visit to a UCC were identified and served as their own controls. The ED and clinic usage 6 months before and 6 months after a UCC visit were examined. After the UCC visit for adults there was a 48% reduction in ED visits (P = .0001) and 49% increase in clinic visits (P = .0001). After the UCC visit for children there was a 28% reduction in ED visits (P < .005) and 65% increase in clinic visits (P = .0001). Moreover the majority of clinic visits occurred within 90 days after the UCC visit. There was no substantial change in patterns of hospitalization 6 months after the UCC visit. We conclude that UCC usage decreases nonemergent ED use without adverse effects of increased patient hospitalization. PMID- 10750914 TI - Patterns of use of emergency medical transport: a population-based study. AB - The objective of this study was to characterize population-based emergency medical service (EMS) use rates and examine some of the factors associated with usage of prehospital services. The design was a population-based observational study with multiple regression analysis. Transports reported by prehospital services to the Kentucky Emergency Medical Services Information System from Kentucky counties in which all EMS units submit computerized data was the data source. There were 102,321 emergent transports reported. The overall rate of use of EMS transports was 51.7 +/- 24.1/1,000/year, but the rate varied between different communities (range 11 to 139/1,000/year). Rate of use was highest in those older than 65 (178.5 +/- 84.2/1,000/year), with the rate increasing exponentially with increasing age in this age group. Reasons for transport were age dependent. There was an association between increasing poverty level and use of EMS. In addition, the absence of 911 service (odds ratio [OR] 1.18, 95% confidence interval [CI; 1.14,1.22]) and the absence of a hospital in the county (OR 1.27, 95% CI [1.24,1.30]) were also associated with increasing rates of ambulance use. Rates of use of EMS are most dependent on age and cause, but use is also correlated with increasing levels of poverty. Wide variations in use between communities suggests that point estimates using one community may over- or underestimate EMS usage. PMID- 10750913 TI - Intraosseous is faster and easier than umbilical venous catheterization in newborn emergency vascular access models. AB - The purpose of this study is to compare the speed and ease of establishing newborn emergency vascular access using intraosseous (IO) versus umbilical venous catheterization (UVC). The study is an experimental design. A total of 42 medical students, without prior IO and UVC experience, were recruited as study subjects. All subjects performed the UVC procedure and were randomized (by a coin flip) to perform the IO procedure in one of two models: (1) turkey bone or (2) plastic infant leg. Each subject performed an initial trial for both the IO and UVC procedures without practice ("Inexperienced attempt") and a second trial in both procedures after practice ("Experienced attempt"), such that in total, each subject completed four attempts (two IO and two UVC). IO and UVC placement times were measured, and placement difficulty scores for IO and UVC were measured using a 10 cm visual analog scale (VAS). The averaged elapsed time to successful access was significantly shorter for the IO procedure on both the initial "inexperienced" attempt (52 versus 134 seconds, P < .001) as well as the "experienced" attempt (45 versus 95 seconds, P = .011). Procedure difficulty scores were lower in the IO procedure for both "inexperienced" and "experienced" attempts (3.5 versus 5.5, P = .001 and 2.6 versus 4.7, P < .001) as measured on a 10 cm VAS. Although UVC may be preferred by neonatologists, this model suggests that IO results in easier and more rapid vascular access in those who do not frequently perform newborn resuscitation. As such, the benefit of teaching UVC in pediatric resuscitation courses should be reconsidered. The recommended method of emergency newborn vascular access should be reconsidered pending further studies on this subject. PMID- 10750915 TI - Comparison of a new screw-tipped intraosseous needle versus a standard bone marrow aspiration needle for infusion. AB - The purpose of this study is to compare the speed and ease of establishing intraosseous infusion using a standard bone marrow needle (SBMN; $8) and a new screw-tipped intraosseous needle (Sur-Fast; $42). The study is an experimental design. A total of 42 medical students, without prior IO experience, were recruited as study subjects. Subjects were randomized to perform the IO procedures in one of two models: (1) turkey femur or (2) pork ribs. Each subject performed an initial trial using both IO needles without practice (inexperienced) and a second trial using both IO needles after practice (experienced attempt), such that in total, each subject completed four attempts (two with each needle type). IO placement times were measured, and placement difficulty scores were measured using a 10 cm visual analog scale (VAS). The averaged elapsed time to successful IO completion was significantly shorter for the SBMN in the initial "inexperienced" attempt (33 versus 54 seconds, P = .019), but there was no significant difference in the postpractice "experienced" attempt. VAS difficulty scores were lower (easier) for the SBMN for both inexperienced and experienced trials. Success rates were significantly higher for the Sur-Fast needle during the experienced attempt (95% versus 79%, P < .05), but there was no significant difference in success rates during the inexperienced attempt. The Sur-Fast screw tipped intraosseous needle does not show superiority over the SBMN in this intraosseous model, therefore its higher cost is difficult to justify based on this study. PMID- 10750916 TI - Acute diverticulitis in patients 40 years of age and younger. AB - Acute colonic diverticulitis typically occurs in patients older than 60 years of age but is uncommon in patients under the age of 40, which may lead to a delay in diagnosis. Because abdominal pain is a very common presenting symptom in emergency department patients, we retrospectively analyzed the cases of 21 patients 40 years of age and younger diagnosed with acute diverticulitis and characterized the presenting signs and symptoms, laboratory and radiographic findings, treatment, and outcome. There were 17 men and 4 women with a mean age of 34.1 +/-5.9 years. All patients had abdominal pain, with 14 (67%) patients noting pain in the left lower quadrant (LLQ) and 5 (24%) patients noting right lower quadrant (RLQ) pain. Nausea was present in 18 (86%) patients and fever in 15 (71%) patients. The mean pulse rate was 103 +/- 16 and the mean temperature was 100.7 +/- 1.4 F. Leukocytosis was present in 19 (90%) patients. Plain abdominal radiographs were obtained in 19 (91%) patients and were normal in 15 (79%) of these cases. Computed tomographic (CT) scans were obtained in 15 (71%) patients which revealed findings consistent with acute diverticulitis in 14 (93%) patients. The admitting diagnosis was diverticulitis in 10 of the 12 patients with LLQ tenderness and appendicitis in 4 of the 6 patients with RLQ tenderness. Overall, six patients were taken to surgery: three patients had cecal diverticulitis and three patients had perforated colonic diverticulitis. General treatment measures included bowel rest in 18 (86%) patients, and intravenous fluids and antibiotics in all patients. All patients survived. In conclusion, acute diverticulitis is uncommon in patients under 40 years of age; however, this condition may be confused with other conditions, usually acute appendicitis. As a result, clinicians should consider acute diverticulitis in young patients with acute abdominal pain, especially if they are male with nausea, fever, tachycardia, and leukocytosis, and consider obtaining a CT scan to aid in the diagnosis. PMID- 10750918 TI - Survey of moonlighting practices and work requirements of emergency medicine residents. AB - We conducted an anonymous moonlighting and academic practice survey of all emergency medicine residents enrolled in accredited programs during 1997. Expanding on previous work, this survey included specific details and practice trends of moonlighting emergency medicine residents and for comparison also included academic work requirements. The typical emergency residency program requires residents to work 204 hours monthly. However, the range of required work hours is strikingly large (120-300). Half of emergency medicine residents moonlight. The typical moonlighting resident works as a solo emergency department practitioner in multiple facilities outside of residency-affiliated institutions. Moonlighting salaries generally double a resident's annual income and are used to pay off student loans and other debt. Residents with higher student debt are more likely to moonlight. Despite the fact that most residency programs restrict moonlighting, a majority of moonlighting residents have violated an Accreditation Council for Graduate Medical Education prohibition restricting work within one period of a regular residency-scheduled shift. Half of all residents surveyed, whether involved in moonlighting practice or not, would violate a ban on the practice. Residents universally felt that moonlighting enhanced residency performance and was a positive educational experience. Use of these data may aid in the development of formal guidelines regarding emergency medicine moonlighting practice. PMID- 10750917 TI - Prevalence and risk factors for multidrug resistant uropathogens in ED patients. AB - The purpose of this study was to describe resistance patterns of infecting organisms and determine risk factors for multidrug resistance in patients with urinary tract infections. Retrospective case series of 435 patients age > or =16 with urinary tract infection. Multidrug resistance was defined as resistance to > or = two classes of antibiotics. Demographic, historical, and microbiological data were collected. Univariate analysis and multivariate logistic regression were used to determine risk factors for multidrug resistance. Multidrug resistance was seen in 37% of isolates. Univariate analysis revealed numerous associations with resistance. Multivariate analysis found three independent factors associated with multidrug resistance: urinary catheter use (odds ratio [OR] 2.6, 95% confidence interval [CI] 1.4 to 4.8), age > or = 65 years (OR 3.0, 95% CI 1.7 to 5.4) and antibiotic use (OR 4.6, 95% CI 2.8 to 7.5). Diabetes was also a risk factor when patients with urinary catheters were excluded (OR 2.4, 95% CI 1.1 to 5.3). Resistance was seen in all groups of patients, but was particularly common in older patients and those who used a urinary catheter. Antibiotic use was highly associated with multidrug resistance. PMID- 10750919 TI - Percentage of emergency medicine residency graduates who got their first choice of jobs did not change between 1995 and 1997. AB - The objective of this study was to compare the number of emergency medicine (EM) graduates unable to find a job in the city/area of their first choice in 1995 and 1997. Self-administered questionnaires were distributed to EM residents who graduated in both 1995 and 1997. The survey ascertained resident's practice city and state, whether their job was in the city/area of first choice and how satisfied they were with their practice selection. Eighty (83%) programs and 507 (70%) of eligible residents participated. Thirty-eight percent of graduates chose to practice in the city or metropolitan area where they trained and 75% of respondents were very satisfied with their practice selection. Nineteen percent stated they would not be practicing in the city/area of their first choice; one third indicated there were no jobs available and two-thirds stated that jobs were available but not desirable. These numbers were similar to the 1995 data (P = .79). Job selection was more important than liking (P < .001) or having lived in (P < .001) a desired city/area of practice location. In conclusion, 1997 EM residency graduates were as successful as 1995 graduates in obtaining their first choice of jobs. PMID- 10750920 TI - Frequency and costs of laboratory and radiograph repetition in trauma patients undergoing interfacility transfer. AB - Receiving trauma centers often duplicate laboratory and radiograph testing performed by referring institutions. Our objective was to quantify frequency and costs of this practice. In this prospective study of 104 consecutive interfacility-transported adult trauma patients flown by an emergency medical service to an urban level I center, flight crew noted which labs and radiographs were done at referring hospitals, which tests were sent with patients, and which were repeated on trauma center arrival. Overall, results from 246 of 283 (86.9%) laboratory tests and 241 of 249 (96.8%) radiographs done at referring hospitals were sent with patients. Repetition of laboratory tests at the receiving hospital was frequent regardless of whether initial results were sent (P = .6 by chi2), and radiograph repetition was unrelated to whether sent films were originals or copies (P = .2 by chi2). For these 104 patients, the receiving hospital charged $66,463 for repetition of work-up done at referring facilities. PMID- 10750921 TI - Use of a complete neurological examination to screen for significant intracranial abnormalities in minor head injury. AB - Indications for head computed tomography (CT) scans are unclear in patients with nonpenetrating head injury and Glasgow Coma Scale (GCS) scores of 15. We performed a prospective study to determine if significant intracranial injury could be excluded in patients with GCS-15 and a normal complete neurological examination. A prospective trial of clinically sober adult patients with GCS = 15 on emergency department (ED) presentation after closed head injury with loss of consciousness or amnesia was conducted from May 1996 through April 1997. All subjects underwent a standardized neurological examination including mental status evaluation, and assessment of motor, sensory, cerebellar and reflex function before CT scan. During the study period, 58 patients met inclusion criteria. Fifty-five patients (95%) had normal CT scans and 23 (42%) had focal neurological abnormalities. Three patients (5%) had CT scan findings of acute intracranial injury, two of whom had normal neurological examinations. One patient had an acute subdural hematoma requiring emergent surgical decompression; the other had both an epidural hematoma and pneumocephalus that did not require surgery. Significant brain injury and need for CT scanning cannot be excluded in patients with minor head injury despite a GCS = 15 and normal complete neurological examination on presentation. PMID- 10750923 TI - EMT domestic violence knowledge and the results of an educational intervention. AB - The objective of this study was to determine levels of knowledge regarding domestic violence (DV), and the effectiveness of formal instruction about DV. A general knowledge survey of DV was given before and approximately 4 to 6 months after 3 hours of instruction given by Emergency Medicine and Law Enforcement faculty. A Emergency medical service (EMS) consisting of 73% paramedic-level providers in a metropolitan urban/suburban area. Differences in DV knowledge before and after the instruction were the main outcome measures. In the preinstruction series, 46 emergency medical technicians (EMTs) participated. After the instruction 19 EMTs participated (42%). Thirty-five percent of EMTs before instruction and 37% after instruction correctly identified the prevalence of DV against women as 15% to 30%. Thirty-five percent of EMTs before instruction and 63% after instruction (P < .05) correctly identified the prevalence of DV against men as 0% to 15%. Before instruction 54% knew that DV is equal among races, and 79% after instruction (P < .05). Before instruction 37% of EMTs knew that DV is equal in different socioeconomic groups and 68% after instruction (P < .05). The percent of EMTs who knew that the victim is not responsible for the abuse was 50% before instruction and 89% after instruction (P < .05). Before instruction, the results on a knowledge questionnaire were 54% correct, after instruction, results improved to 71% correct. Improvement in understanding of DV was shown for 4 of 11 questions after 3 hours of instruction. These results indicate the need for more instruction on DV for EMTs. PMID- 10750922 TI - Dispensing home nebulizers for acute wheezing from the hospital is cost effective. AB - The objective was to investigate the use characteristics of home nebulizers and to measure the benefit gained from dispensing home nebulizers (compared with their cost) to patients from the hospital. During the study period, August 28,1996 to May 17,1997, a sample of 232 of the 291 entries from a log of home nebulizers dispensed by the hospital respiratory care department were surveyed over the telephone. Of the 232 study subjects under the age of 21, a telephone interview of a guardian or supervising adult was completed in 106 subjects (46%) a mean of 43 weeks after the home nebulizer was prescribed (47% of the cohort received their home nebulizers from the inpatient service and another 47% were discharged with home nebulizers from the emergency department (ED)). An average of 3.6 estimated additional ED visits and 5.4 office/clinic visits for each patient were prevented by the home nebulizer. The benefit (savings from reduced ED and office visits alone) to cost ratio estimates range from $855:$90 to $1710:$90 or more. The overwhelming majority of the patients felt that the home nebulizer was a good idea, it was easy to use, they had no problems with the nebulizer and they received adequate training for home nebulizer use. Home nebulizers are a cost-effective means of providing home nebulized albuterol for selected outpatients. Hospital inpatient units and EDs which have the ability to dispense a home nebulizer, have an additional therapeutic option available for selected patients who may benefit from it. Medical insurance companies should fully support (ie, pay for) home nebulizers because it is cost effective. If there is any concern about the reliability of the patient to follow-up with their primary physician, the patient's primary physician should be contacted to discuss the feasibility of discharging the patient with a home nebulizer. PMID- 10750924 TI - An incident of hydrogen cyanide poisoning. AB - Seven cases of hydrogen cyanide gas poisoning which occurred in an industrial building in Hong Kong are presented here. Two of them were more severely injured and required specific antidotal treatment. The other five were mild and responded to supportive treatment alone. All except one patient recovered completely. Cyanide poisoning is relatively uncommon in urbanized area, so high index of suspicion is important for early diagnosis and treatment. We believe that prevention of cyanide poisoning can be achieved by proper storage of chemicals, and by enforcing rescuers to wear special chemical protective clothing to avoid systemic poisoning because of dermal absorption of hydrogen cyanide gas. As there are newer and safer cyanide antidotes available, each emergency department should have a stock of updated products such as hydroxocobalamin. PMID- 10750925 TI - A mortality index for postmarketing surveillance of new medications. AB - The rate of introduction of new pharmaceuticals is growing as a result of advances in molecular pharmacology and targeted drug development. The Fatal Toxicity Index (FTI) has been proposed as a means for monitoring drug toxicity through post-marketing surveillance. The FTI requires data regarding the general availability of a particular agent in the community which, in the US, is proprietary. The authors propose a Mortality Index as an alternative method for calculating relative lethality that does not rely on proprietary information for postmarketing surveillance. Using data from the Toxic Exposure Surveillance System (TESS) a Mortality Index was calculated from the proportion of deaths occurring among all patients who present to a health care facility with an overdose on the same agent or class of agents. The average Mortality Index for various drugs or drug classes for the years 1989 to 1997 is reported. Because the Mortality Index for desipramine appeared much greater than that for the other tricyclics, a chi-squared analysis was performed. The authors conclude, based on this analysis, that desipramine is significantly more likely to lead to death after overdosage than any other tricyclic antidepressant in the study. Also, the Mortality Index appeared to identify the impact of pediatric formulations on overdose lethality. We conclude that the Mortality Index may be a useful tool for determining the safety of agents during the postmarketing surveillance phase. PMID- 10750926 TI - Low molecular weight dextran attenuates increase in extravascular lung water caused by ARDS. AB - We studied the effect of low molecular weight dextran (mean molecular weight 40,000, Dextran 40; LMD) on the accumulation of extravascular lung water (EVLW), and also on hemodynamics and blood gases, in the oleic acid (OA)-injured lung in pentobarbital anesthetized rats. Starting just before the OA injection (0.01 mL/kg via femoral vein), 10% LMD in lactated Ringer's solution was infused throughout the experiment (5 mL/kg/h) instead of lactated Ringer's solution. OA caused acute lung injury leading to decreased oxygenation (PaO2: 87 +/- 11 mmHg versus control group 128 +/- 11) and an increased permeability of the alveolar capillary membrane, as shown by increases in EVLW (4.89 +/- 0.54 versus control group 4.07 +/- 0.14), and albumin leakage (0.043 +/- 0.015 versus control group 0.010 +/- 0.004). LMD protected against the increase in EVLW (4.14 +/- 0.10) and the hypoxemia (112 +/- 19 mmHg), but it did not reduce the albumin leakage into the alveolar space (0.052 +/- 0.009). These data suggest that LMD may limit the fluid accumulation that is secondary to OA-induced lung injury. PMID- 10750927 TI - Insertion of percutaneous ECMO cannula. AB - Extracorporeal membrane oxygenation plays a very important role in resuscitation when patients are approaching impending death, because it can provide adequate cardiac and pulmonary support immediately. But percutaneous tunnel creation is a critical step for set-up of extracorporeal membrane oxygenation by percutaneous Seldinger technique. A guidewire dilator forceps used in percutaneous tracheostomy was tried to create the femoral subcutaneous tunnel. We found it could make easy the advancement of the percutaneous cannula into the vessels and over-dilatation of the vessels could also be prevented by controlled jaw opening. PMID- 10750928 TI - Ultrasound interpretation of hydronephrosis is improved by a brief educational intervention. AB - Nineteen emergency medicine (EM) physicians (14 residents and 3 attendings) from an EM residency program which teaches ultrasound as part of the curriculum, were asked to rate 40 ultrasound scans showing different degrees of kidney hydronephrosis, first solely on the basis of their prior knowledge and experience. One week later, after a brief 15 minute lecture on a new objective method to read degrees of hydronephrosis, the same EM physicians were again asked to rate the 40 ultrasounds. One month later, to assess retention of the method, the same physicians were asked to read the same scans using the objective method presented 1 month prior. The three readings were compared with each other, and then each with a gold standard established for the study. Agreement of the group regarding scan interpretation improved and was maintained after the educational intervention (multirater kappa + .19, .32, and .32 for the three tests administered). When the differences between each week's readings and the gold standard were assessed, differences decreased with each successive test, and were statistically significant with the third test (P = .029). We conclude that our brief educational intervention improves agreement among physicians in readings of ultrasound scans and also significantly increases accuracy in readings when compared with a gold standard. PMID- 10750930 TI - 3D-CT diagnosis for ingested foreign bodies. AB - Ingested foreign bodies can be hard to diagnose but cannot be missed. We report two cases where helical computed tomography (three-dimensional computed tomography) was used for the effective preoperative diagnosis (swallowed fish bone-induced perforation of sigmoid colon and a case of ileus caused by ingested PTP [press-through package]). Other traditional diagnostic methods could not identify the foreign bodies. Three-dimensional computed tomography is useful for the diagnosis of foreign body ingestion and should be used for the difficult cases. PMID- 10750929 TI - Posterior shoulder dislocation: avoiding a missed diagnosis. AB - Posterior shoulder dislocation is a relatively uncommon event, with an incidence of 1% to 4% of all shoulder dislocations. Because of the infrequency of this condition, the diagnosis is often missed, with significant consequences to the patient Injury in the athlete is usually from a direct blow or fall onto an outstretched arm. After such an injury, symptoms may be confused with a shoulder contusion or rotator cuff injury. Significant complications such as chronic posterior dislocation and degenerative disease of the shoulder can occur if the diagnosis is missed. A careful history and physical examination, complete radiographic evaluation, and a high level of suspicion are required to identify posterior shoulder dislocation. Treatment consists of prompt closed reduction, or operative repair if this is unsuccessful. PMID- 10750931 TI - Rib fractures induced by coughing: an unusual cause of acute chest pain. AB - We report three patients with stress fractures of the ribs induced by coughing. Standard radiographs of the chest and ribs did not reveal evidence of rib fractures in any of the patients. Bone scintigraphy, performed 1 to 2 weeks after initial onset of symptoms, showed a focal area of increased uptake along the chest wall in all cases. Thin section angulated helical CT directly visualized the subtle rib fractures. Initial diagnosis of a cough-induced fracture of the rib may be difficult because of the associated underlying disorder, and unnecessary examinations are commonly performed. Identification of a cough induced fracture of the rib using helical CT may be clinically important to avoid unnecessary concern and additional examinations. PMID- 10750933 TI - Cases in electrocardiography. PMID- 10750932 TI - Ultrasonographic diagnosis of testicular torsion by emergency physicians. AB - Testicular torsion has an incidence of one case per 4,000 men/boys. Most frequently affected are pubescent boys. Average testicular salvage rate is only 50%, and infertility can result Testicular torsion remains problematic for emergency physicians (EPs) as clinical diagnosis can be difficult and other testicular pathology can present similarly. In many institutions there are delays in obtaining Doppler or Scintigraphy studies during off-hours. We report two cases of testicular torsion diagnosed by the treating EPs using power Doppler in the emergency department (ED). Rapid diagnosis of torsion led to successful salvage of the affected testicle in both cases. These represent the first cases for emergency screening ultrasound examinations (ESUEs) of testicular torsion in the literature. Rapid technological advances over the last decade have brought portable equipment with high-resolution capability to the bed-side for EPs, who should consider using it more frequently to evaluate testicular torsion. PMID- 10750934 TI - Etomidate-facilitated hip reduction in the emergency department. AB - Joint dislocations are common presenting complaints in emergency departments (EDs). Dislocations of major joints, such as the shoulder, elbow, and hip, are often difficult to gently reduce because of the challenge in obtaining sufficient relaxation of large muscle groups. This is the first reported use of etomidate to assist in the reduction of a major joint in an outpatient setting. We discuss the case of an elderly woman with total hip arthroplasty who experienced four spontaneous posterior hip dislocations in a 5-month period. Narcotics and benzodiazepines failed to facilitate reduction at every encounter, whereas etomidate made the procedure easy the two times it was used in the ED. This article reviews the administration of etomidate for conscious sedation and discusses potential complications. PMID- 10750935 TI - Emergency department presentations of patients with Parkinson's disease. AB - Parkinson's disease (PD) is a chronic progressive neurological disorder characterized by tremor, muscle rigidity, slowness of movement (bradykinesia), and gait instability. In early disease, PD is well managed in an office setting, however, as the disease progresses, a variety of syndromes may result in emergency department visits. The scenarios most likely to require an emergent evaluation are severe motor "off" periods with immobility, involuntary movements (dyskinesia), psychosis, acute confusion, panic disorder, and pain. Other less frequent presentations are also discussed. This article uses illustrative cases to provide a framework to discuss emergency department diagnosis and management issues in caring for these patients. PMID- 10750936 TI - Efficacy of magnesium sulfate in acute adult asthma: a meta-analysis of randomized trials. AB - The purpose of this article was to review of the literature to determine whether MgSO4 provides an additive improvement in adults with acute asthma in the emergency department (ED). English-language published and unpublished studies (1968-1998) were retrieved using MEDLINE, SCIENCE CITATION INDEX, CURRENT CONTENTS, bibliographic reviews of primary research, review articles, consultation with experts, and the register of Medical Editors' Trial Amnesty. Meta-analysis of randomized, placebo controlled trials. The five selected trials included 374 patients with acute asthma (mean age 38.3 +/- 13.4 years, 30% men, 70% women). The main outcome measure was pulmonary function; admission rate was evaluated as a secondary goal. Individual and pooled effect sizes (ES) were determined for pulmonary function data. Binary outcome (admission rate) was combined and reported as odds ratio (OR). The overall summary ES (95% confidence interval [CI]) of the five trials satisfying all inclusion criteria, weighted by sample size was nonsignificant (ES = 0.02, -0.20 to 0.24, P = .84). This pooled ES was equivalent to 1.7%, 95% CI: -9.8 to 13.1%) greater in pulmonary function the MgSO4 compared with the control group. On the other hand, four studies addressed the effect of MgSO4 administration on hospital admissions. Pooled results revealed that MgSO4 did not decrease significantly admission rates (OR = 0.68, 95% CI: 0.41 to 1.15). The existing evidence reveals that the addition of MgSO4 to ED patients with moderate to severe asthmatic exacerbations does not alter treatment outcomes. Nevertheless, the number and size of studies being pooled remain small. PMID- 10750937 TI - The uninsured: emergency medicine's challenge to our political leaders. PMID- 10750938 TI - The moonlighting paradox. PMID- 10750939 TI - Prolonged QT interval and torsades de pointes caused by the combination of fluconazole and amitriptyline. PMID- 10750940 TI - Rhabdomyolysis after correction of severe hyponatremia. PMID- 10750941 TI - Role of Alvarado score in diagnosis and treatment of suspected acute appendicitis. PMID- 10750942 TI - Acute St. John's wort toxicity. PMID- 10750943 TI - Myasthenic crisis presenting as isolated vocal cord paralysis. PMID- 10750944 TI - Group A streptococcal pharyngitis and scarlatiniform rash in an 8-week-old infant. PMID- 10750945 TI - Fetal bradycardia after IV adenosine for maternal PSVT. PMID- 10750946 TI - Premixed buffered lidocaine retains efficacy after prolonged room temperature storage. PMID- 10750947 TI - Seat belt injury to the abdominal aorta. PMID- 10750948 TI - On collective dose. PMID- 10750949 TI - Collective dose: kill or cure? PMID- 10750951 TI - In defence of collective dose. AB - Recent proposals for a new scheme of radiation protection leave little room for collective dose estimations. This article discusses the history and present use of collective doses for occupational, ALARA, EIS and other purposes with reference to practical industry papers and government reports. The linear no threshold (LNT) hypothesis suggests that collective doses which consist of very small doses added together should be used. Moral and ethical questions are discussed, particularly the emphasis on individual doses to the exclusion of societal risks, uncertainty over effects into the distant future and hesitation over calculating collective detriments. It is concluded that for moral, practical and legal reasons, collective dose is a valid parameter which should continue to be used. PMID- 10750950 TI - Can we put aside the LNT dilemma by the introduction of the controllable dose? AB - Recently, Professor R Clarke, ICRP Chairman, published his proposal for a renewal of the basic radiation protection concept (1999 J. Radiol. Prot. 19 107-15). The two main points of his proposed system are: (a) the term 'controllable dose' is introduced and (b) the protection philosophy is based on the individual. For practical use terms like 'action level', 'investigation level' etc are introduced. The outline of the new system promises a much less complex frame; no distinction between practices and interventions and unified treatment for occupational, medical and public exposures. There is, however, an inconsistency within the new system: though linearity is not assumed, the relations between the definitions of the new terms of the system of protection and the doses assigned to them are still based on the LNT hypothesis. To avoid this inconsistency a new definition of action level is recommended as a conservative estimate of the lowest dose where harmful effects have ever been demonstrated. Other levels should be defined by the action level and safety factors applied on the doses. PMID- 10750952 TI - Radiological risk in perspective: risks and benefits for comforters and carers. AB - This paper discusses the meaning and origins of the concept of 'comforters and carers' and cites some examples. The application of the Ionising Radiations Regulations 1985 to this group of individuals is described, and the requirements of the European Council directives on protection against ionising radiations are outlined. The paper looks forward to the way these individuals are likely to be treated under the forthcoming revised Ionising Radiations Regulations and describes, with examples, HSE's approach to 'comforters and carers'. PMID- 10750953 TI - Environmental effects of radionuclides--observations on natural ecosystems. AB - To better quantify risk to non-human species from exposure to environmental radioactivity, understanding of the behaviour of radionuclides in the biosphere needs to be increased. This study outlines current thinking on ecological risk assessment (ERA) methodology and applies the indicator species or critical groups approach to biota inhabiting a semi-natural coniferous woodland contaminated with the radionuclides 137Cs, 238Pu, 239+240Pu and 241Am. The majority of these radionuclides originate from routine aerial emissions from the nuclear fuel reprocessing plant at BNFL, Sellafield, Cumbria, UK. Radionuclide activity concentrations have been determined in biota from the woodland and estimates of absorbed dose rates (mGy d(-1)) have been calculated using the dosimetric models outlined. Dose rates to the key indicator species, Oniscus asellus, Carabus violaceous and Apodemus sylvaticus (detritivorous invertebrate, predatory invertebrate and the granivorous wood mouse) have been determined at 3.0 x 10(-3) mGy d(-1), 2.2 x 10(-3) mGy d(-1) and 1.0 x 10(-3) mGy d(-1) respectively. The values are at least three orders of magnitude lower than the 1 mGy d(-1) level below which no observable effects on populations in a terrestrial ecosystem are thought to occur. Limitations of this approach are discussed. PMID- 10750954 TI - Natural radioactivity and radiation exposure at the Minjingu phosphate mine in Tanzania. AB - In this paper the results of studies on activity and ambient radiation background around the Minjingu phosphate mine in Tanzania are presented. The outdoor dose rate in air and the activity levels of samples from and outside the mine were determined by thermoluminiscent dosimeters and a gamma spectrometer system with a Hyper Pure germanium detector system respectively. The determination of activity was made for the 226Ra, 228Ra, 228Th and 40K radionuclides. High concentrations of radium-226 were observed in phosphate rock (5760+/-107 Bq kg(-1)), waste rock (4250+/-98 Bq kg(-1)), wild leaf vegetation (650+/-11 Bq kg(-1)), edible leaf vegetation (393+/-9 Bq kg(-1)), surface water (4.7+/-0.4 mBq l(-1)) and chicken feed (4+/-0.1 Bq kg(-1)) relative to selected control sites. These findings suggest a radiation health risk particularly when the samples are ingested, because the internal exposure may give rise to an effective dose exceeding 20 mSv which is the annual limit of intake of natural radionuclides recommended by the ICRP. On the other hand, the radiation dose from ambient air over five years at the phosphate mine ranges from 1375 to 1475 nGy h(-1) with an average of 1415 nGy h(-1). The average is about 28 times that of the global average background radiation from terrestrial sources, and about 12 times the allowed average dose limit for public exposure over five consecutive years. Future investigations on the occupancy factor, external dose rate and radon and radon progeny exposure in drinking water, buildings and activity content in the locally grown foodstuffs are proposed, for the realistic quantification of the overall exposure of workers and public at Minjingu, and remedial measures for future radiation safety. PMID- 10750955 TI - A study of energy and effective atomic number dependence of the exposure build-up factors in biological samples. AB - A theoretical method is presented to determine the gamma-radiation build-up factors in various biological materials. The gamma energy range is 0.015-15.0 MeV, with penetration depths up to 40 mean free paths considered. The dependence of the exposure build-up factor on incident photon energy and the effective atomic number (Zeff) has also been assessed. In a practical analysis of dose burden to gamma-irradiated biological materials, the sophistication of Monte Carlo computer techniques would be applied, with associated detailed modelling. However, a feature of the theoretical method presented is its ability to make the consequences of the physics of the scattering process in biological materials more transparent. In addition, it can be quickly employed to give a first-pass dose estimate prior to a more detailed computer study. PMID- 10750957 TI - Bridging radiation policy and science. PMID- 10750956 TI - Preliminary fact finding mission following the accident at the nuclear fuel processing facility in Tokaimura, Japan (Vienna: IAEA) PMID- 10750958 TI - Second-event theory reviewed. PMID- 10750959 TI - SRP Meeting: Land Remediation--Liabilities and Practicalities. PMID- 10750960 TI - Acute suppurative thyroiditis due to foreign body-induced retropharyngeal abscess presented as thyrotoxicosis. AB - Acute suppurative thyroiditis is an uncommon condition. Most patients have preexisting oropharyngeal fistulae. Penetrating oropharyngeal injuries resulting from swallowed foreign bodies provide an acquired channel of infection spreading into the relatively resistant thyroid gland. The authors describe a patient with infective thyroiditis complicating retropharyngeal abscess caused by a chicken bone that perforated the upper esophagus. Transient thyrotoxicosis complicating acute suppurative thyroiditis is very rare. Pertechnetate and Ga-67 scans confirmed extensive inflammation of the thyroid gland and the release of hormones as the cause, as distinct from concurrent Graves' disease. Awareness of this unusual complication is important to avoid inappropriate treatment for hyperthyroid disease. PMID- 10750961 TI - Dual thyroid ectopy: case report and review of the literature. AB - Dual ectopic thyroid glands rarely occur, and only six cases have been reported in the literature. The authors describe a 14-year-boy who had a midline neck swelling for 8 to 9 years. The swelling increased gradually, but he had no pressure symptoms. The thyroid hormone profile showed a moderately increased thyroid-stimulating hormone value and normal T3 and T4 levels. A Tc-99m sodium pertechnetate thyroid scan showed dual ectopic thyroid glands in the sublingual and subhyoid regions. PMID- 10750962 TI - Underestimation of regional myocardial perfusion with Tc-99m sestamibi single-day rest-stress SPECT: a "drug washout" pitfall? AB - Myocardial perfusion scintigraphy with a Tc-99m sestamibi single-day SPECT protocol is a widely used technique to examine patients with possible or known coronary artery disease. A 76-year-old man with a clinical history suggestive of ischemic heart disease underwent Tc-99m sestamibi myocardial SPECT imaging with a same-day rest and stress protocol after temporary discontinuation of his current therapy, which included calcium channel and beta blockers and nitrates. The scintigraphic pattern was consistent with an asymptomatic infarction of the posterolateral myocardial wall and periinfarct ischemia. One week later, the patient had a Tc-99m sestamibi myocardial SPECT study at rest without discontinuing therapy, and scintigraphic images showed normalization of the posterolateral wall perfusion defect. The angiographic study showed a 90% stenosis of the circumflex artery. This case suggests that, during a 1-day cardiac SPECT protocol, washout of therapeutic pharmaceuticals may be responsible for underestimation of myocardial rest perfusion in territory supplied by a coronary artery with a critical stenosis. PMID- 10750963 TI - Determination of glomerular filtration rate using a dual-detector gamma camera and the geometric mean of renal activity: correlation with the Tc-99m DTPA plasma clearance method. AB - PURPOSE: In this study, the authors studied the use of a dual-detector gamma camera to measure the glomerular filtration rate (GFR). METHODS: Thirty-three patients with a wide range of renal function participated in this study. The GFR was measured using a dual-detector gamma camera by calculating the geometric mean of activity from each kidney and using an outline background. These results were compared with the GFR estimates obtained from Tc-99m DTPA plasma clearance using a multiple blood sample method. RESULTS: Correlation was excellent between GFR estimated using the dual-detector gamma camera and GFR measured using the plasma clearance of Tc-99m DTPA with multiple blood samples (r = 0.89). The correlation was especially strong in children younger than 13 years (r = 0.94). CONCLUSION: Measuring the GFR using a dual-detector gamma camera and calculating the geometric mean of renal activity yields relatively accurate results. PMID- 10750964 TI - Coexisting renal vein thrombosis and bilateral adrenal hemorrhage: renoscintigraphic demonstration. AB - A rare case of simultaneous renal vein thrombosis and bilateral adrenal hemorrhage is presented, showing the utility of Tc-99m DTPA and DMSA renal scans to identify the condition and to assess renal function. The characteristic appearances of suprarenal tracer-free areas encircled by peripheral radioactive rims over the inferiorly displaced kidneys on a Tc-99m DTPA renal scan, and that of the flattened upper poles of the kidneys on a Tc-99m DMSA scan, were pathognomonic and strongly indicative of bilateral adrenal hemorrhage. These two scans also showed markedly diminished tracer uptake in the poorly functioning right thrombosed kidney. Follow-up imaging using these two renal scans well demonstrated the functional and morphologic alterations and recovery of the kidneys. PMID- 10750965 TI - A qualitative analysis of brain SPECT for prognostication of gross motor development in children with cerebral palsy. AB - PURPOSE: In this report, the authors assessed the clinical significance of decreased regional cerebral blood flow (rCBF) in the thalamus or cerebellar hemispheres in relation to gross motor performance in the children with cerebral palsy. MATERIALS AND METHODS: Thirty-six children with bilateral spastic cerebral palsy (BSCP) underwent brain SPECT. Visual analysis was used for the brain SPECT interpretation. The rCBF in the thalamus or cerebellum was graded as normal, mildly decreased, or severely decreased. A marked decrease or near absence of rCBF in the thalamus or cerebellum was considered as severely decreased. RESULTS: All 36 children with BSCP had hypoperfusion in the thalamus or cerebellar hemispheres. Eight of 20 children (40%) with mildly decreased rCBF on brain SPECT had mild developmental delays. On the other hand, only 1 of 16 children (6.3%) with severe hypoperfusion in the thalamus or cerebellum had a mild developmental delay, and the remaining 15 of 16 children (93.8%) had severe developmental delays. There was good correlation between the degree of developmental delay and the severity of hypoperfusion in the thalamus or cerebellum (P = 0.023). CONCLUSION: The measurement of rCBF by Tc-99m ethyl cysteinate dimer brain SPECT appears to be valuable in prognostication of gross motor development in children with BSCP. PMID- 10750966 TI - F-18 fluorodeoxyglucose chest uptake in lung inflammation and infection. AB - PURPOSE: F-18 fluorodeoxyglucose (FDG) may accumulate at sites of inflammation or infection, making interpretation of whole-body scans difficult in patients with cancer. METHODS: More than 650 whole-body positron emission tomographic (PET) scans performed to examine patients with cancer were reviewed to identify uptake in pulmonary infection or inflammation based on the appearance of F-18 FDG chest uptake, chest radiographs, computed tomography, or all of these. RESULTS: Ten patients had uptake in benign lung disease. Eight patients had head and neck tumors and two patients had breast cancer. Intense focal or multifocal F-18 FDG chest uptake was seen in 6 of 10 scans. This was difficult to distinguish from pulmonary metastases based on the scan appearance. However, in the remaining patients, the uptake was atypical for malignancy and displayed an apical, segmental, or lobar pattern. In all patients, the F-18 FDG lung uptake corresponded to benign radiologic changes (infiltration, consolidation, or atelectasis), and the final diagnosis was pulmonary inflammation or infection. Nine patients were asymptomatic and one patient had clinical aspiration pneumonia. Follow-up PET scans were performed in five patients to evaluate their conditions. Chest uptake disappeared completely in three patients and partially in two patients, and there were no new findings. Variable degrees of F-18 FDG chest uptake have been reported with more than 40 different benign causes. They can be classified based on the underlying mechanism into four major categories: 1) Inflammation or infection, 2) benign tumor, 3) physiologic activity, and 4) iatrogenic. Most of these false-positive cases are included in the first category. CONCLUSIONS: Pulmonary infection or inflammation might predispose patients to localized F-18 FDG chest uptake mimicking pulmonary metastases and limiting the specificity of whole-body scans performed in patients with cancer. PMID- 10750967 TI - Reduced F-18 fluorodeoxyglucose uptake within marrow after external beam radiation. AB - PURPOSE: The authors examined the delayed effects of external beam radiation on the metabolism of intravertebral marrow cavities. MATERIALS AND METHODS: Two neurologically normal persons who had completed a full course of external beam radiotherapy for squamous cell carcinoma of the head and neck had positron emission tomography (PET) scans with F-18 fluorodeoxyglucose (FDG) to identify residual neoplasm. RESULTS: Abnormally low uptake of F-18 FDG was noted within the central marrow cavities of the cervical vertebral bodies in both cases. CONCLUSIONS: Diminished metabolic activity within irradiated marrow can be revealed by F-18 FDG PET imaging techniques. Further correlative MRI PET studies are needed to determine if this postradiation phenomenon is related to fatty infiltration of the marrow cavity. PMID- 10750968 TI - Exclusion of chronic osteomyelitis with F-18 fluorodeoxyglucose positron emission tomographic imaging. AB - PURPOSE: Excluding the diagnosis of chronic osteomyelitis is often difficult with noninvasive techniques, especially when bone anatomy and structure have been altered by trauma, surgery, or soft-tissue infection. It has been reported that fluorodeoxyglucose (FDG) positron emission tomography (PET) has excellent potential to diagnose osteomyelitis. In this study, the accuracy of FDG PET in the diagnosis of chronic osteomyelitis was determined. METHOD: Twenty-two patients with possible osteomyelitis (5 in the tibia, 5 in the spine, 4 in the proximal femur, 4 in the pelvis, 2 in the maxilla, and 2 in the feet) who underwent FDG PET imaging and on whom operative or clinical follow-up data were available were included in this analysis. The final diagnosis was made by surgical exploration or clinical follow-up during a 1-year period. RESULTS: FDG PET correctly diagnosed the presence or absence of chronic osteomyelitis in 20 of 22 patients. Six had chronic osteomyelitis and 16 proved to be free of osteomyelitis. FDG PET correctly identified all six patients with chronic osteomyelitis but produced two false-positive results. This study had a sensitivity rate of 100%, a specificity rate of 87.5%, and an accuracy rate of 90.9%. CONCLUSION: FDG PET is a highly effective imaging method to exclude osteomyelitis when a negative scan result is obtained. However, positive results can be caused not only by true osteomyelitis but also by inflammation in the bone or surrounding soft tissues as a result of other causes. Overall, FDG PET may prove to be the preferred study in the management of patients with possible chronic osteomyelitis. PMID- 10750969 TI - A case of local recurrent pheochromocytoma: usefulness of I-123 MIBG early SPECT and maximum intensity projection images. AB - It has been reported that 10% of patients with pheochromocytoma experience recurrence despite surgery. The authors administered I-123 MIBG to patients with recurrent pheochromocytoma and found that early images are more useful than delayed images to detect recurrence. Early imaging with I-123 MIBG is useful for postoperative follow-up studies in patients with pheochromocytoma and those with possible tumor recurrence. PMID- 10750970 TI - Sequestered collection in association with infected arthroplasty. PMID- 10750972 TI - Venous bypass after deep venous thrombosis visible on an early-phase bone scan. PMID- 10750971 TI - Intrathoracic mass simulated by ectopic kidney. PMID- 10750973 TI - Mediastinal neurilemmoma demonstrated by positive Tc-99m(V) DMSA SPECT and negative Ga-67 uptake. PMID- 10750974 TI - Tc-99m MDP uptake by an advanced colon cancer lesion in a juvenile patient. PMID- 10750975 TI - Sign of the school knapsack: myositis of the trapezius muscles caused by overload. PMID- 10750976 TI - An avascular crisis in Gaucher's disease. PMID- 10750977 TI - Bile duct stenosis seemingly caused by a giant pedunculated hemangioma with hypogastric growth. PMID- 10750979 TI - Gastric volvulus detected with iodine-131 whole-body imaging. PMID- 10750978 TI - Uterine visualization on Tc-99m DTPA HSA scintigraphy to detect gastrointestinal protein loss. PMID- 10750980 TI - Pleural activity in a patient with a lumbopleural shunt after intrathecal injection of Tc-99m DTPA. PMID- 10750981 TI - The Colyte renal scan. PMID- 10750982 TI - F-18 fluorodeoxyglucose positron emission tomographic imaging: in search of an unknown primary tumor. PMID- 10750983 TI - Nonvisualization of the common bile duct and normal biliary to bowel transit: an indirect sign of bilioenteric fistula. PMID- 10750984 TI - Hepatobiliary imaging in a patient with liver abscess: interesting diagnostic findings. PMID- 10750986 TI - Current readings in nuclear medicine. PMID- 10750985 TI - Abnormal uptake of I-131 mimicking salivary gland uptake in a patient with diffuse dental disease. PMID- 10750987 TI - Letters to the editor PMID- 10750988 TI - Biomechanical analysis of a double-bundle posterior cruciate ligament reconstruction. AB - The objective of this study was to experimentally evaluate a single-bundle versus a double-bundle posterior cruciate ligament reconstruction by comparing the resulting knee biomechanics with those of the intact knee. Ten human cadaveric knees were tested using a robotic/universal force-moment sensor testing system. The knees were subjected to a 134-N posterior tibial load at five flexion angles. Three knee conditions were tested: 1) intact knee, 2) single-bundle reconstruction, and 3) double-bundle reconstruction. Posterior tibial translation of the intact knee ranged from 4.9 +/- 2.7 mm at 90 degrees to 7.2 +/- 1.5 mm at full extension. After the single-bundle reconstruction, posterior tibial translation increased to 7.3 +/- 3.9 mm and 9.2 +/- 2.8 mm at 90 degrees and full extension, respectively, while the corresponding in situ forces in the graft were up to 44 +/- 19 N lower than those in the intact ligament. Conversely, with double-bundle reconstruction, the posterior tibial translation did not differ significantly from the intact knee at any flexion angle tested. This reconstruction also restored in situ forces more closely than did the single bundle reconstruction. These data suggest that a double-bundle posterior cruciate ligament reconstruction can more closely restore the biomechanics of the intact knee than can the single-bundle reconstruction throughout the range of knee flexion. PMID- 10750989 TI - The effect of a geographic lateral bone bruise on knee inflammation after acute anterior cruciate ligament rupture. AB - We prospectively evaluated 40 patients who had knee inflammation after isolated anterior cruciate ligament rupture with or without an associated "geographic" bone bruise/subchondral fracture of the lateral femoral condyle. All patients with acute ruptures documented by magnetic resonance imaging within 1 week of injury were evaluated for a geographic bone bruise/subchondral fracture of the lateral femoral condyle. Two groups of 20 patients each (bone bruise versus no bone bruise) were then enrolled. Variables measured at 1, 2, 3, and 4 weeks after injury included pain, range of motion, effusion, and number of days with an antalgic gait. Patients with a bone bruise had increased size and duration of effusion, increased number of days required to nonantalgic gait without external aids, increased days to achieve normal range of motion, and increased pain scores at measured time intervals. This study confirms results of previous clinical and histologic studies showing an associated articular cartilage lesion, otherwise known as bone bruise/subchondral fracture, is clinically significant. There appears to be an association between a geographic bone bruise and increased disability in patients with acute anterior cruciate ligament ruptures. Patients with a geographic bone bruise may require longer to reach normal homeostasis (range of motion, pain, neuromuscular control) before undergoing anterior cruciate ligament reconstruction. PMID- 10750990 TI - Open revision stabilization surgery for recurrent anterior glenohumeral instability. AB - Fifty patients (average age, 27 years) who underwent revision anterior stabilization surgery for failed anterior glenohumeral instability procedures were retrospectively reviewed. Failure of the original procedure occurred subsequent to significant trauma in only 17 of 50 shoulders. At revision, 49 shoulders underwent an anteroinferior capsular shift procedure and 23 underwent concurrent repair of a Bankart lesion. One shoulder was treated with a coracoid transfer to reconstruct the anteroinferior glenoid. At an average follow-up of 4.7 years (range, 2 to 10), there were 36 excellent and 3 good results (78%). Eleven shoulders were considered unsatisfactory (22%); 7 of these 11 patients had a diagnosis of voluntary dislocation. All 17 patients who had failed results after significant trauma had excellent results after revision surgery. However, only 22 of the 33 patients (67%) with atraumatic recurrent instability achieved excellent or good results after revision surgery. This difference was statistically significant. No patients had radiographic evidence of osteoarthritis at the most recent follow-up. Range of motion, return to function, and glenohumeral stability can be reliably restored in a high percentage of patients after revision anterior stabilization surgery for glenohumeral instability. However, the results are not as predictable as for primary surgery. Factors associated with poor results of revision repair included an atraumatic cause of failure, voluntary dislocations, and multiple prior stabilization attempts. PMID- 10750991 TI - Quantification of anterior translation of the humeral head in the throwing shoulder. Manual assessment versus stress radiography. AB - Clinical evaluation of humeral head translation relies mainly on manual tests to measure laxity in the human shoulder. The purposes of this study were to determine whether side-to-side differences exist in anterior humeral head translation in professional baseball pitchers, to compare manual laxity testing with stress radiography for quantifying humeral head translation, and to test intrarater reliability of the manual humeral head translation and stress radiography tests. Twenty professional baseball pitchers underwent bilateral manual anterior humeral head translation and stress radiographic tests. Stress radiography was performed by imparting a 15-daN anterior load to the shoulder in 90 degrees of abduction with both neutral and 60 degrees of external rotation and recording the glenohumeral joint translation at rest and under stress in each position. Eight subjects were retested to assess the reliability of these methods. Results showed no significant difference between the dominant and nondominant extremity in the amount of anterior humeral head translation measured manually and with stress radiography, nor significant correlation between anterior humeral head translation measured manually and by stress radiography. Test-retest reliability was moderate-to-poor for the manual humeral head translation test and moderate for stress radiography. PMID- 10750992 TI - Anterior cruciate ligament reconstruction in adolescents with open physes. AB - The purpose of this study was to evaluate anterior cruciate ligament reconstructions performed in adolescents with open physes and a skeletal age of at least 14 years. At one center, from 1992 to 1996, 19 adolescents (ages, 11 to 15 years) with open physes and a skeletal age of at least 14 years underwent arthroscopic anterior cruciate ligament reconstruction using an Achilles tendon allograft placed through drill holes across the open physes in both the distal femur and proximal tibia. Fifteen patients returned for reevaluation at an average of 25 months postoperatively (range, 12 to 60 months); the remaining four patients were interviewed by telephone. There were no significant leg-length discrepancies or angular deformities as determined by scanograms and anteroposterior and lateral radiographs of the femur and tibia. The mean Lysholm knee score was 97 (range, 94 to 100) and the mean KT-1000 arthrometer side-to side difference at 20 pounds of anterior force was 1.7 mm (range, 0.0 to 3.0). All patients were satisfied with the results of surgery, and 16 of 19 patients returned to the same sport they were participating in before the injury. This study demonstrates that anterior cruciate ligament reconstruction using an Achilles tendon allograft is a viable treatment option for skeletally immature patients with a skeletal age of 14 years who have sustained midsubstance tears of the anterior cruciate ligament. PMID- 10750993 TI - Does nitric oxide help explain the differential healing capacity of the anterior cruciate, posterior cruciate, and medial collateral ligaments? AB - This study compared the ability of rabbit medial collateral ligament, posterior cruciate ligament, and anterior cruciate ligament tissue to synthesize nitric oxide, and determined its effects on matrix synthesis, an important component of ligament repair. It is not known whether ligament cells can produce nitric oxide and, if so, whether it influences healing of ligament injuries. The anterior cruciate and posterior cruciate ligament tissue produced large amounts of nitric oxide in response to the inflammatory cytokine interleukin-1. Medial collateral ligament, in contrast, produced only modest amounts of nitric oxide. Furthermore, anterior cruciate ligament and, to some degree, posterior cruciate ligament synthesized nitric oxide spontaneously in culture, whereas medial collateral ligament never did so. When nitric oxide was supplied to these tissues, it strongly inhibited collagen synthesis by the two cruciate ligaments, but had little effect on collagen synthesis by the medial collateral ligament. Endogenously synthesized nitric oxide was also able to inhibit collagen synthesis as well as proteoglycan synthesis by the two cruciate ligaments, but had little effect on matrix synthesis by the medial collateral ligament. We propose a novel hypothesis, based on nitric oxide production and matrix synthesis, that may help explain why the two cruciate ligaments have such limited healing capacity compared with the medial collateral ligament. PMID- 10750994 TI - Open and arthroscopic patellar tenotomy for chronic patellar tendinopathy. A retrospective outcome study. Victorian Institute of Sport Tendon Study Group. AB - Chronic patellar tendinopathy often requires surgical treatment. We compared the outcomes in 25 subjects (29 tendons) who had had open patellar tenotomy and 23 subjects (25 tendons) who had had arthroscopic patellar tenotomy at a mean follow up of 3.8 and 4.3 years, respectively. At follow-up, outcomes in the open and arthroscopic groups were as follows: 1) symptomatic benefit was seen in 81% of open and 96% of arthroscopic tenotomy patients, 2) sporting success was seen in 54% of open and 46% of arthroscopic tenotomy patients, 3) median time to return to preinjury level of activity was 10 months for open and 6 months for arthroscopic tenotomy patients, and 4) median Victorian Institute of Sport Assessment score at follow-up was 88 for open and 77 for arthroscopic tenotomy patients. There were no significant differences between groups for all outcomes. The appearance of the tendon on sonography remained abnormal in over 70% of subjects at follow-up, and sonographic appearance did not correlate with clinical outcome. Thus, arthroscopic patellar tenotomy was as successful as the traditional open procedure. Both procedures provided virtually all subjects with symptomatic benefit, but only about half the subjects who underwent either open or arthroscopic patellar tenotomy were competing at their former sporting level at follow-up. PMID- 10750995 TI - The magnetic resonance imaging appearance of individual structures of the posterolateral knee. A prospective study of normal knees and knees with surgically verified grade III injuries. AB - The purpose of this study was to contrast the magnetic resonance imaging appearance of uninjured components of the posterolateral knee with that of injured structures, and to assess the accuracy of magnetic resonance imaging in identifying posterolateral knee complex injuries. Thin-slice coronal oblique T1 weighted images through the entire fibular head were used to identify the posterolateral structures in seven uninjured knees. The appearance of corresponding grade III injuries to these structures was identified prospectively in 20 patients and verified at the time of surgical reconstruction. The sensitivity, specificity, and accuracy of imaging for the most frequently injured posterolateral knee structures in this series were as follows: iliotibial band deep layer (91.7%, 100%, and 95%), short head of the biceps femoris-direct arm (81.3%, 100%, and 85%), short head of the biceps femoris-anterior arm (92.9%, 100%, and 95%), midthird lateral capsular ligament-meniscotibial (93.8%, 100%, and 95%), fibular collateral ligament (94.4%, 100%, and 95%), popliteus origin on femur (93.3%, 80%, and 90%), popliteofibular ligament (68.8%, 66.7%, and 68%), and the fabellofibular ligament (85.7%, 85.7%, and 85.7%). Magnetic resonance imaging of the knee was accurate in the identification of these injuries. PMID- 10750996 TI - Ligamentous restraints to external rotation of the humerus in the late-cocking phase of throwing. A cadaveric biomechanical investigation. AB - The late-cocking phase of throwing is characterized by extreme external rotation of the abducted arm; repeated stress in this position is a potential source of glenohumeral joint laxity. To determine the ligamentous restraints for external rotation in this position, 20 cadaver shoulders (mean age, 65 +/- 16 years) were dissected, leaving the rotator cuff tendons, coracoacromial ligament, glenohumeral capsule and ligaments, and coracohumeral ligament intact. The combined superior and middle glenohumeral ligaments, anterior band of the inferior glenohumeral ligament, and the entire inferior glenohumeral ligament were marked with sutures during arthroscopy. Specimens were mounted in a testing apparatus to simulate the late-cocking position. Forces of 22 N were applied to each of the rotator cuff tendons. An external rotation torque (0.06 N x m/sec to a peak of 3.4 N x m) was applied to the humerus of each specimen with the capsule intact and again after a single randomly chosen ligament was cut (N = 5 in each group). Cutting the entire inferior glenohumeral ligament resulted in the greatest increase in external rotation (10.2 degrees +/- 4.9 degrees). This was not significantly different from sectioning the coracohumeral ligament (8.6 degrees +/- 7.3 degrees). The anterior band of the inferior glenohumeral ligament (2.7 degrees +/- 1.5 degrees) and the superior and middle glenohumeral ligaments (0.7 degrees +/- 0.3 degrees) were significantly less important in limiting external rotation. PMID- 10750997 TI - Assessment of the infraspinatus spinal stretch reflex in the normal, athletic, and multidirectionally unstable shoulder. AB - To examine neural aspects of motor control in the glenohumeral joint, this study evaluates utilization of an innate spinal segmental pathway, the spinal stretch reflex, as an investigational tool that reflects neural circuitry. The purpose of this study was to determine if this reflex could be evoked from the infraspinatus muscle, if the testing apparatus and protocol for elicitation were reliable, and if the reflex response varies between groups of subjects and therefore could be useful clinically. These reflex characteristics were evaluated in the infraspinatus muscle, since rotator cuff muscle activity in subjects with glenohumeral instability exhibits differences in electromyographic activity and coordination patterns, implicating its role in dynamic stability. Normal shoulders were compared with athletic shoulders and shoulders with multidirectional instability. The spinal stretch reflex was elicited in a controlled and reliable manner. Shoulders with multidirectional instability exhibited a more-prominent spinal stretch reflex response than normal shoulders, whereas athletic shoulders exhibited a more-quiescent spinal stretch reflex response. As the spinal stretch reflex probably plays a role in motor control, variation in this reflex profile may reflect some differences in development that contribute to the variable expression of dynamic glenohumeral stability. This study suggests that the spinal stretch reflex profile may be a useful clinical tool to assist in discriminating between the normal and pathologic state. This information may also be useful in the evaluation of new treatment approaches exploiting spinal cord plasticity and spinal stretch reflex mutability through neuromuscular training. PMID- 10750998 TI - Association of burners with cervical canal and foraminal stenosis. AB - The purpose of this study was to determine whether the burner phenomenon is associated with cervical canal and foraminal stenosis in a scholastic population. Lateral cervical radiographs were reviewed for 64 athletes, 15 to 18 years of age, who had sustained at least one burner. Controls consisted of age-matched athletes who had sustained head or neck trauma without evidence of the burner phenomenon (N = 32). Pavlov ratios were calculated for levels C-3 through C-6; both mean minimum and mean average ratios were determined. Available oblique radiographs from both the study (N = 31) and control (N = 15) groups were then used to calculate the foramen/vertebral body ratio--a measure of relative foraminal height. Significant differences were found between the burner and control groups for the mean minimum and mean average Pavlov ratios and foramen/vertebral body ratios. Scholastic athletes sustaining the burner phenomenon have an increased risk of cervical canal and foraminal stenosis as measured by the Pavlov and foramen/vertebral body ratios, respectively. The foramen/vertebral body ratio is an easily reproducible and reliable means of assessing foraminal dimensions from oblique radiographs and controls for x-ray magnification and rotation. Foraminal stenosis assessment may prove useful in predicting burner risk, especially in athletes with extension-compression injuries. PMID- 10750999 TI - A radiographic and histologic evaluation of the patellar tendon after harvesting its central third. AB - Nineteen consecutive patients undergoing anterior cruciate ligament reconstruction using the central third of the ipsilateral patellar tendon were included in the study. Serial magnetic resonance images revealed that the donor site gap in the tendon decreased with time (mean follow-up, 26 months). The thickness was significantly increased compared with the intact contralateral patellar tendon, regardless of when the magnetic resonance imaging was performed. Ultrasonography showed the same findings at a mean follow-up of 26 months. Histologic evaluation of the repair tissue in the central part of the tendon, as well as the tissue in the peripheral part of the patellar tendon at the donor site, revealed a significant increase in cellularity and vascularity as compared with normal control tendons. Thus, 2 years after the harvesting procedure, the patellar tendon displayed significant radiographic and histologic abnormalities. On the basis of these findings, reharvest of the patellar tendon, at least up to 2 years after primary harvest, cannot be recommended. PMID- 10751000 TI - Comparison of soccer shin guards in preventing tibia fracture. AB - The goal of this study was to evaluate the effectiveness of a number of shin guards in protecting against tibia fracture in soccer players. A secondary purpose was to determine the relationship between the material and structural differences in shin guard design and the protection provided. Twenty-three commercially available shin guards were tested on a model leg containing a synthetic tibia that had been calibrated against human cadaver specimens. Each guard was categorized into one of four material types: plastic (N = 9), fiberglass (N = 6), compressed air (N = 4), and Kevlar (N = 4). The maximum combined force at the ends of the tibia, the principal strain on the posterior side of the tibia, and the contact time of the impact were measured using a drop track impact simulation. Shin guards provided significant protection from tibia fracture at all drop heights. The average guard reduced force by 11% to 17% and strain by 45% to 51% compared with the unguarded leg. At the higher drop heights, material composition and structural characteristics of the shin guards showed significant differences in protective abilities. These findings indicate that all shin guards provide some measure of protection against tibia fracture, although the level of protection may vary significantly among the different guards. PMID- 10751001 TI - Electromyographic and kinematic analysis of cutting maneuvers. Implications for anterior cruciate ligament injury. AB - The objective of this study was to qualitatively characterize quadriceps and hamstring muscle activation as well as to determine knee flexion angle during the eccentric motion of sidestep cutting, cross-cutting, stopping, and landing. Fifteen healthy collegiate and recreational athletes performed the four movements while knee angle and electromyographic activity (surface electrodes) of the vastus lateralis, vastus medialis obliquus, rectus femoris, biceps femoris, and medial hamstring (semimembranosus/semitendinosus) muscles were recorded. The results indicated that there is high-level quadriceps muscle activation beginning just before foot strike and peaking in mid-eccentric motion. In these maneuvers, the level of quadriceps muscle activation exceeded that seen in a maximum isometric contraction. Hamstring muscle activation was submaximal at and after foot strike. The maximum quadriceps muscle activation for all maneuvers was 161% maximum voluntary contraction, while minimum hamstring muscle activity was 14%. Foot strike occurred at an average of 22 degrees of knee flexion for all maneuvers. This low level of hamstring muscle activity and low angle of knee flexion at foot strike and during eccentric contraction, coupled with forces generated by the quadriceps muscles at the knee, could produce significant anterior displacement of the tibia, which may play a role in anterior cruciate ligament injury. PMID- 10751002 TI - The effects of chlorhexidine irrigation solution on contaminated bone-tendon allografts. AB - The purpose of this study was to determine an expedient and effective method for disinfecting contaminated human bone-tendon allografts. The first part of this study used beef muscle and cadaveric human tissues to determine the most effective solution and volume to decontaminate tissues inoculated with four different organisms: Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Of the solutions tested (benzalkonium chloride, castile soap, castile soap followed by benzalkonium chloride, triple antibiotic, chlorhexidine gluconate, and chlorhexidine gluconate/triple antibiotic), only the 4% chlorhexidine power irrigation solution and 4% chlorhexidine/triple antibiotic bath completely disinfected all tissues. Work in part 2 revealed that a 2% chlorhexidine irrigation solution was equally effective as the 4% solutions. Part 3 of the study involved human Achilles tendon calcaneus allografts. We found similar results: 3 liters of 2% chlorhexidine power irrigation solution thoroughly removed all microorganisms from the contaminated tissues. All control allografts irrigated with normal saline solution alone revealed positive bacterial growth for all four organisms after 72 hours' growth on sheep blood agar. Total decontamination time was 10 to 12 minutes. Two percent chlorhexidine irrigation solution may be an effective method for decontaminating human bone-tendon allografts challenged with a polymicrobial inoculum. This method of disinfecting bone-tendon allografts is at least five times more expeditious than methods in previously reported studies. PMID- 10751003 TI - Using bone's adaptation ability to lower the incidence of stress fractures. AB - In three prospective epidemiologic studies of the effect of pre-military induction sport activities on the incidence of lower extremity stress fractures during infantry basic training, recruits who played ball sports (principally basketball) regularly for at least 2 years before basic training had a significantly lower incidence of stress fractures (13.2%, 16.7%, and 3.6% in the three studies, respectively) than recruits who did not play ball sports (28.9%, 27%, and 18.8%, respectively). Preinduction running was not related to the incidence of stress fracture. To assess the tibial strain environment during these sport activities, we made in vivo strain measurements on three male volunteers from the research team. Peak tibial compression and tension strain and strain rates during basketball reached levels 2 to 5.5 times higher than during walking and about 10% to 50% higher than during running. The high bone strain and strain rates that occurred in recruits while playing basketball in the years before military induction may have increased their bone stiffness, according to Wolff's Law. The stiffer bone could tolerate higher stresses better, resulting in lower strains for a given activity and a lower incidence of stress fractures during basic training. PMID- 10751004 TI - Subcutaneous migration of meniscal arrows after failed meniscus repair. A report of two cases. PMID- 10751005 TI - Delayed repair of a ruptured pectoralis major muscle. A case report. AB - Repair of a pectoralis major muscle rupture at the tendinous insertion into the humerus was successfully performed 13 years after the initial injury. Repair was possible only because the ruptured sternal portion of the muscle was scarred to the intact clavicular portion and therefore had not retracted. The attachment of the avulsed sternal head to the intact clavicular head enabled successful restoration of strength and function, as well as normal contour and appearance of the pectoralis major muscle complex. PMID- 10751006 TI - Spinal process apophysitis mimics spondylolysis. Case reports. PMID- 10751007 TI - Stress lesion of the proximal medial ulna in a throwing athlete. A case report. PMID- 10751008 TI - Injuries to the shoulder in the throwing athlete. Part one: Biomechanics/pathophysiology/classification of injury. AB - Over the last decade, significant advances have been made in the study and understanding of shoulder mechanics. Much of this may be attributed to the use of more sophisticated technology to improve our ability to assess the shoulder in real-time athletics. As a consequence of these advances, our understanding of the pathophysiology of injury has also increased. Our manual examination skills have improved and our noninvasive diagnostic techniques have advanced greatly. New insight into forces at play during actions as complex as the throwing motion has allowed us to develop better protocols for the prevention and treatment of the most common injuries. Additionally, paralleling improvements in the understanding of shoulder kinematics and the pathophysiology of injury, advances in surgical techniques, particularly arthroscopy, have aided in the diagnosis of and the development of less invasive surgical treatments for injuries that do not respond to nonoperative measures. Undoubtedly, an up-to-date understanding of the developments in shoulder biomechanics, pathophysiology of injury, diagnostic techniques, and surgical management is necessary for the clinician who wishes to continue to apply proper skills in the sports medicine setting. PMID- 10751009 TI - "Biomechanical analysis of flat and oblique tibial tubercle osteotomy for recurrent patellar instability". PMID- 10751010 TI - Update on genetic risk factors for systemic lupus erythematosus and rheumatoid arthritis. AB - The results of twin and family studies clearly implicate an important role for genetic factors in the etiology of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). However, the complex nature of these diseases has hampered progress in defining the genetic determinants. Recent advances in molecular genetic and statistical methodology offer new hope to overcome these challenges. This review highlights recent efforts to identify genetic risk factors for SLE and RA using allele sharing and other linkage methods. In spite of striking differences between these studies, some agreement in terms of the regions providing evidence of linkage also exists. Thus, together these studies highlight regions of the genome that are likely to contain SLE and RA susceptibility genes. In addition, the results of these studies, in conjunction with progress in other complex human diseases, suggest several important considerations for future studies. PMID- 10751011 TI - Update on outcome assessment in rheumatic disorders. AB - Outcome assessment in rheumatic disorders is getting more and more attention. A series of Outcome Measures in Rheumatology (OMERACT) conferences has provided a good impulse for further research in the field. In this chapter we will review the results of the last OMERACT 4 conference in detail. This conference was focused on longitudinal/observational studies, rheumatoid arthritis (response criteria and imaging), and core sets for ankylosing spondylitis and systemic lupus erythematosus. Moreover, an overview of recent literature on measures of disease activity, quality of life measures, and imaging is presented. For the various rheumatic disorders, several new instruments and/or further validation steps are described. PMID- 10751012 TI - Health services in rheumatology. AB - Studies of the costs associated with rheumatic diseases, the referral of patients to rheumatology subspecialty care, rheumatology practice patterns, and the relation between medical care and patient outcomes are reviewed. Direct medical costs in patients with rheumatoid arthritis (RA) are higher among those with more functional disability. Direct medical costs in patients with systemic lupus erythematosus (SLE) did not differ among Canadian, American, and British patients, despite substantial differences in the mechanisms by which medical care is financed and delivered in these three countries. The diagnostic accuracy of rheumatic complaints by primary care physicians may be low, and concomitant psychiatric disorders may not be uncommon among patients referred to rheumatologists. Most patient visits to rheumatologists involve patients with rheumatic diseases or musculoskeletal complaints, and few visits involve primary care. Fewer than half of elderly patients with RA or SLE are seen by a rheumatologist in a given year; access is particularly limited among black women. Early access to rheumatology subspecialty care may be associated with improved health status in patients with RA, and mortality among patients with SLE varies with the experience a hospital has in treating patients with SLE. PMID- 10751013 TI - Update on the epidemiology of systemic lupus erythematosus: new spins on old ideas. AB - Systemic lupus erythematosus is a serious autoimmune disease of obscure etiology with a propensity for widespread organ involvement. Its manifestations range from ephemeral symptoms to life-threatening events such as thromboses and strokes. Although once considered rare, SLE now appears to be relatively common in certain subsets of the population. Considerable publicity for this disorder as a "silent killer" has heightened public awareness and may have contributed to the increases in the number of individuals reporting this diagnosis in surveys. Epidemiologic studies face numerous challenges in disease classification and case ascertainment. Despite this, such studies have increased in size and number, and have generated novel etiologic hypotheses, ranging from environmental pollutants to viral infections. PMID- 10751014 TI - Fibromyalgia, chronic fatigue syndrome, and myofascial pain syndrome. AB - Fibromyalgia and widespread pain were common in Gulf War veterans with unexplained illness referred to a rheumatology clinic. Increased tenderness was demonstrated in the postmenstrual phase of the cycle compared with the intermenstrual phase in normally cycling women but not in users of oral contraceptives. Patients with fibromyalgia had high levels of symptoms that have been used to define silicone implant-associated syndrome. Tender points were found to be a common transient finding associated with acute infectious mononucleosis, but fibromyalgia was an unusual long-term outcome. The common association of fibromyalgia with other rheumatic and systemic illnesses was further explored. A preliminary study revealed a possible linkage of fibromyalgia to the HLA region. Patients with fibromyalgia were found to have an impaired ability to activate the hypothalamic pituitary portion of the hypothalamic pituitary adrenal axis as well as the sympathoadrenal system, leading to reduced corticotropin and epinephrine response to hypoglycemia. Much interest has been expressed in the literature on the possible role of autonomic dysfunction in the development or exacerbation of fatigue and other symptoms in chronic fatigue syndrome. Mycoplasma genus and mycoplasma fermentans were detected by polymerase chain reaction in patients with chronic fatigue syndrome. It was reported that myofascial temporomandibular disorder does not run in families. No major therapeutic trials in fibromyalgia, chronic fatigue syndrome, or myofascial pain syndrome were reported over the past year. The effectiveness of cognitive behavioral therapy and behavior therapy for chronic pain in adults was emphasized. A favorable outcome of fibromyalgia and chronic fatigue syndrome in children and adolescents was reported. PMID- 10751015 TI - Work-related musculoskeletal disorders. AB - Work-related musculoskeletal disorders continue to be extremely common and to present an important challenge to clinicians. Debate regarding terminology and case definitions has discouraged practitioners from aggressively approaching the diagnosis and management of these conditions. Considerable progress has, however, been made recently. Previously more commonly referred to as repetitive strain injuries or cumulative trauma disorders, the new term work-related musculoskeletal disorders has fewer etiological implications. These disorders, affecting the back, lower limbs, and especially upper limbs and neck, can be extremely costly if not addressed appropriately. Generally resulting from a combination of physical factors (including repetition, force, and awkward postures) as well as other workplace environmental or organizational factors (including excessive work rates or durations, inadequate breaks, and a variety of psychosocial workplace characteristics), work-related musculoskeletal disorders can often be remediated when these factors are appropriately assessed and addressed. Clinicians must play a positive role in ensuring that this approach prevails. PMID- 10751016 TI - Morton neuroma and metatarsalgia. AB - Morton neuroma is most likely a mechanically-induced degenerative neuropathy that predilects the third common digital nerve in middle-aged women who frequently wear fashionable shoes that are not designed for the physiology of the foot. A compression test of the affected web space is quite specific for its diagnosis, and an ultrasonograph can tell its exact size. If conservative means fail to relieve the painful symptoms of a Morton neuroma, surgical removal can produce dramatic pain relief. Metatarsalgia means pain in the metatarsal head region, and exists in three general forms: metatarsalgia of the first metatarsal head region, metatarsalgia of the fourth lateral metatarsal head region, and generalized metatarsalgia. There are numerous causes of metatarsalgia; a selected and important group of causes is discussed in this article. When conservative means fail to relieve metatarsalgia, specific surgical operations are quite effective for relief of pain, and are briefly described in the text. PMID- 10751017 TI - Epidemiology, etiology, diagnostic evaluation, and treatment of low back pain. AB - Low back pain is a common medical problem but has decreased in frequency in the occupational setting over the past decade. The weather affects low back pain but to a minor degree. Physical factors, as well as job satisfaction, play a role in the development and perpetuation of low back pain. In contradistinction to previous measurements, intradiscal pressure has been determined in vivo to be greater in the standing than the sitting position. Adenovirus-mediated gene transfer to nucleus pulposus cells may be the initial stage of a new form of therapy for degenerative disc disease. Bed rest is not more helpful than activity as tolerated for the treatment of sciatica. The outcome of spinal stenosis surgery is more closely associated with the patient's perception of improvement than with the degree of canal narrowing. PMID- 10751018 TI - Sports and other soft tissue injuries, tendinitis, bursitis, and occupation related syndromes. AB - This review highlights three areas: plantar fasciitis, Achilles tendinitis, and carpal tunnel syndrome. The diagnosis and treatment of plantar fasciitis are reviewed; nonsurgical treatments remain the mainstay of management. Several recent articles support the use of night splints. Some novel treatments recently investigated, including low intensity laser irradiation and extracorporeal shock wave lithotripsy, are reviewed, as well as the effectiveness of steroid injection. Novel treatments for Achilles tendinitis are also reviewed, including the use of injection therapy and the treatment approach of one author for the management of Achilles tendon rupture. Nonsurgical techniques in the treatment of carpal tunnel syndrome, such as yoga, ultrasound, noninvasive laser neurolysis, manipulation, nerve and tendon gliding exercises, and medications, are reviewed. Prednisolone was shown to be effective in the treatment of mild to moderate disease and nonsteroidal anti-inflammatory drugs were found to be ineffective. PMID- 10751020 TI - Bibliography. Current world literature. Epidemiology and health-related services. PMID- 10751019 TI - Muscle strain injuries. AB - Muscle injuries--lacerations, contusions or strains--are by far the most common injuries in sports. After first aid following the RICE principle (Rest, Ice, Compression and Elevation), therapy must be tailored according to the severity of the injury and based on the knowledge gained from experimental studies on regeneration of injured muscle. Most muscle injuries can be treated conservatively with excellent recovery, but complete ruptures with complete loss of function should be managed surgically. Immediately after the injury, a short period of immobilization is needed to accelerate formation of the scar between the stumps of the ruptured myofibers, to which the stumps adhere. The optimal length of immobilization depends on the grade of the injury, and should not be longer than needed for the scar to bear the pulling forces without rerupture. Early mobilization is required to invigorate adhesion, orientation of the regenerating muscle fibers, revascularization and resorption of the connective tissue scar. Another important aim of early mobilization, especially in clinical sports medicine, is to minimize inactivity-induced atrophy as well as loss of strength and extensibility, which are rapidly appearing adverse sequelae of prolonged immobilization. PMID- 10751021 TI - Bibliography. Current world literature. Nonarticular rheumatism, sport-related injuries, and related conditions. PMID- 10751022 TI - NIGMS glue grants. PMID- 10751023 TI - On the heterogeneity of drug dissolution and release. PMID- 10751024 TI - Cationic polymer based gene delivery systems. AB - Gene transfer to humans requires carriers for the plasmid DNA which can efficiently and safely carry the gene into the nucleus of the desired cells. A series of chemically different cationic polymers are currently being investigated for these purposes. Although many cationic polymers indeed condense DNA spontaneously, which is a requirement for gene transfer in most types of cells, the physicochemical and biopharmaceutical behavior of the current generation of polyplexes severely limits an efficient gene transfer in vitro and especially in vivo. This paper summarizes recent physicochemical and biological information on polyplexes and aims to provide new insights with respect to this type of gene delivery system. Firstly, the chemical structure of frequently studied cationic polymers is represented. Secondly, the parameters influencing condensation of DNA by cationic polymers are described. Thirdly, the surface properties, solubility, aggregration behavior, degradation and dissociation of polyplexes are considered. The review ends by describing the in vitro and in vivo gene transfection behavior of polyplexes. PMID- 10751025 TI - An isolated in-situ rat head perfusion model for pharmacokinetic studies. AB - PURPOSE: To develop a viable, single pass rat head perfusion model useful for pharmacokinetic studies. METHODS: A viable rat head preparation, perfused with MOPS-buffered Ringer's solution, was developed. Radiolabelled markers (red blood cells, water and sucrose) were injected in a bolus into the internal carotid artery and collected from the posterior facial vein over 28 minutes. The double inverse Gaussian function was used to estimate the statistical moments of the markers. RESULTS: The viability of the perfusion was up to one hour, with optimal perfusate being 2% bovine serum albumin at 37 degrees C, pH 7.4. The distribution volumes for red blood cells, sucrose and water (from all studies, n = 18) were 1.0 +/- 0.3 ml, 6.4 +/- 4.2 ml and 18.3 +/- 11.9 ml, respectively. A high normalised variance for red blood cells (3.1 +/- 2.0) suggests a marked vascular heterogeneity. A higher normalised variance for water (6.4 +/- 3.3) is consistent with additional diffusive/permeability limitations. CONCLUSIONS: Analysis of the physiological parameters derived from the moments suggested that the kinetics of the markers were consistent with distribution throughout the head (weight 25 g) rather than just the brain (weight 2 g). This model should assist in studying solute pharmacokinetics in the head. PMID- 10751026 TI - Evaluation of using dog as an animal model to study the fraction of oral dose absorbed of 43 drugs in humans. AB - PURPOSE: To conduct a retrospective evaluation of using dog as an animal model to study the fraction of oral dose absorbed (F) of 43 drugs in humans and to briefly discuss potential factors that might have contributed to the observed differences in absorption. METHODS: Mean human and dog absorption data obtained under fasted state of 43 drugs with markedly different physicochemical and pharmacological properties and with mean F values ranging from 0.015 to 1.0 were obtained from the literature. Correlation of F values between humans and dogs was studied. Based on the same references, additional F data for humans and rats were also obtained for 18 drugs. RESULTS: Among the 43 drugs studied, 22 drugs were virtually completely absorbed in both dogs and humans. However, the overall correlation was relatively poor (r2 = 0.5123) as compared to the earlier rat vs. human study on 64 drugs (r2 = 0.975). Several drugs showed much better absorption in dogs than in humans. Marked differences in the nonliner absorption profiles between the two species were found for some drugs. Also, some drugs had much longer Tmax values and prolonged absorption in humans than in dogs that might be theoretically predicted. Data on 18 drugs further support great similarity in F between humans and rats reported earlier from our laboratory. CONCLUSIONS: Although dog has been commonly employed as an animal model for studying oral absorption in drug discovery and development, the present study suggests that one may need to exercise caution in the interpretation of data obtained. Exact reasons for the observed interspecies differences in oral absorption remain to be explored. PMID- 10751027 TI - Microdialysis technique as a method to study the percutaneous penetration of methyl nicotinate through excised human skin, reconstructed epidermis, and human skin in vivo. AB - PURPOSE: The aim was to assess the feasibility of cutaneous microdialysis as a method to study percutaneous penetration of methyl nicotinate through human skin in vitro and in vivo. METHODS: Microdialysis was applied in vitro in excised human skin, in isolated dermis, in reconstructed human epidermis and in vivo in the volar forearm skin of volunteers using methyl nicotinate (MN) as a model compound. After topical application of MN, aliquots of the perfusate were collected and analyzed for the presence of MN spectrophotometrically and by HPLC. In vivo, visual scoring and laser Doppler perfusion imaging (LDPI) were used to monitor the effects on skin blood flow. RESULTS: In vitro, MN was detected in the dialysate after a 1 min exposure of excised skin to concentrations as low as 25 mM. Higher concentrations up to 500 mM showed increased levels. Prolongation of the application time to 60 min resulted in increased levels of MN in the perfusate as the duration of application increased. Reconstructed epidermis and isolated dermis showed an almost 2- and 20-fold higher penetration compared to excised skin, respectively. In vivo, LDPI measurements showed a rapid increase in skin blood flow after application of 25 to 100 mM MN for 1 min. MN was only detectable in the microdialysate after application of 100 mM for 10 min (two of three subjects). CONCLUSIONS: Cutaneous microdialysis may be a tool for comparative studies linking responses in human skin in vivo to in vitro data using the same technique and endpoint. PMID- 10751028 TI - Distribution of gacyclidine enantiomers in spinal cord extracellular fluid. AB - PURPOSE: Determination of the pharmacokinetics of gacyclidine enantiomers, a non competitive NMDA antagonist, in plasma and spinal cord extracellular fluid (ECF) of rats. METHODS: Implantation of microdialysis probes in spinal cord (T9). Serial collection of plasma samples and ECF dialysates over 5 hours after IV bolus administration of (+/-)-gacyclidine (2.5 mg/kg). Plasma protein binding determined in vivo by equilibrium dialysis. Chiral GC/ MS assay. RESULTS: Plasma concentrations of (+)-gacyclidine were approximately 25% higher than those of (-) gacyclidine over the duration of the experiment in all animals. Plasma concentrations decayed in parallel in a biphasic manner (t1/2alpha approximately 9 min; t1/2beta approximately 90 min) with no significant difference between enantiomers. Clearance and volume of distribution of (-)-gacyclidine were approximately 20% higher than those of its optical antipode (CL: 248 vs 197 ml.kg(-1)x min(-1); Vdbeta: 31.6 vs 23.5 1/kg). Protein binding (approximately 90%) was not stereoselective. Both gacyclidine enantiomers were quantifiable in spinal cord ECF 10 min after drug administration and remained stable over the duration of the experiment in spite of changing blood concentrations. Penetration of (-)-gacyclidine was significantly higher (approximately 40%) than that of (+) gacyclidine in all animals. Yet, exposure of spinal cord ECF was similar for both enantiomers, and not correlated with plasma AUCs. CONCLUSIONS: The disposition of gacyclidine enantiomers is stereoselective. Both enantiomers exhibit a high affinity for spinal cord tissue and their distribution may involve a stereoselective and active transport system. This hypothesis could also explain the discrepancy between drug concentrations in plasma and spinal cord ECE PMID- 10751029 TI - Comparison of zafirlukast (Accolate) absorption after oral and colonic administration in humans. AB - PURPOSE: This study characterized the gastrointestinal (GI) absorption of zafirlukast after oral and colonic administration in humans. METHODS: Five healthy subjects received zafirlukast solution (40 mg) orally and via an oroenteric tube into the colon in a randomized, crossover fashion. Two additional subjects were dosed into the distal ileum. Serial blood samples were obtained and plasma concentrations were quantitated by HPLC. RESULTS: Mean +/- SD pharmacokinetic parameters after oral vs. colonic administration were: AUC infinity of 2076 +/- 548 vs. 602 +/- 373 ng x h/mL, respectively, and Cmax of 697 +/- 314 vs. 194 +/- 316 ng/mL, respectively. Mean colon:oral AUCalpha and Cmax were 0.29 and 0.30, respectively. Median tmax values were 2.0 and 1.35 hr after oral and colonic administration. First-order absorption rate constants (Ka and Kac) were estimated from a two-compartment model with first-order elimination. Kac:Ka was <0.5 in 4 of the 5 subjects dosed in the colon. CONCLUSIONS: Zafirlukast was absorbed at multiple sites in the GI tract. The rate and extent of zafirlukast absorption was less after colonic than oral administration. Zafirlukast was significantly absorbed in the distal ileum. This study demonstrated that gamma scintigraphy, digital radiography, and fluoroscopy can be used to track the movement and confirm the location of the oroenteric tube in the GI tract. PMID- 10751030 TI - Application of a biomagnetic measurement system (BMS) to the evaluation of gastrointestinal transit of intestinal pressure-controlled colon delivery capsules (PCDCs) in human subjects. AB - PURPOSE: For determination of the transit time through various parts of the gastrointestinal (GI) tract, we developed a method that provides the location of disintegration and drug release. This method involves GI magnetomarkergraphy (GIMG) using a 129-channel Shimadzu vector biomagnetic measurement system (BMS). METHODS: To magnetically label the pressure-controlled colon delivery capsule (PCDC) containing 75.0 +/- 0.5 mg of caffeine as a tracer drug, small capsule caps containing 90 mg of ferric oxide powdered magnetite (Fe2O3) were attached to PCDCs. After orally administration to fasted human volunteers, saliva samples were collected hourly and salivary caffeine concentration was measured. At the same time, locations of the magnetic PCDC were detected by BMS just after the PCDCs were magnetized with the coils of a magnetic resonance imaging (MRI) system. The magnetic field distributions were analyzed and the estimated positions were shown on the MRI picture of the same subject's abdominal structure. RESULTS: We magnetized PCDC with permanent magnets or an electromagnet before ingestion and the estimated locations of PCDC in the GI tract exhibited high estimation error. In order to increase the precision of estimated localization of PCDCs, PCDCs were magnetized within the coils of the MRI. As a result, these PCDCs had strong magnetic dipoles that were parallel to the sensor unit of BMS in every measurement, and therefore the spatial resolution of the PCDC's two-dimensional positions in the organs of the GI tract was within a range of several millimeters. CONCLUSIONS: GIMG is a powerful tool for the study of colon delivery efficiencies of PCDCs. The main advantage of GIMG is the capability to obtain even more detailed knowledge of the behavior and fate of solid pharmaceutical formulations during GI passage. PMID- 10751031 TI - Hollow porous particles in metered dose inhalers. AB - PURPOSE: To assess the physical stability and aerosol characteristics of suspensions of hollow porous microspheres (PulmoSpheres) in HFA-134a. METHODS: Cromolyn sodium, albuterol sulfate, and formoterol fumarate microspheres were prepared by a spray-drying method. Particle size and morphology were determined via electron microscopy. Particle aggregation and suspension creaming times were assessed visually, and aerosol performance was determined via Andersen cascade impaction and dose uniformity studies. RESULTS: The hollow porous particle morphology allows the propellant to permeate freely within the particles creating a novel form of suspension termed a homodispersion, wherein the dispersed and continuous phases are identical, separated by an insoluble interfacial layer of drug and excipient. Homodispersion formation improves suspension stability by minimizing the difference in density between the particles and the medium, and by reducing attractive forces between particles. The improved physical stability leads to excellent dose uniformity. Excellent aerosolization efficiencies are also observed with PulmoSpheres formulations, with fine particle fractions of about 70%. CONCLUSIONS: The formation of hollow porous particles provides a new formulation technology for stabilizing suspensions of drugs in hydrofluoroalkane propellants with improved physical stability, content uniformity, and aerosolization efficiency. PMID- 10751033 TI - Dissolution of hydrocortisone in human and simulated intestinal fluids. AB - PURPOSE: To compare solubility and dissolution rate of hydrocortisone in aspirated human intestinal fluids (HIFs) with simulated intestinal fluids (SIFs) and buffer. METHODS: Solubility and flux from a rotating disk of hydrocortisone were measured. The bile salt content, pH and osmotic pressure were determined in HIFs. RESULTS: In fasted state the solubility of hydrocortisone was higher in HIFs than in the buffer and SIFs. The flux of hydrocortisone in HIFs was similar to the flux in the buffer but lower than the flux in SIFs at fasted state. Addition of intestinal surfactants in SIFs increased solubility and flux at both fasted and fed state. The increase in solubility was caused by micelle formation in SIFs. The increase in flux may partly be explained by increased solubility. The bile salt content of the HIFs did not correlate with the solubility or the flux but pH in the HIFs seems to have some effect on the components of the HIFs resulting in increased solubility. CONCLUSIONS: It is possible to perform comparable dissolution tests in HIFs and SIFs. The lack of correlation between the results in HIFs and the bile salt content may be explained by the relatively low lipophilicity of the model drug. PMID- 10751032 TI - Formulation development and antitumor activity of a filter-sterilizable emulsion of paclitaxel. AB - PURPOSE: Paclitaxel is currently administered i.v. as a slow infusion of a solution of the drug in an ethanol:surfactant:saline admixture. However, poor solubilization and toxicity are associated with this drug therapy. Alternative drug delivery systems, including parenteral emulsions, are under development in recent years to reduce drug toxicity, improve efficacy and eliminate premedication. METHODS: Paclitaxel emulsions were prepared by high-shear homogenization. The particle size of the emulsions was measured by dynamic light scattering. Drug concentration was quantified by HPLC and in vitro drug release was monitored by membrane dialysis. The physical stability of emulsions was monitored by particle size changes in both the mean droplet diameter and 99% cumulative distribution. Paclitaxel potency and changes in the concentration of known degradants were used as chemical stability indicators. Single dose acute toxicity studies were conducted in healthy mice and efficacy studies in B 16 melanoma tumor-bearing mice. RESULTS: QW8184, a physically and chemically stable sub-micron oil-in-water (o/w) emulsion of paclitaxel, can be prepared at high drug loading (8-10 mg/mL) having a mean droplet diameter of <100 nm and 99% cumulative particle size distribution of <200 nm. In vitro release studies demonstrated low and sustained drug release both in the presence and absence of human serum albumin. Based on single dose acute toxicity studies, QW8184 is well tolerated both in mice and rats with about a 3-fold increase in the maximum tolerated-dose (MTD) over the current marketed drug formulation. Using the B16 mouse melanoma model, a significant improvement in drug efficacy was observed with QW8184 over Taxol. CONCLUSIONS: QW8184, a stable sub-micron o/w emulsion of paclitaxel has been developed that can be filter-sterilized and administered i.v. as a bolus dose. When compared to Taxol, this emulsion exhibited reduced toxicity and improved efficacy most likely due to the composition and dependent physicochemical characteristics of the emulsion. PMID- 10751034 TI - Metal-catalyzed oxidation of brain-derived neurotrophic factor (BDNF): analytical challenges for the identification of modified sites. AB - PURPOSE: We examined the metal-catalyzed oxidation of brain-derived neurotrophic factor (BDNF) using the Cu(II)/ascorbate/O2 model oxidative system. METHODS: Electrospray ionization mass spectrometry, peptide mapping and amino acid analysis were utilized to determine the nature of the covalent modification induced by the metal-catalyzed oxidative system. Additionally, analytical ultracentrifugation, the Bradford assay, circular dichroism and ANSA dye-binding were used to determine the nature of any conformational changes induced by the oxidation. RESULTS: Exposure of BDNF to the Cu(II)/ascorbate/O2 system led to the modification of ca. 35% of Met92 to its sulfoxide, and to subsequent conformational changes. The proteolytic digestion procedure was sensitive to this conformational change, and was unable to detect the modification. Chemical digestion with CNBr, however, was not sensitive to this change, and allowed for the identification of the site of modification. CONCLUSIONS: The modification of Met92 to its sulfoxide rendered the oxidized BDNF inaccessible to proteolytic digestion, due to conformational changes associated with the oxidation. PMID- 10751035 TI - Oxidation of the N-terminal gly-residue of peptides: stress study of pexiganan acetate in a drug formulation. AB - PURPOSE: The purpose of this study was to identify four major degradation products, which were formed during a stress study of pexiganan (a 22-mer peptide) in a 1% formulation. METHODS: The degradation products were isolated and characterized by LC/MS, tryptic and aminopeptidase digests. RESULTS: One of the degradation products was shown to be des-glyl-pexiganan. The other three are structural isomers of N-glyoxylyl-desgly1-pexiganan. These isomers undergo reversible inter-conversions, as well as decompose irreversibly to des-gly1 pexiganan. Thus, all the impurities were formed from a single oxidation product of pexiganan, N-glyoxylyl-des-gly1-pexiganan. The aldehyde group of the glyoxylyl residue and the NH-amide of the adjacent isoleucine residue form a piperazinedione derivative of des-gly1-pexiganan. This heterocyclic compound rearranges to other tautomers or back to the N-glyoxylyl compound (see Fig. 3). Tryptic digests of the three degradation products showed that their N-terminal segment produced N-glyoxylyl-I-G-K whereas the N-terminal segment of pexiganan produced G-I-G-K. All the other tryptic-digest segments were identical to those formed in pexiganan. The LC/MS of the N-terminal segment and of synthetic N glyoxylyl-I-G-K were identical. The enzymatic resistance of the three impurities to undergo aminopeptidase-M cleavage further supported the conclusion that their N-terminal amino residues are substituted. CONCLUSIONS: After a year under stress conditions 1% pexiganan cream lost about 15% of the active component to oxidative deamination, where the N-terminal glycine residue was oxidized to N-glyoxylyl desgly1-pexiganan. The other nine epsilon-amino lysine-residues of the peptide stayed intact. This oxidation product inter-converted and formed two additional impurities, tautomers of piperazinedionyl-des-gly-pexiganan, and decomposed to des-gly1-pexiganan, the forth impurity. PMID- 10751036 TI - Apparent lack of effect of P-glycoprotein on the gastrointestinal absorption of a substrate, tacrolimus, in normal mice. AB - PURPOSE: To study the contribution of P-glycoprotein (P-gp) to the oral absorption of a substrate, tacrolimus, by comparing the extent and rate of bioavailability in normal and mdr1a knockout mice. Methods. Intravenous and oral (2 mg/kg) blood concentration data of tacrolimus in normal and knockout mice were obtained from a study by K. Yokogawa et al. in Pharm. Res. 16:1213-1218 (1999). Mean bioavailability (F), mean hepatic first-pass extraction ratio (Fh), mean bioavailability rates, mean oral clearance, and mean total hepatic intrinsic clearance were calculated using standard pharmacokinetic methods. RESULTS: The mean F of tacrolimus (an apparently highly permeable compound) was increased from 0.22 in normal mice to 0.72 in knockout mice. These values were consistent with mean predicted Eh (based on intravenous data) of 0.77 and 0.27 in normal and knockout mice, respectively. Great similarity in the relative bioavailability profile (such as short Tmax) between normal and knockout mice was also found. Mean oral clearance and mean total or unbound hepatic intrinsic clearance of tacrolimus in knockout mice were found to be about 10 times lower compared to those in normal mice. CONCLUSIONS: The above results suggest an apparent lack of effect of P-gp on the gastrointestinal absorption of tacrolimus in normal mice under the study condition. It is postulated that the effect of P-gp on the rate and extent of oral absorption should be more pronounced for those more slowly or incompletely absorbed drugs (i.e., drugs with relatively low permeabilities) as illustrated by talinolol in humans. The clearance data also suggest a very dominant role of P-glycoprotein in controlling the rate of hepatic metabolism of tacrolimus in normal mice, and P-glycoprotein may serve as an effective efflux pump for direct transport of metabolites formed in hepatocytes into the blood circulation. PMID- 10751037 TI - Atorvastatin transport in the Caco-2 cell model: contributions of P-glycoprotein and the proton-monocarboxylic acid co-transporter. AB - PURPOSE: The purpose of this study was to elucidate the mechanisms by which an HMG-CoA reductase inhibitor, atorvastatin (an organic acid with a pKa of 4.46), was transported in the secretory and absorptive directions across Caco-2 cell monolayers. METHODS: Caco-2 cells were grown on polycarbonate membrane inserts in 6-well Snapwell plates (Costar). The permeability of radiolabeled compounds across Caco-2 cell monolayers was determined using a side-by-side diffusion apparatus (NaviCyte) and an automated liquid handler (Hamilton Microlab 2200). The apical uptake of 14C-atorvastatin was also determined in Caco-2 cells. Cyclosporin A (20 microM) was present in the uptake media to block potential P glycoprotein-mediated atorvastatin efflux. RESULTS: Polarized permeation of atorvastatin was observed with the basolateral-to-apical (B-to-A) permeability being 7-fold greater than the A-to-B permeability (35.6 x 10(-6) and 4.9 x 10(-6) cm/s, respectively). The secretion of atorvastatin was a saturable process with an apparent Km of 115 microM. The B-to-A permeability of atorvastatin was significantly reduced by cyclosporin A (10 microM), verapamil (100 microM), and a P-glycoprotein specific monoclonal antibody, UIC2(10 microg/ml) (43%, 25%, and 13%, respectively). Furthermore, both CsA and verapamil significantly increased the A-to-B permeability of atorvastatin by 60%; however, UIC2 did not affect the A-to-B permeability of atorvastatin. CsA uncompetitively inhibited the B-to-A flux of atorvastatin with a Ki of 5 microM. In addition, atorvastatin (100 microM) significantly inhibited the B-to-A permeability of vinblastine by 61%. The apical uptake of atorvastatin increased 10.5-fold when the apical pH decreased from pH 7.4 to pH 5.5 while the pH in the basolateral side was fixed at pH 7.4. A proton ionophore, carbonylcyanide p-trifluoro-methoxyphenylhydrazone (FCCP) significantly decreased atorvastatin uptake. In addition, atorvastatin uptake was significantly inhibited by benzoic acid, nicotinic acid, and acetic acid each at 20 mM (65%, 14%, and 40%, respectively). Benzoic acid competitively inhibited atorvastatin uptake with a Ki of 14 mM. Similarly, benzoic acid, nicotinic acid, and acetic acid significantly, inhibited the A-to-B permeability of atorvastatin by 71%, 21%, and 66%, respectively. CONCLUSION: This study demonstrated that atorvastatin was secreted across the apical surface of Caco-2 cell monolayers via P-glycoprotein-mediated efflux and transported across the apical membrane in the absorptive direction via a H(+)-monocarboxylic acid cotransporter (MCT). In addition, this study provided the first evidence that negatively charged compounds, such as atorvastatin, can be a substrate for P glycoprotein. PMID- 10751038 TI - Structure activity relationship of human microsomal epoxide hydrolase inhibition by amide and acid analogues of valproic acid. AB - PURPOSE: The purpose of this study was to evaluate the in vitro inhibitory potency of various amide analogues and derivatives of valproic acid toward human microsomal epoxide hydrolase (mEH). METHODS: mEH inhibition was evaluated in human liver microsomes with 25 microM (S)-(+)-styrene oxide as the substrate. Inhibitory potency expressed as the median inhibitory concentration (IC50) was calculated from the formation rate of the enzymatic product, (S)-(+)-1-phenyl-1,2 ethanediol. RESULTS: Inhibitory potency was directly correlated with lipophilicity and became significant for amides with a minimum of eight carbon atoms. Branched eight-carbon amides were more potent inhibitors than their straight chain isomer, octanamide. N-substituted valproylamide analogues had reduced or abolished inhibition potency with the exception of valproyl hydroxamic acid being a potent inhibitor. Inhibition potency was not stereoselective in two cases of chiral valpromide isomers. Valproyl glycinamide, a new antiepileptic drug currently undergoing phase II clinical trials and its major metabolite valproyl glycine were weak mEH inhibitors. Acid isomers of valproic acid were not potent mEH inhibitors. CONCLUSIONS: The structural requirements for valproylamide analogues for potent in vitro mEH inhibition are: an unsubstituted amide moiety; two saturated alkyl side chains; a minimum of eight carbons in the molecule. PMID- 10751039 TI - Nasal epithelial permeation of thymotrinan (TP3) versus thymocartin (TP4): competitive metabolism and self-enhancement. AB - PURPOSE: To investigate concentration dependent permeabilities and metabolism kinetics of thymotrinan (TP3) versus thymocartin (TP4) in nasal epithelium in vitro. METHODS: Excised bovine nasal mucosa was used as an in vitro model. Permeabilities were studied in a diffusion chamber, metabolism kinetics in a reflection kinetics set-up. Studies were performed at various TP3 and TP4 concentrations. The 3H-mannitol flux was measured to monitor junctional permeability. Potential Ca(2+)-complexation was investigated using a Ca(2+) selective electrode. RESULTS: Permeability of TP3 was negligible at 0.1 and 0.2 mM and increased drastically above 0.4 mM up to -2 X 10(-5) cm s(-1). In the presence of 2 mM TP4 the TP3 permeabilites were significantly above (approximately 4 x 10(-5) cm s(-1)) the level of TP3 without TP4, and TP3 metabolism was totally inhibited. TP3 and TP4 showed a significant concentration dependent effect on the permeability of 3H-mannitol. A hyperosmolarity effect of the peptide solutions was excluded. Transepithelial electrical resistance (TEER; approximately 30 ohms cm2) was unchanged by either TP3 or TP4. At 1 mM TP3 the mucosal-to-serosal permeability was four times higher than serosal-to-mucosal, indicating enzyme polarization. In reflection kinetics studies, TP3 degradation was slightly higher on the mucosal than on the serosal side. TP3 and TP4 followed the same non-linear metabolism kinetics. CONCLUSIONS: Increase in permeability at high TP concentrations involves competitive enzyme saturation combined with self enhanced paracellular permeation. PMID- 10751040 TI - Histone H1-mediated transfection: serum inhibition can be overcome by Ca2+ ions. AB - PURPOSE: One of the drawbacks of polycationic and cationic liposomal gene transfer is its sensitivity to serum. Gene therapy requires the transfectant-DNA complex to be resistant to serum as well as blood. Since Ca2+ has proved to be an efficient cofactor of polycationic gene transfer, we decided to investigate its effects on transfection in the presence of serum. METHODS: We studied transgene expression of luciferase gene (pCMV Luc) on ECV 304 human endothelial cells using H1 histone and DOSPER as transfectants in the presence of 0-100% fetal calf serum. RESULTS: H1-and DOSPER-mediated transfection was found to be inhibited by serum above the concentration of 10%. If 2 mM Ca2+ or 2 mM Ca2+/0.1 mM chloroquine was included in the culture medium which replace the transfection mixture and was left on the cells for 24 hours postincubation, the inhibiting effect of even 100% serum was overcome. CONCLUSIONS: A high serum level does not interfere with binding and uptake of H1- and DOSPER-DNA complexes, but inhibits subsequent steps such as endosomal escape. Ca2+ in the form of nascent calcium phosphate microprecipitates and other lysosomolytical agents facilitate endosomal/lysosomal release by their fusigenic and membranolytic activity. PMID- 10751041 TI - Transepithelial transport of levofloxacin in the isolated perfused rat kidney. AB - PURPOSE: The transepithelial transport of levofloxacin was evaluated in the isolated perfused kidney to investigate its renal secretory mechanisms. METHODS: Levofloxacin was instantaneously administered into the renal artery together with inulin and Evans blue-labeled albumin, and the single-pass dilution curves of the renal venous and urinary outflow were determined in the absence or presence of various compounds. Kinetic parameters were computed based on non-compartment moment analysis. RESULTS: The ratio of fractional excretion to filtration fraction (FE/FF) for levofloxacin was 2.99 +/- 0.18, indicating the involvement of tubular secretion. In the presence of cimetidine and quinolones, the FE/FF of levofloxacin was significantly decreased and the transepithelial mean transit time (Tcell) of levofloxacin was prolonged. The Tcell showed a negative correlation with renal secretion of levofloxacin, while the volume of distribution of levofloxacin showed no correlation. CONCLUSIONS: Transport on the brush-border membrane plays a determining step in the renal secretion of levofloxacin, and cimetidine and quinolones interact with levofloxacin transport on the brush-border membrane. PMID- 10751042 TI - Development and characterization of a rapid and comprehensive genotyping assay to detect the most common variants in cytochrome P450 2D6. PMID- 10751044 TI - Evaluations of aggressive women: the effects of gender, socioeconomic status, and level of aggression. AB - Research finds gender differences in aggression and suggests that female violence is viewed differently from male violence. Participants were 94 female and 38 male students from a mid-size public university in the Southeast. Participants read a mock trial and answered questions about their attitudes concerning an aggressor in the scenario. The study was a 2 (male or female) by 2 (high socioeconomic status or low socioeconomic status) by 2 (verbal aggression or physical aggression) between-subjects factorial design. The participants responded to a revised version of the Attitudes Toward Women Scale (Nelson, 1988). As hypothesized aggressive women were evaluated more negatively than aggressive men and participants evaluated the female aggressor who used physical aggression more negatively than the female who used verbal aggression. The hypothesis that the female aggressor would be perceived as more in need of counseling than the male aggressor was not supported. Contrary to another hypothesis, respondents did not evaluate higher socioeconomic status aggressors more negatively than those of lower status. As hypothesized people with traditional views of women evaluated the female aggressor more negatively than people with more liberal views of women, and liberal participants evaluated the male and female aggressors similarly. The more negative evaluation of female aggressors and, in particular, females who use physical aggression, may result in unfair treatment of such females. These social biases may cloud perceptions of aggressive females, suggesting that their actions are more inappropriate than those of a male who committed the same act. PMID- 10751043 TI - Gun injury and mortality: the delinquent backgrounds of juvenile victims. AB - Serious injuries and deaths of three samples of boys were examined in an inner city, longitudinal study. By age 19, almost 1 in 10 of the 506 participants in the oldest sample had been seriously wounded or killed. Two thirds of this group had been either wounded or killed by guns. Victims, compared to controls, tended to have a history of engaging in serious delinquency, gang fights, and selling drugs. They also tended to carry guns. The delinquent lifestyle of the victims was also evident from their court records. Victims tended to do poorly academically in school, received poorer parental supervision, had poorer communication with their parents, and had a long history of behavior problems. PMID- 10751045 TI - Risk factors and correlates of dating violence: the relevance of examining frequency and severity levels in a college sample. AB - To determine whether categorizing levels of violence along dimensions of frequency and severity would result in informative distinctions among individuals using dating violence, reported use of physical violence, along with variables theorized to be related to use of force in intimate relationships, was assessed in a sample of 617 college students (males = 290; females = 327). When participants' scores were analyzed by dichotomizing them along the lines of ever versus never using dating violence, numerous past findings were replicated. However, participants using a higher frequency of force were distinguished by needing to control their partner and by less inhibition in expressing their anger. Higher severity of force by an individual was predicted only by a need to control his/her dating partner. Implications for these findings were discussed. PMID- 10751046 TI - Normative advice to campus crime victims: effects of gender, age, and alcohol. AB - Three studies investigated the appropriateness of calling the police as a function of crime, victim, and subject factors. In particular, the studies focused on whether and how the victim's consumption of alcohol affected normative advice to report the crime, as opposed to other options. Across the three studies, subjects viewed reporting as more appropriate for female victims, for victims who were 21 or older, and for victims who had not been drinking. In addition, females were more likely than males to believe reporting to the police was appropriate whereas males were more likely than females to favor some type of private action. Subjects viewed reporting as particularly inappropriate when the victim was underage and had been drinking. Results suggest that, because of the perceived stigma attached to victims who have been drinking, even serious victimizations may go unreported. PMID- 10751047 TI - Neuropsychological correlates of domestic violence. AB - Neuropsychological functioning was assessed in 39 males who had committed domestic violence (batterers) and compared to 63 nonviolent (both maritally discordant and satisfied) subjects recruited by advertisement. Subjects were subsequently divided into two groups (head injured, nonhead injured) and these groups were also contrasted as a function of batterer status. Tests were administered to assess for cognitive and behavioral functions, including executive dysfunction, hypothesized to be a factor contributing to propensity for violence. Questionnaires and structured clinical interviews were used to assess marital discord, emotional distress, and violent behaviors. Batterers differed from nonbatterers across several cognitive domains: executive, learning, memory, and verbal functioning. Batterers were reliably discriminated from nonbatterers based on three neuropsychological tasks: Digit Symbol, Recognition Memory Test Words, Wisconsin Card Sorting Test. Neuropsychological performance was the strongest correlate of domestic violence of all clinical variables measured. However, the inclusion of two other variables, severity of emotional distress and history of head injury, together with the neuropsychological indices provided the strongest correlation with batterers status. Among batterers, neuropsychological performance did not vary as a function of head injury status, indicating that while prior head injury was correlated with batterer status, it was not the sole basis for their impairments. The findings suggest that current cognitive status, prior brain injury, childhood academic problems, as well as psychosocial influences, contribute along with coexisting emotional distress to a propensity for domestic violence. PMID- 10751048 TI - Comparing violence over the life span in samples of same-sex and opposite-sex cohabitants. AB - Using data from a nationally representative telephone survey that was conducted from November 1995 to May 1996, this study compares lifetime experiences with violent victimization among men and women with a history of same-sex cohabitation and their counterparts with a history of marriage and/or opposite-sex cohabitation only. The study found that respondents who had lived with a same-sex intimate partner were significantly more likely than respondents who had married or lived with an opposite-sex partner only to have been: (a) raped as minors and adults; (b) physically assaulted as children by adult caretakers; and (c) physically assaulted as adults by all types of perpetrators, including intimate partners. The study also confirms previous reports that intimate partner violence is more prevalent among gay male couples than heterosexual couples. However, it contradicts reports that intimate partner violence is more prevalent among lesbian couples than heterosexual couples. Overall study findings suggest that intimate partner violence is perpetrated primarily by men, whether against same sex or opposite-sex partners. PMID- 10751049 TI - Obstacles to victims' cooperation with the criminal prosecution of their abusers: the role of social support. AB - Over the last 20 years, diminishing tolerance for domestic violence has triggered significant criminal justice reforms designed to facilitate the prosecution of abusers. Prosecutors, for example, have adopted policies requiring that cases go forward even if the victim later has second thoughts. Although increasingly common, these "no drop" policies reflect a profound irony about domestic cases that is well known but little understood: the most formidable problem in prosecuting such cases is often the victim's own unwillingness to bring the abuser to justice. This prospective study explored a range of factors potentially predictive of domestic violence victims' cooperation with the prosecution of their abusers. Although the study focused on interpersonal and institutional social support, it also investigated the influence of violence severity, victim demographic factors, and victim mental health characteristics, including the presence of depressive symptoms, emotional dependence on the abuser, and substance abuse. Findings showed that tangible support, severity of violence in the relationship, and the presence of children in common with the abuser all significantly predicted victims' cooperation with the prosecution of their abusers. Substance abuse significantly predicted victims' noncooperation with prosecution. The research and policy implications of these findings are discussed. PMID- 10751050 TI - Histological factors of 1-h biopsy influencing the delayed renal function and outcome in cadaveric renal allografts. AB - The morphological characteristics of a kidney biopsy specimen taken 1 h after reperfusion of blood into the graft (1-h biopsy) during a cadaveric transplant operation were studied. The aim of the 1-h biopsy is to evaluate the pre transplant risk factors for the delayed graft function, assess the renal function of the graft, and predict long-term graft survival. The total number of 1-h biopsies was 113, consisting of 86 male and 27 female donors. The mean age of the donors is 39.5 +/- 17.3 yr. Arteriosclerosis (AS) and tubulo-interstitial injury (TI) were both estimated using a semi-quantitative scale. AS score was graded into four categories, according to the severity of the thickening of interlobular artery: 0: none, 1: mild, 2: moderate and 3: severe. No biopsy revealed severe AS of grade 3. The TI score was graded from 0 to 5, according to the morphological injury: 0: none, 1-2 non-specific tubulo-interstitial injury (NSTI), and 3-5 compatible with acute tubular necrosis (ATN) in terms of pathological diagnosis. The mean ages of donors showing as AS of score 0, 1, and 2 were 30.6 +/- 14.6, 49.7 +/- 13.5, and 56.9 +/- 6.30, respectively. The mean donor age of the AS 1 group and AS 2 group was significantly lower than for the AS 0 group. The lowest serum creatinine values after operation (best Cr) of recipients with AS scores of 0, 1, and 2 were 1.31 +/- 0.45, 1.60 +/- 0.70, and 1.84 +/- 0.71 mg/dL, respectively; the best Cr of AS scores of 1 and 2 was significantly higher than in the AS score 0 group. The mean creatinine level at the final point of the AS 0 group was significantly lower than in the combined AS 1 and AS 2 group (serum creatinine 1.44 +/- 1.03 vs 1.87 +/- 1.53 mg/dL: p < 0.01). The duration of severe hypotension less than 50 mmHg or 80 mmHg was significantly shorter in the NSTI group than in the ATN group (less than 50 mmHg was 29.7 +/- 124 vs 72.5 +/- 174, less than 80 mmHg 105 +/- 234 vs 193 +/- 261 min: p < 0.01). The post operative (po) day expressing diuresis in excess of 1000 mL of urine per d was 8.28 +/- 17.5 and 13.7 +/- 23.3 (p < 0.01) in the NSTI and ATN group, respectively. The po-d of the last hemodialysis and the po-d showing serum creatinine less than 2.0 mg/dL in NSTI and ATN group was 7.74 +/- 17.4 and 13.3 +/- 23.2 (p < 0.01), and 25.0 +/- 30.5 and 38.0 +/- 35.2 (p < 0.01), respectively. We concluded that 1-h renal biopsy is useful for assessing the outcome of renal allograft. AS of a donor kidney is one of the most important risk factors for both short and long-term outcome of the graft. The TI score was useful to predict the outcome of delayed graft function. PMID- 10751051 TI - Case study of paired cadaver renal allografts from the same donor: influence of local DIC kidney and concomitant acute rejection on early graft outcome. AB - We report the clinical course of 2 recipients whose renal allografts were obtained from the same cadaver donor after cardiac arrest. The recipients showed different outcomes after transplantation. Graft biopsy after reperfusion revealed disseminated intravascular coagulation (so-called DIC kidney) and severe acute tubular necrosis (ATN) in both recipients. While one graft showed primary nonfunction, the other graft became functional after a post-operative anuric period. Serial graft biopsies performed during the oligo-anuric period revealed recovery of ATN and no intra-glomerular fibrin thrombi, but development of acute rejection was detected in both recipients. The left kidney graft showed more severe local DIC kidney than the right kidney, as well as more severe acute rejection in the oligo-anuric period. Despite aggressive anti-rejection therapy, the left kidney graft showed primary nonfunction. Therefore, severe acute rejection leading to primary nonfunction might have been related to more severe ischemic injury and more extensive local DIC kidney in the left kidney. PMID- 10751052 TI - Significance of interstitial lesions as the early indicator for acute vascular rejection in human renal allografts. AB - To evaluate the relevance between interstitial lesions and acute vascular rejection of renal allograft, we examined 129 renal allograft biopsy samples obtained from 60 transplant patients within 90 d post-transplantation. Plasma cells, eosinophils and polymorphonuclear cells (PMNs) in the cortical interstitium were counted, respectively, and the number of each cells per 10 fields at a magnification of 200x was calculated. Then, the existence of interstitial edema was judged. We categorized 129 biopsy specimens into specimens before (Pre), on (R) and after acute rejection (Post). An increased number of eosinophils (mean +/- SE; 15.4 +/- 9.2 per 10 fields) was observed in R rather than in Pre (0.6 +/- 0.3, p < 0.05). The increasing number of plasma cells depends on a time course following the onset of acute rejection. The number is significantly larger in acute vascular rejection (4.3 +/- 1.1) than in non vascular rejection (1.3 +/- 0.4, p < 0.01). In living donor transplants, the number of PMNs in patients before vascular rejection (median, 16.7 per 10 fields; range, 5.0-20.0 per 10 fields) was significantly larger than in normal or borderline patients (2.2; 0.0-36.7, p < 0.01). The incidence of interstitial edema in patients before vascular rejection (55.6%) was significantly higher than in normal or borderline patients (8.0%, p < 0.01). This study suggested that acute vascular rejection is relevant to interstitial lesions, such as for a plasma cell, eosinophil and PMN infiltrate, and edema. Also, these lesions seem to be the indicator of acute vascular rejection. These findings may contribute to the refinement of histological diagnosis of acute vascular rejection. PMID- 10751053 TI - Electron microscopic peritubular capillary lesions: a new criterion for chronic rejection. AB - Typical chronic rejection showing prominent glomerular and/or arterial lesions is less common in renal allograft patients treated with cyclosporin A (CsA). We investigated the value of peritubular capillary lesions as a criterion for chronic rejection in the CsA era. A total of 129 renal graft biopsies, taken from recipients showing graft dysfunction after more than 2 months post-operatively, were examined by electron microscopy, and peritubular capillary lesions were studied, especially multi-layered basement membrane lesions (MLPTC). Eighty-two biopsy specimens taken from non-transplantation patients were also studied as a control. Five biopsies (6%) showed mild and atypical MLPTC in the control group. Of the 129 allograft biopsies, MLPTC was seen in 55 (42.6%). The prevalence showed no significant relationship to the interval from operation to biopsy. MLPTC was either typical (n = 10) or incomplete (n = 45). Concomitant membrane disruption, edema and lymphocyte infiltration of the subendothelial space, reflecting acute cellular rejection, were occasionally noted in both groups. Incomplete MLPTC often developed within 1 yr after surgery, in association with acute rejection. It was found in 26-50% of biopsies at any time up to 5 yr post operatively. The incidence of typical MLPTC was 5.7-12.8% over 1 yr post operatively. These findings suggest that the development of chronic rejection is closely related to relapsing acute tubulo-interstitial allograft rejection which is often clinically silent. We concluded that MLPTC is useful as a specific criterion for chronic rejection. PMID- 10751054 TI - Peritubular capillary deposition of C4d complement fragment in ABO-incompatible renal transplantation with humoral rejection. AB - In ABO-incompatible renal transplantation complement activation may be related to antibody-associated humoral rejection. However, immune deposits within the vasculature have been infrequently demonstrated in biopsy specimens. Whether deposition of complement fragment C4d is correlated with graft outcome and pathological findings (as measured by the severity of antibody-associated humoral rejection) is investigated in this study. Nineteen ABO-incompatible and 9 ABO compatible renal graft biopsy specimens were selected. Four out of 19 ABO incompatible patients lost their grafts within 1 yr. Ten out of 19 ABO incompatible and just 1 out of 9 ABO-compatible patients, had prominent C4d deposition in peritubular capillaries. ABO-incompatible patients with predominant C4d deposition showed few tubulitis, accumulation of polymorphonuclear cells and thrombosis in peritubular and glomerular capillaries. The severity of the humoral rejection was correlated to C4d deposition in peritubular capillaries. Three out of four graft losses in ABO-incompatible renal transplantation showed severe humoral rejection and profuse deposition of C4d complement fragments in peritubular capillaries. Immunosuppression therapy was discontinued in the 4th patient, who lost his graft because of his lethal intestinal bleeding. C4d deposition in peritubular capillaries would be helpful for differential diagnosis between humoral rejection and drug-induced nephrotoxicity, and may serve as a sensitive marker of ABO-incompatible humoral rejection for patients with unsatisfactory (no glomeruli) biopsy specimens. PMID- 10751055 TI - A case of IgA nephropathy after ABO-incompatible living kidney transplantation. AB - A 39-yr-old Japanese man underwent living related kidney transplantation. Because the graft was ABO-incompatible, he was treated with double filtration plasmapheresis before transplantation and his immunosuppressive therapy was stronger than usual. However, immunoglobulin A nephropathy, accompanied by cellular crescents and necrotizing lesions, developed after 18 months. To our knowledge, the association of IgA nephropathy with ABO-incompatible kidney transplantation has not been reported previously. PMID- 10751056 TI - A case of ABO-incompatible renal transplant patient with non-insulin-dependent diabetes mellitus; long-standing observation of serial glomerular change by protocol biopsy. AB - A 41-yr-old patient with non-insulin-dependent diabetes mellitus (NIDDM), before and after ABO-incompatible renal transplant, is reviewed using serial protocol biopsy. Although she recovered from delayed hyperacute rejection (DHAR) immediately post-transplantation, her graft function deteriorated gradually. A mild acute transplant glomerulitis, noted at the 155th day post-transplantation, progressed to pronounced chronic transplant glomerulopathy over 5 yr. In the specimen of the last biopsy, at 5 yr post-transplantation, glomeruli demonstrated an exudative hyaline lesion, which was characteristic of diabetic nephropathy in addition to chronic transplant glomerulopathy. Therefore, we made a diagnosis of this glomerular lesion as chronic transplant glomerulopathy complicated by diabetic glomerulopathy. Considering the result of this case, the protocol biopsy is a useful procedure to diagnose an accurate cause of graft dysfunction in individual cases. It is concluded that the protocol biopsy is apparently useful for the detection of various pathological processes occurring in allograft and may contribute to a strategy for improvement of graft survival. PMID- 10751057 TI - Clinical and histological analysis of acute tacrolimus (TAC) nephrotoxicity in renal allografts. AB - Tacrolimus (TAC) is an effective primary immunosuppressive agent in kidney transplantation. Acute nephrotoxicity due to TAC in kidney transplant patients is either similar to, or significantly more frequent than that of cyclosporin A (CsA). In this study we describe the clinico-morphologic characteristics of acute TAC nephrotoxicity in kidney transplant recipients. PATIENTS AND METHODS: We studied retrospectively the clinical courses of 67 patients who underwent kidney transplantation under TAC immunosuppression at Tokyo Women's Medical University between July 1990 and January 1997. We compared the recipient's characteristics with and without acute TAC nephrotoxicity. We also studied retrospectively the clinico-morphologic profiles of the acute TAC nephrotoxicity with 16 kidney transplant recipients under TAC immunosuppression, who were diagnosed with acute TAC nephrotoxicity by allograft biopsies between January 1996 and January 1997. RESULTS: There were no significant differences between acute TAC nephrotoxicity and non-nephrotoxicity groups about age, sex, donor source, age of donor, the proportion of ABO incompatible and the number of human leukocyte antigen (HLA) mismatches. Of 27 acute TAC nephrotoxicity recipients, 7 patients (26%) had moderate-to-severe grade arteriosclerosis in their allograft arteries. The onset of TAC nephrotoxicity occurred 8-69 days post-operatively. The baseline creatinine level was 1.92 mg/dL (range 0.4-5.1 mg/dL) and rose by 38.4% (range 0 84.6) during episodes of nephrotoxicity. The mean peak of the whole blood TAC trough level during the toxic episodes was 32.5 ng/mL (range 21.2-58.5 ng/mL). The rise in creatinine level preceded the highest TAC level in 10 cases. A mean reduction in TAC dosage of 18.9% (range 0-50% led to a fall of 17.2% (range 0 43%) in serum creatinine levels. The moderate-to-severe arteriosclerosis in allograft arteries was seen in 5 (31%) patients. CONCLUSION: The high trough level of the whole blood TAC and the existence of moderate-to-severe arteriosclerosis in allograft arteries have the potential of causing TAC nephrotoxicities. A reduction of TAC dosage may be effective in improving allograft functions. PMID- 10751058 TI - A case of late onset cyclosporine-induced hemolytic uremic syndrome resulting in renal graft loss. AB - A case of late onset hemolytic uremic syndrome (HUS) associated with cyclosporine (CYA) is described in this report. A 50-yr-old man with end-stage renal failure due to immunoglobulin A (IgA) nephropathy received a renal transplant from his wife. Human leucocyte antigen was completely unmatched. Immunosuppressant was a combination of prednisolone, azathioprine, and CYA. He was discharged 1 month after transplantation, with no episode of acute rejection. Twenty-one months after transplantation, his platelet count and hematocrit began to decrease and lactate dehydrogenase began to increase. Graft biopsy showed thrombotic microangiopathy and recurrent IgA nephropathy. Graft function was rapidly deteriorated and methylprednisolone pulse therapy was not effective. Twenty-five months after transplantation, he returned to a regular hemodialysis. Hemolysis was immediately improved after a reduction of the dose of CYA to 50 mg/d. The trough level of CYA was less than 200 ng/mL in most periods of his clinical course. Blood pressure was high throughout the clinical course. Although acute vascular rejection or malignant hypertension could also cause a thrombotic microangiopathy, CYA was most likely a cause of HUS in the present case because of the following reasons: neither anti-acute rejection therapy nor an adequate control of his blood pressure was effective in improving clinical features of HUS; hemolysis and thrombocytopenia disappeared immediately after the reduction of the dose of CYA to 50 mg/d. It has been reported that HUS carried poor prognosis only when occurring shortly after transplantation in cadaver kidney recipients. The present transplant was from a living donor and HUS occurred 21 months after transplantation and was severe enough to result in graft loss. High blood pressure might be one of the predisposing factors of HUS associated with CYA in the present case. CYA should be stopped and other alternative immunosuppressants should be given in cases of acute graft deterioration with hemolysis and thrombocytopenia, irrespective of the interval from transplantation, CYA dose, or CYA trough level. PMID- 10751059 TI - Recurrence of IgA nephropathy 17 months after renal transplantation in the allograft transmitted thin basement membrane disease (TBMD) from donor. AB - Recurrence of IgA nephropathy (IgAN) following renal transplantation has been described in 40-50% of such patients and it usually has a good outcome. We present the case of a 20-yr-old woman with IgAN who developed end-stage renal failure in 1995. In November 1996, she received a kidney from a living-related donor and was treated with tacrolimus, azathioprine and steroids. Zero- and one hour biopsies were performed, which revealed minor glomerular abnormalities in light microscopy, thin basement membrane disease (TBMD) in electron microscopy. Eight months later she developed microscopic hematuria and proteinuria; however, the graft function was normal. Renal biopsy revealed an IgAN that is thought to be due to recurrence of the original disease. PMID- 10751060 TI - Renal transplantation in systemic amyloidosis. AB - Renal transplantation was performed in a 24-yr-old woman with type AA systemic amyloidosis. Renal biopsy at 11 yr post-transplantation showed no amyloid recurrence, but the presence of mesangial proliferative glomerulonephritis and focal segmental glomerular sclerosis. Renal function was satisfactory with the exception of mild proteinuria. Symptoms related to systemic amyloidosis including goiter and cardiac deposition, improved post-transplantation. Renal transplantation is the recommended therapy for type AA systemic amyloidosis. PMID- 10751061 TI - Good news from India. PMID- 10751062 TI - The fruits of the labor: reinvesting the savings from good tuberculosis control in the United States. PMID- 10751063 TI - The status and prospects of tuberculosis control in India. AB - SETTING: India, where much of the global strategy for tuberculosis control was established, but where, every year, there are an estimated 2 million cases of tuberculosis. OBJECTIVE: To describe the policies, initial results, and lessons learned from implementation of a Revised National Tuberculosis Control Programme using the principles of DOTS (Directly Observed Treatment, Short-course). DESIGN: A Revised National Tuberculosis Control Programme (RNTCP) was designed and implemented starting in 1993. With funding from the Government of India, State Governments, the World Bank and bilateral donors, regular supply of drugs and logistics was ensured. Persons with chest symptoms who attend health facilities are referred to microscopy centres for diagnosis. Diagnosed cases are categorized as per World Health Organization guidelines, and treatment is given by direct observation. Systematic recording and cohort reporting is done. RESULTS: From October 1993 through mid-1999, 146,012 patients were put on treatment in the programme. The quality of diagnosis was improved, with the ratio of smear positive to smear-negative patients being maintained at 1:1. Case detection rates varied greatly between project sites and correlated with the percentage of patients who were smear-positive among those examined for diagnosis, suggesting heterogeneous disease rates. Treatment success was achieved in 81% of new smear positive patients, 82% of new smear-negative patients, 89% of patients with extra pulmonary tuberculosis, and 70% of re-treatment patients. CONCLUSION: The RNTCP has successfully treated approximately 80% of patients in 20 districts of 15 states of India. Treatment success rates are more than double and death rates are less than a seventh those of the previous programme. Starting in late 1998, the programme began to scale up and now covers more than 130 million people. Maintaining the quality of implementation during the expansion phase is the next challenge. PMID- 10751064 TI - Modeling the epidemiology and economics of directly observed therapy in Baltimore. AB - SETTING: From 1958 to 1978, Baltimore maintained one of the highest pulmonary tuberculosis (TB) rates in the US. But, from 1978 to 1992 its TB rate declined by 64.3% and its ranking for TB fell from second highest among large US cites to twenty-eighth. This TB trend coincided with the implementation of an aggressive directly observed therapy (DOT) program by Baltimore's Health Department. OBJECTIVES: We used modeling to estimate the range of TB cases prevented in Baltimore under DOT. Case estimates equal the difference between the observed number of TB cases in Baltimore versus the expected number if Baltimore's TB trend was replaced by the TB trend for the US (low estimate) or the TB trend for all US cities with over 250,000 residents (high estimate). Economic savings are estimated. RESULTS: Without DOT we estimate there would have been between 1,577 (53.6%) and 2,233 (75.9%) more TB cases in Baltimore, costing $18.8 million to $27.1 million. Cases prevented and expenditures saved increased with increased DOT participation. CONCLUSION: Our model predicts that Baltimore's TB decline accompanying DOT resulted in health care savings equal to twice the city's total TB control budget for this period. These results are most plausibly due to DOT, since it was the only major change in Baltimore's TB control program, and rising TB risk factors-AIDS, injection drug use, poverty-in a city where TB had been epidemic should have triggered a TB increase as in comparable US cities, rather than the observed decline. As national TB rates continue to decline it will be important to identify ways to capture and reinvest these savings to support effective TB control programs. PMID- 10751065 TI - Predictors of death from pulmonary tuberculosis: the case of Veracruz, Mexico. AB - OBJECTIVES: To identify factors (particularly social, economic and cultural), associated with the risk of death from pulmonary tuberculosis in Mexico. METHODS: A case-control study of patients receiving medical attention from the official health services of Veracruz, Mexico. Cases were deaths from pulmonary tuberculosis in 1993. Controls were survivors randomly selected from the State Tuberculosis Case Registry. Next of kin provided information for both cases and controls. RESULTS: Multivariate analysis of 161 cases and 161 controls showed an increased risk of dying for those patients who withdrew from treatment (odds ratio [OR] = 3.52), who were refused medical attention during some period of time in any health center (OR = 4.45), and who had a concomitant disease at the time of diagnosis (OR = 2.62). A linear trend with age was observed (OR = 1.02 per year), as well as a lower risk for those patients who were compliant with treatment and optimistic about surviving the disease (OR = 0.17). The risk of death was not associated with the presence of a health care unit in the town, time spent to get to the health center, or the residence of a patient in an urban area. CONCLUSIONS: These findings indicate that deaths due to tuberculosis in this area are not related to the geographical distribution of health services but to delays in treatment after the onset of disease and to the low adherence of patients to the treatment regimen. PMID- 10751066 TI - BCG coverage and the annual risk of tuberculosis infection over a 14-year period in the Philippines assessed from the Nationwide Prevalence Surveys. AB - SETTING: A prevalence survey of tuberculosis (TB) infection was undertaken in the Philippines, a developing country in the Western Pacific region. OBJECTIVE: To determine the bacille Calmette Guerin (BCG) vaccination rate, the prevalence of TB infection and the annual risk of TB infection (ARTI). METHODS: A nationwide stratified multi-stage cluster survey of 21,960 individuals. BCG scar verification and tuberculin test were performed on those aged > or =2 months. The ARTI was calculated using the prevalence rates of TB infection in children aged 5 9 years. RESULTS: BCG scars were noted in 66% of the study population. The prevalence of TB infection was 63.4% among unvaccinated individuals. The prevalence rate was higher in males in both urban and rural areas. With both sexes combined, urban and rural communities had similar prevalence rates. In children aged 5-9 years, the prevalence rate was 16.1% (males 17.4%, females 14.9%), corresponding to an ARTI of 2.3% (males 2.5%, females 2.1%). CONCLUSION: BCG coverage increased substantially between 1981-1983 and 1997. The ARTI, however, was virtually unchanged, indicating that morbidity due to TB continued to be high. PMID- 10751068 TI - Tuberculosis surveillance in immigrants through health undertakings in Western Australia. AB - SETTING: Tuberculosis Control Program, Western Australia. OBJECTIVE: To assess the effectiveness and efficiency of tuberculosis (TB) surveillance in immigrants. DESIGN: Retrospective descriptive analysis of records of immigrants who first registered with the Tuberculosis Program from January 1994 to December 1995, having entered Western Australia on health undertakings (nonlegal contracts signed by those determined during premigration screening to require post-arrival evaluation). RESULTS: Of 1,344 immigrants on health undertakings for TB in the period, 587 (44%) had findings directly related to TB through pre-migration screening, 69 of whom required treatment for active disease. Another 443 (33%) had chest X-ray changes for which TB could not be excluded. Of the remaining 314 (23%), 172 had poor quality X-rays. Post-arrival assessment and follow-up of the whole group detected four of seven additional cases of active TB, 373 persons (28%) requiring on-going surveillance, 667 (50%) with non-TB conditions and 280 (21%) with normal chest X-rays. CONCLUSIONS: Migrant surveillance for TB in Australia is effective in detecting active disease and identifying a high-risk subgroup requiring further evaluation. Efficiency can be improved and high compliance achieved with simple administrative changes. Routine pre-migration Mantoux testing is inappropriate, and its inclusion in post-arrival assessments should be a national policy decision. PMID- 10751067 TI - Anti-tuberculosis drug resistance in Portugal. AB - SETTING: A survey based upon a representative sample of smear-positive pulmonary tuberculosis patients was undertaken in Portugal, as part of the World Health Organization's Global Project on Anti-Tuberculosis Drug Resistance Surveillance. OBJECTIVE: To determine the level of primary antituberculosis drug resistance at both national and regional levels, and to assess its relative weight within the performance of the National Tuberculosis Programme (NTP). DESIGN: Mycobacterium tuberculosis isolates from 1,105 patients with smear-positive pulmonary tuberculosis admitted to 46 randomly stratified treatment centres all over mainland Portugal were submitted to susceptibility testing with four drugs. Human immunodeficiency virus (HIV) testing was included in the patients' evaluation scheme. RESULTS: Of the strains isolated, 197 (17.8%) were resistant to at least one drug. Primary resistance to isoniazid was 7.7% and to rifampicin 1.9%. Acquired drug resistance was 39.2% in total, any acquired resistance to isoniazid 31.1% and to rifampicin 20.9%. Primary multidrug resistance (MDR) was 1.8% and acquired MDR was 20.9%. HIV testing was positive in 29.2% of MDR-TB cases. CONCLUSIONS: Drug resistance in Portugal is high. Primary MDR and particularly acquired MDR occur in a high proportion of cases, indicating a need for improvement in NTP performance. PMID- 10751069 TI - Erythrocyte sedimentation rate in childhood tuberculosis: is it still worthwhile? AB - OBJECTIVE: To evaluate the utility of the erythrocyte sedimentation rate (ESR) in the diagnosis of childhood tuberculosis. DESIGN: Data were collected retrospectively from the Qatar National Tuberculosis (TB) Registry for children (birth to 14 years of age) from 1983 to 1996. The diagnosis of active tuberculosis was based on positive sputum cultures (or histology) or an abnormal chest radiograph that responded to anti-tuberculosis chemotherapy. RESULTS: Of 144 childhood TB patients, 68 (47%) had an ESR documented at the time of diagnosis. Twenty-two children (33%) had a normal ESR (<10 mm/hour) and 46 children (67%) had an elevated ESR (> or =10 mm/hour) at the time of diagnosis. Culture positive and symptomatic children had significantly higher ESR values than culture negative and asymptomatic children, respectively, at the time of diagnosis. There was no significant difference in ESR values for children with extrapulmonary versus pulmonary disease, and likewise no significant correlation between either age or size of tuberculin skin test reactivity and ESR values. CONCLUSION: Although an elevated ESR may be expected in children with tuberculosis, this study found that one-third of children with TB had a normal ESR at the time of diagnosis, and consequently there would seem to be little value in using ESR as a diagnostic test for childhood tuberculosis. PMID- 10751070 TI - Diagnosing HIV-associated tuberculosis: reducing costs and diagnostic delay. AB - SETTING: University-affiliated hospital in South Africa. OBJECTIVE: To assess the time to diagnosis and the yield and laboratory cost of diagnostic procedures in human immunodeficiency virus (HIV) associated tuberculosis. DESIGN: Cohort study. PATIENTS: Adult HIV-infected patients with newly-diagnosed tuberculosis admitted over a 2-year period. RESULTS: A total of 141 admissions fulfilled the case definition. Sputum smear yield (43% overall) correlated strongly with chest radiograph appearance but not with CD4+ lymphocyte count. Sputum smear yield was approximately 40% per sample sent, resulting in a high cumulative yield when > or = three samples were sent. Smear of sputum or wide needle lymph node aspirates were the most cost-effective diagnostic methods. Significant diagnostic delay occurred in sputum smear-negative patients. Most patients with sputum smear negative tuberculosis had either pleural effusions or lymphadenopathy. Lymph node biopsy had a high diagnostic yield even in patients with symmetrical nodes, but was under-utilised in this group. There was unnecessary expenditure on cultures, with many patients having several positive cultures. CONCLUSION: Repeated sputum smear examination produces a high cumulative yield in HIV-associated tuberculosis. Considerable savings in laboratory utilisation and bed occupancy would have been made if a streamlined diagnostic approach with greater use of lymph node aspirate and early pleural or lymph node biopsy had been followed. PMID- 10751071 TI - An incremental cost-effectiveness analysis of the first, second and third sputum examination in the diagnosis of pulmonary tuberculosis. AB - SETTING: St. Francis Hospital in Katete District, Eastern Province, Zambia. OBJECTIVE: To compare the incremental cost-effectiveness of examining serial sputum smears for screening suspects for pulmonary tuberculosis at a rural district hospital in Zambia. DESIGN: An incremental cost-effectiveness analysis of serial sputum smear examinations for diagnosing pulmonary tuberculosis based on laboratory results collected during 1997 and 1998 in a rural district hospital in Zambia. The cost analysis took a health service provider perspective, and used the ingredients approach. The cost-effectiveness is expressed in terms of the incremental cost per tuberculosis case diagnosed. Relevant information was obtained from various sources, including administrative records, interviews and direct observation. RESULTS: Of a total of 166 acid-fast bacilli positive suspects who had three sputum smears examined sequentially, 128 (77.1%) were found on the first smear, a further 25 (15%) on the second smear and 13 (7.9%) additional cases were identified on the third smear. The economic analysis shows that the incremental cost of performing a third test, having already done two, increases rapidly with only a small gain in terms of additional cases of tuberculosis identified. CONCLUSION: A policy of examining two samples should be considered in resource-poor settings, if the remaining steps of the national diagnostic algorithm can be adhered to with respect to smear-negative suspects. PMID- 10751072 TI - Clinical aspects of miliary tuberculosis in Saudi adults. AB - OBJECTIVE: To review all cases of miliary tuberculosis (MT) over a period of 7 years in a Saudi Arabian population, to determine the clinical and laboratory characteristics and diagnostic methods, and to compare our data with others reported in the literature. DESIGN: A retrospective case review. RESULTS: Of 780 cases of active tuberculosis seen over period of 7 years, 47 (6%) had MT. The majority of the patients were aged over 60 years (68%). The most common symptoms were fever and weight loss. Risk factors were present in 70% of cases, mainly diabetes mellitus, history of prior tuberculosis, chronic liver and renal disease, immunosuppressive drug therapy and malignancy. Miliary pattern on chest radiography was seen in 89%. The most common laboratory abnormalities were anemia (66%), lymphopenia (89%), low albumin (87%), and high alkaline phosphatase (80%). Fiberoptic bronchoscopy was diagnostic in 7/10 (70%), bone marrow examination in 8/11 (73%), and liver biopsy in 14/16 (88%). High alkaline phosphatase and lymphopenia were associated with high yield of liver biopsies and bone marrow examination: 13/14 (93%) and 7/8 (88%), respectively. Death occurred in 10/47 (21%); all of these had radiological evidence of miliary pattern, and 80% had comorbid conditions. CONCLUSION: These data confirm and extend the results of other studies and suggest that MT is a disease of the elderly and immunocompromised, and is associated with high mortality. A high index of suspicion is required for diagnosis. PMID- 10751073 TI - Slow N-acetyltransferase 2 genotype affects the incidence of isoniazid and rifampicin-induced hepatotoxicity. AB - SETTING: Japanese in-patients with pulmonary tuberculosis and normal liver function receiving treatment with isoniazid and rifampicin (INH + RMP). OBJECTIVE: To elucidate the relationship between N-acetyltransferase 2 (NAT2) genotype and the incidence of isoniazid + rifampicin-induced hepatotoxicity. DESIGN: Prospective study. After NAT2* genotyping, 77 patients were classified into three groups according to their NAT2* genotypes: rapid-type (a homozygote of NAT2*4), intermediate-type (a heterozygote of NAT2*4 and mutant alleles) and slow type (a combination of mutant alleles). Their biochemical profiles of liver function test were investigated for 3 months to assess the development of serum aminotransferase elevation. RESULT: Of the 77 patients, 18.2% developed adverse hepatic reaction within the first month of INH + RMP treatment. A significant association was observed between hepatotoxicity and NAT2* genotype: compared with rapid-type, the relative risk was 4.0 (95% CI 1.94-6.06) for intermediate-type and 28.0 (95%CI 26.0-30.0) for slow-type. Especially in slow-type, the incidence of hepatotoxicity and serum aminotransferase elevation was significantly higher than in the other two types. CONCLUSION: Slow NAT2* genotype significantly affected the development of INH + RMP-induced hepatotoxicity. This suggests the possibility that NAT2* genotyping prior to medication may be useful in evaluating patients with high risk for INH + RMP-induced hepatotoxicity. PMID- 10751074 TI - Rifapentine and isoniazid in the continuation phase of a 6-month regimen. Interim report: no activity of isoniazid in the continuation phase. AB - SETTING: Clinical trial amongst 762 patients with newly diagnosed pulmonary tuberculosis in Hong Kong. After an initial 2 months of a four-drug intensive phase consisting of streptomycin, isoniazid, rifampicin and pyrazinamide (SHRZ), a random allocation in continuation to once-weekly rifapentine + isoniazid (HRp1), HRp1 given in 2 of every 3 weeks (HRp1.2/3), or to three times weekly isoniazid + rifampicin (HR3). OBJECTIVE: Interim report evaluating progress of study and the role of isoniazid in the continuation phase. METHODS: Kaplan-Meier analysis and response of patients related to susceptibility of pretreatment organisms to isoniazid and to rate of isoniazid acetylation determined by NAT2 genotyping. RESULTS: In the 30-month follow-up, rates for adverse treatment events (failure and relapse) were 4.2% in the HR3, 10.2% in the HRp1 and 11.2% in the HRp1.2/3 series (P = 0.02 for HR3 vs HRp1 and P = 0.01 for HR3 vs HRp1.2/3). Occurrence of adverse events was not related to initial susceptibility to isoniazid nor to the rate of acetylation of isoniazid. CONCLUSIONS: The two rifapentine regimens had similar final rates of adverse events which were unsatisfactory. Isoniazid had little or no activity in the continuation phase, indicating that no improvement of the continuation regimen is likely to be obtained by alteration of the isoniazid dosage. PMID- 10751076 TI - Viability of stored sputum specimens for smear microscopy and culture. AB - A laboratory study was performed to determine how long sputum specimens from smear-positive tuberculosis patients can be stored at room temperature or in the refrigerator and retain a positive acid-fast bacilli (AFB) smear or a positive mycobacterial culture. Sputum samples from 30 patients were examined up to 4 weeks and samples from 13 patients examined up to 8 weeks. Provided samples had not dried out, all sputum smears remained AFB positive up to 4 and 8 weeks. In both patient groups, at 4 weeks 37-39% of specimens at room temperature grew mycobacteria compared with 54-67% of specimens stored in the refrigerator. These results have implications for tuberculosis programme policy. PMID- 10751075 TI - Affordability of inhaled corticosteroids as a potential barrier to treatment of asthma in some developing countries. AB - SETTING: The cost and availability of the medications required for the treatment of asthma may represent potential barriers to effective management. METHOD: A survey of prices and policies for components of asthma treatment in 1998, in Algeria, Burkina Faso, Ivory Coast, Guinea, Mali, Syria, Turkey and Vietnam. RESULTS: Medications were consistently available in only four of the eight countries studied. The cost of essential medications for standard case management varied by over five times for beclomethasone and by over three times for inhaled salbutamol. In all but two countries, the cost of one year of drugs for treatment of a moderate, persistent case exceeded the monthly salary of a nurse in that country. The essential drugs list included inhaled salbutamol in five of eight countries and beclomethasone in three of eight. The costs of medications were lower where generic preparations were available and, to a lesser extent, where the medications are on the essential drugs list. CONCLUSIONS: The cost and availability of medications vary widely, and may represent an important barrier to effective management in some low and middle income countries. PMID- 10751077 TI - Association of prevalence of tuberculin reactions with closeness of contact among household contacts of new smear-positive pulmonary tuberculosis patients. AB - OBJECTIVES: Among household contacts of newly diagnosed smear-positive pulmonary tuberculosis (TB) patients, to examine the association of apparent tuberculosis transmission with the closeness and duration of contact. METHODS: Household contacts of newly diagnosed patients with smear-positive pulmonary TB were identified, as well as a healthy population sample. Household contacts were categorized into three groups based on closeness, regularity, and duration of contact with index cases. All subjects underwent tuberculin testing. RESULTS: Among the household contacts, 47 of 112 (42%) of very close/intimate contacts had positive tuberculin tests, compared to 121 of 356 (34%) close/ regular contacts and 23 of 178 (13%) not close/sporadic contacts and 55 of 355 (16%) of a healthy population sample. CONCLUSIONS: Even among household contacts of smear-positive pulmonary TB patients, there were major differences in the proximity and duration of contact which were strongly associated with differences in the prevalence of positive tuberculin reactions. PMID- 10751078 TI - Restriction fragment length polymorphism analysis of Mycobacterium avium isolates from animal and human sources. AB - Restriction fragment length polymorphism (RFLP) analysis using probes derived from the insertion sequences IS901, IS1245 and IS1311, was carried out on Mycobacterium avium isolates obtained from 18 human patients, 44 deer, 14 pigs and five cattle in the Republic of Ireland. Forty-two of the cervine isolates and two of the bovine isolates contained IS901, while this insertion sequence was absent from all of the human and porcine isolates. RFLP analysis with IS901 probe differentiated the 44 field isolates which contained this element into three types. All of the IS901-positive isolates had a characteristic three-band IS1245 hybridisation pattern and a characteristic single-band IS1311 hybridisation pattern. The IS901-negative isolates exhibited highly polymorphic IS1245 and IS1311 hybridisation patterns which differentiated the human and porcine isolates into a wide diversity of strain types. PMID- 10751079 TI - Culture examination--an essential step to increase diagnostic yield for pulmonary tuberculosis in developing countries. PMID- 10751080 TI - DOTS in Haiti. PMID- 10751081 TI - Distress in MEN 2 family members and partners prior to DNA test disclosure. Multiple endocrine neoplasia type 2. AB - Multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominant early-onset cancer disorder. In the Netherlands presymptomatic genetic testing for MEN 2 is offered to testees from the age of five years. We report on adults requesting testing for themselves (n=90) and on parents who want an at-risk child to be tested (n=26). Sociodemographic, personality, and attitude characteristics, and levels of psychological distress, were determined for applicants and their partners in the predisclosure phase of testing. These participants showed only mildly increased levels of psychological distress, defined as heightened scores on measures of general and test-related anxiety, and of psychological complaints. Compared with a normal population, high levels of anxiety and health complaints were found in applicants who were younger than 25 years and single, and in persons who generally tended to react to distressful situations with anxiety or depression. These characteristics were particularly evident in young applicants (<25 years). Our study shows that people who feel ambivalent towards DNA testing and who are more vulnerable to psychological distress are more likely to agree to participate in the test as part of a collective application by members of a hereditary cancer family. PMID- 10751082 TI - Quantitative approach to identifying abnormal variation in the human face exemplified by a study of 278 individuals with five craniofacial syndromes. AB - We have two objectives in this study: to demonstrate the utility of two summary anthropometric measures for quantifying craniofacial variation and to explore some of their potential uses by physicians and clinical morphologists in general. The Craniofacial Variability Index (CVI) is a summary anthropometric measure of facial "harmony." The mean z-score, based on craniofacial anthropometry, is a measure of overall facial size. Both add an objective component to the assessment of individual facial variation and allow us to place the individual along a scale of continuous variation with predetermined limits of "normality" based on a reference or control series. Our results suggest that these summary measures coincide well with clinical assessments of abnormality in 278 individuals representing five distinct syndromes (Brachmann-de Lange, Prader-Willi, Rubinstein-Taybi, Smith-Magenis, and Sotos), each of which has an associated craniofacial component. Although craniofacial variation is continuous and the normal and syndromic populations overlap to varying degrees, the syndromic cases can be characterized in a variety of ways by using CVI as a measure of facial harmony and Mean-Z as an indicator of overall facial size. Thus, these two objective measures offer a novel and efficient means of assessing craniofacial variation, whether they are used as tools in the clinical evaluation of subjects or as a means of exploring the nature of craniofacial variation within or between syndromes. PMID- 10751083 TI - Duplication of chromosome region 8p23.1-->p23.3: a benign variant? AB - Chromosome analysis was performed in a 34-year-old man who was phenotypically normal except for oligoasthenozoospermia. In this patient, analysis of GTG-banded chromosomes showed in one chromosome 8 additional chromosomal material of unknown origin. To characterize the aberrant chromosome more precisely, a paint specific for chromosome region 8pter-->8p23.1 was generated by microdissection and degenerated oligonucleotide primed-polymerase chain reaction (DOP-PCR) and used as fluorescence in situ hybridization (FISH) paint. After reverse painting, hybridization signals were only found on the short arm of the two chromosomes 8, with an enlarged signal on the derivative chromosome 8. The duplication was characterized further with band-specific FISH probes. We concluded that (part of) chromosome region 8p23.1-->p23.3 was duplicated. Chromosome analysis of the parents showed that the dup(8) was of maternal origin and that the fertile brother of the index patient also was a carrier of the chromosome aberration. There was no history of miscarriages. We suggest that duplication of region 8p23.1-->p23.3 can be regarded as euchromatic variant or duplication with no phenotypic effect. PMID- 10751085 TI - A case of Perlman syndrome: fetal gigantism, renal dysplasia, and severe neurological deficits. AB - We report on a neonate presenting with polyhydramnios; macrosomia; macrocephaly; visceromegaly including bilateral nephromegaly, hepatomegaly, cardiomegaly; thymus hyperplasia; cryptorchidism; generalized muscle hypotonia; and a distinctive facial appearance. The clinical course was marked by severe neurodevelopmental deficits combined with progressive respiratory decompensation leading to death at the age 6 months. Magnetic resonance imaging (MRI) disclosed a generalized cerebral atrophy with a marked deficit of the white matter. Renal ultrasound and MRI showed markedly enlarged kidneys with multiple small cystic lesions, a pattern indistinguishable from polycystic kidney disease. The postmortem kidney biopsy revealed dysplastic changes, microcysts, and a focal nephrogenic rest, characteristic features of the Perlman syndrome. In children with fetal gigantism, renal abnormalities, and neurological deficits, Perlman syndrome should be considered and may be confirmed by kidney biopsy. PMID- 10751084 TI - Fetomaternal cell trafficking: a new cause of disease? AB - Isolation of fetal cells from maternal blood is under active investigation as a noninvasive method of prenatal diagnosis. In the context of studying cell surface antigens expressed on fetal cells we discovered that fetal cells from a prior pregnancy also could be detected. This led to the appreciation of the persistence of fetal cells in maternal blood for as long as 27 years postpartum, and the realization that following pregnancy, a woman becomes a chimera. Quantitative polymerase chain reaction analyses have shown that a term pregnancy is not required for the subsequent development of fetal cell microchimerism. As many as 500,000 fetal nucleated cells are transfused following an elective first trimester termination of pregnancy. The relationship between fetal cell microchimerism and maternal disease is currently being explored. During pregnancy, fetal cells in the maternal skin are related to polymorphic eruptions of pregnancy and increased fetomaternal trafficking is detectable in cases of preeclampsia. After delivery, more male DNA of presumed fetal origin is present in the blood and skin of women with scleroderma as compared with healthy controls. Scleroderma is of particular interest because it shows a strong female predilection and it is an autoimmune disease with clinical similarities to graft versus-host disease. Fetomaternal cell trafficking provides a potential explanation for the increased prevalence of autoimmune disorders in adult women following their childbearing years. PMID- 10751086 TI - Familial hypercholesterolemia study in Sardinia using 6 LDLR polymorphic markers based on PCR. AB - Twenty-two Sardinian families with multiple cases of hypercholesterolemia were investigated with six polymorphic markers of the low-density lipoprotein receptor (LDLR) gene that could be quickly analyzed by PCR-based methods. Five single nucleotide polymorphisms (SNP) in exons 8, 10, 13, 15, and 18 and a microsatellite marker flanking the 3' end of the LDLR gene were used to define the haplotypes at the LDLR locus for familial hypercholesterolemia (FH) diagnosis within families. No significant differences were observed between the allele frequencies of the normal and mutant chromosomes. In two families, hypercholesterolemia did not cosegregate with the LDLR locus. In the remaining 20 FH chromosomes, seven different haplotypes were identified. The same haplotypes were found with a similar frequency among the 61 normal chromosomes. Other five haplotypes were characteristic only of normal chromosomes. These data provide no evidence for a gene founder effect in the Sardinian population and, instead, highlight a pattern of genetic heterogeneity comparable with that found in mainland European populations. The replacement of the restriction fragment length polymorphisms currently used in the genetic analysis of FH with PCR-based markers proved to be a simple and less time-consuming method and did not reduce informativity in the molecular analysis of FH families. PMID- 10751087 TI - Pathogenesis of chromosomal mosaicism and its effect on early human development. AB - Constitutional chromosomal mosaicism is the result of postfertilization mitotic error, the mechanism of which is not fully understood. The distribution of mosaicism in the conceptus depends on the timing, cell lineage(s) involved, cell viability, and chromosome involved. The developmental consequences of mosaicism also are related to its meiotic or somatic type. Meiotic mosaicism often is associated with a more severely adverse effect on the conceptus (see trisomy zygote rescue) due to the presence of uniparental disomy in the embryo/fetus and/or to dysfunction of a trisomic placenta. As mosaicism can be tissue specific, the result of a normal karyotype in cultured lymphocytes does not exclude the presence of mosaicism elsewhere in the conceptus. Mosaicism can best be detected by a combination of traditional cytogenetic analysis with molecular cytogenetic techniques such as comparative genomic hybridization and fluorescence in situ hybridization. PMID- 10751088 TI - Imprinted H19 gene expression in embryogenesis and human cancer: the oncofetal connection. AB - Cancer cells resemble embryonal cells morphologically and share with them characteristics such as reduced differentiation, rapid proliferation rate, and increased motility. Genes expressed in embryogenesis, down-regulated with tissue maturation and reexpressed in cancer, are designated as oncofetal genes, and many of them are used as tumor markers. The H19 gene is an imprinted gene that is expressed from the maternal allele and functions as an RNA molecule. It is abundantly expressed in fetal life and down-regulated postnatally. We have shown oncofetal expression of H19 in human cancer. The study of H19 expression in testicular germ cell tumors of adolescents and young adults, which follow lines of differentiation of the conceptus, demonstrates dissociation between level of expression and monoallelic versus biallelic expression, which are two independent oncofetal characteristics of cancer. Expression of the maternally expressed H19 from the paternal allele in the villous cytotrophoblastic cells of the androgenetic complete hydatidiform mole is designated relaxation of imprinting. H19 is abundantly expressed in the fetal bladder mucosa and in carcinoma of the urinary bladder. It is a marker of early recurrence and may be used as a potential basis for gene therapy. PMID- 10751089 TI - Detection of submicroscopic subtelomeric chromosome translocations: a new case study. AB - Two sisters presented with multiple congenital abnormalities and developmental delay; abnormalities elsewhere in their extended family suggested that their father carried a balanced translocation. G-banded chromosome analysis showed apparently normal karyotypes. Fluorescence in situ hybridisation (FISH) with whole chromosome paints revealed no apparent abnormality in the father. However, further FISH studies, using multiple subtelomeric probes, demonstrated a derivative chromosome 16 in one sister. Subsequent studies showed that her sister also had a derivative 16 which had been inherited in an unbalanced form from their father, who carried a balanced reciprocal translocation between chromosomes 1 and 16. This report describes the detection of this submicroscopic translocation and the clinical findings in the two sisters. PMID- 10751090 TI - Possible case of Rubinstein-Taybi syndrome in a prehistoric skeleton from west central Illinois. AB - I describe an adult female skeleton (#Yo3-96) from the Yokem Mound skeletal series that had microcephaly; mild micrognathia; a broad nasal bridge; orbital hypertelorism; hypoplasia of the first metacarpals; pes planus; anomalies of the cervical spine, ribs, and sternum; and delayed epiphyseal remodeling of the radii and tibiae. Because the Yokem series has been subject to extensive measurement in previous studies, Yo3-96 can be characterized metrically and compared with others from her population. A differential diagnosis is made by comparing the available skeletal evidence with the known syndromes characterized by radial ray dysplasia. It is suggested that Yo3-96 represents the earliest known case of Rubinstein Taybi syndrome, a mental retardation syndrome that has been described in individuals from several modern populations. PMID- 10751092 TI - Diagnostic criteria for hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber syndrome). AB - Hereditary Hemorrhagic Telangiectasia (HHT) is easily recognized in individuals displaying the classical triad of epistaxis, telangiectasia, and a suitable family history, but the disease is more difficult to diagnosis in many patients. Serious consequences may result if visceral arteriovenous malformations, particularly in the pulmonary circulation, are unrecognized and left untreated. In spite of the identification of two of the disease-causing genes (endoglin and ALK-1), only a clinical diagnosis of HHT can be provided for the majority of individuals. On behalf of the Scientific Advisory Board of the HHT Foundation International, Inc., we present consensus clinical diagnostic criteria. The four criteria (epistaxes, telangiectasia, visceral lesions and an appropriate family history) are carefully delineated. The HHT diagnosis is definite if three criteria are present. A diagnosis of HHT cannot be established in patients with only two criteria, but should be recorded as possible or suspected to maintain a high index of clinical suspicion. If fewer than two criteria are present, HHT is unlikely, although children of affected individuals should be considered at risk in view of age-related penetration in this disorder. These criteria may be refined as molecular diagnostic tests become available in the next few years. PMID- 10751091 TI - Did Robert Louis Stevenson have hereditary hemorrhagic telangiectasia? AB - Chronic illness played a major role in the life and literary success of Robert Louis Stevenson. However, the exact nature of his chronic illness remains unclear. It is possible that Stevenson had hereditary hemorrhagic telangiectasia (Osler-Rendu-Weber Syndrome). This would explain his chronic respiratory complaints, recurrent episodes of pulmonary hemorrhage, and his death, at age 44 years, of probable cerebral hemorrhage. It would also explain his mother's hitherto unreported but apparent stroke, at age 38 years. Further support for this hypothesis might come from new details about the health of Stevenson and his relatives or from molecular analysis of tissue specimens remaining from him. PMID- 10751093 TI - Characterization of four arylsulfatase A missense mutations G86D, Y201C, D255H, and E312D causing metachromatic leukodystrophy. AB - Metachromatic leukodystrophy is a lysosomal storage disease caused by the deficiency of arylsulfatase A. Here we describe a hitherto unknown arylsulfatase A allele carrying a E312D missense mutation and characterize the effects of this and three previously described missense mutations, G86D, Y201C, and D255H, on arylsulfatase A. In transfection experiments no enzyme activity can be expressed from arylsulfatase A cDNAs coding for the D255H substituted enzyme, whereas Y201C and E312D mutations were associated with low amounts of residual enzyme activity. All amino acid substitutions lead to a decreased stability of the mutant enzyme, and metabolic labeling experiments indicated that except for the E312D substitution the mutations cause arrest of the mutant arylsulfatase A polypeptides in a prelysosomal compartment. PMID- 10751094 TI - Molecular cytogenetic studies in three patients with partial trisomy 2p, including CGH from paraffin-embedded tissue. AB - We report on three cases of partial trisomy 2p in which the identification and exact localization of the duplicated chromosome segment was possible only by application of molecular cytogenetic techniques. These included fluorescence in situ hybridization by use of wcp2, N-myc, and subtelomeric 2p probes and comparative genomic hybridization with DNA isolated from blood samples, frozen fetal tendon, and formalin fixed, paraffin-embedded fetal lung tissue. Two of the cases concerned fetuses of gestational week 20 and 24 with duplication of nonoverlapping terminal (2pter-->p24) and more proximal (2p25-->p23) segments and with distinctly different phenotypes. The third case was due to a de novo inverted duplication of 2p25-->p23, with loss of the subtelomeric region of 2p. This 53-month-old girl was a Bloom syndrome carrier. The patient had prenatal growth failure, borderline microcephaly, dilated lateral horns of the cerebral ventricles, transient cortical blindness, myopia, muscle hypotonia, and dilatation of the left renal collecting system. Dermal cysts were found on the glabella, the soles of both feet, and the vocal cord, causing respiratory embarrassment. Previously reported cases of pure trisomy 2p are reviewed, in an attempt to correlate clinical findings to overlapping regions in 2p. These cases illustrate the effectiveness of molecular cytogenetic methods in resolving subtle chromosomal aberrations in order to coordinate more accurately a chromosome regionspecific phenotype. PMID- 10751095 TI - Identification of a mutation in the XNP/ATR-X gene in a family reported as Smith Fineman-Myers syndrome. PMID- 10751096 TI - Position sense acuity is diminished following repetitive low-intensity work to fatigue in a simulated occupational setting. AB - Repetitive work to fatigue is soundly associated with work-related musculoskeletal disorders (WMSD), although the underlying mechanisms are poorly understood. In the present study, we tested the hypothesis that fatiguing work leads to proprioceptive deficits, which can be an initiating factor for the occurrence of WMSD. Thus, the position sense of the shoulder was determined for 13 males and 13 females before and after performing repetitive low-intensity arm work to fatigue in a simulated occupational setting. From a starting position of 45 degrees to the sagittal plane, position sense tests consisted of subjects attempting to actively reproduce target positions of horizontal movements to 15 degrees and 30 degrees (shoulder adduction) and to 60 degrees and 75 degrees (shoulder abduction). An analysis of variance revealed that the absolute error was significantly increased following fatigue for the subjects as a group (P < 0.001). Furthermore, females had an overall higher error than males (P < 0.01). No difference in error was detected for the shorter movements versus the longer movements. However, the overall absolute error for adduction was significantly higher than for abduction (P < 0.001). The results of the present study support the hypothesis of diminished proprioceptive acuity following low-intensity work to fatigue. A reduction in position sense acuity could lead to impairment in motor control, which would further impact on position sense. Thus, a vicious cycle may be activated that might result in WMSD. The poorer position sense acuity observed for females may contribute to the explanation of why females demonstrate a higher incidence of WMSD than males. PMID- 10751097 TI - Breathing pattern and exercise endurance time after exhausting cycling or breathing. AB - The aim of the present study was to investigate whether the changes in breathing pattern that frequently occur towards the end of exhaustive exercise (i.e., increased breathing frequency, fb, with or without decreased tidal volume) may be caused by the respiratory work itself rather than by leg muscle work. Eight healthy, trained subjects performed the following three sessions in random order: (A) two sequential cycling endurance tests at 78% peak O2 consumption (VO2peak) to exhaustion (A1, A2); (B) isolated, isocapnic hyperpnea (B1) at a minute ventilation (VE) and an exercise duration similar to that attained during a preliminary cycling endurance test at 78% VO2peak, followed by a cycling endurance test at 78% VO2peak (B2); (C) isolated, isocapnic hyperpnea (C1) at a VE at least 20% higher than that of the preliminary cycling test and the same exercise duration as the preliminary cycling test, followed by a cycling endurance test at 78% VO2peak (C2). Neither of the two isocapnic hyperventilation tasks (B1 or C1) affected either the breathing pattern or the endurance times of the subsequent cycling tests. Only cycling test A2 was significantly shorter [mean (SD) 26.5 (8.3) min] than tests A1 [41.0(9.0) min], B2 [41.9 (6.0) min], and C2 [42.0 (7.5) min]. In addition, compared to test A1, only the breathing pattern of test A2 was significantly different [i.e., VE: + 10.5 (7.6) 1 min(-1), and fb: + 12.1 (8.5) breaths min(-1)], in contrast to the breathing patterns of cycling tests B2 [VE: -2.5 (6.2) 1 min(-1), f(b): +0.2 (3.6) breaths min(-1)] and C2 [VE: -3.0 (7.0) 1 min(-1), fb: +0.6 (6.1) breaths min(-1)]. In summary, these results suggest that the changes in breathing pattern that occur towards the end of an exhaustive exercise test are a result of changes in the leg muscles rather than in the respiratory muscles themselves. PMID- 10751098 TI - Tension regulation during lengthening and shortening actions of the human soleus muscle. AB - In the present study we investigated tension regulation in the human soleus (SOL) muscle during controlled lengthening and shortening actions. Eleven subjects performed plantar flexor efforts on an ankle torque motor through 30 degrees of ankle displacement (75 degrees -105 degrees internal ankle angle) at lengthening and shortening velocities of 5, 15 and 30 degrees. S(-1). To isolate the SOL from the remainder of the triceps surae, the subject's knee was flexed to 60 degrees during all trials. Voluntary plantar flexor efforts were performed under two test conditions: (1) maximal voluntary activation (MVA) of the SOL, and (2) constant submaximal voluntary activation (SVA) of the SOL. SVA trials were performed with direct visual feedback of the SOL electromyogram (EMG) at a level resulting in a torque output of 30% of isometric maximum. Angle-specific (90 degrees ankle angle) torque and EMG of the SOL, medial gastrocnemius (MG) and tibialis anterior (TA) were recorded. In seven subjects from the initial group, the test protocol was repeated under submaximal percutaneous electrical activation (SEA) of SOL (to 30% isometric maximal effort). Lengthening torques were significantly greater than shortening torques in all test conditions. Lengthening torques in MVA and SVA were independent of velocity and remained at the isometric level, whereas SEA torques were greater than isometric torques and increased at higher lengthening velocities. Shortening torques were lower than the isometric level for all conditions. However, whereas SVA and SEA torques decreased at higher velocities of shortening, MVA torques were independent of velocity. These results indicate velocity- and activation-type-specific tension regulation in the human SOL muscle. PMID- 10751099 TI - Cardiac, arterial and venous adaptation to weightlessness during 6-month MIR spaceflights with and without thigh cuffs (bracelets). AB - The objectives of this investigation were to study the effects of thigh cuffs (bracelets) on cardiovascular adaptation and deconditioning in 0 g. The cardiovascular parameters of six cosmonauts were measured by echocardiography, Doppler, and plethysmography, during three 6-month MIR spaceflights. Measurements were made at rest during preflight (-30 days), inflight (1, 3-4, and 5-5.5 months) without cuffs (morning) and after 5 h with cuffs, and during postflight (+3 and +7 days). Lower-body negative pressure (LBNP) measurements were performed 1 day after each resting session. Inflight values of left ventricle end-diastolic volume and stroke volume measured without the thigh cuffs (-8 to -24% and -10 to 16%, respectively, both P < 0.05) were lower than corresponding preflight values. The jugular and femoral vein cross-sectional areas (Ajv and Afv, respectively) were enlarged (Ajv: by 23-30%, P < 0.001; Afv: by 33-70% P < 0.01). The renal and femoral vascular resistances (Rra and Rfa, respectively) decreased (Rra: by -15 to -16%, P < 0.01; Rfa: by -5 to -11%, P < 0.01). Inflight, the thigh cuffs reduced the Ajv (by -12 to -20%, P < 0.02), but enlarged the Afv (Afv: by 9-20%, P < 0.02) and increased the vascular resistance (Rra: by 8-13%, P < 0.05; Rfa: by 10-16%, P < 0.01) compared to corresponding inflight, without-cuffs values. During LBNP (-45 mmHg, where 1 mmHg = 133.3 N/ m2), Rfa and the ratio between cerebral and femoral blood flow (Qca/Qfa) increased less inflight and postflight (+25% for Rfa and +30% for Qca/Qfa) than during preflight (60% for Rfa and 75% for Qca/Qfa, P < 0.01). This reduced vasoconstrictive response and less efficient flow redistribution toward the brain was associated with orthostatic intolerance during postflight stand tests in all of the cosmonauts. The calf circumference increased less inflight and postflight (6% P < 0.05) than preflight (9% P < 0.05). The vascular response to LBNP remained similarly altered throughout the flight. The thigh cuffs compensated partially for the cardiovascular changes induced by exposure to 0 g, but did not interfere with 0 g deconditioning. PMID- 10751100 TI - Effects on dogs of surface-induced hypothermia and rewarming on the right heart function and pulmonary circulation. AB - Hypothermia is commonly found in accidents on land and at sea, yet its pulmonary circulatory effects have not been studied before. To study the effects of hypothermia on the right heart function and pulmonary circulation, cardiac catheterization was carried out on nine anaesthetized beagle dogs. The dogs were cooled between ice bags until the temperature in the pulmonary artery was 25 degrees C and then rewarmed using a heating box especially constructed for this purpose. Heart rate decreased significantly (P < 0.01) during cooling. Cardiac output also diminished mainly because of decreased heart rate. Total pulmonary resistance increased in the cold (P < 0.05) and returned to the initial level during rewarming. The peak rate of increase in pressure (dP/dtmax) of the right ventricular pressure curve did not show any significant change. Retardation in relaxation in hypothermia was indicated by an increase (P < 0.01) in the peak negative dP/dt of the right ventricular pressure curve. According to our results, the contraction rate did not change, but the relaxation rate decreased significantly during cooling. No signs of heart failure were observed and all parameters returned to normal during rewarming. In conclusion, right ventricular function was not compromised even during deep hypothermia. PMID- 10751101 TI - Thermal responses to cold wind of thermoneutral and cooled subjects. AB - The effects of initial thermal state on thermoregulatory responses to cold (-10 degrees C) in a 0.2 (still air), 1.0, and 5.0 m. S(-1) wind speed were studied. Eight young male subjects were first preconditioned in thermoneutral (+20 degrees C, TN) or cool (-5 degrees C, CO) environment for 60 min. After preconditioning the subjects were exposed to wind at -10 degrees C in a standing position, facing the wind, for 30 min. Precooling decreased mean skin temperature (Tsk) by 4.0 (SEM 0.1) degrees C (P < 0.001) and increased heat flux by 57 (SEM 2) W x m(-2) (P < 0.001) in comparison to TN. Cooling rate of Tsk was faster (P < 0.001) in TN than in CO at every wind speed. Even so, Tsk ended up at a lower level in CO (P < 0.001-0.01) than in TN at every wind speed. Local skin temperatures of hand, finger, foot and toe were significantly lower in CO than in TN at the end of all exposures to wind. Heat flux from the skin was 8% higher (NS) in TN at 5.0 m x s( 1) wind speed in comparison to CO. A 5.0 m x s(-1) wind speed increased oxygen consumption significantly (P < 0.001) in both CO and TN in comparison to still air. At 5.0 m x s(-1) wind speed the general thermal sensation was the same (cold) in both TN and CO, despite the higher Tsk in TN. In conclusion, Tsk decreased more rapidly in TN, probably due to rapid skin vasoconstriction and redistribution of circulation to the central body. Probably for the same reason, dry heat loss from the skin was at nearly the same level in both TN and CO. Although the initial thermal state did not affect the amount of heat loss, it significantly affected the peripheral temperatures and thermal sensations and should therefore be taken into consideration in the prediction of thermophysiological responses to wind. PMID- 10751102 TI - Motor unit synchronization in young and elderly adults. AB - In an effort to determine whether aging might alter the manner in which pairs of motor units are coactivated, the extent of motor unit synchronization was assessed in the first dorsal interosseous (FDI) muscle in seven young (mean 28 years) and eight older adults (mean 75 years). During constant-force isometric contractions at either 50% or 100% of maximal effort, motor units were recorded using a four-wire needle electrode and a multi-channel recording technique. Customized software was utilized to identify the occurrences of motor unit action potentials from the resultant three-channel signals. The magnitude of motor unit synchronization was determined using six variables that have been described previously in the literature. The extent of motor unit synchronization was similar in both young and older adults, occurring in nearly every motor unit pair observed. During the 50% maximum voluntary contractions, the CIS synchronization measure (the number of synchronized discharges per unit time) averaged 2.5 in the young subjects and 2.4 in the older individuals. The intensity of motor unit synchronization was somewhat greater during maximal force isometric contractions. We conclude that motor unit synchronization in the FDI muscle is a ubiquitous phenomenon that is not affected by the aging process. PMID- 10751103 TI - Continuous monitoring of haemodynamic parameters in humans during the early phase of simulated diving with and without breathholding. AB - This study examined the integrative changes of blood pressure (BP) and stroke volume (SV) leading to the initial biphasic heart rate (fc) response (first 15 s) in simulated diving manoeuvres with and without breathholding (BH). Simulated diving was studied in ten young healthy volunteers by application of a gel-filled pack at 0 degree C and 18 degrees C on the forehead with and without BH. Beat-by beat and second-to-second fc, BP, SV, and total peripheral vascular resistance (TPR) were followed by continuous non-invasive monitoring. In all conditions (BH with forehead cooling at 0 degree and 18 degrees C) there was an early rise in BP triggering the first tachycardial response (fc acceleration) which was immediately counteracted by the concurrent further increase of SV leading to the second phase of early bradycardic response (fc deceleration). Furthermore, the continuous beat-by-beat and second-to-second monitoring allowed the documentation of a highly significant increase of TPR within the first few seconds of the manoeuvres. Our data further indicated that the differences in haemodynamics observed during the stimuli at different temperatures was overruled by BH. Detailed comparisons of the beat-by-beat and second-to-second analyses were unable to show that one method was better than the other. Using continuous non invasive monitoring of haemodynamic variables during simulated diving manoeuvres it was possible to provide better insights into the physiological principles and meaning of the diving reflex in humans. PMID- 10751104 TI - Effect of concurrent strength and endurance training on skeletal muscle properties and hormone concentrations in humans. AB - The purpose of this study was to investigate the effect of concurrent strength and endurance training on strength, endurance, endocrine status and muscle fibre properties. A total of 45 male and female subjects were randomly assigned to one of four groups; strength training only (S), endurance training only (E), concurrent strength and endurance training (SE), or a control group (C). Groups S and E trained 3 days a week and the SE group trained 6 days a week for 12 weeks. Tests were made before and after 6 and 12 weeks of training. There was a similar increase in maximal oxygen consumption (VO2max) in both groups E and SE (P < 0.05). Leg press and knee extension one repetition maximum (1 RM) was increased in groups S and SE (P < 0.05) but the gains in knee extension 1 RM were greater for group S compared to all other groups (P < 0.05). Types I and II muscle fibre area increased after 6 and 12 weeks of strength training and after 12 weeks of combined training in type II fibres only (P < 0.05). Groups SE and E had an increase in succinate dehydrogenase activity and group E had a decrease in adenosine triphosphatase after 12 weeks of training (P < 0.05). A significant increase in capillary per fibre ratio was noted after 12 weeks of training in group SE. No changes were observed in testosterone, human growth hormone or sex hormone binding globulin concentrations for any group but there was a greater urinary cortisol concentration in the women of group SE and decrease in the men of group E after 12 weeks of training (P < 0.05). These findings would support the contention that combined strength and endurance training can suppress some of the adaptations to strength training and augment some aspects of capillarization in skeletal muscle. PMID- 10751105 TI - Simulated moderate altitude elevates serum erythropoietin but does not increase reticulocyte production in well-trained runners. AB - The purpose of this study was to investigate whether the modest increases in serum erythropoietin (sEpo) experienced after brief sojourns at simulated altitude are sufficient to stimulate reticulocyte production. Six well-trained middle-distance runners (HIGH, mean maximum oxygen uptake, VO2max = 70.2 ml x kg( 1) x min(-1) spent 8-11 h per night for 5 nights in a nitrogen house that simulated an altitude of 2650 m. Five squad members (CONTROL, mean VO2max= 68.9 ml x kg(-1) x min(-1) undertook the same training, which was conducted under near sea-level conditions (600 m altitude), and slept in dormitory-style accommodation also at 600 m altitude. For both groups, this 5-night protocol was undertaken on three occasions, with a 3-night interim between successive exposures. Venous blood samples were measured for sEpo after 1 and 5 nights of hypoxia on each occasion. The percentage of reticulocytes was measured, along with a range of reticulocyte parameters that are sensitive to changes in erythropoiesis. Mean serum erythropoietin levels increased significantly (P < 0.01) above baseline values [mean (SD) 7.9 (2.4) mU x ml(-1)] in the HIGH group after the 1st night [11.8 (1.9) mU x ml(-1), 57%], and were also higher on the 5th night [10.7 (2.2) mU x ml(-1), 42%] compared with the CONTROL group, whose erythropoietin levels did not change. After athletes spent 3 nights at near sea level, the change in sEpo during subsequent hypoxic exposures was markedly attenuated (13% and -4% change during the second exposure; 26% and 14% change during the third exposure; 1st and 5th nights of each block, respectively). The increase in sEpo was insufficient to stimulate reticulocyte production at any time point. We conclude that when daily training loads are controlled, the modest increases in sEpo known to occur following brief exposure to a simulated altitude of 2650 m are insufficient to stimulate reticulocyte production. PMID- 10751106 TI - Human physiological responses to immersion into water of different temperatures. AB - To differentiate between the effect of cold and hydrostatic pressure on hormone and cardiovascular functions of man, a group of young men was examined during 1-h head-out immersions in water of different temperatures (32 degrees C, 20 degrees C and 14 degrees C). Immersion in water at 32 degrees C did not change rectal temperature and metabolic rate, but lowered heart rate (by 15%) and systolic and diastolic blood pressures (by 11 %, or 12%, respectively), compared to controls at ambient air temperature. Plasma renin activity, plasma cortisol and aldosterone concentrations were also lowered (by 46%, 34%, and 17%, respectively), while diuresis was increased by 107%. Immersion at 20 degrees C induced a similar decrease in plasma renin activity, heart rate and systolic and diastolic blood pressures as immersion at thermoneutrality, in spite of lowered rectal temperature and an increased metabolic rate by 93%. Plasma cortisol concentrations tended to decrease, while plasma aldosterone concentration was unchanged. Diuresis was increased by 89%. No significant differences in changes in diuresis, plasma renin activity and aldosterone concentration compared to subjects immersed to 32 degrees C were observed. Cold water immersion (14 degrees C) lowered rectal temperature and increased metabolic rate (by 350%), heart rate and systolic and diastolic blood pressure (by 5%, 7%, and 8%, respectively). Plasma noradrenaline and dopamine concentrations were increased by 530% and by 250% respectively, while diuresis increased by 163% (more than at 32 degrees C). Plasma aldosterone concentrations increased by 23%. Plasma renin activity was reduced as during immersion in water at the highest temperature. Cortisol concentrations tended to decrease. Plasma adrenaline concentrations remained unchanged. Changes in plasma renin activity were not related to changes in aldosterone concentrations. Immersion in water of different temperatures did not increase blood concentrations of cortisol. There was no correlation between changes in rectal temperature and changes in hormone production. Our data supported the hypothesis that physiological changes induced by water immersion are mediated by humoral control mechanisms, while responses induced by cold are mainly due to increased activity of the sympathetic nervous system. PMID- 10751107 TI - Influence of temperature on the distribution of blood in humans as assessed by electrical impedance. AB - This study investigated whether ambient temperature influences the distribution of blood as indicated by electrical impedance. In ten supine humans, the room temperature was raised from 14 to 35 degrees C. Skin temperature and blood flow on the thorax increased by 3.6 (SD 0.3) degrees C and 84 (SD 40)%, respectively, and by 9.8 (SD 1) degrees C and 115 (SD 45)%, respectively, on the extremities (P < 0.05). Cardiac output remained unchanged, ear temperature and heart rate became elevated, and the oesophageal temperature and mean arterial pressure decreased (P < 0.05). At five discrete frequencies (1.5. 5, 50, 100, 200 kHz) thoracic impedance was increased by 1.2 (SD 1) to 1.5 (SD 1) omega (P < 0.05). In contrast, total body impedance was reduced by 16.4 (SD 5) omega and leg impedance was reduced by 4.0 (SD 2) omega, while an index of intracellular water within the thorax (the difference between the admittances at 100 kHz and 1.5 kHz) was decreased by 10 (SD 1) x 10(-4)S (P < 0.05). The results would suggest that total body impedance is dominated by the impedance of the extremities. The increase in thoracic impedance and a decrease in leg impedance (as in total body impedance) could be explained by a redistribution of blood from the thorax to the extremities during heating. Such a translocation of blood was confirmed by a reduced impedance based index of intracellular water within the thorax. PMID- 10751108 TI - Vascular risk factors in dementia. AB - This review describes differing profiles of vascular risk factors in different types of dementia. Although vascular risk factors are related to various types of strokes, their independent effect on the occurrence of poststroke dementia appears to be small. Various risk factors have been identified for microangiopathy-related cerebral abnormalities, such as white matter changes and lacunae, which are the core lesions for the development of a vascular dementia syndrome without stroke symptoms. Most consistently, arterial hypertension and diabetes mellitus have been found to be associated with such brain abnormalities. Diastolic blood pressure seems to be of particular importance as recent investigations demonstrate that this factor is related to the course of multiple lacunar strokes and the progression of white matter disease. Epidemiological studies report that various vascular risk factors including arterial hypertension, diabetes mellitus, and atrial fibrillation may also be associated with Alzheimer's disease. There is also evidence of a direct relationship between Alzheimer's disease and general atherosclerosis. Further investigations are needed to determine whether these associations are due to the weakness of diagnostic criteria, or whether vascular risk factors indeed modulate the clinical expression of primary degenerative dementia. Common susceptibility genes leading to shared risk factors may be one of the reasons for a higher coincidence of Alzheimer's disease and vascular dementia than can be expected by chance. A modulatory effect of vascular risk factors in the development of primary degenerative dementia may extend treatment options. PMID- 10751109 TI - Olfaction in Parkinson's disease: methods of assessment and clinical relevance. AB - Several neurological conditions have been reported to be associated with peripheral or central deficits of olfactory system. In recent years particular emphasis has been placed on the early and severe olfactory impairment in Parkinson's disease (PD), in which limited neuropathological studies have revealed a marked dopaminergic deficit in the olfactory tubercles. Moreover, indirect evidence suggests that dysfunction of the dopaminergic pathways from mesencephalon to the piriform cortex may play a role in olfactory impairment in PD. A large number of clinical studies have reported that olfactory loss in idiopathic PD is bilateral, present in hemiparkinsonism, unrelated to the stage or clinical subtype of the disease, and independent of antiparkinsonian medication. In addition, major olfactory alterations have been reported in familial PD and dementia with Lewy bodies but not in progressive supranuclear palsy and essential tremor. These findings might stimulate further research targeted to determine the biological substrate of dissimilar olfactory performances in these movement disorders. The present review summarizes standardized procedures for the assessment of olfactory acuity (detection threshold), identification (multiple choice odor naming), discrimination (differentiation between similar/dissimilar odorants), and memory (recognition of a substance previously smelled). Specific suggestions concerning the psychometric and neuropsychological evaluation of PD patients are provided. PMID- 10751110 TI - A comparison of neuropsychological deficits in primary and secondary progressive multiple sclerosis. AB - Neuropsychological deficits and the relationship to brain pathology were examined in 13 primary progressive (PP) and 12 secondary progressive (SP) multiple sclerosis patients with a similar duration of the progressive phase and comparable physical disability. A battery of neuropsychological tests to assess attention, short-term and working memory was administered to the patients, and their performance was compared to that of 20 healthy controls matched for age and premorbid IQ. Total cerebral lesion load on T2-weighted magnetic resonance imaging was measured in the patients. Both PP and SP patients performed significantly worse than controls in most of the neuropsychological tests. There were only subtle differences between SP and PP on the working memory task although magnetic resonance imaging lesion load was significantly higher in SP than in PP patients. In this exploratory study only subtle differences in cognitive impairment were detected between SP and PP patients matched for physical disability and relevant illness features. The results also suggest that the severity of cognitive impairment cannot be fully explained by the extent of abnormalities detected on conventional T2-weighted magnetic resonance images, and that other pathological abnormalities such as in normal-appearing white matter are likely to be involved. PMID- 10751111 TI - Oral pulsed high-dose dexamethasone for myositis. AB - To study the short-term effect of oral pulsed high-dose dexamethasone for myositis we treated eight newly diagnosed patients with three 28-day cycles of oral dexamethasone. Primary outcome measures were muscle strength, pain, and serum creatine kinase activity. Six patients responded. Side effects were mild. At follow-up five responders were still in remission, without medication. Pulsed high-dose dexamethasone seems beneficial in myositis. A larger, prednisone controlled trial is justified to analyze long-term efficacy. PMID- 10751112 TI - Three-dimensional power-mode ultrasound for quantification of the progression of carotid artery atherosclerosis. AB - Elucidation of the dynamic nature of plaque progression has important implications for clinicians. The present study sought to establish an in vivo method for visualizing structural changes in carotid plaques. Three-dimensional reconstruction of parallel two-dimensional gray-scale B-mode ultrasound combined with power-mode examination of 38 carotid artery plaques was performed in a prospective study of 32 patients (18 men, 14 women; mean age 67.5 +/- 7.6 years). Initial mean plaque volume was 391 microl. After a mean of 18.9 months carotid artery plaque progression had occurred in 15% of carotid artery plaques, with plaque volume increasing 59% in these cases. Plaque volume remained constant, within a range of +/- 20% in 85% of cases. Progressive plaques were predominantly hypoechoic (3/5 cases) or had an ulcerated surface in cases of a hyperechoic echogenicity (2/5 cases). Risk factors and drug therapy were unrelated to plaque progression. This study illustrates that the combination of three-dimensional ultrasound with power-mode imaging improves the separation of the intraluminal plaque surface from the vessel lumen. Three-dimensional reconstruction of atherosclerotic carotid artery plaques enables the reproducible quantification of plaque volume and is therefore an excellent technique for longitudinal trials assessing progression or regression of carotid artery disease. PMID- 10751113 TI - Acute confusional state as presenting feature in aneurysmal subarachnoid hemorrhage: frequency and characteristics. AB - In many patients with subarachnoid hemorrhage (SAH) there is a delay between the onset of symptoms and admission to hospital. An important cause for the delay is an initially erroneous diagnosis. The goal of this study was to determine the frequency of acute confusional state (ACS) as a presenting symptom of SAH and to describe the clinical and radiological characteristics of these patients. We studied all 717 patients registered from January 1989 to July 1997 in the SAH database of the University Medical Center Utrecht. For patients who presented with ACS we reviewed the computed tomography scans for baseline characteristics: the amount of cisternal blood, intraventricular or intracerebral hemorrhage, and hydrocephalus. Details about features of onset were known for 646 patients. Nine patients (1.4%) presented with ACS. In five patients ACS was either preceded by a period of loss of consciousness or accompanied by severe headache. Subtle focal deficits were found at initial neurological examination in four patients. Computed tomography demonstrated a frontal hematoma in three patients and hydrocephalus in four. The site of the ruptured aneurysm was at the anterior communicating artery in four patients, at the internal carotid artery in two, and at the basilar artery in two. In our series, one per 70 patients with SAH presents with ACS. Keys to early diagnosis of SAH in patients presenting with ACS are a preceding period of loss of consciousness and severe headache on neurological assessment. PMID- 10751114 TI - Treatment and outcome of severe intraventricular extension in patients with subarachnoid or intracerebral hemorrhage: a systematic review of the literature. AB - Severe intraventricular hemorrhage caused by extension from subarachnoid hemorrhage or intracerebral hemorrhage leads to hydrocephalus and often to poor outcome. We conducted a systematic review to compare conservative treatment, extraventricular drainage, and extraventricular drainage combined with fibrinolysis. We carried out a search in Medline of the literature between January 1966 and December 1998 and an additional hand-search from January 1990 to December 1998. Pharmaceutical companies were contacted to gather unpublished data. We reviewed the reference lists of all relevant articles. Two authors independently assessed eligibility of the studies and extracted data on characteristics of study design, patients, and treatment. Patients with primary intraventricular hemorrhage were excluded. Main outcome measures were death and poor outcome (defined as death or dependency) at the end of follow-up. No randomized clinical trial has yet been conducted so far, and we therefore reviewed only observational studies. The case fatality rate for conservative treatment (ten studies) was 78%. For extraventricular drainage (seven studies) it was 58% [relative risk versus conservative treatment (RR) 0.74; 95% confidence interval (CI) 0.55-0.99]. For extraventricular drainage with fibrinolytic agents (five studies) the case fatality rate was 6% (RR 0.08; 95% CI 0.02-0.24). The poor outcome rate for conservative treatment was 90%, that for extraventricular drainage 89% (RR 0.98; 95% CI 0.75-1.30) and that for extraventricular drainage with fibrinolytic agents 34% (RR 0.38; 95% CI 0.21-0.68). All RR values remained essentially the same after adjusting for age, sex, World Federation of Neurological Surgeons scale, study design, and year of publication for the studies that provided these data. Outcome is thus poor in patients with intraventricular extension of subarachnoid or intracerebral hemorrhage. This meta analysis suggests that treatment with ventricular drainage combined with fibrinolytics may improve outcome for such patients, although this impression is derived only from an indirect comparison between observational studies. A randomized clinical trial is warranted. PMID- 10751115 TI - HLA class I and class II profiles of patients presenting with Sydenham's chorea. AB - Sydenham's chorea (SC) may occur in rheumatic fever (RF) patients without arthritis and carditis. In this study we typed HLA antigens and alleles in patients presenting with the distinct major clinical manifestations of RF, i.e., chorea, carditis, or arthritis, in population and family studies. We evaluated 91 patients with RF for HLA-A, HLA-B, and HLA-DR antigens; of these, 33 had pure chorea, 26 pure carditis, 16 pure arthritis, and 16 carditis plus arthritis. We also typed 24 SC patients and their unaffected siblings for HLA-DRB1 and HLA-DQB1 alleles using molecular methods. HLA-B49 and HLA-DR1 antigens were overrepresented in the total group of patients with RF and in all the subgroups studied, excluding the SC subgroup in which the frequency of HLA-DR1 antigen was not increased. The frequencies of the HLA-DRB1 and HLA-DQB1 alleles in patients with pure chorea were not significantly different from those observed in controls. Similarly, the frequencies of HLA class II alleles in SC patients did not differ significantly from those observed in unaffected siblings. These findings show that immunogenetic susceptibility to RF varies according to the major clinical manifestation presented by the patient. PMID- 10751116 TI - Multiple sclerosis in North Norway, and first appearance in an indigenous population. AB - This study was performed to determine the prevalence of multiple sclerosis (MS) in 1993 and annual incidence rates 1983-1992, and to examine whether the disease occurs among the Sami people. According to earlier reports the two northernmost counties of Norway, Troms and Finnmark with 225,000 inhabitants, have a relatively low prevalence of MS: 20.6 per 100,000 in 1973 and 31.5 in 1983. Also no person who is of pure Sami heritage (i.e., with both parents speaking Sami natively) has been found with the disease. Except for the introduction of magnetic resonance imaging as a diagnostic tool, there has been no significant change in the neurological service in the area during the past 20 years. Files of patients with the diagnosis of MS were reviewed, and questionnaires were sent to all patients alive on the prevalence day of 1 January 1993. The prevalence in 1993 was 73.0 per 100,000. The mean crude annual incidence rate was 3.5 per 100,000 during the period 1983-1992 compared with 3.0 during 1974-1982. In 1983 there were no pure Sami among the MS patients, but one had a Sami father. On 1 January 1993 there were three patients with both Sami parents and three with only one Sami parent, which is a rate that is still lower than would be expected if the prevalence of MS among the Sami were similar to that in the rest of the Norwegian population. The study shows that the incidence of MS in Troms and Finnmark has been increasing over the past 10 years, but is still lower than on the western coast and in the eastern part of Norway. The lowest incidence is found in Finnmark, where the Sami population is highest. During the past 10 years MS has also been diagnosed among the Sami population. PMID- 10751117 TI - Cytarabine and highly active antiretroviral therapy in HIV-related progressive multifocal leukoencephalopathy. AB - We assessed survival in AIDS-related progressive multifocal leukoencephalopathy (PML) and the effect of cytarabine and antiretroviral therapy in a retrospective analysis of a series of consecutive 35 patients with AIDS-related PML in an academic AIDS referral center over 15 years. Treatment regimens consisted of highly active antiretroviral treatment (HAART), intravenous cytarabine, or both. Median survival after diagnosis in the overall series was 88 days. Patients with low CD4 cell count tended to have shorter survival. Seven patients (20%) had prolonged survival (> 1 year). Cytarabine did not affect survival. Seven patients were treated with HAART, which did not significantly improve survival. We conclude that the prognosis of AIDS-related PML is still poor, with a median survival of 3 months. PMID- 10751118 TI - Trigeminal neuralgia in pontine cavernous angioma. PMID- 10751119 TI - European brain death codes: Portuguese guidelines. PMID- 10751120 TI - Unusual ulnar sensory innervation and Martin-Gruber anastomosis in a patient with a carpal tunnel syndrome. PMID- 10751121 TI - Chronic progressive primary lumbosacral plexus neuritis: MRI findings and response to immunoglobulin therapy. PMID- 10751122 TI - Intramuscular phenol injection for severe cervical dystonia. PMID- 10751123 TI - Phenotype variability of dystonia in monozygotic twins. PMID- 10751124 TI - Thomas Willis (1621-1675). PMID- 10751125 TI - Beta-amyloid of Alzheimer's disease activates an apoptotic pathway via caspase-8. PMID- 10751126 TI - Refractory spinal cord injury induced gastroparesis: resolution with erythromycin lactobionate, a case report. AB - Erythromycin lactobionate (ERY), a macrolide antibiotic, has been the focus of investigation as a new gastrointestinal prokinetic agent. In individuals who are able-bodied (AB), ERY has shown promise in various forms of gastroparesis (GP). Recent evidence suggests that medications used to stimulate intestinal motility in individuals who are AB have had similar results in those individuals with spinal cord injury (SCI). Medications that have been used in the past for GP in SCI include metaclopramide, neostigmine, and bethanechol. In this observation, a patient with T-6 paraplegia, who developed GP secondary to acute SCI, is presented. During his hospital stay, the patient was treated with gastric decompression, bowel rest, H2 blockers, intravenous metaclopramide, and eventually required parenteral nutritional support. ERY was started and symptoms abated. At this point, the nasogastric tube was removed and oral feeding was successfully started. This case report is the first to describe a patient with refractory SCI-induced GP who responded to intravenous ERY. Further study in this area is warranted. PMID- 10751127 TI - The effect of pulsed electromagnetic fields on osteoporosis at the knee in individuals with spinal cord injury. AB - The purpose of this study was to determine the effects of pulsed electromagnetic fields on osteoporotic bone at the knee in individuals with chronic spinal injury. The study consisted of 6 males with complete spinal cord injury at a minimum of 2 years duration. Bone mineral density (BMD) was obtained at both knees at initiation, 3 months, 6 months, and 12 months using dual energy X-ray absorptiometry. In each case, 1 knee was stimulated using The Bone Growth Stimulator Model 3005 from American Medical Electronics, Incorporated and the opposite knee served as the control. Stimulation ceased at 6 months. At 3 months BMD increased in the stimulated knees 5.1% and declined in the control knees 6.6% (p < .05 and p < .02, respectively). By 6 months the BMD returned to near baseline values and at 12 months both knees had lost bone at a similar rate to 2.4% below baseline for the stimulated knee and 3.6% below baseline for the control. There were larger effects closer to the site of stimulation. While the stimulation appeared useful in retarding osteoporosis, the unexpected exaggerated decline in the control knees and reversal at 6 months suggests underlying mechanisms are more complex than originally anticipated. The authors believe a local as well as a systemic response was created. PMID- 10751128 TI - Spinal cord injury and snowboarding--the British Columbia experience. AB - Snowboarding is an alpine sport growing in popularity, particularly among male youth. This study of 10 consecutive cases admitted to the Vancouver Hospital and Health Sciences Centre Acute Spinal Cord Injury Unit, over the 1997 to 1998 winter season, analyzes the epidemiology of snowboarding spinal injury. Information was collected retrospectively on the mechanism, location, and pattern of injury, and personal details of the individuals who suffered the injuries. The average age at time of injury was 22.4 years, with a range of 16 to 29. All but 1 of the cases were self-acknowledged expert-level snowboarders, with an average of 6.25 years experience. Nine of the injured were male. There was only 1 cervical injury, with the majority being low thoracolumbar, and 4 incidences of L-1 fracture. Compression and burst fracture were the predominant vertebral fracture patterns and there was a 50% incidence of significant neurologic injury and deficit. The most common mechanism of injury was axial loading following a failed jump or fall from a height, varying from 2 to 25 feet. The lack of associated injuries in 9 of the cases suggests a limited ability of the extremities to offset such falls. Contributing factors included the inherent riskiness of the sport, participant characteristics, lack of formal instruction, and self constructed jumps. Poor weather conditions, unfamiliarity with a run, collisions, and consumption of alcohol also played lesser roles. The frequent association between spinal fracture and significant neurologic deficit in this group has not previously been reported in other studies. PMID- 10751129 TI - Effectiveness of neomycin/polymyxin bladder irrigation to treat resistant urinary pathogens in those with spinal cord injury. AB - Individuals with spinal cord injury (SCI) will sometimes develop bacterial organisms in the bladder that are resistant to oral antibiotics. This study evaluated the effectiveness of a 5-day course of intermittent neomycin/polymyxin bladder irrigation at eradicating or changing the bacterial sensitivity from parenteral to oral antibiotics. A chart review of individuals with SCI who were treated with neomycin/polymyxin bladder irrigations was performed. Inclusion criteria included the use of an indwelling catheter and the presence of asymptomatic bacteria resistant to oral antibiotics. The most common reason for treatment was eradication of resistant organisms prior to urologic testing. Bladder irrigation consisted of 3 rinses with 30 ml 3 times a day for 5 days. Pre and post-urine samples were compared for white blood cells (WBCs), colony count and culture, and sensitivity. Chi-square tests were used to determine whether the proportion of changes in resistance or sensitivities was different from zero. The Wilcoxon Signed Rank Test was used to determine differences in bacteria, colony counts, and WBCs. Ten individuals were identified. A total of 12 neomycin/polymyxin irrigation treatments were evaluated because 2 individuals had a second series of irrigations at least 6 months apart. Nine of the 12 (75%) were considered to have successful irrigations because there was a change in culture sensitivity so that oral antibiotics would be effective post irrigation. This was statistically significant. There were no significant changes in colony counts or the number of WBCs. The authors concluded that while neomycin/polymyxin bladder irrigation did not change the type of organism, it was effective in changing resistance of most organisms. Individuals could then be treated with oral rather than intravenous or intramuscular antibiotics. Further work is needed to determine whether other variables, such as increased length of time of irrigation or increased frequency of irrigations, may actually eradicate the organisms. PMID- 10751130 TI - Systematic review of risk factors for urinary tract infection in adults with spinal cord dysfunction. AB - Recurrent urinary tract infections (UTIs) occur frequently in persons with neurogenic bladders due to spinal cord dysfunction, resulting in increased morbidity and cost. The authors conducted a systematic review to identify risk factors for UTI using controlled clinical trials, cohort, and cross-sectional studies that assessed risk factors for UTI and included bacteriuria or UTI as an outcome. Twenty-two studies met the inclusion criteria. Quality assessment revealed important methodological deficiencies. Two studies provide evidence supporting increased bladder residual volume as a risk factor. Most of the retrieved studies investigated method of drainage with the consistent finding that persons using intermittent catheterization had fewer infections than those with indwelling catheters. There is conflicting evidence over the value of sterile or "no touch" catheter techniques compared with clean intermittent catheterization. There is insufficient evidence to assess risk due to psychosocial, behavioral, and hygiene factors; sex; level of function; and time since injury. PMID- 10751131 TI - Heterotopic ossification: diagnosis and management, current concepts and controversies. AB - HO is a pathologic process in which soft tissues undergo ossification. The etiology is unknown. Patients with neurologic deficits develop HO in the proximal joints. An incidence of 20% to 30% is commonly reported; 8% to 10% of these patients develop severe functional limitations. Routine prophylaxis in patients with SCI cannot be justified. Early diagnosis and treatment with EHDP or other anti-inflammatory medications have been shown to result in good functional outcomes. Elevated SAP levels and positive radionuclide scan, in addition to restriction of joint ROM, are indicative of HO formation. Role of other anti inflammatories, radiation, and early surgical excision without bone maturity in these patients need to be studied further. A small percentage of patients require surgery to improve function. Joint manipulation to produce pseudoarthrosis is not indicated in patients with SCI. PMID- 10751132 TI - Depression and spinal cord injury: a monozygotic twin study. AB - Although, in earlier work, depression in individuals with spinal cord injury (SCI) was attributed to difficulties adjusting to SCI, more recent articles have emphasized the importance of constitutional and environmental factors not specific to SCI, as well as established theoretical models of depression. To further explore this question, 11 pairs of monozygotic twins, where one of each pair was spinal cord injured, were studied. Measures included the Beck Depression Inventory, the depression scale of the SCL-90R, and the Rosenberg Self-Esteem Scale. Using pairwise t tests, the authors did not find any significant differences between SCI and non-SCI co-twins. These findings are consistent with the idea that the occurrence of SCI does not inevitably lead to increased depression. PMID- 10751133 TI - FK506 increases the regeneration of spinal cord axons in a predegenerated peripheral nerve autograft. AB - The authors examined the ability of FK506 to accelerate axonal regeneration of rat spinal cord axons in a peripheral nerve (PN) graft. Predegenerated autografts were produced by transecting the left tibial nerve 1 week prior to spinal cord implantation into the lumbar (L-3-L-4) spinal cord. Rats were given daily injections of either FK506 (5 mg/kg, subcutaneous) or vehicle for 21 days. The PN grafts from FK506-treated rats contained larger sized regenerating axons compared with vehicle-treated controls, and mean axonal areas increased by 25% at 7.5 mm along the PN graft. Fluoro-Gold retrograde labeling confirmed that the regenerating axons originated from the central nervous system. Unexpectedly, the majority (>50%) of neurons in the red nucleus were retrogradely labeled in the FK506-treated animals only. The results indicate that FK506 not only accelerates the elongation of spinal cord axons but also promotes regeneration of rubrospinal neurons. PMID- 10751134 TI - Breathlessness and exercise in spinal cord injury. AB - After spinal cord injury (SCI), breathlessness during daily activities is common. In 308 individuals with SCI, the authors measured pulmonary function and administered a survey regarding health status, participation in wheelchair athletics, and breathlessness during different activities. The following questions were included: A. Are you troubled by shortness of breath when hurrying on the level or going up a slight hill?; B. Do you have to go slower than people of your own age on the level because of breathlessness?; C. Do you ever have to stop for breath when going at your own pace on the level?; and D. Do you ever have to stop for breath after going about 100 yards on the level? The analysis was restricted to 183 subjects with neurologically motor complete or incomplete SCI who, to get around, used hand-propelled wheelchairs more than 50% of the time. Of these, 56 (31%) reported breathlessness during some types of activities. Subjects with neurologically motor complete cervical or high thoracic SCI (T-6 and above) were more likely to report breathlessness than others (39% compared with 25%, p = .039). Among wheelchair athletes, the prevalence of breathlessness was 8/49 (16%) versus 48/134 (36%) for non-athletes (p = .011). Adjusting for smoking, neurological level, and history of obstructive lung disease, non athletes were 2.3 times more likely to report breathlessness than athletes were (p = .049 to .075, depending on regression model). This relationship persisted when adjusted for percent predicted forced expiratory volume (FEV1) and maximal expiratory and inspiratory pressures. Therefore in SCI, wheelchair athletes are less likely to report breathlessness than non-athletes, but the mechanism does not appear to be improvement in respiratory muscle performance or pulmonary function. PMID- 10751135 TI - A simplified pharmacologic erection program for patients with spinal cord injury. AB - INTRODUCTION AND OBJECTIVES: Intracavernosal injection (ICI) of vasoactive agents has been successfully used in the treatment of erectile dysfunction (ED). The authors' pharmacologic erection program, using a fixed combination of prostaglandin E1 (PGE1) and papaverine, is notable for its simplicity and acceptance by patients who have suffered spinal cord injuries (SCIs). METHODS: Patients undergo baseline questionnaire, physical examination, and hormone profile followed by instruction and injection of a fixed combination of PGE1 and papaverine. On successive visits, the patient injects himself and the dosage is titrated until a satisfactory erection is obtained. Patients who do not respond to injection of 1.0 cc are considered treatment failures. Patients return periodically for routine follow-up. RESULTS: From May 1994 to March 1997, 37 patients with SCI underwent initial evaluation and 28 (76%) responded to injection therapy and were successfully using self-injection therapy at 3-month follow-up. Twenty-three patients are still on injection therapy. Five patients have dropped out for several reasons including a lack of a current sexual partner (60%) and pain with injection (40%). Patient age ranges from 24 to 72. The dosage range was 0.10 to 0.50 cc (mean = 0.29 cc). The average duration of erection was 43 minutes. At 3-month follow-up, 85% of the patients rated their erections as good or excellent. Forty-three percent of patients are using ICI 1 or more times per week. Seventy-seven percent of patients are moderately or extremely satisfied with their treatment and 89% said that they would recommend this program to a friend. CONCLUSIONS: This simplified pharmacologic erection program offers safe, well accepted, and effective therapy for ED to a SCI population with very high patient satisfaction. PMID- 10751136 TI - Human immunodeficiency virus type 1 p24 concentration measured by boosted ELISA of heat-denatured plasma correlates with decline in CD4 cells, progression to AIDS, and survival: comparison with viral RNA measurement. AB - Human immunodeficiency virus type 1 (HIV-1) RNA and p24 antigen concentrations were determined in plasma samples from 169 chronically infected patients (median CD4 cell count, 140 cells/microL; range, 0-1500 cells/microL). p24 quantification involved heat-mediated immune complex dissociation and tyramide signal amplification-boosted ELISA, which has a diagnostic sensitivity similar to that of RNA quantification by a commercial polymerase chain reaction kit. In Cox's proportional hazard models adjusted for CD4 cell count, both RNA (P<.005) and p24 (P=.043) levels were significant predictors of progression to AIDS. Measurement of p24 was superior to measurement of RNA in the model for survival (P=.032 vs. P=.19). p24 level was a significant predictor of CD4 cell decline in models adjusted for CD4 cell counts and was superior or equivalent to RNA level, depending on the group analyzed. Stratification by CD4 cell counts at baseline showed that the superiority of p24 measurement was more pronounced at lower levels of CD4 cells (<200/microL). p24 level may be of interest as a simple and inexpensive predictive marker of disease progression. PMID- 10751137 TI - Strong human immunodeficiency virus (HIV)-specific CD4+ T cell responses in a cohort of chronically infected patients are associated with interruptions in anti HIV chemotherapy. AB - Virus-specific CD4+ T-helper cell function is important in controlling human immunodeficiency virus (HIV) infection but is impaired in patients with progressive HIV disease. It has been reported that after highly active antiretroviral therapy (HAART), HIV-specific lymphoproliferative responses remain absent, whereas responses to non-HIV microbial antigens are restored. However, in analyzing immune responses in a cohort of chronically infected adults on HAART, we observed strong HIV-specific CD4+ T cell responses of Th-1 phenotype in 11 of 22 patients. The magnitude and frequency of HIV-specific lymphoproliferative responses was strongly associated with previous interruptions in HAART (P=.001). In contrast, the magnitude of CD8+ T cell responses to HIV Gag, Pol, Env, and Nef was similar in patients who had and those who had not interrupted HAART. We conclude that (1) a significant proportion of chronically HIV-infected patients on HAART can generate strong HIV-specific CD4+ and CD8+ T cell immunity and (2) transient interruptions in antiviral treatment may prime or boost HIV-specific CD4+ T-helper responses. PMID- 10751138 TI - Analysis of measles virus binding sites of the CD46 gene in patients with subacute sclerosing panencephalitis. AB - Measles virus (MV) binding sites of the CD46 gene were sequenced in patients with subacute sclerosing panencephalitis (SSPE) and in controls. There were 3 novel polymorphisms, including C/T at nucleotide position 38 (C/T38), G/A at position 176 (G/A176), and C/T at position 453 (C/T453), at allele frequencies of.97:.03, .99:.01, and.97:.03, respectively. The G/A176 polymorphism causes an Arg/Gln amino acid change within the essential binding sites of MV, whereas the C/A38 polymorphism causes a Ser/Phe change outside the MV binding sites. The C/T453 polymorphism does not produce an amino acid change. Two of the 40 SSPE patients and 2 of the 32 controls had both C/T38 and C/T453 polymorphisms in heterozygous patterns. One control subject, but no SSPE patients, had the G/A176 polymorphism in a heterozygous pattern. Thus, it is not likely that CD46 gene alteration has a role as a host susceptibility factor in the development of SSPE. PMID- 10751139 TI - The emergence of another tickborne infection in the 12-town area around Lyme, Connecticut: human granulocytic ehrlichiosis. AB - Human granulocytic ehrlichiosis (HGE) is an emerging tickborne infection, increasingly recognized in areas in which Lyme disease is endemic, but there are few data on the incidence of HGE. Prospective population-based surveillance was conducted in the 12-town area around Lyme, Connecticut, by means of both active and passive methods, from April through November of 1997, 1998, and 1999. Five hundred thirty-seven residents presenting to their primary care provider with an acute febrile illness suggestive of HGE were identified. Of these, 137 (26%) had laboratory evidence (by indirect fluorescent antibody staining or polymerase chain reaction) of HGE; 89 were confirmed cases, and 48 were probable cases. The incidence of confirmed HGE was 31 cases/100,000 in 1997, 51 cases/100,000 in 1998, and 24 cases/100,000 in 1999. A subset of sera was tested by use of immunoblot assays, and results were in agreement with indirect fluorescent antibody methods for 86% of samples analyzed. Thus, HGE is an important cause of morbidity and is now the second most common tickborne infection in southeastern Connecticut. PMID- 10751141 TI - The role of normal flora in Giardia lamblia infections in mice. AB - The presence of normal bacterial flora in the intestinal tract is thought to protect against colonization by pathogens. Only a few specific examples of this protection have been demonstrated for bacterial pathogens and protozoan infections. Mice from one commercial breeding farm were found to be less susceptible to infection with Giardia lamblia than were isogenic mice from another facility. When mice were housed together, resistance to infection was readily transferred to normally susceptible mice. After resistant mice were treated with neomycin, differences in susceptibility to infection were shown to be due to differences in the resident flora present in these mice. These results suggest the possible use of probiotic therapy for prevention of G. lamblia infections and may help explain some of the variability of outcomes seen in G. lamblia infections in humans. PMID- 10751142 TI - Tying the knot: linking cytokinesis to the nuclear cycle. AB - For the survival of both the parent and the progeny, it is imperative that the process of their physical division (cytokinesis) be precisely coordinated with progression through the mitotic cell cycle. Recent studies in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are beginning to unravel the nature of the links between cytokinesis and the nuclear division cycle. The cyclin-dependent kinases and a novel surveillance mechanism that monitors cytokinesis and/or morphogenesis appear to play important regulatory roles in forging these links. It is becoming increasingly clear that the inactivation of the mitosis-promoting cyclin-dependent kinase, which marks the completion of the nuclear division cycle, is essential for actomyosin ring constriction and division septum assembly in both yeasts. Additionally, the spindle pole bodies are emerging as important transient locale for proteins that might play a key role in coupling the completion of mitosis to the onset of cytokinesis. PMID- 10751144 TI - VEGF nuclear accumulation correlates with phenotypical changes in endothelial cells. AB - Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that plays a prominent role in normal vascular biology and pathology. In an experimental wound model, the mechanical disruption of monolayers of cultured endothelial cells resulted in two phenotypically distinct cell subpopulations in which VEGF was internalized by alternative endocytotic pathways and delivered to different subcellular compartments. In the cells away from the wound, VEGF was internalized via the classical receptor-mediated endocytosis pathway and accumulated in the endosomal compartment, whereas in the cells situated at the edges of a wound, VEGF was rapidly taken up and translocated to the nucleus. VEGF internalization and subsequent nuclear accumulation only occurred for a short period of time after the wounding and was specifically abolished by antibodies that bind to the KDR binding site of VEGF. In the cells with VEGF nuclear accumulation, the levels of wound healing related proteins, such as Factor VIII (FVIII), tissue factor (TF) and tissue plasminogen activator, rapidly and dramatically increased compared to the cells that internalized VEGF via the classical endocytotic pathway. The increase in FVIII and TF was abolished when the nuclear transport is blocked. These data suggest that nuclear VEGF accumulation may be involved in modulating the levels of the proteins of the coagulation and fibrinolysis pathways. PMID- 10751145 TI - Cadherin repertoire determines partner-specific gap junctional communication during melanoma progression. AB - Reduced gap junction activity has long been implicated in tumorigenesis. To elucidate the potential role of intercellular communication in melanoma development, we examined gap junctional capability of melanocytic cells from various stages of tumor progression in coculture models using dye transfer assays. Normal melanocytes coupled with keratinocytes by gap junctional formation, whereas melanoma cells did not. Instead, melanoma cells communicated among themselves and with fibroblasts. This switch in communication partners coincided with a shift from E-cadherin to N-cadherin expression during melanoma development. Forced expression of E-cadherin by adenoviral gene transfer in N cadherin-expressing melanoma cells restored gap junctional compatibility with keratinocytes. Our data suggest that (1) melanocyte transformation is associated with loss of the pre-existing gap junctional activity with keratinocytes but a concomitant gain of communication with a newly juxtaposed cell type, the fibroblasts, (2) the specificity of gap junctional formation during melanoma development is determined by the cadherin profile on the melanocytic cells and (3) the overall gap junctional activity of melanocytic cells is not reduced with transformation. PMID- 10751143 TI - Lysosome-endosome fusion and lysosome biogenesis. AB - Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can fuse directly with late endosomes to form a hybrid organelle. This has a led to a hypothesis that dense core lysosomes are in essence storage granules for acid hydrolases and that, when the former fuse with late endosomes, a hybrid organelle for digestion of endocytosed macromolecules is created. Lysosomes are then re-formed from hybrid organelles by a process involving condensation of contents. In this Commentary we review the evidence for formation of the hybrid organelles and discuss the current status of our understanding of the mechanisms of fusion and lysosome re-formation. We also review lysosome biosynthesis, showing how recent studies of lysosome-like organelles including the yeast vacuole, Drosophila eye pigment granules and mammalian secretory lysosomes have identified novel proteins involved in this process. PMID- 10751146 TI - Cell cycle-dependent localization of the CDK2-cyclin E complex in Cajal (coiled) bodies. AB - We have found that CDK2 and cyclin E, but not cyclin A, accumulates within Cajal bodies (CBs) in a cell cycle-dependent fashion. In the absence of cyclin E, CDK2 is not enriched in the CB compartment, suggesting that the translocation of CDK2 to CBs is dependent on cyclin E. CDK2 and cyclin E could be recruited to CBs as a functional complex or CBs may serve as 'docking stations' for CDK2-cyclin E activation by CAKs during the G(1)/S transition. Notably, CDK7-cyclin H-Mat1 complexes are known to accumulate in CBs. Treatment of cells with inhibitors of either CDKs (olomoucine, 200 microM) or RNA polymerase I (actinomycin D, 0.05 microgram/ml), results in a striking reorganization of CDK2 and p80 coilin to the nucleolar periphery. Furthermore, we demonstrate that p80 coilin can be phosphorylated by purified CDK2-cyclin E complexes in vitro. Thus coilin and other CB proteins appear to be downstream targets of CDK2-cyclin E complex mediated signaling pathways regulating cell cycle progression and controlling aspects of CB function. Possible roles for CDK2 and cyclin E in the well documented association of CBs, histone gene clusters and RNA 3' end processing factors are discussed. PMID- 10751147 TI - The muscle regulatory and structural protein MLP is a cytoskeletal binding partner of betaI-spectrin. AB - Muscle LIM protein (MLP) is a striated muscle-specific factor that enhances myogenic differentiation and is critical to maintaining the structural integrity of the contractile apparatus. The ability of MLP to regulate myogenesis is particularly interesting since it exhibits multiple subcellular localizations, being found in both nuclear and cytoplasmic compartments. Despite extensive biochemical analyses on MLP, the mechanism(s) by which it influences the myogenic program remains largely undefined. To further examine the role of MLP as a positive myogenic regulator, a yeast two-hybrid screen was employed to identify cytoplasmic-associated MLP binding partners. From this screen, the cytoskeletal protein betaI-spectrin was isolated. Protein interaction assays demonstrate that MLP and betaI-spectrin associate with one another in vivo as well as when tested under several in vitro binding conditions. betaI-spectrin binds specifically to MLP but not to the MLP related proteins CRP1 and CRP2 or to other LIM domain containing proteins. The MLP:beta-spectrin interaction is mediated by the second LIM motif of MLP and by repeat 7 of beta-spectrin. Confocal microscopy studies also reveal that MLP co-localizes with beta-spectrin at the sarcolemma overlying the Z- and M-lines of myofibrils in both cardiac and skeletal muscle tissue. Given that beta-spectrin is a known costamere protein, we propose that sarcolemma associated MLP also serves as a key costamere protein, stabilizing the association of the contractile apparatus with the sarcolemma by linking the beta spectrin network to the alpha-actinin crosslinked actin filaments of the myofibril. PMID- 10751148 TI - Large-scale chromatin organization of the major histocompatibility complex and other regions of human chromosome 6 and its response to interferon in interphase nuclei. AB - The large-scale chromatin organization of the major histocompatibility complex and other regions of chromosome 6 was studied by three-dimensional image analysis in human cell types with major differences in transcriptional activity. Entire gene clusters were visualized by fluorescence in situ hybridization with multiple locus-specific probes. Individual genomic regions showed distinct configurations in relation to the chromosome 6 terrritory. Large chromatin loops containing several megabases of DNA were observed extending outwards from the surface of the domain defined by the specific chromosome 6 paint. The frequency with which a genomic region was observed on an external chromatin loop was cell type dependent and appeared to be related to the number of active genes in that region. Transcriptional up-regulation of genes in the major histocompatibility complex by interferon-gamma led to an increase in the frequency with which this large gene cluster was found on an external chromatin loop. Our data are consistent with an association between large-scale chromatin organization of specific genomic regions and their transcriptional status. PMID- 10751149 TI - A new specialized cell-matrix interaction in actively resorbing osteoclasts. AB - We have identified a novel cell-matrix interaction in activated osteoclasts. Resorbing osteoclasts maintain a barrier adjacent to the bone surface that prevents the leakage of secreted protons and proteases from the resorption area. Using a series of fluorescent dyes of known molecular mass and different surface charge we established that negatively charged molecules with M(r )up to 10,000 rapidly accumulate underneath actively resorbing osteoclasts. Live cell imaging shows that staining could be detected underneath the osteoclasts as early as 30 seconds after the addition of the low molecular mass markers. We provide evidence that the actin cytoskeleton and the adhesion substrate in contact with the cells are critically involved in the maintenance of the sealing barrier. These data taken together suggest that the accumulation under resorbing osteoclasts is by diffusion rather than transcytotic delivery. Our results indicate that the net concentration of secreted and resorbed components is a balance between generation rate and limited diffusion rather than the presence of an impermeable barrier as previously suggested. This dynamic osteoclast sealing zone may, thus, provide the mechanism by which osteoclast migration and resorption can occur simultaneously. PMID- 10751150 TI - Glycocalyx modulation is a physiological means of regulating cell adhesion. AB - Here we present experimental evidence that phagocytic cells use modulation of specific components of their glycocalyx to regulate their binding capacity. Particles coated with antibodies specific for the CD32 medium affinity IgG receptor were driven along human monocytic THP-1 cells (expressing CD32) in a flow chamber operated at low shear rate. Surprisingly, only minimal adhesion was observed. However, when cells were activated by exposure to fibronectin-coated surfaces and/or soluble &ggr; interferon, adhesion efficiency was dramatically increased, whereas the apparent glycocalyx thickness displayed 20% decrease, and the surface density of CD43/leukosialin carbohydrate epitopes displayed 30-40% decrease on activated cells. The existence of a causal link between adhesion increase and glycocalyx alteration was strongly supported by the finding that (i) both phenomena displayed similar kinetics, (ii) an inverse relationship between THP-1 cell binding capacity and glycocalyx density was demonstrated at the individual cell level, and (iii) adhesion enhancement could not be ascribed to an increased binding site density or improved functional capacity of activated cells. Additional experiments revealed that cell-to-particle adhesion resulted in delayed (i.e. more than a few minutes) egress of CD43/leukosialin from contact areas. Since the time scale of particle attachment was less than a second, surface mobility should not affect the potential of CD43 to impair the initial step of adhesion. Finally, studies performed with fluorescent lectins suggested that THP-1 cell activation and increased adhesive potential were related to a decrease of O-glysosylation rather than N-glycosylation of surface glycoproteins. PMID- 10751151 TI - Activation of TrkA tyrosine kinase in embryonal carcinoma cells promotes cell compaction, independently of tyrosine phosphorylation of catenins. AB - Cadherins are transmembrane receptors whose extracellular domain mediates homophilic cell-cell interactions, while their cytoplasmic domain associates with a family of proteins known as catenins. Although the mechanisms that regulate the assembly and functional state of cadherin-catenin complexes are poorly understood, current evidence supports a role for protein tyrosine kinase activity in regulating cell adhesion and migration. Tyrosine phosphorylation of catenins is thought to mediate loss of intercellular adhesion promoted by activation of receptor tyrosine kinases in epithelial cells. Here, we show that activation of ectopically expressed TrkA, the tyrosine kinase receptor for nerve growth factor (NGF), stimulates embryonal carcinoma P19 cells to develop extensive intercellular contacts and to assemble into closely packed clusters. Thus, activation of receptor tyrosine kinases can differentially regulate adhesiveness by cell-type-specific mechanisms. Furthermore, activation of TrkA in P19 and epithelial MDCK cells induces tyrosine phosphorylation of p120(ctn) and of beta catenin, irrespective of the elicited cellular response. The selective Src tyrosine kinase inhibitor PP2, however, suppresses NGF- or HGF-induced tyrosine phosphorylation of catenins in both P19 and MDCK cells without interfering with the acquisition of a compacted or scattered phenotype. These findings provide a cogent argument for considering that tyrosine phosphorylation of catenins is dispensable for their interaction with cadherins and, ultimately, for the modulation of cadherin-based cell adhesion by receptor tyrosine kinases. PMID- 10751152 TI - Determination of cell polarity in germinated spores and hyphal tips of the filamentous ascomycete Ashbya gossypii requires a rhoGAP homolog. AB - In the filamentous ascomycete Ashbya gossypii, like in other filamentous fungi onset of growth in dormant spores occurs as an isotropic growth phase generating spherical germ cells. Thereafter, a switch to polarized growth results in the formation of the first hyphal tip. The initial steps of hyphal tip formation in filamentous fungi, therefore, resemble processes taking place prior to and during bud emergence of unicellular yeast-like fungi. We investigated whether phenotypic similarities between these distinct events extended to the molecular level. To this end we isolated and characterized the A. gossypii homolog of the Saccharomyces cerevisiae BEM2 gene which is part of a network of rho-GTPases and their regulators required for bud emergence and bud growth in yeast. Here we show that the AgBem2 protein contains a GAP- (GTPase activating protein) domain for rho-like GTPases at its carboxy terminus, and that this part of AgBem2p is required for complementation of an Agbem2 null strain. Germination of spores resulted in enlarged Agbem2 germ cells that were unable to generate the bipolar branching pattern found in wild-type germ cells. In addition, mutant hyphae were swollen due to defects in polarized cell growth indicated by the delocalized distribution of chitin and cortical actin patches. Surprisingly, the complete loss of cell polarity which lead to spherical hyphal tips was overcome by the establishment of new cell polarities and the formation of multiple new hyphal tips. In conclusion these results and other findings demonstrate that establishment of cell polarity, maintenance of cell polarity, and polarized hyphal growth in filamentous fungi require members of &rgr;-GTPase modules. PMID- 10751154 TI - During both interphase and mitosis, DNA topoisomerase II interacts with DNA as well as RNA through the protein's C-terminal domain. AB - DNA topoisomerase II (topo II) is thought to be a nuclear enzyme; during interphase most was insoluble and could be recovered in the pellet after centrifugation of cell homogenates at 10,000 g (P-10). Upon entry into mitosis, the majority of topo II did not associate with condensed chromosomes but was apparently solubilized and redistributed throughout the cell. Although two non chromosomal subfractions of mitotic topo II were defined by centrifugation at 130,000 g, the vast majority (>90%) was recovered in the pellet (P-130). In vivo nucleic acid interactions with topo II were monitored by a recently developed approach of UV-photo-crosslinking, immunoprecipitation and (32)P-labeling. P-10 (interphase) topo II was largely associated with DNA. P-130 (mitotic non chromosomal) topo II was primarily associated with RNA. These nucleic acid interactions with both interphase and mitotic topo II occurred through the catalytically inert and as yet, poorly understood C-terminal domain of the protein. P-10 topo II was highly active enzymatically. Activity, measured by the ability of topo II to decatenate kDNA minicircles, was reduced by treatment with phosphatase. In contrast, P-130 topo II was relatively inactive but activity could be increased by phosphatase treatment. In vivo, P-130 topo II was more heavily phosphorylated than P-10 topo II; in both, only the C-terminal domain of topo II was detectably modified. Our observations suggest that cell cycle dependent changes in the distribution, nucleic acid interactions and enzymatic activity of topo II are regulated, at least in part, by phosphorylation/dephosphorylation. PMID- 10751153 TI - Two domains of p80 katanin regulate microtubule severing and spindle pole targeting by p60 katanin. AB - The assembly and function of the mitotic spindle requires the activity of a number of microtubule-binding proteins. Some microtubule-binding proteins bind microtubules in vitro but do not co-localize with microtubules in interphase cells. Instead these proteins associate with specific subregions of the mitotic spindle. Katanin, a heterodimeric microtubule-severing ATPase, is found localized at mitotic spindle poles. In this paper we demonstrate that human p60 katanin and the C-terminal domain of human p80 katanin both bind microtubules in vitro. Association of these two proteins results in an increased microtubule affinity and increased microtubule-severing activity in vitro. Association of these subunits in transfected HeLa cells increases microtubule disassembly activity and targeting to spindle poles. The N-terminal WD40 domain of p80 katanin acts as a negative regulator of microtubule disassembly activity and is also required for spindle pole localization, possibly through interactions with another spindle pole protein. These results support a model in which katanin is targeted to spindle poles through a combination of direct microtubule binding by the p60 subunit and through interactions between the WD40 domain and an unknown protein. We propose that both domains of p80 are essential in precisely regulating katanin's activity in vivo. PMID- 10751155 TI - Oxygen-sensitive membrane transporters in vertebrate red cells. AB - Oxygen is essential for all higher forms of animal life. It is required for oxidative phosphorylation, which forms the bulk of the energy supply of most animals. In many vertebrates, transport of O(2) from respiratory to other tissues, and of CO(2) in the opposite direction, involves red cells. These are highly specialised, adapted for their respiratory function. Intracellular haemoglobin, carbonic anhydrase and the membrane anion exchanger (AE1) increase the effective O(2)- and CO(2)-carrying capacity of red cells by approximately 100 fold. O(2) also has a pathological role. It is a very reactive species chemically, and oxidation, free radical generation and peroxide formation can be major hazards. Cells that come into contact with potentially damaging levels of O(2) have a variety of systems to protect them against oxidative damage. Those in red cells include catalase, superoxide dismutase and glutathione. In this review, we focus on a third role of O(2), as a regulator of membrane transport systems, a role with important consequences for the homeostasis of the red cell and also the organism as a whole. We show that regulation of red cell transporters by O(2) is widespread throughout the vertebrate kingdom. The effect of O(2) is selective but involves a wide range of transporters, including inorganic and organic systems, and both electroneutral and conductive pathways. Finally, we discuss what is known about the mechanism of the O(2) effect and comment on its physiological and pathological roles. PMID- 10751156 TI - Three-dimensional odor tracking by Nautilus pompilius. AB - The 'living fossil' Nautilus pompilius is thought to use olfaction as its primary sensory system during foraging, yet neither the organs responsible for olfaction nor the mechanisms or behaviors associated with odor tracking have been subjected to experimentation. Flume testing under dark conditions revealed that Nautilus could consistently detect and follow turbulent odor plumes to the source over distances up to 10 m, exhibiting two types of orientation behavior while sampling in three dimensions. The paired rhinophores were necessary for orientation behavior: when they were temporarily blocked either uni- or bilaterally, Nautilus detected odor but could not track the plume and locate the source. Animals that were tested post-blockage were able to track and locate the source. The role of the 90 thin tentacles remains enigmatic; they seemed to be able to detect odor, but they were not capable of guiding orientation behavior towards a distant odor source. Bilateral chemical sensing by rhinophores in three dimensions may have been the Umwelt of ammonites and belemnites before the evolution of complex eyes and fast locomotion in modern coleoids. PMID- 10751157 TI - Cranial kinesis in geckoes: functional implications. AB - Although it is generally assumed that cranial kinesis is a plesiomorphic characteristic in squamates, experimental data tend to contradict this hypothesis. In particular, coupled kinesis (i.e. streptostyly and mesokinesis) presumably arose independently in only a limited number of highly specialised groups. In this study, we investigated cranial kinesis in one of the most specialised of these groups: geckoes. On the basis of cineradiographic and electromyographic data, the fast opening and the slow closing/power stroke phases were modelled to elucidate possible functions of the observed kinesis. The results of these analyses show that the retraction of the muzzle unit during crushing is a self-reinforcing system that increases bite force and reduces the joint forces; the active protraction of the kinetic system during jaw opening, in contrast, enhances opening speed through the coupling of the intracranial units. It can be argued that cranial kinesis in geckoes is probably not an adaptive trait as such but, instead, a consequence of the 'Bauplan' of the cranial system in these animals. Presumably as a result of constructional constraints on the size of the jaw musculature and eyes, the supratemporal and postorbital bars were lost, which resulted in enormous mobility in the skull. To counteract the potential negative factors associated with this (decrease in bite force, skull damage), the kinetic system may have become coupled, and thus functional. PMID- 10751159 TI - Changes in electric organ discharge after pausing the electromotor system of Gymnotus carapo. AB - During their entire lives, weakly electric fish produce an uninterrupted train of discharges to electrolocate objects and to communicate. In an attempt to learn about activity-dependent processes that might be involved in this ability, the continuous train of discharges of intact Gymnotus carapo was experimentally interrupted to investigate how this pausing affects post-pause electric organ discharges. In particular, an analysis was conducted of how the amplitude and relative timing of the three major deflections of the complex discharge change over the course of the first 1000 post-pause discharges. The dependence of these variables on the duration of the preceding pause and on water temperature is analysed. In addition, pause-induced small reverberations at the end of the discharge are described. Common to all amplitude changes is a fast initial decrease in amplitude with a slow recovery phase; amplitude changes scale with the duration of the preceding pause and are independent of the interdischarge interval. The absence of changes in the postsynaptic-potential-derived first phase of the discharge together with changes in the amplitude ratio of the third and fourth deflections suggest that the amplitude changes are mainly due to pause induced changes in the inner resistance of the electric organ. A model is formulated that approximates the pattern of amplitude changes. The post-pause changes described here may provide a new way to test current models of complex discharge generation in Gymnotus carapo and illustrate the speed at which changes of an electric organ discharge can take place. PMID- 10751158 TI - In search of molecular dovetails in mussel byssus: from the threads to the stem. AB - We recently described the cDNA sequence for a unique collagenous protein, preCol P, in the byssal threads of the marine mussel Mytilus edulis. The translated amino acid sequence encodes an unprecedented block-copolymer-like sequence with a central collagenous domain flanked by elastin-like sequences. Here, we report on the presence of two additional variants of preCol-P. The distribution of these variants in M. edulis foot tissue was examined by reverse transcription followed by polymerase chain reaction (RT-PCR) and in situ hybridization techniques. One of the variants, P33, exhibits a graded distribution with decreasing concentrations along the longitudinal axis of the foot. The second variant, P22, is expressed only at the base of the mussel's foot. In situ hybridization confirms the exclusive expression of preCol-P variant P22 in the stem gland. We propose that this variant may represent a molecular 'dovetail' between the proximal thread and the byssal stem, imparting extensibility and elastic recoil to the ring portion of the stem. PMID- 10751160 TI - Graded neuromuscular transmission in the heart of the isopod crustacean Ligia exotica. AB - We present several lines of evidence for the occurrence of graded synaptic transmission in addition to impulse-mediated transmission at the neuromuscular junction between cardiac ganglion (CG) neurones and the myocardium in the isopod crustacean Ligia exotica. In the heart of adult Ligia exotica, the CG acts as a primary pacemaker for the heartbeat by generating periodic bursts of impulses and entrains the myogenicity of the myocardium via impulse-mediated excitatory junctional potentials. When impulse generation was blocked by tetrodotoxin (TTX; 50 nmol l(-)(1)), the CG neurones and the myocardium periodically exhibited synchronized slow depolarizing potentials. The association between the slow depolarizing potentials in the neurone and the myocardium was eliminated by application of Joro spider toxin (JSTX), a specific glutamate antagonist. When the CG neurone was made quiescent by a higher dose of TTX (1.0 micromol l(-)(1)), sinusoidal current injected into the CG neurone induced similar sinusoidal membrane potential responses in the myocardium. The sinusoidal muscle responses were eliminated by application of either JSTX or low-Ca(2+) saline. Under voltage clamp conditions, the myocardium exhibited periodic inward current responses to sinusoidal current stimuli applied to the CG neurone. The reversal potential for the current response of the myocardium was similar to that of the impulse mediated excitatory junctional current (EJC). Extracellular macropatch recordings of EJCs made at the neuromuscular junctional site revealed the spontaneous appearance of miniature EJCs asynchronous with the CG spikes in addition to large spike-evoked EJCs. The miniature EJCs were present in saline containing TTX, and their frequency was strongly affected by the slow membrane potential change in the CG neurone. These results suggest that the CG neurones drive the myocardium by graded neuromuscular transmission in addition to impulse-mediated transmission in the heart of Ligia exotica. PMID- 10751161 TI - Central role of the apical membrane H+-ATPase in electrogenesis and epithelial transport in Malpighian tubules. AB - The effects of bafilomycin A(1), a blocker of V-type H(+)-ATPases, were investigated in Malpighian tubules of Aedes aegypti. Bafilomycin A(1) reduced rates of transepithelial fluid secretion and the virtual short-circuit current (vI(sc)) with an IC(50) of approximately 5 micromol l(-)(1). As vI(sc) decreased, the electrical resistance increased across the whole epithelium and across the apical membrane, indicating effects on electroconductive pathways. Bafilomycin A(1) had no effect when applied from the tubule lumen, pointing to the relative impermeability of the apical membrane to bafilomycin A(1). Thus, bafilomycin A(1) must take a cytoplasmic route to its blocking site in the proton channel of the H(+)-ATPase located in the apical membrane of principal cells. The inhibitory effects of bafilomycin A(1) were qualitatively similar to those of dinitrophenol in that voltages across the epithelium (V(t)), the basolateral membrane (V(bl)) and the apical membrane (V(a)) depolarized towards zero in parallel. Moreover, V(bl )always tracked V(a), indicating electrical coupling between the two membranes through the shunt. Electrical coupling allows the H(+)-ATPase to energize not only the apical membrane, but also the basolateral membrane. Furthermore, electrical coupling offers a balance between electroconductive entry of cations across the basolateral membrane and extrusion across the apical membrane to support steady-state conditions during transepithelial transport. PMID- 10751162 TI - The effect of metabolic depression on proton leak rate in mitochondria from hibernating frogs. AB - Futile cycling of protons across the mitochondrial inner membrane accounts for 20 % or more of the total standard metabolic rate of a rat. Approximately 15 % of this total is due to proton leakage inside the skeletal muscle alone. This study examined whether the rate of proton leak is down-regulated as a part of a coordinated response to energy conservation during metabolic depression in cold submerged frogs. We compared the proton leak rate of skeletal muscle mitochondria isolated from frogs at different stages of hibernation (control, 1 month and 4 months of submergence in normoxia and hypoxia). The kinetics of mitochondrial proton leak rate was unaltered throughout normoxic and hypoxic submergence. The state 4 respiration rates did not differ between control animals and frogs hibernating in normoxia. In contrast, the state 4 respiration rates obtained from frogs submerged in hypoxic water for 4 months were half those of control animals. This 50 % reduction in respiration rate in hypoxic hibernation was due to a reduction in electron transport chain activity and consequent decrease in mitochondrial membrane potential. We conclude that proton leak rate is reduced during metabolic depression as a secondary result of a decrease in electron transport chain activity, but that the proton conductance is unchanged. In addition, we show that the rate of proton leakage and the activity of the electron transport chain are lower in frogs than in rats, strengthening the observation that mitochondria from ectotherms have a lower proton conductance than mitochondria from endotherms. PMID- 10751163 TI - The inhibitory effects of (gamma)-aminobutyric acid (GABA) on growth hormone secretion in the goldfish are modulated by sex steroids. AB - Double-labelling studies at the electron microscopic level demonstrated that gamma-aminobutyric acid (GABA)-immunoreactive nerve endings are associated with growth-hormone-secreting cells in the proximal pars distalis of the goldfish pituitary gland, suggesting that GABA may be important for the control of growth hormone release in this species. An in vitro assay for GABA-transaminase activity demonstrated that the pituitary is a site for the metabolism of GABA to succinic acid. In vitro, GABA or the GABA antagonists bicuculline and saclofen did not affect the rate of growth hormone release from dispersed pituitary cells in static incubation. In contrast, intracerebroventricular injection of GABA reduced serum growth hormone levels within 30 min. During the seasonal gonadal cycle, intraperitoneal injection of GABA was without effect in sexually regressed goldfish, but caused a significant decrease in serum growth hormone levels in sexually recrudescent animals. Intraperitoneal implantation of solid silastic pellets containing oestradiol increased serum GH levels fivefold in sexually regressed and recrudescent goldfish; in both groups, GABA suppressed the oestradiol-stimulated increase in circulating growth hormone levels. The effect of oestradiol on basal serum growth hormone levels was specific since progesterone and testosterone were without effect. However, in recrudescent animals treated with progesterone and testosterone, the inhibitory effects of GABA on serum growth hormone levels were absent, indicating a differential role for these steroids in growth hormone release. Taken together, these results demonstrate that GABA has an inhibitory effect on growth hormone release in goldfish. PMID- 10751164 TI - The effects of acute hypoxia on chemically or neuronally induced catecholamine secretion in rainbow trout (Oncorhynchus mykiss) in situ and in vivo. AB - The potential direct and modulating effects of acute hypoxia on catecholamine secretion in rainbow trout (Oncorhynchus mykiss) were assessed in situ, using a perfused cardinal vein preparation, and in vivo, using chronically cannulated fish. Acute (10 min) perfusion with hypoxic (P(O2)<10 mmHg) saline or homologous hypoxic blood did not have a statistically significant effect on basal (non stimulated) catecholamine secretion. A field stimulation technique was used to excite the sympathetic nerves innervating the chromaffin cells electrically in situ under conditions of high-P(O2) (saline P(O2)=152 mmHg; 1 mmHg=0.133 kPa) or low-P(O2) (saline P(O2)<10 mmHg) perfusion at constant P(CO2) (2.3 mmHg). The results demonstrated that neuronally evoked catecholamine secretion was significantly lowered by 50 % during perfusion with hypoxic saline. To assess whether the inhibitory effect of hypoxia during neuronal stimulation in situ resulted from modulation of nicotinic and/or muscarinic receptor-linked pathways, perfused posterior cardinal vein preparations were injected with selective nicotinic (10(-)(7) or 10(-)(6 )mol kg(-)(1) nicotine) or muscarinic (10(-)(3 )mol kg(-)(1) methacholine) receptor agonists. For both doses of nicotine, catecholamine secretion was significantly lowered during hypoxia by 55 %. During muscarinic receptor stimulation, perfusion with hypoxic saline caused a 42 % reduction in the rate of catecholamine secretion. In contrast, catecholamine secretion elicited by depolarising levels of KCl (60 mmol l(-)(1)) was unaffected by the oxygen status of the perfusate. In vivo, intra-arterial injections of nicotine (300-600 nmol kg(-)(1)) into normoxic (water P(O2)=155 mmHg) or moderately hypoxic fish (water P(O2)=80 mmHg) caused a dose-dependent elevation of circulating catecholamine levels. However, despite the inhibitory influence of localised hypoxia on chromaffin cell responsiveness previously demonstrated in situ, the increase in plasma catecholamine levels after intra-arterial injection of nicotine was significantly enhanced in the hypoxic fish. The differences between the results from the in vivo and in situ experiments may reflect the contribution of higher control centres and modulating factors in vivo that are absent in situ. PMID- 10751166 TI - Effects of fish size and temperature on weakfish disturbance calls: implications for the mechanism of sound generation. AB - To categorize variation in disturbance calls of the weakfish Cynoscion regalis and to understand their generation, we recorded sounds produced by different sized fish, and by similar-sized fish at different temperatures, as well as muscle electromyograms. Single, simultaneous twitches of the bilateral sonic muscles produce a single sound pulse consisting of a two- to three-cycle acoustic waveform. Typical disturbance calls at 18 degrees C consist of trains of 2-15 pulses with a sound pressure level (SPL) of 74 dB re 20 microPa at 10 cm, a peak frequency of 540 Hz, a repetition rate of 20 Hz and a pulse duration of 3.5 ms. The pulse duration suggests an incredibly short twitch time. Sound pressure level (SPL) and pulse duration increase and dominant frequency decreases in larger fish, whereas SPL, repetition rate and dominant frequency increase and pulse duration decreases with increasing temperature. The dominant frequency is inversely related to pulse duration and appears to be determined by the duration of muscle contraction. We suggest that the lower dominant frequency of larger fish is caused by a longer pulse (=longer muscle twitch) and not by the lower resonant frequency of a larger swimbladder. PMID- 10751165 TI - The oxalate/sulfate antiporter in lobster hepatopancreas: internal and external binding constants. AB - Utilizing a purified basolateral plasma membrane vesicle (BLMV) preparation containing a sulfate/oxalate antiporter, it was demonstrated that sulfate exhibited similar binding characteristics to the transporter whether bound internally or externally. It was also demonstrated that oxalate had similar binding characteristics to the antiporter whether it was bound internally or externally. Oxalate had a greater affinity to the transporter than did sulfate. Several organic anions affected binding and, therefore, overall transport by the antiporter. Most notably, sulfate was the only anion that stimulated oxalate uptake into BLMVs, which suggests a conservative binding specificity for the antiporter. 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and/or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited the transport rate, confirming the existence of oxalate/sulfate exchange by the transporter. These results suggest that oxalate, not sulfate, regulates the transport rate because of its greater affinity to the transporter. PMID- 10751167 TI - Microtubules and mitotic cycle phase modulate spatiotemporal distributions of F actin and myosin II in Drosophila syncytial blastoderm embryos. AB - We studied cyclic reorganizations of filamentous actin, myosin II and microtubules in syncytial Drosophila blastoderms using drug treatments, time lapse movies and laser scanning confocal microscopy of fixed stained embryos (including multiprobe three-dimensional reconstructions). Our observations imply interactions between microtubules and the actomyosin cytoskeleton. They provide evidence that filamentous actin and cytoplasmic myosin II are transported along microtubules towards microtubule plus ends, with actin and myosin exhibiting different affinities for the cell's cortex. Our studies further reveal that cell cycle phase modulates the amounts of both polymerized actin and myosin II associated with the cortex. We analogize pseudocleavage furrow formation in the Drosophila blastoderm with how the mitotic apparatus positions the cleavage furrow for standard cytokinesis, and relate our findings to polar relaxation/global contraction mechanisms for furrow formation. PMID- 10751168 TI - The development of Xenopus tropicalis transgenic lines and their use in studying lens developmental timing in living embryos. AB - The generation of reporter lines for observing lens differentiation in vivo demonstrates a new strategy for embryological manipulation and allows us to address a long-standing question concerning the timing of the onset of differentiation. Xenopus tropicalis was used to make GFP reporter lines with (gamma)1-crystallin promoter elements directing GFP expression within the early lens. X. tropicalis is a close relative of X. laevis that shares the same ease of tissue manipulation with the added benefits of a diploid genome and faster life cycle. The efficiency of the Xenopus transgenic technique was improved in order to generate greater numbers of normal, adult transgenic animals and to facilitate in vivo analysis of the crystallin promoter. This transgene is transmitted through the germline, providing an accurate and consistent way to monitor lens differentiation. This line permitted us to distinguish models for how the onset of differentiation is controlled: by a process intrinsic to differentiating tissue or one dependent on external cues. This experiment would not have been feasible without the sensitivity and accuracy provided by the in vivo reporter. We find that, in specified lens ectoderm transplanted from neural tube stage donors to younger neural-plate-stage hosts, the onset of differentiation, as measured by expression of the crystallin/GFP transgene, is delayed by an average of 4.4 hours. When specified lens ectoderm is explanted into culture, the delay was an average of 16.3 hours relative to control embryos. These data suggest that the onset of differentiation in specified ectoderm can be altered by the environment and imply that this onset is normally controlled by external cues rather than by an intrinsic mechanism. PMID- 10751169 TI - The morphogenetic role of midline mesendoderm and ectoderm in the development of the forebrain and the midbrain of the mouse embryo. AB - The anterior midline tissue (AML) of the late gastrula mouse embryo comprises the axial mesendoderm and the ventral neuroectoderm of the prospective forebrain, midbrain and rostral hindbrain. In this study, we have investigated the morphogenetic role of defined segments of the AML by testing their inductive and patterning activity and by assessing the impact of their ablation on the patterning of the neural tube at the early-somite-stage. Both rostral and caudal segments of the AML were found to induce neural gene activity in the host tissue; however, the de novo gene activity did not show any regional characteristic that might be correlated with the segmental origin of the AML. Removal of the rostral AML that contains the prechordal plate resulted in a truncation of the head accompanied by the loss of several forebrain markers. However, the remaining tissues reconstituted Gsc and Shh activity and expressed the ventral forebrain marker Nkx2.1. Furthermore, analysis of Gsc-deficient embryos reveals that the morphogenetic function of the rostral AML requires Gsc activity. Removal of the caudal AML led to a complete loss of midline molecular markers anterior to the 4th somite. In addition, Nkx2.1 expression was not detected in the ventral neural tube. The maintenance and function of the rostral AML therefore require inductive signals emanating from the caudal AML. Our results point to a role for AML in the refinement of the anteroposterior patterning and morphogenesis of the brain. PMID- 10751170 TI - The essential Mcm7 protein PROLIFERA is localized to the nucleus of dividing cells during the G(1) phase and is required maternally for early Arabidopsis development. AB - PROLIFERA (PRL) encodes a homologue of the DNA replication licensing factor Mcm7, a highly conserved protein found in all eukaryotes. Insertions in the PROLIFERA gene are lethal, resulting in decreased transmission through the female gametophyte, and homozygous embryonic lethality. We show here that PROLIFERA is specifically expressed in populations of dividing cells in sporophytic tissues of the plant body, such as the palisade layer of the leaf and founder cells of initiating flower primordia. Gene fusions with the green fluorescent protein (GFP) reveal that the PROLIFERA protein accumulates during the G(1) phase of the cell cycle, and is transiently localized to the nucleus. During mitosis, the fusion protein rapidly disappears, returning to daughter nuclei during G(1). PROLIFERA::GUS fusions are strongly expressed in the central cell nucleus of mature megagametophytes, which have a variety of arrest points reflecting a leaky lethality. Expression is also observed in the endosperm of mutant prl embryo sacs that arrest following fertilization. Crosses with wild-type pollen result in occasional embryonic lethals that also stain for GUS activity. In contrast, embryos resulting from crosses of wild-type carpels with PRL::GUS pollen do not stain and are phenotypically normal. In situ hybridization of GUS fusion RNA indicates transcription is equivalent from maternally and paternally derived alleles, so that accumulation of maternally derived gametophytic protein is likely to be responsible for the 'maternal' effect. PMID- 10751171 TI - Chip is an essential cofactor for apterous in the regulation of axon guidance in Drosophila. AB - LIM-homeodomain transcription factors are expressed in subsets of neurons and are required for correct axon guidance and neurotransmitter identity. The LIM homeodomain family member Apterous requires the LIM-binding protein Chip to execute patterned outgrowth of the Drosophila wing. To determine whether Chip is a general cofactor for diverse LIM-homeodomain functions in vivo, we studied its role in the embryonic nervous system. Loss-of-function Chip mutations cause defects in neurotransmitter production that mimic apterous and islet mutants. Chip is also required cell-autonomously by Apterous-expressing neurons for proper axon guidance, and requires both a homodimerization domain and a LIM interaction domain to function appropriately. Using a Chip/Apterous chimeric molecule lacking domains normally required for their interaction, we reconstituted the complex and rescued the axon guidance defects of apterous mutants, of Chip mutants and of embryos doubly mutant for both apterous and Chip. Our results indicate that Chip participates in a range of developmental programs controlled by LIM-homeodomain proteins and that a tetrameric complex comprising two Apterous molecules bridged by a Chip homodimer is the functional unit through which Apterous acts during neuronal differentiation. PMID- 10751172 TI - Temporal and spatial gradients of Fgf8 and Fgf17 regulate proliferation and differentiation of midline cerebellar structures. AB - The midbrain-hindbrain (MHB) junction has the properties of an organizer that patterns the MHB region early in vertebrate development. Fgf8 is thought to mediate this organizer function. In addition to Fgf8, Fgf17 and Fgf18 are also expressed in the MHB junction. Fgf17 is expressed later and broader than either Fgf8 or Fgf18. Disrupting the Fgf17 gene in the mouse decreased precursor cell proliferation in the medial cerebellar (vermis) anlage after E11.5. Loss of an additional copy of Fgf8 enhanced the phenotype and accelerated its onset, demonstrating that both molecules cooperate to regulate the size of the precursor pool of cells that develop into the cerebellar vermis. However, expression patterns of Wnt1, En2, Pax5 and Otx2 were not altered suggesting that specification and patterning of MHB tissue was not perturbed and that these FGFs are not required to pattern the vermis at this stage of development. The consequence of this developmental defect is a progressive, dose-dependent loss of the most anterior lobe of the vermis in mice lacking Fgf17 and in mice lacking Fgf17 and one copy of Fgf8. Significantly, the differentiation of anterior vermis neuroepithelium was shifted rostrally and medially demonstrating that FGF also regulates the polarized progression of differentiation in the vermis anlage. Finally, this developmental defect results in an ataxic gait in some mice. PMID- 10751174 TI - The transcription factor Lmx1b maintains Wnt1 expression within the isthmic organizer. AB - Cells in the caudal mesencephalon and rostral metencephalon become organized by signals emanating from the isthmus organizer (IsO). The IsO is associated with the isthmus, a morphological constriction of the neural tube which eventually defines the mesencephalic/ metencephalic boundary (MMB). Here we report that the transcription factor Lmx1b is expressed and functions in a distinct region of the IsO. Lmx1b expression is maintained by the glycoprotein Fgf8, a signal capable of mediating IsO signaling. Lmx1b, in turn, maintains the expression of the secreted factor Wnt1. Our conclusions are substantiated by the following: (i) Lmx1b mRNA becomes localized to the isthmus immediately after Fgf8 initiation, (ii) Wnt1 expression is localized to the Lmx1b expression domain, but with slightly later kinetics, (iii) Fgf8-soaked beads generate similar domains of expression for Lmx1b and Wnt1 and (iv) retroviral-mediated expression of Lmx1b (Lmx1b/RCAS) maintains Wnt1 expression in the mesencephalon. Ectopic Lmx1b is insufficient to alter the expression of a number of other genes expressed at the IsO, suggesting that it does not generate a new signaling center. Instead, if we allow Lmx1b/RCAS infected brains to develop longer, we detect changes in mesencephalic morphology. Since both ectopic and endogenous Lmx1b expression occurs in regions of the isthmus undergoing morphological changes, it could normally play a role in this process. Furthermore, a similar phenotype is not observed in Wnt1/RCAS-infected brains, demonstrating that ectopic Wnt1 is insufficient to mediate the effect of ectopic Lmx1b in our assay. Since Wnt1 function has been linked to the proper segregation of mesencephalic and metencephalic cells, we suggest that Lmx1b and Wnt1 normally function in concert to affect IsO morphogenesis. PMID- 10751173 TI - Integration of FGF and TWIST in calvarial bone and suture development. AB - Mutations in the FGFR1-FGFR3 and TWIST genes are known to cause craniosynostosis, the former by constitutive activation and the latter by haploinsufficiency. Although clinically achieving the same end result, the premature fusion of the calvarial bones, it is not known whether these genes lie in the same or independent pathways during calvarial bone development and later in suture closure. We have previously shown that Fgfr2c is expressed at the osteogenic fronts of the developing calvarial bones and that, when FGF is applied via beads to the osteogenic fronts, suture closure is accelerated (Kim, H.-J., Rice, D. P. C., Kettunen, P. J. and Thesleff, I. (1998) Development 125, 1241-1251). In order to investigate further the role of FGF signalling during mouse calvarial bone and suture development, we have performed detailed expression analysis of the splicing variants of Fgfr1-Fgfr3 and Fgfr4, as well as their potential ligand Fgf2. The IIIc splice variants of Fgfr1-Fgfr3 as well as the IIIb variant of Fgfr2 being expressed by differentiating osteoblasts at the osteogenic fronts (E15). In comparison to Fgf9, Fgf2 showed a more restricted expression pattern being primarily expressed in the sutural mesenchyme between the osteogenic fronts. We also carried out a detailed expression analysis of the helix-loop helix factors (HLH) Twist and Id1 during calvaria and suture development (E10 P6). Twist and Id1 were expressed by early preosteoblasts, in patterns that overlapped those of the FGF ligands, but as these cells differentiated their expression dramatically decreased. Signalling pathways were further studied in vitro, in E15 mouse calvarial explants. Beads soaked in FGF2 induced Twist and inhibited Bsp, a marker of functioning osteoblasts. Meanwhile, BMP2 upregulated Id1. Id1 is a dominant negative HLH thought to inhibit basic HLH such as Twist. In Drosophila, the FGF receptor FR1 is known to be downstream of Twist. We demonstrated that in Twist(+/)(-) mice, FGFR2 protein expression was altered. We propose a model of osteoblast differentiation integrating Twist and FGF in the same pathway, in which FGF acts both at early and late stages. Disruption of this pathway may lead to craniosynostosis. PMID- 10751175 TI - Migration of cardiac neural crest cells in Splotch embryos. AB - Pax3 encodes a transcription factor expressed during mid-gestation in the region of the dorsal neural tube that gives rise to migrating neural crest populations. In the absence of Pax3, both humans and mice develop with neural crest defects. Homozygous Splotch embryos that lack Pax3 die by embryonic day 13.5 with cardiac defects that resemble those induced by neural crest ablation in chick models. This has led to the hypothesis that Pax3 is required for cardiac neural crest migration. However, cardiac derivatives of Pax3-expressing precursor cells have not been previously defined, and Pax3-expressing cells within the heart have not been well demonstrated. Hence, the precise role of Pax3 during cardiac development remains unclear. Here, we use a Cre-lox method to fate map Pax3 expressing neural crest precursors to the cardiac outflow tract. We show that although Pax3 itself is extinguished prior to neural crest populating the heart, derivatives of these precursors contribute to the aorticopulmonary septum. We further show that neural crest cells are found in the outflow tract of Splotch embryos, albeit in reduced numbers. This indicates that contrary to prior reports, Pax3 is not required for cardiac neural crest migration. Using a neural tube explant culture assay, we demonstrate that neural crest cells from Splotch embryos show normal rates of proliferation but altered migratory characteristics. These studies suggest that Pax3 is required for fine tuning the migratory behavior of the cardiac neural crest cells while it is not essential for neural crest migration. PMID- 10751176 TI - The Drosophila homeobox gene optix is capable of inducing ectopic eyes by an eyeless-independent mechanism. AB - optix is a new member of the Six/so gene family from Drosophila that contains both a six domain and a homeodomain. Because of its high amino acid sequence similarity with the mouse Six3 gene, optix is considered to be the orthologous gene from Drosophila rather than sine oculis, as previously believed. optix expression was detected in the eye, wing and haltere imaginal discs. Ectopic expression of optix leads to the formation of ectopic eyes suggesting that optix has important functions in eye development. Although optix and sine oculis belong to the same gene family (Six/so) and share a high degree of amino acid sequence identity, there are a number of factors which suggest that their developmental roles are different: (1) the expression patterns of optix and sine oculis are clearly distinct; (2) sine oculis acts downstream of eyeless, whereas optix is expressed independently of eyeless; (3) sine oculis functions synergistically with eyes absent in eye development whereas optix does not; (4) ectopic expression of optix alone, but not of sine oculis can induce ectopic eyes in the antennal disc. These results suggest that optix is involved in eye morphogenesis by an eyeless-independent mechanism. PMID- 10751177 TI - Functional analysis of the Drosophila diaphanous FH protein in early embryonic development. AB - The Drosophila Formin Homology (FH) protein Diaphanous has an essential role during cytokinesis. To gain insight into the function of Diaphanous during cytokinesis and explore its role in other processes, we generated embryos deficient for Diaphanous and analyzed three cell-cycle-regulated actin-mediated events during embryogenesis: formation of the metaphase furrow, cellularization and formation of the pole cells. In dia embryos, all three processes are defective. Actin filaments do not organize properly to the metaphase and cellularization furrows and the actin ring is absent from the base of the presumptive pole cells. Furthermore, plasma membrane invaginations that initiate formation of the metaphase furrow and pole cells are missing. Immunolocalization studies of wild-type embryos reveal that Diaphanous localizes to the site where the metaphase furrow is anticipated to form, to the growing tip of cellularization furrows, and to contractile rings. In addition, the dia mutant phenotype reveals a role for Diaphanous in recruitment of myosin II, anillin and Peanut to the cortical region between actin caps. Our findings thus indicate that Diaphanous has a role in actin cytoskeleton organization and is essential for many, if not all, actin-mediated events involving membrane invagination. Based on known biochemical functions of FH proteins, we propose that Diaphanous serves as a mediator between signaling molecules and actin organizers at specific phases of the cell cycle. PMID- 10751178 TI - Alteration of the retinotectal projection map by the graft of mesencephalic floor plate or sonic hedgehog. AB - The floor plate plays crucial roles in the specification and differentiation of neurons along the dorsal-ventral (DV) axis of the neural tube. The transplantation of the mesecephalic floor plate (mfp) into the dorsal mesencephalon in chick embryos alters the fate of the mesencephalon adjacent to the transplant from the tectum to the tegmentum, a ventral tissue of the mesencephalon. In this study, to test whether the mfp is involved in the specification of the DV polarity of the tectum and affects the projection patterns of retinal fibers to the tectum along the DV axis, we transplanted quail mfp into the dorsal mesencephalon of chick embryos, and analyzed projection patterns of dorsal and ventral retinal fibers to the tectum. In the embryos with the mfp graft, dorsal retinal fibers grew into the dorsal part of the tectum which is the original target for ventral but not dorsal retinal fibers and formed tight focuses there. In contrast, ventral retinal fibers did not terminate at any part of the tectum. Transplantation of Sonic hedgehog (Shh)-secreting quail fibroblasts into the dorsal mesencephalon also induced the ectopic tegmentum and altered the retinotectal projection along the DV axis, as the mfp graft did. These results suggest that some factors from the mesencephalic floor plate or the tegmentum, or Shh itself, play a crucial role in the establishment of the DV polarity of the tectum and the retinotectal projection map along the DV axis. PMID- 10751179 TI - The conditional medaka mutation eyeless uncouples patterning and morphogenesis of the eye. AB - In early vertebrate eye development, the retinal anlage is specified in the anterior neuroectoderm. During neurulation, the optic vesicles evaginate from the lateral wall of the prosencephalon. Here we describe the temperature-sensitive mutation eyeless in the Japanese medakafish. Marker gene analysis indicates that, whereas, specification of two retinal primordia and proximodistal patterning takes place in the mutant embryo, optic vesicle evagination does not occur and subsequent differentiation of the retinal primordia is not observed. The mutation eyeless thus uncouples patterning and morphogenesis at early steps of retinal development. Temperature-shift experiments indicate a requirement for eyeless activity prior to optic vesicle evagination. Cell transplantation shows that eyeless acts cell autonomously. PMID- 10751180 TI - The Iroquois homeobox genes function as dorsal selectors in the Drosophila head. AB - The Iroquois complex (Iro-C) genes are expressed in the dorsal compartment of the Drosophila eye/antenna imaginal disc. Previous work has shown that the Iro-C homeoproteins are essential for establishing a dorsoventral pattern organizing center necessary for eye development. Here we show that, in addition, the Iro-C products are required for the specification of dorsal head structures. In mosaic animals, the removal of the Iro-C transforms the dorsal head capsule into ventral structures, namely, ptilinum, prefrons and suborbital bristles. Moreover, the Iro C(-) cells can give rise to an ectopic antenna and maxillary palpus, the main derivatives of the antenna part of the imaginal disc. These transformations are cell-autonomous, which indicates that the descendants of a dorsal Iro-C(-) cell can give rise to essentially all the ventral derivatives of the eye/antenna disc. These results support a role of the Iro-C as a dorsal selector in the eye and head capsule. Moreover, they reinforce the idea that developmental cues inherited from the distinct embryonic segments from which the eye/antenna disc originates play a minimal role in the patterning of this disc. PMID- 10751181 TI - Basic fibroblast growth factor positively regulates hematopoietic development. AB - Recently identified BLast Colony Forming Cells (BL-CFCs) from in vitro differentiated embryonic stem (ES) cells represent the common progenitor of hematopoietic and endothelial cells, the hemangioblast. Access to this initial cell population committed to the hematopoietic lineage provides a unique opportunity to characterize hematopoietic commitment events. Here, we show that BL-CFC expresses the receptor tyrosine kinase, Flk1, and thus we took advantage of the BL-CFC assay, as well as fluorescent activated cell sorter (FACS) analysis for Flk1(+) cells to determine quantitatively if mesoderm-inducing factors promote hematopoietic lineage development. Moreover, we have analyzed ES lines carrying targeted mutations for fibroblast growth factor receptor-1 (fgfr1), a receptor for basic fibroblast growth factor (bFGF), as well as scl, a transcription factor, for their potential to generate BL-CFCs and Flk1(+) cells, to further define events leading to hemangioblast development. Our data suggest that bFGF-mediated signaling is critical for the proliferation of the hemangioblast and that cells expressing both Flk1 and SCL may represent the hemangioblast. PMID- 10751182 TI - Specification of neuropeptide Y phenotype in visual cortical neurons by leukemia inhibitory factor. AB - Building the complex mammalian neocortex requires appropriate numbers of neurochemically specified neurons. It is not clear how the highly diverse cortical interneurons acquire their distinctive phenotypes. The lack of genetic determination implicates environmental factors in this selection and specification process. We analysed, in organotypic visual cortex cultures, the specification of neurons expressing neuropeptide Y (NPY), a potent anticonvulsant. Endogenous brain-derived neurotrophic factor and neurotrophin 4/5 play no role in early NPY phenotype specification. Rather, the decision to express NPY is made during a period of molecular plasticity during which differentiating neurons with the potential to express NPY compete for the cytokine leukemia inhibitory factor which is produced in the cortex, but is negatively regulated by thalamic afferences. The neurons that fail in this competition are parvalbuminergic basket and chandelier neurons, which express NPY transiently, but will not acquire a permanent NPY expression. They switch into a facultative NPY expression mode, and remain responsive to the neurotrophins which modulate NPY expression later in development. PMID- 10751183 TI - Laser-induced gene expression in specific cells of transgenic zebrafish. AB - Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle. PMID- 10751184 TI - The regulation of proliferation and differentiation in oligodendrocyte progenitor cells by alphaV integrins. AB - We have previously shown that oligodendrocyte progenitor cells exhibit developmental switching between alphav-associated beta integrin subunits to sequentially express alphavbeta1, alphavbeta3 and alphavbeta5 integrins during differentiation in vitro. To understand the role that alphavveta3 integrin may play in regulating oligodendrocyte progenitor cell behaviour, cells of the rat cell line, CG-4, were genetically engineered to constitutively express alphavbeta3 integrin by transfection with full-length human beta3 integrin subunit cDNA. Time-lapse videomicroscopy showed no effect of beta3 expression on cell migration but revealed enhanced proliferation on vitronectin substrata. Comparison of mitotic indices, as measured by 5-bromo-2'-deoxyuridine incorporation, confirmed that human beta3 integrin-expressing cells exhibited enhanced proliferation, as compared to both vector-only transfected, and wild type CG-4 cells when switched to differentiation medium from growth medium, but only in cultures grown on vitronectin and not on poly-D-lysine. The effects on proliferation were inhibited by a function-blocking antibody specifically directed against the human beta3 integrin subunit. Human beta3 integrin expressing cells also exhibited reduced differentiation. This differentiation could be reduced still further by a function-blocking monoclonal antibody against alphavbeta5 integrin, as could differentiation in the wild-type CG-4 cells. Taken together, these results suggest that alphavbeta3 integrin may regulate oligodendroglial cell proliferation and that both downregulation of alphavbeta3 integrin expression and signalling through alphavbeta5 integrin may be critical to continued differentiation in vitro. PMID- 10751185 TI - The concentric structure of the developing gut is regulated by Sonic hedgehog derived from endodermal epithelium. AB - The embryonic gut of vertebrates consists of endodermal epithelium, surrounding mesenchyme derived from splanchnic mesoderm and enteric neuronal components derived from neural crest cells. During gut organogenesis, the mesenchyme differentiates into distinct concentric layers around the endodermal epithelium forming the lamina propria, muscularis mucosae, submucosa and lamina muscularis (the smooth muscle layer). The smooth muscle layer and enteric plexus are formed at the outermost part of the gut, always some distance away from the epithelium. How this topographical organization of gut mesenchyme is established is largely unknown. Here we show the following: (1) Endodermal epithelium inhibits differentiation of smooth muscle and enteric neurons in adjacent mesenchyme. (2) Endodermal epithelium activates expression of patched and BMP4 in adjacent non smooth muscle mesenchyme, which later differentiates into the lamina propria and submucosa. (3) Sonic hedgehog (Shh) is expressed in endodermal epithelium and disruption of Shh-signaling by cyclopamine induces differentiation of smooth muscle and a large number of neurons even in the area adjacent to epithelium. (4) Shh can mimic the effect of endodermal epithelium on the concentric stratification of the gut. Taken together, these data suggest that endoderm derived Shh is responsible for the patterning across the radial axis of the gut through induction of inner components and inhibition of outer components, such as smooth muscle and enteric neurons. PMID- 10751186 TI - The putative wnt receptor Xenopus frizzled-7 functions upstream of beta-catenin in vertebrate dorsoventral mesoderm patterning. AB - We have isolated one member of the frizzled family of wnt receptors from Xenopus (Xfz7) to study the role of cell-cell communication in the establishment of the vertebrate axis. We demonstrate that this maternally encoded protein specifically synergizes with wnt proteins in ectopic axis induction. Embryos derived from oocytes depleted of maternal Xfz7 RNA by antisense oligonucleotide injection are deficient in dorsoanterior structures. Xfz7-depleted embryos are deficient in dorsal but not ventral mesoderm due to the reduced expression of the wnt target genes siamois, Xnr3 and goosecoid. These signaling defects can be restored by the addition of beta-catenin but not Xwnt8b. Xfz7 thus functions upstream of the known GSK-3/axin/beta-catenin intracellular signaling complex in vertebrate dorsoventral mesoderm specification. PMID- 10751187 TI - Calmodulin and son of sevenless dependent signaling pathways regulate midline crossing of axons in the Drosophila CNS. AB - The establishment of axon trajectories is ultimately determined by the integration of intracellular signaling pathways. Here, a genetic approach in Drosophila has demonstrated that both Calmodulin and Son of sevenless signaling pathways are used to regulate which axons cross the midline. A loss in either signaling pathway leads to abnormal projection of axons across the midline and these increase with roundabout or slit mutations. When both Calmodulin and Son of sevenless are disrupted, the midline crossing of axons mimics that seen in roundabout mutants, although Roundabout remains expressed on crossing axons. Calmodulin and Son of sevenless also regulate axon crossing in a commissureless mutant. These data suggest that Calmodulin and Son of sevenless signaling pathways function to interpret midline repulsive cues which prevent axons crossing the midline. PMID- 10751188 TI - Endurance training, expression, and physiology of LDH, MCT1, and MCT4 in human skeletal muscle. AB - To evaluate the effects of endurance training on the expression of monocarboxylate transporters (MCT) in human vastus lateralis muscle, we compared the amounts of MCT1 and MCT4 in total muscle preparations (MU) and sarcolemma enriched (SL) and mitochondria-enriched (MI) fractions before and after training. To determine if changes in muscle lactate release and oxidation were associated with training-induced changes in MCT expression, we correlated band densities in Western blots to lactate kinetics determined in vivo. Nine weeks of leg cycle endurance training [75% peak oxygen consumption (VO(2 peak))] increased muscle citrate synthase activity (+75%, P < 0.05) and percentage of type I myosin heavy chain (+50%, P < 0.05); percentage of MU lactate dehydrogenase-5 (M4) isozyme decreased (-12%, P < 0.05). MCT1 was detected in SL and MI fractions, and MCT4 was localized to the SL. Muscle MCT1 contents were consistent among subjects both before and after training; in contrast, MCT4 contents showed large interindividual variations. MCT1 amounts significantly increased in MU, SL, and MI after training (+90%, +60%, and +78%, respectively), whereas SL but not MU MCT4 content increased after training (+47%, P < 0.05). Mitochondrial MCT1 content was negatively correlated to net leg lactate release at rest (r = -0.85, P < 0.02). Sarcolemmal MCT1 and MCT4 contents correlated positively to net leg lactate release at 5 min of exercise at 65% VO(2 peak) (r = 0.76, P < 0.03 and r = 0. 86, P < 0.01, respectively). Results support the conclusions that 1) endurance training increases expression of MCT1 in muscle because of insertion of MCT1 into both sarcolemmal and mitochondrial membranes, 2) training has variable effects on sarcolemmal MCT4, and 3) both MCT1 and MCT4 participate in the cell cell lactate shuttle, whereas MCT1 facilitates operation of the intracellular lactate shuttle. PMID- 10751189 TI - Endurance exercise training attenuates leucine oxidation and BCOAD activation during exercise in humans. AB - We studied the effects of a 38-day endurance exercise training program on leucine turnover and substrate metabolism during a 90-min exercise bout at 60% peak O(2) consumption (VO(2 peak)) in 6 males and 6 females. Subjects were studied at both the same absolute (ABS) and relative (REL) exercise intensities posttraining. Training resulted in a significant increase in whole body VO(2 peak) and skeletal muscle citrate synthase (CS; P < 0.001), complex I-III (P < 0.05), and total branched-chain 2-oxoacid dehydrogenase (BCOAD; P < 0.001) activities. Leucine oxidation increased during exercise for the pretraining trial (PRE, P < 0.001); however, there was no increase for either the ABS or REL posttraining trial. Leucine oxidation was significantly lower for females at all time points during rest and exercise (P < 0.01). The percentage of BCOAD in the activated state was significantly increased after exercise for both the PRE and REL exercise trials, with the increase in PRE being greater (P < 0.001) compared with REL (P < 0.05). Females oxidized proportionately more lipid and less carbohydrate during exercise compared with males. In conclusion, we found that 38 days of endurance exercise training significantly attenuated both leucine oxidation and BCOAD activation during 90 min of endurance exercise at 60% VO(2 peak) for both ABS and REL exercise intensities. Furthermore, females oxidize proportionately more lipid and less carbohydrate compared with males during endurance exercise. PMID- 10751191 TI - Oxidation of glutamine by the splanchnic bed in humans. AB - [1,2-(13)C(2)]glutamine and [ring-(2)H(5)]phenylalanine were infused for 7 h into five postabsorptive healthy subjects on two occasions. On one occasion, the tracers were infused intravenously for 3.5 h and then by a nasogastric tube for 3.5 h. The order of infusion was reversed on the other occasion. From the plasma tracer enrichment measurements at plateau during the intravenous and nasogastric infusion periods, we determined that 27 +/- 2% of the enterally delivered phenylalanine and 64 +/- 2% of the glutamine were removed on the first pass by the splanchnic bed. Glutamine flux was 303 +/- 8 micromol. kg(-1). h(-1). Of the enterally delivered [(13)C]glutamine tracer, 73 +/- 2% was recovered as exhaled CO(2) compared with 58 +/- 1% of the intravenously infused tracer. The fraction of the enterally delivered tracer that was oxidized specifically on the first pass by the splanchnic bed was 53 +/- 2%, comprising 83% of the total tracer extracted. From the appearance of (13)C in plasma glucose, we estimated that 7 and 10% of the intravenously and nasogastrically infused glutamine tracers, respectively, were converted to glucose. The results for glutamine flux and first pass extraction were similar to our previously reported values when a [2 (15)N]glutamine tracer [Matthews DE, Morano MA, and Campbell RG, Am J Physiol Endocrinol Metab 264: E848-E854, 1993] was used. The results of [(13)C]glutamine tracer disposal demonstrate that the major fate of enteral glutamine extraction is for oxidation and that only a minor portion is used for gluconeogenesis. PMID- 10751190 TI - Transgenic mice overexpressing GLUT-1 protein in muscle exhibit increased muscle glycogenesis after exercise. AB - The purpose of the present study was to determine the rates of muscle glycogenolysis and glycogenesis during and after exercise in GLUT-1 transgenic mice and their age-matched littermates. Male transgenic mice (TG) expressing a high level of human GLUT-1 and their nontransgenic (NT) littermates underwent 3 h of swimming. Glycogen concentration was determined in gastrocnemius and extensor digitorum longus (EDL) muscles before exercise and at 0, 5, and 24 h postexercise, during which food (chow) and 10% glucose solution (as drinking water) were provided. Exercise resulted in approximately 90% reduction in muscle glycogen in both NT (from 11.2 +/- 1.4 to 2. 1 +/- 1.3 micromol/g) and TG (from 99.3 +/- 4.7 to 11.8 +/- 4.3 micromol/g) in gastrocnemius muscle. During recovery from exercise, the glycogen concentration increased to 38.2 +/- 7.3 (5 h postexercise) and 40.5 +/- 2.8 micromol/g (24 h postexercise) in NT mice. In TG mice, however, the increase in muscle glycogen concentration during recovery was greater (to 57.5 +/- 7.4 and 152.1 +/- 15.7 micromol/g at 5 and 24 h postexercise, respectively). Similar results were obtained from EDL muscle. The rate of 2-deoxyglucose uptake measured in isolated EDL muscles was 7- to 10-fold higher in TG mice at rest and at 0 and 5 h postexercise. There was no difference in muscle glycogen synthase activation measured in gastrocnemius muscles between NT and TG mice immediately after exercise. These results demonstrate that the rate of muscle glycogen accumulation postexercise exhibits two phases in TG: 1) an early phase (0-5 h), with rapid glycogen accumulation similar to that of NT mice, and 2) a progressive increase in muscle glycogen concentration, which differs from that of NT mice, during the second phase (5-24 h). Our data suggest that the high level of steady-state muscle glycogen in TG mice is due to the increase in muscle glucose transport activity. PMID- 10751192 TI - Meal-induced insulin secretion in dogs is mediated by both branches of the autonomic nervous system. AB - We investigated the relationship between autonomic activity to the pancreas and insulin secretion in chronically catheterized dogs when food was shown, during eating, and during the early absorptive period. Pancreatic polypeptide (PP) output, pancreatic norepinephrine spillover (PNESO), and arterial epinephrine (Epi) were measured as indexes for parasympathetic and sympathetic nervous activity to the pancreas and for adrenal medullary activity, respectively. The relation between autonomic activity and insulin secretion was confirmed by autonomic blockade. Showing food to dogs initiated a transient increase in insulin secretion without changing PP output or PNESO. Epi did increase, suggesting beta(2)-adrenergic mediation, which was confirmed by beta-adrenoceptor blockade. Eating initiated a second transient insulin response, which was only totally abolished by combined muscarinic and beta-adrenoceptor blockade. During absorption, insulin increased to a plateau. PP output showed the same pattern, suggesting parasympathetic mediation. PNESO decreased by 50%, suggesting withdrawal of inhibitory sympathetic neural tone. We conclude that 1) the insulin response to showing food is mediated by the beta(2)-adrenergic effect of Epi, 2) the insulin response to eating is mediated both by parasympathetic muscarinic stimulation and by the beta(2)-adrenergic effect of Epi, and 3) the insulin response during early absorption is mediated by parasympathetic activation, with possible contribution of withdrawal of sympathetic neural tone. PMID- 10751193 TI - Deactivation of TSH receptor signaling in filter-cultured pig thyroid epithelial cells. AB - Thyrotropin [thyroid-stimulating hormone (TSH)] receptor on-off signaling was studied in polarized monolayers of pig thyrocytes cultured on permeable support. Transepithelial resistance (R) and potential difference (PD) were used as parameters to monitor the effect of altered TSH concentrations on vectorial electrolyte transport. TSH induced rapid but long-lasting changes in R (decrease) and PD (increase) that were cAMP-dependent and related to enhanced transcellular conductance of sodium and chloride. Withdrawal of TSH from cultures prestimulated with TSH (0.1 mU/ml) for 48 h resulted in restitution of R to control level within 30 min. Such deactivation was markedly accelerated by mild trypsinization, which degraded receptor-bound ligand without affecting TSH receptor responsiveness or ion transporting capacity. Small alterations in the TSH concentration (0.01-0.1 mU/ml) were followed almost instantaneously by adjustments of R. In contrast, the reversal of R after acute TSH stimulation (30 min) and subsequent TSH washout was delayed for several hours independently of cell surface trypsinization. The observations indicate that, during continuous exposure to physiological concentrations, TSH exerts a close minute-to-minute surveillance of thyroid function and the rate-limiting step of deactivation is the dissociation of ligand from the TSH receptor at the cell surface. TSH deprived cells briefly exposed to TSH are refractory to rapid deactivation, probably because of altered metabolism downstream of TSH receptor signal transduction. PMID- 10751194 TI - Resistance exercise acutely increases MHC and mixed muscle protein synthesis rates in 78-84 and 23-32 yr olds. AB - We determined whether short-term weight-lifting exercise increases the synthesis rate of the major contractile proteins, myosin heavy chain (MHC), actin, and mixed muscle proteins in nonfrail elders and younger women and men. Fractional synthesis rates of mixed, MHC, and actin proteins were determined in seven healthy sedentary 23- to 32-yr-old and seven healthy 78- to 84-yr-old participants in paired studies done before and at the end of a 2-wk weight lifting program. The in vivo rate of incorporation of 1-[(13)C]leucine into vastus lateralis MHC, actin, and mixed proteins was determined using a 14-h constant intravenous infusion of 1-[(13)C]leucine. Before exercise, the mixed and MHC fractional synthetic rates were lower in the older than in the younger participants (P < or = 0.04). Baseline actin protein synthesis rates were similar in the two groups (P = not significant). Over a 2-wk period, participants completed ten 1- to 1. 5-h weight-lifting exercise sessions: 2-3 sets per day of 9 exercises, 8-12 repetitions per set, at 60-90% of maximum voluntary muscle strength. At the end of exercise, MHC and mixed protein synthetic rates increased in the younger (88 and 121%) and older participants (105 and 182%; P < 0.001 vs. baseline). These findings indicate that MHC and mixed protein synthesis rates are reduced more than actin in advanced age. Similar to that of 23-32 yr olds, the vastus lateralis muscle in 78-84 yr olds retains the capacity to increase MHC and mixed protein synthesis rates in response to short-term resistance exercise. PMID- 10751196 TI - Determination of protein synthesis in human ileum in situ by continuous [1 (13)C]leucine infusion. AB - Efficient protein synthesis plays an important role in the physiology and pathophysiology of the human gastrointestinal tract. Because of methodological restrictions, no studies on ileal protein synthesis in situ are available in humans. We used advanced mass spectrometry techniques (capillary gas chromatography/combustion isotope ratio mass spectrometry) to determine directly the incorporation rate of [1-(13)C]leucine into ileal mucosal protein in control subjects and postoperative patients. All subjects had an ileostomy, which allowed easy access to the ileal mucosa. To examine changes in ileal protein synthesis during prolonged isotope infusion (0.16 micromol. kg(-1). min(-1), 9.6 micromol/kg prime), studies were performed over a 10-h period. Mucosal biopsies were performed after 3, 6, and 10 h of infusion. Protein synthesis was calculated separately between hour 3 and hour 6 (period 1) and hour 6 and hour 10 (period 2). Control subjects demonstrated an ileal protein fractional synthetic rate of 0.62 +/- 0.06%/h in period 1 and of 0. 52 +/- 0.08%/h in period 2 (not significant). In postsurgical subjects, ileal protein synthesis was significantly higher (1.11 +/- 0.14%/h in period 1, P < 0.01 vs. controls in period 1) but declined markedly in period 2 (0.39 +/- 0.13, P < 0.01 vs. period 1 after surgery). The rate of protein synthesis in the small bowel of control subjects is, thus far, among the lowest measured in mammals and reflects the comparably slow turnover of human ileal mucosa. Postoperative disturbances of gut integrity lead to an accelerated anabolic response. During prolonged isotope infusion, stimulated protein synthesis declines because of diurnal variations or is erroneously reduced by tracer loss due to an accelerated cell turnover. PMID- 10751195 TI - Insulin increases NO-stimulated guanylate cyclase activity in cultured VSMC while raising redox potential. AB - Insulin acutely stimulates cyclic guanosine monophosphate (cGMP) production in primary confluent cultured vascular smooth muscle cells (VSMC) from canine femoral artery, but the mechanism is not known. These cells contain the inducible isoform of nitric oxide (NO) synthase (iNOS), and insulin-stimulated cGMP production in confluent cultured cells is blocked by the NOS inhibitor, N(G) monomethyl-L-arginine (L-NMMA). In the present study, it is shown that iNOS is also present in freshly dispersed VSMC from this artery, indicating that iNOS expression in cultured VSMC is not an artifact of the culture process. Insulin did not stimulate NOS activity in primary confluent cultured cells because it did not affect citrulline or combined NO(-)(3)/NO(-)(2) production. To see whether insulin required the permissive presence of NO to stimulate cGMP production, iNOS and basal cGMP production were inhibited with L-NMMA, and the cells were incubated with or without 1 nM insulin and/or the NO donor, S-nitroso-N-acetyl D,L-penicillamine (SNAP) at a concentration (0.1 microM) that restored cGMP production to the basal value. In the presence of L-NMMA, insulin no longer affected cGMP production but when insulin was added to L-NMMA plus SNAP, cGMP production was increased by 69% (P < 0.05 vs. L-NMMA plus SNAP). Insulin, which increases glucose uptake by these cells, increased the cell lactate content and the lactate-to-pyruvate ratio (LPR) by 81 and 97%, respectively (both P < 0.05), indicating that the hormone increased aerobic glycolysis and the redox potential. The effects of insulin on LPR and cGMP production were blocked by removing glucose or by adding 2-deoxyglucose to the incubation media and were duplicated by the reducing substrate, beta-hydroxybutyrate. We conclude that insulin does not acutely affect iNOS activity in these VSMC but it does augment cGMP production induced by the NO already present in the cell while increasing aerobic glycolysis and the cell redox potential. PMID- 10751197 TI - Tolbutamide and diazoxide modulate phospholipase C-linked Ca(2+) signaling and insulin secretion in beta-cells. AB - Arginine vasopressin (AVP), bombesin, and ACh increase cytosolic free Ca(2+) and potentiate glucose-induced insulin release by activating receptors linked to phospholipase C (PLC). We examined whether tolbutamide and diazoxide, which close or open ATP-sensitive K(+) channels (K(ATP) channels), respectively, interact with PLC-linked Ca(2+) signals in HIT-T15 and mouse beta-cells and with PLC linked insulin secretion from HIT-T15 cells. In the presence of glucose, the PLC linked Ca(2+) signals were enhanced by tolbutamide (3-300 microM) and inhibited by diazoxide (10-100 microM). The effects of tolbutamide and diazoxide on PLC linked Ca(2+) signaling were mimicked by BAY K 8644 and nifedipine, an activator and inhibitor of L-type voltage-sensitive Ca(2+) channels, respectively. Neither tolbutamide nor diazoxide affected PLC-linked mobilization of internal Ca(2+) or store-operated Ca(2+) influx through non-L-type Ca(2+) channels. In the absence of glucose, PLC-linked Ca(2+) signals were diminished or abolished; this effect could be partly antagonized by tolbutamide. In the presence of glucose, tolbutamide potentiated and diazoxide inhibited AVP- or bombesin-induced insulin secretion from HIT-T15 cells. Nifedipine (10 microM) blocked both the potentiating and inhibitory actions of tolbutamide and diazoxide on AVP-induced insulin release, respectively. In glucose-free medium, AVP-induced insulin release was reduced but was again potentiated by tolbutamide, whereas diazoxide caused no further inhibition. Thus tolbutamide and diazoxide regulate both PLC linked Ca(2+) signaling and insulin secretion from pancreatic beta-cells by modulating K(ATP) channels, thereby determining voltage-sensitive Ca(2+) influx. PMID- 10751198 TI - Metabolic regulation of Na(+)/P(i)-cotransporter-1 gene expression in H4IIE cells. AB - We showed that the rat Na(+)/P(i) cotransporter-1 (RNaPi-1) gene was regulated by insulin and glucose in rat hepatocytes. The aim of this work was to elucidate signaling pathways of insulin-mediated metabolic regulation of the RNaPi-1 gene in H4IIE cells. Insulin increased RNaPi-1 mRNA abundance in the presence of glucose and decreased RNaPi-1 mRNA in the absence of glucose, clearly establishing an involvement of metabolic signals for insulin-induced upregulation of the RNaPi-1 gene. Pyruvate and insulin increased RNaPi-1 expression but downregulated L-pyruvate kinase, indicating the existence of gene-specific metabolic signals. Although fructose, glycerol, and lactate could support insulin induced upregulation of the RNaPi-1 gene, compounds entering metabolism beyond pyruvate oxidation, such as acetate and citrate, could not, suggesting that RNaPi 1-specific metabolic signals are generated at or above pyruvate oxidation. Wortmannin, LY-294002, and rapamycin abolished the insulin effect on the RNaPi-1 gene, whereas expression of dominant negative Asn(17) Ras and mitogen-activating protein kinase (MAPK) kinase (MEK) inhibitor PD-98059 exhibited no effect. Thus we herein propose that metabolic regulation of RNaPi-1 expression by insulin is mediated through the phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase pathways, but not the Ras/MAPK pathway. PMID- 10751199 TI - Protein kinase C-beta mediates lipoprotein-induced generation of PAI-1 from vascular endothelial cells. AB - Elevated levels of low-density lipoproteins (LDL) and lipoprotein(a) [Lp(a)] have been considered strong risk factors for atherosclerotic cardiovascular disease. Increased production of plasminogen activator inhibitor-1 (PAI-1) has been implicated in the development of thrombosis and atherosclerosis. Previous studies by our group and others demonstrated that oxidation enhances LDL- and Lp(a) induced production of PAI-1 in human umbilical vein endothelial cells (HUVEC). The present study examined the involvement of protein kinase C (PKC) and its isoform in vascular endothelial cells (EC) induced by native or oxidized LDL and Lp(a). Treatment with Lp(a) or LDL transiently increased PKC activity at 15 min and 5.5 h after the start of lipoprotein treatment in EC. Copper-oxidized LDL and Lp(a) induced greater PKC activation in EC compared with comparable forms of those lipoproteins. Additions of 1 microM calphostin C, a PKC-specific inhibitor, at the beginning or > or =5 h, but not > or = 9 h, after the initiation of lipoprotein treatment, blocked native and oxidized LDL- or Lp(a)-induced increases in PKC activity and PAI-1 production. Treatment of LDL, Lp(a), or their oxidized forms was induced in translocation of PKC-beta1 from cytosol to membrane in HUVEC. Treatments with 60 nM 379196, a PKC-beta-specific inhibitor, effectively prevented PAI-1 production induced by LDL, Lp(a), or their oxidized forms in HUVEC and human coronary artery EC. The results suggest that activation of PKC-beta may mediate the production of PAI-1 in cultured arterial and venous EC induced by LDL, Lp(a), or their oxidized forms. PMID- 10751201 TI - Effects of plasma epinephrine on fat metabolism during exercise: interactions with exercise intensity. AB - This study determined the effects of elevated plasma epinephrine on fat metabolism during exercise. On four occasions, seven moderately trained subjects cycled at 25% of peak oxygen consumption (VO(2 peak)) for 60 min. After 15 min of exercise, subjects were intravenously infused with low (0.96 +/- 0.10 nM), moderate (1.92 +/- 0.24 nM), or high (3.44 +/- 0.50 nM) levels (all P < 0.05) of epinephrine to increase plasma epinephrine above control (Con; 0.59 +/- 0.10 nM). During the interval between 35 and 55 min of exercise, lipolysis [i.e., rate of appearance of glycerol] increased above Con (4.9 +/- 0.5 micromol. kg(-1). min( 1)) with low, moderate, and high (6.5 +/- 0.5, 7.1 +/- 0.8, and 10.6 +/- 1.2 micromol. kg(-1). min(-1), respectively; all P < 0.05) levels of epinephrine despite simultaneous increases in plasma insulin. The release of fatty acid into plasma also increased progressively with the graded epinephrine infusions. However, fatty acid oxidation was lower than Con (11.1 +/- 0.8 micromol. kg(-1). min(-1)) during moderate and high levels (8.7 +/- 0.7 and 8.1 +/- 0.9 micromol. kg(-1). min(-1), respectively; P < 0.05). In one additional trial, the same subjects exercised at 45% VO(2 peak) without epinephrine infusion, which produced a plasma epinephrine concentration identical to low levels. However, lipolysis was lower (i.e., 5.5 +/- 0.6 vs. 6.5 +/- 0.5 micromol. kg(-1). min(-1); P < 0.05). In conclusion, elevations in plasma epinephrine concentration during exercise at 25% of VO(2 peak) progressively increase whole body lipolysis but decrease fatty acid oxidation. Last, increasing exercise intensity from 25 to 45% VO(2 peak) attenuates the lipolytic actions of epinephrine. PMID- 10751200 TI - Mechanism of muscle glycogen autoregulation in humans. AB - To examine the mechanism by which muscle glycogen limits its own synthesis, muscle glycogen and glucose 6-phosphate (G-6-P) concentrations were measured in seven healthy volunteers during a euglycemic ( approximately 5.5 mM) hyperinsulinemic ( approximately 450 pM) clamp using (13)C/(31)P nuclear magnetic resonance spectroscopy before and after a muscle glycogen loading protocol. Rates of glycogen synthase (V(syn)) and phosphorylase (V(phos)) flux were estimated during a [1-(13)C]glucose (pulse)-unlabeled glucose (chase) infusion. The muscle glycogen loading protocol resulted in a 65% increase in muscle glycogen content that was associated with a twofold increase in fasting plasma lactate concentrations (P < 0.05 vs. basal) and an approximately 30% decrease in plasma free fatty acid concentrations (P < 0.001 vs. basal). Muscle glycogen loading resulted in an approximately 30% decrease in the insulin-stimulated rate of net muscle glycogen synthesis (P < 0.05 vs. basal), which was associated with a twofold increase in intramuscular G-6-P concentration (P < 0.05 vs. basal). Muscle glycogen loading also resulted in an approximately 30% increase in whole body glucose oxidation rates (P < 0.05 vs. basal), whereas there was no effect on insulin-stimulated rates of whole body glucose uptake ( approximately 10.5 mg. kg body wt(-1). min(-1) for both clamps) or glycogen turnover (V(syn)/V(phos) was approximately 23% for both clamps). In conclusion, these data are consistent with the hypothesis that glycogen limits its own synthesis through feedback inhibition of glycogen synthase activity, as reflected by an accumulation of intramuscular G 6-P, which is then shunted into aerobic and anaerobic glycolysis. PMID- 10751203 TI - Islet amyloid polypeptide (amylin)-deficient mice develop a more severe form of alloxan-induced diabetes. AB - To examine whether islet amyloid polypeptide (IAPP), other than through amyloid formation, may be of importance in diabetes pathogenesis, IAPP-deficient mice (IAPP(-/-)) were challenged with alloxan (day 0). Diabetes in IAPP(-/-) mice was more severe at day 35, indicated by greater weight loss; glucose levels were higher in alloxan-treated IAPP(-/-) mice, whereas insulin levels were lower, indicating a greater impairment of islet function. Accordingly, glucose levels upon intravenous glucose challenges at days 7 and 35 were consistently higher in alloxan-treated IAPP(-/-) mice. At day 35, insulin mRNA expression, but not beta cell mass, was lower in untreated IAPP(-/-) mice. Yet, upon alloxan administration, beta-cell mass and numbers of beta-cell-containing islets were significantly more reduced in IAPP(-/-) mice. Furthermore, they displayed exaggerated beta-cell dysfunction, because in their remaining beta-cells, insulin mRNA expression was significantly more impaired and the localization of glucose transporter-2 was perturbed. Thus the lack of IAPP has allowed exaggerated beta cell cytotoxic actions of alloxan, suggesting that there may be beneficial features of IAPP actions in situations of beta-cell damage. PMID- 10751202 TI - Effects of fructose and glucose on plasma leptin, insulin, and insulin resistance in lean and VMH-lesioned obese rats. AB - To determine the influence of dietary fructose and glucose on circulating leptin levels in lean and obese rats, plasma leptin concentrations were measured in ventromedial hypothalamic (VMH)-lesioned obese and sham-operated lean rats fed either normal chow or fructose- or glucose-enriched diets (60% by calories) for 2 wk. Insulin resistance was evaluated by the steady-state plasma glucose method and intravenous glucose tolerance test. In lean rats, glucose-enriched diet significantly increased plasma leptin with enlarged parametrial fat pad, whereas neither leptin nor fat-pad weight was altered by fructose. Two weeks after the lesions, the rats fed normal chow had marked greater body weight gain, enlarged fat pads, and higher insulin and leptin compared with sham-operated rats. Despite a marked adiposity and hyperinsulinemia, insulin resistance was not increased in VMH-lesioned rats. Fructose brought about substantial insulin resistance and hyperinsulinemia in both lean and obese rats, whereas glucose led to rather enhanced insulin sensitivity. Leptin, body weight, and fat pad were not significantly altered by either fructose or glucose in the obese rats. These results suggest that dietary glucose stimulates leptin production by increasing adipose tissue or stimulating glucose metabolism in lean rats. Hyperleptinemia in VMH-lesioned rats is associated with both increased adiposity and hyperinsulinemia but not with insulin resistance. Dietary fructose does not alter leptin levels, although this sugar brings about hyperinsulinemia and insulin resistance, suggesting that hyperinsulinemia compensated for insulin resistance does not stimulate leptin production. PMID- 10751204 TI - Stimulation by iodide of H(2)O(2) generation in thyroid slices from several species. AB - The regulation of thyroid metabolism by iodide involves numerous inhibitory effects. However, in unstimulated dog thyroid slices, a small inconstant stimulatory effect of iodide on H(2)O(2) generation is observed. The only other stimulatory effect reported with iodide is on [1-(14)C]glucose oxidation, i.e., on the pentose phosphate pathway. Because we have recently demonstrated that the pentose phosphate pathway is controlled by H(2)O(2) generation, we study here the effect of iodide on basal H(2)O(2) generation in thyroid slices from several species. Our data show that in sheep, pig, bovine, and to a lesser extent dog thyroid, iodide had a stimulatory effect on H(2)O(2) generation. In horse and human thyroid, an inconstant effect was observed. We demonstrate in dogs that the stimulatory effect of iodide is greater in thyroids deprived of iodide, raising the possibility that differences in thyroid iodide pool may account, at least in part, for the differences between the different species studied. This represents the first demonstration of an activation by iodide of a specialized thyroid function. In comparison with conditions in which an inhibitory effect of iodide on H(2)O(2) generation is observed, the stimulating effect was observed for lower concentrations and for a shorter incubation time with iodide. Such a dual control of H(2)O(2) generation by iodide has the physiological interest of promoting an efficient oxidation of iodide when the substrate is provided to a deficient gland and of avoiding excessive oxidation of iodide and thus synthesis of thyroid hormones when it is in excess. The activation of H(2)O(2) generation may also explain the well described toxic effect of acute administration of iodide on iodine-depleted thyroids. PMID- 10751205 TI - Fetal growth and the physiological control of glucose tolerance in adults: a minimal model analysis. AB - Although there is now substantial evidence linking low birthweight with impaired glucose tolerance and type 2 diabetes in adult life, the extent to which reduced fetal growth is associated with impaired insulin sensitivity, defective insulin secretion, or a combination of both factors is not clear. We have therefore examined the relationships between birth size and both insulin sensitivity and insulin secretion as assessed by an intravenous glucose tolerance test with minimal model analysis in 163 men and women, aged 20 yr, born at term in Adelaide, South Australia. Birth size did not correlate with body mass index or fat distribution in men or women. Men who were lighter or shorter as babies were less insulin sensitive (P = 0.03 and P = 0.01, respectively), independently of their body mass index or body fat distribution. They also had higher insulin secretion (P = 0.007 and P = 0.006) and increased glucose effectiveness (P = 0.003 and P = 0.003). Overall glucose tolerance, however, did not correlate with birth size, suggesting that the reduced insulin sensitivity was being compensated for by an increase in insulin secretion and insulin-independent glucose disposal. There were no relationships between birth size and insulin sensitivity or insulin secretion in women. These results show that small size at birth is associated with increased insulin resistance and hyperinsulinemia in young adult life but that these relationships are restricted to the male gender in this age group. PMID- 10751206 TI - Influences of IGF-I gene disruption on the cellular profile of the diaphragm. AB - The impact of a targeted disruption of the Igf1 gene, encoding the insulin-like growth factor I (IGF-I), on diaphragm (DIA) cellularity was studied in 2-mo-old homozygous mutant [IGF-I(-/-)] mice and their wild-type [WT; i.e., IGF-I(+/+)] littermates. DIA fiber types were classified histochemically. DIA fiber cross sectional areas (CSA) were determined from digitized muscle sections, and fiber succinate dehydrogenase (SDH) activity was determined histochemically using a microdensitometric procedure. An acidic ATPase reaction was used to visualize capillaries. Myosin heavy chain (MyHC) isoforms were identified by SDS-PAGE, and their proportions were determined by scanning densitometry. The body weight of IGF-I(-/-) animals was 32% that of WT littermates. DIA fiber type proportions were unchanged between the groups. The CSAs of types I, IIa, and IIx DIA fibers of IGF-I(-/-) mutants were 63, 68, and 65%, respectively, those of WT animals (P < 0.001). The DIA thickness and the number of fibers spanning its entire thickness were reduced by 36 and 25%, respectively, in IGF-I(-/-) mice (P < 0. 001). SDH activity was significantly increased in all three types of DIA fibers of IGF-I(-/-) mutants (P < 0.05). The number of capillaries per fiber was reduced approximately 30% in IGF-I(-/-) animals, whereas the capillary density was preserved. The proportions of MyHC isoforms were similar between the groups. Muscle hypoplasia likely reflects the importance of IGF-I on cell proliferation, differentiation, and apoptosis (alone or in combination) during development, although reduced cell size highlights the importance of IGF-I on rate and/or maintenance of DIA fiber growth in the postnatal state. Reduced capillarity may result from both direct and indirect influences on angiogenesis. Improved oxidative capacity likely reflects DIA compensatory mechanisms in IGF-I(-/-) mutants. PMID- 10751207 TI - Interstitial glucose concentration and glycemia: implications for continuous subcutaneous glucose monitoring. AB - The changes in plasma glucose concentration and in interstitial glucose concentration, determined with a miniaturized subcutaneous glucose sensor, were investigated in anesthetized nondiabetic rats. Interstitial glucose was estimated through two different calibration procedures. First, after a glucose load, the magnitude of the increase in interstitial glucose, estimated through a one-point calibration procedure, was 70% of that in plasma glucose. We propose that this is due to the effect of endogenous insulin on peripheral glucose uptake. Second, during the spontaneous secondary decrease in plasma glucose after the glucose load, interstitial glucose decreased faster than plasma glucose, which may also be due to the effect of insulin on peripheral glucose uptake. Third, during insulin-induced hypoglycemia, the decrease in interstitial glucose was less marked than that of plasma glucose, suggesting that hypoglycemia suppressed transfer of glucose into the interstitial tissue; subsequently, interstitial glucose remained lower than plasma glucose during its return to basal value, suggesting that the stimulatory effect of insulin on peripheral glucose uptake was protracted. If these observations obtained in rats are relevant to human physiology, such discrepancies between plasma and interstitial glucose concentration may have major implications for the use of a subcutaneous glucose sensor in continuous blood glucose monitoring in diabetic patients. PMID- 10751208 TI - Different effects of IGF-I on insulin-stimulated glucose uptake in adipose tissue and skeletal muscle. AB - The effect of insulin-like growth factor I (IGF-I) on insulin-stimulated glucose uptake was studied in adipose and muscle tissues of hypophysectomized female rats. IGF-I was given as a subcutaneous infusion via osmotic minipumps for 6 or 20 days. All hypophysectomized rats received L-thyroxine and cortisol replacement therapy. IGF-I treatment increased body weight gain but had no effect on serum glucose or free fatty acid levels. Serum insulin and C-peptide concentrations decreased. Basal and insulin-stimulated glucose incorporation into lipids was reduced in adipose tissue segments and isolated adipocytes from the IGF-I-treated rats. In contrast, insulin treatment of hypophysectomized rats for 7 days increased basal and insulin-stimulated glucose incorporation into lipids in isolated adipocytes. Pretreatment of isolated adipocytes in vitro with IGF-I increased basal and insulin-stimulated glucose incorporation into lipids. These results indicate that the effect of IGF-I on lipogenesis in adipose tissue is not direct but via decreased serum insulin levels, which reduce the capacity of adipocytes to metabolize glucose. Isoproterenol-stimulated lipolysis, but not basal lipolysis, was enhanced in adipocytes from IGF-I-treated animals. In the soleus muscle, the glycogen content and insulin-stimulated glucose incorporation into glycogen were increased in IGF-I-treated rats. In summary, IGF-I has opposite effects on glucose uptake in adipose tissue and skeletal muscle, findings which at least partly explain previous reports of reduced body fat mass, increased body cell mass, and increased insulin responsiveness after IGF-I treatment. PMID- 10751209 TI - Thyroid hormone-induced stimulation of the sarcoplasmic reticulum Ca(2+) ATPase gene is inhibited by LIF and IL-6. AB - We investigated the effects of the leukemia inhibitory factor (LIF) and interleukin-6 (IL-6) on 3,3', 5-triiodo-L-thyronine, or thyroid hormone (T(3)) stimulated sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) gene expression on cultured neonatal rat cardiac myocytes. A reduction of T(3) induced increases in SERCA2 mRNA levels after co-treatment with LIF or IL-6. To investigate for the molecular mechanism(s) responsible for the blunted gene expression, a 3.2-kb SERCA2 promoter construct containing a reporter gene was transfected into cardiac myocytes. T(3) treatment stimulated transcriptional activity twofold, whereas co treatment with T(3) and either of the cytokines caused an inhibition of T(3) induced SERCA2 transcriptional activity. A T(3)-responsive 0.6-kb SERCA2 construct also showed a similar inhibition by cytokines. Cytokine inhibition of SERCA2 transcriptional activity was also evident when a 0.6-kb SERCA2 mutant, T(3)-unresponsive promoter construct was used. Treatment with T(3) and cytokines showed a significant decrease in transcription when a reporter construct was used that was comprised of direct repeats of SERCA2 thyroid response element I. These data provide evidence for cytokine-mediated inhibitory effects on the SERCA2 promoter that may be mediated by interfering with T(3) action. PMID- 10751211 TI - Variable imprinting of the heterotrimeric G protein G(s) alpha-subunit within different segments of the nephron. AB - The heterotrimeric G protein G(s) is required for hormone-stimulated intracellular cAMP generation because it couples hormone receptors to the enzyme adenylyl cyclase. Hormones that activate G(s) in the kidney include parathyroid hormone, glucagon, calcitonin, and vasopressin. Recently, it has been demonstrated that the G(s)alpha gene is imprinted in a tissue-specific manner, leading to preferential expression of G(s)alpha from the maternal allele in some tissues. In the kidney, G(s)alpha is imprinted in the proximal tubule but not in more distal nephron segments, such as the thick ascending limb or collecting duct. This most likely explains why in both humans and mice heterozygous mutations in the maternal allele lead to parathyroid hormone resistance in the proximal tubule whereas mutations in the paternal allele do not. In contrast, heterozygous mutations have little effect on vasopressin action in the collecting ducts. In mice with heterozygous null G(s)alpha mutations (both those with mutations on the maternal or paternal allele), expression of the Na-K-2Cl cotransporter was decreased in the thick ascending limb, suggesting that its expression is regulated by cAMP. The G(s)alpha genes also generate alternative, oppositely imprinted transcripts encoding XLalphas, a G(s)alpha isoform with a long NH(2)-terminal extension, and NESP55, a chromogranin-like neurosecretory protein. The role, if any, of these proteins in renal physiology is unknown. PMID- 10751210 TI - Inhibin is an important factor in the regulation of FSH secretion in the adult male hamster. AB - We investigated the importance of inhibin and testosterone in the regulation of gonadotropin secretion in adult male golden hamsters (Mesocricetus auratus). After castration, plasma concentrations of inhibin and testosterone were reduced to undetectable, whereas plasma follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were increased. After hemicastration, plasma FSH and LH increased moderately and plasma inhibin decreased to one-half its initial level. Plasma testosterone levels in hemicastrated animals decreased 3 h after hemicastration but returned to those in sham-operated animals at 6 h. Plasma LH in the castrated hamster declined comparably to intact animals with testosterone treatment; plasma FSH also decreased but still remained at levels higher than those in intact animals. After treatment with inhibin in long-term-castrated animals, plasma FSH decreased, whereas plasma LH was not altered. Intact males treated with flutamide, an anti-androgen, showed a significant increase in plasma LH but not in FSH. On the other hand, treatment with anti-inhibin serum induced a significant elevation in plasma FSH, but not in LH. Using immunohistochemistry, we showed that the inhibin alpha-subunit was localized to both Sertoli and Leydig cells. The present study in adult male hamsters indicates that FSH secretion is regulated mainly by inhibin, presumably from Sertoli and Leydig cells, and that LH secretion is controlled primarily by androgens produced from the Leydig cells. This situation is more similar to that of primates than of rats. PMID- 10751212 TI - Cell cycle regulatory proteins in renal disease: role in hypertrophy, proliferation, and apoptosis. AB - The response to glomerular and tubulointerstitial cell injury in most forms of renal disease includes changes in cell number (proliferation and apoptosis) and cell size (hypertrophy). These events typically precede and may be responsible for the accumulation of extracellular matrix proteins that leads to a decrease in renal function. There is increasing evidence showing that positive (cyclins and cyclin-dependent kinases) and negative (cyclin-dependent kinase inhibitors) cell cycle regulatory proteins have a critical role in regulating these fundamental cellular responses to immune and nonimmune forms of injury. Data now show that altering specific cell cycle proteins affects renal cell proliferation and improves renal function. Equally exciting is the expanding body of literature showing novel biological roles for cell cycle proteins in the regulation of cell hypertrophy and apoptosis. With increasing understanding of the role for cell cycle regulatory proteins in renal disease comes the hope for potential therapeutic interventions. PMID- 10751214 TI - Urothelial pathophysiological changes in feline interstitial cystitis: a human model. AB - Unique barrier properties of the urothelial surface membrane permit urine storage. Interstitial cystitis causes disabling dysuria, and frequency. Similarly, feline interstitial cystitis (FIC) occurs in cats. These studies define the permeability and structural properties of normal and FIC urothelium. To determine the effects of bladder filling, groups were studied before and after hydrodistention. Normal urothelium with or without hydrodistention exhibited high transepithelial resistances (TER) and low water and urea permeabilities, resembling other species. Fluorescence confocal microscopy revealed localization of the marker AE-31 to the apical surface of all umbrella cells in normal urothelium, with the tight junction protein ZO-1 localized to tight junctions. Scanning and transmission electron microscopy revealed uniform distribution of luminal cells with characteristic apical membrane and tight junction morphology. Urothelium in FIC animals displayed reduced TER and increased water and urea permeability following hydrodistention. Structural studies in FIC revealed denuded urothelium, with appearance of AE-31 in underlying epithelial cells. The results demonstrate severe epithelial damage and dysfunction in FIC and suggest novel approaches toward examining the etiology and therapy of IC. PMID- 10751213 TI - Localization of epithelial sodium channel and aquaporin-2 in rabbit kidney cortex. AB - The amiloride-sensitive epithelial sodium channel (ENaC) and the vasopressin dependent water channel aquaporin-2 (AQP2) mediate mineralocorticoid-regulated sodium- and vasopressin-regulated water reabsorption, respectively. Distributions of ENaC and AQP2 have been shown by immunohistochemistry in rats. Functional data from rabbits suggest a different distribution pattern of these channels than in rats. We studied, by immunohistochemistry in the rabbit kidney cortex, the distributions of ENaC and AQP2, in conjunction with marker proteins for distal segments. In rabbit cortex ENaC is restricted to the connecting tubule (CNT) cells and cortical collecting duct (CCD) cells. The intracellular distribution of ENaC shifts from the apical membrane in the most upstream CNT cells to a cytoplasmic location further downstream in the CNT and in the CCD cells. AQP2 is detected in the CCD cells exclusively. The anatomic subdivisions in the rabbit distal nephron coincide exactly with distributions of apical transport systems. The differences between rabbits and rats in the distribution patterns of ENaC and AQP2 may explain functional differences in renal salt and water handling between these species. PMID- 10751215 TI - Growth factor ultrafiltration in experimental diabetic nephropathy contributes to interstitial fibrosis. AB - Glomerular proteinuria is a risk factor for progression of chronic renal failure and contributes to renal interstitial fibrosis. In experimental diabetic glomerular sclerosis, there is translocation of high-molecular-weight growth factors, namely, hepatocyte growth factor (HGF) and transforming growth factor (TGF)-beta, from plasma into tubular fluid, both of which act on tubular cells through apical membrane receptors. In the present studies, the hypothesis is examined that ultrafiltered HGF and TGF-beta induce increased expression of extracellular matrix (ECM) proteins directly in tubular cells, or induce increased expression of cytokines that may act on interstitial myofibroblasts. Incubation of cultured tubular cells with recombinant human (rh) TGF-beta modestly raises expression of collagen type III, but rhHGF dose dependently blocks expression of this ECM protein. Both growth factors raise fibronectin expression up to fourfold and increase expression of platelet-derived growth factor (PDGF)-BB up to sixfold, but not of fibroblast growth factor-2. Pooled, diluted glomerular ultrafiltrate that had been collected by nephron micropuncture from rats with diabetic nephropathy (24-30 wk) also raises expression of fibronectin as well as PDGF-BB in proximal tubular cells. In the presence of neutralizing antibodies that block actions of HGF and TGF-beta, diabetic rat glomerular ultrafiltrate fails to increase tubular cell PDGF-BB expression. In NRK-49F renal interstitial myofibroblasts, rhPDGF-BB, in turn, raises the expression of collagen type III but not type I or fibronectin. The findings provide evidence for ultrafiltered HGF and TGF-beta to contribute to interstitial accumulation of ECM proteins by direct effects on tubular cells as well as indirect mechanisms, via PDGF-BB and its action on myofibroblasts. These events may be important mechanisms of proteinuria-induced renal interstitial fibrosis and accelerated progression of chronic renal failure in diabetic nephropathy and perhaps other proteinuric glomerular diseases. PMID- 10751217 TI - Postglomerular vasoconstriction induced by dopamine D(3) receptor activation in anesthetized rats. AB - In the present study we investigated the renal hemodynamic effects of dopamine D(3) receptor activation by R(+)-7-hydroxy-dipropylaminotetraline (7-OH-DPAT) in thiopental-anesthetized Sprague-Dawley rats. In clearance experiments infusion of 7-OH-DPAT (0.01-1.0 microg. kg(-1). min(-1)) dose-dependently elevated glomerular filtration rate (GFR) without affecting mean arterial blood pressure (MAP). In renal blood flow experiments 7-OH-DPAT infusion (1.0 microg. kg(-1). min(-1)) increased GFR by 16 +/- 2%, associated with an unexpected fall in renal blood flow by 20 +/- 3% and a significant elevation of renal vascular resistance by 18 +/- 3%. The renal hemodynamic changes were not influenced by pretreatment with the D(2)-receptor antagonist S(-)-sulpiride but were completely abolished during D(3) receptor inhibition by 5,6-dimethoxy-2-(di-n-propylamino)indane (U-99194A). In micropuncture experiments 7-OH-DPAT (1.0 microg. kg(-1). min(-1)) significantly elevated stop-flow pressure measured in the early proximal tubules and reduced hydrostatic pressure at the first branching point of the efferent arteriole without altering MAP. We conclude from these data that pharmacological activation of dopamine D(3) receptors affects renal hemodynamics in anesthetized rats by preferential postglomerular vasoconstriction. PMID- 10751216 TI - Effects of Ca(2+) channel activity on renal hemodynamics during acute attenuation of NO synthesis in the rat. AB - In cultured vascular muscle cells, nitric oxide (NO) has been shown to inhibit voltage-dependent Ca(2+) channels, which are involved in renal blood flow (RBF) autoregulation. Therefore, our purpose was to specify in vivo the effects of this interaction on RBF autoregulation. To do so, hemodynamics were investigated in anesthetized rats during Ca(2+) channel blockade before or after acute NO synthesis inhibition. Rats were treated intravenously with vehicle (n = 10), 0.3 mg/kg body wt N(G)-nitro-L-arginine-methyl ester (L-NAME; n = 7), 4.5 microg. kg body wt(-1). min(-1) nifedipine (n = 8) alone, or with nifedipine infused before (n = 8), after (n = 8), or coadministered with L-NAME (n = 10). Baseline renal vascular resistance (RVR) averaged 14.0 +/- 1.2 resistance units and did not change after vehicle. RVR increased or decreased significantly by 27 and 29% after L-NAME or nifedipine, respectively. Nifedipine reversed, but did not prevent, RVR increase after or coadministered with L-NAME. RBF autoregulation was maintained after L-NAME, but the autoregulatory pressure limit (P(A)) was significantly lowered by 15 mmHg. Nifedipine pretreatment or coadministration with L-NAME limited P(A) resetting or suppressed autoregulation at higher doses. Results were similar with verapamil. Intrarenal blockade of Ca(2+)-activated K(+) channels also prevented autoregulatory resetting by L-NAME (n = 8). These findings suggest NO inhibits voltage-dependent Ca(2+) channels and thereby modulates RBF autoregulatory efficiency. PMID- 10751218 TI - IP(3), IP(3) receptor, and cellular senescence. AB - Herein we demonstrate that replicative cellular senescence in vitro results in sharply reduced inositol 1,4,5-trisphosphate (IP(3)) receptor levels, reduced mitogen-evoked IP(3) formation and Ca(2+) release, and Ca(2+) store depletion. Human diploid fibroblasts (HDFs) underwent either 30 mean population doublings [mean population doublings (MPDs) thymidine labeling index (TI) >92% ("young") or between 53 and 58 MPDs (TI < 28%; "senescent")]. We found that the cytosolic Ca(2+) release triggered by either ionomycin or by several IP(3)-generating mitogens, namely bradykinin, thrombin, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF), was attenuated markedly in senescent HDFs. Notably, the triggered cytosolic Ca(2+) transients were of a smaller magnitude in senescent HDFs. However, the response latency seen with both PDGF and EGF was greater for senescent cells. Finally, a smaller proportion of senescent HDFs showed oscillations. In parallel, IP(3) formation in response to bradykinin or EGF was also attenuated in senescent HDFs. Furthermore, senescent HDFs displayed a sharply diminished Ca(2+) release response to intracellularly applied IP(3). Finally, to compare IP(3) receptor protein levels directly in young and senescent HDFs, their microsomal membranes were probed in Western blots with a highly specific anti-IP(3) receptor antiserum, Ab(40). A approximately 260-kDa band corresponding to the IP(3) receptor protein was noted; its intensity was reduced by approximately 50% in senescent cells. Thus, we suggest that reduced IP(3) receptor expression, lowered IP(3) formation, and Ca(2+) release, as well as Ca(2+) store depletion, all contribute to the deficient Ca(2+) signaling seen in HDFs undergoing replicative senescence. PMID- 10751219 TI - High dietary sodium chloride consumption may not induce body fluid retention in humans. AB - A commonly accepted hypothesis is that a chronically high-sodium diet expands extracellular volume and finally reaches a steady state where sodium intake and output are balanced whereas extracellular volume is expanded. However, in a recent study where the main purpose was to investigate the role of natriuretic peptides under day-to-day sodium intake conditions (Heer M, Drummer C, Baisch F, and Gerzer R. Pflugers Arch 425: 390-394, 1993), our laboratory observed increases in plasma volume without any rise in extracellular volume. To scrutinize these results that were observed as a side effect, we performed a controlled, randomized study including 32 healthy male test subjects in a metabolic ward. The NaCl intake ranged from a low level of 50 meq NaCl/day to 200, 400, and 550 meq/day, respectively. Plasma volume dose dependently increased (P < 0.01), being elevated by 315 +/- 37 ml in the 550-meq-NaCl-intake group. However, in contrast to the increased plasma volume, comparable to study I, total body water did not increase. In parallel, body mass also did not increase. Mean corpuscular volume of erythrocytes, as an index for intracellular volume, was also unchanged. We conclude from the results of these two independently conducted studies that under the chosen study conditions, in contrast to present opinions, high sodium intake does not induce total body water storage but induces a relative fluid shift from the interstitial into the intravascular space. PMID- 10751221 TI - Renal angiotensin II receptors and protein kinase C in diabetic rats: effects of insulin and ACE inhibition. AB - It has been shown that glomerular ANG II receptors are downregulated and protein kinase C (PKC) activity is enhanced in diabetes mellitus. Therefore, we investigated glomerular and preglomerular vascular ANG II receptors and PKC isoform regulation in streptozotocin (STZ)-diabetic rats treated with insulin and/or captopril. Diabetic rats were prepared by injecting STZ (60 mg/kg). Those that developed diabetes after 48 h were treated with low or high doses of insulin, or with a low dose of insulin as well as captopril, and killed 14 days later. Their glomeruli and preglomerular vessels were purified, competitive binding studies were performed by using the ANG II antagonists losartan and PD 123319, and PKC analysis was carried out by Western blotting. Competitive binding studies showed that the AT(1) receptor was the only ANG II receptor detected on both glomeruli and preglomerular vessels of all groups. Preglomerular vascular AT(1) receptor density (B(max)) was significantly upregulated in low insulin treated STZ rats, whereas glomerular AT(1) B(max) was downregulated. Furthermore, both the captopril- and high insulin-treated groups had less glomerulosclerosis and vascular damage than the low insulin-treated group. PKCalpha, PKCdelta, PKCepsilon, and PKCmu isoforms found in preglomerular vessels were upregulated by captopril and high insulin doses, respectively, whereas no such regulation occurred in glomeruli. We conclude that in STZ-diabetic rats ANG II receptors and PKC isoforms on preglomerular vessels and glomeruli are differentially regulated by treatment with insulin and/or captopril. PMID- 10751220 TI - Maturation of proximal straight tubule NaCl transport: role of thyroid hormone. AB - We have recently demonstrated that the rates of both active and passive proximal straight tubule (PST) NaCl transport in neonatal rabbits were less than in adults. In this segment NaCl entry across the apical membrane is via parallel Na(+)/H(+) and Cl(-)/OH(-) exchangers, which increases in activity with maturation. The present in vitro microperfusion study examined whether thyroid hormone plays a role in the maturational increase in PST NaCl transport. Neonatal and adult PST were perfused with a high-chloride-low bicarbonate solution without organic solutes, simulating late proximal tubule fluid. Thyroid hormone-treated neonates had a higher rate of PST total and passive NaCl transport. In 8-wk-old animals that were hypothyroid since birth, the maturational increase in total and passive NaCl transport was prevented. Thyroid treatment for 4 days in hypothyroid 8-wk-old rabbits increased the rate of both total and passive NaCl transport. The maturational increases in both Na(+)/H(+) and Cl(-)/OH(-) exchange activities were blunted in 8-wk-old hypothyroid animals and increased to control levels with thyroid treatment. This study demonstrates that thyroid hormone is a factor responsible for the maturational increase in both active and passive PST NaCl transport. PMID- 10751222 TI - Regulation of sgk by aldosterone and its effects on the epithelial Na(+) channel. AB - Aldosterone is the major corticosteroid regulating Na(+) absorption in tight epithelia and acts primarily by activating the epithelial Na(+) channel (ENaC) through unknown induced proteins. Recently, it has been reported that aldosterone induces the serum- and glucocorticoid-dependent kinase sgk and that coexpressing ENaC with this kinase in Xenopus laevis oocytes increases the amiloride-sensitive Na(+) current (Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, and Pearce D. Proc Natl Acad Sci USA 96: 2514-2519, 1999). The present study was done to further characterize regulation of sgk by aldosterone in native mammalian epithelia and to examine its effect on ENaC. With both in vivo and in vitro protocols, an almost fivefold increase in the abundance of sgk mRNA has been demonstrated in rat kidney and colon but not in lung. Induction of sgk by aldosterone was detected in kidney cortex and medulla, whereas the papilla expressed a constitutively high level of the kinase. The increase in sgk mRNA was detected as early as 30 min after the hormonal application and was independent of de novo protein synthesis. The observed aldosterone dose-response relationships suggest that the response is mediated, at least in part, by occupancy of the mineralocorticoid receptor. Coexpressing sgk and ENaC in Xenopus oocytes evoked a fourfold increase in the amiloride-blockable Na(+) channel activity. A point mutation in the beta-subunit known to impair regulation of the channel by Nedd4 (Y618A) had no significant effect on the response to sgk. PMID- 10751223 TI - Long-term regulation of urea transporter expression by vasopressin in Brattleboro rats. AB - Regulation of urea concentration in the renal medullary interstitium is important for maintenance of hypertonicity and therefore the osmotic driving force for water reabsorption. Studies in Sprague-Dawley rats showed that restriction of water intake for 3 days results in upregulation of urea transporter (UT) mRNA in the inner stripe of outer medulla of the kidney (2.9-kb UT2) but not in the inner medulla (4.0-kb UT1). The present study was performed to investigate the role of vasopressin in long-term regulation of UT1 and UT2 in neurogenic diabetes insipidus (Brattleboro) rats treated with a 7-day continuous infusion of [Arg(8)] vasopressin (AVP), [deamino-Cys(1), D-Arg(8)]-vasopressin (dDAVP) or vehicle. Northern analysis showed that water restriction alone had no effect on the level of UT2 mRNA in vehicle-treated Brattleboro rats but UT2 mRNA markedly increased and UT1 mRNA modestly decreased after treatment with dDAVP. In situ hybridization further demonstrated that the UT2 signal is upregulated and spread along the descending thin limbs of loops of Henle and that UT1 signal is downregulated in the inner medullary collecting ducts in vasopressin-treated rats, with a greater response for dDAVP compared with the AVP-treated group. Immunocytochemistry studies revealed that the UT1 and UT2 proteins are also modified in the same pattern as the transcript changes. Our studies reveal the role of vasopressin in long-term regulation of UT1 and UT2 expression during water restriction. PMID- 10751224 TI - Therapy with antisense TGF-beta1 oligodeoxynucleotides reduces kidney weight and matrix mRNAs in diabetic mice. AB - Inhibition of gene expression by antisense oligodeoxynucleotides (ODNs) relies on their ability to bind complementary mRNA sequences and prevent translation. The proximal tubule is a suitable target for ODN therapy in vivo because circulating ODNs accumulate in the proximal tubule in high concentrations. Because increased proximal tubular transforming growth factor- beta1 (TGF-beta1) expression may mediate diabetic renal hypertrophy, we investigated the effects of antisense TGF beta1 ODN on the high-glucose-induced proximal tubular epithelial cell hypertrophy in tissue culture and on diabetic renal hypertrophy in vivo. Mouse proximal tubular cells grown in 25 mM D-glucose and exposed to sense ODN as control (1 microM) exhibited increased (3)[H]leucine incorporation by 120% and total TGF-beta1 protein by 50% vs. culture in 5.5 mM D-glucose. Antisense ODN significantly decreased the high-glucose-stimulated TGF-beta1 secretion and leucine incorporation. Continuous infusion for 10 days of ODN (100 microg/day) was achieved via osmotic minipumps in diabetic and nondiabetic mice. Sense ODN treated streptozotocin-diabetic mice had 15.3% increase in kidney weight, 70% increase in alpha1(IV) collagen and 46% increase in fibronectin mRNA levels compared with nondiabetic mice. Treatment of diabetic mice with antisense ODN partially but significantly decreased kidney TGF-beta1 protein levels and attenuated the increase in kidney weight and the alpha1(IV) collagen and fibronectin mRNAs. In conclusion, therapy with antisense TGF-beta1 ODN decreases TGF-beta1 production and attenuates high-glucose-induced proximal tubular cell hypertrophy in vitro and partially prevents the increase in kidney weight and extracellular matrix expression in diabetic mice. PMID- 10751225 TI - Developmentally regulated expression of organic ion transporters NKT (OAT1), OCT1, NLT (OAT2), and Roct. AB - Several xenobiotic (organic cation and anion) transporters have recently been identified, although their endogenous substrates, if such exist, remain unknown. When we initially identified NKT, also known as OAT1, the first member of the organic anion transporter (OAT) family (Lopez-Nieto CE, You G, Bush KT, Barros EJ, Beier DR, and Nigam SK. J Biol Chem 272: 6471-6478, 1997), we noted its expression in the embryonic kidney. We have now demonstrated its transporter function and more fully examined the spatiotemporal expression patterns of representative organic ion transporters, [NKT (OAT1), Roct, OCT1, and NLT, also known as OAT2] during murine development. In the kidney, NKT (OAT1), OCT1, and Roct transcripts appeared at midgestation, coinciding with proximal tubule differentiation, and gradually increased during nephron maturation. A similar pattern was observed for NLT (OAT2) in the liver and kidney, although, in the kidney, NLT (OAT2) transcription did not increase as dramatically. The roughly cotemporal expression of these related transporters in the developing proximal tubule may indicate common transcriptional regulation. Expression during embryogenesis in extrarenal sites could suggest a role in the formation and maintenance of nonrenal tissues. Importantly, all four genes were expressed in unexpected places during nonrenal organogenesis: Roct in the fetal liver (temporally coinciding with the onset of hematopoiesis) and neural tissue; NKT (OAT1) in the fetal brain; OCT1 in the ascending aorta and atrium; and NLT (OAT2) in the fetal lung, intestine, skin, and developing bone. Because these gene products mediate the transport of a broad range of metabolites and toxins, it seems likely that, apart from their known functions, these transporters play a role in transport of organic molecules, perhaps including those with morphogenetic activity. These genes could also play important developmental roles independent of transport function. PMID- 10751226 TI - Renal interstitial Ca(2+). AB - Renal interstitial fluid Ca(2+) concentration ([Ca(2+)](isf)) was measured in anesthetized Wistar rats by using in situ microdialysis. During perfusion of 20 cm of the proximal small intestine with Ca(2+)-free buffer, renal [Ca(2+)](isf) was 1.63 +/- 0.19 mmol/l in the cortex (n = 6) and 1.93 +/- 0.12 mmol/l in the medulla (n = 5, P = 0.223). When Ca(2+) in the intestinal lumen was increased to 3 mmol/l, no change was seen in total or ionized serum Ca(2+) (S(Ca)), urinary Ca(2+) excretion (U(Ca)), or Ca(2+) in a microdialysate of the kidney cortex. Increasing intestinal Ca(2+) further, to 6 mmol/l, was without effect on S(Ca) but significantly increased U(Ca) by 38% and microdialysate Ca(2+) by 36% (1.25 +/- 0.0.09 vs. 1.70 +/- 0. 14 mmol/l, n = 4, P < 0.05). Intravenous infusion of 28 ng. kg(-1). min(-1) of parathyroid hormone for 1 h during perfusion of the intestinal lumen with 1 mmol/ Ca(2+)caused a 7-10% rise in S(Ca), a 40% fall in U(Ca), and a 32% increase in microdialysate Ca(2+) (1.32 +/- 0.13 vs. 1.74 +/- 0.13 mmol/l, n = 6, P < 0.05). Interlobar arteries with a mean diameter of 120 microm were studied by using a wire myograph to determine whether changes in extracellular Ca(2+) affect muscle tone. When precontracted with 5 micromol/l serotonin, the arteries relaxed in response to cumulative addition of Ca(2+) (1-5 mmol/l) with an ED(50) value for Ca(2+) of 3.30 +/- 0.08 mmol/l, n = 3. These data demonstrate that [Ca(2+)](isf) changes dynamically during manipulation of whole-animal Ca(2+) homeostasis and that intrarenal arteries relax in response to extracellular Ca(2+) varied over the range measured in vivo. PMID- 10751227 TI - 5-HT(2A) receptors stimulate mitogen-activated protein kinase via H(2)O(2) generation in rat renal mesangial cells. AB - Serotonin (5-HT) stimulates mitogenesis in rat renal mesangial cells through a G protein-coupled 5-HT(2A) receptor. We tested the hypothesis that oxidants might be involved in the signal transduction pathway linking the receptor to extracellular signal-regulated protein kinase (ERK). 5-HT rapidly increased the activity and phosphorylation of ERK. These effects were blocked by the 5-HT(2A) receptor antagonist ketanserin. The peak effect was noted at 5-10 min, and half maximal stimulation was achieved at 10-30 nM 5-HT. Chemical inhibitor and activator studies supported the involvement of phospholipase C, protein kinase C (PKC), and reactive oxygen species (ROS, i.e., H(2)O(2) and superoxide) generated by an NAD(P)H oxidase-like enzyme in the ERK activation cascade. Mapping studies supported a location for the NAD(P)H oxidase enzyme and the ROS downstream from PKC. Our studies are most consistent with an ERK activation pathway as follows: 5 HT(2A) receptor --> G(q) protein --> phospholipase C --> diacylglycerol --> classical PKC --> NAD(P)H oxidase --> superoxide --> superoxide dismutase --> H(2)O(2) --> mitogen-activated extracellular signal-regulated kinase --> ERK. These studies demonstrate a role for the 5-HT(2A) receptor in rapid, potent, and efficacious activation of ERK in rat renal mesangial cells. They support a role for oxidants in conveying the stimulatory signal from 5-HT, because 1) chemical antioxidants attenuate the 5-HT signal, 2) oxidants and 5-HT selectively activate ERK to a similar degree, 3) 5-HT produces superoxide and H(2)O(2) in these cells, and 4) a specific enzyme [NAD(P)H oxidase] has been implicated as the source of the ROS, which react selectively downstream of classical PKC. PMID- 10751228 TI - Rat homolog of sulfonylurea receptor 2B determines glibenclamide sensitivity of ROMK2 in Xenopus laevis oocyte. AB - Recent studies showed that coexpression of Kir6.1 or Kir6.2 with the sulfonylurea receptor (SUR1, SUR2A, or SUR2B) reconstituted an inwardly rectifying, ATP sensitive K(+) channel that was inhibited by glibenclamide (2, 15-17). Here we report the isolation of a rat homolog of mouse SUR2B (denoted rSUR2B) from a rat kidney cDNA library. The rSUR2B sequence contains a 4,635-bp open reading frame that encodes a 1,545-amino acid polypeptide, showing 67% shared identity with SUR1 (a pancreatic beta-cell isoform) and 98% with both SUR2A (a brain isoform) and SUR2B (a vascular smooth muscle isoform). Consistent with the predicted structures of other members of the ATP-binding cassette (ABC) superfamily, the sequence of rSUR2B contains 17 putative membrane-spanning segments. Also, predicted Walker A and B consensus binding motifs, present in other ABC members, are conserved in the rSUR2B sequence. RT-PCR revealed that rSUR2B is widely expressed in various rat tissues including brain, colon, heart, kidney, liver, skeletal muscle, and spleen. The intrarenal distribution of the rSUR2B transcript was investigated using RT-PCR and Southern blot of microdissected tubules. The rSUR2B transcript was detected in proximal tubule, cortical thick ascending limb, distal collecting tubule, cortical collecting duct, and outer medullary collecting duct, but not medullary thick ascending limb. This distal distribution overlaps with that of ROMK. Coexpression of rSUR2B with ROMK2 cRNA (in 1:10 ratio) in Xenopus laevis oocytes resulted in whole cell Ba(2+)-sensitive K(+) currents that were inhibited by glibenclamide (50% inhibition with 0.2 mM glibenclamide). In contrast, rSUR2B did not confer significant glibenclamide sensitivity to oocytes coinjected with ROMK1 or ROMK3. The interaction between ROMK2 and rSUR2B was further studied by coimmunoprecipitation of in vitro translated rSUR2B and ROMK2. In agreement with the functional data, the rSUR2B protein was coimmunoprecipitated with ROMK2 in the ROMK2-rSUR2B cotranslated samples. Our data demonstrate that ROMK2, but not ROMK1 and ROMK3, can interact with rSUR2B to confer a sulfonylurea-sensitive K(+) channel, implicating SUR proteins in forming and regulating renal ATP-sensitive K(+) channels. The ROMK isoform specificity of glibenclamide effects suggests that the NH(2) terminus of the ROMK protein mediates rSUR2B-ROMK2 interactions. PMID- 10751229 TI - Etomoxir-induced PPARalpha-modulated enzymes protect during acute renal failure. AB - Regulation of fatty acid beta-oxidation (FAO) represents an important mechanism for a sustained balance of energy production/utilization in kidney tissue. To examine the role of stimulated FAO during ischemia, Etomoxir (Eto), clofibrate, and WY-14,643 compounds were given 5 days prior to the induction of ischemia/reperfusion (I/R) injury. Compared with rats administered vehicle, Eto-, clofibrate-, and WY-treated rats had lower blood urea nitrogen and serum creatinines following I/R injury. Histological analysis confirmed a significant amelioration of acute tubular necrosis. I/R injury led to a threefold reduction of mRNA and protein levels of acyl CoA oxidase (AOX) and cytochrome P4A1, as well as twofold inhibition of their enzymatic activities. Eto treatment prevented the reduction of mRNA and protein levels and the inhibition of the enzymatic activities of these two peroxisome proliferator-activated receptor-alpha (PPARalpha) target genes during I/R injury. PPARalpha null mice subjected to I/R injury demonstrated significantly enhanced cortical necrosis and worse kidney function compared with wild-type controls. These results suggest that upregulation of PPARalpha-modulated FAO genes has an important role in the observed cytoprotection during I/R injury. PMID- 10751231 TI - Colorectal cancer: still a major killer despite progress on many fronts. PMID- 10751230 TI - AGEs induce oxidative stress and activate protein kinase C-beta(II) in neonatal mesangial cells. AB - Increased activation of specific protein kinase C (PKC) isoforms and increased nonenzymatic glycation of intracellular and extracellular proteins [the accumulation of advanced glycation end products (AGEs)] are major mechanistic pathways implicated in the pathogenesis of diabetic complications. Blocking PKC beta(II) has been shown to decrease albuminuria in animal models of diabetes. To demonstrate a direct relationship between AGEs and the induction and translocation of PKC-beta(II), studies were carried out in rat neonatal mesangial cells, known to express PKC-beta(II) in association with rapid proliferation in post-natal development. Oxidative stress was studied by using the fluorescent probe dichlorfluorescein diacetate. Translocation of PKC-beta(II) was demonstrated by using immunofluorescence and Western blotting of fractionated mesangial cells. Induction of intracellular oxidative stress, increase in intracellular calcium, and cytosol to membrane PKC-beta(II) translocation (with no change in PKC-alpha) were demonstrated after exposure to AGE-rich proteins. These data support the hypothesis that AGEs cause mesangial oxidative stress and alterations in PKC-beta(II), changes that may ultimately contribute to phenotypic abnormalities associated with diabetic nephropathy. PMID- 10751232 TI - Cardiovascular risk in women: the cardiologist's perspective. PMID- 10751233 TI - Substance abuse and the kidney. AB - Substance abuse has been increasing steadily in the UK and some other countries. Recent evidence suggests more than 40% of young people have tried illicit drugs at some time. There are numerous medical consequences to recreational drug use, and a physician should always consider substance abuse in any unexplained illness. The renal complications of drug abuse are also becoming more frequent, and may encompass a spectrum of glomerular, interstitial and vascular diseases. Although some substances are directly nephrotoxic, a number of other mechanisms are also involved. These effects are often chronic and irreversible, but occasionally acute with possible recovery. The rapid growth of illicit drug use is clearly a major public health problem. We review the commonly used substances of abuse and their associations with renal disease. PMID- 10751234 TI - Effects of diet and serotonergic agonist on hepatic apolipoprotein B-100 secretion and endothelial function in obese men. AB - We studied the effects of a hypocaloric diet with or without a serotonergic agonist (dexfenfluramine, Df) on the hepatic secretion of very-low-density lipoprotein (VLDL) apoB and endothelial function of the forearm microcirculation in 20 viscerally obese men. The kinetics of VLDL apoB were studied using an infusion of 1-((13)C)-leucine. Isotopic enrichment of apo B was measured using gas-chromatography mass spectrometry, and a multicompartmental model was used to estimate kinetic functions. Forearm vasodilatation was measured following an ischaemic stimulus using strain-gauge plethysmography, and visceral adipose tissue mass using magnetic resonance imaging. Compared with leaner subjects, the obese men had significantly higher hepatic apoB secretion (p<0.05) and lower forearm flow debt repayment (p<0.001). Both treatments produced similar decreases (p<0.05) in body weight, waist circumference, visceral adipose tissue and fasting plasma insulin. With diet alone, there was a significant decrease (p<0.05) in the plasma concentration and pool size hepatic secretion rate of VLDL apoB, as well as a significant increase (p<0.05) in post-ischaemic flow debt repayment. With diet plus Df, there were parallel responses in these variables, but only decreased forearm vascular resistance (p<0.05) was statistically significant. Combining both data sets, there was a highly significant reduction in hepatic apoB secretion rate (20. 9+/-2.0 vs. 14.7+/-1.6 mg/kg fat-free mass/day, p=0.005), as well as an increase in both maximal forearm blood flow (16.8+/-7.5 vs. 22. 2+/-8.5 ml/100 ml/min, p=0.006) and flow debt repayment (3.5+/-2.1 vs. 5.4+/-2.8 ml/100 ml, p=0.01), and a decrease in vascular resistance (6.7+/-3.7 vs. 5.1+/-4.4 mmHg/ml/100 ml/min, p=0.007). Obese men have increased hepatic secretion of apoB and endothelial dysfunction of the forearm microcirculation, and decreasing their visceral adipose tissue mass by diet (with or without a serotonergic agonist) improves these abnormalities. This may provide a mechanistic basis for the reduction in cardiovascular risk in obese patients who lose weight. PMID- 10751235 TI - Diastolic blood pressure is related to urinary sodium excretion in hypertensive Chinese patients. AB - We studied 70 Hong Kong Chinese patients with untreated hypertension and 47 normotensive controls. Blood pressure measurements and 24-h urine collection were performed for each patient, and were repeated 12 weeks later in 14 hypertensive patients who remained untreated. Twenty-two hypertensive patients underwent ambulatory blood pressure monitoring. The primary hypothesis tested was a correlation between diastolic blood pressure and 24-h urinary sodium excretion. In the hypertensive patients, diastolic blood pressure correlated with 24-h urinary sodium excretion (r=0.41, p<0.001), even after adjustment for age, gender, body mass index, ethanol intake and season (r=0.34, p=0.02). In normotensive controls, diastolic blood pressure did not correlate with sodium excretion (r=0.21, p=0.16). A correlation between diastolic blood pressure and sodium excretion was also observed in the patients who underwent ambulatory blood pressure monitoring (r=0.47, p=0.026), and in repeat measurements in untreated patients (r=0.60, p=0.02). Systolic blood pressure did not correlate with sodium excretion, although it increased with patient age (0.6+/-0.1 mmHg/year, p<0.001). In a multiple regression analysis with diastolic blood pressure as the dependent variable, the regression coefficient was 0.06+/-0.02 mmHg/mmol Na. The regression coefficients for ambulatory diastolic blood pressure and diastolic pressure repeated at 12 weeks were 0.07+/-0.03 and 0. 09+/-0.04 mmHg/mmol Na, respectively. Urinary sodium excretion was related to diastolic blood pressure in our hypertensive patients, accounting for 17% of the variance of diastolic blood pressure. PMID- 10751236 TI - Risk factors for central nervous system involvement in systemic lupus erythematosus. AB - We investigated risk factors for central nervous system (CNS) involvement in systemic lupus erythematosus (SLE), in 32 such patients individually matched 1 : 3 to 96 control SLE patients without CNS events. Univariate analysis showed that CNS involvement was significantly associated with the antiphospholipid syndrome (APS) as well as its features: arterial thrombosis, recurrent fetal loss, livedo reticularis and IgG anticardiolipin (aCL) antibodies in high titres. Other potential associations included cutaneous vasculitic lesions, thrombocytopenia, positive ANA, anti-SS-B/La and low serum levels of C(3) and C(4) complement components, while articular manifestations and discoid rash were significantly less common in patients with neuropsychiatric (NP) disease. In multivariate modeling, CNS involvement was strongly associated with cutaneous vasculitic lesions OR 33, 95% CI 1.5-720) and arterial thromboses (OR 13, 95%CI 0.82-220), and negatively related to the presence of articular manifestations (OR 0.015, 95%CI 0.00-0.17) and discoid rash (OR 0.004, 95%CI 0.00-0.35). Associations with APS-related arterial thromboses and vasculitis point to the importance of arterial vascular pathophysiology in the pathogenesis of NP disease in SLE. Patients with articular manifestations and discoid rash are at very low risk of NP events. Patients with an adverse SLE disease profile may require closer observation and may be the target group for studying pre-emptive interventions. PMID- 10751237 TI - Gastric acid suppression does not promote clostridial diarrhoea in the elderly. AB - Gastric acid prevents bacterial colonization of the stomach and suppression of its secretion might predispose to Clostridium difficile (CD) diarrhoea. We retrospectively studied elderly patients admitted to medical wards of an acute hospital to determine whether the incidence of CD diarrhoea was greater among those previously treated with gastric acid suppressants. From records of stool CD toxin tests undertaken in 1995 and 1996, we found 126 cases with positive results, and selected 126 controls with negative results. Information about pre morbid illness, predisposing factors for CD and medication received in the preceding 16 weeks was obtained from case-notes. A greater number of CD positive cases had received antibiotics such as Cefuroxime, ciprofloxacin or macrolides with or without metronidazole, were more severely disabled, required assistance for feeding, or had hypoalbuminaemia before the onset of diarrhoea. A greater number of controls had received lactulose, suggesting either that its laxative effect resembled CD infection prompting frequent stool tests, or that it offered protection against CD in this group. Both groups were similar for the use of proton-pump inhibitors or H2-receptor antagonists, suggesting that susceptible elderly patients are not more likely to develop CD diarrhoea after receiving gastric acid suppression therapy. PMID- 10751238 TI - Glycaemic control in type 2 diabetes: the impact of body weight, beta-cell function and patient education. AB - We examined the determinants of glycaemic control in a consecutive cohort of 562 newly-referred Chinese type 2 diabetic patients (57% women) during a 12-month period. All patients underwent a structured assessment with documentation of clinical and biochemical characteristics. Pancreatic beta-cell function was assessed by fasting plasma C-peptide concentration. Insulin deficiency was defined as fasting plasma C-peptide <0.2 pmol/ml. Insulin resistance (IR) was calculated using the homeostasis model assessment (HOMA) based on a product of fasting plasma glucose and insulin concentrations. Treatment was considered appropriate when insulin-deficient patients were treated with insulin and non insulin-deficient patients were treated with oral agents or diet. Mean (+/-SD) age was 54.3+/-13.8 years (range 17-87 years) and disease duration was 5.0+/-5.9 years. At the time of referral, 70.5% (n=396) were on drug therapy (9% on insulin and 62.8% on oral agents), 20.6% (n=116) were on diet and 9% (n=50) had not received any form of treatment. The mean HbA(lc) was 8.4+/-2.3%. The geometric mean (x// antilog SD) of IR was 4.62x//2.51 (range 0. 63-162.7) and correlated only with waist : hip ratio (WHR, p=0.008). The geometric mean of plasma C peptide was 0.47x//2.89 nmol/l and correlated with BMI (p<0.001). Glycated haemoglobin was correlated positively with age (p=0.013), disease duration (p<0.001), IR (p<0. 001) and negatively with BMI (p<0.001). Glycated haemoglobin was lower in patients who had seen a dietitian (7.9% vs. 8.7%, p<0.001) or diabetes nurse (7.8% vs. 8.7%, p<0.001) or who performed self blood glucose monitoring (7.9% vs. 8.6%, p=0.001) and higher among smokers (8.9% vs. 8.2%, p=0.003). Compared to insulin-deficient patients (n=118), non-insulin-deficient patients (n=413) had features resembling that of the Metabolic Syndrome with increased WHR (p=0.005), blood pressure (p<0.001), BMI (p=0.001) and were older (p=0.04). Amongst the insulin-deficient patients, 27% were treated with oral agents or diet. Patients receiving appropriate therapy (n=362) had a lower HbA(lc) than those treated inappropriately (n=173) (8.2% vs. 8.7%, p=0.02). On multivariate analysis, short disease duration (p<0.001), low IR (p<0.001), high BMI (p=0.001), diabetes education (p<0.001), lack of smoking (p=0. 014) and choice of appropriate treatment (p=0.009) were the independent determinants of good glycaemic control. PMID- 10751239 TI - Cheating sudden death: how to do it, and what life's like after it. PMID- 10751240 TI - Seasonal variation in CHD. PMID- 10751243 TI - Serum transferrin receptor assay in iron deficiency anaemia and anaemia of chronic disease in the elderly. PMID- 10751242 TI - Recurrent and prolonged fever in asplenic patients with human granulocytic ehrlichiosis. PMID- 10751244 TI - Ataxias on the march from Quebec to Tunisia. PMID- 10751245 TI - Understanding low-grade glioma: a decade of progress. PMID- 10751246 TI - Therapeutic dilemma: the use of anticonvulsants in HIV-positive individuals. AB - ARTICLE ABSTRACT: The concurrent use of anticonvulsants and antiretrovirals is a poorly studied area that poses a therapeutic dilemma for the clinician caring for HIV-positive patients requiring both classes of medications. Anticonvulsants and antiretrovirals may interact through multiple mechanisms including competition for protein binding, enhanced or reduced liver metabolism, and increased viral replication. The authors present many of the challenges faced by clinicians caring for HIV-positive patients who may require anticonvulsant therapy. PMID- 10751247 TI - Access www.neurology.org now for full-text articles PMID- 10751248 TI - Linkage to chromosome 13q11-12 of an autosomal recessive cerebellar ataxia in a Tunisian family. AB - OBJECTIVE: To report the clinical findings and the genetic linkage mapping of an autosomal recessive cerebellar ataxia associated to peripheral neuropathy, showing an early onset cerebellar ataxia with retained tendon reflexes (EOCA) phenotype. BACKGROUND: EOCA is a clinical syndrome delimited by Harding distinguished from Friedreich's ataxia (FA) mainly by the preservation of tendon reflexes. Molecular genetic study of patients with EOCA has demonstrated genetic heterogeneity. A form of autosomal recessive spastic ataxia has been described in Charlevoix Saguenay area in Quebec (ARSACS); the gene responsible has been mapped to chromosome 13q. METHODS: Genetic linkage analysis was performed on 18 members of a large family including 8 of 9 members with EOCA. After exclusion of FA and ataxia with vitamin E deficiency loci as well as loci of autosomal dominant cerebellar ataxias, we performed a linkage analysis to markers of 13q11-12 region. RESULTS: The 9 affected members of this family showed stereotyped clinical features with cerebellar ataxia, pyramidal syndrome, and a variable degree of axonal peripheral neuropathy. Linkage was detected between the disease locus and the microsatellite marker D13S232. Surrounding markers to D13S232 confirmed the linkage and showed the homozygosity of the affected members. CONCLUSION: The family reported here showed the same locus as autosomal recessive spastic ataxia Charlevoix Saguenay disease. PMID- 10751249 TI - A phase I trial of solubilized DR2:MBP84-102 (AG284) in multiple sclerosis. AB - OBJECTIVE: To assess the safety, tolerability, and biologic and clinical activity of a solubilized complex comprised of human leukocyte antigen-DR2 with myelin basic protein84-102 (AG284)in patients with secondary progressive MS. BACKGROUND: Soluble species-specific major histocompatibility complex myelin basic protein91 103 complexes ameliorate disease in a dose-dependent manner when administered to SJL/J mice with chronic relapsing experimental allergic encephalomyelitis. Preincubation with AG284 reduces the proliferative response of a DR2-restricted, myelin basic protein84-102-reactive T cell clone, derived from a MS patient, to myelin basic protein84-102 in the presence of autologous antigen-presenting cells. METHODS: Thirty-three patients with secondary progressive MS were randomly assigned to receive three alternate day IV doses of AG284 or placebo in a double masked dose escalation study. The primary outcome was safety and tolerability. Secondary outcomes included a comparison of pre- and post-treatment gadolinium enhanced brain MRI activity, Kurtzke Expanded Disability Status Scale, and Nine Hole Peg Test scores. RESULTS: The frequency of adverse events was similar in the AG284 and placebo recipients. No significant treatment effect was detected by Expanded Disability Status Scale, Nine Hole Peg Test, or number of new gadolinium enhancing MRI lesions. CONCLUSIONS: AG284 as administered during this study was safe and well tolerated. Further studies are warranted to determine the biologic activity and clinical efficacy of this potential treatment for MS. PMID- 10751250 TI - Whole-brain diffusion MR histograms differ between MS subtypes. AB - OBJECTIVE: To determine whether quantitative whole-brain MR diffusion histograms in patients with MS differ from those of normal control subjects. BACKGROUND: MRI detects macroscopic cerebral lesions in MS, but the white matter lesion burden on MRI correlates imperfectly to clinical disability. Previous reports have further suggested abnormalities in white matter of MS patients with no visible lesions on conventional MRI. METHODS: A total of 25 subjects (13 with MS [9 relapsing remitting, 4 secondary progressive] and 12 healthy control subjects) underwent diffusion-weighted echoplanar MRI encompassing the entire brain. The average apparent diffusion coefficient (ADCave, or diffusion trace) was calculated on a pixel-by-pixel basis after segmentation of intracranial space from calvarium and extracranial soft tissues. Whole-brain ADCave histograms were calculated and plotted for statistical comparison. RESULTS: Mean whole-brain MR ADCave in MS patients was elevated and histograms were shifted to higher values compared with normal control subjects. Mean whole-brain ADCave of secondary progressive patients was shifted to higher values compared with relapsing-remitting patients. Whole-brain ADCave histograms of relapsing-remitting patients showed no significant difference from normal control subjects. CONCLUSION: Whole-brain MR diffusion histograms may quantitate overall cerebral lesion load in patients with MS and may be able to discern differences between clinical subgroups. PMID- 10751251 TI - Open-ring imaging sign: highly specific for atypical brain demyelination. AB - OBJECTIVE: To test the specificity for demyelination of a new neuroimaging sign: contrast enhancement shaped as an open ring or a crescent circumscribed to the white matter. BACKGROUND: Brain demyelination can cause ring enhancement mimicking neoplasm or infection on CT or MRI. METHODS: A MEDLINE search of pathology-proved demyelination yielded 32 illustrated cases of ring-enhancing lesions published between 1981 and 1995. Controls consisted of the same number of published images of neoplasms and infections, pathology proved, and matched by year of publication, and age and gender of the patient. Two neuroradiologists read the images twice independently 1 year apart. RESULTS: Interrater agreement was good (kappa = 0.64 and 0.66 for either reading). Test-retest reliability was high (kappa = 0.75 and 0.74 for either rater). The open-ring sign clearly distinguished demyelinating lesions from neoplasms and infections. For demyelination versus neoplasm or infection, the specificity of the reading by the first neuroradiologist was 93.8 (95% CI, 86 to 98), and that of the second was 84.4 (95% CI, 74 to 92). The likelihood ratio of demyelination versus neoplasm averaged 5.2, and versus infection, 17.2. That is, if the lesions had the same incidence in the population, in the presence of an open-ring sign demyelination would be five times more likely than neoplasm and 17 times more likely than infection. However, given the much higher incidence of neoplasms and infections, these lesions are still frequently responsible for open-ring enhancement. CONCLUSIONS: The open-ring sign is often present in large, contrast-enhancing demyelinating lesions and helps to differentiate them from neoplasms and infections. PMID- 10751252 TI - Encephalomyelitis-associated antimyelin autoreactivity induced by streptococcal exotoxins. AB - OBJECTIVE: After implicating Streptococcus pyogenes as causing acute disseminated encephalomyelitis (ADEM) in a child, we wanted to prove that in vivo activation of autoreactive T lymphocytes by superantigens of this Streptococcus contributed to the dramatic demyelination. BACKGROUND: ADEM is a demyelinating disorder of the CNS sharing many similarities with MS. Demyelination in MS is considered to be the result of an autoimmune process mediated by autoreactive T lymphocytes with specificity for myelin antigens. METHODS: Phenotypic analysis and proliferation assays on blood monocytes, as well as isolation of myelin basic protein (MBP)-reactive T-cell lines/clones; and TCR repertorium analysis by PCR ELISA and cytokine production. RESULTS: 1) The blood T-cell receptor (TCR) repertoire was compatible with in vivo expansion induced by S. pyogenes exotoxins. 2) TCR expression analysis indicated clonal expansion of CD8+ MBP reactive T cells, suggesting in vivo activation. MBP-reactive T cells showed crossreactivity to S. pyogenes supernatant and exotoxins. 3) Cytokine mRNA quantification of the mononuclear cells revealed a Th2-biased profile. CONCLUSION: In vivo exposure to S. pyogenes may have induced activation of pathogenic myelin reactive T cells, contributing to the dramatic inflammatory demyelination. PMID- 10751253 TI - Cerebellar and medullar histoplasmosis PMID- 10751254 TI - Long-term outcome of low-grade oligodendroglioma and mixed glioma. AB - BACKGROUND: Low-grade oligodendrogliomas and mixed gliomas can be indolent and remain unchanged for years. Optimal timing and effectiveness of initial treatment is uncertain and therapy can be associated with toxicity. METHODS: Retrospective review of patients diagnosed between 1979 and 1997 with low-grade oligodendroglioma or mixed glioma. Time to progression, survival, prognostic factors, and treatment toxicities were evaluated. RESULTS: A total of 106 patients (77 oligodendroglioma, 29 mixed glioma) were identified; median age was 36.7 years. Initial presenting symptoms were seizures in 76 (72%) and headache in 11 (10%); tumor was diagnosed as an incidental finding in five patients. Tumor progression was diagnosed in 72 patients (68%). Overall median time to progression (MTTP) was 5.0 years (range 0.5 to 14.2). Median overall survival (OS) was 16.7 years. No prognostic factors reached statistical significance. MTTP and OS were not significantly affected by treatment. Of 62 patients who received radiation therapy, 9 (15%) developed radiation necrosis and 13 developed radiation therapy-related cognitive changes, requiring ventriculoperitoneal shunting in six. Significant myelosuppression was seen in 35 of 76 (46%) patients treated with chemotherapy. CONCLUSIONS: Low-grade oligodendroglioma and mixed glioma have a long median overall survival. There were no apparent differences in either immediate versus deferred treatment or choice of initial therapy on disease-free or overall survival. Chemotherapy was associated with significant acute toxicity in almost one half of patients; radiation therapy produced late neurotoxicity in one third, justifying deferred treatment until clinically necessary. PMID- 10751255 TI - Antisulfatide antibodies in neuropathy: clinical and electrophysiologic correlates. AB - OBJECTIVE: To investigate the clinical and electrophysiologic characteristics of the neuropathy associated with elevated serum antisulfatide antibodies. METHODS: Clinical, electrophysiologic, morphologic, and laboratory data of 25 patients with significantly elevated (>25,600) antisulfatide antibodies were reviewed. RESULTS: Four groups were distinguished based on clinical and electrophysiologic data: Group 1, eight patients with predominantly small fiber sensory neuropathy (32%); Group 2, five patients with mixed large and small fiber sensory neuropathy (20%); Group 3, seven patients with axonal sensorimotor neuropathy (28%); and Group 4, three patients with demyelinating sensorimotor neuropathy (12%). One additional patient had mononeuritis multiplex and one had ALS. An immunoglobulin M (IgM) monoclonal gammopathy was found in 30% of the patients tested, but not in any of the Group 1 patients with small fiber sensory neuropathy. Serum IgM level was elevated in 12 patients, of whom six had a concomitant monoclonal gammopathy. Morphologic studies in five patients showed predominantly axonal degeneration, with three of the patients also exhibiting additional features of demyelination. CONCLUSIONS: Antisulfatide antibodies are associated with several subtypes of peripheral neuropathy. Predominantly sensory or sensorimotor axonal neuropathies are most common in this series, with the sensory component either small fiber or mixed fiber type. A smaller demyelinating group indistinguishable from patients with chronic inflammatory demyelinating polyradiculopathy was also seen. One third of patients had a concomitant IgM monoclonal gammopathy, and approximately one half had elevated serum IgM. PMID- 10751256 TI - Activate your online subscription PMID- 10751257 TI - Cross-reactive antibodies against GM2 and CMV-infected fibroblasts in Guillain Barre syndrome. AB - OBJECTIVE: To investigate whether anti-GM2 antibodies in patients with Guillain Barre syndrome (GBS) are induced by molecular mimicry with cytomegalovirus (CMV). BACKGROUND: Antibodies against ganglioside GM2 are frequently present in the serum from GBS patients with an antecedent infection with CMV. METHODS: The authors detected inhibition of anti-GM2 reactivity after incubation of GM2 reactive serum samples with fibroblasts infected with a GBS-associated CMV strain. Control sera consisted of GQ1b-reactive samples, and control antigens included uninfected fibroblasts and fibroblasts that were infected with other herpes viruses. RESULTS: Serum immunoglobulin M reactivity with GM2 was decreased in a dose-dependent manner after incubation with CMV-infected fibroblasts. Incubation of anti-GM2-positive serum samples with uninfected fibroblasts and fibroblasts infected with varicella zoster virus did not inhibit anti-GM2 reactivity, whereas this reactivity was slightly decreased after incubation with herpes simplex virus type 1 in one patient. Antibodies against ganglioside GQ1b did not react with CMV-infected fibroblasts. CONCLUSIONS: CMV-infected fibroblasts express gangliosidelike epitopes that recognize specifically anti-GM2 antibodies. These results support the hypothesis that antiganglioside antibodies in CMV-infected GBS patients are induced by molecular mimicry between GM2 and antigens that are induced by a CMV infection. PMID- 10751258 TI - Glycosphingolipid antibodies and blood-nerve barrier in autoimmune demyelinative neuropathy. AB - OBJECTIVE: To evaluate morphologic changes in small endoneurial vessels in patients with autoimmune demyelinative neuropathy and antiglycosphyngolipid antibodies. BACKGROUND: Although a humoral immune cause has been postulated for various demyelinating neuropathies, the mechanism by which large molecules like immunoglobulins can traverse the blood-nerve barrier (BNB) to enter the endoneurium is not understood. METHODS: We examined histologic and ultrastructural changes in small endoneurial vessels in sural nerve biopsy specimens from autoimmune demyelinative neuropathy patients, with or without anti glycosphingolipid (GSL) antibodies. Density of small endoneurial vessels, mean endothelial area, mean luminal area, and the percentage of endothelial cell junctions (that make up the BNB) that showed evidence of disruption were evaluated. RESULTS: Only junctional disruption showed a significant difference: autoimmune demyelinative neuropathy patients with anti-GSL antibodies showed more BNB disruption than autoimmune demyelinative neuropathy patients without antibodies or control patients with nonautoimmune neuropathies. The most commonly observed endoneurial change in antibody-positive autoimmune demyelinative neuropathy patients was the finding of continuous, patent spaces lacking tight junctions between endothelial cells. CONCLUSIONS: Brain endothelial cells and endoneurial endothelial cells share various GSL antigens, including GM1 and GD1b gangliosides, with peripheral nerve tissues. Circulating anti-GSL antibodies could damage cell-to-cell attachments in endoneurial endothelium. This barrier disruption may permit the large molecules like immunoglobulins to enter the endoneurial space, contributing to development of autoimmune demyelinative neuropathy. PMID- 10751259 TI - Medial temporal lobe neuronal damage in temporal and extratemporal lesional epilepsy. AB - OBJECTIVE: To assess the extent of medial temporal lobe (TL) abnormalities of the neuronal marker N-acetylaspartate (NAA) in TL and extra-TL lesional partial epilepsy, and to determine whether decreases in NAA are related to lesion location, to lesion pathology, or to the seizures themselves. METHODS: The authors studied 19 patients with intractable partial epilepsy and an isolated structural cerebral lesion (10 TL, 9 extra-TL; 10 cortical dysplasia [CD], 9 non CD lesions). Proton MRS imaging was used to determine the average relative resonance intensity of NAA for the TL regions of the left and right hemispheres. Values less than two SDs below the mean of normal control subjects were considered abnormal. RESULTS: Fourteen patients (74%) had abnormally low NAA relative to creatine (NAA/Cr) in at least one TL. Three-way analysis of variance (ANOVA; lesion pathology, lesion location, side of NAA/Cr decrease) showed that ipsilateral NAA/Cr was lower than contralateral (p = 0. 04). Three-way ANOVA (lesion location, generalized tonic-clonic seizures, side of NAA/Cr decrease) showed that generalized tonic-clonic seizures were associated with lower TL NAA/Cr (p = 0. 02). Lesion location and pathology showed no main effect on the NAA-to-Cr ratio in either analysis (p > 0.05). Linear regression analyses between seizure duration and NAA/Cr decrease was not significant. CONCLUSION: The authors demonstrated abnormally low TL NAA/Cr in the majority of patients with structural cerebral lesions. This abnormality did not differ with lesion location or pathology. They propose that the altered function of neuronal networks by an isolated structural cerebral lesion results in remote "functional dual pathology." PMID- 10751260 TI - EEG predicts surgical outcome in lesional frontal lobe epilepsy. AB - BACKGROUND: Because of the relatively poor results of frontal lobe epilepsy (FLE) surgery, identification of prognostic factors for surgical outcome is of great importance. METHODS: To identify predictive factors for FLE surgery, we analyzed the data of 61 patients (mean age at surgery 19.2) who had undergone presurgical evaluation and resective surgery in the frontal lobe. Postoperative follow-up ranged from 0.5 to 5 years (mean 1.78). Fifty-nine patients had MRI-detectable lesions. Histopathologic examination showed dysplasia (57.4%), tumor (16.4%), or other lesions (26.2%). Thirty postoperatively seizure-free patients were compared with 31 non-seizure-free patients with respect to clinical history, seizure semiology, EEG and neuroimaging data, resected area, and postoperative data including histopathology. RESULTS: Three preoperative and two postoperative variables were related to poor outcome: generalized epileptiform discharges, generalized slowing, use of intracranial electrodes, incomplete resection detected by MRI, and postoperative epileptiform discharges. The only preoperative factor associated with seizure-free outcome was the absence of generalized EEG signs. Multivariate analysis showed that only the absence of generalized EEG signs predicts the outcome independently. Moreover, the occurrence of a somatosensory aura, secondarily generalized seizures, and negative MRI was identified as additional independent risk factors for poor surgical results. CONCLUSIONS: The absence of generalized EEG signs is the most predictive variable for a seizure-free outcome in FLE surgery. Furthermore, nonlesional MRI, somatosensory aura, and secondarily generalized seizures are risk factors for poor surgical results. PMID- 10751261 TI - The role of somatosensory evoked potentials in the diagnosis of AIDS-associated myelopathy. AB - BACKGROUND: Although AIDS-associated vacuolar myelopathy is detected in >50% of autopsy cases, it is often unrecognized during life. The clinical assessment is often difficult because of concurrent peripheral neuropathy and lack of specific diagnostic markers. Somatosensory evoked potentials (SEPs) have been successfully used to evaluate central conduction in a number of diseases involving the spinal cord. OBJECTIVES: To assess the diagnostic yield of SEPs in AIDS-associated myelopathy. METHODS: We recorded tibial and median nerve SEPs in 69 HIV-infected subjects referred for evaluation of lower extremity neurologic abnormalities. Stimulation of the peroneal nerve at the popliteal fossa was performed in patients with absent response to ankle stimulation. RESULTS: HIV-infected subjects had significantly delayed latencies of both peripheral and central potentials, suggesting a combination of peripheral and CNS abnormalities. Analysis of peripheral and central latencies allowed us to discriminate between neuropathy and myelopathy in individual patients. Abnormalities of tibial central conduction time (CCT) correlated with clinical diagnosis of myelopathy. There was no significant difference in median CCTs between patients and controls, suggesting that conduction abnormalities were restricted to the thoracolumbar spinal cord. A derived spinal conduction time was a sensitive indicator of central conduction abnormalities in AIDS patients with myelopathy. CONCLUSIONS: The combination of median, posterior tibial, and peroneal SEPs is a valuable tool in the diagnosis of AIDS-associated myelopathy, particularly when myelopathy and peripheral neuropathy coexist. The use of a derived spinal conduction time improves the diagnostic yield of SEPs in AIDS-associated myelopathy. PMID- 10751262 TI - Extrastriatal dopamine D2 and D3 receptors in early and advanced Parkinson's disease. AB - OBJECTIVE: To investigate whether dopamine D2 and D3 receptor subtypes (D2/3Rs) outside the caudate-putamen are affected in PD. BACKGROUND: Alterations in striatal D2-like dopamine receptors in PD have been extensively demonstrated using PET, but there are no studies focusing on extrastriatal D2/3Rs. METHODS: Fourteen unmedicated patients with idiopathic early PD with predominantly left sided symptoms, 14 levodopa-medicated patients with advanced PD, and 20 normal age-matched controls were examined using PET. PET scanning was performed with a novel high-affinity D2/3R radioligand ([11C]FLB 457) and a PET scanner in three dimensional mode. RESULTS: In advanced PD, the binding potential of [11C]FLB 457 in the dorsolateral prefrontal cortex was decreased by 40% (p < 0.01), in the anterior cingulate cortex by 20% (p < 0.01), and in the medial thalamus by 17% (p < 0.05) compared with healthy controls. In early PD, the extrastriatal [11C]FLB 457 binding potentials were not significantly different compared with the control group. However, the binding potential in the anterior cingulate cortex (29%; p < 0. 05) was higher in early PD compared with advanced PD. CONCLUSIONS: These results imply that the D2/3 receptor subtypes outside the striatum are affected in advanced PD but not in the early stages of the disease, and that this receptor decline is present in the anterior cingulate cortex, the dorsolateral prefrontal cortex, and the thalamus. PMID- 10751263 TI - NeuroImages. Cerebral shotgun pellet embolism. PMID- 10751264 TI - Video analysis of acute motor and convulsive manifestations in sport-related concussion. AB - OBJECTIVES: To describe the motor and convulsive manifestations in acute sports related head injury. METHODS: A total of 234 cases of concussive injuries during the 1995 through 1997 football seasons were obtained from the Australian Football League Medical Officers Association injury survey. Of these, 102 cases were recorded adequately on television videotape and were analyzed by two independent observers using a standardized recording form detailing injury mechanics and clinical features of the episodes. Motor and convulsive features were correlated with mechanical variables and with duration of loss of consciousness using linear modeling techniques. RESULTS: Tonic posturing occurred in 25 subjects, clonic movements in 6, righting movement in 40, and gait unsteadiness in 42. In one subject the tonic and clonic features were sufficiently prolonged to be deemed a concussive convulsion. The only risk factor for tonic posturing using logistic regression was the presence of loss of consciousness (p = 0.0001). There was a trend toward facial impact being an independent predictor of tonic posturing but this did not reach significance. No other independent variable predicted the development of clonic movements, righting movements, or gait unsteadiness. CONCLUSIONS: Subtle motor manifestations such as tonic posturing and clonic movements commonly occur in concussion; the main predictive factor for tonic posturing is the presence of loss of consciousness. The authors speculate that these clinical features are due to brainstem dysfunction secondary to biomechanical forces inducing a transient functional decerebration. PMID- 10751265 TI - APOE-epsilon4 is associated with memory decline in cognitively impaired elderly. AB - OBJECTIVE: To investigate whether the association between APOE-epsilon4 and memory decline is modified by baseline cognition and age in a population-based elderly sample. METHODS: The study sample consisted of 1,243 subjects, 62 to 85 years old, with a Mini-Mental State Examination (MMSE) score between 21 and 30 and known APOE phenotypes. Memory performance was measured with an abbreviated Auditory Verbal Learning Test (AVLT) at baseline and repeated after 3 years (n = 854). Memory decline was defined as a decrease of at least 1 SD from the mean change score on immediate recall (IR), delayed recall (DR), and retention, based on the AVLT. RESULTS: Multivariate logistic regression analyses showed that APOE epsilon4 is associated with memory decline in cognitively impaired subjects (MMSE score, 21 to 26) (OR for decline on IR adjusted for age, sex, education, and baseline recall score, 3.8; 95% CI, 1.4 to 10.0; adjusted OR for decline on DR, 2.9; 95% CI, 1.2 to 7.0; adjusted OR for decline on retention, 3.3; 95% CI, 1.1 to 10. 1), but not in cognitively normal subjects (MMSE score, 27 to 30) (adjusted OR for decline on IR, 1.1; 95% CI, 0.6 to 2.0; adjusted OR for decline on DR, 1.0; 95% CI, 0.6 to 1.8; adjusted OR for decline on retention, 1.5; 95% CI, 0.7 to 3.0). In particular, cognitively impaired epsilon4 carriers older than 75 years were at high risk of memory decline (adjusted OR for decline on IR, 4.5; 95% CI, 1.4 to 13.8; adjusted OR for decline on DR, 3.6; 95% CI, 1.2 to 10.8; adjusted OR for decline on retention, 6.6; 95% CI, 1.5 to 29.7). CONCLUSIONS: APOE-epsilon4 was associated with memory decline in subjects with cognitive impairment, but not in normally functioning subjects. Contrary to AD studies, our study suggests that the risk of APOE-epsilon4 on memory decline does not decrease at higher ages. PMID- 10751266 TI - Combined assessment of tau and neuronal thread protein in Alzheimer's disease CSF. AB - OBJECTIVE: Comparative study of CSF levels of tau and AD7C-neuronal thread protein (NTP) in patients with AD and control subjects. BACKGROUND: AD is characterized by neurofibrillary tangles composed of the abnormally hyperphosphorylated microtubule-associated protein tau. AD7C-NTP is a proposed AD marker expressed at early stages of neurofibrillary degeneration. METHODS: Enzyme linked immunosorbent assays specific for tau and AD7C-NTP. CSF samples were obtained from 35 demented patients (25 with antemortem clinical diagnosis of probable AD, 5 with neuropathologic diagnosis of definite AD, 5 with Lewy body pathology), 29 nondemented patients with PD, and 16 elderly healthy control subjects. Receiver operating characteristics (ROC) and multivariate discriminant analysis for AD versus controls. Correlational analysis of CSF tau and AD7C-NTP and of each marker with Mini-Mental State Examination (MMSE) scores was performed. RESULTS: Levels of both tau and AD7C-NTP were significantly elevated in the AD patients compared with control subjects. ROC analysis showed that CSF tau distinguished between patients with AD and nondemented control subjects with 63% sensitivity and 89% specificity, AD7C-NTP with 70% sensitivity and 87% specificity. Combined evaluation of both markers with discriminant analysis raised the specificity to 93% at a 63% sensitivity level. Both markers positively correlated with each other within the AD group, but not among control subjects. CSF levels of AD7C-NTP, but not of tau, showed a small but significant inverse correlation (r = -0.43) with MMSE scores of AD patients. CONCLUSIONS: CSF levels of tau and AD7C-NTP may be useful biomarkers for AD. PMID- 10751267 TI - Quantitative neurologic assessment of ataxia-telangiectasia. AB - BACKGROUND: Ataxia telangiectasia (A-T) is a rare disorder with many distinctive neurologic features. Although there is substantial individual variation in the rate of progression of these features, their relationship to one another or to age has not been characterized. METHODS: We formulated and tested multiple elements that assess different neurologic functions known to be affected by A-T. The overall index was applied to 52 patients with A-T, 2 to 29 years of age. RESULTS: Seven elements items proved to be informative, and three elements were added based on face validity. In a linear regression model of individuals under 19 years of age, controlled for correlation within sibships, age accounted for 87% of the variation in the A-T Index. CONCLUSION: Despite substantial individual variability of the phenotypic elements of A-T, scores on this multidimensional index have a very high correlation with age, indicating that there is a characteristic rate of progression of the disease, although functional domains in the brain are differentially affected. The pattern of scores suggests that a severe and a mild form of A-T may be distinguished by this quantitative measure. With further development this index may become useful as an outcome measure for treatment studies and prognosis. PMID- 10751268 TI - Age-related cognitive decline in hereditary spastic paraparesis linked to chromosome 2p. AB - OBJECTIVES: To investigate whether cognitive decline is part of the phenotype of SPG4-linked hereditary spastic paraparesis (HSP) and to determine whether cognitive changes are present in haplotype carriers before the onset of paraparesis. BACKGROUND: The major locus for "pure" autosomal dominant HSP is the SPG4 locus on chromosome 2p. Cognitive impairment linked to this locus has been described in two families. METHODS: The authors identified 19 families with "pure" autosomal dominant HSP. Five had linkage to the SPG4 locus (maximum lod score for D2S2374: 5.99 at zero recombination in four smaller families and 3.86 at zero recombination in the largest family). Haplotype construction identified a disease haplotype for all families; 41 individuals carried this haplotype (30 affected by HSP, 11 unaffected). All haplotype carriers and 41 matched controls underwent Cambridge Cognitive (CAMCOG) examination. Nonparametric significance tests were used comparing total and subset scores. RESULTS: Haplotype carriers affected by HSP had lower total CAMCOG scores than control subjects (85.86/107 versus 96.2/107; p < 0.0005). The subsets of orientation, memory, language expression, and comprehension were also significantly lower. Ten individuals had scores < or =80/107, indicating mild dementia. Unaffected haplotype carriers had mean total CAMCOG scores lower than control subjects (91.82/107 versus 98. 09/107; p = 0.016). In both groups cognitive decline was age-dependent and scores diverged from control subjects from age 40. All SPG4-linked families showed the effect. CONCLUSION: Mild, age-related cognitive impairment is a feature common to these families. It illustrates variable phenotypic expression at this locus and may be the first manifestation of the disease gene in individuals as yet unaffected by paraparesis. PMID- 10751269 TI - Treatment of multifocal motor neuropathy with interferon-beta1A. AB - Nine patients with multifocal motor neuropathy who had previously responded favorably to IV immunoglobulins (IVIg) were treated with interferon-beta1a. Muscle strength and disability were evaluated. In six patients there was no effect of treatment. Four patients deteriorated in such a way that IVIg had to be restarted during the study. Three patients showed an improvement that was more pronounced than on IVIg. These patients had a shorter disease duration and were less affected clinically and electrophysiologically than those who did not respond. PMID- 10751270 TI - Irreversible spinal cord injury as a complication of subarachnoid ethanol neurolysis. AB - Subarachnoid neurolysis using ethanol to destroy selectively the posterior roots of the spinal cord is a method for providing pain relief in patients with advanced cancer. Weakness of the extremities is a complication of the procedure that has been attributed to spread of the neurolytic agent to the anterior roots. The authors provide evidence of spinal cord injury as a cause of lower extremity weakness in a patient after subarachnoid ethanol neurolysis. PMID- 10751271 TI - Chronic paroxysmal hemicrania-tic syndrome. AB - The association of chronic paroxysmal hemicrania and ticlike pain-chronic paroxysmal hemicrania-tic (CPH-tic)-is a recently described syndrome. The authors found only two previously reported cases. They report three new cases of this rare syndrome with both chronic paroxysmal hemicrania and ticlike pain concurrently and ipsilaterally. The trigeminal-autonomic cephalalgias (TAC) are considered as differential diagnoses. CPH-tic syndrome could be a different clinical entity. PMID- 10751272 TI - APOE-epsilon4 predicts incident AD in Japanese-American men: the honolulu-asia aging study. AB - The authors assessed the 3-year incidence of dementia, including subtypes, in 2,603 Japanese-American men 71 to 93 years of age who were dementia free at baseline. There were 137 new cases of dementia according to the Diagnostic and Statistical Manual of Mental Disorders, 3rd edition, revised, including 51 with a primary diagnosis of AD. The rates for all subtypes increased with age. Men with an APOE4 allele had a significantly increased risk of AD of 2.39 (95% CI, 1.07, 5.31), after adjusting for age and education. There was no significant relationship of APOE4 with other subtypes of dementia. PMID- 10751273 TI - Reduction of human visual cortex excitability using 1-Hz transcranial magnetic stimulation. AB - The effects of low-frequency (1-Hz) repetitive transcranial magnetic stimulation on visual cortex excitability were investigated by measuring phosphene thresholds (PTs) and stimulus-response curves. Stimulation over the visual cortex led to significantly decreased visual cortex excitability, expressed as an increase in PT. The motor threshold of the hand muscles did not change, indicating the topographic specificity of this effect. This intervention may be useful in situations associated with a hyperexcitable visual cortex. PMID- 10751274 TI - Functional MRI in double cortex: functionality of heterotopia. AB - A 12-year-old boy with epilepsy and subcortical laminar heterotopia (band heterotopia) underwent a functional MRI protocol to study voluntary motor activity in the hand. Finger tapping produced an activation of a contralateral limited and focused frontal cortical area both in the subcortical band heterotopia and the overlying cortex. Despite its epileptogenic activity, subcortical laminar heterotopia seems to be responsible for part of the functional activity of the brain. This has to be pointed out for epilepsy surgery resecting cortical dysplasia. PMID- 10751275 TI - Autonomic failure in ALS with a novel SOD1 gene mutation. AB - The authors report a patient with ALS and a novel SOD1 gene mutation who was in the totally locked-in state and developed autonomic failure followed by sudden cardiac arrest. A neuropathologic study showed widespread multisystem degeneration, including involvement of the autonomic nuclei in the medulla and spinal cord. SOD1 gene analysis detected a missense mutation of V118L in exon 4. These findings show notable phenotypic heterogeneity for SOD1-associated ALS. PMID- 10751276 TI - Quetiapine for l-dopa-induced psychosis in PD. PMID- 10751277 TI - Variability of the mini-mental state examination in dementia. PMID- 10751278 TI - Detection of 14-3-3 protein in the CSF of a patient with Hashimoto's encephalopathy. PMID- 10751279 TI - Combined VIth and XIIth cranial nerve palsies: a clival syndrome. PMID- 10751280 TI - Intravenous heparin for acute stroke: what can we learn from the megatrials? PMID- 10751281 TI - Electrodiagnostic studies in ulnar neuropathy at the elbow. PMID- 10751282 TI - Neurologic side effects in neuroleptic-naive patients treated with haloperidol or risperidone. PMID- 10751283 TI - Clinical correlations of occipital epileptiform discharges in children. PMID- 10751285 TI - Editorial: making the best of your data PMID- 10751284 TI - Lethal capillary leak syndrome after a single administration of interferon beta 1b. PMID- 10751286 TI - Potent protective effects of melatonin on experimental spinal cord injury. AB - STUDY DESIGN: Experimental biochemical, behavioral, and histologic investigations of spinal cord injury in rats. OBJECTIVE: To investigate the effects of melatonin, a pineal hormone, in compression ischemic-induced spinal cord injury. SUMMARY OF BACKGROUND DATA: The implication of activated neutrophils in the worsening of spinal cord injury has been shown. Melatonin was shown to play an important role in protecting animal cells from neutrophil-induced toxicity and damage by free radicals. There is no report on using melatonin for spinal cord injury. METHODS: Spinal cord injury was induced by placing 25 g of weight extradurally on the rat spinal cord at T12 for 20 minutes. The rats were randomly divided into three groups. Sham rats had only laminectomy. Melatonin rats were injected with melatonin (2.5 mg/kg) intraperitoneally (intraperitoneal) five times: at 5 minutes, then 1, 2, 3, and 4 hours after the injury. Correspondingly, the control rats were injected with saline. Measured levels of lipid peroxidation estimated thiobarbituric acid reactive substances (TBARS) and the accumulation of leukocytes at the site of trauma, which were evaluated by measuring tissue myeloperoxidase activity. The recovery was assessed by using three clinical scoring systems, and histologic changes of the damaged spinal cord were examined. RESULTS: The thiobarbituric acid reactive substances content in the spinal cord increased after the injury, with two peaks (at 1 and 4 hours), and nitrogen mustard-induced leukocytopenia significantly attenuated the thiobarbituric acid reactive substances content in four 4 after injury. Also in these 4 hours, myeloperoxidase activity increased and melatonin injection reduced thiobarbituric acid reactive substances content and myeloperoxidase activity, which attenuated the motor deficits as well. Histologic findings showed that the melatonin group had less cavity formation than the control group. CONCLUSION: Results showed that injection of melatonin reduced thiobarbituric acid reactive substances content and myeloperoxidase activity, facilitating recovery of the damaged spinal cord. PMID- 10751288 TI - Anterior cervical plating reverses load transfer through multilevel strut-grafts. AB - STUDY DESIGN: In vitro biomechanical study using a programmable testing apparatus that replicated physiologic flexion/extension cervical spine motion and loading mechanics. OBJECTIVE: To determine the influence of anterior plating on multilevel cervical strut-graft mechanics in vitro. SUMMARY OF BACKGROUND DATA: The addition of anterior instrumentation does not prevent construct failure in multilevel cervical corpectomy. METHODS: Six fresh human cadaveric cervical spines (C2-T1) were tested in the four following sequential conditions: harvested, C4-C6 corpectomy, strut-grafted, and strut-grafted with an anterior cervical plate. A force-sensing strut-graft was used to measure compression/tension, flexion/extension and lateral bending moments, and axial torsion. Parameters of stiffness, vertebral motion, and strut-graft loads were compared to determine differences between the four spine conditions. RESULTS: Application of the anterior plate significantly increased the global stiffness (P < 0.01) and decreased the local motion (P < or = 0.01) of the instrumented levels (C3-C7). Flexion of the strut-grafted spine loaded the strut-graft, whereas extension unloaded the strut-graft. With the anterior plate, flexion of the plated spine unloaded the strut-graft. Extension significantly loaded the strut graft more than similar degrees of flexion in the strut-grafted condition (P = 0.01). Strut-graft loading end limits of 225 N were reached with a mean 7.5 degrees extension in the plated spines. CONCLUSIONS: Anterior multilevel cervical plating effectively increases stiffness and decreases local cervical motion after corpectomy. However, anterior cervical plating also reverses graft loads and excessively loads the graft in extension, which may promote pistoning and failure of multilevel constructs. PMID- 10751287 TI - Sensory innervation of the lumbar dura mater passing through the sympathetic trunk in rats. AB - STUDY DESIGN: Sensory innervation of the lumbar dura mater passing through the sympathetic trunk was investigated by neuronal tracing and immunohistochemical methods. OBJECTIVE: To assess an anatomic basis indicating that sympathetic block in the higher lumbar region is effective for patients with low back pain. SUMMARY OF BACKGROUND DATA: Low back pain is elicited by disorder or mechanical stimulation of the lumbar dura mater. Clinically, the authors often have observed patients in whom root block at the responsible level or sympathetic block at the higher level could relieve low back pain. Therefore, there may be two different sensory routes, the segmental innervation at the same level and nonsegmental fibers from higher dorsal root ganglia. METHODS: The tracers were injected into the sympathetic trunk between L3 and L4 of rats. The lumbar dorsal root ganglia and dura mater were examined, and labeled cells were measured in size and the distribution. To establish the sensory property, the materials were processed in immunohistochemistry for calcitonin gene-related peptide. RESULTS: Many small- to medium-sized neurons were retrogradely labeled L1 and L2 dorsal root ganglia after injection into the sympathetic trunk. The anterogradely labeled fibers were found in the dura mater at L4 and L5. Some of the labeled neurons and fibers were immunoreactive for calcitonin gene-related peptide. CONCLUSION: Sensory fibers from the upper lumbar ganglia innervated the lower lumbar dura mater directly. These sensory nerves may mediate low back pain and possibly interact with sympathetic nerves. PMID- 10751289 TI - Cervical myelopathy caused by C3-C4 spondylosis in elderly patients: a radiographic analysis of pathogenesis. AB - STUDY DESIGN: A radiographic analysis of elderly patients with cervical spondylotic myelopathy, particularly those with involvement of the C3-C4 level. OBJECTIVES: To elucidate the potential contributors to the higher incidence of pathology at C3-C4 in the elderly. SUMMARY OF BACKGROUND DATA: In this study, the elder patients showed a greater predilection for involvement of the C3-C4 lesion compared with their younger counterparts. No previous study has addressed C3-C4 pathology in elderly patients. METHODS: This study included 18 patients, 10 men and 8 women, with cervical spondylotic myelopathy caused by C3-C4 disorders (group I). For the purpose of comparison, 18 younger patients (less than 50 years of age) with myelopathy (group II) and 30 volunteers over the age of 65 (group III) were also investigated. Mean age at admission was 73.5 years for group I, 42.4 years for group II, and 73.4 years for group III. Radiographic analysis, using static and dynamic radiographs, was performed to evaluate the morphologic features. RESULTS: The mean spinal canal diameter for groups I and II was significantly smaller than that for group III. Group I exhibited greater C2-C7 lordosis. The aged population, group I and group III, showed greater C3-C4 angulation associated with C4 forward inclination in neutral standing position as compared with younger patients. Regarding dynamic factors, group I showed the largest segmental motion at C3-C4, and, conversely, the smallest mobility at the lower segments, with significant differences. CONCLUSIONS: Using radiographic analysis, morphologic features that predispose patients to disorders of the C3-C4 motion segment were evaluated. These features included 1) greater C3-C4 angulation associated with age-related postural change and 2) hypermobility at the C3-C4 segment compensating for decreased mobility at the lower segments. PMID- 10751290 TI - Health outcome assessment before and after anterior cervical discectomy and fusion for radiculopathy: a prospective analysis. AB - STUDY DESIGN: A prospective assessment, performed using the Health Status Questionnaire, of the outcomes for 28 patients with cervical radiculopathy treated with one- or two-level anterior cervical discectomy and fusion. OBJECTIVE: To assess patient outcome using the Health Status Questionnaire after one- or two-level anterior cervical discectomy and fusion. SUMMARY OF BACKGROUND DATA: Although outcomes for many types of surgical procedures already have been evaluated, few have focused on the results of cervical surgery. METHODS: Before and after anterior cervical discectomy and fusion for cervical radiculopathy, 28 patients filled out the Health Status Questionnaire. The average follow-up interval was 21.8 months. There were 10 men and 18 women, with an average age of 44 years. All outcome instruments were graded for individual scores of general health, physical function, role limitation because of physical health problems, role limitation because of emotional problems, social function, mental health, bodily pain, and energy. Data were analyzed using the age (< 55 vs. > 55), worker's compensation status, and education status of the patient. Preoperative and postoperative scores were compared for each subscale. RESULTS: Statistically significant improvements were found in postoperative scores for bodily pain (P < 0.001), vitality (P = 0.003), physical function (P = 0.01), role function/physical (P = 0.0003), and social function (P = 0.0004). No significant differences were found before and after surgery for three health scales: general health, mental health, and role function associated with emotional limitations. Age, educational status, and history of compensation litigation did not appear to affect outcome measures. CONCLUSIONS: Although this is a preliminary report involving 28 patients, it would appear, based on the results of the Health Status Questionnaire, that anterior cervical discectomy and fusion performed on appropriately selected patients is a highly reliable surgical procedure for the management of cervical radiculopathy. Additional disease-specific questions may provide more sensitivity in evaluating radiculopathy after surgical and nonsurgical intervention. PMID- 10751291 TI - Chondrosarcoma of the mobile spine: report on 22 cases. AB - STUDY DESIGN: A retrospective review of 22 cases of chondrosarcoma arising from the mobile spine. OBJECTIVE: To evaluate the role of oncologic and surgical staging in correlating management and outcome of chondrosarcoma involving the spine. SUMMARY OF BACKGROUND DATA: Approximately 10% of chondrosarcomas arise from the mobile spine, occurring mainly in adults, particularly elderly men. The course of the disease depends on the aggressiveness of the tumor, but also is influenced by the management. Intralesional surgery is followed almost constantly by local recurrence even with adjuvant therapy. METHODS: All charts, radiographs, and images were reviewed. The composite information provided by this review allowed for oncologic and surgical staging of the reviewed cases. According to Enneking criteria, the surgical procedures were defined as curettage (piecemeal excision) or en blocexcision. The margins were submitted to histologic study and reported as intralesional, marginal, or wide. - As primary management, 10 intralesional curettages (follow-up period, 2-119 months; average, 61 months) and 12 en bloc excisions (follow-up period, 39-207 months; average, 97 months) were performed. A total of 33 procedures were performed, including the management of the recurrences (18 curettages and 15 en bloc excisions: one for soft tissue recurrence). A clinical and radiographic follow-up period of of 2 to 236 months (average, 81 months; minimal follow-up period for survivors, 30 months; average follow-up period for survivors, 115 months) was available for all the patients. RESULTS: Three recurrences occurred in 14 patients treated by en bloc excision at onset or for recurrence, two in cases of histologically proven contaminated or intralesional margins. All but one patient were alive at final follow-up evaluation. Conversely, all the patients treated by one or more curettages (with or without adjuvant radiation therapy) had at least one recurrence, and 8 of 10 of these patients died of the disease. At final follow-up evaluation, nine patients had died of the disease; nine were continuously disease free (but one had died of another unrelated malignancy); and four were symptom free after management for recurrences (one was found alive 155 months after a soft-tissue metastasis en-bloc excision). CONCLUSIONS: En bloc excision, with wide or marginal histologic margins, is the suggested management for chondrosarcomas of the spine. Early diagnosis and careful surgical staging and planning are necessary for conducting adequate management. However, tumor contamination of the specimen margins, even in a small area, or spreading of the tumor myxoid content can worsen the prognosis. PMID- 10751292 TI - The effect of continued posterior spinal growth on sagittal contour in patients treated by anterior instrumentation for idiopathic scoliosis. AB - STUDY DESIGN: Retrospective analysis of radiographs on a prospective cohort of patients undergoing anterior instrumentation for thoracic idiopathic scoliosis. OBJECTIVES: To analyze the change in sagittal profile after growth. SUMMARY OF BACKGROUND DATA: The authors previously reported some advantages of anterior instrumentation for treatment of thoracic idiopathic scoliosis. However, postsurgery hyperkyphosis has resulted in some patients, especially those who were skeletally immature at the time of surgery. METHODS: Inclusion criteria required that participants have thoracic idiopathic scoliosis treated with anterior instrumentation and a confirmed solid fusion, no rod breakage, and a minimum follow-up period of 2 years. The 47 patients meeting the criteria were divided into a study group of 10 patients who were Risser 0 at the time of surgery and a control group of 37 patients who were Risser 1 to 5. Progressive sagittal kyphosis was defined as an increase of 10 degrees or more (T5-T12) after surgery. RESULTS: Sagittal progression greater than 10 degrees (average, 15 degrees ) occurred in 6 of 10 patients (60%) in the study group (Risser 0). Five patients progressed from 10 degrees to 19 degrees, and one patient from 20 degrees to 30 degrees. In contrast, sagittal progression occurred in only 10 of 37 patients (27%) in the control group (Risser 1 to 5). CONCLUSIONS: Some patients with thoracic adolescent idiopathic scoliosis treated with anterior instrumentation may be at risk for progressive sagittal kyphosis secondary to growth. Skeletal immaturity (Risser 0) appears to be a risk factor. In these immature patients, preserving the sagittal profile with intervertebral spacers, rigid rods, and bone graft (allowing for an average 15 degrees increase of kyphosis with growth) may be appropriate. PMID- 10751293 TI - Associations between patient report of symptoms and anatomic impairment visible on lumbar magnetic resonance imaging. AB - STUDY DESIGN: A cross-sectional study comparing the relationship of symptoms with anatomic impairment visible on lumbar magnetic resonance imaging in 408 symptomatic subjects. OBJECTIVE: To determine how various anatomic impairments, including the magnitude and location of nerve compression visible on lumbar magnetic resonance imaging, are associated with patient reports of pain, weakness, and dysesthesia. SUMMARY AND BACKGROUND DATA: Anatomic impairments of the intervertebral disc, radicular canal, and associated soft tissues are prevalent in people with and those without low back pain or lower extremity radiculopathy. This has led to confusion in differentiating between symptom generators and benign variation visible on lumbar magnetic resonance imaging. Recent literature has suggested that the presence of nerve compression is an important finding in the prediction of symptoms. However, the threshold for meaningful nerve compression has not been described. METHODS: In this study, 408 participants undergoing a diagnostic workup for low back pain, radiculopathy, and/ or completed a survey and pain drawing. Participants underwent standardized lumbar magnetic resonance imaging using a 1.5-T scanner. Two classification systems describing the spatial distribution of symptoms were developed. An additional system to quantify the magnitude of nerve and thecal sac compression was created. All systems were assessed for reliability, after which comparisons among variables were performed using Chi2 as well as simple and multiple logistic regression analysis. RESULTS: The reliability coefficients for categorizing patients on the basis of pain drawing ranged from 0. 75 to 0.88. The S1-S2 segmental distribution was the most commonly reported location of symptoms, followed by L4-L5. The most common magnetic resonance imaging diagnosis was "unremarkable," followed by "disc impairment without nerve compression." Disc extrusion was present in 10.8% of participants. The reliability of classifying nerve compression visible on magnetic resonance imaging ranged from 0.27 to 1. Nerve compression was present in 37% of participants, and 18% had severe nerve compression. There were no significant associations between segmental distribution of symptoms and the presence of anatomic impairment. However, according to a collapsed classification scale, severe nerve compression and disc extrusion were predictive of pain distal to the knee (odds ratios, 2.72 and 3. 34). The self-report of weakness was associated mildly with severe nerve compression and disc extrusion, but not with other findings. Magnetic resonance imaging findings did not predict self-reports of dysesthesia. CONCLUSIONS: The presence of disc extrusion and/or ipsilateral, severe nerve compression at one or multiple sites is strongly associated with distal leg pain. Mild to moderate nerve compression, disc degeneration or bulging, and central spinal stenosis are not significantly associated with specific pain patterns. Although segmental distributions of pain can be determined reliably from pain drawings, this finding alone is of little use in predicting lumbar impairment. The self-report of lower extremity weakness or dysesthesia is not significantly related to any specific lumbar impairments. [Key words: back pain, diagnosis, magnetic resonance imaging, nerve compression, pain drawing, pathology] PMID- 10751294 TI - Abnormalities in muscle function during gait in relation to the level of lumbar disc herniation. AB - STUDY DESIGN: A comparison between gait in patients undergoing surgery for L4 and L5 lumbar disc herniations and that in an age- and weight-matched control group. OBJECTIVES: To study whether changes in the moments produced at the ankle and knee joints during walking reflect the neurologic level of a herniated nucleus pulposus. SUMMARY OF BACKGROUND DATA: Lumbar herniated discs often cause muscle weakness, reduced motor function, and change in walking capacity. The specific effects of a disc herniation on muscle function during gait is poorly documented. METHODS: Conventional physical examination and kinetic analysis of gait were performed on 16 patients who subsequently underwent surgery for herniated discs (eight with L4-L5 and eight with L5-S1 disc herniations) and 16 healthy control subjects. The three components of the external moment at the ankle and knee were computed. The peak magnitudes of specific components of the external moments were compared with those of the control group. RESULTS: Reduced external ankle plantar flexion moment, indicating a decreased function of the ankle dorsiflexors, was found in patients with herniated nucleus pulposus of both L4-L5 and L5-S1. Reduced external ankle dorsiflexion moment, indicating a decreased function of the ankle plantar flexors, was found only in patients with a lesion to the L5-S1 disc, but not in those with herniations at L4-L5. CONCLUSIONS: Preoperative gait analysis identified functional deficits of the muscles about the ankle and foot that relate to the level of the herniation. Kinetic measurements can assist in understanding the functional limitations associated with specific levels of a herniation. PMID- 10751295 TI - The relation between pain intensity, disability, and the episodic nature of chronic and recurrent low back pain. AB - STUDY DESIGN: An observational study on the course of chronic and recurrent low back pain and its relation to disability and medication use performed on the basis of daily diary recording. OBJECTIVES: To provide a description of daily pain reporting by individuals with self-reported chronic and recurrent low back pain, to study how the intensity and episodic nature of low back pain is related to disability and medication use, and to classify subjects according to Von Korff's categories of chronic low back pain. SUMMARY OF BACKGROUND DATA: The natural history of low back pain has been described, and some classification schemes have been proposed, but little has been reported on pain characteristics and their relation to self-report of disability. METHODS: Daily self-reports of pain intensity, social and work disability, and medication use were collected from 94 participants with self-reported chronic or recurrent low back pain over a 6-month period. A metric for describing the episodic nature of chronic low back pain was developed. RESULTS: A significant effect of pain intensity on disability was found. During an episode, participants had significantly greater disability and medication use. Work-related disability and medication use was significantly greater in the latter half of an episode. CONCLUSIONS: Pain intensity can affect disability, but the episodic nature of low back pain also affects the ability to function in both work and personal life. Intermittent increases in pain can markedly alter disability. Chronic low back pain should not be treated as a static phenomenon. PMID- 10751296 TI - Sudden and unexpected loading generates high forces on the lumbar spine. AB - STUDY DESIGN: A cross-sectional study of spinal loading in healthy volunteers. OBJECTIVES: To measure the bending and compressive forces acting on the lumbar spine, in a range of postures, when unknown loads are delivered unexpectedly to the hands. SUMMARY OF BACKGROUND DATA: Epidemiologic studies suggest that sudden and unexpected loading events often lead to back injuries. Such incidents have been shown to increase back muscle activity, but their effects on the compressive force and bending moment acting on the spine have not been fully quantified. Furthermore, previous investigations have focused on the upright posture only. METHODS: In this study, 12 volunteers each stood on a force plate while weights of 0, 2, 4, and 6 kg (for men, 40% less for women) were delivered into their hands in one of three ways: 1) by the volunteer holding an empty box with handles, into which an unknown weight was dropped; 2) by the same way as in 1, but with volunteer wearing a blindfold and earphones to eliminate sensory cues; or 3) by the volunteer sliding a box of unknown weight off a smooth table. Experiments were carried out with participants standing in upright, partially flexed, and moderately flexed postures. Spinal compression resulting from muscular activity was quantified using electromyographic signals recorded from the back and abdominal muscles. The axial inertial force acting up the long axis of the spine was calculated from the vertical ground reaction force. The bending moment acting on the osteoligamentous spine was quantified by comparing measurements of lumbar curvature with the bending stiffness properties of cadaveric lumbar spines. RESULTS: The contribution from abdominal muscle contraction to overall spinal compression was small (average, 8%), as was the axial inertial force (average, 2.5%), and both were highest in the upright posture. Peak bending moments were higher in flexed postures, but did not increase much at the moment of load delivery in any posture. Peak spinal compressive forces were increased by 30% to 70% when loads were suddenly and unexpectedly dropped into the box, and by 20% to 30% when they were slid off the table, as compared with loads simply held statically in the same posture (P < 0.001). The removal of audiovisual cues had little effect. CONCLUSIONS: Sudden and alarming events associated with manual handling cause a reflex overreaction of the back muscles, which substantially increases spine compressive loading. Manual handling regulations should aim to prevent such events and limit the weight of objects to be lifted. PMID- 10751297 TI - Anterior lumbar fusion improves discogenic pain at levels of prior posterolateral fusion. AB - STUDY DESIGN: A descriptive case review. OBJECTIVES: To assess the outcomes of anterior lumbar interbody fusion for painful discs within a solid posterolateral spinal fusion. SUMMARY OF BACKGROUND DATA: Some patients continue to have pain after posterolateral spinal fusion despite apparently solid arthrodesis. One potential etiology is pain that arises from a disc within the fused levels. METHOD: Retrospective review of 176 patients with anterior interbody fusion, which located 20 who had anterior interbody fusion levels of prior posterolateral spinal fusion. All had low back pain, solid posterolateral spinal fusion, and painful disc(s) at the posterolateral spinal fusion level(s) but not elsewhere. Pain was measured by the Numerical Rating Scale, function by Oswestry Disability Questionnaire, and patient satisfaction by the North American Spine Society Outcome Questionnaire. RESULTS: Follow-up data were available for 18 patients (90%). Mean follow-up was 58 months (25 to 102). There were 10 men and 8 women. Mean age was 45 years (26 to 72). Diagnoses were degenerative discs, herniated nucleus pulposus, spondylolisthesis, and spinal stenosis. Eight patients had injuries after the previous posterolateral spinal fusion that precipitated new symptoms. Two patients had one level fusion, 14 had two levels, and 1 each had three and four levels. Four patients had one prior surgery, 5 had two, and 9 had three or more. All patients had solid anterior interbody fusion by radiograph. Mean Numerical Rating Scale improved from 7.9 before surgery to 4.7 after (P< 0.001). Mean Oswestry Disability Questionnaire improved from 56.3 before surgery to 47.9 after (P = 0.04). Of 15 patients unable to work before anterior interbody fusion, 5 returned to work. Sixteen patients (89%) were satisfied with their results. CONCLUSION: Low back pain that continues or recurs after apparently solid posterolateral spinal fusion may be caused by painful disc(s) at motion segment(s) within the fusion. A solid posterolateral spinal fusion may not protect the residual disc(s) from injury. Anterior interbody fusion can provide significant improvements in pain and function and a high degree of patient satisfaction in this clinical setting. PMID- 10751298 TI - Can insertional torque predict screw loosening and related failures? An in vivo study of pedicle screw fixation augmenting posterior lumbar interbody fusion. AB - STUDY DESIGN: An investigation of the relation between intraoperative insertional torque of pedicle screws, bone mineral density of the vertebra, and development of screw loosening in vivo. OBJECTIVES: To determine the usefulness of intraoperative measurement of the insertional torque of pedicle screws. SUMMARY OF BACKGROUND DATA: Some biomechanical studies have demonstrated that the insertional torque is highly correlated with bone mineral density and the stability of pedicle screws in vitro. METHODS: Pedicle screw fixation was performed with posterior lumbar interbody fusion in 62 consecutive patients. The mean age of the patients at the time of surgery was 58 years. The insertional torque of pedicle screws was measured intraoperatively in all patients. The mean follow-up period was 2.7 years. RESULTS: The mean insertional torque was 1.28 +/- 0.37 Nm in patients with screw loosening and 1.50 +/- 0. 40 Nm in patients without the problem. The mean insertional torque in patients with compression fractures in the upper vertebra adjacent to the fixed segment was 0.83 +/- 0.23 Nm. There was no significant difference between the mean insertional torque in patients with screw loosening and those without the condition. The mean insertional torque in patients without screw loosening was significantly greater than in patients with compression fractures (P < 0.01). A high correlation was found between insertional torque and bone mineral density (P < 0.01). CONCLUSIONS: Although a high correlation was found between the insertional torque of pedicle screws and bone mineral density in vivo, the insertional torque could not objectively predict screw loosening. PMID- 10751299 TI - Patient outcomes after minimally destabilizing lumbar stenosis decompression: the "Port-Hole" technique. AB - STUDY DESIGN: A prospective evaluation of the outcome of a decompressive procedure for lumbar spinal stenosis designed to preserve spinal stability. OBJECTIVES: To determine whether decompression could be achieved without subsequent fusion for spinal stenosis with and without degenerative spondylolisthesis. SUMMARY OF BACKGROUND DATA: The traditional surgical decompression of spinal stenosis involves removal of the posterior elements. Success occurs in 64% of cases, on the average, with results deteriorating over time. Concomitant spinal fusion is associated with higher costs and complication rates. METHODS: This prospective study included 54 consecutive patients treated surgically by one surgeon. Patients were contacted 21/2 and 4 years, on the average, after surgery. Patients with spondylolisthesis were evaluated for worsening of the listhesis after surgery. RESULTS: At a mean of 4 years after surgery, all patients were satisfied with their treatment. Concerning their symptoms, 80% reported relief of back pain; 96% had improvement of leg pain; 93% experienced relief of leg numbness; and 97% had relief of lower extremity weakness. Before surgery, only 1 patient could walk for longer than 15 minutes. After surgery, 98% (47/48) could walk for more than 15 minutes. Overall clinical results were graded as good to excellent (88%), fair (8%), or poor (4%). Clinical outcomes were comparable between those with and without degenerative spondylolisthesis (P = 0.08). Patients with degenerative spondylolisthesis showed no change in the amount of slip in 13/15 patients (87%). CONCLUSIONS: Degenerative spinal stenosis, even with nonlytic spondylolisthesis, can be decompressed effectively without violating the integrity of the posterior elements. PMID- 10751301 TI - A new system for the anterior restoration and fixation of thoracic spinal deformities using an endoscopic approach. AB - STUDY DESIGN: A report on the results of animal experiments in which the authors' new system that enables the anterior correction and fixation of thoracic spinal deformity by use of a thoracic endoscope and small incisions was used. The results suggest that the new approach involving thoracoscopic anterior correction and fusion for thoracic spinal deformity could be adapted successfully for the clinic. OBJECTIVE: To develop a new system that enables the anterior correction and fixation of thoracic spinal deformity by use of a thoracic endoscope and small incisions. SUMMARY OF BACKGROUND DATA: Anterior correction and fusion through open thoracotomy have been applied mainly for thoracic scoliosis because this approach provides effective correction with short fusion. However, excellent visualization of the spine during thoracic surgical procedures led to the development of thoracoscopic spine surgery. Therefore, the authors postulated that a thoracoscopic approach could allow not only discectomy but also correction and fusion of the deformed thoracic spine in a single surgical event. METHODS: The vertebral columns and attached thoracic walls were dissected from fresh porcine cadavers and used in the experimental surgery to evaluate the new system. As a next step, thoracoscopic surgery using this new system was performed on four or five vertebrae of five live pigs with an average weight of 50 kg. Initially, the discs and rib heads were removed through the thoracic wall. For each vertebra, a screw connected to a shaft used as a screw holder was inserted through the thoracic wall in a posterior to anterior direction. Each shaft then was linked outside the body to an outrigger. This outrigger was used to both create and restore scoliosis. A rod then was introduced through the thoracic wall and fixed to the screw heads. RESULTS: The animal experiments clearly showed that it was possible to change the Cobb angles of the spine through the use of the outrigger apparatus. In cadaver experiments, it was possible to create scoliosis and re-store it by 25 degrees to 35 degrees. Also, surgery on live pigs resulted in scoliosis of approximately 30 degrees, which means approximately 5 degrees to 10 degrees for each disc space. The procedures used also demonstrated that it was possible to fix a rod, introduced into the pleural cavity through a port, with screw heads. CONCLUSIONS: The use of this system successfully changed the Cobb angle of the spine, which suggests that its use should make it possible to correct spinal deformities. This apparatus also succeeded in fixing the rods in the screw heads, which raises the possibility of its use in spinal fixation. The authors believe that this apparatus could be adapted successfully for clinical use. Studies currently are under way in clinics using the new system. PMID- 10751300 TI - Influence of macrophage infiltration of herniated lumbar disc tissue on outcome after lumbar disc surgery. AB - STUDY DESIGN: An immunohistochemical examination of the presence of inflammatory cells in routinely processed resection specimens of the lumbar disc, and a comparison of the histologic results with clinical data collected prospectively before and after surgery. OBJECTIVES: To assess the influence of inflammatory reactions in herniated lumbar disc specimens on the outcome after lumbar disc surgery. SUMMARY OF BACKGROUND DATA: Histologic and biochemical studies on herniated lumbar disc tissue led to the notion of inflammation-induced sciatic pain. At this writing, no investigations have sought to discover how outcome after lumbar disc surgery is influenced by histologically described inflammation. METHODS: Disc specimens from 79 patients who underwent surgery for lumbar disc herniation were studied immunohistologically with regard to the presence of inflammatory reactions. Of these, 92% were followed up approximately 7 months after surgery. The histologic results were compared with the outcome at follow-up evaluation. RESULTS: A statistically significant correlation was found between the histologically proven inflammation and the outcome, as shown by the pain grading scale. CONCLUSIONS: The results from this study seem to support the theory of a foreign body reaction to the herniated material. This reaction may result in inflammation-induced sciatic pain. PMID- 10751302 TI - Lumbosacral radiculopathy secondary to metastatic uterine leiomyosarcoma: a case report. AB - STUDY DESIGN: A retrospective case report. OBJECTIVES: To increase awareness of the fact that very serious and potentially devastating conditions can be associated with lumbosacral radiculopathy. To reinforce the need to have a definitive diagnosis before performing epidural injections in patients with radicular pain who are not responsive to conservative treatment. SUMMARY OF BACKGROUND DATA: To the authors' knowledge, this is the first reported case of uterine leiomyosarcoma presenting with a lumbosacral radiculopathy. METHODS: The authors describe the treatment and the radiologic, surgical, and pathologic findings in this patient. RESULTS: Proper diagnostic work-up led to a diagnosis of metastatic uterine leiomyosarcoma, which was managed with decompressive laminectomy, radiotherapy, and chemotherapy. CONCLUSIONS: This is the first reported case of a uterine leiomyosarcoma presenting with radicular pain. When a patient has not responded to conservative care, a definitive etiology for radiculopathy needs to be established before epidural steroid injection. PMID- 10751303 TI - Concomitant post-traumatic craniocervical junction epidural hematoma and pontomedullary junction infarction: clinical, neurophysiologic, and neuroradiologic features. AB - STUDY DESIGN: A case report. OBJECTIVES: To report and discuss a case of post traumatic epidural hematoma of the craniocervical junction with concomitant brain stem infarction. SUMMARY OF BACKGROUND DATA: Post-traumatic epidural hematoma of the cervical spine and brain stem post-traumatic infarction are very rare disorders. Post-traumatic epidural hematoma is usually located dorsally in the epidural space. METHODS: The clinical, neuroradiologic, and neurophysiologic findings in one patient with post-traumatic epidural hematoma located ventrally at the cervicomedullary junction and associated with medial infarction at the pontomedullary junction are reported. RESULTS: The main clinical finding in this patient was bilateral corticospinal and corticobulbar tract involvement. A magnetic resonance image showed displacement and flattening of the medulla oblongata and of the most cranial portion of cervical cord, which were caused by the epidural hematoma associated with an ischemic lesion of the pontomedullary junction. Results of central motor conduction studies indicated that the abnormality of the central motor pathways was localized at brain stem level, and that there was normal conduction from the cervicomedullary junction to spinal cord. CONCLUSION: This is the first reported case of spinal epidural hematoma located ventrally in the cervical spine at the cervicomedullary junction level and concomitant infarction at the pontomedullary junction resulting from whiplash injury. PMID- 10751304 TI - Bending of the Cotrel-Dubousset instrumentation after direct trauma: a case report. AB - STUDY DESIGN: Case report. OBJECTIVES: To describe a fracture through the fusion mass of a spine that had been corrected previously with Cotrel-Dubousset rods. These rods had failed in bending after direct trauma. SUMMARY OF BACKGROUND DATA: Nine years after successful treatment of scoliosis with Cotrel-Dubousset instrumentation, the patient was in a motor vehicle accident and sustained a hyperextension spine injury with complete L1-L2 paraplegia and disruption of the fusion mass. The Cotrel-Dubousset instrumentation rods, which failed in bending, could not be corrected in situ, and the angulated segments had to be resected. The spine then became extremely unstable, and the patient consulted the authors for definitive stabilization. RESULTS: The spine was stabilized by attaching the proximal and distal retained Cotrel-Dubousset instrumentation to supplemental rods in a "domino" fashion. Crosslinks were added to improve the torsional stability. Intraoperatively, the fracture was well reduced, and the fixation was stable. A posterolateral fusion was performed with allogenic bone graft. CONCLUSION: Bent Cotrel-Dubousset instrumentation rods are still very strong and may not correct in situ.- If resection is required, the retained portions of Cotrel-Dubousset instrumentation can serve as attachments to restore stable fixation a "domino"technique. PMID- 10751306 TI - Imagery PMID- 10751305 TI - Iatrogenic spondylolysis leading to contralateral pedicular stress fracture and unstable spondylolisthesis: a case report. AB - STUDY DESIGN: A case report of iatrogenic spondylolysis as a complication of microdiscectomy leading to contralateral pedicular stress fracture and unstable spondylolisthesis. OBJECTIVE: To improve understanding of this condition by presenting a case history and roentgenographic findings of a patient that differ from those already reported and to propose an effective method of surgical management. METHODS: A 67-year-old woman with no history of spondylolysis or spondylolisthesis underwent an L4-L5 microdiscectomy for a left herniated nucleus pulposus 1 year before the current consultation. For the preceding 8 months, she had been experiencing low back and bilateral leg pain. Imaging studies revealed a left L4 spondylolytic defect and a right L4 pedicular stress fracture with an unstable Grade I spondylolisthesis. RESULTS: The patient was treated with posterior spinal fusion, which resulted in complete resolution of her clinical and neurologic symptoms. CONCLUSIONS: Iatrogenic spondylolysis after microdiscectomy is an uncommon entity. However, it can lead to contralateral pedicular stress fracture and spondylolisthesis, and thus can be a source of persistent back pain after disc surgery. Surgeons caring for these patients should be aware of this potential complication. PMID- 10751307 TI - Identification of a fourth subunit of mammalian DNA polymerase delta. AB - A 12-kDa and two 25-kDa polypeptides were isolated with highly purified calf thymus DNA polymerase delta by conventional chromatography. A 16-mer peptide sequence was obtained from the 12-kDa polypeptide which matched a new open reading frame from a human EST () encoding a hypothetical protein of unknown function. The protein was designated as p12. Human EST was identified as the putative human homologue of Schizosaccharomyces pombe Cdm1 by a tBlastn search of the EST data base using S. pombe Cdm1. The open reading frame of human EST encoded a polypeptide of 107 amino acids with a predicted molecular mass of 12.4 kDa, consistent with the experimental findings. p12 is 25% identical to S pombe Cdm1. Both of the 25-kDa polypeptide sequences matched the hypothetical KIAA0039 protein sequence, recently identified as the third subunit of pol delta. Western blotting of immunoaffinity purified calf thymus pol delta revealed the presence of p125, p50, p68 (the KIAA0039 product), and p12. With the identification of p12 mammalian pol delta can now be shown to consist of four subunits. These studies pave the way for more detailed analysis of the possible functions of the mammalian subunits of pol delta. PMID- 10751308 TI - Editorial Page. PMID- 10751310 TI - "It's ugly, but there it is." PMID- 10751311 TI - Age- and tissue-specific induction of NHE3 by glucocorticoids in the rat small intestine. AB - Of the two known apical isoforms of the Na(+)/H(+) exchanger (NHE) family, only the NHE3 gene is regulated by glucocorticoids. The aim of these studies was to investigate the mechanisms underlying the effects of methylprednisolone (MP) on expression of NHE3 in the proximal and distal small intestine of suckling and adult rats. Immunoblots showed that the glucocorticoid responsiveness in the proximal small intestine was greatest in suckling animals (NHE3/beta-actin: 0.43 +/- 0.09 control vs. 1.57 +/- 0.15 MP; P < 0. 001), and responsiveness decreased with age with no effect in adults (0.56 +/- 0.14 vs. 0.64 +/- 0.17). Distal small intestine was responsive only in adult rats (0.49 +/- 0.13 vs. 1.65 +/- 0.09; P < 0.001). This pattern was confirmed at the mRNA level and by (22)Na(+) uptake. Western blot and [(3)H]dexamethasone mesylate binding showed that the responsiveness of NHE3 to glucocorticoids is directly related to the expression of glucocorticoid receptor (GR) in the small intestine. These studies suggest that loss and gain of glucocorticoid responsiveness in the proximal and distal small intestine, respectively, are related to age- and segment-dependent expression of GR. PMID- 10751312 TI - Potentiation of rat brain sodium channel currents by PKA in Xenopus oocytes involves the I-II linker. AB - Functional modulation of voltage-gated sodium channels affects the electrical excitability of neurons. Protein kinase A (PKA) can decrease sodium currents by phosphorylation at consensus sites in the cytoplasmic I-II linker. Once the sites are phosphorylated, however, additional PKA activity can increase sodium currents by an unknown mechanism. When the PKA sites were eliminated by substitutions of alanine for serine, peak sodium current amplitudes were increased by 20-80% when PKA was activated in Xenopus oocytes either by stimulation of a coexpressed beta(2)-adrenergic receptor or by perfusion with reagents that increase cAMP. Potentiation required the I-II linker of the brain channel, in that a chimeric channel in which the brain linker was replaced with the comparable linker from the skeletal muscle channel did not demonstrate potentiation. Using a series of chimeric and deleted channels, we demonstrate that potentiation is not dependent on any single region of the linker and that the extent of potentiation varies depending on the total length and the residues throughout the linker. These data are consistent with the hypothesis that potentiation by PKA is an indirect process involving phosphorylation of an accessory protein that interacts with the I-II linker of the sodium channel. PMID- 10751313 TI - Eicosatetraynoic and eicosatriynoic acids, lipoxygenase inhibitors, block meiosis via antioxidant action. AB - We previously showed that nordihydroguaiaretic acid (NDGA) and other antioxidants inhibit the resumption of meiosis in oocyte-cumulus complexes (OCC) and denuded oocytes (DO). Because NDGA is well known to be an inhibitor of lipoxygenases (LOX), we assessed whether other LOX inhibitors influence spontaneous germinal vesicle breakdown (GVBD) in OCC and DO. Spontaneous GVBD in rat OCC obtained from preovulatory follicles was significantly and reversibly inhibited by the minimum effective doses of 80 and 100 microM 5,8,11, 14-eicosatetraynoic acid (ETYA) and 5,8,11-eicosatriynoic acid (ETI), respectively. In DO, GVBD was significantly inhibited by 100 microM ETYA or ETI. The minimum effective concentrations of ETYA and ETI for inhibition of GVBD in either OCC or DO are approximately 30- to 50 fold higher than the concentrations necessary to inhibit LOX activity by 50% in intact cells. Because we previously showed that NDGA and other antioxidants inhibit the spontaneous resumption of meiosis, we assessed whether ETYA and ETI may act similarly as scavengers of reactive oxygen species (ROS). Luminol amplified chemiluminescence showed that 50 microM of either ETYA or ETI markedly and significantly reduced ROS generated with 10 mM 2, 2'-azobis(2 amidinopropane)dihydrochloride (AAPH). Moreover, incubation of DO with 30 mM AAPH reversed the inhibition of GVBD produced by 100 microM ETYA or ETI. These findings support the conclusion that ETYA and ETI inhibit oocyte maturation by acting as antioxidants rather than by inhibiting LOX. PMID- 10751314 TI - Alternatively spliced isoforms of the rat eye sodium/calcium+potassium exchanger NCKX1. AB - We have investigated the structure, function, and expression of the rat eye sodium/calcium+potassium exchanger NCKX1. The sequence of independent rat NCKX1 clones and the analysis of rat eye mRNA by RT-PCR revealed a region of alternative splicing that comprised four exons and encoded a stretch of 113 amino acids near the beginning of the large cytosolic loop. In comparison with other NCKX1 molecules and the rat NCKX2 protein, rat NCKX1 was highly conserved within the hydrophobic regions but was quite divergent in the two large hydrophilic loops. The only exception was the region of the cytosolic loop encoded by the second alternatively spliced exon, which was approximately 60% identical. Similar to bovine, but different from human, rat NCKX1 possessed an acidic motif that was repeated 14 times in the cytoplasmic loop. Analysis of NCKX1 expression in different rat tissues by Northern blot revealed a very high level of expression of a 7-kb transcript in the eye but also lower levels of transcripts of various lengths in other tissues. The recombinant rat NCKX1 protein was tagged in the extracellular loop with the FLAG epitope and expressed in HEK-293 cells. Surface delivery and potassium-dependent sodium/calcium exchange activity were observed for each spliced variant. PMID- 10751315 TI - Interaction of PIP(2) with the XIP region of the cardiac Na/Ca exchanger. AB - The sarcolemmal Na/Ca exchanger undergoes an inactivation process in which exchange activity decays over several seconds following activation by the application of Na to the intracellular surface of the protein. Inactivation is eliminated by an increase in membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)). Inactivation is also strongly affected by mutations to a basic 20-amino acid segment of the exchanger known as the endogenous XIP region. The hypothesis that PIP(2) directly interacts with the XIP region of the exchanger was tested. First, we investigated the ability of a peptide with the same sequence as the XIP region to bind to immobilized phospholipid vesicles. (125)I-labeled XIP bound avidly to vesicles containing only a low concentration (<3%) of PIP(2). The binding was specific, in that binding was not displaced by other basic peptides. The effects of altering the sequence of XIP peptides also indicated binding specificity. Second, we examined the functional response to PIP(2) of exchangers with mutated XIP regions. Outward Na/Ca exchange currents were measured using the giant excised patch technique. The mutated exchangers either had no inactivation or accelerated inactivation. In both cases, the exchangers no longer responded to PIP(2) or to PIP(2) antibodies. Overall, the data indicate that the affinity of the endogenous XIP region for PIP(2) is an important determinant of the inactivation process. PMID- 10751316 TI - Ca(2+)-dependent activation of Cl(-) currents in Xenopus oocytes is modulated by voltage. AB - Ca(2+)-activated Cl(-) currents (I(Cl,Ca)) were examined using fluorescence confocal microscopy to monitor intracellular Ca(2+) liberation evoked by flash photolysis of caged inositol 1,4, 5-trisphosphate (InsP(3)) in voltage-clamped Xenopus oocytes. Currents at +40 mV exhibited a steep dependence on InsP(3) concentration ([InsP(3)]), whereas currents at -140 mV exhibited a higher threshold and more graded relationship with [InsP(3)]. Ca(2+) levels required to half-maximally activate I(Cl,Ca) were about 50% larger at -140 mV than at +40 mV, and currents evoked by small Ca(2+) elevations were reduced >25-fold. The half decay time of Ca(2+) signals shortened at increasingly positive potentials, whereas the decay of I(Cl,Ca) lengthened. The steady-state current-voltage (I-V) relationship for I(Cl,Ca) exhibited outward rectification with weak photolysis flashes but became more linear with stronger stimuli. Instantaneous I-V relationships were linear with both strong and weak stimuli. Current relaxations following voltage steps during activation of I(Cl,Ca) decayed with half-times that shortened from about 100 ms at +10 mV to 20 ms at -160 mV. We conclude that InsP(3)-mediated Ca(2+) liberation activates a single population of Cl(-) channels, which exhibit voltage-dependent Ca(2+) activation and voltage independent instantaneous conductance. PMID- 10751317 TI - Malignant gliomas display altered pH regulation by NHE1 compared with nontransformed astrocytes. AB - Malignant gliomas exhibit alkaline intracellular pH (pH(i)) and acidic extracellular pH (pH(e)) compared with nontransformed astrocytes, despite increased metabolic H(+) production. The acidic pH(e) limits the availability of HCO(-)(3), thereby reducing both passive and dynamic HCO(-)(3)-dependent buffering. This implies that gliomas are dependent upon dynamic HCO(-)(3) independent H(+) buffering pathways such as the type 1 Na(+)/H(+) exchanger (NHE1). In this study, four rapidly proliferating gliomas exhibited significantly more alkaline steady-state pH(i) (pH(i) = 7.31-7.48) than normal astrocytes (pH(i) = 6.98), and increased rates of recovery from acidification, under nominally CO(2)/HCO(-)(3)-free conditions. Inhibition of NHE1 in the absence of CO(2)/HCO(-)(3) resulted in pronounced acidification of gliomas, whereas normal astrocytes were unaffected. When suspended in CO(2)/HCO(-)(3) medium astrocyte pH(i) increased, yet glioma pH(i) unexpectedly acidified, suggesting the presence of an HCO(-)(3)-dependent acid loading pathway. Nucleotide sequencing of NHE1 cDNA from the gliomas demonstrated that genetic alterations were not responsible for this altered NHE1 function. The data suggest that NHE1 activity is significantly elevated in gliomas and may provide a useful target for the development of tumor-selective therapies. PMID- 10751318 TI - Involvement of estrogen receptors alpha and beta in the regulation of cervical permeability. AB - Estrogen increases the permeability of cultured human cervical epithelia (Gorodeski, GI. Am J Physiol Cell Physiol 275: C888-C899, 1998), and the effect is blocked by the estrogen receptor modulators ICI-182780 and tamoxifen. The objective of the study was to determine involvement of estrogen receptor(s) in mediating the effects on permeability. In cultured human cervical epithelial cells estradiol binds to high-affinity, low-capacity sites, in a specific and saturable manner. Scatchard analysis revealed a single class of binding sites with a dissociation constant of 1.3 nM and binding activity of approximately 0.5 pmol/mg DNA. Estradiol increased the density of estrogen-binding sites in a time- and dose-related manner (half time approximately 4 h, and EC(50) approximately 1 nM). RT-PCR assays revealed the expression of mRNA for the estrogen receptor alpha (alphaER) and estrogen receptor beta (betaER). Removal of estrogen from the culture medium decreased and treatment with estrogen increased the expression of alphaER and betaER mRNA. In cells not treated with estrogen, ICI-182780 and tamoxifen increased betaER mRNA. In cells treated with estrogen, neither ICI 182780 nor tamoxifen had modulated significantly the increase in alphaER or betaER mRNA. The transcription inhibitor actinomycin D blocked the estrogen induced increase in permeability, and it abrogated the estradiol-induced increase in estrogen binding sites. These results suggest that the estrogen-dependent increase in cervical permeability is mediated by an alphaER-dependent increase in transcription. PMID- 10751320 TI - MHC polymorphism in rodent plantaris muscle: effects of mechanical overload and hypothyroidism. AB - In a previous study, it was shown that a combined treatment of hyperthyroidism and hindlimb suspension effectively converted the slow-twitch soleus muscle to a fast-twitch muscle. The objective of this study was to test the hypothesis that hypothyroidism [absence of triiodothyronine (-T(3))] and mechanical overload (OV) would convert the plantaris (Plan) muscle from a fast- to a slow-twitch muscle. Single-fiber analyses demonstrated that the normal rodent Plan muscle was composed of approximately 13 different fiber types as defined by myosin heavy chain (MHC) isoform content. The largest proportion of fibers ( approximately 35%) coexpressed the fast type IIX and IIB MHC isoforms (i.e., type IIX/IIB fibers). In this context, the combined intervention of -T(3) and OV produced a significant reduction in the relative proportion of the fast type IIB MHC isoform and a concomitant increase in the slow type I MHC isoform. These transitions were manifested by a large decrease in the proportion of type IIX/IIB fibers and a large increase in fibers coexpressing all four MHC protein isoforms. The mechanical consequences of these transitions, however, were modest, producing a 15% decrease in maximal shortening velocity. The findings of this study demonstrate that -T(3) + OV does produce a partial shift toward a slower phenotype; however, the high degree of polymorphism found in the Plan muscle represents a unique design that appears to minimize the functional consequences of these significant MHC transitions. PMID- 10751321 TI - Phosphorylation of caldesmon by ERK MAP kinases in smooth muscle. AB - Phosphorylation of h-caldesmon has been proposed to regulate airway smooth muscle contraction. Both extracellular signal-regulated kinase (ERK) and p38 mitogen activated protein (MAP) kinases phosphorylate h-caldesmon in vitro. To determine whether both enzymes phosphorylate caldesmon in vivo, phosphorylation-site selective antibodies were used to assay phosphorylation of MAP kinase consensus sites. Stimulation of cultured tracheal smooth muscle cells with ACh or platelet derived growth factor increased caldesmon phosphorylation at Ser789 by about twofold. Inhibiting ERK MAP kinase activation with 50 microM PD-98059 blocked agonist-induced caldesmon phosphorylation completely. Inhibiting p38 MAP kinases with 25 microM SB-203580 had no effect on ACh-induced caldesmon phosphorylation. Carbachol stimulation increased caldesmon phosphorylation at Ser789 in intact tracheal smooth muscle, which was blocked by the M(2) antagonist AF-DX 116 (1 microM). AF-DX 116 inhibited carbachol-induced isometric contraction by 15 +/- 1.4%, thus dissociating caldesmon phosphorylation from contraction. Activation of M(2) receptors leads to activation of ERK MAP kinases and phosphorylation of caldesmon with little or no functional effect on isometric force. P38 MAP kinases are also activated by muscarinic agonists, but they do not phosphorylate caldesmon in vivo. PMID- 10751319 TI - EGF stimulates gastrin promoter through activation of Sp1 kinase activity. AB - Epidermal growth factor (EGF) receptor activation stimulates gastrin gene expression through a GC-rich element called gastrin EGF response element (gERE). This element is bound by Sp1 family members and is a target of the ras extracellular signal-regulated kinase (Erk) signal transduction cascade. This raised the possibility that Sp1 may be phosphorylated by kinases of this signaling pathway. Erk is capable of phosphorylating other mitogen-inducible transcription factors, e.g., Elk and Sap, suggesting that Erk may also mediate EGF-dependent phosphorylation of Sp1. This possibility was tested by studying Sp1 dependent kinase activity in extracts prepared from EGF-activated AGS cells by use of solid-phase kinase assays and immunoprecipitation of metabolically labeled Sp1. The results revealed that Sp1 kinase activity (like gastrin promoter activation) is inhibited by PD-98059 and, therefore, is dependent on mitogen activated protein kinase kinase 1 (Mek 1). However, EGF-dependent activation of endogenous Erk did not account for most of the Sp1 kinase activity, since Erk and additional Sp1 kinase activity analyzed in a solid-phase kinase assay eluted from an ion-exchange column in different fractions. Phosphoamino acid analysis of in vivo radiolabeled Sp1 demonstrated that the kinase phosphorylates Sp1 on Ser and Thr in response to EGF. Therefore, most EGF-stimulated Sp1 kinase activity is Mek 1 dependent and distinct from Erk. PMID- 10751323 TI - Cellular basis for contractile dysfunction in the diaphragm from a rabbit infarct model of heart failure. AB - Abnormal respiratory muscle function is thought to contribute to breathlessness and exercise intolerance in heart failure but little is known about possible alterations in the function of such muscle. We have measured tetanic force and intracellular Ca(2+) concentration ([Ca(2+)](i)) in isolated, arterially perfused hemidiaphragm preparations from a rabbit coronary artery ligation model of heart failure. Increasing stimulation frequency (10-100 Hz) caused a progressive increase of force and [Ca(2+)](i) in control preparations, whereas force and [Ca(2+)](i) only increased between 10 and 25 Hz stimulation (decreasing at higher frequencies) in preparations from ligated animals. Cyclopiazonic acid produced a dose-dependent shift in the relationship between stimulation frequency and [Ca(2+)](i) in control preparations that was similar to the shift observed in the diaphragm of coronary-ligated animals. These data indicate that the in vitro contractile characteristics of the diaphragm are significantly altered in our model and that altered [Ca(2+)](i) regulation contributes to the reduced diaphragm strength observed in heart failure. PMID- 10751322 TI - Interaction of alpha- and beta-subunits in native H-K-ATPase and cultured cells transfected with H-K-ATPase beta-subunit. AB - The assembly of the beta-subunit of the gastric H-K-ATPase (HKbeta) with the alpha-subunit of the H-K-ATPase or the Na-K-ATPase (NaKalpha) was characterized with two anti-HKbeta monoclonal antibodies (MAbs). In fixed gastric oxyntic cells, in H-K-ATPase in vitro, and in Madin-Darby canine kidney (MDCK) cells transfected with HKbeta, MAb 2/2E6 was observed to bind to HKbeta only when interactions between alpha- and beta-subunits were disrupted by various denaturants. The epitope for MAb 2/2E6 was mapped to the tetrapeptide S(226)LHY(229) of the extracellular domain of HKbeta. The epitope for MAb 2G11 was mapped to the eight NH(2)-terminal amino acids of the cytoplasmic domain of HKbeta. In transfected MDCK cells, MAb 2G11 could immunoprecipitate HKbeta with alpha-subunits of the endogenous cell surface NaKalpha, as well as that from early in the biosynthetic pathway, whereas MAb 2/2E6 immunoprecipitated only a cohort of unassembled endoglycosidase H-sensitive HKbeta. In HKbeta-transfected LLC-PK(1) cells, significant immunofluorescent labeling of HKbeta at the cell surface could be detected without postfixation denaturation or in live cells, although a fraction of transfected HKbeta could also be coimmunoprecipitated with NaKalpha. Thus assembly of HKbeta with NaKalpha does not appear to be a stringent requirement for cell surface delivery of HKbeta in LLC-PK(1) cells but may be required in MDCK cells. In addition, endogenous posttranslational regulatory mechanisms to prevent hybrid alpha-beta heterodimer assembly appear to be compromised in transfected cultured renal epithelial cells. Finally, the extracellular epitope for assembly-sensitive MAb 2/2E6 may represent a region of HKbeta that is associated with alpha-beta interaction. PMID- 10751324 TI - Regulation of mitochondrial respiration in heart cells analyzed by reaction diffusion model of energy transfer. AB - The purpose of this study is to investigate theoretically which intracellular factors may be important for regulation of mitochondrial respiration in working heart cells in vivo. We have developed a model that describes quantitatively the published experimental data on dependence of the rate of oxygen consumption and metabolic state of working isolated perfused rat heart on workload over its physiological range (Williamson JR, Ford G, Illingworth J, Safer B. Circ Res 38, Suppl I, I39-I51, 1976). Analysis of this model shows that for phosphocreatine, creatine, and ATP the equilibrium assumption is an acceptable approximation with respect to their diffusion in the intracellular bulk water phase. However, the ADP concentration changes in the contraction cycle in a nonequilibrium workload dependent manner, showing the existence of the intracellular concentration gradients. The model shows that workload-dependent alteration of ADP concentration in the compartmentalized creatine kinase system may be taken, together with the changes in P(i) concentration, to be among the major components of the metabolic feedback signal for regulation of respiration in muscle cells. PMID- 10751325 TI - Proliferative activity and tumorigenic conversion: impact on cellular metabolism in 3-D culture. AB - Oxygen consumption, glucose, lactate, and ATP concentrations, as well as glucose and lactate turnover rates, have been studied in a three-dimensional carcinogenesis model of differently transformed rat embryo fibroblasts (spontaneously immortalized Rat1 and myc-transfected M1, and the ras-transfected, tumorigenic descendants Rat1-T1 and MR1) to determine metabolic alterations that accompany tumorigenic conversion. Various bioluminescence techniques, thymidine labeling, measurement of PO(2) distributions with microelectrodes, and determination of cellular oxygen uptake rates (Qc(O(2))) have been applied. In the ras-transfected, tumorigenic spheroid types, the size dependencies of some of the measured parameters exhibited sharp breaks at diameters of approximately 830 microm for Rat1-T1 and approximately 970 microm for MR1 spheroids, respectively, suggesting that some fundamental change in cell metabolism occurred at these characteristic diameters (denoted as "metabolic switch"). Qc(O(2)) decreased and lactate concentration increased as functions of size below the characteristic diameters. Concomitantly, glucose and lactate turnover rates decreased in MR1 spheroids and increased in Rat1-T1. Spheroids larger than the characteristic diameters (exhibiting cell quiescence and lactate accumulation) showed an enhancement of Qc(O(2)) with size. Systematic variations in the ATP and glucose levels in the viable cell rim were observed for Rat1-T1 spheroids only. Proliferative activity, Qc(O(2)), and ATP levels in small, nontumorigenic Rat1 and M1 aggregates did not differ systematically from those recorded in the largest spheroids of the corresponding ras transfectants. Unexpectedly, respiratory activity was present not only in viable but also in the morphologically disintegrated core regions of M1 aggregates. Our data suggest that myc but not ras transfection exerts major impacts on cell metabolism. Moreover, some kind of switch has been detected that triggers profound readjustment of tumor cell metabolism when proliferative activity begins to stagnate, and that is likely to initiate some other, yet unidentified energy consuming process. PMID- 10751326 TI - Mxi2, a splice variant of p38 stress-activated kinase, is a distal nephron protein regulated with kidney ischemia. AB - Mxi2 is one of three known alternative spliced forms of the stress-activated mitogen-activated protein kinase p38 (CSBP). Mxi2 was originally identified as a Max-interacting protein and is the smallest member of the family of stress activated kinases isolated to date. Mxi2 lacks most of the XI domain found in p38 and instead has a distinct COOH-terminal sequence of 17 amino acids. Here we present the genomic structure of the Mxi2/p38 locus on human chromosome 6q21.2/21.3 and establish the origin of the three spliced forms of p38. Using Mxi2-specific antibodies in mouse organs, we found the Mxi2 protein to be present exclusively in the kidney. Mxi2 is present predominantly in the distal tubule of the nephron and the level of the protein decreased during kidney ischemia reperfusion. Stress signals or other known activators of the p38 pathway including MAP kinase-kinase 3 and MAP kinase-kinase 6 did not induce the kinase activity of Mxi2 using ATF-2 as a substrate. With the use of hybrid proteins encoding different portions of Mxi2 and p38 polypeptides, the different properties of Mxi2 can be assigned to its unique COOH terminus. PMID- 10751327 TI - Aquaporin 2 is a vasopressin-independent, constitutive apical membrane protein in rat vas deferens. AB - Aquaporin 2 (AQP2), the vasopressin-regulated water channel, was originally identified in renal collecting duct principal cells. However, our recent description of AQP2 in the vas deferens indicated that this water channel may have extra-renal functions, possibly related to sperm concentration in the male reproductive tract. In this study, we have examined the regulation and membrane insertion pathway of AQP2 in the vas deferens. The amino acid sequence of vas deferens AQP2 showed 100% identity to the renal protein. AQP2 was highly expressed in the distal portion (ampulla) of the vas deferens, but not in the proximal portion nearest the epididymis. It was concentrated on the apical plasma membrane of vas deferens principal cells, and very little was detected on intracellular vesicles. Protein expression levels and cellular localization patterns were similar in normal rats and vasopressin-deficient Brattleboro homozygous rats, and were not changed after 36 h of dehydration, or after 3 days of vasopressin infusion into Brattleboro rats. AQP2 was not found in apical endosomes (labeled with Texas Red-dextran) in vas deferens principal cells, indicating that it is not rapidly recycling in this tissue. Finally, vasopressin receptors were not detectable on vas deferens epithelial cell membranes using a [(3)H]vasopressin binding assay. These data indicate that AQP2 is a constitutive apical membrane protein in the vas deferens, and that it is not vasopressin regulated in this tissue. Thus AQP2 contains targeting information that can be interpreted in a cell-type-specific fashion in vivo. PMID- 10751328 TI - Sorting of metabolic pathway flux by the plasma membrane in cerebrovascular smooth muscle cells. AB - We used beta-escin-permeabilized pig cerebral microvessels (PCMV) to study the organization of carbohydrate metabolism in the cytoplasm of vascular smooth muscle (VSM) cells. We have previously demonstrated (Lloyd PG and Hardin CD. Am J Physiol Cell Physiol 277: C1250-C1262, 1999) that intact PCMV metabolize the glycolytic intermediate [1-(13)C]fructose 1,6-bisphosphate (FBP) to [1 (13)C]glucose with negligible production of [3-(13)C]lactate, while simultaneously metabolizing [2-(13)C]glucose to [2-(13)C]lactate. Thus gluconeogenic and glycolytic intermediates do not mix freely in intact VSM cells (compartmentation). Permeabilized PCMV retained the ability to metabolize [2 (13)C]glucose to [2-(13)C]lactate and to metabolize [1-(13)C]FBP to [1 (13)C]glucose. The continued existence of glycolytic and gluconeogenic activity in permeabilized cells suggests that the intermediates of these pathways are channeled (directly transferred) between enzymes. Both glycolytic and gluconeogenic flux in permeabilized PCMV were sensitive to the presence of exogenous ATP and NAD. It was most interesting that a major product of [1 (13)C]FBP metabolism in permeabilized PCMV was [3-(13)C]lactate, in direct contrast to our previous findings in intact PCMV. Thus disruption of the plasma membrane altered the distribution of substrates between the glycolytic and gluconeogenic pathways. These data suggest that organization of the plasma membrane into distinct microdomains plays an important role in sorting intermediates between the glycolytic and gluconeogenic pathways in intact cells. PMID- 10751329 TI - Folate deficiency reduces the GPI-anchored folate-binding protein in rat renal tubules. AB - A folate-binding protein (FBP) anchored to cell membranes by a glycosyl phosphatidylinositol (GPI) adduct is constitutively expressed in some transformed and cultured cell lines. Its expression is upregulated when these cells are grown in medium containing low folate, but whether this occurs in vivo with nutritional folate deficiency is unknown. To address this question, the GPI-FBP in the liver, kidney, and brain of rats on control and folate-deficient (FD) diets was measured. The GPI-FBP in the kidney of FD rats decreased significantly in contrast to the upregulation of this protein in cultured cells. Northern blot analysis and nuclear run-on assays indicated that transcription of the GPI-FBP gene in the kidney was not reduced by folate deficiency. This decrease of the GPI FBP appears to result from its proteolysis, similar to the enzymatic degradation of the apoprotein that occurs in vitro. Because the GPI-FBP is on the brush borders of the proximal renal tubules and provides for the reabsorption of folate, this function diminishes when the protein decreases in folate deficiency. PMID- 10751330 TI - Coordinate expression of secretory phospholipase A(2) and cyclooxygenase-2 in activated human keratinocytes. AB - PGE(2) levels are altered in human epidermis after in vivo wounding; however, mechanisms modulating PGE(2) production in activated keratinocytes are unclear. In previous studies, we showed that PGE(2) is a growth-promoting autacoid in human primary keratinocyte cultures, and its production is modulated by plating density, suggesting that regulated PGE(2) synthesis is an important component of wound healing. Here, we examine the role of phospholipase A(2) (PLA(2)) and cyclooxygenase (COX) enzymes in modulation of PGE(2) production. We report that the increased PGE(2) production that occurs in keratinocytes grown in nonconfluent conditions is also observed after in vitro wounding, indicating that similar mechanisms are involved. This increase was associated with coordinate upregulation of both COX-2 and secretory PLA(2) (sPLA(2)) proteins. Increased sPLA(2) activity was also observed. By RT-PCR, we identified the presence of type IIA and type V sPLA(2), along with the M-type sPLA(2) receptor. Thus the coordinate expression of sPLA(2) and COX-2 may be responsible for the increased prostaglandin synthesis in activated keratinocytes during wound repair. PMID- 10751331 TI - Gender-specific reduction in contractility and [Ca(2+)](i) in vascular smooth muscle cells of female rat. AB - The hypothesis that vascular protection in females and its absence in males reflects gender differences in [Ca(2+)](i) and Ca(2+) mobilization mechanisms of vascular smooth muscle contraction was tested in fura 2-loaded aortic smooth muscle cells isolated from intact and gonadectomized male and female Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. In WKY cells incubated in Hanks' solution (1 mM Ca(2+)), the resting length and [Ca(2+)](i) were significantly different in intact males (64.5 +/- 1.2 microm and 83 +/- 3 nM) than in intact females (76.5 +/- 1.5 microm and 64 +/- 7 nM). In intact male WKY, phenylephrine (Phe, 10(-5) M) caused transient increase in [Ca(2+)](i) to 428 +/- 13 nM followed by maintained increase to 201 +/- 8 nM and 32% cell contraction. In intact female WKY, the Phe-induced [Ca(2+)](i) transient was not significantly different, but the maintained [Ca(2+)](i) (159 +/- 7 nM) and cell contraction (26%) were significantly less than in intact male WKY. In Ca(2+)-free (2 mM EGTA) Hanks', Phe and caffeine (10 mM) caused transient increases in [Ca(2+)](i) and contraction that were not significantly different between males and females. Membrane depolarization by 51 mM KCl caused 31% cell contraction and increased [Ca(2+)](i) to 259 +/- 9 nM in intact male WKY, which were significantly greater than a 24% contraction and 214 +/- 8 nM [Ca(2+)](i) in intact female WKY. Maintained Phe- and KCl-stimulated cell contraction and [Ca(2+)](i) were significantly greater in SHR than WKY in all groups of rats. Reduction in cell contraction and [Ca(2+)](i) in intact females compared with intact males was significantly greater in SHR ( approximately 30%) than WKY ( approximately 20%). No significant differences in cell contraction or [Ca(2+)](i) were observed between castrated males, ovariectomized (OVX) females, and intact males, or between OVX females with 17beta-estradiol implants and intact females. Exogenous application of 17beta-estradiol (10(-8) M) to cells from OVX females caused greater reduction in Phe- and KCl-induced contraction and [Ca(2+)](i) in SHR than WKY. Thus the basal, maintained Phe- and depolarization-induced [Ca(2+)](i) and contraction of vascular smooth muscle triggered by Ca(2+) entry from the extracellular space exhibit differences depending on gender and the presence or absence of female gonads. Cell contraction and [Ca(2+)](i) due to Ca(2+) release from the intracellular stores are not affected by gender or gonadectomy. Gender specific reduction in contractility and [Ca(2+)](i) in vascular smooth muscle of female rats is greater in SHR than WKY rats. PMID- 10751333 TI - VEGF expression in an osteoblast-like cell line is regulated by a hypoxia response mechanism. AB - Angiogenesis is essential for the increased delivery of oxygen and nutrients required for the reparative processes of bone healing. Vascular endothelial growth factor (VEGF), a potent angiogenic growth factor, has been implicated in this process. We have previously shown that hypoxia specifically and potently regulates the expression of VEGF by osteoblasts. However, the molecular mechanisms governing this interaction remain unknown. In this study, we hypothesized that the hypoxic regulation of VEGF expression by osteoblasts occurs via an oxygen-sensing mechanism similar to the regulation of the erythropoietin gene (EPO). To test this hypothesis, we examined the kinetics of oxygen concentration on osteoblast VEGF expression. In addition, we analyzed the effects of nickel and cobalt on the expression of VEGF in osteoblastic cells because these metallic ions mimic hypoxia by binding to the heme portion of oxygen sensing molecules. Our results indicated that hypoxia potently stimulates VEGF mRNA expression. In addition, we found that nickel and cobalt both stimulate VEGF gene expression in a similar time- and dose-dependent manner, suggesting the presence of a hemelike oxygen-sensing mechanism similar to that of the EPO gene. Moreover, actinomycin D, cycloheximide, dexamethasone, and mRNA stabilization studies collectively established that this regulation is predominantly transcriptional, does not require de novo protein synthesis, and is not likely mediated by the transcriptional activator AP-1. These studies demonstrate that hypoxia, nickel, and cobalt regulate VEGF expression in osteoblasts via a similar mechanism, implicating the involvement of a heme-containing oxygen-sensing molecule. This may represent an important mechanism of VEGF regulation leading to increased angiogenesis in the hypoxic microenvironment of healing bone. PMID- 10751332 TI - Cellular mechanisms involved in carotid body inhibition produced by atrial natriuretic peptide. AB - Atrial natriuretic peptide (ANP) and its analog, atriopeptin III (APIII), inhibit carotid body chemoreceptor nerve activity evoked by hypoxia. In the present study, we have examined the hypothesis that the inhibitory effects of ANP and APIII are mediated by cyclic GMP and protein kinase G (PKG) via the phosphorylation and/or dephosphorylation of K(+) and Ca(2+) channel proteins that are involved in regulating the response of carotid body chemosensory type I cells to low-O(2) stimuli. In freshly dissociated rabbit type I cells, we examined the effects of a PKG inhibitor, KT-5823, and an inhibitor of protein phosphatase 2A (PP2A), okadaic acid (OA), on K(+) and Ca(2+) currents. We also investigated the effects of these specific inhibitors on intracellular Ca(2+) concentration and carotid sinus nerve (CSN) activity under normoxic and hypoxic conditions. Voltage dependent K(+) currents were depressed by hypoxia, and this effect was significantly reduced by 100 nM APIII. The effect of APIII on this current was reversed in the presence of either 1 microM KT-5823 or 100 nM OA. Likewise, these drugs retarded the depression of voltage-gated Ca(2+) currents induced by APIII. Furthermore, APIII depressed hypoxia-evoked elevations of intracellular Ca(2+), an effect that was also reversed by OA and KT-5823. Finally, CSN activity evoked by hypoxia was decreased in the presence of 100 nM APIII, and was partially restored when APIII was presented along with 100 nM OA. These results suggest that ANP initiates a cascade of events involving PKG and PP2A, which culminates in the dephosphorylation of K(+) and Ca(2+) channel proteins in the chemosensory type I cells. PMID- 10751334 TI - Molecular profiling of clinical tissue specimens: feasibility and applications. PMID- 10751336 TI - Lymphoid tissue homing chemokines are expressed in chronic inflammation. AB - Secondary lymphoid tissue chemokine (SLC) and B lymphocyte chemoattractant (BLC) are homing chemokines that have been implicated in the trafficking of lymphocytes and dendritic cells in lymphoid organs. Lymphotoxin-alpha (LTalpha), a cytokine crucial for development of lymphoid organs, is important for expression of SLC and BLC in secondary lymphoid organs during development. Here we report that transgenic expression of LTalpha induces inflammation and ectopic expression of SLC and BLC in the adult animal. LTbeta was not necessary for induction of BLC and SLC in inflamed tissues, whereas, in contrast, tumor necrosis factor receptor 1 was found to be important for the LTalpha-mediated induction of these chemokines. The ectopic expression of LTalpha is associated with a chronic inflammation that closely resembles organized lymphoid tissue and this lymphoid neogenesis can also be seen in several chronic inflammatory diseases, including in the pancreas of the prediabetic nonobese diabetic (NOD) mouse. Expression of SLC was also observed in the pancreas of prediabetic NOD mice. This study implicates BLC and SLC in chronic inflammation and presents further evidence that LTalpha orchestrates lymphoid organogenesis both during development and in inflammatory processes. PMID- 10751335 TI - Rous-Whipple Award Lecture. Viruses, immunity, and cancer: lessons from hepatitis B. PMID- 10751337 TI - Breakdown of the blood-retinal barrier induced by activated T cells of nonneural specificity. AB - The cellular and microvascular responses of JC Lewis rats to an intravenous injection of activated T cells specific for ovalbumin were examined with the retinal whole mount technique. The retina was examined at various times post injection (pi) with the use of antibodies to the alphabeta T cell receptor (TCR) or to major histocompatibility complex class II (MHC II), the monoclonal antibody ED1, and intravascular tracers. By 12 hours pi, small numbers of TCR(+), ED1(+), and MHC II(+) cells were present within the lumen of retinal vessels, and minor breakdown of the blood-retinal barrier (BRB) and microglial activation were evident. The intensity of these responses had increased by 1 day pi, when small numbers of TCR(+) cells had also undergone extravasation. By 2 to 3 days pi, the numbers of TCR(+), ED1(+), and MHC II(+) cells in the retinal parenchyma had increased, but the BRB breakdown and microglial activation had subsided. Thus, in the absence of target antigen, activated T cells induced limited and transient breakdown of the BRB, microglial activation, and the extravasation of ED1(+), MHC II(+) monocytes. In contrast, the retina of rats that received an intraocular injection of ovalbumin in addition to the intravascular injection of T cells showed massive cellular recruitment and breakdown of the BRB. These results indicate that an increase in the number of activated T cells in the circulation, such as that which occurs during viral or bacterial infection, has the potential to result in transient breakdown of the BRB and a mild local microglial response. PMID- 10751338 TI - Paired helical filaments of inclusion-body myositis muscle contain RNA and survival motor neuron protein. AB - Sporadic inclusion-body myositis (s-IBM) is the most common progressive muscle disease of older persons. Pathologically, the muscle biopsy manifests various degrees of inflammation and specific vacuolar degeneration of muscle fibers characterized by paired helical filaments (PHFs) composed of phosphorylated tau. IBM vacuolated fibers also contain accumulations of several other Alzheimer characteristic proteins. Molecular mechanisms leading to formation of the PHFs and accumulations of proteins in IBM muscle are not known. We report that the abnormal muscle fibers of IBM contained (i) acridine-orange-positive RNA inclusions that colocalized with the immunoreactivity of phosphorylated tau and (ii) survival motor neuron protein immunoreactive inclusions, which by immuno electron microscopy were confined to paired helical filaments. This study demonstrates two novel components of the IBM paired helical filaments, which may lead to better understanding of their pathogenesis. PMID- 10751339 TI - Gastrointestinal stromal tumors may originate from a subset of CD34-positive interstitial cells of Cajal. AB - Most gastrointestinal stromal tumors (GISTs), a subgroup of mesenchymal neoplasms of the gut wall, express both Kit (CD117) and CD34 proteins. It has been suggested that GISTs originate from or differentiate into interstitial cells of Cajal (ICC), after several reports indicated that ICC are likely the only cells in the gut which express both Kit and CD34. ICC are among the few cell types resident in the gut which express Kit, together with mast cells. However, the question whether or not ICC express CD34 is currently disputed. Using single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) on cultured murine intestinal cells, single ICC were selected by morphology and tested for the expression of c-kit and CD34 mRNA. Most ICC were only c-kit-positive, however a subset (7 out of 43) were double positive for both c-kit and CD34. In the human small intestine, sequential immunohistochemical staining for Kit and CD34 proteins on the same 3-microm sections showed that some of the ICC surrounding Auerbach's plexus and ICC within the circular muscle layer of the small intestine were positive for both Kit and CD34. In addition, CD34(+)Kit(-) cells were seen adjacent to ICC. These data from two different techniques indicate that ICC can be double positive for Kit and CD34. Thus, GISTs with the Kit(+)CD34(+) phenotype may arise from a subpopulation of CD34(+) Kit(+) ICC. PMID- 10751340 TI - The adhesion molecule CEACAM1 (CD66a, C-CAM, BGP) is specifically expressed by the extravillous intermediate trophoblast. AB - CEACAM1 (CD66a, C-CAM, BGP) is an adhesion molecule of the carcinoembryonic antigen family which has been shown to be normally expressed at the apical pole of epithelial cells, including the apical pole of endometrial surface and glandular epithelia. The purpose of the present study was to investigate its expression pattern at the maternal-fetal interface, and thus to determine whether CEACAM1 could be implicated in the human implantation process. For this purpose, we performed immunohistochemistry using the 4D1/C2 monoclonal antibody (mAb) as well as flow cytometry and Western blot on isolated trophoblast populations. On the maternal side of the maternal-fetal interface, CEACAM1 was present in epithelial cells of pregnancy endometrium as well as in small endometrial vessels, whereas it was absent from decidual cells. On the fetal side, CEACAM1 was strongly expressed by the extravillous (intermediate) trophoblast at the implantation site, as well as by extravillous trophoblast cells with invasive phenotype in primary culture, as shown by flow cytometry and Western blot. Expression was also observed in placental villous core vessels but was absent from both villous cyto- and syncytiotrophoblasts throughout the pregnancy. We conclude that, given its specific expression pattern, CEACAM1 can be a useful marker for extravillous intermediate trophoblast and might be functionally implicated in mediating trophoblast/endometrial and/or trophoblast/endothelial interactions during the trophoblastic invasion of the endometrium. PMID- 10751341 TI - Tumor necrosis factor induces tumor necrosis via tumor necrosis factor receptor type 1-expressing endothelial cells of the tumor vasculature. AB - Activation of endothelial cells, fibrin deposition, and coagulation within the tumor vasculature has been shown in vivo to correlate with the occurrence of tumor necrosis factor (TNF)-induced tumor necrosis in mice. In the present study we investigated which target cells mediate the TNF-induced necrosis in fibrosarcomas grown in wild type (wt), TNF receptor type 1-deficient (TNFRp55-/ ), and TNF receptor type 2-deficient (TNFRp75-/-) mice. TNF administration resulted in tumor necrosis exclusively in wt and TNFRp75-/-, but not in TNFRp55-/ mice, indicating a dependence of TNF-mediated tumor necrosis on the expression of TNF receptor type 1. However, using wt and TNFRp55-/- fibrosarcomas in wt mice, we found that TNF-mediated tumor necrosis was completely independent of TNF receptor type 1 expression in tumor cells. Thus we could exclude any direct tumoricidal effect of TNF in this model. Soluble TNF induced leukostasis in wt and TNFRp75-/- mice but not in TNFRp55-/- mice. TNF-induced leukostasis in TNFRp55-/- mice was restored by adoptive bone marrow transplantation of wt hematopoietic cells, but TNF failed to induce tumor necrosis in these chimeric mice. Because TNF administration resulted in both activation and focal damage of tumor endothelium, TNF receptor type 1-expressing cells of the tumor vasculature, likely to be endothelial cells, appear to be target cells for mediating TNF induced tumor necrosis. PMID- 10751342 TI - Microvascular effects of oral interleukin-6 on ischemia/reperfusion in the murine small intestine. AB - Oral administration of interleukin-6 (IL-6) has been shown to reduce hemorrhage induced bacterial translocation from the gut in mice and rats. To examine the intestinal microvasculature, mice were given the electron-dense tracer horseradish peroxidase (HRP) after hemorrhage and IL-6 or vehicle administration. In normal mice and in those hemorrhaged and given IL-6, the electron-dense marker, administered intravenously, could be found in intestinal capillaries and between mucosal epithelial cells, suggesting that the microvasculature was patent. In mice given saline after shock, however, no marker was present in the gut, suggesting that the intestinal microvasculature was unable to deliver the marker to the epithelia. When mice were given HRP intralumenally (il) the tracer was able to penetrate between intestinal epithelial cells only in mice given vehicle after hemorrhage. This finding suggests that hemorrhaged mice were susceptible to sepsis and endotoxic shock from the leaky gut. In normal and IL-6 treated mice, the tracer was unable to pass from the lumen between mucosal epithelial cells, because the presence of an intact zonula occludens prevented passage. Functional studies supported the electron microscopy findings. Bacteria were cultured from the livers of mice fed vehicle after hemorrhage, but not from those fed IL-6. These data support the conclusions that parts of the intestinal microvasculature remain diminished after hemorrhage and resuscitation and that oral IL-6 restores this circulation. PMID- 10751344 TI - Quantitation of DNA extracted after micropreparation of cells from frozen and formalin-fixed tissue sections. AB - Quantitation of DNA from microdissected fresh-frozen or paraffin-embedded tissue sections would be not only a valuable tool for ensuring optimum reaction conditions for many types of qualitative polymerase chain reaction (PCR) analyses, but also a prerequisite for any kind of subsequently performed genetic analyses aimed at the absolute quantitation of target sequences. The present study describes the quantitation of DNA after microdissection and extraction of cells with the PicoGreen fluorescence method. The limits of detection and of quantitative determination, respectively, have been determined by measuring dilutional series of three different DNA extractions, using either a medium-scale preparation from a solid tissue specimen or a known number of leukocytes or microdissected cells from frozen tumor sections. As corresponding limits of detection, 26, 24, and about 40 diploid genomes, and as limits of quantitative determination, 80, 73, and about 120 diploid genomes were obtained. Furthermore, it was shown that formalin fixation as well as hematoxylin staining of frozen sections with Delafield's and Mayer's alum or Weigert's iron hematoxylin before microdissection significantly diminishes the amount of extractable DNA and may lead to less reliable results, even of qualitative PCR analysis. In conclusion, the PicoGreen method allows precise quantitation of DNA corresponding to a minimum of about 120 diploid cells. It provides the basis for reliable qualitative analyses as well as the precondition for further quantitative genetic measurements from microdissected frozen or formalin-fixed and paraffin-embedded tissue sections. PMID- 10751343 TI - Incidence and subtype specificity of API2-MALT1 fusion translocations in extranodal, nodal, and splenic marginal zone lymphomas. AB - The t(11;18)(q21;q21) is thought to represent an important primary event in the development of marginal zone lymphomas, although an accurate estimation of the frequency and distribution of this genetic alteration among nodal, splenic, and extranodal marginal zone lymphoma types has yet to be determined. Recently, molecular genetic studies have shown that this translocation results in the fusion of the API2 gene on chromosome 11 and a novel gene termed MALT1 on chromosome 18. To investigate the incidence of API2-MALT1 fusion transcripts among marginal zone lymphomas and to determine possible marginal zone lymphoma subtype associations, we used reverse transcriptase-polymerase chain reaction to analyze RNAs extracted from frozen tissue samples of 99 marginal zone lymphomas. Fifty-seven involved diverse extranodal sites including 14 stomach, 11 lung, 7 orbit, 7 parotid, 5 thyroid, 5 lacrimal gland, 3 small intestine, 2 large intestine, 1 kidney, 1 paraspinal region and 1 skin. Twenty-one primary splenic and twenty-one primary nodal marginal zone lymphomas were also studied. API2 MALT1 fusion transcripts were detected in 12 of 57 extranodal marginal zone lymphomas (21%), but in none of the nodal or splenic cases. The cDNA sequences of the fusion transcripts were determined, revealing variation in the coding sequence fusion point for both API2 and MALT1. The findings suggest that t(11;18)(q21;q21) is restricted to extranodal marginal zone lymphomas and that these tumors have distinct genetic etiologies in comparison with their splenic and nodal counterparts. PMID- 10751346 TI - Proliferation in HHV-8-positive primary effusion lymphomas is associated with expression of HHV-8 cyclin but independent of p27(kip1). AB - Primary effusion lymphoma (PEL) develops in immunodeficient patients, selectively localizes to the serous body cavities, and harbors infection by human herpesvirus type-8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. HHV-8 encodes a viral (v)-cyclin homologous to cellular D-type cyclins, a class of positive cell-cycle regulators that are physiologically modulated by the p27(Kip1) cell cycle inhibitor. The aims of the present study were: 1) to establish the expression pattern of p27(Kip1) in PEL; and 2) to address the relationship between p27(Kip1) expression, proliferation index, and expression of cellular cyclin D1 and v-cyclin in PEL. Expression of p27(Kip1) was detected in all (n = 18) PEL samples analyzed by both immunocytochemistry and Western blot. All PELs displayed a high proliferation index as assessed by Ki-67 staining. Expression of cellular cyclin D1 was absent in all PELs tested, which conversely expressed (14 out of 14 samples) v-cyclin by immunocytochemistry and/or Western blot. In contrast to PELs, HHV-8-negative lymphomatous effusions secondary to a tissue-based lymphoma generally failed to express p27(Kip1). Overall, these data show that PELs consistently express p27(Kip1) protein despite the high proliferative rate of the lymphoma clone, suggesting that p27(Kip1) may be unable to drive cell-cycle arrest in PEL cells. The co-existence of p27(Kip1) expression and high proliferative index is a selective feature of PEL among lymphomas involving the serous body cavities, because lymphomatous effusions secondary to a tissue-based lymphoma generally display the inverse relationship between p27(Kip1) positivity and growth fraction observed in normal lymphoid tissues and in most other lymphomas. Expression of p27(Kip1) in PEL associates with expression of HHV-8 v-cyclin, but not of cellular cyclin D1. The fact that HHV-8 v-cyclin is resistant to p27(Kip1)-modulated inhibition, whereas cellular cyclin D1 is sensitive, may explain, at least in part, the co-existence of p27(Kip1) expression and high proliferative index observed in PEL. PMID- 10751345 TI - Rapid shift from virally infected cells to germinal center-retained virus after HIV-2 infection of macaques. AB - Lymphoid tissues are the primary target during the initial virus dissemination that occurs in HIV-1-infected individuals. Recent advances in antiretroviral therapy and techniques to monitor virus load in humans have demonstrated that the early stages of viral infection and host response are major determinants of the outcome of individual infections. Relatively little is known about immunopathogenic events occurring during the acute phase of HIV infection. We analyzed viral dissemination within lymphoid tissues by in situ hybridization and by combined immunohistochemistry/in situ hybridization during the acute infection phase (12 hours to 28 days) in pig-tailed macaques (Macaca nemestrina), challenged intravenously with a virulent strain of HIV-2, HIV-2(287). Two stages in viral dissemination were clearly evident within the first 28 days after HIV 2(287) infection. First, a massive increase in individual HIV-2-infected cells, mostly CD3+ T lymphocytes and a smaller percentage of macrophages and interdigitating dendritic cells, was identified within lymph nodes which peaked on the 10th day after HIV-2 infection. A shift of HIV-2 distribution was demonstrable between day 10 and day 14 after HIV-2 infection. Coincident with a marked reduction in individual HIV-2 RNA+ cells by day 14 postinfection, there was a dramatic increase in germinal center-associated HIV-2 RNA. High concentrations of HIV-2 RNA persisted in germinal centers in all animals by days 21 and 28 postinfection. Thus, HIV-2 appears to go through an initial, highly disseminated cellular phase followed by localization in the follicular dendritic cell network with relatively few infected cells. In this nonhuman primate model of HIV-associated immunopathogenesis, using a virus derived from a human pathogen, we identified a significant shift in the pattern of HIV-2 localization within a narrow time frame (day 10 to day 14). This shift in virus localization and behavior indicates that there may be a discrete but remarkably narrow window for therapeutic interventions that interrupt this stage in the natural course of HIV infection. Reproducibility and the accelerated time course of disease development make this model an excellent candidate for such intervention studies. PMID- 10751347 TI - Molecular and functional analysis of the human prothrombinase gene (HFGL2) and its role in viral hepatitis. AB - In the present studies, we report the cloning and structural characterization of the HFGL2 gene and its functional role in human fulminant hepatitis. The HFGL2 gene is approximately 7 kb in length with 2 exons. The putative promoter contains cis element consensus sequences that strongly suggest the inducibility of its expression. From the nucleotide sequence of the human gene, a 439-amino acid long protein is predicted. The overall identity between the murine fgl2 and hfgl2 coded proteins is over 70%. About 225 amino acids at the carboxyl end of these molecules are almost 90% identical, and correspond to a well-conserved fibrinogen related domain. Both HFGL2 and FGL2 encode a type II transmembrane protein with a predicted catalytic domain toward the amino terminus of the protein. Transient transfection of Chinese hamster ovary (CHO) cells with a full-length cDNA of HFGL2 coding region resulted in high levels of prothrombinase activity. Livers from 8 patients transplanted for fulminant viral hepatitis were examined for extent of necrosis, inflammation, fibrin deposition, and HFGL2 induction. In situ hybridization showed positive staining of macrophages in areas of active hepatocellular necrosis. Fibrin stained positively in these areas and was confirmed by electron microscopy. These studies define a unique prothrombinase gene (HFGL2) and implicate its importance in the pathogenesis of fulminant viral hepatitis. PMID- 10751348 TI - Detection of TT virus DNA in liver biopsies by in situ hybridization. AB - A novel hepatitis-associated virus named TT virus (TTV) has been isolated. However, its hepatotropism has not been proven. We have retrospectively analyzed the presence of TTV-DNA by polymerase chain reaction (PCR) and in situ hybridization in liver biopsies from 30 patients with liver disease (15 TTV-DNA positive and 15 TTV-DNA-negative in serum), and prospectively in serum and liver from eight patients with normal liver histology. TTV-DNA was detected by PCR in the liver from the 15 patients with serum TTV-DNA and in serum and liver of two of the eight patients without liver disease. TTV-DNA titers in liver were 10 times higher than in serum, although no correlation between TTV-DNA titers in serum and liver were observed. In situ hybridization shows positive signals in the hepatocytes of the 17 patients infected by TTV but in none of the TTV-DNA negative patients by PCR. No morphological changes were observed in the hepatocytes showing hybridization signals. The percentage of positive hepatocytes ranged from 2.1% to 30% and correlated with the TTV-DNA titers in liver (r = 0.54; P = 0.037). In conclusion, our results show that TTV is able to infect liver cells although they do not support a role for TTV in causing liver disease. PMID- 10751349 TI - Hepatic stellate cells express the low affinity nerve growth factor receptor p75 and undergo apoptosis in response to nerve growth factor stimulation. AB - We have examined the expression of p75, a member of the TNF receptor superfamily in hepatic stellate cells (HSC) and pancreatic stellate cells (PSC). Activated HSC and PSC were demonstrated by Western blot analysis to express p75. p75 was immunolocalized to cells with a myofibroblast-like morphology in the fibrotic bands of six fibrotic and cirrhotic liver biopsies and three biopsies of fibrotic human pancreas. Immunostaining of parallel sections indicated that these cells were alpha-smooth muscle actin-positive, identifying them as activated HSC and PSC, respectively. HSC apoptosis in tissue culture in the presence of serum was quantified after addition of 0.1 to 100 ng/ml of nerve growth factor (NGF) a ligand for p75, by in situ counting of apoptotic bodies after addition of acridine orange. HSC demonstrated a significant increase in apoptosis in response to 100 ng/ml NGF (0.05 > P by Wilcoxon's rank; n = 7) after 24 hours. NGF 100 ng/ml had no effect on HSC proliferation, but reduced total HSC DNA by 19% relative to control after 24 hours (n = 3). These data demonstrate that activated HSC express p75 and respond to NGF stimulation by undergoing apoptosis. We therefore report p75 as a novel marker of activated HSC and suggest that signaling via ligand binding to p75 may provide a mechanism for selective apoptosis of HSC. PMID- 10751351 TI - The Fn14 immediate-early response gene is induced during liver regeneration and highly expressed in both human and murine hepatocellular carcinomas. AB - Polypeptide growth factors stimulate mammalian cell proliferation by binding to specific cell surface receptors. This interaction triggers numerous biochemical responses including the activation of protein phosphorylation cascades and the enhanced expression of specific genes. We have identified several fibroblast growth factor (FGF)-inducible genes in murine NIH 3T3 cells and recently reported that one of them, the FGF-inducible 14 (Fn14) immediate-early response gene, is predicted to encode a novel, cell surface-localized type Ia transmembrane protein. Here, we report that the human Fn14 homolog is located on chromosome 16p13.3 and encodes a 129-amino acid protein with approximately 82% sequence identity to the murine protein. The human Fn14 gene, like the murine Fn14 gene, is expressed at elevated levels after FGF, calf serum or phorbol ester treatment of fibroblasts in vitro and is expressed at relatively high levels in heart and kidney in vivo. We also report that the human Fn14 gene is expressed at relatively low levels in normal liver tissue but at high levels in liver cancer cell lines and in hepatocellular carcinoma specimens. Furthermore, the murine Fn14 gene is rapidly induced during liver regeneration in vivo and is expressed at high levels in the hepatocellular carcinoma nodules that develop in the c myc/transforming growth factor-alpha-driven and the hepatitis B virus X protein driven transgenic mouse models of hepatocarcinogenesis. These results indicate that Fn14 may play a role in hepatocyte growth control and liver neoplasia. PMID- 10751350 TI - Exaggerated hepatic injury due to acetaminophen challenge in mice lacking C-C chemokine receptor 2. AB - Monocyte chemoattractant protein-1 is one of the major C-C chemokines that has been implicated in liver injury. The C-C chemokine receptor, CCR2, has been identified as the primary receptor that mediates monocyte chemoattractant protein 1 (MCP-1) responses in the mouse. Accordingly, the present study addressed the role of CCR2 in mice acutely challenged with acetaminophen (APAP). Mice genetically deficient in CCR2 (CCR2(-/-)) and their wild-type counterparts (CCR2(+/+)) were fasted for 10 hours before receiving an intraperitoneal injection of APAP (300 mg/kg). Liver and serum samples were removed from both groups of mice before and at 24 and 48 hours post APAP. Significantly elevated levels of MCP-1 were detected in liver samples from CCR2(+/+) and CCR2(-/-) mice at 24 hours post-APAP. Although CCR2(+/+) mice exhibited no liver injury at any time after receiving APAP, CCR2(-/-) mice exhibited marked evidence of necrotic and TUNEL-positive cells in the liver, particularly at 24 hours post-APAP. Enzyme linked immunosorbent assay analysis of liver homogenates from both groups of mice at the 24 hours time point revealed that liver tissue from CCR2(-/-) mice contained significantly greater amounts of immunoreactive IFN-gamma and TNF alpha. The in vivo immunoneutralization of IFN-gamma or TNF-alpha significantly attenuated APAP-induced liver injury in CCR2(-/-) mice and increased hepatic IL 13 levels. Taken together, these findings demonstrate that CCR2 expression in the liver provides a hepatoprotective effect through its regulation of cytokine generation during APAP challenge. PMID- 10751352 TI - Cytokeratin 8 protects from hepatotoxicity, and its ratio to cytokeratin 18 determines the ability of hepatocytes to form Mallory bodies. AB - In alcoholic hepatitis, a severe form of alcohol-induced toxic liver injury, as well as in experimental intoxication of mice with the porphyrinogenic drugs griseofulvin and 3,5-diethoxycarbonyl-1, 4-dihydrocollidine, hepatocytes form cytoplasmic protein aggregates (Mallory bodies; MBs) containing cytokeratins (CKs) and non-CK components. Here we report that mice lacking the CK8 gene and hence CK intermediate filaments in hepatocytes, but still expressing the type I partner, ie, the CK18 gene, do not form MBs but suffer from extensive porphyria and progressive toxic liver damage, leading to the death of a considerable number of animals (7 of 12 during 12 weeks of intoxication). Our observations show that 1) in the absence of CK8 as well as in the situation of a relative excess of CK18 over CK8 no MBs are formed; 2) the loss of CK8 is not compensated by other type II CKs; and 3) porphyria and toxic liver damage are drastically enhanced in the absence of CK8. Our results point to a protective role of CKs in certain types of toxic liver injury and suggest that MBs by themselves are not harmful to hepatocytes but may be considered as a product of a novel defense mechanism in hepatocytes. PMID- 10751354 TI - Transition from squamous cell carcinoma to adenocarcinoma in adenosquamous carcinoma of the lung. AB - The heterogeneity of tumor cells is frequently observed in lung cancer, but the clonality of these cells has not yet been established. The distinct components of 12 lung adenosquamous carcinomas were compared by genetic alterations of p53 and K-ras, chromosomal abnormalities at 9p21 and 9q31-32, and immunohistochemical reactions. The immunoreactivity of p53 was consistent in both adenocarcinomatous and squamous cell carcinomatous components as well as in the transitional areas, retaining the morphological characteristics of the distinct components. The same p53 mutation was found in both components of each tumor with p53 overexpression. No K-ras mutations were detected in any of the tumors examined. Three of the four tumors with chromosomal abnormalities detected, one at 9p21 and two at 9q31-32, had coincident abnormalities between the distinct components, whereas one tumor deleted homozygously at 9p21 (D9S259) in the adenocarcinomatous component with loss of heterozygosity in the other component. The expression of squamous cell carcinoma-related antigen in adenocarcinomatous components was significantly higher than that of lung adenocarcinomas (57 +/- 5.8% vs. 1.0 +/- 0.5%, P < 0.0001), whereas Mucin 1 expression is less in these components (9.0 +/- 4.9% vs. 55 +/- 8.2%, P = 0.003). These results suggest monoclonal transition from squamous cell carcinoma to adenocarcinoma in lung adenosquamous carcinoma. PMID- 10751355 TI - Dual-hit hypothesis explains pulmonary hypoplasia in the nitrofen model of congenital diaphragmatic hernia. AB - Pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH) remains a major therapeutic problem. Moreover, the pathogenesis of pulmonary hypoplasia in case of CDH is controversial. In particular, little is known about early lung development in this anomaly. To investigate lung development separate from diaphragm development we used an in vitro modification of the 2, 4 dichlorophenyl-p-nitrophenylether (Nitrofen) animal model for CDH. This enabled us to investigate the direct effects of Nitrofen on early lung development and branching morphogenesis in an organotypic explant system without the influence of impaired diaphragm development. Epithelial cell differentiation of the lung explants was assessed using surfactant protein-C and Clara cell secretory protein 10 mRNA expression as markers. Furthermore, cell proliferation and apoptosis were investigated. Our results indicate that Nitrofen negatively influences branching morphogenesis of the lung. Initial lung anlage formation is not affected. In addition, epithelial cell differentiation and cell proliferation are attenuated in lungs exposed to Nitrofen. These data indicate that Nitrofen interferes with early lung development before and separate from (aberrant) diaphragm development. Therefore, we postulate the dual-hit hypothesis, which explains pulmonary hypoplasia in CDH by two insults, one affecting both lungs before diaphragm development and one affecting the ipsilateral lung after defective diaphragm development. PMID- 10751353 TI - Rat pulmonary cyclooxygenase-2 expression in response to endotoxin challenge: differential regulation in the various types of cells in the lung. AB - Cyclooxygenase (Cox), the key enzyme of prostanoid synthesis, consists of the two isoforms Cox-1 and Cox-2, both recently noted to be constitutively expressed in rat lungs with a distinct profile of cellular distribution. The responsiveness of pulmonary Cox-1 and Cox-2 expression to intravascular endotoxin lipopolysaccharide (LPS) administration was investigated in isolated, ventilated rat lungs, buffer-perfused with or without admixture of rat plasma. Immunohistochemical staining intensity was measured by a previously described method of silver enhancement and epipolarization image analysis. Both the Cox-1 mRNA, quantified in the whole lung homogenate, and the cellular localization of Cox-1 were unchanged in response to LPS. In contrast, time- and dose-dependent up regulation of Cox-2 mRNA (lung homogenate) occurred, and differential LPS reactivity at the cellular level was observed. Up-regulation of Cox-2 in cell types expressing this enzyme already under baseline conditions was noted in bronchial epithelial cells, bronchial and vascular smooth muscle cells, cells within the BALT and myocytes of the large hilar veins. De novo induction of Cox-2 occurred in endothelial cells and the majority of alveolar macrophages. Down regulation of Cox-2 was observed in perivascular and peribronchial macrophage like cells. Moreover, differential impact of plasma components was noted: for the large majority of cells, CD14 surface expression correlated with Cox-2 responsiveness to LPS independent of plasma, whereas the presence of plasma components was a prerequisite for the LPS response in CD14-negative cells. LPS did not provoke physiological changes in the perfused lungs, but markedly enhanced baseline prostanoid generation. We conclude that LPS-induced Cox-2 regulation occurs in a complex, cell-specific manner, which may be relevant for pathogenetic sequelae in septic lung injury and acute respiratory failure. PMID- 10751356 TI - Increased phosphorylated extracellular signal-regulated kinase immunoreactivity associated with proliferative and morphologic lung alterations after chrysotile asbestos inhalation in mice. AB - Activation of extracellular signal-regulated kinases (ERK) has been associated with the advent of asbestos-associated apoptosis and proliferation in mesothelial and alveolar epithelial cells and may be linked to the development of pulmonary fibrosis. The objective of studies here was to characterize the development of inflammation, cellular proliferation, and fibrosis in asbestos-exposed C57Bl/6 mice in relationship to patterns of ERK phosphorylation. Inflammation occurred after 10 and 20 days of asbestos exposure as evidenced by increases in total protein and neutrophils in bronchoalveolar lavage fluid. Increases in cell proliferation were observed at 30 days in bronchiolar epithelia and at 4, 14, and 30 days in the alveolar compartment of the lung. Trichrome-positive focal lesions of pulmonary fibrosis developed at 30 days in the absence of elevations in lung hydroxyproline or procollagen mRNA levels. Striking increases in ERK phosphorylation were observed within pulmonary epithelial cells at sites of developing fibrotic lesions after 14 and 30 days of inhalation. In addition to characterizing a murine inhalation model of asbestosis, we provide the first evidence showing activation of ERK signaling within lung epithelium in vivo, following inhalation of asbestos fibers. PMID- 10751358 TI - Cell differentiation and matrix gene expression in mesenchymal chondrosarcomas. AB - Mesenchymal chondrosarcomas are small-cell malignancies named as chondrosarcomas due to the focal appearance of cartilage islands. In this study, the use of in situ detection techniques on a large series of mesenchymal chondrosarcoma specimens allowed the identification of tumor-cell differentiation pathways in these neoplasms. We were able to trace all steps of chondrogenesis within mesenchymal chondrosarcoma by using characteristic marker genes of chondrocytic development. Starting from undifferentiated cells, which were negative for vimentin and any other mesenchymal marker, a substantial portion of the cellular (undifferentiated) tumor areas showed a chondroprogenitor phenotype with an onset of expression of vimentin and collagen type IIA. Cells in the chondroid areas showed the full expression panel of mature chondrocytes including type X collagen indicating focal hypertrophic differentiation of the neoplastic chondrocytes. Finally, evidence was found for transdifferentiation of the neoplastic chondrocytes to osteoblast-like cells in areas of neoplastic bone formation. These results establish mesenchymal chondrosarcoma as the very neoplasm of differentiating premesenchymal chondroprogenitor cells. The potential of neoplastic bone formation in mesenchymal chondrosarcoma introduces a new concept of neoplastic (chondrocytic) osteogenesis in musculoskeletal malignant neoplasms, which qualifies the old dogma that neoplastic bone/osteoid formation automatically implies the diagnosis of osteosarcoma. PMID- 10751357 TI - Differential expression of cytokeratin after orthotopic implantation of newly established human tongue cancer cell lines of defined metastatic ability. AB - Two human tongue squamous cell carcinoma cell lines, SQUU-A and SQUU-B, were established from the same patient. Cervical lymph node metastasis was detected in the mice orthotopically implanted with SQUU-B (86.7%, 13/15), but not in those with SQUU-A (0/13). Histologically, SQUU-B showed invasive growth and intravasation in the tongue, whereas SQUU-A simply demonstrated expansive growth without intravasation. By Western blot analysis, nonmetastatic clone SQUU-A expressed cytokeratin (CK)13/4, 14, 16/6, 18/8, and 19, whereas a high metastatic clone SQUU-B expressed CK18/8 and 19. The reverse transcription-polymerase chain reaction technique showed that CK13/4 mRNA was expressed in both cell lines, but CK14 and 16 mRNA was expressed only in SQUU-A. CK13 was immunohistochemically expressed in both SQUU-A and SQUU-B transplanted into the tongues of nude mice; CK14 and 16 were detected in SQUU-A of the tongues, but not in SQUU-B. As seen in SQUU-B cell line, SQUU-B of the cervical lymph node metastasis did not exhibit CK13, 14, or 16. These results suggest that the loss or down-regulation of CK13, 14, or 16 is related to the invasive and metastatic ability of cancer. The cytoskeletal system is thus considered to be closely related to the malignant phenotype. PMID- 10751359 TI - Vascular endothelial growth factor and hepatocyte growth factor levels are differentially elevated in patients with advanced retinopathy of prematurity. AB - Although the roles of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in angiogenesis are well described, the putative roles of these factors in retinopathy of prematurity (ROP) remain unknown. We evaluated VEGF and HGF protein levels in subretinal fluid of eyes with ROP, and expression of their corresponding receptors in retrolental membranes associated with stage 5 ROP. We examined subretinal fluid samples from eyes using rhegmatogenous retinal detachment as a control. VEGF and HGF were differentially elevated in eyes with ROP. In Stage 5 ROP (n = 22), the mean VEGF and HGF levels were 14.77 +/- 14.01 ng/ml and 16.56 +/- 9.62 ng/ml, respectively. Interestingly, in patients with active stage 4 ROP, mean VEGF levels were highly elevated (44.16 +/- 18.72 ng/ml), whereas mean HGF levels remained very low (4.77 +/- 2.50 ng/ml). Next, we investigated in vivo expression of VEGF receptor-2 and HGF receptor in retrolental membranes from 16 patients with stage 5 ROP. Both VEGF receptor-2 and HGF receptor proteins were detected mainly in posterior portions of the membrane as well as in vessel walls and along the retinal interface where angiogenesis was active. These findings together suggest that VEGF and HGF play important roles in the pathogenesis of ROP. PMID- 10751361 TI - Openings between defective endothelial cells explain tumor vessel leakiness. AB - Leakiness of blood vessels in tumors may contribute to disease progression and is key to certain forms of cancer therapy, but the structural basis of the leakiness is unclear. We sought to determine whether endothelial gaps or transcellular holes, similar to those found in leaky vessels in inflammation, could explain the leakiness of tumor vessels. Blood vessels in MCa-IV mouse mammary carcinomas, which are known to be unusually leaky (functional pore size 1.2-2 microm), were compared to vessels in three less leaky tumors and normal mammary glands. Vessels were identified by their binding of intravascularly injected fluorescent cationic liposomes and Lycopersicon esculentum lectin and by CD31 (PECAM) immunoreactivity. The luminal surface of vessels in all four tumors had a defective endothelial monolayer as revealed by scanning electron microscopy. In MCa-IV tumors, 14% of the vessel surface was lined by poorly connected, overlapping cells. The most superficial lining cells, like endothelial cells, had CD31 immunoreactivity and fenestrae with diaphragms, but they had a branched phenotype with cytoplasmic projections as long as 50 microm. Some branched cells were separated by intercellular openings (mean diameter 1.7 microm; range, 0.3 4.7 microm). Transcellular holes (mean diameter 0.6 microm) were also present but were only 8% as numerous as intercellular openings. Some CD31-positive cells protruded into the vessel lumen; others sprouted into perivascular tumor tissue. Tumors in RIP-Tag2 mice had, in addition, tumor cell-lined lakes of extravasated erythrocytes. We conclude that some tumor vessels have a defective cellular lining composed of disorganized, loosely connected, branched, overlapping or sprouting endothelial cells. Openings between these cells contribute to tumor vessel leakiness and may permit access of macromolecular therapeutic agents to tumor cells. PMID- 10751360 TI - Regulation of angiogenesis in vivo by ligation of integrin alpha5beta1 with the central cell-binding domain of fibronectin. AB - Angiogenesis depends on the cooperation of growth factors and cell adhesion events. Although alphav integrins have been shown to play critical roles in angiogenesis, recent studies in alphav-null mice suggest that other adhesion receptors and their ligands also regulate this process. Evidence is now provided that the integrin alpha5beta1 and its ligand fibronectin are coordinately up regulated on blood vessels in human tumor biopsies and play critical roles in angiogenesis, resulting in tumor growth in vivo. Angiogenesis induced by multiple growth factors in chick embryos was blocked by monoclonal antibodies to the cell binding domain of fibronectin. Furthermore, application of fibronectin or a proteolytic fragment of fibronectin containing the central cell-binding domain to the chick chorioallantoic membrane enhanced angiogenesis in an integrin alpha5beta1-dependent manner. Importantly, antibody, peptide, and novel nonpeptide antagonists of integrin alpha5beta1 blocked angiogenesis induced by several growth factors but had little effect on angiogenesis induced by vascular endothelial growth factor (VEGF) in both chick embryo and murine models. In fact, these alpha5beta1 antagonists inhibited tumor angiogenesis, thereby causing regression of human tumors in animal models. Thus, fibronectin and integrin alpha5beta1, like integrin alphavbeta3, contribute to an angiogenesis pathway that is distinct from VEGF-mediated angiogenesis, yet important for the growth of tumors. PMID- 10751362 TI - Inhibition of angiogenesis and vascular tumor growth by interferon-producing cells: A gene therapy approach. AB - We developed an in vivo gene therapy approach to characterize and optimize the anti-angiogenic activity of class I interferons (IFNs), using packaging cell lines producing an amphotropic LXSN-based retrovirus expressing either IFN-alpha1 (alpha1Am12), IFN-beta (betaAm12) murine cDNAs, or the vector alone (neoAm12). Pretreatment of endothelial-like Eahy926 cells in vitro with conditioned media (CM) from alpha1Am12 or betaAm12 cells for 48 hours significantly inhibited their migration and invasion as compared to neoAm12-CM-treated cells. betaAm12-CM also inhibited the formation of capillary-like structures on Matrigel by EAhy926 cells. In vivo, inclusion of the betaAm12 cells strongly inhibited, and alpha1Am12 partially inhibited, the angiogenic response in the Matrigel sponge model in both immune-competent and athymic nude mice. Electron microscopy showed a reduction of host cell infiltration in alpha1Am12- and betaAm12-containing sponges and reduction of invading tubular clefts of host cells as compared to controls. Finally, inoculation of either alpha1Am12 or betaAm12 cells (10%) along with a highly angiogenic Kaposi's sarcoma cell line (90%) resulted in a powerful reduction of tumor growth in nude mice in vivo, as did infection with the interferon-alpha-producing retroviruses. These data suggest that a gene therapy approach using class I interferons can effectively inhibit tumor angiogenesis and growth of vascular tumors. PMID- 10751364 TI - Cystic fibrosis transmembrane conductance regulator does not affect neutrophil migration across cystic fibrosis airway epithelial monolayers. AB - Recent studies have shown that airway inflammation dominated by neutrophils, ie, polymorphonuclear cells (PMN) was observed in infants and children with cystic fibrosis (CF) even in the absence of detectable infection. To assess whether there is a CF-related anomaly of PMN migration across airway epithelial cells, we developed an in vitro model of chemotactic migration across tight and polarized CF(15) cells, a CF human nasal epithelial cell line, seeded on porous filters. To compare PMN migration across a pair of CF and control monolayers in the physiological direction, inverted CF(15) cells were infected with increasing concentrations of recombinant adenoviruses containing either the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, the DeltaF508 CFTR cDNA, or the beta-galactosidase gene. The number of PMN migrating in response to N-formyl-Met-Leu-Phe across inverted CF(15) monolayers expressing beta galactosidase was similar to that seen across CF(15) monolayers rescued with CFTR, whatever the proportion of cells expressing the transgene. Moreover, PMN migration across monolayers expressing various amounts of mutated CFTR was not different from that observed across matched counterparts expressing normal CFTR. Finally, PMN migration in response to adherent or Pseudomonas aeruginosa was equivalent across CF and corrected monolayers. The possibility that mutated CFTR may exert indirect effects on PMN recruitment, via an abnormal production of the chemotactic cytokine interleukin-8, was also explored. Apical and basolateral production of interleukin-8 by polarized CF cells expressing mutated CFTR was not different from that observed with rescued cells, either in baseline or stimulated conditions. CF(15) cells displayed a CF phenotype that could be corrected by CFTR containing adenoviruses, because two known CF defects, Cl(-) secretion and increased P. aeruginosa adherence, were normalized after infection with those viruses. Thus, we conclude that the presence of a mutated CFTR does not per se lead to an exaggerated inflammatory response of CF surface epithelial cells in the absence or presence of a bacterial infection. PMID- 10751363 TI - Overexpression of Bcl-2 protects from ultraviolet B-induced apoptosis but promotes hair follicle regression and chemotherapy-induced alopecia. AB - Hair follicle (HF) growth and regression is an exquisitely regulated process of cell proliferation followed by massive cell death and is accompanied by cyclical expression of the apoptosis regulatory gene pair, Bcl-2 and Bax. To further investigate the role of Bcl-2 expression in the control of hair growth and keratinocyte apoptosis, we have used transgenic mice that overexpress human Bcl-2 in basal epidermis and in the outer root sheath under the control of the human keratin-14 promoter (K14/Bcl-2). When irradiated with ultraviolet B (UVB) light, K14/Bcl-2 mice developed about 5-10-fold fewer sunburn cells (ie, apoptotic keratinocytes) in the basal layer of the epidermis, compared to wild-type mice, whereas cultures of primary keratinocytes from transgenic mice were completely resistant to UVB-induced histone formation, at doses that readily induced histone release from wild-type cells. K14/Bcl-2 mice show no alteration of neonatal hair follicle morphogenesis or of the onset of the first wave of HF regression (catagen). However, compared to wild-type controls, K14/Bcl-2 mice subsequently displayed a significant acceleration of spontaneous catagen progression. During chemotherapy-induced alopecia, follicular dystrophy was promoted in K14/Bcl-2 mice. Thus, although K14-driven overexpression of Bcl-2 protected murine epidermal keratinocytes from UVB-induced apoptosis, it surprisingly promoted catagen- and chemotherapy-associated keratinocyte apoptosis. PMID- 10751365 TI - ErbB2 immune response in breast cancer patients with soluble receptor ectodomain. AB - Investigation of ErbB2 immunity in human breast cancer employing recombinant expression sources in immunoblot analysis revealed ErbB2-specific antibodies of the IgG isotype in sera of 14 of 71 cancer patients and 1 of 31 normal donors. Reactivity was confirmed on ErbB2-specific immunoprecipitates. Independent evidence of existing ErbB2 immunity was obtained after in vitro transformation of peripheral blood leukocytes from six positive patients. Furthermore, in vitro immortalization of B-lymphocytes unmasked existent ErbB2 immunity in 1 of 8 patients negative for ErbB2 serum antibodies. Determining shed ErbB2 extracellular domain as an indirect measure of tumor burden in ErbB2-positive malignancy, elevated serum levels were observed in 16 of 71 breast cancer and 1 of 31 normal donor sera. Strikingly, existing ErbB2 immunity correlated significantly with elevated shed ErbB2 ectodomain among the patients analyzed. Incidence of both ErbB2 immunity and elevated ErbB2 extracellular domain increased with a progressed disease stage and was significantly associated with metastatic breast cancer. These observations implicate soluble ErbB2 amounts in vivo in the development of ErbB2 immunity in breast cancer. They further project serum analysis of ErbB2 immunity and soluble ectodomain as potential markers of disease progression in ErbB2-positive malignancy. PMID- 10751366 TI - Telomerase activity in melanocytic lesions: A potential marker of tumor biology. AB - Telomerase activation, being a cardinal requirement for immortalization, is a crucial step in the development of malignancy. With a view toward diagnostic and biological aspects in melanocytic neoplasia, we investigated the relative levels of telomerase activity in 72 nevi and 16 malignant melanomas by means of a modified telomeric repeat amplification protocol (TRAP) assay, including an internal amplification standard. We further compared telomerase activity with the expression of two different proliferation-specific proteins, Ki-67 and repp86, a protein expressed exclusively in the cell cycle phases S, G2, and M. Telomerase activity was associated with the overall growth fraction (Ki-67) but showed a closer correlation with the expression of repp86. Both telomerase activity and proliferation indices discriminated clearly between malignant melanomas and nevi, but not between common and dysplastic nevi. Nonetheless, a portion of nevi exhibited markedly elevated telomerase activity levels without proportionally increased proliferation. This was independent of discernible morphological changes. Clinicopathological correlations showed an association between high telomerase activity and early metastatic spread in melanomas, linking telomerase to tumor biology. Our results provide arguments in favor of an occasional progression from nevi to melanomas and imply that proliferation measurements in combination with telomerase assays may help to elicit early malignant transformation that is undetectable by conventional morphology. PMID- 10751367 TI - The product of the t(11;18), an API2-MLT fusion, marks nearly half of gastric MALT type lymphomas without large cell proliferation. AB - Recently we demonstrated that the t(11;18)(q21;q21) associated with extranodal marginal zone B cell lymphomas of MALT type results in the expression of a chimeric transcript fusing 5' API2 on chromosome 11 to 3' MLT on chromosome 18. Here we report the development of an RT-PCR approach for the detection of the API2-MLT fusion transcript and its application for the analysis of 58 cases of gastric lymphoma. Initially nested PCR amplification was combined with Southern analysis using internal API2 and MLT probes. A genuine API2-MLT fusion transcript of variable length was demonstrated in 11 out of 58 cases. Sequence analysis revealed that in all cases the breakpoint on chromosome 11 occurred between exons 7 and 8 of the API2 gene. In contrast, the breakpoints on chromosome 18 appeared to be heterogeneous as fusions to bp 814, 1123, and 1150, respectively, of MLT were observed. These observations allowed us to work out a highly sensitive diagnostic test for the API2-MLT fusion on an ABI Prism 7700 sequence detector that confirmed the results of our initial approach. The API2-MLT fusion was found in 48% of gastric marginal zone cell lymphomas of MALT type that did not contain a large cell component and it was lacking in all other lymphomas of the stomach. PMID- 10751368 TI - Chemokine and chemokine-receptor expression in human glial elements: induction by the HIV protein, Tat, and chemokine autoregulation. AB - Human immunodeficiency virus (HIV) encephalitis is a prominent pathology seen in children infected with HIV. Immunohistochemical analyses of pediatric brain tissue showed distinct differences in expression of C-C chemokines and their receptors between children with HIV encephalitis and those with non-CNS-related pathologies. Evidence suggests that soluble factors such as HIV Tat released from HIV-infected cells may have pathogenic effects. Our results show Tat effects on chemokines and their receptors in microglia and astrocytes as well as chemokine autoregulation in these cells. These results provide evidence for the complex interplay of Tat, chemokines, and chemokine receptors in the inflammatory processes of HIV encephalitis and illustrate an important new role for chemokines as autocrine regulators. PMID- 10751371 TI - The role of theory in evidence-based health promotion practice. PMID- 10751369 TI - Biomaterial-induced sarcoma: A novel model to study preneoplastic change. AB - In the study of carcinogenesis most interest has focused on carcinomas, as they represent the majority of human cancers. The recognition of the adenoma-carcinoma sequence both in humans and in animal experimental models has given the field of basic oncology the opportunity to elucidate individual mechanisms in the multistep development of carcinoma. The relative scarcity of human sarcomas coupled with the lack of adequate animal models has hampered understanding of the molecular genetic steps involved. We present an experimental model in the rat in which a high incidence of malignant mesenchymal tumors arise around a subcutaneously implanted biomaterial. Nine commercially available biomaterials were implanted in a total of 490 rats of the Fischer strain for 2 years. On average, macroscopic tumors were found in 25.8% of implantation sites over a period from 26 to 110 weeks after implantation. The most frequent tumors were malignant fibrous histiocytomas and pleomorphic sarcomas, although fibrosarcomas, leiomyosarcomas, and angiosarcomas readily developed, the latter especially around polyurethane implants. Of particular interest are the results of a detailed histological study of the capsules around the implanted biomaterials without tumors. Here a spectrum of change from focal proliferative lesions through preneoplastic proliferation to incipient sarcoma could be observed. A parallel immunohistochemical study of peri-implant capsules showed that proliferating cell nuclear antigen was of particular help in identifying these atypical proliferative lesions. To our knowledge this is the first description of a sarcoma model in which preneoplastic lesions can be readily identified and also reproducibly induced. This model provides the molecular biologist with defined stages in the development of mesenchymal malignancy, with which the multistage tumorigenesis hypothesis can be tested, analogous to the well-known adenoma carcinoma sequence. PMID- 10751370 TI - Overexpression of VEGF 121 in immortalized endothelial cells causes conversion to slowly growing angiosarcoma and high level expression of the VEGF receptors VEGFR 1 and VEGFR-2 in vivo. AB - Vascular endothelial growth factor (VEGF or vascular permeability factor) is an important angiogenic factor that is up-regulated in numerous benign and malignant disorders, including angiosarcoma, hemangiomas, and solid tumors. To determine the functional role of VEGF in the development of endothelial tumors, we expressed primate VEGF 121 in an endothelial cell line, MS1, derived from primary murine cells by immortalization with a temperature-sensitive SV40 large T antigen. This cell line expresses the VEGFR-2 (Flk-1/Kdr) receptor for VEGF. Expression of VEGF 121 led to the development of slowly growing endothelial tumors, which were histologically well-differentiated angiosarcomas. The angiosarcomas generated from MS1 VEGF cells demonstrated up-regulation of the VEGF receptors VEGFR-2 and VEGFR-1 (Flt-1) in vivo compared with benign hemangiomas generated from MS1 cells. Treatment of these cells with the VEGFR-2 tyrosine kinase inhibitor SU 1498 led to decreased expression of ets-1, a transcription factor which has been shown to be stimulated by VEGF. These results suggest that high level expression of VEGF in endothelial cells may result in malignant transformation. This transformation process likely involves both autocrine and paracrine pathways. PMID- 10751373 TI - An attempt to reduce negative stereotyping of obesity in children by changing controllability beliefs. AB - The purpose of the present study was to investigate whether changing children's beliefs about the controllability of obesity would reduce their negative attitudes toward fat people. The participants were 74 children from Grades 4-6, 42 in the experimental group and 32 in the control group. The experimental group were presented with a brief intervention which focussed on the uncontrollability of weight. The study found that the intervention was successful in reducing the amount of controllability that children assigned to obesity, but was not successful in reducing negative stereotyping of the obese among the experimental group compared to the control group. These results indicate that while children's beliefs about the controllability of obesity can be changed, reducing their negative stereotyping is more difficult. PMID- 10751372 TI - Are perceptions of a family history of heart disease related to health-related attitudes and behaviour? AB - It has been argued that perceptions of familial tendencies to disease are common and important in decisions about health-related behaviours. Indeed, it has been suggested that the increased 'geneticization' of society may lead to an increased fatalism about health, which could undermine initiatives aimed at reducing coronary-prone behaviour. To date, much of the research on lay perceptions of inheritance has been based on people at high risk of particular genetic disorders or on qualitative research with small general population samples. Here we investigate perceptions of a family history of heart disease, using quantitative techniques, to test hypotheses about the relationship between a perceived family history (pFH), coronary 'candidacy' and adherence to health promotion advice which were raised by earlier anthropological work. We find that reported perceptions of a family history of heart disease are common, particularly amongst women in middle-age. In isolation a pFH is not related to current smoking; however, the odds of smoking are lower for those with a pFH of heart disease when account is also taken of other attitudinal factors (the 'salience' of heart disease and the strength of adherence to conventional coronary health promotion. PMID- 10751374 TI - 'I'll worry about that when it comes along': osteoporosis, a meaningful issue for women at mid-life? AB - This paper reports findings from a qualitative study of the health concerns and perceptions of health risks and osteoporosis of women in the age group 40-55 years. Osteoporosis has been increasingly put forward in the popular and scientific press as an important issue for women in mid-life. A variety of preventive measures, including use of hormone replacement therapy (HRT), are suggested. The medicalization of women's experiences and associated use of HRT at this point in the life course is the subject of considerable debate in the medical, social scientific and feminist literatures, although, to date, this issue has received less attention in health promotion. Much of this debate is informed by quantitative and survey data, and there is a lack of in-depth qualitative information on women's own views. This study casts doubt on the salience of osteoporosis for women at mid-life. Our qualitative research suggests that, unless they had experiential knowledge which had rendered osteoporosis particularly salient, most women in this study evidenced a surprising degree of disinterest in this health issue. To make sense of this disinterest we examined women's wider accounts of their lives, health and the lifecourse, and the menopause. These findings would appear to present a challenge for those in health promotion who might wish to emphasize early preventive strategies for osteoporosis. PMID- 10751375 TI - Demographic variation in nutrition knowledge in England. AB - This paper describes a nutrition knowledge survey carried out on a cross-section of the adult population of England (n = 1040), looking at knowledge relating to current dietary recommendations, sources of nutrients, healthy food choices and diet-disease links. Serious gaps in knowledge about even the basic recommendations were discovered, and there was much confusion over the relationship between diet and disease. Significant differences in knowledge between socio-demographic groups were found, with men having poorer knowledge than women, and knowledge declining with lower educational level and socio economic status. Possible reasons for these differences and implications for public education campaigns and socio-economic inequalities in health are discussed. PMID- 10751377 TI - Development of an instrument for monitoring adolescent health issues. AB - The aim of the project was to develop a survey instrument to monitor relevant health status and health-related behaviors among secondary school students. The development of the instrument occurred in three main phases: collection of existing surveys, workshops with relevant health professionals and focus groups with adolescents. The topics for inclusion were refined using Health Goals and Targets for Australian Children and Youth and consultations with health professionals, and included alcohol/illicit drug use, smoking, nutrition, exercise, injury, mental health, violence and sexual health. Content validity was demonstrated through a comprehensive literature review, review and application of existing instrumentation, dialog and exchange with health professionals, and focus groups with adolescents. The process of peer review through correspondence with health professionals, and the coordination of workshops and focus groups established face validity. Responses from students also indicated that they interpreted the questions as intended. The instrument was piloted in five secondary schools during class periods. Process evaluation was also conducted to determine the appropriateness of the survey and the procedures used in administering the survey. Feedback from school staff was supportive and favorable with respect to the choice of issues. Reliability was assessed by a test-re-test procedure 2 weeks apart. In general, most of the questions showed moderate to high reliability (kappa > 0.5) indicating agreement of 50% or greater. This instrument was developed as a monitoring instrument and places emphasis on determining prevalence levels of a range of health issues and health behaviors to assist with identifying clustering patterns of negative health outcomes. Although the instrument is primarily for use with students in school hours, the nature of the instrument allows modification for use in older groups of adolescents and out of-school youth. The final version of the questionnaires for senior and junior students can be accessed via the Internet (http://;+www.spmed.uq.edu.au/CHPCPR /qu estionnaire). PMID- 10751376 TI - Tobacco growers' knowledge of revenue distribution and foreign prices: implications for health education. AB - This study examined tobacco growers' knowledge of tobacco economics, particularly their knowledge of how tobacco revenue is distributed and their knowledge of the price of foreign tobacco. Tobacco growers (n = 1236) from 14 tobacco-dependent counties in North Carolina were interviewed by telephone and asked to estimate how much of a hypothetical $2.00 for a pack of cigarettes is received by each of four market segments: tobacco growers, retailers, government and manufacturers. Respondents were also asked to estimate the price of foreign raw tobacco. USDA data were used to assess the accuracy of the respondents' estimates. Respondents were within the 'correct' range as follows: growers 31%, retailers 15%, government 23%, manufacturers 43% and foreign prices 25%. Knowledge was positively related to education and farm size for the growers' share and foreign tobacco prices. Knowledge of the government's and manufacturers' shares was positively associated with younger age. North Carolina growers lacked knowledge of the distribution of the tobacco dollar. This may influence the positions that growers take on health policy and provide significant opportunities for health educators to become involved in grassroots efforts to educate growers. PMID- 10751378 TI - School-based scalds prevention: reaching children and their families. AB - Preventing children's thermal injuries requires changes to both the home environment and the behaviour of family members. Two pilot studies were undertaken of a school-based programme that taught children aged 7-11 years about burns and scalds hazards, and encouraged changes to the home environment and family practices through a take-home exercise. Both studies took place at ethnically diverse schools from low/middle-income areas of Waitakere City, New Zealand. Study 1 involved 55 children who received the programme, and Study 2 involved 64 children who received the programme and 71 children from a control school. The children's ability to identify the burns and scalds hazards illustrated in a series of pictures was measured before and after the programme. Children who received the programme showed considerable improvement in hazard identification at the post-test, while children at the control school showed minimal improvement. The take-home exercise was completed by 85% of the children and their families in Study 1, and 61% of the participants from the intervention school in Study 2. In both studies families reported positive safety changes as a result of the programme. The programme appeared equally effective with all the ethnic groups involved. Future development of the programme is discussed. PMID- 10751380 TI - The practice of community development approaches in heart health promotion. AB - There has been a recent shift in public health policy towards population-based approaches to the reduction of cardiovascular disease. This shift has been accompanied by a re-examination of strategies appropriate to the goal. Often, community development approaches, designed to affect socio-environmental change, are suggested as the most appropriate strategy for affecting community-wide change. Despite the fact that community development approaches have been used by several of the major community-based heart health initiatives, evidence of their use and usefulness remains sparse. This paper presents the findings of a qualitative study of the factors (i.e. community context, facilitators, barriers) affecting the use of community development approaches to heart health promotion in Ontario, Canada. Key informant interviews (n = 30) were conducted with stake holders representing voluntary agencies, community health providers, boards of education and local coalitions in eight of the 42 health unit areas across Ontario. The qualitative analysis reveals (1) that the use of comprehensive community development approaches is limited and (2) that community agencies typically employ elements of community development approaches (e.g. community organization, community-based), often in combination and adapted to suit local conditions. The resulting landscape of community development approaches is characterized by a continuum of collaborative practices indicating that no one type of community approach is appropriate for all initiatives and in all communities. Therefore, from a programmatic perspective, it may not be realistic to advocate community development as the goal to which all communities should strive. PMID- 10751379 TI - Evaluating 'Safe in the Sun', a curriculum programme for primary schools. AB - Eleven schools in the south of England took part in a trial of 'Safe in the Sun', a curriculum programme for primary school aged pupils. Case study methodology and the 'draw and write' technique were combined to evaluate changes in pupils' perceptions of the effects of the sun on their skin. Teachers were free to use the materials, consisting of a teacher's handbook and video, as they wished. Pupils in all schools showed higher levels of awareness of sun-safety measures in post-intervention studies compared with the levels recorded 4 months earlier. Chi square analysis revealed that those pupils whose teachers had used the materials as recommended by the authors had significant increases in awareness and knowledge about keeping safe in the sun, compared with other levels of intervention. More pupils also appeared able to transfer their awareness to the context of the school playground, although the differences were not significant for any level of intervention. The discussion focuses on the value of the 'draw and write' technique as a tool for illuminative evaluation, on the factors which contributed to the pupils increased awareness of sun safety and on the importance of a whole school approach to sun safety. PMID- 10751381 TI - Peptide-directed suppression of a pro-inflammatory cytokine response. AB - Signal-dependent nuclear translocation of transcription factor nuclear factor kappaB (NF-kappaB) is required for the activation of downstream target genes encoding the mediators of immune and inflammatory responses. To inhibit this inducible signaling to the nucleus, we designed a cyclic peptide (cSN50) containing a cell-permeable motif and a cyclized form of the nuclear localization sequence for the p50-NF-kappaB1 subunit of NF-kappaB. When delivered into cultured macrophages treated with the pro-inflammatory agonist lipopolysaccharide, cSN50 was a more efficient inhibitor of NF-kappaB nuclear import than its linear analog. When delivered into mice challenged with lipopolysaccharide, cSN50 potently blocked the production of proinflammatory cytokines (tumor necrosis factor alpha and interferon gamma) and significantly reduced the lethality associated with ensuing endotoxic shock. Based on specificity studies conducted with a mutated form of cSN50, a functional nuclear localization motif is required for this protective effect. Taken together, our findings demonstrate effective targeting of a cell-permeable peptide that attenuates cytokine signaling in vivo. This new class of biological response modifiers may be applicable to the control of systemic inflammatory reactions. PMID- 10751382 TI - Bcl-2 inhibits a Fas-induced conformational change in the Bax N terminus and Bax mitochondrial translocation. AB - Members of the Bcl-2 family of proteins control the cellular commitment to apoptosis, although their role in Fas-induced apoptosis is ill-defined. In this report we demonstrate that activation of the Fas receptor present on a human breast epithelial cell line resulted in a conformational change in the N terminus of the pro-apoptotic protein Bax. This conformational change appeared to occur in the cytosol and precede Bax translocation to the mitochondria. Overexpression of the anti-apoptotic protein Bcl-2 inhibited both the conformational change of Bax as well as its relocalization to the mitochondria. Bcl-2 overexpression did not, however, inhibit Fas-induced cleavage of both procaspase-8 and the pro-apoptotic protein Bid, indicating that Bcl-2 functions downstream of these events. These results suggest that the mechanism by which Bcl-2 inhibits Bax mitochondrial translocation and subsequent amplification of the apoptotic cascade is not by providing a physical barrier to Bax, but rather by inhibiting an upstream event necessary for Bax conformational change. PMID- 10751383 TI - The orientation of helix 4 in apolipoprotein A-I-containing reconstituted high density lipoproteins. AB - The three-dimensional structure of the high density lipoprotein (HDL) component apolipoprotein (apo) A-I and the molecular basis for its protection against coronary artery disease are unknown. In terms of discoidal HDL particles, there has been a debate as to the orientation of the apoA-I alpha-helices around the disc edge. The "picket fence" model states that the alpha-helical repeats, separated by turns, are arranged parallel to the phospholipid acyl chains of the enclosed lipid bilayer. On the other hand, the "belt" model states that the helical segments run perpendicular to the acyl chains. To distinguish between these models, we used nitroxide spin labels present at various depths in the bilayer of reconstituted HDL (rHDL) to measure the position of Trp residues in single Trp mutants of human proapoA-I. Two mutants were studied; the first contained a Trp at position 108, which was located near the center of helix 4. The second contained a Trp at position 115, two turns along the same helix. The picket fence model predicts that these Trp residues should be at different depths in the bilayer, whereas the belt model predicts that they should be at similar depths. Different sized rHDL particles were produced that contained 2, 3, and >4 molecules of proapoA-I per complex. In each case, parallax analysis indicated that Trp-108 and Trp-115 were present at similar depths of about 6 A from the center of the bilayer, consistent with helix 4 being oriented perpendicular to the acyl chains (in agreement with the belt model). Similar experiments showed that control transmembrane peptides were oriented parallel to the acyl chains in vesicles, demonstrating that the method was capable of distinguishing between the two models. This study provides one of the first experimental measurements of the location of an apoA-I helix with respect to the bilayer edge. PMID- 10751384 TI - The calcimimetic R-467 potentiates insulin secretion in pancreatic beta cells by activation of a nonspecific cation channel. AB - The extracellular, G protein-linked Ca(2+)-sensing receptor (CaSR), first identified in the parathyroid gland, is expressed in several tissues and cells and can be activated by Ca(2+) and some other inorganic cations and organic polycations. Calcimimetics such as NPS (R)-N-(3-phenylpropyl)-alpha-methyl-3 methoxybenzylamine hydrochloride (R-467), a phenylalkylamine, are thought to activate CaSR by allosterically increasing the affinity of the receptor for Ca(2+). When tested for its effect on insulin release in C57BL/6 mice, R-467 had no effect under basal conditions but enhanced both phases of glucose-stimulated release. The betaHC9 cell also responded to R-467 and to the enantiomer S-467 with a stimulation of insulin release. In subsequent studies with the betaHC9 cell, it was found that the stimulatory effect was due to activation of a nonspecific cation channel, depolarization of the beta-cell, and increased Ca(2+) entry. No other stimulatory mechanism was uncovered. The depolarization of the cell induced by the calcimimetic could be due to a direct action on the channel or via the CaSR. However, it appeared not to be mediated by G(i), G(o), G(q/11), or G(s). The novel mode of action of the calcimimetic, combined with the glucose dependence of the stimulation on islets, raises the possibility of a totally new class of drugs that will stimulate insulin secretion during hyperglycemia but which will not cause hypoglycemia. PMID- 10751385 TI - Ubiquitination of neuronal nitric-oxide synthase in vitro and in vivo. AB - It is established that suicide inactivation of neuronal nitric-oxide synthase (nNOS) with guanidine compounds, or inhibition of the hsp90-based chaperone system with geldanamycin, leads to the enhanced proteolytic degradation of nNOS. This regulated proteolysis is mediated, in part, by the proteasome. We show here with the use of human embryonic kidney 293 cells transfected with nNOS that inhibition of the proteasome with lactacystin leads to the accumulation of immunodetectable higher molecular mass forms of nNOS. Some of these higher molecular mass forms were immunoprecipitated by an anti-ubiquitin antibody, indicating that they are nNOS-polyubiquitin conjugates. Moreover, the predominant nNOS-ubiquitin conjugate detected in human embryonic kidney 293 cells, as well as in rat brain cytosol, migrates on SDS-polyacrylamide gels with a mobility near that for the native monomer of nNOS and likely represents a conjugate containing a few or perhaps one ubiquitin. Studies in vitro with the use of (125)I-ubiquitin and reticulocyte extracts could mimic this ubiquitination reaction, which was dependent on ATP. The heme-deficient monomeric form of nNOS is preferentially ubiquitinated over that of the heme-sufficient functionally active homodimer. Thus, we have shown for the first time that ubiquitination of nNOS occurs and is likely involved in the regulated proteolytic removal of non-functional enzyme. PMID- 10751386 TI - Equilibrium and kinetic studies of substrate binding to 6-hydroxymethyl-7,8 dihydropterin pyrophosphokinase from Escherichia coli. AB - 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the pyrophosphorylation of 6-hydroxymethyl-7,8-dihydropterin (HMDP) by ATP to form 6 hydroxymethyl-7,8-dihydropterin pyrophosphate, an intermediate in the pathway for folic acid biosynthesis. The enzyme has been identified as a potential target for antimicrobial drugs. Equilibrium binding studies showed that Escherichia coli HPPK-bound ATP or the nonhydrolyzable ATP analogue alpha, beta-methyleneadenosine triphosphate (AMPCPP) with high affinity. The fluorescent ATP analogue 2'(3')-O (N-methylanthraniloyl) adenosine 5'-triphosphate (MANT-ATP) exhibited a substantial fluorescence enhancement upon binding to HPPK, with an equilibrium dissociation constant comparable with that for ATP (10.4 and 4.5 micrometer, respectively). The apoenzyme did not bind the second substrate HMDP, however, unless AMPCPP was present, suggesting that the enzyme binds ATP first, followed by HMDP. Equilibrium titration of HPPK into HMDP and AMPCPP showed an enhancement of fluorescence from the pterin ring of the substrate, and a dissociation constant of 36 nm was deduced for HMDP binding to the HPPK.AMPCPP binary complex. Stopped flow fluorimetry measurements showed that the rate constants for the binding of MANT-ATP and AMPCPP to HPPK were relatively slow (3.9 x 10(5) and 1.05 x 10(5) m(-1) s(-1), respectively) compared with the on rate for binding of HMDP to the HPPK.AMPCPP binary complex. The significance of these results with respect to the crystal structures of HPPK is discussed. PMID- 10751387 TI - Oxidation of methionine residues to methionine sulfoxides does not decrease potential antiatherogenic properties of apolipoprotein A-I. AB - The initial stage of oxidation of high density lipoproteins (HDL) is accompanied by the lipid hydroperoxide-dependent, selective oxidation of two of the three Met residues of apolipoprotein A-I (apoA-I) to Met sulfoxides (Met(O)). Formation of such selectively oxidized apoA-I (i.e. apoA-I(+32)) may affect the antiatherogenic properties of HDL, because it has been suggested that Met(86) and Met(112) are important for cholesterol efflux and Met(148) is involved in the activation of lecithin:cholesterol acyl transferase (LCAT). We therefore determined which Met residues were oxidized in apoA-I(+32) and how such oxidation of apoA-I affects its secondary structure, the affinity for lipids, and its ability to remove lipids from human macrophages. We also assessed the capacity of discoidal reconstituted HDL containing apoA-I(+32) to act as substrate for LCAT, and the dissociation of apoA-I and apoA-I(+32) from reconstituted HDL. Met(86) and Met(112) were present as Met(O), as determined by amino acid sequencing and mass spectrometry of isolated peptides derived from apoA-I(+32). Selective oxidation did not alter the alpha-helicity of lipid-free and lipid-associated apoA-I as assessed by circular dichroism, and the affinity for LCAT was comparable for reconstituted HDL containing apoA-I or apoA-I(+32). Cholesteryl ester transfer protein mediated the dissociation of apoA-I more readily from reconstituted HDL containing apoA-I(+32) than unoxidized apoA-I. Also, compared with native apoA-I, apoA-I(+32) had a 2- to 3-fold greater affinity for lipid (as determined by the rate of clearance of multilamellar phospholipid vesicles) and its ability to cause efflux of [(3)H]cholesterol, [(3)H]phospholipid, and [(14)C]alpha-tocopherol from lipid-laden human monocyte-derived macrophages was significantly enhanced. By contrast, no difference was observed for cholesterol and alpha-tocopherol efflux to lipid-associated apolipoproteins. Together, these results suggest that selective oxidation of Met residues enhances rather than diminishes known antiatherogenic activities of apoA-I, consistent with the overall hypothesis that detoxification of lipid hydroperoxides by HDL is potentially antiatherogenic. PMID- 10751388 TI - GalNAc-alpha -O-benzyl inhibits sialylation of de Novo synthesized apical but not basolateral sialoglycoproteins and blocks lysosomal enzyme processing in a post trans-Golgi network compartment. AB - Glycosylation plays an important role in glycoprotein traffic. Our previous work has shown that long term treatment of mucus-secreting HT-29 cells with GalNAc alpha-O-benzyl reversibly inhibits sialylation and causes the accumulation of apical glycoproteins in cytoplasmic vesicles. We have analyzed at the biochemical level the effects of GalNAc-alpha-O-benzyl on glycoprotein processing. Both apical and basolateral membrane glycoproteins were sialylated, but GalNAc-alpha-O benzyl selectively inhibited the sialylation of apical glycoproteins. In addition, lysosomal alpha-glucosidase, which is partially targeted to the apical membrane, was abnormally processed leading to the accumulation of an immature molecular species. Several findings support the conclusion that accumulation of this protein occurs in a post-trans-Golgi network (TGN) compartment: 1) it is partially sialylated; 2) it does not occur when glycoprotein exit from the TGN is blocked at 20 degrees C; 3) upon Triton X-114 partition, it distributes to the aqueous phase, a characteristic that is acquired in a post-TGN compartment; and 4) its appearance is inhibited when cells are cultured in the presence of NH(4)Cl. The processing of cathepsin D was also found to be affected by GalNAc alpha-O-benzyl treatment. In conclusion, GalNAc-alpha-O-benzyl selectively inhibits sialylation of apical glycoproteins and perturbs lysosomal enzyme processing; these effects occur in a post-TGN acidic compartment and are reminiscent of the alterations found in sialic acid storage diseases. PMID- 10751389 TI - The F420H2 dehydrogenase from Methanosarcina mazei is a Redox-driven proton pump closely related to NADH dehydrogenases. AB - The F(420)H(2) dehydrogenase is part of the energy conserving electron transport system of the methanogenic archaeon Methanosarcina mazei Go1. Here it is shown that cofactor F(420)H(2)-dependent reduction of 2-hydroxyphenazine as catalyzed by the membrane-bound enzyme is coupled to proton translocation across the cytoplasmic membrane, exhibiting a stoichiometry of 0.9 H(+) translocated per two electrons transferred. The electrochemical proton gradient thereby generated was shown to drive ATP synthesis from ADP + P(i). The gene cluster encoding the F(420)H(2) dehydrogenase of M. mazei Go1 comprises 12 genes that are referred to as fpoA, B, C, D, H, I, J, K, L, M, N, and O. Analysis of the deduced amino acid sequences revealed that the enzyme is closely related to proton translocating NADH dehydrogenases of respiratory chains from bacteria (NDH-1) and eukarya (complex I). Like the NADH-dependent enzymes, the F(420)H(2) dehydrogenase is composed of three subcomplexes. The gene products FpoA, H, J, K, L, M, and N are highly hydrophobic and are homologous to subunits that form the membrane integral module of NDH-1. FpoB, C, D, and I have their counterparts in the amphipathic membrane-associated module of NDH-1. Homologues to the hydrophilic NADH-oxidizing input module are not present in M. mazei Go1. Instead, the gene product FpoF may be responsible for F(420)H(2) oxidation and may function as the electron input part. Thus, the F(420)H(2) dehydrogenase from M. mazei Go1 resembles eukaryotic and bacterial proton translocating NADH dehydrogenases in many ways. The enzyme from the methanogenic archaeon functions as a NDH-1/complex I homologue and is equipped with an alternative electron input unit for the oxidation of reduced cofactor F(420) and a modified output module adopted to the reduction of methanophenazine. PMID- 10751390 TI - Pancreatic homeodomain transcription factor IDX1/IPF1 expressed in developing brain regulates somatostatin gene transcription in embryonic neural cells. AB - Hox-like homeodomain proteins play a critical role during embryonic development by regulating the transcription of genes that are important for the generation of specific organs or cell types. The homeodomain transcription factor IDX1/IPF1, the expression of which was thought until recently to be restricted to the pancreas and foregut, is required for pancreas development and for the expression of genes controlling glucose homeostasis. We report that IDX1/IPF1 is also expressed in embryonic rat brain at a time coincident with active neurogenesis. Electrophoretic mobility shift assays with nuclear extracts of embryonic brains indicated that IDX1/IPF1 binds to two somatostatin promoter elements, SMS-UE-B and the recently discovered SMS-TAAT3. The requirement of these elements for IDX1/IPF1 transactivation of the somatostatin gene in neural cells was confirmed in transfection studies using embryonic cerebral cortex-derived RC2.E10 cells. Immunohistochemical staining of rat embryos showed IDX1/IPF1-positive cells located near the ventricular surface in germinative areas of the developing central nervous system. Cellular colocalization of IDX1/IPF1 and somatostatin was found in several areas of the developing brain, including cortex, ganglionic eminence, hypothalamus, and inferior colliculus. These results support the notion that IDX1/IPF1 regulates gene expression during development of the central nervous system independent of its role on pancreas development and function. PMID- 10751391 TI - GABAA and alpha-amino-3-hydroxy-5-methylsoxazole-4-propionate receptors are differentially affected by aging in the rat hippocampus. AB - We have investigated the age-dependent modifications in the expression of eight different subunits of the gamma-aminobutyric acid, type A (GABA(A)) receptor (alpha1, alpha2, alpha3, alpha5, beta2, beta3, gamma2S, and gamma2L) and all four subunits of the alpha-amino-3-hydroxy-5-methylsoxazole-4-propionate (AMPA) receptor (GluR1-4) in the hippocampus of 24-month-old rats. All aged hippocampi displayed a remarkable increase (aged/adult ratio, 3.53 +/- 0.54) in the mRNA levels of the short version of the gamma2 subunit in parallel with a similar increase in the gamma2 subunit protein (aged/adult ratio, 2.90 +/- 0.62). However, this increase was not observed in the mature receptor. On the other hand, the expression of the different alpha subunit mRNAs increased moderately with aging, displaying a heterogeneous pattern. The most frequent modification consisted in an increase in the expression of the alpha1 subunit mRNA (aged/adult ratio, 1.26 +/- 0.18), in parallel with a similar increase on the alpha1 protein (aged/adult ratio, 1. 27 +/- 0.12) and in the alpha1 incorporated to the assembled GABA(A) receptor (tested by immunoprecipitation; aged/adult ratio, = 1.20 +/- 0.10). However, in the same hippocampal samples, no major modifications were observed on the expression of the AMPA receptor subunits. As a whole, these results indicated the existence of an increased expression of the GABA(A) receptor subunits and a preservation of the AMPA receptor at the hippocampal formation. These modifications could reflect the existence of specific deficiencies (neuronal loss and/or deafferentiation) on the GABAergic system in the aged rats. PMID- 10751392 TI - Relationship between expression levels and atherogenesis in scavenger receptor class B, type I transgenics. AB - Both in vitro and in vivo studies of scavenger receptor class B type I (SR-BI) have implicated it as a likely participant in the metabolism of HDL cholesterol. To investigate the effect of SR-BI on atherogenesis, we examined two lines of SR BI transgenic mice with high (10-fold increases) and low (2-fold increases) SR-BI expression in an inbred mouse background hemizygous for a human apolipoprotein (apo) B transgene. Unlike non-HDL cholesterol levels that minimally differed in the various groups of animals, HDL cholesterol levels were inversely related to SR-BI expression. Mice with the low expression SR-BI transgene had a 50% reduction in HDL cholesterol, whereas the high expression SR-BI transgene was associated with 2-fold decreases in HDL cholesterol as well as dramatic alterations in HDL composition and size including the near absence of alpha migrating particles as determined by two-dimensional electrophoresis. The low expression SR-BI/apo B transgenics had more than a 2-fold decrease in the development of diet-induced fatty streak lesions compared with the apo B transgenics (4448 +/- 1908 micrometer(2)/aorta to 10133 +/- 4035 micrometer (2)/aorta; p < 0.001), whereas the high expression SR-BI/apo B transgenics had an atherogenic response similar to that of the apo B transgenics (14692 +/- 7238 micrometer(2)/aorta) but 3-fold greater than the low SR-BI/apo B mice (p < 0.001). The prominent anti-atherogenic effect of moderate SR-BI expression provides in vivo support for the hypothesis that HDL functions to inhibit atherogenesis through its interactions with SR-BI in facilitating reverse cholesterol transport. The failure of the high SR-BI/apo B transgenics to have similar or even greater reductions in atherogenesis suggests that the changes resulting from extremely high SR-BI expression including dramatic changes in lipoproteins may have both pro- and anti-atherogenic consequences, illustrating the complexity of the relationship between SR-BI and atherogenesis. PMID- 10751393 TI - Histidine 20, the crucial proximal axial heme ligand of bacterial heme oxygenase Hmu O from Corynebacterium diphtheriae. AB - The hemin complex of Hmu O, a 24-kDa soluble heme degradation enzyme in Corynebacterium diphtheriae, is coordinated axially to a neutral imidazole of a proximal histidine residue in Hmu O. To identify which of the eight histidines in Hmu O is the proximal heme ligand, we have constructed and expressed the plasmids for eight His --> Ala Hmu O mutants. Reconstituted with hemin, the active site structures and enzymatic activity of these mutants have been examined by EPR, resonance Raman, and optical absorption spectroscopy. EPR of the NO-bound ferrous heme-Hmu O mutant complexes reveals His(20) as the proximal heme ligand in Hmu O, and this is confirmed by resonance Raman results from the ligand-free ferrous heme-H20A. All eight His --> Ala mutants bind hemin stoichiometrically, proving that none of the histidines is essential for hemin-Hmu O formation. However, His(20) is crucial to Hmu O catalysis. Its absence by point mutation has inhibited the conversion of hemin to biliverdin. The ferric heme-H20A complex is pentacoordinate. Resonance Raman of the CO-bound ferrous heme-H20A corroborates this and reveals an Fe-C-O bending mode, delta(Fe-C-O), the first reported for a pentacoordinate CO-bound hemeprotein. The appearance of delta(Fe-C-O) in C. diphtheriae Hmu O H20A but not mammalian HO-1 mutant H25A indicates that the heme environment between the two heme oxygenases is different. PMID- 10751394 TI - Cholesterol movement in Niemann-Pick type C cells and in cells treated with amphiphiles. AB - Cholesterol accumulates to massive levels in cells from Niemann-Pick type C (NP C) patients and in cells treated with class 2 amphiphiles that mimic NP-C disease. This behavior has been attributed to the failure of cholesterol released from ingested low density lipoproteins to exit the lysosomes. However, we now show that the rate of movement of cholesterol from lysosomes to plasma membranes in NP-C cells is at least as great as normal, as was also found previously for amphiphile-treated cells. Furthermore, the lysosomes in these cells filled with plasma membrane cholesterol in the absence of lipoproteins. In addition, we showed that the size of the endoplasmic reticulum cholesterol pool and the set point of the homeostatic sensor of cell cholesterol were approximately normal in NP-C cells. The plasma membrane cholesterol pools in both NP-C and amphiphile treated cells were also normal. Furthermore, the build up of cholesterol in NP-C lysosomes was not a physiological response to cholesterol overload. Rather, it appeared that the accumulation in NP-C lysosomes results from an imbalance in the brisk flow of cholesterol among membrane compartments. In related experiments, we found that NP-C cells did not respond to class 2 amphiphiles (e.g. trifluoperazine, imipramine, and U18666A); these agents may therefore act directly on the NPC1 protein or on its pathway. Finally, we showed that the lysosomal cholesterol pool in NP-C cells was substantially and preferentially reduced by incubating cells with the oxysterols, 25-hydroxycholesterol and 7 ketocholesterol; these findings suggest a new pharmacological approach to the treatment of NP-C disease. PMID- 10751395 TI - Biosynthesis of PF1022A and related cyclooctadepsipeptides. AB - PF1022A belongs to a recently identified class of N-methylated cyclooctadepsipeptides (CODPs) with strong anthelmintic properties. Described here is the cell-free synthesis of this CODP and related structures, as well as the purification and enzymatic characterization of the responsible synthetase. For PF1022A synthesis extracts of Mycelia sterilia were incubated with the precursors L-leucine, D-lactate, D-phenyllactate, and S-adenosyl-L-methionine in the presence of ATP and MgCl(2). A 350-kDa depsipeptide synthetase, PFSYN, responsible for PF1022A synthesis was purified to electrophoretic homogeneity. Like other peptide synthetases, PFSYN follows a thiotemplate mechanism in which the substrates are activated as thioesters via adenylation. N-Methylation of the substrate L-leucine takes place after covalent binding prior to peptide bond formation. The enzyme is capable of synthesizing all known natural cyclooctadepsipeptides of the PF1022 type (A, B, C, and D) differing in the content of D-lactate and D-phenyllactate. In addition to PF1022 types A, B, C, and D, the in vitro incubations produced PF1022F (a CODP consisting of D-lactate and N-methyl-L-leucine), as well as di-, tetra-, and hexa-PF1022 homologs. PFSYN strongly resembles the well documented enniatin synthetase in size and mechanism. Our results suggest that PFSYN, like enniatin synthetase, is an enzyme with two peptide synthetase domains and forms CODP by repeated condensation of dipeptidol building blocks. Due to the low specificity of the d-hydroxy acid binding site, D lactate or D-phenyllactate can be incorporated into the dipeptidols depending on the concentration of these substrates in the reaction mixture. PMID- 10751397 TI - The crystal structure and amino acid sequence of dehaloperoxidase from Amphitrite ornata indicate common ancestry with globins. AB - The full-length, protein coding sequence for dehaloperoxidase was obtained using a reverse genetic approach and a cDNA library from marine worm Amphitrite ornata. The crystal structure of the dehaloperoxidase (DHP) was determined by the multiple isomorphous replacement method and was refined at 1.8-A resolution. The enzyme fold is that of the globin family and, together with the amino acid sequence information, indicates that the enzyme evolved from an ancient oxygen carrier. The peroxidase activity of DHP arose mainly through changes in the positions of the proximal and distal histidines relative to those seen in globins. The structure of a complex of DHP with 4-iodophenol is also reported, and it shows that in contrast to larger heme peroxidases DHP binds organic substrates in the distal cavity. The binding is facilitated by the histidine swinging in and out of the cavity. The modeled position of the oxygen atom bound to the heme suggests that the enzymatic reaction proceeds via direct attack of the oxygen atom on the carbon atom bound to the halogen atom. PMID- 10751396 TI - The mouse mammary tumor virus promoter adopts distinct chromatin structures in human breast cancer cells with and without glucocorticoid receptor. AB - Steroid receptors represent a class of transcription regulators that act in part by overcoming the often repressive nature of chromatin to modulate gene activity. The mouse mammary tumor virus (MMTV) promoter is a useful model for studying transcriptional regulation by steroid hormone receptors in the context of chromatin. The chromatin architecture of the promoter prevents the assembly of basal transcription machinery and binding of ubiquitous transcription factors. However, in human breast carcinoma T47D cells lacking the glucocorticoid receptor (GR), but expressing the progesterone receptor (PR), nucleosome B (nuc B) assumes a constitutively hypersensitive chromatin structure. This correlation led us to test the hypothesis that the chromatin structure of nuc B was dependent on GR expression in T47D cells. To examine this possibility, we stably co-transfected the MMTV promoter and the GR into T47D cells that lacked both the GR and the PR. We found that in T47D cells that lack both the GR and the PR or express only the GR, nuc B assumes a constitutively "open" chromatin structure, which allows hormone independent access by restriction endonucleases and transcription factors. These results suggest that in GR(+)/pr(-) T47D cells, the MMTV chromatin structure permits GR transcriptional activation, independent of chromatin remodeling. PMID- 10751398 TI - Involvement of two NF-kappa B binding elements in tumor necrosis factor alpha -, CD40-, and epstein-barr virus latent membrane protein 1-mediated induction of the cellular inhibitor of apoptosis protein 2 gene. AB - The antiapoptotic function of NF-kappaB is believed to be mediated through the induction of antiapoptotic genes. Among the antiapoptotic genes, cellular inhibitor of apoptosis protein 2 (c-IAP2/HIAP-1/MIHC) is originally identified as a molecule recruited to the tumor necrosis factor (TNF) receptor complex, and its expression is preferentially up-regulated by TNF and other stimuli activating NF kappaB. However, direct evidence of transcriptional regulation of NF-kappaB on the c-IAP2 gene is still missing. Here, we have cloned and characterized the promoter region required for NF-kappaB-dependent transcription of the c-IAP2 gene. Sequencing of a 3.5-kilobase fragment of the 5'-flanking region of the c IAP2 gene has identified a TATA-like sequence and potential binding sites for nuclear factor of activated T cells, interferon regulatory factor 1, activator protein 1, glucocorticoid response element, and three putative NF-kappaB binding elements. Deletion and mutational analysis of the 5'-flanking region linked to the luciferase gene revealed that transcriptional activation by TNF or interleukin 1 is mediated cooperatively by two NF-kappaB binding sites. Electrophoretic mobility shift assays characterized that the two NF-kappaB sites can be recognized and bound by the NF-kappaB p50/p65 heterodimer. In addition, the transcription of c-IAP2 promoter was strongly up-regulated when CD40 or Epstein-Barr virus latent membrane protein 1 was overexpressed. PMID- 10751399 TI - HIV-1 reverse transcriptase-pseudoknot RNA aptamer interaction has a binding affinity in the low picomolar range coupled with high specificity. AB - Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful method for the identification of small oligonucleotides that bind with high affinity and specificity to target proteins. Such DNAs/RNAs are a new class of potential chemotherapeutics that could block the enzymatic activity of pathologically relevant proteins. We have conducted a detailed biochemical study of the interaction of human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) with a SELEX-derived pseudoknot RNA aptamer. Electron paramagnetic resonance spectroscopy of site-directed spin-labeled RT mutants revealed that this aptamer was selected for binding to the "closed" conformation of the enzyme. Kinetic analysis showed that the RNA inhibitor bound to HIV RT in a two-step process, with association rates similar to those described for model DNA/DNA and DNA/RNA substrates. However, the dissociation of the pseudoknot RNA from RT was dramatically slower than observed for model substrates. Equilibrium binding studies revealed an extraordinarily low K(d), of about 25 pm, for the enzyme-aptamer interaction, presumably a consequence of the slow off-rates. Additionally, this pseudoknot aptamer is highly specific for HIV-1 RT, with the closely related HIV-2 enzyme showing a binding affinity close to 4 orders of magnitude lower. PMID- 10751400 TI - Kinetics of manganese lipoxygenase with a catalytic mononuclear redox center. AB - Manganese lipoxygenase was isolated from the take-all fungus, Gaeumannomyces graminis, and the oxygenation mechanism was investigated. A kinetic isotope effect, k(H)/k(D) = 21-24, was observed with [U-(2)H]linoleic acid as a substrate. The relative biosynthesis of (11S)-hydroperoxylinoleate (11S-HPODE) and (13R)-hydroperoxylinoleate (13R-HPODE) was pH-dependent and changed by [U (2)H]linoleic acid. Stopped-flow kinetic traces of linoleic and alpha-linolenic acids indicated catalytic lag times of approximately 45 ms, which were followed by bursts of enzyme activity for approximately 60 ms and then by steady state (k(cat) approximately 26 and approximately 47 s(-1), respectively). 11S-HPODE was isomerized by manganese lipoxygenase to 13R-HPODE and formed from linoleic acid at the same rates (k(cat) 7-9 s(-1)). Catalysis was accompanied by collisional quenching of the long wavelength fluorescence (640-685 nm) by fatty acid substrates and 13R-HPODE. Electron paramagnetic resonance (EPR) of native manganese lipoxygenase showed weak 6-fold hyperfine splitting superimposed on a broad resonance indicating two populations of Mn(II) bound to protein. The addition of linoleic acid decreased both components, and denaturation of the lipoxygenase liberated approximately 0.8 Mn(2+) atoms/lipoxygenase molecule. These observations are consistent with a mononuclear Mn(II) center in the native state, which is converted during catalysis to an EPR silent Mn(III) state. We propose that manganese lipoxygenase has kinetic and redox properties similar to iron lipoxygenases. PMID- 10751401 TI - Human NRAMP2/DMT1, which mediates iron transport across endosomal membranes, is localized to late endosomes and lysosomes in HEp-2 cells. AB - NRAMP2 (natural resistance-associated macrophage protein 2)/DMT1 (divalent metal transporter 1) is a divalent metal transporter conserved from prokaryotes to higher eukaryotes that exhibits an unusually broad substrate range, including Fe(2+), Zn(2+), Mn(2+), Cu(2+), Cd(2+), Co(2+), Ni(2+), and Pb(2+), and mediates active proton-coupled transport. Recently, it has been shown that the microcytic anemia (mk) mouse and the Belgrade (b) rat, which have inherited defects in iron transport that result in iron deficiency anemia, have the same missense mutation (G185R) in Nramp2. These findings strongly suggested that NRAMP2 is the apical membrane iron transporter in intestinal epithelial cells and the endosomal iron transporter in transferrin cycle endosomes of other cells. To investigate the cellular functions of NRAMP2, we generated a polyclonal antibody against the N terminal cytoplasmic domain of human NRAMP2. The affinity-purified anti-NRAMP2 N terminal antibody recognized a 90-116-kDa membrane-associated protein, and this band was shifted to 50 kDa by deglycosylation with peptide N-glycosidase F. Subcellular fractionation revealed that NRAMP2 co-sedimented with the late endosomal and lysosomal membrane proteins and LAMP-1 (lysosome-associated membrane protein 1), but not with the transferrin receptor in early endosomes. The intracellular localization of endogenous NRAMP2 and recombinant green fluorescent protein (GFP)-NRAMP2 was examined by immunofluorescence staining and by native fluorescence of GFP, respectively. Both endogenous and GFP-NRAMP2 were detected in vesicular structures and were colocalized with LAMP-2, but not with EEA1 (early endosome antigen 1) or the transferrin receptor. These results indicated that NRAMP2 is localized to the late endosomes and lysosomes, where NRAMP2 may function to transfer the endosomal free Fe(2+) into the cytoplasm in the transferrin cycle. PMID- 10751402 TI - The activation state of alphavbeta 3 regulates platelet and lymphocyte adhesion to intact and thrombin-cleaved osteopontin. AB - Cleavage of osteopontin by thrombin has been reported to enhance cell adhesion. We asked whether thrombin could regulate the alpha(v)beta(3)-mediated adhesion of platelets and B lymphocytes to this substrate. Although there was no difference in the extent or the avidity of thrombin- and ADP-stimulated platelet adhesion to intact or thrombin-cleaved human osteopontin, both the extent and avidity of phorbol ester-stimulated B cell adhesion to thrombin-cleaved osteopontin was significantly increased. Thus, these data suggest that the ability of alpha(v)beta(3) to recognize osteopontin can be differentially regulated in a cell-specific manner. To localize the alpha(v)beta(3) binding site on osteopontin, we measured cell adhesion to the two thrombin cleavage products of osteopontin and to a series of nested RGD-containing osteopontin peptides cross linked to albumin. Whereas ADP-stimulated platelets adhered to the amino-terminal but not the carboxyl-terminal osteopontin fragment and to the osteopontin peptide RGDSVVYGLR, phorbol ester-stimulated B cells did not adhere to this peptide, although they did so in the presence of 1 mm Mn(2+). Thus, our data confirm that thrombin cleavage enhances the accessibility of the binding motif for alpha(v)beta(3) on osteopontin, but this enhancement is also a function of the activation state of alpha(v)beta(3). Moreover, they indicate that the sequence RGDSVVYGLR contains sufficient information to specify activation-dependent alpha(v)beta(3)-mediated platelet and lymphocyte adhesion. PMID- 10751403 TI - Autoactivation of type-1 parathyroid hormone receptors containing a tethered ligand. AB - Interactions between the N-terminal residues of parathyroid hormone (PTH) and the region of the PTH receptor containing the extracellular loops and transmembrane domains are thought to be critical for receptor activation. We evaluated this hypothesis by replacing the large N-terminal extracellular domain of the human type 1 PTH receptor (hP1Rc-WT) with residues 1-9 of PTH (AVSEIQLMH) using a tetraglycine linker between His-9 of the ligand and Glu-182 of the receptor near the extracellular terminus of transmembrane domain-1. Expression of this construct, hP1Rc-Tether(1-9), in COS-7 cells resulted in basal cAMP levels that were 10-fold higher than those seen in control cells transfected with hP1Rc-WT. Extending the ligand sequence to include Asn-10 and the activity-enhancing substitution of Leu-11 --> Arg yielded hP1Rc-[Arg(11)]Tether(1-11), for which we observed basal cAMP levels that were 50-fold higher than those seen with P1Rc-WT. An alanine-scan analysis of hP1Rc-[Arg(11)]Tether(1-11) revealed that Gln-6 and His-9 were not critical for autoactivation, whereas Val-2, Ile-5, and Met-8 were. The data show that tethered PTH/PTH receptors can autoactivate. Analysis of the structure-activity relationships in these tethered receptor constructs can provide new information concerning how the N-terminal residues of PTH interact with the extracellular loops and transmembrane regions of the PTH-1 receptor, particularly in regard to receptor activation. PMID- 10751404 TI - Overlapping sites of tetratricopeptide repeat protein binding and chaperone activity in heat shock protein 90. AB - The sequential binding of different tetratricopeptide repeat (TPR) proteins to heat shock protein 90 (hsp90) is essential to its chaperone function in vivo. We have previously shown that three basic residues in the TPR domain of PP5 are required for binding to the acidic C-terminal domain of hsp90. We have now tested which acidic residues in this C-terminal domain are required for binding to three different TPR proteins as follows: PP5, FKBP52, and Hop. Mutation of Glu-729, Glu 730, and Asp-732 at the C terminus of hsp90 interfered with binding of all three TPR proteins. Mutation of Glu-720, Asp-722, Asp-723, and Asp-724 inhibited binding of FKBP52 and PP5 but not of Hop. Mutation of Glu-651 and Asp-653 did not affect binding of FKBP52 or PP5 but inhibited both Hop binding and hsp90 chaperone activity. We also found that a conserved Lys residue required for PP5 binding to hsp90 was critical for the binding of FKBP52 but not for the binding of Hop to hsp90. These results suggest distinct but overlapping binding sites on hsp90 for different TPR proteins and indicate that the binding site for Hop, which is associated with hsp90 in intermediate stages of protein folding, overlaps with a site of chaperone activity. PMID- 10751405 TI - Identification of a yeast transcription factor IID subunit, TSG2/TAF48. AB - The RNA polymerase II general transcription factor TFIID is a complex containing the TATA-binding protein (TBP) and associated factors (TAFs). We have used a mutant allele of the gene encoding yeast TAF(II)68/61p to analyze its function in vivo. We provide biochemical and genetic evidence that the C-terminal alpha-helix of TAF(II)68/61p is required for its direct interaction with TBP, the stable incorporation of TBP into the TFIID complex, the integrity of the TFIID complex, and the transcription of most genes in vivo. This is the first evidence that a yeast TAF(II) other than TAF(II)145/130 interacts with TBP, and the implications of this on the interpretation of data obtained studying TAF(II) mutants in vivo are discussed. We have identified a high copy suppressor of the TAF68/61 mutation, TSG2, that has sequence similarity to a region of the SAGA subunit Ada1. We demonstrate that it directly interacts with TAF(II)68/61p in vitro, is a component of TFIID, is required for the stability of the complex in vivo, and is necessary for the transcription of many yeast genes. On the basis of these functions, we propose that Tsg2/TAF(II)48p is the histone 2A-like dimerization partner for the histone 2B-like TAF(II)68/61p in the yeast TFIID complex. PMID- 10751406 TI - Interactions of CCCH zinc finger proteins with mRNA. Binding of tristetraprolin related zinc finger proteins to Au-rich elements and destabilization of mRNA. AB - Macrophages derived from tristetraprolin (TTP)-deficient mice exhibited increased tumor necrosis factor alpha (TNFalpha) release as a consequence of increased stability of TNFalpha mRNA. TTP was then shown to destabilize TNFalpha mRNA after binding directly to the AU-rich region (ARE) of the 3'-untranslated region of the TNFalpha mRNA. In mammals and in Xenopus, TTP is the prototype of a small family of three known zinc finger proteins containing two CCCH zinc fingers spaced 18 amino acids apart; a fourth more distantly related family member has been identified in Xenopus and fish. We show here that representatives of all four family members were able to bind to the TNFalpha ARE in a cell-free system and, in most cases, promote the breakdown of TNFalpha mRNA in intact cells. Because the primary sequences of these CCCH proteins are most closely related in their tandem zinc finger domains, we tested whether various fragments of TTP that contained both zinc fingers resembled the intact protein in these assays. We found that amino- and carboxyl-terminal truncated forms of TTP, as well as a 77 amino acid fragment that contained both zinc fingers, could bind to the TNFalpha ARE in cell-free cross-linking and gel shift assays. In addition, these truncated forms of TTP could also stimulate the apparent deadenylation and/or breakdown of TNFalpha mRNA in intact cells. Alignments of the tandem zinc finger domains from all four groups of homologous proteins have identified invariant residues as well as group-specific signature amino acids that presumably contribute to ARE binding and protein-specific activities, respectively. PMID- 10751408 TI - The allosteric regulation of pyruvate kinase. AB - Pyruvate kinase (PK) is critical for the regulation of the glycolytic pathway. The regulatory properties of Escherichia coli were investigated by mutating six charged residues involved in interdomain salt bridges (Arg(271), Arg(292), Asp(297), and Lys(413)) and in the binding of the allosteric activator (Lys(382) and Arg(431)). Arg(271) and Lys(413) are located at the interface between A and C domains within one subunit. The R271L and K413Q mutant enzymes exhibit altered kinetic properties. In K413Q, there is partial enzyme activation, whereas R271L is characterized by a bias toward the T-state in the allosteric equilibrium. In the T-state, Arg(292) and Asp(297) form an intersubunit salt bridge. The mutants R292D and D297R are totally inactive. The crystal structure of R292D reveals that the mutant enzyme retains the T-state quaternary structure. However, the mutation induces a reorganization of the interface with the creation of a network of interactions similar to that observed in the crystal structures of R-state yeast and M1 PK proteins. Furthermore, in the R292D structure, two loops that are part of the active site are disordered. The K382Q and R431E mutations were designed to probe the binding site for fructose 1, 6-bisphosphate, the allosteric activator. R431E exhibits only slight changes in the regulatory properties. Conversely, K382Q displays a highly altered responsiveness to the activator, suggesting that Lys(382) is involved in both activator binding and allosteric transition mechanism. Taken together, these results support the notion that domain interfaces are critical for the allosteric transition. They couple changes in the tertiary and quaternary structures to alterations in the geometry of the fructose 1, 6-bisphosphate and substrate binding sites. These site-directed mutagenesis data are discussed in the light of the molecular basis for the hereditary nonspherocytic hemolytic anemia, which is caused by mutations in human erythrocyte PK gene. PMID- 10751409 TI - Kinetic and mutational analyses of the regulation of phosphoribulokinase by thioredoxins. AB - Despite little supportive data, differential target protein susceptibility to redox regulation by thioredoxin (Trx) f and Trx m has been invoked to account for two distinct Trxs in chloroplasts. However, this postulate has not been rigorously tested with phosphoribulokinase (PRK), a fulcrum for redox regulation of the Calvin cycle. Prerequisite to Trx studies, the activation of spinach PRK by dithiothreitol, 2-mercaptoethanol, and glutathione was examined. Contrary to prior reports, each activated PRK, but only dithiothreitol supported Trx dependent activation. Comparative kinetics of activation of PRK showed Trx m to be more efficient than Trx f because of its 40% higher V(max) but similar S(0.5). Activations were insensitive to ribulosebisphosphate carboxylase, which may complex with PRK in vivo. To probe the basis for superiority of Trx m, we characterized site-directed mutants of Trx f, in which unique residues in conserved regions were replaced with Trx m counterparts or deleted. These changes generally resulted in V(max) enhancements, the largest (6-fold) of which occurred with T105I, reflective of substitution in a hydrophobic region that opposes the active site. Inclusive of the present study, activation kinetics of several different Trx-regulated enzymes indicate redundancy in the functions of the chloroplastic Trxs. PMID- 10751410 TI - Identification of a new type of mammalian peroxiredoxin that forms an intramolecular disulfide as a reaction intermediate. AB - Peroxidases of the peroxiredoxin (Prx) family contain a Cys residue that is preceded by a conserved sequence in the NH(2)-terminal region. A new type of mammalian Prx, designated PrxV, has now been identified as the result of a data base search with this conserved Cys-containing sequence. The 162-amino acid PrxV shares only approximately 10% sequence identity with previously identified mammalian Prx enzymes and contains Cys residues at positions 73 and 152 in addition to that (Cys(48)) corresponding to the conserved Cys. Analysis of mutant human PrxV proteins in which each of these three Cys residues was individually replaced with serine suggested that the sulfhydryl group of Cys(48) is the site of oxidation by peroxides and that oxidized Cys(48) reacts with the sulfhydryl group of Cys(152) to form an intramolecular disulfide linkage. The oxidized intermediate of PrxV is thus distinct from those of other Prx enzymes, which form either an intermolecular disulfide or a sulfenic acid intermediate. The disulfide formed by PrxV is reduced by thioredoxin but not by glutaredoxin or glutathione. Thus, PrxV mutants lacking Cys(48) or Cys(152) showed no detectable thioredoxin dependent peroxidase activity, whereas mutation of Cys(73) had no effect on activity. Immunoblot analysis revealed that PrxV is widely expressed in rat tissues and cultured mammalian cells and is localized intracellularly to cytosol, mitochondria, and peroxisomes. The peroxidase function of PrxV in vivo was demonstrated by the observations that transient expression of the wild-type protein, but not that of the Cys(48) mutant, in NIH 3T3 cells inhibited H(2)O(2) accumulation and activation of c-Jun NH(2)-terminal kinase induced by tumor necrosis factor-alpha. PMID- 10751411 TI - Identification and characterization of a novel protein from Sertoli cells, PASS1, that associates with mammalian small stress protein hsp27. AB - hsp27 is involved in development of tolerance to stress, possibly by its involvement in molecular chaperoning, maintenance of glutathione status, and/or modulation of microfilament structure and function. We hypothesize that hsp27 function depends on specific association with other proteins. To discover proteins that associate with hsp27, we made a differentiated rat Sertoli cell cDNA expression library and screened it using the yeast two-hybrid system. We obtained a cDNA coding for a novel protein of 428 amino acids that we have named PASS1 (protein associated with small stress proteins 1). BLAST searches did not reveal major similarity of PASS1 to any known protein, but the cDNA sequence matched several mouse EST clones and shares 34% homology with a Caenorhabditis elegans genomic sequence. In vitro, bacterially expressed glutathione S transferase-PASS1 fusion protein bound to hsp27, and hsp27 was co immunoprecipitated with c-Myc-tagged PASS1 overexpressed in several cell lines. The region of PASS1 responsible for association with hsp27 was identified as existing predominantly between amino acids 108 and 208 of PASS1. Northern hybridization and Western blot analysis demonstrated that PASS1 is expressed in several tissues, with the highest expression occurring in testis, primarily in Sertoli cells. The presence of a 1.4-kilobase PASS1 mRNA in kidney as well as the 1. 8-kilobase mRNA seen in other tissues suggests that alternate splicing may occur in this organ. Ectopic expression of PASS1 in two cultured cell lines was observed to inhibit the ability of hsp27 to protect cells against heat shock, indicating that PASS1 does interact with hsp27 in the live cell. PMID- 10751412 TI - The catalytic Tyr-9 of glutathione S-transferase A1-1 controls the dynamics of the C terminus. AB - The glutathione S-transferase enzymes (GSTs) have a tyrosine or serine residue at their active site that hydrogen bonds to and stabilizes the thiolate anion of glutathione, GS(-). The importance of this hydrogen bond is obvious, in light of the enhanced nucleophilicity of GS(-) versus the protonated thiol. Several A class GSTs contain a C-terminal segment that undergoes a ligand-dependent local folding reaction. Here, we demonstrate the effects of the Y9F substitution on binding affinity for glutathione conjugates and on rates of the order-disorder transition of the C terminus in rat GST A1-1. The equilibrium binding affinity of the glutathione conjugate, GS-NBD (NBD-Cl, 7-chloro-4-nitrobenzo-2-oxa-1, 3 diazole), was decreased from 4.09 microm to 0.641 microm upon substitution of Tyr 9 with Phe. This result was supported by isothermal titration calorimetry, with K(d) values of 1.51 microm and 0.391 microm for wild type and Y9F, respectively. The increase in binding affinity for the mutant is associated with dramatic decreases in rates for the C-terminal order-disorder transition, based on a stopped-flow kinetic analysis. The same effects were observed, qualitatively, for a second GSH conjugate, GS-ethacrynic acid. Apparently, the phenolic hydroxyl group of Tyr-9 is critical for orchestrating C-terminal dynamics and efficient product release, in addition to its role in lowering the pK(a) of GSH. PMID- 10751413 TI - Natural resistance of human beta cells toward nitric oxide is mediated by heat shock protein 70. AB - Human beta cells exhibit increased resistance against nitric oxide (NO) radicals as compared with rodent islet cells. Here we tested whether endogenous heat shock protein 70 (hsp70) accounts for the resistance of human cells. Stable transfection of the human beta cell line CM with an antisense hsp70 mRNA expressing plasmid (ashsp70) caused selective suppression (>95%) of spontaneously expressed hsp70 but not of hsc70 or GRP75 protein. ashsp70 transfection abolished the resistance of CM cells to the NO donors (Z)-1- (2-(2-aminoethyl)-N-(2 ammonioethyl)amino)diazen-1-ium -1,2-diolate and sodium nitroprusside and increased the proportions of necrotic cells 3-5-fold (p < 0.05) and of apoptotic cells about 2-fold (p < 0.01). Re-induction of hsp70 expression by heat shock re established resistance to NO toxicity. hsp70 did not exert its protective effect at the level of membrane lipid integrity because radical induced lipid peroxidation appeared independent of hsp70 expression. However, after NO exposure only hsp70-deficient cells showed significantly decreased mitochondrial activity, by 40-80% (p < 0.01). These results suggest a key role of hsp70 in the natural resistance of human beta cells against NO induced injury, by preserving mitochondrial function. These findings provide important implications for the development of beta cell protective strategies in type 1 diabetes and islet transplantation. PMID- 10751414 TI - Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type 2 isoform. AB - Sphingosine-1-phosphate (SPP) has diverse biological functions acting inside cells as a second messenger to regulate proliferation and survival, and extracellularly, as a ligand for G protein-coupled receptors of the endothelial differentiation gene-1 subfamily. Based on sequence homology to murine and human sphingosine kinase-1 (SPHK1), which we recently cloned (Kohama, T., Oliver, A., Edsall, L. , Nagiec, M. M., Dickson, R., and Spiegel, S. (1998) J. Biol. Chem. 273, 23722-23728), we have now cloned a second type of mouse and human sphingosine kinase (mSPHK2 and hSPHK2). mSPHK2 and hSPHK2 encode proteins of 617 and 618 amino acids, respectively, both much larger than SPHK1, and though diverging considerably, both contain the conserved domains found in all SPHK1s. Northern blot analysis revealed that SPHK2 mRNA expression had a strikingly different tissue distribution from that of SPHK1 and appeared later in embryonic development. Expression of SPHK2 in HEK 293 cells resulted in elevated SPP levels. d-erythro-dihydrosphingosine was a better substrate than d-erythro sphingosine for SPHK2. Surprisingly, d, l-threo-dihydrosphingosine was also phosphorylated by SPHK2. In contrast to the inhibitory effects on SPHK1, high salt concentrations markedly stimulated SPHK2. Triton X-100 inhibited SPHK2 and stimulated SPHK1, whereas phosphatidylserine stimulated both type 1 and type 2 SPHK. Thus, SPHK2 is another member of a growing class of sphingolipid kinases that may have novel functions. PMID- 10751416 TI - Oncostatin M and transforming growth factor-beta 1 induce post-translational modification and hyaluronan binding to CD44 in lung-derived epithelial tumor cells. AB - CD44, a receptor for hyaluronan (HA), has been implicated in tumor growth and metastasis. Most CD44-positive cells fail to exhibit constitutive HA receptor function but CD44-mediated HA binding on hematopoetic cells can be induced by antibody cross-linking of the receptor and by physiologic stimuli, including cytokines. We now demonstrate that oncostatin M (OSM) and transforming growth factor-beta1, cytokines known to regulate the growth of tumor cells, stimulate HA binding in lung epithelial-derived tumor cells. In lung epithelial-derived tumor cells, cytokine-induced binding resulted from post-translational modification of the receptor. OSM-induced HA binding was associated with a reduction in N-linked carbohydrate content of CD44. In addition, OSM induced HA binding via a novel mechanism requiring sulfation of chondroitin sulfate chains linked to CD44. The mechanism underlying transforming growth factor-beta1 induced HA binding was distinct from the effects of OSM. The data presented indicate that modulation of the glycosylation and sulfation of CD44 by cytokines provides mechanisms for regulating cell adhesion during tumor growth and metastasis. PMID- 10751418 TI - Chemokines are the main proinflammatory mediators in human monocytes activated by Staphylococcus aureus, peptidoglycan, and endotoxin. AB - It is widely believed that the cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, and IL-6 are the main proinflammatory mediators induced in the host by bacteria and their cell wall components. To test this hypothesis, we compared the level of expression of 600 genes activated in human monocytes by Staphylococcus aureus, peptidoglycan, endotoxin, and interferon-gamma. These stimulants induced expression of over 120 genes, as identified by cDNA arrays. The highest activated genes for proinflammatory mediators induced by all three bacterial stimulants were chemokine genes (IL-8 and macrophage inflammatory protein (MIP)-1alpha), whereas cytokine genes (TNF-alpha, IL-1, and IL-6) were induced to a lower extent. Genes for other chemokines (MIP-2alpha, MIP-1beta, and monocyte chemoattractant protein-1) were also induced higher than the cytokine genes by peptidoglycan, and as high or higher than the cytokine genes by S. aureus and endotoxin. This high induction of chemokine genes was confirmed by quantitative RNase protection assay, and high secretion of chemokines was confirmed by enzyme-linked immunosorbent assays. Although genes for chemokines were the highest and genes for cytokines were the second highest induced genes by all three bacterial stimulants, each stimulus induced a unique pattern of gene expression. By contrast, expression of a completely different gene pattern was induced by a nonbacterial stimulus, interferon-gamma. These results establish chemokines as the main mediators induced by both Gram-positive and Gram-negative bacteria and are consistent with the highly inflammatory nature of bacterial infections. PMID- 10751417 TI - Overexpression of protein-tyrosine phosphatase-1B in adipocytes inhibits insulin stimulated phosphoinositide 3-kinase activity without altering glucose transport or Akt/Protein kinase B activation. AB - Previous studies suggested that protein-tyrosine phosphatase 1B (PTP1B) antagonizes insulin action by catalyzing dephosphorylation of the insulin receptor (IR) and/or other key proteins in the insulin signaling pathway. In adipose tissue and muscle of obese humans and rodents, PTP1B expression is increased, which led to the hypothesis that PTP1B plays a role in the pathogenesis of insulin resistance. Consistent with this, mice in which the PTP1B gene was disrupted exhibit increased insulin sensitivity. To test whether increased expression of PTP1B in an insulin-sensitive cell type could contribute to insulin resistance, we overexpressed wild-type PTP1B in 3T3L1 adipocytes using adenovirus-mediated gene delivery. PTP1B expression was increased approximately 3 5-fold above endogenous levels at 16 h, approximately 14-fold at 40 h, and approximately 20-fold at 72 h post-transduction. Total protein-tyrosine phosphatase activity was increased by 50% at 16 h, 3-4-fold at 40 h, and 5-6-fold at 72 h post-transduction. Compared with control cells, cells expressing high levels of PTP1B showed a 50-60% decrease in maximally insulin-stimulated tyrosyl phosphorylation of IR and insulin receptor substrate-1 (IRS-1) and phosphoinositide 3-kinase (PI3K) activity associated with IRS-1 or with phosphotyrosine. Akt phosphorylation and activity were unchanged. Phosphorylation of p42 and p44 MAP kinase (MAPK) was reduced approximately 32%. Overexpression of PTP1B had no effect on basal, submaximally or maximally (100 nm) insulin stimulated glucose transport or on the EC(50) for transport. Our results suggest that: 1) insulin stimulation of glucose transport in adipocytes requires 0.05). In contrast, tenotomy of the regenerating soleus increased its relative levels of MyHC-2b (P < 0.05) and MyHC-2x/d (P < 0.01), and decreased its relative level of MyHC-1 (P < 0.01). Tenotomy of the synergistic gastrocnemius (overload) tended to decrease the relative level of MyHC-2b in regenerating plantaris (P < 0.07). The effect of tenotomy of the synergistic gastronecmius on the regenerating soleus was different: a decrease in the relative levels of MyHC 1 (P < 0.05) and an increase in the relative level of MyHC-neonatal (P < 0.01). In conclusion, and in contrast to a regenerating slow muscle, a change of mechanical loading by tenotomy did not seem to markedly alter the expression of mature MyHC phenotype in a fast regenerating muscle. PMID- 10751516 TI - Growth induced by incremental static stretch in adult rabbit latissimus dorsi muscle. AB - Incrementally applied static stretch over 3 weeks resulted in a 72 % increase in the weight of the in situ latissimus dorsi muscle in rabbits. True growth rather than tissue oedema was confirmed by increases in the protein content (130 %), the cross-sectional area of the type I fibres (30 %) and the muscle length (i.e. number of sarcomeres in series increased 25 %). Despite an increase in the proportion of fibres staining positive for the enzyme succinate dehydrogenase (SDH), the myosin ATPase stain showed no appreciable fibre type transformation. While total power output in the stretched muscle was unchanged, its maximum mass specific power output, as determined by oscillatory work loops, was decreased by 50 %. The cross-sectional area that was occupied by connective tissue increased from 15 to 19 % in the stretched muscles, with a concomitant increase in passive energy dissipation. Some incrementally stretched muscles were then allowed an additional 3 weeks of maintained stretch to determine whether the adaptive changes would be preserved or reversed. Previous gains in muscle weight, length and area of type I fibres all remained. In contrast, the connective tissue content and the passive properties returned to control values during this period. PMID- 10751517 TI - The role of nitric oxide in the coronary vasoconstriction caused by growth hormone in anaesthetized pigs. AB - Intravenous injection of growth hormone in anaesthetized pigs has been shown to cause coronary vasoconstriction by antagonizing the vasodilatory effects of 2 adrenergic receptors. Because nitric oxide is believed to modulate or mediate 2 adrenergic effects, the present study was undertaken in the same experimental model to determine the role of nitric oxide in the above response to growth hormone. In fourteen pigs anaesthetized with sodium pentobarbitone, changes in left circumflex or anterior descending coronary blood flow caused by intravenous injection of 0.05 i.u. kg-1 of growth hormone at constant heart rate and arterial blood pressure were assessed using electromagnetic flowmeters. In a first control group of six pigs, growth hormone caused a decrease in coronary blood flow which averaged 13.1 % of the baseline values. In a second group of eight pigs, intravenous administration of N-nitro-L-arginine methyl ester (L-NAME) was used to block the endothelial release of nitric oxide. In these pigs, the subsequent injection of growth hormone did not cause any significant changes in coronary blood flow, even when performed after reversing the increase in arterial blood pressure and coronary vascular resistance caused by L-NAME with continuous intravenous infusion of papaverine. These results indicated that the coronary vasoconstricting effect of growth hormone, known to involve antagonism of 2 adrenergic vasodilatory effect, was mediated by inhibition of nitric oxide release. PMID- 10751518 TI - Absence of selective brain cooling in free-ranging zebras in their natural habitat. AB - We used implanted miniature data loggers to measure brain and arterial blood temperatures in three free-ranging zebras (Equus burchelli) in their natural habitat, every 5 min for 9 days. The animals experienced globe temperatures exceeding 40 C, and radiant heat load of about 1000 W m-2. Arterial blood exhibited a moderate amplitude (1.7 C) nychthemeral rhythm, with an acrophase at 19.00 h and a nadir late in the morning, at 10.00 h. Brain temperature consistently exceeded blood temperature, on average by 0.2-0.4 C, and changes in brain temperature closely tracked changes in blood temperature. There was no evidence of selective brain cooling, even during the hyperthermia which followed surgery or that associated with intense, short-duration exercise. The relationship between brain and arterial blood temperatures in free-ranging zebras was unlike that reported for horses in the laboratory. Our results do not support the view that mammals lacking a carotid rete can achieve selective brain cooling. PMID- 10751519 TI - Modelling the influence of age, body size and sex on maximum oxygen uptake in older humans. AB - The purpose of this study was to describe the influence of body size and sex on the decline in maximum oxygen uptake (O2,max) in older men and women. A stratified random sample of 152 men and 146 women, aged 55-86 years, was drawn from the study population. Influence of age on O2,max, independent of differences in body mass (BM) or fat-free mass (FFM), was investigated using the following allometric model: O2,max = BMb (or FFMb) exp(a + (c ' age) + (d ' sex)) [epsilon]. The model was linearised and parameters identified using standard multiple regression. The BM model explained 68.8 % of the variance in O2,max. The parameters (+/- s.e.e., standard error of the estimate) for lnBM (0.563 +/- 0.070), age (-0.0154 +/- 0.0012), sex (0.242 +/- 0.024) and the intercept (-1.09 +/- 0.32) were all significant (P < 0.001). The FFM model explained 69.3 % of the variance in O2,max, and the parameters (+/- s.e.e) lnFFM (0.772 +/- 0.090), age ( 0.0159 +/- 0.0012) and the intercept (-1.57 +/- 0.36) were significant (P < 0.001), while sex (0.077 +/- 0.038) was significant at P = 0.0497. Regardless of the model used, the age-associated decline was similar, with a relative decline of 15 % per decade (0.984 exp(age)) in O2,max in older humans being estimated. The study has demonstrated that, for a randomly drawn sample, the age-related loss in O2,max is determined, in part, by the loss of fat-free body mass. When this factor is accounted for, the loss of O2,max across age is similar in older men and women. PMID- 10751520 TI - Body position and cardiac dynamic and chronotropic responses to steady-state isocapnic hypoxaemia in humans. AB - Neural mediation of the human cardiac response to isocapnic (IC) steady-state hypoxaemia was investigated using coarse-graining spectral analysis of heart rate variability (HRV). Six young adults were exposed in random order to a hypoxia or control protocol, in supine and sitting postures, while end-tidal PCO2 (PET,CO2) was clamped at resting eucapnic levels. An initial 11 min period of euoxia (PET,O2 100 mmHg; 13.3 kPa) was followed by a 22 min exposure to hypoxia (PET,O2 55 mmHg; 7.3 kPa), or continued euoxia (control). Harmonic and fractal powers of HRV were determined for the terminal 400 heart beats in each time period. Ventilation was stimulated (P < 0.05) and cardiac dynamics altered only by exposure to hypoxia. The cardiac interpulse interval was shortened (P < 0.001) similarly during hypoxia in both body positions. Vagally mediated high-frequency harmonic power (Ph) of HRV was decreased by hypoxia only in the supine position, while the fractal dimension, also linked to cardiac vagal control, was decreased in the sitting position (P < 0.05). However, low-frequency harmonic power (Pl) and the HRV indicator of sympathetic activity (Pl/Ph) were not altered by hypoxia in either position. These results suggest that, in humans, tachycardia induced by moderate IC hypoxaemia (arterial O2 saturation Sa,O2 85 %) was mediated by vagal withdrawal, irrespective of body position and resting autonomic balance, while associated changes in HRV were positionally dependent. PMID- 10751521 TI - Telemetry facilitates long-term recording of gastrointestinal myoelectrical activity in pigs. AB - A method of telemetric measurement of antroduodenal myoelectrical activity (EMG) in freely moving pigs has been developed. Silver bipolar electrodes were sutured on the antrum and duodenum and connected to a telemetry transmitter implanted extraperitoneally between the abdominal muscles. The EMG signals were collected by a receiver, filtered, amplified and archived by a computer. The telemetric EMG signal did not differ from that obtained with a conventional wire method. Substantial differences between day and night migrating myoelectric complex (MMC) patterns were found. The advantages of telemetric EMG recording make it a useful tool for long-term recording of antroduodenal EMG. PMID- 10751522 TI - [Information behavior of microsatellite loci in genome scanning]. AB - OBJECTIVE: To determine the heterozygosity and polymorphism information content of 139 microsatellite loci in Han population. METHODS: Multiplex approach was used to analyze the 139 loci. The amplified fragments were subjected to electrophoresis in PAGE gel and analyzed with Genescan( TM) and Genotyper(TM). RESULTS: The heterozygosities are between 0.35 to 0.89, with the heterozygosities of 88% loci >0.60. PICs are in the range of 0.32 and 0.88, with PICs of 95% loci >0.50. CONCLUSION: The polymorphism information content of microsatellite loci is very high and their distribution varies in different races and populations. PMID- 10751523 TI - [Cytogenetics and clinical studies of hematologic malignancies with 11q23 abnormalities in China]. AB - OBJECTIVE: To evaluate the association between 11q23 abnormalities and the clinical, hematologic, prognostic aspects of hematologic malignancies. METHODS: A total of 6000 cases of hematologic malignancies from our hospital and near regions in China were investigated between October 1985 and November 1998. Chromosome preparations were made on bone marrow cells by using direct method and/or unstimulated short-term cultures. Karyotypes were analyzed by R-banding technique and expressed according to ISCN(1995). RESULTS: 11q23 abnormalities were found in 28 of 6000 cases with hematologic malignancies(0.47%). It may be separated into seven cytonetic categories: t(4;11)(q21;q23)(ten cases), t(11;19)(q23;p13)(five cases), t(9;11)(p12;q23)(two cases), t(10;11)(p15;q23)(two cases), t(6;11)(q27;q23) (one case), del(11)(q23)(seven cases), and t(11;?)(q23;?)(one case). The diagnoses included acute myeloid leukemia in 14 cases (M(2a), three; M(4), two; M(5a), one; M(5b), eight), acute lymphoblastic leukemia(ALL) in 10 cases, myelodysplastic syndrome in three cases and malignant histiocytosis in one case. All 10 cases with t(4;11) anomaly were ALL. Follow-up data were available for 22 of them. Their median survival was 113 days. The patients may be grouped according to the presence or absence of additional abnormalities and/or normal karyotype. The median survival was 75 days for five patients with 11q23 anomaly only, 18 days for two patients with 11q23 and additional abnormalities, 135 days for 10 patients with 11q23 anomaly, additional abnormalities and normal karyotype, and 150 days for 5 patients with 11q23 anomaly and normal karyotype. CONCLUSION: 11q23 abnormalities were mainly seen in ALL and acute monocytic leukemia. Their prognosis was very poor. Both additional abnormalities and lack of normal karyotype had adverse effects on the survival of patients. Thus, they represent a unique clinical-cytogenetic association. 11q23 abnormality; hematologic malignancy; acute monocytic leukemia; acute lymphoblastic leukemia; prognosis PMID- 10751524 TI - [The common C677T polymorphism in the methylenetetrahydrofolate reductase gene is associated with neural tube defects and preeclampsia]. AB - OBJECTIVE: To assess whether methylenetetrahydrofolate reductase(MTHFR) gene C677T polymorphism is associated with neural tube defects and preeclampsia. METHODS: Twenty- four mothers who had given birth to normal children, 27 mothers who had given birth to NTDs children, 120 normal women, and 57 women who had suffered from preeclampsia were genotyped for C677T polymorphism by using PCR/RFLP method. RESULTS: (1) VV genotype frequency in the normal mothers group it was 0.13, while in the NTDs mothers group it was 0.33; mothers with VV genotype were at 3 times increased risk to have a NTDs child, compared with the normal mothers. (2) In the normal women group VV genotype frequency was 0.15, while in the preeclampsia women group it was 0.31; women with VV genotype were at 2.5 times increased risk to have preeclampsia, compared with the normal women. CONCLUSION: The authors believe that MTHFR VV genotype is associated with the development of NTDs and preeclampsia. PMID- 10751525 TI - [Bone marrow transplantation for beta-thalassemia major]. AB - OBJECTIVE: To examine the feasibility of bone marrow hematopoietic stem cell transplantation for thalassemia major. METHODS: Bone marrow transplantation was performed on a 4-year-old patient with beta-thalassemia major, whose gene mutation was CD41-42/654. The donor was his brother. In the 850ml bone marrow, the nucleated cells were 5.6x10(8)/kg, CD(+)(34) cells 7.8 x 10(6)/kg, and colony forming-unit-granulocyte-macrophages(CFU-GM) 5.7x10(5)/kg. HLA was one site mismatched, while RBC type matched. Pre-transplant conditions included Busulfan 16mg/kg, cyclophosphamide 200mg/kg and lymphoglobuline 88mg/kg. Cyclosporine and methotrexate were used for prevention of graft-versus-host disease(GVHD). RESULTS: After transplantation, the patient had grade II acute GVHD and cytomegalovirus(CMV) interstitial pneumonia; he was cured. White blood cells(WBC) and neutrophil granulocytes recovered to 1.1 x 10(9)/L and 0.4 x10(9)/L respectively on day 14 post transplantation. WBC was 4.5 x 10(9)/L on day 18, and descended for a time during treating interstitial pneumonia with Ganciclovir. Platelet(Plt) exceeded 50 x 10(9)/L on day 86 and recovered to normal 5 mothers and a half post transplantation. Hb reached 106g/L on day 128. The patient received the last blood transfusion on day 103. Before transplantation, the patient had to receive 200ml blood infusion per month for maintaining Hb 60 90g/L; after transplantation, his Hb kept up >110g/L for 6 months without blood transfusion. The gene type had changed to donor's CD41-42/N heterozygen. CONCLUSION: This is the first report on successful transplantation of bone marrow stem cell for thalassemia major in China, suggesting a new way to treat the disease and to expand the use of BMT. For treatment of CMV interstitial pneumonia post-transplantation, Ganciclovir should be dose enough and be used for at least 2 months. PMID- 10751526 TI - [One of the etiological factors of digestive tract cancers in Chinese: the missense mutation Val384Asp in the hMLH1 gene]. AB - OBJECTIVE: To investigate the etiological role of Val384Asp in the hMLH1 gene, which may be a polymorphism in Chinese, in colorectal, gastric and esophageal cancers. METHODS: Genomic DNA extracted from normal tissues were subjected to analysis in exon 12 of the hMLH1 gene by single strand conformation polymorphism(SSCP) followed by DNA sequencing of aberrant bands in 101 colorectal, 79 gastric and 76 esophageal cancer patients; in 79 and 76 first degree relatives of gastric cancer and esophageal cancer patients respectively; and in 100 healthy control individuals. RESULTS: Six percent of Chinese healthy individuals were the carriers of Val3 84Asp in the hMLH1 gene. There were significant differences in the frequencies of Val384Asp in the hMLH1 gene between the patients with colorectal cancer at young age(<45 years) and the control individuals (P<0.05), and between the gastric cancer patients with family history, the first degree relatives and the control group (P<0.05 and P<0.01 respectively). No difference was found between the esophageal cancer patients, their relatives and healthy controls. CONCLUSION: The alleles frequency of Val384Asp in the hMLH1 gene in Chinese is three percent. This missense mutation may play a part in the etiology of colorectal and gastric cancers in Chinese. PMID- 10751527 TI - [IL-4 gene transfer induces the differentiation of cells and inhibits the activity of telomerase in hepatoblastoma cells]. AB - OBJECTIVE: To investigate the effects of human interleukin-4(hIL-4) gene transfer on the differentiation and the activities of telomerase in hepatoblastoma cells. METHODS: Retroviral vector (PL-IL-4-SN) was employed to introduce hIL-4 gene into human hepatoblastoma cell line(Hep G2)cells. Trypan blue and Wright's stain, radioimmunoassay, in situ hybridization, flow cytometry, PCR-ELISA were used to determine the change in morphology and cell cycle, the expression of proto oncogenes, the synthesis of AFP and the activities of telomerase in IL-4 gene transferred tumor cells. RESULTS: The shape of the hepatoblastoma cells tended to become relatively mature; the growth of the cells was significantly suppressed(P<0.05); the synthesis of AFP was reduced from 15.36+/-0. 67 ng x10(6) cells(-1); x24h(-1); to 3.26 +/- 1.43ng x10(6) cells(-1); x 24h(-1); the proliferation of the cells was significantly suppressed(P<0.05); the cell cycle was arrested at G(0)/ G(1) stage; the expression of c-fos, c-jun, c-myc and the activities of telomerase were remarkably decreased in IL-4 gene-modified Hep G2 cells. CONCLUSION: hIL-4 gene transfer could induce the differentiation of heptoblastoma cells and down-regulate the activity of tolemarase in Hep G2 cells. PMID- 10751528 TI - [Low expression of nm23-H1 mRNA and its protein in breast cancer]. AB - OBJECTIVE: To explore the expression of nm23-H1 mRNA and its protein in breast cancer and its correlation to the biologic behavior. METHODS: RT-PCR and immunohistochemical method were employed to detect the expression of nm23-H1 mRNA and its protein in 58 breast cancer tissues. RESULTS: Low level expression of nm23-H1 mRNA and protein was shown in 44.8% and 48.2% of examined cases respectively. The low expression of nm23-H1 mRNA was correlated, but not always parallel to the protein expression. The low level expression of nm23-H1 mRNA or the protein was significantly associated with lymphatic metastasis of the cancer. CONCLUSION: Examination of nm23-H1 expression may be of significance in evaluating metastatic potentiality and prognosis of breast cancer. PMID- 10751529 TI - [A new potential promoter region of the human connexin 26 gene]. AB - OBJECTIVE: To explain regulatory mechanism of the human connexin 26(Cx26) gene. METHODS: A DNase-1 hypersensitive 1.6 kb fragment upstream of the 5'-terminus of the Cx26 gene was sequenced and assayed with CAT reporter system. RESULTS: The Cx26-1.6 kb sequence serves as a powerful promoter. CONCLUSION: The Cx26-1.6 kb fragment contains two GT boxes (centering at-6158 and -6213 bp), and a TATA-less TTAAAA box (-6237/-6232 bp) which is another promoter region of the human Cx26. PMID- 10751530 TI - [The association between A55V variant in UCP2 gene and body fat distribution, serum lipid profile in Chinese]. AB - OBJECTIVE: Uncoupling protein 2(UCP2) could play an important role in energy metabolism and body weight regulation. The aim of this study was to investigate Ala55Val(A55V) variant in the UCP2 gene has effects on serum lipid profile, body fat and its distribution in Chinese. METHODS: The genotypes of A55V variant in the UCP2 gene were determined by a PCR-RFLP assay in 359 unrelated Chinese [including 193 normal glucose tolerance(NGT) and 166 type 2 diabetic subjects by ADA 97' criteria]. The parameters for regional adipose tissue distribution were measured by magnetic resonance imaging(MRI). RESULTS: In NGT group, an association between A55V variant in the UCP2 gene and body mass index(BMI) (P=0.0246), as well as femoral subcutaneous adipose tissue area (FA) (P=0.0017), was noted in females. A55V variant in the UCP2 gene was also associated with serum triglyceride (TG) level (P=0.0072) in males. However, in type 2 diabetes group, an association between A55V variant in the UCP2 gene and FA (P=0.0150) was replicated in females too. Those females who were homozygotes of AA in the UCP2 gene had decreased FA not only in NGT group but also in type 2 diabetes group. Furthermore, logistic regression analysis indicated that FA (P=0.0098 in NGT females, P=0.0071 in type 2 diabetic females) and BMI (P=0.0016 in NGT females), as well as TG level (P=0.0040 in NGT males) were associated with this variant in the UCP2 gene. CONCLUSION: A55V variant in the UCP2 gene is associated not only with FA (in NGT females and type 2 diabetic females) but also with BMI (in NGT females). Therefore A55V variant in the UCP2 gene appears to play a role in body fat and its distribution in Chinese females. PMID- 10751531 TI - [Study on the p53 gene mutation and microsatellite instability of gastric carcinoma]. AB - OBJECTIVE: To observe the relationship between p53 gene mutation and microsatellite instability (MSI) of the preoperative staging of gastric carcinoma by endoscopic ultrasonography(EUS). METHODS: A total of 73 cases of gastric carcinoma were taken for the preoperative staging with EUS. Silver staining PCR SSCP method was used to detect mutations in exons 5, 6, 7, 8 of p53 gene and MSI at 4 loci on chromosomes 2, 5, 17 in the 73 paraffin-embedded biopsy specimens, and the relationship between them was studied further. RESULTS: The overall mutated rate of p53 gene was 54.8%, with 6.8%, 15.1%, 19.2% and 13.7% in exons 5 to 8, respectively. Analysis of the relation of the mutation with the preoperative staging by EUS showed that it was significantly higher in T3(64.3%) and T4(67.9%) than in T1(0) or T2(25.0%)(P<0.05), and it was 67.6% in cases with lymph node metastasis, which was significantly higher than that without metastasis (41.7%)(P<0.05). The overall detective rate of MSI was 37.0%. The detective rate of MSI had no relation with the depth of invasion, which was 40.0%, 33.3%, 39.3% and 34.5% at T1 to T4 stages (P<0.05), respectively, but it was significantly higher in cases with lymph node metastasis(51.4%) than those without metastasis(22.2%)(P<0.05). Although the detective rate of MSI showed an increasing trend in the mutated cases of p53 gene as compared with the no mutation cases, there was no relationship between MSI and p53 gene mutation (P<0.05). CONCLUSION: These results suggest that the gene mutation of p53 and MSI may represent the different mechanisms of carcinogenesis. They can reflect the cytobiologic malignant behavior of gastric carcinoma in varying degrees, which may be of reference significance in analysing the prognosis of the patients in clinics. PMID- 10751532 TI - [Effect of lipoprotein lipase gene polymorphism on plasma lipid levels,BMI and subcutaneous fat distribution in simple obesity children]. AB - OBJECTIVE: To assess the effect of Hind III DNA polymorphism in the lipoprotein lipase(LPL) gene on plasma lipid levels, body mass index(BMI) and subcutaneous fat distribution in simple obesity children. METHODS: The polymerase chain reaction(PCR) and restriction fragment length polymorphism(RFLP) techniques were used to detect the Hind III-LPL genotypes in 92 children with simple obesity. The levels of the plasma lipid, plasma lipoproteins, BMI and skinfold thickness at three measuring points(biceps, subscapular and abdominal wall) were also measured. RESULTS: The levels of TG, TC, LDL-C, Apo B, BMI, biceps and subscapular skinfold thickness with the average value of three measuring points in the obesity children with H(+) H(+)-LPL genotype were significantly higher than those in the obesity children with H(+) H(-)-LPL genotype. CONCLUSION: LPL Hind III polymorphism may modify the levels of plasma lipid. plasma lipoproteins and BMI in children with simple obesity, and in the mean while it may affect the distribution of subcutaneous fat. PMID- 10751533 TI - [The application of polymerase chain reaction-single strain conformation polymorphism in the pedigree analysis of familial hypercholerolemia patients]. AB - OBJECTIVE: To discuss the value of polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) in the pedigree analysis of familial hypercholesterolemia(FH) patients. METHODS: For 4 patients with FH detected by PCR-SSCP and DNA sequence analysis (1 homozygote has point mutation in exon 7, 1 heterozygote in exon 14, and 2 heterozygotes in 3' part of exon 4 ), members of each pedigree, altogether 23 individuals, were analysed using PCR-SSCP. RESULTS: Every member of the 4 pedigrees was examined genetically. Besides the 4 probands, another 1 homozygote and 8 heterozygotes were found. CONCLUSION: The PCR-SSCP method can be used in the pedigree analysis of FH probands. The early diagnosis of siblings and relatives can help to provide genetic consultation and direction so as to pay attention to development of hypercholesterolemia. PMID- 10751534 TI - [Genetic polymorphism of 5 STR loci on chromosome 14 in Chinese Han]. AB - OBJECTIVE: To understand the distribution of genes and genotypes of 5 STR loci on chromosome 14 in Chinese Han. METHODS: PCR and polyacrylamide gel electrophoresis were used to analyze the polymorphism of 5 STR loci (D14S742, D14S306, D14S606, D14S617, D14S611) on chromosome 14 in Chinese Han. RESULTS: 5 alleles and 13 genotypes, 5 alleles and 13 genotypes, 6 alleles and 13 genotypes, 9 alleles and 21 genotypes, and 6 alleles and 13 genotypes were observed at D14S742, D14S306, D14S606, D14S617 and D14S611, respectively. The frequencies of the most common allele at these five loci were 0.31, 0.31, 0.47, 0.35 and 0.29 respectively. CONCLUSION: These five loci are highly polymorphic in Chinese Han and their allele distribution is in good agreement with Hardy-Weinberg equilibrium. PMID- 10751535 TI - [A molecular cytogenetic study on chromosome anomalies of chronic myeloid leukemia]. AB - OBJECTIVE: To study the bcr/abl fusion gene in chronic myeloid leukemia (CML) cases with Philadelphia chromosome(Ph) negative, and variant Ph translocations and other chromosome anomalies. METHODS: The status of bcr/abl fusion gene in 8 kinds of chromosome anomalies was tested with dual-color fluorescence in situ hybridization (D-FISH) technique. RESULTS: The positive hybridization was demonstrated in 3 cases of variant Ph CML and other 7 cases of standard Ph, respectively. In 2 cases of Ph-negative CML, one case that had karyotype such as 46,XY,t(9;18) showed positive hybridization, the other case with 46,XY,inv(2) showed negative result. CONCLUSION: The result demonstrates that D-FISH technique is reliable for detecting bcr/abl fusion gene. The existence of negative bcr/abl fusion gene CML implies that some other initial events inducing CML might have occurred in such cases. PMID- 10751537 TI - [The influence of some factors on DNA cycle sequencing]. AB - OBJECTIVE: To study the influence of some factors on DNA cycle sequencing. METHODS: The effects of DNA templates, primers, cycle sequencing reaction conditions as well as purification methods were comparatively analyzed. RESULTS: When the DNA concentration was low, the nucleotide curve showed low ratio of signal to noise, even there were no fluorescent signal. While DNA concentration was too high, the readable length of nucleotides was short. Ions in the DNA template could result in bad sequencing reaction. The Tm value, length and G+C content of primers had no obvious influences on sequencing reaction. The alteration of the denaturing, annealing and extension temperature, or the addition of dimethl sulfoxide or glycerin facilitated sequencing some DNA templates. The fluorescent residues in the purified products of the sequence reaction could interfere the automatic reading of the sequencer, but did not influence the manually proofreading of the sequence. CONCLUSION: The purity and concentration of DNA templates are closely related to sequence data quality. The modification of reaction parameters and usage of additives can help to obtain good result of sequencing some DNA with certain structure. The use of 70% ethanol is recommended to precipitate the extension product. PMID- 10751536 TI - [Effect of laminin antisense RNA on the expression of five intracellular proteins stimulated by transforming growth factor beta(1)]. AB - OBJECTIVE: To investigate the effect of laminin antisense RNA on laminin antisense RNA on the expression of E-cadherin, alpha-, beta-, gamma-catenins and focal adhesion kinase(FAK) stimulated by transforming growth factor beta(1)(TGFbeta(1)). METHODS: Western blot technique was used to study their expression at protein level with ECL reagent. RESULTS: The expression of alpha- and gamma-catenins was up regulated by TGF beta(1) without the interference of laminin antisense RNA. The up regulation of E-cadherin stimulated by TGFbeta(1) was inhibited in the presence of laminin antisense RNA. No change was found in the expression of FAK when stimulated by TGFbeta(1), alone, but its expression was down regulated with the help of laminin antisense RNA. CONCLUSION: The inhibition of TGFbeta (1) on the malignancy of colon cancer cell may partially be mediated by the up regulation of E-cadherin and catenins. The effect of TGFbeta(1) on the expression of E-cadherin and FAK may be the secondary results of the change in laminin expression. PMID- 10751538 TI - [Development of a mouse cell line containing stably integrated copies of pMCLacI/Neo plasmid: a model for studying mutations in vitro]. AB - OBJECTIVE: To establish a suitable model for studying the different mechanisms of mutation between expressed and non-expressed genes in mammalian cells. METHODS: The NIH3T3 cells were transfected with the linearized pMCLacI/Neo DNAs by liposome-mediated transfection, and grew in the presence of G418. One drug resistant cell clone was selected to proliferate and to be analyzed with Southern blot and RT-PCR analyses on its genomic DNAs. RESULTS: (1) Multiple copies of pMCLacI/Neo plasmid DNA were intactly integrated in the genomic DNAs of the cell clone. (2) One of lac I target genes in the integrated plasmid could be transcribed in the NIH3T3 cells while the other could not. (3) The pMCLacI/Neo plasmid DNA could be efficiently rescued from the genomic DNAs of the cell clone with the average rescue efficiency of 410 cfu/microg DNA. CONCLUSION: The NIH3T3 cell line containing copies of a stably integrated pMCLacI/Neo has been established. The two lacI target genes in the cell line could imitate the functional states of expressed and non-expressed genes in mammalian cells respectively. The cell line will be a useful model for studying the different mechanisms of mutation between expressed and non-expressed genes in mammalian cells. PMID- 10751539 TI - [Amplification and sequence of human CD14 gene from human mononuclear cell in peripheral blood]. AB - OBJECTIVE: To introduce a new method for getting human CD14 gene. METHODS: Based on the character of CD14 gene, the authors amplified the CD14 gene directly from genome DNA of human mononuclear cell in peripheral blood by PCR for the first time. RESULTS: A human CD14 gene about 1.1 kb was successfully amplified from genome DNA of human mononuclear cell in peripheral blood, and the sequence analysis has indicated that the CD14 gene cloned in this way is the same as the sequence reported by other authors. CONCLUSION: It is a simple and effective method to obtain human CD14 gene from genome DNA of human mononuclear cell in peripheral blood. PMID- 10751540 TI - [Progress in researches on the function of PML gene]. AB - This paper was aimed to review and expound the progreses in studies on biological fuction of wild-type PML gene and hence to have a gain in knowledge about the carcinogenesis of PML-RARalpha protein. Related reports issued at home and abroad and some research results from this institute were collected and reviewed. According to the data collected PML gene has many biological activites. High expression of PML gene can inhibit the growth of cells and induce apoptosis; PML gene is possibly involved in the regulation of gene expression. Moreover, the function of PML gene is related to its three important domains and its nuclear location; PML gene is associated with carcinogenesis of APL. PMID- 10751541 TI - Advances in immunopharmacology of asthma. AB - Asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness and recurrent reversible airway obstruction. As there appears to be a preponderance of T-helper 2 (Th2) cells over Th1 cells in asthma, more attention has been focused on the role of Th2-derived cytokines such as interleukin (IL)-4 and IL-5 and their corresponding signaling pathways in the pathophysiology of the disease. These complex pathways may involve the activation of signal transducers and activators of transcription (STATs) and nuclear factor kappaB (NF-kappaB). On the other hand, immunoglobulin (Ig) E-mediated mechanisms and the protein tyrosine kinase signaling cascade are important in triggering the release of mediators from inflammatory cells. In spite of all of these, host regulatory mechanisms exist to limit the inflammation. An increase in the 3', 5' cyclic adenosine monophosphate (cAMP) level generally suppresses the activities of immune and inflammatory cells, and the level of cAMP is closely regulated by a family of phosphodiesterases (PDEs). Heparin, a glycosaminoglycan released exclusively from mast cells, also is believed to possess anti-inflammatory actions. Many new therapeutic agents have been developed either to attenuate the pro-inflammatory processes in asthma or to augment the host anti-inflammatory mechanisms. In this article, we discuss the immunopharmacology of several of these agents, which include heparin and inhibitors of PDEs, tyrosine kinases, and NF-kappaB, as well as antibodies and soluble receptors directed against IgE, IL 4, and IL-5. PMID- 10751542 TI - Inactivation of artemisinin by thalassemic erythrocytes. AB - Plasmodium falciparum infecting alpha-thalassemic erythrocytes (Hb H or Hb H/Hb Constant Spring) is resistant to artemisinin derivatives. Similar resistance, albeit at a much lower level, is shown by the parasite infecting beta thalassemia/Hb E erythrocytes. The resistance is due to host-specific factors, one of which is the higher uptake of the drugs by thalassemic erythrocytes than normal erythrocytes, due to binding with Hb H. In addition to higher drug binding, incubation of artemisinin with alpha-thalassemic erythrocytes resulted in preferential inactivation of the drug. Both thalassemic and normal erythrocytes have the capability to inactivate the drug. Addition of serum can protect against inactivation by normal erythrocytes, but not by thalassemic erythrocytes. Incubation with either the hemolysate or the membrane fraction from these erythrocytes also resulted in preferential inactivation of the drug. The drug was also inactivated by purified Hb H. It is concluded that the ineffectiveness of artemisinin derivatives against P. falciparum infecting thalassemic erythrocytes is due partly to competition of the host cell components for binding with the drugs, and partly to inactivation of the drugs by the cell components. PMID- 10751543 TI - On the mechanism of plasmin-induced platelet aggregation. Implications of the dual role of granule ADP. AB - Plasmin-induced platelet aggregation has been considered to be a cause of reocclusion after thrombolytic treatment with plasminogen activators. However, little is known regarding the mechanism and regulation of plasmin-induced platelet aggregation. In this study, we demonstrated that plasmin causes the degranulation of platelets, and that ADP released from granules plays a crucial role in the induction of platelet aggregation. This conclusion is supported by results showing that both ADP antagonists and ADPase can inhibit the effect of plasmin on platelets. We also demonstrated that pretreatment of platelets with ADP makes the platelets more sensitive to plasmin, and plasmin-induced platelet aggregation is, therefore, observed at lower concentrations where no aggregation occurs in quiescent platelets. In other words, it is thought that ADP potentiates the plasmin-induced aggregation. The effect of ADP was inhibited by N(6)-[2 (methylthio)-ethyl]-2-(3,3, 3-trifluoropropyl)thio-5'-adenylic acid, monoanhydride with dichloromethylenebisphosphonic acid (AR-C69931), a selective antagonist for the P2T(AC) subtype of P2 receptor, but not by the P2Y1 receptor selective antagonist adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS). The P2X1 receptor agonist alpha, beta-methylene adenosine 5'-triphosphate (alpha,beta MeATP) did not mimic the action of ADP. These data indicate that ADP potentiates plasmin-induced platelet aggregation via the P2T(AC) receptor. In addition, epinephrine, a typical G(i) agonist against platelets, could potentiate the plasmin-induced platelet aggregation, suggesting that the signal via the G(i) protein is involved in potentiating the plasmin-induced platelet aggregation, ADP is secreted from platelet granules, and concomitantly works in conjunction with plasmin in a P2T(AC) receptor-mediated manner. PMID- 10751544 TI - Carnitine transport into muscular cells. Inhibition of transport and cell growth by mildronate. AB - Carnitine is involved in the transfer of fatty acids across mitochondrial membranes. Carnitine is found in dairy and meat products, but is also biosynthesized from lysine and methionine via a process that, in rat, takes place essentially in the liver. After intestinal absorption or hepatic biosynthesis, carnitine is transferred to organs whose metabolism is dependent on fatty acid oxidation, such as heart and skeletal muscle. In skeletal muscle, carnitine concentration was found to be 50 times higher than in the plasma, implicating an active transport system for carnitine. In this study, we characterized this transport in isolated rat myotubes, established mouse C2C12 myoblastic cells, and rat myotube plasma membranes and found that it was Na(+)-dependent and partly inhibited by a Na(+)/K(+) ATPase inhibitor. L-carnitine analogues such as D carnitine and gamma-butyrobetaine interfere with this system as does acyl carnitine. Among these inhibitors, the most potent was mildronate (3-(2,2,2 trimethylhydrazinium)propionate), known as a gamma-butyrobetaine hydroxylase inhibitor. It also induced a marked decrease in carnitine transport into muscle cells. Removal of carnitine or treatment with mildronate induced growth inhibition of cultured C2C12 myoblastic cells. These data suggest that myoblast growth and/or differentiation is dependent upon the presence of carnitine. PMID- 10751545 TI - Protection of erythrocytes against oxidative damage and autologous immunoglobulin G (IgG) binding by iron chelator fluor-benzoil-pyridoxal hydrazone. AB - Iron is released in a free desferrioxamine-chelatable form when erythrocytes are challenged by an oxidative stress. The release of iron is believed to play an important role in inducing destructive damage (lipid peroxidation and hemolysis) or in producing membrane protein oxidation and generation of senescent cell antigens (SCA). In this report, we further tested the hypothesis that intracellular chelation of iron released under conditions of oxidative stress prevents erythrocyte damage or SCA formation. Fluor-benzoil-pyridoxal hydrazone (FBPH), an iron-chelating molecule of the family of aromatic hydrazones, was prepared by synthesis and used for the above purpose after the capacity of the product to enter cells had been ascertained. GSH-depleted mouse erythrocytes were incubated with the oxidant drug phenylhydrazine in order to produce iron release, lipid peroxidation, and hemolysis. FBPH at a concentration of 200 microM prevented lipid peroxidation and hemolysis in spite of equal values of iron release. FBPH was active even at a lower concentration (100 microM) when the erythrocytes were preincubated with it for 15 min. No preventive effect was seen when FBPH saturated with iron was used. Prolonged aerobic incubation (60 hr) of erythrocytes produced iron release and formation of SCA as determined by autologous immunoglobulin G (IgG) binding. The IgG binding was detected by using an anti-IgG antibody labeled with fluorescein and by examining the cells for fluorescence by confocal microscopy. FBPH prevented SCA formation in a dose related manner. These results lend further support to the hypothesis that iron release is a key factor in erythrocyte ageing. PMID- 10751546 TI - Role of glutathione in the biliary excretion of the arsenical drugs trimelarsan and melarsoprol. AB - After administration of the inorganic sodium arsenite or arsenate to rats, the biliary excretion of arsenic is rapid, is accompanied by the biliary output of large amounts of GSH, and is completely arrested by the GSH depletor diethyl maleate (DEM). We studied the biliary excretion of trimelarsan (TMA) and melarsoprol (MAP) in rats in order to determine whether biliary excretion is also significant in the disposition of these trivalent organic arsenicals that are used as therapeutic agents and whether GSH is also involved in their hepatobiliary transport. After injection of either drug (100 micromol/kg, i.v.), arsenic was rapidly excreted in bile (up to 1 micromol/kg. min, approximately 55% of dose/100 min). Concurrently, TMA and MAP increased the biliary output of GSH 3 and 6 fold, and lowered the hepatic GSH content by 24% and 27%, respectively. In TMA-injected rats, pretreatment with DEM or buthionine sulfoximine decreased the initial biliary excretion of arsenic by 75% and 40%, respectively, whereas in MAP injected rats these GSH depletors diminished arsenic output by 45% and 20%. Both arsenicals reacted with GSH in vitro, giving rise to the same product, which was also shown by HPLC analysis to be a major biliary metabolite of both TMA and MAP. This metabolite was sensitive to gamma-glutamyltranspeptidase in vitro and its biliary excretion was virtually prevented by the GSH depletors, confirming that it is a GSH conjugate (purportedly melarsen-diglutathione). Some TMA was excreted in the bile unchanged, whereas a significant amount of MAP also appeared there as two glucuronides. The biliary excretion of unchanged TMA and MAP glucuronides was increased by experimental depletion of GSH. These studies indicate that the biliary excretion of TMA and MAP (1) is very significant in their disposition, (2) is partially dependent on the hepatic availability of GSH, as these arsenicals are excreted in part as a GSH conjugate, and (3) is concomitant with the increased appearance of GSH in bile, probably originating from dissociation of the unstable GSH conjugate of these arsenicals. Thus, conjugation with GSH is important in the elimination of both TMA and MAP, although glucuronidation is also involved in the fate of MAP. PMID- 10751547 TI - Pharmacological profile of apigenin, a flavonoid isolated from Matricaria chamomilla. AB - Dried flowers of Matricaria chamomilla L. are largely used to provide sedative as well as spasmolytic effects. In the present study, we examined in particular the pharmacological property of a fraction isolated from a methanolic extract of M. chamomilla, which was identified by HPLC-MS-MS analysis as apigenin. By radioreceptor binding assays, we demonstrated the ability of the flavone to displace a specific radioligand, [(3)H]Ro 15-1788, from the central benzodiazepine binding site. Electrophysiological studies performed on cultured cerebellar granule cells showed that apigenin reduced GABA (gamma-aminobutyric acid)-activated Cl(-) currents in a dose-dependent fashion. The effect was blocked by co-application of Ro 15-1788, a specific benzodiazepine receptor antagonist. Accordingly, apigenin reduced the latency in the onset of picrotoxin induced convulsions. Moreover, apigenin injected i.p. in rats reduced locomotor activity, but did not demonstrate anxiolytic, myorelaxant, or anticonvulsant activities. The present results seem to suggest that the inhibitory activity of apigenin on locomotor behaviour in rats cannot be ascribed to an interaction with GABA(A)-benzodiazepine receptor but to other neurotransmission systems, since it is not blocked by Ro 15-1788. PMID- 10751548 TI - Relationship of mu opioid receptor binding to activation of G-proteins in specific rat brain regions. AB - This study investigated the relationship between mu receptor binding and mu agonist activation of G-proteins in the rat brain. To directly compare agonist potencies in receptor binding (K(i) values) and G-protein activation (K(s) values), both agonist-stimulated [(35)S]guanosine-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) and [(3)H]naloxone binding assays were conducted under identical conditions, using the full mu agonist [d-Ala(2), N-Me(4), Gly(5)-ol] enkephalin (DAMGO). DAMGO exhibited biphasic competition of [(3)H]naloxone binding and stimulation of [(35)S]GTPgammaS binding in most regions. Whereas the high-affinity component represented a large percentage (50-80%) of total receptor sites, the high-affinity component of DAMGO-stimulated [(35)S]GTPgammaS binding was much lower, <30% of the total, and in most regions significant stimulation of [(35)S]GTPgammaS binding did not occur until the high-affinity binding sites were completely occupied. Moreover, the low-affinity potencies for DAMGO in receptor binding and G-protein activation were the same across different regions. Receptor transducer amplification factors were calculated by the ratio of the apparent B(max) of net agonist-stimulated [(35)S]GTPgammaS binding to the B(max) of receptor binding. Amplification factors for the nine regions examined were relatively high and varied significantly across regions, from a ratio of 8 in the thalamus to 38 in the cortex, suggesting that the efficiency of mu opioid receptor coupling to G-proteins varies across brain regions. PMID- 10751549 TI - Capsaicin-stimulated release of substance P from cultured dorsal root ganglion neurons: involvement of two distinct mechanisms. AB - Capsaicin, the pungent component of "hot" chili peppers, selectively activates a distinct population of primary sensory neurons responsive to noxious stimuli. Many of these fibres express neuropeptides including the tachykinin, substance P. Using cultured dorsal root ganglion neurons, we found that capsaicin (10 microM) stimulated a 2-fold increase in release of substance P in the absence of extracellular Ca(2+). Elevated potassium (75 mM) was unable to induce release under these conditions. The introduction of Ca(2+) enhanced capsaicin-induced release and brought about a robust response to potassium. Preincubation of cells with botulinum neurotoxin A (100 nM) completely blocked potassium-induced release but the capsaicin response, in the absence of Ca(2+), was unaffected. However, toxin treatment dramatically reduced capsaicin-stimulated release in the presence of Ca(2+). It is concluded that capsaicin induces release of substance P from dorsal root ganglion neurons via two mechanisms, one requiring extracellular Ca(2+) and the intact synaptosomal-associated protein 25 kDa (SNAP-25), and the other independent of extracellular Ca(2+) and not involving SNAP-25. PMID- 10751550 TI - Modification of the plasma clearance and liver uptake of steroid ester-conjugated oligodeoxynucleotides by association with (lactosylated) low-density lipoprotein. AB - Low-density lipoprotein (LDL) has been proposed as carrier for the selective delivery of anticancer drugs to tumor cells. We reported earlier the association of several lipidic steroid-conjugated anticancer oligodeoxynucleotides (ODNs) with LDL. In the present study, we determined the stability of these complexes. When the complexes were incubated with a mixture of high-density lipoprotein and albumin, or with rat plasma, the oleoyl steroid-conjugated ODNs appeared to be more stably associated with LDL than the cholesteryl-conjugated ODN. Intravenously injected free lipid-ODNs were very rapidly cleared from the circulation of rats. The area under the curve (AUC) of the lipid-ODNs in plasma was <0.4 microg x min/mL. After complexation with LDL, plasma clearance of the lipid-ODNs was delayed. This was most evident for ODN-5, the ODN conjugated with the oleoyl ester of lithocholic acid (AUC = 6.82 +/- 1.34 microg x min/mL). The AUC of ODN-4, a cholesteryl-conjugated ODN, was 1.49 +/- 0.37 microg x min/mL. In addition, the liver uptake of the LDL-complexed lipid-ODNs was reduced. The lipid ODNs were also administered as a complex with lactosylated LDL, a modified LDL particle that is selectively taken up by the liver. A high proportion of ODN-5 was transported to the liver along with lactosylated LDL (69.1 +/- 8.1% of the dose at 15 min after injection), whereas much less ODN-4 was transported (36.6 +/ 0.1% of the dose at 15 min after injection). We conclude that the oleoyl ester of lithocholic acid is a more potent lipid anchor than the other steroid lipid anchors. Because of the stable association, the oleoyl ester of lithocholic acid is an interesting candidate for tumor targeting of anticancer ODNs with lipoproteins. PMID- 10751551 TI - Transcriptional suppression of cytochrome P450 2C11 gene expression by 3 methylcholanthrene. AB - Aromatic hydrocarbon receptor-mediated transcriptional up-regulation of cytochrome P450 (CYP) enzymes of the CYP1A subfamily by polycyclic aromatic hydrocarbons (PAHs) such as 3-methylcholanthrene (MC) is accompanied by down regulation of rat hepatic CYP2C11 expression at the catalytic activity, protein, and mRNA levels. To gain insight into the molecular mechanism of this CYP2C11 suppression response, we have used a nuclear run-on assay to assess directly the effect of MC on the hepatic transcription rate of the CYP2C11 gene following in vivo administration of MC to adult male rats. A single intraperitoneal dose of MC (40 mg/kg) caused a 179-fold increase in the rate of CYP1A gene transcription at 6 hr, and the rate of CYP2C11 gene transcription was reduced by 51% at this time point, compared with vehicle controls. By 48 hr after MC treatment, the rates of CYP1A and CYP2C11 gene transcription were no longer significantly different from the corresponding vehicle controls. These results indicate for the first time that the suppression of hepatic CYP2C11 caused by in vivo administration of PAHs to adult male rats is at least partially due to a decrease in the rate of transcription of the CYP2C11 gene. PMID- 10751552 TI - Effect of starvation and chlormethiazole on cytochrome P450s of rat nasal mucosa. AB - Cytochrome P450 (CYP) enzymes of nasal tissue are relatively resistant to induction by classical inducers. In the present study, the effects of starvation on the expression of CYP1A, 2A, 2B, 2C, 2E, 2G, and 3A subfamilies in the nasal mucosa of rat were studied. Fasting for 72 hr caused an increase in 2E1-dependent p-nitrophenol hydroxylase and 1A-dependent ethoxy- (or methoxy) resorufin dealkylase activities, but did not affect either 2A-linked coumarin hydroxylase or the testosterone hydroxylase activity, the latter reaction being a marker of several CYPs including 2G1. Whereas increases in 2E1- and 1A- associated catalytic activities were accompanied by a concomitant increase in the corresponding apoproteins as determined by immunoblotting, immunoactive protein bands reactive with antibodies raised against rat 1A1, 2B1, 2C11, 3A1 or rabbit nasal 2A10/11 and 2G1 were not altered. Fasting also increased CYP2E1 and CYP1A2 on the mRNA level, but did not alter CYP1A1 mRNA as determined by hybridization with cDNA probes selective for these cytochromes. A reiterative administration of chlormethiazole, a specific inhibitor of 2E1 in liver, strongly inhibited many CYPs, including 2E1, 1A2, 2G1, and 2A in the nasal mucosa, but did not influence expression of 2B or 3A as determined by immunoblotting or catalytic activities. The chlormethiazole-mediated inhibition of 1A1 and 2E1 was demonstrated to be at the mRNA level. These results suggest that fasting induces the gene expression of 2E1 and 1A2 and that the mechanisms involved in the regulation of CYPs in the nasal mucosa are tissue-specific. The inducibility of the above-mentioned isoforms may have a significant role in the clearance of drugs and bioactivation of inhaled compounds. PMID- 10751553 TI - Nrf2 and c-Jun regulation of antioxidant response element (ARE)-mediated expression and induction of gamma-glutamylcysteine synthetase heavy subunit gene. AB - gamma-Glutamylcysteine synthetase (gamma-GCS) is a rate-limiting enzyme in the de novo synthesis of glutathione, a known scavenger of electrophiles and reactive oxygen species (ROS). The gamma-GCS gene is expressed ubiquitously and induced coordinately with NAD(P)H:quinone oxidoreductase(1) (NQO1) and glutathione S transferase Ya (GST Ya) in response to xenobiotics and antioxidants. The antioxidant response element (ARE) is required for expression and induction of these genes. In the current report, we demonstrated that ARE-mediated gamma-GCS gene expression and induction is regulated by similar Nrf and Jun factors as reported earlier for the NQO1 and GST Ya genes. The gamma-GCS gene ARE competed with the binding of nuclear proteins (Nrf + Jun) to the NQO1 gene ARE (hARE). In addition, the overexpression of Nrf2 and Nrf1 with c-Jun significantly up regulated gamma-GCS ARE-mediated basal expression and beta-naphthoflavone induction of the chloramphenicol acetyltransferase gene in transfected HepG2 cells. Interestingly, Nrf2 + c-Jun was more effective than Nrf1 + c-Jun in the regulation of ARE-mediated gamma-GCS gene expression. Further experiments demonstrated that the c-Jun level within the cells is an important determinant of the level of ARE-mediated gamma-GCS gene expression. Therefore, at higher concentrations of c-Jun, gamma-GCS gene expression is repressed, presumably due to generation of a sufficient amount of c-Jun + c-Fos complex that interferes with the binding of Nrf2 + c-Jun complex to the ARE. PMID- 10751554 TI - In situ hybridization studies of UDP-glucuronosyltransferase UGT1A6 expression in rat testis and brain. AB - UDP-glucuronosyltransferases (UGTs), in addition to their role in overall pharmacokinetics, play important roles in local protection of cells against toxins and in the control of endogenous receptor ligands. UGT1A6, which conjugates planar phenols, appears to be expressed in many organs, but information on cell-specific expression in these organs is controversial or absent. Therefore, a non-isotopic in situ hybridization method was developed and applied to localize UGT1A6 expression in rat testis and brain. It was found that UGT1A6 is expressed in Sertoli cells and spermatogonia of rat testis and in brain neurons, in particular in hippocampal pyramidal cells and Purkinje cells of the cerebellum. PMID- 10751555 TI - Oroxylin A inhibition of lipopolysaccharide-induced iNOS and COX-2 gene expression via suppression of nuclear factor-kappaB activation. AB - Polyphenols are major components of many traditional herbal remedies, which exhibit several beneficial effects including anti-inflammation. The exact mechanism of the anti-inflammatory action of polyphenols, however, has not been determined. In the present study, we examined the effects of eight different polyphenols isolated from Chinese herbs, including two flavonoids (myricitrin and oroxylin A), four ellagitannins (penta-O-galloyl-beta-glucopyranose, woodfordin C, oenothein B, and cuphiin D1), and two anthraquinones (emodin and physcion), on lipopolysaccharide (LPS)-induced nitric oxide (NO) production, and inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expression in RAW264.7 macrophages. The results indicated that only oroxylin A and emodin concentration-dependently inhibited LPS-induced NO production. The remaining compounds slightly inhibited LPS-induced NO production only at the highest concentration examined. Furthermore, oroxylin A inhibited the expression of LPS induced iNOS and COX-2 proteins and mRNAs without an appreciable cytotoxic effect on RAW264.7 cells. Emodin also inhibited LPS-induced iNOS protein as potently as oroxylin A, but it inhibited LPS-induced iNOS mRNA expression only slightly and did not affect COX-2 mRNA and proteins. This was consistent with the findings that oroxylin A but not emodin or physcion inhibited prostaglandin E(2) synthesis induced by LPS. The inhibitory effects of oroxylin A on LPS-induced iNOS and COX 2 gene expression were also demonstrated in Bcl-2-overexpressing RAW264.7 macrophages, suggesting that oroxylin A inhibition of iNOS and COX-2 expression was not due to its antioxidant effect. Furthermore, oroxylin A but not emodin blocked nuclear factor-kappaB (NF-kappaB) binding and transcriptional activation associated with decreased p65 proteins in the nucleus induced by LPS. These results indicated that oroxylin A, an active component in Huang Qin, inhibited LPS-induced iNOS and COX-2 gene expression by blocking NF-kappaB activation, whereas emodin inhibition of LPS-induced iNOS expression may be mediated by a different transcription factor. PMID- 10751556 TI - Strategies to antagonise the cyclosporine A-induced proliferation of human pulmonary artery smooth muscle cells: anti-endothelin-1 antibodies, verapamil, and octreotide. AB - The present study investigated the mechanisms mediating the actions of the immunosuppressive drug cyclosporine A (CsA) on human pulmonary artery smooth muscle cell (PASMC) proliferation. The new hydroxyethyl derivative of D-serine(8) cyclosporine, SDZ IMM 125, was used for comparison. CsA-induced proliferation was determined by incorporation of [(3)H]thymidine ([(3)H]Thy). CsA in the concentration range between 0.1 nM and 0.1 microM induced a concentration dependent increase in proliferation after 24, 48, and 72 hr of incubation. Higher CsA concentrations were cytotoxic. When proliferation experiments were performed in the presence of a monoclonal antibody against endothelin-1 (ET-1), CsA-induced proliferation was totally inhibited. No inhibition occurred in the presence of the same antibody when heat-inactivated or a non-specific monoclonal antibody. In parallel, CsA increased the production of ET-1, as determined by radioimmunoassay. Incubation of PASMCs with ET-1 at the concentration range at which the latter was released by CsA induced cell proliferation. The somatostatin derivative Sandostatin (SDT; octreotide), which is an inhibitor of the growth of smooth muscle cells as well as a potent inhibitor of ET-1 secretion, inhibited both the CsA-induced ET-1 release and the increase in [3H]Thy incorporation by PASMCs. A similar effect was observed for the calcium channel blocker verapamil (VP). SDZ IMM 125 induced weaker effects than CsA in terms of PASMC proliferation and ET-1 secretion. In conclusion, CsA increased the rate of proliferation of PASMCs, while SDZ IMM 125 induced a weaker effect. Anti-ET-1 antibody, VP, and SDT significantly inhibited CsA-induced PASMC proliferation. PMID- 10751557 TI - Paracetamol (acetaminophen) cytotoxicity in rat type II pneumocytes and alveolar macrophages in vitro. AB - Paracetamol (acetaminophen, APAP) liver and kidney cytotoxicity is associated with bioactivation by P450 and/or prostaglandin H synthetase (PGHS) to a reactive metabolite, which depletes GSH, covalently binds to proteins, and leads to oxidative stress. Although APAP may also damage the lung, little is known about the mechanism by which this occurs. We studied the in vitro 24-hr-old type II pneumocytes. A time- and concentration-dependent decrease in intracellular GSH occurred in freshly isolated type II pneumocytes and alveolar macrophages exposed to subtoxic (OH degrees +OH(-)+ Fe(3+)) is partly responsible for the production of CAs by this compound. On the other hand, the addition of PNT to CHO and mosquito cell cultures did not prevent the induction of SCEs by STZ. Therefore, it is valid to assume that the induction of CAs and SCEs by STZ occurs by different mechanisms. PMID- 10751604 TI - Transcriptional slippage of p53 gene enhanced by cellular damage in rat liver: monitoring the slippage by a yeast functional assay. AB - The Long-Evans Cinnamon (LEC) rat is a mutant strain characterized by abnormal copper metabolism and a high incidence of hepatitis and hepatoma. Using a yeast based assay which scores mutants in p53 gene transcripts as red colonies, we detected frequent mutations in the liver of LEC rats. The majority (50-60%) of these were frameshift mutations caused by the insertion of an extra adenine (A) in the regions containing six consecutive adenines. The rate of A insertion was calculated to be 6.9-9.0% of the total p53 cDNA. Insertions of an extra adenine were found almost exclusively in the mRNA (cDNA), especially in the (A)(6) tract located at the most 5'-side (exon 4) among the three (A)(6) tracts (exons 4, 7, and 8), but rarely in the corresponding sites of genomic DNA. Wild-type p53 cDNA was transcribed in vitro into mRNA with the use of SP6 RNA polymerase and tested by the yeast functional assay. Subsequent sequencing detected A insertions at an overall rate of 1.6% in exons 7 and 8 but none in exon 4. This indicates that the A insertion in the exon 4 (A)(6) tract was an in vivo phenomenon rather than an artifact in reverse transcription or polymerase chain reaction. The percentage of red colonies increased sharply to about 20% of the liver samples in the acute hepatitis stage, and returned to control level of those in the chronic hepatitis stage, and increased again slightly to those in the neoplastic stage. The percentage of red colonies correlated with the serum GOT level (r=0.96, p<0.001) but not with the contents of copper and 8-hydroxydeoxyguanosine in the liver of LEC rats. Ethanol treatment of hepatic cell lines also increased the rate of transcriptional slippage at the (A)(6) tract. These findings indicate that cellular damage is responsible for the increase in the rate of mutation at the transcriptional level, and suggest that cellular damage degrades transcriptional fidelity, thereby further impairing cellular functions. PMID- 10751606 TI - A comparison of the roles of p53 mutation and AraC inhibition in the enhancement of bleomycin-induced chromatid aberrations in mouse and human cells. AB - Previous studies have shown that p53 is involved in the repair of bleomycin induced DNA damage, and that the frequency of bleomycin-induced chromatid aberrations is elevated in G(2)-treated p53 null transgenic mouse embryo fibroblasts (MEF) as compared to isogenic controls. To further characterize p53 mediated DNA repair, we studied the effect of p53 status on the ability of the DNA repair inhibitor 1-ss-D-arabinofuranosylcytosine (AraC) to sensitize MEF to bleomycin-induced chromatid aberrations. Both p53+/+ and p53-/- MEF were treated in G(2) with 0 to 7.5 microg/ml bleomycin in the presence or absence of AraC (5x10(-5) M). The frequency of bleomycin-induced chromatid aberrations was significantly higher in p53-/- cells than wild-type cells in the absence of AraC. AraC treatment significantly increased the frequency of bleomycin-induced chromatid aberrations in p53+/+ MEF to the levels in p53-/- (no AraC) but had no effect in p53-/- MEF. These results suggest that an AraC-sensitive DNA repair component is altered or absent in p53-/- cells. Similar results were observed in p53-mutant WTK1 and wild-type TK6 human lymphoblast cells exposed to 0 to 3 microg/ml bleomycin in G(2). However, AraC did cause a small increase in bleomycin sensitivity in WTK1 cells. This difference from the p53-/- MEF response may be due to differences in p53-mutant phenotype. To determine whether mutation of p53 alters DNA replication fidelity, p53+/+ and p53-/- MEF were exposed to 0 to 1 microg/ml mitomycin C (MMC). MMC did not induce chromosome aberrations in either cell line treated in G(2) but did with the same effectiveness in both cell lines treated in S-phase. Thus, p53 deficiency does not affect DNA replication fidelity or the repair of MMC-induced DNA damage. PMID- 10751608 TI - Seminal plasma reduces exogenous oxidative damage to human sperm, determined by the measurement of DNA strand breaks and lipid peroxidation. AB - Exposure of spermatozoa to reactive oxygen species (ROS) has been associated with cellular injury, that includes DNA damage and lipid peroxidation. In addition, sperm preparation techniques such as centrifugation, commonly used prior to in vitro fertilization and scientific studies, are associated with the generation of ROS and an increase in the level of DNA damage. The preservation, therefore, of sperm in vitro that might decrease the potential for oxidative DNA damage to arise and allow for an improvement in semen quality used for artificial insemination, is of importance. Seminal plasma is a rich source of antioxidants, which, potentially, safeguards sperm from oxidative attack during storage and once ejaculated. We have investigated the protection of human spermatozoa from ROS afforded by seminal plasma. Sperm were exposed to exogenous ROS by incubating the cells with hydrogen peroxide in the presence of ferrous sulfate and ADP. Aliquots of seminal plasma were added to the incubation mixture in differing amounts, and the generation of DNA strand breaks and thiobarbituric acid reactive species (TBARS), indicative of lipid peroxidation, determined. Incubation of sperm with exogenous ROS resulted in a significant generation of DNA strand breaks and lipid peroxidation compared to basal levels of damage (P<0.05). Addition of seminal plasma to the incubation media produced a significant decrease in DNA strand breaks and TBARS (P<0. 05), when the amount of plasma added exceeded 60% v/v. The results indicate that spermatozoal oxidative damage induced by exogenous ROS, specifically DNA damage and lipid peroxidation, is reduced by the presence of seminal plasma. PMID- 10751607 TI - Comparison of the mutational spectra of the lacZ transgene in four organs of the MutaMouse treated with benzo[a]pyrene: target organ specificity. AB - We recently demonstrated that not all organs with a high rate of induction of mutation in the lacZ transgene develop tumors in the lambdalacZ transgenic mice (MutaMouse) used for a long-term carcinogenicity study with benzo[a]pyrene (BP). To better understand the role of chemical-induced in vivo mutations in carcinogenesis, we compared the mutational spectra of the lacZ transgene in four organs of the MutaMouse obtained 2 weeks after five daily consecutive oral treatments with 125 mg/kg/day BP. lacZ transgenes were analyzed in two target organs (forestomach and spleen) and two non-target organs (colon and glandular stomach) for BP-induced carcinogenesis in MutaMouse, and all of these organs were highly mutated in the lacZ transgene. The sequence data showed similar mutational spectra of the lacZ transgene between the two target organs; the predominant mutations were G:C-->T:A transversions (55% and 50% for forestomach and spleen, respectively), followed by deletions (20% and 21% for forestomach and spleen, respectively) mainly at G:C site. The frequent G:C-->T:A transversions are consistent with reports of the mutational spectra produced in the p53 gene in tumors generated in rats and mice exposed to BP. In contrast, the mutational spectra of the lacZ transgene in the two non-target organs are different from those in the target organs, and are also suggested to differ from one another. These findings suggest an organ/tissue-specific mechanism of mutagenesis. PMID- 10751609 TI - Toxicity, mutation frequency and mutation spectrum induced by dacarbazine in CHO cells expressing different levels of O(6)-methylguanine-DNA methyltransferase. AB - The toxicity and mutagenicity (including the mutation spectrum induced) of dacarbazine, a methylating cytostatic drug, was examined in CHO cells expressing different levels of the repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). Expression of low or high levels of a transfected human MGMT gene under the control of the metallothionein promoter protected the cells against dacarbazine-induced toxicity and mutagenesis. In the absence of MGMT expression, the mutation spectrum in the HPRT locus was dominated by GC-->AT transitions (mostly found at 5'Pu-G sequences), while there were also a few AT-->GC transitions. Expression MGMT was associated with a substantial decrease of GC- >AT mutations, suggesting that these mutations arose primarily via O(6) methylguanine. These data illustrate the important role of the latter lesion in the drug's mutagenic and cytotoxic activity. PMID- 10751610 TI - Reduction of diepoxybutane-induced sister chromatid exchanges by glutathione peroxidase and erythrocytes in transgenic Big Blue mouse and rat fibroblasts. AB - We have investigated the effect of glutathione peroxidase (GSH-Px) and mammalian erythrocytes (RBCs) on spontaneous and diepoxybutane (DEB)-induced sister chromatid exchange (SCE) in primary Big Blue(R) mouse (BBM1) and Big Blue(R) rat (BBR1) fibroblasts. DEB is the putative carcinogenic metabolite of 1,3-butadiene (BD) for which inhalation exposure yields a high rate of malignancies in mice but not in rats. BD is metabolized differently in mice and rats, producing much higher levels of DEB in mice than in rats, which may partly explain the different carcinogenic responses. However, other factors may contribute to the observed differences in the rodent carcinogenic response to BD. DEB is a highly reactive compound. Upon epoxide hydrolysis, DEB can covalently bind to DNA bases. Likewise, DEB generates reactive oxygen species that, in turn, can either damage DNA or produce H(2)O(2). Reduced glutathione (GSH) is known to play a role in the metabolism and detoxification of DEB; and GSH is reduced by GSH-Px in the presence of H(2)O(2). GSH-Px is a constitutive enzyme that is found at high concentrations in mammalian RBCs. Therefore, we were interested in examining the role of RBCs and GSH-Px on DEB-induced SCE in rat and mouse cells for detection of possible differences in the species response. Transgenic BBM1 and BBR1 fibroblasts were treated with either 0, 2 or 4 microM DEB plus 0, 2 or 20 units of GSH-Px with and without 2x10(8) species-specific RBCs. DEB effectively induced SCEs in both rat and mouse cells. The relative induction of SCEs in both cell types was comparable. Both GSH-Px and RBCs alone and in combination were effective in significantly reducing DEB-induced SCEs in both mouse and rat fibroblasts, although there was more variability in the SCE response in rat cells. The present study suggests that GSH-Px may be important in the detoxification of DEB-induced DNA damage that results in the formation of SCEs. PMID- 10751611 TI - Mutation induction by mechanical irritation caused by uracil-induced urolithiasis in Big Blue rats. AB - Some chronic mechanical irritations induce cancers, and it is speculated that mutations are induced by increased rate of cell proliferation caused by the irritation. In this study, it was investigated using chronic mechanical irritation to urothelium caused by urolithiasis, whether mutations are really induced by such cell proliferation or not. Male rats transgenic for lacI (Big Blue(R) rats), in which lacI mutations accumulated in tissue can be measured, were fed 3% uracil, a component of RNA, to induce urolithiasis associated with papillomatosis, and eventually with bladder cancers. The frequency of independent mutations in the bladders of the treated rats showed 3-5 fold increases at weeks 10, 20, and 51 (P=0.01 at week 51) while the frequency was not elevated at week 2. The mutation frequencies in the control bladders ranged from 3 to 9x10(-6). In both groups, G to A transitions at CpG sites, indicative of spontaneous mutations, constituted the most prevalent mutations. Mechanical irritation caused by uracil was shown to induce a 3-5 fold increase of mutations, possibly through an elevation of spontaneous mutations by vigorous cell proliferation. PMID- 10751612 TI - Tumorigenicity of morphologically distinct transformed foci induced by 3 methylcholanthrene in BALB/c-3T3 cells. AB - 4 mm in diameter), invasiveness (smooth vs. invading margins) and other properties (piling vs. spread). In our previous report, we showed that cells from all five types grew in soft agar, transformed normal NIH 3T3 cells and formed foci on normal layer of BALB/c-3T3 cells. In this study, the neoplastic/tumorigenic potential of cells from the five different types of transformed foci was investigated in nude mice. About two million cells from each transformed focus were injected into 4-week-old nude mice. Non-transformed BALB/c 3T3 cells were used as control. The results of this study indicate that all the 45 athymic mice injected with different transformants developed tumors between 2 and 4 weeks after injection. Tumors were not observed in eight mice injected with non-transformed BALB/c-3T3 cells. All tumors were histopathologically confirmed fibrosarcomas. These findings indicate that all five morphologically different foci show tumorigenicity and that any foci of size > or =2 mm regardless of invasiveness and piling could be scored as positive during the cell transformation assay. PMID- 10751613 TI - Peroxynitrite-induced DNA damage in the supF gene: correlation with the mutational spectrum. AB - Tissue inflammation and chronic infection lead to the overproduction of nitric oxide and superoxide. These two species rapidly combine to yield peroxynitrite (ONOO(-)), a powerful oxidizing and nitrating agent that is thought be involved in both cell death and an increased cancer risk observed for inflamed tissues. ONOO(-) has been shown to induce single-strand breaks and base damage in DNA and is mutagenic in the supF gene, inducing primarily G to T transversions clustered at the 5' end of the gene. The mutagenicity of ONOO(-) is believed to result from chemical modifications at guanine nucleobases leading to miscoding DNA lesions. In the present work, we applied a combination of molecular and analytical techniques in an attempt to identify biologically important DNA modifications induced by ONOO(-). pUC19 plasmid treated with ONOO(-) contained single-strand breaks resulting from direct sugar damage at the DNA backbone, as well as abasic sites and nucleobase modifications repaired by Fpg glycosylase. The presence of carbon dioxide in the reaction mixture shifted the ONOO(-) reactivity towards reactions at nucleobases, while suppressing the oxidation of deoxyribose. To further study the chemistry of the ONOO(-) interactions with DNA, synthetic oligonucleotides representing the mutation-prone region of the supF gene were treated with ONOO(-), and the products were analyzed by liquid chromatography negative ion electrospray ionization mass spectrometry (LC-ESI(-) MS) and tandem mass spectrometry. 8-Nitroguanine (8-nitro-G) was formed in ONOO(-)-treated oligonucleotides in a dose-dependent manner with a maximum at a ratio of [ONOO( )]: [DNA]=10 and a decline at higher ONOO(-) concentrations, suggesting further reactions of 8-nitro-G with ONOO(-). 8-Nitro-G was spontaneously released from oligonucleotides (t(1/2)=1 h at 37 degrees C) and, when present in DNA, was not recognized by Fpg glycosylase. To obtain more detailed information on ONOO(-) induced DNA damage, a restriction fragment from the pSP189 plasmid containing the supF gene (135 base pairs) was [32P]-end-labeled and treated with ONOO(-). PAGE analysis of the products revealed sequence-specific lesions at guanine nucleobases, including the sites of mutational "hotspots." These lesions were repaired by Fpg glycosylase and cleaved by hot piperidine treatment, but they were resistant to depurination at 90 degrees C. Since 8-nitro-G is subject to spontaneous depurination, and 8-oxo-guanine is not efficiently cleaved by piperidine, these results suggest that alternative DNA lesion(s) contribute to ONOO(-) mutagenicity. Further investigation of the identities of DNA modifications responsible for the adverse biological effects of ONOO(-) is underway in our laboratory. PMID- 10751614 TI - Accumulation of DNA damage in the organs of mice deficient in gamma glutamyltranspeptidase. AB - We have used a differential alkaline single cell gel electrophoresis assay of DNA ("omet assay" at pH 13 and 12.3) to evaluate DNA damage as a function of age in mice with an inherited defect in gluthathione (GSH) metabolism. The mice are homozygous null for gamma-glutamyltranspeptidase (GGT), the enzyme responsible for initiating the catabolism of GSH, and paradoxically have reduced levels of GSH and cysteine in many organs. We found an accumulation of DNA damage in lung, liver and kidney in these mice as a function of age. The largest differences were in assays run at pH 13, suggesting that the accumulation of apurinic/apryrimidinic (AP) sites and oxidative damage of DNA was largely responsible. In contrast, little if any accumulation of these lesions was detected in wild-type mice. Although these findings do not allow a precise analysis of the molecular basis of damage accumulation in GGT-deficient mice, they implicate low GSH and cysteine levels as a cause of accumulative DNA damage in the intact mammal. PMID- 10751615 TI - A molecular pathway for UV-induced CC to TT mutations. PMID- 10751616 TI - The study of antigenotoxic effects of dietary fibre is lost in a confused concept. PMID- 10751617 TI - Kinetics of excision repair of UV-induced DNA damage, measured using the comet assay. AB - The kinetics of UV- (254 nm) irradiation-induced DNA single-strand breaks (SSBs), generated during the excision repair of UV-induced DNA damage, in leukemic lymphocytes and in normal blood mononuclear cells (MNCs) were studied using the alkaline comet assay. The cells were isolated by density gradient centrifugation from peripheral blood of patients with chronic lymphocytic leukemia (CLL) and from healthy study subjects. The cytotoxicity of UV irradiation was determined in vitro in peripheral blood mononuclear lymphocytes from 36 CLL patients and from eight healthy donors using the incorporation of radioactive leucine in 4-day cultures. A remarkable difference in excision repair capability was observed between normal and leukemic lymphocytes. In contrast to normal lymphocytes, there was always a subpopulation of CLL cells that did not complete the repair of UV induced DNA damage during the 24-h repair period. Furthermore, differences were also recorded between UV-sensitive and UV-resistant CLL cases. The differences in DNA migration between the maximum increase (59-77 microm) and that at 24 h after irradiation (21-66 microm) was statistically significant in two of three patients exhibiting UV-resistance. Correspondingly, only in one of three patients exhibiting UV-sensitivity was the difference in DNA migration statistically significant (maximum increase: 44-107 microm, vs. 24 h after: 42-100 microm). Our results confirm an abnormal pattern of the CLL cell response to UV irradiation. Furthermore, we identified defective processing of UV-induced DNA damage in CLL versus normal lymphocytes, particularly in UV-sensitive cases. PMID- 10751618 TI - Characterization and antimutagenic activity of soybean saponins. AB - An extract was prepared from a commercial soybean-processing by-product (soybean molasses) and was fractionated into purified chemical components. In previous work, this extract (phytochemical concentrate, PCC) repressed induced genomic DNA damage, whole cell clastogenicity and point mutation in cultured mammalian cells. In the current study, a chemical fraction was isolated from PCC using preparative high-performance liquid chromatography (HPLC). This fraction, PCC100, repressed 2 acetoxyacetylaminofluorene (2AAAF)-induced DNA damage in Chinese hamster ovary (CHO) cells as measured by single cell gel electrophoresis (alkaline Comet assay). Using liquid chromatography-electrospray ionization-mass spectroscopy and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, PCC100 was shown to consist of a mixture of group B soyasaponins and 2,3-dihydro-2,5-dihydroxy-6 methyl-4H-pyran-4-one (DDMP) soyasaponins. These include soyasaponins I, II, III, IV, V, Be, betag, betaa, gammag and gammaa. Purified soyasapogenol B aglycone prepared from fraction PCC100 demonstrated significant antigenotoxic activity against 2AAAF. To our knowledge, these data demonstrate for the first time the antimutagenic activity of soybean saponins in mammalian cells. PMID- 10751619 TI - Transforming and carcinogenic potential of cadmium chloride in BALB/c-3T3 cells. AB - A large number of workers are potentially exposed to cadmium during mining and processing. Therefore, there is a concern regarding the potential carcinogenic hazards of cadmium to exposed workers. Studies have been performed to determine if cadmium chloride (CdCl(2)) can induce morphological cell transformation, DNA from CdCl(2)-induced transformed cells can transform other mammalian cells, and the transformed cells induced by CdCl(2) can form tumors in nude mice. BALB/c-3T3 cells were treated with different concentrations of CdCl(2) for 72 h. The frequency of transformed foci from each treatment was determined after cells were cultured for 4 to 5 weeks. DNAs from five CdCl(2)-induced transformed cell lines were isolated and gene transfection assay was performed using NIH-3T3 cells. Non transformed BALB/c-3T3 cells and cells from 10 transformed cell lines induced by CdCl(2) were injected into both axillary regions of nude mice. Mice were screened once a week for the appearance and size of tumors. CdCl(2) caused a statistically significant, concentration-related increase in the transformation frequency. DNA from all five CdCl(2)-induced transformed cell lines tested was found to induce varying degrees of transfection-mediated transformation in NIH-3T3 cells. All 10 CdCl(2)-induced transformed cell lines formed fibrosarcomas in nude mice within 39 days of inoculation. Within this time period, no tumors were found in nude mice injected with non-transformed BALB/c-3T3 cells. These results indicate that CdCl(2) is capable of inducing morphological cell transformation and that the transformed cells induced by CdCl(2) are potentially tumorigenic. PMID- 10751620 TI - Yield of SCEs and translocations produced by 3 aminobenzamide in cultured Chinese hamster cells. AB - Different concentrations of 3-aminobenzamide (3AB), a strong inhibitor of poly(ADP-ribose) polymerase (PARP), were used to study their effect on the BrdU substituted DNA of the Chinese hamster AA8 cell line. The frequencies of sister chromatid exchanges (SCEs) and translocations were determined using the fluorescence plus Giemsa (FPG) and fluorescence in situ hybridization (FISH) techniques, respectively. The results indicate that 3AB effectively induced a dose-dependent increase in the frequency of SCEs, but this enhancement in the yield of SCEs was not paralleled by an increase in translocations. These results are discussed in terms of the as yet poorly understood molecular mechanisms of action of the enzyme PARP. PMID- 10751621 TI - Mutations induced by some DNA minor groove binding alkylators in AS52 Chinese hamster cells. AB - Nitrogen mustards are commonly used in cancer chemotherapy. They interact with DNA at electronegative sites, primarily forming N7 guanine mono-adducts and interstrand cross-links. Targeting nitrogen mustards to DNA by attachment of a DNA minor groove binding carrier such as the bisbenzimidazoles Hoechst 33258 (pibenzimol) or Hoechst 33342 (HOE) makes it possible to direct DNA alkylation to more specific stretches of DNA. We have performed a detailed molecular analysis of 6-thioguanine resistant clones arising in Chinese hamster AS52 cells after treatment with HOE, in comparison with a mono- and bifunctional pair of bisbenzimidazole-targeted nitrogen mustards (MGBs). HOE showed no significant ability to induce 6-thioguanine resistant mutants, possibly because drug-treated cells are highly susceptible to apoptosis within very short times. Neither of the MGBs caused the rapid cell death seen with the bisbenzimidazole. However, both MGBs were weaker mutagens than previously found for undirected mustards in the same system, an effect that we suggest could relate to greater structure-directed binding to less mutable DNA sites in the minor groove. Additionally, the nature of some of the mutants suggested there may be a small component of topo I and/or II-mediated events in the mutagenicity of the MGBs. Both MGBs showed high activity in causing deletion mutations, which may be due to errors in attempted repair of the complex lesions formed by minor groove targeted alkylators. PMID- 10751623 TI - Correlation between cell survival and micronuclei formation in V79 cells treated with vindesine before exposure to different doses of gamma-radiation. AB - Effect of 20 nM vindesine sulphate (VDS) treatment was studied on cell survival, growth kinetics and micronuclei induction in V79 cells exposed to 0-300 cGy of gamma-radiation at 16, 22 and 28 h post-irradiation. Treatment of V79 cells with VDS before exposure to different doses of gamma radiation resulted in a significant decline in cell survival and growth kinetic when compared with the concurrent PBS+irradiation group. The decline in cell survival and growth kinetics was dose related. Similarly, the cell proliferation indices also declined in a dose dependent manner in both PBS+irradiation and VDS+irradiation groups and this decline was higher in VDS+irradiation group in comparison with the PBS+irradiation group. In contrast, the frequency of micronuclei increased in a dose related manner in both PBS+irradiation and VDS+irradiation groups. However, the frequency of micronuclei was significantly greater in the VDS+irradiation group when compared to the PBS+irradiation group at all the post irradiation time periods studied and the dose response for both groups was linear for all the scoring time periods. The biological response was determined by plotting surviving fraction and micronuclei frequencies on X- and Y-axes, respectively. The plot between surviving fraction and micronuclei induction showed a close correlation. The surviving fraction of V79 cells reduced with the increasing frequency of micronuclei in both groups and the relationship between micronuclei induction and cell survival could be fitted on a linear quadratic model. PMID- 10751622 TI - The frequency of mutators in populations of Escherichia coli. AB - Owing to occasional spontaneous mutations in genes encoding DNA repair, any population of a reasonable size is expected to harbor a sub-population of genetic mutators. Using a genetically modified strain of Escherichia coli K-12, we have estimated the frequency of mutators to be about 3x10(-5). By and large, this corresponds to a mutation rate from non-mutators to mutators of 5x10(-6) per bacterium per generation. Using a mutS∷Tn10 derivative as representative for mutators, we estimated the increase in mutation rates in mutators to be 19- to 82-fold, depending on the test-mutation under consideration. The load associated with this increase in mutation rate resulted in a growth inhibition of 1%. From these data, we estimated that the rate of detrimental mutations in the non-mutators to be 2x10(-4)-8x10(-4). The situations where adaptive mutations may result in an increase in the frequency of mutators are discussed. PMID- 10751624 TI - Subchronic administration of phenobarbital alters the mutation spectrum of lacI in the livers of Big Blue transgenic mice. AB - Phenobarbital (PHE) is a liver carcinogen in B6C3F1 mice and a weak mutagen that does not appear to form DNA adducts. To investigate PHE mutagenicity in vivo, B6C3F1 Big Blue(R) male transgenic mice harboring the lambdaLIZ shuttle vector containing the lacI target gene were fed PHE at 2500 ppm for 180 days. A modest increase in the mutant frequency (MF) from 5.02+/-2.4x10(-5) in the control group to 6.88+/-0.754x10(-5) in the PHE-treated group, which was marginally different (p<0.05), was obtained. To better assess the relevance of this increase in MF, a random collection of mutants from each PHE-exposed mouse was sequenced. After correcting for clonal expansion, which is the most conservative approach, the MF in the PHE-treated mice decreased to 6.39+/-1.02x10(-5), an insignificant difference (p=0.10) from that in control group. Despite this modest increase in MF, the mutation spectrum obtained from the PHE-exposed group was significantly different (pA:T transitions remained the same in the two spectra. It is postulated that the increase in transversions at G:C base pairs found in the PHE derived spectrum is likely due to oxidative damage as a result of induction of CYP2B isozymes by the chronic administration of PHE. Results from this study demonstrate that PHE alters the spectrum of mutations, rather than inducing a significant global increase in the MF. The PHE-derived spectrum of lacI mutants from the liver of Big Blue(R) B6C3F1 male mice was remarkably similar (p=0.8) to that generated by oxazepam (OX), a compound which also induces CYP2B isozymes following chronic administration of the drug. PMID- 10751625 TI - Selective elimination of acentric double minutes from cancer cells through the extrusion of micronuclei. AB - Several lines of evidences from us or other authors had shown that tumor cells revert their phenotypes and differentiate by the elimination of oncogenes amplified on the acentric double minutes (DMs). The selective incorporation of DMs into the cytoplasmic micronuclei was thought to be involved in this elimination, however, the mechanism by which the content of micronuclei was eliminated from the cells remains to be discovered. In this report, we show the finding and the characterization of the extruded micronuclei in the culture fluid of human COLO 320DM tumor line, and suggest that the extrusion of micronuclei mediates the selective elimination of DMs. The extracellular micronuclei enriched with DMs had an apparently normal cytoplasmic membrane, decondensed chromatin and nuclear lamin protein, and their DNA did not suffer any extensive degradation. These characteristics were closely related to their cytoplasmic counterpart and clearly differentiated from the apoptotic bodies. We also developed a method for purifying the extracellular micronuclei. In this paper, the implications of the micronuclear extrusion are discussed. PMID- 10751626 TI - Genetic analysis of thiopurine methyltransferase polymorphism in a Japanese population. AB - Thiopurine methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathiopurine. Several mutations in the TPMT gene have been identified which correlate with a low activity phenotype. The molecular basis for the genetic polymorphism of TPMT has been established for European Caucasians, African-Americans, Southwest Asians and Chinese, but it remains to be elucidated in Japanese populations. The frequency of the four allelic variants of the TPMT gene, TPMT*2 (G238C), TPMT*3A (G460A and A719G), TPMT*3B (G460A) and TPMT*3C (A719G) were determined in Japanese samples (n=192) using polymerase chain reaction (PCR)-RFLP and allele-specific PCR-based assays. TPMT*3C was found in 0.8% of the samples (three heterozygotes). The TPMT*2, TPMT*3A and TPMT*3B alleles were not detected in any of the samples analyzed. This study provides the first analysis of TPMT mutant allele frequency in a sample of Japanese population and indicates that TPMT*3C is the most common allele in Japanese subjects. PMID- 10751627 TI - Copper, zinc superoxide dismutase enhances DNA damage and mutagenicity induced by cysteine/iron. AB - Oxidative DNA damage caused by a cysteine metal-catalyzed oxidation system (Cys MCO) comprised of Fe(3+), O(2), and a cysteine as an electron donor was enhanced by copper, zinc superoxide dismutase (CuZnSOD) in a concentration-dependent manner, as reflected by the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and strand breaks. Unlike CuZnSOD, manganese SOD (MnSOD) as well as iron SOD (FeSOD) did not enhance DNA damage. The capacity of CuZnSOD to enhance damage to DNA was inhibited by a spin-trapping agent, 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) and a metal chelator, diethylenetriaminepentaacetic acid (DETAPAC). The deoxyribose assay showed that hydroxyl free radicals were generated in the reaction of CuZnSOD with Cys-MCO. We found that the Cys-MCO system caused the release of free copper from CuZnSOD. CuZnSOD also caused the two-fold enhancement of a mutation in the pUC18 lacZ' gene in the presence of Cys-MCO when measured as a loss of alpha-complementation. Based on these results, we interpret the effects of CuZnSOD on Cys-MCO-induced DNA damage and mutation as due to reactive oxygen species, probably hydroxyl free radicals, formed by the reaction of free Cu(2+), released from oxidatively damaged CuZnSOD, and H(2)O(2) produced by the Cys-MCO system. PMID- 10751628 TI - Assessment of DNA damage induced by high-LET ions in human lymphocytes using the comet assay. AB - The alkaline single-cell gel electrophoresis assay (comet assay) was used to analyze DNA damage induced in human lymphocytes by irradiation with high linear energy transfer (LET) ions. Our aim was to measure DNA breaks and to demonstrate the heterogeneity of the damage levels in a lymphocyte population irradiated with ions of different energies and LETs. Four experiments with heavy ions (Ar, C and U), as well as gamma-ray exposure, were conducted to enable comparisons. We demonstrated that the comet assay is able to assess the variability in DNA damage induced at the single cell level. The amount of DNA damage and its heterogeneity increased with particle fluence and LET, but saturated at high LETs. However, when expressed in terms of the mean dose, gamma-rays were more efficient than most of the ions used. The comet assay also allowed the detection of highly damaged cells (HDC), which were previously described as cells in late apoptotic stages. The rapid emergence of HDC in this study suggests that they were generated following ion irradiation-induced creation of DNA break clusters induced by ion exposure. Another clue was that the proportion of HDC increased with LET and fluence. We hypothesized that the LET threshold observed and the higher efficiency of low-LET radiation might be linked to the impossibility of measuring small DNA fragments in HDC. PMID- 10751629 TI - Molecular basis of albinism in the rhesus monkey. AB - Sequence analysis of the tyrosinase (TYR) coding region from one albino rhesus monkey (Macaca mulatta) family revealed that the two monkeys with phenotype similar to human TYR-negative oculocutaneous albinism (OCA) were homozygous for a missense mutation (S184TER) in exon 1 at codon 184. The offspring of one of the albino monkey ("Kangkang") are all heterozygous for the S184TER mutation, but the S184TER mutation was not observed in 93 control individuals. We conclude that the point mutation is responsible and sufficient to generate the albino rhesus monkey phenotype. The rough age of the S184TER nonsense mutation may be about 0.8 million years using a rate of 0.16% per million years. PMID- 10751630 TI - Chromosomal aberrations of blood lymphocytes induced in vitro by radon-222 daughter alpha-irradiation. AB - Blood samples were irradiated in vitro with alpha-rays emitted from short-lived radon decay products dissolved in the culture medium at doses between 0.03 and 41.4 mGy. The data were collected from experiments conducted during the period 1984-1992 and comprise a total of about 64000 scored metaphases. For statistical reasons, only 60,022 metaphases were used for the subsequent analysis. The results for total chromosome aberrations and dicentrics indicate a linear dose dependence in the dose range above about 10 mGy, consistent with other experimental observations. At doses below about 10 mGy, aberration frequencies cannot be linearly extrapolated from higher doses, suggesting that there is no dependence on dose within a certain low-dose range. In addition, a statistically significant minimum has been observed at a dose of about 0.03 mGy, which is consistently lower than the related control values. The behavior of the aberration frequencies in the low-dose region seems to be influenced by the control values, which also depend on the environmental radiation burdens to the donors before blood sampling and thus were significantly affected by the Chernobyl fallout. PMID- 10751631 TI - Overexpression of wild-type and nuclear-targeted catalase modulates resistance to oxidative stress but does not alter spontaneous mutant frequencies at APRT. AB - Animal cells generate hydrogen peroxide as a byproduct of energy metabolism. In the presence of reduced metals H(2)O(2) can decompose to a highly reactive hydroxyl radical that attacks essentially all organic molecules, including DNA. We wished to determine if overexpression of catalase and/or the targeting of the enzyme to the nucleus could protect cells from oxidative stress and reduce the frequency of mutation. Wild-type human catalase, which localizes to peroxisomes, and a modified construct, which targets catalase to the nucleus, were overexpressed in a murine line of embryonic carcinoma cells (P19). Both constructs enhanced the resistance of the cells to hydrogen peroxide, but sensitized them to bleomycin. Overexpression of wild-type catalase protected cells against paraquat, while nuclear targeting sensitized them to this agent. Expression of neither construct significantly altered spontaneous mutant frequencies at the endogenous murine adenosine phosphoribosyl transferase (APRT) locus; however, nuclear-targeted catalase prevented an increase in mutant frequency after H(2)O(2) treatment. These results suggest that endogenous levels of hydrogen peroxide may not generate DNA damage in vivo, or that such damage may be efficiently repaired in murine embryonic carcinoma cells. PMID- 10751632 TI - Effect of exogenous melatonin on the ovarian follicles in gamma-irradiated mouse. AB - The present study was performed to obtain the evidence of the radioprotective function of melatonin on gamma-radiation-induced follicular atresia in mouse ovary. Three-week-old immature mice received 10 and 100 microg of melatonin dissolved in 100 microl of the alcoholic saline. Two hours after the treatments, they were whole-body irradiated with a dose of LD(80(30)) (8.3 Gy). The ovaries were dissected out of the animals at -2, 2, 8, 14 h after the onset of irradiation. The total number of follicles including the normal and atretic follicles examined in the largest cross sections was 125. The number was reduced to 103 in the irradiated group. The number of primordial follicles of the irradiation group or the melatonin-treated group was smaller than that of the control group. However, the number of primary, preantral, and antral follicles was not different from that of the control group. In the group pretreated with 100 microg of melatonin before irradiation, the ratio of normal primordial follicles was significantly higher than that of the irradiation group at any time point after irradiation. The high concentration of melatonin also reduced the radiation-induced degeneration of the primary follicles at 14 h after irradiation. On the other hand, the pretreatment of 10 microg of melatonin had little or no effect on the radiation-induced degeneration of primary follicles. However, it gave a protective effect on the radiation-induced degeneration in the primordial follicles at 2 h after irradiation, and 14 h after irradiation in preantral and antral follicles. From the above results, it is concluded that the exogenous melatonin has different functions depending on the follicle stages, and that the radioprotective effect of exogenous melatonin on the follicular degeneration is related to its concentration. PMID- 10751633 TI - Absence of point mutations at codon 17 of the mdm2 gene (serine 17) in human primary tumors. AB - Mdm2 is a phosphoprotein that interacts with protein p53, inhibiting its activity. A serine located in position 17 of Mdm2, has been implicated in its phosphorylation process. We hypothesize that point mutations at serine 17 could block its phosphorylation and thereby increase the p53-Mdm2 interaction. This mechanism could increase the p53 degradation and cause a loss of the protective effect of p53 against tumorigenesis. This hypothesis was based on recent studies in vitro, demonstrating that when serine 17 is mutated, the DNA-dependent protein kinase, activated by genomic damage, is unable to phosphorylate it. Thus, we investigated whether structural point mutations at exon 3 of the Mdm2 gene, affecting codon 17, were present in 162 human primary tumors, 70 breast carcinomas, 14 bladder tumors, 18 colon adenocarcinomas and 60 testicular tumors. Direct sequencing of a fragment (204 bp) of exon 3 of the Mdm2 gene that contains the codon 17 showed no mutations at this position, independently of the presence or absence of p53 gene mutations in the same tumors. These results do not support the hypothesis that mutations in the Mdm2 gene at this level are involved in the tumorigenic process of human cancers. PMID- 10751634 TI - Plants experiencing chronic internal exposure to ionizing radiation exhibit higher frequency of homologous recombination than acutely irradiated plants. AB - Ionizing radiation (IR) is a known mutagen responsible for causing DNA strand breaks in all living organisms. Strand breaks thus created can be repaired by different mechanisms, including homologous recombination (HR), one of the key mechanisms maintaining genome stability [A. Britt, DNA damage and repair in plants, Annu. Rev. Plant. Phys. Plant Mol. Biol., 45 (1996) 75-100; H. Puchta, B. Hohn, From centiMorgans to basepairs: homologous recombination in plants, Trends Plant Sci., 1 (1996) 340-348.]. Acute or chronic exposure to IR may have different influences on the genome integrity. Although in a radioactively contaminated environment plants are mostly exposed to chronic pollution, evaluation of both kinds of influences is important. Estimation of the frequency of HR in the exposed plants may serve as an indication of genome stability. We used previously generated Arabidopsis thaliana and Nicotiana tabacum plants, transgenic for non-active versions of the beta-glucoronidase gene (uidA) [P. Swoboda, S. Gal, B. Hohn, H. Puchta, Intrachromosomal homologous recombination in whole plants, EMBO J., 13 (1994) 484-489; H. Puchta, P. Swoboda, B. Hohn, Induction of homologous DNA recombination in whole plants, Plant, 7 (1995) 203 210.] serving as a recombination substrate, to study the influence of acute and chronic exposure to IR on the level of HR as example of genome stability in plants. Exposure of seeds and seedlings to 0.1 to 10.0 Gy 60Co resulted in increased HR frequency, although the effect was more pronounced in seedlings. For the study of the influence of chronic exposure to IR, plants were grown on two chemically different types of soils, each artificially contaminated with equal amounts of 137Cs. We observed a strong and significant correlation between the frequency of HR in plants, the radioactivity of the soil samples and the doses of radiation absorbed by plants (in all cases r0.9, n=6, P<0.05). In addition, we noted that plants grown in soils with different chemical composition, but equal radioactivity, exhibited different levels of HR, dependent upon the absorbed dose of radiation. Remarkably, we observed a much higher frequency of HR in plants exposed to chronic irradiation when compared to acutely irradiated plants. Although acute application of 0.1-0.5 Gy did not lead to an increase of frequency of HR, the chronic exposure of the plants to several orders of magnitude lower dose of 200 muGy led to a 5-6-fold induction of the frequency of HR as compared to the control. PMID- 10751635 TI - Adaptively responsive hypermutation and its configurational-based regulation due to global position effect. PMID- 10751636 TI - Estrogen receptor interaction with co-activators and co-repressors. PMID- 10751637 TI - Synthesis and biological activities of the two C(23) epimers of 1alpha,23,25 trihydroxy-24-oxo-19-nor-vitamin D(3): novel analogs of 1alpha,23(S),25 trihydroxy-24-oxo-vitamin D(3), a natural metabolite of 1alpha,25 dihydroxyvitamin D(3). AB - In a previous report, we indicated that 1alpha,23(S), 25-trihydroxy-24-oxovitamin D(3) [1alpha,23(S), 25(OH)(3)-24-oxo-D(3)], a natural metabolite of 1alpha, 25 dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is almost equipotent to 1alpha,25(OH)(2)D(3) in suppressing parathyroid hormone (PTH) secretion (Lee et al., 1997. Biochemistry 36, 9429-9437). Also, 1alpha,23(S),25(OH)(3)-24-oxo-D(3) has been shown to possess only weak in vivo calcemic actions. Thus, vitamin D(3) analogs structurally related to 1alpha,23(S),25(OH)(3)-24-oxo-D(3) may have therapeutic value. Furthermore, biologic activity studies of various synthetic analogs of 1alpha,25(OH)(2)D(3) showed that the removal of carbon-19 (C-19) reduces the calcemic activity of 1alpha, 25(OH)(2)D(3.) Therefore, in an attempt to produce vitamin D(3) analogs with a better therapeutic index, we synthesized C(23) epimers of 1alpha,23,25(OH)(3)-24-oxo-19-nor-vitamin D(3) [1alpha,23, 25(OH)(3)-24-oxo-19-nor-D(3)]. The two epimers were compared to 1alpha,25(OH)(2) 19-nor-D(3) and 1alpha,25(OH)(2)D(3) in their ability to generate biologic activities in several in vitro assay systems. In the assay measuring the suppression of parathyroid hormone (PTH) secretion in bovine parathyroid cells, 1alpha,23(S), 25(OH)(3)-24-oxo-19-nor-D(3) was as potent as 1alpha, 25(OH)(2)-19 nor-D(3) but was less potent than 1alpha,25(OH)(2)D(3). In the same assay 1alpha,23(R),25(OH)(3)-24-oxo-19-nor-D(3) exhibited greater potency than 1alpha,23(S), 25(OH)(3)-24-oxo-19-nor-D(3). In the assays measuring the ability of vitamin D compounds to inhibit clonal growth and to induce differentiation of human promyelocytic leukemia (HL-60) cells, 1alpha,23(S),25(OH)(3)-24-oxo-19-nor D(3) was less potent than 1alpha,25(OH)(2)-19-nor-D(3) but was equipotent to 1alpha, 25(OH)(2)D(3). More importantly, in the same assays, 1alpha,23(R), 25(OH)(3)-24-oxo-19-nor-D(3) was more potent than 1alpha,23(S), 25(OH)(3)-24-oxo 19-nor-D(3) and was equipotent to 1alpha, 25(OH)(2)-19-nor-D(3). Also, the vitamin D receptor-mediated transcriptional activity of 1alpha,23(R), 25(OH)(3) 24-oxo-19-nor-D(3) was almost equal to that of 1alpha, 25(OH)(2)-19-nor-D(3), but higher than that of 1alpha,23(S), 25(OH)(3)-24-oxo-19-nor-D(3). This finding explains in part the greater in vitro biologic activities of 1alpha,23(R), 25(OH)(3)-24-oxo-19-nor-D(3). In summary, our results indicate that 1alpha,23(R),25(OH)(3)-24-oxo-19-nor-D(3) and to a lesser extent 1alpha,23(S),25(OH)(3)-24-oxo-19-nor-D(3) are potent 19-nor vitamin D(3) analogs, which suppress PTH secretion in bovine parathyroid cells and strongly inhibit clonal growth and induce differentiation of HL-60 cells in vitro. PMID- 10751639 TI - Characterization of the oligosaccharides of plasma sex hormone binding globulin from noncirrhotic alcoholic patients. AB - In previous reports we have demonstrated high plasma levels of sex hormone binding globulin (SHBG) in asymptomatic alcoholic men. In the present work the physicochemical properties of SHBG from plasma of noncirrhotic alcoholic patients have been further compared with SHBG of control subjects. Steroid binding to SHBG was similar for the two groups: alcoholic men, K(d) of 0.62 +/- 0.07 nM and control individuals, K(d) of 0.70 +/- 0.10 nM. The structure of oligosaccharides attached to SHBG from controls and alcoholic men were determined by using serial chromatography. Our data indicated that 7% of SHBG of control individuals was not retarded by the Con-A column, whereas approximately 30% of SHBG of alcoholic men eluted in the void volume of Con A. Approximately 46% of SHBG of alcoholics applied to Con A, possessed biantennary complex oligosaccharides, as indicated by the fact that it could be eluted with methyl-alpha-D-glucopyranoside and by its retention on wheat germ agglutinin; in contrast, when SHBG from control men was analyzed, approximately 51% was eluted with methyl-alpha-D-glucopyranoside. Approximately 9% of the biantennary complex oligosaccharides on SHBG of control men and none of those on SHBG from alcoholic men were fucosylated on the chitobiose core, as determined by chromatography on Lenn culinaris lectin. Galactosylated oligosaccharides were also present on the SHBG fraction as indicated by its interaction with Ricinus communis-I. Approximately 24% of SHBG of alcoholic men and 39% of those on SHBG from control individuals applied to Con A were retained and could be eluted with methyl-alpha-D-mannopyranoside. Evidence based on the binding on mannoside-eluted SHBG to Con-A, wheat germ agglutinin, and R. communis-I indicated that at least the SHBG in this fraction, from alcoholics or controls, contained two glycosylation sites and that the sites were differentially glycosylated. PMID- 10751638 TI - Synthesis and biological activity of a new progestogen, 16-methylene-17alpha hydroxy-18-methyl-19-norpregn-4-ene-3, 20-dione acetate. AB - The progestational activity of second- and third-generation progestins in oral contraceptives were markedly increased by addition of an 18-methyl group. A new progestin, the 18-methyl analog of Nestorone, 16-methylene-17alpha-hydroxy-18 methyl-19-norpregn-4-ene-3,2 0-dione acetate (10), was synthesized. The relative binding affinity and biologic activity of 10 was compared with Nestorone, levonorgestrel, and progesterone using a binding assay for rat progesterone receptors, the Clauberg assay in the rabbit, and by assessing pregnancy maintenance in the rat. These studies, as summarized in Table 4, show that 10 is three to ten times more potent than Nestorone. The addition of the 18-methyl group to Nestorone markedly increased its potency as noted above, but is unlikely to change its rate of delivery from sustained release systems. 10 should be ideally suited for administration by implants or small skin patches. PMID- 10751640 TI - Characterization of new conjugated metabolites in bile of rats administered 24,25 dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3). AB - The characterization of new conjugated vitamin D metabolites in rat bile was performed using HPLC, liquid chromatography/tandem mass spectrometry combined derivatization, and GC-MS. After the administration of 24,25-dihydroxyvitamin D(3) to rats, 23, 25-dihydroxy-24-oxovitamin D(3) 23-glucuronide, 3-epi-24, 25 dihydroxyvitamin D(3) 24-glucuronide, and 24,25-dihydroxyvitamin D(3) 3-sulfate were obtained as new biliary metabolites together with 24,25-dihydroxyvitamin D(3) 3- and 24-glucuronides. The above metabolites, except 24,25-dihydroxyvitamin D(3) 3-glucuronide, were obtained from rats dosed with 25-hydroxyvitamin D(3). 23, 25-Dihydroxyvitamin D(3) 23-glucuronide was also obtained from the bile of rats administered 25-hydroxyvitamin D(3) in addition to its 3-glucuronide, 25 glucuronide, and 3-sulfate. Thus, it was found that 24,25-dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3) were directly conjugated as glucuronide and sulfate, whereas at the C-23 position, they were hydroxylated and then conjugated. Furthermore, we found that the C-3 epimerization acts as one of the important pathways in vitamin D metabolism. PMID- 10751641 TI - Endothelial vasodilator production by uterine and systemic arteries. V. Effects of ovariectomy, the ovarian cycle, and pregnancy on prostacyclin synthase expression. AB - Prostacyclin (PGI(2)) is a potent vasodilator, the level of which is increased during pregnancy, and is the main eicosanoid of which production is elevated in the endothelium and vascular smooth muscle (VSM) of both uterine and omental (systemic) arteries. We tested the hypothesis that during physiologic states that have high uterine blood flow, such as pregnancy and the follicular phase of the ovarian cycle (versus luteal phase and ovariectomized ewes), there is an increased level of prostacyclin synthase (PGIS) expression in ovine uterine and omental artery endothelium and VSM. To investigate this, the cellular localization and PGIS protein expression level in uterine and systemic arteries was examined by immunohistochemistry as well as by Western immunoblot analysis of endothelial-isolated protein and denuded vessels (VSM). Whole uterine, but not omental (systemic), arteries from the pregnant ewes showed an increase (P < 0.001) in PGIS expression. Further localization of PGIS protein by immunohistochemistry and quantification by Western analysis showed PGIS to be somewhat higher in the uterine artery VSM (69 +/- 7%) than endothelium (31 +/- 7%). PGIS protein levels in uterine and omental artery endothelial isolated protein were not altered by ovariectomy or the ovarian cycle, although they were both significantly elevated by pregnancy. Uterine and omental artery VSM PGIS expression levels also were not altered by ovariectomy or the ovarian cycle, whereas PGIS expression, in uterine but not omental artery VSM showed a significant elevation during pregnancy. Thus, the rise in PGI(2) production by uterine arteries observed in ovine pregnancy is paralleled by an elevation in PGIS expression in both endothelium and VSM, whereas those seen in omental arteries is associated with increases in endothelial PGIS. PMID- 10751642 TI - Expression of cytosolic and secreted forms of phospholipase A(2) and cyclooxygenases in human placenta, fetal membranes, and chorionic cell lines. AB - Lipid mediators play a crucial role in human parturition and phospholipase A(2) (PLA(2)) is a key regulator of the production of these compounds. We have investigated by PCR the expression of different groups of PLA(2) and COX enzymes in human fetal membranes (amnion and chorion), placenta and three chorionic cell lines (JEG-3, Jar, BeWo). Our data show that the cytosolic Group IV PLA(2) and COX-1 are expressed in all of them, whereas the secretory forms of PLA(2), (Groups IIA, and V), have a more restricted expression. Group IIA mRNA is most abundant in placenta and chorion, whereas Group V PLA(2) mRNA is most abundant in placenta and amnion. On the other hand, COX-2 is present in placenta, chorion and amnion, but was not detected in any of the chorionic cell lines. These results suggest that both cytosolic and distinct secreted forms of PLA(2) could be involved in arachidonic acid (AA) release preceding prostaglandin production at the fetal/maternal interface. PMID- 10751643 TI - Modulation of PAF production by incorporation of arachidonic acid and eicosapentaenoic acid in phospholipids of human leukemic monocyte-like cells THP 1. AB - Stimulated leukocytes generate platelet-activating factor (PAF) from membrane 1-O alkyl-2-acyl-sn-glycerophosphocholine through hydrolysis of fatty acid and subsequent acetylation at the sn2 position of glycerol. Since the enzymes involved in the hydrolysis step of PAF biosynthesis have relative selectivity for arachidonic acid (AA), the fatty acid composition of PAF precursors might modulate PAF production. We studied the effect of AA and eicosapentaenoic acid (EPA) incorporation on PAF biosynthesis, by measuring the incorporation of [(3)H]acetate, in Ca(2+) ionophore (A23187)-stimulated human leukemic monocyte like cells, THP-1. Supplementation of THP-1 with AA (25 microM, 1 week) or EPA (25 microM, 1 week) led to their efficient incorporation, in comparable quantities and with similar distributions, into phosphatidylcholine and phosphatidylethanolamine, and to a lesser extent into phosphatidylinositol. THP-1 cells supplemented with AA or with EPA synthetized similar amounts of PAF and of acyl analog of PAF under resting condition. However, AA-supplemented cells responded to A23187 stimulation by important raises of PAF (+125.71%) and of acyl analog of PAF (+381.75%) productions, whereas the same stimulation had little effect or no effect at all in cells supplemented with EPA. These results show that both EPA and AA may influence PAF production through their incorporation into PAF precursors, indicating that PAF production might be modulated by the fatty acid composition of its precursors. PMID- 10751644 TI - Chronic exercise training preserves prostaglandin-induced dilation of epicardial coronary artery during development of heart failure in awake dogs. AB - Although it has been shown that long-term exercise training preserves endothelium mediated nitric oxide vasodilator function in chronic heart failure (CHF), whether exercise training exerts similar beneficial effects on endothelial/prostaglandin-mediated vasodilator capacity in coronary circulation during the development of CHF has not been determined. Fifteen mongrel dogs were surgically instrumented for measurement of left ventricular pressure, aortic pressure, coronary blood flow and left circumflex coronary artery diameter. Dogs (n = 5) who underwent 4 weeks of cardiac pacing (210 b/min for 3 weeks and 240 b/min for the 4th week) developed CHF as characterized by significant reduction in left ventricular systolic pressure, mean arterial pressure and left ventricular dP/dt, increases in left ventricular end-diastolic pressure and heart rate, as well as clinical signs of CHF. Endothelial prostaglandin-mediated vasodilation of the epicardial coronary artery was impaired, as manifested by an attenuated arachidonic acid (AA)-induced dilation of the artery (epicardial artery diameter increased by: 0.78 +/- 0. 84% in CHF versus 4.6 +/- 0.89% in normal, P < 0.05); however, prostacyclin (PGI(2))-induced and nitroglycerin induced vasodilation of the coronary circulation were not altered. In contrast, dogs (n = 6) with cardiac pacing plus daily exercise training (4.4 +/- 0.3 km/h, 2 h/day) only developed mild cardiac dysfunction, and the response of the epicardial coronary artery diameter to AA was preserved (epicardial artery diameter increased by 4.2 +/- 0.98% from baseline, P 0.05 compared to its respective control). Thus, long-term exercise training preserves endothelial/prostaglandin-mediated dilation of epicardial coronary artery during development of CHF. PMID- 10751645 TI - Pharmacokinetics of oral iloprost in patients with Raynaud's phenomenon secondary to systemic sclerosis. AB - Iloprost is a chemically stable, pharmacologically highly potent prostacyclin minietic. The therapeutic efficacy of the intravenous preparation was proven in patients with peripheral arterial occlusive disease or with Raynaud's phenomenon (RP). Recently, a sustained release oral preparation was developed for outpatient therapy. The purpose of the current study was to investigate whether the oral drug has a different pharmacokinetic profile in patients with RP secondary to systemic sclerosis (SSc) in comparison with healthy volunteers. Ten patients with RP secondary to SSc and 10 healthy volunteers (matched for age and sex) participated. Oral iloprost 50 microg was given twice daily for 8 days with dosing intervals of 5 h and plasma levels were taken over 10 h on Day 1 and 8. Plasma levels of iloprost were determined by a validated specific and sensitive radio-immunoassay. Compared with healthy volunteers, patients with SSc exhibited higher AUC values (by mean factors of 2. 1 and 2.0 on Day 1 and 8) and maximum plasma levels (by mean factors of 1.6 and 1.8 on Day 1 and 8). The increased systemic iloprost exposure was observed after both daily doses and on both monitored study days. Mean AUC values did not show accumulation over the 8 days. These findings are in agreement with a reduced total clearance of iloprost given by i.v. route in SSc patients compared to healthy volunteers, although no participant with severe renal impairment was included. A weak but significant correlation was found between individual creatinine clearance and AUC values. In conclusion, RP secondary to SSc is associated with an increased systemic iloprost exposure which is probably caused by changes of the metabolic clearance of iloprost. These effects cannot be explained by changes of renal function alone. PMID- 10751646 TI - One year follow-up of patients treated with misoprostol in acute phase of viral hepatitis B. AB - The study was undertaken to determine the long-term effect of misoprostol, on hepatitis B virus (HBV) elimination in patients treated during acute phase of viral hepatitis B. Forty male patients were evaluated 12 months after treatment with misoprostol (M-group) or sylimarin (S-group). HBsAg clearance, as an indicator of HBV elimination, and serum bilirubin concentration, prothrombin index, and activities of alanine transaminase, aspartate transaminase, alkaline phosphatase as well as gamma glutamyltranspeptidase were analysed. At the end of treatment phase, improvement of liver function demonstrated through these biochemical indices was faster in M-group. After 12 months of follow-up HBsAg was cleared in all misoprostol treated patients and in 85% among S-group. Moreover misoprostol treatment resulted with normalization of bilirubin concentration and enzymes activity in all patients. Two among sylimarin treated patients (both HBsAg positive), had transaminases activities elevated over 100 U/l, that resulted with significantly higher values than in M-group. These results confirm beneficial effect of misoprostol treatment in patients with liver injury, but promising effect on HBV clearance should be confirmed in a study involving more patients. PMID- 10751647 TI - Directional secretion of prostaglandin F(2alpha) by polarized luminal epithelial cells from pig endometrium. AB - In swine, endometrial prostaglandin F(2alpha) (PGF(2alpha)) is the luteolysin. The capacity of luminal epithelial cells isolated from the endometrium of day 16 cyclic pigs, to secrete PGF(2alpha)500 Omega/cm(2)), they were treated on the apical, basal or both surfaces with 0 or 100 nM oxytocin (OT) in Experiment 1 or phorbol 12-myristate 13-acetate (PMA) in Experiment 2. In the absence of OT or PMA, PGF(2alpha) secretion occurred primarily from the basal surface and was approximately 12-fold greater (P < 0.001) than from the apical surface. Treatment with OT did not stimulate PGF(2alpha) secretion from either surface regardless of which surface was treated. In contrast, PMA increased PGF(2alpha) secretion from both surfaces. Treatment of the apical surface or both surfaces with PMA increased (P < 0.001) PGF(2alpha) secretion similarly from both surfaces. Treatment of only the basal surface with PMA increased (P < 0.01) PGF(2alpha) secretion from both surfaces, but tended (P = 0. 06) to increase its secretion from the basal surface more than from the apical surface. These results indicated that PGF(2alpha) secretion by luminal epithelial cells obtained from cyclic pigs occurs primarily toward a basal direction and is not stimulated by oxytocin. Activation of protein kinase C stimulates directional secretion of PGF(2alpha) from both surfaces of the epithelial cells. PMID- 10751648 TI - Thromboxane A(2) receptors: where have you gone? PMID- 10751649 TI - Muscle plasticity and metabolism: effects of exercise and chronic diseases. PMID- 10751650 TI - Introduction PMID- 10751652 TI - Hiss Martins-Ferreira. PMID- 10751651 TI - Michael V.L. Bennett and the cellular study of neural systems at Albert Einstein and Woods Hole. PMID- 10751653 TI - David Spray and science in Brazil. PMID- 10751654 TI - Electrical synapses, a personal perspective (or history). AB - Gap junctions are the morphological substrate of one class of electrical synapse. This memoir records the author's involvement in the development of our knowledge of the physiology and ultrastructure of electrical synapses. The answer to whether neurotransmission is electrical or chemical is either. One lesson is that Occam's razor sometimes cut too deep; the nervous system does its operations in a number of different ways and a unitarian approach can lead one astray [M.V.L. Bennett, Nicked by Occam's razor: unitarianism in the investigation of synaptic transmission, Biol. Bull. 168 (1985) 159-167]. Electrical synapses can do many things that chemical synapses can do, and do them just as slowly. The new molecular, cellular and physiological techniques will clarify where gap junctions and electrical coupling do and do not occur and permit experimental manipulation with high specificity. PMID- 10751655 TI - Connexins and gap junctions of astrocytes and oligodendrocytes in the CNS. AB - This review article summarizes early and recent literature on the structure, distribution and composition of gap junctions between astrocytes and oligodendrocytes, and the differential expression of glial connexins in adult and developing mammalian CNS. In addition to an overview of the topic, discussion is focused on the organization of homologous gap junctional interactions between astrocytes and between oligodendrocytes as well as on heterologous junctional coupling between astrocytes and oligodendrocytes. The homotypic and heterotypic nature of these gap junctions is related to the connexins known to be produced by glial cells in the intact brain and spinal cord. Emphasis is placed on the ultrastructural level of analysis required to attribute gap junction and connexin deployment to particular cell types and subcellular locations. Our aim is to provide a firm basis for consideration of anticipated rapid advances in understanding of structural relationships of gap junctions and connexins within the glial gap junctional syncytium. Conclusions to date suggest that the glial syncytium is more complex than previously appreciated and that glial pathways of junctional communication may not only be determined by the presence of gap junctions, but also by the connexin composition and conductance regulation of junctional channels. PMID- 10751656 TI - Connexin43 null mice reveal that astrocytes express multiple connexins. AB - The gap junction protein connexin43 (Cx43) is the primary component of intercellular channels in cardiac tissue and in astrocytes, the most abundant type of glial cells in the brain. Mice in which the gene for Cx43 is deleted by homologous recombination die at birth, due to profound hypertrophy of the ventricular outflow tract and stenosis of the pulmonary artery. Despite this significant cardiovascular abnormality, brains of connexin43 null [Cx43 (-/-)] animals are shown to be macroscopically normal and to display a pattern of cortical lamination that is not detectably different from wildtype siblings. Presence of Cx40 and Cx45 in brains and astrocytes cultured from both Cx43 (-/-) mice and wildtype littermates was confirmed by RT-PCR, Northern blot analyses and by immunostaining; Cx46 was detected by RT-PCR and Northern blot analyses. Presence of Cx26 in astrocyte cultures was indicated by RT-PCR and by Western blot analysis, although we were unable to resolve whether it was contributed by contaminating cells; Cx30 mRNA was detected by Northern blot in long term (2 weeks) but not fresh cultures of astrocytes. These studies thus reveal that astrocyte gap junctions may be formed of multiple connexins. Presumably, the metabolic and ionic coupling provided by these diverse gap junction types may functionally compensate for the absence of the major astrocyte gap junction protein in Cx43 (-/-) mice, providing whatever intercellular signaling is necessary for brain development and cortical lamination. PMID- 10751658 TI - Expression of Cx36 in mammalian neurons. AB - Cx36 is the first mammalian member of a novel subgroup of the connexin family, characterized by a long cytoplasmic loop, a peculiar gene structure and a preferential expression in cell types of neural origin. In the present review we summarize the evidence in favour of its predominant expression in neuronal cells in the mammalian central nervous system, such as results from experiments with specific neurotoxins and co-localization of Cx36 mRNA and a neuronal marker. We also report a detailed description of Cx36 mRNA distribution in the rat and human central nervous system by in situ hybridization and, for each brain region, we correlate the novel findings with previous morphological or functional demonstrations of neuronal gap junctions in the same area. PMID- 10751657 TI - Temporal expression of neuronal connexins during hippocampal ontogeny. AB - Communication through gap junction channels provides a major signaling mechanism during early brain histogenesis, a developmental time during which neural progenitor cells are inexcitable and do not express ligand-gated channel responses to the major CNS neurotransmitters. Expression of different gap junction types during neurogenesis may therefore define intercellular pathways for transmission of developmentally relevant molecules. To better understand the molecular mechanism(s) by which growth and differentiation of neurons are modulated by gap junction channels, we have been examining the developmental effects of a specific set of cytokines on differentiation and gap junction expression in a conditionally immortalized mouse embryonic hippocampal neuronal progenitor cell line (MK31). When multipotent MK31 cells are in an uncommitted state, they uniformly express the neuroepithelial intermediate filament class VI marker, nestin, are strongly coupled by gap junctions composed of connexin43 (Cx43) and express connexin45 (Cx45) at the mRNA level. As these cells undergo neuronal lineage commitment and exit from cell cycle, they begin to express the early neurofilament marker, NF66, and coupling strength and expression of Cx43 begin to decline with concurrent expression of other connexin proteins, including Cx26, Cx33, Cx36, Cx40 and Cx45. Terminal neuronal differentiation is heralded by the expression of more advanced neurofilament proteins, increased morphologic maturation, the elaboration of inward currents and action potentials that possess mature physiological properties, and changing profiles of expression of connexin subtypes, including upregulation of Cx36 expression. These important developmental transitions are regulated by a complex network of cell cycle checkpoints. To begin to examine the precise roles of gap junction proteins in traversing these developmental checkpoints and in thus regulating neurogenesis, we have focused on individual members of two classes of genes involved in these seminal events: ID (inhibitor of differentiation)-1 and GAS (growth arrest specific gene)5. When MK31 cells were maintained in an uncommitted state, levels of ID-1 mRNA were high and GAS5 transcripts were essentially undetectable. Application of cytokines that promote neuronal lineage commitment and cell cycle exit resulted in down-regulation of ID-1 and upregulation of GAS5 transcripts, whereas additional cytokine paradigms that promoted terminal neuronal differentiation resulted in the delayed down-regulation of GAS5 mRNA. Stable MK31 transfectants were generated for ID-1 and GAS5. In basal conditions, cellular proliferation was enhanced in the ID-1 transfectants and inhibited in the GAS5 transfectants when compared with control MK31 cells. When cytokine-mediated neurogenesis was examined in these transfected cell lines, constitutive expression of ID-1 inhibited and constitutive expression of GAS5 enhanced initial and terminal stages of neuronal differentiation, with evidence that terminal neuronal maturation in both transfectant lines was associated with decreased cellular viability, possibly due to the presence of conflicting cell cycle associated developmental signals. These experimental reagents will prove to be valuable experimental tools to help define the functional interrelationships between changing profiles of connexin protein expression and cell cycle regulation during neuronal ontogeny in the mammalian brain. The present review summarizes the current state of research involving the temporal expression of such connexin types in differentiating hippocampal neurons and speculates on the possible role of these intercellular channels in the development and plasticity of the nervous system. In addition, we describe the functional properties and expression pattern of the newly discovered neuronal-specific gap junctional protein, Cx36, in the developing mouse fetal hippocampus and in the rat retina and brain. PMID- 10751659 TI - Neurotransmitters and gap junctions in developing neural circuits. AB - A growing body of evidence suggests that highly correlated, spontaneous neural activity plays an important role in shaping connections in the developing nervous system prior to the maturation of sensory afferents. In this article we discuss the mechanisms involved in the generation and the regulation of spontaneous activity patterns in the developing retina and the developing neocortex. Spontaneous activity in the developing retina propagates across the ganglion cell layer as waves of action potentials and drives rhythmic increases in intracellular calcium in retinal neurons. Retinal waves are mediated by a combination of chemical synaptic transmission and gap junctions, and the circuitry responsible for generating retinal waves changes with age and between species. In the developing cortex, spontaneous calcium elevations propagate across clusters of cortical neurons called domains. Cortical domains are generated by a regenerative mechanism involving second messenger diffusion through gap junctions and subsequent calcium release from internal stores. The neocortical gap junction system is regulated by glutamate-triggered second messenger systems as well as neuromodulatory transmitters, suggesting extensive interactions between synaptic transmission and information flow through gap junctions. The interaction between gap junctions and chemical synaptic transmission observed in these developing networks represent a powerful mechanism by which activity across large groups of neurons can be correlated. PMID- 10751660 TI - Gap junctions in the eye: evidence for heteromeric, heterotypic and mixed homotypic interactions. AB - Some of the best evidence that different types of gap junction proteins (connexins) interact with each other in vivo has been found in the eye. This review focuses on three diverse ocular tissues that may contain heterotypic or heteromeric gap junction channels. Each of the tissues uses gap junctions in a superlative fashion: The crystalline lens has an exceptionally high density of gap junctions; the ciliary body expresses a surprising variety of connexins; the neural retina shows remarkable specificity in the patterns of intercellular coupling. PMID- 10751661 TI - Modulation of coupling between retinal horizontal cells by retinoic acid and endogenous dopamine. AB - The regulation of electrical coupling between retinal neurons appears to be an important component of the neuronal mechanism of light adaptation, which enables the retina to operate efficiently over a broad range of light intensities. The information about the ambient light conditions has to be transmitted to the neuronal network of the retina and previous evidence has indicated that dopamine is an important neurochemical signal. In addition, recent studies suggest that another important chemical signal is retinoic acid, which is a light-correlated byproduct of the phototransduction cycle. This review summarizes the latest findings about the effects of dopamine and retinoic acid on gap junctional coupling in the retinas of mouse, rabbit and fish. PMID- 10751662 TI - Intercellular communication in the eye: clarifying the need for connexin diversity. AB - In the vertebrate eye, virtually every cell type is directly coupled to its neighbors by intercellular channels present in gap junctions. Although these structures share the common property of allowing adjacent cells to directly exchange ions, second messengers and small metabolites, intercellular channels in the eye also play a specific role in distinct functions such as neuronal transmission at electrotonic synapses in the retina, and the maintenance of homeostasis in the avascular lens. The structural proteins comprising these channels, the connexins (Cx), are a multigene family of which many members are expressed in the eye, even in the same cell type. This molecular heterogeneity poses the crucial question of whether and how a diversity in gap junctional structural proteins influences intercellular communication in ocular tissues. This review will focus on two recent advances in the understanding of connexin diversity in regard to the eye. First, connexin knockouts have demonstrated that postnatal development and homeostasis in the lens requires multiple connexin proteins. Secondly, functional characterization of new connexins that are abundantly expressed in the retina has revealed biophysical properties that mimic those recorded from retinal neurons. PMID- 10751663 TI - Connexin expression in the retina. AB - Here, we review recent results from our laboratory on connexin expression in mouse retina in the context of previous results with other vertebrate species. In mouse retina, four different connexin proteins were detected by immunoblot and immunofluorescence: connexin (Cx)-36, -37, -43 and -45. Cx36 and Cx45 immunoreactive signals were found in the inner and outer plexiform layer, both of which are known to show interneuronal gap junctions. Cx43 was detected in the ganglion cell layer, presumably in astrocytes, where it appeared to be colocalized with glial fibrillary acid protein. Cx37 was expressed in retinal endothelial cells. Additionally, Cx26, -31, -32 and -40 mRNAs were detected in retina by RT-PCR but none of the corresponding proteins were found. In order to exclude cross reactions of the corresponding antibodies, retinae from targeted connexin-deficient mice (Cx31 -/-, Cx32 -/- and Cx40 -/-) were used as negative controls for immunoblot and immunofluorescence analyses of wild-type retina. Further detailed investigation of cell type specific connexin expression in the mouse retina will be necessary for functional analyses of targeted mouse mutants with defects in connexins expressed in retinal neuronal cells. PMID- 10751664 TI - The anti-death league: associative control of apoptosis in developing retinal tissue. AB - Apoptosis, the major form of programmed cell death (PCD), is executed through a proteolytic cascade that can be differentially engaged by various extracellular signals. Modulation of both the sensitivity to PCD and of the actual sequence of apoptotic events is, therefore, strongly dependent on cell interactions. This paper reviews the use of a retinal explant preparation as a model of the organized nervous tissue, to study the effects of neural messengers in the control of sensitivity to apoptosis. Studies of retinal explants showed that dopamine, glutamate and nitric oxide may have anti-apoptotic effects upon developing retinal cells. At least the effects of nitric oxide are clearly paracrine. In addition, preliminary evidence has been gathered of a role for gap junctional communication in the control of sensitivity of retinal cells to the induction of apoptosis. These findings underscore the importance of selective cell interactions in the control of PCD in the developing nervous system. PMID- 10751665 TI - Gap junction systems in the mammalian cochlea. AB - Recent findings that a high proportion of non-syndromic hereditary sensorineural hearing loss is due to mutations in the gene for connexin 26 indicate the crucial role that the gene product plays for normal functioning of the cochlea. Excluding sensory cells, most cells in the cochlea are connected via gap junctions and these gap junctions appear to play critical roles in cochlear ion homeostasis. Connexin 26 occurs in gap junctions connecting all cell classes in the cochlea. There are two independent systems of cells, which are defined by interconnecting gap junctions. The first system, the epithelial cell gap junction system, is mainly composed of all organ of Corti supporting cells, and also includes interdental cells in the spiral limbus and root cells within the spiral ligament. The second system, the connective tissue cell gap junction system, consists of strial intermediate cells, strial basal cells, fibrocytes in the spiral ligament, mesenchymal cells lining the bony otic capsule facing the scala vestibuli, mesenchymal dark cells in the supralimbal zone, and fibrocytes in the spiral limbus. One function of these gap junctional systems is the recirculation of K(+) ions from hair cells to the strial marginal cells. Interruption of this recirculation, which may be caused by the mutation in connexin 26 gene, would deprive the stria vascularis of K(+) and result in hearing loss. PMID- 10751666 TI - Cell coupling in Corti's organ. AB - The mammalian organ of Corti is responsible for the initial analysis of sound; injury leads to hearing loss. During the last two decades, the characteristics of cellular coupling in this specialized epithelium have been studied. In this review, data on both electrical and mechanical coupling are covered. While electrical coupling likely contributes to homeostasis in the organ, this concept is far from proven. PMID- 10751667 TI - Immunolocalization of connexin 26 in the developing mouse cochlea. AB - Gap junctions play a pivotal role in embryonic development by forming specialized regions of cell-cell communication. In this study, we demonstrate the temporal spatial distribution of connexin 26 in the embryonic and early postnatal mouse cochlea. Our results show localization of this gap junction protein to specific cochlear structures, including the inner and outer sulcus cells, the supporting cells of the inner hair cells, the mesenchyme derived portion of the stria vascularis, and the cells of the spiral ligament that interface with the basal cells of the stria vascularis. This suggests that this gap junction protein of served patterns of connexin 26 distribution is important for the differentiation and development of these structures (e.g., the role of the inner sulcus cells in producing the tectorial membrane). PMID- 10751668 TI - Functional analysis of human Cx26 mutations associated with deafness. AB - Mutations in the connexin26 (Cx26) gene are not only a major cause of nonsyndromic deafness, but can also cause syndromic forms of hearing loss that are associated with palmoplantar keratoderma (PPK, i.e., Vohwinkel's syndrome). It is not clear how two very distinct pathologies can arise from different mutations within the same connexin gene. This review summarizes the available data on wildtype and mutant Cx26 channel behavior that has been obtained in the paired Xenopus oocyte assay. These results suggest that dominant and recessive loss of function mutations in Cx26 can cause nonsyndromic deafness, but cannot easily explain the syndromic forms exhibiting PPK. Dominant Cx26 mutations that can cause both PPK and deafness must show some additional alteration of function beyond a simple inhibition of Cx26 activity. PMID- 10751669 TI - Connexin 26: required for normal auditory function. AB - A single base deletion mutation, 35delG, in the gene (GJB2/DFNB1)(OMIM 121011/220290) encoding the gap junction protein, connexin 26 is the most important single cause of genetic hearing loss in European and American populations. It is the cause of one of the most common human genetic disorders with a frequency similar to cystic fibrosis. Mutations in this connexin are associated with skin disorders. PMID- 10751670 TI - Connexin channels in Schwann cells and the development of the X-linked form of Charcot-Marie-Tooth disease. AB - Charcot-Marie-Tooth disease comprises a group of genetically heterogenous disorders of the peripheral nervous system. The X-linked form of Charcot-Marie Tooth (CMTX) is associated with mutations in the gene encoding the gap junction protein connexin32 (Cx32), which is expressed in Schwann cells. Immunocytochemical evidence suggests that Cx32 is localized to the incisures of Schmidt-Lanterman and the paranodes of myelinating Schwann cells, where it appears to form reflexive gap junctions. It is currently thought that this cytoplasmic continuity provides a much shorter diffusion pathway for the transport of ions, metabolites and second messenger molecules through intracellular channels between the adaxonal and peri-nuclear regions of Schwann cells, across the myelin sheath. This review summarizes our current understanding of the role of connexins in Schwann cells and focuses on the lessons for channel function and disease pathophysiology derived from the functional analysis of Cx32 mutations. One of the most intriguing aspects emerging from this work is that several mutations retain functional competence, although the mutated channels exhibit altered gating properties. This suggests that partial and/or selective disruption of the radial communication pathway formed by Cx32 is sufficient to cause a functional deficit and lead to the development of CMTX. The next challenge will be to define, at the molecular level, the sequence of events involved in the disease process. The presence of a group of functional mutations should help understand the cellular basis of CMTX, by allowing the identification of the specific molecules that need to be exchanged through Cx32 channels, but are excluded from the mutated ones. PMID- 10751671 TI - Mutations in connexin 32: the molecular and biophysical bases for the X-linked form of Charcot-Marie-Tooth disease. AB - The connexins are a family of homologous integral membrane proteins that form channels that provide a low resistance pathway for the transmission of electrical signals and the diffusion of small ions and non-electrolytes between coupled cells. Individuals carrying mutations in the gene encoding connexin 32 (Cx32), a gap junction protein expressed in the paranodal loops and Schmidt-Lantermann incisures of myelinating Schwann cells, develop a peripheral neuropathy - the X linked form of Charcot-Marie-Tooth disease (CMTX). Over 160 different mutations in Cx32 associated with CMTX have been identified. Some mutations will lead to complete loss of function with no possibility of expression of functional channels. Some mutations in Cx32 lead to the abnormal accumulation of Cx32 proteins in the cytoplasm, particularly in the Golgi apparatus; CMTX may arise due to incorrect trafficking of Cx32 or to interference with trafficking of other proteins. On the other hand, many mutant forms of Cx32 can form functional channels. Some functional mutants have conductance voltage relationships that are disrupted to a degree which would lead to a substantial reduction in the available gap junction mediated communication pathway. Others have essentially normal steady-state g-V relations. In one of these cases (Ser26Leu), the only change introduced by the mutation is a reduction in the pore diameter from 7 A for the wild-type channel to less than 3 A for Ser26Leu. This reduction in pore diameter may restrict the passage of important signaling molecules. These findings suggest that in some, if not all cases of CMTX, loss of function of normal Cx32 is sufficient to cause CMTX. PMID- 10751672 TI - Perspectives on spreading depression. AB - Spreading depression (SD) consists of a transient suppression of all neuronal activity that spreads slowly across regions of gray matter. The paper is divided into three parts. Martins-Ferreira describes 30 years of research on SD in the isolated retina. Much of this work has relied on the prominent intrinsic optical signals that accompany SD in the retina. By inducing SD to propagate in circles with a velocity of 3.7 mm min(-1), it is possible to investigate the finely balanced electrochemical equilibrium that maintains the traveling wave. SD is accompanied by a slow negative extracellular voltage and ion movements that are greatest in the inner plexiform layer of the retina. Nedergaard discusses the role of astrocytes in SD propagation. Astrocytes mediate slowly moving waves of intracellular Ca(2+) increase, for which gap junctions are essential. SD is accompanied by entry of Ca(2+) into cells and fails when gap junctions are blocked. SD, however, is blocked by glutamate receptor antagonists but glial Ca(2+) waves are not. Astrocytic Ca(2+) waves are probably involved in the initiation of SD but other factors, including K(+), glutamate and purinergic receptors, are necessary for sustained propagation. Nicholson describes studies on the different preparations that helped clarify the role of extracellular space in SD. It has long been known that extracellular K(+) reaches levels of 50 mM or more during SD. Studies with ion-selective microelectrodes showed that extracellular Na(+) and Cl(-) fall by as much as 100 mM during SD, and water leaves the extracellular space. Further work showed that extracellular Ca(2+) falls 10-fold during SD and significant changes in extracellular pH and ascorbate occur. These studies imply that large perturbations of the extracellular milieu occur during SD and are an essential part of the interlocking cascade of events that produce this still mysterious phenomenon. PMID- 10751673 TI - The role of gap junctions in seizures. AB - Electrotonic synaptic communication between neurons via gap junctions (gjs) is increasingly recognized as an important synchronizing mechanism in the brain. At the same time, the biology of central nervous system (CNS) gjs is being unravelled. The pathogenesis of the abnormal neuronal synchrony underlying seizures, formerly thought to be based mainly on chemical synaptic transmission, now includes a role for gap junctional communication. This concept has been strengthened by evidence from several in vitro seizure models, in which pharmacological manipulations of gap junctional communication predictably affect the generation of seizures: blockers diminishing seizures and enhancers increasing the seizures. Evidence for interneurons, coupled in part by gjs, generating synchronous neural network activity including seizures, is presented. Also neuromodelling studies, which have enhanced our ability to understand the functional role that gap junctional communication plays in the generation and maintenance of neural synchrony and seizures, are presented. Gap junctional communication appears to be a promising target for the development of future anticonvulsant therapy. PMID- 10751674 TI - Gap junctional communication among developing and injured motor neurons. AB - The functional significance of gap junctional coupling among neurons is poorly understood. We are studying gap junctions among spinal motor neurons as a model for understanding roles of interneuronal gap junctional communication during development and after injury. Electrical and dye coupling is widespread among neonatal motor neurons but is transient, disappearing by the end of the first postnatal week. Reverse transcription polymerase chain reaction (RT-PCR) analysis, in situ hybridization and immunohistochemistry show that five rodent connexins, Cx36, Cx37, Cx40, Cx43 and Cx45, are expressed by developing motor neurons. These gap junction proteins remain expressed in some motor neurons through adult life, with the exception of Cx40, whose expression appears to decrease shortly after birth. After nerve injury in adult animals, motor neurons once again become dye coupled, and this appears to occur without dramatic changes in connexin expression. The transient gap junctional coupling present among developing motor neurons, which is re-capitulated after axotomy, may mediate electrical or biochemical signaling that shapes neuronal function. PMID- 10751675 TI - Regulation of astrocyte gap junctions by hypoxia-reoxygenation. AB - Confluent cultures of rat cortical astrocytes were subjected to 12-h hypoxia (<1% O(2)) followed by reoxygenation. Just after hypoxia, the cellular distribution, phosphorylation state and levels of connexin43 (Cx43), as well as the extent of dye coupling were as in control conditions. Nonetheless, 15-30 min after reoxygenation, dye coupling was transiently reduced by approximately 70%. The reduction in dye coupling occurred without changes in the state of phosphorylation or levels of Cx43. Nevertheless, it was correlated with a decrease in Cx43 reactivity found at membrane appositions and the appearance of intracellular Cx43-positive vesicle-like structures of variable size, suggesting internalization of gap junction channels. Reoxygenation-induced cellular uncoupling and redistribution of Cx43 were prevented by melatonin (500 microM), a potent-free radical scavenger, or indomethacin (50 microM), an inhibitor of the cyclooxygenase-dependent arachidonic acid metabolism. In astrocytes cultured under normoxia, the state of phosphorylation of Cx43 was not affected by antimycin A, a blocker of the mitochondrial oxidative metabolism, but phosphorylation was drastically reduced by iodoacetate, a blocker of anaerobic glycolysis. Thus, these results strongly suggest that reoxygenation-induced uncoupling is mediated by arachidonic acid byproducts that induce, at least, disorganization of Cx43 gap junction channels. PMID- 10751676 TI - Identification of genes differentially expressed in C6 glioma cells transfected with connexin43. AB - Astrocytes are characterized by extensive gap junctional intercellular communication (GJIC) mediated primarily by channels composed of connexin43. In contrast, C6 glioma cells are deficient in connexin expression and gap junctional communication. Transfection of these glioma cells with connexin cDNAs results in changes in cellular phenotype following increased GJIC. Specifically, connexin expression correlates with reduced cellular proliferation and tumorigenicity. To characterize the role of gap junctions in this growth control, we have screened for changes in gene expression by differential display. We have observed that these changes in GJIC are associated with changes in expression of several genes, including those coding for a number of secreted factors which may play a role in modulating the tumor phenotype of these cells. These include the immediate early gene cyr61, ostoepontin and the KC gene (murine homologue of the human gro gene). PMID- 10751677 TI - Epileptogenesis in immature rats following recurrent status epilepticus. AB - Strong evidences link status epilepticus (SE) in childhood with the later development of epilepsy. Pilocarpine-induced SE in developing rats leads to late appearance of spontaneous epileptic seizures only when SE is induced after the 18th day of life. We examined the possibility that 3 consecutive episodes of pilocarpine-induced SE on postnatal days 7, 8 and 9 could induce behavioral, electrographic and histological epileptic changes in adult life. The animals also underwent behavioral tests (inhibitory step-down avoidance, skinner box, rota rod, open field and elevated plus-maze). EEG recordings made at the age of 30, 60 and 90 days showed the occurrence of several episodes of spikes and/or polyspikes appearing simultaneously in hippocampus and cortex. Only three isolated spontaneous seizures were observed during the whole period of observation (120 days). The long-term effects of three consecutive episodes of SE include increased spontaneous exploratory activity, learning impairment, and reduced anxiety when tested on P60. Our findings provide evidence for EEG changes and cognitive deficits in adult life following recurrent SE on postnatal days 7-9. PMID- 10751678 TI - Brain dystrophin, neurogenetics and mental retardation. AB - Duchenne muscular dystrophy (DMD) and the allelic disorder Becker muscular dystrophy (BMD) are common X-linked recessive neuromuscular disorders that are associated with a spectrum of genetically based developmental cognitive and behavioral disabilities. Seven promoters scattered throughout the huge DMD/BMD gene locus normally code for distinct isoforms of the gene product, dystrophin, that exhibit nervous system developmental, regional and cell-type specificity. Dystrophin is a complex plasmalemmal-cytoskeletal linker protein that possesses multiple functional domains, autosomal and X-linked homologs and associated binding proteins that form multiunit signaling complexes whose composition is unique to each cellular and developmental context. Through additional interactions with a variety of proteins of the extracellular matrix, plasma membrane, cytoskeleton and distinct intracellular compartments, brain dystrophin acquires the capability to participate in the modulatory actions of a large number of cellular signaling pathways. During neural development, dystrophin is expressed within the neural tube and selected areas of the embryonic and postnatal neuraxis, and may regulate distinct aspects of neurogenesis, neuronal migration and cellular differentiation. By contrast, in the mature brain, dystrophin is preferentially expressed by specific regional neuronal subpopulations within proximal somadendritic microdomains associated with synaptic terminal membranes. Increasing experimental evidence suggests that in adult life, dystrophin normally modulates synaptic terminal integrity, distinct forms of synaptic plasticity and regional cellular signal integration. At a systems level, dystrophin may regulate essential components of an integrated sensorimotor attentional network. Dystrophin deficiency in DMD/BMD patients and in the mdx mouse model appears to impair intracellular calcium homeostasis and to disrupt multiple protein-protein interactions that normally promote information transfer and signal integration from the extracellular environment to the nucleus within regulated microdomains. In DMD/BMD, the individual profiles of cognitive and behavioral deficits, mental retardation and other phenotypic variations appear to depend on complex profiles of transcriptional regulation associated with individual dystrophin mutations that result in the corresponding presence or absence of individual brain dystrophin isoforms that normally exhibit developmental, regional and cell-type-specific expression and functional regulation. This composite experimental model will allow fine-level mapping of cognitive-neurogenetic associations that encompass the interrelationships between molecular, cellular and systems levels of signal integration, and will further our understanding of complex gene-environmental interactions and the pathogenetic basis of developmental disorders associated with mental retardation. PMID- 10751679 TI - Gap junctions: the "kiss of death" and the "kiss of life". AB - Cells expressing herpes simplex-thymidine kinase (HSV-tk) can be killed "in vitro" within 5 days of treatment with 20 microM ganciclovir (GCV) and transmit this toxicity to adjacent cells lacking HSV-tk; this phenomenon was termed "bystander effect" or "kiss of death". On testing a large number of cell lines in vitro, a wide range of sensitivity to GCV-mediated bystander killing has been reported. Although intercellular transfer of GCV metabolites through gap junction channels seems to be a likely mechanism for the "kiss of death", some studies suggest that other pathways may contribute to induced apoptosis of neighboring cells. To further investigate the mechanism underlying cell death mediated by HSV tk and to evaluate the efficacy of gap junction channels formed by different connexins in this process, we have stably transfected a virtually uncoupled mouse neuroblastoma cell line (N2A cells) with different connexin-types expressed by neural cells (Cx32, Cx37, Cx40, Cx43) and co-cultured these cells with N2A cells stably transfected with Cx37 and HSV-tk. Here, we confirm our previous studies and those of others that the extent of cell death and sensitivity to GCV depend on the degree of connexin expression in transfectants. Further, we show that the bystander effect also depends on which connexin is expressed; reported disparities regarding the extent of GCV-mediated cellular apoptosis are likely due both to the degree of functional coupling and the type of connexin expressed. These results support the notion that gap junction hemichannels formed of certain connexins are more likely than others to pair functionally with Cx37, and suggest co-transfection strategies that might prove effective in sensitizing tumor cell populations to GCV. In addition, potential applications are discussed for use of the "good Samaritan effect", a mechanism by which bystander cells have been suggested to prevent cytotoxicity. PMID- 10751680 TI - Alfentanil-induced activation: a promising tool in the presurgical evaluation of temporal lobe epilepsy patients. AB - Pharmacologic activation of epileptic foci has been used experimentally with the hope that it may accelerate the presurgical evaluation of patients with medically intractable epilepsy. In this article, we will review the existing literature on these activating tests giving emphasis on the opioid analogs, and particularly alfentanil. Alfentanil is an opioid analog with rapid anesthetic effect, which has been known to trigger epileptiform discharges in epilepsy patients. 58 temporal lobe epilepsy (TLE) patients were studied with alfentanil activation during electrocorticography, at the Epilepsy Surgery Unit (ING, Brazil). An increase of the interictal epileptiform discharges was observed originating from hippocampal and parahippocampal regions (96.5%). To a lesser extent, alfentanil activated the basal and lateral temporal regions. Electrographic seizures were observed in 38%. In addition, we performed continuous video-EEG (VT/EEG) monitoring, with scalp and bilateral foramen ovale electrodes, in 12 TLE patients. The results of spontaneously observed seizures were compared with the electrographic changes following alfentanil activation (50-75 microg/kg, i.v.). In seven cases, alfentanil triggered focal electrographic seizures, ipsilaterally to the side generating the spontaneous seizures and in two patients it produced bilateral sequential activation of the temporal lobes. Ictal SPECTs during the alfentanil test showed hyperperfusion at the lateral temporal regions, ipsilaterally to the activated area or bilaterally. In summary, our study confirms the activating effect of alfentanil, and provides a strong evidence for its selective activating effect on the temporal lobes of TLE patients. The ictal SPECT during alfentanil activation did not offer any additional advantage for the localization of the ictal onset. PMID- 10751681 TI - Simultaneous intrastriatal and intranigral grafting (double grafts) in the rat model of Parkinson's disease. AB - Experimental and clinical studies of neural transplantation in Parkinson's disease have focused on the placement of fetal dopaminergic grafts not in their ontogenic site (substantia nigra) but in the main nigral target area (striatum). The reason for this is the apparent inability of intranigral nigral grafts to extend axons for long distances reinnervating the ipsilateral striatum. This review presents previous work by our laboratory [I. Mendez, M. Hong, Reconstruction of the striato-nigro-striatal circuitry by simultaneous double dopaminergic grafts: a tracer study using fluorogold and horseradish peroxidase, Brain Res. 778 (1997) 194-205; I. Mendez, D. Sadi, M. Hong., Reconstruction of the nigrostriatal pathway by simultaneous intrastriatal and intranigral dopaminergic transplants, J. Neurosci. 16 (1996) 7216-7227] using a new transplantation strategy aimed at restoring dopaminergic innervation of the nigra and striatum by simultaneous dopaminergic transplants placed in the substantia nigra and ipsilateral striatum (double grafts) in the 6-hydroxydopamine lesioned adult rat brain. These double grafts achieve not only greater striatal reinnervation than the standard intrastriatal grafts but also produce a faster and more complete behavioural recovery six weeks after transplantation. Injection of the retrograde tracer fluorogold into the striatum and nigra resulted in fluorescent labeled cells within the intranigral graft and the intrastriatal graft and surrounding striatum, respectively suggesting that these double grafts promote at least partial reconstruction of the nigrostriatal dopaminergic pathway. This double graft strategy may have potential implications in clinical neural transplantation for Parkinson's disease. PMID- 10751683 TI - The role SWISS-PROT and TrEMBL play in the genome research environment. AB - SWISS-PROT, a curated protein sequence data bank, contains not only sequence data but also annotation relevant to a particular sequence. The annotation added to each entry is done by a team of biologists and comes, primarily, from articles in journals reporting the actual sequencing and sometimes characterisation. Review articles and collaboration with external experts also play a role along with the use of secondary databases like PROSITE and Pfam in addition to a variety of feature prediction methods. Annotation added by these methods is checked for relevance and likelihood to a particular sequence. The onset of genome sequencing has led to a dramatic increase in sequence data to be included in SWISS-PROT. This has led to the production of TrEMBL (Translation of the EMBL database). TrEMBL consists of entries in a SWISS-PROT format that are derived from the translation of all coding sequences in the EMBL nucleotide sequence database, that are not in SWISS-PROT. Unlike SWISS-PROT entries those in TrEMBL are awaiting manual annotation. However, rather than just representing basic sequence and source information, steps have been taken to add features and annotation automatically. In taking these steps it is hoped that TrEMBL entries are enhanced with some indication as to what a protein is, could or may be. PMID- 10751682 TI - Implication of gene distribution in the bacterial chromosome for the bacterial cell factory. AB - As bacterial genome sequences accumulate, more and more pieces of data suggest that there is a significant correlation between the distribution of genes along the chromosome and the physical architecture of the cell, suggesting that the map of the cell is in the chromosome. Considering sequences and experimental data indicative of cell compartmentalisation, mRNA folding and turnover, as well as known structural features of protein and membrane complexes, we show that preliminary in silico analysis of whole genome sequences strongly substantiates this hypothesis. If there is a correlation between the genome sequence and the cell architecture, it must derive from some selection pressure in the organisms growing in the wild. As a consequence, the underlying constraints should be optimised in genetically modified organisms if one is to expect high product yields. Consequences in terms of gene expression for biotechnology are straightforward: knocking genes out and in genomes should not be randomly performed, but should follow the rules of chromosome organisation. PMID- 10751684 TI - PPMdb: a plant plasma membrane database. AB - PPMdb is a proteome database dedicated to proteins from plant plasma membranes. It provides comprehensive two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) maps, partial amino acid sequences and expression data. All this information is gathered and structured in a relational database, after being analyzed and annotated. PPMdb includes active links to related biological databases (EMBL, GenBank, GenPep, and SWISS-PROT and TrEMBL) as well as to MEDLINE abstracts. Information on specific protein spots can be displayed by clicking on the 2-D maps. In addition, users can query the database by accession number, protein name, pI and MW, and cellular location. Access to PPMdb is available at the following URL: http://sphinx.rug. ac.be:8080. PMID- 10751685 TI - Analysis of large gene databases for discovery of novel therapeutic agents. AB - During the 1980s and early 1990s the recombinant DNA revolution provided a vital source of therapeutic targets and agents for pharmaceutical research. However, during the early 1990s, it became apparent that the identification and cloning of novel human cDNAs was a rate limiting step in drug discovery and that new technological approaches were required to address the challenge. There was an increasing realisation that the new science of 'genomics', together with the associated large gene sequence databases, would provide a radically new means of generating targets. SmithKline Beecham has been at the forefront of this breakthrough in pharmaceutical research. The productivity of this strategy is illustrated by reference to our work on novel enzymes, chemokines and receptors and new approaches linking genes to pathological processes. PMID- 10751686 TI - Genomics, molecular genetics and the food industry. AB - The production of foods for an increasingly informed and selective consumer requires the coordinated activities of the various branches of the food chain in order to provide convenient, wholesome, tasty, safe and affordable foods. Also, the size and complexity of the food sector ensures that no single player can control a single process from seed production, through farming and processing to a final product marketed in a retail outlet. Furthermore, the scientific advances in genome research and their exploitation via biotechnology is leading to a technology driven revolution that will have advantages for the consumer and food industry alike. The segment of food processing aids, namely industrial enzymes which have been enhanced by the use of biotechnology, has proven invaluable in the production of enzymes with greater purity and flexibility while ensuring a sustainable and cheap supply. Such enzymes produced in safe GRAS microorganisms are available today and are being used in the production of foods. A second rapidly evolving segment that is already having an impact on our foods may be found in the new genetically modified crops. While the most notorious examples today were developed by the seed companies for the agro-industry directed at the farming sector for cost saving production of the main agronomical products like soya and maize, its benefits are also being seen in the reduced use of herbicides and pesticides which will have long term benefits for the environment. Technology driven advances for the food processing industry and the consumer are being developed and may be divided into two separate sectors that will be presented in greater detail: 1. The application of genome research and biotechnology to the breeding and development of improved plants. This may be as an aid for the cataloging of industrially important plant varieties, the rapid identification of key quality traits for enhanced classical breeding programs, or the genetic modification of important plants for improved processing properties or health characteristics. 2. The development of advanced microorganisms for food fermentations with improved flavor production, health or technological characteristics. Both yeasts and bacteria have been developed that fulfill these requirements, but are as yet not used in the production of foods. PMID- 10751687 TI - A fast and convenient MALDI-MS based proteomic approach: identification of components scaffolded by the actin cytoskeleton of activated human thrombocytes. AB - A recently developed concentration and purification method (Gevaert, K., Demol, H., Puype, M., Broekaert, D., De Boeck, S., Houthaeve, T., Vandekerckhove, J., 1997. Electrophoresis 18, 2950-2960) for the analysis of diluted peptide samples by matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) is compared with conventional MALDI sample preparation methods. In the procedure developed, reverse-phase chromatographic beads are added to diluted peptide solutions and act as a peptide-trapping device. Peptides concentrated on the added beads are subsequently harvested, transferred to the MALDI-target disc and efficiently on target desorbed from the beads in a very small volume of an organic-aqueous mixture containing the aromatic MALDI-matrix components. Using this procedure, we show that it is possible to use the totality of in gel protein digests without negative interference of buffers and chaotropes that may be present in the digestion mixture. This method links MALDI-MS peptide analysis more efficiently to 2-D gel electrophoresis in the concept of proteome analysis. The procedure is illustrated by the identification of a class of proteins, which translocate to the actin cytoskeleton of human platelets upon thrombin stimulation. PMID- 10751688 TI - The application of DNA microarrays in gene expression analysis. AB - DNA microarray technology is a new and powerful technology that will substantially increase the speed of molecular biological research. This paper gives a survey of DNA microarray technology and its use in gene expression studies. The technical aspects and their potential improvements are discussed. These comprise array manufacturing and design, array hybridisation, scanning, and data handling. Furthermore, it is discussed how DNA microarrays can be applied in the working fields of: safety, functionality and health of food and gene discovery and pathway engineering in plants. PMID- 10751690 TI - Gene prediction and gene classes in Arabidopsis thaliana. AB - Gene prediction methods for eukaryotic genomes still are not fully satisfying. One way to improve gene prediction accuracy, proven to be relevant for prokaryotes, is to consider more than one model of genes. Thus, we used our classification of Arabidopsis thaliana genes in two classes (CU(1) and CU(2)), previously delineated according to statistical features, in the GeneMark gene identification program. For each gene class, as well as for the two classes combined, a Markov model was developed (respectively, GM-CU(1), GM-CU(2) and GM all) and then used on a test set of 168 genes to compare their respective efficiency. We concluded from this analysis that GM-CU(1) is more sensitive than GM-CU(2) which seems to be more specific to a gene type. Besides, GM-all does not give better results than GM-CU(1) and combining results from GM-CU(1) and GM CU(2) greatly improve prediction efficiency in comparison with predictions made with GM-all only. Thus, this work confirms the necessity to consider more than one gene model for gene prediction in eukaryotic genomes, and to look for gene classes in order to build these models. PMID- 10751689 TI - Progress in Arabidopsis genome sequencing and functional genomics. AB - Arabidopsis thaliana has a relatively small genome of approximately 130 Mb containing about 10% repetitive DNA. Genome sequencing studies reveal a gene-rich genome, predicted to contain approximately 25000 genes spaced on average every 4.5 kb. Between 10 to 20% of the predicted genes occur as clusters of related genes, indicating that local sequence duplication and subsequent divergence generates a significant proportion of gene families. In addition to gene families, repetitive sequences comprise individual and small clusters of two to three retroelements and other classes of smaller repeats. The clustering of highly repetitive elements is a striking feature of the A. thaliana genome emerging from sequence and other analyses. PMID- 10751691 TI - Target selected insertional mutagenesis on chromosome IV of Arabidopsis using the En-I transposon system. AB - Reverse genetics using insertional mutagenesis is an efficient experimental strategy for assessing gene functions. The maize Enhancer-Inhibitor (En-I) transposable element system was used to develop an effective reverse genetics strategy in Arabidopsis based on transposons. To generate insertion mutations in a specific chromosomal region we developed a strategy for local transposition mutagenesis. A small population of 960 plants, containing independent I transpositions was used to study local mutagenesis on chromosome IV of Arabidopsis. A total of 15 genes, located on chromosome IV, were tested for I insertions and included genes identified by the European ESSA I sequencing programme. These genes were of particular interest since homologies to other genes and gene families were identified, but their exact functions were unknown. Somatic insertions were identified for all genes tested in a few specific plants. Analysis of these progeny plants over several generations revealed that the ability to generate somatic insertions in the target gene were heritable. These genotypes that show high levels of somatic insertions can be used to identify germinal insertions in the progeny. PMID- 10751692 TI - Management of scrap computer recycling in Taiwan. AB - It is estimated that approximately 300,000 scrap personal computers are generated each year in Taiwan [S.-L. Chang, A Study on the Scrap Computer Treatment Cost, Environment Protection Administration of Taiwan, December 1998 (in Chinese)]. The disposal of such a huge number of scrap computers presents a difficult task for the island due to the scarcity of landfills and incineration facilities available locally. Also, the hazardous materials contained (i.e., phosphor coatings of cathode ray tubes (CRTs), batteries, polychlorinated biphenyl capacitors, mercury containing parts, liquid crystal display, high-lead content CRT funnel glass, and plastic containing flame-retardant bromine, etc.) in the scrap computers may seriously pollute the environment if they are not properly disposed of. Therefore, the EPA of Taiwan declared scrap personal computers the producer's recycling responsibility as of July 1997. Under this decree, the manufacturers, importers and sellers of personal computers have to properly recover and recycle the scrapped computers which they originally sell. On June 1, 1998, a producer responsibility recycling program for scrap computers was officially implemented in Taiwan. Under this program, consumers can bring their unwanted personal computers to the designated collection points and receive reward money. Currently, only six computer items are mandated to be recycled in this recycling program. They are notebooks, monitors, hard disks, power supplies, printed circuit boards and main frame shells. This article outlines the current scrap computer recycling system in Taiwan. PMID- 10751693 TI - Rapid delineation of humic and non-humic organic matter fractions in water. AB - Dissolved organic matter (DOM) in water is often characterized by aggregate parameters like dissolved organic carbon (DOC). DOM from conventional surface water treatment plant in Northern New Jersey was isolated and fractionated using resin adsorption chromatography into six different fractions, which were operationally categorized as hydrophobic acid, hydrophobic neutral, hydrophobic base, hydrophilic acid, hydrophilic neutral and hydrophilic base. The spectral fluorescent signatures (SFS) technique was developed for the quantitative identification of the six fractions by post-processing analysis that includes a statistical model. The SFS is the total sum of emission spectra of a sample at different excitation wavelengths, recorded as a matrix of fluorescent intensity in coordinates of excitation and emission wavelengths, in a definite spectral window. High sensitivity and rapid identification and quantification of DOM fractions are among the main features of the technique. Since hydrophobic and hydrophilic substances are considered more humic and non-humid in nature, respectively, the technique provided an opportunity to rapidly delineate source waters in terms of such categories. PMID- 10751694 TI - Evaluation of malonanilides as new stabilizers for double-base propellants. AB - Five malonanilide derivatives (M1-M5) were prepared by the reaction of ethyl malonate with aniline derivatives. These compounds were investigated as new stabilizers for double-base propellants (DBPs). The evaluation process has been performed through thermal stability tests, thermal analyses measurements (TGA and DSC) and kinetic parameters calculations (E(a)). The results of the new stabilizers were compared with the results of the classical stabilizer N, N diethyldiphenyl urea. It has been found that o- and p-dinitromalonanilides, in particular, showed better stability effect for DBPs than the classical one. PMID- 10751695 TI - Supercritical water oxidation of high concentrations of phenol. AB - Decomposition of phenol at a concentration as high as 2 wt.% was effected by supercritical water oxidation at 25 MPa. Reaction temperatures ranged from 623 723 K and residence times were varied from 6.5 to 26 s. Oxygen was added in an equivalent amount to investigate reaction intermediates. The degree of phenol decomposition and reaction product were measured. Although tarry material production was observed, phenol decomposition conversion was predicted well by the reaction rate equations developed by previous researchers who conducted experiments at lower concentrations. Difference from low concentration phenol oxidation was found in the reaction product distribution and tarry material production. One possible explanation for this result is that the initiation of phenol decomposition is the same regardless of phenol concentration but that the succeeding radical reactions are different. The additive reaction between aromatic compounds was enhanced by high phenol concentration. PMID- 10751696 TI - Investigation of the stability of hardened slag paste for the stabilization/solidification of wastes containing heavy metal ions. AB - We have studied the effect of chromium ions and lead ions on the chemical stability of hardened slag paste with toxic wastes during the stabilization/solidification process. The influences of Cr and Pb ions on the hydration of slag were also investigated. Sodium silicate (Na(2)SiO(3)), 5 wt.% of slag, was used as an alkali activator for slag hydration. The physical stability of hardened paste containing partial replacement of slag with fly ash and gypsum was also examined. When gypsum was added to slag, the compressive strength of hardened slag paste developed, accompanying the activation of alumino ferrite-tricalciumsulfate (Al(2)O(3)-Fe(2)O(3)-3CaSO(4), AFt) and alumino-ferrite monocalciumsulfate (Al(2)O(3)-Fe(2)O(3)-CaSO(4), AFm) phase generation. Those phases caused densification of the microstructure. Concurrently, the leaching amount of heavy metal ions was decreased. When fly ash was added to slag, the compressive strength increased and the leaching amount decreased with both active formation of aluminate hydrates and ion substitution. Lead ions were mostly stabilized through physical encapsulation by the hardened slag paste's hydrate matrix. In the case of chromium ions, we observed that it was mainly solidified through the formation of a substitutional solid solution with aluminum atoms in the structure of aluminate hydrates. PMID- 10751697 TI - Separation of chloride and sulfate ions in univalent and divalent cation forms from aqueous streams. AB - The precipitation and separation of chloride and sulfate in several cation forms (sodium, potassium, magnesium, calcium, strontium, and barium) from aqueous streams were studied using isopropylamine (IPA) and ethylamine (EA) as precipitation solvents. The precipitation fractions (P) of the tested chloride salts at 5000 and 10,000 ppm by both IPA and EA over the studied range of solvents volume ratio (V(R)) were relatively identical (18-60%) and their small variations were within their experimental uncertainty. The P of combined sulfate at 1000 ppm (56-99.5%) and chloride at 5000 ppm (28-62%) in the form of calcium by IPA over the studied range of V(R) were appreciably higher than the P of sulfate (10-98.5%) from calcium sulfate in the absence of calcium chloride, or the P of chloride (18-58%) from calcium chloride in the absence of calcium sulfate. The P of chloride from oil-field-produced waters at 106,654 ppm (20-88%) by both IPA and EA were higher than the P of chloride from diluted produced water at 20,000 (17-68%) and 10,000 ppm (16-65%) over the studied range of V(R). The small amounts of sulfate present in the produced waters (e.g., 435 ppm) were completely removed at V(R) of 0.1 (the first stage of precipitation). Consistency tests performed on the acquired data indicated a good level of experimental consistency. Two model equations (2-Suffix and 3-Suffix) derived from thermodynamic principles of solid-liquid equilibrium (SLE) criteria were employed to correlate the acquired data. While both equations were adequate for correlating the precipitation data, the 3-Suffix equation was more accurate. PMID- 10751698 TI - Maternal and child psychological sequelae in paediatric burn injuries. AB - It is a commonly held belief that many children suffer psychological sequelae following burn injury. This six month controlled, prospective, follow up study was designed to investigate psychological sequelae in children and their mothers following paediatric burn injury. The study employed a sample of 40 children with burn injuries, and their mothers with three control groups, each of 40 children and their mothers: an acutely ill group, a fracture group and a non ill/injured group. Measures at initial contact and 6 month follow up included the Child Behaviour Checklist and the Hospital Anxiety and Depression Scale. The results demonstrated higher initial maternal anxiety scores in the burn, compared to the fracture and non ill/injured groups, which remained comparatively high 6 months later even though they decreased over time. Children with burn injuries, of the type included in this study, did not appear to develop significant psychological or behavioural sequelae. Following paediatric burn injury mothers appear to be at higher risk than children for developing psychological sequelae, which may have longer term implications for the child's outcome such as affecting compliance with treatment. PMID- 10751699 TI - A survey into toxic shock syndrome (TSS) in UK burns units. AB - Toxic shock syndrome (TSS) is a rare complication of a Staphylococcus aureus infection and is primarily seen in children with small burns. The true incidence of TSS in burns patients is not known and the number of presumptive cases rarely reported. This survey was undertaken to determine if the incidence of TSS in children with burns could be related to the type of dressing used to cover the wound. A questionnaire was compiled and sent to the Senior Nurse in charge of each of the UK burns units. General information on the number of admissions, age of the patient, cause of injury and burn wound management was sought. An 81% response was obtained after two mailshots and follow up telephone calls. Seventy percent (23/33) of units which answered the survey nursed children. Of these, eight units had either not encountered TSS previously or not had a case within the past two years. These units were small, admitting a maximum of 50 patients each year. Of the units where TSS was encountered, approximately 2.5% of children admitted showed symptoms of TSS. Of the units who nursed both adults and children, seven units had seen TSS in burned adult patients which has not been reported in the literature. Of the eight units where TSS had not been recently encountered, four routinely administered prophylactic antibiotics to prevent infection whereas routine administration of antibiotics occurred in only two of the 15 units where TSS was seen. Although wound management procedures differed slightly there were many similarities. These included wound cleaning with normal saline, covering with either silver sulphadiazine (1%) or povidone iodine (10%), depending upon the infection status, and dressing with a paraffin tulle, gauze and crepe bandages. No association between the management of the burn wound and subsequent development of TSS could be established. PMID- 10751700 TI - The delays in intestinal motility and neutrophil infiltration following burn injury in rats involve endogenous endothelins. AB - This study was carried out to investigate the role of endogenous endothelins in intestinal motility following bum injury by using a nonselective endothelin-1 (ET 1) antagonist and to evaluate the ET-1-mediated reactive oxygen metabolite formation and neutrophil infiltration following burn injury. In 2 h and 3 day postburn groups, transit indices were significantly decreased as compared to corresponding sham groups. Transit index was not significantly changed by PD156252 pretreatment in the 2 h postburn group, whereas the delay in transit was abolished in the ET-antagonist treated 3 day postbum group. In the 2 h postburn group, tissue-associated myeloperoxidase (MPO) activity value was found to be increased compared to corresponding sham group, while PD156252 pretreatment partially reversed this effect. Although MPO activity levels were not significantly different between 3 day postburn and corresponding sham groups, MPO levels showed a significant increase in ET antagonist-treated group as compared to the corresponding burn group. In the early phase of the burn, there was no significant difference in protein oxidation levels among the groups. In the 3 day postburn group, protein oxidation levels in ET-antagonist-treated group showed an increase compared to its corresponding burn group. In conclusion, the results demonstrate that endogenous endothelins have an important role in the systemic response to burn injury, as observed by a delay in intestinal motility and an infiltration of neutrophils. Although the results of the animal studies are not readily applicable to burned patients, the present study may suggest that the burned patient's condition should be carefully evaluated to secure a proper and early enteral feeding. PMID- 10751702 TI - The continuing risk of domestic hot water scalds to the elderly. AB - Elderly people are particularly at risk of accidents in the home and with an ageing population, they constitute a large proportion of scald victims. We have seen several elderly patients every year in our own unit who had sustained domestic hot water burns. We decided to ascertain the temperature of water in the private homes of elderly people and in residential care homes in our community. Twenty-five private homes and 14 residential care homes were visited and peak water temperatures were measured in the kitchens and bathrooms. Only one private home and one residential home had water below 45 degrees C. It is recommended that water should be delivered at less than 43 degrees C. We conclude that water is dangerously hot. However, if water is stored at a temperature of less than 60 degrees C there is a risk of infection with Legionella pneumophila. Existing regulations governing the use of 'fail safe' thermostatic mixing devices must be enforced and extended to cover domestic hot water systems. PMID- 10751701 TI - Ibuprofen modulates tissue perfusion in partial-thickness burns. AB - INTRODUCTION: The anti-inflammatory and anticoagulant effects of ibuprofen and heparin may enhance skin perfusion in cutaneous scald burns. To test this hypothesis, skin perfusion and edema formation in scald burned rabbit ears were measured. METHOD: Eighteen rabbits (3.5-4.5 kg) received partial-thickness scald burns to one ear and then were given normal saline, n = 6, 20 mg/kg ibuprofen, n = 6, or 700 IU/kg heparin, n = 6. Skin perfusion, blood flow and edema formation in the burned ear were measured with laser Doppler, ultrasound flowmeter and skin calipers, respectively. Statistical analysis was performed using repeated measures ANOVA with post hoc Scheffe's test for comparison between groups. RESULTS: Blood flow to the scald burned ear increased 10-15 times that of baseline with tissue perfusion increasing by 70% within 0.5 h compared to pre burn. Ibuprofen maintained the elevated tissue perfusion for 5 h while the heparin and saline groups showed decreases to 95 and 35% of pre-burn values, respectively. The heparin and ibuprofen groups demonstrated significant increases in ear perfusion at 4 and 5 h postburn. Ibuprofen also showed a significant difference within the first hour postburn, p<0.01. Wet to dry weight ratios in burned ear tissue were greater in rabbits receiving saline or heparin compared to ibuprofen at 3.6+/-0.2 and 2.9+/-0.3 vs. 2.1+/-0.1, respectively (p<0.001). CONCLUSION: Ibuprofen increases tissue perfusion and reduces edema formation in scald burned rabbit ears. PMID- 10751703 TI - Differences between burns in rural and in urban areas: implications for prevention. AB - This study aimed to identify typical features of burns in rural areas and to improve their prevention by comparing the characteristics of burnt patients and their burns in rural and urban areas. A cross-sectional survey was conducted in 19 of the 23 French burns units over one year, using a structured questionnaire. We analysed the resulting database. Of the 1422 patients, excluding 188 burnt in unclassified areas, 420 (34%) were burnt in rural areas. Burns were more frequent in rural than in urban areas. Occupational burns were more frequent in rural than in urban areas. The characteristics of patients and burns did not differ between the areas. Patients burnt during everyday activities were older and more frequently had predisposing factors in rural than in urban areas. Burns occurred outdoors more often in rural than in urban areas and were frequently due to flames or explosions or to the use of barbecues or open fires. The hands and perineum were burnt more frequently in rural than in urban areas. Rural burns were more severe than urban burns: they were deeper, involved a larger body surface area and caused more deaths. Preventive measures in rural areas should take into account occupation and everyday activities. They should be specifically adapted to the profiles of burnt patients. PMID- 10751704 TI - Staphylococcal septicaemia in burns. AB - This study analyses staphylococcal septicaemia in a series of 1516 burn patients who were admitted to the burn unit of the Al-Babtain Centre for Burns and Plastic Surgery, Ibn Sina Hospital, Kuwait over a period of 6.5 years (1 June 1992-31 December 1998). One hundred and nine patients (7.2%) developed clinically and microbiologically proven septicaemia, of which 80 (73.4%) showed one or the other type of Staphylococcus in their blood. Fifty (62.5%) of them were males and 30 (37.5%) females, with a mean age of 26 years and the mean total body surface area of burns (TBSA) of 45% (range 1-93%). Preschool age children comprised 27.5% of the patients. Flame was the dominant (80%) cause of burn. Of the 80 patients who had 91 episodes of septicaemia, 52 (65%) had MRSA, 8 (10%) MSSA, 11 (13.8%) MRSE and 5 (6.2%) MSSE and 4 (5%) others had mixed organisms. Only the patients with MRSA had multiple episodes. Eight patients (10%) showed septicaemic episodes within only 48 h of admission; however, the majority of the patients (77.5%) had a septicaemic attack within 2 weeks postburn. Of the 52 MRSA septicaemic cases, 39 (75%) survived and 13 (25%) died. Four patients with septicaemia due to mixed infections died. A total of 19 patients were intubated, 14 due to inhalation injury and 5 because of septicaemia; all in the former group died. Glycopeptide therapy (vancomycin/teicoplanin) was instituted immediately following the detection of staphylococci in the blood. No significant difference was noted in relation to mortality amongst the septicaemic patients, whether or not on prophylactic antibiotic. Fifty-six (70%) of the 80 patients had 139 sessions of skin grafting and survived. Of the 52 MRSA patients, 40 had 101 sessions of skin grafting and 33 of them survived. The apparent low mortality was probably due to early detection of the organism, appropriate antibiotic therapy, care for nutrition and early wound cover. This study indicates a high incidence of staphylococcal septicaemia (especially due to MRSA) in the burn unit. A surface wound is the likely source of entry to the blood stream in these immunocompromised patients. The organism could be detected in blood as early as 48 h postburn and in as little TBSA burn as 1% in this MRSA endemic unit. Inhalation injury with major burns and added staphylococcal septicaemia invariably proved to be fatal. PMID- 10751706 TI - Cultured epithelial autografts in extensive burn coverage of severely traumatized patients: a five year single-center experience with 30 patients. AB - OBJECTIVE: We report recent five-year experience in a large, single center series of severely burned and otherwise traumatized patients given cultured epithelial autografts (CEA) from a single commercial laboratory. SUMMARY BACKGROUND DATA: Initial optimism over CEA application has been tempered by subsequent reports asserting that this modality is unreliable and expensive. Discussion continues over its clinical role. METHODS: From 1991 to 1996, CEA were applied to a mean 37+/-17% of total body surface area (TBSA) of 30 patients. These patients had 78+/-10% average burn size, 65+/-16% average third-degree burn size, 90% prevalence of endoscopically confirmed inhalation injury and 37% prevalence of other serious conditions. RESULTS: CEA achieved permanent coverage of a mean 26+/ 15% of TBSA, an area greater than that covered by conventional autografts (a mean 25+/-10% of TBSA). Survival was 90% in these severely burned and otherwise traumatized patients. Final CEA take was a mean 69+/-23%. In subset analyses, only younger age was significantly associated with better CEA take (p = 0.0001 in univariate analysis, p<0.04 in multivariate analysis, Student's t-test). CONCLUSIONS: Epicel CEA successfully provided extensive, permanent burn coverage in severely traumatized patients, proving an important adjunct to achievement of a high survival rate in a patient population whose prognosis previously had been poor. In our experience CEA appear to have a very high beneficial value in the management of bur ns >60% TBSA. In some cases studied it is very likely that CEA was a life-saving treatment. PMID- 10751705 TI - Effect of storage and preservation methods on viability in transplantable human skin allografts. AB - This study compared the metabolic activity of fresh skin samples to that of cadaver human skin allografts processed and stored by current tissue banking methods. We chose to use two metabolic assays as surrogate measures for viability in these grafts. Skin allografts stored either in liquid media at 4 degrees C for varying periods of time or stored by cryopreservation were quantitatively assessed for viability by tetrazolium reduction and oxygen consumption assays. These measurements were compared to viability assessments of fresh autograft skin. Human cadaver skin grafts, after procurement and just prior to further tissue bank processing, exhibited approximately 60% of the metabolic activity found in fresh skin samples obtained from living surgical donors. If allowed an overnight (18-24 h) incubation period at 37 degrees C, cadaver samples showed a recovery of their metabolic activity to 95% of that found in the autograft skin samples. When stored in liquid media at 4 degrees C, the cadaver skin declined steadily in cellular metabolic activity, arriving in less than 5 days storage at a measurement below that of cryopreserved skin. The cryopreserved skin was measured both immediately after thawing and dilution of cryoprotectant, as well as after equilibration and overnight incubation. Skin cryopreserved with dimethylsulfoxide Me(2)SO retained higher viability than glycerol cryopreserved skin. Residual concentrations of cryoprotectants were determined following typical recommendations for thawing and diluting skin allografts. The implications of these findings for transplantation and tissue banking are discussed. PMID- 10751707 TI - Octylcyanoacrylate versus polyurethane for treatment of burns in swine: a randomized trial. AB - In order to determine whether the enhanced reepithelialization of second-degree burns treated with octylcyanoacrylate (OCA) was due to its occlusive nature we compared reepithelialization (REP) and infection rates of second degree burns treated with OCA and polyurethane film (Tegaderm) in swine. Forty-four standardized partial thickness burns were created by applying an aluminum bar preheated to 80 degrees C to the backs of pigs for 20 s and randomly treated with OCA or Tegaderm. Full thickness biopsies were taken at 7, 10 and 14 days for blinded histopathological evaluation of rates of infection and reepithelialization. T-tests and chi(2) tests were used for group comparisons. There were no between group difference in the rates of reepithelialization and infection. All wounds were reepithelialized by day 14 and there were no infections in either group. We conclude that treatment of partial thickness burns with OCA spray or Tegaderm results in similar rates of reepithelialization and infection, suggesting that the beneficial effects of OCA on reepithelialization are due to its occlusive nature. PMID- 10751708 TI - Unusual donor site reactions to calcium alginate dressings. AB - Calcium alginate dressings have been used as the standard dressing for split skin donor sites at the Women's and Children's Hospital burn unit for the last 12 yr. This method has proven satisfactory with good haemostasis, reliable healing, low infection rate and is well accepted by the children and nursing staff. We have recently had 5 cases with the unusual and unexplained phenomenon of dermal calcification in the donor site following the use of two new varieties of calcium alginate dressing. PMID- 10751709 TI - Car-tyre friction injuries of the foot in children. AB - In a prospective study, 25 cases of car-tyre friction injuries of the foot in children were included. The mechanism of injury was similar in all patients: As the car-tyre impacted on the child's foot, the driver was usually trying to stop the car resulting in an injury of variable severity, depending on the amount of energy absorbed by the foot. The site of injury varied according to the position of the foot in relation to the tyre at the time of impact. Sixteen out of the 25 patients presented with acute injuries. The severity of foot injury was classified into five grades and the plan of management in these patients was guided by the grade of the acute injury. The remaining nine patients presented with secondary deformity and principles of secondary reconstruction were also demonstrated with clinical examples. Prevention of these injuries are discussed. PMID- 10751710 TI - Liquid ammonia injury. AB - The toxic effects of a gas depend on the time of exposure, concentration and its chemical nature. Pressurized liquids and gases exert an additional cold thermal injury and this may complicate the clinical picture. A patient who had an accidental exposure to liquid ammonia over a prolonged period, manifesting in cutaneous, respiratory and ocular damage in addition to a severe cold thermal injury (frostbite) with a fatal outcome is presented. The patient had flaccid quadriparesis and episodes of bradycardia, which has not been reported previously. These manifestations raise the possibility of the systemic toxicity in patients with prolonged exposure to ammonia. PMID- 10751711 TI - Improved dose homogeneity to the intact breast using three-dimensional treatment planning: technical considerations. AB - The current consensus is that breast-conservative treatment is superior to mastectomy because it provides survival equivalent to total mastectomy and axillary dissection while preserving the breast. This technique still has several technical issues that can adversely influence the successful outcome of breast conservative treatment. Specifically, dose coverage and homogeneity must be maintained throughout the breast while reducing the hot-volume magnitude and normal tissue complications. A random retrospective three-dimensional treatment planning study was conducted using computed tomography scans of 20 female patients with early-stage breast cancer. Two- and three-dimensional homogenous and heterogeneous treatment planning was conducted using all possible hard-wedge combinations and effective photon energies, with the goal of reducing the hot volumes in the breast below 110 percent of the prescribed dose. The hard-wedge combination that minimized the hot volumes uses either 15-degree wedges on the medial and lateral beams or a 30-degree wedge on the medial beam and a 15-degree wedge on the lateral beam. For patients with bridge distances less than 20 cm, this wedge combination reduces the hot volumes below 110 percent of the prescribed dose. For patients with bridge distances greater than 20 cm, low- and high-energy photon beams must be mixed to lower the maximum dose below 110 percent of the prescribed dose. The hot volumes in the breasts of 20 random patients was reduced below 110 percent of the prescribed dose without a significant reduction in tumor coverage. PMID- 10751712 TI - Dose attenuation through a titanium alloy hip prosthesis. AB - The problem of dose perturbation due to hip prostheses during pelvic irradiation can be significant. Dose attenuation by a commonly used titanium alloy hip prosthesis was measured. Depending on the energy of the radiation beam and the thickness along the prosthesis, the attenuation was between 32% and 64%. This attenuation effect should be considered in treatment planning, especially in a patient with bilateral hip prostheses and when undergoing treatment for prostate cancer where the tumor typically lies between prostheses. PMID- 10751713 TI - A phantom for dosimetric characterization of small radiation fields: design and use. AB - An acrylic phantom was designed and constructed for the acquisition and verification of basic dosimetric data of narrow fields in stereotactic radiotherapy/radiosurgery (SRT/SRS) using thermoluminescent (TL) dosimetry. An array of holes to accommodate up to 426 dosimeters was used to allow the assessment of dose distribution in circular fields with a 1-mm spatial dose resolution with minimal field perturbation. It was found experimentally that there must be a minimum gap of 1 mm between neighboring dosimeters in 6-MV photon fields. Most of the dosimetric characteristics of a 6-MV x-ray SRS/SRT unit assessed using TL dosimetry and ion chamber dosimetry were in good agreement when the longitudinal axis of the chamber was parallel to the central beam axis. TL dosimetry showed that the penumbra width increased with increasing collimator aperture. The low cost of the phantom and the widespread use and familiarity of TL dosimetry in radiotherapy departments offer a significant advantage in the use of the proposed methodology. PMID- 10751714 TI - Comparison of interpolated vs. calculated micromultileaf settings in dynamic conformal arc treatment. AB - Stereotactic radiosurgery has developed into a technique where patient positioning and treatment delivery can be performed with submillimeter precision. Achievement of this level of precision has allowed margins to be significantly reduced, and in some cases, removed altogether. Joined with these reductions in treatment margin has come a desire to shape the radiation beam, further limiting dose to normal tissues. Initial applications of shaped radiosurgery fields utilized circular blocking apertures in an attempt to shape the beam to these small volumes. The resultant dose distributions conformed well to spherical treatment volumes but were inadequate for situations where the volume of interest was irregular in shape. Other techniques, such as applying these circular apertures through multiple isocenter positions to a single volume, have been investigated as possible ways to better conform dose distributions to these irregularly-shaped volumes. Recent technological advances allow the use of micromultileaf collimators which dynamically shape the beam by adjustment of individual leaves as the gantry rotates through the are. With margins potentially so tight, accurate evaluation of these dynamically adjusting treatment parameters becomes critical. Our current treatment planning software evaluates adjustments of the leaf positions in increments of 10 degrees and then does a linear interpolation between increments. Treatment delivery, however, is performed with adjustment in leaf position more consistent with a 1 degree increment. This paper compares the individual position of each leaf as determined for the 10 degrees interpolation to required changes in leaf position when the calculation is performed at increments of less than 10 degrees. Our data suggest that there are instances where improvements can be seen when corrections in leaf positions are made at these smaller increments. PMID- 10751715 TI - Dosimetric assessment of nonperfectly abutted fields using asymmetric collimators. AB - The abutment of adjacent fields has been facilitated through the use of asymmetric collimators. Conceptually, the abutment yields a perfectly uniform dose distribution across the junction, provided the asymmetric jaw is set precisely at the beam central axis. However, the asymmetric jaw has an associated tolerance, which can cause the abutment to be misaligned. This study examined the dose distribution at the junction of nonperfectly abutted fields. The abutment of fields was carried out using an asymmetric collimation of 5 x 10 cm, with an asymmetric jaw positioned at the beam central axis. A film was initially exposed using this field with the collimator set at 90 degrees. The collimator was then rotated 180 degrees and the same film was exposed for the second time to create the field abutment. Positioning the asymmetric jaw with respect to the beam central axis set the amount of gap and overlap between the abutted fields. The dose distribution was measured for asymmetric jaw positioning of -2, -1, 0, + 1, and +2 mm from the beam central axis. In addition, the dose distribution was also computed mathematically by summing the 2 dose profiles with defined gap or overlap. A field mismatch of +/-1 mm would result in a dose nonuniformity of 17%, and a +/-2 mm mismatch would produce a 35% dose nonuniformity. PMID- 10751716 TI - Designing attenuators for total-body irradiation using virtual simulation. AB - In total-body photon irradiation, the lungs are the most commonly shielded organ. Lung compensators are often designed by using high-energy portal films. Other organs, such as the kidneys and liver, are poorly visualized in portal films due to their unit-density composition. A computed tomography-based technique to design kidney and liver attenuators involves outlining these organs in a virtual simulation. The position and the shape of the attenuator are then determined from a digitally-reconstructed radiograph. Appropriate attenuator thickness is determined from measured transmission curves. This article provides a summary of this technique for total-body photon irradiation in a 4-MV photon beam. PMID- 10751717 TI - Cesium-137 source strength verification. AB - Over the years, the protocol for Cesium-137 source calibration has undergone a number of revisions based on updated data. The 3M Corporation issued product alerts and a revision of the calibration protocol in the early 1980s. We verified the activity of clinically used cesium tubes and found the difference with the activity stated by 3M in the range of 6-13%, which exceeds the recommended by 3M adjustment of 5% for all sources issued before 1979. Therefore, the verification and adjustment of activity should be recommended for each affected tube individually. PMID- 10751718 TI - Conformal prostate treatment planning using a low-energy (6-MV) beam. AB - A 4-field noncoplanar technique for treatment of prostate cancer developed at the University of Michigan was modified for use with low-energy (6 MV) beams. These modifications include the use of wedges on the 2 anterior inferior-superior oblique fields and adjusting the weights of the oblique and lateral fields appropriately. A margin of 1.5 cm around the physician-defined target region was used to define the blocks on each beam's-eye view. Dose distributions produced using this technique with 6-MV and 24-MV beams were compared visually on several dose planes (transverse and sagittal) and quantitatively by dose volume histograms (target, rectum, and bladder). These comparisons showed insignificant differences between the high-energy and low-energy treatment plans. Much larger differences were observed in comparisons of 2 types of coplanar plans with the noncoplanar setup for the 6-MV photon machine. Rectal doses measured in situ were used to help validate the dose distribution predicted by the treatment planning system for the 6-MV noncoplanar technique. PMID- 10751719 TI - Effect of the photocatalytic activity of TiO(2) on plasmid DNA. AB - We investigated the photodynamic DNA strand-breaking activity of TiO(2). A solution of super-coiled pBR 322 DNA was irradiated with 5 J/cm(2) of UVA in the presence of TiO(2) and the products were analyzed by using gel electrophoresis. The ratio of open-circular DNA to super-coiled circular DNA was calculated from the resulting peak areas as a DNA strand-breaking index (SBI). The SBI of anatase structure TiO(2) (band gap=3.23 eV) was greater than that of rutile structure (band gap=3.06 eV), and the level of SBI correlated with the photocatalytic activity for degradation of 2-propanol. The inhibitory effects of active oxygen scavengers, including DMSO, glutathione and histidine, on the DNA strand-breaking activity were examined. All of the scavengers except ascorbic acid showed inhibitory effects, as did several polyhydric alcohols including mannitol, a well known hydroxyl radical scavenger. These results suggest that the photodynamic DNA strand-breaking activity of TiO(2) is due to active oxygen species, especially hydroxyl radicals. Polyhydric alcohols showed an inverse correlation between the inhibitory effect on DNA strand-breaking activity and the octanol/water partition coefficient (logP). PMID- 10751720 TI - A public domain image-analysis program for the single-cell gel-electrophoresis (comet) assay. AB - The single-cell gel electrophoresis (or comet) assay has gained widespread acceptance as a cheap and simple genotoxicity test, but it requires a computer assisted image-analysis system. As commercial programs are expensive and inflexible, we decided to develop an image-analysis system based on public domain programs and make it publicly available for the scientific community. Our system is based on the scientific image-processing program NIH Image, and was written in its Pascal-like macro language. User interaction was kept as simple as possible, to enable the measurement of a large number of cells with a few keystrokes. Therefore, the time for image analysis is very low, even on slow computers. The comet macro can be obtained from http://mailbox.univie.ac.at/christoph.helma++ +/comet/, NIH Image is available at http://rsb.info.nih.gov/nih-image/. Both programs are free of charge. PMID- 10751722 TI - Identification of mutagenic heterocyclic amines (IQ, Trp-P-1 and AalphaC) in the water of the Danube river. AB - Three mutagenic heterocyclic amines, 2-amino-3-methylimidazo-[4, 5-f]quinoline (IQ), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-9H pyrido[2,3-b]indole (AalphaC), were isolated and identified in water from the Danube River in Vienna. Heterocyclic amines were extracted from river water by the blue rayon hanging method, and analyzed by gas chromatography with a nitrogen phosphorous detector (GC-NPD) and GC-mass spectrometry (GC-MS) after conversion into their N-dimethylaminomethylene derivatives. Identity of IQ, Trp-P-1 and AalphaC in the river water was confirmed by GC-MS. The contents of IQ, Trp-P-1 and AalphaC were estimated by GC-NPD at 1.78+/-0.17, 0.14+/-0.02 and 0.44+/-0.02 ng/g blue rayon equivalent (n=3), respectively. The total amounts of these amines accounted for 26% of the mutagenicity of blue rayon extracts evaluated by the Ames test using TA98 with metabolic activation. PMID- 10751721 TI - Liver subcellular fractions from rats treated by organosulfur compounds from Allium modulate mutagen activation. AB - The effects of in vivo administration of naturally occurring organosulfur compounds (OSCs) from Allium species were studied on the activation of several mutagens. Male SPF Wistar rats were given p.o. one of either diallyl sulfide (DAS), diallyl disulfide (DADS), dipropyl sulfide (DPS) or dipropyl disulfide (DPDS) during 4 consecutive days and the ability of hepatic S9 and microsomes from treated rats to activate benzo[a]pyrene (BaP), cyclophosphamide (CP), dimethylnitrosamine (DMN), N-nitrosopiperidine (N-PiP) and 2-amino-1-methyl-6 phenylimidazo[4,5-b]pyridine (PhIP) was determined in the Ames test. Administration of DAS, DPS and DPDS resulted in a significant increase of the activation of BaP, CP, N-PiP and PhIP mediated by S9 and microsomes while DADS treatment only increased the mutagenicity of PhIP. In contrast, S9 from DADS treated rats significantly inhibited the mutagenicity of N-PiP and BaP. DAS, DADS and DPS strongly inhibited DMN mutagenicity while DPDS enhanced it. To understand the mechanisms underlying these effects, the modifications of the activities of specific isozymes of CYP involved in the activation of these mutagens were studied. DAS, DPS and DPDS strongly enhanced pentoxyresorufin O-dealkylase (PROD) activity related to CYP2B and slightly increased ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities related to CYP1A family. DADS exerted the same effects than other OSCs but to a lesser extent. p Nitrophenol hydroxylase (PNPH) activity related to CYP2E1 was inhibited by DAS and DADS, whereas DPDS significantly increased this activity. Hence, the effects of OSCs on the mutagenicity of several genotoxic compounds are mediated by modification (enhancement or inhibition) of specific CYP involved in their activation. PMID- 10751723 TI - Analysis of chromosomal aberration frequencies in the peripheral blood lymphocytes of smokers exposed to uranyl compounds. AB - One hundred fifteen smokers working in a nuclear fuel manufacturing facility were analysed for various types of chromosomal aberrations. They experienced exposure for a period of 1-25 years. Their age ranges from 23 to 52 years. A total of 94 smokers and 118 non-smokers who were not exposed to uranyl compounds or to any other known mutagens and belong to the same age group formed the control subjects. The results showed that there is a significant increase in the frequency of chromosomal aberrations in the exposed smokers when compared to the control smokers. In the control group, the smokers showed a high frequency of chromosomal aberrations when compared to non-smokers suggesting clastogenic effect of smoking. Chromosomal aberrations observed in the exposed smokers could be due to the cumulative effect of both smoking and exposure to uranyl compounds. PMID- 10751724 TI - Quinacrine dihydrochloride, the non-surgical female sterilant induces dicentrics, rings, and marker chromosomes in human peripheral blood lymphocytes treated in vitro: a preliminary report. AB - During the last decade, quinacrine dihydrochloride (QDH) has been promoted for clinical trials as a much needed non-surgical female sterilant, largely in the Third World. Recently, however, these human trials have come under severe criticism due to lack of adequate evidence of biological safety of QDH, particularly of its genotoxicity in mammalian systems. In the present study, the cytogenetic analysis of QDH-treated human lymphocytes, grown as whole blood cultures in vitro, surprisingly showed a wide range of chromosomal aberrations. At a concentration of 3.0 and 6.0 microg/ml in culture, QDH was cytotoxic, as shown by the very few analyzable metaphases that could be observed. G(0) lymphocytes, treated with 0. 6 microg/ml QDH, exhibited chromosome aberrations including dicentrics, ring configurations, translocations, inversions, and marker chromosomes. Near haploid, polyploid, and endoreduplicated cells were also observed. All the rings appeared to be formed as a result of telomere fusion/association. Twenty percent of the dicentrics observed also indicated telomere fusion/association in the D and G groups of chromosomes. Overall, a frequent involvement of chromosomes 1, 2, and 3 in both unstable and stable chromosome rearrangements was also observed. Exposure of 72-h cultures to 0.45 microg/ml QDH at 69 h resulted in an accumulation of C-metaphases, suggesting that probably QDH behaves as a mitotic spindle inhibitor. The G(2) lymphocytes from two donors exposed to 0.6, 1.5 or 3.0 microg/ml of QDH showed no increase in chromatid aberrations in two donors. However, QDH at 0.6 microg/ml increased the frequency of micronucleated binucleate cells. No increase in sister chromatid exchanges was observed at this concentration. Though preliminary, these observations demonstrate the chromosome damaging ability of QDH in human lymphocytes treated in vitro. Surprisingly, like ionizing radiation, QDH acted by an S-phase-independent mechanism, unlike most of the chemical mutagens. These results warrant detailed investigations on the cytogenetic effects of QDH in vitro, as well as among women exposed to this agent during clinical trials for non-surgical sterilization. The interesting cytogenetic profile of QDH deserves to be pursued and the underlying mechanisms, in particular, the DNA topoisomerase II inhibitory effect, if any, needs to be elucidated. PMID- 10751725 TI - Clastogenic effect of copper deficiency in cattle. AB - Copper plays an essential role as a micronutrient. Deficiency of this element (hypocuprosis) in experimental and domestic animals has a severe impact on growth as well as on reproduction. The occurrence of lesions during hypocuprosis is correlated with the depletion of an enzymatic group in which copper takes part. The aim of this work was to analyse chromosomal aberrations in Aberdeen Angus cows of the province of Buenos Aires in relation with the Cu plasma levels. Short term lymphocyte cultures were made from samples obtained from four groups of animals: two groups with normal levels of copper in plasma and two groups with severe hypocupremia. This analysis showed a significant increase of the frequency of chromosomal aberrations (p<0.001) in the hypocupremic groups in relation with control groups. Finally, the Spearman correlation analysis showed a significant negative association (p<0.05) between copper levels and the yield of chromosomal aberrations. The increase of the frequencies of chromosomal aberrations found in the hypocupremic groups could be explained by the higher oxidative stress suffered by these animals. A lower catalytic activity of enzymes such as Cu/Zn superoxide dismutase (Cu/Zn-SOD) and cytochrome-c oxidase could increase the intracellular production of active oxygen species (O(2)(-), H(2)O(2) and OH(o)) with the consequent clastogenic effects. PMID- 10751726 TI - Induction of vaginal Lactobacillus phages by the cigarette smoke chemical benzo[a]pyrene diol epoxide. AB - Because smoking increases a woman's risk of contracting bacterial vaginosis (BV), which is manifested by a reduction of vaginal lactobacilli and an overgrowth of anaerobic bacteria, chemicals contained in cigarette smoke were analyzed in vitro to determine their role in reducing lactobacilli. The result showed that trace amounts of benzo[a]pyrene diol epoxide (BPDE), which can be found in vaginal secretion of women who smoke, significantly increased phage induction in lactobacilli. This finding implies that smoking may reduce vaginal lactobacilli by promoting phage induction. PMID- 10751727 TI - Comet assay: rapid processing of multiple samples. AB - The present study describes modifications to the basic comet protocol that increase productivity and efficiency without sacrificing assay reliability. A simple technique is described for rapidly preparing up to 96 comet assay samples simultaneously. The sample preparation technique allows thin layers of agarose embedded cells to be prepared in multiple wells attached to a flexible film of Gelbond, which improves the ease of manipulating and processing samples. To evaluate the effect of these modifications on assay sensitivity, dose-response curves are presented for DNA damage induced by exposure of TK6 cells to low concentrations of hydrogen peroxide (0-10 microM) and for exposure of human lymphocytes to X-irradiation (0-100 cGy). The limit of detection of DNA damage induced by hydrogen peroxide in TK6 cells was observed to be 1 uM for all parameters (tail ratio, tail moment, tail length and comet length) while the limit of detection of DNA damage in human lymphocytes was 10 cGy for tail and comet length parameters, but 50 cGy for tail ratio and tail moment parameters. These results are similar to those previously reported using the conventional alkaline comet assay. The application of SYBR Gold for detection of DNA damage was compared to that of propidium iodide. Measurements of matching samples for tail length and comet length were similar using both stains. However, comets stained with SYBR Gold persisted longer and were much brighter than those obtained with propidium iodide. SYBR Gold was found to be ideal for measuring tail length and comet length but, under present assay conditions, impractical for measuring tail ratio or tail moment due to saturation of staining in the head region of the comets. PMID- 10751729 TI - 5-Azacytidine- and Hoechst-induced aneuploidy in Indian muntjac. AB - Hoechst 33258 (bis-benzimidazole) and 5-azacytidine (5-AC) cause decondensation of the pericentric heterochromatin in mouse and aberrations in the sequence of centromere separation apparently by different mechanisms. We treated the male Indian muntjac cells (2n=7), which do not undergo decondensation of the pericentric heterochromatin, to study if these chemicals would result in induction of aneuploidy limited to the Y(2) chromosome. This paper reports that both agents result in aneuploidy primarily limited to one chromosome, the Y(2). It is likely that other chromosomes are not tolerated in aneuploid condition because every chromosome carries some household genes including those essential for mitotic progression. The loss/gain of the Y(2) chromosome is tolerated because it is the smallest chromosome and is almost entirely composed of constitutive heterochromatin. Since Indian muntjac has only three pairs of large chromosomes comprising its basic genome, which can be clearly viewed under high dry objective, these cells are very suitable for the preliminary analysis of aneuploidy-inducing ability of various chemicals. PMID- 10751728 TI - Mutagenic activity of 4-nitroquinoline-N-oxide in upper aerodigestive tissue in lacZ mice (MutaMouse) and the effects of 1, 4 phenylenebis(methylene)selenocyanate. AB - 4-Nitroquinoline-N-oxide (4-NQO) was administered to lacZ mice at a concentration of 20 microg/ml in drinking water for 2 weeks, and the mutagenic fractions in a number of organs were assayed. The mutant fractions in tongue, esophagus and other pooled oral tissues were, respectively, 117+/-26, 73+/-15, and 48+/-15 mutants/10(5) plaque-forming units (pfu) (ca. 15-40xbackground). 4-NQO was not mutagenic in lung, liver or colon at conditions used here. We had previously demonstrated that the synthetic organoselenium compound, 1,4 phenylenebis(methylene)selenocyanate (p-XSC), an established chemopreventive agent, greatly reduced carcinogenicity in 4-NQO in rat tongue, and we observed here that administration of p-XSC (10 ppm se) in the diet for 6 weeks (2 weeks before, during, and 2 weeks after 4-NQO) resulted in a 33% decrease in mutagenesis in oral tissue, a 17% decrease in esophagus, and a slight increase in tongue. Only the decrease in oral tissue reached statistical significance (p<0.04). The results reported here demonstrate that 4-NQO was extremely mutagenic in lacZ mouse tongue, with lower, but highly significant activities in esophagus and other pooled oral tissues. The high activity of 4-NQO in lacZ mouse tongue is consistent with the organ specificity of 4-NQO in the rat. Inhibition of 4-NQO-induced mutagenesis by p-XSC was observed mainly in pooled oral tissues, other than tongue. Possible reasons for the difference between inhibition of mutagenesis and carcinogenesis in tongue are discussed, as well as advantages and disadvantages of in vivo mutagenesis assays as surrogates for carcinogenicity assays in chemoprevention studies. PMID- 10751730 TI - Impact of p53 status on heavy-ion radiation-induced micronuclei in circulating erythrocytes. AB - Transgenic mice that differed in their p53 genetic status were exposed to an acute dose of highly charged and energetic (HZE) iron particle radiation. Micronuclei (MN) in two distinct populations of circulating peripheral blood erythrocytes, the immature reticulocytes (RETs) and the mature normochromatic erythrocytes (NCEs), were measured using a simple and efficient flow cytometric procedure. Our results show significant elevation in the frequency of micronucleated RETs (%MN-RETs) at 2 and 3 days post-radiation. At 3 days post irradiation, the magnitude of the radiation-induced MN-RET was 2.3-fold higher in the irradiated p53 wild-type animals compared to the unirradiated controls, 2.5 fold higher in the p53 hemizygotes and 4.3-fold higher in the p53 nullizygotes. The persistence of this radiation-induced elevation of MN-RETs is dependent on the p53 genetic background of the animal. In the p53 wild-type and p53 hemizygotes, %MN-RETs returned to control levels by 9 days post-radiation. However, elevated levels of %MN-RETs in p53 nullizygous mice persisted beyond 56 days post-radiation. We also observed elevated MN-NCEs in the peripheral circulation after radiation, but the changes in radiation-induced levels of MN NCEs appear dampened compared to those of the MN-RETs for all three strains of animals. These results suggest that the lack of p53 gene function may play a role in the iron particle radiation-induced genomic instability in stem cell populations in the hematopoietic system. PMID- 10751732 TI - Genotoxic effects of estradiol-17beta on human lymphocyte chromosomes. AB - The cytogenetic effect of a hormonal steroid, estradiol-17beta, was assessed in peripheral blood human lymphocyte culture. Sister chromatid exchanges (SCE) and chromosome aberrations (CA) were scored as genetic end points. Significant induction of CA was observed at 25 microg/ml and 50 microg/ml concentrations of estradiol-17beta in the absence of microsomal activation. The drug was effective in all treatments in the presence of rat liver S(9) microsomal fraction (S(9) mix) and exhibited increased frequency of chromosomal aberrations. The drug was effective in increasing the SCE frequency which was found to be maximum at the dose of 50 microg/ml concentration (i.e., 4.34+/-1.22) both with and without metabolic activation. It was found that estradiol-17beta itself and possibly its metabolites are potent mutagens beyond a particular dose in human lymphocytes. PMID- 10751733 TI - Cytogenetic biomonitoring in a Mexican floriculture worker group exposed to pesticides. AB - The cytogenetic damage in floriculturists of Morelos State, Mexico, exposed to pesticides, was evaluated by mean of biological tests based on sister chromatid exchanges (SCE) in lymphocytes of peripheral blood and micronuclei (MN) in exfoliated cells of the buccal mucosa. Besides the cytogenetic analysis, the effects of pesticides exposure on the cell proliferation kinetics (CPK) by the replication index (RI) were also studied. The mitotic index (MI) to detect cytotoxic effects was also determined. Greenhouses of the towns of Santa Catarina, Jiutepec and Yecapixtla were selected for the study, because the application of chemicals to the flowers is uncontrolled. As non-exposed group, people of the town of Temisco were chosen; their activity was not related to pesticides. The SCE were analyzed in the peripheral blood of 30 persons, 22 women and 8 men, with 10 and 1.5 years of exposure to pesticides, respectively, and of 30 persons, 28 women and 2 men, that were considered as the non-exposed group. Samples of buccal mucosa were also taken from each person. Significant differences between exposed and non-exposed groups were found in SCE, CKP and MI. Besides, the MN frequencies in the exposed group were three times higher than in the non-exposed group. PMID- 10751731 TI - Improved bacterial SOS promoter∷lux fusions for genotoxicity detection. AB - Escherichia coli strains containing plasmid-borne fusions of Vibrio fischeri lux to the recA promoter-operator region were previously shown to be potentially useful for detecting genotoxicants. In an attempt to improve past performance, the present study examines several modifications and variations of this design, singly or in various combinations: (1) modifying the host cell's toxicant efflux capacity via a tolC mutation; (2) incorporating the lux fusion onto the bacterial chromosome, rather then on a plasmid; (3) changing the reporter element to a different lux system (Photorhabdus luminescens), with a broader temperature range; (4) using Salmonella typhimurium instead of an E. coli host. A broad spectrum of responses to pure chemicals as well as to industrial wastewater samples was observed. Generally, fastest responses were exhibited by Sal94, a S. typhimurium strain harboring a plasmid-borne fusion of V. fischeri lux to the E. coli recA promoter. Highest sensitivity, however, was demonstrated by DPD3063, an E. coli strain in which the same fusion was integrated into the bacterial chromosome, and by DPD2797, a plasmid-bearing tolC mutant. Overall, the two latter strains appeared to perform better and seemed preferable over the others. The sensor strains retained their sensitivity following a 2-month incubation after alginate-embedding, but at the cost of a significantly delayed response. PMID- 10751734 TI - Recent advances in cardiovascular pharmacology. PMID- 10751735 TI - Color and power Doppler sonography of liver hemangiomas: a dream unfulfilled? AB - PURPOSE: The aim of this study was to determine whether color Doppler or power Doppler sonography can aid in the diagnosis of hepatic cavernous hemangiomas. METHODS: We imaged 25 hepatic cavernous hemangiomas in 17 patients with gray scale, color Doppler, and power Doppler sonography. Five malignant lesions were also imaged in the same manner for reference. Hemangiomas had been previously diagnosed by biopsy in 8 patients (15 lesions) and by CT, MRI, and/or tagged red blood cell scanning in 9 patients (10 lesions). RESULTS: Of the 25 hemangiomas, color or power Doppler imaging showed no internal blood flow in 23. Of these 23 lesions, 11 showed a peripheral flow pattern believed to represent flow in displaced blood vessels. This pattern was better visualized with power Doppler imaging in 3 lesions and equally well visualized with color and power Doppler imaging in 8 lesions. Two hemangiomas that had unusual central fibrosis with large vessels in 1 patient showed diffusely increased blood flow on power Doppler study. All 5 malignant lesions showed flow in peripheral vessels, and 1 showed internal vascularity as well. CONCLUSIONS: Neither color nor power Doppler imaging improved the capability of sonography for making a specific diagnosis of benign hepatic cavernous hemangioma. PMID- 10751737 TI - Indications for pelvic sonography-Do patients and doctors agree? AB - PURPOSE: Often, it seems that the patient history provided by the referring clinician on the sonography requisition form differs from that given by the patient during the sonographic examination. Because such a discrepancy in the history may delay the scan and disrupt the daily work flow while the referring clinician is contacted for clarification, we sought to document the incidence and cause of such discrepancies at our institution. METHODS: During a 3-month period, all outpatients who were referred for a pelvic sonographic examination were asked to indicate their understanding of why the examination had been requested. The health care providers' reasons for requesting sonography were recorded using a computer order entry system. Each pair of responses (health care provider and patient) were classified as either concordant or discordant. Patient and physician characteristics were fit into a logistic regression model with concordance of history as the outcome variable. RESULTS: One hundred fifty-six (90%) of the 173 patients enrolled in our study indicated that their health care provider had discussed with them the reason for ordering the sonographic examination. The histories provided by the patient and health care provider were concordant in 134 (77%) of 173 cases. The histories provided by the patient and health care provider were more likely to be concordant if the patient's insurance was a managed care plan or if the patient had a college or graduate level education, had been cared for by the same health care provider for more than 2 years, or had been seen by a female health care provider. Concordance of history was not associated with a higher incidence of abnormal sonographic findings. CONCLUSIONS: It appears that health care providers, despite increased demands on their time, adequately discuss with their patients the reasons for ordering a pelvic sonographic examination. However, our study suggests that health care providers may need to spend additional time with patients whose education is limited and that male physicians may need to pay particular attention to their communication with female patients. PMID- 10751736 TI - Sonographic assessment of ceftriaxone-associated biliary pseudolithiasis in children. AB - PURPOSE: Ceftriaxone is a widely used third-generation cephalosporin. In this prospective study, we used sonography to investigate the incidence and outcome of biliary complications in children receiving ceftriaxone therapy. METHODS: Ceftriaxone was administered intravenously at a dosage of 100 mg/kg/day for 1-3 weeks to 118 children hospitalized for severe infection. Serial gallbladder sonograms were obtained on days 1, 5-7, and 10-14 of therapy and the day after therapy ended if it had lasted more than 2 weeks. When sonographic abnormalities were found, additional sonograms were obtained every 3 days until the abnormalities had completely resolved. RESULTS: Twenty patients (17%), all asymptomatic, demonstrated sonographic abnormalities: 8 had gallbladder sludge, defined as echogenic material without associated acoustic shadowing, and 12 had pseudolithiasis, defined as echogenic material with acoustic shadowing. These abnormalities spontaneously resolved within 2 weeks of stopping the ceftriaxone (mean time to disappearance, 8.2 +/- 3.4 days). No significant differences were found between patients with normal versus abnormal sonographic findings in sex, age, duration of treatment, or other risk factors for drug precipitation. CONCLUSIONS: Ceftriaxone-associated biliary pseudolithiasis is usually asymptomatic and was rapidly reversible after cessation of therapy in this group of Turkish children. PMID- 10751739 TI - Metaplastic carcinoma of the breast: mammographic and sonographic findings. AB - PURPOSE: We investigated the mammographic, sonographic, and pathologic findings in metaplastic carcinoma of the breast. METHODS: The mammographic (n = 16) and sonographic (n = 11) findings in 16 patients with metaplastic carcinoma of the breast were analyzed retrospectively along with pathologic findings. Whenever possible, results of preoperative fine-needle aspiration biopsy and immunohistochemical studies were obtained. RESULTS: All patients presented with a palpable breast mass. The mean size of the lesions at pathologic examination was 4.2 cm. On mammography, 15 patients had a mass (1 patient had 2 masses), and 1 patient had only clustered microcalcifications without an associated mass. The mean longest diameter of the 16 masses on mammography was 4.6 cm. Eleven lesions (69%) were round to ovoid in shape, 13 lesions (81%) showed ill-defined or obscured margins, and 10 lesions (63%) showed associated architectural distortion. On sonography, 6 (55%) of 11 lesions were round to ovoid, 9 lesions (82%) had well-defined margins, and 6 lesions (55%) showed complex echogenicity with solid and cystic components. At pathologic examination, 4 of these 6 lesions showed hemorrhagic or cystic necrosis. Axillary lymph nodes were positive in 6 (40%) of 15 patients in whom axillary node dissection was performed. CONCLUSIONS: Metaplastic carcinoma of the breast manifests as a rapidly growing, mammographically ill-defined round mass with associated architectural distortion on mammograms. Complex echogenicity with solid and cystic components may be seen sonographically and is related to hemorrhagic or cystic necrosis seen pathologically. PMID- 10751738 TI - Endometrial sonographic findings in asymptomatic, hypertensive postmenopausal women. AB - PURPOSE: In this retrospective study, the sonographically measured endometrial thickness in asymptomatic, hypertensive postmenopausal women was compared with that in normotensive postmenopausal women. METHODS: We reviewed clinical and sonographic data on 511 consecutive, unselected, asymptomatic postmenopausal women who attended our hospital for routine gynecologic examinations during a 6 month period. Two hundred nineteen patients (mean age, 60.2 years; age range, 49 81 years) were included in the study. Reasons for exclusion were: clinical data about hypertension were not available (n = 159); the patient had received or was receiving hormonal treatment (n = 78); the patient had undergone a hysterectomy (n = 25); and endometrial thickness could not be determined (n = 30). All patients had been examined using transvaginal or transabdominal sonography. Endometrial thickness was measured at the level of its maximum thickness in the uterine sagittal plane. RESULTS: Fifty-six (26%) of 219 patients were hypertensive. Of these 56 patients, 41 (73%) were receiving drug treatment. The mean endometrial thickness in the hypertensive patients receiving treatment [6.2 mm; 95% confidence interval (CI), 5.1-7.4 mm] was significantly greater than in both the untreated, hypertensive patients (4.3 mm; 95% CI, 3.1-5. 5 mm) (p = 0.008) and the normotensive patients (3.6 mm; 95% CI, 3. 4-3.8 mm) (p < 0.0001). Endometrial thickness was equal to or greater than 5 mm in 59% of the hypertensive patients receiving drug treatment compared with 40% of the untreated, hypertensive patients and 18% of the normotensive patients (p < 0.001). An endometrial stripe was sonographically detected in 22% of the hypertensive patients undergoing treatment, 7% of the hypertensive patients undergoing no treatment, and 1% of the normotensive patients (p < 0. 0001). CONCLUSIONS: Our data indicate that endometrial thickness, which can be determined sonographically, is frequently greater in asymptomatic, hypertensive postmenopausal women receiving antihypertensive drugs than in untreated hypertensive and normotensive patients. This conclusion could have clinical relevance when interpreting endometrial sonographic findings in asymptomatic, hypertensive postmenopausal patients. PMID- 10751740 TI - Incisional hernia: an unusual cause of acute pain and swelling following renal transplant. AB - We present a case in which a strangulated incisional hernia following a renal transplant was sonographically diagnosed. The patient presented with acute pain and swelling over the transplant site 6 weeks after surgery. Sonograms showed a normal-sized kidney with normal echotexture, no evidence of hydronephrosis, and no perinephric collections. Color Doppler sonography and spectral analysis demonstrated normal blood flow throughout the kidney. Sonograms showed that the palpable mass was a dilated loop of fluid-filled small bowel. Sonography allowed the correct diagnosis to be established, and early surgical intervention allowed revascularization of viable bowel. PMID- 10751741 TI - Scrotal hematocele as an unusual presentation of blunt abdominal trauma in three male infants. AB - Three infants presented with bluish discoloration and swelling of the scrotum with no history of scrotal trauma. Sonography revealed unilateral hematoceles in 2 cases and bilateral hematoceles in the third. All 3 infants had intact testes. The communicating hematoceles were the result of hemoperitoneum due to splanchnic hematoma. PMID- 10751742 TI - Prenatal sonographic detection of pericardial teratoma. AB - Patients with pericardial teratomas usually present shortly after birth with cardiorespiratory distress or in utero with hydrops fetalis. We report a case in which the tumor was diagnosed in utero using sonography during a routine obstetric examination. Sonograms showed a 3-cm echogenic mass compressing the right atrium. The lesion was inhomogeneous with solid and cystic areas. The fetus was followed to term, and additional radiologic studies were performed before the infant underwent surgery. Prenatal identification of a pericardial teratoma is essential for planning fetal management and delivery and potential prenatal surgery. PMID- 10751743 TI - Sonographic appearances of conglomerated polyps (giant polyposis) in patients with Crohn's disease. AB - Giant polyposis is a rare presentation of Crohn's disease (CD) of the colon and can be misdiagnosed as colon cancer. To our knowledge, the sonographic characteristics of conglomerated polyps in colonic CD have not been published. The purpose of this article is to describe sonographic findings in 3 patients with giant polyposis and evaluate the contribution of sonography in establishing this diagnosis. We conclude that sonography can facilitate the diagnosis of giant polyposis in CD by demonstrating associated findings in the large and small bowels that are suggestive of CD. PMID- 10751745 TI - In this issue PMID- 10751744 TI - Congenital ileal atresia presenting as a single cyst-like lesion on prenatal sonography. AB - A routine prenatal sonographic examination at 37 weeks' menstrual age revealed a large sonolucent lesion with peristaltic movement in the abdomen of a fetus. After birth, the female infant showed progressive abdominal distention, and radiography showed a bubble-like dilatation of the small intestine. Exploratory laparotomy revealed ileal atresia with nearby partial torsion of the dilated small bowel. The incomplete torsion may have functioned as a check valve, inducing segmental dilatation of the ileum without proximal dilatation. PMID- 10751746 TI - Scanning force microscopy reveals structural alterations in diabetic rat collagen fibrils: role of protein glycation. AB - BACKGROUND: The main functional property of collagen is to provide a supporting framework to almost all tissues: the effects of non-enzymatic glycation on this protein are deleterious and in diabetes mellitus contribute to the mechanism of late complications. The aim of this work is to provide evidence by scanning force microscopy of modifications in collagen structure caused by high glucose concentration, in vivo and in vitro, and to correlate the data with markers of non-enzymatic glycation. METHODS: Tendon fibrils were obtained from the tails of 8-month-old rats (BB/WOR/MOL?BB) which developed diabetes spontaneously at least 12 weeks before they were killed, and from diabetes-resistant rats of the same strain (BB/WOR/MOL?WB). A scanning force microscope (SFM; Nanoscope III) equipped with a Contact Mode Head was used for imaging. Band interval, diameter and depth of D-band gap were measured in non-diabetic and diabetic tail tendon fibrils and in fibrils incubated with glucose (0.5 M for 2 weeks). Fructosamine was determined in the tendon fibrils by a colorimetric method and pentosidine was evaluated in acid-hydrolyzed samples by coupled reverse phase-ionic exchange column HPLC. RESULTS: Incubated fibrils revealed modifications in radius (228+/-5 nm) and gap depth (3.65+/-0.10 nm) that closely reproduce diabetes-induced damage (236+/-3 and 3.20+/-0.04 nm respectively) and were significantly different from the pattern seen in non-diabetic fibrils (151+/-1 and 2.06+/-0.03 nm; p<0.001). Both fructosamine and pentosidine were higher in diabetic (3.82+/-1.43 nmol/mg and 2.23+/-0.24 pmol/mg collagen respectively) and in glucose-incubated fibrils (9.27+/-0.55 nmol/mg and 5.15+/-0.12 pmol/mg collagen respectively) vs non diabetic tendons (1.29+/-0.08 nmol/mg and 0.88+/-0.11 pmol/mg collagen respectively; p<0.01); during the time course of incubation, an early increase in fructosamine was seen, whereas pentosidine increased later. The D-band parameter was similar in all three groups, indicating that axial organization is not modified by non-enzymatic glycation. CONCLUSION: This is the first description obtained with SFM of diabetes-induced ultrastructural changes in collagen fibrils. Moreover, the data presented are consistent with the concept that chronic exposure of collagen to glucose in vivo or in vitro leads to similar structural modifications in collagen fibrils, probably through crosslinks. The correlation between morphologic parameters and both markers of glycation provides strong evidence for a crucial role of this non-enzymatic modification. PMID- 10751747 TI - Pravastatin compared to bezafibrate in the treatment of dyslipidemia in insulin treated patients with type 2 diabetes mellitus. AB - BACKGROUND: Both HMG-CoA reductase inhibitors and fibric acid derivates are used for the treatment of dyslipidemia in Type 2 diabetes patients. The aim of this study was to compare the lipid lowering effect of 40 mg pravastatin, a HMG-CoA reductase inhibitor, and 400 mg bezafibrate, a fibric acid derivate, on serum lipids, lipoproteins and lipoprotein composition in 45 (22 men and 23 women) dyslipidemic, insulin-treated Type 2 diabetes patients. METHOD: The study used a double-blind, cross-over design. RESULTS: Pravastatin treatment was more effective in reducing total cholesterol, LDL-cholesterol, LDL-triglycerides, LDL ApoB and LDL/HDL-cholesterol ratio (all p<0.001 between groups) and total/HDL cholesterol and ApoA1/LDL-ApoB ratios (both p<0.01) and always induced a decrease in LDL-cholesterol concentrations and LDL/HDL-cholesterol ratio irrespective of baseline triglyceride concentration. Bezafibrate was more effective in increasing HDL-cholesterol (p<0.01 between groups), ApoA1 lipoprotein and decreasing triglycerides (both p<0.001 between groups) but induced an increase in LDL cholesterol concentration particularly in patients with baseline triglyceride concentrations exceeding 2.0 mmol/l. With bezafibrate treatment the LDL cholesterol/LDL-ApoB ratio showed a tendency to rise, suggesting a change in the LDL particle composition to a less small and dense form, while pravastatin treatment induced a decrease in this ratio suggesting a change in the LDL particle to a more dense form. With pravastatin treatment a small rise in HbA(1c) was observed. CONCLUSION: Pravastatin treatment is superior in lowering cholesterol-enriched lipoprotein subpopulations and improving cardiovascular risk factors. Bezafibrate is more effective in raising HDL-cholesterol and alters LDL particle composition to a more favorable form. PMID- 10751748 TI - Birth weight and the metabolic syndrome: thrifty phenotype or thrifty genotype? AB - BACKGROUND: Inverse correlations have been reported between birth weight and the Metabolic Syndrome (abdominal obesity, insulin resistance, hyperinsulinemia, glucose intolerance, dyslipidemia, and hypertension). These correlations are thought to reflect primarily nutritional inadequacies during fetal and early life. We explored familial influences on these correlations. METHODS: Using birth weight data on 602 subjects from 65 pedigrees, we partitioned phenotypic correlations into familial and non-familial. The former are usually regarded as reflecting primarily genetic influences, although they may also reflect environmental influences that are shared by family members, and the latter reflect random environmental influences. RESULTS: A consistent pattern of positive familial and inverse non-familial correlations were observed. The strongest familial correlations were between birth weight and fasting insulin (r = 0.58, p = 0.002), leptin (r = 0.48, p = 0.021), split proinsulin (r=0.51, p = 0.090), and heart rate (r = 0.39, p = 0.037). An inverse familial correlation was observed with HDL cholesterol (r = -0.28, p = 0.018). Non-familial correlations were weaker and only two-- subscapular-to-triceps skinfold ratio and fasting insulin--were statistically significant. CONCLUSION: Since the familial and non familial correlations were in opposite directions, we attribute the former to the pleiotropic effects of genes. Specifically, we conclude that genes that increase birth weight also worsen the Metabolic Syndrome in adult life. Since the inverse correlations reported in the literature reflect mainly cohorts born in the early part of the 20th century, improved maternal nutrition since then may have allowed the expression of genetic influences in our participants, all of whom were born after 1950. PMID- 10751749 TI - Overnutrition in spiny mice (Acomys cahirinus): beta-cell expansion leading to rupture and overt diabetes on fat-rich diet and protective energy-wasting elevation in thyroid hormone on sucrose-rich diet. AB - PREVIOUS STUDIES: The investigation of diabetes propensity in spiny mice, performed in Geneva and Jerusalem colonies, is reviewed. Spiny mice live in semi desert regions of the eastern Mediterranean countries. Those transferred to Geneva in the 1950s were maintained on a rodent diet supplemented by fat-rich seeds. They became obese, exhibited pancreatic islet hyperplasia and hypertrophy. Low insulin secretion response was characteristic of this species, despite ample pancreatic content of insulin. After a few months, diabetes with ketosis occurred, often suddenly, in association with islet cell disintegration. In Jerusalem the spiny mice were collected from their native habitat and placed on diets containing 50% sucrose or fat-rich seed diets. On a sucrose-rich diet, spiny mice developed hepatomegaly, lipogenic enzyme hyperactivity, and elevation in very low density lipoproteins as a result of metabolism of the fructose component mainly in the liver. No overt diabetes or pancreatic islet disintegration were observed, although insulin content and beta-cell hypertrophy and hyperplasia were apparent. On a fat-rich diet, spiny mice exhibited marked weight gain, adipose tissue growth and low hepatic lipogenesis. The obesity was accompanied by mild hyperglycemia and hyperinsulinemia with glucose intolerance leading to an occasional glucosuria after several months on the diet. NOVEL EXPERIMENTS: The sucrose diet induced an extrathyroidal elevation of triiodothyronine (T(3)). Serum T(3) level and hepatic T(4)-T(3) conversion were increased, while serum T(4) levels tended to decrease. The activity of the T(3) inducible hepatic mitochondrial FAD-glycerophosphate oxidase and K(+)/Na(+) ATPase, as well as body temperature were increased, indicating that the sucrose diet was associated with enhanced thermogenesis and energy-wasting metabolic cycling. The sucrose-rich diet might exert an adaptive thermogenesis-mediated defense mechanism, protecting against excessive weight gain and disruptive pancreatic islet lesion. After 18 months maintenance on sucrose-rich versus fat rich diets the number of animals surviving was significantly higher on the sucrose diet whereas on the fat diet a significant number of animals succumbed to expansive islet cell disruption and diabetes. PMID- 10751750 TI - Advanced glycosylation end products (AGEs), insulin-like growth factor-1 (IGF-1) and IGF-binding protein-3 (IGFBP-3) in patients with type 2 diabetes mellitus. AB - BACKGROUND: Advanced glycosylation end product (AGE) formation is a major mechanism for the development of complications in diabetes, and the possible roles of insulin-like growth factor 1 (IGF-1) and IGF binding protein 3 (IGFBP-3) are not clearly established. METHODS: We examined the associations of AGEs, free IGF-I and IGFBP-3 in Type 2 diabetes mellitus (DM) patients under diverse conditions. In a cross-sectional design we studied 110 subjects (67 women and 43 men): non-diabetic controls in group 1, (n = 15) and diabetes patients as follows: group 2, without complications (n = 25); group 3, with chronic complications (n = 25); group 4, with acute or chronic infections (n = 24); group 5, hospitalized for reasons unrelated to diabetes (n = 9); group 6, with end stage renal disease (ESRD) (n = 12). AGEs were determined by a spectrofluorometric method (HPLC). Insulin and IGFBP-3 were measured by RIA and free IGF-1 with an IRMA method. RESULTS: AGEs were 13-fold higher in patients with ESRD (p<0.001), and lower in healthy individuals. Free IGF-1 was lower in the patients with complications (p = 0.017), with infections (p = 0.006) and hospitalized (p = 0.04). IGFBP-3 was higher in hospitalized patients (p=0.017). AGEs were associated with free IGF-1 (r = 0.41, p = 0.04) in the group with complications, and with HbA(1c) (r = -0.90, p = 0.002) in hospitalized patients. In the total group, free IGF-1 (r = -0.25, p = 0.008), and IGFBP-3 (r = -0.22, p = 0.021) were associated with HbA(1c). CONCLUSION: We concluded that AGEs were markedly increased in diabetic patients with ESRD, IGF-1 was decreased in patients with infections and hospitalized, and was negatively associated with HbA(1c). IGFBP-3 was increased in hospitalized patients, with higher levels in patients with long bone fractures. A complex interaction of humoral factors may participate in the acceleration of complications of diabetes. PMID- 10751751 TI - Antiobesity pharmacotherapy in the management of type 2 diabetes. AB - Obesity is a well-known risk factor for the development of Type 2 diabetes mellitus. The management of the obese diabetic patient remains a challenge for the clinician but, in any case, weight reduction should be considered as a key objective. In this respect, several antiobesity drugs have demonstrated potential. However, while fenfluramine and dexfenfluramine have been shown to promote weight loss and to directly improve insulin sensitivity, being two mechanisms contributing to better blood glucose control in obese Type 2 diabetic patients, they were recently withdrawn due to safety problems. Sibutramine, a new selective norepinephrine and serotonin reuptake inhibitor, promotes weight loss by decreasing food intake, an effect which leads to a mild improvement (significant in patients losing > or =5% of initial body weight) of blood glucose control in obese diabetic patients. Similarly, orlistat, a selective gastrointestinal lipase inhibitor which increases faecal fat losses, enhances diet-induced weight reduction and improves both blood glucose control and vascular risk profile, especially dyslipidaemia, in obese Type 2 diabetic patients. Further studies are required to better identify good responders to pharmacotherapy and specify the role of antiobesity agents in the overall long term management of obese subjects with Type 2 diabetes. Other novel pharmacological approaches deserve further consideration, for instance beta-3 agonists aiming to increase energy expenditure, drugs interfering with tumor necrosis factor-alpha (TNF-alpha) or free fatty acid release by the adipose tissue or agents that slow gastric emptying. However, until now, results regarding efficacy and/or safety have been disappointing or preliminary in humans. PMID- 10751752 TI - The post-prandial state and cardiovascular disease: relevance to diabetes mellitus. AB - There is increasing evidence that the post-prandial state is an important contributing factor to the development of atherosclerosis. In non-diabetic subjects the atherosclerotic risk factors comprised in the categories of lipids, coagulation system and endothelial function may be adversely modified in the post prandial phase. The generation of an oxidative stress may be the common pathway through which eating may induce these alterations. In diabetic patients these phenomena may be amplified by post-prandial hyperglycemia. PMID- 10751754 TI - Current awareness AB - In order to keep subscribers up-to-date with the latest developments in their field, John Wiley & Sons are providing a current awareness service in each issue of the journal. The bibliography contains newly published material in the field of diabetes/metabolism. Each bibliography is divided into 17 sections: 1 Books, Reviews & Symposia; 2 General; 3 Genetics; 4 Epidemiology; 5 Immunology; 6 Prediction; 7 Prevention; 8 Intervention: a&rpar General; b&rpar Pharmacology; 9 Pathology: a&rpar General; b&rpar Cardiovascular; c&rpar Neurological; d&rpar Renal; 10 Endocrinology & Metabolism; 11 Nutrition; 12 Animal Studies; 13 Techniques. Within each section, articles are listed in alphabetical order with respect to author (9 Weeks journals - Search completed at 29th Dec. 1999) PMID- 10751753 TI - Difficult therapeutic decisions in the management of diabetic retinopathy. AB - Management decisions for patients with diabetic eye disease can remain difficult despite the presence of guidelines. The cases below illustrate the dilemmas about the timing of instituting insulin in patients needing laser photocoagulation for improvement of glycaemic control. The use of angiotensin-converting enzyme inhibitors for diabetic eye disease is also discussed. PMID- 10751755 TI - Anatomic variations of the radial artery in patients undergoing transradial coronary intervention. AB - Anatomic variations of the radial artery and their effect on the use of the radial artery as a route for transradial coronary intervention (TRI) were studied. Ultrasonography of the radial artery was performed prospectively in 115 patients selected to undergo elective TRI. Anatomic variations were observed in 11 of 115 patients (9.6%). Variations included six tortuous configurations (5. 2%), two stenoses (1.7%), two hypoplasias (1.7%), and one radioulnar loop (0.9%). The hypoplastic radial arteries and the radioulnar loop were inaccessible for catheterization, and coronary intervention was planned via the femoral artery. The transradial approach was attempted in the remaining 112 patients (97.4%) with only one instance of access failure, in a patient who had a stenotic vessel. These findings indicate that anatomic variations of the radial artery is not rare, and that preoperative ultrasound examination may help to exclude patients with inaccessible arteries and those at high risk for access failure. PMID- 10751756 TI - Cardiac catheterization reduces resource utilization in patients with chronic chest pain. AB - The evaluation of patients with recurrent chest pain accounts for a significant proportion of the $274 billion annual cost of cardiovascular services in the United States. Our investigation examines the impact of coronary angiography on subsequent use of medical resources for evaluation of chest pain symptoms. The study seeks to determine whether a finding of noncritical coronary artery disease on cardiac catheterization leads to a reduced use of resources for subsequent evaluation and treatment of chest pain syndromes. Our study included 22 consecutive patients who had sought evaluation for chest pain symptoms, and who had persistence of symptoms after functional testing. Cardiac catheterization demonstrated angiographically mild coronary artery disease (stenosis less than 50%) in these patients. The patient cohort accounted for 22 emergency room evaluations and 41 ambulatory clinic evaluations in the 2.5 years before cardiac catheterization. In the 2.5-year period after catheterization, these patients had only 3 emergency room visits and 1 ambulatory clinic visit for chest pain evaluation (P < 0.001). There was a significant reduction in the number of prescriptions written for topical and oral nitrates (32% precatheterization vs. 5% postcatheterization, P < 0.04), but not of beta-blockers (26% vs. 21%, P = 0.53) or calcium blockers (32% vs. 32%, P = 1.0). Furthermore, most of the 21 surviving patients were found subsequently to have a noncardiac basis for their pain: pericarditis was felt to be the cause of chest pain in 4 patients, pulmonary disease in 7 patients, and gastrointestinal conditions in 8 patients. Diagnostic coronary arteriography may identify a subset of patients in whom a finding of noncritical coronary artery disease leads to a reduction in physician visits for evaluation of chest pain syndromes and reduced use of nitrates. In addition, when coronary artery disease is known to be mild, a noncardiac etiology for the chest pain can be sought. These results may decrease the use of expensive medical resources and encourage full occupational and lifestyle rehabilitation. PMID- 10751757 TI - Cost-effective evaluation of chest pain syndromes. PMID- 10751758 TI - Physiologically guided angioplasty in support to a provisional stenting strategy: immediate and six-month outcome. AB - The results of an observational multicenter angioplasty study suggested that stenting decisions may be facilitated by physiologic data. The purpose of this study was to evaluate the early and long-term clinical and angiographic outcome of prospective physiologically guided provisional stenting. Coronary angioplasty using a Doppler-tipped angioplasty guidewire was performed in 68 patients. The provisional stent strategy dictated that balloon angioplasty was to be continued until a coronary flow reserve was >/= 2.2 with a residual diameter stenosis by quantitative coronary angiography < 35%. Repeat coronary angiography was obtained at 6 months. Based on the study criteria, 32/68 patients (47%) received a stent. Compared to the stent group, the angioplasty alone group had higher postprocedural stenosis (23% +/- 13% vs. 13% +/- 10%; P < 0. 05) and lower coronary vasodilatory reserve (2.3 +/- 0.4 vs. 2.6 +/- 0.7; P < 0.05). At follow up (6.0 +/- 1.5 months), the angiographic restenosis rate was 39% in the angioplasty group and 35% in the stent groups (P = NS). Adverse cardiac events (unstable angina, target lesion revascularization, myocardial infarction, death) occurred in 19% and 18% (P = NS) of the angioplasty and stent patients, respectively. A prospective application of a physiologically guided provisional stent strategy for coronary angioplasty indicated that stent implantation may be required in approximately 50% of patients, an approach that produces similar clinical and angiographic long-term outcomes for stenting and guided angioplasty. These data support a role of coronary physiology as an adjunct in conducting an angioplasty procedure without obligatory stenting. PMID- 10751760 TI - In-stent restenosis: is the "stent-wich" the answer? PMID- 10751759 TI - Stenting for in-stent restenosis. AB - Intravascular ultrasound studies have shown that additional stent implantation is the only percutaneous technique that allows for recovery of all the lumen area of the original implantation procedure. Despite this theoretical advantage, information on systematic additional stent implantation is still forthcoming, especially concerning the impact of new stent designs. This prospective study evaluated the efficacy of routine additional stent implantation for treatment of in-stent restenosis in 68 consecutive patients. Repeat stenting was successful in all cases, and second-generation tubular stents were used in 84% of patients. The mean additional stent length was 19.2 +/- 9.4 mm, and 15% of patients had multiple stent implantation. The postprocedure minimum lumen diameter was 3.11 +/ 0.41 mm, and the percentage residual stenosis was 2% +/- 7%. At a mean clinical follow-up of 10 +/- 8 months (follow-up rate 100%), the incidence of major adverse events was 21% (1 death, 13 target vessel revascularizations). Overall, angiographic restenosis rate was 32% (angiographic follow-up rate 79%). By multivariate analysis, the only predictors of recurrence after additional stenting were unstable angina at the second procedure (OR 8.70, 95% CI 1.50 50.33, P = 0.019), and early clinical recurrence after the first stent procedure (OR 4.83, 95% CI 1.13-20.71, P = 0.038). Additional stenting is a safe and effective treatment modality for the majority of patients with in-stent restenosis. Alternative treatments should be considered only for patients with in stent restenosis presenting as unstable angina or early recurrence after a first stent procedure. PMID- 10751761 TI - Coronary stenting without predilatation (SWOP): applicable technique in everyday practice. AB - To evaluate the feasibility of stenting without predilatation, we registered all interventional procedures over a 6-month period. Six hundred patients were registered, and 684 lesions were treated. Interventions were divided into four groups: stenting without predilatation (SWOP), 221 lesions (32.4%); primary stenting with predilatation (PDS), 161 lesions (23.5%); provisional stenting (PRS), 131 lesions (19.2%); and plain-old balloon angioplasty (POBA), 171 lesions (25%). Interventional strategy was at the discretion of the operator based on few simple angiographic criteria and his clinical judgment. Procedural success was similar in all stent groups. We conclude that when primary stenting is planned, about 60% of lesions can be treated by SWOP effectively with excellent procedural results and considerable cost saving. PMID- 10751763 TI - Catheter debris: it's a jungle in there! PMID- 10751762 TI - Potential embolization by atherosclerotic debris dislodged from aortic wall during cardiac catheterization:: histological and clinical findings in 7,621 patients. AB - Embolic events during cardiac catheterization have been attributed to atherosclerotic aortic debris dislodged by catheter manipulation. We evaluated the frequency and the histologic morphology of atherothrombotic material retrieved during placement of coronary catheters in patients undergoing diagnostic or interventional cardiac procedures. Over a 4-year period, macroscopically visible aortic debris from coronary catheters, if present after advancement to the ascending aorta, was obtained for histologic examination. In 41 of 7,621 patients (0.54%), visible atherothrombotic material was present in the backflow of catheters. Debris occurred most frequently with 8 Fr guiding catheters (98%). Histologic examination showed foam cells, cholesterol crystals, and amorphic lipoid substance as markers of atheromatous material from atherosclerotic plaques in 38/41 patients (93%) with former plaque hemorrhage in 55% of them. In three patients, fresh thrombus material was observed (7%). None of these patients showed in-hospital ischemic complications. Although visible atheromatous material is a rare phenomenon in cardiac catheterization, an increased risk of scraping debris is associated with large-lumen guiding catheters. In order to avoid vascular embolization, the use of smaller guiding catheters and sufficient free backflow of catheters after advancement are recommended. PMID- 10751764 TI - Directional coronary atherectomy in intermediate sized vessels: final results of the intermediate vessel atherectomy trial (IVAT). AB - Revascularization options for intermediate sized vessels (2.5-2.9 mm) have historically been limited. IVAT is a pilot study to assess the efficacy and safety of debulking intermediate sized vessels using directional coronary atherectomy (DCA). Between March 1996 and June 1997, 50 patients were enrolled at seven hospitals in the United States. Of those patients, 70% presented with unstable angina and 52% had single vessel disease. Of the lesions treated, 96% were de novo. Adjunctive PTCA after DCA was performed in 90% of cases at the discretion of the investigator to maximize luminal diameter. The GTO DCA device was used in 90% of cases. Procedural success (residual stenosis <50% without major complications) was 94%. Stents were placed in 12% of patients. The only complications were three non-Q wave MIs. Mean reference vessel diameter increased from 2.49 mm pre-procedure to 2.57 mm after DCA and 2.61 post-procedure; mean MLD increased from 0.76 mm to 2.03 mm to 2.31 mm; and mean stenosis decreased from 70% to 21% post DCA and to 11% post procedure. At six months follow-up, 18.0% of target lesions required revascularization. Total revascularization, including non target vessels, was 32%. These results suggest that DCA has a high procedural success rate and a low target lesion revascularization rate in intermediate sized vessels. PMID- 10751765 TI - Angiographic and clinical outcome of a new self-expanding intracoronary stent (RADIUS): results from multicenter experience in Japan. AB - The RADIUS coronary stent featuring a multisegmented slotted tube design and self expanding nitinol delivery system has a high radial force and flexibility, uniform expansion, and contours to the shape of the vessel. Successful stent deployment was achieved in 104 stable angina patients (106 lesions; 44% LAD, 19% circumflex, and 37% RCA). Mean minimal lumen diameter (MLD) increased from 0.77 +/- 0.46 mm to 2.88 +/- 0.61 mm and mean percent diameter stenosis (% DS) decreased from 73 +/- 14% to 6 +/- 13% immediately after the procedure. At 6 month follow-up, two patients (2%) underwent urgent target revascularization, and cerebral bleeding occurred in one patient (1%). Angiographic follow-up was performed in 94 lesions (89%) and mean MLD and mean % DS were 2.08 +/- 0.92 mm and 30% +/- 24%, respectively. Stent restenosis (>50% diameter stenosis at follow up) was observed in 16 (17%) of all lesions. The high success rate for stent deployment, low incidence of major adverse cardiac event, and lower restenosis rate after stent implantation indicate that the RADIUS stent is useful for coronary intervention. PMID- 10751766 TI - Unique properties of self-expanding stents. PMID- 10751767 TI - Influence of residual stenosis in determining restenosis after cutting balloon angioplasty. AB - The cutting balloon is a new device for coronary angioplasty, which, by the combination of incision and dilatation of the plaque, is believed to minimize arterial wall trauma, the neoproliferative response, and subsequent restenosis. In this study, we sought to determine predictors of the restenosis using this technique. Seventy-seven patients underwent successful coronary angioplasty with cutting balloon alone. In 67 of these patients (87%), we performed a control angiogram at 6-month follow-up. Pre-, post-, and late angiographic results were evaluated by quantitative coronary analysis. Clinical and angiographic variables were correlated with restenosis as a binary variable and a continuous variable (late loss and late minimum luminal diameter). Univariate analysis showed that the immediate postprocedure minimum luminal diameter (MLD) was smaller in the restenotic group (defined as MLD > 50% by quantitative coronary angiography) than in the nonrestenotic group (1.90 +/- 0.47 mm vs. 2.19 +/- 0.56 mm, P < 0.05). In addition, the immediate percentage of stenosis was higher in the restenotic group than in the nonrestenotic group (37% +/- 10% vs. 27% +/- 11%, P < 0. 003). Multivariate analysis identified the immediate postcutting balloon percentage of stenosis as an independent determinant of binary restenosis (P < 0.008). When restenosis was defined as a continuous variable, the immediate postprocedure MLD was an independent predictor of late loss (P < 0.02) and of late MLD (P < 0. 0002). No clinical, preprocedure angiographic, or technical variables tested were associated with restenosis. The degree of postprocedural residual stenosis after cutting balloon angioplasty is predictive of late restenosis. PMID- 10751768 TI - Diagnosis and catheter treatment of innominate artery stenosis following stage I Norwood procedure. AB - Four infants aged 20-115 days (mean, 57.8 days) who had undergone stage I Norwood procedure for hypoplastic left heart syndrome came to early cardiac catheterization (6-112; mean, 47.3 days) following surgery because of significant arterial desaturation (pulse oximetry indicating oxygen saturations consistently in the 40%-70% range). Cardiac catheterization demonstrated a significant systolic pressure gradient between the ascending aorta and innominate artery (30 65; mean, 51 mm Hg) as the likely cause of diminished pulmonary blood flow in these patients. Routine angiography by itself was not conclusive in identifying a discrete area of obstruction, but selective angiography coupled with a knowledge of the obstruction did reveal the stenosis. All patients were successfully treated with balloon dilatation of the stenotic area, with the pressure gradient being reduced to 7-25 (mean, 17 mm Hg) immediately following dilatation. On follow-up catheterization in three patients, the systolic gradients were 3, 6, and 9 mm Hg. Arterial oxygen saturations rose from 63.5% predilatation to 77.3% immediately postdilatation and 81% on follow-up evaluation. In conclusion, innominate artery stenosis is an important cause of diminished blood flow through a modified right Blalock-Taussig shunt. Routine angiography will often miss the diagnosis. Pressure gradients and selective angiograms are necessary in order to make the diagnosis, although careful noninvasive assessment should also be diagnostic of this problem. Catheter dilatation is therapeutic in this situation and can be performed early after surgery in the absence of a fresh suture line. PMID- 10751769 TI - Percutaneous transluminal angioplasty of the aorta in children with nonspecific aortoarteritis: acute and follow-up results with special emphasis on left ventricular function. AB - Nonspecific aortoarteritis (NSAA) is a chronic disease of unknown etiology and may result in stenosing and obstructive lesions of the aorta. Transluminal balloon angioplasty was carried out in 17 children, 4-12 years of age (mean 8.9 +/- 2.7 years) with symptomatic stenosis of thoracic or abdominal aorta due to NSAA. Hypertension was present in 15 children, and 11 had significant left ventricular dysfunction. All children had a clinically inactive disease. The stenosis was in descending thoracic aorta in 10, thoracoabdominal in 3 and only abdominal in 4. Symptomatic improvement with relief of stenosis was seen in 16 of 17 children, although in 3, the results were suboptimal. The peak systolic gradient fell from 70.9 +/- 19.3 mmHg to 32.0 +/- 19.2 mmHg immediately after dilation (P < 0.0001). Angiographically measured luminal diameter stenosis also fell from 82.5 +/- 7.7% to 19.4 +/- 11.8% (P < 0.0001). Dissection flap was seen in 13 cases. It was large in cases with long, diffuse and eccentric stenosis of the thoracic or thoracoabdominal aorta. On follow-up (available for 14 of 16 cases for a period of 2-87 months mean 20.7 +/- 21.5 months, median 15.5 months) two patients developed restenosis. Left ventricular function improved in all 8 children that had sustained benefit of angioplasty. Blood pressure normalized in 9 of 13 children and the drug requirements fell in 4 others. Further remodeling of the lesion with decrease in gradients and luminal stenosis was observed in 11 children that have been restudied. Balloon angioplasty of aorta in children with NSAA is a feasible and safe procedure. Small intimal flap is seen in the majority, extensive dissection is more likely in the long diffuse lesions in descending thoracic aorta. Hypertension and left ventricular dysfunction improve after relief of obstruction. PMID- 10751770 TI - Aortitis: dilation and/or stent. PMID- 10751771 TI - Closure of a large pulmonary arteriovenous malformation using multiple Gianturco Grifka vascular occlusion devices. AB - A large pulmonary arteriovenous malformation was successfully occluded using multiple Gianturco-Grifka vascular occlusion devices. The filler wire protruded from one of the devices and was successfully retrieved 3 weeks after implantation. PMID- 10751772 TI - Stenting of stenosed aortopulmonary collaterals and shunts for palliation of pulmonary atresia/ventricular septal defect. AB - Patients with unrepaired pulmonary artery atresia and ventricular septal defect (PA/VSD) depend on aortoplumonary collaterals and surgically created shunts for pulmonary blood flow. These vessels frequently develop stenoses with time, leading to hypoperfusion of lung segments and systemic hypoxemia. The purpose of this article is to describe catheter palliation of hypoxemic patients with PA/VSD who were not candidates for surgical repair. We present our experience with stent implantation for stenosis of aortopulmonary collaterals and shunts in these patients. Three patients with hypoplastic pulmonary arteries underwent stent placement in aortopulmonary collateral arteries (APCAs) or their shunts. Technical aspects of the interventional catheterization procedure are discussed in detail. Case 1 underwent placement of five stents in collateral vessels and one stent in the Blalock-Taussig shunt (BT) with dramatic increase in vessel size and improvement in saturations from 70% to 89%. Case 2 underwent placement of two overlapping stents in a collateral vessel with an increase in diameter of the collateral vessel from 2.3 to 6 mm and an improvement in saturation from 68% to 88%. Case 3 underwent placement of three overlapping stents in a BT shunt with an increase in diameter of the shunt from 2.2 to 6.6 mm and an improvement in saturation from 71% to 89%. All three patients had excellent clinical improvement and stable saturation at follow-up. Stent placement for maintaining patency of APCAs and aortopulmonary shunts is feasible and safe. PMID- 10751773 TI - Use of a bow tie stent occluder for transcatheter closure of a large anomalous vein. AB - Percutaneous occlusion of superfluous vascular connections, which are short and have large caliber, is technically difficult. We report use of a composite device (consisting of a Palmaz stent, a Gianturco Grifka vascular occlusion device, and multiple platinum detachable embolization coils) to close a large left superior vena cava draining to the pulmonary venous atrium. PMID- 10751774 TI - Internal mammary artery perfusing Leriche's syndrome in association with significant coronary arteriosclerosis: four case reports and review of literature. AB - Four cases of collateral perfusion of a lower extremity by way of an internal mammary artery in the presence of Leriche's syndrome are described. Angiographic documentation preceding coronary artery bypass grafting prevented an acutely ischemic leg in two of the cases. PMID- 10751775 TI - Winslow's pathway--it's not the only way. PMID- 10751776 TI - Percutaneous transluminal coronary angioplasty and stenting for cocaine-induced acute myocardial infarction: a case report and review. AB - Cocaine-induced myocardial infarction has been well reported. Likewise, there are numerous reports of patients with cocaine-induced myocardial infarction being treated conservatively with nitroglycerin, verapamil, and thrombolytics. However, based on a Medline search from 1977 to 1998 (with the keywords cocaine and angioplasty), there have been no reports in English of cocaine-induced myocardial infarction being treated with catheter-based intervention. We report such a case, as well as review what is known about the pathophysiology of cocaine-induced coronary arteriopathy and myocardial infarction. PMID- 10751777 TI - Coronary air embolism treated by bubble aspiration. AB - We describe a case of coronary air embolism following cardiac catheterization, with all the signs and symptoms of an acute coronary event. Thanks to the rapid and effective aspiration of the air bubble from the distal artery, blood flow was restored and the clinical picture was resolved. PMID- 10751778 TI - Coronary sinus compression as a sign of cardiac tamponade. AB - Coronary perforation and resultant cardiac tamponade are well-known but rare complications of percutaneous coronary interventions. We present a case that demonstrates coronary sinus compression caused by increasing pericardial pressure as a new sign of impending cardiac tamponade. This previously unreported angiographic sign preceded hemodynamic, symptomatic, and echocardiographic evidence of tamponade. PMID- 10751779 TI - Precipitous labor in association with percutaneous mitral valvuloplasty: successful delivery in the catheterization laboratory. PMID- 10751780 TI - Local delivery of heparin post-PTCA: a multicenter randomized pilot study. AB - Bailout stenting for major dissection and threatened closure has high rates of ischemic complications. We performed a randomized trial of local heparin delivery using the infusion sleeve before bailout stenting for suboptimal angioplasty results. In phase I, 20 patients were randomized to local delivery with either 40 or 100-psi infusion pressure. In phase II, 37 patients were randomized to local delivery at 100 psi or standard therapy. Local delivery succeeded in all but one patient; overall there was no significant worsening of intimal dissection. One patient treated with 100-psi drug infusion suffered a perforation after stent placement. There were no significant differences in the composite endpoint of death, MI, CABG, urgent repeat angioplasty, and stent thrombosis at 30 days (21% vs. 0%; P = 0.18). At 6 months, the rates of myocardial infarction in phase II were 27% with local delivery vs. 10% with standard treatment (P = 0.4). Local heparin delivery in dissected vessels may be associated with increased complications and should be approached with caution. PMID- 10751781 TI - Intravascular low-power red laser light as an adjunct to coronary stent implantation: initial clinical experience. AB - Low-power red laser light (LPRLL) irradiation enhances endothelial cell growth in vitro and in vivo and reduces restenosis in animal models. The present study reports the preliminary clinical experience in our center. Eighty-one patients were treated with LPRLL, 30 mW/1 min, for in-stent restenosis (n = 27), elective stenting for recurrent restenosis (n = 16), and stenting for treatment of a suboptimal PTCA result (n = 38). All interventions were successful and no major adverse events due to LPRLL therapy were observed. At follow-up, 12 patients (14.8%) underwent an early control coronarogram due to target vessel restenosis. At 6 months, another 20 patients showed a significant restenosis of the target vessel. Preliminary clinical evaluation demonstrates that LPRLL is feasible and safe. The preliminary results suggest that LPRLL results in a decrease of in stent restenosis when used during primary stenting. PMID- 10751782 TI - Preclinical safety testing of percutaneous transatrial access to the normal pericardial space for local cardiac drug delivery and diagnostic sampling. AB - The safety of a percutaneous method and streamlined catheter system to access the normal pericardial space via the right atrial appendage for drug delivery and diagnostic sampling was demonstrated in 20 anesthetized pigs. Access was successfully accomplished in all animals within 3 min of guide catheter positioning and was documented by fluoroscopic imaging and pericardial fluid sampling. The animals were sacrificed at 24 hr (n = 10) and 2 weeks (n = 10) for histopathologic analysis. Mean pericardial hematocrit was 1.1% +/- 0.3% at initial sampling, 4.3% +/- 1.4% at 24 hr (P = 0.005 vs. baseline), and 0.4% +/- 0.2% at 2 weeks (P = 0.13 vs. baseline). At 24 hr, there was local inflammatory reaction in the atrial wall and a small thrombus at the site of puncture. At 2 weeks, no significant inflammatory changes or pericarditis were evident. The technique is well tolerated with no apparent adverse complications. Advances in intrapericardial therapeutics and diagnostics will direct the clinical application of this novel approach in human subjects. PMID- 10751783 TI - PASTA is good, but SUSHI is better. PMID- 10751784 TI - Late total occlusions following restenting for in-stent restenosis: to restent or not to restent? PMID- 10751785 TI - Advances in Alliteration in Acute Myocardial Infarction: From "Time to Treatment" to "Onset to Opening" PMID- 10751786 TI - The National Heart Attack Alert Program: Progress at 5 Years in Educating Providers, Patients, and the Public and Future Directions. AB - The National Heart Attack Alert Program (NHAAP) was launched by the National Heart, Lung, and Blood Institute in 1991 with the goal of reducing morbidity and mortality from acute myocardial infarction (AMI) through the rapid identification and treatment of individuals with symptoms and signs of an AMI. To achieve this goal, the NHAAP established objectives for each of three phases of action where treatment delays can occur: in the hospital, the prehospital setting, and the patient/bystander arena. The NHAAP initially directed its educational efforts toward emergency department professionals. Recommendations for reducing delays in emergency department identification of patients presenting with heart attack symptoms were developed by a working group convened in late 1991. These recommendations were published in February 1994 in a peer-reviewed journal reaching more than 17,000 emergency physicians. The NHAAP worked in a partnership with its coordinating committee, representing 40 health professional, voluntary, and government organizations, to extend the reach of the report's recommendations to their members. Strategies for promoting the emergency department recommendations included publication of excerpts in newsletters and journals of the medical, nursing, and prehospital provider organizations represented on the NHAAP Coordinating Committee, and through symposia at annual meetings. Industry assisted with dissemination efforts and with implementing a continuous quality improvement program based on the paper's recommendations. The NHAAP also developed, with the Joint Committee on Accreditation of Health Care Organizations, a time-to-treatment indicator for thrombolytic therapy to be incorporated into their Indicator Measurement System (IMSystem). To track achievement of the objectives related to the Hospital Action Phase, national data sources for emergency department management of patients with AMI were evaluated at the 5-year point of the NHAAP. Data from a national registry showed that the median time from presentation at the emergency department to receiving thrombolytic therapy declined by about one third between 1992 and the last half of 1995. The percentage of all Medicare patients receiving thrombolytic therapy within the recommended 30 minutes after emergency department arrival nearly doubled between 1992 and 1995. Based on these and other results presented at the 5-year juncture of the program, the NHAAP Coordinating Committee assessed progress and identified new areas of focus for the next 5 years. Improvements in emergency departments' ability to identify and treat AMI patients progressed during the first 5 years of the NHAAP, when the program was highlighting this as a priority. This model is continuing to be used to address delays in the Prehospital Action Phase. Further research from a National Heart, Lung, and Blood Institute (NHLBI) community intervention trial will guide the program in its plans for full-scale public education to address the Patient/Bystander Recognition and Action Phase. PMID- 10751787 TI - Clinical Features of Emergency Department Patients Presenting with Symptoms Suggestive of Acute Cardiac Ischemia: A Multicenter Study. AB - Identification of patients with acute cardiac ischemia (ACI) remains challenging. The object of this study was to examine the role of clinical findings in the diagnosis/triage of emergency department (ED) patients with symptoms suggestive of ACI. The study was designed as a secondary data analysis of a multicenter prospective controlled clinical trial. It was set in 10 midwest, southeast, and northeast U.S. hospitals, and 10,689 patients with chest pain or other symptoms suggesting ACI presenting from May 1993 to December 1993, participated. The results indicated that ACI patients were more likely to have chest pain as a chief complaint or presenting symptom (P = 0.001). The presenting symptom of nausea was more commonly associated with a final diagnosis of ACI (P = 0.003). Shortness of breath as the chief complaint and presenting symptoms of abdominal pain, nausea, dizziness, and fainting were less frequent among patients with a final diagnosis of ACI (P = 0.001). A past history of diabetes mellitus, myocardial infarction, or angina pectoris was more frequently associated with a final diagnosis of ACI (P = 0.001). A lower pulse rate in patients with a final diagnosis of ACI (P = 0.001) was not considered clinically significant. Median first and highest systolic blood pressures (SBPs) were higher, median lowest SBPs were lower, median diastolic blood pressure of the lowest SBPs were lower, and initial and highest pulse pressures were wider in patients with a final diagnosis of ACl (P = 0.001). On arrival, these blood pressure variables in AMI patients, subsequently classified as Killip class 4, were above the threshold for this classification. Rales were more commonly present in patients with a final diagnosis of ACI (P = 0.001). All primary ST-segment abnormalities, Q waves, and T-wave abnormalities, except T-wave flattening, were seen more frequently in patients with a final diagnosis ACI (P = 0.001). Normal ECGs were more frequently associated with a non-ACI final diagnosis, yet 20% of AMI patients and 37% of Unstable Angina Pectoris (UAP) patients had normal ECGs. It can be concluded that certain clinical features can help to identify ED patients with ACI. Initially normal ECGs can be seen in 20% of patients with AMI and 37% of patients with UAP. Patients with ACI can present with "normal" blood pressures and develop cardiogenic shock. Clinical outcome data for ACI patients are presented. PMID- 10751788 TI - Targeted Coronary Thrombolysis via "Pericardial" Administration of Lytic Agents? PMID- 10751789 TI - Protection Conferred by Preinfarct Angina is Manifest in the Aged Heart: Evidence from the TIMI 4 Trial. AB - There has been debate regarding the issue of whether ischemic preconditioning is effective in the aging and diseased heart. Therefore, we retrospectively analyzed the effect of preinfarction angina in patients less than versus greater than 60 years of age in the TIMI 4 study. Preinfarction angina was defined as an episode of typical angina pectoris that occurs prior to the time of index chest pain associated with the myocardial infarction itself. Patients who were 60 years and older had a higher rate of death and the combined endpoints of death, heart failure/shock, and/or reinfarction compared with younger patients. However, patients 60 years or older who had preinfarction angina had lower rates of the combined endpoints of death, heart failure/shock, and/or reinfarction (11%) compared with patients without angina (23%; P = 0.04). They also had lower creatine kinase (CK) values. Therefore, preinfarction angina was protective in patients 60 years or older in the TIMI 4 study. PMID- 10751790 TI - P-Selectin: Basic Aspects. PMID- 10751791 TI - Comparison of Argatroban and Hirudin for the Reperfusion of Thrombotic Arterial Occlusion by Tissue Plasminogen Activator. AB - Despite theoretical advantages of direct thrombin inhibitors, recent clinical studies failed to show the superiority of hirudin over heparin in patients with acute coronary syndromes. However, these inhibitors have important in vitro differences for the inhibition of clot-bound thrombin that may translate into different in vivo relative efficacy. The effects of two direct thrombin inhibitors, argatroban and hirudin, on the reperfusion of thrombotic arterial occlusion by t-PA were compared. In anesthetized rabbits thrombotic occlusion was induced in the femoral artery. t-PA, aspirin, and various doses of argatroban (1.25, 2.5, and 5.0 mg/kg/h) or hirudin (2.5 and 5.0 mg/kg/h) were administered (six animals in each group). Blood flow was measured for 4 hours. Animals treated with 2.5 mg argatroban more rapidly achieved full reperfusion than those treated with high-dose argatroban or hirudin (P < 0.05). At the doses that induced comparable prolongation of bleeding time, argatroban showed a significantly faster and higher level of reperfusion than hirudin. In animals treated with hirudin, there was a positive correlation between the aPTT and the mean reperfusion blood flow (r = 0.70, P < 0.05). In animals treated with argatroban, this correlation did not exist and the high-dose argatroban was paradoxically less effective in promoting thrombolysis despite greater anticoagulation effects. In this animal model of arterial thrombosis, argatroban was more effective than hirudin in inducing rapid, full reperfusion with t-PA. Although they are both direct thrombin inhibitors, these two agents showed important dose-related differences in efficacy and anticoagulant effects. PMID- 10751792 TI - Influence of Sulfation on Platelet Aggregation and Activation with Differentially Sulfated Hyaluronic Acids. AB - A number of sulfated hyaluronic acid derivatives (HyalS(2.5), HyalS(3), and HyalS(4)) were prepared by sulfation of the -OH groups present on hyaluronic acid and were generically termed HyalS(x). The anticoagulant properties of this series of compounds has previously been shown to be good in terms of their whole blood clotting inhibition and factor Xa and thrombin inactivation. The purpose of the present study was to investigate whether the use of these compounds would be beneficial to patients who would normally be given heparin, and to perform some preliminary investigations into their effects on platelets. The three compounds were thus studied by investigating their ability to inhibit von Willebrand factor dependent platelet agglutination in comparison with unfractionated heparin. Agglutination was determined turbidometrically after the addition of ristocetin to stirred formaldehyde-fixed platelets and was demonstrated to be dependent on the presence of sulfate groups on the polysaccharide chain and correlated with the degree of HyalS(x) sulfation. Interactions possibly important in low shear environments were investigated by measuring the pharmacological action of the HyalS(x) on spontaneous platelet activation and aggregate formation by flow cytometry. The data indicate that platelet activation is not correlated with the number of sulfate or hydroxyl groups on HyalS(x), suggesting that activation occurs not via electrostatic interactions or H bonding, but via some other mechanism. A differentiation between low and high glycosaminoglycan sulfation densities is observed with respect to platelet aggregation, which is correlated with the number of sulfated groups per disaccharide unit. The ability of HyalS(x) to inhibit platelet aggregation induced by ADP and thrombin was measured by aggregometry. HyalS(4) resisted thrombin stimulation to a similar extent as heparin. All Hyal derivatives, however, were better at inhibiting ADP-induced aggregation than was heparin. We conclude, therefore, that clinical use of HyalS(x) in place of heparin may be beneficial because ristocetin-dependent agglutination, and therefore resistance to platelet aggregation in high shear environments, in addition to resistance to stimulation by ADP, has been shown to be superior to heparin. Spontaneous platelet activation and aggregation are induced at an overall low level, even at high HyalS(x) concentrations, and are comparable with that of heparin. PMID- 10751793 TI - Tyrosine Phosphorylation of Moesin in Arachidonic Acid-Stimulated Human Platelets. AB - Moesin, a member of the ezrin/radixin/moesin (ERM) family of cytoskeletal proteins, has been implicated in dynamic membrane-based processes such as the formation and stabilization of filopodia. Ezrin is known to be a substrate of tyrosine kinases in activated T cells and epithelial growth factor-stimulated A431 cells. For the closely related 77-kD protein moesin, which shares 72% identity with ezrin on the basis of their amino acid sequences, a reversible phosphorylation on tyrosine residues has not yet been described. Because our scanning electron microscopy studies revealed the appearance of multiple, up to 3 um long filopodia on the surface of activated human platelets, we investigated the participation of moesin in dynamic shape changes on platelet stimulation with arachidonic acid. Antimoesin immunoprecipitates obtained under denaturing conditions from lysates of resting platelets contained only low amounts of tyrosine-phosphorylated moesin. In lysates of arachidonic acid-stimulated platelets, the level of tyrosine phosphorylation was significantly increased. This activation-dependent phosphorylation of moesin was verified by probing antiphosphotyrosine immunoprecipitates from unstimulated and stimulated platelets with antimoesin antibodies. Tyrosine-phosphorylated moesin was detectable only in the presence of the tyrosine phosphatase inhibitor vanadate, suggesting that a coordinated balance between kinase and phosphatase activities controls the steady state level of moesin phosphorylation. PMID- 10751794 TI - Cooling System Permits Effective Transcutaneous Ultrasound Clot Lysis In Vivo Without Skin Damage. AB - Previous in vivo studies have shown that transcutaneous ultrasound enhances clot dissolution in the presence of either streptokinase or microbubbles. However, ultrasound-induced skin damage has been a major drawback. The objective was to evaluate the effect of a cooling system to prevent the skin damage that has heretofore been associated with transcutaneous low-frequency, high-intensity ultrasound clot dissolution. After thrombi were induced in both iliofemoral arteries in 15 rabbits, streptokinase (25,000 U/kg) was given intravenously and dodecafluoropentane was injected slowly (2 mL/15 min) through an infusion catheter into the abdominal aorta. One iliofemoral artery was randomized to receive ultrasound treatment, and the contralateral artery was treated as a control (receiving streptokinase and dodecafluoropentane alone). In six rabbits (group 1), the skin below the ultrasound transducer was protected by the use of a balloon cooling system, and in the other nine rabbits (group 2), ultrasound was used without a cooling system. Seven of nine (78%) arteries treated without the cooling system, and six of six (100%) arteries treated with the cooling system were angiographically recanalized after ultrasound + streptokinase + dodecafluoropentane treatment. Thermal damage was present in the skin and soft tissues of all nine rabbits treated without a cooling system. However, the skin and soft tissues were grossly and histologically normal in the six rabbits in which the transcutaneous ultrasound was used with the cooling system. Low frequency, high-intensity ultrasound energy can be delivered transcutaneously for clot dissolution without concomitant tissue damage when coupled with the use of a cooling system to prevent thermal injury. PMID- 10751795 TI - Combination Hemotherapy and Mortality Prevention (CHAMP) Study Rationale and Design. AB - It is now agreed that the majority of acute myocardial infarctions result from intracoronary thrombosis at sites of atherosclerotic plaque that have been disrupted. In 1947 Nicol and Fassett published the first clinical paper suggesting that agents interfering with blood coagulation could prevent myocardial infarction in patients at risk. Scores of subsequent clinical trials were performed to assess the efficacy of anticoagulants and antiplatelet agents in preventing death and reinfarction in survivors of acute myocardial infarction. Despite these efforts no agreement exists on whether these strategies are beneficial and, if so, which is superior. The primary obstacle to progress in this field has been the failure of nearly all trials to enroll the large numbers of subjects required to demonstrate a survival benefit. The large sample size requirement derives from two inescapable facts: mortality rates following acute infarction, though variable, are generally low and the potential benefit of these agents in preventing mortality is small. Combining oral anticoagulants with antiplatelet agents (combination hemotherapy) may significantly enhance their antithrombotic effect. Clinical trials of combination hemotherapy have demonstrated superiority over anticoagulant monotherapy in the setting of stroke prevention in patients with prosthetic heart valves. Similar benefit was not observed in trials studying stroke prevention in nonvalvular atrial fibrillation and vascular morbidity in patients surviving an acute myocardial infarction. The failure of these latter studies may relate to the particularly low intensity of warfarin administered in combination with aspirin. This trial proposes to demonstrate that the combination of oral anticoagulation, administered in a moderate dose intensity, and antiplatelet therapy is superior to aspirin monotherapy in reducing overall mortality following acute myocardial infarction. PMID- 10751796 TI - Platelet and Thrombin Activity Following Cardiac Catheterization Despite Treatment with Aspirin. AB - Thromboembolic complications are reported to occur in up to 0.5-2% of left cardiac catheterizations and angiographies. Activation of the hemostatic system may contribute to their onset. To prevent platelet and thrombin activity during catheterization, aspirin or systemic heparin are often used in addition to heparinized flush solutions. We investigated whether aspirin alone can prevent platelet and thrombin activity induced by catheterization in ten consecutive patients (nine males, mean 50 +/- 8 years) undergoing elective left cardiac catheterization after at least 5 days of oral aspirin (75-300 mg/d). Anticoagulant drugs were not given. Peripheral venous samples were drawn before, immediately after (time 0), and at 15, 60, and 180 minutes after the procedure for measurement of thrombin-antithrombin (TAT), prothrombin fragment 1.2 (F 1.2), fibrinopeptide A (FPA), and beta-thromboglobulin (beta-TG). TAT, F1.2, and FPA increased significantly at time 0 compared with both before and 180 minutes after the procedure (P < 0.04); beta-TG values were higher at time 0 compared with 180 minutes later (P = 0.01). TAT levels were related to those of FPA (r = 0.66; P = 0.0003), F1.2 (r = 0.35; P = 0.01), and beta-TG (r = 0.37; p = 0.04). Thus, routine left cardiac catheterization is associated with transient, systematically detectable, activation of coagulation and platelets, despite aspirin therapy. Newer antiplatelet agents may be more effective in preventing hemostatic activation induced by catheterization. PMID- 10751797 TI - Bolus Administration of Saruplase in Europe (BASE), a Pilot Study in Patients with Acute Myocardial Infarction. AB - To study the safety and efficacy of the thrombolytic agent saruplase as a bolus, the angiographic and clinical outcomes of three bolus regimens were investigated in a pilot study conducted in 192 patients with an acute myocardial infarction and were compared with the standard regimen. Fifty-two patients received a double bolus of 40 mg and 40 mg after 30 minutes, 51 patients a bolus of 80 mg, and 36 patients a bolus of 60 mg. Fifty-three patients received the standard regimen (a bolus of 20 mg and 60 mg IV infusion over 1 hour). At 60 minutes TIMI 2 and 3 flow were, respectively, 9.6% and 61.5% with the 40/40-mg bolus, 15.7% and 51.0% with the 80-mg bolus, 16.7% and 30.6% with the 60-mg bolus, and 7.5% and 54.7% with the standard 20/60-mg infusion. At 90 minutes TIMI 2 and 3 flow improved to 9.6% and 73.1%, 15.7% and 56.9%, 13.9% and 36.1%, and 5.7% and 71.7%, respectively. The primary endpoint, persistent patency (TIMI 2 + 3) at 24-45 hours, was seen in 69.2%, 64.7%, 44.4%, and 67.9% of patients who had no rescue PTCA, respectively. Inclusion in the 60-mg bolus group was prematurely stopped because of their low patency rates. The 40/40-mg bolus group had the highest mortality rate (13.5%), whereas the 60-mg bolus group had no deaths. Other adverse event rates were similar in the four groups. This clinical outcome is highly influenced by rescue PTCA of patients with insufficient TIMI flow. This pilot study indicates that in patients with an acute myocardial infarction, a double bolus of 40/40 mg resulted in the highest patency but also had the highest complication rate. The 80-mg single bolus is an attractive alternative for further evaluation because of its acceptable patency and event profile, and its easy form of administration. PMID- 10751798 TI - Interobserver Reproducibility and Biological Variability of the Surgicutt II Bleeding Time. AB - The bleeding time is a readily and easily performed clinical test with immediate results, but there is a degree of subjectivity in its performance and interpretation. We performed a study on 27 volunteers designed to determine the normal range, interobserver reproducibility, and biological variability of the test. Bleeding times in these normal subjects ranged from as low as 129 seconds to as high as 803 seconds. The interobserver variability was 106 seconds (2 standard deviations of the mean of the differences of paired results of repeated measurements), and the coefficient of variation was 18%. For bleeding times taken on the same subjects 6 weeks apart, when the same nurse performed the test at both visits, the difference was 150 seconds (2 standard deviations of the mean of the differences of paired samples) and the coefficient of variation was 27%, and they were essentially the same if a different nurse performed the tests at each visit. There is a wide range in the bleeding times among subjects. However, within individuals there is little biological variability, and most of the difference over time is due to interobserver variability. This suggests that changes in bleeding time are clinically useful in predicting platelet responsiveness in individual patients. PMID- 10751800 TI - Treatment of Deep Vein Thrombosis with Low Molecular Weight Heparin at Patient's Home. AB - We have applied the low molecular weight heparin dalteparin as once-daily subcutaneous injections to the treatment of deep vein thrombosis (DVT) on an outpatient basis since 1994. Until today, 377 consecutive patients with DVT below the inguinal ligament have been treated at home with dalteparin. The patients administered the injections themselves or with the help of either a relative or a primary nurse. Here we report the outcome of the 212 patients treated during 1994 1995, which has been followed for 2 years after the start of treatment. At the 2 year follow-up only 13 patients (6.6%) had suffered a recurrent DVT and of these none were on continuous dicumarol treatment. No cases of major bleedings were seen. This new therapeutic approach for the treatment of DVT is safe, most of the patients are able to treat themselves, and the patients are satisfied with the home treatment model. PMID- 10751799 TI - Hemostasis Laboratory: Assays for Platelet Function and Von Willebrand Disease. PMID- 10751803 TI - Comparison of Baseline Characteristics and Outcome of Patients with Chest Pain Brought to the Hospital by Private Versus Public Mobile Intensive Care Units. AB - The baseline characteristics and outcome of patients with acute chest pain who were brought to the hospital by public versus private mobile intensive care units were compared. Of 283 patients brought to the Department of Emergency Medicine at the Tel Aviv Sourasky Medical Center by mobile units in July and September of 1995, 163 were transported by the private service and 120 by the public service. Patients who were brought by the private service were older and had significantly more comorbidities, especially cardiac history. However, a significantly higher proportion of patients brought by the public service suffered confirmed myocardial infarction, and were hospitalized in the intensive coronary care unit. In addition, mortality in this group was significantly higher (4.0% vs. 2.2%). Possible explanations for this finding are greater accessibility or bias in the prehospital management of patients channeled through the private mobile service. PMID- 10751802 TI - Effects of Intravenous Enoxaparin and Intravenous Inogatran in an Electrolytic Injury Model of Venous Thrombosis in the Dog. AB - The objective of this study was to compare the effectiveness of the low molecular weight heparin, enoxaparin (Lovenox((R))), and inogatran (a direct thrombin inhibitor) in a canine electrolytic injury model of venous thrombosis. Effectiveness was defined as the ability of either drug to prolong the following parameters: activated partial thromboplastin time (aPTT), thrombin time (TT), prothrombin time (PT), and time to formation of an occlusive thrombus in the vein. There were 5 dogs and 10 vessels for each group (the right and left femoral veins were used in each dog to measure time to occlusion). Dogs were randomly assigned to one of six groups: (1) saline controls; (2) low-dose inogatran (0.075 mg/kg IV bolus followed by a 5 ug/kg/min infusion); (3) mid-dose inogatran (0.25 mg/kg IV bolus followed by a 20 ug/kg/min infusion); (4) high-dose inogatran (0.75 mg/kg IV bolus followed by a 50 ug/kg/min infusion); (5) low-dose enoxaparin (100 units/kg IV bolus followed by a 50 U/kg/h infusion); and (6) high dose enoxaparin (200 U/kg IV bolus followed by a 100 U/kg/h infusion). Administration of inogatran resulted in dose-dependent increases in aPTT, TT, and PT, and administration of enoxaparin resulted in dose-dependent increases in aPTT and TT. There were no changes in hemodynamics. The time to occlusion in the control group averaged 81.7 +/- 9.9 minutes compared with 141.8 +/- 12.7, 185.8 +/- 17.6, and 226.9 +/- 8.8 minutes with the low, mid, and high doses of inogatran, and 131 +/- 20.3, and 183.0 +/- 19.0 minutes with the low and high doses of enoxaparin. Bleeding times were elevated by inogatran and enoxaparin, but no appreciable differences were detected between the two compounds. In summary, the direct thrombin inhibitor inogatran, administered intravenously, was as effective as the low molecular weight heparin enoxaparin in a canine model of venous thrombosis induced by electrolytic injury, supporting the conclusion that direct antithrombins may prove useful for prevention and treatment of deep venous thrombosis. PMID- 10751801 TI - Management of Acute Ischemic Coronary Syndromes: The Present and Future. AB - This article begins by discussing the pathogenesis of acute ischemic syndromes reviewing the cascade of ischemic events. We then go on to discuss the interventional management of acute ischemic syndromes and the benefits of early conservative therapy versus aggressive management with either PTCA or stenting. A discussion is then held regarding the pharmacological management of acute ischemic syndromes including the various agents involved including thrombolytic agents, aspirin, heparin and other measures. The main body of the article reviews the role of the 2B/3A inhibitors and the management of acute ischemic syndromes, including those studies that have been conducted to date, reviewing the role of the 2B/3A inhibitors and acute ischemic syndromes and the outcome from their application. PMID- 10751804 TI - Pharmacological Therapy of Cardiac Arrhythmias. PMID- 10751805 TI - Noninvasive Cardiac Imaging in Chest Pain Syndromes. PMID- 10751806 TI - Mechanical Prosthetic Valve Thrombosis: Case Report and Review of the Literature. AB - Mechanical prosthetic valve thrombosis is a life-threatening complication necessitating immediate intervention. The presenting signs and symptoms of this illness are somewhat variable, but physical examination and transesophageal echocardiography enable rapid diagnosis. To avoid catastrophic complications, valve replacement or debridement, or thrombolysis in the correct setting, must be performed without delay. It is not entirely clear which therapy is superior. For any given patient, the risks of thrombolytic therapy, including bleeding, systemic embolism, and failure to restore valvular function, must be weighed against the risks of surgical intervention. Once the decision is made to operate, the choice of valve replacement versus debridement is one best made intraoperatively, upon visual inspection of the valve apparatus. Despite aggressive therapy, morbidity and mortality from prosthetic valve thrombosis and its treatment are not trivial. Fortunately, with current prosthetic devices and aggressive prophylactic anticoagulation, the incidence of prosthetic valve thrombosis remains low. Antiplatelet therapy may offer additional benefit to patients being prophylaxed with warfarin. This report details the case of a woman with aortic and mitral prosthetic valves who presented with heart failure and evidence of severe aortic prosthetic dysfunction after a period of suboptimal anticoagulation. She successfully underwent debridement of the mitral prosthesis and replacement of the aortic valve. The relevant literature is reviewed. PMID- 10751807 TI - Stereotactic surgery in India: the past, present and the future. AB - The achievements of Indian neurosurgeons in different fields of stereotactic surgery over the past decades have been discussed. This covers diverse areas like Parkinson's disease, abnormal movements, cerebral palsy, spasticity, pain relief, and sedative and functional neurosurgery. Recently, technological advances have made stereotactic surgery useful in many fields like deep biopsies, minimally invasive surgery and radiosurgery. Apart from these areas, there is still a big scope for revival of surgery on deep structures of the brain, as was practised earlier. This will lead to newer knowledge about brain function and also give relief to many patients. The future is bright, provided Indian neurosurgeons show a paradigm shift in their thinking and bring out new ideas. Interaction with other scientific disciplines is necessary in the future if new knowledge has to be added or new techniques have to be devised. PMID- 10751808 TI - Anterior surgical approaches to the sub-axial cervical spine. AB - Anterior cervical spine surgery has come of age, as a golden route for treating anteriorly placed cervical compressions ranging from simple prolapsed disc to long segment pathologies like ossification of posterior longitudinal ligaments and cervical spondylotic myelopathy. Numerous technical modifications of the procedure are described. The role of stabilisation established for several pathologies, is still debateable in surgery for cervical disc. Bone is the ideal tissue for fusion. Hydroxyapetite implants are goods, but costly for our set up. Methylmethacrylate has a limited role in elderly patients with malignancy and a short life expectancy. Anterior cervical instrumentation has mushroomed over the last decade. Acceptable as methods of immediate stabilisation, the choice of the system varies with the surgeon. The authors use simple titanium plates with locking screws for the purpose. PMID- 10751809 TI - Centronuclear myopathy--morphological relation to developing human skeletal muscle: a clinicopathological evaluation. AB - Centronuclear myopathy (CNM), an uncommon condition, is one of the congenital myopathies. It is believed to arise as a result of maturational arrest, with persistence of myotubes postnatally. However, denervation being the basic disease process and its possible influence on central nervous system causing defect in nuclear migration has also been postulated. Keeping in view these existing controversies, we have studied 17 cases of CNM (neonatal - 1, childhood - 13, adulthood - 3) during the last twelve and a half years. Diagnosis was based on histological and enzyme histochemical findings of muscle biopsy along with clinical data. Ultrastructural characterstics of muscle have been studied in 10 cases. The affected muscle fibres showed a central nucleus (40-99%) with perinuclear halo. Type I fibre predominance with hypoplasia was consistently seen. Fibre type disproportion was noticed in 7 cases. The neonatal form revealed dense oxidative enzyme reaction product in the centre. The morphological features of CNM were compared with foetal skeletal muscles obtained at gestational ages ranging from 9 weeks - 36 weeks (n = 18). In the severe neonatal form th myofibres resembled the foetal myotubes. In the less severe childhood and adult form of CNM, aberrant organization of cytoskeletal network might have played a pathogenetic role in causing the disease. PMID- 10751810 TI - Usefulness of short term video-EEG monitoring in children with frequent intractable episodes. AB - A prospective study was done to evaluate the role of short-term Video EEG (VEEG) recording in assessing the nature of clinical behaviour and in classification of seizures in children with frequent intractable seizure episodes. Forty five children upto 12 years of age with frequent intractable seizure episodes (> or =3/week) were included in the study. VEEG was done on an outpatient basis until an event was recorded or for a minimum period of 6 hours. The events were detected in 78% of cases. The seizures were classified in all children with recorded events and seizure classification was changed in 22%. Anti-epileptic drugs could be stopped or reduced in 11%. Short term VEEG monitoring was useful in characterising events and in classification of seizures in children with frequent, intractable seizure episodes. Use of this investigation as a screening procedure for diagnosis in children with frequent episodes is stressed, thereby reducing the cost associated with prolonged VEEG recordings and disability associated with misdiagnosis of epilepsy. PMID- 10751811 TI - Motor dysfunction on the nonhemiplegic side in patients with intracerebral haemorrhage. AB - This study was undertaken to evaluate the motor dysfunction on the nonhemiplegic side in patients with acute intracerebral haemorrhage (ICH) and correlate these with radiological and motor evoked potentials findings. 28 patients (23 males, 5 females), with CT proven ICH within 10 days of ictus were subjected to clinical evaluation and central motor conduction studies (CMCT) of upper limbs. The patient's age ranged between 28 and 84 years. Motor signs on the nonhemiplegic side were present in 17 patients in the form of increased tone (10), hyperreflexia (13) and extensor plantar response (10). Sixteen of these patients had severe weakness on the hemiplegic side. Most of the patients had putaminal haemorrhage (13) and thalamic and lobar haemorrhage was seen in 2 patients each. The motor dysfunction on the nonhemiplegic side correlated with midline shift but not with the size of haematoma. CMCT findings correlated with motor signs on the nonhemiplegic side in 6 patients. In the patients without any signs on the nonhemiplegic side, CMCT was normal. Out of 17 patients with motor dysfunction on the nonhemiplegic side 9 revealed improvement in CMCT at 1 month followup. The patients with CMCT abnormalities on the nonhemiplegic side either died (2) or had poor outcome (9). Motor dysfunction on the nonhemiplegic side may be due to tentorial herniation and suggests a poor outcome. PMID- 10751812 TI - Effect of pipecuronium and pancuronium on intracranial pressure and cardiovascular parameters in patients with supratentorial tumours. AB - A prospective, randomised, single blind study was conducted to evaluate and compare the intracranial pressure (ICP) and cardiovascular effects of pipecuronium (PPC) and pancuronium (PNC) in 20 patients undergoing supratentorial surgery. Patients were randomly divided into two groups. Patients in Group I (n = 10) received pancuronium (0.1 mg kg(-1)) and in Group II (n = 10) pipecuronium (0.07 mg kg(-1)) for intubation. Intracranial pressure (ICP), heart rate (HR), systolic, diastolic and mean arterial pressures (SAP, DAP, MAP), central venous pressure (CVP), nasopharyngeal temperature and arterial blood gases (ABG) were monitored at the following time periods: before induction (0 minutes); 3 minutes after thiopentone and muscle relaxant; immediately after intubation; and 4, 6, 8, 10, 20 and 30 minutes following intubation. The rise in intracranial pressure at intubation was significantly greater in group I (21.10+/-3.97 torr, 122.59%) when compared to group II patients (1.80+/-0.70 torr, 10.04%) (p<0.0 1). Cardiovascular parameters also showed a significantly greater degree of rise in group I when compared to group II patients. Heart rate increased by 29+/-6.32 beats min(-1) (33.52%) and systolic arterial pressure by 11.60+/-7.37 torr (9.47%) in group I. These parameters did not change significantly in group II. No significant alterations were observed in the other measured parameters in either of the two groups. PMID- 10751813 TI - A mathematical outcome prediction model in severe head injury: a pilot study. AB - 103 patients of head injury, with a Glasgow coma scale (GCS) score of 8 or less, were studied prospectively. GCS score, brain stem reflexes, motor score, reaction level scale, and Glasgow Liege scale were evaluated as prognostic variables. Linear logistic regression analysis was used to obtain coefficients of these variables and mathematical formulae developed to predict outcome in individual patients. PMID- 10751814 TI - Characterization of gait parameters in patients with Charcot-Marie-Tooth disease. AB - The gait of five patients with Charcot-Marie-Tooth(CMT) disease was analyzed using light-emitting diodes and a force plate. The flexion-extension motions of the hips, knees, and ankles, as well as their moments (vector sums of forces acting at the joints) in the flexion-extension and abduction-adduction planes, were quantified. The gait of the CMT patients showed abnormalities consistent with both distal weakness (ankle dorsi- and plantar-flexors) and weakness of the hip abductor muscles. The latter weakness appeared to produce asymmetric hip moments and truncal instability in the mediolateral plane during ambulation. However, the extent to which the gait was abnormal appeared not to be exclusively related to the severity of the sensorimotor conduction deficits in the peripheral nerves. In the four patients for whom nerve conduction velocity studies were available, decrease in the lower-extremity distal conduction velocities and evoked motor amplitude potentials did not correlate with the severity and extent of the gait abnormalities. PMID- 10751815 TI - Role of emergency surgery to reduce mortality from rebleed in patients with aneurysmal SAH. AB - Potentially salvageable patients with aneurysmal subarachnoid hemorrhage may rebleed and die after admission to a hospital, awaiting an angiogram or surgery. In an attempt to reduce the number of patients thus lost, we are operating on such patients on an emergency basis. This report is a retrospective analysis of patients operated early after aneurysmal SAH, but during routine working hours, versus patients operated on an emergency basis. During an 18 month period 109 patients underwent surgery for intracranial aneurysm. Fifty two (Group 1) patients were subjected to emergency clipping of the aneurysm, and 57 (Group 2) patients underwent early clipping but during routine working hours. There was no selection bias between the two groups. Another 10 patients in Hunt and Hess Grade II and III rebled and died, after admission, awaiting an angiogram or surgery. The overall mortality for the two groups (Grade I to IV) was 11.9%. Mortality in Groups 1 and 2 was 9.6% and 14.0% respectively. At 1 month, good outcome was recorded in 86.6% patients in grade I, 69.2% patients in grade II and 42.8% patients in grade III in group I compared to 68.7% patients in grade I, 80% patients in grade II and 31.5% patients in grade III in group 2. However, the difference was not statistically significant. There was also no significant difference between the incidence of intraoperative brain swelling and delayed ischaemic neurological deficit between the two groups. Grade IV patients had a poor outcome in both the groups. Angiographic vasospasm was associated with poor out come, in either group, in grade IV patients. Patients in Grades I to III should undergo emergency surgery, if the surgeon is experienced and willing to operate at odd hours and necessary support facilities of neuroradiology and neuroanaesthesiology are available. This would avoid deaths in patients awaiting angiograms or surgery. PMID- 10751816 TI - Safety of acute normovolemic haemodilution with hydroxyethyl starch during intracranial surgery. AB - The effect of acute normovolemic haemodilution on haemodynamics, serum osmolality and coagulation parameters was studied in 20 patients undergoing intracranial surgical procedures. After induction of anaesthesia, 740+/-153 ml of blood was collected and the same was replaced with an equal volume of 6% hexaethyl starch. Heart rate (HR), blood pressure (BP), central venous pressure (CVP) and end tidal carbon dioxide tension (Et CO2) were monitored for 45 min. Haemoglobin concentration (Hb), haematocrit (Hct), serum osmolality (Osm), bleeding time (BT), prothrombin time (PT) and platelet count were determined before and 45 min after haemodilution. Hb and Hct were significantly lower following haemodilution (13.1+/-1.8 and 10.3+/-1.7 g/dL for Hb and 38.0+/-4.6%. and 30.1+/-4.5% for Hct). There was no significant change in the HR, BP and Et CO2 throughout the study period. CVP increased marginally from 35 to 45 min but was within normal limits. There was no significant change in serum osmolality, bleeding time and prothrombin time following haemodilution. Platelet count decreased following haemodilution but the values were within normal limits. The brain relaxation, as assessed by a semiquantitative scale, was satisfactory in all cases. None of the patients developed intraoperative brain swelling. In conclusion, acute normovolemic haemodilution with hexaethyl starch is tolerated well haemodynamically. It does not cause changes in serum osmolality which can increase brain oedema. It has no adverse effect on intraoperative haemostasis. It is a safe technique to decrease homologous blood transfusion during intracranial surgery. PMID- 10751817 TI - Detection of deletion in the dystrophin gene of a patient with quadriceps myopathy. AB - A 43 year old male presented with slowly progressive weakness of limbs and hypertrophy of triceps, brachioradialis and calf muscles for four years. There was thinning of quadriceps muscles in both thighs. Histological study was compatible with Becker muscular dystrophy (BMD). Genomic DNA analysis showed a deletion of the Hind III fragments, spanning exons 45-47. A junction fragment of 11.0 kb was observed along with a deletion of a 3.4 kb PstI fragment containing exon 51 in the patient, and in one of his two sisters. The clinical and laboratory characteristics in this patient are in keeping with what has been described 'quadriceps myopathy' and fall within the phenotypic variants of BMD as has been shown by others. PMID- 10751818 TI - Pituitary adenoma and parasagittal meningioma: an unusual association. AB - Simultaneous detection of an intracranial meningioma with a pituitary tumour prior to radiotherapy is an extremely uncommon occurrence. Authors have managed an elderly acromegalic lady with an acidophilic pituitary adenoma, who also harboured an asymptomatic anterior third parasagittal meningioma. There were no features of neurofibromatosis. Both tumours were concurrently excised. PMID- 10751819 TI - Intramedullary spinal epidermoid cyst. AB - Intramedullary epidermoid cysts of the spinal cord are rare tumours, especially those not associated with spinal dysraphism. Around 50 cases have been reported in the literature. Of these, only seven cases have had magnetic resonance imaging studies. We report two cases of spinal intramedullary epidermoid cysts with MR imaging. Both were not associated with spina bifida. In one patient the tumour was located at D4 vertebral level, while in other within the conus medullaris. The clinical features, MR imaging characteristics and surgical treatment of such rare intramedullary benign tumours are discussed, and the relevant literature reviewed. PMID- 10751820 TI - Episodic ataxia: a case report and review of literature. AB - This report describes the clinical features of a 29 year female presenting with a 3 years history of episodes of cerebellar ataxia, dysarthria and nystagmus lasting 3-5 days, recurring almost every month. Sleep disturbance and buzzing in ears were noted 3-4 days before each episode. No other precipitant factor was present. Family history was negative. She was diagnosed as a case of episodic ataxia type-2 and was successfully treated with acetazolamide, a carbonic anhydrase inhibitor. She was asymptomatic at 2 year followup. PMID- 10751821 TI - Retrocerebellar arachnoid cyst with syringomyelia: a case report. AB - Association of syringomyelia with retrocerebellar arachnoid cysts is rare. A case of 14 year old female is being reported, who presented with hydrocephalus caused by a large midline retrocerebellar infravermal arachnoid cyst leading to obstruction of the outlet foramina of the fourth ventricle. There was associated syringomyelia. The pathogenesis of syringomyelia is discussed. The need to evaluate cervical spinal cord by taking T1 weighted sagittal sections in all the patients of large posterior fossa mass lesions causing obstruction to the outlet foramina of the fourth ventricle has been stressed, in order to detect associated syringomyelia. PMID- 10751822 TI - Acute alcoholic myopathy, rhabdomyolysis and acute renal failure: a case report. AB - A case of middle aged male who developed swelling and weakness of muscles in the lower limbs following a heavy binge of alcohol is being reported. He had myoglobinuria and developed acute renal failure for which he was dialyzed. Acute alcoholic myopathy is not a well recognized condition and should be considered in any intoxicated patient who presents with muscle tenderness and weakness. PMID- 10751823 TI - Enlarging single CT lesions can also spontaneously resolve. AB - Computed tomography in two patients, aged 9 and 14 years, with history of focal seizures, revealed single, small, enhancing CT lesions. These patients were treated with albendazole and anticonvulsants. Follow-up CT scans revealed an increase in the size of the solitary lesions. They were managed conservatively and further follow-up CT scans revealed complete resolution of the lesions. The report suggests that some enlarging CT lesions may also spontaneously resolve. The most likely cause of the enlarging lesions was albendazole therapy. PMID- 10751824 TI - A massive extradural haematoma at the vertex. PMID- 10751825 TI - Shunt migration into a Dandy Walker cyst. PMID- 10751826 TI - Malaria in epileptics--an additional hazard. PMID- 10751827 TI - Systemic lupus erythematosus causing cerebral infarction and basal ganglion calcification: a case report. PMID- 10751828 TI - Tuberculous osteomyelitis of the bone flap following craniotomy for a glioma. PMID- 10751829 TI - Fracture occipital condyle with isolated 12th nerve paresis. PMID- 10751831 TI - Spinal cord tuberculoma and misleading MR imaging. PMID- 10751830 TI - Ectopic meningioma: a case report. PMID- 10751833 TI - Medically refractory epilepsy PMID- 10751832 TI - Pontine and extrapontine myelinolysis following rapid correction of hyponatremia. PMID- 10751834 TI - Epilepsy : the indian perspective PMID- 10751835 TI - Update on Botulinum Toxin. AB - Botulinum toxin type-A (BTX-A) is a neurotoxin which blocks presynaptic release of acetylcholine. It interferes with neuromuscular transmission, temporarily paralyzing the affected muscle. Of special interest for dermatologists is the unlabelled cosmetic applications, for conditions such as wrinkles and hyperhidrosis. Labelled indications in Europe are for cervical dystonia and cerebral palsy. In the US, it is approved for treatment of strabismus, blepharospasm and hemifacial spasm in adults. After repeated use of high doses, antibodies can develop in some individuals, making further treatment ineffective indefinitely. Even when used in high does for neurological conditions, the development of antibodies occurs in < 5&percnt of patients. In 1997, the US FDA approved a new bulk toxin source for use in the manufacture of BTX-A. It has a higher specific potency than original BTX-A formulations, reducing the amount of utilized neurotoxin protein, and thereby reducing antibody production. Another form of this neurotoxin (type B) also appears to be effective in patients who have developed antibodies to BTX-A. It is awaiting US FDA approval for treatment of cervical dystonia. PMID- 10751836 TI - N-2-butylcyanoacrylate (GluStitch). AB - Cyanoacrylates are surgical adhesives that provide another option for wound closure. When compared to sutures, cyanoacrylates were found to be as effective as sutures in low tension lacerations and for the attachment of some full thickness skin grafts. In addition, clinical practitioners have found cyanoacrylates easier to apply, time saving, and more economical. There are a number of surgical adhesives either currently available or under development. The presently available butylcyanoacrylates and octylcyanoacrylates are reviewed. PMID- 10751837 TI - Soft Tissue Augmentation With Silicone. AB - Adatosil is a viscous silicone oil which was granted FDA approval in 1994. This designation removes the legal obstacles to usage for soft tissue augmentation. However, physicians are advised to exercise discretion when using this product. There are several caveats which govern its use, including several contraindications, as well as difficult technical application. PMID- 10751838 TI - New Corticosteroids. AB - Corticosteroids have dominated the class of anti-inflammatory agents for the past 50 years. In the last ten years, seven new corticosteroids have been developed for topical use. Characteristics common to these several chemically different corticosteroids are their class III, or high potency (USP) designation and their improved safety profile. Allergic contact dermatitis is an unexpected adverse effect that is caused by some of them, in particular budesonide and, to a somewhat lesser degree, the labile "prodrug" corticosteroids, such as prednicarbate and prednisone acepontate. PMID- 10751839 TI - Electrolysis for the treatment of hypertrichosis and hirsutism. AB - There are three electrolysis modalities. In galvanic electrolysis, a direct current is passed down a needle inserted into the hair follicle, destroying the follicle. In thermolysis a high frequency alternating current is passed down the needle and produces destructive heat. The blend is the third modality, which combines galvanic electrolysis and thermololysis. Electrolysis satisfactorily removes hair from women and men with hypertrichosis, but women with hirsutism require concomitant hormonal management. Scarring does not occur with properly performed electrolysis. Shaving one to five days before electrolysis greatly increases efficacy because it ensures that only growing anagen hairs are epilated. The judicious use of ice packs and the recent availability of EMLA (lidocaine/prilocaine cream) have been beneficial in reducing the sensations of electrolysis. PMID- 10751840 TI - Current treatments for scabies and pediculosis. AB - Scabies and lice have afflicted man since ancient times. Permethrin is generally the treatment of choice for head lice and scabies, because of its residual effect. Toxicity and absorption are minimal. Ivermectin should be reserved for cases where permethrin fails. PMID- 10751841 TI - Update on topical acne treatments. AB - Topical acne treatment can positively benefit patients with acne. This review summarizes clinical and prescribing information on currently available topical agents. The efficacy of the medications included in this report is supported by properly designed randomized clinical trials. PMID- 10751842 TI - Topical antipsoriatics. AB - Topical corticosteroids are important in psoriasis therapy. However, there are other worthwhile options available including tar, anthralin, tazarotene, calcipotriol, topical PUVA, and topical porphyrin derivatives. With growing public reluctance to use systemic medications, topical treatments for psoriasis could become increasingly important in the future. PMID- 10751843 TI - Treatment options for localized scleroderma. AB - Localized scleroderma, or morphea, is a chronic disease that causes a thickening and induration of the skin. For plaque type morphea, the treatments of choice include super-potent corticosteroids and calcipotriol. For the more generalized forms, as well as the linear forms, UVA is currently the best therapeutic modality. Patients with localized scleroderma are managed by both rheumatologists and dermatologists. There is still much therapeutic uncertainty in this disease. PMID- 10751845 TI - Drug treatments for skin disease introduced in 1999 PMID- 10751844 TI - Synopsis of laser assisted hair removal systems. AB - Conventional treatment options for hypertrichosis and hirsutism are tedious and time consuming. Laser hair removal offers an efficient way to permanently reduce excessive hair growth. Hair is damaged using the principle of selective photothermolysis with wavelengths of light well absorbed by follicular melanin and pulse durations that selectively thermally damage the target without damaging surrounding tissue. Patients with dark hair and light skin are ideal candidates. Multiple treatments (3 to 6) performed every 6-8 weeks are necessary to achieve a permanent reduction of hair growth. As the field develops, a better sense of the effectiveness of laser hair removal will evolve and reasonable expectations will be determined. PMID- 10751846 TI - Saw palmetto for the treatment of men with lower urinary tract symptoms. AB - PURPOSE: A comprehensive review of the literature on the use of saw palmetto in men with lower urinary tract symptoms is provided. MATERIALS AND METHODS: A literature search of studies that have assessed the mechanism of action and clinical results of saw palmetto in men with benign prostatic hyperplasia was performed. RESULTS: A variety of potential mechanisms of action of saw palmetto have been demonstrated through in vitro studies, including 5-alpha reductase inhibition, adrenergic receptor antagonism and intraprostatic androgen receptor blockade. Clinical evidence of the relevance of these effects is largely unavailable. The use of saw palmetto in men with benign prostatic hyperplasia is safe with no recognized adverse effects. No effect on serum prostate specific antigen has been noted. Placebo controlled trials and meta-analyses have suggested that saw palmetto leads to subjective and objective improvement in men with lower urinary tract symptoms. However, most studies are significantly limited by methodological flaws, small patient numbers and brief treatment intervals. CONCLUSIONS: Evidence suggests that saw palmetto may have a significant effect on urinary flow rates and symptom scores compared to placebo in men with lower urinary tract symptoms. However, large scale, placebo controlled trials are needed to assess the efficacy of saw palmetto. PMID- 10751847 TI - The effect of cystectomy, and perioperative methotrexate, vinblastine, doxorubicin and cisplatin chemotherapy on the risk and pattern of relapse in patients with muscle invasive bladder cancer. AB - PURPOSE: Trials have demonstrated decreased relapse with perioperative methotrexate, vinblastine, doxorubicin and cisplatin (M-VAC) chemotherapy in patients with muscle invasive bladder cancer. We evaluated whether the benefit of chemotherapy correlates with its effects on distant or pelvic relapse. MATERIALS AND METHODS: We retrospectively evaluated the records of all 107 patients who underwent cystectomy for muscle invasive bladder cancer at our institution between 1988 and 1994. Factors predicting relapse were identified and used to group patients at high or low risk. The outcome in each group with and without M VAC chemotherapy was then analyzed in terms of overall, metastatic and pelvic relapse. Univariate analysis was performed using the Kaplan-Meier method and log rank statistic, and multivariate analysis was done using the Cox proportional hazards model. Median survival was 29 months for patients free of disease. RESULTS: Pathological stage T3 or greater according to the American Joint Committee on Cancer, tumor greater than 3 cm. and creatinine greater than 1.5 fold normal were independent poor prognostic factors in patients treated with cystectomy only. Patients with any of these factors or metastatic involvement of the pelvic lymph nodes were considered at high risk. All 35 low risk patients were treated with cystectomy only and had an excellent outcome with a 3-year relapse-free survival plus or minus standard error of 93% +/- 5%. The 3-year rates in 52 and 20 high risk patients treated without and with chemotherapy, respectively, were 42% +/- 8% versus 57% +/- 13% for relapse-free survival (p = 0.17), 38% +/- 9% versus 8% +/- 8% for pelvic failure (p = 0.02) and 39% +/- 9% versus 38% +/- 13% for distant metastases (not significant). Multivariate analysis of patients who underwent pelvic lymphadenectomy revealed that perioperative chemotherapy improved relapse-free survival and pelvic control but not metastatic control (p = 0.03, 0.02 and 0.31, respectively). CONCLUSIONS: Low risk patients have excellent disease control when treated with cystectomy only. Those with high risk features are at substantial risk for pelvic failure (38% at 3 years) after cystectomy only. Perioperative M-VAC chemotherapy has a profound impact on pelvic but not on metastatic failure. PMID- 10751848 TI - Medical treatment of cystinuria: critical reappraisal of long-term results. AB - PURPOSE: We evaluated long-term results of a contemporary medical therapeutic regimen in patients with cystinuria and analyzed factors predictive of therapeutic success. MATERIALS AND METHODS: A total of 27 adults with cystine urolithiasis were treated at our institution for 1.3 to 32 years (mean 11.6, overall 312 patient-years). We obtained data on the pre-referral period for 274 patient-years overall. Basic therapy included hyperdiuresis and alkalization. The thiols D-penicillamine or tiopronin were added when standard therapy failed to prevent new stones and stone growth or dissolve preexisting stones. X-ray and echography were performed every 4 months during the initial 2 years and every 6 months thereafter. RESULTS: In the pre-referral period 256 stone episodes occurred and 81 urological procedures were performed in 24 patients (0.93 and 0. 29 per patient-year, respectively). Nine patients were treated with added thiols. During the therapeutic period the incidence of stone episodes decreased to 66 (0.20 per patient-year, p <0.001), while the need for urological procedures decreased to 44 (0.14 per patient-year, p <0.001). No further urological procedures were required in 15 patients, including 4 treated with thiols. However, the remaining 12 patients, including 5 treated with thiols, underwent 1 to 7 procedures each (mean 0.26 per patient-year). In the 2 groups mean daily cystine excretion plus or minus standard deviation at baseline (863 +/- 253 versus 761 +/- 270 mg. daily) and mean urinary pH of about 7.4 did not differ significantly. However, daily urine volume was significantly higher in patients with arrested stone formation (3,151 +/- 587 versus 2,446 +/- 654 ml./24 hours, p = 0.006). CONCLUSIONS: Our study provides evidence that a regularly followed medical program based on high diuresis and alkalization with second line addition of thiols may arrest or markedly decrease cystine stone formation and preclude the need for urological procedures in more than half of the patients. However, patients poorly compliant with hyperdiuresis remain at risk for recurrence. We suggest that maintaining a daily urine volume of greater than 3 l. is essential for therapeutic success regardless of whether thiol derivatives are administered. PMID- 10751849 TI - A phase 2 study of radio frequency interstitial tissue ablation of localized renal tumors. AB - PURPOSE: Small renal tumors are frequently detected during the screening of patients with a hereditary type of renal cancer. The development of nonsurgical treatment modalities would greatly improve quality of life in these patients. We present our experience with radio frequency interstitial tissue ablation, a heating device approved by the Food and Drug Administration for treating soft tissue tumors. MATERIALS AND METHODS: Patients underwent radio frequency interstitial tissue ablation of small renal tumors just before surgical excision. Pathological examination of the renal tumors was done to evaluate the treatment effect. Computerized tomography and renal function testing were performed before and after therapy to evaluate toxicity. RESULTS: Four patients underwent treatment of a total of 14 tumors with the radio frequency interstitial tissue ablation device just before surgical removal of the tumors. All lesions were brown after ablation, in contrast to the normal pink appearance of untreated lesions that were resected. On color Doppler ultrasound blood flow to each tumor evident before was not visualized after treatment. The Wilcoxon rank sum test demonstrated no difference preoperatively and postoperatively in blood urea nitrogen, serum creatinine, creatinine clearance or differential renal function. We identified no toxicity associated with radio frequency interstitial tissue ablation. Of the excised tumors 11 were renal cell carcinoma and 3 fibrotic hemorrhagic cysts. For renal cell carcinoma the treatment effect involved the loss of nuclear detail and nonvisualization of nucleoli. These changes were not observed in any tumors resected without radio frequency interstitial tissue ablation. The treatment effect was noted in 10 of the 11 lesions, and in 1 case the treatment effect involved 35% of the tumor. CONCLUSIONS: No toxicity was associated with radio frequency interstitial tissue ablation. Percutaneous treatment of renal tumors is planned to evaluate the treatment effect better and further evaluate toxicity. PMID- 10751850 TI - Simplified technique with short and long-term followup of conversion of an ileal conduit to an Indiana pouch. AB - PURPOSE: We report a simplified technique for converting an existing conduit to an Indiana pouch as well as short and long-term results. MATERIALS AND METHODS: From May 1988 to February 1998 we evaluated short and long-term outcome and complications in 23 patients 14 to 82 years old (average age 51.8) who underwent conversion of a conduit to an Indiana pouch. When no obstruction of the existing ureteroileal anastomoses was identified, the conduit was freed from the abdominal wall and surrounding bowel. The proximal conduit and ureteral anastomoses were not dissected. The conduit was opened along the antimesenteric wall proximal to the ureteral anastomoses and attached to 25 to 28 cm. of detubularized right colon as a refluxing Studer limb. The pouch was completed in the usual fashion and the stoma was matured at a virgin site. RESULTS: Surgical indications included stomal complications in 10 patients, an infected nonfunctioning kidney in 2 and patient preference in 11. There were no perioperative deaths although 3 patients died of cancer progression. Average operative time was 6.6 hours, estimated blood loss 518 cc and length of stay 7.8 days. Average followup after conversion was 4.7 years (range 0.2 to 11.0). Six late complications developed in 4 cases, including pyelonephritis in 2, severe pouchitis in 1, dehydration in 1 and stomal revision in 2. Renal function was well preserved with an average preoperative and postoperative creatinine of 0.91 and 1.14 mg./dl., respectively. CONCLUSIONS: This technique simplifies conversion and decreases bowel requirements. The low complication rate and stable serum creatinine support the finding that conversion of a conduit to an Indiana pouch is a safe, viable procedure. PMID- 10751851 TI - Urinary diversion. PMID- 10751852 TI - A prospective study of interstitial cystitis: results of longitudinal followup of the interstitial cystitis data base cohort. The Interstitial Cystitis Data Base Study Group. AB - PURPOSE: We present baseline characteristics and longitudinal profiles of symptoms in the Interstitial Cystitis Data Base study, a prospective cohort study of patients with interstitial cystitis. MATERIALS AND METHODS: A total of 637 eligible patients were entered into the study and followed for symptoms of pain, urgency and urinary frequency. Median followup was 31 months. RESULTS: More than 90% of patients were white women with a median age of 43 years. Using the overall pain-urgency-frequency score 7% of participants presented with mild, 44% with moderate and 49% with severe symptoms. Severe urgency in 41% of cases and severe 24-hour frequency in 41% were more common than severe pain in 29%. Of the patients 51% reported nighttime frequency of 2 or more voids. Median duration of interstitial cystitis symptoms was 8 years and 68% of participants were previously diagnosed with the condition. The 36% of patients who withdrew from study or were lost to followup were more likely to have had more severe symptoms at baseline. Patterns of change with time suggest initial symptom improvement due to regression to the mean, and an intervention effect associated with the increased followup and care of cohort participants. Although all symptoms fluctuated, there was no evidence of significant long-term change in overall disease severity. CONCLUSIONS: Our observations support the clinical observation that interstitial cystitis is a chronic disease and no current treatments have a significant impact on symptoms with time. These results provide a foundation for the design and performance of future clinical trials in interstitial cystitis using these end points in a similar patient population. PMID- 10751854 TI - Application of pericardial graft in the surgical management of Peyronie's disease. AB - PURPOSE: We investigated the use of Tutoplast cadaveric pericardium as an alternative material for grafting the tunica albuginea defect after Peyronie's plaque excision. MATERIALS AND METHODS: A total of 11 patients with significant penile curvature interfering with sexual intercourse were evaluated after at least 12 months of conservative therapy. Preoperative evaluation in all cases included penile duplex Doppler ultrasound studies. Chemically processed and gamma irradiated pericardium was used to graft the cavernosal defect after surgical excision of the penile plaque. In 3 patients a penile prosthesis was simultaneously placed secondary to documented erectile dysfunction at duplex Doppler ultrasound evaluation. RESULTS: Penile curvature resolved in all patients, allowing for normal sexual function after a mean followup of 14 months (range 9 to 19). No postoperative evidence of tissue rejection or infection was noted. In 1 patient urethro-corporal communication developed and a single penile cylinder was explanted. Another patient had a mild dorsal bulge at the site of graft placement when the prosthesis was fully inflated. CONCLUSIONS: Polymeric silicone material used to provide satisfactory results in the surgical treatment of Peyronie's disease but it is no longer available for biomedical use. We demonstrate the effective use of cadaveric pericardium as a graft material for cavernosal defects after Peyronie's plaque excision. Overall patient satisfaction, absence of a second surgical incision, relative low risk and ease of surgical placement make this material ideal for the surgical treatment of this disease process. PMID- 10751853 TI - Bladder stretch alters urinary heparin-binding epidermal growth factor and antiproliferative factor in patients with interstitial cystitis. AB - PURPOSE: The etiology of interstitial cystitis is unknown. Urine from patients with interstitial cystitis has been shown to inhibit urothelial proliferation through a putative antiproliferative factor and to contain decreased levels of heparin-binding epidermal growth factor-like growth factor (HB-EGF) compared to controls. Stretch of detrusor smooth muscle cells is known to stimulate HB-EGF production. Because bladder hydrodistention sometimes alleviates the symptoms of interstitial cystitis, we determined whether the stretch stimulus of hydrodistention alters antiproliferative factor activity and/or HB-EGF in interstitial cystitis urine specimens. MATERIALS AND METHODS: Urine was collected immediately before, and 2 to 4 hours and 2 weeks after hydrodistention from 15 patients with symptoms and cystoscopic findings compatible with interstitial cystitis and 13 controls. Hydrodistention was performed with the subject under general or regional anesthesia and bladders were distended to 80 cm. water 3 times. Urinary HB-EGF was measured by enzyme-linked immunosorbent assay and urinary antiproliferative factor activity was determined by measuring 3H thymidine uptake by normal human bladder urothelial cells. RESULTS: Hydrodistention significantly increased urinary HB-EGF in patients with interstitial cystitis toward normal control values (before distention p = 0.003, 2 weeks after distention p = 0.67). Urine antiproliferative factor activity decreased significantly after hydrodistention in patients with interstitial cystitis. However, antiproliferative factor activity in interstitial cystitis and control specimens was still statistically different 2 weeks after distention (before distention p = 0.0000004, 2 weeks after distention p = 0.04). CONCLUSIONS: Bladder stretch increased HB-EGF and conversely reduced antiproliferative factor activity in urine from patients with interstitial cystitis but not controls up to 2 weeks after distention. These results provide additional evidence for the possible role of antiproliferative factor and decreased HB-EGF in the pathophysiology of interstitial cystitis. To our knowledge this is also the first human study to show that in vivo bladder stretch can alter urinary factors that regulate cell growth. PMID- 10751855 TI - The ultrasonic description of postpubertal testicles in men who have undergone prepubertal orchiopexy for cryptorchidism. AB - PURPOSE: We provide a sonographic description of postpubertal testicles in patients who have undergone prepubertal orchiopexy with placement of a suture through the tunica albuginea. MATERIALS AND METHODS: Testicular ultrasound was performed in men who had undergone prepubertal testicular fixation for cryptorchidism using a suture passed through the tunica albuginea. Comparison was made to the sonographic appearance of cryptorchid testicles not secured with a fixating suture through the tunica albuginea. The operative report for each patient was reviewed with specific attention to use and type of suture material, and age at orchiopexy. RESULTS: Ultrasonography was performed on 22 men in whom a tunica albuginea fixating suture was placed during orchiopexy for cryptorchidism. Average patient age at orchiopexy was 5.6 years and median time from orchiopexy to this examination was 14.8 years. Right cryptorchidism existed in 64% of the men. No significant difference was noted in size between the 2 testicles in any patient. Ultrasonography did not identify any parenchymal abnormalities. A tunica albuginea calcification with posterior shadowing on the side of fixation was noted in 32% of patients, of whom 3 had calcified areas evident on physical examination. An area of subtunical hypoechogenicity was noted in 14% of patients. All tunical abnormalities were associated with the use of chromic suture. The remaining 12 patients (54%) had normal sonographic and physical examinations. The control group comprised 6 men who underwent orchiopexy without a fixating suture through the tunica albuginea. These men were examined identically and no sonographic abnormalities were noted in the tunica albuginea or testicular parenchyma. At a median of 16 months no patient had been diagnosed with testicular cancer or had a change in testicular self-examination. CONCLUSIONS: Tunica albuginea calcifications and hypoechogenic cysts are common ultrasonic findings in men who have undergone orchiopexy using a suture passed through the tunica albuginea. Parenchymal lesions should not be considered secondary to this procedure and must be treated as de novo abnormalities. These changes seem to be induced by the use of a fixating suture through the tunica albuginea. PMID- 10751856 TI - Effects of a saw palmetto herbal blend in men with symptomatic benign prostatic hyperplasia. AB - PURPOSE: We tested the effects of a saw palmetto herbal blend in men with symptomatic benign prostatic hyperplasia (BPH) via a randomized, placebo controlled trial. MATERIALS AND METHODS: We randomized 44 men 45 to 80 years old with symptomatic BPH into a trial of a saw palmetto herbal blend versus placebo. End points included routine clinical measures (symptom score, uroflowmetry and post-void residual urine volume), blood chemistry studies (prostate specific antigen, sex hormones and multiphasic analysis), prostate volumetrics by magnetic resonance imaging, and prostate biopsy for zonal tissue morphometry and semiquantitative histology studies. RESULTS: Saw palmetto herbal blend and placebo groups had improved clinical parameters with a slight advantage in the saw palmetto group (not statistically significant). Neither prostate specific antigen nor prostate volume changed from baseline. Prostate epithelial contraction was noted, especially in the transition zone, where percent epithelium decreased from 17.8% at baseline to 10.7% after 6 months of saw palmetto herbal blend (p <0.01). Histological studies showed that the percent of atrophic glands increased from 25. 2% to 40.9% after treatment with saw palmetto herbal blend (p <0.01). The mechanism of action appeared to be nonhormonal but it was not identified by tissue studies of apoptosis, cellular proliferation, angiogenesis, growth factors or androgen receptor expression. We noted no adverse effects of saw palmetto herbal blend. When the study was no longer blinded, 41 men elected to continue therapy in an open label extension. CONCLUSIONS: Saw palmetto herbal blend appears to be a safe, highly desirable option for men with moderately symptomatic BPH. The secondary outcome measures of clinical effect in our study were only slightly better for saw palmetto herbal blend than placebo (not statistically significant). However, saw palmetto herbal blend therapy was associated with epithelial contraction, especially in the transition zone (p <0.01), indicating a possible mechanism of action underlying the clinical significance detected in other studies. PMID- 10751857 TI - High energy transurethral microwave thermotherapy for the treatment of patients in urinary retention. AB - PURPOSE: We evaluated the efficacy of high energy transurethral microwave thermotherapy for treating urinary retention due to benign prostatic hyperplasia. MATERIALS AND METHODS: Between October 1993 and March 1999, 41 patients with urinary retention were treated with high energy transurethral microwave thermotherapy. Initial evaluation consisted of a history, clinical examination, urethrocystoscopy, transrectal prostate ultrasonography and urodynamic investigation with a pressure flow study. Followup visits at 12, 26 and 52 weeks included International Prostate Symptom Score (I-PSS), uroflowmetry and post-void residual urine volume determination. At 26 weeks the urodynamic study was repeated. Kaplan-Meier plots were constructed to evaluate the risk of re treatment adjusted for patients lost to followup. RESULTS: At baseline median patient age was 74 years and median prostate size was 67 ml. Median 133 kJ. were administered. Nine patients underwent re-treatment and 11 were lost to followup. The re-treatment rate after 1 year was 25% (95% confidence interval 11 to 40). In good responders at 12, 26 and 52 weeks median maximal urine free flow was 15, 11 and 15 ml. per second, post-void residual urine volume was 61, 8 and 35 ml., and I-PSS was 7, 5 and 2, respectively. Quality of life (I-PSS question 8) was 1 at all visits. CONCLUSIONS: In patients with severe co-morbidity transurethral microwave thermotherapy is often the only alternative to an indwelling catheter with obvious quality of life advantages. High energy transurethral microwave thermotherapy appears to be effective in these patients. PMID- 10751858 TI - Differences between urologists in the United States and Canada in the approach to prostate cancer. AB - PURPOSE: We examine differences in screening, detection, staging and treatment of prostate cancer between urologists in the United States and Canada. MATERIALS AND METHODS: An anonymous questionnaire was developed and mailed to 700 randomly selected American and 350 Canadian urologists. The 7 domains of prostate cancer management comprised screening/case identification, radical prostatectomy indications, staging evaluations, neoadjuvant therapy, nerve sparing techniques, postoperative management and treatment of biochemical recurrence. The Dillman method of questionnaire administration was used. All data were stratified by country and analyzed using the generalized chi-square test. RESULTS: Surveys were adequately completed by 45% and 79% of American and Canadian urologists, respectively. Practice experience and clinical volumes were not significantly different between the 2 cohorts. Overall, there were few differences in prostate cancer screening, staging, postoperative management, biochemical failure and use of neoadjuvant therapy. However, practicing American urologists tended to pursue more aggressive case finding practices, such as a higher age cutoff for prostate specific antigen testing (p = 0.0001) and more frequent use of transition zone biopsies (p = 0.0001). American urologists also displayed a tendency toward more liberal indication for extirpative surgery. They were more likely to perform radical prostatectomy in men older than 70 years, those with higher prostate specific antigen and those with node positive disease. Among both national cohorts there was considerable variation in management patterns for all domains of prostate cancer. Variation was most common among treatment of patients with adverse pathological conditions (positive margins, seminal vesicle involvement) and postoperative biochemical failure. Even when credible evidence exists (biopsy technique, preoperative staging) significant proportions of urologists in both countries continued to practice contrary to existing data. CONCLUSIONS: American and Canadian practice patterns for prostate cancer differ significantly only in the domains of case identification and surgical indications. In addition, considerable intra-national variation in practice patterns exists. These data highlight the necessity to support randomized clinical trials in prostate cancer. PMID- 10751859 TI - Percent free prostate specific antigen and cancer detection in black and white men with total prostate specific antigen 2.5 to 9.9 ng./ml. AB - PURPOSE: The ratio of free-to-total prostate specific antigen (PSA), or percent free PSA, is a useful adjunct to total PSA for estimating the risk of prostate cancer when total PSA is 2.5 to 9.9 ng./ml. Relationships between cancer detection and total PSA are influenced by race but to our knowledge relationships between cancer detection and percent free PSA have not been studied. MATERIALS AND METHODS: A total of 222 black and 298 white consecutive and evaluable men with total PSA 2.5 to 9.9 ng./ml. underwent prostate biopsy for suspected cancer at a Veterans Affairs Medical Center. Clinical measurements included digital rectal examination, total and free serum PSA, prostate volume, PSA density and Gleason score of malignant biopsy specimens. RESULTS: Median percent free PSA was 14.1 (range 3.6 to 49.2) in 201 men with prostate cancer and 21.9 (range 5.7 to 83.3) in 319 without detectable cancer (p <0.0001). Significant racial differences in demographic characteristics and clinical measurements were limited to total PSA, which was higher in black men (p = 0.03). Cancer was detected in 156 black (47%) and 206 white (33%) men (p = 0.001). Areas under receiver operating characteristics curves for percent free PSA and total PSA were 0.66 and 0.58, respectively, for black men (p = 0.15), and 0.76 and 0.58, respectively, for white men (p <0.00001). Percent free PSA was 35.2 in black men and 29.2 in white men, and specificity was 9.1% and 28.7%, respectively, when sensitivity for percent free PSA was set at 95%. Of 156 black and 206 white men with percent free PSA less than 25, 83 (53%) and 85 (41%), respectively, had detectable cancer (p = 0.03). Of 66 black and 92 white men with percent free PSA 25 or greater 21 (32%) and 12 (13%), respectively, had detectable cancer (p = 0.005). CONCLUSIONS: Our study demonstrates racial differences in relationships between percent free PSA and cancer detection in men with suspected prostatic carcinoma and total PSA 2.5 to 9.9 ng./ml. Clinical application of the commonly used percent free PSA cutoff of less than 25 to determine the advisability of prostate biopsy may lead to under diagnosis of early stage prostate cancer in black men, who are at greater risk of morbidity and mortality from disease than white men. PMID- 10751860 TI - Positive and negative biopsies in the pre-prostate specific antigen and prostate specific antigen eras, 1980 to 1997. AB - PURPOSE: We assess temporal trends in prostate biopsy incidence, utilization and cancer yield in the community before and after the introduction of serum prostate specific antigen (PSA) to the community medical practice MATERIALS AND METHODS: Study subjects comprised all Olmsted County men with a first prostate biopsy performed between January 1, 1980 and December 31, 1997. Medical records of all study subjects (1,729) were reviewed for clinical information from the first and all subsequent biopsies. RESULTS: Annual age adjusted prostate biopsy incidence increased from 113/100, 000 (95% confidence interval 76, 150) in 1980 to 487/100,000 (421, 554) in 1992 and decreased to 264/100,000 (219, 309) in 1997. For men 50 to 59 years old biopsy incidence increased 400% from 137/100, 000 in 1980 to 1986 to 686/100,000 in 1993 to 1997. Overall, there were 93/100,000 more negative biopsies and 49/100,000 more positive biopsies in 1993 to 1997 than in 1980 to 1986. The overall cancer yield of 36% was essentially unchanged across periods (p = 0.6). However, by age cancer yield decreased from 29% to 21% (1980 to 1986 versus 1993 to 1997) for men 50 to 59 years old but increased from 38% to 45% for those 70 to 79 years old. CONCLUSIONS: Overall cancer yield from prostate biopsies has changed little during the last 15 years. Increased cancer yield for men 70 years old or older has been offset by the decreased yield in younger men. Attention must now be given to diagnostic techniques which might reduce the incidence of negative biopsies and improve cancer yield in younger men. PMID- 10751861 TI - Complexed prostate specific antigen provides significant enhancement of specificity compared with total prostate specific antigen for detecting prostate cancer. AB - PURPOSE: Determining serum total prostate specific antigen (PSA) has proved to be a valuable diagnostic aid for detecting prostatic carcinoma, although the lack of specificity has limited its usefulness. Studies indicate that the use of percent free PSA would improve specificity while maintaining sensitivity. Since complexed PSA represents the major proportion of measurable PSA in serum, we determined whether it represents a single test alternative to the use of percent free PSA for the early detection of prostate cancer. MATERIALS AND METHODS: Archival serum was obtained from 385 men with no evidence of malignancy on biopsy and 272 with biopsy confirmed prostate cancer. We determined the concentration and proportion of total, complexed and free PSA. RESULTS: Receiver operating characteristics analysis using total PSA results from all samples (range 0.32 to 117 ng./ml.) indicated that the areas under the curve for complexed PSA alone as well as the free-to-total and complexed-to-total PSA ratios were similar and significantly greater than those for total PSA alone. Within the range of 85% to 95% sensitivity receiver operating characteristics analysis revealed that the specificity of complexed PSA was higher than that of total PSA and equivalent to that of the free-to-total PSA ratio. We noted a similar improvement in specificity in the 4 to 10 ng./ml. total PSA range. Using published cutoff values for complexed, total and percent free PSA when total PSA was in the 4 to 10 ng./ml. range the sensitivity and specificity of complexed and percent free PSA were similar. Within the 4 to 10 ng./ml. total PSA range the population of patients with no evidence of malignancy and complexed PSA below the upper limit was different with respect to total PSA from that with no evidence of malignancy and free PSA greater than 25%. CONCLUSIONS: The measurement of complexed PSA represents an alternative to the use of percent free PSA, although the patient populations identified by the 2 tests are different. PMID- 10751862 TI - Prostate specific antigen response to mitoxantrone and prednisone in patients with refractory prostate cancer: prognostic factors and generalizability of a multicenter trial to clinical practice. AB - PURPOSE: We determine prostate specific antigen (PSA) response and durability, and prognostic factors associated with response and survival in patients with symptomatic hormone refractory prostate cancer treated with mitoxantrone and prednisone at a single institution. We then compare the results with those of a randomized phase III clinical trial. MATERIALS AND METHODS: A retrospective review of all 133 patients treated with mitoxantrone and prednisone at Princess Margaret Hospital since 1994 was performed. PSA response and duration, and overall survival were determined as well as the influence of baseline factors on these outcome parameters. Results were compared to those for patients randomized to receive mitoxantrone and prednisone in the Canadian clinical trial which demonstrated palliative benefit of this regimen. RESULTS: Patients treated after trial closure had shorter survival (p = 0.003) but represented a poorer prognosis cohort. PSA response of the trial and post-trial cases was 34% and 28%, respectively (p = 0.36), and median duration of response was 118 and 175 days or greater, respectively. Factors predictive of PSA response in the non-trial cohort were longer time from diagnosis of prostate cancer (p = 0. 027) and higher baseline PSA (p = 0.013). Factors predictive of increased survival in both groups were younger age (p <0.04), better baseline Eastern Cooperative Oncology Group performance status (p <0. 02), and higher hemoglobin (p /= 90% of EMG activity recordings. The duration of activity was approximately 5 seconds. Such an excited segment of ureter led to a contraction which occluded the ureter and could prevent retrograde leakage of intraluminal contents. PMID- 10751891 TI - The association of elevated urinary total to sulfated glycosaminoglycan ratio and high molecular mass hyaluronic acid with interstitial cystitis. AB - PURPOSE: A decrease in the glycosaminoglycan (GAG) layer on the urothelium is believed to be one of the possible causes of interstitial cystitis. Consequently, GAG-like substances and hyaluronic acid (HA) have been prescribed for treating this condition. To delineate the possible role of GAG and HA in the interstitial cystitis disease process, we compared the urinary levels of total GAGs (sulfated + non-sulfated), sulfated GAGs and HA in interstitial cystitis patients and normal controls. We also examined different HA species present in the urine of interstitial cystitis patients. MATERIALS AND METHODS: The total GAG and sulfated GAG levels in urine specimens of normal individuals (n = 20) and interstitial cystitis patients (n = 25) were determined by utilizing the carbazole reaction assay and the Farndale method, respectively, and were expressed as microg./mg. creatinine. Urinary HA levels were measured by applying the HA test and were expressed as ng./mg. creatinine. Gel filtration column chromatography was used to examine the profile of urinary GAGs and HA species. RESULTS: Total urinary GAGs were 2.5 to 4-fold elevated in interstitial cystitis patients with moderate to severe symptoms (Group 2; 76.2 +/- 24.8) when compared with those in normal individuals (19.9 +/- 2.5) and patients with mild symptoms (Group 1; 30.4 +/- 5.1) (p <0.001). Three urinary GAG peaks were detected in both normal and interstitial patients. However, each GAG peak from interstitial cystitis patient urine was 3 to 5-fold higher than that from normal patient urine. The sulfated GAG levels, however, remained unchanged among normal individuals (1.4 +/- 0.22), Group 1 (2.2 +/- 0.96) and Group 2 (1.6 +/- 0.38) patients (p >0.05). Consequently, the ratio of total GAGs to sulfated GAGs was elevated 3 to 3.5-fold in Group 2 patients (49.9 +/- 13.9) in comparison to that in normal individuals (16.7 +/- 2.5) and group 1 patients (14.4 +/- 4.6) (p <0.001). Urinary HA levels were marginally elevated in Group 2 patients (821. 4 +/- 247.9) when compared with those in the normal group (337.3 +/- 106.1) and Group 1 patients (540.9 +/- 166.5). In addition, a distinct high molecular mass HA species was present only in Group 2 patients. CONCLUSIONS: The increased ratio of total GAGs to sulfated GAGs and marginally elevated HA levels in urine indicate that the GAG layer is altered in interstitial cystitis patients. However, these results are in contrast to the accepted concept that a reduction in urothelial GAGs causes interstitial cystitis. The high molecular mass HA species detected in patients with severe symptoms may play a role in the pathophysiology of this disease. PMID- 10751892 TI - Frequent genotype changes at -308, and 488 regions of the tumor necrosis factor alpha (TNF-alpha) gene in patients with prostate cancer. AB - PURPOSE: Tumor necrosis factor-alpha (TNF-alpha) is involved in oncogenesis of several cancers. The purpose of this study was to investigate whether genotype changes of TNF-alpha promoter regions (-238, -308) and at the 488 region are associated with human prostate cancer. MATERIALS AND METHODS: The DNA from 73 cases of human prostate cancer was analyzed by allele-specific polymerase chain reaction to characterize the genotype changes of three regions of the TNF-alpha gene in prostate cancer patients. We also determined the genotype frequency in these patients. The relative risk of variant genotype was calculated by comparing with our previous data from healthy controls. RESULTS: Genetic changes were detected in 15.1% (11/73) of prostate cancer samples at 488 region of TNF-alpha. Seventy-three percent (53/73) of the patients showed genotype GA at -308 region of TNF-alpha. Genotype GA at 488 region in TNF-alpha was observed in 73% (53/73) of the cancer and 71% (52/73) of the normal tissue. The relative risks of incidence for prostate cancer was 14-fold higher in people with genotype GA at 308 region of TNF-alpha. The relative incidence for prostate cancer was a 17-fold higher in-patient with genotype GA at 488 region of TNF-alpha. Genotype GA at 308 of TNF-alpha was related to higher clinical tumor stage of prostate cancer than genotype G (p <0.05). CONCLUSIONS: The present study demonstrates, for the first time, that the genotype changes in -308 and 488 regions of TNF-alpha are associated with prostate cancer. PMID- 10751893 TI - Induction of mycobacteremia by intravesical bacillus Calmette-Guerin instillation in an experimental animal model and detection with polymerase chain reaction. AB - PURPOSE: The aim of this study was to detect mycobacteremia by polymerase chain reaction (PCR), induced by the instillation of bacillus Calmette-Guerin (BCG) to guinea pig bladder. We also investigated the peak time and the effect of the dose of BCG in injured and non-injured bladder. The sensitivities of routine culture and PCR were also compared. MATERIALS AND METHODS: Five different doses (0, 0.069, 0.69, 6.9 and 69 mg.) of BCG were instilled into 5 injured and 5 non injured bladders. Blood samples were collected at 0, 5, 15, 30 and 60 minutes following instillation for routine culture and PCR for each dose. A total of 50 female guinea pigs were used. RESULTS: Three of 5 samples (60%) obtained 30 minutes after the instillation of 69 mg. BCG into injured bladders were PCR positive. Furthermore, 4 of 5 samples (80%) were PCR positive when samples were obtained at the 60th minute following instillation. All the other samples were negative for PCR and routine culture. All the routine tuberculosis culture results were negative, including those which were PCR positive. CONCLUSIONS: Mycobacteremia was detected only in injured bladders and with high doses of BCG. PCR is a highly sensitive and rapid diagnostic method for detection of mycobacteremia. PMID- 10751894 TI - A blood-prostate barrier restricts cell and molecular movement across the rat ventral prostate epithelium. AB - INTRODUCTION: Blood-epithelial barriers have been described in the testis and epididymis, but the possibility of such barriers in other regions of the male genitourinary tract has received little investigation. The purpose of this study was to use in vivo micro-puncture to determine if the blood-epithelial barrier exists in the rat ventral prostate. In addition, using a model of prostatic inflammation, we sought to examine the effect of inflammation on the passage of blood borne molecules and leukocytes into the prostatic ductal lumen. MATERIALS AND METHODS: Adult Sprague-Dawley rats were divided into two groups, control and 24-hour lipopolysaccharide (LPS)-induced inflammation. Both groups were subjected to vascular infusion of radiolabeled 3H dextran, 14C urea, and 3H water. Contemporaneous in vivo micropuncture sampling of prostatic ductal fluid (DF) and arterial blood occurred at multiple time points over 120 minutes. Transepithelial movement of radiolabeled compounds at each sampling time point was quantified by the expression of DF isotope concentrations as a percentage of serum isotope concentrations at that time point. Histology of representative specimens of control and inflamed prostates was used to confirm the inflammatory response and to examine for the presence of leukocytes into the ductal lumen. RESULTS: The transepithelial movement of radiolabeled compounds from blood to prostatic lumen varied in direct relationship to the compound's molecular weight. 3H-water (MW = 18) movement into the ductal lumina was relatively rapid plateauing at 70-80% of serum values. 14C urea (MW = 60) achieved intermediate penetration into ductal fluid (50-60% of serum values) and 3H dextran (MW = 2 x 106) was essentially excluded from entry (<2% of serum). These results were not altered by LPS-induced inflammation. Histology revealed a diffuse leukocyte infiltrate in the inflamed prostatic interstitium, but penetration of inflammatory cells into the ductal lumen was very restricted. CONCLUSIONS: Our findings demonstrate a blood-prostate barrier in the rat ventral prostate with characteristics similar to the blood testis barrier. This blood-prostate barrier is not affected by LPS-induced acute inflammation. Further, this persistent barrier apparently restricts the passage of leukocytes into prostate DF even in the presence of pronounced interstitial inflammation. This observation may help to explain the observation that expressed prostatic secretions in human males are often free of leukocytes in clinical prostatitis. PMID- 10751895 TI - [The upper esophageal sphincter and its basal pressure]. PMID- 10751897 TI - [Multicenter study on the prevalence of hepatitis C virus infection in blood donors in the city of Curitiba, Brazil]. AB - In order to check the presence of anti-HCV in the blood of voluntary blood donors in four of the most active blood banks of Curitiba, PR, Brazil a third generation immunoassay (Murex--version III, ABBOTT AxSYM HCV version 3.0) was used. The study detected 347 positive cases, i.e. a median prevalence of 0.80% (range 0.64% 1.11%). Only 44 from the 347 anti-HCV positive blood donors had elevated values of serum aminotransferases (12.68%). Such low values of ALT suggest that the confirmatory RIBA should be applied to the remnant 303 anti-HCV positive cases (87.32%) which did not show elevation of ALT, with the expectation that 30% to 40% of them might be positive. The prevalence of anti-HCV in blood donors in Curitiba seems similar to published data in the Brazilian medical literature. PMID- 10751896 TI - Evaluation of esophageal motility in laryngectomized patients. AB - Laryngectomy for treatment of laryngeal-pharyngeal carcinomas may impair the sensation in the larynx and epiglottis, with consequent impairment of esophageal motility. Our aim in the present study was to investigate the esophageal motility of laryngectomized patients. Esophageal manometry was performed on 17 patients submitted to laryngectomy 2 to 71 months (median 29 months) before the examination. Eleven were rehabilitated with esophageal voice and six could not speak. Ten swallows of a 5 ml bolus of water were recorded at the lower esophageal sphincter and at 5, 10 and 15 cm above it. The lower esophageal sphincter pressure was measured by the rapid pull-through method and the upper esophageal sphincter pressure by the station pull-through method. The results were compared with those obtained for a control group of 40 healthy volunteers. The amplitude of contractions was lower and the number of nonperistaltic contractions was higher in laryngectomized patients than in volunteers (P < 0.05). The duration of lower esophageal sphincter relaxation (7.4 +/- 1.5 s) was shorter in laryngectomized patients than in volunteers (8.8 +/- 1.6 s, P < 0.05). The upper esophageal sphincter pressure was lower (34.9 +/- 29.1 mm Hg) in laryngectomized patients than in volunteers (61.2 +/- 20.8 mm Hg, P < 0.05). There was no difference between groups in contraction duration or velocity, in the numbers of multipeaked or failed contractions, lower esophageal sphincter pressure or in the number of swallows followed by complete lower esophageal sphincter relaxation. In conclusion, laryngectomy causes esophageal motility impairment characterized by low contraction amplitude, nonperistaltic contraction and shorter lower esophageal sphincter relaxation duration. PMID- 10751898 TI - [Determination of proliferative activity using Ki67 and expression of p53 in colorectal cancer]. AB - BACKGROUNDS AND PURPOSE: Classically, neoplasia has been considered a disturbance in the regulation of proliferation. In the search of a prognostic discriminant, the distribution of Ki67 as a marker of cell proliferation and the expression of p53 was studied in patients with colorectal cancer, to see whether this marker correlate with clinicopathological aspects. PATIENTS AND METHODS: Formalin-fixed paraffin-embedded tissues from 38 surgically resected adenocarcinomas of colon and rectal (5 Dukes A, 15 B and 18 C) were examined for p53 and Ki67 reactivity by immunohistochemistry (Dako). Nuclear staining of 5% of the cells was the criterion for a positive p53 reaction. For the study of Ki67 10 or more fields were chosen until 1000 nuclei per specimen were counted. The Ki67 labelling indices was determined by the number of positive nuclei/1000 cells. Correlation was done with the Dukes stage, grade of differentiation and percentual of recurrence. RESULTS: The mean Ki67 labelling indices was 40. The p53 was positive in 79% of the patients. The mean of Ki67-Li was higher in the positive p53 patients. However we did not lymphatic find differences between Ki67 or p53 expression and sex, age, Dukes stage, grade of differentiation, neural, venous or lymphatic invasion or recurrence. CONCLUSION: Ki67 and p53 could not be correlated to clinical and pathologic parameters in this study and was of limited use as a prognostic discriminant. The positive p53 cancers have more proliferative activity detected by a higher index of Ki67. PMID- 10751900 TI - Polypectomies: colonic endoscopic resections. AB - The authors present their experience with 924 colonoscopic polypectomies in 549 patients. Postpolypectomy bleeding was 0.36% without perforations. Complications associated with the procedure are analyzed; evaluating the risk, diagnosis and treatment based on a survey of the literature and personal experience. PMID- 10751899 TI - [Pancreatic anatomopathologic changes in chronic chagasic women. Preliminary data]. AB - The aim of this study was to compare the frequency of pancreas morphologic and morphometric changes in chagasic women (n = 6) aged > or = 40 years, with non chagasic controls (n = 4), paired concerning to age (59.8 +/- 11.5 versus 59.0 +/ 6.3 years), white color (33.3% versus 50.0%) and body mass index (22.3 +/- 7.0 versus 18.8 +/- 2.1 kg/m2). There were no pancreatic morphometric differences between necropsied chagasic and control groups. However, there was a smaller intrapancreatic neuronal population and a higher frequency of leukocyte perineural infiltrate in the pancreas of chagasic women. This possible intrapancreatic denervation could be associated with changes in carbohydrate metabolism, including hyperglycemia and diabetes mellitus, in some chronic chagasic women. PMID- 10751901 TI - [Mechanism of action and control in the digestion of proteins and peptides in humans]. AB - This review aims to report the major control mechanisms of protein and peptides digestion of special interest in human patients. Regarding protein assimilation its digestive process begins at the stomach with some not so indispensable actions comparatively to those of duodenal/jejunal lumen. However even the intestine processes are partially under gastric secretion control. Proteolytic enzyme activities are related to protein structure and amino acid constituents, tertiary and quartenary structures need HCl denaturation prior to enzymatic hydrolysis. Thereafter the exopeptidases are guided by either NH2 (aminopeptidases) or COOH (carboxypeptidases) terminals of the molecule while endopeptidases are oriented by the specific amino acids constituents of the peptide. Both dietary and luminal secreted proteins and polypeptides undergo to either limited or complete proteolysis resulting basic or neutral free-amino acids (40%) or dioctapeptides. The brush border peptidases continue to degrade oligopeptide to di-tripeptides and neutral free-amino acids. Some peptides are uptaked by the enterocytes whose cytosolic peptidases complete the hydrolysis. Hence the digestive products flowing in the portal vein are mainly free-amino acids from either luminal or cytosolic hydrolysis and some di-tripeptides intactly absorbed. Both mechanical and chemical processes of digestion are under neural (vagal), neuroendocrinal (acetilcholine), endocrinal (gastrin, secretin and cholecystokinin) or paracrinal (histamine) controls. The gastric phase (hydrochloric acid and pepsinogen secretions) is activated by gastrin, histamine and acetilcholine which respond to both dietary-amino acids (tryptophan and phenylalanine) and mechanic distention of stomach. The pancreatic secretion is stimulated by either cephalic or gastric phases and has influence on the intestinal phase of digestion. The intestinal types of cells S and I release secretin and cholecystokinin respectively in response of acid quimo (cells S) or amino acids and peptides (cells I) in the lumen. Secretin stimulates the releasing of water, bicarbonate and enteropeptidases whereas cholecystokinin acts on pancreatic enzymes. PMID- 10751902 TI - Nutritional status improvement in neurologically impaired patients by percutaneous endoscopic gastrostomy feeding. AB - With increased use of percutaneous endoscopic gastrostomy, it became clear that neurologically impaired patients might benefit from its use. From August 1996 to July 1997, we performed 19 percutaneous endoscopic gastrostomies in patients with neurological sequelae, who were incapable to maintain their nutritional status by oral ingestion or had repeated episodes of aspiration. Sixteen patients were followed prospectively, from 30 days to 11 months (median: 6.4 months). Average weight (38.2 kg to 44.8 kg), BMI (14.8 kg/m2 to 17.8 kg/m2), weight/height ratio (23.5 kg/cm to 28 kg/cm), mid-upper arm circumference (19.4 cm to 21.6 cm) and triceps skinfold thickness (10.3 mm to 12.6 mm) were significantly increased (P < 0.01). Before percutaneous endoscopic gastrostomy, there were 10 (10/16, 62.5%) patients with grade III thinness. In this group, 3/10 patients (30%) showed improvement to grade I (two individuals) and II (one patient). All but five patients (68.75%) were below the fifth percentile of normal distribution for mid upper arm circumference. One patient (6.2%) showed improvement of her status (between 25th and 50th percentiles). Four patients (25%) started the follow-up below the fifth percentile for normal distribution of triceps skinfold thickness, and showed no improvement. There were no early complications secondary the procedure. Late complications included granulation tissue on ostomy site (18.8%) and ostomy infection (6.2%). Statistical analysis showed significant improvement of anthropometric data. Percutaneous endoscopic gastrostomy is a simple, highly successful and safe procedure, when performed in neurologically impaired patients. It is efficient as a long-term enteral feeding method. PMID- 10751903 TI - [Videolaparoscopy gastrectomy for complicated peptic ulcer: technique and case report]. AB - Since the introduction of H2 receptor antagonists and inhibitors of the acid pump, the indications for the surgical management of peptic disease have decreased significantly. However some patients presenting bleeding, perforation, gastric outlet obstruction and intractable peptic ulcer still need surgical treatment. The first laparoscopic Billroth II gastrectomy was performed in 1992. To date, laparoscopic gastrectomy has been performed by a small number of surgeons around the world. The aim of this study was to present a case of totally laparoscopic Billroth II gastrectomy and to describe an alternative technique using endoscopic stapling devices. We present a case of a 48-year-old man, complaining of severe epigastralgia, who had a 20-year history of peptic ulcer. Gastroscopy had revealed a duodenal ulcer and a deformity of the bulbus. A diagnosis of intractable peptic ulcer was made, and the patient underwent laparoscopic Billroth II gastrectomy with side-to-side intracorporeal gastrojejunostomy using endoscopic stapling devices. On postoperative day 1, he was able to walk. On postoperative day 3, he started on a clear liquid diet and was discharged on postoperative day 6. During his postoperative recovery, the patient experienced little pain and did not request narcotic analgesia. Laparoscopic gastric resection is an alternative to open procedure in well selected cases. PMID- 10751905 TI - Muscle involvement in leprosy. Study of the anterior tibial muscle in 40 patients. AB - The involvement of skeletal striated muscle in leprosy is considered secondary due to peripheral neuropathy, but some studies point it to a primary muscle lesion. In order to investigate the muscle involvement in leprosy, we studied 40 patients (lepromatous 23, tuberculoid 13, borderline 2 and indeterminate 2). The motor nerve conduction of the peroneal nerves had a reduction of the velocity, decreased compound muscle action potential and sometimes absence of potentials. The electromyographic study of the anterior tibial muscle showed signs of recent and chronic denervation in 77.5% of the cases and no myopathic potentials. The anterior tibial muscle biopsy revealed denervation in 45% of the cases, interstitial inflammatory myopathy in 30% and mixed (myopathic and neuropathic) pattern in 12.5%. Acid fast bacillus was detected in 25% of the cases, always in the interstitial tissue. Inflammatory reaction was present in the interstitial space and in patients with the lepromatous type. The histological findings clearly defined the presence of the so-called "Leprous Interstitial Myositis" on the top of denervation signs. PMID- 10751904 TI - Increased sleeping time after pentobarbital anesthesia in rats treated with intravenous injection of BCG. AB - Intravenous injection of BCG in rats induces protection against liver cell necrosis produced by CCl4. Impairment of hepatic mixed function oxidases by cytokines produced by activated Kupffer cells is the mechanism proposed to explain that protection. To verify the function of hepatic mixed function oxidases after Kupffer activation, the sleeping time after sodium pentobarbital anesthesia was evaluated in rats after intravenous injection of BCG. Male adult albino rats received BCG (50 micrograms, intravenous) and 48 h or 6 days after were anestethized with sodium pentobarbital (33 or 66 mg/g i.p.). The sleeping time was measured from the beginning of sleep until the animal started having spontaneous movement and stand up on the forepaws. The results showed that the animals treated with BCG presented a significative increase in the sleeping time, indicating reduced inactivation of the pentobarbital, an indirect evidence of inhibition of mixed function oxidase system. BCG treated rats showed hepatic and splenomegaly, both 48 and 6 days after treatment. Histology showed an increase in number of mononuclear cells in the sinusoids in the liver and in the red pulp of the spleen 48 h after injection. Small epitheliod granulomas scattered in the hepatic lobules and in the red pulp were observed in rats killed six days after the BCG injection. Hepatocyte injury, induced by activated macrophages, would be not responsible for the reduced pentobarbital inactivation, because at six days there were several granulomas scattered in lobules, but the increase of sleep time in this group was similar to that observed in rats 48 h after injection of BCG. These results demonstrate that activation of Kupffer cells with BCG induces impairment of mixed function oxidase system soon as 48 h after injection of activator, probably due to production of IL-1, IL-6 and TNF alpha by activated Kupffer cells and other mononuclear cells migrated to the liver. PMID- 10751906 TI - Morphology of the walls of the cavernous sinus of Cebus apella (tufted capuchin monkey). AB - The morphology of the dura mater and its relationship with the structures of the cavernous sinus were analyzed in five tufted capuchin monkeys (Cebus apella) using histological sections, showing that the walls of the cavernous sinus of this species are similar to those of other primates, including man. Except for the medial wall of the cavernous sinus, the remaining walls consist of two distinct dura mater layers. The deep layer of the lateral wall of the cavernous sinus is contiguous to the sheath of the oculomotor, trochlear and ophthalmic nerves. Arterioles, venules, venous spaces, neuronal bodies and nervous fiber bundles are found on this lateral wall. PMID- 10751907 TI - Number and size of myenteric neurons of the duodenum of adult rats with acute diabetes. AB - This study had as its purpose to assess the effects of acute diabetes induced by streptozotocin (35 mg/kg body weight) on the number and size of the myenteric neurons of the duodenum of adult rats considering equally the antimesenteric and intermediate regions of the intestinal circumference. Experimental period extended for a week. Neuronal counts were carried out on the same number of fields of both regions of the duodenal circumference and measurements of neuronal and nuclear areas on equal numbers of cells. Number and size of the myenteric neurons stained with Giemsa were not significantly different between groups. On the other hand, the proportion of NADH-positive neurons increased from 18.54% on the controls to 39.33% on the diabetics. The authors discuss that this increased reactivity probably results from a greater NADH/NAD+ ratio, described in many tissues of diabetic animals, which has consequences on the modulation of the enzymes that use these cofactors and whose activity is detected by the NADH diaphorase technique. PMID- 10751908 TI - Behavioral changes of Wistar rats with experimentally-induced painful diabetic neuropathy. AB - With the purpose of studying data on spontaneous customary changes in diabetic rats, we induced diabetes in 28 Wistar rats with streptozotocin. The animals were observed for 27 weeks in an attempt to characterize spontaneous customary changes that could suggest signs of chronic pain. Morphine, as a central-acting potent analgesic and its specific antagonist naloxone, were used. Our results evidenced in the animals a clinical syndrome similar to human diabetes. Long-term customary analysis revealed a significant (p < 0.05) increase of scratching and resting/sleeping behaviors, but diminished motor, eating and grooming customs. Moreover, the thermal tests revealed hyperalgesia in 43% of the animals, what may corroborate the meaning of scratching as a sign of pain. Pharmacological tests with morphine showed a significant (p < 0.05) inhibition of scratch, with concomitant increase of motor and eating activities and diminished rest/sleep capacity. Naloxone antagonized the effects induced by morphine. Such results suggest that these animals exhibit evoked behavior of hyperalgesia and that scratch may possibly be a spontaneous manifestation of chronic pain also in Wistar rats with this experimental model of painful diabetic neuropathy. PMID- 10751909 TI - The effect of baclofen on spontaneous and evoked behavioural expression of experimental neuropathic chronic pain. AB - Baclofen (beta-p-chlorophenyl-GABA) has been used in humans to treat spasticity, as well as trigeminal neuralgia. Since GABA (gamma-aminobutyric acid) has been implicated in inhibitory and analgesic effects in the nervous system, it was of interest to study the effect of baclofen in experimental neuropathic pain. With this purpose, experiments were carried out in 17 neuropathic rats with constrictive sciatic injury, as described by Bennet and Xie (1988), taking as pain parameters scratching behaviour and the latency to the thermal nociceptive stimulus. The results showed that baclofen induces, in a dose-dependent manner, significant decrease (p < 0.05) of scratching behaviour and significant increase (p < 0.05) of the latency to the nociceptive thermal stimulus. The absence of antagonism of naloxone suggested a non-participation of an opioid-mediated mechanism in this analgesic effect of baclofen on experimental neuropathic pain. PMID- 10751910 TI - Amyotrophic lateral sclerosis. Clinical analysis of 78 cases from Fortaleza (northeastern Brazil). AB - We report on the clinical characteristics of amyotrophic lateral sclerosis (ALS) in Fortaleza (Northeastern Brazil). For this, we analyzed retrospectively (from 1980 to 1999) 78 cases of ALS from the Service of Neurology of the University Hospital of Fortaleza diagnosed clinically and laboratorially (EMG, muscle biopsy, myelography, blood biochemistry, muscle enzymes and cranio-cervical X ray). The results showed that they were mostly sporadic ALS (76/78), and they were divided into definite (n = 36), probable (n = 20), possible (n = 15) and suspected (n = 7), according to the level of diagnostic certainty. They were also subdivided into juvenile (n = 17), early-onset adult (n = 18), age-specific (n = 39) and late-onset (n = 4) groups. Clinically, they presented as initials symptoms, principally, asymmetrical (30/78) and symmetrical (24/78) weakness of extremities, besides bulbar signs, fasciculations, and atrophy. Curiously, pain as first symptom occurred in an expressive fashion (17/78). The predominant initial anatomic site, in this series, was the spinal cord, and mainly affecting the arms. As to the symptom accrual from region to region, this occurs more quickly in contiguous areas, and fasciculations are predominant when bulbar region was associated. PMID- 10751911 TI - Cognitive profile of patients with relapsing remitting multiple sclerosis. AB - Multiple sclerosis (MS) is a common disease in Western countries of temperate/cold climate, but in tropical countries an increasing number of cases have been diagnosticated. Moved by the lack of information about cognitive dysfunction of Brazilian MS patients, the present study attempted to describe features of neuropsychological alterations in patients with relapsing remitting MS living in the city of Sao Paulo. They were compared to healthy volunteers, matched for age and education. In the absence of global intellectual deterioration, the patients had a deficit: a) in learning and verbal long-term memory tasks and in visual long-term memory of complex figure; b) in timed tasks, accounted for by a slowness of mental processes; c) in tasks with a motor component. Tendency to depression was observed; anxiety levels were normal. PMID- 10751912 TI - Phenytoin as the first option in female epileptic patients? AB - OBJECTIVE: Phenytoin (PHT) is one of the first-choice drugs in several epileptic syndromes, mostly in partial epilepsies, in which case it is effective as carbamazepine and phenobarbital. However, like any other anti-epileptic drug (AED), unpleasant side-effects are not rare. The aim of this study is the evaluation of dermatological troubles related to chronic PHT usage in female patients. METHOD: Between 1990-93, 731 new patients underwent investigation for epilepsy at the Multidisciplinary Clinic for Epilepsy in our State. In this sample 283 were AED users at the time of the first assessment. Sixty one female patients taking PHT were identified. They were taking PHT in a dosage ranging from 100 to 300 mg daily, in mono or polytherapy regimen, during 1-5 previous years. RESULTS: More than 50% of the sample showed coarse facial features made by the combination of several degrees of acne, hirsutism and gingival hyperplasia. CONCLUSION: Except in emergency situations, PHT should not be prescribed as the first option to the treatment of female epileptic patients, because not uncommonly the cosmetic side-effects are more socially handicapping than the epileptic syndrome by itself. PMID- 10751913 TI - [Causes of late-onset epilepsy in an epilepsy clinic of Santa Catarina--Southern Brazil]. AB - OBJECTIVE: To identify the most common etiologies of late-onset epilepsy in our society. METHOD: Retrospective and descriptive study in the CME/SUS of Florianopolis from 1990 to 1998, evaluating 120 handbooks of patients with late onset epilepsy, that is, those that had initiated epileptic seizures of the 18 years in ahead. The collected variables were: age of the first seizure, etiologies and morbid familial history. RESULTS: The prevalence rate found for late-onset epilepsy was 29.48%. Most of the sample had partial symptomatic epilepsy and the most frequent and important were: cysticercosis with 20%, head trauma with 15%, febrile convulsions in infancy with 5% and 35% were classified as idiopatic. CONCLUSION: Prevalence of late-onset epilepsy in our society is 29.48%, value slightly upper to the told one in developed countries (25%). Very probably, this difference is consequence of the epidemic character of cysticercosis among us. Moreover, head trauma and febrile convulsions are common etiologies. Prophylaxis of epilepsy is viable and urgently necessary in our society. PMID- 10751914 TI - [Age of appearance and disappearance of rolandic spikes of 160 children treated in ambulatory. Actuarial study]. AB - This study aims to determine the age at which the rolandic spikes (RS) appear and disappear in routine EEGs. METHOD: It has been carried out a hospital based prospective study of 412 EEGs records of 160 children who had been assisted at the neuropediatric out-patient department during the period between March, 1989 and March, 1998. Recordings were made on 8-channel instruments and 10/20 system has been to place the electrodes. The children have been divided into 4 groups, according to their age (1 to 4; 5 to 8; 9 to 16; 1 to 16), and the actuarial curve method has been used to show the disappearance of the rolandic spikes. RESULTS: RS distribution between boys and girls has been 64/36 ratio. RS have been found at average age of 7 years (boys, 7.2 and girls, 6.6). In a small group of children with normal EEGs previous to the RS appearance, the average age has been 6.8 years old. After this 7-year follow up period, the percentage of patients free from RSs was 60% for 1 to 4 year old group at admission; 84% for the 5 to 8 year old group and for the 9 to 16 year old group. For the total group (1 to 16 year old), the percentage of those free from RSs after this 7 year follow up period was 78%. CONCLUSION: This study shows that RSs appear at the average age of 7 years old, and are likely to disappear up to the end of a 7-year follow up period, independently on the appearance age. PMID- 10751915 TI - [Sexual disorders in epilepsy. Results of a multidisciplinary evaluation]. AB - Eleven epileptic men who complained of epilepsy and sexual dysfunction were submitted to a multidisciplinary evaluation. Mean age was 27 years (20-34), mean epilepsy duration was 19 years (0.5-32) and the mean seizure frequency was two by week (0-7). Ten patients had partial seizures and one other had myoclonic epilepsy. Ten patients were treated with antiepileptic drugs (phenytoin--1, carbamazepine--8, clonazepam--3, clobazam--2, valproic acid--3, vigabatrin--1). As defined in the DSM III-R, the complaints were: erectile disorder (9), hypoactive sexual desire disorder (4), frotteurism (4), inhibited orgasm (3), premature ejaculation (3), fetishism (2), voyeurism (2), exhibitionism (2), pedophilia (1) and sexual aversion disorder (1). Two patients showed hypogonadotropic hypogonadism on endocrinologic screening. Urological evaluation disclosed organic erectile dysfunction in other two. One patient had a diagnosis of psychogenic sexual disorder. In six patients a conclusive etiologic diagnosis was not reached. This report shows the multifactorial nature of sexual disorder in epilepsy and underlies the need of a multidisciplinar evaluation. PMID- 10751916 TI - [Audiological assessment and follow-up after bacterial meningitis]. AB - Hearing loss is the more frequent sequel of bacterial meningitis in children. This study describes the audiological profile (peripheric and central) of 89 children admitted to the hospital wards with the diagnosis of bacterial meningitis. Those children attended audiological follow up, after their hospital discharge, and were submitted to audiological tests. The results showed that 85.4% among them presented normal hearing in both ears 10.1% presented bilateral neurosensorial hearing loss and 4.5% presented unilateral neurosensorial hearing loss. The results from the auditory processing skills assessment showed that 10% of those children presented auditory localization and recognition of sentences with competitive messages (Paediatric Sentences Identification-ipsilateral) disorders. PMID- 10751918 TI - [Complications of pituitary surgery. Analysis of 120 operations at HUCFF-UFRJ]. AB - We evaluated 120 pituitary operations performed at Hospital Universitario Clementino Fraga Filho of the Universidade Federal do Rio de Janeiro from 1979 to July 1998 with the aim of analysing the immediate post operative morbidity and mortality. The ages ranged from 15 to 70 years and the clinical diagnoses were: nonsecreting adenomas, 46 (38.34%); acromegaly, 30 (25%); prolactinomas, 29 (24.16%) and Cushing's disease 15 (12.5%). The main endocrine complications were: panhypopituitarism, 16 (13.34%); diabetes insipidus, 15 (12.5%) and adrenocortical insufficiency, 4 (3.34%). Neurological complications: infection, 13 (10.84%); and cerebrospinal fluid leakage, 6 (5%). Two patients died. Our results agree with those of the international literature. We identified a progressive decrease of morbidity and mortality due to surgeon's skill increase and to the uniformity of the clinical team which managed those patients. PMID- 10751917 TI - [Headache treatment in an emergency unit of the city of Ribeirao Preto, Brazil]. AB - Headache is one of the most common symptoms observed in clinical practice. It has a considerable economic impact and overburdens emergency rooms. In Brazil, most emergency rooms have no tryptans. The present study analyses the treatment provided by the Emergency Room of the University Hospital of Ribeirao Preto. In 1996, 1254 patients were treated for headache and 64 of them required hospitalization. Of the non-hospitalized (NH) patients, 77% had primary headache, as opposed to 29.7% of hospitalized patients. Of the patients with migraine, 83.6% improved with intravenous dipyrone, 66.7% improved with intramuscular diclofenac and 81.8% improved with intravenous chlorpromazine. The percentages of patients with tension-type headache who improved with the same drugs were 77.8%, 80% and 100%, respectively. Among NH patients, 16.3% improved without any medication. We conclude that the drugs used have similar efficacy profiles and costs and can be used at basic health unities. The major drawback is parenteral administration. PMID- 10751919 TI - [Transfontanellar Doppler ultrasound measurement of cerebral blood velocity before and after surgical treatment of hydrocephalus]. AB - Twenty-seven children with hydrocephalus of different etiologies diagnosed by clinical examination, neurosonography and computerized brain tomography were submitted to transfontanellar US-Doppler evaluation for measurement of blood flow velocity and for the calculation of resistance index (RI) in the anterior and middle cerebral arteries and internal carotids. All children were submitted to evaluation before surgery and on the 1st, 30th and 60th postoperative days. We conclude that neurosonography and US-Doppler technique is useful for determination of hydrocephalus, indication and control of cerebrospinal fluid shunts and monitoring of changes in RI, comparing data obtained immediately before and after surgery and during the late postoperative period. The results obtained when comparing the RI values for the various arteries during the different stages of the study also permitted us to conclude that the anterior cerebral arteries are representative of the maximal alterations that occur in cerebral vascular resistance in pediatric patients with hydrocephalus. PMID- 10751920 TI - [Prognostic factors related to gunshot wounds to the spine in patients submitted to laminectomy]. AB - The spinal trauma related to civilian gunshot missile still remains a serious neurological event that carries a dismal prognosis almost in all cases. Its surgical indication also is a matter of discussion. Our goal is to identify the aspects that could influence the prognosis after surgery to this kind of lesions. We conducted a retrospective study of 45 consecutive patients submitted to laminectomy at Hospital de Base do Distrito Federal (Brasilia, Brazil), testing the following aspects: initial neurological status, level of the deficit, surgical timing, use of methilprednisolone and presence of dural tearing. Among those, the initial clinical presentation and the level of the lesion (60% of the patients with cauda equina syndrome and 53% of that with lesions in the lombar region improved their neurological status after laminectomy) were the most important factors affecting the outcome. Seventy percent of the patients experienced a pain relief after the surgical procedure. PMID- 10751921 TI - [Intracranial hypertension in victims of fatal traffic accidents]. AB - A morphological study, macro and microscopical, was made of brain lesions in 120 victims of fatal road traffic accidents. Parahipoccampal hernia, Duret haemorrhage and infarction in the medio-basal occipital lobe, clear evidence of raised intracranial pressure, occurred in 43 (35.8%) patients. The increased intracranial pressure from the head injury is caused by brain swelling and by intracranial haematomas. In this series 3 cases of extradural haematomas (7.0%), 9 of subdural haematomas (20.9%), 6 of intracerebral haematomas (14%), 6 of burst lobe (14%) and 9 of brain swelling (20.9%) were observed. PMID- 10751922 TI - [Cerebral contusion in victims of fatal traffic accidents. Frequency and association with other craniocerebral lesions]. AB - A morphological study, macro and microscopical, was made of brain lesions in 120 victims of fatal road traffic accidents. Contusions of the brain were identified in 67 (55.8%) of the patients. The contusions especially affected the orbital surfaces of the frontal lobes (27.8%) and the temporal poles (19.8%). The recent brain contusion was associated with a skull fracture in 70.1% of the cases and with a diffuse axonal injury in 89.5%. These can be explained by the association of contact and inertia from the road traffic accidents. Old contusions were identified in six patients (5.0%). PMID- 10751923 TI - [Magnetic resonance imaging and clinical features in adults with monophasic demyelinating disease. Acute disseminated encephalomyelitis or a variant of multiple sclerosis?]. AB - Acute disseminated encephalomyelitis (ADEM) is a widespread monophasic inflammatory disease affecting the central nervous system, that usually follows an infection or vaccination. In this study, we present an analysis of magnetic resonance imaging (MRI), cerebrospinal fluid (CSF) and clinical aspects in four patients with clinical diagnosis of ADEM. The presence of MRI demyelinating lesions was crucial, but not in itself sufficient for definitive diagnosis. Clinical and MRI follow up, in order to exclude new lesions and to reevaluate the former ones, as well as CSF, were important for the differential diagnosis with other demyelinating diseases, particularly multiple sclerosis. In addition, we have shown that early treatment with methylprednisolone after the initial symptoms was effective for improving clinical manifestations as well as for reducing MRI lesions. PMID- 10751925 TI - Brain SPECT imaging in Huntington's disease before and after therapy with olanzapine. Case report. AB - Olanzapine, an atypical antipsychotic drug, was administered to a patient with Huntington's disease (HD) with marked choreiform movements. Brain SPECT with 99mTc-HMPAO was performed before and after treatment. Brain SPECT imaging has been performed in patients with HD in order to determine the status of basal ganglia perfusion. The use of brain SPECT with 99mTc-HMPAO before and after treatment in patients with HD has not been yet reported. The marked hypoperfusion of the basal ganglia on brain SPECT performed before therapy with olanzapine improved significantly after treatment. PMID- 10751924 TI - [Dupuytren's and Ledderhose's diseases associated with chronic use of anticonvulsants. Case report]. AB - We present the case of a patient that after chronic use of anticonvulsant drugs without proven epilepsy, developed Dupuytren's and Ledderhose's diseases. We discuss the most frequent predisposing factors, and their relationship with chronic use of anticonvulsants, particularly phenobarbitone. PMID- 10751926 TI - Juvenile Huntington's disease confirmed by genetic examination in twins. AB - Early-onset Huntington's disease (HD) occurs in approximately 10% of HD's cases. We report juvenile HD in phenotypically identical twins, evaluated by history, clinical and neurologic examination, minimental state examination, blood laboratory exams, cerebrospinal fluid examination, skull computed tomography, and genetic examination for HD. Patients had the akinetic-rigid variety (Westphal variant) of the disease and paternal inheritance. The laboratory workup confirmed the clinical diagnosis of HD, which adds this report to the rare cases of HD in twins reported in the literature. PMID- 10751927 TI - [Tropical spastic paraparesis/HTLV-I associated myelopathy. Report of 2 cases diagnosed in Cuiaba, Mato Grosso, Brazil]. AB - We describe two cases of tropical spastic paraparesis/HTLV-I associated myelopathy, according to the criteria of World Health Organization-1989. These are the first cases diagnosed in Cuiaba (Mato Grosso State, Brazil). One of them had a good response with the treatment with prednisone. PMID- 10751928 TI - [Guillain-Barre syndrome associated with acute pancreatitis. Case report]. AB - We report a clinical case of Guillain-Barre syndrome associated with acute pancreatitis, and perform a review in literature about clinical complications related to Guillain-Barre syndrome. We suggest the possibility of being the events immunologically related. PMID- 10751929 TI - [Parinaud syndrome and Collier signal. Initial neurological manifestation in an HIV-1 positive patient]. AB - We describe the third case in the literature of Parinaud's syndrome as the sole manifestation of brainstem toxoplasmosis, in a positive HIV-1 patient and review the literature concerning the possible etiologies of Parinaud's syndrome, enforcing that it is a rare manifestation of central nervous system toxoplasmosis. We refer to its occurrence without simultaneous obstructive hydrocephalus and increased intracranial pressure, pointing out the Collier signal which is not very well known, but has great semiotic value once it locates the lesion in the brainstem. PMID- 10751930 TI - [Syndrome of Munchausen and pseudoparaplegia. Case report]. AB - We present a 36 years old female, with a 8 years history of "paraplegia", who was admitted to our department to have "some needles withdrawn" from her back. She referred previous admittances to general and psychiatric hospitals in order to treat several episodes of renal failure, combined with other clinical conditions like pulmonary tuberculosis and esophagus, ovary and breast cancer. On the psychiatric evaluation, we found a patient who seemed to be indifferent to her disease, giving accurate and very elaborated description of her symptoms. She was always complaining about previous medical therapies. She moved her both legs simultaneously, without any impairment on reflexes and sensibility. We did not find evidence of tumor processes. The electromyography, cranial CT scan, abdominal ultrasound scan and chest X-ray were normal. Lumbosacral X-ray and CT scan showed 16 sewing needles widespread in the lumbosacral soft tissues. A diagnosis of Munchausen syndrome was formed. After a 15-day trial of anti depressive drug, physiotherapy and psychotherapy, her clinical status significantly improved, reverting its neurological picture after treatment. PMID- 10751931 TI - [Serotonin syndrome associated to the use of paroxetine. Case report]. AB - We report on a case of serotonin syndrome associated to the use of the paroxetine, a serotonin reuptake inhibitor drug. Serotonin syndrome related to this drug not combined with other drugs had not yet been described in literature. PMID- 10751932 TI - [Common iliac artery false aneurysm as complication of lumbar disc hernia surgery. Case report]. AB - Surgery for discogenic disease of lumbar region is a challenging and diverse field. The significance of complications in disc surgery arises from the possibility that failure of primary treatment may lead to a permanently disable patient. The most common iatrogenic lesions in the posterior approach to lumbar disc herniation are neurologic, vascular, enteric, ureteral, and sexual dysfunction. Abramovitz reports that vascular complications of the posterior approach occur in at least 0.1% of cases. The vascular complications may present during surgery as severe bleeding leading to hypotension. The most common late complication is arteriovenous fistula followed by false aneurysms. We describe the case of a 39 year-old woman who was operated on for a lumbar disc herniation in other hospital. She came to us with a persistent pain in lumbar area. A MRI scan of lumbosacral region revealed a paravertebral lesion suggesting abscess. An angiography was performed and showed a false aneurysm of left common iliac artery, which was surgically treated successfully. We discuss the mechanisms of lesion and precautions against vascular ones during lumbar disc surgery. PMID- 10751933 TI - [Spontaneous extradural spinal hematoma. Case report]. AB - We report a case of a 55-year-old woman, that has a mild hypertension, in use of a betablocker drugs, who had a sudden spinal cord compression syndrome with intense cervical pain, tetraplegia and sensitivity level in C4. Extradural spinal hematoma of the cervical spine was diagnosed by magnetic resonance. The patient underwent a laminectomy and aspiration of the hematoma. The patient is on physiotherapy and presents progressive motor and sensitivity improvement. PMID- 10751934 TI - [Professor Antonio Austregesilo. The pioneer of neurology and of the study of movement disorders in Brazil]. AB - Professor Antonio Austregesilo was the pioneer of neurology in Brazil, creating the first neurological school, in Rio de Janeiro, of which he was the first professor. He was also the first to study the movement disorders in Brazil, publishing several works on this subject, primarily in "Revue Neurologique", and "L'Encephale", including a rival sign of Babinski and the first description of a posttraumatic dystonia. PMID- 10751935 TI - [Neurophysiologic evaluation of the carpal tunnel syndrome]. PMID- 10751936 TI - Effect of Trypanosoma lewisi (Kinetoplastida: Trypanosomatidae) on the infection of white rats with Toxoplasma gondii (Eucoccidia: Sarcocystidae) oocysts. AB - White rats were inoculated with 10(6) trypomastigotes of Trypanosoma lewisi, simultaneously or two days before and after inoculation with 10(5) oocysts of T. gondii. A greater number of cysts was found in the brain of the animals having concomitant inoculations, as compared with rats inoculated with either one of the two parasites. An apparent immunosuppressive effect is likely. Since both organisms can be found in rats, it is possible that infections with T. lewisi, could make this rodent another intermediate host for Toxoplasma infections. PMID- 10751937 TI - [Serum monitoring of IgG and IgM Babesia bigemina (Haemosporidia: Babesiidae) antibodies in calves from the Mexican tropics]. AB - Antibody dynamics (IgG and IgM) against Babesia bigemina was studied on 41 under 15 days of age from three ranches (R1, R2 and R3) in Yucatan, Mexico. Blood samples were collected every 30 days, for eight months. Sera were tested by the indirect fluorescent antibody method to detect IgG and IgM. Overall IgM seroprevalence during the calves first eight months of life was 17.1% without relation to age. Overall IgG seroprevalence was 66.8%, increasing with age. Seroprevalence in R1, R2 and R3 were 87.5%, 77.1% and 31.8% respectively. Ranches 1 and 2 were in enzootic stability. In Yucatan, the modification of management factors in ranches with enzootic instability, could increase the risk of clinical babesiosis. Cattle mobilization from ranches with enzootic instability must be strictly controlled. PMID- 10751938 TI - [Cardiovascular alterations induced by the venom of Lachesis muta (serpentes: Viperidae) and its fibrinogenolitic enzyme]. AB - The fibrinogenolytic activity of Lachesis muta stenophyrs venom was studied. Wistar rats catheterized at carotid artery and jugular vein were inoculated with crude venom or enzyme and changes in arterial pressure, cardiac frequency and electrocardiogram were monitored. The enzyme induced a greater fibrinogen reduction than the crude venom without any cardiovascular or histological alteration. In vitro crude venom coagulated blood whereas the enzyme reduced fibrinogen in 23%. Results suggest the potential use of the fibrinogenolytic enzyme as antithrombotic agent. PMID- 10751939 TI - The biology of some intraerythrocytic parasites of fishes, amphibia and reptiles. AB - Fishes, amphibia and reptiles, the ectothermic vertebrates, are hosts for a variety of intraerythrocytic parasites including protists, prokaryotes, viruses and structures of uncertain status. These parasites may experience host temperature fluctuations, host reproductive strategies, population genetics, host habitat and migratory behaviour quite unlike those of endothermic hosts. Few blood infections of fishes, amphibia and reptiles have proven pathogenicity, in contrast to the many intraerythrocytic parasites of mammals and some birds which harm their hosts. Although not given the attention afforded to intraerythrocytic parasites of endotherms, those of ectotherms have been studied for more than a century. This review reports on the diversity, general biology and phylogeny of intraerythrocytic parasites of ectotherms. The existence of taxonomic confusion is emphasized and the main taxonomic features of most of the 23 better characterized genera, particularly the kinetoplastid and apicomplexan protists, are summarized. Transmission of protistan infections of aquatic ectotherms is also discussed. Leeches can transfer sporozoties or merozoites to the vertebrate host during feeding. Dormant sporozoites of Lankesterella may permit transmission of species of this genus between vertebrates by predation. The fish haemogregarine, Haemogregarina bigemina, probably has gnathiid isopods, rather than leeches, as its definitive hosts. Hepatozoon spp. in aquatic hosts, and Progarnia of caiman, may also use invertebrate hosts other than leeches. Protistan infections of terrestrial or semi-terrestrial hosts are transmitted by a variety of arthropods, or, in some cases, leeches, contaminated paratenic hosts, or sporocysts free in water. Transfer of protists between vertebrates by predation and congenitally may also occur. The biology of the host cells of these infections, the red blood cells of ectotherm vertebrates, is summarized and compared with that of mammalian erythrocytes. Erythropoiesis, the nature of the surface molecules (especially the possible existence of a major histocompatibility complex), the haemoglobins, and the shape and size of erythrocytes are discussed. The exoerythrocytic sites in which protists, prokaryotes, viruses and structures of uncertain status exist before erythrocyte entry are described. Tissue merogony, tissue cysts and invasion of the white cell series occur in a variety of protistan infections. Intraerythrocytic stages of protistan infections are also discussed, including modes of entry to erythrocytes, survival mechanisms, and multiplication. The impact of infection on host populations is difficult to assess, in part because there is no agreement in the literature on the criteria used to evaluate parasite-induced cost to the host. Almost all studies have been on haemogregarine and Plasmodium infections in, mainly, lizards, but also fishes and snakes. Some infections may be responsible for mortality in their hosts, but hosts themselves may be short lived, or have a limited ability to recover from infection. PMID- 10751940 TI - The range and biological activity of FMRFamide-related peptides and classical neurotransmitters in nematodes. AB - Nematodes include both major parasites of humans, livestock and plants in addition to free-living species such as Caenorhabditis elegans. The nematode nervous system (especially in C. elegans) is exceptionally well defined in terms of the number, location and projections of the small number of neurons in the nervous system and their integration into circuits involved in regulatory behaviours vital to their survival. This review will summarize what is known about the biological activity of neurotransmitters in nematodes: the biosynthetic pathways and genes involved, their receptors, inactivation mechanisms and secondary messenger signalling systems. It will cover the 'classical' transmitters, such as acetylcholine (ACh), GABA, glutamate, serotonin, dopamine, octopamine, noradrenaline and nitric oxide. The localization of peptides throughout the nematode nervous system is summarized, in addition to the isolation of nematode neuropeptides by both traditional biochemical techniques and more modern genetic means. The major contribution of the completion of the C. elegans genome-sequencing program is highlighted throughout. Efforts to unravel neurotransmitter action in various physiological actions such as locomotion, feeding and reproduction are detailed as well as the various inactivation mechanisms for the current complement of nematode transmitters. PMID- 10751941 TI - The immunobiology of gastrointestinal nematode infections in ruminants. AB - The major gastrointestinal nematode parasites of ruminants all belong to the Order Strongylida and the family Trichostrongyloidea. Despite this close evolutionary relationship, distinct differences exist in the microenvironmental niches occupied by the developmental stages of the various parasites, which may account for the variable susceptibility of the different parasite species to the immune effector mechanisms generated by the host. In addition, different manifestations of resistance have been observed against the adult and larval stages of the same parasite species, and even against the same parasite stage. In particular, both rapid and delayed rejection of infective larval stages of gastrointestinal nematode parasites has been documented. This review will give an overview of the various manifestations of resistance to gastrointestinal nematode parasites of ruminants, as well as the immune mechanisms and antigens associated with the generation of immunity by the ruminant hosts to these parasites. In addition, a working model is provided aimed at reconciling most of the present knowledge on the different immune responses generated during infection with the various parasite rejection profiles. Extrapolation of these results to field conditions will need to take into account the variability imposed by seasonal changes and management practices, as well as the individual variability in immune responsiveness present in outbred animal populations. PMID- 10751942 TI - Autonomous protein folding units. PMID- 10751943 TI - The relationship between sequence and structure in elementary folding units. PMID- 10751944 TI - The energy landscape theory of protein folding: insights into folding mechanisms and scenarios. PMID- 10751945 TI - Barriers in protein folding reactions. PMID- 10751946 TI - Role of the molten globule state in protein folding. PMID- 10751947 TI - Comparison of equilibrium and kinetic approaches for determining protein folding mechanisms. PMID- 10751948 TI - Folding and association of oligomeric and multimeric proteins. PMID- 10751949 TI - [The fight against nosocomial infections in anesthesia and intensive care: a rational medical strategy]. PMID- 10751951 TI - [Performance studies of 6 new anesthesia ventilators: bench tests]. AB - OBJECTIVE: To assess the pneumatic performances of six new anaesthesia ventilators. STUDY DESIGN: Bench test study. MATERIAL: The study included one ventilator operated by an electric motor: ABT 5300 (Kontron); four ventilators of "bellows-in-bottle" category: ADU version 97 (Datex-Ohmeda); Aestiva 3000 (Datex Ohmeda), Kion (Siemens), the two versions of Julian (Drager); and an original ventilator devised for quantitative, or self-regulating target controlled inhalation anaesthesia, with a totally closed circuit, made of four ventilating chambers: PhysioFlex (Drager). METHODS: The bench test included a passive lung model with adjustable compliance and resistances, and flow and pressure gauges. The accuracy of volume and pressure measurements was tested in various conditions of resistance and compliance. RESULTS: Pneumatic performance and accuracy were satisfactory, even in severe ventilatory conditions. All the ventilators, except ABT 5300 and Julian 1, have a compliance compensation system permitting to deliver and to maintain a constant tidal volume under various conditions of downstream load, particularly under maximal load condition. Variations of tidal volume with the increase of the fresh gas flow are negligible. CONCLUSION: Recent technological progress has improved pneumatic performance of anaesthesia ventilators and the marketed models are more homogeneous at present. Ergonomics and training for the use of the machine are becoming major criteria for the global assessment and the choice of a ventilator. PMID- 10751950 TI - [Sciatic nerve block: an new lateral mediofemoral approach. The value of its combination with a "3 in 1" block for invasive surgery of the knee]. AB - OBJECTIVE: To describe a new midfemoral lateral approach for the sciatic nerve block. Its combination with the "3 in 1" block was tested for postoperative analgesia following major surgery of the knee. STUDY DESIGN: Descriptive, anatomical and clinical study prospective. PATIENTS: After testing in four unembalmed corpses the new approach was applied to 42 ASA 1-2 patients, in combination with a continuous "3 in 1" block. METHODS: The new approach was analysed for reliability of the surface landmarks (a line drawn from the posterior margin of the greater trochanter towards the knee and parallel to the femur) and block extent assessed on the foot. Its combination with the "3 in 1" block was evaluated with a visual analogue scale (VAS) scoring, for postoperative analgesia after total knee arthroplasty. RESULTS: The sciatic nerve was located in less than 10 min. A block of the sciatic nerve was fully achieved in all patients. Its median duration was 16 h. The median VAS score at rest was 0 mm (sciatic bloc + continuous block "3 in 1"), but increased to 40 mm (block "3 in 1" alone). CONCLUSION: The new lateral midfemoral sciatic block is easy to master. Combined with a continuous "3 in 1" block, it provides excellent analgesia during the early postoperative period after major surgery of the knee. PMID- 10751953 TI - [Acute pulmonary edema associated with the use of beta2-mimetic tocolytic agents]. AB - A case of severe pulmonary oedema during beta2-adrenergic agonist tocolytic therapy (salbutamol) in a pregnant woman admitted for preterm labor at 32 weeks of amenorrhoea is reported. Echocardiography and haemodynamic investigations did not show any left ventricular systolic or diastolic dysfunction. Pulmonary oedema is an exceptional complication of beta2-adrenergic agonist tocolytic therapy. The diagnosis is considered in pregnant patients presenting with respiratory distress, associated with or following tocolytic therapy. Anaesthetic management of patients treated with a beta2-adrenergic agonist should take into consideration the delay between discontinuation of tocolytic therapy and anaesthetic induction, as well as volume expansion. PMID- 10751952 TI - [Catheter-related nosocomial urinary infections in intensive care: physiopathology, epidemiology and prevention]. AB - OBJECTIVES: Nosocomial urinary tract infections associated with bladders catheters are common and poorly understood. Data on the prevention of urinary tract infections are numerous and heterogenous. This update article aimed at analysing mechanisms, epidemiology and prevention of these infections. DATA SOURCES: We searched in the Medline database for articles in English or French, without limiting date of publication, using the following key words separely or in combination: urinary tract infection, nosocomial, catheter, infection urinaire, sonde urinaire. STUDY SELECTION: We considered all categories of articles. DATA EXTRACTION: Data on prevention of nosocomial urinary tract infections were analysed in depth. DATA SYNTHESIS: The data on pathogenesis of nosocomial urinary tract infections are still controversial. Various means for preventing urinary tract infections have been recommended: addition of antibacterial agents to urinary drainage system, inclusion of antimicrobial components into the catheter itself, antibiotic prophylaxis or closed sterile drainage system. Their efficiency in intensive therapy unit has not yet been fully assessed. The therapy of these infections is still under debate and requires additional prospective studies to establish the optimal management. CONCLUSION: Catheter-associated urinary tract infections reflect the general hygiene policy, starting with nurse practice patterns at catheter insertion, and ending with antibio-therapy prescriptions by medical staff. PMID- 10751954 TI - [Radial neuropathy after peripheral venous puncture]. AB - We report a case of left radial neuropathy from a venipuncture, in a 59-year-old woman. She had a history of amputation of the four last fingers of the right hand, when she was 27-year-old. One year before, she had a difficult venipuncture of the left radial vein, for a preanaesthetic assessment for cataract surgery. The puncture elicited an excruciating pain, associated with hypoaesthesia in the area of the radial nerve, at forearm and the wrist. Conventional therapeutic means had only a minor efficiency. The concept of nervous system sensitizing is discussed. Prevention is essential. PMID- 10751955 TI - [Mesenteric vein and inferior vena cava thrombosis: disseminated intravascular coagulation and/or G 20210 A mutation of the prothrombin gene?]. AB - The authors report the case of a 30-year-old man who died from pulmonary embolism and multiorgan failure caused by mesenteric and inferior vena cava thrombosis. The patient was found heterozygous for the prothrombin gene variant (G 20210 A). The family study showed the same asymptomatic anomaly in his brother. This recently described mutation is associated with an increased risk for venous thrombosis. The investigations and treatment of mesenteric venous thrombosis are discussed. PMID- 10751956 TI - [Surveillance of nosocomial infections related to anesthesia. A multicenter study]. AB - OBJECTIVES: To determine incidence rate, main characteristics and risk factors of nosocomial infections associated with anesthesia (NIAA). STUDY DESIGN: Prospective, descriptive multicentre survey. PATIENTS: All patients aged more than 15 years and undergoing surgery (except cardio-thoracic, ENT or ambulatory surgery) under general or regional anaesthesia. METHODS: Voluntary participation of surgical units from public or private hospitals. Use of pre-established definitions of infections and a 72 hours postanesthetic follow-up. Anaesthesia and operation related risk factors collected. End point based on occurrence, or not, of clinical infection. Record, control, treatment and analysis of the data by Epi Info--5.0 software. Statistics used: Fischer's exact test, Mantel-Haenszel test, Anova method, Kruskall-Wallis test. RESULTS: Among 7,300 patients belonging to 13 hospitals, 25 developed an infection (nine vascular catheter related infections, 12 respiratory tract infections, two infections of the eye and two of the mouth). Only two infections have been bacteriologically documented. The overall incidence of NIAA was 3.4 per 1,000 patients. It was significantly higher after an anaesthetic of more than 2 hours and after transfusion. CONCLUSIONS: This first prospective survey of NIAA confirmed that nosocomial infections are a real problem in the practice of anaesthesia and the necessity to use preventive measures. A survey with a larger sample size would allow to specify the respective part of the various risk factors and to develop a risk index. PMID- 10751957 TI - [Postoperative mortality a university Department of General Surgery: incidence of cardiac-related deaths]. AB - OBJECTIVES: To evaluate the incidence of cardiac deaths following noncardiac nonvascular surgery. STUDY DESIGN: Retrospective survey. PATIENTS: All patients undergoing mainly endocrinous and digestive surgery in a University department of general surgery between 1991 and 1996. METHODS: Analysis of all deaths occurring intra- and postoperatively, until discharge of the patients. Demographic and medical data, including patent myocardial ischaemia and risk factors for coronary artery disease, were recorded and compared with a control group including all patients undergoing surgery from January to September 1996. RESULTS: In the 8,700 patients who underwent mainly endocrine neck surgery (66%), or intra-abdominal surgery (31%), the mortality rate (n = 96) was 1.1% (95% confidence interval [95% CI] = 0.9-1.3%). Patent myocardial ischaemia or high risk factors for coronary artery disease were existing in 24% of patients with neck surgery, 31% of those with intra-abdominal surgery, and in 60% of the deceased patients (P < 0.01 vs control group). Those who died were older, were in a higher ASA physical class, and had undergone an emergency procedure more often than patients of the control group (P < 0.002 for each parameter). Two cardiac deaths, in patients with a patent cardiopathy, were recorded (cardiac mortality: 0.02%; 95% CI = 0.003 0.08%). The main cause of death was infection (n = 46), followed by haemorrhage (n = 12). Seven deaths remained unexplained. CONCLUSION: This study suggests that cardiac morbidity is a rare cause of death after noncardiac nonvascular surgery. PMID- 10751959 TI - [Eructation disclosing an accidental gastric perforation during the creation of a pneumoperitoneum for laparoscopic surgery. The contributory role of the anesthesiologist]. PMID- 10751958 TI - [The effect of "seniorization" on the prescription of biological tests in an admissions and emergency service]. AB - This prospective study compared the prescriptions of biological tests (PBT) for patients admitted to an Emergency medical service (EMS) of a University hospital, according as to whether the staff consisted either of medical residents only (study period P1) or of residents trained in emergency medicine and continuously supervised by a senior specialist (study period P2). During the one-month study periods, a total of 5,948 patients were admitted (2,781 during P1 and 3,167 during P2 respectively). Between P1 and P2 the rate of PBT decreased by 38.8% (P < 0.0001), the mean expenses for PBT per patient by 13.8% (P < 0.0001) and the total expenses by 53.4%. Teaching and training of residents in emergency medicine and supervision by a senior specialist improve the quality of care and decrease health care cost. PMID- 10751960 TI - [Bilateral pneumothorax caused by dilatation of laryngeal stenosis]. PMID- 10751961 TI - [Hematoma of the right rectus abdominis in relation to treatment with low molecular-weight heparin]. PMID- 10751962 TI - [Application of the Heimlich maneuver with instructions over the telephone by the chief medical officer of the emergency medical services department]. PMID- 10751963 TI - Parenting and its effects on children: on reading and misreading behavior genetics. AB - There is clear evidence that parents can and do influence children. There is equally clear evidence that children's genetic makeup affects their own behavioral characteristics, and also influences the way they are treated by their parents. Twin and adoption studies provide a sound basis for estimating the strength of genetic effects, although heritability estimates for a given trait vary widely across samples, and no one estimate can be considered definitive. This chapter argues that knowing only the strength of genetic factors, however, is not a sufficient basis for estimating environmental ones and indeed, that attempts to do so can systematically underestimate parenting effects. Children's genetic predispositions and their parents' childrearing regimes are seen to be closely interwoven, and the ways in which they function jointly to affect children's development are explored. PMID- 10751964 TI - The pain of being sick: implications of immune-to-brain communication for understanding pain. AB - This review focuses on the powerful pain facilitatory effects produced by the immune system. Immune cells, activated in response to infection, inflammation, or trauma, release proteins called proinflammatory cytokines. These proinflammatory cytokines signal the central nervous system, thereby creating exaggerated pain as well as an entire constellation of physiological, behavioral, and hormonal changes. These changes are collectively referred to as the sickness response. Release of proinflammatory cytokines by immune cells in the body leads, in turn, to release of proinflammatory cytokines by glia within the brain and spinal cord. Evidence is reviewed supporting the idea that proinflammatory cytokines exert powerful pain facilitatory effects following their release in the body, in the brain, and in the spinal cord. Such exaggerated pain states naturally occur in situations involving infection, inflammation, or trauma of the skin, of peripheral nerves, and of the central nervous system itself. Implications for human pain conditions are discussed. PMID- 10751965 TI - Thought suppression. AB - Although thought suppression is a popular form of mental control, research has indicated that it can be counterproductive, helping assure the very state of mind one had hoped to avoid. This chapter reviews the research on suppression, which spans a wide range of domains, including emotions, memory, interpersonal processes, psychophysiological reactions, and psychopathology. The chapter considers the relevant methodological and theoretical issues and suggests directions for future research. PMID- 10751966 TI - Social cognition: thinking categorically about others. AB - In attempting to make sense of other people, perceivers regularly construct and use categorical representations to simplify and streamline the person perception process. Noting the importance of categorical thinking in everyday life, our emphasis in this chapter is on the cognitive dynamics of categorical social perception. In reviewing current research on this topic, three specific issues are addressed: (a) When are social categories activated by perceivers, (b) what are the typical consequences of category activation, and (c) can perceivers control the influence and expression of categorical thinking? Throughout the chapter, we consider how integrative models of cognitive functioning may inform our understanding of categorical social perception. PMID- 10751967 TI - Are there kinds of concepts? AB - Past research on concepts has focused almost exclusively on noun-object concepts. This paper discusses recent research demonstrating that useful distinctions may be made among kinds of concepts, including both object and nonobject concepts. We discuss three types of criteria, based on structure, process, and content, that may be used to distinguish among kinds of concepts. The paper then reviews a number of possible candidates for kinds based on the discussed criteria. PMID- 10751968 TI - New perspectives and evidence on political communication and campaign effects. AB - We review recent empirical evidence that shows political campaigns are more potent than widely believed, focusing on the conceptual and methodological advances that have produced these findings. Conceptually, a broader definition of effects--that includes learning and agenda-control, as well as vote choice- characterizes contemporary research. This research also features two kinds of interactive models that are more complex than the traditional hypodermic (message based) approach. The resonance model considers the relationship between message content and receivers' predispositions, while the strategic model highlights the interactions between competing messages. Finally, we attribute the emergence of stronger evidence in favor of campaign effects to the use of new methodologies including experimentation and content analysis, as well as the more sophisticated use of sample surveys. PMID- 10751969 TI - Goal theory, motivation, and school achievement: an integrative review. AB - The purpose of this review is to document the directions and recent progress in our understanding of the motivational dynamics of school achievement. Based on the accumulating research it is concluded that the quality of student learning as well as the will to continue learning depends closely on an interaction between the kinds of social and academic goals students bring to the classroom, the motivating properties of these goals and prevailing classroom reward structures. Implications for school reform that follow uniquely from a motivational and goal theory perspective are also explored. PMID- 10751970 TI - Applications of structural equation modeling in psychological research. AB - This chapter presents a review of applications of structural equation modeling (SEM) published in psychological research journals in recent years. We focus first on the variety of research designs and substantive issues to which SEM can be applied productively. We then discuss a number of methodological problems and issues of concern that characterize some of this literature. Although it is clear that SEM is a powerful tool that is being used to great benefit in psychological research, it is also clear that the applied SEM literature is characterized by some chronic problems and that this literature can be considerably improved by greater attention to these issues. PMID- 10751971 TI - The environmental psychology of capsule habitats. AB - Capsule habitats make it possible for human beings to survive and function in environments that would otherwise be lethal, such as space, the ocean depths, and the polar regions. The number of people entering capsules in the course of their work or for purposes of recreation is constantly increasing. However, long-term living in such habitats imposes physical and psychological risks as well as offering opportunities and benefits. This paper reviews what is known about the environmental, social, and personality aspects of adaptation to capsules, including sources of stress, selection criteria, obstacles to and facilitators of adequate coping, changes in group interaction, the role of temporal factors, and post-mission consequences. PMID- 10751972 TI - Food intake and the regulation of body weight. AB - This chapter reviews the recent literature on hormonal and neural signals critical to the regulation of individual meals and body fat. Rather than eating in response to acute energy deficits, animals eat when environmental conditions (social and learned factors, food availability, opportunity, etc.) are optimal. Hence, eating patterns are idiosyncratic. Energy homeostasis, the long-term matching of food intake to energy expenditure, is accomplished via controls over the size of meals. Individuals who have not eaten sufficient food to maintain their normal weight have lower levels of adiposity signals (leptin and insulin) in the blood and brain, and one consequence is that meal-generated signals (such as CCK) are less efficacious at reducing meal size. The converse is true if individuals are above their normal weight, when they tend to eat smaller meals. The final section reviews how these signals are received and integrated by the CNS, as well as the neural circuits and transmitters involved. PMID- 10751973 TI - Negotiation. AB - The first part of this paper traces a short history of the psychological study of negotiation. Although negotiation was an active research topic within social psychology in the 1960s and 1970s, in the 1980s, the behavioral decision perspective dominated. The 1990s has witnessed a rebirth of social factors in the psychological study of negotiation, including social relationships, egocentrism, motivated illusions, and emotion. The second part of this paper reviews five emerging research areas, each of which provides useful insight into how negotiators subjectively understand the negotiation: (a) mental models in negotiation; (b) how concerns of ethics, fairness, and values define the rules of the game being played; (c) how the selection of a communication medium impacts the way the game is played; (d) how cross-cultural issues in perception and behavior affect the negotiation game; and (e) how negotiators organize and simplify their understandings of the negotiation game when more than two actors are involved. PMID- 10751974 TI - Parental and child cognitions in the context of the family. AB - Parent and child family-related cognitions are reviewed with respect to (a) their origins, (b) their linkage to affect and behavior, (c) their transmission and perpetuation, (d) their alteration on the basis of first-hand experience, and (e) their collaborative negotiation and renegotiation. A distinction is offered between the functioning of implicit, relatively unaware, schematic cognitions and relatively aware, explicit, event-dependent cognitions. Consideration is also given to the differential content (or topics) of cognitions. As a positive outcome of recent research, many new insights have emerged with respect to the linkage of family members' cognitions and their individual and shared patterns of behavior. However, several limitations remain, including too little consideration of the shared influences of parents' and children's cognitions and the changes in these cognitions over time. As a growth area, there is emerging interest in the application of our knowledge of cognitions to the clinical context in programs designed to remediate and prevent family problems. PMID- 10751975 TI - Evaluation methods for social intervention. AB - Experimental design is the method of choice for establishing whether social interventions have the intended effects on the populations they are presumed to benefit. Experience with field experiments, however, has revealed significant limitations relating chiefly to (a) practical problems implementing random assignment, (b) important uncontrolled sources of variability occurring after assignment, and (c) a low yield of information for explaining why certain effects were or were not found. In response, it is increasingly common for outcome evaluation to draw on some form of program theory and extend data collection to include descriptive information about program implementation, client characteristics, and patterns of change. These supplements often cannot be readily incorporated into standard experimental design, especially statistical analysis. An important advance in outcome evaluation is the recent development of statistical models that are able to represent individual-level change, correlates of change, and program effects in an integrated and informative manner. PMID- 10751976 TI - Adult psychopathology: issues and controversies. AB - This review discusses issues and controversies with respect to the construct of a mental disorder, models of etiology and pathology, and domains of psychopathology. Fundamental to the science of psychopathology is a conceptualization of mental disorder, yet inadequate attention is being given to the differentiation of normal and abnormal psychological functioning in current research. The boundaries between mental and physical disorders are equally problematic. Neurophysiological models are receiving particular emphasis in large part because of the substantial progress being made in documenting and clarifying the important role of neurophysiological structures and mechanisms in etiology and pathology. However, this attention might be at the expense of the recognition of equally valid psychological models. Problematic diagnostic boundaries are also considered, including those within and between different classes of disorder. Dimensional models may offer a more precise and comprehensive classification of psychopathology. PMID- 10751977 TI - Scientific and social significance of assessing individual differences: "sinking shafts at a few critical points". AB - This chapter reviews empirical findings on the importance of assessing individual differences in human behavior. Traditional dimensions of human abilities, personality, and vocational interests play critical roles in structuring a variety of important behaviors and outcomes (e.g. achieved socioeconomic status, educational choices, work performance, delinquency, health risk behaviors, and income). In the review of their importance, the construct of general intelligence is featured, but attributes that routinely add incremental validity to cognitive assessments are also discussed. Recent experimental and methodological advances for better understanding how these dimensions may contribute to other psychological frameworks are reviewed, as are ways for determining their scientific significance within domains where they are not routinely assessed. Finally, some noteworthy models are outlined that highlight the importance of assessing relatively distinct classes of individual-differences attributes simultaneously. For understanding fully complex human phenomena such as crime, eminence, and educational-vocational development, such a multifaceted approach is likely to be the most productive. PMID- 10751978 TI - The effects of family and community violence on children. AB - This review examines theoretical and empirical literature on children's reactions to three types of violence--child maltreatment, community violence, and interparental violence. In addition to describing internalizing and externalizing problems associated with exposure to violence, this review identifies ways that violence can disrupt typical developmental trajectories through psychobiological effects, post-traumatic stress disorder (PTSD), cognitive consequences, and peer problems. Methodological challenges in this literature include high rates of co occurrence among types of violence exposure, co-occurrence of violence with other serious life adversities, heterogeneity in the frequency, severity, age of onset, and chronicity of exposure, and difficulties in making causal inferences. A developmental psychopathology perspective focuses attention on how violence may have different effects at different ages and may compromise children's abilities to face normal developmental challenges. Emphasis is placed on the variability of children's reactions to violence, on outcomes that go beyond diagnosable disorders, and on variables that mediate and moderate children's reactions to violence. PMID- 10751979 TI - Toward a psychology of memory accuracy. AB - There has been unprecedented interest in recent years in questions pertaining to accuracy and distortion in memory. This interest, catalyzed in part by real-life problems, marks a significant departure from the quantity-oriented approach that has characterized much of traditional memory research. We outline a correspondence metaphor of memory underlying accuracy-oriented research, and show how the features of this metaphor are manifested across the disparate bodies of research reviewed here. These include work in the Gestalt tradition, spatial memory, memory for gist, schema theory, source monitoring, fluency misattributions, false recall and recognition, postevent misinformation, false memories, eyewitness research, and autobiographical memory. In examining the dynamics of memory accuracy, we highlight the importance of metacognitive monitoring and control processes. We end by discussing some of the methodological, theoretical, and metatheoretical issues inherent in accuracy oriented research, attempting to prepare the groundwork for a more coherent psychology of memory accuracy. PMID- 10751980 TI - Attitude change: persuasion and social influence. AB - This chapter reviews empirical and theoretical developments in research on social influence and message-based persuasion. The review emphasizes research published during the period from 1996-1998. Across these literatures, three central motives have been identified that generate attitude change and resistance. These involve concerns with the self, with others and the rewards/punishments they can provide, and with a valid understanding of reality. The motives have implications for information processing and for attitude change in public and private contexts. Motives in persuasion also have been investigated in research on attitude functions and cognitive dissonance theory. In addition, the chapter reviews the relatively unique aspects of each literature: In persuasion, it considers the cognitive and affective mechanisms underlying attitude change, especially dual mode processing models, recipients' affective reactions, and biased processing. In social influence, the chapter considers how attitudes are embedded in social relations, including social identity theory and majority/minority group influence. PMID- 10751981 TI - Cultural psychopathology: uncovering the social world of mental illness. AB - We review cultural psychopathology research since Kleinman's (1988) important review with the goals of updating past reviews, evaluating current conceptualizations and methods, and identifying emerging substantive trends. Conceptual advances are noted, particularly developments in the definition of culture and the examination of both culture-specific and cultural-general processes. The contributions of the Culture and Diagnosis Task Force for DSM-IV and the World Mental Health Report are reviewed and contrasted. Selected research on anxiety, schizophrenia, and childhood disorders is examined, with particular attention given to the study of ataque de nervios, social factors affecting the course of schizophrenia, and cross-national differences in internalizing and externalizing problems in children. Within the last ten years, cultural psychopathology research has become a significant force. Its focus on the social world holds promise to make significant inroads in reducing suffering and improving people's everyday lives. PMID- 10751982 TI - Memory systems in the brain. AB - The operation of different brain systems involved in different types of memory is described. One is a system in the primate orbitofrontal cortex and amygdala involved in representing rewards and punishers, and in learning stimulus reinforcer associations. This system is involved in emotion and motivation. A second system in the temporal cortical visual areas is involved in learning invariant representations of objects. A third system in the hippocampus is implicated in episodic memory and in spatial function. Fourth, brain systems in the frontal and temporal cortices involved in short term memory are described. The approach taken provides insight into the neuronal operations that take place in each of these brain systems, and has the aim of leading to quantitative biologically plausible neuronal network models of how each of these memory systems actually operates. PMID- 10751983 TI - Personnel selection: looking toward the future--remembering the past. AB - This chapter reviews personnel selection research from 1995 through 1999. Areas covered are job analysis; performance criteria; cognitive ability and personality predictors; interview, assessment center, and biodata assessment methods; measurement issues; meta-analysis and validity generalization; evaluation of selection systems in terms of differential prediction, adverse impact, utility, and applicant reactions; emerging topics on team selection and cross-cultural issues; and finally professional, legal, and ethical standards. Three major themes are revealed: (a) Better taxonomies produce better selection decisions; (b) The nature and analyses of work behavior are changing, influencing personnel selection practices; (c) The field of personality research is healthy, as new measurement methods, personality constructs, and compound constructs of well known traits are being researched and applied to personnel selection. PMID- 10751984 TI - Emotion, regulation, and moral development. AB - Research and theory on the role of emotion and regulation in morality have received considerable attention in the last decade. Much relevant work has concerned the role of moral emotions in moral behavior. Research on differences between embarrassment, guilt, and shame and their relations to moral behavior is reviewed, as is research on the association of these emotions with negative emotionality and regulation. Recent issues concerning the role of such empathy related responses as sympathy and personal distress to prosocial and antisocial behavior are discussed, as is the relation of empathy-related responding to situational and dispositional emotionality and regulation. The development and socialization of guilt, shame, and empathy also are discussed briefly. In addition, the role of nonmoral emotions (e.g. anger and sadness), including moods and dispositional differences in negative emotionality and its regulation, in morally relevant behavior, is reviewed. PMID- 10751985 TI - Neural basis of hearing in real-world situations. AB - In real-world situations animals are exposed to multiple sound sources originating from different locations. Most vertebrates have little difficulty in attending to selected sounds in the presence of distractors, even though sounds may overlap in time and frequency. This chapter selectively reviews behavioral and physiological data relevant to hearing in complex auditory environments. Behavioral data suggest that animals use spatial hearing and integrate information in spectral and temporal domains to determine sound source identity. Additionally, attentional mechanisms help improve hearing performance when distractors are present. On the physiological side, although little is known of where and how auditory objects are created in the brain, studies show that neurons extract behaviorally important features in parallel hierarchically arranged pathways. At the highest levels in the pathway these features are often represented in the form of neural maps. Further, it is now recognized that descending auditory pathways can modulate information processing in the ascending pathway, leading to improvements in signal detectability and response selectivity, perhaps even mediating attention. These issues and their relevance to hearing in real-world conditions are discussed with respect to several model systems for which both behavioral and physiological data are available. PMID- 10751987 TI - Universal HIV testing in pregnancy. PMID- 10751986 TI - Understanding the effect of domestic violence on pregnancy, labour, and delivery. PMID- 10751988 TI - End-stage renal disease. Timely, appropriate treatment is crucial. PMID- 10751989 TI - Equal access to health care. PMID- 10751990 TI - Are physicians treated equally? PMID- 10751991 TI - Is this good palliative care? PMID- 10751992 TI - Plus ca change.... PMID- 10751993 TI - Can we use anxiolytics during pregnancy without anxiety? AB - QUESTIONOne of my patients suffers from anxiety and was using lorazepam to treat it. When she became pregnant, she stopped the medication immediately, but now she is worried about the potential effect on the baby because she was using the drug just after conception. Is this class of drugs safe during pregnancy? What should she do if she needs antianxiety treatment during the rest of her pregnancy?ANSWEREvidence to date from cohort studies did not identify a notable association between use of benzodiazepines and increased risk of major malformations, including oral cleft. In contrast, data from case-control studies show a slightly increased risk of oral cleft. Hence, level 2 ultrasonography is recommended to rule out visible forms of cleft lip. Using benzodiazepines late in pregnancy could cause withdrawal syndrome in newborns. PMID- 10751994 TI - Dermacase. Granuloma annulare. PMID- 10751995 TI - Radiographic imaging during pregnancy. PMID- 10751996 TI - Anonymous calls. PMID- 10751997 TI - Practice tips. Bedside cystometry. Simple diagnostic skill for family physicians. PMID- 10751998 TI - Intrapartum care by general practitioners and family physicians. Provincial trends from 1984-1985 to 1994-1995. AB - OBJECTIVE: To determine provincial trends in provision of intrapartum care by general practitioners and family physicians (GP/FPs) for the 11 years from 1984 to 1995. DESIGN: Analyses of provincial Medical Care Fee-for-Service Utilization data for births from 1984-1985 to 1994-1995. SETTING: 10 provinces of Canada. MAIN OUTCOME MEASURES: Proportion of vaginal births billed by GP/FPs (expressed as total number of vaginal births billed by GP/FPs divided by total number of vaginal births). RESULTS: In 1994-1995, the proportion of vaginal births billed by GP/FPs ranged from 77.2% in British Columbia and 70.8% in Nova Scotia to 28.9% in Ontario and 23.6% in Prince Edward Island. These proportions have remained relatively high and stable during the period studied in some provinces, such as British Columbia and Nova Scotia, and have declined steadily and notably in others. CONCLUSIONS: Data show that GP/FPs' involvement in vaginal births in most Canadian provinces is decreasing. This trend demonstrates a shift in GP/FPs' practice patterns and could indicate a coming shortage of obstetrical care providers. PMID- 10751999 TI - Family physicians in maternity care. Still in the game? Report from the CFPC's Janus Project. AB - OBJECTIVE: To describe family doctors' contribution to maternity care in Canada and to observe the influence of age, sex, region of the country, and practice population on provision of maternity care. DESIGN: SURVEY: College of Family Physicians of Canada's Janus Project national family physician survey. SETTING: All 10 provinces and two territories. PARTICIPANTS: Random sample of family physicians and general practitioners, both members and non-members of the College. MAIN OUTCOME MEASURES: Proportion of family doctors participating in prenatal, intrapartum, postpartum, and newborn care, and proportion of doctors involved in intrapartum care by age, sex, location in Canada, and practice population. RESULTS: Overall response rate was 58%. Just over 50% of all family doctors in Canada are involved in some aspect of maternity care; 19% do intrapartum care; and 33% are involved in prenatal (shared) care. Similar proportions of men and women still do intrapartum care, but women care for more pregnancies than men. More family doctors serving rural areas are doing intrapartum care compared with doctors in urban areas, although those in urban areas tend to do more deliveries. The western provinces have the highest percentages of intrapartum caregivers. A gradual decline in percentage of intrapartum caregivers by age group increases among the 55- to 64-year-old cohort. Almost a quarter of women doctors younger than 35 years are doing intrapartum care. Most physicians doing prenatal (shared) care look after women until the third trimester. CONCLUSIONS: Family doctors are still providing a large proportion of maternity care in Canada. This contribution must be nurtured by the College through its Maternity and Newborn Care Committee and other contacts to encourage family doctors to continue offering this essential service to childbearing women in Canada. PMID- 10752000 TI - Women's experience of maternal serum screening. AB - OBJECTIVE: To explore the ideas, opinions, feelings, and experiences of women regarding prenatal genetic screening, specifically maternal serum screening (MSS). DESIGN: Qualitative technique of focus groups. SETTING: Northern, rural, inner-city, urban, and suburban communities in Ontario. PARTICIPANTS: Women who had given birth to babies from January 1994 to May 1996, but who were not currently pregnant (n = 60). METHOD: Six focus groups composed of women living in various communities who had recently given birth to babies explored the experience of MSS. MAIN FINDINGS: Women want informed choice about prenatal genetic screening. Three factors influenced women's decisions to undergo or decline prenatal genetic screening: their personal values, including their philosophy of life, moral, and religious values, and attitudes regarding Down syndrome and disability; social support including their partners, families, and friends; and quality of information from health care providers. Women want their providers to give them information personally; they want to receive the information as early as possible in prenatal care to allow time for reflection; and they want unbiased, accurate information in order to make a decision that is in keeping with their personal values and beliefs. CONCLUSIONS: Knowledge of women's ideas, opinions, feelings, and experiences regarding MSS suggests specific ways health care providers can facilitate informed decision making in prenatal screening. Providing information about genetic testing needs to be individualized, with women actively participating in the decision-making process. Information needs described by these women could apply to other prenatal genetic tests that might be available in the future. PMID- 10752001 TI - Are SSRIs safe for pregnant and breastfeeding women? AB - OBJECTIVE: To summarize the literature on use of the selective serotonin reuptake inhibitor (SSRI) class of antidepressants for pregnant and breastfeeding women. DATA SOURCES AND STUDY SELECTION: MEDLINE was searched over the past 9 years. An examination of the literature over the last 8 years was included in this review. Primary studies consist of prospective investigations and case studies. Evidence for the safety of SSRIs is limited, but some good studies describe the effects of untreated depression. SYNTHESIS: All studies report that infants are exposed to SSRIs; the drugs can be measured in their plasma and urine. Some evidence shows an increase in minor perinatal complications among infants exposed to SSRIs late in gestation or while nursing. No studies, however, have found an increase in major fetal malformations or pregnancy-related complications. The only investigation of long-term neurodevelopmental outcomes found no negative outcomes among infants exposed to SSRIs during pregnancy. Data are scarce, and readers are cautioned to take into consideration the limitations of the studies reviewed before making definite treatment decisions. CONCLUSIONS: Major fetal malformations and exposure to SSRIs during pregnancy and lactation do not appear to be associated. Some minor perinatal complications have been reported. Data on the long-term developmental outcomes of children exposed to SSRIs in utero and during breastfeeding are limited. PMID- 10752002 TI - Diabetic nephropathy. Prevention and early referral. AB - OBJECTIVE: To review the clinical and pathophysiologic features of diabetic nephropathy and to examine evidence supporting primary, secondary, and tertiary treatment strategies. QUALITY OF EVIDENCE: The medical literature provides both level 1 and level 2 evidence on treatment of diabetic nephropathy, including randomized controlled trials, well-designed clinical trials without randomization, consensus papers, and cohort and case-control analytic studies. MAIN MESSAGE: Diabetes is the most common cause of end-stage renal failure in Canada and the United States, and both diabetes and its renal complications are increasing. Diabetic nephropathy, in both type 1 and type 2 diabetes, usually progresses through five stages. Treatment and prevention strategies depend on stage of disease. Primary prevention includes addressing hyperglycemia, hypertension, and smoking. Secondary prevention adds angiotensin-converting enzyme inhibitors, cholesterol lowering, and perhaps restrictions on dietary protein. Tertiary care, including dialysis or transplantation, is generally managed by nephrologists, but family physicians continue to play an important role in the care of these patients. CONCLUSIONS: Diabetic nephropathy is a serious cause of morbidity and mortality for patients with type 1 and type 2 diabetes. To reduce end-stage diabetic nephropathy and its complications, both specialists and family physicians need to focus efforts on primary and secondary prevention strategies. PMID- 10752003 TI - Preparing patients to travel abroad safely. Part 2: Updating vaccinations. AB - OBJECTIVE: To provide, for family physicians without access to a travel clinic, evidence-based recommendations on vaccinating infants and children, adults, pregnant women, and immunocompromised patients traveling to non-Western countries. QUALITY OF EVIDENCE: Searches were undertaken of MEDLINE from 1990 to November 1998 (372 articles); the Cochrane Collaboration Library; publications of the National Action Committee on Immunization and the Committee to Advise on Tropical Medicine and Travel in Canada Communicable Disease Reports; the Canadian Immunization Guide; and Laboratory Centre for Disease Control, United States Centres for Disease Control, and World Health Organization websites. Evidence based statements, randomized controlled trials, systematic reviews, and meta analyses were selected. Vaccination recommendations are based on this evidence. MAIN MESSAGE: Physicians should complete vaccination schedules for children whose primary series is incomplete and vaccinate unvaccinated adults. Hepatitis A is widespread, and travelers to areas where it is endemic should be vaccinated. The elderly should be vaccinated against influenza and pneumococcal disease. Pregnant women should receive vaccines appropriate to their trimester. Immunocompromised patients should be vaccinated, but BCG and live vaccines are contraindicated. Travelers to areas where meningitis, typhoid, cholera, Japanese encephalitis, and rabies are endemic should be vaccinated if they are likely to be exposed. Those traveling to areas where tuberculosis is endemic should take precautions and should have skin tests before traveling and 2 to 4 months after return. CONCLUSIONS: Family physicians can administer all necessary vaccinations. They can advise pregnant women and immunocompromised people about the balance of risk of disease and benefits of vaccination. PMID- 10752004 TI - Elevated levels of serum creatinine. Guidelines for management and referral. Canadian Society of Nephrology. PMID- 10752005 TI - New vaccine for an old problem. Laboratory Centre for Disease Control. PMID- 10752006 TI - Acetaminophen intoxication during treatment: what you don't know can hurt you. AB - For over two decades, pediatricians have been made aware of the potential risk associated with the acute ingestion of large single and/or multiple doses of acetaminophen (APAP). Clearly, APAP-induced hepatotoxicity remains as a recognized medical emergency which, when treated promptly with appropriate gastrointestinal decontamination and when indicated, with the antidote N acetylcysteine, has a uniformly good clinical outcome. Recently, the hepatotoxic potential associated with "therapeutic" APAP administration has been brought to the attention of the pediatric community. This review explores the issue of APAP toxicity with therapeutic intent by examining both the clinical literature and also, relevant information concerning the basic pharmacology and toxicology of this old and widely used nonprescription drug. A "risk profile" is developed with regard to factors that may predispose infants and children to this iatrogenic form of toxicity so that the awareness of physicians and other caregivers (including parents) can be heightened and preventative education administered. As is true for most all potentially beneficial medicines used in pediatrics, awareness of the actual amount of drug received from all sources and caution to not exceed the age-appropriate dosing guidelines (i.e., both amount and duration) contained in the approved labeling for all products containing APAP will insure safe and effective therapy. PMID- 10752007 TI - Neurodevelopmental outcome in ECMO vs near-miss ECMO patients at 5 years of age. AB - The objective of this study was to compare the outcome of children at 5 years of age who were treated with extracorporeal membrane oxygenation (ECMO) and those who were critically ill but did not meet ECMO criteria, identified as near-miss ECMO. In one of the longest studies of its kind, we compared the neurodevelopmental outcome of 76 5-year-old ECMO-treated children with 20 5-year old near-miss ECMO patients with similar primary diagnoses. The two groups were compared for demographic data, level of ventilatory support, and degree of hyperventilation. The comprehensive assessment protocol included an assessment of intelligence (IQ), attainment of preacademic and early academic skills, and parents' report of adaptive behavior. Both groups had similar demographic data and primary diagnosis. The near-miss ECMO patients required increased ventilatory support but not significantly more than the ECMO patients. The cognitive outcome was similar in both groups with mean estimated Full-Scale IQ in the normal range for near-miss and ECMO groups (89 and 97, respectively). Rates of severe mental handicap (FSIQ < 70) (near-miss = 11%, ECMO = 12%) and risk for school failure (near-miss = 38%, ECMO = 37%) were also similar. More parents of near-miss ECMO patients reported immature adaptive skills than did parents of ECMO patients, although the numbers were small in each group. Rates of parent-reported child behavior problems were similar in both groups. ECMO and near-miss ECMO patients have similar cognitive and adaptive outcomes at 5 years of age. A significant number in each group are at risk of school failure and should be closely followed up. PMID- 10752009 TI - Adolescents and sports: risk or benefit? AB - In order to begin to compensate for a lack of data on the effects of athletic participation in the development of adolescent mental health patterns, as well as to assess general health of the adolescent population, the Juvenile Wellness and Health Inventory (JWHS-76) was administered to 1,769 high school students. Our results indicate that sports participation is associated with self-reported lower total risk scores, mental and physical health benefits, and an increased risk of injury. This suggests a positive role for organized sports participation in youth populations. Prospective studies are needed to assess the impact of different sports, mounting performance pressure, and transition into collegiate levels of participation. PMID- 10752010 TI - Momma, don't let your babies grow up to be academics! PMID- 10752008 TI - The ketogenic diet in refractory epilepsy: the experience of Children's Hospital of Pittsburgh. AB - The ketogenic diet appears to be effective in reducing seizure frequency in patients with epilepsy refractory to antiepileptic drug therapy. Reported seizure frequencies before and after the diet was initiated were obtained for 48 patients started on the ketogenic diet between December 1994 and January 1998 at Children's Hospital of Pittsburgh. The majority of patients (71%) were able to achieve > or = 50% reduction in seizure activity. Of these, more than half (53%) had > 90% reduction in seizures after 45 days of diet therapy. Complications included gastrointestinal complaints and infrequent lipid abnormalities. The ketogenic diet appears to be an effective method of treatment for children with epilepsy refractory to drug therapy. PMID- 10752011 TI - Morbid obesity in a young child. PMID- 10752012 TI - Behaviors associated with Neisseria gonorrhoeae and Chlamydia trachomatis: cervical infection among young women attending adolescent clinics. PMID- 10752013 TI - Meconium periorchitis--a cause of scrotal mass in the newborn. PMID- 10752014 TI - Extreme thrombocytosis in response to PEG-ADA: early therapeutic and risk indicator. PMID- 10752015 TI - Rapid advancement of feedings after pyloromyotomy for pyloric stenosis. PMID- 10752016 TI - Gastrointestinal bleeding in older people. AB - Aging is associated with an increased rate of comorbidity, greater medication use, and atypical clinical presentations. The aging of the population makes the evaluation and management of gastrointestinal bleeding in older people a special and increasingly common clinical challenge. The unique features and common causes of upper and lower gastrointestinal bleeding in older people are reviewed. Important management issues considered include hemodynamic resuscitation, anticoagulation, and endoscopic and surgical therapy. PMID- 10752017 TI - Gastrointestinal bleeding in infancy and childhood. AB - Gastrointestinal (GI) bleeding is an alarming problem in children. Although many causes of GI bleeding are common to children and adults, the frequency of specific causes differs greatly, and some lesions, such as necrotizing enterocolitis or allergic colitis, are unique to children. This article reviews the spectrum of GI bleeding in infants and children. The causes, diagnostic evaluation, and management are discussed, and differences with adult medicine are highlighted. PMID- 10752018 TI - Small intestinal bleeding. AB - Bleeding from the small intestine may be difficult to diagnose, because of the organ's length, free intraperitoneal location, and the nature of the lesions that bleed in the small bowel. Although there are several causes of intestinal bleeding, angiodysplasias are most common. Several different tests can be used to identify a bleeding site preoperatively and intraoperatively, including enteroscopy. PMID- 10752019 TI - Diagnosis and treatment of nonsteroidal anti-inflammatory drug-associated upper gastrointestinal toxicity. AB - Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely prescribed in the United States to treat pain and reduce inflammation from chronic inflammatory disorders such as rheumatoid arthritis and osteoarthritis. Approximately 40% of older Americans take NSAIDs. Chronic NSAID use carries a risk of peptic ulcer and other gastrointestinal disturbances. This article reviews the diagnosis of medication induced ulcers based on clinical presentation, laboratory tests, and endoscopic findings to assist the clinician in early diagnosis and appropriate therapy. Risk factors for NSAID-induced ulcers include old age, poor medical status, prior ulcer, alcoholism, smoking, high NSAID dosage, prolonged NSAID use, and concomitant use of other drugs that are gastric irritants, such as alendronate, a bone resorption inhibitor prescribed for osteoporosis. Appropriate treatment options for patients with medication-induced ulcers include dosage reduction, medication substitution, medication withdrawal, antiulcer therapy, and discontinuation of other gastrotoxic drugs. PMID- 10752020 TI - Management of gastrointestinal bleeding induced by gastrointestinal endoscopy. AB - Therapeutic gastrointestinal endoscopy has a much greater risk of inducing gastrointestinal hemorrhage than diagnostic endoscopy. For example, colonoscopic polypectomy has a risk of approximately 1.6% of inducing bleeding, compared with a risk of approximately 0.02% for diagnostic colonoscopy. Higher-risk procedures include colonoscopic polypectomy, endoscopic biliary sphincterotomy, endoscopic dilatation, endoscopic variceal therapy, percutaneous endoscopic gastrostomy, and endoscopic sharp foreign body retrieval. The risk of inducing hemorrhage is decreased by meticulous endoscopic technique. Hemorrhage from endoscopy may be immediate or delayed. Immediate hemorrhage should be immediately treated by endoscopic hemostatic therapy, including injection therapy, thermocoagulation, or electrocoagulation. Delayed hemorrhage generally requires repeat endoscopy for diagnosis and for therapy, using the same hemostatic techniques. PMID- 10752021 TI - Hematologic management of gastrointestinal bleeding. AB - The hematologic management of gastrointestinal (GI) bleeding requires evaluation of the underlying cause of bleeding, associated diseases that can exacerbate the bleeding, and identification of related and unrelated coagulation abnormalities. Erythrocyte transfusions are given to increase oxygen carrying capacity; however, there is limited information on the level of anemia that places a patient at increased risk of adverse events after a GI bleed and when patients should receive erythrocyte transfusion. Isolated thrombocytopenia is uncommon in patients with GI bleeding, and there is little evidence documenting the degree of thrombocytopenia associated with an increased risk of bleeding. Platelets are often administered when the count is 50,000 per cu/mL in a bleeding patient. The coagulopathy of liver disease is the most common abnormality seen in the setting of GI bleeding. Fresh-frozen plasma (FFP) should be given in a dose equivalent to the underlying abnormality and the common practice of administering 2 units of FFP is often insufficient in a bleeding patient. PMID- 10752022 TI - Surgical management of gastrointestinal bleeding. AB - Severe gastrointestinal bleeding has historically been a clinical problem primarily under the purview of the general surgeon. Diagnostic advances made as the result of newer technologies, such as fiberoptic and video endoscopy, selective visceral arteriography, and nuclear scintigraphy, have permitted more accurate and targeted operations. More importantly, they have led to safe, effective nonoperative therapeutic interventions that have obviated the need for surgery in many patients. Today, most gastrointestinal bleeding episodes are initially managed by endoscopic or angiographic control measures. Such interventions are often definitive in obtaining hemostasis. Even temporary cessation or attenuation of massive bleeding in an unstable patient permits a safer, more controlled operative procedure by allowing an adequate period of preoperative resuscitation. Despite the less frequent need for surgical intervention, traditional operative approaches, such as suture ligation, lesion or organ excision, vagotomy, portasystemic anastomosis, and devascularization procedures, continue to be life-saving in many instances. The proliferation of laparoscopic surgery has fostered the application of minimally invasive techniques to highly selected patients with gastrointestinal bleeding. Intraoperative endoscopy has greatly facilitated the accuracy of laparoscopic surgery by endoscopic localization of bleeding lesions requiring excision. It is anticipated that the evolving technologies pertinent to the diagnosis and management of gastrointestinal bleeding will continue to promote collaboration and cooperation between gastroenterologists, radiologists, and surgeons. PMID- 10752023 TI - Fifty landmark discoveries in gastroenterology during the past 50 years. A brief history of modern gastroenterology at the millennium: Part I. Gastrointestinal procedures and upper gastrointestinal disorders. AB - During the last half century, many outstanding discoverers have revolutionized the clinical practice and science of gastroenterology. Whereas the scientific results are widely disseminated, the discoverers have received inadequate recognition, and the history of their discoveries is poorly known. At the millennium, a committee selected the 50 landmark discoveries in gastroenterology during the past 50 years. A brief history of each landmark discovery is presented. Part I presents the landmark discoveries in gastrointestinal (GI) procedures and in upper GI disorders. Part II of this presentation, which covers landmark discoveries in other areas of gastroenterology, will publish in Part II of the volume on High Risk Gastrointestinal Bleeding. PMID- 10752024 TI - [Generation of Kruppel phenocopies by injecting into Drosophila embryos RNA complementary to mRNA in parallel orientation]. AB - RNA preparations synthesized in vitro were used to study the influence of RNA interference on the Kruppel gene activity in Drosophila embryos. RNA complementary in parallel orientation to the mRNA fragment proved to induce the development of Kruppel phenocopies. The data obtained indicate that mechanisms of specific regulation of gene activity exist in Drosophila cells, which are sensitive to the formation of both parallel and antiparallel RNA-RNA duplexes that include mRNA of the corresponding gene. PMID- 10752025 TI - [Polymorphism of the SUP35 gene and its product in the Saccharomyces cerevisiae yeasts]. AB - The product of the SUP35 gene of the saccharomycete yeast, the translation termination eRF3 factor, can be converted in prion, the heritable determinant of protein nature. The nucleotide sequence of this gene from the strain belonging to Peterhof genetic lines of the yeast Saccharomyces cerevisiae was determined. A comparison of the identified sequence with SUP35 sequences in the database of GenBank allowed the detection of polymorphic sites both in the SUP35 gene and its product. The location of polymorphic sites in the evolutionarily nonconserved N terminal protein region confirmed that this eRF3 fragment lacks functions vital to life activity. Nevertheless, these sites are located in the vicinity of sites, whose role in the prion conversion of eRF3 has been established. Based on this, natural polymorphism of the primary eRF3 structure is assumed to be connected with the existence of different variants (strains) of its prion analog. PMID- 10752026 TI - [Mutants of the nitrogen-fixing rhizospheric bacteria Pseudomonas sp. 418 with loss of the ability to colonize roots]. AB - Mutants of nitrogen-fixing rhizospheric bacterium Pseudomonas sp. 418, which lacked the competitive ability to colonize roots, were induced by random Tn5 mutagenesis. By means of Southern blot analysis, it was shown that single transposon insertions occurred in eight mutants, whereas in two mutants, the Tn5 insertion occurred twice in different DNA regions. Analysis of these mutants revealed the following disturbances of characters having adaptive significance: weakened attachment to the root surface; a defect in chemotaxis; impaired motility as a result of the loss of flagella or nondisjunction of cells after division; and alterations in the synthesis of exopolysaccharides. The effect of Tn5 insertions was, as a rule, pleiotropic, which suggests the coordinated expression of traits essential for the survival of bacterial cells in the root region. PMID- 10752027 TI - [Generation of Y-chromosome insertions into left arm of chromosome 2 of Drosophila melanogaster]. AB - An experimental procedure describing production of insertions of Y-chromosome material into autosomes of Drosophila is presented. Irradiated Y;2 translocations served as source material. The insertion selection scheme was based on the emergence of additional progeny classes in the case of independent segregation of the detached fragments of the Y chromosome and autosome. A total of seven insertions of Y-chromosome material into the left arm of chromosome 2, specifically in regions 29F, 34A, and 36B, were obtained. All insertions were lethal in the homozygous state and caused crossing-over suppression in the left arm of chromosome 2. In addition, these mutations induced the formation of loops between the chromocenter and the region of insertion, as well as breaks in one or both homologs, which are frequently observed in preparations of polytene chromosomes. The selection scheme suggested can be used to produce insertions in any region of Drosophila melanogaster chromosomes 2 and 3, for which the Y;2 translocations exist. PMID- 10752028 TI - [Frequency of chromosome aberrations after irradiation of gametes of cabbage root fly (Delia brassicae Bouche) with various doses of X rays]. AB - Chromosome rearrangements were isolated via crossing of F1 offspring from X irradiated male and female Delia brassicae with intact insects. An enhanced (more than 30%) rate of late embryonic lethality (LEL) in clutches was the primary criterion of rearrangements. When males alone were irradiated at 2.5, 5, and 15 Gy, a total of 7.8, 9.6, and 23.6% of their offspring, respectively, inherited semisterility. After irradiation of females alone, the LEL rate in F1 was insignificant. Hybridization analysis of F2 revealed 18 lines with autosomal inheritance of semisterility. Fourteen lines (43.7%) exhibited an increased rate of LEL linked to the male sex. PMID- 10752029 TI - [Pericentromeric and intercalating alpha-heterochromatin of polytene chromosomes of the malaria mosquito]. AB - The properties of heterochromatin from polytene chromosomes of the malaria mosquito Anopheles messeae Fal. and A. atroparvus V. Tiel. were studied by various methods of differential staining and by hybridization in situ with two repetitive DNA sequences of Drosophila melanogaster. In malaria mosquito, the heterochromatin was heterogeneous. Two forms of alpha-heterochromatin were revealed: pericentromeric and intercalary heterochromatin, which is localized within the internal chromosome regions. PMID- 10752030 TI - [Effect of genotypic environment on phenotypic manifestation of radius incompletus mutation in Drosophila melanogaster]. AB - Genetic analysis of marked regions of Drosophila chromosome 3 was performed in order to localize the "effective factors" of the polygene system that controls the expression of the limiting mutation in radius incompletus, the major-effect gene. The marked homozygous strain with genotype th st ri sr ca was crossed with the "selection" riSN strain. Contributions of the marked regions of chromosome 3 to the expression of the proximal and distal fragments of the wing radial vein were estimated. It was demonstrated that the th-st region of the marked strain contained a polygene determining a large positive contribution to the lengths of both fragments, whereas the st-ri region contained a polygene determining a large negative contribution to the length of the distal fragment compared to the riSN strain. Crossings were performed between strains that contained Mendelian mutations of the ri, ve, and vn major-effect genes of the wing vein patterns. Unexpectedly, a strong, non-additive effect of the interaction between these mutations was found. This effect was expressed as a complete disarrangement of the wing vein pattern. Each participant gene may be regarded as a large-effect polygene relative to the other genes. PMID- 10752031 TI - [Distribution of cytoplasmically inherited bacteria Spiroplasma causing female bias in the Eurasian populations of Adalia bipunctata]. AB - Two-spot ladybirds Adalia bipunctata were collected from the populations of Western and Eastern Europe and Central Asia. The agent killing males at the early embryonic stage in these populations was identified as bacteria of the genus Spiroplasma. Bacteria found in A. bipunctata proved to be phylogenetically related to Spiroplasma ixodetis (typical line Y-32) found in tick Ixodes pacificus but not to Spiroplasma causing the death of male embryos in Drosophila. PMID- 10752032 TI - [Analysis of genetic diversity of wheat using genetic resources information system GRIS]. AB - The possibilities of using the Genetic Resources Information and Analysis System (GRIS) 3.2, a database and software package, in studies of various aspects of genetic diversity in wheat are demonstrated. A model of genetic diversity was analyzed; diversity levels were estimated in wheat from various regions at different times. The genetic basis of Russian winter and spring common wheat cultivars was investigated. Transmission of powdery mildew resistance genes was traced in cultivars with identified Pm genes. For some genes, patterns of geographical distribution were characterized. Description of GRIS 3.2 is given: the package consists of a database on the global gene pool of wheat and software on genealogical and statistic analyses of genetic diversity. PMID- 10752033 TI - [The role of phytohormones in tumor formation in radish]. AB - Tumor formation was studied in inbred radish lines that produce tumors on plant roots during flowering. In all radish lines under consideration, the sequences homologous to oncogenes tmr/tml of Agrobacterium tumefaciens were revealed by Southern hybridization. No sequences homologous to the tms locus of A. tumefaciens and the oncogenes of A. rhizogenes were determined. It was found that auxin sensitivity and the tumor-producing capacity were coinherited. We suggest that tumor phenotype arise as a result of a combination between agrobacterial "cytokinin" oncogenes and certain alleles of "auxin" radish genes. PMID- 10752034 TI - [Chromosomal variability of ginseng cells transformed with plant oncogene rolC]. AB - Chromosome numbers were was studied in ginseng cell line 1c transformed with Agrobacterium rhizogenes strain A4, which carried plasmid pRiA4, and with A. tumefaciens strain GV3101, which carried vector pPCV002-35S rolC. As compared with the nontransformed cell line 1c, tumor cell cultures 1c-A4 and 1c-rolC and the tissues of rolC teratoma (excluding leaves) displayed higher polyploidy and aneuploidy. The 1c-A4 and 1c-rolC hairy-root cultures also had aneuploid and polyploid cells, but the chromosome variation was lower than in tumor cells or the initial culture 1c. Generally, an increase of chromosome variation in cultivated cells was the main effect of the integration of several oncogenes, which were in the A. rhizogenes A4 T-DNA, or of the individual rolC gene in the ginseng genome. Another effect consisted in stabilization of the chromosome number in some differentiated transgenic tissues. Possible reasons for this effect are discussed. PMID- 10752035 TI - [Genetic diversity of the European bison herd from the Prioksko-Terrasny nature reserve]. AB - Maintaining the existing genetic diversity in populations is one of the most important measures for species conservation. Some components of this diversity in the bison population of the Prioksko-Terrasny Nature Reserve, including the level of preservation of the founder genetic diversity or the founder genomic equivalent (fge) of the current bison stock and the indices of similarity (mk) and genome uniqueness (gu), as well as their effects on viability of the animals from 1950-1995 were studied. The data obtained were treated and the diversity parameters were calculated with the use of the SPARKS software package. This was performed in collaboration with the Institute for Zoo Biology and Wildlife Research (Berlin, Germany). The results of the analysis indicated that, in breeding groups of the European bison, the fge, mk, and gu values should be maintained at levels higher than 1.30, lower than 40%, and higher than 10%, respectively. When selecting males for specific groups of females, the male gu should be at least 20%, and mk should be lower than 40%. PMID- 10752036 TI - [Chromosomal variability of the field mouse Apodemus agarius (Rodentia, Muridae)]. AB - Chromosome sets of 114 Apodemus agrarius mice from 29 localities in Moldova, Ukraine, Siberia, and Far East were studied by means of G-, C-, and NOR-banding. In all populations studied, the Y chromosome was shown to be a medium-size acrocentric chromosome consisting of heterochromatin. Chromosome polymorphism observed in populations from Primorskii krai concerned (1) the morphology of the first two autosome pairs (variants A/A, A/ST, and ST/ST), (2) the number of metacentric chromosomes (from 6 to 8), and (3) heterochromatin localization in the pericentromeric regions of two metacentric chromosome pairs. A karyotype with an additional heterochromatic microchromosome found in all the metaphases studied was described in one mouse from a locality of western Primorye that has not been studied previously. In the karyotype of 15 mice from four populations of Primorye, the pool of nucleolus organizer regions is distributed over three autosome pairs rather than over four, as is the case A. agrarius from Europe. Based on the analysis of literature sources and our own data, the problem of chromosome polymorphism in the field mouse is discussed. PMID- 10752037 TI - [Polymorphism and divergence of karyotypes in taimens of the Hucho genus]. AB - Karyotypes of Siberian taimen Hugo taimen from the Manoma River (Amur basin) were investigated. The karyotypes examined differed in chromosome number from 2n = 82 to 2n = 83; chromosome arm number was NF = 112. These differences, as well as the difference in the karyotype of Siberian taimen from the lower flow of the Amur River (2n = 84) described earlier, are due to Robertsonian polymorphism of one pair of large submetacentric chromosomes. The nucleolus organizer regions are located on the short arms of one or two subtelocentric chromosomes of different pairs. The probable sequence of karyotype divergence in taimens of the Hugo genus is discussed. PMID- 10752038 TI - [Analysis of allele frequency of seven microsatellite loci of Y chromosome in three Tuva populations]. AB - The allele frequency distribution of seven microsatellite loci of the nonrecombining region of the Y chromosome (Y-STRs) was analyzed in three geographically distant indigenous populations of the Tuva Republic. The populations did not differ in allele frequency distribution of the seven Y-STRs. The Y-chromosome microsatellite loci in Tuvinians showed a high diversity (H = 0.575) that was nearly identical in all three populations. The genetic distance Ddm between the three populations was low, suggesting no subdivision of the modern male population of Tuva. Estimates of the period of linear changes in Ddm showed that Y-chromosome microsatellites can be used to reconstruct evolutionary events dating back no more than 40,000-50,000 years. The problems of human population phylogeny are discussed on the basis of data on Y-chromosome STRs. PMID- 10752039 TI - [Analysis of genealogical structure of populations. I. A method of collecting genealogical data in numerical form]. AB - A method for collecting genealogical data with respect to an individual, a family, and members of the whole population is suggested. The essence of vertical pedigree construction consists of the same type of steps for filling in data (in the fixed order which excludes skips in the enumeration of lines of descent) about the father and the mother of the next ancestor. Each number in the received ordered list of ancestors uniquely determines a path (line of descent) to the given pedigree member. The path is explicitly described by a sequence of digits 0 and 1 (that corresponds to the sequence of fathers and mothers in the line of descent) at binary notation of this number. As a result, a pedigree is presented as a set of numbered rows that contain information, which uniquely identifies direct ancestors as individual persons. Results of joining separate pedigrees are recorded as a family list that contains lists of children for each parental pair. A pair of parents (more exactly, pointers of their families in the previous generation and numbers of pair members in their families) plays the role of the family "heading." Such a family list permits one to trace lines of descent and relationships for any population members presented in the list. It contains all genealogical information within the bounds of the study in a compact form. Here the process of collection requires considerably less time than traditional graphic representation of pedigrees. In addition, due to repeated checks of data during accumulation of material, error is minimized. Using pedigrees that have been collected, it is possible to calculate the coefficient of inbreeding manually. In connection with the wide prevalence of personal computers at present, it is also important that the data received are in fact ready to direct input to a computer for further automated data processing. PMID- 10752040 TI - [Analysis of genealogical structure of populations. II. The use od numerical pedigrees for calculation of inbreeding coefficient]. AB - A method for calculation of inbreeding coefficient F in a numerical pedigree with no reference to its graphic representation is suggested. For calculation of F, a formula that does not take into account inbreeding coefficients of common ancestors and admits intersections in a loop is used. An advantage of this method is that it automatically finds all loops formed by paths to common ancestors. Detecting these loops via their tracing in a graphic pedigree with intersecting lines of descent creates a possibility of errors. A criterion of existence of at least one common link for two numerical paths is presented. It enables one to exclude pairs of paths to common ancestors that do not form loops. The methods considered for computing F in a given pedigree give exact values of the inbreeding coefficient for autosomal and sex-linked loci and generalize the known approximate approaches. The methods are illustrated by examples. PMID- 10752041 TI - [Analysis of genetic heterogeneity of bronchial asthma in relation with the age at the onset of disease]. AB - Earlier, the distribution of bronchial asthma (BA) morbidity with respect to the age of onset (AO) in the Moscow population was found to be bimodal. The distribution had two peaks (before and after 25 years of age) and a significant (P < 0.001) minimum between them. Based on these data, genetic heterogeneity of BA with respect to AO was hypothesized. The purpose of this study was to test this hypothesis via analysis of BA morbidity in families of probands with different AOs. The BA morbidity at different ages and the total recurrent risk of BA were estimated in 1518 relatives of 815 BA probands registered in several district outpatient clinics of Moscow. Based on the data obtained, phenotypic between relatives and correlation by genotype between early-onset and late-onset BA cases (with AOs under and over 25 years, respectively) were estimated. It was demonstrated for the first time that the age distribution of BA morbidity in families of probands was also bimodal. Moreover, when probands with early and late AOs were analyzed separately, proband relatives in each of the two groups exhibited these two peaks of morbidity. This suggests that BA that begins in adolescence and BA of adults are not genetically independent forms of the disease. This agrees with the data on the correlation by genotype between the "forms" with the early and late AOs, which does not significantly differ from 1. However, the prevalence of BA was higher in relatives of those probands who developed BA under the age of 25 compared to relatives of those who developed BA over the age of 25 (11.28 and 7.31%, respectively; P < 0.05). Therefore, patients with early-onset BA are more "burdened" genetically with respect to this disease. Since the BA genetic heterogeneity connected with AO has not been confirmed in this study, it is assumed that the observed bimodal distribution of BA morbidity with respect to age is accounted for by the effect of age itself. In other words, it is hypothesized that ontogenetic factors affect susceptibility to BA so that the susceptibility threshold varies with age. PMID- 10752042 TI - [Geography of genetic processes in populations: gene migrations in Siberia and Far East]. AB - A map of gene migration rate m in the indigenous population of Siberia and the Russian Far East was constructed on the basis of data obtained from questionnaires of 1960 to 1990. The mean gene migration rate weighted with respect to the region area and averaged over 3951 grid nodes was m = 0.0083. Weighting with respect to population density yielded a significantly lower rate (m = 0.0053), which reflected a more intense gene exchange in less populous regions of traditional nomadism. The association between gene migration rate m and genetically effective population size Ne was analyzed. The parameter Nem, which characterizes the interpopulation gene diversity, was used to identify regions where this parameter is autoregulated and those where the autoregulatory mechanisms were disrupted. A tree of ethnolinguistic types was constructed. Its analysis did not reveal any association between migration structure and linguistic characteristics, suggesting that the spreading of cultural elements is not necessarily associated with migration. The tree was also used to construct a map of ethnos-forming migration; its major element reflected migration from the Baikal and Altai regions to the ethnic region of modern Yakuts. PMID- 10752043 TI - [Radiation hybrid mapping: statistical criteria and their properties]. AB - Statistical tests specially developed for radiation hybrid (RH) mapping, modified likelihood-ratio test and Akaike's information content, are proposed. They allow several most likely gene orders to be identified to a certain accuracy. The properties of the proposed tests that characterize the accuracy of gene ordering as dependent on the number of genes mapped, the position of the selective gene, and the sample size have been studied. Recommendations as to the practical use of these tests are presented. PMID- 10752044 TI - [Multiloci DNA fingerprinting in population studies: primary analysis of blot hybridization results]. AB - The principles of primary treatment of blot-hybridization patterns obtained by means of multilocus DNA fingerprinting method are suggested. Final results, represented as a matrix of fragment sizes, are characterized by sufficiently high fidelity and reproducibility. PMID- 10752045 TI - In support of AIDS activism. PMID- 10752046 TI - La desesperacion: migrant and seasonal farm workers living with HIV/AIDS. AB - Migrant seasonal farm workers (MSFWs) in south Texas are predominantly Mexican American and represent one of the most impoverished and medically underserved populations in the United States. La Frontera is a collaborative partnership between the University of Texas Health Science Center at San Antonio, migrant organizations, and HIV service delivery organizations to study and address special needs of MSFWs in Hidalgo and Maverick counties of south Texas. The purpose of this qualitative study is to explore the health-seeking experiences of HIV-positive MSFWs living in the La Frontera service area. Denzin's interpretive interactionism methodology guided the study. Thirteen participants were interviewed and audiotaped as they described their experiences. Findings from content analysis of the interviews revealed a description of the life of an HIV positive MSFW as la desesperacion (a separate way of life). After their diagnosis of HIV/AIDS, the major themes in their lives were living in secrecy (a silent cry), finding and receiving health care, and accepting the disease (may it be the will of God). PMID- 10752047 TI - HIV-related lipodystrophy: a clinical syndrome with implications for nursing practice. AB - Since the clinical introduction of protease inhibitors for the treatment of HIV disease in 1996, optimism has grown for the long-term survival of persons living with HIV disease. With the addition of protease inhibitors to highly active antiretroviral therapy, the number of deaths from AIDS has decreased dramatically. However, along with reports of dramatic clinical improvement, many cases of a novel lipodystrophy syndrome associated with high triglyceride levels, diabetes, accumulations of fatty tissue, and alterations in body shape have been reported by researchers, clinicians, and persons living with HIV disease. This article reviews the literature on the defining characteristics and pathogenesis of HIV-related lipodystrophy, summarizes the current state of the science, and discusses related research and clinical implications. PMID- 10752048 TI - Substance abuse and HIV: considerations with regard to methamphetamines and other recreational drugs for nursing practice and research. AB - Substance use continues to be closely associated with both HIV infection and treatment considerations in all at-risk populations. Among those groups heretofore not well characterized epidemiologically or clinically are those dual risk men who have sex with other men (MSM) and use and/or inject drugs. Of particular current concern with regard to drug-using MSM is the growth in popularity of a group of recreational or so-called party drugs associated with specific social and sexual environments and networks. Chief among these drugs are hallucinogens, such as MDMA, ketamine, and GHB, and stimulants, such as cocaine, amphetamines, and methamphetamine. Increased methamphetamine use by MSM is particularly alarming because of its reported associations with high-risk injecting and sexual behaviors. Preliminary data are reported from an ethnographic exploration of MSM methamphetamine users in the Pacific Northwest of the United States. Case studies drawn from the data illustrate the complex and variable patterns of methamphetamine use among MSM. Finally, implications for nursing are discussed, and "upstream nursing" is suggested as a means of patient advocacy for HIV nurses working with substance-using populations. PMID- 10752050 TI - Continuous quality improvement project: decreasing the potential for the development in the inpatient setting of drug resistance by improving nursing practice for HAART administration. AB - HIV/AIDS patients' dissatisfaction with highly active antiretroviral therapy (HAART) medication administration in the inpatient setting was the impetus for a continuous quality improvement (CQI) project. The purpose of the CQI project was to initiate a change in nursing practice for HAART medication administration. The goal of the project was to decrease the potential for development of drug resistance in the inpatient setting related to nonadherence with food requirements for drug administration and to incomplete or "missed" doses of prescribed HAART. A secondary goal was to increase the provision of patient education on HAART medications by nurses. The interdisciplinary CQI team found that medication administration in the inpatient setting involved more than nurses simply "passing meds." Inpatient medication administration was a complex process involving a variety of hospital systems, departments, and traditions, all of which had an impact on patient care. The article describes the CQI methodology that was used for the project and how each step of the project was planned and implemented. Specific problems related to administering HAART in the hospital setting are listed as areas for needed nursing research. PMID- 10752049 TI - Support from health care providers and parental uncertainty during the diagnosis phase of perinatally acquired HIV infection. AB - Support from health care providers (HCPs) can reduce parental uncertainty during the diagnosis of serious childhood conditions, but the effect may change when the parent is chronically ill. The purpose of this study was to learn whether a mother's uncertainty about her HIV diminished the effect of support from HCPs on parental uncertainty during the diagnosis of perinatally acquired HIV infection. Participants were 25 women who accompanied their biological children to pediatric HIV screening clinics at two urban sites. Questionnaires were read to participants in a private area of clinic. To test for mediation, three models for parental uncertainty were examined using regression procedures. The results suggest that when a mother has HIV infection, HCPs can maximize the effect of their support on parental uncertainty by also addressing mother's uncertainty about her HIV during the baby's screening visits. PMID- 10752051 TI - Stress reduction and HIV disease: a review of intervention studies using a psychoneuroimmunology framework. AB - The field of psychoneuroimmunology (PNI) posits that relationships exist between stress, immunological impairment, and health outcomes. Accumulating evidence suggests that stress may hasten HIV disease progression by increasing viral replication, suppressing immune response, and inducing deleterious health-related behaviors. Interventions that attenuate the effects of stress are postulated to operate by altering cognitive perception and/or modulating neuroendocrine and sympathetic reactivity. A review of HIV/PNI intervention studies is presented as a guide for the inclusion of stress reduction interventions in comprehensive plans of care for HIV-infected individuals. Although effect and sample sizes are small, the results of these studies provide support for a positive effect of various interventions on immunological and health-related indices in HIV-infected individuals. PMID- 10752053 TI - Mothers. PMID- 10752052 TI - Immune reconstitution in the HAART era, Part 2: Implications for practice. AB - As discussed in Part 1 of this series, HIV infection alters the function of the immune system. These alterations include declines in CD4-positive lymphocyte counts, disturbances in the function of the CD4-positive lymphocyte, diminished delayed-type hypersensitivity (DTH) skin test responses and cell-mediated immune responses, phenotypic changes in lymphocytes, and changes in cytoxic T-lymphocyte and natural killer cell function. Although highly active antiretroviral therapy (HAART) has dramatically improved CD4-positive lymphocyte counts and quality of life, there have been unforeseen consequences of immune reconstitution. For example, cytomegalovirus (CMV) and hepatitis C flares have been observed in HAART treated patients coinfected with HIV and CMV and hepatitis C (HCV). Part 2 of this series will examine the clinical consequences of immune reconstitution in persons with HIV infection. PMID- 10752054 TI - A recent study shows selenium supplementation benefits HIV patients: Selenomax decreases risk of development of depressed-dejected mood state. PMID- 10752055 TI - New data show opiates take lead over cocaine in treatment admissions: opiates lead all other illicit drugs in treatment admissions. PMID- 10752056 TI - Gender and motivation. Introduction. PMID- 10752057 TI - The science and politics of gender research: the meanings of difference. PMID- 10752058 TI - Hidden feelings: emotionality in boys and men. PMID- 10752059 TI - Childhood aggression and gender: a new look at an old problem. PMID- 10752060 TI - Challenging sexual naturalism, the shibboleth of sex research and popular sexology. PMID- 10752061 TI - Gender and competitive motivation: from the recreation center to the Olympic arena. AB - Gender makes a difference; we do gender in sport. Gender is a pervasive social force in society, and the sport world reflects society's gender hierarchy in the extreme. Gender is so ingrained in our sport structure and practice that we cannot simply treat all athletes the same. Nor can we assume that male and female athletes are dichotomous opposites, and treat all males one way and all females another way. Biology is part of the mix, but biology is not destiny. Gender is a dynamic, social influence that varies with the individual, situation, and time. PMID- 10752062 TI - Rhythmic transcription: the molecular basis of oscillatory melatonin synthesis. AB - Pulsatile hormone synthesis and secretion are characteristic features of various oscillatory biological systems. Circadian rhythms are critical in the regulation of most physiological functions, and much interest has been centred on the understanding of the molecular mechanisms governing them. Adaptation to a changing environment is an essential feature of physiological regulation. The day night rhythm is translated into hormonal oscillations governing the metabolism of all living organisms. In mammals the pineal gland is responsible for the circadian synthesis of the hormone melatonin in response to signals originating from the endogenous clock located in the hypothalamic suprachiasmatic nucleus (SCN). The molecular mechanisms involved in rhythmic synthesis of melatonin involve the CREM gene, which encodes transcription factors responsive to activation of the cAMP signalling pathway. The CREM product, ICER, is rhythmically expressed and participates in a transcriptional autoregulatory loop which also controls the amplitude of oscillations of serotonin N-acetyl transferase, the rate-limiting enzyme of melatonin synthesis. Thus, a transcription factor modulates the oscillatory levels of a hormone. PMID- 10752063 TI - The frequency encoding of pulsatility. AB - Examples of pulsatile signalling abound in intercellular communication, suggesting that this phenomenon represents a major function of biological rhythms. Pulsatile signals can be encoded in terms of their frequency and prove more efficient than monotonous ones whenever constant stimulation induces desensitization of target cells. We address the main examples of frequency encoding of pulsatility, besides those of neuronal nature. Considered in turn are cAMP oscillations in the slime mould Dictyostelium discoideum, the pulsatile secretion of hormones such as gonadotropin-releasing hormone or growth hormone, intracellular Ca2+ oscillations, and circadian rhythms. Models based on receptor desensitization show the possibility of optimizing cellular responses to cAMP signals in Dictyostelium or to pulsatile hormonal stimulation. The models indicate how the optimal duration of the pulsatile signal and the optimal interval between successive pulses vary as a function of the rates or receptor desensitization and resensitization and of the maximum ligand level during stimulation. The frequency encoding of intracellular Ca2+ oscillations appears to rely on another molecular mechanism. Models based on protein phosphorylation by a Ca(2+)-calmodulin activated kinase show that the mean level of phosphorylated protein increases with the frequency of calcium spikes--which itself rises with the degree of stimulation--and that distinct levels of different phosphorylated proteins can be reached for a Ca2+ signal of given frequency. PMID- 10752064 TI - Timing-dependent modulation of insulin mitogenic versus metabolic signalling. AB - This chapter will not deal sensu stricto with the mechanisms and biological significance of pulsatile hormone secretion, the general theme of this book. Rather, we will attempt to demonstrate that timing events at the receiving end of the hormonal signal, i.e. the kinetics and duration of receptor activation in target cells and subsequent downstream signalling, can play an equally important role as that of the timing aspects of secretion, in determining the qualitative and quantitative aspects of hormonal responses. We will focus on the mechanisms that determine signalling specificity by the receptor tyrosine kinases, especially the insulin receptor and the type I insulin-like growth factor receptors (IGF-I receptor). We will be succinct and refer the reader to our recent reviews and publications on this topic and references therein. PMID- 10752066 TI - Orderliness of hormone release. AB - ApEn, approximate entropy, is a recently formulated family of parameters and statistics quantifying regularity (orderliness) in serial data, with developments both within theoretical mathematics, as well as numerous applications to multiple biological contexts. ApEn appears to have broad application to hormone pulsatility analysis within endocrinology, bringing a new perspective to the assessment of secretory patterns. ApEn is complementary to pulse detection algorithms widely employed to evaluate hormone secretion time-series--it is scale invariant and model-independent, evaluates both dominant and subordinant patterns in data, discriminates series for which clear pulse recognition is difficult, and often provides a direct barometer of feedback between subsystems. ApEn is applicable to systems with at least 50 data points and to broad classes of models: it can be applied to discriminate both general classes of correlated stochastic processes, as well as noisy deterministic systems. Moreover, ApEn is complementary to spectral and autocorrelation analyses, providing effective discriminatory capability in instances in which the aforementioned measures exhibit minimal distinctions. We present some basic background on the above, and illustrate various facets of ApEn utility via several representative endocrinological studies. PMID- 10752065 TI - Growth hormone pulse-activated STAT5 signalling: a unique regulatory mechanism governing sexual dimorphism of liver gene expression. AB - Growth hormone (GH) exerts sexually dimorphic effects on liver gene transcription that are regulated by the temporal pattern of pituitary GH release; this release is intermittent in male rats and nearly continuous in females. Comparisons of liver nuclear protein tyrosine phosphorylation in male and female rats have led to the discovery that the liver transcription factor STAT5b is tyrosine phosphorylated in male but not female rats in response to GH pulses. Intermittent plasma GH pulses trigger a rapid and repeated tyrosine phosphorylation and nuclear translocation of liver STAT5b in intact male rats, while the more continuous pattern of GH exposure down-regulates the STAT5b signalling pathway in female rat liver. The central importance of STAT5b for the physiological effects of GH pulses has been verified using a mouse gene knockout model. STAT5b gene disruption leads to a major loss of multiple sexually differentiated responses associated with the sexually dimorphic pattern of pituitary GH secretion. Male characteristic body growth rates and male-specific liver gene expression are decreased to wild-type female levels in STAT5b-/- males, while female-predominant liver gene products are increased in males to near female levels. STAT5b is thus a liver-expressed, latent cytoplasmic transcription factor that undergoes repeated tyrosine phosphorylation and nuclear translocation in response to intermittent plasma GH stimulation, and is a key intracellular mediator of the stimulatory effects of GH pulses on male-specific liver gene transcription. Other studies indicate, however, that STAT5a and STAT5b are both required for constitutive expression in female, but not male liver, of certain GH-regulated CYP enzymes. GH activation of both STAT5 proteins, which in turn form distinct homodimeric and heterodimeric DNA-binding complexes, is thus an important determinant of the sex-dependent and gene-specific effects that GH has on the liver. PMID- 10752067 TI - Prediction and significance of the temporal pattern of hormone secretion in disease states. AB - Comparison of the temporal pattern of hormone secretion in health and disease reveals distinct differences in many systems. Analysis of these visually apparent differences conventionally rests on computer-assisted programs based on either model assumptions, or estimations of hormonal decay rates or threshold values, all of which may not accurately reflect physiological and/or pathophysiological states. Only recently have new methods evolved which are independent of preexisting knowledge of the system under study. Apart from the widely used approximate entropy statistic (ApEn), a measure for the regularity of a time series, artificial neural networks are able to capture temporal structures in endocrine rhythms without any previous assumptions. In particular, non-linear dynamical systems may be delineated and separated from random behaviour. This is achieved by mapping complex input data to a given complex output by propagating data from the input layer to the output layer through a larger number of interconnections, so-called hidden layers. The networks are capable to extract relevant features from training samples and store this information in the distributed structure of interconnections. Using this approach on growth hormone (GH) rhythms of healthy controls, fasted healthy subjects, untreated acromegalic patients and acromegalics under octreotide suppressive therapy we were recently able to demonstrate the power of this approach to differentiate the temporal pattern of GH secretion following normalization of the data for absolute amplitudes. In a second approach we were able to significantly reduce the number of data points required to characterize the temporal structure of these rhythms. This latter quality of the networks may help to transfer analysis of changes in the temporal pattern of hormone secretion on a more routine basis. PMID- 10752068 TI - Therapeutic implications of circadian rhythms in cancer patients. AB - Drug absorption, transport, metabolism and/or elimination usually show 24 h changes in both laboratory rodents and human beings. These variations in target cell exposure to drugs, as well as the rhythms which modulate cellular detoxification functions, account for the chronopharmacology of most medications, including anticancer agents, and have warranted the exploration of the relevance of the chronotherapy principle. Most of the cellular detoxication rhythms appear to be coupled to the rest-activity cycle, both in nocturnally active rodents and in diurnally active healthy subjects as well as in cancer patients. For instance, a 24 h rhythm was found in the activity of dehydropyrimidine dehydrogenase (DPD), both in rodent liver and in human circulating mononuclear cells, with a maximum located in the early rest span in either species. This cellular enzyme catabolizes 5-fluorouracil (5-FU), hence protects normal cells against damage produced by this widely used antimetabolite drug. Although DPD amplitude was nearly threefold in rodent liver and 40% in human lymphocytes, the adaptation of 5-FU administration to this rhythm largely improved tolerability both in rodents and in patients. The results thus support the coupling of the DPD rhythm and other chronopharmacology mechanisms to the average rest-activity cycle across species. The clinical relevance of such group chronotherapy has been further validated in Phase I, II and III clinical trials involving nearly 1500 patients. Multicentre randomized clinical trials have demonstrated that chronotherapy was both better tolerated and more effective than constant rate infusion in patients with metastatic colorectal cancer. Nevertheless 24 h rhythms in plasma cortisol or rest-activity could be altered in nearly 30% of cancer patients. Results from a prospective study performed in 200 patients with metastatic colorectal cancer indicated that poor circadian coordination constitutes an independent prognostic factor of both treatment tolerability and efficacy. Novel chronotherapeutic approaches targeted at circadian system coordination should then be devised in these patients. PMID- 10752069 TI - Pathophysiology of human circadian rhythms. AB - The 24 h profiles of hormonal secretions represent a good model for the study of the human circadian system. Diurnal hormonal variations generally reflect the modulation of ultradian or pulsatile release at 1-2 h intervals by signals occurring at nearly 24 h periods and result from the interaction of an internal timekeeping system--or circadian clock--with the sleep-wake homeostasis and various environmental factors, including the light-dark cycle, periodic changes in activity levels and the meal schedule. This temporal organization is altered in many pathophysiological conditions, including ageing, sleep loss, night or shift work, jet lag, affective disorders and endocrine diseases. Both photic and non-photic stimuli may affect the regulation of the circadian pacemaker and, therefore, the diurnal pattern of hormonal secretions. Appropriately timed stimuli may induce either a phase-advance or a phase-delay of the circadian clock, according to the timing of administration. Phase-shifting effects have been shown in humans for light and for dark pulses, physical exercise, melatonin and melatonin agonists, and benzodiazepine hypnotics. These results open new perspectives for the treatment of a variety of disorders involving dysregulation of the circadian rhythmicity. PMID- 10752070 TI - Nature of altered pulsatile hormone release and neuroendocrine network signalling in human ageing: clinical studies of the somatotropic, gonadotropic, corticotropic and insulin axes. AB - Recent clinical investigations have implemented an array of new analytical tools to evaluate the neuroregulation of endocrine axes. These studies demonstrate multifold disruption within the growth hormone (GH), luteinizing hormone (LH) testosterone, adrenocorticotropin (ACTH)-cortisol and the insulin axes in healthy ageing men and women. Novel research strategies in ageing include such developments as the indirect in vivo assessment of neuroendocrine network integration, via the approximate entropy (ApEn) statistic to monitor the unihormonal orderliness and bihormonal synchronicity of hormone release, and thus infer stability of network-integrative processes. For example, ApEn calculations show that the individual orderliness of GH, insulin or LH release falls progressively in older men and women, and the conditional synchrony between LH and testosterone (or LH and follicle-stimulating hormone/prolactin) release, and LH secretion and the neurogenically organized signal, nocturnal penile tumescence (NPT), all decline markedly in older men. Evaluation of the ACTH-cortisol axis points additionally to disrupted bihormonal synchrony within this stress responsive system in healthy ageing. A complementary investigative tool, viz. a stochastic differential equation random-effects feedback construct of the interactive male gonadotropin-releasing hormone-LH-testosterone axis, predicts that only certain extant postulates of ageing in the male reproductive axis will give rise to the observed erosion of LH-testosterone synchrony. Collectively, available clinical data suggest a general model of early neuroendocrine ageing in the human, in both the male and female, wherein ageing is marked by variable disruption in the time-delayed feedback and feedforward interconnections among neuroendocrine glands, which constitute an integrated axis and which control the joint synchrony of hormone release. PMID- 10752071 TI - Pulsatile insulin secretion. AB - Insulin is secreted in a pulsatile manner. Recently it has been shown that almost all (approximately 70-100%) of insulin is secreted in discrete insulin secretory bursts occurring approximately every 6 min. Furthermore, it has been revealed that regulation of the rate of insulin secretion is achieved primarily through modulation of the mass of these discrete insulin bursts. Thus meal ingestion increases insulin secretion by enhancing insulin burst mass by approximately 50% but also increases pulse frequency by approximately 50%. Interestingly, the hepatic clearance of insulin is also apparently closely related to the pattern of insulin delivery to the liver. It has been known for many years that the pattern of insulin delivery is abnormal in patients with type 2 diabetes. Recently, with new more sensitive insulin assays (ELISA) and validated methods for pulse detection, it has been possible to examine more precisely the abnormalities of pulsatile insulin in patients with type 2 diabetes. These recent studies suggest that the principal defect of insulin secretion is a deficient pulse mass of insulin with no changes in pulse frequency, and that this defect can be overcome by a period of beta cell rest. PMID- 10752072 TI - Control of growth hormone (GH) release by GH secretagogues. AB - Despite its long-term role in postnatal growth and metabolism, pituitary growth hormone (GH) is secreted in a short-term highly episodic pulsatile pattern in all species in which it has been examined. This pattern is tightly controlled by the interplay of GH-releasing hormone (GHRH) and somatostatin (SRIF), the primary hypothalamic factors that determine GH secretion from the somatotroph and which also regulate GH synthesis and secretory reserve. The discovery of a endogenous receptor for synthetic GH secretagogues (GHS)s that differ from GHRH implies the existence of at least one other endogenous GHS system, though the physiological role of the hypothetical GHS ligand remains unclear. The GH secretory pattern is sexually dimorphic with sex differences at many levels in the hypothalamo pituitary somatotroph axis. Studies in transgenic animals have shown that GH output is also highly sensitive to feedback control by GH itself, as well as by insulin-like growth factor I. Peripheral responses to GH are markedly dependent on the pattern of GH exposure, which is further modified after secretion by interaction with GH binding proteins and with GH receptors, both also regulated by the pattern of GH exposure. Although the hypothalamic GH pulse generator is of central importance in the control of GH output, its origin, location and mechanisms remain to be elucidated. PMID- 10752073 TI - Pulsatile parathyroid hormone secretion in health and disease. AB - In humans plasma parathyroid hormone (PTH) fluctuates episodically at a frequency of 6-7 bursts per hour. Approximately 30% of circulating PTH is attributable to pulsatile secretion and 70% to tonic secretion. PTH release is tightly controlled by Ca2+. Acute hypocalcaemia elicits a biphasic wave of PTH release, with an initial selective amplification and acceleration of the pulsatile component followed by proportionate stimulation of pulsatile and tonic secretion. Acute hypercalcaemia submaximally suppresses the frequency and mass of PTH bursts as well as tonic PTH release. Patients with primary hyperparathyroidism exhibit proportionate increases in pulsatile and tonic secretion, with no change in pulse frequency. In secondary hyperparathyroidism due to renal insufficiency, tonic secretion and pulsatile burst mass are also proportionately amplified, and burst frequency is increased. Moreover, the hypocalcaemia-induced increase in burst frequency and mass as well as their suppression during hypercalcaemia is diminished, suggesting partial uncoupling of hyperplastic parathyroids from physiological regulatory mechanisms. While the secretory pattern of PTH and its dysregulation in disease states is now well defined, the functional significance of pulsatile PTH signalling for target tissues is still largely unexplored. Preliminary work indicates that intermittent, in contrast to continuous, PTH administration stimulates bone formation. Cell culture studies suggest PTH receptor down-regulation with tonic exposure. PMID- 10752074 TI - Significance of pulsatility in the HPA axis. AB - A stress-free automated blood sampling system has been employed to demonstrate pulsatile hypothalamo-pituitary-adrenal (HPA) activity in the rat. In females, pulses of corticosterone secretion occur approximately once/hour throughout the 24 h cycle, with variation in pulse amplitude underlying a diurnal rhythm. Males show smaller pulses of secretion which become widely spaced during the early light phase nadir. Ageing does not affect the occurrence of pulses but the diurnal variation is lost. Analysis of the relationship between the HPA response to an acute noise stress and its coincidence with the various phases of the pulse, suggests that pulsatile activity arises from alternating periods of activation and suppression. Responses to i.v. corticotropin-releasing factor are not affected by pulse phase, indicating that this relationship is not generated at the pituitary-adrenal level. This phase relationship holds for all strains of rat except the hyperresponsive Fischer-344 in which an exaggerated stress response arises from a lack of phase-dependent suppression. Patterns of pulsatile activity are also modulated by neonatal programming or chronic HPA activation arising from adjuvant-induced arthritis, with consequent impact upon the response to acute stimuli. Thus, variations in the patterns of pulsatile activity are important determinants of both basal secretion and acute responses of the HPA axis. PMID- 10752075 TI - [Polyclonal antiserum against noncatalytic part of cellobiohydrolase I from Trichoderma reesei]. AB - A specific antiserum to the noncatalytic part of cellobiohydrolase I from Trichoderma reesei was obtained by exhaustion of rabbit antiserum to the native enzyme with its catalytic domain prepared by papain treatment of cellobiohydrolase I tightly adsorbed onto microcrystalline cellulose. PMID- 10752076 TI - [Preparative dephosphorylation of calcium salts of 2'(3)-mononucleotides by extracellular phosphatase from Spicaria violacea]. AB - The properties of the phosphatase present in the culture liquid of Spicaria violacea were investigated. Based on these results, a method for preparative dephosphorylation of calcium salts of 2'(3')-mononucleotides was proposed. A 96 98% yield was achieved at a substrate concentration of 100 mg/ml. Mild quantitative hydrolysis of RNA to nucleosides can be performed by RNA digestion to mononucleotides with Ca2+ followed by the proposed dephosphorylation procedure. PMID- 10752077 TI - [Restriction endonucleases from various bacterial strains exhibit an ice nucleating activity]. AB - Six strains containing site-specific endonucleases II were selected from a collection of 45 ice-nucleating bacterial strains isolated from rhizosphere of plants growing in various geographical regions. Endonucleases Pfl211I, Psp8I, and Psp23I were isolated and purified from two Pseudomonas sp. strains and a Pseudomonas fluorescens strain. Restriction endonucleases Pfl21I and Psp23I were shown to recognize and cleave the DNA nucleotide sequence 5'-CTGCA decrease G-3'. Endonuclease Psp8I recognized and cleaved the DNA nucleotide sequence 5'-G decrease GATCC-3'. These endonucleases were found to be true isoschizomers of PstI and BamHI, respectively. PMID- 10752078 TI - [The Use of the abundance ratio of 13C and 12C isotopes for characteristic of the origin of ethyl alcohol]. AB - During alcohol fermentation, the carbon isotope composition of ethyl alcohol produced depended on the substrate used and was characterized by the value of delta 13C equal to -24.7 +/- 0.8/1000 (wheat grain), -22 +/- 0.1/1000 (rye grain), -22 +/- 0.5/1000 (products of wood hydrolysis), -15.3 +/- 0.3/1000 (maize grain) and -10 +/- 0.1/1000 (sugar cane). The isotope composition of carbon of ethyl alcohol obtained during catalytic hydroxylation of ethylene has a delta 13C of -30.6 +/- 0.3/1000. The possibility of quantitative determination of specific components in mixtures of ethanol samples with various isotope compositions (chemical synthesis and alcohol fermentation of raw material from C3- or C4 plants) was shown. PMID- 10752079 TI - [Adaptation of acrylamide producer Rhodococcus rhodochrous M8 to change in ammonium concentration in medium]. AB - The mechanism of adaptation of the acrylamide producing strain Rhodococcus rhodochrous M8 to changes in ammonium concentrations in the medium was studied. An increase in the content of ammonium in the medium changed the activity of glutamine synthetase (GS) (EC 6.3.1.2) and glutamine dehydrogenase (GD) (EC 1.4.1.4), the enzymes of ammonium assimilation, as well as the activities of enzymes responsible for nitrile utilization: nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4). This also caused inhibition of activation of GS induced by phosphodiesterase (EC 3.1.4.1). Increases in the activities of nitrile hydratase and amidase and resistance of these enzymes to ammonium were observed in mutant of R. rhodichrous resistant to phosphotricine, an inhibitor of GS. An important role of GS in the mechanism of adaptation is suggested. PMID- 10752080 TI - [Enzymatic utilization of cotton soap stock]. AB - Enzymatic hydrolysis of neutral fat of cotton oil soap stock with a nonspecific lipase produced by Oospora lactis F-500 was designed. The culture liquid and a preparation of enzyme obtained by precipitation with isopropanol from a filtrate of the culture liquid were used. Utilization of cotton oil soap stock as the only source of carbon during cultivation of the fungus was studied. The rate of hydrolysis of soap stock fat strongly depended on the way of biological conversion of cotton oil soap stock. The most effective utilization was observed during cultivation of the fungus in the medium containing soap stock. PMID- 10752081 TI - [Optimization of conditions for cultivation of the basidiomycete Coriolus hirsutus--producer of extracellular laccase]. AB - The effects of various factors on the biosynthesis of extracellular laccase (EC 1.14.18.1) by the basidiomycete Coriolus hirsutus (Wulf.: Fr.) Quel. no. 072 during submerged cultivation were examined. Optimal parameters for cultivation in a fermenter of 10 l were determined: temperature, 28 degrees C; stirrer rotation speed, 160 rpm; and the inoculum volume, 15% of the working volume of the fermenter. The filtrate contained peroxidase, laccase, and phenol oxidase activities and displayed a high thermal stability. PMID- 10752082 TI - [Effect of stress on the composition of yeast lipids]. AB - Pigmented (Rhodotorula glutinis) and nonpigmented (Lipomyces starkeyi) yeasts were studied. Exogenous stressors (UV irradiation and methylene blue) were shown to change the composition of yeast lipids (especially the ratio of unsaturated fatty acids) and to increase the content of lipid peroxidation products formed (particularly in nonpigmented yeasts). In carotene-synthesizing yeasts, these stressors decreased the amount of carotenoids produced and did not affect the ratio between carotenoid pigments (beta-carotene, torulene, and torularhodin). PMID- 10752083 TI - [Formation of pyrogallol ether during oxidative destruction of oak lignin with air oxygen]. AB - Formation of pyrogallol ether during thermal oxidation of oak (Quercus iberica L.) lignin in the presence of air oxygen was investigated. A model reaction demonstrated that the substance identified by us as pyrogallol ether (1,2-dioxy-3 methoxybenzene) is derived from syringic aldehyde. PMID- 10752084 TI - [Spectrophotometric and fluorometric study of albumins and hemin interactions with aromatic antioxidants]. AB - Difference spectrophotometry and fluorescence quenching of human and bovine serum albumins were used to determine their association constants (Ka) with hemin in buffered physiological saline (pH 7.4) supplemented with 2% dimethylsulfoxide or in 40% aqueous dimethylformamide (pH 7.4). Ka values depended on the medium, the extent of albumin delipidation, and on the method of determination. The formation of hemin complexes with o-phenylenediamine, tetramethylbenzidine, gallic acid, its polydisulfide, and two substituted di-tert-butyl pyrocatechols was studied by difference spectrophotometry in the same media; Ka values for the complexes were calculated and compared to each other. The formation of complexes of these aromatic ligands with albumins was studied fluorometrically; Ka values were of order of approximately 10(-5) M-1 and decreased with the ligand hydrophobicity. PMID- 10752086 TI - [Storage of industrial microorganisms entrapped into polymer matrices]. AB - Our study of the techniques of long-term storage of the biomass of various strains of microorganisms, which cause breakdown or transformation of synthetic organic compounds, demonstrates that desiccated agar beads with immobilized microbial cells can be used for this purpose. In addition, the cells can be stored in desiccated matrices of agar or polyvinyl alcohol, coating synthetic cords. Such dry biocatalysts may be used for quick starting of bioreactors and in other biotechnological processes. The technique is applicable to storage of various strains of Pseudomonas, Corynebacterium, Rhodococcus, and, to a lesser extent, Enterobacteriaceae. PMID- 10752085 TI - [Isolation of highly active strain producing the antistaphylococcal antibiotic batumin]. AB - The use of chemical and UV-induced mutageneses allowed us to increase the biosynthetic activity of the strain capable of producing new antistaphylococcal antibiotic, batumin. The strain of Pseudomonas batumici N17 producing 87-100 mg batumin per liter culture liquid was selected. Its activity was 3.5-5 times higher than the activity of the most potent natural strain. P. batumici N17 was shown to be stable in relation to the synthesis of batumin. PMID- 10752087 TI - [Alpha-chymotrypsin immobilized on ferromagnetic particles coated with titanium oxide: production and catalytic properties]. AB - Immobilization of alpha-chymotrypsin on magnetic particles with stable coat with titanium oxides as a main constituent allowed the biocatalytic system to be quickly and qualitatively separated into the components after completion of the enzymatic reaction. X-ray phase analysis demonstrated that the coat of magnetic particles is composed mainly of titanium dioxide in brookite modification. The maximal capacity of the particles amounted to 0.3 mg protein/mg particles. It was demonstrated that the reaction catalyzed by immobilized alpha-chymotrypsin proceeds in a kinetic mechanism due to a high dispersion of the ferromagnetic particles. The catalytic constant (25 s-1) and KM (0.17 mM) for the immobilized enzyme for the hydrolysis of N-acetyl-L-tyrosine ethyl ester are comparable to the corresponding characteristics for the free enzyme. PMID- 10752088 TI - [Reduction of nitro-substituted compounds by native and immobilized Escherichia coli cells]. AB - Reduction of nitro-substituted compounds, 1,4-benzodiazepine-2-ones, dibenzo[b,f] 1,4-diazepines, quinolones, and quinoxalinones, by Escherichia coli cells was studied. Physicochemical methods demonstrated the formation of corresponding amines. 4-(p-Nitrophenyl)-1H-6-R-quinolones-2 were nor reduced by Escherichia coli cells. Regiospecific reduction of 2,4-dinitro-5H-11-(p-R-phenyl) dibenzo[b,f]-1,4-diazepines and 4-(2'-R-3',5'-dinitro)-benzoyl-3,4 dihydroquinoxalinones-2 was shown to result in the formation of 2-nitro-4-amino 5H-11-(p-R-phenyl)-dibenzo[b,f]-1,4-diazepines and 4-(2'-R-3'-nitro-5'-amino) benzoyl-3,4-dihydroquinoxalinones-2, respectively. Methods for microbiological reduction of nitro compounds and immobilization of Escherichia coli cells into carrageenan and its modified forms were elaborated. PMID- 10752089 TI - [Effect of various methods of immobilization on stability of a microbial biosensor based on Pseudomonas rathonis T during detection of surfactants]. AB - The operating and storage stability of a receptor element of an amperometric biosensor based on the Pseudomonas rathonis strain T capable of degrading surfactants was tested. Microbial cells were immobilized by incorporation in gels (agar, agarose, and calcium-alginate), polyvinyl alcohol membrane, adhesion to the chromatographic paper GF/A, or by the cross-linking induced by glutaric aldehyde. Incorporation of microbial cells in agar gel provides the long-standing conservation of their activity and viability during measurements of high concentrations of surfactants and allows the receptory element of the biosensor to be rapidly recovered after the measurements. PMID- 10752090 TI - [Immobilization of cells by entrapping in aubasidan]. AB - A new technique of immobilization of microbial cells by entrapping in aubasidan, a microbial polysaccharide, was developed. This technique was applied to three cultures: Erwinia aroidea, Pseudomonas sp., and Alcaligenes faecalis, the producers of aspartase and L-aspartate beta-decarboxylase. The new method is effective. After immobilization, microbial cells retained 79-91% of their initial enzymatic activity. PMID- 10752091 TI - [Thiol oxidase and disulfide reductase activities of the wheat Triticum aestivum L. caryopsis and its technological quality]. AB - Activities of oxygen-dependent thiol: O2 oxidoreductase (EC 1.8.3.2) and glutathione-dependent thiol: protein-disulfide oxidoreductase (EC 1.8.4.2) as well as technological value of seven soft spring wheat cultivars grown on different soil under contrasting climatic conditions (Krasnodar krai and Irkutsk oblast) were studied. It was found that the ratio of these enzymatic activities correlated positively with dough physical properties and flour bread-baking quality. PMID- 10752092 TI - [Immunoenzyme test system for detection of aflatoxin B1]. AB - Indirect enzyme immunoassay based on immobilized conjugate of aflatoxin B1 carboxymethyloxime with bovine serum albumin and polyclonal rabbit antibodies allows determining aflatoxin B1 with a low relative cross-reactivity against aflatoxin B2, G1, G2, M1, B2a and G2a and sterigmatocystin (15.5, 15.5, 1.7, 1.0, 0.03, 0.03 and 0.01%, respectively) with a sensitivity of 0.04 ng per well or 4.0 ng per ml organic solvent. PMID- 10752093 TI - [Effect of exogenic folic acid on the yield and amino acid composition of the seeds of Pisum sativum L. and Hordeum vulgare L]. AB - Effects of exogenous folic acid (FA) on the productivity of Pisum sativum L. and Hordeum vulgare L. have been studied. After flowering, the plants were treated with optimum concentration of FA (25 mg per 1 water). This treatment increased the weight of the seeds by 17-19% (samplings of 1000 pcs were compared), whereas the yield became 26-29% higher. Amino acid analysis revealed a notable increase in the content of folate-dependent amino acids (e.g., glutamate, glycin, and methionine). Analysis of total folate content demonstrated that tetrahydrofolic coenzymes were significantly increased in experimental seeds. Treatment of the plants with exogenous FA increased both the content of chlorophyll in the leaves and their continuance of function. The results obtained led to the conclusion that FA treatment increases the productivity of pea and barley, by affecting the yield, weight, and quality of the seed. PMID- 10752094 TI - [Production of the therapeutic and prophylactic preparation enterobifidin on the basis of Bifidobacterium adolescentis MC-42]. AB - Production of Enterobifidin comprises preparation of culture media, reparation of lyophilized Bifidobacterium adolescentis MS-42 culture, preparation of starters, cultivation of bacteria in fermenters, biomass conservation, and its biological control. The preparation contains physiologically active bifidobacterium cells with high activities of growth (mu = 0.7 h-1, g = 1.0 h) and acid formation (titratable acidity is approximately 120-140 degrees T; acetate concentration, 0.50-0.75%; and lactate concentration, 0.33-0.50%). The antagonistic activity of these bacteria towards Escherichia coli 08, E. coli 086, E. coli 015, E. coli 0115, and E. coli 0101 amounts to 98.2;, to Proteus vulgaris 102, to 87.2; and Staphylococcus aureus 209p, to 83.2%. The bifidobacteria (with a titer of 10(9) CFU/ml) remained viable for two to five months. PMID- 10752095 TI - Quality and its dimensions. PMID- 10752096 TI - Effect of loading rate and hydration on the mechanical properties of the disc. AB - STUDY DESIGN: The mechanical response of bovine intervertebral discs to axial compression at different loading rates and hydration levels was quantified. OBJECTIVES: To quantify the effects of hydration and loading rate on the mechanical response of the intervertebral disc to compressive axial load. SUMMARY OF BACKGROUND DATA: The disc is known to be viscoelastic, but there are few experimental data showing the effect of loading rate and hydration on its response to compression. METHODS: Hydration level reduced by creep-loading from a fully hydrated starting point. Four groups were tested: Group A: fully hydrated (n = 5), six loading rates, from 0.3 kPa/sec to 30 MPa/sec; Group B: after 30 minutes of creep (n = 4); and Group C: after 2 hours of creep (n = 4) under a static load of 1 MPa, loading rates 3 MPa/sec, 30 kPa/sec, and 0.3 kPa/sec; Group D: at 5-minute intervals, during an 8-hour period of creep (n = 3) under a static load of 1 MPa, loading rate 3 MPa/sec. Data normalized by disc area and height: nominal stress, strain, and modulus calculated. RESULTS: Group A: Modulus increased with load and rate of loading, with significant differences among the lower three loading rates. The highest three loading rates were significantly different from the lower rates, but not from each other. Group B: At the two higher loading rates, modulus was greater than in group A. At the lowest loading rate the modulus was similar to that in Group A. Group C: At the highest loading rate, the modulus was less than that of Groups A and B. At the lower two loading rates, the modulus was similar to that in Group A. Group D: The modulus increased in the first 30 minutes and decreased in the interval from 60 to 480 minutes. CONCLUSIONS: Intervertebral disc compressive mechanical properties are significantly dependent on loading rate and hydration. PMID- 10752097 TI - Outcome of patients treated for cervical myelopathy. A prospective, multicenter study with independent clinical review. AB - STUDY DESIGN: This Cervical Spine Research Society (CSRS) Study is a prospective, multicenter, nonrandomized investigation of patients with cervical spondylosis and disc disease. In this analysis, only patients with cervical myelopathy as the predominant syndrome were considered. OBJECTIVES: To determine demographics, surgeon treatment practices, and outcomes in patients with symptomatic myelopathy. SUMMARY OF BACKGROUND DATA: Current data on patient demographics and treatment practices of surgeons do not exist. There are no published prospective studies in which neurologic, functional, pain, and activities of daily living outcomes are systematically quantified. METHODS: Patients were recruited by participating CSRS surgeons. Demographic information, patients' symptoms, and patients' functional data were compiled from patient and physician surveys completed at the time of initial examination, and outcomes were assessed from patient surveys completed after treatment. Data were compiled and statistically analyzed by a blinded third party. RESULTS: Sixty-two (12%) of the 503 patients enrolled by 41 CSRS surgeons had myelopathy. Patients (48.4% male; mean age, 48.7 +/- 12.03 years) had a mean duration of symptoms of 29.8 months (range, 8 weeks to 180 months). Surgery was recommended for 31 (50%) of these patients. Forty three patients (69%) returned for follow-up and completed the questionnaire adequately for analysis. Twenty (46%) of the 43 patients on whom follow-up data are available underwent surgery, and 23 (54%) received medical treatment. Surgically treated patients had a significant improvement in functional status and overall pain, with improvement also observed in neurologic symptoms. Patients treated nonsurgically had a significant worsening of their ability to perform activities of daily living, with worsening of neurologic symptoms. CONCLUSIONS: When medical and surgical treatments are compared, surgically treated patients appear to have better outcomes, despite exhibiting a greater number of neurologic and nonneurologic symptoms and having greater functional disability before treatment. Randomized studies, if feasible, should be performed to address outcome in cervical myelopathy further. PMID- 10752099 TI - Symptoms, signs, and functional disability in adult spondylolisthesis. AB - STUDY DESIGN: A cross-sectional clinical study. OBJECTIVES: To determine whether there are specific symptoms, signs, and functional disability associated with adult spondylolisthesis. SUMMARY OF BACKGROUND DATA: In spite of the common occurrence of adult spondylolisthesis, the symptoms, signs, and disability associated with it have not been analyzed in a large, well-defined group of patients. METHODS: The symptoms, signs, and disability of 111 consecutive patients with adult spondylolisthesis, before randomized treatment with fusion or physiotherapy, were compared with those of 39 patients with nonspecific low back pain before lumbar fusion. The patients completed a questionnaire covering clinical history and symptoms and submitted a pain drawing. The signs were documented. Functional disability and pain were quantified by 12-function and 2 pain visual analog scales, respectively. RESULTS: Sixty-two percent of the patients reported low back pain as well as sciatica, 7% sciatica only, and 31% low back pain only. Specific signs were infrequent. A positive straight leg raising test result in 12% and an L5 sensory disturbance in 13% were the most common. The symptoms were similar in patients with spondylolisthesis and chronic low back pain, but the chronic low back pain group reported more functional disability. Patients with a nonorganic pain drawing (widespread, nonspecific pain) were more often blue collar workers; were more often and longer on sick leave; and reported reduced mental condition, sexual function, functional ability, and more pain than patients with an organic pain drawing (localized, specific pain). CONCLUSIONS: The clinical pattern and functional disability in adult spondylolisthesis and in low back pain of nonspecific origin are similar. Sciatica in adult spondylolisthesis is typically not associated with a positive straight leg raising test result. PMID- 10752098 TI - Increased signal intensity of the spinal cord on magnetic resonance images in cervical compressive myelopathy. Does it predict the outcome of conservative treatment? AB - STUDY DESIGN: Correlation between a lesion of the spinal cord that elicits increased signal intensity (ISI) on magnetic resonance images (MRIs) and the outcome of conservative treatment for cervical compressive myelopathy was retrospectively investigated. OBJECTIVE: To investigate whether ISI could predict the outcome of conservative treatment for cervical compressive myelopathy. SUMMARY OF BACKGROUND DATA: It is unknown whether ISI is related to the outcome of conservative treatment for cervical compressive myelopathy. METHODS: Fifty-two patients with mild cervical myelopathy underwent conservative treatment with a cervical brace. The compressive lesions were spondylosis in 29 patients, disc herniation in 12, and an ossification of the longitudinal ligament in 11. They also underwent MRI (1.5 T), and ISI was evaluated on T2-weighted sagittal and axial images. The ISI areas were classified as focal or multisegmental. Thirty nine patients underwent follow-up MRI after a mean interval of 2 years, 4 months. The transverse area of the spinal cord was also measured on T2-weighted axial images. The outcome of conservative treatment was assessed using the Japanese Orthopedic Association Score (JOA score). Patients showing either an improvement in the JOA score or with a JOA score of 15 or more were considered to have a satisfactory outcome. RESULTS: The average JOA score was 14.0 +/- 1.4 (range, 10 16) before conservative treatment and 14.4 +/- 1.9 (range, 10-17) at follow-up. The average gain in the JOA score was 0.4 points +/- 1.9 (range, -5 to +6). The outcome was satisfactory in 36 patients (69%). An area of ISI was observed in 34 patients (65%) before treatment (24 focal and 10 multisegmental). A satisfactory outcome was obtained in 78% of the patients without ISI, in 63% of those with focal ISI, and in 70% of those with multisegmental ISI. No statistically significant difference was seen among these three groups in the percentages of patients with satisfactory outcome, JOA scores before and after treatment or transverse spinal cord area. Of the 39 patients who were re-examined by MRI, 28 showed an area of ISI. The ISI regressed in five patients (18%). Satisfactory outcome was obtained in all 5 patients with regression of ISI, in 16 (70%) of the 23 patients without regression of ISI, and in 10 (91%) of the 11 patients without ISI apparent on the the first images (difference, not significant). CONCLUSIONS: Increased signal intensity was not related to a poor outcome of conservative treatment or severity of myelopathy in the patients with mild cervical myelopathy. PMID- 10752100 TI - Safety of sublaminar wires with Isola instrumentation for the treatment of idiopathic scoliosis. AB - STUDY DESIGN: To investigate the incidence of acute neurologic complications of use of sublaminar wires with third-generation spine instrumentation for the treatment of idiopathic scoliosis. OBJECTIVES: To assess the safety of sublaminar wires in the surgical treatment of idiopathic scoliosis. SUMMARY OF BACKGROUND DATA: The use of sublaminar wires in spine deformity for neuromuscular scoliosis and the Luque system has been reported. Use of sublaminar wires is an integral part of the technique in the surgical treatment of spine deformity with Isola instrumentation (AcroMed, Cleveland, OH). To date, the safety of this technique has not been documented. METHODS: The average age of the patients was 37 years (range, 11-74 years). Preoperative diagnosis was adolescent idiopathic scoliosis in 75 patients and adult idiopathic scoliosis in 66. One hundred nine were primary surgeries, and 32 were revision. Detailed evaluation of the curve type, curve magnitude, number of vertebrae instrumented, level of vertebrae wired, postoperative neurologic deficit, and the findings of intraoperative spinal cord monitoring was performed. Wires were always passed just before corrective maneuvers were performed. RESULTS: A total of 1366 wires were placed, 65% (n = 888) in the thoracic region, 22% (n = 300) in the thoracolumbar, and 13% (n = 178) in the lumbar. No permanent change in intraoperative spinal cord monitoring was detected. Stagnara wake-up test was performed in all patients. No patient with adolescent idiopathic scoliosis had neurologic complication. Two adults underwent revision surgery and had transient dysesthesia in the leg, which completely resolved with observation. CONCLUSION: Despite the increasing complexity of spinal instrumentation systems, sublaminar wire placement is a safe and useful adjunct in the surgical treatment of neurologically intact patients with idiopathic scoliosis. PMID- 10752101 TI - Anterior fusion for idiopathic scoliosis. AB - STUDY DESIGN: A review of the charts and radiographs of 22 patients with idiopathic lumbar, thoracolumbar, and thoracic scoliosis who underwent single stage anterior fusion with rigid third-generation instrumentation and titanium surgical mesh implants. OBJECTIVES: To validate a new technique for scoliosis correction by assessing the initial correction of deformity, trunk shift, and rotation. Perioperative statistics as well as complications were reviewed. The maintenance of sagittal balance and rate of fusion were also determined after a minimum of 2 years' follow-up. SUMMARY OF BACKGROUND DATA: Posterior correction of scoliosis has been the preferred method of treatment since the introduction of Harrington rods. Recent advances in instrumentation and surgical techniques have allowed surgeons to approach scoliosis correction through the chest and abdomen. These new techniques must be validated before they become accepted alternatives to the standard of care. METHODS: One male and 21 female patients, aged 11-18 years, were observed for an average of 44 months. Matched-pairs t tests were used to compare pre- and postoperative curve measurements. RESULTS: Statistically significant curve improvements were seen when postoperative radiographs were compared with preoperative ones. The instrumented coronal plane curve was corrected 82%. A 65% spontaneous correction of the uninstrumented coronal plane curve was observed. Sagittal kyphosis improved 7 degrees, and apical rotation and tilt angles improved 71% and 80%, respectively. A mean loss of 4 degrees of correction was seen on final follow-up radiographs. Lumbar lordosis did not change significantly, and postoperative hyperlordosis was not detected. Ninety six percent of all instrumented levels fused within 6 months. Although the perioperative complication rate was low, five patients (23%) had asymptomatic idiopathic retrolisthesis develop at the caudal end of the fused vertebrae. CONCLUSIONS: Accepted correction of idiopathic scoliosis can be achieved with anterior instrumented fusion alone. PMID- 10752102 TI - Electrical thresholds for biomechanical response in the ankle to direct stimulation of spinal roots L4, L5, and S1. Implications for intraoperative pedicle screw testing. AB - STUDY DESIGN: A comparison of electrical thresholds for biomechanical response in the ankle and for evoked electromyographic signals from specific leg muscles during intraoperative extradural direct stimulation of roots L4, L5, and S1. OBJECTIVE: To determine whether a biomechanical response in the ankle to direct root stimulation occurs before evoked electromyographic signals and to determine differences in electrical excitability of the roots circumferentially. SUMMARY OF BACKGROUND DATA: Stimulus intensities of 1.2-5.7 mA are reported to evoke electromyographic response in corresponding muscles to direct stimulation of normal roots. Stimulus intensities of 6-8 mA were suggested to detect bony pedicular compromise by stimulation of a hole or a screw during pedicle instrumentation. Electrical thresholds of three-dimensional torque response in the ankle to direct root stimulation have not yet been evaluated and compared with thresholds of evoked electromyogram. METHODS: Direct monopolar stimulation of the surgically exposed roots L4, L5, and S1 was performed from different sites around the root by a cuff multielectrode. Biomechanical response was measured as an isometric torque in the ankle at each of three orthogonal axes. Compound muscle action potentials (CMAPs) from root-specific muscles were detected by a pair of surface or wire electrodes. RESULTS: Mean threshold for biomechanical response in the ankle to stimulation of roots L4, L5, and S1 was 0.72 +/- 0.39 mA and for CMAP response was 1.09 mA +/- 0.36 (N = 13). Thresholds for biomechanical responses were significantly lower than for CMAP responses (P = 0.0004; paired t test). Nerve roots were electrically most excitable on their ventral aspects. CONCLUSION: The biomechanical response in the joint to root stimulation can be used to test all root-related muscles crossing that joint at their individual innervation pattern and their residual innervation and to detect electrical excitation of the root at electric thresholds lower than those for detecting CMAP from single standard root-specific muscle. However, this method does not provide sufficient root specificity. It will be valuable in conjunction with multimodality neurophysiologic monitoring of the roots for earlier and more reliable detection of pedicle bone breakthrough or integrity. Further clinical investigations are suggested. PMID- 10752103 TI - The radiologic anatomy of the lumbar and lumbosacral pedicles. AB - STUDY DESIGN: An anatomic and radiologic study of lumbar and lumbosacral pedicle anatomy. OBJECTIVES: To define the radiologic anatomy of the lumbar and first sacral pedicle in the coaxial projection. SUMMARY OF BACKGROUND DATA: Fluoroscopic assistance for pedicle screw placement requires radiologic landmarks. The radiologic landmarks have previously been assumed. Detailed study of the correlation between anatomy and radiology is required. METHODS: Lumbar vertebrae and sacra were marked with radiopaque material to demonstrate the pedicle cortical borders. The vertebrae were then imaged in the coaxial projection to determine the correlation between the pedicle cortex and the radiologic image. Pedicle dimensions were recorded. RESULTS: Pedicle dimensions were consistent with known measurements, yet the long axis of the L4 and L5 pedicle ellipse was oblique to the vertical. Consequently, the minor diameter of the pedicle ellipse was considerably less than the measured pedicle width at L5. The radiologic pedicle image was consistently within the true pedicle cortex, by up to 3 mm, and probably represents the inner cortical border of the pedicle. The S1 pedicle has reliable anatomic landmarks, yet only the medial and superior borders were visualized. CONCLUSIONS: The radiologic pedicle image in the lumbar and lumbosacral spine is a reliable guide to the true bony cortex of the pedicle. At S1 the pedicle image is less well correlated with the cortical borders of the pedicle, yet other reliable anatomic landmarks exist. PMID- 10752104 TI - Spinal cord injury in Mississippi. Findings and evaluation, 1992-1994. AB - STUDY DESIGN: The Mississippi spinal cord injury surveillance system is both active and passive, designed to capture all cases of spinal cord injury through mandated reporting by multiple sources. Each case is confirmed by medical record review. OBJECTIVES: To describe the development of a state-wide spinal cord injury surveillance system, discuss findings from the system, and evaluate sensitivity. SUMMARY OF BACKGROUND DATA: In the United States, the annual incidence rate of spinal cord injury requiring hospital admission has been estimated at 32-50 per million. With prehospital fatalities included, the estimated incidence rate ranges from 43 to 55 per million population annually. METHODS: In the current study all cases identified during the first 2 years of operation of the spinal cord injury (SCI) system were included. To evaluate the sensitivity of the system, International Classification of Disease, Ninth Revision, Clinical Modification (ICD-9-CM) codes from each hospital's discharge database were used. RESULTS: The incidence rate among patients in hospitals and prehospital fatal cases was 77 per million. The rate for patients in hospitals was 59 per million. The incidence rate of spinal cord injury among males was 4.4 times higher than among females. Rates of spinal cord injury were highest among persons 20-24 years of age. Rates were similar for whites and blacks. The most frequent causes of spinal cord injury were motor vehicle collisions, violence, and falls. Additional cases were identified during the evaluation, resulting in a 94% sensitivity. CONCLUSIONS: Mississippi's spinal cord injury incidence rates are substantially higher than rates reported for other states except Alaska. The surveillance system was found to be very complete. Prevention efforts should focus on increasing safety belt usage, increasing alcohol awareness, and reducing violence. PMID- 10752105 TI - Patient satisfaction after circumferential lumbar fusion. AB - STUDY DESIGN: A review of 141 consecutive patients who underwent instrumented circumferential lumbar fusions. Outcome was assessed by an independent third party after a minimum follow-up of 2 years. OBJECTIVES: To evaluate clinical outcomes and assess patient satisfaction with circumferential lumbar fusions. SUMMARY OF BACKGROUND DATA: Circumferential lumbar fusion has been used as a salvage procedure for revision spine surgery. METHODS: Data were collected from patient interviews, patient satisfaction questionnaire, and review of records and radiographs of 141 consecutive patients who underwent circumferential lumbar fusion under a single anesthetic. RESULTS: There were follow-up data in 133 of the 141 patients (92%). Mean duration of follow-up was 37.2 months. There were 41 (31%) primary and 92 (69%) revision surgeries. There were 73 (55%) workers' compensation and 60 (45%) non-workers' compensation patients. There were radiographs available at 12-month follow-up in 86 patients (65%). There was solid radiographic fusion in 85 (99%) of the 86 patients. The overall complication rate was 20%. Clinical outcome was determined by the patient satisfaction questionnaire. There were 14 (11%) who chose the statement, "surgery met my expectations"; 68 (51%) who chose, "surgery improved my condition enough that I would go through it again for the same outcome"; 26 (20%) who chose, "surgery helped me but I would not go through it again for the same outcome"; and 25 (19%) who chose, "I am the same or worse compared with before surgery." There was no statistical difference in patient satisfaction between primary and revision surgeries or between workers' compensation versus non-workers' compensation groups. CONCLUSIONS: Circumferential lumbar fusion is a useful procedure for a patient with difficult reconstructive disease. There is a very high fusion rate. Overall, 62% of patients are satisfied with the result, especially those with a diagnosis of pseudarthrosis or spondylolisthesis with stenosis. Patients who are working before surgery and patients who are not injured workers also tended to progress well. PMID- 10752106 TI - Review of 36 cases of spinal cord meningioma. AB - STUDY DESIGN: Thirty-six consecutive patients with histologically confirmed spinal cord meningioma were presented to evaluate clinical, diagnostic, therapeutic options and to correlate treatment methods and outcome. OBJECTIVE: To present the incidence, clinical presentation, localization, techniques, and long term results of surgically treated spinal meningiomas. SUMMARY OF BACKGROUND DATA: Meningiomas are common tumors of spinal neoplasm. They are generally benign and slow-growing. Advanced in radiologic and surgical techniques have brought about better surgical results. The goal of surgical treatment must be total resection if possible. However, spinal meningiomas may recur, especially as a result of incomplete resection. METHODS: Thirty-six consecutive patients with histologically confirmed spinal meningiomas were treated from 1980 to 1997. Neuroradiological diagnosis was made through myelogram in 20 patients, CT scan in 15 patients, and MRI in 16 patients. All patients were operated on via the posterior approach and using microsurgical technique and when necessary Cooper Ultrasonic surgical aspirator (CUSA) and CO2 laser were also applied. The patients were followed for 2 to 15 years (mean 9 years). Radiotherapy was not undertaken except in recurrent tumors. RESULTS: The most frequent site of spinal meningiomas was in the thoracic region. In 30 (83%) patients tumors were found to be completely intradural extramedullary during surgery. Total tumor resection was achieved in 35 (97%) of patients. In the follow-up period, 30 cases (83%) improved when compared to their preoperative conditions. There was one operative mortality (3%). A 66-year-old women died of pulmonary emboli. CONCLUSIONS: Magnetic resonance imaging is the best imaging technique for diagnosis. Total tumor resection improved the surgical results of spinal meningiomas. If total removal of the tumor cannot be achieved, or in the case of early recurrence followed by total resection, radiotherapy should be performed in adjuvant therapy. PMID- 10752107 TI - Radiographic analysis of lumbar motion in relation to lumbosacral stability. Investigation of moderate and maximum motion. AB - STUDY DESIGN: This in vivo study was performed to examine active lumbar motion without any support. OBJECTIVES: To establish the behavior of segmental flexibility according to the degree of whole lumbar motion and to clarify the correlation between bony characteristics of the lumbosacral junction and stability in the segment. SUMMARY OF BACKGROUND DATA: In previous studies, the full mobility of the lumbar segments has been investigated. The details of motion commonly seen with the activities of daily living have not been clarified. It has been reported that the iliolumbar ligaments have an influence on lumbosacral stability and that the relative thickness of the transverse process of L5 could indicate the functional strength of the iliolumbar ligaments. However, the effects of the iliolumbar ligaments on the lumbosacral range of motion have not been studied in vivo. METHODS: Ninety adults, aged 20-39 years, were requested to perform motion commonly associated with activities of daily living, defined as moderate motions of the lumbar spine. The subjects then were asked to perform maximal motion of the lumbar spine. The segmental ranges of motion, segmental flexion, and extension at every level of the lumbar spine were calculated by using functional radiographs. The correlation between the relative thickness of the transverse process of L5 and the motion seen at the lumbosacral junction was also determined. RESULTS: The greatest segmental range of motion was found at L2 L3 in moderate motion and at L4-L5 in maximal motion. It shifted gradually from the upper to lower lumbar levels with the increase in total lumbar motion. With an increase in lumbar spine motion, maximum segmental flexion shifted from L2-L3 to L3-L4, then to L4-L5. Segmental extension changed only at L5-S1, increasing with total lumbar spine motion. There was an inverse statistical correlation between lumbosacral motion and relative thickness of the L5 transverse process. CONCLUSIONS: The greatest segmental flexibility induced by the moderate lumbar motion, usually seen with the activities of daily living, occurred more in the upper segments of the lumbar spine, especially in flexion. Further, the iliolumbar ligaments regulate lumbosacral motion especially flexion. PMID- 10752108 TI - Bridging the gap between science and practice in managing low back pain. A comprehensive spine care system in a health maintenance organization setting. AB - STUDY DESIGN: A case study of spine care system changes in a multispecialty group practice health maintenance organization setting. OBJECTIVES: To reduce unnecessary use of imaging and specialty referrals for low back pain in the primary care setting and to reduce spine surgery rates. SUMMARY OF BACKGROUND DATA: Results of previous research indicate that diagnostic and therapeutic procedures for low back pain are frequently used even though there is no scientific evidence of their efficacy. This indicates that low back pain care can be made more efficient by reducing the use of unproven diagnostic and therapeutic interventions for low back pain. METHODS: Rates of diagnostic imaging and specialty referral rates for low back pain were monitored for 9 months before and 9 months after primary care physician education regarding appropriate low back pain evaluation and management. Spine surgery rates were also monitored before and after implementation of a nonsurgical spine clinic. RESULTS: Large reductions in rates of imaging and specialty referrals for low back pain were achieved after primary care physician education. After spine clinic implementation, visits to spine surgeons dropped by approximately 50%, and spine surgery rates per thousand health plan members were reduced by 35%. CONCLUSIONS: Primary care physician education regarding low back pain management can reduce use of imaging and specialty referrals without reductions in patient satisfaction, and implementation of a nonsurgical spine clinic for complex or chronic spine patients can significantly reduce spine surgery consultations and spine surgery rates. PMID- 10752109 TI - Synovial cyst of the transverse ligament of the atlas in a patient with os odontoideum and atlantoaxial instability. AB - STUDY DESIGN: A case report and review of the literature. OBJECTIVE: To describe the diagnosis and successful treatment of a synovial cyst arising from the transverse ligament in a patient with os odontoideum and atlantoaxial instability. SUMMARY OF BACKGROUND DATA: Synovial cysts arising from the transverse ligament of the atlas are extremely rare. Development of a synovial cyst is thought to be attributable to degenerative changes of the C1-C2 facet joints or to microtrauma. Direct excision of the cyst is the only treatment cited in previous reports. METHODS: A synovial cyst arising from the transverse ligament of the atlas in a 45-year-old man with os odontoideum and atlantoaxial instability was treated surgically with posterior atlantoaxial fusion alone. The magnetic resonance images, surgical treatment, and related literature are reviewed. RESULTS: Preoperative magnetic resonance images of the cervical spine showed a large cystic mass located ventral to the cord arising at the level of the transverse ligament of the atlas: the mass was of low signal intensity on T1 weighted images, was of high signal intensity on T2-weighted images, and was enhanced marginally with gadolinium-DTPA on T1-weighted images. Spontaneous regression of the cyst was identified on the follow-up magnetic resonance images taken 3 months after C1-C2 posterior wiring and fusion. CONCLUSIONS: A patient with a synovial cyst arising at the C1-C2 junction ventrally at the level of the transverse ligament showed spontaneous regression of the lesion after C1-C2 posterior wiring and fusion. PMID- 10752110 TI - Aggressive thoracic actinomycosis complicated by vertebral osteomyelitis and epidural abscess leading to spinal cord compression. AB - STUDY DESIGN: Report of a successfully diagnosed and treated case of spinal cord compression due to epidural actinomycosis. OBJECTIVE: To illustrate that proper use of imaging strategy can greatly facilitate diagnosis and management of this rare condition. SUMMARY OF BACKGROUND DATA: Spinal actinomycosis causing epidural abscess and significant spinal cord compression is an uncommon condition. Although diagnosis is difficult, favorable results are widely reported when specific therapy is instituted. METHODS: A 32-year-old Chinese man had extensive dorsal thoracic soft tissue swelling and lower limb weakness. Collapse of the T5 vertebral body was found on plain radiographs with mediastinal infiltrates on chest radiograph. It took magnetic resonance imaging (MRI) to fully delineate the epidural abscess and dorsal muscular abscesses, which were not depicted by computed tomographic (CT) scan. Diagnosis was made by examination of CT-guided aspirate and tissue recovered during surgery by a microbiologist. The patient received high-dose intravenous penicillin and prompt spinal decompression once diagnosis of actinomycosis was confirmed. RESULTS: The dorsal muscular abscesses and upper thoracic epidural abscess resolved rapidly after intravenous antibiotics and surgical drainage. This was well documented by follow-up MRI and the full recovery of motor power and lower limb sensation in the patient. CONCLUSIONS: High clinical suspicion and proper use of imaging data led to timely diagnosis of this rare case of mediastinal, epidural, and intramuscular thoracic actinomycosis. Specific antibiotic therapy and timely, well-targeted surgical intervention greatly improve the outcome of this condition. PMID- 10752111 TI - An unusual presentation of solitary fibrous tumor. AB - STUDY DESIGN: Case report of a lumbar extradural solitary fibrous tumor. OBJECTIVE: To describe the clinical manifestations and successful treatment of a patient with solitary fibrous tumor of the lumbar spine. SUMMARY OF BACKGROUND DATA: Solitary fibrous tumor usually occurs on the pleura but has been described in a growing number of sites. Although this is the first case in a lumbar extradural location, clinicians should be aware of the many guises in which this entity appears. Most cases are benign and cure is effected by surgical extirpation. METHODS: The diagnosis, treatment, and outcome of a patient with a lumbar extradural solitary fibrous tumor are reviewed. RESULTS: A patient showing signs of myeloradiculopathy was found, by means of magnetic resonance imaging, to have an intensely enhancing extradural lesion in the region of L1. Excision biopsy revealed features of a solitary fibrous tumor. The patient made a rapid, complete recovery. CONCLUSIONS: Solitary fibrous tumor has protean clinical manifestations and should be be considered in the differential diagnosis of intraspinal enhancing lesions on either side of the dura. PMID- 10752112 TI - Malignant spinal neurofibrosarcoma. AB - STUDY DESIGN: A report of a case of metastatic spinal neurofibrosarcoma. OBJECTIVE: To document metastatic neurofibrosarcoma as a cause of spinal cord compression and to review the literature. SUMMARY OF BACKGROUND DATA: Three previously reported cases of metastatic neurofibrosarcoma of the spine were reviewed. METHODS: The patient's clinical record and radiologic investigations as well as the result of a search of the English literature are reported. Magnetic resonance images, computed tomographic scans, and histology photomicrographs are displayed. RESULTS: Paraparesis developed in this patient, due to a posterior extradural thoracic spinal cord compression by a neurofibrosarcoma believed to be metastatic from a neurofibrosarcoma of the femoral nerve. CONCLUSIONS: Malignant spinal metastasis remains a rare complication of neurofibromatosis, with a very poor prognosis. PMID- 10752113 TI - Traumatic lumbosacral dislocation. AB - STUDY DESIGN: Case report of a patient with lumbosacral dislocation. OBJECTIVES: To report a case of traumatic lumbosacral dislocation treated nonoperatively, with 10 years of follow-up. SUMMARY OF BACKGROUND DATA: Lumbosacral dislocation is rare, with only 48 cases reported in the literature. Surgical treatment by means of open reduction and fusion is advisable. However, the literature shows six cases of patients with complete lumbosacral dislocation treated nonoperatively with good results. METHODS: A 38-year-old man was involved in a highspeed vehicle accident. The lumbosacral dislocation (anterior displacement of L5 on S1, 44% slippage) was initially missed, and the patient was treated conservatively. RESULTS: Slippage did not progress, and the patient returned to full, normal activity. Ten years later he is asymptomatic and is able to work and play sports. CONCLUSIONS: This study reports a rare injury of the lumbosacral junction that was detected 3 months after injury. Satisfactory results were obtained with nonoperative treatment in this case. Although a surgical approach is advisable in acute cases to decompress the neurologic structures and to stabilize the lumbosacral junction, conservative management may be the treatment of choice in inveterate lesions. PMID- 10752114 TI - Re: Accuracy of blind versus fluoroscopically guided caudal epidural injection. PMID- 10752115 TI - [Hormonal regulation of gametogenesis in insect]. AB - The review discusses the role of juvenile hormone (JH), ecdysone and brain in the regulation of oogenesis and spermatogenesis in insects. The early period of gametogenesis (gonial mitoses, the meiotic prophase) in both sexes is controlled mainly by ecdysone and neurosecretory cells of the brain. In periods of cytoplasmic growth of oocytes and vitellogenesis the main role in the regulation belongs to JH. The modern views on hormonal regulation of vitellogenin synthesis and follicular epithelium differentiation are under consideration with a special reference of the role of ecdysteroids in Diptera and Lepidoptera oogenesis. PMID- 10752116 TI - [Characteristics of oogenesis in the Barents sea sponge Leucosolenia complicata]. AB - Ovogenesis of the Barents sea sponge Leucosolenia complicata Mont. having asconoid canal system was studied at light (using histochemical methods) and ultrastructural levels. It has been established that the reproduction period "arranges" for autumn-winter season. The ovogenesis lasts for approximately two months. Grand accroissement is the longest stage of ovogenesis, lastiing in L. complicata from the end of Sptember to the middle of November. At this stage ovocytes lose their amebic movement, which coincides with the beginning of nurse cell formation. The nurse cells are derived from choanocytes. The choanocytes contacting with the ovocyte surface lose the collar and flagellum, grow in size and transform into nurse cells. Any cell of nurse-cell complex is capable of seizing a spermium and being transformed in situ into a carrier-cell. The specific role of nurse-cell complex is to synthesize sudanophilic (lipid) granules for the carrier cell and ovocyte. In the whole, a weak phagocytic activity of ovocytes, is characteristic of the ovogenesis of L. complicata. During vitellogenesis the ovocyte synthesises endogenic mucopolysaccharide granules from oligosaccharides originated from the mesohyl. Lipid granules pass to the ovocyte from nurse cells. Ribosomal RNAs are accumulated as a result of the own activity of nucleolus-nucleus apparatus of the ovocyte. PMID- 10752117 TI - [Oogenesis in turbellarians of the genus Geocentrophora studied by light and electron microscopy. V. Oocyte nuclear structures containing factors of pre-mRNA splicing and pre-rRNA processing]. AB - An immunoelectron study of nuclear distribution of pre-mRNA splicing and pre-rRNA processing factors was carried out for oocytes of two turbellarian species: the Baikal endemic Geocentrophora wagini and a cosmopolitan G. baltica. Using monoclonal antibodies against Sm-epitope of small nuclear RNPs (snRNPs) and SR protein SC35, it has been shown that on different stages of oocyte growth splicing factors (snRNPs and SC35) are distributed within the whole nucleus. A fibrogranular material located near heterochromatin clumps is labeled with these antibodies. A fibrillar part of this material seems to represent perichromatin fibrils. The features of intranuclear distribution of splicing factors in Geocentrophora oocyte nuclei and their ultrastructural features suggest that pre mRNA synthesis and splicing may occur up to the end of diplotene. In Geocentrophora oocyte nuclei a few nuclear bodies (NBs) were found. Splicing factors (snRNPs and SC35) and fibrillarin were revealed in these NBs. Homology of Geocentrophora oocyte NBs to coiled bodies of oocyte and somatic cell nuclei of other animals is discussed. During diplotene, Geocentrophora oocyte nucleoli were found to lose their granular component and to change to large fibrillar structures named "postnucleoli". The postnucleoli contain both fibrillarin and non-nucleolar spliceosomal components (snRNPs and SC35). Geocentrophora oocyte postnucleoi are compared with similar structures of mammalian oocyte nuclei, taken as an example of morphological convergence of nuclear structure organization in phylogenetically distant animal species. PMID- 10752118 TI - [Mitotic activity, ploidy and ultrastructure of cardiomyocytes from human embryo and fetuses]. AB - There is an evidence that mitotic activity of human cardiomyocytes in late fetal and early postnatal ontogenesis is very low. But little is known of the division of human cardiomyocytes at earlier stages of development. In this study mitotic activity of ventricular and atrial human cardiomyocytes of 4-8-week-old embryos and 17-32-week-old fetuses has been studied. On these stages the mitotic index is relatively low to reduce moderately within the 1st to the 3rd trimester of pregnancy from 1.4 to 0.7%. These findings are consistent with the data on cell ploidy demonstrating the presence of relatively small share of myocytes with 3c and 4c DNA in ventricles of 6-8-week-old embryos and 12-22-week-old fetuses. The share of such cells in the 1st and 2nd trimesters of pregnancy varies from 19 to 24% and from 8 to 18%, respectively. Cells with 3c and 4c DNA are most likely to be in mitotic cycle. This assumption is supported by electron microscope pictures showing all phases of typical mitosis. Cyclic changes of myofibrillar ultrastructure during mitosis of prenatal human cardiomyocytes are the same as during mitosis of low differentiated myocytes in mouse and rat hearts. These results suggest that in prenatal human cardiomyogenesis the level of myocyte differentiation and the cell number increase at slow rate. PMID- 10752120 TI - [Kinetics of the reaction of nitroblue tetrazolium reduction by human blood neutrophils]. AB - Kinetics of Nitro blue tetrazolium (NBT) reduction to diformasan by neutrophils was investigated using 27 samples of human blood. Analysis of alteration in the share of activated neutrophils (ANP) and activated neutrophil index (ANI) was done in relation to the reaction time. The former reaction is an irreversible reaction of zero (pseudozero) order, while the latter is an irreversible reaction of the first (pseudofirst) order. It has been found out that an induced NBT reduction occurs in parallel with a spontaneous reaction, and that neutrophils have essentially different oxidizing power. The kinetic approach enabled us to discover some indices (NBT quantity involved in the reaction, and reaction speed constant of the first order) which in different samples varied within broader limits than ANP or ANI (within the limits of an order), i.e. provided a possibility to make a more delicate analysis of processes in neutrophils. PMID- 10752119 TI - [The study of the Ca2+ role in cytotoxic response of human cells in culture to the action of xenobiotics]. AB - The role of Ca2+ in mechanisms of cell death, necrosis and apoptosis is diverse and generally recognized. The purpose of this work was to study Ca2+ participation in a cytotoxic response of human cultured cells in the presence of toxic concentrations of cationic antiseptic substance poly(hexamethylene guanidine), anionic surfactant SDS and monomeric methyl methacrylate (a component of bone cement applied in surgery). Human cell line U-937 grown in suspension was used for this study. A fluorescent probe chlortetracycline was used, as an indicator of Ca2+ transport through biologic membranes. Our results show that weakly toxic concentrations of xenobiotics under study, close to the minimum toxic doses, nearly always provoke a fair but statistically significant drop in Ca2+ binding by cells. At the same time, higher toxic doses lead to significant increase in Ca2+ influx. The latter event well compares with the majority of literary data, while the mentioned decrease in Ca2+ influx at low toxic concentrations of xenobiotics presumably correlates with the initial stage of acute cytotoxic response, accompanied by a metabolic activation and enhanced resistance of cells to injuring stimuli, demonstrated by the authors elsewhere. In parallel, a possible effect of Ca(2+)-channel antagonist nifedipine was explored under conditions of cytotoxic response of cell lines U-937, A-549 and human embryonic lung fibroblasts to poly(hexamethylene guanidine). Nifedipine (10 microM) was introduced in the incubation medium simultaneously with the toxic agent, and the cells were further maintained for 5 or 24 h in culture; their viability was monitored with the microtetrasolium test or by assessment of LDH leakage into the incubation medium. The effect of nifedipine proved to be dual, depending on the applied concentration of toxic agent: at low toxic concentrations the improvement of viability could be noticed, while at more pronounced toxic doses aggravation of viability was evident. From our point of view the explanation of this result could be the following. In weakly toxic conditions, as in intact cells, Ca2+ influx is brought about by specific mechanisms, mainly through Ca(2+)-channels, that is why nifedipine partly abolishes Ca(2+)-dependent cytotoxic response. At high concentrations, cell plasma membrane is directly damaged by toxic agent, Ca2+ enters cells mainly non specifically, so that Ca2+ antagonist cannot protect cell injury. The reason of toxic effect aggravation by nifedipine in these conditions is still waiting for its explanation. PMID- 10752121 TI - [Effect of a short-term moderate whole body hyperthermia of healthy humans on their peripheral lymphocytes]. AB - A short (75-110 min) mild (39.5 degrees C) whole body hyperthermia of humans does not alter the absolute amount of peripheral lymphocytes immediately after heating, but significantly reduces their relative amount due to neutrophil increase. Body heating does not induce lymphocyte degeneration: their potential viability does not reduce. The total level of lymphocyte oxidative metabolism remains unaltered, while SDG and LDG activities are seen reduced and increased, respectively. Lymphocytes adapt to body heating by inducing thermotolerant state and activating anaerobic metabolism. PMID- 10752122 TI - [Nitric oxide production by normal and injured neurons of Nodose ganglion and Nucleus dorsalis of Vagus nerve]. AB - Nodosum ganglion and nucleus dorsalis contain from 19.2 to 22.65% of NO-positive neurons. Their amount increases up to almost 77.4 and 68.8% in the ganglion and the nucleus dorsalis, respectively, in the damaged nervus vagus due to i-NOS transcription. It has been shown that NO participates in desorganization and recovery of the traumatic neuron, as auto- and paracrine regulator. PMID- 10752123 TI - [The role of topoisomerase II in mammalian cells response to ionizing radiation. II. Effect of a small concentration of novobiocin on cell cycle in x-rays irradiated HeLa cells]. AB - Flow cytometry was used to study cell cycle recovery in X-irradiated HeLa cells after the action of novobiocin, an inhibitor of topoisomerase II. A prolonged treatment with 0.4 mM novobiocin (30 h) of intact cells did not affect cell cycle progression. Novobiocin treatment of 5 Gy-irradiated cells resulted in a significant decrease of G2-block, if compared with X-irradiated cells. These data, together with our previous results on the effect of 1 mM novobiocin on cell cycle in X-irradiated CHO K1 cells, allow to suggest an involvement of topoisomerase II in cell response to ionizing radiation. But one cannot exclude a concurrent effect of novobiocin with proteins other than topoisomerase II, for example, with protein p34cdc2-kinase, known to be involved in cell cycle regulation. PMID- 10752125 TI - [The effect of mycoplasmal contamination of human embryonic lung cell line MRC-5 on the karyotypic variability]. AB - Karyotypic variability has been investigated for nonimmortalized human embryonic lung cell line MRC-5, cultivated with Acholeplasma laidlawii strain PG-8 for 15 45 days. The character of cell distribution for chromosome number did not change during this time. In all investigated variants the number of polyploid cells increased considerably with the lengthening of the term after decryoconservation. The number of chromosomal aberrations in 15-45 days contaminated cells increased significantly as compared to the control at the expense of dicentrics (telomeric associations). The number of dicentrics had a tendency to increase with the lengthening of the term of contamination. Thus, in 45 days the number of dicentrics increased twice as much as that in 15 days. The increase of polyploids may be due presumably to the specific character of karyotypic variability in nonimmortalized cell lines with the long-term cultivation. Our present and previous results made it possible to suppose that the formation of dicentrics (telomeric associations) in nonimmortalized "markerless" cell line, following the long-term mycoplasmal contamination, may prove additionally the role played by dicentrics in cell adaptation to in vitro conditions whatever the degree of transformation may be--nonimmortalized line or immortalized nontumorogenic or high tumorogenic lines. PMID- 10752124 TI - [UV-induced immobilization of DNA repair protein XPA in different human cell line]. AB - XPA repair protein is absolutely needed for nucleotide excision repair (NER). It preferentially binds UV-irradiated DNA in vitro and possibly takes place in the recognition of pyrimidine dimers, the main type of UV-lesions in DNA. Using immunofluorescent microscopy and immunoblotting technique we have found that XPA protein is fully extractable by Triton X-100 solution from non-irradiated normal human fibroblasts, but after UV-irradiation its extractability decreases in UV dose dependent manner. UV-induced XPA-immobilization was observed in human cell lines with different types of repair defects, but XPA-extractability from unirradiated cells of these lines was significantly lower in comparison with normal fibroblasts. These data do not permit to make conclusion concerning the distinct connection of this phenomenon with different pathways of NER. Histone deacetylase inhibitor, sodium butyrate, did not change the level of extractability in unirradiated and UV-irradiated normal human cells and CHO cells, defective in global genome repair, that indicated the independence of XPA immobilization from the level of histone acetylation. It was established with the help of confocal microscopy that XPA-foci in detergent-treated UV-irradiated cell were partially colocalized with the focal sites of PCNA, an auxiliary protein of DNA polymerases delta and epsilon. It may mean that a part of detergent-resistant XPA foci correspond to DNA repair synthesis sites, but the major part of immobilized XPA reflects the early step of repair proteins assembly formation needed for the repair of the lesions. PMID- 10752126 TI - [Cytogenetic characteristics of seed progeny of trees under condition of antropogenic contamination in Voronezh town]. AB - It has been shown that in seed progeny of Quercus robur L., Pinus sylvestris L. and Betula pendula Roth. some cytogenetical characteristics vary under conditions of contamination. Such changes may be common or specific type. Thus, the frequency of pathological mitosis increases under such conditions in all the investigated species of trees. Inhibition of mitosis was found in the progeny of the pine, and variability in the number of nucleoli was detected in the pine and oak. However, in some cases the level of pathological mitosis in the oak progeny did not differ from the control, but the mitotic activity was higher due to the presence of much more cells being at the prophase stage. In the birch progeny under conditions of contamination the mitotic index increased, with a simultaneous shifts in the peaks of mitotic activity. The possibility of using these cytological characteristics for the aims of cytogenetical monitoring is considered. PMID- 10752127 TI - [The use of in situ polymerase chain reaction for detection the intracellular localization of Mycoplasma hominis in cultured HeLa cells]. AB - Mycoplasma hominis was incubated with HeLa cells up to 72 h. Infection of HeLa cells was estimated by in situ polymerase chain reaction with a subsequent use of computer digital image analysis for result processing. The localization of mycoplasma in the cell nucleus was demonstrated after a 48 h incubation. The quantity of infected HeLa cells was increased with time to make 58 and 75% on days 2 and 3, respectively. No M. hominis was found in the cytoplasma of HeLa cells. PMID- 10752128 TI - [Disorders of hormone reception and intracellular signaling in hypertension]. AB - This article discusses the disruption taking place due to hypertension in regulatory cell systems. These can either participate in development of the pathology or, on the opposite, provide compensation, be part of the mechanism that brings to normal the functional and metabolic state of cells under this disease. Data on increased level of Ca2+, higher reactivity of cells in relation of Ca2+ mobilizing hormones, alkalinisation of cytoplasma, activation of phosphoinozitide metabolism and stimulation of proteinkinaze C evidence in favour of the suggestion that these disruptions of regulatory cell systems are the material basis of the phenomenon of "structural support" to arterial hypertension. PMID- 10752129 TI - [The regulatory effects of opioid peptides--enkephalins--in controlling the activities of the cardiovascular system]. AB - In this review we analyse the experimental and clinical findings demonstrating important regulatory significance of met-enkephalin, leu-enkephalin and their derivatives in the control of cardiovascular system activity. Enkephalin-positive immunoreactivity is revealed in the heart of different species of animals, and their cardiovascular effects are established in numerous investigations. It is determined that cardiac effects of enkephalins are essentially associated with modulatory influence at the presynaptic and postsynaptic levels on the activity of extracardiac neural regulation. Cardiovascular effects of endogenous opioid system are extremely important in developing of myocardial ischemia, cardiac arrhythmias and congestive heart failure. The cellular mechanisms of opioid effects are associated with stimulation of mu- and delta-subtypes of opiate receptors which stimulation of mu- and delta-subtypes of opiate receptors which are coupled with conductivity of ion channels, adenylate cyclase activity, phosphoinositide turnover and calcium-calmodulin-dependent protein kynases. PMID- 10752130 TI - [Neuropeptide Y: its diversity and the apparent contradictoriness of its functions. An analysis of its possible mediated effects]. AB - The review of NPY functions summarizes so fare accumulated data of its physiological effects. The estimation of heterogeneity of NPY receptors in responding reactions is made. The analysis of possible direct and indirect NPY effects is performed including cascade mechanism of other peptides releasing. Peptide cascade regulation modeling software created on database programming language Visual FoxPro 5.0 allows to trace several induction steps and estimate possible contribution of each peptide into final spectrum of biological activity. PMID- 10752131 TI - [The energy "budget" of the heart (a review of the literature)]. AB - An attempt is made on the grounds of literary information to represent the contemporary picture of the energy distribution in the man heart left ventricle in the different "budget items". About 20% of energy of the normally working left ventricle are spent without connection with contractions (on the "basal metabolism" of the myocardium). About 10% of energy are spent on the work of Na+/K(+)- and Ca(2+)-ATPases in the processes of excitation and electromechanical coupling. About 70% of energy are spent on the mechanical activity of the left ventricle. Further details of these basic components of the energy expenditure are examined. The Different kinds of the left ventricle efficiency are determined. PMID- 10752132 TI - [Immediate-early genes in the cerebral mechanisms of emotional stress]. AB - Immediate-early genes (IEGs) induction is one of the primary neuronal responses to emotional stress. In experiments with different types of stressors the brain structures with the most pronounced immediate early genes expression were identified. Biochemical, physiological and genetical mechanisms of the IEGs expression in the central nervous system during emotional stress are discussed in the article. Investigation of characteristics and conditions of the IEGs induction showed that immediate early genes induction is crucial for organization of neuroendocrine and hormonal responses during emotional stress. In situations that require adaptation and learning IEGs induction in brain is especially strong. The present data permit us to think that immediate early genes participate in formation of the long-lasting circulation of the negative emotional excitation in limbic system. Animals resistant and predisposed to psycho-emotional stress have different patterns of IEGs expression in conflict situations leading to emotional stresses. Immediate-early genes induction is especially pronounced in brains of sensitive to stress individuals. It is proved that differential approach with regard to individual differences in IEGs expression is preferential in emotional stress research. Neglect of this fact has led to contradictions in results of various authors. The investigation of the IEGs properties can help to define prospective ways of the effective coping with emotional stress and prevention of its dangerous consequences. PMID- 10752134 TI - [The systems organization of human functions: the theoretical aspects]. AB - On the basis of the theory of functional systems, suggested by P. K. Anokhin, leading principals of system organization of human functions are regarded. General characteristics of functional systems are stated. Some peculiarities of intrasystem and intersystem organization of functional systems of human organism are discovered. The role of functional systems in organization of normal human live activity as well as under psychoemotional stress and pathology is shown. System principals of compensation of disordered functions during rehabilitation of persons undergone stresses and ecologically unfavorable loads are considered. PMID- 10752133 TI - [The experimental development of the concept of O. S. Adrianov on the correlation of functional and neurochemical processes: regulatory peptides in mediator system dysfunction]. AB - The article is devoted to commemoration of full member of Russian Academy of Medical Sciences, Oleg Andreevich Adrianov, who would have celebrated his 75-th anniversary in 1998. O. S. Adrianov, author of numerous works on physiology and morphology of central nervous system, in the recent years of his was studying the problem of the processes relationship at macro and micro levels of brain organization. Further to the concept created by O.S. Adrianov, data on action of two peptides: delta-sleep and tafcine, on behavior, neurophysiological and neurochemical processes have been consolidated. Experimental data were obtained for rabbits, cats, and dogs, both intact and in the state of pathology (psychomotoric excitement, bradykinesia, penicillin epilepsy). Impact of peptides on convergation processes is discussed: peptide of delta-sleep depresses reactions of brain structures to photo- and phono-stimulation, and activates the serotoninergic system in general; tafcine enforces the convergation processes and activates the dopaminergic system. PMID- 10752135 TI - Equine T-cell cytokines. Protection and pathology. AB - The ultimate reason for better characterizing the immune response to infectious agents is the hope that this knowledge may lead to the development of better preventative or therapeutic measures. As more information becomes available, it becomes possible to incorporate these findings into the design of better vaccines and treatments. Likewise, attempts to either enhance or suppress specific helper T-cell responses may be required to control immunopathologic reactions. Although cytokine intervention in the clinical setting remains theoretic at this time, future manipulation based on the TH1/TH2 paradigm is probable. PMID- 10752136 TI - Inflammation in horses. AB - After inflammation is initiated by detection of antigen, plasma components and activated leukocytes are concentrated at the inflammatory site. Cellular and chemical effectors of inflammation are focused on the offending antigen, usually resulting in its destruction and elimination. Activation of endogenous counter regulatory systems damps down the inflammatory process and is the first stage of repair. In addition to local effects, the inflammatory focus may initiate a continuum of systemic acute phase responses ranging from the systemic inflammatory response syndrome (SIRS) to generalized immunosuppression. PMID- 10752137 TI - Equine immunity to bacteria. AB - The remarkable ability of the horse and other animals to prevent infection by most bacterial pathogens encountered is the result of a complex set of distinct but overlapping defense mechanisms. This article summarizes the current state of knowledge on innate and adaptive immunity to bacterial pathogens and reviews various ways in which some bacteria have evolved in order to evade components of the host response. PMID- 10752138 TI - Equine immunity to viruses. AB - The identification of some of the adaptive immune responses to infection with equine viruses has been the first step toward rational immunoprophylactic design. Sufficient knowledge of infection-induced immunity and informed estimates of the requirements for long-term immunity for EIV have now been obtained. Thus, the future for inactivated EIV vaccines is promising now that new adjuvants have been applied to induce cellular immunity and safe methods have been designed to stimulate virus-neutralizing (VN) antibody at mucosal surfaces. Adenoviruses induce circulating VN antibody, the presence of which appears to correlate with protection from reinfection. Therefore, the potential of vaccines to induce VN antibody and protect from challenge is an important next step with this virus. With persistent viruses such as EHV-1, antibody-mediated protection from infection can be achieved only at the site of initial infection, that is, the nasopharynx and upper respiratory tract. Systemic dissemination is very rapid and consequently VN antibody is unlikely to play a major role in prevention of disease once the initial infection event has occurred. Cellular immune responses, particularly CTLs, play a dominant role in protection and recovery and are important in immune surveillance and determination of the outcome of reactivation of latent virus. Therefore, the key to future EHV-1 vaccine design is to focus on stimulation of CTL responses, and this requires the successful presentation of vaccine-derived antigenic peptides to MHC class I molecules that are recognized by specific receptors on CTL. There is some evidence that stimulation of EHV-1 specific CTL precursors may correlate with immunity to this virus. By analogy with gamma herpesviruses in humans, CTL precursor frequency may also function as an immune correlate for EHV-2. Although EAV infection induces strong immunity in females and geldings, persistent infection of the genital tract is an important route of dissemination from stallions. Although inactivated vaccines induce strong immunity (which depends upon VN activity of serum antibody) to first infection, the immunologic control of persistent infection is currently poorly understood; however, analogy with other persistent viruses suggests that CTLs are also likely to play an important role in the control of persistent EAV infections. PMID- 10752139 TI - Equine immunity to parasites. AB - Helminths are among the most significant parasites of horses in developed countries. This article examines immune responses against helminth parasites and the implications that immunologic investigations have on vaccine development, improvement of diagnostic procedures, and disease eradication. PMID- 10752140 TI - Immunodiagnostic testing in horses. AB - Technologic advances in immunodiagnostic testing have enhanced our understanding of the pathogenesis of a broad array of diseases, including infectious diseases, immunodeficiency disorders, and immune-mediated disorders. If applied rationally, with an understanding of the questions the tests answer as well as the limitations that constrain their use, these tests can serve as valuable aids in the diagnosis and management of equine diseases. PMID- 10752141 TI - Primary immunodeficiencies of horses. AB - Primary immunodeficiency disorders are genetically determined failures of immune defense that increase susceptibility to infectious agents. This article reviews the salient features of equine primary immunodeficiency disorders, summarizes the molecular mechanisms of each disorder, and updates information that facilitates diagnosis and management of affected horses. The central theme is to encourage clinicians to ask, "I wonder if this horse has an underlying primary immunodeficiency disorder?" when caring for horses suffering from chronic and recurring infections and responding poorly to standard therapy. PMID- 10752142 TI - Secondary immunodeficiencies of horses. AB - FPT of immunoglobulin in foals is the commonest form of acquired immunodeficiency in horses. FPT predisposes foals to bacterial infections and septicemia and easily is preventable and treatable if breeding farms and veterinarians are attentive to optimum foaling management practices. Other forms of acquired immunodeficiencies are uncommon in horses, although immune function may be transiently suppressed by a wide variety of drugs, infections, or other conditions. As immunologic testing becomes more sophisticated and more readily available to equine practitioners, acquired immunodeficiencies are likely to be characterized more frequently in horses. PMID- 10752143 TI - Hypersensitivity disorders in horses. AB - Hypersensitivity is an exaggerated immunologic response to a foreign agent that results in inflammation and organ dysfunction. Hypersensitivity disorders are broadly divided into antibody-mediated and T-cell-mediated reactions. The inflammatory pathways that result in disease are initiated in an antigen-specific manner through Fab portions of antibodies or the T-cell receptor, causing the up regulation of effector mechanisms designed to clear the offending agent. Effector mechanisms include the generation of inflammatory chemicals such as cytokines and chemokines and the attraction of leukocytes and potentiation of their function. This article reviews current concepts in the immunopathogenesis of hypersensitivity disorders and demonstrates these mechanisms as they apply to equine disease. PMID- 10752144 TI - Equine autoimmunity. AB - Although relatively little is known about autoimmunity and autoimmune mechanisms specifically in horses, the similarities between clinical syndromes with identifiable effector mechanisms in horses and other species suggest that comparable mechanisms may be applicable. Our understanding of equine autoimmunity undoubtedly will benefit from the extensive study of autoimmunity in other species. PMID- 10752145 TI - Leukoproliferative disorders in horses. AB - Leukoproliferative disorders reported in horses include lymphoma, lymphocytic leukemia, plasma cell myeloma, granulocytic leukemia, monocytic leukemia, myelomonocytic leukemia, and eosinophilic leukemia. Lymphoma affects horses of all ages, whereas leukemias often occur in younger horses. Clinical signs are often nonspecific including depression, anorexia, fever, and weight loss. Specialized diagnostic techniques such as cytochemistry and immunophenotyping better define the cellular origin of leukoproliferative disorders, which is essential for developing appropriate therapeutic protocols and rendering an accurate prognosis. PMID- 10752146 TI - Immunomodulation in horses. AB - Nonspecific immunomodulators are substances that induce non-antigen-specific enhancement of the body's native or acquired defense mechanisms. Immunomodulant preparations are most often used for treatment of chronic, viral, or bacterial infection with evidence of secondary immunosuppression. The proposed mechanism of action of these products is macrophage activation and subsequent release of cytokines that enhance humoral and cell-mediated immunity. In equine medicine, nonspecific immunostimulant products are used for prevention and treatment of infectious respiratory disease and treatment of sarcoid skin tumors. PMID- 10752147 TI - Equine vaccination. AB - Equine infectious disease remains a constant and important threat to the health of domesticated horses. Vaccination plays a critical role in protecting against such disease, but at the present time the efficacy of some equine vaccination strategies is in doubt. The best strategy for resolving these concerns is an improved knowledge of the immunologic basis of successful vaccination, combined with the appropriate integration of effective vaccines into well-designed disease control policies. PMID- 10752148 TI - [Calcium-activated neutral proteinase in gametes of the Strongylocentrotus droebachiensis]. PMID- 10752149 TI - [Spectrophotometric parameters of conformations of hemoglobin and its complexes with ligands in the sturgeon]. PMID- 10752150 TI - [Species specific characteristics of the functional response of sodium pump to phosphorylation with protein kinase A]. PMID- 10752151 TI - [Study of histamine binding with H2 membrane receptors in the gastric mucosa in Rana ridibunda]. PMID- 10752152 TI - [Effect of water and food deprivation during pregnancy in rats on mitochondrial respiration and gluconeogenesis in their offspring]. PMID- 10752153 TI - [Lactate dehydrogenase isoenzymes during seasonal adaptation of predatory fur animals]. PMID- 10752154 TI - [Fatty acid composition of the liver phospholipids in the cow Bos taurus infected with the trematode Orientobilharzia turkestanica]. PMID- 10752155 TI - [Stress dynamics of sodium, potassium, calcium, and magnesium levels in the fresh water mollusk Dreissenia polymorpha]. PMID- 10752156 TI - [Effect of sublethal concentrations of mercury, cadmium, and copper salts on the lysozyme level in tissues from the Lena sturgeon Asipenser baeri brood]. PMID- 10752157 TI - [Characteristics of osmoregulation of the circulating erythrocytes in steno- and euryhaline sea fishes in the hypoosmotic medium]. PMID- 10752158 TI - [Sex hormone levels in the enteral medium in female ruminant animals]. PMID- 10752159 TI - [Localization of serotonin and FMRF-amide in the bivalve mussel Mytilus edulis during early development]. PMID- 10752160 TI - [Morphofunctional study of statocyst of the Cuban crayfish Procambarus cubensis]. PMID- 10752161 TI - [Calcium reservoirs in the Drosophila retina: electron-cytochemical calcium determination]. PMID- 10752162 TI - [Relation between carotenoid concentration and anaerobic respiration capability in mollusks Lymnaea stagnalis and Unio pectorum]. PMID- 10752163 TI - [Effect of the hydra peptide morphogen on proliferation of spermatogonia in the scallop Mizuhopecten yessoensis]. PMID- 10752164 TI - Fatal pulmonary embolism in hospitalized patients. Clinical diagnosis versus pathological confirmation. AB - OBJECTIVE: To assess the incidence of fatal pulmonary embolism (FPE), the accuracy of clinical diagnosis, and the profile of patients who suffered an FPE in a tertiary University Hospital. METHODS: Analysis of the records of 3,890 autopsies performed at the Department of General Pathology from January 1980 to December 1990. RESULTS: Among the 3,980 autopsies, 109 were cases of clinically suspected FPE; of these, 28 cases of FPE were confirmed. FPE accounted for 114 deaths, with clinical suspicion in 28 cases. The incidence of FPE was 2.86%. No difference in sex distribution was noted. Patients in the 6th decade of life were most affected. The following conditions-were more commonly related to FPE: neoplasias (20%) and heart failure (18.5%). The conditions most commonly misdiagnosed as FPE were pulmonary edema (16%), pneumonia (15%) and myocardial infarction (10%). The clinical diagnosis of FPE showed a sensitivity of 25.6%, a specificity of 97.9%, and an accuracy of 95.6%. CONCLUSION: The diagnosis of pulmonary embolism made on clinical grounds still has considerable limitations. PMID- 10752165 TI - Analysis of the effects of the continuous electrode paste band on precordial leads of the electrocardiogram. AB - OBJECTIVE: To determine if the application of a continuous electrode paste band on precordial leads results in alteration of the electrocardiographic tracing as compared with an adequate amount of electrode paste, and if the former condition does not cause uniform morphologies from V1 to V6. METHODS: The amplitude and morphology of the electrocardiographic waves on the precordial leads in electrocardiographic tracings, which were performed with standard (control group) or excessive (continuous band) application of the electrode paste, were compared. RESULTS: None of the 106 patients studied showed uniformity of the QRS morphology from V1 to V6. The electrocardiographic alterations identified in the tracings performed with a continuous electrode paste band that showed statistical significance in relation to the control group were the following: inversion of the P wave in V1; inversion of the T wave in V1, V2, and V3; appearance of R' waves in V1 and V2; disappearance of S waves in V1; appearance of S waves in V5 and V6; alterations in the amplitude of almost all waves, in all leads. CONCLUSION: Application of a continuous electrode paste band in the precordial leads may cause significant alterations in the electrocardiographic tracing obtained. PMID- 10752166 TI - Mitral valve repair. Quadrangular resection of the posterior leaflet in patients with myxomatous degeneration. AB - OBJECTIVE: To analyze the immediate and late results of mitral valve repair with quadrangular resection of the posterior leaflet without the use of a prosthetic ring annuloplasty. METHODS: Using this technique, 118 patients with mitral valve prolapse who underwent mitral repair from January '84 through December '96 were studied. Age ranged from 30 to 86 (mean = 59.1 +/- 11.8) years and 62.7% were males. An associated surgery was performed in 22% of the patients, and coronary artery bypass graft was the most frequently performed surgery (15 patients- 12.7%). In 20 (16.9%) patients other associated techniques of mitral valve repair were used and shortening of elongated chordae tendineae was the most frequent one (6 patients). RESULTS: Immediate mortality was 0.9% (one patient). Long-term rates for thromboembolism, endocarditis, reoperation and death in the late postoperative period were 0.4%, 0.4%, 1.7% and 2.2% patients/year, respectively. The actuarial curve of survival was 83.8 +/- 8.6% over 12 years; survival free from re-operation was 91.8 +/- 4.3%, free from endocarditis was 99.2 +/- 0.8% and free from thromboembolism was 99.2 +/- 0.8%. In the late postoperative period, 93.8% of the patients were in functional class 1 (NYHA), with a complete follow up in 89.7% of the patients. CONCLUSION: Patients with mitral valve prolapse who undergo mitral valve repair using this technique have a satisfactory prognosis over 12 years. PMID- 10752167 TI - Cardiac abnormalities in the acquired immunodeficiency syndrome. A prospective study with a clinical-pathological correlation in twenty-one adult patients. AB - OBJECTIVE: To evaluate the cardiac abnormalities and their evolution during the course of the acquired immunodeficiency syndrome, as well as to correlate clinical and pathological data. METHODS: Twenty-one patients, admitted to the hospital with the diagnosis of acquired immunodeficiency syndrome, were prospectively studied and followed until their death. Age ranged from 19 to 42 years (17 males). ECG and echocardiogram were also obtained every six months. After death, macro- and microscopic examinations were also performed. RESULTS: The most frequent causes of referral to the hospital were: diarrhea or repeated pneumonias, tuberculosis, toxoplasmosis or Kaposi sarcoma. The most frequent findings were acute or chronic pericarditis (42%) and dilated cardiomyopathy (19%). Four patients died of cardiac problems: infective endocarditis, pericarditis with pericardial effusion, bacterial myocarditis and infection by Toxoplasma gondii. CONCLUSION: Severe cardiac abnormalities were the cause of death in some patients. In the majority of the patients, a good correlation existed between clinical and anatomical-pathological data. Cardiac evaluation was important to detect early manifestations and treat them accordingly, even in asymptomatic patients. PMID- 10752168 TI - Effects of propranolol on the QT dispersion in congestive heart failure. AB - OBJECTIVE: Studies have shown that therapy with beta-blockers reduces mortality in patients with heart failure. However, there are no studies describing the effects of propranolol on the QT dispersion in this population. The objective of this study was to assess the electrophysiological profile, mainly QT dispersion, of patients with heart failure regularly using propranolol. METHODS: Fifteen patients with heart failure and using propranolol were assessed over a period of 12 months. Twelve-lead electrocardiograms (ECG) were recorded prior to the onset of beta-blocker therapy and after 3 months of drug use. RESULTS: A significant reduction in heart rate, in QT dispersion and in QTc dispersion was observed, as was also an increase in the PR interval and in the QT interval, after the use of propranolol in an average dosage of 100 mg/day. CONCLUSION: Reduction in QT dispersion in patients with heart failure using propranolol may explain the reduction in the risk of sudden cardiac death with beta-blocker therapy, in this specific group of patients. PMID- 10752169 TI - Stenting of unprotected left main coronary artery in a patient with cardiogenic shock. AB - A 64-year-old female presented with pulmonary edema and cardiogenic shock after coronary arteriography that showed severe subocclusive lesion in the left main coronary artery (LMCA) in a dominant left coronary system. The patient successfully underwent urgent angioplasty with stent deployment in the LMCA. After an uneventful period, the patient was discharged at day six. PMID- 10752170 TI - Total agenesis of the left pericardium. AB - This is the report of a 46-year-old patient with the preoperative diagnosis of an atrial septal defect (ASD) of the ostium secudum type. After sternectomy, partial agenesis of the left pericardium was diagnosed. It is our opinion that, if the radiographic picture is suggestive of this entity, a clinical search for cardiopulmonary anomalies should be performed, because the majority of these associated anomalies can and should be surgically corrected. PMID- 10752171 TI - The greatest medical discovery of the millennium (fundamental steps to the understanding of cardiac performance). PMID- 10752172 TI - Are apolipoproteins A and B better than lipoproteins for assessing risk of obstructive coronary heart disease? AB - OBJECTIVE: To evaluate whether apolipoproteins A-I (Apo A-I) and B (Apo B) have, higher ensitivity (SN), specificity (SP) and positive predictive value (PPV) than lipoproteins (LP), total cholesterol (TC), high density lipoprotein (HDL), low density lipoprotein (LDL), very low density lipoprotein (VLDL), and triglycerides (TGL) in assessing the risk of coronary heart disease (CHD). METHODS: This is a transversal case-control study of 241 patients, who were divided into two groups: 1) 145 patients with CHD, and 2) 96 patients without coronary disease. A model of logistic regression to evaluate the relation between the LPs and CHD was developed in which variables with a p-alpha < 0.1 were included. RESULTS: Apo A-I levels were higher in the patients without CHD, (OR 2.08, CI 1.20-3.57). There were no statistical differences between the values of Apo A-I and the remaining lipid fractions (Apo A-I: 67%; Apo B: 100%; PPV: TC = 71%; TGC = 71%; HDL = 71%; LDL = 71%). The costs of the tests in Reais were as follows: Apo A-I: R$ 56.60; Apo B-100: R$ 56.60; TC: R$ 9.94; HDL: R$ 21.30; LDL: R$ 28.40; TGL: R$ 14.20. CONCLUSION: Levels of Apo A-I and Apo B have no advantage over conventional lipoproteins in predicting the risk of CHD, despite the statistical association between Apo A-I and CHD; in addition, their costs are higher than those of the conventional lipoproteins. PMID- 10752173 TI - Difference in the in-hospital mortality of unstable angina pectoris between men and women. AB - PURPOSE: To assess differences in the in-hospital mortality (HM) rate between men and women with unstable angina pectoris (UA) according to age, depression of the ST segment, history of previous acute myocardial infarction (AMI), and risk factors for coronary heart disease. METHODS: From October 96 to March 98, 261 patients with UA were selected. Logistic regression models were developed to adjust the association between sex and HM for possible influence of covariables, such as hypertension, diabetes mellitus, dyslipidemia, sedentary lifestyle, smoking, and familial history of early coronary heart disease. RESULTS: HM due to UA was approximately three times higher in women (9.3%; 12/129) than in men (3.0%; 4/132) accounting for a relative risk of 3.07; 95% confidence interval (CI) = 1.02-9.27. In logistic regression models, the association between sex and death was not significantly altered when the following parameters were considered: age, depression of the ST segment, history of previous AMI and risk factors for coronary heart disease. The nonadjusted and adjusted odds ratio (OR) for the distinct covariables were 3.28 (CI 95% = 1.03-10.45) and 3.14 (CI = 95% = 0.88-11.20), respectively. CONCLUSION: Similarly to AMI, HM in UA is higher in women than in men. Age, risk factors for coronary heart disease, and depression of the ST segment in the electrocardiogram on patients' admission to the hospital did not significantly influence the association between sex and death. PMID- 10752174 TI - Myocardial perfusion by contrast echocardiography. Establishment of normal pattern of intracoronary injection and safety in humans. AB - OBJECTIVE: To establish the normal pattern and safety of echocardiographic contrast in patients with no significant obstruction of epicardial coronary arteries. METHODS: 67 patients with normal coronary arteries or obstructions < 50% were selected from 277 patients who underwent coronary angiography (CA). Mean age was 56 +/- 11 years and 36 were males. At the end CA, echocardiographic contrast was selectively injected into each coronary artery. The parasternal short axis of the left ventricle (LV) was divided into six segments: anterior (A), antero-lateral (AL), postero-lateral (PL), posterior (P), infero-septal (IS) and antero-septal (AS). Anterolateral (ALPM) and posteromedial papillary muscles (PMPM) were also considered. The pattern and intensity of the appearance of the myocardial contrast was visually analyzed. RESULTS: The right coronary artery (RCA) was dominant in 60 patients. Contrast appearance was sudden and simultaneous in the 3 muscle layers. All segments could be contrasted after the injection in both coronary arteries. 100% of the AS, A and AL segments, 97% of the PL and 98% of the ALPM were perfused by the left coronary artery (LCA). P and IS segments were perfused by the RCA in 85% and 82%, respectively, and by a dominant LCA in 71% of the cases. The PMPM was perfused by a dominant RCA in 77% and by a dominant LCA in 86%. There were no symptoms. CONCLUSION: Intracoronary injection of the sonicated solution is a safe procedure that allows for an excellent opacification of the myocardium and can potentially be used during routine CA. PMID- 10752175 TI - Signal averaged electrocardiogram in top deficient athletes. PMID- 10752176 TI - Radiofrequency catheter ablation in patients with atrial fibrillation. PMID- 10752177 TI - Catheter-induced 3:1 second degree atrioventricular nodal block during atrioventricular nodal reentrant tachycardia. PMID- 10752178 TI - Hypoperfusion of the left ventricle in the absence of changes in segmental contractility as observed through echocardiography by using microbubbles during dobutamine infusion. PMID- 10752179 TI - Case 3/99--a 45-year-old woman with hypertrophic cardiomyopathy and cardiogenic shock. Heart Institute-Incor-HCFMUSP-Sao Paulo-Brazil. PMID- 10752181 TI - Medically unexplained syncope and its relationship to psychiatric disorders. PMID- 10752180 TI - Detection of functional changes of the fetal heart in the first trimester of gestation. AB - The authors consider the possibility of using color Doppler of the ductus venosus and the measurement of nuchal translucency as a screening test for alterations in fetal cardiac functions in the first trimester of gestation. Review of the literature suggests that the combination of the ultrasonographic measurement of nuchal translucency and Doppler at 10 and 14 weeks of gestation can be effective in detecting certain cardiac abnormalities. This conclusion, however, is preliminary and needs to be further investigated. PMID- 10752182 TI - Invasive hemodynamic monitoring in the postoperative period of cardiac surgery. AB - OBJECTIVE: To assess the hemodynamic profile of cardiac surgery patients with circulatory instability in the early postoperative period (POP). METHODS: Over a two-year period, 306 patients underwent cardiac surgery. Thirty had hemodynamic instability in the early POP and were monitored with the Swan-Ganz catheter. The following parameters were evaluated: cardiac index (CI), systemic and pulmonary vascular resistance, pulmonary shunt, central venous pressure (CVP), pulmonary capillary wedge pressure (PCWP), oxygen delivery and consumption, use of vaso active drugs and of circulatory support. RESULTS: Twenty patients had low cardiac index (CI), and, 10 had normal or high CI. Systemic vascular resistance was decreased in 11 patients. There was no correlation between oxygen delivery (DO2) and consumption (VO2), p = 0.42, and no correlation between CVP and PCWP, p = 0.065. Pulmonary vascular resistance was decreased in 15 patients and the pulmonary shunt was increased in 19. Two patients with CI < 2 L/min/m2 received circulatory support. CONCLUSION: Patients in the POP of cardiac surgery frequently have a mixed shock due to the systemic inflammatory response syndrome (SIRS). Therefore, invasive hemodynamic monitoring is useful in handling blood volume, choice of vasoactive drugs, and indication for circulatory support. PMID- 10752184 TI - In-stent restenosis. Acute and long-term outcomes after excimer laser coronary angioplasty. AB - OBJECTIVE: With the increased use of intracoronary stents, in-stent restenosis has become a clinically significant drawback in invasive cardiology. We retrospectively assessed the short- and long-term outcomes after excimer laser coronary angioplasty of in-stent restenosis. METHODS: Twenty-five patients with 33 incidents of in-stent restenosis treated with excimer laser coronary angioplasty (ELCA) were analyzed. Sixty-six percent were males, mean age of 73 +/ 11 years, and 83% were functional class III-IV (NYHA). ELCA was performed using 23 concentric and 10 eccentric catheters with a diameter of 1.6-2.2 mm, followed by balloon angioplasty (PTCA) and ultrasound monitoring. The procedure was performed in the following vessels: left anterior descending artery, 10; left circumflex artery, 8; right coronary artery, 6; left main coronary artery, 2; and venous bypass graft, 7. RESULTS: The ELCA was successful in 71% of the cases, and PTCA was 100% successful. The diameter of the treated vessels was 3.44 +/- 0.5 mm; the minimal luminal diameter (MLD) increased from 0.30 mm pre-ECLA to 1.97 mm post-ELCA, and to 2.94 mm post-PTCA (p < 0.001). The percent stenosis was reduced from 91.4 +/- 9.5% before ECLA to 42.3 +/- 14.9% after ELCA and to 14.6 +/- 9.3% after PTCA (p < 0.001). Seventeen (68%) patients were asymptomatic at 6 months and 15 (60%) at 1 year. New restenosis rates were 8/33 (24.2%) at 6 months and 9/33 (27.3%) at 12 months. CONCLUSION: ELCA is safe and effective for the treatment of in-stent restenosis. In the present sample, a slight increase in new restenotic lesions between 6 and 12 months was found. PMID- 10752183 TI - Results of the surgical treatment of chronic atrial fibrillation. AB - OBJECTIVE: Report clinical experience in surgical treatment of atrial fibrillation (AF) by Cox-maze procedure. METHODS: 61 patients underwent surgical treatment for AF. Two had primary AF and 59 AF secondary to heart disease (2 atrial septal defects, 57 mitral). Ages ranged from 20 to 74 years (mean = 49). There were 44 females (72%). The surgical technique employed was Cox 3 without cryoablation. The patients were follow-up in specific at patient clinics and underwent periodical ECG, exercise tests, echocardiogram and Holter monitoring. RESULTS: In-hospital mortality was 4.9% and late mortality 1.6%. A temporary pacemaker was used in 28 (46%) and a definitive in 7 patients (11.4%). On hospital discharge, AF remained in 17%; 63.9% had sinus rhythm, 6.9% atrial rhythm, 1.7% junctional rhythm, and 10.3% had pacemaker rhythm. In the last evaluation, AF was present in 19.5%; (70.5% sinus rhythm, 4% atrial rhythm, 2% atrial tachycardia, and 4% pacemaker rhythm). There was no report of thromboembolic episodes. Chronotropic response was considered adequate in 19%, intermediate in 29%, and inadequate in 42%. In Holter monitoring, the mean heart rate was 82 +/- 8 bpm, with a minimum of 57 +/- 7 bpm and maximum of 126 +/- 23 bpm, with supraventricular extrasystoles in 2.3 +/- 5.5% of the total heartbeats and ventricular extrasystoles in 0.8 +/- 0.5%. In the echocardiogram, the A wave was present in the left atrium in 87.5%. CONCLUSION: Maze procedure is effective and has acceptable surgical risk. Atrial or sinus rhythms remain stable with a small but remarkable frequency of atrial and ventricular arrhythmias. Left atrial contraction is present, although attenuated, as well as the chronotropic response to exercise. PMID- 10752185 TI - Doppler echocardiographic evaluation of HIV-positive patients in different stages of the disease. AB - OBJECTIVE: To evaluate by Doppler echocardiography (DE) early abnormalities of ventricular function in HIV-positive patients, as well as other cardiac abnormalities that can be detected by this method, with special emphasis on mitral valve flow. METHODS: 84 HIV-positive patients, 59 with CD4 cell count > 500/mm3 (Group A) and 25 with CD4 cell count < 500/mm3 (Group B), were analyzed. CD4 cells were counted and matched with structural data and systolic and diastolic function of the left ventricle (LV), as analyzed by DE. The results were compared with those obtained in 47 healthy individuals (Group C). RESULTS: 8% of patients in Group B had mild pericardial effusion; 31.5% showed decreased systolic function of the LV, and 12% had moderate mitral regurgitation. A wave velocity from the mitral inflow was different among the 3 groups, being higher in Group B, where the deceleration time of the E wave of the mitral inflow and the E/A ratio were significantly lower with a normal value of the isovolumic relaxation time (IVRT). CONCLUSION: HIV-positive patients with a CD4 cell count > 500/mm3 had no abnormalities by DE. Patients with a more advanced infection (those with a CD4 cell count < 500/mm3), had a significantly abnormal LV systolic function and a higher incidence of pericardial effusion and mitral regurgitation. Mitral valve inflow by Doppler did not indicate diastolic dysfunction. PMID- 10752186 TI - Ventricular evoked response in patients with hypertrophic obstructive cardiomyopathy treated with DDD pacing. AB - OBJECTIVE: To assess the changes in ventricular evoked responses (VER) produced by the decrease in left ventricular outflow tract gradient (LVOTG) in patients with hypertrophic obstructive cardiomyopathy (HOCM) treated with dual-chamber (DDD) pacing. METHODS: A pulse generator Physios CTM (Biotronik, Germany) was implanted in 9 patients with severe drug-refractory HOCM. After implantation, the following conditions were assessed: 1) Baseline evaluation: different AV delay (ranging from 150 ms to 50 ms) were sequentially programmed during 5 to 10 minutes, and the LVOTG (as determined by Doppler echocardiography) and VER recorded; 2) standard evaluation, when the best AV delay (resulting in the lowest LVOTG) programmed at the initial evaluation was maintained so that its effect on VER and LVOTG could be assessed during each chronic pacing evaluation. RESULTS: LVOTG decreased after DDD pacing, with a mean value of 59 +/- 24 mmHg after dual chamber pacemaker, which was significantly less than the gradient before pacing (98 + 22 mmHg). An AV delay > 100 ms produced a significantly lower decrease in VER depolarization duration (VERDD) when compared to an AV delay < or = 100 ms. Linear regression analyses showed a significant correlation between the LVOTG values and the magnitude of VER (r = 0.69; p < 0.05) in the 9 studied patients. CONCLUSION: The telemetry obtained intramyocardial electrogram is a sensitive means to assess left ventricular dynamics in patients with HOCM treated with DDD pacing. PMID- 10752187 TI - Anomalous origin of the left coronary artery from the right pulmonary artery with intramural aortic trajectory. Clinicosurgical diagnostic implications. AB - OBJECTIVE: Anomalous origin of the left coronary artery from the right pulmonary artery (AOLCARPA), is a rare entity that is usually associated with other defects. Of the 20 cases of AOLCARPA reported in the literature, 14 (70%) had associations. We describe four patients with AOLCARPA without associated defects, but with a peculiar intramural aortic trajectory. METHODS: Fifty-five patients with anomalous origin of the left coronary artery were operated upon at INCORFMUSP. Four of the patients had the anomalous origin from the right pulmonary artery (RPA) without associated defects but with intramural aortic trajectory. Clinical and laboratory examinations were analyzed, as well as surgical findings. RESULTS: All patients had congestive heart failure (CHF) and 3 also had angina pectoris. Two patients had a murmur of mitral regurgitation, signs of myocardial infarction on the ECG and cardiomegaly. The shortening fraction varied from 9% to 23%. The hemodynamic study confirmed the diagnosis of anomalous origin of the coronary artery, but the intramural trajectory and the origin from the RPA were established only at surgery. In 3 patients, the technique of side-to-side anastomosis was performed with a good outcome. One patient, who underwent end-to-side anastomosis, died 6 months after the surgery. CONCLUSION: Association with other defects usually occurs in the AOLCARPA, and the intramural aortic trajectory is difficult to clinically diagnose but easy to surgically correct. PMID- 10752188 TI - Intraaortic balloon pump support during coronary angioplasty. Initial experience. AB - OBJECTIVE: To evaluate the use of the intraaortic balloon (IAoB) in association with coronary angioplasty in high-risk patients. METHODS: Fourteen high-risk patients unresponsive to clinical therapy and with formal contraindication to surgical revascularization were treated by coronary angioplasty, most of which was followed by stenting. All procedures were performed with circulatory support with the IAoB. This study reports the early results and the late findings after 12 months of follow-up. Six patients had multivessel coronary disease; of these, four had left main equivalent lesions and two had unprotected left main coronary artery disease, one of whom had severe "end-vessel" stenosis and the other was a patient with Chagas' disease with single-vessel lesion. Eleven patients had a left ventricular ejection fraction < 30%. RESULTS: In 100% of the patients, the procedures were initially successful. Two patients had severe bleeding during the withdrawal of the left femoral sheath. At the end of twelve months, 4 patients were asymptomatic and the others were clinically controlled. There were two late deaths in the 7th and 11th months. CONCLUSION: The combined use of the intraaortic balloon pump and percutaneous coronary angioplasty in high-risk patients with acute ischemic syndromes provides the necessary hemodynamic stability to successfully perform the procedures. PMID- 10752189 TI - Laboratory assessment of the hypertensive individual. Value of the main guidelines for high blood pressure. AB - OBJECTIVE: To determine if abnormal laboratory findings are more common in individuals with hypertension and in those with other risk factors, such as obesity, smoking and alcohol ingestion. METHODS: A study was carried out in the general outpatient clinics of a university hospital (145 individuals without previous diagnosis of hypertension) and the following variables were assessed: high blood pressure (as defined by the VI Joint National Committee on Prevention, Detection and Treatment of High Blood Pressure--VI JNC), obesity [calculated using body mass index (BMI)], tobacco use, and alcoholic ingestion. The laboratory examinations consisted of the following tests: hemogram, glycemia, uric acid, potassium, total/HDL-fraction cholesterol, triglycerides, calcium and creatinine. RESULTS: High blood pressure was not associated with a higher number of abnormal laboratory tests. Hypertensive individuals with a BMI > or = 25 kg/m2 or normotensive obese individuals, however, had a higher frequency of diabetes (12X), hypertriglyceridemia (3X), and hypercholesterolemia (2X), as compared with hypertensive individuals with BMI < 25 kg/m2 and preobese/normal weight normotensive individuals. CONCLUSION: High blood pressure is not associated with a higher frequency of abnormal laboratory tests. The association of high blood pressure and obesity, however, increases the detection of diabetes and dyslipidemias. PMID- 10752190 TI - Recurrence of atrial septal defect in three generations. AB - Beginning with a patient presenting with an atrial septal defect (ASD) of the secundum type, the genealogy was identified in four affected individuals who belonged to three successive generations of the same family. The defects were visually confirmed in all individuals and were found to be anatomically similar. No other congenital malformations were present in these individuals. The genealogy was identified in 1972, when ASD recurred in two generations, and it was concluded that the mechanism of transmission was autosomal recessive. The fifth individual, identified 21 years later, and having an anomaly identical to that of the others, was the child of a couple who had no consaguinity and whose mother was a member of the previously studied genealogy. Considering the absence of phenotype in the parents and the rarity of the ASD gene in the general population, the occurrence of the uniparental disomy for this family nucleus, and the same autosomal recessive mechanism of transmission by this affected individual is possible. This study reports the familial occurrence of ASD by genetic mechanisms of transmission, emphasizing the necessity for genetic clinical studies in members of the familial nucleus in order to detect new carriers, who usually are asymptomatic, thereby allowing for early and adequate treatment of individuals who may be affected. PMID- 10752192 TI - Clinicopathologic session. (Case 4/99--a 63-year-old man with heart failure 3 years after bioprosthetic replacement of mitral and aortic heart valves- Instituto do Coracao do Hospital das Clinicas-FMUSP, Sao Paulo) PMID- 10752191 TI - Ebstein's anomaly with coarctation of the aorta. An unusual association. AB - Ebstein's anomaly with coarctation of the aorta is an extremely unusual condition. In this report, the clinical and surgical features of 3 male patients, aged 7 months, 4 years and 14 years, are discussed. All patients were in situs solitus. The first 2 patients had atrioventricular and ventriculoarterial discordance and progressed to heart failure in the neonatal period. The third had atrioventricular and ventriculoarterial concordance, as well as Wolf-Parkinson White syndrome, with frequent episodes of paroxysmal tachycardia. The 3 patients underwent surgery for correction of the coarctation of the aorta. The patient with atrioventricular and ventriculoarterial concordance underwent tricuspid valvuloplasty using a DeVega-like technique. In addition, ablation of 2 anomalous pathways (Kent bundle), which were detected by the electrophysiologic study, was also subsequently performed. The 3 patients showed a good postoperative outcome for 2 years, although, in those with discordance, the surgical procedure did not influence the dysplasia of the tricuspid valve, because this valve showed light to moderate dysfunction. PMID- 10752193 TI - The role of depression in coronary artery disease. PMID- 10752195 TI - [Analysis of hemopoietic progenitor cells in bone marrow and peripheral blood samples which have undergone different periods of cryopreservation]. AB - Bone marrow (BM) and peripheral blood stem cell (PBSC) samples from patients undergoing autologous transplant were tested to evaluate the effects of cryopreservation. Cell viability was assessed as well as the proliferative capability of CFU-GM and BFU-E (myeloid and erythroid progenitors respectively). Moreover, long term culture (LTC) of stromal cells was used to test their functionality. A total of 23 samples were studied: 5 from AML patients, 7 MM, 6 NHL, 3 ALL and 2 HL. Nine patients received autologous bone marrow transplant (ABMT) and the remaining 14 PBSC. The cells were frozen during 24 to 33 days before infusion and 16 to 40 months before culture. Forty percent of AML and MM samples gave rise to colonies in vitro while the other hematology diseases tested showed colony growth in almost 100% of the cases. Samples from patients with lymphoid diseases exhibited a good correlation between the percentage of CD34+ cells and the number of colonies developed in culture. Nevertheless, there was no correlation when ALL and MM were tested suggesting that the underlying disease may have affected the growth in culture. The stromal layer was fully developed on BM samples, but on PBSC samples it only generated macrophages and fibroblasts. Our results suggest that the efficacy of cryopreservation of hematopoietic cells can be measured by means of CFU-GM and BFU-E culture and that the period the samples remained frozen did not affect the growth capability of the cells. PMID- 10752194 TI - [Rotavirus diarrhea. Impact in a pediatric hospital of Buenos Aires]. AB - Anticipating the use of the rotavirus vaccine, we performed this study in order to estimate the rotavirus disease burden in a pediatric hospital. We studied 648 children < 3 years of age between September/97 and August/98, assisted at the Outpatient Diarrhea Unit, or hospitalized due to acute diarrhea in our Hospital. We found rotavirus associated to 36% of the diarrhea cases studied in the Outpatient Clinics, and in 45% of the hospitalized children. We estimate the assistance of 1674 rotavirus diarrheas per year in the Outpatient Clinics, but only 14 of them required hospitalization. The study describes a peak of rotavirus diarrheas between March and June, and another peak of rotavirus-negative diarrheas between January and March (probably due to bacterial diarrheas). Rotavirus disease presented a higher frequency between 6 to 23 months of age; only 10% of the 233 rotavirus cases occurred in children older than 24 months and 13% in infants less than 6 months of age. The situation described is significant because the recently licensed rotavirus vaccine is being used in 3 doses at 2, 4 and 6 months and could have prevented most of the rotavirus cases observed during this study. PMID- 10752196 TI - [Tuberculosis in the V Sanitary Zone of Buenos Aires Province. Analysis of its trends between 1984 and 1996]. AB - Tuberculosis (TB) continues to be a serious health problem in most of the districts in the 5th Sanitary Region of Buenos Aires Province (RSV) which is located in North Buenos Aires City suburbs. The aim of this study was to analyse and compare the evolution of the TB epidemiological situation in ten districts, in a thirteen year period (1984-96) and to analyse possible reasons for differences. The average annual variation (VAP) of morbidity rate was calculated in order to estimate the trends of TB in these communities along time. Reporting to the National and Provincial TB Control Programmes of TB cases is mandatory for physicians and these data were used as a source of information for the number of cases and rates. A statistical analysis was performed. Our results showed that the VAP value for the whole RSV was lower than -5.0%. On this basis several districts were identified as having a TB critical situation. The proportion of smear positive cases among TB patients reported decreased during this period, suggesting that smear microscopy was being poorly used as a diagnostic tool for TB. Furthermore, the cure rates of the patients attained--in average--only 60.0% and several warning indicators (IA)--like TB meningitis cases and mortality due to TB in people younger than thirty years old, as well as TB--HIV/AIDS association--have been observed to grow in several districts. Previous world global experience and those obtained in other provinces in our own country, suggest that the unique possible strategy for modifying this situation, is to set up the directly observed treatment (DOTS), according to World Health Organization/International Union Against Tuberculosis and Lung Diseases (WHO IVATLD) recommendations, in order to achieve the control of TB. PMID- 10752197 TI - [Exogenous adenosine and postischemic dysfunction in the isolated rabbit heart]. AB - It is recognized that adenosine lessens the systolic alterations of the postischemic ventricular dysfunction ("stunned myocardium"), but little is known about the drug's effects on the diastolic phase of the cardiac cycle. The aim of this work was to determine the effect of adenosine when it was administered: a) before ischemia and during reperfusion, and b) from the early reperfusion period to the end of the experiment on the systolic and diastolic function of the "stunned myocardium". An additional objective was to determine whether adenosine modifies the release of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH), in the "stunned myocardium". Rabbit isolated isovolumic hearts were perfused according to Langendorff's technique, and subjected to 15 minutes global ischemia and 30 minutes reperfusion. A small latex balloon was inserted into the left ventricle via the left atrium which allowed to measure the ventricular end diastolic pressure (diastolic stiffness) and calculate the developed pressure, the maximal rate of pressure generation and maximal rate of pressure decay (+dP/dtmax and -dP/dtmax), the ratio between these two variables (+P/-P), and the time constant of isovolumic relaxation (tau, Tau). The adenosine administered both before the ischemia period, and at the beginning of reperfusion, attenuated the systolic and diastolic stiffness alterations without modifying the isovolumic relaxation. The administration of adenosine did not diminish the CPK and LDH release significantly when it was given before the ischemia period or the beginning of reperfusion. PMID- 10752198 TI - [The equivalent renal urea clearance. Its relationship with mortality in chronic hemodialysed patients]. AB - The Equivalent Renal Urea Clearance (EKR) integrates the residual renal function (KR) and the dialysis dose (Kt/V). The present study was performed with these objectives: to calculate EKR in our hemodialysis (HD) patients during a three year follow up, to define its relationship with mortality and to compare its importance as a risk factor among others and to calculate actuarial survival. We analyzed 267 chronic HD patients. We measured Kt/V single pool, TACu, albumin, creatinine, hemoglobin and HD time and we calculated KR, EKR, KRc and EKRc (the last two corrected for V* 401--to compare clearances of different size patients). The EKRc median was 14.20 ml/min and it was taken as cut off point. The mortality OR was 2.17. The multivariated analysis showed, as independent mortality predictors, the albumin (the most significant), the EKRc and the HD time. The actuarial survival of EKRc, Kt/V and albumin showed marked similarity of their curves. The significant differences between the predictor curves began on the 2nd year of HD, for albumin they began in levels lesser than 3.5 g/dl and higher than 3.4 g/dl. Therefore, we consider that the best advantage of EKRc, compared to other parameters derived from the urea kinetics model, was the possibility to valuate the weekly HD adequation (once, twice or three times a week) and this, depending on the KR and the Kt/V of each HD treatment. Besides, the EKRc could also be used as adequacy criterion in CAPD considering daily Kt/V so that we could employ EKRc as adequacy parameter for both replacement therapies. PMID- 10752199 TI - [Soluble intercellular adhesion molecule-1 (ICAM-1) levels in HIV infected children]. AB - Intercellular adhesion molecule-1 (ICAM-1) is a membrane bound molecule that is involved in cell to cell adhesive interactions within the immune system. The aim of this study was to measure the concentrations of soluble ICAM-1 (s-ICAM-1) in 25 HIV-1 infected pediatric patients. We compared s-ICAM-1 values to parameters of immune activation--such as IgA and beta 2 microglobulin (beta 2 m) and viral replication such as adenosine deaminase (ADA). s-ICAM-1 levels were found to be significantly increased in HIV-1 infected children when compared with healthy controls. Levels of s-ICAM-1 were higher in patients with severe forms of HIV-1 infection in comparison with those with a mild form of the disease or non symptomatic infection. No differences in titers of s-ICAM-1 were recorded between seroreverters and healthy controls. A positive correlation between levels of s ICAM-1 and IgA, beta 2 m or ADA concentrations was detected. Similarly, there was statistically significant correlation between levels of IgA, beta 2 m or ADA. In conclusion, increased s-ICAM-1 levels in HIV-1 pediatric patients appeared to be another important feature among the immune disturbances triggered by HIV-1 infection. s-ICAM-1 might be involved in the development of the immunologic dysfunction during the progression of the disease. PMID- 10752200 TI - [Survey among chest physicians regarding the diagnosis and management of asthma]. AB - A survey on asthma management was conducted in 300 chest physicians randomized from a national list. Diagnosis procedures, methods for recognizing life threatening asthma attacks and patient education about his or her disease were reviewed. Ninety-eight responses were obtained (32.7% of the questionnaires mailed). 71% of the responders were specialized in respiratory medicine (RM), 12% in RM + Internal Medicine (IM), 6% in RM + Allergy (A), 5% in A, 4% in IM, and 2% in IM + A. As a diagnostic test, immediate response to bronchodilator was seldom used and a trial course of oral steroids was even less used. Blood eosinophilia and specific IgE RAST were frequently used and more than in other compared countries in spite of its doubtfulness for the diagnosis of asthma. Severity of asthma attack was assessed less than in other countries in relation to symptoms, lack of response to inhaled bronchodilators and with practically no use of any objective method for the assessment of airways obstruction (PEFR). The latter was seldom employed for chronic control of the disease. The assessment of the correct technique of metered dose inhaler or dry powder inhaler use was rarely done. Only the questions referred to patient education about their disease, teaching about the difference between relieving (bronchodilator) and anti-inflammatory treatment, communication to the patient about the severity of his or her disease and the frequency of giving a written action plan in case of severe asthma attacks qualified equal or better than countries that always qualified well. These responses, together with other data of the literature, makes us suspect, as is common in these audits, that the interviewed person sometimes replies what he or she should do, but not necessarily what he or she does. PMID- 10752201 TI - In vivo down regulation of HIV replication after hepatitis C superinfection. AB - There are increasing molecular and clinical evidences that the effects of human immunodeficiency virus (HIV) infection can be modified by coinfection with other viruses. The objective was to investigate the viral interaction between HIV and hepatitis C virus (HCV) after HCV superinfection. A 16 year-old pregnant woman was evaluated because of icteric acute hepatitis. Admission laboratory tests showed the following results: ALT 877 IU/L; AST 1822 IU/L; bilirubin 6.79 mg/dl. Diagnosis of acute HCV was based on detection of serum HCV RNA by PCR and anti HCV seroconversion. ELISA for anti HIV testing was positive and confirmed by western blot. Serum markers for other viruses were negative. The patient was followed during 19 months; serum samples were taken monthly during this period for detection of plasma HIV and HCV RNA. Levels of plasma HIV-RNA were positive in all samples tested before and after the onset of acute hepatitis C. Six months later and a for two month period, and 13 months later for a period of one month HIV viremia was undetectable; then HIV-RNA in plasma was detectable again. In conclusion, HCV superinfection may have temporarily interfered with HIV replication in our patient. The following observations support our hypothesis: it has been demonstrated that HIV-1 replication is suppressed by HCV core protein which has transcriptional regulation properties of several viral and cellular promoters. Clinical implications of this event are not generally known and the interaction between these two viruses in dual infections is worth considering. PMID- 10752203 TI - [Great vessel and temporal artery involvement in temporal arteritis]. AB - The importance of this case report resides in the unusual initiation of an arterial inflammatory disease. The patient presented ischemic symptoms and signs with bilateral upper limb claudication and Raynaud's phenomenon, with ulcerations on her left hand. We describe the patient's clinical history, diagnosis and therapy. PMID- 10752204 TI - [Serious depression and cancer of the pancreas]. PMID- 10752205 TI - [Subclinical atherosclerosis. An objective index of susceptibility and vascular risk]. AB - Clinical data suggest that the individual susceptibility to atherosclerosis is not accounted for only by exposure to classical or "emerging" vascular risk factors. Although recent investigations with transgenic animals have revealed new genetic determinants of susceptibility, little is known concerning this situation in human beings. Even though the human genome project might uncover specific genetic markers in the future, the only early and objective method to identify the susceptible individual at present is to detect atherosclerosis non invasively. High resolution B-mode ultrasonography of superficial arteries coupled with advanced computer-assisted image processing systems is a highly reproducible method to perform a quali-quantitative early evaluation of already developed wall lesions. Clinically more significant, the detection of silent atherosclerosis has an additional value for risk factor assessment in the prediction of global vascular risk, a relevant index for decision-making in cardiovascular prevention. It is conceivable that the introduction of non invasive measures of the atherosclerotic burden in risk stratification of asymptomatic subjects will help to target interventions for more rational risk factor control and to reduce the cost/benefit ratio of primary prevention. PMID- 10752206 TI - [Current status of the treatment of HIV infection]. AB - The antiretroviral therapeutic strategy changed in 1996, when the prognostic value of the plasma viral load and the CD4 cell counts were confirmed. Two nucleosides, plus a protease inhibitor or a non-nucleoside, are currently the initial preferred options. Viral resistance, and pharmacokinetic problems, however, have limited the efficacy of these schemes. It should be emphasized that continued use of triple drug therapy regimens have been associated with a decrease of morbidity and mortality associated to AIDS. We have analyzed the different drug combinations used in clinical trials and their results, the definition of therapeutic failure, and its consequences to control the treatment. With the currently available regimens, the virus cannot be totally eradicated from the organism and treatment should be continued for life. The combination of drugs in a single tablet, and the decrease in the number of daily intakes can help the patient with adherence, and allow for supervised treatment programs like the ones already applied with success in the treatment of tuberculosis. The use of virus resistance assays and pharmacokinetic assays will help to select the most appropriate treatment in the near future. PMID- 10752207 TI - [On the numbers and duration of a physiologic phenomenon]. PMID- 10752208 TI - [In defense of aminophylline]. PMID- 10752202 TI - [Salami worker's lung]. AB - Hypersensitivity pneumonitis (HP) is the clinical manifestation of an pulmonary immunological reaction to inhaled antigens. The list of provocative antigens and specific illnesses grows permanently. A woman of 56 years consulted for dyspnea and cough of two months of evolution. She worked in a factory of sausages removing the dust that recovers the salamis during their stationing. She presented hypoxemia and the pulmonary function test revealed a predominantly obstructive pattern. The x-ray of the thorax was normal and the high resolution computed tomography showed a mosaic pattern. A lung biopsy confirmed the diagnosis of HP. She improved with systemic corticosteroids and the avoidance of antigen exposure. In the cultures of the material that recovers the salamis, Penicillium spp. was recovered and was interpreted as the probable etiological agent of this HP, which we have denominated "the salami worker's lung". PMID- 10752209 TI - [Kidney and pancreas transplantation: initial experience at a single transplant center in Argentina]. AB - After more than 10,000 cases reported all over the world until 1998, simultaneous kidney and pancreas transplantation has become a safe clinical practice, and it may probably represent the best treatment available for diabetic patients in end stage renal disease. Here we present our results after 12 cadaveric pancreas transplants (8 whole organ, and 4 islet transplants), performed on insulin dependent diabetic patients. Eleven of these patients received a kidney simultaneously, and one of them required a kidney retransplantation. All vascularised pancreatic grafts were positioned intraperitoneally, anastomosed to the iliac vessels, and bladder drained. One year patient, whole pancreas, and kidney survival rates were 86%, 86% and 71%, respectively. All of these patients remain insulin and dialysis-free, the longest for 37 months. Islets for transplantation were obtained from single cadaveric donors. Fresh, unpurified cells were transplanted intraperitoneally by laparoscopy (equivalent islet yields: 3 x 10(5), 4 x 10(5), 1 x 10(6) and 5 x 10(5)). None of the islet recipients resulted insulin-independent but they all reduced daily requirements in about 40%, with better metabolic control (mean HbA1c pretransplant 9.4 +/- 1.8, vs 7.9 +/- 1.6 posttransplant). One kidney graft was lost due to venous thrombosis. Simultaneous kidney and pancreas transplantation offers the diabetic patient in end-stage renal disease a chance of independence both from dialysis and exogenous insulin. Whole pancreas transplantation has better functional outcome than islet transplantation. Nevertheless, for those diabetic patients who do not meet the criteria to receive a vascularised graft, pancreatic cells may still improve carbohydrate metabolism with minor surgical risk. PMID- 10752210 TI - [Serological diagnosis of celiac disease: anti-gliadin peptide antibodies and tissue anti-transglutaminase]. AB - Serological markers currently used for the diagnosis of celiac disease are anti gliadin (AG) and anti-endomysium (AE) antibodies. Recently tissue transglutaminase (tTG) was identified as the specific autoantigen for endomysial antibodies. The aim of this work was to determine sensitivity and specificity of ELISA tests developed by using defined molecular structures as capture antigen for AG and AE antibodies. Three synthetic peptides, from the amino terminal region of alpha gliadin, were used as immobilized antigens for AG, and the transglutaminase from guinea pig liver for AE. A total of 80 sera from celiac patients, non celiac disease controls and healthy controls were examined. Age range was 7 months to 14 years. A sensitivity of 97% and a specificity of 86% was obtained for IgG determined by using as antigen one of the three synthetic peptides (corresponding to residues 31-55 of alpha gliadin). Therefore, this peptide appears as a highly sensitive antigen and more specific than gliadin. The best result, showing 100% of sensitivity and specificity, was obtained for IgA anti-tTG, thus pointing out the relevance of these antibodies as serological markers for celiac disease. PMID- 10752211 TI - Management of overt and subclinical hypothyroidism. Factors influencing L thyroxine dosage. AB - With the aim of establishing optimal dosage schedules, 171 women with either overt (OH, n = 80) or subclinical (SCH, n = 91) hypothyroidism were assessed before and 6 months after starting L-thyroxine (LT4) replacement therapy. Each group was further classified into four subgroups according to post-therapy serum TSH level, as follows: A) complete suppression; B) partial suppression; C) normal range and D) above normal range (insufficient response). In all subgroups, LT4 doses were higher for OH than for SCH, whether expressed as total daily dose (micrograms) or as a function of either actual or ideal body weight (micrograms/kg BW). In OH, LT4 dose was higher for subgroups A or B as compared with either C or D. In SCH, subgroup A received a larger dose than the other subgroups. Post-treatment serum thyroxine levels showed the same pattern for both OH and SCH. Mean LT4 dose was similar in patients with high and normal antithyroid antibodies and in patients with goiter and in those without it. In goitrous patients thyroid volume decreased in subgroup B, particularly in those patients that had elevated antithyroid antibodies, but not in subgroup C. In OH patients a significant negative correlation was found between daily LT4 dose per kg actual BW and actual BW, especially in subgroup C for patients with a body mass index > 27 kg/cm2 (r = -0.90, p < 0.001). In subgroup C of the SCH group, a negative correlation between LT4 dose and age was noticed. Both in OH and in SCH, LT4 dose per kg actual BW required to obtain a serum TSH within the normal range was lower in women with a body mass index (BMI) > 27 kg/m2 than in those with a BMI < or = 27 kg/m2. LT4 doses for subgroup C did not differ from those needed in hypothyroid patients with previous Graves' disease, in either OH or SCH patients. PMID- 10752212 TI - [Respiratory failure in the Guillain Barre syndrome]. AB - Severe involvement of the respiratory muscles is seen in 25% of patients with a diagnosis of Guillain Barre syndrome. In order to evaluate the clinical characteristics and evolution of this disorder we reviewed the clinical records of patients admitted with this diagnosis to our Hospital between January 1987 and December 1996. We identified 44 patients with respiratory failure. The age was 34.0 +/- 14.1 years, 31 patients were male and 13 female; 70.5% required mechanical ventilation (MV). The time elapsed between the beginning of symptoms and MV was 9.4 +/- 8.0 days. Total duration of MV was 1,224 +/- 1,208 hours. Patients who required ventilatory support during the first 48 hours of evolution had a significantly longer duration of MV compared to the rest of the group (2,100 +/- 2,076 vs 934 +/- 735 hours, p < 0.05). Two of the survivors needed ventilatory support for more than 6 months. All patients showed quadriparesia, 55% had cranial nerve involvement and 43% had autonomic dysregulation. Twenty four percent had a positive serologic titer for cytomegalovirus infection. The vital capacity measured before the beginning of MV was 1,050 +/- 378 ml and at discharge 2,837 +/- 1,080 ml. Mortality was 18%, with a higher mean age among those who died (44.9 +/- 17.5 years vs 31.9 +/- 12.5, p < 0.02). Mortality was also related to sepsis, barotrauma and severe autonomic dysfunction. In our group, we identified male preponderance, a high percentage of MV with an extended duration, and a longer MV time in the group of patients with a faster evolution. PMID- 10752213 TI - [Multiparametric analysis for the diagnosis of iron deficiency anemia]. AB - The serum transferrin receptor (sTR) as a marker of iron depletion was evaluated in two groups: 50 normal adults of both sexes living at sea level and 50 iron deficiency anemias (secondary to nutritional, gastrointestinal or gynecologic diseases). Mean values were 16.6 nmol/L (interval of reference 8.8 to 26.2), for controls, without variations related to age and sex, and 66.3 nmol/L (16.1 to 148.8) for anemic patients. Statistical analysis (receiver operating characteristics, ROC) determined an optimal reference interval of 8.8 to 25.8 nmol/L. Predictive values as a diagnostic tool were 97.5%, PV (+) and 97.7%, PV ( ); diagnostic efficiency was 97.7%. In both controls and anemics it was observed: 1) an inverse relationship between sTR and serum ferritin (F) (r2 72%; p < 0.001); 2) wide variations of sTR when plasma hemoglobin (Hb) was < 100 g/L (r2 71%; p < 0.001); 3) values for the sTR/logarithm of serum ferritin ratio (sTR/F index) much higher in anemics (75.8) than in controls (9.6). In the former group, iron supplementation normalized sTR levels but did not change ferritin values. We conclude that sTR is a specific and sensitive index of functional iron deficiency and therefore a quick, accurate and non invasive quantitative parameter for the diagnosis of iron deficient erythropoiesis. PMID- 10752214 TI - A seroepidemiological survey of HTLV-I/II carriers in the Puna Jujena. AB - Human T-cell leukemia virus type I (HTLV-I) carriers are clustered in limited groups in the world. One of these groups is the Andean native population of South America. As part of an international collaborative study devoted to explore the clustering of HTVL-I carriers in different countries, the aim of this paper was to evaluate the seroprevalence of HTLV-I/II virus in the native population of Puna Argentina in Jujuy. Blood samples of individuals of three populations of Puna Jujena (Susques, Rinconada, Cochinoca) were screened with particle agglutination (PA), immunofluorescence (IF) and western immunoblotting analysis (WB) tests. Two out 86 (2.32%) individuals examined in the Puna Jujena showed positive results for HTLV-I antibodies. It is concluded that the Province of Jujuy, in particular its less miscegenated highest altitude areas, constitute the northern and southern Andean natural geographical clustering of HTLV-I. This distribution is probably linked both to a history of prehistoric human dispersal in the Andes and to high mother- to-child transmission of the virus under close conditions of each group. PMID- 10752215 TI - [Chronic inflammatory demyelinating polyneuropathy. Findings in 30 patients]. AB - Chronic demyelinating inflammatory polineuropathy (CIDP) is a disease which was recognized several years ago. However, the mechanism underlying its pathogenesis remains poorly understood. Nevertheless, there are some clues which strongly suggest that it constitutes an autoimmune disease. Since 1992 we have studied 30 cases. All them were clinically assessed and submitted to laboratory investigations encompassing nerve conduction studies, sera proteins immunoelectrophoresis, spinal fluid analysis and sural nerve biopsies. Upon clinical examination the usual findings were weakness, muscle atrophy, absence or diminished tendon jerks, paresthesias and hyposthesias. Electrophysiological studies disclosed marked slowing of the nerve conduction velocities, suggesting demyelination. Sera immunoelectrophoresis detected monoclonal gammopathy in 17% of the studied patients, which was not associated with lymphoproliferative illnesses. Of the patients 79% had increased levels of spinal fluid proteins. Seventeen patients gave their consent for performing a sural nerve biopsy; all the samples showed demyelination. In conclusion, we think that CDIP is a disease which can be recognized when the clinical assessment, the nerve conduction studies and the spinal fluid findings suggest the diagnosis. Although nerve biopsy may be strongly supporting, we believe that it has to be performed only if doubts arise from the clinical, electrophysiological or spinal fluid observations. It is worth noting that its early detection may benefit the patient through the administration of the right therapy precluding the eventual sequelae of the disease. PMID- 10752216 TI - [Heart rate and physical activity to assess energy expenditure in children]. AB - The aim of the present study was to validate assessment of oxygen consumption (VO2) from heart rate (HR) and physical activity (PA) electronically recorded. Every minute, mean VO2 were validated with indirect calorimetry. We studied 25 children (12 girls, 13 boys), 12.1 +/- 0.7 years old. Measurements were made during about 60 minutes while kids were supine, sitting, standing and walking at four intensities. Minute by minute heart rate was converted to VO2 and energy expenditure using 2 different functions for active and inactive situations. A linear function (VO2 = a + beta HR) was used when counts were 7 or higher and HR higher than a prefixed point (intersection between the 2 line functions). A nonlinear equation (VO2 = a + beta HR3) was used in the remaining minutes. Mean predicted VO2 for every minute were similar to measured VO2 (2 ways interaction ANOVA, p = 0.99). Mean VO2 correlated significantly with VO2 predicted by equation (r = 0.99, p < 0.01). High degree of agreement was found (Bland-Altman comparisons). Combined heart rate and physical activity predicted oxygen consumption with a precision similar to the indirect calorimetric method. PMID- 10752217 TI - [Community-acquired pneumonia in the elderly requiring hospitalization. Clinical features and prognosis]. AB - We evaluated retrospectively 96 patients older than 64 years admitted with the diagnosis of Community Acquired Pneumonia (CAP) in order to describe the clinical features, evaluate severity and assess prognostic factors. During an 18-month period 100 cases of CAP were included. Average age was 82.3 years +/- 8.3 (+/- SD). By the time of admission, cough and fever were found in 35% of cases and 48% had altered mental status. Fourteen per cent needed mechanical ventilation. Etiology was determined in 21% of cases. Most common pathogens were S. pneumoniae (38.1%), S. aureus (19%) and H. infuenzae (14.3%). Overall mortality was 29%. The most commonly present criteria of severity were tachypnea (respiratory rate > 30) and a PaO2/FIO2 ratio < 250. Severe pneumonia was found in 60% of patients and mortality in that group was 40%. Multivariate analysis demonstrated that some independent prognostic factors were associated with higher mortality: requirement of vasopressors (Odds Ratio [OR] = 22.0; 95% confidence interval [CI] = 1.9 249.5), oliguria (OR = 9.9; CI = 1.5-66.2), previous neurologic disease (OR = 8.2; CI = 1.8-36.6), PaCO2 > 44 mm/Hg (OR = 6.9; CI = 1.1-43.2), and creatinine > 1.4 mg/dl (OR = 4.7; CI = 1.2-19.1). We conclude that CAP features in elderly patients requiring hospitalization are atypical, severe presentations are frequent and mortality is high. Prognostic factors as found in this study can help the evaluating physician to identify those who require special care. PMID- 10752218 TI - [Thrombotic thrombocytopenic purpura and systemic lupus erythematous. Three cases presenting simultaneously]. AB - Thrombotic thrombocytopenic purpura (TTP) is a clinical syndrome characterized by microangiopathic hemolytic anemia, thrombocytopenia, fever, neurological symptoms, and renal involvement. The relationship of TTP to systemic lupus erythematosus (SLE) has been recognized in the medical literature since 1939. The differential diagnosis is difficult because both diseases have similar clinical features. The mainstay for recognizing TTP in the context of active SLE is the presence of helmet red cells, marked reticulocytosis, and negative direct Coomb's test. We report three female patients with simultaneous presentation of TTP and SLE. We suggest combined treatment with immunosuppressive therapy and plasma exchange using fresh frozen plasma. PMID- 10752219 TI - [Catastrophic antiphospholipid syndrome. Communication of 2 forms of presentation]. AB - Antiphospholipid syndrome is characterized by recurrent fetal loss, arterial and venous thromboses, thrombocytopenia and circulating antiphospholipid antibodies. Few patients have a rapidly progressive, fatal outcome. We report two young patients with systemic lupus erythematosus and antiphospholipid antibodies who died after a short course of disease. Although clinical and laboratory findings differed in both patients--small vessel thromboses and microangiopathic hemolytic anemia mimicking thrombotic thrombocytopenic purpura predominated in one of the patients while small and medium size vessel thromboses without hemolysis were present in the other case--autopsy revealed widespread visceral thromboses in both of them, features consistent with a diagnosis of catastrophic antiphospholipid syndrome. This syndrome has not been reported to occur in association with Pneumocistis carinii pneumonia as we describe in one of our patients. PMID- 10752220 TI - [Intestinal alterations and reduction of growth in prepuberal rats fed with soybean]. AB - Weanling rats from the inbred lines alpha and beta were fed with a soybean-cereal mixture used for human consumption (AN). A group fed with laboratory rat chow was used as reference (AC). Growth and other nutritional parameters as well as intestinal morphohystometry were evaluated from 22 to 44 days of age. Growth rate and final weight were greater with AC in both lines of rats. The greater initial food conversion efficiency of AN diet, compared to AC, decreased rapidly with the progression of age, particularly in the beta strain. Nitrogen (N) and lipid fecal contents were significantly larger for AN in both lines. Apparent nitrogen digestibility, expressed as the difference between N intake and fecal N with respect to N intake was AC < AN (p < 0.01) for line alpha and AC > AN (p < 0.01) for line beta. The weight and the mucosal total width of the small intestine were AC > AN in both lines. AN produced a significant decrease of villi goblet cells in both strains (p < 0.005). Cecum weight was AC > AN (p < 0.01) for the beta strain. These results alert about uncontrolled consumption of soybean products without adequate inhibition of antinutritional factors, a potential risk for growing animal populations. PMID- 10752221 TI - Oligobodies: bench made synthetic antibodies. AB - Using synthetic peptides and a combinatorial library of 56 mer random oligonucleotides, we have developed reagents that behave as "synthetic antibodies". The results obtained with the protein phosphatase 2A as a model system are shown here. The specificity of these reagents, named "oligobodies", has been demonstrated by Western blot analysis and immunohistochemistry. The oligobodies have enormous advantages compared to antibodies: their production is independent of the immune system, they can be prepared in a few days and there is no need for a purified target protein. These reagents can be produced even if the corresponding protein was never isolated or purified, since only a partial DNA sequence from a database provides enough information to make them. PMID- 10752222 TI - [Toxoplasma gondii infection in Amerindians of Venezuelan Amazon]. AB - We conducted a serological survey for Toxoplasma gondii in 121 Amerindians of the Guajibo ethnic group, 4 to 45 years of age, inhabiting Amazonas State, in the Venezuelan rain forest. The overall prevalence was 88%. Pattern of prevalence and antibody titres were compatible with constant transmission. Sex differences in antibody prevalence were not detected but antibody titres were significantly higher in females. The study is consistent with the presence of risk factors, which favour a frequent exposure of these Amerindians to T. gondii since childhood. PMID- 10752223 TI - [Acquired renal cystic disease. Polysyndromatic entity as cause of non immunological progression of renal failure]. AB - We present a patient with rapidly progressive glomerulonephritis who after immunosuppression and hemodialysis treatment showed an improvement in his condition. Eight years later a computed tomography discovered an acquired renal cystic disease (ARCD) characterized by the development of 3 or more cysts in both kidneys of patients with chronic renal disorders and no history of hereditary cystic disease. ARCD may be asymptomatic or as it occurred in this patient, associated with several complications related to renal cysts such as polyuria polydipsia syndrome, renal hemorrhagic cyst, perinephric hemorrhage and renal cell carcinoma. Along 12 years of follow-up the renal function showed a very slow declination which could be attributed to ARCD. It is suggested that ARCD can be considered as a non-immunological factor of renal progression when it develops in patients with mild chronic renal failure. PMID- 10752224 TI - [Images in medicine. Pleural lymphoma]. PMID- 10752225 TI - [Images in medicine. Prolonged febrile syndrome and inferior vena cava thrombosis]. PMID- 10752226 TI - [Purpura, lung disease and necrotizing glomerulonephritis]. PMID- 10752227 TI - [Study of 2 methods to avoid the nephropathy associated with radiologic contrast]. PMID- 10752228 TI - [Post-transplantation osteoporosis]. AB - In the last two decades organ transplantation has become an effective and established therapy for end-stage disease of various organs. The increase in survival has been due to the greater immunosuppressive capacity of regimens that include cyclosporin. During the first few months after transplantation cyclosporin is associated with high-dose steroids, which produce deleterious effects on bone and mineral metabolism. These effects are superimposed on the previous bone lesions produced by the underlying chronic diseases. Rapid bone loss occurs specially during the first 6 to 12 months after transplantation, when the incidence of fractures is greater. The majority of the fractures involve the spine. Fracture rates are lower after renal transplantation (7 to 11% in nondiabetic renal transplant recipients) and higher in the recipients of other organ transplants: 17.2 to 42% after liver transplantation, 18 to 50% after cardiac transplantation and 25 to 29% after lung transplantation. No pretransplant densitometric or biochemical parameter can adequately predict fracture risk in the individual patient. Despite this, patients with low bone mineral density at the hip, particularly in women, tend to have an increased risk of fracture. Patients can have vertebral fractures despite normal bone mineral density at the spine. Pathogenesis of bone loss is multifactorial. Patients with renal and liver diseases have either renal or hepatic osteodystrophy prior to transplantation that predispose to bone loss, and many patients awaiting pulmonary transplantation already have osteoporosis due to the use of corticosteroids for their lung disease. Rapid bone loss after transplantation depends, as suggested by prospective biochemical parameters, on a decrease in bone formation (reduction in osteocalcin levels) and an increase in bone resorption. Steroids seem to be the principal determinants of these derangements, although some role of cyclosporin cannot be excluded. Other factors that contribute to bone loss are secondary hyperparathyroidism and hypogonadism. Calcium supplementation and vitamin D administration as the only preventive measures do not seem to reduce fracture risk. The most promising regimens to prevent bone loss after transplantation seem to be the use of bisphosphonates immediately prior to and during the first year after transplantation. PMID- 10752229 TI - [Heart failure in Argentina]. AB - Heart failure is a common and costly clinical entity that implies high morbidity and mortality. The aim of this study was to establish the impact of the heart failure syndrome in our country. We analyzed: 1) the data-base of vital statistics from the National Program of Health Statistics, Ministry of Health, between 1980 and 1997, 2) the registers from two national surveys on heart failure patients performed by the Argentine Society of Cardiology and the Argentine Council of Residents in Cardiology. Cardiovascular syndromes have constituted the first cause of death in our country for the last twenty years. Among these, heart failure represents the most frequent entity. From 1980 to 1997 a progressive reduction of 31% in the rate of cardiovascular mortality was observed. From 1990 to 1997, a decrease in the mortality rate due to heart failure of 22.4% was registered. Age and sex adjusted mortality from heart failure suffered a steady increase in older groups, specially above 65 years of age. Hospital discharge data showed that heart failure and cerebrovascular illness are the highest prevalent entities. National surveys on heart failure demonstrate a high prevalence of hypertension, as an associated risk factor, and non compliance with the medication and diet as causes of decompensation. A trend towards an increase in pharmacological prescriptions was also observed. PMID- 10752230 TI - [Betablockers and the treatment of chronic heart failure]. PMID- 10752231 TI - [Hundred years later in scientific investigation]. PMID- 10752232 TI - [Nobel prize in physiology or medicine for 1999. The signals directing traffic in the cells]. PMID- 10752233 TI - [Ethical aspects on human experimentation included in the instructions for the authors in Brazilian scientific journals]. PMID- 10752234 TI - [Sensitivity and specificity of stereotaxic core biopsy in the diagnosis of non palpable lesions of the breast]. AB - BACKGROUND: Recently the stereotaxic core biopsy has been proposed as an alternative method for the initial evaluation of non palpable breast lesions. The authors verify the accuracy of core biopsy in diagnosis and discuss the applicability of tests used for nominal variables, like sensibility and specificity, to evaluate grading of quality such as benign, borderline and malignant. MATERIAL AND METHODS: Among 700 stereotaxic core biopsies of the breast the authors selected the first 567 cases, examined from May, 1993 to May, 1996 to check the accuracy of the histopathologic diagnosis. These cases were followed-up for a minimum of six months and a maximum of thirty months. For the evaluation, the diagnosis by core biopsies was compared with the findings of the surgical resection specimens for the malignant and atypical hyperplasia cases, and mammographic/clinical examinations for the cases not submitted to surgery. RESULTS: The sensibility and specificity were respectively 90.72% e 98.36% and the Spearman correlation test was 0.92. CONCLUSIONS: The stereotaxic core biopsy is an accurate method for the evaluation of breast non-palpable lesions. These results are similar to others in the literature. PMID- 10752235 TI - [Analysis of ethical aspects on human experimentation included in the instructions for the authors in 139 Brazilian scientific journals]. AB - The concern with ethical standards during research in human subjects has been improving in Brazil during the last decade. Analysis of the ethical recommendations in instructions to authors may contribute to understand Brazilian Scientific Journals policies. OBJECTIVE: To evaluate the recommendations related to the ethics of investigation in human subjects in instructions to authors of Brazilian Scientific Journals. MATERIAL AND METHODS: Instructions to authors of 139 Brazilian Scientific Journals in medicine, biomedicine, nursery, odontology and general sciences were analysed. RESULTS: In one hundred ten (79.1%) of the 139 journals no recommendations related to ethics were found; 17 (12.2%) required previous approval by the institutional committee; 3 (2.1%) the procedures should be in accordance with the Declaration of Helsinki; 1 (0.7%) recommends the application of the informed consent; 5 (3.5%) recommend the use of Uniform requirements for manuscripts submitted to biomedical journals and 3 (2.1%) follow no specified rules. Twenty nine journals ask for references in ethics standards: in 15 (51.7%) the information must be included in the text; in 2 (6.8%) the authors must send a letter informing how the ethics standards were followed; in 1 (3.4%) a copy of the approval by the institutional committee must be included; in 1 (3.4%) believed the authors followed the standards and in 10 (34.4%) no specific reference was made. CONCLUSION: These results indicated that the majority of Brazilian Scientific Journals have little concern related to ethical aspects of research in human subjects included in instructions to authors. Even journals that make references to the ethical aspects the recommendations are quite different. PMID- 10752236 TI - [Chlamydia trachomatis infection in the neonatal period: clinical and laboratory aspects. Experience of a decade: 1987-1998]. AB - Chlamydia trachomatis infection is adquired by the newborn infant during the delivery, 25 to 50% of them may develop conjunctivitis and 10 to 20% pneumonia. BACKGROUND: To verify the incidence of ocular infection by C. trachomatis in the newborn infants with conjunctivitis. To observe the association between ocular infection and intersticial pneumonia.-Study the epidemiological aspects and laboratorial methods of criterial diagnosis. CASUISTICS AND METHODS: We studied the newborn infants admitted in the intensive neonatal care with diagnostic of conjunctivitis and/or interstitial pneumonia during the period of ten years. The diagnostic methods were direct exam of etiologic agent in conjunctival material, X ray chest and serologic test by imunofluorescence method for IgG and IgM antibodies. RESULTS: We studied the clinical characteristics of 20 newborns infants with chlamydial trachomatis infection: 15 (75%) were terms newborns and 5 (25%) pre-terms. We observed the predominance of infection in females (60%); pneumonia was observed in 15/20 (75%) and 12 of them had both: conjunctivitis and pneumonia. We did not observe significant association among type of delivery, age of the mother, number of partner and infection. Leukorrhea was present em 50% of the mothers The serologic test was positive in 100% of the newborn with pneumonia and none with conjunctivitis alone, and the direct exam in conjuntival material was positive in newborns with conjunctivitis. The incidence of C. trachomatis in the newborns admitted in this period with conjunctivitis were 17/100 (17%). CONCLUSION: Chlamydia trachomatis is an important pathogenical agent and the research of it is essential in newborn infants with conjunctivitis and/or interstitial pneumonia even there were not risk factors for sexually transmitted diseases. The direct exam of conjunctival material and serologic test are very important to diagnosis. PMID- 10752237 TI - [Health care: a critical review]. AB - Historic review of the medical care in a marketing context as part o Welfare State as claimed by economists. The medical work in our century may be inserted in services domain and it is no more a preoccupation of administrative neoliberal schedule. Instead, the financial issues entirely hold heart and mind of our governors. PMID- 10752238 TI - [Late results of Heller operation and fundoplication for the treatment of the megaesophagus: analysis of 83 cases]. AB - BACKGROUND: Retrospective study of the late results of the Heller's cardiomyotomy and fundoplication for the treatment of the megaesophagus. MATERIAL AND METHODS: Were studied 83 patients with a follow-up from one to 186 months (average 40.0 +/ 47.4 months). The fundoplications used were in three plans in 15.7% and posterior in 83.1%. The main pre-operatory complain was dysphagia followed by regurgitation and loss of weight. Chagas, Disease as the etiology was confirmed in 72.3% of the patients. RESULTS: In the follow-up 55.4% of the patients were assymptomatic, 34.9% complained of sporadic dysphagia, 14.4% of heartburn, 8.4% of regurgitation and 2.4% did not changed the dysphagia, these being re-operated and had improvement the symptoms. Gastro-esophageal reflux was noted in 8.4% of the patients. Other late complications were par-esophageal hernia, sliping of the fundoplication, Barrett esophagus and cancer. CONCLUSIONS: The necessity of a long-term clinical and endoscopical follow-up, even after surgery, owing to the possibility of late complications, especially cancer was emphasized. The late results are good in relation to the dysphagia. Myotomy is proposed as an alternative to patients with advanced megaesophagus with bad clinical conditions who can not be submitted to an esophagectomy. PMID- 10752239 TI - [Ocular manifestations in HIV infected patients attending the department of ophthalmology of Universidade Federal de Sao Paulo]. AB - PURPOSE: To evaluate ocular manifestations of HIV-infected patients. MATERIAL AND METHODS: A retrospective study of 1,100 HIV positive out patients in 3 years with a standard complete ophthalmological exam. RESULTS: Eight hundred and sixty nine (79%) of the 1,100 patients studied were male and 231 (21%) female. The most important risk factors were: male homosexuality in 303 cases (27.55%), heterosexuality in 272 (24.73%), endovenous drugs use in 168 (15.27%), and unknown cause in 246 (26.36%). Related to ocular complains, we found 432 (39.27%) patients with decreased visual acuity, 138 (12.55%) with visual hazy, 64 (5.82%) with ocular pain, 186 (16.9%) with inspecific complaints, and 235 (21.36%) with no complains. The most frequent ocular manifestations were: CMV retinitis in 246 (22.36%), cotton wool spots in 110 (10%), blepharitis in 51 (4.64%), and toxoplasmosis in 48 (4.36%). CONCLUSION: The increase of disease ratio was observed among heterosexual and females in this specific population. Patients with ocular complains were 79.42%. The most frequent ocular manifestations was CMV retinitis, in 246 (22.36%). We found also 10% of cotton wool spots. PMID- 10752241 TI - [Training of surgeons in laparoscopic surgery of the digestive system. Experience in 1,818 interventions without accidents and mortality]. AB - Since the beginning or laparoscopic surgery on University of Sao Paulo Medical School Clinics Hospital, the Digestive Surgery Division established an educational program for surgeons of the alimentary tract. PURPOSE: The course structure includes the information on medical school, extension in laparoscopic surgery league, and surgical formation during the residence, mainly in the fourth year, with a three months period in the Laparoscopic Surgery Unit. METHOD: This model of surgical formation is certainly responsible for the excellent results obtained. RESULTS: An example is the performing of 1818 cholecystectomies in the Laparoscopic Surgery Unit from 1990 till 1998, with 0.9% convertion, and no mortality. The absence of operative accidents or complications give support to the orientation assumed in our Division. CONCLUSION: In a university hospital there is the necessity of professional formation in a well structured program, with humanistic and technical education, in a large period of rotation, but with a solid information and practical structure. PMID- 10752240 TI - [Frequency of adenocarcinomas in serous effusions]. AB - BACKGROUND: Metastatic adenocarcinomas in effusions are frequently observed. Cytologic diagnosis of adenocarcinoma is often precise; unfortunately, the classification of the primary site is not possible in a great number of cases. The scope of this study was to report the frequency of adenocarcinomas in effusions according to the primary site. MATERIAL: We studied 2317 sequential cases: 1146 pleural effusions (943 women and 203 men), 1168 ascitic (727 women and 441 men), and 3 pericardial (2 women and 1 men). METHOD: Retrospective analysis of the records of the patients and correlations of the data of clinical follow up and morphological findings. RESULTS: The primary sites more frequently seen in pleural cavity were: breast (N = 586--51.1%), lung (N = 185--16.1%: 102 men and 83 women), and ovary (N = 124--10.8%); in ascites: stomach (N = 473- 40.5%: 300 men and 173 women), ovary (N = 306--26.2%) and breast (N = 83--7.1%). Regarding only the samples of malignant effusions we observed 555 in pleural cavity: breast (N = 288--51.9%), lung (N = 92, 16.6%: 45 men and 47 women) and ovary (N = 54, 9.7%). In ascites we reported: ovary (N = 205--37.9%), stomach (N = 202--37.3%: 119 men and 83 women) and breast (N = 31--6.8%). In 47 pleural effusions (8.5%) and 37 ascites (6.8%), the original site of the neoplasia remained undetermined. CONCLUSION: The knowledge of the relative frequencies, associated with clinical information, can be helpful in the selection of further investigation for the metastatic adenocarcinomas. PMID- 10752242 TI - [The role of surgery in the treatment of Wilms tumor: analysis of cooperative study]. AB - OBJECTIVES: To detect the effects of the surgical treatment and staging on the obtained results and to check the possible relationship between these results and the compliance, or not, with the preset surgical approach protocol (in accordance with the National Wilms Tumor Study--NWTS). MATERIAL AN METHOD: One hundred and sixty six operated on patients entered between October 1986 and December 1988, with the data updated until February 1992 were studied. The minimum follow-up period was 24 months for 147 patients (average 36 months). The remaining 19 patients were followed in the outpatient clinic for three to 18 months. RESULTS: After submitting these data to statistical analysis and the obtained results compared to those in the literature we observed that: previous ligature of the renal vessels had no discernible effect on the ultimate outcome (relapses and mortality); intra-operative tumoral rupture with contamination of the peritoneal cavity unfavorably interferes with the mortality rate; even when the adrenal and the perirenal fat are normal from the surgeon's point of view, the histological findings showed tumor contamination in a number of cases; there is a worse prognosis when the tumor weight exceeds 500 g; according to the lympho-node evaluation, surgeon's performance was not in accordance with the protocol recommendations, considering that the node evaluation was neglected in 56.6% of the patients. This led to a surgical staging error and has contributed to some unexpected results like: lower relapses rate in the patients with affected lymph nodes; higher incidence of lung metastases in patients without lymph nodes analysis; stage II patients with better prognosis, compared to stage I patients and patients with stage I and III having similar relapse rates. CONCLUSION: From the above-referred results, we concluded that some omissions have happened jeopardizing accuracy of the surgical staging in a significant way. In this way, in our environment the oncologist in charge of the treatment of Wilms tumor bearers, must establish a close integration with a surgical team, whose members have a precise understanding of the surgeon's importance and his fundamental role, not only of the surgical treatment itself, but who are aware on the minimal details on the surgical staging of this neoplasm, and of its importance in relation to the multidisplinary approach. PMID- 10752243 TI - [Cyclospora cayetanensis, a new protozoan to be researched]. PMID- 10752244 TI - [Urticaria and systemic diseases]. AB - Chronic urticaria and concurrent angioedema are disappointing problems for both physicians and patients. The disease can result from multiple causes and probably does not have a single etiology. Several factors have been identified that appear to be important in the pathogenesis of individual cases, some drugs, food additives, physical factors and internal diseases. In some cases no pathogenesis are identified and those cases are classified as idiopathic. In recent years several articles has emphasized autoimmunity and infections due to Helicobacter pylori. Our article reviewed the etiology of chronic urticaria at current concepts. PMID- 10752245 TI - [Administrative Processes and quality of health certification]. PMID- 10752246 TI - [Abuse of anabolic-androgenic steroids in sports]. PMID- 10752248 TI - [Helicobacter pylori. The bacteriological revolution]. AB - The finding of II pylori in the gastric mucosa has changed the understanding of the pathogenesis of common diseases of the stomach and duodenum. The bacteria causes acute and chronic gastritis and is the precursor of peptic ulcers, gastric lymphoma and gastric cancer. The most common test used to diagnose the infection is the urease test on a gastric biopsy but an endoscopy must be performed to obtain the biopsy. Determination of serum levels of IgG appears the most suitable test to perform in large population studies. Patients with peptic ulcers, gastric lymphoma and early gastric cancer should be treated. Combinations of antibiotics such as amoxicillin, chlarithromycin and a proton pump inhibitor have yielded the best results in irradicating H pylori. Unfortunately this therapy is expensive and new combinations should be explored. Vaccination should be the treatment of choice for prevention and irradication in countries like Chile with high infection rate. Preliminary results are encouraging. PMID- 10752247 TI - [Bacterial endocarditis as a complication of neonatal sepsis: case report]. AB - The authors reported on a 11 day-old child, admitted in Neonatal Intensive Care Unit for multiple congenital malformations, who had sepsis and bacterial endocarditis. Among the risk factors for endocarditis were outstanding: the central venous catheterism, hemoculture with growth of Staphylococcus aureus and mechanical ventilation. The diagnosis was made in the 61st day after admission owing to the presence of persistent fever and appearance of systolic murmur. The echocardiogram revealed a thrombus in the right atrium measuring 1.9 x 0.7 mm. Antibiotic therapy and surgical resection being performed, with clinical improvement. On the 125st day after admission the patient died owing sepsis and cerebral abscess. At necropsy, heart malformations were not observed. The authors concluded to be very important the knowledge of the potential risks of invasive procedures currently used to care for critically ill newborns. The clinical suspicion of endocarditis should be considered in all neonates with sepsis and receiving intensive care for long time. PMID- 10752249 TI - [Percutaneous revascularization of chronic iliac artery occlusions with primary stent placement]. AB - BACKGROUND: Percutaneous transluminal angioplasty is a well established treatment method for aorto iliac stenoses. However its success is limited in arterial occlusions and vascular stent placement can improve the results. AIM: To assess the effectiveness of percutaneous revascularization with stent placement in patients with chronic iliac artery occlusions. PATIENTS AND METHODS: Twenty six patients (18 male) aged 47 to 82 years, with iliac artery occlusions lasting six or more weeks were treated. Fourteen had involvement of common iliac artery, five had involvement of external iliac artery and seven of both. The occluded segment length ranged from 3.5 to 12 cm. According to the Society of Cardiovascular Surgery/International Society for Cardiovascular Surgery classification, 22 patients had category 1 claudication, 10 were in category 2, 12 in category 3 and two in category 4. RESULTS: A technical success was obtained in 23 patients and clinical success in 21. After the intervention, the anklearm index improved from 0.49 +/- 0.12 to 0.88 +/- 0.18 (p < 0.001). Permeability after 12 months of follow up was 81% and after 36 months, 65%. Four patients had complications; one had a vascular perforation, two had an acute occlusion and one had an asymptomatic distal embolization. All these were solved using endovascular techniques. CONCLUSIONS: Percutaneous revascularization with stent placement is a valid alternative to surgery in selected patients with chronic iliac artery occlusion. PMID- 10752250 TI - [Nutritional status of school children from indigenous and non indigenous ancestry]. AB - BACKGROUND: The few studies in Chile assessing the nutritional status of indigenous children show a high prevalence of stunting, excess weight and feeding problems. AIM: To compare anthropometric indices in children from indigenous and non indigenous ancestry. SUBJECTS AND METHODS: School children aged 6 to 8 years old, living in locations with three clear cut levels of social vulnerability were studied. Children were considered indigenous if their last names, as well as those of their parents were of Mapuche origin. Non indigenous were those whose last names were of Spanish origin. RESULTS: Four hundred and fifty indigenous and 684 non indigenous children were studied. Indigenous children from high vulnerability communities were approximately 0.5 z score shorter than those of non indigenous origin. Heights of indigenous and non indigenous children were similar in communities with intermediate and low social vulnerability. The proportion of the lower segment followed the same trend. Weight/height ratios were higher among indigenous children in the three vulnerability levels. Among indigenous children coming from areas of low vulnerability arm circumference was 1 cm broader than that of their non indigenous counterparts. CONCLUSIONS: Stunting is prevalent among school children from areas of high socioeconomic vulnerability, mainly rural, and independent from ethnicity. Among indigenous school children overweight and a broader arm circumference are frequent. These results urgently call for located and specific nutrition interventions. PMID- 10752251 TI - [Antihypertensive efficacy of monotherapy in increasing doses versus therapy associated in low doses]. AB - BACKGROUND: When hypertension treatment does not achieve the expected reduction in blood pressure levels, experts recommended increasing the dose of the initially used drug or the addition of a new medication. AIM: To compare the efficacy of increasing doses of losartan or the addition of hydrochlorothiazide to achieve adequate blood pressure levels in patients with hypertension. PATIENTS AND METHODS: Seventy- three patients aged 64.4 +/- 5.3 years, with stage 1 or 2 essential hypertension were studied. If after four weeks of treatment with losartan 50 mg od, blood pressure levels were still high, the dose was increased to 100 mg od. After four weeks with this new schedule, the treatment was switched to losartan 50 mg and hydrochlorothiazide 12.5 mg for another four weeks. RESULTS: Thirty- seven patients normalized blood pressure with losartan 50 mg od. Of the 36 patients that did not respond, 69% achieved a normal blood pressure with losartan 100 mg od and 81% did so with the combination of losartan and hydrochlorothiazide. Combination therapy resulted in a better blood pressure lowering than monotherapy (33.2 +/- 3.2 and 29.5 +/- 3.4 mm Hg for systolic blood pressure respectively, 16.4 +/- 3.2 and 13.2 +/- 3.4 mm Hg for diastolic blood pressure, p < 0.05). No changes in blood glucose, total and HDL cholesterol, triglycerides, urea nitrogen and uric acid were observed with the combination therapy. CONCLUSIONS: In this group of patients, combination therapy achieved better blood pressure levels than monotherapy in high doses. PMID- 10752252 TI - [Surveillance of gram positive cocci susceptibility to betalactams, glycopeptides, and other antimicrobials]. AB - BACKGROUND: During the last decade, there has been a progressive increase in the resistance of gram (+) cocci to betalactamics and other antimicrobials. Therefore, vancomycin and teicoplanin have incorporated as alternative antimicrobial drugs. AIM: To assess the susceptibility of gram (+) cocci to different antimicrobials including vancomycin and teicoplanin. MATERIAL AND METHODS: We studied 447 strains of gram (+) cocci coming from ambulatory and hospitalized patients. These included 308 Enterococcus sp strains, 99 Staphylococcus aureus strains and 40 coagulase negative Staphylococci strains. Enterococci susceptibility was measured using minimal inhibitory concentrations in agar and that of Staphylococci, through diffusion. Susceptibility to vancomycin and teicoplanin was measured using minimal inhibitory concentrations in all strains. RESULTS: Enterococcus faecalis was 100% susceptible to ampicillin, penicillin, vancomycin and teicoplanin, 23% susceptible to tetracyclin and 47% to chloramphenicol. Susceptibility of E faecium was 61% to penicillin, 49% to chloramphenicol, 41% to tetracyclin, 100% to vancomycin and teicoplanin. Of 19 Enterococcus spp strains, 90% were susceptible to ampicillin, 80% to penicillin, 55% to chloramphenicol and 45% to tetracyclin. Only one E casseiflavus strain had a low level resistance to vancomycin and was susceptible to teicoplanin. No Staphylococcus aureus strain was resistant to vancomycin or teicoplanin. CONCLUSIONS: A permanent surveillance of gram (+) cocci antimicrobial susceptibility is required to update therapeutic schemes. PMID- 10752253 TI - [Antimicrobial resistance of different Acinetobacter baumannii biotypes isolated in the northern region of Chile]. AB - BACKGROUND: Acinetobacter baumannii nosocomial outbreaks are common and the microorganism is frequently resistant to multiple antimicrobials. There is little information about Acinetobacter baumannii antimicrobial susceptibility in the northern region of Chile. AIM: To identify different Acinetobacter baumannii biotypes isolated from clinical samples and to determine their antimicrobial susceptibility. MATERIAL AND METHODS: One hundred twenty three Acinetobacter baumannii isolates were studied. The identification and typing of Acinetobacter baumannii was based on phenotypic characteristics. Antimicrobial susceptibility was investigated using agar dilution techniques. RESULTS: Most Acinetobacter baumannii strains were isolated from wounds, urinary and respiratory infections. Seven biotypes were isolated, being biotype 9 the most frequent. Imipenem was the antimicrobial with the higher activity against the microorganism. Amikacin, cefoperazonesulbactam, ampicillinsulbactam and ceftazidime had a moderate activity. There were high resistance levels to ampicillin and older cephalosporins. CONCLUSIONS: Acinetobacter baumannii is emerging as a significant nosocomial pathogen in Chile and shows high resistance rates to multiple antibiotics. PMID- 10752254 TI - [Adult T cell leukemia lymphoma in Chile. A clinical pathologic and molecular study of 26 patients]. AB - BACKGROUND: Adult T cell leukemia lymphoma is a lymphoproliferative syndrome etiologically associated to human T cell lymphotropic virus type I. AIM: To describe the clinical and laboratory features of 26 Caucasian Chilean patients, with HTLV-I positive adult T-cell leukemia lymphoma (ATLL). MATERIAL AND METHODS: Diagnostic criteria included clinical features, cell morphology, immunophenotype, HTLV-I serology and/or DNA analysis by Southern blot or PCR. RESULTS: According to the clinical presentation, 12 cases had the acute ATLL form, 6 had a lymphoma, 4 the chronic form and 4 had smoldering ATLL. The median presentation age was 50 years, younger than the Japanese patients, but significantly older than patients from other South American countries (e.g. Brasil, Jamaica, Colombia). The main clinical features: lymphadenopathy, skin lesions and hepatosplenomegaly, were similar in frequency to those of patients from other countries, except for the high incidence of associated neurological disease. Tropical Spastic Paraparesis (TSP) in our series of ATLL, was seen in one third of the patients (8/26). A T cell immunophenotype was shown in all 26 cases and HTLV-I serology was positive in 25/26 patients. Molecular analysis on the seronegative patient showed clonal integration of proviral HTLV-I DNA into the lymphocytes DNA, and thus he may have been a poor responder to the retroviral infection. Proviral DNA integration was also demonstrated in 15/16 patients being clonal in 10, polyclonal in 3 (all smoldering cases) and oligoclonal in one. CONCLUSIONS: ATLL in Chile has similar clinical and laboratory features than the disease in other parts of the world, except for a younger age than Japanese patients but older than those from other Latin American countries and a high incidence of patients with associated TSP. Detailed morphological and immunophenotypic analysis of the abnormal circulating lymphocytes, together with the documentation of HTLV-I by serology and/or DNA analysis are key tests for the identification of this disease. PMID- 10752255 TI - [HTLV-I tax gene on the etiological identification of tropical spastic paraparesis. A clinical, serological and polymerase chain reaction (PCR) study in 72 patients]. AB - BACKGROUND: Tropical spastic paraparesis (TSP) is an endemic disease in Chile. In most countries, only 50% of patients are seropositive to HTLV-I. However, new studies suggest that seronegative TSP is also associated with HTLV-I. AIM: To describe clinical and virological features of seronegative patients with TSP. PATIENTS AND METHODS: Seventy two Chilean patients with TSP, studied by clinical, radiological and laboratory methods during 1998, are reported. The determination of antibodies to HTLV-I was accomplished by ELISA, immunofluorescence and Western blot analysis. Polymerase chain reaction for tax and 5'Ltr genes was made using primers SK 43-44, LTR1 and LTR6. RESULTS: Thirty one patients were HTLV-I positive and 41 were negative. No clinical, radiological or laboratory differences were observed between both groups. In seropositive patients, tax and 5'ltr viral gene sequences of the HTLV-I provirus were detected in DNA of peripheral blood mononuclear cells. In seronegative cases, sequences of tax gene were detected, exclusively, in 18 of 41 patients. CONCLUSIONS: These results confirm an association with HTLV-I infection in 43.9% of the TSP seronegative patients. These findings support the hypothesis that a defective provirus infects peripheral blood mononuclear cells in seronegative cases of TSP. The importance tax gene in the diagnosis of the TSP is also emphasized. PMID- 10752256 TI - [Preliminary results of horizontal gastroplasty with Roux in Y anastomosis in patients with severe and morbid obesity]. AB - BACKGROUND: Morbidly obese subjects have a high incidence of complications. The poor results of dietary treatments, has prompted the search of new therapies for obesity and among these, surgical procedures. AIM: To report the long term results of horizontal gastroplasty with Roux en Y anastomosis in morbidly obese subjects. PATIENTS AND METHODS: Fifty patients with an initial body mass index of 41.3 +/- 6 kg/m2 have been subjected to a horizontal gastroplasty with Roux en Y anastomosis. During the study period, surgical techniques were modified, reducing the gastric pouch size, adding a truncal vagotomy, cholecystectomy, and increasing the length of the Roux en Y loop from 70 to 100 cm. Twenty five patients have been followed for two years. RESULTS: There was no operative mortality and one patient had an anastomotic leak that required 35 days of hospitalization. During follow up, in one patient, the stapled suture line loosened. After two years of follow up, weight decreased from 112 +/- 19 to 77.2 +/- 14 kg. CONCLUSIONS: Horizontal gastroplasty with Roux en Y anastomosis achieved an adequate weight loss with a low rate of complications in this group of morbidly obese subjects. PMID- 10752257 TI - [Primary ciliary dyskinesia. Experience in 6 patients]. AB - BACKGROUND: Primary ciliary dyskinesia is characterized by a congenital alteration of the ciliary ultrastructure and function. As a consequence, their respiratory tract sweeping action is lost and recurrent respiratory infections ensue. AIM: To analyze a clinical series of patients with primary ciliary dyskinesia, their clinical and laboratory features. PATIENTS AND METHODS: A retrospective review of patients with primary ciliary dyskinesia seen a University Hospital, between 1994 and 1998. Bronchial biopsies were obtained with 3.6 mm diameter Olympus fibrobronchoscope, using a cayman type forceps. Ultrastructural alterations of respiratory tract ciliated cells were recorded. RESULTS: Six patients (four male) aged 9 months to 13 years old were reviewed. Three patients had situs inversus. All had repeated bouts of obstructive bronchitis and pneumonia, five had sinusitis, four atelectasis, three recurrent otitis and three had bronchiectasis. Cystic fibrosis and immunological alterations were ruled out in five children. Ultrastructural analysis revealed absence of dynein arms in three cases, absence of the internal dynein arm in one, additional peripheral microtubules and absence of dynein arms in one case. CONCLUSIONS: Primary ciliary dyskinesia must be considered in the differential diagnosis of recurrent respiratory infections. Ultrastructural analysis of ciliary structure can be done in bronchial biopsies obtained through bronchoscopy. PMID- 10752258 TI - [Down syndrome and hyperthyroidism. Report of 3 cases]. AB - We report three patients with Down syndrome that developed a hyperthyroidism. A 25 years old female and a 18 years old male had Basedow Graves disease and were treated with radioiodine. A 19 years old male had a Hashitoxicosis and is presently being treated with propylthiouracyl. Clinical and subclinical thyroid dysfunction is frequent in patients with Down syndrome and the risk increases with age. Therefore a surveillance with yearly TSH measurements should be done in these patients, since signs and symptoms of thyroid disease are barely detected in them. Hypothyroidism is the most frequent dysfunction but hyperthyroidism is also associated to Down syndrome. PMID- 10752259 TI - [Crescentic glomerulonephritis with anti neutrophil cytoplasmic antibody in children. Cases report]. AB - Anti neutrophil cytoplasmic antibodies are associated to vasculitis and crescentic glomerulonephritis in adults. However, this association has been seldom reported in children. We report two girls aged 12 and 15 years old with ANCA + glomerulonephritis. Both were subjected to a percutaneous kidney biopsy. One girl had to enter a chronic hemodialysis program. The other patient recovered her renal function and after 12 months of treatment with steroids and cyclophosphamide microscopic hematuria and proteinuria persist but with normal kidney function. ANCA should be measured in children with vasculitis and glomerulonephritis. PMID- 10752260 TI - [Anterior cervical bronchogenic cyst simulating a thyroid lesion. Report of case]. AB - Bronchogenic cysts are congenital malformations of the tracheo-bronchial tree usually located intrathoraxically. We report a 44 years old male with a bronchogenic cyst located exceptionally in the pre tracheal anterior cervical region, simulating a thyroid lesion. PMID- 10752262 TI - [The scope of moral responsibility due to therapeutic actions]. AB - Besides utilitarianism, there are different ways to understand Medicine and its role. The opinion of the author is that the pursuit of mankind welfare is not the role of physicians and that their responsibility is not referred to the world. On the contrary, their bumble mission is to cure or mitigate pain through therapeutic actions. Any other way of proceeding means that the medical action is becoming orchestrated, destroying the fundamental value of patient physician relationship. This article intends to demonstrate the insufficiency of the Aristotelian idea of technique applied to medicine and that, on the other hand, the modern ideal of a principle free science does not account for the complexity of medical practice, mainly in the field of Psychiatry. Finally the author wonders if the moral convictions of physicians may play a role in the treatment of specific diseases caused by morally inadequate behaviors. PMID- 10752261 TI - [Molecular medicine: present and future]. AB - The genetic background of individuals is recognized as an important clue in the analysis of classical hereditary and multifactorial acquired diseases. This new concept derives from the development and increasing use of molecular genetics in clinical medicine. The application of molecular biology techniques in biomedical investigation has encompassed the identification of the pathogenesis and etiology of diseases, prenatal diagnosis the production of new therapeutic agents, gene therapy and the development of pharmacogenetics. The impact on the fundamentals and practice of clinical medicine that will have the use of molecular biology is analyzed in this review. PMID- 10752263 TI - [New pieces in the puzzle of atherogenesis]. AB - The contribution of high serum levels of cholesterol to atherogenesis has been widely recognized, but the mechanisms are not completely clear. Numerous publications have emphasized that oxidized, but not native low-density lipoproteins, are the particles incorporated into the arterial wall. A group of receptors generically called "scavenger" (SR), actively bind these modified lipoproteins and incorporate them into monocytes-macrophages, in the arterial intima. SR are not down regulated by intracellular concentrations of cholesterol, thus accumulating huge amounts of lipids, transforming monocyte-macrophages into foam cells, predominant cell type of the fatty streak. The simultaneous cytokine production and migration of other cellular types progressively transform this initial lesion into the organized atherosclerotic plaque. In this setting SR, which are up-regulated by oxidized LDL, play a central promoting role. Its presence has been demonstrated in arterial plaques both in human and animal models, and its blockade protects animals from development or progression of atherosclerosis. In humans, elevated antibody titers to oxidized LDL in patients with coronary stenosis, and increased SR activity, in pro-atherogenic conditions such as haemodialysis, indicate that this model may operate as well, but the evidences are still not solid enough to definitively conclude that the oxidized LDL-SR hypothesis is a finished puzzle. PMID- 10752264 TI - [Risk factors for non communicable diseases: methods and global results of the CARMEN program basal survey]. AB - BACKGROUND: In the last decades, chronic non communicable diseases are becoming the main cause of disability and mortality among adults. The risk factor surveillance and management is the most efficient mean of reducing the impact of these diseases. AIM: To report the results of a non communicable disease risk factor surveillance program in Valparaiso, Chile. MATERIAL AND METHODS: A random samples of people aged 25 to 64 years old living in Valparaiso, Chile was studied. Subjects were questioned about smoking and physical activity habits. Blood pressure, height and weight were measured using standardized techniques at their homes and blood samples were obtained to measure serum lipid levels and oral glucose tolerance test at the nearest outpatient clinic. RESULTS: Of the initial 3852 homes selected, 752 individuals did no agree to answer the inquiry, therefore 3120 subjects were finally interviewed. Of these, 40.6% were smokers, 15% drank alcohol in two or more occasions per month, 84.6% were physically inactive, 19.7% had a body mass index over 30 kg/m2, 11.1% had high blood pressure, 3.9% were diabetic and 46.9% had high serum cholesterol levels. CONCLUSIONS: The basal survey for the CARMEN program shows a high prevalence of cardiovascular risk factors among Chileans. PMID- 10752265 TI - [Antimicrobial resistance of agents causing urinary tract infections in 11 Chilean hospitals. PRONARES project]. AB - BACKGROUND: The computer program WHONET generates a common database to analyze local or general antimicrobial resistance of bacteria. A surveillance of agents causing urinary tract infections in Chile has been performed using this program. AIM: To report the results after 12 months of urinary tract infection agent surveillance. MATERIAL AND METHODS: Since November, 1997, a surveillance of in vitro antimicrobial resistance, using agar diffusion techniques, has been performed in 20 to 40 bacterial strains per month, isolated from 11 hospitals in the country. Results have been analyzed using WHONET program. RESULTS: In first 12 months, 3144 strains, 1625 coming from outpatients, have been studied. Seventy four percent of isolated strains were E coli, 19% were other enterobacteria, 4.1% were non fermenting bacilli and 2.1% were Gram (+) cocci. Sixty five percent of E coli strains were resistant to ampicillin, 11% to cefazolin, 2.5% to cefuroxime, 19% to ceftriaxone, 9% to ceftazidime, 4.2% to gentamicin 1.3% to amikacin, 5.6% to ciprofloxacin, 8.4% to grepafloxacin, 4.3% to nitrofurantoin and 43% to trimeproprim/sulphamethoxazole. Eighty two percent of other enterobacteria strains were resistant to ampicillin, 45.5% to cefazolin, 33.5% to cefuroxime, 26.6% to ceftriaxone, 21.5% to ceftazidime, 30.3% to gentamicin 17.2% to amikacin, 21% to ciprofloxacin, 16.3% to grepafloxacin, 48.2% to nitrofurantoin and 44.6% to trimeproprim/sulphamethoxazole. There were differences in betalactamic resistance among hospitals. CONCLUSIONS: Noteworthy is the high resistance rates to third generation cephalosporins, evidenced when the new cutoff values for E coli and Klebsiella spp are used. This national surveillance provides updated information on antimicrobial resistance of agents causing urinary tract infections. PMID- 10752266 TI - [Gastric cancer and cerebrovascular diseases. A natural history with common elements? Analysis of the salt theory in Chile (1955-1994)]. AB - BACKGROUND: In the last forty years, a sustained reduction in the gastric cancer and cerebrovascular disease mortality has been recorded. Joossens has postulated that sodium intake has an influence in the natural history of both diseases. AIM: To analyze the mortality caused by both diseases in Chile. MATERIAL AND METHODS: The mortality of people aged 35 to 64 years old, due to gastric cancer and cerebrovascular diseases in the period 1955-1994, was analyzed. An analysis according to regions, using temporal series and correlation techniques was performed. RESULTS: In the studied period, the mortality due to gastric cancer decreased by 71.6% in men and by 79.5% in women. Mortality due to cerebrovascular diseases decreased by 56.9% in men and by 63.9% in women. There was a correlation coefficient of +0.91 between the mortality due to both diseases. Cerebrovascular disease mortality appears as a predictor of gastric cancer mortality in temporal series analysis. CONCLUSIONS: Joossens theories are supported by the present data. Prospective studies should be designed to confirm the hypothesis. PMID- 10752267 TI - [Microbiological study of gallbladder bile in a high risk zone for gallbladder cancer]. AB - BACKGROUND: Gallbladder cancer is frequent in Chile and there is sparse information about the association between this type of cancer and the presence of bacteria in the gallbladder bile. AIM: To determine the presence of aerobic bacteria in gallbladder bile in patients with and without gallbladder cancer. MATERIAL AND METHODS: A microbiological analysis of bile and pathological study was performed in 608 gallbladders, obtained during to cholecystectomies performed to 513 women and 95 men aged 44 years old as a mean. RESULTS: Pathological study showed a chronic cholecystitis in 468 cases (77%), an acute cholecystitis in 140 (33%), cancer in 24 (3.9%) and dysplasia in 5 cases (0.8%). A positive culture was obtained in 22.5% of women and 28.5% of males. Twenty seven percent of women over 30 years old had positive cultures compared with 10% of younger women (p < 0.001). Thirty two percent of acute cholecystitis had positive cultures, compared with 24% of chronic cholecystitis (p = 0.03). E Coli was isolated in 51% of positive cases, Streptococci-Enterococci in 24%, Enterobacter sp in 9%, Klebsiella and Proteus in lower proportions. Salmonella sp was isolated in 4 cases, being all women with chronic cholecystitis. Thirteen of 29 cases with cancer or dysplasia had positive cultures (45%), compared with 25% of patients with inflammatory gallbladder diseases (p = 0.02). Streptococci-Enterococci were isolated in 7 cases and Enterobacter sp in three. CONCLUSIONS: The presence of Salmonella sp in gallbladder bile was not frequent in the studied patients. Its role in the pathogenesis of gallbladder cancer must be reassessed. PMID- 10752268 TI - [Paternity analysis using four DNA markers amplified by polymerase chain reaction]. AB - BACKGROUND: DNA typing in forensic analysis is a useful tool to analyze paternity due to its high discrimination power. AIM: To report the experience of Servicio Medico Legal in Santiago, resolving cases of dubious paternity. SUBJECTS AND METHODS: Four highly polymorphic loci, amplified by polymerase chain reactions, were analyzed in 153 cases of uncertain paternity. The paternity index was calculated for each case. RESULTS: The four genetic markers analyzed provided an exclusion probability of 0.933 for the general population in Santiago. Thirty seven cases were excluded as parents. In 31 cases, the paternity index ranged from 19 to 100, considered as probable paternity and 77 cases had an index of over 100, considered as almost certain paternity. Eight cases had an index between 0.5 and 19, considered as inconclusive. All loci met Hardy-Weinberg expectations and their frequencies were similar to other data from people living in Santiago. CONCLUSIONS: The use of these genetic markers proved to be very useful, reliable and with a high exclusion power for paternity analysis. PMID- 10752270 TI - [Influence of respiratory viruses, cold weather and air pollution in the lower respiratory tract infections in infants children]. AB - BACKGROUND: All winters, there is an increase in the number of pediatric consultations, associated to three factors: cold weather, air pollution and respiratory virus epidemics. AIM: To study the influence of these three factors in the demand for pediatric consultations between March and September, in an area of Metropolitan Santiago. PATIENTS AND METHODS: The number of consultations between March and September 1998 in the emergency room and the number of hospital discharges due to lower respiratory tract infections, were registered in a public pediatric hospital of Santiago. A respiratory virus surveillance (respiratory syncytial virus, adenovirus, influenza and parainfluenza virus) was done among children admitted for lower respiratory infections. Atmospheric temperature values and air pollution, measured as the number of particles of 10 microns or more per m3 (MP 10), were obtained from local health services. RESULTS: Two elevation waves of outpatient consultations were detected at weeks 19 and 26, that coincided with the periods of maximal detection of influenza and syncytial respiratory virus, respectively. The epidemics of respiratory syncytial virus coincided with the maximal number of hospital admissions for lower respiratory tract infections at week 27. There was no correlation between air pollution and the number of pediatric consultations. The lower ambient temperatures coincided with the higher detection of respiratory syncytial virus at week 28, moment in which the demand for consultations or hospital admissions was descending. CONCLUSIONS: There is a direct relationship between respiratory virus epidemics and the demand for pediatric consultations. There is also a minor influence of ambient temperature. PMID- 10752269 TI - [Systematic surveillance of influenza, syncytial respiratory, parainfluenza and adenovirus in children with acute respiratory infections]. AB - BACKGROUND: The efficacy of influenza vaccination programs depends on the antigenic similitude between vaccine and the influenza virus circulating in the community. Therefore the surveillance of clinical activity and antigenic features of influenza virus is of utmost importance. AIM: To perform a systematic surveillance of clinical activity and antigenic characteristics of influenza virus. MATERIAL AND METHODS: Since 1996 and during the cold months (May to September), 20 samples of upper respiratory secretions per week, were obtained from children with acute respiratory infections consulting to the emergency room of a public hospital. Using indirect immunofluorescence and cellular cultures, the presence of influenza, syncytial respiratory, parainfluenza and adenovirus was assessed. The weekly number of consultations in the emergency room and the number of hospital discharges due to acute respiratory infections, were registered. RESULTS: Influenza and syncytial respiratory were the predominant virus detected since 1996. In 1996 and 1998, the weekly detection of influenza virus followed a single seasonal curve. The maximal weekly positively results reached 85 and 80% of the obtained samples, respectively. During 1997, two curves of influenza virus activity were observed, but none reached more than 50% of weekly positive samples. The demand for outpatient care evolved in parallel to the weekly detection of influenza virus. The hospital discharges due to acute respiratory infections paralleled the syncytial respiratory virus detection rates. CONCLUSIONS: This surveillance model is effective for the detection of influenza and other virus responsible for acute respiratory infections and their relationship with the demand for health care during the cold months. PMID- 10752271 TI - [Influence of body posture in the prevalence of craniomandibular dysfunction]. AB - BACKGROUND: Postural alterations of the shoulders, dorsal spine and hips could have an influence on the development of craniomandibular dysfunctions. AIM: To study the influence of body posture on the prevalence of craniomandibular dysfunction. SUBJECTS AND METHODS: One hundred thirty six dental students and 41 patients assisting to the temporomandibular joints (TMJ) clinic at the Freie Universitat at Berlin, were studied. Masticator, cervical muscles, temporomandibular joints and occlusions were clinically examined. The position of shoulders and hips was measured with the use of an acromiopelvimeter. RESULTS: No relationship was found between postural alterations of the hips and shoulders, articular noises and sensibility or pain while palpating the temporomandibular joints. Among students, a relationship between postural alterations of the shoulders and the sensibility or pain while palpating the TMJ, was observed. When all muscles were considered, a significant relationship between asymmetric shoulders or hips and muscular pain while palpating was observed among students. CONCLUSIONS: Some symptoms, especially muscular sensibility is more pronounced in people with hip and shoulder asymmetries. This relation is more pronounced in dental students than in patients. PMID- 10752272 TI - [Non linear analysis of infection diseases dynamics in Chile]. AB - BACKGROUND: The incidence of infectious diseases generally has large fluctuations, probably due to interactions between seasonal fluctuations and those secondary to case-susceptible host interactions. AIM: To analyze the complexity and attractant topological resemblance of seven infectious diseases in Chile. MATERIAL AND METHODS: The annual incidence of measles, whooping cough, scarlet fever, meningococcal meningitis, diphtheria, typhoid fever and poliomyelitis was obtained from the annual reports of diseases. Correlation dimensions and the largest Lyapunov series exponents were estimated. The resemblance among their attractants was assessed by Hausdorff distance. The measures were performed both before and after seasonal filtering. RESULTS: All series showed a dynamics near low dimensional chaos. The correlation dimensions ranged between 2.12 and 2.76. The correlation dimensions did not change after seasonal differentiation. Apart from one, all disease dynamics had large Lyapunov exponents, near 0.6 Bits/year. These decreased if series were differentiated. Before differentiation, the topological resemblance was mainly caused by the seasonal component of the dynamics but thereafter, the resemblance increased. In spite of different transmission mechanisms and etiologies, all analyzed infectious diseases conformed a truly single group, during cluster analyses. CONCLUSIONS: These results suggest that beneath the dynamics of infectious diseases, obscured by seasonal environmental factors, lays a very consistent nonlinear agent-susceptible host dynamics. PMID- 10752274 TI - [Coronary angioplasty and stent placement through the radial artery. Report of a case]. AB - Coronary angiography and percutaneous treatment of stenotic lesions have expanded in the last few years, due to availability of better diagnostic equipment. The femoral technique applied to this aims has prevailed, considering its efficacy, safety and wide acceptance. Since the beginning of this decade, an alternative access has been developed, in relation to miniaturization of the required elements to perform coronary diagnostic and therapeutic procedures. This new radial artery access is supported by multiple reports from many centers around the world that are increasingly using the technique. With this access it is possible to perform all the regular procedures done regularly through the femoral route. This case report illustrates a coronary angiography study through the radial access, followed by a stent implantation, through the same route. PMID- 10752273 TI - [Long-term results of reconstructive surgery for mitral insufficiency]. AB - BACKGROUND: Surgical repair is the procedure of choice for mitral insufficiency since it preserves better left ventricular structure and function. AIM: To assess the long term clinical and echocardiographic results of mitral valve reconstructive surgery. MATERIAL AND METHODS: A review of clinical and echocardiographic data of 68 patients (34 male, age range 17 to 82 years), subjected to surgical mitral valve repair between December 1991 and March 1998. Preoperative functional capacity of these patients was 2.96 +/- 0.7. Surgical repair was assessed using transesophagic echocardiography in all subjects. RESULTS: The etiology of mitral insufficiency was degenerative in 43 patients, rheumatic in 10, infectious in 6, ischemic in 5 and miscellaneous in 4. The most frequent pathological findings were dilatation of the mitral ring in 42% of patients, chordae tendinae rupture in 32% and enlargement in 24%. A mitral anuloplasty was done in 90% of patients, a cuadrilateral resection of posterior leaflet in 52% and chordae tendinae transference in 12%. An additional surgical procedure was done in 34% of subjects. Three patients died during hospitalization (4.4%). During the follow up of 36.5 +/- 22.3 months, five patients died and one required a mitral valve replacement. The actuarial survival probability was 95.3 +/- 2.6% at one year and 83.5 +/- 6.5% at five years. The reoperation free survival was 100% at one year and 97.4 +/- 2.5% at five years. At the end of follow up the functional capacity improved to 1.25 +/- 0.4. Echocardiography showed absence of mitral insufficiency in 48.4% of patients, minimal, mild and moderate insufficiency in 35.5, 14.5 and 1.6% of patients respectively. CONCLUSIONS: Surgical valve reconstruction in mitral insufficiency has satisfactory long term results and should be the procedure of choice for eligible patients. PMID- 10752275 TI - [Chronic myeloid leukemia and non Hodgkin lymphoma in the same patient. Clinical case]. AB - Chronic myeloid leukemia is a myeloproliferative disorder caused by a clonal disturbance of the trunk cell and the accumulation of granulocytic series in the marrow, blood and other organs. We report a 63 years old male, carrier of a chronic myeloid leukemia whose clinical condition was complicated by the appearance of a T cell lymphoma. He was subjected to chemotherapy, that reduced the size of adenopathies and improved his general condition. Further studies are required to determine if there is a relationship between these two clinical entities. PMID- 10752277 TI - [Identification of molecular defects in liver diseases. Recent advances]. AB - Recent molecular studies have resulted in the identification of genetic alterations underlying several hereditary disorders of the liver. Cloning of disease genes are increasing our understanding of the basic defects in liver diseases. This review focuses on selected inherited liver diseases such as hyperbilirubinemic syndromes, hemochromatosis, Wilson disease and genetic cholestatic syndromes and illustrate the knowledge gained on these disorders from molecular studies. Potential implications of the identification of disease genes such as practical applications for diagnosis, information on prognosis and the possibility to design new therapies are discussed. PMID- 10752276 TI - [Neck hydatidosis. Thyroid and submaxillary gland involvement in 2 cases]. AB - Neck located hydatid cysts are of rare occurrence. We report two patients with such condition. A 66 years old male with a slowly progressive painless 4 cm nodule located in the right thyroid. It did not concentrate 131I and a fine needle aspiration cytology was informed as an acute thyroiditis. A 5 years old boy presented with a 5 cm painless right submaxillary cyst. Ultrasound examination showed that it was unilocular, and fine needle aspiration biopsy disclosed unspecific findings. In both cases surgical findings and the pathological study showed hydatid cysts. Both patients had normal chest x ray and abdominal ultrasound examinations. They had an uneventful postoperative evolution. Echinococcosis must be considered in the differential diagnosis of cervical cysts in endemic area. PMID- 10752278 TI - [Cesarean section and vaginal delivery. An ethical perspective]. AB - The increasing number of cesarean sections is worrisome. In this article, several causes that could cause this phenomenon are invoked and a primary cause that explains all others is proposed. The hierarchical analysis of different values such as safety, costs and mother autonomy at the moment of medical decision, is discussed. PMID- 10752279 TI - [Metabolic and dietary factors involved in the etiology of obesity in Chilean women]. AB - This review discusses the factors involved in the aetiology of obesity. The effect of dietary macronutrient composition on the metabolic fate of fat (oxidation or storage) is emphasised. Available information on dietary intake in adults from food balance sheets or dietary surveys, show that females eat a relatively low amount of fat (50-70 g/d on average), although this seems to be rising. The increasing prevalence of obesity in our population could be related to the glycemic index of meals, their fatty acid composition and the time interval between meals. This paper compares the oxidation rates of different fatty acids, particularly polyunsaturated fatty acids (PUFA), and among these, the high oxidation rates found for alpha-linolenic and linoleic acids. Medium chain saturated fatty acids (C8 to C12) are more likely to be oxidised in comparison with PUFA and longer chain saturated fatty acids. The latter are more likely to be directed to fat storage, particularly when they are combined with highly glycemic carbohydrates (CHO) in the same meal. Given the large proportion of CHO in the usual diet, the relevance of de-novo lipogenesis from CHO is questioned. It is concluded that this route is not readily used in humans consuming normal diets. Therefore, this mechanism can not be responsible for the rising prevalence of obesity. PMID- 10752280 TI - [Trends in current medicine]. AB - Predicting the future of medicine is daring. One can speculate about some of its future traits at the most. The spectacular progress in biological sciences has nurtured the hope that medicine will be able to dominate all ailments, improve the quality of life and longevity. Physicians are uncomfortable with the weak knowledge that they have about some diseases such as cancer, connective tissue diseases, degenerative diseases, mental and psychosocial conditions. They are also worried about the aggressive and mutilating surgical procedures that are required nowadays. One can foresee that molecular medicine and applied technology will advance at a great speed and will modify the therapy of several diseases and the social organization of health care. Scientific progress will also change our values and will pose new political and economical challenges. I believe that medical ethics and bioethics will become a growing concern for medical education and professional organizations. The so called biotechnology century will also be the bioethics century. The revision and elucidation of the fundamentals of medicine will differentiate, in the future, a medicine devoted to mankind with a solid ethical background from an impersonal health care that considers man as an object or maybe a merchandise. The second option will cast medical care through the abyss of decadence, to its end. PMID- 10752281 TI - [The physician-patient relationship and the health care system]. PMID- 10752282 TI - [A tribute to Professor Mario Plaza de los Reyes, MD]. AB - Revista Medica de Chile renders a tribute to Professor Mario T Plaza de los Reyes, one of the founders of Nephrology as a specialty in Chile, a brilliant teacher and scholar. As Member of the Sociedad Medica de Santiago (Chilean Society of Internal Medicine) and former President (1964-1965) he had an active role as contributor, reviewer and reader of our journal. PMID- 10752283 TI - [Posthumous tribute to Hector Ducci, MD]. AB - Hector Ducci, MD (1915-1959) was a bright and energetic young physician who became one of the best known Chilean physicians abroad. Trained with Cecil Watson, MD, they both devised a method to measure conjugated bilirubin in serum ("direct-reacting bilirubin") and Dr. Ducci developed several laboratory techniques, mainly liver function tests, and proposed a practical clinical classification of jaundice. Probably one of his major contributions in hepatology was the use of high doses of cortisone in fulminant hepatic failure due to viral hepatitis, that standard worldwide for at least two decades as the life-saving alternative. He modernized medical care in the Internal Medicine wards, at the Hospital del Salvador in Santiago, Chile, becoming an example soon followed by other hospitals and specialties. Medical Residencies and Nurses School were also organized or modernized under his leadership. His unexpected and premature death shocked the medical environment not only in Chile but also in other countries. PMID- 10752284 TI - [The effect of emotionally significant visual stimuli on cortical evoked potentials]. PMID- 10752285 TI - [The psychophysiological and neurophysiological characteristics of the organization of the visual-spatial activities in right- and left-handed children 6 to 7 years old]. PMID- 10752286 TI - [The measurement of visual acuity and the crowding effect in children from the age of 3 to 9]. PMID- 10752287 TI - The contribution of the cerebellum to mental and social functions in developmental age. AB - Here are present the data concerning the intellectual, language and frontal performances of 24 children who had undergone surgery because of cerebellar hemispheric or vermis tumors and one girl with viral cerebellitis. The children with right cerebellar tumors presented auditory sequential memory and language processing disturbances; those with left cerebellar tumors showed deficits in spatial tests and visual sequential memory. The girl with cerebellitis showed a complex neuropsychological picture with impairment in processing language and in general sequential functions. Lesions of the vermis lead to two pictures: 1) a postsurgical mutism that could be subdivided into speech disorders (even to the extent of anarthria) and true language disturbances similar to frontal aphasia; and 2) behavioral disturbances ranging from irritability to a truly autistic response. These data seem to support the recently attributed role of the cerebellum as a modulator of the superior mental and social functions. PMID- 10752288 TI - [The reflection in the interhemispheric distribution of the EEG frequency amplitude parameters of the force of emotional experience, the strength of drive and the probability of its satisfaction]. PMID- 10752289 TI - [An analysis of the effect of reverse masking in a complex model of a network of neuron-like elements]. PMID- 10752290 TI - [The synchronization of the human circadian rhythm by social time sensors: the role of motivation. IV. The individual characteristics of the free course of the sleep-waking circadian rhythm under simulated conditions of vital activity]. PMID- 10752291 TI - [The prognostic significance of the adaptation potential of the cardiovascular system in 10- to 11-year-old children]. PMID- 10752292 TI - [The age-related characteristics of pulmonary venous blood flow (based on data from pulsed dopplerography)]. PMID- 10752293 TI - [Does a respiratory function exist for human blood thrombocytes?]. PMID- 10752294 TI - [The dynamics of cardiac activity during isometric loads in athletes]. PMID- 10752295 TI - [Changes in the fibrinolytic properties of the erythrocytes under the influence of a physical load]. PMID- 10752296 TI - [The physiological phenotypes of immune resistance and their significance in evaluating the level of health]. PMID- 10752298 TI - [The antihypoxic and antioxidant action of medicinal plants as the basis for their use in destructive brain diseases]. PMID- 10752297 TI - [Systemic and regional neuroimmune processes in focal CNS lesions]. PMID- 10752299 TI - [The dynamics of ion osmolarity and concentration in the blood serum of women during pregnancy]. PMID- 10752300 TI - [The red blood system of 10- to 12-year-old children in the Far North]. PMID- 10752302 TI - [The spectral characteristics of short-latency auditory evoked potentials]. PMID- 10752301 TI - [Changes in the electrokinetic properties of human cell nuclei during reflexotherapy]. PMID- 10752303 TI - [The effect of ongoing visual control on the spatial-temporal structure of cyclic graphic movements. The age-related aspect]. PMID- 10752304 TI - [The dynamic characteristics of skin elasticity during cyclic loading]. PMID- 10752305 TI - Reperfusion in acute ischemic myocardium by transmyocardial revascularization using CO2 laser. AB - Transmyocardial laser revascularization (TMLR) is currently applied to provide clinical benefits in the patients with end-stage coronary artery disease. However, this method is so far indicated only for chronic status of ischemic heart disease. In this study, we have investigated in the canine model whether acute ischemic myocardium could be reperfused by TMLR using CO2 laser. A CO2 laser was used to create transmural myocardial channels. The ischemic areas of 3 cm in diameter were created on the left ventricle with multiple coronary ligations. Laser procedure was carried out 30 minutes after coronary ligation in TMLR group (n = 6), while laser treatment was not performed after coronary ligation in acute myocardial infarction (AMI) group (n = 6). The level of MB isozyme of creatinine kinase (CK-MB) derived from coronary sinus was measured at 0, 3, 6, 12, 18, 24, and 48 hours after coronary ligations, and the pattern of serial CK-MB changing was analyzed. Animals were sacrificed 48 hours after treatment and histologically investigated. The time to peak level of CK-MB in TMLR group appeared significantly earlier (13.0 +/- 2.4 hours) than that in AMI group (22.0 +/- 3.1 hours). The value of CK-MB of 24 hours after ligation in TMLR group (1985 +/- 805 IU/L) was significantly lower than that in AMI group (4759 +/ 778 IU/L). The channels on the gross section after 48 hours of TMLR were patent with some of fibrin network. Red blood cells were scattered in the lumens. It was suggested that acute ischemic myocardium was directly reperfused through the open laser channels from the left ventricular chamber in the canine model. PMID- 10752306 TI - Peroneus longus can not be fully activated during ankle complex exercises by uninjured subjects. AB - This study characterized, for the first time, the extent to which the peroneus longus muscle can be voluntarily activated by uninjured subjects during common ankle rehabilitative exercises. Ten healthy subjects performed isometric maximum voluntary contractions (MVCs) of eversion and plantarflexion on a dynamometer and against manual resistance, and performed dynamic whole-body activities that place large loads on the ankle complex (vertical jumps and side-cuts). During these activities, surface EMG signals were collected from the peroneus longus and expressed as a percentage of the maximum motor responses elicited by electric stimulation. Voluntary activation levels of the peroneus longus during isometric MVCs performed on the dynamometer and performed against manual resistance were not significantly different. Side-cuts produced significantly larger activation levels than those maximum vertical jumps and the isometric exercises. Nevertheless, the activation levels were substantially lower than those reported for other lower extremity muscles. These results serve as the basis for further questions related to whether voluntary peroneus longus activation level can be affected by specific rehabilitative efforts, whether activation levels are influenced by acute and chronic ankle complex injuries and, if so, whether this effect influences the functional outcome of rehabilitation efforts following ankle injuries. PMID- 10752307 TI - Experimental investigations on relationships between myocardial damage and laser type used in transmyocardial laser revascularization (TMLR). AB - Transmyocardial laser revascularization (TMLR) using a CO2 laser is clinically attempted in end-stage ischemic heart disease that is not treated by conventional bypass grafting or transluminal angioplasty. Besides, clinical trials of TMLR using a Ho:YAG laser have started recently. In this study, we compared the degree of damage to normal myocardium using these 2 types of lasers. Hearts of adult mongrel dogs were exposed under general anesthesia. Dogs were divided into 2 groups; those with channels made in the left ventricle by CO2 laser (CO2 group, n = 5) and those with channels made by Ho:YAG laser (Ho:YAG group, n = 5). The chest was temporarily closed, then serum MB isozyme of creatinine kinase (CK-MB) and troponin T (TnT) were measured sequentially. Twenty-four hours after laser irradiation, hearts were isolated for pathological studies with hematoxylin-eosin and Masson's trichrome stains. The CO2 group produced CK-MB with a peak of 1162.2 +/- 462.2 IU/l and the Ho:YAG group 1804.0 +/- 992.4 IU/l after 12 hours, and there was a significant difference between two groups (p < 0.01). The CO2 group produced TnT with a peak of 1.2 +/- 0.4 ng/ml and the Ho:YAG group 11.6 +/- 4.1 ng/ml after 6 hours, and the peak value in Ho:YAG group was significantly higher than that in the CO2 group (p < 0.001). Thirty channels were confirmed histologically in the CO2 group, and the width of thermal damage layer around the channel lumen was 249 +/- 83 microns. Twenty-seven channels were confirmed histologically in the Ho:YAG group, and the width of thermal damage layer was 760 +/- 288 microns. Thermal damage in the Ho:YAG group was significant greater than that in the CO2 group (p < 0.01). We concluded that TMLR using a CO2 laser is more suitable for end-stage myocardial ischemia than a Ho:YAG laser in terms of myocardial damage. PMID- 10752308 TI - Evaluation of local platelet deposition during laser thermal angioplasty. AB - Laser thermal angioplasty is one of the brand-new transluminal interventions for arterial occlusive disease. And one of the most important prognostic factors of this intervention is the degree of local platelet deposition which causes the acute platelet thrombus and increases the proliferation of smooth muscle cells. The purpose of this study is to assess the degree of platelet deposition on the laser ablated area and to investigate the optimal conditions of laser ablation from the point of thrombogenesis. First of all, the laser ablations of various delivered energy were carried out on canine femoral arteries, then thrombus formation after laser ablation was evaluated with angioscopy. No thrombus was recognized on all ablated areas in the cases with a laser energy of 16 Joule (J) and 20 J. On the other hand, small thrombus on the ablated area was observed in 1 of 4 cases (25%) with that of 24 J, and in 2 of 4 cases (50%) with that of 30 J. Then, the degree of platelet deposition on the laser ablated area was evaluated with platelet labeled radioimmunoassay. Laser ablations were employed in canine femoral arteries varying delivered laser energy. There were no differences in the numbers of platelet deposition when the laser energy for one shot was within 24 J. The numbers of platelet deposition with a laser energy of 30 J and 45 J were significantly (p < 0.05) higher than those with a laser energy within 24 J. The numbers of platelet deposition were gradually increased related to the frequency of laser ablations, and the numbers of platelet depositions in 10 repeated laser ablations of 16 J and 20 J were significantly (p < 0.05) higher than that in single laser ablation of 16 J and 20 J. From the aspect of local platelet deposition, the optimal laser energy for one shot should be restricted within 24 J and the repeated ablation was the most appropriate method of laser thermal ablation. PMID- 10752309 TI - Effect of various properties of hydroxyapatite ceramics on osteoconduction and stability. AB - Hydroxyapatite ceramics (HA) are widely used for clinical applications as a bone substitute or dental implant because they have been shown to be biocompatible and exhibit excellent osteoconductivity when grafted into the bone tissue. However, the influence of the physical properties of HA on the osteoconduction and stability remains unclear. We examined here the effect of various properties of HA granules on osteoconduction and stability using 6 types of HA granules. The HA granules were grafted into the rabbit tibia and the initial bone formation and long-term stability of the new bone were studied histologically. The following results were obtained; 1. Osteoconduction of the HA granule was influenced by the shape. Multi-nuclear granules with continuous pores ranging from 30 to 200 microns in diameter showed both excellent osteoconduction and stability. 2. Solubility behavior of HA granules, which was effected by the relative surface area sizes, also seemed to be an important factor for osteoconduction and stability. 3. Stable bi-phase ceramics with HA and beta TCP were produced at the sintering temperature of 1200 degrees C when Mg was added. The beta TCP and Mg content did not negatively influence the initial osteoconduction or long-term stability. PMID- 10752310 TI - Effects of subthreshold transcranial magnetic stimulation on choice reaction time and correlation with motor cortical activation. AB - To study the effect of subthreshold transcranial magnetic stimulation (TMS), we measured choice reaction time (RT), with or without TMS, in 7 healthy participants. TMS over the hand motor area was randomly delivered at variable delays after the imperative signal, while participants performed right or left abduction of the thumb. Lateralized movement-related potentials (MRPs) were recorded in a separate session to link the TMS effect with the motor cortical circuitry. For the right hand, the coexistence of a motor specific and non specific effects of TMS was clearly evidenced, by the RT shortening at delays of 0 and 150 ms. The response dependency of the specific TMS effect was also demonstrated through the response-locked analysis, showing a maximal shortening at 120-ms bin before EMG onset. Furthermore, the lateralized MRP commenced at about 80 ms before EMG onset, indicating that TMS influences the cortical motor circuitry around 30-40 ms before the activation of the primary motor cortex. In contrast, for the left hand, we were faced with some uncertain concepts as human handedness and hemisphere asymmetries in both measures of RT and MRP, and thus it was not possible to substantiate about the motor specific effect of subthreshold TMS. PMID- 10752311 TI - Characterization of phospholipase D activation by GM2 activator in a cell-free system. AB - Phospholipase D (PLD) activator which synergistically activates the enzyme with ADP ribosylation factor has recently been shown homologous to GM2 activator (Nakamura, S. et al.: Proc. Natl. Acad. Sci. USA 1998. 95, 12249/12253). The present studies were undertaken to further clarify the identity of the activator by immunological technique and to characterize the mechanism of activation of PLD by enzymological approach. The activator was further confirmed as GM2 activator by immunoblot analysis. Kinetic analysis showed Vmax for the PLD reaction was 16 fold elevated by GM2 activator, whereas Km for phosphatidylcholine remained constant by GM2 activator. These results strongly suggest that GM2 activator might activate enzyme by protein-protein interaction not by substrate modification. These results facilitate the understanding how the metabolism of both phospholipids and gangliosides is regulated by the same protein. PMID- 10752312 TI - The effect of the two-layer cold storage method on islet isolation from ischemically damaged pancreas. AB - Previous studies have shown that the two-layer cold storage method (TL) resuscitates the function of warm ischemically (WI) damaged pancreas when transplanted as a segmental graft in a dog model. In this study, we evaluated the effect of TL on yield and viability of islets isolated from WI damaged pancreas. In group 1 (n = 10), dog pancreases were processed into islets immediately after total pancreatectomy. In group 2 (n = 5), excised pancreases were left in the abdominal cavity for 60 minutes as WI then processed. In group 3 (n = 5), WI damaged pancreases were preserved in TL at 4 degrees C for 24 hours before islet isolation. Islets were isolated by the digestion methods described by Ricordi, et al. And purified on Euro-Ficoll discontinuous gradients. We compared three groups in terms of islet yields before and after purification, expressed as islet equivalents (IEQ, diameter standardizing to 150 microns) per pancreas weight (IEQ/g pancreas). Viability of islets was assessed with in vitro static incubation test. Islet yields after purification were 2500 +/- 1100 (mean +/- SD) in group 1, 1200 +/- 800 in group 2, (p = 0.04 vs. group 1, p = 0.03 vs. group 3), and 2200 +/- 300 in group 3 (p = 0.38 vs. group 1). Static incubation test revealed that islets in group 1 and 3 showed sufficient insulin release in response to glucose stimuli. These data clearly demonstrated that TL can regain islet yield from WI damaged pancreas up to similar level to that from fresh pancreas. Viability of those islets was well maintained. Thus temporary TL applied before islet isolation may allow to use islets from non-heartbeating cadavers in the clinical setting. PMID- 10752313 TI - [Decrease in serum propofol concentrations after acute autologous blood letting]. AB - A change in serum propofol concentrations associated with acute autologous blood letting during anesthesia was investigated in seven scheduled surgical patients. Anesthesia was induced with propofol 2 mg.kg-1 and maintained with infusion of propofol 6 mg.kg-1.hr-1 at a constant rate. After achieving a stable anesthesia, about 10 g.kg-1 of autologous blood was withdrawn in about 15 minutes and 20 ml.kg-1 of acetated Ringer's solution was infused to manage the hypotension caused by withdrawal. A blood sample each 4 ml was taken before and 0, 5, 15, 30 minutes after blood withdrawing. Another 7 patients were anesthetized with the same procedure without blood letting to distinguishing the effect of blood letting from rapid infusion therapy of crystalloid. Assay of serum concentration of propofol was performed with HPLC-spectrofluorometry. Concentrations of propofol were significantly decreased from 2.8 micrograms.ml-1 to 2.3 micrograms.ml-1 just after blood letting, and remained at 2.3 micrograms.ml-1 after 30 minutes from letting. Rapid infusion therapy also decreased the concentrations of propofol from 2.4 micrograms.ml-1 to 1.7 micrograms.ml-1. Continuous infusions of propofol may become a major method of general anesthesia with target controlled infusion techniques (TCI) in clinical settings for the accuracy and reliability of prediction of blood concentrations. However, this study demonstrated unexpected decreases of concentration of propofol during acute autologous blood letting similar to surgical mass bleeding, which might be mainly caused by rapid infusion therapy. The rate of infusion of anesthetic should be readjusted to counteract the effect of acute blood loss or volume replacement. PMID- 10752314 TI - [Intraoperative motor evoked potential monitoring: a review of 115 cases]. AB - We reviewed our experiences of intraoperative motor evoked potentials (MEPs) monitoring for 115 operations on the spine or spinal cord. We observed compound muscle action potentials from bilateral anterior tibial muscles by electrical transcranial stimulation of the motor cortex under general anesthesia induced and maintained with intravenous anesthetics (ketamine, propofol, or droperidol), fentanyl, and 50% nitrous oxide. Partial neuromuscular blockade was obtained with continuous infusion of vecuronium. MEPs were recorded bilaterally in 91 cases (79%) and laterally in 18 cases (16%). Postoperative deterioration of motor function was observed in 2 cases and amplitude of MEPs decreased more than 50% of control values in both cases. Intraoperative monitoring of MEPs might be a reliable indicator of spinal cord motor function. PMID- 10752315 TI - [The necessity and the efficacy of the second administration of midazolam for sedation during spinal anesthesia]. AB - We investigated the necessity of the second additional administration of intravenous midazolam for sedation during spinal anesthesia and the adequate dose if necessary. Fifty patients with ages between 35 to 70 years for spinal anesthesia were administered midazolam 0.05 mg.kg-1 intravenously during surgery. Thirty-four patients opened their eyes spontaneously and to these patients midazolam 1 mg was administered every three minutes until patients closed their eyes. All 34 patients closed their eyes with 1 mg of midazolam. Patients opened their eyes again at 47 +/- 16 minutes (mean +/- SD) after the first dose (0.05 mg.kg-1) and at 24 +/- 14 minutes after the second dose (1 mg). The decreases in blood pressure, heart rate and percutaneous arterial oxygen saturation after the administration of midazolam were less after the second administration than those after the first one. The percent of patients with amnesia was larger in the patients with only first administration than those with the second dose. These results suggest that the additional dose of midazolam is not necessary after intravenous midazolam 0.05 mg.kg-1 until patients open their eyes and the additional 1 mg is useful even after patients have opened their eyes. PMID- 10752316 TI - [Splenic natural killer cell activity is suppressed by ligation of unilateral mental nerve in rats]. AB - It has been reported that surgical procedures and postoperative pain suppress immune activities of the patient. But it is not clear if chronic pain in a small area affects immune activities. We prepared rats with chronic neuralgia of the mental branch originating from the mandibular nerve (a division of the trigeminal nerve) and examined the change of splenic NK-cell activity. Surgical procedures to prepare rat models for the study were as follows: one mental nerve was exposed and ligated at the mental foramen in order to create hypersensitivity in the ipsilateral innervated area. Splenic NK-cell activity 3 weeks after the surgery was reduced significantly in the operation group than that of the sham-operation group and the non-operated control group. The result suggests that the immune functions are remarkably affected by chronic pain evoked in a limited area such as the area innervated by the mental nerve. PMID- 10752317 TI - [Differential effects of ketamine enantiomers on anesthetic levels and glutamate release in the hippocampus]. AB - The anesthetic effects of ketamine enantiomers (40 and 100 mg.kg-1, i.p.) were evaluated in the rat by behavioral parameters such as the orienting response, the tail-pinch test and the spontaneous motor behavior including stereotyped behavior (circling and nodding). The intercellular glutamate concentration in the dorsal hippocampus was measured by brain microdialysis method. The analgesic and sedative effects of the S(+)-ketamine were more potent than those of the R(-) ketamine at each dose. The incidence of stereotyped behaviors in R(-)-ketamine groups was higher than that of S(+)-ketamine groups. The basal release of glutamate under perfusion with normal artificial cerebrospinal fluid (ACSF) was unchanged after the administration of these enantiomers. Repeat perfusion with high-potassium ACSF (100 mM K+) for 20 min increased the glutamate content in vehicle-treated rats. This depolarization-related glutamate release was suppressed by ketamine, except in the 100 mg R(-)-ketamine group in which the stimulated glutamate release tended to increase after the treatment. The inhibitory effect on this stimulated glutamate release was marked in S(+) ketamine groups. These results suggest that the anesthetic effects of ketamine enantiomers may be mediated at least in part, via regulation of depolarization released glutamate release. PMID- 10752318 TI - [The effect of preoperative factors on intraoperative oxyhemoglobin dissociation curve]. AB - The position of oxyhemoglobin dissociation curve (ODC) expressed with P50, has a large influence on the oxygen supply. It has been reported that during hypoxia with increased oxygen demand, P50 can increase with a reduction in oxygen affinity. In this study, we investigated the effect of preoperative factors on intraoperative P50 and the relation between the P50 values of the mixed venous blood and those of internal jugular bulb venous blood in seventy patients for cardiac surgery. Preoperative reduction in percentage vital capacity (%VC) and reduced hemoglobin concentration were associated significantly with an increase in intraoperative P50 value. The P50 act (P50 value with only temperature adjusted to 37 degrees C) of the internal jugular bulb venous blood was significantly higher than that of mixed venous blood. These results suggest that intraoperative tolerance to hypoxia and the increase in oxygen demand might be reduced in patients with preoperative abnormality of %VC and the hemoglobin concentration. And this tolerance might be limited in brain. PMID- 10752319 TI - [Perioperative management for hemicolectomy using propofol infusion in an elderly patient with a mechanical heart valve prosthesis]. AB - An 81-year-old female (145 cm, 39 kg) with a mechanical heart valve prosthesis underwent right hemicolectomy for cecal cancer under total intravenous anesthesia. She had received anticoagulation therapy [international normalized ratio (INR) 4.3] with warfarin for two years before surgery due to replaced aortic valve and atrial fibrillation. Three days before surgery warfarin was switched to heparin infusion (400 U.h-1) continued until 3 hours before the surgery. The activated partial thromboplastin time (APTT) was maintained above 1.5 times of the control (45 sec). Before induction of anesthesia, the activated clotting time (ACT) was 166 seconds. Following propofol infusion (50 ml.h-1; 38 mg) and vecuronium, anesthesia was maintained with propofol infusion (5.9 mg.kg 1.h-1), buprenorphine i.v. (0.08 mg) and appropriate doses of vecuronium. The total dose of propofol used was 950 mg. Heparin infusion (80 U.h-1) was restarted at the onset of surgery. Intraoperative ACT levels were between 148 and 156 seconds. However, at the end of surgery, APTT was prolonged (60.7 sec). Heparin infusion was reduced to 40 U.h-1 and APTT became normal 3 hours after the surgery. On the 4th postoperative day, heparin infusion was returned to 400 U.h-1 (APTT 32-38 sec). Two days after warfarin restoration on the 15th postoperative day, heparin was discontinued (INR 2.1-2.5). The thromboembolism and bleeding tendency did not occur. This case suggests that despite the suspected bleeding tendency via platelet inhibition under propofol infusion, the reduced heparin infusion can be continued with close coagulation monitoring. PMID- 10752320 TI - [Anesthetic management of bilateral lung lavage for pulmonary alveolar proteinosis--comparison between sevoflurane and propofol]. AB - A 55-year-old man with pulmonary alveolar proteinosis underwent lung lavage under general anesthesia using sevoflurane three years ago. Although transient hypoxia occurred postoperatively, there were no complications. Because of the recent deterioration of his symptoms, he was rescheduled for lung lavage. Anesthesia was induced with propofol 120 mg and fetanyl 0.2 mg. Vecuronium 7 mg was administered to facilitate tracheal intubation using a double-lumen tube. Anesthesia was maintained with propofol 4 mg.kg-1.h-1. Electrocardiogram, blood pressure, SPO2, EtCO2 and rectal temperature were monitored intraoperatively. We also checked PaO2 when necessary. Although transient hypoxia occurred after the procedure, it receded spontaneously. Since inhalation anesthetics inhibit hypoxic pulmonary vasoconstriction, intravenous anesthetics may be more useful for patients with severe pulmonary alveolar proteinosis. PMID- 10752321 TI - [Tracheal tube/laryngeal mask exchange to prevent coughing in lung volume reduction surgery]. AB - We report the use of laryngeal mask airway (LMA) to facilitate smooth emergence from anesthesia in 3 patients who underwent lung volume reduction surgery (LVRS) for severely symptomatic emphysema. To prevent coughing during emergence we replaced the endotracheal tube with LMA while patient was still in deep anesthesia. Emergence was smooth without coughing in all three patients, and ventilation with facemask was not needed after removal of LMA. We believe this technique is safe and beneficial for anesthetic management of LVRS. PMID- 10752322 TI - [Anesthetic management of an emergency surgery for panperitonitis during an asthmatic attack]. AB - We report anesthetic management of an emergency surgery for panperitonitis during an asthmatic attack in a patient with angina pectoris. A 71-year-old male patient, complaining of abdominal pain and dyspnea, was diagnosed as having panperitonitis and asthmatic attack by surgeons in the emergency room. General anesthesia was induced by intravenous injection of propofol (30 mg), ketamine (30 mg), fentanyl (200 micrograms), suxamethonium (60 mg) and diltiazem (5 mg) following cannulation of the left radial artery for continuous monitoring of direct arterial pressure. Anesthesia was maintained by continuous infusion of propofol (4-10 mg.kg-1.h-1) and ketamine (1 mg.kg-1.h-1) in combination with intermittent epidural injection of local anesthetics. Although sudden onset of increased peak airway pressure occurred 45 minutes after starting operation, 50 mg of propofol injection and 500 mg of aminophyline infusion could relieve this high airway pressure. Because increased peak airway pressure appeared frequently and this could not be relieved by bolus injection of propofol, we changed the intravenous anesthesia to nitrous oxide-oxygen-isoflurane (GOI). After this change, no asthmatic attack occurred during the operation. While the mechanical ventilation was required during the early postoperative period along with infusion of aminophyline and inhalation of beta-stimulants, the patient was weaned successfully from the mechanical ventilation 12 hours postoperatively. It was speculated that the intraoperative asthmatic attack might have been caused by light level of anesthesia with propofol and ketamine. We concluded that other analgesics, such as fentanyl or epidural local anesthetics, must have been supplemented at proper timing during the continuous infusion of propofol and ketamine during the surgery. PMID- 10752324 TI - [Fluid management after off-pump CABG in a patient receiving preoperative continuous ambulatory peritoneal dialysis]. AB - Perioperative management of off-pump CABG in a patient receiving continuous ambulatory peritoneal dialysis (CAPD) was reported. Prior to the procedure, we considered that postoperative management could be performed by CAPD alone in the case of off-pump CABG. However, due to an unexpected increase in the circulating blood volume and dilution of blood after surgery, congestive cardiac failure developed and active dehydration was required for 3 days after the operation using CHDF and HD. Despite this treatment, the central venous pressure and the cardiothoracic ratio in the chest X-ray were elevated, and a state of volume overload continued. However, cardiac contractility was not disturbed as shown by echocardiography. A possible cause of this condition was that body fluid had accumulated in the third space such as the peritoneum and intestine due to long term use of CAPD before surgery, and the fluid was mobilized after surgery. PMID- 10752323 TI - [Sick sinus syndrome (SSS) after induction of anesthesia in a patient with increased intracranial pressure and ischemic heart disease]. AB - An 84-year-old male with increased intracranial pressure (ICP) and ischemic heart disease was scheduled to undergo brain tumor resection. After induction of anesthesia using midazolam, fentanyl and vecuronium, sinus bradycardia, junctional rhythm and escape-capture bigeminy were observed. In spite of intravenous administration of lidocaine and atropine, the bigeminy did not disappear. As he awoke from anesthesia, his cardiac rhythm changed from a bigeminal to a regular sinus rhythm. After extubation, sinus arrest, which continued for up to 4.8 seconds, appeared frequently. A temporary pacing catheter was inserted and ventricular pacing was started (mode VVI rate 50 min-1). Four days later, the patient was again scheduled to undergo brain tumor resection. After induction of anesthesia with the same drugs as used before, his cardiac rhythm gradually became bradycardic, and a complete pacing rhythm was observed throughout the surgical procedure. After the operation had been completed, his cardiac rhythm changed to a regular sinus rhythm. In addition to degenerative and ischemic changes in the SA node, an increase in ICP secondary to a brain tumor is thought to have induced sick sinus syndrome after the induction of anesthesia. PMID- 10752325 TI - [Cesarean section using a temporary IVC filter in a pregnant woman with several histories of deep vein thrombosis]. AB - A 31-year-old pregnant woman with a history of several episodes of deep vein thrombosis was scheduled for Caesarian section. The patient had an increased risk of intraoperative pulmonary thromboembolism due to thrombi isolated from deep veins. This is because a thrombus can be formed easily in deep veins by the influence of hypercoagulability in the third trimester and by the disturbance of venous return due to the pressure of the enlarged pregnant uterus. A temporary IVC filter (Filtre Thery, France) was inserted preoperatively. The filter basket was set in the IVC cephalad from the left renal vein and caudad from the hepatic vein. The operation was completed without any trouble. Rehabilitation was safely initiated just after the surgery by insertion of the filter and then the filter was removed on the third postoperative day. We conclude that the preoperative insertion of a temporary IVC filter is a useful technique to prevent intraoperative pulmonary thromboembolism in a patient with a history of deep vein thrombosis. PMID- 10752326 TI - [Intravenous sedation with target-controlled infusion (TCI) in patients with difficult airways]. AB - Tracheal intubation was facilitated with an intubating laryngeal mask (ILM) in two patients with difficult airways. Target-controlled infusion (TCI) of propofol and fentanyl was used for sedation during placement of an ILM. An ILM was inserted smoothly. Spontaneous ventilation and oxygenation were well maintained throughout the induction. Both patients were satisfied with intravenous sedation using TCI for awake instrumentation of their airways. PMID- 10752328 TI - [A case of hypothyroidism found by delayed awakening after the operation]. AB - We report a case of hypothyroidism found by delayed awakening after surgery. A 55 year-old male patient had been suffering from elevated creatine phosphokinase (CPK) and cartinoembryonic antigen (CEA) of unknown origin before the operation. Laparoscopic cholecystectomy was performed under general anesthesia combined with epidural block. Hypotension, low arterial oxygen saturation, hypothermia and metabolic acidosis developed and continued during the operation. Awakening was delayed for about two hours postoperatively. Specific examination resulted in a definitive diagnosis of hypothyoidism. We should pay careful attention to any patient with elevated CPK and CEA of unknown origin before surgery, continuous hypotension, respiratory and circulatory failure and metabolic disorder during surgery, and delayed awakening after surgery that may be the result of hypothyroidism. PMID- 10752327 TI - [Anesthetic management of a patient with deep venous thrombosis using temporary inferior vena cava filter]. AB - A patient with deep venous thrombosis caused by a huge uterine leiomyoma underwent abdominal hysterectomy. To prevent pulmonary thromboembolism, the patient received anticoagulant therapy until 6 hr before surgery and temporary inferior vena cava filter was placed. A combination of preoperative anticoagulant therapy and the filter placement during perioperative period enabled this patient to be successfully-managed. PMID- 10752329 TI - [Postoperative pancreatitis after total hip replacement under general anesthesia]. AB - A 61 year old male patient developed postoperative pancreatitis after total hip replacement under general anesthesia with sevoflurane. The patient had chronic renal failure and was receiving hemodialysis. The estimated intraoperative blood loss was 1500 ml, and 1200 ml of blood was administered. The intraoperative mean blood pressure was 60 to 70 mmHg and the central venous pressure at the end of anesthesia was 0 mmHg. Postoperatively he complained of severe upper abdominal pain. On the 1st postoperative day serum amylase level increased to fifteen times of the normal level. He complained again of severe abdominal pain on hemodialysis. From these episodes, we estimate that the circulatory disturbance of pancreas is the cause of this postoperative pancreatitis. PMID- 10752330 TI - [Equilibration period for PaO2 following alteration of FIO2 in mechanically ventilated patients]. AB - To determine arterial oxygen tension (PaO2) in mechanically ventilated patients, we repeatedly measured arterial blood gas (ABG) to examine changes in PaO2 until equilibration following alteration of fractional concentrations of oxygen in inspired gas (FIO2). Forty anesthetized patients with normal arterial oxygenation were randomly assigned to one of four groups. Group 1 (n = 10): Arterial blood samples were drawn every one minute during a 15-min period of equilibration after change of FIO2 from 1.0 to 0.21. Then FIO2 was returned to 1.0 and the similar sampling was repeated. Group 2 (n = 10): The protocol was same as in Group 1 except for FIO2 targeted for 0.8 and 0.4. Group 3 (n = 10): Blood sampling was every two minutes during a 14-min period and repeated while FIO2 was altered as in Group 1. Group 4 (n = 10): The protocol was same as in Group 3 except for the target FIO2 similar to Group 2. PaO2 was stabilized in 4 min at earliest and in 6 min at latest after change of FIO2 in every settings of this study. In mechanically ventilated patients with normal arterial oxygenation, PaO2 could be stabilized after a 6-min period of equilibration following alteration of FIO2. PMID- 10752331 TI - [Usefulness of laryngeal mask for difficult intubation in a child with congenital laryngeal webs]. AB - We report a case of difficult intubation with congenital laryngeal web. An 11 year-old boy was scheduled for urinary surgery. After anesthetic induction, the anesthesiologist encountered resistance at the vocal cord preventing satisfactory intubation. A size 3 laryngeal mask was immediately inserted, and an adequate airway was obtained. We found a laryngeal web at the vocal cord by bronchoscope inserted thorough the laryngeal mask. The peroperative course was uneventful. Immediately after removal of the laryngeal mask, the patient developed airway obstruction due to sputum and/or laryngeal spasm. The obstruction was relieved through application of positive pressure ventilation and aspiration of the sputum. PMID- 10752332 TI - [On-line data management system using a portable blood gas analyzer in the operating room]. AB - It is very important to establish a clinical testing system which is not only prompt, simple and accurate but also safe for the patients and medical staff in the operating room, emergency room and intensive care unit. In our institution an i-STAT portable blood gas analyser has been widely used for point of care testing in all the operating rooms. This clinical testing system has been upgraded by adding an i-STAT communication protocol to our online data management system. The analysed data transmitted by the i-STAT as an infrared signal is transformed to an electronic signal through the IR link and sent to the central data station (CDS) via RS232C. The data received by the CDS is then sent to the upper grade computer system where the data is recorded on the hard disk. One advantage of this system is that it is connected to the hospital computer system. Not only does this new system meet the need for accurate, safe, effective and economical laboratory testing, but also retrospective and multifactorial analysis of intraoperative events can be easily carried out. In the future this system can be applied to telemedicine through the Internet and contribute to the treatment of critically ill patients. PMID- 10752333 TI - [Retinal photic injury--history and prospect]. PMID- 10752334 TI - [Electrophysiological evaluation of the protective effect of dimethylthiourea against retinal photic injury]. AB - PURPOSE: The Protective effect of dimethylthiourea (DMTU) against photic injury of the retina was evaluated by electroretinogram (ERG). METHODS: In the DMTU treated group, 250, 500, or 750 mg/kg DMTU was administered intraperitoneally to albino rabbits at 24 hours and immediately before starting light exposure to the eye. In the control group, physiological saline was injected intraperitoneally instead of DMTU. Preservation rates of ERG a-, b- and c-wave amplitudes were defined as the percentages of the post-photic injury values to the pre-photic injury ones, and were compared between the control and the DMTU-treated groups. RESULTS: In 750 mg/kg DMTU-treated group, the preservation rate of the a-wave was significantly higher than that in the control group 24 hours after the photic injury. While the preservation rate of the c-wave was remarkably low in the control group 24 hours after the photic injury, it was significantly higher in the 500 and 750 mg/kg DMTU-treated groups. Furthermore, better preservation rates of the c-wave were noted with higher doses of DMTU. CONCLUSION: These results suggest that DMTU protects against photic injury of the retinal pigment epithelium and photoreceptors. PMID- 10752335 TI - [Thickness of retinal nerve fiber layer decreases after vitreous surgery for idiopathic macular hole]. AB - PURPOSE: To investigate the alteration of the retinal nerve fiber thickness (NFLT) before and after vitreous surgery for a macular hole in cases with or without visual field defects. METHODS: The NFLT of 23 eyes with idiopathic macular hole was measured with scanning laser polarimeter upto 12 months after surgery. The NFLT was divided into superior, inferior, nasal, and temporal quadrants. The mean NFLT of each quadrant was analyzed statistically and compared to the results of visual field tests. RESULTS: In all cases, the NFLT decreased significantly upto 3 months after surgery, but increased gradually upto 12 months. In cases with visual field loss, the thickness of the nerve fiber layer that corresponds to the visual field defect diminished strongly, in contrast with other quadrants. Even in cases without visual field loss, the NFLT decreased significantly a month after surgery, especially in the nasal and inferior quadrants. CONCLUSION: This study demonstrates that the thickness of retinal nerve fiber layer decreases at least one month after surgery, even in cases without visual field defect. This fact may suggest the existence of a subclinical complication of vitreous surgery. PMID- 10752336 TI - [Optic disc blood flow measured by scanning laser-Doppler flowmetry using a new analysis program]. AB - PURPOSE: Using a new analysis program for scanning laser-Doppler flowmetry (SLDF) by a Heidelberg retina flowmeter (HRF), we studied the relation between flow and visual field or disc morphology. SUBJECTS AND METHODS: In 42 eyes of 21 patients with normal tension glaucoma (NTG) the mean-flow of the HRF blood flow parameters at the disc rim was measured and analyzed by a new analysis program for perfusion maps (the SLDF analysis tool), to minimize the influence of large vessels or/and artifacts caused by small eye movements. We investigated whether difference of the mean-flow between a pair of eyes had any relation to differences between a pair of eyes in visual field indices and those in disc morphological measurements of the Heidelberg retina tomograph. RESULTS: We found statistically significant correlations between the mean-flow and optic disc parameters (Disk Area, Cup Area, Height Variation Contour, Cup Volume, Rim Volume, Mean RNFL Thickness). We found no statistically significant correlations between the mean-flow and visual field parameters (mean deviation, corrected pattern standard deviation). CONCLUSION: The results suggested that eyes with less flow in the optic disc rim have more advanced glaucomatous morphological changes. PMID- 10752337 TI - [Sinusotomy with intraoperative mitomycin C administration in selected eyes with advanced glaucomatous damage]. AB - PURPOSE: To study the surgical outcome of sinusotomy with intra-operative mitomycin C administration (0.04%, 3 minutes) in a total of 17 selected glaucoma eyes from 14 patients with advanced glaucomatous damage. Selection criteria were 1. an eye with visual field defect threatening the central fixation, while the central visual acuity of the contralateral eye was already impaired; and/or 2. an aphakic or pseudophakic eye with advanced glaucomatous damage in a patient of advanced age. MATERIALS AND METHODS: Patients' age, preoperative mean deviation (Humphrey 30-2 program) and intraocular pressure (IOP) averaged 63.5 +/- 11.1 (mean +/- standard deviation) years, -18.6 +/- 6.7 (mean +/- standard deviation) dB, and 20.7 +/- 4.1 (mean +/- standard deviation) mmHg, respectively. RESULTS: No postoperative hypotony (< 5 mmHg) or deterioration of visual acuity equal to or more than 2 lines of Ishihara Visual Acuity Chart were encountered and the mean postoperative IOP ranged between 11 and 13 mmHg up to 18 months postoperatively with less medication than preoperatively. The success was defined as IOP < or = 15 mmHg without oral carbonic anhydrase inhibitors and with less medication than preoperatively. The life-table method analysis according to the above criteria yielded a success probability of 70.6 +/- 11.1 (mean +/- standard deviation) % (standard error) at 18 months postoperatively. PMID- 10752339 TI - [Intraocular lens calculation for cataract treated with photorefractive keratectomy using ray tracing method]. AB - PURPOSE: Conventional methods (such as the SRK-II formula) do not accurately calculate the power of the intraocular lens (IOL) after refractive surgery. Therefore, we compared a new formula including a ray tracing method to the conventional method for foldable IOL lens implantation. METHOD: Foldable IOLs (MA 60 BM) were implanted in 26 patients (32 eyes) using the phakoemulsification technique. The power of the IOL was measured preoperatively using the SRK-II formula in all cases. From the results of postoperative refractive errors of these cases, the power of IOL calculated by the ray tracing method was compared to the SRK-II formula. Cataract patients first treated with photorefractive keratectomy (PRK) received IOL implants using our ray tracing method and their postoperative refraction was measured. RESULTS: The average postoperative refractive error was 1.32 D in SRK-II formula, 0.95 D in the ray tracing method with Ray 1 used and 0.89 D with Ray 2 used. Postoperative refraction of both eyes first treated with PRK was--1.00 D. CONCLUSION: The average postoperative refractive error was reduced in the ray tracing method using Olsen's predicted ACD (Ray 2) compared to SRK-II formula. This new tracing method appears to be useful for determination of IOL power and it may be applied for IOL calculation for cataract surgery after refractive surgery. PMID- 10752338 TI - [Clinical results of selective laser trabeculoplasty]. AB - PURPOSE: Selective laser trabeculoplasty (SLT) is a new technique aimed to developed to impact pigmented trabecular cells selectively. Compared with ordinary argon laser trabeculoplasty, it is expected to have fewer complications with more efficacy for open-angle glaucoma. In this study we performed SLT on 17 eyes of 10 patients with primary open-angle glaucoma and 1 eye with capsular glaucoma. METHODS: Follow-up period was up to 10 months. Average energy irrachieted was 28.14 mJ (0.47 mJ x 59 spots) against pigmented trabecular band over the half circumference of anterior chamber angle. RESULTS: Preoperative mean intraocular pressure (IOP) was 22.8 mmHg and postoperative mean IOP was decreased significantly to 8.6, 17.3, and 16.1 mmHg at 1, 3, and 6 months after treatment, respectively. The average maximum IOP reduction was 8.8 (3-18) mmHg after SLT. Among 11 eyes showing transient IOP elevation, 6 eyes had an elevation of more than 5 mmHg. No remarkable postoperative complications were noted. CONCLUSION: SLT is a safe and effective modality for the treatment of open-angle glaucoma such as primary open-angle glaucoma (POAG) and capsular glaucoma. PMID- 10752340 TI - [The contribution of inflammatory cell infiltration to conjunctival inclusion cyst formation]. AB - PURPOSE: The incidence of inclusion cysts was examined histopathologically in conjunctival disorders where inflammatory cell infiltration was seen in the subepithelial connective tissue. METHOD: The incidence of inclusion cysts was examined histopathologically in pterygium, vernal conjunctivitis, pyogenic granuloma and pingueculitis. The specimens of pinguecula were used as control. RESULTS: Inclusion cysts were recognized in 5/55 cases of pterygium, 2/12 cases of vernal conjunctivitis, 1/4 cases of pyogenic granuloma, and 1/2 cases of pingueculitis. On the other hand no inclusion cyst was recognized in 20 cases of pinguecula in which no inflammatory cell infiltration was seen. CONCLUSIONS: Inflammatory cell infiltration may contribute to the formation of conjunctival inclusion cysts in pterygium, pingueculitis, vernal conjunctivitis and pyogenic granuloma. PMID- 10752341 TI - [A case of proliferative sarcoid retinopathy with sarcoid nodules in tissue obtained during vitrectomy]. AB - BACKGROUND: In previous studies, intraocular proliferative tissues obtained from proliferative sarcoid retinopathy cases during vitrectomy have been examined histopathologically. However, there is no report of identification of sarcoid nodules in examined tissues. We performed vitrectomy for a case of proliferative sarcoid retinopathy with extensive proliferative changes. Histopathologically, sarcoid nodules were identified in the fibrovascular membranes. CASE: A 25-year old man was treated for sarcoid uveitis in the right eye by his local ophthalmologist. Lens aspiration was performed for complicated cataract. He was referred to our hospital with vitreous opacity and traction retinal detachment which occurred after the surgery. FINDINGS: The vitreous opacity was so severe that details of the fundus were not visible. Traction retinal detachment was suspected from findings of B-mode echography. Vitrectomy was performed, and total retinal detachment due to contraction of the fibrovascular membrane around the optic disc and posterior pole was observed. Sarcoid nodules were identified histopathologically in fibrovascular membranes obtained during vitrectomy. CONCLUSION: In proliferative sarcoid retinopathy cases, sarcoid lesions may be one of the causes of fibrovascular membrane formation. PMID- 10752342 TI - [A pediatric case of ophthalmoplegic migraine with recurrent oculomotor nerve palsy]. AB - PURPOSE: A young patient of ophthalmoplegic migraine with recurrent oculomotor nerve palsy is reported. CASE: A 4-year-old girl came to our hospital complaining of recurrent left ptosis and double vision which completely resolved within a few days. She had a history of drowsiness after vomiting every two months. Left oculomotor nerve palsy with internal ophthalmoplegia was observed. Electroencephalography revealed mild changes, but computed tomography, single photon emission computed tomography, and cerebrovascular Doppler-ultrasound examination demonstrated no abnormalities. This oculomotor nerve palsy completely improved on the next day. RESULTS: Thereafter, left episodic mydriasis and oculomotor nerve palsy occurred twice each. However, the frequency and symptoms of migraine were remarkably reduced by topical administration of 0.25% timolol maleate twice daily to both eyes. CONCLUSION: In isolated oculomotor nerve palsy in childhood, ophthalmoplegic migraine should be also considered. We emphasize that a detailed case history is very important to its diagnosis. PMID- 10752343 TI - [A case of tuberculous uveitis complicated by myelodysplastic syndrome]. AB - BACKGROUND: Recently, the incidence of tuberculosis in compromised hosts has increased. CASE: A 52-year-old man suffering from myelodysplastic syndrome (MDS) had pulmonary tuberculosis. On June 22, 1996, we found a disc-sized choroidal lesion with milky appearance on the paramacular in his right eye. After 1 month, a similar choroidal lesion was found near the disc in the same eye. In spite of chemotherapy for MDS and antituberculosis medications, those choroidal lesions gradually enlarged. In November, satellite lesions were found around them. Some retinal exudates and hemorrhage were also detected in both eyes. He died on April 6, 1997. We found Langhans' giant cells in the choroid on the specimen of his eyes. So we judged the choroidal lesions to be tuberculous uveitis. CONCLUSION: This case showed choroidal tuberculosis and choroidal miliary tuberculosis in the same eye that were resistant to medications. We thought resistance to medication was due to destruction of the immune system by MDS. PMID- 10752344 TI - [A case report of neovascular glaucoma due to carotid artery occlusive disease- diagnosis of neovascular glaucoma and histological characteristics]. AB - PURPOSE: The methods of diagnosing neovascular glaucoma were evaluated in a case with carotid artery occlusive disease. A trabeculectomy specimen taken from this patient was examined histologically to find the characteristic picture of this slowly progressive neovascular glaucoma. MATERIALS AND METHODS: The left eye of a 48-year old man with neovascular glaucoma due to carotid artery occlusive disease had been previously diagnosed as uveitis with ocular hypertension. His left eye was examined by fluorescein fundus and gonioangiography. Trabeculectomy was performed because of uncontrolled intraocular pressure and the trabeculectomy specimen was examined histologically including immunohistochemistry with antibodies against von Willebrand factor antigen. RESULTS: The characteristic picture of fluorescein fundus angiography was filling delay in the choroid and stained walls of the retinal arteries and veins. Fluorescein gonioangiography using a fundus camera clearly demonstrated new vessels in the pupil and angle with intense fluorescein leak in spite of faint neovascularization in those areas when observed by slit lamp with gonioscopy. Histological examination of the trabeculectomy specimen revealed proliferation of new vessels in the spaces of the trabecular meshwork and focal occlusion of Schlemm's canal. CONCLUSION: Fluorescein fundus angiography and gonioangiography by fundus camera is very useful for diagnosis of neovascular glaucoma due to carotid artery occlusive disease. Neovascular proliferation in the trabecular meshwork with slight of peripheral anterior synechia and focal occlusion of Schlemm's canal may be peculiar to such slowly progressive neovascular glaucoma. PMID- 10752345 TI - The burdens of professionalism. Patients' rights and social justice. PMID- 10752346 TI - The right to die mad. PMID- 10752347 TI - Death of a nation: the AIDS crisis in Zimbabwe. PMID- 10752348 TI - Dressed to kill: the mid-nineteenth century crinoline craze. PMID- 10752349 TI - MAST: what went wrong? An essay on changes in medical practice. PMID- 10752350 TI - Response: the MAST lesson: contemporary example of society and academic responses to paradigm shifts. PMID- 10752351 TI - Response: does the emperor have clothes? PMID- 10752352 TI - Eavesdropping on the womb: the advent of fetal ausculatation. PMID- 10752353 TI - Angelical conjunction: religion, reason, and inoculation in Boston, 1721-1722. PMID- 10752354 TI - The physician at the movies PMID- 10752355 TI - "Have we come a long way, baby?". PMID- 10752356 TI - "Have we come a long way, baby?". PMID- 10752357 TI - "Have we come a long way, baby?". PMID- 10752358 TI - Introduction to the special section on the use of computers for making judgments and decisions. AB - Computers have the potential to greatly increase the accuracy of judgments and decisions that are made in the area of psychological assessment. The validity of present-day computer programs are described in the first 2 articles of this Special Section. Both computer-based test interpretation programs and mechanical prediction rules (e.g., statistical prediction rules) are described. Limitations of present-day computer programs are described in the 3rd article along with methodological recommendations for building more powerful rules. In the 4th article, a statistical analysis (neural network modeling) is described that may allow us to build better rules. PMID- 10752359 TI - Validity and utility of computer-based test interpretation. AB - Computers have been important to applied psychology since their introduction, and the application of computerized methods has expanded in recent decades. The application of computerized methods has broadened in both scope and depth. This article explores the most recent uses of computer-based assessment methods and examines their validity. The comparability between computer-administered tests and their pencil-and-paper counterparts is discussed. Basic decision making in psychiatric screening, personality assessment, neuropsychology, and personnel psychology is also investigated. Studies on the accuracy of computerized narrative reports in personality assessment and psychiatric screening are then summarized. Research thus far appears to indicate that computer-generated reports should be viewed as valuable adjuncts to, rather than substitutes for, clinical judgment. Additional studies are needed to support broadened computer-based test usage. PMID- 10752361 TI - Computers will become increasingly important for psychological assessment: not that there's anything wrong with that! AB - Though one can expect that computer programs will become increasingly important for psychological assessment, current automated assessment programs and statistical-prediction rules are of limited value. Validity has not been clearly established for many automated assessment programs. Statistical-prediction rules are of limited value because they have typically been based on limited information that has not been demonstrated to be optimal and they have almost never been shown to be powerful. Recommendations are made for building and evaluating new computer programs. Finally, comments are made about the ethics of using computers to make judgments. PMID- 10752360 TI - Clinical versus mechanical prediction: a meta-analysis. AB - The process of making judgments and decisions requires a method for combining data. To compare the accuracy of clinical and mechanical (formal, statistical) data-combination techniques, we performed a meta-analysis on studies of human health and behavior. On average, mechanical-prediction techniques were about 10% more accurate than clinical predictions. Depending on the specific analysis, mechanical prediction substantially outperformed clinical prediction in 33%-47% of studies examined. Although clinical predictions were often as accurate as mechanical predictions, in only a few studies (6%-16%) were they substantially more accurate. Superiority for mechanical-prediction techniques was consistent, regardless of the judgment task, type of judges, judges' amounts of experience, or the types of data being combined. Clinical predictions performed relatively less well when predictors included clinical interview data. These data indicate that mechanical predictions of human behaviors are equal or superior to clinical prediction methods for a wide range of circumstances. PMID- 10752363 TI - Computer-assisted judgment: defining strengths and liabilities. AB - Clinicians often fail to recognize limitations in their own subjective judgments, make use of well-developed mechanical-prediction methods, or carefully evaluate which computer-based aids warrant their consideration. This article addresses issues regarding computer-based test interpretations (CBTIs) and computer-based decision making. Comments highlight conclusions reached by other contributors to this Special Section, additional literature bearing on these observations, and implications for consumers of computer-assisted techniques and researchers developing or evaluating these methods. The future of computer-assisted assessment depends on educating clinicians and researchers to be better consumers of existing as well as emerging technologies in this domain. PMID- 10752362 TI - Applying artificial neural network models to clinical decision making. AB - Because psychological assessment typically lacks biological gold standards, it traditionally has relied on clinicians' expert knowledge. A more empirically based approach frequently has applied linear models to data to derive meaningful constructs and appropriate measures. Statistical inferences are then used to assess the generality of the findings. This article introduces artificial neural networks (ANNs), flexible nonlinear modeling techniques that test a model's generality by applying its estimates against "future" data. ANNs have potential for overcoming some shortcomings of linear models. The basics of ANNs and their applications to psychological assessment are reviewed. Two examples of clinical decision making are described in which an ANN is compared with linear models, and the complexity of the network performance is examined. Issues salient to psychological assessment are addressed. PMID- 10752364 TI - Acute Stress Disorder Scale: a self-report measure of acute stress disorder. AB - The Acute Stress Disorder Scale (ASDS) is a self-report inventory that (a) indexes acute stress disorder (ASD) and (b) predicts posttraumatic stress disorder (PTSD). The ASDS is a 19-item inventory that is based on Diagnostic and Statistical Manual of Mental Disorders (4th ed.; DSM-IV, American Psychiatric Association, 1994) criteria. The ASDS possessed good sensitivity (95%) and specificity (83%) for identifying ASD against the ASD Interview on 99 civilian trauma survivors. Test-retest reliability of the ASDS scores between 2 and 7 days was strong (r = .94). The ASDS predicted 91% of bushfire survivors who developed PTSD and 93% of those who did not; one third of those identified by the ASDS as being at risk did not develop PTSD, however. The ASDS shows promise as a screening instrument to identify acutely traumatized individuals who warrant more thorough assessment for risk of PTSD. PMID- 10752365 TI - Measurement properties of the Center for Epidemiologic Studies-Depression Scale in a homeless population. AB - The measurement properties of the Center for Epidemiologic Studies--Depression Scale (CES-D; L. S. Radloff, 1977) were evaluated in a probability sample of homeless adults residing in a large and demographically diverse community. The findings from this investigation suggest that the CES-D is a reliable measure of depressive symptoms among homeless adults and that the factor structure of the scale replicates the factor structure found in the general population. Change in the CES-D scores was associated with change in residential status, with participants who had made a transition from homelessness to regular domicile, reporting significantly lower levels of depressive symptomatology. This result indicates the scale's sensitivity to current depressive mood, as affected by significant life events encountered by homeless persons. PMID- 10752366 TI - Development and validation of the Stephenson Multigroup Acculturation Scale (SMAS). AB - This article describes the development and psychometric evaluation of the Stephenson Multigroup Acculturation Scale (SMAS). Three studies were conducted to describe its development and refinement, examine its psychometric properties with 436 participants from 5 ethnic groups, and examine the robustness of the factor structure with a new sample. Exploratory factor analyses generated a 2-factor solution: ethnic society immersion and dominant society immersion. Item refinement resulted in a 32-item version of the SMAS. Findings indicated a robust factor structure across groups. Confirmatory factor analysis indicated that the 2 factor model provided a close approximation to the observed data. Studies indicated high reliability and validity indexes. Findings support the role of acculturation as a mediator between ethnic group affiliation and standardized assessment results. PMID- 10752367 TI - Increasing the reliability of Wechsler Intelligence Scale for Children--Third Edition difference scores with reliable component analysis. AB - Wechsler Intelligence Scale for Children--Third Edition index score differences are generally interpreted cautiously, if at all, primarily because of their poor reliability. On the basis of prior analyses with the Wechsler Adult Intelligence Scale--Third Edition (J. C. Caruso & N. Cliff, 1999), it was hypothesized that differences between scores defined by reliable component analysis would have higher reliability than those defined by traditional equal weighting. Differences between the reliable component scores showed substantially higher reliability than equally weighted score differences. The differences between reliable component scores were also substantially more reliable than those derived from the weighted scores suggested by K. C. H. Parker and L. Atkinson (1994). Using the weights provided in this article will allow researchers and practitioners to compute the RCA scores and have the assurance of high reliability with its attractive consequences. PMID- 10752368 TI - Evaluation of the validity of the Wechsler Intelligence Scale for Children--Third Edition comprehension and picture arrangement subtests as measures of social intelligence. AB - Clinical lore suggests that the Wechsler Intelligence Scale for Children--Third Edition (D. M. Wechsler, 1991) Comprehension and Picture Arrangement scores may be used as indices of social intelligence. This study evaluated this supposition by comparing these subtest scores with mother- and teacher-reported social functioning in 142 children with ADHD and 30 control children. After general intelligence was partialled out, the Comprehension subtest related to some aspects of social functioning, but the clinical significance of this was limited. The Picture Arrangement subtest was unrelated to social functioning, once general intelligence was controlled. These findings were consistent across study groups. PMID- 10752369 TI - On the sins of short-form development. AB - The empirical short-form literature has been characterized by overly optimistic views of the transfer of validity from parent form to short form and by the weak application of psychometric principles in validating short forms. Reviewers have thus opposed constructing short forms altogether, implying researchers are succumbing to an inappropriate temptation by trying to abbreviate measures. The authors disagree. The authors do not oppose the development of short forms, but they do assert that the validity standards for short forms should be quite high. The authors identify 2 general and 9 specific methodological sins characterizing short-form construction and offer methodological suggestions for the sound development of short forms. They recommend a set of 6 a priori steps researchers should consider and 9 methodological procedures researchers can use to develop valid abbreviated forms of clinical-assessment procedures. PMID- 10752370 TI - The reason we do what we do. PMID- 10752371 TI - Eye doctor's vision helps poor see, live better. PMID- 10752372 TI - No health insurance, no problem. PMID- 10752373 TI - Medical schools spearhead assessment of Wisconsin's health needs. PMID- 10752374 TI - Assessing health concerns and priorities: process and results. PMID- 10752376 TI - Medical schools develop plans to address public health needs. PMID- 10752375 TI - Phone poll gets at the heart of citizen concerns about modern health care. PMID- 10752378 TI - The future of public health in Wisconsin, revisited. PMID- 10752377 TI - Assessing Wisconsin's health needs and priorities. PMID- 10752379 TI - Medicine and public health: imagine... PMID- 10752380 TI - Building effective partnerships with Wisconsin communities. PMID- 10752381 TI - Wisconsin's population health. PMID- 10752382 TI - The rural women's health study: health and behavior of farm and nonfarm resident women. PMID- 10752383 TI - Behavior changes & disease prevention: MCW research shows effectiveness of HIV/AIDS risk reduction interventions. Medical College of Wisconsin. AB - Many of the diseases that cause premature illness and death--including some cancers, heart disease, and AIDS--could be prevented if persons made behavior changes to reduce their risk for developing the illnesses. Over the past two decades, there have been great advances in our scientific understanding of how to promote health risk behavior change. This paper briefly reviews elements and examples of effective behavior change interventions, including programs that can be offered in service settings as well as community-level interventions. The prevention of diseases through behavioral public health interventions requires the investment of funds but can reduce burdens on health care systems, reduce the human toll caused by premature deaths, and be highly cost-effective. A remarkable number of diseases could be prevented if individuals were effectively assisted in changing the risk behaviors responsible for those illnesses. The causal association between cigarette smoking and lung cancer, other pulmonary diseases, and cardiovascular disease is well-known, and millions of premature deaths could be prevented if people stopped smoking cigarettes. Deaths due to cardiovascular disease could be dramatically reduced if persons made behavioral and lifestyle changes to improve their fitness through exercise, obesity reduction, and maintenance of low blood cholesterol levels. The World Health Organization estimates that over 45 million persons worldwide have already contracted HIV infection, and nearly 1 million of these cases are in the United States. Over 40,000 Americans continue to contract HIV infection each year. Virtually every new case of HIV infection is preventable if individuals at risk made changes in their sexual or drug use practices. While lung cancer, cardiovascular disease, and AIDS are three of the clearest examples, persons' behavior plays a direct or a contributing role in the development of many other diseases that cause premature death or that worsen health and life quality. Recognition of the link between behavior and preventable illness--and recognition that enormous health, economic, and quality of life benefits could be realized through healthier behavior patterns--is not new. We have known all of this for a long time. We have also known for a very long time that helping people to successfully change risky behavior habits is often very difficult. Over the past 20 years, a field of scientific study and applied practice has developed with the purpose of better understanding why persons engage in health risk behavior patterns and developing approaches to help people change these patterns. Under the rubric of "behavioral medicine", this field makes use of behavioral science theory and behavior change techniques applied to health and disease prevention. PMID- 10752384 TI - School-based/school-linked health centers expanding points of access. AB - Health providers and leaders in urban Milwaukee collectively acted to expand school-based health services to children attending Milwaukee Public Schools (MPS). This School-Based/School-Linked Health Centers' (SB/SLHCs) Collaboration was initiated to increase points of access for children, primarily to working poor families, through mobilizing community resources among local leaders and statewide health systems. Systematic steps such as needs assessment, sharing data, seeking funds and prioritization of school-based sites facilitated the establishment of more than 30 additional SB/SLHCs. This has resulted in approximate 700% increase in school-based health care and health promotion services. This collaboration illustrates how communities and health care systems can effectively advocate and impact local services to benefit a population having high social risk factors. As welfare reform efforts evolve, SB/SLHCs have significantly advanced access to mainstream health services through effective local collaborations. PMID- 10752385 TI - Public health: a successful paradigm applied to firearm injuries. AB - Firearm injuries are a major contributor to injury mortality and morbidity in the United States and account for substantial loss of productive years of life. A public health approach to injury reduction, and particularly firearm injury prevention, has promise because it emphasizes prevention rather than reaction, it utilizes a scientific approach to policies and programs, it brings an integrative, multi-disciplinary approach to the effort, and it emphasizes the role of communities in solving health problems. The new Firearm Injury Center at the Medical College of Wisconsin exemplifies the public health approach to this vexing problem. PMID- 10752386 TI - Native American infant mortality in Wisconsin. AB - An infant mortality rate is an important public health measure because it may indicate unmet health needs or unfavorable environmental factors. The infant mortality rate for Native Americans in Wisconsin has decreased over the past several years, although there remains a discrepancy between Native Americans, all races combined and the Healthy People 2000 goal. The Wisconsin American Indian Infant Mortality Review Project (WAIIMR) conducted a study to understand local underlying factors that contributed to infant deaths and to recommend local health initiatives that could be developed to have a positive impact on the health of infants and mothers. The results from the WAIIMR Project and more recent Wisconsin birth record data are discussed in this paper. Two risk factors, levels of prenatal care and maternal smoking during pregnancy, show discrepancies between races and warrant further investigation. Native American communities in Wisconsin are addressing risk factors affecting maternal and child health through projects such as Honoring Our Children and The Michigan/Wisconsin Epidemiology Center. PMID- 10752387 TI - Six years in the making: OIG publishes 8 new anti-kickback safe harbors. Office of Inspector General. PMID- 10752388 TI - A clinical technique for giving painless injections. PMID- 10752389 TI - Laughing all the way. PMID- 10752390 TI - Cuspal fracture with conventional Class II amalgam. PMID- 10752391 TI - Baby boomers make cosmetic dentistry boom. AB - Baby Boomers want to stay younger longer. As they age, a healthy attractive smile becomes much more important to their appearance. Nothing conveys good health and maintenance like aesthetically pleasing teeth. Because the timing is perfect for cosmetic dentistry, there is no reason for people to not have a great smile. Smile-Vision and the latest cosmetic dentistry techniques and materials helped the case patient first see and then become the person he wanted to be. His teeth look and feel better, resulting in a more confident appearance. (Figures 14 through 16). PMID- 10752392 TI - Laser use in endodontics. PMID- 10752393 TI - The magic of magnification. PMID- 10752394 TI - A laser for hard and soft tissue applications. PMID- 10752395 TI - A new generation restorative material. Interview by Phillip Bonner. PMID- 10752397 TI - Aesthetic excellence through comprehensive diagnosis and tissue management. PMID- 10752396 TI - Aesthetic dilemmas: Part 2.. Interview by Phillip Bonner. PMID- 10752398 TI - Step into action. PMID- 10752399 TI - Open your vertical and smile: metal-free, full-mouth rehabilitation. PMID- 10752400 TI - New techniques create a new smile. PMID- 10752401 TI - Measurement of the apical foramen. PMID- 10752402 TI - The problem with second molars. PMID- 10752403 TI - The anatomy of the intraosseous injection. PMID- 10752404 TI - Root coverage grafting: the aesthetic subepithelial connective tissue graft, Part 2. PMID- 10752405 TI - Laser sulcular debridement: the newest weapon in fighting periodontitis. PMID- 10752406 TI - Aesthetic dilemmas: Part 1. Interview by Phillip Bonner. PMID- 10752407 TI - The crown coping partial denture. PMID- 10752409 TI - Restoring faith in restorative dentistry: advanced techniques and technology. PMID- 10752408 TI - Conservative tooth-colored restorations for the 21st century. AB - Within the past 5 years, the development of tooth-colored restorative materials has allowed dentists to restore teeth to proper form and function, while achieving great aesthetics (Figures 8 through 11). Adhesive resins enable dentists to strengthen teeth, while providing the patient with strong, long lasting restorations. Dentists should become comfortable with placing these types of restorations and offer them as alternatives to patients who are looking for brighter, healthier smiles. PMID- 10752410 TI - Taking the mystery out of endodontics, Part 3: The truth about single-visit endodontics. PMID- 10752411 TI - The resurgence of air abrasion into restorative dentistry, Part 2. PMID- 10752412 TI - New horizons in local anesthesia. AB - The computer-controlled local anesthesia system and the TEA system present 21st century alternatives to the traditional syringe. The TEA system is a non-invasive form of anesthesia that blocks pain electronically, using the same cellular mechanism as local chemical anesthesia. Targeted electronic anesthesia provides pain control for restorative dental procedures without the use of needles or postoperative discomfort, numbness, and swelling. The computer-assisted system outperforms syringes for traditional injections. This new system generates a precisely controlled anesthetic flow rate that eliminates the need for the operator to use thumb pressure to administer the injection. The lightweight pen grasp handle results in greater tactile feedback, precision, operator ease, and patient comfort. The greatest advantage may be in the new techniques that it makes available. With these techniques, a dentist can target the teeth to achieve profound pulpal anesthesia, often without the annoying side effects of facial numbness. With this new advanced system in the maxillary arch, the AMSA injection offers clinical advantages over traditional anesthesia techniques, according to Dr. Mark Friedman, whom I consulted with earlier this year. In the mandibular arch, a safe and predictable PDL injection technique may replace the need for an inferior alveolar block in numerous clinical situations. The use of these modified injection techniques can have a positive influence on patient safety, patient comfort, and office productivity. Both of these systems take the fear and anxiety out of dental injections. They offer exciting advanced technology for local pain control. Significantly, if patient stress and anxiety are reduced, the operator immediately benefits. New horizons in local anesthesia offer improved opportunities for patient comfort using computer-controlled local anesthetic systems and TEA. PMID- 10752413 TI - Root coverage grafting: the aesthetic subepithelial connective tissue graft, Part 1. PMID- 10752414 TI - A new era in halitosis and periodontal treatment. AB - An effective, 12 hour, anti-halitosis remedy is a spectacular breakthrough in the dental field and a significant step toward improving the lives of millions of people in the United States who suffer from chronic halitosis and the resultant psychological trauma. The technology contained in TriOral Anti-Halitosis Treatment Rinse, along with its significant research, will provide dentists with the ability to accelerate their halitosis treatment success curve. I am excited to present the science of this product and resulting research in my upcoming seminar series, and I view this as one of my greatest contributions to other dental professionals and their patients. PMID- 10752415 TI - Advances in orthognathic surgery. PMID- 10752416 TI - The art of the endodontic referral, Part 1: Would you do this root canal for your mother? PMID- 10752417 TI - Influence of occlusion on the degradation of posterior composite resins. PMID- 10752418 TI - Attaining consistent aesthetic success with veneers. PMID- 10752419 TI - Something to smile about. PMID- 10752420 TI - Aggressive aesthetics: becoming the artist. PMID- 10752421 TI - Techniques for managing the pediatric patient. Interview by Phillip Bonner. PMID- 10752422 TI - Aesthetic enhancement of pontic sites for fixed partial dentures. AB - The ridge expansion technique using tapered osteotomes can be used anywhere in the maxilla when a change in external ridge morphology would be advantageous for both aesthetics and/or proper dental implant placement. Usually subsequent to tooth loss, the maxilla is generally somewhat undercut in form. This ridge expansion technique can be used to reduce the undercut by bulging out the base of the facial/buccal plate. This would recreate the illusion of root prominences (Figure 12) or permit implant fixtures to be inserted in a more upright position. The tapered osteotomes can therefore predictably be used to expand the buccal bone to simulate the presence of a root prominence for optimal aesthetics for placement of a fixed partial denture prosthesis (Figure 13). PMID- 10752423 TI - Laser ENAP for periodontal ligament regeneration. PMID- 10752424 TI - Endocarditis prophylaxis. AB - This article attempts to simplify the decision-making process regarding endocarditis prophylaxis. Cardiac conditions are stratified as to risk and oral procedures for which prophylaxis is or is not recommended are outlined. The cardiac conditions are paired with various oral procedures to clarify which require prophylaxis. Antibiotic regimens are described. A distinction is made between MVP patients who require prophylaxis and those who do not. Finally, the value of professional oral healthcare for cardiac patients is confirmed. PMID- 10752425 TI - Closing the gap between specialists and GPs: technologies and interdisciplinary therapy. PMID- 10752426 TI - When to modify treatment plans. PMID- 10752427 TI - Stress in dentistry: how can you C-O-N-T-R-O-L it? AB - Stress cannot be eliminated. The challenge and opportunity lies in the ability to control or manage your response to stress. Those who choose to control their stress by actively and constructively managing it can learn to turn its potentially harmful force into high-powered energy. PMID- 10752429 TI - Anterior direct-ceramic restorations using computer-aided technology. PMID- 10752428 TI - Aesthetic treatment of tetracycline stains. PMID- 10752430 TI - Conservative rehabilitation. PMID- 10752431 TI - The coming of age of lasers in dentistry. PMID- 10752432 TI - Condensable composites: the new paradigm in amalgam alternatives. PMID- 10752433 TI - Hydraulic and hydrophobic impressions. PMID- 10752434 TI - The use of glass ionomers in pediatric patients. PMID- 10752435 TI - Incorporating nickel titanium instrumentation into your practice. PMID- 10752436 TI - Taking the mystery out of endodontics, Part 4: Cleaning and shaping, pure and simple. PMID- 10752437 TI - Aesthetics in implantology. AB - A young man presented himself with a missing maxillary central incisor and a depressed defect due to a trauma. The trauma caused the loss of No. 8 and its surrounding supportive structure. The defective results were seen by the labial bone loss in the area and a depression. The depression became a food-and-plaque trap and an unaesthetic eyesore. Before the tooth loss, iatrogenic factors from a root canal or retrograde amalgam caused a permanent gingival tattoo. The bonded temporary pontic was larger mesio-distally than the adjacent teeth, and this was very noticeable. It was dull in its finish and poor aesthetically. The patient requested an implant to avoid excess drilling of natural teeth. A combined effort was used to achieve the pleasing final result. To correct the area and improve the physiology and aesthetics, several techniques had to be understood. These techniques also need to be mastered, recalled, and used almost instinctively, and done in a proper team-like sequence. In this case we performed an aesthetic ridge augmentation and tissue surgical manipulation to acquire the form, shape, and correct color background. Deciding upon which implant and material involved choosing the size, type, and shape for the area involved. The techniques used included implant insertion, surgical second stage abutment, mucosal periosteal flap design, various suturing and gingival grafting techniques, ridge buildup augmentation, and GTR. Other choices included which resorbable membrane to use, what bone graft materials, and what oral hygiene to recommend to maintain final health. The restorative technique included full crown coverage, laminate veneers, provisional pontic design and bonding, control of porcelain materials, computer imaging, occlusion, cementing, smile designs, and implant prosthodontics. PMID- 10752438 TI - Modulation of the host response in the treatment of periodontitis. AB - Periodontitis has two distinct but interconnected etiologic components: periodontopathic bacteria adjacent to the periodontal tissues, and host-mediated connective tissue-destructive responses to the specific causative bacteria and their metabolic products. Although past and existing therapies have focused primarily on the causative microbial challenge, the host component of periodontal destruction has recently been intensely studied. New treatment strategies that focus on attenuating destructive host responses are emerging. Host modulatory agents, when used adjunctively, may enhance clinical therapeutic responses and make these responses more predictable in the susceptible host. The ongoing development of safe, effective pharmacotherapies that specifically target host response mechanisms, and the introduction of such pharmacotherapies as adjuncts to traditional, antimicrobial interventions may represent a new, integrated approach in the long-term treatment and management of this chronic disease. PMID- 10752439 TI - Time-tested botanical remedies for modern periodontal therapy. PMID- 10752440 TI - Divide and conquer: extracting maxillary first molars. PMID- 10752441 TI - Techniques and technologies for the advanced aesthetic practice. PMID- 10752442 TI - Are dental school graduates prepared for the real world?. Interview by Phillip Bonner. PMID- 10752443 TI - Improving TMD treatment and protecting restorative dentistry. PMID- 10752444 TI - Case presentation. Periapical cemental dysplasia. PMID- 10752445 TI - Advantages of implant overdentures. PMID- 10752446 TI - Child abuse assessment for Mississippi dentists. PMID- 10752447 TI - Tooth loss related to age. PMID- 10752448 TI - Clinical dental research center: an ongoing study. Salivary detection of prostate cancer. The expanding role of dentists in health care. PMID- 10752449 TI - Case presentation. Squamous cell carcinoma. PMID- 10752450 TI - Fluoride: a review--Part II: Topical fluorides. AB - Fluoride therapy continues to be the best defense in the battle against dental caries. The decision to utilize topical fluorides is no longer age dependent. Changing disease patterns require dentists to critically evaluate the caries risk of each patient and develop a fluoride treatment plan based upon the needs of the individual patient. A variety of professional and self-applied fluoride products are available and new fluoride delivery systems have recently entered the market. A critical review of literature, combined with an understanding of the advantages and disadvantages of each topical fluoride system will assist the dentist in selecting the product best suited for each patient. PMID- 10752451 TI - The effect of delayed photocuring on the hardness of resin-modified glass ionomers. PMID- 10752452 TI - When should my child see a pediatric dentist? PMID- 10752453 TI - Prosthodontic considerations in complicated restorative and esthetic dental problems. AB - An axiom of prosthetic dentistry has always been that the restoration of esthetics, speech and function requires the successful arrangement of teeth in space. The road to this success often requires the restorative dentist be a visionary who can coordinate multidisciplined planning in the presence of such obstacles as deficient alveolar bone and gingival tissues, poor occlusal plane, and jaw malrelations. Dr. Cook would like to thank the many Albuquerque generalists and specialists whose experienced hands contributed to the successful treatment of these patients. PMID- 10752454 TI - Esthetic periodontal therapy: recognizing conditions requiring periodontal plastic surgery. PMID- 10752455 TI - GBR and sinus augmentation using autogenous and DFDB Allograft and Alloderm as the barrier. PMID- 10752456 TI - Problems in the care management of the aging patient: legal, financial and practical implications. AB - The graying of America poses many challenges to the dental professional. As the population ages, most dental practices are experiencing growth in percentage of older people served. The dental practitioner must be on the alert to special problems associated with the treatment of the older patient. Failure to identify these common concerns may result in patient non-compliance, failure to achieve treatment goals and heightened risk of malpractice. In addition, dental professionals who are aware of the multiplicity of factors that impact the aging process can better promote their own successful aging. This article focuses on identifying some of the key "hidden" issues typically facing older patients which can adversely impact dental treatment; and, practical techniques for managing these problem areas. Also covered are common financial and social concerns associated with aging and suggestions for how dental professionals can avoid complications from compounding in their own lives. PMID- 10752457 TI - Mainstream or sidelined? The choice for dentistry. AB - The 1998 white paper 'The New NHS: Modern, Dependable' announced changes to the way in which healthcare will be commissioned in the future, stimulating debate on how dentistry fits in with these developments. In this paper the authors examine first the mechanism by which healthcare is commissioned under the existing arrangements and then look at how this might develop in the future. The authors conclude that oral health will continue to be a priority for the NHS and therefore dental care will continue to be included in the portfolio of commissioned healthcare. This will however necessitate a continued dialogue between commissioners and providers and the opportunity for dental services to stay within the mainstream of the NHS by working within the new system has to be seized by the profession. PMID- 10752458 TI - Morbidity, mortality and local anaesthesia. AB - Two cases of local anaesthetic overdose and death are described. The patients, a four-year-old child and a 68-year old female, received local anaesthetic doses greatly in excess of those recommended. Their overdose reactions are described as well as subsequent management. The paper reviews the causes of local anaesthetic overdose, its signs and symptoms, and the recommended management of these reactions. Specific discussion as to the cause of death of these two patients and of the means to prevent such occurrences in the future concludes the paper. PMID- 10752459 TI - Reasons for the placement and replacement of restorations in vocational training practices. AB - AIM: The reasons for placement and replacement of restorations are central to clinical practice. It is the purpose of this paper to assess the reasons for placement and replacement of restorations in the practices of vocational dental practitioners (VDPs) and their trainers. METHOD: VDPs and their trainers were invited to take part in the study. VDPs were asked to record the principal reason for the placement or replacement of all restorations which they provided for a one-month period and, also, to record the age, Class and materials used for each restoration replaced (if known). Trainers were also asked to participate. The data thereby collected were analysed statistically. RESULTS: 56 VDPs and 17 trainers participated. Data were obtained on a total of 9031 restorations, 4423 (49%) being initial placements and 4608 (51%) replacements. Of the total number of restorations assessed, 54% were amalgam, 30% composite and 16% glass ionomer. Principal reasons for placement/replacement of the restorations were primary caries (41%), secondary caries (22%), tooth fracture (6%), margin fracture/degradation (6%), non-carious defects (6%) and bulk fracture (5%). The type of existing restorative material was recorded for 4196 restorations: secondary caries was the most common reason for the replacement of restorations- for 46.0%, 39.6% and 39.8% of amalgam, composite and glass-ionomer restorations respectively. VDPs placed more restorations as a consequence of caries than their trainers. CONCLUSION: Primary caries is the most common reason for the placement of restorations by the participating dentists, and secondary caries is the most common reason for the replacement of restorations of amalgam, composite and glass ionomer. VDPs placed more restorations as a consequence of caries than their trainers. PMID- 10752460 TI - The provision of resin-bonded bridgework within the General Dental Services 1987 1997. AB - The purpose of this study was to investigate the performance of resin-bonded bridgework provided within the General Dental Services of the National Health Service. The basic procedure involved analysis of data collected from the administering authorities in England, Wales and Scotland between 1987 and 1997. These figures included the cost of the provision of such bridgework and the incidence and cost of refitting bridges which had become debonded in service. The investigation found that while the number of resin-bonded bridges provided had peaked in the early 1990s the number of claims for rebonding was still increasing at a high rate. In the most recent year of the study (1996/97) the number of claims for rebonding bridges was 50% more than the number of bridges actually fitted during that year. It was concluded that resin-bonded bridgework provided under the General Dental Services of the National Health Service was experiencing a high failure rate and that this problem was worthy of further investigation. PMID- 10752461 TI - Dealing with sensitive teeth. AB - Dental pain is extremely common in the UK and can severely reduce the quality of life when it is associated with eating, drinking or speech. Patients often present complaining of a short, sharp, stabbing pain from hot or cold air, foods or drinks: while this may be caused by tooth decay, failing fillings or cracks in teeth, a major cause is sensitivity from exposed dentine. Treatment varies from dietary control, desensitising toothpastes to protection with dentine-bonding agents and glass ionomers. In extreme cases elective devitalisation is necessary. PMID- 10752462 TI - The HIV patient in general dental practice. AB - General practitioners, especially those working in areas where there is a high incidence of HIV, will inevitably have to face patients who may or may not know that they are HIV antibody positive. This paper gives some guidelines on how to approach the problem, using two case scenarios. PMID- 10752463 TI - Oral health promotion and caries prevention. AB - This paper is based on a paper presented at a British Dental Association caries prevention symposium. It represents the views of the authors and does not necessarily represent the views of any organisation with which they, or the work reported, are associated. PMID- 10752464 TI - Leading edge treatment using Emdogain. PMID- 10752466 TI - Changes in maternal attitudes towards baby bottle tooth decay PMID- 10752465 TI - An interdisciplinary approach to address tobacco issues in elementary schools through peer training. PMID- 10752467 TI - Latex glove hypersensitivity and irritation: a literature review. AB - Latex hypersensitivity is an emerging health problem that is reaching epidemic proportions. Since the introduction of universal precautions, latex glove manufacture and use has risen dramatically. With more and more individuals exposed to latex, the incidence of acquired latex sensitivity is increasing, especially in the health care professions. Latex is a natural product and is processed in a manner that retains protein allergens and leaves residual chemicals on the latex gloves. The reactions to these allergens and irritants are varied and can be classified as allergic or non-allergic. Dental health care professionals are among the populations at risk for developing these problems, the most dangerous of which is an immediate allergy to latex proteins. To increase awareness of this important issue, several studies have been completed with results showing an average prevalence of about ten percent in selected populations of dental professionals. PMID- 10752468 TI - Tailoring your services. Part II: Oral health needs assessment of long-term care facilities and nursing homes in Perth County. PMID- 10752471 TI - Dental hygienists in multidisciplinary health care. AB - The evolving roles and responsibilities of the dental hygiene profession are placing very different demands on its members. Collaboration with other health professionals will become necessary as the profession moves forward into more interdisciplinary settings. The purpose of this paper is to describe a recently published study involving collaboration between dental hygienists, physician assistants and physical therapists. Multidisciplinary teams comprised of representatives from each of the three professions participated in both a simulated problem-based case assessment and a real life patient assessment. Results showed favourable learning experiences by all three disciplines which could serve as a potential model for future multidiscipline projects. Collaborations between health professionals have the potential to enhance client care and ultimately improve their quality of life. PMID- 10752469 TI - Results of a seniors' oral health survey in rural Alberta. AB - A questionnaire and oral screening were conducted with seniors in a rural Alberta health authority to determine their oral health needs. Both the dentate and the edentulous had high levels of treatment need. Of the dentate respondents, 41.8 percent had coronal caries, averaging .88 caries per dentate person. All dentate respondents had calculus and/or pocketing as measured by the Community Periodontal Index of Treatment Needs (CPITN). Among denture wearers, 64.4 percent were found to have calculus on one or both dentures, and 61.2 percent of lower dentures exhibited poor retention. In assessing their own oral health, 76.4 percent rated themselves somewhere between good and excellent. Among the dentate, 83.6 percent brushed daily; however 57 percent never flossed. Although 40.6 percent had been to a dentist/denturist within the last year, 70.1 percent saw a dentist/denturist only when they experienced pain or problems. Of those who had not been to a dentist/denturist within a year, 83.9 percent said the primary reason was because there was nothing wrong. Although treatment needs in this group were high, lasting change in the oral health status of this group requires more than immediate treatment. Enduring effects can only be facilitated by educating seniors as to what optimal oral health is, appropriate preventive behaviours, and the effective use of available professional services. Unless these issues are addressed there is not likely to be a great deal of improvement within each cohort. PMID- 10752470 TI - Demonstrating the need for oral health education in geriatric institutions. AB - OBJECTIVES: This review paper discusses the need for an educational component of a preventive oral health care program in geriatric institutions. The content of an education component that can improve the awareness of health care professionals in geriatric institutions with respect to the positive relationship of good oral health to good general health, thereby motivating them to provide adequate oral care, is investigated. METHODS: A literature review was conducted of the following areas: dental health education in nurse training; dental care for the elderly; prevention of dental caries, periodontal diseases and oral cancer; oral hygiene; nursing homes; long-term care facilities, and homes for the aged. The search was conducted through Medline from 1966 to 1998. RESULTS: Overall, there is little information and agreement about the content and structure of oral health education programs in geriatric institutions, although information exists with respect to the effectiveness of methods of oral hygiene and the efficacy of oral care agents. The oral health care needs of the institutionalized elderly are not being met due to health care professionals' demanding workload, a lack of knowledge of the importance of oral health care and apathy, or lack of interest, towards the practical application of oral health care principles. CONCLUSIONS: There is a need to educate all stakeholder groups, including health care professionals, in the importance of mouth care for elderly clients. Dental professionals working in geriatric care should address this need by implementing a preventive oral health care program consisting, not only of examinations and preventive care, but also of an educational component for the allied health care professionals and members of the patient's family. The ultimate goal is for the medical and dental professionals and caregivers to collaborate with an integrated approach to preventing oral disease, thereby improving overall health and quality of life for the institutionalized elderly. PMID- 10752472 TI - How is your water? An interdisciplinary approach to management of biofilm in dental unit waterlines. AB - Much new information has become available as interest has grown in issues of biofilm formation and dental unit water quality. This interest is combined with a growing concern for occupational safety and for patients with diminished resistance. A review of recent literature spurred interest amongst the staff of The Hamilton-Wentworth Regional Public Health Department to carry out a quality audit of the dental unit water quality in clinics operated by the Department's Dental Services. Water testing was carried out, and problems with biofilm formation and water quality were identified. An interdisciplinary consultation process helped identify problems, offered possible solutions and made decisions on appropriate action to be taken. Some conservative interventions were successfully implemented even while it was recognized that more involved interventions were also needed. Repeat testing was carried out until the water quality in the clinical waterlines reached acceptable levels. Biofilm management to ensure water quality for both patients and staff, is now an integral part of quality management in the Hamilton-Wentworth Regional Dental Clinics. PMID- 10752473 TI - A psychological intervention for reducing pain during dental hygiene treatment. AB - Previous research has shown that individuals who "catastrophize" are more likely to experience high levels of pain and emotional distress during dental treatment. In the present study, we examined the effects of emotional disclosure on the pain and emotional distress experienced by "catastrophizers" and "noncatastrophizers" during dental hygiene treatment. Eighty undergraduate students were randomly assigned to a dental-worry disclosure condition or a control condition prior to scaling. In the disclosure condition, participants were asked to write about their dental worries before having dental hygiene treatment. In the control condition, participants were asked to write about their activities of the previous day. Consistent with previous research, catastrophizers in the control condition reported significantly more pain and emotional distress than noncatastrophizers, F (1, 76) = 9.86, P < .001. Disclosure reduced the pain and emotional distress of catastrophizers, but increased the pain and emotional distress of noncatastrophizers, F (1, 76) = 6.1, P < .01. While catastrophizers benefited from disclosure in regard to their immediate physical and emotional experience, their levels of catastrophizing and dental anxiety remained essentially unchanged. Clinical implications of the findings are discussed. PMID- 10752474 TI - Melanomas with concordant loss of multiple melanocytic differentiation proteins: immune escape that may be overcome by targeting unique or undefined antigens. AB - Melanoma-reactive HLA-A x 0201-restricted cytotoxic T lymphocyte (CTL) lines generated in vitro lyse autologous and HLA-matched allogeneic melanoma cells and recognize multiple shared peptide antigens from tyrosinase, MART-1, and Pme117/gp100. However, a subset of melanomas fail to be lysed by these T cells. In the present report, four different HLA-A x 0201+ melanoma cell lines not lysed by melanoma-reactive allogeneic CTL have been evaluated in detail. All four are deficient in expression of the melanocytic differentiation proteins (MDP) tyrosinase, Pme117/gp100, gp75/ trp-1, and MART-1/Melan-A. This concordant loss of multiple MDP explains their resistance to lysis by melanoma-reactive allogeneic CTL and confirms that a subset of melanomas may be resistant to tumor vaccines directed against multiple MDP-derived epitopes. All four melanoma lines expressed normal levels of HLAA x 0201, and all were susceptible to lysis by xenoreactive-peptide-dependent HLA-A x 0201-specific CTL clones, indicating that none had identifiable defects in antigen-processing pathways. Despite the lack of shared MDP-derived antigens, one of these MDP-negative melanomas, DM331, stimulated an effective autologous CTL response in vitro, which was restricted to autologous tumor reactivity. MHC-associated peptides isolated by immunoaffinity chromatography from HLA-A1 and HLA-A2 molecules of DM331 tumor cells included at least three peptide epitopes recognized by DM331 CTL and restricted by HLA-A1 or by HLA-A x 0201. Recognition of these CTL epitopes cannot be explained by defined, shared melanoma antigens; instead, unique or undefined antigens must be responsible for the autologous-cell-specific anti-melanoma response. These findings suggest that immunotherapy directed against shared melanoma antigens should be supplemented with immunotherapy directed against unique antigens or other undefined antigens, especially in patients whose tumors do not express MDP. PMID- 10752475 TI - Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells. AB - Anti-CD20 monoclonal antibodies have been successfully employed in the clinical treatment of non-Hodgkin's lymphomas in both unmodified and radio-labeled forms. Previous publications have demonstrated that the antitumor effects of unmodified anti-CD20 mAb are mediated by several mechanisms including antibody-dependent cellular cytotoxicity, complement-mediated cell lysis, and induction of apoptosis by CD20 cross-linking. In this report, we demonstrate induction of apoptosis by three anti-CD20 monoclonal antibodies [1F5, anti-B1, and C2B8 (Rituximab)]. The magnitude of apoptosis induction was greater with the chimeric Rituximab antibody than with the murine 1F5 and anti-B1 antibodies. Apoptosis could be enhanced with any of the antibodies by cross-linking with secondary antibodies (or Fc-receptor bearing accessory cells). The signaling events involved in anti-CD20-induced apoptosis were investigated, including activation of protein tyrosine kinases, increases in intracellular Ca2+ concentrations, caspase activation, and cleavage of caspase substrates. Our results indicate that anti-CD20-induced apoptosis can be attenuated by PP1, an inhibitor of protein tyrosine kinases Lck and Fyn, chelators of extracellular or intracellular Ca2+, and inhibitors of caspases, suggesting that anti-CD20-induced apoptosis may involve modulation of these signaling molecules. We also demonstrated that varying the expression of Bc1-2 did not affect the magnitude of anti-B1-induced apoptosis, possibly because of the sequestering effects of other Bc1-2 family members, such as Bad. These studies identify several of the signal-transduction events involved in the apoptosis of malignant B cells that transpire following ligation of CD20 by anti CD20 antibodies in the presence of Fc-receptor-expressing cells or secondary goat anti-(mouse Ig) antibodies and which may contribute to the tumor regressions observed in mouse models and clinical trials. PMID- 10752476 TI - Microsatellite instability analysis in tumors with different mechanisms for total loss of HLA expression. AB - Down-regulation of the expression of major histocompatibility complex molecules is a frequent event that is associated with the poor immunogenicity of tumor cells. Acquired resistance to T-cell-based immunotherapy has been associated with loss of functional beta2-microglobulin expression. This anomaly appears to be particularly relevant in tumors exhibiting a defect in DNA-mismatch repair, and induces structural abnormalities in HLA cell-surface expression that are not reversible by cytokine treatment. We examined HLA expression in 118 melanoma, colon or larynx tumors to identify total loss of HLA class I expression with or without somatic beta2-microglobulin gene mutation. Microsatellite instability was investigated in these tumors to determine whether a replication error phenotype (RER+) implied a particular alteration in HLA phenotype. A total of 7.6% of the tumors showed the RER+ phenotype, and 12.7% were HLA-ABC-negative. In the RER+ group, only one tumor was HLA-ABC-negative and no beta2-microglobulin mutation was identified. In contrast, in the HLA-ABC-negative group, only one tumor showed microsatellite instability. None of the three melanomas that contained beta2 microglobulin mutation exhibited the mutator phenotype. These findings suggest that beta2-microglobulin mutation in human melanoma tumors may arise through a mechanism that does not necessarily involve microsatellite instability. Our results also indicate that somatic mutations of the beta2-microglobulin gene are not the main mechanism of total loss of HLA expression. PMID- 10752477 TI - Phage-selected primate antibodies fused to superantigens for immunotherapy of malignant melanoma. AB - The high-molecular-weight melanoma-associated antigen, HMW-MAA, has been demonstrated to be of potential interest for diagnosis and treatment of malignant melanoma. Murine monoclonal antibodies (mAb) generated in response to different epitopes of this cell-surface molecule efficiently localise to metastatic lesions in patients with disseminated disease. In this work, phage-display-driven selection for melanoma-reactive antibodies generated HMW-MAA specificities capable of targeting bacterial superantigens (SAg) and cytotoxic T cells to melanoma cells. Cynomolgus monkeys were immunised with a crude suspension of metastatic melanoma. A strong serological response towards HMW-MAA demonstrated its role as an immunodominant molecule in the primate. Several clones producing monoclonal scFv antibody fragments that react with HMW-MAA were identified using melanoma cells and tissue sections for phage selection of a recombinant antibody phage library generated from lymph node mRNA. One of these scFv fragments, K305, was transferred and expressed as a Fab-SAg fusion protein and evaluated as the tumour-targeting moiety for superantigen-based immunotherapy. It binds with high affinity to a unique human-specific epitope on the HMW-MAA, and demonstrates more restricted cross-reactivity with normal smooth-muscle cells than previously described murine mAb. The K305 Fab was fused to the superantigen staphylococcal enterotoxin A (D227A) [SEA(D227A)], which had been mutated to reduce its intrinsic MHC class II binding affinity, and the fusion protein was used to demonstrate redirection of T cell cytotoxicity to melanoma cells in vitro. In mice with severe combined immunodeficiency, carrying human melanoma tumours, engraftment of human lymphoid cells followed by treatment with the K305Fab SEA(D227A) fusion protein, induced HMW-MAA-specific tumour growth reduction. The phage-selected K305 antibody demonstrated high-affinity binding and selectivity, supporting its use for tumour therapy in conjunction with T-cell-activating superantigens. PMID- 10752478 TI - Observed localization of the long-term cultured rat adherent natural killer cells in mammary tumor tissues. AB - Adherent natural killer (A-NK) cells were isolated from splenic lymphocytes and treated with long-term culture in the presence of recombinant interleukin-2 (rIL 2). Immunocytochemical and flow-cytometric analysis revealed that most of the A NK cells strongly expressed lymphocyte-function-associated antigen 1 (LFA-1alpha, and LFA-1beta) throughout the incubation. All A-NK cells from 8- to 150-day cultures, particularly those cultured for 8 days, showed significant cytolytic activity against all targets. Analysis of the tissue distribution of the injected [3H] uridine-labeled A-NK cells demonstrated that, in the first 3 h, most (over 60%) cells localized in the lungs, and that most cells remained temporally within the cavities of blood capillaries of the lungs and moved gradually into lymphoid and other tissues. Peritumoral injection of various kinds of adjuvant, particularly Freund's complete adjuvant (FCA) plus bacillus Calmetee-Guerin (BCG), resulted in a marked accumulation of [3H]A-NK cells in mammary tumor tissues 24 h after injection, and simultaneously in the formation of vessels resembling high-endothelium venules (HEV), infiltration of LFA-1+ lymphocytes and expression of the ICAM-1 molecule on the tumor cells in the sites of tumor tissues. When 30 x 10(6) A-NK cells were intravenously administered, significant retardation of tumor growth and prolongation of survival of tumor-bearing rats were observed in the groups that received the prior injection of adjuvants, especially FCA + BCG and Freund's incomplete adjuvant (FIA) + BCG. The suppressive effect of combination therapy on tumor growth was blocked effectively by the injection of anti-ICAM-1 mAb. These results indicate that the prior injection of proper adjuvant into the peritumoral region is effective for the selective accumulation or infiltration of A-NK cells into the sites of tumor tissues, and results in the marked retardation of tumor growth. PMID- 10752479 TI - Quantitation of in vivo measurements with carbon fiber microelectrodes. AB - Fast-scan cyclic voltammetry (FSCV) at carbon fiber disk microelectrodes and quantitative microdialysis were used to measure striatal concentration changes of N-acetyl-p-aminophenol (APAP, acetaminophen) following an intraperitoneal injection of 75 mg/kg APAP in rats. The goal of this work was to determine which in vitro calibration procedure, precalibration or postcalibration, gave the most accurate results when using carbon fiber microelectrodes in vivo. Voltammetric detection of APAP in vivo was complicated with normal electrodes by interference from pH changes. An electrode treatment was used to minimize electrode sensitivity to pH and this allowed successful APAP detection. In vitro calibrations of the treated carbon fiber disk microelectrodes before and after the in vivo experiment were used to calculate APAP concentration changes measured in vivo and compared to microdialysis results. The maximal striatal APAP concentration determined by microdialysis, adjusted for in vitro recovery, was 23.1 microM. The electrochemical results were approximately two times greater (postcalibration) or smaller (precalibration) than the microdialysis result. PMID- 10752480 TI - Fast blue and diamidino yellow as retrograde tracers in peripheral nerves: efficacy of combined nerve injection and capsule application to transected nerves in the adult rat. AB - Capsule application of Diamidino Yellow (DY) to the cut end of the sciatic nerve immediately followed by capsule application of Fast Blue (FB) resulted in approximately 95% double-labelled dorsal root ganglion neurones (DRGn) and motoneurones (Mn). Nerve injection of DY followed either immediately or 2 months later by capsule application of FB resulted in approximately 90% double-labelled DRGn and Mn, indicating that DY and FB label similar populations of DRGn and Mn, and that insignificant DY fading occurred during this period. Inversing the order of application, however, i.e. nerve injection of FB followed immediately by capsule application of DY, resulted in double labelling in only approximately 10% of the DRGn and Mn. These percentages increased to 70% of the DRGn and 60% of the Mn when the FB injection was followed 1 or 2 months after by the DY application, indicating that DY uptake is blocked by recent administration of FB. The results indicate that DY and FB might be useful for sequential labelling before and after nerve injury as a tool to investigate the accuracy of sensory and motor regeneration. PMID- 10752481 TI - Spatial spectral analysis of human electrocorticograms including the alpha and gamma bands. AB - Spatial spectral analysis is essential for deriving spatial patterns from simultaneous recordings of electrocorticograms (ECoG), in order to determine the optimal interval between electrodes in arrays, and to design spatial filters, particularly for extraction of information about the dynamics of human gamma activity. ECoG were recorded from up to 64 electrodes 0.5 mm apart in a linear array 3.2 cm long, which was placed on the exposed superior temporal gyrus or motor cortex of volunteers undergoing diagnostic surgery. Visual displays of multiple traces revealed broad spectrum oscillations in episodic bursts having a common aperiodic wave form with recurring patterns of spatial amplitude modulation (AM patterns) on selected portions of the array. The one-dimensional spatial spectrum of the human ECoG was calculated at successive time samples and averaged over periods of up to 20 s. Log power decreased monotonically with increasing log spatial frequency in cycles/mm (c/mm) to the noise level approximately 2 log units below maximal power at minimal frequency (0.039+/-0.002 c/mm). The inflection point at 0.40+/-0.05 c/mm specified an optimal value for a low pass spatial filter to remove noise, and an optimal interelectrode spacing of 1.25 mm to avoid undersampling and aliasing. An 8 x 8 array with that spacing would be 10 x 10 mm. PMID- 10752482 TI - An efficient, reliable and inexpensive device for the rapid homogenization of multiple tissue samples by centrifugation. AB - Homogenization of tissue samples is a common first step in the majority of current protocols for RNA, DNA, and protein isolation. This report describes a simple device for centrifugation-mediated homogenization of tissue samples. The method presented is applicable to RNA, DNA, and protein isolation, and we show examples where high quality total cell RNA, DNA, and protein were obtained from brain and other tissue samples. The advantages of the approach presented include: (1) a significant reduction in time investment relative to hand-driven or individual motorized-driven pestle homogenization; (2) easy construction of the device from inexpensive parts available in any laboratory; (3) high replicability in the processing; and (4) the capacity for the parallel processing of multiple tissue samples, thus allowing higher efficiency, reliability, and standardization. PMID- 10752483 TI - Quick sex determination of mouse fetuses. AB - We designed a rapid, simple and accurate PCR method to determine sexual identity of mouse fetuses collected on embryonic day 15. A multiplex PCR amplification was used to detect male-specific sequence (Sry) in DNA extracted from fetal livers through SDS denaturation followed by high salt extraction and precipitation. This extraction method resulted in sufficiently purified DNA in < 1 h and was suitable for PCR. The DNA obtained was amplified using a robot thermal cycler for 33 cycles. The reaction was performed in 50 microl, using two sets of primers specific for Sry gene (chromosome Y) and IL3 gene (chromosome 11). Amplification duration was 1.5 h. The assessment of the results was done by electrophoresis in 3% agarose run at high voltage. The 402 bp band (Sry) obtained identifies the male fetuses and the 544 bp product (IL3) confirms the correct amplification of the template DNA. The entire procedure took < 4 h. The specificity of the method was confirmed by fluorescent in situ hybridization using a specific male probe on cultured male and female neural stem cells. This method allowed the preparation and culture of pure male and female neural stem cells from fetal tissue. PMID- 10752484 TI - Body modeling and model-based tracking for neuroethology. AB - The accurate tracking of an animal's movements and postures through time has broad applicability to questions in neuroethology and animal behavior. In this paper we describe methods for precision body modeling and model-based tracking of non-rigid animal movements without the use of external markers. We describe the process of obtaining high-fidelity urethane casts of a model organism, the weakly electric knifefish Apteronotus albifrons, and the use of a stylus-type 3-D digitizer to create a polygonal model of the animal from the cast. We describe the principles behind markerless model-based tracking software that allows the user to translate, rotate, and deform the polygon model to fit it to digitized video images of the animal. As an illustration of these methods, we discuss how we have used model-based tracking in the study of prey capture in nocturnal weakly electric fish to estimate sensory input during behavior. These methods may be useful for bridging between the analytical approaches of quantitative neurobiology and the synthetic approaches of integrative computer simulations and the building of biomimetic robots. PMID- 10752485 TI - Morphological and immunocytochemical characterization of electrophysiologically investigated neurons in the PVN of the rat. AB - This study was carried out to characterize angiotensin II (ANG II) sensitive neurons in the hypothalamic paraventricular nucleus (PVN) of the rat. An approach was chosen in which a combination of an electrophysiological, a morphological, and an immunocytochemical method was focused on one single neuron. The cell's reaction to an application of ANG II and its specific antagonist Losartan (Dup753) was investigated using the technique of intracellular recording inside 450-microm-thick brain slices. A final injection of a fluorescent dye labelled the neurons. Optical sections were taken through the marked cells by a confocal laser-scanning microscope and made into a three-dimensional cell model on a computer. One-micrometer thin sections were cut from the thick slice at the level of the electrophysiologically characterized and marked cell body for immunocytochemical tests with different antibodies. Our results show an example of such a neuron inside the PVN excited by ANG II. It was possible to block this excitation with the specific ANG II receptor subtype 1 (AT1) antagonist Losartan. The result indicated that the ANG II reaction was mediated by the AT1 receptor subtype. Immunocytochemical studies show that this ANG II-sensitive neuron contains ANG II but no vasopressin. The combination of the results enables us to gain improved information on interactions of peptidergic systems. PMID- 10752486 TI - Improved post-embedding immunocytochemistry of myelinated nervous tissue for electron microscopy. AB - The particularly high lipid content of normal mature adult myelin sheaths, together with the light fixation protocols usually necessary to retain antigenicity, combine to make white matter nervous tissue an especially problematical subject for post-embedding immuno-electron microscopy using modern acrylic resins. Fixation and infiltration modifications to standard processing schedules for Lowicryl were found to greatly improve the embedding and therefore the resulting morphology. This in turn improved the signal to noise ratio by reducing the high non-specific backgrounds usually found in poorly infiltrated areas. Using Lowicryl HM20, we have been able to obtain satisfactory immunostaining for myelin basic protein with good retention of structural integrity in the myelin of both normal and lesioned adult cortico spinal tract. PMID- 10752487 TI - Pulmonary function affects the quantification of rCBF by non-invasive xenon methods. AB - Estimates of regional cerebral blood flow (rCBF) by non-invasive xenon methods (133-xenon inhalation, xenon-enhanced computed tomography (Xe/CT) and 133-xenon iv injection) are frequently applied in the diagnosis and evaluation of patients suffering from diseases which cause disturbances in the cerebrovascular circulation. These methods all depend on an estimate of the arterial xenon concentration curve derived non-invasively from measurements of the end-tidal xenon concentration curve and used as brain input function in the Kety equation. We have studied the influence of impaired pulmonary gas exchange on the end-tidal and arterial xenon concentration curves in nine anaesthetized pigs by simultaneously measurements of both the end-tidal xenon and arterial xenon concentration curves. Computer simulations were performed to determine the deviations in the calculated rCBF values when using the end-tidal as compared to the arterial xenon concentration curve as brain input function. The results indicated that impairment of the pulmonary gas exchange caused a significant further 'delay' in the arterial xenon concentration curve in comparison to the end-tidal xenon concentration curve. The time constants of arterial curve delay were 11.9 s in the normal pulmonary group, 21 s in the right lung atelectasis group, and 19.7 s in the left pulmonary artery occlusion group. Accordingly, computer simulations indicated a statistically significant 'underestimation' of rCBF due to: (1) pulmonary gas exchange; (2) high or low levels of rCBF; (3) partition coefficient (lambda) of gray and white matter; and (4) xenon inhalation protocols. Our results indicate that quantitative measurements of rCBF by non invasive xenon methods are markedly affected by deviations between the end-tidal and arterial xenon concentration curve, so that estimates of flow thresholds for infarction are problematic under conditions of impaired pulmonary gas exchange. PMID- 10752488 TI - Unchanged total number of neurons in motor cortex and neocortex in amyotrophic lateral sclerosis: a stereological study. AB - Modern stereological methods provide precise and reliable estimates of the number of neurons in specific regions of the brain. The total number of neurons in the neocortex and motor cortex from eight patients suffering from amyotrophic lateral sclerosis (ALS) and nine controls was estimated. No attempt was made to estimate subpopulations of neurons such as the number of giant pyramidal cells of Betz. No difference was found in the average number of neurons in neocortex in ALS and controls, 21.7 and 22.3 x 10(9), respectively, and 1.33 and 1.29 x 10(9) in motor cortex, respectively. In the light of our stereological measurements, results obtained from in-vivo proton magnetic resonance spectroscopy (1H-MRS), suggesting neuronal loss in ALS, may instead be due to neuronal metabolic dysfunction and/or alteration in the size or the volume fraction of the neurons. PMID- 10752490 TI - What happens to the fibromyalgia concept? PMID- 10752491 TI - Treatment of fibromyalgia with tropisetron, a 5HT3 serotonin antagonist: a pilot study. AB - In this pilot study we investigated 10 women suffering from primary fibromyalgia. All patients received 5 mg of tropisetron in the evening, for a period of 4 weeks. Clinical disease variables included the measurement of a pain score, fatigue, sleep disturbances and measurement of the number of tender points. Five of our patients (50%) showed a statistical clinical improvement of all the above parameters starting after the first week of treatment. Two patients did not respond to the therapy and three discontinued the study because of side-effects. We conclude that administration of tropisetron in fibromyalgia patients could be useful in the management of this difficult and incurable syndrome. PMID- 10752489 TI - Green fluorescent protein as a quantitative tool. AB - Manipulating the expression of a protein can provide a powerful tool for understanding its function, provided that the protein is expressed at physiologically-significant concentrations. We have developed a simple method to measure (1) the concentration of an overexpressed protein in single cells and (2) the covariation of particular physiological properties with a protein's expression. As an example of how this method can be used, teratocarcinoma cells were transfected with the neuron-specific calcium binding protein calretinin (CR) tagged with green fluorescent protein (GFP). By measuring GFP fluorescence in microcapillaries, we created a standard curve for GFP fluorescence that permitted quantification of CR concentrations in individual cells. Fura-2 measurements in the same cells showed a strong positive correlation between CR-GFP fusion protein expression levels and calcium clearance capacity. This method should allow reliable quantitative analysis of GFP fusion protein expression. PMID- 10752492 TI - The effect of melatonin in patients with fibromyalgia: a pilot study. AB - The aim of the study was to determine the possible effect of melatonin treatment on disturbed sleep, fatigue and pain symptoms observed in fibromyalgia (FM) patients. Twenty-one consecutive patients with FM were included in an open 4-week duration pilot study. Before and after treatment with melatonin 3 mg at bedtime, patients were evaluated using tender point count by palpation of 18 classic anatomical regions, pain score in four predesignated areas, pain severity on a 10 cm visual analogue scale (VAS), sleep disturbances, fatigue, depression, anxiety, and patient and physician global assessments, also by a VAS. Urine 6 sulphatoxymelatonin levels (aMT-6S) were measured in the patients and 20 age- and sex-matched controls. Nineteen patients completed the study. One patient withdrew because of migraine and another was lost to follow-up. At day 30, median values for the tender point count and severity of pain at selected points, patient and physician global assessments and VAS for sleep significantly improved with melatonin treatment. Other variables improved but did not reach statistical significance. Adverse events were mild and transient. Lower levels of aMT-6S were found in FM patients compared with normal median controls (+/-SD, 9.16 +/- 7.9 microg/24 h vs 16.8 +/- 12.8 microg/24 h) (p = 0.06). Although this is an open study, our preliminary results suggest that melatonin can be an alternative and safe treatment for patients with FM. Double-blind placebo controlled studies are needed. PMID- 10752493 TI - Levels of rheumatoid factor isotypes, metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 in synovial fluid from various arthritides. AB - When synovial effusion is the only symptom, it is often difficult to make an exact diagnosis of the arthritic disease. To distinguish various types of arthritis with synovial effusion, we measured the levels of matrix metalloproteinase-3 (MMP-3, Stromelysin), tissue inhibitor of metalloproteinase-1 (TIMP-1) and rheumatoid factor (RF) isotypes in synovial fluid (SF) from patients with rheumatoid arthritis (RA), osteoarthritis (OA), pyogenic arthritis (PA), pseudogouty arthritis (PG), gouty arthritis (GA) and traumatic arthritis (TA). SF was aspirated from the knee joint or the ankle joint. Levels of IgG-, IgM- and IgA-RF isotypes were measured by ELISA. Levels of MMP-3 and TIMP-1 in SF were simultaneously determined by a one-step EIA system. Levels of IgG-RF, IgM-RF and MMP-3 in SF from RA patients were significantly higher than those in OA, PA, PG, GA and TA. However, IgA-RF in SF from RA patients, when compared with PA and GA, did not show a significantly increased level. In addition, TIMP-1 in SF from RA, when compared with PA and TA, also has not shown a significantly increased level. Therefore, in addition to analysing clinical data, measurements of IgG-RF, IgM-RF and MMP-3 in SF may contribute in distinguishing RA from other arthritic diseases. PMID- 10752494 TI - How is gout managed in primary care? A review of current practice and proposed guidelines. AB - Twelve practices with a total list of 74,111 patients were audited; 429 patients were identified with a diagnosis of gout. A wide variation in various clinical and laboratory assessments was detected. Similar variations were also noted regarding dietary advice and medical treatment. Monitoring of patients was infrequent. As a result of this audit, guidelines are proposed to improve the diagnosis and management of gout in the community. PMID- 10752495 TI - Prediction of creatinine clearance from serum creatinine in patients with rheumatoid arthritis: comparison of six formulae and one nomogram. AB - The estimation of glomerular filtration rate is important for the medical treatment of patients with rheumatoid arthritis (RA). However, the determination of endogenous creatinine clearance (Clcr) from a 24-h urine collection is an unreliable and time-consuming procedure. We therefore tested the accuracy of six equations and one nomogram for the prediction of Clcr from serum creatinine (Scr) in 38 patients with RA and 20 controls. A positive correlation was found for all methods in the controls (r = 0.83-0.94) and RA patients (r = 0.51-0.69). The methods did not overestimate Clcr in RA. In the RA group the simple formula published by Cockcroft [Clcr = ((140 - age) x body weight)/(72 x Scr), x 0.85 for females] showed the best correlation with the measured Clcr. In RA the Cockroft formula can reliably be used to predict Clcr from Scr. PMID- 10752496 TI - Arthritis impact measurement scales in a community-based rheumatoid arthritis population. AB - The objective of this study was to use the Arthritis Impact Measurement Scales (AIMS) in a community-based rheumatoid arthritis (RA) population to describe the patient population, analyse health status changes and predict survival. The AIMS was assessed in 91 RA patients in a community-based RA population in Kuusamo, Northern Finland. A 5-year follow-up study was performed. The mean AIMS scores in this series of RA patients were comparable to those reported in previous studies. Dexterity correlated with disease duration. Lower extremity function was well preserved and only slightly dependent on disease duration. The AIMS scores changed for the worse in all the subscales over the follow-up period. Of all the AIMS subscale scores, poor functional status, as measured by the AIMS lower extremity function and social activity subscales, best predicted mortality. PMID- 10752497 TI - The differing patterns of subclinical pulmonary involvement in connective tissue diseases as shown by application of factor analysis. AB - To explore common patterns of interstitial lung disease (ILD) in symptomless patients with connective tissue disease (CTD), we applied factorial analysis to determine the relationship among the factors. A selected cohort of 71 non-smoking patients with a confirmed diagnosis of CTD [24 with primary Sjogren's syndrome (pSS), 21 with systemic sclerosis (SS), 20 with rheumatoid arthritis (RA) and six with polymyositis/ dermatomyositis (PM/DM)] were identified. The diagnostic techniques included pulmonary function tests, bronchoalveolar lavage (BAL), chest radiographs and high-resolution computed tomography (HRCT). Disease extent and severity were assessed by a radiological and HRCT grading system. Three factors, accounting for 67% of the total variance, were extracted. The first factor (disease duration, diffusing lung capacity, neutrophils and CD8+ T cells on BAL, radiographic score and HRCT reticular score), with the highest percentage of variance (36.5%), defines a fibrotic lung pattern. The second factor (17.9% of variance) identifies an inflammatory lung pattern (macrophages, lymphocytes and eosinophils on BAL and HRCT ground-glass score). The third factor (12.6% of variance) represents a ventilatory function pattern (forced vital capacity, total lung capacity and forced respiratory volume in 1 s). The negative correlation between the fibrotic lung pattern and ventilatory function pattern, but not with the inflammatory lung pattern, suggests the presence of a significant derangement of the alveolar structures. In conclusion, application of factor analysis reveals various lung disease patterns in patients with CTD that might have different prognostic implications. PMID- 10752498 TI - Autoimmune thyroid diseases in patients with undifferentiated connective tissue disease. AB - Previous data have indicated that organ-specific and non-organ-specific autoimmune diseases may occur in the same patient. We report here our study on the type and prevalence of endocrine autoimmune diseases in undifferentiated connective tissue disease (UCTD). A retrospective analysis revealed five out of 75 UCTD cases (6.6%) with cytology-verified autoimmune thyroiditis (associated with insulin-dependent diabetes mellitus in one case). Other UCTD patients had Graves' disease (one case), non-toxic multinodular goitre (two cases) and central hypothyroidism (one case). In a prospective study, thyroid function was evaluated in 15 consecutive UCTD patients with neither clinical nor laboratory signs of thyroid involvement. Basal and post-TRH stimulation TSH levels were significantly higher in UCTD patients than in healthy subjects. PMID- 10752499 TI - Calcitonin versus clodronate in the prevention of ovariectomy-induced osteopenia in rats. AB - The effects of salmon calcitonin and clodronate were compared in ovariectomised rats. Sixty female Wistar rats ( 260 g in weight) were fed the same diet and had the same living conditions. The rats were divided into the following groups: 15 rats with sham ovariectomy and no drug treatment (Sham-OVX); 45 rats with bilateral ovariectomy subdivided into 15 rats not receiving drug treatment (OVX group), 15 rats treated with subcutaneous salmon calcitonin, 2 U/kg/day every 2 days (OVX + CT group) and 15 rats treated with subcutaneous clodronate, 5 mg/kg/day every 2 days (OVX + Cl group). Sixty days after surgery, the rats were sacrificed and their femurs and fifth lumbar vertebrae were dissected and cleaned of soft tissue. Femur length, vertebral height, and bone mineral content and bone mineral density of the femur and fifth lumbar vertebra by dual-energy X-ray absorptiometry were measured. Calcitonin had a significant and stronger effect in preventing ovariectomy-induced osteopenia in the femur (OVX + CT vs OVX groups, p < 0.0001); both calcitonin and clodronate had a significant effect on the fifth lumbar vertebra, which was greater in the calcitonin group (OVX + CT vs OVX + Cl groups, p<0.005). These findings indicate that calcitonin has a protective effect on both the axial (trabecular bone) and peripheral (cortical bone) skeletons, but clodronate only has a protective effect on the axial skeleton. PMID- 10752500 TI - Imaging of sacroiliitis. AB - Inflammation of one or both sacroiliac joints is a characteristic feature of patients with spondyloarthropathies (SpA). Sacroiliitis often leads to inflammatory back pain (IBP). IBP and asymmetric peripheral arthritis of the lower limbs are the main clinical symptoms and criteria for classification and diagnosis of SpA in which sacroiliac joints are uni- or bilaterally affected with an intensity ranging from mild to very severe inflammation resulting in partial or complete ankylosis Sacroiliitis is a very frequent feature of undifferntiated SpA. In ankylosing spondylitis (AS) inflammation in the axial skeleton occurs rarely in the absence of sacroiliitis. Objective evidence of sacroiliitis obtained by imaging procedures, especially x-rays, has always been part of diagnostic and classification criteria for AS. This is in contrast to spinal radiography which, however, has been recently included in a core set of outcome items to be assessed in clinical studies. In early and acute stages of sacroiliitis the diagnosis can be difficult because conventional radiographs -- which are known to have considerable intra- and interobserver variability -- may be normal. Since IBP is not a specific indicator of sacroiliitis there is need for valuable imaging techniques. Scintigraphy lacks specificity. Computed tomography (CT) is a very good method to demonstrate already established bony changes and magnetic resonance imaging (MRI) has the advantage of combining a good visualisation of the complicated anatomy of the sacroiliac joint with the ability to localise different degrees of inflammation and oedema and prove a possible spread to muscles as it occurs in septic sacroiliitis, an important differential diagnosis. PMID- 10752501 TI - Interferon-alpha-associated monoarthritis. AB - The therapeutic indications for interferons (IFNs) have increased in recent years to include many different diseases of viral and malignant origin. Autoimmune phenomena may occur with IFN therapy, but arthritis is uncommon. In this case report, we describe a patient who developed monoarthritis in the knee after IFN alpha therapy for chronic hepatitis B infection. A synovial fluid assay by polymerase chain reaction was negative for hepatitis B virus DNA and serum transaminase levels were normal as the arthritis developed. The patient was thought to have arthritis due to IFN-alpha treatment. A connection between IFN alpha therapy and an induced autoimmune disorder is discussed. PMID- 10752502 TI - Filtration of cerebrospinal fluid for acute demyelinating neuropathy in systemic lupus erythematosus. AB - We report a patient with systemic lupus erythematosus complicated by an acute demyelinating neuropathy. Conventional therapy with intravenous immunoglobulins and immunoadsorption complemented by pulse methylprednisolone and cyclophosphamide failed. Institution of filtration of the cerebrospinal fluid was followed by a rapid improvement of the paresis. PMID- 10752504 TI - Gouty arthritis in a female patient with mixed connective tissue disease. AB - Gout with systemic lupus erythematosus (SLE) or progressive systemic sclerosis (PSS) has rarely been reported, whereas mixed connective tissue disease (MCTD) with the demonstration of intra-articular monosodium urate crystals has never been reported. We describe an unusual case of MCTD (SLE-PSS) in a 37-year-old woman who developed acute gouty arthritis. Arthrocentesis and synovianalysis may be necessary to differentiate gout from the arthropathy of MCTD. PMID- 10752503 TI - High bone mass and hypocalcaemic myopathy in a patient with idiopathic hypoparathyroidism. AB - The clinical manifestations of hypoparathyroidism are mainly characterised by increased neuromuscular irritability as a consequence of hypocalcaemia. Occasionally, elevation of the muscle enzymes may mimic polymyositis. Reduced parathyroid hormone production, but also vitamin D treatment and calcium supplementation, may contribute to the increased bone mass found in patients with postsurgical hypoparathyroidism. We report the case of a 36-year-old woman with untreated idiopathic hypoparathyroidism and a high bone mass despite severe muscle impairment due to hypocalcaemic myopathy. PMID- 10752505 TI - Dysphagia in a patient with giant osteophytes: case presentation and review of the literature. AB - A patient with increasing dysphagia due to external bone compression of the oesophagus is presented. Radiographic evaluation revealed the underlying condition to be a diffuse idiopathic skeletal hyperostosis with exuberant and bumpy change within the anterior longitudinal ligament. PMID- 10752506 TI - Giant cell arteritis with an erythrocyte sedimentation rate lower than 50. AB - A high erythrocyte sedimentation rate (ESR) is considered to be a hallmark of giant cell arteritis (GCA) and one of the 1990 American College of Rheumatology classification criteria. We studied 248 patients with GCA to assess the frequency and clinical features of patients with GCA and an ESR <50 mm/h. Ten patients had a low ESR (43.1 +/- 4.9 mm/h) and in the remaining 238, the ESR was > or = 50 mm/h (96.4 +/-23.6). None of the patients with an ESR less than 50 had a completely normal ESR. The spectrum of the disease in both groups was very similar. Both groups required similar corticosteroid therapy and had a similar outcome. The ESR, analysed as a continuous variable, showed a significant positive correlation with other parameters reflecting the acute-phase response such as presence of anaemia, weight loss and fever. We suggest that in patients with a clinical picture compatible with GCA, the use of an ESR > or = 30 mm/h as the main laboratory parameter to consider the possibility of GCA would be enough to arise the suspicion and prevent cases of GCA being missed. PMID- 10752508 TI - Systemic adverse effect of antithyroid drugs. PMID- 10752507 TI - Serum chondrex values in knee osteoarthritis (OA). The effect of arthroscopy. PMID- 10752509 TI - Gonadal cryopreservation in the young patient with gynaecological malignancy. AB - For patients who are planning to have chemotherapy, radiotherapy or to undergo bilateral oophorectomy, the loss of ovarian function will result in premature ovarian menopause and loss of fertility. Embryo preservation is not an option for single women or married women because delaying treatment for at least 2 months of in-vitro fertilization cycles is inappropriate and may be life-threatening. This study reports on the indications for ovarian tissue cryobanking and the state of the art of this method in preserving fertility in women with iatrogenic premature menopause. PMID- 10752510 TI - Role of routine human papillomavirus subtyping in cervical screening. AB - Human papillomavirus testing has been evaluated as an adjunct to cervical cytologic screening. There is a well-established association between high-risk malignant subtypes of human papillomavirus infection and the development of neoplastic change. Although human papillomavirus infection is a necessary condition for malignant transformation, it is not a sufficient condition. Multiple cofactors are necessary for progression to dysplasia and then to invasive cervical cancer. Because of the multifactorial nature of malignant transformation, current methods of human papillomavirus testing do not add sufficient predictive and diagnostic information over that obtained by cytology and colposcopic biopsy to warrant routine clinical use. PMID- 10752512 TI - Quality of life as an outcome measure in gynecologic malignancies. AB - Health-related quality of life instruments for cancer patients and specifically for gynecologic cancer patients have been developed, validated and applied in clinical trials. More focused investigations of specific symptoms, side-effects and both short- and long-term toxicities are also being pursued. Assessments are now included as part of many cancer treatment trials and some studies are looking at quality of life in survivors. Another area of investigation seeks to define the types of stress and coping mechanisms being used in order to identify predictors of psychologic distress. PMID- 10752511 TI - Innovations in the management of vulvar carcinoma. AB - Radical surgery has resulted in impressive cure rates in women with locally advanced vulvar carcinoma. Unfortunately, morbidity mostly related to inguinofemoral lymphadenectomy, is common. The present review discusses innovations in the management of vulvar disease with attempts to reduce attendant morbidity. PMID- 10752513 TI - Surveillance of the endometrium in tamoxifen treated women. AB - There is much debate about the risks and benefits of tamoxifen, most specifically about the incidence of associated endometrial cancer. Nearly all of the published trials on the subject have been criticized for methodological flaws and various forms of bias, making resolution of this controversy difficult. There is a consensus, however, that tamoxifen results in an increased incidence of both premalignant and malignant lesions of the endometrium. As the indications for tamoxifen continue to broaden, a larger number of women will be subjected to the potential adverse effects of tamoxifen. Many techniques for screening patients on tamoxifen for the development of endometrial abnormalities have been suggested. None of these methods appears to be consistently clinically or cost effective. We have reviewed the literature on endometrial surveillance in tamoxifen treated women with a focus on the larger publications reported within the past year. From this, we have provided what we hope to be safe and cost-effective recommendations for the management of these patients. PMID- 10752514 TI - Atypical glandular cells of undetermined significance Pap smears: appropriate evaluation and management. AB - This review focuses on atypical glandular cells of undetermined significance Pap smears, with attention given to the classifications of atypical glandular cells of undetermined significance, the clinical and histopathologic correlation, and recent advances in triage strategies for this entity. The management of atypical glandular cells of undetermined significance Pap smears will emphasize the significance of this finding in terms of diagnosing dysplasia and carcinoma of the lower female genital tract. PMID- 10752515 TI - Recent developments in ovarian cancer screening. AB - There has been growing interest in the possibility of screening for ovarian cancer. This article addresses papers published following a systematic review of all prospective ovarian cancer screening studies since 1998. In the past year, new markers have been reported and previous strategies have been refined. A randomized controlled trial of ovarian cancer screening has shown a survival benefit in women who developed ovarian cancer in the screened group. Although the results do not justify ovarian cancer screening in the general population, the data support the need for a larger randomized trial powered to assess the impact of screening on mortality. PMID- 10752517 TI - Bibliography. Current world literature. Gynecologic oncology and pathology. PMID- 10752516 TI - Reproductive technologies and risk of ovarian cancer. AB - Use of artificial technologies is increasing within the United States. Unfortunately, data describing the possible effects of 'superovulation' and potential risk of subsequent ovarian carcinoma are not well defined. This discussion focuses on current data regarding the potential for increased risk for ovarian carcinoma in patients who have undergone infertility evaluation and subsequent treatments. PMID- 10752518 TI - Identification, cellular distribution and potential function of the metalloprotease-disintegrin MDC9 in the kidney. AB - The complex interactions of glomerular and tubular epithelial cells with the basal laminae play a critical role in renal function. Disruption of these interactions has been widely implicated in glomerular diseases and acute renal failure. MDC are a large family of membrane-bound proteins containing metalloprotease, disintegrin (integrin interaction sites), and cysteine-rich domains. Little information is available concerning the presence of MDC in the kidney or their role in renal pathophysiology. Using degenerate PCR primers for the conserved metalloprotease and disintegrin domains of this protein family, cDNA templates from tubules, whole glomeruli, and glomerular epithelial cells (GEC) yielded a single, 195-bp product, which on sequence analysis corresponded to a region in the disintegrin domain of MDC9. Northern analysis of poly(A)+ RNA from tubules, whole glomeruli, and GEC revealed a 3.9-kb transcript, identical to that of mouse MDC9. Using antibodies generated against a 21-amino acid peptide present in the metalloprotease domain of MDC9, Western analysis of concanavalin A enriched glomerular microsomal extracts demonstrated both processed (76 kD) and unprocessed (116 kD) forms of MDC9, which upon reduction changed to the corresponding 84- and 124-kD forms. Histochemical studies revealed a basolateral localization of intrinsic MDC9 protein in renal cortical tubule cells and glomerular visceral epithelial cells, which colocalized with the beta1 integrin chain. Expression of green fluorescence protein MDC9 chimeric constructs in GEC or polarized Madin-Darby canine kidney epithelial cells revealed a similar punctate basolateral surface localization. Transient overexpression of the soluble disintegrin domain-green fluorescence protein chimera in GEC led to dramatic changes in cellular morphology with rounding and detachment from cell monolayers. These studies document the presence of MDC9 in renal epithelial cells and suggest an important role for MDC9 in renal epithelial cellular interactions with the basal lamina and adjoining cells. PMID- 10752519 TI - Collecting duct (Na+/K+)-ATPase activity is correlated with urinary sodium excretion in rat nephrotic syndromes. AB - In puromycin aminonucleoside (PAN)-treated nephrotic rats, sodium retention is associated with increased (Na+/K+)-ATPase activity in the cortical collecting ducts (CCD). This study was undertaken to determine whether stimulation of (Na+/K+)-ATPase in the CCD is a feature of other experimental nephrotic syndromes, whether it might be responsible for renal sodium retention, and whether it is mediated by increased plasma vasopressin levels or activation of calcineurin. For this purpose, the time courses of urinary excretion of sodium and protein, sodium balance, ascites, and (Na+/K+)-ATPase activities in microdissected CCD were studied in rats with PAN or adriamycin nephrosis or HgCl2 nephropathy. The roles of vasopressin and calcineurin in PAN nephrosis were evaluated by measuring these parameters in Brattleboro rats and in rats treated with cyclosporin or tacrolimus. Despite different patterns of changes in urinary sodium and protein excretion in the three nephrotic syndrome models, there was a linear relationship between CCD (Na+/K+)-ATPase activities and sodium excretion in all three cases. The results also indicated that there was no correlation between proteinuria and sodium retention, but ascites was present only when proteinuria was associated with marked reduction of sodium excretion. Finally, the lack of vasopressin in Brattleboro rats or the inhibition of calcineurin by administration of either cyclosporin or tacrolimus did not prevent development of the nephrotic syndrome in PAN-treated rats or stimulation of CCD (Na+/K+)-ATPase. It is concluded that stimulation of Na(+/K+)-ATPase in the CCD of nephrotic rats might be responsible for sodium retention and that this phenomenon is independent of proteinuria and vasopressin and calcineurin activities. PMID- 10752520 TI - Locally activated renin-angiotensin system associated with TGF-beta1 as a major factor for renal injury induced by chronic inhibition of nitric oxide synthase in rats. AB - Chronic inhibition of nitric oxide synthase (NOS) is known to cause renal parenchymal injury with systemic hypertension. To elucidate the pathogenetic mechanism in renal damage induced by NOS inhibition, N(omega)-nitro-L-arginine methyl ester (L-NAME) was given orally for 12 wk in Wistar rats, and the roles of tissue renin-angiotensin system and transforming growth factor-beta1 (TGF-beta1) were investigated. BP and urinary protein excretion increased significantly in L NAME rats compared with control rats, and glomerulosclerosis and interstitial fibrosis developed. In L-NAME rats, the cortical tissue levels of angiotensin converting enzyme activity and angiotensin II were significantly higher than those in control rats. The cortical mRNA expressions of both TGF-beta1 and fibronectin were significantly elevated in L-NAME rats. Immunohistochemically, increased expressions of both fibronectin and alpha-smooth muscle actin were also revealed in L-NAME rats. In L-NAME rats, these histologic injuries and the increased expression of TGF-beta1 were equally ameliorated by either angiotensin converting enzyme inhibitor or angiotensin II type 1 receptor antagonist, but not by hydralazine. In conclusion, the locally activated renin-angiotensin system in connection with the increased TGF-beta1 expression is a major pathogenetic feature of renal injury in chronically NOS-inhibited rats. PMID- 10752521 TI - Role of dietary potassium in the hyperaldosteronism and hypertension of the remnant kidney model. AB - The remnant kidney model of progressive renal disease is marked by arterial hypertension, especially when produced by nephrectomy and partial infarction. Hyperaldosteronism sustains much of the hypertension, but the stimuli to the increased aldosterone levels are uncertain. It is hypothesized that the hyperaldosteronism attending this model stems from the combination of fixed dietary potassium load in the face of reduced filtration on the one hand, and persistent renin secretion from the scarred remnant kidney on the other. This hypothesis predicted that dietary potassium restriction would lower aldosterone and BP in this model. To test this prediction, two groups of rats with a remnant kidney were studied. Group 1 consumed 0.4 +/- 0.06 mEq (mean +/- SD) of potassium chloride daily, and group 2 ate 4.8 +/- 1.0 mEq daily. Two sham-operated groups with intact kidneys also were studied. Group 3 consumed 1.7 +/- 0.2 mEq daily and group 4 ate 15.2 +/- 1.4 mEq daily. These levels of intake were designed to provide at least as much potassium per liter of GFR in the sham groups as in the remnant kidney rats. Systolic BP (SBP), 24-h protein excretion, plasma aldosterone levels, 24-h urinary aldosterone excretion, and plasma renin activity (PRA) were determined in all groups at 2 wk. At 4 wk, after SBP and protein excretion measurements, remnant kidneys were perfusion-fixed for morphometric analysis. SBP was normal in both sham-operated groups and was not different between the groups (113 +/- 13 versus 117 +/- 2 mmHg, group 3 versus group 4). In the remnant animals, SBP at 2 wk followed potassium intake: Group 1 had a lower SBP than group 2 (140 +/- 26 versus 170 +/- 34 mmHg, P = 0.005). The same SBP pattern persisted at 4 wk (153 +/- 25 versus 197 +/- 27 mmHg, group 1 versus group 2, P = 0.0006). However, 24-h urinary protein excretion was not different between the two groups with remnant kidneys at either 2 or 4 wk. Both plasma and 24-h urinary aldosterone excretion at 2 wk followed potassium intake (120 +/- 124 versus 580 +/- 442 pg/ml for plasma aldosterone, group 1 versus group 2, P = 0.03, and 2.6 +/- 1.8 versus 23.2 +/-9.8 ng/d for urinary aldosterone, group 1 versus group 2, P = 0.0001). PRA, however, followed a reverse pattern in which dietary potassium restriction resulted in higher levels (16 +/- 6 versus 6 +/- 3 ng angiotensin I/ml per h, group 1 versus group 2, P = 0.01). A similar pattern for PRA and aldosterone excretion was also observed in the sham groups, in which lower potassium intake also resulted in a significantly higher PRA and lower aldosterone excretion. The constancy of BP in the sham groups likely reflects their lack of nephron reduction and greater sodium excretory capacity. Morphometric analysis in remnant animals revealed no significant difference between the two dietary groups in the prevalence of glomerular sclerosis, glomerular volume, or interstitial volume. It is concluded that dietary potassium is a potent determinant of hypertension in the remnant kidney model probably through the actions of aldosterone and that the high aldosterone secretion in this model is a function of the dietary potassium load. In this model, reduction in nephron number is also critical in promoting hypertension in conjunction with hyperaldosteronism. PMID- 10752522 TI - Characterization and localization of the neonatal Fc receptor in adult human kidney. AB - The binding of Fc fragments of Ig on glomerular epithelial cells (GEC) was observed previously, but the receptor could not be identified. In immunofluorescence and immunohistochemical studies using normal adult human kidney sections, the presence of the so-called neonatal Fc receptor (FcRn) was demonstrated on GEC as well as in the brush border of proximal tubular cells. FcRn transcripts were also detected on isolated glomeruli by reverse transcription-PCR. Using an immortalized GEC line, the presence of the FcRn was confirmed by flow cytometry, reverse transcription-PCR, Western blotting, and by the pH dependence of the binding of heat-aggregated IgG. Because it is well established that the FcRn is involved in IgG transcytosis, it is hypothesized that the FcRn in the kidney may play a role in the reabsorption of IgG. Ongoing studies should clarify the role of the FcRn as a potential target for immune complexes on GEC and should assess its relevance in physiology and pathology. PMID- 10752523 TI - Proteinase 3 interacts with a 111-kD membrane molecule of human umbilical vein endothelial cells. AB - Proteinase 3 (PR3) is the major autoantigen of antineutrophil cytoplasmic antibodies in Wegener's granulomatosis. Previously, it was demonstrated that PR3 induces apoptosis of human endothelial cells and that PR3 contributes to endothelial cell activation by enhancing interleukin-8 production. The present study demonstrates that PR3 binds specifically to human umbilical vein endothelial cells (HUVEC). Digoxigenin (DIG)-labeled PR3 bound readily to HUVEC cultured on coverslips. By fluorescence-activated cell sorter analysis, a homogeneous binding of PR3 to HUVEC, using either DIG-labeled or unlabeled PR3, was observed. No detectable membrane expression of PR3 was observed after either tumor necrosis factor-alpha stimulation or in nonstimulated HUVEC. The binding of PR3-DIG to HUVEC was dose-dependent and was inhibited by unlabeled PR3. Scatchard analysis revealed 2000 binding sites per cell, with a Kd of 0.1 microM. Affinity precipitation of biotin-labeled HUVEC membrane proteins with protein G-Sepharose bearing PR3 resulted in specific precipitation of a membrane molecule with a molecular weight of 111 kD under nonreducing conditions and 52 and 63 kD under reducing conditions. It is hypothesized that PR3, either released systemically or locally at inflammatory sites following activation of primed polymorphonuclear neutrophils, may lead to endothelial cell injury and activation of endothelial cells. PMID- 10752524 TI - X-linked Alport syndrome: natural history in 195 families and genotype- phenotype correlations in males. AB - Alport syndrome (AS) is a type IV collagen hereditary disease characterized by the association of progressive hematuric nephritis, hearing loss, and, frequently, ocular changes. Mutations in the COL4A5 collagen gene are responsible for the more common X-linked dominant form of the disease. Considerable allelic heterogeneity has been observed. A "European Community Alport Syndrome Concerted Action" has been established to delineate accurately the AS phenotype and to determine genotype-phenotype correlations in a large number of families. Data concerning 329 families, 250 of them with an X-linked transmission, were collected. Characteristics of the 401 male patients belonging to the 195 families with COL4A5 mutation are presented. All male patients were hematuric, and the rate of progression to end-stage renal failure and deafness was mutation dependent. Large deletions, non-sense mutations, or small mutations changing the reading frame conferred to affected male patients a 90% probability of developing end-stage renal failure before 30 yr of age, whereas the same risk was of 50 and 70%, respectively, in patients with missense or splice site mutation. The risk of developing hearing loss before 30 yr of age was approximately 60% in patients with missense mutations, contrary to 90% for the other types of mutations. The natural history of X-linked AS and correlations with COL4A5 mutations have been established in a large cohort of male patients. These data could be used for further evaluation of therapeutic approaches. PMID- 10752526 TI - Early mechanisms of renal injury in hypercholesterolemic or hypertriglyceridemic rats. AB - Hyperlipidemia in conjunction with uninephrectomy leads to renal injury in rats. It is unknown whether this is due to mesangial cell or podocyte injury and whether the injuries induced by hypercholesterolemia and hypertriglyceridemia share a similar pathogenesis. Therefore, renal effects of hypercholesterolemia were studied in male rats with dietary hypercholesterolemia compared with rats on a regular diet. Renal effects of hypertriglyceridemia were studied in female Nagase analbuminemic rats (NAR). Hypertriglyceridemia was reduced in NAR by ovariectomy. Both models were studied after uninephrectomy or sham operation. Dietary hypercholesterolemia had little effect on plasma triglycerides, whereas ovariectomy in the NAR had no effect on plasma cholesterol. However, an increase in intermediate density lipoprotein cholesterol was common to both models. Dietary hypercholesterolemia and uninephrectomy separately induced a similar increase in proteinuria after 13 wk, which was additive when these interventions were combined. At this stage, only a minimal increase was present in glomerular alpha-smooth muscle actin staining, a marker of mesangial cell activation, or in mesangial matrix expansion. Moreover, platelet-derived growth factor-B chain, a marker of mesangial cell proliferation, was not increased. However, podocyte injury was prominent as evidenced by podocytic de novo expression of desmin and ultrastructural changes. Glomerular macrophage counts were increased by hypercholesterolemia but not by uninephrectomy, and were not related to the level of proteinuria. Hypertriglyceridemia and uninephrectomy in female NAR induced an increase in proteinuria after 24 wk, which was also associated with an increase in podocyte desmin expression without any mesangial activation and proliferation or matrix accumulation. Hypertriglyceridemia, proteinuria, and the increase in desmin staining were largely prevented by ovariectomy. Interstitial myofibroblast activation and tubulointerstitial injury accompanied proteinuria in both models. These findings indicate that both hypercholesterolemia and hypertriglyceridemia aggravate renal injury primarily via podocyte rather than via mesangial cell damage. Such podocyte injury is accompanied by tubulointerstitial cell activation and injury. PMID- 10752525 TI - Identification of two alternatively spliced forms of human tubulointerstitial nephritis antigen (TIN-Ag). AB - Tubulointerstitial nephritis antigen (TIN-Ag) is a recently described basement membrane glycoprotein reactive with autoantibodies in some forms of immunologically mediated human tubulointerstitial nephritis. This report presents the complete cDNA and predicted amino acid sequences of two human TIN-Ag mRNA species referred to as TIN1 and TIN2. Translation through the open reading frames of these clones indicates the presence of a signal peptide and putative pre propeptide. TIN1 additionally contains a characteristic laminin-like epidermal growth factor (EGF) motif and significant homology within the carboxy terminus with the cysteine proteinase family of enzymes. The EGF motif bears important similarities in the positions of cysteines with two motifs in the propeptide of von Willebrand factor. The EGF motif and part of the region that is homologous with the cysteine proteinase family are removed from the TIN2 cDNA. However, the rest of the sequence is identical in these two forms, indicating an alternatively spliced TIN-Ag mRNA product. Both forms contain putative calcium-binding sites. Secondary structure predictions strongly suggest differences between TIN1 and TIN2 leading to the hypothesis that these two forms of TIN-Ag may exhibit differences in their function. Expression studies with appropriate probes demonstrate expression mainly in the kidney and in the intestinal epithelium and lack of expression in other tissues. In the kidney, both TIN1 and TIN2 transcripts are detected, however, TIN1 appears to be the predominant form. PMID- 10752527 TI - Inhibition of neutrophil superoxide production by uremic concentrations of guanidino compounds. AB - In uremia, diminished reactive oxygen intermediate production is an important consequence of impaired neutrophil function. The effects of guanidino compounds, which are known uremic toxins, on neutrophil reactive oxygen intermediate production in vitro were studied. Neutrophils from healthy volunteers were exposed for 3 h to individual guanidino compounds or mixed guanidino compounds (GCmix), at concentrations observed in uremic plasma. After removal of the guanidino compounds, the neutrophils were activated by adhesion, N formylmethionylleucylphenylalanine, phorbol myristate acetate, or opsonized zymosan, and superoxide production was measured by monitoring lucigenin chemiluminescence. The direct effects of guanidino compounds on superoxide production in activated neutrophils were also measured. The energy status (ATP and creatine phosphate), antioxidant status (total glutathione), and glycolytic flux (lactate production) were measured. GCmix pretreatment decreased superoxide production in activated neutrophils (activated by N formylmethionylleucylphenylalanine or zymosan) by 50% (P < 0.01), decreased ATP concentrations by 60% (P < 0.05), and inhibited glycolytic flux (lactate production) by 45% (P < 0.01) but did not alter glutathione concentrations. Simultaneous GCmix exposure and activation did not inhibit NADPH oxidase activity in cell lysates but inhibited superoxide formation in zymosan-activated intact neutrophils; this inhibition was reversed after removal of the guanidino compounds. Guanidinosuccinic acid, guanidinopropionic acid, and guanidinobutyric acid, when tested individually, were each as potent as GCmix. The inhibition of neutrophil superoxide generation by guanidino compounds results from decreased energy status. Micromolar concentrations of guanidino compounds significantly inhibit neutrophil metabolism, with serious implications for the functions of neutrophils in host defenses. PMID- 10752528 TI - High prevalence and adverse effect of hepatitis C virus infection in type II diabetic-related nephropathy. AB - Over a 4-yr period in the northeast region of Japan (Tohoku), 3643 patients for whom a renal biopsy was available were screened. In addition, 2370 biopsied patients for whom hepatitis C virus (HCV) serology was available were evaluated. The prevalence of HCV infection was investigated in the 2370 biopsied patients. The highest prevalence of HCV infection was found in type II diabetic-related glomerulosclerosis (II-DGS) (24 of 123; 19.5%). At renal biopsy, clinical and laboratory findings and histologic parameters were comparable between the HCV positive and -negative II-DGS groups. After renal biopsy, the decline of renal function reflected by the slope of reciprocal serum creatinine (1/S(Cr)) was significantly greater in the HCV-positive group than in the HCV-negative group (P = 0.001). The log-rank test performed on the renal survival curves showed a significant difference in the two groups (P = 0.019). According to a multiple linear regression analysis adjusted for the effect of age, gender, BP, HbA1c, urinary protein excretion, and histologic parameters as covariates, urinary protein excretion (P = 0.011), severe arteriolar hyalinosis (P = 0.006), and HCV infection (P < 0.001) were significantly associated with 1/S(Cr) slope. Finally, HCV infection was randomly examined in 545 outpatients and inpatients with type II diabetes mellitus who did not undergo renal biopsy. Of these, 56 patients were positive for HCV antibody (10.3%), and their proteinuria was heavier than in 489 HCV-negative patients (P = 0.001). This study reveals that HCV infection is present at a high rate in type II diabetic-related nephropathy and may have an adverse effect on the progression of the disease. PMID- 10752529 TI - Complement activation products in the urine from proteinuric patients. AB - The presence of plasma proteins in the tubular lumen has variety of adverse effects on the tubular cells. Among various plasma proteins filtered through glomerular barrier, complement has been proven as the possible candidate inducing tubulointerstitial injury. To study the role of intratubular complement activation in proteinuric patients, complement activation products (CAP) at C3 level (iC3b and Bb) and C9 level (membrane attack complex) were measured in both plasma and urine of patients with minimal change nephrotic syndrome (MCNS), focal glomerular sclerosis, IgA nephropathy, membranous nephropathy, and diabetic nephropathy. For evaluation of the effect of metabolic acidosis on the intratubular complement activation, urinary CAP were measured before and after sodium bicarbonate administration in patients with renal insufficiency. The following results were obtained: (1) Patients with focal glomerular sclerosis and diabetic nephropathy showed the highest level of urinary CAP excretion rate (unit/creatinine), while MCNS revealed no increase. (2) Patients with membranous nephropathy showed a unique finding, i.e., isolated increase of membrane attack complex excretion. (3) There was no significant correlation between urine and plasma levels of CAP. (4) Except for MCNS patients, the urinary excretion rate of CAP significantly increased when the level of proteinuria exceeded the nephrotic range, and it was significantly correlated with the serum creatinine level. (5) Urinary CAP excretion rate significantly decreased 2 wk after sodium bicarbonate administration without affecting the level of proteinuria or plasma CAP. These results suggest that the degree of intratubular complement activation correlates with the level of proteinuria, type of glomerular disease, impairment of renal function, and metabolic acidosis. PMID- 10752530 TI - Nutrition and outcome on renal replacement therapy of patients with chronic renal failure treated by a supplemented very low protein diet. AB - Protein-restricted diets are prescribed in patients with chronic renal failure (CRF) to alleviate uremic symptoms and to slow the progression of CRF. The potential deleterious effects of protein restriction on nutritional status and clinical outcome of patients with CRF have raised concern. In this study, data were collected from 1985 to 1998 on 239 consecutive patients (age 50.2 +/- 15.6 yr) with advanced CRF (GFR 13.1 +/- 4.8 ml/min) to whom a supplemented very low protein diet (SVLPD) providing 0.3 g protein, 35 kcal, and 5 to 7 mg of inorganic phosphorus per kg per day was administered for a mean duration of 29.6 +/- 25.1 mo. The diet was supplemented with essential amino acids and ketoanalogs, calcium carbonate, iron, and multivitamins. During SVLPD, protein intake decreased from 0.85 +/- 0.23 to 0.43 +/- 0.11 g/kg per d, and body mass index and serum albumin concentration remained unchanged overall. Fourteen patients died during SVLPD; death was unrelated to nutritional parameters. Hemodialysis was initiated after SVLPD in 165 patients at a mean GFR of 5.8 +/-1.5 ml/min. During an average of 54 mo on hemodialysis, mortality was low (2.4% after 1 yr) and correlated to age only, not to nutritional parameters observed at the end of SVLPD. Similar results were obtained in 66 transplanted patients (12 were not dialyzed before transplantation). SVLPD can be safely used in patients with CRF without adverse effects on the clinical and nutritional status of the patients. Due to the preservation of nutritional status and the correction of uremic symptoms, the initiation of dialysis was deferred in these patients. The outcome of patients on renal replacement therapy is not affected by prior treatment with SVLPD during the predialysis phase of CRF. PMID- 10752531 TI - Vascular proliferation and enhanced expression of endothelial nitric oxide synthase in human peritoneum exposed to long-term peritoneal dialysis. AB - Long-term peritoneal dialysis (PD) is associated with alterations in peritoneal permeability and loss of ultrafiltration. These changes originate from increased peritoneal surface area, but the morphologic and molecular mechanisms involved remain unknown. The hypothesis that modifications of activity and/or expression of nitric oxide synthase (NOS) isozymes might play a role in these modifications, via enhanced local production of nitric oxide, was tested in this study. NOS activities were measured by the L-citrulline assay in peritoneal biopsies from seven control subjects, eight uremic patients immediately before the onset of PD, and 13 uremic patients on short-term (<18 mo, n = 6) or long-term(>18 mo, n = 7) PD. Peritoneal NOS activity is increased fivefold in long-term PD patients compared with control subjects. In uremic patients, NOS activity is positively correlated with the duration of PD. Increased NOS activity is mediated solely by Ca(2+)-dependent NOS and, as shown by immunoblotting, an upregulation of endothelial NOS. The biologic relevance of increased NOS in long-term PD was demonstrated by enhanced nitrotyrosine immunoreactivity and a significant increase in vascular density and endothelial area in the peritoneum. Immunoblotting and immunostaining studies demonstrated an upregulation of vascular endothelial growth factor (VEGF) mostly along the endothelium lining peritoneal blood vessels in long-term PD patients. In the latter, VEGF colocalized with the advanced glycation end product pentosidine deposits. These data provide a morphologic (angiogenesis and increased endothelial area) and molecular (enhanced NOS activity and endothelial NOS upregulation) basis for explaining the permeability changes observed in long-term PD. They also support the implication of local advanced glycation end product deposits and liberation of VEGF in that process. PMID- 10752532 TI - Effect of glucose degradation products on human peritoneal mesothelial cell function. AB - Bioincompatibility of conventional glucose-based peritoneal dialysis fluids (PDF) has been partially attributed to the presence of glucose degradation products (GDP) generated during heat sterilization of PDF. Most previous studies on GDP toxicity were performed on animal and/or transformed cell lines, and the impact of GDP on peritoneal cells remains obscure. The short-term effects of six identified GDP on human peritoneal mesothelial cell (HPMC) functions were examined in comparison to murine L929 fibroblasts. Exposure of HPMC to acetaldehyde, formaldehyde, glyoxal, methylglyoxal, furaldehyde, but not to 5 hydroxymethyl-furfural, resulted in dose-dependent inhibition of cell growth, viability, and interleukin-1beta (IL-1beta)-stimulated IL-6 release; for several GDP, this suppression was significantly greater compared with L929 cells. Although the addition of GDP to culture medium at concentrations found in PDF had no major impact on HPMC function, the exposure of HPMC to filter-sterilized PDF led to a significantly smaller suppression of HPMC proliferation compared to that induced by heat-sterilized PDF. The growth inhibition mediated by filter sterilized PDF could be increased after the addition of clinically relevant doses of GDP. These effects were equally evident in L929 cells. In conclusion, GDP reveal a significant cytotoxic potential toward HPMC that may be underestimated in test systems using L929 cells. GDP-related toxicity appears to be particularly evident in experimental systems using proliferating cells and the milieu of dialysis fluids. Thus, these observations may bear biologic relevance in vivo where HPMC are repeatedly exposed to GDP-containing PDF for extended periods of time. PMID- 10752533 TI - Hospital utilization among chronic dialysis patients. AB - Factors driving inpatient and outpatient utilization were studied among patients who began dialysis for chronic renal failure at the New England Medical Center (NEMC) between 1992 and 1997. Clinical, laboratory, and hospital resource utilization data were obtained from patient records and electronic databases. There were 2.2 hospitalizations and 14.8 hospital days per patient year at risk (PYAR). The number of hospitalizations and hospital days per PYAR were higher in the first 3 mo of initiating dialysis (4.3 and 28.3, respectively) compared to after 3 mo (1.9 and 12.9, respectively). Factors associated with increased risk of hospital days within the first 3 mo included non-health maintenance organization insurance, ischemic heart disease, late referral to the nephrologist, and use of temporary vascular access for the first dialysis. Patients with ischemic heart disease and who received dialysis during the years 1992-1994 compared with 1996-1997 had an increased risk of hospital days after 3 mo of initiating dialysis. There were 16.6 outpatient visits per PYAR, with significant differences in utilization between the first 3 mo and after 3 mo of initiating dialysis. Thus, hospital utilization was significantly higher in the first 3 mo compared to after 3 mo, and factors associated with hospital utilization depended on duration of dialysis. In particular, delayed referral to the nephrologist and lack of permanent vascular access were independently associated with increased risk of hospital utilization in the first 3 mo of dialysis. Greater attention to timely referral to the nephrologist and timely placement of vascular access could result in reduced utilization and cost savings. PMID- 10752535 TI - Cigarette smoking in renal transplant recipients. AB - Cigarette smoking increases the risk for cancer and cardiovascular disease in the general population, but the effects of smoking in renal transplant recipients are unknown. The effects of smoking were investigated among patients transplanted at Hennepin County Medical Center between 1963 and 1997. Information on smoking was available in 1334 patients. The 24.7% prevalence of smoking at the time of transplantation was similar to that in the general population. After adjusting for multiple predictors of graft failure, smoking more than 25 pack-years at transplantation (compared to smoking less than 25 pack-years or never having smoked) was associated with a 30% higher risk of graft failure (relative risk 1.30; 95% confidence interval [CI], 1.04 to 1.63; P = 0.021). Having quit smoking more than 5 yr before transplantation reduced the relative risk of graft failure by 34% (relative risk 0.66; 95% CI, 0.52 to 0.85; P < 0.001). The increase in graft failure was due to an increase in deaths (adjusted relative risk 1.42; 95% CI, 1.08 to 1.87; P = 0.012). The relative risk for major cardiovascular disease events with smoking 11 to 25 pack-years at transplant was 1.56 (95% CI, 1.06 to 2.31; P = 0.024), whereas that of smoking more than 25 pack-years was 2.14 (95% CI, 1.49 to 3.08; P < 0.001). The relative risk of invasive malignancies was 1.91 (95% CI, 1.05 to 3.48; P = 0.032). Smoking had no discernible effect on the rate of return to dialysis or on serum creatinine during the first year after transplantation. Thus, cigarette smoking is associated with an increased risk of death after renal transplantation. The effects of smoking appear to dissipate 5 yr after quitting. These results indirectly suggest that greater efforts to encourage patients to quit smoking before transplantation may decrease morbidity and mortality. PMID- 10752534 TI - CTLA4Ig gene transfer prolongs survival and induces donor-specific tolerance in a rat renal allograft. AB - Organ transplantation requires lifelong antirejection therapy, which carries the risk of infection and cancer. A revolutionary approach is to transduce the organ graft with immunomodulatory genes to render them tolerated with no need of systemic immunosuppression. Prolonged allograft survival was achieved by adenovirus-mediated transduction of the cold-preserved kidney with sequences encoding CTLA4Ig, a recombinant fusion protein that blocks T cell activation. Organ expression of the transgene was achieved associated with mild infiltration of mononuclear cells in the transfected kidney. Mixed lymphocyte reaction as well as the production of both Thl and Th2 cytokines were reduced. Thus, the gene transfer technique to prolong graft survival is indeed effective and safe and can induce donor-specific unresponsiveness. Pending appropriate large animal testing, ex vivo genetic manipulation of the organ before surgery may hopefully represent a major step forward in human transplant medicine. PMID- 10752536 TI - Proximal tubular cysts in fetal human autosomal recessive polycystic kidney disease. AB - Standard texts describe human autosomal recessive polycystic kidney disease (ARPKD) as a cystic kidney disease in which lesions are localized to collecting tubules. Murine models of ARPKD consistently demonstrate an early phase of proximal tubular (PT) cystic involvement, which disappears shortly after birth. This is followed by a phase of collecting tubular (CT) cyst formation and progressive enlargement leading to compromise of renal function and death. Because the description of cystic lesions in human ARPKD has been largely based on postnatal specimens, PT cyst formation was hypothesized to be a characteristic feature of fetal human, as well as murine, ARPKD. This study examines nephron segment-specific cyst localization histochemically by lectin binding in 11 human ARPKD specimens obtained at different fetal and postnatal ages. PT cysts were found in human fetal specimens from gestational age 14 wk to 26 wk. The percentage of cysts involving PT segments ranged from 2 to 41%. The cystic index of PT cysts ranged from 2 to 5. In all specimens in which PT cysts were found, both the percentage of CT cysts and their cystic index were equal to or greater than the percentage of PT cysts and the associated PT cystic index. PT cysts were absent in all kidney specimens older than 34 wk gestational age. It is concluded that human ARPKD, like murine ARPKD, has a transient phase of PT cyst formation during early fetal development. PMID- 10752537 TI - Homer W. Smith award lecture. Aquaporin water channels in kidney. PMID- 10752538 TI - Calcification of the aortic valve in the dialyzed patient. PMID- 10752539 TI - Fluid absorption and active and passive ion flows in the rabbit superficial pars recta. PMID- 10752540 TI - Opposing effects on infarction of delta and kappa opioid receptor activation in the isolated rat heart: implications for ischemic preconditioning. AB - Delta-opioid receptors are known to participate in the protection found following ischemic preconditioning (IPC), but the role of kappa-receptors in IPC is currently controversial. Langendorff-perfused rat hearts received 35 min regional ischemia and 2 h reperfusion. PC (2 cycles 5 min global ischemia) substantially reduced infarct size. Pharmacological PC with the delta-agonist DADLE (10 nmol/L) had similar protective effects. However, higher dose DADLE (1 micromol/L) had a less beneficial effect, and in conjunction with the delta-antagonist naltrindole unexpectedly increased infarct size (61.5 +/- 2.0%, p<0.05 v 45.9 +/- 2.4% in controls) suggesting a non-delta effect. The universal kappa-opioid agonist bremazocine (30 nmol/L) increased infarct size (61.3 +/- 1.6%, p<0.05 v controls), an effect abrogated by the selective kappa1-antagonist nor binaltorphimine (BNI). Since opiates are known to have anti-adrenergic effects, which hypothetically may help to mediate IPC, cyclic AMP levels were measured in DADLE and in bremazocine-treated hearts. Decreased levels of cyclic AMP at the start of the regional ischemic period were found in low dose DADLE hearts (0.485 +/- 0/020, n = 8, vs controls, 0.654 +/- 0.025 nmol/g wet weight, p<0.001), but not in high dose DADLE nor in bremazocine treated hearts. Thus, in the isolated rat heart kappa1-opioid receptor activation exacerbates infarct size through an as yet unknown mechanism, suggesting that there could be an "antipreconditioned state". In contrast, delta-activity mediates protection which may be associated with a reduction of tissue cyclic AMP levels. PMID- 10752542 TI - Hyperosmotic perfusion of the beating rat heart and the role of the Na+/K+/2Cl- co-transporter and the Na+/H+ exchanger. AB - The aim of the present study was to investigate the role of the Na+/K+/2Cl- co transporter and the Na+/H+ exchanger on contractile function and electrolyte regulation during hyperosmotic perfusion of the heart. Langendorff perfused rat hearts were subjected to hyperosmolal perfusion in 10-min intervals. Perfusates were made hyperosmotic by adding mannitol to the buffer (370, 450 and 600 mOsmol/kg H2O). Cardiac contractile function was monitored with a balloon in the left ventricle (LV) coupled to a pressure transducer. Cardiac effluent was sampled repeatedly throughout and after hyperosmotic perfusion and analysed for content of Na+, K+, and Cl-. All three hyperosmotic perfusates initially reduced LV developed pressure (LVDP), but for 370 and 450 mOsmol/kg H2O, LVDP recovered to baseline within 4 min of perfusion. With 600 mOsmol/kg H2O, LVDP recovered slowly and was 50% below baseline after 10 min of hyperosmotic perfusion. Inhibition of the Na+/H+ exchanger with 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and 3-methylsulfonyl-4-piperidinobenzoyl-guanidine methanesulfonate (HOE 694) abolished the recovery of LVDP to the 600 mOsmol/kg H2O perfusate, whereas inhibition of the Na+/K+/2Cl- co-transporter had no impact on LVDP. Potassium was taken up by the heart during hyperosmotic perfusion and this uptake was significantly reduced with inhibition of the Na+/H+ exchanger. Intracellular pH was assessed with 31p magnetic resonance spectroscopy and hyperosmolality induced a significant alkalosis that was dependent upon the Na+/H+ exchanger. The rat heart responds to moderate elevations in osmolality with a transient reduction in contractile function, whereas an elevation of 300 mOsmol/kg H2O persistently reduces contractile function. The Na+/H+ exchanger, but not the Na+/K+/2Cl- co transporter, is of importance in contractile recovery and electrolyte regulation during hyperosmotic perfusion in the rat heart. PMID- 10752541 TI - The enhanced contractility in phospholamban deficient mouse hearts is not associated with alterations in (Ca2+)-sensitivity or myosin ATPase-activity of the contractile proteins. AB - Work performing heart preparations from hypercontractile, phospholamban deficient mouse hearts showed no change in parameters of contraction or relaxation in response to isoproterenol stimulation. Thus, the aim of the present study was to investigate whether or not changes at the level of the contractile apparatus occur in addition to the altered expression of Ca2+-regulating proteins observed in these mouse models, e.g., phospholamban, ryanodine receptors. Triton-X skinned fiber preparations from phospholamban deficient (n = 9) and wild-type (n = 10) mice were used and the Ca2+-activated force as well as the myosin ATPase-activity were simultaneously measured. The tension dependent ATPase-activity was unchanged in phospholamban deficient animals when compared to controls. The SERCA 2a inhibitor cyclopiazonic acid did not affect myosin ATPase-activity in this system. The Ca2+-sensitivity of Ca2+-activated force and myosin ATPase were unchanged as well. Comparison of the concentrations needed to achieve half maximal activation of the myosin ATPase-activity and force demonstrated that the Ca2+-sensitivity of the myosin ATPase was higher compared to the Ca2+-sensitivity of tension development. This holds true for phospholamban deficient mice (EC50 ATPase: 0.9 +/- 0.2 micromol/l; tension: 1.7 +/- 0.3 micromol/l; p < 0.001) and wild-type controls (1.1 +/- 0.01 micromol/l; 2.2 +/- 0.4 micromol/l; p < 0.01). The myosin ATPase-activity and force were correlated to each other in both, phospholamban deficient mice and controls and did not change at submaximal Ca2+ concentrations. The ATPase/ force-ratio, as a parameter of tension cost, was similar in either phospholamban deficient mice or controls. Thus, the present study provides evidence that at the level of the contractile proteins regulation of Ca2+-activated force and energy demand of force development are not altered in phospholamban deficient mice with enhanced myocardial performance. At the level of the regulation of crossbridge interaction, no adaptive or compensatory mechanisms have been initiated by ablation of phospholamban. PMID- 10752543 TI - Cyclic GMP attenuates cyclic AMP-stimulated inotropy and oxygen consumption in control and hypertrophic hearts. AB - We tested the hypothesis that increasing myocardial cyclic GMP would attenuate cyclic AMP induced positive inotropy and O2 consumption, in part, through changes in cyclic AMP and that renal hypertension-induced cardiac hypertrophy (HYP) would alter this relationship. Anesthetized, open chest rabbits (N = 48) were divided into four groups of control (CON) and HYP animals which received vehicle (VEH), isoproterenol 10(-6)M (ISO), 3-morpholinosyndnonimine 10(-4)M, (SIN-1), or a combination of ISO+SIN-1. Coronary blood flow (microspheres) and O2 extraction (microspectrophotometry) were used to determine O2 consumption in both subepicardium (EPI) and subendocardium (ENDO). Left ventricular change in wall thickness (%) was increased significantly by ISO in both CON (16 +/- 4 to 31 +/- 6) and HYP (17 +/- 2 to 24 +/- 3). Percent change in wall thickness was similar in the CON, SIN-1, and ISO+SIN-1 groups. Myocardial O2 consumption (ml O2/min/100 g) was increased by ISO in CON (10.3 +/- 1.0 to 13.6 +/- 2.0 EPI; 10.9 +/- 1.0 17.1 +/- 1.7 ENDO) and HYP (8.2 +/- 1.4 to 12.3 +/- 2.2 EPI; 6.6 +/- 1.4 to 14.8 +/- 1.8 ENDO). Oxygen consumption was unaffected by SIN-1 in CON and HYP animals. ISO+SIN-1 caused attenuated ISO-induced increases in O2 consumption in CON in EPI and ENDO, and in EPI in HYP. Cyclic GMP (pmol/g) was unchanged by ISO in CON and HYP, and increased by SIN-1 in CON (8.1 +/- 1.3 to 19.2 +/- 2.3 EPI) and HYP (9.1 +/- 1.5 to 12.8 +/- 2.0 EPI). Cyclic GMP remained elevated with ISO+SIN-1 in both groups. Cyclic AMP (pmol/g) was increased significantly by ISO in CON (496 +/- 43 to 725 +/- 106 EPI; 534 +/- 44 to 756 +/- 148 ENDO) and insignificantly in HYP (435 +/- 50 to 566 +/- 35 EPI; 497 +/- 51 to 583 +/- 47 ENDO). Cyclic AMP levels were unaffected by SIN-1 in either group. Isoproterenol induced increases in cyclic AMP were blunted by ISO+SIN-1 in CON (496 +/- 43 to 537 +/- 59 EPI) and not affected in HYP. The current study demonstrated attenuation of cyclic AMP mediated increased inotropy and O2 consumption by increasing cyclic GMP, which appeared, in part, related to cyclic GMP-induced reduction in cyclic AMP. This effect of cyclic GMP on cyclic AMP was not observed in myocardial hypertrophy. PMID- 10752544 TI - Glucose uptake increases relative to oxygen consumption during short-term hibernation. AB - BACKGROUND: Although glucose uptake is increased in chronically hypoperfused, viable myocardium, the dynamic changes in glucose uptake relative to oxygen consumption in "short-term" models of hibernation have not been fully explored. METHODS: 14 anesthetized swine were instrumented with an hydraulic occluder and flow probe on the proximal LAD artery. Blood flow was reduced approximately 30% for 1 hour. Myocardial blood flow and uptake of oxygen, free fatty acids, glucose and lactate were determined in the LAD region at baseline and at 10, 30, and 60 minutes of ischemia. Transmural biopsies for ATP and creatine phosphate (CP) were obtained in the LAD region prior to and at 15 and 45 minutes of ischemia. In 5 animals, glycogen was assayed at baseline and at the end of 60 minutes of ischemia. RESULTS: In the LAD region, myocardial oxygen consumption was reduced from 2.06 +/- 0.16 micromol/min/gram to 1.46 +/- 0.13 micromol/min/gram (P < 0.05). By 15 minutes of ischemia, transmural creatine phosphate fell from 7.48 +/ 0.76 micromol/g-wet weight at baseline to 6.19 +/- 0.32 micromol/g-wet weight (P < 0.05) but normalized by 45 minutes of ischemia (7.39 +/- 0.56 micromol/g-wet weight; NS). Between 10 and 60 minutes of constant flow reduction, glucose uptake as a percentage of MVO2 increased from 3 +/- 2% to 10 +/- 2% (P < 0.05) while lactate uptake increased from -9 +/- 9% to -1 +/- 2% (P < 0.05). Glycogen decreased from 27.8 +/- 3.7 at baseline to 16.9 +/- 1.2 micromol/g-wet weight at end-ischemia. CONCLUSIONS: In this model of short-term hibernation, glucose and lactate uptake increase relative to oxygen consumption during sustained ischemia, and temporally coincide with the recovery of bioenergetics. The findings are consistent with the notion that glycolytically derived ATP is important for the maintainance of energy supply during sustained ischemia. PMID- 10752545 TI - Leukemia Inhibitory Factor and Interleukin-6 downregulate sarcoplasmic reticulum Ca2+ ATPase (SERCA2) in cardiac myocytes. AB - Alterations in gene expression are a hallmark of cardiac hypertrophy and heart failure. Among these, the decreased expression of the sarcoplasmic reticulum calcium ATPase (SERCA2) has been described. Elevated levels of cytokines in particular, Leukemia Inhibitory Factor (LIF) and Interleukin-6 (IL-6) have been shown to have the capacity to elicit hypertrophic responses in cultured cardiac myocytes. In this study, we investigated the effects of these cytokines (LIF & IL 6) on the regulation of SERCA2 levels in cardiac myocytes. Cultured neonatal rat ventricular myocytes were transfected with a 3.2 kb promoter plasmid construct containing the SERCA2 promoter linked to a chloramphenicol acetyltransferase (CAT) reporter gene, and subsequently treated with 10 ng/ml LIF or 10 ng/ml IL-6. LIF and IL-6 independently caused a significant (p < or = 0.05) 23-36% inhibition in SERCA2 promoter activity. LIF and IL-6 induced inhibition was also evident in SERCA2 mRNA levels as assessed by Northern analysis. Time course of inhibition of SERCA2 mRNA levels showed the most prominent decrease occurring after 48 hours of treatment, with both cytokines having a dose dependent effect on the inhibitory response. Western analysis using a polyclonal antibody to SERCA2 protein indicate a significant, 60% decrease in the amount of total SERCA2 protein in cultured myocytes treated with 10 ng/ml LIF or IL-6. In conclusion, the cytokines LIF and IL-6 downregulate SERCA2 gene expression and protein levels. The molecular mechanism responsible for cytokine induced downregulation of SERCA2 is at least partly transcriptional. PMID- 10752546 TI - Histologic evidence that basic fibroblast growth factor enhances the angiogenic effects of transmyocardial laser revascularization. AB - OBJECTIVES: To determine whether addition of basic fibroblast growth factor (bFGF), an angiogenic growth factor, enhances the angiogenic effects of transmyocardial laser revascularization (TMR). BACKGROUND: TMR is an investigational therapy for treating patients with medically refractory angina not amenable to traditional therapies. Histologic and blood flow studies in animals have suggested that TMR enhances angiogenesis above that normally seen in ischemic myocardium. We tested the hypothesis that bFGF administered into TMR channels further enhance the angiogenic effects of TMR. METHODS: Chronic ischemia was created in 3 groups of dogs using an ameroid constrictor on the proximal LAD. In the bFGF group (n = 5) non-transmyocardial channels were created in the LAD territory and bFGF, (100 ng/ml) dissolved in pluronic gel was injected into the each channel. In the TMR group (n = 7), transmyocardial channels were created without bFGF. A control group (n = 7) had ischemia without TMR of bFGF. 5-bromo 2'-deoxyuridine (BrdU) was administered to mark proliferating cells. After 8 weeks survival, colored microspheres were injected to assess the regional myocardial blood flow. RESULTS: TMR and TMR+bFGF increased total vascular density by approximately 40% over that observed in the control group. However, the number of large vessels (internal diameter > or = 50 microm) was doubled by the addition of bFGF, and this correlated with a 50% increase in the density of proliferating vascular cells and a tripling of the total estimated vascular cross sectional area. Blood flow to the LAD territory was increased by TMR compared to controls, with no further benefit observed in the bFGF group. CONCLUSIONS: On a histologic basis, basic fibroblast growth factor further enhances angiogenesis following TMR in ischemic myocardium mainly by increasing the size but not the total number of vessels. PMID- 10752547 TI - Role of angiotensin II AT1 and AT2 subtype receptors in the regulation of atrial natriuretic peptide expression in salt-restricted rats. AB - Previous studies have suggested that angiotensin II modulates ANP secretion and this action appears to be largely independent from its hemodynamic effects. In order to explore the contribution of angiotensin II AT1 (AT1r) and AT2 (AT2r) receptor subtypes in the regulation of cardiac ANP, we studied the effects of selective antagonists of these receptors on ANP mRNA levels in the cardiac chambers of salt-restricted rats. Thirty-one Sprague-Dawley rats (12 weeks-old) weighing 250-350 g were studied during a low salt regimen and randomly assigned to the following treatment groups: AT1r-blockade (losartan) (10 mg/kg/day) (n = 18), AT2r-blockade (PD123319) (50 microg/kg/min) (n = 6), Control (salt restriction) (n = 7). Treatments were maintained for 7 days; subsequently, 12 rats from the AT1r-blockade group were subdivided in to two groups: AT1r/AT2r blockade (losartan +PD123319) (n = 6) and AT1r-blockade/vehicle (losartan+vehible) (n = 6), and treated for 7 additional days. Systolic blood pressure was significantly reduced by AT1r-blockade (p < 0.001), while it was not affected by AT2r-blockade. Concomitant treatment with both antagonists (AT1r/AT2r blockade) restored blood pressure values to baseline (p < 0.001 vs. AT1r blockade, p = n.s. vs Control). Atrial ANP mRNA was reduced by AT1r-blockade ( 42%, p < 0.05) and did not change during AT1r-blockade alone. On the contrary, concomitant treatment with both antagonists resulted in a further significant inhibition of ANP expression (-65% and -36% vs Control and AT1r-blockade, respectively, both p < 0.05). ANP expression in ventricles was not affected by any of these treatments. Our results demonstrate that angiotensin II tonically modulates cardiac ANP expression in our experimental model. In particular, angiotensin II receptor subtypes AT1r and AT2r regulate atrial ANP mRNA levels through a synergic action and independently from blood pressure changes. PMID- 10752548 TI - Interaction between transient metabolically mediated dilatation and pressure induced constriction in the canine coronary artery. AB - This study explored the interaction between metabolically mediated vasodilatation (ventricular extra-activation) and pressure induced vasoconstriction (transient augmentation of aortic diastolic pressure). Eight dogs having formalin-induced heart block were chronically instrumented with aortic and left ventricular catheters and an electromagnetic flow probe on the left circumflex coronary artery. At a heart rate of 60 beats/min a single ventricular extra-activation introduced at 200 ms after the normal paced beat resulted in a 13 +/- 1% decrease in diastolic coronary vascular resistance index (DCVRI) in the first response beat (D1) and a persistent vasodilatation lasting for five beats (D1-D5). An increase in aortic diastolic pressure (32 +/- 3% for 520 +/- 15 ms) resulted in 13 +/- 2% increase in DCVRI in the D1 which was not evident in subsequent beats. Following a combined intervention, DCVRI in D1 was not significantly different from control but DCVRI did decrease to a greater degree in the subsequent response beats (D2-D7). These data indicate that the responses of two opposing vasoactive stimuli, i.e., pressure induced vasoconstriction and metabolic vasodilatation, were negated in the first response beat. The metabolically mediated vasodilatation was unaltered in the subsequent response beats. PMID- 10752549 TI - Molecular analysis of the prototype coxsackievirus B5 genome. AB - To facilitate studies of the phylogenetic relationship between enteroviruses, in particular the prototype strain (Faulkner) of coxsackievirus B5 (CVB5F) and other CVB5 isolates and to facilitate studies of the interactions between CVB5F and the target cell, the complete nucleotide sequence of the prototype has been determined. The complete sequence was collected from three overlapping reverse transcription polymerase chain reaction (RT-PCR) generated amplicons. Molecular analysis of the CVB5F genome verified that this strain is more related to other CVB5 isolates and to swine vesicular disease virus strains than to other enteroviruses. In addition, comparison of the amino acid sequence derived from the structural genes indicated a division of group B coxsackievirus into subgroups, where CVB1, CVB3 and CVB5 constitute one group, CVB2 and CVB4 from a second group and CVB6 prototype forms a branch of its own. This observation, supported by reports describing the interactions between CVB and the cell surface, may reflect that these subgroups of group B coxsackieviruses have evolved to use slightly different approaches to carry out the complete infectious cycle within the cell. PMID- 10752550 TI - Genetic polymorphism across regions of the three open reading frames of "Norwalk like viruses". AB - Genomic characterization of Norwalk-like human caliciviruses (NLVs) originating from outbreaks and sporadic cases of acute gastroenteritis has revealed surprisingly high levels of diversity, even in the RNA polymerase gene, which is anticipated to be highly conserved. Since information on antigenic relationship is limited, due to the lack of a tissue culture system for these viruses, strains mostly are described on the basis of their genetic relatedness. However, the lack of uniformly applied criteria has led to a confusing array of strains with different groups employing different names for similar genetic lineages. Our goal was to conduct a structured analysis of genomic relationships among NLV strains in an attempt to provide an interim framework for genotyping. We assembled a panel of 31 potentially distinct genogroup I (GGI) and genogroup II (GGII) NLVs that reflected the diversity seen in strains detected by our laboratories and in published sequences. Phylogenetic analysis of sequences from regions of the open reading frames (ORF) 1, 2 and 3 was performed in order to investigate genomic relationships. The strains sequenced fell into seven phylogenetic groups in GGI and at least five phylogenetic groups in GGII, based on greater than 80% nucleotide identity in the region of ORF2 encoding the N-terminus of the capsid protein, and consistent clustering with high bootstrap values irrespective of the method used. Analysis of the ORF1 and ORF3 regions supported for most strains the clustering as established for those derived from ORF2. In the ORF1 region, used by most laboratories for diagnostic RT-PCR, clustering was consistent when a putative genotype border was set at 15% nucleotide mismatches for viruses in GGI and at 10% for viruses in GGII. Two strains grouped within different clusters based on ORF1 and ORF2 indicating that recombination may have occurred. We discuss the implications of these observations for the classification and typing of NLVs. PMID- 10752551 TI - Comparison between acute oral/respiratory and chronic stomatitis/gingivitis isolates of feline calicivirus: pathogenicity, antigenic profile and cross neutralisation studies. AB - Feline calicivirus (FCV) is a major oral and respiratory pathogen of cats, able to induce subclinical infection as well as acute disease. It is also characterized by a high degree of antigenic variation. This work sought to address the question of the existence of distinct biotypes of FCV. Eight French, 6 British and 9 American FCV isolates, responsible for acute oral/respiratory disease or chronic gingivitis/stomatitis, were compared for their pathogenicity, antigenic profiles and serological relationships. Antigenic profiles were assessed by an indirect immunofluorescence assay with a large panel of characterized monoclonal antibodies. Cross-neutralisation assays were performed with specific cat antisera collected at 30 days p.i., then analysed by calculation of antigenic bilateral relatedness and dominance. Whatever their pathogenic origin, all the isolates induced an acute upper-respiratory tract infection in oronasally infected SPF kittens. Their antigenic profiles were different and did not correlate with their geographical or pathological origin. Cross-neutralisation studies and calculation of the mean bilateral relatedness allowed us to distinguish chronic original isolates from acute original ones. This study did not confirm the existence of FCV biotypes but showed that the chronic carrier state is related to the emergence of antigenically distant viruses. PMID- 10752552 TI - Protein analysis of geographic isolates of shrimp white spot syndrome virus. AB - Six geographic isolates of the white spot syndrome virus (WSSV) of penaeid shrimp, from China, India, Thailand, South Carolina, Texas, as well as from crayfish kept at the US National Zoo in Washington D. C, were compared by electron microscopy and sodium sulfate polyacrylamine gel electrophoresis (SDS PAGE). Amino acid compositions of four of the major structural polypeptides of the South Carolina WSSV were analyzed, and three of the four polypeptides were partially sequenced from their NH2 termini. The morphologies of purified virions of the six geographic isolates of WSSV were indistinguishable by transmission electron microscopy. By SDS-PAGE, the protein profiles of the six isolates were very similar, but not identical. They all contained three major polypeptides with sizes of approximately 25, 23 and 19 kDa. A fourth major polypeptide at the 14.5 kDa position was observed in four of the geographic isolates. The WSSV isolated from crayfish presented a slightly different structural protein profile, particularly with regard to the protein in the 19 kDa range that appeared larger in size than those of the other isolates. The NH2 terminal amino acids of the 25, 23 and 14.5 kDa polypeptides of the South Carolina WSSV were sequenced as MDLSFTLSVVTA, MEFGNLTNLDVA, and VARGGKTKGRRG, respectively. No significant homologous sequence was found in the GenBank. These protein sequences have been submitted to the SWISS-PROT Protein Data Bank and assigned the accession numbers P82004, P82005 and P82006. PMID- 10752553 TI - Inter- and intra-site genetic diversity of natural field populations of rice tungro bacilliform virus in the Philippines. AB - The genetic structure of rice tungro bacilliform virus (RTBV) populations within and between growing sites was analyzed in a collection of natural field isolates from different rice varieties grown in eight tungro-endemic sites of the Philippines. Total DNA extracts from 345 isolates were digested with EcoRV restriction enzyme and hybridized with a full-length probe of RTBV, a procedure shown in preliminary experiments capable of revealing high levels of polymorphism in RTBV field isolates. In the total population, 17 distinct EcoRV-based genome profiles (genotypes) were identified and used as indicators for virus diversity. Distinct sets of genotypes occurred in Isabela and North Cotabato provinces suggesting a geographic isolation of virus populations. However, among the sites in each province, there were few significant differences in the genotype compositions of virus populations. The number of genotypes detected at a site varied from two to nine with a few genotypes dominating. In general the isolates at a site persisted from season to season indicating a genetic stability for the local virus population. Over the sampling time, IRRI rice varieties, which have green leafhopper resistance genes, supported similar virus populations to those supported by other varieties, indicating that the variety of the host exerted no apparent selection pressures. Insect transmission experiments on selected RTBV field isolates showed that dramatic shifts in genotype and phenotype distributions can occur in response to host/environmental shifts. PMID- 10752554 TI - Relationship between serotypes and genotypes based on the hypervariable region of the S1 gene of infectious bronchitis virus. AB - To group infectious bronchitis virus (IBV) isolates, a genetic grouping method based on hypervariable region 1 (HVR 1, nucleotides 168 to 197) was compared with that based on the whole S1 gene. Both methods resulted in the same grouping data. So the grouping method based on HVR 1 could represent the grouping method based on the whole S1 gene. Taiwan isolates could not be placed within the existing groups. In order to test the correlation between genotype and serotype, a one-way neutralization test was used to compare 9 Taiwan isolates selected from different genotypes with Massachusetts (Mass) (H120) and Connecticut (Conn) standard strains. In addition, a two-way cross-neutralization test was performed in embryonated eggs with the beta method (constant-virus, diluted-serum) and the reciprocal neutralization titers were calculated to give the relatedness (r) values. The results of two kinds of neutralizing tests showed that the serotypes of 9 isolates were different from Mass or Conn. Based on the r-values, 9 isolates were divided into two serotypes which were correlated with their genotypes. From pathogenicity tests, IBV Taiwan isolates could be divided into high, intermediate, and low pathogenicity according to their pathogenicity indexes. However, no relationship exists between pathotype and genotype. In conclusion, the genetic typing method based on HVR 1 can be used for typing IBV isolates. PMID- 10752555 TI - Comparative studies of piscine and amphibian iridoviruses. AB - A total of 30 iridoviruses collected from Australia, South-East Asia, North America, South America and Europe were characterised. With the exception of the South-East Asian iridoviruses all viruses were found to belong to the genus Ranavirus. All viruses, except those originating from South-East Asia, cross reacted with antisera against epizootic haematopoietic necrosis virus (EHNV). Viruses or virus-infected cells were examined using electron microscopy, SDS PAGE, restriction endonuclease (RE) digestion, DNA hybridisation, and DNA sequencing. Data from RE digestion of genomic DNA, and from the sequencing of PCR products indicated that the viruses generally grouped according to their geographic and taxonomic (i.e. amphibian or fish) origin. The one exception to this was the viruses from the United Kingdom that grouped with the North American ranaviruses. The differences between specified genomic regions were small. To assess the validity of the differences in sequence homology, similar studies were performed with different isolates from two viruses (EHNV and Guatopo virus (GV), collected from different animals at different locations and time). The sequence data showed complete homology for the isolates for any one virus over the 200 and 586 bp regions examined. Collectively, the data showed that the coding region for the major coat protein (MCP) is stable for any one species (e.g. EHNV). PMID- 10752556 TI - The extracellular part of glycoprotein E of bovine herpesvirus 1 is sufficient for complex formation with glycoprotein I but not for cell-to-cell spread. AB - Glycoproteins gE and gI of bovine herpesvirus 1 (BHV-1) are type I transmembrane proteins that can form a complex that is involved in cell-to-cell spread mechanisms. The extracellular domains of both proteins have cysteine-rich regions that are also found in the homologous proteins of other alpha-herpesviruses. The extracellular domain of gE has two conserved cysteine-rich regions: C1 and C2. The other conserved regions in gE are located between C2 and transmembrane region and in the cytoplasmic domain of gE. We studied the complex formation between gE and gI using a series of truncated gE proteins and a full length form and a secreted form of gI. All proteins were expressed in recombinant baculoviruses. To analyse the complex formation between these polypeptides we used monoclonal antibodies (MAbs 67 and 75) that specifically react with the gE/gI complex and not with separately expressed glycoproteins gE and gI alone. This analysis showed that the BHV-1 gE/gI complex can be formed in insect cells after a co-infection with baculoviruses expressing gE and gI in their full length form. When secreted forms of gE and gI were expressed after co-infection, the gE/gI complex was still formed and could also be detected in the tissue culture medium. This gE/gI complex was also formed after mixing the tissue culture media of insect cells expressing the secreted form or gE or gI separately. The smallest part of gE that still formed a complex is encoded by the first 246 residues of gE. This extracellular domain contains only the C1 region, showing that the C2 region is not essential for gE/gI complex formation. Shorter forms of gE encoding the C1 region did not form a detectable complex. We also found that the formation of gE/gI complex is not sufficient for normal cell-to-cell spread of BHV-1. A recombinant BHV-1 gE TM-virus, expressing a truncated glycoprotein E from which the transmembrane and cytoplasmic domain were removed, forms plaques as small as a gE null mutant. PMID- 10752557 TI - Expression and cytoplasmic localisation of deoxyuridine triphosphate pyrophosphatase encoded by a human endogenous retrovirus. AB - Many lentiviruses encode a dUTPase which may protect against toxic misincorporation of dUTP into cDNA during reverse transcription. However, the primate lentiviruses HIV and SIV do not express a dUTPase. Significantly, the host genomes of these lentiviruses contain a multicopy endogenous retrovirus which is absent in non-primate genomes. In humans, this endogenous retrovirus is known as HERV-K and encodes a potential dUTPase sequence. Previously, we have suggested that HIV infection is complemented by a cytosolic dUTPase derived from the dUTPase gene encoded by HERV-K. This study demonstrates expression of HERV-K dUTPase transcripts and protein in human cell lines using RT-PCR and western blot analysis. Immunocytochemistry showed that HERV-K dUTPase was predominantly located in cell cytoplasm when transiently expressed in COS-1 cells. These data provide substantiation and support for the hypothesis above and is the first documentation of expression of an enzyme of nucleotide metabolism expressed by an endogenous retrovirus. PMID- 10752558 TI - Fine characterization of the antigenic site within the flap region in the protease protein of HIV-1. AB - The aspartate protease encoded by human immunodeficiency virus type 1 is essential for cleavage of the gag and gag-pol precursor proteins. The majority of HIV-1-antibody-positive sera react with the protease. In this study we used a substitution set of peptides for detailed characterization of the earlier defined antigenic site (aa 44-58) within the central "flap" region, also important in the context of conformational flexibility during protease inhibitor binding. We found that isoleucine at position 54 was important for creating an antigenic site required for binding of anti-HIV-1 sera. The identification of structurally essential amino acids in the flap region of HIV-1 PR may have important implications in future development of antiviral drugs. PMID- 10752559 TI - Species specificity and interspecies relatedness of NSP4 genetic groups by comparative NSP4 sequence analyses of animal rotaviruses. AB - Previous sequence analyses of the rotavirus nonstructural NSP4 from human and some animal rotavirus strains revealed the presence of three distinct NSP4 alleles or genetic groups. To examine the species of origin relatedness and diversity of NSP4, the nucleotide and deduced amino acid sequences of the gene encoding the NSP4 from 15 animal rotavirus strains of porcine, equine, bovine, lapine and canine origin were determined and compared to human and other animal strains sequenced previously. Lapine and equine strains were shown to belong to the NSP4 genotype A. Murine NSP4 sequences formed a previously unrecognized fourth distinct NSP4 genotype (genotype D) that was more divergent compared to NSP4 genotype A, B, and C than the latter three are among each other. Within NSP4 genotypes, strains isolated from rabbits, horses, cows (genotype A) and pigs (genotype B) clustered according to species of origin, suggesting a conserved pattern of evolution within species. NSP4 sequence comparison among one wildtype and two tissue culture-adapted lapine strains, known to cause disease in neonatal rabbits, failed to identify amino acid changes within the variable region spanning amino acids 130 to 141, suggesting that disease in rabbits is the result of the lapine virus infection and replication, including production of the NSP4 enterotoxin. PMID- 10752560 TI - Herpes simplex virus type-1 and -2 pathogenesis is restricted by the epidermal basement membrane. AB - Murine flank scarification with HSV-1 and -2 results in primary lesions at the site of inoculation within three days and lesions at secondary sites within four days. The severity of the infection can be given a numerical value or "score" which is derived from the number and size of these lesions. Using this model, we investigated the role of the epidermal basement membrane in HSV pathogenesis. We exposed murine epidermis to 5 x 10(4) plaque forming units of HSV-1 and -2, which by day 8 produced inoculation site (primary site) disease scores of 27 and 12.4 respectively, and secondary site disease scores of 29 and 30 respectively. In contrast, intradermal injection of HSV below the epidermal basement membrane did not cause disease. To determine if the basement membrane restricts HSV spread in vitro, Vero cells were cultured in the lower well of a dual well system. The upper well was separated from the lower well by a filter coated with the artificial basement membrane, matrigel. Addition of virus to the upper well failed to result in either viral accumulation in the lower well or infection of the cells in the lower well. These data suggest that the basement membrane is a barrier to the passage and spread of HSV. PMID- 10752561 TI - Infectious cDNA clones of two grapevine viruses. AB - Full-length cDNA copies of the genomes of Grapevine virus A (GVA) and Grapevine virus B (GVB) under the control of bacteriophage T7 promoter have been synthesized, which were refractory to cloning in Escherichia coli. However, both transcribed cDNAs were infectious when mechanically inoculated to Nicotiana plants. A full-length cDNA copy of GVB was engineered in pCass2, a plasmid containing a partially duplicated copy of the Ca35S promoter, but was rather unstable in Escherichia coli. No infection of Nicotiana plants was obtained following mechanical inoculation but detached Nicotiana leaves, inoculated by particle bombardment, supported the multiplication of a GVB isolate seemingly identical to the wild-type used for cloning. Nicotiana seedling inoculated with sap expressed from these leaves became infected showing typical GVB symptoms. Transient transcription of Ca35S driven cDNA clones was also detected by RT-PCR in leaves of the grapevine hybrid LN33 following inoculation by particle bombardment. The availability of infectious cDNA clones of GVA and GVB constitutes a tool for the study of genome expression and pathogenesis, and for the ultimate establishment of the aetiological role of these viruses. PMID- 10752562 TI - Evidence for translation of VP3 of avian polyomavirus BFDV by leaky ribosomal scanning. AB - Due to several incomplete splicing reactions, budgerigar fledgling disease virus (BFDV) late mature mRNAs are either bicistronic or polycistronic with an agnogene located upstream of viral protein (VP) genes. While the bicistronic mRNAs code for the vast majority of VP1, the polycistronic mRNAs contain the coding sequences of VP2, VP3, and VP1 (as the most distal cistron relative to VP2 and VP3). In this work, the translation initiation mechanism of VP3 was investigated in chicken embryo fibroblast cells by transfection of a series of BFDV mutant clones and transient reporter gene chloramphenicol acetyltransferase (CAT) expression assay, leading to the conclusion that BFDV VP3 was translated by leaky ribosomal scanning. Furthermore, thanks to the high sensitivity of CAT assay experiment, we were able to demonstrate that ribosomes could reach VP1-AUG and initiate translation after scanning through 900 nucleotides on the unspliced polycistronic mRNA. PMID- 10752563 TI - Herpes simplex virus UL17 protein is associated with B capsids and colocalizes with ICP35 and VP5 in infected cells. AB - A previous study using a mutant lacking the UL17 gene has suggested that the UL17 protein of herpes simplex virus type 1 (HSV-1) is required for the cleavage/packaging of viral DNA. In this study, we have raised a rabbit polyclonal antiserum which specifically reacted with the UL17 protein which has an apparent molecular mass of 78-kDa in the lysates of HSV types 1- and 2 infected Vero cells. Western blot analysis of intracellular capsids demonstrates that the UL17 protein was associated with B and C capsids. Indirect immunofluorescence studies reveal that it colocalized with the major capsid protein VP5 and the scaffolding protein ICP35 within the nucleus. These results suggest that the association of the UL17 protein with immature B-type capsids is important for its role in cleavage/packaging. PMID- 10752564 TI - Constructing and writing the names of virus species--an editorial note. PMID- 10752565 TI - Structure and typography of virus names. PMID- 10752566 TI - On the relative merits of italics, Latin and binomial nomenclature in virus taxonomy. PMID- 10752567 TI - Atypical antipsychotics in the treatment of drug-induced psychosis in Parkinson's disease. AB - Our experience with atypical antipsychotics in patients with PD is that their motor effects are not predictable. The multiple reports concerning clozapine's beneficial effects on tremor, dystonia, nocturnal akathisia, and dyskinesias all underscore this observation. However, the appearance of even minor degrees of parkinsonism in normal volunteers or schizophrenics should suggest that an antipsychotic will not be well-tolerated in patients with PD. The treatment of PD is probably the most stringent test of a drug's freedom from parkinsonian side effects. The data from trials in schizophrenia concerning parkinsonian effects cannot always be confidently interpreted. Virtually all subjects in these trials have been treated with typical neuroleptics until shortly before study entry. Because the parkinsonian side effects of these drugs may persist for several months, patients may still show declining levels of parkinsonism even when placed on a drug that induces it if this effect is milder than that induced by the pre study neuroleptic. Depending on the pre-study drug used and the duration of the study, distinguishing placebo from a low-potency neuroleptic may be impossible. Furthermore, the standard measure of parkinsonism in psychiatric studies is the Simpson-Angus scale which is heavily weighted toward rigidity and may underscore bradykinesia, gait, and posture abnormalities. The prolactin response to an antipsychotic drug may turn out to be a good predictor of its freedom from parkinsonian side effects. That would fit with the data presented above of clozapine and quetiapine having less parkinsonian effects, olanzapine having more but variable effects and risperidone being poorly tolerated. With the data presented above, comprising a current review of all reports of the use of atypical antipsychotics in PD that we could locate, we can say little with certainty. The only drug with confirmed benefit without worsening parkinsonism is clozapine. Open-label trials involving over 400 patients and two multicenter, placebo-controlled, double-blind trials have demonstrated that it is effective in treating the psychosis. It improves tremor, does not worsen other motor functions to any significant extent, and is safe at low doses. Limited data provide conflicting information on both risperidone and olanzapine. Quetiapine seems to be well-tolerated with some, but definitely less, worsening of PD motor features than risperidone and olanzapine. Based on the current literature, our personal experience, and anecdotal experience of other PD specialists which we solicited, we will venture our own interpretation and recommendations. We think risperidone is poorly tolerated and should be used only as a last resort; that olanzapine is better than risperidone but will, in a majority of patients with PD, worsen motor function. We are optimistic, but not yet convinced, that quetiapine may prove to be as effective and better tolerated than clozapine. It will not require cumbersome monitoring because it does not induce a blood dyscrasia. We therefore recommend that DP be treated in the following manner. First, the anti-PD medications should be simplified and reduced as much as tolerated. We think, in general, side effects multiply more with increasing numbers of drugs than with drug dose, so that patients are more likely to tolerate a higher dose of levodopa than a lower dose of levodopa combined with other adjunctive anti-PD medications. In reducing anti-PD medications, we recommend tapering and stopping, if necessary, the drugs with the highest risk-to-benefit ratio first. Anticholinergics are stopped first, then selegiline, dopamine agonists, amantadine, and finally COMT inhibitors, which have no psychotomimetic action of their own. Finally, levodopa is reduced. Generally, a point is reached at which the anti-PD medications cannot be reduced without jeopardizing motor function. If psychosis persists at this point, then an antipsychotic is added. (ABS PMID- 10752569 TI - The quality of life in Parkinson's disease. AB - The objective of this study was to examine the quality of life in patients with Parkinson's disease (PD) in a community-based sample (n = 228 patients) using a Medical Outcomes Study 36-Item Short Form Health Survey (SF-36) as a measure. Associations to the variables age, age at onset, duration, clinical stage (Hoehn and Yahr), depression (Zung), and dementia (MMSE) were studied. Women scored significantly lower on five of the eight dimensions of SF-36. Depression, as measured in this study, was more common among parkinsonian women than men. Depression was the factor that was associated most significantly with the experienced quality of life, according to SF-36. With physical functioning, only the clinical stage had a more significant association than depression. To improve the quality of life in patients with PD, it is necessary to make every effort to recognize and relieve the depression of patients with PD. PMID- 10752570 TI - Health-related quality of life following bilateral intrastriatal transplantation in Parkinson's disease. AB - Intrastriatal transplantation of embryonic dopaminergic tissue is a new, experimental approach for the treatment of Parkinson's disease (PD). Clinical trials have shown longterm graft survival and therapeutically valuable improvements with decreased L-dopa dose and time spent in the "off"-phase, and reduced rigidity and hypokinesia. We have measured health-related quality of life (HRQoL) using the Nottingham Health Profile (NHP) in five patients subjected to bilateral transplantation in the caudate and putamen to explore the influence of intrastriatal grafts on HRQoL and the value of such measures in trials of restorative therapies. The results demonstrate improved HRQoL following transplantation, with individual patients showing striking improvements within different dimensions of the NHP as well as the NHP distress index (NHPD). The most pronounced improvements after grafting were observed for physical mobility along with emotional reactions and energy. These results indicate that intrastriatal transplantation of embryonic dopaminergic tissue can give rise to improvements within most areas of HRQoL, and that HRQoL measurements provide important information additional to that obtained by traditional, symptom oriented assessment protocols. However, the optimal approach to HRQoL measurement in PD remains to be determined. PMID- 10752568 TI - Waking up to sleep episodes in Parkinson's disease. PMID- 10752571 TI - Treatment of advanced Parkinson's disease by unilateral posterior GPi pallidotomy: 4-year results of a pilot study. AB - To assess the long-term outcome following unilateral pallidotomy for advanced Parkinson's disease, we performed nonblinded Core Assessment Program for Intracerebral Transplantations protocol assessments in 10 of the original 15 patients in our pilot study for 4 years following surgery. Although Unified Parkinson's Disease Rating Scale motor examination scores returned to baseline levels at 3 and 4 years, most patients continued to show sustained improvements in contralateral tremor, akinesia, and drug-induced dyskinesias. Contralateral tremor was absent at 4 years in all seven patients with preoperative tremor. Contralateral "off" arm movement times (averaged for three tasks) decreased by 37% at 1 year and by 30% at 4 years. Contralateral dyskinesia scores improved by 82% at 1 year and by 64% at 4 years. In contrast, after reaching speeds equal to the contralateral side at 1 year, ipsilateral "off" movement times increased by 13% over baseline levels at 4 years. Although most gait and postural stability measures showed modest initial improvement followed by a return to baseline values, "on" stand-walk-sit task performance declined significantly at 4 years. Despite the restriction of our surgeries to one side and the expected natural progression of Parkinson's disease, the results of patient self-assessments suggest that 4 years after unilateral pallidotomy, most patients continue to experience a quality of life above preoperative levels. PMID- 10752572 TI - Motor unit changes in sporadic idiopathic Parkinson's disease. AB - We studied motor unit changes in 20 patients with Parkinson's disease (PD) and 20 age-matched control subjects to look for evidence of motorneuron degeneration in sporadic idiopathic PD. Patients and control subjects were screened by clinical criteria and nerve conduction studies to exclude those with peripheral neuropathic processes. Changes in motor unit morphology were investigated with subjective and computerized quantitative electromyography (QEMG) of the anterior tibialis (AT) and first dorsal interosseous. Multivariate comparisons showed a significant difference in the QEMG analysis for motor unit enlargement in patients with PD versus control subjects. Some of the univariate comparisons for both the subjective and QEMG analyses of the AT were also significant. These results demonstrate that motorneuron drop-out with reinnervation occurs in sporadic idiopathic PD. In summary, our findings provide evidence that clinically silent motorneuron disease occurs in typical cases of sporadic idiopathic PD, suggesting that it may be a normal part of the pathologic picture of PD. Any hypothesis concerning the pathogenic mechanism of PD would need to take into account such a finding. PMID- 10752573 TI - Action tremor during object manipulation in Parkinson's disease. AB - In previous studies of fingertip forces during precision grip in subjects with Parkinson's disease (PD), we observed regular oscillations in isometric force. The present study characterizes the nature of these oscillations. Fingertip forces were recorded from the index finger and thumb during precision grip-lifts with a 300 g and 900 g object in 10 subjects with PD and 20 healthy control subjects. Fourier analysis confirmed that all subjects with PD exhibited force oscillations with a clearly definable frequency (approximately 7-11 Hz). Five of these subjects also exhibited a second lower frequency peak (approximately 5 Hz). Approximately half of the 20 control subjects displayed a single frequency peak (approximately 8-12 Hz), which was generally lower in amplitude than in the subjects with PD (representing enhanced physiological tremor), whereas the remaining control subjects had low-amplitude, broad-based spectra (representing physiological tremor). The amplitude of the force oscillations was higher for lifts with the heavier object in both the control subjects and subjects with PD. L-Dopa resulted in a decreased tremor amplitude but did not influence the frequency. The force oscillations of the two opposing digits normal to the grip surfaces were in phase, whereas the oscillations tangential to the grip surfaces were often out of phase. The results suggest that the multipeaked force rate trajectories reported previously are caused by action tremor. The similarity of force oscillations in subjects with PD and healthy control subjects suggests common tremor-generating mechanisms and supports the notion that the parkinsonian action tremor (AT) is an exaggerated form of physiological tremor. These findings provide insight into the impaired hand function observed in individuals with PD. PMID- 10752574 TI - Pushing and pulling with the upper extremities while standing: the effects of mild Alzheimer dementia and Parkinson's disease. AB - Eleven patients with mild dementia of Alzheimer type, 12 patients with mild to moderate Parkinson disease, and 27 control subjects of comparable age, education, and gender pushed or pulled on a rigid horizontal bar while maintaining stable erect stance. A target window (target force +/-10% maximum force) and a bar force cursor were displayed on a video screen, and subjects were asked to place the bar force cursor within the target window as quickly and as accurately as possible holding the target window for at least 1 sec. The target forces were 50% and 75% maximum force for each person, and three 4.0-sec push trials and three 4.0-sec pull trials were performed for each target force. Moments of force (torque), body motion, and extremity electromyography were measured with a computerized motion analysis system. The patients with Alzheimer's disease had only slightly lower Mini Mental State Examination (MMSE) scores (mean +/- standard deviation [SD] = 25.0 +/- 2.3) than the patients with Parkinson's disease (28.8 +/- 1.5) and control subjects (28.7 +/- 1.3). The patients with Alzheimer's disease had upper limb reaction times (0.827 +/- 0.399 sec) that were greater than those of the patients with Parkinson's disease (0.672 +/- 0.315 sec) and control subjects (0.606 +/- 0.263 sec). Furthermore, the patients with Alzheimer's disease achieved the designated target in only 46.2% of trials, which was comparable to the performance of the patients with Parkinson's disease (55.6%) but inferior to the control subjects (80.6%). Movement times did not differ significantly. The patients and control subjects initiated movement with comparable anticipatory postural activity in the lower limbs. The poor success rates of the patients with Alzheimer's disease and the patients with Parkinson's disease were attributable to inadequate visually guided adjustments in force after the initial movement began. This difficulty in making quick motor adjustments may be relevant to the tendency of patients with Alzheimer's disease to fall. PMID- 10752575 TI - Altered expression of calcium- and apoptosis-regulating proteins in multiple system atrophy Purkinje cells. AB - The expression patterns of the calcium binding proteins calbindin and parvalbumin and of the apoptosis modulating proteins Bcl-2, Bax, and Bcl-x were studied in the cerebellum of patients with multiple system atrophy (MSA). Calbindin and parvalbumin immunoreactivity was markedly decreased in MSA Purkinje cells whereas Bax and Bcl-x protein expression was increased. Bcl-2 expression was restricted to a subpopulation of granule neurons, but no decrease of Bcl-2 was evident in MSA. Additional DNA end-labeling (ISEL) studies revealed only one possible apoptotic Purkinje cell nucleus, but nuclei in the cerebellar white matter, probably oligodendrocytes, in the cerebellum of patients with MSA. The present results suggest that a diminished calcium binding capacity of MSA Purkinje cells might lead to a change in the regulation of proteins of the bcl-2 family that could favor the pathologic initiation of apoptosis. PMID- 10752576 TI - Application of the Unified Parkinson's Disease Rating Scale in progressive supranuclear palsy: factor analysis of the motor scale. AB - An important criterion in scale validation is the demonstration of a stable factor structure. The Unified Parkinson's Disease Rating Scale (UPDRS) is widely used to assess Parkinson's disease (PD). The reliability and applicability of the motor subscale of the UPDRS (UPDRSm) when applied to patients diagnosed with progressive supranuclear palsy (PSP) is unknown. In a sample of 175 patients with PSP, factor analysis revealed five clinically distinct factors: two independent bradykinesia factors (axial/gait and extremities), one rigidity factor, and two independent tremor factors (rest and action). Two items (posture and rest head tremor) did not reach criteria for factor loadings. There was a high degree of internal consistency. These results suggest that UPDRSm is a reliable and applicable scale for assessing most aspects of PSP function as well as severity measures of five clinical disability domains. PMID- 10752577 TI - Benign hereditary chorea--entity or syndrome? AB - The existence of "benign hereditary chorea" (BHC), a rare disorder of childhood onset familial chorea without other neurologic features or progression, has increasingly been questioned, because many patients with this disorder were subsequently diagnosed with different conditions. We therefore analyzed all published reports of families with BHC and contacted their authors to obtain follow-up information. In addition, we reviewed all patients in whom at least one of the authors had at some stage considered a possible diagnosis of BHC. Of 42 families reported to have BHC in the literature, we obtained follow-up information on 11 families, three of which had been seen by us. An additional seven new, unreported families and four sporadic cases, in which this diagnosis was suspected by at least one of us at one point, were reviewed and videotaped. On reviewing the videotapes of the 11 families in the literature, the diagnosis of BHC was changed in nine. In the remaining two families, atypical features suggesting different diagnoses were present in the original reports. In none of our own previously unreported patients (seven familial and four sporadic) was BHC diagnosed unequivocally by all evaluators after review of their video recordings. In three of these families and all four sporadic patients the diagnosis was changed; in one family multifocal myoclonus could not be differentiated from chorea by any author, and in the remaining three families no consensus between the raters was found. Apart from the 11 families in whom we obtained follow-up information, analysis of the remaining 31 reports on families with BHC also revealed atypical features in the majority. We conclude that BHC is not a diagnosis, but a syndrome that requires further investigation. Whether there is a distinct entity "BHC" with a single gene abnormality remains to be proven. PMID- 10752578 TI - Arm restlessness in patients with restless legs syndrome. AB - Restless legs syndrome (RLS) is a neurologic disorder characterized principally by leg paresthesia and motor restlessness. Several clinical reports indicated that many patients with RLS also have arm paresthesia and restlessness. In the present study, the incidence of arm restlessness was assessed by questionnaire in 230 patients diagnosed with idiopathic RLS. Arm restlessness was reported by 48.7% of the patients. Patients with and without arm restlessness were compared with regard to clinical and polysomnographic parameters. No between-group differences were found for age at onset of RLS, duration of illness, gender, presence of a family history of RLS, sleep latency, total sleep time, sleep efficiency, and periodic leg movements index during sleep. However, arm restlessness was more frequent in patients with severe RLS. Interestingly, the polysomnographic data failed to differentiate the patients with arm restlessness from those without arm restlessness, except for sleep efficiency, which tended to be lower in patients with arm restlessness. In conclusion, the results of the present study are in agreement with clinical observations that a large proportion of patients with RLS have arm restlessness. PMID- 10752579 TI - Abnormal activity of membrane phospholipid synthetic enzymes in the brain of patients with Friedreich's ataxia and spinocerebellar atrophy type-1. AB - Much evidence, derived from biochemical studies of both blood and autopsied brain, has suggested that phospholipid metabolism is abnormal in patients with Friedreich's ataxia (FA), a disorder characterized by severe neuronal loss in the spinal cord and lower brain stem with no, or only modest, damage in other brain regions. To establish the cause of our recent finding of reduced brain levels of phospholipids in FA, we assayed activities of 10 phospholipid-metabolizing enzymes in the autopsied cerebellar cortex of patients with the disorder and, for comparison, in a group of patients with spinocerebellar ataxia type 1 (SCA-1), a disease characterized, unlike FA, by marked neuronal loss in the cerebellar cortex. Enzyme activities were also measured in four brain areas which are relatively unaffected morphologically in both FA and SCA-1. We found that ethanolamine kinase activity was increased in multiple brain regions of patients with FA (increased 31%-137%) and, more modestly, in SCA-1 (increased 39%-60%), suggesting a nonspecific enhancement of phosphoethanolamine production in both disorders. In contrast, the activity of phosphoethanolamine cytidylyltransferase (PECT), the rate-limiting enzyme of phosphatidylethanolamine synthesis, was significantly and markedly decreased by 35%-78% in the cerebellar, frontal, and occipital cortices of patients with FA but was normal in SCA-1. Reduced PECT activity in FA may explain the lower brain levels of phosphatidylethanolamine in the disorder. Moreover, because decreased PECT activity in FA occurs in brain regions having no, or only modest, morphologic damage, this may represent a systemic change consequent to the frataxin gene defect. Our data also suggest that therapeutic intervention in FA designed to increase synthesis of membrane phospholipids may warrant further investigation. PMID- 10752580 TI - Risperidone treatment of drug-related psychosis in patients with parkinsonism. AB - Risperidone, a novel neuroleptic with approximately equal D2 and 5HT2A receptor blocking properties, has been used to treat drug-related hallucinations in patients with Parkinson's disease. However, the results of only small numbers of patients have been reported with the drug demonstrating limited usefulness. We report our experience with this drug in 39 patients (25 women and 19 men) with parkinsonism. Monitored clinical data included duration of disease, Hoehn and Yahr score, Mini-Mental State Score, Unified Parkinson's Disease Rating Scale (UPDRS) prior to drug administration and after 3 and 6 months of treatment, and response to treatment. Twenty-three patients with Parkinson's disease had either complete or near-complete resolution of hallucinations whereas an unsatisfactory response (N = 6) or worsening of parkinsonism (N = 6) was noted in 12 patients, only six of whom had Parkinson's disease. Excluding patients with diffuse Lewy body disease, there was no significant worsening of the UPDRS scores after either 3 or 6 months of treatment. The presence of dementia did not predict response to treatment. Our results suggest that risperidone is a useful treatment for hallucinations in patients with parkinsonism. PMID- 10752581 TI - Sildenafil citrate (Viagra) for the treatment of erectile dysfunction in men with Parkinson's disease. AB - Sildenafil citrate (Viagra) is a phosphodiesterase type V inhibitor used to treat erectile dysfunction. Ten men with idiopathic Parkinson's disease (PD) and erectile dysfunction were prescribed 50-100 mg sildenafil citrate to use in eight sexual encounters over a 2-month period. Patients underwent Unified Parkinson's Disease Rating Scale (UPDRS) evaluations and completed a Beck's Depression Inventory (BDI) and a Sexual Health Inventory-M version (SHI-M) at baseline and after 8 weeks. There was statistically significant improvement in total SHI-M scores (23.8 +/- 2.0 vs 16.6 +/- 2.8; p = 0.01), overall sexual satisfaction (p = 0.03), satisfaction with sexual desire (p = 0.04), ability to achieve erection (p = 0.02), ability to maintain erection (p = 0.03), and ability to reach orgasm (p = 0.04) with use of sildenafil citrate. UPDRS and BDI scores were not significantly changed. Side effects included headache in one patient during three sexual encounters. In this open-label study, sildenafil citrate significantly improved sexual function in men with PD and erectile dysfunction. PMID- 10752582 TI - "On" freezing in Parkinson's disease: resistance to visual cue walking devices. AB - OBJECTIVE: To measure "on" freezing during unassisted walking (UW) and test if two devices, a modified inverted stick (MIS) and a visual laser beam stick (LBS) improved walking speed and number of "on" freezing episodes in patients with Parkinson's disease (PD). BACKGROUND: Multiple visual cues can overcome "off' freezing episodes and can be useful in improving gait function in parkinsonian patients. These devices have not been specifically tested in "on" freezing, which is unresponsive to pharmacologic manipulations. METHODS: Patients with PD, motor fluctuations and freezing while "on," attempted walking on a 60-ft track with each of three walking conditions in a randomized order: UW, MIS, and LBS. Total time to complete a trial, number of freezes, and the ratio of walking time to the number of freezes were compared using Friedman's test. RESULTS: Twenty-eight patients with PD, mean age 67.81 years (standard deviation [SD] 7.54), mean disease duration 13.04 years (SD 7.49), and mean motor Unified Parkinson's Disease Rating Scale score "on" 32.59 (SD 10.93), participated in the study. There was a statistically significant correlation of time needed to complete a trial and number of freezes for all three conditions (Spearman correlations: UW 0.973, LBS 0.0.930, and MIS 0.842). The median number of freezes, median time to walk in each condition, and median walking time per freeze were not significantly different in pairwise comparisons of the three conditions (Friedman's test). Of the 28 subjects, six showed improvement with the MIS and six with the LBS in at least one outcome measure. CONCLUSION: Assisting devices, specifically based on visual cues, are not consistently beneficial in overcoming "on" freezing in most patients with PD. Because this is an otherwise untreatable clinical problem and because occasional subjects do respond, cautious trials of such devices under the supervision of a health professional should be conducted to identify those patients who might benefit from their long-term use. PMID- 10752583 TI - Posttraumatic akinetic-rigid syndrome resembling Parkinson's disease: a report on three patients. AB - We describe three patients who developed a rapidly evolving posttraumatic akinetic-rigid syndrome (ARS), the clinical manifestations of which were similar to Parkinson's disease, including response to levodopa. Despite initial imaging studies showing traumatic damage to the substantia nigra, the ARS appeared after a delay of 1-5 months after the injury. We stress the importance of magnetic resonance imaging to illustrate nigral damage in all patients in whom head trauma precedes an ARS. PMID- 10752584 TI - Sign language tics in a prelingually deaf man. AB - Gilles de la Tourette syndrome is characterized by vocal and motor tics starting in childhood. Vocal tics may be either noises or words, and the vocal language tics may consist of obscenities (coprolalia) and repetitions of speech that has been heard (echolalia). We describe a prelingually deaf man who has the full array of tics seen in Gilles de la Tourette syndrome, but in whom vocal language tics are replaced by equivalent sign language tics. This is, to our knowledge, the first report of sign language tics in a person with prelingual deafness. The implications of this phenomenon for the separation between language and ideas in tics and the equivalence of sign language to spoken language is discussed. PMID- 10752587 TI - The movement disorder of Nicolas Poussin (1594-1665) [historical article]. PMID- 10752586 TI - Amantadine is beneficial in restless legs syndrome. AB - Twenty-one patients (mean age 70 yrs) with restless legs syndrome (RLS) were treated with amantadine in an open-label trial. Amantadine was started at 100 mg per day and was increased every 3-5 days by 100 mg (up to a maximum of 300 mg per day) until significant relief of symptoms or intolerable side effects were experienced. Patients were rated pre- and posttreatment using an RLS rating scale (0-10). Each patient also rated the degree of response in a continuous scale from 0% (no improvement) to 100% (complete improvement). Eleven of 21 (52%) had subjective benefit to amantadine, with degree of response ranging from 25%-100% (mean 69%) among responders. Six had 95%-100% improvement. The RLS score for all 21 patients dropped from a mean (+/- standard deviation) of 9.8 +/- 0.6 (range, 8 10) pretreatment to 6.6 +/- 3.8 (range, 0-10) posttreatment (p = 0.001). The duration of response was 0-13 months (mean, 3.6 +/- 4.5), with nine responders still remaining on the drug as of last follow up. The mean effective dose was 227 mg per day. The most common side effects were drowsiness (3), fatigue (2), and insomnia (2); only two stopped amantadine because of side effects. We conclude that amantadine is an effective and well-tolerated drug for RLS. PMID- 10752585 TI - Rheumatologic serologies in secondary restless legs syndrome. AB - Diagnostic criteria for restless legs syndrome (RLS) have been established; however, the pathophysiology of this common condition remains elusive. Several secondary forms of RLS potentially include renal failure, iron deficiency, pregnancy, and neuropathy. RLS has also been reported in approximately 25% of patients diagnosed with rheumatoid arthritis and Sjogren's syndrome. We performed clinical and serologic evaluations on 68 patients diagnosed with RLS to determine how many may have concurrent rheumatologic disease that could be causing their RLS symptoms. We compared these with other postulated secondary causes of RLS. No patient had clinical evidence of rheumatologic disease, and only four had any positive serologic evaluations (two positive SSA/SSB and two mildly elevated RF titers). Three of these had a positive family history for RLS. Patients without a family history of RLS did have lower ferritin levels, more cases of neuropathy, and an older age at symptom onset. We do not think rheumatologic disease represents a significant secondary cause of RLS and do not recommend serologic investigation unless there are overt clinical signs. In contrast, our study suggests that neuropathy and serum iron deficiency do represent secondary forms of RLS. PMID- 10752588 TI - Schwab & England: standardization of administration. PMID- 10752589 TI - Idazoxan is ineffective for levodopa-induced dyskinesias in Parkinson's disease. PMID- 10752590 TI - Visuomotor ataxia in corticobasal degeneration. PMID- 10752591 TI - Levodopa-induced dyskinesias in autopsy-proven cortical-basal ganglionic degeneration. PMID- 10752592 TI - Acquired and persistent stuttering as the main symptom of striatal infarction. PMID- 10752593 TI - Unilateral arm tremor as the sole feature of ischemic stroke: a 5-year follow up. PMID- 10752594 TI - Focal task-specific dystonia induced by peripheral trauma. PMID- 10752596 TI - Botulinum toxin for the treatment of oro-facial-lingual-masticatory tardive dyskinesia. PMID- 10752597 TI - Propriospinal myoclonus in ischemic myelopathy secondary to a spinal dural arteriovenous fistula. PMID- 10752595 TI - Paraneoplastic choreic syndrome during non-Hodgkin's lymphoma. PMID- 10752599 TI - The initial treatment of Parkinson's disease. PMID- 10752598 TI - Marked improvement in a stiff-limb patient treated with intravenous immunoglobulin. PMID- 10752600 TI - Treatment of Parkinson's disease should begin with a dopamine agonist. PMID- 10752601 TI - Cortical and brain stem hyperexcitability in a pathologically confirmed case of multiple system atrophy. PMID- 10752602 TI - Hemifacial spasm and hypertension: how strong is the association? PMID- 10752603 TI - Mycophenolate in the prevention of recurrent neutralizing botulinum toxin A antibodies in cervical dystonia. PMID- 10752604 TI - Atypical and typical cranial dystonia following dental procedures. PMID- 10752605 TI - Detection of intact megaDalton protein assemblies of vanillyl-alcohol oxidase by mass spectrometry. AB - Well-resolved ion signals of intact large protein assemblies, with molecular masses extending above one million Dalton, have been detected and mass analyzed using electrospray ionization mass spectrometry, with an uncertainty in mass of <0.2%. The mass spectral data seem to reflect known solution-phase behavior of the studied protein assembly and have therefore been directly used to probe the protein assembly topology and stability as a function of ionic strength and pH. PMID- 10752606 TI - The prion domain of yeast Ure2p induces autocatalytic formation of amyloid fibers by a recombinant fusion protein. AB - The Ure2 protein from Saccharomyces cerevisiae has been proposed to undergo a prion-like autocatalytic conformational change, which leads to inactivation of the protein, thereby generating the [URE3] phenotype. The first 65 amino acids, which are dispensable for the cellular function of Ure2p in nitrogen metabolism, are necessary and sufficient for [URE3] (Masison & Wickner, 1995), leading to designation of this domain as the Ure2 prion domain (UPD). We expressed both UPD and Ure2 as glutathione-S-transferase (GST) fusion proteins in Escherichia coli and observed both to be initially soluble. Upon cleavage of GST-UPD by thrombin, the released UPD formed ordered fibrils that displayed amyloid-like characteristics, such as Congo red dye binding and green-gold birefringence. The fibrils exhibited high beta-sheet content by Fourier transform infrared spectroscopy. Fiber formation proceeded in an autocatalytic manner. In contrast, the released, full-length Ure2p formed mostly amorphous aggregates; a small amount polymerized into fibrils of uniform size and morphology. Aggregation of Ure2p could be seeded by UPD fibrils. Our results provide biochemical support for the proposal that the [URE3] state is caused by a self-propagating inactive form of Ure2p. We also found that the uncleaved GST-UPD fusion protein could polymerize into amyloid fibrils by a strictly autocatalytic mechanism, forcing the GST moiety of the protein to adopt a new, beta-sheet-rich conformation. The findings on the GST-UPD fusion protein indicate that the ability of the prion domain to mediate a prion-like conversion process is not specific for or limited to the Ure2p. PMID- 10752607 TI - Entropic barriers, transition states, funnels, and exponential protein folding kinetics: a simple model. AB - This paper presents an analytically tractable model that captures the most elementary aspect of the protein folding problem, namely that both the energy and the entropy decrease as a protein folds. In this model, the system diffuses within a sphere in the presence of an attractive spherically symmetric potential. The native state is represented by a small sphere in the center, and the remaining space is identified with unfolded states. The folding temperature, the time-dependence of the populations, and the relaxation rate are calculated, and the folding dynamics is analyzed for both golf-course and funnel-like energy landscapes. This simple model allows us to illustrate a surprising number of concepts including entropic barriers, transition states, funnels, and the origin of single exponential relaxation kinetics. PMID- 10752609 TI - Conservation among HSP60 sequences in relation to structure, function, and evolution. AB - The chaperonin HSP60 (GroEL) proteins are essential in eubacterial genomes and in eukaryotic organelles. Functional regions inferred from mutation studies and the Escherichia coli GroEL 3D crystal complexes are evaluated in a multiple alignment across 43 diverse HSP60 sequences, centering on ATP/ADP and Mg2+ binding sites, on residues interacting with substrate, on GroES contact positions, on interface regions between monomers and domains, and on residues important in allosteric conformational changes. The most evolutionary conserved residues relate to the ATP/ADP and Mg2+ binding sites. Hydrophobic residues that contribute in substrate binding are also significantly conserved. A large number of charged residues line the central cavity of the GroEL-GroES complex in the substrate-releasing conformation. These span statistically significant intra- and inter-monomer three dimensional (3D) charge clusters that are highly conserved among sequences and presumably play an important role interacting with the substrate. Unaligned short segments between blocks of alignment are generally exposed at the outside wall of the Anfinsen cage complex. The multiple alignment reveals regions of divergence common to specific evolutionary groups. For example, rickettsial sequences diverge in the ATP/ADP binding domain and gram-positive sequences diverge in the allosteric transition domain. The evolutionary information of the multiple alignment proffers attractive sites for mutational studies. PMID- 10752608 TI - An additional aromatic interaction improves the thermostability and thermophilicity of a mesophilic family 11 xylanase: structural basis and molecular study. AB - In a general approach to the understanding of protein adaptation to high temperature, molecular models of the closely related mesophilic Streptomyces sp. S38 Xyl1 and thermophilic Thermomonospora fusca TfxA family 11 xylanases were built and compared with the three-dimensional (3D) structures of homologous enzymes. Some of the structural features identified as potential contributors to the higher thermostability of TfxA were introduced in Xyl1 by site-directed mutagenesis in an attempt to improve its thermostability and thermophilicity. A new Y11-Y16 aromatic interaction, similar to that present in TfxA and created in Xyl1 by the T11Y mutation, improved both the thermophilicity and thermostability. Indeed, the optimum activity temperature (70 vs. 60 degrees C) and the apparent Tm were increased by about 9 degrees C, and the mutant was sixfold more stable at 57 degrees C. The combined mutations A82R/F168H/N169D/delta170 potentially creating a R82-D169 salt bridge homologous to that present in TfxA improved the thermostability but not the thermophilicity. Mutations R82/D170 and S33P seemed to be slightly destabilizing and devoid of influence on the optimal activity temperature of Xyl1. Structural analysis revealed that residues Y11 and Y16 were located on beta-strands B1 and B2, respectively. This interaction should increase the stability of the N-terminal part of Xyl1. Moreover, Y11 and Y16 seem to form an aromatic continuum with five other residues forming putative subsites involved in the binding of xylan (+3, +2, +1, -1, -2). Y11 and Y16 might represent two additional binding subsites (-3, -4) and the T11Y mutation could thus improve substrate binding to the enzyme at higher temperature and thus the thermophilicity of Xyl1. PMID- 10752610 TI - Characterization of a discontinuous epitope of the human immunodeficiency virus (HIV) core protein p24 by epitope excision and differential chemical modification followed by mass spectrometric peptide mapping analysis. AB - A combination of epitope excision, epitope extraction, and differential chemical modification followed by mass spectrometric peptide mapping was used for the characterization of a discontinuous epitope that is recognized by the mouse anti HIV-p24 monoclonal antibody 5E2.A3. In epitope excision, the protein is first bound to an immobilized antibody and then digested with proteolytic enzymes. In epitope extraction, the protein is first digested and subsequently allowed to react with the antibody. After epitope excision of the p24-5E2.A3 complex with endoproteinase Lys-C, a large fragment remained affinity bound corresponding to amino acids 1-158 of HIV-p24 (fragment 1-158). Further digestion, however, resulted in loss of affinity. Moreover, no affinity-bound fragments were observed after an epitope extraction experiment. These data from the epitope excision and extraction experiments suggest that the epitope is discontinuous. For the further characterization of the epitope, amino groups in the epitope-containing fragment were acetylated in both the affinity bound and free states followed by mass spectrometric analysis. Two successive acetylation reactions were performed: (1) the first used a low molar excess of acetic anhydride, and (2) the second, after separation from the antibody, a high molar excess of its hexadeuteroderivative. This isotopic labeling procedure, in combination with high resolution mass spectrometry, allowed the precise determination of relative reactivities of amino groups. In this study, no differences were observed in the ranking of the relative reactivities of five lysine residues. However, the N-terminal amino group was found to be part of the discontinuous epitope. PMID- 10752611 TI - A "structural" water molecule in the family of fatty acid binding proteins. AB - A single water molecule (w135), buried within the structure of rat intestinal fatty acid binding protein (I-FABP), is investigated by NMR, molecular dynamics simulations, and analysis of known crystal structures. An ordered water molecule was found in structurally analogous position in 24 crystal structures of nine different members of the family of fatty acid binding proteins. There is a remarkable conservation of the local structure near the w135 binding site among different proteins from this family. NMR cross-relaxation measurements imply that w135 is present in the I-FABP:ANS (1-sulfonato-8-(1')anilinonaphthalene) complex in solution with the residence time of >300 ps. Mean-square positional fluctuations of w135 oxygen observed in MD simulations (0.18 and 0.13 A2) are comparable in magnitude to fluctuations exhibited by the backbone atoms and result from highly constrained binding pocket as revealed by Voronoi volumes (averages of 27.0 +/- 1.8 A3 and 24.7 +/- 2.2 A3 for the two simulations). Escape of w135 from its binding pocket was observed only in one MD simulation. The escape process was initiated by interactions with external water molecules and was accompanied by large deformations in beta-strands D and E. Immediately before the release, w135 assumed three distinct states that differ in hydrogen bonding topology and persisted for about 15 ps each. Computer simulations suggest that escape of w135 from the I-FABP matrix is primarily determined by conformational fluctuations of the protein backbone and interactions with external water molecules. PMID- 10752612 TI - The effect of nucleotide bias upon the composition and prediction of transmembrane helices. AB - Transmembrane helices are the most readily predictable secondary structure components of proteins. They can be predicted to a high degree of accuracy in a variety of ways. Many of these methods compare new sequence data with the sequence characteristics of known transmembrane domains. However, the known transmembrane sequences are not necessarily representative of a particular organism. We attempt to demonstrate that parameters optimized for the known transmembrane domains are far from optimal when predicting transmembrane regions in a given genome. In particular, we have tested the effect of nucleotide bias upon the composition and hence the prediction characteristics of transmembrane helices. Our analysis shows that nucleotide bias of a genome has a strong and predictable influence upon the occurrences of several of the most important hydrophobic amino acids found within transmembrane helices. Thus, we show that nucleotide bias should be taken into account when determining putative transmembrane domains from sequence data. PMID- 10752613 TI - Analysis of the dynamic properties of Bacillus circulans xylanase upon formation of a covalent glycosyl-enzyme intermediate. AB - NMR spectroscopy was used to search for mechanistically significant differences in the local mobility of the main-chain amides of Bacillus circulans xylanase (BCX) in its native and catalytically competent covalent glycosyl-enzyme intermediate states. 15N T1, T2, and 15N[1H] NOE values were measured for approximately 120 out of 178 peptide groups in both the apo form of the protein and in BCX covalently modified at position Glu78 with a mechanism-based 2-deoxy-2 fluoro-beta-xylobioside inactivator. Employing the model-free formalism of Lipari and Szabo, the measured relaxation parameters were used to calculate a global correlation time (tau(m)) for the protein in each form (9.2 +/- 0.2 ns for apo BCX; 9.8 +/- 0.3 ns for the modified protein), as well as individual order parameters for the main-chain NH bond vectors. Average values of the order parameters for the protein in the apo and complexed forms were S2 = 0.86 +/- 0.04 and S2 = 0.91 +/- 0.04, respectively. No correlation is observed between these order parameters and the secondary structure, solvent accessibility, or hydrogen bonding patterns of amides in either form of the protein. These results demonstrate that the backbone of BCX is well ordered in both states and that formation of the glycosyl-enzyme intermediate leads to little change, in any, in the dynamic properties of BCX on the time scales sampled by 15N-NMR relaxation measurements. PMID- 10752614 TI - Early intermediates in the PDI-assisted folding of ribonuclease A. AB - The oxidative refolding of ribonuclease A has been investigated in several experimental conditions using a variety of redox systems. All these studies agree that the formation of disulfide bonds during the process occurs through a nonrandom mechanism with a preferential coupling of certain cysteine residues. We have previously demonstrated that in the presence of glutathione the refolding process occurs through the reiteration of two sequential reactions: a mixed disulfide with glutathione is produced first which evolves to form an intramolecular S-S bond. In the same experimental conditions, protein disulfide isomerase (PDI) was shown to catalyze formation and reduction of mixed disulfides with glutathione as well as formation of intramolecular S-S bonds. This paper reports the structural characterization of the one-disulfide intermediate population during the oxidative refolding of Ribonuclease A under the presence of PDI and glutathione with the aim of defining the role of the enzyme at the early stages of the reaction. The one-disulfide intermediate population occurring at the early stages of both the uncatalyzed and the PDI-catalyzed refolding was purified and structurally characterized by proteolytic digestion followed by MALDI-MS and LC/ESIMS analyses. In the uncatalyzed refolding, a total of 12 disulfide bonds out of the 28 theoretical possible cysteine couplings was observed, confirming a nonrandom distribution of native and nonnative disulfide bonds. Under the presence of PDI, only two additional nonnative disulfides were detected. Semiquantitative LC/ESIMS analysis of the distribution of the S-S bridged peptides showed that the most abundant species were equally populated in both the uncatalyzed and the catalyzed process. This paper shows the first structural characterization of the one-disulfide intermediate population formed transiently during the refolding of ribonuclease A in quasi-physiological conditions that mimic those present in the ER lumen. At the early stages of the process, three of the four native disulfides are detected, whereas the Cys26 Cys84 pairing is absent. Most of the nonnative disulfide bonds identified are formed by nearest-neighboring cysteines. The presence of PDI does not significantly alter the distribution of S-S bonds, suggesting that the ensemble of single-disulfide species is formed under thermodynamic control. PMID- 10752615 TI - Cytochrome c folds through a smooth funnel. AB - A dominant feature of folding of cytochrome c is the presence of nonnative His heme kinetic traps, which either pre-exist in the unfolded protein or are formed soon after initiation of folding. The kinetically trapped species can constitute the majority of folding species, and their breakdown limits the rate of folding to the native state. A temperature jump (T-jump) relaxation technique has been used to compare the unfolding/folding kinetics of yeast iso-2 cytochrome c and a genetically engineered double mutant that lacks His-heme kinetic traps, H33N,H39K iso-2. The results show that the thermodynamic properties of the transition states are very similar. A single relaxation time tau(obs) is observed for both proteins by absorbance changes at 287 nm, a measure of solvent exclusion from aromatic residues. At temperatures near Tm, the midpoint of the thermal unfolding transitions, tau(obs) is four to eight times faster for H33N,H39K iso-2 (tau(obs) approximately 4-10 ms) than for iso-2 (tau(obs) approximately 20-30 ms). T-jumps show that there are no kinetically unresolved (tau < 1-3 micros T-jump dead time) "burst" phases for either protein. Using a two-state model, the folding (k(f)) and unfolding (k(u)) rate constants and the thermodynamic activation parameters standard deltaGf, standard deltaGu, standard deltaHf, standard deltaHu, standard deltaSf, standard deltaSu are evaluated by fitting the data to a function describing the temperature dependence of the apparent rate constant k(obs) (= tau(obs)(-1)) = k(f) + k(u). The results show that there is a small activation enthalpy for folding, suggesting that the barrier to folding is largely entropic. In the "new view," a purely entropic kinetic barrier to folding is consistent with a smooth funnel folding landscape. PMID- 10752616 TI - The X-ray structure of a chitinase from the pathogenic fungus Coccidioides immitis. AB - The X-ray structure of chitinase from the fungal pathogen Coccidioides immitis has been solved to 2.2 A resolution. Like other members of the class 18 hydrolase family, this 427 residue protein is an eight-stranded beta/alpha-barrel. Although lacking an N-terminal chitin anchoring domain, the enzyme closely resembles the chitinase from Serratia marcescens. Among the conserved features are three cis peptide bonds, all involving conserved active site residues. The active site is formed from conserved residues such as tryptophans 47, 131, 315, 378, tyrosines 239 and 293, and arginines 52 and 295. Glu171 is the catalytic acid in the hydrolytic mechanism; it was mutated to a Gln, and activity was abolished. Allosamidin is a substrate analog that strongly inhibits the class 18 enzymes. Its binding to the chitinase hevamine has been observed, and we used conserved structural features of the two enzymes to predict the inhibitors binding to the fungal enzyme. PMID- 10752617 TI - Direct evidence by H/D exchange and ESI-MS for transient unproductive domain interaction in the refolding of an antibody scFv fragment. AB - The refolding kinetics of a single-chain Fv (scFv) fragment, derived from a stabilized mutant of the phosphorylcholine binding antibody McPC603, was investigated by H/D exchange and ESI-MS and compared with the folding kinetics of its constituting domains V(H) and V(L). Both V(H) and V(L) adopt essentially native-like exchange protection within the dead time of the manual-mixing H/D exchange experiment (10 s) and in the case of V(L), which contains two cis prolines in the native conformation, this fast protection is independent of proline cis/trans isomerization. At the earliest time point resolvable by manual mixing, fewer deuterons are protected in the scFv fragment than in the two isolated domains together, despite the fact that the scFv fragment is significantly more stable than V(L) and V(H). Full H/D exchange protection in the scFv fragment is gained on a time scale of minutes. This means that the domains in the scFv fragment do not refold independently. Rather, they associate prematurely and in nonnative form, a kinetic trap. Unproductive domain association is observed both after equilibrium- and short-term denaturation. For the equilibrium-denatured scFv fragment, whose native structure formation is dependent on a cis conformation of an interface proline in V(L), this cis/trans isomerization reaction proceeds about one order in magnitude more slowly than the escape from the trap to a conformation where full H/D exchange protection is already achieved. We interpret these data in terms of a general kinetic scheme involving intermediates with and without domain association. PMID- 10752618 TI - Fluorinated alcohol, the third group of cosolvents that stabilize the molten globule state relative to a highly denatured state of cytochrome c. AB - The effects of 1,1,1,3,3,3-hexafluoro-isopropanol (HFIP) on the conformation of cytochrome c (cyt c) at pH 1.9 were studied using a combination of spectroscopic and physical methods. Analysis varying the HFIP concentration showed that a compact denatured conformation (M(HF)) accumulates in a low concentration range of HFIP in the middle of structural transition from the highly unstructured acid denatured state to the highly helical alcohol-denatured state of cyt c. This contrasts clearly with the effect of isopropanol (IP), in which no compact conformation accompanied with the transition. Analysis varying concentrations of HFIP and NaCl concurrently showed that the M(HF) state of cyt c is essentially identical to the salt-induced molten-globule (M(G)) state, and the M(G) state in the presence of salt was also stabilized by a low concentration of HFIP. Furthermore, 2,2,2-trifluoroethanol stabilized M(HF) similarly to HFIP, supporting the proposition that the specific effect observed for HFIP is caused by fluorination of alcohol. The mechanism stabilizing compact conformation by HFIP remains unclear, but is probably distinct from that of salts and polyols, which are also known to stabilize the M(G)-like state. PMID- 10752619 TI - Alternative modes of binding of proteins with tandem SH2 domains. AB - The issue of specificity in tyrosine kinase intracellular signaling mediated by src homology 2 (SH2) domains has great importance in the understanding how individual signals maintain their mutual exclusivity and affect downstream responses. Several proteins contain tandem SH2 domains that, on interacting with their ligand, provide a higher level of specificity than can be afforded by the interaction of a single SH2 domain. In this study, we focus on the comparison of two proteins ZAP70 and the p85 subunit of PI 3-kinase, which although distinctly different in function and general structure, possess tandem SH2 domains separated by a linker region and which bind to phosphorylated receptor molecules localized to the cell membrane. Binding studies using isothermal titration calorimetry show that these two proteins interact with peptides mimicking their physiological ligands in very different ways. In the case of the SH2 domains from ZAP70, they interact with a stoichiometry of unity, while p85 is able to make two distinct interactions, one with a stoichiometry of 1:1 and the other with two p85 molecules interacting with one receptor. The observation of two different modes of binding of p85 might be important in providing different cellular responses based on fluctuating intracellular concentration regimes of this protein. Thermodynamic data on both proteins suggest that a conformational change occurs on binding. On investigation of this structural change using a truncated form of p85 (including just the two SH2 domains and the inter-SH2 region), both NMR and circular dichroism spectroscopic studies failed to show significant changes in secondary structure. This suggests that any conformational change associated with binding is small and potentially limited to loop regions of the protein. PMID- 10752620 TI - Optimal region of average side-chain entropy for fast protein folding. AB - Search and study the general principles that govern kinetics and thermodynamics of protein folding generates new insight into the factors that control this process. Here, we demonstrate based on the known experimental data and using theoretical modeling of protein folding that side-chain entropy is one of the general determinants of protein folding. We show for proteins belonging to the same structural family that there exists an optimal relationship between the average side-chain entropy and the average number of contacts per residue for fast folding kinetics. Analysis of side-chain entropy for proteins that fold without additional agents demonstrates that there exists an optimal region of average side-chain entropy for fast folding. Deviation of the average side-chain entropy from the optimal region results in an anomalous protein folding process (prions, alpha-lytic protease, subtilisin, some DNA-binding proteins). Proteins with high or low side-chain entropy would have extended unfolded regions and would require some additional agents for complete folding. Such proteins are common in nature, and their structure properties have biological importance. PMID- 10752621 TI - Cloning, expression, purification, and preliminary characterization of a putative hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803. AB - The genome of the unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a gene (slr2097, glbN) encoding a 123 amino-acid product with sequence similarity to globins. Related proteins from cyanobacteria, ciliates, and green algae bind oxygen and have a pronounced tendency to coordinate the heme iron with two protein ligands. To study the structural and functional properties of Synechocystis sp. PCC 6803 hemoglobin, slr2097 was cloned and overexpressed in Escherichia coli. Purification of the hemoglobin was performed after addition of hemin to the clarified cell lysate. Recombinant, heme-reconstituted ferric Synechocystis sp. PCC 6803 hemoglobin was found to be a stable helical protein, soluble to concentrations higher than 500 microM. At neutral pH, it yielded an electronic absorption spectrum typical of a low-spin ferric species, with maxima at 410 and 546 nm. The proton NMR spectrum revealed sharp lines spread over a chemical shift window narrower than 40 ppm, in support of low-spin hexacoordination of the heme iron. Nuclear Overhauser effects demonstrated that the heme is inserted in the protein matrix to produce one major equilibrium form. Addition of dithionite resulted in an absorption spectrum with maxima at 426, 528, and 560 nm. This reduced form appeared capable of carbon monoxide binding. Optical data also suggested that cyanide ions could bind to the heme in the ferric state. The spectral properties of the putative Synechocystis sp. PCC 6803 hemoglobin confirmed that it can be used for further studies of an ancient hemoprotein structure. PMID- 10752622 TI - Steady state and time resolved effects of guanidine hydrochloride on the structure of Humicola lanuginosa lipase revealed by fluorescence spectroscopy. AB - Effects of guanidine hydrochloride (GdnHCl) on the structure and dynamics of wild type Humicola lanuginosa lipase (HLL) and its two mutants were studied. The latter were S146A (with the active site Ser replaced by Ala) and the single Trp mutant W89m, with substitutions W117F, W221H, and W260H. Steady-state, stopped flow, and time-resolved laser-induced fluorescence spectroscopy were carried out as a function of [GdnHCl]. The maximum emission wavelength and fluorescence lifetimes revealed the microenvironment of the tryptophan(s) in these lipases to become more polar upon increasing [GdnHCl]. However, significant extent of tertiary structure in GdnHCl is suggested by the observation that both wild-type HLL and W89m remain catalytically active at rather high GdnHCl concentrations of >6 and 4.0 M, respectively. Changes in steady-state emission anisotropy, as well as variation in rotational correlation times and residual anisotropy values, demonstrate that upon increasing [GdnHCl] the structure of the lipases became more loose, with increasing amplitude of structural fluctuations. Finally, intermediate states in the course of exposure of the proteins to GdnHCl were revealed by stopped-flow fluorescence measurements. PMID- 10752623 TI - Structural similarities and differences in Staphylococcus aureus exfoliative toxins A and B as revealed by their crystal structures. AB - Staphylococcal aureus epidermolytic toxins (ETs) A and B are responsible for the induction of staphylococcal scalded skin syndrome, a disease of neonates and young children. The clinical features of this syndrome vary from localized blisters to severe exfoliation affecting most of the body surface. Comparison of the crystal structures of two subtypes of ETs-rETA (at 2.0 A resolution), rETB (at 2.8 A resolution), and an active site variant of rETA, Ser195Ala at 2.0 A resolution has demonstrated that their overall topology resembles that of a "trypsin-like" serine protease, but with significant differences at the N- and C termini and loop regions. The details of the catalytic site in both ET structures are very similar to those in glutamate-specific serine proteases, suggesting a common catalytic mechanism. However, the "oxyanion hole," which is part of the catalytic sites of glutamate specific serine proteases, is in the closed or inactive conformation for rETA, yet in the open or active conformation for rETB. The ETs contain a unique amphipathic helix at the N-terminus, and it appears to be involved in optimizing the conformation of the catalytic site residues. Determination of the structure of the rETA catalytic site variant, Ser195Ala, showed no significant perturbation at the active site, establishing that the loss of biological and esterolytic activity can be attributed solely to disruption of the catalytic serine residue. Finally, the crystal structure of ETs, together with biochemical data and mutagenesis studies, strongly confirms the classification of these molecules as "serine proteases" rather than "superantigens." PMID- 10752624 TI - Role of residue Y99 in tissue plasminogen activator. AB - The crystal structure of the fibrinolytic enzyme tissue plasminogen activator (tPA) shows that the bulky side chain of Y99 hinders access to the active site by partially occluding the S2 site and may be responsible for the low catalytic activity of tPA toward plasminogen. We have tested the role of Y99 by replacing it with Leu, the residue found in more proficient proteases like trypsin and thrombin. The Y99L replacement results in an increase in the k(cat)/Km for chromogenic substrates due to enhanced diffusion into the active site. The increase is modest (threefold) for substrates specific for tPA that carry Pro or Gly at P2, but reaches 80-fold for less specific substrates carrying Arg at P2. On the other hand, the Y99L mutation has no effect on the activity of tPA toward the natural substrate plasminogen, that carries Gly at P2, and reduces more than 10-fold the inhibition of tPA by plasminogen activator inhibitor-1 (PAI-1), that carries Ala at P2. We conclude that the steric hindrance provided by Y99 in the crystal structure affects mostly nonphysiological substrates with bulky residues at P2. In addition, residue Y99 plays an active role in the recognition of PAI-1, but not plasminogen. Mutations of Y99 could therefore afford a resistance to inhibition by PAI-1 without compromising the fibrinolytic potency of tPA, a result of potential therapeutic relevance. PMID- 10752625 TI - Convergent evolution of major histocompatibility complex molecules in humans and New World monkeys. AB - In both Old World and New World monkeys Mhc-DRB sequences have been found which resemble human DRB1*03 and DRB3 genes in their second exon. The resemblance is shared sequence motifs and clustering of the genes or the encoded proteins in phylogenetic trees. This similarity could be due to common ancestry, convergence at the molecular level, or chance. To test which of these three explanations applies, we sequenced segments of New World monkey and macaque genes which encompass the entire second exon and large parts of both flanking introns. The test strongly supports the monophyly of New World monkey DRB intron sequences. The phylogenies of introns 1 and 2 from DRB1*03-like and DRB3-like genes are congruent, but both are incongruent with the exon 2-based phylogeny. The matching of intron 1- and intron 2-based phylogenies with each other suggests that reciprocal recombination has not played a major role in exon 2 evolution. Statistical comparisons of exon 2 from different DRB1*03 and DRB3 lineages indicate that it was neither gene conversion (descent), nor chance, but molecular convergence that has shaped their characteristic motifs. The demonstration of convergence in anthropoid Mhc-DRB genes has implications for the classification, age, and mechanism of generation of DRB allelic lineages. PMID- 10752626 TI - The SH2D2A gene encoding the T-cell-specific adapter protein (TSAd) is localized centromeric to the CD1 gene cluster on human Chromosome 1. AB - The SH2D2A gene encoding the T-cell-specific adapter protein (TSAd), was isolated from a human Chromosome (Chr) 1 cosmid library (LLNL, UK HGMP). The gene spans 11 kilobases and contains nine exons and eight introns. Four alternative transcript variants were observed in activated T cells. Three single-nucleotide polymorphisms were identified within intron 2. A variable number of GA repeats was found at position -340 from the first coding ATG. Linkage analysis using this marker in eight CEPH families showed that the SH2D2A gene is located close to the D1S2624 marker on Chr 1q21-1q22. Physical mapping of a PAC and BAC contig containing the CD1 gene cluster telomeric to D1S2624 failed to identify a clone harboring the SH2D2A gene. Thus the SH2D2A gene is located centromeric to the CD1 gene cluster on Chr 1. PMID- 10752627 TI - Trans-species polymorphism of the major histocompatibility complex-encoded proteasome subunit LMP7 in an amphibian genus, Xenopus. AB - LMP7 (PSMB8) is a major histocompatibility complex (MHC)-encoded catalytic subunit of 20S immunoproteasome, which is responsible for the production of antigenic peptide to be presented by the MHC class I molecules. Two highly diverged allelic lineages of LMP7, termed LMP7A and LMP7B, have been identified previously in an amphibian, Xenopus laevis. Fourteen Xenopus species were analyzed by genomic Southern hybridization using LMP7A- and LMP7B-specific probes. Ten had both LMP7A and LMP7B, and the other 4 had only LMP7A. Identification of LMP7A and LMP7B was confirmed by reverse transcription polymerase chain reaction/sequencing analysis of LMP7 mRNA including eight diagnostic amino acid residues that discriminate the two allelic lineages. These data suggest that these two allelic lineages were established more than 80 million years ago, and were transmitted from species to species. Trans-species evolution has so far been reported for MHC class I and II molecules in mammals and teleost fish, and is believed to be a basis for the extraordinary polymorphism of these molecules. A similar mode of evolution of the LMP7 alleles in Xenopus provides a possible explanation for the linkage of the LMP7 gene with the MHC in all vertebrates analyzed to date. PMID- 10752628 TI - A novel truncated isoform of the mannose-binding lectin-associated serine protease (MASP) from the common carp (Cyprinus carpio). AB - The mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs) play crucial roles in activation of the lectin pathway of the complement system. Mammals and Xenopus possess two distinct MASPs, MASP1 and MASP2, with different substrate specificity. Recently, a truncated form named MAp19 or sMAP, composed of N-terminal C1r/C1s/Uegf/bone morphogenetic protein (CUB)-1 and epidermal growth factor domains of MASP2, has been shown to be generated by alternative polyadenylation and splicing from the MASP2 gene. In the present study, we isolated cDNA encoding a novel MASP-related protein, designated MRP, from carp. MRP is distinct from MAp19/sMAP in containing two additional domains, CUB-2 and short consensus repeat (SCR)-1, followed by a unique C-terminal 21 amino acids, but resembles it by also lacking the serine protease domain, suggesting that carp MRP is a functional homologue of human MAp19/sMAP. Analyses of polymerase chain reaction (PCR)-amplified carp genomic DNA, from CUB-2 to SCR-2 of MASP, indicated that carp possess duplicated MASP genes, designated MASP-A and MASP-B, both of which contain an exon encoding the MRP-specific C-terminal stretch between the exons coding for SCR-1 and SCR-2 domains. Reverse transcription-PCR analysis showed that both MASP genes of carp produce the two MASP isoforms, MASP and MRP, through alternative polyadenylation and splicing. The conservation of MASP isoforms that lack the catalytic domain in both carp and human implies that they meet an essential requirement in the MBL-MASP complex of the lectin pathway. PMID- 10752629 TI - Association of TNFA gene polymorphism at position -308 with susceptibility to irritant contact dermatitis. AB - Mechanisms underlying susceptibility to skin irritants are not clearly understood. Cytokines play a key role in inflammation, and functional polymorphisms in cytokine genes may affect responses to irritants. We investigated the relationship between polymorphism in the tumor necrosis factor (TNF) alpha-chain gene and responses to irritants. Volunteers (n=221) tested with sodium dodecyl sulfate (SDS) and benzalkonium chloride (BKC) were divided into responders and nonresponders and high and low irritant-threshold groups. DNA was assayed for the TNF-308 polymorphism by a polymerase chain reaction-restriction fragment length polymorphism method. There was a significant increase in the A allele (P=0.030) and AA genotype (P=0.023) in both the SDS low irritant-threshold group and in SDS responders (A allele P=0.022, AA genotype P=0.048). In the BKC low irritant-threshold group, we found a significant increase in the A allele (P=0.002) and AA genotype (P=0.016). Individuals with a low threshold to both irritants demonstrated a significant increase (P=0.002) in the A allele. This is the first description of a nonatopic genetic marker for irritant susceptibility in normal individuals. Genotyping for the TNF-308 polymorphism may thus contribute to screening of individuals deemed at risk of developing irritant contact dermatitis. PMID- 10752630 TI - Mouse FcgammaRI: identification and functional characterization of five new alleles. AB - The mouse Fcgr1 gene encoding the high-affinity IgG receptor (FcgammaRI) exists as two known alleles, FcgammaRI-BALB and FcgammaRI-NOD, and these alleles exhibit functional differences. To determine whether other alleles exist in mouse strains, Fcgr1 coding regions from 35 strains of mice were sequenced and a further five alleles were identified. The FcgammaRI-BALB and NOD alleles are now designated the "a" and "d" alleles, respectively. Analysis of the five new alleles revealed that although no polymorphisms were observed in the two leader exons, nucleotide and subsequent amino acid changes were observed in the exons encoding the extracellular domains, and transmembrane and cytoplasmic tail. The cDNA of the seven alleles (a-g) were isolated and transiently transfected into COS cells, and IgG-binding studies were performed. Receptors encoded by four of the five new alleles (b, c, f, g) bound IgG2a with high affinity, displaying IgG binding characteristics similar to the a allele (previously FcgammaRI-BALB). The d allele (previously FcgammaRI-NOD) and the e allele [derived from Mus spretus (SPRET/Ei)] encoded receptors which showed broader specificity by binding monomeric IgG2a, IgG2b, and IgG3. PMID- 10752632 TI - Sequence and diversity of DRB genes of Aotus nancymaae, a primate model for human malaria parasites. AB - The New World primate Aotus nancymaae is susceptible to infection with the human malaria parasite Plasmodium falciparum and Plasmodium vivax and has therefore been recommended by the World Health Organization as a model for evaluation of malaria vaccine candidates. We present here a first step in the molecular characterization of the major histocompatibility complex (MHC) class II DRB genes of Aotus nancymaae (owl monkey or night monkey) by nucleotide sequence analysis of the polymorphic exon 2 segments. In a group of 15 nonrelated animals captivated in the wild, 34 MHC DRB alleles could be identified. Six allelic lineages were detected, two of them having human counterparts, while two other lineages have not been described in any other New World monkey species studied. As in the common marmoset, the diversity of DRB alleles appears to have arisen largely by point mutations in the beta-pleated sheets and by frequent exchange of fixed sequence motifs in the alpha-helical portion. Pairs of alleles differing only at amino acid position b86 by an exchange of valine to glycine are present in Aotus, as in humans. Essential amino acid residues contributing to MHC DR peptide binding pockets number 1 and 4 are conserved or semiconserved between HLA DR and Aona-DRB molecules, indicating a capacity to bind similar peptide repertoires. These results support fully our using Aotus monkeys as an animal model for evaluation of future subunit vaccine candidates. PMID- 10752631 TI - Immunoglobulin kappa light-chain V, J, and C gene sequences of the owl monkey Aotus nancymaae. AB - Sequences of Aotus nancymaae immunoglobulin kappa light-chain rearrangements were analyzed after reverse transcription polymerase chain reaction. Among 22 in-frame rearrangements analyzed, 12 IGKV genes belonging to the families 1, 2, or 3 were identified. Aotus counterparts for all five human IGKJ genes were found. The identity of the deduced human and Aotus amino acid sequences was between 83% and 92% for junctional regions and 74% for the constant region. Sequence comparisons between rearrangements indicated that somatic mutations, the addition of nongermline-encoded nucleotides, and exonuclease trimming contribute to the generation of diversity of Aotus immunoglobulin kappa chains. The high identity of Aotus and human IGK genes is comparable to that of T-cell receptor genes and further supports the proposal to use the Aotus Plasmodium falciparum infection model for the evaluation of malaria vaccine candidates. PMID- 10752633 TI - Predominance of one T-cell antigen receptor BV haplotype in African populations. AB - The human T-cell antigen receptor (TCR) is the counter-receptor for the HLA/peptide complex displayed on the surface of antigen-presenting cells. It confers antigen specificity on T lymphocytes and therefore plays a central role in pathogen recognition and host response. The most frequently used form of the TCR is a heterodimer composed of variable alpha and beta chains. We investigated allele frequencies for four variable-region gene segments of the beta chain (2S1, 3S1, 8S3, and 15S1) in 146 Caucasians and 165 Africans. The results reveal significant unexpected differences between the two populations for allele frequencies, phenotypes, genotypes, and haplotypes. Among Caucasians, there are 43 phenotypes, whereas there are 31 among the Africans studied. There are 17 haplotypes in the Caucasian sample but only 10 in Africans. This loss of diversity is largely due to the high frequency of one haplotype in the African sample which represents 65% of the informative chromosomes. At least one copy of this haplotype is present in 90% of informative individuals. As a result, 29% of Africans are homozygous for the common haplotype. Less genetic diversity at TCRBV is unexpected, since Africans usually show greater genetic diversity than other ethnic groups. For example, there are approximately twice as many HLA haplotypes in Africans compared to Caucasians. Homozygosity is also unexpected because it reduces the number of TCR variants available to recognize HLA pathogen-derived peptide complexes. PMID- 10752634 TI - Lack of association between atopic asthma and polymorphisms of the histamine H1 receptor, histamine H2 receptor, and histamine N-methyltransferase genes. PMID- 10752635 TI - Germline sequences of V(H)7183 gene family members in C57BL/6 mice demonstrate natural selection of particular sequences during recent evolution. PMID- 10752636 TI - A single amino acid substitution causes loss of expression of a MICA allele. PMID- 10752637 TI - The HLA-DRB4 gene does not explain genetic susceptibility in HLA-DQ2-negative celiac disease. PMID- 10752638 TI - Regioselective hydroxylation of debrisoquine by cytochrome P4502D6: implications for active site modelling. AB - 1. Debrisoquine, a prototypic probe substrate for human cytochrome P4502D6 (CYP2D6), is hydroxylated at the alicyclic C4-position by this enzyme. Phenolic metabolites of debrisoquine (5-, 6-, 7- and 8-hydroxydebrisoquine) have also been reported as in vivo metabolites, but the role of CYP2D6 in their formation is unclear. 2. As part of studies to develop a predictive model of the active site of CYP2D6 using pharmacophore and homology modelling techniques, it became important to determine the precise regioselective hydroxylation of debrisoquine by CYP2D6. 3. Data from studies with human liver microsomes and yeast microsomes containing cDNA-derived CYP2D6 demonstrated unequivocally that debrisoquine was hydroxylated by CYP2D6 at each aromatic site in the molecule, as well as at the alicyclic 4-position. The four phenolic metabolites amounted to > 60% of the total identified products and the pattern of regioselective hydroxylation (4-HD > 7-HD > 6-HD > 8-HD > 5-HD) was similar in both in vitro systems. 4. A pharmacophore model for CYP2D6 indicated that while the hydroxylation of debrisoquine at alternative positions could arise from the substrate adopting multiple binding orientations, the energy constraints for the aromatic hydroxylations were unfavourable. An alternative proposal involving essentially a single binding orientation and a mechanism of hydroxylation based on benzylic radical spin delocalization could satisfactorily rationalize all the hydroxylations of debrisoquine. 5. This latter proposal demonstrates the need to consider the mechanism of oxidation as well as the spatial orientation of the substrate in the development of a predictive model of the active site of CYP2D6. PMID- 10752639 TI - In vitro inhibition of human P450 enzymes by prenylated flavonoids from hops, Humulus lupulus. AB - 1. Several unique flavonoid compounds have recently been isolated from hops, Humulus lupulus, and their presence has been detected in beer. Their chemical structures are similar to other plant-derived compounds, many present in the human diet, that have been shown to have cancer chemopreventive properties due, in part, to inhibition of cytochrome P450 enzymes that activate carcinogens. Additionally, preliminary studies have shown these flavonoids (at 100 microM) to be inhibitory of P450-mediated activation reactions in a variety of in vitro systems. Thus, the in vitro effects of these phytochemicals on cDNA-expressed human CYP1A1, CYP1B1, CYP1A2, CYP3A4 and CYP2E1 were currently examined by the use of diagnostic substrates and the carcinogen AFB1. 2. At 10 microM, the prenylated chalcone, xanthohumol (XN), almost completely inhibited the 7 ethoxyresorufin O-deethylase (EROD) activity of CYP1A1. At the same concentration, other hop flavonoids decreased the EROD activity by 90.8-27.0%. 3. At 10 microM, XN completely eliminated CYP1B1 EROD activity, whereas the other hop flavonoids showed varying degrees of inhibitory action ranging from 99.3 to 1.8%. 4. In contrast, the most effective inhibitors of CYP1A2 acetanilide 4 hydroxylase activity were the two prenylated flavonoids, 8-prenylnaringenin (8PN) and isoxanthohumol (IX), which produced > 90% inhibition when added at concentrations of 10 microM. 5. CYP1A2 metabolism of the carcinogen AFB1 was also inhibited by IX and 8PN as shown by decreased appearance of dihydrodiols and AFM1 as analysed by hplc. IX and 8PN also decreased covalent binding of radiolabelled AFB1 to microsomal protein in a concomitant manner. 6. XN, IX and 8PN, however, were poor inhibitors of CYP2E1 and CYP3A4 as measured by their effect on chorzoxazone hydroxylase and nifedipine oxidase activities respectively. 7. These results suggest that the hop flavonoids are potent and selective inhibitors of human cytochrome P450 and warrant further in vivo investigations. PMID- 10752640 TI - Sulphonation of N-hydroxy-2-acetylaminofluorene by human dehydroepiandrosterone sulphotransferase. AB - 1. The aim was to determine which human recombinant sulphotransferase (ST) isoform(s) were responsible for the sulphonation and, thus, potential further bioactivation of the classical hepatic procarcinogen N-hydroxy-2 acetylaminofluorene (N-OH-2AAF). 2. N-OH-2AAF was incubated together with the cosubstrate 3'-phosphoadenosine-5'-phosphosulphate (PAPS) and either human liver cytosol or recombinant P-form phenolsulphotransferase (P-PST), M-form PST, dehydroepiandrosterone-ST (DHEA-ST) or oestrogen ST (EST). Formation of 3' phosphoadenosine-5'-phosphate (PAP) from PAPS, measured by HPLC, was used as the assay for determination of sulphoconjugation rates. 3. The liver cytosol produced a 100% increase in PAP formation in the presence of 200 microM N-OH-2AAF as compared with baseline levels (p < 0.01), corresponding to a rate of 19 pmol/min/mg protein. Recombinant P-PST, however, was without effect. This is in contrast to previous suggestions using crude enzyme preparations. Like P-PST, recombinant M-PST and EST did not sulphonate N-OH-2AAF. On the other hand, recombinant DHEA-ST produced a 161% increase in PAP formation in the presence of 200 microM N-OH-2AAF as compared with baseline values (p < 0.001). 4. Kinetic studies of N-OH-2AAF sulphonation by DHEA-ST and human liver cytosol gave similar apparent Kms. Interestingly, the Vmax for N-OH-2AAF sulphonation by DHEA-ST was very similar to that of DHEA, the natural substrate for DHEA-ST. 5. This is the first paper to demonstrate the involvement of the human DHEA-ST in the sulphonation of an N-hydroxylated aromatic amide carcinogen. PMID- 10752641 TI - Metabolism and disposition of luminol in the rat. AB - 1. The metabolism and disposition of Luminol (LMN, 3-aminophthalhydrazide), a widely used forensic and laboratory reagent that chemiluminesses upon oxidation, was determined as part of its overall toxicological characterization. 2. Radiolabelled LMN was well absorbed, metabolized and excreted following p.o. administration of a range of doses. About 90% of the total dose was recovered within 24 h of administration in urine in the form of two metabolites identified as LMN N8-glucuronide and LMN N8-sulphamic acid. 3-Aminophthalic acid, the oxidative product of LMN in the light-emitting reaction, was apparently not formed in vivo. 3. Metabolism and disposition of an i.v. administered dose was similar to that following gavage. Little or no LMN-derived radioactivity was present in tissue within 12 h post-dosing. Excretion of radioactivity in bile following i.v. injection was minimal (approximately 8% of the total dose in 6 h) and consisted of the same urinary-excreted glucuronide and sulphate conjugates. 4. LMN was not absorbed dermally in rat, potentially a major route of exposure to human. If the fate of LMN is similar between species, this compound should have little potential for either dermal absorption, bioaccumulation in tissues following other routes of exposure or chronic toxicity in humans. PMID- 10752643 TI - Ethnic-related differences in the frequency distribution of genetic polymorphisms in the CYP1A1 and CYP1B1 genes in Japanese and Caucasian populations. AB - 1. Race-related differences in the frequency distribution of genetic polymorphisms in the CYP1A1 and CYP1B1 genes were studied in 39 Japanese and 45 Caucasians. 2. Four types of CYP1A1 polymorphism, namely m1 (a nucleotide change at T6235C in the 3'-flanking region), m2 (A4889G at exon 7), m3 (T5639C in the 3' flanking region) and m4 (C4887A at exon 7), and three types of CYP1B1 genetic polymorphism, namely m1 (C488G and G701T leading to Arg48Gly and Ala119Ser exchanges respectively), m2 (C1294G leading to a Leu432Val exchange) and m3 (A1358G leading to an Asn453Ser exchange) were studied. 3. The distribution of the m1-, m2-, m3-, and m4-types of CYP1A1 polymorphism in the Japanese population was 30.8, 17.9, 0 and 0% respectively; those in Caucasians were 3.3, 6.7, 0 and 2.2% respectively. Two types (m1, and m2) of CYP1B1 polymorphism were expressed at 14.1 and 21.8% respectively in the Japanese, and by 28.9 and 37.5% respectively in the Caucasian. Ethnic differences were also noted in the m3-type CYP1B1 polymorphism in which the incidence in Caucasians was 23.9%, whereas no cases in the 39 Japanese subjects were observed. 4. No apparent association was found in the incidence in each of the genetic polymorphisms of CYP1A1 and CYP1B1 genes, nor in methylenetetrahydrofolate reductase gene, except that the occurrence of the m2-type of CYP1A1 genetic polymorphism was related to that of the m1-type CYP1A1 polymorphism in the Japanese population. 5. These results suggest that there are race-related differences in the occurrence of genetic polymorphisms in both CYP1A1 and CYP1B1 genes in Japanese and Caucasian populations and that these differences in P450 genetic polymorphisms may, in part, cause differences in the occurrence of lung and breast cancers in these ethnic groups. PMID- 10752642 TI - Effect of troglitazone on cytochrome P450 enzymes in primary cultures of human and rat hepatocytes. AB - 1. Troglitazone was the first thiazolidinedione approved for clinical use in the treatment of non-insulin-dependent diabetes mellitus. During clinical investigations of drug-drug interactions with therapeutics (terfenadine and cyclosporine) known to be metabolized by CYP3A4, pharmacokinetic interactions were noted upon troglitazone multiple-dose treatments. The nature of the interactions suggested induction of CYP3A enzymes. 2. Primary cultures of human hepatocytes were used to investigate the induction potential of troglitazone with respect to CYP3A4, CYP2B6 and CYP1A1/2. In human hepatocytes, troglitazone induced both immunoreactive CYP3A4 protein and testosterone 6beta-hydroxylase activity in a dose-dependent fashion (EC50 = 5-10 microM), accompanied by an increase in CYP3A4 mRNA. The capacity of troglitazone to induce CYP3A4 was between that of rifampin (EC50 = 0.8 microM) and dexamethasone (40-50 microM). Troglitazone increased CYP2B6 immunoreactive protein but did not significantly effect CYP1A1/2 activity, immunoreactive protein or mRNA. 3. Troglitazone produced significant increases in CYP3A message, protein and activity in primary rat hepatocytes, a slight increase in CYP2B1/2 activity and no change in CYP1A1/2 message or activity. 4. These results provide evidence that troglitazone can induce CYP3A and CYP2B enzymes while apparently not altering CYP1A. This provides a rationale for the clinically observed interactions of troglitazone with selected CYP3A4 substrates. PMID- 10752644 TI - Metabolism of N,N-dialkylated amphetamines, including deprenyl, by CYP2D6 expressed in a human cell line. AB - 1. Five N,N-dialkylated amphetamines, N-methyl-N-propargylamphetamine (deprenyl; DEP), N-benzyl-N-methylamphetamine (benzphetamine; BPA), N-allyl-N methylamphetamine (AMA), N,N-diallylamphetamine (DAA) and N-methyl-N propylamphetamine (MPA), were metabolized in vitro with a microsomal preparation from cells expressing human CYP2D6 to determine what influence the N,N-dialkyl substituents had on the extent of N-dealkylation and/or aromatic ring oxidation. 2. The results obtained from experiments with the first two substrates, DEP and BPA, were surprisingly different. Whereas DEP was N-demethylated and N depropargylated by the CYP2D6 enzyme system, no metabolites were formed from BPA. Subsequently, it was determined that AMA, DAA and MPA also underwent CYP2D6 catalysed N-dealkylation. Both N-methyl- and N-allylamphetamine were identified as products of AMA metabolism; similarly, metabolism of MPA produced both N methyl- and N-propargylamphetamine, and N-allylamphetamine was the sole metabolite of DAA. 3. No N,N-didealkylated product (i.e. amphetamine) was isolated from incubates of any of the five substrates, and none of the N,N dialkylated substrates was metabolized to a ring-hydroxylated product. 4. Rates of these CYP2D6-catalysed reactions were dependent on the nature and degree of unsaturation of the N-substituents. PMID- 10752645 TI - Absorption, distribution, metabolism and excretion of p-bromophenylacetylurea in the female rat. AB - 1. This study has investigated absorption, distribution, metabolism and excretion of p-bromophenylacetylurea (BPAU) in the F344 female rat. BPAU and its metabolites were determined by HPLC. 2. Following a single p.o. dose of 150 mg/kg BPAU, the absorbed fraction of dosed BPAU was 65.9% and its half-life in the blood was 9.4 h. The relative distribution of BPAU (tissue/serum ratio) at 6 h (peak time point) after a single i.p. dose of 150 mg/kg BPAU was spinal cord (4.6+/-0.2) > liver (3.7+/-0.1) > brain (2.9+/-0.1) (mean+/-SD, n = 5), and they were significantly different from each other (p < 0.05). BPAU in spinal cord reached the highest level. 3. Absorbed BPAU was metabolized in vivo into three major metabolites. N'-hydroxy-p-bromophenylacetylurea (M1) was a dominant metabolite in tissues, whereas 4-(4-bromophenyl)-3-oxapyrrolidine-2,5-dione (M2) reached a high concentration in blood. N'-methyl-p-bromophenylacetylurea (M3) was mainly found in the urine. All three metabolites were excreted via the urine and together accounted for 87% of absorbed BPAU. 4. This study provides a basic understanding of BPAU absorption, distribution, metabolism and elimination in rat. PMID- 10752646 TI - Hippuric acid as a major excretion product associated with black tea consumption. AB - 1. Nine habitual tea-drinking volunteers were recruited and asked to follow a low polyphenol and low-caffeine diet for 6 days and to provide daily 24-h urine samples. On day 4 of the experiment strong black tea brewed under standardized conditions was re-introduced to the volunteers' diet. 2. 1H-NMR and HPLC profiling of the urine samples indicated that consumption of black tea (6-10 mugs per day) was associated with a significant (p = 0.00017) increase in hippuric acid excretion relative to control, increasing from 153-512 to 742-1374 mg day( 1). The excretion of substantial amounts of hippuric acid has not previously been associated with black tea consumption. 3. For some volunteers, the quantity of benzoic acid processed exceeded the acceptable daily intake (ADI), but this is not considered to constitute any hazard. 4. A mass-balance analysis indicated that the necessary quantity of benzoic acid could not be obtained from the contents of gallic acid, flavanols, flavonol glycosides and theaflavins in black tea even if 100% transformation was obtained, suggesting that the thearubigins (the major and chemically ill-defined polyphenols of black tea) may be an important source. PMID- 10752647 TI - Is 'bedside medicine' returning to oncology? PMID- 10752648 TI - Are healthcare providers adequate raters of patients' quality of life--perhaps more than we think? PMID- 10752649 TI - Patient and staff perceptions of cancer patients' psychological concerns and needs. AB - This paper reviews patient and staff perceptions of cancer patients' psychological concerns and needs. An extensive literature search resulted in a total of 22 studies. Six of these concern staff ratings of hypothetical 'average' patients, and the main findings are that staff and patients are in relative agreement about the overall spectrum of concerns relevant to cancer patients, but less so regarding which concerns are of most importance to patients. Sixteen studies compare patient ratings with staff ratings of individual patients. The overall pattern of results indicates that staff tend to overestimate patient anxiety and emotional distress and show limited ability adequately to assess patient distress in terms of rank. Staff were able correctly to detect a majority of anxiety 'cases', while the identification of depression was less accurate. High detection rates for anxiety were obtained partly at the expense of staff also presuming anxiety in many non-distressed patients. Generally, there was better patient-staff agreement on the absence than the presence of patient anxiety and depression. Many studies were found to have serious methodological deficiencies and there is no clear evidence that patient-staff agreement is markedly better in studies fulfilling important methodological criteria. Several explanations of patient-staff discrepancies are discussed. Future research should be aimed at critically testing possible reasons for patient-staff disagreement and at investigating the consequences of such discrepancies. PMID- 10752650 TI - Developing psychosocial aftercare for children surviving cancer and their families. AB - More children than ever are surviving childhood cancer. However, the medical and psychosocial consequences of their longer survival are becoming clear for former patients and their families, underlining the need for aftercare. Whereas the need for medical follow-up programs is widely recognized, psychosocial aftercare is still in its infancy. This paper presents a systematic approach to developing psychosocial aftercare based on evidence from research studies. First, the family member most in need of aftercare is identified. Research findings indicate that the child surviving cancer is most seriously affected by the cancer experience, followed by mothers and fathers, whereas siblings are least affected. Accordingly, former patients and parents should be the primary target groups. Second, different types of interventions need to be developed based on the nature of the problems of the target groups and the individual's level of adjustment. Finally, psychosocial aftercare programs need to demonstrate their effectiveness in reducing late effects and improving the quality of life after treatment ends. If our aim is to cure the child truly of cancer, than the development of psychosocial aftercare, its clinical application, and evaluation of its effectiveness will be the challenge for paediatric psycho-oncology in years to come. PMID- 10752651 TI - Adjuvant cyclophosphamide, methotrexate, fluorouracil (CMF) in breast cancer--is it cost-effective? AB - Adjuvant chemotherapy (ACT) may expose patients to morbidity, with little gain in outcome. Treatment with CMF (cyclophosphamide, methotrexate, fluorouracil) has been the standard ACT in several countries for decades. In this model, efficacy, tolerability and quality of life data from the English-language literature were incorporated with Norwegian standard ACT practice and cost data in a cost effectiveness/cost-utility approach. The CMF efficacy was calculated as 2.45 years saved per patient treated. The quality of life was assumed diminished by 0.33 (0-1 scale) for 6 months and the life years gained were valued Q = 0.86. An 85% dose intensity was employed, one British pound ( 1) was calculated as 12 NOK and a 5% discount rate was used. The total cost of adjuvant CMF, including amounts spent on drugs, administration, travelling and production loss, was calculated to 2365- 6253, depending on the method chosen. Money spent on drugs alone constituted 13-34%. The cost per life year saved was measured as 2170- 5737. A cost-utility approach revealed a cost per quality-adjusted life year (QALY) of 2973- 7860. Adjuvant CMF in breast cancer is cost-effective in Norway. PMID- 10752652 TI - Histologic grading in breast cancer--reproducibility between seven pathologic departments. South Sweden Breast Cancer Group. AB - Histologic grade, including tubular formations, nuclear grade, and mitotic activity, is a well-documented prognostic factor in breast cancer. In comparison with other prognostic parameters, the evaluation of histologic grade is cheap and can be performed, in principle, in all cases of breast cancer. One possible disadvantage is that the evaluation may vary between different pathological departments. The aim of the present work was therefore to study the reproducibility of the histologic grading system by distributing haematoxylin erythrosin-stained slides from 93 invasive breast cancers to the seven pathology departments within the southern healthcare region of Sweden. The evaluation was performed blindly and without any knowledge of other clinical parameters. In 31% of the cases the same histologic grade was obtained for all departments. The overall mean kappa was 0.54, indicating a moderate reproducibility. Of the three factors included in histologic grade, the agreement was best for tubular formations and poorest for nuclear grade and mitotic activity. The overall moderate reproducibility should be considered when the clinical usefulness of histologic grading is compared with other prognostic instruments. PMID- 10752653 TI - Paclitaxel-containing high-dose chemotherapy in high-risk breast cancer patients. AB - Despite standard-dose adjuvant chemotherapy, the prognosis for patients with breast cancer and extensive axillary lymph node involvement at diagnosis is poor. The efficacy of a paclitaxel-containing, high-dose chemotherapy protocol in 21 high-risk breast cancer patients is assessed. After standard-dose chemotherapy followed by peripheral blood stem cell (PBSC) mobilization, high-dose therapy with paclitaxel, carboplatin, and cyclophosphamide and CD34-selected PBSC rescue was given. Hematologic reconstitution after high-dose therapy was rapid. Main toxicity included diarrhea grade I or II in about half of the patients and infections were observed in 19%. Five-year probabilities for relapse and failure free survival were 32% and 62%, respectively. High-dose consolidation with paclitaxel, carboplatin, and cyclophosphamide achieves a high failure-free survival in patients with high-risk breast cancer with acceptable toxicities and stable, long-term hematopoietic reconstitution. Evaluation of the benefit of high dose therapy in these patients in larger prospective, randomized trials is warranted and currently under way. PMID- 10752654 TI - Changes in thymidylate synthase mRNA in blood leukocytes from patients with colorectal cancer after bolus administration of 5-fluorouracil. AB - 5-fluorouracil (5-FU) is considered the standard antineoplastic drug of choice for metastatic colorectal cancer. It has been suggested that 5-FU administered as bolus infusion is cytotoxic mainly through an RNA damaging effect. We investigated the effect of i.v. bolus 5-FU 500-600 mg/m2 on the 5-FU target enzyme, thymidylate synthase (TS) mRNA, in blood leukocytes before and after courses 1 and 3 in 21 patients with colorectal cancer. TS mRNA expression was quantified using an RT-PCR assay with an internal RNA standard. Median TS mRNA expression decreased significantly 30 min after course no. 1 (p = 0.004), and both 15 min and 30 min after course 3 (p = 0.01). After course 1, the median TS mRNA expression decreased by 31% and after course 3 by 24%. Pharmacokinetic parameters were similar for individual patients during the two courses but did not correlate with the degree of TS mRNA inhibition. The present results indicate that TS mRNA in blood leukocytes may be an early indicator of an RNA damaging effect after i.v. bolus infusion of 5-FU. PMID- 10752655 TI - A pharmacokinetic study of 5-FU/leucovorin and alpha-interferon in advanced cancer. AB - The present study was designed to study 5-FU pharmacokinetics after interferon. Weekly bolus 5-FU (500 mg/m2), immediately followed by leucovorin (60 mg/m2) was given in 14 weekly cycles to 55 gastrointestinal and breast cancer patients. Interferon-alpha was given on days 2, 4 and 6, starting from cycle 2 at a dose of 0.5 million units (MU) and stepwise increased to 12 MU in cycles 12 and 13. Five patients could not tolerate the treatment even at the lowest dose of interferon and 22 patients were unavailable for the pharmacokinetic analysis because of dose reductions of 5-FU. Five patients were able to follow the protocol to 12 MU, whereas most patients were unable to continue owing to toxicity. 5-FU pharmacokinetics was analysed every second cycle. Peak concentration and AUC were increased after 12 MU of interferon, but no other significant influence of interferon on pharmacokinetic parameters of 5-FU was observed. PMID- 10752657 TI - Gynaecological infections as risk determinants of subsequent cervical neoplasia. AB - A longitudinal cohort study was carried out to determine whether gynaecological infections other than human papillomavirus (HPV) are also related to the subsequent increased risk of cervical neoplasia. The study comprised 19114 women attending the organized mass screening in Finland in 1985-1990 with cytologically detected HPV, Actinomyces, herpes simplex, Trichomonas vaginalis, or yeast. The women were followed-up for subsequent preinvasive lesions and invasive cancers until the end of 1994 by linkage to the nation-wide Cancer Registry. Standardized incidence ratios (SIR) with rates for the whole of Finland as reference and 95% confidence intervals (CI) were estimated. Trichomonas vaginalis and HPV were associated with a high relative risk of cervical cancer, SIR 6.4 (CI 3.7-10, preinvasive lesion and invasive cancer combined) and SIR 5.5 (CI 4.2 7.2, preinvasive lesion and invasive cancer combined), respectively. Herpes simplex was rarely detected, but the highest and statistically most significant point estimate was observed (SIR 12, CI 2.4-34, preinvasive lesion and invasive cancer combined). Neither Actinomyces nor yeast was associated with a significantly increased risk of cervical cancer. None of these results could be accounted for by the confounding effect of the other infections. Our results, based on a prospective design, lead us to propose that Trichomonas vaginalis and herpes simplex virus are also predictors for cervical neoplasia. PMID- 10752656 TI - Telomerase, p53 and human papillomavirus infection in the uterine cervix. AB - Human papillomavirus infection is postulated to be a major risk factor for cervical cancer, while more recent data have stressed the clinical significance of telomerase expression during tumorigenesis. This study therefore looked for any relationship between telomerase expression, presence of human papillomavirus (HPV) and expression of the high-risk HPV E6 protein at various phases of tumor progression in the uterine cervix. In addition, accumulation of the p53 protein and total tissue proliferative fraction were also studied. Telomerase was detected using a modified TRAP (telomerase repeat amplification protocol) assay. Expression of p53, Ki 67 and E6 protein was evaluated by immunocytochemistry. Presence of mutant p53 was detected using a mutant-specific ELISA. Type of HPV infection was determined by polymerase chain reaction and Southern blot using type-specific primers and probes. There was a significant correlation between the expression of telomerase with histological grade (r = 0.646, p = 0.00003). Fisher's exact test analysis revealed that the odds ratio of a tissue sample expressing telomerase being a case (high-grade squamous intraepithelial lesion or invasive cancer) was 28.93 (p = 0.0001, 95% CI: 7.22, to 115.94). High-risk HPV infected tissues and those expressing E6 showed increased telomerase expression (r = 0.555, p = 0.00001). Similarly, accumulation of p53 protein and increased cell proliferation (Ki 67 index) also correlated to the presence of telomerase (r = 0.661, p = 0.000004 for p53 and r = 0.647, p = 0.000003 for Ki 67). There was no correlation between telomerase expression and presence of p53 mutation. Activation of telomerase thus appears to be associated with high-risk-HPV infection, accumulation of inactive p53 protein and increased cell proliferation in cervical lesions. PMID- 10752658 TI - Is the subsite distribution of laryngeal cancer related to smoking habits? AB - In Finland traditionally as many as two-thirds of laryngeal cancers have been considered to be supraglottic, while in other countries the majority are glottic. The Finnish observation is based on clinical series diagnosed and treated mainly before the 1960s. The aim of our study was to evaluate the present situation. This study consisted of 279 patients treated in Eastern Finland between 1975 and 1994 and included 145 (52%) glottic, 124 (44%) supraglottic and 10 (4%) subglottic tumours in 260 (93%) men and 19 (7%) women. During this study period, the proportion of glottic tumours remained stable in men but increased in women. The change in smoking habits seems to be the most important reason for the shift from supraglottic to glottic tumours. PMID- 10752659 TI - Therapeutic efficacy of intralesional 131I-labelled hyaluronectin in grafted human glioblastoma. AB - The grafted human glioblastoma cell CB109 was used as a model for intralesional therapy with 131I-labelled hyaluronectin glycoprotein (131I-HN).131I-HN bound specifically to in situ hyaluronic acid (HA), a main component of the extracellular matrix which is involved in tumour invasion. Labelling experimental conditions were determined and, finally, 25 microCi/microgHN, 1 microg chloramine T/microgHN and a 60-s stirring period provided a 131I-HN preparation with an optimal affinity for HA (64% compared to unlabelled HN). Following intratumoral injection, 131I-HN was retained with a limited diffusion outside the tumour. On day 4 the radioactivity concentrated in the tumour was still 25 times greater than that in the liver, spleen and kidneys combined. For therapeutic assays, 65 microCi 131I-HN was injected into the tumour, resulting in a delivery of 6.8 Gy over a 7-day period. Controls received unlabelled HN, heat-inactivated HN, a mixture of inactivated HN plus free 131I or no treatment (six animals per group). Tumour volumes were evaluated every second day from treatment day and the rate of tumour growth was expressed as a ratio of tumour size at time intervals to the tumour size at the time of injection. Growth curves were compared: heat inactivated with or without free 131I had no anti-tumour effect. Unlabelled HN injected tumours had a slightly slower growth rate than untreated tumours (p < 0.02) and growth rate of 131I-HN-injected tumours was much lower (p < 0.00002). A pronounced inhibitory effect with intralesional 131I-labelled HN injection resulted from a combination of a) blockage of HA, a proliferation facilitating factor, and b) local irradiation of tumoral tissue, while uptake in normal tissues was minimized. PMID- 10752660 TI - O6-(fluorobenzyl)guanine and chloroethylnitrosourea in xenografted rat brain tumor in vivo. AB - O6-methylguanine-DNA methyltransferase (MGMT), one of the DNA repair enzymes, potently repairs DNA damage induced by chloroethylnitrosoureas (CENUs). Depletion of MGMT activity after treatment with MGMT inhibitors increases the sensitivity of tumor cells to CENUs. We tested the effect of O6-(4-, 3- and 2 fluorobenzyl)guanines (4F, 3F and 2F, respectively), three newly synthesized MGMT inhibitors, on 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nit rosoureahydrochloride (ACNU) therapy in C6 tumor xenografts. Treatment with 4F + ACNU and 3F + ACNU significantly decreased tumor volume and extended the delay of growth in comparison to untreated mice (control group, p < 0.05). Both groups showed significantly lower proliferating indices than the control group (p < 0.05) 12 h after treatment. In contrast, 2F did not enhance the ACNU anti-tumor effect. These results indicate that O6-(4- and 3-fluorobenzyl)guanines as well as O6-benzylguanines enhance the effect of ACNU on the growth of C6 tumor xenografts in vivo. PMID- 10752661 TI - Intracranial ependymomas in childhood--a retrospective review of sixty-two children. AB - Of the 818 tumours of the central nervous system diagnosed between 1972 and 1991, 62 patients (35 males and 27 females) with histopathologically confirmed ependymomas were treated and followed-up at the Children's Hospital of Hacettepe University during that period. The median age was 6 years (range 1-17 years). Headache, nausea and vomiting were the most frequent symptoms; papilledema was the most common sign in our patients. Tumour sites were in the posterior fossa in 47 patients and supratentorial in 15 patients. All patients underwent surgery. Gross- total resection was performed in 27 patients, subtotal resection in 32 patients and biopsy in the remaining 3 patients. Initially, 53 patients were given postoperative radiotherapy. Four patients did not receive radiotherapy because of their young age, whereas five patients died prior to starting radiotherapy. Two slightly different types of chemotherapy protocols were applied for an average of one year in 47 patients. Event-free and overall survival rates at 10 years were 36% and 50%, respectively. Twenty children suffered relapse 4 to 55 months after diagnosis (median 16 months). Relapses were distant in 3 cases and local in 17. Age was the only statistically significant prognostic factor, patients younger than 5 years of age having a poorer outcome. Sex, histopathologic type, localization of the tumour, extent of surgery, and chemotherapy did not influence the prognosis in our study. Because the majority of recurrences were local, better local tumour control is required. New treatment strategies should be developed in order to improve local control. PMID- 10752662 TI - Long-term survival of prostate cancer in southeastern Netherlands. AB - With the increase in detection at an early stage, only a few and conflicting results have been reported on the long-term outcome for prostate cancer beyond 10 years. The vast majority of prostate cancer patients diagnosed between 1955 and 1984 in southeastern Netherlands, with a population of almost one million inhabitants, did not receive any curative treatment. We calculated the prognosis for 10-year survivors of prostate cancer diagnosed in the era preceding prostate specific antigen (PSA) testing to determine how long these patients exhibited excess mortality. All patients under age 70 diagnosed with prostate cancer and registered in the population-based Eindhoven Cancer Registry between 1955 and 1984 were included in the study. Relative survival was calculated for those who survived for at least 10 years (n = 174). Initially, these patients still exhibited an almost 25% excess mortality risk, but this decreased with time and no excess mortality was found after 15 years. PMID- 10752663 TI - False-negative biopsy for testicular intraepithelial neoplasia and high-risk features for testicular cancer. AB - The purpose of this report is to emphasize the possibility of false-negative biopsies for testicular intraepithelial neoplasia (TIN) in men with high-risk features of testicular cancer and to review the relevant literature. At the Norwegian Radium Hospital patients in this category are offered the chance to undergo a testicular biopsy. A patient is described who had a normal testicular biopsy a decade before presenting with an invasive testicular cancer. Furthermore, this patient is the first case reported with a false-negative biopsy for TIN and a family history of testicular cancer. The evaluation of the biopsies included immunohistochemical staining for c-kit and PIAP (placental-like alkaline phosphatase) in order to diagnose early TIN. Though multifocal or diffuse extension seems to be the most frequent pattern of distribution of TIN, the presented case and another 14 cases from the literature review indicate that the focality of TIN may be a reason for a TIN-negative biopsy. PMID- 10752664 TI - Neoadjuvant chemotherapy for Ewing's sarcoma of bone in patients older than thirty-nine years. AB - The aim of this study was to determine whether the behaviour of Ewing's sarcoma of bone in adult patients is the same as that observed in children and adolescents. We reviewed the clinical features and outcomes of 23 patients over the age of 39 (17 males, 6 females) who had been treated with neoadjuvant chemotherapy between 1983 and 1995 at our institution. The most common primary sites of tumor were the extremities (16 cases); tumor volume was more than 100 ml in 17 patients, and elevated serum LDH levels were found in 6 cases. The local treatment was surgery in 8 cases, surgery plus radiotherapy in 8, and radiotherapy alone in 7 cases. Chemotherapy comprised a 4-drug regimen in 10 patients, while the other 13 patients received 6 drugs. At a follow-up of 8.8 years (3.5-15) 13 patients remained continuously free of disease and 10 relapsed. The 5-year disease-free survival and overall survival rates were 53% and 59%, respectively. Clinical features, dose intensity, and toxicity of chemotherapy, as well as the outcome of these 23 patients were found to be exactly comparable to the findings observed in 327 patients younger than 40 years treated at our institution in the same period, with the same therapy. We conclude that Ewing's sarcoma of bone in adults is no different from that occurring in children, and we therefore recommend the inclusion of all adult patients in multidisciplinary treatment trials of this tumor. PMID- 10752665 TI - Multifocal demyelinative leukoencephalopathy associated with 5-fluorouracil and levamisole. PMID- 10752666 TI - Paraquat-induced membrane dysfunction in pulmonary microvascular endothelial cells. AB - Membrane dysfunction monitored by lactate dehydrogenase release from cultured pulmonary microvascular endothelial cells of pigs, which were exposed to paraquat at different concentrations (0.1-2 mM), was examined. Paraquat caused a time dependent increase in lactate dehydrogenase release. Lactate dehydrogenase releases after 72 hr, 32, 58, and 84% by 0.1, 0.5, and 2 mM paraquat, respectively, were well correlated with cell viability measured by cell adherence. In contrast, reductions of two tetrazolium compounds were depleted profoundly by 72 hr after exposure to 0.5 mM paraquat, suggesting depletion of intracellular reductive substances. Extracellular hydrogen peroxide began to significantly increase 56 hr or 32 hr after exposure to 0.5 mM or 1.5 mM paraquat, respectively, preceding the initial increase of lactate dehydrogenase release (64 hr by 0.5 mM or 48 hr by 1.5 mM). Lactate dehydrogenase release 72 hr after exposure to 0.5 mM paraquat was prevented strongly by catalase (1000 units/ml), but weakly by superoxide dismutase (1000 units/ml). These enzymes failed to restore the reduced acid phosphatase activity. Also, 0.1 mM desferal or alpha,alpha'-dipyridyl protected lactate dehydrogenase release. Similarly, 1 mM thiourea or dimethylthiourea, and 0.5 mM alpha-tocopherol or trolox, were effective, but diethylenetriaminepentaacetic acid (0.1 mM) and probucol (5 or 10 microM) were ineffective. Exposure of 0.5 or 1.5 mM paraquat suppressed levels of lipid peroxidation. These results indicate that membrane dysfunction by paraquat is ascribed to an iron-catalyzed reaction of extracellularly increased hydrogen peroxide. A deleterious species for the membrane dysfunction is discussed. PMID- 10752667 TI - The preservatives ethyl-, propyl- and butylparaben are oestrogenic in an in vivo fish assay. AB - The widely used phenolic preservatives ethylparaben, propylparaben, butylparaben and their common metabolite p-hydroxybenzoic acid were tested for their ability to evoke an oestrogenic response in vivo. Yolk protein induction in sexually immature rainbow trout was used as an oestrogen-specific endpoint after repeated injections of the compounds. All tested parabens were oestrogenic in doses between 100 and 300 mg/kg, while the metabolite showed no activity. Ethylparaben was found to be approximately sixty times weaker than propyl- and butylparaben which had oestrogenic potencies comparable to those previously found for bisphenol A. PMID- 10752668 TI - Ceramides that mediate apoptosis reduce glucose uptake and transporter affinity for glucose in human leukaemic cell lines but not in neutrophils. AB - We have demonstrated that CD95-induced apoptosis in a human leukaemic T-cell line resulted in loss of glucose transporter function (Berridge et al. 1996). To determine whether ceramide, a mediator of CD95 and tumour necrosis factor-alpha induced apoptosis, has similar effects on glucose transport, the human leukaemic cell lines, Jurkat and U937, and human peripheral blood neutrophils were treated with ceramide or sphingomyelinase and the effects on glucose transport determined by measuring [3H]-2-deoxyglucose uptake. We show that in U937 and Jurkat cells, the cell permeable ceramides, C2 (N-acetylsphingosine) and C6 (N hexanoylsphingosine) inhibit glucose uptake within minutes of initiating ceramide treatment, 60-70% inhibition being observed within 2 hr. Loss of glucose transport correlated with loss of proliferative response, but metabolic activity as measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction, was affected to a much lesser extent. With Jurkat and U937 cells, the inhibitory effects of ceramides on glucose transport were associated with reduced affinity of glucose transporters for glucose (Km). Similar effects were observed with sphingomyelinase. With human peripheral blood neutrophils, C2 and C6 ceramides inhibited glucose uptake by 70-80% within 30 min. without affecting transporter affinity for glucose, but the maximum velocity of uptake (Vmax) was reduced. These results show that acute regulation of glucose transport is an early effector mechanism of cell death induced by ceramides in human leukaemic cell lines and peripheral blood neutrophils. This is the first study which describes ceramide-induced early physiological/biochemical events leading to cell death in human cells. PMID- 10752669 TI - Minimal toxicity to myeloid progenitor cells of weekly 24-hr infusion of high dose 5-fluorouracil: direct evidence from colony forming unit-granulocyte and monocyte (CFU-GM) clonogenic assay. AB - Although very high doses of 5-fluorouracil was used in the weekly 24-h infusion, high-dose 5-fluorouracil (2600 mg/m2/week) and leucovorin (500 mg/m2/week) protocol, myelosuppression was surprisingly low. The current study was conducted to investigate the possible mechanism underlying the low myelosuppression. To mimic the clinical situation, peripheral blood progenitor cells collected from 12 patients were used for colony forming unit-granulocyte and monocyte clonogenic assay; and 2 representative modes of 5-fluorouracil exposure (30 min. versus 24 hr) were examined for cytotoxic effects on human myeloid progenitor cells. Previous pharmacokinetic studies have estimated the concentrations of 5 fluorouracil in the bone marrow to be 200-400 microM and 1-2 microM for the 30 min. infusion (600-900 mg/m2) and the 24 hr-infusion (1000-2000 mg/m2) regimens, respectively. The results of our colony-forming unit-granulocyte and monocyte clonogenic assay showed that 24-hr exposure to 5-fluorouracil (2 microM) and 30 min. exposure to 5-fluorouracil (100 microM) resulted in 27.2% and 78.2% inhibition of the colony formation, respectively. Our data provided direct evidence which may explain why myelotoxicity is significantly less in weekly 24 hr infusion of fluorouracil than in the conventional bolus regimens. PMID- 10752670 TI - Hypotensive activity of the pineal indoleamine hormones melatonin, 5 methoxytryptophol and 5-methoxytryptamine. AB - Injection of the pineal indoles melatonin, 5-methoxytryptophol and 5 methoxytryptamine via the external jugular vein elicited a dose-dependent depression in mean arterial pressure. Melatonin and 5-methoxytryptophol were approximately equipotent and a dose of 150 micromol/kg brought about a reduction of about 40 mmHg in mean arterial pressure. Methoxytryptamine exerted a much more potent hypotensive action. An abrupt decrement in mean arterial pressure by 30 mmHg occurred when the dose was only 2 nmol/kg. Subsequent increases in the dose further lowered the mean arterial pressure, but more gently. The other pineal indoles tested including 5-methoxyindoleacetic acid and 5-hydroxyindoleacetic acid, as well as 6-methoxy-2-benzoxazolinone, did not affect the mean arterial pressure when tested up to 80 micromol/kg. Methylene blue, a guanylate cyclase inhibitor, was not able to antagonize the hypotensive activity of melatonin, suggesting that the mechanism of action of melatonin does not involve guanylate cyclase. Lidocaine, which blocks sodium channels in perivascular nerves, antagonized the hypotensive action of melatonin. PMID- 10752671 TI - Evaluation of flutamide genotoxicity in rats and in primary human hepatocytes. AB - Flutamide, an effective competitive inhibitor of the androgen receptor used orally for palliative treatment of prostatic carcinoma and regulation of prostatic hyperplasia was evaluated for its genotoxic effects in the intact rat and in primary cultures of human hepatocytes. Negative responses were obtained in all the in vivo assays as well as in the in vitro assay. In rats given a single oral dose of 500 mg/kg flutamide, fragmentation and repair of liver DNA were absent, and no increase was observed in the frequency of micronucleated hepatocytes. In the liver of rats given flutamide as initiating agent at the dose of 500 mg/kg/week for 6 successive weeks, gamma-glutamyltraspeptidase-positive foci were detected only in 3 of 10 rats. There was no evidence of a promoting effect on the development of aberrant crypt foci in rats given 100 mg/kg flutamide on alternate days for 8 successive weeks. In primary cultures of human hepatocytes from one male and one female donor DNA fragmentation as measured by the Comet assays, and DNA repair synthesis as revealed by quantitative autoradiography, were absent after a 20 hr exposure to flutamide concentrations ranging from 18 to 56 microM. Taken as a whole, our results seem to indicate that flutamide is a non-genotoxic drug. PMID- 10752672 TI - Effects of various aluminium compounds given orally to mice on Al tissue distribution and tissue concentrations of essential elements. AB - To evaluate the risk of gastrointestinal long-term aluminium (Al) exposure, aluminium distribution and the levels of the following essential elements: Ca, Mg, Zn, Cu, and Fe in tissue were studied. Aluminium was administered in drinking water as aluminium chloride, dihydroxyaluminium sodium carbonate or aluminium hydroxide. Mice (strain Pzh:SFIS) were exposed to a total dose of 700 mg Al in long-term treatment (for each Al compound n = 15). Concentrations of Al, Ca, Mg, Zn, Cu, and Fe in stomach, kidneys, bone and liver were analyzed by atomic absorption spectrometry. After AlCl3 treatment, aluminium was found to accumulate in all tested tissues. A significant decrease in Fe concentration in liver and Zn in kidneys was observed in comparison to concentrations of these elements in the control group. In the Al(OH)3-treated group, accumulation of aluminium was observed in bone only and decline of Fe concentration in stomach and Cu in liver and kidney. In the NaAl(OH)2CO3-treated group the increase in Al concentration was significant in bone; there was no change in concentration of essential elements in the examined tissues. The observed aluminium accumulation was not accompanied by changes in Ca and Mg concentration except for bone. This study showed that oral administration as a route of Al exposure can result in diverging accumulation of aluminium in tissues, the concentration depending on the chemical form. PMID- 10752673 TI - Nitric oxide protects murine embryonic liver cells (BNL CL.2) from cytotoxicity induced by glucose deprivation. AB - We investigated the protective effects of nitric oxide on cell death of murine embryonic liver cells (BNL CL.2) after glucose deprivation. Endogenous nitric oxide production by BNL CL.2 cells was induced by 6 hr pretreatment with interferon-gamma and lipopolysaccharide. We used sodium nitroprusside and S nitroso-L-glutathione as exogenous nitric oxide-generating compounds. All agents were used at doses that did not show direct cytotoxicity as measured by crystal violet staining assay. In the BNL CL.2 cells, the viability dropped very steeply after 24 hr incubation with glucose-free media. Endogenous nitric oxide produced by treatment of the cells with interferon-gamma and lipopolysaccharide protected the cells from glucose deprivation-induced cytotoxicity, but did not protect them in the presence of the nitric oxide synthesis inhibitor, N(G)-monomethyl-L arginine. Exogenous nitric oxide protected the cells from glucose deprivation induced cytotoxicity in a concentration-dependent manner. Cytoprotection by nitric oxide donors was abolished by the use of nitric oxide scavenger, 2-phenyl 4,4,5,5,-tetramethylimidazole, but not by the soluble guanosine cyclase inhibitor, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. In addition, cytoprotective effects comparable to endogenous or exogenous nitric oxide were not observed when the cells were incubated with dibutyl guanosine 3',5'-cyclic monophosphate. Based upon these results, we suggest that nitric oxide may enhance the cell survival of BNL CL.2 cells after glucose deprivation via a guanosine 3',5'-cyclic monophosphate-independent pathway. PMID- 10752674 TI - Involvement of CYP3A4 in the metabolism of bromperidol in vitro. AB - In this study, human cytochrome P450 isoenzymes (CYP1A2, CYP2C19, CYP2D6, and CYP3A4) expressed in a cell line were used to elucidate their roles in the metabolism of bromperidol. We found that CYP3A4 catalyzes the N-dealkylation of bromperidol and its metabolite, reduced bromperidol. CYP3A4 also catalyzes the dehydration of bromperidol to bromperidol 1,2,3,6-tetrahydropyridine, metabolizes bromperidol to bromperidol pyridinium, and catalyzes the oxidation of reduced bromperidol back to bromperidol. CYP1A2, CYP2C19, and CYP2D6 do not catalyze these reactions. PMID- 10752675 TI - The diagnostic and prognostic utility of prostate-specific antigen for diseases of the breast. AB - Although prostate-specific antigen (PSA) is the most valuable tumor marker for the diagnosis and management of prostate carcinoma, it is widely accepted that PSA is not prostate specific. Numerous studies have shown that PSA is present in some female hormonally regulated tissues, principally the breast and its secretions. In this review, we summarize the findings of PSA in the breast, and focus on its potential for clinical applications in breast disease. PSA is produced by the majority of breast tumors and is a favorable indicator of prognosis in breast cancer. Low levels of PSA are released into the female circulation, and while the level of serum PSA is elevated in both benign and malignant breast disease, the molecular form of circulating PSA differs between women with and without breast cancer. These findings indicate that PSA may have potential diagnostic utility in breast cancer. PSA may also have a clinical application in benign breast disease, as both the level and molecular form of PSA differ between Type I and II breast cysts. High levels of PSA have been reported in nipple aspirate fluid (NAF) and recent studies have shown that the concentration of PSA in NAF is inversely related to breast cancer risk, indicating that NAF PSA may represent a clinical tool for breast cancer risk assessment. Thus, PSA represents a marker with numerous potential clinical applications as a diagnostic and/or prognostic tool in breast disease. PMID- 10752676 TI - Overexpression of the glucose-regulated stress gene GRP78 in malignant but not benign human breast lesions. AB - The 78 kDa glucose-regulated stress protein GRP78 is induced by physiological stress conditions such as hypoxia, low pH, and glucose deprivation which often exist in the microenvironments of solid tumors. Activation of this stress pathway occurs in response to several pro-apoptotic stimuli. In vitro studies have demonstrated a correlation between induced expression of GRP78 and resistance to apoptotic death induced by topoisomerase II-directed drugs. We were interested in characterizing this protein in human breast lesions for potential implications in chemotherapeutic intervention. Surgical specimens of human breast lesions and paired normal tissues from the same patients were flash frozen for these studies. Total RNA and/or protein were extracted from these tissues and used in northern and/or western blot analyses, respectively, to quantify the relative expression of GRP78. Northern blot analysis indicated that 0/5 benign breast lesions, 3/5 estrogen receptor positive (ER+) breast tumors, and 6/9 estrogen receptor negative (ER-) breast tumors exhibited overexpression of GRP78 mRNA compared to paired normal tissues, with fold overexpressions ranging from 1.8 to 20. Western blot analyses correlated with these findings since 0/5 benign breast lesions, 4/6 ER+ breast tumors, and 3/3 ER- breast tumors overexpressed GRP78 protein with fold overexpressions ranging from 1.8 to 19. Immunohistochemical analysis of these tissues demonstrated that the expression of GRP78 was heterogeneous among the cells comprising different normal and malignant glands, but confirmed the overexpression of GRP78 in most of the more aggressive ER- tumors. These results suggest that some breast tumors exhibit adverse microenvironment conditions that induce the overexpression of specific stress genes that may play a role in resistance to apoptosis and decreased chemotherapeutic efficacy. PMID- 10752677 TI - Cyclin D1 overexpression in a model of human breast premalignancy: preferential stimulation of anchorage-independent but not anchorage-dependent growth is associated with increased cdk2 activity. AB - Cyclin D1 is frequently overexpressed in human breast ductal carcinoma in situ (DCIS) specimens, which confer a high risk for the development of infiltrating ductal carcinoma. If causally involved in the genesis of human breast malignancy, cyclin D1 may represent an interesting target for chemopreventive approaches, as it sits at the junction of many growth factor and hormonal pathways. We have used the MCF-10A human breast cell line, derived from a mastectomy containing a low risk premalignant lesion, as a model system. Three cyclin D1 transfectants exhibited physiologically relevant levels of transgene overexpression, but no coordinate overexpression of other cell cycle related genes. Proliferation assays, flow cytometry, and cdk enzymatic assays of anchorage-dependent proliferation indicated only a minimal and transient effect of cyclin D1. In contrast, cyclin D1 overexpression significantly stimulated anchorage-independent colonization in soft agar or methylcellulose, accompanied by greater Gl-S progression. The cdk4 activity of the control- and cyclin D1 transfectants in colonization assays was comparable, but the cdk2 activity was higher in the latter. Injection of control- and cyclin D1 transfected MCF-10A cells in matrigel into nude mice failed to produce tumors within 1.5 years. The data suggest that cyclin D1 overexpression is an early feature of breast neoplastic progression, and can contribute to cancer development through the promotion of colonization. PMID- 10752678 TI - Isolation of genes overexpressed in freshly isolated breast cancer specimens. AB - A number of approaches have been used to identify genes important in breast cancer. In one approach the genes already shown to be involved in other tumors, such as p53 and Her2neu, were examined. A second approach examined genes detected through genetic screening of families with a high incidence of breast cancer, for example, BRCA-1 and BRCA-2. We used a third approach, subtractive hybridization, to identify and clone genes that were preferentially expressed in breast cancer cells compared to normal mammary epithelium. Instead of analyzing breast cancer cell lines, we examined fresh human breast cancer specimens. By subtracting normal mammary epithelial cDNA from breast cancer cDNA, we were able to clone several genes overexpressed in breast cancer. Two of these genes, L19 and MLN70, were previously reported to be overexpressed in breast cancer. Three of these genes, L19, L34, and MLN70, were localized to a region on chromosome 17 where Her2/neu and BRCA-1 are found. In addition, we isolated a gene we call breast cancer associated gene-1 that was expressed almost exclusively in fresh breast cancer tissue and not in normal mammary epithelium or breast cancer cell lines. We were unable to detect expression of breast cancer associated gene-1 in cell lines from melanoma, renal cell carcinoma, lymphoma, or leukemia. The full-length sequence from two separate breast cancer specimens revealed one amino acid difference compared to the sequence from normal breast epithelial tissue. Further studies are necessary to determine whether these genes contribute to breast cancer development or can be used as therapeutic targets. PMID- 10752679 TI - The influence of infiltrating lobular carcinoma on the outcome of patients treated with breast-conserving surgery and radiation therapy. AB - BACKGROUND: The role of conservative surgery and radiation therapy (CS and RT) in the treatment of patients with infiltrating ductal carcinoma is well established. However, the efficacy of CS and RT for patients with infiltrating lobular carcinoma is less well documented. The goal of this study was to examine treatment outcome after CS and RT for patients with infiltrating lobular carcinoma and to compare the results to those of patients with infiltrating ductal carcinoma and patients with mixed ductal-lobular histology. METHODS: Between 1970 and 1986, 1624 patients with Stage I or II invasive breast cancer were treated with CS and RT consisting of a complete gross excision of the tumor and > or = 6000 cGy to the primary site. Slides were available for review for 1337 of these patients (82%). Of these, 93 had infiltrating lobular carcinoma, 1089 had infiltrating ductal carcinoma, and 59 had tumors with mixed ductal and lobular features; these patients constitute the study population. The median follow-up time for surviving patients was 133 months. A comprehensive list of clinical and pathologic features was evaluated for all patients. Additional histologic features assessed for patients with infiltrating lobular carcinoma included histologic subtype, multifocal invasion, stromal desmoplasia, and the presence of signet ring cells. RESULTS: Five and 10-year crude results by site of first failure were similar for patients with infiltrating lobular, infiltrating ductal, and mixed histology. In particular, the 10-year crude local recurrence rates were 15%, 13%, and 13% for patients with infiltrating lobular, infiltrating ductal, and mixed histology, respectively. Ten-year distant/regional recurrence rates were 22%, 23%, and 20% for the three groups, respectively. In addition, the 10-year crude contralateral breast cancer rates were 4%, 13% and 6% for patients with infiltrating lobular, infiltrating ductal and mixed histology, respectively. In a multiple regression analysis which included established prognostic factors, histologic type was not significantly associated with either survival or time to recurrence. CONCLUSIONS: Patients with infiltrating lobular carcinoma have a similar outcome following CS and RT to patients with infiltrating ductal carcinoma and to patients with tumors that have mixed ductal and lobular features. We conclude that the presence of infiltrating lobular histology should not influence decisions regarding local therapy in patients with Stage I and II breast cancer. PMID- 10752680 TI - Liarozole fumarate (R85246): a novel imidazole in the treatment of receptor positive postmenopausal metastatic breast cancer. AB - This phase II study of liarozole fumarate (R85246, liarozole), a novel imidazole with retinomimetic and aromatase inhibitory effects, was designed to determine the efficacy and tolerability in postmenopausal women with advanced breast cancer in progression, to correlate these effects with hormonal levels, and to evaluate quality of life. Twenty-nine women with ER-positive or unknown metastatic disease who received > or = 2 prior hormonal therapies were treated with 150-300 mg liarozole twice daily until disease progression. All patients were evaluable for toxicity and 25 for response. Four patients (16.0%, 95% CI 5.3-37.4%) had partial remission (PR) of their disease for a median of 7.4 months (range 1.2-12.9) and 7 (28%) had disease stabilization for a median of 4.8 months (1.6-16.0). Estradiol decreased from pre-treatment levels of 9.2-52 pM (mean 17.1) to below detection (9.2 pM, p = 0.0005) after 1 month. Similarly estrone levels fell from 14-307 pM (mean 92.7) to below detection (9.2 pM, p = 0.0001). The most common toxicity was dermatological (96.6%) with features compatible with hypervitaminosis A syndrome such as rash, pruritus, dry skin, and brittle nails. The majority of these were mild to moderate in severity. No significant improvement in quality of life scores (FLI-C) were noted. Liarozole is an active new treatment for breast cancer in patients heavily pre-treated with hormone therapies. Further studies are needed to confirm its relative efficacy in both receptor positive and negative postmenopausal breast cancer. PMID- 10752681 TI - Results of two or five years of adjuvant tamoxifen correlated to steroid receptor and S-phase levels. South Sweden Breast Cancer Group, and South-East Sweden Breast Cancer Group. AB - A Swedish cooperative trial demonstrated that 5 years of adjuvant tamoxifen was more beneficial than 2 years of tamoxifen in the treatment of postmenopausal women with estrogen receptor (ER) positive, early stage, invasive breast cancer. The main aim of the present study was to investigate the importance of progesterone receptor (PgR) and ER concentration levels for patients participating in the trial and still distant recurrence free two years after the primary operation. Subgroup analyses revealed that only patients with ER positive and PgR positive breast cancer had improved distant recurrence free survival (DRFS) by prolonged tamoxifen therapy (p = 0.0016). Patients with ER negative and PgR negative as well as ER positive and PgR negative tumors showed no significant effect of prolonged tamoxifen (p = 0.53 and p = 0.80, respectively). The percentage of ER negative and PgR positive breast cancers was too small (2.2%) for any meaningful subgroup analysis. There was a significant positive trend that the concentration level of PgR (high positive vs. low positive vs. negative) decreased the recurrence rate for those with prolonged therapy. No corresponding pattern was found for the ER content. S-phase fraction did not correlate to the recurrence rate of PgR positive breast cancers. Patients recurring during tamoxifen therapy had receptor negative tumors to a greater extent than those recurring after tamoxifen treatment. In conclusion, prolonged tamoxifen therapy for 5 years instead of 2 years was found to be beneficial for patients with ER positive and PgR positive breast cancer, whereas three extra years of tamoxifen had little or no effect for patients with ER positive but PgR negative tumors as well as for steroid receptor negative patients. PMID- 10752682 TI - The axillary arch of Langer--the most common muscular variation in the axilla. AB - A case of an 'Axillary Arch of Langer' is reported during a routine axillary lymphadenectomy for breast carcinoma. The features of this muscle are described together with other less common axillary muscular anomalies. Knowledge of these anomalies is important for surgeons performing safe axillary surgery [1]. PMID- 10752683 TI - Eradication of murine mammary adenocarcinoma through HSVtk expression directed by the glucose-starvation inducible grp78 promoter. AB - Gene therapy strategies employing the HSVtk/ganciclovir (GCV) suicide gene offer promising approaches towards the treatment of metastatic breast cancer. These include bystander effects on non-transduced tumor cells, lower systemic toxicity, and the possibility of inducing immunity against the tumor. Previously we have demonstrated the ability of the grp78 stress-inducible promoter to stimulate expression of reporter genes within the tumor microenvironment. However, experimental evidence demonstrating the ability of this promoter to activate therapeutic agents within the breast cancer environment causing tumor eradication is needed prior to clinical trials. In this report, we test the efficacy of the grp78 promoter in a retroviral system to drive the expression of the HSVtk suicide gene in a murine mammary adenocarcinoma cell line (TSA) in syngeneic, immune-competent hosts. Our results show that under glucose-starvation conditions in vitro, the expression of HSVtk and GCV induced cell death are enhanced in tumor cells in which the HSVtk gene is driven by the internal grp78 promoter compared to cells in which the Moloney murine leukemia virus LTR drives HSVtk. In in vivo studies, in tumors in which the HSVtk gene is driven by the grp78 promoter, GCV treatment causes complete tumor eradication, whereas tumors persist when the HSVtk gene is driven by the retroviral LTR. Our study suggests that the grp78 promoter may be useful to enhance the effectivity of therapeutic agents within a breast tumor. In addition, it is shown that immune memory is induced in syngeneic, immune-competent hosts. This new retroviral vector might therefore be useful for breast cancer gene therapy. PMID- 10752684 TI - Basement membrane laminin-5 is deposited in colorectal adenomas and carcinomas and serves as a ligand for alpha3beta1 integrin. AB - Interplay between laminin-5 (Ln-5) and its integrin (Int) receptors alpha2beta1, alpha3beta1 and alpha6beta4 has been implicated in the progression and invasion of carcinomas. In this study we found abundant immunoreactivity for chains of Ln 5 (alpha3-beta3-gamma2) and Ln-10 (alpha5-beta1-gamma1), as well as for type VII collagen, in basement membranes (BM) of colorectal adenomas. In carcinomas of all differentiation grades, Lns were seen in tumor BMs, whereas type VII collagen was almost absent. Ln-5 appeared to accumulate along the invading edges of carcinomas, while Ln-10 was mostly absent. Immunoreactivity for Ln al chain, a component of Lns-1 and -3, was not seen in adenomas or carcinomas. Immunoreactivity for alpha2, alpha6, beta1 and beta4 Ints was found in all tumors and that for alpha3 Int in all adenomas and most of the carcinomas, often in colocalization with Ln-5. Immunoblotting of carcinoma tissues showed that the gamma2 chain of Ln-5 was present as typical Mr 105000 and 155000 isoforms. Immunoprecipitation experiments showed production of Ln-5 by cultured colon carcinoma cells. In quantitative cell adhesion experiments, function-blocking MAbs to alpha3 and beta1 Int subunits, but not those to Int alpha2 or alpha6 subunits, significantly inhibited the adhesion of cells to Ln-5. Our results suggest that BM composition in colorectal adenomas reflects the properties of surface epithelial BM of colorectal mucosa. In invading carcinomas, trimeric Ln 5, produced by carcinoma cells, is a major BM component and the cells use the alpha3beta1 Int complex for adhesion to Ln-5. PMID- 10752685 TI - Well-differentiated adenocarcinoma of the gallbladder mimicking minimal deviation adenocarcinoma of the cervix. AB - We report a case of well-differentiated adenocarcinoma of the gallbladder, histologically mimicking minimal deviation adenocarcinoma (MDA) of the cervix. A 71-year-old Japanese male underwent cholecystectomy because of the suggestion of gallbladder carcinoma. The resected gallbladder showed a localized thickening of the gallbladder wall with a polypoid lesion measuring 12x7 mm in diameter. Microscopically, the polypoid lesion proved to be a well-differentiated adenocarcinoma composed of columnar cells with a clear cytoplasm. In the thickened gallbladder wall, well-formed glands were extensively distributed; they were surrounded by a slightly desmoplastic reaction instead of lamina propria, or were directly in contact with smooth muscle cells. The diagnostic criteria for cervical MDA may be useful in distinguishing well-differentiated adenocarcinoma of the gallbladder from benign conditions, such as Rokitansky-Aschoff sinus and adenomyomatosis. It is remarkable that the tumor cells of the present case expressed gastric type mucin which is characteristic of mucinous type cervical MDA. PMID- 10752686 TI - Mucosal and systemic antibody responses after peroral or intranasal immunization: effects of conjugation to enterotoxin B subunits and/or of co-administration with free toxin as adjuvant. AB - The mucosa-binding molecules cholera toxin (CT) from Vibrio cholerae and heat labile enterotoxin (LT) from Escherichia coli have previously been used as mucosal adjuvants and carriers for many types of antigen. However, since these molecules are toxic and cannot be used in human vaccines, it is important to study whether their non-toxic mucosa-binding B subunits, CTB and LTB, can be used as alternative safe mucosal adjuvants and/or carrier molecules. We have as a model protein antigen used human gammaglobulin (HGG) for admixture with or chemical conjugation to recombinantly produced CTB and LTB, respectively, and measured antigen-specific local secretory IgA antibodies in saponin extracts from intestine and lung tissue by ELISA following intra-nasal (i.n.) or per-oral (p.o.) immunization. The results show that local antibody formation against HGG was increased after immunization with conjugated as compared to free HGG. However, while the conjugates alone gave rise to significant immune responses in the lung and also, to a lesser degree, in the intestine after i.n. immunization, co-administration of a small amount of free CT/LT as adjuvant was needed to induce a significant immune response in the intestine after p.o. immunization. We also found that following i.n. immunization, the addition of CTB to HGG, without coupling, increased the mucosal immune response to some extent, indicating that CTB by itself can work as an adjuvant by the i.n. route of immunization. A striking finding was that, as a carrier, CTB was superior to LTB when the conjugates were used by the oral but not by the i.n. route of immunization. In conclusion, conjugation of an antigen to mucosa-binding molecules such as CTB and/or LTB can dramatically increase their mucosal immunogenicity. This approach may thus be useful in the preparation of mucosal vaccines. PMID- 10752687 TI - Septic arthritis caused by a gram-negative bacterium representing a new species related to the Bordetella-Alcaligenes complex. AB - A knee-joint exudate culture yielded on two occasions a gram-negative bacterium. Regular methods for speciation did not provide an identification. The infection was successfully treated with ciprofloxacin. The unknown isolate, CCUG 36768, was subjected to further investigation, including 16S rDNA sequencing, protein profiling, cellular fatty acid analysis, and various biochemical tests, in order to produce a species identification. The 1469 bp-long 16S rDNA sequence did not reveal identity with any known species sequence. CCUG 36768 clustered in a group of species, including Alcaligenes defragrans, Denitrobacter permanens, Taylorella equigenitalis, Alcaligenes faecalis, and four strains of Alcaligenes species without a specific species name. Bordetella species also showed a high degree of similarity with CCUG 36768. Protein profiling, cellular fatty acid analysis and computer-assisted analysis of biochemical profiles indicated similarity with Bordetella-Alcaligenes species, often close to B. holmesii and B. avium. API 20 NE indicated the profile of Moraxella species of poor identity. It is concluded that CCUG 36768 represents a new bacterial species of pathogenic potential in humans. It is related to the Bordetella-Alcaligenes group. Powerful new methods for speciation are available and it is recommended that unknown isolates from normally sterile sites be submitted for further analysis. Several isolates are required for the definition of new species. PMID- 10752688 TI - Absence of mutations in the kinase domain of the Met gene and frequent expression of Met and HGF/SF protein in primary gastric carcinomas. AB - Hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, Met, are known to play important roles in tumor cell migration, invasion, and metastasis. We analyzed the expression of these genes and the mutations in the kinase domain of the Met gene in 43 gastric carcinomas. Of the 43 cases, the Met and HGF/SF protein were expressed in 29 (67%) and 22 (51%), respectively. All of the cases with HGF/SF immunopositivity also expressed Met. Of 22 cases with HGF/SF immunopositivity, 13 (59%) expressed HGF/SF in tumor cells as well as fibroblasts. We detected no aberrant single-strand conformational polymorphism patterns, suggesting that there are no genetic alterations in the kinase domain of the Met gene. These results indicate that HGF/SF-mediated autocrine and/or paracrine stimulation and overexpression rather than structural alteration of its receptor may contribute to the development and progression of gastric carcinoma, and that expression of Met and HGF/SF may confer a growth advantage to neoplastic cells. PMID- 10752689 TI - The bactericidal/permeability-increasing protein (BPI) is membrane-associated in azurophil granules of human neutrophils, and relocation occurs upon cellular activation. AB - Neutrophilic granulocytes contain the 55 kDa bactericidal/permeability-increasing protein (BPI). BPI binds to lipopolysaccharides (LPS), and exerts bacteriostatic and bactericidal effects against a wide variety of Gram-negative bacterial species. We have investigated the subcellular location of BPI in immature and mature neutrophils using cryotechnique for immunoelectron microscopy. BPI was found to colocate with myeloperoxidase (MPO), a marker for azurophil granules, and it also showed the same pattern of distribution as CD63, a transmembrane anchored protein. This suggests that BPI is membrane-associated in the azurophil granules in neutrophils. Its presence in azurophil granules was further confirmed by the finding of BPI in the azurophil granules of neutrophil promyelocytes of the bone marrow. Induction of selective release of azurophilic granules by the Na ionophore monensin resulted in fusion of endosomes with azurophil granules, leading to the formation of large vacuoles containing MPO, CD63, and BPI. After phagocytosis of serum-treated zymosan (STZ), BPI was detected in phagosomes, both in association with membranes as well as in the lumen, suggesting the release of BPI into activated compartments. The results show that BPI is present in azurophil granules, is probably primarily membrane-associated, and is relocated after activation, following the same route as MPO and CD63. PMID- 10752690 TI - Expression of topoisomerase II and Ki-67 in cervical carcinoma- clinicopathological study using immunohistochemistry. AB - AIM: To study the correlation between the expression of topoisomerase II and Ki 67 antigen and disease outcome in cervical squamous cell carcinomas. EXPERIMENTAL DESIGN: Forty-nine cervical carcinomas, 10 cases of high-grade cervical intraepithelial neoplasia (CIN II-III) and 5 control cervices were stained by monoclonal antibodies for topoisomerase II and Ki-67 (MIB-1 clone). Nuclear counts were correlated with patient age, tumor stage, histological grade and survival. RESULTS: Thirteen patients died of disease, 35 remained free of disease, and one patient was lost to follow up. Ki-67 counts were higher in CIN lesions, when compared to both invasive carcinomas and control cervices. Topoisomerase II counts were comparable for CIN and invasive tumors. No immunoreactivity for topoisomerase was detected in control cases. Neither stage nor grade was associated with nuclear counts using either marker. In multivariate survival analysis, stage (p=0.001), grade (p=0.03) and older patient age (p=0.02) predicted poor survival. Ki-67 counts predicted survival with borderline significance (p=0.07), while topoisomerase II counts were not related to survival. CONCLUSION: Ki-67 and topoisomerase II counts do not appear to have a significant role in the prediction of survival in cervical squamous cell carcinoma. PMID- 10752691 TI - Histopathological and morphometric analyses of late rectal injury after irradiation. AB - The aim of the present study was to assess and quantify changes in the structural components of the rectal wall after irradiation with varying single doses of x rays. A total of 70 CDF1/Bom male mice were irradiated at a selective 1.5 cm of the distal rectum with varying single doses of 0-30 Gy. At 32 weeks postirradiation the mice were sacrificed and the rectum was removed. Sampling of the specimens was based on unbiased stereological principles using systematic random sampling. Vertical tissue sections were used to estimate mucosal surface area density and to measure the thickness and volume fraction of the different intestinal wall layers. The surface area density decreased with increasing dose (p<0.02) due to pronounced injury of the crypts and mucosal morphology. The thickness and the volume fraction of the submucosa were significantly increased in the 20, 25 and 30 Gy treatment groups (p<0.0001 and p<0.0001), whereas the thicknesses of the mucosa, the lamina muscularis propria and the serosa did not change after irradiation. It is concluded that irradiation causes histopathological changes in the rectal mucosa and submucosa, and changes in the thickness of the submucosal intestinal layer. These changes were found to be dose dependent. PMID- 10752692 TI - Human cytomegalovirus induces apoptosis in the hematopoietic cell line MO7e. AB - Several studies have shown that human cytomegalovirus (HCMV) induces growth suppression of hematopoietic progenitors. In vitro studies have demonstrated that the HCMV-induced suppression is independent of viral protein production. Previous studies have indicated a link between HCMV infection and apoptosis in human cells. The purpose of our study was to investigate whether the observed inhibitory effect of HCMV on the human myeloid progenitors could be connected to the induction of apoptosis. The growth and cell death of the hematopoietic cell line MO7e was investigated following infection with HCMV virions and dense bodies. Both virions and dense bodies inhibited the growth of MO7e cells, and induced cell death measured by trypan blue staining. In addition, both HCMV virions and dense bodies caused an increased amount of apoptosis-characteristic DNA fragmentation in the MO7e cells compared to mock-treated cells. The HCMV virions were also able to induce an increased expression of phosphatidylserine on the cell surface, which is an early event in the initiation of apoptosis in most cell types. In conclusion, HCMV and HCMV dense bodies are able to induce apoptosis in the myeloid progenitor cell line MO7e. PMID- 10752693 TI - Application of four molecular techniques for typing outbreak-associated Mycobacterium tuberculosis strains. AB - We applied four molecular techniques for the typing of strains of Mycobacterium tuberculosis associated with outbreaks: RFLP of the IS6110 insertion sequence, spoligotyping, RAPD, and PCR-IS6110. All 4 techniques were applied to 18 strains which were shown by epidemiological data to be involved in 6 outbreaks. All the methods classified the strains into the same groups as the classical epidemiological data did, but RFLP of the IS6110 insertion sequence and spoligotyping are laborious techniques requiring more than a full day's work, whilst RAPD and PCR IS6110 are simple methods easily incorporated into the daily routine. Nevertheless, a large-scale process of standardization and evaluation is necessary in order to be able to establish the true value of the latter two methods as intraspecific characterization markers for M. tuberculosis isolates. PMID- 10752694 TI - Comparison of screening methods for detection of extended-spectrum beta lactamases and their prevalence among Escherichia coli and Klebsiella species in Hong Kong. AB - Three tests, the disk diffusion test, the double-disc synergy test and the inhibitor-potentiated disc diffusion test, were compared for their abilities to detect production of extended-spectrum beta-lactamases (ESBL) in 702 Escherichia coli and 472 Klebsiella spp. strains from four hospitals. Eleven percent E. coli and 13% Klebsiella spp. were found to produce ESBL. As an indicator of ESBL activity, the sensitivities of the five extended-spectrum beta-lactams were as follows: cefotaxime (100%), cefpodoxime (99.3%), ceftriaxone (98.6%), aztreonam (93%) and ceftazidime (57.7%) when interpreted using the National Committee for Clinical Laboratory Standards criteria. Their positive predictive values ranged from 67.8-83.8%. Both the inhibitor-potentiated disc diffusion test and the double-disc synergy test (at three inter-disc widths of 20, 25 and 30 mm) were capable of identifying all the ESBL-producers. However, at a single inter-disc width of 30 mm, the double-disc synergy test has limited sensitivity (83.8%). As a second test for confirming ESBL activity in strains with reduced susceptibility to beta-lactams, the inhibitor-potentiated disc diffusion test is therefore a simple and reliable option. PMID- 10752695 TI - Medicine's rude awakening to the commercial world. PMID- 10752696 TI - Antibodies to tumour necrosis factor alpha as treatment for Crohn's disease. PMID- 10752697 TI - Triptans and migraine. PMID- 10752698 TI - Early laparoscopy to help diagnose acute non-specific abdominal pain. PMID- 10752699 TI - Implications of discontinuation of doxazosin arm of ALLHAT. Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial. PMID- 10752700 TI - Is there a case for school-based screening for sexually transmitted diseases? PMID- 10752702 TI - Variation and trends in incidence of childhood diabetes in Europe. EURODIAB ACE Study Group. AB - BACKGROUND: To study the epidemiology of childhood-onset type 1 insulin-dependent diabetes in Europe, the EURODIAB collaborative group established in 1988 prospective geographically-defined registers of new cases diagnosed under 15 years of age. This report is based on 16 362 cases registered during the period 1989-94 by 44 centres representing most European countries and Israel and covering a population of about 28 million children. METHODS: Multiple sources of ascertainment were used in most centres to validate the completeness of registration by the capture-recapture method. Trends in incidence during the period were analysed by Poisson regression, the data from centres within each country being pooled. FINDINGS: The standardised average annual incidence rate during the period 1989-94 ranged from 3.2 cases per 100000 per year in the Former Yugoslav Republic of Macedonia to 40.2 cases per 100000 per year in two regions of Finland. By pooling over all centres, the annual rate of increase in incidence was 3.4% (95% CI 2.5-4.4%), but in some central European countries it was more rapid than this. Pooled over centres and sexes, the rates of increase were 6.3% (4.1-8.5%) for children aged 0-4 years, 3.1% (1.5-4.8%) for 5-9 years, and 2.4% (1.0-3.8%) for 10-14 years. INTERPRETATION: The results confirm a very wide range of incidence rates within Europe and show that the increase in incidence during the period varied from country to country. The rapid rate of increase in children aged under 5 years is of particular concern. PMID- 10752701 TI - Risks of untreated and treated isolated systolic hypertension in the elderly: meta-analysis of outcome trials. AB - BACKGROUND: Previous meta-analysis of outcome trials in hypertension have not specifically focused on isolated systolic hypertension or they have explained treatment benefit mainly in function of the achieved diastolic blood pressure reduction. We therefore undertook a quantitative overview of the trials to further evaluate the risks associated with systolic blood pressure in treated and untreated older patients with isolated systolic hypertension METHODS: Patients were 60 years old or more. Systolic blood pressure was 160 mm Hg or greater and diastolic blood pressure was less than 95 mm Hg. We used non-parametric methods and Cox regression to model the risks associated with blood pressure and to correct for regression dilution bias. We calculated pooled effects of treatment from stratified 2 x 2 contingency tables after application of Zelen's test of heterogeneity. FINDINGS: In eight trials 15 693 patients with isolated systolic hypertension were followed up for 3.8 years (median). After correction for regression dilution bias, sex, age, and diastolic blood pressure, the relative hazard rates associated with a 10 mm Hg higher initial systolic blood pressure were 1.26 (p=0.0001) for total mortality, 1.22 (p=0.02) for stroke, but only 1.07 (p=0.37) for coronary events. Independent of systolic blood pressure, diastolic blood pressure was inversely correlated with total mortality, highlighting the role of pulse pressure as risk factor. Active treatment reduced total mortality by 13% (95% CI 2-22, p=0.02), cardiovascular mortality by 18%, all cardiovascular complications by 26%, stroke by 30%, and coronary events by 23%. The number of patients to treat for 5 years to prevent one major cardiovascular event was lower in men (18 vs 38), at or above age 70 (19 vs 39), and in patients with previous cardiovascular complications (16 vs 37). INTERPRETATION: Drug treatment is justified in older patients with isolated systolic hypertension whose systolic blood pressure is 160 mm Hg or higher. Absolute benefit is larger in men, in patients aged 70 or more and in those with previous cardiovascular complications or wider pulse pressure. Treatment prevented stroke more effectively than coronary events. However, the absence of a relation between coronary events and systolic blood pressure in untreated patients suggests that the coronary protection may have been underestimated. PMID- 10752703 TI - Accuracy of assessment of pregnancy duration by women seeking early abortions. AB - BACKGROUND: Advances in medical abortion might allow women seeking early abortions to terminate their pregnancies safely and effectively without medical supervision. We investigated whether such women can calculate pregnancy duration accurately, a key step in unsupervised use. METHODS: 422 women seeking first trimester abortions in two clinics (Pune, India, and Atlanta, USA) used a simple worksheet and calendar to calculate the duration of gestation from the date of last menstrual period (LMP) and/or of unprotected intercourse. Clinicians then used standard clinic practices to estimate pregnancy duration. We compared the two sets of estimates, focusing on women who fell into the "caution zone" (ie, had pregnancy durations >8 weeks according to providers, but < or =8 weeks by their own estimates). FINDINGS: The participants were generally representative of the women seeking abortion at the two clinics. 217 (97.7%) of 222 women in Atlanta and 173 (86.5%) of 200 in Pune could produce an estimate of pregnancy duration. Most (85.4% in Atlanta; 93.6% in Pune) of these estimates were within 2 weeks of those made by providers. For estimates based on LMP, only 10.0% (exact 95% CI 6.2-15.0) of women in Atlanta and 9.8% (5.8-15.3) in Pune fell into the caution zone. For estimates based on a date of intercourse, just 7.7% (4.0-13.1) of women in Atlanta and 3.4% (0-17.8) in Pune fell into the caution zone, although fewer women could use this method. INTERPRETATION: The vast majority of women seeking first-trimester abortion in this study could accurately calculate pregnancy duration within a margin of error clinically inconsequential for safe use of unsupervised medical abortion. PMID- 10752704 TI - Community study of people who live in squalor. AB - BACKGROUND: The reasons why people live in squalor have been the subject of much debate but little systematic research other than reports of case series from secondary health-care services. We did a study in the community using standardised instruments to investigate the relation between squalor and mental and physical disorders. METHODS: We did a cross-sectional study of the clients of a local-authority special cleaning service. Levels of domestic squalor and self neglect were measured with the living conditions rating scale, and diagnoses of mental disorder were made by use of WHO's schedules for clinical assessment in neuropsychiatry (SCAN). FINDINGS: 91 individuals were eligible for inclusion; 81 from 76 households consented and were interviewed (a response rate of 89%). 41 (51%) were younger than 65 years of age. 57 individuals (70%) were diagnosed as having a mental disorder at interview, as defined by the SCAN, and 21 participants (26%) had a physical health problem which contributed significantly to the unclean state of their living environment. Those with a contributory physical disorder had a lower severity of domestic squalor. People older than 65 years were less likely to have a mental disorder than those younger than 65 years, but a contributory physical disorder was not associated with the presence of active mental disorder. Only 30 (53%) of the 57 individuals with active mental disorder had had any contact with mental-health services in the previous year. INTERPRETATION: People who live in squalor and who receive special cleaning services have high rates of mental disorder, and squalor affects younger as well as older people. Living in squalid conditions in the group was generally associated with a mental or physical disorder, and there were possible deficits in the health care received. The extent to which these disorders might respond to more assertive treatment from health services requires further study, but questions are raised about the adequacy of their current health care. PMID- 10752706 TI - Relation between tissue structure and imposed electrical current flow in cervical neoplasia. AB - BACKGROUND: When an electrical potential is applied to human tissue, the pattern of the resulting current flow is determined by the shapes, arrangements, and internal structure of the tissue cells. By measurement of the electrical current patterns over a range of frequencies, and use of an inverse modelling procedure, electrical variables describing the tissue structure can be calculated. We used this method to develop a screening technique for the detection of cervical precancers. METHODS: We used a pencil probe (diameter 5 mm) to measure electrical impedance spectra from eight points on the cervix in 124 women with abnormal cervical smears. Variables that should be sensitive to the expected tissue changes were calculated. These were compared with the colposcopic results. FINDINGS: The measured electrical impedance changes were those predicted on the basis of the expected tissue structures. Measurements made on normal squamous tissues were well separated from those made on precancerous tissues. We constructed receiver-operating-characteristic curves, comparing measurements made on normal tissue and that showing cervical intraepithelial neoplasia grade 2/3; the area under the curve was 0.951. These groups of women could be separated with a sensitivity of 0.92 and a specificity of 0.92. INTERPRETATION: Characteristics of the electrical impedance spectra of tissues can be explained by changes in cell arrangements (layering) and in the size of the nuclei. This relation opens the way to deriving tissue structure from electrical impedance spectral measurements. We show that this approach can be used to give good separation of normal and precancerous cervical tissues. PMID- 10752705 TI - The role of parenteral antischistosomal therapy in the spread of hepatitis C virus in Egypt. AB - BACKGROUND: The population of Egypt has a heavy burden of liver disease, mostly due to chronic infection with hepatitis C virus (HCV). Overall prevalence of antibody to HCV in the general population is around 15-20%. The risk factor for HCV transmission that specifically sets Egypt apart from other countries is a personal history of parenteral antischistosomal therapy (PAT). A review of the Egyptian PAT mass-treatment campaigns, discontinued only in the 1980s, show a very high potential for transmission of blood-borne pathogens. We examine the relative importance of PAT in the spread of HCV in Egypt. METHODS: The degree of exposure to PAT by cohort was estimated from 1961-86 Ministry of Health data. A cohort-specific exposure index for PAT was calculated and compared with cohort specific HCV prevalence rates in four regions. FINDINGS: HCV prevalence was calculated for 8499 Egyptians aged 10-50 years. A significant association between seroprevalence of antibodies to HCV and the exposure index (1.31 [95% CI 1.08 1.59]; p=0.007) was identified across four different regions. In all regions cohort-specific HCV prevalence was lowest in children and young adults than in older cohorts. These lower prevalence rates coincided with the gradual and final replacement of PAT with oral antischistosomal drugs at different points in time in the four regions. INTERPRETATION: The data suggest that PAT had a major role in the spread of HCV throughout Egypt. This intensive transmission established a large reservoir of chronic HCV infection, responsible for the high prevalence of HCV infection and current high rates of transmission. Egypt's mass campaigns of PAT may represent the world's largest iatrogenic transmission of blood-borne pathogens. PMID- 10752707 TI - A hazard of paint spraying. PMID- 10752708 TI - Allergic reactions to the polymeric glucose-based peritoneal dialysis fluid icodextrin in patients with renal failure. AB - A new glucoee polymer, icodextrin, related to maltose, is in increasing use as a peritoneal dialysis fluid. We report on adverse events seen in our unit during a 12-month period after the introduction of this reagent. PMID- 10752710 TI - Intraocular pressure changes and alterations in intracranial pressure. AB - We show that although there is a significant correlation between intraocular pressure and intracranial pressure in neurosurgical patients, changes in intraocular pressure are a poor predictor of changes in intracranial pressure. PMID- 10752709 TI - Low serum IGF-1 and occurrence of osteoporotic fractures in postmenopausal women. AB - The link between serum insulin-like growth factor 1 (IGF-1) and postmenopausal osteoporosis remains controversial. In this study of healthy postmenopausal women, decreased serum concentrations of IGF-1 were strongly associated with an increased risk of osteoporotic fractures independently of bone-mineral density. PMID- 10752711 TI - Fat absorption in cystic fibrosis after correction of hypokalaemia. AB - Extracellular potassium up-regulates exocrine pancreatic function in tissue preparations of mammalian pancreatic lobules. We report on a child with cystic fibrosis in whom clinical evidence suggested that this laboratory observation may be of relevance to clinical practice. PMID- 10752712 TI - 14-3-3 protein in the cerebrospinal fluid of patients with acute transverse myelitis. AB - The eventual recovery of patients with acute transverse myelitis is variable and can be difficult to predict on the basis of the presenting clinical and radiographic features. We find that an early accumulation of the 14-3-3 protein occurs exclusively in the cerebrospinal fluid of patients who show little or no recovery of neurological function and therefore could serve as a helpful prognostic indicator in this disorder. PMID- 10752713 TI - Surface tension, proteinuria, and the urine bubbles of Hippocrates. AB - Hippocrates noted that bubbles in urine were associated with kidney disease. We examined changes in surface tension responsible for bubble formation in urine, to investigate whether surface tension could be a more accurate and continuous linear predictor of 24-h proteinuria than currently available tests on spot urine. PMID- 10752714 TI - No easy answer to high native suicide rates. PMID- 10752715 TI - Lebanese medicine--still struggling against the odds. PMID- 10752716 TI - Pharmaceutical companies pledge vaccines for developing countries. PMID- 10752717 TI - Calls made for improvements in Bangladeshis' physical health. PMID- 10752718 TI - SNaRIs, NaSSAs, and NaRIs: new agents for the treatment of depression. AB - A major goal of antidepressant development is to improve on preceding drug classes with agents with greater specificity (and therefore fewer unwanted side effects) and with more rapid onset of antidepressant action. To this end, four antidepressants with significantly distinct pharmacological characteristics have been recently introduced: venlafaxine, nefazodone, mirtazapine, and reboxetine. Venlafaxine is the first antidepressant in a new drug class referred to as the serotonin noradrenergic reuptake inhibitors (SNaRIs). Nefazodone is a weaker serotonin and norepinephrine reuptake inhibitor, but a potent serotonin 5-HT2 receptor antagonist. Mirtazapine is a potent antagonist of central 2alpha adrenergic autoreceptors, and heteroreceptors and is an antagonist of serotonin 5 HT2 and 5-HT3 receptors. The result of these actions is to increase both noradrenergic and specific (5-HT1) serotonergic transmission, and mirtazapine has therefore been termed a noradrenergic and specific serotonergic antidepressant (NaSSA). Reboxetine is the first selective noradrenaline reuptake inhibitor (NaRI) to be introduced since the tricyclics, and lacks immediate serotonergic effects. PMID- 10752719 TI - Insulin for the world's poorest countries. AB - In the industrialised world, type 1 diabetes rarely results in death from ketoacidosis. The same is not true in many countries in the developing world where insulin availability is intermittent, and insulin may not even be included on national formularies of essential drugs. The life expectancy for a newly diagnosed patient with type 1 diabetes in some parts of Africa may be as short as 1 year. The World Bank has identified 40 highly indebted poor countries (HIPCs) whose national debt substantially exceeds any possibility of repayment without heavy impact on health and social programmes. Incidence and prognosis of type 1 diabetes in HIPCs are lower than in most industrialised countries, and 0.48% of the world's current use of insulin is estimated to be sufficient to treat all type 1 diabetic patients in these countries. A proposal is made for the major insulin manufacturers to donate insulin, at an estimated cost of US$3-5 million per year, as part of a distribution and education initiative for type 1 diabetic patients in the HIPCs. No type 1 diabetic patient in the world's poorest countries need then die because they, or their government, cannot afford insulin. PMID- 10752720 TI - Nuisance or natural and healthy: should monthly menstruation be optional for women? PMID- 10752721 TI - Overoptimism over cancer. PMID- 10752722 TI - Overoptimism over cancer. PMID- 10752723 TI - Male circumcision and HIV infection. PMID- 10752724 TI - Male circumcision and HIV infection. PMID- 10752725 TI - Male circumcision and HIV infection. PMID- 10752726 TI - Male circumcision and HIV infection. PMID- 10752727 TI - Pathophysiology of hypertrophic cardiomyopathy. PMID- 10752728 TI - Role of microvascular decompression in trigeminal neuralgia. PMID- 10752729 TI - Therapeutic efficiency of tirofiban in acute coronary syndromes. PMID- 10752730 TI - Therapeutic efficiency of tirofiban in acute coronary syndromes. PMID- 10752731 TI - Toxins and genetically modified food. PMID- 10752732 TI - Surgeon experience. PMID- 10752733 TI - Pain in neonates. PMID- 10752734 TI - Drunk driving and mental disorders. PMID- 10752735 TI - Stalkers and the definition of violence. PMID- 10752736 TI - High necropsy rates: just a ritualistic mantra? PMID- 10752737 TI - Danger from rabies-infected bats. PMID- 10752738 TI - A potted history of specimen-taking. PMID- 10752739 TI - A biopsy of the use of the Baxter formula to resuscitate burns or do we do it like Charlie did it? AB - The Baxter formula is commonly used to calculate fluid requirements. Baxter reported that 12% of patients would require more than 4.3 mL/kg per percentage of total body surface area (%TBSA). We anecdotally observed that we frequently exceeded the predictions of the formula, and we wondered if this was unique to our practice. We studied our last 11 burn-related resuscitations and collected fluid resuscitation data from US burn centers. Twenty-eight centers were queried, and 6 centers shared data. We were therefore able to study the resuscitation data of 50 adult patients. For 29 patients (58%), 4.3 mL/kg/%TBSA was exceeded compared with the 12% reported by Baxter. These findings suggest that in actual practice, fluid volumes administered are larger than the Baxter formula predicts. This survey does not explain why. Possible reasons for the larger fluid volumes are as follows: (1) the sample is not representative; (2) the formula is used improperly; (3) burns have changed and require more fluids; (4) burn care has changed. PMID- 10752740 TI - The hair color-highlighting burn: a unique burn injury. AB - A unique, preventable, 2.8 x 3.7-cm, full-thickness scalp burn resulted after a woman underwent a professional color-highlighting procedure at a hair salon. The burn appeared to result from scalp contact with aluminum foil that had been overheated by a hair dryer during the procedure. The wound required debridement and skin grafting and 3 subsequent serial excisions to eliminate the resulting area of burn scar alopecia. The preventive aspects of this injury are discussed. PMID- 10752741 TI - Routine assessment of viability in split-thickness skin. AB - Effective quality control of allograft skin that is cryopreserved for transplantation requires a simple, reproducible technique for the assessment of cell viability. Tetrazolium reduction assays and an oxygen consumption technique have been the two methods of choice to determine the metabolic function of allograft skin after it has been thawed. In this study, we investigated the use of a novel tetrazolium salt, WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5 tetrazolio]-1,3-benzen e disulfonate), that is cleaved to a water-soluble formazan product. Porcine split-thickness skin in minimal essential medium without cryoprotectant was subjected to a graded freezing protocol to generate progressive amounts of cryoinjury. Recovery as determined with WST-1 was compared with measurements made with the use of the oxygen consumption technique. The similarity of the resulting recovery curves indicates that WST-1 is a simple, effective, and convenient technique for the assessment of metabolic function in porcine split-thickness skin. The WST-1 assay is applicable for the routine assessment of tissue viability in cryopreserved allograft skin. PMID- 10752742 TI - Post-traumatic stress symptoms and distress 1 year after burn injury. AB - The occurrence and predictors of post-traumatic stress symptoms 1 year after a burn injury were assessed in a large prospective sample (N = 172). Participants completed a self-report post-traumatic stress symptom checklist at 3 time points: within 24 hours of admission to a burn center, 1 month after the injury, and 1 year after the injury. A notable number of participants had a range of post traumatic stress symptoms both at 1 month and at 1 year after the burn injury. More than half of the sample reported recurrent intrusive recollections of the burn injury at 1 month and at 1 year. Other commonly endorsed symptoms were sleep disturbance, avoidance of thoughts or feelings associated with the burn, and distress at reminders of the burn. The number of post-traumatic stress symptoms endorsed at 1 month was the only significant predictor of post-traumatic stress symptoms at 1 year. These results suggest that it is common for patients to have some post-traumatic symptoms 1 year after a burn injury and that early experiences of post-traumatic stress symptoms may be associated with the development or maintenance of post-traumatic stress disorder. We recommend that burn care professionals identify and intervene with patients who have clinically significant distress as a result of their burn injuries. PMID- 10752743 TI - Vietnamese "coining": a burn case report and literature review. AB - "Coining," "coin rubbing," or cao gio, is an ancient Vietnamese folk remedy that is practiced by many Vietnamese-Americans to treat minor ailments. There are few reported cases of serious complications of cao gio; most of the complications have been minor burns. We present a case report of a 45-year-old woman who sustained 22% partial-thickness and full-thickness burns after she caught on fire during a coining treatment. She underwent fluid resuscitation, wound care management, and, eventually, burn debridement and autografting. She was discharged 10 days after the operation, with excellent take of all of her grafts. However, a contracture of her right axilla did develop and will require surgical release. This case represents a serious injury that can result from traditional cultural forms of medicine. PMID- 10752744 TI - Antithrombin(H) concentrate infusions are safe and effective in patients with thermal injuries. AB - An antithrombin (AT) deficiency develops in patients with thermal injuries as a result of the subclinical disseminated intravascular coagulation. We conducted a pilot study to assess AT(Human [H]) concentrate infusions for safety and efficacy in thermal injury. Nine patients with burns who received Thrombate (Bayer Corporation, Berkeley, Calif) AT(H) concentrate infusions every 8 hours to raise the plasma level to 175% in the first 72 hours after injury were compared with 9 control patients with burns. Admission AT plasma levels were 45%+/-10% in patients treated with AT(H) versus 49%+/-18% in control patients (normal, 100%+/ 20%). Day-2 to day-4 levels were 120%+/-25% in patients treated with AT(H) patients versus 50%+/-12% in the control patients (P < .002). In the group treated with AT(H), the time to wound healing was shorter for all body regions and was significantly shorter for the hand (P < .02). Compared with control patients, patients treated with AT(H) had similar blood loss per grafted area. A trend toward fewer autografting procedures, a shorter meshed autograft healing time, and a decreased hospital stay was found for the patients treated with AT(H). AT(H) concentrate infusions are safe with thermal injury and are a viable option to shorten the length of hospitalization and to promote graft viability and survival. Clinical trials to confirm the benefit of this medication in the acute phase of thermal injury would be worthwhile. PMID- 10752745 TI - Transforming growth factor-beta1 and splenocyte apoptotic cell death after burn injuries. AB - Transforming growth factor (TGF)-beta1 is a multifunctional cytokine that mediates apoptotic cell death in human lymphocytes in vitro. To better understand the mechanism through which TGF-beta1 exerts its apoptotic effect, we investigated the role of TGF-beta1 in the relationship between burn injury and cell death of splenocytes in a mouse model of either 0%, 25%, or 40% full thickness burns. Mice were killed and spleens were harvested at 15 and 30 minutes and at 1, 2, 4, 8, 12, and 24 hours after the burn. The spleens were divided and used for both histologic analyses with H-E stain and TUNEL stain and for total messenger RNA isolation and reverse transcriptase-polymerase chain reaction amplification. Amplified polymerase chain reaction products were analyzed for signal strength by electrophoresis. TGF-beta1 RNA expression was highest at 2 hours after the burn injuries in the 40% full-thickness burns and at 4 hours after the burn injuries in the 25% full-thickness burns. The relative increase in TGF-beta1 RNA was 3 times greater with the larger burn than with the smaller burn. In histologic analysis, splenocyte apoptotic cell death was observed at 4 to 24 hours after the burn in the 40% full-thickness burns but at only 4 to 12 hours in the 25% full-thickness burns. TGF-beta1 RNA peak expression was observed at different times after the burn in 25% and 40% full-thickness burns. Histologic analysis showed apoptotic cell death in proportion with respective messenger RNA expressions. This suggests that TGF-beta1 may be associated with apoptosis of splenocytes in vivo and that the effect of TGF-beta1 after a burn injury may be important in the immune system. PMID- 10752747 TI - Altered interleukin-6 expression in fibroblasts from hypertrophic burn scars. AB - Hypertrophic burn scars (HBSs) are characterized by a net accumulation of collagen. The role of growth factors and various cytokines in the pathogenesis of these lesions has not been fully characterized. Interleukin-6 (IL-6) is an important immunoregulatory cytokine that can affect fibroblast function. This study showed increased expression of the IL-6 gene in fibroblasts isolated from patients with HBSs compared with control fibroblasts when the ribonuclease protection assay was used. A subsequent increase in unstimulated and stimulated IL-6 secretion was noted in HBS fibroblasts compared with normal control fibroblasts (unstimulated, 0.3627+/-0.2017 pg/cell vs 0.0662+/-0.0786 pg/cell, P = .0054; serum, 0.770+/-0.523 pg/cell vs 0.233+/-0.231 pg/cell, P = .0381; serum and interferon-gamma, 0.912+/-0.650 pg/cell and 0.244+/-0.199 pg/cell, P = .0327). These results are further evidence of the role of inflammatory cytokines in the development of HBSs. PMID- 10752746 TI - Gene expression and cytokine and enzyme activation in the liver after a burn injury. AB - The liver plays a critical role in the inflammatory response to injury; however, the mechanisms by which the liver is affected and how it influences the rest of the immune system are not well understood. Partial hepatectomy is a direct injury to the liver, whereas a burn is an indirect injury to liver, but both injuries appear to produce damage to the liver. In this study, we used a mouse model of 25% total body surface area and 40% total body surface area full-thickness burns to investigate the mechanism of liver damage and response to burn injury by measuring levels of c-Jun messenger (m)RNA, NFkappaB nuclear protein, interleukin 6, transaminases, and liver tissue histology over time. c-Jun and NFkappaB are 2 transcription factors that are induced by partial hepatectomy and related to hepatocyte injury and growth. In both groups of mice with burns, expression of c Jun mRNA and NFkappaB nuclear protein was activated within 30 minutes after the burn injury, followed by increased levels of interleukin-6 and, finally, elevated enzyme levels. Liver injuries were similar in both groups despite the magnitude of the burns. We believe that these gene products are initiated in the hepatocyte injury after a burn and that they precede other inflammatory responses such as cytokine release, plasma transaminase levels, and histologic changes. PMID- 10752748 TI - The relationship between oxygen delivery and oxygen consumption during fluid resuscitation of burn-related shock. AB - Although burn-related shock resuscitation based on invasive hemodynamic monitoring has been reported at an increased rate, little is known about appropriate hemodynamic end points. Shock resuscitation based on oxygen transport criteria has been widely used for patients with trauma and patients who undergo surgery, and supranormal values of oxygen delivery (DO2) have been reported in association with an improved survival rate. This improved survival rate has been attributed to a shifting of the critical threshold of DO2 to higher values in these patients. In patients with thermal injuries, the effects of the manipulation of hemodynamics to optimize oxygen transport have not been proven. It is still unclear whether these patients exhibit delivery-dependent oxygen consumption (VO2) during the shock phase. The goal of this study was to evaluate the existence of oxygen supply dependency and to determine critical levels of DO2 in patients with burns. In a prospective study that included 16 patients with serious thermal injuries, we studied the effects of volume loading on DO2 and VO2. A transpulmonary double dilution technique was used for hemodynamic monitoring, and resuscitation end points included a normalization of preload and cardiac output parameters within 24 hours of the thermal injury. Fluid loading with crystalloids and colloids, according to our resuscitation protocol, was used to augment cardiac output and DO2. Of the 16 patients with a mean of 46% total body surface area burned (range, 22%-80%), 8 patients survived and 8 patients died. With the use of progressive fluid loading, cardiac index was restored within 24 hours of admission in all of the patients. Successful resuscitation was associated with increased levels of DO2 and VO2 and with declining serum lactate levels. VO2 appeared to be dependent on DO2 during the resuscitation period (r = 0.596), and the correlation was significantly stronger in the patients who survived (r = 0.744) than in the patients who died (r = 0.368; P < .05). A critical threshold of oxygen supply could not be identified. We concluded that increasing DO2 by fluid resuscitation increases VO2 during hypovolemic shock after a severe burn injury. PMID- 10752749 TI - Pain and anxiety with burn dressing changes: patient self-report. AB - Pain and anxiety are integral parts of burn dressing care. Descriptions of pain and anxiety from the patients' perspectives enhance our understanding of their experiences. This descriptive study measured relationships between self-reported pain and anxiety. Twenty-four patients who had an uncomplicated flame or scald wound reported scores immediately before, immediately after, and 1/2 hour after dressing changes. Results indicated that pain and anxiety increased until day 4. Pain correlated with anxiety. Self-report scores showed that pain and anxiety are perceived by patients in a similar manner. Self-report scores are of great value because they indicate the degree of relief achieved with medication. Through the descriptive study, we found that the pain score immediately after dressing changes was greater than 3 on all study days. This finding indicates a need to further examine the way pain is managed with dressing changes. PMID- 10752750 TI - The fire-safe cigarette: a burn prevention tool. AB - Cigarettes are the most common ignition source for fatal house fires, which cause approximately 29% of the fire deaths in the United States. A common scenario is the delayed ignition of a sofa, chair, or mattress by a lit cigarette that is forgotten or dropped by a smoker whose alertness is impaired by alcohol or medication. Cigarettes are designed to continue burning when left unattended. If they are dropped on mattresses, upholstered furniture, or other combustible material while still burning, their propensity to start fires varies depending on the cigarette design and content. The term "fire-safe" has evolved to describe cigarettes designed to have a reduced propensity for igniting mattresses and upholstered furniture. Legislative interest in the development of fire-safe smoking materials has existed for more than 50 years. Studies that showed the technical and economic feasibility of commercial production of fire-safe cigarettes were completed more than 10 years ago. Despite this, commercial production of fire-safe smoking materials has not been undertaken. The current impasse relates to the lack of consensus on a uniform test method on which to base a standard for fire-safe cigarettes. Although the fire-safe cigarette is a potentially important burn prevention tool, commercial production of such cigarettes will not occur until a standard against which fire-starting performance can be measured has been mandated by law at the state or federal level. The burn care community can play a leadership role in such legislative efforts. PMID- 10752751 TI - Deficiency in peripheral glutamine production in pediatric patients with burns. AB - Plasma glutamine levels decrease in association with severe injury, which suggests that the consumption of glutamine exceeds the production of glutamine or possibly represents a deficit in the release of glutamine from skeletal muscle. The goal of this study was to assess the peripheral glutamine kinetic response to prolonged stress in children with critical injuries. To accomplish this purpose, we quantitated peripheral glutamine kinetics in vivo with the use of 5N15 glutamine in 5 children with severe burns (total body surface area, 74%+/-14%; mean +/- SEM) and 3 children who underwent elective scar reconstruction. In the children with severe burns, leg blood flow was significantly elevated (16.2+/-2.1 vs 7.5 +/-0.3 mL/min/100 mL leg volume, P < .02) and the arterial concentration of glutamine was significantly reduced (0.31+/-0.04 vs 0.84+/-0.05 mmol/L, P < .001). The rate of glutamine turnover within the leg was significantly reduced in the patients with acute burns, whereas the net efflux of glutamine was similar between the 2 groups. These findings suggest that plasma glutamine concentrations decrease during severe stress as a result of a deficit in peripheral glutamine release in conjunction with an increased central consumption. This preliminary study supports the notion that exogenous glutamine supplementation in pediatric patients with severe injuries may be needed because of this inadequate skeletal muscle response. PMID- 10752752 TI - Evaluating the psychosocial adjustment of 2- and 3-year-old pediatric burn survivors. AB - Very little information has been published about 2- and 3-year-old children who have experienced major burns. This study used a standardized instrument to measure the behavioral adjustment of these young burn survivors, and the results were compared with those of a nonclinical normative sample. Thirty-three pediatric burn survivors with 50%+/-28% total body surface area burns were evaluated 1.2+/-0.7 years postburn. Parental observations were assessed with the use of the Child Behavior Checklist for 2- and 3-Year-Olds, a 99-item standardized checklist designed to identify behavior problems. Forty of the questions are specific to 2- and 3-year-olds, and the scores of male and female children are not differentiated. The raw scores of the children with burns were statistically compared with the reported normative sample for this version of the Child Behavior Checklist. Pediatric burn survivors in this sample exhibited significantly more internalizing behaviors than the children in the normative group. Parents reported children who had been burned to be more depressed and to have more somatic complaints and sleep problems. Determining the relationship of behavior problems to posttrauma sequelae and preburn environmental factors would assist with the establishment of appropriate psychosocial interventions. PMID- 10752753 TI - Immunohistochemistry and burn depth. PMID- 10752754 TI - Role of transplant induction therapy on recurrence rate of focal segmental glomerulosclerosis. AB - Individuals with focal segmental glomerulosclerosis (FSGS) are at risk for recurrence of disease following renal transplantation. The rate of recurrence has been estimated to range from 20% to 30%. The factors associated with an increased probability of recurrence are not known, although the rapidity of progression of disease, age at onset, and the presence of diffuse mesangial proliferation in the native kidney have all been implicated. We analyzed the data from 35 patients with FSGS who received 37 renal transplants at this institution between October 1968 and December 1997. Recurrence was diagnosed by the development of nephrotic range proteinuria and a transplant biopsy compatible with the diagnosis. Sixteen recurrences were noted, with an overall recurrence rate of 43%. The risk of recurrence was associated with the use of antilymphocytic serum (ALS) for initial induction therapy; being 11% in those who received no induction therapy versus 53% in those who received ALS. Furthermore, in the latter group, the rate of recurrence was 88% in those who received antithymocyte globulin (ATGAM) versus 40% in those who received Minnesota antilymphocytic globulin. Factors such as race, sex, age at time of diagnosis, rapidity of progression to end-stage renal disease (ESRD), response to alkylating agents and/or cyclosporin therapy prior to ESRD, age at time of transplant, donor source, and triple or double immunosuppressive therapy did not appear to have an effect on the rate of recurrence. We conclude that induction therapy with ALS at time of transplantation increases the risk of recurrence of FSGS following renal transplantation. PMID- 10752755 TI - Reproducible erythroid aplasia caused by mycophenolate mofetil. AB - Anemia secondary to mycophenolate mofetil (MMF) was recently described in experimental animals. A clinical association between MMF and anemia has been observed, but there are no proven reports. We describe a girl with chronic graft failure who developed erythroid aplasia under immunosuppression with MMF. She showed prompt resolution when MMF was discontinued and a recurrence of this clinical course when MMF was restarted. As re-challenge with a medication is the most definitive approach for showing a direct relationship between the drug and the side effect, this case clearly demonstrates that MMF can cause erythroid aplasia. PMID- 10752756 TI - Management of hemodialysis catheter-related bacteremia--a 10-year experience. AB - Between January 1986 and December 1995, 18 episodes of bacteremia occurred in our pediatric patients undergoing chronic hemodialysis on an outpatient basis. Seven episodes were caused by coagulase-negative Staphylococcus, 6 by Staphylococcus aureus, 2 by Mycobacterium, and 1 each by Pseudomonas, Xanthomonas, and Enterococcus. In 6 cases, the catheter was retained with antimicrobial therapy alone, whereas 12 cases required removal of the catheter after some period of time. The subset of cases in which catheter removal was necessary included 2 cases of Mycobacterium fortuitum complex and 5 cases of Staphylococcus aureus. We found that Staphylococcus aureus bacteremia may be cleared with antibiotic therapy alone in a minority of cases (17%). In the 6 cases in which catheters were retained and infections cleared, the maximum length of time to sterilization of blood with appropriate antibiotics was 48 h. PMID- 10752757 TI - Phrenic nerve palsy: a rare complication of indwelling subclavian vein catheter. AB - The use of central venous catheters as access for hemodialysis has become common in children with end-stage renal disease. Phrenic nerve palsy is an unusual complication of this procedure. We report a case of delayed right diaphragmatic palsy due to phrenic nerve damage resulting from an indwelling right subclavian catheter in a 3-year-old child. PMID- 10752758 TI - Analysis of chromosome 6p in Spanish families with recessive polycystic kidney disease. AB - Several previous reports have suggested that autosomal recessive polycystic kidney disease (ARPKD) is caused by mutations in a single gene (the PKDH1 gene). Linkage analysis showed a positive linkage for polymorphic markers at the short arm of chromosome 6 (6p) in all families. PKHD1 has not been cloned. Recombinants in the critical region would permit the narrowing of the 6p interval containing the PKHD1 gene, thus facilitating the final identification (cloning) of this gene. Our study included 30 Spanish families. Each family consisted of both parents and at least two children, with at least one diagnosed with ARPKD by clinical and pathological parameters. DNA was obtained and 6p microsatellite markers were used to establish haplotypes for each family. A positive linkage to chromosome 6p was found for all families. In 2 cases, recombinants in the region containing the PKHD1 gene were found. These families will help narrow the size of the 6p region, facilitating the efforts to position and clone the PKHD1 gene. In conclusion, our analysis of Spanish ARPKD families confirms the lack of linkage heterogeneity. This suggests that mutations at a single locus on chromosome 6p21.1-p12 are responsible for the broad clinical spectrum of variable phenotypes. PMID- 10752759 TI - Wegener granulomatosis in children and young adults. A case study of ten patients. AB - This retrospective study reports seven children and three young adults (aged 11 30 years) who suffered from Wegener granulomatosis. Nine represent consecutive patients admitted to the Division of Nephrology over a period of 23 years. All patients had respiratory tract symptoms and renal involvement on admission. In several patients infiltrates on chest X-ray developed within 2 weeks of onset of symptoms. All patients survived. The median observation period was 9 years (range 13 months to 23 years). One patient progressed to end-stage renal disease. Nine patients initially received cyclophosphamide and steroids. After a median period of 9 months (range 6-31 months) the cyclophosphamide was replaced by azathioprine. Relapses occurred after a median of 28 months (range 4-120 months) in 80% of patients, in six of the eight patients causing a definite decrease in kidney function. We believe that early diagnosis and initiation of therapy reduce the extent of organ damage. Since relapses are frequent, these patients should be evaluated frequently. PMID- 10752760 TI - Long-term clinical and pathological effects of cyclosporin in children with nephrosis. AB - The clinical course of eight children with minimal change disease (MCNS) who were treated with cyclosporin (CYA) was retrospectively reviewed (group A). Five children had frequently relapsing (FRNS) and three had steroid-resistant (SRNS) primary nephrotic syndrome (PNS). The mean age (+/-SEM) of the patients at the time of initiation of CYA therapy was 8.01+/-1.30 years. Twelve follow-up renal biopsies, obtained from patients in group A, were compared with baseline 3.36+/ 0.76 years after the initiation of CYA. Follow-up renal biopsies from group A were compared with another cohort of eight children with PNS who did not receive CYA (group B, controls). In this later group four children had FRNS and four had SRNS, and all had MCNS on the initial renal biopsy. In group B, the time between the initiation of CYA and the last renal biopsy was 4.07+/-0.82 years. All 36 baseline and follow-up renal biopsies, from group A and B, were retrospectively reviewed by the same pathologist who was blinded to the clinical course and therapy. CYA decreased the number of relapses in patients from group A from 5.20+/-1.02 to 1.14+/-0.63 episodes per year (P<0.05). All patients with SRNS went into remission after initiation of CYA. Estimated creatinine clearance before CYA therapy was unchanged at the end of the observation period, 133+/-10 vs. 131+/-8 ml/min per 1.73m2, respectively. One child developed reversible acute renal failure while on CYA therapy. Attempts to wean three patients off CYA after 3.89+/-0.87 years of CYA therapy were unsuccessful. Mild but increasing tubular atrophy and interstitial fibrosis was observed in serial biopsies of 75% of the patients in group A compared with 25% of the patients in group B, all of whom had MCNS on initial biopsy. In addition, the percentage of renal cortex showing interstitial fibrosis and tubular atrophy in biopsies from group A patients was slightly greater than that of the group B patients (P<0.05). Hence, CYA therapy in children with MCNS is associated with mild renal interstitial fibrosis and tubular atrophy similar to that noted in a minority of the patients with primary MCNS who were not treated with CYA. However, the mild chronic interstitial damage is more frequent and extensive in MCNS patients treated with CYA, suggesting drug related interstitial alteration. Despite its efficacy and minimal nephrotoxicity in most patients with MCNS, CYA therapy carries the potential for significant morbidity. PMID- 10752761 TI - Polymorphism of the ACE gene in Henoch-Schonlein purpura nephritis. AB - Individuals with IgA nephropathy (IgAN) who are homozygous for the deletion (D) polymorphism of the gene for angiotensin converting enzyme (ACE) are reported to be at increased risk of progressive renal damage. Since IgAN and Henoch-Schonlein purpura with associated nephritis (HSPN) share a common aetiology, we have investigated this influence in 31 children with HSPN. The distribution of genotypes was as follows: II: 4, ID: 17 and DD: 10 patients. Median length of follow-up was 4.5 years (range 0.5-15.75 years). Severe onset with nephrotic oedema and crescent formation on renal biopsy was seen in 10 of 17 patients with ID genotype and 5 of 10 patients with DD genotype. In the ID group, 2 patients have undergone renal transplantation and 4 have persistent proteinuria 4, 7, 9 and 10 years after presentation. One patient in the DD group has been transplanted and 1 patient has proteinuria and a reduced glomerular filtration rate 5 years after initial presentation. All other patients have either made a complete recovery or have microscopic haematuria alone. These results do not support an association between disease severity and DD genotype in children with HSPN; however larger studies are required to confirm this. PMID- 10752762 TI - Prognostic factors in Saudi children with posterior urethral valves. AB - Posterior urethral valves (PUV) account for a sizeable proportion of children with chronic renal failure. Several criteria have been identified as predictive of future renal function in children with PUV. We compared the presenting features and initial treatment in two groups of Saudi children treated for PUV, with the aim of identifying any factors that might account for the differences observed in their renal function. One group (group A, 19 patients) had a serum creatinine of 80 micromol/l or less at follow-up and the other (group B, 13 patients) had higher levels. There was no significant difference in age or weight at presentation, incidence and severity of reflux, urinary tract infection, or type of primary treatment (valve ablation versus vesicostomy) between the two groups. However, after 5-7 days of catheter drainage, the mean serum creatinine level was 88+/-62 micromol/l for patients in group A compared with 172+/-77 micromol/l for those in group B (P<0.0001). This difference was highly significant. Linear regression analysis of post-drainage creatinine and serum creatinine at last follow-up showed a correlation coefficient of 0.7171 (P<0.0001). Hence the serum creatinine level after catheter drainage correlated strongly with renal function during short-term follow-up of Saudi children with PUV. This might help in the selection of the optimal initial therapy and provide some basis for prognostication. PMID- 10752763 TI - Remission of relapsing childhood nephrotic syndrome with mycophenolate mofetil. AB - We report a 21-year-old male with childhood-onset familial nephrotic syndrome and frequent relapses who manifested toxicity or treatment resistance to corticosteroids, cyclophosphamide, cyclosporin-A, and tacrolimus. Monotherapy with mycophenolate mofetil (MMF) resulted in maintenance of clinical remission for 14 months without noticeable toxicity, while allowing resolution of steroid induced side effects. Our observation suggests that MMF may be useful in maintaining remission in nephrotic patients who manifest toxicity to standard immunosuppressive agents. PMID- 10752764 TI - The stressed neonatal kidney: from pathophysiology to clinical management of neonatal vasomotor nephropathy. AB - The healthy term, and particularly the premature infant, is born with a very low glomerular filtration rate (GFR), controlled by a delicate balance of intrarenal vasoconstrictor and vasodilator forces. Vasoactive disturbances can easily further reduce the already low GFR. The newborn infant is thus prone to develop vasomotor nephropathy (VMNP) or acute renal failure (ARF). The main causes for ARF at this young age are prerenal mechanisms, and include hypotension, hypovolemia, hypoxemia perinatal asphyxia, and neonatal septicemia. Other causes include the administration of angiotensin converting enzyme inhibitors, indomethacin and tolazoline. The most-important factors governing the ultimate renal prognosis are the severity of the underlying disorder, the rapidity of an accurate diagnosis, prompt treatment, and avoidance of severe iatrogenic complications. The immediate treatment is of particular importance in VMNP, i.e., prerenal ischemic ARF, and consists of correcting abnormalities in fluid homeostasis and reduction of the complications of the acute azotemic state (uremia, hyperkalemia, acidosis, and hypertension). In severe and prolonged (established) ARF, temporary dialysis therapy may be indicated. Prerenal ARF with oliguria or anuria warrants immediate volume resuscitation. Special attention should be given to infants with congestive heart failure (CHF). The sick neonate with persistent oliguria and CHF should be treated with intravenous dopamine. Furosemide (FM) is the second line of therapy for babies with indomethacin induced ARF. In most other conditions, the therapeutic effect of FM is limited to a transient increase in urine flow, without improving basic renal function. The special conditions of the maturing kidney have to be appreciated in order to protect babies from undue renal injury. With the increasing knowledge of the mechanisms governing the development of ARF, progress has been made in the development of new treatment modalities. For example theophylline, calcium antagonists, ATP-MgCl2, thyroxine, and a variety of cytokines may in the near future be used to prevent or ameliorate VMNP and/or recently established ARF. With a combination of time-honored and new therapeutic strategies, there may well be a brighter future for neonates with vasomotor, prerenal, ischemic ARF. PMID- 10752765 TI - Regulatory molecules in kidney development. AB - The molecular regulation of the complex inductive events associated with formation of the vertebrate excretory system has been progressively elucidated as a result of both genetic and tissue culture approaches. Kidney organogenesis is initiated and maintained by a series of reciprocal inductive interactions between different tissues derived from the intermediolateral mesoderm to form the nephrons and collecting system of the metanephric kidney. Recent progress in this area has resulted in the identification of regulatory systems controlling branching morphogenesis of the ureteric bud, formation of the early renal vesicle and the glomerulus. These events are controlled by genes that regulate pattern formation, cellular proliferation, and differentiation in other tissues. Although it is not yet possible to completely identify a complete genetic pathway required for any one of the many steps in nephrogenesis, it is now evident that pathways previously identified in studies of mesenchymal-epithelial inductive mechanisms in limb bud, neural tissues, lung, and gut have direct relevance to the study of these processes in kidney development. For instance, a primary system for pattern formation involving retinoic acid, homeobox genes, sonic hedgehog, fibroblast growth factor (FGF), and an FGF receptor all appear to function in limb, lung, and kidney organogenesis. A major challenge is determining how this common cast of signalling molecules plays a specific role in kidney development essential to nephrogenesis, which results in the unique structural organization of the adult kidney. From this more-sophisticated understanding will come important insights relevant to understanding the molecular basis of developmental malformations of the kidney necessary for the prevention and treatment of these disorders. PMID- 10752766 TI - Signal transduction through protein kinase C. PMID- 10752768 TI - Third International Symposium on Perinatal Nephrology, 25-26 June 1999, Strasbourg, France. PMID- 10752767 TI - A 16-year-old boy with medullary sponge kidneys, osteoporosis, and premature loss of all teeth. PMID- 10752769 TI - Pseudotumor cerebri in a child with familial hypomagnesemia-hypercalciuria. PMID- 10752770 TI - Post-transplant ascitic fluid--hypothesis for its origin. PMID- 10752771 TI - The treatment of primary palmar hyperhidrosis: a review. AB - Primary palmar hyperhidrosis (HH) is a pathological condition of overperspiration caused by excessive secretion of the eccrine sweat glands, the etiology of which is unknown. This disorder affects a small but significant proportion of the young population all over the world. Neither systemic nor topical drugs have been found to satisfactorily alleviate the symptoms. Although the topical injection of botulinum has recently been reported to reduce the amount of local perspiration, long-term results are required before a definitive evaluation of this method can be made. Hypnosis, psychotherapy, and biofeedback have been beneficial in a limited-number of cases. While radiation achieves atrophy of the sweat glands, its detrimental effects prohibit its use. Iontophoresis has attained some satisfactory results but it has not been assessed long term. Percutaneous computed tomography-guided phenol sympathicolysis achieves excellent immediate results, but its long-term failure rate is prohibitive. Furthermore, percutaneous radiofrequency sympathicolysis may be an effective procedure, but its long-term results are not superior to surgical sympathectomy. On the other hand, surgical upper dorsal (T2-T3) sympathectomy achieves excellent long-term results and the thoracoscopic approach has supplanted the open procedures. Despite some sequelae, mainly in the form of neuralgia and compensatory sweating which cannot be predicted and may be distressing, surgical sympathectomy remains the best treatment for palmar hyperhidrosis. PMID- 10752772 TI - Laparoscopic-assisted surgery for Crohn's disease: reduced surgical stress following ileocolectomy. AB - Recent progress in laparoscopic techniques has enabled operations for various intestinal disorders to be performed under laparoscopic assistance. This study was conducted to assess the benefits of performing laparoscopic-assisted surgery (LAS) in patients with Crohn's disease. LAS was performed in 24 selected patients with Crohn's disease, most of whom underwent ileocolic resection for ileitis and/or colitis with stenosis. To determine the benefits of LAS, the postoperative inflammatory parameters of these patients were examined and compared with those of 17 patients who underwent conventional open surgery. Despite giving all patients total parenteral nutrition (TPN) for more than 2 weeks preoperatively, two patients with large inflammatory masses involving enteroenteric fistulas required conversion to laparotomy. No laparoscopic procedure was converted for adhesions after previous resection or intraoperative complications. The maximum C reactive protein values and body temperatures were significantly lower, and the time taken to normalize body temperature and leukocyte counts was significantly shorter in the LAS group than in the laparotomy group. LAS should be performed for patients with Crohn's disease in the inactive phase after appropriate nutritional support. Patients with terminal ileitis without a fistula are considered to have the highest indication for this procedure. PMID- 10752773 TI - The incidence and outcome of pelvic sepsis following handsewn and stapled ileal pouch anal anastomoses. AB - The incidence and outcome of pelvic sepsis was analyzed in 210 patients who underwent restorative proctocolectomy for ulcerative colitis (UC) in 197 patients, and for familial adenomatous polyposis (FAP) in 13 patients. Pelvic sepsis developed in 18 patients (8.6%) and a significantly higher incidence was seen in men than in women, at 13.6% vs 3.7%, respectively (P < 0.05). The incidence of pelvic sepsis in patients with UC complicated by toxic megacolon and/or fulminant colitis was significantly higher that in those without any preoperative complications, at 36.4% vs 7.4% (P < 0.05). The incidence of pelvic sepsis following handsewn anastomosis was significantly higher than that following stapled anastomosis, at 15.6% vs 5.5% (P < 0.05). The outcome of pelvic sepsis in patients with a stapled anastomosis was better than that in those with a handsewn anastomosis. The prognosis of women who developed pelvic sepsis was better than that of men who developed pelvic sepsis. The risk factors predisposing to pelvic sepsis were UC, especially when complicated by toxic megacolon and/or fulminant colitis, and male sex, while a handsewn anastomosis was more vulnerable than a stapled anastomosis. PMID- 10752775 TI - Results of surgical treatment for vascular injuries. AB - The purpose of this study was to clarify the current etiology of and prognosis for iatrogenic vascular injuries. The study involved patients who underwent surgery for vascular injury. The patient record was retrospectively reviewed to collect information on the patient profile and type of vascular injury. The surgical techniques used were classified as simple or complex. The outcome was measured as morbidity and 30-day hospital mortality due to vascular injuries. The average age of the group with iatrogenic injury was significantly older than the group without. The number of males and females with iatrogenic injuries were almost equal, whereas males predominated in the group with noniatrogenic injuries. During the first half of the study period, more injuries were noniatrogenic; during the second half, most were iatrogenic. Most injuries due to angiographic procedures could be treated by a simple repair. Injuries due to operative procedures and noniatrogenic traumas had to be repaired by complex techniques. The overall morbidity rate was 3.8%, and the 30-day hospital mortality rate was 1.9%. The recently developed minimally invasive surgical techniques tend to increase the risk of vascular injuries whereas catheterization remains the most important cause of iatrogenic vascular injuries. However, as long as such injuries are treated promptly, the prognosis is good. PMID- 10752776 TI - Video-assisted blebectomy using a flexible scope and a bleb implement. AB - We describe herein our new technique of performing video-assisted thoracic surgery (VATS) using a flexible scope and a bleb implement which allows for a reduction in the risk of overlooking small and flat blebs in patients with a spontaneous pneumothorax (SP). This technique was performed in 26 consecutive patients with a SP. A skin incision about 18 mm long was made in the area over which the patients had specified preoperatively, and meticulous observation of the apex and superior segment was performed using a bleb implement. Partial pneumonectomy with endoscopic staplers and laser ablation were carried out for pleural lesions. We found that VATS combined with a flexible scope and a bleb implement resulted in better visibility for detection and removal of any small and flat blebs. PMID- 10752774 TI - The effects of intraoperative glucose infusion on portal blood insulin concentration and hepatic mitochondrial redox state during surgery: comparison of short-term and continuous infusions. AB - The relationships between the blood glucose level, portal blood immunoreactive insulin (IRI) concentration, ketogenesis, and hepatic mitochondrial redox state associated with intraoperative glucose administration were evaluated in patients undergoing total gastrectomy. A total of 26 patients were randomly allocated to two groups according to the type of infusion given; group 1 was given a short term glucose infusion of 25 g in 30 min and group 2 was given a continuous glucose infusion of 10 g/h. The blood glucose concentration peaked 30 min after the glucose infusion was commenced, then decreased in group 1, despite a continuous rise in group 2. A temporary but significantly higher blood glucose level was observed in group 1 than in group 2, 30 and 60 min after the infusion was commenced. The portal blood IRI concentrations and arterial ketone body ratio (AKBR) continued to increase and the blood ketone body concentrations continued to decline after the start of the glucose infusion in both groups; however, after 60 and 120 min, the portal blood IRI and AKBR levels were significantly higher, and the blood ketone body levels significantly lower in group 1 than in group 2. These findings suggest that intraoperative glucose administration is beneficial for insulin secretion, ketogenesis, and the hepatic mitochondrial redox state, and that short-term infusion is superior to continuous infusion. PMID- 10752777 TI - The efficacy and side effects of gelatin-resorcinol formaldehyde-glutaraldehyde (GRFG) glue for preventing and sealing pulmonary air leakage. AB - This study was conducted to examine the efficacy and side effects of gelatin resorcinol formaldehyde-glutaraldehyde (GRFG) glue, when used for preventing or sealing pulmonary air leakage during lung surgery. Formaldehyde-glutaraldehyde (FG) jelly was prepared by mixing FG fluid with 2.5% carboxymethyl cellulose to increase its viscosity. A GRFG glue-spreading stapler was employed to prevent air leakage from the staple line when cutting emphysematous lung tissue in 26 patients, 25 of whom underwent video-assisted thoracoscopic surgery (VATS) and 1, open thoracotomy. A glue-sealing procedure was employed to stop existing pulmonary air leakage in a further 36 patients, 28 of whom underwent VATS and 8, open thoracotomy. The glue-spreading stapler technique prevented air leakage in all 26 patients (100%), while the glue-sealing procedure for pulmonary air leakage stopped air leakage in 31 of the 36 patients (86%). Two patients who underwent glue sealing for air leakage from a deeply cut lung surface suffered massive pulmonary air leakage on postoperative day (POD) 1 and 6, respectively, and required repeat surgery to suture the lung fistula. A transient fever of 38 degrees-39 degrees developed on POD 7 in 6 of the 62 patients (9.7%), but no other complications were observed. These results demonstrated that GRFG is safe and effective in preventing or stopping pulmonary air leakage in the peripheral lung, but not for stopping air leakage from a deeply cut lung. PMID- 10752778 TI - Pathological assessment of the response of locally advanced breast cancer to neoadjuvant chemotherapy and its implications for surgical management. AB - The effectiveness of breast-conserving surgery for patients with locally advanced breast cancer (LABC) after neoadjuvant chemotherapy (NACT) is still a controversial issue, and variable incidences of locoregional failures have been reported. The present study was conducted to pathologically assess the response of LABC to NACT, and also to evaluate the efficacy of preoperative clinical examination and mammography in detecting these pathological changes. A total of 38 patients with LABC received NACT in the form of three cycles of fluorouracil/adriamycin/cyclophosphamide and were then subjected to a mastectomy. The residual tumors in the mastectomy specimens were measured, mapped, and compared to the pretreatment and preoperative clinical and mammographic findings for evaluation. An objective response to NACT was observed in 70.4% of the patients; however, only 26.7% of them were suitable candidates for conservative surgery. The rest of the responders showed an increased incidence of multifocality and in situ lesions localized within the original tumor-bearing area. Both clinical examinations and mammography were inadequate for the selection of candidates for breast conservation. Tumor regression by NACT is probably induced by a process of tumor segmentation. It is also associated with an increased incidence of multifocality and in situ lesions. PMID- 10752779 TI - The prognostic value of quality-of-life scores: preliminary results of an analysis of patients with breast cancer. AB - This study was conducted to elucidate the prognostic value of patient-assessed quality-of-life (QL) scores in cancer patients. QL was assessed in 47 consecutive patients with advanced or end-stage breast cancer using the Quality of Life Questionnaire for Cancer Patients Treated with Anticancer Drugs (QOL-ACD). The data collected from 19 of the 47 patients, who completed QL questionnaires more than twice before dying of cancer, were analyzed. The relationships between the QL scores and subsequent survival were examined at two assessment points, being the first and last assessment points of each of the 19 patients; corresponding respectively to median survival times of 14 and 4 months. The prognostic significance of the changes in QL scores that occurred over 3 months before the last assessment point was also examined. At the last assessment point, the scores of the physical aspects of QL were significantly related to survival. The change in scores of both overall QL and the physical aspects of QL were also significant predictors of survival. On the other hand, neither the scores nor the change in scores of the psychological and social aspects of QL was significant. This study indicates that both QL scores and changes in QL scores are promising prognostic predictors. PMID- 10752780 TI - Crohn's disease of the esophagus: report of a case. AB - We report herein the case of a 27-year-old man with Crohn's disease of the esophagus. The patient presented with large ulcers in the esophagus for which treatment based on a diagnosis of reflux esophagitis was commenced. Although his symptoms were initially resolved, the ulcers did not improve and he was readmitted to hospital 3 months later for progressive heartburn. An esophagoscopy revealed large ulcers in the esophagus, and a colonoscopy revealed a longitudinal ulcer in the terminal ileum. Histological examination of specimens from the terminal ileum showed severe inflammation without granuloma formation, which led to a diagnosis of Crohn's disease. The oral administration of prednisolone and salazosulfapyridine controlled his symptoms and the esophageal ulcers were observed to be healing 2 weeks after this treatment was initiated. A review of the English literature revealed only 77 cases of this disease. Isolated esophageal lesions were reported in ten patients (13.0%), none of which were able to be diagnosed as Crohn's disease preoperatively. Ileocolic lesions developed after esophageal lesions in only five patients (6.5%) including ours. In the remaining 62 patients (80.5%), ileocolic lesions had existed synchronous with or prior to the esophageal lesions. This suggests that ileocolic lesions may often coexist in Crohn's patients with esophageal lesions, and that examination of the terminal ileum must be performed to confirm a diagnosis of Crohn's disease of the esophagus. PMID- 10752781 TI - Congenital hepatoportal arteriovenous fistula: report of a case. AB - Fistulae between the hepatic artery and portal vein, known as hepatoportal arteriovenous fistula (HPAVF), most commonly occur secondary to trauma or malignancy. Congenital HPAVF is an extremely rare anomaly and only 11 pediatric cases have been documented to date HPAVF causes portal hypertension with the reversal of flow in the portal circulation, leading to hyperemia and congestion of the bowel, causing severe ascites, gastrointestinal bleeding, anemia, and malabsorption. The diagnosis is first made by Doppler sonography, then confirmed by angiography. HPAVF is most effectively treated by performing either ligation of the hepatic artery or percutaneous transcatheter embolization. We describe herein the case of an 8-month-old boy diagnosed to have congenital HPAVF by duplex Doppler sonography and confirmed by digital subtraction angiography, who was successfully managed by percutaneous transcatheter embolization of the feeding right hepatic artery. A review of the previously reported 11 cases of infants with this unusual anomaly, examining presentation, management, and outcome, is also presented. PMID- 10752782 TI - Choledochocele demonstrated by computed tomographic cholangiography: report of a case. AB - Choledochocele is a rare cystic congenital malformation of the distal common bile duct, the precise pathogenesis of which remains unclear. It is usually diagnosed in adulthood after many examinations for unclear digestive symptoms, as in the patient whose case is described herein. Of all the diagnostic techniques available, endoscopic retrograde cholangiography (ERCP) seems to be the best; however, it is an invasive procedure associated with some morbidity. Spiral computed tomographic cholangiography with three-dimensional reconstruction is also an interesting screening technique. Malignant transformation of a choledochocele occurs very rarely, compared with other cystic malformations of the biliary tract. If a choledochocele is small and symptomatic, the lesion can be treated by endoscopic sphincterotomy, but larger cysts are more effectively removed by a surgical cystoduodenostomy. We present herein the case of a 25-year old woman admitted to our hospital in March 1998 with upper right quadrant abdominal pain caused by a choledochocele, who was successfully treated by a cystoduodenostomy. PMID- 10752784 TI - Stromal sarcoma of the breast with lung metastasis successfully treated by radiotherapy: report of a case. AB - We report herein the case of a 42-year-old woman in whom a solitary lung metastasis from stromal sarcoma of the breast was effectively treated by radiotherapy. The original breast tumor had been extirpated in a local hospital, and she was subsequently referred to our outpatient clinic for nonsurgical treatment. Pathological examination of the breast tumor had confirmed a diagnosis of stromal sarcoma. Radiation and systemic chemotherapy for the remnant disease were administered at our hospital; however, 8 months later, local recurrence and a solitary lung metastasis were recognized on a chest X-ray. A second lumpectomy of the recurrent breast tumor along with radiation for the lung metastasis and systemic chemotherapy were performed. After a 4-year disease-free period, there is still no sign of recurrence. This case serves to demonstrate the effectiveness of radiotherapy for the treatment of lung metastasis from stromal sarcoma of the breast. PMID- 10752783 TI - Nonspecific ulcerated jejunitis as an unusual complication of laparoscopic cholecystectomy: report of a case. AB - We describe herein the case of a 65-year-old man in whom a lower gastrointestinal hemorrhage developed a few days after he underwent an elective laparoscopic cholecystectomy. A laparotomy was performed on postoperative day 16 and a jejunal segment containing mucosal changes and oozing ulcers was resected. Pathologic examination of the specimen revealed "nonspecific ulcerated jejunitis." There is no explanation for the etiopathogenesis of this pathology; however, we concluded that this clinical picture may be attributed to ischemia-reperfusion injury that occurred following an ischemic period caused by the pneumoperitoneum during laparoscopic surgery. PMID- 10752785 TI - Long-term survival of a poor-risk octogenarian following wedge resection under VATS for small-cell lung cancer: report of a case. AB - We describe herein the case of an 81-year-old man who has remained disease-free for more than 3 years after undergoing a wedge resection of cStage I small-cell lung cancer (SCLC) under video-assisted thoracoscopic surgery (VATS), with no adjunct chemotherapy or radiotherapy. The patient had compromised pulmonary function and was a poor surgical risk. As he could not have endured a conventional lobectomy or intensive chemotherapy, a nonanatomical wedge resection of the area of lung involved by the primary tumor was carried out under VATS. Cancer cells from the resected tumor were cultured and the growth characteristics and sensitivity to 12 anticancer drugs were examined. The majority of primary cultured cells proliferated in a monolayer, like paving stones, resembling the growth pattern of non-small-cell carcinoma cells in vitro. The subcultured cells were resistant to most of the drugs, but showed weak sensitivity to cisplatin (CDDP), adriamycin (ADR), and vincristine (VCR). Therefore, the patient was discharged with no adjunct postoperative therapy and was followed up at an outpatient clinic. He has remained alive and disease-free for more than 3 years. Thus, we considered that performing wedge resection under VATS for a primary tumor could be appropriate treatment for selected patients with cStage I SCLC in a peripheral region, especially if they are elderly and a poor surgical risk. PMID- 10752786 TI - Occlusion of the common iliac artery secondary of fungal endocarditis: report of a case. AB - Fungal endocarditis becomes complicated by peripheral arterial embolization of the lower limbs in 33%-75% of cases. Although the prognosis of patients with fungal endocarditis has improved somewhat over recent years, it remains poor, especially when the disease is associated with peripheral arterial embolization. We report herein our experience of treating a patient in whom occlusion of the right common iliac artery developed secondary to endocarditis caused by Candida parapsilosis, and review the literature on this subject. PMID- 10752787 TI - Recurrence of giant adrenocortical carcinoma in the contralateral adrenal gland 6 years after surgery: report of a case. AB - We report herein the case of a patient in whom a giant adrenocortical carcinoma was found to have recurred in the contralateral adrenal gland and intrapelvic cavity 6 years after his initial operation. A 52-year-old man had consulted our hospital complaining of right upper abdominal pain and weight loss, and was subsequently diagnosed as having a giant adrenal tumor by computed tomography scans and echography. A laparotomy was performed and the tumor, located in the right retroperitoneal cavity and infiltrating the liver and right kidney, was surgically removed. The lesion, 29 x 19 x 10 cm in size and 4700 g in weight, was histopathologically diagnosed as an adrenocortical carcinoma. Adjuvant chemotherapy with mitotane was given for 3 months and his postoperative course was uneventful until a recurrence in the contralateral adrenal gland and peritoneal cavity was found 6 years later. The second resection was successful, and he is currently alive with no further sign of recurrence 8 years after his first operation. We report this unusual case as it provides much useful information on the biological features of adrenocortical carcinomas and the state of tumor dormancy. PMID- 10752788 TI - A malignant rhabdoid tumor of the kidney occurring concurrently with a brain tumor: report of a case. AB - Malignant rhabdoid tumor of the kidney (MRTK) is one of the most lethal neoplasms to occur in young infants. Cases of MRTK accompanying an embryonal tumor in the central nervous system have occasionally been described. We present herein an interesting case of MRTK that was clinically diagnosed preoperatively. A male infant aged 6 months with both a midline brain tumor and a renal neoplasm was transferred to our institution. Although roentgenographic evaluation suggested that the renal lesion was a Wilms' tumor, midkine (MK), a growth and differentiation factor characteristically present in the urine of patients with Wilms' tumor, was not detected. A preoperative diagnosis of MRTK was established based on the lack of urinary MK in addition to the typical clinical features of the young age and the concurrent brain tumor. PMID- 10752789 TI - Dermatomyositis accompanied by rectal cancer: report of a case. AB - Dermatomyositis (DM) is a rare inflammatory disorder of the skin and muscles associated with an increased incidence of malignancy. We describe herein the case of a 59-year-old woman with DM accompanied by rectal cancer. Following excision of the rectal cancer, the characteristic features of the skin rash such as the heliotrope eyelid rash and Gottron's papules, and proximal muscle weakness, improved. Moreover, the elevated preoperative serum levels of muscle-associated enzymes, including aspartate transaminase, creatine phosphokinase, lactate dehydrogenase, and aldolase, decreased from 38 to 16 (IU/1), 138 to 42 (IU/1), 672 to 515 (IU/1), and 32.2 to 4.3 (IU/1), respectively. The current concepts of the correlation between DM and malignancy are discussed with regard to the present case. PMID- 10752790 TI - Primary ovarian lymphoma in an infant: report of a case. AB - Primary ovarian malignant lymphomas are rarely encountered in children. We present herein the unusual case of an 11-month-old female infant with primary bilateral non-Hodgkin's lymphoma of the ovaries who was treated by bilateral salpingo-oophorectomy followed by chemotherapy. The clinicopathological features of this rare entity are discussed, focusing special emphasis on the diagnostic and therapeutic strategies. PMID- 10752791 TI - Leukemia and non-Hodgkin's lymphoma in childhood and exposure to pesticides: results of a register-based case-control study in Germany. AB - Previous studies have suggested an association between exposure to pesticides and different types of childhood cancer. This paper presents results from a population-based case-control interview study of parents of children less than 15 years of age, which was conducted in the states of West Germany from 1993 to 1997. Cases were 1,184 children with leukemia, 234 with non-Hodgkin's lymphoma, and 940 with a solid tumor; 2,588 controls were also included. Parental occupational exposures were found to be related to childhood cancer regardless of the time period of exposure and the type of cancer. This finding might partially be explained by different recall of past exposures by the parents of cases and controls. Residential use of insecticides was associated with childhood lymphoma: both extermination of insects by professional pest controllers (odds ratio (OR) = 2.6, 95% confidence interval (CI): 1.2, 5.7) and frequency of parental use of household insecticides (p for trend = 0.02) were significant risk factors for this diagnosis. The use of pesticides on farms was weakly related to childhood leukemia (OR = 1.5, 95% CI: 1.0, 2.2), while their use in gardens was not associated with childhood leukemia (OR = 1.0, 95% CI: 0.8, 1.2). The major strengths of this study were the population base and the large number of cases and controls included; a drawback was assessment of exposure on the basis of parental interviews. The data provide some evidence for an increased leukemia risk for children living on farms and for an association between use of household pesticides and risk of childhood leukemia or lymphoma. PMID- 10752792 TI - Alcohol intake and mortality: findings from the National Health Interview Surveys (1988 and 1990). AB - The authors used prospective data from two supplemental studies of the National Health Interview Survey, the 1988 Alcohol Supplement and the 1990 Health Promotion and Disease Prevention Supplement, to examine the relation between alcohol intake and mortality. Their study included 17,821 men and 25,874 women aged 40 years or older at baseline; during an average of 6 years of follow-up, 5,540 deaths occurred. The alcohol-mortality relation was U-shaped for men and J shaped for women. On the basis of categorical analyses adjusted for age, race, smoking, and baseline diseases, men who drank 2 drinks per day had a significantly lower risk of death compared with abstainers (relative risk = 0.60, 95% confidence interval (CI): 0.45, 0.82). The relative risk was 0.75 (95% CI: 0.55, 1.03) after further adjustment for marital status, education, and self perceived health status. For women, the corresponding relative risks were 0.69 (95% CI: 0.61, 0.78) and 0.79 (95% CI: 0.70, 0.90) for those who drank less than 1 drink per day. When drinking category was considered as an ordinal variable and fitted with a quadratic function in the Cox model, the estimated optimal alcohol intake was approximately less than 1 to 1 drink per day for men and lifetime infrequent to less than 1 drink per day for women. Data from these representative US cohorts demonstrated that less than 2 drinks per day for men and less than 1 drink per day for women are associated with the lowest all-cause mortality. PMID- 10752793 TI - Underweight and overweight in relation to mortality among men aged 40-59 and 50 69 years: the Seven Countries Study. AB - This study investigated the relation between body mass index (BMI) and the all cause mortality rate among 7,985 European men. Starting around 1960, when all men were aged 40-59 years, mortality was followed for 15 years (1960-1975); starting around 1970, the survivors were followed for an additional 15 years (1970-1985). For the first and second follow-up periods, a BMI of 18.5-25 kg/m2 around 1960 and 1970, respectively, was considered the reference category. The authors found that the hazard ratios of mortality for a BMI of <18.5 kg/m2 was 2.1 (95% confidence interval (CI): 1.5, 2.8) for the first follow-up period and 1.7 (95% CI: 1.3, 2.2) for the second. A BMI of 25-30 kg/m2 was not related to increased mortality. Among never smokers, the hazard ratios for a BMI of >30 kg/m2 were 1.8 (95% CI: 1.2, 2.8) for the 1960-1975 follow-up period and 1.4 (95% CI: 1.0, 1.9) for the 1970-1985 follow-up period. A BMI of >30 kg/m2 was not related to increased mortality among current smokers. When mortality was followed for more than 15 years, the hazard ratio for a BMI of <18.5 kg/m2 declined and the hazard ratios for a BMI of >30 kg/m2 did not change. Underweight among those in all smoking categories and severe overweight in never smokers remained predictors of increased mortality when middle-aged men became older. PMID- 10752794 TI - Characteristics of older men who lose weight intentionally or unintentionally. AB - In most prospective studies involving older subjects, weight loss is associated with increased mortality. The authors examined the characteristics and health status of middle-aged men from 24 towns (the British Regional Heart Study), who reported that they had lost weight intentionally or unintentionally as they aged. Questionnaires were completed at screening (1978-1980), 5 years later, and in 1992 and 1996. This paper concerns the 4,713 men who reported their weight in 1992 and 1996 and their perceived weight change over this period. Weight loss was reported by 847 men (18%), of whom 39% stated that it was intentional. Compared with intentional weight loss, unintentional weight loss was associated with lower social class, more smoking, less obesity, and less physical activity and with the highest rates of "poor health,"long-standing disability, cancer, and respiratory diseases. The rates of recalled heart attack, stroke, diabetes, and cardiovascular hospitalization were higher than among men whose weight was stable. Men who lost weight intentionally had been more obese than those who lost weight unintentionally and had high rates of obesity-related conditions. Thus, both intentional and unintentional weight loss may follow the development of disease. These findings probably account for the lack of benefit and the increased mortality associated with weight loss observed in most large-scale prospective studies. PMID- 10752795 TI - Cognitive impairment and mortality in the community-dwelling elderly. AB - The effects on mortality of cognitive impairment and 3-year declines in cognitive function were examined among community-dwelling adults aged 68 years or more. Data were taken from a population-based cohort study that enrolled noninstitutionalized elderly residents of New Haven, Connecticut, and followed them by conducting in-home interviews in 1982, 1985, 1988, and 1994. The cognitive function of 1,997 respondents was assessed by using the 30-point Mini Mental State Examination in 1985; 1,372 respondents (86% of those alive) were retested in 1988. Responses were classified as high normal (28-30), low normal (24-27), mild impairment (18-23), or severe impairment (0-17); cognitive decline was defined as a transition to a lower category. After control for multiple potential confounders, both severe and mild cognitive impairment were strongly predictive of subsequent mortality among respondents aged less than 80 years. Upon closer examination, the elevated mortality risk was observed primarily among respondents whose cognitive decline was recent rather than among those whose cognitive performance was compromised but stable. Among respondents aged 80 years or more, declines to severe cognitive impairment were predictive of mortality, but it was not clear whether the decline per se signaled an unfavorable prognosis not accounted for by the resulting impairment level. Cognitive declines, especially those in the young elderly, have a marked adverse impact on survival. PMID- 10752796 TI - Elderly cohort study subjects unable to return for follow-up have lower bone mass than those who can return. AB - Longitudinal studies of osteoporosis in older persons may underestimate bone loss because of a lack of follow-up measurements on subjects too frail to return. The authors addressed this possible bias as part of the population-based Framingham Study; in 1996-1997, they used quantitative ultrasound to assess the bone status of elderly subjects regardless of their ability to return to the clinic. Broadband ultrasound attenuation (BUA) and speed of sound of the calcaneus (heel) were measured in 433 subjects at the Framingham, Massachusetts, clinic and in 167 subjects at their homes or nursing homes. All ultrasound parameters were measured with intramachine coefficients of variation of <6.0%. The mean BUA for those subjects evaluated at the clinic was higher than for those measured at home (9.2% higher for men, p = 0.081; 8.6% higher for women, p = 0.034). After adjustment for age and weight, the differences in BUA were no longer significant. Among the elderly subjects participating in this longitudinal cohort study, those who were unable to return for follow-up were older, weighed less, and had a lower BUA than those who did return, suggesting that longitudinal studies of changes in bone mass with aging may underestimate the true population values. PMID- 10752797 TI - Diet and bladder cancer: a meta-analysis of six dietary variables. AB - In 1996, more than 300,000 new cases of bladder cancer were diagnosed worldwide. Besides tobacco smoking, occupation, and other factors, diet may play a role in causation of this illness. The authors performed a meta-analytical review of epidemiologic studies linking six dietary factors to bladder cancer. These factors include retinol, beta-carotene, fruits, vegetables, meat, and fat. Increased risks of bladder cancer were associated with diets low in fruit intake (relative risk (RR) = 1.40, 95% confidence interval (CI): 1.08, 1.83), and slightly increased risks were associated with diets low in vegetable intake (RR = 1.16, 95% CI: 1.01, 1.34). Elevated risks were identified for diets high in fat intake (RR = 1.37, 95% CI: 1.16, 1.62) but not for diets high in meat intake (RR = 1.08, 95% CI: 0.82, 1.42). No increased risks were found for diets low in retinol (RR = 1.01, 95% CI: 0.83, 1.23) or beta-carotene (RR = 1.10, 95% CI: 0.93, 1.30) intake. These results suggest that a diet high in fruits and vegetables and low in fat intake may help prevent bladder cancer, but the individual dietary constituents that reduce the risks remain unknown. PMID- 10752798 TI - Hormone-related factors and risk of breast cancer in relation to estrogen receptor and progesterone receptor status. AB - Risk factors were examined for subgroups of breast cancer characterized by estrogen receptor (ER) and progesterone receptor (PR) status. Data from the Carolina Breast Cancer Study, a population-based, North Carolina case-control study of 862 breast cancer cases aged 20-74 years diagnosed during 1993-1996 and 790 controls frequency matched on race and age, were obtained by personal interview. ER and PR status was retrieved from medical records (80%) or was determined in the authors' laboratory (11%) but was missing for 9% of cases. The receptor status distribution was as follows: 53% ER+PR+, 11% ER+PR-, 8% ER-PR+, and 28% ER-PR-. Several hormone-related factors were associated with stronger increased risks for ER+PR+ than for ER-PR- breast cancer: the elevated odds ratios were strongest for ER+PR+ breast cancer among postmenopausal women who had an early age at menarche (odds ratio (OR) = 1.6, 95% confidence interval (CI): 1.0, 2.4), nulliparity/late age at first full-term pregnancy (OR = 1.7, 95% CI: 0.9, 3.2 and OR = 1.6, 95% CI: 1.0, 2.7, respectively), or a high body mass index (OR = 1.6, 95% CI: 0.9, 3.0) and among pre-/perimenopausal women who had a high waist-hip ratio (OR = 1.9, 95% CI: 1.2, 3.1). In contrast, family history of breast or ovarian cancer and medical radiation exposure to the chest produced higher odds ratios for ER-PR- than for ER+PR+ breast cancer, especially among pre /perimenopausal women. PMID- 10752799 TI - Age at any full-term pregnancy and breast cancer risk. AB - The authors analyzed data from two multistate, population-based case-control studies to investigate the association between age at any full-term pregnancy (FP) and breast cancer risk. Study subjects included breast cancer cases aged 20 79 years identified from four statewide cancer registries and randomly selected controls interviewed from 1988 to 1996. Complete information on a comprehensive set of risk factors for breast cancer was available for 9,891 cases and 12,271 controls. The large number of subjects enabled simultaneous adjustment of the covariates and efficient application of various modeling approaches. Overall, each 5-year increase in age at first FP was associated with an odds ratio of 1.07 (95% confidence interval (CI): 1.01, 1.13) for breast cancer. The corresponding estimates were odds ratio = 1.02 (95% CI: 1.00, 1.05) for age at second through ninth FPs. For age at last FP, the effect estimate (odds ratio = 1.01, 95% CI: 0.97, 1.06) was indistinguishable from that for other FPs after the first. In this analysis, a modest and transient increase in breast cancer risk after childbirth was also observed. The relatively greater effect of age at first FP is consistent with the existence of a long-term effect of early first FP on the differentiation of mammary cells, causing them to become less susceptible to carcinogenesis. PMID- 10752800 TI - Increased childhood morbidity after measles is short-term in urban Bangladesh. AB - In a 1995-1996 cohort study in the city of Dhaka, Bangladesh, morbidity in 117 hospitalized and 137 acute measles cases compared with age-matched children without measles (unexposed) was determined by weekly interview for 6 months. Compared with unexposed children, there were higher incidences of hospitalization (adjusted rate ratio (RR) = 3.1, 95% confidence interval (CI): 1.3, 7.6) and bloody diarrhea (adjusted RR = 2.7, 95% CI: 1.4, 5.1) in hospital measles cases during the 6 weeks after recruitment. Among community cohorts, there were higher incidences of bloody diarrhea (adjusted RR = 4.1, 95% CI: 1.1, 14.6), watery diarrhea (adjusted RR = 1.6, 95% CI: 0.9, 2.7), fast breathing (adjusted RR = 3.8, 95% CI: 2.1, 6.9), and the weekly point prevalence of pneumonia (adjusted prevalence ratio = 3.1, 95% CI: 1.0, 9.8) in measles cases during the same period. All measles cases regained lost weight within about 6 weeks. The prevalence of anergy to seven recall antigens 6 weeks after recruitment was higher in both hospital (adjusted odds ratio = 2.8, 95% CI: 1.2, 6.4) and community (adjusted odds ratio = 3.1, 95% CI: 1.1, 8.9) measles cases. Morbidity increased during the first 6-8 weeks after measles, but the authors found no consistent evidence of longer-term morbidity or wasting. The results support recent findings that measles is not associated with increased delayed mortality. PMID- 10752801 TI - Re: "Physical activity and cardiovascular disease risk in middle-aged and older women". PMID- 10752802 TI - Resuscitation of critically ill patients based on the results of gastric tonometry: a prospective, randomized, controlled trial. AB - OBJECTIVE: To determine whether additional therapy aimed at correcting low gastric intramucosal pH (pHi) improves outcome in conventionally resuscitated, critically ill patients. DESIGN: Prospective, randomized, controlled study. SETTING: General intensive care unit (ICU) of a university teaching hospital. PATIENTS: A total of 210 adult patients, with a median Acute Physiology and Chronic Health Evaluation II score of 24 (range, 8-51). INTERVENTIONS: All patients were resuscitated according to standard guidelines. After resuscitation, those patients in the intervention group with a pHi of <7.35 were treated with additional colloid and then dobutamine (5 microg/kg/min then 10 microg/kg min) until 24 hrs after enrollment. MEASUREMENTS AND MAIN RESULTS: There were no significant differences (p > .05) in ICU mortality (39.6% in the control group vs. 38.5% in the intervention group), hospital mortality (45.3% in the control group vs. 42.3% in the intervention group), and 30-day mortality (43.7% in the control group vs. 40.2 in the intervention group); survival curves; median modified maximal multiorgan dysfunction score (10 points in the control group vs. 13 points in the intervention group); median modified duration of ICU stay (12 days in the control group vs. 11.5 days in the intervention group); or median modified duration of hospital stay (60 days in the control group vs. 42 days in the intervention group). A subgroup analysis of those patients with gastric mucosal pH of > or =7.35 at admission revealed no difference in ICU mortality (10.3% in the control group vs. 14.8% in the intervention group), hospital mortality (13.8% in the control group vs. 29.6% in the intervention group), or 30 day mortality (10.3% in the control group vs. 26.9% in the intervention group). CONCLUSIONS: The routine use of treatment titrated against pHi in the management of critically ill patients cannot be supported. Failure to improve outcome may be caused by an inability to produce a clinically significant change in pHi or because pHi is simply a marker of disease rather than a factor in the pathogenesis of multiorgan failure. PMID- 10752803 TI - The incidence of major morbidity in critically ill patients managed with pulmonary artery catheters: a meta-analysis. AB - INTRODUCTION: The impact of pulmonary artery (PA) catheters on patient outcome has been questioned and their usage has become controversial. Meta-analysis on mortality has shown a trend for improved survival with PA catheter-guided therapy. We now perform a meta-analysis on morbidity from PA catheters in the published literature. METHODS: We did a search of the medical database (Medline) from 1970 through 1996, using the headings "pulmonary artery catheterization," "Swan-Ganz catheterization" and "right heart catheterization," and restricting the results to "effectiveness" and "usefulness." We also consulted with other experts regarding published randomized controlled trials (RCTs). This yielded 16 RCTs addressing the question of effectiveness of PA catheter-guided treatment. Of these, 12 were found to include data on morbidity. Major morbidity, defined as organ failures as per the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference criteria, from these trials was entered into a formal meta-analysis. RESULTS: A total of 1,610 patients from the 12 trials were analyzed. Morbidity events were observed in 62.77% of the PA catheter treatment group, and in 74.34% of the control group. A relative risk ratio of 0.78074 was obtained, with a 95% confidence interval of 0.6459-0.94374 and a corresponding p of .0168, a lower morbidity in the PA catheter treatment group. Those with PA catheter-guided treatment had a mean protective effect of 21.9% for risk of morbidity. Other important covariates such as acuity of illness, quality score of trials, year of publication, type of PA catheter-guided treatment used (PA catheter vs. no PA catheter, or PA catheter vs. PA catheter for supranormal hemodynamic values), and surgical or mixed patient population, all increased variability and were not statistically significant predictors for risk ratio of morbidity. CONCLUSIONS: Meta-analysis of RCTs included in this study shows that there is a statistically significant reduction in morbidity using PA catheter-guided strategies. PMID- 10752804 TI - Detection of capillary protein leakage by indocyanine green and glucose dilutions in septic patients. AB - OBJECTIVE: To determine whether indocyanine green (ICG) and glucose dilutions can detect generalized capillary protein leakage in septic patients without requiring repeated measurements. DESIGN: Prospective, clinical study. SETTING: General intensive care unit. PATIENTS: Twelve consecutive patients who met the criteria of sepsis and 16 consecutive acute myocardial infarction (AMI) patients without any underlying pathology inducing generalized protein capillary leakage. INTERVENTIONS: Both ICG 25 mg and glucose 5 g were administered simultaneously, to calculate the plasma volume determined by the ICG dilution method (PV-ICG) and the initial distribution volume of glucose (IDVG), on day 1 of sepsis or on day 1 of hospitalization for the AMI patients. The relationship between these two volumes and the PV-ICG/IDVG ratio was evaluated in two patient groups. MEASUREMENTS AND MAIN RESULTS: Although the IDVG of the two patient groups was not statistically different, the PV-ICG in the septic patients was higher than that in the AMI patients (p < .01). Consequently, the PV-ICG/IDVG ratio in the septic patients was higher than that in the AMI patients (p < .01). Eight of the 12 septic patients had a PV-ICG/IDVG ratio of >0.45, which was not observed in any of the AMI patients. The PV-ICG/IDVG ratio in the septic patients correlated inversely with the total plasma protein concentration (r2 = .46, p < .025) and mean arterial pressure (r2 = .42, p < .05). CONCLUSIONS: Our results indicate that overestimation of the PV-ICG can occur in septic patients and, further, suggest that simultaneous measurement of the two distribution volumes would help predict generalized capillary protein leakage in septic patients without repeated measurement. PMID- 10752805 TI - Measuring blood volume with fluorescent-labeled hydroxyethyl starch. AB - OBJECTIVES: To develop and evaluate a method for measuring blood volume using the dilution of a fluorescent-labeled hydroxyethyl starch. DESIGN: Laboratory and clinical investigation. SETTING: Biochemistry laboratory at the University of Cardiff. Hematology clinic, surgical ward and intensive care unit of the University Hospital of Wales. PATIENTS: Seventeen patients with suspected polycythemia. Eight patients who had undergone major surgery and/or were receiving intensive postoperative care. INTERVENTIONS: All surgical and postoperative care was provided by clinicians not involved in the study. Patients with suspected polycythemia were referred for blood volume measurement using labeled albumin and red blood cells. MEASUREMENT AND MAIN RESULTS: A proprietary brand of hydroxyethyl starch (Elohaes) was labeled with fluorescein isothiocyanate. Dilution of this compound in vivo was used for measuring blood volume, and the results were compared with those obtained using radiolabeled albumin and the considered criterion, radiolabeled red cells. The elimination of the labeled starch follows the same progress as that of the parent compound, indicating that the fluorescent tag is stable in vivo. The volume of distribution of the labeled starch is 2.5 mL/kg lower than that for labeled albumin (p = .05). Blood volume, measured from the dilution of fluorescent starch, is lower (4.9 mL/kg) than that measured with albumin (p = .048) but higher (6.61 mL/kg) than that measured with red blood cells (p = .0007). This latter difference may be even smaller at marginally higher doses of the fluorescent starch. CONCLUSION: These data support the view that hydroxyethyl starch provides a valid alternative to red cell labels as a means of calculating blood volume in patients. Labeling the starch with a fluorescent marker makes the assay procedure more sensitive and infinitely easier. The dose required is not high enough to affect the hemodynamic status of the patient. PMID- 10752806 TI - Pharmacokinetics of meropenem in intensive care unit patients receiving continuous veno-venous hemofiltration or hemodiafiltration. AB - OBJECTIVE: To evaluate an intravenous meropenem dosage regimen in adult intensive care patients with acute renal failure treated by continuous renal replacement therapy. DESIGN: A prospective, clinical study. SETTING: General intensive care unit of a university hospital. PATIENTS: Ten critically ill adult patients being treated with meropenem and receiving continuous veno-venous hemofiltration (hemofiltration rates, 1-2 L/hr) (n = 5) or continuous venovenous hemodiafiltration (hemofiltration rates, 1-1.5 L/hr; dialysis rates, 1-1.5 L/hr) (n = 5) via a polyacrylonitrile hollow fiber 0.9-m2 filter. INTERVENTIONS: Patients received a meropenem dose of 1 g iv every 12 hrs as a 5-min bolus. MEASUREMENTS AND MAIN RESULTS: Meropenem concentrations were measured by high performance liquid chromatography in serum taken at timed intervals and in ultrafiltrate/dialysate to determine serum concentration-time profiles, derive pharmacokinetic variable estimates, and determine sieving coefficients and filter clearances. The serum concentrations were examined to see whether they were above the minimum inhibitory concentrations (MICs) for pathogens that may be encountered in intensive care patients. Serum concentrations exceeded 4 mg/L (MIC90 for Pseudomonas aeruginosa) during 67% of the dosage period in all patients. Sub-MIC90 concentrations were obtained in three patients immediately before treatment and in one patient 12 hrs after treatment. Mean (SD) (n = 10) pharmacokinetic variable estimates were as follows: elimination half-life, 5.16 hrs (1.83 hrs); volume of distribution, 0.35 L/kg (0.10 L/kg); and total clearance, 4.30 L/hr (1.38 L/hr). A sieving coefficient of 0.93 (0.06) (n = 9) indicated free flow across the filter. The fraction cleared by the extracorporeal route was 48% (13%) (n = 9), which is clinically important. CONCLUSIONS: A meropenem dose of 1g iv every 12 hrs provides adequate serum concentrations in the majority of patients receiving continuous veno-venous hemofiltration or continuous venovenous hemofiltration with a 0.9-m2 polyacrylonitrile filter at combined ultrafiltrate/dialysate flow rates of up to 3 L/hr. A lower dose would not be sufficient for the empirical treatment of potentially life-threatening infections in all patients. PMID- 10752807 TI - Phase II multicenter clinical study of the platelet-activating factor receptor antagonist BB-882 in the treatment of sepsis. AB - OBJECTIVE: To evaluate the safety and efficacy of the platelet-activating factor receptor antagonist BB-882 in the treatment of patients with sepsis. DESIGN: Double-blind, placebo-controlled, randomized, multi-centered study. SETTING: Thirty-four European intensive care units. PATIENTS: One hundred fifty-two patients with clinical suspicion of infection and a mean APACHE II score between 15 and 35 in the 24 hrs before entry into the trial. INTERVENTIONS: Patients received either a loading dose of 4 mg of BB-882 on the first day, followed by an intravenous infusion of 96 mg/24 hrs for up to 120 hrs, or placebo. MEASUREMENTS: Hemodynamic, respiratory and oxygen transport variables, blood lactate concentrations, interleukin-6, interleukin-8, tumor necrosis factor (TNF)-alpha, soluble TNF receptor concentrations, organ failure score, 28-day mortality rate, Acute Physiology And Chronic Health Evaluation (APACHE) II score within 24 hrs of entry. RESULTS: Sixty-nine patients (42 male, 27 female) received placebo and 83 (59 male, 24 female) received BB-882. Patients ranged in age from 16 to 89 yrs (mean, 60 yrs). No important differences existed between the two groups in terms of gender distribution, age, or initial APACHE II score. Sepsis was identified as Gram-positive in 49 patients, Gram-negative in 40, mixed in 37, and unknown in 26. No important differences were shown in hemodynamic, respiratory, or oxygen transport variables between groups during the study. Organ failure scores were similar in the two groups throughout the study. Cytokine concentrations were not significantly different in the two groups. Within 28 days of entering the study, 75 patients died, including 31 (45%) in the placebo group and 44 (53%) in the treatment group, p = .32. The median time to death in the placebo group was 6.0 days, and in the treatment group, it was 4.5 days (p = .30). CONCLUSION: Treatment of sepsis with the platelet-activating factor antagonist BB-882 offers no advantage over placebo on survival, hemodynamic status, respiratory function, or organ failure scores. PMID- 10752808 TI - An immune-enhancing enteral diet reduces mortality rate and episodes of bacteremia in septic intensive care unit patients. AB - OBJECTIVE: To determine whether early enteral feeding in a septic intensive care unit (ICU) population, using a formula supplemented with arginine, mRNA, and omega-3 fatty acids from fish oil (Impact), improves clinical outcomes, when compared with a common use, high protein enteral feed without these nutrients. DESIGN: A prospective, randomized, multicentered trial. SETTING: ICUs of six hospitals in Spain. PATIENTS: One hundred eighty-one septic patients (122 males, 59 females) presenting for enteral nutrition in an ICU. INTERVENTIONS: Septic ICU patients with Acute Physiology and Chronic Health Evaluation (APACHE) II scores of > or =10 received either an enteral feed enriched with arginine, mRNA, and omega-3 fatty acids from fish oil (Impact), or a common use, high protein control feed (Precitene Hiperproteico). MEASUREMENTS AND MAIN RESULTS: One hundred seventy-six (89 Impact patients, 87 control subjects) were eligible for intention to-treat analysis. The mortality rate was reduced for the treatment group compared with the control group (17 of 89 vs. 28 of 87; p < .05). Bacteremias were reduced in the treatment group (7 of 89 vs. 19 of 87; p = .01) as well as the number of patients with more than one nosocomial infection (5 of 89 vs. 17 of 87; p = .01). The benefit in mortality rate for the treatment group was more pronounced for patients with APACHE II scores between 10 and 15 (1 of 26 vs. 8 of 29; p = .02). CONCLUSIONS: Immune-enhancing enteral nutrition resulted in a significant reduction in the mortality rate and infection rate in septic patients admitted to the ICU. These reductions were greater for patients with less severe illness. PMID- 10752809 TI - Energy expenditure in acetaminophen-induced fulminant hepatic failure. AB - OBJECTIVE: To determine energy expenditure in critically ill patients suffering from acetaminophen-induced fulminant hepatic failure and compare it with values obtained in matched, healthy control subjects and in patients studied during the anhepatic period of elective liver transplantation. DESIGN: Prospective, controlled, observational study. SETTING: A ten-bed intensive therapy unit and a liver transplant unit at a University teaching hospital. PATIENTS AND SUBJECTS: Sixteen patients suffering from acetaminophen-induced fulminant hepatic failure who were sedated, paralyzed, and mechanically ventilated; 16 age-, gender-, and weight-matched, awake, healthy control subjects; and 16 patients with chronic liver disease, undergoing elective liver transplantation, who were studied during the anhepatic period of surgery. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The mean energy expenditure was calculated in each case for a 30-min period, using indirect calorimetry. In the patients undergoing liver transplantation, measurements were performed after clamping the hepatic veins and recipient hepatectomy. Energy expenditure was markedly increased in the fulminant hepatic failure group (mean energy expenditure, 4.05 [SD 0.52] kJ x kg(-1) x hr( 1)), in comparison with healthy control subjects (mean, 3.44 [0.27] kJ x kg(-1) x hr(-1); mean difference, 18%; p < .001) and in comparison with patients during the anhepatic period of liver transplantation (mean, 3.15 [0.61] kJ x kg(-1) x hr(-1); mean difference, 29%; p < .001). These differences were even more pronounced when a correction factor for differences in core temperature was included in the calculation. Harris-Benedict predictions of energy expenditure were unreliable in the patients with acute liver failure. No correlations were found among energy expenditure and hemodynamic variables, the requirement for vasoconstrictors, or the presence of renal failure. CONCLUSIONS: Despite the loss of functioning liver cell mass, the metabolic rate is substantially increased in patients with acetaminophen-induced fulminant hepatic failure. This finding is consistent with the marked systemic inflammatory response, which accompanies acute hepatic failure. The Harris-Benedict equation is unreliable when an estimation of energy expenditure is required in patients with this condition. PMID- 10752810 TI - Carbamezapine for pain management in Guillain-Barre syndrome patients in the intensive care unit. AB - OBJECTIVE: To evaluate carbamezapine (CBZ) for neuritic pain relief in Guillain Barre syndrome (GBS) patients in the intensive care unit (ICU). DESIGN: Prospective, double-blind, randomly allocated cross-over study days. SETTING: ICU in a tertiary care university hospital. PARTICIPANTS: Twelve consecutive, conscious adult (22-54 yrs) patients with GBS during recovery from the muscular weakness and receiving pressure-support ventilation in the ICU. All patients complained of severe backache and/or leg cramps and tenderness in muscles, and they required opioids for pain relief. INTERVENTIONS: CBZ (100 mg every 8 hrs) or equivalent placebo was given to nursing staff in coded powder form. Medication was given to patients through a nasogastric feeding tube. The same coded medicine was given for 3 days, and after a 1-day omission, a second set of coded powder was given for the next 3 days in a randomized, double-blind, crossover fashion. Pethidine (1 mg x kg(-1)) was given intravenously in between, if the pain score was >2. Group 1 (n = 6) patients were given a placebo on the first 3 days, followed by CBZ. Group 2 (n = 6) patients were given CBZ on the first 3 days, followed by a placebo. MEASUREMENTS AND MAIN RESULTS: In these two study periods of different medications, we observed and scored pain (1, no pain; 5, severe pain), sedation (1, alert; 6, asleep, does not respond to verbal command), and total pethidine requirement per day. In group 1 patients, a significant (p < .001) improvement in the sedation score and a low requirement for pethidine was observed 3 days later, when CBZ was started. However, in group 2 patients, a gradual increase in the pethidine requirement and a high sedation score were noteworthy in the later days of placebo medication. Observations were also analyzed for CBZ days vs. placebo days. Overall, the pain score (1.7 +/- 0.8) during the CBZ period of both regimens was significantly (p < .001) lower than during the placebo days (3.1 +/- 0.9). Significantly higher doses of pethidine (3.7 +/- 0.9 mg/kg/day) were used on the placebo days than on the CBZ days (1.7 +/- 1.0 mg/kg/day). CONCLUSION: The pain in GBS has a dual origin, and we recommend CBZ as an adjuvant to treat pain in GBS patients, during the recovery phase in the ICU, to reduce the narcotic requirement. PMID- 10752811 TI - Airway accidents in intubated intensive care unit patients: an epidemiological study. AB - OBJECTIVE: To assess the rate of occurrence and nature of airway accidents in intubated patients. DESIGN: Prospective recording of all airway accidents in a 16 bed multidisciplinary intensive care unit. PATIENTS: A total of 5,046 ventilated patients intubated for 9,289 days during 4 yrs. MEASUREMENTS AND MAIN RESULTS: We determined the number and diagnoses of intubated and ventilated patients, the number and timing of airway accidents, the type of tracheal tube used and duration for which the tube was in situ, the description of the type of accident, the severity of the accident, and its impact on the course of the patient's illness, whether the patient needed reintubation, and whether the accident was preventable. The total accident rate was 36 of 5,046 patients during 9,289 intubated patient days; 26 occurred in 5,043 endotracheally intubated patients during 8,446 patient endotracheal tube days. There were 10 tracheostomy-related accidents from a total of 79 patients with tracheostomies during 843 tracheostomy patient days. Six had severe consequences and one resulted in death. Eleven were completely preventable, 17 partly preventable, and 8 were considered unpreventable. Self-extubation was the most common accident. Seven of 13 self extubations occurred in patients due for elective extubation in the next few hours. Twelve of 15 patients with self- or accidental extubation of an endotracheal tube accidents did not require reintubation. CONCLUSIONS: Airway accidents occurred at low levels with even lower rates of resultant morbidity and mortality. Tracheostomy accidents are more common than those with an endotracheal tube. PMID- 10752812 TI - Safety, efficacy, and cost-effectiveness of mechanical ventilation with humidifying filters changed every 48 hours: a prospective, randomized study. AB - OBJECTIVE: To determine whether three hydrophobic and hygroscopic heat and moisture exchangers (HMEs) retain their heating and humidifying properties (assessed by psychrometric measurements of absolute humidity, relative humidity, and tracheal temperature) for 48 hrs without any drop in their bacteriologic efficiency. DESIGN: Prospective randomized clinical trial. PATIENTS: Sixty-one consecutive unselected mechanically ventilated intensive care unit patients. INTERVENTIONS: Patients were randomly allocated to one of the three HMEs studied (Hygrobac-Dar from Mallinckrodt, n = 21; Humid-Vent from Gibeck, n = 20; and Clear-Thermal from Intersurgical, n = 20). MEASUREMENTS AND MAIN RESULTS: Hygrometric parameters were measured by psychrometry after 3, 24, and 48 hrs of use. Peak airway pressure was recorded every 6 hrs and averaged over 24 hrs. Bacterial colonization of both patients and circuits was studied. Patients in all three groups were similar in terms of age, indications for, and overall duration of mechanical ventilation. Tracheal tube occlusion never occurred. Hygrometric data included 371 measurements whereas bacteriologic data included >700 samples and cultures. The Hygrobac-Dar HMEs gave a significantly higher absolute humidity whatever the time of measurement (3, 24, or 48 hrs) than the other two HMEs (p < .001). The Clear-Thermal HMEs gave the poorest hygrometric parameters (p < .01); five of them were replaced prematurely (24 hrs) because the absolute humidity was <25 mg H2O/L. This did not occur for the other HMEs. Mean peak airway pressures were identical in the three groups. The bacterial colonizations of both patient and circuit were similar (and negligible for circuits) for all three groups. CONCLUSION: Some HMEs may be used safely for 48 hrs without change. However, this does not pertain to every brand of HME. Objective in vivo evaluation of their humidifying performances is decisive before extending their duration of use. PMID- 10752813 TI - Right atrial pressure predicts hemodynamic response to apneic positive airway pressure. AB - OBJECTIVE: To evaluate if the preexistant filling state, assessed by right atrial pressure (RAP), pulmonary artery occlusion pressure (PAOP), and right ventricular end-diastolic volume index (EDVI), would define the subsequent hemodynamic effects of increases in airway pressure (Paw). DESIGN: Prospective open clinical study. SETTING: Postoperative intensive care unit, university hospital. PATIENTS: Twenty-two consecutive ventilator-dependent patients with mild to severe acute lung injury with Murray scores (scoring infiltrates on chest radiograph, oxygenation index, lung compliance, and the level of positive end-expiratory pressure) ranging from 0.5 to 3.0 without history of preexisting cardiopulmonary disease. INTERVENTIONS: Paw varied during apnea from 0 to 10, 20, and 30 cm H2O using inspiratory hold maneuvers of 15 secs. MEASUREMENTS AND MAIN RESULTS: Cardiac index and right ventricular ejection fraction were measured by the thermodilution technique. We made measurements in triplicate using manual injection of iced saline. Right ventricular volumes were calculated. Increasing Paw induced variable changes in cardiac index among subjects (+6% to -43% change from baseline 0 cm H2O Paw values), which correlated with percentage changes in both stroke index (r2 = .89) and right ventricular EDVI (r2 = .75), whereas heart rate and right ventricular ejection fraction did not change. The change in cardiac index from 0 to 30 cm H2O Paw correlated with baseline values for RAP, PAOP, and right ventricular EDVI (r2 = .68, .43, and .34, respectively, p < 0.01). Increases in RAP correlated with lung compliance if baseline RAP was >10 mm Hg but did not if it was < or =10 mm Hg. Similarly, patients with baseline RAP < or =10 mm Hg had a greater decrease in cardiac index than patients with a RAP >10 mm Hg (for 30 cm H2O Paw: -30% +/- 9% vs. -8% +/- 7%, p < .01). CONCLUSIONS: Apneic positive Paw decreased cardiac output mainly by reducing venous return. From the investigated filling variables, RAP was most sensitive in predicting the hemodynamic response, followed by PAOP and right ventricular EDVI. Patients with RAP < or =10 mm Hg, if subjected to aggressive positive pressure ventilation, are at risk of hemodynamic deterioration and organ hypoperfusion. PMID- 10752814 TI - Incentive spirometry does not enhance recovery after thoracic surgery. AB - OBJECTIVE: To investigate the additional effect of incentive spirometry to chest physiotherapy to prevent postoperative pulmonary complications after thoracic surgery for lung and esophageal resections. DESIGN: Randomized controlled trial. SETTING: University hospital, intensive care unit, and surgical department. PATIENTS: Sixty-seven patients (age, 59 +/- 13 yrs; forced expiratory volume in 1 sec, 93% +/- 22% predicted) undergoing elective thoracic surgery for lung (n = 40) or esophagus (n = 27) resection. INTERVENTIONS: Physiotherapy (breathing exercises, huffing, and coughing) (PT) plus incentive spirometry (IS) was compared with PT alone. MEASUREMENTS AND MAIN RESULTS: Lung function, body temperature, chest radiograph, white blood cell count, and number of hospital and intensive care unit days were all measured. Pulmonary function was significantly reduced after surgery (55% of the initial value) and improved significantly in the postoperative period in both groups. However, no differences were observed in the recovery of pulmonary function between the groups. The overall score of the chest radiograph, based on the presence of atelectasis, was similar in both treatment groups. Eight patients (12%) (three patients with lobectomy and five with esophagus resection) developed a pulmonary complication (abnormal chest radiograph, elevated body temperature and white blood cell count), four in each treatment group. Adding IS to regular PT did not reduce hospital or intensive care unit stay. CONCLUSIONS: Pulmonary complications after lung and esophagus surgery were relatively low. The addition of IS to PT did not further reduce pulmonary complications or hospital stay. Although we cannot rule out beneficial effects in a subgroup of high-risk patients, routine use of IS after thoracic surgery seems to be ineffective. PMID- 10752815 TI - Naloxone decreases tolerance to hypotensive, hypovolemic stress healthy humans. AB - OBJECTIVE: In animal studies, naloxone, an opioid receptor antagonist, improves tolerance to hemorrhagic shock. The purpose of this study was to determine whether naloxone would augment tolerance to hypotensive hypovolemic stress (lower body negative pressure [LBNP]) in healthy human males. DESIGN: This study was a repeated measures design. SETTING: The experiments were conducted in a laboratory setting. SUBJECTS: Eight healthy male subjects were tested. The subjects' ages were 30 +/- 4.0 yrs, height = 177 +/- 7.0 cm, and weight = 75.5 +/- 3.5 kg (mean +/- SEM). INTERVENTIONS: Subjects underwent two LBNP exposures terminated by the onset of vasodepression. At each of the exposures, using a double-blind procedure, the subjects received an intravenous injection of either saline placebo or naloxone in a dosage totaling 0.4 mg/kg. PMID- 10752816 TI - Evaluation of antimicrobial treatment in mechanically ventilated patients with severe chronic obstructive pulmonary disease exacerbations. AB - OBJECTIVE: To study microbial and susceptibility patterns and antimicrobial treatment responses in patients with severe, acute exacerbations of chronic obstructive pulmonary disease requiring mechanical ventilation. DESIGN: Microbial investigation using tracheobronchial aspirates, bronchoscopy with a protected specimen brush, and bronchoalveolar lavage, as well as paired serologies. Evaluation of antimicrobial treatment by results of the initial investigation, susceptibility testing, and a repeated microbial investigation (tracheobronchial aspirates, bronchoscopy with a protected specimen brush, and bronchoalveolar lavage) after 72 hrs. SETTING: A respiratory intensive care unit of a 1,000-bed teaching hospital. PATIENTS: Fifty severely exacerbated and mechanically ventilated patients with chronic obstructive pulmonary disease. INTERVENTIONS: Initial empirical antimicrobial treatment according to clinical judgment. MEASUREMENTS AND MAIN RESULTS: Overall, 36 of 50 patients (72%) had evidence of a microbial origin. Community-acquired endogenous pathogens were present in 70% of patients, and Gram-negative enteric bacilli and Pseudomonas/Stenotrophomonas species were present in 30%. All five isolates of Streptococcus pneumoniae were resistant to penicillin (three intermediately and two highly), and three were resistant to multiple antibiotics. Pseudomonas species revealed multiresistance in four of nine isolates (44%), and Stenotrophomonas maltophilia revealed multiresistance in one of two isolates. Antimicrobial treatment was modified according to diagnostic results in 11 of 31 patients (36%) with potentially pathogenic microorganisms. In patients who underwent a repeat investigation after 72 hrs, 24% of the initially present and potentially pathogenic microorganisms persisted. Inappropriate initial antimicrobial therapy was associated significantly with bacterial persistence (p < .002). CONCLUSIONS: Considering the diversity of microbial pathogens and the resistance rates especially to S. pneumoniae in this patient population, antimicrobial treatment should be based on the constant study of local microbial and susceptibility patterns along with routine microbial investigation of the individual patient. PMID- 10752817 TI - Evaluation of a thoracic bioimpedance cardiac output monitor during cardiac catheterization. AB - OBJECTIVE: To evaluate the accuracy and precision of an advanced thoracic bioimpedance cardiac output monitor by comparing it with conventional thermodilution. DESIGN: Prospective data collected from 47 patients undergoing routine cardiac catheterization. The new bioimpedance system differs from its predecessors in electrode system configuration, advanced signal processing, use of a modified Kubicek equation, and a reliable estimate of left ventricular ejection time from the time derivative bioimpedance signals. SETTING: A cardiac catheterization laboratory in a university affiliated teaching hospital. PATIENTS: A series of 47 relatively homogenous patients undergoing routine cardiac catheterization for suspected cardiac disease. MEASUREMENTS AND MAIN RESULTS: The data from the first 20 patients was used to determine optimal values for coefficients in the bioimpedance cardiac output equations. The coefficients found were used when the system was tested in the subsequent 27 patients. For the last 27 patients, a total of 80 simultaneous pairs of cardiac output measurements were made by conventional thermodilution and by thoracic bioimpedance. The mean difference between the two methods was -0.31 L/min and the standard deviation of the differences was (0.76 L/min). The correlation coefficient was r2 = .72 (p < .001). CONCLUSIONS: The correlation between conventional thermodilution and thoracic bioimpedance cardiac output estimates was good and the standard deviation of the differences was lower than that reported for commercially available devices. The system can be used in the cardiac catheterization lab for reliable and continuous noninvasive measurement of cardiac output. PMID- 10752818 TI - Evidence for the need of bedside accuracy of pulse oximetry in an intensive care unit. AB - OBJECTIVE: To compare pulse oximetry saturation (Spo2 with arterial blood gas saturation (SaO2) obtained during clinical routine to determine the optimal lowest reliable value of SpO2 in ventilator-dependent patients before setting up a nurse-directed protocol of FIO2 titration. DESIGN: Prospective clinical study. SETTING: Surgical intensive care unit in a university hospital. PATIENTS: Thirty three patients with a pulse oximeter probe in whom arterial blood gas was measured with a radial artery line. INTERVENTIONS: SPO2 was recorded by the nurses and compared with SaO2 obtained by blood gas analysis with a co-oximeter. Two sensors currently used in our surgical intensive care unit and connected to a monitor (HP OmniCare M1165/66A; Hewett Packard, Andover, MA) were tested. In group I, the Durensor DS 100A (Nellcor Puritan Bennett, Pleasanton, CA), a reusable sensor, was used. In group II, the Oxisensor D25L (Nellcor Puritan Bennett), a nonreusable sensor, was used. MEASUREMENTS AND MAIN RESULTS: In group 1, 64 data pairs were obtained. In this group, SaO2 ranged from 87 to 98% and SpO2 ranged from 92 to 100%. The bias was -1.90% and the limits of agreement ranged from -5.56 to 1.76%. In group 11, 47 data pairs were obtained. In this group, SaO2 ranged from 87 to 99% and SpO2 ranged from 92 to 100%. The bias was 2.49% and the limits of agreement ranged from -6.62 to 1.64%. CONCLUSIONS: In the range of SaO2 tested, regardless of the sensor used, SpO2 overestimated SaO2. Large limits of agreement were found. Based on this result, the authors concluded that before defining a nurse-directed protocol of FIO2 titration with SpO2, the material used daily must be evaluated. A minimum threshold SpO2 value of 96% in both groups I and II is more reliable to ensure SaO2 > or = 90%. PMID- 10752819 TI - Introduction of sedative, analgesic, and neuromuscular blocking agent guidelines in a medical intensive care unit: physician and nurse adherence. AB - OBJECTIVE: To determine physician and nurse adherence with sedative, analgesic, and neuromuscular blocking agent guidelines in the management of mechanically ventilated patients in a medical intensive care unit. DESIGN: Prospective cohort study. SUBJECTS: One hundred consecutively admitted patients to a medical intensive care unit who required mechanical ventilatory support. A sample of 29 nurses, residents, and attending physicians were interviewed regarding their attitudes and perceptions of the guidelines. MEASUREMENT: Data were collected from concurrent medical records and included the following: demographic characteristics; clinical variables; physician prescriptions of sedative, analgesic, and/or neuromuscular blocking agents; nurse administration of these medications; documentation of monitoring; and assessment of patient hemodynamic status and behaviors. A semistructured interview was elicited from both nurses and physicians about their rationale for the use or nonuse of the guidelines. RESULTS: Patients ranged in age from 24 to 87 yrs, mean 60.7 (+15.3) yrs. Admission Acute Physiology and Chronic Health Evaluation III scores ranged from 36 to 192, mean 93.8 ( 30.5) and median 88. Length of mechanical ventilatory support ranged from 1 to 112 days, mean 14.8 ( 20.0) days, and median 8 days; medical intensive care unit length of stay ranged from 1 to 46 days, with a mean of 9.8 ( 8.1) days and a median of 8 days. Of the 100 patients, 47% died, 28% returned home, and 25% were discharged to a nursing facility. Eighty-five patients were administered one or more sedative, analgesic, and/or neuromuscular blocking agent, range 1-9 drugs, mean 2.5 (+1.5) drugs. Physicians prescribed 14 different medications; the most commonly administered drug was lorazepam (n = 71), followed by morphine (n = 39). Physicians and nurses had partial or total adherence to the guidelines in 58% of patients. The initial choice of the drug followed the guidelines in 60% of patients; the overall guideline was followed in 23% of patients. The most common rationales for nonadherence to the guidelines stated by both physicians and nurses were patient-specific factors, resident guideline learning curve, and physician medication preferences. CONCLUSION: Most patients required treatment for agitated behaviors. The majority of treatment regimens partially or totally adhered to the guidelines. Factors such as patient specific disease states, resident guideline learning curve, and physician preferences of medications may have decreased adherence. Improving adherence to the guidelines is essential to assess their effectiveness in improving clinical outcomes. PMID- 10752820 TI - Changing heat and moisture exchangers after 96 hours rather than after 24 hours: a clinical and microbiological evaluation. AB - OBJECTIVE: The aim of this study was to determine whether changing heat and moisture exchangers (HMEs) every 96 hrs rather than 24 hrs would affect their efficacy to preserve the heat and moisture of inspiratory gases. The impact of a prolonged use of the HME on its microbial colonization was also assessed. DESIGN: Prospective cohort observational study. SETTING: Intensive care unit of a university hospital. PATIENTS: Thirteen consecutive patients with no previous history of respiratory disease requiring controlled mechanical ventilation with an HME for >4 days were evaluated. INTERVENTIONS: The same HME was used for 96 hrs in each patient. MEASUREMENTS AND MAIN RESULTS: In each patient, during the inspiration phase, the following measurements were performed: peak and mean airway pressures, mean values of temperature, and relative and absolute humidity of inspired gases. In each patient, measurements were performed after 1 hr of HME use and then daily up to the fourth day. On days 1 and 4, microbiological samples were obtained from the patients' bronchial secretions and the ventilator side of the HME. After 96 hrs of ventilation with the same HME, tracheal tube occlusion was never observed. Using the same HME for 96 hrs rather than 24 hrs did not affect its technical performances: temperature at 24 hrs: 32.2 +/- 1.5 degrees C (90.0 +/- 34.7 degrees F), at 96 hrs: 32.1 +/- 1.6 degrees C (89.8 +/- 34.9 degrees F); relative humidity at 24 hrs: 97.9 +/- 2%, at 96 hrs: 98.1 +/- 1.7%; absolute humidity at 24 hrs: 33.1 +/- 2.4 mg H2O/L, at 96 hrs: 33.0 +/- 2.5 mg H2O/L. This analysis was based on a total of 312 measurements performed on the 13 patients. Peak and mean airway pressures did not change during the 96-hr study period, with identical tidal and minute volumes in the study patients. On day 1, ten patients had a positive culture of their tracheal secretions at a colony count of > or = 10(3) colony forming units/mL. After 96 hrs of use with the same HME, only seven patients had a positive culture of their tracheal secretions. Cultures from the ventilator sides of the HMEs were all sterile (13/13) after 96 hrs of use. CONCLUSIONS: In patients free from previous chronic respiratory disorder and ventilated for neurologic reasons, changing the HME after 96 hrs rather than 24 hrs did not affect its technical performance in terms of heat and water preservation of ventilatory gases. There is also some indirect evidence of very little, if any, changes in the HME resistance. No bacterial colonization of the ventilator sides of the HMEs was observed after 96 hrs of use. However, other large clinical trials should be undertaken to confirm the safety of extending the time between HME changes. PMID- 10752821 TI - Improved outcome prediction in unconscious cardiac arrest survivors with sensory evoked potentials compared with clinical assessment. AB - OBJECTIVE: To compare the prognostic ability of sensory evoked potentials in cardiac arrest survivors with the outcome predicted by a panel of experienced emergency physicians based on detailed prehospital, clinical, and laboratory data. DESIGN: Inception cohort study. SETTING: Medical intensive care unit and department of emergency medicine at a university hospital. PATIENTS: A total of 162 unconscious, mechanically ventilated patients who survived > or =24 hrs after resuscitation from cardiac arrest. INTERVENTIONS: Recording of sensory evoked potentials and outcome prediction after review of detailed clinical and laboratory data by emergency physicians within 24 hrs after cardiac arrest. MEASUREMENTS AND MAIN RESULTS: At 6 months, the outcome of 36 patients was classified as favorable and 126 patients were rated as poor. After review of prehospital data, emergency physicians predicted favorable vs. poor outcome with a sensitivity of 70% and a specificity of 65%. After additional assessment of data 1 hr after cardiac arrest, the sensitivity of emergency physician predictions increased to 80%, whereas the specificity decreased to 48%. Outcome prediction by emergency physicians was most accurate after obtaining detailed patient data 24 hrs after cardiac arrest (sensitivity, 81%; specificity, 58%). In 35 of 36 patients with favorable outcomes, the cortical evoked potential N70 peak was detected between 72 and 128 msec. Of 113 patients with an N70 peak latency >130 msec or an absent N70 peak, all except one had a poor outcome. By using a cutoff of 130 msec, the N70 peak latency alone had a sensitivity of 94% and a specificity of 97%. The predictive accuracy of the N70 peak latency was significantly higher than the clinical assessment 24 hrs after cardiac arrest (91% vs. 76%, p = .0003). CONCLUSION: In unconscious cardiac arrest survivors, a recording of long-latency sensory evoked potentials is more accurate in predicting individual outcome than an emergency physician review of clinical data. PMID- 10752822 TI - Thrombus formation on the balloon of heparin-bonded pulmonary artery catheters: an ultrastructural scanning electron microscope study. AB - OBJECTIVE: To investigate heparin-bonded pulmonary artery catheters with respect to thrombus formation and platelet aggregation at the balloon and the shaft using a scanning electron microscope in critically ill patients. DESIGN: Prospective study. SETTINGS: Critical care unit and research laboratories. PATIENTS: Pulmonary artery catheters were inserted in critically ill patients (n = 10). INTERVENTIONS: Pulmonary artery catheters were removed after 24, 48, 72, or 120 hrs, and the ultrastructure was investigated in specialized research laboratories. MEASUREMENTS AND MAIN RESULTS: Balloon and shaft were investigated using a scanning electron microscopic technique. Area of thrombus formation was quantified using image analysis. Heparin release of the catheters was measured. The frequency of balloon inflations was investigated in in vitro experiments by inflating catheters different times (0, 10, 20, and 30 times). Twenty-four hours after catheter insertion, scanning electron microscopic images showed thrombus formation and platelet aggregation at the site of the balloon. Seventy-two hours after catheter insertion, a thrombus started to detach. The areas of thrombus formation did not differ, but thrombus organization changed dramatically 72 and 120 hrs after catheter insertion. The shaft was colonized by single cells only. Cracks of the balloon could be observed after 72 hrs, whereas no cracks could be found in in vitro controls. In vitro, heparin release of the pulmonary artery catheters decreased significantly after 24 hrs. CONCLUSIONS: Scanning electron microscopic images of heparin-bonded pulmonary artery catheters demonstrate thrombus formation on the balloon 24 hrs after pulmonary artery catheter insertion, increasing dramatically at 72 and 120 hrs. The shaft was colonized by single cells only. The thrombus size is not significantly different during the observation time, but the grade and quality of thrombus formation differ. PMID- 10752823 TI - Serum amyloid A versus C-reactive protein in acute pancreatitis: clinical value of an alternative acute-phase reactant. AB - OBJECTIVES: The acute-phase reactant C-reactive protein (CRP) is currently the serum variable of choice for an early, accurate, and cost-effective severity assessment of acute pancreatitis in the daily clinical routine. Serum amyloid A (SAA) proteins comprise a family of apolipoproteins that constitute another major acute-phase reactant and thus could be a potential alternative to CRP assessment. In the present study we investigated the clinical usefulness of SAA determinations in acute pancreatitis using an automated immunoassay technique. DESIGN: Cohort study, comparing patients with complicated and mild acute pancreatitis; control groups included individuals with further abdominal disorders and healthy volunteers. SETTING: A collaborative study between the department of general surgery and the routine laboratory of the department of clinical chemistry/pathobiochemistry. PATIENTS: We enrolled 66 patients with acute pancreatitis in the present study. Control groups consisted of healthy subjects (n = 30), patients with chronic pancreatitis (n = 20), patients with pancreatic carcinoma (n = 20), and patients with acute appendicitis (n = 20). INTERVENTIONS: Blood samples were collected during 14 consecutive days in patients with acute pancreatitis. A single blood specimen was taken in all control groups after the diagnosis was established. MEASUREMENTS AND MAIN RESULTS: SAA concentrations were 3 mg/L (median; range, 3-93) in healthy subjects. Although SAA and CRP both reached their maximum within 4 days after onset of symptoms in patients with acute pancreatitis, SAA concentrations rose faster above normal ranges and reached 676 mg/L (median; range, 12-1880), higher than CRP, which reached 313 mg/L (median; range, 29-613). As observed for CRP, SAA was significantly higher in patients who developed complications such as necrosis, infection of necrosis, or multiple organ dysfunction syndrome or in patients who died. SAA achieved best results in discriminating between necrotizing pancreatitis and interstitial edematous pancreatitis. However, CRP provided an earlier differentiation between both entities and a significantly better overall accuracy, as shown by receiver operating characteristics analysis. SAA concentrations in patients with chronic pancreatitis were 6 mg/L (median; range, 3-756). In patients with pancreatic carcinoma, SAA concentrations were 7 mg/L (median; range, 3-492), and in patients with acute appendicitis, they were 50 mg/L (median; range, 3-2140). CONCLUSION: SAA is a nonspecific and rapidly produced variable in inflammatory abdominal disorders with a wider dynamic range than CRP. The current assay technique renders SAA an applicable and readily available variable under clinical routine conditions. In cases of acute pancreatitis, however, CRP is still superior to SAA for early and accurate stratification of patients with a complicated course. PMID- 10752824 TI - Effects of the phosphodiesterase III inhibitors olprinone, milrinone, and amrinone on hepatosplanchnic oxygen metabolism. AB - OBJECTIVE: To measure the hepatic venous oxygen saturation in patients after cardiac surgery and to compare the effects of olprinone (OLP), a newly synthesized phosphodiesterase III inhibitor, with those of milrinone (MIL) and amrinone (AMR) on hepatosplanchnic oxygen dynamics. Phosphodiesterase III inhibitors are used to improve the hemodynamic state after cardiac surgery. However, the effect of these agents on the hepatosplanchnic circulation has not been investigated thoroughly. DESIGN: Prospective, randomized study. SETTING: University hospital intensive care unit (ICU). PATIENTS: Twenty-nine patients undergoing elective cardiac surgery. MEASUREMENTS AND MAIN RESULTS: In each patient, a 7.5-Fr oximeter catheter was placed in the hepatic vein via the right femoral vein. Catheterization was completed before admission to the ICU, and the study was performed 8 to 24 hrs after surgery, after obtaining stable systemic hemodynamics in the ICU. The patients were assigned randomly to three groups, and they received one of three drugs for 2 hrs (OLP group, 0.3 microg/kg/min of OLP; MIL group, 0.5 microg/kg/min of MIL; AMR group, 10 microg/kg/min of AMR). The authors did not change the patient's hemodynamic interventions, including catecholamines and vasodilators, throughout the study period. Arterial and hepatic venous blood gas data and hemodynamic data (via a pulmonary artery catheter) were obtained before and after drug infusion. Using these data, the authors calculated systemic oxygen delivery and consumption, the systemic oxygen extraction ratio and the hepatosplanchnic oxygen extraction ratio, and the change in hepatosplanchnic blood flow using Fick's equation. Although the increases in cardiac index were not significantly different among the three groups, hepatic venous oxygen saturation increased significantly only in the OLP group (from 47.1% +/-2.6% to 57.0% +/- 1.5% in the OLP group, from 48.4% +/- 2.3% to 50.9% +/ 2.6% in the MIL group, and from 49.8% +/- 3.6% to 50.8% + +/-.7% in the AMR group). The calculated hepatosplanchnic blood flow change was significantly larger in the OLP group than in the other groups (30.1% +/- 5.7% in the OLP group, 9.3% +/- 5.1% in the MIL group, and 2.6% +/- 6.5% in the AMR group). CONCLUSIONS: These results suggest that OLP enhances hepatosplanchnic blood flow and thus may be beneficial in protecting the hepatosplanchnic organs after cardiac surgery. PMID- 10752825 TI - The effect of vigorous fluid resuscitation in uncontrolled hemorrhagic shock after massive splenic injury. AB - OBJECTIVE: Using a standardized massive splenic injury model of uncontrolled hemorrhagic shock, we studied the effect of vigorous fluid resuscitation on the hemodynamic response and survival time in rats. DESIGN: Randomized, controlled study. Duration of follow-up was 4 hrs. SETTING: University research laboratory. SUBJECTS: Adult male Sprague-Dawley rats, weighing 240-430 g. INTERVENTIONS: Standardized massive splenic injury was induced by two transverse incisions in the rat's spleen. The animals were randomized into four groups: group 1 (n = 8) underwent sham operation; in group 2 (n = 15), massive splenic injury was untreated; in group 3 (n = 15), massive splenic injury was treated with 41.5 mL/kg 0.9% sodium chloride (large-volume normal saline); and in group 4 (n = 15), massive splenic injury was treated with 5 mL/kg 7.5% sodium chloride (hypertonic saline). MEASUREMENTS AND MAIN RESULTS: The hemodynamic and metabolic variables in the sham-operated group 1 were stable throughout the experiment. Mean arterial pressure in group 2 decreased from 86.5 +/- 4.0 to 50.3 +/- 6.3 mm Hg (p < .001) in the first 15 mins after massive splenic injury. Mean survival time in group 2 was 127.5 +/- 17.0 mins; total blood loss was 33.8% +/-2.6% of blood volume; and the mortality rate at 1 hr was 13.3%. Bolus infusion of large-volume normal saline after 15 mins resulted in an early increase in mean arterial pressure from 48.6 +/-7.4 to 83.3 +/- 7.2 mm Hg (p < .01); it then rapidly decreased to 24.6 +/ 8.6 mm Hg (p < .001) after 60 mins. The mean survival time (95.3 +/- 16.4 mins) was significantly lower than in group 2 (p < .01); total blood loss (48.0% +/- 4.3%) was significantly higher than in group 2 (p < .01); and mortality rate in the first hour was 33.3% (p < .05). Bolus infusion of hypertonic saline also decreased survival time to 93.3 +/- 20.3 mins (p < .01), but total blood loss was 35.2% +/- 3.0%, which was not significantly different from the blood loss in group 2. The mortality rate in the first hour (60.0%) was significantly higher than in group 2 (p < .005). CONCLUSIONS: Vigorous infusion of normal saline after massive splenic injury resulted in a significant increase in intra-abdominal bleeding and decreased survival time. The hemodynamic response to crystalloid infusion in blunt abdominal trauma is primarily dependent on the severity of injury and the rate of blood loss. PMID- 10752826 TI - Effects of lidocaine administration on hemodynamics and cytokine responses to endotoxemia in rabbits. AB - OBJECTIVE: To investigate the effects of lidocaine administration on hemodynamics and cytokine concentrations in Escherichia coli endotoxemia in rabbits. DESIGN: Randomized, prospective laboratory study. SETTING: University laboratory. SUBJECTS: Thirty-two Japanese rabbits anesthetized with urethane and ventilated mechanically. INTERVENTIONS: Animals were randomly assigned to one of four groups: endotoxemic controls (n = 8), receiving intravenous E. coli endotoxin (0.5 mg/kg bolus) via the mesenteric vein; laparotomy controls (n = 8), treated identically to the endotoxemic controls except for the substitution of 0.9% saline for endotoxin; lidocaine controls (n = 8), treated identically to the laparotomy controls with the addition of intravenous lidocaine (3 mg/kg bolus followed by infusion at 2 mg/kg/hr) administered immediately after the injection of 0.9% saline; and lidocaine-treated rabbits (n = 8), treated identically to the endotoxemic controls with the addition of intravenous lidocaine (3 mg/kg bolus followed by infusion at 2 mg/kg/hr) administered immediately after the injection of endotoxin. MEASUREMENTS AND MAIN RESULTS: We compared the cardiac output, systemic vascular resistance, blood gases, and plasma cytokine concentrations (tumor necrosis factor, interleukin [IL]-6, and IL-8) for each group. After endotoxin injection, the mean arterial pressure, cardiac output, and systemic vascular resistance decreased progressively in the endotoxemic controls. At 4 hrs after injection, all of the variables except the heart rate and central venous pressure were lower in the endotoxemic controls than in the other groups. At 4 hrs after endotoxin injection, both IL-6 and IL-8 concentrations increased in all groups. However, the mean concentrations of IL-6 and IL-8 in the endotoxemic controls significantly exceeded those in the other groups. No significant differences existed between the laparotomy controls and lidocaine-treated rabbits. CONCLUSIONS: Lidocaine had a profound inhibitory effect on the hemodynamic and cytokine responses to endotoxemia when it was administered immediately after exposure to endotoxin. Our results demonstrate the potential usefulness of lidocaine as an anti-inflammatory agent in endotoxemia. PMID- 10752827 TI - Single-breath CO2 analysis as a predictor of lung volume change in a model of acute lung injury. AB - OBJECTIVE: To examine the utility of single-breath CO2 analysis as a measure of lung volume change in a model of acute lung injury. SETTING: Animal laboratory in a university-affiliated medical center. DESIGN: Prospective, animal cohort study comparing 21 variables derived from single-breath CO2 analysis with lung volume measurements determined by nitrogen washout. SUBJECTS: Seven lambs with saline lavage-induced acute lung injury. METHODS: Animals were treated with repetitive saline lavage to achieve a uniform degree of acute lung injury (PaO2 < 100 torr [13.32 kPa] on FiO2 of 1.0). Twenty-one derived components of the CO2 expirogram were evaluated as predictors of lung volume change. Lung volume was manipulated by 3-cm H2O incremental increases in positive end-expiratory pressure from 0 to 21 cm H2O and ranged between 90 and 765 mL. MEASUREMENTS AND MAIN RESULTS: Fifty five measurements of lung volume were available for comparison with derived variables from the CO2 expirogram. Stepwise linear regression identified five variables that were most predictive of lung volume change: a) dynamic lung compliance; b) the slope of phase III; c) the slope of phase II divided by the mixed expired CO2 concentration; d) airway deadspace; and e) PaO2/FIO2 ratio. The multivariate equation was highly statistically significant and explained 94% of the variance (adjusted r2 = .94, p < .0001). The bias and precision of the calculated lung volume were 10.9 and 55.9, respectively. The mean percentage difference for the lung volume estimate derived from the single-breath CO2 analysis station was 3.3%. CONCLUSIONS: Our data indicate that analysis of the CO2 expirogram can yield accurate information about lung volume in animals with saline lavage-induced acute lung injury. Specifically, five variables derived from a plot of expired CO2 concentration vs. expired volume predict changes in lung volume in healthy lambs with an adjusted coefficient of determination of 0.94. We hope to further define the utility of this technique by prospective application of this methodology in the clinical setting. PMID- 10752828 TI - Hepatic and splanchnic oxygen consumption during acute hypoxemic hypoxia in anesthetized pigs. AB - OBJECTIVE: To compare the hepatosplanchnic oxygen consumption (VO2) with the hepatic and splanchnic VO2 and to calculate the critical oxygen delivery (DO2crit) below which VO2 decreases in the hepatic, splanchnic, and hepatosplanchnic regions in a model of hypoxemic hypoxia. DESIGN: Prospective animal study. SETTING: University research laboratory. SUBJECTS: Anesthetized and ventilated pigs (n = 7). INTERVENTIONS: The right carotid artery was cannulated to measure mean arterial pressure. A pulmonary artery catheter was inserted to measure mean pulmonary arterial pressure and cardiac output. After a midline abdominal incision, two flow probes were positioned around the portal vein and the hepatic artery to measure portal vein blood flow and hepatic artery blood flow. Oxygen and lactate contents in the carotid artery, the portal vein, and the hepatic vein were measured in blood samples obtained from the appropriate catheters. MEASUREMENTS AND MAIN RESULTS: After a 2-hr stabilization period, hemodynamic and biological variables were recorded during acute hypoxemic hypoxia (FIO2 = 0.5, 0.4, 0.3, 0.21, 0.15, 0.10, and 0.07). VO2, DO2, and DO2crit were determined in the hepatic, splanchnic, and hepatosplanchnic regions. The hepatosplanchnic VO2 was 48 +/- 5 mL/min at high FIO2 (40% for the liver and 60% for the splanchnic organs) and decreased below FIO2 of 0.15. Lactate uptake in the whole hepatosplanchnic region remained steady at FIO2 values of 0.5 to 0.15 and then switched to a lactate release at low FIO2. However, the splanchnic region released lactate, whereas lactate was taken up by the liver. DO2crit in the hepatic, splanchnic, and hepatosplanchnic regions was 24 +/- 3, 38 +/- 2, and 49 +/- 4 mL/min, but the systemic DO2crit, below which regional VO2 became oxygen supply dependent, did not differ in the liver, splanchnic, and hepatosplanchnic regions. CONCLUSIONS: The variables of oxygenation and lactate flux measured in the hepatosplanchnic region summarize the metabolic changes of various organs that may vary in different ways during hypoxemic hypoxia. PMID- 10752829 TI - Comparison of intratracheal pulmonary ventilation and hybrid intratracheal pulmonary ventilation with conventional mechanical ventilation in a rabbit model of acute respiratory distress syndrome by saline lavage. AB - OBJECTIVES: To study changes in PaCO2 and PaO2 during intratracheal pulmonary ventilation (ITPV) and hybrid intratracheal pulmonary ventilation (h-ITPV) compared with conventional mechanical ventilation (CMV) in a rabbit model of respiratory failure, and to define the technique of h-ITPV that combines conventional mechanical ventilation and ITPV. DESIGN: Prospective, interventional study. SUBJECTS: Twelve adult New Zealand White rabbits. INTERVENTIONS: Surfactant deficiency was induced by saline lavage, and rabbits were randomized to either ITPV or h-ITPV. The study consisted of four phases: phase 0, CMV after saline lavage, ventilator rate 30 breaths/min; phase I, ITPV or h-ITPV initiated at the same pressure and rate as in phase 0; phase II, ITPV or 1.0 L/min h-ITPV bias flow, with peak inspiratory pressure (PIP) decreased and ventilator rate increased to achieve the lowest tidal volume while maintaining adequate gas exchange; and phase III, animals returned to CMV. MEASUREMENTS AND MAIN RESULTS: In phase I, no difference in PaCO2 was observed between ITPV, h-ITPV, or CMV. There was a decrease in PaO2 when switching from CMV to ITPV but not to h-ITPV. In phase II, it was possible to decrease PIP (average of 37% for ITPV and 36% for h-ITPV) and tidal volume (average of 64% for ITPV and 53% for h-ITPV) without compromising gas exchange (p < .05). Oxygenation tended to improve from phase 0 to the end of phase II. In phase III, PaCO2 increased (average of 71% for ITPV and 79% for h-ITPV) and pH decreased (p < .05). Normocapnia was achieved using significantly higher PIP and tidal volume, compared with phase 0 (p < .05). CONCLUSIONS: ITPV and h-ITPV can effectively ventilate and oxygenate rabbits with surfactant-deficient lungs at tidal volumes and therefore pressures lower than required with CMV. Maximum benefit appears to occur at high ventilator rates. These findings suggest that both modes of ventilation may represent powerful new tools in the management of patients with acute respiratory failure. (Crit Care Med 2000; 28:774-781) PMID- 10752830 TI - Diaspirin cross-linked hemoglobin improves mucosal perfusion in the ileum of septic rats. AB - OBJECTIVE: To determine the effect of a bolus infusion of diaspirin cross-linked hemoglobin (DCLHb or hemoglobin crosfumaril) on the ileal mucosal microcirculation in septic rats. DESIGN: Prospective, randomized, single-blinded study. SETTING: University-affiliated animal research laboratory. SUBJECTS: Twenty-four male Sprague-Dawley rats, weighing 320-380 g. INTERVENTIONS: Under inhalational anesthesia, arterial and venous catheters were inserted and sepsis was created by cecal ligation and perforation (CLP). Twenty-four hours later, animals were reanesthetized and ventilated. Via midline abdominal incision, the ileum was mobilized and prepared for intravital microscopy. Post-CLP hemodynamic values were obtained, and videomicroscopy was performed on four to ten villi. Animals were then randomized to receive 2 mL of DCLHb solution (100 mg/mL; n = 12) or pentastarch (n = 12) intravenously, and measurements were repeated after 20 mins. Rats treated with DCLHb then received nitroprusside to restore mean arterial pressure to post-CLP levels, and final measurements were obtained 15 mins later. MEASUREMENTS AND MAIN RESULTS: Cardiac index increased with both treatments (p < .001), whereas systemic vascular resistance index and mean arterial blood pressure were augmented only with DCLHb (p < .0001 compared with pentastarch). Intercapillary areas (ICA; inversely related to capillary density) were determined using computerized image analysis. ICA size decreased after treatment, from 974 +/- 79 to 791 +/- 106 microm2 with DCLHb and from 1044 +/- 90 to 840 +/- 82 microm2 with pentastarch (both p < .05). Red blood cell velocity in terminal arterioles, as assessed by velocimetry from the recorded images, increased by 15% with both treatments (p < .05). Restoration of mean arterial pressure to post-CLP levels in DCLHb animals by nitroprusside infusion abolished the effects of the hemoglobin solution on ICA size and red blood cell velocity. CONCLUSION: Both DCLHb and pentastarch infusion improved microcirculatory perfusion in the ileum of septic rats. In addition, DCLHb also exhibited vasopressor properties, which in combination with improved perfusion may be particularly useful in the treatment of sepsis. PMID- 10752831 TI - Peritoneal cytokine concentrations and survival outcome in an experimental bacterial infusion model of peritonitis. AB - OBJECTIVE: To correlate the dynamics of peritoneal cytokines with systemic concentrations and survival outcome. DESIGN: Randomized, controlled study using a recently developed rat model of peritonitis. SETTING: Government research facility. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Infected animals (INF) received an intraperitoneal infusion of 6.5 x 10(8) colony-forming units of Escherichia coli over 12 hrs, whereas control rats (CON) received a sterile inoculum. Peritoneal fluid and plasma samples were obtained from all rats at the end of the 12-hr infusion period as well as from all animals that survived the 7 day study (SURV). MEASUREMENTS AND MAIN RESULTS: Interleukin (IL)-1beta concentration in the peritoneal fluid at 12 hrs tended to be higher in nonsurvivors (NONSURV) than in SURV. Tumor necrosis factor-alpha and IL-6 peritoneal concentrations at 12 hrs were significantly greater in NONSURV than in SURV. There were no significant differences in IL-2 and IL-4 peritoneal concentrations at 12 hrs between SURV and NONSURV. Although the concentrations of IL-1beta and tumor necrosis factor-alpha in the peritoneal fluid of INF decreased gradually during the study, these concentrations remained significantly higher than those of CON at 7 days. In contrast, peritoneal IL-2 concentrations remained lower in INF than in CON for most of the experiment. Peritoneal IL-6 concentrations in INF were transiently elevated above those of CON for 12 hrs. Cytokine concentrations in the peritoneal fluid of INF were always higher than those in plasma, which remained relatively unchanged throughout the study. For most of the variables as. sessed, CON showed no significant changes compared with INF. CONCLUSIONS: This model of peritonitis is associated with a significant and prolonged peritoneal inflammatory response that is adversely correlated with survival outcome. Our data would suggest that to be effective, novel immunotherapies should target mainly the peritoneal compartment. PMID- 10752832 TI - Additive effects of inhaled nitric oxide and intravenous milrinone in experimental pulmonary hypertension. AB - OBJECTIVE: To determine whether inhaled nitric oxide (IN0) and intravenous milrinone have additive pulmonary vasodilator effects in a rat model of pulmonary hypertension. DESIGN: Prospective, experimental study. SETTING: Animal laboratory of a university medical center. SUBJECTS: Male New Zealand White rabbits. INTERVENTIONS: Anesthetized rabbits were mechanically ventilated and instrumented for measurement of systemic mean arterial pressure (MAP), pulmonary artery pressure (PAP), left atrial pressure, and cardiac output (CO). After baseline measurements, the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (30 mg/kg iv) was administered. Pulmonary hypertension was produced by the continuous infusion of U46619, a thromboxane A2 mimetic. INO (40 ppm) was added to the inspired gas, and hemodynamic measurements were obtained before and after INO. Milrinone was administered sequentially as a 30-mg/kg bolus followed by a 3 microg/kg/min infusion, a 100-mg/kg bolus followed by a 10-microg/kg/min infusion, and a 300-mg/kg bolus followed by a 30-microg/kg/min infusion (M3). Hemodynamic measurements were obtained with and without INO at each dose of milrinone. MEASUREMENTS AND MAIN RESULTS: During U46619-induced pulmonary hypertension, INO decreased PAP and pulmonary vascular resistance (PVR) but did not affect MAP, systemic vascular resistance (SVR), or CO. Milrinone dose dependently decreased PAP, PVR, MAP, and SVR and increased CO. At each dose of milrinone, INO further decreased PVR but not SVR. M3 decreased PVR 49%, and the addition of INO decreased PVR an additional 19% so that PAP and PVR decreased to baseline values. CONCLUSIONS: Milrinone and INO both decrease pulmonary hypertension individually, and the combination produces additive effects. Combination therapy may produce potent and selective pulmonary vasodilation during the treatment of pulmonary hypertension. PMID- 10752833 TI - The hemodynamic effects of inhaled nitric oxide and endogenous nitric oxide synthesis blockade in newborn piglets during infusion of heat-killed group B streptococci. AB - OBJECTIVE: To determine the effects of therapy with inhaled nitric oxide (NO) gas and partial or complete blockade of endogenous NO synthesis with N(omega)nitro-L arginine (L-NA) on the hemodynamic responses to group B streptococci infusion in newborn piglets. DESIGN: Randomized, acute intervention study. SETTING: Animal research laboratory. SUBJECTS: Twenty-five anesthetized piglets younger than 3 days of age divided into five groups. INTERVENTIONS: Heat-killed group B streptococci (GBS) were infused systemically until a 50% increase in pulmonary artery pressure (PAP) was obtained, and the infusion was continued for another 2 hrs. The five groups were designed as follows: group 1, sepsis control: continuous GBS infusion, with two brief trials (10 mins) of inhaled NO given after the initial development of pulmonary hypertension and again 2 hrs later; group 2, continuous inhaled NO: NO was given at 40 ppm for 2 hrs during GBS infusion; group 3, high-dose L-NA pretreatment: 10 mg/kg L-NA bolus followed by 1 mg/kg/min before, and continuing throughout, GBS infusion; group 4, high-dose L NA: same dose as in group 3, but given after the start of the GBS infusion with continuous inhaled NO at 40 ppm; and group 5, low-dose L-NA: 3 mg/kg bolus given after start of GBS infusion with continuous inhaled NO at 40 ppm. MEASUREMENTS AND MAIN RESULTS: The sepsis controls, group 1, had an increase in PAP, which took 15-45 mins to develop, from a mean of 3.4 (SD 0.7) to 5.9 (1.9) kPa (p < .05), at which time the cardiac index had decreased from 169 (28) to 146 (46) mL/kg/min (p < .05). Brief inhaled NO during the early phase decreased PAP to normal. Two hours later, PAP had increased to 6.1 (0.2) kPa and cardiac index had decreased to 88 (31) mL/kg/min. Inhaled NO after 2 hrs decreased PAP to 3.2 (0.5) kPa and increased cardiac index to 106 (44) ml/kg/min (p < .05). Continuous inhaled NO (group 2) ameliorated the deterioration in cardiac index, which at 2 hrs was 140 (30) mL/kg/min (significantly greater than in the sepsis controls) (p < .05). The L-NA-pretreated animals (group 3) had a greater increase in PAP and pulmonary vascular resistance index when GBS infusion was started. PAP increased from 3.0 (0.7) to 7.3 (1.5) kPa within 15 mins, and cardiac index simultaneously decreased to 68 (20) mL/kg/min. Cardiac index subsequently rapidly deteriorated to 48 (21) mL/kg/min, and only one of five animals survived for 2 hrs. Group 4 animals also developed a rapid deterioration in cardiac output, and only two of five survived for 2 hrs. Group 5 animals had results indistinguishable from group 2 animals. CONCLUSION: Pulmonary hypertension and shock resulting from GBS infusion in newborn piglets are much worse if endogenous NO production is completely inhibited. Continuous inhaled NO with or without low-dose L-NA inhibits the decrease in cardiac output. PMID- 10752834 TI - Protective effect of heme oxygenase induction in ischemic acute renal failure. AB - OBJECTIVE: To examine the role of heme oxygenase-1 (HO-1) induction in the recovery of renal function in rats with ischemic acute renal failure. DESIGN: Randomized, masked, controlled animal study. SETTING: University-based animal research facility. SUBJECTS: Sprague-Dawley male rats, weighing 200-250 g. INTERVENTIONS: Anesthetized rats were subjected to bilateral flank incisions, and the right kidney was removed. Renal ischemia was performed by left renal microvascular clamping, followed by reflow of the blood. MEASUREMENTS AND MAIN RESULTS: Ischemia of the kidney in the uninephrectomized rat significantly induced HO-1 messenger RNA, protein, and enzyme activity, reaching a maximum at 6 hrs, which was mediated in part through an increase in microsomal heme concentration. Heat shock protein 70 was induced extremely rapidly, reaching a maximum at 1 hr, suggesting that HO-1 and heat shock protein 70 gene expression are regulated separately. Inhibition of HO activity by tin mesoporphyrin, which resulted in an increase in microsomal heme concentration, significantly exacerbated renal function, as judged by the sustained increase in serum creatinine concentration and extensive tubular epithelial cell injuries. In contrast, animals that did not receive tin mesoporphyrin showed normal creatinine concentration and microsomal heme concentration 24 hrs after reperfusion, as well as restoration of abnormal renal histology. CONCLUSION: These findings indicate that the expression of HO-1 in the ischemic kidney may be critical in the recovery of renal cell function in this animal model. These findings also suggest that H0-1 induction may play an important role in conferring protection on renal cells from oxidative damage caused by heme. PMID- 10752836 TI - Early predictors of outcome in patients receiving hypervolemic and hypertensive therapy for symptomatic vasospasm after subarachnoid hemorrhage. AB - OBJECTIVE: Symptomatic vasospasm after subarachnoid hemorrhage (SAH) is associated with a high incidence of permanent disability and death. For early identification of patients who are at risk for poor outcome, we determined the predictors of outcome in patients with symptomatic vasospasm after SAH. DESIGN: We retrospectively determined the prognostic value of clinical characteristics and computed tomographic scan both at admission and at the time of initiation of hypervolemic and hypertensive therapy. SETTINGS: Neurosciences critical care unit at a University hospital. PATIENTS: A total of 70 consecutive patients who developed symptomatic vasospasm after SAH. INTERVENTION: Treatment with oral nimodipine, hypervolemic therapy, and hypertensive therapy. Angioplasty and intra arterial papaverine were used in patients with vasospasm resistant to standard treatment. MEASUREMENTS AND MAIN RESULTS: Poor outcome, defined as Glasgow Outcome Scale Score of 3-5 at 2 months or discharge, was observed in 32 (46%) patients. In the logistic regression analysis, a Glasgow Coma Scale (GCS) score of < or =11 (odds ratio, 11.0; 95% confidence interval, 3.6-39.3) and hydrocephalus (odds ratio, 4.3; 95% confidence interval, 1.2-18.2) at the time of initiation of hypervolemic and hypertensive therapy were significantly associated with poor outcome. Poor outcome was observed in 91% of the patients who had both a GCS score of < or =11 and hydrocephalus compared with 15% of patients with a GCS score of >11 and no hydrocephalus at the time of initiation of hypervolemic and hypertensive therapy. A GCS score of < or =11 was also independently associated with length of intensive care unit stay (F ratio = 18.0; p = .0011) and hospital stay (F ratio = 9.2; p = .0034) after initiation of hypervolemic and hypertensive therapy. CONCLUSIONS: The results of this study suggest that outcome in patients with symptomatic vasospasm can be effectively predicted by routinely available information, including GCS score at the time of initiation of hypervolemic and hypertensive therapy. This information can be used for selection and stratification of patients in future treatment studies of patients with symptomatic vasospasm. PMID- 10752835 TI - Specific angiotensin II receptor blockage improves intestinal perfusion during graded hypovolemia in pigs. AB - OBJECTIVE: To investigate the potential of specific angiotensin II subtype 1 (AT1) receptor blockade to modify the mesenteric hemodynamic response to acute hypovolemia and retransfusion. DESIGN: Prospective, randomized, controlled experimental study. SETTING: University-affiliated animal research laboratory. SUBJECTS: Fasted, anesthetized, ventilated, juvenile domestic pigs of both sexes. INTERVENTIONS: Acute, graded hypovolemia by 20% and 40% of the total estimated blood volume followed by retransfusion in control animals (CTRL; n = 10) and animals pretreated with the AT1 receptor blocker candesartan (CAND; n = 10). MEASUREMENTS AND MAIN RESULTS: Invasive monitoring of arterial and central venous blood pressures, cardiac output, portal venous blood flow, and jejunal mucosal blood flow. Blood gases were repeatedly analyzed to calculate oxygen delivery and consumption. Thirty minutes after each level of hypovolemia at 20% and 40%, cardiac output was decreased in CTRL animals from a baseline of 2.9 +/- 0.1 to 1.8 +/- 0.2 and 1.1 +/- 0.2 L/min, with no differences compared with CAND animals. Cardiac output was restored to 3.0 +/- 0.3 L/min 30 mins after retransfusion in CTRL animals, with no significant intergroup differences. Baseline portal venous blood flow (Q(MES)) and jejunal mucosal perfusion (PU(JEJ)) were greater in CAND animals compared with CTRL animals. During graded hypovolemia, CAND animals maintained Q(MES) and PU(JEJ) at significantly higher levels compared with CTRL animals, particularly after 40% hemorrhage (+221% and + 244%, respectively, relative to the mean values in CTRL animals). The same pattern was observed after retransfusion. Moreover, the calculated mesenteric critical oxygen delivery was significantly greater in CTRL animals (74 mL/min) compared with CAND animals (34 mL/min). No animals died in the CAND group, whereas four animals died during 40% hypovolemia or retransfusion in the CTRL group. CONCLUSIONS: Specific AT1 blockade before acute hypovolemia significantly ameliorated mesenteric and, in particular, jejunal mucosal hypoperfusion. In addition, cardiovascular stability was improved, and mortality in conjunction with acute hypovolemia and retransfusion could be completely avoided. These findings support a fundamental role of the renin-angiotensin system in the mesenteric response to acute hypovolemia and indicate a substantial interventional potential for candesartan in conjunction with circulatory stress. PMID- 10752837 TI - Outcome of children who require mechanical ventilatory support after bone marrow transplantation. AB - OBJECTIVE: To identify clinically measurable factors that could predict outcome for pediatric patients undergoing mechanical ventilatory support after bone marrow transplant. DESIGN: Cohort study. SETTING: A referral center for bone marrow transplant patients in Seattle, Washington. PATIENTS: Children <17 yrs old who received a bone marrow transplant and subsequently required mechanical ventilatory support for > or =24 hrs between 1983 and 1996. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Data were abstracted from the charts of 121 pediatric patients who received a bone marrow transplant and subsequently required mechanical ventilatory support. A total of 19 patients (16%) survived to be extubated and survived for > or =30 days postextubation. Major risk factors for death included respiratory failure as the reason for endotracheal intubation (4% survival), the presence of pulmonary infection (6% survival), and impairment of more than one organ system (2% survival if more than one organ system was dysfunctional on day 7 postintubation). CONCLUSIONS: Although the prognosis generally is poor among pediatric bone marrow transplant recipients who subsequently require mechanical ventilatory support, there appear to be some groups within this population in whom the likelihood of survival is close to 0. Because the chance of survival was so small for children with dysfunction of more than one organ system on day 7 after intubation, a recommendation to limit medical support for these children could be considered pending the results of other studies. PMID- 10752838 TI - Fentanyl-induced chest wall rigidity and laryngospasm in preterm and term infants. AB - OBJECTIVE: To assess the occurrence of muscle rigidity after fentanyl administration in premature and term neonates. DESIGN: Prospective case series, observational study. SETTING: A university hospital neonatal intensive care unit. PATIENTS: 8/89 preterm and term infants (25-40 wks gestational age) who received fentanyl for perioperative analgesia and sedation or intensive care procedures. INTERVENTIONS: Mechanical or bag mask ventilation and antagonization with naloxone. MEASUREMENTS AND MAIN RESULTS: We observed chest wall rigidity in 8 patients after low dosage of fentanyl (3-5 microg/kg body weight). All patients presented with respiratory distress, hypercapnia, and hypoxemia leading to bradycardia. In two patients, laryngospasm was noted and associated with muscle rigidity, thus making intubation impossible. Naloxone (20-40 microg/kg body weight) reversed the laryngospasm and muscle rigidity immediately, allowing restitution within 1 min. In our patient population, we found fentanyl-induced chest wall rigidity in 4% of neonates after fentanyl administration. CONCLUSION: Even low doses of fentanyl can lead to thoracic rigidity in neonates. Additionally, we observed laryngospasm in two patients and speculate that it might be a variant of muscle rigidity. PMID- 10752839 TI - Repeat computed tomographic scan within 24-48 hours of admission in children with moderate and severe head trauma. AB - OBJECTIVE: To asses the yield and contribution of a routine predetermined repeat head computed tomographic (CT) scan within 24-36 hrs in pediatric patients with moderate to severe head trauma. DESIGN: Records review. SETTING: Five pediatric intensive care units. PATIENTS: We reviewed the charts of 173 consecutive pediatric patients with moderate to severe head trauma (Glasgow Coma Scale score of < or = 11) that survived the first 24 hrs after being admitted to five Israeli trauma centers. Clinical data collected included status at admission, at the time between the first and second CT scans, and after the second scan. Head details of the first, second, and, if performed, third CT scan were collected. Treatment strategy during each period was recorded, including any change in treatment after each CT scan. MEASUREMENTS AND MAIN RESULTS: A total of 47 (27%) of the second CT scans showed new lesions including six intracranial hemorrhages, 17 cases of worsening brain edema, and 18 newly diagnosed brain contusions. However, none of these findings necessitated surgical intervention or any change in therapy. Of the 67 patients who underwent a third CT scan, two cases required surgical intervention because of new findings in the third CT. CONCLUSIONS: A second routine prescheduled head CT scan within 24-36 hrs after admission in pediatric patients with moderate to severe head trauma is unlikely to yield any change in therapy. Clinically and intracranial pressure-oriented CT scan may better select and diagnose patients who require changes in therapy, including surgery. Studies aimed to determine the ideal timing for the second are warranted. PMID- 10752840 TI - Septic emboli caused by vascular catheters after surgery for congenital heart disease. AB - OBJECTIVE: To review the incidence, diagnosis, and management of septic emboli caused by vascular catheters after surgery for congenital heart disease. DESIGN: Retrospective clinical review. All patients were computer registered. Our database includes daily follow-up and every sign of infection registered. SETTING: Pediatric cardiac surgery intensive care unit in a university hospital. PATIENTS: A total of 720 consecutive pediatric cardiac operations performed in 108 neonates and 612 older children from 1995 to 1997 are reviewed. MEASUREMENTS AND MAIN RESULTS: Septic emboli were defined as erythematous non-tender papulonodular hemorrhagic lesions restricted to the limb and distal to the monitoring catheter. Four patients (0.55%) with catheter-related septic emboli after congenital heart surgery were identified, three neonates (0.41%) and one older infant (0.14%). The incidence of catheter-related septic emboli in our patients was significantly higher in the neonatal group compared with older infants (p = .0076; odds ratio=17.45). All infants with catheter-associated septic emboli were severely ill and required prolonged intensive care management postoperatively for periods ranging from 27 to 90 days (mean, 50 days). The catheters involved were in place for periods ranging from 5 to 7 days. All patients were treated by catheter removal and intravenous antibiotics without surgical intervention in the vascular access area. The affected limbs healed well without residual damage. CONCLUSIONS: Septic emboli are a rare complication of infected vascular catheters in neonates and small infants undergoing prolonged postoperative intensive care management (0.55%). They may indicate the source of unexplained sepsis involving mainly Gram-negative bacilli. Generally, treatment consists of removal of the offending catheter and antibiotic administration with no need for surgical intervention. PMID- 10752841 TI - The impact of prematurity: a perspective of pediatric intensive care units. AB - OBJECTIVE: To evaluate the relative resource use of pediatric intensive care unit (PICU) patients who had been born prematurely. DESIGN: Nonconcurrent cohort study. SETTING: Consecutive admissions to 16 voluntary PICUs. PATIENTS: A total of 431 formerly premature patients (FPP) and 5,319 nonpremature patients. INTERVENTIONS: None METHODS: Patients with a history of prematurity and a prematurity-related complication or an anatomical deformity were compared for demographic and resource requirements to a group of non-premature patients by a bivariable logistic regression analysis that controlled for age as a co-morbid factor. RESULTS: Compared with other patients, FPP were younger (34.9 +/- 2.2 months vs. 72.4 +/- 1.0 months; p < .001), readmitted to the PICU more often during the same hospitalization (11.1% vs. 5.5%; p < .001), used more chronic technologies (ventilators, gastrostomy tubes, tracheostomy tubes, and parenteral nutrition; 30.3% vs. 5.6%; p < .001), and had longer lengths of stay (5.98 +/ 0.59 days vs. 3.56 +/- 0.12 days; p = .004). FPP had significantly higher use of ventilators (45.5% vs. 35.0%; p < .007) and lower use of arterial catheters (27.8% vs. 35.9%, p = .006) and central venous catheters (16.9% vs. 20.9%, p = .026) than nonprematures. The need for other PICU resources, including vasopressors, were similar. CONCLUSIONS: FPP used more chronic and acute care resources than patients who were not prematurely born. Continued improvements in neonatal care will influence change in many aspects of the health care system. This will also affect the delivery of care to the current patient base of the PICU. PMID- 10752842 TI - Sedation in the intensive care unit. AB - OBJECTIVE: To describe the goals of sedative use in the intensive care unit and review the pharmacology of commonly used sedative drugs as well as to review pertinent publications in the literature concerning the comparative pharmacology of these drugs, with emphasis on outcomes related to sedation and comparative pharmacoeconomics. DATA SOURCES: Publications in the scientific literature. DATA EXTRACTION: Computer search of the literature with selection of representative articles. SYNTHESIS: Proper choice and use of sedative drugs is based on knowledge of the pharmacology of commonly used agents and is an essential component of caring for patients in the intensive care unit. The large variability in pharmacokinetics and pharmacodynamics in the critically ill make it difficult to directly compare agents. Midazolam provides rapid and reliable amnesia, even when administered for low levels of sedation. Propofol may be useful when deeper levels of sedation and more rapid awakening are required. Lorazepam can be used for long-term sedation in more stable patients if rapidity of effect is not required. Further investigation in assessment of depth of sedation in the critically ill is needed. Continued study of costs, side effects, and appropriate dosing strategies of all sedative agents is needed to answer questions not sufficiently addressed in the current literature. CONCLUSION: An individualized approach to sedation based on knowledge of drug pharmacology is needed because of confounding variables including concurrent patient illness, depth of sedation, and concomitant use of analgesic agents. (Crit Care Med 2000; 28:854-866) PMID- 10752843 TI - Use of a screen log to audit patient recruitment into multiple randomized trials in the intensive care unit. Canadian Critical Care Trials Group. AB - OBJECTIVE: To develop and evaluate a screen log for monitoring enrollment in multiple randomized clinical trials conducted in a single center. SETTING: University-affiliated 20-bed tertiary care medical-surgical intensive care unit (ICU). PATIENTS: Consecutive ICU patients admitted between April 1995 and March 1997. METHODS: We developed a screen log for multicentered studies conducted in our ICU. Using a multiple-project, unicenter perspective, we evaluated the screen log as a tool for monitoring eligibility and enrollment of patients in four multicentered randomized trials focused on stress ulcer prophylaxis, blood transfusion thresholds, immunotherapy for sepsis and mechanical ventilation strategies. RESULTS: The screen log was used as an instrument to monitor trial execution. We recorded all aspects of study enrollment and created a taxonomy of reasons for nonenrollment into each trial. We calculated enrollment efficiency rates and used these data to develop strategies to maximize accrual. The screen log became a communication tool that fostered research-oriented continuous quality improvement initiatives for the management of concurrently conducted randomized trials in our ICU. CONCLUSIONS: Intensivists participating in several clinical trials may be interested in monitoring and maximizing enrollment when conducting multiple studies and understanding the influence of each trial on enrollment into the others. The unicenter, multiple-project screen log is one tool that may help to achieve these goals. PMID- 10752845 TI - Critical care medicine education of surgeons: recommendations from the Surgical Section of the Society of Critical Care Medicine. AB - PERSPECTIVE: The role of surgeons in critical care medicine has a long and esteemed past. The presence of surgeons in intensive care units provides specific insights and perspectives to the care of surgical patients sometimes not fully appreciated by the non-surgical practitioners caring for the same patients. The training and education of surgeons is becoming more complex, fragmented, and lengthy. The knowledge base and skill set required to manage critically ill or injured surgical patients is also becoming more extensive but has the potential of becoming lost in the process of providing the overall educational program for surgical trainees. Simultaneously, nonsurgical specialties are continuing to train individuals with special skills in critical care medicine and the concept of "hospitalists" is becoming more accepted by institutions across the United States. The certification exams in critical care medicine remain under the aegis of the individual medical specialty boards, and there is still not a unified examination process in critical care. Surgeons, in particular, have tremendous pressures these days to spend more clinical time in the operating room, and the task of consistently conducting high quality research is also becoming arduous. This list of reasons could continue but are simply examples for why surgeons need to spend focused attention on how best to train and educate upcoming surgical trainees in regards to the principles of critical care medicine. The critically ill or injured patients need this focused attention and the specialty of surgical critical care medicine needs this attention. The Surgical Section of the Society of Critical Care Medicine has developed this position statement in the hopes that ongoing discussion and refinement of this particular aspect of surgery will continue on several levels. PMID- 10752844 TI - A cost-effectiveness analysis of the application of nitric oxide versus oxygen gas for near-term newborns with respiratory failure: results from a Canadian randomized clinical trial. AB - OBJECTIVE: To conduct a cost-effectiveness analysis of the use of inhaled nitric oxide (NO) vs. oxygen administered to near-term (gestational age > or =34 wks) newborns with severe respiratory illness that were referred for consideration of extracorporeal membrane oxygenation (ECMO). DESIGN: The cost-effectiveness analysis is based on outcome and utilization data from two multicentered randomized clinical trials conducted by the Canadian Inhaled Nitric Oxide Study group, one for patients with congenital diaphragmatic hernia (CDH) and one for patients without CDH. Data from the western Canadian ECMO center were used to establish costs. SETTING: Patients were cared for in Canadian regional neonatal intensive care units, including two ECMO centers. Air transport was used for transporting patients between centers. PATIENTS: Term and near-term newborns with severe respiratory illness who were receiving maximum conventional therapy and whose oxygenation index was >40. INTERVENTIONS: Patients randomly received NO or oxygen. If their conditions deteriorated, they qualified for ECMO. Not all that qualified for ECMO received it because of individual parent/ physician preferences. MEASUREMENTS AND MAIN RESULTS: The cost-effectiveness ratio was the ratio of net cost (including neonatal intensive care, ECMO, and transport) to net outcome (survival) for the two interventions. For non-CDH cases, the cost effectiveness ratio was $36,613 (Canadian) per life saved; the confidence intervals were wide and the results were not statistically significant. For CDH patients, the death rate was lower for oxygen and the oxygen patients cost less; the results were not statistically significant. CONCLUSIONS: The small numbers of patients in the trials precluded significant results. Further, our results have a short-term time horizon (discharge to home or death). Thus, for non-CDH patients, the favorable ratio provides very qualified evidence in favor of NO. PMID- 10752846 TI - The right heart catheter: leaner, fitter patient management. PMID- 10752847 TI - Microvascular leakage in sepsis. PMID- 10752848 TI - Is it time to use blood volume measurements as a clinical tool? PMID- 10752849 TI - Immunonutrition comes of age. PMID- 10752850 TI - Energy expenditure and the liver: acute fulminant hepatitis. PMID- 10752851 TI - Naloxone therapy in shock: the controversy continues. PMID- 10752852 TI - Guidelines, pathways, and the end result. PMID- 10752853 TI - Pulmonary artery catheter under the microscope. PMID- 10752854 TI - Regardless of origin, uncontrolled hemorrhage is uncontrolled hemorrhage. PMID- 10752855 TI - Hemoglobin solutions: an "all-in-one" therapeutic strategy in sepsis? PMID- 10752856 TI - Combination therapy for pulmonary hypertension: a glimpse into the future? PMID- 10752857 TI - Improving natural selection. PMID- 10752858 TI - Mechanical ventilation for bone marrow transplant patients: when does it become futile? PMID- 10752859 TI - Has the increased survival of premature infants affected resource utilization in pediatric intensive care units? PMID- 10752860 TI - Inhaled nitric oxide: plenty of data, no consensus. PMID- 10752861 TI - Immune-enhancing diets. PMID- 10752862 TI - Does immunonutrition make an impact? It depends on the analysis. PMID- 10752863 TI - Low-dose dopamine: what benefit? PMID- 10752864 TI - Potential carry-over effect because of non-randomization of intervention order. PMID- 10752865 TI - Sedation of children by adult intensivists. PMID- 10752866 TI - Single kidney outcome and management in persons with spinal cord injury. AB - This case study examined the outcomes of persons with spinal cord injury (SCI) who had a single kidney. A Urologic Database, including 1655 persons with SCI between 1969 and 1997, was examined and 22 persons were identified with single kidneys. Twenty persons had adequate follow-up. Renal function was measured by total and individual kidney effective renal plasma flow (ERPF). Of 11 persons who had a single kidney prior to injury or as a result of an associated injury, all maintained a normal ERPF for an average of 8.6 years. Of 9 persons who had removal of a kidney following their injury for other diseases or urinary complications, 3 were deceased, but 2 had a normal ERPF in the remaining kidney prior to death. One with vesicoureteral reflux had decreased renal function in the remaining kidney. Recurrent renal calculi in a single kidney carries risks for decreasing renal function, urosepsis, and death. PMID- 10752867 TI - Latex allergy in adults with spinal cord injury: a pilot investigation. AB - Latex allergy is a well-known complication of repeated exposure to natural rubber latex (NRL) products. The medical literature is replete with studies investigating the prevalence of NRL allergy in myelodysplastic children. However, the prevalence of NRL allergy in adults with spinal cord injury (SCI) has received little attention. Patients with a history of NRL exposure secondary to long-term indwelling urinary catheter usage were recruited as subjects. The presence of NRL allergy was established using the radioallergosorbent assay technique (RAST). Serum from 15 subjects who had been injured an average of 23.8 +/- 11.9 years and who had used an indwelling urinary catheter an average of 17.1 +/- 11.5 years was obtained. RAST for NRL was positive in 7 of the 15 (47%). Of note, serum obtained in a control group of 4 subjects who had been injured an average of 54.4 +/- 3.1 years and had no significant history of long-term indwelling urinary catheter usage were all RAST negative. This study suggests that adults with SCI and significant NRL exposure via long-term indwelling urinary catheter usage may be at risk for the development of NRL allergy. PMID- 10752868 TI - Systemic lupus erythematosus: a unique cause of central cord syndrome, a case report. AB - Central cord syndrome has classically been defined by disproportionately more motor impairment of the upper than the lower extremities, bladder dysfunction, and varying degrees of sensory loss below the level of the lesion. Review of the literature indicates that the majority of causes of central cord syndrome have a traumatic etiology and few are a consequence of connective tissue disorders. This is a case of a 53-year-old female with systemic lupus erythematosus who developed central cord syndrome due to an exacerbation of her disease. The patient presented with upper extremity motor and sensory deficits but only sensory deficits of the lower extremities. She had laboratory abnormalities consistent with lupus, and magnetic resonance imaging of the cervical spine revealed decreased attenuation from C-1-T-2. High-dose intravenous steroids were started in the acute care hospital with some return of proximal upper extremity strength. Upon transfer to the acute rehabilitation unit, the patient was noted to have neurogenic bladder dysfunction requiring a catheterization program. The patient was slowly tapered down on her oral prednisone with minimal improvement of strength but with return of bladder function. A review of the literature revealed few cases of central cord syndrome secondary to a connective tissue disorder or a non-traumatic etiology. PMID- 10752869 TI - Vertebral osteomyelitis in paraplegia: incidence, risk factors, clinical picture. AB - PURPOSE: Because vertebral osteomyelitis (VOM) appears to be relatively common among patients paralyzed due to myelopathy, a population of these patients was surveyed to A) estimate the incidence of VOM, B) identify risk factors, and C) review clinical features. METHOD: The cohort of 537 patients with myelopathy paralyzed prior to January 1, 1989 and subsequently hospitalized at the authors' facility over an 11-year survey period was surveyed. All were male, 93% had a traumatic etiology of paralysis, 50% were > or = 50 years of age, 53% were paraplegic, 54% were motor and sensory complete, and 56% had been paralyzed for 15 years or more. VOM was diagnosed by computerized tomographic demonstration of intervertebral end plate destruction and a positive culture of that site. The relative incidences of VOM were calculated as cases per spinal cord injury (SCI) and general hospitalizations; potential risk factors were assessed by rates of VOM in the myelopathy cohort and clinical features of VOM in these patients were noted. RESULTS: Ten patients with myelopathy developed VOM, an incidence of 20.9 cases/10,000 SCI versus 1.8 cases/10,000 general hospitalizations. The risk ratio was 7.1 (p = .05) for paraplegia, 7.9 (p = .04) for motor and sensory completeness, and 4.4 (p = .06) for diabetes mellitus. The risk ratio for the 3 factors combined was 14.5 (p = .001). Initial clinical presentations were notable for back pain and plain radiographic films revealing paraspinal masses and pleural effusion. CONCLUSIONS: Patients with myelopathy are uniquely at risk for VOM if their paralysis is at the paraplegic level and is motor and sensory complete. PMID- 10752870 TI - Gastroesophageal reflux disease in patients with spinal cord injury. AB - Due to certain risk factors that pertain to patients with spinal cord injury (SCI), the prevalence and severity of gastroesophageal reflux disease (GERD) may be greater among patients with SCI than the general population. In this retrospective study, the prevalence of GERD in patients with SCI as compared with age- and sex-matched controls, using pharmacy data from a large health care system, and the adequacy of the diagnostic evaluation process are evaluated and the symptom and disease severity in patients with SCI who were treated for GERD are assessed. The findings show a 22% prevalence of GERD in patients with SCI versus 28% in General Medicine Clinic controls. Although the symptom severity is similar in patients with SCI and controls, patients with SCI who have GERD symptoms undergo endoscopy less frequently. Disturbingly, the prevalence of severe, stage IV esophagitis among patients with SCI is higher than controls (p = .03). These results should alert clinicians caring for patients with SCI to more aggressively evaluate and treat such patients with reflux symptoms of heartburn and regurgitation before the development of complications. PMID- 10752871 TI - An unusual complication during electroejaculation in an individual with tetraplegia. AB - Infertility in males is a common problem after spinal cord injury, often requiring interventional methods. In the authors' fertility program they have experienced 4 pregnancies and 3 live births using both electroejaculation (EEJ) and vibratory stimulation techniques. EEJ is a relatively safe procedure with few complications reported in the literature. Here is a case of a 23-year-old with C 6 ASIA A tetraplegia who, while receiving EEJ, developed autonomic dysreflexia that was followed by new onset atrial fibrillation. The patient's cardiac condition required management in the Emergency Department with pharmacologic conversion to a normal sinus rhythm. Although autonomic dysreflexia is a common adverse effect during EEJ, atrial fibrillation has not been previously reported as a complication. It is proposed that the patient's atrial fibrillation developed as a result of cardiac changes induced through autonomic dysreflexia. PMID- 10752872 TI - Systemic administration of domoic acid-induced spinal cord lesions in neonatal rats. AB - Domoic acid (Dom) is a glutamate analog and a seafood toxin that has caused neurological disturbance and death in humans. Brain lesions caused by Dom have been documented in the literature, but the effect of Dom on the spinal cord has not been investigated as extensively. Systemic administration of glutamate agonists (i.e., homocysteate, kainate, and a-amino-3-hydroxy-5-methylisoxazole-4 propionic acid) caused spinal cord lesions in infant rats. In the present study, the toxic effects of Dom on the developing spinal cord are examined. Neonatal rats on Postnatal Day 7 were administered Dom subcutaneously at doses of 0.10, 0.17, 0.25, 0.33, 0.42, and 0.50 mg/kg, respectively. Motor seizures characterized by scratching, tail flicking, and swimming-like movement were induced by Dom at all doses. High doses of Dom (> or = 0.33 mg/kg) further induced a hindlimb paralysis, a forelimb tremor, and death that occurred in less than 2 hours. The percentages of death and paralysis induced by 0.33 mg/kg Dom were 47% and 65%, respectively (n = 17). At this dose, electrocorticogram was recorded and synchronized interrupted electrical activities in brains of these animals were detected. However, no brain damage was detected in these rats. Spinal cord lesions characterized by focal hemorrhage, neuronal swelling, and neuronal vacuolization were found in 73% of the animals that had shown the paralysis/tremor in their extremities, as examined 1 to 2 hours after Dom injection. These lesions were seen at all spinal cord levels. Neuronal degeneration was mainly found in the ventral and intermediate gray matter, whereas cells in the dorsal portion of the spinal cord were relatively spared. Data suggest that observed behavioral changes were due to spinal cord damage rather than seizures or brain lesions. PMID- 10752873 TI - The effect of etidronate on late development of heterotopic ossification after spinal cord injury. AB - Forty patients with spinal cord injury (SCI) and heterotopic ossification (HO) were treated with etidronate and followed after therapy to determine the effects of long-term medication on heterotopic bone formation. Eighteen patients had tetraplegia and 22 had paraplegia. Early diagnosis of HO (positive bone scintigraphy and negative radiographic findings of HO) was established by 3-phase bone scintigraphy using 99m technetium-labeled methylene diphosphonate. All patients underwent treatment with etidronate, first with intravenous administration of 300 mg/day for 3 days followed by an oral administration of 20 mg/kg/day for 6 months. Eleven patients (27.5%) developed radiographic evidence of HO from 1.5 to 6 years after therapy. A low degree of HO was found in these patients; 8 had grade I and 3 had grade II ectopic ossification (Brooker's scale). The analysis of data showed that 2 different types of ectopic bone may form in the later stages after SCI. In 5% of patients, HO was found in the same anatomical site initially and finally, suggesting a "rebound" in mineralization of bone matrix not prevented by the administration of etidronate. The other type of HO was found in the majority of patients (95%) where the localization of HO showed different involvement of joints than initially, indicating de novo appearance of HO following SCI. The data suggest that etidronate given for a prolonged period in higher doses has, in addition to an inhibitory effect on crystal formation, a cellular effect on bone-forming cells. PMID- 10752874 TI - Suppression of uninhibited rectal detrusor by functional magnetic stimulation of sacral root. AB - PURPOSE: To demonstrate the effect of sacral magnetic stimulation (MS) on the uninhibited rectal detrusor aiming at suppression of unstable rectal contractions. METHODS: The study was comprised of 6 patients (3 women, 3 men, ages between 32 and 49 years) with supraconal spinal cord injury who complained of fecal incontinence. Rectal sensation for defecation was felt after the onset of involuntary detrusor contraction in 4 patients and not perceived in 2. Rectal and rectal neck pressures were normal. Rectal hyperreflexia was provoked by rapid saline infusion into the rectum (100 to 150 ml; 15 ml/s). Sacral MS was effected by a magnetic coil and a stimulator. The coil was applied to the back between L-4 and L-5. RESULTS: During the provocative saline test, the rectal pressure showed a significant increase (p < .001) and the subjects evacuated the infused saline involuntarily; the rectal neck pressure showed no significant change (p > .05). Intermittent MS during the induced rectal pressure rise using 70% of maximal intensity (i.e., 175 Joules per pulse, 40 Hz frequency, and 2 second burst length with 2 seconds off) affected rectal pressure decline (p < .01) and no saline evacuation. CONCLUSIONS: Sacral MS produced inhibition of provoked rectal hyperreflex contractions in patients with supraconal cord injuries. The method is simple, easy, noninvasive, and with no adverse effects. PMID- 10752875 TI - Spinal cord injury: facts and figures at a glance. PMID- 10752876 TI - Visualization modes for CT colonography using cylindrical and planar map projections. AB - PURPOSE: The purpose of this study was to demonstrate the limitations to the effectiveness of CT colonography, colloquially called virtual colonoscopy (VC), for detecting polyps in the colon and to describe a new technique, map projection CT colonography using Mercator projection and stereographic projection, that overcomes these limitations. METHOD: In one experiment, data sets from nine patients undergoing CT colonography were analyzed to determine the percentage of the mucosal surface visible in various visualization modes as a function of field of view (FOV). In another experiment, 40 digitally synthesized polyps of various sizes (10, 7, 5, and 3.5 mm) were randomly inserted into four copies of one patient data set. Both Mercator and stereographic projections were used to visualize the surface of the colon of each data set. The sensitivity and positive predictive value (PPV) were calculated and compared with the results of an earlier study of visualization modes using the same CT colonography data. RESULTS: The percentage of mucosal surface visualized by VC increases with greater FOV but only approaches that of map projection VC (98.8%) at a distorting, very high FOV. For both readers and polyp sizes of > or =7 mm, sensitivity for Mercator projection (87.5%) and stereographic projection (82.5%) was significantly greater (p < 0.05) than for viewing axial slices (62.5%), and Mercator projection was significantly more sensitive than VC (67.5%). Mercator and stereographic projection had PPVs of 75.4 and 78.9%, respectively. CONCLUSION: The sensitivity of conventional CT colonography is limited by the percentage of the mucosal surface seen. Map projection CT colonography overcomes this problem and provides a more sensitive method with a high PPV for detecting polyps than other methods currently being investigated. PMID- 10752877 TI - Focal lesions in the cirrhotic liver: high resolution ex vivo MRI with pathologic correlation. AB - Cirrhosis is a progressive, diffuse process of liver fibrosis that is characterized by architectural distortion and the development of a spectrum of nodules ranging from benign regenerative nodules to premalignant dysplastic nodules to overtly malignant hepatocellular carcinoma. The purpose of this essay is to demonstrate the ex vivo MR and pathology findings of these nodules as well as other masses that can be seen in the cirrhotic liver. The optimal conditions under which ex vivo imaging can be performed allow greater spatial resolution than that achieved with in vivo imaging, without artifacts that may degrade image quality. Clearly, contrast-enhanced MRI is essential for both the diagnosis and the characterization of focal lesions in the cirrhotic liver. However, the use of ex vivo imaging precludes the evaluation of these important in vivo pulse sequences. PMID- 10752878 TI - Hepatic metastases from breast carcinoma: comparison of noncontrast, arterial dominant, and portal-dominant phase spiral CT. AB - PURPOSE: The purpose of this work was to determine the relative value of noncontrast (NC), arterial-dominant (AD), and portal-dominant (PD) phase images in spiral CT of the liver for breast cancer metastases. METHOD: Forty-four spiral CT scans in 18 patients with hepatic metastases from breast cancer were retrospectively reviewed by three radiologists. Subjective evaluations of overall lesion conspicuity and margination were graded on a 5 point scale for NC, AD, and PD phase images, and the three phases were also ranked for demonstration of overall tumor volume. Those scans with hypervascular lesions were separately analyzed, resulting in three groups (all, hypervascular, hypovascular). RESULTS: For lesion conspicuity and margination for the entire study group, AD phase images showed the lowest grades (1.97 and 1.83), whereas the PD phase showed the highest grade (3.34 and 3.14; p < 0.0001) followed by NC (2.36 and 2.42; p < 0.0001). For the hypervascular subgroup, the AD phase also showed the lowest grades (2.39 and 2.24). In no case did the AD phase show more lesions than the combination of NC and PD phases. For depiction of overall tumor volume, the AD phase had the lowest ranking (2.51) compared with the NC and PD phases (1.71 and 1.78; p < 0.001). For the hypervascular subgroup, the AD phase had the lowest ranking (2.33) compared with the NC and PD phases (1.39 and 2.27; p < 0.0001). CONCLUSION: The AD phase is not required for lesion detection in spiral CT for hepatic metastases from breast carcinoma. The NC phase depicts the maximal tumor volume. PMID- 10752879 TI - Signal characteristics of focal liver lesions on double echo T2-weighted conventional spin echo MRI: observer performance versus quantitative measurements of T2 relaxation times. AB - PURPOSE: The purpose of this work was to evaluate the ability of expert readers to differentiate benign from malignant liver lesions based on visual assessment of lesion signal intensity on double echo T2-weighted conventional spin echo (CSE) MR images and to compare reader performance with quantitative measurements of T2 relaxation times. METHOD: Sixty-seven MR examinations demonstrating 85 liver lesions (37 hemangiomas, 32 malignancies, 15 cysts, and 1 focal nodular hyperplasia) on double echo T2-weighted CSE sequences (TR 3,600 ms/TE 50, 160 ms) were qualitatively reviewed by three independent readers. T2 relaxation times were calculated for each lesion. Receiver operating characteristic (ROC) analyses of expert readers were compared with calculated T2 relaxation times. RESULTS: T2 values performed significantly better than subjective reader analysis for liver lesion characterization (area under ROC = 0.93 vs. 0.81, 0.78, and 0.75; p < 0.0001). With use of a T2 threshold of 125 ms, the sensitivity of T2 values for malignant lesions was 100%, specificity 71%, and accuracy 84%. By comparison, the sensitivity of the three readers for malignant lesions was 76-83%, with a specificity of 61-72% and an overall accuracy of 71-80%. CONCLUSION: Despite expert reader analyses, subjective evaluations of liver lesion signal characteristics are prone to inaccuracy and lack certainty and consistency when intermediate TEs (50/160 ms) are used. Quantitative measurements of T2 relaxation times should be performed to accurately and confidently differentiate benign from malignant liver lesions. Use of a higher T2 threshold than previously recommended is required to avoid misclassification of malignancies. PMID- 10752880 TI - Nodular hepatocellular carcinoma: variation of tumor conspicuity on single-level dynamic scan and optimization of fixed delay times for two-phase helical CT. AB - PURPOSE: The purpose of this work was to determine the optimal delay times in two phase helical CT for nodular hepatocellular carcinoma (HCC). METHOD: Twenty-four patients with nodular HCC (size 2.1-6.7 cm, mean 4.2 cm) were divided into three groups to undergo single-level dynamic CT with 150 ml of contrast material (iodine load of 45 g) at a rate of 3 ml/s. CT acquisition started 10, 30, or 60 s after the injection for each group, respectively, and lasted for 110 or 120 s. The optimal 20 s windows that allowed a tumor-to-liver contrast of >10 HU were determined in the pooled tumor-to-liver contrast curve. RESULTS: The determined temporal windows were 36-56 and 130-150 s, respectively. However, each window was not appropriate in seven (33%) and five (36%) patients because of the individual variations of the contrast curve. CONCLUSION: There is no optimal fixed delay time that is appropriate in all individual patients. The best delay times are 36 and at least 130 s with our injection protocol. PMID- 10752881 TI - Helical CT of the liver with computer-assisted bolus-tracking technology: scan delay of arterial phase scanning and effect of flow rates. AB - PURPOSE: The purpose of this work was to assess the scan delay and the effect of flow rates on arterial phase scanning of hepatic CT. METHOD: One hundred twenty patients suspected of having hepatocellular carcinoma were examined by three phase helical CT using computer-assisted bolus-tracking technology. We set the region of interest (ROI) in the abdominal aorta at the level of the celiac artery as a baseline. The triggering threshold was set at 100 HU. A volume of 100 ml of iomeprol (350 mg of I/ml) was administered at 2, 2.5, or 3 ml/s i.v. RESULTS: In all cases, helical CT scanning began after reaching the ROI threshold. Then, portal venous phase scanning was initiated 50 s after arterial phase initiation. The mean delay time from the initiation of contrast agent administration to the beginning of arterial phase scanning was 29.2 +/- 3.8 s (mean +/- SD, range 22-39 s). A faster injection rate significantly shortened the scan delay (p < 0.01). In portal venous phase scanning, calculated areas under the hepatic enhancement curves were almost equal among different injection rates. CONCLUSION: The computer-assisted bolus-tracking technology is a useful method for determining an individual scan delay of arterial phase CT. PMID- 10752882 TI - CT-guided aspiration core needle biopsy of gastrointestinal wall lesions. AB - PURPOSE: The purpose of this work was to examine the accuracy and safety of CT guided core biopsy of gastrointestinal wall lesions. METHOD: We reviewed over 1,200 CT-guided abdominal core biopsy results from 1989 through 1998. Forty-five were from gastrointestinal wall lesions (16 gastric, 3 duodenal, 7 small intestine, and 19 colon). RESULTS: A definitive histologic diagnosis was obtained from the core biopsy in 41 patients (91%) without complication. The gastric lesion diagnoses were mesenchymal tumor of smooth muscle origin (eight), lymphoma (one), adenocarcinoma (three), and normal (one). Duodenal core biopsy diagnoses were inflammation (one) and normal (one). Small intestinal core biopsy diagnoses were non-Hodgkin lymphoma (four), metastatic leiomyosarcoma (one), carcinoma (one), and tuberculosis (one). Colon core biopsy diagnoses were carcinoma (11), lymphoma (2), actinomycosis (2), granulomatous inflammation (1), metastatic squamous cell carcinoma (1), chronic abscess (1), and mesenchymal tumor of smooth muscle origin (1). CONCLUSION: Percutaneous CT-guided core needle biopsy from gastrointestinal wall lesions is safe and accurate, especially in submucosal lesions, and should be considered in selected cases with negative endoscopic biopsy or in which endoscopic biopsy is not possible. PMID- 10752883 TI - MR cholangiopancreatography before and after oral blueberry juice administration. AB - Blueberry juice was used in 37 patients who underwent MR cholangiopancreatography (MRCP) examination. Quantitative and qualitative comparison between pre- and post blueberry juice oral administration was made. Common bile duct and main pancreatic duct depiction improved statistically significantly (p < 0.05) after blueberry juice administration. Blueberry juice may be used routinely in MRCP studies as a natural, safe, and inexpensive negative contrast agent with high efficacy. PMID- 10752884 TI - CT manifestations of respiratory syncytial virus infection in lung transplant recipients. AB - PURPOSE: The purpose of our study was to evaluate CT findings during respiratory syncytial virus (RSV) infection in lung transplant recipients and to identify sequelae. METHOD: Thirty-nine CT scans prior to, during, and following acute infection in 10 lung transplant recipients were reviewed. Abnormalities that were new from baseline observations and occurred within 4 weeks of diagnosis were defined as acute. Chronic findings were defined as those present >4 weeks after diagnosis. RESULTS: Findings in nine patients were ground-glass (seven), air space (five), and tree-in-bud (four) opacities and acute bronchial dilatation (four) and wall thickening (four). Patients lacked pleural effusions or lymph node enlargement. Five of seven patients with follow-up exams had new air trapping (three), persistent bronchial dilatation (three), and thickening (two). Three and 2 of the 10 patients developed bronchiolitis obliterans syndrome and obliterative bronchiolitis, respectively. CONCLUSION: During acute infection, patients commonly had ground-glass opacities but lacked pleural effusions and lymph node enlargement. There can be chronic sequelae after infection. PMID- 10752885 TI - Videotaped helical CT images for lung cancer screening. AB - PURPOSE: The goal of this work was to determine a radiologist's ability to detect solitary pulmonary nodules on helical CT using both video (cine) viewing and film based viewing. METHOD: Sixty-five chest helical CT studies were reviewed. Six radiologists searched for 40 lung nodules on CT images presented in three formats. Film-based viewing of images at 10 and 5 mm increments was performed with a light box. Video viewing of the same examinations was performed in 5 mm increments at 2 frames/s. The area under the receiver operating characteristic curve (Az) measured the observer's ability to detect nodules. RESULTS: The Az was 0.948 for the video viewing, 0.844 for 5 mm increment film-based viewing, and 0.879 for 10 mm increment film-based viewing. There were no statistically significant differences. CONCLUSION: Lung nodules can be detected with similar detection rates when viewing conventional film or videotaped helical CT images. Videotaped images incur a lower cost, an important consideration in mass screening for lung cancer. PMID- 10752886 TI - Dynamic and quantitative assessment of tracheomalacia by electron beam tomography: correlation with clinical symptoms and bronchoscopy. AB - PURPOSE: The purpose of this study was to evaluate the performance of electron beam tomography (EBT) in the dynamic and quantitative assessment of tracheal collapse in tracheomalacia. METHOD: Eight patients with suspected tracheomalacia were evaluated by EBT, and the results were correlated with both clinical symptoms and bronchoscopy. To validate this technique, an EBT phantom study preceded the patients' examination. A dynamic imaging sequence consisting of 20 50-ms scans obtained at 0.5 s intervals during a 10 s period was performed while the patient followed an instructed breathing maneuver. RESULTS: Good correlation between EBT data and clinical symptoms was found in all patients. In comparison to bronchoscopic findings, EBT results correlated well in three, showed limited correlation in three patients, and no correlation in one case. CONCLUSION: EBT with its short scanning time may be regarded as an accurate noninvasive method to dynamically evaluate tracheomalacia. PMID- 10752887 TI - Coronary artery motion in electron beam tomography. AB - PURPOSE: The purpose of this work was to evaluate coronary artery motion characteristics and determine optimal electron beam tomography (EBT) scan time during the cardiac cycle to image the coronary arteries. METHOD: This study evaluated the movement of coronary arteries in 20 EBT cine studies, at rest and during stress, obtained for evaluating coronary artery disease. The proximal, middle, and distal segments of each coronary artery were measured at multiple times during the cardiac cycle. The motion distance (mm) and velocity (mm/s) of each segment of the coronary arteries were then measured to establish the motion that occurs in the x and y axes during different times in the cardiac cycle. RESULTS: Coronary artery velocity ranged from 22.4 to 108.6 mm/s. The least motion (and slowest speed) occurred between 30-50 and 40-60% of the R-R interval at rest and stress, respectively. The right coronary artery moved the greatest in the x and y planes (highest speed and spatial change), followed in decreasing order by the circumflex, left main, and left anterior descending arteries. The phase of the cardiac cycle with the greatest coronary artery motion was between 0 and 20% of the R-R interval. CONCLUSION: Coronary artery motion varies greatly throughout the cardiac cycle. To minimize cardiac motion during tomographic imaging of the coronary arteries, we recommend 40-50% R-R interval as an electrocardiographic trigger time and avoiding the use of image acquisition times of >100 ms. PMID- 10752889 TI - A case of localized solitary microscopic honeycombing in the lung: an unusual appearance on CT. PMID- 10752888 TI - Nitrofurantoin-induced lung disease: two cases demonstrating resolution of apparently irreversible CT abnormalities. AB - A reticular pattern on high resolution CT (HRCT) invariably represents significant lung pathology and is the dominant feature of irreversible fibrosis. We present two cases of nitrofurantoin-induced pulmonary toxicity in which the initial HRCT showed a widespread reticular pattern and associated distortion of the lung parenchyma, thought to represent established fibrosis. Follow-up HRCT scans after withdrawal of the drug showed resolution of this supposedly irreversible pattern. PMID- 10752890 TI - Percutaneous CT-guided biopsy: improved confirmation of sampling site and needle positioning using a multistep technique at CT fluoroscopy. AB - We describe a new multistep procedure for CT fluoroscopy-guided core biopsy, which allows confirmation of the biopsy-sampling trough and the final needle tip position prior to sampling. Twelve lesions measuring < or =2 cm or in close proximity to vital structures were biopsied using commercially available biopsy systems. Diagnostic biopsies were obtained in all cases. Mean fluoroscopy time was 11.1 +/- 2.8 s per biopsy. This technique may have the potential to improve the accuracy and safety of CT-guided biopsy. PMID- 10752891 TI - Helical CT for the evaluation of suspected acute pulmonary embolism: diagnostic pitfalls. AB - Helical CT is being increasingly utilized for the evaluation of suspected pulmonary embolism (PE). Proper scan interpretation depends on the awareness of several diagnostic pitfalls that may simulate PE, including normal bronchovascular structures such as pulmonary veins, bronchi, and lymph nodes, technical considerations such as improper bolus timing and streak artifacts, and patient-related factors such as motion artifacts, pulmonary arterial catheters, and vascular shunts. An understanding of these pitfalls facilitates accurate diagnosis. PMID- 10752893 TI - Pseudotumoral bilateral involvement of the breast in Erdheim-Chester disease: CT appearance. AB - We report a case of pseudotumoral involvement of the breast in Erdheim-Chester disease. CT shows an enlargement of both breasts with inhomogeneous structure, microcalcifications, and foci of fatty density. PMID- 10752892 TI - Comparison of FDG-PET with MIBI-SPECT in the detection of breast cancer and axillary lymph node metastasis. AB - PURPOSE: The purpose of this work was to compare [18F]2-deoxy-2-fluoro-D-glucose (FDG) PET and 99mTc-methoxyisobutylisonitrile (MIBI) SPECT in the detection of breast cancer and axillary lymph node metastasis in the same patients. METHOD: FDG-PET and MIBI-SPECT were performed within 3 days for 40 women (age range 25-86 years old) with suspected breast cancer, in whom biopsies and/or mastectomies were performed. Both images were visually assessed, and the count ratio between tumor and normal tissue (T/N ratio) was calculated. RESULTS: Thirty-eight patients had breast cancer, and the remaining two had benign breast lesions. The sensitivities of FDG-PET and MIBI-SPECT were 78.9 and 76.3% for breast cancer and 50.0 and 37.5% for axillary lymph node metastasis, respectively. The T/N ratio of breast cancer was significantly higher in FDG-PET (6.01 +/- 3.08 mean +/- SD) than that in MIBI-SPECT (3.48 +/- 1.21) (p = 0.01). Nonmalignant diffuse uptake of FDG in the breasts and the accumulation of MIBI in heart and liver occasionally obscured tumor uptake. CONCLUSION: These results indicate that MIBI SPECT is comparable with FDG-PET in detecting breast cancer. Neither FDG-PET nor MIBI-SPECT is sufficiently sensitive to rule out axillary lymph node metastasis. PMID- 10752894 TI - Screening for avascular necrosis of the hip with rapid MRI: preliminary experience. AB - PURPOSE: The purpose of this work was to compare rapidly acquired MR images with routinely employed SE and turbo SE (TSE) images in screening for hip avascular necrosis (AVN). METHOD: Twelve patients with findings suspicious for radiographically occult AVN of one or both hips were studied with our routine screening protocol (imaging time >7 min) and similarly weighted, rapidly acquired MR sequences (imaging time <1 min). RESULTS: The rapidly acquired MR images were judged to be similar to the routine protocol in demonstrating marrow edema, irregular lines within the femoral head characteristic of AVN, and osteoarthritis. CONCLUSION: The rapidly acquired MR sequences that we studied reliably revealed the presence or absence of AVN, marrow edema, and osteoarthritis of the hip in our sample population when compared with SE and TSE sequences that we routinely perform. Further investigation of rapidly acquired MR sequences is warranted, as imaging time may be dramatically reduced and patient throughput increased. PMID- 10752895 TI - Atypical appearance of elastofibroma dorsi on MRI: case reports and review of the literature. AB - Three patients with histologically proven elastofibroma dorsi underwent MRI, using T1-weighted, T2-weighted, STIR (short inversion time inversion recovery), and contrast-enhanced SE sequences. All lesions typically displayed low signal intensity masses interspersed with areas of high signal intensity on T1- and T2 weighted SE images. Contrary to prior reports, two patients showed marked enhancement of the mass after administration of Gd-DTPA. Although the characteristic signal intensity on conventional T1- and T2-weighted images may lead to the early diagnosis of this rare tumor, radiologists should be aware that marked contrast enhancement may be representative in elastofibroma dorsi. PMID- 10752896 TI - MRI of eccrine cystadenoma. PMID- 10752897 TI - Focal cortical dysplasia: comparison of MRI and FDG-PET. AB - PURPOSE: The purpose of this work was to compare the use of MRI and [18F]fluorodeoxyglucose (FDG) positron emission tomography (PET) in the diagnosis of focal cortical dysplasia (FCD). METHOD: Nineteen patients with surgically proven FCD were analyzed retrospectively. MRI was performed in all patients, and FDG-PET was performed in 17 patients. We compared the MR and FDG-PET findings of FCD according to the histologic findings that were classified into three grades. RESULTS: Four cases were classified as Grade I, 4 cases as Grade II, and 11 cases as Grade III FCD. The lesions were detected on MRI in 9 (82%) of the 11 patients with Grade III FCD and in only 1 (13%) of the 8 patients with Grade I and II FCD. Cortical hypometabolism of the lesion was revealed on FDG-PET in 6 (86%) of the 7 patients with Grade I and II FCD and in 9 (90%) of the 10 patients with Grade III FCD. The extent of the cortical abnormality was larger on FDG-PET than on MRI in 11 (65%) of the 17 patients. CONCLUSION: FDG-PET is more useful in delineating the cortical abnormality in patients with mild degrees of FCD. The extent of the lesion was larger or similar on FDG-PET compared with that of the MRI. PMID- 10752898 TI - MRI of influenza encephalopathy in children: value of diffusion-weighted imaging. AB - PURPOSE: Our purpose was to describe the MR findings of influenza encephalopathy and assess the value of diffusion-weighted imaging for its diagnosis. METHOD: We examined a total of five patients diagnosed as having influenza encephalopathy or encephalitis by MRI, including diffusion-weighted imaging. We analyzed the conventional images and compared them with diffusion-weighted images. RESULTS: Abnormally hyperintense lesions of varying extent and location were noted on T2 weighted and fluid-attenuated inversion recovery images in the cortex and adjacent white matter in every case. They showed no contrast enhancement. Diffusion-weighted imaging demonstrated the lesions as areas of restricted proton diffusion more clearly than conventional imaging. CONCLUSION: Influenza encephalopathy is depicted as areas of T2 elongation having a rather nonspecific distribution. Diffusion-weighted imaging can demonstrate the lesions sensitively and serve as a valuable adjunct to conventional MRI. PMID- 10752900 TI - Investigating demyelination in the brain in a canine model of globoid cell leukodystrophy (Krabbe disease) using magnetization transfer contrast: preliminary results. AB - PURPOSE: This study was designed to examine the use of quantitative magnetization transfer imaging (MTI) in naturally occurring globoid cell leukodystrophy (GLD) in the Cairn terrier. METHOD: A model of GLD was established via a breeding colony, and a total of seven animals were studied with MTI, including two dogs with GLD, one of which underwent whole-body irradiation (725 cGy) and bone marrow transplantation from a genotypically normal littermate. The remaining dogs served as untreated, irradiated, and unirradiated controls. RESULTS: Region-of-interest (ROI) analysis of the MTI showed a decrease in MT ratio (MTR) in the internal capsule of the untreated/affected dog compared with age-matched controls but revealed similar results in the two other study animals. On MT contour plotting, inside-to-out gradients of MTR mimicked the demyelination pathology of the disease in the untreated/affected dog. CONCLUSION: MT contour plotting demonstrated patterns of MT abnormality in the untreated/affected dog that were consistent with histopathology, establishing a clear relationship between pathology-proven demyelination and MTR as well as a striking contrast to the patterns of radiation damage. PMID- 10752899 TI - MRA of intracranial aneurysm models: a comparison of contrast-enhanced three dimensional MRA with time-of-flight MRA. AB - PURPOSE: The purpose of our study was to compare contrast-enhanced 3D MR angiography (MRA) using ultrafast spoiled gradient-recalled acquisition in the steady state (SPGR) with 2D and 3D time-of-flight (TOF) MRA for visualization of intracranial lateral saccular aneurysm models. METHOD: We used lateral saccular aneurysm models with a height of 10 mm and neck sizes of 2.5 and 10 mm. Imaging was performed using a 1.5 T MR system with a head coil. Contrast-enhanced 3D MR angiograms were obtained using 3D ultrafast SPGR sequence with and without the MR Smartprep technique. Two-dimensional and 3D TOF MR angiograms were also obtained. RESULTS: Contrast-enhanced multiphase 3D MR angiograms taken every 5 s after injection of contrast medium proved superior to the other MRA techniques for delineating lateral saccular aneurysm models. Contrast-enhanced 3D MRA images taken with inadequate delay after MR Smartprep trigger showed poor visualization of the aneurysm model. CONCLUSION: Use of contrast-enhanced multiphase 3D MRA with ultrafast SPGR with shorter TR and TE resulted in clear images of the lateral saccular aneurysm model. PMID- 10752901 TI - Correlation of CT values, iodine concentration, and histological changes in the thyroid. AB - PURPOSE: We have reported that in the thyroid, there is a linear correlation between iodine concentrations and CT values. However, the slope of the regression line was about three times as large as that in KI solutions. We investigated the factor(s) contributing to the increment of the slope of the regression line in the thyroid. METHOD: Solutions of NH4I and thyroid hormones were used to investigate the regression line. Thirty-six thyroids were evaluated to investigate the correlation between the iodine concentrations and the area ratio of thyroid follicles, which were measured by preoperative CT and from tissue slices, respectively. RESULTS: The slopes of the regression lines in the solutions were almost identical to those in KI solutions. In the thyroid, iodine concentrations were logarithmically correlated with the area ratio of follicles. CONCLUSION: The decrease in CT values not only revealed a decrease of iodine concentration in the thyroid but also represented an increase of follicular cells and/or interstitial structures in the volume ratio secondary to it. PMID- 10752902 TI - Gadolinium-enhanced magnetic resonance dacryocystography in patients with epiphora. AB - PURPOSE: Digital subtraction dacryocystography (DS-DCG) is considered the "gold standard" in the assessment of the nasolacrimal duct system but fails to delineate the soft tissue structures that surround the lacrimal drainage apparatus. The goal of this study was to compare high resolution MR surface coil imaging with DS-DCG to determine the value of gadolinium-enhanced MR dacryocystography (MR-DCG) in patients with epiphora. METHOD: We performed bilateral MR-DCG and unilateral DS-DCG in 11 patients (aged 3-70 years) with epiphora using Gd-DTPA-containing eyedrops (Magnevist; 78.63 mg of Gd/ml diluted in a sterile 0.9% NaCl solution 1:100) and a water-soluble contrast medium (Omnipaque; 677 mg of Iohexol/ml). Radiographic and MR findings were separated and reviewed by two radiologists who were blinded to patients' names and histories. Afterwards, the passage of the contrast material was compared in the two procedures. The gold standard was defined as the distal-most point of the contrast material identified on either study. RESULTS: Obstruction of the nasolacrimal duct system was diagnosed with a sensitivity of 100% with both MR DCG and DS-DCG. With MR-DCG, the two readers correctly diagnosed the location of an obstruction in 67 and 89% and with DS-DCG in 56 and 67%. With MR-DCG, contrast material was traced further distally than with DS-DCG in three patients. DS-DCG was superior in only one case in which MR-DCG failed to delineate fistulas distal to the nasolacrimal sac. CONCLUSION: Our data suggest that MR-DCG is suitable for assessing drainage problems of the nasolacrimal duct system, giving additional information concerning the surrounding soft tissue structures. Further studies are required to show whether MR-DCG can replace DS-DCG as the gold standard in the assessment of the nasolacrimal duct system. PMID- 10752903 TI - Pilomatricoma in the head and neck: CT findings in three patients. AB - We report three cases of pilomatricoma, one in the infrahyoid neck and the others in the preauricular area. In all cases, CT showed well marginated soft tissue masses that were located mainly in the subcutaneous fat, partly attaching to the overlying skin. There was no evidence of infiltration to deeper structures. Substantial amounts of calcification were found in one tumor. Pilomatricoma should be included in diagnostic consideration when CT shows a well marginated subcutaneous soft tissue mass adherent to the skin with or without visible calcification in the head and neck region. PMID- 10752904 TI - Chordoid glioma of the third ventricle: CT and MR findings. AB - We present a case of chordoid glioma involving the third ventricle in a 42-year old woman. CT and MR showed a homogeneously enhancing mass occupying the third ventricle, with a cystic component. Chordoid glioma should be included in the differential diagnosis of uncommon masses of the third ventricle in middle-aged women. PMID- 10752906 TI - A guide to the venous drainage of the anterior Sylvian fissure. PMID- 10752905 TI - Metastatic chordoma to the larynx in a patient presenting with hoarseness. PMID- 10752908 TI - Terminology used for allergen immunotherapy. PMID- 10752907 TI - Aunt Minnie's corner. Hepatic hemangioma. PMID- 10752909 TI - Why pursue a career in allergy and immunology? PMID- 10752910 TI - New medications for use in patients with rheumatoid arthritis. AB - INTRODUCTION: Several new medications have become available to physicians for the treatment of patients with rheumatoid arthritis (RA). LEARNING OBJECTIVES: To familiarize the reader with these new medications, including their benefits and side effects. DATA SOURCES: Data sources include published articles regarding the use of these medications. Study selection includes available information obtained from an online literature search (Mayo Search) regarding these agents, with additional information from the manufactures and our pharmacy. CONCLUSION: The results of this review indicate that leftunomide (Arava) and etanercept (Enbrel) are useful new agents for treatment of patients with rheumatoid arthritis. PMID- 10752911 TI - Vomiting and syncope in a 14-year-old female. PMID- 10752912 TI - Isolated late cutaneous reactions to allergen skin testing in children. AB - BACKGROUND: Occasionally parents report a reaction developing at the site of an allergen skin test several hours after application of the test, despite there having been no immediate reaction. The medical literature contains little information regarding isolated late reactions (ILRs) to allergen skin testing. OBJECTIVE: The goal of this project was to determine the incidence of ILRs in children undergoing allergen skin testing. METHODS: Prick and intradermal (ID) skin testing was performed for routine clinical indications in an allergy clinic. Children with a positive histamine control, and at least one negative immediate reaction to allergen skin testing were enrolled in the study. The parents were given detailed instructions to examine the skin test sites 6 hours later, and to record the size of any erythematous indurated sites. Circles of various diameters were included on the report form to assist the parents' size estimates. RESULTS: Fifty-seven children enrolled in the study and 50 returned the forms. No patients reported ILRs to prick skin tests. Eighteen of the 50 respondents reported 40 ILRs to ID tests, of > or = 5 mm diameter; 7 of these were > or = 10 mm. The most common allergen causing ILR was cockroach, accounting for 20% of the ILRs. Each of the other allergens also caused ILRs. The clinical history did not show a definite correlation of symptoms with exposure to the allergens causing ILRs, although all 14 patients with ILRs to indoor allergens had year-round symptoms. There was no correlation between the incidence of ILRs and age, gender, or diagnosis of asthma. CONCLUSION: ILRs to allergen skin testing occurred in 36% of pediatric allergy clinic patients. The clinical significance of such reactions is unknown. PMID- 10752913 TI - Superior clinical outcomes of inner city asthma patients treated in an allergy clinic. AB - BACKGROUND: Asthma morbidity and mortality continue to increase especially in the inner cities despite medical advances in disease management. OBJECTIVE: To investigate the clinical outcomes of inner city asthma patients treated in an allergy clinic. METHODS: Phase 1 involved random review of medical records of 100 asthma patients treated in an allergy clinic for 2 consecutive years, assessing the frequency of hospitalizations, emergency room visits (ERV) and asthma severity during three periods; 1 year prior to initial visit (year 0) and during the first (year 1) and second (year 2) years of intervention. Phase 2 involved administration of quality of life (QOL) survey to 23 patients volunteered from allergy clinic (group I), and 21 patients volunteered from emergency room (group II), treated by primary care or emergency room physicians during the previous year. RESULTS: The frequency of hospitalizations and ERV significantly declined over time (P < .001) with greatest declines during year 1. Disease severity of all patients significantly declined over time (P < .001); good compliers had significant improvement over poor compliers (P < .023). Quality of life scores were significantly lower for both groups than for the general population; and although the scores were higher in the allergy clinic group than in the non allergy clinic group, significant differences were achieved only in mental health and social functioning domains. CONCLUSIONS: Patients treated in an allergy clinic demonstrate superior clinical outcomes. PMID- 10752914 TI - Partial mattress encasing significantly reduces house dust mite antigen on bed sheet surface: a controlled trial. AB - BACKGROUND: The most effective measure in house dust mite antigen reduction is mattress encasing with an impermeable membrane. A reduction in encasing costs will help increase patients' compliance in mite antigen avoidance. OBJECTIVE: To investigate the effectiveness of partial mattress encasing with a nylon sheet produced in Thailand on the reduction of group I mite antigens from beddings. METHODS: Sixty regularly-used beds from the house officers' dormitory of the Siriraj Hospital Mahidol University, Thailand, were randomly matched into two groups according to mite antigen levels. The control group (CG) used only regular cotton bed sheets whereas the partial encasing group (PG) used mattresses partially covered with a locally produced nylon sheet underneath the regular cotton bed sheets. Dust collection from the beddings was performed at baseline, 2, 4 and 6 months after application of the nylon sheet. Mite antigen levels were detected by a two step monoclonal antibody ELISA. RESULTS: Mite antigen levels in both groups were not different at the beginning of the study. The PG had significantly lower group I antigen levels on regular bed sheet surfaces than the CG (P < .004) at the 2, 4 and 6 month timepoints. However, antigen levels on the mattress surface of the PG was significantly higher than the CG at the end of the study (P < .004). The barrier efficacy of the nylon sheet in preventing migration of group I mite antigens from the mattress to the surface of the regular cotton bed sheet was 94% whereas that of the regular cotton bed sheet was 66% (P = .007). CONCLUSION: Partial mattress encasing with a locally made nylon sheet can reduce mite antigens on the regular cotton bed sheet surfaces for up to 6 months. PMID- 10752915 TI - Comparison of efficacy and safety between flunisolide/AeroChamber and budesonide/turbuhaler in patients with moderate asthma. AER-MD-04 Study Group. AB - BACKGROUND: There is a limited body of evidence comparing the clinical effects of different inhaled corticosteroids in the treatment of asthma. This study compared the safety and efficacy of inhaled flunisolide and budesonide, both with unique delivery systems that may affect clinical response. OBJECTIVE: This multicenter study was carried out to compare the efficacy and safety of flunisolide, administered via AeroChamber, with budesonide, administered via Turbuhaler, in the treatment of moderate asthma. METHODS: Patients with moderate asthma, defined as an FEV1 of 40% to 85% of predicted, underwent a 2-week run-in period during which beclomethasone, 750 microg twice daily by MDI, was administered, along with salbutamol prn. Patients (n = 176) were then randomized into two groups. One group received flunisolide administered via AeroChamber, 750 microg (3 puffs), twice daily. The second group received budesonide administered via Turbuhaler, 600 microg (3 puffs), twice daily. All patients took salbutamol prn. RESULTS: At the end of the 6-week treatment period, there were no significant differences (P > .05 for all comparisons) in efficacy between the groups as evaluated by any efficacy parameter. The treatment groups also did not differ significantly in the number of adverse events or in the incidence of oropharyngeal Candida infection. CONCLUSION: Flunisolide administered by AeroChamber and budesonide administered via Turbuhaler demonstrate similar efficacy and safety in the treatment of moderate asthma. PMID- 10752916 TI - Foundational readings concerning pollen for allergists and aerobiologists. AB - BACKGROUND: In a medical culture being revolutionized by MEDLINE, MD-Consult, and full-text electronic journals, books are frequently dismissed as antiquated and hopelessly out of date. For those involved in the study of atmospheric pollen, however, books--many several decades old--still represent foundational reading. OBJECTIVES: This paper will present and briefly review 17 books dealing with the ecology of plants that cause pollinosis, pollen morphology, pollen movement in the atmosphere, and air sampling techniques. CONCLUSIONS: This small collection of books provides a foundation with which to approach the study of atmospheric pollen. Allergists and aerobiologists are encouraged to become familiar with these titles, particularly if they intend to publish pollen data in medical journals. PMID- 10752917 TI - Effect of salmeterol on patients' adherence to their prescribed refills for inhaled corticosteroids. AB - BACKGROUND: Optimal treatment for persistent asthma requires multiple classes of medication, including antiinflammatory agents and bronchodilators. Inhaled corticosteroids are the most effective antiinflammatory agents available and are recommended by recent guidelines as first-line treatment. Salmeterol, a long acting inhaled bronchodilator, is recommended as adjunctive therapy to inhaled corticosteroids. Non-adherence to prescribed medication is prevalent and has been implicated in asthma exacerbations. Salmeterol's benefits in terms of asthma control are readily perceived by patients whereas the benefits of inhaled corticosteroid therapy may be less apparent. OBJECTIVE: To evaluate whether the addition of salmeterol to a medication regimen affects patient adherence to prescription refills for inhaled corticosteroids. METHODS: A retrospective medical chart and pharmacy claims record review before and after the addition of salmeterol was used. Medication adherence rates were calculated for 67 patients requiring inhaled corticosteroids for at least 8 months before and after the addition of salmeterol. RESULTS: Adherence with inhaled corticosteroid therapy before (49.7% +/- 29.3%) and after (56.5% +/- 28.6%) the introduction of salmeterol was not significantly different (P = .0785, pre versus post). Adherence with salmeterol was significantly higher (58.7% +/- 28.3%) than inhaled corticosteroids at baseline (P = .0202), but not with concurrent use. Dosing frequency of inhaled corticosteroid administration was not a significant factor in adherence, but increasing age was (r = 0.41788, P = .0048). CONCLUSIONS: The addition of salmeterol does not adversely affect the adherence rates to prescription refills for prescribed inhaled corticosteroid therapy. On average, important antiinflammatory treatment should not be supplanted with salmeterol if prescribed in combination. PMID- 10752918 TI - Clinical approach to penicillin-allergic patients: a survey. AB - BACKGROUND: Approximately 10% of individuals report a history of penicillin allergy. OBJECTIVE: We chose to survey various physician groups to determine how they would manage penicillin-allergic patients who present with an infectious process for which penicillin is the drug of choice. METHODS: In situations where penicillin was the drug of choice, physicians were asked to choose the type of antibiotic treatment for patients who presented with diseases of varying severity and who had either vague or convincing penicillin-allergic histories. RESULTS: A total of 601 surveys were mailed and 93 (16%) were returned. For those patients who present with a vague history of penicillin allergy, 58% and 59% of the physicians surveyed stated that they would choose cephalosporins for individuals with mild and moderate diseases, respectively. In contrast, in the vague penicillin history/severe disease scenario, physicians were split between choosing cephalosporins (42% of responders) and vancomycin (40% of responders). For those patients who present with a convincing history of penicillin allergy, 55% of the physicians chose erythromycin for individuals with mild disease; 44% chose quinolones for individuals with moderate disease, and 63% chose vancomycin for individuals with severe disease. In each of the three disease severities, physicians were significantly more likely to choose cephalosporins for patients with a vague history of penicillin allergy than for patients with a convincing history (each P<10-5). CONCLUSION: The choice of antibiotics is influenced both by the type of penicillin allergic history and by the severity of the disease process to be treated. To decrease the use of broad-spectrum antibiotics in patients labeled "penicillin-allergic", an effort should be made to identify, by skin testing, those patients who lack penicillin-specific IgE antibodies. PMID- 10752919 TI - Comparison of powder and aerosol formulations of salmeterol in the treatment of asthma. AB - BACKGROUND: The efficacy and safety of the aerosol metered-dose inhaler (MDI) formulation of salmeterol for asthma symptoms have been established. Recently, salmeterol has been introduced as a micronized powder formulation administered via a breath-activated multidose powder inhaler (Diskus). OBJECTIVE: A multicenter, randomized, double-blind, double-dummy, parallel-group, placebo controlled study involving 498 adolescents and adults with mild-to-moderate asthma was conducted to compare the efficacy and safety of salmeterol powder 50 microg twice daily via Diskus, salmeterol aerosol 42 microg twice daily via MDI, and placebo. METHODS: Patients were randomized to one of the three treatment groups for 12 weeks. Efficacy was assessed by serial measurements of forced expiratory volume in one second (FEV1) over 12 hours, daily peak expiratory flow (PEF), self-rated asthma symptom scores, nighttime awakenings, and supplemental albuterol use. Safety of each treatment was evaluated by monitoring vital signs, electrocardiograms, Holter monitoring, and occurrence of adverse events. RESULTS: As compared with placebo, both salmeterol powder and aerosol produced significant improvement in FEV1 and PEF and decreased nighttime awakenings and supplemental albuterol use. There were no significant differences in the efficacy of the two salmeterol formulations. The magnitude of improvement in pulmonary function was undiminished over the 12-week study. Both formulations of salmeterol were well tolerated, with safety profiles not significantly different from placebo. CONCLUSION: Results of this study indicate that salmeterol, administered either as a powder 50 microg twice daily via Diskus or as an aerosol 42 microg twice daily via MDI, produces clinically significant and comparable improvement in pulmonary function and is well tolerated in patients with mild-to-moderate persistent asthma. PMID- 10752920 TI - Sensitization to gelatin in children with systemic non-immediate-type reactions to varicella vaccines. AB - BACKGROUND: We recently found that four children who experienced systemic immediate-type reactions to varicella vaccine with gelatin had anti-gelatin IgE. We also found systemic non-immediate-type allergic reactions, which mainly consist of systemic cutaneous signs, appearing several hours or more after the vaccination. OBJECTIVE: To investigate the relationship between immune responses to gelatin and non-immediate-type reactions to gelatin-containing varicella vaccines, we measured anti-gelatin IgE and IgG in the sera of the children with these allergic reactions. METHODS: Serum samples were taken from 21 children who showed non-immediate-type reactions to varicella vaccines. As a positive control, serum samples were taken from 33 children who showed immediate-type reactions to varicella vaccine and had anti-gelatin IgE. As a negative control, serum samples were taken from 50 children who showed no reaction to the vaccine. We then examined anti-gelatin IgE and IgG in sera of the children. RESULTS: Of 21 children with non-immediate-type reactions, two (10%) had anti-gelatin IgE and six (29%) had anti-gelatin IgG. In the positive control group, all 33 children with immediate-type reactions had anti-gelatin IgG as well as IgE. In the negative control group, all 50 children who showed no allergic reaction to varicella vaccines had neither anti-gelatin IgE nor IgG. CONCLUSIONS: These results suggest that the possibility exists that some non-immediate-type reactions to varicella vaccine are caused by immune reactions to gelatin. PMID- 10752921 TI - Changes of serum levels of interleukin-2, intercellular adhesion molecule-1, endothelial leukocyte adhesion molecule-1 and Th1 and Th2 cell in severe atopic dermatitis after intravenous immunoglobulin therapy. AB - OBJECTIVE: To evaluate the effect of intravenous immunoglobulin (IVIG) in the treatment of severe intractable atopic dermatitis (AD) in children and to investigate the inflammatory markers used to measure their disease activity. MATERIALS AND METHODS: Serum levels of interleukin-2 receptor (sIL-2R), intercellular adhesion molecule-1 (ICAM-1), endothelial leukocyte adhesion molecule (ELAM-1), and eosinophil cationic protein (ECP) were measured in five children with AD (group A) who had a mean age of 9.4 months (range 7 to 12 months) before and after IVIG therapy. Seven age-matched patients with similar severity of AD who only received topical corticosteroid therapy served as the control group (group B). Ten normal control serum samples were collected from well-baby clinic (group C). T helper 1 (Th1) was defined by IFN-gamma/CD4+ and Th2 by IL4/CD4+, using 3-colored flow cytometry. Clinical severity of AD was evaluated with the SCORAD index. Intravenous immunoglobulin (2 g/kg/dose) was administered monthly for a total of 3 doses. RESULTS: The serum levels of ICAM-1, ELAM-1, and IL-2R in patients with AD were significantly higher than normal control infants. After IVIG therapy, the SCORAD index and the inflammatory markers (ICAM-1, ELAM-1, and ECP) in group A were significantly decreased (P = .01 for SCORAD index; .034, .043, and .03 for ICAM-1, ELAM-1 and ECP, respectively). The serum levels of ICAM-1, ELAM-1, ECP and IL-2R in group B did not show a significant reduction after 3 months of topical corticosteroid therapy. In comparison to normal healthy children, patients with AD had decreased Th2 cells (P = .009) and higher ratio of Th1/Th2 (P = .009) in peripheral blood mononuclear cells (PBMC). There was no significant difference of Th1, Th2 cells, and ratio of Th1/Th2 in PBMC before and after IVIG therapy in patients with AD. CONCLUSION: Intravenous immunoglobulin can be safely and effectively given for the treatment of severe intractable AD. The determination of ICAM-1, ELAM-1, and ECP levels may be useful in monitoring disease activity of AD in childhood. The IVIG may play a role in treatment. PMID- 10752923 TI - Effect of misoprostol on the secretion of histamine from basophils of whole blood. AB - BACKGROUND: Misoprostol (MSP), the synthetic prostaglandin E1 (PGE1) analog, possesses multifunctional features, including modulating some inflammatory aspects of immune and allergic disorders. OBJECTIVES: To investigate the effect of MSP on histamine release (HR) from basophils of whole blood using anti-IgE, specific allergens, and calcium ionophore. METHODS: The study was performed using the automated glass fiber-based whole blood leukocyte histamine release test (LHRT). RESULTS: Very low concentrations of MSP produced a marked inhibition of HR induced with anti-IgE. Maximum inhibition was observed at 10-9 M. It was also shown that the levels of HR inhibition with MSP varied at different incubation times. The greatest inhibition of HR was noted at 1 to 2 hours of incubation at MSP concentrations of 10-8 and 10-9 M, respectively. Incubation of blood from allergic patients at the optimal MSP concentration and optimal elapsed time (2 hours) resulted in significant reductions of allergen-specific HR induced by both Timothy pollen grass allergen and D.pteronissinus. Incubation of blood with varying concentrations of MSP and subsequent stimulation with calcium ionophore A23187 also inhibited HR from basophils. In the latter case, the most effective concentrations of MSP ranged from 10-8 to 10-6 M. CONCLUSIONS: This study demonstrated that MSP can inhibit basophil HR indicating a potentially beneficial role of PGE1 analogs as pharmacotherapy for allergic diseases. PMID- 10752922 TI - Evaluation of the presence of bovine proteins in human milk as a possible cause of allergic symptoms in breast-fed children. AB - BACKGROUND: It is generally believed that the elimination of certain foods from the diet of mothers during the lactation period produces a significant improvement in breast-fed children who develop allergic symptoms. Several studies have shown the presence of food proteins in human milk; on the other hand, no study has been able to correlate unequivocally the presence of these allergens in human milk with newborn sensitization. OBJECTIVE: The aim of this study was to evaluate the presence of bovine proteins in breast milk. METHODS: Milk samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). To detect bovine proteins in human milk, immunoblotting was performed by using monoclonal antibodies (MA) specific for beta-lactoglobulin and bovine caseins. RESULTS: The results of this study do not confirm the presence of bovine proteins in breast milk suggested by other authors and shows unequivocally that the conflicting results reported in the literature about the presence of betalactoglobulin in human milk are due to cross-reactivity between bovine milk proteins and human proteins. CONCLUSIONS: Components other than bovine betalactoglobulin or caseins could be involved in the induction of allergic symptoms in exclusively breast-fed children. PMID- 10752924 TI - Comparison of skin prick test with serially diluted wild-type and genetically engineered recombinant Der f2. AB - BACKGROUND: C8/119S, a genetically engineered less allergenic mutant of group II allergen (Der f2) of house dust mite, Dermatophagoidesfarinae, was constructed in order to reduce the risk of anaphylactic reactions of allergen specific immunotherapy. OBJECTIVE: To further evaluate, with a larger number of mite allergic patients, the safety of C8/119S for the treatments of humans. METHOD: We tested the dose-dependent responses of 20 mite-allergic volunteers to a skin prick test with C8/119S and wild-type recombinant Der f2 and compared the biologic potentials of these allergens to induce type I allergic reactions. In a separate experiment, we compared IgE binding capacities to C8/119S and to wild type recombinant Der f2 in individual sera from 34 mite-allergic donors. RESULTS: The concentration of C8/119S needed to induce positive skin prick test (SPT) reaction was at least 100 times more than that of recombinant Der f2 in 95% of the volunteers tested. Consistent with this result, IgE binding data showed that 85% of the mite-allergic donors had little or no detectable IgE bound to C8/119S. Our data also shows that a minority of mite-allergic patients responded in a similar manner to both C8/119S and wild-type recombinant Der f2. CONCLUSION: Our data confirms that C8/119S is much less allergenic and thus can be used safely for immunotherapy of most of mite-allergic patients. Care should still be taken because, in a minority of patients, C8/119S may cause similar type I allergic reactions as does wild-type recombinant Der f2. PMID- 10752925 TI - Blomia tropicalis sensitization as a risk factor for the development of allergic rhinitis. PMID- 10752926 TI - Successful outcome of montelukast overdosage in an asthmatic child. PMID- 10752927 TI - Practice parameter. PMID- 10752928 TI - Drug delivery for posterior segment eye disease. PMID- 10752929 TI - The cavernous body of the human efferent tear ducts: function in tear outflow mechanism. AB - PURPOSE: To determine the structure and function of a system of large blood vessels integrated in the bony canal between the orbit and the inferior nasal duct. METHODS: Thirty-one dissected lacrimal systems of adults were analyzed by using gross anatomy, histology, and electron microscopy as well as corrosion vascular casts. RESULTS: More than two thirds of the bony canal between orbit and inferior nasal duct is filled by a plexus of wide-lumened veins and arteries. The vascular system is embedded in the wall of the lacrimal sac and nasolacrimal duct and is connected to the cavernous tissue of the inferior turbinate. Three types of blood vessels can be distinguished inside the vascular tissue that surrounds the lumen of the lacrimal passage: barrier arteries, capacitance veins, and throttle veins. CONCLUSIONS: The surrounding vascular plexus of the lacrimal sac and nasolacrimal duct is comparable to a cavernous body. While regulating the blood flow, the specialized blood vessels permit opening and closing of the lumen of the lacrimal passage, effected by the bulging and subsiding of the cavernous body, and at the same time regulate tear outflow. Other functions such as drainage of absorbed tear fluid components and a role in immunologic response are under discussion as well. Malfunctions in the cavernous body may lead to disturbances in the tear outflow cycle, ocular congestion, or total occlusion of the lacrimal passages. Variations in the conditions for swelling of the cavernous tissue may have led to the (mistaken) description of valves in the lacrimal passage. PMID- 10752930 TI - The spatial and temporal expression of outer segment proteins during development of Macaca monkey cones. AB - PURPOSE: To characterize the spatial and temporal expression of key structural and phototransduction cascade proteins in the monkey cone outer segment (OS). METHODS: Retinas from Macaca monkeys from ages fetal day (Fd) 89 through adulthood were double labeled using immunofluorescence for short (S) or long/medium (L/M) wavelength-sensitive cone opsin and either a structural protein (peripherin) or a phototransduction cascade protein (alpha-transducin [alpha-T], phosphodiesterase [PDE], or rhodopsin kinase [RK]). The spatial and temporal patterns of expression for each protein at each age were determined and graphed as a percentage of retinal coverage. RESULTS: In both cone types, opsins and phototransduction proteins appear first in the fovea and last at the retinal edge. Peripherin appears concomitantly with opsin in both S and L/M cones, but S cones express peripherin and opsin 1 to 3 weeks before neighboring L/M cones. Alpha-T, PDE, and RK are expressed together in the L/M cone OS shortly after L/M opsin appears. Phototransduction proteins are not expressed in S cones until 1 to 3 weeks after the appearance of S opsin and at the same time that neighboring cones are expressing both L/M opsin and phototransduction proteins. CONCLUSIONS: The concomitant appearance of opsin and peripherin strongly suggests roles in promoting the structural integrity of the developing OS. Phototransduction cascade proteins appear in the developing OS at the same time as one another, but after opsin. The significant lag between their expression and that of S cone opsin indicates that phototransduction proteins are not essential for OS formation, nor does opsin expression trigger their expression. The different temporal but similar spatial expression patterns of phototransduction proteins within S and L/M cones suggests that some local signal(s) coordinates their appearance. PMID- 10752931 TI - Muscle fiber types of human extraocular muscles: a histochemical and immunohistochemical study. AB - PURPOSE: To classify muscle fibers of human extraocular muscle (hEOM) and to compare them to previous studies on hEOM, as well as to nonhuman EOM classification schemes and skeletal muscle fiber types. METHODS: Muscle fibers cut in different muscle planes were followed on consecutive cross sections and typed with regard to their oxidative profile in combination with their myosin immunohistochemical characteristics. RESULTS: Three zones were observed. In the global layer three muscle fiber types were observed: global layer singly innervated granular fibers, 79.4 +/- 8.1 microm (perimeter [values at midmuscle region] +/- SD); 59%; global layer singly innervated coarse fibers (80.3 +/- 10.8 microm; 21%); and global layer multiply innervated muscle fibers (4.1 +/- 9.7 microm; 21%). Two muscle fiber types were detected in the orbital layer: orbital layer singly innervated muscle fibers (54.1 +/- 8.5 microm; 83%) and orbital layer multiply innervated muscle fibers (53.5 +/- 7.6 microm; 17%). Three muscle fiber types were differed in the marginal zone: marginal zone singly innervated muscle fibers (83.1 +/- 15.8 microm; 56%), marginal zone multiply innervated low oxidative muscle fibers (84.4 +/- 23.3 microm; 7%), and marginal zone multiply innervated high oxidative muscle fibers (88.4 +/- 14.5 microm; 37%). Coexpressions of developmental myosin heavy chain isoforms and fast myosin heavy chain isoform were detected mainly in the marginal zone. CONCLUSIONS: hEOMs resemble mammalian EOM with regard to their organization. However, in addition to an inner global layer and an orbital layer an external marginal zone was described for the first time in hEOM in the present study. PMID- 10752932 TI - Cholestanol induces apoptosis of corneal endothelial and lens epithelial cells. AB - PURPOSE: To determine whether cholestanol induces cornea endothelial and lens epithelial cell death in vitro. METHODS: Cornea endothelial and lens epithelial cells were cultured in minimum essential media with 10% fetal bovine serum containing 10 microg/ml cholesterol in ethanol, 10 microg/ml cholestanol in ethanol, or 1% ethanol. These cells, stained using the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) method, were analyzed by laser cytometer. The activities of ICE and CPP32 proteases in cells were also measured. RESULTS: Both cornea endothelial and lens epithelial cells cultured with 10 microg/ml cholestanol showed a significant loss of viability. The nuclei of these cells cultured with 10 microg/ml cholestanol were more frequently stained than those exposed to 10 microg/ml cholesterol or 1% ethanol. Quantitative analysis of apoptotic DNA fragmentation confirmed that the cholestanol induced apoptosis of these cells in a time-dependent manner. The activities of interleukin-1beta converting enzyme (ICE) and CPP32 proteases for cells cultured with 10 microg/ml cholestanol were significantly higher than those observed in control cells. CONCLUSIONS: In vitro, cholestanol was taken up by corneal endothelial cells and lens epithelial cells, an event that led to apoptosis of these cells. PMID- 10752933 TI - Expression of adenylate cyclase subtypes II and IV in the human outflow pathway. AB - PURPOSE: It has been demonstrated that low doses of pilocarpine and other muscarinics substantially increase outflow facility in the isolated human outflow system devoid of ciliary muscle. These cholinergic-induced facility responses were thought possibly to be due to elevation of cAMP as a result of the presence of adenylate cyclases II (AC-II) and IV (AC-IV). Therefore, whether these isoforms are present in outflow tissues was examined. METHODS: Human anterior segments were perfused with carbachol (10(-9)-10(-5) M), and outflow facility and cAMP levels in the perfusate were measured simultaneously. Isolated trabecular meshwork (TM) were incubated with carbachol (10(-7) M), and the subsequent changes in cAMP were measured by radioimmunoassay. AC-II and AC-IV were characterized in ocular tissue with reverse transcription-polymerase chain reaction and in situ hybridization. RESULTS: Outflow facility increased, in a dose-dependent manner, by 10%, 16%, and 27% in response to 10(-9), 10(-7), and 10(-5) M carbachol, respectively. Similarly, cAMP increased by 9%, 70%, and 210% in response to 10(-9), 10(-7), and 10(-5) M carbachol, respectively. In addition, cAMP levels significantly increased by 39% in isolated TM strips incubated with 10(-7) M carbachol. AC-II was detected in most normal tissue examined, but not in any cultured cell lines or any glaucomatous tissue. AC-IV was also widely expressed in most normal tissues, faintly detected in some glaucoma tissue, but not detected in most cultured cells. CONCLUSIONS: The presence of AC-II and AC-IV in outflow tissues supports the hypothesis that cholinergics may indeed exert an effect on outflow facility, mediated by cAMP, which is independent of muscle contraction. PMID- 10752934 TI - Plasminogen activator inhibitor-1 mRNA is localized in the ciliary epithelium of the rodent eye. AB - PURPOSE: To identify in the adult and developing rodent eye cells expressing the gene encoding plasminogen activator inhibitor-1 (PAI-1), an important component of the fibrinolytic system. METHODS: PAI-1 mRNA was localized in cryostat thin eye sections via in situ hybridization analysis using specific 35S-labeled riboprobes. PAI-1 activity was tested in the aqueous humor using one-phase reverse zymography. RESULTS: In the adult eye, PAI-1 mRNA was detected exclusively in epithelial cells of the ciliary processes, primarily in the apexes. In addition, PAI-1 activity was detected in the aqueous humor. PAI-1 mRNA was first found in the ciliary epithelium in embryonic day 18.5, when the ciliary body has reached an advanced developmental stage. PAI-1 mRNA was also detected in the ganglion cell layer of the retina at postnatal days 1 to 4, when angiogenesis takes place. CONCLUSIONS: During development, PAI-1 is likely to be involved in retina vascularization, in agreement with other cases of angiogenesis. Results for the adult eye indicate that the ciliary epithelium is the source for PAI-1 activity found in the aqueous humor. The results suggest that PAI-1 plays a role in balancing fibrinolysis and proteolysis specifically in the anterior segment of the eye, implying that PAI-1 overproduction in the ciliary epithelium could shift the balance against proteolysis and thus may interfere with aqueous outflow. PMID- 10752935 TI - A comparison of the pattern- and total deviation-based Glaucoma Change Probability programs. AB - PURPOSE: To compare the rates of progression of visual field defects in glaucoma patients, by using the Glaucoma Change Probability program based on pattern deviation and total deviation probability maps. METHODS: The incidence of progression of visual field loss among 67 eyes of 56 glaucoma patients with an average of 6 years of follow-up was estimated by applying the criteria set by the Early Manifest Glaucoma Treatment study, which uses the output from the Glaucoma Change Probability (GCP) program of the Humphrey Field Analyzer (San Leandro, CA) based on pattern deviation probability maps. This incidence estimate was compared with one obtained by applying the same criteria but using the GCP program based on total deviation probability maps. RESULTS: The 6-year incidence of progression among patients with glaucoma was 23.2% and 35.7% using the GCP program based on pattern deviation and total deviation probability maps, respectively. Not all patients in whom visual field loss progressed according to pattern deviation also showed progression according to total deviation. CONCLUSIONS: The GCP program based on pattern deviation probability maps appears to screen out patients in whom progression of visual field defects may be due to diffuse loss from cataract, but the pattern deviation maps may also be identifying other types of field loss not detected by the total deviation maps. PMID- 10752937 TI - Peripheral refraction and ocular shape in children. AB - PURPOSE: To evaluate the relation between ocular shape and refractive error in children. METHODS: Ocular shape was assessed by measuring relative peripheral refractive error (the difference between the spherical equivalent cycloplegic autorefraction 30 degrees in the nasal visual field and in primary gaze) for the right eye of 822 children aged 5 to 14 years participating in the Orinda Longitudinal Study of Myopia in 1995. Axial ocular dimensions were measured by A scan ultrasonography, crystalline lens radii of curvature by videophakometry, and corneal power by videokeratography. RESULTS: Myopic children had greater relative hyperopia in the periphery (+0.80 +/- 1.29 D), indicating a prolate ocular shape (longer axial length than equatorial diameter), compared with relative peripheral myopia and an oblate shape (broader equatorial diameter than axial length) for emmetropes (-0.41 +/- 0.75 D) and hyperopes (-1.09 +/- 1.02 D). Relative peripheral hyperopia was associated with myopic ocular component characteristics: deeper anterior and vitreous chambers, flatter crystalline lenses that were smaller in volume, and steeper corneas. Lens thickness had a more complex association. Relative peripheral hyperopia was associated with thinner lenses between refractive error groups but changed in sign to become associated with thicker lenses when analyzed within each refractive error group. Receiver operator characteristics analysis of the ocular components indicated that vitreous chamber depth was the most important ocular component for characterizing the myopic eye, but that peripheral refraction made a significant independent contribution. CONCLUSIONS: The eyes of myopic children were both elongated and distorted into a prolate shape. Thinner crystalline lenses were associated with more hyperopic relative peripheral refractions across refractive error groups, but failure of the lens to thin may account for the association between thicker lenses and more hyperopic relative peripheral refractions within a given refractive group. Increased ciliary-choroidal tension is proposed as a potential cause of ocular distortion in myopic eyes. PMID- 10752938 TI - Screening for refractive errors with the Topcon PR2000 Pediatric Refractometer. AB - PURPOSE: The PR2000 (Topcon, Tokyo, Japan) is a photorefractor that has been used in a population study comparing different methods of screening preschool children. The present study was conducted to determine the accuracy of the device in a largely clinical population. METHODS: Two hundred twenty-two children less than 8 years of age were included. All children were examined by an orthoptist using the PR2000 without inducing cycloplegia. All children then underwent retinoscopy with cycloplegia by an examiner who was unaware of the results from the PR2000 examination. RESULTS: The PR2000 gave a numerical reading for 90% of the children's right eyes and the message "Out of range" for a further 5%. The readings underestimated the amount of hypermetropic or astigmatic refractive error found on retinoscopy by an amount proportional to the magnitude of the refractive error. Agreement with retinoscopy for the axis of astigmatism more than 0.75 D was moderately good (intraclass correlation coefficient [ICC] = 0.63). The PR2000 was more useful as a screener, especially for anisometropia for which it was 91% sensitive and 92% specific. The repeatability was good for sphere (ICC = 0.74), less so for astigmatism (ICC = 0.59), and better than the optometrist for anisometropia (ICC = 0.38). The presence of nonrefractive diagnoses and the age of the children examined made little difference in the screening results. CONCLUSIONS: The PR2000 underestimated hypermetropic refractive errors when used without cycloplegia. However, it was at least as good a screening device as other similar instruments, especially when judged by its ability to detect anisometropia and the repeatability of the results. PMID- 10752936 TI - The National Eye Institute Visual Function Questionnaire: experience of the ONTT. Optic Neuritis Treatment Trial. AB - PURPOSE: To describe the health-related quality of life, measured with the National Eye Institute Visual Function Questionnaire (NEI-VFQ), of patients several years after the onset of optic neuritis, according to their neurologic and visual status; to assess the relationship between the NEI-VFQ subscales and clinical measures of visual function; and to assess the internal consistency reliability of the NEI-VFQ subscales. METHODS: The NEI-VFQ was administered to 244 patients 5 to 8 years after treatment for an episode of acute optic neuritis as part of the Optic Neuritis Treatment Trial. Visual acuity, visual field, contrast sensitivity, and color vision were measured at the same time as questionnaire completion. RESULTS: The NEI-VFQ scores generally were lower than those reported for a disease-free group. Reported dysfunction was greater when multiple sclerosis was present and when visual acuity was abnormal, supporting the construct validity of the NEI-VFQ. Rank correlations between the NEI-VFQ subscales and clinical measures of visual function were moderate at best. Internal consistency reliability was generally high for most of the NEI-VFQ subscales. CONCLUSIONS: These findings add support to the use of the NEI-VFQ as a valuable measure of self-reported visual impairment. PMID- 10752939 TI - Conjunctival fibroblasts enhance the survival and functional activity of peripheral blood eosinophils in vitro. AB - PURPOSE: To examine the effect of human conjunctival fibroblasts on the survival and functional activity of human peripheral blood eosinophils. METHODS: Eosinophils were purified by negative immunoselection [magnetic activated cell sorter (MACS), purity > 97%] from volunteers with mild atopia. Fibroblasts were cultured from conjunctival specimens of healthy donors. Eosinophils were cultured on confluent monolayers of conjunctival fibroblasts or in culture medium alone. Eosinophil survival was evaluated by the trypan blue exclusion test. Eosinophil adherence was assessed by counting the attached cells after washing the cultures. Eosinophil viability and adherence in coculture were also assessed in the presence of anti-granulocyte-macrophage colony-stimulating factor (GM-CSF), anti interleukin (IL)-3, and anti-IL-5 neutralizing antibodies. Cocultured eosinophils were activated by lipopolysaccharide (LPS) after 4 days in culture, and eosinophil peroxidase (EPO) release was determined as a marker of their activation. RESULTS: Eosinophils cocultured with conjunctival fibroblasts had a significantly increased viability of 35.9% (P = 0.004) and 12.8% (P = 0.003) on days 4 and 8, respectively. Fibroblast-conditioned medium did not enhance the survival of eosinophils. The increase in eosinophil survival in coculture was partially inhibited by anti-GM-CSF (P = 0.019), anti-IL-3 (P = 0.033), or anti-IL 5 (P = 0.011), whereas eosinophil adherence was reduced by anti-GM-CSF alone (P = 0.034). LPS activation of eosinophils cultured for 4 days with conjunctival fibroblasts induced higher EPO release than in freshly isolated eosinophils (P = 0.01). CONCLUSIONS: Human conjunctival fibroblasts induced prolonged survival and increased secretory function of human peripheral blood eosinophils. Increased survival is partially mediated by IL-3, IL-5, and GM-CSF. The coculture of conjunctival fibroblasts with eosinophils can serve as an in vitro system for the study of eosinophil behavior in the ocular surface and of cellular interactions in allergic eye diseases. PMID- 10752940 TI - Disruption of the basement membrane after corneal debridement. AB - PURPOSE: To determine whether the native basement membrane left behind after manual debridement wounding is retained throughout healing in the Balb/c mouse. METHODS: Mouse corneas were subjected to either 1.5 mm (small) or limbus-to limbus (large) epithelial debridement wounds and allowed to heal for times ranging from 12 hours to 3 days. For the larger wounds, care was taken to leave an approximately 0.5-mm zone of epithelial cells near the limbal border. Unwounded corneas served as control specimens. At each time point, confocal immunofluorescence microscopy was used to localize several proteins found in the basement membrane including laminin-5, entactin, and perlecan. In addition, ultrastructural studies were performed using transmission electron microscopy (TEM) to assess the basement membrane zone (BMZ) of the corneas at various times after injury. RESULTS: The smaller (1.5-mm) wounds healed within 24 hours, and the larger wounds healed at approximately 48 hours. Both wound sizes healed with little scarring or neovascularization. At all time points after 1.5-mm wounding, immunofluorescence confocal microscopy and TEM showed that both basement membrane proteins and the lamina densa were retained at the BMZ throughout healing. For the larger wounds, at time points after 24 hours, confocal microscopy showed patches along the denuded corneal stroma where there was a partial or complete loss of basement membrane markers at the BMZ. TEM confirmed that the lamina densa was partly or completely absent along the anterior surface of the exposed cornea at time points of more than 24 hours after the larger wounds. CONCLUSIONS: The denuded epithelial basement membrane was shown to be partially disassembled in response to manual debridement wounds when re-epithelialization took more than 24 hours. Regulated disassembly of the epithelial basement membrane probably plays a role in the healing of large-diameter debridement wounds. PMID- 10752941 TI - Expression patterns of retinoblastoma and E2F family proteins during corneal development. AB - PURPOSE: To determine the expression patterns of the retinoblastoma protein and the E2F transcription factor families in limbal and corneal epithelia and in corneal keratocytes in situ during corneal development and differentiation. METHODS: Retinoblastoma protein (pRb) and its family members p107 and p130; E2F 1, -2, and -4, members of the E2F family of transcription factors; and Ki67, a marker of actively cycling cells, were localized by indirect immunofluorescence microscopy, in corneas of neonatal, juvenile, and adult rats. Presence of mRNA for pRb, p107, p130, and E2F types 1 to 5 in adult corneal epithelium was determined by reverse transcription-polymerase chain reaction. RESULTS: mRNA for all members of pRb and E2F families was present in adult corneal epithelium. The greatest number of Ki67-positive corneal and limbal epithelial cells were present at days 13 to 19, and Ki67-positive stromal keratocytes at day 2. pRb and E2F-2 were localized to all cells in neonatal, juvenile, and adult corneas. With age, p130 localization became more intense and nuclear in stromal keratocytes and suprabasal cells of corneal and limbal epithelia; p107, initially nuclear in limbal and corneal epithelia, became increasingly cytoplasmic in corneal epithelium. E2F-1 was initially nuclear in keratocytes and diminished after day 10. E2F-1 was localized in the basal cell layer of limbal and corneal epithelia after day 10. E2F4 was always nuclear in limbal epithelium and cytoplasmic in corneal epithelium. CONCLUSIONS: Expression patterns of pRb and E2F family proteins vary with corneal cell differentiation, but are most apparent with p130 and p107. Nuclear localization of p130 appears to correlate with terminal differentiation in epithelium and entrance into a quiescent state by keratocytes. In contrast, p107 is nuclear in the undifferentiated limbal basal cells and is cytoplasmic in the remainder of the corneal epithelial cells. PMID- 10752942 TI - Nerve growth factor promotes corneal healing: structural, biochemical, and molecular analyses of rat and human corneas. AB - PURPOSE: A recent clinical report demonstrated that topical nerve growth factor (NGF) treatment in patients affected by corneal neurotrophic ulcers induced epithelial and stromal healing restoring corneal integrity. Mechanisms(s) undergoing these clinical NGF actions are still unclear. The aim of this study was to investigate the role of NGF in human and rat cornea physiopathology. METHODS: Expression of high-affinity NGF receptors, NGF-mRNA, and NGF protein was evaluated in human and rat normal corneas, in human and rat corneal epithelial cell cultures, in human corneal organ culture, and in the rat cornea after an experimental model of epithelial injury, by means of immunohistochemistry, in situ hybridization reverse transcription-polymerase chain reaction, and enzyme linked immunosorbent assay. RESULTS: The resultant data demonstrated that NGF is a constitutive molecule present and produced in normal human and rat corneas. In vitro human and rat corneal epithelial cells produce, store, and release NGF and also express high-affinity NGF receptors (TrkA). In human organ culture, epithelium, keratocytes, and endothelium have been shown to bind exogenous radiolabeled NGF, and the epithelial cells' binding was increased after epithelium injury. In vivo, after rat corneal epithelial injury, a transient increase of corneal NGF levels was observed. Inhibition of endogenous NGF activity by neutralizing anti-NGF antibodies delayed the corneal epithelial healing rate, whereas exogenous administration of NGF accelerated healing. CONCLUSIONS: Taken together, the above findings show that NGF plays an important role in corneal physiopathology and suggest that this neurotrophin may exert therapeutic action in wide-spectrum corneal diseases. PMID- 10752943 TI - Relationship of telomeres and p53 in aging bovine corneal endothelial cell cultures. AB - PURPOSE: To demonstrate a relationship between telomere lengths and levels of p53 in cultured bovine corneal endothelial cells (CECs) during aging. METHODS: Bovine CECs were grown and aged as long-term cultures. Telomere lengths were determined directly on gels with 32P probes after treatment of isolated DNA with RsaI and HinfI. Protein p53 was determined using an enzyme-linked immunosorbent sandwich assay. Cellular aging and the development of replicative senescence were monitored by the appearance of senescent morphology and the beta-galactosidase assay. RESULTS: Bovine CEC telomeres lost 4 kb (from 12.8 to 8.8 kb) over 1 year (89 population doublings [PDs]). The p53 levels in bovine CECs were initially small (approximately 60 pg/million cells), but rose 3.5-fold by culture age of 260 days (64 PDs). On initiation, cultured bovine CECs did not stain for the senescent marker beta-galactosidase. However, these cells stained at 89 PDs and senescent morphology was observed in the cultures at 64 PDs. CONCLUSIONS: The data indicate an inverse relationship between telomere lengths (decreasing) and levels of p53 (increasing) in bovine CECs during aging. These properties may influence the ability of these cells to divide as they enter into replicative senescence. PMID- 10752944 TI - Effect of sustained cyclovergence on eye alignment: rapid torsional phoria adaptation. AB - PURPOSE: To describe adaptive changes in torsional alignment that follow sustained cyclovergence in healthy humans. METHODS: Eye movements were recorded binocularly from four healthy subjects using dual-coil scleral annuli. Cyclovergence movements were evoked over periods of 30 to 150 seconds using a stereoscopic display, presenting gratings of lines arranged horizontally, vertically, or at 45 degrees, subtending angles of up to 48 degrees. In- and excyclodisparities of 5 degrees were introduced and removed in a single-step fashion. After stimulation, the time course and magnitude of the decay in cyclovergence was compared with the subject either in darkness or viewing a baseline stimulus of zero cyclodisparity. RESULTS: As reported previously, the cyclovergence response to incyclodisparities was greater than to excyclodisparities. After sustained excyclovergence, however, in all subjects and in response to all orientations of the gratings, the decay in darkness was incomplete, implying an adaptive change in torsional alignment. In response to the horizontal gratings, for incyclovergence there was also an incomplete decay in darkness but to a lesser degree than in response to excyclovergence, and in only three of four subjects. The incyclovergence evoked by the oblique and vertical gratings was of small magnitude, and its decay was unaffected by the presence or absence of a visual stimulus. CONCLUSIONS: After sustained cyclovergence, its decay in the absence of a visual stimulus may be incomplete. The residual component may be interpreted, by analogy with horizontal and vertical vergence, as reflecting so-called phoria adaptation for torsional alignment. PMID- 10752945 TI - Prospects for treating acquired pendular nystagmus with servo-controlled optics. AB - PURPOSE: To determine whether a device featuring electronically controlled motor driven prisms can reduce oscillopsia and improve acuity in patients with acquired pendular nystagmus (APN). METHODS: A device was developed that senses eye movements and, by the use of motor-driven prisms, oscillates the image of the world in lockstep with the pathologic nystagmus, to negate its deleterious visual effects. Unlike existing optical and surgical treatments for nystagmus, the device negates only the pathologic movements. Voluntary and normal reflex eye movements required for normal vision are unaffected. The benefits of the device were assessed by its impact on acuity in five patients with medication-refractory APN. RESULTS: All patients reported decreases in oscillopsia when the device was in operation. Averaged across patients, the device increased the percentage of time in which retinal image velocity was within +/-4 degrees/sec from 12.8% to 33.3%. Acuities improved in four of five patients, by an average of 0.21 logMAR units. CONCLUSIONS: The symptoms of pendular nystagmus can be treated with a servomechanical device. Further refinements in the device should result in greater improvements in acuity, and a portable, wearable version is feasible using existing technologies. PMID- 10752946 TI - Aqueous humor stimulates the migration of human trabecular meshwork cells in vitro. AB - PURPOSE: Depletion of trabecular meshwork cell numbers is a feature of the outflow system in aging and in primary open-angle glaucoma. It is possible that migration stimulated by factors present in aqueous humor may contribute to the cell loss. This investigation assessed the chemoattractant potential of glaucomatous and nonglaucomatous human aqueous humor and fibronectin, one of its constituents, on a range of cultured trabecular meshwork cell lines. METHODS: Migration was assessed in 48-well modified Boyden chambers. The potential migratory stimulants were soluble fibronectin and glaucomatous and nonglaucomatous aqueous humor. The glaucomatous aqueous samples were collected from patients undergoing trabeculotomy for primary open-angle glaucoma and the normal aqueous from normal bovine eyes and patients undergoing cataract surgery. The target cell types were normal human and bovine meshwork cells grown from explants and two human transformed meshwork cell lines from a normal (HTM-5) and a glaucomatous (HTM-3) source. RESULTS: Soluble fibronectin stimulated all the target cells to migrate with an optimal concentration ranging from 1 to 30 microg/ml, and Zigmond Hirsch checkerboard analysis indicated that both chemotaxis and chemokinesis took place. All the aqueous humor samples stimulated migration of the meshwork cell lines at an optimal concentration of 200 microl/ml. Glaucomatous aqueous humor stimulated a greater migratory response than nonglaucomatous aqueous for two of the four target cell types (P < or = 0.03). Neutralization of the fibronectin content of nonglaucomatous and glaucomatous aqueous by addition of excess anti-fibronectin antibody indicated that fibronectin could account for 35% to 80% of the migratory activity of the aqueous. CONCLUSIONS: Aqueous humor contains potentially powerful chemoattractants for trabecular meshwork cells. The activity of one of these constituents, fibronectin, has been accounted for by this study. Glaucomatous aqueous appears to be as good and in some cases a better migratory stimulant than nonglaucomatous aqueous in vitro. The migratory evidence points to a trend that may help to explain cell loss in the aging meshwork and possibly some of the extra loss in primary open-angle glaucoma. PMID- 10752947 TI - Multivariate approach for quantification of morphologic and functional damage in glaucoma. AB - PURPOSE: To determine the usefulness of confirmatory factor analysis in examination of morphometric, electrophysiological, and psychophysical quantitative methods that measure the extent of global glaucomatous damage without referring to a preselected gold standard. METHODS: In a cross-sectional clinical study, 406 eyes of 203 glaucoma patients and 200 eyes of 100 normal control subjects 18 to 70 years old underwent optic disc morphometry, automated perimetry, measurement of temporal contrast sensitivity by a full-field flicker test, blue-on-yellow visually evoked potential (VEP), and black-and-white pattern reversal electroretinogram (ERG). Diagnosis of glaucoma was based on a qualitative classification of the optic nerve head and retinal nerve fiber layer independent of intraocular pressure and visual field. Confirmatory factor analysis was performed in the patient group as a whole and in a subgroup showing moderate to advanced glaucomatous optic nerve head damage. RESULTS: The confirmatory factor analysis models explained the data satisfactorily (P > 0.18, all patients; P > 0.34, subgroup). Global glaucomatous damage was quantified best by the mean defect of automated perimetry (r = 0.81; r = 0.87), followed by the area of the neuroretinal rim (r = 0.64; r = 0.73), the full-field flicker test (r = 0.59; r = 0.65), the pattern-reversal ERG amplitude (r = 0.54; r = 0.55), and the VEP peak time (r = 0.55; r = 0.54). CONCLUSIONS: Confirmatory factor analysis allows quantification of the validity of established and new procedures that measure global glaucomatous damage using cross-sectional data. The results are not dependent on the preselection of a specific gold standard. Psychophysical testing and morphometry quantified glaucomatous damage best, compared with electrophysiological procedures. PMID- 10752948 TI - The effects of FK506 on retinal ganglion cells after optic nerve crush. AB - PURPOSE: The purpose of this study was twofold: to determine whether immunophilins were present in the rat retina and to determine the physiologic consequence of their presence. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were performed on rat retinal tissue, and the immunophilin FKBP12 was found to be present in retina. Immunohistochemical studies showed the presence of FKBP12 in retinal ganglion cells (RGCs). In rats, optic nerve crush was performed on one side and a sham operation on the other side. By gavage, animals were given 5 mg/kg per day of the FKBP12 ligand FK506 in sterile phosphate-buffered saline (PBS) or in PBS alone. Eight days after nerve crush, the total number of back-labeled RGCs was estimated from retinal wholemounts. RESULTS: In control eyes, the number of labeled ganglion cells was 74,104 +/- 4,166 (mean +/- SEM) in rats receiving vehicle and 74,993 +/- 3,098 in animals receiving FK506 daily. Eight days after optic nerve crush, 27,775 +/- 3,332 labeled ganglion cells were counted in retinas of animals receiving vehicle (n = 11), whereas 33% more ganglion cells (37,118 +/- 2,475) were counted in animals receiving FK506 daily (n = 11). This difference was statistically significant (P < 0.05). CONCLUSIONS: The data presented demonstrate that the immunophilin FKBP12 is present in retina and specifically in RGCs. In addition, the FKBP12 ligand FK506 confers neuroprotection on RGCs after optic nerve crush. This neuroprotection may occur as a result of FK506's ability to interfere with apoptotic mechanisms after optic nerve crush. PMID- 10752949 TI - Differences in virulence between two Candida albicans strains in experimental keratitis. AB - PURPOSE: To study the differences in disease caused by two wild-type strains of Candida albicans in a model of contact lens-facilitated keratitis in rabbits. METHODS: Two strains, SC5314 and VE175, were examined. Standardized inocula were placed on the debrided corneal surface of one eye in Dutch belted rabbits and covered with a contact lens. A temporary tarsorrhaphy was opened after 24 hours with removal of the contact lens. Six days later, corneas were photographed and animals killed. Corneas were bisected with one half for quantitative isolate recovery and the other for stromal penetration by hyphae. RESULTS: Strain SC5314 was significantly more virulent. The mean hyphal penetration into the cornea was 24.4% +/- 8.5% of the corneal thickness, and in three of six corneas hyphae penetrated through the entire cornea. In contrast, for VE175, the mean hyphal penetration was 2.6% +/- 1.2%. The difference between these two strains was statistically significant (P = 0.0297). Hyphae did not penetrate into the deep layers of the cornea in any of the six rabbits infected with VE175. The grading of clinical disease was consistent with histology, in that strain SC5314 caused more severe infection than VE175 and the difference was statistically significant (P = 0.0048). There was no difference in isolate recovery. CONCLUSIONS: Wild-type strains of C. albicans can differ significantly in virulence as measured by depth of fungal invasion into corneas and clinical evaluation of infection. Further characterization of the intrinsic genetic differences between such strains may help identify factors responsible for fungal virulence. PMID- 10752950 TI - Alpha-smooth muscle actin expression in cultured lens epithelial cells. AB - PURPOSE: Lens epithelial cells transdifferentiate to myofibroblasts during the formation of anterior subcapsular cataracts and secondary cataracts. One of the defining characteristics of myofibroblasts is the expression of alpha-smooth muscle actin (alpha-SMA). This study investigated some of the factors that influence alpha-SMA expression in lens epithelial cells. METHODS: Bovine, rabbit, and human lens epithelial explants or cells were cultured with or without serum. Immunohistochemistry and immunoblotting were used to detect and quantitate alpha SMA expression. RESULTS: Cells from all species studied expressed alpha-SMA in primary explant culture with or without serum. Immunostaining for alpha-SMA first appeared in a diffuse granular pattern, then accumulated at the cell cortex, and eventually was detected along stress fibers. When lens epithelial cells migrated onto cell-free regions of the capsule or were transferred to a plastic culture dish, alpha-SMA expression increased significantly. Expression of alpha-SMA positively correlated with cell size and cell migration. CONCLUSIONS: Expression of alpha-SMA is a common feature of cultured mammalian lens epithelial cells. Because alpha-SMA expression occurred without the addition of exogenous factors, the fibrosis seen in anterior subcapsular cataracts or secondary cataracts may reflect the intrinsic properties of lens epithelial cells. Interaction between lens epithelial cells and their substratum appears to be an important regulator of myofibroblast formation. Understanding the factors that regulate alpha-SMA expression in lens epithelial cells could lead to the development of methods for preventing secondary cataracts and anterior subcapsular cataracts. PMID- 10752951 TI - Lens cell populations studied in human donor capsular bags with implanted intraocular lenses. AB - PURPOSE: Posterior capsule opacification is an ongoing cellular redistribution process. The level of viable cell coverage was therefore determined in human donor capsular bags with implanted intraocular lenses, and cellular morphology and ultrastructure were investigated in relation to cell type and level of differentiation. METHODS: Donor capsular bags, retrieved at intervals of 4 months to 13 years after surgery, were investigated by phase optics before fixation. Postfixation techniques included scanning electron microscopy and transmission electron microscopy of sections and immunofluorescent staining of cytoskeletal proteins in wholemounts. RESULTS: All the capsular bags contained a large population of viable cells on the capsular surfaces. Cells on the anterior face of the anterior capsule and in the spaces around the intraocular lens had an elongated morphology and expressed alpha-smooth muscle actin. The cells formed light-scattering, multilayered aggregates and strands that were surrounded by layers of extracellular matrix. The regions between the intraocular lens and the equator of the bags were populated by monolayers of epithelial cells of normal morphology and ultrastructure, on both the anterior and posterior capsules. In some regions the apical surfaces of the two epithelial monolayers were in contact, and in some parts of the equatorial regions, differentiation of cells into well-organized fiberlike cells was evident. CONCLUSIONS: Human capsular bags contain a large population of viable cells for many years after cataract surgery. Cells in the regions around the intraocular lens undergo transition to a mesenchymal type. Cells peripheral to these regions can form a stable closed microenvironment in which both normal epithelial morphology and differentiation to fiberlike cells are maintained. PMID- 10752952 TI - Cholinergic and adrenergic modulation of the Ca2+ response to endothelin-1 in human ciliary muscle cells. AB - PURPOSE: To determine the cholinergic (carbachol, CCH) and adrenergic (norepinephrine, NE) modulation of Ca2+ response to endothelin-1 in human ciliary smooth muscle (HCSM) cells. METHODS: Intracellular calcium levels were measured using the Fura-2 calcium imaging system in HCSM cells treated either singly with endothelin-1 (ET-1; 2-200 nM), CCH (1-100 microM), NE (0.1-10 microM) or isoproterenol (ISO; 1 microM) or in combinations of CCH, NE, or ISO with ET-1. Intracellular cAMP levels after NE and ISO treatments were also measured using a radioimmunoassay. RESULTS: Endothelin-1 dose-dependently increased [Ca2+]i and was characteristically biphasic (peak [Ca2+]i for ET-1: 2 nM, 517 +/- 73 nM; 20 nM, 785 +/- 65 nM; and 200 nM, 2564 +/- 359 nM). Carbachol also dose-dependently increased [Ca2+]i; however, subsequent additions of ET-1 (200 nM) resulted in lower [Ca2+]i (100 microM CCH + ET-1; 300 +/- 21 nM) compared with that observed with 200 nM ET-1 alone (2564 +/- 359 nM). Norepinephrine pretreatment also decreased ET-1-induced [Ca2+]i (10 microM NE + ET-1; 619 +/- 64 nM) compared with ET-1 alone, and NE's effect could be reversed by propranolol (beta-adrenergic antagonist) treatment. Neither CCH nor NE was able to completely abolish ET-1's ability to mobilize calcium in HCSM cells. Isoproterenol (a beta-agonist) mimicked NE's effect on ET-1-induced [Ca2+]i (1 microM ISO + ET-1; 254 +/- 56 nM). Both ISO and NE elevated [cAMP] in HCSM cells. CONCLUSIONS: In HCSM cells, CCH and ET-1 can activate common as well as specific [Ca2+]i pools. The reduction in ET-1-induced [Ca2+]i after NE/ISO treatment appears to be due to elevated cAMP levels via beta-receptor activation, suggesting the existence of receptor cross talk. The ability of CCH and NE to modulate ET-1's actions on HCSM may be relevant to the regulation of ciliary muscle contraction and aqueous humor outflow. PMID- 10752953 TI - Effect of nitric oxide synthase inhibitor on optic nerve head circulation in conscious rabbits. AB - PURPOSE: To study the effect of a nitric oxide synthase inhibitor on tissue circulation in the optic nerve head (ONH) of conscious rabbits. METHODS: N(G) nitro-L-arginine methyl ester (L-NAME) (1, 10, or 100 mg/kg), D-NAME (10 mg/kg), or physiological saline was administered intravenously to albino rabbits. A quantitative index of blood velocity, the normalized blur (NB), was measured in the ONH by laser speckle tissue circulation analyzer. The intraocular pressure (IOP) and blood pressure (BP) were also measured. L-arginine (10 mg/kg) was intravenously administered 20 minutes after L-NAME (10 mg/kg) injection. Acetylcholine (ACh; 10 microg/kg per minute) was infused for 15 minutes, with or without pretreatment of L-NAME (1 mg/kg). RESULTS: L-NAME induced a continuous decrease of the NB in a dose-dependent manner, but D-NAME caused no significant change. At 100 mg/kg, L-NAME significantly increased the IOP, mean BP, and ocular perfusion pressure, but the other doses caused no significant changes. When L arginine was administered after L-NAME injection, the NB returned to its initial level and remained there. Pretreatment with L-NAME inhibited the increase of NB induced by ACh. CONCLUSIONS: These results indicate that nitric oxide regulates basal tissue circulation in the ONH of conscious rabbits and suggest that ACh increases the circulation by promoting nitric oxide synthesis. PMID- 10752955 TI - Characterization and localization of the rabbit ocular calcitonin gene-related peptide (CGRP)-receptor component protein (RCP). AB - PURPOSE: To determine whether the calcitonin gene-related peptide (CGRP) receptor component protein (RCP), a novel signal transduction molecule, is required for CGRP signaling in the eye and to determine potential ocular sites of CGRP action. METHODS: The cDNA for the rabbit ocular RCP homologue was cloned using a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Function of the rabbit ocular RCP was assessed using a sensitive oocyte-based assay, which utilizes the protein kinase A (PKA) sensitive cystic fibrosis transmembrane conductance regulator (CFTR) as a sensor of cAMP formation. RCP expression in the rabbit eye was localized using immunohistochemistry. RESULTS: A 2063-bp cDNA for the rabbit ocular RCP was cloned and sequenced. Expression of the rabbit RCP cDNA confers CGRP responsiveness in a sensitive oocyte-based assay. Antisense oligonucleotides made to the ocular RCP abolishes CGRP responsiveness of ciliary body and iris mRNA in the oocyte-CFTR assay. Localization of RCP protein in the rabbit eye using immunohistochemistry demonstrated RCP immunoreactivity in the ciliary body and iris blood vessels, as well as in layers of the ciliary epithelium. CONCLUSIONS: The rabbit ocular RCP appears to be required for signal transduction at ocular CGRP receptors and is localized to sites previously reported to bind CGRP, which affect intraocular pressure and neurogenic inflammation. PMID- 10752954 TI - Integrin-mediated neutrophil adhesion and retinal leukostasis in diabetes. AB - PURPOSE: A critical early event in the pathogenesis of diabetic retinopathy is leukocyte adhesion to the diabetic retinal vasculature. The process is mediated, in part, by intercellular adhesion molecule-1 (ICAM-1) and results in blood retinal barrier breakdown and capillary nonperfusion. This study evaluated the expression and function of the corresponding ICAM-1-binding leukocyte beta2 integrins in experimental diabetes. METHODS: Diabetes was induced in Long Evans rats with streptozotocin. The expression of the surface integrin subunits CD11a, CD11b, and CD18 on rat neutrophils isolated from peripheral blood was quantitated with flow cytometry. In vitro neutrophil adhesion was studied using quantitative endothelial cell-neutrophil adhesion assays. The adhesive role of the integrin subunits CD11a, CD11b, and CD18 was tested using specific neutralizing monoclonal antibodies. CD18 bioactivity was blocked in vivo with anti-CD18 F(ab')2 fragments, and the effect on retinal leukocyte adhesion was quantitated with acridine orange leukocyte fluorography. RESULTS: Neutrophil CD11a, CD11b, and CD18 surface integrin levels were 62% (n = 5, P = 0.006), 54% (n = 5, P = 0.045), and 38% (n = 5, P = 0.009) greater in diabetic versus nondiabetic animals, respectively. Seventy-five percent more neutrophils from diabetic versus nondiabetic animals adhered to rat endothelial cell monolayers (n = 6, P = 0.02). Pretreatment of leukocytes with either anti-CD11b or anti-CD18 antibodies lowered the proportion of adherent diabetic neutrophils by 41% (n = 6, P = 0.01 for each treatment), whereas anti-CD11a antibodies had no significant effect (n = 6, P = 0.5). In vivo, systemic administration of anti-CD18 F(ab')2 fragments decreased diabetic retinal leukostasis by 62% (n = 5, P = 0.001). CONCLUSIONS: Neutrophils from diabetic animals exhibit higher levels of surface integrin expression and integrin-mediated adhesion. In vivo, CD18 blockade significantly decreases leukostasis in the diabetic retinal microvasculature. Integrin adhesion molecules may serve as therapeutic targets for the treatment and/or prevention of early diabetic retinopathy. PMID- 10752956 TI - LEDGF: survival of embryonic chick retinal photoreceptor cells. AB - PURPOSE: Lens epithelium-derived growth factor (LEDGF) is a novel adhesive, survival, and growth factor for lens epithelial cells, keratinocytes, fibroblasts, and cos7 cells. In the presence of LEDGF, these cells acquire resistance to environmental stresses, and in the absence of LEDGF they die. The effects of LEDGF on survival of embryonic chick retinal photoreceptor cells under serum starvation and heat stress were studied. METHODS: The expression pattern of LEDGF in embryonic chick retinal photoreceptor cells was investigated with protein blot analysis and immunohistochemistry using antibodies (Abs) to LEDGF. Retinal cells were cultured in serum-free medium for up to 6 days in the presence of varying amounts of LEDGF at 37 degrees or 41 degrees C. The photoreceptor cells were immunostained with Abs to arrestin and counted to evaluate the photoreceptor cell viability. Heat shock proteins in the cultured cells were quantified by protein blot analysis with Ab probes and semiquantitative reverse transcription-polymerase chain reaction analysis. RESULTS: LEDGF was found predominantly in the nucleus of neuroretinal cells, including photoreceptor cells. In the presence of LEDGF, photoreceptor cells manifested increased resistance to serum starvation and thermal stress and survived for a longer period. The levels of heat shock protein 90 were elevated in those cells. Most retinal cells died in the absence of LEDGF. CONCLUSIONS: LEDGF enhanced survival of retinal photoreceptor cells under serum starvation and heat stress. Thus, LEDGF has a potency to enhance survival of neuronal cell types against environmental stresses, and it may be applicable as a therapeutic agent for those cells. PMID- 10752957 TI - Aminoguanidine and the effects of modified LDL on cultured retinal capillary cells. AB - PURPOSE: Compared with normal low density lipoprotein (N-LDL), LDL minimally modified in vitro by glycation, minimal oxidation, or glycoxidation (G-, MO-, GO LDL) decreases survival of cultured retinal capillary endothelial cells and pericytes. Similar modifications occurring in vivo in diabetes may contribute to retinopathy. The goal of this study was to determine whether low concentrations of aminoguanidine might prevent cytotoxic modification of LDL and/or protect retinal capillary cells from previously modified LDL. METHODS: Minimal in vitro modification of LDL (3 days, 37 degrees C) was achieved with glucose (0, 50 mM), under antioxidant conditions (for N-LDL, G-LDL), or under mild oxidant conditions (for MO-, GO-LDL) in the presence/absence of aminoguanidine (0, 1, 10, 100 microM). Glucose and aminoguanidine were then removed by dialysis. Confluent bovine retinal capillary endothelial cells (n = 13) and pericytes (n = 14) were exposed to LDL (100 mg/l) for 3 days, with and without aminoguanidine (100 microM) in media. Cell counts were determined by hemocytometer. RESULTS: A decrease in cell counts after exposure to modified compared with N-LDL was confirmed (P < 0.001) but was significantly mitigated if LDL had been modified in the presence of aminoguanidine (P < 0.001). Aminoguanidine was as effective at 1 microM as at the higher concentrations. Aminoguanidine (100 microM) present in culture media conferred no additional protection, and showed slight evidence of toxicity. Aminoguanidine present during LDL modification had no effect on measured glycation or oxidation products, or on LDL oxidizability. CONCLUSIONS: Very low concentrations of aminoguanidine mitigate toxicity of LDL exposed to stresses that simulate the diabetic environment. This action may contribute to the beneficial effects of aminoguanidine observed in experimental diabetic retinopathy. PMID- 10752958 TI - Diffusion of high molecular weight compounds through sclera. AB - PURPOSE: To determine the in vitro permeability of the sclera to high molecular weight compounds and the relationship between scleral permeability and molecular size. METHODS: Fresh rabbit sclera was mounted in a two-chamber diffusion apparatus, and its permeability to sodium fluorescein, fluorescein isothiocyanate (FITC)-conjugated bovine serum albumin, FITC-IgG, and FITC dextrans ranging in molecular weight from 4 to 150 kDa was determined by fluorescence spectrophotometry. Electron microscopy was used to assess the impact of the experimental design on scleral ultrastructural integrity. The effect of the diffusion apparatus on scleral hydration was examined. Rabbit scleral permeability was compared with previously reported data for human and bovine sclera. RESULTS: Scleral permeability decreased with increasing molecular weight and molecular radius, consistent with previous human and bovine data. Molecular radius was a better predictor of scleral permeability than molecular weight. The sclera was more permeable to globular proteins than to linear dextrans of similar molecular weight. The experimental apparatus did not alter scleral ultrastructure. Permeability of rabbit sclera was similar to human sclera but greater than bovine sclera. CONCLUSIONS: Large molecules, such as IgG, diffuse across sclera in a manner consistent with porous diffusion through a fiber matrix. Transscleral delivery of immunoglobulins and other large compounds to the choroid and retina may be feasible. PMID- 10752959 TI - Transscleral delivery of bioactive protein to the choroid and retina. AB - PURPOSE: To investigate the feasibility of transscleral drug delivery to the choroid and retina. METHODS: An osmotic pump was used to deliver IgG across the sclera of pigmented rabbits, and levels were measured in the choroid, retina, vitreous humor, aqueous humor, orbit, and plasma over 28 days. This method was then used to deliver an anti-intercellular adhesion molecule-1 (ICAM-1) monoclonal antibody (mAb), and its effect on inhibiting vascular endothelial growth factor (VEGF)-induced leukostasis in the choroid and retina was determined by measuring tissue myeloperoxidase (MPO) activity. RESULTS: Levels of retinal and choroidal IgG were significantly higher than baseline at all points up to 28 days (P < or = 0.01). IgG levels in the orbit, vitreous humor, aqueous humor, and plasma were negligible (P > 0.05). MPO activity in the choroid of eyes treated with anti-ICAM-1 mAb was 80% less (P = 0.01) than in eyes receiving an equal rate of delivery of an isotype control antibody. Inhibition of MPO activity in the retina was 70% (P = 0.01). The plasma concentration of anti-ICAM-1 mAb was 31,000 fold less than the concentration in the osmotic pump. CONCLUSIONS: Minimally invasive transscleral delivery can be used to deliver therapeutic levels of bioactive drugs to the choroid and retina with negligible systemic absorption. This method of ocular drug delivery may be used in the treatment of a variety of chorioretinal disorders. PMID- 10752960 TI - Angiotensin II-stimulated vascular endothelial growth factor expression in bovine retinal pericytes. AB - PURPOSE: Angiotensin II (AII) has been shown to play a role in many vascular diseases. In the study described, the effect of AII on vascular endothelial growth factor (VEGF) expression and related intracellular signaling mechanism was investigated in bovine retinal microcapillary pericytes. METHODS: Cultured bovine retinal microvascular endothelial cells and pericytes were prepared. VEGF expression was determined by Northern blot analysis and immunoprecipitation assay. Cell proliferation was assessed by DNA content growth assay. Reporter gene studies were performed to identify the AII responsible transcription-activating region of VEGF gene. RESULTS: Angiotensin II induced a significant increase in VEGF mRNA in a time- and dose-dependent manner. Angiotensin II type I receptor antagonist inhibited this effect. Angiotensin II activates the transcription of VEGF gene without changing the mRNA half-life, and the AII responsible region was found in the 5'-flanking region of the VEGF gene. Angiotensin II also increased the expression of c-fos and c-jun mRNA, and antisense oligonucleotides against c Fos blocked the AII-induced VEGF mRNA expression. The conditioned media of AII stimulated pericyte cultures had a growth-promoting effect on endothelial cells, and this effect was inhibited almost completely by VEGF neutralizing antibody. CONCLUSIONS: These findings suggest that AII might induce angiogenic activity through a paracrine function of VEGF in retinal microvascular cells. PMID- 10752961 TI - Lutein and zeaxanthin concentrations in rod outer segment membranes from perifoveal and peripheral human retina. AB - PURPOSE: In addition to acting as an optical filter, macular (carotenoid) pigment has been hypothesized to function as an antioxidant in the human retina by inhibiting the peroxidation of long-chain polyunsaturated fatty acids. However, at its location of highest density in the inner (prereceptoral) layers of the foveal retina, a specific requirement for antioxidant protection would not be predicted. The purpose of this study was to determine whether lutein and zeaxanthin, the major carotenoids comprising the macular pigment, are present in rod outer segment (ROS) membranes where the concentration of long-chain polyunsaturated fatty acids, and susceptibility to oxidation, is highest. METHODS: Retinas from human donor eyes were dissected to obtain two regions: an annular ring of 1.5- to 4-mm eccentricity representing the area centralis excluding the fovea (perifoveal retina) and the remaining retina outside this region (peripheral retina). ROS and residual (ROS-depleted) retinal membranes were isolated from these regions by differential centrifugation and their purity checked by polyacrylamide gel electrophoresis and fatty acid analysis. Lutein and zeaxanthin were analyzed by high-performance liquid chromatography and their concentrations expressed relative to membrane protein. Preparation of membranes and analysis of carotenoids were performed in parallel on bovine retinas for comparison to a nonprimate species. Carotenoid concentrations were also determined for retinal pigment epithelium harvested from human eyes. RESULTS: ROS membranes prepared from perifoveal and peripheral regions of human retina were found to be of high purity as indicated by the presence of a dense opsin band on protein gels. Fatty acid analysis of human ROS membranes showed a characteristic enrichment of docosahexaenoic acid relative to residual membranes. Membranes prepared from bovine retinas had protein profiles and fatty acid composition similar to those from human retinas. Carotenoid analysis showed that lutein and zeaxanthin were present in ROS and residual human retinal membranes. The combined concentration of lutein plus zeaxanthin was 70% higher in human ROS than in residual membranes. Lutein plus zeaxanthin in human ROS membranes was 2.7 times more concentrated in the perifoveal than the peripheral retinal region. Lutein and zeaxanthin were consistently detected in human retinal pigment epithelium at relatively low concentrations. CONCLUSIONS: The presence of lutein and zeaxanthin in human ROS membranes raises the possibility that they function as antioxidants in this cell compartment. The finding of a higher concentration of these carotenoids in ROS of the perifoveal retina lends support to their proposed protective role in age-related macular degeneration. PMID- 10752962 TI - IL-10 and antibodies to TGF-beta2 and PDGF inhibit RPE-mediated retinal contraction. AB - PURPOSE: Retinal pigment epithelial (RPE) cells are believed to play a pivotal role in the formation and contraction of epiretinal membranes in proliferative vitreoretinopathy (PVR). In the present study, an organ culture method was used that mimics the contractile stage of PVR, to investigate the contribution of a variety of growth factors in human RPE cell-mediated contraction of the retina. METHODS: Cultured human RPE cells were seeded onto bovine retinal explants. After attachment, cultures received one of the following exogenous growth factors: platelet-derived growth factor (PDGF)-AB, PDGF-BB, basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-beta1, TGF-beta2, or interleukin (IL) 10; or a neutralizing antibody to PDGF and/or TGF-beta2. Control explants were either untreated or received a null antibody. Contraction was assessed by image analysis and expressed as percentage reduction in retinal area. RESULTS: RPE cells produced a more than 50% contraction of the retina after 7 days in untreated samples. PDGF and TGF-beta2 stimulated RPE-mediated contraction by a further 20% at 100 ng/ml. IL-10 decreased contraction by 63%, whereas the other growth factors gave rise to similar contraction to untreated controls. Neutralizing antibodies against PDGF and TGF-beta2 reduced RPE-mediated contraction by up to 70% in comparison with untreated controls. The neutralizing antibodies also inhibited the effects of exogenous PDGF and TGF-beta2 on RPE mediated contraction of the retina (P < 0.01). CONCLUSIONS: These findings confirm a role for both PDGF and TGF-beta2 in RPE cell-mediated contraction of the retina. Such contraction can be inhibited by neutralizing antibodies against PDGF and TGF-beta2, which, together with IL-10, are putative candidates for therapeutic intervention in PVR. PMID- 10752963 TI - Vascularization of the human fetal retina: roles of vasculogenesis and angiogenesis. AB - PURPOSE: To characterize the topography of and the cellular processes that underlie vascularization of the human retina. METHODS: The vasculature of human eyes obtained from fetuses ranging in age from 14 to 38 weeks of gestation (WG) was examined in Nissl-stained, whole-mount preparations and by anti-CD34 immunohistochemistry. RESULTS: The first event in retinal vascularization, apparent before 15 WG, was the migration of large numbers of spindle-shaped mesenchymal precursor cells from the optic disc. These cells proliferated and differentiated to produce cords of endothelial cells. By 15 WG, some cords were already patent and formed an immature vascular tree in the inner retinal layers that was centered on the optic disc. These processes are consistent with vessel formation by vasculogenesis. Angiogenesis then increased the vascular density of this immature plexus and extended it peripherally and temporally. Maturation of the plexus was characterized by substantial remodeling, which involved the withdrawal of endothelial cells into neighboring vascular segments. The outer plexus was formed as a result of the extension of capillary-sized buds from the existing inner vessels, a process that began around the incipient fovea between 25 and 26 WG. CONCLUSIONS: These observations suggest that the formation of primordial vessels in the central retina is mediated by vasculogenesis, whereas angiogenesis is responsible for increasing vascular density and peripheral vascularization in the inner retina. In contrast, the outer plexus and the radial peripapillary capillaries are formed by angiogenesis only. These mechanisms of retinal vascularization appear similar to those of vascularization of the central nervous system during development. PMID- 10752964 TI - Pupil perimetry using M-sequence stimulation technique. AB - PURPOSE: M-sequence stimulation technique allows mapping of the retinal function by multifocal electroretinographic (ERG) recordings. However, the information provided about visual field is limited to retinal function. Optic nerve diseases and diseases of the higher visual pathways usually show normal multifocal ERGs. Using pupillary responses instead of the electrical retinal responses might enhance the diagnostic possibilities of this system. The problems of local ERG recordings are very similar to those encountered in pupil perimetry: Local stimuli have to be dim to avoid or at least reduce stray-light responses. Dim stimuli, close to the absolute threshold, elicit only subtle pupillomotor responses. Therefore, techniques that are able to detect small focal responses are promising. METHODS: Pupillography was done by means of an infrared video camera and real time image processing (50 Hz) using a custom-designed videoboard in a personal computer (486). Recording conditions: The stimulus was presented on a monitor (75 Hz) in 26 cm distance from the patient's eyes. It contained 37 hexagons in a 25 degrees visual field. Each element changed between black (1.6 cd/m2) and white (160 cd/m2) after a binary M-sequence independently from other elements. Four thousand ninety six different stimulus pictures of 120-msec duration were shown during a single pupillogram recording. Thirty-seven local pupillograms were calculated in a cross-correlation of stimulus sequence and the pupil diameter. RESULTS: The pupillomotor fields in normals showed a shape and sensitivity distribution as known from conventional pupil perimetry techniques. Artificial paracentral scotomas (5 degrees) created by masking different locations could be demonstrated convincingly. Even in patients with optic nerve lesions it was possible to demonstrate visual field defects. CONCLUSIONS: Pupil perimetry using the M-sequence technique is a promising method of objective perimetry that may find its entrance into clinical application. PMID- 10752965 TI - Visual field losses in subjects with migraine headaches. AB - PURPOSE: To characterize the visual fields of subjects with migraine headaches using static and temporal modulation perimetry. METHODS: Sixteen subjects with migraines (15 with aura, 1 without) and 15 nonheadache controls were tested. Perimetry was conducted 7 days after the offset of a headache with both static and temporally modulated targets using the Medmont M-600 automated perimeter (Medmont Pty Ltd., Camberwell, Victoria, Australia). Flicker thresholds were measured using the autoflicker test, which varies flicker rate with eccentricity. A subset of four subjects with migraines (3 with aura, 1 without) had the temporal tuning characteristics of their loss evaluated using fixed temporal frequencies (4, 6, 9, 12, and 16 Hz). RESULTS: Field losses were identified with temporal modulation perimetry in 11 of 16 migraine subjects. The majority of these losses occurred in the presence of normal static thresholds (8/11). The deficits displayed temporal tuning, being greatest for higher temporal frequencies (> or =9 Hz). None of the subjects revealed deficits typical of cortical lesions. The migraine-without-aura subject displayed a selective loss to temporally modulated stimuli, which was consistent with the aura group. This defect altered over time, decreasing for 30 to 40 days but remaining, to a smaller extent, for up to 75 days after the headache event. CONCLUSIONS: Visual dysfunction that is selective for temporally modulated targets occurs in migraine subjects. The migrainous pattern of dysfunction shares some features with that identified in early stages of glaucoma and raises the possibility for a common precortical vascular involvement in these two conditions. PMID- 10752966 TI - Are the goose and gander cooked in the same sauce? PMID- 10752967 TI - Relation of gender and health insurance to cardiovascular procedure use in persons with progression of chronic renal disease. AB - BACKGROUND: Women often are less likely than men to receive diagnostic and therapeutic invasive procedures for coronary disease. OBJECTIVE: To examine the relation between gender, health insurance, and access to cardiovascular procedures over time in persons with chronic illness. RESEARCH DESIGN: Seven-year longitudinal analyses in a cohort from the United States Renal Data System. SUBJECTS: National random sample of women and men who progressed to end-stage renal disease (ESRD) in 1986 to 1987 and were treated at 303 dialysis facilities (n = 4,987). MEASURES: Medical history and utilization records, physical examination, and laboratory data. MAIN OUTCOME MEASURES: Receipt of a coronary catheterization or revascularization procedure before (baseline) and after (follow-up) the development of ESRD and acquisition of Medicare, adjusted for clinical and socioeconomic variables. RESULTS: At baseline, 5.2% of women and 9.2% of men had undergone a cardiac procedure; the odds of women receiving a procedure were one third lower than for men (adjusted odds ratio 0.66 [95% CI 0.49-0.88]). During follow-up, women were just as likely as men to undergo a procedure (adjusted odds ratio 0.94 [95% CI 0.74-1.20]). Compared with men with baseline private insurance, men and women with other and no insurance had 34% to 81% lower odds of receiving procedures at baseline. Women with private insurance had 42% lower odds of having a procedure at baseline compared with men (adjusted odds ratio 0.58 [95% CI 0.42-0.78]) but had the same odds at follow-up (adjusted odds ratio 1.09 [95% CI 0.82-1.45]). At follow-up, gender differences in procedure use were eliminated for groups with baseline Medicaid or no insurance. CONCLUSIONS: Overall gender differences in cardiac procedure use were narrowed markedly after progression of a serious illness, the assurance of health insurance, and entry into a comprehensive care system. Gender disparities in procedure use for different baseline insurance groups were largely equalized in follow-up. These findings suggest that provision of insurance with disease managed care for a chronic disease can provide equalized access to care for women. PMID- 10752968 TI - Are preferences for equity over efficiency in health care allocation "all or nothing"? AB - BACKGROUND: In a previous study we showed that within a budget constraint, most people would rather offer a less effective screening test to 100% of a Medicaid population, thereby saving 1,000 lives, than a more effective test to 50% of the population, thereby saving 1,100 lives. We present here a study exploring whether this preference for equity over efficiency persists when neither test can be offered to the entire population. METHODS: Members of Physicians' Online and prospective jurors at the Philadelphia County Courthouse randomly received 1 of 3 questionnaires (Q1, Q2, or Q3) describing a limited budget to screen Medicaid enrollees for colon cancer. In all questionnaires, test 1 was said to save 1,000 lives, and test 2, a more effective and more expensive test, was said to save 1,100. In Q1, test 1 was offered to 100% and test 2 to 50% of the population. In Q2, the 2 tests could be offered to 50% and 25%, respectively; in Q3, to 90% and 40%, respectively. Respondents indicated which test they recommended and provided justification. RESULTS: The majority of physicians (59%) and the general public (56%) receiving Q1 favored the less effective screening test However, of those receiving Q2, only 26% of physicians and 27% of the general public recommended the less effective screening test. And of those receiving Q3, only 38% of physicians and 28% of the general public recommended the less effective test. Justifications for these recommendations were based largely on concerns for equality of treatment among those who chose the less effective test and concerns for saving the most lives among those who preferred the more effective test. CONCLUSIONS: Although most respondents show a preference for equity over efficiency when equity means that 100% of a population can receive a service, many fewer respondents maintain this preference when equity is no longer absolute. This result suggests that the preference for equity is sometimes "all or none." PMID- 10752969 TI - A comparison of response error by adolescents and adults: findings from a health care study. AB - OBJECTIVES: Most assessments of response error have focused only on reports by adult respondents; we know little about the propensity to misreport or the magnitude or direction of response error for self-reports by adolescents. To the extent that adults and adolescents differ in the magnitude or direction of response error, comparisons between adults and adolescents will be confounded. This study compares the quality of self-response among adolescents and adults in their reporting of health conditions and health care utilization. METHODS: Approximately 2,000 members of an HMO were interviewed, and by use of medical record information, the accuracy of their responses was assessed for several measures: number of outpatient visits, date of the most recent outpatient visit, site of care, type of professional seen during the last visit, and an indicator as to whether the respondent had asthma. RESULTS: The findings indicate that both the magnitude and the direction of response error vary by the age of the respondent. In multivariate models predicting erroneous response, adolescents were more likely to misreport than were adult respondents. In addition, among erroneous respondents, adolescents tended to err in the direction of overreporting, whereas adult respondents tended to err in the direction of underreporting. CONCLUSIONS: Although the results should be viewed cautiously because of limitations in both the sample design and the survey topic, the findings suggest that substantive comparisons of health care utilization between adolescents and adults may be confounded by differential measurement error. PMID- 10752970 TI - Can history and physical examination be used as markers of quality? An analysis of the initial visit note in musculoskeletal care. AB - BACKGROUND: The medical record serves as an important source of information regarding the care process, but few studies have examined whether thoroughness of documentation is associated with outcomes. OBJECTIVE: The objectives of this study were to analyze the initial visit note for 513 patients presenting with acute musculoskeletal pain, compare thoroughness of documentation by physician specialty, and determine whether thoroughness of documentation was associated with clinical improvement or patient satisfaction. METHODS: A structured medical record abstraction was performed to examine whether treating physicians documented key historical and physical exam findings. Satisfaction with care, symptom relief, and functional improvement were assessed after 3 months with validated survey instruments. RESULTS: In the initial visit note, 43+/-16% of selected historical findings and 28+/-17% of physical examination findings were documented. Orthopedic surgeons documented 2 to 4 more historical and physical examination items (P <0.01) and assigned more specific diagnoses (P <0.01) than rheumatologists and general internists. Multivariate models showed a very weak association between all aspects of documentation and patient satisfaction with the provider-patient interaction (all partial R2 <0.016) and no association between documentation and 3-month pain relief or functional status. Patients' perception of physician communication was more highly associated with patient satisfaction (P = 0.0001) than was documentation. CONCLUSIONS: No provider types consistently documented many important historical items and physical examination findings. While thoroughness of documentation was not associated with clinical outcomes, there was a very weak relationship between documentation and patient satisfaction with provider-patient interactions. PMID- 10752971 TI - The role of physician recommendation in women's mammography use: is it a 2-stage process? AB - OBJECTIVE: To explore the use of a 2-stage model in explaining the role of physician recommendation in women's use of mammography screening and to provide an integrated framework for understanding the mammography-use process. METHODS: Data on 1,301 women aged > or =52 years from the North Carolina Breast Cancer Screening Program (NC-BCSP) questionnaire were combined with information from 91 of their physicians from the North Carolina Medical Board. A 2-stage system of equations using women's characteristics (demographic, health, access), physicians' characteristics (demographic, practice), women's beliefs, and women's report of a physician recommendation was created and statistically tested. The model was estimated using 2-stage logistic and probit estimation. RESULTS: The 2 stage approach produced different results compared with the single pooled model. In the second-stage mammography-use model, younger age, family history of breast cancer, and a woman's having ever requested a mammogram retained significance (P < or = 0.05) in addition to the predicted value of physician recommendation obtained from the first stage. Women's characteristics significantly associated with physician recommendation in the first stage included some access, health risk, and demographic variables as well as physician age and race (P < or = 0.05). CONCLUSIONS: A 2-stage model for estimating mammography use among women with regular physicians may be more informative than a single model. These results imply that programs designed to increase mammography rates should focus on ensuring appropriate physician recommendations in addition to encouraging women to request screening. Future research should consider using an integrated framework for evaluating utilization of mammography and other preventive services. PMID- 10752972 TI - Relationship between CD4 count, viral burden, and quality of life over time in HIV-1-infected patients. AB - BACKGROUND: Although surrogate markers such as CD4 counts and viral burden (HIV-1 RNA) are predictive of AIDS-related disease progression, little is known about the relationship between changes in surrogate markers and health-related quality of life (HRQOL) outcomes. This study investigated how changes in CD4/mm3 and viral burden (RNA copies/mL) are related to changes in HRQOL as indexed by the Medical Outcomes Study HIV Health Survey (MOS-HIV-30). METHODS: Subjects were HIV 1-infected patients with CD4 counts <300/mm3 enrolled in a double-blind, randomized clinical trial of delavirdine. As part of the clinical protocol, patients completed the MOS-HIV-30, from which the Physical Health (PHS) and Mental Health (MHS) summary scores were used for analyses. HRQOL and surrogate marker data assessed up to 2 years after randomization were analyzed for a total of 1,112 patients. RESULTS: Individual patients' initial status (intercepts) and rates of change (slopes) over time for log CD4, log RNA, PHS, and MHS were estimated with the use of empirical Bayes. Early response to treatment correlated with HRQOL better for RNA than for CD4. However, the relationship between weekly change and HRQOL was stronger for CD4 than for RNA. CONCLUSIONS: Surrogate markers are significantly associated with HRQOL outcomes. Improvements in HRQOL over time are associated with lower initial viral load and with increases in CD4 counts. Limitations concerning the restricted variability of the change scores are addressed. PMID- 10752973 TI - Agreement of Medicare claims and tumor registry data for assessment of cancer related treatment. AB - BACKGROUND: Although health claims data are increasingly used in evaluating variations in patterns of cancer care and outcomes, little is known about the comparability of these data with tumor registry information. OBJECTIVES: To evaluate the agreement between Medicare claims and tumor registry data in measuring patterns of diagnostic and therapeutic procedures for older cancer patients. RESEARCH DESIGN: Analysis of a database linking Surveillance, Epidemiology and End Results (SEER) registry data and Medicare claims in patients aged > or =65 years with cancer. SUBJECTS: 361,255 Medicare patients with invasive breast, colorectal, endometrial, lung, pancreatic, and prostate cancer diagnosed between 1984 and 1993. MEASURES: Concordance of SEER files with corresponding Medicare claims. RESULTS: Medicare claims generally identified patients who underwent resection and radical surgery according to SEER (ie, concordance > or =85%-90%) but less likely biopsy or local excision (ie, concordance < or =50%). In some instances, claims also categorized patients as having more invasive surgery than was listed in SEER and also provided incremental information about the use of surgical treatment after 4 months. SEER files and, to a lesser degree, Medicare claims identified radiation therapy not included in the other data source, and Medicare files also captured a significant number of patients with codes for chemotherapy. CONCLUSIONS: Medicare files may be appropriate for studies of patterns of use of surgical treatment, but not for diagnostic procedures. The potential benefit of Medicare claims in identifying delayed surgical intervention and chemotherapy deserves further study. PMID- 10752974 TI - Comparison of risk-adjustment systems for the medicaid-eligible disabled population. AB - OBJECTIVE: The objective of this study was to compare 2 approaches for subjecting capitation rates for disabled Medicaid-eligible patients in managed care plans to risk adjustment, the Disability Payment System (DPS) and the Ohio Prior Expenditure System (OPES). DESIGN: This was a retrospective cohort. SETTING AND SUBJECTS: The subjects were 157,142 nonelderly disabled individuals eligible for > or =1 month during state fiscal year 1995 (SFY95) for a 3-county Ohio Medicaid managed care demonstration project. DATA SOURCE: Data were from the Ohio Medicaid eligibility and fee-for-service claims files. ANALYSIS: As per OPES policy, individuals were classified by the duration of their eligibility in SFY93 as "old" eligibles (> or =6 months) or "new" eligibles (<6 months). Published relative payment weights for each system were adjusted and used to predict SFY95 expenditures in a budget-neutral comparison. Measures were variance in SFY95 expenditures explained by predicted payments (R2) and predictive ratios (predicted payment/actual SFY95 expenditure). Individuals with HIV/AIDS and hematological conditions, who enrolled disproportionately across the demonstration counties, were analyzed separately. RESULTS: Of the 157,142 individuals, 56.4% were new eligibles; 40.1% of the old eligibles had no claims documented chronic disease diagnosis in the baseline year. The overall R2 was 0.091 with OPES and 0.057 with DPS. Neither system predicted >1% of individual level expenditures for new eligibles. OPES severely underpaid for eligibles in the top percentile of predicted expenditures; DPS had mixed results. DPS predicted SFY95 expenditures substantially better than OPES for the enrollment bias categories. CONCLUSIONS: Before Medicaid programs move to full-risk capitation for disabled populations, better risk-adjustment methods are needed, especially for eligible patients with little claims experience, high predicted expenditures, or enrollment-bias conditions. PMID- 10752975 TI - Effects of medicaid managed care on adults. AB - BACKGROUND: Despite the rapid growth in Medicaid managed care (MMC) during the 1990s, only limited research exists on how such care affects beneficiaries. OBJECTIVE: The objective of this study was to assess how switching from a fee-for service (FFS) delivery system to managed care affects Medicaid beneficiaries' access to, use of, quality of, and satisfaction with health care services. METHODS: Using a quasi-experimental design framework, we compared the experiences of 540 Minnesota Medicaid recipients living in counties that had switched to managed care with those of 528 recipients living in counties operating under FFS. The data for the analysis came from a 1998 survey of Minnesota Medicaid clients. Data were analyzed by logit regression. RESULTS: We find limited effects of MMC on access to, use of, quality of, and satisfaction with health care. Among others, we found no significant differences between the share of managed care and FFS enrollees (78.5% versus 76%) who had a health care visit during the last year. We also found no evidence of a significant reduction in the proportion of managed care and FFS enrollees (17.6% versus 17%) who had had a hospital stay during the past year. The results did show some negative effects of MMC on satisfaction with care, the most consistent being that managed care enrollees are somewhat less satisfied with their health care than their FFS counterparts. CONCLUSIONS: Our results suggest that a shift from FFS to MMC did not fundamentally change the patterns of health care service use, the location at which care was delivered, or quality. PMID- 10752976 TI - Experience of primary care by racial and ethnic groups. PMID- 10752977 TI - Hematopoietic cell transplantation for primary systemic amyloidosis: what have we learned. AB - Dose-intensive therapy with hematopoietic cell transplantation is effective at reversing AL amyloidosis but is not without risk. Guidelines have been developed for patient selection in order to maximize benefit and minimize treatment-related mortality. Identification of a patient's clonal germline light chain variable region gene may become relevant to patient selection, and development of less morbid approaches to stem cell mobilization and collection would be helpful. While there is room for discussion regarding the design of future therapeutic trials, it is reasonable to attempt to improve the complete response rate for good risk patients by continuing efforts on the phase II level. Attempts to improve outcomes for patients with symptomatic cardiac or advanced multisystem disease may require serial solid organ and stem cell transplantation as well as the development of less toxic approaches using lower doses of melphalan, improved supportive care measures and specific organ-system prophylaxis. If outcomes can be improved, issues related to clonotypic contamination of stem cells will need to be revisited. PMID- 10752978 TI - Interleukin 8 (IL-8) and the release of leukocytes from the bone marrow. AB - Interleukin 8 (IL-8) is produced by various cells upon stimulation and influences a variety of functions of leukocytes in particular neutrophils. Systemic administration of IL-8 induces a rapid neutropenia associated by sequestration of neutrophils in the lung that is followed by a neutrophilia characterized by the rapid release of neutrophils from the bone marrow. These cells are released predominantly from the bone marrow venous sinusoids. In addition, several studies have shown the potential role of IL-8 in hematopoiesis and trafficking of hematopoietic stem cells. Systemic administration of IL-8 induces a rapid mobilization of progenitors from the bone marrow with long-term myelo-lymphoid repopulation capacity. It has been employed clinically to mobilize hematopoietic progenitor cells into the peripheral blood and used for autologous or allogeneic bone marrow transplantation. The mechanism for these effects of IL-8 is largely speculative. This report summarizes current ideas on the possible mechanisms how IL-8 influences cell trafficking in and from the bone marrow. PMID- 10752980 TI - The role of FAS-mediated apoptosis in chronic myelogenous leukemia. AB - Clinical observation and laboratory evidence suggest that immune mechanisms play an important role in the natural control of evolution of the Ph+ clone in chronic phase as well as during progression of chronic myelogenous leukemia (CML). The understanding of these mechanisms could facilitate development of innovative therapeutic approaches. Due to bcr-abl translocation, CML cells carry an intrinsic resistance to apoptotic signals. However, resistance to apoptosis is not absolute and can be overcome through enhancement of immune-mediated pathways, e.g., during graft vs. leukemia reaction after allogeneic bone marrow transplantation, or during interferon-alpha (IFN-alpha) therapy. Among the effector mechanisms, T-lymphocyte-mediated killing of target cells via Fas receptor (Fas-R) triggering plays an important role in the elimination of malignant cells, including CML cells. Although CML Ph+ progenitor cells express Fas-R, the expression levels are variable and do not correlate with clinical parameters. In addition, CML progenitor cells also express functional Fas-ligand (Fas-L), which may be an important immune surveillance escape factor. IFN-alpha can greatly upmodulate Fas-R expression, an effect that seems to be more pronounced in CML compared to normal cells, while Fas-L expression levels are not affected by IFN-alpha, thereby improving their susceptibility to elimination by the immune system. Responsiveness to Fas-induced apoptosis following stimulation with IFN-alpha correlates with the clinical effects of IFN-alpha therapy. This effect seems to be associated with decreased bcr-abl protein levels, which are influenced by Fas via posttranscriptional modulation. In comparison to the chronic phase, CML cells derived from patients in blast crisis are refractory to Fas-mediated apoptosis, regardless of the expression levels of Fas, suggesting that an immune-mediated selection pressure could result in acquisition of Fas resistance. In the future, enhancement of immunological recognition and elimination of CML cells may prove to be an effective therapeutic approach directed towards the cure of CML. PMID- 10752979 TI - Matrix metalloproteinases in multiple myeloma. AB - Multiple myeloma is a deadly malignancy characterized by plasma cell infiltration of bones. The resulting effect is painful "punched-out" lesions where bone is eroded and filled with myeloma cells that suppress and replace the normal marrow components. Recently it has been shown that myeloma cells produce matrix metalloproteinase-9 (MMP-9) and MMP-2 and that accumulation of MMP-9 protein is suppressed upon expression of the heparan sulfate proteoglycan, syndecan-1. In this review, we briefly consider the potential roles for MMPs in the pathogenesis of multiple myeloma. MMPs likely have major roles in: 1) the infiltration of bone and other tissues by the myeloma cells; 2) the osteolytic bone destruction caused by overly active osteoclasts, 3) extracellular matrix remodeling by bone marrow stromal cells; 4) promoting the invasion of the endothelial cells that form neoangiogenic blood vessels necessary to sustain tumor foci; and 5) promoting the growth of myeloma cells. Effective and safe synthetic inhibitors of MMPs are available and these may prove useful in limiting the growth and spread of myeloma cells. In addition, recent insights into the suppression of MMP-9 by syndecan-1 may suggest new strategies for treatment of myeloma. PMID- 10752981 TI - Gab-family adapter molecules in signal transduction of cytokine and growth factor receptors, and T and B cell antigen receptors. AB - Gab1 and Gab2 (Grb2 associated binder 1 and 2) are scaffolding adapter molecules that display sequence similarity with Drosophila DOS (daughter of sevenless), which is a potential substrate for the protein tyrosine phosphatase, Corkscrew, Both Gab1 and Gab2, like DOS, have a pleckstrin homology domain and potential binding sites for SH2 and SH3 domains. Gab1 and Gab2 are phosphorylated on tyrosine upon the stimulation of various cytokines, growth factors, and antigen receptors, and interact with signaling molecules, such as Grb2, SHP-2, and PI-3 kinase. Overexpression of Gab1 or Gab2 mimics or enhances growth factor or cytokine-mediated biological processes and activates ERK MAP kinase. These data imply that Gab1 and Gab2 act downstream of a broad range of cytokine and growth factor receptors, as well as T and B antigen receptors, and link these receptors to ERK MAP kinase and biological actions. PMID- 10752982 TI - FLUDAP: salvage chemotherapy for relapsed/refractory aggressive non-Hodgkin's lymphoma. AB - The aim of this study was to investigate the combination of fludarabine phosphate, dexamethasone, cytosine arabinoside and cis-platinum (FLUDAP) in the treatment of patients with relapsed/refractory aggressive non-Hodgkin's lymphoma (NHL). This regimen comprises: dexamethasone 100 mg/d continuous infusion (cont. inf.) d1-3; cytosine arabinoside (ara-C) 1 g/m2/d cont. inf. d 2,3; fludarabine phosphate 30 mg/m2 short inf. 4hr prior to each 24hr ara-C inf.; cis-platinum 50 mg/m2 4hr inf. at the start of each 24hr ara-C inf. G-CSF (lenograstim, Granocyte) is given at 263 microg s.c. daily from day 7 until the neutrophil count reaches 1.0x10(9)/l. The regimen repeats at 21 day intervals. A total of 33 patients were registered (median age 47 years; 24 males, 9 females); the majority (73%) were refractory to their previous treatment and most had advanced disease by Ann Arbor stage. Thirteen (39%) of the 33 enrolled patients (52% of the 25 fully evaluable patients who received at least 2 courses of FLUDAP) responded to treatment. A maximum response of complete remission was achieved in 5 patients, good partial remission in 3, and partial remission in 5. Twelve patients went on to successful stem cell supported intensification therapy. Median survival times were higher in the responding patients, and in those patients transplanted post FLUDAP. The toxicity associated with the FLUDAP regimen was generally predictable; frequently reported severe events included haematological toxicity and infection. In conclusion, the FLUDAP regimen shows promise as a salvage regimen and increases the available therapeutic options in the treatment of recurrent/refractory aggressive NHL. PMID- 10752984 TI - Soluble cytokine levels correlate with the activity and clinical stage of Hodgkin's disease at diagnosis. AB - The serum levels of some cytokines seem to correlate with outcome in Hodgkin's disease (HD) and may be helpful in formulating new and better prognostic systems. The aim of this study was to analyse the correlations between the serum levels of different cytokines and the clinico-hematological features suggestive of a worse prognosis. The study involved 31 pts with a "de novo" diagnosis of HD (median age: 30 yrs; M/F: 13/18; stage I/II vs III/IV: 19/12; B symptoms: 12; bulky disease and extranodal disease: 9). The serum levels of sCD30, TNFalpha, TNF receptor I and II, IL6, IL6 receptor, IL10, sICAM-1 were evaluated at diagnosis, and correlated with gender, age (< or =/> 30), stage (I-II vs III-IV), systemic symptoms, bulky disease, ESR ( or = 40), serum copper (< or =/> 170 microg/dL), WBC counts (< or =/> 15x10(9)/L), prognostic scores (PS) according to Hasenclever ( or = 2), and the presence of extranodal disease. Stages III/IV were associated with significantly higher levels of sCD30 and TNF-RI (p=0.03), systemic symptoms with significantly higher levels of sCD30, TNFalpha, IL6, TNF RI (p=0.027, 0.03, 0.0005, 0.002), bulky disease with TNF-RI (p=0.03), high ESR with IL6 and TNF-RI (p=0.0011, 0.0001), high WBC counts with sCD30, IL6, TNF-RI (p=0.03, 0.002, 0.01), high serum copper with sCD30 and IL6 (p=0.05, 0.0004), higher PS with sCD30, IL6, TNF-RI (p=0.002, 0.0003, 0.005), extranodal disease with TNFalpha and IL6 (p=0.05, 0.01). It was possible to define cut-off levels for some cytokines (sCD30 > 33.15 U/mL, TNFalpha > 29.71 pg/mL, IL6 > 12.43 pg/mL, TNF-RI > 3.23 ng/mL, IL6-R > 57 ng/mL) that significantly correlate with systemic symptoms, higher disease stages, ESR, serum copper, WBC counts and PS. Our study shows that high sCD30, TNFalpha, IL6 and TNF-RI levels are associated with advanced disease or a worse prognostic score. In the context of multiparametric HD staging, cytokine evaluation may be useful for identifying candidates for more intensive therapies. PMID- 10752983 TI - Terminal deoxynucleotidyl transferase expression in acute myelogenous leukemia and myelodysplasia as determined by flow cytometry. AB - The significance of terminal deoxynucleotidyl transferase (TdT) expression in acute myelogenous leukemia (AML) remains controversial. Therefore, we studied TdT expression by flow cytometry in 120 previously untreated patients with AML or myelodysplastic syndrome (MDS) to determine the distribution of TdT-positive blasts and the intensity of TdT expression and to seek clinically significant associations. TdT expression measured by flow cytometry (flow TdT%) was heterogeneous, ranging from 0.1% to 87% (median, 8.5%), and 74 patients (62%) had at least 5% TdT-positive blasts. TdT positivity was associated with the M0 or M1 subtype and with expression of CD34 and CD7. No significant correlation was found between TdT expression and type of cytogenetic abnormality or rearrangement of immunoglobulin or T-cell receptor genes. Remission lasted longer in patients with a flow TdT% < 5 than in patients with a flow TdT% > 5 (median, 95 weeks vs 55 weeks, p = 0.02); however, complete remission rates did not differ when patients were classified by initial flow TdT%. Survival was slightly better for patients with flow TdT% less than 5%. Among patients with a flow TdT% > 5%, those with a higher TdT intensity survived longer than those with a lower intensity. These data suggest that quantitative TdT measurement may contribute to prognostic estimate in AML patients. PMID- 10752985 TI - Smoking and risk of non-Hodgkin lymphoma subtypes in a cohort of older women. AB - Although non-Hodgkin lymphoma (NHL) has not been considered to be a smoking related malignancy, recent investigations suggest otherwise. We evaluated this association in a cohort of 37,336 women, aged 55-69 years, who reported in a mailed questionnaire in 1986 information regarding smoking history as well as demographic, medical history and dietary factors. Cancer and mortality experience through 1996 was determined by linkage to the Iowa Cancer Registry and other databases; there were 200 incident cases of NHL during the 380,231 total person years of follow-up. Compared to never smokers, former (age-adjusted RR = 1.0; 95% CI 0.8-1.5) and current smokers (age-adjusted RR = 1.0; 95% CI 0.7-1.5) were not at elevated risk of NHL, and there was no trend with pack-years smoked (Ptrend = 0.3). Multivariate adjustment for other NHL risk factors did not alter these findings. Age-adjusted analysis by NHL subtype revealed a suggestive positive association of smoking with follicular NHL [(RRformer = 1.3; 95% CI 0.6-2.8), (RRcurrent = 1.8; 95% CI 0.8-3.8)], which strengthened after multivariate adjustment [(RRformer = 1.6; 95% CI 0.7-3.4), (RRcurrent = 2.3; 95% CI 1.0-5.0)]; there was no association for diffuse or small cleaved-cell NHL. Our study findings, which are consistent with other recent investigations, suggest that smoking may be associated with an increased risk of follicular NHL. PMID- 10752986 TI - A predictive model for life-threatening neutropenia and febrile neutropenia after the first course of CHOP chemotherapy in patients with aggressive non-Hodgkin's lymphoma. AB - The purpose of this study was to develop a model for predicting the occurrence of life-threatening neutropenia (LN, ANC < or = 0.5 x 10(9)/l) and febrile neutropenia (FN, an ANC < 0.5x10(9)/l in association with a body temperature of > or = 38.3 degrees C) after the first cycle of CHOP therapy in patients newly diagnosed with aggressive NHL. One hundred and forty-five patients, aged > or = 15 years, with newly diagnosed diffuse mixed, diffuse large-cell or large-cell immunoblastic lymphoma (IWF categories, F, G, H), who had been treated with CHOP at King Chulalongkorn Memorial Hospital between June 1994 and December 1998, were entered into the study. The criteria for eligibility included complete work-up for baseline evaluation, treatment with standard CHOP chemotherapy, at least one complete blood count performed during days 8-14 post-treatment or if at any time the patients experienced a BT of > or = 38.3 degrees C and were not treated with any colony-stimulating factors (CSFs). The median age of the patients was 47 years (range, 17-78). Forty-eight percent of the patients were in stage III/IV, 36% had ECOG performance status (PS) II-IV, 30% had > or = 2 extranodal diseases, 59% had serum LDH > 1 x normal and 23% had bone marrow involvement. The frequencies of patients in the low-, low-intermediate, high-intermediate and high risk groups according to the international index were 29%, 28%, 17% and 26%, respectively. Thirty-nine percent of the patients had LN at nadir and 33% developed FN after the first course of CHOP. By using stepwise logistic regression analysis, the pretreatment variables independently predictive of the LN at nadir and the FN were serum albumin concentration of < or = 3.5 g/dl, serum LDH > 1 x normal and whether there was bone marrow involvement of lymphoma at presentation. The model, based on the incorporation of these three factors, identified three risk groups of patients with a predicted probability of developing LN at nadir of 81.5% (95% CI, 68.5-90.7) (high risk), 23.9% (95% CI, 12.6-38.8) (intermediate risk) and 4.4% (95% CI, 0.5-15.1) (low risk). The predicted rate of FN in the three groups were 72.2% (95% CI, 58.4-83.5), 17.4% (95% CI, 7.8-31.4) and 2.2% (95% CI, 0.05-11.8), respectively. In conclusion, our model could be used as a means to identify patients with newly diagnosed aggressive NHL, treated with CHOP, who are at high risk (> or = 50% probability) of developing post-first course LN and FN, in whom CSF and/or antibiotic prophylaxis might be indicated. PMID- 10752987 TI - Human herpesviruses in primary ocular lymphoma. AB - Several human lymphotropic herpesviruses have been found in certain lymphoproliferative disorders and implicated as possible etiologic factors or as modulating elements of the diseases. To assess a possible association of the human herpesviruses with lymphomas arising from mucosa-associated lymphoid tissue (MALT), we evaluated the presence of four human herpesviruses, Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), HHV-7, and HHV-8, in the biopsied specimens from 14 patients with primary ocular MALT lymphomas. EBV DNA sequences were detected in four specimens by polymerase chain reaction (PCR), and four cases were positive for HHV-6 DNA. In situ hybridization showed that three and two of 14 cases were positive for EBV mRNA and HHV-6 DNA, respectively. Neither HHV-7 nor HHV-8 sequences could be detected by PCR. These findings would stimulate further investigation as to the involvement of these lymphotropic viruses in the pathogenesis of a subset of low-grade primary ocular lymphomas. PMID- 10752989 TI - Leukemia and myelodysplasia effect of multiple cytotoxic therapy in essential thrombocythemia. AB - Essential thrombocythemia (ET) is a chronic myeloproliferative disorder characterized by increased risk of thrombosis and/or hemorrhages. Cytotoxic drugs are mostly used in patients at high risk for thrombotic complications, while their use is still debated in low risk patients because of the risk of leukemia or secondary neoplasm. We discuss the leukemic risk of available treatment strategies in a large cohort of patients. Over a 12 years period we treated 23 patients with busulfan (BU), 1 with pipobroman (Pi), 6 with 32P, 48 with hydroxyurea (HU) in 62 cases associated with acetyl salicylic acid (ASA) while 77 patients received ASA alone and 33 did not receive any therapy. We observed 2 cases of acute leukemia (AL) and 1 of myelodysplastic syndrome (MDS). One of these patients had been treated with 32P and Pi these after with and the other two with BU and HU. They represented 23% of all patients treated with more than 1 cytotoxic agent, 16.6% of 32P treated subjects, 4% of those with HU and 6.4% of those with BU. The case of MDS occurred in a 81 years old female and represents 4% of cases of ET over the 70 years of age. No cases of AL or MDS were observed in patients not receiving cytotoxic therapy (with or without ASA). According to our experience the use of more than one cytotoxic agent in ET confirms the increase in the risk of leukemia in these cases. However, none of the patients treated with HU alone, even for more than 10 years (12 cases) developed AL. No treatment or therapy with ASA alone may be the best choice in young patients with ET with a low risk of thrombotic complications. PMID- 10752988 TI - A prospective randomized study of alpha-2b interferon plus hydroxyurea or cytarabine for patients with early chronic phase chronic myelogenous leukemia: the International Oncology Study Group CML1 study. AB - A prospective randomized international study of 143 patients showed no apparent early survival advantage conferred by combining cytarabine, rather than hydroxyurea, with INF as first-line CML therapy. Combinations of alpha-interferon (INF) and chemotherapeutic agents are currently first-line therapy for the majority of patients with chronic myeloid leukemia (CML). The International Oncology Study Group conducted a prospective randomized study comparing INF combined with hydroxyurea or cytarabine. The primary study aim was to compare the survival durations in these patient cohorts. Patients with early chronic phase CML were randomized to receive INF 5 million units (Mu) given five times per week subcutaneously plus hydroxyurea or cytarabine as required to achieve a complete hematologic response and to maintain a WBC count between 2x10(9)/L and 10x10(9)/L and a platelet count between 75x10(9)/L and 100x10(9)/L. Therapy continued as tolerated unless progressive or blast phase disease occurred. At 36 months, the actuarial survival rate was equivalent in both groups: HI group (79 patients) survival was 85% (95% CI, 68-100%), as compared to 95% (95% CI, 79-100%) in the CI group (64 patients). In conclusion if seems that there is no apparent early survival advantage conferred by combining cytarabine, rather than hydroxyurea, with INF as first-line CML therapy. PMID- 10752990 TI - T-cell infiltrate after monoclonal anti-CD20 antibody therapy for B-cell lymphoma. PMID- 10752991 TI - An in vivo and in vitro comparison of the effects of b2-a2 and b3-a2 p210BCR-ABL splice variants on murine 32D cells. AB - The Philadelphia (Ph) chromosome, a characteristic cytogenetic marker of chronic myeloid leukaemia (CML), is caused by a reciprocal translocation juxtaposing the 3' region of the ABL gene onto the 5' region of the BCR gene. Due to conservation of the reading frame, but depending on the site of the breakpoint in the BCR gene, two alternatively spliced variants of the p210BCR-ABL mRNA (known as b2-a2 and b3-a2) are produced. To investigate whether there are any biological differences between these splice variants we have transfected the b3-a2 or b2-a2 cDNA into a murine myeloid cell line, 32D. We have also included the previously prepared 32Dp210 cell line (which expresses the b3-a2 transcript) in all of our comparisons. RT-PCR analysis indicated that transcription levels were comparable between the variants. Morphological examination of the cells expressing either of the BCR-ABL transcripts indicated that these cells were more mature with increased cytoplasm:nuclear ratios compared to the 32D parental and 32Dneo vector control cells. However, the 32Dp210 cells had a very different appearance from the other panel members and flow karyotyping indicated a clonal evolution and cytogenetic instability in these cells alone. At 10(6) and 10(7) cell doses all 32D cells expressing BCR-ABL caused ill health and tissue infiltration in SCID mice with such rapidity that statistical analysis was not informative. However, at the 10(5) and 10(4) dosage levels there were similar survival rates between mice injected with 32Db2-a2 or 32Db3-a2 while mice injected with 32Dp210 had a significantly shorter survival time. The study of this 32D cell line panel indicated that there were no overt differences in the biological properties conferred by the b3-a2 or b2-a2 transcripts to the 32D cells although these transcripts were able to confer in vitro and in vivo biological effects. This panel of BCR-ABL expressing 32D cells provides a useful model for CML disease progression studies. PMID- 10752992 TI - Negative regulators of hemopoiesis and stroma function in patients with myelodysplastic syndrome. AB - The mechanism that leads to hemopoietic failure in patients with myelodysplastic syndrome (MDS) is not well understood. There is evidence, however, that regulatory molecules such as tumor necrosis factor (TNF)-alpha, Fas (CD95), and Fas-ligand, which negatively affect hemopoiesis by way of apoptosis are upregulated. Here we analyzed marrow samples from 80 patients with MDS in regard to TNF-alpha and Fas-ligand levels and a possible correlation with various disease parameters and risk factors. TNF-alpha levels were elevated in comparison to samples from normal marrow donors, however, no significant correlation with FAB subtype, cytogenetic risk group or score by the International Prognostic Scoring System (IPSS) was observed. However, there was an inverse correlation between the cytogenetic risk category (low, intermediate, high) and levels of soluble Fas-ligand. The major source of TNF-alpha were mononuclear (non-stromal) cells which appeared to produce TNF-alpha at maximum levels. Limiting dilution analysis of CD34+ precursor cells showed that individually assayed cells, removed from companion cells that presumably provided negative signals such as TNF-alpha or Fas-ligand, were able to generate progressively increasing numbers of colonies. Stromal layers derived from MDS marrow did not have an inhibitory effect. In fact, higher colony numbers were obtained from both normal and MDS marrow derived hemopoietic precursors propagated on irradiated stromal layers from MDS marrow than on stromal layers from normal marrow. These results show that substantial numbers of normal hemopoietic precursors persist in MDS marrow. However, differentiation into mature cells is inhibited by negative signals originating from accessory or abnormal hemopoietic precursors in the non-adherent marrow fraction. PMID- 10752994 TI - Association of EBV strains, defined by multiple loci analyses, in non-Hodgkin lymphomas and reactive tissues from HIV positive and HIV negative patients. AB - Epstein-Barr virus (EBV) associated with lymphoid neoplasms demonstrates preferential association with certain viral strains. Previous subtyping studies have however been confined to analysis of sequence variability within a single locus in EBV. Variations have now been reported for several latently expressed EBV genes, including, EBNAs-1, 2 and LMP-1. Variant EBNA-1 strains have been identified in Burkitt's lymphomas and clustering of subtypes for LMP and EBNA-2 have been associated with either malignancy and/or clinical disease. To investigate the linkage between the variability in these three loci in EBV associated with lymphoid malignancies, we subclassified EBV-associated lymphoproliferations (9 reactive and 24 malignant) from HIV-negative and HIV positive patients by analysis of the EBNA-1, LMP1, and EBNA-2 genes. Our results demonstrate that (1) EBV identical to the prototype B95.8 strain (Type 1 EBNA-2, wild type EBNA-1 and LMP-1) is very rarely associated with tumors. (2) The EBNA-1 variant V-leucine, restricted to malignant lymphomas in immunocompetent patients, was readily identified in non-malignant lesions in HIV infected patients. (3) Variations of EBNA-1 occur independent of variations at other loci. PMID- 10752993 TI - Midkine expression in Reed-Sternberg cells of Hodgkin's disease. AB - Midkine (MK) was originally cloned as a product of a retinoic acid-responsive gene. The rationale for studying MK expression is based on previous reports showing that it transforms 3T3 cells, and that it acts as an autocrine growth factor in Wilm's tumors, and that its overexpression has been associated with worse outcome in bladder carcinoma. Besides bladder carcinoma, its expression was reported in various solid tumors. We investigated the expression of MK protein and/or MK gene in biopsied specimens from 40 patients with primary malignant lymphoma, 21 with Hodgkin's disease (HD) and 19 with non-Hodgkin's lymphoma (NHL). Reed-Sternberg (R-S) cells were stained positive in 10 of 16 HD cases evaluated by immunohistochemical method, whereas 18 of 19 NHL cases did not stain, and one B-cell NHL stained weakly positive. Immunostaining analysis was extended to established cell lines and to normal lymphocytes with or without lectin stimulation or with EB virus transformation. Among hematopoietic cells examined, erythro- or megakaryoblastic leukemia cell lines (K562, MEG-01 and UT7) were positive, while normal lymphocytes (except the EB virus-transformed one) and most myeloid and lymphoid cell lines (except Raji cells) were negative. On the contrary, solid tumor cell lines showed high and strongly positive staining including cell lines derived from of lung gastric, colon, and a pancreatic cancer. Using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), which is suitable for the detection of weakly expressed mRNA, the relative ratio of MK mRNA to beta-actin mRNA of samples was measured and compared in cases where RNA was available. The mean values of relative ratio (MK/beta actin) of HD were almost twice as those of NHL samples, peripheral blood T cells, and spleen B cells. Our findings showed that MK is expressed in Reed-Sternberg cells of HD, and that MK might play a role in the pathogenesis of HD. PMID- 10752995 TI - Acute blast crisis with EBV-infected blasts, in a patient with chronic myeloid leukemia, and vasculitis. AB - Unless they undergo transplantation, all patients with chronic myeloid leukemia (CML) will eventually develop a late phase of acute blast crisis (ABC). Although additional chromosomal abnormalities to the Philadelphia (Ph) chromosome may herald ABC in many CML cases, the mechanisms leading to this fatal event are obscure. Viral etiology, including the Epstein-Barr virus (EBV) has never been implicated in the pathogenesis of ABC in CML. Iloprost is an analogue of epoprostenol (prostacyclin; PGI2) commonly used for the treatment of peripheral vascular diseases and acts via inhibition of platelet activation, and by vasodilation. A case of ABC with blasts of undetermined lineage showing EBV infection in a male patient with Ph positive CML is described here. This unusual event developed during a course of treatment with the prostacyclin analogue, iloprost administered for vasculopathic leg ulcers. The proliferating blasts stained positively by immunohistochemistry only for the leukocyte common antigen (LCA/CD-45), and the EBV-latent membrane protein 1 (LMP-1). The only chromosomal abnormality detected by cytogenetic analysis was the conventional Ph-chromosome. It is suggested that ABC in this case of CML, was associated with EBV-activated blasts of undetermined lineage. PMID- 10752997 TI - Carboxypeptidase G2 rescue in delayed methotrexate elimination in renal failure. AB - We report here the case of a 68-year-old woman who presented severe renal failure following the first cycle of high dose methotrexate (HDMTX) for the treatment of a cerebral malignant lymphoma. Before HDMTX administration, serum creatinine value was normal and three days after HDMTX, it reached 457 micromol/L. Leucovorin rescue, hemodialysis and cholestyramine did not increase MTX clearance. Because of the persistence of renal failure, and the high risk of important hematological side-effects associated with high MTX plasma levels, the patient received carboxypeptidase G2 (CPDG2). This allowed MTX plasma levels to decrease by 80% in 15 minutes. No side effects were observed and renal function normalized rapidly. In some patients, when high-dose leucovorin associated with hemodialysis and cholestyramine are unable to restore normal MTX clearance, CPDG2 should be considered because it may represent a safe and efficient alternative for the management of MTX intoxication. PMID- 10752996 TI - Seropositive polyarthritis and skin manifestations in T-prolymphocytic leukemia/Sezary cell leukemia variant. AB - Sezary cell leukemia (SCL) is a rare T cell neoplasia that has been suggested to be a variant of T-prolymphocytic leukemia (T-PLL). Both disorders have an aggressive clinical course, lymphocytosis with characteristic morphology, lymphadenopathy, hepatomegaly, characteristic cytogenetic abnormalities and mature T cell phenotypes. Skin lesions, however, are mainly found in T-PLL. We describe a patient with T-PLL/SCL, who atypically presented with severe seropositive polyarthritis and skin lesions, responding to treatment with human CD52 antibody, CAMPATH-1H and pentostatin. Meningeal leukemia and an assumed myocardial infiltration subsequently developed. Polyarthritis is common in T large granular lymphocyte leukemia and adult T cell lymphoma-leukemia, but both entities could be ruled out in the present case. In rheumatoid arthritis, an expansion of CD4+ and/or CD8+ T lymphocytes is well documented and this phenomenon is believed to be of pathogenetic importance. We speculate that the T cell clone in the present case had special homing properties or cytokine effects resulting in synovitis. PMID- 10752998 TI - Pyogenic granuloma of the tongue early after allogeneic bone marrow transplantation for multiple myeloma. AB - Oral complications occur frequently after bone marrow transplantation (BMT). Some of them are caused by regimen-related toxicity of the preparative regimen, and others by infections. In addition, oral tissues are targets of graft-versus-host disease (GVHD). Oral granulomatous lesions are not a common complication after BMT, and are especially rare on the tongue. Such rare lesions reported in the literature, developed late after BMT with oral chronic GVHD. We present here a patient who developed pyogenic granuloma of the tongue early after allogeneic BMT done for multiple myeloma. Regimen-related mucositis, oral acute GVHD, the administration of cyclosporine A, and the preexisting macroglossia might be responsible for the formation of granuloma. PMID- 10752999 TI - Primary Hodgkin's disease of the vagina. AB - We describe a patient with primary Hodgkin's disease (HD) of the vagina presenting with stage IEB. To our knowledge, this is the first case reported so far. Based on morphological and immunophenotypic features, the HD was classified as nodular sclerosis subtype, "syncytial" variant. The patient, a 64-year old woman, received chemotherapy followed by radiation therapy. She is still disease free 14 months after diagnosis. PMID- 10753000 TI - A misleading pulmonary lesion in a patient with acute monoblastic leukemia. PMID- 10753001 TI - Electroencephalographic coherence in Alzheimer's disease: comparisons with a control group and population norms. AB - Previous research from independent laboratories has shown reduced electroencephalographic coherence in patients diagnosed with dementia of the Alzheimer type (DAT). This study added to this work by comparing interhemispheric and intrahemispheric coherence in nonmedicated DAT patients (n = 35) with that of a normal control group (n = 30), as well as with a data bank of population norms. Raw and Z-score transformed values showed reduced coherence, interhemispherically (in delta, theta, alpha, and beta bands) and intrahemispherically (delta and theta bands) in DAT patients with both comparison procedures. Discriminant analysis correctly classified 73% to 75% of patients. The results are discussed in relation to earlier research, "trait" versus "state" factors, the cholinergic system, and cognitive processes in dementia. PMID- 10753002 TI - Metrifonate enhances the ability of Alzheimer's disease patients to initiate, organize, and execute instrumental and basic activities of daily living. AB - The objective of this analysis was to evaluate comprehensively the efficacy of metrifonate, a long-acting acetylcholinesterase inhibitor, in improving the ability of mild-to-moderate Alzheimer's disease (AD) patients to perform activities of daily living (ADLs). Alzheimer's disease patients with Mini-Mental State Examination scores of 10 to 26 were enrolled in three 26-week trials to receive once-daily placebo (n = 430) or metrifonate 30 to 60 mg (by weight, n = 650) or 60/80 mg (by weight, n = 197). Metrifonate efficacy was assessed using the Disability Assessment for Dementia scale. Data from the three studies were pooled and analyzed retrospectively. The intent-to-treat analysis (last observation carried forward) at 26 weeks demonstrated that metrifonate significantly improved the ability of AD patients to perform ADLs when compared with placebo (30-60 mg dose, delta = 3.03; P = .002; 60/80 mg dose, delta = 5.25; P = .0002). Metrifonate significantly improved the ability of the AD patients to perform instrumental ADLs, those abilities typically lost first during the disease process (30-60 mg dose, delta = 3.88, P = .002; 60/80 mg dose, delta = 5.79, P = .003). Metrifonate also tended to improve, relative to placebo, the ability of AD patients to use three levels of executive skills when performing ADLs: initiation (30-60 mg dose, delta = 3.45, P = .001; 60/80 mg dose, delta = 5.44, P = .003), planning/organization (30-60 mg dose, delta = 4.50, P = .004; 60/80 mg dose, delta = 4.89, P = .014), and effective execution (30-60 mg dose, delta = 1.80, P = .076; 60/80 mg dose, delta = 4.06, P = .030). These results indicate that metrifonate has a beneficial effect on the ADLs in mild-to-moderate AD patients. PMID- 10753003 TI - An actigraphic comparison of sleep restriction and sleep hygiene treatments for insomnia in older adults. AB - We compared the efficacy of sleep restriction therapy combined with sleep hygiene, nap modification of sleep restriction therapy combined with sleep hygiene, and sleep hygiene alone as treatments for insomnia in 39 community dwelling men and women 55 years and older. We used the wrist actigraph as an objective outcome measure for all subjects at baseline, end of treatment, and 3 month follow-up; polysomnography (PSG) was conducted in a subgroup of subjects. Although subjects appeared to follow restriction instructions through follow-up, we found few between-group differences in treatment efficacy. Lack of treatment effect might be explained by the efficacy of HYG as a treatment in itself and the relatively low symptom level in these healthy older poor sleepers. At baseline, actigraphic results were found to correlate more highly than sleep log data with PSG in our sample. Actigraphic total sleep time, in particular, was highly correlated with PSG. PMID- 10753004 TI - Clinical experience with quetiapine in elderly patients with psychotic disorders. AB - Quetiapine fumarate is a recently marketed atypical antipsychotic medication proved to be effective in the treatment of schizophrenia and schizoaffective disorder in the younger population. There is a paucity of studies of this drug in the elderly and more data are needed on the effects of quetiapine in this population, especially those with comorbid medical illnesses. Quetiapine was used to treat seven elderly hospitalized patients between 61 and 72 years of age who manifested signs of psychosis related to schizophrenia, schizoaffective disorder, or bipolar disorder. All patients had been treated previously with conventional antipsychotics or other atypical antipsychotics. Response was assessed by observation of patient's behavior. Four patients responded to treatment; three did not respond. Positive symptoms decreased markedly in all four responders. Negative symptoms showed marked decrease in two patients and moderate decrease in one patient. Preexisting extrapyramidal symptoms (EPS) diminished in three patients. Transient hypotension, dizziness, and somnolence occurred in two patients. No other side effects were noted. No adverse consequences occurred when lithium, carbamazepine, valproic acid, or venlafaxine was given concurrently. The reduction of positive and negative symptoms of schizophrenia and lack of significant EPS and minimal sedative, hypotensive, and anticholinergic side effects indicate that quetiapine may be a safe and effective medication for the elderly. PMID- 10753005 TI - Nonalcohol-related use disorders in geropsychiatric patients. AB - The authors determined the prevalence and demographic features of drug use disorders in a database of 565 veteran geropsychiatric inpatients. Twenty-four patients (4%) were diagnosed with nonalcoholic substance use disorders, including 18 (3%) with prescription drug use disorders and 6 (1%) with illegal drug use disorders. The patients diagnosed with prescription drug use disorders were predominantly Caucasian, whereas those diagnosed with illegal substance use disorders were predominantly African American. No other significant demographic differences were noted. Nonalcohol substance use disorders represent an unappreciated problem in psychiatrically hospitalized older adults, with illegal substance use disorders making up a larger than expected proportion of this group. PMID- 10753006 TI - Etiologic and outcome profiles in hypoactive and hyperactive subtypes of delirium. AB - The existence of hyperactive, hypoactive, or mixed clinical subtypes of delirium is widely accepted. But relationships between these motor profiles and etiology or outcome remain unclear. The aim of this study was to compare etiologic and outcome profiles in a case series of 183 elderly patients (mean age = 84.1 years, SD = 5.9) consecutively admitted into the geriatric wards of two French university hospitals or referred to a geriatric psychiatry consultation-liaison unit within a Swiss university hospital. All patients met DSM-III-R criteria for delirium and were classified into clinical subtypes according to the results of a previous factor analysis of scores on a 19-item checklist rating a wide range of delirium symptoms. The hyperactive subtype was more frequent (n = 85, 46.5%) than the unspecified (n = 50, 27.3%) and hypoactive subtypes (n = 48, 26.2%). There was no significant difference in terms of etiologic or outcome profile between clinical subtype groups. The presence of acute metabolic disorders, cardiovascular disease, and hyperthermia as etiologic factors was significantly associated with full recovery of the episode at 3 weeks follow-up, whereas probable preexisting dementia was significantly associated with partial recovery or failure to recover. PMID- 10753007 TI - Fluvoxamine pharmacotherapy of anxiety disorders in later life: preliminary open trial data. AB - The authors present data from an open trial of fluvoxamine (median daily dosage: 200 mg) in the treatment of generalized anxiety disorder, panic disorder, and obsessive-compulsive disorder in 19 older outpatients (mean age = 66.8). Of the 12 subjects completing the 21-week trial, 8 achieved a good response (50% reduction in symptom measures) and 7 were rated as much or very much improved. Fluvoxamine pharmacotherapy also had a significant effect in reducing comorbid depressive symptoms and in increasing levels of functioning. These data support the effectiveness of fluvoxamine in older subjects with anxiety disorders (particularly generalized anxiety disorder) and warrant further double-blind, placebo-controlled evaluation. PMID- 10753008 TI - Behavioral disturbances in geropsychiatric inpatients across dementia types. AB - The objective of this study was to compare differences in behavioral, psychiatric, and cognitive status among geropsychiatric inpatients with Alzheimer's, vascular, alcohol-induced, and mixed dementia. Participants included 150 patients with dementia consecutively admitted to an acute geropsychiatric inpatient unit. Measures included the Mini-Mental State Examination, Cohen Mansfield Agitation Inventory, Cumulative Illness Rating Scale, Basic and Independent Activities of Daily Living, Positive and Negative Syndrome Scale for Schizophrenia, and the Initiation/Perseveration subscale of the Dementia Rating Scale. No significant differences existed in the character or severity of agitation among patients with Alzheimer's, vascular, alcohol-related and mixed dementia. Interestingly, patients with vascular dementia compared to patients with other dementias admitted for behavioral disturbances were less cognitively impaired and more medically burdened. PMID- 10753009 TI - Atopic dermatitis: from the genes to skin lesions. PMID- 10753010 TI - Bacterial vaccines: anything but placebo. PMID- 10753011 TI - Distribution of dust-mite allergens (Lep d 2, Der p 1, Der f 1, Der 2) in pig farming environments and sensitization of the respective farmers. AB - BACKGROUND: The aim of the present study was to determine the distribution of mite allergens in pig-farming environments in comparison to urban homes and the relationship between exposure to mite allergens and sensitization to the respective allergens in 100 pig farmers with work-related respiratory symptoms. METHODS: The concentration of storage mite (Lep d 2) and house-dust-mite (Der p 1, Der f 1, and Der 2) allergens in dust collected from five different sampling sites (floor, wall, grain mill, transit area between confinement house and farmer's kitchen, and farmers' mattresses) was studied in relationship to the respective sensitization rates. Allergen concentrations in the mattresses were compared to those determined in mattresses from 22 urban dwellers. RESULTS: Median concentrations of Der p 1 and Der 2 in the mattresses of the farmers were significantly higher than in the urban dwellers' samples (53.4 microg/g dust vs 1.05 microg/g dust, P=0.001; 19.6 microg/g dust vs 2.2 microg/g dust, P<0.0001, respectively). Allergen concentrations in the transit areas were strongly related to bedroom exposure. In a multiple logistic regression model, a weak but significant relationship between Der p 1 exposure and sensitization to Der p 1 was found. Despite these findings, the prevalence of sensitization to mite allergens in the farmers (18%) was comparable to the prevalence in the general population. CONCLUSIONS: Allergen exposure at the workplace is strongly related to the concentration of allergens in farmers' beds. Exposure to domestic mite allergens should be taken into account when assessing occupational exposure to allergens and the respiratory health of farmers. PMID- 10753012 TI - Quantitative flow cytometric analysis of the effects of cetirizine on the expression of ICAM-1/CD54 on primary cultured nasal cells. AB - An in vitro flow cytometric model has been developed to evaluate the effects of antiallergic drugs such as cetirizine (CTZ) on the expression of surface molecules on primary cultured normal cells. Quantitative analysis demonstrated that HLA class I and ICAM-1/CD54 molecules are present on both epithelial and stromal cells, and that their expression is strongly enhanced by treatment with interferon-gamma (IFN-gamma). Nevertheless, the IFN-gamma-mediated upregulation of ICAM-1/CD54 was inhibited by treatment with CTZ, demonstrating a direct effect on both cell types. This finding is particularly interesting because ICAM-1/CD54 is the main rhinovirus receptor, and rhinoviruses are the principal cause of asthma exacerbation in children. Thus, according to data derived from this in vitro model, CTZ should have an important role in the reduction of infectious exacerbation of asthma in atopic patients. PMID- 10753013 TI - Trends in hospital admissions for childhood asthma in Oslo, Norway, 1980-95. AB - BACKGROUND: The prevalence of asthma and quality of asthma care both influence hospital admission rates for childhood asthma. Therefore, we aimed to assess possible changes in the hospital admission rate for acute asthma in Oslo, Norway, from 1980 to 1995, as well as evaluate the possible effect of changes in asthma treatment upon hospitalization for acute asthma in this period. METHODS: All pediatric patient records from the two municipal hospitals in Oslo from 1980 through 1995 with the discharge diagnoses (ICD-9) acute asthma, acute bronchitis/bronchiolitis, pneumonia, and/or atelectasis were thoroughly reviewed. RESULTS: Of the 3,538 children admitted for acute asthma, 66% were boys and 75% were younger than 4 years, and the admittance rate increased significantly among children aged 0-3 years. First admissions increased throughout the study, whereas readmissions, as well as the mean duration of hospital stay, decreased significantly. Prophylactic treatment with inhaled steroids prior to admission increased over 1980-89, but stabilized thereafter. The use of a short course of systemic steroids during admission increased markedly from 1991. CONCLUSIONS: The findings of increasing first admission rate as well as overall admission rate for acute asthma in children under 4 years of age, but decreasing readmissions as well as number of treatment days in hospital, probably reflect changes in the management of the disease, as well as an increasing prevalence of childhood asthma. PMID- 10753014 TI - Development of allergies and asthma in infants and young children with atopic dermatitis--a prospective follow-up to 7 years of age. AB - BACKGROUND: The prognosis of atopic dermatitis is usually good, but the risk of developing asthma and allergic rhinitis is very high. The aim of this study was to follow children with atopic eczema up to school age to chart the course of sensitization and development of clinical allergy, as well as to study risk factors of sensitization. METHODS: Ninety-four children with atopic dermatitis were followed up to 7 years of age. The children were examined twice a year up to 3 years of age, and thereafter once yearly. At each visit, a clinical examination was performed, and a blood sample was taken. After 3 years of age, skin prick tests (SPTs) with inhalation allergens were performed at each visit. Information was obtained about atopy in the family, feeding patterns during infancy, symptoms of atopic disease, infections, and environmental factors. RESULTS: During the follow-up, the eczema improved in 82 of the 94 children, but 43% developed asthma and 45% allergic rhinitis. The risk of developing asthma was higher in children with a heredity of eczema. Presence of severe eczema at the time of inclusion in the study was associated with an increased tendency to produce food-specific IgE. An early onset of eczema was associated with an increased risk of sensitization to inhalant allergens, and development of urticaria. Early allergic reactions to food were associated with later reactions to food, allergic rhinitis, urticaria, and sensitization to both food and inhalant allergens. Early feeding patterns, time of weaning, and introduction of solid food did not influence the risk of development of allergic symptoms. A large number of periods or days with fever during the follow-up was associated with an increased risk of developing allergic rhinitis and urticaria. CONCLUSIONS: Our results confirm the good prognosis for the dermatitis and the increased risk of developing asthma and allergic rhinitis. Development of other allergic symptoms or sensitization was associated with the following factors: a family history of eczema, age at onset of eczema and its severity, early adverse reactions to foods, and proneness to infections. PMID- 10753015 TI - The IgE-binding epitopes of rPar j 2, a major allergen of Parietaria judaica pollen, are heterogeneously recognized among allergic subjects. AB - Pollen allergens are multivalent proteins that cross-link IgE antibodies on mast or basophil cells, inducing secretion of biologic mediators, and resulting in various allergic symptoms. The IgE-binding regions of the Parietaria judaica (Pj) pollen major allergen rPar j 2 were investigated. Twenty-nine single sera from Pj allergic subjects were tested by Western blot against five recombinant peptides. At least four putative IgE-binding epitopes were identified. The analysis of their diffusion suggested a heterogeneous IgE-binding response. In fact, 75% of the sera reacted with peptide 1-54, 48% with peptide 48-101, 24% with peptide 1 30, 7% with peptide 29-54, and none with peptide 48-76. These five peptides were analyzed with the histamine-release assay. Only peptide 48-101 was capable of inducing degranulation and release of histamine. These results suggest that the recombinant rPar j 2 allergen contains IgE epitopes that are heterogeneously recognized by sensitive patients, and that therefore the therapeutic approach based on the use of haptenic peptides needs a careful evaluation. PMID- 10753016 TI - Comparison of antibody responses to hen's egg and cow's milk proteins in orally sensitized rats and food-allergic patients. AB - BACKGROUND: No adequate enteral sensitization models are available to study food allergy and the allergenicity of food proteins. To further validate an enteral brown Norway (BN) rat sensitization model under development, we studied specific protein recognition to determine whether a comparable pattern of proteins is recognized by the rat immune system and the human immune system. METHODS: The animals were exposed to either ovalbumin as a positive reference control, hen's egg-white-protein extract, or a cow's milk preparation by daily gavage dosing (0.5, 1, 2.5, 5, 10, or 15 mg protein per rat/day) for 9 weeks. No adjuvants were used during the sensitization studies. The specificities of antibodies against hen's egg-white proteins or cow's-milk proteins in sera from orally sensitized rats and food-allergic patients were studied and compared by immunoblotting. RESULTS: The IgG and IgE antibodies to hen's egg-white proteins and cow's-milk proteins present in sera from orally sensitized rats and food-allergic patients showed a comparable pattern of protein recognition. CONCLUSIONS: Upon daily intragastric exposure to food allergens, the specificities of the induced antibody responses in the BN rat resemble those found in food-allergic patients. These studies add further support to the hypothesis that the BN rat may provide a suitable animal model for food allergy research and research on the allergenicity of food proteins. PMID- 10753017 TI - Different airway responsiveness profiles in atopic asthma, nonatopic asthma, and Sjogren's syndrome. BHR Study Group. Bronchial hyperresponsiveness. AB - BACKGROUND: Different mechanisms may underlie bronchial hyperresponsiveness (BHR) in different diseases. The aim of this study was to investigate the bronchial responsiveness profile produced by three different challenge tests, methacholine, a direct stimulus, and two indirect stimuli, adenosine 5'-monophosphate (AMP) and cold air, in subjects with asthma and patients with Sjogren's syndrome. METHODS: The study population comprised 40 adult patients with asthma, 18 subjects with Sjogren's syndrome, and 20 controls. Blood samples were collected before each challenge for measurements of serum eosinophil peroxidase (S-EPO) and eosinophil cationic protein (S-ECP). The investigated subjects recorded peak expiratory flow and kept a symptom diary. RESULTS: Atopic subjects with asthma were significantly more hyperresponsive to AMP than nonatopic subjects with asthma (P=0.01) and subjects with Sjogren's syndrome (P=0.02). No difference was seen between atopic and nonatopic subjects with asthma in the case of challenges with methacholine or cold air. In atopic subjects with asthma, a significant correlation was found between challenges with methacholine and AMP (r=0.91, P=0.0001) and methacholine and cold air (r=0.83, P=0.004), but, in nonatopic subjects with asthma, no significant correlation was seen between methacholine and AMP or cold air challenges. In atopic subjects with asthma, the dose-response slope for AMP was correlated to S-EPO (r= -0.56; P = 0.01) and S-ECP (r= -0.51, P = 0.02), while no correlation between BHR and inflammation markers was found in the two other patient groups. CONCLUSIONS: The results of this study suggest that patients with asthma and subjects with Sjogren's syndrome display different bronchial responsiveness profiles for different challenge agents. Atopic subjects with asthma are more hyperresponsive to AMP than nonatopic subjects and patients with Sjogren's syndrome. More than one challenge may be required to detect different aspects of bronchial responsiveness. PMID- 10753018 TI - IgE reactivity to patatin-like latex allergen, Hev b 7, and to patatin of potato tuber, Sol t 1, in adults and children allergic to natural rubber latex. AB - BACKGROUND: Patients allergic to natural rubber latex (NRL) frequently show positive skin prick tests (SPT) and hypersensitivity reactions to various fruits, such as avocado, banana, and kiwi, as well as to vegetables such as potato. METHODS: Hev b 7 was purified from NRL "C-serum" and Sol t 1 from potato extract, and they were detected by immunoblotting. IgE antibodies to Hev b 7 and Sol t 1 were measured with ELISA in sera from 35 adults and 35 children allergic to NRL. ELISA inhibition and immunoblotting were used to study allergen cross-reactivity. The in vivo reactivity of Hev b 7 and Sol t 1 were demonstrated in the SPT. RESULTS: Seventeen (49%) of the 35 NRL-allergic adults had IgE antibodies to Hev b 7, in contrast to only one of the 35 NRL-allergic children. Fifteen (43%) of the NRL-allergic adults and 29 (83%) of the NRL-allergic children had IgE antibodies to Sol t 1. Ten (29%) of the adult sera showed IgE binding to both Sol t 1 and Hev b 7, and crosswise inhibition tests with pooled sera revealed marked cross-reactivity. In the SPT, both natural Hev b 7 and Sol t 1 were able to produce a wheal and flare reaction. CONCLUSIONS: One-half of the NRL-allergic adults, but only one of the NRL-allergic children, had IgE antibodies to natural Hev b 7. These results suggest that Hev b 7 is an important NRL allergen for adults, but not for children. Elucidation of the clinical importance of the observed cross-reactivity between Hev b 7 and Sol t 1 requires further studies. PMID- 10753019 TI - Allergens of Epicoccum nigrum grown in different media for quality source material. AB - BACKGROUND: The Epicoccum nigrum (EN) extract used in allergy disorders exhibits batch-to-batch variations in protein composition and allergenic potency. In this study, the allergens of EN grown in different media were investigated. METHODS: EN was grown in five different nutrient media as stationary cultures at 25 degrees C for 5-23 days. The growth pattern was characterized by measuring dry weight, protein and carbohydrate content. The antigenic and allergenic content of EN extract was evaluated with EN-positive patients' sera and antibodies raised in rabbit. RESULTS: The growth of EN in Czapeck Dox medium yielded insufficient material, while Sabouraud's broth with yeast extract (SBY) gave maximum spore mycelial mass and protein content. Potato dextrose broth (PDB) and potato dextrose agar (PDA) showed higher dry weight and protein in 7-9-day cultures. SDS PAGE resolved 26, 22, and 21 protein bands in EN extracts from cultures of day-13 SBY, day-7 PDB, and day-9 PDA, respectively. IgE/IgG immunoblots showed more allergenic (25)/antigenic (25) bands in EN cultured in SBY than in the others. Specific IgE ELISA and intradermal tests showed EN extract from day-13 culture in SBY to be the most potent. CONCLUSIONS: The day-13 culture of EN in SBY was the most potent and may be selected for preparing EN extracts for diagnosis of allergy and future studies. PMID- 10753020 TI - The atopy patch test (APT)-- a useful tool for the diagnosis of food allergy in children with atopic dermatitis. AB - BACKGROUND: While immediate-type clinical reactions to food can quite easily be identified by history or measurement of specific IgE in combination with positive oral food challenges, the evaluation of food allergy in the absence of immediate clinical reactions still presents diagnostic difficulties--particularly in children with atopic dermatitis. The objective of this study was to evaluate the diagnostic value of the atopy patch test (APT) with regard to late-phase reactions observed in double-blind, placebo-controlled food challenges with cow's milk, hen's egg, wheat, and soybean. METHODS: We investigated 75 children (median age 2.1 years) with suspected food allergy by double-blind, placebo-controlled food challenges, specific IgE in serum, skin prick test, and APT. Of the subjects, 69/75 suffered from atopic dermatitis. RESULTS: Of 209 oral challenges, 133 were performed with allergen and 76 with placebo. We assessed 77/133 allergen and 2/76 placebo challenges as positive. In 66 of 77 (86%) positive oral challenges, specific IgE in serum to the corresponding allergen was positive; in 64/77 (83%) the skin prick test, and in 42/77 (55%) the APT was positive. While immediate-type reactions were associated with positive skin prick test and proof of specific IgE in serum, late-phase clinical reactions were associated with a positive APT (sensitivity 76%, specificity 95%). CONCLUSIONS: The APT seems to be a valuable additional tool in the diagnostic work-up of food allergy in children with atopic dermatitis - especially with regard to late-phase clinical reactions. The APT may help to prevent unnecessary restrictive diets which may be the consequence of misjudging late reactions by clinical assessment alone. PMID- 10753022 TI - A 17-kDa allergen detected in pine nuts. AB - BACKGROUND: Few cases of allergy to pine nuts have been described. We report a case of anaphylactic reaction to pine nuts. The patient needed to be treated in the emergency room due to a systemic reaction immediately after eating pine nuts. METHODS: The patient was studied by prick tests and prick by prick tests. Specific IgE was measured by CAP and by SDS-PAGE/immunoblotting by a diffusion method. RESULTS: The patient showed positive prick by prick tests to pine nuts (12 mm of maximum wheal diameter). Specific IgE was positive (0.79 kU/l). The patient's serum recognized several proteins by immunoblot. However, a 17-kDa allergen band was detected with high intensity. This protein was found to be sensitive to reducing agents, losing its IgE-binding properties after reduction. CONCLUSIONS: The patient presented an IgE-mediated reaction and detected a 17-kDa protein from pine nuts not previously described. PMID- 10753021 TI - Relationships between natural T cells, atopy, IgE levels, and IL-4 production. AB - BACKGROUND: Th2 cells govern allergic disorders. Mechanisms leading to the Th2 commitment are dominated by the requirement of IL-4. A potential source of this triggering IL-4 could be the CD4 + subset of a small population of T cells, natural T (NT) cells. Indeed, this subset is involved in IgE responses in mice and produces promptly high amounts of IL-4 in both mice and man. METHODS: NT cells were identified in peripheral blood by flow cytometry with antibodies against Valpha24 and Vbeta11, recognizing the T-cell receptor specific for NT cells. Simultaneous staining with anti-CD3, anti-CD4, or anti-CD8 antibodies was performed. The frequency of NT cells in man was studied according to the presence of atopy defined by the positivity of skin tests, according to total IgE levels in serum, and according to IL-4 concentration of whole-blood culture supernatants determined by a flow cytometer microsphere-based assay. RESULTS: Seventy subjects were included, of whom 30 were atopic. The number of CD4+ NT cells was higher in atopics than in nonatopics (P=0.009). This number was correlated to the total IgE levels (r = 0.34, P = 0.03). In addition, the number of CD4 + NT cells, but also of CD8 + NT cells, was correlated to the levels of IL-4 (r=0.71, P=0.01, and r=0.6, P=0.03, respectively). CONCLUSIONS: These results show that the number of NT cells, particularly the CD4+ subset, is related to atopy, IL-4 production, and IgE levels. Therefore, this population of T cells is likely to play a role in the Th2 commitment initiating atopic diseases. PMID- 10753023 TI - Immediate contact allergy from hydrolyzed wheat in a cosmetic cream. AB - Immediate contact allergy to cosmetics seems to be rare, since only a few case reports on it have been published. We report on a case of IgE-mediated allergic contact urticaria caused by hydrolyzed wheat in a body cream. PMID- 10753024 TI - Fixed drug eruption caused by dimenhydrinate. PMID- 10753025 TI - Allergenic cross-reactivity in the Liliaceae family. PMID- 10753027 TI - Pediatric allergology in Austria. PMID- 10753026 TI - Fern allergy. PMID- 10753028 TI - Effect of SIT on quality of life. PMID- 10753030 TI - Anaphylaxis to E466. PMID- 10753029 TI - Anaphylaxis after Argas reflexus bite. PMID- 10753031 TI - Urticaria induced by meperidine allergy. PMID- 10753032 TI - Chronic urticaria: a mystery. PMID- 10753033 TI - Obligatory information on patient-oriented end point outcome. PMID- 10753034 TI - Nitric oxide and vascular responses in Type I diabetes. AB - Vascular complications are major causes of morbidity and mortality in patients with diabetes mellitus. The mechanisms underlying the development of microvascular and macrovascular angiopathy in Type I (insulin-dependent) diabetes mellitus are complex and incompletely understood. The discovery of endothelium derived nitric oxide has greatly improved our understanding of vascular biology. Nitric oxide has an important role in the regulation of vascular tone and impaired nitric oxide activity could be implicated in the development of diabetic vasculopathy. Vascular studies of endothelial function in Type I diabetes have produced conflicting results. The role of nitric oxide in diabetic vasculopathy is still not clear. PMID- 10753035 TI - Cardiovascular risk factors clustering with endogenous hyperinsulinaemia predict death from coronary heart disease in patients with Type II diabetes. AB - AIMS/HYPOTHESIS: Information on the association of hyperinsulinaemia with coronary heart disease (CHD) in patients with Type II (non-insulin-dependent) diabetes is limited and controversial. Therefore, we carried out a prospective study to examine the predictive value of fasting plasma insulin and "hyperinsulinaemia cluster" with regard to the risk of CHD mortality. METHODS: At baseline risk factors for CHD were determined in 902 patients aged 45 to 64 years with Type II diabetes not treated by insulin (499 men and 403 women). These patients were followed up to 7 years for CHD mortality. RESULTS: Coronary heart disease mortality (16.2% in men, 9.2% in women) increased significantly in men with increasing plasma insulin tertiles (p = 0.006) and in both sexes combined (p = 0.010) but not in women (p = 0.090). The predictive value of hyperinsulinaemia with regard to death from CHD was independent of conventional cardiovascular risk factors but not of risk factors clustering with hyperinsulinaemia. By applying factor analysis and principal component analysis we showed that "hyperinsulinaemia cluster" (a factor having high positive loadings for body mass index, triglycerides and insulin; and a high negative loading for high-density lipoprotein cholesterol) was predictive of death from CHD in patients with Type II diabetes (hazard ratio with 95% confidence intervals 1.43 (1.18, 1.73), p < 0.001). CONCLUSION/INTERPRETATION: Our results support the notion that cardiovascular risk factors clustering with endogenous hyperinsulinaemia increase the risk of death from CHD in patients with Type II diabetes not treated with insulin. PMID- 10753036 TI - Impaired glucose tolerance, Type II diabetes mellitus and carotid atherosclerosis: prospective results from the Bruneck Study. AB - AIMS/HYPOTHESIS: Cardiovascular disease is a well-known severe complication of impaired glucose tolerance and Type II (non-insulin-dependent) diabetes mellitus. The independent contribution of glucose intolerance to cardiovascular disease and the underlying pathogenic mechanisms are still, however, not clear. METHODS: In this prospective population-based study, 826 subjects aged 40-79 years underwent high resolution duplex ultrasound examinations of carotid arteries and extensive clinical and laboratory screenings for potential vascular risk factors at baseline and 5 years later. The ultrasound protocol involved measurements of maximum axial diameter of atherosclerotic plaques, if any, in common and internal carotid arteries on both sides and enable differentiation of two main stages in carotid artery disease, termed early non-stenotic and advanced stenotic atherosclerosis. Intima-media thickness was assessed at the follow-up examination. RESULTS: Type II diabetes and, to a lesser extent, impaired glucose tolerance were found to be statistically significant risk predictors of 5-year changes in carotid atherosclerosis. These associations were in part independent of other vascular risk factors typically clustering with glucose intolerance. Both impaired glucose tolerance and Type II diabetes mellitus were not independently related to early non-stenotic atherosclerosis. In contrast, Type II diabetes mellitus was the strongest single risk predictor of advanced stenotic atherosclerosis [odds ratio 5.0 (95% confidence intervals 2.3-11.1)] and impaired glucose tolerance was of relevance as well [odds ratio 2.8 (1.2-6.4)] (p < 0.001). CONCLUSION/INTERPRETATION: Impaired glucose tolerance and, to a greater extent, Type II diabetes were strong independent predictors of advanced carotid atherosclerosis in our prospective population-based study. PMID- 10753037 TI - 1H- and 31P-magnetic resonance spectroscopy and imaging as a new diagnostic tool to evaluate neuropathic foot ulcers in Type II diabetic patients. AB - AIMS/HYPOTHESIS: We studied 36 Type II (non-insulin-dependent) diabetic patients without occlusive arterial diseases in the lower extremities and 12 age-matched and sex-matched non-diabetic subjects to clarify the association between diabetic polyneuropathy and foot ulcers using 1H- and 31P-magnetic resonance spectroscopy and imaging. METHODS: The 36 diabetic patients consisted of 12 patients with superficial foot ulcers and 24 patients free from this disease. We measured fat to water and phosphocreatine to inorganic phosphate (PCr:Pi) ratios and calculated the intracellular pH of resting plantar muscles by depth-resolved surface-coil spectroscopy using an 1H-31P double tuned coil. Furthermore, foot vasculature, fat and PCr contents of plantar muscles were visualised by phase contrast angiography, T1-weighted spin-echo imaging and 31P-chemical shift imaging. RESULTS: The 12 foot ulcer patients showed a reduced PCr to Pi ratio (p < 0.001) and peripheral nerve functions (p < 0.01-0.001) but an increased fat to water ratio (p < 0.001) and intracellular pH (p < 0.001) compared with the 24 patients without ulcers. From stepwise multiple regression analyses, motor nerve function as well as severity of nephropathy was associated with both fat to water and PCr to Pi ratios. When these patients were categorised into three groups based on their level of motor nerve function, the frequency of foot ulcers of the lowest group was higher than that of the highest group. CONCLUSION/INTERPRETATION: Our findings indicated that motor nerve dysfunction in diabetic patients was closely associated with impaired energy metabolism, fatty infiltration and increased intracellular pH of plantar muscles and high frequency of foot ulcers. These new techniques could contribute to help clarify the predisposing factors for foot ulcers. PMID- 10753038 TI - Hyperglycaemia-induced subcellular redistribution of GLUT1 glucose transporters in cultured human term placental trophoblast cells. AB - AIMS/HYPOTHESIS: We have recently shown that hyperglycaemia down-regulates the GLUT1 glucose transport system of term placental trophoblast. The reduction in GLUT1 protein alone was, however, not sufficient to explain the decrease in net glucose uptake, suggesting additional mechanisms. Therefore, we hypothesised that hyperglycaemia in vitro leads to a GLUT1 translocation from the trophoblast surface to intracellular sites. METHODS: This was tested in our study by determining the subcellular distribution of GLUT1 in human term placental trophoblast (n = 5 placentas) cultured for 48 h with 5 compared with 25 mmol/l D glucose in vitro using immunogold labelling. RESULTS: Electron microscopic examination of cell profiles showed that 73% of total GLUT1 molecules reside in the trophoblast plasma membrane under basal conditions. The reduced GLUT1 expression (-20%; p < 0.05) after culture of the cells with 25 mmol/l glucose was accompanied by an internalisation of plasma membrane GLUT1, resulting in a loss of 40% (p < 0.05) in cell surface transporter labelling. Western blotting identified a characteristically broad band between 55-65 kDa, confirming the specificity of the GLUT1 antiserum. CONCLUSION/INTERPRETATION: We postulate that in addition to down-regulating human GLUT1 protein concentrations, glucose exerts its autoregulatory effect on hexose transport in term placental trophoblast by altering GLUT1 partitioning between the plasma membrane and intracellular sites in favour of the latter. PMID- 10753039 TI - The tumour necrosis factor alpha -238 G --> A and -308 G --> A promoter polymorphisms are not associated with insulin sensitivity and insulin secretion in young healthy relatives of Type II diabetic patients. AB - AIMS/HYPOTHESIS: Tumour necrosis factor-alpha (TNF-alpha) is believed to influence skeletal muscle insulin resistance. Two G --> A transitions in the promoter region of TNF-alpha at position -238 and -308 have been identified that could play a part in transcriptional regulation of the gene. Insulin resistance is an independent familial trait that predicts the development of Type II (non insulin-dependent) diabetes mellitus. We investigated the influence on insulin sensitivity and insulin secretion of both polymorphisms in a cohort of young healthy relatives of patients with Type II diabetes. METHODS: We examined 109 first-degree relatives of Caucasian patients with a history of Type II diabetes, who underwent extensive metabolical and anthropometrical phenotyping, and determined the TNF-alpha -238 and -308 G --> A promoter polymorphisms. RESULTS: For the -238 polymorphism, 3 probands (76.1%) were homozygous for the G-allele, 25 probands (22.9%) were heterozygous and 1 proband (0.9%) was homozygous for the A-allele. For the -308 polymorphism, 83 probands (76.1%) were homozygous for the G-allele, 24 probands (22.0%) were heterozygous and 2 probands (1.18%) were homozygous for the A-allele. Probands with and without the polymorphism did not differ in insulin sensitivity (p = 0.78), insulin-concentrations and C-peptide concentrations in oral glucose tolerance tests (p > 0.05). CONCLUSIONS/INTERPRETATION: We could not detect an association between insulin sensitivity or insulin secretion and TNF-alpha promoter polymorphisms in our cohort. The polymorphisms occur at the same frequencies in probands with either low or high insulin sensitivity. PMID- 10753041 TI - Glucose intolerance is predicted by low insulin secretion and high glucagon secretion: outcome of a prospective study in postmenopausal Caucasian women. AB - AIMS/HYPOTHESIS: To study the pathophysiological importance of changes in insulin sensitivity and islet function over time for alterations in glucose tolerance in a randomly selected large group of non-diabetic women aged 57-59 years over a 3 year period. METHODS: At baseline and at the 3-year follow-up, glucose tolerance (WHO 75 g oral glucose), insulin sensitivity (euglycaemic, hyperinsulinaemic clamp) and insulin and glucagon secretion (2 to 5-min responses to 5 g i.v. arginine at fasting, 14 and > 25 mmol/l glucose) were measured. RESULTS: At baseline, women with impaired glucose tolerance (IGT, n = 28) had lower insulin sensitivity (p = 0.048) than normal women (NGT, n = 58). The arginine-induced insulin responses (AIR) were inversely associated with insulin sensitivity (r > or = -0.55, p < 0.001). When related to the 3-year follow-up, the baseline product of AIR at 14 mmol/l glucose times insulin sensitivity, insulin effect index (IE) (r = -0.40, p < 0.001) and the arginine-induced glucagon response at 14 mmol/l glucose (AGR, r = 0.28, p = 0.009) both correlated with follow-up 2-h glucose. In a multiple regression model, baseline 2-h glucose, insulin effect index and arginine-induced glucagon response independently predicted 2-h glucose at follow-up (total r = 0.668, p < 0.001). Furthermore, delta insulin sensitivity (i. e. follow-up minus baseline) correlated with delta insulin secretion (r = 0.30, p = 0.006), whereas delta glucagon secretion correlated with delta2-h glucose (r = 0.30, p = 0.006) over the 3 years. In a multiple regression, alterations in 2-h glucose over the 3 years were independently determined by changes in fasting insulin and glucagon secretion (r = 0.424, p < 0.001). CONCLUSION/INTERPRETATION: Low insulin secretion, when judged in relation to insulin sensitivity, and high glucagon secretion, determine glucose tolerance over time in the individual subject. These processes are therefore potential targets for prevention of deterioration in glucose tolerance. PMID- 10753040 TI - Insulin and non-esterified fatty acid relations to alimentary lipaemia and plasma concentrations of postprandial triglyceride-rich lipoproteins in healthy middle aged men. AB - AIMS/HYPOTHESIS: Enhanced and prolonged postprandial lipaemia is related to cardiovascular disease but how postprandial lipaemia is regulated is poorly known. We therefore determined the relations of fasting insulin concentrations to fasting and postprandial lipids, lipoproteins and non-esterified fatty acids in middle-aged men. METHODS: The subjects, 99 healthy 50-year-old men with an apolipoprotein E3/3 genotype, ate a mixed meal. The apolipoprotein B-48 and B-100 contents were determined in triglyceride-rich lipoproteins as a measure of chylomicron remnant and very low density lipoprotein particle concentrations. RESULTS: Fasting plasma insulin was associated with the triglyceride response to the test meal, independently of body mass index, waist-to-hip circumference ratio, blood glucose and the insulin effect on fasting plasma triglycerides. Exaggerated and prolonged postprandial lipaemia in subjects in the upper quartile of the plasma insulin distribution was largely accounted for by large (Svedberg flotation rate > 60) very low density lipoproteins and chylomicron remnants. Insulin relations to large postprandial triglyceride-rich lipoproteins exclusively reflected the association between plasma insulin and the fasting plasma concentrations of these lipoprotein species, whereas plasma insulin and late postprandial plasma concentrations of small (Svedberg flotation rate 20-60) chylomicron remnants were related, independently of insulin influences on fasting concentrations. Strong positive relations were found between the late increases in large triglyceride-rich lipoproteins and plasma non-esterified fatty acid concentrations after 6 h. CONCLUSION/INTERPRETATION: The degree of insulin sensitivity is a major determinant of postprandial lipaemia in healthy middle aged men and could add to the regulation of the basal production of large triglyceride-rich lipoproteins. PMID- 10753042 TI - Islet autoimmunity in infants with a Type I diabetic relative is common but is frequently restricted to one autoantibody. AB - AIMS/HYPOTHESIS: To determine the sequence of development of islet autoantibodies and their relation to HLA genes in infants at risk for Type I diabetes followed from birth. METHODS: We followed 357 (189 male, 168 female) infants, with a first degree relative with Type I diabetes for a mean of 3 years from birth. Human leukocyte antigen typing and assays for insulin autoantibodies (IAA), glutamic acid decarboxylase antibodies (GADAb) and tyrosine phosphatase IA2 (IA2Ab) antibodies were done on cord blood, and venous blood was sampled every 6 months for IAA, GADAb and IA2Ab. RESULTS: We did not find any antibodies in 263 (73%) infants; 50 (14%) were positive for a single antibody once, 19 (5%) for a single antibody more than once and 25 (7%) for two or more antibodies. Of the latter, 10 (2.8% overall) were persistently positive; they had higher frequencies of HLA DR4 (p < 0.01) and HLA DR3, 4 (p < 0.05). Of the group persistently positive for two or more antibodies four infants developed diabetes. Insulin autoantibodies were the first ones to develop in 64% of infants with two or more antibodies. CONCLUSION/INTERPRETATION: Infants with high risk HLA-DR alleles and multiple antibodies at high risk for diabetes were identified. A much larger group of infants had transient low level increases usually of a single antibody. Whereas transient low level positivity could be attributed to difficulties with assay technique and cut off levels for normality, the results overall support the phenomenon of transient 'self limited' islet autoimmunity in at risk infants. PMID- 10753043 TI - Epitope spreading and a varying but not disease-specific GAD65 antibody response in Type I diabetes. The Childhood Diabetes in Finland Study Group. AB - AIMS/HYPOTHESIS: The aim of this study was to analyse the conformational and linear epitope profiles of glutamic acid decarboxylase antibody (GAD65-ab) positive sera to find disease-specific epitope profiles and to study, whether GAD65-ab epitope recognition changes or spreads during the prediabetic period and, thus, can provide markers to differentiate early from later stages of progression to diabetes. METHODS: Sera from subjects before (n = 21), at onset (n = 44), or at increased risk of Type I (insulin-dependent) diabetes mellitus (n = 20) and from patients with stiff-man syndrome (SMS, n = 18) or polyendocrine autoimmune syndrome (PAS, n = 21) were analysed for conformational and linear GAD65 epitope recognition by an immunohistochemical blocking test based on human monoclonal GAD65-ab (MICA 1-10) and western blotting of a GAD65 epitope-cDNA library. RESULTS: A redundant reactivity of many GAD65-ab positive sera to three major conformational (EP-1, EP-2, EP-3) and two dominant linear epitope clusters (amino acid 1-124 and 535-585) was observed in diabetes, polyendocrine autoimmune syndrome and stiff-man syndrome and no disease-specific epitopes or epitope profiles were detected. Epitope recognition broadened with higher titres and with the vulnerability of patients to acquire additional autoimmune diseases apart from diabetes. Low GAD65-ab serum titres (< 1200 arbitrary units) and EP-1 recognition in the absence of EP-2 binding characterised the early immune response. Changing epitope profiles combined stable recognition of EP-1 with gain or loss of reactivity to C-terminal epitopes during follow-up. CONCLUSION/INTERPRETATION: A maturing autoantibody response, which could spread from EP-1-recognition to other regions of GAD65, resulted in titre-related rather than disease-specific epitope profiles which were not sufficient to predict whether GAD65-ab positive subjects will progress to Type I diabetes, autoimmune polyendocrine syndrome or stiff-man syndrome. PMID- 10753044 TI - The impact of diabetes mellitus on survival after myocardial infarction: can it be modified by drug treatment? Results of a population-based myocardial infarction register follow-up study. AB - AIMS/HYPOTHESIS: Mortality of diabetic patients after myocardial infarction remains high despite recent improvement in their management. This study population-based evaluates the impact of cardiovascular drug therapy on mortality within 28 days and during 5-year follow-up in diabetic compared with non-diabetic patients. METHODS: Using the MONICA Augsburg register from 1985 to 1992, 2210 inpatients with incident Q-wave myocardial infarction aged 25-74 years were included, of whom 468 had diabetes. Primary end point was mortality within 28 days and over 5 years. General linear model procedures were used for age adjustment, controlling for sex, and testing significance; hazard risk ratios were calculated using multivariable Cox proportional hazards model procedures. RESULTS: During the 5-year follow-up, 598 subjects died (396 diabetic, 202 non diabetic). The mortality rate within 28 days was 12.6% in diabetic patients (women 18.0%, men 9.9%) and 7.3% in non-diabetic patients (p = 0.001). Mortality in diabetic patients over 5 years was increased by 64% (95% confidence interval 1.39-1.95) compared with non-diabetic patients. This was considerably reduced (p < 0.001) in patients treated with thrombolytic drugs (risk ratio: diabetes 0.57, no diabetes 0.65) and with beta blockers (0.62 and 0.64) and antiplatelets (0.76 and 0.74) at hospital discharge. Mortality of diabetic patients treated with these drugs was reduced to that of non-diabetic patients without such treatment (risk ratio 1.01 to 1.27; p > 0.1). CONCLUSION/INTERPRETATION: Diabetic patients after myocardial infarction are at particularly high risk of dying, but benefit clearly from treatment with thrombolytics, beta blockers and antiplatelets. This study does not, however, allow any inferences to be drawn for treatment with angiotensin converting enzyme inhibitors or the impact of left ventricular function. PMID- 10753045 TI - Contrasting renal functional reserve in very long-term Type I diabetic patients with and without nephropathy. AB - AIMS: This study was to determine whether renal functional reserve (RFR) is present in patients who have suffered long-lasting Type I (insulin-dependent) diabetes mellitus. METHODS: Renal functional reserve was elicited by a 3-h amino acid infusion (4.5 mg x kg(-1) x min(-1)) in 10 patients with nephropathy (DN+) and 10 patients without nephropathy (DN-) who had lived with diabetes for 24 +/- 3 and 27 +/- 3 years, respectively and in 15 healthy control subjects. Renal functional reserve was calculated as the difference between amino acid-stimulated and baseline glomerular filtration rates (GFR). RESULTS: Baseline glomerular filtration rate in DN- patients (106 +/- 8) and control subjects (112 +/- 3 ml x min(-1) x (1.73m2)(-1)) was significantly higher (p < 0.01) than in DN+ patients (79 +/- 7 ml x min(-1) x (1.73m2)(-1)). Renal functional reserve was absent in DN+ patients, whereas it represented 26 +/- 4% of the baseline in DN- patients and 23 +/- 2% in control subjects. Renal vascular resistance decreased statistically significantly during amino acid infusion in DN- patients and control subjects but not in DN+ patients. CONCLUSIONS/HYPOTHESIS: These results indicate that very long-term Type I diabetic patients without diabetic nephropathy still have a normal renal functional reserve. In contrast, this reserve is suppressed in similarly long-term macroalbuminuric and hypertensive patients with overt nephropathy in spite of their remarkably maintained glomerular filtration rate. This opposite impairment supports the interpretation that glomerular hyperfiltration is a determining mechanism in human diabetic nephropathy. PMID- 10753046 TI - Comparison of progression of macrovascular diseases after kidney or pancreas and kidney transplantation in diabetic patients with end-stage renal disease. AB - AIMS/HYPOTHESIS: The aim of the study was to examine the effect of pancreas kidney transplantation on the progression of macrovascular diseases in Type I diabetic patients with end-stage renal disease. METHODS: The progression of cerebrovascular disease, coronary heart disease and peripheral vascular disease in uraemic patients with Type I (insulin-dependent) diabetes mellitus and who had had simultaneous pancreas-kidney transplantation was compared with that of recipients of a kidney transplant alone. Between 1986 and 1998 a total of 11 uraemic diabetic patients received a simultaneous pancreas-kidney transplantation and 10 diabetic patients a kidney transplant alone. All transplants functioned for at least 24 months, the mean observation period was 69 +/- 37 compared with 70 +/- 33 months in both patient groups. Macroangiopathic diseases were classified in four stages as described earlier. RESULTS: In the group with simultaneous pancreas-kidney transplantation progression of cerebrovascular and coronary heart disease was observed in four patients (36%) and progression of peripheral vascular disease in five subjects (45%). In the cohort with kidney transplant alone four patients (40%) showed progression of cerebrovascular and coronary heart disease and five progression of peripheral vascular disease (50%); the difference is not significant. Mean values of HbA1c (5.8 +/- 0.2 vs 7.5 +/- 0.6%, p < 0.001) and serum triglycerides (1.2 +/- 0.4 vs 2.0 +/- 1.0 mmol/l, p < 0.05) were significantly lower in the patients with pancreas-kidney transplantation than in the patient group with kidney transplant alone. Serum cholesterol concentrations and blood pressures were similar in both cohorts. CONCLUSION/INTERPRETATION: From our results we concluded that pancreas-kidney transplantation reduces risk factors for the development of macroangiopathy but fails to halt progression of macrovascular diseases similar to Type I diabetic patients with kidney transplant alone. PMID- 10753047 TI - Albumin permeability in isolated glomeruli in incipient experimental diabetes mellitus. AB - AIMS/HYPOTHESIS: The pre-clinical phase of diabetic nephropathy is characterised by increased glomerular filtration rate and episodes of microalbuminuria. The cause of the microalbuminuria has been variably ascribed to alterations of the size or charge selective barriers of the glomerulus or both or as a consequence of the haemodynamic changes. Our aim was to investigate very early albumin permeability alterations in isolated glomeruli which were not subject to perfusion pressure. METHODS: Isolated glomeruli were studied from 120 male Wistar rats, divided into three groups: streptozotocin-treated, streptozotocin-treated with insulin pellet implants, and controls. From each group ten animals were killed at 7, 14, 28, and 56 days after induction. Study variables included blood pressure, proteinuria, iopamidol clearance, albumin permeability and glomerular area. Subsequently, albumin permeability, proteinuria, and iopamidol clearance were determined in an additional group of 40 diabetic animals studied at 24, 72, 96, and 120 h after induction. RESULTS: Albumin permeability increased steadily from induction in streptozotocin-treated animals, reaching a plateau at approximately 120 h. Glomerular filtration rate was shown to increase significantly at approximately 7 days and proteinuria correlated with it. Glomerular hypertrophy was observed both in streptozotocin-treated animals and in streptozotocin-treated rats with insulin pellet implants. Strict blood glucose control delayed the appearance of the permeability defect in isolated glomeruli and inhibited the increase in glomerular filtration in intact animals. It did not prevent glomerular hypertrophy. CONCLUSION/INTERPRETATION: An albumin permeability defect exists early in isolated non-perfused glomeruli from streptozotocin-treated rats and seems to be independent of glomerular filtration rate alterations. PMID- 10753048 TI - Association of -3826 G variant in uncoupling protein-1 with increased BMI in overweight Australian women. AB - AIMS/HYPOTHESIS: To determine whether genetic variation in uncoupling protein-1 (UCP-1) is associated with obesity or obesity-related risk factors in overweight women. METHODS: We genotyped 526 overweight/obese women (mean body mass index 34.1 kg/m2, range 25.0 to 47.5 kg/m2) for the -3826 A-->G uncoupling protein-1 polymorphism. Of the 526 women genotyped 144 had fasting blood samples analysed for glucose and lipid measurements. RESULTS: The -3826 G allele was found with a frequency of 0.23 and was associated with higher BMI (p = 0.02). A higher frequency of this polymorphism (0.33) was found in subjects with Type II (non insulin-dependent) diabetes mellitus (p = 0.02), though adjustment for BMI weakened this significance (p = 0.06). The -3826 G variant was associated with increased fasting glucose (p = 0.01). This was, however, a result of a greater proportion of women with Type II diabetes also having the G variant (p = 0.10, adjusted for Type II diabetes). The -3826 G variant of uncoupling protein-1 did not have an effect on other metabolic variables associated with obesity. CONCLUSION/INTERPRETATION: In overweight Australian women the -3826 G variant of UCP-1 increased the susceptibility to obesity indicating that UCP-1 could be involved in weight regulation. PMID- 10753049 TI - A genetic variation in the 5' flanking region of the UCP3 gene is associated with body mass index in humans in interaction with physical activity. AB - AIMS/HYPOTHESIS: In obese French Caucasian subjects we previously described a silent UCP3 Tyr99Tyr mutation, associated with body mass index. We hypothesised that an unknown polymorphism in the vicinity of the gene could contribute to obesity. METHODS: Morbidly obese subjects were screened for mutations in 1 kb upstream from the UCP3 gene. Association studies were done between a variant and obesity in 401 morbidly obese and 231 control subjects. RESULTS: We detected three rare genetic variants and one polymorphism: a +5 G-->A in exon 1, a -155 C- >T, a -439 A insertion and a -55 C-->T located 6 bp from the putative TATA box. This variant was in linkage disequilibrium with the Tyr99Tyr polymorphism. Frequencies of the variant allele at the -55 locus were similar in the obese and control groups (0.23 vs 0.21). The -55 polymorphism was associated with BMI in the obese group (p = 0.0031): BMI was higher in TT than in CC or CT patients. Likewise control subjects with a TT genotype had a higher BMI (p = 0.03). In the obese group, homozygosity for this variant is a risk factor for high BMI (odds ratio: 1:75, p = 0.02). Obese patients were divided into tertiles according to physical activity. In the group with a wild C/C genotype, BMI was negatively associated with physical activity (p = 0.015). CONCLUSION/INTERPRETATION: The C- >T polymorphism in the 5' sequences of the UCP3 gene might contribute to the corpulence in obese and normal weight subjects and alter the benefit of physical activity. The UCP3 gene can be considered as a gene modifying corpulence. PMID- 10753050 TI - A high prevalence of glucokinase mutations in gestational diabetic subjects selected by clinical criteria. AB - AIMS/HYPOTHESIS: Patients with glucokinase mutations are characterised by mild, persistent fasting hyperglycaemia, a small increment in glucose in response to an oral load and a dominant family history. These patients frequently present with gestational diabetes and often require insulin treatment during pregnancy. We assessed whether the selection of gestational diabetic subjects by clinical criteria would result in a high detection rate of glucokinase mutations. METHODS: Caucasian gestational diabetic subjects from the United Kingdom who had fasting hyperglycaemia in pregnancy but did not meet the diagnostic criteria for maturity onset diabetes of the young (MODY) were selected for direct sequencing of the glucokinase gene if they fulfilled the following four criteria; (1) persisting fasting hyperglycaemia outside pregnancy (5.5-8 mmol/l) (2) a small increment (< 4.6 mmol/l) during a 2-h oral glucose tolerance test (3) insulin treatment during at least one pregnancy but subsequently controlled on diet and (4) a history of Type II (non-insulin-dependent) diabetes mellitus, gestational diabetes or fasting hyperglycaemia (> 5.5 mmol/l) in a first-degree relative. RESULTS: Of the 15 subjects 12 (80%) with all these clinical criteria had glucokinase gene mutations. These included four previously unreported mutations (N180K, R191W, Y215X and L288-1G --> A). CONCLUSION/INTERPRETATION: Phenotypic selection of subjects with gestational diabetes greatly increases the likelihood of detecting a mutation in the glucokinase gene as previous studies have suggested a prevalence of 2.5% (range 0-6%). Our study in gestational diabetes to successfully used clinical criteria to assist in the definition of a genetic subgroup. PMID- 10753051 TI - Randomized controlled trials remain fundamental to clinical decision making in Type II diabetes mellitus: a comment to the debate on randomized controlled trials (For debate) [corrected]. PMID- 10753053 TI - Genetic heterogeneity by age at onset of Type I diabetes: higher prevalence of patients with 0 susceptible heterodimers in adults than in children in the registry of Turin, Italy. PMID- 10753052 TI - Diabetes in a population-based series of twin pairs discordant for leisure sedentariness. PMID- 10753054 TI - Insulin-dependent diabetes mellitus induced by the antitussive agent dextromethorphan. PMID- 10753055 TI - Amylin and hypertension: the status of the current debate. PMID- 10753056 TI - Study of the protective effects of hyperimmune immunoglobulins G and M against endotoxin in mice and rats. AB - We prepared solutions of human IgM and IgG to various lipopolysaccharide (LPS) species. These were then tested, along with solutions of non-LPS specific human IgG or IgM, for their ability to confer passive immunity against experimental endotoxemia in two animal models. The immunoglobulins were first tested for an effect on the lethality induced by seven different LPSs in actinomycin-D sensitized mice, or by three different bacteria in normal mice. When the immunoglobulins were administered 1 h before challenge, a small protective effect was observed. This protection was dependent upon both the anti-LPS agent, the chemical composition of the LPS, or the strain of gram-negative bacteria used for injection. The anti-LPS IgM and IgG preparations reduced the mortality induced by Escherichia coli but not by Serratia marcescens or Klebsiella pneumoniae, indicating protection by strain-specific antibodies. When the antibodies were preincubated with LPS or bacteria for 30 min before administration, almost complete protection was seen. The influence of these immunoglobulin preparations or of human albumin (as a control) on the hypotensive and vascular-permeabilizing effects of LPS in rats was then studied. A dose-dependent inhibitory effect was observed with IgG preparations and albumin. At 200 mg/kg, anti-LPS IgG reduced the effects of LPS, while at 400 mg/kg, both anti-LPS and normal IgG preparations showed protection, as did human albumin used at the same dose. The IgM-enriched preparation worsened the initial hypotensive phase after LPS, whereas the anti LPS IgM significantly reduced the second phase of the hypotension, but only at the largest dose of 400 mg/kg. In this second model using the rat, a clear difference between the activity of IgG and IgM was thus observed. We conclude that pretreatment with human immunoglobulins from large plasma pools modestly, but significantly, attenuated the effects of murine and rat Gram-negative sepsis, but that protection was incomplete. Our results suggest that single regimen intervention strategies may not be sufficient to influence the course of the disease. PMID- 10753058 TI - Induction of apoptosis in human renal proximal tubular epithelial cells by Escherichia coli verocytotoxin 1 in vitro. AB - Verocytotoxin 1 and 2 (VT1 and 2) produced by verocytotoxin-producing Escherichia coli have been considered to play an important role in the pathogenesis of glomerular and tubular damage in the epidemic form of hemolytic uremic syndrome (HUS). VTs are known to be cytotoxic to culture cells by inhibiting cellular protein synthesis. In this in vitro study, the mechanism(s) of tubular damage in HUS and the ability of VT1 to induce apoptosis in normal human renal proximal tubular epithelial cells (HRPTEC) were examined. VTI markedly reduced cell viability of HRPTEC and rapidly inhibited overall protein synthesis. VT1 directly induced apoptotic cell death in HRPTEC in a dose- and time-dependent fashion, and co-incubation with tumor necrosis factor-alpha enhanced the VT1-induced apoptosis. These results suggest that apoptosis induced by VT1, possibly in concert with host cytokines, in renal tubular cells may contribute to the tubular damage in HUS. PMID- 10753057 TI - Influence of human anti-lipopolysaccharide immunoglobulins on tissue distribution and clearance of lipopolysaccharide in rats. AB - To examine the influence of passive immunization on the biological fate of injected lipopolysaccharide (LPS), we used a human IgG preparation (anti-LPS IgG) rich in antibodies to a large panel of smooth and rough purified LPS extracts as well as a normal IgG preparation (standard IgG). Our approach was to compare the uptake of 125I-labeled LPS by the tissues of saline or IgG-treated rats. After intravenous injection, one fraction of 125I-labeled Escherichia coli O55:B5 LPS is rapidly taken up by tissues, while another fraction remained in the blood. Uptake of 125I-labeled LPS was principally observed into the liver and spleen. In rats treated prophylactically with standard IgG, these tissues accumulated significantly larger amount of LPS than the tissues of rats treated with anti-LPS IgG. Nevertheless, both IgG preparations increased the specific binding of LPS by the liver and spleen. High levels of homologous unlabeled LPS decreased the uptake of LPS by the liver, presumably by occupying tissue receptors, whereas in the presence of E. coli O127:B8 LPS, an increase of the uptake of 125I-labeled LPS by the liver and lungs was observed. The pharmacokinetics and tissue distribution of LPS-IgG complexes pre-formed in vitro were compared. In the presence of standard IgG, a unexpected increase of the uptake of LPS by the tissues was recorded, whereas LPS-anti-LPS IgG complexes decreased the binding of 125I-labeled LPS to the tissues. On the other hand, the vascular effects induced by LPS did not appear to be modified in rats pretreated with either IgG preparation. In conclusion, although passive immunization against LPS slightly modified the uptake and clearance of LPS, neither in vitro nor in vivo formation of LPS-anti-LPS IgG complexes afforded a very significant protection against the toxic effects of LPS. PMID- 10753059 TI - Isolation of monoclonal antibodies directed against the V protein of human parainfluenza virus type 2 and localization of the V protein in virus-infected cells. AB - Two monoclonal antibodies (mAbs) specific for the human parainfluenza virus type 2 (hPIV-2) V protein were obtained by immunizing mice with the V protein recombinantly expressed in Escherichia coli. Both mAbs were found to react with the V protein in ELISA and in Western blot analysis. Using these mAbs and previously obtained mAbs specific for hPIV-2 nucleoprotein (NP) or hPIV-2 phospho (P) protein, we examined the intracellular distributions of the V, P and NP proteins in hPIV-2-infected cells by indirect immunofluorescence analyses. The P and NP proteins were organized in numerous granules in the cytoplasm of hPIV-2 infected cells. In contrast, the V protein showed diffuse nuclear and cytoplasm distributions. PMID- 10753060 TI - The prevalence of TT virus (TTV) infection and its relationship to hepatitis in children. AB - TT virus (TTV) is a newly discovered virus from a patient with post-transfusion hepatitis. We investigated the frequency and pathogenesis of TTV infection in children. A semi-nested PCR assay was used to amplify TTV-DNA in serum samples from 254 ambulatory children without liver disease, 20 with hepatitis of unknown etiology, and 18 transfusion recipients or hemophiliacs. In positive samples, TTV DNA was quantified by real-time quantitative PCR using a fluorescent probe. We detected TTV-DNA in 20% of children with hepatitis of unknown etiology, which was not statistically different from the 23% prevalence in ambulatory children. In transfusion recipients or hemophiliacs, the frequency was higher (50%) than that in ambulatory children (P = 0.01). Among ambulatory children, TTV-DNA was frequently detected in children with acute gastroenteritis (36%). TTV-DNA was detected in 10% of the infants under 6 months old, and 20% of the children from 7 to 12 months old. The prevalence was constant after the age of 1 year; however, the copy number of TTV-DNA was significantly higher in children under 1 year of age (mean: 10(5.4) versus 10(3.8) copies/ml, P= 0.008). Finally, TTV-DNA was quantified serially in three children with chronic hepatitis who were positive for TTV-DNA. The presence or amount of TTV-DNA was unrelated to the serum alanine aminotransferase level. These results indicate that TTV infection is common in children. The larger quantity of TTV-DNA in infants and the high prevalence of TTV in children of all ages suggest that TTV may be transmitted in early childhood. Its relationship to hepatitis is doubtful in children. PMID- 10753061 TI - Highly resistant Burkholderia pseudomallei small colony variants isolated in vitro and in experimental melioidosis. AB - Burkhloderia pseudomallei is the causative agent of melioidosis, a disease in which treatment failures and relapses are common. This study reports on slow growing B. pseudomallei 'small colony variants' (SCVs), isolated either in vitro after exposure to ceftazidime, ciprofloxacin or gentamicin or from the spleen and liver in a mouse model of melioidosis after treatment with ceftazidime. Interestingly, SCVs isolated by either method or antimicrobial agent showed a significant increase in the minimal inhibitory concentrations of various unrelated classes of antimicrobial agents. B. pseudomallei SCVs did not differ from their parental strains in standard biochemical profiles, nor by pulsed field gel electrophoresis or electron microscopy. Although the SCV phenotype was stable throughout numerous passages on antibiotic-free solid media, revertants with the parental colony morphology and, most importantly, with the parental susceptibility pattern occurred. These revertants led to rapid overgrowth of SCVs in liquid media without added antibiotics. Future studies will have to determine the clinical relevance of B. pseudomallei SCVs especially in treatment failure and relapse of infection. PMID- 10753062 TI - Cysteine protease SpeB expression in group A streptococci is influenced by the nutritional environment but SpeB does not contribute to obtaining essential nutrients. AB - Group A streptococcal (GAS) cysteine protease is a major virulence factor involved in the pathogenesis of purulent and invasive infections. The secreted enzyme cleaves a number of different bacterial and host proteins which could contribute to different stages of the infective processes. It has been proposed that, among these functions, SpeB plays a role in obtaining nutrients during late growth phases. In the present study, speB mutants of various GAS serotypes were found to exhibit unaltered growth characteristics in several complex and chemically defined media (CDM). When amino acid-depleted CDM was prepared, neither SpeB activity on whole proteins added to the medium during incubation nor the addition of SpeB-digested proteins was able to support bacterial growth. SpeB also was unable to liberate iron from iron-containing protein sources added to iron-deficient CDM. However, SpeB levels in culture supernatants changed in response to the protein and glucose content of the media. Using a speB promoter luciferase reporter, speB expression levels were found to correspond to peptide concentrations in the culture media. The effect appeared to be specific for peptides since addition of peptides derived from various proteins had an affect on expression, while addition of the whole proteins had no effect. Addition of glucose to CDM had no effect on speB expression, while glucose addition to complex medium decreased speB expression. Overall, SpeB did not appear to be directly involved in providing the bacteria with nutritional factors but expression of the speB gene responded to ratios of peptides and carbohydrates in the culture medium. PMID- 10753063 TI - Liver repopulation with hepatocyte transplantation: new avenues for gene and cell therapy. AB - Liver-directed gene therapy is appropriate for many conditions. Recent work established that liver repopulation with transplanted cells can be effective in treating genetic disorders. Although hepatocytes express therapeutic genes with considerable efficiency, correction of genetic disorders is constrained by limitations in permanent gene transfer into hepatocytes and repopulation of the liver with transplanted cells. Adenoviral vectors are highly efficient for hepatic gene transfer but the onset of deleterious host immune responses against adenoviral vectors, along with clearance of transduced hepatocytes have caused problems. Nonetheless, recent work concerning engraftment and proliferation of transplanted hepatocytes in the liver has provided significant new information, which should refocus interest in hepatocyte-based therapies. Moreover, hepatocyte transplantation systems offer creative tools for defining critical mechanisms in gene regulation and survival of transduced cells. PMID- 10753064 TI - Extended tropism of an adenoviral vector does not circumvent the maturation dependent transducibility of mouse skeletal muscle. AB - BACKGROUND: Efficient adenoviral gene delivery to mature skeletal muscle has been hindered by different factors. The low levels of adenoviral attachment receptor (CAR) that have been reported in this tissue may be a limiting factor. Therefore, adenoviral transduction of mature muscle may be improved by extending the tropism of the adenoviral vectors to attachment receptors that are highly expressed in mature myofibers. In this study, we have investigated whether an extended tropism adenoviral vector which additionally attaches to the broadly expressed heparan containing receptors (AdPK) can bypass the maturation-dependent adenoviral transducibility of mouse skeletal muscle. METHODS: The adenoviral vector AdPK carrying the LacZ gene was evaluated as a gene delivery vehicle in mouse skeletal muscle at different maturities in vitro and in vivo. The viral transduction efficiencies were determined by histochemical and ONPG analysis of the beta galactosidase activity level. RESULTS: Higher transduction efficiencies were detected in immature muscle from normal mice, and in mature muscle from merosin deficient dy/dy mice (carrying myofibers with an impaired extracellular matrix) and dystrophin-deficient mdx mice (showing a high level of myoblast activity) when compared to mature muscle from normal mice. CONCLUSION: Despite the enhanced attachment characteristics, the extended tropism adenoviral vector is, similarly to the wild-type adenoviral vector in previous studies, still hindered by both a protective extracellular matrix and the diminished myoblast-mediation in mature muscle. PMID- 10753065 TI - Dendritic cell-based genetic immunization in mice with a recombinant adenovirus encoding murine TRP2 induces effective anti-melanoma immunity. AB - BACKGROUND: The induction of cellular immune responses to melanocyte-specific enzymes such as the tyrosinase family of proteins is the goal of various clinical studies for the immunotherapy of melanoma. Tyrosinase-related protein-2 (TRP2) is an attractive model antigen for preclinical studies in C57BL/6 mice because it is naturally expressed by the murine B16 melanoma and can be recognized by self reactive cytolytic T lymphocytes (CTL). Here we describe efforts to develop genetic immunization with dendritic cells (DC) for the immunotherapy of melanoma in this clinically relevant system. METHODS: Recombinant adenoviruses encoding green fluorescent protein (Ad-EGFP) and murine TRP2 (Ad-mTRP2) were constructed using Cre-loxP-mediated recombination. DC were generated in vitro from precursors in bone marrow and transduced with Ad-EGFP or Ad-mTRP2. Mice were immunized by direct injection of adenovirus or by injection of Ad-transduced DC. Induction of tumor immunity was assessed by intravenous challenge with B16 melanoma cells and enumeration of experimentally induced lung metastases. RESULTS: Flowcytometric analysis of DC transduced with Ad-EGFP demonstrated endogenous fluorescence due to cytoplasmatic expression of EGFP in 30-60% of cells. Ad-EGFP-transduced DC simultaneously displayed the DC-specific marker NLDC145 and high levels of MHC and costimulatory molecules on their cell surface. Transduction of DC with Ad mTRP2 resulted in strong intracellular expression of TRP2 which could be readily detected by immunostaining. Importantly, immunization of mice with cultured Ad mTRP2-transduced DC completely prevented the development of lung metastases following an intravenous challenge with B16 melanoma cells. This striking protective effect was observed with both the intravenous and the subcutaneous route of DC immunization. In vivo depletion of T-cell subsets suggested that the protective effect of an immunization with Ad-mTRP2-transduced DC involved both CD8+ and CD4+ T-cells. CONCLUSIONS: Our results demonstrate that DC-based genetic immunization of mice with TRP2, a clinically relevant melanocyte-specific self antigen, induces effective cellular immunity and prevents metastatic growth of B16 melanoma cells in vivo. PMID- 10753066 TI - Cationic polymeric gene delivery of beta-glucuronidase for doxorubicin prodrug therapy. AB - BACKGROUND: An approach to improve current chemotherapy is the selective transduction of tumor cells with suicide genes to sensitize these cells to prodrugs of cytostatic agents. METHODS: In this study, gene transfer was accomplished with the cationic polymer poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), able to condense plasmid-DNA by electrostatic interaction. OVCAR-3 cells were transfected with plasmids encoding E. coli-derived or human beta glucuronidase and the transfection efficiency and inhibition by serum was determined. Next, we measured the sensitivity of OVCAR-3 cells transiently expressing beta-glucuronidase to the glucuronide prodrug of doxorubicin (DOX-GA3) or to doxorubicin. RESULTS: OVCAR-3 cells were efficiently transfected with a plasmid encoding E. coli-derived beta-glucuronidase. The degree of transfection (30% of cells) was higher than that achieved with commercially available cationic lipids (DOTAP, Lipofectamine) without inhibition by serum. OVCAR-3 cells transiently expressing beta-glucuronidase were equally sensitive to the glucuronide prodrug of doxorubicin (DOX-GA3) or to doxorubicin itself, indicating complete conversion of prodrug to drug. Similar studies were performed with the plasmid encoding for human beta-glucuronidase, which is likely to be less immunogenic. Also in this case, OVCAR-3 cells showed an increased sensitivity to the prodrug DOX-GA3, although less pronounced than when the bacterial enzyme was used. A strong bystander effect was observed when OVCAR-3 cells transfected with beta-glucuronidase were mixed with non-transfected cells at different ratios. Complete tumor cell growth inhibition was already observed when only 15% of the cells expressed the activating enzyme. CONCLUSION: These studies suggest that beta-glucuronidase gene therapy using PDMAEMA as a carrier system and DOX-GA3 as the prodrug has a potential application in cancer gene therapy. PMID- 10753067 TI - Intramuscular administration of expression plasmids encoding interferon-gamma receptor/IgG1 or IL-4/IgG1 chimeric proteins protects from autoimmunity. AB - BACKGROUND: Interferon gamma (IFNgamma) is an inflammatory cytokine that promotes autoimmune insulitis and diabetes in NOD mice, while interleukin-4 (IL-4) is protective. We constructed plasmids encoding either an IFNgamma receptor/IgG1 (IFNgammaR/IgG1) chimeric protein which inhibits IFNgamma, or an IL-4/IgG1 chimeric protein with IL-4 activity, for therapeutic gene transfer into NOD mice. METHODS: Murine IFNgammaR/IgG1 and IL-4/IgG1 cDNA segments were cloned into the VICAL VR1255 expression plasmid. Naked plasmid DNA was injected i.m. into young NOD mice, which were then observed for development of insulitis and diabetes. RESULTS: After transient transfection of COS-7 cells, IFNgammaR/IgG1 and IL 4/IgG1 fusion proteins are secreted in vitro as disulfide-linked homodimers, with the expected biological activity. Intramuscular injection of these vectors results in the production of the respective fusion proteins locally in muscle. In serum, the IFNgammaR/IgG1 protein is present at >200 ng/ml over 130 days after the last of five DNA injections, but IL-4/IgG1 is undetectable in our assays (<10 pg/ml) at all time points. Both vectors protect NOD mice from autoimmune insulitis and diabetes, but the IL-4/IgG1 vector is more effective. Neutralization of IFNgamma with IFNgammaR/IgG1 was most protective when treatment was begun early (3 weeks of age). CONCLUSION: Gene therapy by i.m. injection of these plasmids protects NOD mice from autoimmunity, and the IL-4/IgG1 vector is more effective despite low circulating protein levels. These chimeric proteins consist of nonimmunogenic self elements and are suitable for long-term therapy of autoimmune disorders. PMID- 10753068 TI - Therapeutic plasma concentrations of human factor IX in mice after gene delivery into the amniotic cavity: a model for the prenatal treatment of haemophilia B. AB - BACKGROUND: Several groups including our own have reported gene delivery to fetal organs by vector administration into the amniotic cavity. Based on these studies we hypothesised that the large surface of the fetal skin may be exploitable for high level production of systemically required gene products to be released into the fetal circulation. METHODS: We administered E1/E3-deleted adenoviral vectors carrying a bacterial beta-galactosidase gene or the human coagulation factor IX gene into the amniotic cavities of mid- to late-gestation mouse fetuses. The concentrations of human factor IX in the plasma of fetal or new-born mice were determined by ELISA. Reverse transcription PCR was used to identify sites of transgene expression. RESULTS: Application of 5 x 10(8) infectious units of the factor IX gene vector in utero resulted in plasma concentrations of human factor IX of up to 1.2 microg/ml without significant decrease in fetal survival. Transgenic protein was found to be produced in the fetal skin, mucosae and amniotic membranes and was shown to be present for several days after birth of healthy pups. CONCLUSION: As ultrasound-guided amniocentesis in humans is a well established diagnostic procedure, delivery of the factor IX gene into the amniotic cavity appears to be a safe route for prenatal treatment of haemophilia B and may prevent haemorrhagic complications such as intracranial bleeding during delivery. Our study allowed for the first time a quantification of the expression of a potentially therapeutic transgene in rodents after prenatal gene delivery. It thus provides a model for the prenatal treatment of haemophilia B, but may also serve as a pathfinder to gene therapy of inheritable skin disorders such as epidermolysis bullosa. PMID- 10753069 TI - Closed hollow-fiber bioreactor: a new approach to retroviral vector production. AB - BACKGROUND: The ability to obtain high-titer and large quantities of retroviral vector production in a 'closed' system would have profound implications in clinical and experimental gene therapy. METHODS: We studied the cell growth and vector production of three retroviral packaging cell lines in a variety of conditions using hollow-fiber bioreactors designed as an 'artificial capillary system' (ACS) and enhanced with the application of a hermetically sealing device for sterile welding of connecting plastic tubings. Vector titer, fetal bovine serum (FBS) concentration, volume and the duration of productivity were assessed to optimize vector production. RESULTS: In this pilot study, we observed that retroviral vector production (frozen-and-thawed) from cultures containing as low as 2.5% FBS yielded titers up to 2.2 x 10(7) cfu/ml, 14.4-fold higher than titers obtained from control dish cultures. Up to 3 liters of vector supernatant were generated during a 2-month large-scale production run. There was a potential to double this volume of higher-titer supernatant by increasing the frequency of harvest. It seemed that a lower metabolic rate (i.e. lactate production) in the packaging cell culture was associated with higher vector producing ability. CONCLUSIONS: These data demonstrated the feasibility of producing retroviral vector with enhanced titers and clinically useful quantities in a 'closed' ACS. Thus a new approach for large-scale retroviral vector production is developed. PMID- 10753070 TI - Non-HIV lentiviral vectors: interview with Alan Kingsman. PMID- 10753071 TI - The IVth workshop on Duchenne muscular dystrophy gene therapy. PMID- 10753072 TI - Detecting the Geographical Pattern of Speciation from Species-Level Phylogenies. AB - We introduce a general approach for investigating the role of geography in speciation, based on analyzing the geography of sister clades across all nodes in a species-level phylogeny. We examine the predictions of allopatric, sympatric, and peripatric models of speciation in several animal groups, using patterns of range overlap and range size symmetry between sister clades. A simple model of cladogenesis incorporating random movements of species' ranges is used to illustrate the effects of range changes on expected patterns. We find evidence for a predominantly allopatric mode of speciation in our study groups, with sympatry arising through postspeciational range changes. In addition, we find that relatively recent speciation events are characterized by greater asymmetry in range size between sister clades than expected under our null models, providing potential support for the peripatric model of speciation. We discuss the possible confounding effects of postspeciational range changes on our conclusions. PMID- 10753073 TI - The Interaction between Competition and Predation: A Meta-analysis of Field Experiments. AB - Ecologists working with a range of organisms and environments have carried out manipulative field experiments that enable us to ask questions about the interaction between competition and predation (including herbivory) and about the relative strength of competition and predation in the field. Evaluated together, such a collection of studies can offer insight into the importance and function of these factors in nature. Using a new factorial meta-analysis technique, we combined the results of 20 articles reporting on 39 published field experiments to ask whether the presence of predators affects the intensity of competitive effects and to compare the average effects of competition and predation. Across all studies, the effects of competition in the presence of predators were less than in the absence of predators, and the interaction between competition and predation for most response variables was statistically significant. Removal of competitors had much more positive effects on organisms' growth and mass than did exclusion of predators. Predator exclusion had much more beneficial effects on organisms' survival than did competition. The mean effects of competition and predation on density did not differ from one another. The results differed among trophic levels. Further understanding would benefit greatly from more field experiments that manipulate both competition and predation, that focus on a wider range of organisms and environments, that focus on population-level parameters such as density, and that report results more completely, including data such as sample sizes and variances. PMID- 10753074 TI - Reproductive Effort and Reproductive Values in Periodic Environments. AB - Life-history theory concerns the optimal spread of reproduction over an organism's life span. In variable environments, there may be extrinsic differences between breeding periods within an organism's life, affecting both offspring and parent and giving rise to intergenerational trade-offs. Such trade offs are often discussed in terms of reproductive value for parent and offspring. Here, we consider parental life-history optimization in response to varying offspring values of a population regulated by territoriality, where the quality of the environment varies periodically. Periods are interpreted as either within year (seasonality) or between-years variation (cyclicity). The evolutionarily stable strategy in a general model with two-phased periodicity in the environment can generate either higher or lower effort in the more favorable of the two phases; hence knowing survival prospects of offspring does not suffice for predicting reproductive effort-the future of all descendants and the parent must be tracked. We also apply our method to data on the Ural owl Strix uralensis, a species preying on cyclically fluctuating voles. The observed dynamics are best predicted by assuming delayed reproductive costs and Type II functional response. Accounting for varying offspring values can lead to cases where both reproductive effort and recruitment of offspring are higher in the phase when voles are not maximally abundant, a pattern supported by our data. PMID- 10753075 TI - Forest Canopy Stratification-Is It Useful? AB - It has long been recognized that the forest canopy has a complex structure that is significant for environmental interactions, regeneration, growth, and biotic habitat. Not only is the structure variously complex, but also there are many ways to conceptualize that complexity. Yet the persistent theme when considering the structure of canopies continues to be that of stratification: whether structural units are arranged in layers above the ground. We examined the use of the terms "stratification," "layering," and others in connection with canopy structure and found they had various meanings (often only implied) that were difficult to reconcile and to measure. We applied the definitions to the structure of a single, well-studied canopy located in Virginia, U.S.A., and found they failed to define consistently and clearly the presence, number, or location of strata. Additionally, we found the concept had limitations related to scale dependence, point of reference, and spatial averaging. Thus, asserting that a forest is stratified or naming the number of layers generally provided no guide to its structure. We propose alternative ways of conceptualizing and studying the forest canopy that avoid most of the problems associated with stratification. Among these are direct measurement and mapping of structural and environmental variables that have clear potential connections with canopy functions and viewing the distribution of structures or environmental conditions within the canopy as ecological gradients. PMID- 10753076 TI - Evolutionarily Stable Dispersal Rates Do Not Always Increase with Local Extinction Rates. AB - Earlier models on the evolution of dispersal have suggested that evolutionarily stable dispersal rates should increase with the frequency of local extinctions. Most metapopulation models assume site saturation (i.e., no local population dynamics), yet the majority of species distributed as metapopulations rarely attain carrying capacity in all occupied patches. In this article, we relax this assumption and examine the evolutionarily stable dispersal rate under nonsaturated but still competitive demographic conditions. Contrary to previous predictions, we show that increasing local extinction rates may allow decreasing dispersal rates to evolve. PMID- 10753077 TI - A General and Dynamic Species Abundance Model, Embracing the Lognormal and the Gamma Models. AB - One aspect of community ecology that has been given particular attention is the pattern of species abundances in a community. The species may have a wide range of abundances; some are very common and others rare. When species abundance models are fitted to observations, the lognormal model and one of the gamma models (e.g., the log-series model) are usually applied. The model that gives the best fit according to some goodness-of-fit test is then chosen. By applying a diffusion approximation for each species' dynamics with density regulation of the theta-logistic type, we here present a general species abundance model that embraces the two most widely applied species abundance models, the lognormal and the gamma. Our general model will, therefore, provide a better fit than the two special cases, except when it corresponds to one of them. In contrast to the classical models, ours is also dynamic, making it possible to evaluate the fluctuations in species abundance over time through both biotic and abiotic factors. The model is fitted to several species abundance data sets and our results compared to previous attempts to fit a model, usually either the lognormal or the log-series. PMID- 10753078 TI - Optimal Floating and Queuing Strategies: The Logic of Territory Choice. AB - This is a response to a recent article by Hanna Kokko and William J. Sutherland (American Naturalist 152:354-366), who consider evolutionarily stable territory acceptance rules for animals that face the decision between settling on a poor territory now (which is then retained for life) or waiting for better habitat to become available later (taking a chance of dying before reproducing). In contrast to these authors, we argue that the evolutionarily stable threshold quality above which territories are acceptable does depend on whether individuals compete for a single territory (queuing) or for multiple territories (floating) and also on whether access to territories is determined by a hierarchy among waiting individuals. More specifically, we show the following: First, if the choice is between floating and settling, the evolutionarily stable acceptance threshold is such that threshold territories yield an expected lifetime reproductive success (LRS) of [Formula: see text], the survival probability of a floater. Second, if the choice is between queuing and settling, the evolutionarily stable threshold may correspond to any LRS between [Formula: see text] and unity. Third, the number of nonbreeding individuals in the population is maximized at a threshold of unity. In other words, the evolutionarily stable threshold does not maximize the nonbreeding fraction of the population. We argue that models of territory choice should carefully specify the mechanism of choice because some choice processes (e.g., indiscriminate habitat use above the threshold) do not admit an evolutionarily stable acceptance rule. PMID- 10753079 TI - Animal Guts as Ideal Chemical Reactors: Maximizing Absorption Rates. AB - I solved equations that describe coupled hydrolysis in and absorption from a continuously stirred tank reactor (CSTR), a plug flow reactor (PFR), and a batch reactor (BR) for the rate of ingestion and/or the throughput time that maximizes the rate of absorption (=gross rate of gain from digestion). Predictions are that foods requiring a single hydrolytic step (e.g., disaccharides) yield ingestion rates that vary inversely with the concentration of food substrate ingested, whereas foods that require multiple hydrolytic and absorptive reactions proceeding in parallel (e.g., proteins) yield maximal ingestion rates at intermediate substrate concentrations. Counterintuitively, then, animals acting to maximize their absorption rates should show compensatory ingestion (more rapid feeding on food of lower concentration), except for the lower range of diet quality for complex diets and except for animals that show purely linear (passive) uptake. At their respective maxima in absorption rates, the PFR and BR yield only modestly higher rates of gain than the CSTR but do so at substantially lower rates of ingestion. All three ideal reactors show milder than linear reduction in rate of absorption when throughput or holding time in the gut is increased (e.g., by scarcity or predation hazard); higher efficiency of hydrolysis and extraction offset lower intake. Hence adding feeding costs and hazards of predation is likely to slow ingestion rates and raise absorption efficiencies substantially over the cost-free optima found here. PMID- 10753080 TI - Animal Guts as Nonideal Chemical Reactors: Partial Mixing and Axial Variation in Absorption Kinetics. AB - Animal guts have been idealized as axially uniform plug-flow reactors (PFRs) without significant axial mixing or as combinations in series of such PFRs with other reactor types. To relax these often unrealistic assumptions and to provide a means for relaxing others, I approximated an animal gut as a series of n continuously stirred tank reactors (CSTRs) and examined its performance as a function of n. For the digestion problem of hydrolysis and absorption in series, I suggest as a first approximation that a tubular gut of length L and diameter D comprises [Formula: see text] tanks in series. For [Formula: see text], there is little difference between performance of the nCSTR model and an ideal PFR in the coupled tasks of hydrolysis and absorption. Relatively thinner and longer guts, characteristic of animals feeding on poorer forage, prove more efficient in both conversion and absorption by restricting axial mixing. In the same total volume, they also give a higher rate of absorption. I then asked how a fixed number of absorptive sites should be distributed among the n compartments. Absorption rate generally is maximized when absorbers are concentrated in the hindmost few compartments, but high food quality or suboptimal ingestion rates decrease the advantage of highly concentrated absorbers. This modeling approach connects gut function and structure at multiple scales and can be extended to include other nonideal reactor behaviors observed in real animals. PMID- 10753081 TI - Fluctuating Environments and Phytoplankton Community Structure: A Stochastic Model. AB - Spatial heterogeneity in organism and resource distributions can generate temporal heterogeneity in resource access for simple organisms like phytoplankton. The role of temporal heterogeneity as a structuring force for simple communities is investigated via models of phytoplankton with contrasting life histories competing for a single fluctuating resource. A stochastic model in which environmental and demographic stochasticity are treated separately is compared with a model with deterministic resource variation to assess the importance of stochasticity. When compared with the deterministic model, the stochastic model allows for coexistence over a wider range of parameter values (or life-history types). The model suggests that demographic stochasticity alone is far more important in increasing the possibility of coexistence than environmental stochasticity alone. However, the combined effects of both types of stochasticity produce the largest likelihood of coexistence. Finally, the influence of relative nutrient levels and nutrient pulse frequency on these results is addressed. We relate our findings to variable environment theory with evidence for both relative nonlinearity and the storage effect acting in this model. We show for the first time that temporal dynamics generated by demographic stochasticity may operate like the storage effect at particular spatial scales. PMID- 10753082 TI - Daily Patterns of Optimal Producer and Scrounger Use under Predation Hazard: A State-Dependent Dynamic Game Analysis. AB - Feeding in groups often gives rise to joining: feeding from other's discoveries. The joining decision has been modeled as a producer-scrounger game where the producer strategy consists of searching for one's food and the scrounger strategy consists of searching for food discovered by others. Previous models revealed that the evolutionarily stable proportion of scrounging mostly depends on the fraction of each food patch available only to its producer. These early models are static and state independent and are therefore unable to explore whether the time of day, the animal's state, and the degree of predation hazard influence an individual's decision of whether to use the producer or scrounger strategy. To investigate these issues, we developed a state-dependent dynamic producer scrounger game model. The model predicts that, early in the day, low reserves promote a preference for the scrounger strategy, while the same condition late in the day favors the use of the producer strategy. Under rich and clumped food, the availability of scrounging can improve the daily survival of any average group member. The model suggests only weak effects of predation hazard on the use of scrounging. Future developments should consider the effects of dominance asymmetries and allowing foragers a choice between foraging alone or in a group harboring an evolutionarily stable frequency of scrounger. PMID- 10753083 TI - The environmental genome project: ethical, legal, and social implications. AB - The National Institute of Environmental Health Sciences is supporting a multiyear research initiative examining genetic influences on environmental response. Proponents of this new initiative, known as the Environmental Genome Project, hope that the information learned will improve our understanding of environmentally associated diseases and allow clinicians and public health officials to target disease-prevention strategies to those who are at increased risk. Despite these potential benefits, the project presents several ethical and social challenges. Of immediate concern is the protection of individual research participants. Other ethical issues relate to the application of research results and how study findings could affect social priorities. Clarifying these emerging areas of concern, many of which have not received adequate attention in the existing bioethics literature, is an important step toward minimizing potential research-related risks and defining research needs. PMID- 10753084 TI - Associations between mortality and air pollution in central Europe. AB - Increased mortality has been observed in association with elevated concentrations of air pollutants in European cities and in the United States. We reassessed the effects of particulate matter in Central Europe. Mortality and air pollution data were obtained for a highly polluted region of the Czech Republic and a rural region in Germany. Poisson regression analyses were conducted considering trend, season, meteorology, and influenza epidemics as confounders in both a parametric and a nonparametric approach. The Czech Republic had a 3.8% increase in mortality [95% confidence interval (CI), 0.8-6.9%] in association with 100 microg/m(3) total suspended particles (TSP) (lagged 2 days) for the time period 1982-1994. During the last 2 years of study, 68% of the TSP consisted of particulate matter [less than/equal to] 10 microm in aerodynamic diameter (PM(10)). An increase of 100 microg/m(3) TSP (lagged 1 day) was associated with a 9.5% increase in mortality (CI, 1.2-18.5%) and 100 microg/m(3) PM(10 )(lagged 1 day) showed a 9.8% increase in mortality (CI, 0.7-19.7%). We found no evidence for an association between mortality and particulate matter in the rural area in Germany at the Czech border. Data from the coal basin in the Czech Republic suggested an increase in mortality associated with the concentration of particulate matter in a highly polluted setting in Central Europe that is consistent with the associations observed in other western European cities and in the United States. PMID- 10753085 TI - Phlebotomy increases cadmium uptake in hemochromatosis. AB - The intestinal absorption of the nephrotoxic environmental pollutant cadmium increases markedly when iron stores are depleted. This may be mediated by an up regulation of the recently identified mucosal transporter DMT1 (Nramp2 or DCT1) for divalent cations. We tested whether the highly increased iron absorption in hereditary hemochromatosis (HH) was accompanied by an enhanced absorption of cadmium and lead. Cadmium and lead in blood and iron status markers were determined in 21 nonsmoking subjects with HH genetically tested for the HFE mutations and in 21 nonsmoking controls matched for age and sex. In subjects with HH on maintenance phlebotomy treatment, blood concentrations of cadmium, but not lead, were significantly higher than in paired controls. There was a strong age independent positive association between blood cadmium and the number of years of phlebotomy treatment. Blood lead showed a similar but less pronounced consequence of treatment. All HH subjects with lower blood cadmium than the corresponding controls had either no mutation in the HFE gene, were not phlebotomized, or were phlebotomized for only a limited time. Our findings indicate that the treatment rather than the disease increased the cadmium uptake in homozygous HH. Further studies are needed to confirm whether the disease decreased cadmium absorption and whether the absorption was dependent on the genotype. PMID- 10753086 TI - Neurologic function among termiticide applicators exposed to chlorpyrifos. AB - Chlorpyrifos is a moderately toxic organophosphate pesticide. Houses and lawns in the United States receive a total of approximately 20 million annual chlorpyrifos treatments, and 82% of U.S. adults have detectable levels of a chlorpyrifos metabolite (3,5, 6-trichloro-2-pyridinol; TCP) in the urine. The U.S. Environmental Protection Agency has estimated that there are 5,000 yearly reported cases of accidental chlorpyrifos poisoning, and approximately one-fourth of these cases exhibit symptoms. Organophosphates affect the nervous system, but there are few epidemiologic data on chlorpyrifos neurotoxicity. We studied neurologic function in 191 current and former termiticide applicators who had an average of 2.4 years applying chlorpyrifos and 2.5 years applying other pesticides, and we compared them to 189 nonexposed controls. The average urinary TCP level for 65 recently exposed applicators was 629.5 microg/L, as compared to 4.5 microg/L for the general U.S. population. The exposed group did not differ significantly from the nonexposed group for any test in the clinical examination. Few significant differences were found in nerve conduction velocity, arm/hand tremor, vibrotactile sensitivity, vision, smell, visual/motor skills, or neurobehavioral skills. The exposed group did not perform as well as the nonexposed group in pegboard turning tests and some postural sway tests. The exposed subjects also reported significantly more symptoms, including memory problems, emotional states, fatigue, and loss of muscle strength; our more quantitative tests may not have been adequate to detect these symptoms. Eight men who reported past chlorpyrifos poisoning had a pattern of low performance on a number of tests, which is consistent with prior reports of chronic effects of organophosphate poisoning. Overall, the lack of exposure effects on the clinical examination was reassuring. The findings for self-reported symptoms raise some concern, as does the finding of low performance for those reporting prior poisoning. Although this was a relatively large study based on a well-defined target population, the workers we studied may not be representative of all exposed workers, and caution should be exercised in generalizing our results. PMID- 10753087 TI - Socioeconomic predictors of high allergen levels in homes in the greater Boston area. AB - In the United States, childhood asthma morbidity and prevalence rates are the highest in less affluent urban minority communities. More than 80% of childhood asthmatics are allergic to one or more inhalant allergens. We evaluated whether socioeconomic status was associated with a differential in the levels and types of indoor home allergens. Dust samples for an ELISA allergen assay were collected from the homes of 499 families as part of a metropolitan Boston, Massachusetts, longitudinal birth cohort study of home allergens and asthma in children with a parental history of asthma or allergy. The proportion of homes with maximum home allergen levels in the highest category was 42% for dust mite allergen (> or = 10 microg/g Der p 1 or Der f 1), 13% for cockroach allergen (> or = 2 U/g Bla g 1 or Bla g 2), 26% for cat allergen (> or = 8 microg/g Fel d 1), and 20% for dog allergen (> or = 10 microg/g Can f 1). Homes in the high-poverty area (> 20% of the population below the poverty level) were more likely to have high cockroach allergen levels than homes in the low-poverty area [51 vs. 3%; OR, 33; 95% confidence interval (CI), 12-90], but less likely to have high levels of dust mite allergen (16 vs. 53%; OR, 0.2; CI, 0.1-0.4). Lower family income, less maternal education, and race/ethnicity (black or Hispanic vs. white) were also associated with a lower risk of high dust mite levels and a greater risk of high cockroach allergen levels. Within a single U.S. metropolitan area we found marked between-community differences in the types of allergens present in the home, but not necessarily in the overall burden of allergen exposure. PMID- 10753088 TI - Lead in calcium supplements. AB - Intercalibrated measurements of lead in calcium supplements indicate the importance of rigorous analytical techniques to accurately quantify contaminant exposures in complex matrices. Without such techniques, measurements of lead concentrations in calcium supplements may be either erroneously low, by as much as 50%, or below the detection limit needed for new public health criteria. In this study, we determined the lead content of 136 brands of supplements that were purchased in 1996. The calcium in the products was derived from natural sources (bonemeal, dolomite, or oyster shell) or was synthesized and/or refined (chelated and nonchelated calcium). The dried products were acid digested and analyzed for lead by high resolution-inductively coupled plasma-mass spectrometry. The method's limit of quantitation averaged 0.06 microg/g, with a coefficient of variation of 1.7% and a 90-100% lead recovery of a bonemeal standard reference material. Two-thirds of those calcium supplements failed to meet the 1999 California criteria for acceptable lead levels (1.5 microg/daily dose of calcium) in consumer products. The nonchelated synthesized and/or refined calcium products, specifically antacids and infant formulas, had the lowest lead concentrations, ranging from nondetectable to 2.9 microg Pb/g calcium, and had the largest proportion of brands meeting the new criteria (85% of the antacids and 100% of the infant formulas). PMID- 10753089 TI - Balancing the risks and benefits of drinking water disinfection: disability adjusted life-years on the scale. AB - To evaluate the applicability of disability adjusted life-years (DALYs) as a measure to compare positive and negative health effects of drinking water disinfection, we conducted a case study involving a hypothetical drinking water supply from surface water. This drinking water supply is typical in The Netherlands. We compared the reduction of the risk of infection with Cryptosporidium parvum by ozonation of water to the concomitant increase in risk of renal cell cancer arising from the production of bromate. We applied clinical, epidemiologic, and toxicologic data on morbidity and mortality to calculate the net health benefit in DALYs. We estimated the median risk of infection with C. parvum as 10(-3)/person-year. Ozonation reduces the median risk in the baseline approximately 7-fold, but bromate is produced in a concentration above current guideline levels. However, the health benefits of preventing gastroenteritis in the general population and premature death in patients with acquired immunodeficiency syndrome outweigh health losses by premature death from renal cell cancer by a factor of > 10. The net benefit is approximately 1 DALY/million person-years. The application of DALYs in principle allows us to more explicitly compare the public health risks and benefits of different management options. In practice, the application of DALYs may be hampered by the substantial degree of uncertainty, as is typical for risk assessment. PMID- 10753090 TI - Levels of methyleugenol in a subset of adults in the general U.S. population as determined by high resolution mass spectrometry. AB - We developed a sensitive and accurate analytical method for quantifying methyleugenol (ME) in human serum. Our method uses a simple solid-phase extraction followed by a highly specific analysis using isotope dilution gas chromatography-high resolution mass spectrometry. Our method is very accurate; its limit of detection is 3.1 pg/g and its average coefficient of variation is 14% over a 200-pg/g range. We applied this method to measure serum ME concentrations in adults in the general U.S. population. ME was detected in 98% of our samples, with a mean ME concentration of 24 pg/g (range < 3.1-390 pg/g). Lipid adjustment of the data did not alter the distribution. Bivariate and multivariate analyses using selected demographic variables showed only marginal relationships between race/ethnicity and sex/fasting status with serum ME concentrations. Although no demographic variable was a good predictor of ME exposure or dose, our data indicate prevalent exposure of U.S. adults to ME. Detailed pharmacokinetic studies are required to determine the relationship between ME intake and human serum ME concentrations. PMID- 10753091 TI - Embryonic treatment with xenobiotics disrupts steroid hormone profiles in hatchling red-eared slider turtles (Trachemys scripta elegans). AB - Many compounds in the environment capable of acting as endocrine disruptors have been assayed for their developmental effects on morphogenesis; however, few studies have addressed how such xenobiotics affect physiology. In the current study we examine the effects of three endocrine-disrupting compounds, chlordane, trans-nonachlor, and the polychlorinated biphenyl (PCB) mixture Aroclor 1242, on the steroid hormone concentrations of red-eared slider turtle (Trachemys scripta elegans) hatchlings treated in ovo. Basal steroid concentrations and steroid concentrations in response to follicle-stimulating hormone were examined in both male and female turtles treated with each of the three compounds. Treated male turtles exposed to Aroclor 1242 or chlordane exhibited significantly lower testosterone concentrations than controls, whereas chlordane-treated females had significantly lower progesterone, testosterone, and 5[alpha]-dihydrotestosterone concentrations relative to controls. The effects of these endocrine disruptors extend beyond embryonic development, altering sex-steroid physiology in exposed animals. PMID- 10753092 TI - The contribution of benzene to smoking-induced leukemia. AB - Cigarette smoking is associated with an increased risk of leukemia; benzene, an established leukemogen, is present in cigarette smoke. By combining epidemiologic data on the health effects of smoking with risk assessment techniques for low dose extrapolation, we assessed the proportion of smoking-induced total leukemia and acute myeloid leukemia (AML) attributable to the benzene in cigarette smoke. We fit both linear and quadratic models to data from two benzene-exposed occupational cohorts to estimate the leukemogenic potency of benzene. Using multiple-decrement life tables, we calculated lifetime risks of total leukemia and AML deaths for never, light, and heavy smokers. We repeated these calculations, removing the effect of benzene in cigarettes based on the estimated potencies. From these life tables we determined smoking-attributable risks and benzene-attributable risks. The ratio of the latter to the former constitutes the proportion of smoking-induced cases attributable to benzene. Based on linear potency models, the benzene in cigarette smoke contributed from 8 to 48% of smoking-induced total leukemia deaths [95% upper confidence limit (UCL), 20-66%], and from 12 to 58% of smoking-induced AML deaths (95% UCL, 19-121%). The inclusion of a quadratic term yielded results that were comparable; however, potency models with only quadratic terms resulted in much lower attributable fractions--all < 1%. Thus, benzene is estimated to be responsible for approximately one-tenth to one-half of smoking-induced total leukemia mortality and up to three-fifths of smoking-related AML mortality. In contrast to theoretical arguments that linear models substantially overestimate low-dose risk, linear extrapolations from empirical data over a dose range of 10- to 100 fold resulted in plausible predictions. PMID- 10753093 TI - Inhaled crocidolite mutagenicity in lung DNA. AB - We used transgenic mice carrying the lacI reporter gene to study the mutagenesis potential of asbestos crocidolite. The animals were exposed by nose-only inhalation to an aerosol containing 5.75 mg/m(3) crocidolite dust for 6 hr/day and 5 consecutive days. After 1, 4, and 12 weeks, we examined four end points: the cytology of bronchoalveolar lavage, the lung load of crocidolite, the hydrophobic DNA adducts, and the mutations in the lacI reporter gene. Twelve weeks after exposure, nearly 10% of the inhaled fibers remained in the lung (227 +/- 103 ng/mg lung). There was evidence of a typical inflammatory response consisting of multinucleate macrophages at weeks 4 and 12, whereas immediately after the exposure, we observed numerous polymorphonuclear neutrophils. The mutant frequency significatively increased during the fourth week after the exposure: 13.5 [time] 10(-5) in the exposed group versus 6. 9 10(-5) in the control group. The induction factor, defined by the ratio of checked mutants of exposed mice to checked mutants of control mice, was 1.96. The mutation spectrum of control lung DNA and exposed lung DNA was similar, suggesting the possible involvement of a DNA repair decrease in crocidolite-treated animals. We used the (32)P-postlabeling method and did not detect any increase of either 5 mC or bulky adduct in treated mice. This is the first study that demonstrates asbestos mutagenicity in vivo after a nose-only inhalation. PMID- 10753094 TI - Associations between air pollution and mortality in Phoenix, 1995-1997. AB - We evaluated the association between mortality outcomes in elderly individuals and particulate matter (PM) of varying aerodynamic diameters (in micrometers) [PM(10), PM(2.5), and PM(CF )(PM(10) minus PM(2.5))], and selected particulate and gaseous phase pollutants in Phoenix, Arizona, using 3 years of daily data (1995-1997). Although source apportionment and epidemiologic methods have been previously combined to investigate the effects of air pollution on mortality, this is the first study to use detailed PM composition data in a time-series analysis of mortality. Phoenix is in the arid Southwest and has approximately 1 million residents (9. 7% of the residents are > 65 years of age). PM data were obtained from the U.S. Environmental Protection Agency (EPA) National Exposure Research Laboratory Platform in central Phoenix. We obtained gaseous pollutant data, specifically carbon monoxide, nitrogen dioxide, ozone, and sulfur dioxide data, from the EPA Aerometric Information Retrieval System Database. We used Poisson regression analysis to evaluate the associations between air pollution and nonaccidental mortality and cardiovascular mortality. Total mortality was significantly associated with CO and NO(2) (p < 0.05) and weakly associated with SO(2), PM(10), and PM(CF) (p < 0. 10). Cardiovascular mortality was significantly associated with CO, NO(2), SO(2), PM(2.5), PM(10), PM(CF) (p < 0.05), and elemental carbon. Factor analysis revealed that both combustion-related pollutants and secondary aerosols (sulfates) were associated with cardiovascular mortality. PMID- 10753095 TI - Adolescent health and the environment. AB - The effects of toxicants depend on the dose and the time in the life span when exposure occurs. The biology of adolescence is distinctive and provides opportunities for unique actions of toxicants both in terms of disruption of function and disruption of maturation. Maturation of a number of organ systems occurs during this period, including not only the reproductive system but also the respiratory, skeletal, immune, and central nervous systems. Adolescence is a time of increased risk for infectious disease and accidental injury, making the effects of toxicants on the immune and central nervous systems particularly harmful. Differences in blood volume, respiratory parameters, metabolic needs, and capacity all contribute to altered pharmacokinetics. Exposures can also change. Increased food intake associated with rapid adolescent growth alters exposure to food contaminants. Voluntary drug consumption increases, including drinking; smoking; substance abuse; and the use of over-the-counter, prescription, and performance-enhancing drugs. At the same time, adolescents are introduced to toxicants in the workplace. Basic research in the toxicology of adolescence needs to take into account the appropriateness of animal models for this distinctive human developmental stage; risk assessment must take into account pharmacokinetic and lifestyle factors. Screening methodologies that would identify toxic effects unique to adolescence would also be valuable. PMID- 10753096 TI - Recurrent acute respiratory tract infections in areas with high nitrate concentrations in drinking water. AB - A review of the literature indicated an association among high nitrate ingestion, methemoglobinemia, and pathologic changes in bronchi and lung parenchyma. The present study examined a possible correlation among drinking water nitrate concentration, methemoglobin levels, cytochrome b(5) reductase activity, and acute respiratory tract infection with a history of recurrence (RRTI). Our study was conducted in five village units in the state of Rajasthan, India, with nitrate concentrations of 26, 45, 95, 222, and 459 mg NO(3) ion/L. We randomly selected 88 children. The children were up to 8 years of age, age matched, and represented 10% of the total population of these areas. We obtained detailed RRTI histories and conducted medical examinations. Methemoglobin levels and cytochrome b(5) reductase activity were estimated biochemically. The data collected were statistically analyzed using spreadsheet software on a personal computer. We observed strong interdependence between methemoglobin levels and RRTI in children up to 8 years of age. Methemoglobin levels alone explained 80% of the variation in the RRTI cases. This study indicates that methemoglobinemia, secondary to high nitrate ingestion in drinking water, causes RRTI. Increased production of methemoglobin and free radicals of nitric oxide and oxygen due to nitrate metabolism in the body lead to alveolar damage and mismatching of ventilation and perfusion, which may be the reason for high mortality in children due to RRTI. PMID- 10753097 TI - The potential health impacts of climate variability and change for the United States: executive summary of the report of the health sector of the U.S. National Assessment. AB - We examined the potential impacts of climate variability and change on human health as part of a congressionally mandated study of climate change in the United States. Our author team, comprising experts from academia, government, and the private sector, was selected by the federal interagency U.S. Global Change Research Program, and this report stems from our first 18 months of work. For this assessment we used a set of assumptions and/or projections of future climates developed for all participants in the National Assessment of the Potential Consequences of Climate Variability and Change. We identified five categories of health outcomes that are most likely to be affected by climate change because they are associated with weather and/or climate variables: temperature-related morbidity and mortality; health effects of extreme weather events (storms, tornadoes, hurricanes, and precipitation extremes); air-pollution related health effects; water- and foodborne diseases; and vector- and rodent borne diseases. We concluded that the levels of uncertainty preclude any definitive statement on the direction of potential future change for each of these health outcomes, although we developed some hypotheses. Although we mainly addressed adverse health outcomes, we identified some positive health outcomes, notably reduced cold-weather mortality, which has not been extensively examined. We found that at present most of the U.S. population is protected against adverse health outcomes associated with weather and/or climate, although certain demographic and geographic populations are at increased risk. We concluded that vigilance in the maintenance and improvement of public health systems and their responsiveness to changing climate conditions and to identified vulnerable subpopulations should help to protect the U.S. population from any adverse health outcomes of projected climate change. PMID- 10753098 TI - A 37-year-old mechanic with multiple chemical sensitivities. AB - A 37-year-old heating, ventilation, and air-conditioning mechanic developed respiratory, musculoskeletal, and central nervous system symptoms associated with a variety of odorous environmental chemicals. Organic disease was not evident, but the patient was distressed by these symptoms and was at risk for becoming disabled by them. His symptoms fit broadly into the condition recognized as multiple chemical sensitivity. Multiple chemical sensitivity is a diagnostic term for a group of symptoms without demonstrated organic basis. The symptoms are characteristic of dysfunction in multiple organ systems, they increase and decrease according to exposure to low levels of chemical agents in the patient's environment, and they sometimes occur after a distinct environmental change or insult such as an industrial accident or remodeling. Although traditional medical organizations have not agreed on a definition for this syndrome, it is being increasingly recognized and makes up an increasing percentage of the caseload at occupational and environmental medicine clinics. Although there is often dispute about whether the symptoms have a functional or organic basis, an informed approach to evaluation, diagnosis, and management and a careful assessment of impairment, disability, and work relatedness are necessary. Careful exclusion of organic causes is critical, and this should be followed by a judicious approach to coping with symptoms. PMID- 10753099 TI - Cumulative impact. PMID- 10753100 TI - Chinese fridges keep food and the planet cool. PMID- 10753101 TI - The core of the candle problem. PMID- 10753102 TI - FAA turns down the volume. PMID- 10753103 TI - Reducing radon state by state. PMID- 10753104 TI - Breathtaking research. AB - Although the air we breathe is necessary for life, certain factors may make this same air detrimental to our health. For instance, a seemingly endless list of compounds can be toxic at certain concentrations, and inhaling such compounds may lead to damage in different parts of the body. To investigate the particular effects of inhaled toxicants, the NIEHS developed the Respiratory Toxicology Group. The group, which consists of toxicologist Dan Morgan, biologist Cassandra Shines, and engineer Michael Moorman, has been conducting experiments for about a decade at the NIEHS inhalation facility. Morgan says the group typically conducts studies of chemicals that have been nominated to the National Toxicology Program for investigation into their carcinogenic potential and other end points. "We provide research support and conduct special studies on these chemicals," he says. "We also do collaborative studies with other NIEHS investigators, other [federal] agencies such as the Environmental Protection Agency and the Department of Energy, universities, and industry." PMID- 10753105 TI - Winds of change: reducing transboundary air pollutants. AB - Sulfur dioxide, nitrogen oxides, volatile organic compounds, persistent organic pollutants, particulate matter, and heavy metals---air pollutants once thought to be problems that could be solved locally, where the effects occur---are all currently being discussed in international forums. A spate of meetings and agreements in recent months has shown many international governments to be more willing than ever to try to limit the amount of their air pollution that drifts into other countries. Prompting this policy shift are increasing emissions in some parts of the world, better monitoring, and an improved understanding of air pollution transport and the effects of air pollution. In most regions of the world, however, no international agreements on air pollution exist at all, while in others, many overlapping local, multilateral, and global agreements address the problem simultaneously. According to the World Health Organization, air pollution causes nearly 3 million deaths per year, and the U.S. Environmental Protection Agency estimates that ground-level ozone causes damage to U.S. crops totaling $1-2 billion each year. PMID- 10753106 TI - EPA: airing on the side of caution or pulling standards out of thin Air? AB - In May 1999, a federal appeals court ruled that the U.S. Environmental Protection Agency (EPA) had violated the Constitution when it strengthened regulations for ground-level ozone and particulate matter (PM). Although the court did not question the validity of the EPA's scientific basis for establishing the PM and ozone standards, it did challenge how the EPA selected the particular levels since the scientific record in both rules did not indicate unequivocally where the standards should be set. The agency failed to identify an "intelligible principle" that would guide such choices, the court said, and thereby exceeded the power it was granted by Congress. Because scientific uncertainty attends so much rule making, the ruling leaves open the question of when the EPA may make what is essentially a policy determination versus when those determinations must be made by Congress. For this reason, observers consider the ruling to have potentially significant implications beyond just the ozone and PM standards that may affect other EPA regulations and regulations by other agencies. PMID- 10753107 TI - The trickle-down theory of cleaner air. AB - The 1990 Clean Air Act Amendments prompted an increased urgency to find new ways to treat airstreams containing volatile organic compounds, which affect the nitrogen photolytic cycle and help produce ground-level ozone, hazardous air pollutants, and odorous air emissions such as hydrogen sulfide. Scientists at the New Jersey company Envirogen have adapted traditional biofiltration technology to perform airborne waste stream cleanup. Preliminary research on pollutants such as phenol, methylene chloride, benzene, and toluene indicates that Envirogen's biotrickling filter may remove an average of about 94% of total hazardous air pollutants. Scientists are working to identify microbes that will clean up more stubborn pollutants. PMID- 10753108 TI - How animals move: an integrative view. AB - Recent advances in integrative studies of locomotion have revealed several general principles. Energy storage and exchange mechanisms discovered in walking and running bipeds apply to multilegged locomotion and even to flying and swimming. Nonpropulsive lateral forces can be sizable, but they may benefit stability, maneuverability, or other criteria that become apparent in natural environments. Locomotor control systems combine rapid mechanical preflexes with multimodal sensory feedback and feedforward commands. Muscles have a surprising variety of functions in locomotion, serving as motors, brakes, springs, and struts. Integrative approaches reveal not only how each component within a locomotor system operates but how they function as a collective whole. PMID- 10753109 TI - Structure of the S15,S6,S18-rRNA complex: assembly of the 30S ribosome central domain. AB - The crystal structure of a 70-kilodalton ribonucleoprotein complex from the central domain of the Thermus thermophilus 30S ribosomal subunit was solved at 2.6 angstrom resolution. The complex consists of a 104-nucleotide RNA fragment composed of two three-helix junctions that lie at the end of a central helix, and the ribosomal proteins S15, S6, and S18. S15 binds the ribosomal RNA early in the assembly of the 30S ribosomal subunit, stabilizing a conformational reorganization of the two three-helix junctions that creates the RNA fold necessary for subsequent binding of S6 and S18. The structure of the complex demonstrates the central role of S15-induced reorganization of central domain RNA for the subsequent steps of ribosome assembly. PMID- 10753110 TI - Monolithic microfabricated valves and pumps by multilayer soft lithography. AB - Soft lithography is an alternative to silicon-based micromachining that uses replica molding of nontraditional elastomeric materials to fabricate stamps and microfluidic channels. We describe here an extension to the soft lithography paradigm, multilayer soft lithography, with which devices consisting of multiple layers may be fabricated from soft materials. We used this technique to build active microfluidic systems containing on-off valves, switching valves, and pumps entirely out of elastomer. The softness of these materials allows the device areas to be reduced by more than two orders of magnitude compared with silicon based devices. The other advantages of soft lithography, such as rapid prototyping, ease of fabrication, and biocompatibility, are retained. PMID- 10753111 TI - Chain mobility in the amorphous region of nylon 6 observed under active uniaxial deformation AB - A specially designed solid-state deuterium nuclear magnetic resonance probe was used to examine the effect of uniaxial elongation on the chain mobility in the amorphous region of semicrystalline nylon 6. In measurements conducted near the glass transition temperature, there was measurable deformation-induced enhancement of the mobility of the amorphous chains up to the yield point. This enhanced mobility decayed once deformation was stopped. Enhanced mobility was not observed in deformation at room temperature. The mechanics of deformation can be explained by the Robertson model for glassy polymers near the glass transition temperature, which states that applied stress induces liquid-like behavior in the polymer chains. PMID- 10753112 TI - Low (Sub-1-volt) halfwave voltage polymeric electro-optic modulators achieved by controlling chromophore shape AB - Electro-optic (EO) modulators encode electrical signals onto fiber optic transmissions. High drive voltages limit gain and noise levels. Typical polymeric and lithium niobate modulators operate with halfwave voltages of 5 volts. Sterically modified organic chromophores have been used to reduce the attenuation of electric field poling-induced electro-optic activity caused by strong intermolecular electrostatic interactions. Such modified chromophores, incorporated into polymer hosts, were used to fabricate EO modulators with halfwave voltages of 0.8 volts (at a telecommunications wavelength of 1318 nanometers) and to achieve a halfwave voltage-interaction length product of 2.2 volt-centimeters. Optical push-pull poling and driving were also used to reduce halfwave voltage. This study, together with recent demonstrations of exceptional bandwidths (more than 110 gigahertz) and ease of integration (with very large scale integration semiconductor circuitry and ultra-low-loss passive optical circuitry) demonstrates the potential of polymeric materials for next generation telecommunications, information processing, and radio frequency distribution. PMID- 10753113 TI - Dilational processes accompanying earthquakes in the long valley caldera AB - Regional distance seismic moment tensor determinations and broadband waveforms of moment magnitude 4.6 to 4.9 earthquakes from a November 1997 Long Valley Caldera swarm, during an inflation episode, display evidence of anomalous seismic radiation characterized by non-double couple (NDC) moment tensors with significant volumetric components. Observed coseismic dilation suggests that hydrothermal or magmatic processes are directly triggering some of the seismicity in the region. Similarity in the NDC solutions implies a common source process, and the anomalous events may have been triggered by net fault-normal stress reduction due to high-pressure fluid injection or pressurization of fluid saturated faults due to magmatic heating. PMID- 10753114 TI - Nonbiological fractionation of iron isotopes. AB - Laboratory experiments demonstrate that iron isotopes can be chemically fractionated in the absence of biology. Isotopic variations comparable to those seen during microbially mediated reduction of ferrihydrite are observed. Fractionation may occur in aqueous solution during equilibration between inorganic iron complexes. These findings provide insight into the mechanisms of iron isotope fractionation and suggest that nonbiological processes may contribute to iron isotope variations observed in sediments. PMID- 10753115 TI - Carbon isotopic evidence for methane hydrate instability during quaternary interstadials AB - Large (about 5 per mil) millennial-scale benthic foraminiferal carbon isotopic oscillations in the Santa Barbara Basin during the last 60,000 years reflect widespread shoaling of sedimentary methane gradients and increased outgassing from gas hydrate dissociation during interstadials. Furthermore, several large, brief, negative excursions (up to -6 per mil) coinciding with smaller shifts (up to -3 per mil) in depth-stratified planktonic foraminiferal species indicate massive releases of methane from basin sediments. Gas hydrate stability was modulated by intermediate-water temperature changes induced by switches in thermohaline circulation. These oscillations were likely widespread along the California margin and elsewhere, affecting gas hydrate instability and contributing to millennial-scale atmospheric methane oscillations. PMID- 10753116 TI - Sink or swim: strategies for cost-efficient diving by marine mammals. AB - Locomotor activity by diving marine mammals is accomplished while breath-holding and often exceeds predicted aerobic capacities. Video sequences of freely diving seals and whales wearing submersible cameras reveal a behavioral strategy that improves energetic efficiency in these animals. Prolonged gliding (greater than 78% descent duration) occurred during dives exceeding 80 meters in depth. Gliding was attributed to buoyancy changes with lung compression at depth. By modifying locomotor patterns to take advantage of these physical changes, Weddell seals realized a 9.2 to 59.6% reduction in diving energetic costs. This energy conserving strategy allows marine mammals to increase aerobic dive duration and achieve remarkable depths despite limited oxygen availability when submerged. PMID- 10753117 TI - Identification of a coordinate regulator of interleukins 4, 13, and 5 by cross species sequence comparisons. AB - Long-range regulatory elements are difficult to discover experimentally; however, they tend to be conserved among mammals, suggesting that cross-species sequence comparisons should identify them. To search for regulatory sequences, we examined about 1 megabase of orthologous human and mouse sequences for conserved noncoding elements with greater than or equal to 70% identity over at least 100 base pairs. Ninety noncoding sequences meeting these criteria were discovered, and the analysis of 15 of these elements found that about 70% were conserved across mammals. Characterization of the largest element in yeast artificial chromosome transgenic mice revealed it to be a coordinate regulator of three genes, interleukin-4, interleukin-13, and interleukin-5, spread over 120 kilobases. PMID- 10753118 TI - Specialized fatty acid synthesis in African trypanosomes: myristate for GPI anchors. AB - African trypanosomes, the cause of sleeping sickness, need massive amounts of myristate to remodel glycosyl phosphatidylinositol (GPI) anchors on their surface glycoproteins. However, it has been believed that the parasite is unable to synthesize any fatty acids, and myristate is not abundant in the hosts' bloodstreams. Thus, it has been unclear how trypanosomes meet their myristate requirement. Here we found that they could indeed synthesize fatty acids. The synthetic pathway was unique in that the major product, myristate, was preferentially incorporated into GPIs and not into other lipids. The antibiotic thiolactomycin inhibited myristate synthesis and killed the parasite, making this pathway a potential chemotherapeutic target. PMID- 10753119 TI - Unfolding pathways of individual bacteriorhodopsins. AB - Atomic force microscopy and single-molecule force spectroscopy were combined to image and manipulate purple membrane patches from Halobacterium salinarum. Individual bacteriorhodopsin molecules were first localized and then extracted from the membrane; the remaining vacancies were imaged again. Anchoring forces between 100 and 200 piconewtons for the different helices were found. Upon extraction, the helices were found to unfold. The force spectra revealed the individuality of the unfolding pathways. Helices G and F as well as helices E and D always unfolded pairwise, whereas helices B and C occasionally unfolded one after the other. Experiments with cleaved loops revealed the origin of the individuality: stabilization of helix B by neighboring helices. PMID- 10753120 TI - Specification of Drosophila hematopoietic lineage by conserved transcription factors. AB - Two major classes of cells observed within the Drosophila hematopoietic repertoire are plasmatocytes/macrophages and crystal cells. The transcription factor Lz (Lozenge), which resembles human AML1 (acute myeloid leukemia- 1) protein, is necessary for the development of crystal cells during embryonic and larval hematopoiesis. Another transcription factor, Gcm (glial cells missing), has previously been shown to be required for plasmatocyte development. Misexpression of Gcm causes crystal cells to be transformed into plasmatocytes. The Drosophila GATA protein Srp (Serpent) is required for both Lz and Gcm expression and is necessary for the development of both classes of hemocytes, whereas Lz and Gcm are required in a lineage-specific manner. Given the similarities of Srp and Lz to mammalian GATA and AML1 proteins, observations in Drosophila are likely to have broad implications for understanding mammalian hematopoiesis and leukemias. PMID- 10753121 TI - CD1 expression and the nature of CD1-expressing cells in human atherosclerotic plaques. PMID- 10753122 TI - Literature on BRCA1 mutations not cited. PMID- 10753123 TI - Positioning of longitudinal nerves in C. elegans by nidogen. AB - Basement membranes can help determine pathways of migrating axons. Although members of the nidogen (entactin) protein family are structural components of basement membranes, we find that nidogen is not required for basement membrane assembly in the nematode Caenorhabditis elegans. Nidogen is localized to body wall basement membranes and is required to direct longitudinal nerves dorsoventrally and to direct axons at the midlines. By examining migration of a single axon in vivo, we show that nidogen is required for the axon to switch from circumferential to longitudinal migration. Specialized basement membranes may thus regulate nerve position. PMID- 10753124 TI - Receptors for dopamine and somatostatin: formation of hetero-oligomers with enhanced functional activity. AB - Somatostatin and dopamine are two major neurotransmitter systems that share a number of structural and functional characteristics. Somatostatin receptors and dopamine receptors are colocalized in neuronal subgroups, and somatostatin is involved in modulating dopamine-mediated control of motor activity. However, the molecular basis for such interaction between the two systems is unclear. Here, we show that dopamine receptor D2R and somatostatin receptor SSTR5 interact physically through hetero-oligomerization to create a novel receptor with enhanced functional activity. Our results provide evidence that receptors from different G protein (heterotrimeric guanine nucleotide binding protein)-coupled receptor families interact through oligomerization. Such direct intramembrane association defines a new level of molecular crosstalk between related G protein coupled receptor subfamilies. PMID- 10753125 TI - The way things move: looking under the hood of molecular motor proteins. AB - The microtubule-based kinesin motors and actin-based myosin motors generate motions associated with intracellular trafficking, cell division, and muscle contraction. Early studies suggested that these molecular motors work by very different mechanisms. Recently, however, it has become clear that kinesin and myosin share a common core structure and convert energy from adenosine triphosphate into protein motion using a similar conformational change strategy. Many different types of mechanical amplifiers have evolved that operate in conjunction with the conserved core. This modular design has given rise to a remarkable diversity of kinesin and myosin motors whose motile properties are optimized for performing distinct biological functions. PMID- 10753126 TI - Motility powered by supramolecular springs and ratchets. AB - Not all biological movements are caused by molecular motors sliding along filaments or tubules. Just as springs and ratchets can store or release energy and rectify motion in physical systems, their analogs can perform similar functions in biological systems. The energy of biological springs is derived from hydrolysis of a nucleotide or the binding of a ligand, whereas biological ratchets are powered by Brownian movements of polymerizing filaments. However, the viscous and fluctuating cellular environment and the mechanochemistry of soft biological systems constrain the modes of motion generated and the mechanisms for energy storage, control, and release. PMID- 10753127 TI - "Chest pain-please admit": is there an alternative?. A rapid cardiological assessment service may prevent unnecessary admissions. PMID- 10753128 TI - Vaccines and medicines for the world's poorest. Public-private partnerships seem to be essential. PMID- 10753129 TI - Managing status epilepticus. New drug offers real advantages. PMID- 10753130 TI - Practitioners of evidence based care. Not all clinicians need to appraise evidence from scratch but all need some skills. PMID- 10753131 TI - Systems for emergency care. Integrating the components is the challenge. PMID- 10753132 TI - Tobacco companies in US to pay smoker $22m (pound13.8m) PMID- 10753133 TI - GMC ruling upheld despite records obtained "without consent". PMID- 10753134 TI - Scientists build a peptide to stop toxic shock syndrome. PMID- 10753135 TI - Wales launches health promotion strategy. PMID- 10753136 TI - In brief PMID- 10753137 TI - Unsupervised nurses may soon give anaesthetics in United States. PMID- 10753138 TI - Tuberculosis is spreading in central and eastern Europe. PMID- 10753139 TI - Managers accuse ministers of spin over money for NHS. PMID- 10753140 TI - Research needed into effects of IVF. PMID- 10753141 TI - Blair must raise taxes to eradicate child poverty, academics say. PMID- 10753142 TI - Health minister announces initiatives on men's health. PMID- 10753143 TI - Ireland faces shortage of doctors. PMID- 10753144 TI - Services for disabled people are "unacceptably poor". PMID- 10753145 TI - Altering a gene in mice prevents obesity. PMID- 10753146 TI - Systematic review of comparative efficacy and tolerability of calcipotriol in treating chronic plaque psoriasis. AB - OBJECTIVES: To evaluate the comparative efficacy and tolerability of topical calcipotriol in the treatment of mild to moderate chronic plaque psoriasis. DESIGN: Quantitative systematic review of randomised controlled trials. SUBJECTS: 6038 patients with plaque psoriasis reported in 37 trials. MAIN OUTCOME MEASURES: Mean difference in percentage change in scores on psoriasis area and severity index, and response rate ratios for both patients' and investigators' overall assessments of marked improvement or better. Adverse effects were estimated with the rate ratio, rate difference, and number needed to treat. RESULTS: Calcipotriol was at least as effective as potent topical corticosteroids, calcitriol, short contact dithranol, tacalcitol, coal tar, and combined coal tar 5%, allantoin 2%, and hydrocortisone 0.5%. Calcipotriol caused significantly more skin irritation than potent topical corticosteroids (number needed to treat to harm for irritation 10, 95% confidence interval 6 to 34). Calcipotriol monotherapy also caused more irritation than calcipotriol combined with a potent topical corticosteroid (6, 4 to 8). However, the number needed to treat for dithranol to produce lesional or perilesional irritation was 4 (3 to 5). On average, treating 23 patients with short contact dithranol led to one more patient dropping out of treatment owing to adverse effects than if they were treated with calcipotriol. CONCLUSIONS: Calcipotriol is an effective treatment for mild to moderate chronic plaque psoriasis, more so than calcitriol, tacalcitol, coal tar, and short contact dithranol. Only potent topical corticosteroids seem to have comparable efficacy at eight weeks. Although calcipotriol caused more skin irritation than topical corticosteroids this has to be balanced against the potential long term effects of corticosteroids. Skin irritation rarely led to withdrawal of calcipotriol treatment. Longer term comparative trials of calcipotriol versus dithranol and topical corticosteroids are needed to see whether these short term benefits are mirrored by long term outcomes such as duration of remission and improvement in quality of life. PMID- 10753147 TI - Association between postnatal catch-up growth and obesity in childhood: prospective cohort study. AB - OBJECTIVE: To identify predictors of postnatal catch-up growth from birth to two years and its relation to size and obesity at five years. DESIGN: Regional prospective cohort study. SETTING: Avon longitudinal study of pregnancy and childhood, United Kingdom. SUBJECTS: 848 full term singletons from a 10% random sample of the Avon longitudinal study of pregnancy and childhood. MAIN OUTCOME MEASURES: Maternal birth weight, prepregnancy weight, pregnancy weight gain, height, smoking, and parity, and paternal height. Weight and length of infants at birth, two years, and five years expressed as standard deviation (SD) scores from the UK reference scores for 1990. Percentage fat mass and total fat mass (estimated from skinfolds) and waist circumference at five years. RESULTS: Size at birth was representative of the national reference. Overall, 30. 7% (260 of 848) of infants showed a gain in SD score for weight greater than 0.67 SD scores between zero and two years, indicating clinically significant catch-up growth. These children had lower weight, length, and ponderal index at birth than other children, and were more often from primiparous pregnancies. They also had taller fathers than other children, and their mothers had lower birth weights and were more likely to smoke during pregnancy. Children who showed catch-up growth between zero and two years were heavier, taller, and fatter (body mass index, percentage body fat, and waist circumference) at five years than other children. CONCLUSIONS: In this contemporary well nourished cohort, catch-up growth was predicted by factors relating to intrauterine restraint of fetal growth. Children who showed catch-up growth between zero and two years were fatter and had more central fat distribution at five years than other children. Mechanisms that signal and regulate early catch-up growth in the postnatal period may influence associations between small size at birth and risks for disease in adulthood. PMID- 10753148 TI - Factors underlying the effect of organisational downsizing on health of employees: longitudinal cohort study. AB - OBJECTIVE: To explore the underlying mechanisms between organisational downsizing and deterioration of health of employees. DESIGN: Longitudinal cohort study. Data were assembled from before downsizing (time 1); during major downsizing affecting some job categories (time 2); and after downsizing (time 3). Contributions of changes in work, support, and health related behaviours between time 1 and time 2 to the relation between downsizing and sickness absence at time 3 were assessed by multilevel modelling. Mean length of follow up was 4.9 years. SETTING: Raisio, a town in Finland. SUBJECTS: 764 municipal employees who remained in employment after downsizing. MAIN OUTCOME MEASURES: Records of absences from work from all causes with medical certificate. RESULTS: Downsizing was associated with negative changes in work, impaired support from spouse, and increased prevalence of smoking. Sickness absence rate from all causes was 2.17 (95% confidence interval 1.54 to 3.07) times higher after major downsizing than after minor downsizing. Adjustment for changes in work (for instance, physical demands, job control, and job insecurity) diminished the relation between downsizing and sickness absence by 49%. Adjustments for impaired social support or increased smoking did not alter the relation between downsizing and sickness absence. The findings were unaffected by sex and income. CONCLUSIONS: The exploration of potential mediating factors provides new information about the possible causal pathways linking organisational downsizing and health. Downsizing results in changes in work, social relationships, and health related behaviours. The observed increase in certificated sickness absence was partially explained by concomitant increases in physical demands and job insecurity and a reduction in job control. A considerable proportion of the increase, however, remained unexplained by the factors measured. PMID- 10753149 TI - Use of consensus development to establish national research priorities in critical care. AB - OBJECTIVES: To test the feasibility of using a nominal group technique to establish clinical and health services research priorities in critical care and to test the representativeness of the group's views. DESIGN: Generation of topics by means of a national survey; a nominal group technique to establish the level of consensus; a survey to test the representativeness of the results. SETTING: United Kingdom and Republic of Ireland. SUBJECTS: Nominal group composed of 10 doctors (8 consultants, 2 trainees) and 2 nurses. MAIN OUTCOME MEASURE: Level of support (median) and level of agreement (mean absolute deviation from the median) derived from a 9 point Likert scale. RESULTS: Of the 325 intensive care units approached, 187 (58%) responded, providing about 1000 suggestions for research. Of the 106 most frequently suggested topics considered by the nominal group, 37 attracted strong support, 48 moderate support and 21 weak support. There was more agreement after the group had met-overall mean of the mean absolute deviations from the median fell from 1.41 to 1.26. The group's views represented the views of the wider community of critical care staff (r=0.73, P<0.01). There was no significant difference in the views of staff from teaching or from non-teaching hospitals. Of the 37 topics that attracted the strongest support, 24 were concerned with organisational aspects of critical care and only 13 with technology assessment or clinical research. CONCLUSIONS: A nominal group technique is feasible and reliable for determining research priorities among clinicians. This approach is more democratic and transparent than the traditional methods used by research funding bodies. The results suggest that clinicians perceive research into the best ways of delivering and organising services as a high priority. PMID- 10753151 TI - An unexpected present PMID- 10753150 TI - Ethnicity and prescription of analgesia in an accident and emergency department: cross sectional study. PMID- 10753152 TI - Association between illegal drugs and weapon carrying in young people in Scotland: schools' survey. AB - OBJECTIVES: To identify the type and extent of weapons being carried among young people in Scotland, and to determine the relation between use of illegal drugs and weapon carrying. DESIGN: Questionnaire school survey. SETTING: Independent schools in central Scotland and schools in Lanarkshire and Perth and Kinross. PARTICIPANTS: 3121 students aged 11 to 16 in 20 schools. MAIN OUTCOME MEASURES: Self completion questionnaire reporting history of drug use and weapon carrying. RESULTS: Overall, 34.1% of males and 8. 6% of females reported having carried a weapon (P<0.0001), ranging from 29.2% of boys aged 11-13 (classes S1 to S2) to 39.3% of boys aged 13-15 (S3 to S4). These values are higher than those in a recent survey of young people in England. Weapon carrying in Lanarkshire was 70% higher for males than in the rural area of Perth and Kinross. Both males and females who had taken drugs were more likely to carry weapons (63.5% of male drug users versus 20.5% of non-users and 22.8% of female drug users versus 3.7% of non users; both P<0.0001). The proportions of males carrying weapons who used none, one, two, three or four, or five or more illegal drugs were 21%, 52%, 68%, 74%, and 92% respectively. A similar trend was found among females. CONCLUSIONS: Better information is needed on the nature and extent of weapon carrying by young people in the United Kingdom, and better educational campaigns are needed warning of the dangers of carrying weapons. PMID- 10753153 TI - Measuring brain natriuretic peptide in suspected left ventricular systolic dysfunction in general practice: cross-sectional study. PMID- 10753154 TI - Fortuitous-ambiguously inappropriate to describe maternal death? PMID- 10753156 TI - Cheque your spelling PMID- 10753155 TI - Science, medicine, and the future: Pharmacogenetics. PMID- 10753157 TI - ABC of arterial and venous disease. Secondary prevention of transient ischaemic attack and stroke. PMID- 10753158 TI - Role of external evidence in monitoring clinical trials: experience from a perinatal trial. PMID- 10753159 TI - Developing learning organisations in the new NHS. PMID- 10753160 TI - Analgesic effects of sweet solutions and pacifiers in term neonates. Suckling at the breast is better than sweet solutions and pacifiers. PMID- 10753161 TI - Diagnose and be damned. Corroboration is important when children's illnesses are diagnosed. PMID- 10753162 TI - "Correcting" bmj.com. What happened to the false allegation in bmj. com? PMID- 10753163 TI - How much to do at the accident scene? Paramedic agrees with most of comments about prehospital care. PMID- 10753164 TI - Predicted impact of intravenous thrombolysis. Patients who died or recovered fully should have been included in analysis. PMID- 10753165 TI - Intention to treat analysis is related to methodological quality. PMID- 10753166 TI - Using anticoagulation or aspirin to prevent stroke. Research was methodologically flawed. PMID- 10753167 TI - Evidence of a CJD epidemic may still be missed. PMID- 10753169 TI - Talks continue on intensity payments for consultants PMID- 10753168 TI - Obituaries PMID- 10753170 TI - The evolution of hypnotism PMID- 10753172 TI - Clouding the AIDS issue PMID- 10753171 TI - Video casebook: medicine-The toy boy and the burgundy Car PMID- 10753173 TI - Men's health PMID- 10753174 TI - Medical tourism can do harm. PMID- 10753176 TI - On the house PMID- 10753177 TI - Calcipotriol is a useful drug for treating mild to moderate psoriasis PMID- 10753175 TI - I saw satan fall like lightning PMID- 10753178 TI - Early catch-up growth is a risk factor for childhood obesity PMID- 10753179 TI - What makes organisational downsizing a risk to the health of employees? PMID- 10753180 TI - Nominal group technique is reliable for deciding research priorities PMID- 10753181 TI - Weapon carrying by young people is linked to illegal drug use PMID- 10753182 TI - Threshold mechanisms and site specificity in chromium(VI) carcinogenesis. AB - Ten years have elapsed since the International Agency for Research on Cancer (IARC) evaluated the carcinogenicity of chromium and chromium compounds. Further studies performed during the last decade have provided further epidemiological, experimental and mechanistic data which support the IARC conclusions. A wealth of results indicate that, at variance with chromium(0) and chromium(III), chromium(VI) can induce a variety of genetic and related effects in vitro. The lack of carcinogenicity of chromium(0) and chromium(III) compounds in experimental animals is well established, and only a minority of animal carcinogenicity data with chromium(VI) compounds were positive (30 out of 70, i.e. 42.9%). Moreover, most positive studies used administration routes which do not mimic any human exposure and by-pass physiological defense mechanisms. Typically, positive results were only obtained at implantation sites and at the highest dose tested. Exposure to chromium(VI) has been known for more than a century to be associated with induction of cancer in humans. Carcinogenicity requires massive exposures, as is only encountered in well defined occupational settings, and is site specific, being specifically targeted to the lung and, in some cases, to the sinonasal cavity. Increased death rates for cancers at other sites, which were occasionally reported in some epidemiological studies, were almost invariably not statistically significant, and inconsistent (being counterbalanced by other studies which apparently showed decreased rates for the same cancers). As we recently quantified in human body compartments, chromium(VI) can be reduced in body fluids and non-target cells, which results in its detoxification, due to the poor ability of chromium(III) to cross cell membranes. In target cells, chromium(VI) tends to be metabolized by a network of mechanisms leading to generation of reduced chromium species and reactive oxygen species, which will result either in activation or in detoxification depending on the site of the intracellular reduction and its proximity to DNA. When introduced by the oral route, chromium(VI) is efficiently detoxified upon reduction by saliva and gastric juice, and sequestration by intestinal bacteria. If some chromium(VI) is absorbed by the intestine, it is massively reduced in the blood of the portal system and then in the liver. These mechanisms explain the lack of genotoxicity, carcinogenicity, and induction of other long-term health effects of chromium (VI) by the oral route. Within the respiratory tract, chromium(VI) is reduced in the epithelial-lining fluid, pulmonary alveolar macrophages, bronchial tree and peripheral lung parenchyma cells. Hence, lung cancer can only be induced when chromium(VI) doses overwhelm these defense mechanisms. The efficient uptake and reduction of chromium(VI) in red blood cells explains its lack of carcinogenicity at a distance from the portal of entry into the body. All experimental and epidemiological data, and the underlying mechanisms, point to the occurrence of thresholds in chromium(VI) carcinogenesis. PMID- 10753183 TI - High cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2) contents in mouse lung tumors. AB - Mouse lung tumorigenesis is a convenient model for examining all stages of lung adenocarcinoma (AC) progression. Because enhanced cyclooxygenase 2 (COX-2) expression has been observed in advanced human AC, we investigated the intracellular concentrations of the two cyclooxygenases, cyclooxygenase 1 (COX-1) and COX-2, at different times after carcinogen administration to A/J mice. The concentrations of both proteins were much higher in urethane-induced adenomas and carcinomas compared with control A/J mouse lung tissue (P < 0.03 and P < 0.01 in adenomas and AC, respectively, for COX-1; P < 0.003 and P < 0.004 in adenomas and AC, respectively, for COX-2). Small benign tumors that arose spontaneously in 13 month-old mice also stained for COX-1 and COX-2, showing that this elevated enzyme content does not depend on chemical induction. COX-1 and COX-2 immunostaining was observed in normal bronchiolar and alveolar epithelia, alveolar macrophages and bronchiolar smooth muscle. This is the first report of the cellular distribution of COX-1 and COX-2 in murine lungs and the first in any species to demonstrate their co-localization. COX content in isolated bronchiolar Clara cells, a putative cell of tumor origin, was equal to that found in tumors, suggesting that the high enzyme content in neoplasms is due to their proportionally high concentration of these tumor precursor cells. Different patterns of COX-1 and COX-2 expression were observed in tumors of different growth patterns; only occasional small foci stained in solid adenomas, while most cells in papillary adenomas were immunoreactive. This staining pattern was also seen in adenocarcinomas, but some of the papillary portions also included focally stained and unstained regions. The continued expression during neoplastic progression of these specialized enzymes present in normal cells of tumor origin suggests their function in maintenance of the neoplastic state. PMID- 10753184 TI - XPD polymorphisms: effects on DNA repair proficiency. AB - XPD codes for a DNA helicase involved in transcription and nucleotide excision repair. Rare XPD mutations diminish nucleotide excision repair resulting in hypersensitivity to UV light and increased risk of skin cancer. Several polymorphisms in this gene have been identified but their impact on DNA repair is not known. We compared XPD genotypes at codons 312 and 751 with DNA repair proficiency in 31 women. XPD genotypes were measured by PCR-RFLP. DNA repair proficiency was assessed using a cytogenetic assay that detects X-ray induced chromatid aberrations (breaks and gaps). Chromatid aberrations were scored per 100 metaphase cells following incubation at 37 degrees C (1.5 h after irradiation) to allow for repair of DNA damage. Individuals with the Lys/Lys codon 751 XPD genotype had a higher number of chromatid aberrations (132/100 metaphase cells) than those having a 751Gln allele (34/100 metaphase cells). Individuals having greater than 60 chromatid breaks plus gaps were categorized as having sub-optimal repair. Possessing a Lys/Lys751 genotype increased the risk of sub-optimal DNA repair (odds ratio = 7.2, 95% confidence interval = 1.01-87.7). The Asp312Asn XPD polymorphism did not appear to affect DNA repair proficiency. These results suggest that the Lys751 (common) allele may alter the XPD protein product resulting in sub-optimal repair of X-ray-induced DNA damage. PMID- 10753186 TI - High frequency in esophageal cancers of p53 alterations inactivating the regulation of genes involved in cell cycle and apoptosis. AB - Somatic mutations of the tumor suppressor gene p53 have been frequently detected in esophagal cancers, but their biological significance remains to be established. The tumor suppressor activity of p53 results in part from its ability to transactivate genes involved in the cell cycle and apoptosis, such as p21, bax and PIG3, and some p53 mutations may have a differential effect on the transactivation of these target genes. We developed yeast strains in which the activation by wild-type p53 of reporter plasmids containing p53 binding sites present within these target genes induces a change in the color of the colonies (red/white). Using these strains, we analyzed 56 esophageal cancers from patients residing in Normandy, France, a high incidence geographic area. Forty-seven tumors (84%), scored as mutant with the p21, bax and PIG3 reporter strains and in most of the cases (76%), the percentage of red colonies suggested that both p53 alleles were inactivated. Sequencing analysis allowed the identification of a p53 mutation in each positive sample, and the spectrum of mutations was in agreement with the etiological role of tobacco and alcohol. These results confirm the high frequency of biallelic p53 mutations in esophageal carcinoma and strongly suggest that their biological consequence is the complete alteration of the transactivation of genes involved in the cell cycle and apoptosis, which indicates that p53 alteration is a key event in esophagus carcinogenesis. PMID- 10753185 TI - Risk assessment in first degree female relatives of breast cancer patients using the alkaline Comet assay. AB - First degree female relatives (FDFRs) of breast cancer patients have been reported to have a 2- to 3-fold increase in breast cancer risk as compared with the general population. Assessment of genetic instability (DNA damage and repair efficiency) is an important parameter concerning mutagenesis and carcinogenesis. In an attempt to identify individuals at high risk of breast cancer in the FDFRs of breast cancer patients, two tests were used: the alkaline Comet assay on leucocytes and the micronucleus test (MNT) on buccal epithelial cells. In addition to FDFRs, two other categories of subjects were included: breast cancer patients and controls. The Comet assay was used to study basal DNA damage, DNA susceptibility to a mutagen (N-methyl N-nitro N-nitrosoguanidine) and DNA repair efficiency. In addition, the MNT served as an indicator of chromosome breakage/aneuploidy. A significant increase in DNA damage (basal and after treatment with a mutagen, as well as after allowing repair to take place) and micronucleus frequency was observed from controls to FDFRs and from FDFRs to breast cancer patients. There was considerable variability in the subjects with respect to both of these parameters. Outliers identified among the FDFRs based on 3 SD limits of DNA damage and micronucleus frequency were considered as high risk individuals. PMID- 10753187 TI - TGFalpha is required for full expression of the transformed growth phenotype of NIH 3T3 cells overexpressing ornithine decarboxylase. AB - Ornithine decarboxylase (ODC) overexpressed from a heterologous promoter drives the tumorigenic transformation of NIH 3T3 cells and provides a model to investigate the underlying molecular mechanisms. These transformed cells, designated NODC cells, exhibit elevated levels of epidermal growth factor receptor (EGFR) tyrosine kinase (Tyr-k) activity relative to control transfected cells and inhibition of EGFR Tyr-k activation suppresses the transformed growth phenotype of these cells. Thus, ODC-induced transformation of NIH 3T3 cells appears to be mediated, at least in part, by enhanced signaling through the EGFR pathway. Here we extend these studies by evaluating: (i) the effects on growth regulation of overexpressing ODC in EGFR-deficient NIH 3T3 cells; (ii) the potential role of TGFalpha in mediating the EGFR-dependent transformation of NIH 3T3 cells by ODC. Disruption of EGFR-TGFalpha interactions either by deleting EGFR, by treatment with anti-TGFalpha neutralizing antibody or by transfection with a TGFalpha antisense expression vector suppressed acquisition of the full transformed growth phenotype. Specifically, the loss of contact inhibition and the capacity for clonogenic growth appear more dependent on EGFR-TGFalpha interactions than anchorage-independent growth in ODC-overexpressing cells. ODC overexpression does not alter the amount, localization or secretion of TGFalpha. Thus, TGFalpha is not the ODC-responsive component of the EGFR signaling pathway but appears to be critically involved in development of the transformed phenotype of NODC cells. PMID- 10753188 TI - Upregulation of telomerase activity by X-irradiation in mouse leukaemia cells is independent of Tert, Terc, Tnks and Myc transcription. AB - X-irradiation of two mouse myeloid leukaemia cell lines was found to lead to increased telomerase activities. Maximal increases in activity at 24 h post irradiation were approximately three times control unirradiated cell levels. These maxima were reached at between 3-5 Gy depending upon cell line. Peak activity was reached at 8h, remained elevated to 24 h and returned to control levels by 48 h. In contrast, X-irradiation did not activate telomerase in a telomerase-negative human fibroblast line, while in cultured normal mouse bone marrow cells irradiation appeared to reduce activities. No simple relationship between radiation-induced increases in telomerase activity in the myeloid leukaemia lines and the proportions of cells in the S or M phases of the cell cycle was apparent. Radiation-induced increases in activity were significantly reduced by inhibitors of transcription (actinomycin D, alpha-amanatin) and protein synthesis (cycloheximide). These data are consistent with two possibilities: (i) X-irradiation leads to increased transcription and/or translation of a component of telomerase, thus increasing activities; or (ii) X irradiation induces the transcription of a positive regulator of telomerase activity. Northern blot analysis did not indicate that transcription of mTert, the catalytic subunit of telomerase, or mTerc, the RNA component, was elevated after irradiation. Similarly, no significant changes in the expression of Myc or Tnks, the tankyrase gene, two suspected telomerase regulators, were detected. These data are therefore consistent with the induction by X-irradiation of a positive regulator of telomerase activity other than Tnks or Myc or the core protein and RNA components of the enzyme. PMID- 10753189 TI - Role of MAP kinase signalling pathways in the mode of action of peroxisome proliferators. AB - Peroxisome proliferators (PPs) are a class of non-genotoxic chemicals that cause rodent liver enlargement and hepatocarcinogenesis. In primary rat hepatocytes, PPs cause cell proliferation, suppression of apoptosis and peroxisome proliferation. We have investigated the role of different families of mitogen activated protein (MAP) kinases in the mode of action of PPs. Addition of 50 microM nafenopin to primary rat hepatocyte cultures caused weak activation of extracellular signal regulated kinases and p38 MAP kinase. However, incubation of primary hepatocytes with the p38 MAP kinase inhibitor SB203580 or the MAP kinase kinase (MEK) inhibitor PD098059 prevented the induction of DNA synthesis and the suppression of transforming growth factor beta(1)-induced apoptosis by the PP nafenopin. In contrast, in the presence of these MAP kinase inhibitors, nafenopin still induced palmitoyl CoA oxidation, a measure of peroxisome proliferation. We have shown previously that PPs such as nafenopin require tumour necrosis factor alpha (TNF-alpha) to exert their effects on cellular proliferation and apoptosis. Here we show that treatment of primary rat hepatocyte cultures with nafenopin causes an increase in bioactive TNF-alpha and that this process requires p38 MAP kinase activity. PMID- 10753190 TI - Development of a syngeneic mouse model for events related to ovarian cancer. AB - Mouse ovarian surface epithelial cells (MOSEC) were obtained from virgin, mature mice by mild trypsinization and were repeatedly passaged in vitro. Early passage cells (<20 passages) exhibited a cobblestone morphology and contact inhibition of growth. After approximately 20 passages in vitro, cobblestone morphology and contact inhibition of growth was lost. Tumor forming potential was determined by s.c. and i.p. injection of early and late passage cells into athymic and syngeneic C57BL6 mice. Subcutaneous tumors formed in approximately 4 months and were present only at the injection site. Intraperitoneal injection of late passage MOSEC into athymic and syngeneic mice resulted in growth of tumor implants throughout the abdominal cavity, and production of hemorrhagic ascitic fluid. Early passage MOSEC did not form tumors in vivo. Histopathologic analysis of tumors revealed a highly malignant neoplasm containing both carcinomatous and sarcomatous components. Late passage MOSEC expressed cytokeratin and did not produce ovarian steroids in response to gonadotropin stimulation in vitro. Ten clonal lines were established from late passage MOSEC. Each clone formed multiple peritoneal tumors and ascitic fluid after i.p. injection into C57BL6 mice. Three cell lines examined cytogenetically were polyploid with near-tetraploid modal chromosome numbers. Common clonal chromosome gains and losses included +5, +15, +19 and -X, -3, -4. One cell line had a clonal translocation between chromosomes 15 and 18 and another had a small marker chromosome; common structural abnormalities were not observed. These data describe the development of a mouse model for the study of events related to ovarian cancer in humans. The ability of the MOSEC to form extensive tumors within the peritoneal cavity, similar to those seen in women with Stage III and IV cancer, and the ability of the MOSEC to produce tumors in mice with intact immune systems, makes this model unique for investigations of molecular and immune interactions in ovarian cancer development. PMID- 10753191 TI - In vivo administration of O(6)-benzylguanine does not influence apoptosis or mutation frequency following DNA damage in the murine intestine, but does inhibit P450-dependent activation of dacarbazine. AB - Clinically relevant cancer chemotherapeutic alkylating agents such as temozolomide and dacarbazine induce apoptosis and are mutagenic via the formation of O(6)-alkylguanine adducts in DNA. The DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) functions by dealkylating such adducts and can thus prevent apoptosis and mutagenesis. In attempts to maximize the clinical effectiveness of these alkylating agents, inhibitors of AGT such as O(6) benzylguanine (BeG) have been developed. We show here that within murine small intestinal crypt cells, BeG administration does not alter the apoptotic response to the direct-acting methylating agents N-methyl-N-nitrosurea (MNU), temozolomide and N-methyl-N'-nitro-N-nitrosoguanidine. Furthermore, we show that BeG pretreatment fails to elevate the mutation frequency at the murine Dlb-1 locus following exposure to MNU. Consistent with these results, we show that intestinal AGT activity is effectively abolished by administration of 100 mg/kg temozolomide, even in the absence of BeG. In contrast, pretreatment with BeG transiently abolished the apoptotic response to the methylating prodrug dacarbazine. Activation of dacarbazine to its reactive intermediate has previously been shown to be cytochrome P450 dependent and we show here that pretreatment of mice with the cytochrome P450 inhibitor metyrapone also inhibits dacarbazine-induced apoptosis. Thus BeG increases neither the prevalence of apoptosis nor mutation frequency in the murine small intestine, but is capable of inhibiting P450-dependent prodrug activation. The positive implication from this study is that BeG treatment may not exacerbate the toxic and mutagenic effects of methylating agents within normal cells, although it may engender other adverse reactions through the suppression of cytochrome P450-dependent processes. PMID- 10753192 TI - Overexpression of a fish CDKN2 gene in a hereditary melanoma model. AB - The fish genus Xiphophorus provides a vertebrate model useful in etiological studies of cancer. Hybrid fish can spontaneously develop melanomas deriving from the inheritance of melanistic pigment patterns and the simultaneous absence of proper genetic regulation. A cyclin-dependent kinase inhibitor gene, termed CDKN2X, was mapped to a genomic region that is implicated in fish melanoma tumor suppression. The related human tumor suppressor locus CDKN2A (P16, INK4A, MTS1) is deleted, mutated or transcriptionally repressed through methylation of cytosine bases within the 5' CpG island in a variety of neoplasms, including melanoma. The fish CDKN2X locus harbors a CpG island within its promoter and first exon, analogous in location to CpG islands in human CDKN2A and CDKN2B loci. The methylation state of individual CpG dinucleotides was investigated in genomic DNA derived from control tissues and melanomas within the CDKN2X 5' CpG island. The studied genomic area was found to be virtually unmethylated in all tested tissues including melanomas. In addition, RNA expression studies of the fish CDKN2X locus revealed that it is significantly overexpressed in melanoma, in contrast to what has been reported for the human CDKN2A locus in melanoma. Such overexpression may be a consequence of the pronounced upregulation of the Xmrk-2 receptor tyrosine kinase oncogene reported in several Xiphophorus melanoma models. PMID- 10753193 TI - Breast cancer, heterocyclic aromatic amines from meat and N-acetyltransferase 2 genotype. AB - Breast cancer risk has been hypothesized to increase with exposure to heterocyclic aromatic amines (HAAs) formed from cooking meat at high temperature. HAAs require enzymatic activation to bind to DNA and initiate carcinogenesis. N acetyltransferase 2 (NAT2) enzyme activity may play a role, its rate determined by a polymorphic gene. We examined the effect of NAT2 genetic polymorphisms on breast cancer risk from exposure to meat by cooking method, doneness and estimated HAA [2-amino-1-methyl-6-phenylimidazole[4,5-b]pyridine (PhIP), 2-amino 3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8 trimethylimidazo[4,5-f]quinoxaline (DiMeIQx)] intake. Women were recruited with suspicious breast masses and questionnaire data were collected prior to biopsy to blind subjects and interviewers to diagnoses. For 114 cases with breast cancer and 280 controls with benign breast disease, NAT2 genotype was determined using allele-specific PCR amplification to detect slow acetylator mutations. HAAs were estimated from interview data on meat type, cooking method and doneness, combined with a quantitative HAA database. Logistic regression models controlled for known risk factors, first including all controls, then 108 with no or low risk (normal breast or no hyperplasia) and finally 149 with high risk (hyperplasia, atypical hyperplasia, complex fibroadenomas). Meat effects were examined within NAT2 strata to assess interactions. We found no association between NAT2 and breast cancer. These Californian women ate more white than red meat (control median 46 versus 8 g/day). There were no significant associations of breast cancer with red meat for any doneness. White meat was significantly protective (>67 versus <26 g/day, OR 0.46, 95% CI 0.23-0.94, P for trend = 0.02), as was chicken, including well done, pan fried and barbecued chicken. MeIQx and DiMeIQx were not associated with breast cancer. A protective effect of PhIP was confounded after controlling for well done chicken. Results were unchanged using low or high risk controls or dropping 30 in situ cases. There was no interaction between NAT2 and HAAs. These findings do not support a role for HAAs from meat or NAT2 in the etiology of breast cancer. Further research is needed to explain the white meat association. PMID- 10753194 TI - Chemoprevention of familial adenomatous polyposis development in the APC(min) mouse model by 1,4-phenylene bis(methylene)selenocyanate. AB - Epidemiological and experimental studies have suggested that dietary supplementation with selenium can inhibit the development of cancers at several organ sites. We have consistently shown that 1, 4-phenylene bis(methylene) selenocyanate (p-XSC) is a highly effective cancer chemopreventive agent against the development of chemically induced cancers in several laboratory animal species. This is the first report describing the preventive effects of p-XSC in an animal model of familial adenomatous polyposis (FAP) containing a germline mutation of the APC gene. Six-week old male (heterozygous) C57BL/6J-APC(min) or wild-type mice were fed high fat diets containing 0, 10 or 20 p.p.m. p-XSC. After 80 days, the mice were killed and their intestines were excised and evaluated for polyps. Multiple samples were also harvested from normal appearing small intestine and colon for molecular analysis. Both the mucosa and polyps from the intestine and colon were assayed for beta-catenin, cyclooxygenase (COX)-2 expression and COX isoform activities. Administration of p-XSC in the diet significantly decreased the rate of formation of small intestinal tumors (P < 0. 0001) and colon tumors (P < 0.002) in APC(min) mice. p-XSC produced a dose dependent inhibition of tumors in both small intestine (P < 0. 0001) and colon (P < 0.035). Mice fed 20 p.p.m. p-XSC had significantly lower levels of beta-catenin expression and COX-2 activity in polyps. These observations demonstrate for the first time that the synthetic organoselenium compound p-XSC possesses antitumor activity against genetically predisposed neoplastic lesions, such as FAP. While the exact mechanism(s) for this antitumor activity of p-XSC remains to be elucidated, it appears that modulation of beta-catenin expression and COX-2 activity is associated with inhibition of intestinal polyps. PMID- 10753195 TI - Estrogen receptor alpha gene polymorphisms and endometrial cancer risk. AB - Since the estrogen receptor alpha (ER) is an important mediator of hormonal responses such as proliferation in estrogen-sensitive tissues, we hypothesized that polymorphisms in the ER gene could be functional and associated with endometrial cancer risk. We performed a population-based case-control study in Sweden, focusing on restriction fragment length polymorphisms for XbaI and PvuII and an upstream TA repeat polymorphism. In the main analysis, 154 cases and 205 controls who never used hormone replacement therapy took part and we calculated age-adjusted and multivariate odds ratios (OR) and 95% confidence intervals (CI) using unconditional logistic regression. The XbaI X allele appeared to confer a reduced risk for endometrial cancer. The multivariate OR for the XX genotype was 0.52 (95% CI 0.21-1.29) compared to the xx genotype and there were suggestions of decreasing risk with increasing number of X alleles (P for trend = 0.07). The PvuII PP genotype was also associated with a non-significantly decreased risk for endometrial cancer (multivariate OR 0.70, 95% CI 0.34-1.44) compared with the pp genotype (P for trend = 0.43). The multivariate OR for two short TA (<19 repeats) alleles versus two long alleles was 1.54 (95% CI 0. 73-3.27) and there were suggestions of increasing risk with increasing number of short alleles (P for trend = 0.26). We observed the same pattern of results in an expanded group of subjects, which included women who had used hormone replacement (in total 288 cases and 392 controls). Our data suggest that variants of the ER gene may be associated with an altered risk of endometrial cancer. PMID- 10753196 TI - Correlation between DNA or protein adducts and benzo[a]pyrene diol epoxide I triglyceride adduct detected in vitro and in vivo. AB - In this study, we demonstrated the in vitro and in vivo formation of carcinogen lipid adduct and its correlation with DNA or protein adducts. The lipids from serum or hepatocyte membranes of Sprague-Dawley rats, human serum and standard major lipids were in vitro reacted with benzo[a]pyrene (B[a]P) and B[a]P metabolites. 7, 8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE-I), an ultimate carcinogenic form of B[a]P, was covalently bound to triglyceride (TG). BPDE-I-TG adducts isolated by thin-layer chromatography (TLC) were further detected by high-performance liquid chromatography. TGs, including triolein, tripalmitin and tristearin, showed positive reactions with BPDE-I. However, cholesterol, phospholipids (phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidyl-inositol and sphingomyelin) and non-esterified fatty acids (palmitic acid, oleic acid, linoleic acid and stearic acid) did not react with BPDE-I. In addition, other B[a]P metabolites (B[a]P-phenols and -diols) did not react with TG. TG appeared to be the most reactive lipid yet studied with respect to its ability to form an adduct with BPDE-I. There was a clear-cut dose-related formation of [1,3-(3)H]BPDE-I-lipid adducts in vitro between TG and [1,3 (3)H]BPDE-I. In an animal study, BPDE-I-TG was also formed in the serum of rats orally treated with B[a]P (25 mg/rat). Also, obvious correlations between [(3)H]B[a]P related-biomolecule adducts (DNA or protein) or lipid damage and the BPDE-I-TG adducts were obtained in various tissues of mice i.p. treated with [(3)H]B[a]P. These data suggest that TG can form an adduct with BPDE-I, as do other macromolecules (DNA, RNA and protein). Therefore, a carcinogen-lipid adduct would be a useful biomarker for chemical carcinogenesis research and cancer risk assessment. PMID- 10753197 TI - Increase in wild-type p53 stability and transactivational activity by the chemopreventive agent apigenin in keratinocytes. AB - Apigenin, a naturally occurring, non-mutagenic flavonoid, has been shown to inhibit UV-induced skin tumorigenesis in mice when topically applied. In this report we have used the mouse keratinocyte 308 cell line, which contains a wild type p53 gene, to study the effect of apigenin treatment on p53 protein levels and the expression of its downstream partner, p21/waf1. Cells were treated with 70 microM apigenin for various times and levels of p53 and p21/waf1 protein were assessed by western blot analysis. The level of p53 protein was induced 27-fold after 4 h of apigenin treatment and levels remained elevated through 10 h of exposure. After 24 h of exposure to 70 microM apigenin, p53 protein levels returned to control levels. p21/waf1 protein levels increased approximately 1. 5 2-fold after 4 h and remained elevated at 24 h. To investigate the mechanism of p53 protein accumulation, we compared the half-life of p53 protein in vehicle- and apigenin-treated cells. Cells were incubated for 4 h in the presence of apigenin, then cycloheximide was added to inhibit further protein synthesis and p53 protein levels were measured by western blot. The half-life of p53 protein was found to be increased an average of 8-fold in apigenin-treated cells compared with vehicle-treated cells (t(1/2) = 131 min versus 16 min in apigenin- versus vehicle-treated cells, respectively). The mechanism of p53 protein stabilization is currently being investigated. To determine whether p53 was transcriptionally active, we also performed gel mobility shift assays and transient transfection studies using a luciferase plasmid under the control of the p21/waf1 promoter. Both p53 DNA-binding activity and transcriptional activation peaked after 24 h of exposure to apigenin. These studies suggest that apigenin may exert anti tumorigenic activity by stimulating the p53-p21/waf1 response pathway. PMID- 10753198 TI - Development and initial characterization of several new inbred strains of SENCAR mice for studies of multistage skin carcinogenesis. AB - The development and initial characterization of five new inbred strains of SENCAR mice are described in this paper. Ten randomly selected pairs of outbred SENCAR mice were mated and offspring from each separately maintained parental line were sib mated at each successive generation to result in inbred strains. Due to poor reproductive performance only five of the original 10 lines were bred to homogeneity. Initial characterization of the five remaining lines (referred to as SL2/sprd, SL5/sprd, SL7/sprd, SL8/sprd and SLl0/sprd) at F12 for their responsiveness to a two-stage carcinogenesis protocol (10 nmol 7,12 dimethylbenz[a]anthracene and 0.25 microg 12-O-tetradecanoylphorbol-13 acetate) revealed three groups of responders in terms of the number of papillomas per mouse: SL2/sprd and SL8/sprd > SL7/sprd and SL10/sprd >> SL5/sprd. The papilloma responses in SL2/sprd and SL8/sprd were very similar to SENCAR B/Pt compared at the same doses. Papillomas induced on SL2/sprd had the highest propensity to progress to squamous cell carcinomas, similar to that observed in outbred SENCAR and SENCAR B/Pt mice. More detailed comparison of the responsiveness of SL2/sprd and SL5/sprd at Fl5 showed that these two inbred strains differed in their sensitivity to TPA-induced epidermal hyperplasia and that the dose of TPA required to produce a tumor response in SL5/sprd in comparison with that in SL2/sprd was 4-20 times higher. Overall, the availability of the different inbred SENCAR strains will greatly aid mechanistic studies of multistage skin carcinogenesis as well as studies to understand the underlying genetic basis of resistance to tumor promotion and progression in this model system. PMID- 10753199 TI - Apoptosis, mitosis and cyclophilin-40 expression in regressing peroxisome proliferator-induced adenomas. AB - Chronic exposure to peroxisome proliferators (PP), including certain industrial and pharmaceutical chemicals, causes liver cancer in rodents. Continuous exposure to PP is needed for tumor development since the frequency of hepatocellular neoplasms is decreased in animals returned to control diet. To determine cellular and molecular events responsible for enhanced growth in PP-induced liver tumors, we evaluated the relationships of WY-14,643 levels, apoptosis, mitosis and cyclophilin-40 (Cyp-40) expression in regressing tumors induced by WY-14,643, a potent PP. Male F344 rats were fed WY-14,643 (0.1%) in the diet for 43 weeks and then switched to control diet for 2, 3, 5 or 36 days. Mean serum and hepatic concentrations of WY-14,643 were decreased as early as 2 days following removal of WY-14,643 as compared with rats continuously fed WY-14,643. Adenomas from rats maintained on WY-14,643 markedly compressed surrounding parenchyma. Evidence of adenoma regression was observed by 3 days of WY-14,643 withdrawal and was characterized by loss of compression. Decreased compression corresponded to increases in the apoptotic index and decreases in the mitotic index in regressing adenomas at 2, 3, and 5 days following the switch to control diet. Cyclophilins are multifunctional receptor proteins involved in numerous signal transduction pathways, including those mediated by cyclosporin, a liver tumor promoter in rats. Cyp-40 expression was markedly increased in adenomas from continuously exposed rats, but expression returned to levels similar to surrounding parenchyma in adenomas after 5 days of WY-14,643 withdrawal. Taken together, these results indicate that WY-14, 643-induced adenomas regress rapidly following withdrawal of the PP in association with declining liver WY-14,643 levels, suggesting that peroxisome proliferator-activated receptor alpha may mediate PP-induced alterations in mitogenic and/or apoptotic regulation in growing tumors, in conjunction with alterations in Cyp-40 signal transduction. PMID- 10753200 TI - Increase in mutation frequency in lung of Big Blue rat by exposure to diesel exhaust. AB - Exposure to diesel exhaust (DE) is known to cause lung tumors in rats. To clarify the mutagenicity of DE, we estimated mutant frequency (MF) and determined the mutation spectra in rat lung after exposure to DE using lambda/lacI transgenic rats (Big Blue system). Male Big Blue rats (6 weeks old) were exposed for 4 weeks to 1 or 6 mg/m(3) DE, which contains suspended particulate matter. Control rats were maintained in filtered clean air. After exposure to 6 mg/m(3) DE, MF in lung was 4.8-fold higher than in control rats (P < 0.01), but no increase in MF was observed in rats exposed to 1 mg/m(3) DE. Sixty-nine mutants were identified after exposure to 6 mg/m(3) DE. The major mutations were A:T-->G:C (18 mutations) and G:C-->A:T (19 mutations) transitions. Remarkably, G-->T transversion of the lacI gene at site 221 was a hot-spot induced by exposure to DE, and there were complex mutations in which multiple mutations occurred in a single mutant, especially in the rats exposed to 6 mg/m(3) DE. DNA adducts formed by DE were analyzed using a (32)P-post-label TLC method and the amount of 8 hydroxydeoxyguanosine (8-OHdG) was measured using HPLC. Relative adduct level and amount of 8-OHdG were significantly increased in the rats exposed to 6 mg/m(3) DE compared with the controls (3.0- and 2.2-fold, respectively; P < 0.01). The level of cytochrome P450 1A1 mRNA was shown by northern blot analysis to be significantly increased in the lungs of rats exposed to 6 mg/m(3) DE (5.5-fold; P < 0.01). These results indicate that DE causes lesions in genomic DNA and acts as a mutagen in rat lung. PMID- 10753201 TI - Increased salivary acetaldehyde levels in heavy drinkers and smokers: a microbiological approach to oral cavity cancer. AB - The pathogenetic mechanisms behind alcohol-associated carcinogenesis in the upper digestive tract remain unclear, as alcohol is not carcinogenic. However, there is increasing evidence that a major part of the tumour-promoting action of alcohol might be mediated via its first, toxic and carcinogenic metabolite acetaldehyde. Acetaldehyde is produced from ethanol in the epithelia by mucosal alcohol dehydrogenases, but much higher levels derive from microbial oxidation of ethanol by the oral microflora. In this study we investigated factors that might alter the composition and quantities of the oral microflora and, consequently, influence microbial acetaldehyde production. Information about dental health, smoking habits, alcohol consumption and other factors was obtained by a questionnaire from 326 volunteers with varying social backgrounds and health status, e.g. oral cavity malignancy. Paraffin-induced saliva was collected and the microbial production of acetaldehyde from ethanol was measured. Smoking and heavy drinking were the strongest factors increasing microbial acetaldehyde production. Whether poor dental status may alter local acetaldehyde production from ethanol remained unanswered. Bacterial analysis revealed that mainly gram positive aerobic bacteria and yeasts were associated with higher acetaldehyde production. Increased local microbial salivary acetaldehyde production due to ethanol among smokers and heavy drinkers could be a biological explanation for the observed synergistic carcinogenic action of alcohol and smoking on upper gastrointestinal tract cancer. It offers a new microbiological approach to ethanol-associated carcinogenesis at these anatomic sites. PMID- 10753202 TI - CYP1A1 and GSTM1 polymorphisms affect urinary 1-hydroxypyrene levels after PAH exposure. AB - Certain human biotransformation enzymes have been implicated in the formation and scavenging of the ultimate reactive metabolites, the diolepoxides, from polycyclic aromatic hydrocarbons (PAHs). In the present study, performed on aluminum smelter workers, we have analyzed airborne PAH, the pyrene metabolite 1 hydroxypyrene (1-OHP) in urine, and genotypes for biotransformation enzymes involved in PAH metabolism. The aim was to evaluate the correlation between external exposure and biomarkers of exposure and to investigate to what extent genetic polymorphism in metabolic enzymes can explain interindividual variation in urinary 1-OHP levels. DNA was prepared from blood samples from 98 potroom workers and 55 controls and altogether eight polymorphisms in the CYP1A1, mEH, GSTM1, GSTP1 and GSTT1 genes were analyzed. The 1-OHP excretion was found to correlate significantly (P 100-fold) and univariate and multivariate regression analyses were used to find the variables that could determine differences in excretion. The variation could, to some degree, be explained by differences in exposure to airborne particulate-associated PAHs, the use of personal respiratory protection devices, smoking habits and genetic polymorphisms in the cytochrome P450 1A1, GSTM1 and GSTT1 enzymes. The part of the variance that could be explained by differences in biotransformation genotypes seemed to be of the same order of magnitude as the variance explained by differences in exposure. In the control group as well as in the occupationally exposed group, the highest 1-OHP levels were observed in individuals carrying the CYP1A1 Ile/Val genotype who were also of the GSTM1 null genotype. The results show that urinary 1-OHP is a sensitive indicator of recent human exposure to PAHs and that it may also to some extent reflect the interindividual variation in susceptibility to PAHs. PMID- 10753203 TI - Sex-dependent regulation of hepatic peroxisome proliferation in mice by trichloroethylene via peroxisome proliferator-activated receptor alpha (PPARalpha). AB - The mechanism of trichloroethylene-induced liver peroxisome proliferation and the sex difference in response was investigated using wild-type Sv/129 and peroxisome proliferator-activated receptor alpha (PPARalpha)-null mice. Trichloroethylene treatment (0.75 g/kg for 2 weeks by gavage) resulted in liver peroxisome proliferation in wild-type mice, but not in PPARalpha-null mice, suggesting that trichloroethylene-induced peroxisome proliferation is primarily mediated by PPARalpha. No remarkable sex difference was observed in induction of peroxisome proliferation, as measured morphologically, but a markedly higher induction of several enzymes and PPARalpha protein and mRNA was found in males. On the other hand, trichloroethylene induced liver cytochrome P450 2E1, the principal enzyme responsible for metabolizing trichloroethylene to chloral hydrate, only in males, which resulted in similar expression levels in both sexes after the treatment. Trichloroethylene influenced neither the level of catalase, an enzyme involved in the reduction of oxidative stress, nor aldehyde dehydrogenase, the main enzyme catalyzing the conversion to trichloroacetic acid. These results suggest that trichloroethylene treatment causes a male-specific PPARalpha-dependent increase in cellular oxidative stress. PMID- 10753204 TI - Molecular alterations of p73 in human esophageal squamous cell carcinomas: loss of heterozygosity occurs frequently; loss of imprinting and elevation of p73 expression may be related to defective p53. AB - p73 is structurally and functionally related to p53 and is possibly a tumor suppressor gene. Using 15 surgically resected frozen esophageal specimens containing both squamous cell carcinomas (ESCC) and neighboring normal epithelia, we studied p73 gene alterations and mRNA expression. Loss of heterozygosity of the p73 loci was found in nine of 14 informative cases (64%). A polymorphism at codon 173 (Thr) of p73 was identified (eight samples had ACC and seven had ACT), but mutation was not detected in tumor samples. Nine of the 15 ESCC samples (60%) displayed significantly elevated expression of p73 over the neighboring normal epithelium; of these nine samples, four displayed loss of imprinting (LOI) and one switched the expressed allele. Hypermethylation of exon 1 of the p73 gene was not detected, using the bisulfite modification method, in normal or tumor samples. Twelve of the 15 (80%) ESCC samples contained p53 defects, including missense mutation, non-frameshift small deletion or insertion, non-detectable transcripts and protein accumulation. The ESCC samples with p53 defects were significantly correlated with those which had elevated expression of p73 (Fisher's exact test, P < 0.05). The results suggest that increased expression of p73, including that by LOI, could be a partial compensatory mechanism for defective p53. PMID- 10753205 TI - Significant overexpression of metallothionein and cyclin D1 and apoptosis in the early process of rat urinary bladder carcinogenesis induced by treatment with N butyl-N-(4-hydroxybutyl)nitrosamine or sodium L-ascorbate. AB - Effects of a genotoxic bladder carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) and a non-genotoxic bladder promoter, sodium L-ascorbate (Na-AsA), on protein expression, cell proliferation and apoptosis of the bladder epithelium with or without the influence of testicular castration were investigated. Male F344 rats were divided into six groups (groups 1-6). BBN was given with 0.05% drinking water to groups 1 and 4 for 8 weeks, groups 2 and 5 received diet with 5% Na-AsA. Then the animals were treated without any chemicals. Groups 3 and 6 were non-treated controls. Testicular castration was carried out 2 weeks before commencement of chemical treatment on groups 4-6. The total observation period was 18 weeks. Overexpression of cyclin D1 was induced by BBN but not Na-AsA and the degree of overexpression was higher in the order simple hyperplasia, papillary or nodular hyperplasia, papilloma and carcinoma. Metallothionein (MT) was also overexpressed in bladder epithelium treated with BBN but not Na-AsA, but was decreased in papillomas and never found in a carcinoma. Cyclin D1-positive cells were essentially MT-negative. Therefore, it is speculated that MT protects genes from insult by genotoxic carcinogens and its lack is associated with tumor development. Apoptotic cell death occurred during treatment with BBN and Na-AsA and after their withdrawal. Chromatin condensation of many G0/G(1) cells was particularly marked on flow cytometry analysis 1 week after cessation of treatment, this being considered as an early apoptotic change. Although testicular castration had no influence on the above events, it resulted in decreased tumor formation as compared with the case of similarly treated intact animals. Our data demonstrate that overexpression of MT and cyclin D1 is specific for treatment with a genotoxic carcinogen, and suggest that MT overexpression may play an important suppressive role in the early stages of rat urinary bladder carcinogenesis. PMID- 10753206 TI - Arsenite induces DNA-protein crosslinks and cytokeratin expression in the WRL-68 human hepatic cell line. AB - The induction of DNA-protein crosslinks (DPC) has been proposed as an indicator of early biological effects due to the fact that known or suspected carcinogens induce an increased proportion of proteins tightly bound to DNA. Arsenic, a human carcinogen, is reduced and methylated mainly in liver cells generating a number of intermediate reactive forms which could lead to the formation of DNA-protein crosslinks. The induction of DPC by arsenite [As(III)] was investigated in the WRL-68 human hepatic cell line, testing the possibility that cytokeratins or cytokeratin-like proteins, due to their high content of SH groups, could participate in DPC. The formation and decay of DPC was dose-related. Arsenite was the only intracellular species present since no methylated As forms could be detected. Thus, DPC can be attributed to the presence of arsenite, an important species present in liver during As exposure, whose permanence in the tissue would depend on the methylation rate of the organism. Several cytokeratins were identified by immunoblotting among the proteins crosslinked with DNA, including cytokeratin 18 (CK18), a specific liver intermediate filament. An augmented presence of CK18 was detected in treated cultures by immunoblotting of total protein PAGE. In liver cells cytokeratin synthesis is tightly correlated with differentiation programs, thus arsenite could not only be damaging DNA but also modifying differentiation patterns in this tissue. PMID- 10753207 TI - Dramatic synergism between excess soybean intake and iodine deficiency on the development of rat thyroid hyperplasia. AB - The effects of defatted soybean and/or iodine-deficient diet feeding were investigated in female F344 rats. Rats were divided into four groups, each consisting of 10 animals, and fed basal AIN-93G diet in which the protein was exchanged for 20% gluten (Group 1), iodine-deficient gluten (Group 2), 20% defatted soybean (Group 3) and iodine-deficient defatted soybean (Group 4). At week 10, relative thyroid gland weights (mg/100 g body wt) were significantly (P < 0.01) higher in Groups 2 (15.5 +/- 1.3) and 4 (81.7 +/- 8.6) than in Group 1 (8.4 +/- 2.0) and pituitary gland weights (mg/100 g body wt) were significantly (P < 0.01) higher in Groups 3 (9.1 +/- 0. 6) and 4 (9.7 +/- 1.5) than in Group 1 (6.5 +/- 1.5). Serum biochemical assays revealed thyroxine to be significantly (P < 0.05) lower in Groups 2 and 4 than in Group 1. On the other hand, serum thyroid stimulating hormone (TSH) was significantly (P < 0.01) higher in Groups 3 and 4 than in Group 1. This was particularly striking for TSH (ng/ml) at week 10 in Group 4 (126 +/- 11) as compared with Groups 1 (4.36 +/- 0.30), 2 (4.84 +/- 0.80) and 3 (5. 78 +/- 0.80). Histologically, marked diffuse follicular hyperplasia of the thyroid was evident in Group 4 rats. Proliferating cell nuclear antigen labeling indices (%) were significantly higher (P < 0.05) in Groups 2 (4.8 +/- 2.5) and 4 (13.2 +/- 1.1) than in Group 1 (0.4 +/- 0.5). Ultrastructurally, severe disorganization and disarrangement of mitochondria were apparent in thyroid follicular cells of Group 4. In the anterior pituitary, dilated rough surfaced endoplasmic reticulum and increased secretory granules were remarkable in this group. Our results thus strongly suggest that dietary defatted soybean synergistically stimulates the growth of rat thyroid with iodine deficiency, partly through a pituitary-dependent pathway. PMID- 10753208 TI - LacI mutation spectra following benzo[a]pyrene treatment of Big Blue mice. AB - The mutation spectrum of the lacI gene from the liver of C57Bl6 Big Blue transgenic mice treated with benzo[a]pyrene (B[a]P) has been compared with the spectrum of spontaneous mutations observed in the liver of untreated Big Blue mice. Mice were treated with B[a]P for 3 days followed by a partial hepatectomy one day after the last injection. Liver tissue was removed for analysis at hepatectomy and, again, 3 days later at the time of sacrifice. Earlier, we reported that the lacI mutant frequency in these B[a]P-treated mice was elevated in the liver both at the time of hepatectomy and at sacrifice; however, a statistically significant increase in the mutant frequency was observed only at sacrifice. In this study, the DNA sequence spectra of lacI mutations observed in the liver of B[a]P-treated Big Blue mice at hepatectomy and at time of sacrifice were compared with each other and with the spectrum of spontaneous liver mutations. No differences were observed between the two B[a]P-treatment spectra. However, mutation frequencies of both GC-->TA and GC-->CG at the time of hepatectomy and at sacrifice were significantly elevated compared with the spontaneous frequency of these same transversions. Also, the frequency of AT-->TA transversions was significantly higher than the spontaneous frequency at the time of hepatectomy but not at sacrifice. The frequency of all other classes of mutations scored was not significantly different from the frequency of these same events in the spontaneous spectra. These data support the view that B[a]P treatment results in the induction of GC-->TA and GC-->CG transversions within 1 day of the last injection and they provide insights regarding the relative roles of benzo[a]pyrene-7,8-diol-9, 10-epoxide and radical cations of B[a]P in B[a]P induced mutagenesis in vivo. Finally, these data provide evidence for B[a]P induced mutagenesis under conditions where no statistical increase in mutant frequency could be shown. PMID- 10753209 TI - Acrylonitrile-induced morphological transformation in Syrian hamster embryo cells. AB - Acrylonitrile (ACN) is a monomer used in the synthesis of rubber, fibers and plastics. Previous studies demonstrated that ACN induces brain neoplasms (predominately astrocytomas) in rats following chronic treatment. While the mechanisms of ACN-induced glial cell carcinogenicity have not been completely elucidated, investigations by our group and others have suggested a role for the induction of oxidative stress and the resultant oxidative damage in this process. In vitro cell transformation models are useful for detecting and studying the mechanisms of chemical carcinogenesis. Cell transformation by chemical carcinogens in Syrian hamster embryo (SHE) cells exhibits a multistage process similar to that observed in vivo, for both non-genotoxic and genotoxic carcinogens. In the present study, the ability of ACN to induce morphological transformation and oxidative damage was examined in SHE cells. ACN induced an increase in morphological transformation at doses of 50, 62.5 and 75 microg/ml (maximum sub-toxic dose tested) following 7 days of continuous treatment. SHE cells exposed to ACN for 24 h failed to increase morphological transformation. Morphological transformation by ACN was inhibited by co-treatment with the antioxidants alpha-tocopherol and (-)-epigallocathechin-3 gallate (EGCG) for 7 days. Treatment of SHE cells with 75 microg/ml ACN produced a significant increase in 8-hydroxy-2'-deoxyguanosine that was also inhibited by co-treatment with alpha-tocopherol or EGCG. These results support the proposal that oxidative stress and the resulting oxidative damage is involved in ACN-induced carcinogenicity. PMID- 10753210 TI - Induction of mammalian cell transformation and genotoxicity by 2 methoxyestradiol, an endogenous metabolite of estrogen. AB - 2-methoxyestradiol (2-MeOE(2)) is an endogenous metabolite of 17beta-estradiol and a proposed inhibitor of tumor growth and angiogenesis. However, 2-MeOE(2) is also an inhibitor of microtubule assembly and other microtubule inhibitors, e.g. colcemid and diethylstilbestrol, induce aneuploidy and cell transformation in cultured mammalian cells. To assess the in vitro carcinogenicity and related activity of 2-MeOE(2), the abilities of this metabolite to induce cell transformation and genetic effects were studied simultaneously using Syrian hamster embryo (SHE) fibroblasts. Growth of these cells was reduced by treatment with 2-MeOE(2) at 0.1-1.0 microg/ml in a concentration-dependent manner. Treatment of SHE cells with 2-MeOE(2) at 0.3 or 1.0 microg/ml for 2-48 h also resulted in a concentration- and treatment time-related increase in the mitotic index and the percentage of multinucleated cells. Treatment with 2-MeOE(2) at 0.1 1.0 microg/ml for 48 h induced a statistically significant increase in the frequencies of morphological transformation of SHE cells in a concentration dependent manner. A statistically significant increase in the frequencies of somatic mutations at the Na(+)/K(+) ATPase or hprt locus was also observed in cells treated with 2-MeOE(2) for 48 h at 0.1 or 0.3 microg/ml, respectively. Treatment of SHE cells with 2-MeOE(2) at 0.3 or 1.0 microg/ml for 24 h induced chromosome aberrations, mainly breaks, exchanges and chromosome pulverization. The incidence of chromosome aberrations was not affected by co-treatment with alpha-naphthoflavone, an inhibitor of 2-hydroxylase that inhibits oxidative conversion of 2-MeOE(2) to 2-hydroxyestradiol, but the incidence was slightly increased by co-treatment with L-ascorbic acid. Numerical chromosomal changes in the near diploid range and in the tetraploid and near tetraploid ranges were also detected in 2-MeOE(2)-treated cells. These findings indicate that 2-MeOE(2) has cell transforming and genotoxic activities in cultured mammalian cells and potential carcinogenic activity. PMID- 10753211 TI - Fanconi anemia, complementation group A, cells are defective in ability to produce incisions at sites of psoralen interstrand cross-links. AB - The hypersensitivity of Fanconi anemia, complementation group A, (FA-A) cells to agents which produce DNA interstrand cross-links correlates with a defect in their ability to repair this type of damage. In order to more clearly elucidate this repair defect, chromatin-associated protein extracts from FA-A cells were examined for ability to endonucleolytically produce incisions in DNA at sites of interstrand cross-links. A defined 140 bp DNA substrate was constructed with a single site-specific monoadduct or interstrand cross-link produced by 4,5',8 trimethylpsoralen (TMP) plus long wavelength (UVA) light. Our results show that FA-A cells are defective in ability to produce dual incisions in DNA at sites of interstrand cross-links. Specifically, there is defective incision on the 3'- and 5'-sides of both the furan and pyrone sides of the cross-link. This defect is corrected in FA-A cells transduced with a retroviral vector expressing FANCA cDNA. At the site of a TMP monoadduct, FA-A cells can introduce incisions on both the 3'- and 5'-sides of the furan side monoadduct, but are defective in ability to produce these incisions on the pyrone side monoadduct. These studies also indicate that XPF is involved in production of the 5' incision by the normal extracts on these substrates. These results correlate with our previous work, which showed that FA-A cells are mainly defective in ability to repair psoralen interstrand cross-links with a lesser defect in ability to repair psoralen monoadducts. This defect in endonucleolytic incision at sites of TMP interstrand cross-links could be related to reduced levels of non-erythroid alpha spectrin (alphaSpIISigma*) in the extracts from FA-A cells. alphaSpIISigma* could act as a scaffold to align proteins involved in cross-link repair and enhance their interactions; a deficiency in alphaSpIISigma* could thus lead to reduced efficiency of repair and the decreased levels of incisions we observe at sites of interstrand cross-links in FA-A cells. PMID- 10753212 TI - Promotion of intestinal carcinogenesis by Streptococcus bovis. AB - The involvement of Streptococcus bovis, an member of the human gut flora, in colorectal neoplastic diseases is an object of controversy. The aim of this study was to determine the effects of S.bovis and of antigens extracted from the bacterial cell wall on early preneoplastic changes in the intestinal tract. Adult rats received i. p. injections of azoxymethane (15 mg/kg body weight) once per week for 2 weeks. Fifteen days (week 4) after the last injection of the carcinogen, the rats received, by gavage twice per week during 5 weeks, either S.bovis (10(10) bacteria) or wall-extracted antigens (100 microg). One week after the last gavage (week 10), we found that administration of either S.bovis or of antigens from this bacterium promoted the progression of preneoplastic lesions through the increased formation of hyperproliferative aberrant colonic crypts, enhanced the expression of proliferation markers and increased the production of IL-8 in the colonic mucosa. Our study suggests that S.bovis acts as a promoter of early preneoplastic lesions in the colon of rats. The fact that bacterial wall proteins are more potent inducers of neoplastic transformation than the intact bacteria may have important implications in colon cancer prevention. PMID- 10753213 TI - Dysplasia and cancer in the dextran sulfate sodium mouse colitis model. Relevance to colitis-associated neoplasia in the human: a study of histopathology, B catenin and p53 expression and the role of inflammation. AB - Animal models of colitis, which develop dysplasia and cancer similar to human ulcerative colitis are needed to further investigate the dysplasia cancer sequence. This study describes the expression of B-catenin and p53 along with the histopathology and inflammation scores as they relate to dysplasia and cancer in the dextran sulfate sodium (DSS) colitis model. Swiss Webster mice were fed with 5% DSS as follows: group A, four cycles of DSS, 84 days total (1 cycle = 7 days DSS + 14 days H(2)O); group B, four cycles DSS followed by 120 days H(2)O, 204 days total; group C, 7 days DSS followed by 180 days H(2)O, 187 days total; group D, 7 days DSS followed by 90 days H(2)O, 97 days total. The incidences of dysplasia and/or cancer were 15.8, 37.5, 18.1 and 0% in groups A-D, respectively. Dysplasia and/or cancer occurred as flat lesions or as dysplasia-associated lesion or mass (DALM) as observed in the human. Thirty-three percent of cancers had associated dysplasia. Within group A, inflammation scores were significantly higher in animals with dysplasia and/or cancer compared with those without dysplasia and/or cancer (P < 0. 05-P < 0.0001). Inflammation scores were significantly higher in animals with cancers versus those with dysplasia (P < 0.015) and in flat dysplasia and/or cancer versus DALM (P < 0.0042). B-catenin showed translocation from the cell membrane to the cytoplasm and/or nucleus in 100% of DALM and 5.8% of flat dysplasia and/or cancer. A total of 94.2% of flat dysplasia and/or cancer had exclusive cell membrane expression compared with 0% DALM (P < 0.0001). Only 7.4% of dysplasia and/or cancer showed nuclear expression of p53. In colitis-associated dysplasia and/or cancer in the DSS model: (i) histology resembles that in the human; (ii) inflammation plays a significant role in the dysplasia cancer sequence and whether dysplasia and/or cancer grows as a flat lesion or a DALM; (iii) the early molecular pathways are different for flat dysplasia and/or cancer versus DALM, with nuclear/cytoplasmic translocation of B catenin as an early event in DALM but not flat dysplasia and/or cancer; and (iv) p53 has little or no role in dysplasia and/or cancer. This well characterized model provides an excellent vehicle for studying the roles of inflammation, the molecular events and the role of chemopreventive agents in colitis-associated neoplasia. PMID- 10753214 TI - Establishment of a radiation- and estrogen-induced breast cancer model. AB - It is well accepted that cancer arises in a multistep fashion in which exposure to environmental carcinogens is a major etiological factor. The aim of this work was to establish an experimental breast cancer model in order to understand the mechanism of neoplastic transformation induced by high LET radiation in the presence of 17beta-estradiol (E). Immortalized human breast cells (MCF-10F) were exposed to low doses of high LET alpha particles (150 keV/microm) and subsequently cultured in the presence or absence of E for periods of up to 10 months post-irradiation. MCF-10F cells irradiated with either a single 60 cGy dose or 60/60 cGy doses of alpha particles showed gradual phenotypic changes including altered morphology, increase in cell proliferation relative to the control, anchorage-independent growth and invasive capability before becoming tumorigenic in nude mice. In alpha particle-irradiated cells and in those cells subsequently cultured in the presence of E, increased BRCA1, BRCA2 and RAD51 expression were detected by immunofluorescence staining and quantified by confocal microscopy. These studies showed that high LET radiation such as that emitted by radon progeny, in the presence of estrogen, induced a cascade of events indicative of cell transformation and tumorigenicity in human breast epithelial cells. PMID- 10753215 TI - Immunohistochemical detection of 1,N(6)-ethenodeoxyadenosine, a promutagenic DNA adduct, in liver of rats exposed to vinyl chloride or an iron overload. AB - Etheno adducts in DNA bases are formed from exogenous agents such as vinyl chloride and urethane, but also via endogenous lipid peroxidation products like trans-4-hydroxy-2-nonenal. An immunohistochemical method was developed to localize the promutagenic 1,N(6)-ethenodeoxyadenosine DNA adduct in liver of rats exposed to vinyl chloride or an iron overload with or without carbon tetrachloride. Six monoclonal antibodies, previously produced through collaborative efforts, were screened for their optimal adduct recognition and low background formation. The antibody generated by clone EM-A-4 was found to be most suitable. Semi-quantitative image analysis of relative pixel intensity showed approximately 1.5 times higher adduct levels (P < 0.05) in the livers of rats treated with vinyl chloride or an iron overload when compared with untreated controls. Significantly elevated adduct levels persisted in vinyl chloride treated rat liver 14 days after cessation of exposure, suggesting that this adduct is not rapidly eliminated from rat liver DNA. Using the new immunohistochemical method it is possible to visualize this promutagenic etheno DNA adduct that may play a role in oxidative stress and lipid peroxidation induced DNA damage in carcinogenesis. PMID- 10753217 TI - Tamoxifen induces endometrial and vaginal cancer in rats in the absence of endometrial hyperplasia. AB - Tamoxifen was administered orally to neonatal rats on days 2-5 after birth and the subsequent effects on the uterus were characterized, morphometrically, over the following 12 months. Tamoxifen inhibited development of the uterus and glands in the endometrium, indicating a classical oestrogen antagonist action. Between 24 and 35 months after tamoxifen treatment there was a significant increase in the incidence (26%) of uterine adenocarcinomas and a 9% incidence of squamous cell carcinomas of the vagina/cervix in the absence of any oestrogen agonist effect in the uterus. This demonstrates that an oestrogen agonist effect is not an absolute requirement for the carcinogenic effect of tamoxifen in the reproductive tract of the rat. The unopposed oestrogen agonist effect of tamoxifen on the endometrium may not be the only factor involved in the development of endometrial cancers. It is possible that tamoxifen causes these tumours via a genotoxic mechanism similar to that seen in rat liver. However, using (32)P-post-labelling we failed to find evidence of tamoxifen-induced DNA adducts in the uterus. Tamoxifen may affect hormonal imprinting of oestrogen receptor responses in stem cells of the uterus, causing reproductive tract cancers to arise at a later time, in the same way as has been proposed for diethylstilbestrol. If these rodent data extrapolate to humans, then women who are taking tamoxifen as a chemopreventative may have an increased risk of vaginal/cervical cancer, as well as endometrial cancer. PMID- 10753216 TI - Calcium phosphate-containing precipitate and the carcinogenicity of sodium salts in rats. AB - Sodium saccharin, ascorbate and other sodium salts fed at high doses to rats produce urinary bladder urothelial cytotoxicity with consequent regenerative hyperplasia. For sodium salts that have been tested, tumor activity is enhanced when administered either alone or after a brief exposure to a known genotoxic bladder carcinogen. These sodium salts alter urinary composition of rats resulting in formation of an amorphous precipitate. We examined the precipitate to ascertain its composition and further delineate the basis for its formation in rat urine. Using scanning electron microscopy with attached X-ray energy dispersive spectroscopy, the principal elements present were calcium, phosphorus, minor amounts of silicon and sulfur. Smaller elements are not detectable by this method. Infrared analyses demonstrated that calcium phosphate was in the tribasic form and silicon was most likely in the form of silica. Small amounts of saccharin were present in the precipitate from rats fed sodium saccharin (<5%), but ascorbate was not detectable in the precipitate from rats fed similar doses of sodium ascorbate. Large amounts of urea and mucopolysaccharide, apparently chondroitin sulfate, were detected in the precipitate by infrared analysis. Chemical analyses confirmed the presence of large amounts of calcium phosphate with variably small amounts of magnesium, possibly present as magnesium ammonium phosphate crystals, present in urine even in controls. Small amounts of protein, including albumin and alpha(2u)-globulin, were also detected (<5% of the precipitate). Calcium phosphate is an essential ingredient of the medium for tissue culture of epithelial cells, but when present at high concentrations (>5 mM) it precipitates and becomes cytotoxic. The nature of the precipitate reflects the unique composition of rat urine and helps to explain the basis for the species specificity of the cytotoxic and proliferative effects of high doses of these sodium salts. PMID- 10753218 TI - DNA damage in human breast milk cells and its induction by 'early' and 'late' milk extracts. AB - Environmental and dietary factors are thought to be significant in breast cancer aetiology. The alkaline single-cell gel electrophoresis ('Comet') assay was used to examine breast milk cells for DNA damage and to measure the activity of extracts of the milk in causing such damage. UK-resident women were recruited as donors (n = 16) and provided 'early' ( approximately 4 weeks post-partum) and/or 'late' ( approximately 4 months post-partum) milk samples. Cells (79-94% viable, trypan blue exclusion) were either examined immediately for DNA damage or were cultured for 1 week prior to treatment with a breast milk extract. DNA damage in the form of single-strand breaks was quantified as comet tail length (CTL). Cell preparations examined immediately exhibited interindividual variation in median CTL (range 2.0-40.0 microm) with or without the DNA repair inhibitors hydroxyurea (HU) and cytosine arabinoside (ara-C). DNA damage decreased following culture, suggesting either DNA repair or death of DNA-damaged cells. Some donors' breast milk extracts induced DNA damage in their cultured cells and increases in median CTL were significantly greater with HU/ara-C (range 4.0-72.5 microm) than without (range 2.5-27.5 microm). Genotoxicity occurred without cytotoxicity (81-97% viability after treatment). Comparisons between cells and extracts from 'early' and 'late' milk samples did not support the idea of a progressive clearance of genotoxins from mammary lipid during lactation. Donors whose untreated cells contained the most DNA damage tended to yield genotoxic breast milk extracts. Cells isolated from milk activated the rodent mammary carcinogens o-toluidine and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The relevance of genotoxic exposures to breast cancer initiation requires further investigation. PMID- 10753219 TI - Evidence for a selective loss of somatostatin receptor subtype expression in male germ cell tumors of seminoma type. AB - Somatostatin (SRIF) is a potent antiproliferative signal for both normal and tumoral mammalian cells and an alteration in the SRIF receptor expression pattern has been associated with carcinogenesis. In the present study, the relevance of SRIF signaling to human male germ cell tumors was assessed at the receptor level. The expression of five SRIF receptor (sst1-sst5) mRNAs was estimated by RT-PCR and compared between normal and tumoral testes. All 12 normal testicular tissues studied contained sst3 and sst5 receptor transcripts whereas sst4 was present in almost all (11 of 12). sst1 transcripts were consistently absent while the majority (11/12) of normal samples studied did not contain sst2 mRNA. Parallel assessment of SRIF receptor mRNAs in 10 seminoma testicular germ cell tumors showed expression of a single receptor type, sst5, in all samples analyzed. All seminoma samples were depleted in transcripts corresponding to sst1 and sst2 receptors while either sst3 or sst4 mRNAs were absent in almost all (9 of 10) tumoral samples studied. The comparison of SRIF receptor expression between normal tissue and seminoma tumors thus points to a selective loss of sst3 and sst4 mRNA expression in seminomas. Altogether these data indicate that: (i) normal human testes are putative SRIF targets; (ii) loss of sst3 and sst4 SRIF receptor expression might be associated with seminoma carcinogenesis. PMID- 10753220 TI - Inhibitory effect of a flavonoid antioxidant silymarin on benzoyl peroxide induced tumor promotion, oxidative stress and inflammatory responses in SENCAR mouse skin. AB - In this communication, we investigate the preventive effect of a flavonoid antioxidant, silymarin, on free radical-generating skin tumor promoting agent benzoyl peroxide (BPO)-induced tumor promotion, oxidative stress and inflammatory responses in SENCAR mouse skin. Topical application of silymarin at a dose of 6 mg prior to BPO resulted in a highly significant protection against BPO-induced tumor promotion in 7,12-dimethylbenz[a]anthracene-initiated SENCAR mouse skin. The preventive effect of silymarin was evident in terms of a 70% reduction (P < 0.001) in tumor incidence, a 67% reduction (P < 0.001) in tumor multiplicity and a 44% decrease (P < 0.001) in tumor volume/tumor. In oxidative stress studies, topical application of BPO resulted in 75, 87 and 61% depletion in superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX) activities in mouse epidermis, respectively. These decreases in antioxidant enzyme activities were significantly (P < 0.005-0.001) reversed by pre-application of silymarin in a dose-dependent manner. The observed effects of silymarin were 18-66, 32-72 and 20 67% protection against BPO-induced depletion of SOD, catalase and GPX activity in mouse epidermis, respectively. Silymarin pre-treatment also resulted in a dose dependent inhibition (35-87%, P < 0.05-0. 001) of BPO-induced lipid peroxidation in mouse epidermis. In inflammatory response studies, silymarin showed a strong inhibition of BPO-induced skin edema (62-85% inhibition, P < 0.001), myeloperoxidase activity (42-100% inhibition, P < 0.001) and interleukin-1alpha protein level in epidermis (36-81% inhibition, P < 0.001). These results, together with our other recent studies, suggest that silymarin could be useful in preventing a wide range of carcinogen and tumor promoter-induced cancers. PMID- 10753221 TI - Characterization of the murine p19(ARF) promoter CpG island and its methylation pattern in primary lymphomas. AB - The INK4a/ARF locus encodes two different proteins involved in cell cycle control. Both molecules, p16(INK4a) and p19(ARF), inhibit cell cycle progression and have been shown to act as tumor suppressors in a variety of models. Their expression is controlled by separate promoters responding to different stimuli and they therefore show independent transcriptional regulation. We have cloned and characterized a 2.5 kb region upstream of the murine p19(ARF) gene to determine the role of DNA methylation in suppressing p19(ARF) transcription in a wide panel of murine primary T cell lymphomas. This region contains a DNA fragment with the characteristics of a CpG island similar to those described for the murine p16(INK4a) and p15(INK4b) genes. Expression of p19(ARF) is decreased in a significant number (20%) of the murine lymphomas analyzed. Overexpression of the p19(ARF) transcript is also frequent, suggesting alterations in molecules of the retinoblastoma or p53 pathways that are involved in p19(ARF) regulation. Although hypermethylation of the INK4a and INK4b promoters is frequently involved in murine lymphomas, the p19(ARF) CpG island is infrequently methylated in the murine primary lymphomas studied in this work. Since loss of p19(ARF) expression cannot be explained as the result of homozygous deletions or hypermethylation of the ARF gene, other regulatory mechanisms seem to be altered in these malignancies. PMID- 10753222 TI - Peroxisome proliferator-activated receptor alpha is restricted to hepatic parenchymal cells, not Kupffer cells: implications for the mechanism of action of peroxisome proliferators in hepatocarcinogenesis. AB - Peroxisome proliferators increase hepatocyte proliferation and cause liver tumors in rodents, yet the mechanism of action is not understood. Based on studies with null mice it is known that peroxisome proliferator-activated receptor-alpha (PPARalpha) is involved. There is also evidence that Kupffer cells play a central role in peroxisome proliferator-induced carcinogenesis, most likely via mechanisms involving increases in superoxide, activation of nuclear factor kappaB and production of tumor necrosis factor-alpha (TNFalpha). However, it is not known whether PPARalpha is constitutively expressed in Kupffer cells. Therefore, the expression of PPAR isoforms in rat Kupffer and parenchymal cells was examined. Kupffer cells and hepatocytes of >99% purity were isolated from rats fed either a control diet or one containing 0.1% WY-14,643 for 1 week. Protein and RNA were obtained and PPAR expression was analyzed using northern and western blots. PPARalpha, PPARbeta and PPARgamma mRNA was detected in purified hepatocytes. In Kupffer cells, mRNA encoding PPARgamma was present while transcripts for PPARalpha and PPARbeta were not detected. Immunoblots were consistent with the results found by northern analysis. Moreover, when Kupffer cells from wild-type or PPARalpha-null mice were treated with WY-14,643 in vitro, superoxide production was similar. Combined, these results show that PPARalpha is expressed in rat parenchymal cells but not in Kupffer cells. These data are consistent with the hypothesis that parenchymal cells respond to Kupffer cell derived TNFalpha via mechanisms dependent on PPARalpha within the parenchymal cells. PMID- 10753223 TI - Inhibition of N-methyl-N-nitrosourea- and 7,12-dimethylbenz[a] anthracene-induced rat mammary tumorigenesis by dietary cholesterol is independent of Ha-Ras mutations. AB - Dietary cholesterol has previously been shown to inhibit rat mammary tumorigenesis but the mechanisms remain unclear. Uptake of serum low density lipoprotein cholesterol by tissues leads to down-regulation of 3-hydroxy-3 methylglutaryl-CoA (HMG-CoA) reductase, the rate limiting enzyme in cholesterol biosynthesis that catalyzes the formation of mevalonate. In addition to being a precursor of cholesterol, mevalonate is necessary for DNA synthesis and cell proliferation. Isoprenoids, also derived from mevalonate, are required for the post-translational modification of Ras proteins that are mutated in a number of carcinogen-induced rat mammary tumors. The purpose of this study, therefore, was to determine whether inhibition of tumorigenesis by cholesterol is dependent on the frequency of mutations in the Ha-ras gene. Female Sprague-Dawley rats (30/group) were given a single dose of either N-methyl-N-nitrosourea (MNU, 50 mg/kg i.p.) or 7, 12-dimethylbenz[a]anthracene (DMBA, 100 mg/kg intragastrally), carcinogens that produce tumors with either a high (MNU) or low (DMBA) frequency of Ha-ras mutations in codon 12 or 61, respectively. Rats were fed either a control AIN-93G diet or the control diet supplemented with 0.3% cholesterol for 14 weeks. Dietary cholesterol significantly decreased the final tumor incidence in rats given DMBA (83 versus 100%, P < 0.05) or MNU (53 versus 77%, P < 0.05). HMG-CoA reductase activity was higher in mammary tumors than in normal mammary glands, but the activity of this enzyme was reduced by cholesterol feeding only in mammary glands and not in tumors. Tumors induced by MNU had a high frequency of Ha-ras mutations in both the control (65%) and cholesterol-fed (68%) groups. Tumors induced by DMBA had a low frequency of Ha-ras mutations that also did not differ between the control (21%) and cholesterol-fed (18%) groups. These findings show that dietary cholesterol inhibits mammary tumorigenesis induced by either MNU or DMBA and that the inhibition is independent of the type or extent of mutations in the Ha-ras gene. PMID- 10753225 TI - Enantiomeric composition of N'-nitrosonornicotine and N'-nitrosoanatabine in tobacco. AB - The tobacco-specific nitrosamines N'-nitrosonornicotine (NNN) and N' nitrosoanatabine (NAT) are found in substantial quantities in unburned tobacco. Although this has been documented in many previous studies, no data are available on the enantiomeric composition of these nitrosamines, which both have a chiral center at their 2'-positions. We used chiral stationary phase gas chromatography with nitrosamine-selective detection to determine the enantiomeric composition of NNN and NAT in moist snuff, chewing tobacco, and cigarette tobacco. (S)-NNN comprised 75.0 +/- 8.83% (SD) (n = 12) of total NNN while (S)-NAT comprised 82.6 +/- 1.44% (n = 12) of total NAT. Levels of the (S)-enantiomers of NNN and NAT were generally similar to those of the corresponding secondary amines, nornicotine and anatabine, suggesting a precursor to product relationship. Nitrosation of (S)-nicotine at pH 7.0 produced >99% (S)-NNN. These results suggest that nornicotine is a significant precursor of NNN in tobacco. The results of this study provide new insights into the structures and precursors of tobacco-specific nitrosamines in tobacco products. PMID- 10753224 TI - Heterozygous DNA mismatch repair gene PMS2-knockout mice are susceptible to intestinal tumor induction with N-methyl-N-nitrosourea. AB - PMS2-deficient (PMS2(-/-)) mice are hypersensitive to N-methyl-N-nitrosourea (MNU)-induced thymic lymphomas based on the failure to initiate mismatch repair (MMR) at O(6)-methylguanine:T mismatches formed after MNU exposure. However, heterozygous PMS2 knockout (PMS2(+/-)) mice do not develop spontaneous tumors, suggesting that they have sufficient MMR function to prevent genomic instability. We hypothesized that in PMS2(+/-) mice, exogenous carcinogens may either mutationally knockout the remaining normal allele leading cells to develop tumors or introduce sufficient DNA adducts and mismatches to overload the lower capacity for MMR, leading in either case to an increased rate of tumor production. In the present study, PMS2(+/-) mice and their littermate PMS2(+/+) mice were monitored for tumor incidence following MNU treatment. Mice were given 50 mg MNU/kg i.p. when 5 weeks old. They demonstrated a similar incidence of thymic lymphomas, suggesting that expression of the single normal PMS2 allele is sufficient to protect the thymus and implying that a single dose of MNU may not efficiently knock out the remaining PMS2 allele in the thymus. Surprisingly, PMS2(+/-) mice were significantly more likely to develop intestinal tumors-both adenomas and adenocarcinomas-after MNU than were PMS2(+/+) mice (2.34 +/- 0.34 tumors per mouse versus 1.34 +/- 0.25 tumors per mouse; P < 0.05). The intestinal tumors were located mainly in the small intestine. However, these tumors in both the PMS2(+/-) mice and PMS2(+/+) mice did not show microsatellite instability characteristic of loss of MMR. These results suggest that a single normal PMS2 allele can protect thymus but not intestine from MNU carcinogenesis. Organ specific factors might influence MMR- mediated resistance to methylating agents. Heterozygous PMS2 knockout mice may be used as a promising animal model for intestinal tumorigenesis studies involving environmental carcinogens. PMID- 10753226 TI - Lack of evidence for tamoxifen- and toremifene-DNA adducts in lymphocytes of treated patients. AB - Tamoxifen (TAM) is used for the adjuvant treatment of women with breast cancer and has also been recommended as a chemopreventive agent. Among unwanted side effects, TAM was shown to increase endometrial cancer in treated women by mechanisms that are not yet clearly understood. We studied DNA adducts in lymphocytes of female breast cancer patients treated with TAM or toremifene (TOR), a TAM analogue and compared them with adducts formed by TAM in rat liver, where the drug induces tumours. DNA adducts were measured by TLC-(32)P-post labelling assays. After TLC, all DNA samples including DNA from untreated healthy women showed a faint radioactive zone, where the positive control DNA adducts isolated from the liver of rats treated with TAM migrated. The relative adduct levels were calculated from the radioactivity present in this zone. Means +/- SD of adduct levels per 10(8) nucleotides (associated with this area) were for untreated volunteers (control) 1.83 +/- 1.41 (n = 13), for TAM treatment 2.17 +/- 3.04 (n = 25) and for TOR treatment 1.18 +/- 1.05 (n = 8). Most of the human samples were further analysed by HPLC after labelling with (32)P in order to compare adducts in human DNA with those in liver DNA isolated from TAM-treated rats. None of the human samples showed any peaks at retention times where putative TAM-DNA adducts were eluted. In conclusion, lymphocyte DNA from female patients treated at therapeutic levels did not show evidence of the formation of TAM- or TOR-DNA adducts. PMID- 10753227 TI - Re: Hemminki,K., Dickey,C., Karlsson,S., Bell,D., Hsu,Y., Tsai,W.-Y., Mooney,L.A., Savela,K. and Perera,F.P. (1997) Aromatic DNA adducts in foundry workers in relation to exposure, lifestyle and CYP1A1 and glutathione transferase M1 genotype. Carcinogenesis, 18, 345-350. PMID- 10753228 TI - Absolute configuration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol formed metabolically from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. PMID- 10753229 TI - Cooperative coevolution: an architecture for evolving coadapted subcomponents. AB - To successfully apply evolutionary algorithms to the solution of increasingly complex problems, we must develop effective techniques for evolving solutions in the form of interacting coadapted subcomponents. One of the major difficulties is finding computational extensions to our current evolutionary paradigms that will enable such subcomponents to "emerge" rather than being hand designed. In this paper, we describe an architecture for evolving such subcomponents as a collection of cooperating species. Given a simple string-matching task, we show that evolutionary pressure to increase the overall fitness of the ecosystem can provide the needed stimulus for the emergence of an appropriate number of interdependent subcomponents that cover multiple niches, evolve to an appropriate level of generality, and adapt as the number and roles of their fellow subcomponents change over time. We then explore these issues within the context of a more complicated domain through a case study involving the evolution of artificial neural networks. PMID- 10753230 TI - Information characteristics and the structure of landscapes. AB - Various techniques for statistical analysis of the structure of fitness landscapes have been proposed. An important feature of these techniques is that they study the ruggedness of landscapes by measuring their correlation characteristics. This paper proposes a new information analysis of fitness landscapes. The underlying idea is to consider a fitness landscape as an ensemble of objects that are related to the fitness of neighboring points. Three information characteristics of the ensemble are defined and studied. They are termed: information content, partial information content, and information stability. The information characteristics of a range of landscapes with known correlation features are analyzed in an attempt to reveal the advantages of the information analysis. We show that the proposed analysis is an appropriate tool for investigating the structure of fitness landscapes. PMID- 10753231 TI - Fitness landscapes, memetic algorithms, and greedy operators for graph bipartitioning. AB - The fitness landscape of the graph bipartitioning problem is investigated by performing a search space analysis for several types of graphs. The analysis shows that the structure of the search space is significantly different for the types of instances studied. Moreover, with increasing epistasis, the amount of gene interactions in the representation of a solution in an evolutionary algorithm, the number of local minima for one type of instance decreases and, thus, the search becomes easier. We suggest that other characteristics besides high epistasis might have greater influence on the hardness of a problem. To understand these characteristics, the notion of a dependency graph describing gene interactions is introduced. In particular, the local structure and the regularity of the dependency graph seems to be important for the performance of an algorithm, and in fact, algorithms that exploit these properties perform significantly better than others which do not. It will be shown that a simple hybrid multi-start local search exploiting locality in the structure of the graphs is able to find optimum or near optimum solutions very quickly. However, if the problem size increases or the graphs become unstructured, a memetic algorithm (a genetic algorithm incorporating local search) is shown to be much more effective. PMID- 10753232 TI - Variable length representation in evolutionary electronics. AB - This work investigates the application of variable length representation (VLR) evolutionary algorithms (EAs) in the field of Evolutionary Electronics. We propose a number of VLR methodologies that can cope with the main issues of variable length evolutionary systems. These issues include the search for efficient ways of sampling a genome space with varying dimensionalities, the task of balancing accuracy and parsimony of the solutions, and the manipulation of non coding segments. We compare the performance of three proposed VLR approaches to sample the genome space: Increasing Length Genotypes, Oscillating Length Genotypes, and Uniformly Distributed Initial Population strategies. The advantages of reusing genetic material to replace non-coding segments are also emphasized in this work. It is shown, through examples in both analog and digital electronics, that the variable length genotype's representation is natural to this particular domain of application. A brief discussion on biological genome evolution is also provided. PMID- 10753233 TI - ERRATA: Crossover Accelerates Evolution in GAs with a Babel-like Fitness Landscape: Mathematical Analysis. AB - Corrections to Figure 6 and Figure 8 from their article in Evol Comput 7:3 PMID- 10753234 TI - Editorial Introduction. AB - There is exciting news! The International Society for Genetic Algorithms (ISGA) and Genetic Programming, Inc. have joined efforts, recombined, and organized a new society: The International Society for Genetic and Evolutionary Computation (ISGEC). This follows the merger of the International Conference on Genetic Algorithms and the Genetic Programming Conference into a single conference: The Genetic and Evolutionary Computation Conference (GECCO). This is exciting because the new society will have dues that include a subscription to Evolutionary Computation as well as the new journal Genetic Programming and Evolvable Machines. This will help to link Evolutionary Computation more directly to the community that founded it and to associate the journal with a large body of researchers and application developers that would appear to be its natural readership. The journal Evolutionary Computation was founded as a grassroots effort and with a spirit of cooperation that embraced all aspects of the field. The new society builds on this theme. PMID- 10753235 TI - Antithrombotic therapy in pediatrics: time for higher levels of evidence. PMID- 10753236 TI - Disparate outcomes for very low birth weight infants: genetics, environment, or both? PMID- 10753237 TI - Aerosol treatment of acute asthma: and the winner is... PMID- 10753238 TI - Cerebral syncope: new insights into an emerging entity. PMID- 10753239 TI - Brain damage in preterm newborns: biological response modification as a strategy to reduce disabilities. AB - Substances that promote the growth and maturation of oligodendrocytes and their precursors might protect against white matter injury. We suggest that neuroprotection can also be provided by such modulators of fetal and neonatal inflammatory responses as antiinflammatory cytokines, cytokine-binding proteins, and cytokine-receptor blockers. We briefly describe inflammatory responses in the fetus and newborn and show how they might contribute to brain damage. We conclude with the possibility that so-called biological response modifiers, which are drugs that modulate these inflammatory responses, might reduce the risk of brain damage and disabilities. PMID- 10753240 TI - The use of low molecular weight heparin in pediatric patients: a prospective cohort study. AB - OBJECTIVE: Low molecular weight heparins (LMWHs) offer several advantages over standard anticoagulant therapy (unfractionated heparin/warfarin) including predictable pharmacokinetics, minimal monitoring, and subcutaneous administration. Our objective was to determine the safety and efficacy of LMWHs in children. METHODS: A prospective cohort of children treated with the LMWH enoxaparin (Rhone Poulenc Rorer) was monitored at the Hospital for Sick Children, Toronto, Canada, from March 1994 until July 1997. RESULTS: There were 146 courses of LMWH administered for treatment and 31 courses for prophylaxis of thromboembolic events (TEs). Clinical resolution of TEs occurred in 94% of children receiving therapeutic doses of LMWH, and 96% of children receiving prophylactic doses of LMWH had no symptoms of recurrent or new TEs. Major bleeding occurred in 5% of children receiving therapeutic doses. Recurrent or new TEs occurred in 1% and 3% of children receiving therapeutic and prophylactic doses of LMWH, respectively. CONCLUSION: LMWH appears to be efficacious and safe for both management and prophylaxis of TEs. The results of this cohort study justify a randomized controlled trial comparing LMWH with standard therapy for the management of TEs in children. PMID- 10753242 TI - Racial differences in respiratory-related neonatal mortality among very low birth weight infants. AB - OBJECTIVE: To examine racial differences in the secular trends in respiratory related neonatal mortality among very low birth weight (VLBW) infants in the United States, temporally associated with surfactant availability. DESIGN: Comparison of time trends in African American and non-Hispanic white (NHW) VLBW infants of cause-specific neonatal mortality and neonatal and infant mortality for 2 consecutive 3-year periods. RESULTS: From 1985 to 1988 there was no racial difference in the rate of decline of each mortality outcome. From 1988 to 1991 rates of decline in neonatal mortality caused by respiratory distress syndrome and by all respiratory causes were significantly greater for NHWs compared with African Americans. However, the rate of decline in nonrespiratory neonatal mortality was similar for African Americans and NHWs. Compared with African American VLBW infants, NHWs had a greater rate of decline in both neonatal (31% vs 20%; P <.01) and infant mortality (32% vs 21%; P <.01) during this period. CONCLUSIONS: Between 1988 and 1991, declines in neonatal mortality risks caused by respiratory distress syndrome and all respiratory causes were greater for NHW infants than for African American VLBW infants. The decline in nonrespiratory mortality risk showed no racial differences. These findings suggest possible racial disparities in timely access or racial differences in the efficacy of respiratory treatments for VLBW infants. PMID- 10753241 TI - Combined thrombolytic and anticoagulant therapy for venous thrombosis in children. AB - OBJECTIVES: To evaluate safety, efficacy, and outcome after combination thrombolytic and anticoagulant therapy. STUDY DESIGN: An open nonrandomized clinical protocol with prospective standardized monitoring and data collection. Children with a documented first episode of deep vein thrombosis were treated with urokinase 4400 U/kg load and per hour with unfractionated heparin at 10 U/kg/h. At 48 hours heparin infusions were increased to achieve a therapeutic level for 5 days. Children were given therapeutic warfarin for at least 3 months. Outcome was assessed at 48 hours and > or =1 year with history, physical examination, high-resolution imaging, and Doppler ultrasonography +/- impedance and photo plethysmography. RESULTS: Thirty-two children were treated. There was 1 thrombotic death, 1 nonfatal thrombus progression, and 1 pulmonary embolism. At 48 hours half of the children showed substantial clot lysis, and on follow-up these children had complete resolution and had no symptoms. Three children with poor early clot lysis had recurrent thromboemboli, pulmonary embolism, or both, 2 had limb pain and swelling, and 2 had asymptomatic swelling. Two children had minor bleeding, whereas systemic reactions were common. CONCLUSIONS: Combination therapy in children (urokinase and unfractionated heparin) was safe and efficacious. A prospective, randomized, controlled study in children is needed. PMID- 10753243 TI - Hyper-releasability of mast cells in family members of infants with sudden infant death syndrome and apparent life-threatening events. AB - BACKGROUND: An allergic reaction with increased degranulation of mast cells has been suggested as a possible mechanism in sudden infant death syndrome (SIDS). STUDY DESIGN: Mast cell releasability was assessed in 2 study groups: A, 22 subjects, 16 first-degree relatives of infants who had had an apparent life threatening event (ALTE) and 6 infants after ALTE and B, 46 first-degree relatives of SIDS cases. These groups were compared with 71 members of an age matched control group. In each subject the skin wheal and flare reactions were measured after an intradermal injection of 0.02 mL of each of the following substances dissolved in phenol-saline solution: histamine 0.0001 mg/mL, histamine 0.001 mg/mL, codeine 0.5 mg/mL, codeine 1 mg/mL, compound 48/80 0.05 mg/mL, compound 48/80 0. 1 mg/mL, and phenol-saline solution. The size of wheal and flare skin reaction was assessed by computerized planimetry after the shape of the cutaneous response was copied onto a paper. RESULTS: The wheal and flare skin reaction to each of the substances was significantly larger in the 2 study groups compared with the control group (P <.05) except for the wheal reaction to compound 48/80 0.1 mg/mL, codeine 0.5 mg/mL, and histamine in both concentrations for group A and the wheal reaction to codeine 1 mg/mL and histamine in both concentrations for group B. All individuals with increased reaction belonged to 3 (50%) of 6 families with ALTE history and to 8 (73%) of 11 families with SIDS history. CONCLUSIONS: Increased mast cell hyper-releasability and degranulation take place in family members of some SIDS and ALTE cases and in some infants with ALTE, supporting a possible role for an immunologic mechanism in the pathophysiology of these entities. PMID- 10753244 TI - Circulating neutrophil concentration and respiratory distress in premature infants. AB - BACKGROUND: The acute disappearance of neutrophils from the circulation can be associated with pulmonary leukostasis, lung injury, and respiratory distress. OBJECTIVE: To determine whether a low concentration of mature neutrophils in the peripheral blood soon after birth is associated with an increase in subsequent respiratory distress in premature infants. DESIGN: A cohort study performed by chart review at a tertiary medical center. SUBJECTS: Premature infants (birth weight 500 to 1250 g) who had a complete blood count obtained within 2 hours of delivery (n = 237). Patients in the lowest quartile of mature neutrophil concentrations (early neutropenia, < or =0.90 x 10(9) neutrophils/L blood) were compared with patients in the remaining 3 quartiles (control group). RESULTS: Low neutrophil concentrations were transient in the early neutropenia group. The concentration of mature circulating neutrophils rose from 0.49 +/- 0.25 x 10(9) cells/L at an average of 1 hour after delivery to 2.8 +/- 2.2 x 10(9) cells/L within 6 to 8 hours in the early neutropenia group and from 4.6 +/- 4.8 x 10(9) cells/L to 8.2 +/- 8. 0 x 10(9) cells/L in the control group during the same time period. Respiratory support immediately after birth was similar in both groups of infants, but by 12 hours patients who had early neutropenia required significantly greater inflation pressures and concentrations of inspired oxygen. By 1 week after birth patients who had early neutropenia were more likely to require mechanical ventilation and supplemental oxygen. Pulmonary interstitial emphysema, serious intraventricular hemorrhage, and chronic lung disease occurred more frequently in patients with early neutropenia. CONCLUSION: A low concentration of mature neutrophils in the systemic circulation of premature infants within 2 hours of birth is associated with more severe respiratory distress during the first postnatal week and with an increased risk of serious complications of prematurity. PMID- 10753245 TI - The effect of selenium supplementation on outcome in very low birth weight infants: a randomized controlled trial. The New Zealand Neonatal Study Group. AB - BACKGROUND: Low selenium (SE) status has been documented in preterm infants and has been suggested to be a risk factor for chronic lung disease. METHODS: A total of 534 infants with birth weight <1500 g were enrolled in 8 New Zealand centers in a double-blind placebo-controlled randomized trial of SE supplementation from week 1 of life until 36 weeks' postmenstrual age or discharge home. Supplemented infants received 7 microg/kg/d of SE when fed parenterally and 5 microg/kg/d when fed orally. Plasma SE and glutathione peroxidase concentrations were measured in mothers after delivery and in infants before randomization and at 28 days and 36 weeks' postmenstrual age. Primary outcome measures were oxygen dependency at 28 days and total days oxygen dependency. RESULTS: No significant differences were seen between the groups with respect to primary or secondary outcome measures, with the exception that fewer supplemented infants had an episode of sepsis after the first week of life (P <.038). Mean plasma SE concentrations were 0.33 micromol/L before randomization in both groups and at 28 days had risen in the supplemented group (0.56 micromol/L) but fallen in the control group (0.29 micromol/L) (P <.0001). There was no association between outcome measures and SE concentrations at 28 days or 36 weeks' postmenstrual age. However, lower maternal and infant prerandomization SE concentrations were associated with increased respiratory morbidity. CONCLUSIONS: Postnatal SE supplementation in very low birth weight infants did not improve neonatal outcome. Further investigation of SE supplementation of mothers from the second half of pregnancy is warranted. PMID- 10753246 TI - Cost-effectiveness and implications of newborn screening for prolongation of QT interval for the prevention of sudden infant death syndrome. AB - OBJECTIVE: To determine the cost-effectiveness of universal and high-risk neonatal electrocardiographic (ECG) screening for QT prolongation as a predictor of sudden infant death syndrome (SIDS) risk in a theoretical group of neonates. STUDY DESIGN: Incremental cost-effectiveness analysis with decision analytic modeling. A hypothetical cohort of healthy, term infants was modeled, comparing options of no screening, high-risk neonate screening, and universal screening. The high-risk strategy is speculative, because no currently accepted methodology is known for identifying infants at high risk for SIDS. Given the uncertain mechanisms of association between prolonged corrected QT interval (QTc) and SIDS, analyses were repeated under different assumptions. Sensitivity analyses were also performed on all input variables for both costs and effectiveness. RESULTS: Under the assumption that neonatal electrocardiographic screening detects long QT syndrome responsive to conventional therapy, the cost-effectiveness of high-risk screening was $3403 per life year gained, whereas universal screening cost $18,465 per additional life year gained. However, if the effectiveness of SIDS therapy falls below 10%, the cost-effectiveness deteriorates to $28,376 per life year saved for the high-risk strategy and $118,900 for universal screening. The analyses were robust to a broad array of sensitivity analyses. CONCLUSIONS: The acceptability of the cost-effectiveness of neonatal electrocardiographic screening is heavily dependent on the pathophysiologic mechanism of SIDS and on the efficacy of monitoring and antiarrhythmic treatment. The nature of this association must be elucidated before routine neonatal electrocardiographic screening is warranted. PMID- 10753247 TI - Pubertal growth and development and prenatal and lactational exposure to polychlorinated biphenyls and dichlorodiphenyl dichloroethene. AB - OBJECTIVES: Polychlorinated biphenyls (PCBs) and dichlorodiphenyl dichloroethene (DDE) are ubiquitous toxic environmental contaminants. Prenatal and early life exposures affect pubertal events in experimental animals. We studied whether prenatal or lactational exposures to background levels of PCBs or DDE were associated with altered pubertal growth and development in humans. STUDY DESIGN: Follow-up of 594 children from an existing North Carolina cohort whose prenatal and lactational exposures had previously been measured. Height, weight, and stage of pubertal development were assessed through annual mail questionnaires. RESULTS: Height of boys at puberty increased with transplacental exposure to DDE, as did weight adjusted for height; adjusted means for those with the highest exposures (maternal concentration 4+ ppm fat) were 6.3 cm taller and 6.9 kg larger than those with the lowest (0 to 1 ppm). There was no effect on the ages at which pubertal stages were attained. Lactational exposures to DDE had no apparent effects; neither did transplacental or lactational exposure to PCBs. Girls with the highest transplacental PCB exposures were heavier for their heights than other girls by 5.4 kg, but differences were significant only if the analysis was restricted to white girls. CONCLUSIONS: Prenatal exposures at background levels may affect body size at puberty. PMID- 10753248 TI - Costs and effectiveness of spacer versus nebulizer in young children with moderate and severe acute asthma. AB - OBJECTIVE: To compare the costs and effectiveness of albuterol by metered dose inhaler (MDI) and spacer versus nebulizer in young children with moderate and severe acute asthma. DESIGN: Randomized, double-blind, placebo-controlled trial in an emergency department at a children's hospital. The participants were children 1 to 4 years of age with moderate to severe acute asthma. Patients assigned to the spacer group received albuterol (600 microg) by MDI by spacer (AeroChamber) followed by placebo by nebulizer (n = 30). The nebulizer group received placebo MDI by spacer followed by 2.5 mg albuterol by nebulizer (n = 30). Treatments were repeated at 20-minute intervals until the patient was judged to need no further doses of bronchodilator, or a total of 6 treatments. RESULTS: Clinical score, heart rate, respiratory rate, auscultatory findings, and oxygen saturation were recorded at baseline, after each treatment, and 60 minutes after the last treatment. Baseline characteristics and asthma severity were similar for the treatment groups. The spacer was as effective as the nebulizer for clinical score, respiratory rate, and oxygen saturation but produced a greater reduction in wheezing (P =.03). Heart rate increased to a greater degree in the nebulizer group (11.0/min vs 0.17/min for spacer, P <.01). Fewer children in the spacer group required admission (33% vs 60% in the nebulizer group, P =.04, adjusted for sex). No differences were seen in rates of tremor or hyperactivity. The mean cost of each emergency department presentation was NZ$825 for the spacer group and NZ$1282 for the nebulizer group (P =.03); 86% of children and 85% of parents preferred the spacer. CONCLUSION: The MDI and spacer combination was a cost effective alternative to a nebulizer in the delivery of albuterol to young children with moderate and severe acute asthma. PMID- 10753249 TI - Plant stanol ester margarine lowers serum total and low-density lipoprotein cholesterol concentrations of healthy children: the STRIP project. Special Turku Coronary Risk Factors Intervention Project. AB - OBJECTIVE: To investigate cholesterol-lowering efficacy and safety of plant stanol ester margarine in healthy 6-year-old children already consuming a low saturated-fat, low-cholesterol diet. STUDY DESIGN: Eighty-one intervention children from the STRIP project, a randomized prospective trial aimed at reducing exposure of young children to the known environmental atherosclerosis risk factors, were recruited to this double-blind crossover study at 6 years of age. In randomized order the families were advised to replace daily 20 g of the child's dietary fat intake with plant stanol ester margarine or control margarine for 3 months. The washout period lasted 6 weeks. Statistical analysis was performed according to intention-to-treat principle with analysis of variance for crossover design. RESULTS: The mean daily plant stanol ester margarine consumption was 18.2 g (1.5 g plant stanol). The well-tolerated plant stanol ester margarine reduced serum total and low-density lipoprotein cholesterol concentrations by 5.4% and 7.5%, respectively (P =.0001 for both). The serum high density lipoprotein cholesterol and triglyceride concentrations and alpha tocopherol to low-density lipoprotein cholesterol ratio remained unchanged. The serum beta-carotene to low-density lipoprotein cholesterol ratio decreased by 19% (P =.003). CONCLUSION: Plant stanol ester margarine significantly diminishes serum total and low-density lipoprotein cholesterol concentration without adverse clinical effects in healthy children who already consume a low-saturated-fat, low cholesterol diet but decreases the serum beta-carotene to low-density lipoprotein cholesterol ratio. PMID- 10753250 TI - Correlations of cardiovascular disease risk factors between African American siblings. AB - OBJECTIVES: This study examines intrasibling correlations at 2 points during childhood for African American siblings with the same father, different fathers, a father present in the home, and no father present in the home. STUDY DESIGN: Cardiovascular disease (CVD) risk factors were assessed in 267 pairs of African American siblings (visit 1) and in 79 of these siblings approximately 28 months later (visit 2). RESULTS: As a group, correlations of CVD risk factors between African American siblings with the same father were greater than those for African American siblings with different fathers in visit 1 (P <.05). However, having a father present in the home was associated with significantly lower intrasibling correlations for girth and total cholesterol in visit 2 (P <.005). Intrasibling correlations for the 4 family subgroups suggest that CVD risk factors were most similar in siblings who shared the same father but who had no father present in the home. CONCLUSIONS: Intrasibling correlations for African American children were influenced by whether they shared the same father and whether a father was present in their home, reflecting both genetic and environmental influences. Family composition should be considered when family CVD risk factors are used to predict CVD risk in children. PMID- 10753251 TI - Ambulatory blood pressure monitoring in children with aortic coarctation and kidney transplantation. AB - BACKGROUND: Ambulatory blood pressure monitoring (ABPM) has been found to be of significant importance in clinical practice because numerous blood pressure (BP) measurements may be made throughout the 24-hour period. OBJECTIVE: To assess the clinical utility of ABPM in children with secondary hypertension. METHODS: We studied 37 patients (21 boys and 16 girls), with a mean age of 16.4 +/- 4.1 years, after kidney transplantation and 38 patients (27 boys and 11 girls), with a mean age of 10.2 +/- 2.1 years, after surgical correction of aortic coarctation. Data, expressed as mean +/- SD, were analyzed after dividing the patients into 4 groups. Group A consisted of 25 patients receiving antihypertensive therapy; group B included 12 patients not receiving antihypertensive therapy. Group C included 25 patients operated on before 3 years of age (8 +/- 7 months of age); group D included 13 patients operated on after 3 years of age (74 +/- 29 months of age). RESULTS: In groups A and B, casual BP measurement showed that 16 of 37 patients (43%) were hypertensive; 24-hour ABPM detected a larger number of patients who were hypertensive (23 of 37, 62%); there were 18 in group A and 5 in group B. In groups C and D, casual BP measurement identified 6 of 38 (15%) patients as hypertensive, whereas 24-hour ABPM again identified a higher number (13 of 38, 34%). CONCLUSIONS: Our findings confirm that 24-hour ABPM is more sensitive than casual BP in detecting abnormal BP in patients at high risk for secondary hypertension. PMID- 10753252 TI - Absent circadian variation of blood pressure in patients with anorexia nervosa. AB - OBJECTIVE: To examine whether circadian rhythm of blood pressure (BP) is altered in patients with anorexia nervosa (AN), and if so, to determine whether it is reversible after refeeding. STUDY DESIGN: Ambulatory BP monitoring was performed on 17 female inpatients with AN (mean age, 13.3 +/- 1.9 years) at the time of admission and serially during refeeding; 17 age-matched normal weight, normotensive female inpatients served as control subjects. RESULTS: Patients with AN had lost an average of 23.4% +/- 11.5% of body weight before the illness. Weight after refeeding was 105.6% +/- 9. 2% of that before illness. Mean 24-hour systolic BP (SBP) (96.5 +/- 8.6 mm Hg) and diastolic BP (DBP) (53.4 +/- 5.8 mm Hg) were significantly lower in patients with AN compared with those of control subjects (SBP, 106.1 +/- 6.5 mm Hg; DBP, 60.2 +/- 5.8 mm Hg). Although awake SBP and DBP were also lower in patients with AN, asleep SBP and DBP were not statistically different from those of control subjects. Night/day BP ratio in the control group was 0.93 +/- 0.06 in systolic and 0.92 +/- 0.09 in diastolic. Those values were significantly elevated in patients with AN (systolic 1.00 +/- 0. 09 and diastolic 1.00 +/- 0.09). After refeeding, the ratio decreased to 0.88 +/- 0.09 and 0.90 +/- 0.08,respectively (both P <. 05 vs baseline). CONCLUSIONS: In patients with AN, circadian variation of BP is absent. This reverts to normal after refeeding. PMID- 10753253 TI - Rumination syndrome in adolescents. AB - OBJECTIVES: To evaluate the clinical presentation and to assess the usefulness of antroduodenal manometry (ADM) and the results of multidisciplinary team management in 12 neurologically normal adolescents (9 girls) with rumination. STUDY DESIGN: All patients had extensive investigations that ruled out other causes of their chronic symptoms. We performed ADM in all patients. A multidisciplinary approach was used for the nutritional and behavioral rehabilitation of these patients. RESULTS: The median age at presentation was 14 years (range, 9-19 years), and the average duration of symptoms was 17 months. All patients complained of postprandial, effortless regurgitation, and the majority had weight loss and abdominal pain. Results of fasting ADM were normal in all. The postprandial ADM showed brief, simultaneous pressure increases at all recording sites, associated with regurgitation in 8 patients. No emesis was observed in the other 4 children during the study. Treatment included nutritional support in combination with antidepressants and anxiolytics (n = 6), cognitive therapy with biofeedback or relaxation techniques (n = 7), and pain management (n = 2). Resolution or improvement of symptoms was seen in 10 of the 12 patients, and successful transition to oral feedings was achieved in all during the follow up period, which ranged from 5 to 36 months. CONCLUSIONS: Rumination is a distinct functional gastrointestinal disorder of otherwise healthy children and adolescents, which can be diagnosed on the basis of clinical features. The ADM shows a characteristic pattern and rules out motility disorders that are often confused with rumination. A multidisciplinary team approach is associated with satisfactory recovery in most patients. PMID- 10753254 TI - Telephoning the patient's pharmacy to assess adherence with asthma medications by measuring refill rate for prescriptions. AB - OBJECTIVE: To determine whether a prescription refill history obtained by telephoning patients' pharmacies identifies poor adherence with asthma medications more frequently than physician assessment. METHODS: The study population consisted of 116 children with persistent asthma who were Medicaid recipients; patients who received medication samples were excluded. During a clinic visit pulmonologists interviewed patients, caretakers, or both and estimated adherence on a checklist. A nurse asked the caretakers where they obtained medications and telephoned 66 identified pharmacies for refill histories. The maximum possible adherence was calculated as the number of doses refilled/number of doses prescribed x 100 for a mean duration of 163 days (range, 63 to 365 days). The accuracy of the refill information was determined from Medicaid reimbursement records. RESULTS: Information provided by pharmacies was 92% accurate. The mean (95% CI) of maximum potential adherence was 72% (65%,77%) for theophylline, 61% (55%,68%) for inhaled corticosteroids, and 38% (23%,53%) for cromolyn; only cromolyn and theophylline were significantly different. Physicians were able to identify 21 (49%) of 43 patients who refilled 0.55, P <.001). Responsiveness was demonstrated by means of a significant reduction in the score on the ulcer questionnaire as ulcers healed at 6 and 11 weeks (P <.05). CONCLUSION: Good evidence exists that a clinically derived measure for patients with venous ulcers has validity to measure the quality of life. PMID- 10753272 TI - Improving walking ability and ankle brachial pressure indices in symptomatic peripheral vascular disease with intermittent pneumatic foot compression: a prospective controlled study with one-year follow-up. AB - PURPOSE: Intermittent pneumatic foot compression (IPC(foot)) augments arterial leg inflow. It has been suggested that prolonged use of impulse leg compression at home might ameliorate claudication caused by peripheral vascular disease by improving collateral circulation. The purpose of this study was to determine the effect of IPC(foot) treatment on claudication distance and arterial hemodynamics in patients with intermittent claudication caused by peripheral vascular disease. METHODS: Thirty-seven patients with stable intermittent claudication were admitted to this prospective controlled study. Of these, 25 patients received IPC(foot) (>4 hr/d) for 4.5 months (group 1), and the other 12 patients acted as control patients (group 2). Both groups were advised to exercise unsupervised for a minimum of 1 hour daily and received aspirin (75 mg/d). Groups were matched for age, sex, risk factors, claudication distances, and ankle pressures at baseline. In each patient, initial claudication distance (ICD), absolute claudication distance (ACD), resting ankle brachial index (r-ABI), ankle brachial pressure index after exercise (p-eABI), and popliteal artery volume flow were measured at day 0, 2 weeks, and 1, 2, 3, and 4.5 months. On completion of the treatment period (4.5 months), both groups continued with aspirin (75 mg/d) and unsupervised exercise and were re-examined after 12 months. Data analysis is based on nonparametric statistics, the Wilcoxon signed ranks test, and the Mann Whitney test for intragroup and intergroup comparisons, respectively. Results are expressed as median and interquartile ranges. RESULTS: Over the 4.5 months of active treatment, (1) median ICD in group 1 increased by 146% (P <.001), from 78 m (interquartile range, 65-102 m) at baseline to 191.5 m (interquartile range, 127-254 m); ICD did not significantly increase in group 2; (2) median ACD in group 1 improved by 106% (P <.001), from 124 m (interquartile range, 100-160 m) to 255 m (interquartile range, 149-398 m); no significant changes were documented in group 2; (3) median r-ABI in group 1 rose by 18% (P <.001), from 0.57 (interquartile range, 0.48-0.62) to 0.67 (interquartile range, 0.64-0.70); no improvement was noted in group 2; (4) median p-eABI in group 1 rose by 110% (P <.001), from 0.21 (interquartile range, 0.07-0.27) to 0.44 (interquartile range, 0. 36-0.52); no changes were noted in group 2; and (5) median popliteal artery volume flow in group 1 improved by 36% (P <.001), from 100 mL/min (interquartile range, 59-163 mL/min) to 136 mL/min (interquartile range, 99.5-173.4 mL/min); no significant changes were found in group 2. At 4.5 months, ICD, ACD, r-ABI, and p eABI in group 1 were all significantly better than those in group 2 (P <.01). Twelve months' posttreatment, walking ability and ABIs in group 1 were not statistically different from those at 4.5 months and remained significantly better than those of control subjects. CONCLUSION: Intermittent pneumatic foot compression used at home for 4.5 months increases claudication distance by over 100%. Associated increases in r-ABI by 18%, p-eABI by 110%, and arterial calf inflow by 36% suggest an improved collateral circulation. Maximum benefit seems to be offered over the initial 3 months. Treatment benefits are maintained 1 year after treatment. A multicenter study is indicated to quantify actual benefits and to demonstrate cost effectiveness. PMID- 10753273 TI - Influence of upper- and lower-limb exercise training on cardiovascular function and walking distances in patients with intermittent claudication. AB - PURPOSE: The effects of upper-limb (arm cranking) and lower-limb (leg cranking) exercise training on walking distances in patients with intermittent claudication was assessed. METHODS: Sixty-seven patients (33 to 82 years old) with moderate to severe intermittent claudication were recruited, and the maximum power generated during incremental upper- and lower-limb ergometry tests was determined, as were pain-free and maximum walking distances (by using a shuttle walk test). Patients were randomly assigned to an upper-limb training group (n = 26) or a lower-limb training group (n = 26). An additional untrained group (n = 15) was recruited on an ad hoc basis in parallel with the main trial by using identical inclusion criteria. This group was subsequently shown to possess a similar demographic distribution to the two exercise groups. Supervised training sessions were held twice weekly for 6 weeks. RESULTS: Both training programs significantly improved the maximum power generated during the incremental upper- and lower-limb ergometry tests (P <. 001), which may reflect an increase in central cardiovascular function that was independent of the training mode. More importantly, pain-free and maximum walking distances also improved in both training groups (P <.001). The improvements in the training groups were similar; there were no changes in the untrained control group. These findings suggest that the symptomatic improvement after upper-limb exercise training may result, in part, from systemic cardiovascular effects rather than localized metabolic or hemodynamic changes. CONCLUSION: Carefully prescribed upper-limb exercise training can evoke a rapid symptomatic improvement in patients with claudication, while avoiding the physical discomfort experienced when performing lower-limb weight-bearing exercise. PMID- 10753274 TI - Effects of exercise rehabilitation on cardiovascular risk factors in older patients with peripheral arterial occlusive disease. AB - OBJECTIVE: The purpose of this study was to determine whether a 6-month exercise rehabilitation program can improve cardiovascular risk factors in patients with peripheral arterial occlusive disease (PAOD). METHODS: Thirty-four patients (mean age, 68 +/- 8 years; range 54-84 years) with PAOD with intermittent claudication (Fontaine stage II) and 14 longitudinal controls of comparable age with stage II PAOD enrolled in an exercise intervention at the University Medical Center and Veterans Affairs Medical Center at Baltimore, Maryland. The main outcome measures were lipid profile (total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol [LDL-C]), fasting glucose, blood pressure, body mass index, treadmill times to onset and maximal claudication pain, cardiopulmonary function (peak oxygen uptake), and ankle/brachial index. RESULTS: With exercise rehabilitation, treadmill times to onset and maximal claudication pain increased by 106% and 64% (P <.0001), whereas peak oxygen uptake increased 7% (P <.05). Exercise rehabilitation lowered total cholesterol and LDL-C levels by 5.2% (P <.005) and 8% (P <.01), respectively. Systolic blood pressure declined by 5.7% (P <.05) with no change in diastolic blood pressure. These changes in cholesterol and LDL-C concentrations were related to their initial values. All other cardiovascular risk factors measured did not change. There was no correlation between improvement of cardiovascular risk factors and functional performance measurements. None of the variables measured changed significantly in the control group. CONCLUSIONS: Exercise rehabilitation not only improves functional performance, but also results in favorable alterations in cardiovascular risk factor profile, which is an important element in the management of PAOD. PMID- 10753275 TI - Intraoperative duplex monitoring of infrainguinal vein bypass procedures. AB - PURPOSE: The purpose of this study was to evaluate intraoperative duplex scanning of infrainguinal vein bypass grafts to detect technical and hemodynamic problems, monitor their repair, and correlate findings with the incidence of thrombosis and stenosis repair rates within 90 days of operation. METHODS: Color duplex scanning was used at operation to assess vein/anastomotic patency and velocity spectra waveforms of 626 infrainguinal vein bypass grafts (in situ saphenous, 228 grafts; nonreversed translocated saphenous, 170 grafts; reversed saphenous, 147 grafts; alternative [arm, lesser saphenous], 81 grafts) to the popliteal (n = 267 grafts), infrageniculate (n = 323 grafts), or pedal artery (n = 36 grafts). The entire bypass graft was scanned after intragraft injection of papaverine hydrochloride (30-60 mg) to augment graft flow. Vein/anastomotic/artery segments with velocity spectra that indicate highly disturbed flow (peak systolic velocity, >180 cm/sec; spectral broadening; velocity ratio at site, >3) were revised. Grafts with a low peak systolic velocity less than 30 to 40 cm/s and high outflow resistance (absent diastolic flow) underwent procedures (distal arteriovenous fistula, sequential bypass grafting) to augment flow; if this was not possible, the grafts were treated with an antithrombotic regimen, including heparin, dextran, and antiplatelet therapy. RESULTS: Duplex scanning prompted revision of 104 lesions in 96 (15%) bypass grafts, including 82 vein/anastomotic stenoses, 17 vein segments with platelet thrombus, and 5 low-flow grafts. Revision rate was highest (P <.01) for alternative vein bypass grafts (27%) compared with the other grafting methods (reversed vein bypass grafts, 10%; nonreversed translocated, 13%; in situ, 16%). A normal intraoperative scan on initial imaging (n = 464 scans) or after revision (n = 67 scans) was associated with a 30-day thrombosis rate of 0.2% and a revision rate of 0.8% for duplex detected stenosis (peak systolic velocity, >300 cm/s; velocity ratio, >3.5). By comparison, 20 of 95 bypass grafts (21%) with a residual (n = 29 grafts) or unrepaired duplex stenosis (n = 53 grafts) or low flow (n = 13 grafts) had a corrective procedure for graft thrombosis (n = eight grafts) or stenosis (n = 12 grafts; P <.001). Overall, 8% of patients with bypass grafts underwent a corrective procedure within 90 days of operation. Secondary graft patency was 99.4% at 30 days and 98.8% at 90 days (eight graft failures). CONCLUSION: The observed 15% intraoperative revision rate coupled with a low 90-day failure/revision rate (2.5%) for bypasses with normal papaverine-augmented duplex scans supports the routine use of this diagnostic modality to enhance the precision and early results of infrainguinal vein bypass procedures. PMID- 10753276 TI - Hemorheologic variables in critical limb ischemia before and after infrainguinal reconstruction. AB - PURPOSE: Plasma and whole blood viscosity are elevated in patients with intermittent claudication. The objectives of this study were to investigate whether critical limb ischemia influences hemorheologic variables and whether the rheologic variables in blood from the affected limb differ from the general circulation. We also intended to study whether successful infrainguinal reconstruction improved hemorheologic variables. METHODS: Ten consecutive patients with critical limb ischemia (CLI) underwent arterial reconstruction, one patient with profundaplasty and nine patients with bypass procedures. Venous blood was sampled from the antecubital vein (arm) and the femoral vein (leg) of the affected limb 1 day before and 1 month after surgery. Ten control subjects (matched according to age, sex, diabetic status, and renal insufficiency) were also sampled. Whole blood viscosity, plasma viscosity, erythrocyte aggregation tendency, and erythrocyte fluidity (the latter variable describing the deformability of the erythrocytes) were measured by means of rotational viscometry. Erythrocyte volume fraction was also determined. Fibrinogen was measured in the patients with CLI. RESULTS: Erythrocyte fluidity, blood viscosity, and erythrocyte volume fraction were lower in patients with CLI than in control subjects (P <.01, P <.01, and P <.05, respectively). No major differences between cubital and femoral vein blood were seen before or after the operation in patients with CLI or in control subjects. Successful revascularization did not influence the hemorheologic variables, except for a decrease in blood viscosity in femoral vein blood (P <.05). CONCLUSION: Hemorheologic properties was impaired in patients with CLI. Because no differences were seen between the systemic and local circulation and because no major improvement occurred 1 month after arterial reconstruction, other mechanisms besides local tissue ischemia may play a role. PMID- 10753277 TI - Adjunctive techniques to improve patency of distal prosthetic bypass grafts: polytetrafluoroethylene with remote arteriovenous fistulae versus vein cuffs. AB - PURPOSE: The long-term patency for infrapopliteal bypass grafting with prosthetic material is less than optimal. Our experience demonstrates a 40% patency at 2 years for these grafts. Several adjuvant techniques have been developed to improve patency rates, two of which are a remote distal arteriovenous fistula and the creation of a distal vein cuff. This study summarizes our experience with these two techniques. METHODS: Between 1987 and 1998, 107 bypass graftings were performed to the below-knee popliteal or tibial vessels with the use of polytetrafluoroethylene. One group (48 bypass grafts) had polytetrafluoroethylene with adjuvant distal arteriovenous fistula (DAVF), and a second group (59 bypass grafts) was reconstructed with a distal vein cuff (DVC). The type of bypass grafting that was performed was based on surgeon experience and preference. Indications and demographics were similar in the two groups. All patients underwent the operation for limb-threatening ischemia, including gangrene (DAVF, 23%; DVC, 9%), ulceration (DAVF, 27%; DVC, 51%), and rest pain (DAVF, 50%; DVC, 40%). RESULTS: The primary patency rate was 48% and 38% at 3 years for DAVF and DVC, respectively. Secondary patency was 48% and 47% at 3 years, with limb salvage rates of 76% and 92% for DAVF and DVC, respectively (P <.05). Attempted thrombectomy without continuation of patency was undertaken in two patients with a failed DAVF. Attempts at restoration after thrombosis were made in eight patients with failed DVCs. Five patients underwent thrombectomy, of which four procedures were successful. Three patients had thrombolytic therapy, and two of these remained patent. CONCLUSION: Adjuvant techniques, including DAVF and DVC, produce acceptable long-term patency and limb salvage rates in bypass grafts performed to the below-knee popliteal and tibial vessels. This study suggests that DVCs may offer improved limb salvage rates and a greater opportunity for revision when bypass graft failure occurs. PMID- 10753278 TI - Extracranial carotid artery aneurysms: Texas Heart Institute experience. AB - BACKGROUND AND PURPOSE: Aneurysms of the extracranial carotid artery (ECA) are rare. Large single-institution series are seldom reported and usually are not aneurysm type-specific. Thus, information about immediate and long-term results of surgical therapy is sparse. This review was conducted to elucidate etiology, presentation, and treatment for ECA aneurysms. METHODS: We retrospectively reviewed the case records of the Texas Heart Institute/St Luke's Episcopal Hospital, Houston, and found 67 cases of ECA aneurysms treated surgically (the largest series to date) between 1960 and 1995: 38 pseudoaneurysms after previous carotid surgery and 29 atherosclerotic or traumatic aneurysms. All aneurysms were surgically explored, and all were repaired except two: a traumatic distal internal carotid artery aneurysm and an infected pseudoaneurysm in which the carotid artery was ligated. RESULTS: Four deaths (three fatal strokes and one myocardial infarction) and two nonfatal strokes were directly attributed to a repaired ECA aneurysm (overall mortality/major stroke incidence, 9%); there was one minor stroke (incidence, 1.5%). The incidence of cranial nerve injury was 6% (four cases). During long-term follow-up (1.5 months-30 years; mean, 5.9 years), 19 patients died, mainly of cardiac causes (11 myocardial infarctions). CONCLUSION: The potential risks of cerebral ischemia and rupture as well as the satisfactory long-term results achieved with surgery strongly argue in favor of surgical treatment of ECA aneurysms. PMID- 10753280 TI - A fifteen-year experience with carotid endarterectomy after a formal operative protocol requiring highly frequent patch angioplasty. AB - PURPOSE: The early and late outcomes of carotid endarterectomy (CEA) following a rigid protocol of patch angioplasty or occasionally interposition bypass grafting, when the arteriotomy required to obtain a complete internal carotid end point extended distal to the bulb segment, and primary closure, when it was limited to the bulb, were studied. METHODS: From November 1983 to August 1998, 1360 consecutive primary CEAs were performed on 1133 patients (621 men, 512 women), with a mean age of 67 years. Of these patients, 3.8% (51) had primary closure, 66.4% (903) had greater saphenous vein patch angioplasty, 28.4% (386) had synthetic (359 Dacron, 27 polytetrafluoroethylene) patch angioplasty, and 1.4% (20) had vein interposition bypass grafting procedures. Indications were transient ischemic attack in 34.7% of patients (472), stroke in 16.6% of patients (226), nonlateralizing symptoms in 10.9% of patients (148), and asymptomatic stenosis 70% or greater in 37.8% of patients (514). The mean follow-up period was 4.6 years. RESULTS: The 30-day mortality rate was 1.0% (13 patients; 11 cardiac related deaths, 2 strokes). The 30-day stroke rate was 1.3% (18 patients; 13 ipsilateral strokes, 5 major, 8 minor). The combined 30-day stroke and death rate was 2.1%. Four of the strokes (1 death) were caused by the hyperperfusion syndrome. The 30-day ipsilateral major stroke or mortality rate was 1.2% (16 patients). The 30-day rate of ipsilateral major stroke or death from stroke was 0.4% (5 patients). There were two synthetic and one vein patch internal carotid occlusions in 30 days. Synthetic-patched CEAs were predicted by means of Cox proportional hazards analysis to have higher risk ratios than saphenous vein patched CEAs for early and late stroke (1. 3; 95% CI, 1.7 to 1.0; P =.04), for 50% or greater restenosis (2.4; 95% CI, 3.4 to 1.6; P <.001), and for 70% or greater restenosis (2. 5; 95% CI, 3.6 to 1.7; P <.001). The cumulative mortality rate (Kaplan-Meier) was 13% at 5 years and 31% at 10 years. The cumulative stroke rate was 7% at 5 years and 14% at 10 years. The 50% or greater restenosis rate was higher in women than in men at 5 years (9% versus 5%; P =.02, Wilcoxon), but tended to equalize later. The 50% or greater restenosis rate was higher in synthetic-patched CEAs than in saphenous vein-patched CEAs (12% versus 1% at 1 year; 17% versus 3% at 4 years; and 24% versus 10% at 8 years; P <.001 by means of log-rank and Wilcoxon). Restenosis after 5 years was more frequently located in the distal common carotid artery (13 of 20 cases). Late reoperations were more frequent and occurred earlier in synthetic-patched CEAs (eight cases at a mean of 1.6 years) than vein-patched CEAs (14 cases at a mean of 6.9 years; P =.01). No strokes and one restenosis of 50% or greater occurred in the 51 primarily closed CEAs. CONCLUSION: Patch angioplasty reconstruction of CEAs with arteriotomies that extend distal to the carotid bulb gives excellent early and long-term outcomes. Saphenous vein-patched CEAs are superior to synthetic patched CEAs for stroke and restenosis prevention. Primary closure is safe and durable when complete end points and arteriotomies are within the carotid bulb. PMID- 10753279 TI - Surgical treatment of extracranial internal carotid artery aneurysms. AB - PURPOSE: Extracranial internal carotid artery aneurysms (EICAs) can be treated by carotid ligation or surgical reconstruction. In the consideration of the risk of stroke after internal carotid artery (ICA) occlusion, the aim of this study was to report the results of reconstructive surgery for these aneurysms, including lesions located at the base of the skull. METHODS: From 1980 to 1997, 25 ICA reconstructions were performed for EICA: 22 male patients and 3 female patients (mean age, 54.4 years). The cause was atherosclerosis (n = nine patients), dysplasia (n = 12 patients), trauma (n = three patients), and undetermined (n = one patient). The symptoms were focal in 15 cases (12 hemispheric, three ocular), nonfocal in three cases (trouble with balance and visual blurring), and glossopharyngeal nerve compression in one case. Six cases were asymptomatic, including three cases that were diagnosed during surveillance after ICA dissection. In nine cases, the upper limit of the EICA reached the base of the skull. A combined approach with an ear, nose, and throat surgeon allowed exposure and control of the ICA. RESULTS: After operation, there were no deaths, one temporary stroke, two transient ischemic attacks, and 11 cranial nerve palsies (one with sequelae). The ICA was patent on the postoperative angiogram in all but one case. During follow-up (mean, 66 months), there were two deaths (myocardial infarction), one occurrence of focal epileptic seizure at 2 months, and one transient ischemic attack at 2 years. In December 1998, duplex scanning showed patency of the reconstructed ICA in all but one surviving patient. CONCLUSION: Surgical reconstruction is a satisfactory therapeutic choice for EICA, even when located at the base of the skull. PMID- 10753281 TI - Use of ultrasound contrast in the diagnosis of carotid artery occlusion. AB - OBJECTIVE: The purpose of this study was to evaluate the use of an echo-enhancing agent in patients with carotid artery occlusion to improve the sensitivity and specificity of carotid color flow ultrasonography. METHOD: Between January 1997 and December 1998, a prospective study involving 85 cases of carotid artery occlusion in 84 patients was carried out. After a baseline duplex ultrasonography (DU) diagnosis, a second (DU) along with an echo-enhancement agent (SHU-508-A [Levovist]) study was carried out (echo enhancement ultrasonography diagnosis [DUEE]). In 82 cases, a contrast angiography was performed to confirm the diagnosis, whereas in the other three cases the diagnoses were confirmed with surgery. RESULTS: From the 85 internal carotid artery occlusions diagnosed at the initial DU examination, seven came out to be false occlusions in the DUEE examination (8,2%). There was a 100% correlation of the cases between the DUEE examination and the contrast angiography in the 82 cases in which this had been done. In three of the cases, the diagnosis was confirmed surgically because these displayed severe stenoses according to the DUEE studies in symptomatic patients, and so they required urgent treatment. CONCLUSIONS: The DUEE study is a potent diagnosis tool that allows the differentiation between true carotid artery occlusions and pseudo-occlusions. PMID- 10753282 TI - Myocardial injury in major aortic surgery. AB - PURPOSE: The purpose of this study was to examine the effects of major aortic surgery and its associated oxidative stress and injury on the myocardium. METHODS: Plasma from 27 patients who underwent thoracoabdominal aortic aneurysm (TAAA) repair and 17 patients who underwent infrarenal aortic aneurysm (AAA) repair was collected at incision, aortic crossclamping, and reperfusion and 1, 8, and 24 hours thereafter. Samples were assayed for the myocardial specific protein troponin-T, total antioxidant status, and lipid hydroperoxides. RESULTS: Ten patients experienced cardiac dysfunction in the first 24 hours after surgery (eight patients in the TAAA group and two patients in the AAA group). Immediately after reperfusion, total antioxidant status levels dropped in all patients with TAAA and with AAA; this was more marked in patients with TAAA, leading to a significant difference between the two groups at this time point and for up to 1 hour thereafter (P <.01). Patients with TAAA showed a sharp rise in lipid hydroperoxide levels immediately after reperfusion, and levels were significantly higher than in patients with AAA (P =.0007). In patients with AAA, no significant change in troponin-T was observed throughout the study period; whereas in patients with TAAA, levels were significantly elevated at 8 and 24 hours after reperfusion (P <.01). Troponin-T levels significantly correlated with total antioxidant status (r = -0.5) and lipid hydroperoxides (r = 0.78) but not with systolic blood pressure. CONCLUSION: Supracoeliac aortic crossclamping is associated with a significant release of the myocardial injury marker troponin-T. This seems to correlate with the severity of oxidative rather than hemodynamic stresses. Ameliorating oxidative injury during TAAA surgery may therefore have a cardioprotective effect. PMID- 10753284 TI - Wall stress distribution on three-dimensionally reconstructed models of human abdominal aortic aneurysm. AB - PURPOSE: Abdominal aortic aneurysm (AAA) rupture is believed to occur when the mechanical stress acting on the wall exceeds the strength of the wall tissue. Therefore, knowledge of the stress distribution in an intact AAA wall could be useful in assessing its risk of rupture. We developed a methodology to noninvasively estimate the in vivo wall stress distribution for actual AAAs on a patient-to-patient basis. METHODS: Six patients with AAAs and one control patient with a nonaneurysmal aorta were the study subjects. Data from spiral computed tomography scans were used as a means of three-dimensionally reconstructing the in situ geometry of the intact AAAs and the control aorta. We used a nonlinear biomechanical model developed specifically for AAA wall tissue. By means of the finite element method, the stress distribution on the aortic wall of all subjects under systolic blood pressure was determined and studied. RESULTS: In all the AAA cases, the wall stress was complexly distributed, with distinct regions of high and low stress. Peak wall stress among AAA patients varied from 29 N/cm(2) to 45 N/cm(2) and was found on the posterior surface in all cases studied. The wall stress on the nonaneurysmal aorta in the control subject was relatively low and uniformly distributed, with a peak wall stress of 12 N/cm(2). AAA volume, rather than AAA diameter, was shown by means of statistical analysis to be a better indicator of high wall stresses and possibly rupture. CONCLUSION: The approach taken to estimate AAA wall stress distribution is completely noninvasive and does not require any additional involvement or expense by the AAA patient. We believe that this methodology may allow for the evaluation of an individual AAA's rupture risk on a more biophysically sound basis than the widely used 5-cm AAA diameter criterion. PMID- 10753283 TI - The effect of elevated homocysteine levels on adrenergic vasoconstriction of human resistance arteries: the role of the endothelium and reactive oxygen species. AB - PURPOSE: This study investigated the effect of elevated homocysteine levels on adrenergic contraction of human resistance arteries and tested the hypothesis that homocysteine-induced generation of reactive oxygen species contributes to vascular reactivity changes. METHODS: Small (<200 microm) subcutaneous arteries were cannulated and pressurized in an arteriograph chamber that allowed the measurement of lumen diameter. Two arteries from the same patient were obtained; one was perfused and superfused (intraluminal pressure = 50 mm Hg) with physiologic saline solution (control, n = 6), and the other was perfused and superfused with physiologic saline solution plus 200 micromol/L homocysteine (HC, n = 6); the reactivity to adrenergic stimulation was assessed. Another group of arteries was incubated in 200 micromol/L homocysteine plus 1200 U/mL superoxide dismutase and 120 U/mL catalase (HC + SC, n = 6), and the reactivity to norepinephrine was determined. The vasoreactivity of homocysteine was further assessed in intact (n = 6) and denuded (n = 6) arteries that were precontracted with an intermediate concentration of norepinephrine and homocysteine (20-200 micromol/L) added to the bath while the lumen diameter was continuously recorded. RESULTS: Sensitivity to norepinephrine was diminished in HC arteries, which increased the median effective concentration (EC(50)) from 0.24 +/- 0.06 micromol/L in control arteries to 0.65 +/- 0.10 micromol/L in HC arteries (P <.01). Homocysteine also caused concentration-dependent vasodilation of arteries contracted with an intermediate concentration of norepinephrine that was greater in intact than denuded arteries, with the half-maximum responses occurring at 61 +/- 6 micromol/L (intact) and 90 +/- 11 micromol/L (denuded; P <.05). There was no significant difference in sodium nitroprusside sensitivity between control and homocysteine arteries (EC(50) = 61 +/- 3 nmol/L vs 50 +/- 19 nmol/L; P >.05) or in sensitivity to acetylcholine (EC(50) = 19 +/- 7 nmol/L vs 12 +/- 3 nmol/L; P >.05). Arteries in the presence of superoxide dismutase and catalase had similarly impaired reactivity to norepinephrine as did homocysteine arteries (EC(50), 0.58 +/- 0.15 micromol/L; P >.05 vs HC, P <.01 vs control). CONCLUSION: An elevated homocysteine level in vitro diminishes adrenergic contraction, with a differential endothelial versus smooth muscle influence that appears unrelated to the generation of reactive oxygen species. PMID- 10753285 TI - Adenoviral-mediated expression of antisense RNA to basic fibroblast growth factor reduces tangential stress in arterialized vein grafts. AB - PURPOSE: The purpose of this study was to test whether basic fibroblast growth factor (bFGF) participates in arterialized vein graft remodeling. METHODS: Rabbits underwent in vivo gene transfer and carotid interposition vein grafting. Segments of external jugular vein were infected with an adenovirus that expressed antisense bFGF RNA (Ad.ASbFGF) at 1 x 10(10) PFU/mL to inhibit new synthesis of bFGF by cells in the vein graft wall. Control rabbits were treated with either adenovirus that encoded beta-galactosidase (Ad.lacZ) at 1 x 10(10) PFU/mL or vehicle (phosphate-buffered saline solution [PBS]). At 3 days, 3 grafts per treatment group were harvested for the determination of gene expression of ASbFGF RNA by reverse transcriptase-polymerase chain reaction. Rabbits were killed, and perfusion was fixed 2 months after the grafting. Total wall thickness and lumen circumference of vein grafts and normal arteries were measured in cross sections. Calculated mean tangential stress (+/-SD) for the ASbFGF-treated group and controls was compared for significance. Grafts were immunohistochemically stained to assess bFGF protein production. RESULTS: Only the grafts infected with the Ad.ASbFGF gene expressed ASbFGF RNA. Grafts that were treated with Ad.ASbFGF displayed lower tangential stress (10.9 +/- 2.3 dynes/cm(2)) than PBS alone (22 +/- 2.8 dynes/cm(2)) or Ad. lacZ-treated controls (20.6 +/- 5.4 dynes/cm(2); P <.001). Tangential stress in the Ad.ASbFGF group was comparable to a normal carotid artery (13.9 +/- 2.1 dynes/cm(2)). The difference in mean total wall thickness was significant among the 3 treatment groups: Ad.ASbFGF, 164 +/- 3.4 microm); Ad.lacZ, 100 +/- 3.3 microm; and PBS, 96 +/- 3.6 microm; P <.01). Luminal circumference was not different among the groups. The Ad.ASbFGF-treated vein graft wall was composed of thick layers of concentric smooth muscle cells and elastin fibers in contrast to the sponge-like appearance observed in control arterialized vein grafts. Reduction in bFGF protein was noted only in the Ad.ASbFGF-treated group. CONCLUSION: Inhibition of bFGF synthesis in vivo with the use of adenoviral gene transfer of antisense RNA to bFGF promotes a vein graft with decreased tangential stress while maintaining the luminal area. The vein graft wall is remodeled and qualitatively resembles an artery so that wall tangential stress in Ad.ASbFGF and normal artery are not significantly different. The lack of significant difference in lumen circumference among groups suggests that wall thickening in the Ad. ASbFGF grafts is not at the expense of luminal narrowing. Our results suggest that ASbFGF RNA expression may represent an effective strategy in limiting the failure of arterialized venous conduits. PMID- 10753286 TI - Vascular smooth muscle cell effect on endothelial cell endothelin-1 production. AB - Endothelin-1 (ET-1) is a potent mitogen secreted by endothelial cells (ECs) in culture and is a putative factor in vascular lesion development. The purpose of this study was to examine whether smooth muscle cells (SMCs) inhibit EC secretion of ET-1. The effect of SMCs on EC ET-1 and constitutively expressed nitric oxide (NO) synthase activity was examined by using a bilayer co-culture model. SMCs inhibited both EC ET-1 protein and RNA levels, compared with ECs cultured alone. SMCs increased EC NO production when compared with ECs cultured alone. In addition, SMC inhibition of EC ET-1 production could be blocked by the NO synthase inhibitor N(G)-nitro-L-arginine-methyl ester. ECs stimulated SMC proliferation, and the ET-1 AB and B receptor blockers inhibited EC stimulation of SMC proliferation. The ET-1 A blocker had no effect on SMC proliferation. We conclude that SMCs regulate EC ET-1 and ecNOS synthase transcript levels and protein levels. SMC inhibition of ET-1 production by ECs may be mediated through SMC-modulated changes in EC NO activity. Finally, EC stimulation of SMC proliferation in bilayer co-culture is mediated by ET-1 through the ET-1 B receptor. PMID- 10753287 TI - Nitroglycerin to control blood pressure during endovascular stent-grafting of descending thoracic aortic aneurysms. AB - Temporary asystole induced with adenosine or electrically induced ventricular fibrillation has previously been proposed to prevent hypertension during transluminal placement of thoracic endovascular stent-grafts. Nitroglycerin is a safe and less invasive alternative to control blood pressure and, in contrast to the methods mentioned, can also be used during stent-grafting performed under local anesthesia. PMID- 10753288 TI - Successful percutaneous endovascular treatment of a ruptured popliteal artery aneurysm. AB - A rupture of the popliteal artery is a rare but dangerous complication of aneurysmal disease. It accounts for 4% of all popliteal artery aneurysms encountered and threatens the loss of the extremity and, infrequently, is also life-threatening.(1) when this clinical entity is confronted, a prompt operative intervention is indispensable for increasing the chances of limb salvage. We report the first, to our knowledge, successful endovascular treatment of a ruptured popliteal artery aneurysm with a new polytetrafluoroethylene stent-graft in a patient who was unfit for a conventional surgical approach because of his severe pulmonary disease. PMID- 10753289 TI - Postirradiation aortic sarcoma demonstrated by magnetic resonance angiography. AB - This is the first ever reported case of a radiation-induced aortic sarcoma. This patient had symptoms and signs initially interpreted as a pulmonary embolus. The extent of the disease was demonstrated with magnetic resonance imaging and magnetic resonance angiography, in particular, allowing rapid surgical intervention. PMID- 10753290 TI - Abdominal aortic aneurysm with aorta-left renal vein fistula with left varicocele. AB - Abdominal aortic aneurysm with spontaneous aorto-left renal vein fistula is a rare but well-described clinical entity usually with abdominal pain, hematuria, and a nonfunctioning left kidney. This report describes a 44-year-old man with left-sided groin pain and varicocele who was treated with conservative measures only. The diagnosis was eventually made when he returned with microscopic hematuria, elevated serum creatinine level, and nonfunction of the left kidney; computed tomography scan demonstrated a 6-cm abdominal aortic aneurysm, a retroaortic left renal vein, and an enlargement of the left kidney. This patient represents the youngest to be reported with aorto-left renal vein fistula and the second case with a left-sided varicocele. PMID- 10753291 TI - Baroreflex failure syndrome after bilateral excision of carotid body tumors: an underestimated problem. AB - Carotid body tumors (CBTs) are relatively rare paragangliomas that develop from neural crest cells at the bifurcation of the common carotid artery. They are generally slow growing and benign. Excision is currently considered the treatment of choice, although vascular and especially neural injuries are still relatively frequent in patients with large or bilaterally resected tumors. The baroreflex failure syndrome (BFS) has recently been identified as a severe, rarely recognized, and certainly underestimated complication after the bilateral excision of CBTs. The present report describes a case of a bilateral CBT followed by BFS and reviews the experiences reported in the literature. In light of the low incidence of malignancy of these tumors, their biologic behavior, their very high rate of cranial nerve palsy, and the occurrence of BFS in bilaterally resected paragangliomas, the current practice of bilaterally removing these tumors is questioned. PMID- 10753292 TI - Peripheral arterial embolization: Doppler ultrasound scan diagnosis. AB - Use of intraoperative monitoring with transcranial Doppler scanning during carotid endarterectomy has enabled identification of embolus signals in the ultrasound spectrum. Extension of this technique to preoperative screening has enabled identification of actively embolizing lesions and correlation with neurologic deficits. We report embolus signals in the peripheral circulation before operation, which aided diagnosis and decision to operate. The patient had been transferred from another institution after multiple revascularization procedures, including posterior tibial artery thrombectomy. Angiography performed on arrival at our institution confirmed an open bypass graft, although a small indentation was noted at the site of the previous posterior tibial artery thrombectomy. Runoff was intact to the plantar arch where there was attenuation of that vessel and occlusion of most digital branches. Duplex monitoring revealed no embolic signals in the graft or in the posterior tibial artery proximal to the previous arteriotomy. Distal to this site, embolic signals were detected. At the time of operation, a large platelet thrombus was identified at the site of the previous arteriotomy, and platelet thrombus was obtained from the plantar artery. It is concluded that doppler ultrasound scanning enables detection of peripheral embolization and the identification and location of lesions with such embolic activity. Diagnostic accuracy may be improved when there is clinical suspicion of embolization, enabling better patient selection for surgical procedures. This report provides the first clinicopathologic characterization of the emboli detected. PMID- 10753293 TI - Internal jugular vein thrombosis in association with the ovarian hyperstimulation syndrome. AB - Thrombosis of the internal jugular vein is a rare entity with the potential for serious consequences. Most of the reported cases of jugular venous thrombosis have occurred in the presence of an indwelling venous catheter, an established hypercoagulable state, or in association with head and neck sepsis. This report presents a case of a patient in whom jugular venous thrombosis developed during the first trimester of pregnancy after in vitro fertilization. Thromboembolism in these circumstances can be related to a condition known as the ovarian hyperstimulation syndrome. The presentation of severe neck pain in pregnant women, especially in those who have undergone assisted reproduction procedures, should prompt evaluation by duplex scan to evaluate the jugular veins for thrombosis. Anticoagulation is the treatment of choice. PMID- 10753294 TI - Lengthening the greater saphenous vein with the use of the lateral femoral cutaneous vein. AB - Besides quality, the length of the greater saphenous vein dictates the feasibility of vein bypass grafts in femorodistal popliteal or tibial revascularization. A simple and effective technique of lengthening the greater saphenous vein is described that allows the use of the laterofemoral cutaneous vein in continuity. This technique can be applied when the distal segment of the greater saphenous vein is inadequate or unavailable. PMID- 10753295 TI - Pneumatic limb compression: a free lunch? PMID- 10753296 TI - Extracranial carotid aneurysms: a new look at an old problem. PMID- 10753297 TI - Collaboration between vascular surgeons and interventional radiologists: reflections after two years. PMID- 10753298 TI - The discipline of vascular surgery at the close of the millennium, the American Board of Surgery Sub-Board for Vascular Surgery, and the wisdom of evolving a conjoint board of vascular surgery: one surgeon's perspective. PMID- 10753299 TI - A clinical perspective on the management of endoleaks after abdominal aortic endovascular aneurysm repair. PMID- 10753300 TI - Regarding "in situ replacement of infected aortic grafts with rifampicin-bonded prostheses: the Leicester experience (1992 to 1998)". PMID- 10753301 TI - Reply. PMID- 10753302 TI - Regarding "A rational algorithm for duplex scan surveillance after carotid endarterectomy". PMID- 10753303 TI - Reply. PMID- 10753304 TI - Distribution of choline acetyltransferase immunoreactivity in the brain of an elasmobranch, the lesser spotted dogfish (Scyliorhinus canicula). AB - Although the distribution of cholinergic cells is remarkably similar across the vertebrate species, no data are available on more primitive species, such as cartilaginous fishes. To extend the evolutionary analysis of the cholinergic systems, we studied the distribution of cholinergic neurons in the brain and rostral spinal cord of Scyliorhinus canicula by immunocytochemistry using an antibody against the enzyme choline acetyltransferase (ChAT). Western blot analysis of brain extracts of dogfish, sturgeon, trout, and rat showed that this antibody recognized similar bands in the four species. Putative cholinergic neurons were observed in most brain regions, including the telencephalon, diencephalon, cerebellum, and brainstem. In the retrobulbar region and superficial dorsal pallium of the telencephalon, numerous small pallial cells were ChAT-like immunoreactive. In addition, tufted cells of the olfactory bulb and some cells in the lateral pallium showed faint immunoreactivity. In the preoptic-hypothalamic region, ChAT-immunoreactive (ChAT-ir) cells were found in the preoptic nucleus, the vascular organ of the terminal lamina, and a small population in the caudal tuber. In the epithalamus, the pineal photoreceptors were intensely positive. Many cells of the habenula were faintly ChAT-ir, but the neuropil of the interpeduncular nucleus showed intense ChAT immunoreactivity. In the pretectal region, ChAT-ir cells were observed only in the superficial pretectal nucleus. In the brainstem, the somatomotor and branchiomotor nuclei, the octavolateral efferent nucleus, and a cell group just rostral to the Edinger Westphal (EW) nucleus contained ChAT-ir neurons. In addition, the trigeminal mesencephalic nucleus, the nucleus G of the isthmus, some locus coeruleus cells, and some cell populations of the vestibular nuclei and of the electroreceptive nucleus of the octavolateral region exhibited ChAT immunoreactivity. In the reticular areas of the brainstem, the nucleus of the medial longitudinal fascicle, many reticular neurons of the rhombencephalon, and cells of the nucleus of the lateral funiculus were immunoreactive to this antibody. In the cerebellum, Golgi cells of the granule cell layer and some cells of the cerebellar nucleus were also ChAT-ir. In the rostral spinal cord, ChAT immunoreactivity was observed in cells of the motor column, the dorsal horn, the marginal nucleus (a putative stretch-receptor organ), and in interstitial cells of the ventral funiculus. These results demonstrate for the first time that cholinergic neurons are distributed widely in the central nervous system of elasmobranchs and that their cholinergic systems have evolved several characteristics that are unique to this group. PMID- 10753305 TI - Enzymatic removal of polysialic acid from neural cell adhesion molecule perturbs the migration route of luteinizing hormone-releasing hormone neurons in the developing chick forebrain. AB - During development in the chick embryo, luteinizing hormone-releasing hormone (LHRH) neurons migrate along the olfactory nerve from the olfactory epithelium to the forebrain. At embryonic day 5.5 (E5.5) to E6.0, the majority of LHRH neurons begin to enter the medial forebrain and then course dorsocaudally along the forebrain substance just beneath the pia matter in association with the somatostatin (SST)-positive fibers, which branch medially from the SST-positive olfactory nerve. By E6.5, the neurons and SST-positive medial branch of the olfactory nerve have proceeded toward the septal area. Intense immunoreactivity for the polysialylated form of neural cell adhesion molecule (PSA-NCAM) on both the LHRH neurons and the SST-positive fibers during this period suggests that this less adhesive form of NCAM is involved in the migratory process. This possibility was examined by using a polysialic acid (PSA)-specific endoneuraminidase. PSA removal did not alter the behavior or appearance of the SST-positive olfactory fibers within the migration pathway. However, it induced a significant deviation of migrating LHRH neurons from the regular path in the forebrain. The effect of PSA removal is more likely to involve changes in the interaction of the migrating neurons with a subset of the SST-positive olfactory fibers and/or other elements in the forebrain rather than an alteration in the pattern of their axonal substrate. On the basis of these results, it is suggested that PSA contributes to the specific pattern of LHRH neuronal migration in the forebrain by limiting interaction of these LHRH neurons with their surrounding environment. PMID- 10753306 TI - alpha-dystroglycan isoforms are differentially distributed in adult rat retina. AB - alpha-Dystroglycan (alpha -DG) is a laminin/agrin receptor expressed in skeletal muscle as well as in nervous system and other tissues. Glycosylation of the core protein of alpha-DG is extensive, variable from tissue to tissue, and functionally relevant. To address differential glycosylation of alpha-DG in the retina, we have investigated the distribution of this protein using two different antibodies: 1B7 directed against the core protein of alpha-dystroglycan, and IIH6 directed against a carbohydrate moiety (Ervasti and Campbell [1993] J Cell Biol 122:809-823). Monoclonal antibody 1B7 recognizes a broader band than IIH6, which seems to recognize only a subset of alpha-DG forms in retina. These data reflect the existence of differentially glycosylated isoforms of alpha-DG. Monoclonal antibody 1B7 shows an extensive staining for alpha-DG in the inner limiting membrane as well as in the ganglion cell and inner plexiform layers labeling Muller cell processes, whereas monoclonal antibody IIH6 staining is restricted to the inner limiting membrane and blood vessels. Our data indicate that there are distinct isoforms of alpha-DG that are localized in apposition to basal lamina in the inner limiting membrane and blood vessels or within the parenchyma of the retina along Muller glia. Both isoforms are expressed in a Muller cell line in culture and coimmunoprecipitate with beta-dystroglycan. These data suggest that DGs may participate in organizing synapses and basement membrane assembly in the retina. PMID- 10753307 TI - Organization of efferent peripheral synapses at mechanosensory neurons in spiders. AB - The mechanosensory neurons of arachnids receive diverse synaptic inputs in the periphery. The function of most of these synapses, however, is unknown. We have carried out detailed electron microscopic investigations of the peripheral synapses at sensory neurons in the compound slit sense organ VS-3 of the spider Cupiennius salei. Based on the localization of discrete presynaptic vesicle populations, it is possible to discriminate at least four different synapse types, containing either: (1) small round, electron-lucent vesicles 32 nm in diameter; (2) large round, clear 42-nm vesicles; (3) a mixture of small and large clear, round vesicles, similar in size to those in Type 1 and Type 2 synapses, respectively, and granular and dense-core vesicles; or (4) clear, round 37- to 65 nm vesicles. Combined immunocytochemical labeling at the light and the electron microscopic level suggests that gamma-aminobutyric acid (GABA) is the transmitter in many of the 32-nm vesicle synapses, and glutamate in many of the 42-nm ones. Based on vesicle type and particular synaptic configuration, various forms of presumed efferent synaptic contacts are distinguishable with the sensory neurons, the surrounding glia, and between the putative efferent fibers themselves. These include simple unidirectional synapses, reciprocal synapses, serial synapses, and convergent as well as divergent dyads. These various synaptic microcircuits are suited to serve a variety of functions. Among these are direct postsynaptic inhibition or excitation of the mechanosensory neurons, and disinhibition or sensitization via presynaptic inhibition or excitation. The observed synaptic configurations are compared with those at the crustacean muscle receptor organ. They reveal a remarkable complexity of synaptic microcircuits at spider sensilla and suggest manifold possibilities for subtle, efferent control of sensory activity. PMID- 10753308 TI - Immunolocalization of the cocaine- and antidepressant-sensitive l-norepinephrine transporter. AB - Norepinephrine (NE) transporters (NETs) constitute the primary mechanism for inactivation of synaptically released NE, are targets for multiple antidepressants and psychostimulants, and have been reported to be deficient in affective and autonomic disorders. Although the regional distribution of NETs has been defined through synaptosomal transport and autoradiographic approaches, NET protein expression has yet to be characterized fully in the central nervous system (CNS). We identified a cytoplasmic NET epitope (amino acids 585-602) and corresponding antibody (43411) that permits cellular localization of endogenous NET expression in the CNS and periphery. In the adult rat brain, NET labeling was confined to noradrenergic neuronal somata, axons, and dendrites, including extensive arborizations within the hippocampus and cortex, but was absent from epinephrine- and dopamine-containing neurons. Intracerebroventricular anti dopamine beta-hydroxylase/saporin, a treatment that destroys a majority of noradrenergic neurons and their projections, validated the specificity of the 43411 antibody. At the level of light microscopy, 43411 labeling colocalized with the axonal markers syntaxin, synaptophysin, and SNAP-25. Indirect immunofluorescence revealed a nonuniform pattern of NET expression along axons, particularly evident within sympathetic fibers of the vas deferens, reflecting a high degree of spatial organization of NE clearance. NET labeling in somata was intracellular and absent from plasma membranes. Among nonneuronal cells, glial cells lacked NET immunoreactivity, whereas CNS ependymal cells were an unexpected site of labeling. NET immunoreactivity was also evident in a subset of adrenal chromaffin cells where labeling appeared to be predominantly associated with intracellular vesicles. Initial ultrastructural evaluation via preembedding immunogold techniques also revealed substantial cytoplasmic NET immunoreactivity in axon terminals within the prelimbic prefrontal cortex, consistent with postulates of regulated trafficking controlling neurotransmitter clearance. NET visualization should be of significant benefit in evaluating neuronal injury resulting from chronic drug exposure and in disease states. PMID- 10753309 TI - Thalamic projections from the whisker-sensitive regions of the spinal trigeminal complex in the rat. AB - This study investigated the axonal projections of whisker-sensitive cells of the spinal trigeminal subnuclei (SP5) in rat oral, interpolar, and caudal divisions (SP5o, SP5i, and SP5c, respectively). The labeling of small groups of trigeminothalamic axons with biotinylated dextran amine disclosed the following classes of axons. 1) Few SP5o cells project to the thalamus: They innervate the caudal part of the posterior group (Po) and the region intercalated between the anterior pretectal and the medial geniculate nuclei. These fibers also branch profusely in the tectum. 2) Two types of ascending fibers arise from SP5i: Type I fibers are thick and distribute to the Po and to other regions of the midbrain, i.e., the prerubral field, the deep layers of the superior colliculus, the anterior pretectal nucleus, and the ventral part of the zona incerta. Type II fibers are thin; branch sparsely in the tectum; and form small-sized, bushy arbors in the ventral posterior medial nucleus (VPM). Accordingly, a statistical analysis of the distribution of antidromic invasion latencies of 96 SP5i cells to thalamic stimulation disclosed two populations of neurons: fast-conducting cells, which invaded at a mean latency of 1.23 +/- 0. 62 msec, and slow-conducting cells, which invaded at a mean latency of 2.97 +/- 0.62 msec. 3) The rostral part of SP5c contains cells with thalamic projections similar to that of type II SP5i neurons, whereas the caudal part did not label thalamic fibers in this study. A comparison of SP5i projections and PR5 projections in the VPM revealed that the former are restricted to ventral-lateral tier of the nucleus, whereas the latter terminate principally in the upper two tiers of the VPM. These results suggest a functional compartmentation of thalamic barreloids that is defined by the topographic distribution of PR5 and type II SP5i afferents. PMID- 10753310 TI - Connections of a motor cortical region in zebra finches: relation to pathways for vocal learning. AB - The lateral magnocellular nucleus of the anterior neostriatum (lMAN) is necessary for both initial learning of vocal patterns in developing zebra finches, as well as for modification of adult song under some circumstances. Lateral MAN is composed of two subregions: a core of magnocellular neurons and a surrounding shell composed primarily of parvocellular neurons. Neurons in lMAN(core) project to a region of motor cortex known as robust nucleus of the archistriatum (RA), whereas neurons in lMAN(shell) project to a region adjacent to RA known as dorsal archistriatum (Ad). We studied the axonal connections of Ad in adult male zebra finches. In contrast to RA, Ad neurons make a large number of efferent projections, which do not include direct inputs to vocal or respiratory motor neurons. The major efferent projections of Ad are to: (1) the striatum of avian basal ganglia; (2) a dorsal thalamic zone (including the song-control nuclei dorsomedial nucleus of the posterior thalamus [DMP] and dorsolateral nucleus of the medial thalamus [DLM]); (3) restricted regions within the lateral hypothalamus (stratum cellulare externum [SCE]), which may also relay information to the same dorsal thalamic zone; (4) a nucleus in the caudal thalamus (medial spiriform nucleus [SpM]); (5) deep layers of the tectum, which project to the thalamic song-control nucleus Uva; (6) broad regions of pontine and midbrain reticular formation; and (7) areas within the ventral tegmental area and substantia nigra (ventral tegmental area [AVT], substantia nigra [SN]), which overlap with regions that project to Area X, a song-control nucleus of avian striatum. Inputs to Ad derive not only from lMAN(shell), but also from a large area of dorsolateral caudal neostriatum (dNCL), which also receives input from lMAN(shell). That is, lMAN(shell) neurons project directly to Ad, and also multisynaptically to Ad via dNCL. Double-labeling studies show that lMAN(shell) contains two different populations of projection neurons: one that projects to Ad and another to dNCL. These results are exciting for two main reasons. The first is that some of these projections represent potential closed-loop circuits that could relay information back to song-control nuclei of the telencephalon, possibly allowing diverse types of song-related information to be both integrated between loops and compared during the period of auditory-motor integration. Because both auditory experience with an adult (tutor) song pattern and auditory feedback are essential to vocal learning, closed-loop pathways could serve as comparator circuits in which efferent commands, auditory feedback, and the memory of the tutor song are compared in an iterative fashion to achieve a gradual refinement of vocal production until it matches the tutor song. In addition, these circuits seem to have a strong integrative and limbic flavor. That is, the axonal connections of Ad neurons clearly include regions that receive inputs not only from somatosensory, visual, and auditory areas of cortex, but also from limbic regions, suggesting that they may be involved in higher order sensory processing, arousal, and motivation. PMID- 10753311 TI - delta-catenin is a nervous system-specific adherens junction protein which undergoes dynamic relocalization during development. AB - delta-catenin is a member of the Armadillo repeat family and component of the adherens junction discovered in a two-hybrid assay as a bona fide interactor with presenilin-1 (Zhou et al., [1997], NeuroReport 8:2085-2090), a protein which carries mutations that cause familial Alzheimer's disease. The expression pattern of delta-catenin was mapped between embryonic day 10 (E10) and adulthood by Northern blots, in situ hybridization and immunohistochemistry in the mouse. In development, delta-catenin is dynamically regulated with respect to its site of expression. It is first expressed within proliferating neuronal progenitor cells of the neuroepithelium, becomes down-regulated during neuronal migration, and is later reexpressed in the dendritic compartment of postmitotic neurons. In the mouse, delta-catenin mRNA is expressed by E10, increases and peaks at postnatal day (P)7, with lower levels in adulthood. In the developing neocortex, delta catenin mRNA is strongly expressed in the proliferative ventricular zone and the developing cortical plate, yet is conspicuously less prominent in the intermediate zone, which contains migrating cortical neurons, delta-catenin protein forms a honeycomb pattern in the neuroepithelium by labeling the cell periphery in a typical adherens junction pattern. By E18, delta-catenin expression shifts primarily to nascent apical dendrites, a pattern that continues through adulthood. The dynamic relocalization of delta-catenin expression during development, taken together with previously published data which described a role for delta-catenin in cell motility (Lu et al., [1999] J. Cell. Biol. 144:519 532), suggests the hypothesis that delta-catenin regulation is closely linked to neuronal migration and may play a role in the establishment of mature dendritic relationships in the neuropil. PMID- 10753312 TI - Access to Timely and Optimal Care of Patients with Acute Coronary Syndromes - Community Planning Considerations: A Report by the National Heart Attack Alert Program. AB - Age-adjusted mortality due to cardiovascular disease (CVD) has declined by more than 50% over the past three decades; however, CVD continues to be the leading cause of death in the United States. In 1994, 1.25 million people experienced an acute myocardial infarction (AMI). Nearly 500,000 Americans died from CVD, and more than half of these deaths occurred suddenly, within 1 hour of symptom onset, outside the hospital setting. The National Heart Attack Alert Program (NHAAP) endorses the view of the American Heart Association that the community should be recognized as the "ultimate coronary care unit." Rapid identification and early treatment are supported by research that demonstrates time is a fundamental factor in reducing morbidity and mortality from AMI and cardiac arrest. A dramatic relationship has been shown between the onset of AMI symptoms, reperfusion treatment, and outcome for patients treated within the first hour after the onset of symptoms. The golden hour has become a widely recognized term in the trauma field, and communities and states are encouraged to develop and implement regional and statewide plans to ensure that trauma patients receive appropriate care within 1 hour of injury. The primary premise of this report - that planning by communities for rapid recognition and triage of patients with symptoms and signs of acute coronary syndromes will result in better outcomes for patients with AMI, including sudden cardiac arrest - is largely based on experience with trauma patients, a population that is benefitting from similar community planning efforts. This NHAAP report reviews community planning considerations and the essential components of an effective community plan (i.e., action plans and protocols, equipment and resources, education and training, and continuous quality improvement evaluation and research) and provides recommendations for each component. The report also presents strategies to guide communities in developing community cardiac emergency action plans. PMID- 10753314 TI - Thinking about Tinnitus. AB - In 1989, I did a thorough search of the medical records and histopathologic features of the temporal bones in the collection of the Massachusetts Eye and Ear Infirmary. During their lifetimes, these patients had had significant tinnitus. The goal was to find a histopathologic correlate for tinnitus in the cochlea. Although no such correlate could be found, some thought-provoking facts were verified. In this paper I want to share these thoughts hoping that others will follow their suggested investigational lines. PMID- 10753315 TI - On the Functional State of Central Vestibular Structures in Monoaural Symptomatic Tinnitus Patients (BEAM-VbEP Study). AB - Clinical evidence suggests that over-excitation or disinhibition of structures in the brain occurs in tinnitus patients.(1,2) Brain electrical activity mapping of vestibular evoked potentials (BEAM-VbEP method) provides an electrophysiologic approach of quantification of function in brain cortex. The effect of tinnitus on the BEAM-VbEP image was examined in two groups of acoustic tumor patient Group A (n = 24) reported tinnitus and Group B (n = 22) did not. Statistically significant differences in the VbEP parameters have been identified between the two groups. The amplitude of the III/IV peak-to-peak component elicited by the rotation to the affected side, is higher (P < 0.05) in the tinnitus group than in the non-tinnitus group. Latencies of the late VbEP components (III, IV, and V) are shorter. In subgroup III, latency was 317.9 +/- 37.5 ms in the tinnitus group versus 335.5 +/- 30.9 ms in non-tinnitus group (p < 0.05); subgroup IV's component of 437.1 +/- 35.4 ms versus 470.9 +/- 43.5 ms ( p < 0.01), V 622.1 +/- 32.6 versus 655.5 +/- 46.6 ms (p < 0.05). Amplitude mapping of the most prominent VbEP component, subgroup III, demonstrates a well expressed negativity shift of the evoked brain electrical activity. The character of the electrophysiologic VbEP changes in the group of tinnitus patients is irritative. We consider the above described BEAM-VbEP images in tinnitus patients to reflect an electrophysiologic correlative of a state of cortical disinhibition, caused by either hyperactive or hypersensitive neural structures. Tinnitus is an aberrant perception of sound unrelated to an external source of acoustic stimulation; a dysynchrony within the auditory system.(3-5) Does tinnitus originate from hyperactive nerve fibers in the cochlea or is it a consequence of overexcited or disinhibited brain structures?(6,7) This basic question still has no acceptable solution. One working hypothesis is that tinnitus represents periodic or aperiodic excitation in the spontaneous activity of hair cells or nerve fibers originating from a restricted place on the basilar membrane.(8) In most clinical cases, the complaint of tinnitus is an accompanying symptom of other central or peripheral disorders (cerebrovascular and circulatory diseases, acoustic neuromas, sudden hearing loss, intoxication, side effects of drugs, among others which frequently cause various degrees of hearing loss with a corresponding decrease in the spontaneous activity of the auditory nerve.)(9,10) Thus, one could imagine such cases of tinnitus can be caused by some abnormal form of spontaneous activity in the central nervous system. Such considerations influenced us to evaluate the functional state of the brain in patients in whom tinnitus is the chief complaint. The method used in this study is the BEAM-VbEP method. This approach for vestibular stimulation and recording is suggested by the original concept of Shulman and colleagues,(5,11) that in some cases tinnitus can originate at the site of vestibular dysfunction. Furthermore, the BEAM-VbEP method provides data restricted not only to vestibular brain centers and pathways, but also to a much broader perspective to sensory pathways and associative cortical areas. PMID- 10753313 TI - Educational Strategies to Prevent Prehospital Delay in Patients at High Risk for Acute Myocardial Infarction: A Report by the National Heart Attack Alert Program. AB - An estimated 13 million people in the United States have coronary heart disease (CHD), peripheral vascular disease, or cerebrovascular disease. The risk for subsequent myocardial infarction (MI) and death in these patients is fivefold to sevenfold higher than for the general population. Many effective therapies are now available for patients with unstable angina, acute myocardial infarction (AMI), potentially fatal arrhythmias, and cardiogenic shock if they seek and receive care expeditiously. However, delays in accessing and receiving care are a continuing problem, threatening the effectiveness of available treatments. Patients with previously diagnosed CHD, including a previous MI, have the same or greater delay times as those without prior MI or CHD. Because of the high-risk status of these patients, combined with the problem of delay in seeking care, this Working Group of the National Heart Attack Alert Program Coordinating Committee advises physicians and other healthcare providers of their important role in reducing treatment delay in these patients. The Working Group recommends that primary care clinicians in the office and in inpatient settings provide these patients and their family members or significant others with contingency counseling about actions to take in response to symptoms of an AMI. The counseling should address the emotional aspects (e.g., fear and denial) that patients and those around them may experience, as well as barriers that may be associated with the healthcare delivery system. Assistance from other healthcare providers (e.g., nurses) should be solicited to initiate, reinforce, and supplement the counseling. A Patient Advisory Form is offered as an aid to providers in counseling their high-risk patients about these issues. Other materials and aids should be considered as well. Physicians' offices and clinics should devise a system to triage patients rapidly when they call or walk in seeking advice for possible AMI symptoms. Further research is needed to learn more about effective counseling strategies; symptom manifestation in high-risk groups, including the elderly, women, and minorities; and healthcare delivery systems that enhance access to timely care for patients with prior CHD or other clinical atherosclerotic disease. PMID- 10753316 TI - SPECT Imaging of Brain and Tinnitus-Neurotologic/Neurologic Implications. AB - Single Photon Emission Computed Tomography (SPECT) of brain with technetium-99m hexamethyl propyleneamine oxine (Tc-HMPAO) is an imaging technique which has been introduced for the identification of abnormalities of regional cerebral blood flow (rCBF) in patients with a central type of subjective idiopathic tinnitus, which was characterized as severe and disabling. These patients demonstrate a negative clinical history, physical examination does not evidence central nervous system disease, and CT/MRI studies of brain were negative. Two typical cases are presented which demonstrate significant regional abnormalities in cerebral perfusion bilateral of temporal, frontal, parietal and hippocampal amygdala regions when compared with normative Tc-HMPAO SPECT of brain data. Neurotologic and neurologic implications are suggested which include cerebrovascular disease, neurodegenerative disorder and dementia, and neuropsychiatric mood disorder. SPECT results of brain demonstrate for the first time the in vivo significance of the organicity of brain for a central type of tinnitus. PMID- 10753317 TI - Objective Tinnitus and the Tensor Tympani Muscle. AB - Objective tinnitus (OT) may be caused by contraction of the tensor tympani muscle (TTM). The more forcefully the TTM contracts, the greater the intensity of the OT heard. Forceful closure of both eyelids can reflexively cause OT by contracting the TTM. The Forceful Eyelid Closure Syndrome (FECS) was reported at the Proceedings of the Second International Tinnitus Seminar in 1983.(1) FECS consists of several factors: (1) Objective tinnitus (2) An associated waning of hearing primarily of the lower frequencies, as much as 45 dB at 125 Hz, 30 to 40 dB at 250 Hz ascending to the patient's norm at 2000 Hz and approximately a 5 to 10 dB at 4000 Hz and 5 to 20 dB at 8000 Hz (3) Retraction of the manubrium and posterior mid-third of the tympanic membrane (TM) at the malleus-umbo area as seen under the otomicroscope (OM) in 25% (108) of 432 ears examined (4) These same ears were 75% (324) positive for increased impedance at maximum compliance with FEC. Of the patients studied, 25% had no response under the otomicroscope or by impedance audiometry. PMID- 10753318 TI - Some Remarks on the Classification of Subjective Idiopathic Tinnitus (SIT) - An Essay toward Establishing a Cross-matched Grading System. AB - To better quantify levels of individual impairment caused by SIT, two different sets of classification were cross matched. This combination factored in the inaccuracy caused by terminologic inexactitude. Although the system which developed historically had a subjective bias, the newer tests created to analyze tinnitus specifically were far more objective. By combining the findings of both tests using a numeric score, rating tinnitus should become more exact. Classification of tinnitus began long ago in ancient Babylon. In the library of the court of King Assurbanipal (668 to 626 B.C.), clay slates were found which gave the first written classification of tinnitus. Later Pliny, Paracelsus, Itard, and Politzer developed separate systems of tinnitus classification.(2) All these systems can be broken down into the branches of epidemiology, etiology, and legal requirements. Epidemiology gives information on tinnitus in "terms of time, place, and persons." Etiology provides a means to summarize the relationships between the subjective findings of tinnitus and relationships in terms of pathoetiology. It is the large numbers of single complaints about tinnitus and possible associated relationships of pathologic relationships that cause the different clinical classification of tinnitus. In terms of legal disability, a more accurate classification will permit graduated comprehensions of the irritation experienced by the patient and related personal, social and occupational interferences. Measuring these grades of impairment will allow comparison of different types of tinnitus. Unlike other audiologic failures such as generalized hearing loss and disturbances of equilibrium resulting from vestibular affections, assessment of the intensity of tinnitus and its involvement in personal life is restricted by limitations, because each classification system contains inaccuracies per se. PMID- 10753319 TI - Dietary Management for Tinnitus Control in Patients with Hyperinsulinemia-A Retrospective Study. AB - A retrospective study in 205 patients with a chief complaint of tinnitus and associated hyperinsulinemia/euglycemia is presented. Dietary control if umpaired insulin metabolism has resulted in a significant degree of tinnitus control. The methods and research are reported. PMID- 10753320 TI - Hyperinsulinemia: The Common Denominator of Subjective Idiopathic Tinnitus and Other Idiopathic Central and Peripheral Neurootologic Disorders. AB - Hyperinsulinemia as determined by glucose/insulin tolerance identified an etiologic relationship to idiopathic Meniere's disease. This was subsequently concurred with internationally by others. Proctor identified hyperinsulinemia in Subjective Idiopathic Tinnitus (SIT). Hyperinsulinemia and migraine with tinnitus and/or vertigo were also correlated. PMID- 10753321 TI - Idiopathic Subjective Tinnitus Treated by Amitriptyline Hydrochloride/Biofeedback. AB - The efficiency of two treatment modalities for subjective/idiopathic tinnitus (SIT): biofeedback (BF) and amitriptyline hydrochloride (AT) was investigated in 225 randomly selected subjects. Findings show that after 10 weeks of treatment in the BF group, 43.5% of the patients reported an improvement of tinnitus during activity. In the AT group, 27.5% of patients reported subjective improvement of tinnitus at rest although only 15.8% of the AT patients reported improvement during activity. Biofeedback during rest had a significantly better effect on tinnitus disturbance than AT. No objective diminishment of tinnitus loudness was found as a result of any of the treatment modalities. We believe that BF can help tinnitus patients especially during periods of rest and we also suggest trying tricyclic antidepressant drugs such as AT for treatment of tinnitus patients, in small doses, however, to minimize the side effects of this drug. Subjective tinnitus (ST) is one of the most common and yet most unclear of otologic symptoms.(1-4) ST can accompany any type of hearing loss including both sensorineural as well as conductive hearing loss, and may originate from any part of the auditory pathway.(1,5) Treatment of ST must be primarily directed to the basic illness diagnosed after a thorough general ear-nose-throat and neurologic evaluation.(6) Severity of ST is evaluated both objectively, by determining the pitch and intensity of the tinnitus,(7) and subjectively as described by the patient. Because of the relatively high incidence of ST and in some patients, the severe personal reaction to it, many different treatments have been suggested, but generally only small to moderate success has been achieved in reducing tinnitus and its consequences, if any at all.(8) In this study we examined the effect of two treatment modalities: amitriptyline hydro-chloride and biofeedback. PMID- 10753322 TI - The Evolution of Cognitive-Behavioral Therapy as an Approach to Tinnitus Patient Management. AB - Many forms of tinnitus management exist, some directed toward causative factors, others toward symptom relief. In this paper, I describe the evolution of a University-hospital-based multidisciplinary team approach that focuses on the affective component of tinnitus in addition to the sensory component. Cognitive behavioral therapy is the primary intervention method used in conjunction with other management procedures. The rationale and principles underlying this management technique are described. PMID- 10753323 TI - The Concept of Causality (Kausalitat) with respect to the Diagnosis of Head-Neck Trauma and Tinnitus in German Law. PMID- 10753324 TI - Tinnitology, a Search for a Modern Identity of Tinnitus. PMID- 10753326 TI - Past, Present, Future. PMID- 10753325 TI - In Which Direction Do We Go - A Commentary. PMID- 10753327 TI - Tinnitus Synthesis: Fluctuant and Stable Matches to the Pitch of Tinnitus. AB - Five subjects mimicked the sensation caused by their tinnitus with a complex sound pattern consisting of the sum of sine waves. The imitation tinnitus was generally broadband, spanning an average of 2.94 kHz. Measures of the pitch of the tinnitus were made using both a forced-choice double-staircase (FCDS) task and a method of adjustment (MOA) task. Even tinnitus portrayed as broadband was matched reliably using a FCDS task, presumably because all but one component of the tinnitus was ignored. With the MOA task, successive matches to the predominant tinnitus pitch were fluctuant, presumably because disparate components of the tinnitus were subsequently matched. Because tinnitus is a broad band signal, clinical trials evaluating the effect of a treatment on tinnitus should involve assessing changes occurring at any location in tinnitus spectrum. PMID- 10753328 TI - Coping with Tinnitus: Two Studies of Psychological and Audiological Characteristics of Patients with High and Low Tinnitus-Related Distress. AB - Two studies are described which were designed to investigate the relationship between psychological distress and tinnitus. In the first study, four groups of subjects (N=72) who differed in degree of tinnitus-related distress were compared on audiological measures. No differences were found between the four groups on loudness, pitch and minimum masking level when employing the Bonferroni correction which controls for inflation of the Type I error rate when conducting multiple statistical tests on the same set of data. The univariate tests indicated that the more severely distressed tinnitus sufferers experienced loud tinnitus as measured by a loudness match procedure. In the second study, 81 tinnitus patients were categorised as either displaying high or low tinnitus related distress. The two groups were compared on various measures including level of depressive symptomatology, reported use of coping strategies, perceived benefits from these coping strategies, reported engagement in depression-related negative cognitions, and tinnitus-specific dysfunctioning thinking. High distress subjects were found to have elevated scores on the Beck Depression Inventory. These subjects also reported engaging in more dysfunctional thinking specifically in relation to tinnitus. The implications of the findings of the two studies are discussed. PMID- 10753329 TI - Neurootological Evaluation of Tinnitus. AB - We analyse the neurootological data of patients attended in the Ear, Nose and Throat (E.N.T.) department of the Centro Hospitalar de Vila Nova de Gaia for a tinnitus problem. We submitted these patients to our neurootological routine evaluation. This approach is justified in a tinnitus patient because the cochleovestibular system is a unit and acts as a whole. We characterise the population that came to us by sex and mean age. The profile of these patients is then analysed by our neurootological routine evaluation, which comprises the history taking and audiometric as well as equilibriometric investigations. The results of the audiovestibulometric examinations - pure tone audiometry, vocal audiometry, and Brain Evoked Response Audiometry (B.E.R.A.), Cranio-Corpo-Graphy (C.C.G.), Electronystagmography (E.N.G.) - are discussed. We concluded that many tinnitus patients, even those who had no vestibular symptoms, showed some disturbances in the vestibular tests. This fact highlights the need for a complete cochleovestibular investigation in all patients complaining about tinnitus. PMID- 10753330 TI - Furosemide Distinguishes Central and Peripheral Tinnitus. AB - The response of patients with tinnitus to the suppressive effects of IV furosemide is about 50%. Since furosemide is a drug without known effects on the central nervous system but with well documented effects in the auditory periphery, we hypothesized that it suppressed tinnitus of peripheral origin and that the response rate was due to that selectivity. To test this hypothesis we recruited 14 patients with unilateral tinnitus who had previously undergone either a labyrinthectomy or acoustic neuroma removal in the complaint ear. Tinnitus in these patients would most likely be of central origin. The first 12 patients tested were negative in response to IV furosemide as compared to the 50% response rate already documented. The last two patients had acoustic neuromas removed and were positive to IV furosemide. meaning that their tinnitus was suppressed. Examination of the case records of these latter two patients revealed that their cochlear portion of the VIIIn had been spared during surgery. We therefore suggest that IV furosemide selectively suppresses tinnitus of peripheral etiology. PMID- 10753331 TI - Tinnitus in Whiplash Injury. PMID- 10753332 TI - A Final Common Pathway for Tinnitus - The Medial Temporal Lobe System. AB - A final common pathway for tinnitus is hypothesized to exist for all patients with tinnitus. Its function is the transition of the sensory to the affect component of the symptom of tinnitus. Single Photon Emission Computerized Tomography (SPECT) with the radio isotope TC99-HMPAO has identified side to side perfusion asymmetries highlighted by that of the amygdala - hippocampal complex. Adjacent perfusion asymmetries involving the frontal, temporal and parietal lobes suggest a interneuronal network resulting in the transition of the sensory to the affect components of the symptom of tinnitus. It is hypothesized that a fundamental function of the amygdala - hippocampal structures is the establishment of a paradoxical auditory memory for tinnitus. It is a result of alteration in auditory masking found in all tinnitus patients. A paradoxical memory for an aberrant auditory signal i.e., tinnitus, is considered to be the initial process in the transition of the sensory to the affect component. Underlying mechanisms are hypothesized to exist and to be highlighted by a diminution of inhibition mediated by gamma aminobutyric acid (GABA) due to disconnection from excitatory (glutamate) inputs. Blockage of GABA mediated inhibition results in Tinnitogenesis, a epileptiform auditory phenomena. The overall hypothesis of a final common pathway for tinnitus; the role of the MTLS; and clinical support for this hypothesis is presented. PMID- 10753333 TI - Objective Pulse-Synchronous "Essential" Tinnitus due to Narrowing of the Transverse Dural Venous Sinus. AB - Subjective tinnitus is a common problem with many etiologies. Objective tinnitus, in which the sound is perceived by both the patient and the examiner, is less common. Objective tinnitus of the vascular type, in which a pulse synchronous bruit is heard by an independent observer, is frequently related to an underlying arterial or arteriovenous malformation, most commonly a dural arteriovenous fistula (DAVF) involving the transverse and sigmoid sinuses. The remaining cases are usually termed "essential" vascular tinnitus, and are presumed to have a venous etiology. In these cases, the audible noise is generally assumed to be produced within the sino-jugular connection, or within an enlarged jugular bulb. We present four documented cases of objective pulse synchronous tinnitus due to focal narrowing (acquired and developmental) of the mid-portion of the transverse dural sinus. In all cases, a bruit was audible directly over a focal constriction in the sinus, demonstrated by cerebral angiography or direct catheter venography. In one case, selective venography revealed a distensible sinus narrowing, associated with a jet of contrast marking fast flow within a developmental sinus segmentation. In another case, a loud pulse synchronous bruit was heard directly over a focal transverse sinus stenosis, which was detected by angiography at the site of a vascular surgical clip. In this case, magnetic resonance (MR) falsely predicted sinus occlusion. In two other cases, an audible bruit was also heard directly overlying a narrowed transverse sinus, seen in the venous phase of angiography. Transverse sinus stenosis is an unappreciated cause of objective pulsatile tinnitus, and we believe that this mechanism may underlie many cases of "essential" or venous etiology tinnitus not otherwise anatomically explained. Non invasive testing, computed tomography (CT) and MR and non-directed angiography may overlook it. Conventional catheter arteriography or venography should be performed in such cases, with attention to the dural sinuses, if other tests fail to define the anatomic basis of the audible bruit. PMID- 10753334 TI - Surgical Decompression of the Eighth Nerve for Tinnitus. AB - This article reports two cases of vascular decompression for tinnitus, both of which were successful. The pathophysiology of vascular compression syndromes in general and specifically eighth nerve vascular compression syndromes is reviewed. The anatomy of the anterior inferior cerebellar artery and its relationship to the eighth nerve complex is discussed in detail and the literature of vascular compression syndrome reviewed. Although no unequivocal definitive test procedure is available for preoperative diagnosis of vascular compression syndrome, the presence of the following features makes the diagnosis more likely: I-III interpeak latency abnormalities on auditory brain stem response (ABR), abnormalities of stapedius reflex, unilateral sensorineural hearing loss, ipsilateral electronystagmography (ENG) abnormalities, especially spontaneous nystagmus, and radiographic visualization of a vessel contacting the eighth nerve complex. PMID- 10753335 TI - Can a Persistent Stapedial Artery be Safely and Effectively Removed? A Case Report with Therapeutic Implications. AB - In the past, a persistent stapedial artery was frequently cited as a reason to discontinue stapes surgery, however, several authors have had success operating on the oval window despite the presence of a persistent artery. We present a case of a patient with conductive hearing loss and tinnitus successfully treated with removal of a persistent stapedial artery that was filling the obturator foramen. This experience, in conjunction with a review of the literature and a discussion with several neurotologic colleagues, leads us to suggest that a stapedial artery can be safely removed allowing unhindered access to the oval window. PMID- 10753336 TI - Tinnitus as a Presenting Symptom in Secondary Neuropathy: a Case Report. AB - The authors describe a case of neuropathy characterized by involvement of most of the right cranial nerves, the initial symptom of which was tinnitus. The most likely etiopathogenetic hypothesis seems to be the paraneoplastic syndrome in which a primitive neurogenic axon lesion is more likely, although a myelinopathic lesion cannot be ruled out. It is feasible to suggest that the tinnitus was caused by a central neuron demyelination associated with concomitant peripheral nervous system involvement. Observation of present case allows the authors to point out that tinnitus may be the early sign of neurological disease which can evolve into the central and/or peripheral neurologic disease. PMID- 10753337 TI - Tinnitus in Children - Still a Neglected Problem. AB - In general, pediatric tinnitus and head trauma, induce tinnitus in children, in particular, which are a neglected problem in Ear, Nose, and Throat and pediatric medicine. A case report and the pertinent literature demonstrating this issue are presented. It is recommended to perform controlled studies regarding pediatric tinnitus in order to identify, and treat it, so as to minimize its damage. Subjective tinnitus which is often reported as being very disturbing is rarely mentioned in the pediatric population.(l) Nodar(2) mentioned in his literature review of 11 years, that he found only two publications dealing with tinnitus in children. This fact is even more amazing as tinnitus is not uncommon in the pediatric population; 13%(3) to 29%(4) of normal hearing children and 59% of children who had their hearing screened at school(3) complained of tinnitus. Tinnitus was reported in 66% of children with moderate to severe deafness and in 29% of deaf children.(5) Out of those with moderate to severe deafness, 30% complained of a very disturbing tinnitus (every day in frequency, of at least 30 minutes duration and grade III out of three severity degrees of loudness). Although tinnitus in children is as common as in the adult population, children generally do not complain spontaneously of having tinnitus.(5) Out of 403 children with tinnitus only 3% complained spontaneously.(4) This discrepancy between the high incidence of tinnitus and low rate of spontaneous complaint in children may be explained by the fact that the child considers tinnitus to be a normal event, as it has usually been present for a long period of time.(6) A second explanation of this discrepancy lies in the fact that the child may not distinguish between the psychological impact of the tinnitus and its medical significance.(6) Even if a child does not mention the existence of tinnitus, nevertheless, it may cause difficulties in concentration and bring about behavioral problems.(5) Pediatric tinnitus, despite its incidence and behavioral sequela, has not received adequate consideration in the otolaryngological and pediatric literature; furthermore, there is complete disregard concerning the post traumatic tinnitus in children. There is no reason why such an important etiologic factor reported in the adult population(7,8) would not be present in the pediatric population, in which head trauma is, at least, as frequent as in the adult one. PMID- 10753338 TI - Rating the Annoyance of Synthesized Tinnitus. AB - Ten tinnitus sufferers mimicked the sensation caused by their tinnitus with a complex sound pattern consisting of the sum of sine waves. The annoyance of these ten sounds was then rated on a scale from 1 (not annoying) to 10 (very annoying) by some of the tinnitus sufferers and by 50 normal-hearing subjects. For the normal-hearing subjects, the number assigned to rate the annoyance of each sound varied widely from subject to subject, while the rank ordering of the ratings was in rough agreement. Because rankings have less inter-subject variability than ratings, ranking are likely preferable as a dependent variable in the clinical assessment of tinnitus. The correlation between the ratings of the ten stimuli by the subjects with normal hearing and the subjects with tinnitus averaged 0.90, indicating that the tinnitus sufferers and the normal-hearing subjects tended to judge the annoyance of the sounds in a similar fashion. Further, the rating of the subject's own tinnitus lay near the regression line, indicating that the subject judged his own tinnitus imitation no differently than he judged the annoyance of other sounds. PMID- 10753339 TI - The Psychological Management of Tinnitus: Comparison of a Combined Cognitive Educational Program, Education Alone and a Waiting-List Control. AB - Sixty subjects with chronic tinnitus were randomly allocated to one of three experimental conditions: (1) cognitive coping skills training (attention diversion, imagery training and thought management skills) combined with education. (2) education-only, or (3) waiting-list control. The two treatment groups improved significantly more than the waiting-list control on measures of frequency of use of coping strategies, benefits derived from the use of coping strategies, irrational beliefs and knowledge about tinnitus. Subjects who received the combined cognitive/education intervention demonstrated significantly greater reductions in distress and handicaps associated with tinnitus, and engagement in dysfunctional cognitions, than the subjects who received education alone. No significant effects were obtained on measures of depression, locus of control, or on daily ratings of subjective loudness, noticeability or bothersomeness of the tinnitus. At the 12-month follow-up, the differential treatment effects had dissipated. Although the treatment resulted in statistically significant effects, the size of the clinical effects is rather modest. Implications for the further development of treatment techniques are discussed. PMID- 10753340 TI - Surgical Method for Implanted Tinnitus Suppressor. AB - A surgical method of fully internal tinnitus suppressor is presented. The surgical approach for the placement of the electrode is very simple and causes less injury to the cochlea. This surgical approach is quite similar to the exploratory tympanotomy. The unit is located at the seat which is drilled in the mastoid bone just behind the external ear canal. The stimulating electrode is introduced to the middle ear beneath the skin of the ear canal on the posterior bony wall. The tip of the electrode located on the promontory is secured by the fascia The primary coil is contained in the Behind-the-Ear case. PMID- 10753341 TI - Vestibular Evoked Potentials (VestEP) and Brain Electrical Activity Mapping - A Test of Vestibular Function - A Review (1990 - 1996). AB - Brain Electrical Activity Mapping of Vestibular Evoked Potentials (BEAM-VestEP) is a new technology for investigation of the spatial and temporal properties of a rotationally-induced brain electrical events. The method consists of multichannel EEG registration and mapping of the brain isoelectrical contours during short lasting repetitive angular accelerations. A special data bank containing more than 400 BEAM-VestEP investigations on more than 300 persons, either symptom free volunteers or neurotological patients suffering from vertigo, tinnitus, sudden hearing loss, acoustic tumors, balance disorders, has been created for this study. The VestEP wave set consists of 5 - 7 positive/negative wave components, appearing within the time interval of 70 - 850 ms after the onset of the acceleratory step stimulus. The principle components analysis reveals that the shortest latencies and the highest amplitudes of the VestEPs can be registered from the central transversal line of electrodes, T3-C3-Cz-C4-T4. The later components are generated from the more frontally located cortical areas. The VestEP is a compound electrical phenomenon. The initial complex ( waves I - III) is related to the activation of specific (vestibular) cortical areas. The later complex (waves IV - VI) reflects the supramodal cortical proceedings with sensory information (cognitive components). PMID- 10753342 TI - Vestibular Evoked Potentials in Two Patients with Bilateral Vestibular Loss. AB - The Brain Electrical Activity Mapping (BEAM) of Vestibular Evoked Potentials (VestEP) is a new method in the toolbox of the neurootologist. Two cases of bilateral vestibular loss, diagnosed by classical neurootological methods (highlighted by caloric and optokinetic tests' electronystagmography recording), are reported. The additional information, provided by the Vestibular Evoked Potentials (VestEP) and its high clinical significance for a more accurate neurootologic diagnosis are discussed. PMID- 10753343 TI - SPECT of Brain and Vertigo - A Case Report. AB - Vertigo has been identified as a complaint associated with the symptom of tinnitus; as well as a specific clinical type of tinnitus i.e., vestibular tinnitus. Classically, the clinical diagnosis of Meniere's Disease includes a quadrad of complaints, highlighted by episodic vertigo, ear blockage, a gradual progressive sensorineural hearing loss, and an associated complaint of a "low frequency tinnitus". Diagnostic and etiologic difficulties for Meniere's Disease have resulted in multiple diagnostic entities, e.g., Meniere's Syndrome, Cochlear Meniere's Disease; Vestibular Meniere's Disease, etc. Single Photon Emission Computed Tomography (SPECT) imaging of brain is a technique which has been reported to improve the diagnostic accuracy of patients with the symptom of tinnitus; and has now been applied for recurrent persistent vertigo. A case report, diagnosed classically as Meniere's Disease, is presented to demonstrate the increased diagnostic accuracy provided by SPECT of brain for the chief complaint of vertigo. SPECT revealed significant perfusion asymmetries in brain. The medical significance of the SPECT findings and its application for both diagnosis and treatment are presented. PMID- 10753344 TI - Increased Parasympathetic Nerve Tone in Tinnitus Patients Following Electrical Promontory Stimulation. AB - Cutaneous digital blood flow (CDBF) during electrical promontory stimulation was measured by laser Doppler flowmetry in 46 tinnitus patients. In patients with tinnitus suppression. CDBF was increased. In contrast, patients in whom treatment did not suppress tinnitus experienced no change in digital blood flow. The cutaneous digital blood flow of patients who experienced slight relief did not differ significantly from patients who experienced no relief. The ratio of CDBF before and after treatment did not correlate with patient age, audiogram pattern. Our results suggest that relief of tinnitus was closely related to increased parasympathetic nerve tone. PMID- 10753345 TI - Noise Induced Hearing Loss and the Individual Susceptibility to the Noise. AB - Although there is no specific treatment for modality of occupational noise induced hearing loss (NIHL), the best way to manage this problem is the prevention. The fundamental prevention of NIHL is to find the persons who are more susceptible to the noise and to avoid work in the noisy environment prior to or during employment. The purpose of this study is to investigate the actual status of NIHL in the industrial complex of Pusan and Kyongnam area in Korea and to evaluate the individual susceptibility to the noise and to set-up the preventive indices of NIHL, using temporary threshold shift (TTS) and its recovery time after noise stimulation. PMID- 10753346 TI - Tinnitus - Hyperacusis and the Loudness Discomfort Level Test - A Preliminary Report. AB - This paper is a preliminary report from the Tinnitus Center, HSCB-SUNY, of hyperacusis in a patient population who request consultation for tinnitus of the severe disabling type. Forty-two consecutive patients seen from January to August 1995 were reviewed for this preliminary report. There is a positive correlation between tinnitus and hyperacusis as well as a positive correlation between hyperacusis and the loudness discomfort level test (LDL). Hyperacusis is an increased sensitivity to sound that occurs with/without a hearing loss in individuals with tinnitus of the severe disabling type. The present method of assessment for hyperacusis includes pure tone audiometry, LDL's, Feldmann Masking Curves and the Metz test for recruitment. A classification system exists for hearing loss and a similar system is suggested for hyperacusis. PMID- 10753347 TI - Masking Patterns for Partially Masked Tinnitus. AB - Tinnitus isomasking contours were determined for unmasked tinnitus and for tinnitus partially masked by high-pass noise. The noise was selected so that it masked all but the low frequency components of the tinnitus. As a control, the masking pattern of an external tone in the tinnitus region and in the presence of the high-pass noise was compared with that of the partially masked tinnitus. Frequency-specific masking was obtained for the external tone but not for the partially masked tinnitus. This suggests that even narrowband tinnitus is not masked in the cochlea. Thus, tinnitus maskers need not include the frequency region presumed to contain the tinnitus. PMID- 10753348 TI - "C.A.P.P.E."-A Strategy for Counselling Tinnitus Patients. AB - This paper presents a simple, straightforward vehicle for counseling tinnitus patients when discussing probable cause. The five stress factors are: Chemical, Acoustic, Pathologic, Physical, and Emotional (C.A.P.P.E.). PMID- 10753349 TI - Improved Selective Attention and Word Perception in Tinnitus Patients Treated with Electrical Stimulation. AB - Grammatically correct but nonsense twenty 4-segment sentences mixed with multiple talk recorded on CD were delivered to ears tested in 47 tinnitus patients at a comfortable level via a headphone. The signal-to-noise ratios were 0dB, 5dB and 10dBSPL. Patients were requested to repeat what they heard before and after electrical treatment. A sinusoidal wave of 10kHz at the intensity of about 200mA was delivered to ears for 30 minutes by using a plate electrode for ECG at the tragus or a stimulating Pt-Ir electrode on the middle ear. Alternatively, 0.5mA DC was delivered to patients using a iontophoretic instrument. Improved word perception under noises was observed in most patients with tinnitus relief following electrical stimulation of the ear, demonstrating that electrical stimulation improved auditory selective attention. There may be a relationship between tinnitus relief and improved selective attention. It may be electrical stimulation of the ears that produced improved selective attention, inducing tinnitus relief and improved word perception according to our previous reports. PMID- 10753350 TI - Surgery and Tinnitus for Otosclerotic Patients. AB - In our experience, tinnitus for otosclerosis may be due to the following: poor vibration of the inner ear fluids, fixation of the footplate, destruction of the Corti hair cells by the proteolytic enzymes, acoustic trauma, or to poor inner ear blood supply due to the hyalinization of the spiral ligament of the stria vascularis. The aim of this article is to suggest some explanations to enhance future research work carried out by other authors on this important issue, and to show what are the results of surgery in our experience, as far as tinnitus is concerned. Only complete release of the tinnitus one year after surgery is taken into account to assess the results. PMID- 10753351 TI - Treatment of Cochlear-Tinnitus with Dexamethasone Infusion into the Tympanic Cavity. AB - Intratympanic dexamethasone infusion was performed as a treatment for cochlear tinnitus and its efficacy was investigated. This is a Steroid Targeting Therapy (STT) for cochlear-tinnitus. The overall effective rate for the 1214 patients with 1466 affected ears was 71%. In this study, cochlear tinnitus was seen frequently in the age group of 50 - 60 years old, a relatively older population. The results of the treatment for tinnitus in different age groups did not show a correlation between age and efficacy rate of treatment. In the investigation of the treatment results with different underlying ear diseases, the efficacy rate was high for tinnitus accompanying chronic otitis media, Meniere's disease, and labyrinth syphilis. The efficacy rate tended to decrease more with longer disease duration. When the mean hearing level and treatment results were evaluated, a high effective rate was found in patients who had mild deafness. The efficacy rate was high in tinnitus of low tone pitch, and low tinnitus with high tone pitch. On the other hand, different degreees of loudness of tinnitus did not correlate with treatment effects. This treatment method is useful as a local therapy for cochlear-tinnitus in an outpatient setting. PMID- 10753352 TI - A Final Common Pathway for Tinnitus - Implications for Treatment. AB - A final common pathway for tinnitus is hypothesized to exist for all patients with tinnitus. Its function is the transition of the sensory to the affect component of the symptom of tinnitus. Single Photon Emission Computerized Tomography (SPECT) with the radio isotope TC99-HMPAO has identified side to side perfusion asymmetries highlighted by that of the amygdala - hippocampal complex. It is hypothesized that a fundamental function of the amygdala - hippocampal structures is the establishment of a paradoxical auditory memory for tinnitus i.e., an aberrant auditory signal. It is a result of alteration in normal auditory masking; and found in all tinnitus patients. Underlying mechanisms are hypothesized to exist and to be highlighted by a diminution of inhibition mediated by gamma aminobutyric acid (GABA) due to disconnection from excitatory (glutamate) inputs. Blockage of GABA mediated inhibition results in Tinnitogenesis, a epileptiform auditory phenomenon. Significant neurochemistry implications for treatment are suggested by the Final Common Pathway for Tinnitus. The overall hypothesis of a final common pathway for tinnitus; and the clinical implications for treatment are presented. PMID- 10753353 TI - Portable Videonystagmography. AB - Portable videonsystagmography is equipment to detect nystagmus or other eye movements independent from time, place and medical specialists. It has been demonstrated that patients suffering from Meniere's disease have been diagnosed earlier using portable videonystagmography. PMID- 10753354 TI - Tinnitus and Post-Traumatic Vertigo - A Review. AB - Fifteen neuro-otological patients have been investigated for post-traumatic vertigo, hearing loss and tinnitus. Clinical examination and laboratory tests included audiometry and equilibriometry in all patients. Statistically significant findings are reported for tinnitus, vertigo, hearing loss. Correlations for vertigo with the type of head trauma, nystagmus frequency Slow Phase Velocity (SPV) and associated complaints of hearing loss and tinnitus were analyzed. The most frequent symptom reported was vertigo (73.3%); lift sensation (46.6%); tilting and falling (26.6%); and tinnitus (40%). PMID- 10753356 TI - Medico-Legal Aspects of Tinnitus in Switzerland and the Federal Republic of Germany. AB - Issues of impairment, hand and eye disabilities have worldwide significance. This paper presents the issues from a medical-legal perspective in Switzerland and the Federal Republic of Germany. PMID- 10753355 TI - Cortical and Brainstem Topognostic Testing in Tinnitus Patients - A Preliminary Report. AB - Preliminary electrophysiologic topognostic test findings suggest a method for the functional localization and quantification of tinnitus; and provides a basis for the selection and monitoring of treatment. Numerous and different locations of the tinnitus symptom have been identified. In our series approximately 24% of tinnitus patients were identified to have a peripheral origin; 35% originated in the brainstem; and 41% at a supratentorial level. PMID- 10753357 TI - Tinnitus: Report of Ten Cases of Perilymphatic Fistula and/or Endolymphatic Hydrops Improved by Surgery. AB - Presented are ten cases of patients with perilymphatic fistula and/or endolymphatic hydrops who had tinnitus as a major complaint. Tinnitus and its degree of severity often correlate closely with the state of health or hydrodynamic integrity of the inner ear, as these cases illustrate. PMID- 10753358 TI - Tinnitus: Clinical Overview. PMID- 10753360 TI - Tinnitus Habituation Therapy: The University of Maryland Tinnitus and Hyperacusis Center Experience. PMID- 10753359 TI - Use of Masking for Tinnitus. AB - The use of masking for tinnitus has been a primary method of treatment of patients with severe tinnitus since 1976. Over 5,000 patients have been treated in the Tinnitus Clinic at Oregon Health Sciences University since that time. The paper discusses the results of management of new patients followed for one year from January 1, 1992 through December 31, 1995. Of the 373 patients who could be accurately followed and evaluated for this period of time, 180 patients were recommended for tinnitus maskers or tinnitus instruments, and 83 were provided with masking tapes. The results of this study, along with the overall success of the tinnitus program utilizing masking, are discussed. PMID- 10753362 TI - Pulsatile Tinnitus. PMID- 10753361 TI - Electrical Suppression of Tinnitus via Cochlear Implants. AB - Electrical suppression of tinnitus via a cochlear implant is a secondary benefit for many cochlear implant recipients. In this study, a sample of 78 adult cochlear implant users were surveyed. Data was compiled from 64 completed questionnaires. A high prevalence of preoperative tinnitus was documented in profoundly deaf adult cochlear implant users. Although only a few subjects reported that their tinnitus was totally eliminated after implantation, many users reported improvement or stabilization. Duration of tinnitus appeared to be a significant factor as all subjects who reported a significant improvement had less than a 20-year history of tinnitus. PMID- 10753363 TI - Biochemical Evaluation of the Patient with Tinnitus. PMID- 10753364 TI - Medical Significance of Tinnitus. AB - The medical significance of a symptom or disease process in a patient is defined as a clinical manifestation of abnormal function of a living cell, tissue, organ or organ system(s). Tinnitus, an aberrant perception of sound unrelated to an external source of sound, has been identified to have a medical significance. The medical significance of tinnitus has been identified with a Medical Audiologic Tinnitus Patient Protocol since 1979. The highlights of this clinical experience include for diagnosis: tinnitus not to be a unitary symptom; the identification of clinical type and subtypes of tinnitus and a Final Common Pathway for Tinnitus of all clinical types particularly of the severe disabling type. For treatment a multidisciplinary approach has evolved including neurology, otology and psychiatry; a combined treatment protocol of drug therapy and instrumentation, based on differentiation between the components of the symptom of tinnitus i.e. sensory, affect, and psychomotor. Two diagnostic categories are identified: otologic/neurotologic and neurologic. The application of SPECT Imaging of Brain which has identified a Final Common Pathway for tinnitus and provides an increased diagnostic accuracy for tinnitus and a basis for selection of a neuropharmacology for tinnitus is discussed. PMID- 10753365 TI - Tinnitus Outcome in Surgery for Vertigo. AB - The effect of surgery for vertigo on tinnitus was evaluated in 90 patients who underwent surgery for disabling vertigo for Menieres disease. The three procedures studies included a destructive labyrinthectomy, endolymphatic sac decompression and shunt, and vestibular neurectomy. Patients who underwent a vestibular neurectomy had a significantly better tinnitus outcome than those who underwent shunt surgery (50% vs 22.7%). The labyrinthectomy and the neurectomy groups showed an equal positive response of 50%. The severity of the preoperative hearing loss did not correlate with the tinnitus outcome in all groups. Patients who have serviceable hearing and require surgery for disabling vertigo appear to have a better chance of tinnitus control with the vestibular neurectomy rather than an endolymphatic sac decompression and shunt. PMID- 10753366 TI - The Relationship between Tinnitus and Temporomandibular Disorder (TMD) Therapy. AB - Many TMD patients with coexisting tinnitus find TMD therapy improves or resolves their tinnitus in conjunction with their TMD symptoms (Table I). Forty TMD patients rating their tinnitus as moderate or severe, were asked questions and participated in clinical tests. Upon completion of TMD therapy, 21, 12, 7 and 0 reported their tinnitus was resolved, significantly improved, unchanged and worse, respectively. The subjects' ages, question results and clinical test results were statistically evaluated for an association with their tinnitus change. The following were identified as significantly associated with tinnitus improvement when the patient reports: they are younger in age, their tinnitus is moderate rather than severe, their hearing is normal, their tinnitus began approximately when their TMD symptoms began, their tinnitus is worse when their TMD symptoms are worse, their tinnitus is related to stress, their tinnitus is unrelated to loud noise and their tinnitus is reproduced or intensified from one minute of maximum voluntary clenching on their posterior teeth. These findings may help forecast which tinnitus patients with concomitant TMD may obtain tinnitus improvement through TMD therapy. PMID- 10753367 TI - Calpain Inhibitors as Neuroprotective Agents in Neurodegenerative Disorders. AB - It seems plausible to hypothesize that in all forms of neurodegeneration or other forms of tissue degeneration, a common pathway exists which when deciphered could lead to our understanding of a variety of diseases which result in tissue necrosis as well as offer potential for therapeutic intervention. A relatively recent interest has been our preliminary studies on the role of neurodegeneration in hearing loss and tinnitus, particularly that associated with noise. These studies grew out of a collaboration emanating from early discussions with Professor Abraham Shulman of the State University of New York, Health Science Center at Brooklyn, Department of Otolaryngology, and Dr Richard J. Salvi, of the Center for Communication Disorders and Sciences, Hearing Research Laboratories, State University of New York at Buffalo. Further studies in this very promising area of research are continuing for noise induced hearing loss protection and tinnitus control. A brief review of calpain is presented. PMID- 10753368 TI - Neuroprotective Drug Therapy: A Medical and Pharmacological Treatment for Tinnitus Control. AB - The role of neuroprotective agents for maintaining or improving inner ear function, specifically for the symptom of tinnitus, is presented on the basis of neurootological, neurological, and neurosurgical clinical experiences. These clinical experiences involved the use of calcium channel blockers, free radical scavengers, corticosteroids, antagonists of glutamate at N-methyl-D-aspartate (NMDA) and non-NMDA receptors, and various thrombolytic agents for the etiologies of ischemia, trauma, and hemorrhage. A pharmacological basis for such drug efficacy includes a property described as neuroprotection. A pathology-based protocol for drug selection is proposed for tinnitus control. The goal of this article is to introduce to the neurootologist or otologist and other professionals attempting tinnitus control neuroprotective drug therapies that are being applied to such central nervous system pathological processes as ischemia, trauma, hemorrhage, and neurodegeneration. The innovative application of such drug therapies for treating the symptom of tinnitus of the severe disabling type is considered. The use of neuroprotective drugs in intratympanic drug therapy via the round window, for treatment of inner ear complaints of hearing loss, tinnitus, and vertigo, is discussed. PMID- 10753369 TI - Treatment of Tinnitus and Tinnitus Sufferers in Norway: What Is Our Present Standing? Where Do We Go from Here? PMID- 10753370 TI - Pediatric and Geriatric Tinnitus. AB - The subject of tinnitus in the population extremes-children and the elderly-is ignored by the literature, probably because children do not complain of tinnitus spontaneously, whereas it is only one challenge among other major health problems in the elderly. A short review of the literature on this subject is presented. Presbytinnitus, defined as tinnitus that accompanies the progressive hearing loss of presbycusis is classified as: type 1 (normal aging affecting the cochlea), and type II (preexistent sensorineural hearing loss accompanied by multiple systemic complaints, especially of sensory ones). The incidence of tinnitus in presbycusis is 11%. Like in other age groups, there is no significant gender predilection in the prevalence of tinnitus, but a correlation was demonstrated between the severity of tinnitus and exposure to noise. Hypertension was associated with a lower incidence of tinnitus, as compared to normotension and hypotension. Several treatment modalities of geriatric tinnitus are reviewed: the superiority of the band-noise masker in patients with presbycusis, as compared to electrical promontory stimulation; amino-oxyacetic in presbycusis and Meniere's disease; zinc supplementation in marginally zinc-deficient elderly patients in improving sensorineural hearing loss and tinnitus; aeration of the middle ear in presbycusis caused by secretory otitis media. Pediatric tinnitus has an incidence of 13% in children who passed an audiometric screening test, and 23-60% in those with hearing loss, 44% in secretory otitis media, but only 3% complain spontaneously because that the child considers tinnitus to be a normal event. There is no significant difference between children with tinnitus and those without in terms of hearing level, age, gender, or etiology of the deafness. Despite the fact that often children do not mention it, tinnitus may incite behavioral problems. PMID- 10753371 TI - Evaluation of Tullio Phenomenon by Computerized Dynamic Posturography. PMID- 10753372 TI - IMx (Abbott) Immunoassay of Insulin: A Practical Alternative to RIA Hyperinsulinemia Identification in Idiopathic Neurootology and Other Hyperinsulin Metabolic Disorders. AB - Hyperinsulinemia identification as defined by glucose/insulin tolerance has been established as the prime etiological factor in idiopathic neurootological disorders. Insulin assays by radioimmunoassay (RIA) and the IMx (Abbott) immunoassay yielded in 558 of 595 glucose/insulin tolerance a concurrence of 93.7%. The latter measures insulin without cross-reaction with proinsulin. The RIA methodology includes proinsulin. The IMx (Abbott), a micro-particle enzyme immunoassay (MEIA), gave lower values due to its nondetection of proinsulin. Based upon defined insulin values, the dynamic patterns of euinsulinemia, hyperinsulinemia with elevated fasting insulin levels and hyperinsulinemia with impaired and/or hyperglycemia glucose tolerance were concurred 100% by MEIA. All of the nonconcurrences were with normal glucose tolerances when the second and/or third hour insulin values were borderline. The limited utilization of RIA technology and the potential availability of enzymatic immunoassay which requires less technical skills presents MEIA as a practical and precise alternative to RIA hyperinsulinemia identification. The increasing world-wide significance of the clinical pathology of hyperinsulinemia becoming manifest in all disciplines of medicine, warrants the identification and/or exclusion of hyperinsulinemia by cost-effective technology. PMID- 10753373 TI - Clinical Experiences of Steroid Targeting Therapy to Inner Ear for Control of Tinnitus. AB - We summarize our long-term clinical experiences with an inner-ear drug delivery system (DDS) for control of tinnitus. The inner-ear DDS consist of transtympanic perfusion of 2 or 4 mg dexamethasone to the round window via the middle ear. Therapeutic results of steroid targeting therapy to the inner ear were evaluated in 3,978 ears of 3,041 patients. Tinnitus improved in 75% of these patients immediately after treatment and in 68% after 6 months. The effects of such therapy on tinnitus varied depending on the underlying diseases. The improvement of tinnitus in the presence of labyrinthine hydrops was good, whereas the effects on noise- or drug-induced tinnitus were poor. Our results indicate that in high tone tinnitus, the effects were poor, but they were good for low-pitched tinnitus. No correlation existed between the efficacy of management and loudness of the tinnitus. The dangers of perforating the eardrum and of inciting the discomfort of vertigo as a result of the transtympanic injection of dexamethasone are minimal. We believe that the transtympanic infusion technique is an effective DDS for control of inner-ear diseases and symptoms. This technique may be performed by any neurootologist in an outpatient clinic. PMID- 10753374 TI - Evaluation of Implanted Tinnitus Suppressor Based on Tinnitus Stress Test. AB - An electrical tinnitus suppressor developed at the Hokkaido University was implanted in two women and five men, aged 44-77 years old. To evaluate the efficacy of the suppressor, a self-administered tinnitus stress test (TST), annoyance index (AI), and tinnitus intensity index (TII) were conducted 1 1/2-3 years after implantation of the device. Residual inhibition results found at outpatient clinics and at the homes of patients with implanted suppressors were closely correlated except in one patient in whom the device's electrode was free from the promontorium tympani. The AI registered at a severe level in five patients and a moderate level in two patients before implantation of the suppressor. However, the AI improved after the operation, being moderate in three patients and mild in two, and achieving no level in two patients. After the operation, the TST improved except in one patient whose device had electrode trouble. The TII registered as extreme in all patients before implantation of the suppressor, although the intensity of tinnitus varied from patient to patient according to the loudness balance test. After device implantation, the TII did not register any level in two patients, was mild in another two patients, was moderate in yet two more patients, and was severe in a patient whose device had electrode trouble. After the operation, at TST, AI, and TII results were positively correlated (p =.01), though there was no correlation among these parameters before the operation. PMID- 10753375 TI - Gentamicin Cures Vertigo, but What Happens to Hearing? AB - Instillation of gentamicin in the middle ear to cure severe Meniere's disease is of considerable value in the control of vertigo. The drug causes diminished endolymph production and, most often, a peripheral vestibular loss. After vestibular rehabilitation, led by a physiotherapist, the ataxia should be resolved as a result of central compensation. Fifteen to twenty percent of the patients experience an added hearing loss in the treated ear, although this percentage is reduced if the treatment is given as a single instillation on 2 consecutive days. Using this strategy, the mean added hearing loss for the treated group is zero. There is no correlation between the pretreatment hearing level and the added hearing loss. PMID- 10753376 TI - Treating Tinnitus with Hyperbaric Oxygenation. AB - Hyperbaric Oxygenation permits a controlled increase of the partial oxygen pressure in the blood. This technique can be used in cases of tinnitus and sudden deafness when the development in the inner ear and the brain lead to a lack of oxygen and so to a limited energy provision. The results to date allow the recommendation to apply an oxygen high pressure therapy when standard treatments have failed. One can work on an improvement rate of 60-65% with tinnitus. HBO therapy should start as soon as possible. Especially in cases of sudden deafness the success depends on a speedy application of HBO. The HBO therapy broadens the spectrum of treatment possibilities for tinnitus and sudden deafness. PMID- 10753377 TI - Complex Therapy of Neck-Related Tinnitus, Hypacusia, and Vertigo. AB - The authors report the strategy of examination and treatment in patients suffering from tinnitus, hypacusia, and vertigo. In addition to the customary examinations, a routine cervical radiological examination and hemorrheological examination are recommended. The authors propose appropriate complex treatment and report their experience. PMID- 10753378 TI - Tinnitology: Recent Advances for Both Diagnosis and Treatment. PMID- 10753379 TI - Protecting the Inner Ear from Acoustic Trauma. AB - Calcium activated proteases, or calpains, play an important role in neurodegeneration. In some cases, neural degeneration can be significantly reduced by leupeptin, a potent calpain inhibitor. To determine if leupeptin could protect against noise-induced hearing loss and hair cell loss, we infused leupeptin into scala tympani of one cochlea before, during and after a 14-day exposure to a 100 dB SPL, octave band noise centered at 4.0 kHz. Hearing loss, assessed with the auditory evoked response, was less in the leupeptin-treated ear than in the control ear during the early stage of recovery from acoustic trauma. In addition, hair cell loss in the leupeptin-treated ear was significantly less than in the control ear. These preliminary results suggest that leupeptin may protect against noise-induced hearing loss. PMID- 10753380 TI - Change of Chemosensory Event-Related Potentials on Olfactory Stimulation as a Function of Odorant Concentrations. AB - Change of chemosensory event-related potentials on olfactory stimulation as a function of odorant concentrations is discussed. According to the ascending method, from undetectable level to clearly detectable level, the dependency of shortening of the peak latency on the concentration of odorant was recognized as a rapid decrease of amplitude from the threshold level when results of our ascending trial were averaged. However, a much higher concentration of odorant did not always evoke a positive response and a shorter peak latency. PMID- 10753381 TI - Tinnitus Cognitions Questionnaire: Development and Psychometric Properties of a Measure of Dysfunctional Cognitions Associated with Tinnitus. AB - The development of the Tinnitus Cognitions Questionnaire (TCQ), a scale designed to assess positive and negative cognitions associated with tinnitus, is described. Psychometric analyses of the TCQ are examined, with a total of 189 subjects from three separate samples. The results indicate good test-retest reliability (r = 0.88) and internal consistency (Cronbach's alpha = 0.91). Factor analysis revealed two factors that were interpreted as positive cognitions and negative cognitions. TCQ negative items and positive items were found to be unrelated (r = 0.09). Correlations between the TCQ and other self-report indices of depression, automatic negative thoughts, and tinnitus-specific symptomatology are reported. The TCQ may provide a useful index of the cognitive responses of tinnitus sufferers and may be employed as a measure of change in outcome research on psychological management of tinnitus. PMID- 10753382 TI - Cervical Trauma and Tinnitus. PMID- 10753383 TI - Change in Serotonin Level in the Organism in Cases of Hypoxia. AB - This study was performed on both volunteers and test animals subjected to different extreme effects, causing different types of hypoxia. It is found that at high initial values of blood serotonin, the organism is more resistant to hypoxia. In individuals in whom the serotonin level is increased after extreme impacts, the hypoxic state is less readily sustained. The results of this study are used for determining the individual reactivity of an organism to hypoxia and for selecting for hire persons who may be required to work in extreme conditions. PMID- 10753384 TI - Neurootological Findings in Meningiomas of the Internal Auditory Canal and Cerebellopontine Angle. PMID- 10753385 TI - Prevalence of Vestibulocochlear Diseases in 4,825 Patients. AB - A study of the prevalence of vestibulocochlear disorders in 4,825 patients showed that these disorders occurred during middle age and mostly affected women. The most frequent pathologies are vascular labyrinthitis, emotional disturbance, and the cervical syndrome. The study also demonstrated that each disease has a preferential age group, which varies according to the gender. PMID- 10753386 TI - Contralateral Acoustic Effect of Transient Evoked Otoacoustic Emissions in Neonates. AB - Contralateral acoustic stimulation (CAS) has the effect of reducing the amplitude of transient evoked otoacoustic emissions (TEOAE) of the opposite cochlea. This phenomenon is considered to be mediated via the efferent pathway, from the superior olivary complex through the medial olivocochlear system to the contralateral cochlea. The assessment of this suppressive effect provides an objective and noninvasive technique for exploring the function of the efferent auditory system in humans. Two previous studies investigated the suppression effect of TEOAE in newborns and revealed a significant effect in 18 full-term neonates. In this study, the effect of contralateral acoustic stimulation on TEOAE was investigated in 13 full-term neonates (gestational age, 40-42 weeks). The TEOAE were recorded alternately with and without simultaneous, contralateral white noise. The CAS effect of TEOAE was present in all subjects; a mean of 2.21 dB +/- 1.7 (21% +/- 9.3%) was found. Our study demonstrated additional support for the functional maturity of the medial olivocochlear efferent system from birth. PMID- 10753387 TI - Evaluation of Balance Disorders After Minor Head and Whiplash Injuries. PMID- 10753388 TI - Evaluation of Balance Disorders During the First Month After Whiplash Injury. PMID- 10753389 TI - No Effects of Acute Alcohol Ingestion on Subjective Visual Horizontal Determination During Eccentric Rotation. AB - Evaluating the function of the vestibular part of the inner ear comprises more than the classic analysis of the lateral semicircular canal function. In healthy subjects, positional alcohol nystagmus may be seen after acute alcohol ingestion. Posturography has shown a deteriorated equilibrium after even moderate doses of alcohol, which speculatively could be an effect of otolith disturbance or a central integrative effect. We tested the possibility of an otolith effect by using linear acceleration in the lateral direction by means of eccentric rotation, stimulating mainly the outermost ear's otolith organ. The subject is seated eccentrically in a rotatory chair facing the direction of rotation. Thus, the otolith organs are stimulated in steady-state rotation. The subject experiences a lateral tilt and, in darkness, is instructed to put a short light bar in the position thought to be that of a water surface, which is identical to the perceived tilt. Twenty healthy subjects (10 men, 10 women) aged 20-29 years were tested before and approximately 1 hour after ingestion of alcohol, the amounts consumed corresponding to an approximate blood alcohol level of 0.05%, well above the maximum permissible level for driving in Sweden. No significant effects of alcohol were found. The otolith function probably is not affected by moderate alcohol intoxication levels. From this point of view, equilibrium deterioration due to alcohol ingestion in the erect position is caused by a central integrative deficit and not by an otolith effect. PMID- 10753390 TI - Temperature Order Effects in the Caloric Reaction. AB - Does it matter in what order the four irrigations are conducted in the caloric test? It has been suggested that warm water (44 degrees C) elicits stronger reactions than does cold water (30 degrees C), which might be explained simply by slight differences in water temperatures. However, if it can be shown that starting with cold water diminishes the difference between the warm and cold irrigations, it might be worthwhile to apply the cold irrigations first. Binaural, bithermal caloric irrigation was conducted in darkness using electrooculography. Maximum slow-phase velocity was measured. One hundred and eighteen consecutive patients from our laboratory were tested with warm water first and then with cold irrigations. In a second run, 108 subjects were irrigated with cold water before the warm. Employing cold irrigations first produces a smaller difference between the warm and the cold irrigations than does the opposite order. However, the lateral and directional preponderances, according to the formulas of Jongkees, are not affected by this change of irrigation order. The fact that initial warm-water irrigations cause the temperature effect differences to be larger than do initial cold-water irrigations is a phenomenon having only marginal clinical implications. PMID- 10753391 TI - Subjective Visual Horizontal Determination During Otolith Stimulation by Eccentric Rotation in Conservatively Treated Meniere's Disease. AB - Peripheral vestibular equilibrium disorders may originate in various parts of the labyrinth or of the vestibular nerve. Traditionally, the function of the lateral semicircular canals has been assessed by caloric irrigation and has been interpreted (sometimes falsely) as demonstrating a vestibular nerve lesion. The vertical semicircular canals are not assessed easily. Caloric testing with the head in various positions is not very helpful, but the canals may be tested in pairs using specific rotational techniques. Often, the otolith organs, detecting linear acceleration forces, are forgotten as a source of vertigo and dizziness. The extent of otolith involvement in Meniere's disease is not well understood. The tested subject is seated eccentrically in a rotatory chair and faces the direction of rotation. Thus, the otolith organs are stimulated in steady-state rotation. The subject experiences a lateral tilt and, in darkness, is instructed to point a short light bar in the position that he or she thinks a water surface would have (identical to the perceived tilt). Patients with conservatively treated unilateral Meniere's disease were tested. In the eccentric rotation test, the patients with unilateral Meniere's disease showed highly variable, sometimes even paradoxical, responses. No correlation was noted between the eccentric otolith test and pure-tone audiometry or the side difference of the caloric responses. Otolith and lateral semicircular canal functions may differ in patients with Meniere's disease, the nature of which remains to be elucidated in further studies. PMID- 10753392 TI - Three-Dimensional Fast Spin-Echo T2-Weighted Magnetic Resonance Images of the Cerebellopontine Angle. AB - In patients experiencing dizziness and hearing disorders, it is essential to rule out the possibility of an intracranial affection, typically a cerebellopontine angle (CPA) process. Clinically, this often is accomplished using audiology and otoneurology and by interpreting the time course of the symptoms. However, in many cases, we must resort to imaging of the skull base and the CPA. The use of thin-section, fast spin-echo, heavily T2-weighted images can eliminate the need for gadolinium contrast administration, offering a cheaper and quicker investigation as compared with the customary T1-weighted images using gadolinium. With three-dimensional volume sampling, slice thickness can be reduced to 0.7 mm. Data reconstruction in every desired direction is possible. Ninety-two patients with hearing and balance disorders were examined using this method. In subjects free of tumor in the CPA, the seventh and eighth nerves could be followed accurately from the internal auditory meatus to the brainstem. The tumors could be outlined with reasonable accuracy even without gadolinium contrast. Three dimensional, fast spin-echo, T2-weighted images are useful in evaluating the CPA in cases of suspected tumor. Most often the use of gadolinium can be avoided. This seems to be a cheap alternative for magnetic resonance screening of patients experiencing dizziness and hearing disorders. PMID- 10753393 TI - Opening Ceremony: Address of the President of the NES, March 19th, 1998. PMID- 10753395 TI - Honor Acceptance. PMID- 10753394 TI - Homage to Prof. Dr. Abraham Shulman: Tinnitus-A Life with Tinnitology. PMID- 10753397 TI - A Systematic Classification of Tinnitus Generator Mechanisms. AB - Largely unknown causes and innumerable assumed mechanisms conceivably could be involved in the generation of tinnitus. Indeed, tinnitus is so complex and so manifold that a systematic classification could prove helpful. Here represented is a systematic classification that is suitable for scientific communication, for explaining tinnitus models to affected patients in the course of tinnitus counseling, and as a basis for rational diagnosis of and therapy for tinnitus. PMID- 10753396 TI - Tinnitology, Tinnitogenesis, Nuclear Medicine, and Tinnitus Patients. AB - Since the 1970s, clinical interest in otolaryngology and audiology for both diagnosis and treatment of the symptom of tinnitus has witnessed the evolution of a new discipline: tinnitology. Tinnitology is an integrated discipline of basic sciences, neuroscience, and clinical medicine for the understanding of aberrant auditory phenomena unrelated to an external source of sound. To patients with subjective idiopathic tinnitus, nuclear medicine techniques of positron emission tomography and single-photon emission computed tomography provide correlation of structure and function, which improves the accuracy of the tinnitus diagnosis. Additionally, they provide a monitoring system to establish the efficacy of modalities of therapy attempting to provide tinnitus relief. Further, they provide information of neuroreceptors and neurochemistry in brain underlying or accompanying basic mechanism for production of specific clinical types and subtypes of tinnitus. This study reports the application of nuclear medicine techniques for a new clinical neuropharmacology protocol for tinnitus treatment highlighted by intratympanic drug therapy in predominantly cochlear-type tinnitus. We further report a neuroprotective drug therapy to control tinnitogenesis, an auditory epileptiform phenomenon. Additionally, we report the hypothesis of a benzodiazepine deficiency syndrome. PMID- 10753398 TI - The Psychometric Properties of Two Measures of Tinnitus Complaint and Handicap. AB - We describe two studies that examined the psychometric properties of the Tinnitus Handicap Questionnaire (THQ) and Tinnitus Effects Questionnaire (TEQ) with an Australian sample of tinnitus patients. The results indicated that both the THQ and the TEQ have good test-retest reliability and internal consistency. The factorial structure of the THQ is well captured by a three-factor solution. Its major constituents are (1) Emotional, social, and physical effects of tinnitus; (2) hearing acuity and communication; and (3) appraisal of tinnitus. This factorial structure is similar to the one reported previously for a sample of tinnitus patients in the United States. Overall, the results obtained with the TEQ yielded findings similar to the work conducted on this measure in the United Kingdom and Germany. Correlations among the THQ, TEQ, and the Tinnitus Reaction Questionnaire (TRQ), and a selection of other psychological and psychoacoustic measures are reported. It would appear that the THQ, TEQ, and TRQ tend to measure a number of different dimensions of response to tinnitus. All three scales are likely to be far superior to simple visual analog scales, which presently are not understood well in terms of psychometric properties. PMID- 10753399 TI - Magnetic Resonance Angiography in Pulsatile Tinnitus: The Role of Anatomical Variations. AB - Pulsatile tinnitus (PT) is a perception of a rhythmical sound that is synchronous with the heartbeats. Despite being seen rarely in daily practice, frequently it is associated with identifiable causes, thus warranting special attention in regard to the etiological diagnosis. PT results from blood flow turbulence, which in turn results from changes in flow velocity or in the vessel lumen. One of the most important causes of PT is the paraganglioma, a vascular tumor that appears as a reddish retrotympanic mass. However, a normal tympanic membrane mandates differentiating among other diagnoses, such as arteriovenous malformations or fistulas, intracranial or extracranial aneurysms, a high or dehiscent jugular bulb, and persistent stapedial artery. Owing to the progress of radiological evaluation, magnetic resonance angiography (MRA) has proven to be excellent for evaluating vascular diseases. From January 1995 to June 1997, the authors prospectively studied 16 patients with PT and normal otoscopic examination. The study comprised 1 male and 15 female patients (ages 25-71 years; mean age, 42.5 years). All were subjected to MRA evaluation, which revealed the etiological diagnosis in 13 cases (81.25%), including 2 aneurysms and 1 case of intracranial hypertension. Of the 13 patients 9(69.23%) presented with at least one variation of vascular anatomy of the skull, showing a close correlation, in most cases, with the side on which PT occurred. Our results confirm that MRA is an excellent primary screening modality for patients with PT and normal otoscopic findings. The authors point out the importance of making etiological diagnoses in such cases, suggesting that variations of the vascular anatomy of the skull are a possible etiology. PMID- 10753400 TI - Hyperinsulinemia: A Merging History with Idiopathic Tinnitus, Vertigo, and Hearing Loss. AB - The history of neurootology and the history of diabetes mellitus have their earliest but separate recognition in ancient Egyptian medicine. Both the polyuric condition resembling diabetes and "humming in the ear" now known as tinnitus were described. Yallow's refinement of a radioimmunoassay for insulin demonstrated increased insulin (hyperinsulinemia) in known diabetics. Glucose-insulin tolerances corroborated Yallow's findings. Specific hyperinsulinemia patterns of non-insulin-dependent diabetes mellitus, type II (NIDDM) have been identified. Hyperinsulinemia precedes hyperglycemia. Hyperinsulinemia with normal glucose tolerance is the earliest identifier of NIDDM. In 1977, Updegraff identified hyperinsulinemia with idiopathic Meniere's disease. Sustained clinical response was achieved in all who maintained nutritional management. This finding was the first major impact of hyperinsulinemia in the clinical arena. Subsequently, Updegraff's studies were substantiated by others. As a result, the clinical pathology of hyperinsulinemia has become a major factor in multiple medical disciplines. The hyperinsulinemia associated with idiopathic tinnitus, vertigo, and hearing loss and the hyperinsulinemia of NIDDM, without regard for glycemia status, are one and the same entity. The merging relationship preceded the clinical recognition of both entities. A retrospective relationship to ancient Egyptian medicine and before is considered to be most probable. PMID- 10753401 TI - Tinnitus in Hereditary Meniere's Syndrome. AB - In 1997, the authors described a large family with classic Meniere's syndrome and migraine inherited as an autosomal dominant trait. Using a special questionnaire applicable to all the affected members of this family, the majority of respondents convincingly demonstrated true migraine preceding or after the Meniere's attacks. Now we have used another questionnaire designed to describe the natural history of tinnitus and have posed the questions therein to all members of this sibship affected by migraine, Meniere's syndrome, or both. In this way, we obtained a clear description of tinnitus regarding volume, intensity, and degree of annoyance; time of onset and duration; variations with time; and so on. The relations of tinnitus with the other Meniere's symptoms and with the migraine also were explored. Only headache appears more often than tinnitus in these patients, and the latter is the only symptom constantly present, with little variation during migraine and Meniere's attacks. These findings are discussed. PMID- 10753402 TI - Noise, Calpain, Calpain Inhibitors, and Neuroprotection: A Preliminary Report of Tinnitus Control. AB - Frequently, noise-induced hearing loss is associated with the symptom of tinnitus. Preliminary results in the animal model after noise exposure suggest that the calpain inhibitor leupeptin may protect against noise-induced hearing loss. A final common pathway for cell destruction and cell death (i.e., apoptosis) is the calpain hypothesis. Calpain is a normal, intracellular, cytosolic protease activated by excess intracellular calcium. Calpain inhibitors (AK275, AK295) have been shown to provide neuroprotection in the central nervous system. A collaboration of basic science and clinical research efforts focusing on calpain antagonists and inhibitors was established in New York in 1997; the initiators are attempting to develop neuroprotective drug therapy regimens for hearing and balance system complaints, particularly hearing loss, tinnitus, and vertigo. Both calpain inhibitors and antagonists are being developed and are being investigated wth perfusion techniques of the inner ear, in vitro and in vivo, for their effects on peripheral and central portions of the cochleovestibular system. PMID- 10753404 TI - Intratympanic and Round-Window Drug Therapy: Effect on Cochlear Tinnitus. AB - Tinnitus is a common symptom for which few existing therapeutic approaches can produce reliable reduction or elimination. Chemical perfusion involves the delivery of medication directly into the inner ear via the round-window membrane. This report discusses the use of dexamethasone or gentamicin in 20 individuals who had inner ear diseases in which disturbing cochlear tinnitus was one of the symptoms. Preliminary results indicate that chemical perfusion is a promising option for the treatment of cochlear tinnitus. PMID- 10753403 TI - Round-Window Microcatheter-Administered Microdose Gentamicin: Results from Treatment of Tinnitus Associated with Meniere's Disease. AB - In this study, we review the results of Meniere's disease treatment using microdose gentamicin delivered directly to the round window using the Round Window Microcatheter (IntraEar, Inc., Denver, CO). A total of 18 patients were studied, with follow-up ranging from 6 to 18 months. In 15 of 18 patients (83%), tinnitus was improved significantly throughout the follow-up period. Vertigo was eliminated in all patients, and pressure was relieved in 17 of 18 (94%). Ihese preliminary data suggest that Round Window Microcatheter-delivered gentamicin is a safe and effective treatment for the reduction of tinnitus, vertigo, and pressure associated with Meniere's disease. PMID- 10753405 TI - Glutamate Antagonists, Steroids, and Antioxidants as Therapeutic Options for Hearing Loss and Tinnitus and the Use of an Inner Ear Drug Delivery System. AB - A wealth of anecdotal, empirica], and double-blind placebo-controlled data exists on medicines that may have a beneficial role in the management of patients with tinnitus. Tinnitus is a symptom that affects between 40 and 45 million Americans alone; this represents approximately 14% of the US population. Data exist for Japan (population: 125,732,794), Europe (population: 503 million), and Australia (population: 18,426,900), and estimates suggest that tinnitus affects a similar percentage of those populations (B. Tabachnick, personal communication, 1998). Thus, in those industrialized nations, approximately 90 million may experience tinnitus to some degree. One to two percent of the population expriences debilitating tinnitus, severely limiting the quality of life of affected individuals. All too often, the response from well-trained medical professionals is, "Learn to live with it" or "There is no cure." Although the author does not dispute that currently no cure exists, I contend that help is available. This article discusses the use of glutamate antagonists, steroids, and antioxidants for the management of hearing loss and tinnitus. Additionally, the results of using an inner ear drug delivery system on nine patients with a variety of inner ear disorders are reviewed briefly. PMID- 10753406 TI - Acoustic Tumor Surgery and Tinnitus. AB - A total of 311 patients with a unilateral acoustic neurinoma were operated on via the enlarged middle cranial fossa approach. A total tumor removal was achieved in 98% of cases. The mortality was 0.6%. Overall in acoustic neurinoma surgery, the percentage rates of meningitis (1.6%), cerebrospinal fluid fistula (1.6% requiring surgery), and neurological deficits were fairly low. The facial nerve could be preserved anatomically in 99%. A House I or House II classification was demonstrated in 91% of all tumors and in 98% of small- and medium-sized tumors. Preservation of the hearing function was possible in 49% of all patients (71% in small tumors). A positive effect on tinnitus usually occurred in 45% of cases. The enlarged middle cranial fossa approach allows function-preserving surgery of the cranial nerves (e.g., the facial and cochlear nerves) and cerebral structures, even including total removal of large acoustic neurinomas of up to 3 4 cm. PMID- 10753407 TI - The AllHear Cochlear Implant and Tinnitus. AB - The authors have studied the effect of AllHear cochlear implants on tinnitus. We describe five cases of deaf patients with tinnitus and show that the implant device had a good effect on tinnitus in four. We also discuss one case in which the tinnitus was caused by electrical stimulation. The authors conclude that AllHear cochlear implants have a good effect on tinnitus in the majority of patients. PMID- 10753408 TI - Tinnitus in Cochlear Implant Users: The Freiburg Experience. AB - Cochlear implantation is a well-accepted method of aural rehabilitation in deaf or severely hearing-impaired adults and children. A majority of patients not only suffer from hearing impairment but from tinnitus. The high rate of preoperative tinnitus in adults (68.1%) stands in contrast to assumed lower rates in children. Unknown are such factors as how tinnitus develops in children, how they realize what tinnitus is, and whether the mechanism of development of tinnitus differs from that in adults, respectively. Electrical stimulation of the auditory pathway is followed by loss, or at least reduction, of tinnitus in most cases (75%). Also, the insertional trauma alone is able to stop tinnitus in some patients. Attention must be paid to the low risk of developing tinnitus postoperatively. No reports are available regarding tinnitus in children. Though younger children may not be able to report, some adolescent patients report preoperative or postoperative tinnitus (or both) that is reduced by electrical stimulation at the rates seen in adults. Further investigations are needed to define the mechanism of tinnitus development in children and to define optimal stimulation modes and rates for tinnitus reduction with best auditory performance. PMID- 10753409 TI - The Influence of Atlas Therapy on Tinnitus. AB - We present questions and thoughts of an outsider on the proprioceptive input of the posterior small cervical muscles to the brainstem as a source of tinnitus. After treatment of whiplash injuries and other muscular conditions with Arlen's atlas therapy, some patients reported that their tinnitus had abated with the muscular tensions. Atlas therapy has been proven to slacken the muscles and seems to quiet to normal levels afferent impulses to the brainstem. This has been proven to apply to nystagmus. We sought to determine whether it has the same effect on some forms of tinnitus. PMID- 10753410 TI - A tinnitus objectivization: how we do it. AB - The medical therapy of tinnitus should be oriented by objective measurement of the disorder. Preferably, it should be qualitative, indicating the exact neural mechanism to be neuromodulated by neuroprotective medication. The neurophysiological approach in objectivization of tinnitus is presented by means of auditory brainstem response and middle latency response. These tests could be applied in functional follow-up of medical therapy, as these are more sensitive and harmless methods as compared to standard morphological methods. PMID- 10753411 TI - Central auditory speech test findings in individuals with subjective idiopathic tinnitus. AB - This study reports central auditory speech test performance of 25 consecutive patients with subjective idiopathic tinnitus of the severe disabling type. A preliminary study of 14 individuals who had subjective idiopathic tinnitus and complained of difficulty in hearing and understanding revealed a high incidence of abnormal central auditory speech test performance (71%), despite satisfactory peripheral hearing. The results (1) identify objectively for the first time that tinnitus affects specific components of the auditory pathway; (2) provide a basis for monitoring methods of tinnitus control; and (3) provide a basis for understanding "the interference effect" and problem of communication difficulties in patients with tinnitus of the severe disabling type. PMID- 10753412 TI - Tinnitus, heart rate variability, and some biochemical indicators. AB - Twenty-three tinnitus patients between the ages of 25 and 66 years (mean age, 47.6) were examined completely in the neurootological clinic of the Medical University, Sofia. Heart rate variability measurements were obtained to reveal the relationship between tinnitus and the autonomic equilibrium. Some biochemical indicators (serotonin and the colored sedimentary uric reaction of Kimbarovski [CSURK]) also were followed up. For 19 patients with transitional disturbances in the vertebrobasilar blood circulation, the sympathetic portion of the autonomic nervous system predominates, and heart rate variability is suppressed. The higher suppression of heart rate variability and higher values of the health risk in four occupationally exposed patients are attributable to the higher expressed level of stress connected with the adverse work conditions. Changes in the health risk, CSURK, and serotonin blood values in four tinnitus patients also are discussed. PMID- 10753413 TI - Changes in blood serotonin in patients with tinnitus and other vestibular disturbances. AB - The role of serotonin as a mediator and stress hormone and its function in vessel tonus regulation is proved. The aim of the work is to study the concentration of serotonin in blood in patients with vestibular disturbances before and after vestibular provocation and in patients with tinnitus. The study was performed on 134 persons distributed among three groups: group I, 35 patients with vestibular disturbances; group II, 75 healthy persons; group III, 24 patients with tinnitus. Serotonin was examined twice in patients with vestibular disturbances (group I) and in healthy persons (group II), before and 15 minutes after vestibular provocation realized by a cold caloric test, and once in patients with tinnitus (group III). Vestibular loading provokes changes in blood serotonin: In vestibularly stable persons, serotonin decreases and comparatively weaker vestibular vegetative reactions develop. The role of serotonin in the compensatory mechanisms of the organism related to vestibular crisis is discussed. We recommend its inclusion with routine neurootological examinations for selecting candidates for work in conditions that overload the vestibular analyzer. Patients with tinnitus have serotonin blood values that significantly exceed the referent ones. PMID- 10753414 TI - Tinnitus suppression via cochlear implants: review and remarks. PMID- 10753415 TI - The treatment of annoying tinnitus with electrical stimulation. PMID- 10753416 TI - Cochlear implantation in the twenty-first century. PMID- 10753417 TI - Effects of tinnitus on posture: a study of electrical tinnitus suppression. AB - We investigated the effects of tinnitus on postural responses using posturography. Thirty-three tinnitus patients (19 female and 14 male) ranging in age from 33 to 67 years (mean age, 53) were selected randomly at our outpatient clinic. Nobody complained of dizziness. Posturographic examination was given before and after 30-minute electrical stimulation. Items tested were envelope area, area (root mean square), total length, total length-area, mean amplitude of lateral body sway (mx), and anteroposterior sway (my). Those parameters did not improve in patients without tinnitus relief. Two postural measures (envelope area and mx) showed significant improvement in patients with tinnitus relief. These effects appeared only on positions involving restricted visual feedback. In a comparison of postural measures in patients with and without tinnitus relief, all parameters except total length and mx improved significantly in patients with eyes open. Our study showed that aside from the auditory system, tinnitus can affect balance, implying that tinnitus may be a factor in increasing unsteadiness in patients with tinnitus. PMID- 10753418 TI - Effects of greater occipital nerve block on tinnitus and dizziness. AB - We analyzed relationships among tinnitus relief, dizziness, and results of vertical autocorrelation studies of head movement to investigate the effect of greater occipital nerve block on the sensory and motor system in 2 patients with abnormal head movement. Tinnitus improved in 14 (52%) of 28 tinnitus patients after occipital nerve block. The percentage of patients reporting tinnitus improvement (54%, or 7 patients) among 13 patients without a history of trauma was not significantly higher than among 12 patients with trauma (33% or 4 patients). However, tinnitus improvement in patients with dizziness but without trauma was more frequent than that reported by nontraumatic tinnitus patients without dizziness (p<.05). Vertical autocorrelation results while stepping in place were better in patients with tinnitus relief than in those not reporting tinnitus relief (p =.001). Dizziness improved in 8 of 13 patients (62%) with trauma, similar to improvement in 8 of 14 patients without trauma (57%). Improved word perception test results in the presence of noise suggested that improvement in tinnitus and dizziness after occipital nerve block was related to improved attention. PMID- 10753419 TI - Prediction of vertigo recurrences in Meniere's disease by the head-shaking test. AB - Researchers have observed that when head-shaking nystagmus (HSN) is provoked in patients with peripheral vestibular disorders, usually (in more than 75% of cases) it beats toward the normal or unaffected ear. The reverse of this pattern occurs commonly in patients with Meniere's disease. This finding presumably reflects the changeable pathophysiological state of the labyrinth of Meniere's disease. We retrospectively analyzed clinical records of eight patients who had unilateral Meniere's disease and came to Gunma University Hospital for consultation in the period from 1984 through 1989. All patients satisfied the following condition: In the period prior to the attacks of vertigo, for which a 10-day period preceding the attack was arbitrarily considered (the forerunning period), HSN reversed its direction, appeared, or disappeared. When HSN showed a biphasic pattern, only the first phase was considered in this present analysis. In the period before the attack, HSN reversed its direction from the normal to the morbid ear five times in four patients, appeared toward the morbid ear in three patients, and disappeared from one beating toward the normal ear before the forerunning period of vertigo attacks in one patient. These findings suggest that the occurrence of HSN directed to the morbid ear in the recuperation period in Meniere's disease might indicate the impending recurrence of a vertigo attack in a few days. In the present group of patients, vertigo attacks occurred from 6 hours to 8 days (average, 3.2 days) after the observation of HSN beating toward the morbid ear. In three of these patients, the immediate administration of isosorbide (a hyperosmotic diuretic) in this stage successfully suppressed the recurrence of vertigo attacks. PMID- 10753420 TI - The migraine: benign paroxysmal vertigo of childhood complex. AB - The migraine--a benign paroxysmal vertigo of childhood complex is the most frequent etiology of pediatric dizziness, with an incidence of 34.7%. We present a series of 34 children in whom this entity was diagnosed. We describe the most important characteristics and discuss the possible etiological factors. We review the theories about a common pathophysiological origin for migraine, benign paroxysmal vertigo of childhood, and paroxysmal torticollis. Evolution of these entities confirms the idea of a common origin and a different vestibular symptomatology, depending on the age of the child experiencing dizziness. PMID- 10753421 TI - The medical audiological evaluation of tinnitus patients. AB - We outline our routine approach to tinnitus patients, highlighting the different aspects of our examination: the clinical history, objective ear, nose, and throat and general examination, and audiovestibular testing. We emphasize the interest in testing not only the cochlear but the vestibular function of the inner ear. In our view, this testing is of paramount importance, for the cochleovestibular system is a unit, acting as a whole. All these data are stored in a database bank. Then we present our preliminary results. Currently, we have 83 patients suffering from tinnitus, and their results are statistically presented. We characterize the population in terms of its gender distribution and mean age. The localization and characteristics of tinnitus and the associated symptoms also are noted. The results of the audiovestibulometric examinations-pure-tone audiometry, vocal audiometry, and brain evoked response audiometry craniocorpography and electronystagmography are discussed. We concluded that many tinnitus patients, even those who had no vestibular symptoms, showed some disturbances in the vestibular tests. This fact highlights the need for a complete cochleovestibular investigation in all patients complaining of tinnitus. PMID- 10753422 TI - Experiences with a computer-based electronystagmography system in examining totally deaf patients. PMID- 10753423 TI - The efficacy of Arlevert therapy for vertigo and tinnitus. AB - We were interested in determining the efficacy of Arlevert therapy in vertigo and tinnitus patients, especially as it compared to the efficacy of the drug's two component agents, dimenhydrinate and cinnarizine. We conducted a comparative, randomized, double blind, multicenter, parallel group study involving a group of patients (n = 122) with vertigo and tinnitus of peripheral or central origin. The three test groups, nearly equal in size, were administered three times daily for 4 weeks one of the three agents being evaluated. To evaluate therapeutic success, we studied patients' vertigo symptoms, concomitant vegetative symptoms, CCG parameters, and electronystagmographic and audiometric parameters. We also obtained subjective assessments of outcomes from the test patients. Our results led us to conclude that Arlevert is more effective than either of its component drugs alone in treating vertigo and tinnitus. The drug also was well tolerated. PMID- 10753424 TI - Medicolegal aspects of tinnitus. PMID- 10753425 TI - Forensic aspects of tinnitus in Belgium and France. AB - In Belgium and in France, tinnitus has to be considered together with the study of hearing loss to determine a degree of disability. PMID- 10753426 TI - Endogenous dynorphins: possible role in peripheral tinnitus. AB - Tinnitus has been defined as the perceptual correlate of altered spontaneous neural activity occurring without an external auditory stimulus. Hyperacusis, defined as a collapse of tolerance to sound, is present in 40-86% of those who suffer from disabling forms of tinnitus. Both phenomena often are induced or exacerbated by physical or psychological stress. Biological systems known to regulate the body's overall response to stress use and release endogenous neuroactive opioid peptides. These stress-related neuromodulators consist of products derived from three genetically distinct precursor hormones. Two of these precursor hormones are proenkephalin and prodynorphin. Enkephalin and dynorphin related peptides exist within the efferent olivocochlear systems (lateral and medial) of several mammalian species, including humans. Prodynorphin derivatives, however, may be restricted exclusively to lateral efferent neurons. Descending lateral efferent axons terminate solely on primary (type I) auditory dendrites innervating cochlear inner hair cells in most species. This action indicates that they play an important role in modulating auditory nerve sensitivity and spontaneous discharge. In a fashion similar to that exhibited by the observed excitatory mechanism of action of dynorphins in the spinal cord, sodium salicylate (aspirin) recently was shown to facilitate the excitatory effects of glutamate in the cochlea. This article provides support for a neurochemical model in which endogenous dynorphins may induce hyperacusis and can contribute to the induction, maintenance, or exacerbation of tinnitus in the auditory periphery by altering auditory type I neural excitability to glutamate. PMID- 10753427 TI - Descending auditory system/cerebellum/tinnitus. AB - The cerebellum and the descending auditory system (DAS) are considered clinically significant for influencing the development of the clinical course of tinnitus of the severe disabling type. It is hypothesized that the SPECT of Brain perfusion asymmetries in cerebellum, demonstrated since 1993, reflect clinically the influence of an aberrant auditory stimulus i.e. tinnitus, on the activity and function of the descending auditory system highlighted by the cerebellum and the acousticomotor systems. SPECT of Brain perfusion asymmetries in the cerebellum have been demonstrated in 60-70% of tinnitus patients of the central type. Electrophysiologic support for this finding includes interference in ocular fixation suppression of the vestibulocular (VOR) with rotation and position testing. Abnormalities in cerebellar function are considered to reflect the psychomotor component of tinnitus. Support for the hypothesis is demonstrated with one patient with a predominantly central type tinnitus of the severe disabling type with cerebellar perfusion asymmetries and associated electrophysiologic evidence of interference in the VOR with rotation testing. PMID- 10753428 TI - Correlations between audiogram and objective hearing tests in sensorineural hearing loss. AB - Owing to its subjective nature, behavioral pure-tone audiometry often is an unreliable testing method in uncooperative subjects, and assessing the true hearing threshold becomes difficult. In such cases, objective tests are used for hearing-threshold determination (i.e., auditory brainstem evoked potentials [ABEP] and frequency-specific auditory evoked potentials: slow negative response at 10 msec [SN-10]). The purpose of this study was to evaluate the correlation between pure-tone audiogram shape and the predictive accuracy of SN-10 and ABEP in normal controls and in patients suffering from sensorineural hearing loss (SNHL). One-hundred-and-fifty subjects aged 15 to 70, some with normal hearing and the remainder with SNHL, were tested prospectively in a double-blind design. The battery of tests included pure-tone audiometry (air and bone conduction), speech reception threshold, ABEP, and SN-10. Patients with SNHL were divided into four categories according to audiogram shape (i.e., flat, ascending, descending, and all other shapes). The results showed that ABEP predicts behavioral thresholds at 3 kHz and 4 kHz in cases of high-frequency hearing loss. Also demonstrated was that ABEP threshold estimation at 3 kHz was not affected significantly by audiogram contour. A good correlation was observed between SN-10 and psychoacoustic thresholds at 1 kHz, the only exception being the group of subjects with ascending audiogram, in which SN-10 overestimated the hearing threshold. PMID- 10753429 TI - Stress electrocochleography. AB - Electrocochleographic recordings after changes of middle-ear and intracranial pressure were studied in a group of otologically normal subjects and in patients with suspected Meniere's disease. Electrocochleography performed under these conditions was called stress electrocochleography. It was useful to distinguish among Meniere's disease, benign positional vertigo, and a round-window fistula. Changes of middle-ear pressure, whether positive or negative, always resulted in a rise of the SP/AP ratio. Raised intracranial pressure after Trendelenburg positioning showed fluctuations of the SP/AP ratio in both normal subjects and symptomatic patients. However, in normal subjects, a congruent pattern of these fluctuations was apparent, whereas symptomatic patients exhibited a discordant pattern. The discordant pattern, typical for Meniere's disease, showed an increase of the SP voltage, whereas the AP voltage decreased. In benign positional vertigo, the SP voltage decreased, and the AP voltage increased, once during Trendelenburg positioning and again at the end of the test when starting position was resumed. During raised intracranial pressure after the Queckenstedt maneuver, round-window fistulas showed an increase of the SP voltage, whereas the AP voltage decreased considerably. At our clinic, stress electrocochleography has become an important neurootological test in the differential diagnosis of Meniere's disease. PMID- 10753430 TI - Importance of behavior in response to tinnitus symptoms. AB - Among subjects affected by tinnitus, two groups are distinguished: patients who can cope positively with the symptom and patients who cannot cope with it. These differing attitudes suggest the necessity to study affected patients' "illness behavior" (i.e., a subjective interpretation of symptoms concerning body functioning). Our study considered 125 idiopathic tinnitus sufferers who requested a visit by an otorhinolaryngologist expressly for this symptom. All patients were invited to complete the illness behavior questionnaire (IBQ). IBQ mean score results were lower for affective inhibition and irritability and resulted in higher denial. Patients with more psychological suffering presented higher levels of hypochondria, disease convinction, and dysphoria. Results revealed a correlation between psychological suffering and tinnitus intensity: The group of patients with stronger psychological suffering included more subjects with a higher intensity level. The other group included more subjects with a moderate intensity level. Within the psychological evaluation of tinnitus sufferers, the IBQ results demonstrated particular sensitivity in revealing patients' nonadaptation area in coping with the symptom. PMID- 10753431 TI - The cervicogenic otoocular syndrome: a suspected forerunner of Meniere's disease. AB - Over a period of 4 years, 420 patients were observed with fullness in the ear, episodic vertigo, fluctuating hearing, and tinnitus. Of them, 182 patients showed normal hearing levels, a mild eustachian tube dysfunction, normal SP/AP ratios, mydriasis on the side of the affected ear, and a functional disorder of the upper cervical spine. These patients responded to conservative management, particularly physiotherapy. Additionally, 51 patients showed normal hearing levels, a mild eustachian tube dysfunction, an elevated SP/AP ratio suggesting endolymphatic hydrops, mydriasis on the side of the affected ear, and a functional disorder of the upper cervical spine. These patients were given a diagnosis of cervicogenic otoocular (COO) syndrome. Within this group, 43 patients did not respond to preceding conservative management, and 3 patients developed Meniere's disease within 12 months. Also within this group, 8 patients responded to physiotherapy only, with return of the SP/AP ratio to normal levels. A total of 187 patients had a sensorineural hearing loss, an elevated SP/AP ratio, mydriasis on the side of the affected ear, a functional disorder of the upper cervical spine; 186 had a mild eustachian tube dysfunction and had diagnosed Meniere's disease. Patients with a COO syndrome invariably responded to the insertion of a middle-ear ventilation tube, with return of the SP/AP ratio to normal levels and relief of symptoms. Patients with Meniere's disease had a mixed response. The COO syndrome is suspected to be a forerunner of Meniere's disease. PMID- 10753432 TI - Antiorthostatic posture as an earth model of the effect of microgravity on the human body. AB - We examined the horizontal and vertical component of diagonal optokinetic nystagmus (DOKN) in sitting posture and in the last 15 minutes of antiorthostatic posture at -30 degrees lasting 3 hours. The antiorthostatic posture is suited to the earthy model of the fluid shifting observed in microgravity. We found that the frequency of the vertical component of DOKN is rarer in antiorthostatic posture than in sitting posture. Moreover, the amplitude of the vertical component of DOKN is lower in antiorthostatic posture than is the amplitude of the horizontal component. According to our examinations, we suppose that the frequency and amplitude of vertical OKN are directed by different cerebral structures. Furthermore, we found that the heart rate becomes slower and the diastolic pressure is augmented after antiorthostatic posture and visual stimulation. PMID- 10753433 TI - Gustometry of diabetes mellitus patients and obese patients. AB - The sensation of adequate taste detection can be associated with satisfaction of food intake. The impairment of taste detection may be associated with the development of obesity. Taste detection is determined hereditarily, but it can be influenced also by the occurrence of neuropathy. To find an explanation for these phenomena, we investigated 73 patients with diabetes mellitus (DM) 2 (i.e., non insulin-dependent DM); 11 patients with DM 1 (i.e., insulin-dependent DM); 12 obese patients (body-mass index >30) without DM; and 29 control patients. All subjects underwent electrogustometric examination with Hortmman's electrogustometer. During this examination, we obtained electrical thresholds of taste by stimulating appropriate parts of the tongue. We stimulated the apex, middle, and near tongue radix areas on both sides. The resulting value is the average on the left and right sides of the mentioned areas. We considered a value of less than 40 microA to be normal. Values in excess of 100 microA are considered as hypogeusia. Values between 40 and 100 microA are taken as borderline, and ageusia is in excess of 500 microA. According to these criteria, in the DM 2 group, we found 40% of patients with hypogeusia, whereas in the DM 1 group, we found 33% of patients; 25% of patients were in the obese group. Among normal subjects (people without obesity or DM), no hypogeusia was found. We found ageusia in 5% of patients with DM 2, in 3% of patients with DM 1, and in 14% of obese patients. Among normal subjects, we found no ageusia. These results support the hypothesis that diminished taste detection can evoke hyperphagia and later obesity. PMID- 10753435 TI - Nutrition, biochemistry, and tinnitus. AB - Biochemical mechanisms are a significant factor in neurootological problems. Investigation of biochemical causes of neurootological problems, such as tinnitus, are warranted and can be cost-effective. Dietary, nutritional, chemical, hormonal, immunological, and stress factors are involved directly in neurootological problems and must be evaluated and considered in designing the treatment regimen for patients complaining of tinnitus. PMID- 10753436 TI - Bulbar myasthenia presenting as acute respiratory failure--a case report. PMID- 10753434 TI - Tinnitus program at Brasilia University Medical School. AB - Over the last 6 months, all patients seen at the otologic clinic of Brasilia University Medical School answered a questionnaire designed to identify and describe the symptom of tinnitus. A total of 500 patients reported and described this symptom. They underwent physical examination, laboratory tests and audiological evaluation. In their order of frequency, presbycusis, chronic otitis media, otosclerosis, acoustic trauma, Meniere's disease, ototoxicity, and vestibular schwannoma were found. Tinnitus was rated as minor in 81%, moderate in 18%, and severely disabling in 1%. Those who requested treatment for tinnitus were treated medically. Central vasodilators, vestibular suppressants, calcium channel blockers, anticholinergic drugs, anticonvulsant drugs, and gingko biloba were used with variable results. Tinnitus maskers were not used, but hearing prostheses were fitted when indicated. Treatment failed in the 1% with severe disabling tinnitus, and they were entered in a double-blind, randomized protocol for intratympanic dexamethasone injection. Under topical anesthesia, 0.2 ml of a 4-mg/ml dexamethasone solution (0.8 mg per injection) or 0.2 ml of normal saline was injected just posterior to the umbo. Patients remained supine for 20 minutes with the injected ear up and received four injections at 1-week intervals. Preliminary results are reported. Tinnitus is a very frequent symptom among our otologic patients, but most of them would not mention the symptom spontaneously, probably because for 81% it was mild. Curiously, the 5% of the severely disabling type tend to exhibit no clear cause, whereas the mild and moderate cases usually have an identifiable etiology. PMID- 10753437 TI - Optimization of tryptophan production in bacteria. Design of a strategy for genetic manipulation of the tryptophan operon for tryptophan flux maximization. AB - In the present work we have applied the indirect optimization method (Torres, N. V. et al. Biotechnol. Bioeng. 1996, 49, 247-258) to the maximization of tryptophan biosynthesis in Escherichia coli. The optimization procedure is applied to an updated model of this biochemical system (Xiu, Z-L et al., J. Biotechnol. 1997, 58, 125-140) and thus extended to a problem that includes the processes of transcription and translation. The model representation used by these authors is first translated into the corresponding S-system version. Then, to guarantee cell viability, we impose a set of constraints on some variable and parameter values, all of which are able to be modulated by available techniques. Our results show that it is possible to attain a stable and robust steady state with a rate of tryptophan production increased more than 4 times. This is achieved by changing four key parameters related to the efflux of tryptophan, the growth rate, the inhibition constant, and the tryptophan repressor level. Moreover, it is demonstrated that we can reach this optimum state in a sequential manner, each step leading us to a better situation in relation to the previous one. Thus, only by doubling the tryptophan excretion we can triplicate the rate of tryptophan production. A further, although lesser, improvement can be attained by increasing 4-fold the rate of growth and subsequently by weakening the inhibitory feedback interaction of tryptophan on the enzymes leading to its synthesis. Finally, a significant jump in the rate of production can be obtained if the level of the trp operon could be decreased. When a second approach was considered, in which the growth rate is kept constant in the optimized profile, we found out that by modulation of the parameters it is possible to increase more than 2-fold the rate of tryptophan production. PMID- 10753439 TI - Solid substrate fermentation of Monascus purpureus: growth, carbon balance, and consistency analysis. AB - Solid substrate fermentation (SSF) of Monascus purpureus on rice is a promising new technology for obtaining natural pigments. However, before attempts can be made at maximizing pigment yield, all significant macroscopic compounds should be assayed. Here, Monascus purpureus has been grown on rice in batch mode, and the evolution of the main components, biomass, residual rice, O(2), CO(2), ethanol, acetic acid, and pigments, have been followed. This set of data, never previously studied for Monascus SSF, allowed both the performance of a macroscopic elemental balance, which accounted for 83-94% of the initial substrate carbon, and a check of data consistency. Standard consistency analysis showed a significant underestimation of the nitrogen fraction of biomass, but it was unable to discriminate the errors in the carbon balance as a result of the simultaneous presence of two gross errors in the system. A simple stoichiometric model in tandem with consistency analysis explained unaccounted carbon as an underestimation of CO(2) and ethanol. Using the simplified method to estimate ethanol, the macroscopic balance accounted for 87-99% of the initial carbon. PMID- 10753438 TI - Production of functional hepatocyte growth factor (HGF) in insect cells infected with an HGF-recombinant baculovirus in a serum-free medium. AB - Three insect cell lines, SL-7B cells derived from Spodoptera litura, Sf9, and High Five (Hi-5) cells, were used for the production of pro-hepatocyte growth factor (pro-HGF). Cells were cultured and then infected with a recombinant HGF containing baculovirus in a serum-free medium. In SL-7B cells, pro-HGF is synthesized and excreted from the cells and late in infection is converted to a heterodimeric form of HGF even when the cells are grown in serum free medium. Conversion of a single-chain form of HGF (pro-HGF) into an HGF heterodimer was unexpected, as pro-HGF is normally cleaved by a serum protease called HGF activator. The proliferation activity of heparin-affinity-purified HGF from serum free culture supernatant of SL-7B cells is comparable to that obtained from HGF converted by serum proteases, suggesting that SL-7B cells produce a functionally analogous protease to correctly process pro-HGF. This work reports, for the first time, on the feasibility of properly processing pro-HGF to form functional HGF by proteases from invertebrate cells in serum-free media. Avoiding the supplementation of sera provides the advantages of a low production cost, zero contamination of infectious agents from sera, and simple downstream product purification. Experimental results further demonstrate that the conversion of pro HGF by insect cells is cell-line-dependent, because proteases in Hi-5 or Sf9 cells could not process pro-HGF as efficiently and properly as those in SL-7B cells. PMID- 10753440 TI - Glass transition temperatures and fermentative activity of heat-treated commercial active dry yeasts. AB - Differential scanning calorimetry thermograms of various samples of commercial instant active dry yeasts revealed a clear glass transition typical of amorphous carbohydrates and sugars. The resulting glass transition temperatures were found to decrease with increasing moisture content. The observed glass curve was similar to that of pure trehalose, which is known to accumulate in large amounts in baker's yeast. The effect of heat treatment at various temperatures on the fermentative activity (as measured by the metabolic production of CO(2)) of dry yeast was studied. First-order plots were obtained representing the loss of fermentative activity as a function of heating time at the various temperatures assayed. Significant losses of fermentative activity were observed in vitrified yeast samples. The dependence of rate constants with temperature was found to follow Arrhenius behavior. The relationship between the loss of fermentative activity and glass transition was not verified, and the glass transition was not reflected on the temperature dependence of fermentative activity loss. PMID- 10753441 TI - 13C NMR evidence for pyruvate kinase flux attenuation underlying suppressed acid formation in Bacillus subtilis. AB - When batch and continuous Bacillus subtilis cultures are provided with a small amount of citrate, acid production ceases, carbon yield increases by more than 2 fold, and the productivity of recombinant protein increases. It has been hypothesized that pyruvate kinase activity is attenuated, which in turn lowers glucose flux and minimizes the acid overflow prompted by low Krebs cycle capacity. To complement existing enzyme activity, linear programming, and metabolite pool studies, (13)C NMR studies were performed. Atom mapping and isotopomer mapping matrix methods were used to select the best glucose label. "Best" was defined such that the NMR spectra of glutamate associated with metabolizing labeled glucose via the different candidate metabolic trafficking scenarios would differ considerably in fine structure (e.g., relative singlet intensities). When experiments were performed with 1-(13)C glucose, the observed NMR spectra corresponded well to the one predicted to arise when the metabolic trafficking occurs according to a pyruvate kinase attenuation scenario. This evidence further fortifies the prospects for successfully basing a metabolic engineering strategy on reducing pyruvate kinase activity to better match glycolytic and Krebs cycle capacities. PMID- 10753443 TI - NADH-Regulated metabolic model for growth of Methylosinus trichosporiumOB3b. Cometabolic degradation of trichloroethene and optimization of bioreactor system performance. AB - A metabolic model describing growth of Methylosinus trichosporium OB3b and cometabolic contaminant conversion is used to optimize trichloroethene (TCE) conversion in a bioreactor system. Different process configurations are compared: a growing culture and a nongrowing culture to which TCE is added at both constant and pulsed levels. The growth part of the model, presented in the preceding article, gives a detailed description of the NADH regeneration required for continued TCE conversion. It is based on the metabolic pathways, includes Michaelis-Menten type enzyme kinetics, and uses NADH as an integrating and controlling factor. Here the model is extended to include TCE transformation, incorporating the kinetics of contaminant conversion, the related NADH consumption, toxic effects, and competitive inhibition between TCE and methane. The model realistically describes the experimentally observed negative effects of the TCE conversion products, both on soluble methane monooxygenase through the explicit incorporation of the activity of this enzyme and on cell viability through the distinction between dividing and nondividing cells. In growth-based systems, the toxicity of the TCE conversion products causes rapid cell death, which leads to wash-out of suspended cultures at low TCE loads (below microM inlet concentrations). Enzyme activity, which is less sensitive, is hardly affected by the toxicity of the TCE conversion products and ensures high conversions (>95%) up to the point of wash-out. Pulsed addition of TCE (0.014 0.048 mM) leads to a complete loss of viability. However, the remaining enzyme activity can still almost completely convert the subsequently added large TCE pulses (0.33-0.64 mM). This emphasizes the inefficient use of enzyme activity in growth-based systems. A comparison of growth-based and similar non-growth-based systems reveals that the highest TCE conversions per amount of cells grown can be obtained in the latter. Using small amounts of methane (negligible compared to the amount needed to grow the cells), NADH limitation in the second step of this two-step system can be eliminated. This results in complete utilization of enzyme activity and thus in a very effective treatment system. PMID- 10753442 TI - NADH-Regulated metabolic model for growth of Methylosinus trichosporium OB3b. Model presentation, parameter estimation, and model validation. AB - A biochemical model is presented that describes growth of Methylosinus trichosporium OB3b on methane. The model, which was developed to compare strategies to alleviate NADH limitation resulting from cometabolic contaminant conversion, includes (1) catabolism of methane via methanol, formaldehyde, and formate to carbon dioxide; (2) growth as formaldehyde assimilation; and (3) storage material (poly-beta-hydroxybutyric acid, PHB) metabolism. To integrate the three processes, the cofactor NADH is used as central intermediate and controlling factor-instead of the commonly applied energy carrier ATP. This way a stable and well-regulated growth model is obtained that gives a realistic description of a variety of steady-state and transient-state experimental data. An analysis of the cells' physiological properties is given to illustrate the applicability of the model. Steady-state model calculations showed that in strain OB3b flux control is located primarily at the first enzyme of the metabolic pathway. Since no adaptation in V(MAX) values is necessary to describe growth at different dilution rates, the organism seems to have a "rigid enzyme system", the activity of which is not regulated in response to continued growth at low rates. During transient periods of excess carbon and energy source availability, PHB is found to accumulate, serving as a sink for transiently available excess reducing power. PMID- 10753444 TI - Modeling photoheterotrophic growth kinetics of Rhodospirillum rubrum inrectangular photobioreactors. AB - Based on a previously established model for radiant light transfer in photobioreactors (PBR), taking into account absorption and scattering of light, a new knowledge model for coupling radiant light energy available and local growth kinetics in PBRs for the photoheterotrophic bacteria Rhodospirillum rubrum is discussed. A revised method is presented for the calculation of the absorption and scattering coefficients. The specific characteristics of the electron transfer chains in such microorganisms leads to definition of three different metabolic zones in the PBR, explaining the behavior of mean kinetics observed in a wide range of incident light fluxes. The model is validated in rectangular PBRs for five different carbon sources and proved robust and fully predictive. This approach can be considered for simulation and model-based predictive control of PBRs cultivating photoheterotrophic microorganisms. PMID- 10753445 TI - Effect of aeration during cell growth on ketone reactions by immobilized yeast. AB - The effect of aeration during cell growth on the subsequent reduction of 2 hexanone and 2-octanone by yeast cells entrapped in calcium alginate beads was studied. The reactions were conducted using 2-propanol as a sacrificial substrate to regenerate the cofactor NAD(H), and a mixture of (S)- and (R)-alcohols was produced. The use of strictly aerobic conditions when growing the cells resulted in the highest initial reaction rates, as well as the production of only a single product (i.e., the enantiomeric excess of the (S)-alcohols was 1.0). However, initial reaction rates decreased proportionally with fermentation time regardless of whether the yeast were grown aerobically or under both aerobic and anaerobic conditions. The data also suggest that it is the aerobic (or anaerobic) condition, rather than the cell growth phase, which is responsible for the results seen. PMID- 10753446 TI - Modeling retrovirus production for gene therapy. 1. Determination Of optimal bioreaction mode and harvest strategy. AB - Although retroviruses are a promising tool for gene therapy, there are two major problems limiting the establishment of viable industrial processes: retrovirus stability and low final yield in the supernatant. This fact emphasizes the need for an effective process optimization, not only at a genetic level but also at a bioprocess engineering level. In part 1 of this paper a mathematical model was developed to optimize the bioreaction yield by determining the best retrovirus harvest strategy in perfusion cultures. PA317 cells producing recombinant retroviruses were used to develop and test this model. Cell culture was performed in stirred tanks using porous supports. The parameters of the proposed model were experimentally determined for batch and perfusion cultures at 32 and 37 degrees C both with and without additives to enhance production; the model was then validated. This model allowed the determination of the optimal values of all operational variables included: batch and perfusion duration and perfusion rate. The highest productivity (2682 virus cm(-)(3) h(-)(1)) was obtained under the following conditions: batch at 37 degrees C for 53 h followed by perfusion at 32 degrees C for 23 h with a perfusion rate of 0.107 h(-)(1). This value was 3.5 fold higher than the best result obtained in batch cultures for the same conditions of titer and quality. A sensitivity analysis of the parameters showed that the parameters that affect most the final productivity depend on the bioreaction phase: cell growth in batch culture and production and product degradation in perfusion culture. In part 2 of this paper, this model is extended to the first step of downstream processing, and the addition of further steps to the process is discussed in order to achieve global process optimization. PMID- 10753447 TI - Increased heterologous protein production in Aspergillus niger fermentation through extracellular proteases inhibition by pelleted growth. AB - The dependence of filamentous fungal protease secretion on morphology was investigated by employing the recombinant Aspergillus niger strain AB4.1[pgpdAGLAGFP] which contains a gene for the glucoamylase-GFP (green fluorescence protein) fusion protein. Different inoculum levels were used to obtain different sizes of pellet or free mycelia. The extracellular protease activity of the cultures varied with the pellet size and decreased dramatically when the morphology was changed from free mycelia to pellets. The culture with an optimal pellet size of 1.6 mm was obtained from an inoculum of 4 x 10(6) spores/mL. It resulted in a specific protease activity of 158 units/L, only one third of that in free mycelial growth, and a maximum specific GFP yield of 0.98 mg/g (cell mass) compared to 0. 29 mg/g for free mycelial growth with an inoculum of 10(7) spores/mL. The results indicate that this bioprocessing strategy can be effectively used to inhibit protease activity in filamentous fungal fermentation and thereby to enhance heterologous protein production. PMID- 10753448 TI - Optimal design of protein production plants with time and size factor process models. AB - In this work we propose an optimization model for the design of a biotechnological multiproduct batch plant. A first level of detail posynomial model is constructed for each unit, as well as decisions regarding the structural optimization of the plant. A particular feature of this model is that it contains composite units in which semicontinuous items operate on the material contained by batch items. This occurs in the purification steps, in particular with the microfilters operating between retentate and permeate vessels, and with the homogenizer and ultrafilters operating on the material contained in a batch holding vessel. Also, the unit models rely on batch operating time expressions that depend on both the batch size and the size of semicontinuous items. The model takes into account all of the available options to increase the efficiency of the batch plant design: unit duplication in-phase and out-of-phase and intermediate storage tanks. The resulting mathematical model for the minimization of the plant capital cost is a mixed integer non-linear program (MINLP), which is solved to global optimality with an implementation of the outer approximation/ equality relaxation/ augmented penalty (OA/ER/AP) method. A plant that produces four recombinant proteins in eight processing stages is used to illustrate the proposed approach. An interesting feature of this example is that it represents an attempt to standardize a plant for the production of both therapeutic and nontherapeutic proteins; the model applied is generic and can thus be applied to any such modular plant. Results indicate that the best solution in terms of minimal capital cost contains no units in parallel and with intermediate storage tank allocation. PMID- 10753449 TI - Separation of Alcaligenes eutrophus cells containing Poly(3-hydroxybutyrate) from fermentation broth with pretreatment using Al- and Fe-based coagulants. AB - Alcaligenes eutrophus containing intracellular poly(3-hydroxybutyrate) was recovered from fermentation broth by centrifugation and filtration after pretreatment with Al- and Fe-based coagulants. Coagulation efficiency was largely affected by pH, and the optimum pH's for cell recovery were about 4.6-5.6 for the Al-based coagulants and about 5-8 for the Fe-based coagulants. Ammonium ions that combined with metals to form complex compounds increased the coagulant requirement, and the additional requirement of coagulant was found to be proportional to the ammonium concentration. In addition, various ligands in addition to ammonium ions contained in the culture medium interfered with the coagulation reaction and increased the coagulant requirement also. The coagulant requirement increased with the cell concentration regardless of coagulant type. The polymeric coagulants such as PACS, Hi-PAX, and Ferix-3 were more effective than nonpolymeric coagulants of aluminum sulfate and ferrous sulfate. The optimum dosages of the coagulants tested were determined over a broad range of cell concentration of 20.5-210 g/L. It was observed that the energy requirement for centrifugation could be greatly reduced with cell coagulation. PMID- 10753450 TI - Mixing in process vessels used in biopharmaceutical manufacturing. AB - The use of nonbaffled vessels for mixing applications is becoming common in the biopharmaceutical industry but is not sufficiently well studied. Orientation of the impellers off-centered and/or at an angle is necessary to enhance mixing and eliminate swirling that would result without a baffle in a standard tank. This study focuses on characterizing mixing in vessels with the hydrofoil axial flow impellers mounted off-center at 10 degrees to the vertical. Geometrically similar vessels ranging from 100 to 5000 L working volume were used in this study. Mixing performance was successfully correlated to vessel geometric factors. PMID- 10753451 TI - Renaturation of reduced ribonuclease A with a microsphere-induced refolding system. AB - We intended to refold reduced ribonuclease A (RNase A) using polymeric microspheres. Polymeric microspheres were allowed to react with dithiothreitol (DTT) to immobilize the disulfide and thiol moieties on their surface. The fully reduced RNase A was added to the dispersion of the modified microspheres. Protein refolding and renaturation were estimated by the change in the number of disulfide bonds of RNase A and the recovery of the enzymatic activity, respectively. Without microspheres, the activity gradually recovered with the increase in the number of disulfide bonds. However, the formation of disulfide bonds of reduced RNase A was accelerated by adding the modified microspheres, and the rate of renaturation was increased depending on the amount of charged DTT and the reaction time of the immobilization. These results indicate that modified microspheres significantly catalyze the recovery of active RNase A from the reduced form. The protein adsorption data demonstrated that the disulfide moieties of the modified microspheres react with the thiol moieties of the reduced RNase A to form a mixed disulfide. The thiol/disulfide exchange reaction can possibly proceed at the microsphere/protein interface, resulting in the formation of a correct three-dimensional structure. PMID- 10753452 TI - Transfection of cells mediated by biodegradable polymer materials with surface bound polyethyleneimine. AB - Poly(epsilon-CBZ-L-lysine) can be mixed with biodegradable polymers such as poly(D,L-lactic-co-glycolic acid) or poly(L-lactic acid) and formed into films, foams, or microspheres. Surface amino groups may then be deprotected with acid or lithium/liquid ammonia. The amino groups serve as a method to modify the surface by attachment of other molecules. In the present experiments, we show that these polymer materials, as films or foams, may be surface modified by the attachment of polyethyleneimine (PEI). Plasmid DNA attached to the PEI can transfect cells plated on the surface over several days. Covalent atachment of PEI was required for transfection to be efficient. PEI was also attached to surface-bound collagen on cell culture plates and was shown to mediate transfection. PMID- 10753453 TI - Bisphosphonate conjugation to proteins as a means to impart bone affinity. AB - Growth factors are endogenous proteins capable of stimulating new bone formation, but their clinical benefit for systemic stimulation of bone mass has not been demonstrated. The critical challenge is to deliver a significant dose of the proteins to bone after intravenous injection. This challenge may be overcome by derivatizing proteins with ligands that exhibit a high bone affinity (e.g., bisphosphonates). To demonstrate the feasibility of this approach, 1-amino-1,1 diphosphonate methane (aminoBP) was conjugated to a model protein, albumin. The conjugation was performed by (1) converting the amino group of aminoBP to a thiol group using 2-iminothiolane, (2) derivatizing the albumin amino groups with a thiol-reactive sulfosuccinimidyl-4-(N-maleimidomethyl)-1-cyclohexane carboxylate, and (3) reacting the derivatized albumin with thiolated aminoBP. Typically, 1-4 aminoBP molecules per albumin were obtained. The conjugated albumin exhibited a high affinity to hydroxyapatite that was proportional to the extent of conjugation. The conjugates were shown to exhibit a high affinity to bone matrix in vitro in a serum-containing medium. Once bound to bone matrix, the conjugates were found to desorb more slowly than the unmodified albumin, especially from bone whose organic matrix was removed by ashing. In conclusion, conjugation of bisphosphonates to albumin was shown to impart a high bone affinity to the protein, and such conjugates can be potentially targeted to bone. PMID- 10753455 TI - Gene expression profiling by DNA microarrays and metabolic fluxes in Escherichia coli. AB - DNA microarray technology was applied to detect differential transcription profiles of a subset of the Escherichia coli genome. A total of 111 E. coli genes, including those in central metabolism, key biosyntheses, and some regulatory functions, were cloned, amplified, and used as probes for detecting the level of transcripts. An E. coli strain was grown in glucose, acetate, and glycerol media, and the transcript levels of the selected genes were detected. Despite extensive studies on E. coli physiology, many new features were found in the regulation of these genes. For example, several genes were unexpectedly up regulated, such as pps, ilvG, aroF, secA, and dsbA in acetate and asnA and asnB in glycerol, or down-regulated, such as ackA, pta, and adhE in acetate. These genes were regulated with no apparent reasons by unknown mechanisms. Meanwhile, many genes were regulated for apparent purposes but by unknown mechanisms. For example, the glucose transport genes (ptsHI, ptsG, crr) in both acetate and glycerol media were down-regulated, and the ppc, glycolytic, and biosynthetic genes in acetate were also down-regulated because of the reduced fluxes. However, their molecular mechanisms remain to be elucidated. Furthermore, a group of genes were regulated by known mechanisms, but the physiological roles of such regulation remain unclear. This group includes pckA and aspA, which are up regulated in glycerol, and gnd and aspA, which are down- and up-regulated, respectively, in acetate. The DNA microarray technology demonstrated here is a powerful yet economical tool for characterizing gene regulation and will prove to be useful for strain improvement and bioprocess development. PMID- 10753456 TI - Covalent and metal-chelate immobilization of a modified 2-haloacid dehalogenase for the enzymatic resolution of optically active chloropropionic acid. AB - The stereospecific L-2-haloacid dehalogenase DehCI from Pseudomonas CBS3 was tagged with a peptide tail containing six histidines and overexpressed in Escherichia coli. The His-tagged protein was purified after a single-step affinity chromatography on Zn(2+)-chelating sepharose. The activity of the modified protein was tested after immobilization on Zn(2+)-chelating sepharose and on covalently bound acrylic polymer. Both immobilization systems were used for the transformation of racemic 2-chloropropionic acid into D-lactate and D chloropropionic acid. Although immobilization on chelating sepharose produced a limited increase in stability, covalent immobilization on acrylic polymer significantly extended the operational temperature and pH range of the enzyme: up to 60% of activity was recovered at either 80 degrees C or pH 11, whereas no activity could be detected under these conditions in the soluble or chelate immobilized enzyme. Both forms of immobilization extended the enzyme effective storage periods, and after 10 cycles of reutilization, 70% and 20% of the initial activity was recovered in the covalent- and chelate-immobilized enzyme, respectively. PMID- 10753454 TI - The quartz crystal microbalance as a continuous monitoring tool for the study of endothelial cell surface attachment and growth. AB - The quartz crystal microbalance (QCM) was used to monitor endothelial cell (EC) adhesion on the gold surface of an oscillating quartz crystal contained in a QCM device. A number of parameters were investigated. First, we observed differential QCM O-ring toxicities for ECs. Second, appropriate conditions for cell culture and QCM cell environment were identified that can eliminate large-scale frequency oscillations in the measurements. These artifacts are not due to added cells but originate in the time-dependent evaporation of water. Having eliminated these artifacts, we then demonstrated that the measured steady-state crystal frequency shift, Delta f, and motional resistance shift, DeltaR, were determined by the number of firmly attached ECs requiring trypsinization from the crystal surface. Last, following steady-state attachment of ECs, the EC growth stimulation by fibroblast growth factor was monitored in a continuous fashion by measuring f and R values over a 72 h. period. We observed the Delta f values to increase in a way that reflected the increase in EC number bound to the QCM surface. Following addition of ECs to the QCM, the time-dependent increase in DeltaR can be interpreted in terms of increase by the ECs of the energy dissipation properties of the solution at the solution-gold surface interface. This effect is due to their rapid surface attachment and the elaboration of their cytoskeletal properties. These results indicate that the QCM technique can be used for the study of EC attachment and growth and suggest its potential for the real time study of per unit surface area cell mass distribution dynamics and viscoelastic properties and the cells' responses to stresses or perturbations brought about using biologically active molecules. PMID- 10753457 TI - Effect of temperature on the separation of soybean 11 S and 7 S protein fractions during bipolar membrane electroacidification. AB - The purpose of this study was to evaluate the effect of temperature (10 and 27 degrees C) on the efficiency of bipolar membrane electroacidification (BMEA) to fractionate soybean proteins. BMEA is a technology derived from electrodialysis, based on the isoelectric precipitation of proteins. It appears that temperature has a significant effect on the selective precipitation of the soybean protein fractions, mainly 11 S and 7 S, during BMEA. At 27 degrees C, the precipitation profile of the four protein fractions is situated in a pH range from 6.6 to 4.4, with no possibility of separating any of theses fractions. However, at 10 degrees C, the 11 S globulin precipitates at a higher pH than at 27 degrees C, pH 6.7 vs 5.9, allowing the fractionation of 11 S from the other fractions. Using electroacidification it is possible to obtain a precipitate solution enriched in the 11 S fraction (71.8% of 11 S and 10.8% of 7 S) and a supernatant solution enriched in the 7 S fraction (46.6% of 7 S and 4.6% of 11S). PMID- 10753458 TI - Protein purification by ultrafiltration using a beta-galactosidase fusion tag. AB - The use of beta-galactosidase (465 kDa) as a fusion tag for ultrafiltration-based protein purification has been investigated. The target protein studied was thermophilic glucose dehydrogenase (157 kDa, GDH) from Thermoplasma acidophilum. An expression vector was constructed comprising the lacZ gene fused to a factor Xa cleavage sequence that was attached to the 5' end of the GDH gene. This gene fusion was expressed in Escherichia coli JM109 to yield a soluble protein that exhibited activities for both enzymes. Cleavage of this fusion protein (622 kDa) by factor Xa gave two smaller proteins that showed individual beta-galactosidase and GDH activity. A two-stage diafiltration process for protein purification was used in an ultrafiltration stirred cell. In the first stage, a 500 kDa membrane was used to retain the fusion protein and transmit smaller E. coli host proteins. Approximately 80% of the GDH activity was retained in this step. Following cleavage, the second stage utilized a 300 kDa membrane to fractionate the beta galactosidase and GDH. No beta-galactosidase was detected in the permeate solutions, and 97% of the GDH activity was recovered in the permeate. PMID- 10753459 TI - Quantification and characterization of volatiles evolved during extrusion of rice and soy flours. AB - NASA-Johnson Space Center is designing and building a habitat (Bioregenerative Planetary Life Support Systems Test Complex, BIO-Plex) intended for evaluating advanced life support systems developed for long-duration missions to the Moon or Mars where all consumables will be recycled and reused. A food system based on raw products obtained from higher plants (such as soybeans, rice, and wheat) may be a central feature of a biologically based Advanced Life Support System. To convert raw crops to edible ingredients or food items, multipurpose processing equipment such as an extruder is ideal. Volatile compounds evolved during the manufacturing of these food products may accumulate and reach toxic levels. Additionally, off-odors often dissipated in open-air environments without consequence may cause significant discomfort in the BIO-Plex. Rice and defatted soy flours were adjusted to 16% moisture, and triplicate samples were extruded using a tabletop single-screw extruder. The extrudate was collected in specially designed Tedlar bags from which air samples could be extracted. The samples were analyzed by GC-MS with special emphasis on compounds with Spacecraft Maximum Allowable Concentrations (SMACs). Results showed a combination of alcohols, aldehydes, ketones, and carbonyl compounds in the different flours. Each compound and its SMAC value, as well as its impact on the air revitalization system, was discussed. PMID- 10753460 TI - Continuous ethanol production from concentrated wood hydrolysates in an internal membrane-filtration bioreactor. AB - Continuous culture for the production of ethanol from wood hydrolysate was carried out in an internal membrane-filtration bioreactor. The hydrolysate medium was sterilized at a relatively low temperature of 60 degrees C with the intention of reducing the formation of inhibitory compounds during the sterilization. The maximum ethanol concentration and productivity obtained in this study were 76.9 g/L and 16.9 g/L-h, respectively, which were much higher than those (57.2-67 g/L and 0.3-1.0 g/L-h) obtained in batch cultures using hydrolysate media sterilized at 60 degrees C. The productivity was also found to be much higher than that (6.7 g/L-h) obtained in a continuous cell retention culture using a wood hydrolysate sterilized at 121 degrees C. These results show that the internal membrane filtration bioreactor in combination with low-temperature sterilization could be very effective for ethanol production from wood hydrolysate. PMID- 10753462 TI - A novel macromolecular prodrug concept exploiting endogenous serum albumin as a drug carrier for cancer chemotherapy. PMID- 10753461 TI - Isosteric replacement of acidic with neutral residues in extracellular loop-2 of the kappa-opioid receptor does not affect dynorphin A(1-13) affinity and function. PMID- 10753463 TI - N-3-substituted imidazoquinazolinones: potent and selective PDE5 inhibitors as potential agents for treatment of erectile dysfunction. AB - Phosphodiesterase type 5 (PDE5) inhibitors with improved PDE isozyme selectivity relative to sildenafil may result in agents for the treatment of male erectile dysfunction (MED) with a lower incidence of PDE-associated adverse effects. This paper describes the discovery of 14, a PDE5 inhibitor with improved potency and selectivity in vitro compared to sildenafil. This compound shows activity in a functional assay of erectile function comparable to that of sildenafil. PMID- 10753464 TI - Synthesis, conformational analysis, and biological activity of C thioribonucleosides related to tiazofurin. AB - The syntheses of furanthiofurin [5beta-D-(4'-thioribofuranosyl)furan-3 carboxamide, 1] and thiophenthiofurin [5beta-D-(4'-thioribofuranosyl)thiophene-3 carboxamide, 2], two C-thioribonucleoside analogues of tiazofurin, are described. Direct trifluoroacetic acid-catalyzed C-glycosylation of ethyl furan-3 carboxylate with 1-O-acetyl-2,3,5-tri-O-benzyl-4-thio-D-ribofuranose gave 2- and 5-glycosylated regioisomers, as a mixture of alpha and beta anomers. Ethyl 5 (2,3,5-tri-O-benzyl)-beta-D-(4'-thioribofuranosyl)furan-3-carboxylate (6beta) was debenzylated and then converted into the corresponding amide (furanthiofurin) by reaction with ammonium hydroxide. A similar C-glycosylation of ethyl thiophene-3 carboxylate with 1,2,3,5-tetra-O-acetyl-4-thio-D-ribofuranose catalyzed by stannic chloride afforded an anomeric mixture of 2- and 5-glycosylated regioisomers. Deacetylation of ethyl 5-(2,3,5-tri-O-acetyl)-beta-D-(4' thioribofuranosyl)thiophene-3-carboxylate (13beta) with methanolic ammonia and treatment of the ethyl ester with ammonium hydroxide gave thiophenthiofurin. The glycosylation site and anomeric configuration were established by (1)H NMR spectroscopy. Thiophenthiofurin was found to be cytotoxic in vitro toward human myelogenous leukemia K562, albeit 39-fold less than thiophenfurin, while furanthiofurin proved to be inactive. K562 cells incubated with thiophenthiofurin resulted in inhibition of inosine 5'-monophosphate dehydrogenase (IMPDH) and an increase in IMP pools with a concurrent decrease in GTP levels. From computational studies it was deduced that, among the C-nucleoside analogues of tiazofurin, activity requires an electrophilic sulfur adjacent to the C glycosidic bond and an energetically favorable conformer around chi = 0 degrees. Among these, the more constrained (least flexible) compounds (tiazofurin and thiophenfurin) are more active than the less constrained thiophenthiofurin. Those compounds which contain a nucleophilic oxygen in place of the thiazole or thiophene (oxazofurin, furanfurin, and furanthiofurin) show the least activity. PMID- 10753465 TI - Conformational selection of inhibitors and substrates by proteolytic enzymes: implications for drug design and polypeptide processing. AB - Inhibitors of proteolytic enzymes (proteases) are emerging as prospective treatments for diseases such as AIDS and viral infections, cancers, inflammatory disorders, and Alzheimer's disease. Generic approaches to the design of protease inhibitors are limited by the unpredictability of interactions between, and structural changes to, inhibitor and protease during binding. A computer analysis of superimposed crystal structures for 266 small molecule inhibitors bound to 48 proteases (16 aspartic, 17 serine, 8 cysteine, and 7 metallo) provides the first conclusive proof that inhibitors, including substrate analogues, commonly bind in an extended beta-strand conformation at the active sites of all these proteases. Representative superimposed structures are shown for (a) multiple inhibitors bound to a protease of each class, (b) single inhibitors each bound to multiple proteases, and (c) conformationally constrained inhibitors bound to proteases. Thus inhibitor/substrate conformation, rather than sequence/composition alone, influences protease recognition, and this has profound implications for inhibitor design. This conclusion is supported by NMR, CD, and binding studies for HIV-1 protease inhibitors/substrates which, when preorganized in an extended conformation, have significantly higher protease affinity. Recognition is dependent upon conformational equilibria since helical and turn peptide conformations are not processed by proteases. Conformational selection explains the resistance of folded/structured regions of proteins to proteolytic degradation, the susceptibility of denatured proteins to processing, and the higher affinity of conformationally constrained 'extended' inhibitors/substrates for proteases. Other approaches to extended inhibitor conformations should similarly lead to high-affinity binding to a protease. PMID- 10753466 TI - Cyclin-dependent kinase inhibition by new C-2 alkynylated purine derivatives and molecular structure of a CDK2-inhibitor complex. AB - A new series of 2,6,9-trisubstituted purines, characterized by the presence of a common alkynyl substituent at C-2 and a range of different anilino/benzylamino groups at C-6, were synthesized. These compounds were evaluated for their capacity to inhibit cyclin-dependent kinase activity (CDK1-cyclin B) in vitro. Compounds 4e (N-6-p-Cl-benzylamino derivative) and 5e (N-6-m-Cl-anilino derivative) exhibited the strongest inhibitory activity with an IC(50) of 60 nM. The structure of compound 4b (N-6-p-methoxybenzylamino derivative) in complex with human CDK2 was determined by X-ray crystallography, revealing the molecular basis of inhibition by this molecule. Subsequent molecular modeling studies allowed us to rationalize the SAR observed for these compounds. PMID- 10753467 TI - Novel benzofuran and benzothiophene biphenyls as inhibitors of protein tyrosine phosphatase 1B with antihyperglycemic properties. AB - Insulin resistance in the liver and peripheral tissues, together with a pancreatic cell defect, are the common causes of Type 2 diabetes. It is now appreciated that insulin resistance can result from a defect in the insulin receptor signaling system, at a site post binding of insulin to its receptor. Protein tyrosine phosphatases (PTPases) have been shown to be negative regulators of the insulin receptor. Inhibition of PTPases may be an effective method in the treatment of Type 2 diabetes. We have identified two novel series of benzofuran/benzothiophene biphenyl oxo-acetic acids and sulfonyl-salicylic acids as potent inhibitors of PTP1B with good oral antihyperglycemic activity. To assist in the design of these inhibitors, crystallographic studies have attempted to identify enzyme inhibitor interactions. Resolution of crystal complexes has suggested that the inhibitors bind to the enzyme active site and are held in place through hydrogen bonding and van der Waals interactions formed within two hydrophobic pockets. In the oxo-acetic acid series, hydrophobic substitutents at position-2 of the benzofuran/benzothiophene biphenyl framework interacted with Phe182 of the catalytic site and were very critical to the intrinsic activity of the molecule. The hydrophobic region of the catalytic-site pocket was exploited and taken advantage by hydrophobic substituents at either the alpha-carbon or the ortho aromatic positions of the oxo-acetic acid moiety. Similar ortho aromatic substitutions on the salicylic acid-type inhibitors had no effect, primarily due to the different orientation of these inhibitors in the catalytic site. The most active inhibitors of both series inhibited recombinant human PTP1B with phosphotyrosyl dodecapeptide TRDI(P)YETD(P)Y(P)YRK as the source of the substrate with IC(50) values in the range of 20-50 nM. Compound 68 was one of the most active compounds in vivo, normalizing plasma glucose levels at the 25 mg/kg dose (po) and the 1 mg/kg dose (ip). Compound 68 was also selective against several other PTPases. PMID- 10753468 TI - Synthesis and anticonvulsant and neurotoxic properties of substituted N-phenyl derivatives of the phthalimide pharmacophore. AB - A series of compounds including 4-amino (1), 3-amino (2), 4-nitro (3), 2-methyl-3 amino (4), 2-methyl-3-nitro (5), 2-methyl-4-amino (6), 2-methyl-4-nitro (7), 2 methyl-5-amino (8), 2-methyl-5-nitro (9), 2-methyl-6-amino (10), 2-methyl-6-nitro (11), 2,6-dimethyl (12), 2-methyl-3-carboxy (13), 2-methoxycarbonyl (14), 2 methyl-4-methoxy (15), 2,4-dimethoxy (16), 2-chloro-4-amino (17), and 2-chloro-4 nitro (18) N-phenyl substituents of phthalimide were evaluated along with N-[3 methyl-(2-pyridinyl)]phthalimide (19), N-(3-amino-2-methylphenyl)succinimide (20), and phenytoin for anticonvulsant and neurotoxic properties. Initial screening in the intraperitoneal (ip) maximal electroshock-induced seizure (MES) test and the subcutaneous pentylenetetrazol-induced seizure (scPtz) test in mice led to the selection of 1, 2, 4, 10, 12, 17, and 19 for oral MES evaluation in rats. The resultant ED(50) values for 4, 10, 17, and phenytoin were 8.0, 28.3, 5.7 and 29.8 mg/kg, respectively. In the batrachotoxin affinity assay, IC(50) values for 17 and phenytoin were 0.15 and 0.93 microM, respectively, and in the recently validated magnesium deficiency-dependent audiogenic seizure test, ED(50) values of 5.2 and 23 mg/kg were obtained for 17 and phenytoin, respectively. Electrophysiology studies on compound 17 point out its ability to (i) potentiate GABA-evoked current responses with a failure to directly activate the GABAA receptor and (ii) to affect, at 100 microM excitatory non NMDA, but not NMDA, receptors with a 25% block of kainate-evoked response. Electrophysiology measurements on voltage-gated sodium channels in N1E-115 neuroblastoma cells confirm voltage-dependent block of these channels by compound 17. In view of its interaction with multiple ion channels, one would predict that compound 17 might be active in a wide range of seizure models. PMID- 10753469 TI - Statistical molecular design of building blocks for combinatorial chemistry. AB - The reduction of the size of a combinatorial library can be made in two ways, either base the selection on the building blocks (BB's) or base it on the full set of virtually constructed products. In this paper we have investigated the effects of applying statistical designs to BB sets compared to selections based on the final products. The two sets of BB's and the virtually constructed library were described by structural parameters, and the correlation between the two characterizations was investigated. Three different selection approaches were used both for the BB sets and for the products. In the first two the selection algorithms were applied directly to the data sets (D-optimal design and space filling design), while for the third a cluster analysis preceded the selection (cluster-based design). The selections were compared using visual inspection, the Tanimoto coefficient, the Euclidean distance, the condition number, and the determinant of the resulting data matrix. No difference in efficiency was found between selections made in the BB space and in the product space. However, it is of critical importance to investigate the BB space carefully and to select an appropriate number of BB's to result in an adequate diversity. An example from the pharmaceutical industry is then presented, where selection via BB's was made using a cluster-based design. PMID- 10753470 TI - High-affinity, non-peptide agonists for the ORL1 (orphanin FQ/nociceptin) receptor. AB - The discovery of 8-(5,8-dichloro-1,2,3,4-tetrahydro-naphthalen-2-yl)-1-phenyl 1,3,8-triazaspiro[4.5]decan-4-one, 1a, as a high-affinity ligand for the human ORL1 (orphanin FQ/nociceptin) receptor led to the synthesis of a series of optimized ligands. These compounds exhibit high affinity for the human ORL1 receptor, exhibit moderate to good selectivity versus opioid receptors, and behave as full agonists in biochemical assays. In this paper we present the synthesis, structure-activity relationship (SAR), and biochemical characterization of substituted 1-phenyl-1,3,8-triazaspiro[4.5]decan-4-ones culminating in the discovery of 8-(5-methyl-1,2,3,4-tetrahydro-naphthalen-1-yl)-1 phenyl-1,3,8-triazaspiro[4.5]decan-4-one, 1p, and 8-acenaphten-1-yl-1-phenyl 1,3,8-triazaspiro[4.5]decan-4-one 1q, two high-affinity, potent ORL1 receptor agonists with good to moderate selectivity versus the other opioid receptors. PMID- 10753471 TI - Derivatives of (R)-1,11-methyleneaporphine: synthesis, structure, and interactions with G-protein coupled receptors. AB - The design and synthesis of a well-characterized novel ring system, (R) lambda1,11-methyleneaporphine [(R)-4], and 15 derivatives thereof are presented. The addition of various nucleophiles to (R)-lambda1,11-carbonylaporphine [(R)-11] or to the 1,11-hydroxymethyleneaporphine epimers gave separable mixtures of epimers. The epimeric ratios obtained in most reactions seem to be a result of steric factors directing the nucleophilic attack. The structure of the epimers was determined by a combination of X-ray crystallography (5 derivatives), NMR spectroscopy, and chemical correlation. Interesting and diverse pharmacological profiles of the derivatives were revealed through binding studies at serotonin 5 HT(7) and 5-HT(1A) receptors as well as at dopamine D(2A) receptors. Two derivatives appeared to be selective 5-HT(7) receptor antagonists. It is evident from our results that the novel ring system [(R)-4] provides a useful complement to other scaffolds available to medicinal chemists involved in studies of GPC receptors. PMID- 10753472 TI - Bis(phenazine-1-carboxamides): structure-activity relationships for a new class of dual topoisomerase I/II-directed anticancer drugs. AB - Ring-substituted bis(phenazine-1-carboxamides), linked by a (CH(2))(3)NMe(CH(2))(3)- chain, were prepared from the corresponding substituted phenazine-1-carboxylic acids by reaction of the intermediate imidazolides with bis(3-aminopropyl)methylamine. The compounds were evaluated for growth inhibitory activity in a panel of tumor cell lines, including P388 leukemia, Lewis lung carcinoma, and wild-type (JL(C)) and mutant (JL(A) and JL(D)) forms of human Jurkat leukemia. The latter mutant lines are resistant to topoisomerase (topo) II targeted agents because of lower levels of the enzyme. Analogues with small, lipophilic substituents (e.g., Me, Cl) at the 9-position were the most potent inhibitors, superior to the corresponding dimeric bis(acridine-4-carboxamides) (bis-DACA analogues). Several of the compounds were preferentially (up to 2-fold) more cytotoxic toward the mutant Jurkat lines than the wild-type. To test whether this selectivity was related to topoisomerase action, the most potent of the compounds (9-methyl) was evaluated in a cell-free system. It poisoned topo I at drug concentrations of 0.25 and 0.5 microM and inhibited the catalytic activity of both topo I and topo II at concentrations of 1 and 5 microM, respectively. Results from the NCI human tumor cell line panel showed the compounds had preferential activity toward colon tumor lines (on average 9.5-fold more active in the HT29 line than in the cell line panel as a whole). Several analogues produced significant growth delays in the relatively refractory subcutaneous colon 38 tumor model in vivo. In particular, the 9-methyl compound was substantially more potent in this tumor model than the clinical dual topo I/II poison DACA (total dose 90 versus 400 mg/kg) with comparable activity. The bis(phenazine-1-carboxamides) are a new and interesting class of dual topo I/II directed anticancer drugs. PMID- 10753474 TI - Lipopolycationic telomers for gene transfer: synthesis and evaluation of their in vitro transfection efficiency. AB - We report on the synthesis of a series of lipopolyamine telomers I-14,n, I-18,n, and II-18,n and on their in vitro gene-transfer capability. Their structure consists of a polyamine polar moiety, including n primary amine functions (from 1 to 70), connected to a hydrophobic moiety, including two hydrocarbon C14 or C18 chains, through a mercaptopropanoyl or mercaptoglyceryl unit and an amide or ether function. They were obtained by telomerization of N-[2 [(BOC)aminoethyl]]acrylamide with N,N-ditetradecyl- and N,N dioctadecylpropanamide-3-thiol and rac-1,2-dioctadecyloxypropane-3-thiol, respectively, then BOC deprotection. For N/P ratios (N = number of telomer amine equivalents; P = number of DNA phosphates) from 0.8 to 10, these lipopolyamines condensed DNA, with or without the use of DOPE, forming lipopolyplexes or teloplexes of mean sizes less than 200 nm. Some trends, structure-activity and structure-toxicity relationships, were established to achieve both highest in vitro transfection levels and cell viability. Thus, DNA formulations based on telomers I-14,20 and I-18,20 and for N/P ratios lower than 5 led to the most efficient teloplex formulations for plasmid delivery to lung epithelial A549 cells. PMID- 10753473 TI - Deltorphin II analogues with 6-hydroxy-2-aminotetralin-2-carboxylic acid in position 1. AB - Two approaches to the design of very active and highly selective delta opioid peptides were used to obtain new deltorphin analogues with altered hydrophobic and stereoelectronic properties. Deltorphin II analogues were synthesized with the substitution of Ile instead of Val at positions 5 and 6 in the address domain and 6-hydroxy-2-aminotetralin-2-carboxylic acid (Hat) instead of Tyr(1) in the message domain. In the radioreceptor-binding studies, in which type-specific tritiated opioid ligands were used, (R)- and (S)-Hat-deltorphins exhibited similar K(i) values, revealing high delta selectivity. The peptides displayed agonist properties in the in vitro bioassay, with IC(50) values in the subnanomolar range in the mouse vas deferens assay and in the micromolar or higher range in the guinea pig ileum assay, again demonstrating a high selectivity toward delta receptors. The agonist property of the new ligands was confirmed by means of [(35)S]GTPgammaS-binding experiments in membranes of the rat frontal cortex. PMID- 10753475 TI - Tyrosine kinase inhibitors. 17. Irreversible inhibitors of the epidermal growth factor receptor: 4-(phenylamino)quinazoline- and 4-(phenylamino)pyrido[3,2 d]pyrimidine-6-acrylamides bearing additional solubilizing functions. AB - 4-Anilinoquinazoline- and 4-anilinopyrido[3,2-d]pyrimidine-6-acrylamides substituted with solubilizing 7-alkylamine or 7-alkoxyamine side chains were prepared by reaction of the corresponding 6-amines with acrylic acid or acrylic acid anhydrides. In the pyrido[3,2-d]pyrimidine series, the intermediate 6-amino 7-alkylamines were prepared from 7-bromo-6-fluoropyrido[3,2-d]pyrimidine via Stille coupling with the appropriate stannane under palladium(0) catalysis. This proved a versatile method for the introduction of cationic solubilizing side chains. The compounds were evaluated for their inhibition of phosphorylation of the isolated EGFR enzyme and for inhibition of EGF-stimulated autophosphorylation of EGFR in A431 cells and of heregulin-stimulated autophosphorylation of erbB2 in MDA-MB 453 cells. Quinazoline analogues with 7-alkoxyamine solubilizing groups were potent irreversible inhibitors of the isolated EGFR enzyme, with IC(50[app]) values from 2 to 4 nM, and potently inhibited both EGFR and erbB2 autophosphorylation in cells. 7-Alkylamino- and 7-alkoxyaminopyrido[3,2 d]pyrimidines were also irreversible inhibitors with equal or superior potency against the isolated enzyme but were less effective in the cellular autophosphorylation assays. Both quinazoline- and pyrido[3,2-d]pyrimidine-6 acrylamides bound at the ATP site alkylating cysteine 773, as shown by electrospray ionization mass spectrometry, and had similar rates of absorptive and secretory transport in Caco-2 cells. A comparison of two 7-propoxymorpholide analogues showed that the pyrido[3,2-d]pyrimidine-6-acrylamide had greater amide instability and higher acrylamide reactivity, being converted to glutathione adducts in cells more rapidly than the corresponding quinazoline. This difference may contribute to the observed lower cellular potency of the pyrido[3,2 d]pyrimidine-6-acrylamides. Selected compounds showed high in vivo activity against A431 xenografts on oral dosing, with the quinazolines being superior to the pyrido[3,2-d]pyrimidines. Overall, the quinazolines proved superior to previous analogues in terms of aqueous solubility, potency, and in vivo antitumor activity, and one example (CI 1033) has been selected for clinical evaluation. PMID- 10753476 TI - Phosphinic derivatives as new dual enkephalin-degrading enzyme inhibitors: synthesis, biological properties, and antinociceptive activities. AB - The development of dual inhibitors of the two zinc metallopeptidases, neprilysin (neutral endopeptidase) and aminopeptidase N involved in the inactivation of the opioid peptides, enkephalins, represents an attractive physiological approach in the search for new analgesics devoid of the major drawbacks of morphine. Phosphinic compounds, corresponding to the general formula H(3)N(+)-CH(R(1)) P(O)(OH)-CH(2)-CH(R(2))-CONH-CH(R(3))-COO(-), able to act as transition-state analogues and to fit the S(1), S(1)', and S(2)' subsites of both enzymes were designed. Selection of the R(1), R(2), and R(3) residues for optimal recognition of these enzymes led to the first dual competitive inhibitors with K(i) values in the nanomolar range for neprilysin and aminopeptidase N. These compounds induce potent analgesic responses after intracerebroventricular or intravenous administrations in mice (hot plate test), and several of them were shown to be, at least, 10 times more potent than the previously described dual inhibitors. PMID- 10753477 TI - Synthesis and oral antitumor activity of tetrakis(carboxylato)platinum(IV) complexes. AB - A novel class of tetrakis(carboxylato)platinum(IV) complexes, [Pt(O(2)CR)(4)(dach)] (dach = trans-(+/-)-1,2-diaminocyclohexane; R = C(n)H(2n+1), n = 1 approximately 5), was synthesized and studied for physicochemical properties and oral antitumor activity. Lipophilicity and aqueous solubility of the title complexes were greatly dependent on the alkyl chain length of the carboxylate ligand, and their partition coefficient and solubility changed by 4 or 5 orders of magnitude from acetate to hexanoate complexes. On the other hand, the range of their cathodic reduction potential (-546 approximately 403 mV) depending on the chain length of the carboxylate ligand was relatively small. Among the title complexes, the tetrakis(propionato)platinum(IV) complex, [Pt(O(2)CC(2)H(5))(4)(dach)], with appropriate lipophilicity (log P = 0.18) and aqueous solubility (14.6 mg/mL) was found to exhibit better oral antitumor activity than JM216 against the human ovarian tumor xenograft SKOV3 in nude mice. PMID- 10753478 TI - Inhibition of protein kinase C(alpha) by dequalinium analogues: dependence on linker length and geometry. AB - Analogues of a bipartite compound, dequalinium (DECA) (quinolinium, 1,1'-(1,10 decanediyl)bis(4-amino-2-methyl diiodide)), were tested for inhibition of protein kinase C(alpha) (PKC(alpha)). In vitro assays of monomeric and dimeric analogues support a model in which DECA inhibits PKC(alpha) by an obligatory two-point contact, a unique mechanism among PKC inhibitors. The presence of unsaturation in the center of the C(10)-alkyl linker produced geometric isomers with different inhibitory potencies: cis IC(50) = 52 +/- 12 microM and trans IC(50) = 12 +/- 3 microM, where the trans isomer was equipotent to that of the saturated C(10) DECA. DECA analogues with longer, saturated linkers (C(12), C(14), or C(16)) exhibited enhanced inhibitory potencies which reached a plateau with the C(14) linker (IC(50) = 2.6 +/- 0.2 microM). Metastatic melanoma cells treated with 250 nM C(12)-, C(14)-, or C(16)-DECA and irradiated with long-wave UV light (which causes irreversible inhibition of PKC(alpha) by DECA) confirmed the linker dependent inhibition of intracellular PKC(alpha) activity. PMID- 10753479 TI - Protection against oxidative damage by iron chelators: effect of lipophilic analogues and prodrugs of N,N'-bis(3,4,5-trimethoxybenzyl)ethylenediamine- N,N' diacetic acid (OR10141). AB - N,N'-Bis(3,4,5-trimethoxybenzyl)ethylenediamine-N,N'-diacetic acid (1) was recently described as a new type of iron chelator for protection against oxidative damage. It has a low affinity for iron, but the corresponding iron complex undergoes a site-specific oxidation by hydrogen peroxide through intramolecular aromatic hydroxylation into a highly stable iron phenolato complex, which does not catalyze hydroxyl radical formation. The purpose of this local activation process is to minimize toxicity compared to strong iron chelators, which may interfere with normal iron metabolism. 1 efficiently protects biological molecules against oxidative damage in vitro but not intact cells because of poor membrane permeability. We show here that, among a series of prodrug esters and lipophilic analogues, membrane-permeant N,N'-bis(3,4,5 trimethoxybenzyl)ethylenediamine-N,N'-diacetic acid diacetoxymethyl ester (7) protects human skin fibroblasts against hydrogen peroxide toxicity with an IC(50) of 3 microM. These results thus demonstrate that, providing sufficient intracellular chelator concentration is reached, 1 efficiently protects cells against the deleterious effects of hydrogen peroxide. This strategy of oxidative activation should help the design of new chelators with better safety margins, which may be useful against oxidative damage under conditions where a prolonged administration is needed. PMID- 10753480 TI - Alpha(2) adrenoceptor agonists as potential analgesic agents. 2. Discovery of 4 (4-imidazo)-1,3-dimethyl-6,7-dihydro-thianaphthene as a high-affinity ligand for the alpha(2D) adrenergic receptor. PMID- 10753481 TI - Introduction to neurobehavioral issues in pallidotomy and pallidal stimulation. PMID- 10753482 TI - Behavioral complications of early pallidotomy. AB - A review of stereotactic medial pallidotomy of the 1950s in five neurosurgical centers is presented. The surgical technique varied from one center to the other. The results of surgery, however, seemed to be quite equal, being positive in 70 90% of the patients. The surgical mortality ranged from 0 to 13%. Behavioral complications were adequately analyzed and reported from one center only and published by three independent neurologists. The side effects included drowsiness (12%), confusion (13.6%), mental deterioration (5%), memory deficit (13.6%), and dysphasia (7.5-24%, the rate depending on the concomitant brain atrophy). Among permanent side effects, 5% of the patients presented with a mild postoperative mental deterioration, whereas 13.6% had a severe memory deficit. In the four other centers, the results and side effects were analyzed only by the surgeons and were more biased. A comparison of the results and complications between Leksell's early medial pallidotomy of 1951-1957 and recent medial pallidotomies of the 1990s from two centers showed that 40 years ago Leksell had at least as good results as, and less serious complications than, two representative neurosurgeons of today. Even when positive clinical results of GPi pallidotomy have recently been reported from several centers, the patients seem to have improved relatively little, the dyskinesias excepted, and the rate of side effects has been quite high. The author is afraid that medial pallidotomy will soon be abandoned as a method of choice in the surgical treatment of Parkinson's disease, as in fact happened 40 years ago. One should look for better surgical alternatives and targets outside of the medial pallidum. PMID- 10753483 TI - Neuropsychological outcome of GPi pallidotomy and GPi or STN deep brain stimulation in Parkinson's disease. AB - This paper highlights the neuropsychological sequelae of posteroventral pallidotomy (PVP) and deep brain stimulation (DBS) of the subthalamic nucleus (STN) and the internal segment of the globus pallidus (GPi) at 3/6 months postoperatively. Results are based on our extensive experience with PVP and our preliminary observations with DBS. Patients with borderline cognitive or psychiatric functioning risk postoperative decompensation. Nonlateralizing attentional and hemisphere-specific impairments of frontostriatal cognitive functions followed unilateral PVP. "Frontal" behavioral dyscontrol was observed in approximately 25% of patients. Three cases of staged bilateral PVP suggest that premorbid factors may predict outcome, although lesion size and location are also critical. Older patients are at risk for significant cognitive and behavioral decline after bilateral STN DBS, while GPi DBS may be safer. PMID- 10753485 TI - Neuropsychological outcome following unilateral stereotactic pallidotomy in intractable Parkinson's disease. AB - Neuropsychological functioning was examined at baseline and 2- to 3-month follow up in 40 subjects with advanced Parkinson's disease (PD) who underwent unilateral posteroventral pallidotomy. Most subjects demonstrated improved verbal learning, visual memory, confrontation naming, and figural fluency at follow-up. Right pallidotomy was associated with decreased cognitive flexibility and increased verbal fluency, whereas Left pallidotomy uniquely resulted in a decline in verbal fluency. Significant motor improvement was demonstrated in both groups. Pallidotomy appears to be an effective treatment for advanced PD, providing a significant improvement in motor functioning, while resulting in few deleterious neurocognitive changes in most cases. PMID- 10753484 TI - Impaired frontostriatal cognitive functioning following posteroventral pallidotomy in advanced Parkinson's disease. AB - We investigated the long-term effects of posteroventral pallidotomy on tests sensitive to the functional integrity of frontostriatal neural systems in a sample of 11 patients with advanced Parkinson's disease (PD). Patients were assessed within 1 month prior to surgery and at 12 months following pallidotomy. Changes in outcome measures were compared to a control sample of equally performing PD patients receiving nonsurgical medical management assessed over a 12-month period. Measures of cognitive abilities sensitive to frontostriatal functional integrity tested psychomotor processing speed, executive components of working memory, and reasoning. Additional tests of general mental status and semantic memory ability were utilized to assess the specificity of the effect of pallidotomy on cognitive function. Significant declines in performance on all measures sensitive to frontostriatal integrity were found for the surgery group but not the PD control group. No significant changes in performance were found on the measures of general mental status or semantic memory for either the surgery or PD control samples. These results suggest that the posteroventral pallidotomy selectively impairs performance on tests of frontostriatal cognitive abilities. PMID- 10753486 TI - The impact of lesion laterality on neuropsychological change following posterior pallidotomy: a review of current findings. AB - This paper reviews seven studies evaluating the impact of lesion laterality on the neuropsychological sequelae of posterior pallidotomy for treatment of Parkinson's disease. Left lesions of the internal globus pallidus (GPi) were associated with subtle deficits on measures sensitive to frontal lobe function. The findings of a randomized clinical trial including a patient control group indicated that many of these deficits were transient, resolving by 6 months following surgery. Right GPi lesions were not consistently associated with neuropsychological deficit, except in one study that included a significant proportion of demented patients. It is hypothesized that when neuropsychological decline is present following surgery, this reflects impingement of posterior GPi lesions into proximal regions such as anterior GPi or the external pallidum that participate in cognitive basal ganglia-thalamocortical circuits. The findings from neuroimaging will be important for elucidating the relationship between lesion locus and neuropsychological sequelae. PMID- 10753487 TI - Health-related quality of life in Parkinson's disease after pallidotomy and deep brain stimulation. AB - This study explored the multidimensional outcome of three neurosurgical interventions for Parkinson's disease (PD): pallidotomy (N = 23), pallidal deep brain stimulation (DBS) (N = 9), and thalamic DBS (N = 7). All patients completed the Sickness Impact Profile (SIP) and the Beck Depression Inventory. Pallidotomy patients also completed the Profile of Mood States, the Beck Anxiety Inventory, and a disease-specific quality of life (QOL) measure, the Parkinson's Disease Questionnaire (PDQ-39). Three months after surgery, all neurosurgical groups showed significant improvements in mood and function, including physical, psychosocial, and overall functioning. Pallidal DBS and pallidotomy patients who completed additional QOL measures reported decreased anxiety and tension, increased vigor, improved mobility and ability to perform activities of daily living, and decreased perceived stigma. Psychosocial dysfunction scores from the SIP were related to depressed mood both at baseline (r = .42) and at followup (r = .45), but the physical dysfunction subscale was not related to mood at either time point, suggesting that disruption of social relationships due to PD may have more impact on affective distress than physical symptoms alone. Results suggest that neurosurgical interventions for PD improve disabling PD motor symptoms and also improve several domains of quality of life. PMID- 10753488 TI - The behavioral complications of pallidal stimulation: a case report. AB - We report a case of recurrent manic episodes associated with chronic deep brain stimulation (DBS) targeting globus pallidus (GP) in the treatment of Parkinson's disease (PD). Cardinal PD symptoms and dyskinesia improved with DBS, and neuropsychological testing found improvements in visuospatial measures associated with left DBS and in verbal memory with right DBS when compared to the patient's preoperative baseline. Under conditions of right, left, and bilateral DBS, the patient experienced bouts of mania and hypomania lasting several days at a time. Positron emission tomography (PET) with (15)O-labeled water was performed after his first manic episode under four conditions: no stimulation, right DBS, left DBS, and bilateral DBS. Although no manic switch occurred during the course of the PET study, all three DBS conditions were associated with decreases in regional flow in the left parahippocampus and hippocampus and right mid-cingulate gyrus. Increases in flow in left inferior frontal area, bilateral insula, dorsolateral prefrontal cortex, and cuneus were common to all DBS conditions. GP stimulation in PD may be associated with behavioral and cognitive effects. Distributed blood flow changes observed with pallidal DBS support a role for the pallidum in cognition and affective regulation. PMID- 10753489 TI - Time factors in conscious processes: reply to Gilberto Gomes. AB - The critical reinterpretations of Libet's research by G. Gomes make speculative, unwarranted, and untested assumptions. These assumptions and arguments are analyzed and their status relative to Libet's findings is criticized. PMID- 10753490 TI - I like it, but I'm not sure why: can evaluative conditioning occur without conscious awareness? AB - There is good evidence that, in general, autonomic conditioning in humans occurs only when subjects can verbalize the contingencies of conditioning. However, one form of conditioning, evaluative conditioning (EC), seems exceptional in that a growing body of evidence suggests that it can occur without conscious contingency awareness. As such, EC offers a unique insight into what role contingency awareness might play in associative learning. Despite this evidence, there are reasons to doubt that evaluative conditioning can occur without conscious awareness. This paper aims to critically review the EC literature and to draw some parallels to what is known about autonomic conditioning. In doing so, some important general issues about measuring contingency awareness are raised. These issues are illustrated with a brief report of an experiment in which a sensitive measure of contingency awareness is compared against a commonly used measure. PMID- 10753491 TI - I like it, but only when I'm not sure why: evaluative conditioning and the awareness issue. PMID- 10753492 TI - Evaluative conditioning is pavlovian conditioning: issues of definition, measurement, and the theoretical importance of contingency awareness AB - In her commentary of Field (1999), Hammerl (1999) has drawn attention to several interesting points concerning the issue of contingency awareness in evaluative conditioning. First, she comments on several contentious issues arising from Field's review of the evaluative conditioning literature, second she critiques the data from his pilot study and finally she argues the case that EC is a distinct form of conditioning that can occur in the absence of contingency awareness. With reference to these criticisms, this reply attempts to address Hammerl's comments by exploring the issues of how awareness is defined, how it is best measured, and whether it is reasonable to believe that EC uniformly occurs in the absence of contingency awareness. The article concludes that the available evidence supports Field's proposition that EC is, in fact, Pavlovian learning. Copyright 2000 Academic Press. PMID- 10753493 TI - Electroencephalographic registration of low concentrations of isoamyl acetate. AB - Previous research has demonstrated electroencephalogram (EEG) changes in response to low-odor concentrations, resulting in near-chance detection. Such findings have been taken as evidence for olfaction without awareness. We replicated and extended previous work by examining EEG responses to water-water control, 0.0001, 0. 001, 0.01, and 1 ppm isoamyl acetate (IAA) in water paired with water only. Detection was above chance (>50%) for.001 and above, and alpha decreased only to those concentrations, suggesting that EEG changes corresponded to IAA awareness. However, when correct trial EEGs were compared to incorrect trial EEGs during.001 ppm, right posterior/central alpha decreased during incorrect trials and alpha decreased more globally (including frontal sites) during correct trials. These data may not reflect awareness or unawareness per se. Instead, results are discussed regarding activation of perceptual systems in the posterior region during incorrect trials and the activation of frontal action systems during a subset of correct trials. PMID- 10753494 TI - Coherent EEG indicators of cognitive binding during ambiguous figure tasks. AB - We tested the hypothesis that perception of an alternative image in ambiguous figures would be manifest as high-frequency (gamma) components that become synchronized over multiple scalp sites as a "cognitive binding" process occurs. For 171 combinations of data from 19 electrodes, obtained from 17 subjects and 10 replicate stimuli, we calculated the difference in correlation between the response to first seeing an ambiguous figure and when the alternative percept for that figure became consciously realized (cognitively bound). Numerous statistically significant correlation differences occurred in all frequency bands tested with ambiguous-figure stimulation, but not in two kinds of control data (a reaction-time test to sound stimuli and a no-task, mind-wandering test). Statistically significant correlation changes were widespread, involving frontal, parietal, central, and occipital regions of both hemispheres. Correlation changes were evident at each of five frequency bands, ranging up to 62.5 Hz. Most of the statistically significant correlation changes were not between adjacent sites but between sites relatively distant, both ipsilateral and contralateral. Typically, these correlation changes occurred in more than one frequency band. These results suggest that cognitive binding is a distinct mental state that is reliably induced by ambiguous-figure perception tasks. Coherent oscillations at multiple frequencies may reflect the mechanism by which such binding occurs. Moreover, different coherent frequencies may mediate different components of the total cognitive-binding process. PMID- 10753495 TI - Visual awareness due to neuronal activities in subcortical structures: a proposal. AB - It has been shown that visual awareness in the blind hemifield of hemianopic cats that have undergone unilateral ablations of visual cortex can be restored by sectioning the commissure of the superior colliculus or by destroying a portion of the substantia nigra contralateral to the cortical lesion (the Sprague effect). We propose that the visual awareness that is recovered is due to synchronized oscillatory activities in the superior colliculus ipsilateral to the cortical lesion. These oscillatory activities are normally partially suppressed by the inhibitory, GABAergic contralateral nigrotectal projection, and the destruction of the substantia nigra, or the sectioning of the collicular commissure, disinhibits the collicular neurons, causing an increase in the extent of oscillatory activity and/or synchronization between activities at different sites. This increase in the oscillatory and synchronized character is sufficient for the activities to give rise to visual awareness. We argue that in rodents and lower vertebrates, normal visual awareness is partly due to synchronized oscillatory activities in the optic tectum and partly due to similar activities in visual cortex. It is only in carnivores and primates that visual awareness is wholly due to cortical activities. Based on von Baerian recapitulation theory, we propose that, even in humans, there is a period in early infancy when visual awareness is partially due to activities in the superior colliculus, but that this awareness gradually disappears as the nigrotectal projection matures. PMID- 10753496 TI - Vector subtraction implemented neurally: a neurocomputational model of some sequential cognitive and conscious processes. AB - Although great progress in neuroanatomy and physiology has occurred lately, we still cannot go directly to those levels to discover the neural mechanisms of higher cognition and consciousness. But we can use neurocomputational methods based on these details to push this project forward. Here we describe vector subtraction as an operation that computes sequential paths through high dimensional vector spaces. Vector-space interpretations of network activity patterns are a fruitful resource in recent computational neuroscience. Vector subtraction also appears to be implemented neurally in primate frontal eye field activity, which computes dimensions of saccadic eye movements. We use this apparent neural implementation as a model and construct from it a general neurocomputational account of an important type of sequential cognitive and conscious process. We defend the biological plausibility of all components of the general model and show that it yields testable anatomical and physiological predictions. We close by suggesting some interesting consequences for consciousness if our model characterizes correctly the neural mechanisms producing a common type of episode in our conscious streams. PMID- 10753497 TI - Increased burn-induced immunosuppression in lipopolysaccharide-resistant mice. AB - Severe burns induce a state of immunosuppression, and the inflammatory response after burn injury may play a role in this phenomenon. This study examined the effect of the inflammatory response to endotoxin on burn-induced immunosuppression and oxidative stress. An endotoxin-resistant mouse strain (C3H/HeJ) and a normally responding mouse strain (C3H/HeN) were compared. The mice were separated into three groups of five animals for each experimental day: (1) saline, (2) buprenorphine, and (3) buprenorphine and 20% total body surface area burn. All animals were fed ad libitum. The inflammatory response was studied at 1, 4, 7, 10, and 14 days postburn. Proliferation of activated splenocytes in burn mice was significantly lower on days 7, 10, and 14 for the C3H/HeJ strain and on days 4 and 10 for the C3H/HeN strain. Globally, C3H/HeJ presented stronger immune suppression than C3H/HeN. Oxidative stress parameters (liver malonaldehyde, spleen metabolic activity, and thiol concentrations) were higher in endotoxin-resistant mice than in the control strain. Impairment of the inflammatory response was more pronounced and oxidative stress was greater in endotoxin-resistant burn mice than in normal burn controls. Buprenorphine administration was not related to depression of these immune parameters. The inflammatory response following burn injury may be beneficial to the immune system. PMID- 10753498 TI - The autoimmune accelerating yaa mutation does not accelerate murine AIDS. AB - Murine acquired immunodeficiency syndrome (MAIDS) is characterized by lymphoproliferation, polyclonal B cell activation resulting in the production of autoantibodies, and a progressive immunodeficiency. These are all hallmarks of some autoimmune diseases. Yaa is a Y-chromosome-linked gene that accelerates autoimmune diseases in some autoimmune-prone strains of mice. To further elucidate a possible relationship with autoimmunity, the effect of the Yaa gene on MAIDS was investigated. Analysis of phenotypic and functional disease parameters revealed that Yaa does not accelerate MAIDS disease. This is probably due to the generalized activation of most or all lymphoid cells in MAIDS, which cannot be enhanced by the Yaa gene. This result is in accordance with the selective enhancing effect of the Yaa gene on the immune response against self and foreign antigens in a specific genetic background. It suggests that the autoimmune response associated with MAIDS is a secondary phenomenon. Interestingly, even in wild-type C57BL/6 mice, autoantibody production may contribute overproportionally to the hypergammaglobulinemia associated with MAIDS. PMID- 10753499 TI - Soluble tumor necrosis factor receptor type I enhances tumor development and persistence in vivo. AB - Secretion of human soluble tumor necrosis factor receptor type I (sTNFRI) by the mouse fibrosarcoma cell line, L929, previously has been demonstrated to confer resistance to in vitro lysis by TNF and to LAK- and CTL-mediated cytolysis. These findings suggest that, in vivo, sTNFRI contributes to tumor survival by inhibiting these immunologic mechanisms. To evaluate this hypothesis, we compared the growth of sTNFRI-secreting L929 cells with that of the unmodified parental fibrosarcoma in an in vivo mouse transplantation model. Secretion of sTNFRI by L929 cells markedly enhanced their tumorigenicity and persistence in syngeneic recipients. This benefit was abrogated by sTNFRI-neutralizing antibodies induced by immunization prior to tumor challenge. These data demonstrate that sTNFRI directly influences tumor formation and persistence in vivo and suggest the selective removal and/or inactivation of sTNFRI as a promising new avenue for cancer immunotherapy. PMID- 10753500 TI - Interferon-gamma receptor polymorphisms determine strain differences in accessibility of activated lymphocyte NK-triggering antigens to recognition by self-reactive NK cells. AB - The "NK-triggering-antigen regulator" (Nktar) gene is a locus identified in the C57BL/6 genome which regulates the ability of unlabeled activated Con A blasts to compete for recognition of labeled syngeneic Con A blasts by BALB/c NK cells. Linkage analysis on Con A blasts from (BALB/c x CByB6F1) N2 backcross progeny for (1) relative level of competitive inhibition of BALB/c NK lysis of syngeneic Con A blasts and (2) genotypes at polymorphic microsatellite markers distributed throughout the mouse genome mapped the Nktar gene locus to a 5-cM region of chromosome 10 containing the interferon-gamma receptor (Ifngr) gene locus. N2 Con A blasts exhibited an inverse relationship between (a) their cell surface density of IFN-gammaR molecules detected by FACS with monoclonal anti-CD119 and (b) their cold target inhibition of BALB/c NK self-reactivity. Con A blasts from Ifngr(-/-) knockout mice showed a relatively high level of inhibition of BALB/c NK self lysis and a relatively low level of class I MHC, which were both reversed by transient transfection with the Ifngr gene. Sequencing studies showed that Balb/c Ifngr encodes a Gly(69) whereas C57BL/6 Ifngr encodes Glu(69) due to a difference at nucleotide 284. Sequencing studies of N2 progeny demonstrated 100% concordance between their Nktar inhibitory phenotype and their Ifngr genotype. These findings demonstrate that the Nktar and Ifngr loci are identical. They further indicate that polymorphisms related to the Ifngr locus and affecting expression of cell surface IFN-gammaR may underlie genetic differences in the availability of NK triggering antigens (NKTAgs) to recognition by self-reactive BALB/c NK cells by differentially affecting the ability of IFN-gammaR molecules to mediate up regulation of NKTAg-masking class I molecules. PMID- 10753501 TI - Pulmonary eosinophilia and production of MIP-1alpha are prominent responses to infection with pneumonia virus of mice. AB - Human eosinophils secrete two distinct ribonucleases that have antiviral activity against pathogens of the family Paramyxoviridae. To examine the role of eosinophils and their ribonucleases in host defense against paramyxovirus pathogens in vivo, we have developed a mouse model involving a viral pathogen that naturally targets a rodent host. In this work we describe infection of Balb/c mice with pneumonia virus of mice (PVM, strain J3666), a paramyxovirus pathogen found frequently among rodent populations. We show here that pulmonary eosinophilia is an immediate response to infection with PVM, with bronchoalveolar lavage fluid containing 12-14% eosinophils obtained as early as day 3 postinoculation. Infection is accompanied by the production of macrophage inflammatory protein-1-alpha (MIP-1alpha), a chemokine that has been associated with the pulmonary eosinophilia observed in response to respiratory syncytial virus infection in humans and with enhanced clearance of influenza virus in mice. Interestingly, we observed no changes in expression of the chemoattractants eotaxin and RANTES in response to PVM infection, and interleukin-5 remained undetectable throughout. These responses-clinical pathology, viral recovery, pulmonary eosinophilia, and production of MIP-1alpha-will provide a means for exploring the role of eosinophils, eosinophil secretory ribonucleases, and eosinophil chemoattractants in host defense against PVM and related paramyxovirus pathogens in vivo. PMID- 10753502 TI - Seasonal variations in cytokine expression and cell-mediated immunity in male rhesus monkeys. AB - Our objectives in this study were to examine seasonal changes in immune responses including cytokine profiles of male rhesus monkeys housed under natural lighting conditions. We also monitored circannual changes in the secretion of several immunomodulatory hormones as potential mediators of the seasonal shifts in immune status. Retrospectively, the medical records of a large group of rhesus monkeys were examined to determine whether a common disease (campylobacteriosis) in this species shows a seasonal pattern of prevalence. Results of the study showed that there was a seasonal shift in the frequency of cells expressing TH1 cytokines (interleukin-2 and interferon-gamma) versus the TH2 prototype cytokine (interleukin-4) by peripheral blood mononuclear cells (PBMC) collected during the winter and summer. The frequency of TH1-type cytokine synthesis in the summer was markedly greater than in the winter whereas TH2-type cytokine expression did not vary between the two seasons. The proliferative response of PBMC to mitogens and natural killer cell activity of PBMC also varied with the season. Several hormones (testosterone, leptin, and prolactin) that modulate immune function exhibited circannual patterns of secretion. The prevalence of Campylobacter infections was higher in the spring than during the summer, fall, or winter. The data suggest that seasonal fluctuations in immune system status may alter the ability of primates to successfully respond to pathogens, and this may be related to circannual patterns of secretion of immunomodulatory hormones. PMID- 10753503 TI - Nitric-oxide-dependent systemic immunosuppression in animals with progressively growing malignant gliomas. AB - The role of nitric oxide (NO) and adherent spleen cells in systemic immunosuppression developing in animals carrying malignant glioma isografts was analyzed. Rats harboring a subcutaneous glioma isograft for 3 weeks were immunized with glioma cells genetically engineered to express IFN-gamma. One week later spleen cells were tested for immune responsiveness in vitro. A decreased cytotoxic activity of NK-cells and T-cells compared to tumor-free animals immunized in parallel was shown. Spleen cell proliferative responses to tumor cells, SEA, and anti-CD3 were all significantly suppressed, as was the production of IFN-gamma and IL-10. Plastic adherent spleen cells from tumor-bearing rats suppressed the SEA-induced proliferative response and the production of IFN-gamma and IL-10 by nonadherent spleen cells from tumor-free rats. A major part of this suppression appears to be dependent on the production of NO because suppression was efficiently counteracted in vitro by the NO-synthase inhibitor N-nitro-l arginine methyl ester. Moreover, a significantly increased level of nitrite in culture supernatants correlated with the observed suppression. We conclude that the systemic immunosuppression associated with growing gliomas is in part mediated by mechanisms dependent on NO overproduction in adherent spleen cells. PMID- 10753504 TI - Composition, synthesis, and assembly of the embryonic chick retinal basal lamina. AB - To study the biology of basal laminae in the developing nervous system the protein composition of the embryonic retinal basal lamina was investigated, the site of synthesis of its proteins in the eye was determined, and basal lamina assembly was studied in vivo in two assay systems. Laminin, nidogen, agrin, collagen IV, and XVIII are major constituents of the retinal basal lamina. However, only agrin is synthesized by the retina, whereas the other matrix constituents originate from cells of the ciliary body, the lens, or the optic disc. The synthesis from extraretinal tissues infers that the retinal basal lamina proteins must be shed from their tissues of origin into the vitreous body and from there bind to receptor proteins provided by the retinal neuroepithelium. The fact that all proteins typical for the retinal basal lamina are abundant in the vitreous body and a new basal lamina is only formed when the vitreous body was directly adjacent to the retina is consistent with the contention of the vitreous body having a function in retinal basal lamina formation. Basal lamina assembly was also studied after disrupting the retinal basal lamina by intraocular injection of collagenase. The basal lamina regenerated after chasing the collagenase with Matrigel, which served as a collagenase inhibitor. The basal lamina was reconstituted within 6 h. However, the regenerated basal lamina was located deeper in the retina than normal by reconstituting along the retracted neuroepithelial endfeet demonstrating that these endfeet are the preferred site of basal lamina assembly. PMID- 10753505 TI - Ectopic adenoviral vector-directed expression of Sema3A in organotypic spinal cord explants inhibits growth of primary sensory afferents. AB - Sema3A (Sema III, SemD, collapsin-1) can induce neuronal growth cone collapse and axon repulsion of distinct neuronal populations. To study Sema3A function in patterning afferent projections into the developing spinal cord, we employed the recombinant adenoviral vector technique in embryonic rat spinal cord slices. Virus solution was injected in the dorsal aspect of organotypic spinal cord cultures with segmentally attached dorsal root ganglia (sc-DRG). In cultures grown in the presence of nerve growth factor (NGF), injected either with the control virus AdCMVLacZ or with vehicle only, afferent innervation patterns were similar to those of control. However, unilateral injection of AdCMVSema3A/AdCMVLacZ in sc-DRG slices revealed a strong inhibitory effect on NGF dependent sensory afferent growth. Ectopic Sema3A in the dorsal spinal cord, the target area of NGF-responsive DRG fibers in vivo, created an exclusion zone for these fibers and as a result they failed to reach and innervate their appropriate target zones. Taken together, gain of Sema3A function in the dorsal aspect of sc DRG cultures revealed a dominant inhibitory effect on NGF-dependent, nociceptive sensory DRG afferents, an observation in line with the model proposed by E. K. Messersmith et al. (1995, Neuron 14, 949-959), suggesting that Sema3A secreted by spinal cord cells can act to repel central sensory fibers during the formation of lamina-specific connections in the spinal cord. PMID- 10753506 TI - Bromodeoxyuridine specifically labels the regenerative stem cells of planarians. AB - The singular regenerative abilities of planarians require a population of stem cells known as neoblasts. In response to wounding, or during the course of cell turnover, neoblasts are signaled to divide and/or differentiate, thereby replacing lost cell types. The study of these pluripotent stem cells and their role in planarian regeneration has been severely hampered by the reported inability of planarians to incorporate exogenous DNA precursors; thus, very little is known about the mechanisms that control proliferation and differentiation of this stem cell population within the planarian. Here we show that planarians are, in fact, capable of incorporating the thymidine analogue bromodeoxyuridine (BrdU), allowing neoblasts to be labeled specifically during the S phase of the cell cycle. We have used BrdU labeling to study the distribution of neoblasts in the intact animal, as well as to directly demonstrate the migration and differentiation of neoblasts. We have examined the proposal that a subset of neoblasts is arrested in the G2 phase of the cell cycle by double-labeling with BrdU and a mitosis-specific marker; we find that the median length of G2 (approximately 6 h) is sufficient to account for the initial mitotic burst observed after feeding or amputation. Continuous BrdU-labeling experiments also suggest that there is not a large, slow-cycling population of neoblasts in the intact animal. The ability to label specifically the regenerative stem cells, combined with the recently described use of double stranded RNA to inhibit gene expression in the planarian, should serve to reignite interest in the flatworm as an experimental model for studying the problems of metazoan regeneration and the control of stem cell proliferation. PMID- 10753508 TI - Primary neuronal differentiation in Xenopus embryos is linked to the beta(3) subunit of the sodium pump. AB - In amphibian embryos, activation of additional sodium pumps in neural plate cell membranes ensures that neural plate-derived neurons differentiate subsequently in the neural tube. When the sodium pump is inhibited during the mid-neural fold stages, neuronal differentiation fails. The effect is irreversible. We find that these events operate through the Na pump beta(3) subunit. When neural plate specific Na pumps are activated, transcripts for beta(3) decline precipitately during the mid-neural fold stages, first in the neural plate and then in the dorsal mesoderm. As the neural tube closes, beta(3) returns, specifically in motor neurons and interneurons. Inhibition of the Na pump with the cardiac glycoside strophanthidin prevents the normal fall in beta(3) during neurulation: beta(3) is maintained in the neural plate until the neural tube closes, but lost from the dorsal mesoderm. Complete elimination of beta(3) transcripts from dorsal structures then occurs. Inhibiting the Na pump does not induce cell death (assessed by TUNEL staining) in the nervous system. Transcripts for X-Delta, NeuroD, and GSK3beta are not affected by inhibition of the Na pump. Xotch and N tubulin transcripts fall to very low levels and Xotch disappears permanently from the nervous system. When beta(3) transcript expression is maintained throughout neurulation, by over expression of injected mRNAs, Xotch is eliminated from the neural tube and somites and switches to the ectoderm. PMID- 10753507 TI - Targeted misexpression of constitutively active BMP receptor-IB causes bifurcation, duplication, and posterior transformation of digit in mouse limb. AB - Members of bone morphogenetic proteins (BMPs) play important roles in many aspects of vertebrate embryogenesis. In developing limbs, BMPs have been implicated in control of anterior-posterior patterning, outgrowth, chondrogenesis, and apoptosis. These diverse roles of BMPs in limb development are apparently mediated by different BMP receptors (BMPR). To identify the developmental processes in mouse limb possibly contributed by BMP receptor-IB (BMPR-IB), we generated transgenic mice misexpressing a constitutively active Bmpr-IB (caBmpr-IB). The transgene driven by the mouse Hoxb-6 promoter was ectopically expressed in the posterior mesenchyme of the forelimb bud, the lateral plate mesoderm, and the whole mesenchyme of the hindlimb bud. While the forelimbs appeared normal, the transgenic hindlimbs exhibited several phenotypes, including bifurcation, preaxial polydactyly, and posterior transformation of the anterior digit. However, the size of bones in the transgenic limbs seemed unaltered. Defects in sternum and ribs were also found. The bifurcation in the transgenic hindlimb occurred early in the limb development (E10.5) and was associated with extensive cell death in the mesenchyme and occasionally in the apical ectodermal ridge (AER). Sonic hedgehog (Shh) and Patched (Ptc) expression appeared unaffected in the transgenic limb buds, suggesting that the BMPR-IB mediated signaling pathway is downstream from Shh. However, ectopic Fgf4 expression was found in the anterior AER, which may account for the duplication of the anterior digit. An ectopic expression of Gremlin found in the transgenic limb bud would be responsible for the ectopic Fgf4 expression. The observations that Hoxd-12 and Hoxd-13 expression patterns were extended anteriorly provide a molecular basis for the posterior transformation of the anterior digit. Together these results suggest that BMPR-IB is the endogenous receptor to mediate the role of BMPs in anterior-posterior patterning and apoptosis in mouse developing limb. In addition, BMPR-IB may represent a critical component in the Shh/FGF4 feedback loop by regulating Gremlin expression. PMID- 10753509 TI - The ERK-1/2 signaling pathway is involved in the stimulation of branching morphogenesis of fetal mouse submandibular glands by EGF. AB - We have previously reported that epidermal growth factor (EGF) stimulates branching morphogenesis of the fetal mouse submandibular gland (SMG) (M. Kashimata and E. W. Gresik, 1997, Dev. Dyn. 208, 149-161) and that the EGF receptor (EGFR) is localized principally, if not exclusively, on the epithelial components of the fetal SMG (E. W. Gresik, M. Kashimata, Y. Kadoya, R. Mathews, N. Minami, and S. Yamashina, 1997, J. Histochem. Cytochem. 45, 1651-1657). The EGFR is a receptor tyrosine kinase, and after binding of its ligand, it triggers several intracellular signaling cascades, among them the one activating the mitogen-activated protein kinases (MAPK) ERK-1/2. Here we investigated whether EGF utilizes the ERK-1/2 signaling cascade to stimulate branching morphogenesis in the fetal mouse SMG. SMG rudiments were collected as matched pairs at E14, E16, and E18 (E0 = day of vaginal plug); placed into wells of defined medium (BGJb); and exposed to EGF for 5 or 30 min or to medium alone (controls). By Western blotting we found that EGF induced the appearance of multiple bands of phosphotyrosine-containing proteins, including bands at 170 kDa and 44 kDa/42 kDa, presumably corresponding to the phosphorylated forms of EGFR and ERK-1/2, respectively. Other blots showed the specific appearance of the phosphorylated EGFR and of phospho-ERK-1/2 in response to EGF. Immunohistochemical staining for phosphotyrosine increased at the plasma membrane after EGF stimulation for 5 or 30 min. Diffuse cytoplasmic staining for MEK-1/2 (the MAPK kinase that activates ERK-1/2) increased near the cell membrane after EGF stimulation. Phospho-ERK-1/2 was localized in the nuclei of a few epithelial cells after EGF for 5 min, but in the nuclei of many cells after EGF for 30 min. PD98059, an inhibitor of phosphorylation and activation of MEK-1/2, by itself inhibited branching morphogenesis and, furthermore, decreased the stimulatory effect of EGF on branching. Western blots confirmed that this inhibitor blocked phosphorylation of ERK-1/2 in fetal SMGs exposed to EGF. These results show that components of the ERK-1/2 signaling cascade are present in epithelial cells of the fetal SMG, that they are activated by EGF, and that inhibition of this cascade perturbs branching morphogenesis. However, EGF did not cause phosphorylation of two other MAPKs, SAPK/JNK or p38MAPK, in fetal SMGs. These results imply that the ERK-1/2 signaling is responsible, at least in part, for the stimulatory effect of EGF on branching morphogenesis of the fetal mouse SMG. PMID- 10753510 TI - Identification of a proliferating marginal zone of retinal progenitors in postnatal chickens. AB - In warm-blooded vertebrates it is generally accepted that after early stages of development new neurons are not added to the retina. Contrary to this belief, we show here that hatched chickens have a zone of proliferating cells at the peripheral margin of the retina, similar to that of fish and amphibians. We found that cells at the peripheral edge of the retina incorporated the thymidine analog BrdU and expressed the cell cycle regulator proliferating cell nuclear antigen (PCNA). Furthermore, cells in the ciliary epithelium and retinal margin coexpressed the homeodomain transcription factors Pax6 and Chx-10, similar to multipotent progenitors of embryonic retina. Expression of PCNA, Pax6, and Chx-10 in cells at the retinal margin was maintained in adult birds. Double-labeling studies showed that BrdU-labeled cells that were integrated into the retina expressed proteins found only in differentiated neurons. Increased rates of ocular growth, induced by visual deprivation, resulted in increased numbers of BrdU-labeled cells at the retinal margin. Unlike the progenitors in the retinal marginal zone of fish and amphibians, the progenitors of the chick retina do not increase their rate of proliferation in response to acute damage. Furthermore, insulin, insulin-like growth factor-I, and epidermal growth factor increased proliferation of progenitors at the retinal margin, while basic fibroblast growth factor had no effect. These results indicate that the avian retina has a marginal growth zone containing proliferating cells that share similarities with multipotent embryonic retinal progenitors and the retinal stem cells of cold blooded vertebrates. PMID- 10753511 TI - The ecdysone regulatory pathway controls wing morphogenesis and integrin expression during Drosophila metamorphosis. AB - Drosophila imaginal discs are specified and patterned during embryonic and larval development, resulting in each cell acquiring a specific fate in the adult fly. Morphogenesis and differentiation of imaginal tissues, however, does not occur until metamorphosis, when pulses of the steroid hormone ecdysone direct these complex morphogenetic responses. In this paper, we focus on the role of ecdysone in regulating adult wing development during metamorphosis. We show that mutations in the EcR ecdysone receptor gene and crooked legs (crol), an ecdysone-inducible gene that encodes a family of zinc finger proteins, cause similar defects in wing morphogenesis and cell adhesion, indicating a role for ecdysone in these morphogenetic responses. We also show that crol and EcR mutations interact with mutations in genes encoding integrin subunits-a family of alphabeta heterodimeric cell surface receptors that mediate cell adhesion in many organisms. alpha Integrin transcription is regulated by ecdysone in cultured larval organs and some changes in the temporal patterns of integrin expression correlate with the ecdysone titer profile during metamorphosis. Transcription of alpha- and beta integrin subunits is also altered in crol and EcR mutants, indicating that integrin expression is dependent upon crol and EcR function. Finally, we describe a new hypomorphic mutation in EcR which indicates that different EcR isoforms can direct the development of adult appendages. This study provides evidence that ecdysone controls wing morphogenesis and cell adhesion by regulating integrin expression during metamorphosis. We also propose that ecdysone modulation of integrin expression might be widely used to control multiple aspects of adult development. PMID- 10753512 TI - Regulation of germ cell and Sertoli cell development by activin, follistatin, and FSH. AB - We have demonstrated a role for activin A, follistatin, and FSH in male germ cell differentiation at the time when spermatogonial stem cells and committed spermatogonia first appear in the developing testis. Testis fragments from 3-day old rats were cultured for 1 or 3 days with various combinations of these factors, incubated with bromodeoxyuridine (BrdU) to label proliferating cells, and then processed for stereological analysis and detection of BrdU incorporation. Gonocyte numbers were significantly elevated in cultures treated with activin, while the combination of FSH and the activin antagonist, follistatin, increased the proportion of spermatogonia in the germ cell population after 3 days. All fragment groups treated with FSH contained a significantly higher proportion of proliferating Sertoli cells, while activin and follistatin each reduced Sertoli cell division. In situ hybridization and immunohistochemistry on normal rat testes demonstrated that gonocytes, but not spermatogonia, contain the activin beta(A) subunit mRNA and protein. In contrast, gonocytes first expressed follistatin mRNA and protein at 3 days after birth, concordant with the transition of gonocytes to spermatogonia. Collectively, these data demonstrate that germ cells have the potential to regulate their own maturation through production of endogenous activin A and follistatin. Sertoli cells were observed to produce the activin/inhibin beta(A) subunit, the inhibin alpha subunit, and follistatin, demonstrating that these cells have the potential to regulate germ cell maturation as well as their own development. These findings indicate that local regulation of activin bioactivity may underpin the coordinated development of germ cells and somatic cells at the onset of spermatogenesis. PMID- 10753513 TI - Function for Hedgehog genes in zebrafish retinal development. AB - The hedgehog (hh) genes encode secreted signaling proteins that have important developmental functions in vertebrates and invertebrates. In Drosophila, expression of hh coordinates retinal development by propagating a wave of photoreceptor differentiation across the eye primordium. Here we report that two vertebrate hh genes, sonic hedgehog (shh) and tiggy-winkle hedgehog (twhh), may perform similar functions in the developing zebrafish. Both shh and twhh are expressed in the embryonic zebrafish retinal pigmented epithelium (RPE), initially in a discrete ventral patch which then expands outward in advance of an expanding wave of photoreceptor recruitment in the subjacent neural retina. A gene encoding a receptor for the hedgehog protein, ptc-2, is expressed by retinal neuroepithelial cells. Injection of a cocktail of antisense (alphashh/alphatwhh) oligonucleotides reduces expression of both hh genes in the RPE and slows or arrests the progression of rod and cone photoreceptor differentiation. Zebrafish strains known to have mutations in Hh signaling pathway genes similarly exhibit retardation of photoreceptor differentiation. We propose that hedgehog genes may play a role in propagating photoreceptor differentiation across the developing eye of the zebrafish. PMID- 10753514 TI - Expression of a novel receptor tyrosine kinase gene and a paired-like homeobox gene provides evidence of differences in patterning at the oral and aboral ends of hydra. AB - Axial patterning of the aboral end of the hydra body column was examined using expression data from two genes. One, shin guard, is a novel receptor protein tyrosine kinase gene expressed in the ectoderm of the peduncle, the end of the body column adjacent to the basal disk. The other gene, manacle, is a paired-like homeobox gene expressed in differentiating basal disk ectoderm. During regeneration of the aboral end, expression of manacle precedes that of shin guard. This result is consistent with a requirement for induction of peduncle tissue by basal disk tissue. Our data contrast with data on regeneration of the oral end. During oral end regeneration, markers for tissue of the tentacles, which lie below the extreme oral end (the hypostome), are detected first. Later, markers for the hypostome itself appear at the regenerating tip, with tentacle markers displaced to the region below. Additional evidence that tissue can form basal disk without passing through a stage as peduncle tissue comes from LiCl induced formation of patches of ectopic basal disk tissue. While manacle is ectopically expressed during formation of basal disk patches, shin guard is not. The genes examined also provide new information on development of the aboral end in buds. Although adult hydra are radially symmetrical, expression of both genes in the bud's aboral end is initially asymmetrical, appearing first on the side of the bud closest to the parent's basal disk. The asymmetry can be explained by differences in positional information in the body column tissue that evaginates to form a bud. As predicted by this hypothesis, grafts reversing the orientation of evaginating body column tissue also reverse the orientation of asymmetrical gene expression. PMID- 10753515 TI - Vitamin A deficiency results in the dose-dependent acquisition of anterior character and shortening of the caudal hindbrain of the rat embryo. AB - The developing nervous system is particularly vulnerable to vitamin A deficiency. Retinoid has been proposed to be a posteriorizing factor during hindbrain development, although direct evidence in the mammalian embryo is lacking. In the present study, pregnant vitamin A-deficient (VAD) rats were fed purified diets containing varying levels of all-trans-retinoic acid (atRA; 0, 0.5, 1.5, 6, 12, 25, 50, 125, or 250 microg/g diet) or were supplemented with retinol. Hindbrain development was studied from embryonic day 10 to 12.5 ( approximately 6 to 40 somites). Normal morphogenesis was observed in all embryos from groups fed 250 microg atRA/g diet or retinol. The most caudal region of the hindbrain was the most sensitive to retinoid insufficiency, as evidenced by a loss of the hypoglossal nerve (cranial nerve XII) in embryos from the 125 microg atRA/g diet group. Further reduction of atRA to 50 microg/g diet led to the loss of cranial nerves IX, X, XI, and XII and associated sensory ganglia IX and X in all embryos as well as the loss of hindbrain segmentation caudal to the rhombomere (r) 3/4 border in a subset of embryos. Dysmorphic orthotopic otic vesicles or immature otic-like vesicles in both orthotopic and caudally ectopic locations were also observed. As the level of atRA was reduced, a loss of caudal hindbrain segmentation was observed in all embryos and the incidence of otic vesicle abnormalities increased. Perturbations in hindbrain segmentation, cranial nerve formation, and otic vesicle development were associated with abnormal patterning of the posterior hindbrain. Embryos from VAD dams fed between 0.5 and 50 microg atRA/g diet exhibited Hoxb-1 protein expression along the entire neural tube caudal to the r3/r4 border at a time when it should be restricted to r4. Krox-20 protein expression was expanded in r3 but absent or reduced in presumptive r5. Hoxd-4 mRNA expression was absent in the posterior hindbrain, and the rostral limit of Hoxb-5 protein expression in the neural tube was anteriorized, suggesting that the most posterior hindbrain region (r7/r8) had been deleted and/or improperly patterned. Thus, when limiting amounts of atRA are provided to VAD dams, the caudal portion of the hindbrain is shortened and possesses r4/r5 like characteristics, with this region finally exhibiting r4-like gene expression when retinoid is restricted even more severely. Thus, regions of the anterior hindbrain (i.e., r3 and r4) appear to be greatly expanded, whereas the posterior hindbrain (r5-r8) is reduced or absent. This work shows that retinoid plays a critical role in patterning, segmentation, and neurogenesis of the caudal hindbrain and serves as an essential posteriorizing signal for this region of the central nervous system in the mammal. PMID- 10753516 TI - The unique developmental program of the acoel flatworm, Neochildia fusca. AB - Acoel embryos exhibit a unique form of development that some investigators argue is related to that found in polyclad turbellarians and coelomate spiralians, which display typical quartet spiral cleavage. We generated the first cell lineage fate map for an acoel flatworm, Neochildia fusca, using modern intracellular lineage tracers to assess the degree of similarity between these distinct developmental programs. N. fusca develops via a "duet" cleavage pattern in which second cleavage occurs in a leiotropically oblique plane relative to the animal-vegetal axis. At the four-cell stage, the plane of first cleavage corresponds to the plane of bilateral symmetry. All remaining cleavages are symmetrical across the sagittal plane. No ectomesoderm is formed; the first three micromere duets generate only ectodermal derivatives. Endomesoderm, including the complex assemblage of circular, longitudinal, and oblique muscle fibers, as well as the peripheral and central parenchyma, is generated by both third duet macromeres. The cleavage pattern, fate map, and origins of mesoderm in N. fusca share little similarity to that exhibited by other spiralians, including the Platyhelminthes (e.g., polyclad turbellarians). These findings are considered in light of the possible evolutionary origins of the acoel duet cleavage program versus the more typical quartet spiral cleavage program. Finally, an understanding of the cell-lineage fate map allows us to interpret the results of earlier cell deletion studies examining the specification of cell fates within these embryos and reveals the existence of cell-cell inductive interactions in these embryos. PMID- 10753517 TI - Analysis of gene expressions during Xenopus forelimb regeneration. AB - Xenopus laevis can regenerate an amputated limb completely at early limb bud stages, but the metamorphosed froglet gradually loses this capacity and can regenerate only a spike-like structure. We show that the spike formation in a Xenopus froglet is nerve dependent as is limb regeneration in urodeles, since denervation concomitant with amputation is sufficient to inhibit the initiation of blastema formation and fgf8 expression in the epidermis. Furthermore, in order to determine the cause of the reduction in regenerative capacity, we examined the expression patterns of several key genes for limb patterning during the spike like structure formation, and we compared them with those in developing and regenerating limb buds that produce a complete limb structure. We cloned Xenopus HoxA13, a marker of the prospective autopodium region, and the expression pattern suggested that the spike-like structure in froglets is accompanied by elongation and patterning along the proximodistal (PD) axis. On the other hand, shh expression was not detected in the froglet blastema, which expresses fgf8 and msx1. Thus, although the wound epidermis probably induces outgrowth of the froglet blastema, the polarizing activity that organizes the anteroposterior (AP) axis formation is likely to be absent there. Our results demonstrate that the lost region in froglet limbs is regenerated along the PD axis and that the failure of organization of the AP pattern gives rise to a spike-like incomplete structure in the froglet, suggesting a relationship between regenerative capacity and AP patterning. These findings lead us to conclude that the spike formation in postometamorphic Xenopus limbs is epimorphic regeneration. PMID- 10753518 TI - Regulatory mutations of the Drosophila Sox gene Dichaete reveal new functions in embryonic brain and hindgut development. AB - Sox domain proteins encompass a conserved family of transcriptional regulators that are implicated in a variety of developmental processes in eukaryotes from worm to man. The Dichaete gene of Drosophila encodes a group B Sox protein related to mammalian Sox1, -2, and -3 and, like these proteins, it is widely and dynamically expressed throughout embryogenesis. In order to unravel new Dichaete functions, we characterized the organization of the Dichaete gene using a combination of regulatory mutant alleles and reporter gene constructs. Dichaete expression is tightly controlled during embryonic development by a complex of regulatory elements distributed over 25 kb downstream and 3 kb upstream of the transcription unit. A series of regulatory alleles which affect tissue-specific domains of Dichaete were used to demonstrate that Dichaete has functions in addition to those during segmentation and midline development previously described. First, Dichaete has functions in the developing brain. A specific group of neural cells in the tritocerebrum fails to develop correctly in the absence of Dichaete, as revealed by reduced expression of labial, zfh-2, wingless, and engrailed. Second, Dichaete is required for the correct differentiation of the hindgut. The Dichaete requirement in hindgut morphogenesis is, in part, via regulation of dpp, since ectopically supplied dpp can rescue Dichaete phenotypes in the hindgut. Taken together, there are now four distinct in vivo functions described for Dichaete that can be used as models for context dependent comparative studies of Sox function. PMID- 10753519 TI - Brachyury (T) expression in embryos of a larvacean urochordate, Oikopleura dioica, and the ancestral role of T. AB - The Brachyury, or T, gene is required for notochord development in animals occupying all three chordate subphyla and probably also had this role in the last common ancestor of the chordate lineages. In two chordate subphyla (vertebrates and cephalochordates), T is also expressed during gastrulation in involuting endodermal and mesodermal cells, and in vertebrates at least, this expression domain is required for proper development. In the basally diverging chordate subphylum Urochordata, animals in the class Ascidiacea do not employ T during gastrulation in endodermal or nonaxial mesodermal cells, and it has been suggested that nonnotochordal roles for T were acquired in the cephalochordate vertebrate lineage after it split with Urochordata. To test this hypothesis, we cloned T from Oikopleura dioica, a member of the urochordate class Appendicularia (or Larvacea), which diverged basally in the subphylum. Investigation of the expression pattern in developing Oikopleura embryos showed early expression in presumptive notochord precursor cells, in the notochord, and in parts of the developing gut and cells of the endodermal strand. We conclude that the ancestral role of T likely included expression in the developing gut and became necessary in chordates for construction of the notochord. PMID- 10753520 TI - Hox C cluster genes are dispensable for overall body plan of mouse embryonic development. AB - Hox genes encode transcription factors which provide positional information during morphogenesis along the body axis; genetic interaction among Hox genes is thought to be necessary for correct pattern formation. One of the most curious features of the 39 vertebrate Hox genes is that they form four clusters each composed of several genes paralogous between clusters. This raises the question are all four clusters necessary for the development of vertebrates and, if so, what is the function of each cluster? To provide an answer to this question, we prepared Hox C cluster null mice utilizing the Cre-loxP system. Hox C cluster null mice, lacking all nine Hox C genes, die at the time of birth; however, the mutant pups develop to this stage with minor transformations. This development shows that the Hox C genes are dispensable for the overall body plan of mouse embryogenesis. Furthermore, transformations observed in the skeletal system of the Hox C cluster null mice are milder than those observed in the Hoxc-9 null mice, providing further evidence that at least some genes within a cluster exhibit interaction functions with each other. PMID- 10753521 TI - Medial edge epithelial cell fate during palatal fusion. AB - To explain the disappearance of medial edge epithelial (MEE) cells during palatal fusion, programmed cell death, epithelial-mesenchymal transformation, and migration of these cells to the oral and nasal epithelia have been proposed. However, MEE cell death has not always been accepted as a mechanism involved in midline epithelial seam disappearance. Similarly, labeling of MEE cells with vital lipophilic markers has not led to a clear conclusion as to whether MEE cells migrate, transform into mesenchyme, or both. To clarify these controversies, we first utilized TUNEL techniques to detect apoptosis in mouse palates at the fusion stage and concomitantly analyzed the presence of macrophages by immunochemistry and confocal microscopy. Second, we in vitro infected the MEE with the replication-defective helper-free retroviral vector CXL, which carries the Escherichia coli lacZ gene, and analyzed beta galactosidase activity in cells after fusion to follow their fate. Our results demonstrate that MEE cells die and transform into mesenchyme during palatal fusion and that dead cells are phagocytosed by macrophages. In addition, we have investigated the effects of the absence of transforming growth factor beta(3) (TGF-beta(3)) during palatal fusion. Using environmental scanning electron microscopy and TUNEL labeling we compared the MEE of the clefted TGF-beta(3) null and wild-type mice. We show that MEE cell death in TGF-beta(3) null palates is greatly reduced at the time of fusion, revealing that TGF-beta(3) has an important role as an inducer of apoptosis during palatal fusion. Likewise, the bulging cells observed on the MEE surface of wild-type mice prior to palatal shelf contact are very rare in the TGF-beta(3) null mutants. We hypothesize that these protruding cells are critical for palatal adhesion, being morphological evidence of increased cell motility/migration. PMID- 10753522 TI - Overexpression of rice OSH genes induces ectopic shoots on leaf sheaths of transgenic rice plants. AB - Five rice homeobox (OSH) genes were overexpressed under the control of the cauliflower mosaic virus 35S promoter or the rice actin gene promoter in transgenic rice plants. Almost all of the transgenic plants showed abnormal phenotypes, which could be classified into three types according to their severity. Plants with the most severe phenotype formed only green organs, with many shoot apices on their adaxial sides. Plants with an intermediate phenotype formed bladeless leaves with normally developed leaf sheaths. Plants with a mild phenotype formed normal leaf sheaths and blades, but lacked ligules and showed diffusion of the blade-sheath boundary. The leaf structure of this phenotype was similar to that of dominant maize mutants, such as Kn1, Rs1, Lg3, and Lg4. Based on these phenotypes, we suggest that ectopic expression of the rice OSH genes interferes with the development of leaf blades and maintains leaves in less differentiated states. These results are discussed in relation to the leaf maturation schedule hypothesis (M. Freeling et al., 1992, BioEssays 14, 227-236). PMID- 10753523 TI - Two pathways of maternal RNA localization at the posterior-vegetal cytoplasm in early ascidian embryos. AB - A cDNA library prepared from fertilized eggs of the ascidian Halocynthia roretzi was screened for prelocalized mRNAs in the early embryo by means of whole-mount in situ hybridization using a digoxigenin-labeled antisense RNA of each clone. Random mass screening of 150 cDNAs in a fertilized egg yielded six different clones which showed mRNA localization in the posterior-vegetal cytoplasm of the 8 cell embryo. An in situ hybridization study of the detailed spatial distribution of each mRNA in embryos of various stages revealed that there are, in contrast to the identical localization in embryos after the 16-cell stage, two distinct patterns of RNA distribution at earlier stages. One is colocalization with the myoplasm from the prefertilization stage to the 8-cell stage (type I postplasmic RNAs). The other is delayed accumulation of RNA at the posterior-vegetal cytoplasm after fertilization (type II postplasmic RNAs). We found that both types of RNAs associate with the cytoskeleton, but that they show different sensitivities to inhibitors of the cytoskeleton; translocation of the type I RNAs is dependent upon microfilaments during the first phase of ooplasmic segregation and dependent upon microtubules during the second phase of segregation, whereas translocation of the type II RNAs is dependent upon microfilaments throughout ooplasmic segregation. These results show that there are two pathways for the localization of the RNAs at the posterior-vegetal cytoplasm in the 8-cell embryo of the ascidian H. roretzi. PMID- 10753524 TI - Identification of endogenous retinoids, enzymes, binding proteins, and receptors during early postimplantation development in mouse: important role of retinal dehydrogenase type 2 in synthesis of all-trans-retinoic acid. AB - Specific combinations of nuclear retinoid receptors acting as ligand-inducible transcription factors mediate the essential role of retinoids in embryonic development. Whereas some data exist on the expression of these receptors during early postimplantation development in mouse, little is known about the enzymes controlling the production of active ligands for the retinoid receptors. Furthermore, at early stages of mouse development virtually no data are available on the presence of endogenous retinoids. In the present study we have used a recently developed high-performance liquid chromatographic (HPLC) technique to identify endogenous retinoids in mouse embryos down to the egg cylinder stage. All-trans-retinoic acid, a ligand for the retinoic acid receptors, was detected in embryos dissected as early as 7.5 dpc (i.e., a combination of midstreak until late allantoic bud stage embryos). At these stages, we detected mRNA coding for all the retinoid receptors, retinoid binding proteins, and two enzymes able to convert retinol to retinal (retinol dehydrogenase 5 (RDH5) and alcohol dehydrogenase 4 (ADH4)). We also detected retinal dehydrogenase type 2 (RALDH2), an enzyme capable of oxidising the final step in the all-trans-retinoic acid synthesis. In egg cylinder stage mouse embryos no all-trans-retinoic acid was detected. However, at this stage its precursor all-trans-retinal was present. In accordance with these HPLC observations, RDH5 and ADH4 were expressed, but no transcripts coding for enzymes that oxidise retinal to retinoic acid. Therefore, our results suggest that RALDH2 is a key regulator in initiating retinoic acid synthesis sometime between the mid-primitive streak stage and the late allantoic bud stage in mouse embryos. PMID- 10753525 TI - Characterization of polo-like kinase 1 during meiotic maturation of the mouse oocyte. AB - We have characterized plk1 in mouse oocytes during meiotic maturation and after parthenogenetic activation until entry into the first mitotic division. Plk1 protein expression remains unchanged during maturation. However, two different isoforms can be identified by SDS-PAGE. A fast migrating form, present in the germinal vesicle, seems characteristic of interphase. A slower form appears as early as 30 min before germinal vesicle breakdown (GVBD), is maximal at GVBD, and is maintained throughout meiotic maturation. This form gradually disappears after exit from meiosis. The slow form corresponds to a phosphorylation since it disappears after alkaline phosphatase treatment. Plk1 activation, therefore, takes place before GVBD and MAPK activation since plk1 kinase activity correlates with its slow migrating phosphorylated form. However, plk1 phosphorylation is inhibited after treatment with two specific p34(cdc2) inhibitors, roscovitine and butyrolactone, suggesting plk1 involvement in the MPF autoamplification loop. During meiosis plk1 undergoes a cellular redistribution consistent with its putative targets. At the germinal vesicle stage, plk1 is found diffusely distributed in the cytoplasm and enriched in the nucleus and during prometaphase is localized to the spindle poles. At anaphase it relocates to the equatorial plate and is restricted to the postmitotic bridge at telophase. After parthenogenetic activation, plk1 becomes dephosphorylated and its activity drops progressively. Upon entry into the first mitotic M-phase at nuclear envelope breakdown plk1 is phosphorylated and there is an increase in its kinase activity. At the two-cell stage, the fast migrating form with weak kinase activity is present. In this work we show that plk1 is present in mouse oocytes during meiotic maturation and the first mitotic division. The variation of plk1 activity and subcellular localization during this period suggest its implication in the organization and progression of M-phase. PMID- 10753526 TI - Functional analysis of spermatogonial stem cells in Steel and cryptorchid infertile mouse models. AB - Spermatogenesis is a complex and productive process that originates from stem cell spermatogonia and ultimately results in formation of mature spermatozoa. The stem cell undergoes self-renewal throughout life, but study of its biological characteristics has been difficult because a very small number (2 to 3 in 10(4) cells) exist in the testis and they can only be identified by function. Although the development of the spermatogonial transplantation technique has provided an assay system for stem cells, efficient methods to enrich stem cells have not been available. Here, we examined two infertile mouse models, Steel/Steel(Dickie)(Sl/Sl(d)) and experimental cryptorchid, as a source of testis cell populations enriched in stem cells. The Sl/Sl(d) testis showed little enrichment, which raises questions about how adult stem cell number is determined and about the currently accepted belief that adult stem cells are independent of Sl factor. The cells recovered from cryptorchid testes were enriched for stem cells 25-fold (colonies) or 50-fold (area) compared to wild-type testes. The cryptorchid condition does not affect stem cell activity, but eliminates almost all differentiated cells, and about 1 in 200 cells is a stem cell. Thus, cryptorchid testes provide an important approach for purification and characterization of spermatogonial stem cells. PMID- 10753527 TI - The role of ABI3 and FUS3 loci in Arabidopsis thaliana on phase transition from late embryo development to germination. AB - Arabidopsis abi3 and fus3 mutants are defective in late embryo development and their embryos show precocious growth. To understand the function and role of ABI3 and FUS3, we analyzed expression patterns of genes which were normally activated during late embryo development and germination in these mutants. Using the differential display method, both upregulated and downregulated genes were observed in immature siliques of the abi3 fus3 double mutant. Four clones having more abundant expression in the abi3 fus3 double mutant than in wild type were isolated. These genes were activated during wild-type germination, suggesting that some genes that are activated during wild-type germination are precociously activated in the abi3 fus3 mutant during late embryo development. Also, genes that were activated during wild-type germination were isolated and their expression patterns during late embryo development in the wild type and in abi3, fus3, and abi3 fus3 mutants were analyzed. Sixteen such clones were found, and 11 of these showed derepression or precocious activation of gene expression in the mutants. These results indicate that ABI3 and FUS3 negatively regulate a particular set of genes during late embryo development. We also showed that immature fus3 siliques accumulated one-third of the wild-type level of abscisic acid (ABA), but mature fus3 siliques accumulated ABA at a level comparable to that in the wild type. The possible mechanisms of controlling developmental timing in late embryo development as well as collaborative and distinct roles of ABI3 and FUS3 are discussed. PMID- 10753528 TI - The lens organizes the anterior segment: specification of neural crest cell differentiation in the avian eye. AB - During the development of the anterior segment of the eye, neural crest mesenchyme cells migrate between the lens and the corneal epithelium. These cells contribute to the structures lining the anterior chamber: the corneal endothelium and stroma, iris stroma, and trabecular meshwork. In the present study, removal of the lens or replacement of the lens with a cellulose bead led to the formation a disorganized aggregate of mesenchymal cells beneath the corneal epithelium. No recognizable corneal endothelium, corneal stroma, iris stroma, or anterior chamber was found in these eyes. When the lens was replaced immediately after removal, a disorganized mass of mesenchymal cells again formed beneath the corneal epithelium. However, 2 days after surgery, the corneal endothelium and the anterior chamber formed adjacent to the lens. When the lens was removed and replaced such that only a portion of its anterior epithelial cells faced the cornea, mesenchyme cells adjacent to the lens epithelium differentiated into corneal endothelium. Mesenchyme cells adjacent to lens fibers did not form an endothelial layer. The cell adhesion molecule, N-cadherin, is expressed by corneal endothelial cells. When the lens was removed the mesenchyme cells that accumulated beneath the corneal epithelium did not express N-cadherin. Replacement of the lens immediately after removal led to the formation of an endothelial layer that expressed N-cadherin. Implantation of lens epithelia from older embryos showed that the lens epithelium maintained the ability to support the expression of N-cadherin and the formation of the corneal endothelium until E15. This ability was lost by E18. These studies provide evidence that N-cadherin expression and the formation of the corneal endothelium are regulated by signals from the lens. N-cadherin may be important for the mesenchymal-to-epithelial transformation that accompanies the formation of the corneal endothelium. PMID- 10753529 TI - Resection with cryotherapy of colorectal hepatic metastases has the same survival as hepatic resection alone. AB - BACKGROUND: Hepatic resection is well established as a potentially curative treatment for hepatic colorectal cancer metastases. However, only a small proportion of patients with liver metastases are suitable for resection because they either have extrahepatic disease, or the extent and/or the distribution of their hepatic disease would make excision impossible. We have previously described the use of cryotherapy for inadequate resection margins and lesions in the remaining lobe of the liver. Combining such cryodestructive techniques with resection offers the possibility of increasing the proportion of patients to whom potentially curative treatment can be offered. The aim of this study was to compare survival in patients treated with resection and cryotherapy against those of patients treated with resection alone. Potential prognostic variables were also examined. METHOD: Patients undergoing a hepatic resection with or without cryotherapy at our unit between April 1990 and July 1997 were identified from our database and their notes reviewed. Survival was estimated using the Kaplan-Meier method and compared using the Log rank test. RESULTS: One hundred and seven patients were treated in total: 32 underwent resection alone, and 75 underwent resection combined with cryotherapy. There was no significant difference between the survival of patients treated with resection alone and those treated with resection and cryotherapy. CONCLUSIONS: Edge and contralobe cryotherapy can be combined with hepatic resection to allow a greater proportion of patients with hepatic colorectal metastases to be offered treatment, and results in similar survival figures comparable to hepatic resection for at least 3 years. PMID- 10753530 TI - Thymoma--the usefulness of some prognostic factors for diagnosis and surgical treatment. AB - AIMS: The aim of the study was to identify prognostic factors which could help evaluate both the treatment offered to patients with thymoma and late results. METHODS: Forty patients were treated for mediastinal thymoma. The patients were staged clinico-pathologically (according to Masaoka) on the basis of the retrospective analysis of their operation protocols as follows: seven (17.5%) stage I, 19 (22. 5%)-stage II, 17 (42.5%)-stage III, seven (17.5%)-stage IV. Analysis of DNA contents in cell nuclei of 23 thymomas was performed by the flow cytofluorometric method. RESULTS: From the whole group of patients, 65% survived for 5 years, 55% survived for 10 years and 43% survived for 15 years. We noted significant differences in survival time between stage I and stage IV (P<0.0012); stage II and stage IV (P<0.0006), as well as between stage III and stage IV (P<0. 005). Significantly worse prognosis was observed in the case of cortical thymomas as compared with medullary or mixed types (P<0. 0001 P<0.002). Analysis of DNA content showed signficantly higher probability of survival for the patients who had DI=1.0 (diploid), as compared with DNA >1.0 (aneuploid) (P<0.006). Of the 11 patients with diploid tumours, 91% survived for 5 years, but of the 12 aneuploid, only 23% survived. CONCLUSION: The most important positive prognostic factors influencing survival rate in patients with thymoma are: lower stage, medullary type (according to Muller-Hermelink classification), possibility of performing complete resection, diploidal nature of the tumour. Multivariate analysis of survival revealed clinico-pathological stage (according to Masaoka) and histological type (according to Salyer) as significantly independent prognostic factors. PMID- 10753531 TI - Curative liver resection for metastatic breast cancer. AB - AIMS: Hepatic resection is a standard procedure in the treatment of colorectal liver metastases. Liver metastases are frequent in breast cancer, but resectional treatment is rarely possible and few reports have addressed the results of surgical treatment for metastatic breast cancer. The aim of our study was to analyse the outcome of patients with metastatic breast cancer after resection of isolated hepatic secondaries and possibly to identify selection criteria for patients who may benefit from surgery. METHODS: Between 1984 and 1998, 90 patients with a history of breast cancer and suspected liver metastases were referred for surgical evaluation. Fifty-four patients also had extrahepatic disease or metastases from another primary tumour; multiple liver metastases were not amenable to surgical treatment in 20 patients. Five patients were treated by regional chemotherapy via an intra-arterial port catheter; after liver resection two patients were found to have liver metastases from intercurrent colorectal cancer. Thus only nine liver resections for metastatic breast cancer could be performed with curative intent. RESULTS: No patient died post-operatively after liver resection. In the follow-up period, four of the nine patients who were treated with curative intent received systemic chemotherapy. At a median follow up of 29 months, four patients died from tumour recurrence. Five patients are currently alive. Five-year survival in the resection group was calculated as 51% (Kaplan-Meier estimate). Node-negative primary breast cancer and a long interval between treatment of the primary and liver metastases appeared to be associated with long survival after liver resection. CONCLUSIONS: These observations suggest that careful follow-up and adequate patient selection could offer some patients with isolated liver metastases from breast cancer a chance of long-term survival. PMID- 10753532 TI - Isolated limb perfusion with cisplatin and doxorubicin for locally advanced soft tissue sarcoma of an extremity. AB - AIMS: To identify independent adverse clinico-pathological factors for disease free and overall survival in patients undergoing isolated limb perfusion (ILP) with cisplatin and doxorubicin for locally advanced soft tissue sarcoma (STS) of an extremity. METHODS: A retrospective analysis was carried out, using a univariate method and a multivariate analysis, to look at the patient, tumour and treatment associated with prognostic factors in 37 patients with locally advanced STSs of the limbs who underwent ILP with cisplatin and doxorubicin. Patient's age, gender, presenting status, tumour location, tumour grade, tumour stage according to TNM classification, tumour size and radiotherapy (RT) were analysed. Survival curves were calculated according to the Kaplan-Meier method. A Cox proportional hazard model was used to indicate which factors related to overall survival and the recurrence-free interval after ILP. RESULTS: No major systemic toxicity was seen. Regional toxicity was limited. Limb salvage was achieved in 94.6% of the patients. The estimated 5-year overall and disease-free survival rates were 62% and 54%, respectively. It was found that tumour stage, tumour grade, presenting status, RT and tumour size were associated with cumulative overall survival when the Kaplan-Meier method was applied (P<0.05). By Cox proportional hazards model, only tumour grade (P=0. 0254) was found to have significant influence on overall survival; however, tumour stage (P=0.0157) and RT (P=0.0014) were related to disease-free survival. CONCLUSIONS: ILP and delayed excision followed by RT achieves good limb salvage rates and may improve survival. PMID- 10753534 TI - Cell production rates in human tissues and tumours and their significance. Part 1: an introduction to the techniques of measurement and their limitations. AB - In the past two decades, the technology of laser cytometry and use of the halogenated thymidine (HP) analogues bromodeoxyuridine and iododeoxyuridine as proliferation labels, have allowed us to quantify the rate of cell turnover in tissues and tumours, in clinical samples as in laboratory models. The principal studies have used injection of bromo- or iododeoxyuridine to measure cell production rates in vivo. Flow cytometry (FCM) has been used to estimate the S phase labelling index (LI) and the S phase duration (Ts) and calculate the cell production rate, represented by the potential doubling time (Tpot). This has allowed calculation of time-dependent indices of proliferation from single biopsies of HP pulse labelled human tissues and tumours. In the first part of this two-part review, we describe the technique and its limitations as a biological assay. The second part summarizes the knowledge gained about cell production rates and the relevance that this information may have to future investigative, prognostic and treatment strategies. PMID- 10753533 TI - Problems with intraoperative hyperthermic peritoneal chemotherapy for advanced gastric cancer. AB - BACKGROUND: Intraoperative hyperthermic peritoneal chemotherapy (IHPC) after total gastrectomy for advanced, serosa-penetrating gastric cancer has been demonstrated in several studies to reduce the incidence of peritoneal carcinosis and to prolong survival. METHODS: In a prospective pilot study, nine patients with advanced gastric cancer were selected to receive IHPC with Mitomycin and Cisplatin after total gastrectomy and systematic lymphadenectomy. RESULTS: All patients had nodal, and four patients distant, metastases. Six patients (66%) suffered from post-operative complications including renal failure, pancreatitis, pancreatic fistula and anastomotic dehiscence. Thirty-day mortality was zero. Six patients died within 3-10 months after surgery. Five of these deaths were related to peritoneal carcinosis and one patient died from cardiac failure 3 months after surgery. Three patients, respectively, have been alive for 12, 20 and 24 months at present, with suspected peritoneal tumour in the last patient. The 2-year probability of survival among our patients receiving IHPC is 29%. CONCLUSION: Intraoperative hyperthermic peritoneal chemotherapy carries a high risk of peri operative complications and was not able to prevent or delay peritoneal tumour recurrence in patients with advanced gastric cancer. PMID- 10753535 TI - European society of surgical oncology 10th congress held in groningen, the netherlands, from 5-8 april 2000 PMID- 10753536 TI - PROGRAMME OVERVIEW: 10th congress of the european society of surgical oncology PMID- 10753537 TI - PROGRAMME: 10th congress of the european society of surgical oncology PMID- 10753538 TI - SUBJECT INDEX: 10th congress of the european society of surgical oncology PMID- 10753539 TI - Anaphylactic shock following peritumoral injection of patent blue in sentinel lymph node biopsy procedure. AB - Recently, sentinel lymph node dissection has been established for early staging of malignant melanoma. We describe an anaphylactic reaction due to patent blue injection in sentinel lymph node procedure, which was proven by positive results in intradermal testing with patent blue 1:100. PMID- 10753540 TI - A non-insulin secreting malignant insulinoma of the pancreas. AB - We report a rare case of a non-insulin secreting malignant insulinoma of the pancreas and discuss its medical and surgical management. Surgical excision of malignant insulinoma produces good palliation and can increase survival. PMID- 10753541 TI - Adenomyoepithelioma of the breast: a diagnostic dilemma. PMID- 10753542 TI - Lobular carcinoma of the breast in an 85-year-old man. AB - The present study describes a case of the extremely rare histological picture of a lobular carcinoma of the male breast. The 85-year-old patient presented with a tumour in a very advanced stage. The results of genetic studies excluded Klinefelter's syndrome, but a new gene mutation affecting the BRCA1 gene (breast cancer gene 1) was found in the patient. PMID- 10753543 TI - Role of Mimeticism and Spatiality in Textual Recall. AB - Four experiments were conducted to investigate the role of map spatiality and icon mimeticism in facilitating text recall. A secondary goal was to explore an assumption of the conjoint retention hypothesis, that the visuospatial component of working memory is involved in retrieving map information. We manipulated display conditions to evaluate the separate and combined effects of map spatiality and icon mimeticism on text recall. We also utilized a concurrent task paradigm to assess both the recognition of spatial displays and the recall of map feature information. The results of all four experiments point to the mimeticism of icons as the key attribute of maps for facilitating recall, rather than the spatial layout of the map when visual displays and text are presented simultaneously during encoding. We also found no evidence indicating that maps are processed in a more spatial manner than are lists. Copyright 2000 Academic Press. PMID- 10753544 TI - Knowledge Encapsulation and the Intermediate Effect. AB - The present study explored the role of so-called encapsulated knowledge in diagnosing clinical cases outside the expert physicians' domain of expertise. Neurologists and 2nd-year and 6th-year medical students were required to diagnose, recall, and explain the signs and symptoms of two cardiological and two pulmonological clinical case descriptions. Our experiment showed that neurologists diagnosed these clinical cases faster and more accurately than 2nd year and 6th-year medical students. An inverted U-shaped relationship with levels of expertise was found in recall and pathophysiological protocols: 6th-year medical students remembered more information from the cases and produced more elaborated explanations for the described signs and symptoms than both other groups. The proportion of encapsulating concepts in recall and pathophysiological explanations, on the other hand, increased with levels of expertise. This pattern is similar to that found in previous studies on clinical case representations using only cases within the expert physicians' domain of expertise. Therefore, these results suggest that expert physicians process clinical case descriptions both within and outside their domain of expertise in essentially the same way. Copyright 2000 Academic Press. PMID- 10753545 TI - Effect of Students' After-School Activities on Teachers' Academic Expectancies. AB - Teacher expectancies can have an impact on students' academic achievement. These expectancies can be based on diverse student characteristics, only one of which is past academic performance. The present study investigated three student individual differences that teachers may use when forming academic expectancies: the sex of the student, the family socioeconomic status (SES) of the student, and the student's after-school activities. Results indicated teachers held higher grade, graduation, and college attendance expectancies for females than for males and for middle-SES than low-SES students. Also, students who participated in extracurricular activities were expected to achieve more academically than either students who were employed after school or who did nothing after school. The latter two groups did not elicit different teacher expectancies. Interactions revealed that (a) lowest expectations were held for low-SES males who did nothing after school and (b) the difference in graduation expectancies between the SES groups was only half as great for students who took part in extracurricular activities than it was for students who had no involvements after school or who had jobs. Copyright 2000 Academic Press. PMID- 10753546 TI - Incidental Word Learning in Science Classes. AB - The purpose of the project was to investigate students' incidental word learning in science classes that depended on discussion and hands-on activities. In separate studies, 4th- and 8th-grade students were given pretests and posttests that assessed depth of knowledge of topical words used in a single unit. In both studies, students made significant improvement in their knowledge of topical words; knowledge of nontopical words did not improve. Students who started the unit with partial knowledge of topical words were likely to learn meanings appropriate for the unit. Depth of topical word knowledge also contributed significantly to improvement on a test of applied problems. While significant incidental word learning occurred over the science units, students with little or no understanding of topical words at the outset tended to make limited progress in both word learning and learning the ideas and information of the unit. The educational implications are potentially serious and need to be explored in further studies. Copyright 2000 Academic Press. PMID- 10753547 TI - Teaching Elementary Students Who Speak Black English Vernacular to Write in Standard English: Effects of Dialect Transformation Practice. AB - Although nonstandard dialects of English are legitimate forms of spoken language used by many Americans, students in U.S. schools must acquire writing competence using Standard English (SE). Participants in this study were 3rd- and 4th-grade African-American students who exhibited Black English Vernacular (BEV) features in their written work. Six syntactic features differing in BEV and SE were targeted. Students received one of three treatments to increase their use of the SE features in their writing: (1) exposure to SE features in stories; (2) story exposure plus explanation of SE rules; and (3) story exposure, SE rule instruction, and guided practice transforming sentences from BEV to SE features. The third treatment proved most effective in enabling students to translate BEV sentences into SE forms and to employ the targeted SE features in their free writing. Results indicate that having students practice translating nonstandard sentences that typify their own writing and providing corrective feedback are effective for teaching them to use SE forms in their writing. Findings are interpreted to support a social-cognitive view of self-regulated learning. Copyright 2000 Academic Press. PMID- 10753548 TI - Cognitive neuropharmacology: new perspectives for the pharmacology of cognition. AB - Taking its roots both in neuropharmacology and in cognitive science, cognitive neuropharmacology is an emerging approach in the field of psychopharmacology. It attempts to use theoretical knowledge to understand the biochemical bases of cognition and the mode of action of the commonly used drugs and to find new brain targeted therapeutics. The aim of the present article is to throw up the main characteristics of this way of research. It is defined in comparison with its neighbouring approaches and by presenting its own rationale. Its particular methods mainly concern the animal modelling of the highest human cognitive functions and the original means of intra-cerebral drug administration. Finally, we present an illustrative example of a study in cognitive neuropharmacology and propose further perspectives. PMID- 10753549 TI - Regulation of herbal medicines in Japan. AB - In Japan, two overlapping types of traditional herbal medicines coexisted for centuries. The first one was the traditional Japanese and Chinese medicine. These medical systems were damaged by the first Medical Care Law in 1874 that proclaimed the abrogation of traditional Japanese medicine. The second type of herbal medicine used in Japan originated in Europe and south-east Asia and became popular after the law in 1874 was announced. Some of those products are still used today as prescription drugs. Although the renaissance of the traditional medicines has been on the rise since approximately 1960, the confusion and decline of the traditional Japanese medicines was further strengthened by introduction of dietary supplements. Regulation of herbal medicines, except 'Kampo' formulas is the same as the approval for both prescription and OTC drugs. Typical characteristics of the Japanese herbal medicines is the existence of Japanese traditional medicines, 'Kampo' formulas and combinations of the traditional medicines with vitamins and pharmaceuticals. Regulation of quality standards of those herbal products was established in Japanese Pharmacopoeia for more than 90% of them. PMID- 10753550 TI - A preliminary evaluation of antifertility activity of a triterpenoid glycoside (DSS) from Dalbergia saxatilis in female Wistar rats. AB - Antifertility activity of a triterpenoid glycoside, DSS, isolated from the root of Dalbergia saxatilis was investigated in female Wistar rats of breeding age. When administered by gastric intubation at a dose rate of 200 mg kg(-1)body weight at the premating period, conception was inhibited in 71.4% of the treated animals. Fertility Index (FI) for this group was 107.82 compared with 373.5 value for control rats that received 30% aqueous Tween 20 vehicle. DSS, did not significantly alter the fertility of rats at the first and second trimesters of pregnancy but did cause a significant decrease (P<0.05) in the mean Day 20 foetal crown-rump length when administered at the premating period and at the third trimester of pregnancy; with a concurrent decline in the mean maternal body weights. The potential use of DSS as a chemosterilant in fertility control are discussed. PMID- 10753551 TI - Clarithromycin attenuates cyclophosphamide-induced mucositis in mice. AB - No universally recognized agent is available for prophylaxis or therapy of mucositis induced by chemotherapy or chemo-radiotherapy. The effect of clarithromycin on the severity of mucositis induced by cyclophosphamide was investigated using a mouse model. Four cross-sections of small intestine (levels A, B, C, and D) were taken at equivalent intervals at day 5 after cyclophosphamide (400 mg kg(-1)) administration. The sections were stained with haematoxylin and eosin, and were graded for the degree of mucositis histologically. At section level B, the number of mice with no mucositis (grade 0) in the clarithromycin group was significantly greater than that in the ceftriaxone group (P<0.05). At levels B and C, the number of mice with no mucositis (grade 0) in the clarithromycin group was significantly greater than that in the normal saline (NS) group (P<0.05). At level C, the number of mice with grade 2 mucositis in the ceftriaxone group was significantly greater than that in the NS group (P<0.05). When the number of sections at all levels were analyzed together, the number of mice with no mucositis (grade 0) in the clarithromycin group was significantly greater than that in the ceftriaxone and NS groups (P<0.05). The present observation suggests that clarithromycin and ceftriaxone attenuates and aggravates cyclophosphamide-induced mucositis. It prompts clinical trials in bone marrow transplant (BMT) recipients receiving cyclophosphamide for conditioning, and reconsideration in the use of ceftriaxone in the treatment of neutropenic fever in BMT recipients. PMID- 10753552 TI - Possible protective effect of melatonin and/or desferrioxamine against streptozotocin-induced hyperglycaemia in mice. AB - There is a clear link between diabetes and oxidative stress. Hyperglycaemia leads to free radical generation and alteration of endogenous antioxidants. The present study is an attempt to evaluate the possible protective effect of melatonin (MLT) and/or desferrioxamine (DF) against streptozotocin (STZ)-induced hyperglycaemia in mice. Serum lipid profile, pancreatic tissue contents of glutathione (GSH) and malondialdehyde (MDA) were determined. MLT and/or DF were given p.o. in doses of 5 mg kg(-1)day(-1)and 250 mg kg(-1) day(-1), respectively for 15 consecutive days prior to STZ treatment (60 mg kg(-1) day(-1) i.p.) for 3 consecutive days. Results revealed that STZ induced a marked increase in serum glucose, serum triglycerides (TG), cholesterol (CHO) and LDL-cholesterol. On the contrary HDL cholesterol was markedly decreased in STZ-treated group. Moreover, STZ induced a significant decrease in the pancreatic content of GSH with concomitant increase in MDA content. Administration of MLT or (MLT+DF) prior to STZ treatment revealed a marked decrease in serum glucose level by 35.6 and 31.6%, respectively as compared to STZ-treated group. Furthermore, MLT pretreatment of STZ-induced hyperglycemic mice, has not only normalized GSH content of pancreatic tissues but also increased its level more than that of control animals by 110%. On the contrary, MDA content of pancreatic tissues was markedly decreased even lower than normal control group. MLT also, induced a marked protection in terms of decreasing serum CHO, LDL, TG by 21.8, 83.8 and 82.2%, respectively, while HDL was increase by 56% as compared to STZ treated group. DF was found to be less effective than MLT in the protection against STZ-induced hyperglycemia. In conclusion, these data suggest that MLT protects against the damaging consequences induced by hyperglycemia either systemically or in the pancreatic tissues. PMID- 10753553 TI - Phenazone potentiates the local anaesthetic effect of lidocaine in mice. AB - To justify the inclusion of phenazone, independently of its anti-inflammatory properties, in combination with a local anaesthetic, such as lidocaine, in some ear drop medications, we have studied the effect of this compound on the local anaesthetic activity of lidocaine in an animal model, that of sciatic nerve blockade in mice. Lidocaine and phenazone were tested alone and in combination at various concentrations. The local anaesthetic activity was estimated as the loss of motor activity of the hindlimb after topical injection of the drugs in the region of the sciatic nerve. Lidocaine, at concentrations ranging from 0.03 to 0.25%, induced a concentration-dependent anaesthetic effect. Phenazone alone had no effect at 0.25-1%. When combined, the two compounds acted synergistically. The local anaesthesia induced by lidocaine plus phenazone was significantly more intense and longer lasting than that induced by lidocaine alone. Phenazone enhanced the potency of lidocaine in this animal model. It is suggested that the potentiated local anaesthetic effect of the combination may be partly due to enhanced local bioavailability of lidocaine. PMID- 10753554 TI - New esters of succinic acid and mixed molecules formed by such esters and a meglitinide analog: study of their insulinotropic potential. AB - Eighteen novel esters of succinic acid, including three mixed molecules formed of both succinic acid and nateglinide, were examined for their insulinotropic efficiency in isolated rat pancreatic islets. The secretory response to these esters at increasing concentrations of both d -glucose and the ester itself allowed to identify five esters judged of potential interest for further investigations. They include three molecules with CH(3)-O-CO-CH(2)-CH(2)-CO-NH CH(R)-CO-O- sequence and two mixed molecules with a nateglinide moiety. The effects of the most potent molecule in the first group and that of two of the esters with a nateglinide moiety upon islet biosynthetic activity in vitro and insulin release in vivo, after either oral or intravenous administration were also investigated. The results suggested that mixed molecules formed of both a succinic acid ester and a meglitinide analog may efficiently stimulate proinsulin biosynthesis and/or insulin release. Further work is required, however, to improve their modality of in vivo administration. PMID- 10753555 TI - Pumpkin-seed oil modulates the effect of felodipine and captopril in spontaneously hypertensive rats. AB - Natural products like pumpkin-seed oil (PSO) may modify the potency of the calcium antagonist felodipine (FEL) or angiotensin-converting enzyme inhibitor (ACE-inhibitor), captopril (CPT) in modulating the biochemical derangement in blood, heart and kidney as well as blood pressure and heart rate of spontaneously hypertensive rats (SHR) were investigated. SHR were treated orally with FEL at a dose of 0. 45 mg kg(-1) body wt. or CPT at a dose of 9 mg kg(-1) body wt. once daily for 4 weeks. PSO was administered at a dose of 40 mg kg(-1) body wt. alone or with FEL or CPT in the previous respective dose regimen for the same period to SHR. This study showed that hypertension induced increments the content of malondialdehyde (MDA) by 55% and 38% as well as the activity of glutathione peroxidase (GSH-Px) by 26% and 23% in heart and kidney, respectively, accompanied by reductions in the activity of myocardial superoxide dismutase (SOD) from 3.40+/-0.17 to 2.42+/-0.19 U mg protein(-1)and contents of glutathione (GSH) and protein thiols (PrSHs) in different tissues of SHR as compared to normotensive rats. Treatment of SHR with FEL or CPT monotherapy or combined with PSO produced improvement in the measured free radical scavengers in the heart and kidney. Our results also showed that pretreatment of SHR with PSO for 4 weeks then i.v. administration of FEL or CPT produced a significant beneficial hypotensive action. The results were explained in the light of the antioxidant properties of PSO. Therefore, it is concluded that concomitant administration of FEL or CPT with natural antioxidants can yield a beneficial therapeutic effect and retard the progression of hypertension. PMID- 10753556 TI - The effects of chronic L-name and L-arginine administration on beta-adrenergic responsiveness of STZ-diabetic rat atria. AB - Recent studies have shown that NO acts as a negative inotrope and chronotrop in cardiac muscle. In the present study, we investigated the effects of the chronic administration of L-NAME and L -arginine on 14-week streptozotocin (STZ)-diabetic rat atria. To study these effects, we compared the alterations of inotropic and chronotropic responses to isoprenaline (ISO) on electrically-driven left atria and spontaneously beating right atria. In addition, we compared the blood pressures of rats in all groups. The chronic administration of L-arginine resulted in a significant reduction in blood pressure of the diabetic rats. On the other hand, the chronic nitric oxide synthase inhibition resulted in a significant increase in blood pressure of diabetic animals. To our findings, maximum positive inotropic responses of ISO diminished in STZ-diabetic, L arginine and L-NAME treated diabetic groups relative to controls but neither the basal contractility of the left atria nor the pD(2)values were altered significantly in all groups. The basal atrial rate and maximum positive chronotropic responses to ISO were found to be significantly lower in treated and untreated diabetic groups, no significant changes were observed in pD(2)values. Our results demonstrate that the changes in inotropic and chronotropic responses in diabetic rat atria were not influenced by the chronic administration of L arginine and L-NAME treatments. PMID- 10753557 TI - Very low intakes of N-3 fatty acids incorporated into bovine milk reduce plasma triacylglycerol and increase HDL-cholesterol concentrations in healthy subjects. AB - Eight normolipidaemic volunteers, habitual partial skim milk drinkers and non eaters of fish during the study, were given 500 ml day(-1) of partial skim milk for 1 month; they were then switched to 500 ml day(-1) of a novel commercially available milk preparation, supplying 400 mg of N-3 fatty acids-of which 300 mg were EPA+DHA-and 15 mg vitamin E, for 6 weeks. No changes in plasma lipid parameters were observed after the first run-in month; at 3 and 6 weeks on the N 3-rich milk, marked increments of plasma EPA (44 and 31%, respectively) and DHA (13 and 31%, respectively) were observed. Triacylglycerol (TG) concentrations decreased by 19% and high-density lipoprotein (HDL) concentrations increased by 19% at 6 weeks; plasma vitamin E rose by 21% while the susceptibility of plasma to oxidation was unaffected. Correlations were found between plasma EPA or DHA and TG, cholesterol, and HDL. In conclusion, the intake of a milk preparation providing low amounts of EPA+DHA to healthy individuals led to marked increases of N-3 fatty acids and vitamin E in plasma and in associated favourable changes in HDL and TG. PMID- 10753558 TI - Effects of nicotine, dimethylphenylpiperazinium and tetramethylammonium on smooth muscles from feline and human gastric corpus. AB - Both excitatory and inhibitory intrinsic neurons could be found within the gastric wall, both of them receiving innervation from vagal fibres and being sensitive to nicotine. The effects of three nicotine receptor agonists, nicotine, tetramethylammonium (TMA) and 1,1-dimethyl-4-phenylpiperazinium (DMPP), on contractile activity of preparations isolated from feline and human gastric corpus wall were investigated. While DMPP (3.5x10(-8) to 5.9x10(-4)m) did not affect either spontaneous contractions or basal tension of isolated preparations from both species, TMA produced concentration-dependent tonic contractions of both circular and longitudinal isolated preparations from human (3.66x10(-5) to 5.10x10(-3)m) and feline (6. 1x10(-7) to 2.1x10(-3)m) stomach. On the other hand, nicotine (4. 1x10(-8) to 7.0x10(-4)m) produced concentration-dependent relaxation of only circular isolated preparations from feline gastric corpus. The effect of nicotine was sensitive to mecamylamine, and not to pancuronium, while the effect of TMA was sensitive to both mecamylamine and pancuronium. Although in our experiments DMPP had no effect, its excitatory action on gastric intrinsic neurons through the hexamethonium-insensitive pathway had already been described. The results of our study suggest that two different types of ganglion nicotine receptor exist together within the wall of feline stomach: (1) type N(N1)which is involved in relaxation and is sensitive only to nicotine and mecamylamine, and not to DMPP, TMA and pancuronium; (2) and type N(N2)which is involved in contraction of gastric muscle and sensitive to DMPP, TMA, mecamylamine and pancuronium, and not to nicotine. PMID- 10753559 TI - The inhibitory effects of terikalant on the contractile activity of galanin in isolated rat fundus strips. AB - The maximal responses (E(max)s) of isolated rat gastric fundus strips to 300 n m porcine galanin (Gal) were decreased in a concentration-dependent manner by terikalant (RP 62719). EC(50)of the agent equalled 4.39 microm (2.35-8.22). On the contrary the action of 30 n m of carbachol were not affected by the modulator in concentrations up to 30 microm. It is concluded that potassium currents may contribute to the modulation of Gal myotropic activity in the gut. PMID- 10753561 TI - Effects of Manduca diuresin on neonates of the tobacco hornworm, Manduca sexta. AB - Manduca diuresin (MD), a 30 amino acid peptide, was isolated and identified from the type IIb median neurosecretory cells in the brain of adult Manduca sexta (Blackburn et al., Biochem. Biophys. Res. Commun., 181, 927-932, 1991). The synthetic hormone stimulates water loss in the adult moth both in vitro using isolated Malphigian tubule preparations and in vivo using decapitated adult moths as bioassay animals (Blackburn and Ma, Arch. Insect Biochem. Physiol., 27, 3-10, 1994). In the present study, MD was shown to have adverse physiological effects on neonates of Manduca sexta when fed synthetic hormone-treated leaf discs. With a sustained diet of MD-treated discs, neonates exhibited symptoms of reduced food consumption and marginal weight increases. In addition to slowed growth and reduced developmental rates, a high percentage of these neonates failed to molt into second instar larvae and death usually followed shortly thereafter. This is a first report that a synthetic insect neuropeptide has shown detrimental effect on insect larvae by ingestion. The possibility of utilizing this peptide in plant protection is discussed. PMID- 10753560 TI - Hypochlorous acid-induced responses in sheep isolated pulmonary artery rings. AB - The formation of reactive oxygen species (ROS) appears to play a significant role in many pathological states including cystic fibrosis and asthma. Although stimulated inflammatory cells represent a major source of oxygen metabolites and these cells are able to generate the potent oxidant hypochlorous acid (HOCl) effects of HOCl on arteries are not known. HOCl at low concentrations (10(-7)to 10(-4)m) did not affect the resting force or have an action in precontracted sheep pulmonary arteries. HOCl at 10(-4) m concentration reduced histamine induced relaxations in endothelium intact preparations. However, at high concentrations (10(-2) to 1 m) HOCl led to constriction under resting conditions and caused vasodilation in endothelium intact and denuded serotonin (10 microm) precontracted arteries. These effects of HOCl were significantly reduced by pretreatment of l -arginine (10(-3)m), sodium nitroprusside (SNP, 10(-5) m) and N -acetyl-l-cysteine (NAC, 10(-4) m). The effects of SNP and NAC on HOCl-induced responses were due to direct interaction since only these compounds markedly diminished the HOCl-induced luminol chemiluminescence (CL). Lack of contraction with KCl after high concentrations of HOCl showed that HOCl causes irreversible tissue damage. These results suggest that HOCl produce vasoconstriction under resting force and cause vasodilation when the pulmonary arteries precontracted. HOCl may interact with endothelium-derived mediators and contribute to tissue injury and vascular dysfunction seen in disease states. PMID- 10753562 TI - Possible relationship between the B-cell threshold for glucose-induced insulin secretion and blood glucose concentrations in the normal toad. AB - The aim of this work was to gain information on the possible relationship between basal glycemia in the toad and the B-cell threshold for glucose-induced insulin release. Hence, pieces of pancreas from Bufo arenarum were incubated with 2 to 20 mM glucose or preincubated with 2 mM glucose plus the hexokinase and glucokinase inhibitors (50 mM of 2-deoxyglucose and mannoheptulose, respectively) followed by an incubation with different glucose concentrations. The maximal rate of insulin release occurred at 8 mM glucose, while 50% of the release (K(s50)) was observed at 7 mM glucose. Regardless of the glucose concentration employed, pancreas pieces preincubated with 2-deoxyglucose released less insulin than the corresponding controls. On the other hand, mannoheptulose significantly inhibited the release of insulin at high glucose concentrations, having no effect at low glucose concentrations. The blocking effect of these two inhibitors is the first indirect evidence of the existence of the hexokinase/glucokinase enzymic system in the toad pancreas. Since the activity ratio of this system determines the glucose sensitivity of the insulin secretory mechanism, it is concluded that the possible existence of a higher ratio of these enzymes in toad B cells could explain the particular characteristics of glucose sensitivity in this animal, which in turn may explain its low blood glucose concentration. PMID- 10753564 TI - Localization of receptors for natriuretic peptide and endothelin in the duct of the epididymis of the freshwater turtle. AB - The presence of receptor subtypes for natriuretic peptides (NPs) and endothelin (ET) in the epididymis of the freshwater turtle, Amyda japonica, was examined by quantitative in vitro autoradiography using iodinated mammalian-type atrial NP ((125)I-ANP((1-28))), phylogenically conserved C-type NP ((125)I-[Tyr(0)]-CNP((1 22))), and ET-1 ((125)I-ET-1) as radiolabeled ligands. To characterize NP receptor (NPR) subtypes, we also performed an activation of particulate guanylyl cyclase (GC) in membranes of the epididymis by NPs. Specific (125)I-ANP((1-28)) and (125)I-[Tyr(0)]-CNP((1-22)) bindings were localized in surrounding smooth muscle cell layer of the duct of the epididymis with an apparent dissociation constant (K(d)) of 0.84+/-0.15 and 1.74+/-0.39 nM and a maximal binding capacity (B(max)) of 0.47+/-0.11 and 0.08+/-0.01 fmol/mm(2), respectively. Bindings of (125)I-ANP((1-28)) and (125)I-[Tyr(0)]-CNP((1-22)) to these sites were also displaced by des[Gln(18),Ser(19),Gly(20), Leu(21),Gly(22)]ANF((4-23)), a specific ligand of the NP clearance receptor. Production of 3',5'-cyclic guanosine monophosphate by particulate GC in membranes of the epididymis was stimulated by ANP((1-28)), BNP((1-26)), and CNP((1-22)). Receptor subtypes for ET in the epididymis were characterized by competition with BQ 123 and BQ 788 as specific antagonists for ET receptors, type A (ET(A)) and type B (ET(B)) subtypes, respectively. Specific (125)I-ET-1 bindings were localized in the smooth muscle cell layer of the duct of the epididymis with K(d) and B(max) of 0.21+/-0.03 nM and 0.52+/-0.05 fmol/mm(2), respectively. These specific bindings were potently inhibited in a dose-dependent manner by BQ 123, whereas BQ 788 (10 microM) was not in competing for specific (125)I-ET-1 bindings in this structure. Therefore, these results indicate that specific NP and ET receptors are localized in surrounding smooth muscle cells of the duct of the epididymis of the freshwater turtle. It is also suggested that biological and clearance NPR-like subtypes coexist in these cells, and the predominant ET receptor subtype in this tissue is the ET(A)-like receptor. The localization of specific receptors for NPs and ET in the epididymis may be involved in the control of the transport of sperm in the freshwater turtle. PMID- 10753563 TI - Seasonal variations and maturity stages in relation to differences in serum levels of gonadal steroids, vitellogenin, and thyroid hormones in the common dentex (Dentex dentex). AB - Seasonal variations in serum concentrations of 17beta-estradiol (E(2)), vitellogenin (Vg), testosterone (T), 11 ketotestosterone (11-KT), and thyroid hormones (T(4), l-thyroxine; and T(3), 3,5, 3'-triiodo-l-thyronine) were investigated during the first, second, and third reproductive cycles in intensively reared populations of common dentex, Dentex dentex, and correlated with gonadal development and spawning. In females, there were baseline E(2) values (<0.10 ng/ml) and negligible Vg concentrations during the postspawning and pregametogenesis period (June to December), and these increased thereafter to peak during the spawning period. Maximum T(3) and T(4) serum concentrations were found around spawning. There was a positive correlation during vitellogenesis and final maturation between Vg and T(3) (r(2) = 0.366). In addition, Vg and T(3) concentrations were statistically higher in the stages of vitellogenesis and final maturation than at the other stages (P<0.001). Minimum T(3) and T(4) concentrations (October) coincided with the decrease in water temperature and the associated decrease in the daily feeding rate and the specific growth rate. In males, as in females, seasonal changes in serum levels of T and 11-KT were well correlated with gonadal development. The presence of males in the stage of completed spermiogenesis in December coincided with the surge in both androgens and this increase lasted until the end of the spawning period. There were no significant differences in serum T(3) and T(4) levels among the maturity stages. The observed seasonal changes in serum gonadal steroids and Vg reflected the pattern of oocyte development and the spawning behavior of common dentex and were typical of the patterns described in most multiple spawners studied to date. Thyroid hormones may enhance early ovarian development and stimulate vitellogenesis in female dentex. PMID- 10753565 TI - beta-endorphin and gonadotropin-releasing hormone in the forebrain and pituitary of the female catfish, Clarias batrachus: double-immunolabeling study. AB - The role of beta-endorphin in modulating the gonadotropic action of gonadotropin releasing hormone (GnRH) is well established in mammals. Although the information from teleosts also suggests that endogenous opioids modulate GnRH secretion and influence gonadotropic hormone release, the anatomical substrate in which opiate peptides and GnRH may interact has not been studied. Herein we describe the mammalian GnRH- and beta-endorphin-like immunoreactivities in the olfactory system, forebrain, and pituitary of the teleost, Clarias batrachus, using the double immunocytochemical method. While several olfactory receptor neurons showed beta-endorphin- or GnRH-like immunoreactivity, some neurons with dual immunoreactivities were also seen. GnRH- and/or beta-endorphin-like immunolabeled fascicles were seen in the olfactory nerves as they run caudally to the olfactory bulb and spread in the periphery. Several fascicles branch profusely to form tufts organized as spherical neuropils in the glomerular layer. Frequently, the innervation of the glomeruli showed a distinct pattern. While the fascicles on the medial side showed a predominance of beta-endorphin-like fibers, the majority of the fascicles on the lateral side of the bulb showed dual immunoreactivities. Several GnRH- and beta-endorphin-like immunoreactive fibers were seen in the medial olfactory tract as it extends through the telencephalon in the area ventralis telencephali/pars supracommissuralis; individual fibers with dual staining were also seen. The nucleus lateralis tuberis showed beta-endorphin- as well as GnRH-like immunoreactive neurons. While GnRH-containing cells were seen in the proximal pars distalis and pars intermedia, beta-endorphin-like cells were located throughout the pituitary; some cells in the pars intermedia showed dual immunoreactivity. The high degree of overlapping suggests the possibility of profound interplay between GnRH- and beta-endorphin-like immunoreactive systems at different levels of the neuraxis. PMID- 10753566 TI - Catechol concentrations in the hemolymph of the scallop, Placopecten magellanicus. AB - Catecholamines have previously been detected in numerous tissues and are thought to control a wide variety of physiological functions in bivalve molluscs. In the present study, alumina extraction and high-performance liquid chromatography reveal the presence of significant concentrations of 3,4-dihydroxyphenylalanine (DOPA), dopamine, and 3,4-dihydroxyphenylacetic acid (DOPAC) in the hemolymph of the sea scallop, Placopecten magellanicus. The concentration of dopamine in the hemolymph averaged 223.8 ng/ml, (+/-48.4, SEM), equivalent to 10(-7) to 10(-6) M. Neither epinephrine nor norepinephrine was reliably detected in significant quantities. Previous studies have demonstrated physiological responses to dopamine with thresholds of 10(-9) to 10(-6) M, thus suggesting that this catecholamine may have an endocrine function. Furthermore, monitoring hemolymph concentrations of catechols might provide a sensitive measure of the physiological status of bivalves. For example, drugs known to affect catechol concentrations in other tissues also effect hemolymph levels. Administration of monoamine oxidase inhibitors such as pargyline, deprenyl, and clorgyline at 10( 4) M for 1 day of incubation followed by a 2-day wash resulted in decreased hemolymph concentrations of DOPAC and increased concentrations of its precursors, DOPA and dopamine. Incubation in 10(-4) M 3,5-dinitrocatechol, a catecholamine-O methyl transferase blocker, for 1 day followed by a 2-day wash significantly increased the concentration of dopamine and DOPAC in the hemolymph. Scallops incubated in 10(-5) M alpha-methyl-p-tyrosine, a blocker of tyrosine hydroxylase, for 1 day followed by a 3-day wash in artificial seawater had significantly reduced concentrations of DOPA, dopamine, and DOPAC in the hemolymph. In addition to responding to pharmacological agents, dopamine levels also decreased significantly following thermal induction of spawning, thus suggesting that hemolymph concentrations of catechols might provide indices of reproductive activity and/or stress. PMID- 10753567 TI - The distribution of lamprey GnRH-III in brains of adult sea lampreys (Petromyzon marinus). AB - In the sea lamprey, Petromyzon marinus, two forms of GnRH, lamprey GnRH-I and III, have been demonstrated to be neurohormones regulating the pituitary-gonadal axis. The objective of the present study was to determine the distribution of lamprey GnRH-III in the brains of adult sea lampreys and to compare it to the distribution of lamprey GnRH-I. For this purpose, two kinds of immunostaining were employed: one was a single immunostaining by one of two GnRH antibodies using two successive sections; the other was double immunostaining of a single section. A dense accumulation of neuronal cells immunoreactive (ir) to antisera against either lamprey GnRH-I or -III was found in the arc-shaped preoptico anterior hypothalamic area. Additional smaller numbers of irGnRH cells were found in the periventricular zone of the posterior hypothalamus. In the above-mentioned locations, the distribution of both irGnRH-I and -III cells was intermixed and very similar, but the cells exhibiting GnRH-III immunoreactivity were distinctly different from those exhibiting GnRH-I immunoreactivity. The relative numbers of irGnRH-III cells were larger than those of irGnRH-I cells in the preoptico anterior hypothalamic area, and more than 90% of GnRH cells in the posterior hypothalamus were irGnRH-III cells. Both irGnRH-I and -III cells projected their fibers primarily into the neurohypophysis. The relative densities of the accumulated irGnRH-III fibers were similar to those of irGnRH-I fibers in the anterior neurohypophysis but higher than those of irGnRH-I fibers in the posterior neurohypophysis. The present study provides further immunocytochemical data to the already compelling physiological evidence that indicates that both lamprey GnRH-I and -III act through the hypothalamic-pituitary-gonadal axis to modulate reproductive processes in the sea lamprey. PMID- 10753568 TI - Occurrence of immunoreactive Activin/Inhibin beta(B) in thyrotropes and gonadotropes in the bullfrog pituitary: possible Paracrine/Autocrine effects of activin B on gonadotropin secretion. AB - Occurrence of immunoreactive activin/inhibin beta(B) in the bullfrog (Rana catesbeiana) pituitary was investigated immunocytochemically by use of antibody against Xenopus activin/inhibin beta(B) subunit. Thyrotropes were demonstrated to contain activin/inhibin beta(B)-immunoreactive substances. Moreover, immunoelectron microscopy revealed that in the secretory granules of thyrotropes and, to a lesser extent, in those of gonadotropes, activin/inhibin beta(B) immunoreactive substances were present. Based on this observation, we investigated the effect of activin B on the release of gonadotropins from dispersed anterior pituitary cells of the bullfrog. Activin B stimulated the release of not only follicle-stimulating hormone (FSH) but also luteinizing hormone (LH) dose dependently. Under the culture conditions used in this experiment, inhibin B, as well as follistatin, did not affect the basal levels of LH and FSH, but they suppressed the activin-induced release of these hormones. This is the first study on the effect of activin on pituitary hormone secretion in lower tetrapods. PMID- 10753569 TI - Biological and immunological characterization of multiple GnRH in an opisthobranch mollusk, Aplysia californica. AB - Gonadotropin-releasing hormone (GnRH) is a neurohormone central to the regulation of reproductive functions in vertebrates. Recently, several studies have reported the presence of GnRH immunoreactivity (IR) in a number of mollusks, suggesting that the distribution of GnRH may not be restricted to Phylum Chordata. In the present study, we extend our investigations to an opisthobranch mollusk, Aplysia californica, to characterize the source, chemical nature, and biological activity of molluscan GnRH-related molecules. Specific radioimmunoassays (RIAs) of various tissue extracts of Aplysia revealed that only ovotestis, hemocytes, and hemolymph contained significant amounts of GnRH that crossreacts with antisera raised against tunicate-I (tI) and mammalian (m) GnRH. Further RIAs and extractions revealed that the GnRH-IR in the hemolymph is biochemically and immunologically distinct from the GnRH-IR in the hemocytes and ovotestis. Using reverse-phase high-performance liquid chromatography coupled with RIAs, the GnRH-IR in the hemolymph was resolved into two major peaks. The first peak eluted earlier than most known forms of vertebrate GnRH, and the later peak coeluted with m, lamprey I, chicken II, and tI-GnRH. However, both peaks were broad and may contain a heterogeneous mixture of GnRH-IR. Immunocytochemical study showed that tI-GnRH-IR was present in the connective sheath surrounding the central nervous system, with a strong presence in what appeared to be vascular space, again suggesting the close association between Aplysia GnRH-IR and circulation. Finally, treatment of the neuroendocrine bag cells with chicken II GnRH significantly decreased the duration of the afterdischarge (AD, a characteristic pattern of electrical firing in bag cell neurons) and the number of action potentials fired during an AD, indicating the presence of a corresponding GnRH receptor in the Aplysia central nervous system. Overall, the results demonstrated the presence of multiple forms of GnRH-IR that crossreact with tI- and mGnRH antisera in A. californica and the ability of a vertebrate GnRH to alter Aplysia neural activity. Together, these data suggest that GnRH may be a factor released by the ovotestis and hemocytes into the circulation to alter neural functions. GnRH-IR produced by the latter may serve as a novel mediator of the neural and immune functions in Aplysia. PMID- 10753570 TI - Melatonin counteracts the stimulatory effects of blinding or exposure to red light on reproduction in the skipper frog Rana cyanophlyctis. AB - An earlier study reported that blinding or exposure to red light stimulates reproduction in R. cyanophlyctis. The present study investigates whether melatonin counteracted blinding- or red-light-induced ovarian stimulation. The study consisted of two experiments carried out during the prebreeding (March/April) and breeding phase (August/September) of the reproductive cycle of this species. In experiment 1, exposure to red light for 30 days increased (P<0.01) the gonadosomatic index (GSI) and oviductal weights, whereas exposure to continuous darkness for the same duration decreased (P<0.01) the GSI. Data on follicular kinetics revealed an increase in vitellogenic oocytes and decrease in previtellogenic oocyte numbers following exposure to red lights. Exposure to continuous dark on the other hand increased previtellogenic oocyte and decreased vitellogenic oocyte numbers. In experiment 2, the GSI and the number of vitellogenic oocytes increased in blinded and red-light-exposed frogs. However, treatment with melatonin (15 microg sc) for 30 days to both these groups of frogs reduced the GSI, oviductal weights, and the number of vitellogenic oocytes. The results both confirm earlier finding that blinding and exposure to red light stimulate ovarian growth and also demonstrate that melatonin counteracts blinding or red-light-induced stimulation of ovarian activity. This apart, the findings from experiment 1 demonstrate for the first time that exposure to continuous dark inhibits ovarian growth in this species. Ocular melatonin seems to play an important role in regulation of reproduction in this species. PMID- 10753571 TI - Expression of cytochrome P450 cholesterol side chain cleavage and 3beta hydroxysteroid dehydrogenase during embryogenesis in chicken adrenal glands and gonads. AB - Expression of cytochrome P450 cholesterol side chain cleavage (P450scc) and 3beta hydroxysteroid dehydrogenase (3beta-HSD) mRNAs was examined in chicken embryonic adrenal glands and gonads between days 4 and 12 of incubation. In situ hybridization analysis showed that 3beta-HSD mRNA appeared on day 5 of incubation in the adrenal glands and on day 6 in the gonads, while P450scc mRNA was expressed on day 7 in both the adrenal glands and the gonads. Cells expressing both enzyme mRNAs were distributed in the steroidogenic tissues of the adrenal glands and in the medullary cords of the gonads. From days 9 to 11 of incubation, P450scc mRNA expression was not found in the majority of both the adrenal glands and the gonads, but was detected again in both on day 12, although 3beta-HSD mRNA was constitutively expressed during this period. Changes in the expression pattern of P450scc mRNA are paralleled by changes in the plasma corticosterone level reported previously. Therefore, it is suggested that P450scc is essential to embryogenesis. PMID- 10753572 TI - Molecular cloning of proopiomelanocortin cDNA from an elasmobranch, the stingray, Dasyatis akajei. AB - Recently, we have characterized a new MSH (named delta-MSH) which joins the group of MSHs (alpha, beta, gamma) in dogfish proopiomelanocortin (POMC). The present study has confirmed the presence of delta-MSH in POMC of another member of the elasmobranchian order, the stingray, Dasyatis akajei, by cDNA cloning from pituitary mRNAs. Overlapping partial cDNA clones corresponding to stingray POMC were amplified by PCR from single-strand cDNA prepared from pituitary poly (A)(+) RNA. Excluding the poly A tail, stingray POMC cDNA consists of 1077 base pairs (bp). It contains a 912-bp open reading frame encoding a signal peptide of 24 amino acids (aa) and a POMC of 280 aa. gamma-MSH, alpha-MSH, ACTH, delta-MSH, beta-MSH, and beta-endorphin are located at POMC (50-61), (115-127), (115-153), (182-193), (226-242), and (245-280), respectively. The stingray POMC is smaller than that of the dogfish POMC (294 aa) mainly due to the absence of a sequence of 11 consecutive aa between delta-MSH and beta-MSH. delta-MSH has been found only in the elasmobranchs and, therefore, delta-MSH might have evolved after the divergence of chondrichthians from the ancestral vertebrate lineage and before divergence of sharks and rays. PMID- 10753573 TI - Effects of weather on corticosterone responses in wild free-living passerine birds. AB - Harsh weather can have devastating effects on both the survival and the breeding success of wild animals. Corticosterone, released in response to the stress caused by harsh conditions, may trigger physiological and behavioral changes that help ameliorate these effects. We examined three species of Arctic-breeding passerine birds for correlations between circulating plasma corticosterone levels and weather conditions at the time of capture. Furthermore, because persistently poor weather conditions may be required to initiate a stress response, we also looked for a relationship between corticosterone levels and weather conditions over the 24 and 72 h preceding capture. None of the three species showed substantial effects of weather on unstressed corticosterone levels during the breeding season, although one species showed a significant relationship with stress-induced corticosterone levels. In two species, however, variations in weather during molt (an energetically costly period when birds replace their feathers) explained from 35 to 88% of the individual variation in corticosterone levels. In a third molting species, weather conditions in the preceding 24 and 72 h explained between 20 and 30% of the individual variation in corticosterone levels. It thus appears that adverse weather may be a potent stimulator of corticosterone release during molt, but not during the breeding season. Although extreme weather conditions (those experienced once every few years) can disrupt breeding, since none of the birds abandoned breeding we might conclude that the storms in this study were well within the ability of the birds to cope physiologically. PMID- 10753574 TI - Molecular cloning and characterization of Japanese eel ovarian P450c17 (CYP17) cDNA. AB - As a first step in investigating the mechanism underlying the steroidogenic shift from the production of ovarian androgens (vitellogenic stage) to that of 17alpha hydroxylated progestins (maturational stage) in Japanese eel during induced oogenesis, a cDNA encoding Japanese eel (Anguilla japonica) ovarian P450c17 (CYP17: steroid 17alpha-hydroxylase/C(17-20) lyase) was cloned and sequenced. This cDNA contained the complete coding region representing 510 amino acid residues, which showed high sequence homology to those of rainbow trout (74%) and mammals (45-55%). The protein encoded by this cDNA possessed high enzymatic activities of 17alpha-hydroxylase and C(17-20) lyase, thus quickly converting pregnenolone and progesterone to their respective delta(4) and delta(5) C19 products. P450c17 produced a single transcript of 2.4 kb in length, as assessed by Northern blot. Transcript levels of this enzyme significantly increased throughout artificially induced ovarian development. Considering this together with the previous data showing that C(17-20) lyase activity decreased from the vitellogenic to the maturational stage, whereas 17alpha-hydroxylase activity increased, the present data suggest that changes in C(17-20) lyase activity (the production of androgens) do not depend on transcriptional changes of the P450c17 gene. PMID- 10753575 TI - Long-term peripheral treatment of immature coho salmon (Oncorhynchus kisutch) with human leptin has no clear physiologic effect. AB - The effects of long-term peripheral exposure to recombinant human leptin were tested in immature coho salmon under both fed and fasted conditions. We found that high circulating levels of human leptin did not alter growth, energy stores, gonad weight, pituitary content of follicle-stimulating hormone, or plasma levels of insulin-like growth factor-I, insulin, growth hormone, or thyroxine. PMID- 10753576 TI - Phylogenetic sequence analysis, recombinant expression, and tissue distribution of a channel catfish estrogen receptor beta. AB - An estrogen receptor beta (ERbeta) cDNA fragment was amplified by RT-PCR of total RNA extracted from liver and ovary of immature channel catfish. This cDNA fragment was used to screen an ovarian cDNA library made from an immature female fish. A clone was obtained that contained an open reading frame encoding a 575 amino-acid protein with a deduced molecular weight of 63.9 kDa. Maximum parsimony and Neighbor Joining analyses were used to generate a phylogenetic classification of channel catfish ERbeta on the basis of 25 full-length teleost and tetrapod ER sequences. The consensus tree obtained indicated the existence of two major vertebrate ER subtypes, alpha and beta. Within each subtype, and in accordance with established phylogenetic relationships, teleost and tetrapod ER were monophyletic confirming the results of a previous analysis (Z. Xia et al., 1999, Gen. Comp. Endocrinol. 113, 360-368). Extracts of COS-7 cells transfected with channel catfish ERbeta cDNA bound estrogen with high affinity (K(d) = 0.21 nM) and specificity. The affinity of channel catfish ERbeta for estrogen was higher than previously reported for channel catfish ERalpha. As determined by qualitative RT-PCR, the tissue distributions of ERalpha and ERbeta were similar but not identical. Both ER subtypes were present in ovary and testis. ERalpha was found in all other tissues examined from juvenile and mature fish of both sexes. ERbeta was also found in most tissues except, in most cases, whole blood and head kidney. Interestingly, the pattern of expression of ER subtypes in head kidney always corresponded to the pattern in whole blood. In conclusion, we isolated a channel catfish ERbeta with ligand-binding affinity and tissue expression patterns different from ERalpha. Also, we confirmed the validity of our previously proposed general classification scheme for vertebrate ER into alpha and beta subtypes and within each subtype, into teleost and tetrapod clades. PMID- 10753577 TI - The expression of preprosomatostatin II mRNAs in the Brockmann bodies of rainbow trout, Oncorhynchus mykiss, is regulated by glucose. AB - We previously characterized two cDNAs that encode for distinct preprosomatostatin molecules containing [Tyr(7), Gly(10)]-somatostatin-14 at their C-termini (PPSS II' and PPSS II") and found that these cDNAs were differentially expressed in the endocrine pancreas (Brockmann body) of rainbow trout, Oncorhynchus mykiss. In this study, we examined the control of PPSSII' mRNA and PPSS II" mRNA expression by glucose. Fish injected with glucose displayed elevated plasma levels of glucose in association with nearly three-fold higher levels of PPSS II mRNAs compared to saline-injected control animals. Glucose directly stimulated the expression of both PPSS II mRNAs in vitro in a dose-dependent manner; however, glucose was a more potent stimulator of PPSS II" expression than of PPSS II' expression. The hexoses, mannose, galactose, and fructose, as well as glucose, all induced the expression of PPSS II mRNAs, whereas, sucrose and the glucose analogs, 3-o-methylglucose and 2-deoxyglucose, were without effect. In addition, the expression of PPSS II mRNAs was stimulated by dihydroxyacetone, pyruvate, lactate, acetate, and citrate. Furthermore, the expression of PPSS II mRNAs was inhibited by iodoacetate, an inhibitor of glycolysis, but was stimulated by dichloroacetate, a stimulator of Krebs cycle flux via pyruvate dehydrogenase activation. Finally, glucose-stimulated PPSS II expression was inhibited by actinomycin. These results indicate that the expression of PPSS II mRNAs in the Brockmann body of trout is regulated by nutrients such as glucose and suggest that glucose-stimulated expression of PPSS II mRNAs requires the uptake and subsequent metabolism of the sugar and is transcription sensitive. PMID- 10753578 TI - Identification of orcokinin gene-related peptides in the brain of the crayfish Procambarus clarkii by the combination of MALDI-TOF and on-line capillary HPLC/Q Tof mass spectrometries and molecular cloning. AB - We developed a strategy for the exploration of brain peptides in the red swamp crayfish, Procambarus clarkii, utilizing the combined techniques of matrix assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI TOF MS), molecular cloning, and on-line capillary reversed-phase HPLC/quadrupole orthogonal acceleration time-of-flight (Q-Tof)-MS. We initially performed direct MALDI-TOF MS analysis with slices of the brain. The MS spectra from a slice of the olfactory lobe indicated that an orcokinin (NFDEIDRSGFGFN) occurs in this species. Subsequently, its occurrence was confirmed by molecular cloning of the cDNAs encoding the precursor protein of orcokinin. The deduced amino acid sequences indicated that there are two different types of preproorcokinins. Preproorcokinin A (251 residues long) contains not only seven copies of orcokinin but also two copies of NFDEIDRSGFGFV and one copy each of NFDEIDRSGFGFA, NFDEIDRTGFGFH, and FDAFTTGFGHS. The former three peptides were previously isolated from another crayfish, Orconectes limosus, and/or the shore crab, Carcinus maenas, and the latter two were novel. Preproorcokinin B (266) harbors one additional orcokinin. All sequences of the peptides are flanked by dibasic sequences which are the consensus signal for processing. Moreover, brain extract was subjected to Sephadex G-25 and, subsequently, to on-line capillary reversed phase HPLC/Q-Tof MS analysis. From the LC-MS analysis, the molecular weights of orcokinin, NFDEIDRSGFGFV, NFDEIDRSGFGFA, NFDEIDRTGFGFH, and FDAFTTGFGHS were identified as the doubly charged ions at m/z 759.37, 751.92, 737.86, 777.90, and 593. 78, respectively. In addition, the sequences were assigned by the collision induced dissociation spectra using the doubly charged ions in the LC-MS/MS analysis. These data suggest that orcokinin and its related peptides are especially abundant in the olfactory lobe and are synthesized and processed from the two types of preproorcokinins in the crayfish brain. PMID- 10753579 TI - Photoperiodic regulation of the male house sparrow song control system: gonadal dependent and independent mechanisms. AB - The primary and secondary sexual characteristics of many species of passerine birds undergo dramatic seasonal variation in response to the change in the length of photoperiod. Among the many physiological processes that undergo seasonal changes, bird song and the song control system underlying it undergo similar seasonal variation in size and function. The mechanisms of this seasonal variation are largely unknown but are at least partially due to steroidal action from the gonads. The present study determined the relative roles played by the gonads and the photoperiodic timing system that controls gonadal development on song control nuclei in the brain of the male house sparrow, Passer domesticus. Sparrows maintained in short photoperiods (SD) possessed small regressed testes. Transfer to long photoperiods (LD) for 6 weeks evoked a dramatic increase in testes size, but, after 20 weeks under the same conditions (LDLD), testes completely collapsed. Song control nuclei HVC and RA were smaller in SD than in LD but regressed only moderately in LDLD. Castration of sparrows in SD reduced the amplitude of the seasonal variation but did not completely abolish it. The data support the view that the song control system of the house sparrow is regulated by the photoperiodic timing system independently of gonadal influence, but that the gonads augment seasonal regulation of song, presumably via steroidal hormone secretion. PMID- 10753580 TI - Rapid effects of corticosterone on cache recovery in mountain chickadees (Parus gambeli). AB - Environmental perturbations increase adrenal activity in several vertebrates. Increases in corticosterone may serve as a proximate trigger whereby organisms can rapidly adapt their behavior to survive environmental fluctuations. In food caching songbirds, inclement weather may present the need to alter caching and/or retrieval behaviors to ensure food supplies. We hypothesized that corticosterone may increase the rate of caching and/or retrieval behaviors in the mountain chickadee, a food-storing songbird, and tested if these potential effects were mediated by alterations in appetite, activity, or memory for cache sites. Corticosterone or vehicle was administered to subjects 5 min prior to either caching or recovery in a naturalistic laboratory paradigm during which we recorded the number of caching events, sites visited, and seeds eaten (caching) or caches recovered, total sites visited, cache-related visits, and non-cache related visits (recovery). Data were analyzed using nested ANOVA for treatment within sequential trial. There was no effect on any caching behaviors following treatment. However, birds treated with corticosterone during retrieval recovered more seeds and tended to visit more cache-related sites than did controls. Since groups did not differ in the number of seeds eaten or the total number of sites visited, it seems unlikely that corticosterone affected appetite or activity. Rapid surges in corticosterone may increase the efficacy of an underlying memory process for cache sites which is reflected in higher cache recovery in corticosterone-treated birds than in controls. Thus, rapid alterations in plasma corticosterone following environmental change may alter memory-reliant behaviors which promote survival in the food-caching mountain chickadee. PMID- 10753581 TI - Paternal hamsters mechanically assist the delivery, consume amniotic fluid and placenta, remove fetal membranes, and provide parental care during the birth process. AB - We report the first description of a male mammal behaviorally participating in the birth of his young. Djungarian hamsters, Phodopus campbelli, are naturally biparental and males contribute to pup survival and growth through direct paternal care and indirect effects on maternal physiology. Males of the closely related Siberian hamster, P. sungorus, have only seasonal opportunities to interact with pups and neither contribute to pup survival and growth nor provide extensive paternal care under laboratory conditions. Male P. campbelli undergo a series of hormonal changes prior to the birth of their first litter which are not shared with P. sungorus males and may "prime" males to express paternal behavior. We hypothesized that P. campbelli males would behave paternally on first contact with pups during the birth process, whereas P. sungorus males would not. In addition to showing paternal behavior toward neonates, male P. campbelli licked amniotic fluid before the birth, mechanically assisted the delivery, opened an airway by clearing the nostrils (so that pups flushed red with oxygenation), licked and sniffed pups in the moments and minutes after birth to clean the pups of membranes, and ate the placenta. None of these behaviors were observed in P. sungorus. We anticipate that P. campbelli will not be the only species with highly developed paternal behavioral repertoires during the birth. PMID- 10753582 TI - Shield characteristics are testosterone-dependent in both male and female moorhens. AB - The immunocompetence handicap hypothesis proposes that the expression of secondary sexual characteristics is positively related to testosterone levels, but that elevated testosterone levels also impose costs from immune suppression. Hence, testosterone-dependent characteristics should accurately reflect male quality because only high-quality males are able to invest in large sexual characteristics without detrimental effects upon their own immune system. Most studies to date have focused on the role of testosterone in the expression of male ornaments and on the possible immunosuppressant effects of androgens in males. In the moorhen (Gallinula chloropus), a sexually monomorphic monogamous bird species showing a partial sex-role reversal, both sexes have a prominent frontal shield. We implanted both sexes with testosterone-filled implants to examine the effects of testosterone on shield characteristics and immune function. Shield size, thickness, and color were significantly increased by an experimental increase in testosterone concentrations in both males and females. Likewise, removal of the implants led to a rapid decrease in shield size and thickness in both males and females, suggesting that both sexes responded quickly to an increase or a decrease in testosterone. Moorhens implanted with testosterone had higher intensities of ectoparasite infestations than control birds, but other indirect measures of immunocompetence did not differ significantly between the two categories of birds. PMID- 10753583 TI - Progesterone, but not progesterone-independent activation of progestin receptors by a mating stimulus, rapidly decreases progestin receptor immunoreactivity in female rat brain. AB - Recent studies suggest that progestin receptors may be activated in vivo by neurotransmitters in the absence of ligand. More specifically, vaginal-cervical stimulation (VCS) can influence sexual behavior by activating progestin receptors in the absence of progesterone. Another way to test if progestin receptors are influenced by particular stimuli is to examine progestin receptor immunostaining. We report that progestin receptor immunoreactivity is decreased in the forebrain of estradiol-primed ovariectomized (OVX) rats within 1 h after a subcutaneous injection of progesterone, a time by which rapid down-regulation of progestin receptors does not seem to have occurred. In estradiol-primed OVX rats, VCS also decreased progestin receptor immunoreactivity within 1 h in the medial preoptic area, but not in any other area examined. To determine if the decrease in immunoreactivity by VCS was due to adrenal secretions or by ligand-independent activation of progestin receptors, we repeated the experiment in estradiol-primed OVX/adrenalectomized rats. Prior removal of the adrenal glands blocked the rapid decrease in progestin receptor immunoreactivity, even though data from other experiments suggest that progestin receptors are activated by VCS at this time. These studies suggest the possibility that progestin receptors may be affected differentially by progesterone-dependent or by progesterone-independent pathways. This raises the possibility that activation of progestin receptors by these two distinct pathways may lead to different neuronal consequences. PMID- 10753584 TI - Infant vocalization, adult aggression, and fear behavior of an oxytocin null mutant mouse. AB - Previous studies have shown that oxytocin (OT)-deficient female mice produced by homologous recombination fail to lactate but exhibit normal parturition and reproductive behaviors. We examined the ultrasonic vocalizations of infant mice and the subsequent aggressive and fear behavior of adult male OT knockout (OT-KO) mice. Infant OT-KO mice were less vocal than wild-type (WT) control mice during separations from the mother and peers. Adult OT-KO males were generally more aggressive in isolation-induced and resident-intruder tests of aggression and less fearful in the plus maze and acoustic startle reflex tests than WT controls. Although the increase in tests of aggression was robust for OT-KO males from obligate litters (progeny of homozygous x homozygous crossings), the increase in aggression was reduced during tests for OT-KO males derived from nonobligate mating (progeny of heterozygous x heterozygous crossings), suggesting that the OT KO genotype was not, by itself, responsible for the changes in adult behavior. We conclude that the absence of exposure to OT during development was associated with abnormalities in the development of emotional behavior. PMID- 10753585 TI - Adult offspring long-term effects of high salt and water intake during pregnancy. AB - Offspring from dams subjected to hypereninemia, hyperdipsia, and natriophilia by partial aortic ligation (PAL) showed a long-term modification of their ingestive behavior. These rats, upon reaching adulthood, showed an increased appetite for low-concentration saline solutions (0.1 M) when compared to control rats. They also presented a high intake of a medium concentration NaCl solution (0.45 M) after having been offered a very aversive highly concentrated NaCl solution (1.0 M) along with water for 2 days. An increase was also observed in their salt/water intake ratio following two different thirst challenges, 24-h fluid deprivation or sodium depletion by furosemide treatment. The demonstration of the long-term effect of pregnancy history on salt preference in adult offspring draws attention to the possible physiopathological aspects that may be of relevance, considering the well-established relationship between salt intake and hypertension, a disease most commonly occurring in the adult and aged population. PMID- 10753586 TI - The hormonal control of birth behavior in the gray short-tailed opossum (Monodelphis domestica). AB - In all major groups of Australian marsupials, prostaglandin F2alpha (PGF) or oxytocin injection initiates birth behavior in adult females, adult males and pouch young. Because inhibitors of PGF synthesis block this initiation, oxytocin may activate birth behavior via the stimulation of PGF synthesis. In this study, the role of PGF and oxytocin in the activation of birth behavior was examined in an American marsupial, the gray short-tailed opossum (Monodelphis domestica). Adult male and female gray opossums were given PGF, oxytocin, or saline (control) before behavioral observation. On the next day, the animals in the oxytocin group were injected with the PGF inhibitor flunixin meglumide (Finadyne, Schering Corp., U.S.A.) before oxytocin reinjection and behavioral observation. Both males and females showed birth behavior in response to PGF but only females responded to oxytocin. There was no significant difference in the latency of response of females to oxytocin alone versus response to oxytocin after receipt of the PGF inhibitor. These results suggest that, in contrast to Australian species, in this American marsupial, oxytocin initiates birth behavior only in females and does not operate via stimulation of prostaglandin secretion. PMID- 10753587 TI - Actions and interactions of the IGF system in Alzheimer's disease: review and hypotheses. AB - Insulin-like growth factors (IGF) are pleiotrophic polypeptides affecting all aspects of growth and development. The IGF system, including ligands, receptors, binding proteins and proteases is also involved in pathophysiological conditions, such as cancer and degenerative conditions. In this review, the actions and interactions of the IGF system as it relates to Alzheimer's disease will be investigated. PMID- 10753588 TI - Changes in lipoprotein(a) levels measured by six kit methods during growth hormone treatment of growth hormone-deficient adults. AB - Lipoprotein(a) [Lp(a)], an independent risk factor for cardiovascular disease, has previously been reported to increase, decrease or show no change in growth hormone (GH)-deficient individuals receiving GH replacement. To assess whether these inconsistencies could be attributed to differences in immunoassay methods, Lp(a) was measured by six commercial kits at 0, 3, 6 and 9 months in nine GH deficient individuals (median age 68.3 years, six male) during 9 months GH therapy. There was a significant rise in Lp(a) with the INCStar immunoturbidimetric (IT) method and the Mercodia enzyme linked immunosorbent assay (ELISA) (P1000 resting spores/ml. Resting spore production was not affected when isolates were mixed. Glycerol (used for fungal storage), trehalose, and selected amino acids each inhibited resting spore formation. Fetal bovine serum was required for spore production but the presence of >5% yielded lower resting spore densities. A large surface area:volume ratio (12.5 cm(2):ml versus 4.2 cm(2):ml) was required for abundant formation of resting spores. At present, resting spores have only been produced in small volumes with a maximum of 3 x 10(4) resting spores/ml. PMID- 10753596 TI - Effects of fluctuating moisture and temperature regimes on the infection potential of Beauveria bassiana for Rhodnius prolixus. AB - The effect of both moisture and temperature on the infective potential of Beauveria bassiana to the Chagas' disease vector, Rhodnius prolixus, was studied under fluctuating regimes. At constant 25 degrees C, contaminated first-instar nymphs exposed to increasing daily periods of initial exposure to 97% RH, followed by transfer to reduced humidity (43, 53, 75, and 86% RH), showed a significant reduction in mortality when the 97% RH exposure time declined from 12 to 8 h per day. The duration of disease incubation depended on the daily 97% RH exposure time. Under fluctuating regimes of both humidity (97% RH versus 75% RH) and temperature (15/28, 20/25, 25/28, and 25/35 degrees C), first-instar mortality was affected by weather conditions, daily 97% RH exposure time (8, 12, and 16 h per day), and number of temperature and humidity fluctuations before transferring tested insects to constant unfavorable conditions. In most cases, at 12/12 h alternating cycles, high and rapid mortality required five cycles. Under these fluctuating regimes, fungus-induced mortality and mortality time were similarly affected in third- and fifth-instar nymphs by the daily 97% RH exposure time. Despite a lower susceptibility of older larval stages, mortality rates in insects exposed for at least 12 h per day at 97% RH remained very high except at 15 degrees C. Moisture and temperature regimes at 12/12 h cycling significantly affected the dose-mortality response in first-instar nymphs. The most favorable conditions consisted of 97%-20 degrees C combined with either 75%-25 degrees C or 43%-25 degrees C. Under less favorable alternating conditions (lower and higher temperatures) the amounts of inoculum required for killing 50% of first-instar nymphs were 10 or 20 times higher. From a vector control standpoint, daily high humidity appears to be the most crucial climatic constraint. B. bassiana has the potential to control R. prolixus populations with applications made during the rainy seasons when humidity is high. PMID- 10753597 TI - Inhibitory effects of ovoglobulins on bacillary necrosis in larvae of the pacific oyster, Crassostrea gigas. AB - In order to develop an alternative method to antibiotics for preventing bacillary necrosis in bivalve mollusc larvae, we examined the effects of ovoglobulins (proteins derived from the whites of hens' eggs) on the survival of larvae of the Pacific oyster Crassostrea gigas. The pathogenic Vibrio tubiashii (ATCC 19106) was used to infect larvae of the Pacific oyster. V. tubiashii showed strong pathogenicity to oyster larvae, causing 100% mortality after experimental exposure for 24 h at a concentration of 10(5) cfu (colony-forming units)/ml. In contrast, the addition of ovoglobulins at a concentration of 10 microg/ml to larval oysters, challenged with V. tubiashii at 10(5) cfu/ml, led to a marked increase in larval survival of 96.5% at 24 h after infection. The V. tubiashii culture supernatant was also shown to be pathogenic to larval oysters; however, its pathogenicity was completely inhibited by the addition of 10 microg/ml of ovoglobulins. Larval oysters infected by V. tubiashii showed typical symptoms of bacillary necrosis including anomalous swimming and detachment of cilia and/or vela. In contrast, live larvae were actively motile, and their cilia and vela were not necrotized in the ovoglobulins-added group. The addition of ovoglobulins clearly suppressed the growth of V. tubiashii in gelatin-sea water broth, but the number of viable V. tubiashii 24 h after incubation did not decrease to the initial dose level. Findings obtained in this study indicate that ovoglobulins almost completely protect larval oysters from V. tubiashii infection by nonbactericidally inhibiting the growth of V. tubiashii without affecting survival of the oysters. PMID- 10753598 TI - Parasitism-linked block of host plasma melanization. AB - When parasitized by the Ichneumonid parasitoid Campoletis sonorensis, larvae of the Noctuid moth, Heliothis virescens, are unable to mount an effective immune response against parasitoid eggs. Defensive melanization of plasma and cellular encapsulation of parasite eggs are dramatically inhibited by infection with the symbiotic immunosuppressive C. sonorensis ichnovirus (CsIV). This study demonstrates that the CsIV-mediated inhibition of melanization is associated with reduction in the enzymatic activity and protein titer of key enzymes in the melanization pathway, phenoloxidase, dopachrome isomerase, and DOPA decarboxylase. Inhibition of the synthesis of key melanization enzymes leads to reductions in the melanization substrates l-dihydroxyphenylalanine, N acetyldopamine, and N-beta-alanyl dopamine from millimolar to nanomolar levels in parasitized larvae. By contrast, concentration of a precursor catecholamine, dopamine, rises fourfold in these larvae. Thus in CsIV-infected larvae, enzymatic deficiencies in the melanization pathway lead to reduced concentrations of specific enzyme substrates, causing failure of melanization in parasitized insects. PMID- 10753599 TI - Infectivity, speed of kill, and productivity of a baculovirus expressing the itch mite toxin txp-1 in second and fourth instar larvae of Trichoplusia ni. AB - A cDNA clone of the gene coding for the paralytic neurotoxin (tox34) from the female straw itch mite, Pyemotes tritici, was created by RT-PCR and inserted into the genome of the Autographa californica nucleopolyhedrovirus (AcMNPV) under the control of the AcMNPV p10 promoter. This recombinant virus, AcTOX34.4, caused a rigid paralysis in infected larvae. The infectivity of AcTOX34.4 was compared to the wild-type parent strain, AcMNPV-C6, in second and fourth instar larvae of the cabbage looper, Trichoplusia ni. There were no significant differences in LD(50) values between the recombinant virus and its wild-type parent strain but, as expected, the LD(50) was lower for second instar larvae. The mean time to death and yield of occlusion bodies were measured in second and fourth instar T. ni larvae at a high (100% mortality) and low (<50% mortality) doses of the virus. The mean time to death of recombinant infected larvae was reduced by 50-60% compared to larvae infected with the wild-type strain, depending on virus dose and instar, with these larvae becoming paralysed after approximately 60 h and dying 10-20 h later. This is among the fastest speeds of kill recorded for recombinant baculoviruses. Fourth instar larvae were found to succumb to the recombinant virus more quickly than the second instar larvae. The increase in the speed of kill of the recombinant virus was accompanied by a large reduction of approximately 95% in the yield of progeny virus. The yield of virus showed a highly significant relationship with time to death, but this relationship was complex and varied between the different viruses, concentrations, and instars. The yield per unit weight of the larvae was found to be constant at a low virus dose and increased over time at a high virus dose, irrespective of instar and virus. It is predicted that these changes in the performance of the recombinant virus would act toward reducing its fitness, leading to it being outcompeted by the wild type in field situations. PMID- 10753600 TI - Phagocytosis of vairimorpha sp. (Microsporida, Nosematidae) spores by Plutella xylostella and Panorpa vulgaris hemocytes. PMID- 10753601 TI - RAPD analysis of Nomuraea rileyi. PMID- 10753602 TI - Cardiolipin enhances protein C pathway anticoagulant activity. AB - The anticoagulant activity of activated protein C (APC) was studied using factor Xa-1-stage assays of both the procoagulant and anticoagulant activities of phospholipid vesicles containing phosphatidylserine or cardiolipin as active phospholipids. In the absence of APC, phosphatidylserine vesicles showed higher procoagulant activity than cardiolipin vesicles whereas cardiolipin vesicles supported APC-dependent anticoagulant activity better than phosphatidylserine vesicles. Enhancement of APC anticoagulant activity in plasma by cardiolipin was markedly stimulated by the APC cofactor protein S. In purified reaction mixtures, cardiolipin in phospholipid vesicles dose-dependently enhanced APC anticoagulant activity. This effect of cardiolipin was partially dependent on protein S, and immunoblotting studies showed that cardiolipin enhanced the APC-mediated cleavage of the factor Va heavy chain at Arg506 and Arg306. In solid-phase binding assays, increasing amounts of cardiolipin in multicomponent phospholipid vesicles increased the affinity for protein S and to a lesser extent APC. These data are consistent with the hypothesis that cardiolipin stimulates the anticoagulant protein C pathway by increasing the affinity of phospholipid surfaces for protein S:APC and by enhancing inactivation of factor Va by APC due to cleavages at Arg506 and Arg306 in factor Va. Based on this, it is further hypothesized that anti-cardiolipin or anti-oxidized cardiolipin antibodies may be thrombogenic because they inhibit phospholipid-dependent expression of the anticoagulant protein C pathway. PMID- 10753603 TI - Ribosomal protein S19 gene mutations in patients with diamond-blackfan anemia and identification of ribosomal protein S19 pseudogenes. AB - Diamond-Blackfan anemia (DBA) is a rare congenital pure red cell hypoplasia characterized by a selective defect of erythropoiesis with a normochromic macrocytic anemia and reticulocytopenia often accompanied by various congenital anomalies. The critical region responsible for the pathogenesis of DBA has been mapped in some patients to chromosome 19q13.2 (P Gustavsson, E Garelli, N Draptchinskaia, et al. Am. J. Hum. Genet. 63:1388-1395, 1998) and the gene encoding ribosomal protein S19 (RPS19) is believed to be the candidate gene. Here we present molecular analysis of the RPS19 gene in DBA patients from the Czech National DBA Registry. We found that the RPS19 gene was mutated in 25% (5/20) of DBA patients (insertion, deletion, and point mutations, but no nonsense or splice site mutations). Point mutations were localized to hot spots defined by Willig (TN Willig, N Draptchinskaia, I Dianzani, et al. Blood 94:4294-4306, 1999). Moreover, we describe two processed RPS19 pseudogenes, which were not expressed. Possible models of the DBA pathogenesis in the view of RPS19 mutations are discussed. PMID- 10753605 TI - The Rotational Spectrum of SO(2) and the Determination of the Hyperfine Constants and Nuclear Magnetic Shielding Tensors of (33)SO(2) and SO(17)O. AB - Precise frequencies for the 1(11)-2(02) transition of (33)SO(2) and SO(17)O in natural isotopic abundance have been obtained near 12 GHz by microwave Fourier transform spectroscopy in order to yield improved hyperfine constants. Nuclear spin-rotation coupling constants have been determined experimentally for (33)SO(2) for the first time. The spin-rotation constants have been used to derive nuclear magnetic shielding parameters. These parameters are compared with values for the isoelectronic O(3) molecule. The transition mentioned above was also measured for (32)SO(2), (34)SO(2), SO(18)O, and vibrationally excited (v(2) = 1) (32)SO(2). For (33)SO(2), some transitions with large hyperfine splitting were also recorded in the millimeter-wave region. Continuing our investigations of the rotational spectra of SO(2) in the submillimeter region, several transitions of SO(17)O have been recorded with the Cologne terahertz spectrometer between 540 and 840 GHz with J and K(a) up to 63 and 16, respectively. Transitions with high K(a), up to 28, have been recorded with the JPL laser sideband spectrometer between 1.8 and 3.2 THz. Copyright 2000 Academic Press. PMID- 10753604 TI - Clustered CD20 induced apoptosis: src-family kinase, the proximal regulator of tyrosine phosphorylation, calcium influx, and caspase 3-dependent apoptosis. AB - Anti-CD20 antibodies may reduce or eliminate non-Hodgkin's lymphoma B cells in patients, although the mechanism of action is not clear. To explore mechanism(s), we examined the induction of signal transduction events using anti-CD20 monoclonal antibodies (mAb) in the human non-Hodgkin's lymphoma Ramos B cell line. We found that while Rituximab (a human-mouse hybrid mAb) alone induced apoptotic cell death, other murine anti-CD20 mAbs induced apoptosis of Ramos B cells only upon clustering with a secondary antibody. CD20 clustering was accompanied by activation of tyrosine protein kinase activity, PLCgamma2 phosphorylation, influx of Ca(2+), and activation of caspase 3. All signaling events, as well as the subsequent apoptosis, were blocked by PP2, a selective inhibitor of Src-family kinases. Treatment of Ramos with EGTA and BAPTA to block changes in cytoplasmic Ca(2+) likewise prevented CD20-induced apoptosis. Our findings support a model in which CD20 clustering activates members of the Src family of protein tyrosine kinases, leading to phosphorylation of PLCgamma2 and increased cytoplasmic Ca(2+). These early signal transduction events activate caspase 3 to promote apoptotic cell death of NHL B cells. PMID- 10753606 TI - Rotational Analysis of the Ground State and the Lowest Fundamentals nu(3), nu(5), and nu(6) of (13)CH(3)D. AB - The nu(3), nu(5), and nu(6) fundamental bands of the (13)CH(3)D molecule have been studied with Fourier transform infrared spectroscopy. The spectra and results for the parent species (12)CH(3)D (O. N. Ulenikov, G. A. Onopenko, N. E. Tyabaeva, J. Schroderus, and S. Alanko, J. Mol. Spectrosc. 193, 249-259 (1999)) have been used to assign and analyze about 1900 lines belonging to the (13)CH(3)D isotopic species. About 850 ground state combination differences with DeltaK = 0 were calculated, which allowed us to determine the J-dependent ground state rotational constants. The K-dependent constants as well as those describing the a(1)-a(2) (K = 3) splitting were fixed to the values obtained for the (12)CH(3)D species. The (v(3) = 1), (v(5) = 1), and (v(6) = 1) states were fit simultaneously by including the intervibrational interactions in the Hamiltonian. The rotational energies, the rotational and centrifugal distortion constants, as well as the resonance parameters involving the three states have been determined and discussed. Copyright 2000 Academic Press. PMID- 10753607 TI - Far-Infrared Laser Magnetic Resonance Spectroscopic Study of the nu(2) Bending Fundamental of the CCN Radical in Its &Xtilde;(2)Pi(r) State. AB - Bending vibration-rotation transitions between the (010) u(2)Sigma(-) and (000) (2)Pi(r) vibronic states of the CCN radical in its ground electronic state have been observed using far-infrared laser magnetic resonance (FIR LMR) spectroscopy. Thirteen FIR laser lines were used to record 769 resonances. The LMR data, combined with previous data, were used to determine vibrational, Renner-Teller, fine-structure, rotational, hyperfine, and molecular g-factor parameters using a least-squares fitting routine. The model used was an N(2) effective Hamiltonian modified to include the Renner-Teller effect explicitly in a (2)Pi electronic state. The band origin for the (010) u(2)Sigma(-) <-- (000) &Xtilde;(2)Pi(r) transition was determined to be 179.598176 +/- 0.000067 cm(-1). The spin-orbit splitting in the ground state was refined and the complete set of (14)N-hyperfine parameters determined for the first time. PMID- 10753608 TI - Pulse Pyrolysis Infrared Laser Jet Spectroscopy of Chloroketene. AB - The short-lived molecule chloroketene (ClHCCO) was generated by the pyrolysis of chloroacetyl chloride in a high-temperature nozzle and the infrared laser absorption spectrum of its nu(2) fundamental band was measured between 2153 and 2161 cm(-1). The spectrum was greatly simplified by comparison with a room temperature spectrum enabling 230 lines of the (35)ClHCCO isotopomer to be assigned and measured. A least-squares fit of these transitions yielded accurate molecular parameters for the v(2) = 1 state using ground state constants from microwave spectroscopy. The nu(2) band origin of (35)ClHCCO is 2157.19238(16) cm( 1). A comparison of simulated and experimental spectra showed that the rotational temperature of the jet spectrum was around 35 K. Copyright 2000 Academic Press. PMID- 10753609 TI - FT Infrared Spectra of FCN from 1200 to 1800 cm(-1) and from 2800 to 7000 cm(-1) and Global Rovibrational Analysis of the Main Isotopomers. AB - We measured the FT spectrum of natural FCN from 1200 to 1800 cm(-1) and from 2800 to 7000 cm(-1) with a near Doppler resolution and a line-position accuracy between 1 and 8 x 10(-4) cm(-1). Lines were assigned to 91 different bands (75 of (19)F(12)C(14)N, 11 of (19)F(13)C(14)N, and 5 of (19)F(12)C(15)N), of which no more than five had been previously reported at lower resolution. From band-by band analyses we deduced effective state parameters. As those results complete the study of the Fourier transform spectra of natural cyanogen fluoride from 1200 to 7000 cm(-1), global rovibrational analyses of (19)F(12)C(14)N, (19)F(13)C(14)N, and (19)F(12)C(15)N have been performed on the basis of all the available experimental data about their rovibrational energies. We have so determined sets of about 100 molecular parameters for each isotopomer, and a statistical agreement was obtained. A labeling of the vibrational states is proposed on the basis of the eigenvector coefficients. Copyright 2000 Academic Press. PMID- 10753610 TI - Diode-Laser Measurements of N(2)-Broadening Coefficients in the nu(7) Band of C(2)H(4). AB - N(2)-broadening coefficients have been measured for 35 lines of C(2)H(4) in the nu(7) fundamental transition, using a tunable diode-laser spectrometer. These lines with 3 A' (2)Delta Transition of LaO. PMID- 10753625 TI - Intensity of the Simultaneous Vibrational Absorption CO(2) (v(3) = 1) + N(2) (v = 1) <-- CO(2) (v(3) = 0) + N(2) (v = 0) at 4680 cm(-1). PMID- 10753626 TI - Serum amyloid A is a chemotactic agonist at FPR2, a low-affinity N-formylpeptide receptor on mouse neutrophils. AB - Serum amyloid A (SAA) is an acute-phase plasma protein and the source of amyloid A, which accumulates in lesions of secondary amyloidosis. SAA can induce phagocyte migration in vitro and in vivo, and is a specific chemotactic agonist for the human low-affinity N-formylpeptide receptor FPRL1R, a G-protein-coupled receptor expressed on phagocytes. Here we show that FPR2, a mouse counterpart of FPRL1R, is also an SAA receptor. SAA selectively induced calcium flux and chemotaxis in mouse PMN, which express FPR2, as well as in HEK 293 cells expressing recombinant FPR2 but not in HEK 293 cells expressing FPR, a closely related high affinity N-formylpeptide receptor. Consistent with this, SAA activity on PMN from FPR+/+ and FPR-/- mice was indistinguishable. Moreover, the prototype N-formylpeptide fMLF desensitized SAA-induced calcium flux in a dose dependent manner in both mouse neutrophils and HEK 293/FPR2 transfectants. Our results suggest that FPR2 specifically mediates mouse neutrophil migration in response to SAA. PMID- 10753627 TI - Functional analysis of the domain organization of Trypanosoma brucei RNase HI. AB - The structure-function relationship of Trypanosoma brucei RNase HI was investigated by evaluating the abilities of truncated forms of the enzyme to convert RNase H substrate to product. Our studies identify a 42-amino-acid noncanonical RNase HI spacer domain essential for function. We also show that the enzyme's nuclear localization domain is not required for RNase H activity but functions as an RNA binding domain which modulates the enzyme's Mn(2+)-dependent activity. These findings show that the enzyme's RNA binding/nuclear targeting and RNase H activities are organized into discrete N- and C-terminal domains with boundaries established by its spacer domain. This is the first report of the unusual structure to function relationship of a protozoal RNase H. This relationship may be conserved in other eukaryotic RNases H suggesting that criteria preserving their structure and function may be important to their roles in nucleic acid metabolism. PMID- 10753628 TI - Stimulation by eicosapentaenoic acids of leptin mRNA expression and its secretion in mouse 3T3-L1 adipocytes in vitro. AB - Recent evidence indicates that both leptin and eicosapentaenoic acids (EPA) improve insulin sensitivity. In the present study, we examined the effect of EPA on endogenous leptin expression in 3T3-L1 adipocytes to clarify whether the EPA's effect is exerted through leptin expression. EPA caused a time- and dose dependent increase of leptin mRNA levels in 3T3-L1 adipocytes. Leptin mRNA expression was significantly increased up to 309.4 +/- 17.0% of the control by 24 h (P < 0.01; n = 6). Leptin secretion was also significantly increased up to 193.3 +/- 12.1% of the control by 24 h (P < 0.01; n = 6). EPA is a ligand for peroxisome proliferator-activated receptors (PPARs) with the highest affinity to PPARalpha. We examined the effect on leptin expression of clofibrate, a ligand for PPARalpha, bezafibrate, for PPARbeta, or troglitazone, for PPARgamma, to clarify whether these ligands for PPARs could mimic EPA-induced stimulation of leptin expression. Neither clofibrate nor bezafibrate affected leptin mRNA expression, whereas troglitazone significantly suppressed leptin mRNA expression. On the other hand, inhibition by 6-diazo-5-oxo-l-norleucine of the rate-limiting enzyme in hexosamine biosynthesis blunted EPA-induced stimulation of leptin mRNA expression and its secretion. These data suggest that EPA up-regulates leptin gene expression and its secretion probably through a hexosamine biosynthetic pathway. PMID- 10753629 TI - High level of ferritin light chain mRNA in lens. AB - Ferritin is of particular interest with regard to cataract because (i) cataract occurs in individuals with hereditary hyperferritinemia cataract syndrome (HHCS), a condition in which ferritin light chain (L-ferritin) protein is overexpressed systemically, and (ii) ferritin is an important regulator of oxidative stress, a primary factor in the etiology of aging-related cataract. From gene array analysis two novel observations were made with respect to ferritin gene expression: first, lenses from guinea pigs and humans have disproportionately high levels of L-ferritin mRNA relative to the amounts of ferritin protein present, and second, L-ferritin message increased markedly in lenses from guinea pigs with hereditary nuclear cataract. The human lens L-ferritin sequence was identical to previous data from human liver; the guinea pig sequence was 86% identical to the human sequence at the amino acid level. Despite mRNA levels similar to those of major lens crystallins, lens ferritin was undetectable by Western blot techniques. PMID- 10753630 TI - Molecular cloning and characterization of the mouse peroxiredoxin V gene. AB - We have cloned two cDNA isoforms as well as genomic sequences of the mouse Prx V gene and characterized their molecular genetic features. Two isoforms of the mouse Prx V cDNA were identified from liver and testis. The testis-originated long transcripts had extra 1164-bp 5'-UTR sequences compared to the liver originated short transcripts. Primer extension and sequence analyses revealed that the two isoforms were presumably transcribed at the same gene locus. The gene was composed of six exons spanning 3.2 kb. The short transcript was abundantly expressed in the kidney, liver, and heart of the adult mouse tissues and in the extra-membrane of the 10.5 dpc embryos. The long transcript of 1985 bp was abundantly detected in testis with trace amounts in other tissues. Interestingly, in testis and fetus, only mRNA expression of the long form was identified. However, the protein expression was not found in testis, implying that the long form could not properly direct the protein expression. The long Prx V cDNA has eight uORFs in the extra 5'-UTR, which proceed the major ORF. The inability of protein expression for the long-form cDNA in testis suggests that the uORFs might inhibit translation of the major ORF and thereby confer the tissue-specific regulation of the mouse Prx V gene. PMID- 10753631 TI - Pyrimidine morpholino oligonucleotides form a stable triple helix in the absence of magnesium ions. AB - Oligonucleotides can be used as sequence-specific DNA ligands by forming a local triple helix. In order to form more stable triple-helical structures or prevent their degradation in cells, oligonucleotide analogues that are modified at either the backbone or base level are routinely used. Morpholino oligonucleotides appeared recently as a promising modification for antisense applications. We report here a study that indicates the possibility of a triple helix formation with a morpholino pyrimidine TFO and its comparison with a phosphodiester and a phosphoramidate oligonucleotide. At a neutral pH and in the presence of a high magnesium ion concentration (10 mM), the phosphoramidate oligomer forms the most stable triple helix, whereas in the absence of magnesium ion but at a physiological monovalent cation concentration (0.14 M) only morpholino oligonucleotides form a stable triplex. To our knowledge, this is the first report of a stable triple helix in the pyrimidine motif formed by a noncharged oligonucleotide third strand (the morpholino oligonucleotide) and a DNA duplex. We show here that the structure formed with the morpholino oligomer is a bona fide triple helix and it is destabilized by high concentrations of potassium ions or divalent cations (Mg(2+)). PMID- 10753632 TI - Molecular cloning of a novel form (two-repeat) protein related to voltage-gated sodium and calcium channels. AB - Voltage-gated Ca and Na channels share similar structure: four homologous domains (I-IV), each with six transmembrane segments (S1-S6). They may be formed by two rounds of duplication of a single channel domain similar to voltage-activated potassium channels. However, the channels with the intermediate structure, namely, two-domain channels have not yet been identified. We report here the cloning of a novel protein from rat kidney that contains two domains (I-II), each with S1-S6 segments that are found in voltage-gated Ca and Na channels. Because of unusual structure, the protein was named two-pore channel 1 (TPC1). TPC1 encodes 819 amino acids with two conserved positively charged voltage sensor segments (S4) but the pore segments are not conserved. Northern blot analysis showed that TPC1 mRNA (5 kb) was expressed widely. It was expressed at relatively high level in kidney, liver, and lung. Immunohistochemistry of kidney revealed that TPC1 was expressed at inner medullary collecting ducts. In expression studies, no functional currents could be detected in CHO cells and Xenopus oocytes. Based on its primary structure, we propose that TPC1 might be a predecessor of the conventional four repeat voltage-gated Ca and Na channels and will give insights into the evolution of ion channels. PMID- 10753633 TI - Glycoprotein Ibalpha-bound thrombin functions as a serine protease to produce macromolecular activators of phagocytosis from platelets. AB - Production of macromolecular activators of phagocytosis from platelets (MAPPs) was observed when the lysate of fresh platelets was incubated with MAPP precursors and thrombin. An 800-Da MAPP activator (PMA-II) was obtained by Superdex peptide gel filtration of the lysate after thrombin treatment. The necessity of thrombin in MAPP production in fresh platelets was confirmed by the action of anti-thrombin monoclonal antibody or anti-thrombin III and heparin. To specify the thrombin receptor on which the thrombin forming PMA-II binds, the effects of thrombin-receptor-derived peptides and anti-thrombin receptor antibodies on MAPP production by stored platelets which have lost their thrombin content were investigated. DYYPEEDTEGD involved in glycoprotein Ibalpha and anti glycoprotein Ibalpha antibody prevented stored platelets from producing MAPP. These observations suggest that thrombin bound to glycoprotein Ibalpha functions as a serine protease in MAPP formation. PMID- 10753634 TI - Borato-1,2-diaminocyclohexane platinum (II), a novel anti-tumor drug. AB - Borato-1,2-diaminocyclohexane platinum (II) (BDP) was synthesized and compared to cisplatin (cisPt) as a potential anti-tumor drug. BDP and cisPt (0.2-20 microM) dose-dependently inhibited DNA synthesis in L1210 murine leukemia cells, DU-145 prostate cancer cells, A549 lung carcinoma cells, and MCF-7 breast cancer cells, as judged by measuring [(3)H]-thymidine incorporation. BDP and cisPt induced killing of L1210 murine cells by 97 and 78%, respectively. The LD(50) was 35 and 14 mg/kg for BDP and cisPt, respectively. Interestingly, while cisPt (at optimal dose) induced a 100% increase in life span (ILS) of BDF1 mice bearing L1210 tumor cells, BDP (at optimal dose) induced a 200% increase in ILS in the same tumor model. In conclusion, BDP is a novel platinum derivative compound that appears more effective in increasing the ILS than cisplatin against the leukemia tumor mouse model. PMID- 10753635 TI - A conserved residue at the extreme C-terminus of FtsZ is critical for the FtsA FtsZ interaction in Staphylococcus aureus. AB - FtsZ, a tubulin-like protein with GTPase activity, and FtsA, an actin-like protein with ATPase activity, are two proteins known to play critical roles in the later stages of the bacterial cell cycle. It is well documented that FtsA FtsZ co-localization at the septum of dividing bacteria is essential for successful cell division in E. coli. We have validated and characterized this interaction by co-expressing FtsA and FtsZ, from both E. coli and S. aureus, in the yeast two-hybrid system. We demonstrate for the first time a specific association between S. aureus FtsA and FtsZ proteins and self interaction of S. aureus FtsZ. These observations are consistent with the conserved role of FtsA and FtsZ in bacterial cell division. Using deletion mutagenesis, we have shown that a highly conserved motif as small as 10 residues in the extreme C-terminus of S. aureus FtsZ is critical for the interaction with FtsA. Further dissection of this motif by alanine scanning mutagenesis showed that Phe376 likely plays a major role in the FtsA-FtsZ interaction. This work has important implications for the design of antagonists and agonists of the FtsA-FtsZ interaction as such agents could provide a novel approach for tackling multi-resistant Gram positive pathogens. PMID- 10753636 TI - An ARE-selective DNA minor groove binder from a combinatorial approach. AB - A synthetic combinatorial library of 10,000 components mostly containing aromatic amino acids was screened for inhibition of DNase I cleavage at two ARE sequences. Ten amino acid building blocks were used to generate the library in which the N and C terminal residues were fixed and the four central positions of the peptide ligands were varied. The DNase I footprinting assay led, after deconvolution through sublibrary synthesis, to the identification of CGL-6382 as an ARE selective minor groove binder containing a N-terminal nicotinic acid motif adjacent to a N-methylimidazole unit and three N-methylpyrrole units coupled to a C-terminal argininamide residue. The optimized ligand CGL-6382 was found to recognize a 5'-GC(A/T)(A/T) motif within the two cloned androgen receptors responsive elements. The discovery of CGL-6382 as an ARE-selective ligand augurs well for the use of the DNase I footprinting methodology to identify sequence specific DNA recognition ligands from large mixtures of small molecules. PMID- 10753637 TI - Inhibition of human lung cancer cell growth by the peroxisome proliferator activated receptor-gamma agonists through induction of apoptosis. AB - Peroxisome proliferator-activated receptors (PPARs), members of the nuclear hormone receptors superfamily, have an important regulatory role in adipogenesis and inflammation. PPAR-gamma ligands induce terminal differentiation and growth inhibition of human breast cancer cells and prostatic cancer cells. In this study, we demonstrated that PPAR-gamma, but not PPAR-alpha, was expressed in human lung cancer cell lines by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. We also found that the synthetic PPAR-gamma agonist thiazolidinedione compounds (troglitazone) and the endogenous PPAR-gamma ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), inhibited the growth of human lung cancer cells through the induction of apoptosis. However, PPAR-alpha agonist (bezafibrate) and other prostanoids (PGE(2), PGF(2alpha)) did not induce apoptosis. These findings suggest that PPAR-gamma may play an important role in the pathogenesis of lung cancer and that PPAR-gamma agonist may be useful therapeutic agents in the treatment of human lung cancer. PMID- 10753638 TI - Induction of apoptosis and mitosis inhibition by degraded DNA lipotransfection mimicking genotoxic drug effects. AB - Genotoxic damage induces cell cycle arrest and/or apoptosis by activation of p53 oncosuppressor protein. A number of anticancer drugs are genotoxic and their damaging effect upon cells is mediated by this mechanism. Microinjection of defined DNA species directly into nucleus has been reported previously to activate p53 and inhibit cell cycle. Here, we demonstrate that simple addition of heterogeneous degraded DNA to cultured cells (Rat-1 fibroblasts) in combination with lipotransfecting agent DOTAP leads to apoptosis induction and mitosis inhibition by a molecular mechanism which mimics that of the cellular response to genotoxic anticancer agents. Indeed, both cellular effects induced by lipotransfected degraded DNA (essentially, heterogeneous small DNA fragments) are associated to p53 activation and modulated by two apoptosis-related genes, such as bcl-2 and c-myc, which also modulate the apoptotic threshold to anticancer agents. Here we raise the hypothesis of exogenous DNA segment lipotransfection as possible new tool for anticancer therapy. PMID- 10753639 TI - Resistance to apoptosis is correlated with the reduced caspase-3 activation and enhanced expression of antiapoptotic proteins in human cervical multidrug resistant cells. AB - Recent studies have indicated that induction of apoptosis is the primary cytotoxic mechanism of most cancer chemotherapeutic agents, and abnormalities in the control of apoptosis can affect the sensitivity of malignant cells to multiple drugs. Here, we treated cells with cisplatin and other apoptotic stimuli and found that multidrug-resistant (MDR) endocervical HEN-16-2/CDDP cells, compared with drug-sensitive parental cells, were significantly more resistant to apoptosis and exhibited decreased proteolytic activation of caspase-3. The latter was further demonstrated by decreased cleavage of its substrate poly(ADP-ribose) polymerase (PARP). Further, Western blot analysis showed that MDR HEN-16-2/CDDP cells had significantly higher levels of the apoptosis-inhibiting proteins BAG-1 p50 and p33 isoforms and Bcl-X(L). This study provided the first evidence that overexpression of antiapoptotic BAG-1 p50 and p33 and Bcl-X(L) may cause resistance to apoptosis through reduction of caspase-3 activity in human cervical cells having an MDR phenotype. PMID- 10753640 TI - Plicatamide: A lead to the biosynthetic origins of the tunichromes? AB - Plicatamide is a modified octapeptide from the ascidian Styela plicata having the structure Phe-Phe-His-Leu-His-Phe-His-decarboxyDeltaDOPA (where decarboxyDeltaDOPA = decarboxy-(E)-alpha,beta-dehydro-3, 4 dihydroxyphenylalanine). Edman sequencing, tandem mass spectrometry, and proton NMR have been used to characterize 100 microg of the compound. Plicatamide represents an important link between two classes of biomolecules: the tunichromes which share an oxidatively decarboxylated C-terminus and higher molecular weight DOPA-polypeptides. The 8-residue sequence provides the first opportunity to investigate the biosynthetic origins of the tunichrome family by molecular biological techniques. PMID- 10753641 TI - Estrogen receptor beta mRNA in colon cancer cells: growth effects of estrogen and genistein. AB - Knowledge regarding the expression of the recently cloned estrogen receptor beta (ERbeta) in colonic mucosa is limited. In this study, we demonstrated that five human colon cancer cell lines, HT29, Colo320, Lovo, SW480, and HCT116, expressed ERbeta mRNA, but lacked ERalpha mRNA. Results from a cell growth assay demonstrated that these colon cancer cells were not influenced by estrogen, while genistein possessed slight growth inhibitory effects on HT29, Colo320 and Lovo cells at 10 microM, at which concentration is stimulated the growth of ERalpha positive human breast cancer MCF-7 cells. Tamoxifen inhibited the growth of HT29 and Colo320 cells, dose-dependently, as well as MCF-7 cells. A transfected reporter plasmid containing a vitellogenin estrogen response element could be activated by estradiol in Colo320 cells. Taken together with previous reports, these data suggest that ERalpha and ERbeta may have different biological functions in colon cells. PMID- 10753642 TI - Mapping of the human CP49 gene and identification of an intragenic polymorphic marker to allow genetic linkage analysis in autosomal dominant congenital cataract. AB - The CP49 protein is an intermediate filament protein expressed specifically in the lens fibre cells of the lens, where it is an important cytoplasmic structural component. Dominant-negative mutations in other intermediate filament proteins, such as keratins, cause disorders characterised by dense cytoplasmic aggregates in specific cell types. The CP49 gene is therefore a good candidate for dominantly inherited forms of cataract. To allow genetic linkage analysis of families with autosomal dominant cataract with respect to CP49, a highly polymorphic intragenic microsatellite marker for this gene has been developed. In addition, both low and high resolution radiation hybrid mapping of the CP49 gene has been completed, placing it very close to microsatellite marker D3S1290 on human chromosome 3q. Furthermore, using the intragenic CP49 microsatellite, linkage was excluded in four families with genetically uncharacterized forms of autosomal dominant congenital cataract. PMID- 10753643 TI - Nitric oxide inhibits cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi. AB - Nitric oxide (NO) is a pluripotent regulatory molecule showing, among others, an antiparasitic activity. Moreover, NO inhibits cysteine proteinase action by nitrosylating the Cys catalytic residue. In the present study, the inhibitory effect of the substrate N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7 amino-4-methyl coumarin) and of NO on the catalytic activity of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi (the hemoflagellate protozoan parasite which causes the American trypanosomiasis), is reported. In particular, NO-donors S-nitroso-glutathione (GSNO), (+/-)-(E)-4-ethyl-2-[(E) hydroxyimino]-5-nitro-3-hexenamide (NOR-3), 3-morpholinosydnonimine (SIN-1), S nitroso-acetyl-penicillamine (SNAP), and sodium nitroprusside (SNP) dose dependently inhibited cruzipain, this effect being likely attributable to the S nitrosylation of the Cys25 catalytic residue. These results were analyzed in parallel with those concerning the inhibitory effect of the substrate and of NO on the catalytic activity of falcipain, the cruzipain-homologous cysteine proteinase from Plasmodium falciparum. The modulation of the cruzipain and falcipain activity by NO may be relevant in developing new strategies against T. cruzi and P. falciparum in human host. As a whole, the NO-mediated S nitrosylation of pathogenic viral, bacterial, fungal, and parasitic cysteine proteinases may represent a general mechanism of antimicrobial and antiparasitic host defences. PMID- 10753644 TI - Properties of ryanodine receptor in rat muscles submitted to unloaded conditions. AB - Unloading of skeletal muscles by hindlimb unweighting is known to induce muscle atrophy and a shift toward faster contractile properties associated with an increase in the expression of fast contractile proteins, particularly in slow soleus muscles. Contractile properties suggest that slow soleus muscles acquire SR properties close to those of a faster one. We studied the expression and properties of the sarcoplasmic reticulum calcium release (RyR) channels in soleus and gastrocnemius muscles of rats submitted to hindlimb unloading (HU). An increase in RyR1 and a slight decrease in RyR3 expression was detected in atrophied soleus muscles only after 4 weeks of HU. No variation appeared in fast muscles. [(3)H]Ryanodine binding experiments showed that HU neither increased the affinity of the receptors for [(3)H]ryanodine nor changed the caffeine sensitivity of [(3)H]ryanodine binding. Our results suggested that not only RyR1 but also RyR3 expression can be regulated by muscle activity and innervation in soleus muscle. The changes in the RyR expression in slow fibers suggested a transformation of the SR from a slow to a fast phenotype. PMID- 10753645 TI - Lipid oxidation deletes the nanodomain organization of artificial membranes. AB - Nanoscopic domains with different crystal structures have been induced in closed artificial membranes and have been directly imaged by atomic force microscopy at a spatial resolution better than 0.3 nm. These observations provide experimental evidence to the hydrophobic mismatching theory of lateral phase separation phenomena. Under oxidant conditions, the lipid-lipid assembly reorganizes into a new steady-state structure with disappearance of specific nanodomains. This finding may contribute to understanding the mechanism of peroxidative damage to membrane properties. In fact, alterations of specific modes of molecular conformation and packing may lead to perturbation of specific properties. PMID- 10753646 TI - Trichosanthin interacts with and enters cells via LDL receptor family members. AB - The type-I ribosome-inactivating protein trichosanthin displays selective cytotoxicity, suggesting specific mechanisms for entry into cells. Here we show that trichosanthin binds specifically to the endocytic receptors LRP and megalin, and that binding as well as uptake into cells is inhibited by the receptor associated protein (RAP). The results suggest that the known abortifacient and renotoxic actions of trichosanthin are caused by LRP-mediated uptake in trophoblasts and megalin-mediated uptake in proximal tubule epithelial cells, respectively. PMID- 10753647 TI - Metallothionein-null mice express altered genes during development. AB - Metallothionein (MT) can modulate transcriptional activity in vitro. We examined whether the absence of MT affects gene expression in vivo. We compared the hepatic RNA profiles of wild-type and MT-null neonatal mice using improved differential display. The hepatic MT level was maximal during neonatal development. We identified five cDNA fragments that were expressed in MT-null mice at different levels from those in wild-type mice. Two were fragments of MT-I and mutant MT-I cDNA. The sequences of the other cDNA fragments were identical to those of contrapsin, transketolase, and vanin-3. The latter two were up regulated, whereas contrapsin was down-regulated in neonatal MT-null mice. These mRNA levels were remarkably different between the two strains of neonatal mice. Further characterization of the regulated mRNA identified here will determine whether or not they are primary or secondary effects of an MT deficiency. PMID- 10753648 TI - Two functionally distinct forms of NKX2.1 protein are expressed in the pulmonary epithelium. AB - The homeodomain transcriptional factor NKX2.1 is critical for normal morphogenesis of the lung, thyroid, and the brain. In the lung, NKX2. 1 binds to and activates the expression of pulmonary differentiation-specific genes SP-A, SP B, and SP-C. The Nkx2.1 gene is comprised of three exons separated by two introns. In both thyroid and lung, the predominant Nkx2.1 transcript includes exons II and III and is translated into a 371 amino acid protein. A minor transcript also exists which includes all three exons. This transcript encodes a 401 amino acid isoform of NKX2.1. The 30 amino acid extension is highly conserved amongst various mammalian species. In the current study, we demonstrate that the two NKX2.1 isoforms are functionally distinct and their corresponding transcripts are expressed differentially during mouse embryonic lung development. The results demonstrate that the longer isoform of NKX2.1 exhibits reduced activity in transactivating an SP-C target promoter when compared to the truncated major NKX2.1 protein. Site directed mutagenesis of the 30 amino acid peptide extension suggests that this fragment alters the activity of 5E likely by steric interference. PMID- 10753649 TI - ADP-Ribosyl cyclase in rat salivary glands. AB - Both the Ca(2+)-releasing mechanism induced by cyclic ADP-ribose (cADPR) and the ADP-ribosyl cyclase (ADPRC) activity that converts NAD(+) to cADPR were observed in a variety of cell types. We studied the ADPRC activity in rat major salivary glands that include parotid gland (PG), submandiblar gland (SMG), and sublingual gland (SLG). The enzyme activity responsible for cADPR synthesis was detected by spectrofluorometric assay using NGD(+) as a substrate. The enzyme activities in SLG, SMG, and PG were about 400, 30, and 40 nmol/min/g tissue, respectively, in 5 week-old rats. The highest value was observed in SLG and this value was higher than those in other tissues; e.g., spleen (200 nmol/min/g tissue). The enzyme activity in SLG increased gradually after birth and showed a maximum value at 3 weeks. On the other hand, the enzyme activities almost did not change in both PG and SMG between 0 and 9 weeks. In spite of the high ADPRC activity in SLG, we could not detect the cADPR-induced Ca(2+)-release from SLG microsomes. These results suggest that the ADPRC in SLG does not function through Ca(2+)-release observed in various tissues. PMID- 10753650 TI - In vitro translation extracts prepared from Drosophila ovaries and embryos. AB - Translational regulation has emerged as an important feature of animal development, especially in the embryo prior to the onset of zygotic transcription. Specialized forms of control regulate the translation of individual mRNAs, and the factors involved in these mRNA-specific events are expected to be found in only a subset of all tissues. Consequently, homologous in vitro translation systems, prepared from tissues in which important regulatory events occur, are likely to be required to pursue biochemical studies of the underlying mechanisms. Here we describe the characterization of extracts prepared from Drosophila ovaries and embryos that support translation of exogenous reporter mRNAs in vitro. These in vitro systems should prove to be useful in dissecting mechanisms of the numerous translational control events shown to occur during the early stages of Drosophila development. PMID- 10753651 TI - Adhesion-dependent tyrosine phosphorylation of enabled in Drosophila neuronal cell line. AB - Cell culture consisting of Drosophila BG2-c6 cells and laminin revealed its value for the analysis of the integrin-mediated activity of extracellular matrix (Takagi, Y., et al. (1998) Neurosci. Lett. 244, 149-152). To elucidate Drosophila integrin cascade further, we report here our characterization on the tyrosine phosphorylation in BG2-c6 cells, coupling with their spreading on extracellular matrix. Large-scale culture of Drosophila Kc167 cells provided a sufficient amount of extracellular matrix (including laminin) for performing biochemical analysis on the signal transduction in BG2-c6 cells. Several proteins underwent significant tyrosine phosphorylation in an adhesion-dependent manner. Among them, the heavy phosphorylation of Enabled (a substrate for Abelson tyrosine kinase) was noteworthy because of the proposed function of Enabled in cell adhesion. Together with our previous results, we propose a model for signal transduction activated by cell adhesion for the first time in Drosophila. PMID- 10753652 TI - Skeletal muscle CaMKII enriches in nuclei and phosphorylates myogenic factor SRF at multiple sites. AB - We characterized the activity of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in homogenates and nuclear extracts of skeletal muscle and analyzed their capacity to phosphorylate the myogenic factor SRF. Isoforms of CaMKII enriched from skeletal muscle phosphorylated SRF in vitro to high stoichiometries and produced multiple forms on SDS-PAGE, suggesting that SRF was phosphorylated at multiple sites. Phosphopeptide-mapping experiments using truncated SRF proteins located the residues of SRF phosphorylated by recombinant CaMKII within amino acids 1-171, with at least one site residing in amino acids 142-171. Microsequencing of these phosphorylated peptides identified that both Ser-103 and a novel residue, Thr-160 in the MADS box of SRF, were sites of phosphorylation. CaMKII activity was enriched in nuclear extracts relative to crude homogenates from skeletal muscle and similarly phosphorylated the nuclear transcription factor SRF in vitro. The location of Thr-160 in the 3-D structure of SRF suggests that its phosphorylation by nuclear CaMKII may directly influence DNA binding of SRF and other MADS box factors. PMID- 10753653 TI - Increased aquaporin-4 immunoreactivity in rat brain in response to systemic hyponatremia. AB - The present study was undertaken to assess whether the protein and mRNA expression levels of the glial water channel aquaporin-4 (AQP4) undergo downregulation and whether there is a subcellular redistribution of AQP4 protein in rat brain in response to systemic hyponatremia and brain edema. Systemic hyponatremia was induced for 4 or 48 h by combined administration of hypotonic dextrose i.p. and 8-deamino-arginine vasopressin (dDAVP) s.c. Semiquantitative immunoblotting of membrane enriched fractions showed significantly increased immunoreactivity to 164 +/- 12% (n = 6) and 153 +/- 12% (n = 6) of control levels in brain after 4 or 48 h of systemic hyponatremia, respectively. Similarly, immunoblots of cerebellar samples revealed an increase in AQP4 immunoreactivity to 136 +/- 6% (n = 6) and 218 +/- 44% (n = 6) of control levels, after 4 or 48 h of hyponatremia. In contrast, AQP4 mRNA levels were unchanged after 4 h of severe hyponatremia (104 +/- 14% of control levels; n = 17), indicating that there are no changes in AQP4 expression in response to systemic hypoosmolarity. Immunocytochemistry and high-resolution immunogold electron microscopy revealed highly polarized labeling of AQP4 in astrocyte end-feet surrounding capillaries and forming the glia limitans. This pattern of labeling was not changed whereas an increased labeling intensity of AQP4 could be observed in response to hyponatremia. In conclusion, hyponatremia causes a pronounced and rapid increase in AQP4 immunoreactivity that is not accompanied by any increase in AQP4 mRNA expression. The increased AQP4 immunosignal may reflect secondary conformational modifications of AQP4 protein, leading to enhanced antibody binding. This post translational modification of AQP4 may participate in the adaptation of cerebral tissue to systemic hyponatremia. PMID- 10753654 TI - The carboxyl terminal domain of phosducin functions as a transcriptional activator. AB - In previous work, we identified a set of phosducin (Phd) isoforms with unknown function including the phosducin (Phd)-like orphan protein 1 (PhLOP1), an amino terminal truncated isoform of the retinal Phd lacking the Gbetagamma binding domain. To investigate the potential biological function of PhLOP1, PhLOP1 was fused at its amino terminus with the DNA binding domain (BD) of the yeast transcriptional factor, GAL4, and used as bait in a yeast two-hybrid screen. Two potential functional protein partners were identified during the screen: SUG1, a subunit of the 26S proteasome and a putative transcriptional mediator, and CRX, a retina- and pineal-specific transcription factor. Upon localizing the interacting domain of PhLOP1 with one of the new partners, SUG1, we found that a domain of 40 amino acids at the carboxyl terminus of Phd and PhLOP1 had intrinsic transcriptional activation activity in yeast. The transactivation activity was further confirmed in mammalian cells. This region contains an acidic domain that has been shown to be involved in the function of several transcriptional activators. In addition, we showed that Phd is cytoplasmic while PhLOP1 is localized predominantly to the nucleus when fused to an enhanced green fluorescent protein (EGFP) and transiently expressed in transfected cells, suggesting that PhLOP1 may play a distinct functional role in transcriptional regulation independent of the known Phd interaction/regulation of Gbetagamma transduction. PMID- 10753655 TI - Expression of myostatin gene in regenerating skeletal muscle of the rat and its localization. AB - The expression of myostatin mRNA was examined in regenerating skeletal muscle of the rat. Skeletal muscle regeneration was induced by injecting bupivacaine or hypertonic saline solution into the femoral muscle, and the tissues were collected 48 h after the treatment. In situ hybridization analysis revealed that the cells positive for myostatin message were localized in the regenerating area of the bupivacaine-treated tissues, where a numerous number of mononucleated cells were present. The myostatin-positive mononucleated cells contained both myogenic and nonmyogenic cells, as revealed by immunohistochemical staining for desmin and vimentin. Bupivacaine treatment to the testes resulted in no myostatin message expression in the testicular vimentin-positive cells, suggesting that the expression of myostatin message in vimentin-positive cells is a skeletal muscle specific phenomenon. Furthermore, crushed muscle extract prepared from regenerating skeletal muscle had induced myostatin mRNA expression in skeletal muscle-derived fibroblasts in a dose-dependent manner. These results indicated that myostatin is expressed during skeletal muscle regeneration both in myogenic and nonmyogenic cells, and suggested that some factor(s) capable of inducing myostatin expression in fibroblasts are present in regenerating skeletal muscle. PMID- 10753656 TI - Identification of neural progenitors in the adult mammalian eye. AB - We have shown that the embryonic mammalian retina contains neural progenitors which display stem cell properties in vitro. Here we report the characterization of neural progenitors isolated from the adult mammalian eye. These quiescent cells, located in the pigmented ciliary bodies, proliferate in the presence of FGF2 and express the neuroectodermal marker nestin. The proliferating cells give rise to neural spheres and are multipotential; they express cell type-specific markers corresponding to neurons and glia. In addition, neural progenitors can generate secondary neural spheres, thus displaying potential to self-renew. The ciliary body-derived neural progenitors display retina-specific properties; the undifferentiated cells express Chx10, a retinal progenitor marker, and upon differentiation express markers corresponding to specific retinal cell types. Therefore, the pigmented ciliary body in the adult mammalian eye harbors neural progenitors that display stem cell properties and have the capacity to give rise to retinal neurons in vitro. PMID- 10753658 TI - Genomic structures of synaptotagmin II protein: comparison of exon-intron organization of the synaptotagmin gene family. AB - Synaptotagmin (Syt) I (or II) is thought to be a Ca(2+) sensor for neurotransmitter release in the rostral (or caudal) region of the mammalian brain. In this study, we first report the genomic structures and the transcription initiation site of the mouse syt II gene. Syt II protein coding regions consist of 8 exons with a total size of about 7 kbp. Exon 2 encodes the transmembrane domain, and exons 3-5 and 6-8 encode the C2A and C2B domains, respectively, although the precise intron positions within the two C2 domains are different. The syt II gene structures are quite similar to the mouse syt VIII and human syt IX genes, but not Caenorhabditis elegans syt I, human syt VII, or the mouse syt III genes, indicating that the exon-intron patterns of the functional domain of synaptotagmins (especially the C2A domain) have not been as well conserved during evolution as among isoforms. The only conserved feature among the synaptotagmin gene family seems to have a single exon that encodes the whole transmembrane domain. PMID- 10753657 TI - Meltrin beta (ADAM19) gene: cloning, mapping, and analysis of the regulatory region. AB - Meltrin beta (ADAM19) is a member of the metalloprotease-disintegrin family. We report here chromosomal mapping of the mouse and rat meltrin beta genes and cloning and analysis of the mouse upstream regulatory regions. The meltrin beta transcript shows a spatially and temporally restricted expression pattern during morphogenesis, indicating that the actions of this membrane-bound protease are regulated, at least in part, at the transcriptional level. Analysis of the promoter revealed positive and negative regulatory regions upstream of the gene. The former includes a GC-box that appears to be a critical cis-element for activation of the promoter in muscle cells. PMID- 10753659 TI - DNA fragmentation occurs in skeletal muscle during tumor growth: A link with cancer cachexia? AB - In two different experimental models of cancer cachexia, the rat Yoshida AH-130 ascites hepatoma and the mouse Lewis lung carcinoma, the implantation of the tumor caused a loss of body weight which was associated with a reduction in the weight of different skeletal muscles, as well as with their protein content. The decrease in protein content was accompanied by a reduction in DNA content. Interestingly, the protein/DNA ratio was unchanged in the skeletal muscle of the tumor-bearing animals as compared with the non-tumor-bearing controls. Analysis of DNA fragmentation in skeletal muscle clearly showed enhanced laddering in the skeletal muscle of tumor-bearing animals, suggesting an apoptotic phenomenon. Interestingly, the degree of laddering (total DNA fragmented) increased with tumor burden. These results suggest that DNA fragmentation may be a primary event in cancer-associated cachexia. PMID- 10753660 TI - Association of membrane-associated guanylate kinase-interacting protein-1 with Raf-1. AB - Membrane-associated guanylate kinase-interacting protein (MAGUIN)-1 was identified as a protein interacting with synaptic scaffolding molecule (S-SCAM) and postsynaptic density (PSD)-95/synapse-associated protein (SAP)90. MAGUIN-1 has a chimerical molecular structure composed of one sterile alpha motif, one PSD 95/Dlg-A/ZO-1 (PDZ), and one pleckstrin homology (PH) domain, and interacts with the PDZ domains of S-SCAM and PSD-95/SAP90 via its carboxyl-terminal PDZ-binding motif. MAGUIN-1 is considered as a mammalian homologue of Drosophila CNK, which is a Raf-interacting protein implicated in the regulation of eye development. Here we have tested whether MAGUIN-1 interacts directly with Raf-1. MAGUIN-1 and Raf-1 were coimmunoprecipitated from rat brain. MAGUIN-1 binds to the kinase domain of Raf-1, and Raf-1 binds to the middle region of MAGUIN-1 containing the PH domain. However, in contrast to the dominant active mutant of Ki-Ras, which interacts with Raf-1, recruits it to the plasma membrane from the cytosol, and activates it, MAGUIN-1 neither activates Raf-1 nor recruits it to the plasma membrane. MAGUIN-1 may link Raf-1 to components of synapses assembled by PSD 95/SAP90 and S-SCAM. PMID- 10753661 TI - Mutation of the toxin binding site of PP-1c: comparison with PP-2B. AB - The catalytic cores of PP-1c and PP-2B (calcineurin) are structurally conserved. However, PP-2B is resistant to inhibition by toxins of the okadaic acid and cyclic peptide classes, while PP-1c is potently inhibited. Molecular docking of the structure of microcystin-LR onto the catalytic core of PP-2B identified residues that may be responsible for blocking access of toxins to the catalytic site. Amino acids in PP-1c were substituted with these PP-2B residues to investigate their contribution to PP-2B toxin resistance. Mutants of PP-1c were also produced to test the importance of hydrophobic interactions to toxin binding. Our results suggest that different classes of toxin inhibitors interact with the same hydrophobic side chains of PP-1c through different mechanisms. Substitution of amino acids in PP-1c with PP-2B residues demonstrated no highly significant changes in toxin inhibition. We hypothesize that an interaction outside the catalytic core causing the L7 loop of PP-2B to block the catalytic site may be responsible for PP-2B resistance to toxins. PMID- 10753662 TI - Cell adhesion is a prerequisite for osteoclast survival. AB - The present study demonstrates that loss of cell adhesion potently promotes apoptosis in osteoclasts, a process termed "anoikis." When purified mature rabbit osteoclasts were cultured on plastic for 18 h, about 25% of them were spontaneously committed to apoptosis. The death rate increased more than twofold, after osteoclasts were subjected to suspension culture in inverted Terasaki plates. The osteoclast anoikis was significantly prevented by bongkrekic acid, an inhibitor of mitochondrial permeability transition (PT), and z-VAD-FMK, a caspase inhibitor, suggesting involvement of mitochondrial PT and caspase activation in the death process. Colony-stimulating factor-1 (CSF-1), receptor activator of NF kappaB ligand (RANKL), and calcitonin protected adherent osteoclasts, but not floating osteoclasts from anoikis. These data show that adhesion is a primary requirement for osteoclast survival. PMID- 10753663 TI - Two acidic amino acid residues, Asp(470) and Glu(471), contained in the carboxyl cytoplasmic tail of a major lysosomal membrane protein, LGP85/LIMP II, are important for its accumulation in secondary lysosomes. AB - Lysosomal membrane glycoprotein termed LGP85 or LIMP II has a COOH-terminal cytoplasmic tail whose amino acid sequence is R(459)GQGSMDEGTADERAPLIRT(478). Two acidic amino acid residues, D(470) and E(471), in the cytoplasmic tail of LGP85 are crucial for its binding to adaptor-like complex AP-3. In the present study we investigated their role(s) in intracellular distributions of LGP85 using two alanine substitution mutants at D(470) and E(471) (defined as D470A and E471A, respectively). Immunofluorescence analysis showed that D470A and E471A are localized to endocytic organelles as well as wild-type LGP85. However, the subcellular fractionation study revealed that D470A and E471A are different from wild-type LGP85 in the distribution among early endosomes, late endosomes, and lysosomes. A major portion of wild-type LGP85 existed in the densest lysosomal fraction. In contrast, a significant amount of D470A existed in the early endosomal fraction with a light buoyant density, while less D470A resided in the lysosomal fraction. E471A broadened from the early endosomal fraction to the lysosomal fraction without the high lysosomal peak. These findings indicate that the two acidic residues, D(470) and E(471), play an important role in regulation of LGP85 movement within the endocytic pathway, which finally makes the highest concentration of LGP85 in the dense secondary lysosomes. PMID- 10753664 TI - LMW-PTP exerts a differential regulation on PDGF- and insulin-mediated signaling. AB - Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) is able to specifically bind and dephosphorylate activated PDGF and insulin receptors, modulating the onset of mitogenic process. LMW-PTP is present in two distinct intracellular locations. While the cytosolic LMW-PTP pool interacts directly with activated insulin or PDGF receptors, the cytoskeleton-associated LMW-PTP is tyrosine phosphorylated upon PDGF stimulation and is involved in cytoskeleton rearrangement acting on p190Rho-GAP. We investigated the differential role of LMW PTP in PDGF- or insulin-induced mitogenesis and cytoskeleton rearrangement. Dominant negative LMW-PTP influences both PDGF- and insulin-induced mitogenesis with a different extent and it induces a decrease in cellular adhesion and chemotaxis after PDGF but not insulin treatment. PDGF but not insulin stimulation leads to tyrosine phosphorylation of LMW-PTP. We propose that the differential effect of LMW-PTP on PDGF and insulin signaling is mainly due to the fact that during insulin signaling LMW-PTP does not become phosphorylated and thus does not act on its cytoskeleton-associated substrate/s. PMID- 10753665 TI - Interaction between HIV-1 NEF and G(o) proteins in transfected COS-7 cells. AB - Nef protein of HIV/SIV lentiviruses affects G-protein-mediated signaling, and physically associates to Lck, a myristoylated and palmitoylated Src-like tyrosine kinase. To assess whether Nef interacts with alpha-subunits of heterotrimeric G proteins (Galpha), carrying the same lipidation motif as Lck, we transiently expressed Nef and G(o)alpha (wild-type or nonpalmitoylated C3S mutant), individually or in combination, in transfected COS-7 cells. Recombinant Nef was mostly recovered in particulate fractions, and a Nef-Green Fluorescent Protein chimera was localized at the plasmalemma by in vivo fluorescence imaging. Moreover, Nef and C3S were entirely solubilized by cold Triton X-100, and excluded from low buoyant density sucrose gradient fractions, containing caveolin 1, whereas wild-type G(o)alpha was partially resistant to Triton extraction, and colocalized with caveolin-1. After coexpression, Nef recruited soluble C3S to membranes, and the two proteins were coimmunoprecipitated by G(o)alpha and Nef antisera. We conclude that Nef interacts with nonpalmitoylated G(o)alpha, presumably outside caveolin-rich microdomains of the plasma membrane. PMID- 10753666 TI - Rates of evolution of pyridoxal-5'-phosphate-dependent enzymes. AB - The pyridoxal-5'-phosphate-dependent enzymes (B(6) enzymes), that operate in the metabolism of amino acids, are of multiple evolutionary origin. To estimate their rates of evolution, a total of 180 sequences of 21 B(6) enzymes from distantly related eukaryotic species were compared. The enzymes belong to all four evolutionarily independent families of B(6) enzymes with different folds, i.e., the large alpha family, the beta family, the d-alanine aminotransferase family, and the alanine racemase family. Their unit evolutionary periods, i.e., the time for a 1% sequence difference to accumulate between branches, ranged from 4.6 to 45.1 million years. Both, fastest changing serine pyruvate aminotransferase and most slowly changing glutamate decarboxylase are members of the alpha family. The evolutionary rates of the few enzymes belonging to the other three families were interspersed among those of the alpha family members. Enzymes that catalyze the same reaction, e.g., transamination or amino acid decarboxylation, with different substrates show widely varying rates. The absence of correlations of the rate of evolution with either protein fold or type of catalyzed reaction suggests that individual functional constraints have determined the differential rates of evolution of B(6) enzymes. PMID- 10753668 TI - Enzymatic reconstruction of dermatan sulfate. AB - We investigated the enzymatic reconstruction of dermatan sulfate (DS) using the transglycosylation reaction of testicular hyaluronidase. First, in order to insert the IdoA-GalNAc disaccharide unit into chondroitin sulfate chains consisting of GlcA-GalNAc disaccharide units, desulfated DS as a donor and pyridylaminated (PA) chondroitin 6-sulfate (Ch6S) hexasaccharide as an acceptor were subjected to a transglycosylation reaction using testicular hyaluronidase. The products were analyzed by HPLC, mass spectrometry, and enzymatic digestions, and the results indicated that one of the products was IdoA-GalNAc-(GlcA GalNAc6S)(3)-PA. Next, when the resulting PA-Ch6S (hexa-)desulfated DS (di )octasaccharide was used as an acceptor and chondroitin as a new donor, a decasaccharide having a GlcA-GalNAc-IdoA-GalNAc-(GlcA-GalNAc6S)(3) sequence was reconstructed. Using suitable combinations of donors and acceptors, it was possible to custom synthesize DS having any IdoA sequence as its uronic acid component. It is likely that application of this system would facilitate artificial reconstruction of variant DS having different specific functions. PMID- 10753667 TI - Modulation of the Ca(2+)-activated Cl(-) channel by 14-3-3epsilon. AB - We have previously reported an association of 14-3-3epsilon isoform with calmodulin. Using the voltage-clamp technique, the present study investigated the potential role of 14-3-3 in modulating the Ca(2+)-activated Cl(-) channel (CaCC) endogenously expressed in Xenopus oocytes. Injection of 14-3-3epsilon antisense oligodeoxynucleotides resulted in potentiation of the ionomycin-induced Cl(-) current, while 14-3-3 peptide and calmodulin inhibitor, W13, suppressed the antisense-potentiated current. The data suggest that 14-3-3epsilon plays an inhibitory role in modulating the CaCC by interacting with the calmodulin dependent pathway. The potential role of 14-3-3epsilon in other tissues and its therapeutic potential for cystic fibrosis are discussed. PMID- 10753669 TI - Growth hormones reverse desensitization of P2Y(2) receptors in rat mesangial cells. AB - Rat glomerular mesangial cells (GMC) express P2Y(2) purinoceptors and respond to nucleotide stimuli with a transient increase in the cytosolic Ca(2+) concentration and the receptors desensitize upon repeated stimulation with nucleotide. We demonstrate that there is a cross-talk from the signaling of tyrosine kinase to P2Y(2) receptors. For most cells repeated applications of ATP completely abolished the response, as did activation of PKC with 500 nM PMA. In contrast, preincubation with the PKC inhibitor chelerythrine (100 nM) prevented desensitization. Desensitization after application of ATP was reversed by subsequent incubation with PDGF-BB (50 ng/ml) or insulin (660 mU/ml). We conclude that the desensitization is caused by phosphorylation due to PKC and is under the control of growth factors. The findings support the hypothesis that growth hormones potentiate nucleotides as proinflammatory mediators and we hypothesize that they have bearing on the hyperfiltration seen in diabetes. PMID- 10753670 TI - The kinetics and magnesium requirements for the folding of antigenomic delta ribozymes. AB - Using an oligonucleotide hybridization assay to gain insight into the folding of delta ribozymes, we demonstrate a correlation between their folding and catalytic behavior. Together with recent structural information on the crystal structure of self-cleaved genomic delta ribozyme, in which the L3 loop interacts with J1/4 to form the newly proposed stem P1.1, we conclude that it is likely that the P1.1 stem forms only in the presence of Mg(2+). This stem can be detected in both the self-cleaved and trans-acting delta ribozymes. When the trans-acting version of antigenomic delta ribozyme was studied, it is demonstrated that its L3 loop requires magnesium and, apparently, formation of the P1 stem for the subsequently formation of the P1.1 stem. Most importantly, the kinetics were monitored, and provide a significant addition to our understanding of ribozyme tertiary structure formation prior to the chemical cleavage step. Using previous kinetic data and our new findings, we discuss the rate-limiting characteristics of delta ribozyme folding. PMID- 10753671 TI - Functional comparison between YCF1 and MRP1 expressed in Sf21 insect cells. AB - YCF1 is a yeast vacuole membrane transporter involved in resistance to Cd(2+) and to exogenous glutathione S-conjugate precursors. MRP1 contributes to multidrug resistance (MDR) in tumor cells. MRP1 and YCF1 have extensive amino acid sequence homology (63% amino acid similarity). We expressed MRP1 or YCF1 in insect cell membranes and compared their functions to know more about their structure function relationships. YCF1 and MRP1 with His epitopes were expressed in Sf21 insect cells; both of them in the plasma membrane. The ATP-dependent transport of [(3)H]LTC(4) in Sf/YCF1-His vesicles was osmotically sensitive and showed saturable kinetics with an apparent K(m) of 758 nM for LTC(4) and 94 microM for ATP which were similar to those in yeast cells. The K(m) of YCF1 for LTC(4) (758 nM) was sevenfold higher than that of MRP1 (108 nM). MK-571 and ONO-1078, reversing agents for MRP1-mediated MDR, considerably inhibited the transport of LTC(4) by both YCF1 and MRP1. However, PAK-104P, a pyridine analog that reverses MDR associated with P-gp and MRP1, inhibited the transporting activity of MRP1 stronger than that of YCF1. KE1, another MDR reversing agent, moderately inhibited the transport of LTC(4) by MRP1 but not that of YCF1. In conclusion, we successfully expressed yeast YCF1 in Sf21 insect cells and found that the localization of the protein was different from that in yeast. The function of YCF1 in Sf21 insect cells was similar but not identical to that of MRP1. PMID- 10753672 TI - Shear-stress effect on mitochondrial membrane potential and albumin uptake in cultured endothelial cells. AB - Endothelial cells (ECs) that line the inner surface of blood vessels are continuously exposed to shear stress induced by blood flow in vivo, and shear stress affects ATP-dependent macromolecular transport in ECs. However, the relationship between the ATP production and shear stress is still unclear. We, therefore, evaluated mitochondrial ATP synthesis activity in cultured endothelial cells exposed to shear stress, using a confocal laser scanning microscope (CLSM) and a mitochondrial membrane potential probe (5,5',6,6'-tetrachloro-1,1',3, 3' tetraethyl-benzimidazolycarbocyanine iodide, JC-1). Low shear stress (10 dyn/cm(2)) increased mitochondrial membrane potential by 30%. On the contrary, high shear stress (60 dyn/cm(2)) decreased it by 20%. This observation was consistent with the ATP-dependent albumin uptake into endothelial cells. Our results indicate that ATP synthetic activity is related to the albumin uptake into endothelial cells. PMID- 10753673 TI - Effect of a novel vasoconstrictor endothelin-1 (1-31) on human umbilical artery. AB - To clarify the action of a novel endothelin-1 with 31 amino acids, ET-1 (1-31), on fetal circulation, its vasoconstrictive activity on human umbilical and uterine arteries was investigated in comparison with that of a conventional ET-1 (1-21). UFER micro-easy magnus was used for determination of vasoconstriction. The contraction of umbilical artery by KCl was significantly weaker than that of the uterine artery. In ETs, constriction by KCl was set as control, and the rate of constriction of uterine and umbilical arteries was used for comparison. The constriction of human uterine artery induced by ET-1 (1-31) was also significantly weaker than that by ET-1 (1-21). On the contrary, ET-1 (1-31) was a potent constrictor on the umbilical artery equally to ET-1 (1-21). The present study is the first to demonstrate that ET-1 (1-31) has a contractile activity on human vessels. Furthermore, the regulatory mechanism on constriction of umbilical artery is different from that observed in a systemic vessel, indicating a particularly important role of ET-1 (1-31) in fetal circulation. PMID- 10753674 TI - Heterogeneity of catecholamine-containing vesicles in PC12 cells. AB - Vesicular catecholamine release has been measured amperometrically from undifferentiated rat PC12 cells using carbon fiber microelectrodes. During superfusion with high K(+) saline, vesicular release was detected from approximately 50% of 200 cells investigated. On repeated stimulation the releasable pool of vesicles is rapidly depleted, while vesicle contents remains constant. Vesicular catecholamine release is not restored within 1 h after depletion of the releasable pool. Although the distribution of the cube root of vesicle contents of many cells is apparently Gaussian, maximum likelihood analysis of single cell data demonstrates double Gaussian distributions with median vesicle contents of 141 and 293 zeptomole. It is concluded that the releasable pool of vesicles in PC12 cells is heterogeneous. In the presence of l DOPA mean vesicle contents increases, but cessation of release cannot be prevented, indicating that the number of releasable vesicles in PC12 cells is limited by a slow rate of vesicle cycling. PMID- 10753675 TI - Polarized secretion of the regulated secretory protein chromogranin A. AB - Bovine chromogranin A (CgA), together with secreted alkaline phosphatase (SEAP) as an external control for apical secretion were expressed in MDCK cells to test if CgA contains sorting signals for polarized secretion. CgA, SEAP, and the endogenous apical marker GP80 were secreted 75-80% apically. Basolateral secretion of SEAP was inhibited 40% by ammonium chloride. Sulfate labeling and digestion with chondroitinase ABC revealed a 120 kDa proteoglycan-CgA and 75 kDa CgA. Inhibition of proteoglycan synthesis did not affect apical secretion of CgA. As CgA is not N-glycosylated, we used tunicamycin to test if cellular N glycosylation is required for apical sorting. Tunicamycin reversed the polarity of secretion of CgA to the basolateral side. These results suggest that CgA contains dominant apical and recessive basolateral sorting information. PMID- 10753677 TI - Fluid shear stress stimulates prostaglandin and nitric oxide release in bone marrow-derived preosteoclast-like cells. AB - Bone is a porous tissue that is continuously perfused by interstitial fluid. Fluid flow, driven by both vascular pressure and mechanical loading, may generate significant shear stresses through the canaliculi as well as along the bone lining at the endosteal surface. Both osteoblasts and osteocytes produce signaling factors such as prostaglandins and nitric in response to fluid shear stress (FSS); however, these humoral agents appear to have more profound affects on osteoclast activity at the endosteal surface. We hypothesized that osteoclasts and preosteoclasts may also be mechanosensitive and that osteoclast-mediated autocrine signaling may be important in bone remodeling. In this study, we investigated the effect of FSS on nitric oxide (NO), prostaglandin E(2) (PGE(2)), and prostacyclin (PGI(2)) release by neonatal rat bone marrow-derived preosteoclast-like cells. These cells were tartrate-resistant acid phosphatase (TRAP) positive, weakly nonspecific esterase (NSE) positive, and capable of fusing into calcitonin-responsive, bone-resorbing, multinucleated cells. Bone marrow-derived preosteoclast-like cells exposed for 6 h to a well-defined FSS of 16 dynes/cm(2) produced NO at a rate of 7.5 nmol/mg protein/h, which was 10-fold that of static controls. This response was completely abolished by 100 microM N(G)-amino-L-arginine (L-NAA). Flow also stimulated PGE(2) production (3.9 microg/mg protein/h) and PGI(2) production (220 pg/mg protein/h). L-NAA attenuated flow-induced PGE(2) production by 30%, suggesting that NO may partially modulate PGE(2) production. This is the first report demonstrating that marrow derived cells are sensitive to FSS and that autocrine signaling in these cells may play an important role in load-induced remodeling and signal transduction in bone. PMID- 10753676 TI - Ectopic expression of active processed form of atrial natriuretic peptide in skeletal myoblasts. AB - Atrial natriuretic peptide (ANP) is a cardiac hormone that elicits a profound diuresis, natriuresis, and hypotension. As a preliminary study toward ANP gene therapy of cardiovascular disorders, we have cloned a cDNA for mouse preproANP and carried out expression studies in muscle cells. The expression cassette, which was flanked by ITRs from AAV-2, consisted of HCMV IE enhancer/promoter, preproANP gene, and polyadenylation signal from bovine growth hormone. We transfected this expression vector into primary skeletal myoblasts and examined the following points: (1) secretion of immunoreactive ANP, (2) biological activity, and (3) nature of secreted ANP(s). The conditioned media from cells transfected with ANP vector had significantly higher levels of irANP in comparison to mock control. The secreted irANP had biological activity as confirmed by the elevated level of intracellular cGMP in human umbilical vein endothelial cells. Reverse-phase HPLC analysis showed that the processed form of ANP was the predominant form. These results demonstrate that preproANP gene could be ectopically expressed and correctly processed in skeletal myoblasts, which has implications for development of muscle-based ANP gene therapy. PMID- 10753678 TI - Do conjugated eicosapentaenoic acid and conjugated docosahexaenoic acid induce apoptosis via lipid peroxidation in cultured human tumor cells? AB - Conjugated eicosapentaenoic acid (CEPA) and conjugated docosahexaenoic acid (CDHA) with triene structure, isomerized by alkaline treatment, showed intensive cytotoxicity with LD(50) at 12 and 16 microM, respectively, in DLD-1 cells (colorectal adenocarcinoma), while they had no effect on normal human fibroblast cell lines such as MRC-5, TIG-103, and KMS-6 cells. Cytotoxic action of CEPA and CDHA was also demonstrated in other tumor cell lines including HepG2, A549, MCF 7, and MKN-7 cells. alpha-Tocopherol suppressed cytotoxicity of CEPA and CDHA in tumor cells, and the cytotoxicity involved membrane phospholipid peroxidation. CEPA and CDHA induced DNA condensation and fragmentation in DLD-1 cells, indicating the involvement of apoptosis in this cytotoxic mechanism. Furthermore, previous reports have shown that lipid peroxidation product induces cell death, including apoptotic cell death in different cell lines. CEPA and CDHA have been demonstrated in cultured cells to cause cell death via lipid peroxidation and apoptosis in the absence of alpha-tocopherol. PMID- 10753679 TI - Replicative forms of TT virus DNA in bone marrow cells. AB - TT virus (TTV) is a human virus consisting of a single-stranded, circular DNA genome of 3.8 kilobases (kb). To examine whether TTV replicates in peripheral blood mononuclear cells (PBMCs) and bone marrow cells (BMCs), DNA was extracted from the PBMCs and/or BMCs of six TTV-infected individuals and separated by agarose gel electrophoresis. The TTV DNAs from the PBMCs migrated to the 2.0- to 2.5-kb region. The TTV DNAs from the BMCs migrated to the 2.0- to 2. 5-kb and 3.3 to 6.1-kb regions. The faster-migrating TTV DNAs were sensitive to S1 nuclease, while the slower-migrating TTV DNAs were resistant and their position on the agarose gel shifted to the position of the full genomic size upon digestion with restriction enzyme PstI. Full-length inverted polymerase chain reaction on the slower-migrating, double-stranded TTV DNAs from the BMCs amplified a 3.8-kb product. Replicative intermediate forms of TTV DNA are present in BMCs but not in PBMCs. PMID- 10753680 TI - A potent inhibitor of inducible nitric oxide synthase, ONO-1714, a cyclic amidine derivative. AB - (1S,5S,6R,7R)-7-Chloro-3-imino-5-methyl-2-azabicyclo[4.1.0]heptane hydrochloride (ONO-1714), a novel cyclic amidine analogue, inhibits human inducible nitric oxide (iNOS) with a K(i) of 1.88 nM and rodent iNOS with similar potency in vitro. ONO-1714 was found to be 10-fold selective for human iNOS over human endothelial NOS (ecNOS). When the inhibitory activity of ONO-1714 was compared for iNOS, it was found to be 451-fold and >20,000-fold more potent than L-NMMA and aminoguanidine (AG), respectively. In terms of human iNOS selectivity, ONO 1714 was approximately 34- and 2-fold more selective for iNOS than L-NMMA and AG, respectively. ONO-1714 inhibited the LPS-induced elevation of plasma nitrate/nitrite in mice with an ID(50) value of 0.010 mg/kg, s.c. The maximum tolerated dose of ONO-1714 was 30 mg/kg, i.v. Thus, ONO-1714 represents one of the most potent iNOS inhibitors in vitro and in vivo to date and has great potentials for use as an inhibitor for clarifying the pathophysiological roles of iNOS and for use as a therapeutic agent. PMID- 10753681 TI - Further evidence for the presence of "septide-sensitive" tachykinin binding sites in tissues possessing solely NK(1) tachykinin receptors. AB - Binding experiments performed with [(125)I]-NKA allowed us to demonstrate the presence of "septide-sensitive" specific binding sites on membranes from rat CHO cells transfected with the NK(1) receptor cDNA (CHO-rat-NK1 cells), human astrocytoma U373 MG, or mouse cortical astrocytes, cells which express NK(1) but neither NK(2) nor NK(3) receptors. In all cases, [(125)I]-NKA was specifically bound with high affinity (2 to 5 nM) to a single population of sites. In the three preparations, pharmacological characteristics of [(125)I]-NKA binding sites were notably different from those of classical NK(1) binding sites selectively labelled with [(125)I]-BHSP. Indeed, the endogenous tachykinins NKA, NPK, and NKB and the septide-like compounds such as septide, SP(6-11), ALIE-124, [Apa(9 10)]SP, or [Lys(5)]NKA(4-10) had a much higher affinity for [(125)I]-NKA than [(125)I]-BHSP binding sites. Interestingly, differences were also found in the ratio of B(max) values for [(125)I]-NKA and [(125)I]-BHSP specific bindings from one tissue to another. These latter observations suggest that these two types of NK(1) binding sites are present on distinct NK(1) receptor isoforms (or conformers). Finally, while several tachykinins and tachykinin-related compounds stimulated cAMP formation or increased inositol phosphate accumulation in CHO-rat NK1 cells, these compounds only increased the accumulation of inositol phosphates in the two other preparations. PMID- 10753682 TI - Identification of regulatory regions of the putative tumor suppressor gene DMBT1. AB - The lack of expression of the putative tumor suppressor gene DMBT1 has been described in gliomas, lung carcinomas, and other malignancies. To investigate the mechanisms regulating DMBT1 expression we have screened a human genomic library with a 5' cDNA probe and identified a fragment of approximately 3 kb upstream of the proposed coding sequence of DMBT1. This fragment contains subregions that may up- or down-regulate the expression of a reporter gene. These findings may help to elucidate the mechanisms regulating the alternative splicing and the tissue specific expression of DMBT1. PMID- 10753683 TI - Viscoelastic properties of normal and atherosclerotic carotid arteries. AB - BACKGROUND: remodelling of the arterial wall occurs with ageing, even in the absence of atherosclerotic risk factors. With increasing age, arteries dilate, thicken, and get stiffer. The aim of this study was to correlate carotid artery stiffness with wall thickness and plaque presence between healthy individuals and patients with early and advanced atherosclerosis. METHODS: twenty healthy volunteers, 40 carotid segments and 90 patients, 174 carotid segments, with vascular disease were included in the study. The carotid artery was imaged longitudinally and measurements of the intimal-medial thickness (IMT) and plaque were obtained. Systolic and diastolic blood pressures were taken from each arm. The carotid artery stiffness (pressure-strain elastic modulus, Ep) was calculated in all sites from the changes in pressure and diameter. M-mode was used to detect the diameter change (systolic to diastolic) over five cardiac cycles. RESULTS: in the healthy volunteers there was no evidence of plaque or increased IMT. The mean IMT was significantly higher in the patients compared to control (0.83+/-0.27 mm vs. 0.54+/-0.08 mm, p <0.0001). The IMT had a poor correlation with Ep at lower thickness (r=0.24, p=0.08) but this association became stronger with increasing thickness (r=0.62, p<0.001). Arterial segments with an IMT 5 0.88 mm became significantly stiffer compared to the controls (p<0.001) and to patients with an IMT<0.88 mm (p <0.01). Carotid Ep was markedly greater in arterial segments with plaques than in those with increased IMT (p <0.001) and the controls (p<0.0001). CONCLUSIONS: carotid wall areas with small increase in IMT have a poor correlation with carotid artery stiffness. The carotid stiffness increases in areas with marked wall thickening and particularly in segments with plaque. The simultaneous study of vessel-wall elastic behaviour with IMT and plaque changes may increase our understanding of atherosclerotic progression and wall remodelling. PMID- 10753684 TI - The use of preoperative transcranial Doppler variables to predict which patients do not need a shunt during carotid endarterectomy. AB - OBJECTIVES: to analyse whether preoperative transcranial Doppler (TCD) variables can predict intraoperative shunt requirement. DESIGN AND METHODS: the blood-flow velocity (BFV) in the major basal cerebral arteries was measured preoperatively with TCD, in 178 patients scheduled for CEA. Carotid artery compression and CO2 reactivity tests were also performed. Intraoperative electroencephalography was used to decide whether a shunt was needed. Differences in the probability of shunt requirement between the categories of variables were assessed with crosstabs statistics. RESULTS: preoperative TCD criteria clearly identified a subgroup of 59 patients (33%) who did not require a shunt. In general, these patients appeared to have adequate collateral flow through the anterior communicating artery. In contrast, prediction of the need for a shunt was less reliable. TCD variables could predict the need for a shunt with a probability of only 60%. CONCLUSIONS: preoperative TCD can be used to identify patients who do not require a shunt during carotid endarterectomy. PMID- 10753685 TI - A simplified technique for the cryopreservation of vein allografts. AB - OBJECTIVES: we assessed the effects of cryopreservation on smooth-muscle cell injury in human vein. MATERIALS AND METHODS: long saphenous vein was collected during surgery and cryopreserved. Smooth-muscle cell damage was assessed after thawing by in situ detection of fragmented DNA. The presence of cryoprotectant (10% dimethyl sulphoxide, DMSO), cooling and warming rates, and the rate of cryoprotectant removal after thawing were examined. RESULTS: control veins exhibited damage in 8.5% (95% confidence interval (CI) 4.7 to 13.4%,n=13) of smooth-muscle cells compared with 27.7% (95% CI 23.2 to 32.4%, n=115) in vein frozen in 10% DMSO (p=0.001). In the presence of DMSO, damage to smooth-muscle cells was independent of the rates of cooling (p=0.72) and warming (p=0.45). The rate of dilution to remove the cryoprotectant after thawing also had no effect on cell damage (p=0.64). In the absence of cryoprotectant, cell damage was doubled to approximately 50% by slow rather than rapid warming (p=0.01). CONCLUSION: cooling rate, and the presence of a cryoprotectant, has little effect on smooth muscle damage, provided that the tissue is warmed rapidly. Slow warming, in the absence of DMSO, causes substantial damage. These results suggest that simplified methods of vein cryopreservation are feasible. PMID- 10753686 TI - The effects of successful intervention on quality of life in patients with varying degrees of lower-limb ischaemia. AB - OBJECTIVES: to assess the quality of life after successful intervention among patients with varying degrees of lower-limb ischaemia in comparison with healthy controls and the respondents>> degree of sense of coherence. MATERIALS AND METHODS: one hundred and twelve patients and 102 healthy controls were assessed for quality of life (Nottingham Health Profile) and sense of coherence. MAIN RESULTS: successful angioplasty or surgical intervention led to an improved quality of life at 6 months, in particular with regard to pain, sleep, physical mobility, hobbies and holiday and to a level similar to healthy controls in sleep, social isolation, paid employment and family relationships. It remained at a significantly lower level than that of healthy controls with regard to pain, emotional reactions, physical mobility, energy, housework, hobbies, holidays, sex and social life. Critical ischaemia patients did not reach the same level of quality of life as the claudicants or the healthy controls. CONCLUSION: successful treatment for chronic limb ischaemia improved the quality of life significantly, more so in claudicants than in patients with critical ischaemia. The degree to which the quality of life improved was associated with the patients>> sense of coherence and their ankle pressure. PMID- 10753687 TI - Non-operative treatment of advanced limb ischaemia: the decision for palliative care. AB - OBJECTIVES: to identify and describe patients with advanced limb ischaemia who were selected for palliative care, rather than surgical intervention. DESIGN: case-note review of patients identified from a prospective register. MATERIALS AND METHODS: thirty patients (22 female; median age 87 years) were identified during 1993-1998, for whom a clearly documented decision was made for palliative care, rather than major amputation or possible revascularisation. RESULTS: two thirds of the patients had limiting cardiac problems, two-thirds were immobile, and 47% had suffered a stroke. Half had three or more important co-morbidities. Twelve (40%) had unsalvageable acute ischaemia. There were clear records of the decision about non-intervention being made by a consultant in 87%; being discussed with the patient in 43%; and with known relatives in 90%. Survival after this decision ranged from <24 hours to 42 days (median 3.5 days). CONCLUSION: there is a small subgroup of patients with advanced ischaemia who are best treated palliatively, and who have not been well described before. Recognising these patients, recording discussions about their management, and a high standard of terminal care are all important. PMID- 10753688 TI - Enhancing venous outflow in the lower limb with intermittent pneumatic compression. A comparative haemodynamic analysis on the effect of foot vs. calf vs. foot and calf compression. AB - OBJECTIVES: intermittent pneumatic compression (IPC), an established method of deep-vein thrombosis prophylaxis, is also an effective means of leg inflow enhancement, improving the walking capacity and ankle pressure of claudicants, long-term. This study, using duplex ultrasonography, compares the haemodynamic effect of IPC of the (a) foot (at 120 mmHg [IPC(foot/120 mmHg)], and 180 mmHg [IPC(foot/180 mmHg)]), (b) calf (IPC(calf), 120 mmHg) and (c) both simultaneously (IPC(foot+calf), 120 mmHg), on the venous outflow of 20 legs of normals and 25 legs of claudicants. RESULTS: the peak and mean velocities, volume flow and pulsatility index in the superficial femoral and popliteal veins of both groups increased significantly with all IPC modes (p<0.001). IPC(foot+calf)produced the highest enhancement followed by IPC(calf)(p<0.01), which was more effective (p<0.001) than either IPC(foot/180 mmHg)or IPC(foot/120 mmHg). The venous volume expelled with IPC(calf)and IPC(foot+calf)was 2-2.5 and 3-3.5 times that with IPC(foot/180 mmHg)respectively. Velocity enhancement with IPC was similar between groups and the superficial femoral and popliteal veins. IPC(foot/180 mmHg)produced higher (p<0. 01) flow velocities than IPC(foot/120 mmHg)in both groups and veins examined; however, differences were limited. CONCLUSIONS: all IPC modes proved effective, IPC(foot+calf)generating the highest venous outflow enhancement. Higher venous volumes expelled with IPC(foot+calf)explain its reported superiority on leg inflow over the other modes. Increase of applied pressure from 120 to 180 mmHg with IPC(foot)offered only a small outflow improvement. Venous haemodynamics at rest and with IPC in claudicants do not differ significantly from those in healthy subjects. PMID- 10753689 TI - Optimum intermittent pneumatic compression stimulus for lower-limb venous emptying. AB - OBJECTIVE: intermittent pneumatic compression (IPC) of the foot (IPC(foot)), calf (IPC(calf)) or both (IPC(foot+calf)) augments calf inflow, and improves the walking ability and peripheral haemodynamics of claudicants (IPC(foot), IPC(foot+calf)), largely due to venous outflow enhancement. This cohort study, using direct pressure measurements in healthy limbs, determines the optimal combination of frequency (2-4 impulses/minute), applied pressure (60-140 mmHg), mode (IPC(foot)-IPC(calf)-IPC(foot+calf)) and delay time of calf-to-foot impulse (0 s-0.5 s-1 s) that enables IPC to generate an almost complete and sustained decrease in venous pressure. RESULTS: (a) IPC(foot)at 120 and 80 mmHg generated lower venous pressure than that with 100 and 60 mmHg (p=0.036) respectively, for 2-4 impulses/minute; venous pressure differences between applied pressures of 140 and 120 mmHg or between 80 and 100 mmHg were insignificant. (b) Venous pressure with IPC(calf)at 80 mmHg was lower than that with 60 mmHg (p=0.036) (2-4 cycles/minute); differences in venous pressure between applied pressures of 140 and 100 mmHg or between 120 and 80 mmHg were insignificant. (c) At applied pressures 60-140 mmHg, IPC(foot+calf)with one-second delay generated lower venous pressure than that with half-second delay (p=0.036), the latter being more efficient than zero delay; increasing applied pressures produced lower venous pressure, but differences were small. Venous pressure decreased with increasing IPC frequency (from 2 to 3-4/minute), at applied pressures 60-140 mmHg. CONCLUSIONS: IPC(foot+calf)at applied 120-140 mmHg, a frequency of 3-4 impulses/minute and one-second delay, provided the optimum intermittent pneumatic stimulus. PMID- 10753690 TI - Effect of intermittent pneumatic foot compression on popliteal artery haemodynamics. AB - PURPOSE: the aim was to investigate the effect of intermittent pneumatic foot compression (IPC(foot)) on popliteal artery haemodynamics in normal individuals and in patients with intermittent claudication due to peripheral vascular disease (PVD) (Fontaine stage II). MATERIAL AND METHODS: popliteal artery volume flow [vFl], pulsatility index [PI], mean velocity [mV], peak systolic [PSV] and end diastolic velocity [EDV], in 25 limbs of 20 normal subjects and 40 limbs of 32 stable claudicants were obtained in the sitting position before, during and within 30 seconds after the application of IPC(foot)(applied pressure: 120 mmHg; inflation time: 3 seconds; deflation time: 17 seconds) using colour-flow duplex imaging (CFDI). The reproducibility of flow velocity estimations using CFDI in the horizontal [hor] (recovery) and sitting [sit] positions was evaluated in 20 limbs of normal controls and 20 limbs of claudicants. RESULTS: popliteal artery vFl, mV, PSV and PI measurements were performed with a coefficient of variation (CV) of less than 14.6% among claudicants and of less than 13.3% in normal subjects. EDV is the least reproducible parameter with an overall CV range of 10.2-21.5% in normal controls and 9.1-18.6% in arteriopaths. On application of IPC(foot)popliteal artery vFl increased by 111% in the control group (p<0.001) and by 51% in the claudicants (p<0.001). Within 30 seconds of the cessation of pump action flow decreased significantly in both groups (p<0.001), but maintained a significantly higher level than that at baseline (p<0. 001, in both groups). The mV, PSV and EDV showed a similar pattern of significant changes. Both in normals and claudicants, the PI decreased with IPC(foot)(p<0.001) and increased post-compression; however, it was significantly lower than baseline (p<0.005) within 30 seconds of impulse delivery. CONCLUSIONS: current CFDI technology enables a reproducible estimation of popliteal artery flow velocities. IPC(foot)can significantly augment arterial calf inflow on an acute basis both in normals and claudicants. The increase of EDV and decrease of PI indicate that attenuation of peripheral resistance to flow is the main mechanism underlying the popliteal artery vFl enhancement on application of IPC(foot). Prospective trials on the long-term effect of IPC(foot)in the management of patients with PVD are indicated from the results of this study. PMID- 10753691 TI - Remote superficial femoral artery endarterectomy: medium-term results. AB - BACKGROUND: the aim of this study is to determine the medium-term results following successful remote superficial-femoral endarterectomy (RSFE). SETTING: district general hospital in United Kingdom. METHODS: RSFE comprises a single incision over the origin of the superficial femoral artery. The endarterectomy is carried out in a closed fashion from above. The cut end of distal atheroma is secured with a stent. Twenty-five limbs were followed up with three monthly duplex scans and angiography if any abnormality was suggested. All patients presented with intermittent claudication; in addition three had rest pain and three ulceration or gangrene. The length of atheromatous core removed was 10-30 cm. RESULTS: all patients had a follow-up of at least one year (range 12-27 months). Eleven arteries developed 14 stenoses. Nine became apparent within nine months of RSFE. The cumulative risk of stenoses developing in patent arteries was 24% at 6 and 63% at 12 months. Eleven angioplasties (PTA) of these stenoses were undertaken. Nine of these remain patent at a median of 12 months after PTA. At one year primary patency was 10 of 25 (40%), primary-assisted patency 18 of 25 (72%) and secondary patency 19 of 25 (76%) and at two years 29%, 57% and 57% respectively. CONCLUSIONS: RSFE is worth considering for superficial femoral artery occlusive disease, particularly in high-risk patients without suitable vein and with limited life expectancy. Careful duplex surveillance is important. Until stenoses can be prevented, the widespread use of RSFE cannot be recommended. PMID- 10753692 TI - Intraoperative contamination of synthetic vascular grafts. Effect of glove change before graft implantation. A prospective randomised study. AB - OBJECTIVES: to investigate the incidence of intraoperative graft contamination, bacterial species and the influence of change of surgeon's gloves on contamination. DESIGN: a prospective randomised study. MATERIALS AND METHODS: forty patients had implantation of synthetic vascular grafts. All patients received intraoperative cloxacillin (2.0 g) or clindamycin (0.6 g) intravenously. The procedures were randomised to two groups: Group 1 - surgeons changed the gloves before the first contact with the vascular prosthesis and Group 2 - operation without glove change. The growth of all bacterial species from graft segments and from the gloves was recorded. The susceptibility to antibiotics was tested. RESULTS: the number of contaminated grafts was similar in the two groups. Growth of bacteria was recorded from 92.5% (37/40) of the graft segments and 33% (51/156) of glove imprints. Of the cultured species, 75% and 47%, respectively, were identified as coagulase-negative staphylococci (CNS). Twenty-eight per cent of CNS were resistant to cloxacillin, 15% to clindamycin, and 10% to cloxacillin and clindamycin. In all, 25% of the CNS strains were resistant to the prophylactic antibiotic used. In 50% of cases, the antibiogram of the CNS strain recovered from gloves agreed with that of the strain harvested from the graft. CONCLUSIONS: a high incidence of graft contamination was found which was not reduced by changing gloves. However, changing gloves did seem to reduce the number of bacterial species. PMID- 10753693 TI - Ten years' experience of aortic aneurysm associated with systemic lupus erythematosus. AB - BACKGROUND: aortic aneurysm is a rare but life-threatening cardiovascular complication in patients with systemic lupus erythematosus (SLE). The purpose of this study was to clarify the characteristic clinical features and the pathological mechanism of aneurysmal formation in these patients. METHODS: among 429 patients operated on for abdominal aortic aneurysm (AAA) during the past 10 years, five cases with SLE were treated surgically. Their clinical data were reviewed, and the resected aneurysmal wall of the five patients was also examined histologically. RESULTS: the mean age of the patients with SLE was 55 years, which was statistically younger than that of the other patients (mean 77 years, s.d. 7.9, p <0.05). They had received long-term corticosteroid therapy for the treatment of SLE for a mean of 23 years. Histologically, destruction of the medial elastic lamina was characteristic. Four patients had no complications in the postoperative follow-up period (mean 4 years), while the remaining patient died of rupture of a dissecting aneurysm two years after operation. CONCLUSION: prolonged steroid therapy may play a major role in accelerating atherosclerosis, which can result in aortic aneurysmal enlargement, possibly together with primary aortic wall involvement and/or vasculitic damage in patients with SLE. PMID- 10753694 TI - Oral health of patients scheduled for elective abdominal aortic correction with prosthesis. AB - OBJECTIVE: to evaluate the frequency of potential oral foci of infection in patients scheduled for elective abdominal aortic surgery. DESIGN: prospective clinical study. MATERIALS: oral health and dentures of 50 patients (33 males and 17 females, mean age 65 years) were examined before aortic surgery. CHIEF OUTCOME MEASURES: radiographic and clinical examination with special emphasis on identifying acute and chronic oral and ontogenic conditions which may contribute to aortic prosthesis infection. RESULTS: eighty-two per cent of the patients had some oral infection foci. The mean number of remaining teeth in the cohort was 9.3, and 21% of these were potential infectious foci (62% in the patients). Twenty-six per cent of the patients suffered from oral Candida infection. Seventy four per cent of the patients had total or partial dentures, 45% of which were ill-fitting and needed repair. CONCLUSIONS: oral infectious foci occur frequently in patients needing aortic surgery. Untreated foci may contribute to aortic prosthesis infection. Preoperative oral evaluation and elimination of intraoral infection is recommended for patients scheduled for abdominal aortic repair. PMID- 10753695 TI - Abdominal aortic aneurysm: the role of clinical examination and opportunistic detection. AB - OBJECTIVES: to investigate the method of discovery of abdominal aortic aneurysms (AAA) in a district general hospital setting. DESIGN: retrospective study. MATERIALS AND METHODS: we analysed 198 patients with an AAA who presented to our unit over a 3-year period. The method of initial diagnosis, size of the AAA and whether this was palpable, irrespective of the method of detection, were recorded. RESULTS: ninety-five (48%) were discovered clinically, 74 (37.4%) during a radiological investigation, and 29 (14.6%) at laparotomy. Of the 74 AAAs first detected radiologically, subsequent physical examination showed that 28 (37.8%) were in fact palpable and missed at presentation. The average size of those discovered clinically (6. 48+/-1.32 cm) was larger than those found radiologically (5.37+/-1. 44 cm, p<0.001) or at operation (5.43+/-1.48 cm, p=0.039). The average diameter of the palpable AAAs was also greater than that of the non-palpable AAAs (6.42+/-1.24 cm vs. 4.86+/-1.38 cm, p<0.001). CONCLUSIONS: opportunistic detection of a clinically unsuspected aneurysm during clinical examination or investigation for another reason is the most common way the diagnosis of an AAA is made. Almost half of the aneurysms were diagnosed clinically, but physical examination also missed more than a third of those detected radiologically. Despite technological advancement, clinical examination still plays a paramount role in the detection of AAAs. Larger AAAs are usually palpable and more likely to be detected on clinical examination. PMID- 10753696 TI - Can colonoscopy diagnose transmural ischaemic colitis after abdominal aortic surgery? An evidence-based approach. AB - OBJECTIVES: to assess the diagnostic value of colonoscopy in ischaemic colitis following abdominal aortic surgery, based on a literature review, and to introduce the concept of evidence-based medicine. METHOD: a review of the literature according to evidence-based principles was made by all doctors of our department. RESULTS: seven prospective non-randomised reports on routine colonoscopy after abdominal aortic surgery were found. None of the participants found all the reports, and the last was identified by the reviewer. CONCLUSIONS: Endoscopy may disclose ischaemic colitis, but cannot separate transmural from the clinically less important mucosal ischaemia. Endoscopy had no impact on mortality in any of the prospective series. The evidence-based conference was an inspiring teaching modality, and illustrated for the participants the difficulty in performing a targeted literature search. PMID- 10753697 TI - Anatomical risk factors for proximal perigraft endoleak and graft migration following endovascular repair of abdominal aortic aneurysms. AB - INTRODUCTION: proximal perigraft endoleak (PPE) and graft migration are associated with significant morbidity and mortality. Objective data establishing correlation between neck anatomy and these complications are lacking. The aim of this study was to analyse the anatomy of the neck in order to find which variables were significantly associated with PPE and graft migration. METHODS: one hundred and eighty-four patients underwent endovascular repair (EVR) of infrarenal AAA using an in-house custom-made stent graft (Gianturco stents plus Dacron). Thirty-one patients had PPE and fifteen had graft migration. Neck diameter was measured at the level of renal arteries and lower limit of the neck. Necks were classified according to shape. Neck angulation was measured from spiral computed tomography (CT) or magnetic resonance imaging (MRI) reconstructions, or angiograms. Thrombus or atheroma lining and presence of calcifications were recorded. RESULTS: neck angulation was significantly greater in patients who had PPE (50+/-16, p=0. 0005) or graft migration (54+/-20, p=0.003), compared to patients who had none of these two complications (37+/-18). Neck diameter was significantly greater in patients with PPE (p=0.05). Incidence of PPE or graft migration was not significantly higher in the presence of a conical shape, thrombus or atheroma lining and calcifications. CONCLUSION: neck angulation was the risk factor most significantly related to PPE and graft migration. PMID- 10753698 TI - Endoleaks following conventional open abdominal aortic aneurysm repair. AB - OBJECTIVE: to describe the complication of <<<>>> following conventional open abdominal aortic aneurysm (AAA) repair. DESIGN: prospective case study. SETTING: two specialist vascular surgical centres. PATIENTS AND METHODS: six patients who had successful conventional open AAA repair. RESULTS: six patients presented with back or abdominal pain or hypotension between one and eighteen months later. An endoleak at the distal anastomosis was noted in five of the cases and one endoleak at the proximal anastomosis. All six cases were successfully repaired; two of these patients required Dacron graft replacement, whilst in four cases only direct resuturing was needed. There was no evidence of infection. CONCLUSIONS: an endoleak is not a phenomenon confined to stent grafts. It should be considered in all patients who present with back or abdominal pain within eighteen months of open AAA repair. The combination of computed tomography (CT) scan and digital subtraction angiography is most useful for preoperative diagnosis. PMID- 10753699 TI - A prolonged spinal cord ischaemia model in pigs. Passive shunting offers stable central haemodynamics during aortic occlusion. AB - OBJECTIVES: to evaluate the effect of a modified aortic shunt on central haemodynamic variables during experimental thoracic aortic occlusion in a prolonged spinal cord ischaemia model. MATERIAL AND METHODS: central haemodynamic variables were evaluated during aortic cross-clamping. In the shunt group (n=11), after the placement of proximal and distal aortic clamps, distal aortic perfusion was restored through an aortoiliac shunt via the left subclavian artery. In the no-shunt group (n=11), spinal cord ischaemia was achieved with only proximal aortic cross-clamping. The clamping time was 60 minutes in the shunt group and 30 minutes in the no-shunt group. RESULTS: in the no-shunt group, all animals needed inotropic support, vasodilators and buffers during the experiment. None of these drugs were needed in the shunt group. In the no-shunt group, cross-clamping caused a significant increase in mean arterial pressure and heart rate compared to baseline values. These variables were stable in the shunt group during aortic occlusion. In the reperfusion period cardiac output, heart rate and arterial pCO(2)were significantly higher in the no-shunt than in the shunt group. CONCLUSION: the present experimental spinal cord ischaemia model, using double aortic cross-clamping with shunt, offers improved central haemodynamics. This enables the study of prolonged selective spinal cord ischaemia without interaction from vasoactive drugs or systemic reperfusion. PMID- 10753700 TI - Endovascular and surgical techniques - AAA endografting: two straightforward indications? PMID- 10753701 TI - Endovascular and surgical techniques - percutaneous cross-over stent graft from one Y-graft limb to another to exclude an aortic aneurysm. PMID- 10753702 TI - Cell recognition and entry by rhino- and enteroviruses. PMID- 10753704 TI - Dual-viral vector approach induced strong and long-lasting protective immunity against very virulent infectious bursal disease virus. AB - To induce strong protective immunity against very virulent infectious bursal disease virus (vvIBDV) in chickens, two viral vector systems, Marek's disease and Fowlpox viruses expressing the vvIBDV host-protective antigen VP2 (rMDV, rFPV), were used. Most of chickens vaccinated with the rFPV or rMDV alone, or vaccinated simultaneously with both at their hatch (rMDV-rFPV(1d)), were protected against developing clinical signs and mortality; however, only zero to 14% of the chickens were protected against gross lesions. In contrast, gross lesions were protected in 67% of chickens vaccinated primarily with the rMDV followed by boosting with the rFPV 2 weeks later (rMDV-rFPV(14d)). Protection against the severe histopathological lesions of rFPV, rMDV, rMDV-rFPV(1d), and rMDV-rFPV(14d) vaccine groups were 33, 42, 53, and 73%, respectively. Geometric mean antibody titers to VP2 of chickens vaccinated with the rFPV, rMDV, rMDV-rFPV(1d), and rMDV rFPV(14d) before the challenge were 110, 202, 254, and 611, respectively. Persistent infection of the rMDV in chickens after the booster vaccination with rFPV was suggested by detection of the rMDV genes from peripheral blood lymphocyte DNA at 28 weeks of age. These results indicate that the dual-viral vector approach is useful for quickly and safely inducing strong and long-lasting protective immunity against vvIBDV in chickens. PMID- 10753703 TI - Protection of CD3 delta knockout mice from lymphocytic choriomeningitis virus induced immunopathology: implications for viral neuroinvasion. AB - For a virus to establish a neuronal infection, it must spread from its primary site of infection to the central nervous system (CNS) before immune-mediated clearance occurs. Lymphocytic choriomeningitis virus (LCMV) is a murine pathogen that can result in persistent neuronal infection in newborn mice and in adults that lack CD8(+) T cells. To determine the neuroinvasive capacity of LCMV in the presence of an existent, but compromised, cytotoxic T lymphocyte response, the course of LCMV infection was examined in mice that possess 10% of the normal complement of T lymphocytes, due to the lack of the CD3 delta (delta) subunit of the T cell receptor complex (CD3 delta KO mice). Unlike immunocompetent mice that produced a massive immune response that caused death by 6-7 days postinfection, CD3 delta KO mice mounted a weak response and survived. The presence of viral antigen gradually shifted from the class I MHC-positive meninges and ependyma to class I MHC-deficient CNS neurons 10-30 days postinoculation. The infected CD3 delta KO mice developed a delayed T cell response that suppressed virus replication in peripheral tissues but not in the CNS; subsequent adoptive transfer experiments supported the hypothesis that the lack of clearance from neurons was due to sequestration of LCMV in an immune-privileged cell type. Based on these results, we propose that a critical parameter in the pathogenesis of neurotropic viruses is the rate of immune activation; individuals with impaired T cell responses may be more vulnerable to persisting CNS infections. PMID- 10753705 TI - Dox-dependent SIVmac with tetracycline-inducible promoter in the U3 promoter region. AB - An attenuated live vaccine is a candidate in developing vaccines against human immunodeficiency virus type 1 (HIV-1). The study using macaques and simian immunodeficiency virus (SIVmac) showed an attenuated virus to be more effective than any other vaccine candidate. However, development of a safer vaccine is required for clinical application. In this study, we constructed pSIVmac Delta nef with tetracycline inducible promoter (pTet) and tried to control viral expression in a drug-dependent manner. Promoter/enhancer motifs in the U3 region of the long terminal repeats (LTRs) were serially deleted and replaced with pTet. In mutant LTRs, which lack NF-kappaB and Sp1 binding sites, TATA box motifs, and the 5' half of the U3 region, promoter activity was stringently controlled by doxycycline (Dox). Their activities were similar to or higher than that of wild type LTR in the presence of Dox, based on the transient chloramphenicol acetyltransferase reporter assay. Three of these mutant LTRs were introduced into the pSIVmac239 Delta nef genome. Viral protein from these viruses was efficiently expressed in a Dox-dependent manner after transfection to a HeLa cell, which expresses reverse tetracycline transactivator (rtTA). The 2-LTR-form viral DNA of these viruses could be detected in M8166 cells that had been infected with supernatants from the transfected rtTA HeLa cell. These results suggest that pSIVmac Delta nef containing mutant LTRs can proceed through one viral replication cycle consisting of transcription, formation of viral particles, infection to cells, and reverse transcription. Although continuous replication of these Dox-dependent viruses requires a supply of rtTA as a constituent for the pTet-On viral genome, the successful replacement of the original promoter with a drug-dependent promoter suggests a new possibility for developing a safer attenuated live virus. PMID- 10753706 TI - Monoclonal antibodies to the hypervariable region 1 of hepatitis C virus capture virus and inhibit virus adsorption to susceptible cells in vitro. AB - To analyze the neutralizing-related activity of antibodies against the hypervariable region 1 (HVR1) of hepatitis C virus (HCV) in more detail, monoclonal antibodies (mAbs) against HVR1 were raised by immunizing various strains of mice with one of two synthetic HVR1 peptides that had been derived from two isolates of HCV. The epitope specificity of all six mAbs could be assigned by the use of a series of linear peptides in competitive ELISA. It seems that most subregions in the amino acid sequence of HVR1 can induce a humoral immune response in mice. All three mAbs specific to HVR1-6-1 had the ability to capture homologous HCV-6 and inhibit its absorption to susceptible cells in vitro despite the fact that the epitope of each mAb was at a different location in HVR1, whereas the other three mAbs specific to HVR1-7 could not capture HCV-6 nor inhibit the absorption of HCV-6 to susceptible cells. The data in this study suggest that mAbs against HVR1 can prevent the infectivity of HCV in an isolate specific and epitope position-independent manner. PMID- 10753707 TI - Coding changes in the poliovirus protease 2A compensate for 5'NCR domain V disruptions in a cell-specific manner. AB - Polioviruses are single-stranded RNA viruses with an unusually long noncoding region (NCR) at the 5' end predicted to have an elaborate secondary structure made up of six domains. Mutations in domain V of the poliovirus 5'NCR that disrupt secondary structure are responsible for attenuation of the virus and a temperature-sensitive (ts) phenotype in vitro. In addition to direct back mutation or compensatory second site mutation in the 5'NCR as previously documented, the ts phenotype was found to be compensated for in monkey kidney cells in vitro by a coding change in the protease 2A. These coding changes were found throughout the protease with no obvious pattern or trend. They were not all found to be equivalent and limited in ability to compensate for the severest domain V disruption. The compensatory effect of the 2A changes was found to be cell specific, having no effect on monkey neurovirulence and in a mouse cell line but a significant effect in two monkey cell lines and a human epithelial line. PMID- 10753708 TI - CXCR4 mediates entry and productive infection of syncytia-inducing (X4) HIV-1 strains in primary macrophages. AB - CCR5 and CXCR4 are the main coreceptors for non-syncytia-inducing (NSI) and syncytia-inducing (SI) HIV-1 strains, respectively. NSI HIV-1 isolates do not infect either human lymphoid or monocytoid cell lines, and this inability correlates with the absence of CCR5 expression in these cell types. The ability of SI HIV-1 isolates to infect human primary macrophages has been disputed. Here, we report that CXCR4 is expressed in primary blood-derived human mononuclear phagocytes at all stages of differentiation, although the maturation process correlates with downregulation of CXCR4 mRNA. Infection experiments with the SI molecular clone NL4-3 tagged with a mutant of the green fluorescent protein established that both monocytes and attached macrophages are susceptible to infection with CXCR4-restricted HIV-1 strains. NL4-3 entry into primary macrophages could be blocked by SDF-1alpha in a dose-dependent manner, or by the anti-CXCR4 monoclonal antibody 12G5. HIV-1 entry led to productive infection. No evidence of postentry defects or nuclear import delay for CXCR4-restricted HIV-1 strains was detected using a quantitative real-time PCR assay measuring HIV-1 DNA entry into the nucleus. Macrophages infected by HIV-1 and expressing virus were maintained in culture for long periods of time (up to 5 months). These results demonstrate that CXCR4 is the main HIV-1 SI coreceptor in human primary macrophages and underline the importance of the macrophage as a long-living viral reservoir for HIV-1. PMID- 10753709 TI - The N-terminal matrix domain of HIV-1 Gag is sufficient but not necessary for viral protein U-mediated enhancement of particle release through a membrane targeting mechanism. AB - Viral protein U (Vpu) is an 81 amino acid phosphoprotein found in human immunodeficiency virus type 1 (HIV-1)-infected cells. One function of Vpu is to enhance the release of virus particles from the plasma membrane in infected cells. Using subcellular fractionation, we observed that Vpu promotes the targeting of Pr55 Gag to the plasma membrane, the site of viral assembly. Deletions of Pr55, which removed most of the N-terminal matrix domain (p39) or the C-terminal domains of nucleocapsid and p6 (p41), still allowed for virus-like particle production. Moreover, the release of these particles remained Vpu responsive. The N-terminal matrix (MA) domain of Gag, which contains its membrane binding domain, is sufficient for Vpu-mediated enhanced release into the supernatant. Furthermore, a MA-GFP fusion protein showed enhanced membrane binding in the presence of Vpu. This demonstrates that Vpu action may be mediated by allowing Gag, specifically the N-terminal matrix domain, to efficiently associate with the plasma membrane. Thus MA appears sufficient but not necessary for Vpu-mediated enhanced particle release. PMID- 10753710 TI - High prevalence of hypervariable region 1-specific and -cross-reactive CD4(+) T cells in HCV-infected individuals responsive to IFN-alpha treatment. AB - The hypervariable region 1 (HVR1) of the putative envelope 2 protein of the hepatitis C virus (HCV) is the most variable part of the whole HCV polyprotein. Anti-HVR1 antibodies have been shown to protect against HCV infection, indicating that this region contains an important neutralization determinant. Recently we and others have demonstrated that HVR1 is also a T cell determinant able to activate helper T cell responses during HCV infection. In order to investigate the role of the immune response against HVR1 during HCV infection we have evaluated the humoral and lymphoproliferative responses to a panel of HVR1 peptides in HCV-infected patients with different outcomes of the disease following interferon-alpha (IFN-alpha) treatment. We observed that the frequency of anti-HVR1 T cell responses was significantly higher in patients who recovered after IFN-alpha therapy than in those who did not, while no differences in the anti-HVR1 antibody reactivities were detected. In addition, by generating HVR1 specific T cell lines and clones we identified human leukocyte-associated antigens DR4 restricted T cell epitopes in the carboxy-terminus of HVR1 and we demonstrated that broadly cross-reactive HVR1 T cells are elicited by HVR1. PMID- 10753711 TI - The cytoplasmic tails of the influenza virus spike glycoproteins are required for normal genome packaging. AB - Deletion of the cytoplasmic tails of the influenza A virus spike glycoproteins, hemagglutinin (HA) and neuraminidase (NA), has previously been shown to result in markedly defective virion morphogenesis (Jin et al., 1997, EMBO J. 16, 1236 1247). We have found that influenza A virus preparations lacking the HA and NA cytoplasmic tails (HAt-/NAt-) have a reduced vRNA to protein content, contain an increase in cellular RNA contaminants, and exhibit increased resistance to ultraviolet (UV) inactivation. There is also a direct correlation between abnormal virion morphology and reduced infectivity. The data suggest that the HAt /NAt- virion population contains a broader range of number of packaged RNA segments than wild-type (wt) virus. Sucrose gradient centrifugation analysis indicated the presence of a subpopulation of virions with pronounced deformation in virion morphology and reduced infectivity. The role of the HA and NA cytoplasmic tails was examined further by using a trans-complementation assay and it was found that expression of wt HA and NA from cDNAs followed by HAt-/NAt- virus infection caused the formation of a pseudotype virus with wt sedimentation properties. Taken together the data indicate that the HA and NA cytoplasmic tails affect not only virion morphology but also proper genome packaging. PMID- 10753712 TI - Expression and localization of human herpesvirus 8-encoded proteins in primary effusion lymphoma, Kaposi's sarcoma, and multicentric Castleman's disease. AB - To investigate the expression of human herpesvirus 8 (HHV8)-encoded proteins in the cells of primary effusion lymphoma (PEL), Kaposi's sarcoma (KS) and multicentric Castleman's disease (MCD), nine rabbit polyclonal antibodies to K2, ORF26, K8, K8.1, K10, K11, ORF59, ORF65, and ORF73 were developed. Western blot analysis in PEL cell lines (TY-1 and BCBL-1) revealed that the expression of these proteins, except ORF73 (LANA), was induced by tetradecanoylphorbol acetate (TPA) treatment, indicating that these proteins are lytic proteins. Immunofluorescence assay in primary PEL cells derived from pericardial effusion and PEL cell lines with and without TPA treatment revealed that primary PEL cells exhibited the same expression pattern as noninduced PEL cell lines, and the treatment changed localization of K8, ORF59, and ORF65 proteins. Immunohistochemistry revealed that 90% of KS spindle cells expressed the ORF73 protein, whereas a small population of KS cells expressed K8, K10, K11, ORF59, and ORF65 proteins. In MCD, ORF73, ORF59, K8, K2, and K10 proteins were expressed in the cells at mantle zone of the follicle. These data indicate that KS and PEL cells expressed predominantly latent proteins, whereas MCD expressed both latent and lytic proteins, suggesting that HHV8 plays a different role in the pathogenesis of HHV8-associated diseases. PMID- 10753713 TI - The vaccinia virus bifunctional gene J3 (nucleoside-2'-O-)-methyltransferase and poly(A) polymerase stimulatory factor is implicated as a positive transcription elongation factor by two genetic approaches. AB - Vaccinia virus genes A18 and G2 affect the elongation and termination of postreplicative viral gene transcription in opposite ways. Viruses with mutations in gene A18 produce abnormally long transcripts, indicating that A18 is a negative transcription elongation factor. Viruses containing mutations in gene G2 produce transcripts that are abnormally short, truncated specifically from their 3' ends, indicating that G2 is a positive transcription elongation factor. Despite the fact that both A18 and G2 are essential genes, A18-G2 double-mutant viruses are viable, presumably because the effects of the mutations are mutually compensatory. In addition, the anti-poxviral drug isatin-beta-thiosemicarbazone (IBT) seems to enhance elongation during a vaccinia infection: IBT treatment of a wildtype vaccinia infection induces a phenotype identical to an A18 mutant infection, and G2 mutant viruses are dependent on IBT for growth, presumably because IBT restores the G2 mutant truncated transcripts to a normal length. These observations inspire two independent genetic selections that have now been used to identify an additional vaccinia gene, J3, that regulates postreplicative transcription elongation. In the first selection, a single virus that contains an extragenic suppressor of the A18 temperature-sensitive mutant, Cts23, was isolated. In the second selection, several spontaneous IBT-dependent (IBT(d)) mutant viruses were isolated and characterized genetically. Marker rescue mapping and DNA sequence analysis show that the extragenic suppressor of Cts23 contains a point mutation in the J3 gene, while each of seven new IBT(d) mutants contains null mutations in the J3 gene. The J3 protein has previously been identified as a (nucleoside-2'-O-)-methyltransferase and as a processivity subunit for the heterodimeric viral poly(A) polymerase. The nature of the two independent selections used to isolate the J3 mutants strongly suggests that the J3 protein serves as a positive postreplicative transcription elongation factor during a normal virus infection. PMID- 10753714 TI - Transcription elongation activity of the vaccinia virus J3 protein in vivo is independent of poly(A) polymerase stimulation. AB - Prior genetic analysis suggests that the vaccinia virus J3 gene product, previously characterized as a bifunctional (nucleoside-2'-O-)-methyltransferase and poly(A) polymerase stimulatory factor, is a postreplicative positive transcription elongation factor. To test this hypothesis, viruses bearing mutations in the J3 gene were characterized with respect to viral protein and RNA synthesis in infected cells. The analysis reveals that compared to wt virus infections, J3 mutants synthesize reduced amounts of large late viral proteins and shorter-than-normal intermediate and late mRNAs. Structural analysis of one late mRNA shows that it is specifically truncated from the 3' end, thus accounting for its shorter than normal chain length. Thus J3 mutant viruses are defective in elongation of transcription of postreplicative viral genes, strongly suggesting that the J3 gene product normally acts as a positive transcription elongation factor. Biochemical analysis of one J3 missense mutant demonstrates that it retains poly(A) stimulatory activity but is defective in (nucleoside-2'-O )-methyltransferase activity. Thus the elongation factor activity of the J3 gene product is independent of the poly(A) stimulatory activity. It remains to be determined whether the (nucleoside-2'-O-)-methyltransferase and elongation factor activities of the J3 protein are linked or can be uncoupled by mutation. PMID- 10753715 TI - Polyomavirus large T antigen mutants affected in viral DNA replication. AB - We have characterized two polyomavirus large T antigen mutants with different properties in viral DNA replication. dl-97, a mutant active in immortalization, exerts a dominant negative effect in viral DNA replication. 13val, which is defective in both immortalization and viral DNA replication, has a lesion in the putative DnaJ domain affecting the block of Rb function. PMID- 10753716 TI - Preservation of 5'-end integrity of a potyvirus genomic RNA is not dependent on template specificity. AB - Full-length in vitro transcripts of plum pox potyvirus (PPV) genomic RNA with mutations altering the number of 5'-terminal adenosine residues were able to infect Nicotiana clevelandii plants, whereas a mutant with a substitution of adenosine in position 2 by guanosine failed to infect. The genomic 5' end was template-independently repaired during in vivo RNA synthesis producing wild-type viral progeny. Putative models of replication initiation are discussed. PMID- 10753717 TI - Paramyxoviridae use distinct virus-specific mechanisms to circumvent the interferon response. AB - STAT1 and STAT2 are cellular transcription factors involved in interferon (IFN) signaling and are thus critical for the IFN-induced antiviral state. We have previously shown that the paramyxovirus Simian Virus 5 (SV5) blocks both type I and type II interferon (IFN) signaling by targeting STAT1 for proteasome-mediated degradation. To determine whether this is a feature common to all Paramyxoviridae, we examined the abilities of SV5, Sendai virus (SeV), human respiratory syncytial virus (RSV), and human parainfluenza viruses types 2 and 3 (hPIV2 and hPIV3, respectively) to block interferon signaling. The results showed that in reporter assays SV5, SeV, and hPIV3 blocked both type I and type II IFN signaling; hPIV2 blocked type I but not type II IFN-signaling; and RSV failed to block either type I or type II IFN-signaling. In agreement with these results, SV5 and SeV inhibited the formation of the ISGF3 and GAF transcription complexes (essential for type I and type II signaling, respectively). Surprisingly, although hPIV3 inhibited IFN-induction of the ISGF3 complex, GAF complexes were detected in hPIV3-infected cells. hPIV2 also blocked the formation of the ISGF3 complex but not the GAF complex, whereas RSV failed to block the induction of either complex. SV5 was the only virus that caused the degradation of STAT1. Indeed, in SeV- and hPIV3-infected cells STAT1 was phosphorylated on tyrosine 701 (Y701), a characteristic of IFN receptor activation. However, consistent with these viruses blocking IFN signaling downstream of receptor activation, there was a specific reduction in the levels of serine 727 (S727)-phosphorylated forms of STAT1alpha in SeV- and hPIV3-infected cells. In contrast both (Y701)- and (S727) phosphorylated forms of STAT1 were detected in hPIV2-infected cells but there was a specific loss of STAT2. Both STAT1 (including Y701- and S727-phosphorylated forms) and STAT2 could readily be detected in RSV-infected cells. Despite not being able to block type I or type II IFN signaling, RSV was able to replicate in human cells that produce and respond to IFN, suggesting that RSV must have an alternative method(s) for circumventing the IFN response. These results demonstrate that, although interference with IFN signaling is a common strategy among Paramyxovirinae, distinct virus-specific mechanisms are used to achieve this end. PMID- 10753718 TI - Sendai virosomes revisited: reconstitution with exogenous lipids leads to potent vehicles for gene transfer. AB - A reliable new procedure is described for the reconstitution of Sendai viral envelopes suitable for gene transfer. Both fusion and hemagglutinin-neuraminidase glycoproteins were extracted from purified Sendai virus and reconstituted together with DNA in the presence of cholesterol:sphingomyelin:phosphatidylcholine:phosphatidylethanolamin e (Chol:SM:PC:PE) in a molar ratio of 3.5:3.5:2:1. Before reconstitution, the DNA to be transferred was condensed by pretreatment with polylysine. Exogenous lipid addition and the DNA-condensation step were essential for maximal size as well as for fusogenic activity of the resulting virosomes, the analysis of which revealed (1) the absence of any genomic material originating from Sendai virus, (2) the presence of fusogenic spikes in a functional orientation, (3) the encapsulation of reporter genes, and (4) high-transfer activity for plasmids carrying the green fluorescent protein (GFP) gene and double-stranded nucleotides into different mammalian cells. Transfer rates were up to 10-fold higher than those obtained with different cationic lipids. Gene delivery by means of our lipid-enriched Sendai virosomes extends the known gene transfer strategies, including those based on Sendai virus previously published. PMID- 10753720 TI - Activation of promoters for cellular lipogenic genes by hepatitis B virus large surface protein. AB - Hepatitis B virus large surface protein has the unusual property of accumulating in a particulate form within a preGolgi compartment, leading to marked proliferation of intracellular membranes. We show here that large surface protein activates the promoters for two lipogenic genes that code for farnesyl diphosphate synthase and fatty acid synthase. This activation is transduced, in part, by the transcription factor NF-Y. Although NF-Y is also necessary for the transcriptional induction of chaperone proteins residing in the endoplasmic reticulum by unfolded proteins, other inducers of chaperone synthesis do not activate the promoters for farnesyl diphosphate synthase and fatty acid synthase. Our results suggest the presence of a novel signaling pathway from the endoplasmic reticulum to the nucleus that causes the intracellular membrane proliferation seen in the hepatocytes of persons with accumulated large surface protein particles. PMID- 10753721 TI - Mutations in conserved domains IV and VI of the large (L) subunit of the sendai virus RNA polymerase give a spectrum of defective RNA synthesis phenotypes. AB - The Sendai virus RNA polymerase is a complex of two virus-encoded proteins, the phosphoprotein (P) and the large (L) protein. When aligned with amino acid sequences of L proteins from other negative-sense RNA viruses, the Sendai L protein contains six regions of good conservation, designated domains I-VI, which have been postulated to be important for the various enzymatic activities of the polymerase. To directly address the roles of domains IV and VI, 14 site-directed mutations were constructed either by changing clustered charged amino acids to ala or by substituting selected Sendai L amino acids with the corresponding sequence from measles virus L. Each mutant L protein was tested for its ability to transcribe and replicate the Sendai genome. The series of mutations created a spectrum of phenotypes, from those with significant, near wild-type, activity to those being completely defective for all RNA synthesis. The inactive L proteins, however, were still able to bind P protein and form a polymerase capable of binding the nucleocapsid template. The remainder of the mutations reduced, but did not abolish, enzymatic activity and included one mutant with a specific defect in the synthesis of the leader RNA compared with mRNA, and three mutants that replicated genome RNA much more efficiently in vivo than in vitro. Together, these data suggest that even within a domain, the function of the Sendai L protein is likely to be very complex. In addition, SS3 and SS10 L in domain IV and SS13 L in domain VI were shown to be temperature-sensitive. Both SS3 and SS10 gave significant, although not wild-type, activity at 32 degrees C; however, each was completely inactivated for all RNA synthesis at 37 and 39.6 degrees C. SS13 was completely inactive only when synthesized at the higher temperature. Each polymerase synthesized at 32 degrees C could only be partially heat inactivated in vitro at 39.6 degrees C, suggesting that inactivation involves both thermal lability of the protein and temperature sensitivity for its synthesis. PMID- 10753719 TI - While E1A can facilitate epithelial cell transformation by several dominant oncogenes, the C-terminus seems only to regulate rac and cdc42 function, but in both epithelial and fibroblastic cells. AB - Epithelial and fibroblast cells were differentially susceptible to transformation by oncogenic src, ras, mos, raf, rac, and cdc42 and the influence of adenovirus E1A. In contrast to NIH 3T3 cells, which are easily transformed by all the oncogenes tested, epithelial cells were more resistant to transformation by the same oncogenes. Transformation efficiency of both primary and immortal epithelial cells by E1B, V12ras, v-src, v-raf, and v-mos was increased by cotransfection of E1A 12S, which enables these cells to overcome the M1/M2 mortality blocks, which are not present in NIH 3T3 cells. NIH 3T3 cell transformation by these oncogenes was not altered by E1A. Although V12cdc42 or V12rac1 alone could produce foci on NIH 3T3 cells, morphological conversion was observed only in the presence of a hypertransforming E1A mutant and not WT E1A. Epithelial cells were not transformed by V12cdc42 or V12rac1, even in the presence of WT or mutant E1A, but could be transformed by coexpression of mos/raf and rac/cdc42, and the resultant phenotype was affected by the E1A C-terminus. Hypertransformation, which has previously been reported with ras and E1A C-terminal mutants, turns out to be due to a synergy with rac/cdc42, but not ERK/MAPK or PI3K ras effectors. Like V12rac, expression of the E1A hypertransforming mutant resulted in the upregulation of vinculin and VASP, concomitant with the altered organization of the actin cytoskeleton in these cells. The results show that in addition to requiring abrogation of M1/M2 mortality blocks, primary epithelial cells require activation of the ERK MAPK cascade and rearrangement of the actin CSK to achieve transformation. In addition, the E1A C-terminus regulates rac/cdc42 function in both epithelial and fibroblast cells to affect the extent of transformation progression. PMID- 10753722 TI - A gene encoding a polydnavirus structural polypeptide is not encapsidated. AB - Polydnaviruses are symbiotic viruses associated with some parasitic Hymenoptera that are vertically transmitted as proviruses within wasp genomes. To study this symbiotic association a gene encoding an abundant Campoletis sonorensis polydnavirus virion protein was characterized. This gene is not encapsidated but resides in the wasp genome where it is expressed only during virus replication. Immunolocalization studies detected the encoded 44-kDa protein only in oviduct tissue with ultrastructural studies detecting epitopes between or on virion envelopes. Expression and localization of the 44-kDa protein are consistent with its being a viral structural protein but localization of the gene only within the wasp genome is atypical, raising the possibility that this protein is adventitiously packaged during virion assembly. To address this possibility, quantitative dot blot and genomic Southern blot hybridizations were performed to determine whether the copy number of the p44 gene increased disproportionately during replication, as would be expected for a gene encoding a virion protein. The copy number of the p44 gene increases in tissues supporting virus replication but is unchanged in other tissues, suggesting that this gene is amplified in replicative cells. The data indicate that genes encoding polydnavirus virion proteins may be distributed between wasp and encapsidated viral genomes. PMID- 10753723 TI - Rabbit oral papillomavirus complete genome sequence and immunity following genital infection. AB - Rabbit oral papillomavirus (ROPV) infects mucosal tissues of domestic rabbits. The viral genomic sequence has been determined and the most related papillomavirus type was the cutaneous cottontail rabbit papillomavirus (CRPV). Homologies between the open reading frames (ORFs) of ROPV and CRPV, however, ranged from 68% amino acid identity for L1 to only 23% identity for E4. Shared features unique to the two rabbit viruses included a large E6 ORF and a small E8 ORF that overlapped the E6 ORF. Serological responses to ROPV L1 viruslike particles (VLPs) were detected in rabbits infected at either the genital or oral mucosa with ROPV. The antibody response was specific to intact ROPV L1 VLP antigen, was first detected at the time of late regression, and persisted at high levels for several months after complete regression. Both oral and genital lesions regressed spontaneously, accompanied by a heavy infiltrate of lymphocytes. ROPV infection of rabbit genital mucosa is a useful model to study host immunological responses to genital papillomavirus infections. PMID- 10753724 TI - The 23-kDa protein coded by the 3'-terminal gene of citrus tristeza virus is an RNA-binding protein. AB - The 23-kDa protein (p23), encoded by the 3'-proximal gene of the RNA of Citrus tristeza virus (CTV), was overexpressed in Escherichia coli fused to the maltose binding protein and purified by affinity chromatography. Gel retardation and UV crosslinking assays demonstrated that p23 has the ability to cooperatively bind single-stranded RNA in a non-sequence-specific manner. Formation of the p23-RNA complex was dependent on the conformational state of p23 and on the presence of a basic region, but the complex was stable at high salt concentrations, suggesting that interactions other than those between the negatively charged RNA and the basic region of p23 are involved. Competition assays showed that the affinity of p23 for single-stranded and double-stranded RNA was similar but considerably higher than for single-stranded and double-stranded DNA. By use of a series of artificially generated mutants, the RNA-binding domain of p23 was mapped between positions 50-86, a region containing several basic amino acids and a putative zinc-finger domain. Additional p23-derivatives lacking the conserved residues presumably involved in coordinating the zinc ion showed RNA-binding activity, but with an apparent dissociation constant higher than the wild-type protein. These conserved residues might confer binding specificity or increase binding stability in vivo. Within the Closteroviridae family, p23 is the only protein characterized so far showing RNA-binding activity. PMID- 10753725 TI - Comparison of two single-chain antibodies that neutralize canine parvovirus: analysis of an antibody-combining site and mechanisms of neutralization. AB - We cloned the heavy- and light-chain variable domains of two monoclonal antibodies that recognize each of the two major neutralizing antigenic sites of the canine parvovirus (CPV) capsid. After expression in Escherichia coli as single-chain variable domains (scFv) with glycine-serine linker sequences, both scFv bound CPV capsids with the same specificity as the intact IgG, but with 10- to 20-fold lower avidity. Both scFvs neutralized CPV infectivity with efficiency similar to that of the IgG. Although both IgGs inhibited hemagglutination by CPV, only one scFv was inhibiting. The binding of one of the antibodies has previously been analyzed by cryoelectron microscopic reconstruction and the epitope-binding residues predicted. Mutagenesis of predicted contact residues in three heavy chain complementarity-determining regions (CDR) showed that mutants of CDR1 or CDR3 reduced the binding of the scFv by about 10-fold compared with the wild-type scFv, while no effect was seen for one mutant of CDR2. The levels of neutralization of CPV and of hemagglutination inhibition by the scFv mutants were proportional to their reduction in binding affinity compared with the wild type. Neither scFv blocked virus binding to host cells, but they both caused aggregation of the capsids and appeared to affect the process of infection after virus uptake into the cells. PMID- 10753726 TI - Detection of cervical antibodies to human papillomavirus type 16 (HPV-16) capsid antigens in relation to detection of HPV-16 DNA and cervical lesions. AB - A more sensitive luminescence immunoassay (LIA) for human papillomavirus type 16 (HPV-16) was developed and used to measure HPV-16 antibodies in cervical samples from 292 college-aged women who were examined at 4-month intervals. Of the 609 collected samples, IgG, IgA, and secretory piece-associated antibodies to HPV-16 were detected in 12%, 6%, and 8%, respectively, of samples tested. Cervical IgG antibodies were most strongly associated with HPV-16 DNA detected within the previous 12 months (odds ratio, 3.3; 95% confidence interval, 1.4-7.8). Secretory IgA (cervical IgA- and secretory piece-positive) was most strongly associated with detection of a squamous intraepithelial lesions 4-8 months earlier (odds ratio, 6.4; 95% confidence interval, 1.9-21.8). As with serum HPV-16 antibodies, there appears to be a several-month delay between cervical HPV infection and detection of cervical antibodies. PMID- 10753727 TI - A foodborne outbreak of gastroenteritis associated with Norwalk-like viruses: first molecular traceback to deli sandwiches contaminated during preparation. AB - In March 1998, an outbreak of acute gastroenteritis occurred among students at a Texas university. Overall, 125 ill students sought medical care. Case-control studies revealed that illness was significantly associated with eating foods from the university's main cafeteria deli bar on 9 and 10 March. Stool specimens from 9 (50%) of 18 ill students and samples of deli ham showed evidence of Norwalk like viruses (NLVs) by reverse-transcriptase (RT) polymerase chain reaction (PCR) assay. A food handler who prepared sandwiches for lunch on 9 March reported that her infant had been sick with watery diarrhea since just before the outbreak. A stool sample from the infant was positive for NLV by RT-PCR, and the sequence of the amplified product was identical to that of amplified product from deli ham and students' stool specimens. This is the first time RT-PCR and sequence analysis have successfully confirmed viral contamination of a food item likely to have been contaminated by a food handler. PMID- 10753728 TI - Increased inflammatory responses of persons of blood group O to Helicobacter pylori. AB - Persons of blood group O are at increased risk of peptic ulcers. Enhanced binding of Helicobacter pylori to epithelial cells of persons of blood group O has been demonstrated. Release of interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-alpha by human leukocytes from 40 donors (10 from each ABO blood group) was measured after incubation in vitro with outer membrane protein preparations of H. pylori. Isolates DU (from a patient with a duodenal ulcer), GC (from a patient with gastric cancer), NE (from a patient with normal endoscopic findings), and NCTC 11637 bound in significantly higher numbers to group O leukocytes. Bacterial binding correlated with release of IL-6 and TNF-alpha but not of IL-10. Group O cells released significantly more IL-6 in response to DU, NE, and NCTC 11637, and the cells released more TNF-alpha in response to DU and NCTC 11637. Increased density of colonization of epithelial cells and higher inflammatory responses to H. pylori of persons of blood group O might contribute to increased susceptibility to peptic ulceration. PMID- 10753729 TI - Related strains of Mycobacterium avium cause disease in children with AIDS and in children with lymphadenitis. AB - Sequence analysis of the ribosomal internal transcribed spacer of 56 Mycobacterium avium complex isolates from pediatric patients with AIDS or lymphadenitis revealed (similar to the situation in adults) that the closely related Mav-B and Mav-A sequevars caused the vast majority of disease. IS1245 restriction fragment-polymorphism analysis and pulsed-field gel electrophoresis revealed sets of isolates with closely related patterns among strains from patients in the Boston area and among isolates from Los Angeles and Miami patients. The finding of related strains that cause disease in epidemiologically unrelated patients is most consistent with one of two hypotheses: (1) a limited subset of M. avium strains is more virulent and therefore more likely to cause disease in humans, or (2) pathogenic strains are more prevalent in the environment. PMID- 10753730 TI - Acute otitis media caused by antibiotic-resistant Streptococcus pneumoniae in southern Israel: implication for immunizing with conjugate vaccines. AB - The potential coverage of antibiotic-resistant pneumococci causing acute otitis media (AOM) by 7-, 9-, and 11-valent conjugate pneumococcal vaccines was studied in southern Israel. A total of 876 cases of pneumococcal AOM were studied in the context of various clinical conditions. Of the isolates, 68% were resistant to >/=1 drug, 61% were resistant to penicillin, and 13% were resistant to >/=3 antibiotic classes. Antibiotic resistance and coverage by the various candidates were age and population dependent and were higher among those with a complicated clinical course, as indicated by recent antibiotic use and recurrence of AOM. The results suggest that, if efficacious, the conjugate pneumococcal vaccines can substantially reduce the occurrence of pneumococcal AOM in general and complicated pneumococcal AOM in particular. PMID- 10753731 TI - Randomized controlled trial of Mycobacterium vaccae immunotherapy in non-human immunodeficiency virus-infected ugandan adults with newly diagnosed pulmonary tuberculosis. The Uganda-Case Western Reserve University Research Collaboration. AB - Adjunctive immunotherapy with heat-killed Mycobacterium vaccae was studied in a randomized, placebo-controlled trial of 120 non-human immunodeficiency virus infected adults with newly diagnosed pulmonary tuberculosis. Patients were randomized to a single dose of M. vaccae or placebo 1 week after beginning chemotherapy and were followed up for 1 year. M. vaccae was safe and well tolerated. The rate of sputum culture conversion after 1 month of tuberculosis treatment was 35% in the M. vaccae group and only 14% in the placebo group (P=.01) but was comparable at 2 months and thereafter. Patients receiving M. vaccae had greater improvement on chest radiography at 6 months (91% vs. 77% for placebo recipients; P=.04) and 12 months (94% vs. 80%; P=.04) after initiation of tuberculosis treatment. These data provide evidence of an early increase in sputum culture conversion and greater radiographic improvement among patients who received M. vaccae. Further studies are warranted. PMID- 10753732 TI - Virus load and risk of heterosexual transmission of human immunodeficiency virus and hepatitis C virus by men with hemophilia. The Multicenter Hemophilia Cohort Study. AB - A high human immunodeficiency virus (HIV) load may increase the probability of HIV transmission by sexual contact, but the association of virus load of hepatitis C virus (HCV) with risk of HCV transmission is uncertain. HIV and HCV virus loads were examined in hemophilic men, as were risks of HIV and HCV transmission to their female partners in a hemophilia cohort in which most subjects are dually infected. A higher HIV load was associated with an increased risk of HIV transmission (odds ratio [OR], 1.31 per log10 increase in virus load). A higher HCV load was associated, although not significantly, with an increased risk of HCV transmission (OR, 1. 42 per log10). HCV load was higher among dually infected men than in those infected with HCV alone (P=.001). However, much larger studies are needed to clearly show whether HIV/HCV coinfection significantly increases the risk of HCV transmission to female partners. PMID- 10753733 TI - Risk factors for acute symptomatic coccidioidomycosis among elderly persons in Arizona, 1996-1997. AB - Because of the increase in incidence of coccidioidomycosis among the elderly in Arizona between 1990 and 1996, a case-control study was conducted to look at risk factors for disease among these persons. Cases (n=89) were persons aged > or =60 years with laboratory-confirmed coccidioidomycosis; 2 control groups were selected, the first by use of random-digit dialing (geographic controls, n=91) and the second by use of lists of persons with negative serologic coccidioidomycosis tests (laboratory-negative controls, n=58). Elderly persons with coccidioidomycosis had spent significantly less time in Arizona than did persons in either control group and were more likely than geographic controls to have congestive heart failure or cancer, to have smoked, or to have taken corticosteroids. Elderly persons who recently have moved to Arizona or who have chronic illnesses and their physicians need to be aware of their higher risk for coccidioidomycosis in order to improve their chances of early diagnosis and treatment. These persons may benefit from vaccination, once an effective vaccine for coccidioidomycosis is developed. PMID- 10753734 TI - Coccidioidomycosis in human immunodeficiency virus-infected persons in Arizona, 1994-1997: incidence, risk factors, and prevention. AB - From 1 January 1995 through 31 June 1997, 153 cases of coccidioidomycosis in human immunodeficiency virus (HIV)-infected persons were identified in Arizona (incidence, 41/1000 persons living with AIDS). A case-control study was conducted to evaluate risk factors for coccidioidomycosis in HIV-infected persons. A case was defined as laboratory-confirmed, incident coccidioidomycosis in a person infected with HIV for > or =3 months, and each case patient had 3 control patients matched by county, age group, sex, HIV/AIDS status, and CD4 lymphocyte count. Multivariable analysis identified black race and a history of oropharyngeal or esophageal candidiasis to be associated with increased risk of coccidioidomycosis; protease inhibitor therapy was associated with a reduced risk. In persons with previous history of oropharyngeal or esophageal candidiasis, having received an azole drug was associated with a reduced risk (odds ratio, 0.4; 95% confidence interval, 0.2-0.9; P=.04). Physicians may need to consider azole chemoprophylaxis for HIV-infected persons who live in areas of endemicity, have CD4 cell counts <200/microL, are black, or have a history of thrush. PMID- 10753735 TI - Protein folding: versatility of the cytosolic chaperonin TRiC/CCT. AB - Efficient de novo folding of actins and tubulins requires two molecular chaperones, the chaperonin TRiC (or CCT) and its novel cofactor GimC (or prefoldin). Recent studies indicate that TRiC is exquisitely adapted for this task, yet has the ability to interact with and assist the folding of numerous other cellular proteins. PMID- 10753736 TI - Evolution: to seek out new worlds. AB - A recent study of stickleback 'ecomorphs' generated by independent speciation events in different freshwater lakes suggests that, despite historical contingency, natural selection can run in surprisingly similar ways on multiple occasions. PMID- 10753737 TI - Vision: can colour contribute to motion? AB - Whether colour patterns that have no luminance variation can evoke the perception of visual motion has long been a controversial issue. Recent studies using new and old techniques have now provided compelling evidence that colour can indeed contribute to motion perception. PMID- 10753738 TI - Microbiology: intimate strangers. AB - A more robust view of the diversity of prokaryotes has come from sequencing rRNAs amplified directly from environmental samples. This approach has now been used to examine microbial communities in the human body, revealing populations rich in undescribed species whose impact on humans remains to be determined. PMID- 10753739 TI - Hippocampus formation: an intriguing collaboration. AB - Recent genetic studies have shown that the signalling factor Wnt3a is required for formation of the hippocampus; the developmental consequences of Wnt signalling in the hippocampus are mediated by multiple HMG-box transcription factors, with LEF-1 being required just for formation of the dentate gyrus. PMID- 10753740 TI - Centrosomes: Sic transit gloria centri. AB - Centrosomes are thought to ensure spindle bipolarity and thus correct chromosome segregation during mitosis, but recent studies indicate that somatic cells have an alternative mechanism that enables them to form a bipolar spindle and segregate chromosomes independently of centrosomes. PMID- 10753741 TI - Gamma(delta) T cells: non-classical ligands for non-classical cells. AB - A recent study describes direct binding between a gammadelta T-cell receptor and its ligand, T22, a non-classical class I major histocompatibility complex (MHC) molecule. A companion study, solving the crystal structure of T22, highlights the differences between this interaction and those of classical MHC molecules and alphabeta T cells. PMID- 10753742 TI - Cell polarity: fixing cell polarity with Pins. AB - A protein complex is assembled in a step-wise manner at the apical pole of Drosophila neuroblasts. This complex organizes the apical-basal polarity of asymmetrically dividing neuroblasts, and may act via G-protein signalling. PMID- 10753743 TI - Regulation of N-cadherin-mediated adhesion by the p35-Cdk5 kinase. AB - BACKGROUND: The p35-Cdk5 kinase has been implicated in a variety of functions in the central nervous system (CNS), including axon outgrowth, axon guidance, fasciculation, and neuronal migration during cortical development. In p35(-/-) mice, embryonic cortical neurons are unable to migrate past their predecessors, leading to an inversion of cortical layers in the adult cortex. RESULTS: In order to identify molecules important for p35-Cdk5-dependent function in the cortex, we screened for p35-interacting proteins using the two-hybrid system. In this study, we report the identification of a novel interaction between p35 and the versatile cell adhesion signaling molecule beta-catenin. The p35 and beta-catenin proteins interacted in vitro and colocalized in transfected COS cells. In addition, the p35-Cdk5 kinase was associated with a beta-catenin-N-cadherin complex in the cortex. In N-cadherin-mediated aggregation assays, inhibition of Cdk5 kinase activity using the Cdk5 inhibitor roscovitine led to the formation of larger aggregates of embryonic cortical neurons. This finding was recapitulated in p35( /-) cortical neurons, which aggregated to a greater degree than wild-type neurons. In addition, introduction of active p35-Cdk5 kinase into COS cells led to a decreased beta-catenin-N-cadherin interaction and loss of cell adhesion. CONCLUSIONS: The association between p35-Cdk5 and an N-cadherin adhesion complex in cortical neurons and the modulation of N-cadherin-mediated aggregation by p35 Cdk5 suggests that the p35-Cdk5 kinase is involved in the regulation of N cadherin-mediated adhesion in cortical neurons. PMID- 10753744 TI - Interhemispheric switching mediates perceptual rivalry. AB - BACKGROUND: Binocular rivalry refers to the alternating perceptual states that occur when the images seen by the two eyes are too different to be fused into a single percept. Logothetis and colleagues have challenged suggestions that this phenomenon occurs early in the visual pathway. They have shown that, in alert monkeys, neurons in the primary visual cortex continue to respond to their preferred stimulus despite the monkey reporting its absence. Moreover, they found that neural activity higher in the visual pathway is highly correlated with the monkey's reported percept. These and other findings suggest that the neural substrate of binocular rivalry must involve high levels, perhaps the same levels involved in reversible figure alternations. RESULTS: We present evidence that activation or disruption of a single hemisphere in human subjects affects the perceptual alternations of binocular rivalry. Unilateral caloric vestibular stimulation changed the ratio of time spent in each competing perceptual state. Transcranial magnetic stimulation applied to one hemisphere disrupted normal perceptual alternations when the stimulation was timed to occur at one phase of the perceptual switch, but not at the other. Furthermore, activation of a single hemisphere by caloric stimulation affected the perceptual alternations of a reversible figure, the Necker cube. CONCLUSIONS: Our findings suggest that interhemispheric switching mediates perceptual rivalry. Thus, competition for awareness in both binocular rivalry and reversible figures occurs between, rather than within, each hemisphere. This interhemispheric switch hypothesis has implications for understanding the neural mechanisms of conscious experience and also has clinical relevance as the rate of both types of perceptual rivalry is slow in bipolar disorder (manic depression). PMID- 10753745 TI - MAP kinase signaling induces nuclear reorganization in budding yeast. AB - BACKGROUND: During the mating pheromone response in budding yeast, activation of a mitogen-activated protein kinase (MAP kinase) cascade results in well characterized changes in cytoskeletal organization and gene expression. Spatial reorganization of genes within the nucleus has been documented during cell-type differentiation in mammalian cells, but no information was previously available on the morphology of the yeast nucleus during the major transcriptional reprogramming that accompanies zygote formation. RESULTS: We find that in response to mating pheromone, budding yeast nuclei assume an unusual dumbbell shape, reflecting a spatial separation of chromosomal and nucleolar domains. Within the chromosomal domain, telomeric foci persist and maintain their associated complement of Sir proteins. The nucleolus, on the other hand, assumes a novel cup-shaped morphology and a position distal to the mating projection tip. Although microtubules are required for this orientation with respect to the projection tip, neither microtubules nor actin polymerization are necessary for the observed changes in nuclear shape. We find that activation of the pheromone response MAP kinase pathway by ectopic expression of STE4 or STE11 leads to identical nuclear and nucleolar reorganization in the absence of pheromone. Mutation of downstream effector MAP kinases Fus3p and Kss1p, or of the transcriptional regulator Ste12p, blocks nuclear shape changes, whereas overexpression of Ste12p promotes dumbbell-shaped nuclei in the absence of pheromone. CONCLUSIONS: Nuclear remodeling occurs when the MAP kinase cascade is activated by yeast pheromone, but it is independent of the cytoskeletal reorganization regulated by the same signaling pathway. Activation of the Ste12p transcription factor is necessary, and may be sufficient, for the changes in nuclear structure that coincide with developmentally significant changes in gene expression. PMID- 10753747 TI - Genetic deletion of the Pten tumor suppressor gene promotes cell motility by activation of Rac1 and Cdc42 GTPases. AB - Pten (Phosphatase and tensin homolog deleted on chromosome 10) is a recently identified tumor suppressor gene which is deleted or mutated in a variety of primary human cancers and in three cancer predisposition syndromes [1]. Pten regulates apoptosis and cell cycle progression through its phosphatase activity on phosphatidylinositol (PI) 3,4,5-trisphosphate (PI(3,4,5)P(3)), a product of PI 3-kinase [2-5]. Pten has also been implicated in controlling cell migration [6], but the exact mechanism is not very clear. Using the isogenic Pten(+/+) and Pten( /-) mouse fibroblast lines, here we show that Pten deficiency led to increased cell motility. Reintroducing the wild-type Pten, but not the catalytically inactive Pten C124S or lipid-phosphatase-deficient Pten G129E mutant, reduced the enhanced cell motility of Pten-deficient cells. Moreover, phosphorylation of the focal adhesion kinase p125(FAK) was not changed in Pten(-/-) cells. Instead, significant increases in the endogenous activities of Rac1 and Cdc42, two small GTPases involved in regulating the actin cytoskeleton [7], were observed in Pten( /-) cells. Overexpression of dominant-negative mutant forms of Rac1 and Cdc42 reversed the cell migration phenotype of Pten(-/-) cells. Thus, our studies suggest that Pten negatively controls cell motility through its lipid phosphatase activity by down-regulating Rac1 and Cdc42. PMID- 10753746 TI - A protein complex containing Inscuteable and the Galpha-binding protein Pins orients asymmetric cell divisions in Drosophila. AB - BACKGROUND: In the fruit fly Drosophila, the Inscuteable protein localises to the apical cell cortex in neuroblasts and directs both the apical-basal orientation of the mitotic spindle and the basal localisation of the protein determinants Numb and Prospero during mitosis. Asymmetric localisation of Inscuteable is initiated during neuroblast delamination by direct binding to Bazooka, an apically localised protein that contains protein-interaction motifs known as PDZ domains. How apically localised Inscuteable directs asymmetric cell divisions is unclear. RESULTS: A novel 70 kDa protein called Partner of Inscuteable (Pins) and a heterotrimeric G-protein alpha subunit were found to bind specifically to the functional domain of Inscuteable in vivo. The predicted sequence of Pins contained tetratrico-peptide repeats (TPRs) and motifs implicated in binding Galpha proteins. Pins colocalised with Inscuteable at the apical cell cortex in interphase and mitotic neuroblasts. Asymmetric localisation of Pins required both Inscuteable and Bazooka. In epithelial cells, which do not express inscuteable, Pins was not apically localised but could be recruited to the apical cortex by ectopic expression of Inscuteable. In pins mutants, these epithelial cells were not affected, but neuroblasts showed defects in the orientation of their mitotic spindle and the basal asymmetric localisation of Numb and Miranda during metaphase. Although localisation of Inscuteable in pins mutants was initiated correctly during neuroblast delamination, Inscuteable became homogeneously distributed in the cytoplasm during mitosis. CONCLUSIONS: Pins and Inscuteable are dependent on each other for asymmetric localisation in delaminated neuroblasts. The binding of Pins to Galpha protein offers the intriguing possibility that Inscuteable and Pins might orient asymmetric cell divisions by localising or locally modulating a heterotrimeric G-protein signalling cascade at the apical cell cortex. PMID- 10753748 TI - Fission yeast myosin-II isoforms assemble into contractile rings at distinct times during mitosis. AB - Myosin-II is required for cytokinesis in Schizosaccharomyces pombe [1-3], but unlike other unicellular organisms, S. pombe has two structurally distinct myosin IIs, Myo2p and Myp2p, which are required under different conditions [4]. Disruption of myo2(+) is lethal, whereas disruption of myp2(+) leads to defects in cytokinesis when nutrients are limiting and to cold-sensitivity in 1 M KCl. In dividing cells, both myosin-IIs localize to a ring in the center of the cell, which is thought to contract, separating the cytoplasms of the daughter cells. Using deconvolution microscopy, we obtained three-dimensional reconstructions of fission yeast cells expressing green fluorescent protein-labeled (GFP)-myosin-II, providing for the first time detailed images of GFP-myosin-II rings. By time lapse microscopy, we observed ring assembly and contraction in three dimensions using GFP-tubulin as a cell cycle marker. We determined that the Myo2p ring forms in metaphase/anaphase A whereas the Myp2p ring forms much later, at the end of anaphase B. Myo2p initiates ring formation while Myp2p acts later to increase the efficiency of cytokinesis. PMID- 10753749 TI - A requirement for lipid rafts in B cell receptor induced Ca(2+) flux. AB - Although the major biochemical events triggered by ligation of the B-cell receptor (BCR) have been well defined [1] [2], little is known about the spatio temporal organization of BCR signaling components within the cell membrane and the mechanisms by which signaling specificity is achieved. Partitioning of signaling complexes into specialized domains in the plasma membrane may provide a mechanism for channeling specific stimuli into distinct signaling pathways. Here, we report that multiple tyrosine-phosphorylated proteins accumulate transiently upon BCR activation in detergent-insoluble membrane microdomains known as lipid rafts. We found an activation-dependent translocation to the rafts of the BCR itself, as well as phospholipase Cgamma2 (PLCgamma2), an enzyme critical for BCR induced Ca(2+) flux in B cells. An intact raft structure was required for BCR induced tyrosine phosphorylation of PLCgamma2 and the induction of Ca(2+) flux. Taken together, these data provide a functional role for lipid rafts in BCR signaling. PMID- 10753750 TI - Conformational rearrangements of an archaeal chaperonin upon ATPase cycling. AB - Chaperonins are double-ring protein assemblies with a central cavity that provides a sequestered environment for in vivo protein folding. Their reaction cycle is thought to consist of a nucleotide-regulated alternation between an open substrate-acceptor state and a closed folding-active state. The cavity of ATP charged group I chaperonins, typified by Escherichia coli GroEL [1], is sealed off by a co-chaperonin, whereas group II chaperonins--the archaeal thermosome and eukaryotic TRiC/CCT [2]--possess a built-in lid [3-5]. The mechanism of the lid's rearrangements requires clarification, as even in the absence of nucleotides, thermosomes of Thermoplama acidophilum appear open in vitrified ice [6] and closed in crystals [4]. Here we analyze the conformation of the thermosome at each step of the ATPase cycle by small-angle neutron scattering. The apo chaperonin is open in solution, and ATP binding induces its further expansion. Closure seems to occur during ATP hydrolysis and before phosphate release, and represents the rate-limiting step of the cycle. The same closure can be triggered by the crystallization buffer. Thus, the allosteric regulation of group II chaperonins appears different from that of their group I counterparts. PMID- 10753751 TI - Pericentrin anchors protein kinase A at the centrosome through a newly identified RII-binding domain. AB - Centrosomes orchestrate microtubule nucleation and spindle assembly during cell division [1,2] and have long been recognized as major anchoring sites for cAMP dependent protein kinase (PKA) [3,4]. Subcellular compartmentalization of PKA is achieved through the association of the PKA holoenzyme with A-kinase anchoring proteins (AKAPs) [5,6]. AKAPs have been shown to contain a conserved helical motif, responsible for binding to the type II regulatory subunit (RII) of PKA, and a specific targeting motif unique to each anchoring protein that directs the kinase to specific intracellular locations. Here, we show that pericentrin, an integral component of the pericentriolar matrix of the centrosome that has been shown to regulate centrosome assembly and organization, directly interacts with PKA through a newly identified binding domain. We demonstrate that both RII and the catalytic subunit of PKA coimmunoprecipitate with pericentrin isolated from HEK-293 cell extracts and that PKA catalytic activity is enriched in pericentrin immunoprecipitates. The interaction of pericentrin with RII is mediated through a binding domain of 100 amino acids which does not exhibit the structural characteristics of similar regions on conventional AKAPs. Collectively, these results provide strong evidence that pericentrin is an AKAP in vivo. PMID- 10753752 TI - Assembly of an A kinase-anchoring protein-beta(2)-adrenergic receptor complex facilitates receptor phosphorylation and signaling. AB - Phosphorylation of G-protein-coupled receptors by second-messenger-stimulated kinases is central to the process of receptor desensitization [1-3]. Phosphorylation of the beta(2)-adrenergic receptor (beta(2)-AR) by protein kinase A (PKA), in addition to uncoupling adenylate cyclase activation, is obligatory for receptor-mediated activation of mitogen-activated protein kinase (MAP kinase) cascades [4] [5]. Although mechanisms for linking G-protein-coupled receptor kinases to the activated receptor are well established, analogous mechanisms for targeting second messenger kinases to the beta(2)-AR at the plasma membrane have not been elucidated. Here we show that the A-kinase-anchoring protein, AKAP79/150, co-precipitates with the beta(2)-AR in cell and tissue extracts, nucleating a signaling complex that includes PKA, protein kinase C (PKC) and protein phosphatase PP2B. The anchoring protein directly and constitutively interacts with the beta(2)-AR and promotes receptor phosphorylation following agonist stimulation. Functional studies show that PKA anchoring is required to enhance beta(2)-AR phosphorylation and to facilitate downstream activation of the MAP kinase pathway. This defines a role for AKAP79/150 in the recruitment of second-messenger-regulated signaling enzymes to a G-protein-coupled receptor. PMID- 10753753 TI - Tissue-specific expression and subcellular localisation of mammalian delta tubulin. AB - The properties of the microtubule network are regulated at various levels including tissue-dependent isotype switching, post-translational modification of alpha- and beta-tubulin, and by a variety of microtubule-associated molecules (for reviews, see [1-3]). Microtubule nucleation is attributed to gamma-tubulin, which is present in protein complexes at the centrosome and in the cytoplasm [4,5]. A screen for flagellar mutants in the green alga Chlamydomonas reinhardtii has led to the identification of a fourth member of the tubulin gene superfamily, delta-tubulin. In this unicellular organism, the lack of a functional delta tubulin gene copy causes aberrant numbers of flagella, depending on the age of the corresponding basal bodies; mutants also show abnormal ultrastructure of the basal bodies and a misplacement of the cleavage furrow at mitosis [6]. Here, we report the isolation of the mouse delta-tubulin homologue and show that the gene is highly expressed in testis. In the elongating spermatid, delta-tubulin associated with the manchette, a specialised microtubule system present during reshaping of the sperm head. The protein specifically localised at the perinuclear ring of the manchette, at the centriolar vaults and along the principal piece of the sperm flagellum. In somatic cell lines, unlike most other tubulins, mammalian delta-tubulin was both cytoplasmic and nuclear and did not colocalise with microtubules. The protein was enriched at the spindle poles during mitosis and we found that gamma-tubulin coimmunoprecipitated with delta tubulin. Together, the data indicate a specialised role for mammalian delta tubulin that is distinct from other known tubulins. PMID- 10753754 TI - Flapping ears. PMID- 10753755 TI - Electronic publishing: all talk and no action? PMID- 10753756 TI - New tubulins in protozoal parasites. PMID- 10753757 TI - Too much of a good thing? PMID- 10753758 TI - The cancer research campaign. PMID- 10753759 TI - Proteomic analysis. AB - The field of proteomics is becoming increasingly important as genome sequences are being completed and annotated. Recent advances in proteomics include experimental and mathematical proofs of the need to complement microarray analysis with protein analysis, improved sensitivity for mass spectrometric analysis of separated proteins, better informatic tools for gel analysis and protein spot annotation, first steps towards automated experimental procedures, and new technology for quantitation of protein changes. PMID- 10753760 TI - Genomic analysis. AB - Advances in genomic analysis include improved technology for DNA sequencing, routine use of DNA microarray technology for the analysis of gene expression profiles at the mRNA level and improved informatic tools to organize and analyze such data. At the same time, new developments in chip-based analysis of samples and the emergence of models of gene networks hold promise for the future of the 'Genomic Era'. PMID- 10753761 TI - Mathematical modelling of metabolism. AB - Mathematical models of the cellular metabolism have a special interest within biotechnology. Many different kinds of commercially important products are derived from the cell factory, and metabolic engineering can be applied to improve existing production processes, as well as to make new processes available. Both stoichiometric and kinetic models have been used to investigate the metabolism, which has resulted in defining the optimal fermentation conditions, as well as in directing the genetic changes to be introduced in order to obtain a good producer strain or cell line. With the increasing availability of genomic information and powerful analytical techniques, mathematical models also serve as a tool for understanding the cellular metabolism and physiology. PMID- 10753762 TI - Manipulation of hormone biosynthetic genes in transgenic plants. AB - Modification of plant hormone biosynthesis through the introduction of bacterial genes is a natural form of genetic engineering, which has been exploited in numerous studies on hormone function. Recently, biosynthetic pathways have been largely elucidated for most of the plant hormone classes, and genes encoding many of the enzymes have been cloned. These advances offer new opportunities to manipulate hormone content in order to study their mode of action and the regulation of their biosynthesis. Furthermore, this technology is providing the means to introduce agriculturally useful traits into crops. PMID- 10753763 TI - Metabolic engineering applications to renewable resource utilization. AB - Lignocellulosic materials containing cellulose, hemicellulose, and lignin are the most abundant renewable organic resource on earth. The utilization of renewable resources for energy and chemicals is expected to increase in the near future. The conversion of both cellulose (glucose) and hemicellulose (hexose and pentose) for the production of fuel ethanol is being studied intensively, with a view to developing a technically and economically viable bioprocess. Whereas the fermentation of glucose can be carried out efficiently, the bioconversion of the pentose fraction (xylose and arabinose, the main pentose sugars obtained on hydrolysis of hemicellulose), presents a challenge. A lot of attention has therefore been focused on genetically engineering strains that can efficiently utilize both glucose and pentoses, and convert them to useful compounds, such as ethanol. Metabolic strategies seek to generate efficient biocatalysts (bacteria and yeast) for the bioconversion of most hemicellulosic sugars to products that can be derived from the primary metabolism, such as ethanol. The metabolic engineering objectives so far have focused on higher yields, productivities and expanding the substrate and product spectra. PMID- 10753764 TI - Exploiting the full potential of disease-resistance genes for agricultural use. AB - Effective and sustained control of fungal pathogens and nematodes is an important issue for all agricultural systems. Global losses caused by pathogens are estimated to be 12% of the potential crop production [1], despite the continued release of new resistant cultivars and pesticides. Furthermore, fungi are continually becoming resistant to existing resistance genes and fungicides, and a few of the pesticides are being withdrawn from the market for environmental reasons. In addition to reducing crop yield, fungal diseases often lower crop quality by producing toxins that affect humans and human health. Additional methods of disease control are therefore highly desirable. Breeding programs based on plant disease-resistance genes are being optimized by incorporating molecular marker techniques and biotechnology. These efforts can be expected to result in the first launches of new disease-resistant crops within the next five years. PMID- 10753766 TI - Monitoring genome-wide expression in plants. AB - When completed this year, the Arabidopsis genome will represent the first plant genome to be fully sequenced. This sequence information, together with the large collection of expressed sequence tags, has established the basics for new approaches to studying gene expression patterns in plants on a global scale. We can now look at biology from the perspective of the whole genome. This revolution in the study of how all genes in an organism respond to certain stimuli has encouraged us to think in new dimensions. Expression profiles can be determined over a range of experimental conditions and organized into patterns that are diagnostic for the biological state of the cell. The field of genome-wide expression in plants has yet to produce its fruit; however, the current application of microarrays in yeast and human research foreshadows the diverse applications this technology could have in plant biology and agriculture. PMID- 10753765 TI - Production of viral vectors for gene therapy applications. AB - Advances in cell culture engineering, cell metabolism, bioreactor design and operation, and downstream processing will all positively impact the bioprocessing of viral vectors. Design of appropriate vectors and tailoring of packaging cells to support more productive infections will be of paramount importance for production of high-titer and high-quality vectors. Furthermore, quantitative analysis of the infection parameters during virus propagation, such as time of infection, multiplicity of infection, the length of replication cycle, virus half life, and burst size, will also be important to the process optimization. Finally, procedures for separation, purification and formulation of vector preparations have to be further developed. PMID- 10753767 TI - Control of plant growth and development through manipulation of cell-cycle genes. AB - The plant embryo is a relatively simple structure consisting of a primordial shoot and root, whose development is frozen in the form of a seed. Most development of the mature plant takes place post-embryonically, and is the consequence of cell division and organogenesis in small regions known as meristems, which originate in the embryonic shoot and root apices. Significant recent progress has been made in understanding the mechanisms that control the plant cell cycle at a molecular level, and the first attempts have been made to control plant growth through modulation of cell-cycle genes. These results suggest that there is significant potential to control plant growth and architecture through manipulation of cell division rates. However, a full realisation of the promise of such strategies will probably require a much greater understanding of cell division control and how its upstream regulation is co-ordinated by spatial relationships between cells and by environmental signals. PMID- 10753768 TI - Foreign protein production in plant tissue cultures. AB - Foreign proteins synthesised by plants are now in the marketplace, and clinical trials for plant-derived therapeutic proteins are underway. Economic analysis of plant production systems has helped identify the types of protein that would be most suitable for manufacture using tissue culture methods. The major advantages associated with in vitro plant systems include the ability to manipulate environmental conditions for better control over protein levels and quality, the rapidity of production compared with agriculture, and the use of simpler and cheaper downstream processing schemes for product recovery from the culture medium. PMID- 10753769 TI - Plants for delivery of edible vaccines. AB - Over the past decade, scientific advances in molecular biology and immunology have improved understanding of many diseases and led to the development of novel strategies for vaccination. The development of plants expressing vaccine antigens is a particularly promising approach. Plant-derived antigenic proteins have delayed or prevented the onset of disease in animals and have proven to be safe and functional in human clinical trials. Future areas of research should further characterize the induction of the mucosal immune system and appropriate crop species for delivery of animal and human vaccines. PMID- 10753770 TI - Functional genomics in Arabidopsis: large-scale insertional mutagenesis complements the genome sequencing project. AB - The ultimate goal of genome research on the model flowering plant Arabidopsis thaliana is the identification of all of the genes and understanding their functions. A major step towards this goal, the genome sequencing project, is nearing completion; however, functional studies of newly discovered genes have not yet kept up to this pace. Recent progress in large-scale insertional mutagenesis opens new possibilities for functional genomics in Arabidopsis. The number of T-DNA and transposon insertion lines from different laboratories will soon represent insertions into most Arabidopsis genes. Vast resources of gene knockouts are becoming available that can be subjected to different types of reverse genetics screens to deduce the functions of the sequenced genes. PMID- 10753771 TI - Metabolic engineering and directed evolution for the production of pharmaceuticals. AB - The tools of metabolic and enzyme engineering have been well developed in academic laboratories and are now being applied for the optimization of biocatalysts used in the production of a wide range of pharmaceutically important molecules. Engineered microorganisms with a diverse set of modified or non-native enzyme activities are being used both to generate novel products and to provide improved processes for the manufacture of established products, such as in the production of precursors, intermediates, and complete compounds of importance to the pharmaceutical industry, including polyketides, nonribosomal peptides, steroids, vitamins, and unnatural amino acids. The use of directed evolution has rapidly emerged to be the method of choice for the development and selection of mutated enzymes with improved properties. A variety of such methods have been used to alter the activity, stability and availability of an array of enzymes. The industrial practice of these technologies at large scale is, however, in its infancy and stands as an exciting challenge for process scientists today. PMID- 10753772 TI - RNA silencing. AB - Gene silencing through the increased degradation of mRNA appears to represent a novel cellular pathway that is functional in a broad range of organisms. Recent work has established a role for RNA silencing in host antiviral defense and transposon silencing, suggesting a potential application in plant functional genomics. PMID- 10753773 TI - Chemical-inducible systems for regulated expression of plant genes. AB - Chemical regulation of transgene expression presents a powerful tool for basic research in plant biology and biotechnological applications. Various chemical inducible systems based on de-repression, activation and inactivation of the target gene have been described. The utility of inducible promoters has been successfully demonstrated by the development of a marker-free transformation system and large-scale gene profiling. In addition, field applications appear to be promising through the use of registered agrochemicals (e.g. RH5992) as inducers. PMID- 10753774 TI - Applications of nanotechnology to biotechnology commentary. AB - The ability to systematically modify the properties of nanostructures by controlling their structure and their surface properties at a nanoscale level makes them extremely attractive candidates for use in biological contexts, from fundamental scientific studies to commercially viable technologies. PMID- 10753775 TI - Sloppier copier DNA polymerases involved in genome repair. AB - When chromosomal replication is impeded in the presence of DNA damage, members of a newly discovered UmuC/DinB/Rev1/Rad30 superfamily of procaryotic and eucaryotic DNA polymerases catalyze translesion synthesis at blocked replication forks. Although these polymerases share sequence elements essentially unrelated to the standard replication and repair enzymes, some of them (such as the SOS-induced Escherichia coli pol V) catalyze 'error-prone' translesion synthesis leading to large increases in mutation, whereas others (an example being the Xeroderma pigmentosum variant gene product XPV pol eta) carry out aberrant, yet nonmutagenic translesion synthesis. Ongoing studies of these low fidelity polymerases could provide new insights into the mechanism of somatic hypermutation, a key element in the immune response. PMID- 10753776 TI - The HP1 protein family: getting a grip on chromatin. AB - HP1 was first described in Drosophila as a heterochromatin-associated protein with dosage-dependent effects on heterochromatin-induced gene silencing. Recently, membership of the HP1 protein family has expanded tremendously. A number of intriguing interactions between HP1 and other proteins have been described, implicating HP1 in gene regulation, DNA replication, and nuclear architecture. PMID- 10753777 TI - Replication and recombination intersect. AB - A bacterial housekeeping function, which requires both recombination and replication enzymes, has been identified that re-establishes inactivated replication forks under normal growth conditions. Some long-tract gene-conversion events initiated by double-strand breaks in yeast and mammalian cells can be attributed to recombination-directed DNA replication. Double-strand break repair in yeast has been shown to require both leading- and lagging-strand DNA synthesis. These observations suggest that the recombination and replication machinery cooperate to maintain genomic integrity. PMID- 10753778 TI - Locus control regions and epigenetic chromatin modifiers. AB - Locus control regions are defined as gene regulatory sequences that enable chromosomal position-independent gene expression in transgenic mice. Recent studies have shown the ability of such regions to overcome the highly repressive effect of heterochromatin and have identified both trans-acting and cis-acting factors that participate in gene silencing and activation mechanisms. PMID- 10753779 TI - DNA methylation, a key regulator of plant development and other processes. AB - Recent research has demonstrated that DNA methylation plays an integral role in regulating the timing of flowering and in endosperm development. The identification of key genes controlling these processes, the expression of which is altered in plants with low methylation, opens the way to understanding how DNA methylation regulates plant development. PMID- 10753780 TI - The uniqueness of the imprinting mechanism. AB - An epigenetic imprinting mechanism that is based on a gamete-specific methylation imprint restricts expression of a subset of mammalian genes to one parental chromosome. Recent results suggest that imprints may act only indirectly to induce monoallelic expression of coding genes. Instead, atypical non-coding RNAs appear to be a primary target of the imprints, and their parental-specific repression correlates with parental-specific expression of linked coding genes. PMID- 10753781 TI - mRNA stability in eukaryotes. AB - During the past two years, the role of the proteins HuR and hnRNP D in regulated mRNA degradation in humans has become clearer, and a putative mRNA deadenylase, DAN or PARN, has been identified. In yeast, the relationship between translation and mRNA turnover is clearer, but the mRNA decapping process has turned out to be unexpectedly complex. PMID- 10753782 TI - Dynamics of DNA methylation pattern. AB - Methylation patterns are the result of de novo methylation, demethylation, and the maintenance of existing methylation. Although the existence and identity of an active demethylase remain in doubt, recent evidence suggests that protein binding can specify sites of demethylation through a replication-dependent pathway. By using a stable episomal system in human cells, plus the Drosophila system, and mouse embryonic stem cells, we are beginning to understand the function and targets of de novo methyltransferases in murine and human cells. PMID- 10753783 TI - Making noise about silence: repression of repeated genes in animals. AB - Repeated copies of genes, whether in tandem or dispersed, are often recognized by the cell and silenced. Tandem repeat silencing is associated with a heterochromatin-like complex. Dispersed gene silencing can be mediated by the repressive Polycomb Group complex or involve post-transcriptional silencing presumably involving double-stranded RNA. The I retrotransposable element in Drosophila appears to be susceptible to dispersed gene silencing, potentially by both post-transcriptional and transcriptional processes. Some mutations that eliminate RNA interference in Caenorhabditis elegans result in the mobilization of many transposons and two of these mutations desilence tandem repeats in the germline. One challenge for the future is to determine the nature of any relationship between post-transcriptionally and transcriptionally based mechanisms. The silencing mechanisms potentially act as a protection against high expression of transposons and viruses. PMID- 10753784 TI - Mismatch repair defects in cancer. AB - Post-replicative mismatch repair in humans utilises the hMSH2, hMSH6, hMSH3, hMLH1 and hPMS2 genes and possibly the newly identified hMLH3 gene. Recently, a link has been established between hMSH6 mutations and 'atypical' hereditary non polyposis colon cancer (HNPCC) with an increased incidence of endometrial cancers. To satisfy the need for a diagnostic test capable of differentiating between pathogenic mutations and polymorphisms, several functional assays that fulfil these criteria have been described. These should allow for better diagnosis of HNPCC. PMID- 10753785 TI - Think global, act local--how to regulate S phase from individual replication origins. AB - All eukaryotes use similar proteins to licence replication origins but, paradoxically, origin DNA is much less conserved. Specific binding sites for these proteins have now been identified on fission yeast and Drosophila chromosomes, suggesting that the DNA-binding activity of the origin recognition complex has diverged to recruit conserved initiation factors on polymorphic replication origins. Once formed, competent origins are activated by cyclin- and Dbf4-dependent kinases. The latter have been shown to control S phase in several organisms but, in contrast to cyclin-dependent kinases, seem regulated at the level of individual origins. Global and local regulations generate specific patterns of DNA replication that help establish epigenetic chromosome states. PMID- 10753786 TI - Promoter targeting and chromatin remodeling by the SWI/SNF complex. AB - The SWI/SNF complex is a 2 MDa multi-subunit DNA-dependent ATPase that contributes to the regulation of gene transcription by altering chromatin structure. Recent studies have revealed that the SWI/SNF complex is targeted to promoters via direct interactions with transcription activators and have provided insights into mechanisms by which the complex alters nucleosome structure and contributes to the remodeling of chromatin. PMID- 10753787 TI - DNA double strand break repair in mammalian cells. AB - Human cells can process DNA double-strand breaks (DSBs) by either homology directed or non-homologous repair pathways. Defects in components of DSB repair pathways are associated with a predisposition to cancer. The products of the BRCA1 and BRCA2 genes, which normally confer protection against breast cancer, are involved in homology-directed DSB repair. Defects in another homology directed pathway, single-strand annealing, are associated with genome instability and cancer predisposition in the Nijmegen breakage syndrome and a radiation sensitive ataxia-telangiectasia-like syndrome. Many DSB repair proteins also participate in the signaling pathways which underlie the cell's response to DSBs. PMID- 10753788 TI - Telomere transitions in yeast: the end of the chromosome as we know it. AB - Telomere functions vary as the cell cycle progresses. Recent results highlight fluctuating associations between telomeres and DNA polymerases, DNA-damage repair proteins, and centrosome components. These associations reflect diverse roles of telomeres in chromosome maintenance and in the orchestration of chromosome movements during meiosis. PMID- 10753789 TI - Testing neural network models of memory with behavioral experiments. AB - In recent years, a number of computational neural networks have been proposed aimed at describing memory functions associated with different subregions of the hippocampus, namely dentate gyrus, CA3 and CA1. Recent evidence suggests that indeed specific subregions of the hippocampus may subserve different computational functions, such as spatial and temporal pattern separation, short term or working memory, pattern association, and temporal pattern completion. PMID- 10753790 TI - Schizophrenia and cognitive function. AB - Schizophrenia is often associated with cognitive deficits, particularly within the domains of memory and language. Specific cognitive deficits have recently been linked to psychotic phenomena, including verbal hallucinations and disorganized speech. Impairments of working and semantic memory are primarily due to dysfunction of the frontal cortex, temporal cortex, and hippocampus. Cognitive skills in schizophrenia predict social functioning and may serve as outcome measures in the development of effective treatment strategies. PMID- 10753791 TI - Associative components of recognition memory. AB - Recent results indicate that visual recognition memory (as assessed by habituation and dishabituation of the orienting response) is influenced by associative knowledge, and that this influence is mediated by the hippocampus. A standard, associative model of learning has been recently reported to provide a parsimonious explanation for these results. PMID- 10753792 TI - Linking Hebb's coincidence-detection to memory formation. AB - The theoretical foundations of learning and memory were laid by Donald Hebb 50 years ago. Recent genetic experiments that enhanced coincidence-detection of the NMDA receptor (a molecular master-switch in implementing Hebb's rule) and that led to better learning and memory in adult animals have substantially validated Hebb's rule in memory formation in the brain. PMID- 10753793 TI - Neural representation of visual objects: encoding and top-down activation. AB - Knowledge or experiences are voluntarily recalled from memory by reactivation of their neural representations in the association cortex. Mnemonic representations of visual objects, located in the ventral processing stream of visual perception, provide the best indication of how neuronal codes are created, organized and reactivated. Associative codes are created by neurons that have the ability to link the representations of temporally associated stimuli. Recent experiments suggest that not only bottom-up signals from the retina but also top-down signals from the prefrontal cortex can trigger the retrieval of associative codes, which may serve as a neural basis for conscious recall. PMID- 10753794 TI - Neural aspects of cognitive motor control. AB - Traditionally, motor and cognitive functions were studied separately; however, the investigation of processes at the interface between cognition and action has become more and more popular recently. Typical research goals include the identification of the processes involved using experimental psychological methods, and understanding the neural mechanisms underlying these processes using neurophysiological and functional neuroimaging methods. Specifically, there has been a special emphasis during the past few years on timing mechanisms, practice effects, and the application of rules in guiding action. New information concerning the neural mechanisms involved is being acquired at a rapid pace, albeit mostly within a descriptive framework. With respect to specific brain areas, a key finding has been the clear involvement of the primary motor cortex in complex tasks engaging diverse motor and cognitive dimensions. PMID- 10753795 TI - Changes in memory processing with age. AB - Over the years, a large body of literature has shown that humans display losses in memory with age, but that not all types of memory are affected equally. Similarly, recent evidence from functional neuroimaging experiments has revealed that, depending on the task, older adults can display greater or lesser activity in task-relevant brain areas compared with younger adults. Recent behavioral and neurophysiological experiments are furthering our understanding of the effects of aging on cognition. It appears that some brain changes seen with age may be compensatory. PMID- 10753796 TI - Working memory and executive function: evidence from neuroimaging. AB - Traditional theories of working memory and executive function, when mapped in straightforward ways into the neural domain, yield predictions that are only partly supported by the recent neuroimaging studies. Neuroimaging studies suggest that some constituent functions, such as maintaining information in active form and manipulating it, are not discretely localized in prefrontal regions. Some hypothesized executive processes, such as goal management, have effects in several cortical regions, including posterior regions. Such results suggest a more dynamic and distributed view of the cortical organization of working memory and executive functions. PMID- 10753797 TI - Computational models of association cortex. AB - Recent computational models, or mathematical realizations of neurobiological theories, are providing insights into the organization and workings of the association cortex. Such models concern the construction of cortical maps, the neural basis of cognitive functions such as visual perception, reward-motivated learning and some aspects of consciousness. PMID- 10753798 TI - Natural patterns of activity and long-term synaptic plasticity. AB - Long-term potentiation (LTP) of synaptic transmission is traditionally elicited by massively synchronous, high-frequency inputs, which rarely occur naturally. Recent in vitro experiments have revealed that both LTP and long-term depression (LTD) can arise by appropriately pairing weak synaptic inputs with action potentials in the postsynaptic cell. This discovery has generated new insights into the conditions under which synaptic modification may occur in pyramidal neurons in vivo. First, it has been shown that the temporal order of the synaptic input and the postsynaptic spike within a narrow temporal window determines whether LTP or LTD is elicited, according to a temporally asymmetric Hebbian learning rule. Second, backpropagating action potentials are able to serve as a global signal for synaptic plasticity in a neuron compared with local associative interactions between synaptic inputs on dendrites. Third, a specific temporal pattern of activity--postsynaptic bursting--accompanies synaptic potentiation in adults. PMID- 10753799 TI - Relating unilateral neglect to the neural coding of space. AB - Neuropsychological findings on the human neglect syndrome after parietal damage may relate to the physiological properties of single cells that have been studied in monkey parietal cortex and in related brain areas. Human neglect may reflect partial loss or dysfunction of similar cell populations, producing a pathological gradient in the numbers of cells representing particular lateral positions in space, for particular functions. This can explain the graded deficits seen in patients. We relate the patient deficits to cellular properties for several current issues: spatial frames-of-reference; multimodal integration; effective treatments for neglect; motor components to parietal function; and residual unconscious processing. A neural perspective may resolve traditional debates in the neglect literature and outline directions for future research. PMID- 10753800 TI - Classical fear conditioning in functional neuroimaging. AB - Classical conditioning, the simplest form of associative learning, is one of the most studied paradigms in behavioural psychology. Since the formal description of classical conditioning by Pavlov, lesion studies in animals have identified a number of anatomical structures involved in, and necessary for, classical conditioning. In the 1980s, with the advent of functional brain imaging techniques, particularly positron emission tomography (PET), it has been possible to study the functional anatomy of classical conditioning in humans. The development of functional magnetic resonance imaging (fMRI)--in particular single trial or event-related fMRI--has now considerably advanced the potential of neuroimaging for the study of this form of learning. Recent event-related fMRI and PET studies are adding crucial data to the current discussion about the putative role of the amygdala in classical fear conditioning in humans. PMID- 10753801 TI - Memory trace reactivation in hippocampal and neocortical neuronal ensembles. AB - During active behavior, patterns of hippocampal and neocortical neuronal activity reflect ongoing inputs and their contexts. Recent neurophysiological investigations have shown that during 'off-line' periods, traces of these experiences are spontaneously reactivated in both structures. Although the functional importance of this phenomenon remains to be demonstrated, it does provide clues about the nature and mechanisms of memory retrieval and consolidation. PMID- 10753802 TI - Neurobiology of posttraumatic stress disorder. AB - Recent advances on the neurobiology of posttraumatic stress disorder include: the utilization of functional brain imaging; the incorporation of cross-system research including neuroendocrine (hypothalamic-pituitary-adrenal and hypothalamic-pituitary-thyroid axes), neurochemical (corticotropin-releasing factor, norepinephrine, serotonin, endogenous opiates), and neuroimmunological (humoral and cellular immunity) systems; the expansion beyond exclusive study of combat veterans to include posttraumatic stress disorder patients suffering from noncombat traumas; and the development of animal models of traumatic stress. PMID- 10753803 TI - Transcranial magnetic stimulation in cognitive neuroscience--virtual lesion, chronometry, and functional connectivity. AB - Fifteen years after its introduction by Anthony Barker, transcranial magnetic stimulation (TMS) appears to be 'coming of age' in cognitive neuroscience and promises to reshape the way we investigate brain-behavior relations. Among the many methods now available for imaging the activity of the human brain, magnetic stimulation is the only technique that allows us to interfere actively with brain function. As illustrated by several experiments over the past couple of years, this property of TMS allows us to investigate the relationship between focal cortical activity and behavior, to trace the timing at which activity in a particular cortical region contributes to a given task, and to map the functional connectivity between brain regions. PMID- 10753804 TI - Focusing-in on microtubules. AB - A good approximation of the atomic structure of a microtubule has been derived from docking the high-resolution structure of tubulin, solved by electron crystallography, into lower resolution maps of whole microtubules. Some structural interactions with other molecules, including nucleotides, drugs, motor proteins and microtubule-associated proteins, can now be predicted. PMID- 10753805 TI - Clathrin coat construction in endocytosis. AB - Electron cryomicroscopy of the clathrin coat and X-ray crystallography of parts of the clathrin heavy chain combine to give a detailed picture of the clathrin molecule, assembled as a cage. Recently determined domain structures of other components of the endocytic machinery, particularly the mu2 subunit and the alpha appendage domain of the AP2 adaptor complex, provide important information on the sequence of recognition events involved in the dynamic process of clathrin coat assembly. PMID- 10753806 TI - Membrane protein assemblies - towards atomic resolution analysis. PMID- 10753807 TI - Membrane simulations: bigger and better? AB - Molecular dynamics simulations of biological membranes have come of age. Simulations of pure lipid bilayers are extending our understanding of both optimal simulation procedures and the detailed structural dynamics of lipids in these systems. Simulation methods established using simple bilayer-embedded peptides are being extended to a wide range of membrane proteins and membrane protein models, and are beginning to reveal some of the complexities of membrane protein structural dynamics and their relationship to biological function. PMID- 10753808 TI - Electrostatic aspects of protein-protein interactions. AB - Structural and mutational analyses reveal a central role for electrostatic interactions in protein-protein association. Experiment and theory both demonstrate that clusters of charged and polar residues that are located on protein-protein interfaces may enhance complex stability, although the total effect of electrostatics is generally net destabilizing. The past year also witnessed significant progress in our understanding of the effect of electrostatics on protein association kinetics, specifically in the characterization of a partially desolvated encounter complex. PMID- 10753809 TI - Collective protein dynamics in relation to function. AB - Several techniques for the analysis of the internal motions of proteins are available - separating large collective motions from small, presumably uninteresting motions. Such descriptions are helpful in the characterization of internal motions and provide insight into the energy landscape of proteins. The real challenge, however, is to relate large collective motions to functional properties, such as binding and regulation, or to folding. These issues have been recently addressed in several papers. PMID- 10753810 TI - Dis-assembly lines: the proteasome and related ATPase-assisted proteases. AB - Self-compartmentalizing proteases, such as the proteasome and several prokaryotic energy-dependent proteases, are designed to act in the crowded environment of the cell. Proteins destined for degradation are recognized and unfolded by regulatory subcomplexes that invariably contain ATPase modules, before being translocated into another subcomplex, the proteolytic core, for degradation. The sequential actions effected on substrates are reflected in the linear arrangement of these subcomplexes; thus, the holocomplexes are organized as molecular disassembly and degradation lines. PMID- 10753812 TI - Interpretation of chemical shifts and coupling constants in macromolecules. AB - Recent developments in NMR spectroscopy, along with advances in computational techniques, have produced new approaches to the interpretation of chemical shifts and spin-spin coupling constants in biomolecules. Quantum chemical studies of useful accuracy are now becoming more routine and are increasingly being used in conjunction with experimental studies to map out expected structural patterns for peptides and oligonucleotides. Topics of recent special interest include spin couplings across hydrogen bonds and patterns of chemical shift anisotropies, in both diamagnetic and paramagnetic proteins. PMID- 10753811 TI - Effective energy functions for protein structure prediction. AB - Protein structure prediction, fold recognition, homology modeling and design rely mainly on statistical effective energy functions. Although the theoretical foundation of such functions is not clear, their usefulness has been demonstrated in many applications. Molecular mechanics force fields, particularly when augmented by implicit solvation models, provide physical effective energy functions that are beginning to play a role in this area. PMID- 10753813 TI - Theoretical studies of viral capsid proteins. AB - Recent results in structural biology and increases in computer power have prompted initial theoretical studies on capsids of nonenveloped icosahedral viruses. The macromolecular assembly of 60 to 180 protein copies into a protein shell results in a structure of considerable size for molecular dynamics simulations. Nonetheless, progress has been made in examining these capsid assemblies from molecular dynamics calculations and kinetic models. The goals of these studies are to understand capsid function and structural properties, including quarternary structural stability, effects of antiviral compounds that bind the capsid and the self-assembly process. The insight that can be gained from the detailed information provided by simulations is demonstrated in studies of human rhinovirus; an entropic basis for the antiviral activity of hydrophobic compounds, predicted from calculated compressibility values, has been corroborated by experimental measurements on poliovirus. PMID- 10753815 TI - Ab initio protein folding. AB - Ab initio protein folding methods have been developing rapidly over the past few years and, at the last Critical assessment of methods of protein structure prediction (CASP) meeting, it was shown that important progress has been made in generating structure from sequence. Both methods based on statistical potentials and methods using physics-based potentials have shown improvements. Most current methods use statistics-based potentials and the development of these is ongoing. Additionally, the inclusion of multiple sequence data in the algorithms in order to aid in finding the native structure is a common theme. The use of physics based potentials is less developed, which means that less progress has been made in understanding why a sequence forms a structure. PMID- 10753814 TI - Structures of virus and virus-like particles. AB - Virus structures continue to be the basis for mechanistic virology and serve as a paradigm for solutions to problems concerning macromolecular assembly and function in general. The use of X-ray crystallography, electron cryomicroscopy and computational and biochemical methods has provided not only details of the structural folds of individual viral components, but also insights into the structural basis of assembly, nucleic acid packaging, particle dynamics and interactions with cellular molecules. PMID- 10753816 TI - Nucleic acids: theory and computer simulation, Y2K. AB - Molecular dynamics simulations on DNA and RNA that include solvent are now being performed under realistic environmental conditions of water activity and salt. Improvements to force-fields and treatments of long-range interactions have significantly increased the reliability of simulations. New studies of sequence effects, axis bending, solvation and conformational transitions have appeared. PMID- 10753817 TI - Cell surface receptors. AB - During the past year, the database of ligand-receptor complexes has essentially doubled. These new results have immeasurably extended and expanded our view of cell surface receptor structure and function. The flood of data has revealed new models for receptor cross-linking, demonstrating the potential stringency of the extracellular requirements for the initiation of intracellular signalling and highlighting unexpected interactions suggestive of higher order clustering. PMID- 10753818 TI - Unveiling ribosomal structures: the final phases. AB - Unprecedented insights into the structure of the ribosome have been gained recently: X-ray crystallographic studies have yielded 5-9 A resolution structures and cryo-electron microscopy has elucidated the structure of the Escherichia coli ribosome in different functional states. A 7.5 A cryo-electron microscopy structure of the large subunit indicates that this technique is still in the race to determine the ribosome structure. PMID- 10753819 TI - Long timescale simulations. AB - Computers are becoming increasingly fast, making it possible to perform simulations of macromolecules on timescales that were previously inaccessible. Questions have arisen concerning how well we are keeping up with computer power and the state of the art with respect to long molecular dynamics simulations in solvent. More importantly, however, simulations of macromolecules are performed to aid the understanding of biochemical phenomena. So, what are we learning from longer simulations and are they providing reliable insight into protein dynamics, conformational behavior and function? PMID- 10753820 TI - Molecular chaperones: containers and surfaces for folding, stabilising or unfolding proteins. AB - Newly solved chaperone structures include the thermosome, a group II chaperonin, and a small heat-shock protein. Novel ideas on chaperone mechanism are presented in the forced unfolding hypothesis of GroEL action. Structures of chaperone-pilin complexes reveal the mechanism of chaperone interaction in bacterial pilus assembly and there have been major advances in understanding the structure and function of Hsp100 unfoldases. PMID- 10753821 TI - Introduction: functional polarity of motile neutrophils. PMID- 10753822 TI - Neutrophil polarity and locomotion are associated with surface redistribution of leukosialin (CD43), an antiadhesive membrane molecule. AB - This study analyzed the behavior of an antiadhesive membrane molecule, CD43, in neutrophil polarization and locomotion. CD43 cross-linking by antibodies induced neutrophil locomotion, with CD43 molecules clustered at the uropod of polarized neutrophils. In contrast, CD11b/CD18 cross-linking by antibodies did not affect either cell polarization or locomotion. Stimulation of suspended or adherent neutrophils with chemotactic peptide results in cell polarization and locomotion and a concomitant redistribution of CD43 to the uropod. This process is entirely reversible. The study also investigated which actin-binding protein could be involved in CD43 lateral redistribution. alpha-Actinin and moesin are preferentially adsorbed on Sepharose beads bearing a recombinant CD43 intracellular domain. Analysis by immunofluorescence confocal microscopy shows a codistribution of moesin during CD43 lateral redistribution. By contrast, alpha actinin is located at the leading edge, an area devoid of CD43. These results shed new light on the role of CD43 membrane redistribution, which appears to be directly related to neutrophil polarity and locomotion. (Blood. 2000;95:2462 2470) PMID- 10753823 TI - Oriented endocytic recycling of alpha5beta1 in motile neutrophils. AB - During cell migration, integrin attachments to the substratum provide the means to generate the traction and force necessary to achieve locomotion. Once the cell has moved over these attachments, however, it is equally important that integrins detach from the substratum. The fate of integrins after detachment may include release from the cell, lateral diffusion across the cell surface, or endocytosis and redelivery to the cell surface. Polymorphonuclear neutrophils (PMNs) become stuck on the extracellular matrix proteins fibronectin and vitronectin when their intracellular free calcium concentration ([Ca(++)]i) is buffered. Taking advantage of this feature of PMN migration, we investigated the fate of integrins to differentiate among various models of migration. We demonstrate that alpha5beta1, one of the fibronectin-binding integrins, is responsible for immobilization of [Ca(++)](i)-buffered PMNs on fibronectin. We find that alpha5 and beta1 are in endocytic vesicles in PMNs and that alpha5 colocalizes with a marker for an endocytic recycling compartment. When [Ca(++)](i) is buffered, alpha5 and beta1 become concentrated in clusters in the rear of the adherent cells, suggesting that [Ca(++)](i) transients are required for alpha5beta1 detachment from the substratum. Inhibition of alpha5beta1 detachment by buffering [Ca(++)](i) results in the depletion of alpha5 from both endocytic vesicles and the recycling compartment, providing compelling evidence that integrins are normally recycled by way of endocytosis and intracellular trafficking during cell migration. This model is further refined by our demonstration that the endocytic recycling compartment reorients to retain its localization just behind the leading lamella as PMNs migrate, indicating that membrane recycling during neutrophil migration has directionality. (Blood. 2000;95:2471-2480) PMID- 10753824 TI - Introduction: TH2-inducing DC2 for immunotherapy. PMID- 10753825 TI - Granulocyte-colony stimulating factor mobilizes T helper 2-inducing dendritic cells. AB - Peripheral blood stem cells (PBSC) obtained from granulocyte-colony stimulating factor (G-CSF)-mobilized donors are increasingly used for allogeneic transplantation. Despite a 10-fold higher dose of transplanted T cells, acute graft-versus-host disease (GVHD) does not develop in higher proportion in recipients of PBSC than in recipients of marrow. T cells from G-CSF-treated experimental animals preferentially produce IL-4 and IL-10, cytokines characteristic of Th2 responses, which are associated with diminished GVHD inducing ability. We hypothesized that G-CSF-mobilized PBSC contain antigen presenting cells, which prime T-lymphocytes to produce Th2 cytokines. Two distinct lineages of dendritic cells (DC) have been described in humans, DC1 and DC2, according to their ability to induce naive T-cell differentiation to Th1 and Th2 effector cells, respectively. We have used multicolor microfluorometry to enumerate DC1 and DC2 in the peripheral blood of normal donors. G-CSF treatment with 10 to 16 microg/kg per day for 5 days increased peripheral blood DC2 counts from a median of 4.9 x 10(6)/L to 24.8 x 10(6)/L (P =.0009), whereas DC1 counts did not change. Purified DC1, from either untreated or G-CSF treated donors, induced the proliferation of allogeneic naive T cells, but fresh DC2 were poor stimulators. Tumor necrosis factor-alpha (TNF-alpha)-activated DC1 induced allogeneic naive T cells to produce IFN-gamma, which is typical of Th1 responses, whereas TNF-alpha-activated DC2 induced allogeneic naive T cells to produce IL-4 and IL-10, which are typical of Th2 responses. PBSC transplants contained higher doses of DC2 than marrow transplants (median, 2.4 x 10(6)/kg versus 0.5 x 10(6)/kg) (P =.006), whereas the dose of DC1 was comparable. Thus, it is conceivable that transplantation of G-CSF-stimulated PBSC does not result in overwhelming acute GVHD because the graft contains predominantly Th2-inducing DC. Adoptive transfer of purified DC2 may be exploited to induce immune deviation after transplantation of hematopoietic stem cells or organ allografts. (Blood. 2000;95:2484-2490) PMID- 10753826 TI - Structure of the activation domain of the GM-CSF/IL-3/IL-5 receptor common beta chain bound to an antagonist. AB - Heterodimeric cytokine receptors generally consist of a major cytokine-binding subunit and a signaling subunit. The latter can transduce signals by more than 1 cytokine, as exemplified by the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and IL-6 receptor systems. However, often the signaling subunits in isolation are unable to bind cytokines, a fact that has made it more difficult to obtain structural definition of their ligand-binding sites. This report details the crystal structure of the ligand-binding domain of the GM-CSF/IL-3/IL-5 receptor beta-chain (beta(c)) signaling subunit in complex with the Fab fragment of the antagonistic monoclonal antibody, BION-1. This is the first single antagonist of all 3 known eosinophil-producing cytokines, and it is therefore capable of regulating eosinophil-related diseases such as asthma. The structure reveals a fibronectin type III domain, and the antagonist-binding site involves major contributions from the loop between the B and C strands and overlaps the cytokine-binding site. Furthermore, tyrosine(421) (Tyr(421)), a key residue involved in receptor activation, lies in the neighboring loop between the F and G strands, although it is not immediately adjacent to the cytokine-binding residues in the B-C loop. Interestingly, functional experiments using receptors mutated across these loops demonstrate that they are cooperatively involved in full receptor activation. The experiments, however, reveal subtle differences between the B-C loop and Tyr(421), which is suggestive of distinct functional roles. The elucidation of the structure of the ligand-binding domain of beta(c) also suggests how different cytokines recognize a single receptor subunit, which may have implications for homologous receptor systems. (Blood. 2000;95:2491-2498) PMID- 10753827 TI - Development of lentiviral vectors for gene therapy for human diseases. AB - Retroviral vectors derived from murine retroviruses are being used in several clinical gene therapy trials. Recently, progress has been made in the development of vectors based on the lentivirus genus of retroviruses, which ironically includes a major human pathogen, human immunodeficiency virus (HIV). As these vector systems for clinical gene transfer are developed, it is important to understand the rationale behind their design and development. This article reviews the fundamental features of retrovirus replication and of the elements necessary for development of a retroviral vector system, and it discusses why vector systems based on HIV or other lentiviruses have the potential to become important tools in clinical gene therapy. (Blood. 2000;95:2499-2504) PMID- 10753828 TI - Stromal cell-derived factor-1alpha stimulates tyrosine phosphorylation of multiple focal adhesion proteins and induces migration of hematopoietic progenitor cells: roles of phosphoinositide-3 kinase and protein kinase C. AB - The stromal cell-derived factor-1 (SDF-1) is an alpha chemokine that binds to the CXCR4 receptor. Knock-out studies in mice demonstrate that this ligand-receptor pair is essential in hematopoiesis. One function of SDF-1 appears to be the regulation of migration of hematopoietic progenitor cells. We previously characterized signal transduction pathways induced by SDF-1alpha in human hematopoietic progenitors and found tyrosine phosphorylation of focal adhesion components, including the related adhesion focal tyrosine kinase (RAFTK), the adaptor molecule p130 Cas, and the cytoskeletal protein paxillin. To better understand the functional role of signaling molecules connecting the CXCR4 receptor to the process of hematopoietic migration, we studied SDF-1alpha mediated pathways in a model hematopoietic progenitor cell line (CTS), as well as in primary human bone marrow CD34(+) cells. We observed that several other focal adhesion components, including focal adhesion kinase (FAK) and the adaptor molecules Crk and Crk-L, are phosphorylated on SDF-1alpha stimulation. Using a series of specific small molecule inhibitors, both protein kinase C (PKC) and phosphoinositide-3 kinase (PI-3K) appeared to be required for SDF-1alpha-mediated phosphorylation of focal adhesion proteins and the migration of both CTS and primary marrow CD34(+) cells, whereas the mitogen-activated protein kinases ERK-1 and -2 were not. These studies further delineate the molecular pathways mediating hematopoietic progenitor migration and response to an essential chemokine, SDF 1alpha. (Blood. 2000;95:2505-2513) PMID- 10753829 TI - Effects of megakaryocyte growth and development factor on platelet production, platelet life span, and platelet function in healthy human volunteers. AB - The effects of thrombopoietic stimulation on megakaryocytopoiesis, platelet production, and platelet viability and function were examined in normal volunteers randomized to receive single bolus subcutaneous injections of 3 microg/kg pegylated recombinant megakaryocyte growth and development factor (PEG rHuMGDF) or placebo in a 3:1 ratio. PEG-rHuMGDF transiently doubled circulating platelet counts, from 237 +/- 41 x 10(3)/microL to 522 +/- 90 x 10(3)/microL (P <.0001), peaking on day 12. Baseline and day-12 samples showed no differences in responsiveness of platelets to adenosine diphosphate or thrombin receptor agonist peptide (P >.4 in all cases); expression of platelet ligand-induced binding sites or annexin V binding sites (P >.6 in both cases); or density of platelet TPO receptors (P >.5). Platelet counts normalized by day 28. The life span of autologous (111)In-labeled platelets increased from 205 +/- 18 hours (baseline) to 226 +/- 22 hours (P <.01) on day 8. Platelet life span decreased from 226 +/- 22 hours (day 8) to 178 +/- 53 hours (P <.05) on day 18. The theoretical basis for senescent changes in mean platelet life span was illustrated by biomathematical modeling. Platelet turnover increased from 43.9 +/- 11.9 x 10(3) platelets/microL/d (baseline) to 101 +/- 27.6 x 10(3) platelets/microL/d (P =.0009), and marrow megakaryocyte mass expanded from 37.4 +/- 18.5 fL/kg to 62 +/ 17 x 10(10) fL/kg (P =. 015). Although PEG-rHuMGDF initially increased megakaryocyte volume and ploidy, subsequently ploidy showed a transient reciprocal decrease when the platelet counts exceeded placebo values. In healthy human volunteers PEG-rHuMGDF transiently increases megakaryocytopoiesis 2-fold. Additionally, peripheral platelets expand correspondingly and exhibit normal function and viability during the ensuing 10 days. The induced perturbation in steady state thrombopoiesis resolves by 4 weeks. (Blood. 2000;95:2514-2522) PMID- 10753830 TI - Acute onset hemoglobinemia and/or hemoglobinuria and sequelae following Rh(o)(D) immune globulin intravenous administration in immune thrombocytopenic purpura patients. AB - Rh(o)(D) immune globulin intravenous (anti-D IGIV) was licensed by the United States Food and Drug Administration (FDA) in March 1995 to treat patients with immune thrombocytopenic purpura (ITP). Anti-D IGIV induces extravascular hemolysis, an expected adverse reaction that is consistent with the presumed mechanism of action. Between licensure and April 1999, the FDA received 15 reports of hemoglobinemia and/or hemoglobinuria following anti-D IGIV administration that met the case definition for this review. The mechanism responsible for hemoglobinemia and/or hemoglobinuria is unexplained. Review of these reports was prompted by the seriousness and the unexpectedness of treatment associated sequelae experienced by 11 patients. Of these patients, 7 developed sufficient onset or exacerbation of anemia that orders were written for packed red blood cell transfusions, although only 6 patients were transfused. Eight patients experienced the onset or exacerbation of renal insufficiency, and 2 patients underwent dialysis. One patient died due to complications of exacerbated anemia. Six patients experienced 2 to 3 sequelae. Absent validated incidence data, a 1.5% estimated incidence rate from published clinical trial data and a 0.1% estimated reporting rate from FDA and drug utilization data were calculated for reported cases of hemoglobinemia and/or hemoglobinuria. This review presents the first case series of anti-D-IGIV-associated hemoglobinemia and/or hemoglobinuria and provides pretreatment and posttreatment clinical and laboratory findings of the case series patients. The primary purpose of this review is to increase awareness of this potentially serious occurrence among physicians and health care professionals who manage ITP patients treated with anti-D IGIV, thereby enabling prompt recognition and treatment of sequelae. (Blood. 2000;95:2523-2529) PMID- 10753831 TI - A double-blind, placebo-controlled trial of pegylated recombinant human megakaryocyte growth and development factor as an adjunct to induction and consolidation therapy for patients with acute myeloid leukemia. AB - Newly diagnosed patients with acute myeloid leukemia (AML) were randomized to receive either 2.5 or 5 microg/kg/day of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) or a placebo administered subcutaneously after completion of chemotherapy. The study evaluated the toxicity of PEG-rHuMGDF and any effect on the duration of thrombocytopenia. Each of 35 patients under 60 years of age received the following therapy: 45 mg/m(2) daunorubicin on days 1-3, 100 mg/m(2) cytarabine (ARA-C) for 7 days, and 2 gm/m(2) high-dose ARA-C (HIDAC) for 6 doses on days 8-10. The 22 patients 60 years or older received standard daunorubicin and ARA-C without HIDAC. PEG rHuMGDF was well tolerated, and no specific toxicities could be attributed to its use. There was no difference in the time to achieve a platelet count of at least 20 x 10(9)/L among the 3 groups (median 28-30 days for patients less than 60 years old and 21-23 days for patients 60 years or older). Patients receiving PEG rHuMGDF achieved higher platelet counts after remission. However there was no significant difference in the number of days on which platelet transfusions were administered among the 3 groups. The complete remission rate was 71% for patients less than 60 years and 64% for those 60 years or older, with no significant difference among the 3 groups. Postremission consolidation chemotherapy with either placebo or PEG-rHuMGDF was given to 28 patients beginning the day after completion of chemotherapy. There was no apparent difference in the time that was necessary to reach a platelet count of at least 20 or 50 x 10(9)/L or more platelets or in the number of platelet transfusions received. In summary, PEG rHuMGDF was well tolerated by patients receiving induction and consolidation therapy for AML; however, there was no effect on the duration of severe thrombocytopenia or the platelet transfusion requirement. (Blood. 2000;95:2530 2535) PMID- 10753832 TI - Improved muscle-derived expression of human coagulation factor IX from a skeletal actin/CMV hybrid enhancer/promoter. AB - Hemophilia B is caused by the absence of functional coagulation factor IX (F.IX) and represents an important model for treatment of genetic diseases by gene therapy. Recent studies have shown that intramuscular injection of an adeno associated viral (AAV) vector into mice and hemophilia B dogs results in vector dose-dependent, long-term expression of biologically active F.IX at therapeutic levels. In this study, we demonstrate that levels of expression of approximately 300 ng/mL (6% of normal human F.IX levels) can be reached by intramuscular injection of mice using a 2- to 4-fold lower vector dose (1 x 10(11) vector genomes/mouse, injected into 4 intramuscular sites) than previously described. This was accomplished through the use of an improved expression cassette that uses the cytomegalovirus (CMV) immediate early enhancer/promoter in combination with a 1.2-kilobase portion of human skeletal actin promoter. These results correlated with enhanced levels of F.IX transcript and secreted F.IX protein in transduced murine C2C12 myotubes. Systemic F.IX expression from constructs containing the CMV enhancer/promoter alone was 120 to 200 ng/mL in mice injected with 1 x 10(11) vector genomes. Muscle-specific promoters performed poorly for F.IX transgene expression in vitro and in vivo. However, the incorporation of a sequence from the alpha-skeletal actin promoter containing at least 1 muscle specific enhancer and 1 enhancer-like element further improved muscle-derived expression of F.IX from a CMV enhancer/promoter-driven expression cassette over previously published results. These findings will allow the design of a clinical protocol for therapeutic levels of F.IX expression with lower vector doses, thus enhancing efficacy and safety of the protocol. (Blood. 2000;95:2536-2542) PMID- 10753833 TI - GATA-1 interacts with the myeloid PU.1 transcription factor and represses PU.1 dependent transcription. AB - The GATA-1 transcription factor is capable of suppressing the myeloid gene expression program when ectopically expressed in myeloid cells. We examined the ability of GATA-1 to repress the expression and function of the PU.1 transcription factor, a central regulator of myeloid differentiation. We found that GATA-1 is capable of suppressing the myeloid phenotype without interfering with PU.1 gene expression, but instead was capable of inhibiting the activity of the PU.1 protein in a dose-dependent manner. This inhibition was independent of the ability of GATA-1 to bind DNA, suggesting that it is mediated by protein protein interaction. We examined the ability of PU.1 to interact with GATA-1 and found a direct interaction between the PU.1 ETS domain and the C-terminal finger region of GATA-1. Replacing the PU.1 ETS domain with the GAL4 DNA-binding domain removed the ability of GATA-1 to inhibit PU.1 activity, indicating that the PU.1 DNA-binding domain, rather than the transactivation domain, is the target for GATA-1-mediated repression. We therefore propose that GATA-1 represses myeloid gene expression, at least in part, through its ability to directly interact with the PU.1 ETS domain and thereby interfere with PU.1 function. (Blood. 2000;95:2543-2551) PMID- 10753834 TI - Granulocyte-macrophage colony-stimulating factor stimulation results in phosphorylation of cAMP response element-binding protein through activation of pp90RSK. AB - Granulocyte-macrophage colony-stimulating factor (GM-CSF) activates several kinases and transcription factors through interaction with a heterodimeric receptor complex. We previously demonstrated that phosphorylation of the cyclic adenosine monophosphate (cAMP) response element-binding protein, CREB, occurs through a protein kinase A-independent pathway and is required for GM-CSF-induced transcriptional activation of the immediate early gene, early growth response-1 (egr-1). Recent reports indicate that receptor tyrosine kinases can induce CREB phosphorylation through activation of pp90RSK. We performed immune complex kinase assays in the human myeloid leukemic cell line, TF-1, which revealed that GM-CSF induced pp90RSK activation and phosphorylation of CREB within 5 minutes of stimulation. Transfection with the kinase-defective pp90RSK expression plasmid demonstrated a statistically significant decrease in transcriptional activation of a -116 CAT/egr-1 promoter construct in response to GM-CSF. Furthermore, activation of pp90RSK, CREB and egr-1 in GM-CSF-treated cells was inhibited by the presence of the inhibitor, PD98059. In this study, we report that GM-CSF induces CREB phosphorylation and egr-1 transcription by activating pp90RSK through an MEK-dependent signaling pathway. (Blood. 2000;95:2552-2558) PMID- 10753835 TI - Identification and characterization of a bipotent (erythroid and megakaryocytic) cell precursor from the spleen of phenylhydrazine-treated mice. AB - We have identified a cell population expressing erythroid (TER-119) and megakaryocyte (4A5) markers in the bone marrow of normal mice. This population is present at high frequency in the marrows and in the spleens involved in the erythroid expansion that occurs in mice recovering from phenylhydrazine (PHZ) induced hemolytic anemia. TER-119(+)/4A5(+) cells were isolated from the spleen of PHZ-treated animals and were found to be blast-like benzidine-negative cells that generate erythroid and megakaryocytic cells within 24-48 hours of culture in the presence of erythropoietin (EPO) or thrombopoietin (TPO). TER-119(+)/4A5(+) cells represent a late bipotent erythroid and megakaryocytic cell precursors that may exert an important role in the recovery from PHZ-induced anemia. (Blood. 2000;95:2559-2568) PMID- 10753836 TI - Cloning of PRV-1, a novel member of the uPAR receptor superfamily, which is overexpressed in polycythemia rubra vera. AB - Polycythemia vera (PV) is a clonal stem cell disorder characterized by hyperproliferation of the erythroid, myeloid, and megakaryocytic lineages. Although it has been shown that progenitor cells of patients with PV are hypersensitive to several growth factors, the molecular pathogenesis of this disease remains unknown. To investigate the molecular defects underlying PV, we used subtractive hybridization to isolate complementary DNAs (cDNAs) differentially expressed in patients with PV versus normal controls. We isolated a novel gene, subsequently named PRV-1, which is highly expressed in granulocytes from patients with PV (n = 19), but not detectable in normal control granulocytes (n = 21). Moreover, PRV-1 is not expressed in mononuclear cells from patients with chronic myelogenous leukemia (n = 4) or acute myelogenous leukemia (n = 5) or in granulocytes from patients with essential thrombocythemia (n = 4) or secondary erythrocytosis (n = 4). Northern blot analysis showed that PRV-1 is highly expressed in normal human bone marrow and to a much lesser degree in fetal liver. It is not expressed in a variety of other tissues tested. Although PRV-1 is not expressed in resting granulocytes from normal controls, stimulation of these cells with granulocyte colony-stimulating factor induces PRV-1 expression. The PRV-1 cDNA encodes an open reading frame of 437 amino acids, which contains a signal peptide at the N-terminus and a hydrophobic segment at the C-terminus. In addition, PRV-1 contains 2 cysteine-rich domains homologous to those found in the uPAR/Ly6/CD59/snake toxin-receptor superfamily. We therefore propose that PRV-1 represents a novel hematopoietic receptor. (Blood. 2000;95:2569-2576) PMID- 10753837 TI - Induction of apoptosis by extracellular ubiquitin in human hematopoietic cells: possible involvement of STAT3 degradation by proteasome pathway in interleukin 6 dependent hematopoietic cells. AB - The ubiquitin-proteasome pathway is responsible for selective degradation of short-lived cellular proteins and is critical for the regulation of many cellular processes. We previously showed that ubiquitin (Ub) secreted from hairy cell leukemia cells had inhibitory effects on clonogenic growth of normal hematopoietic progenitor cells. In this study, we examined the effects of exogenous Ub on the growth and survival of a series of human hematopoietic cells, including myeloid cell lines (HL-60 and U937), a B-cell line (Daudi), and T-cell lines (KT-3, MT-4, YTC-3, and MOLT-4). Exogenous Ub inhibited the growth of various hematopoietic cell lines tested, especially of KT-3 and HL-60 cells. The growth-suppressive effects of Ub on KT-3 and HL-60 cells were almost completely abrogated by the proteasome inhibitor PSI or MG132, suggesting the involvement of the proteasome pathway in this process. Furthermore, exogenous Ub evoked severe apoptosis of KT-3 and HL-60 cells through the activation of caspase-3. In interleukin-6 (IL-6)-dependent KT-3 cells, STAT3 was found to be conjugated by exogenous biotinylated Ub and to be degraded in a proteasome-dependent manner, whereas expression levels of STAT1, STAT5, or mitogen-activated protein kinase were not affected. Moreover, IL-6-induced the up-regulation of Bcl-2 and c-myc, and JunB was impaired in Ub-treated KT-3 cells, suggesting that the anti apoptotic and mitogenic effects of IL-6 were disrupted by Ub. These results suggest that extracellular Ub was incorporated into hematopoietic cells and mediated their growth suppression and apoptosis through proteasome-dependent degradation of selective cellular proteins such as STAT3. (Blood. 2000;95:2577 2585) PMID- 10753838 TI - Factor XIIIa supports microvascular endothelial cell adhesion and inhibits capillary tube formation in fibrin. AB - Coagulation factor XIIIa is a transglutaminase that catalyzes covalent cross-link formation in fibrin clots. In this report, we demonstrate that factor XIIIa also mediates adhesion of endothelial cells and inhibits capillary tube formation in fibrin. The adhesive activity of factor XIIIa was not dependent on the transglutaminase activity, and did not involve the factor XIIIb-subunits. The adhesion was inhibited by 99% using a combination of monoclonal antibodies directed against integrin alpha(v)beta(3) and beta(1)-containing integrins, and was dependent on Mg(2+) or Mn(2+). Soluble factor XIIIa also bound to endothelial cells in solution, as detected by flow cytometry. In addition, factor XIIIa inhibited endothelial cell capillary tube formation in fibrin in a dose-dependent manner. Furthermore, the extent of inhibition differed in 2 types of fibrin. The addition of 10 to 100 microg/mL factor XIIIa produced a dose-dependent reduction in capillary tube formation of 60% to 100% in gammaA/gammaA fibrin, but only a 10% to 37% decrease in gammaA/gamma' fibrin. These results show that factor XIIIa supports endothelial cell adhesion in an integrin-dependent manner and inhibits capillary tube formation. (Blood. 2000;95:2586-2592) PMID- 10753839 TI - Transcriptional control of the human plasma membrane phospholipid scramblase 1 gene is mediated by interferon-alpha. AB - Interferons (IFNs) mediate their diverse biologic activities through induction of the expression of multiple genes. Whereas the mode of action of certain of these IFN-regulated genes has been well characterized, most of the molecular and cellular events underlying the constellation of biologic responses to the IFNs remain unresolved. This study showed that the newly identified PLSCR1 gene for phospholipid scramblase, previously implicated in remodeling of plasma membrane phospholipids, is regulated at the transcriptional level by IFN-alpha. Analysis of 5' flanking genomic sequence in reporter constructs showed that transcriptional control of PLSCR1 was entirely regulated by a single IFN stimulated response element located in the first exon. A similar induction of PLSCR1 by IFN-alpha2a was also observed in a variety of other human tumor cell lines as well as in human umbilical vein endothelial cells. In these cell lines, the marked IFN-alpha2a-induced increase in PLSCR1 protein expression, ranging as high as 10-fold above basal levels, was not accompanied by increased cell surface exposure of phosphatidylserine, suggesting that remodeling of the cell surface requires both exposure to IFN and a second yet-to-be identified event to stimulate plasma membrane phospholipid scramblase activity and to mobilize phosphatidylserine to the cell surface. (Blood. 2000;95:2593-2599) PMID- 10753840 TI - Cloning of the human platelet F11 receptor: a cell adhesion molecule member of the immunoglobulin superfamily involved in platelet aggregation. AB - This study demonstrates that the human platelet F11 receptor (F11R) functions as an adhesion molecule, and this finding is confirmed by the structure of the protein as revealed by molecular cloning. The F11R is a 32-/35-kd protein duplex that serves as the binding site through which a stimulatory monoclonal antibody causes platelet aggregation and granule secretion. A physiological role for the F11R protein was demonstrated by its phosphorylation after the stimulation of platelets by thrombin and collagen. A pathophysiological role for the F11R was revealed by demonstrating the presence of F11R-antibodies in patients with thrombocytopenia. Adhesion of platelets through the F11R resulted in events characteristic of the action of cell adhesion molecules (CAMs). To determine the structure of this protein, we cloned the F11R cDNA from human platelets. The predicted amino acid sequence demonstrated that it is an integral membrane protein and an immunoglobulin superfamily member containing 2 extracellular C2 type domains. The structure of the F11R as a member of a CAM family of proteins and its activity in mediating adhesion confirm each another. We conclude that the F11R is a platelet-membrane protein involved in 2 distinct processes initiated on the platelet surface. The first is antibody-induced platelet aggregation and secretion that are dependent on both the FcgammaRII and the GPIIb/IIIa integrin and that may be involved in pathophysiological processes associated with certain thrombocytopenias. The second is an F11R-mediated platelet adhesion that is not dependent on either the FcgammaRII or the fibrinogen receptor and that appears to play a role in physiological processes associated with platelet adhesion and aggregation. (Blood. 2000;95:2600-2609) PMID- 10753841 TI - Functional comparison of thymic B cells and dendritic cells in vivo. AB - In this report we present a transgenic mouse model in which we targeted gene expression specifically to B-lymphocytes. Using the human CD19 promoter, we expressed major histocompatibility complex class II I-E molecules specifically on B cells of all tissues, but not on other cell types. If only B cells expressed I E in a class II-deficient background, positive selection of CD4(+) T cells could not be observed. A comparison of the frequencies of I-E reactive Vbeta5(+) and Vbeta11(+) T cells shows that I-E expression on thymic B cells is sufficient to negatively select I-E reactive CD4(+) T cells partially, but not CD8(+) T cells. Thus partial negative but no positive selection events can be induced by B lymphocytes in vivo. (Blood. 2000;95:2610-2616) PMID- 10753842 TI - Tracing uptake of C3dg-conjugated antigen into B cells via complement receptor type 2 (CR2, CD21). AB - Electron microscopy was used to study the internalization and delivery of ligands for complement receptor type 2 (CR2, CD21) to endocytic compartments of B lymphoblastoid Raji cells. Opsonized antigen was mimicked with purified C3dg conjugated to colloidal gold. C3dg-gold bound specifically to the cell surface in a time-dependent manner, and preincubation of the cells with a monoclonal antibody blocking the CR2 ligand-binding site completely inhibited any C3dg-gold binding. Notably, the binding of C3d-gold was confined to cell surface protrusions, eg, microvilli. C3dg-gold was apparently internalized through coated pits located at the bases of microvilli and could be traced to different compartments of the endocytic pathway. The morphologic characteristics and intracellular distribution of these multivesicular or multilaminar structures were compatible with those of compartments known to harbor major histocompatibility complex (MHC) class II molecules. Immunolabeling showed that the internalized C3dg-gold colocalized with MHC class II in these structures. These data provide the first ultrastructural evidence that complement-coated antigens are endocytosed by antigen-nonspecific B cells by CR2 and are delivered to the compartments in which peptide loading for antigen presentation occurs. They support the notion that CR2 may play a role in antigen presentation by B cells regardless of B-cell receptor specificity. (Blood. 2000;95:2617-2623) PMID- 10753843 TI - Sphingosine-1-phosphate is a ligand for the G protein-coupled receptor EDG-6. AB - EDG-6 is a recently cloned member of the endothelial differentiation gene (EDG) G protein-coupled receptor family that is expressed in lymphoid and hematopoietic tissue and in the lung. Homology of EDG-6 to the known sphingosine-1-phosphate (SPP) receptors EDG-1, EDG-3, and EDG-5 and lysophosphatidic acid (LPA) receptors EDG-2 and EDG-4 suggested that its ligand may be a lysophospholipid or lysosphingolipid. We examined the binding of [(32)P]SPP to HEK293 cells, transiently transfected with cDNA encoding EDG-6. Binding of [(32)P]SPP was saturable, demonstrating high affinity (K(D) = 63 nmol/L). Binding was also specific for SPP, as only unlabeled SPP and sphinganine-1-phosphate, which lacks the trans double bond at the 4 position, potently displaced radiolabeled SPP. LPA did not compete for binding of SPP at any concentration tested, whereas sphingosylphosphorylcholine competed for binding to EDG-6, but only at very high concentrations. In addition, SPP activated extracellular signal-regulated kinase (Erk) in EDG-6 transfected cells in a pertussis toxin-sensitive manner. These results indicate that EDG-6 is a high affinity receptor for SPP, which couples to a G(i/o) protein, resulting in the activation of growth-related signaling pathways. (Blood. 2000;95:2624-2629) PMID- 10753844 TI - Vascular endothelial growth factor and interleukin-6 in paracrine tumor-stromal cell interactions in multiple myeloma. AB - Vascular endothelial growth factor (VEGF), a multifunctional cytokine, potently stimulates angiogenesis including tumor neovascularization. Although well established in solid tumors, the role of VEGF in bone marrow neoangiogenesis and paracrine tumor-stromal cell interactions in lymphohematopoietic malignancies has not been fully elucidated. In multiple myeloma (MM), marrow neovascularization parallels disease progression. This parallel prompted us to investigate the expression and secretion of VEGF by myeloma cells and its potential effects in myeloma-marrow stroma interactions. The biologically active splice variants VEGF165 and VEGF121 were expressed and secreted by myeloma cell lines and plasma cells isolated from the marrow of patients with MM. As shown by immunocytochemistry or RT-PCR, myeloma cells did not express or weakly expressed the VEGF receptors FLT-1 and FLK-1/KDR, indicating that autocrine stimulation is unlikely. In contrast, FLK-1/KDR was abundantly expressed by marrow stromal cells. Therefore, we studied the effects of VEGF on marrow stroma, focusing on the secretion of interleukin-6 (IL-6), a potent growth factor for myeloma cells and an inhibitor of plasma cell apoptosis. Exposure of stromal and microvascular endothelial cells to recombinant human (rh) VEGF165 or VEGF121 induced a time- and dose-dependent increase in IL-6 secretion (14- to 27-fold at 50 ng/mL after 24 hours, P <.001). Conversely, rhIL-6 stimulated VEGF expression and secretion in myeloma cell lines (40%-60%; P <.05) and to a variable degree (up to 5.3-fold; P <.005) in plasma cells purified from the marrow of patients with MM. This mutual stimulation suggests paracrine interactions between myeloma and marrow stromal cells triggered by VEGF and IL-6. (Blood. 2000;95:2630-2636) PMID- 10753845 TI - Increased angiogenesis in the bone marrow of patients with acute myeloid leukemia. AB - The importance of angiogenesis for the progressive growth and viability of solid tumors is well established. In contrast, only few data are available for hematologic neoplasms. To investigate the role of angiogenesis in acute myeloid leukemia (AML), bone marrow biopsies from 62 adults with newly diagnosed, untreated AML (day 0) were evaluated. Further studies were done after the completion of remission induction chemotherapy (day 16 of induction chemotherapy, n = 21; complete remission, n = 20). Microvessels were scored in at least 3 areas (x500 field, 0.126 mm(2)) of the highest microvessel density in representative sections of each bone marrow specimen using immunohistochemistry for von Willebrand factor and thrombomodulin. Microvessel counts were significantly higher in patients with AML (n = 62) compared with control patients (n = 22): median (interquartile range) 24.0 (21.0-27.8)/x500 field vs 11.2 (10. 0 12.0)/x500 field, respectively (P <.001). On day 16 of induction chemotherapy, microvessel density was reduced by 60% (44-66) (P <. 001) in hypoplastic marrows without residual blasts, in contrast to only 17% (0-37) reduction in hypoplastic marrows with >/= 5% residual blasts (P <.001 for the difference between both groups). Bone marrow biopsies taken at the time of complete remission displayed a microvessel density in the same range as the controls. In conclusion, there is evidence of increased microvessel density in the bone marrow of patients with AML, which supports the hypothesis of an important role of angiogenesis in AML. Furthermore, these findings suggest that antiangiogenic therapy might constitute a novel strategy for the treatment of AML. (Blood. 2000;95:2637-2644) PMID- 10753846 TI - Abundance of cyclin B1 regulates gamma-radiation-induced apoptosis. AB - gamma-Radiation is a potent inducer of apoptosis. There are multiple pathways regulating DNA damage-induced apoptosis, and we set out to identify novel mechanisms regulating gamma-radiation-induced apoptosis in hematopoietic cells. In this report, we present data implicating the cyclin B1 protein as a regulator of apoptotic fate following DNA damage. Cyclin B1 is the regulatory subunit of the cdc2 serine/threonine kinase, and accumulation of cyclin B1 in late G2 phase of the cell cycle is a prerequisite for mitotic initiation in mammalian cells. We find that abundance of the cyclin B1 protein rapidly increases in several mouse and human hematopoietic cells (Ramos, DP16, HL60, thymocytes) undergoing gamma radiation-induced apoptosis. Cyclin B1 accumulation occurs in all phases of the cell cycle. Antisense inhibition of cyclin B1 accumulation decreases apoptosis, and ectopic cyclin B1 expression is sufficient to induce apoptosis. These observations are consistent with the idea that cyclin B1 is both necessary and sufficient for gamma-radiation-induced apoptosis. (Blood. 2000;95:2645-2650) PMID- 10753847 TI - Immunoglobulin heavy-chain consensus probes for real-time PCR quantification of residual disease in acute lymphoblastic leukemia. AB - Tumor-related immunoglobulin heavy-chain (IgH) rearrangements are markers for polymerase chain reaction (PCR) detection of minimal residual disease (MRD) in B cell malignancies. Nested PCR with patient IgH allele-specific oligonucleotide primers can detect 1 tumor cell in 10(4) to 10(6) normal cells. In childhood acute lymphoblastic leukemia (ALL), persistence of PCR-detectable disease is associated with increased risk of relapse. The clinical significance of qualitative PCR data can be limited, however, because patients can harbor detectable MRD for prolonged periods without relapse. Recent studies indicate that a quantitative rise in tumor burden identifies patients who are at high risk for relapse. Therefore, an efficient and reliable PCR method for MRD quantification is needed for ALL patients. We have developed a real-time PCR method to quantify MRD with IgH V(H) gene family consensus fluorogenically labeled probes. With this method, a small number of probes can be used to quantify MRD in a large number of different patients. The assay was found to be both accurate and reproducible over a wide range and capable of detecting approximately 1 tumor cell in 5 x 10(4) normal cells. We demonstrate that this methodology can discriminate between patients with persistence of MRD who relapse and those who do not. This technique is generally applicable to B-cell malignancies and is currently being used to quantify MRD in a number of prospective clinical studies at our institution. (Blood. 2000;95:2651-2658) PMID- 10753848 TI - Molecular analysis of lineage-specific chimerism and minimal residual disease by RT-PCR of p210(BCR-ABL) and p190(BCR-ABL) after allogeneic bone marrow transplantation for chronic myeloid leukemia: increasing mixed myeloid chimerism and p190(BCR-ABL) detection precede cytogenetic relapse. AB - We studied lineage-specific chimerism and minimal residual disease (MRD) in sequential posttransplant samples from 55 patients who underwent unmanipulated (n = 44) or partially T-cell-depleted (n = 11) allogeneic bone marrow transplantation (BMT) for chronic myeloid leukemia (CML). Chimerism was assessed by polymerase chain reaction (VNTR [variable number of tandem repeats]-PCR) analysis in highly purified CD19+, CD3+, CD15+, and CD56+ cell fractions, whereas MRD was investigated in whole blood by reverse transcriptase-PCR (RT-PCR) of both p210(BCR-ABL) and p190(BCR-ABL) hybrid transcripts. Of 55 patients, 14 (including 6 T-cell-depleted patients) had cytogenetic relapse at 5-80 months and progressed to hematologic relapse, while 41 patients remained in prolonged cytogenetic remission 12-107 months post-BMT. Before leukemia recurrence, patients in the relapse group showed a consistent evolution pattern sequentially featured by persistent p210(BCR-ABL) positivity, increasing mixed chimerism (MC) in myeloid cells, p190(BCR-ABL) positivity, and, finally, cytogenetic relapse. Myeloid MC preceded cytogenetic relapse by 2-12 months, whereas p190(BCR/ABL) was detected 1 6 months prior to cytogenetic relapse in 11 patients and concomitant with cytogenetic relapse in 3 patients. In the remission group, all patients invariably tested negative for p190(BCR-ABL); 10 patients tested positive for p210(BCR-ABL) at variable time-points but showed persistent full donor chimerism (DC), whereas 31 patients tested p210(BCR-ABL) negative and displayed full DC or transient MC due to the persistence of recipient T cells. Two patients in the relapse group were successfully reinduced into molecular remission with donor lymphocyte infusion. Sequential molecular analysis after such treatment showed the inverse pattern to that observed prior to relapse, ie, progressive disappearance of p190(BCR-ABL) transcripts, conversion of myeloid chimerism to donor type, and, finally, p210(BCR-ABL) negativity. We conclude that lineage specific chimerism and p190(BCR-ABL) messenger RNA (mRNA) analyses contribute a better characterization of CML evolution after BMT and enable early identification of patients at the highest risk of relapse. (Blood. 2000;95:2659 2665) PMID- 10753850 TI - Isolation and characterization of an acute promyelocytic leukemia cell line selectively resistant to the novel antileukemic and apoptogenic retinoid 6-[3 adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid. AB - 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) is a novel compound that represents the prototype of a new class of synthetic retinoids with apoptogenic properties in acute promyelocytic leukemia (APL) and other types of leukemia. In this article, using SCID mice xenografted with APL-derived NB4 cells, we demonstrate that CD437 has significant antileukemic activity in vivo. In addition, we report on the isolation and characterization of an APL cell line (NB4.437r) resistant to CD437. The cell line retains expression of PML-RARalpha and is approximately 33-fold more resistant than the parental counterpart to the apoptogenic effects of the retinoid. Resistance is relatively specific to CD437 and structural congeners because the NB4.437r cell line is still sensitive to various types of apoptogenic compounds. The CD437-resistant cell line maintains sensitivity to the antiproliferative and apoptotic action of all-trans-retinoic acid, AM580, and fenretinide, though it shows partial resistance to the cytodifferentiating effects of the first 2 compounds. Resistance to CD437 lays upstream of the CD437-induced release of cytochrome c from the mitochondria and the activation of caspase-3, -7, -8, and -9. Furthermore, NB4.437r cells are deficient in the CD437-dependent activation of nuclear NFkb and AP1-binding activities and in the phosphorylation of the protein kinase Akt. In the case of AP1, deficient assembly of the complex is not caused by the lack of activation of the Jun N-terminal kinase (JNK) family of kinases. The novel cell line will be useful in the elucidation of the molecular mechanisms underlying the apoptogenic action of CD437 and structurally related retinoids. (Blood. 2000;95:2672-2682) PMID- 10753849 TI - MUC1 is activated in a B-cell lymphoma by the t(1;14)(q21;q32) translocation and is rearranged and amplified in B-cell lymphoma subsets. AB - The band 1q21 is among the most common sites affected by chromosomal translocations in lymphoid, myeloid, epithelial, and sarcomatous lesions. In non Hodgkin's lymphoma (NHL), translocations and duplications affecting this chromosomal site are frequently, but not exclusively, seen in association with primary abnormalities such as the t(14;18)(q32;q21) and t(8;14)(q24;q32) translocations, suggesting a role for 1q21 rearrangements in tumor progression. We report here the characterization and cloning of breakpoints in a case of extranodal ascitic B-cell lymphoma with a t(1;14)(q21;q32) translocation. The breakpoints on the der(1) and der(14) chromosomes were mapped by fluorescence in situ hybridization and Southern blot analysis and cloned using an IGHG (Cgamma) probe. The translocation linked the IGHG4 switch (Sgamma4) sequences of the productively rearranged allele to chromosome 1 sequences downstream of MUC1, leaving the MUC1 transcriptional unit intact. MUC1 was markedly overexpressed in the tumor at the mRNA and protein levels relative to lymphoma cell lines lacking a 1q21 rearrangement. Presumably, MUC1 transcription is aberrantly regulated by the IGHA (Calpha) 3' enhancer element retained on the same chromosome. Screening of a panel of B-cell lymphomas by Southern blot analysis identified a subset with a 3' MUC1 breakpoint and another with low-level amplification of MUC1. MUC-1 mucin has previously been shown to be frequently overexpressed in human epithelial cancers and to be associated with tumor progression and poor clinical outcome. Thus, MUC1 activation by chromosomal translocation, rearrangement, and amplification, identified here for the first time in NHL, is consistent with its suggested role in tumorigenesis. (Blood. 2000;95:2666-2671) PMID- 10753851 TI - The t(5;17) acute promyelocytic leukemia fusion protein NPM-RAR interacts with co repressor and co-activator proteins and exhibits both positive and negative transcriptional properties. AB - The t(5;17) variant of acute promyelocytic leukemia (APL) fuses the genes for nucleophosmin (NPM) and the retinoic acid receptor alpha (RARalpha). Two NPM-RAR molecules are expressed as a result of alternative RNA splicing. Both contain RARalpha sequences that encode the DNA binding, heterodimerization, and ligand activation domains of RARalpha. This study was designed to test the ability of these fusion proteins to act as transcriptional activators of retinoic acid responsive promoters. The NPM-RAR fusion proteins bind to retinoic acid response element sequences as either homodimers or as heterodimers with RXR. Transcription of retinoic acid-inducible promoters is activated by the fusion proteins in the presence of retinoic acid. The level of transactivation induced by the NPM-RAR fusions differs from the level of transactivation induced by wild-type RARalpha in both a promoter and cell specific fashion, and more closely parallels the pattern of activation of the PML-RAR fusion than wild-type RARalpha. In addition, NPM-RAR decreases basal transcription from some promoters and acts in a dominant negative fashion when co-transfected with wild-type RARalpha. Both NPM-RAR and PML-RAR interact with the co-repressor protein SMRTe in a manner that is less sensitive than RARalpha to dissociation by retinoic acid. Retinoic acid induces binding of the co-activator protein RAC3. These data indicate that the NPM-RAR fusion proteins can modulate expression of retinoid-responsive genes in a positive or negative manner, depending on context of the promoter, and lend support to the hypothesis that aberrant transcriptional activation underlies the APL phenotype. (Blood. 2000;95:2683-2690) PMID- 10753852 TI - Concurrent activation of a novel putative transforming gene, myeov, and cyclin D1 in a subset of multiple myeloma cell lines with t(11;14)(q13;q32). AB - Through the application of the NIH/3T3 tumorigenicity assay to DNA from a gastric carcinoma, we have identified a novel transforming gene, designated myeov (myeloma overexpressed gene in a subset of t[11;14]-positive multiple myelomas). Sequence analyses did not reveal any homology with sequences present in the GenBank, except the deduced protein structure predicts a transmembrane localization. Myeov was mapped to chromosome 11q13 and localized by DNA fiber fluorescence in situ hybridization (FISH) 360-kilobase (kb) centromeric of cyclin D1. In 3 of 7 multiple myeloma (MM) cell lines with a t(11;14)(q13;q32) and cyclin-D1 overexpression, Northern blot analysis revealed overexpression of myeov as well. In all 7 cell lines, the translocation breakpoint was mapped within the 360-kb region between myeov and cyclin D1. DNA fiber FISH with a contig of probes covering the constant region of the immunoglobulin heavy chain (IgH) revealed that exclusively in the 3 myeov-overexpressing cell lines (KMS-12, KMS-21, and XG 5), either the 5' E(mu) enhancer or the most telomeric 3' Ealpha enhancer was juxtaposed to myeov. Although cyclin D1 overexpression represents a characteristic feature of all MM cell lines with t(11;14), our results demonstrate aberrant expression of a second putative oncogene in a subset of these cases, due to juxtaposition to IgH enhancers. The clinical relevance of this dual activation remains to be elucidated. (Blood. 2000;95:2691-2698) PMID- 10753853 TI - Weak D alleles express distinct phenotypes. AB - The weak D phenotype is caused by many different RHD alleles encoding aberrant RhD proteins, raising the possibility of distinct serologic phenotypes and of anti-D immunizations in weak D. We reported 6 new RHD alleles, D category III type IV, DIM, and the weak D types 4.1, 4.2.1, 4.2.2, and 17. The immunohematologic features of 18 weak D types were examined by agglutination and flow cytometry with more than 50 monoclonal anti-D. The agglutination patterns of the partial D phenotypes DIM, D(III) type IV, and D(IV) type III correlated well with the D epitope models, those of the weak D types showed no correlation. In flow cytometry, the weak D types displayed type-specific antigen densities between 70 and 4000 RhD antigens per cell and qualitatively distinct D antigens. A Rhesus D similarity index was devised to characterize the extent of qualitative changes in aberrant D antigens and discriminated normal D from all tested partial D, including D category III. In some rare weak D types, the extent of the alterations was comparable to that found in partial Ds that were prone to anti-D immunization. Four of 6 case reports with anti-D in weak D represented auto-anti D. We concluded that, in contrast to previous assumptions, most weak D types, including prevalent ones, carry altered D antigens. These observations are suggestive of a clinically relevant potential for anti-D immunizations in some, but not in the prevalent weak D types, and were used to derive an improved transfusion strategy in weak D patients. (Blood. 2000;95:2699-2708) PMID- 10753854 TI - Survival of donor cells 25 years after intrauterine transfusion. AB - Persistence of donor leukocytes in the circulation of recipients of intrauterine transfusion (IUT) has been observed up to 5 years after birth. The aim of this study was to determine whether transfusions with nonirradiated, nonleukocyte depleted donor blood during the fetal period resulted in long-term immunomodulation of the recipient. Twenty-four surviving IUT recipients between 1966 and 1976 were tested for autoimmune disease and autoantibodies at follow-up. Ten had sex-mismatched donors and were therefore informative for chimerism studies using fluorescence in situ hybridization (FISH). Seven female recipients could be tested for chimerism using a Y- chromosome-specific polymerase chain reaction (PCR) because they received at least 1 IUT from a male donor. Nine recipients could be studied for cytotoxic T-lymphocyte precursor (CTLp) and helper T-lymphocyte precursor (HTLp) frequencies because the original donors were available for testing. All surviving IUT recipients were in good health at the time of the examination, and routine laboratory testing revealed no abnormalities. None of the IUT recipients were chimeric as determined by FISH analysis, but Y-chromosome-specific sequences were detected by PCR in 6 of the 7 women. However, the CTLp and HTLp frequencies of the IUT recipients against the donors were comparable to those of the controls. The current study provides evidence that IUT can result in the persistence of donor cells in the recipient for a period longer than 20 years but that it is not associated with immunotolerance or with signs of chronic antigenic stimulation. (Blood. 2000;95:2709-2714) PMID- 10753855 TI - Constitutive activation of LIL-Stat in adult T-cell leukemia cells. AB - The activation status of a recently identified STAT (signal transducers and activators of transcription) factor, LIL-Stat (lipopolysaccharide [LPS]/IL-1 inducible Stat) in adult T-cell leukemia (ATL) cells was investigated by electrophoretic mobility shift assays using nuclear extracts of leukemic cells from 7 patients with ATL and a GAS (gamma interferon activation site)-like element termed LILRE (LPS/IL-1-responsive element), which is found in the human prointerleukin 1beta (IL1B) gene. Spontaneous DNA binding of LIL-Stat was observed in all ATL cells examined. However, in normal human peripheral lymphocytes, DNA binding of LIL-Stat was detected only after stimulation with IL 1. These results demonstrated that LIL-Stat is constitutively activated in ATL cells. Furthermore, our transient transfection studies using LILRE chloramphenicol acetyltransferase (CAT) reporters argue that LIL-Stat in ATL cells functions as a transcriptional activator through binding to the LILRE in the IL1B gene. (Blood. 2000;95:2715-2718) PMID- 10753856 TI - The Ikaros gene, a central regulator of lymphoid differentiation, fuses to the BCL6 gene as a result of t(3;7)(q27;p12) translocation in a patient with diffuse large B-cell lymphoma. AB - The BCL6 gene, isolated from the breakpoints of 3q27-associated chromosomal translocations, has been implicated in diffuse large B-cell lymphomas (DLBL). Here we describe the molecular characterization of novel t(3;7)(q27;p12) translocations in 2 patients with DLBL. Molecular genetic analysis of the breakpoint area involving BCL6 revealed the presence of the Ikaros gene, a central regulator of lymphoid differentiation that had been mapped to human chromosome 7 band p13-p11.1. As a molecular consequence of the translocation, the 5' regulatory region of the BCL6 gene was replaced by the putative 5' regulatory region of the Ikaros gene, probably leading to deregulated expression of the BCL6 gene throughout B-cell differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) analyses of a patient sample established that the t(3;7)(q27;p12) results in fusion of the Ikaros and BCL6 genes. This study provides the first evidence that the Ikaros gene is rearranged in human hematopoietic malignant disorders. (Blood. 2000;95:2719-2721) PMID- 10753857 TI - Presence of clone-specific antigen receptor gene rearrangements at birth indicates an in utero origin of diverse types of early childhood acute lymphoblastic leukemia. AB - There is strong evidence that infant leukemias with a t(4;11) translocation originate in utero. To test whether other subtypes of childhood leukemias are also initiated during fetal life, we used clone-specific genetic markers for the analysis of neonatal blood spots from 5 children aged 6 months to 4 years 8 months at diagnosis of pro-B, common acute lymphoblastic leukemia (ALL), and T ALL. In all children, the clonotypic antigen receptor gene rearrangements were already present at birth. The estimated amount of clonotypic cells was in the range of 10 to 100 cells per blood spot. In 2 infants with a t(4;11) positive ALL, we detected similar amounts of the fusion gene sequences compared with the clonal antigen receptor gene rearrangements, suggesting the presence of both markers in the same cells. Our data indicate that the first leukemogenic event of diverse types of childhood ALL may already occur in utero. (Blood. 2000;95:2722 2724) PMID- 10753858 TI - Expression of Ig-beta (CD79b) by chronic lymphocytic leukemia B cells that lack immunoglobulin heavy-chain allelic exclusion. AB - Because immunoglobulin (Ig)-beta (CD79b) is required for immunoglobulin allelic exclusion, we examined the CD79b expressed by four chronic lymphocytic leukemia (CLL) samples that expressed more than one immunoglobulin heavy-chain allele and five samples that had normal immunoglobulin heavy-chain allelic exclusion. All leukemia cell samples stained poorly with monoclonal antibodies specific for extracellular epitopes of CD79b. However, all samples expressed functional CD79b genes, regardless of whether they did or did not express more than one immunoglobulin heavy-chain allele. We identified variant CD79b genes that had conservative base substitutions restricted to regions encoding the extracellular immunoglobulin-like domain of CD79b. However, these variants were not restricted to samples lacking immunoglobulin heavy-chain allelic exclusion and most likely reflect genetic polymorphism. Collectively, these data indicate that the unusual expression of more than one immunoglobulin heavy allele by CLL B cells is not associated with structural, nonconservative mutations in the signal-transduction domains of CD79b. (Blood. 2000;95:2725-2727) PMID- 10753860 TI - Binding of liganded vitamin D receptor to the vitamin D receptor interacting protein coactivator complex induces interaction with RNA polymerase II holoenzyme. AB - Because the vitamin D receptor interacting protein (DRIP) coactivator complex shares components with the RNA polymerase II (Pol II) holoenzyme complex, we tested whether the two protein complexes associate in cellular extracts. On initial purification steps, the DRIP complex copurified with the Pol II holoenzyme. Pol II was found to bind to the vitamin D receptor in a ligand dependent fashion when either nuclear extracts or partially purified preparations were used as sources of DRIP and Pol II holoenzyme. A subpopulation of holoenzyme complexes bound to the receptor because BRCA1, which associates with the Pol II holoenzyme, did not associate with the liganded receptor, and only in certain of the holoenzyme- and DRIP-containing fractions did Pol II bind to the liganded receptor. Immunoprecipitation experiments revealed that the DRIP complex was not pre-associated with the Pol II holoenzyme, but the interaction between these two complexes was induced only in the presence of receptor and ligand. These data support a model in which the activation of transcription by hormone-bound receptor requires binding to the DRIP coactivator, and this induced ternary complex can then bind to the Pol II holoenzyme to activate transcription. PMID- 10753861 TI - Identification of a receptor for reg (regenerating gene) protein, a pancreatic beta-cell regeneration factor. AB - Reg (regenerating gene) was isolated as a gene specifically expressed in regenerating islets (Terazono, K., Yamamoto, H., Takasawa, S., Shiga, K., Yonemura, Y., Tochino, Y., and Okamoto, H. (1988) J. Biol. Chem. 263, 2111-2114). Rat and human Reg gene products, Reg/REG proteins, have been demonstrated to stimulate islet beta-cell growth in vitro and in vivo and to ameliorate experimental diabetes. In the present study, we isolated a cDNA for the Reg protein receptor from a rat islet cDNA library. The cDNA encoded a cell surface 919-amino acid protein, and the cells into which the cDNA had been introduced bound Reg protein with high affinity. When the cDNA was introduced into RINm5F cells, a pancreatic beta-cell line that shows Reg-dependent growth, the transformants exhibited significant increases in the incorporation of 5'-bromo-2' deoxyuridine as well as in the cell numbers in response to Reg protein. A homology search revealed that the cDNA is a homologue to a human multiple exostoses-like gene, the function of which has hitherto been unknown. These results strongly suggest that the receptor is encoded by the exostoses-like gene and mediates a growth signal of Reg protein for beta-cell regeneration. PMID- 10753862 TI - Evidence for the transfer of sulfane sulfur from IscS to ThiI during the in vitro biosynthesis of 4-thiouridine in Escherichia coli tRNA. AB - IscS from Escherichia coli is a cysteine desulfurase that has been shown to be involved in Fe-S cluster formation. The enzyme converts L-cysteine to L-alanine and sulfane sulfur (S(0)) in the form of a cysteine persulfide in its active site. Recently, we reported that IscS can donate sulfur for the in vitro biosynthesis of 4-thiouridine (s(4)U), a modified nucleotide in tRNA. In addition to IscS, s(4)U synthesis in E. coli also requires the thiamin biosynthetic enzyme ThiI, Mg-ATP, and L-cysteine as the sulfur donor. We now report evidence that the sulfane sulfur generated by IscS is transferred sequentially to ThiI and then to tRNA during the in vitro synthesis of s(4)U. Treatment of ThiI with 5-((2 iodoacetamido)ethyl)-1-aminonapthalene sulfonic acid (I-AEDANS) results in irreversible inhibition, suggesting the presence of a reactive cysteine that is required for binding and/or catalysis. Both ATP and tRNA can protect ThiI from I AEDANS inhibition. Finally, using gel shift and protease protection assays, we show that ThiI binds to unmodified E. coli tRNA(Phe). Together, these results suggest that ThiI is a recipient of S(0) from IscS and catalyzes the ultimate sulfur transfer step in the biosynthesis of s(4)U. PMID- 10753863 TI - In vivo and in vitro kinetics of metal transfer by the Klebsiella aerogenes urease nickel metallochaperone, UreE. AB - The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to deliver Ni(II) to the urease apoprotein during enzyme activation. Native UreE possesses a histidine-rich region at its carboxyl terminus that binds several equivalents of Ni(2+); however, a truncated form of this protein (H144*UreE) binds only 2 Ni(2+) per dimer and is functionally active (Brayman, T. G., and Hausinger, R. P. (1996) J. Bacteriol. 178, 5410-5416). The urease activation kinetics were studied in vivo by monitoring the development of urease activity upon adding Ni(2+) to spectinomycin-treated Escherichia coli cells that expressed the complete K. aerogenes urease gene cluster with altered forms of ureE. Site-specific alterations of H144*UreE decrease the rate of in vivo urease activation, with the most dramatic changes observed for the H96A, H110A, D111A, and H112A substitutions. Notably, urease activity in cells producing H96A/H144*UreE was lower than cells containing a ureE deletion. Prior studies had shown that H110A and H112A variants each bound a single Ni(2+) per dimer with elevated K(d) values compared with control H144*UreE, whereas the H96A and D111A variants bound 2 Ni(2+) per dimer with unperturbed K(d) values (Colpas, G. J., Brayman, T. G., Ming, L.-J., and Hausinger, R. P. (1999) Biochemistry 38, 4078 4088). To understand why cells containing the latter two proteins showed reduced rates of urease activation, we characterized their metal binding/dissociation kinetics and compared the results to those obtained for H144*UreE. The truncated protein was shown to sequentially bind two Ni(2+) with k(1) approximately 18 and k(2) approximately 100 M(-1) s(-1), and with dissociation rates k(-1) approximately 3 x 10(-3) and k(-2) approximately 10(-4) s(-1). Similar apparent rates of binding and dissociation were noted for the two mutant proteins, suggesting that altered H144*UreE interactions with Ni(2+) do not account for the changes in cellular urease activation. These conclusions are further supported by in vitro experiments demonstrating that addition of H144*UreE to urease apoprotein activation mixtures inhibited the rate and extent of urease formation. Our results highlight the importance of other urease accessory proteins in assisting UreE-dependent urease maturation. PMID- 10753864 TI - SOX9 enhances aggrecan gene promoter/enhancer activity and is up-regulated by retinoic acid in a cartilage-derived cell line, TC6. AB - SOX9 is a transcription factor that plays a key role in chondrogenesis. Aggrecan is one of the major structural components in cartilage; however, the molecular mechanism of aggrecan gene regulation has not yet been fully elucidated. TC6 is a clonal chondrocytic cell line derived from articular cartilage. The purpose of this study was to examine whether SOX9 modulates aggrecan gene expression and to further identify molecules that regulate Sox9 expression in TC6 cells. SOX9 overexpression in TC6 cells enhanced by approximately 3-fold the transcriptional activity of the AgCAT-8 construct containing 8-kilobase (kb) promoter/first exon/first intron fragments of the aggrecan gene. SOX9 enhancement of aggrecan promoter activity was lost when we deleted a 4.5-kb fragment from the 3'-end of the 8-kb fragment corresponding to the region including the first intron. In TC6 cells, SOX9 enhanced the transcriptional activity of a reporter construct containing the Sry/Sox consensus sequence >10-fold. SOX9 enhancement of aggrecan gene promoter activity and SOX9 transactivation through the Sry/Sox consensus sequence were not observed in osteoblastic osteosarcoma cells (ROS17/2.8), indicating the dependence on the cellular background. Northern blot analysis indicated that TC6 cells constitutively express Sox9 mRNA at relatively low levels. To examine regulation of Sox9 gene expression, we investigated the effects of calciotropic hormones and cytokines. Among these, retinoic acid (RA) specifically enhanced Sox9 mRNA expression in TC6 cells. The basal levels of Sox9 expression and its enhancement by RA were observed similarly at both permissive (33 degrees C) and nonpermissive (39 degrees C) temperatures. Furthermore, RA treatment enhanced the transcriptional activity of a reporter construct containing the Sry/Sox consensus sequence in TC6 cells. Moreover, RA treatment also enhanced the transcriptional activity of another reporter construct containing the enhancer region of the type II procollagen gene in TC6 cells. These observations indicate that SOX9 enhances aggrecan promoter activity and that its expression is up-regulated by RA in TC6 cells. PMID- 10753865 TI - Antibacterial and antifungal activities of vasostatin-1, the N-terminal fragment of chromogranin A. AB - Vasostatin-1, the natural N-terminal 1-76 chromogranin A (CGA)-derived fragment in bovine sequence, has been purified from chromaffin secretory granules and identified by sequencing and matrix-assisted laser desorption time-of-flight mass spectrometry. This peptide, which displays antibacterial activity against Gram positive bacteria at micromolar concentrations, is also able to kill a large variety of filamentous fungi and yeast cells in the 1-10 microM range. We have found that the C-terminal moiety of vasostatin-1 is essential for the antifungal activity, and shorter active peptides have been synthesized. In addition, from the comparison with the activity displayed by related peptides (human recombinant and rat synthetic fragments), we could determine that antibacterial and antifungal activities have different structural requirements. To assess for such activities in vivo, CGA and CGA-derived fragments were identified in secretory material released from human polymorphonuclear neutrophils upon stimulation. Vasostatin-1, which is stored in a large variety of cells (endocrine, neuroendocrine, and neurons) and which is liberated from stimulated chromaffin and immune cells upon stress, may represent a new component active in innate immunity. PMID- 10753866 TI - Characterization of the human DNA methyltransferase splice variant Dnmt1b. AB - Tissue- and gene-specific patterns of cytosine-DNA methylation are characteristic features of vertebrate genomes. The generation and proper maintenance of DNA methylation patterns are essential for embryonic development, as demonstrated by the lethal phenotypes of mice with either a targeted disruption of Dnmt1, the gene responsible for the maintenance of DNA methylation, or targeted disruption of Dnmt3a or Dnmt3b, the genes involved in generation of newly formed methylation patterns. Recently, a novel mRNA, Dnmt1b, resulting from alternative splicing of Dnmt1 was identified (Hsu, D. W., Lin, M. J., Lee, T. L., Wen, S. C., Chen, X., and Shen, C. K., (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9751-9756). The abundance of Dnmt1b mRNA was estimated by semiquantitative reverse transcription polymerase chain reaction and was suggested to encode a major C-5 DNA methyltransferase isoform. Here we report characterization of this novel DNA methyltransferase transcript, Dnmt1b, and its protein product in human cell lines and in freshly isolated human peripheral blood mononuclear cells. The abundance of Dnmt1b transcript, as determined by quantitative RNase protection analysis, was determined to range from 6% to 25% of Dnmt1 in human cells. Second generation antisense inhibitors targeted to the 5'- and 3'-ends of Dnmt1 inhibited the accumulation of both Dnmt1 and Dnmt1b in cells. Dnmt1b protein purified from a baculovirus expression system was demonstrated to be a functional DNA methyltransferase, and to have Michaelis constants for both DNA and S-adenosyl-L methionine similar to baculovirus-expressed Dnmt1. However, antibodies raised against Dnmt1b epitopes demonstrated that Dnmt1b protein was present at approximately 2-5% of the level of Dnmt1 and therefore represents only a minor DNA methyltransferase isoform in human cells. PMID- 10753867 TI - Akt/protein kinase B up-regulates Bcl-2 expression through cAMP-response element binding protein. AB - In our previous study we showed that insulin-like growth factor-I induces a cAMP response element (CRE) site-containing Bcl-2 promoter through a novel signaling pathway involving mitogen-activated protein kinase kinase 6/p38beta mitogen activated protein kinase/MAP kinase-activated protein kinase-3/cAMP-response element-binding protein (CREB) (Pugazhenthi, S., Miller, E., Sable, C., Young, P., Heidenreich, K. A., Boxer, L. M., and Reusch, J. E.-B. (1999) J. Biol. Chem. 274, 27529-27535). In the present investigation, we define a second pathway contributing to CREB-dependent up-regulation of Bcl-2 expression as a novel anti apoptotic function of Akt signaling. To examine the role of Akt on Bcl-2 expression, a series of transient transfections using a luciferase reporter gene driven by the promoter region of Bcl-2 containing a CRE were carried out. Pharmacological inhibition of phosphatidylinositol (PI) 3-kinase, the upstream kinase of Akt, with LY294002 led to a 45% decrease in Bcl-2 promoter activity. The reporter activity was enhanced 2.3-fold by overexpression of active p110 subunit of PI 3-kinase and inhibited 44% by the dominant negative p85 subunit of PI 3-kinase. Cotransfection with 3-phosphoinositide-dependent kinase (PDK1), which is required for the full activation of Akt, resulted in enhanced luciferase activity. Insulin-like growth factor-I-mediated induction of Bcl-2 promoter activity was decreased significantly (p < 0.01) by the dominant negative forms of p85 subunit of PI 3-kinase, PDK1, and Akt. These data indicate that regulation of Bcl-2 expression by IGF-I involves a signaling cascade mediated by PI 3 kinase/PDK1/Akt/CREB. Furthermore, we measured the Bcl-2 mRNA in PC12 cells overexpressing Akt by real-time quantitative reverse transcription-polymerase chain reaction using the TaqMan(TM) fluorogenic probe system. We observed a 2.1 fold increase in Bcl-2 mRNA levels in the Akt cell line compared with control PC12 cells, supporting the observation that enhanced CREB activity by Akt signaling leads to increased Bcl-2 promoter activity and cell survival. PMID- 10753868 TI - A G protein-coupled receptor for UDP-glucose. AB - Uridine 5'-diphosphoglucose (UDP-glucose) has a well established biochemical role as a glycosyl donor in the enzymatic biosynthesis of carbohydrates. It is less well known that UDP-glucose may possess pharmacological activity, suggesting that a receptor for this molecule may exist. Here, we show that UDP-glucose, and some closely related molecules, potently activate the orphan G protein-coupled receptor KIAA0001 heterologously expressed in yeast or mammalian cells. Nucleotides known to activate P2Y receptors were inactive, indicating the distinctly novel pharmacology of this receptor. The receptor is expressed in a wide variety of human tissues, including many regions of the brain. These data suggest that some sugar-nucleotides may serve important physiological roles as extracellular signaling molecules in addition to their familiar role in intermediary metabolism. PMID- 10753869 TI - Signaling of hepatocyte growth factor/scatter factor (HGF) to the small GTPase Rap1 via the large docking protein Gab1 and the adapter protein CRKL. AB - Hepatocyte growth factor (HGF; scatter factor) is a multipotent protein with mitogenic, motogenic, and developmental functions. Upon activation, the HGF receptor c-Met binds and phosphorylates the multisite docking protein Gab1. Besides binding motifs for phosphatidylinositol 3-kinase and Grb2, Gab 1 contains multiple Tyr-X-X-Pro (YXXP) motifs which, when phosphorylated, are potential binding sites for the adapter proteins c-Crk and Crk-like (CRKL). Stimulation of human embryonic kidney cells (HEK293) with HGF leads to Gab1 association with CRKL. The Gab1-CRKL interaction requires both, the SH2 domain of CRKL and the region containing the YXXP motifs in Gab1. CRKL binds via its first SH3 domain to several downstream signal transducers, including C3G an activator of the small GTPase Rap1. Indeed, Rap1 was rapidly activated after HGF stimulation of HEK293 cells. Rap1 activation through HGF was suppressed through transfection of a truncated C3G protein which only contains the SH3-binding motifs of C3G. Transfection of nonmutated Gab1 led to a strong increase of Rap1.GTP in the absence of HGF. In contrast, transfection of the GabDeltaYXXP mutant abolished the elevation of Rap1.GTP by HGF. A replating assay indicated that HGF decreases the adhesion of HEK293 cells. The results presented here delineate a novel signaling pathway from HGF to the GTPase Rap1 which depends on the interaction of the adapter protein CRKL with the exchange factor C3G and could be linked to cell migration. PMID- 10753870 TI - Regulation of the rapamycin and FKBP-target 1/mammalian target of rapamycin and cap-dependent initiation of translation by the c-Abl protein-tyrosine kinase. AB - The c-Abl protein-tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents. The rapamycin and FKBP-target 1 (RAFT1), also known as FKBP12-rapamycin-associated protein (FRAP, mTOR), regulates the p70S6 kinase (p70(S6k)) and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E BP1). The present results demonstrate that c-Abl binds directly to RAFT1 and phosphorylates RAFT1 in vitro and in vivo. c-Abl inhibits autophosphorylation of RAFT1 and RAFT1-mediated phosphorylation p70(S6k). The functional significance of the c-Abl-RAFT1 interaction is further supported by the finding that eIF4E dependent translation in mouse embryo fibroblasts from Abl(-/-) mice is significantly higher than that compared in wild-type cells. The results also demonstrate that exposure of cells to ionizing radiation is associated with c-Abl mediated binding of 4E-BP1 to eIF4E and inhibition of translation. These findings with the c-Abl tyrosine kinase represent the first demonstration of a negative physiologic regulator of RAFT1-mediated 5' cap-dependent translation. PMID- 10753871 TI - Evidence that translocation of the proteinase precedes its acylation in the serpin inhibition pathway. AB - The inhibition of proteinases by serpins involves cleavage of the serpin, acylation, and translocation of the proteinase. To see whether acylation precedes or follows translocation, we have investigated the pH dependence of the interaction of fluorescein isothiocyanate-elastase with rhodamine alpha(1) proteinase inhibitor (alpha(1)PI) using two independent methods: (i) kinetics of fluorescence energy transfer which yields k(2,f), the rate constant for the fluorescently detected decay of the Michaelis-type complex (Mellet, P., Boudier, C., Mely, Y., and Bieth, J. G. (1998) J. Biol. Chem. 273, 9119-9123); (ii) kinetics of elastase-catalyzed hydrolysis of a substrate in the presence of alpha(1)PI, which yields k(2,e), the rate constant for the conversion of the Michaelis-type complex into irreversibly inhibited elastase. Both rate constants were found to be pH-independent and close to each other, indicating that acylation, a pH-dependent phenomenon, does not govern the decay of the Michaelis type complex and, therefore, follows translocation. On the other hand, anhydro elastase reacts with alpha(1)PI to form a Michaelis-type complex that translocates into a second complex with a rate constant close to that measured with active elastase, confirming that acylation is not a prerequisite for translocation. Moreover, the anhydro-elastase-alpha(1)PI complex was found to be thermodynamically reversible, suggesting that translocation of active elastase might also be reversible. We propose that serpins form a Michaelis-type complex EI(M), which reversibly translocates into EI(tr) whose acylation yields the irreversible complex EI(ac). [see text] PMID- 10753872 TI - Farnesyltransferase inhibitor induces rapid growth arrest and blocks p70s6k activation by multiple stimuli. AB - We have previously shown that the peptidomimetic farnesyltransferase inhibitor L 744,832 (FTI) inhibits p70s6k activation and cell growth in a mouse keratinocyte cell line but only at concentrations of FTI significantly higher than those required for the inhibition of Ras farnesylation. Here we show that the rapid kinetics of FTI inhibition of DNA synthesis (within 1.5 h) in both normal and v-K Ras transformed keratinocytes matches the rapid kinetics of p70s6k inhibition observed previously. It is further shown that FTI inhibits p70s6k activation in response to serum, phorbol myristate acetate, and increased amino acid levels. The phosphatase inhibitor calyculin A partially reverses the FTI-induced dephosphorylation of p70s6k, suggesting that FTI may act upstream of a protein phosphatase. A rapamycin-resistant mutant of p70s6k is shown to be resistant to FTI-induced dephosphorylation of the major rapamycin-sensitive phosphorylation site of p70s6k, Thr(389). Together, these data demonstrate that FTI rapidly inhibits DNA synthesis irrespective of the presence of v-K-Ras and that FTI inhibits p70s6k activation in response to multiple stimuli. Because the FTI L 744,832 mimics many of the effects of rapamycin, this FTI may prove effective against tumors that exhibit inappropriate activation of the mTOR/p70s6k pathway. PMID- 10753873 TI - Induction of the angiogenic modulator fibroblast growth factor-binding protein by epidermal growth factor is mediated through both MEK/ERK and p38 signal transduction pathways. AB - Fibroblast growth factor-binding protein (FGF-BP) is a secreted protein that binds and activates fibroblast growth factors (FGF-1 and FGF-2) and induces angiogenesis in some human cancers. FGF-BP is expressed at high levels in squamous cell carcinoma (SCC) cell lines and tumor samples and has been shown to be rate-limiting for the growth of SCC tumors in vivo. In this study, we examine the regulation of FGF-BP by epidermal growth factor (EGF) and the signal transduction mechanisms that mediate this effect. We found that EGF treatment of the ME-180 SCC cell line caused a rapid induction of FGF-BP gene expression. This induction was mediated transcriptionally through the AP-1 (c-Fos/JunD) and CCAAT/enhancer-binding protein elements as well as through an E-box repressor site in the proximal regulatory region of the FGF-BP promoter. Pharmacological inhibition of protein kinase C and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) completely blocked EGF induction of FGF-BP mRNA, whereas inhibition of phosphatidylinositol 3-kinase had no effect. Additionally, both EGF- and anisomycin-induced FGF-BP mRNA was abrogated by inhibition of p38 mitogen-activated protein kinase, demonstrating a role for p38 in the regulation of FGF-BP. Co-transfection of the FGF-BP promoter with dominant negative forms of MEK2, extracellular signal-regulated kinase 2, and p38 significantly decreased the level of EGF induction, whereas expression of a dominant negative c-Jun N-terminal kinase mutant or expression of c-Jun N terminal kinase inhibitory protein had no effect. Similarly, activation of the p38 pathway by overexpression of wild-type p38 or MKK6 enhanced FGF-BP transcription. These results demonstrate that EGF induction of FGF-BP occurs selectively through dual activation of the stress-activated p38 and the MEK/extracellular signal-regulated kinase mitogen-activated protein kinase pathways, which ultimately leads to activation of the promoter through AP-1 and CCAAT/enhancer-binding protein sites. PMID- 10753874 TI - Nitric oxide reaction with lipid peroxyl radicals spares alpha-tocopherol during lipid peroxidation. Greater oxidant protection from the pair nitric oxide/alpha tocopherol than alpha-tocopherol/ascorbate. AB - The reactions of nitric oxide ((.)NO) and alpha-tocopherol (alpha-TH) during membrane lipid oxidation were examined and compared with the pair alpha TH/ascorbate. Nitric oxide serves as a more potent inhibitor of lipid peroxidation propagation reactions than alpha-TH and protects alpha-TH from oxidation. Mass spectrometry, oxygen and (.)NO consumption, conjugated diene analyses, and alpha-TH fluorescence determinations all demonstrated that (.)NO preferentially reacts with lipid radical species, with alpha-TH consumption not occurring until (.)NO concentrations fell below a critical level. In addition, alpha-TH and (.)NO cooperatively inhibit lipid peroxidation, exhibiting greater antioxidant capacity than the pair alpha-TH/ascorbate. Pulse radiolysis analysis showed no direct reaction between (.)NO and alpha-tocopheroxyl radical (alpha T(.)), inferring that peroxyl radical termination reactions are the principal lipid-protective mechanism mediated by (.)NO. These observations support the concept that (.)NO is a potent chain breaking antioxidant toward peroxidizing lipids, due to facile radical-radical termination reactions with lipid radical species, thus preventing alpha-TH loss. The reduction of alpha-T(.) by ascorbate was a comparatively less efficient mechanism for preserving alpha-TH than (.)NO mediated termination of peroxyl radicals, due to slower reaction kinetics and limited transfer of reducing equivalents from the aqueous phase. Thus, the high lipid/water partition coefficient of (.)NO, its capacity to diffuse and concentrate in lipophilic milieu, and a potent reactivity toward lipid radical species reveal how (.)NO can play a critical role in regulating membrane and lipoprotein lipid oxidation reactions. PMID- 10753875 TI - Bimodal activation of acetyl-CoA carboxylase by glutamate. AB - Acetyl-CoA carboxylase (ACC) catalyzes the formation of malonyl-CoA, an essential substrate for fatty acid biosynthesis and a potent inhibitor of fatty acid oxidation. Here, we provide evidence that glutamate may be a physiologically relevant activator of ACC. Glutamate induced the activation of both major isoforms of ACC, prepared from rat liver, heart, or white adipose tissue. In agreement with previous studies, a type 2A protein phosphatase contributed to the effects of glutamate on ACC. However, the protein phosphatase inhibitor microcystin LR did not abolish the effects of glutamate on ACC activity. Moreover, glutamate directly activated purified preparations of ACC when protein phosphatase activity was excluded. Phosphatase-independent ACC activation by glutamate was also reflected by polymerization of the enzyme as judged by size exclusion chromatography. The sensitivity of ACC to direct activation by glutamate was diminished by treatment in vitro with AMP-activated protein kinase or cAMP-dependent protein kinase or by beta-adrenergic stimulation of intact adipose tissue. We conclude that glutamate, an abundant intracellular amino acid, induces ACC activation through complementary actions as a phosphatase activator and as a direct allosteric ligand for dephosphorylated ACC. This study supports the general hypothesis that amino acids fulfill important roles as signal molecules as well as intermediates in carbon and nitrogen metabolism. PMID- 10753876 TI - Reactive nitrogen and oxygen species attenuate interleukin- 8-induced neutrophil chemotactic activity in vitro. AB - Peroxynitrite, formed by the reaction between nitric oxide and superoxide, has been shown to induce protein nitration, which compromises protein function. We hypothesized that peroxynitrite may regulate cytokine function during inflammation. To test this hypothesis, the neutrophil chemotactic activity (NCA) of interleukin-8 (IL-8) incubated with peroxynitrite was evaluated. Peroxynitrite attenuated IL-8 NCA in a dose-dependent manner (p < 0.01) but did not significantly reduce NCA induced by leukotriene B(4) or complement-activated serum. The reducing agents, dithionite, deferoxamine, and dithiothreitol, reversed and exogenous L-tyrosine abrogated the peroxynitrite-induced NCA inhibition. Papa-NONOate [N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1, 2 dialase or sodium nitroprusside, NO donors, or a combination of xanthine and xanthine oxidase to generate superoxide did not show an inhibitory effect on NCA induced by IL-8. In contrast, small amounts of SIN-1, a peroxynitrite generator, caused a concentration-dependent inhibition of NCA by IL-8. Consistent with its capacity to reduce NCA, peroxynitrite treatment reduced IL-8 binding to neutrophils. Nitrotyrosine was detected in the IL-8 incubated with peroxynitrite by enzyme-linked immunosorbent assay. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of IL-8 binding to neutrophils and a reduction in NCA and suggest that oxidants may play an important role in regulation of IL-8-induced neutrophil chemotaxis. PMID- 10753877 TI - Short term feedback regulation of cAMP in FRTL-5 thyroid cells. Role of PDE4D3 phosphodiesterase activation. AB - Together with a transient accumulation of intracellular cAMP, thyrotropin (TSH) stimulation of the FRTL-5 thyroid cell induces phosphorylation and activation of a cAMP-specific phosphodiesterase (PDE4D3). Here we have investigated the impact of PDE4D3 activation on hormone responsiveness. Stimulation of FRTL-5 cells with TSH caused an increase in PDE activity within 3 min, with a maximal stimulation reached after 5 min. Preincubation with the protein kinase A (PKA) inhibitor H89 or (R(p))-cAMPS, but not with the inactive isomer H85, blocked this activation. Preincubation with PKA inhibitors also blocked the shift in mobility of the PDE4D3 protein. Under these conditions, H89, but not H85, potentiated the cAMP accumulation induced by TSH. Incubation of FRTL-5 cells with the PKA activator 8 (4-chlorophenylthio)adenosine-cAMP caused an increase in PDE activity and a decrease in the endogenous cAMP, confirming the presence of a PKA-PDE feedback loop. MA-10 Leydig tumor cells stably transfected with either a wild type PDE4D3 or a PDE4D3 with mutations in the PKA phosphorylation sites showed an increase in PDE activity when compared with control cells. Human choriogonadotropin or Bt(2)cAMP treatment induced a stimulation of PDE activity in cells transfected with wild type PDE4D3, whereas the activation was absent in mutant- and control transfected cells. The increase in cAMP accumulation elicited by human choriogonadotropin was reduced in cells transfected with the wild type PDE4D3, but not in cells transfected with the mutant PDE. Rolipram, a specific inhibitor of PDE4, restored the cAMP accumulation in the PDE4D3-transfected cells. These data provide evidence that a rapid activation of PDE4D3 is one of the mechanisms determining the intensity of the cAMP signal. PMID- 10753878 TI - Activation of NF-kappaB by FADD, Casper, and caspase-8. AB - Fas-associated death domain protein (FADD), caspase-8-related protein (Casper), and caspase-8 are components of the tumor necrosis factor receptor type 1 (TNF R1) and Fas signaling complexes that are involved in TNF-R1- and Fas-induced apoptosis. Here we show that overexpression of FADD and Casper potently activates NF-kappaB. In the presence of caspase inhibitors, overexpression of caspase-8 also activates NF-kappaB. A caspase-inactive point mutant, caspase-8(C360S), activates NF-kappaB as potently as wild-type caspase-8, suggesting that caspase-8 induced apoptosis and NF-kappaB activation are uncoupled. NF-kappaB activation by FADD and Casper is inhibited by the caspase-specific inhibitors crmA and BD-fmk, suggesting that FADD- and Casper-induced NF-kappaB activation is mediated by caspase-8. FADD, Casper, and caspase-8-induced NF-kappaB activation are inhibited by dominant negative mutants of TRAF2, NIK, IkappaB kinase alpha, and IkappaB kinase beta. A dominant negative mutant of RIP inhibits FADD- and caspase-8 induced but not Casper-induced NF-kappaB activation. A mutant of Casper and the caspase-specific inhibitors crmA and BD-fmk partially inhibit TNF-R1-, TRADD, and TNF-induced NF-kappaB activation, suggesting that FADD, Casper, and caspase-8 function downstream of TRADD and contribute to TNF-R1-induced NF-kappaB activation. Moreover, activation of caspase-8 results in proteolytic processing of NIK, which is inhibited by crmA. When overexpressed, the processed fragments of NIK do not activate NF-kappaB, and the processed C-terminal fragment inhibits TNF-R1-induced NF-kappaB activation. These data indicate that FADD, Casper, and pro-caspase-8 are parts of the TNF-R1-induced NF-kappaB activation pathways, whereas activated caspase-8 can negatively regulate TNF-R1-induced NF-kappaB activation by proteolytically inactivating NIK. PMID- 10753879 TI - Ca(2+)-activated but not G protein-mediated inositol phosphate responses in rat neonatal cardiomyocytes involve inositol 1,4, 5-trisphosphate generation. AB - Inositol phosphate (InsP) responses to receptor activation are assumed to involve phospholipase C cleavage of phosphatidylinositol 4,5-bisphosphate to generate Ins(1,4,5)P(3). However, in [(3)H]inositol-labeled rat neonatal cardiomyocytes (NCM) both initial and sustained [(3)H]InsP responses to alpha(1)-adrenergic receptor stimulation with norepinephrine (100 microM) were insensitive to the phosphatidylinositol 4,5-bisphosphate-binding agent neomycin (5 mM). Introduction of 300 microM unlabeled Ins(1,4, 5)P(3) into guanosine 5'-3-O-(thio)triphosphate (GTPgammaS)-stimulated, permeabilized [(3)H]inositol-labeled NCM increased [(3)H]Ins(1,4,5)P(3) slightly but did not significantly reduce levels of its metabolites [(3)H]Ins(1,4)P(2) and [(3)H]Ins(4)P, suggesting that these [(3)H]InsPs are not formed principally from [(3)H]Ins(1,4,5)P(3). In contrast, the calcium ionophore A23187 (10 microM) provoked [(3)H]InsP responses in intact NCM which were sensitive to neomycin, and elevation of free calcium in permeabilized NCM led to [(3)H]InsP responses characterized by marked increases in [(3)H]Ins(1,4,5)P(3) (2.9 +/- 0.2% of total [(3)H]InsPs after 20 min of high Ca(2+) treatment in comparison to 0. 21 +/- 0.05% of total [(3)H]InsPs accumulated after 20 min of GTPgammaS stimulation). These data provide evidence that Ins(1,4, 5)P(3) generation is not a major contributor to G protein-coupled InsP responses in NCM, but that substantial Ins(1,4,5)P(3) generation occurs under conditions of Ca(2+) overload. Thus in NCM, Ca(2+)-induced Ins(1,4,5)P(3) generation has the potential to worsen Ca(2+) overload and thereby aggravate Ca(2+)-induced electrophysiological perturbations. PMID- 10753880 TI - Reactions of hypochlorous acid with tyrosine and peptidyl-tyrosyl residues give dichlorinated and aldehydic products in addition to 3-chlorotyrosine. AB - The toxicity of hypochlorous acid (HOCl) generated from activated neutrophils has been associated with several pathological processes such as atherosclerosis. Formation of 3-chlorotyrosine (Cl-Tyr) has been used as a marker for assessing the involvement of HOCl in such processes. In this study, we aimed to investigate the formation of Cl-Tyr from reaction of HOCl with tyrosine (both free and peptide-bound) and the fate of Cl-Tyr under such conditions. Tyrosine, N acetyltyrosine, bovine serum albumin, and human low density lipoproteins were incubated with a range of reagent hypochlorite concentrations for varying periods in 10 mM phosphate buffer (pH 7.4) at 22 degrees C. The reaction products, and several biological samples, were hydrolyzed (in the case of proteins), isolated, and purified by high pressure liquid chromatography and characterized or quantitated by mass spectrometry and NMR. A significant amount of 3,5 dichlorotyrosine (diCl-Tyr) was obtained from the bovine serum albumin, low density lipoprotein, and some biological samples, in addition to Cl-Tyr, indicating that Cl-Tyr competes effectively for HOCl even when tyrosine is present in great excess. Cl-Tyr and diCl-Tyr were also formed from free tyrosine but then reacted further with HOCl. This finding differs from a claim in the literature that Cl-Tyr was not formed in such a system. The further reaction products of Cl-Tyr and diCl-Tyr with HOCl were elucidated as their corresponding mono- and dichlorinated 4-hydroxyphenylacetaldehydes. These results indicate the importance of assessing other products of HOCl action in addition to Cl-Tyr. PMID- 10753881 TI - Close association of the N terminus of Kv1.3 with the pore region. AB - The Shaker superfamily encodes voltage-gated potassium (Kv) channels. The N termini of Shaker proteins are located intracellularly and contain several domains shown to regulate important aspects of channel function, such as speed of inactivation, channel assembly (T1 domain), and steady state protein level (T0 domain, amino acids 3-39 in rabbit). Mutations and/or deletion of certain amino acids in the T0 domain lead to a 13-fold amplification of Kv current as compared with wild type channels, primarily by increasing the absolute number of channel proteins present in the membrane (Segal, A. S., Yao, X., and Desir, G. V. (1999) Biochem. Biophys. Res. Commun. 254, 54-64). Although T0 mutants have kinetic properties virtually indistinguishable from wild type, they were noted to have a slightly larger single channel conductance, suggesting that the T0 domain might also interact with the pore region. In the present study we show that although T0 does not affect pore selectivity, it does modulate the binding affinity of the pore blocker, charybdotoxin. These results suggest that the N terminus of Kv1.3 is closely associated with the pore region. PMID- 10753882 TI - Equilibrium binding of single-stranded DNA with herpes simplex virus type I-coded single-stranded DNA-binding protein, ICP8. AB - We have carried out solution equilibrium binding studies of ICP8, the major single-stranded DNA (ssDNA)-binding protein of herpes simplex virus type I, in order to determine the thermodynamic parameters for its interaction with ssDNA. Fluorescence anisotropy measurements of a 5'-fluorescein-labeled 32-mer oligonucleotide revealed that ICP8 formed a nucleoprotein filament on ssDNA with a binding site size of 10 nucleotides/ICP8 monomer, an association constant at 25 degrees C, K = 0.55 +/- 0.05 x 10(6) M(-1), and a cooperativity parameter, omega = 15 +/- 3. The equilibrium constant was largely independent of salt, deltalog(Komega)/deltalog([NaCl]) = -2.4 +/- 0.4. Comparison of these parameters with other ssDNA-binding proteins showed that ICP8 reacted with an unusual mechanism characterized by low cooperativity and weak binding. In addition, the reaction product was more stable at high salt concentrations, and fluorescence enhancement of etheno-ssDNA by ICP8 was higher than for other ssDNA-binding proteins. These last two characteristics are also found for protein-DNA complexes formed by recombinases in their active conformation. Given the proposed role of ICP8 in promoting strand transfer reactions, they suggest that ICP8 and recombinase proteins may catalyze homologous recombination by a similar mechanism. PMID- 10753883 TI - Identification and characterization of a novel inositol polyphosphate 5 phosphatase. AB - We have identified a cDNA encoding a novel inositol polyphosphate 5-phosphatase. It contains two highly conserved catalytic motifs for 5-phosphatase, has a molecular mass of 51 kDa, and is ubiquitously expressed and especially abundant in skeletal muscle, heart, and kidney. We designated this 5-phosphatase as SKIP (Skeletal muscle and Kidney enriched Inositol Phosphatase). SKIP is a simple 5 phosphatase with no other motifs. Baculovirus-expressed recombinant SKIP protein exhibited 5-phosphatase activities toward inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol (PtdIns) 4,5-bisphosphate, and PtdIns 3,4, 5-trisphosphate but has 6-fold more substrate specificity for PtdIns 4,5-bisphosphate (K(m) = 180 microM) than for inositol 1,4, 5-trisphosphate (K(m) = 1.15 mM). The ectopic expression of SKIP protein in COS-7 cells and immunostaining of neuroblastoma N1E-115 cells revealed that SKIP is expressed in cytosol and that loss of actin stress fibers occurs where the SKIP protein is concentrated. These results imply that SKIP plays a negative role in regulating the actin cytoskeleton through hydrolyzing PtdIns 4,5-bisphosphate. PMID- 10753884 TI - The differential regulation of group II(A) and group V low molecular weight phospholipases A(2) in cultured rat astrocytes. AB - In astrocytes, cytokines stimulate the release of secretory phospholipase A(2) (sPLA(2)) activity and group II(A) sPLA(2) expression. This paper reports that two sPLA(2) isoforms, group II(A) and group V, are in fact expressed by astrocytes. Our studies showed that tumor necrosis factor alpha (TNFalpha) enhanced the mRNA of both isoforms, but the time courses of enhancement differed; group V was induced much faster than group II(A). Moreover, TNFalpha stimulated both the NF-kappaB and mitogen-activated protein (MAP) kinase (extracellular signal-regulated kinase, c-Jun NH(2)-terminal kinase, and p38 MAP kinase) signaling pathways in astrocytes. Interestingly, PI 3-kinase activity also was enhanced by TNFalpha, and NF-kappaB pathway was involved in mediating its effect. Specific inhibitors were used to show that both extracellular signal-regulated kinase and p38 MAP kinase may contribute to the effect of TNFalpha and that blocking phosphatidylinositol 3-kinase activity fully reversed the effect of TNFalpha. Furthermore, in astrocytes, TNFalpha-induced release of sPLA(2) activity was partially reversed by thyroid hormone and almost abolished by growth factors. This phenomenon was accompanied by a less marked increase in both group II(A) and group V sPLA(2) mRNA. In the presence of growth factors, the increase in group V mRNA was inhibited early and transiently, in contrast to what was observed with group II(A), which was more persistently inhibited. Although a transcriptional effect of thyroid hormone or growth factors in astrocytes cannot be definitively excluded, both types of factor interfered with sPLA(2) expression in a manner suggesting the existence of regulation of post-transcriptional events. PMID- 10753885 TI - E2F family members are differentially regulated by reversible acetylation. AB - The six members of the E2F family of transcription factors play a key role in the control of cell cycle progression by regulating the expression of genes involved in DNA replication and cell proliferation. E2F-1, -2, and -3 belong to a structural and functional subfamily distinct from those of the other E2F family members. Here we report that E2F-1, -2, and -3, but not E2F-4, -5, and -6, associate with and are acetylated by p300 and cAMP-response element-binding protein acetyltransferases. Acetylation occurs at three conserved lysine residues located at the N-terminal boundary of their DNA binding domains. Acetylation of E2F-1 in vitro and in vivo markedly increases its binding affinity for a consensus E2F DNA-binding site, which is paralleled by enhanced transactivation of an E2F-responsive promoter. Acetylation of E2F-1 can be reversed by histone deacetylase-1, indicating that reversible acetylation is a mechanism for regulation also of non-histone proteins. PMID- 10753886 TI - Spinocerebellar ataxia type 6 mutation alters P-type calcium channel function. AB - Abnormal CAG repeat expansion in the alpha1A voltage-dependent calcium channel gene is associated with spinocerebellar ataxia type 6, an autosomal dominant cerebellar ataxia with a predominant loss of the Purkinje cell. A reverse transcriptase-polymerase chain reaction analysis of mRNA from mouse Purkinje cells revealed a predominant expression of the alpha1A channel lacking an asparagine-proline (NP) stretch in the domain IV (alpha1A(-NP)). Human alpha1A channels carrying various polyglutamine length with or without NP were expressed in HEK293 cells, and channel properties were compared using a whole-cell voltage clamp technique. alpha1A(-NP), corresponding to P-type channel, with 24 and 28 polyglutamines found in patients showed the voltage dependence of inactivation shifting negatively by 6 and 11 mV, respectively, from the 13 polyglutamine control. Contrarily, the alpha1A channel with NP (alpha1A(+NP)), corresponding to Q-type channel, with 28 polyglutamines exhibited a positive shift of 5 mV. These results suggest that altered function of alpha1A(-NP) may contribute to degeneration of Purkinje cells, which express predominantly alpha1A(-NP), due to the reduced Ca(2+) influx resulting from the negative shift of voltage-dependent inactivation. On the other hand, other types of neurons, expressing both alpha1A( NP) and alpha1A(+NP), may survive because the positive shift of voltage-dependent inactivation of alpha1A(+NP) compensates Ca(2+) influx. PMID- 10753887 TI - Polymerase arrest at the lambdaP(R) promoter during transcription initiation. AB - During transcription initiation by Escherichia coli RNA polymerase, a fraction of the homogeneous enzyme population has been kinetically shown to form two types of nonproductive complexes at some promoters: moribund complexes, which produce only abortive transcripts, and fully inactive ternary complexes (Kubori, T., and Shimamoto, N. (1996) J. Mol. Biol. 256, 449-457). Here we report biochemical isolation of the complexes arrested at the lambdaP(R) promoter and an analysis of their structure by DNA and protein footprintings. We found that the isolated promoter-arrested complexes retain a stoichiometric amount of sigma(70) subunit. Exonuclease III footprints of the arrested complexes are backtracked compared with that of the binary complex, and KMnO(4) footprinting reveals a decrease in the melting of DNA in the promoter region. Protein footprints of the retained sigma(70) have shown a more exposed conformation in region 3, compared with binary complexes. This feature is similar to that of the complexes arrested in inactive state during transcription elongation, indicating the existence of a common inactivating mechanism during transcription initiation and elongation. The possible involvement of the promoter arrest in transcriptional regulation is discussed. PMID- 10753888 TI - Plakoglobin regulates the expression of the anti-apoptotic protein BCL-2. AB - Plakoglobin is a cytoplasmic protein and a homologue of beta-catenin and Armadillo of Drosophila with similar adhesive and signaling functions. These proteins interact with cadherins to mediate cell-cell adhesion and associate with transcription factors to induce changes in the expression of genes involved in cell fate determination and proliferation. Unlike the relatively well characterized role of beta-catenin in cell proliferation via activation of c-MYC and cyclin D1 gene expression, the signaling function of plakoglobin in regulation of cell growth is undefined. Here, we show that high levels of plakoglobin expression in plakoglobin-deficient human SCC9 cells leads to uncontrolled growth and foci formation. Concurrent with the change in growth characteristics we observe a pronounced inhibition of apoptosis. This correlates with an induction of expression of BCL-2, a prototypic member of apoptosis regulating proteins. The BCL-2 expression coincides with decreased proteolytic processing and activation of caspase-3, an executor of programmed cell death. Our data suggest that the growth regulatory function of plakoglobin is independent of its role in mediating cell-cell adhesion. These observations clearly implicate plakoglobin in pathways regulating cell growth and provide initial evidence of its role as a pivotal molecular link between pathways regulating cell adherence and cell death. PMID- 10753889 TI - A new platelet receptor specific to type III collagen. Type III collagen-binding protein. AB - Platelet interaction with type III collagen is mediated by several platelet receptors that recognize specific sequences in collagen. We previously described an octapeptide KP*GEP*GPK within the alpha(1)III-CB4 fragment that binds to platelets and specifically inhibits platelet aggregation induced by type III collagen. In this study, we demonstrated that the octapeptide prevented platelet contact and spreading on type III collagen and subendothelium under static and flow conditions. Platelets adhered to the immobilized octapeptide, and anti bodies directed against other platelet collagen receptors (glycoprotein (GP) Ia/IIa, GP IV, p65, p47) did not impair this adhesion. The platelet octapeptide receptor was identified by ligand blotting as a protein doublet with molecular masses of 68 and 72 kDa and does not correspond to any other already known platelet collagen receptors (GP Ia, GP IV GP VI, and p65). Our results indicate that a specific type III collagen receptor, expressed on the platelet surface, is involved in the first stages of platelet type III collagen interaction. PMID- 10753890 TI - Farnesoid X receptor responds to bile acids and represses cholesterol 7alpha hydroxylase gene (CYP7A1) transcription. AB - Cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription is repressed by bile acids. The goal of this study is to elucidate the mechanism of CYP7A1 transcription by bile acid-activated farnesoid X receptor (FXR) in its native promoter and cellular context and to identify FXR response elements in the gene. In Chinese hamster ovary cells transfected with retinoid X receptor alpha (RXRalpha)/FXR, only chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) were able to stimulate a heterologous promoter/reporter containing an ecdysone response element. In HepG2 cells, all bile acids (25 microM) were able to repress CYP7A1/luciferase reporter activity, and only CDCA and DCA further repressed reporter activity when cotransfected with RXRalpha/FXR. The concentration of CDCA required to inhibit 50% of reporter activity (IC(50)) was determined to be approximately 25 microM without FXR and 10 microM with FXR. Deletion analysis revealed that the bile acid response element located between nucleotides -148 and -128 was the FXR response element, but RXRalpha/FXR did not bind to this sequence. These results suggest that bile acid-activated FXR exerts its inhibitory effect on CYP7A1 transcription by an indirect mechanism, in contrast to the stimulation and binding of FXR to intestinal bile acid-binding protein gene promoter. Results also reveal that bile acid receptors other than FXR are present in HepG2 cells. PMID- 10753891 TI - Inhibition of IkappaB kinase activity by sodium salicylate in vitro does not reflect its inhibitory mechanism in intact cells. AB - Sodium salicylate inhibits activation of the transcription factor NF-kappaB by blocking the phosphorylation and degradation of the NF-kappaB inhibitor IkappaBalpha. We previously demonstrated that salicylate inhibits IkappaBalpha degradation induced by tumor necrosis factor (TNF) but not by interleukin-1 (IL 1) and implicated p38 mitogen-activated protein kinase activation by salicylate in the inhibition of TNF-induced IkappaBalpha phosphorylation. Both TNF and IL-1 rapidly activate the IkappaB kinase (IKK) complex, containing the catalytic subunits IKKalpha and IKKbeta, which directly phosphorylates IkappaB proteins. Others have recently suggested that salicylate inhibits NF-kappaB activation by directly binding to IKKbeta. To clarify the mechanism whereby salicylate inhibits IKK activity, we examined its effects upon cytokine-induced IKK activity in intact cells and in vitro. Treatment of intact cells with salicylate inhibited TNF-induced but not IL-1-induced IKK activity, and this inhibition was prevented by the p38 inhibitor SB203580. In contrast, inhibition of IKK activity by salicylate in vitro was neither selective for TNF nor affected by SB203580. In vitro, salicylate treatment comparably inhibited the kinase activity of overexpressed IKKalpha and IKKbeta and also decreased p38 kinase activity. Therefore, direct inhibition of IKK activity in vitro does not reflect the inhibitory mechanism of salicylate in intact cells, which involves interference with TNF signaling. PMID- 10753892 TI - Liver hyperplasia and paradoxical regulation of glycogen metabolism and glucose sensitive gene expression in GLUT2-null hepatocytes. Further evidence for the existence of a membrane-based glucose release pathway. AB - We investigated the impact of GLUT2 gene inactivation on the regulation of hepatic glucose metabolism during the fed to fast transition. In control and GLUT2-null mice, fasting was accompanied by a approximately 10-fold increase in plasma glucagon to insulin ratio, a similar activation of liver glycogen phosphorylase and inhibition of glycogen synthase and the same elevation in phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNAs. In GLUT2-null mice, mobilization of glycogen stores was, however, strongly impaired. This was correlated with glucose-6-phosphate (G6P) levels, which remained at the fed values, indicating an important allosteric stimulation of glycogen synthase by G6P. These G6P levels were also accompanied by a paradoxical elevation of the mRNAs for L-pyruvate kinase. Re-expression of GLUT2 in liver corrected the abnormal regulation of glycogen and L-pyruvate kinase gene expression. Interestingly, GLUT2-null livers were hyperplasic, as revealed by a 40% increase in liver mass and 30% increase in liver DNA content. Together, these data indicate that in the absence of GLUT2, the G6P levels cannot decrease during a fasting period. This may be due to neosynthesized glucose entering the cytosol, being unable to diffuse into the extracellular space, and being phosphorylated back to G6P. Because hepatic glucose production is nevertheless quantitatively normal, glucose produced in the endoplasmic reticulum may also be exported out of the cell through an alternative, membrane traffic-based pathway, as previously reported (Guillam, M.-T., Burcelin, R., and Thorens, B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12317-12321). Therefore, in fasting, GLUT2 is not required for quantitative normal glucose output but is necessary to equilibrate cytosolic glucose with the extracellular space. In the absence of this equilibration, the control of hepatic glucose metabolism by G6P is dominant over that by plasma hormone concentrations. PMID- 10753893 TI - An investigation of the metabolism of isoleucine to active Amyl alcohol in Saccharomyces cerevisiae. AB - The metabolism of isoleucine to active amyl alcohol (2-methylbutanol) in yeast was examined by the use of (13)C nuclear magnetic resonance spectroscopy, combined gas chromatography-mass spectrometry, and a variety of mutants. From the identified metabolites a number of routes between isoleucine and active amyl alcohol seemed possible. All involved the initial decarboxylation of isoleucine to alpha-keto-beta-methylvalerate. The first, via branched chain alpha-ketoacid dehydrogenase to alpha-methylbutyryl-CoA, was eliminated because abolition of branched-chain alpha-ketoacid dehydrogenase in an lpd1 disruption mutant did not prevent the formation of active amyl alcohol. However, the lpd1 mutant still produced large amounts of alpha-methylbutyrate which initially seemed contradictory because it had been assumed that alpha-methylbutyrate was derived from alpha-methylbutyryl-CoA via acyl-CoA hydrolase. Subsequently it was observed that alpha-methylbutyrate arises from the non-enzymic oxidation of alpha methylbutyraldehyde (the immediate decarboxylation product of alpha-keto-beta methylvalerate). Mutant studies showed that one of the decarboxylases encoded by PDC1, PDC5, PDC6, YDL080c, or YDR380w must be present to allow yeast to utilize alpha-keto-beta-methylvalerate. Apparently, any one of this family of decarboxylases is sufficient to allow the catabolism of isoleucine to active amyl alcohol. This is the first demonstration of a role for the gene product of YDR380w, and it also shows that the decarboxylation steps for each alpha-keto acid in the catabolic pathways of leucine, valine, and isoleucine are accomplished in subtly different ways. In leucine catabolism, the enzyme encoded by YDL080c is solely responsible for the decarboxylation of alpha ketoisocaproate, whereas in valine catabolism any one of the isozymes of pyruvate decarboxylase will decarboxylate alpha-ketoisovalerate. PMID- 10753894 TI - E-cadherin is a WT1 target gene. AB - The WT1 tumor suppressor gene encodes a transcription factor that can activate and repress gene expression. Transcriptional targets relevant for the growth suppression functions of WT1 are poorly understood. We found that mesenchymal NIH 3T3 fibroblasts stably expressing WT1 exhibit growth suppression and features of epithelial differentiation including up-regulation of E-cadherin mRNA. Acute expression of WT1 in NIH 3T3 fibroblasts after retroviral infection induced murine E-cadherin expression. In transient transfection experiments, the human and murine E-cadherin promoters were activated by co-expression of WT1. E cadherin promoter activity was increased in cells overexpressing WT1 and was blocked by a dominant negative form of WT1. WT1 activated the murine E-cadherin promoter through a conserved GC-rich sequence similar to an EGR-1 binding site as well as through a CAAT box sequence. WT1 produced in vitro or derived from nuclear extracts bound to the WT1-response element within the murine E-cadherin promoter, but not the CAAT box. E-cadherin, a gene important in epithelial differentiation and neoplastic transformation, represents a downstream target gene that links the roles of the WT1 in differentiation and growth control. PMID- 10753895 TI - Cellular non-heme iron content is a determinant of nitric oxide-mediated apoptosis, necrosis, and caspase inhibition. AB - In this report, we tested the hypothesis that cellular content of non-heme iron determined whether cytotoxic levels of nitric oxide (NO) resulted in apoptosis versus necrosis. The consequences of NO exposure on cell viability were tested in RAW264.7 cells (a cell type with low non-heme iron levels) and hepatocytes (cells with high non-heme iron content). Whereas micromolar concentrations of the NO donor S-nitroso-N-acetyl-DL-penicillamine induced apoptosis in RAW264.7 cells, millimolar concentrations were required to induce necrosis in hepatocytes. Caspase-3 activation and cytochrome c release were evident in RAW264.7 cells, but only cytochrome c release was detectable in hepatocytes following high dose S nitroso-N-acetyl-DL-penicillamine exposure. Pretreating RAW264.7 cells with FeSO(4) increased intracellular non-heme iron to levels similar to those measured in hepatocytes and delayed NO-induced cell death, which then occurred in the absence of caspase-3 activation. Iron loading was also associated with the formation of intracellular dinitrosyl-iron complexes (DNIC) upon NO exposure. Cytosolic preparations containing DNIC as well as pure preparations of DNIC suppressed caspase activity. These data suggest that non-heme iron content is a key factor in determining the consequence of NO on cell viability by regulating the chemical fate of NO. PMID- 10753896 TI - Secondary and tertiary structure changes of reconstituted LmrA induced by nucleotide binding or hydrolysis. A fourier transform attenuated total reflection infrared spectroscopy and tryptophan fluorescence quenching analysis. AB - LmrA, a membrane protein of Lactococcus lactis, extrudes amphiphilic compounds from the inner leaflet of the cytoplasmic membrane, using energy derived from ATP hydrolysis. A combination of total reflection Fourier transform infrared spectroscopy, (2)H/H exchange, and fluorescence quenching experiments was used to investigate the effect of nucleotide binding and/or hydrolysis on the structure of LmrA reconstituted into proteoliposomes. These measurements allowed us to describe secondary structure changes of LmrA during the catalytic cycle. The structure of LmrA is enriched in beta-sheet after ATP binding, and the protein recovers its initial secondary structure after ATP hydrolysis, when P(i) has been released. (2)H/H exchange and fluorescence quenching studies indicate that the protein undergoes two distinct tertiary structure changes during the hydrolysis process. Indeed, the protein alone is poorly accessible to the aqueous medium but adopts a more accessible conformation when ATP hydrolysis takes place. After ATP hydrolysis, but when P(i) is still associated with the protein, the accessibility is intermediate between these two states. PMID- 10753897 TI - ERK1 and ERK2 activation by chemotactic factors in human eosinophils is interleukin 5-dependent and contributes to leukotriene C(4) biosynthesis. AB - Eosinophils, the major immune effector cells contributing to allergic inflammation and asthma, are profoundly affected by interleukin (IL) 5 with respect to their differentiation, viability, recruitment, and cytotoxic effector functions. IL-5 enhances eosinophil responsiveness to a variety of chemotactic factors via a process called priming, although the molecular mechanism is unknown. In this study, we report that, following IL-5 priming of eosinophils, chemotactic agents including fMet-Leu-Phe, IL-8, and RANTES, promote vigorous transient activation of ERK1 and ERK2. In contrast, these chemotactic factors stimulate weak or indiscernible ERK activation in unprimed eosinophils. Furthermore, this intracellular marker of priming is selective for IL-5-related cytokines, in that it is observed following exposure to IL-5 and granulocyte macrophage-colony stimulating factor but not to interferon-gamma, stem cell factor, tumor necrosis factor alpha, or IL-4. Interestingly, priming of chemoattractant-induced ERK activation is accompanied by an increase in association of tyrosine-phosphorylated proteins with the adapter protein Grb2. The biological relevance of ERK activation to IL-5 priming is supported by the observation that inhibition of ERK activity by treatment with the MEK inhibitors PD98059 or U0126 inhibited the release of leukotriene C(4) stimulated by fMet-Leu Phe in IL-5-primed eosinophils. These data provide evidence for a previously undescribed fundamental mechanism by which stimulation of IL-5 family receptors induces a rapid phenotypic alteration in the signal transduction pathways of chemotactic receptors, enabling their activation of the ERK1 and ERK2 pathway and contributing to the capacity of these cells to synthesize LTC(4). PMID- 10753898 TI - Ultraviolet B exposure of whole leaves of barley affects structure and functional organization of photosystem II. AB - This study examines the effects of ecologically important levels of ultraviolet B radiation on protein D1 turnover and stability and lateral redistribution of photosystem II. It is shown that ultraviolet B light supported only limited synthesis of protein D1, one of the most important components of photosystem II, whereas it promoted significant degradation of proteins D1 and D2. Furthermore, dephosphorylation of photosystem II subunits was specifically elicited upon exposure to ultraviolet B light. Structural modifications of photosystem II and changes in its lateral distribution between granum membranes and stroma-exposed lamellae were found to be different from those observed after photoinhibition by strong visible light. In particular, more complete dismantling of photosystem II cores was observed. Altogether, the data reported here suggest that ultraviolet B radiation alone fails to activate the photosystem II repair cycle, as hypothesized for visible light. This failure may contribute to the toxic effect of ultraviolet B radiation, which is increasing as a consequence of depletion of stratospheric ozone. PMID- 10753899 TI - Functional expression of O-linked GlcNAc transferase. Domain structure and substrate specificity. AB - O-GlcNAc transferase (OGT) modifies nuclear pore proteins and transcription factors. In Arabidopsis, the OGT homolog participates in the gibberellin signaling pathway. We and others have proposed that mammalian OGT is the terminal step in a glucose-sensitive signal transduction pathway that becomes disregulated in insulin resistance. To facilitate mutational analysis of OGT in the absence of competing endogenous activity, we expressed the 103-kDa human OGT in Escherichia coli. Kinetic parameters for the purified recombinant enzyme (K(m) = 1.2 microM for Nup 62; K(m) = 0.5 microM for UDP-GlcNAc) are nearly identical to purified mammalian OGT. Deletions in the highly conserved C terminus result in a complete loss of activity. The N-terminal tetratricopeptide repeat domain is required for optimal recognition of substrates. Removal of the first three tetratricopeptide repeats greatly reduces the O-GlcNAc addition to macromolecular substrates. However, this altered enzyme retains full activity against appropriate synthetic peptides. Autoglycosylation of OGT is augmented when the first six tetratricopeptide repeats are removed showing that these repeats are not required for catalysis. Given its proposed role in modulating insulin action, OGT may modify kinases involved in this signaling cascade. Among the many kinases tested, OGT glycosylates glycogen synthase kinase-3 and casein kinase II, two enzymes critical in the regulation of glycogen synthesis. PMID- 10753900 TI - The major myosin-binding site of caldesmon resides near its N-terminal extreme. AB - The primary myosin-binding site of caldesmon was thought to be in the N-terminal region of the molecule, but the exact nature of the caldesmon-myosin interaction has not been well characterized. A caldesmon fragment that encompasses residues 1 240 (N240) was found to bind full-length smooth muscle myosin on the basis of co sedimentation experiments. The interaction between myosin and N240 was not affected by phosphorylation of myosin, but it was weakened by the presence of Ca(2+)/calmodulin. To locate the myosin-binding site, we have designed several synthetic peptides based on the N-terminal caldesmon sequence. We found that a peptide stretch corresponding to the first 27 residues (Met-1 to Tyr-27), but not that of the first 22 residues (Met-1 to Ala-22), exhibited a moderate affinity toward myosin. We also found that a peptide containing the segment from Ile/Leu 25 to Lys-53 bound both myosin and heavy meromyosin more strongly and was capable of displacing caldesmon from myosin. Our results demonstrate that the sequence near the N-terminal extreme of caldesmon harbors a major myosin-binding site of caldesmon, in which both the nonpolar residues and clusters of positively and negatively charged residues confer the specificity and affinity of the caldesmon myosin interaction. PMID- 10753901 TI - Mapping sonic hedgehog-receptor interactions by steric interference. AB - We have defined regions in the Sonic hedgehog (Shh) molecule that are important for Patched (Ptc) receptor binding by targeting selected surface amino acid residues with probes of diverse sizes and shapes and assessing the effects of these modifications on function. Eleven amino acid residues that surround the surface of the protein were chosen for these studies and mutated to cysteine residues. These cysteines were then selectively modified with thiol-specific probes, and the modified proteins were tested for hedgehog receptor binding activity and their ability to induce differentiation of C3H10T1/2 cells into osteoblasts. Based on these analyses, approximately one-third of the Shh surface can be modified without effect on function regardless of the size of the attachment. These sites are located near to where the C terminus protrudes from the surface of the protein. All other sites were sensitive to modification, indicating that the interaction of Shh with its primary receptor Ptc is mediated over a large surface of the Shh protein. For sites Asn-50 and Ser-156, function was lost with the smallest of the probes tested, indicating that these residues are in close proximity to the Ptc-binding site. The epitope for the neutralizing mAb 5E1 mapped to a close but distinct region of the structure. The structure activity data provide a unique view of the interactions between Shh and Ptc that is not readily attainable by conventional mapping strategies. PMID- 10753902 TI - Antibacterial agents that target lipid A biosynthesis in gram-negative bacteria. Inhibition of diverse UDP-3-O-(r-3-hydroxymyristoyl)-n-acetylglucosamine deacetylases by substrate analogs containing zinc binding motifs. AB - UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the second step in the biosynthesis of lipid A, a unique amphiphilic molecule found in the outer membranes of virtually all Gram-negative bacteria. Since lipid A biosynthesis is required for bacterial growth, inhibitors of LpxC have potential utility as antibiotics. The enzymes of lipid A biosynthesis, including LpxC, are encoded by single copy genes in all sequenced Gram-negative genomes. We have now cloned, overexpressed, and purified LpxC from the hyperthermophile Aquifex aeolicus. This heat-stable LpxC variant (the most divergent of all known LpxCs) displays 32% identity and 51% similarity over 277 amino acid residues out of the 305 in Escherichia coli LpxC. Although A. aeolicus LpxC deacetylates the substrate UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine at a rate comparable with E. coli LpxC, a phenyloxazoline-based hydroxamate that inhibits E. coli LpxC with K(i) of approximately 50 nM (Onishi, H. R., Pelak, B. A., Gerckens, L. S., Silver, L. L., Kahan, F. M., Chen, M. H., Patchett, A. A., Galloway, S. M., Hyland, S. A., Anderson, M. S., and Raetz, C. R. H. (1996) Science 274, 980-982) does not inhibit A. aeolicus LpxC. To determine whether or not broad-spectrum deacetylase inhibitors can be found, we have designed a new class of hydroxamate containing inhibitors of LpxC, starting with the structure of the physiological substrate. Several of these compounds inhibit both E. coli and A. aeolicus LpxC at similar concentrations. We have also identified a phosphinate-containing substrate analog that inhibits both E. coli and A. aeolicus LpxC, suggesting that the LpxC reaction proceeds by a mechanism similar to that described for other zinc metalloamidases, like carboxypeptidase A and thermolysin. The differences between the phenyloxazoline and the substrate-based LpxC inhibitors might be exploited for developing novel antibiotics targeted either against some or all Gram-negative strains. We suggest that LpxC inhibitors with antibacterial activity be termed "deacetylins." PMID- 10753903 TI - Purification and structural determination of SCB1, a gamma-butyrolactone that elicits antibiotic production in Streptomyces coelicolor A3(2). AB - Early stationary phase culture supernatants of Streptomyces coelicolor A3(2) contained at least four small diffusible signaling molecules that could elicit precocious antibiotic synthesis in the producing strain. The compounds were not detected in exponentially growing cultures. One of these compounds, SCB1, was purified to homogeneity and shown to be a gamma-butyrolactone of structure (2R, 3R,1'R)-2-(1'-hydroxy-6-methylheptyl)-3-hydroxymethylbutanolide . Bioassays of chemically synthesized SCB1, and of its purified stereoisomers, suggest that SCB1 acts in a highly specific manner to elicit the production of both actinorhodin and undecylprodigiosin, the two pigmented antibiotics made by S. coelicolor. PMID- 10753904 TI - Acylation-dependent protein export in Leishmania. AB - The surface of the protozoan parasite Leishmania is unusual in that it consists predominantly of glycosylphosphatidylinositol-anchored glycoconjugates and proteins. Additionally, a family of hydrophilic acylated surface proteins (HASPs) has been localized to the extracellular face of the plasma membrane in infective parasite stages. These surface polypeptides lack a recognizable endoplasmic reticulum secretory signal sequence, transmembrane spanning domain, or glycosylphosphatidylinositol-anchor consensus sequence, indicating that novel mechanisms are involved in their transport and localization. Here, we show that the N-terminal domain of HASPB contains primary structural information that directs both N-myristoylation and palmitoylation and is essential for correct localization of the protein to the plasma membrane. Furthermore, the N-terminal 18 amino acids of HASPB, encoding the dual acylation site, are sufficient to target the heterologous Aequorea victoria green fluorescent protein to the cell surface of Leishmania. Mutagenesis of the predicted acylated residues confirms that modification by both myristate and palmitate is required for correct trafficking. These data suggest that HASPB is a representative of a novel class of proteins whose translocation onto the surface of eukaryotic cells is dependent upon a "non-classical" pathway involving N-myristoylation/palmitoylation. Significantly, HASPB is also translocated on to the extracellular face of the plasma membrane of transfected mammalian cells, indicating that the export signal for HASPB is recognized by a higher eukaryotic export mechanism. PMID- 10753905 TI - A rationalization of the acidic pH dependence for stromelysin-1 (Matrix metalloproteinase-3) catalysis and inhibition. AB - The pH dependence of matrix metalloproteinase (MMP) catalysis is described by a broad bell-shaped curve, indicating the involvement of two unspecified ionizable groups in proteolysis. Stromelysin-1 has a third pK(a) near 6, resulting in a uniquely sharp acidic catalytic optimum, which has recently been attributed to His(224). This suggests the presence of a critical, but unidentified, S1' substructure. Integrating biochemical characterizations of inhibitor-enzyme interactions with active site topography from corresponding crystal structures, we isolated contributions to the pH dependence of catalysis and inhibition of active site residues Glu(202) and His(224). The acidic pK(a) 5.6 is attributed to the Glu(202).zinc.H(2)O complex, consistent with a role for the invariant active site Glu as a general base in MMP catalysis. The His(224)-dependent substructure is identified as a tripeptide (Pro(221)-Leu(222)-Tyr(223)) that forms the substrate cleft lower wall. Substrate binding induces a beta-conformation in this sequence, which extends and anchors the larger beta-sheet of the enzyme. substrate complex and appears to be essential for productive substrate binding. Because the PXY tripeptide is strictly conserved among MMPs, this "beta-anchor" may represent a common motif required for macromolecular substrate hydrolysis. The striking acidic profile of stromelysin-1 defined by the combined ionization of Glu(202) and His(224) allows the design of highly selective inhibitors. PMID- 10753906 TI - Identification and characterization of all-trans-retinol dehydrogenase from photoreceptor outer segments, the visual cycle enzyme that reduces all-trans retinal to all-trans-retinol. AB - Retinol dehydrogenase (RDH), the enzyme that catalyzes the reduction of all-trans retinal to all-trans-retinol within the photoreceptor outer segment, was the first visual cycle enzymatic activity to be identified. Previous work has shown that this enzyme utilizes NADPH, shows a marked preference for all-trans-retinal over 11-cis-retinal, and is tightly associated with the outer segment membrane. This paper reports the identification of a novel member of the short chain dehydrogenase/reductase family, photoreceptor RDH (prRDH), using subtraction and normalization of retina cDNA, high throughput sequencing, and data base homology searches to detect retina-specific genes. Bovine and human prRDH are highly homologous and are most closely related to 17-beta-hydroxysteroid dehydrogenase 1. The enzymatic properties of recombinant bovine prRDH closely match those previously reported for RDH activity in crude bovine rod outer segment preparations. In situ hybridization and RNA blotting show that the PRRDH gene is expressed specifically in photoreceptor cells, and protein blotting and immunocytochemistry show that prRDH localizes exclusively to both rod and cone outer segments and that prRDH is tightly associated with outer segment membranes. Taken together, these data indicate that prRDH is the enzyme responsible for the reduction of all-trans-retinal to all-trans-retinol within the photoreceptor outer segment. PMID- 10753907 TI - Localization of von willebrand factor-binding sites for platelet glycoprotein Ib and botrocetin by charged-to-alanine scanning mutagenesis. AB - At sites of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion through binding to platelet glycoprotein Ib (GPIb). Previous studies identified clusters of charged residues within VWF domain A1 that were involved in binding GPIb or botrocetin. The contribution of 28 specific residues within these clusters was analyzed by mutating single amino acids to alanine. Binding to a panel of six conformation-dependent monoclonal antibodies was decreased by mutations at Asp(514), Asp(520), Arg(552), and Arg(611) (numbered from the N terminal Ser of the mature processed VWF), suggesting that these residues are necessary for domain A1 folding. Binding of (125)I-botrocetin was decreased by mutations at Arg(629), Arg(632), Arg(636), and Lys(667). Ristocetin-induced and botrocetin-induced binding to GPIb both were decreased by mutations at Lys(599), Arg(629), and Arg(632); among this group the K599A mutant was unique because (125)I-botrocetin binding was normal, suggesting that Lys(599) interacts directly with GPIb. Ristocetin and botrocetin actions on VWF were dissociated readily by mutagenesis. Ristocetin-induced binding to GPIb was reduced selectively by substitutions at positions Lys(534), Arg(571), Lys(572), Glu(596), Glu(613), Arg(616), Glu(626), and Lys(642), whereas botrocetin-induced binding to GPIb was decreased selectively by mutations at Arg(636) and Lys(667). The binding of monoclonal antibody B724 involved Lys(660) and Arg(663), and this antibody inhibits (125)I-botrocetin binding to VWF. The crystal structure of the A1 domain suggests that the botrocetin-binding site overlaps the monoclonal antibody B724 epitope on helix 5 and spans helices 4 and 5. The binding of botrocetin also activates the nearby VWF-binding site for GPIb that involves Lys(599) on helix 3. PMID- 10753908 TI - Molecular and biochemical characterization of a serine proteinase predominantly expressed in the medulla oblongata and cerebellar white matter of mouse brain. AB - A full-length cDNA clone of a serine proteinase, mouse brain serine proteinase (mBSP), was isolated from a mouse brain cDNA library. mBSP, which has been recently reported to be expressed in the hair follicles of nude mice, is most similar (88% identical) in sequence to rat myelencephalon-specific protease. The mBSP mRNA was steadily expressed in the brain of adult mice with a transient expression in the early fetal stage during development. The genomic structure of the mouse gene for mBSP was determined. The gene, which is mapped to chromosome 7B4-B5, is about 7.4 kilobases in size and contains 7 exons. Interestingly, the 5'-untranslated region of the mBSP gene was interrupted by two introns. In situ hybridization analyses revealed that mBSP is expressed in the white matter of the cerebellum, medulla oblongata, and capsula interna and capsula interna pars retrolenticularis of mouse brain. Further, mBSP was immunolocalized to the neuroglial cells in the white matter of the cerebellum. Recombinant mBSP was produced in the bacterial expression system and activated by lysyl endopeptidase digestion, and the activated enzyme was purified for characterization. The enzyme showed amidolytic activities preferentially cleaving Arg-X bonds when 4 methylcoumaryl-7-amide-containing peptide substrates were used. Typical serine proteinase inhibitors, such as diisopropyl fluorophosphates, phenylmethanesulfonyl fluoride, soybean trypsin inhibitor, aprotinin, leupeptin, antipain, and benzamidine, strongly inhibited the enzyme activity. The recombinant mBSP effectively hydrolyzed fibronectin and gelatin, but not laminin, collagens I and IV, or elastin. These results suggest that mBSP plays an important role in association with the function of the adult mouse brain. PMID- 10753910 TI - Rho GTPase control of protein kinase C-related protein kinase activation by 3 phosphoinositide-dependent protein kinase. AB - The protein kinase C-related protein kinases (PRKs) have been shown to be under the control of the Rho GTPases and influenced by autophosphorylation. In analyzing the relationship between these inputs, it is shown that activation in vitro and in vivo involves the activation loop phosphorylation of PRK1/2 by 3 phosphoinositide-dependent protein kinase-1 (PDK1). Rho overexpression in cultured cells is shown to increase the activation loop phosphorylation of endogenous PRKs and is demonstrated to influence this process by controlling the ability of PRKs to bind to PDK1. The interaction of PRK1/2 with PDK1 is shown to be dependent upon Rho. Direct demonstration of ternary (Rho.PRK.PDK1) complex formation in situ is provided by the observation that PDK1 is recruited to RhoB containing endosomes only if PRK is coexpressed. Furthermore, this in vivo complex is maintained after phosphoinositide 3-kinase inhibition. The control of PRKs by PDK1 thus evidences a novel strategy of substrate-directed control involving GTPases. PMID- 10753909 TI - Bacterial lipopolysaccharide activates NF-kappaB through toll-like receptor 4 (TLR-4) in cultured human dermal endothelial cells. Differential expression of TLR-4 and TLR-2 in endothelial cells. AB - A missense mutation in the cytoplasmic domain of the Toll-like receptor-4 (TLR-4) has been identified as the defect responsible for lipopolysaccharide (LPS) hyporesponsiveness in C3H/HeJ mice. TLR-4 and TLR-2 have recently been implicated in LPS signaling in studies where these receptors were overexpressed in LPS non responsive 293 human embryonic kidney cells. However, the signaling role of TLR-4 or TLR-2 in human cells with natural LPS response remains largely undefined. Here we show that human dermal microvessel endothelial cells (HMEC) and human umbilical vein endothelial cells express predominantly TLR-4 but very weak TLR-2 and respond vigorously to LPS but not to Mycobacterium tuberculosis 19-kDa lipoprotein. Transient transfection of non-signaling mutant forms of TLR-4 and anti-TLR-4 monoclonal antibody inhibited LPS-induced NF-kappaB activation in HMEC, while a monoclonal antibody against TLR-2 was ineffective. In contrast to LPS responsiveness, the ability of HMEC to respond to 19-kDa lipoprotein correlated with the expression of TLR-2. Transfection of TLR-2 into HMEC conferred responsiveness to 19-kDa lipoprotein. These data indicate that TLR-4 is the LPS signaling receptor in HMEC and that human endothelial cells (EC) express predominantly TLR-4 and weak TLR-2, which may explain why they do not respond to 19-kDa lipoprotein. The differential expression of TLRs on human EC may have important implications in the participation of vascular EC in innate immune defense mechanisms against various infectious pathogens, which may use different TLRs to signal. PMID- 10753911 TI - Mutation of Arg(273) to Leu alters the specificity of the yeast N-glycan processing class I alpha1,2-mannosidase. AB - Class I alpha1,2-mannosidases (glycosyl hydrolase family 47) involved in the processing of N-glycans during glycoprotein maturation have different specificities. Enzymes in the endoplasmic reticulum of yeast and mammalian cells remove a single mannose from Man(9)GlcNAc(2) to form Man(8)GlcNAc(2) isomer B (lacking the alpha1, 2-mannose residue of the middle alpha1, 3-arm), whereas other alpha1,2-mannosidases, including Golgi alpha1,2-mannosidases IA and IB, can convert Man(9)GlcNAc(2) to Man(5)GlcNAc(2). In the present work, it is demonstrated that with a single mutation in its catalytic domain (Arg(273) --> Leu) the yeast endoplasmic reticulum alpha1,2-mannosidase acquires the ability to transform Man(9)GlcNAc to Man(5)GlcNAc. High resolution proton nuclear magnetic resonance analysis of the products shows that the order of removal of mannose from Man(9)GlcNAc is different from that of other alpha1, 2-mannosidases that remove four mannose from Man(9)GlcNAc. These results demonstrate that Arg(273) is in part responsible for the specificity of the endoplasmic reticulum alpha1,2 mannosidase and that small differences in non-conserved amino acids interacting with the oligosaccharide substrate in the active site of class I alpha1, 2 mannosidases are responsible for the different specificities of these enzymes. PMID- 10753912 TI - Structure, functioning, and assembly of the ATP synthase in cells from patients with the T8993G mitochondrial DNA mutation. Comparison with the enzyme in Rho(0) cells completely lacking mtdna. AB - The structure and functioning of the ATP synthase of human fibroblast cell lines with 91 and 100%, respectively, of the T8993G mutation have been studied, with MRC5 human fibroblasts and Rho(0) cells derived from this cell line as controls. ATP hydrolysis was normal but ATP synthesis was reduced by 60% in the 100% mutants. Both activities were highly oligomycin-sensitive. The levels of F(1)F(0) were close to normal, and the enzyme was stable. It is concluded that the loss of ATP synthesis is because of disruption of the proton translocation step within the F(0) part. This is supported by membrane potential measurements using the dye JC-1. Cells with a 91% mutation load grew well and showed only a 25% loss in ATP synthesis. This much reduced effect for only a 9% difference in mutation load mirrors the reduced pathogenicity in patients. F(1)F(0) has been purified for the first time from human cell lines. A partial complex was obtained from Rho(0) cells containing the F(1) subunits associated with several stalk, as well as F(0) subunits, including oligomycin sensitivity conferring protein, b, and c subunits. This partial complex no longer binds inhibitor protein. PMID- 10753913 TI - Reversible suppression of in vitro biomineralization by activation of protein kinase A. AB - Parathyroid hormone (PTH-(1-34)) potently suppresses apatite deposition in osteoblastic cultures. These inhibitory effects are mediated through signaling events following PTH receptor binding. Using both selective inhibitors and activators of protein kinase A (PKA), this study shows that a transient activation of PKA is sufficient to account for PTH's inhibition of apatite deposition. This inhibition is not a result of reduced cell proliferation, reduced alkaline phosphatase activity, increased collagenase production, or lowering medium pH. Rather, data suggest a functional relationship between matrix assembly and apatite deposition in vitro. Bone sialoprotein (BSP) and apatite co localize in the extracellular matrix of mineralizing cultures, with matrix deposition of BSP temporally preceding that of apatite. Transient activation of PKA by either PTH-(1-34) or short term cAMP analog treatment blocks the deposition of BSP in the extracellular matrix without a significant reduction in the total amount of BSP synthesized and secreted. This effect is reversible after allowing the cultures to recover in the absence of PKA activators for several days. Thus, a transient activation of PKA may suppress mineral deposition in vitro as a consequence of altering the assembly of an extracellular matrix permissive for apatite formation. PMID- 10753914 TI - Structural basis of BFL-1 for its interaction with BAX and its anti-apoptotic action in mammalian and yeast cells. AB - BFL-1 is the smallest member of the BCL-2 family and has been shown to retard apoptosis in various cell lines. However, the structural basis for its function remains unclear. Molecular modeling showed that BFL-1 could have a similar core structure as BCL-xL, consisting of seven alpha helices, although both proteins share only the conserved BCL-2 homology domains (BH1 and BH2 domains), but otherwise have very limited sequence homology, particularly in the N-terminal region. We demonstrated in the yeast two-hybrid system that BFL-1 interacts strongly with human BAX but is not able to form homodimers nor to interact with human BCL-2 or BCL-xL. Overexpression experiments in REF52 rat fibroblasts showed that BFL-1 conferred increased resistance to apoptosis induced by serum deprivation. BFL-1 had also the ability to neutralize BAX lethality in yeast. BAX requires the BH3 domain for interaction with BFL-1. However, the minimal region of BFL-1 for the interaction with BAX in coimmunoprecipitation experiments was not sufficient to protect cells from apoptosis. Further examination of BFL-1 and several other anti-apoptotic proteins suggests a more general type of structure based on structural motifs, i.e. a hydrophobic pocket for the binding of proapoptotic proteins, rather than extended sequence homologies. PMID- 10753915 TI - CLP-36 PDZ-LIM protein associates with nonmuscle alpha-actinin-1 and alpha actinin-4. AB - The PDZ-LIM family of proteins (Enigma/LMP-1, ENH, ZASP/Cypher, RIL, ALP, and CLP 36) has been suggested to act as adapters that direct LIM-binding proteins to the cytoskeleton. Most interactions of PDZ-LIM proteins with the cytoskeleton have been identified in striated muscle, where several PDZ-LIM proteins are predominantly expressed. By contrast, CLP-36 mRNA is expressed in several nonmuscle tissues, and here we demonstrate high expression of CLP-36 in epithelial cells by in situ hybridization analysis. Our subcellular localization studies indicate that in nonmuscle cells, CLP-36 protein localizes to actin stress fibers. This localization is mediated via the PDZ domain of CLP-36 that associates with the spectrin-like repeats of alpha-actinin. Interestingly, immunoprecipitation and matrix-assisted laser desorption/ionization time-of flight mass spectrometry analysis indicate that both nonmuscle alpha-actinin-1 and alpha-actinin-4 form complexes with CLP-36. The high expression of alpha actinin-4 in the colon, together with these results, suggests a specific function for the alpha-actinin-4-CLP-36 complex in the colonic epithelium. More generally, results presented here demonstrate that the association of PDZ-LIM proteins with the cytoskeleton extends to the actin stress fibers of nonmuscle cells. PMID- 10753916 TI - Control of O-glycan branch formation. Molecular cloning and characterization of a novel thymus-associated core 2 beta1, 6-n-acetylglucosaminyltransferase. AB - Core 2 O-glycan branching catalyzed by UDP-N-acetyl-alpha-D-glucosamine: acceptor beta1, 6-N-acetylglucosaminyltransferases (beta6GlcNAc-Ts) is an important step in mucin-type biosynthesis. Core 2 complex-type O-glycans are involved in selectin-mediated adhesion events, and O-glycan branching appears to be highly regulated. Two homologous beta6GlcNAc-Ts functioning in O-glycan branching have previously been characterized, and here we report a third homologous beta6GlcNAc T designated C2GnT3. C2GnT3 was identified by BLAST analysis of human genome survey sequences. The catalytic activity of C2GnT3 was evaluated by in vitro analysis of a secreted form of the protein expressed in insect cells. The results revealed exclusive core 2 beta6GlcNAc-T activity. The product formed with core 1 para-nitrophenyl was confirmed by (1)H NMR to be core 2-para-nitrophenyl. In vivo analysis of the function of C2GnT3 by coexpression of leukosialin (CD43) and a full coding construct of C2GnT3 in Chinese hamster ovary cells confirmed the core 2 activity and failed to reveal I activity. The C2GnT3 gene was located to 5q12, and the coding region was contained in a single exon. Northern analysis revealed selectively high levels of a 5.5-kilobase C2GnT3 transcript in thymus with only low levels in other organs. The unique expression pattern of C2GnT3 suggests that this enzyme serves a specific function different from other members of the beta6GlcNAc-T gene family. PMID- 10753917 TI - Regulatory mechanisms of TRAF2-mediated signal transduction by Bcl10, a MALT lymphoma-associated protein. AB - To elucidate the function of Bcl10, recently cloned as an apoptosis-associated gene mutated in MALT lymphoma, we identified its binding partner TRAF2, which mediates signaling via tumor necrosis factor receptors. In mammalian cells, low levels of Bcl10 expression promoted the binding of TRAF2 and c-IAPs. Conversely, excessive expression inhibited complex formation. Overexpressed Bcl10 reduced c Jun N-terminal kinase activation and induced nuclear factor kappaB activation downstream of TRAF2. To determine whether overexpression of Bcl10 could perturb the regulation of apoptosis in vivo, we generated Bcl10 transgenic mice. In these transgenic mice, atrophy of the thymus and spleen was observed at postnatal stages. The morphological changes in these tissues were caused by acceleration of apoptosis in T cells and B cells. The phenotype of Bcl10 transgenic mice was similar to that of TRAF2-deficient mice reported previously, indicating that excessive expression of Bcl10 might deplete the TRAF2 function. In contrast, in the other organs such as the brain, where Bcl10 was expressed at high levels, no apoptosis was detected. The altered sensitivities to overexpressed Bcl10 may have been due to differences in signal responses to Bcl10 among cell types. Thus, Bcl10 was suggested to play crucial roles in the modulation of apoptosis associated with TRAF2. PMID- 10753918 TI - Discrete signaling regions in the lymphotoxin-beta receptor for tumor necrosis factor receptor-associated factor binding, subcellular localization, and activation of cell death and NF-kappaB pathways. AB - Lymphotoxin-beta receptor (LTbetaR), a member of the tumor necrosis factor receptor superfamily, is essential for the development and organization of secondary lymphoid tissue. Wild type and mutant LTbetaR containing successive truncations of the cytoplasmic domain were investigated by retrovirus-mediated gene transfer into HT29.14s and in 293T cells by transfection. Wild type receptors accumulated in perinuclear compartments and enhanced responsiveness to ligand-induced cell death and ligand-independent activation of NFkappaB p50 dimers. Coimmunoprecipitation and confocal microscopy mapped the TRAF3 binding site to amino acids PEEGDPG at position 389. However, LTbetaR truncated at position Pro(379) acted as a dominant positive mutant that down-modulated surface expression and recruited TRAF3 to endogenous LTbetaR. This mutant exhibited ligand-independent cell death and activated NF-kappaB p50 dimers. By contrast, truncation at Gly(359) created a dominant-negative mutant that inhibited ligand induced cell death and activation of NF-kappaB p50/p65 heterodimers. This mutant also blocked accumulation of wild type receptor into perinuclear compartments, suggesting subcellular localization may be crucial for signal transduction. A cryptic TRAF-independent NF-kappaB activating region was identified. These mutants define discrete subregions of a novel proline-rich domain that is required for subcellular localization and signal transduction by the LTbetaR. PMID- 10753919 TI - Type-specific sorting of G protein-coupled receptors after endocytosis. AB - The beta(2)-adrenergic receptor (B2AR) and delta-opioid receptor (DOR) are structurally distinct G protein-coupled receptors (GPCRs) that undergo rapid, agonist-induced internalization by clathrin-coated pits. We have observed that these receptors differ substantially in their membrane trafficking after endocytosis. B2AR expressed in stably transfected HEK293 cells exhibits negligible (<10%) down-regulation after continuous incubation of cells with agonist for 3 h, as assessed both by radioligand binding (to detect functional receptors) and immunoblotting (to detect total receptor protein). In contrast, DOR exhibits substantial (>/=50%) agonist-induced down-regulation when examined by similar means. Degradation of internalized DOR is sensitive to inhibitors of lysosomal proteolysis. Flow cytometric and surface biotinylation assays indicate that differential sorting of B2AR and DOR between distinct recycling and non recycling pathways (respectively) can be detected within approximately 10 min after endocytosis, significantly before the onset of detectable proteolytic degradation of receptors ( approximately 60 min after endocytosis). Studies using pulsatile application of agonist suggest that after this sorting event occurs, later steps of membrane transport leading to lysosomal degradation of receptors do not require the continued presence of agonist in the culture medium. These observations establish that distinct GPCRs differ significantly in endocytic membrane trafficking after internalization by the same membrane mechanism, and they suggest a mechanism by which brief application of agonist can induce substantial down-regulation of receptors. PMID- 10753920 TI - Interaction in vivo and in vitro of the metastasis-inducing S100 protein, S100A4 (p9Ka) with S100A1. AB - The calcium-binding protein S100A4 (p9Ka) has been shown to cause a metastatic phenotype in rodent mammary tumor cells and in transgenic mouse model systems. mRNA for S100A4 (p9Ka) is present at a generally higher level in breast carcinoma than in benign breast tumor specimens, and the presence of immunocytochemically detected S100A4 correlates strongly with a poor prognosis for breast cancer patients. Recombinant S100A4 (p9Ka) has been reported to interact in vitro with cytoskeletal components and to form oligomers, particularly homodimers in vitro. Using the yeast two-hybrid system, a strong interaction between S100A4 (p9Ka) and another S100 protein, S100A1, was detected. Site-directed mutagenesis of conserved amino acid residues involved in the dimerization of S100 proteins abolished the interactions. The interaction between S100A4 and S100A1 was also observed in vitro using affinity column chromatography and gel overlay techniques. Both S100A1 and S100A4 can occur in the same cultured mammary cells, suggesting that in cells containing both proteins, S100A1 might modulate the metastasis-inducing capability of S100A4. PMID- 10753921 TI - Structural, kinetic, and calorimetric characterization of the cold-active phosphoglycerate kinase from the antarctic Pseudomonas sp. TACII18. AB - The gene encoding the phosphoglycerate kinase (PGK) from the Antarctic Pseudomonas sp. TACII18 has been cloned and found to be inserted between the genes encoding for glyceraldhyde-3-phosphate dehydrogenase and fructose aldolase. The His-tagged and the native recombinant PGK from the psychrophilic Pseudomonas were expressed in Escherichia coli. The wild-type and the native recombinant enzymes displayed identical properties, such as a decreased thermostability and a 2-fold higher catalytic efficiency at 25 degrees C when compared with the mesophilic PGK from yeast. These properties, which reflect typical features of cold-adapted enzymes, were strongly altered in the His-tagged recombinant PGK. The structural model of the psychrophilic PGK indicated that a key determinant of its low stability is the reduced number of salt bridges, surface charges, and aromatic interactions when compared with mesophilic and thermophilic PGK. Differential scanning calorimetry of the psychrophilic PGK revealed unusual variations in its conformational stability for the free and substrate-bound forms. In the free form, a heat-labile and a thermostable domain unfold independently. It is proposed that the heat-labile domain acts as a destabilizing domain, providing the required flexibility around the active site for catalysis at low temperatures. PMID- 10753922 TI - Cell type-specific E2F activation and cell cycle progression induced by the oncogene product Tax of human T-cell leukemia virus type I. AB - The transactivator protein Tax of human T-cell leukemia virus type I plays an important role in the development of adult T-cell leukemia probably through modulation of growth regulatory molecules including p16(INK4a). The molecular mechanism of leukemogenesis induced by Tax has yet to be elucidated. We analyzed Tax function in the cell cycle using an interleukin-2 (IL-2)-dependent human T cell line (Kit 225) that can undergo cell cycle arrest at G(0)/G(1) phase by deprivation of IL-2. Tax activated endogenous E2F activity in IL-2-starved Kit 225 cells, resulting in activation of E2F site-carrying promoters of genes involved in G(1) to S phase transition in a cell type-dependent and p16(INK4a) independent manner. The ability of Tax mutants to activate E2F coincided with that to activate nuclear factors kappaB and AT, sole expression of which, however, did not activate E2F, suggesting involvement of another pathway in activation of E2F. Introduction of Tax by a recombinant adenovirus induced cell cycle progression to G(2)/M phase in resting Kit 225 cells accompanied by endogenous cyclin D2 gene expression. Similarly, Tax-induced cell cycle progression was seen with peripheral blood lymphocytes prestimulated with phytohemagglutinin. Analyses with Tax mutants did not allow Tax-induced cell cycle progression to be differentiated from Tax-dependent activation of E2F, suggesting that Tax induces cell cycle progression presumably through activation of E2F. Nevertheless, infection with an E2F1-expressing virus, which is sufficient for induction of S phase in serum-starved fibroblasts, was not sufficient for either E2F activation or cell cycle progression in IL-2-starved Kit 225 cells, implying differential regulation of E2F activation and cell cycle progression in T-cells that is activated by Tax. PMID- 10753923 TI - Molecular cloning and characterization of a novel chloride intracellular channel related protein, parchorin, expressed in water-secreting cells. AB - We previously reported a 120-kDa phosphoprotein that translocated from cytosol to the apical membrane of gastric parietal cells in association with stimulation of HCl secretion. To determine the molecular identity of the protein, we performed molecular cloning and expression of the protein. Immunoblot analysis showed that this protein was highly enriched in tissues that secrete water, such as parietal cell, choroid plexus, salivary duct, lacrimal gland, kidney, airway epithelia, and chorioretinal epithelia. We named this protein "parchorin" based on its highest enrichment in parietal cells and choroid plexus. We obtained cDNA for parchorin from rabbit choroid plexus coding a protein consisting of 637 amino acids with a predicted molecular mass of 65 kDa. The discrepancy in size on 6% SDS-polyacrylamide gel electrophoresis is considered to be due to its highly acidic nature (pI = 4.18), because COS-7 cells transfected with parchorin cDNA produced a protein with apparent molecular mass of 120 kDa on 6% SDS polyacrylamide gel electrophoresis. Parchorin is a novel protein that has significant homology to the family of chloride intracellular channels (CLIC), especially the chloride channel from bovine kidney, p64, in the C-terminal 235 amino acids. When expressed as a fusion protein with green fluorescent protein (GFP) in the LLC-PK1 kidney cell line, GFP-parchorin, unlike other CLIC family members, existed mainly in the cytosol. Furthermore, when Cl(-) efflux from the cell was elicited, GFP-parchorin translocated to the plasma membrane. These results suggest that parchorin generally plays a critical role in water-secreting cells, possibly through the regulation of chloride ion transport. PMID- 10753924 TI - Elongin from Saccharomyces cerevisiae. AB - Elongin is a transcription elongation factor that was first identified in mammalian systems and is composed of the three subunits, elongin A, B, and C. Sequence homologues of elongin A and elongin C, but not elongin B, were identified in the yeast genome. Neither yeast elongin A nor C sequence homologues was required for cell viability. The two gene products could be purified from yeast as a complex. A recombinant form of the complex, which could only be produced in bacteria if the gene products were co-expressed, was purified over several chromatographic steps. The complex did not stimulate transcription elongation by yeast RNA polymerase II. Using limited proteolysis, the N-terminal 144 residues of yeast elongin A were shown to be sufficient for interaction with yeast elongin C. The purified complex of yeast elongin C/elongin A(1-143) was analyzed using circular dichroism and nuclear magnetic spectroscopy. These studies revealed that yeast elongin A is unfolded but undergoes a dramatic modification of its structure in the presence of elongin C, and that elongin C forms a stable dimer in the absence of elongin A. PMID- 10753925 TI - Monitoring cellular responses to Listeria monocytogenes with oligonucleotide arrays. AB - Listeria monocytogenes is a pathogenic intracellular microorganism whose infection induces pleiotropic biological changes associated with host cell gene expression regulation. Here we define the gene expression profiles of the human promyelocytic THP1 cell line before and after L. monocytogenes infection. Gene expression was measured on a large scale via oligonucleotide microarrays with probe sets corresponding to 6,800 human genes. We assessed and discussed the reproducibility of the hybridization signatures. In addition to oligonucleotide arrays, we also performed the large scale gene expression measurement with two high-density membranes, assaying for 588 and 18,376 human genes, respectively. This work allowed the reproducible identification of 74 up-regulated RNAs and 23 down-regulated RNAs as a consequence of L. monocytogenes infection of THP1. The reliability of these data was reinforced by performing independent infections. Some of these detected RNAs were consistent with previous results, while some newly identified RNAs encode gene products that may play key roles in L. monocytogenes infection. These findings will undoubtedly enhance the understanding of L. monocytogenes molecular physiology and may help identify new therapeutic targets. PMID- 10753926 TI - Enteroviral protease 2A directly cleaves dystrophin and is inhibited by a dystrophin-based substrate analogue. AB - Enteroviruses such as Coxsackievirus B3 can cause dilated cardiomyopathy through unknown pathological mechanism(s). Dystrophin is a large extrasarcomeric cytoskeletal protein whose genetic deficiency causes hereditary dilated cardiomyopathy. In addition, we have recently shown that dystrophin is proteolytically cleaved by the Coxsackievirus protease 2A leading to functional impairment and morphological disruption. However, the mechanism of dystrophin cleavage and the exact cleavage site remained to be identified. Antibody epitope mapping of endogenous dystrophin indicated protease 2A-mediated cleavage at the site in the hinge 3 region predicted by a neural network algorithm (human, amino acid 2434; mouse, amino acid 2427). Using site-directed mutagenesis, peptide sequencing, and fluorescence resonance energy transfer assays with recombinant dystrophin, we demonstrate that this putative site in mouse and human dystrophin is a direct substrate for the Coxsackieviral protease 2A both in vitro and in vivo. The substrate analogue protease inhibitor z-LSTT-fmk was designed based on the dystrophin sequence that interacts with the protease 2A and was found to have an IC(50) of 550 nM in vitro. Dystrophin is the first cellular substrate of the enteroviral protease 2A that was identified using by a bioinformatic approach and for which the cleavage site was molecularly mapped within living cells. PMID- 10753928 TI - Very rare complementation between mitochondria carrying different mitochondrial DNA mutations points to intrinsic genetic autonomy of the organelles in cultured human cells. AB - In the present work, a large scale investigation was done regarding the capacity of cultured human cell lines (carrying in homoplasmic form either the mitochondrial tRNA(Lys) A8344G mutation associated with the myoclonic epilepsy and ragged red fiber (MERRF) encephalomyopathy or a frameshift mutation, isolated in vitro, in the gene for the ND4 subunit of NADH dehydrogenase) to undergo transcomplementation of their recessive mitochondrial DNA (mtDNA) mutations after cell fusion. The presence of appropriate nuclear drug resistance markers in the two cell lines allowed measurements of the frequency of cell fusion in glucose containing medium, non-selective for respiratory capacity, whereas the frequency of transcomplementation of the two mtDNA mutations was determined by growing the same cell fusion mixture in galactose-containing medium, selective for respiratory competence. Transcomplementation of the two mutations was revealed by the re-establishment of normal mitochondrial protein synthesis and respiratory activity and by the relative rates synthesis of two isoforms of the ND3 subunit of NADH dehydrogenase. The results of several experiments showed a cell fusion frequency between 1.4 and 3.4% and an absolute transcomplementation frequency that varied between 1.2 x 10(-5) and 5.5 x 10(-4). Thus, only 0.3-1.6% of the fusion products exhibited transcomplementation of the two mutations. These rare transcomplementing clones were very sluggish in developing, grew very slowly thereafter, and showed a substantial rate of cell death (22-28%). The present results strongly support the conclusion that the capacity of mitochondria to fuse and mix their contents is not a general intrinsic property of these organelles in mammalian cells, although it may become activated in some developmental or physiological situations. PMID- 10753927 TI - The Saccharomyces cerevisiae Rheb G-protein is involved in regulating canavanine resistance and arginine uptake. AB - The new member of the Ras superfamily of G-proteins, Rheb, has been identified in rat and human, but its function has not been defined. We report here the identification of Rheb homologues in the budding yeast Saccharomyces cerevisiae (ScRheb) as well as in Schizosaccharomyces pombe, Drosophila melanogaster, zebrafish, and Ciona intestinalis. These proteins define a new class of G proteins based on 1) their overall sequence similarity, 2) high conservation of their effector domain sequence, 3) presence of a unique arginine in their G1 box, and 4) presence of a conserved CAAX farnesylation motif. Characterization of an S. cerevisiae strain deficient in ScRheb showed that it is hypersensitive to growth inhibitory effects of canavanine and thialysine, which are analogues of arginine and lysine, respectively. Accordingly, the uptake of arginine and lysine was increased in the ScRheb-deficient strain. This increased arginine uptake requires the arginine-specific permease Can1p. The function of ScRheb is dependent on having an intact effector domain since mutations in the effector domain of ScRheb are incapable of complementing canavanine hypersensitivity of scrheb disruptant cells. Furthermore, the conserved arginine in the G1 box plays a role in the activity of ScRheb, as a mutation of this arginine to glycine significantly reduced the ability of ScRheb to complement canavanine hypersensitivity of ScRheb-deficient yeast. Finally, a mutation in the C-terminal CAAX farnesylation motif resulted in a loss of ScRheb function. This result, in combination with our finding that ScRheb is farnesylated, suggests that farnesylation plays a key role in ScRheb function. Our findings assign the regulation of arginine and lysine uptake as the first physiological function for this new farnesylated Ras superfamily G-protein. PMID- 10753929 TI - Tumor necrosis factor employs a protein-tyrosine phosphatase to inhibit activation of KDR and vascular endothelial cell growth factor-induced endothelial cell proliferation. AB - Vascular endothelial cell growth factor (VEGF) binds to and promotes the activation of one of its receptors, KDR. Once activated, KDR induces the tyrosine phosphorylation of cytoplasmic signaling proteins that are important to endothelial cell proliferation. In human umbilical vein endothelial cells (HUVECs), tumor necrosis factor (TNF) inhibits the phosphorylation and activation of KDR. The ability of TNF to diminish VEGF-stimulated KDR activity was impaired by sodium orthovanadate, suggesting that the inhibitory activity of TNF was mediated by a protein-tyrosine phosphatase. KDR-initiated responses specifically associated with endothelial cell proliferation, mitogen-activated protein kinase activation and DNA synthesis, were also inhibited by TNF, and this was reversed by sodium orthovanadate. Stimulation of HUVECs with TNF induced association of the SHP-1 protein-tyrosine phosphatase with KDR, identifying this phosphatase as a candidate negative regulator of VEGF signal transduction. Heterologous receptor inactivation mediated by a protein-tyrosine phosphatase provides insight into how TNF may inhibit endothelial cell proliferative responses and modulate angiogenesis in pathological settings. PMID- 10753930 TI - Characterization of a novel lipid A containing D-galacturonic acid that replaces phosphate residues. The structure of the lipid a of the lipopolysaccharide from the hyperthermophilic bacterium Aquifex pyrophilus. AB - According to the 16 S rRNA phylogenetic tree, the hyperthermophilic bacterium Aquifex pyrophilus represents the deepest and shortest branching species of the kingdom Bacteria. We show for the first time that an organism, which is phylogenetically ancient on the basis of its 16 S rRNA and that exists at extreme conditions, may contain lipopolysaccharide (LPS). The LPS was extracted from dried bacteria using a modified phenol/water method. SDS-polyacrylamide gel electrophoresis and silver stain displayed a ladder-like pattern, which is typical for smooth-form LPS (possessing an O-specific polysaccharide). The molecular masses of the LPS populations were determined by matrix-assisted laser desorption ionization mass spectrometry. Lipid A was precipitated after mild acid hydrolysis of LPS. Its complete structure was determined by chemical analyses, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption ionization mass spectrometry, and one- and two-dimensional NMR spectroscopy. The lipid A consists of a beta-(1-->6)-linked 2,3-diamino-2,3 dideoxy-D-glucopyranose (DAG) disaccharide carrying two residues each of (R)-3 hydroxytetradecanoic acid and (R)-3-hydroxyhexadecanoic acid in amide linkage and one residue of octadecanoic acid in ester linkage. Each DAG moiety carries one residue of each 3-hydroxytetradecanoic and 3-hydroxyhexadecanoic acid. In the nonreducing DAG, the octadecanoic acid is attached to the 3-hydroxy group of 3 hydroxytetradecanoic acid. Each DAG is substituted by one D-galacturonic acid residue, which is linked to O-1 of the reducing and to O-4 of the nonreducing end. This structure represents a novel type of lipid A. PMID- 10753931 TI - Molecular cloning of the full-length cDNA encoding mouse neutral ceramidase. A novel but highly conserved gene family of neutral/alkaline ceramidases. AB - We report here the molecular cloning, sequencing, and expression of the gene encoding the mouse neutral ceramidase, which has been proposed to function in sphingolipid signaling. A full-length cDNA encoding the neutral ceramidase was cloned from a cDNA library of mouse liver using the partial amino acid sequences of the purified mouse liver ceramidase. The open reading frame of 2,268 nucleotides encoded a polypeptide of 756 amino acids having nine putative N glycosylation sites. Northern blot analysis revealed that the mRNA of the ceramidase was expressed widely in mouse tissues, with especially strong signals found in the liver and kidney. The ceramidase activity of lysates of CHOP cells increased more than 900-fold when the cells were transformed with a plasmid containing the cDNA encoding ceramidase. We also cloned the ceramidase homologue from the cDNA library of mouse brain and found that the sequence of the open reading frame, but not the 5'-noncoding region, was identical to that of the liver. Interestingly, phylogenetic analysis of various ceramidases clearly indicated that neutral/alkaline ceramidases form a novel but highly conserved gene family that is evolutionarily different from lysosomal acid ceramidases. PMID- 10753932 TI - A guanylyl cyclase from Paramecium with 22 transmembrane spans. Expression of the catalytic domains and formation of chimeras with the catalytic domains of mammalian adenylyl cyclases. AB - Paramecium has a 280-kDa guanylyl cyclase. The N terminus resembles a P-type ATPase, and the C terminus is a guanylyl cyclase with the membrane topology of canonical mammalian adenylyl cyclases, yet with the cytosolic loops, C1 and C2, inverted compared with the mammalian order. We expressed in Escherichia coli the cytoplasmic domains of the protozoan guanylyl cyclase, independently and linked by a peptide, as soluble proteins. The His(6)-tagged proteins were enriched by affinity chromatography and analyzed by immunoblotting. Guanylyl cyclase activity was reconstituted upon mixing of the recombinant C1a- and C2-positioned domains and in a linked C1a-C2 construct. Adenylyl cyclase activity was minimal. The nucleotide substrate specificity was switched from GTP to ATP upon mutation of the substrate defining amino acids Glu(1681) and Ser(1748) in the C1-positioned domain to the adenylyl cyclase specific amino acids Lys and Asp. Using the C2 domains of mammalian adenylyl cyclases type II or IX and the C2-positioned domain from the Paramecium guanylyl cyclase we reconstituted a soluble, all C2 adenylyl cyclase. All enzymes containing protozoan domains were not affected by Galpha(s)/GTP or forskolin, and P site inhibitors were only slightly effective. PMID- 10753933 TI - The dominant negative LQT2 mutation A561V reduces wild-type HERG expression. AB - HERG(1) K(+) channel mutations are responsible for one form of dominantly inherited long QT syndrome (LQT). Some LQT mutations exert a dominant negative effect on wild-type current expression. To investigate mechanisms of dominant negative behavior, we co-expressed wild-type HERG with the A561V mutant in mammalian cells. Transfection with various cDNA ratios produced HERG K(+) current densities that approached a predicted binomial distribution where mutant and wild type subunits co-assemble in a tetramer with nearly complete dominance. Using C terminus myc-tagged wild-type HERG we specifically followed the mutant's effect on full-length wild-type HERG protein expression. Co-expression with A561V reduced the abundance of full-length wild-type HERG protein comparable to the current reduction. Reduction of wild-type protein was due to decreased synthesis and increased turnover. Conditions facilitating protein folding (growth at 30 degrees C, or in 10% glycerol) resulted in partial rescue from the dominant effect, as did the 26 S proteosome inhibitor ALLN. Thus, for A561V, dominant negative effects result from assembly of wild-type subunits with mutant very early in production leading to rapid recognition of mutant channels and targeting for proteolysis. These results establish protein misfolding, cellular proofreading, and bystander involvement as contributing mechanisms for dominant effects in LQT2. PMID- 10753935 TI - RNase H overproduction corrects a defect at the level of transcription elongation during rRNA synthesis in the absence of DNA topoisomerase I in Escherichia coli. AB - It has been suggested that the major function of DNA topoisomerase I in Escherichia coli is to suppress the formation of R-loops, which could inhibit growth. Although the currently available data suggest that the inhibitory effect of R-loops is exerted at the level of gene expression, this has never been demonstrated. In the present report, we show that rRNA synthesis is significantly impaired at the level of transcription elongation in a bacterial strain lacking DNA topoisomerase I. We found that this inhibition is due to transcriptional blocks. RNase H overproduction is also shown to considerably reduce the extent of such transcriptional blocks during rRNA synthesis. Moreover, one of these transcriptional blockage sites is located within a region where extensive R-loop formation was previously shown to occur on a plasmid DNA in the absence of DNA topoisomerase I. Together, these results allow us to propose that an important function of DNA topoisomerase I is to inhibit the formation of R-loops, which may otherwise translate into roadblocks for RNA polymerases. Our results also highlight the potential regulatory role of DNA supercoiling at the level of transcription elongation. PMID- 10753934 TI - Distinct signalling pathways mediate insulin and phorbol ester-stimulated eukaryotic initiation factor 4F assembly and protein synthesis in HEK 293 cells. AB - Stimulation of serum-starved human embryonic kidney (HEK) 293 cells with either the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), or insulin resulted in increases in the phosphorylation of 4E-BP1 and p70 S6 kinase, eIF4F assembly, and protein synthesis. All these effects were blocked by rapamycin, a specific inhibitor of mTOR. Phosphatidylinositol 3-kinase and protein kinase B were activated by insulin but not by TPA. Therefore TPA can induce eIF4F assembly, protein synthesis, and the phosphorylation of p70 S6 kinase and 4E-BP1 independently of both phosphatidylinositol 3-kinase and protein kinase B. Using two structurally unrelated inhibitors of MEK (PD098059 and U0126), we provide evidence that Erk activation is important in TPA stimulation of eIF4F assembly and the phosphorylation of p70 S6 kinase and 4E-BP1 and that basal MEK activity is important for basal, insulin, and TPA-stimulated protein synthesis. Transient transfection of constitutively active mitogen-activated protein kinase interacting kinase 1 (the eIF4E kinase) indicated that inhibition of protein synthesis and eIF4F assembly by PD098059 is not through inhibition of eIF4E phosphorylation but of other signals emanating from MEK. This report also provides evidence that increased eIF4E phosphorylation alone does not affect the assembly of the eIF4F complex or general protein synthesis. PMID- 10753936 TI - Receptor protein-tyrosine phosphatase RPTPmu binds to and dephosphorylates the catenin p120(ctn). AB - RPTPmu is a prototypic receptor-like protein-tyrosine phosphatase (RPTP) that mediates homotypic cell-cell interactions. Intracellularly, RPTPmu consists of a relatively large juxtamembrane region and two phosphatase domains, but little is still known about its substrate(s). Here we show that RPTPmu associates with the catenin p120(ctn), a tyrosine kinase substrate and an interacting partner of cadherins. No interaction is detectable between RPTPmu and beta-catenin. Furthermore, we show that tyrosine-phosphorylated p120(ctn) is dephosphorylated by RPTPmu both in vitro and in intact cells. Complex formation between RPTPmu and p120(ctn) does not require tyrosine phosphorylation of p120(ctn). Mutational analysis reveals that both the juxtamembrane region and the second phosphatase domain of RPTPmu are involved in p120(ctn) binding. The RPTPmu-interacting domain of p120(ctn) maps to its unique N terminus, a region distinct from the cadherin interacting domain. A mutant form of p120(ctn) that fails to bind cadherins can still associate with RPTPmu. Our findings indicate that RPTPmu interacts with p120(ctn) independently of cadherins, and they suggest that this interaction may serve to control the tyrosine phosphorylation state of p120(ctn) at sites of cell cell contact. PMID- 10753937 TI - Proliferation of intimal smooth muscle cells. Attenuation of basic fibroblast growth factor 2-stimulated proliferation is associated with increased expression of cell cycle inhibitors. AB - Basic fibroblast growth factor (FGF2) is a potent mitogen for medial smooth muscle cells and is necessary for their proliferation after balloon catheter injury; however, intimal smooth muscle cells do not require FGF2 for their proliferation, and they respond only weakly to exogenous FGF2. The present study examined the activation of extracellular signal-regulated kinase (ERK) signaling as well as the expression and activity of cell cycle proteins in FGF2-stimulated intimal smooth muscle cells. FGF2 activates ERKs 1 and 2, and Western blot analysis showed that cyclin D, cyclin E, and cyclin-dependent kinase (CDKs) 2 and 4 were expressed in intimal smooth muscle cells after FGF2 infusion. FGF2 stimulation, however, did not lead to phosphorylation of the retinoblastoma protein (Rb), CDK 2 activation, or expression of cyclin A. Western blot analysis showed that intimal smooth muscle cells express elevated levels of the cell cycle inhibitors p15(INK4b) and p27(Kip1), compared with medial smooth muscle cells, and that FGF2 stimulation does not reduce the level of these inhibitors. These studies suggest that despite activation of ERKs 1 and 2 and expression of the cell cycle activators, cyclin D and cyclin E, high levels of cell cycle inhibitors may inhibit cell cycle transit in FGF2-stimulated intimal smooth muscle cells. PMID- 10753938 TI - High density lipoprotein prevents oxidized low density lipoprotein-induced inhibition of endothelial nitric-oxide synthase localization and activation in caveolae. AB - Oxidized LDL (oxLDL) depletes caveolae of cholesterol, resulting in the displacement of endothelial nitric-oxide synthase (eNOS) from caveolae and impaired eNOS activation. In the present study, we determined if the class B scavenger receptors, CD36 and SR-BI, are involved in regulating nitric-oxide synthase localization and function. We demonstrate that CD36 and SR-BI are expressed in endothelial cells, co-fractionate with caveolae, and co immunoprecipitate with caveolin-1. Co-incubation of cells with 10 microgram/ml high density lipoprotein (HDL) prevented oxLDL-induced translocation of eNOS from caveolae and restored acetylcholine-induced nitric-oxide synthase stimulation. Acetylcholine caused eNOS activation in cells incubated with 10 microgram/ml oxLDL (10-15 thiobarbituric acid-reactive substances) and blocking antibodies to CD36, whereas cells treated with only oxLDL were unresponsive. Furthermore, CD36 blocking antibodies prevented oxLDL-induced redistribution of eNOS. SR-BI blocking antibodies were used to demonstrate that the effects of HDL are mediate by SR-BI. HDL binding to SR-BI maintained the concentration of caveola-associated cholesterol by promoting the uptake of cholesterol esters, thereby preventing oxLDL-induced depletion of caveola cholesterol. We conclude that CD36 mediates the effects of oxLDL on caveola composition and eNOS activation. Furthermore, HDL prevents oxLDL from decreasing the capacity for eNOS activation by preserving the cholesterol concentration in caveolae and, thereby maintaining the subcellular location of eNOS. PMID- 10753939 TI - Arachidonic acid activates mitogen-activated protein (MAP) kinase-activated protein kinase 2 and mediates adhesion of a human breast carcinoma cell line to collagen type IV through a p38 MAP kinase-dependent pathway. AB - Adhesion of metastatic human mammary carcinoma MDA-MB-435 cells to the basement membrane protein collagen type IV can be activated by treatment with arachidonic acid. We initially observed that this arachidonic acid-mediated adhesion was inhibited by the tyrosine kinase inhibitor genistein. Therefore, we examined the role of the mitogen-activated protein (MAP) kinase family tyrosine phosphorylation-regulated pathways in arachidonic acid-stimulated cell adhesion. Arachidonic acid stimulated the phosphorylation of p38, the activation of MAP kinase-activated protein kinase 2 (MAPKAPK2, a downstream substrate of p38), and the phosphorylation of heat shock protein 27 (a downstream substrate of MAP kinase-activated protein kinase 2). Treatment with the p38 inhibitor PD169316 completely and specifically inhibited arachidonic acid-mediated cell adhesion to collagen type IV. p38 activity was specifically associated with arachidonic acid stimulated adhesion; this was demonstrated by the observation that 12-O tetradecanoylphorbol 13-acetate-activated cell adhesion was not blocked by inhibiting p38 activity. Extracellular signal-regulated protein kinases (ERKs) 1 and 2 were also activated by arachidonic acid; however, cell adhesion to collagen type IV was not highly sensitive to PD98059, an inhibitor of MAP kinase kinase/ERK kinase 1 (MEK1) that blocks activation of the ERKs. c-Jun NH(2) terminal kinase was not activated by arachidonic acid treatment of these cells. Together, these data suggest a novel role for p38 MAP kinase in regulating adhesion of breast cancer cells to collagen type IV. PMID- 10753940 TI - Cyclopentenone prostaglandins as potential inducers of phase II detoxification enzymes. 15-deoxy-delta(12,14)-prostaglandin j2-induced expression of glutathione S-transferases. AB - Exposure of cells to a wide variety of chemoprotective compounds confers resistance to a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of protective enzymes, such as glutathione S-transferases (GSTs). In the present study, we developed a cell culture system that potently responds to phenolic antioxidants and found that antitumor prostaglandins (PGs) are potential inducers of GSTs. We screened primary hepatocytes and multiple cell lines for inducing GST activity upon incubation with the phenolic antioxidant (tert-butylhydroquinone) and found that rat liver epithelial RL34 cells most potently responded. Based on an extensive screening of diverse chemical agents on the induction of GST activity in RL34 cells, the J2 series of PGs, 15-deoxy-Delta(12,14)-prostaglandin J2 (15 deoxy-Delta(12,14)-PGJ2) in particular, were found to be potential inducers of GST. Enhanced gene expression of Class pi GST isozyme (GSTP1) by 15-deoxy Delta(12,14)-PGJ2 was evident as a drastic elevation of the mRNA level. Hence, we examined the molecular mechanism underlying the 15-deoxy-Delta(12, 14)-PGJ2 induced GSTP1 gene expression. From functional analysis of various deletion mutant genes, we found that the 15-deoxy-Delta(12, 14)-PGJ2 reponse element was localized in a region containing a GSTP1 enhancer I (GPEI) that consists of two imperfect phorbol 12-O-tetradecanoylphorbol-13-acetate response elements. When the GPEI was combined with the minimum GSTP1 promoter, the element indeed showed an enhancer activity in response to 15-deoxy-Delta(12, 14)-PGJ2. Point mutations of either of the two imperfect 12-O-tetradecanoylphorbol-13-acetate response elements in GPEI completely abolished the enhancer activity. Gel mobility shift assays demonstrated that 15-deoxy-Delta(12,14)-PGJ2 specifically stimulated the binding of nuclear proteins including the transcription factor c-Jun, but not Nrf2, to GPEI. These results suggest that 15-deoxy-Delta(12,14)-PGJ2 induces the expression of the rat GSTP1 gene through binding of proteins, including c-Jun, to a specific GPEI. PMID- 10753941 TI - Biosynthesis of vitamin B(6) in rhizobium. AB - The biosynthetic pathway of pyridoxol (vitamin B(6)) in Rhizobium was clarified by studies on the incorporation of (13)C- or (15)N-labeled precursors into pyridoxol or its biosynthetic intermediates. Pyridoxol was formed by ring closure of two compounds, 1-deoxy-D-xylulose and 4-hydroxy-L-threonine. The former was formed from D-glyceraldehyde and pyruvate through decarboxylation of pyruvate, and the latter from glycine and glycolaldehyde. PMID- 10753942 TI - Analysis of the association of syncollin with the membrane of the pancreatic zymogen granule. AB - Syncollin is a pancreatic zymogen granule protein that was isolated through its ability to bind to syntaxin. Here we show that syncollin has a cleavable signal sequence and can be removed from granule membranes by washing with sodium carbonate. When membranes were subjected to Triton X-114 partitioning, syncollin was found predominantly in the aqueous phase, indicating that it is not sufficiently hydrophobic to be embedded in the membrane. Syncollin has intramolecular disulfide bonds and was accessible to water-soluble cross-linking and biotinylating reagents only when granules were lysed by sonication. These results indicate that syncollin is tightly bound to the luminal surface of the granule membrane. In situ, syncollin was resistant to proteases such as trypsin. When granule membranes were solubilized in ionic detergents such as deoxycholate, this trypsin resistance was maintained, and syncollin migrated on sucrose density gradients as a large (150 kDa) protein. In contrast, in non-ionic detergents such as Triton X-100, syncollin became partially sensitive to trypsin and behaved as a monomer. Syncollin in alkaline extracts of granule membranes was also monomeric. However, reduction of the pH regenerated the oligomeric form, which was insoluble. We conclude that syncollin exists as a homo-oligomer and that its ability to self-associate can be reversibly modulated via changes in pH. In light of our findings, we reassess the likely role of syncollin in the pancreatic acinar cell. PMID- 10753943 TI - beta-arrestin1 interacts with the catalytic domain of the tyrosine kinase c-SRC. Role of beta-arrestin1-dependent targeting of c-SRC in receptor endocytosis. AB - beta-Arrestins can act as adapter molecules, coupling G-protein-coupled receptors to proteins involved in mitogenic as well as endocytic pathways. We have previously identified c-SRC as a molecule that is rapidly recruited to the beta2 adrenergic receptor in a beta-arrestin1-dependent manner. Recruitment of c-SRC to the receptor appears to be involved in pathways leading to receptor internalization and mitogen-activated protein kinase activation. This recruitment of c-SRC to the receptor involves an interaction between the amino-terminal proline-rich region of beta-arrestin1 and the Src homology 3 (SH3) domain of c SRC, but deletion of the proline-rich domain does not totally ablate the interaction. We have found that a major interaction also exists between beta arrestin1 and the catalytic or kinase domain (SH1) of c-SRC. We therefore hypothesized that a catalytically inactive mutant of the isolated catalytic subunit, SH1(kinase dead) (SH1(KD)), would specifically block those cellular actions of c-SRC that are mediated by beta-arrestin1 recruitment to the G-protein coupled receptor. In contrast, the majority of cellular phosphorylations catalyzed by c-SRC, which do not involve interaction with the SH1 domain, would be predicted to be unaffected. The SH1(KD) mutant did indeed block beta2 adrenergic receptor internalization and receptor-stimulated tyrosine phosphorylation of dynamin, actions previously shown to be c-SRC-dependent. In contrast, SAM-68 and whole cell tyrosine phosphorylation by c-SRC was unaffected, indicating that the SH1(KD) mutant did not inhibit c-SRC tyrosine kinase activity in general. These results not only clarify the nature of the beta-arrestin1/c-SRC interaction but also implicate beta-arrestin1 as an important mediator of receptor internalization by recruiting tyrosine kinase activity to the cell surface to phosphorylate key endocytic intermediates, such as dynamin. PMID- 10753944 TI - Smad3 and Smad4 mediate transcriptional activation of the human Smad7 promoter by transforming growth factor beta. AB - Smad7 is an inducible intracellular inhibitor of transforming growth factor-beta (TGF-beta) signaling that is regulated by diverse stimuli including members of the TGF-beta superfamily. To define the molecular mechanisms of negative control of TGF-beta signaling, we have isolated the human SMAD7 gene and characterized its promoter region. A -303 to +672 SMAD7 region contained a palindromic GTCTAGAC Smad binding element (SBE) between nucleotides -179 and -172 that was necessary for the induction of a Smad7 promoter luciferase reporter gene by TGF-beta. Electrophoretic mobility shift assays using oligonucleotide probes demonstrated that TGF-beta rapidly induced the binding of an endogenous SBE-binding complex (SBC) containing Smad2, Smad3, and Smad4. Transfection assays in mouse embryonic fibroblasts (MEFs), with targeted deletions of either Smad2 or Smad3, and the Smad4-deficient cell line MD-MBA-468 revealed that both Smad3 and Smad4, but not Smad2, were absolutely required for induction of the Smad7 promoter reporter gene by TGF-beta. Furthermore, the TGF-beta-inducible SBE-binding complex was diminished in Smad2-deficient MEFs when compared with wild type MEFs and not detectable in Smad3-deficient MEFs and MD-MBA-468 cells. Taken together, our data demonstrate that TGF-beta induces transcription of the human SMAD7 gene through activation of Smad3 and Smad4 transcription factor binding to its proximal promoter. PMID- 10753945 TI - Wild type and mutant p53 differentially regulate the gene expression of human collagenase-3 (hMMP-13). AB - Matrix metalloproteinases (MMPs) are a family of secreted or transmembrane proteins that can degrade all the proteins of the extracellular matrix and have been implicated in many abnormal physiological conditions including arthritis and cancer metastasis. Recently we have shown for the first time that the human MMP-1 gene is a p53 target gene subject to repression by wild type p53 (Sun, Y., Sun, Y. I., Wenger, L., Rutter, J. L., Brinckerhoff, C. E., and Cheung, H. S. (1999) J. Biol. Chem. 274, 11535-11540). Here, we report that cotransfection of fibroblast-like synoviocytes with p53 expression and hMMP13CAT reporter plasmids revealed that (i) hMMP13, another member of the human MMP family, was down regulated by wild type p53, whereas all six of the p53 mutants tested lost the wild type p53 repressor activity in fibroblast-like synoviocytes; (ii) this repression of hMMP-13 gene expression by wild type p53 could be reversed by overexpression of p53 mutants p53-143A, p53-248W, p53-273H, and p53-281G; (iii) the dominant effect of p53 mutants over wild type p53 appears to be a promoter- and mutant-specific effect. An intriguing finding was that p53 mutant p53-281G could conversely stimulate the promoter activity of hMMP13 up to 2-4-fold and that it was dominant over wild type p53. Northern analysis confirmed these findings. Although the significance of these findings is currently unknown, they suggest that in addition to the effect of cytokines activation, the gene expression of hMMP13 could be dysregulated during the disease progression of rheumatoid arthritis (or cancer) associated with p53 inactivation. Since hMMP13 is 5-10 times as active as hMMP1 in its ability to digest type II collagen, the dysregulation or up-modulation of MMP13 gene expression due to the inactivation of p53 may contribute to the joint degeneration in rheumatoid arthritis. PMID- 10753946 TI - Phosphorylation of paxillin via the ERK mitogen-activated protein kinase cascade in EL4 thymoma cells. AB - Intracellular signals can regulate cell adhesion via several mechanisms in a process referred to as "inside-out" signaling. In phorbol ester-sensitive EL4 thymoma cells, phorbol-12-myristate 13-acetate (PMA) induces activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases and promotes cell adhesion. In this study, clonal EL4 cell lines with varying abilities to activate ERKs in response to PMA were used to examine signaling events occurring downstream of ERK activation. Paxillin, a multifunctional docking protein involved in cell adhesion, was phosphorylated on serine/threonine residues in response to PMA treatment. This response was correlated with the extent and time course of ERK activation. PMA-induced phosphorylation of paxillin was inhibited by compounds that block the ERK activation pathway in EL4 cells, primary murine thymocytes, and primary murine splenocytes. Paxillin was phosphorylated in vitro by purified active ERK2. Two-dimensional electrophoresis revealed that PMA treatment generated a complex pattern of phosphorylated paxillin species in intact cells, some of which were generated by ERK-mediated phosphorylation in vitro. An ERK pathway inhibitor interfered with PMA-induced adhesion of sensitive EL4 cells to substrate. These findings describe a novel inside-out signaling pathway by which the ERK cascade may regulate events involved in adhesion. PMID- 10753947 TI - Nitrosative capacity of macrophages is dependent on nitric-oxide synthase induction signals. AB - Nitrosative stress can occur when reactive nitric oxide (NO) species compromise the function of biomolecules via formation of NO adducts on critical amine and thiol residues. The capacity of inducible nitric-oxide synthase (iNOS) to generate nitrosative stress was investigated in the murine macrophage line ANA-1. Sequential activation with the cytokines IFN-gamma and either tumor necrosis factor-alpha or interleukin-1beta resulted in the induction of iNOS and production of nitrite (20 nM/min) but failed to elicit nitrosation of extracellular 2,3-diaminonapthalene. Stimulation with IFN-gamma and bacterial lipopolysaccharide increased the relative level of iNOS protein and nitrite production of ANA-1 cells 2-fold; however, a substantial level of NO in the media was also observed, and nitrosation of 2,3-diaminonapthalene was increased greater than 30-fold. Selective scavenger compounds suggested that the salient nitrosating mechanism was the NO/O(2) reaction leading to N(2)O(3) formation. These data mimicked the pattern observed with a 5 microM concentration of the synthetic NO donor (Z)-1-[N-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium -1,2 diolate (PAPA/NO). The NO profiles derived from iNOS can be distinct and depend on the inductive signal cascades. The diverse consequences of NO production in macrophages may reside in the cellular mechanisms that control the ability of iNOS to form N(2)O(3) and elicit nitrosative stress. PMID- 10753948 TI - Leptin induces insulin-like signaling that antagonizes cAMP elevation by glucagon in hepatocytes. AB - Although many effects of leptin are mediated through the central nervous system, leptin can regulate metabolism through a direct action on peripheral tissues, such as fat and liver. We show here that leptin, at physiological concentrations, acts through an intracellular signaling pathway similar to that activated by insulin in isolated primary rat hepatocytes. This pathway involves stimulation of phosphatidylinositol 3-kinase (PI3K) binding to insulin receptor substrate-1 and insulin receptor substrate-2, activation of PI3K and protein kinase B (AKT), and PI3K-dependent activation of cyclic nucleotide phosphodiesterase 3B, a cAMP degrading enzyme. One important function of this signaling pathway is to reduce levels of cAMP, because leptin-mediated activation of both protein kinase B and phosphodiesterase 3B is most marked following elevation of cAMP by glucagon, and because leptin suppresses glucagon-induced cAMP elevation in a PI3K-dependent manner. There is little or no expression of the long form leptin receptor in primary rat hepatocytes, and these signaling events are probably mediated through the short forms of the leptin receptor. Thus, leptin, like insulin, induces an intracellular signaling pathway in hepatocytes that culminates in cAMP degradation and an antagonism of the actions of glucagon. PMID- 10753949 TI - Mutations in single hairpin units of genetically fused subunit c provide support for a rotary catalytic mechanism in F(0)F(1) ATP synthase. AB - Previously, we generated genetically fused dimers and trimers of subunit c of the Escherichia coli ATP synthase based upon the precedent of naturally occurring dimers in V-type H(+)-transporting ATPases. The c(2) and c(3) oligomers have proven useful in testing hypothesis regarding the mechanism of energy coupling. In the first part of this paper, the uncoupling Q42E substitution has been introduced into the second loop of the c(2) dimer or the third loop of the c(3) trimer. Both mutant proteins proved to be as functional as the wild type c(2) dimer or wild type c(3) trimer. The results argue against an obligatory movement of the epsilon subunit between loops of monomeric subunit c in the c(12) oligomer during rotary catalysis. Rather, the results support the hypothesis that the c epsilon connection remains fixed as the c-oligomer rotates. In the second section of this paper, we report on the effect of substitution of the proton translocating Asp(61) in every second helical hairpin of the c(2) dimer, or in every third hairpin of the c(3) trimer. Based upon the precedent of V-type ATPases, where the c(2) dimer occurs naturally with a single proton translocating carboxyl in every second hairpin, these modified versions of the E. coli c(2) and c(3) fused proteins were predicted to have a functional H(+)-transporting ATPase activity, with a reduced H(+)/ATP stoichiometry, but to be inactive as ATP synthases. A variety of Asp(61)-substituted proteins proved to lack either activity indicating that the switch in function in V-type ATPases is a consequence of more than a single substitution. PMID- 10753950 TI - The yeast ARG7 gene product is autoproteolyzed to two subunit peptides, yielding active ornithine acetyltransferase. AB - Yeast ornithine acetyltransferase has been purified from total yeast extracts as a heterodimer of two subpeptides (Liu, Y., Van Heeswijck, R., Hoj, P., and Hoogenraad, N. (1995) Eur. J. Biochem. 228, 291-296), confirmed to derive from a single ARG7-encoded precursor (Crabeel, M., Abadjieva, A., Hilven, P., Desimpelaere, J., and Soetens, O. (1997) Eur. J. Biochem. 250, 232-241). By Western immunoblotting, we show that Arg7p is also present as two subpeptides in isolated mitochondria, but that processing occurs before targeting to the mitochondria: deletion of the N-terminal leader peptide results in cytosolic accumulation of N-Arg7p, whereas C-Arg7p partially reaches the organelle by itself. When artificially co-expressed from separate genes, the two subpeptides can complement an arg7 mutation; ornithine acetyltransferase activity is measurable. Maturation of Arg7p occurs at threonine 215 (N-side), in the region most conserved among the 17 ornithine acetyltransferases characterized. Changing this conserved residue to alanine completely abolishes maturation. Furthermore, Arg7p is both processed and active in Escherichia coli, a heterologous background, and is also cleaved in vitro when produced by coupled transcription/translation in a reticulocyte lysate. Together, these data suggest classic autoproteolysis initiated by threonine 215. Most importantly, maturation is required for the enzyme to be functional, since the T215A substitution mutant is catalytically inactive and incapable of genetic complementation, despite its correct targeting to the mitochondria. PMID- 10753951 TI - Membrane type 1 matrix metalloproteinase-associated degradation of tissue inhibitor of metalloproteinase 2 in human tumor cell lines. AB - Tissue inhibitor of metalloproteinase 2 (TIMP-2) is required for the membrane type 1 matrix metalloproteinase (MT1-MMP)-dependent activation of pro-MMP-2 on the cell surface. MT1-MMP-bound TIMP-2 has been shown to function as a receptor for secreted pro-MMP-2, resulting in the formation of a trimolecular complex. In the presence of uncomplexed active MT1-MMP, the prodomain of cell surface associated MMP-2 is cleaved, and activated MMP-2 is released. However, the behavior of MT1-MMP-bound TIMP-2 during MMP-2 activation is currently unknown. In this study, (125)I-labeled recombinant TIMP-2 ((125)I-rTIMP-2) was used to investigate the fate of TIMP-2 during pro-MMP-2 activation by HT1080 and transfected A2058 cells. HT1080 and A2058 cells transfected with MT1-MMP cDNA (but not vector-transfected A2058 cells) were able to bind (125)I-rTIMP-2, to activate pro-MMP-2, and to process MT1-MMP into an inactive 43-kDa form. Under these conditions, (125)I-rTIMP-2 bound to the cell surface was rapidly internalized and degraded in intracellular organelles through a bafilomycin A(1) sensitive mechanism, and (125)I-bearing low molecular mass fragment(s) were released in the culture medium. These different processes were inhibited by hydroxamic acid-based synthetic MMP inhibitors and rTIMP-2, but not by rTIMP-1 or cysteine, serine, or aspartic proteinase inhibitors. These results support the concept that the MT1-MMP-dependent internalization and degradation of TIMP-2 by some tumor cells might be involved in the regulation of pericellular proteolysis. PMID- 10753952 TI - Activation of heparin cofactor II by calcium spirulan. AB - Heparin cofactor II (HCII) is a plasma serine protease inhibitor whose ability to inhibit alpha-thrombin is accelerated by a variety of sulfated polysaccharides in addition to heparin and dermatan sulfate. Previous investigations have indicated that calcium spirulan (Ca-SP), a novel sulfated polysaccharide, enhanced the rate of inhibition of alpha-thrombin by HCII. In this study, we investigated the mechanism of the activation of HCII by Ca-SP. Interestingly, in the presence of Ca-SP, an N-terminal deletion mutant of HCII (rHCII-Delta74) inhibited alpha thrombin, as native recombinant HCII (native rHCII) did. The second-order rate constant for the inhibition of alpha-thrombin by rHCII-Delta74 was 2.0 x 10(8) M( 1) min(-1) in the presence of 50 microgram/ml Ca-SP and 10, 000-fold higher than in the absence of Ca-SP. The rates of native rHCII and rHCII-Delta74 for the inhibition of gamma-thrombin were increased only 80- and 120-fold, respectively. Our results suggested that the anion-binding exosite I of alpha-thrombin was essential for the rapid inhibition reaction by HCII in the presence of Ca-SP and that the N-terminal acidic domain of HCII was not required. Therefore, we proposed a mechanism by which HCII was activated allosterically by Ca-SP and could interact with the anion-binding exosite I of thrombin not through the N terminal acidic domain of HCII. The Arg(103) --> Leu mutant bound to Ca-SP Toyopearl with normal affinity and inhibited alpha-thrombin in a manner similar to native rHCII. These results indicate that Arg(103) in HCII molecule is not critical for the interaction with Ca-SP. PMID- 10753953 TI - Sodium channel beta subunits mediate homophilic cell adhesion and recruit ankyrin to points of cell-cell contact. AB - Sodium channels isolated from mammalian brain are composed of alpha, beta1, and beta2 subunits. The auxiliary beta subunits do not form the ion conducting pore, yet play important roles in channel modulation and plasma membrane expression. beta1 and beta2 are transmembrane proteins with one extracellular V-set immunoglobulin (Ig) protein domain. It has been shown recently that beta1 and beta2 interact with the extracellular matrix proteins tenascin-C and tenascin-R. In the present study we show that rat brain beta1 and beta2, but not alphaIIA, subunits interact in a trans-homophilic fashion, resulting in recruitment of the cytoskeletal protein ankyrin to sites of cell-cell contact in transfected Drosophila S2 cells. Whereas alphaIIA subunits expressed alone do not cause cellular aggregation, beta subunits co-expressed with alphaIIA retain the ability to adhere and recruit ankyrin. Truncated beta subunits lacking cytoplasmic domains interact homophilically to produce cell aggregation but do not recruit ankyrin. Thus, the cytoplasmic domains of beta1 and beta2 are required for cytoskeletal interactions. It is hypothesized that sodium channel beta subunits serve as a critical communication link between the extracellular and intracellular environments of the neuron and may play a role in sodium channel placement at nodes of Ranvier. PMID- 10753954 TI - Nitric oxide increases p21(Waf1/Cip1) expression by a cGMP-dependent pathway that includes activation of extracellular signal-regulated kinase and p70(S6k). AB - Nitric oxide (NO) regulates the expression of p21(Waf1/Cip1) in several cell types. The present study examined the role of both the extracellular signal regulated kinase (ERK) and p70 S6 kinase (p70(S6k)) in the NO-induced increase in p21 expression that occurred in adventitial fibroblasts during the cell cycle. Both ERK and p70(S6k) were phosphorylated in response to the NO donor S-nitroso-N acetylpenicillamine (SNAP) and the activation was rapid, transient, and preceded increased p21 expresion under defined conditions where serum was present. Addition of a selective inhibitor of ERK phosphorylation (PD98059) prevented the subsequent phosphorylation of p70(S6k) and the increase in p21 protein. Both cGMP and cAMP activated both ERK and p70(S6k), whereas only selective inhibitors of protein kinase G prevented the activation of the kinases by SNAP. A complex between ERK and p70(S6k) was documented by immunoprecipitation procedures. Rapamycin blocked p70(S6k) phosphorylation induced by NO and also inhibited p53 phosphorylation and p21 expression whereas PD98059 only prevented the NO-induced increase in p21 protein without influencing either p53 activation or p21 mRNA expression. The studies show a unique relationship between NO, ERK, and p70(S6k) and also provide evidence for a novel role of p70(S6k) in the activation of p53. PMID- 10753955 TI - The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2. AB - This study investigates the apoptotic activity of the cyclooxygenase-2 (COX-2) inhibitor celecoxib in prostate carcinoma cells. COX-2 is constitutively expressed in androgen-responsive LNCaP and androgen-nonresponsive PC-3 cells. Exposure of these cells to celecoxib induces characteristic features of apoptosis, including morphological changes, DNA laddering, and caspase-3 activation, whereas piroxicam, a COX-1-specific inhibitor, displays no appreciable effect on either cancer cell line even after prolonged exposure. Moreover, the potency of celecoxib in apoptosis induction is significantly higher than that of other COX-2 inhibitors examined despite the observation that these inhibitors exhibit similar IC(50) in COX-2 inhibition. It is noteworthy that normal human prostate epithelial cells, expressing a marginally detectable level of COX-2, are insensitive to the induction of apoptosis by celecoxib. These data suggest a correlation between COX-2 expression and sensitivity to the apoptotic effect of the COX-2 inhibitor. In an effort to delineate the underlying mechanism, we examined the effect of celecoxib on the expression of Bcl-2 as well as the activation of the key anti-apoptotic kinase Akt. In contrast to an earlier report that attributed the apoptotic activity of NS398 in LNCaP cells to Bcl-2 down-regulation, we provide evidence that the induction of apoptosis by celecoxib in LNCaP and PC-3 cells is independent of Bcl-2. First, treatment with celecoxib does not alter the cellular Bcl-2 level in both cell lines. Second, enforced Bcl 2 expression in PC-3 cells does not confer protection against the induction of apoptosis by celecoxib. Our data show that celecoxib treatment blocks the phosphorylation of Akt. This correlation is supported by studies showing that overexpression of constitutively active Akt protects PC-3 cells from celecoxib induced apoptosis. Nevertheless, how celecoxib down-regulates Akt is not clear because the drug does not adversely affect phosphoinositide 3-kinase activity in vivo and okadaic acid, a protein phosphatase 2A inhibitor, cannot rescue the inhibition. In summary, our data demonstrate that inhibition of Akt activation may play a crucial role in the induction of apoptosis by celecoxib. PMID- 10753956 TI - Estrogen suppresses transcription of lipoprotein lipase gene. Existence of a unique estrogen response element on the lipoprotein lipase promoter. AB - Estrogen exerts a variety of effects not only on female reproductive organs but also on nonreproductive organs, including adipose tissue. Estrogen inhibits obesity triggered by ovariectomy in rodents. We studied the mechanism underlying this estrogen-dependent inhibition of obesity. Estrogen markedly decreased the amounts of fat accumulation and lipoprotein lipase (LPL) mRNA as well as triglyceride accumulation in genetically manipulated 3T3-L1 adipocytes stably expressing the estrogen receptor (ER). A pLPL(1980)-CAT construct, along with an ER expression vector, was introduced into differentiated 3T3-L1 cells, and CAT activities were determined. ER, mostly ligand-dependently, inhibited the basal LPL promoter activity by 7-fold. We searched the LPL promoter for an estrogen responsive suppressive element by employing a set of 5'-deletion mutants of the pLPL-CAT reporter. Although there was no classical estrogen response element, it was demonstrated that an AP-1-like TGAATTC sequence located at (-1856/-1850) was responsible for the suppression of the LPL gene transcription by estrogen. An electrophoretic mobility shift assay probed with the TGAATTC sequence demonstrated formation of a specific DNA-nuclear protein complex. Interestingly, this complex was not affected by the addition of any antibodies against ER, c Jun, c-Fos, JunB, or JunD. Because this TGAATTC element responded to phorbol ester and overexpression of CREB-binding protein abrogated the suppressive effect of estrogen on the LPL promoter, we conclude that a unique protein that is related to the AP-1 transcription factor families may be involved in the complex that binds to the TGAATTC element. PMID- 10753957 TI - A cartilage oligomeric matrix protein mutation associated with pseudoachondroplasia changes the structural and functional properties of the type 3 domain. AB - Cartilage oligomeric matrix protein (COMP) is a member of the thrombospondin family of extracellular matrix glycoproteins. All members of the family contain a highly conserved region of thrombospondin type 3 sequence repeats that bind calcium. A mutation in COMP previously identified in a patient with pseudoachondroplasia resulted in abnormal sequestration of COMP in distinctive rER vesicles. The mutation, Asp-446 --> Asn, is located in the type 3 repeats of the molecule. This region was expressed in a mammalian culture with and without the mutation to study the structural or functional properties associated with the mutation. The biophysical parameters of the mutant peptide were compared with those of the wild type and revealed the following difference: secondary structural analysis by circular dichroism showed more alpha-helix content in the wild-type peptides. The calcium binding properties of the two peptides were significantly different; there were 17 calcium ions bound/wild-type COMP3 peptide compared with 8/mutant peptide. In addition, wild-type COMP3 had a higher affinity for calcium and bound calcium more cooperatively. Calcium bound by the wild-type peptide was reflected in a structural change as indicted by velocity sedimentation. Thus, the effect of the COMP mutation appears to profoundly alter the calcium binding properties and may account for the difference observed in the structure of the type 3 domain. Furthermore, the highly cooperative binding of calcium to COMP3 suggests that these type 3 sequence repeats form a single protein domain, the thrombospondin type 3 domain. PMID- 10753958 TI - Stimulation of p38 mitogen-activated protein kinase is an early regulatory event for the cadmium-induced apoptosis in human promonocytic cells. AB - Pulse treatment of U-937 promonocytic cells with cadmium chloride (2 h at 200 microM) provoked apoptosis and induced a rapid phosphorylation of p38 mitogen activated protein kinase (p38(MAPK)) as well as a late phosphorylation of extracellular signal-regulated protein kinases (ERK1/2). However, although the p38(MAPK)-specific inhibitor SB203580 attenuated apoptosis, the process was not affected by the ERK-specific inhibitor PD98059. The attenuation of the cadmium provoked apoptosis by SB203580 was a highly specific effect. In fact, the kinase inhibitor did not prevent the generation of apoptosis by heat shock and camptothecin, nor the generation of necrosis by cadmium treatment of glutathione depleted cells, nor the cadmium-provoked activation of the stress response. The generation of apoptosis was preceded by intracellular H(2)O(2) accumulation and was accompanied by the disruption of mitochondrial transmembrane potential, both of which were inhibited by SB203580. On the other hand, the antioxidant agent butylated hydroxyanisole-inhibited apoptosis but did not prevent p38(MAPK) phosphorylation. In a similar manner, p38(MAPK) phosphorylation was not affected by the caspase inhibitors Z-VAD and DEVD-CHO, which nevertheless prevented apoptosis. These results indicate that p38(MAPK) activation is an early and specific regulatory event for the cadmium-provoked apoptosis in promonocytic cells. PMID- 10753959 TI - Molecular cloning and characterization of Pals, proteins associated with mLin-7. AB - In Caenorhabditis elegans, three PDZ domain proteins, Lin-2, Lin-7, and Lin-10, are necessary for the proper targeting of the Let-23 growth factor receptor to the basolateral surface of epithelial cells. It has been demonstrated that homologues of Lin-2, Lin-7, and Lin-10 form a heterotrimeric complex in mammalian brain. Using Far Western overlay assay, we have identified additional proteins that can bind to the amino terminus of mLin-7 and cloned the genes encoding these proteins using bacterial expression cloning. We call these proteins Pals, for proteins associated with Lin-7. These proteins, which include mammalian Lin-2, contain a conserved mLin-7 binding domain in addition to guanylate kinase, PDZ (postsynaptic density 95/discs large/zona occludens-1), and Src homology 3 domains. Using site-directed mutagenesis, we have identified the conserved residues among these proteins crucial for mLin-7 binding. Two of these proteins, Pals1 and Pals2, are newly described. Pals1 consists of 675 amino acids and maps to mouse chromosome 12. Pals2 was found to exist in two splice forms of 539 and 553 amino acids and maps to mouse chromosome 6. Like mLin-2, Pals1 and Pals2 localize to the lateral membrane in Madin-Darby canine kidney cells. Pals proteins represent a new subfamily of membrane-associated guanylate kinases that allow for multiple targeting complexes containing mLin-7. PMID- 10753960 TI - Transcriptional analysis of the glutamate dehydrogenase gene in the primitive eukaryote, Giardia lamblia. Identification of a primordial gene promoter. AB - We studied gene expression in the ancient eukaryote, Giardia lamblia, by taking advantage of assays developed recently in our laboratory, which allow new genetic analyses of this organism. We examined the transcription of a 2.2-kilobase segment of the Giardia genome that contains the glutamate dehydrogenase (GDH) gene and a portion of a second open reading frame encoding an uncharacterized gene. Nuclear run-on analyses showed that the genes are transcribed as two separate units spaced less than 200 base pairs apart, and transcription of the GDH gene initiates just 3-6 nucleotides upstream of its translation start codon. We characterized the GDH promoter by transfecting Giardia with DNA constructs that used the GDH upstream sequence to drive the expression of a luciferase reporter gene. By deletion and mutational analyses, we localized promoter function to three motifs within a 50-base pair region of the GDH upstream sequence. Using band shift assays and UV cross-linking, we demonstrated specific binding of a 68-kDa protein from Giardia nuclear extracts to short poly(T) tracts contained within two of the sequence motifs on single-stranded DNA from the promoter region. This report describes one of the first functional gene promoter and its cognate DNA-binding protein in this primitive eukaryote. PMID- 10753961 TI - DNA structure requirements for the Escherichia coli gamma complex clamp loader and DNA polymerase III holoenzyme. AB - The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is highly processive during DNA synthesis. Underlying high processivity is a ring shaped protein, the beta clamp, that encircles DNA and slides along it, thereby tethering the enzyme to the template. The beta clamp is assembled onto DNA by the multiprotein gamma complex clamp loader that opens and closes the beta ring around DNA in an ATP-dependent manner. This study examines the DNA structure required for clamp loading action. We found that the gamma complex assembles beta onto supercoiled DNA (replicative form I), but only at very low ionic strength, where regions of unwound DNA may exist in the duplex. Consistent with this, the gamma complex does not assemble beta onto relaxed closed circular DNA even at low ionic strength. Hence, a 3'-end is not required for clamp loading, but a single stranded DNA (ssDNA)/double-stranded DNA (dsDNA) junction can be utilized as a substrate, a result confirmed using synthetic oligonucleotides that form forked ssDNA/dsDNA junctions on M13 ssDNA. On a flush primed template, the gamma complex exhibits polarity; it acts specifically at the 3'-ssDNA/dsDNA junction to assemble beta onto the DNA. The gamma complex can assemble beta onto a primed site as short as 10 nucleotides, corresponding to the width of the beta ring. However, a protein block placed closer than 14 base pairs (bp) upstream from the primer 3' terminus prevents the clamp loading reaction, indicating that the gamma complex and its associated beta clamp interact with approximately 14-16 bp at a ssDNA/dsDNA junction during the clamp loading operation. A protein block positioned closer than 20-22 bp from the 3' terminus prevents use of the clamp by the polymerase in chain elongation, indicating that the polymerase has an even greater spatial requirement than the gamma complex on the duplex portion of the primed site for function with beta. Interestingly, DNA secondary structure elements placed near the 3' terminus impose similar steric limits on the gamma complex and polymerase action with beta. The possible biological significance of these structural constraints is discussed. PMID- 10753962 TI - Beta(1,2)-xylose and alpha(1,3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens. AB - Primary structures of the N-glycans of two major pollen allergens (Lol p 11 and Ole e 1) and a major peanut allergen (Ara h 1) were determined. Ole e 1 and Ara h 1 carried high mannose and complex N-glycans, whereas Lol p 11 carried only the complex. The complex structures all had a beta(1,2)-xylose linked to the core mannose. Substitution of the proximal N-acetylglucosamine with an alpha(1, 3) fucose was observed on Lol p 11 and a minor fraction of Ole e 1 but not on Ara h 1. To elucidate the structural basis for IgE recognition of plant N-glycans, radioallergosorbent test analysis with protease digests of the three allergens and a panel of glycoproteins with known N-glycan structures was performed. It was demonstrated that both alpha(1,3)-fucose and beta(1,2)-xylose are involved in IgE binding. Surprisingly, xylose-specific IgE antibodies that bound to Lol p 11 and bromelain did not recognize closely related xylose-containing structures on horseradish peroxidase, phytohemeagglutinin, Ole e 1, and Ara h 1. On Lol p 11 and bromelain, the core beta-mannose is substituted with just an alpha(1,6) mannose. On the other xylose-containing N-glycans, an additional alpha(1,3) mannose is present. These observations indicate that IgE binding to xylose is sterically hampered by the presence of an alpha(1,3)-antenna. PMID- 10753963 TI - Isolation and characterization of an acetylene-resistant nitrogenase. AB - A genetic strategy was developed for the isolation of a mutant strain of Azotobacter vinelandii that exhibits in vivo nitrogenase activity resistant to inhibition by acetylene. Examination of the kinetic features of the altered nitrogenase MoFe protein produced by this strain, which has serine substituted for the alpha-subunit Gly(69) residue, is consistent with other studies that indicate the MoFe protein normally contains at least two acetylene binding/reduction sites. The first of these is a high affinity site and is the one primarily accessed during typical acetylene reduction assays. Results of the present work indicate that this acetylene binding/reduction site is not directly relevant to the mechanism of nitrogen reduction because it can be eliminated or severely altered without significantly affecting nitrogen reduction. Elimination of this site also results in the manifestation of a low affinity acetylene binding site to which both acetylene and nitrogen are able to bind with approximately the same affinity. In contrast to the normal enzyme, nitrogen and acetylene binding to the altered MoFe protein are mutually competitive. The location of the alpha-Ser(69) substitution is interpreted to indicate that the 4Fe-4S face of the FeMo cofactor capped by the alpha-subunit Val(70) residue is the most likely region within FeMo cofactor to which acetylene binds with high affinity. PMID- 10753964 TI - Amyloid and non-amyloid forms of 5q31-linked corneal dystrophy resulting from kerato-epithelin mutations at Arg-124 are associated with abnormal turnover of the protein. AB - Mutations in kerato-epithelin are responsible for a group of hereditary cornea specific deposition diseases, 5q31-linked corneal dystrophies. These conditions are characterized by progressive accumulation of protein deposits of different ultrastructure. Herein, we studied the corneas with mutations at kerato-epithelin residue Arg-124 resulting in amyloid (R124C), non-amyloid (R124L), and a mixed pattern of deposition (R124H). We found that aggregated kerato-epithelin comprised all types of pathological deposits. Each mutation was associated with characteristic changes of protein turnover in corneal tissue. Amyloidogenesis in R124C corneas was accompanied by the accumulation of N-terminal kerato-epithelin fragments, whereby species of 44 kDa were the major constituents of amyloid fibrils. R124H corneas with prevailing non-amyloid inclusions showed accumulation of a new 66-kDa species altogether with the full-size 68-kDa form. Finally, in R124L cornea with non amyloid deposits, we found only the accumulation of the 68 kDa form. Two-dimensional gels revealed mutation-specific changes in the processing of the full-size protein in all affected corneas. It appears that substitutions at the same residue (Arg-124) result in cornea-specific deposition of kerato-epithelin via distinct aggregation pathways each involving altered turnover of the protein in corneal tissue. PMID- 10753965 TI - Characterization of calcium release-activated apoptosis of LNCaP prostate cancer cells. AB - Apoptosis inhibition rather than enhanced cellular proliferation occurs in prostate cancer (CaP), the most commonly diagnosed malignancy in American men. Therefore, it is important to characterize residual apoptotic pathways in CaP cells. When intracellular Ca(2+) stores are released and plasma membrane "store operated" Ca(2+) entry channels subsequently open, cytosolic [Ca(2+)] increases and is thought to induce apoptosis. However, cells incapable of releasing Ca(2+) stores are resistant to apoptotic stimuli, indicating that Ca(2+) store release is also important. We investigated whether release of intracellular Ca(2+) stores is sufficient to induce apoptosis of the CaP cell line LNCaP. We developed a method to release stored Ca(2+) without elevating cytosolic [Ca(2+)]; this stimulus induced LNCaP cell apoptosis. We compared the apoptotic pathways activated by intracellular Ca(2+) store release with the dual insults of store release and cytosolic [Ca(2+)] elevation. Earlier processing of caspases-3 and -7 occurred when intracellular store release was the sole Ca(2+) perturbation. Apoptosis was attenuated in both conditions in stable transfected cells expressing antiapoptotic proteins Bclx(L) and catalytically inactive caspase-9, and in both scenarios inactive caspase-9 became complexed with caspase-7. Thus, intracellular Ca(2+) store release initiates an apoptotic pathway similar to that elicited by the dual stimuli of cytosolic [Ca(2+)] elevation and intracellular store release. PMID- 10753966 TI - Novel negative regulatory element in the platelet-derived growth factor B chain promoter that mediates ERK-dependent transcriptional repression. AB - Platelet-derived growth factor (PDGF), which consists of an A and/or B chain, stimulates migration and proliferation in vascular smooth muscle cells as well as a large number of other cell types. Investigations over recent years have defined roles for several positive regulatory transcription factors in the PDGF-B promoter. However, little is known about the transcriptional mechanisms that negatively regulate this gene. Here, we used transient transfection and 5' deletion analysis to define a specific region in the PDGF-B promoter-mediating repression in vascular smooth muscle cells. Gel retardation assays revealed this region is bound by nuclear protein(s) in a specific manner. Supershift assays excluded the direct association of Sp1, Sp3, and Egr-1. Mutation of the negative regulatory element no longer supported nucleoprotein complex formation and, when introduced into the PDGF-B promoter, rescued the promoter from repression. Promoter activity was also restored by transfection of oligonucleotide decoys bearing the repressor binding site. The MEK1/2 inhibitor, PD98059, and a dominant negative construct generating inactive ERK1 increased reporter expression driven by the PDGF-B promoter. In contrast, the MEK inhibitor had no effect on the activity of the mutant PDGF-B promoter. These effects were cell type-specific, since neither suppression of the PDGF-B promoter nor nucleoprotein complex formation was observed in vascular endothelial cells. These findings define a distinct negative regulatory element in the PDGF-B promoter that interacts with nuclear protein(s) and inhibits PDGF-B promoter-dependent gene expression in an ERK-dependent manner. PMID- 10753967 TI - Farnesol stimulates differentiation in epidermal keratinocytes via PPARalpha. AB - The isoprenoids farnesol and juvenile hormone III (JH), metabolites of the cholesterol biosynthetic pathway, have been shown to stimulate fetal epidermal development in rodents. In this study we determined whether this effect might be attributed to a direct induction of keratinocytes differentiation and examined the mechanisms responsible for these effects. Rates of cornified envelope formation, a marker of keratinocyte terminal differentiation, as well as protein and mRNA levels of two proteins required for cornified envelope formation, involucrin (INV) and transglutaminase, increased 2- to 3-fold in normal human keratinocytes (NHK) treated with either farnesol or JH, even at low calcium concentrations (0.03 mM), which otherwise inhibit differentiation. In contrast, neither cholesterol nor mevalonate affected INV or transglutaminase mRNA levels. Effects of farnesol and JH on INV and transglutaminase mRNA levels were additive with high calcium concentrations (1.2 mM) that independently stimulate keratinocyte differentiation. In contrast, keratinocyte DNA synthesis was inhibited by these compounds. Both farnesol and JH stimulated INV and transglutaminase promoter activity, suggesting regulation at the transcriptional level. A series of truncation and deletion experiments revealed a farnesol responsive region (-2452 to -1880 base pairs (bp)) in the INV gene. This region contained an AP-1 site. A single base pair mutation of the AP-1 site at -2116 to 2110 bp abolished farnesol responsiveness, identical to effects by peroxisome proliferator-activated receptor (PPARalpha) activators. Farnesoid X-activated receptor mRNA was not detected in NHK, but farnesol treatment increased activities of both a PPAR response element and PPARalpha mRNA levels in NHK. Furthermore, the increase in PPRE activity by farnesol was dependent upon PPARalpha in CV-1 cells. Finally, topical applications of farnesol increased mRNA and protein levels of the differentiation-specific genes, profilaggrin and loricrin, determined by immunohistochemistry and in situ hybridization, in wild type but not in PPARalpha-/- murine epidermis. These findings suggest a novel role for selected isoprenoid cholesterol intermediates in the regulation of differentiation-specific gene transcription and a convergence of PPARalpha with the cholesterol synthetic pathway. PMID- 10753968 TI - Decreased DNA repair efficiency by loss or disruption of p53 function preferentially affects removal of cyclobutane pyrimidine dimers from non transcribed strand and slow repair sites in transcribed strand. AB - The tumor suppressor protein p53 plays a central role in modulating the cellular responses to DNA damage. Several recent studies, undertaken with the whole genomic DNA or full-length gene segments, have shown that p53 is involved in nucleotide excision repair and it selectively influences the adduct removal from the non-transcribed strand in the genome. In this study, we have analyzed the damage induction at nucleotide resolution by ligase-mediated polymerase chain reaction and compared the repair of ultraviolet radiation-induced cyclobutane pyrimidine dimers within exon 8 of p53 gene in normal and Li-Fraumeni syndrome fibroblasts as well as in normal and human papillomavirus 16 E6 and E7 protein expressing human mammary epithelial cells. The results demonstrate that (i) loss or disruption of p53 function decreases efficiency of DNA repair, by preferentially affecting the repair of non-transcribed strand and of intrinsically slow repair sites in transcribed strand; (ii) mutant p53 protein affects DNA repair, at least of non-transcribed strand, in a dominant negative manner; and (iii) pRb does not have an effect on the repair of DNA damage within transcribed or non-transcribed strand. The overall data suggest that p53 could regulate excision repair or related events through direct protein-protein interaction. PMID- 10753969 TI - Structural organization of distinct domains within the non-collagenous N-terminal region of collagen type XI. AB - Collagen XI is a heterotrimeric molecule found predominantly in heterotypic cartilage fibrils, where it is involved in the regulation of fibrillogenesis. This function is thought to involve the complex N-terminal domain. The goal of this current study was to examine its structural organization to further elucidate the regulatory mechanism. The amino-propeptide (alpha1-Npp) alone or with isoforms of the variable region were recombinantly expressed and purified by affinity and molecular sieve chromatography. Cys-1-Cys-4 and Cys-2-Cys-3 disulfide bonds were detected by liquid chromatography-tandem mass spectrometry. This pattern is identical to the homologous alpha2-Npp, indicating that the recombinant proteins were folded correctly. Anomalous elution on molecular sieve chromatography suggested that the variable region was extended, which was confirmed using rotary shadowing; the alpha1-Npp formed a globular "head" and the variable region an extended "tail." Circular dichroism spectra analysis determined that the alpha1-Npp comprised 33% beta-sheet, whereas the variable region largely comprised non-periodic structure. Taken together, these results imply that the alpha1-Npp cannot be accommodated within the core of the fibril and that the variable region and/or minor helix facilitates its exclusion to the fibril surface. This provides further support for regulation of fibril diameter by steric hindrance or by interactions with other matrix components that affect fibrillogenesis. PMID- 10753970 TI - Post-transcriptional regulation of thyroid hormone receptor expression by cis acting sequences and a naturally occurring antisense RNA. AB - Thyroid hormone (T(3)) coordinates growth, differentiation, and metabolism by binding to nuclear thyroid hormone receptors (TRs). The TRalpha gene encodes T(3) activated TRalpha1 (NR1A1a) as well as an antagonistic, non-T(3)-binding alternatively spliced product, TRalpha2 (NR1A1b). Thus, the TRalpha1/TRalpha2 ratio is a critical determinant of T(3) action. However, the mechanisms underlying this post-transcriptional regulation are unknown. We have identified a non-consensus, TRalpha2-specific 5' splice site and conserved intronic sequences as key determinants of TRalpha mRNA processing. In addition to these cis-acting elements, a novel regulatory feature is the orphan receptor RevErbAalpha (NR1D1) gene, which is transcribed from the opposite direction at the same locus and overlaps the TRalpha2 coding region. RevErbAalpha gene expression correlates with a high TRalpha1/TRalpha2 ratio in a number of tissues. Here we demonstrate that coexpression of RevErbAalpha and TRalpha regulates the TRalpha1/TRalpha2 ratio in intact cells. Thus, both cis- and trans-regulatory mechanisms contribute to cell specific post-transcriptional regulation of TR gene expression and T(3) action. PMID- 10753971 TI - Acetylation of novel sites in the nucleosomal binding domain of chromosomal protein HMG-14 by p300 alters its interaction with nucleosomes. AB - The reversible acetylation of histones is associated with structural alterations in the chromatin fiber that affect various DNA-related activities. Here we show that the histone acetyltransferase p300 specifically acetylates HMG-14, a nonhistone structural protein that binds to nucleosomes and reduces the compactness of the chromatin fiber. We identify 7 major acetylation sites, 6 of which are novel and have not been known to be acetylated in either HMG-14 or the closely related HMG-17 protein. All the acetylation sites involve evolutionarily conserved residues: 3 within the HMG-14/-17 nucleosomal binding domain and 4 in or near the bipartite nuclear localization domains of the proteins. In tissue culture cells the acetylation pattern is indicative of a selective process in which a subfraction of HMG-14 is preferentially acetylated. We find that the nucleosomal binding domain is a major target for acetylation in vivo and that the specific acetylation of HMG-14 by p300 weakens its interaction with nucleosome cores. Our results suggest that p300 modulates the interaction of HMG-14 with nucleosomes. Thus, p300 may affect chromatin-related activities not only by modifying histones or transcription factors but also by targeting structural nonhistone proteins. PMID- 10753972 TI - Evidence for overlapping and distinct functions in protein transport of coat protein Sec24p family members. AB - Budding of transport vesicles from the endoplasmic reticulum in yeast requires the formation, at the budding site, of a coat protein complex (COPII) that consists of two heterodimeric subcomplexes (Sec23p/Sec24p and Sec13p/Sec31p) and the Sar1 GTPase. Sec24p is an essential protein and involved in cargo selection. In addition to Sec24p, the yeast Saccharomyces cerevisiae expresses two non essential Sec24p-related proteins, termed Sfb2p (product of YNL049c) and Sfb3p/Lst1p (product of YHR098c). We here show that Sfb2p and, less efficiently, Sfb3p/Lst1p are able to bind, like Sec24p, the integral membrane cargo protein Sed5p. We also demonstrate that Sfb2p, like Sec24p and Sfb3p/Lst1p, forms a complex with Sec23p in vivo. Whereas the deletion of SFB2 did not affect transport kinetics of various proteins, the maturation of the glycolipid-anchored plasma membrane protein Gas1p was differentially impaired in sfb3 knock-out cells. We generated several conditional-lethal sec24 mutants that, combined with null alleles of SFB2 and SFB3/LST1, led to a complete block of transport between the endoplasmic reticulum and the Golgi (sec24-11/Deltasfb2) or to cell death (sec24-11/Deltasfb3). Of the Sec24p family members, Sfb2p is the least abundant at steady state, but high intracellular concentrations of Sfb2p can rescue sec24 mutants under restrictive conditions. The data presented strongly suggest that the Sec24p-related proteins function as COPII components. PMID- 10753973 TI - Reversible phosphorylation at the C-terminal regulatory domain of p21(Waf1/Cip1) modulates proliferating cell nuclear antigen binding. AB - The p53-inducible gene product p21(WAF1/CIP1) plays a critical role in regulating the rate of tumor incidence, and identifying mechanisms of its post-translational regulation will define key pathways that link growth control to p21-dependent tumor suppression. A eukaryotic cell model system has been developed to determine whether protein kinase signaling pathways that phosphorylate human p21 exist in vivo and whether such pathways regulate the binding of p21 to one of its key target proteins, proliferating cell nuclear antigen (PCNA). Although human p21 expressed in Sf9 cells is able to form a complex with human PCNA, the inclusion of cell-permeable phosphatase inhibitors renders p21 protein inactive for PCNA binding. The treatment of this inactive isoform of p21 with alkaline phosphatase restores its binding to PCNA, suggesting that p21 expressed in Sf9 cells is subject to reversible phosphorylation at a key regulatory site(s). A biochemical approach was subsequently used to map the phosphorylation sites within p21, whose modification in vitro can inhibit p21-PCNA complex formation, to the C-terminal domain at residues Thr(145) or Ser(146). A phospho-specific antibody was developed that only bound to full-length p21 protein after phosphorylation in vitro at Ser(146), and this reagent was further used to demonstrate that the inactive isoform of p21 recovered from Sf9 cells treated with phosphatase inhibitors had been phosphorylated in vivo at Ser(146). These data identify the first phosphorylation site within the C-terminal regulatory domain of p21 whose modification in vivo modulates p21-PCNA interactions and define a eukaryotic cell model that can be used to study post-translational signaling pathways that regulate p21. PMID- 10753974 TI - Retrieval and reconsolidation: toward a neurobiology of remembering. PMID- 10753975 TI - Functional asymmetry of human prefrontal cortex: encoding and retrieval of verbally and nonverbally coded information. AB - There are several views about the organization of memory functions in the human prefrontal cortex. One view assumes a process-specific brain lateralization according to different memory subprocesses, that is, encoding and retrieval. An alternative view emphasizes content-specific lateralization of brain systems involved in memory processes. This study addresses this apparent inconsistency between process- and content-specific lateralization of brain activity by investigating the effects of verbal and nonverbal encoding on prefrontal activations during encoding and retrieval of environmental novel sounds using fMRI. An intentional memory task was applied in which subjects were required either to judge the sounds' loudness (nonverbal encoding task) or to indicate whether or not a sound can be verbally described (verbal encoding task). Retrieval processes were examined in a subsequent yes/no recognition test. In the study phase the right posterior dorsolateral prefrontal cortex (PFC) was activated in both tasks. During verbal encoding additional activation of the left dorsolateral PFC was obtained. Retrieval-related fMRI activity varied as a function of encoding task: For the nonverbal task we detected an activation focus in the right posterior dorsolateral PFC whereas an activation in the left dorsolateral PFC was observed for the verbal task. These findings indicate that the right dorsolateral PFC is engaged in encoding of auditory information irrespective of encoding task. The lateralization of PFC activity during retrieval was shown to depend on the availability of verbal codes, with left hemispheric involvement for verbally and right hemispheric activation for nonverbally coded information. PMID- 10753976 TI - Differential fear conditioning induces reciprocal changes in the sensory responses of lateral amygdala neurons to the CS(+) and CS(-). AB - In classical fear conditioning, a neutral sensory stimulus (CS) acquires the ability to elicit fear responses after pairing to a noxious unconditioned stimulus (US). As amygdala lesions prevent the acquisition of fear responses and the lateral amygdaloid (LA) nucleus is the main input station of the amygdala for auditory afferents, the effect of auditory fear conditioning on the sensory responsiveness of LA neurons has been examined. Although conditioning was shown to increase CS-evoked LA responses, the specificity of the changes in responsiveness was not tested. Because conditioning might induce nonspecific increases in LA responses to auditory afferents, we re-examined this issue in conscious, head-restrained cats using a differential conditioning paradigm where only one of two tones (CS(+) but not CS(-)) was paired to the US. Differential conditioning increased unit and field responses to the CS(+), whereas responses to the CS(-) decreased. Such changes have never been observed in the amygdala except in cases where the CS(-) had been paired to the US before and fear responses not extinguished. This suggests that fear conditioning is not only accompanied by potentiation of amygdalopetal pathways conveying the CS(+) but also by the depression of sensory inputs unpaired to noxious stimuli. PMID- 10753977 TI - The operant and the classical in conditioned orientation of Drosophila melanogaster at the flight simulator. AB - Ever since learning and memory have been studied experimentally, the relationship between operant and classical conditioning has been controversial. Operant conditioning is any form of conditioning that essentially depends on the animal's behavior. It relies on operant behavior. A motor output is called operant if it controls a sensory variable. The Drosophila flight simulator, in which the relevant behavior is a single motor variable (yaw torque), fully separates the operant and classical components of a complex conditioning task. In this paradigm a tethered fly learns, operantly or classically, to prefer and avoid certain flight orientations in relation to the surrounding panorama. Yaw torque is recorded and, in the operant mode, controls the panorama. Using a yoked control, we show that classical pattern learning necessitates more extensive training than operant pattern learning. We compare in detail the microstructure of yaw torque after classical and operant training but find no evidence for acquired behavioral traits after operant conditioning that might explain this difference. We therefore conclude that the operant behavior has a facilitating effect on the classical training. In addition, we show that an operantly learned stimulus is successfully transferred from the behavior of the training to a different behavior. This result unequivocally demonstrates that during operant conditioning classical associations can be formed. PMID- 10753979 TI - Ad hoc reviewers PMID- 10753980 TI - The rtPA (alteplase) 0- to 6-hour acute stroke trial, part A (A0276g) : results of a double-blind, placebo-controlled, multicenter study. Thromblytic therapy in acute ischemic stroke study investigators. AB - BACKGROUND AND PURPOSE: The Thrombolytic Therapy in Acute Ischemic Stroke Study, which started in August of 1991, was designed to assess the efficacy and safety of intravenous rtPA (alteplase) in patients with acute (0 to 6 hours) ischemic stroke. In October 1993 enrollment was halted because of Safety Committee (DMSB) concerns. In December 1993 the time window was changed to 0 to 5 hours, and it was decided to restart enrollment as a separate study (part B). We report here the results of the original study (part A), focusing on evaluating the safety and efficacy of rtPA given between 0 and 6 hours after stroke onset. METHODS: This investigation was a phase II, placebo-controlled, double-blind, randomized study utilizing 0.9 mg/kg IV rtPA or placebo over 1 hour, which was conducted at university and community sites in North America. Except for time to treatment, enrollment criteria were very similar to those of the NINDS rtPA stroke study. Primary efficacy end points were the number of patients with a decrease of 4 or more points on the National Institutes of Health Stroke Scale (NIHSS) at 24 hours and day 30, along with infarct volume at day 30. Secondary end points included mortality and functional recoveries on the Barthel Index and Modified Rankin scale at days 30 and 90. RESULTS: A total of 142 patients were enrolled at 42 sites in North America, including 22 <3 hours (15%) and 46 between 5 and 6 hours (32%). The groups were well matched on baseline characteristics, including NIHSS (mean of 13 for both). For the primary end points, a higher percentage of rtPA patients had a 4-point improvement at 24 hours (placebo 21%, rtPA 40%; P=0.02); however, this early effect was reversed by 30 days, with more placebo patients having a 4-point improvement (75%) than patients treated with rtPA (60%, P=0.05). Treatment with rtPA significantly increased the rate of symptomatic intracerebral hemorrhage within 10 days (11% versus 0%, P<0.01) and mortality at 90 days (23% versus 7%, P<0.01). CONCLUSIONS: This study found no significant rtPA benefit on any of the planned efficacy end points at 30 and 90 days in patients treated between 0 and 6 hours after stroke onset. These negative results apply to patients treated after 3 hours, because only 15% of the patients were enrolled before 3 hours. The risk of symptomatic intracerebral hemorrhage was increased with rtPA treatment, particularly in patients treated between 5 and 6 hours after onset. These results do not support the use of intravenous rtPA for stroke treatment >3 hours after onset. PMID- 10753978 TI - Tactile experience induces c-fos expression in rat barrel cortex. AB - Understanding gene expression that is responsive to sensory stimulation is central to elucidate molecular mechanisms underlying neuronal plasticity. In this study we demonstrate two new methods of stimulating whiskers that provide major sensory input to rat neocortex. In the first paradigm, animals were placed on the top of a cylinder and their vibrissae were brushed by hand. In the second paradigm, animals were placed for a brief period of time into a new, wired cage resulting in vibrissae stimulation when they explored the new environment. Both approaches induced c-Fos expression in barrel cortex corresponding to the stimulated vibrissae, especially in layer IV. Layers II/III and V/VI also showed c-Fos induction, but there were no detectable changes in layer VIb. The majority of c-Fos-expressing cells are probably not inhibitory neurons, because they do not show parvalbumin staining. Both paradigms, in contrast to the previous methods, are simple to use and do not require anesthesia, restraint of animals, or elaborate experimental setups. PMID- 10753981 TI - Optimal intensity of warfarin therapy for secondary prevention of stroke in patients with nonvalvular atrial fibrillation : a multicenter, prospective, randomized trial. Japanese Nonvalvular Atrial Fibrillation-Embolism Secondary Prevention Cooperative Study Group. AB - BACKGROUND AND PURPOSE: The optimal intensity of warfarin therapy for secondary prevention of stroke in nonvalvular atrial fibrillation (NVAF) remains unclear. We studied the efficacy and safety of conventional- and low-intensity warfarin therapy in a prospective, randomized, multicenter trial. METHODS: The study population consisted of patients with NVAF (<80 years old) who had a stroke or transient ischemic attack. The patients were randomly allocated into a conventional-intensity group (international normalized ratio [INR] 2.2 to 3.5) and a low-intensity group (INR 1.5 to 2.1). They were carefully monitored, and the annual rate of recurrent ischemic stroke and major hemorrhagic complications were compared between the groups. RESULTS: We enrolled 115 patients (mean age 66.7+/-6.5 years) into the study. Fifty-five and 60 patients were allocated into the conventional- and low-intensity groups, respectively. The trial was stopped after a follow-up of 658+/-423 days, when major hemorrhagic complications occurred in 6 patients of the conventional-intensity group and the frequency (6.6% per year) was significantly higher than that in the low-intensity group (0% per year, P=0.01, Fisher's exact test). All of the 6 patients with major bleeding were elderly (mean age 74 years), and their mean INR before the major hemorrhage was 2.8. The annual rate of ischemic stroke was low in both groups (1.1% per year in the conventional-intensity group and 1.7% per year in the low-intensity groups) and did not differ significantly. CONCLUSIONS: For secondary prevention of stroke in persons with NVAF, especially in old patients, the low-intensity warfarin (INR 1.5 to 2. 1) treatment seems to be safer than the conventional intensity (INR 2.2 to 3.5) treatment. PMID- 10753982 TI - Adverse outcomes and predictors of underuse of antithrombotic therapy in medicare beneficiaries with chronic atrial fibrillation. AB - BACKGROUND AND PURPOSE: Antithrombotic therapy can prevent strokes and transient ischemic attacks (TIAs) in carefully selected patients who have chronic nonvalvular atrial fibrillation (NVAF). Our objectives were 3-fold: to document the use of warfarin and aspirin therapy in Missouri Medicare beneficiaries with chronic NVAF; to identify factors associated with warfarin and aspirin underuse; and to determine the association between prescription of warfarin and aspirin at hospital discharge and adverse outcomes in this elderly, frail population. METHODS: We linked chart reviews from all Missouri hospitals to Medicare claims data from 1993 to 1996. From chart reviews, we documented Medicare beneficiaries' demographic factors, comorbid conditions, and antithrombotic therapy prescribed at the time of hospital discharge. From Medicare claims, we determined the date of outcomes-death from any cause or hospitalization for an ischemic event (a stroke, a TIA, or a myocardial infarction). RESULTS: Only 328 (55%) of the 597 Medicare beneficiaries were prescribed antithrombotic therapy at hospital discharge: 34% received warfarin and 21% received aspirin. Advanced age, female gender, and rural residency predicted underuse of antithrombotic therapy. After controlling for these factors, as well as stroke risk factors and contraindications to anticoagulation, the prescription of warfarin was associated with a 24% relative risk reduction (RRR) in adverse outcomes (P=0.003). Prescription of aspirin was associated with a nonsignificant 5% RRR in these events (P=0.56). CONCLUSIONS: The underuse of antithrombotic therapy in Medicare beneficiaries who have NVAF is associated with measurable adverse outcomes. The benefit of warfarin therapy may extend to frail, elderly patients, a group that was excluded from randomized controlled trials. The role of antiplatelet therapy in this population deserves further study because many of these patients have relative contraindications to warfarin. PMID- 10753983 TI - Effects of fixed low-dose warfarin, aspirin-warfarin combination therapy, and dose-adjusted warfarin on thrombogenesis in chronic atrial fibrillation. AB - BACKGROUND AND PURPOSE: Recent clinical trials have established that adjusted dose warfarin (international normalized ratio [INR] 2.0 to 3.0) is highly effective in the reduction of ischemic stroke in patients with nonvalvular atrial fibrillation (AF). We hypothesized that the introduction of fixed low-dose warfarin alone or in combination with aspirin (300 mg) could normalize hemostatic markers, namely plasma fibrin D-dimer (an index of thrombogenesis), plasminogen activator inhibitor-1 (PAI-1, an index of fibrinolysis), fibrinogen, and von Willebrand factor (vWf, an index of endothelial dysfunction), in a manner comparable to adjusted-dose warfarin (target INR 2.0 to 3.0). METJODS: Sixty-one patients with AF (44 men, mean+/-SD age 64+/-19 years) who were not receiving any antithrombotic therapy were prospectively randomized into 1 of 3 treatment groups: warfarin (2 mg) (n=23; group 1), combination 1 mg warfarin plus 300 mg aspirin (n=21; group 2) or combination 2 mg warfarin plus 300 mg aspirin (n=17; group 3). Subjects from all 3 AF groups were matched for sex, age, and blood pressure. Blood samples were taken for sequential measurements for changes in plasma fibrin D-dimer, PAI-1, fibrinogen, and vWf before and at 2 and 8 weeks after randomization (phase 1). All patients were subsequently offered adjusted dose warfarin therapy (phase 2), and an additional blood sample was taken 6 weeks later. RESULTS: When pretreatment results were compared with those from 60 age- and sex-matched healthy control subjects in sinus rhythm, there were significant elevations in levels of fibrinogen (P=0.025), vWf (P<0.0001), and fibrin D-dimer (P<0.0001) in patients with AF compared with control subjects. There were no significant changes in the levels of various indices measured after 2 and 8 weeks of therapy in all 3 groups, except for an increase in PAI-1 level (P=0.024) in group 3. After 6 weeks of therapy with dose-adjusted warfarin (INR 2.0 to 3.0), there was a significant decrease in plasma fibrinogen (P=0.023) and fibrin D dimer (P=0.0067) levels. There were no significant changes in the levels of PAI-1 (P=0.198) or vWf (P=0.33). CONCLUSIONS: The present results confirmed that high levels of vWf, fibrinogen, and fibrin D-dimer levels were present in patients with AF compared with control subjects. Moreover, the introduction of 300 mg aspirin plus low-dose warfarin (1 mg/d), low-dose warfarin alone (2 mg/d), or 300 mg aspirin plus low-dose warfarin (2 mg/d) did not significantly reduce any of the hemostatic markers studied (except PAI-1 levels), whereas conventional full dose warfarin (INR 2.0 to 3.0) significantly reduced levels of fibrin D-dimer and fibrinogen. These results are in keeping with the disappointing ineffectiveness of low-intensity warfarin therapy, aspirin-warfarin combination, and ultralow dose warfarin therapy in the recent prematurely terminated clinical trials and the established benefits of conventional adjusted-dose anticoagulation therapy. PMID- 10753984 TI - Stereotactic computed tomographic-guided aspiration and thrombolysis of intracerebral hematoma : protocol and preliminary experience. AB - BACKGROUND AND PURPOSE: We review preliminary experience with patients harboring intracerebral hematoma (ICH) treated by stereotactic computed tomographic (CT) guided thrombolysis and aspiration and assess procedure feasibility and safety. METHODS: Twelve patients with supratentorial ICH >/=25 mL without suspected underlying structural etiology or coagulopathy and an initial Glasgow Coma Scale (GCS) score of >/=5 were treated. A catheter was directed stereotactically or manually into the ICH through a burr hole under CT guidance. Hematoma aspiration was followed by instillation of urokinase (5 000 to 10 000 IU). This was repeated every 6 to 8 hours at bedside, with interval CT imaging, until the ICH volume diminished to <25 mL, less than half of its initial volume, or after a maximum of 10 aspirations/instillations. RESULTS: Mean age was 69 years (range 55 to 82 years). Median initial GCS was 12 (range 5 to 14). There were 7 ganglionic and 5 lobar ICH, and baseline hematoma size ranged 29 to 70 mL (mean 46 mL). Final ICH volume ranged from 14 to 51 mL (mean 21 mL), with ICH volume reduction by an average of 57% (range 38% to 70%). One patient (8. 3%) suffered hematoma expansion during the procedure. At 6 months after the procedure, 3 patients (25%) had achieved a good recovery (Glasgow Outcome Scale [GOS] score of 5), 5 patients (42%) were dependent (GOS 3), and 1 (8.3%) remained vegetative (GOS 2). Three patients (25%) died in hospital (1 from cardiac arrhythmia and 2 from respiratory failure). CONCLUSIONS: CT-guided thrombolysis and aspiration appears safe and effective in the reduction of ICH volume. Further studies are needed to assess optimal thrombolytic dosage and must include controlled comparisons of mortality, disability outcome, time until convalescence, and cost of care in treated and untreated patients. PMID- 10753985 TI - Treatment of intraventricular hemorrhage with urokinase : effects on 30-Day survival. AB - BACKGROUND AND PURPOSE: Intraventricular hemorrhage (IVH) remains associated with high morbidity and mortality. Therapy with external ventricular drainage alone has not modified outcome in these patients. METHODS: Twelve pilot IVH patients who required external ventricular drainage were prospectively treated with intraventricular urokinase followed by the randomized, double-blinded allocation of 8 patients to either treatment or placebo. Observed 30-day mortality was compared with predicted 30-day mortality obtained by use of a previously validated method. RESULTS: Twenty patients were enrolled; admission Glasgow Coma Scale score in 11 patients was 5.0 micromol/mL were significantly associated with early neurological worsening (OR 5.7, 95% CI 1.2 to 27.4; P=0.030) independent of other important factors related to progressing stroke, such as CSF glutamate levels. CONCLUSIONS: Our clinical findings suggest an important role of NO generation in acute ischemic stroke. Increased NO-m in CSF are associated with a greater brain injury and early neurological deterioration. PMID- 10753989 TI - Predictors of death and vascular events in the elderly : the Perth Community Stroke Study. AB - BACKGROUND AND PURPOSE: The goal of the present study was to identify risk factors for vascular disease in the elderly. METHODS: We conducted a prospective study of control subjects from a population-based study of stroke in Perth, Western Australia, that was completed in 1989 to 1990 and used record linkage and a survey of survivors to identify deaths and nonfatal vascular events. Data validated through reference to medical records were analyzed with the use of Cox proportional hazards models. RESULTS: Follow-up for the 931 subjects was 88% complete. By June 24, 1994, 198 (24%) of the subjects had died (96 from vascular disease), and there had been 45 nonfatal strokes or myocardial infarctions. The hazard ratio for diabetes exceeded 2.0 for all end points, whereas the consumption of meat >4 times weekly was associated with a reduction in risk of /=35 years. Prevalence was >2-fold higher in men than in women (247/100 000 and 99/100 000, respectively) and increased rapidly with age. Seven cases were hospitalized and received specific treatment. CONCLUSIONS: Our crude prevalence is lower compared with rates from developed countries, probably because of a high case fatality rate. Our findings are comparable with those reported from other surveys carried out in rural developing countries. PMID- 10753993 TI - Prevalence and impact of urinary symptoms among community-dwelling stroke survivors. AB - BACKGROUND AND PURPOSE: To establish prevalence of urinary symptoms among community-dwelling stroke survivors and how these symptoms affected lives of these survivors compared with a nonstroke population. METHODS: The present study was a community-based postal survey in Leicestershire community, UK (that excluded institutional settings), that was designed to track stroke, urinary, and bowel symptoms and the effect of such symptoms on relationships, social life, daily activities, and overall quality of life. Subjects included 14 600 people who were living in the community and /=4 National Institutes of Health Stroke Scale points within 24 hours of the initial symptom onset. RESULTS: We studied 50 consecutive patients presenting at 165+/-96 minutes from symptom onset. Mean age was 61+/-14 years; 50% were females. All patients had TCD at the time of presentation, and 68% had subsequent angiographic examinations (DSA 10%, CTA 4%, and MRA 44%). Overall, large-vessel occlusion on TCD was found in 16% of patients (n=8); stenosis was found in 18% (n=9); 54% (n=27) had normal studies; and 6 patients (12%) had no temporal windows. DFI occurred in 16% (n=8) of the 50 patients: in 62% of patients with TCD and angiographic evidence of occlusion, in 22% with stenosis, and in 4% with normal vascular studies (P<0.001, Phi=0.523, chi(2)=12.05). DFI occurred in 31% of patients with large-vessel atherosclerosis, 23% with cardioembolism, and 9% with small-vessel disease when stroke mechanisms were determined within 2 to 3 days after admission (P=0.2, NS). CONCLUSIONS: DFI is strongly associated with the presence of large-vessel occlusion or stenosis of either atherosclerotic or embolic origin. Normal vascular studies and lacunar events were associated with stable spontaneous resolution without subsequent fluctuation. Urgent vascular evaluation may help identify patients with resolving deficits and vascular lesions who may be candidates for new therapies to prevent subsequent deterioration. PMID- 10753999 TI - Benefit of echocontrast-enhanced transcranial color-coded duplex ultrasound in the assessment of intracranial collateral pathways. AB - BACKGROUND AND PURPOSE: Although clinically important, proper assessment of intracranial arterial collateral pathways by transcranial color-coded duplex sonography (TCCD) in patients with internal carotid artery (ICA) high-grade stenosis or occlusion is occasionally made difficult by an insufficient temporal bone window, an unfavorable insonation angle, or low flow velocity or volume. In these cases, echocontrast could be helpful to increase the diagnostic confidence or to make the diagnosis at all. METHODS: We investigated 50 temporal windows of 44 patients with ipsilateral high-grade (>/=70%) ICA stenosis or occlusion and insufficient native transtemporal insonation conditions before and after the application of the echo enhancer Levovist with an infusion pump. RESULTS: Compared with the precontrast scans, echocontrast allowed for more segments to be evaluated by pulsed Doppler sonography (P<0. 0001) and for longer lumen segments to be displayed on color mode (P<0.0001). Also, collateral flow via the anterior and posterior communicating artery could be demonstrated in 25 and 32 scans, respectively, compared with only 1 demonstration of each collateral pathway before the application of contrast medium (both P<0.0001). Similarly, with the help of contrast medium, flow velocity in the middle cerebral artery could be measured in 45 cases compared with only 26 cases before contrast was applied (P<0.0001). CONCLUSIONS: In patients with poor precontrast visualization of intracranial arteries, echocontrast-enhanced TCCD is very helpful in the assessment of intracranial collateral pathways recruited downstream to ICA stenoses and occlusions. PMID- 10754000 TI - Reduced vasomotor reactivity in cerebral microangiopathy : a study with near infrared spectroscopy and transcranial Doppler sonography. AB - BACKGROUND AND PURPOSE: Reduction of cerebral blood flow and vasomotor reactivity (VMR) are thought to play an important role in the pathogenesis of cerebral microangiopathy. The aim of our study was to determine whether near-infrared spectroscopy (NIRS) can detect a reduced VMR in patients with microangiopathy, whether NIRS reactivities correlate with VMR assessed by transcranial Doppler sonography (TCD), and whether the differing extents of patients' microangiopathy demonstrated on MRI or CT can be distinguished by both noninvasive techniques. METHODS: We compared the VMR of 46 patients with cerebral microangiopathy with 13 age-matched control subjects. Patients were classified with the Erkinjuntti scale. We monitored cerebral blood flow velocity (CBFV) in both middle cerebral arteries by TCD, changes in concentration of oxyhemoglobin (HbO(2)), deoxyhemoglobin (Hb) and blood volume (HbT) by NIRS, mean arterial blood pressure, and end-tidal CO(2) (EtCO(2)) during normocapnia and hypercapnia. VMRs were calculated as percent change of CBFV (NCR) and as absolute change in concentration of HbO(2), Hb, and HbT per 1% increase in EtCO(2) (CR-HbO(2), CR Hb, CR-HbT). RESULTS: NCR and NIRS reactivities were significantly reduced in patients with cerebral microangiopathy. CR-HbO(2) and CR-Hb showed a close correlation with NCR, and NCR and NIRS reactivities were related to the severity of cerebral microangiopathy according to the Erkinjuntti scale. Validity of NCR and NIRS reactivities were similar. CONCLUSIONS: VMR is reduced in patients with cerebral microangiopathy and can be noninvasively assessed in basal arteries (with TCD) and brain parenchyma (with NIRS). Reduction of CO(2)-induced VMR, as measured by NIRS and TCD, may indicate the severity of microangiopathy. PMID- 10754001 TI - Linkage of familial moyamoya disease (spontaneous occlusion of the circle of Willis) to chromosome 17q25. AB - BACKGROUND AND PURPOSE: Moyamoya disease is a cerebrovascular disease of unknown cause that mainly affects Japanese children. The incidence of familial occurrence accounts for 9% of cases. The characteristic lesions of moyamoya disease are occasionally seen in neurofibromatosis type 1, of which the causative gene (NF1) has been assigned to chromosome 17q11.2. METHODS: To determine whether a gene related to moyamoya disease is located on chromosome 17, we conducted microsatellite linkage analyses on 24 families containing 56 patients with moyamoya disease. Leukocyte DNA extracted from the family members was subjected to polymerase chain reaction for a total of 22 microsatellite markers on chromosome 17. The amplified polymerase chain reaction fragments were analyzed with GeneScan on an automated sequencer. RESULTS: Two-point linkage analysis gave a maximum log(10) odds (LOD) score of 3.11 at the recombination fraction of 0.00 for the marker at locus D17S939. The affected pedigree member method also showed a significantly low P value (<1. 0x10(-5)) for the 5 adjacent markers at 17q25. Multipoint linkage analysis also indicated that the disease gene is contained within the 9-cM region of D17S785 to D17S836, with a maximum LOD score of 4. 58. CONCLUSIONS: A gene for familial moyamoya disease is located on chromosome 17q25. PMID- 10754002 TI - C242T polymorphism of NADPH oxidase p22 PHOX gene and ischemic cerebrovascular disease in the Japanese population. AB - BACKGROUND AND PURPOSE: Superoxide has been implicated in the pathogenesis of ischemic stroke and atherosclerosis. NADPH oxidase, a major source of superoxide generation in neutrophils and the vascular system, plays a critical role in ischemic injury and atherogenesis. Recently, an association between the C242T polymorphism of p22 PHOX, an essential component of NADPH oxidase, and coronary artery disease (CAD) has been reported in several studies. To investigate the relationship between the C242T polymorphism of p22 PHOX and ischemic cerebrovascular disease (CVD), we conducted a case-control study. METHODS: We recruited 226 CVD patients (atherothrombotic infarction, lacunar infarction, and transient ischemic attack) and 301 control subjects and analyzed C242T polymorphism of p22 PHOX by detection of restriction fragment length polymorphism. RESULTS: The TC+TT genotype frequencies in the CVD group and control group were 21.7% and 13.3%, respectively, and the prevalence of the TC+TT genotype was significantly higher in the CVD patients (chi(2)=6.477, P=0.01, OR 1.81, 95% CI 1.15 to 2.86). Analysis by CVD subtypes showed that the OR for the TC+TT genotype was higher in the CVD patients with atherothrombotic infarction than in those with lacunar infarction and transient ischemic attack. CONCLUSIONS: The C242T polymorphism of the NADPH oxidase p22 PHOX gene is a novel pathogenetic risk factor for CVD. PMID- 10754003 TI - Postischemic attenuation of cerebral artery reactivity is increased in the presence of tissue plasminogen activator. AB - BACKGROUND AND PURPOSE: We investigated the combined effect of tissue plasminogen activator and ischemia on middle cerebral artery (MCA) reactivity to determine whether abnormal MCA function after 2 hours of ischemia was worse in arteries perfused with recombinant tissue plasminogen activator (rtPA). METHODS: The intraluminal suture model of focal cerebral ischemia was used to induce 2 hours of ischemia in rats, after which occluded MCAs were removed and studied in vitro with an arteriograph system that allowed control of transmural pressure (TMP) and measurement of lumen diameter. Arteries were either nonischemic (control; n=8), nonischemic and perfused with 400 microg/mL rtPA (rtPA; n=5), ischemic (ISC; n=6), or ischemic and perfused with 400 microg/mL rtPA (ISC-rtPA; n=6). After a 1 hour equilibration at 75 mm Hg, TMP was increased to 125 mm Hg and lumen diameter was recorded at each pressure. Reactivity to acetylcholine (ACh, 0.1 to 10.0 micromol/L) and serotonin (0.01 to 10 micromol/L) was then determined. RESULTS: Control arteries responded myogenically to pressure and increased the amount of tone from 18.5+/-3.8% at 75 mm Hg to 24.8+/-3.0% at 125 mm Hg (P<0.05), which decreased diameter from 241+/-7 to 232+/-6 microm. In contrast, all other groups decreased tone at 125 mm Hg, which demonstrated a loss of myogenicity. The percent tone in each group at 75 versus 125 mm Hg was rtPA, 16.0+/-4.5% versus 11.8+/-3.8%; ISC, 23.5+/-4.5% versus 13. 5+/-3.1%; and ISC-rtPA, 23.5+/-4.2% versus 12.3+/-3.2% (P<0.05 for all). The percent increase in lumen diameter at each concentration of ACh was diminished in all groups compared with control; ISC rtPA arteries responded the least, which suggests an additive effect of rtPA in ischemic arteries. The percent increase in lumen diameter at 10(-5)mol/L ACh was 23+/-4% for control versus 15+/-2% for rtPA; 17+/-3% for ISC arteries (P<0.05), and 8+/-2% for ISC-rtPA arteries (P<0.01). Sensitivity to serotonin was equally diminished in all groups compared with control: EC(50) (micromol/L) was 0.06+/ 0.01 for control, 0.17+/-0.02 for rtPA, 0.22+/-0.07 for ISC, and 0.16+/-0. 04 for ISC-rtPA (P<0.05). CONCLUSIONS: These results demonstrate that both ischemia and rtPA perfusion diminish cerebral artery reactivity and that the combination may produce an additive effect. This impaired reactivity may contribute to reperfusion-induced injury during or after thrombolysis by altering upstream cerebrovascular resistance. PMID- 10754004 TI - Transient and permanent resolution of ischemic lesions on diffusion-weighted imaging after brief periods of focal ischemia in rats : correlation with histopathology. AB - BACKGROUND AND PURPOSE: The early ischemic lesions demonstrated by diffusion weighted imaging (DWI) are potentially reversible. The purposes of this study were to determine whether resolution of initial DWI lesions is transient or permanent after different brief periods of focal brain ischemia and to evaluate histological outcomes. METHODS: Sixteen rats were subjected to 10 minutes (n=7) or 30 minutes (n=7) of temporary middle cerebral artery occlusion or sham operation (n=2). DWI, perfusion-weighted imaging (PWI), and T(2)-weighted imaging (T(2)WI) were performed during occlusion; immediately after reperfusion; and at 0.5, 1.0, 1.5, 12, 24, 48, and 72 hours after reperfusion. After the last MRI study, the brains were fixed, sectioned, stained with hematoxylin and eosin, and evaluated for neuronal necrosis. RESULTS: No MRI or histological abnormalities were observed in the sham-operated rats. In both the 10-minute and 30-minute groups, the perfusion deficits and DWI hyperintensities that occurred during occlusion disappeared shortly after reperfusion. The DWI, PWI, and T(2)WI results remained normal thereafter in the 10-minute group, whereas secondary DWI hyperintensity and T(2)WI abnormalities developed at the 12-hour observation point in the 30-minute group. Histological examinations demonstrated neuronal necrosis in both groups, but the number of necrotic neurons was significantly higher in the 30-minute group (95+/-4%) than in the 10-minute group (17+/-10%, P<0.0001). CONCLUSIONS: Transient or permanent resolution of initial DWI lesions depends on the duration of ischemia. Transient resolution of DWI lesions is associated with widespread neuronal necrosis; moreover, permanent resolution of DWI lesions does not necessarily indicate complete salvage of brain tissue from ischemic injury. PMID- 10754005 TI - Acute carotid occlusion alters the activation flow coupling response to forepaw stimulation in a rat model. AB - BACKGROUND AND PURPOSE: To determine whether the hemodynamic response to functional stimulation is sensitive to proximal arterial occlusion, we measured the activation flow coupling response in a rat model of acute reversible vascular occlusion. METHODS: In alpha-chloralose-anesthetized rats (n=18), laser Doppler measurements were made through a thinned skull over the somatosensory cortex in response to electrical forepaw stimulation. Signal-averaged responses to 4 and 8 seconds of electrical forepaw stimulation were obtained before, during, and shortly after acute unilateral or bilateral carotid occlusion produced with the use of a surgically placed snare. RESULTS: Baseline cerebral blood flow was significantly decreased over the forepaw region of the somatosensory cortex after both occlusion of the carotid contralateral to the stimulated forepaw and bilateral occlusion compared with preocclusion (P<0.05). Postocclusion and ipsilateral occlusion led to a nonsignificant increase in baseline cerebral blood flow compared with preocclusion. Contralateral carotid occlusion and bilateral occlusion significantly prolonged the temporal characteristics of the flow response, especially the delay to peak (P<0.05), compared with preocclusion, whereas ipsilateral carotid occlusion significantly shortened the delay to peak (P<0.05). Only contralateral carotid occlusion produced a significant reduction in the peak amplitude of the flow response compared with preocclusion (P<0.05). CONCLUSIONS: These findings suggest that temporal characteristics of functional activation responses are sensitive to alterations in the proximal arterial supply and, conversely, that functional activation studies must be interpreted with consideration of proximal arterial disease. PMID- 10754006 TI - Regional cerebral blood flow after cortical impact injury complicated by a secondary insult in rats. AB - BACKGROUND AND PURPOSE: Traumatic injury makes the brain susceptible to secondary insults. An uncomplicated mild lateral cortical impact injury (3 m/s, 2.5-mm deformation) that causes little or no permanent sequelae results in a large contusion at the impact site when the traumatic injury is complicated by a secondary insult, such as 40 minutes of bilateral carotid occlusion. METHODS: To determine whether the increased sensitivity to secondary insults in this model is caused by a vascular mechanism, cerebral blood flow (CBF) was measured with (14)C isopropyliodoamphetamine quantitative autoradiography, and brain tissue PO(2) (PbtO(2)) was measured at the impact site and in the contralateral parietal cortex. RESULTS: In animals that underwent bilateral carotid occlusion 1 hour after the impact injury, CBF and PbtO(2) were lower at the impact site than they were in animals that had either the impact injury or the carotid occlusion alone. In the immediate area of the impact, CBF was 14+/-6 mL. 100 g(-1). min(-1) in the animals with the impact injury followed by carotid occlusion compared with 53+/ 24 mL. 100 g(-1). min(-1) in the animals with the impact injury alone and 74+/-14 mL. 100 g(-1). min(-1) in the animals with the carotid occlusion alone (P<0.001). At the time of this very low CBF value in the animals with the carotid occlusion after the impact injury, PbtO(2) at the impact site averaged 1.3+/-1.6 mm Hg and was <3 mm Hg in 5 of the 6 animals. In contrast, PbtO(2) in the animals with the impact injury alone averaged 9.3+/-2.9 mm Hg, and none of the animals had a PbtO(2) of <3 mm Hg (P=0.008). CONCLUSIONS: The CBF and PbtO(2) findings in this model suggest that the reduced CBF after traumatic injury predisposes the brain to secondary insults and results in ischemia when confronted with a reduction in cerebral perfusion pressure. PMID- 10754007 TI - In vivo gene transfer of endothelial nitric oxide synthase to carotid arteries from hypercholesterolemic rabbits enhances endothelium-dependent relaxations. AB - BACKGROUND AND PURPOSE: Hypercholesterolemia is associated with abnormal endothelium-dependent vasorelaxation due to decreased nitric oxide bioavailability. Our aim was to examine the effect of adenovirus-mediated gene transfer of endothelial nitric oxide synthase (eNOS) to the hypercholesterolemic rabbit carotid artery in vivo. In addition, we examined whether adenovirus mediated gene transfer was associated with vascular dysfunction. METHODS: Rabbits were fed a 1% cholesterol diet for 4 weeks followed by a 0.5% cholesterol diet for 6 weeks. Vascular reactivity was assessed in nontransduced carotid arteries from chow- and cholesterol-fed animals. In addition, carotid arteries were surgically isolated, and 2 separate doses of adenoviral vectors encoding eNOS or beta-galactosidase (AdbetaGal) on the contralateral side were delivered to the lumen (1x10(10) and 5x10(10) pfu/mL). RESULTS: Abnormal acetylcholine-mediated endothelium-dependent vasorelaxation was detected in the carotid artery from cholesterol-fed animals, whereas responses to calcium ionophore A23187 and diethylamine NONOate were normal. Vascular reactivity was similar in nontransduced and AdbetaGal-transduced hypercholesterolemic vessels. In vessels transduced with eNOS, transgene expression was demonstrated by immunostaining in both the endothelium and the adventitia and by Western blot analysis. High-dose but not low-dose eNOS gene transfer enhanced endothelium-dependent relaxation in vessels from cholesterol-fed rabbits. CONCLUSIONS: Adenovirus-mediated gene transfer of eNOS to carotid arteries of cholesterol-fed animals improves endothelium-dependent relaxation when an optimal viral titer is administered. PMID- 10754008 TI - sigma(1)-receptor ligand 4-phenyl-1-(4-phenylbutyl)-piperidine affords neuroprotection from focal ischemia with prolonged reperfusion. AB - BACKGROUND AND PURPOSE: We previously showed that the intravenous administration of the potent final sigma(1)-receptor ligand 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) provides neuroprotection against transient focal cerebral ischemia and that the protection depends on treatment duration. We tested the hypothesis that PPBP would provide neuroprotection in a model of transient focal ischemia and 7 days of reperfusion in the rat as assessed with neurobehavioral outcome and infarction volume. METHODS: Under the controlled conditions of normoxia, normocarbia, and normothermia, halothane-anesthetized male Wistar rats were subjected to 2 hours of middle cerebral artery occlusion (MCAO) with the intraluminal suture occlusion technique. We used laser Doppler flowmetry to assess MCAO. At 60 minutes after the onset of ischemia, rats were randomly assigned to 1 of 4 treatment groups in a blinded fashion and received a continuous intravenous infusion of control saline or 0.1, 1, or 10 micromol. kg( 1). h(-1) PPBP for 24 hours. Neurobehavioral evaluation was performed at baseline (3 to 4 days before MCAO) and at 3 and 7 days of reperfusion. Infarction volume was assessed with triphenyltetrazolium chloride staining on day 7 of reperfusion in all rats. RESULTS: Triphenyltetrazolium chloride-determined infarction volume of ipsilateral cortex was smaller in rats treated with 10 micromol. kg(-1). h(-1) PPBP (n=15, 68+/-12 mm(3), 18+/-3% of contralateral structure, P<0.05) (mean+/ SEM) compared with corresponding rats treated with saline (n=15, 114+/-11 mm(3), 31+/-3% of contralateral structure). PPBP did not provide significant neuroprotection in the caudoputamen complex. Although MCAO was associated with several alterations in behavior, the treatment with PPBP had no effect on behavioral outcomes. CONCLUSIONS: The data demonstrate that the potent final sigma(1)-receptor ligand PPBP decreases cortical infarction volume without altering neurobehavior after transient focal ischemia and prolonged reperfusion in the rat. PMID- 10754009 TI - The A677V MTHFR allele is not associated with carotid atherosclerosis in octogenarians. PMID- 10754010 TI - Supplement to the AHA guidelines for the management of transient ischemic attacks. PMID- 10754011 TI - Supplement to the AHA guidelines for the management of transient ischemic attacks PMID- 10754013 TI - Constraint-induced movement therapy and massed practice. PMID- 10754012 TI - Which targets are relevant for therapy of acute ischemic stroke? PMID- 10754015 TI - Abstracts of literature PMID- 10754014 TI - Effects of statins on ischemic stroke: neuroprotection and/or triggering of apoptotic damage? PMID- 10754016 TI - Addendum to the supplement to the guidelines for the management of transient ischemic attacks PMID- 10754017 TI - Measuring and improving quality of care : A report from the american heart Association/American college of cardiology first scientific forum on assessment of healthcare quality in cardiovascular disease and stroke PMID- 10754040 TI - Occupational exposure during lead abatement of steel surfaces by needle gun methodology. PMID- 10754041 TI - UVA light-induced DNA cleavage by selected polycyclic aromatic hydrocarbons. PMID- 10754042 TI - Photodegradation of the triazole fungicide hexaconazole. PMID- 10754043 TI - Chlorobiphenyls, HCB, and organochlorine pesticides in some tissues of Caretta caretta (Linnaeus) specimens beached along the adriatic sea, italy. PMID- 10754044 TI - Distribution of heavy metals in wheat, mustard, and weed grown in field irrigated with industrial effluents. PMID- 10754045 TI - Air sampling during asbestos abatement of floor tile and mastic. PMID- 10754046 TI - Organochlorine pesticide residues in herbal ayurvedic preparations. PMID- 10754047 TI - Pesticides and polychlorinated biphenyls drained into north coast of the Mediterranean Sea, Egypt. PMID- 10754048 TI - Spectroscopic study of the sorption of isoxaflutole and its diketonitrile metabolite in dissimilar soils. PMID- 10754049 TI - Bioaccumulation of cadmium from durum wheat diets in the livers and kidneys of mice. PMID- 10754050 TI - Noninvasive characterization of the effects of diazinon on pigeons. PMID- 10754051 TI - Nitrification inhibition by naphthalene derivatives and its relationship with copper. PMID- 10754052 TI - Heavy metal accumulation in soil and jack pine (Pinus banksiana) needles in Sudbury, Ontario, Canada. PMID- 10754053 TI - Evaluation of aerobic biodegradability of some chemical compounds commonly applied in paper industry. PMID- 10754054 TI - Effect of thallium on the growth of Anacystis nidulans and Chlamydomonas reinhardtii. PMID- 10754055 TI - Sensitivity of Ampelisca araucana juveniles (Crustacea: amphipoda) to organic and inorganic toxicants in tests of acute toxicity. PMID- 10754056 TI - Toxic effects of hydroxylamino intermediates from microbial transformation of trinitrotoluene and dinitrotoluenes on algae Selenastrum capricornutum. PMID- 10754057 TI - Liver enzyme activities of rats exposed to ochratoxin A and T-2 toxin with antioxidants. PMID- 10754058 TI - Electrophysiological effects of chronic lead treatment on synaptic transmission in murine dorsiflexor muscle. PMID- 10754059 TI - Effects of water soluble fractions of crude oil on growth of the Nile tilapia, Oreochromis niloticus (L.). PMID- 10754060 TI - Vanadium-induced leukocytosis. PMID- 10754061 TI - Genotoxicity of lanthanum (III) and gadolinium (III) in human peripheral blood lymphocytes. PMID- 10754062 TI - Divergent evolution of plant NBS-LRR resistance gene homologues in dicot and cereal genomes. AB - The majority of plant disease resistance genes are members of very large multigene families. They encode structurally related proteins containing nucleotide binding site domains (NBS) and C-terminal leucine rich repeats (LRR). The N-terminal region of some resistance genes contain a short sequence called TIR with homology to the animal innate immunity factors, Toll and interleukin receptor-like genes. Only a few plant resistance genes have been functionally analyzed and the origin and evolution of plant resistance genes remain obscure. We have reconstructed gene phylogeny by exhaustive analysis of available genome and amplified NBS domain sequences. Our study shows that NBS domains faithfully predict whole gene structure and can be divided into two major groups. Group I NBS domains contain group-specific motifs that are always linked with the TIR sequence in the N terminus. Significantly, Group I NBS domains and their associated TIR domains are widely distributed in dicot species but were not detected in cereal databases. Furthermore, Group I specific NBS sequences were readily amplified from dicot genomic DNA but could not be amplified from cereal genomic DNA. In contrast, Group II NBS domains are always associated with putative coiled-coil domains in their N terminus and appear to be present throughout the angiosperms. These results suggest that the two main groups of resistance genes underwent divergent evolution in cereal and dicot genomes and imply that their cognate signaling pathways have diverged as well. PMID- 10754063 TI - The molecular evolutionary history of a winged bean alpha-chymotrypsin inhibitor and modeling of its mutations through structural analyses. AB - A serine protease inhibitor of the Kunitz-STI (soybean trypsin inhibitor) family, isolated from the legume seeds of winged bean, was found to inhibit chymotrypsin at a 1:2 stoichiometric ratio. When the structure was determined in our laboratory, it was found to form a characteristic beta-trefoil fold, which is also seen in other proteins from distant families and sources. The folding organization divides the protein into three approximately equal subdomains related by a pseudo-threefold axis of symmetry passing parallel to the barrel axis of the trefoil. Following the now established idea that the present-day genes originated from ancestral minigenes through evolution, the origin of the proteins having this beta-trefoil organization is scrutinized using its subdomain motif as the search probe. The results, based mainly on structural analyses, indicate the independent existence of such a motif, mimicking the unknown ancestral protein(s) that might have been distributed in nature, not only by gene duplication, but also by insertion and permutation in other folds. The understanding led to a hypothesis for the possible origin of the Kunitz-STI family. On the basis of this model of evolution, structurally hypervariable regions were located on the protein where mutations could be designed and a broad range of engineering of the protein's activity could be conceived. PMID- 10754064 TI - A new method for locating changes in a tree reveals distinct nucleotide polymorphism vs. divergence patterns in mouse mitochondrial control region. AB - A new, model-based method was devised to locate nucleotide changes in a given phylogenetic tree. For each site, the posterior probability of any possible change in each branch of the tree is computed. This probabilistic method is a valuable alternative to the maximum parsimony method when base composition is skewed (i.e., different from 25% A, 25% C, 25% G, 25% T): computer simulations showed that parsimony misses more rare --> common than common --> rare changes, resulting in biased inferred change matrices, whereas the new method appeared unbiased. The probabilistic method was applied to the analysis of the mutation and substitution processes in the mitochondrial control region of mouse. Distinct change patterns were found at the polymorphism (within species) and divergence (between species) levels, rejecting the hypothesis of a neutral evolution of base composition in mitochondrial DNA. PMID- 10754065 TI - Characteristic sequence pattern in the 5- to 20-bp upstream region of primate Alu elements. AB - We conducted comprehensive sequence analysis of 5' flanking regions of primate Alu elements. Information contents were computed and frequencies of 1024 pentanucleotides were measured to approximate the location of a characteristic sequence and to specify its pattern(s), which may be involved in the integration of Alu elements into their host genomes. A large number of samples was used, the wide region of the 5' end of Alu elements was analyzed, and comparisons were made among different subfamilies. Through our analyses, "TTTTAAAAA" or "(T)(m)(A)(n)" can be stated as a candidate for the characteristic sequence pattern, which resides around the region 5 to 20 base pairs upstream of the 5' end of Alu elements. This characteristic sequence pattern was more prominent in the sequences of younger Alus, which is a strong indication that the sequence pattern has a role at the time of Alu integration. PMID- 10754066 TI - Concurrent neutral evolution of mRNA secondary structures and encoded proteins. AB - Messenger RNA sequences often have to preserve functional secondary structure elements in addition to coding for proteins. We present a statistical analysis of retroviral mRNA which supports the hypothesis that the natural genetic code is adapted to such complementary coding. These sequences are still able to explore efficiently the space of possible proteins by point mutations. This is borne out by the observation that, in stem regions of retroviral mRNA foldings, silent mutations on one strand are preferentially accompanied by conservative mutations on the other. Distances between amino acids based on physicochemical properties are used to quantify the conservation of protein function under the constraint of maintained RNA secondary structure. We find that preservation of RNA secondary structure by compensatory mutations is evolutionary compatible with the efficient search for new variants on the protein level. PMID- 10754067 TI - Ancient origin of the null allele se(428) of the human ABO-secretor locus (FUT2). AB - In human populations, a null allele having several nucleotide differences from the wild-type allele is segregating at the FUT2 locus (the ABO-Secretor locus) encoding alpha(1,2)fucosyltransferase. To estimate the age of the most recent common ancestor (MRCA) of these two alleles, we sequenced FUT2 homologues from chimpanzee, gorilla, orangutan, and green monkey. Since we did not detect acceleration or any heterogeneity in the substitution rate at this locus among these species, the age of the MRCA was estimated to be around 3 MYA, assuming the divergence time of human and chimpanzee to be 5 MYA. We developed a simple test to examine whether or not the old age of the MRCA of the FUT2 is consistent with that expected for two divergent neutral alleles sampled from a random mating population. An application of the test to the data at FUT2 indicated that the age of the MRCA is too old to be explained by the simple neutral assumptions, although our test depends on accurate estimation of the divergence time of human and chimpanzee in units of twice the human population size. Various possibilities including balancing selection are discussed to explain this old age of the MRCA. PMID- 10754069 TI - The robust statistical bases of the coevolution theory of genetic code origin. AB - A paper (Amirnovin R, J Mol Evol 44:473-476, 1997) seems to undermine the validity of the coevolution theory of genetic code origin by shedding doubt on the connection between the biosynthetic relationships between amino acids and the organization of the genetic code, at a time when the literature on the topic takes this for granted. However, as a few papers cite this paper as evidence against the coevolution theory, and to cast aside all doubt on the subject, we have decided to reanalyze the statistical bases on which this theory is founded. We come to the following conclusions: (1) the methods used in the above referred paper contain certain mistakes, and (2) the statistical foundations on which the coevolution theory is based are extremely robust. We have done this by critically appraising Amirnovin's paper and suggesting an alternative method based on the generation of random codes which, along with the method reported in the literature, allows us to evaluate the significance, in the genetic code, of different sets of amino acid pairs in biosynthetic relationships. In particular, by using this method and after building up a certain set of amino acid pairs reflecting the expectations of the coevolution theory, we show that the presence of this set in the genetic code would be obtained, purely by chance, with a probability of 6x10(-5). This observation seems to provide particularly strong support to the coevolution theory. PMID- 10754068 TI - The contributions of replication orientation, gene direction, and signal sequences to base-composition asymmetries in bacterial genomes. AB - Asymmetries in base composition between the leading and the lagging strands have been observed previously in many prokaryotic genomes. Since a majority of genes is encoded on the leading strand in these genomes, previous analyses have not been able to determine the relative contribution to the base composition skews of replication processes and transcriptional and/or translational forces. Using qualitative graphical presentations and quantitative statistical analyses (analysis of variance), we have found that a significant proportion of the GC and AT skews can be attributed to replication orientation, i.e., the sequence of a gene is influenced by whether it is encoded on the leading or lagging strand. This effect of replication orientation on skews is independent of, and can be opposite in sign to, the effects of transcriptional or translational processes, such as selection for codon usage, amino acid preferences, expression levels (inferred from codon adaptation index), or potential short signal sequences (e.g., chi sequences). Mutational differences between the leading and the lagging strands are the most likely explanation for a significant proportion of the base composition skew in these bacterial genomes. The finding that base composition skews due to replication orientation are independent of those due to selection for function of the encoded protein may complicate the interpretation of phylogenetic relationships, conserved positions in nucleotide or amino acid sequence alignments, and codon usage patterns. PMID- 10754070 TI - Evolutionary lability of context-dependent codon bias in bacteria. AB - In bacteria, synonymous codon usage can be considerably affected by base composition at neighboring sites. Such context-dependent biases may be caused by either selection against specific nucleotide motifs or context-dependent mutation biases. Here we consider the evolutionary conservation of context-dependent codon bias across 11 completely sequenced bacterial genomes. In particular, we focus on two contextual biases previously identified in Escherichia coli; the avoidance of out-of-frame stop codons and AGG motifs. By identifying homologues of E. coli genes, we also investigate the effect of gene expression level in Haemophilus influenzae and Mycoplasma genitalium. We find that while context-dependent codon biases are widespread in bacteria, few are conserved across all species considered. Avoidance of out-of-frame stop codons does not apply to all stop codons or amino acids in E. coli, does not hold for different species, does not increase with gene expression level, and is not relaxed in Mycoplasma spp., in which the canonical stop codon, TGA, is recognized as tryptophan. Avoidance of AGG motifs shows some evolutionary conservation and increases with gene expression level in E. coli, suggestive of the action of selection, but the cause of the bias differs between species. These results demonstrate that strong context-dependent forces, both selective and mutational, operate on synonymous codon usage but that these differ considerably between genomes. PMID- 10754071 TI - Multiple recurrent evolution of trophic types in northeastern Atlantic and Mediterranean seabreams (Sparidae, Percoidei). AB - Seabreams are among the most valuable fish, not only for small-scale and semiindustrial fisheries but also for aquaculture throughout the Mediterranean. Nevertheless, their phylogenetic relationships are not at all clear. The current taxonomy is based solely on trophic morphology and rests on the assumption that each trophic type evolved only once from a less specialized ancestral condition. We analyzed a 486-bp segment of the mitochondrial 16S rDNA of all 24 seabream species described for the northeastern Atlantic and the Mediterranean to elucidate their generic and subfamily-level relationships. Three major mitochondrial lineages, each comprising species of different feeding strategy and dentition, were found that do not agree with the present taxonomic assignments. Most of the investigated genera were resolved paraphyletically, indicating that the structure and arrangement of oral teeth must have repeatedly evolved from a less specialized ancestral condition. Further, the genus Sparus was resolved as distantly related to the genus Pagrus, in that it was assigned to a different major mitochondrial lineage. Oblada melanura was consistently placed within the Diplodus radiation as sister group to Diplodus puntazzo. Our phylogenetic hypothesis thus suggests multiple independent origins of similar trophic specializations within the Sparidae and indicates that the currently recognized three or four subfamilies need to be redefined. PMID- 10754072 TI - Detection of signature sequences in overlapping genes and prediction of a novel overlapping gene in hepatitis G virus. AB - In viruses an increased coding ability is provided by overlapping genes, in which two alternative open reading frames (ORFs) may be translated to yield two distinct proteins. The identification of signature sequences in overlapping genes is a topic of particular interest, since additional out-of-frame coding regions can be nested within known genes. In this work, a novel feature peculiar to overlapping coding regions is presented. It was detected by analysis of a sample set of 21 virus genomic sequences and consisted in the repeated occurrence of a cluster of basic amino acid residues, encoded by a frame, combined to a stretch of acidic residues, encoded by the corresponding overlapping frame. A computer scan of an additional set of virus sequences demonstrated that this feature is common to several other known overlapping ORFs and led to prediction of a novel overlapping gene in hepatitis G virus (HGV). The occurrence of a bifunctional coding region in HGV was also supported by its extremely lower rate of synonymous nucleotide substitutions compared to that observed in the other gene regions of the HGV genome. Analysis of the amino acid sequence that was deduced from the putative overlapping gene revealed a high content of basic residues and the presence of a nuclear targeting signal; these characteristics suggest that a core like protein may be expressed by this novel ORF. PMID- 10754073 TI - The power of relative rates tests depends on the data. AB - One of the most useful features of molecular phylogenetic analyses is the potential for estimating dates of divergence of evolutionary lineages from the DNA of extant species. But lineage-specific variation in rate of molecular evolution complicates molecular dating, because a calibration rate estimated from one lineage may not be an accurate representation of the rate in other lineages. Many molecular dating studies use a "clock test" to identify and exclude sequences that vary in rate between lineages. However, these clock tests should not be relied upon without a critical examination of their effectiveness at removing rate variable sequences from any given data set, particularly with regard to the sequence length and number of variable sites. As an illustration of this problem we present a power test of a frequently employed triplet relative rates test. We conclude that (1) relative rates tests are unlikely to detect moderate levels of lineage-specific rate variation (where one lineage has a rate of molecular evolution 1.5 to 4.0 times the other) for most commonly used sequences in molecular dating analyses, and (2) this lack of power is likely to result in substantial error in the estimation of dates of divergence. As an example, we show that the well-studied rate difference between murid rodents and great apes will not be detected for many of the sequences used to date the divergence between these two lineages and that this failure to detect rate variation is likely to result in consistent overestimation the date of the rodent primate split. PMID- 10754074 TI - Protein tyrosine phosphatases from amphioxus, hagfish, and ray: divergence of tissue-specific isoform genes in the early evolution of vertebrates. AB - Since separation from fungi and plants, multicellular animals evolved a variety of gene families involved in cell-cell communication from a limited number of ancestral precursors by gene duplications in two separate periods of animal evolution. In the very early evolution of animals before the separation of parazoans and eumetazoans, animals underwent extensive gene duplications by which different subtypes (subfamilies) with distinct functions diverged. The multiplicity of members (isoforms) in the same subtype increased by further gene duplications (isoform duplications) in the first half of chordate evolution before the fish-tetrapod split; different isoforms are virtually identical in structure and function but differ in tissue distribution. From cloning and phylogenetic analyses of four subfamilies of the protein tyrosine kinase (PTK) family, we recently showed extensive isoform duplications in a limited period around or just before the cyclostome-gnathostome split. To obtain a reliable estimate for the divergence time of vertebrate isoforms, we have conducted isolation of cDNAs encoding the protein tyrosine phosphatases (PTPs) from Branchiostoma belcheri, an amphioxus, Eptatretus burgeri, a hagfish, and Potamotrygon motoro, a ray. We obtained 33 different cDNAs in total, most of which belong to known PTP subfamilies. The phylogenetic analyses of five subfamilies based on the maximum likelihood method revealed frequent isoform duplications in a period around or just before the gnathostome-cyclostome split. An evolutionary implication was discussed in relation to the Cambrian explosion. PMID- 10754075 TI - Terms and phrases of cardiac morphology: A call for anarchy! PMID- 10754076 TI - Phase-velocity cine magnetic resonance imaging measurement of pulsatile blood flow in children and young adults: in vitro and in vivo validation. AB - Quantification of blood flow in vessels provides valuable information that aids management decisions in a variety of cardiac conditions. Current flow measurement techniques are often limited by accuracy, time resolution, convenience, or anatomic localization. This study examined the accuracy of a commercially available phase-velocity cine magnetic resonance imaging (PVC MRI) technique to quantify flow rate in a pulsatile flow phantom. In addition, the equivalence of PVC MRI measurements of pulmonary and systemic flow was evaluated in children and adults without any pathologic shunt. Using a pulsatile flow phantom, volume flow rates measured by PVC MRI were compared to those by a transit-time ultrasound flowmeter over a range of flow rates (1.25-3.5 L/min, 13 trials). Close agreement was found between these techniques (y = 1.02x - 0.02, r = 0.99, Bland-Altman bias = -0.045 L/min, 95% limits of agreement = -0. 19-0.10 L/min). Twenty subjects (median age 12.8 years, range 0.7-49 years) with no pathologic shunt underwent PVC MRI measurement of blood flow in the main pulmonary artery (Q(p)) and the ascending aorta (Q(s)). Data processing time for each location was 20 minutes. The Q(p)/Q(s) ratio closely approximated unity (mean = 0.99, SD = 0. 10, range 0.85-1.19). Interobserver agreement was excellent (Bland-Altman bias = 0.09 L/min, 95% limits of agreement = 0.15-0.33 L/min). PVC MRI is an accurate technique to quantify pulsatile blood flow at a specific location. It can be used to noninvasively calculate Q(p) and Q(s) under normal flow conditions. PMID- 10754078 TI - The Segmental Approach to the Diagnosis of Congenital Heart Disease. PMID- 10754077 TI - Evaluation of myocardial perfusion using positron emission tomography in infants following a neonatal arterial switch operation. AB - This study was performed to examine the use of positron emission tomography (PET) as a method of evaluating myocardial perfusion after the arterial switch operation for correction of transposition of the great arteries. Eleven asymptomatic patients (median age 2.3 years, range 1.3-4.3 years) post successful neonatal arterial switch repair for transposition underwent cardiac PET scanning using N(13) ammonia before and after dipyridamole infusion. Reconstructed data from static scans were analyzed for regional perfusion defects before and after pharmacological stress. Simultaneous assessment of coronary flow before and after stress was performed using a Patlak graphical analysis of data from dynamic scans. Results obtained from PET scanning were correlated with patterns of coronary artery anatomy, electrocardiogram (ECG) recordings, and echocardiographic evaluation. PET scanning demonstrated normal distribution of myocardial perfusion before and after stress in all but one patient, who was found to have a discrete inferior transmural perfusion defect. The defect was well correlated with perioperative ECG changes and a complicated postoperative course. Myocardial blood flow before dipyridamole (0.690 ml/min/g) was similar to reported adult rest values. There was a small but significant (p < 0.002) increase in myocardial blood flow after dipyridamole stress with a mean coronary flow reserve of 1.19 (+/-0.103). Echocardiographic evaluation failed to demonstrate significant wall motion abnormalities in any of the patients. Cardiac PET scanning is a reliable noninvasive method for evaluation of myocardial perfusion in small children. In this study, the incidence of myocardial perfusion defects after the arterial switch operation is lower than previously reported. The data obtained concerning coronary flow and coronary flow reserve after the arterial switch need to be interpreted with caution because normal data in children are not available. PMID- 10754079 TI - Rheumatic fever in children: a 15-year experience in a developing country. AB - Clinical data from 91 patients with rheumatic fever (RF), who were hospitalized at a tertiary hospital in Lebanon between 1980 and 1995, were reviewed retrospectively. Age on hospitalization was 11.1+/-2.9 years (mean +/- SD, range 3-17 years). Nineteen patients were <6 years of age. Manifestations included carditis (93%), arthritis (39%), Sydenham's chorea (2%), erythema marginatum (4%), subcutaneous nodules (1%), fever (62%), arthralgia (55%), and acute congestive heart failure (CHF) on initial presentation (44%). Pericardial effusion occurred in 11%. There was positive family history of RF in 14%. Mitral insufficiency and aortic insufficiency occurred in 67 and 35%, respectively. Both mitral and aortic valves were involved in 30% of cases. Tricuspid insufficiency developed in 3% and pulmonary insufficiency in 1%. Mitral stenosis developed in 19%. Twenty-eight patients underwent surgical intervention: mitral valve repair and commissurotomy in 9/91 (10%), mitral valve replacement in 18/91 (20%), and aortic valve replacement in 9/91 (10%). Overall mortality was 12%: 5 following surgical intervention (3 after mitral valve surgery and 2 after mitral and aortic valve surgery). All patients that died had CHF on initial presentation (p = 0.006). This study includes hospitalized patients with predominant rheumatic heart disease. Initial presentation with CHF is a risk factor for surgical intervention and mortality. A significant high surgical intervention rate is noted that is probably related to the nature of the selected group studied. This study emphasizes the significant morbidity and death in patients with RF and carditis. PMID- 10754080 TI - Procainamide for rate control of postsurgical junctional tachycardia. AB - This study was conducted to determine the efficacy of procainamide therapy for rapid rate control of postoperative junctional tachycardia (JT). Postoperative JT is one of the most difficult forms of tachycardia to manage. Reported success with a variety of treatments of JT in infants and children has been inconsistent and limited. Rate control using procainamide was achieved in 17 children having rapid JT (heart rate >200 beats/min) between 1986 and 1997. In the first 5 patients (protocol A), following a loading dose of 3 mg/kg over 20 minutes, a continuous procainamide infusion was initiated at a rate of 20 microg/kg/min. The infusion dose was increased in 10 microg/kg steps every 30 minutes to 40-120 microg/kg/min until the heart rate decreased below the target rate of 180 beats/min. In the other 12 patients (protocol B), after a higher loading dose of 10 mg/kg the infusion rate was increased every 10-15 minutes until the heart rate decreased below the target rate of 180 beats/min. Procainamide decreased JT rates in all patients but the response was significantly faster in protocol B. In the patients treated with protocol A, pretreatment JT rates ranged from 203 to 240 (213+/-17) beats/min and decreased to 195+/-10 beats/min at 2 hours (p = ns), 186+/-8.8 at 4 hours (p<0.02), and 179+/-8 at 6 hour postinitiation of PA. In protocol B, pretreatment JT rates ranged from 201 to 240 (218+/-17) beats/min and decreased to 183+/-20 beats/min at 2 hours (p<0.001) and 171+/-12 at 4 hours after starting the procainamide therapy. The mean duration to decrease JT rates below the target rate of 180 beats/min was 3.2+/-1.1 hours in protocol B compared to 6.4+/-3.8 hours in protocol A (p<0.02). Eight of 12 patients in protocol B achieved rate control below the target rate of 180 beats/min within 4 hours despite remaining on significant inotropic support. The procainamide infusion rates to maintain heart rates below 180 beats/min were 40-120 (68.4+/-22.1) microg/kg/min. No proarrhythmia, bradycardia, or significant hypotension was observed. In this series procainamide provided safe, effective, and rapid rate control of JT occurring in the immediate postoperative period. PMID- 10754081 TI - The Segmental Approach in Describing Cardiac Anomalies Is Insufficient! PMID- 10754082 TI - Spontaneous closure of atrial septal defects in premature vs. full-term neonates. AB - Our hypotheses were that the following factors influenced closure of atrial septal defects (ASDs) detected in neonates: estimated gestational age (EGA), the presence of a persistent ductus arteriosus (PDA), severity of pulmonary disease, gender, and the initial size of the ASD. Our population consisted of 82 newborns (38 premature and 44 term) who were found before the age of 1 month to have an ASD. Closure of ASDs was analyzed using both Kaplan-Meier survival analysis and the Cox proportional hazards model, each with the five covariates. The hazard ratio (or ratio of instantaneous closure rates) of term vs preterm infants was 3.60 (95% CI = 1.80-7.20), whereas the hazard ratio for infants with a PDA (compared to infants with no PDA) was 2.41 (95% CI = 1.28-4.54). Multivariate analysis showed that each of these terms (PDA and EGA) were independent predictors of ASD closure. The hazard ratio of ASD closure for each of four levels of pulmonary disease was 0.632 [95% CI = 0.453-0.881]. We conclude that the majority of neonatal ASDs will close, with EGA and PDA acting as important influences on closure. PMID- 10754084 TI - Unambiguous, Accurate, and Succinct Description of Congenital Heart Defects. PMID- 10754086 TI - Flexible Yet Simple. PMID- 10754085 TI - Cardiac manifestations in patients with HLA B27-associated juvenile arthritis. AB - Cardiac involvement is common in adult patients with the presence of HLA B27 with or without the HLA B27-associated spondyloarthropathy ankylosing spondylitis. Most patients with juvenile spondyloarthropathy, which begins at age 16 or younger, do not have spinal involvement and there are only few reports of cardiac involvement. This study sought to assess the prevalence of carditis in patients with HLA B27-associated juvenile arthritis (B27-JA). In a controlled study, 40 patients with B27-JA, among them only 1 with ankylosing spondylitis, were examined by electrocardiogram and echocardiography with pulsed and color-flow Doppler at rest and at the termination of a bicycle exercise and compared to an age- and sex-matched control group negative for HLA B27. Four patients with B27 JA, and none in the control group, had inflammatory aortic regurgitation. Late diastolic flow velocity was significantly increased in patients with B27-JA at the termination of exercise. HLA B27 is a risk factor for endo-/myocardial damage in patients with B27-JA, even in the presence of only short and mild articular disease. Patients with B27-JA should be screened for the presence of aortic regurgitation. PMID- 10754083 TI - Assessment of ventricular repolarization in deaf-mute children. AB - The long QT syndrome is a congenital disease with frequent familial transmission, characterized primarily by prolongation of the QT interval and by the occurrence of life-threatening arrhythmias. The syndrome may be familial, with or without congenital deafness, or it may be idiopathic. We attempted to assess ventricular repolarization and to identify patients with the Jervell and Lange-Nielsen syndrome among 132 deaf-mute school children. Five deaf-mute subjects had Jervell and Lange-Nielsen syndrome. The deaf-mute subjects were divided into two subgroups according to the length of their QT intervals: group 1 included 5 cases with the long QT interval (>440 msec), and group 2 included 127 subjects with the normal QT interval (< or =440 msec). Group 3 was composed of 96 control subjects. The mean QT, QTc, JT, and JTc intervals (418+/-70, 500+/-38, 302+/- 65, and 389+/ 36 msec, respectively) in group 1 were significantly longer than those of group 2 (344+/-23, 408+/-22, 249+/-34, and 291+/-28 msec, respectively) and group 3 (325+/-11, 383+/-26, 228 +/-36, and 269+/-46 msec, respectively). The dispersion (d) values (QT-d, QTc-d, JT-d, and JTc-d; 63+/-10, 73+/-8, 60+/-8, and 62+/-11 msec, respectively) of group 1 were significantly longer than those of group 2 (49+/-16, 43+/-11, 48+/-21, and 45+/-18 msec, respectively) and group 3 (33+/-13, 33+/-14, 28+/-16, and 27+/-14 msec, respectively) at similar mean RR intervals. Also, the mean QT, QTc, JT, and JTc intervals and the dispersion values (QT-d, QTc-d, JT-d, and JTc-d) in group 2 were significantly longer than those of group 3 at similar mean RR intervals. Consequently, in this study, we determined that the deaf-mute children who did not meet the criteria for Jervell and Lange Nielsen syndrome still had evidence of subtle derepolarization abnormalities evidenced by intermediate prolongation of QTc, JTc, and the corresponding measures of dispersion, and we believe an electrocardiogram examination of deaf mute subjects will reveal this potentially life-threatening syndrome. PMID- 10754087 TI - Distribution of symptomatic congenital heart disease in Hong Kong. AB - Racial group studies have identified differences in the occurrence of congenital heart disease (CHD) among ethnic populations. The aim of this study was to characterize the proportionate frequency and clinical profile of children with symptomatic cardiac abnormalities in Hong Kong. The hospital records of 666, mainly Southern Chinese children with symptomatic CHD, who were 4 years of age or younger and who were admitted to Grantham Hospital, Hong Kong, in 1994 and 1995 were analyzed retrospectively. Left-to-right shunting (45.0%) and pulmonary outflow obstruction (34.4%) were the most frequently diagnosed categories, followed by left ventricular outflow obstruction (8.3%), transposition of the great arteries (4.2%), conditions with intracardiac mixing (3.9%), and other cardiac lesions (4.2%). Compared with Western studies, pulmonary outflow obstruction (p<0.0001), particularly tetralogy of Fallot and critical pulmonary stenosis, were more frequent in Chinese children. In contrast with previous reports, coarctation of the aorta (5%) does not seem to be uncommon in Chinese patients. Conversely, aortic stenosis and hypoplastic left ventricle may be rare in these children (1% vs 3% and 3-7%). Other cardiac lesions showed no consistent racial difference in the frequency of occurrence. Chinese patients with Down's syndrome had ventricular septal defect (38%) as the predominant lesion followed by atrioventricular septal defect (25%). Western studies usually report a reverse pattern for these two lesions. The mortality rate for the total cohort was 7.5%. However, of those with conditions with intracardiac mixing and left ventricular outflow tract obstruction many did not survive childhood (20% and 21%, respectively). PMID- 10754088 TI - Rescue of a child with fulminant myocarditis using percutaneous cardiopulmonary support. AB - This report describes a 14-year-old girl with fulminant myocarditis who was successfully treated with a percutaneous cardiopulmonary support (PCPS). She developed progressive cardiac failure after a 3-week history of progressive fatigue, fever, tachypnea, and dyspnea requiring inotropic support, mechanical ventilation, and intra-aortic balloon pumping. Her condition continued to deteriorate, and she was cannulated for PCPS using a right femoral artery/femoral vein approach, which resulted in rapid improvement and hemodynamic stabilization. This case documents that circulatory support with PCPS is effective for treating children with fulminant myocarditis. PMID- 10754089 TI - Diagnostic Codes for Congenital Heart Disease. PMID- 10754090 TI - Atypical Bland-White-Garland syndrome with stenosis of the origin of the left coronary artery: catheter intervention after mammary artery bypass stenosis and residual fistula to the pulmonary trunk. AB - A 16-year-old boy with anomalous origin of the left coronary artery from the pulmonary artery, Bland-White-Garland syndrome, underwent a mammary artery bypass grafting to the left coronary artery (LCA) together with closure of the stenosed origin of the left coronary artery. A residual LCA to pulmonary artery fistula and stenosis of the shunt at the implantation site developed that resulted in a dominant perfusion of the LCA from the right coronary artery. Interventional catheterization was performed with stenting of the LCA mammary artery anastomosis and subsequent coil occlusion of the residual fistula. After this intervention the LCA was exclusively perfused by the mammarian bypass with no residual leak to the pulmonary artery. PMID- 10754091 TI - Transposition of the great arteries, pulmonary atresia, and multiple ventricular septal defects associated with multiple cardiac rhabdomyomas in a case of tuberous sclerosis. AB - Multiple cardiac rhabdomyomas are frequently associated with tuberous sclerosis. However, very few cardiac malformations have been described with tuberous sclerosis. We report an unusual case of tuberous sclerosis with cerebral and renal lesions associated with dextrotransposition of the great arteries, pulmonary atresia, patent ductus arteriosus, multiple ventricular septal defects, and parachute mitral valve deformity. After reviewing other cases of congenital heart diseases associated with tuberous sclerosis, emphasis is made on the potential influence of multiple rhabdomyomas developing very early during fetal life on final cardiac structures. PMID- 10754092 TI - Device closure of an atrial septal defect following successful balloon valvuloplasty in a neonate with critical pulmonary valve stenosis and persistent cyanosis. AB - Persistent cyanosis after successful balloon valvuloplasty for neonatal critical pulmonary valve stenosis is often related to poor right ventricular compliance and right-to-left shunting at the atrial level. A successful catheter closure of an atrial septal defect was performed with a dramatic increase in systemic oxygen saturation alleviating the need for a surgical systemic-to-pulmonary artery shunt. PMID- 10754093 TI - Right coronary artery to left ventricular fistula associated with left ventricular aneurysm. AB - An infant with right coronary artery to left ventricular fistula associated with left ventricular aneurysm successfully underwent a division of fistula under cardiopulmonary bypass. Follow-up angiogram showed the patency of the right coronary artery, no recurrence of fistula, and the regression of the left ventricular aneurysm. Because the lesion may progress with age, early diagnosis and surgical intervention are indicated to prevent later complications. PMID- 10754094 TI - Isolated congenital left ventricular diverticulum with perinatal dysrhythmia: a case report and review of the literature. AB - We report a case of isolated congenital left ventricular diverticulum (LVD) with perinatal dysrhythmia, which disappeared spontaneously 1 week after birth. The LVD arose from the lateral wall of the LV, and the contraction of the LVD was synchronous with the kinetics of the main LV chamber. The LVD changed very little in size during the first 30 months after birth, and its relative size to the growing LV main chamber decreased. The patient had neither any symptoms nor complications during this time. The available literature on prenatal and neonatal cases with isolated LVD or LV aneurysm is also reviewed. PMID- 10754095 TI - A novel use of Amplatzer duct occluder. AB - This report describes the use of the Amplatzer patent ductus arteriosus occluder to close a left ventricle to descending aorta conduit. The patient was a 10-year old male who was born with critical aortic stenosis and left ventricular outflow tract obstruction. After initial valvotomy, he underwent left ventricular to descending aorta conduit placement. At the age of 10, he had a Konno procedure to enlarge the left ventricular outflow tract and 21-mm St. Jude aortic valve placement. Closure of the conduit was not addressed because it was inaccessable from median sternotomy. Postoperatively, echocardiogram revealed significant flow through the conduit with a wide pulse pressure. Cardiac catheterization was performed with the premise to close the conduit with an Amplatzer patent ductus arteriosus occluder device. PMID- 10754096 TI - Patients' age at time of testing for chromosome 22q11 microdeletions: missed opportunities for genetic counseling. PMID- 10754100 TI - Whiskers amiss, a new vibrissae and hair mutation near the Krt1 cluster on mouse chromosome 11. AB - Whiskers amiss (wam) is a new spontaneous recessive mutation in the SELH/Bc strain of mice that causes a phenotype of askew, sometimes kinked or curled, breakable whiskers and disheveled-appearing body hair, apparently owing to disoriented guard hairs. Heterozygotes on three genetic backgrounds are indistinguishable from normal. Using informative SSLPs in the F2 generation after crosses to two normal strains, we have mapped wam to the region of the type I keratin cluster on Chromosome (Chr) 11, within an approximately 6-cM segment according to the current Mouse Genome Database (MGD) map position of flanking SSLPs. Although several other hair mutations also map to the Krt1 region (Re, Rim3, Bdai, Bsk), none has a hair and whisker phenotype similar to that of wam, and, because all are transmitted as dominants, interpretable complementation tests could not be done. Scabbing and tissue loss occur on the rims of the pinnae and tail tip in some aging wam homozygotes, suggesting that wam may be an animal model of a genetic ectodermal disorder. The SELH/Bc strain background appears to have an unusually high rate of spontaneous mutation; wam is the sixth mutation to be described. PMID- 10754099 TI - Molecular and genetic analysis of the mouse homolog of the Drosophila suppressor of position-effect variegation 3-9 gene. AB - The Drosophila melanogaster gene suppressor of position-effect variegation 3-9 [Su(var)3-9] encodes a component of heterochromatin with a chromodomain and a SET domain. Here, we describe the cloning of a mouse homolog called Suv39hl and describe the genomic organization, pattern of expression, and genetic map position. The genomic locus is approximately 10 kb and consists of five exons. The first two exons, 1a and 1b, are alternative first exons and are followed by three common exons. Two mRNAs, encompassing exon 1a or 1b, encode protein isoforms with distinct amino termini, but which are otherwise identical, including the chromodomain and SET domain. Interestingly, only one of the isoforms contains a putative nuclear localization signal. Consistent with other genes encoding proteins associated with chromatin structure, Suv39hl is expressed in a widespread manner. Interspecific backcross mapping localized Suv39hl near tattered (Td) and scurfy (sf) on the proximal X Chromosome (Chr). However, analysis of Td/Y and sf/Y mutant stocks indicated that Suv39hl is not responsible for either mutant phenotype. PMID- 10754101 TI - List of transgenic and knockout mice: behavioral profiles. AB - A list of transgenic and knockout mice, having behavioral profiles, has been compiled. Behavioral and genetic information has been assembled in a simplified fashion so that the reader can easily determine the possible usefulness of each transgenic or knockout strain. Brief descriptions of the behavioral tests are given. A discussion is also presented of the effects of genetic backgrounds on these behavioral profiles. PMID- 10754102 TI - Characterization of mouse Clpp protease cDNA, gene, and protein. AB - Mutations that cause accumulation or rapid degradation owing to protein misfolding are a frequent cause of inherited disease in humans. In Escherichia coli, Clpp protease is one of the components of the protein quality control system that handles misfolded proteins. In the present study, we have characterized the mouse Clpp cDNA sequence, the organization of the mouse gene, the chromosomal localization, and the tissue-specific expression pattern. Moreover. the cellular localization and processing of mouse Clpp was studied by overexpression in transfected eukaryotic cells. Our results indicate that mouse and human Clpp have similar roles, and they provide the molecular basis for establishing a Clpp knockout mouse and to study its phenotype, thereby shedding light on a possible role of Clpp in human disease. PMID- 10754103 TI - Mater encodes a maternal protein in mice with a leucine-rich repeat domain homologous to porcine ribonuclease inhibitor. AB - MATER (Maternal Antigen That Embryos Require) is an ooplasm-specific protein first identified as an antigen (OP1) associated with ovarian autoimmunity in mice. Its primary structure has been deduced from full-length cDNA that encodes a 125-kDa protein required for progression of the mouse embryo beyond two cells. Expression of the gene encoding MATER is restricted to the oocyte, which makes it one of a growing, but still limited, number of maternal-effect genes in mammals. To further investigate the function of MATER during oogenesis and early development, we have characterized the gene and resultant protein. Mater is a single-copy gene in the genome of 129/Sv mice and is located at the proximal end of Chromosome (Chr) 7. The gene, spanning approximately 32 kbp, contains 15 exons ranging in size from 48 to 1576 bp, which together encode the 111 amino acid MATER protein. The first five exons encode 26-27 amino acid hydrophilic repeats and exons 8-14 encode 14 leucine-rich repeats. The three-dimensional structure of the latter domain can be closely modeled on the previously determined X-ray crystallographic coordinates of porcine ribonuclease inhibitor. These characterizations of the gene and protein provide the basis for genetic investigations of MATER function in early mammalian development. PMID- 10754104 TI - Silencing of the Y-chromosomal gene tspy during murine evolution. AB - We have studied the process of tspy gene silencing in murine evolution. We have isolated functional tspy sequences from Apodemus agrarius, A. sylvaticus, A. flavicollis, and Mus platythrix (subgenus Pyromys) and nonfunctional tspy sequences from species of the subgenus Mus. We present two alternative models as to how tspy may have lost its function in the murine lineage. PMID- 10754105 TI - High-resolution FISH mapping of the rat alpha2u-globulin multigene family. AB - The rat alpha2u-globulins are a group of similar proteins that are encoded by a family of approximately 20 genes located a single locus of < or =880 kbp on Chromosome (Chr) 5q. Individual members of this gene family demonstrate complex tissue, hormonal, and developmental expression patterns despite a high degree of sequence similarity among the members and consequently provide an interesting system for studying the evolution of differential gene expression. Hybridization analysis indicated that gene classes, similar to those identified at the homologous MUP locus in the mouse, do not exist within the rat alpha2u-globulin locus. Furthermore, cross-hybridization analysis revealed the presence of conserved sequences in the 5' and 3' regions flanking the alpha2u-globulin genes, some of which were present in an inverted orientation. We have used high resolution fiber FISH to examine the structural organization of the alpha2u globulin locus, and found the genes to be arranged as an array of both direct and inverted repeats. The organization of the rat alpha2u-globulin genes differs from the MUP genes and suggests different evolutionary events have reorganized these homologous sets of genes. PMID- 10754106 TI - Characterization of newly developed SSLP markers for the rat. AB - We have isolated more than 12,000 clones containing microsatellite sequences, mainly consisting of (CA)n dinucleotide repeats, using genomic DNA from the BN strain of laboratory rat. Data trimming yielded 9636 non-redundant microsatellite sequences, and we designed oligonucleotide primer pairs to amplify 8189 of these. PCR amplification of genomic DNA from five different rat strains yielded clean amplification products for 7040 of these simple-sequence-length-polymorphism (SSLP) markers; 3019 markers had been mapped previously by radiation hybrid (RH) mapping methods (Nat Genet 22, 27-36, 1998). Here we report the characterization of these newly developed microsatellite markers as well as the release of previously unpublished microsatellite marker information. In addition, we have constructed a genome-wide linkage map of 515 markers, 204 of which are derived from our new collection, by genotyping 48 F2 progeny of (OLETFxBN)F2 crosses. This map spans 1830.9 cM, with an average spacing of 3.56 cM. Together with our ongoing project of preparing a whole-genome radiation hybrid map for the rat, this dense linkage map should provide a valuable resource for genetic studies in this model species. PMID- 10754107 TI - Localization of 113 anchor loci in pigs: improvement of the comparative map for humans, pigs, and goats. AB - In total, 113 genes that have already been located in humans and goats were cytogenetically mapped in pigs. For this purpose, 165 gene-containing bacterial artificial chromosomes (BACs) isolated in goats were used in heterologous fluorescent in situ hybridization on porcine chromosomes. Among them, 113 (or 69%) gave clear and specific signals, and 52 did not work in heterologous conditions. These localizations are a significant contribution to development of the porcine gene map and also to the comparative map for humans and pigs. They allowed us to specify the information obtained by Zoo-FISH while taking the gene order into account; the number of conserved fragments detected for human and pig chromosomes reached 84. The average size of conserved fragments could be estimated at 33 cM. As these genes had already been mapped in goats, the comparison was extended to ruminants. The previous results obtained in this species, suggesting a correlation between human chromosome abnormalities and evolutionary breakpoints, were confirmed in pigs. PMID- 10754108 TI - Homozygosity mapping of the locus responsible for renal tubular dysplasia of cattle on bovine chromosome 1. AB - Renal tubular dysplasia is a hereditary disease of Japanese black cattle showing renal failure and growth retardation with an autosomal recessive trait. In the present study, we mapped the locus responsible for the disease (RTD) by linkage analysis with an inbred paternal half-sib pedigree obtained from commercial herds. By analyzing segregation of microsatellite markers in the half-sibs, significant linkage was observed between the RTD locus and markers on bovine Chromosome (Chr) 1 with the highest lod score of 11.4. Homozygosity mapping with the inbred pedigree further defined the localization of the RTD locus in a 4-cM region between microsatellite markers BMS4003 and INRA119. Mapping of the RTD locus on bovine Chr 1 will facilitate cloning and characterization of the gene responsible for this disease. PMID- 10754109 TI - Mapping of 16 ESTs expressed in the bovine mammary gland during lactation. AB - A bovine mammary gland cDNA-library was used to characterize and map genes expressed during lactation. Fifty cDNA clones selected by differential hybridization were sequenced from both ends, and sequences were examined for similarities with database sequences. For 34 of the transcripts, the sequences showed more than 80% similarity to previously characterized genes or expressed sequence tags (ESTs). Twenty cDNAs that could be of interest as candidate genes for milk production are selected for genetic or chromosomal mapping. Twenty-three out of the 39 designed primer pairs representing 16 cDNA clones amplified the expected fragments and were used for subsequent fluorescent single-strand conformation polymorphism analysis (F-SSCP) in the International Bovine Reference Panel families (IBRP). Ten polymorphic loci could be identified and used to genotype the IBRP animals, and nine of them were subsequently genetically mapped on nine chromosomes. In addition, eight loci from the 16 cDNA clones could be mapped by somatic cell hybrids, bringing the total number of mapped genes to 16, one of which was mapped genetically as well as physically. The mapped mammary gland ESTs are potentially useful for cloning economic trait loci by a positional candidate gene cloning approach. PMID- 10754110 TI - Cloning and characterization of murine fanconi anemia group A gene: Fanca protein is expressed in lymphoid tissues, testis, and ovary. AB - Fanconi anemia (FA) is an autosomal recessive disorder in humans characterized by bone marrow failure, cancer predisposition, and cellular hypersensitivity to cross-linking agents such as mitomycin C and diepoxybutane. FA genes display a caretaker function essential for maintenance of genomic integrity. We have cloned the murine homolog of FANCA, the gene mutated in the major FA complementation group (FA-A). The full-length mouse Fanca cDNA consists of 4503 bp and encodes a protein with a predicted molecular weight of 161 kDa. The deduced Fanca mouse protein shares 81% amino acid sequence similarity and 66% identity with the human protein. The nuclear localization signal and partial leucine zipper consensus motifs found in the human FANCA protein were also present in the murine homolog. In spite of the species difference, the murine Fanca cDNA was capable of correcting the cross-linker sensitive phenotype of human FA-A cells, suggesting functional conservation. Based on Northern as well as Western blots, Fanca was mainly expressed in lymphoid tissues, testis, and ovary. This expression pattern correlates with some of the clinical symptoms observed in FA patients. The availability of the murine Fanca cDNA now allows the gene to be studied in experimental mouse models. PMID- 10754111 TI - Characterization of the alternate allelic forms of human PAX7. AB - Six different allelic forms of the human neurogenic and myogenic developmental gene, PAX7, have been identified. They are distinguished by the number of tandem tetranucleotide, GAAG, repeats at a polymorphic site within the second intron of the paired box. Within the same intron, a second polymorphic site was found to have variable numbers of a dinucleotide TG repeat. The alleles are identified by a PCR-based method with oligo primers that span the variable regions of the intron. Several of the alleles include a duplicate copy of the entire paired box. Segregation studies demonstrate that the PAX7 alleles are inherited in a Mendelian fashion and that the duplicate copies of the PAX7 paired box region present in some of the alleles are closely linked. This initial study identified differences in the distribution of PAX7 alleles in DNA from patients with the skeletal muscle myopathy, dermatomyositis. Recognition of genetic polymorphism of PAX7 allows new approaches to understanding the role of PAX7 in myogenesis, neurogenesis, and neuromuscular disorders. PMID- 10754112 TI - Genetic mapping and analysis of mouse p27Kip1 gene as Pas1 candidate gene. PMID- 10754113 TI - A viral induced ubiquitin-specific protease (Ubp) localized on porcine chromosome 5. PMID- 10754114 TI - Linkage and physical mapping of the porcine prepro-orexin gene. PMID- 10754115 TI - A QTL for intramuscular fat and backfat thickness is located on porcine chromosome 6. PMID- 10754116 TI - Granulomatous nephritis as a complication of intrarenal bacille Calmette-Guerin therapy. AB - A case of granulomatous nephritis after intrarenal bacille Calmette-Guerin (BCG) therapy is reported. High fever greater than 38.5 degrees C lasted for 1 month, without response to conservative therapy. Standard nephroureterectomy was subsequently carried out. Histopathologic findings from the surgical specimen were compatible with BCG-induced granulomatous nephritis. The use of a syringe pump for retrograde instillation of BCG was thought to be the major cause of this severe complication. PMID- 10754117 TI - Postirradiation sarcoma after external beam radiation therapy for localized adenocarcinoma of the prostate: report of three cases. AB - We report 3 cases of postirradiation sarcoma that arose in the pelvis 8, 15, and 16 years after completion of external beam radiation therapy (RT) for localized adenocarcinoma of the prostate. Although such cases must be regarded as extremely rare, postirradiation sarcoma should be considered as a potential cause of pelvic pain developing after RT. PMID- 10754118 TI - Adult pseudosarcoma of the bladder. AB - Pseudosarcomatous tumor of the urinary bladder is an unusual benign proliferative lesion that poses a diagnostic dilemma for both the urologist and pathologist. Its clinical and histologic features mimic a malignant neoplasm, although simple excision is curative. Although similar lesions have been reported in multiple extravesicle locations, most commonly the lung, bladder lesions have only recently been reported. We describe 2 cases of pseudosarcomatous bladder tumors presenting in patients with no known risk factors. We review clinical, histologic, and management issues in these patients. By recognizing the existence of this lesion, possible extensive and inappropriate surgery may be avoided. PMID- 10754119 TI - Lidocaine toxicity secondary to postoperative bladder instillation in a pediatric patient. AB - The intravesical use of local anesthetics has been described for a variety of urologic procedures with no previous reports of toxicity. We recently took care of a 2.5- year-old girl with systemic lidocaine toxicity following treatment for postoperative bladder spasms. The patient developed a generalized seizure requiring endotracheal intubation but recovered fully with supportive care. We report the clinical details of this case as well as a brief review of lidocaine toxicity. PMID- 10754120 TI - Intra-abdominal pulmonary sequestration: a benign suprarenal mass. AB - A 34-year-old white woman presented with an 8.5-cm left suprarenal mass. Evaluation revealed normal adrenal hormone function. Pathologic examination after surgical removal revealed a rare benign condition, intra-abdominal extralobar pulmonary sequestration (accessory lung). PMID- 10754121 TI - Renal transplantation in patients with complete obstruction of the inferior vena cava. AB - We describe 2 patients with end-stage renal disease who had complete obstruction of the inferior vena cava and were successfully treated with renal transplantation in the usual iliac fossa position. One patient is doing well, with normal renal function more than 20 years after transplantation; the other patient was lost to follow-up after 3 months. Despite some technical difficulties, these patients apparently do well provided that an adequate collateral circulation bypassing the obstruction is confirmed by venography, and the patients receive anticoagulant therapy indefinitely. PMID- 10754122 TI - Duplicated seminal vesicle. AB - We report a case of complete, unilateral seminal vesicle duplication without concomitant reproductive duct or renal anomalies encountered during radical retropubic prostatectomy. We also discuss the possible embryologic origin of this anomaly and the clinical implications. PMID- 10754123 TI - Lambert-eaton myasthenic syndrome in association with transitional cell carcinoma: a previously unrecognized association. AB - The Lambert-Eaton myasthenic syndrome is known to occur with, or precede, a variety of malignancies, most commonly oat cell carcinoma of the lung. We report the first case of this syndrome associated with transitional cell carcinoma of the bladder and ureter. A brief review of published reports on the presentation, diagnosis, and treatment is included. PMID- 10754124 TI - Solitary metachronous contralateral adrenal metastasis from renal cell carcinoma. AB - Malignant involvement of the contralateral adrenal gland in cases of renal cell carcinoma is extremely rare. We report a case of solitary metachronous contralateral adrenal metastasis occurring 7.5 years after radical nephrectomy. The metastasis was treated with adrenalectomy and steroid replacement. Thirty months later, the patient remained without evidence of disease. This very rare presentation can prove to be a diagnostic challenge. Appropriate aggressive surgical treatment is warranted. PMID- 10754125 TI - Behcet's syndrome with left ventricular aneurysm and ruptured renal artery pseudoaneurysm. AB - A 21-year-old man with Behcet's syndrome presented with both a left ventricular aneurysm and a left renal artery pseudoaneurysm. After successful embolization of the ruptured pseudoaneurysm, the patient underwent successful repair of the left ventricular aneurysm. Although multiple aneurysms have been reported previously, we believe this to be the first reported case of both a ventricular aneurysm and a renal artery pseudoaneurysm afflicting a patient with Behcet's syndrome. PMID- 10754126 TI - False-positive pregnancy test associated with gonadoblastoma. AB - Gonadoblastomas are known to be hormonally active tumors that occur in streak or dysgenetic gonads of patients with intersex abnormalities. Several reports document their ability to produce beta-human chorionic gonadotropin (HCG), but none have documented an elevated peripheral serum beta-HCG. We report on the case of a patient with pure gonadal dysgenesis with XY karyotype who was found to have an elevated peripheral serum beta-HCG after a positive pregnancy test. Knowledge of gonadoblastoma's potential to elevate serum beta-HCG levels may prevent unnecessary searches for other causes. PMID- 10754127 TI - Identification of microtacks in the bladder after laparoscopic pelvic surgery. AB - Laparoscopic techniques are simplified and the operative time is reduced with the use of laparoscopic stapling and tacking devices. We present our experience in identifying and removing surgical tacks in 2 patients after laparoscopic pelvic surgery. PMID- 10754128 TI - Metastatic merkel cell tumor to bladder presenting as an encroachment tumor with gross hematuria. AB - Merkel cell carcinoma is an uncommon and aggressive tumor of neuroendocrine and epithelial origin. A case of metastatic Merkel cell tumor with hematuria secondary to invasion into the bladder is presented. This is the second reported case of metastatic Merkel cell tumor to the bladder and the first published cystoscopic image of such a lesion. PMID- 10754129 TI - Primary penile lymphoma: diagnostic difficulties and management options. AB - A 76-year-old man presented with a painless penile ulcer. After an extensive negative workup, CO(2) laser excision was performed with penile reconstruction. Histologic examination revealed an anaplastic, large cell lymphoma with CD30(+) cells. Computed tomography scans of the thorax, abdomen, and pelvis were negative. He received no adjuvant therapy and was without evidence of recurrence after 18 months. We review published reports and discuss the management options for this rare lesion. PMID- 10754130 TI - Cytomegalovirus infection of the native ureter after liver-kidney transplantation. AB - We report a case of invasive cytomegalovirus (CMV) infection in the native ureter of a patient 7 years after liver-kidney transplantation. Previous reports of CMV ureteritis in transplant patients have involved only the allograft ureter, usually within 3 months of transplantation. The common characteristics of these patients, the possible risk factors, and the diagnostic findings of CMV ureteritis are discussed. Combined surgical and medical intervention are required for successful treatment. PMID- 10754131 TI - Stuttering priapism in a liver transplant patient with toxic levels of fk506. AB - This is the first report of stuttering priapism in a liver transplant patient with toxic levels of the immunosuppressive agent FK506. To date, stuttering priapism has only been reported in patients with sickle cell disease and is not currently listed in the toxicity profile of FK506 or cyclosporine, a drug with a similar mechanism of action. The erections resolved when the FK506 levels normalized. We review the possible mechanisms by which FK506 may have caused these erections. PMID- 10754132 TI - Spontaneous resolution of an in utero perirenal urinoma associated with posterior urethral valves. AB - Ultrasound imaging of a 26-week-gestation fetus demonstrated a large, nonemptying bladder. At 27 weeks, a distended, thick-walled bladder, left hydronephrosis, and a perirenal urinoma were present, without ascites. Observation was undertaken, as the amniotic fluid volume was normal. At 29 weeks, the left perirenal fluid collection persisted but, at 30 weeks, was absent. After delivery at 36 weeks, no ultrasound evidence for perirenal urinoma or ascites was present. Isotope renal scan showed preserved renal function bilaterally. This case illustrates that in utero urinomas associated with posterior urethral valves can resolve spontaneously, with preservation of renal function. PMID- 10754133 TI - Conservative management of an ileal neobladder-enteric fistula. AB - We present a case of an early ileal neobladder-enteric fistula after radical cystectomy with Studer pouch creation for muscle-invasive transitional cell carcinoma of the bladder. This patient was treated conservatively on an outpatient basis with prolonged catheter drainage, a low-residue diet, and oral antibiotics, with complete resolution by 8 weeks. The rationale for this approach in selected patients is discussed. PMID- 10754134 TI - Testicular simple cyst and teratoma: asynchronous bilateral occurrence within the first year of life. AB - Benign and malignant testicular tumors are rare in infancy. Moreover, only a few cases of bilateral testicular tumors in children have been reported to date. To our knowledge, we report the first case of an asynchronous bilateral simple testicular cyst and testicular teratoma in an infant. This case demonstrates that although both lesions are benign in the prepubertal child, treatment decisions should be made carefully. PMID- 10754136 TI - Epidermoid cyst of the scrotum extending into the true pelvis. AB - A 9-year-old boy presented with an asymptomatic scrotal mass that was separate from the testes. The workup included a scrotal ultrasound scan and voiding cystourethrogram. On surgical exploration, the mass was solid, separate from the testes, and extended into the pelvis. The mass was removed, and pathologic examination revealed an epidermoid cyst. This is an uncommon scrotal lesion in boys and may represent a monodermal teratoma or abnormal closure of the median raphe. To our knowledge, this is only the second case report of an epidermoid cyst of the scrotum extending into the true pelvis. PMID- 10754135 TI - Persistent cloaca: are we ready for a correct prenatal diagnosis? AB - Cloacal malformations are rare and can present in variable aspects. The importance of ultrasound in detecting these anomalies is well known. Sonographic features vary in accordance with the type of malformation and the gestational age. A positive diagnosis is not possible because of the lack of specific ultrasound findings, which can show similar aspects to other abnormalities. We present 3 cases of prenatal diagnosis of this malformation, emphasizing that in the presence of a plurilobed cystic pelvic fetal mass with associated malformations, such as cardiac, renal, and vertebral anomalies, a persistent cloaca can reasonably be suspected. PMID- 10754137 TI - Detection of adenocarcinoma by urinary microsatellite analysis after augmentation cystoplasty. AB - Augmentation cystoplasty is associated with an increased risk of bladder cancer development between 10 and 20 years after augmentation. Using microsatellite analysis, we analyzed urine obtained before surgical resection of the malignant lesion from a patient who developed invasive adenocarcinoma after augmentation cystoplasty. Loss of heterozygosity was identified in both urine and tumor samples from this patient. This observation suggests that microsatellite urine analysis may be useful as a monitoring tool for patients after augmentation cystoplasty. PMID- 10754138 TI - Mullerian-type epithelial tumor arising within a torsed appendix testis. AB - Because it is a remnant of the mullerian duct system, the appendix testis contains mullerian epithelium that theoretically may produce epithelial tumors similar to those that occur in the female genital tract. Few reports of tumors of mullerian origin arising in the testis exist, and rarely are neoplasms arising from the appendix testis identified. We present a case of a serous cystic neoplasm of low malignant potential derived from mullerian-type epithelium that was located in the torsed appendix testis of a young, otherwise healthy, boy. PMID- 10754139 TI - Ureteral rhabdomyosarcoma. AB - Rhabdomyosarcoma is a malignant tumor well known to urologists. These tumors arise from the genitourinary system in 20% to 25% of cases, most commonly from the bladder, prostate, vagina, and paratesticular region. This is the first reported case of a rhabdomyosarcoma arising from the ureter. The radiographic findings and ureteroscopic appearance of this tumor suggested a benign fibroepithelial polyp; however, a ureteroscopic biopsy and subsequent nephroureterectomy revealed an embryonal rhabdomyosarcoma. PMID- 10754140 TI - Metastasis of gastric carcinoma to the ureter. AB - Metastasis of gastric carcinoma to the ureter is very rare. In most published cases, the diagnosis is only made at postmortem examination or when gross metastasis is present. This report presents a case in which the metastasis occluded the ureteral lumen from inside, and the diagnosis was made by histopathologic examination of a biopsy specimen taken during ureteroscopy. PMID- 10754141 TI - Renal cell carcinoma with an infrarenal vena caval tumor thrombus. AB - Extension of renal cell carcinoma (RCC) along venous drainage pathways is a well recognized entity. All previously reported cases of inferior vena cava (IVC) involvement by RCC have been with tumor thrombus in the suprarenal IVC. We report a 45-year-old man who had RCC arising from the lower pole of the right kidney with a tumor thrombus totally occluding the infrarenal IVC. The patient underwent radical nephrectomy with successful ligation and resection of the infrarenal IVC. PMID- 10754142 TI - Retrograde endopyelotomy in association with active distal ureteral dilation: a contraindication for the use of the 7F ureteral "tail" stent. AB - Since its introduction in 1997, the 7F "Tail" stent has been used after a variety of endourologic procedures. We describe 2 patients who underwent retrograde endopyelotomy with distal ureteral dilation; in both patients, after placement of a 7F "Tail" stent, a clinically significant urinoma developed. We believe that in the setting of extensive distal ureteral manipulation or distal active ureteral dilation to greater than 10F, placement of a "Tail" stent is contraindicated. PMID- 10754143 TI - Xanthogranulomatous pyelonephritis in an infant with an obstructed upper pole renal moiety. AB - We report a case of xanthogranulomatous pyelonephritis in an infant involving the upper renal pole moiety of a duplicated system associated with an obstructed ectopic ureter. It was successfully managed by an upper pole heminephroureterectomy. We also review the published reports of xanthogranulomatous pyelonephritis in pediatric patients. PMID- 10754144 TI - Adenocarcinoma in the exstrophic bladder. AB - We report 2 cases of cancerous transformation in an exstrophic bladder. The histology of these tumors, methods of surveillance, and treatment are discussed in conjunction with a review of published reports. These rare tumors are almost entirely adenocarcinomas. Their treatment is surgical (radical cystectomy) with or without associated radiation therapy. Surveillance for patients with bladder exstrophy, whether surgically corrected or not, is indispensable and rests on cystoscopy and urine cytology. PMID- 10754145 TI - Traumatic testicular dislocation: a case report and review of published reports. AB - Testicular dislocation after blunt scrotal trauma is a rare event. Its diagnosis depends on the awareness of the physician of its possible occurrence. It is usually a late finding during treatment of a motorcyclist brought to the emergency room because of multitrauma injury and is sometimes demonstrated in an abdominal computed tomography scan. We describe a typical case and discuss the chain of events leading toward the correct diagnosis and treatment based on a review of published reports. PMID- 10754146 TI - Squamous cell carcinoma at the prostatectomy site: squamous differentiation of recurrent prostate carcinoma. AB - Adenosquamous carcinoma of the prostate is rare. Even rarer is the subsequent squamous metastasis or recurrence in which only the malignant squamous component is observed in some sites, with the adenocarcinoma present in other sites. We describe a case of squamous cell carcinoma presenting at the prostatic bed 6 years after radical retropubic prostatectomy was performed for adenocarcinoma. Even though the primary tumor showed adenocarcinoma with foci of squamous differentiation, there was no morphologic evidence of adenocarcinoma in the current tissue examined. The suspected origin of the squamous tumor from a recurrence of the prostate tumor is discussed. PMID- 10754147 TI - Drug-seeking behavior in urolithiasis in the noncontrast computed tomography era: 2 cases. AB - Symptomatic urolithiasis is usually treated with narcotic pain management. This leads to the potential for use of its symptoms for personal gain. Historically, the typical presentation of a narcotic-seeking "stone patient" was a history of radiolucent stones and an intravenous contrast allergy. With the increased use of non-contrast-enhanced computed tomography to evaluate patients suspected of having acute urolithiasis, we have seen a change in the strategy of narcotic seeking patients. We report 2 patients with pelvic calcifications on non-contrast enhanced computed tomography feigning symptoms of urolithiasis to receive narcotic drugs. PMID- 10754148 TI - Rapid disease progression after the administration of bicalutamide in patients with metastatic prostate cancer. AB - We report 5 patients with advanced metastatic prostate cancer who took bicalutamide 50 mg/day for "second-line" hormonal manipulation and demonstrated a rapid rise in prostate-specific antigen (PSA) shortly after the initiation of bicalutamide. After discontinuation of the drug, PSA levels declined in 4 patients and stabilized in the fifth. In 2 of the patients, the PSA rise was associated with an increase in pain level, which subsided after the treatment was stopped. The timing of the rapid changes in PSA and pain levels suggests a direct effect of bicalutamide. The most probable explanation for this observation is a very early agonist activation of androgen receptor by bicalutamide, similar to the underlying mechanism of the "antiandrogen withdrawal syndrome." PMID- 10754149 TI - Squamous cell carcinoma arising at the base of the penis in a burn scar(1). AB - Squamous cell carcinoma developed after 32 years in a burn scar located on the ventral surface of the penis at the penoscrotal junction. The origin of squamous cell carcinoma in burn scars is a rare and not completely understood phenomenon. To the best of our knowledge, this is the first documented case of such an event in the penis because of the extreme rarity of genital burns. PMID- 10754150 TI - Vaginal stenosis and hydrometrocolpos: late complication of inadvertent perivaginal placement of an artificial urinary sphincter in prepubertal girls(1). AB - Urinary incontinence in pediatric and adolescent patients has been successfully managed with the artificial urinary sphincter for several decades. Placement of the sphincter can be difficult in the preadolescent girl due to poorly developed vaginal tissue that can result in difficulty establishing the surgical plane between the bladder and vagina. We report 2 patients in whom the sphincter was placed around the urethra and vagina, a complication that has been reported in only 1 patient previously. All 3 patients presented with hematometrocolpos and bloody vaginal discharge. All were successfully managed with replacement of the sphincter cuff around the urethra and delayed vaginoplasty. PMID- 10754151 TI - Clinical features and management of anterior intraurethral prostatic cyst. AB - We report on a symptomatic anterior intraurethral prostatic cyst in a 46-year-old man without clinical evidence of benign prostatic hyperplasia. The anterior location of this cyst makes it unique to all previously reported cases of prostatic cysts which are located posteriorly. Transurethral resection of the cyst with limited resection of the anterior prostatic tissue at the base of the cyst was performed with successful resolution of voiding symptoms. In the absence of lateral lobe hypertrophy, standard transurethral resection of the prostate should be avoided to ensure preservation of erectile and ejaculatory function. PMID- 10754152 TI - Bladder cancer arising in a spina bifida patient. AB - We report the case of a 52-year-old patient with spina bifida, neurologic bladder, and a history of recurrent urinary tract infections (UTIs) in whom a bladder cancer was incidentally discovered. Cytology, cystoscopy, and cystography showed nonspecific, extensive inflammatory lesions. Cystography demonstrated a complex of diverticulae and cellules. Pathologic examination of a diverticulectomy specimen revealed a grade III pT3b transitional and squamous cell carcinoma. Because of the similar disease causation (recurrent UTIs, stones, and indwelling catheterization), we suggest extension of the guidelines proposed for patients with spinal cord injuries (ie, annual serial bladder biopsies) to patients with nontraumatic neurogenic bladder. PMID- 10754153 TI - Erosion of a fascial sling into the urethra. AB - When synthetic materials are used for the construction of pubovaginal slings, urethral erosions may occur. This complication has not been reported with fascial slings. We present a case of a 34-year-old woman who underwent a pubovaginal sling procedure using rectus fascia. After 10 weeks of urinary retention, urethroscopy identified an erosion of the sling at the midurethra. Surgical revision restored normal voiding without recurrent stress incontinence. Although urethral erosions have been reported with synthetic suburethral slings, this case suggests that erosions can also occur with fascial slings. Careful positioning and minimal tension on the sling arms may minimize this risk. PMID- 10754154 TI - Artificial urinary sphincter insertion in renal transplant recipients. AB - The prosthetic urinary sphincter has contributed significantly to the improved management of urinary incontinence during the past 25 years. However, the safety of these devices in immunosuppressed patients is not well reported. We describe the successful insertion of the AMS 800 artificial urinary sphincter in two renal transplant recipients. PMID- 10754155 TI - Late pelvic recurrence of nonseminomatous testicular carcinoma after negative retroperitoneal lymph node dissection. AB - We report a case of pathologic Stage I teratoma recurring in the pelvis as embryonal carcinoma 12 years after radical orchiectomy and bilateral retroperitoneal lymph node dissection (RPLND). The patient received three cycles of chemotherapy (cisplatin, etoposide, bleomycin) followed by complete surgical excision of the pelvic mass. Successful treatment of these rare late recurrences usually requires chemotherapy and complete surgical excision. Pelvic relapse may potentially result from incomplete iliac node resection at the time of RPLND, altered lymphatic drainage from an incompletely resected spermatic cord, or a second primary extragonadal tumor focus. Our case emphasizes the importance of meticulous surgical technique during RPLND and the necessity for follow-up beyond 5 years in patients with testicular cancer. PMID- 10754156 TI - Antenatal detection and management of suprarenal masses. AB - The indications for, and timing of, surgical intervention for suprarenal masses detected prenatally are unclear. Also, the definitive diagnosis of suprarenal masses using imaging studies is difficult at best. We report 2 cases of antenatally detected suprarenal masses. One case represents an initial cystic mass expanding and becoming solid that had benign pathologic features. The second case was a stable solid mass that, on exploration, was malignant. Management options are discussed. PMID- 10754157 TI - Neonatal intervention for severe antenatal pyelocaliectasis. AB - The postnatal management of the antenatally detected ureteropelvic junction obstruction relies on several factors, including the degree of hydronephrosis detected postnatally, the renogram washout curve, and the degree of renal function. It is imperative for the urologist to review all renal scans because of the inherent pitfalls in performing and interpreting these studies. A select population demonstrating severe pyelocaliectasis and poor function exists in which an intraoperative renal biopsy may be a better predictor of future renal function when compared with the preoperative renal scan. We present a patient with poor renal function that normalized with early surgical intervention. PMID- 10754158 TI - Citrobacter diversus urosepsis and cerebral abscess in a child with antenatal hydronephrosis. AB - One percent of all pregnancies are found to have an antenatal abnormality; of these, 20% involve the genitourinary system. Today, controversy still exists regarding the postnatal management of some antenatal abnormalities detected by ultrasound. We present a case in which antenatal hydronephrosis initially detected by ultrasound appeared to resolve in utero. Postnatally, the child developed Citrobacter diversus urosepsis, meningitis, and cerebral abscess. Voiding cystourethrogram obtained after resolution of sepsis revealed grade IV reflux. This case underscores the importance of a full postnatal evaluation for all children with antenatal hydronephrosis and alerts clinicians to a virulent pathogen not commonly associated with urinary tract infection. PMID- 10754160 TI - Prenatally diagnosed autosomal recessive polycystic kidney disease: initial postnatal management. AB - We report a case of autosomal recessive polycystic kidney disease (ARPKD). A presumptive diagnosis was made after a late-term prenatal ultrasound revealed hypoplastic lungs, massive polycystic kidneys, and oligohydramnios. A full-term baby girl was delivered vaginally. Respiratory distress required intubation. Twelve hours after birth, she underwent bilateral nephrectomy and peritoneal dialysis catheter placement. The average kidney size was 150 g and 9.25 cm. Pathologic examination confirmed ARPKD. Peritoneal dialysis was started on the third day of life. The baby had no gross neurologic deficit. At 6 months of age, she was growing well, and the mother was a candidate to be a living-related kidney donor. PMID- 10754159 TI - Diamniotic twin gestation discordant for renal dysplasia and oligohydramnios. AB - Severe oligohydramnios and renal dysplasia were detected in one of diamniotic, monochorionic twins at 19 weeks' gestation. At birth (37 weeks), the affected twin had only minimal extrarenal Potter's features and mild pulmonary hypoplasia, despite severe renal dysplasia due to posterior urethral valves. The effects of virtual absence of amniotic fluid during the latter half of gestation from bilateral renal dysplasia were ameliorated by the presence of a normal co-twin and its normal amniotic fluid levels, even though the affected twin did not share the same amniotic fluid. PMID- 10754161 TI - Prenatal karyotype and ultrasound discordance in intersex conditions. AB - An infant born at 38 weeks' gestation with ambiguous genitalia had a prenatal 45X karyotype but an enlarged phallus on an ultrasound scan at 31 weeks' gestation. The newborn examination demonstrated penoscrotal hypospadias with chordee and two gonads palpable in the scrotum with a right hydrocele. Ultrasound showed a saccular structure containing debris behind the bladder. The postnatal karyotype was revealed to be 45X/46XY, with a pseudodicentric Y chromosome. Cystoscopy/genitography identified a uterus and a right fallopian tube, which were removed along with a dysgenetic right gonad. Biopsy of the descended left gonad revealed rare germ cells. The final diagnosis was 45X/46XY male pseudohermaphroditism with testicular dysgenesis. One should be aware of possible chromosomal mosaicism and combine the prenatal karyotype with the ultrasound genital findings to formulate an intersex differential diagnosis. PMID- 10754162 TI - Subclinical Cushing syndrome due to adrenocorticotropic hormone-independent macronodular adrenocortical hyperplasia: changes in plasma cortisol levels during long-term follow-up. AB - A 63-year-old man with bilateral adrenal tumors was treated for hypertension and diabetes mellitus. Endocrinologic examination during follow-up revealed the production of cortisol to be independent of the regulation of the hypothalamo pituitary-adrenal axis. Therefore, he was diagnosed as having subclinical Cushing syndrome, and these tumors were removed. Adrenocorticotropic hormone-independent macronodular adrenal hyperplasia (AIMAH) was endocrinologically and histopathologically diagnosed. This is the first report demonstrating that endocrinologic changes of AIMAH can be observed during long-term follow-up. PMID- 10754163 TI - Laparoscopic nephrectomy for autotransplantation. AB - Proximal ureteral injuries often require extensive reconstruction to repair. Management options include nephrectomy, ileal ureter interposition, extensive spiral bladder flaps, or autotransplantation. We report a patient who sustained a proximal ureteral avulsion and underwent a less invasive repair by way of a laparoscopic nephrectomy and subsequent autotransplantation. PMID- 10754164 TI - Testicular amyloid deposition as a cause of secondary azoospermia. AB - We present a case of secondary infertility due to familial amyloidosis. The patient presented with azoospermia, and no other sequela of the disease. A testis biopsy revealed tubules demonstrating full spermatogenesis interspersed with hyalinized tubules containing amyloid, confirmed with Congo red stain. A discussion regarding testicular amyloidosis is presented as well. PMID- 10754166 TI - Ureterorectal fistula: a rare cause of recurrent bacteriuria. AB - The differential diagnosis of recurrent bacteriuria includes a host of conditions very familiar to urologists. Ureterorectal fistula represents an unusual clinical entity that can cause recurrent bacteriuria. We present a patient who ultimately proved to have such a fistula. We review the available literature and comment on the significance of this condition. PMID- 10754165 TI - Ventral hernia of the urinary bladder with mixed urinary incontinence: treatment with herniorrhaphy and allograft fascial sling. AB - Abdominal hernias are not rare in women with urinary incontinence, but incisional bladder hernia is uncommon. The presenting symptoms in the rare cases reported included suprapubic discomfort, irritative voiding symptoms, and urinary incontinence. We present a patient with bladder herniation and severe mixed urinary incontinence. The pathophysiology of the urinary symptoms and the surgical alternatives for the correction of this condition are discussed. PMID- 10754167 TI - Angiomyolipoma of the renal sinus: diagnosis by percutaneous biopsy. AB - We report a case of angiomyolipoma of the renal sinus discovered incidentally during an evaluation for microscopic hematuria. Diagnosis was confirmed by percutaneous aspiration biopsy performed with magnetic resonance imaging control allowing differentiation of this entity from other fatty tumors of the renal sinus including liposarcoma, lipoma, and sinus lipomatosis. PMID- 10754168 TI - Numb chin syndrome as the presenting symptom of metastatic prostate carcinoma. AB - We report a case of the numb chin syndrome as the presenting symptom in a patient with metastatic prostate carcinoma. The numb chin syndrome is characterized by facial numbness along the distribution of the mental branch of the trigeminal nerve. Most cases of this syndrome that are not dental in origin have been associated with diffuse metastatic disease, particularly with underlying lymphoproliferative and breast cancer. Although axial and vertebral bone metastases are common in patients with carcinoma of the prostate, mental nerve involvement is rare. We present a case of the numb chin syndrome as the initial clinical manifestation in a patient with metastatic prostate adenocarcinoma. PMID- 10754169 TI - Application of continent retubularized ileal stoma (Monti procedure) to an ileocecal pouch. AB - We describe the application of a retubularized ileal stoma (Monti procedure) to an ileocecal pouch in a patient with a spinal cord injury who required a continent urinary diversion. When constructing a continent diversion with an ileocecal pouch, this technique seems a good choice, with significant advantages over the other alternatives. PMID- 10754170 TI - Positron emission tomography detection of osseous metastases of renal cell carcinoma not identified on bone scan. AB - The clinical utility of positron emission tomography (PET) in renal cell carcinoma (RCC) has not been determined. We describe a case in which metastatic RCC undetected by traditional staging methods was accurately staged by PET. A 77 year-old man presented with a 20-lb weight loss and bilateral renal masses. Plain radiographs, bone scintigraphy, and alkaline phosphatase were normal. PET imaging confirmed the right renal mass and revealed several metastatic bone lesions, confirmed by biopsy. The patient died 7 months after diagnosis. This case illustrates the potential superiority of PET in evaluating skeletal metastases of RCC. PMID- 10754171 TI - Undiagnosed urethral carcinoma: an unusual cause of female urinary retention. AB - Female urinary retention is extremely rare. Two cases of female urethral carcinoma that presented as urinary retention are reviewed and discussed. PMID- 10754172 TI - Gross hematuria secondary to renal vein hypertension from unilateral retroperitoneal fibrosis. AB - We report the first case of localized, unilateral, idiopathic retroperitoneal fibrosis encasing the left renal vein, which resulted in secondary renal vein hypertension. The patient presented with sudden and dramatic gross hematuria. Surgical release and excision of the surrounding localized retroperitoneal fibrosis resulted in prompt resolution of the hematuria. PMID- 10754173 TI - Superior vena cava obstruction due to prostate carcinoma. AB - Superior vena cava obstruction (SVCO) is considered an oncologic emergency commonly associated with lung carcinoma. The case presented here is that of a 48 year-old man presenting with SVCO, which was diagnosed as metastatic prostate carcinoma localized to the chest. He was treated with goserelin and aggressive radiotherapy with a drop in his prostate-specific antigen levels and symptomatic relief that lasted approximately 12 months. SVCO recurred locally in the chest and the patient died 24 months after diagnosis. This represents a rare presentation of prostate carcinoma and underlines the necessity for tissue diagnosis before local radiotherapy. PMID- 10754174 TI - An adult patient with a mixed germ cell tumor of the spermatic cord. AB - Tumors of the spermatic cord are very rare, and approximately one half of all primary spermatic cord tumors are malignant. We report the presentation and treatment of an adult (36-year-old) patient with a mixed germ cell tumor that originated in the spermatic cord. No similar cases of mixed tumors of the spermatic cord in adults have been reported. PMID- 10754175 TI - Undifferentiated sex cord/stromal testis tumor. AB - Completely undifferentiated sex cord/stromal tumors of the testis are rare after puberty. We describe such a tumor in an 18-year-old man presenting with a right testis mass. PMID- 10754176 TI - A monthly period of symptoms associated with benign prostatic hyperplasia. AB - The number of times that a man with benign prostatic hyperplasia awoke each night with the urge to urinate (nocturia) was analyzed for randomness by Bartlett's Kolmogorov-Smirnov white noise test and for a periodic component by Fourier analysis. The data series (n = 1549) was not white noise; it had a peak periodic component of 25 days, with a range of 21 to 37 days. The possibility that the monthly period of nocturia is a general phenomenon and is coupled to a monthly menstrual period, and the implications for more accurate diagnosis and new modes of therapy, are discussed. PMID- 10754177 TI - Symptomatic paraurethral corpus spongiosum cyst in a male patient. AB - The case of a 25-year-old man presenting with lower urinary tract symptoms who was found to have a noninflammatory posterior corpus spongiosum cyst is presented. The case was successfully managed by surgical excision and presents an uncommon cause of lower urinary tract dysfunction through irritation of the posterior urethra. PMID- 10754178 TI - Communicating hematocele in children following splenic rupture: diagnosis and management. AB - Acute hematocele is commonly associated with direct testicular trauma. Blood within the tunica vaginalis may infrequently accompany blunt abdominal injury in the presence of a communicating hydrocele. Optimal management involves early recognition and treatment of the abdominal source of bleeding. Elective repair of the communicating hydrocele/hematocele should follow. We report 2 cases of boys with scrotal swelling due to communicating hematoceles. Both cases were associated with a patent processus vaginalis and splenic laceration secondary to blunt trauma. PMID- 10754179 TI - Prostatic adenocarcinoma metastatic to the breasts: report of a case with diagnosis by fine needle aspiration biopsy. AB - Metastases of tumors of extramammary origin to the breast are extremely uncommon. We report the case of an 81-year-old man with a history of prostatic adenocarcinoma treated with adjuvant estrogen therapy, who presented with bilateral palpable mammary masses. Mammographic study showed irregular solid nodules. Fine needle aspiration (FNA) biopsy was performed. The aspiration smears showed single cells with high nuclear/cytoplasmic ratios, prominent nucleoli, and rare acinar formations. Immunocytochemical studies using antibodies against prostate-specific antigen and prostate-specific acid phosphatase confirmed the diagnosis of metastatic prostatic adenocarcinoma, allowing appropriate treatment. PMID- 10754180 TI - Cystic pelvic pathology presenting as falsely elevated post-void residual urine measured by portable ultrasound bladder scanning: report of 3 cases and review of the literature. AB - Dedicated portable ultrasound devices generally offer a rapid, noninvasive, largely operator-independent means of assessing post-void residual urine (PVR) volume. In most published series, PVR measured by portable ultrasound correlates well with catheterized urine volume. We report 3 cases in which follow-up of falsely elevated PVR measurements on ultrasound resulted in comparatively low catheterized volumes. In all 3 cases, the elevated readings were due to cystic ovarian pathology, which was diagnosed by formal radiologic evaluation and ultimately confirmed operatively in 2 cases. Cystic pathology of the pelvis or lower abdomen may present as an elevated PVR on ultrasound and low urine volume on subsequent catheterization and should prompt further evaluation. PMID- 10754181 TI - Efficacy of epidural anesthesia for retroperitoneoscopic renal biopsy. AB - Laparoscopic procedures are performed using general anesthesia due to the perceived limitations of regional anesthesia in the upper abdomen and retroperitoneum. We present our initial experience with epidural anesthesia for retroperitoneal laparoscopic renal biopsy. PMID- 10754182 TI - A rare simultaneous presentation of testicular mixed germ cell tumor with a contralateral testis torsion. AB - We report a rare case of a 27-year-old man presenting with an acute scrotum with simultaneous occurrence of testicular tumor and contralateral torsion. Preoperative imaging demonstrated the testicular missed torsion, yet findings were equivocal with regard to the testicular tumor. On scrotal exploration the left testis was found to be ischemic due to threefold rotation. The right testis was not identifiable, being composed of numerous necrotic lesions. Frozen section suggested malignancy, hence high right orchiectomy was performed. Histologic examination of the right testis showed mixed germ cell tumor, containing all the subtypes. To our knowledge no similar case has been reported. PMID- 10754184 TI - Skill mix between nurses and doctors working in primary care-delegation or allocation: a review of the literature. AB - The fundamental role of primary health care teams (PHCT) is to deliver effective services to the local population. Demands on the PHCT have increased since the early 1990s and, with the advent of Primary Care Groups (PCGs), will continue to do so in a primary health care led National Health Service (NHS). Rapid changes, however, raise questions about the feasibility and delivery of new services with skill mix and the distribution of workload within the PHCT being key issues. This paper reviews the literature on workload in primary care, attitudes to delegation, inter-professional relationships and teamworking and concludes that in order to deliver the vision of a primary care led NHS, meet the health care needs of users, address the inevitable anxieties of general practitioners and bring forth the professional aspirations of nurses and other health care professionals, more equitable and less hierarchical models of multi-professional teamworking in primary care will be most successful. PMID- 10754183 TI - Multiple drug resistance mutations in human immunodeficiency virus in semen but not blood of a man on antiretroviral therapy. AB - The concept that the male reproductive tract harbors isolated reservoirs of human immunodeficiency virus (HIV) infection has now been widely accepted. The significance of semen viral burden to sexual transmission of HIV is obvious; however, its contribution to disease progression is unknown. We report a case study that demonstrates the emergence of resistance-conferring mutations to antiviral therapy in infected seminal leukocytes from a man with asymptomatic prostatitis associated with leukospermia. This finding demonstrates the potential importance of male reproductive tract organs to the development of therapy resistance in HIV-infected men. PMID- 10754185 TI - Ways of being a father: how first-time fathers and public health nurses perceive men as fathers. AB - The purpose of this paper is to describe the different ways of being a father identified in a sample of Finnish men. The data consisted of 71 interviews with fathers and 175 essays written by public health nurses. The fathers and public health nurses described different ways of being a father: bystander, supporter of spouse, partner, and head of the family. The spouse, relatives and maternity health care workers could encourage the father to choose a specific way to be a father, or discourage them from adopting a specific way. The information obtained in this study will be helpful in supporting the different kinds of fathers and their individual needs in family nursing. PMID- 10754186 TI - Stress in mental health nursing. AB - Although there have been growing concerns about stress in nursing over recent years, research has primarily focused on general nurses. This paper reports on stress in mental health nursing. The data were obtained, via questionnaires, from mental health nurses at the end of their training. Four-hundred and forty-seven questionnaires were returned, an 80% response rate. Open-ended questions were asked about the nurses' experiences of stress during their time as a student, and what they anticipated would be stressful once qualified. A wide range of stresses were identified, many similar to those reported in previous studies. The open ended nature of the questions, however, provided greater detail about the nature of stress in mental health nursing than has been reported hitherto. This detail points to broader perspectives which might be taken in future research which, in turn, may lead to a fuller understanding of nurses' experiences of stress. PMID- 10754187 TI - Generalising to theory: the use of a multiple case study design to investigate needs assessment and quality of care in community nursing. AB - This paper outlines the detail of the case study method used in a project commissioned by the English National Board for Nursing, Midwifery and Health Visiting (ENB) to investigate the changing educational needs of community nurses with regard to needs assessment and quality of care in the context of the NHS and Community Care Act, 1990. It explains the methodological procedures and analytic processes which led to integration of data across the whole study, focusing on the role of a prior theoretical framework in case study design. Recently qualified practitioners (health visitors and district nurses) were observed during a regular shift (N=134 visits), concentrating on their practice of assessing needs, and on liaison and collaboration within teams and across sectors. They were interviewed after the observation period (N=33 practitioners), to determine the extent of formality they attached to each assessment, and elicit information about aspects which may be embedded in everyday practice as well as those recorded for explicit requirements.The preliminary analysis resulted in the modification of a model for assessing service quality, and identified various points where a 'policy-practice gap' might arise between policies and practice in both the health service and education. The practicalities of operationalising a multiple case study design into research are highlighted, and the mechanism for 'generalising to theory' illustrated. PMID- 10754188 TI - Fitting in: maintaining a sense of self during hospitalisation. AB - In this study the patients' perspective of 'fitting in' was gained through participant observation and interviews. Eleven patients' stories confirmed that while hospitalisation can alleviate the suffering caused by illness, it may cause distress. In order to minimise this threat to self patients adjust to hospital life. They are supported by family, friends and health professionals in interpreting the social rules of the ward by conforming to, circumventing and challenging them. PMID- 10754189 TI - Hunting for Heidegger: questioning the sources in the Benner/Cash debate. AB - This paper uses the Cash/Benner debate as an example of how important philosophical arguments can be hidden due to the use of secondary sources. Nurse academics can get lost in arguments about the secondary sources when there is a much more important issue lurking in the background. This paper argues that Benner's philosophy is not grounded in Heidegger but Dreyfus's interpretation of Heidegger. This has the consequence that Heidegger's ontological difference is collapsed and that her nursing ideas are weakened because of this. PMID- 10754190 TI - Influences on length of stay in intermediate care: lessons from the nursing-led inpatient unit studies. AB - A recent study found that intermediate care in a nursing-led in-patient unit (NLIU) led to dramatically increased hospital stay when compared to usual care in a hospital. This is despite a large body of work across a variety of settings, including the pilot for that same study, which suggests that the NLIU is effective. This paper presents a series of exploratory analyses that aim to explore the findings further. A number of changes were identified in the functioning of the NLIU between the main and pilot study including patient population, leadership and staffing levels. Two ANCOVA analyses reveal that while performance of the NLIU declined between the two studies, that of usual care improved dramatically. Extended lengths of stay appear to be associated with the NLIU's location on a satellite site rather than the model of care per se. Reduction in the staffing levels on the NLIU and a change in patient population may well explain the findings. PMID- 10754191 TI - A transcultural study of Jordanian nursing students' care encounters within the context of clinical education. AB - Clinical education is an integral part of nursing education. Clinical teachers are the vital link in this teaching-learning process. The quality and quantity of student-teacher interactions in the clinical area can either facilitate or hinder students' learning. This paper presents a part of a larger study that discovered, described, explained and compared Australian and Jordanian nursing students' caring and non-caring encounters with their clinical teachers within the context of clinical education. The study was guided by Leininger's theory of culture care universality and diversity and Leininger's ethnonursing research method was utilised. The informants consisted of 12 key informants and 35 general informants. Three major themes emerged from the analysis of the data: (1) clinical teacher's caring behaviours; (2) student-teacher caring encounters; and (3) caring encounter consequences. Under these themes, care constructs emerged which gave light to the Jordanian nursing students' care meanings, expressions and values within their cultural environment, social structures and world view. The overall findings revealed that Jordanian nursing students found their clinical experiences as beneficial when their encounters with the clinical teacher were conducted through mothering, translating, sustaining, negotiating and transforming processes. PMID- 10754192 TI - Patient autonomy during rehabilitation: the experiences of stroke patients in nursing homes. AB - This article describes the results of a grounded theory study among stroke patients (N=17, aged 50-85) in rehabilitation wards in nursing homes. Patient autonomy (dimensions: self-determination, independence and self-care) increases during rehabilitation due to patient factors (conditions and strategies of patient) and environmental factors (nursing home and strategies of health professionals and family). During rehabilitation patients are in a state of transition regarding autonomy: patients need support to enhance autonomy, gradually regain autonomy, and thereby need less support. Although facilitating environmental factors were discovered, patients also experienced constraining factors regarding patient autonomy. Health professionals should give more attention to self-determination and independence; the nursing home should offer stroke patients more opportunities to do familiar activities autonomously. PMID- 10754193 TI - Alpha oscillations as an indicator of dynamic memory operations - anticipation of omitted stimuli. AB - Elaborating on a paradigm of Basar in experiments with periodically presented auditory or visual stimuli, 10 subjects performed tasks in which omitted stimuli had to be anticipated and their (virtual) onset time had either to be marked (a) mentally ('mental marking' MM) or (b) by pressing a button ('button pressing' BP). EEG was obtained from 11 sites in frontal, central, temporal, parietal and occipital areas. The experiments were carried out in triplets of MM, BP, MM measurements. Mean temporal errors of motor responses served as the basis for a functional interpretation of EEG patterns. Correlation analysis of sweeps for each individual revealed brief intervals of phase ordering of EEG patterns in the alpha range lasting approximately three periods. For frontal and vertex derivations a close congruence of the location of the phase-ordered patterns to mean errors of motor responding was shown. These results corroborate the claim that 'emitted' alpha oscillations represent intentional processing. The close agreement between temporal locations of phase-ordered EEG segments and those of the motor responses strongly suggests that EEG phases represent memory retrieval of period duration as a common functional component of MM and BP. A lack of topographical correspondence with results from a passive 'missing stimulus' paradigm underlines the specific intentional origin of the observed phase ordering. Theoretical implications of the results are discussed. PMID- 10754194 TI - Habituation: an event-related potential and dipole source analysis study. AB - The goal of this study was to investigate habituation processes in the brain, and in particular, to identify the brain structures involved in these processes. Therefore, event-related potentials (ERPs) were recorded in response to a series of repeated, task-irrelevant, salient stimuli presented against the background of a cognitive task which required the subjects to attend to a fixation sign on a TV screen. Stimuli were presented randomly in one of four possible quadrants of the TV screen. Dipole analyses were performed in order to localize the source of stimulus repetition effects (habituation). As expected, there was a decrease in amplitude of the N1 and P3 components as a function of stimulus repetition; habituation of N1 occurred faster than that of the P3 component. It is suggested from the results of dipole analyses that the N1 effect concerns diffuse cortical activation. The P3 habituation effect seemed to involve the temporal cortex especially. PMID- 10754195 TI - Event-related potentials (ERPs) to hemifield presentations of emotional stimuli: differences between depressed patients and healthy adults in P3 amplitude and asymmetry. AB - Depression may involve dysfunction of right parietotemporal cortex, a region activated during perception of affective stimuli. To further test this hypothesis, event-related brain potentials (ERPs) were measured in a paradigm previously shown to produce ERP asymmetries to affective stimuli over parietal sites in healthy adults. Pictures of patients with dermatological diseases showing disordered or healed facial areas before (negative) or after (neutral) surgical treatment were briefly exposed for 250 ms to either the left or right hemifield. ERPs of 30 unmedicated, unipolar depressed patients and 16 healthy adults, all right-handed, were recorded from 30 electrodes. A principal components analysis extracted factors which closely corresponded to distinctive ERP components previously reported for this task (N1, N2, early P3, late P3, slow wave). Significant effects of emotional content, i.e. enhanced amplitudes to negative than neutral stimuli, were found for early and late P3. Control subjects showed significant hemispheric asymmetries of emotional processing for late P3 (peak latency 460 ms), with the largest emotional content effects over the right parietal region. In striking contrast to control subjects, depressed patients did not show an increase in late P3 for negative compared to neutral stimuli over either hemisphere and had smaller late P3 amplitude than control subjects. Patients did, however, show larger early P3 (peak latency 330 ms) to negative than neutral stimuli. Results suggest intact early discrimination but abnormal late appraisal of affective content in depression, which may arise from selective inhibition of right parietal regions integral for perceiving and evaluating emotional stimuli. PMID- 10754196 TI - The dimensional complexity of the EEG during cognitive tasks reflects the impaired information processing in schizophrenic patients. AB - The aim of the present study was to investigate whether schizophrenic patients show a different change of the dimensional complexity of the EEG, as represented by the Grassberger-Procaccia correlation dimension D(2,) under cognitive challenge compared to normal control subjects. With respect to results reported in the literature, it was expected that the complexity of the signal under cognitive challenge is higher in schizophrenic patients than in normal control subjects reflecting the impaired information processing abilities of the patients. Eighty-seven schizophrenic and 30 matched control subjects performed two different types of the continuous performance task. The results revealed differences between schizophrenic patients and control subjects for the performance as well as the complexity measures. Schizophrenic patients produced more omission errors than normal subjects did. For the EEG complexity measure no differences occurred under the baseline condition. In contrast, during the first minute under task conditions the control subjects showed a decrease of the dimension while no changes were found for the schizophrenic group. These results occurred for both types of the cognitive task but they reached clear significance only in one of them. The results are interpreted as reflecting the ability of normal subjects to adapt their information processing system to the cognitive challenge and to focus their attention on the task while schizophrenic subjects do not show this adaptation to the task. PMID- 10754197 TI - Two types of cardiovascular autonomic regulatory responses to the war. AB - In war, many people are not directly hurt, but they feel fear of various intensities for a long time. The response of the cardiovascular autonomic system to these conditions, produced by the bombardment of Yugoslav cities by NATO for 78 days in spring 1999, was investigated. A group of 12 active medical professionals was examined 1 and 5 weeks after the beginning of bombardment and 10 days after the end of bombardment by spectral analysis of heart rate variability. Two types of cardiovascular autonomic responses emerged. One type included a fast reaction and then adaptation; the increased activity of symphaticus compared to parasymphaticus in the beginning of war was followed by a balance of these activities after 1 month and a small increase immediately after the end of bombardment. The other type did not show an increased activity of symphaticus compared to parasymphaticus during the war, but at the end of bombardment the prevalence of vagal tone was observed. PMID- 10754198 TI - Trait and state EEG indices of information processing in developmental dyslexia. AB - L asymmetry in beta activity in the dyslexic group, again in both tasks. Theta activity did discriminate between the two tasks in the dyslexic group. In the phonological task, task-related frontal theta in the dyslexic group was significantly different from the control group, with the former showing an increase in amplitude and the latter a decrease. In the visual task, there was no significant difference between the dyslexic and the control group, with both showing a task-related decrease in amplitude. The inter-task variations in EEG response in the dyslexic group paralleling variations in task performance are interpreted in terms of the varying engagement of a frontally-based attentional system. Inter-task consistencies of EEG response despite variations in performance are interpreted in terms of the continued application of a specific cognitive strategy. PMID- 10754199 TI - Momordin I, a compound of ampelopsis radix, inhibits AP-1 activation induced by phorbol ester. AB - Recently, we reported that momordin I from Ampelopsis radix is an activator protein-1 (AP-1) function inhibitor. Mormordin I showed both inhibition of AP-1 transcriptional activity and cellular cytotoxicity. Moreover, its inhibitory action was also identified in the animal experiments. The present study attempted to determine the mechanism of the action of momordin I more clearly. In mouse fibroblast cells, momordin I suppressed the AP-1 activity induced by phorbol 12 myristate 13-acetate (PMA), as well as blocked the de novo synthesis of AP-1 protein. Momordin I also showed cell cytotoxic activities on various cell lines and blocked the uptake of [(3)H]thymidine in cell proliferation induced by PMA. From these results, the inhibitory action of momordin I on AP-1 was clearly identified. Through the competitive binding assays, it appears that the inhibitory site of momordin I might be in the Jun/Fos dimer, and not in the DNA. Momordin I also showed the inhibitory action on the Jun/Jun homodimer, as well as on the Jun/Fos heterodimer. It also inhibited the autoregulated de novo synthesis of AP-1 by the Jun/Jun homodimer. In addition, since the truncated AP-1 used in this study only has the bZip regions of c-Jun and c-Fos, it appears that the inhibitory action site of momordin I may be the basic region of c-Jun instead of on the same region of c-Fos. PMID- 10754200 TI - Cloning, sequence analysis and expression of the cDNAs encoding the canine and equine homologues of the mouse double minute 2 (mdm2) proto-oncogene. AB - The mdm2 oncogene is amplified and overexpressed in a variety of human tumours and the oncogenic potential of MDM2 is partly due to its ability to inactivate tumour suppressor p53 function. In the present communication we describe the cloning, sequence analysis and expression of the complete wildtype canine and equine mdm2 cDNAs. The encoded full-length canine and equine cDNAs show strong sequence homology with MDM2 proteins from other species and both cDNAs generate recombinant proteins of approximately 90 kDa. These data will allow for the role of this oncogene to be established in companion animal oncology. PMID- 10754201 TI - Diminished expression of ING1 mRNA and the correlation with p53 expression in breast cancers. AB - p33(ING1) is a novel candidate tumor suppressor and its overexpression induces growth arrest or apoptosis in different cell lines. These functions of p33(ING1) depend largely on the activity of p53, and p53-dependent activation of the transcription from the p21/WAF1 promoter also requires p33(ING1). We examined the expression of ING1 mRNA in breast cancer cell lines and clinical breast cancer tissues, using quantitative RT-PCR and real time TaqMan technology. In breast cancer cell lines, ING1 mRNA was expressed at almost the same level. However, in a comparison between the cancer and matched normal tissues, a significant decrease in ING1 mRNA expression was found in 17 of 24 (70.8%) breast cancer tissues. We also examined the correlation between ING1 mRNA expression and p53 expression. There was a significant decrease of ING1 mRNA in nine of 15 tumors negative for p53 immunostaining, most of which were considered to have wild type p53. In these tumors, p53 may not function in case of a decreased expression of p33(ING1), and the lack of cell cycle regulation may correlate with the carcinogenesis and tumor progression. PMID- 10754202 TI - The marine compound spisulosine, an inhibitor of cell proliferation, promotes the disassembly of actin stress fibers. AB - Spisulosine is a novel antiproliferative (antitumoral) compound of marine origin. In this work the molecular target for this toxic agent has been analyzed. In the presence of spisulosine, cultured cells change their morphology, first acquiring a fusiform morphology, and later becoming rounded without focal adhesions. Analysis of the cytoskeleton of treated cells indicate the absence of actin stress fibers. PMID- 10754203 TI - Suppression by Chai-hu-gui-zhi-tang of the development of liver lesions induced by N-nitrosomorpholine in Sprague-Dawley rats. AB - The effects of Chai-hu-gui-zhi-tang (TJ-10) on the development of liver lesions induced by N-nitrosomorpholine and on labeling and apoptotic indices were investigated in male Sprague-Dawley rats. Rats were given chow pellets containing 0.5 or 1.0% TJ-10 and, from the beginning of the experiment, were given drinking water containing N-nitrosomorpholine for 8 weeks. Visible white liver nodules, cellular alteration foci, and hepatic foci staining positively for glutathione-S transferase, placental type, were examined macroscopically or histochemically. In week 16, TJ-10 at both dosages significantly reduced the incidence, and/or number of visible white nodules and hepatic lesions. Quantitative histologic analysis also showed that prolonged feeding of TJ-10 at both dosages significantly reduced the number of hepatic foci positive for glutathione-S-transferase, placental type. Administration of TJ-10 at both dosages also significantly decreased the labeling index of adjacent liver and significantly increased the apoptotic index of adjacent liver but had no significant effect on those of neoplastic lesions. These findings indicate that TJ-10 suppresses the development of liver lesions and suggest that this effect might be related to TJ-10's inhibition of cell proliferation and induction of apoptosis in adjacent hepatocytes. PMID- 10754204 TI - Promotion of hepatocarcinogenesis by hexachlorobenzene in energy-restricted rats. AB - The interaction between dietary energy restriction and low dose of the fungicide hexachlorobenzene (HCB) was evaluated in a rat liver medium-term bioassay for carcinogenesis. Male Wistar rats were fed a control or a 50% energy-restricted diet, both added or not with 50 ppm HCB, for 6 weeks. HCB exposure or energy restriction separately did not exert any influence on the development of glutathione S-transferase placental form (GST-P(+)) foci of hepatocytes. Simultaneous HCB exposure and energy restriction induced a significant increase in liver centrilobular hypertrophy and GST-P(+) foci development. Our findings suggest that energy restriction increases liver response to low dose of HCB, unmasking the promoting potential of this fungicide. PMID- 10754205 TI - Somatic mutations of beta-catenin play a crucial role in the tumorigenesis of sporadic hepatoblastoma. AB - Hepatoblastoma (HB) is the most common malignant hepatic tumor during early childhood. Its molecular pathogenesis is still poorly understood. Mutations of adenomatous polyposis coli (APC) gene have been identified in sporadic cases and in individuals associated with familial adenomatous polyposis syndrome. beta catenin is a key element in the cadherin-mediated cell adhesion system and Wnt/wingless pathway, and is controlled by APC. APC affects the degradation of beta-catenin by its NH(2)-terminal phosphorylation on the serine/threonine residues of exon 3. Mutations of these phosphorylation sites are primary targets for activating mutations in several types of human cancer and lead to nuclear accumulation of beta-catenin protein. In this study, we examined nine patients with HB using immunohistochemistry and direct DNA sequencing. All nine cases showed predominant nuclear expression of beta-catenin. Eight cases (89%) showed mutations involving exon 3 of the beta-catenin gene, including five with deletions and three with missense mutations. All five deletions were in-frame deletions without frameshift. The very high frequency of mutations in the beta catenin gene suggests that beta-catenin mutations are crucial in the tumorigenesis of HB. PMID- 10754206 TI - Interactive effects of methyl-deficiency and dietary restriction on liver cell proliferation and telomerase activity in Fischer 344 rats pretreated with aflatoxin B(1). AB - The effects of methyl-deficiency and dietary restriction (DR) on hepatic cell proliferation and telomerase activity was studied in male Fischer 344 rats pretreated with aflatoxin B(1) (AFB(1)). Five-week-old rats were gavaged 5 days per week for 3 weeks with AFB(1) (25 microg/rat per day) or solvent (100 microl 75% dimethylsulfoxide). Rats were then divided into four groups. Two groups were fed a methyl-sufficient (MS) diet either ab libitum (AL) or with DR. The other two groups were fed a methyl-deficient (MD) diet either AL or with DR. At 15, 20, and 32 weeks of age, hepatic cell proliferation, telomerase activity, and the number of glutathione S-transferase-P positive (GST-P(+)) foci were determined. DR reduced hepatic cell proliferation, while the MD diet and AFB(1) pretreatment increased cell proliferation. Telomerase activity was decreased by DR and increased by the MD diet and AFB(1) pretreatment. The same trend was observed with GST-P(+) foci: in AFB(1)-pretreated rats, methyl deficiency increased the number of foci, while DR decreased the number. These results are consistent with a role of telomerase in hepatocarcinogenesis. PMID- 10754207 TI - Loss of heterozygosity at 3p24-p25 as a prognostic factor in breast cancer. AB - Differences in clinical course and biological characteristics among breast cancers will probably be explained ultimately by variations in the pattern of genetic alterations among the many genes that can play roles in carcinogenesis. Loss of heterozygosity (LOH) of a particular chromosomal region in a tumor, which presumably indicates loss of a growth-regulating 'tumor-suppressor' gene in that region, may represent a useful marker for postoperative prognosis. In earlier work we observed LOH at chromosomal regions 3p14-p21 and/or 3p24-p25 in a large proportion of breast cancers. To examine whether allelic losses in either of those regions might correlate with postoperative survival, we tested tumors from a cohort of 504 breast cancer patients for allelic losses of microsatellite markers in the relevant portions of chromosome 3p. Five years postoperatively, patients whose tumors had undergone LOH at 3p24-p25 were found to have borne significantly higher risks of mortality than women whose tumors retained both alleles at that locus; i.e. the 5-year mortality rate was 22% among patients with losses at 3p24-p25 vs. 9% with retentions of heterozygosity at that locus (P=0.0014). These data indicate that LOH at 3p24-p25 is a significant predictive factor for postoperative survival of patients who have undergone surgery for breast cancer. PMID- 10754208 TI - Absence of p16, p21 and p53 gene alterations in hepatocellular carcinomas induced by N-nitrosodiethylamine or a choline-deficient L-amino acid-defined diet in rats. AB - To clarify the involvement of tumor suppressor genes in exogenous and endogenous liver carcinogenesis, alterations of p16, p21 and p53 in hepatocellular carcinomas (HCCs) induced by N-nitrosodiethylamine (DEN) and a choline deficient L-amino acid-defined (CDAA) diet in rats were investigated. Male Fischer 344 rats received DEN at 6-week of age followed by partial hepatectomy (PH), with colchicine to induce cell cycle disturbance, and a selection pressure regimen. Sacrifice was after 42 weeks. Other animals continuously received a CDAA diet for 75 weeks and were then killed. Eleven and 15 HCCs were obtained, respectively. Total RNA was extracted from and cDNA was synthesized with reverse transcriptase to allow investigation of mutations in p16, p21 and p53 by polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) analysis. Expression of p16 and p21 mRNA was also analyzed by reverse transcription (RT)-PCR. The results showed no mutations or deletions of p16, p21 and p53 in any of the HCCs induced by DEN or CDAA. Loss or decrease of p16 and p21 expression were also not found, suggesting that p16, p21 and p53 alteration may not be necessary for either exogenous or endogenous liver carcinogenesis in rats. PMID- 10754209 TI - The role of orally administered dimethylarsinic acid, a main metabolite of inorganic arsenics, in the promotion and progression of UVB-induced skin tumorigenesis in hairless mice. AB - The effect of dimethylarsinic acid (DMA) on skin tumorigenesis by UVB irradiation was examined. Hairless mice (Hos: HR-1) irradiated with UVB at a dose of 2 kJ/m(2) twice weekly, were fed with drinking water containing 1000 ppm DMA, a main metabolite of inorganic arsenics, produced more skin tumors than DMA untreated mice. Histopathological examination revealed that the mouse malignant tumors with severe atypism appeared only in the treatment group of UVB plus 1000 ppm DMA. These positive results point out the importance of dimethylated metabolites of inorganic arsenic in the process of skin carcinogenesis. PMID- 10754210 TI - Analysis of serum ErbB-2 protein and HLA-DRB1 in Japanese patients with lung cancer. AB - We investigated the relationship between ErbB-2 and HLA in order to clarify the clinical and genetic factors related to Japanese patients with lung cancer. Thirty-nine of the 73 lung cancer patients (53.4%) had elevated levels of ErbB-2. Only seven of 23 (30. 4%) patients with small cell carcinoma had elevated ErbB-2 levels. The prevalence of ErbB-2 positivity was highest (23 of 32; 71.8%) in patients with adenocarcinoma, while that in patients with squamous cell carcinoma was 50% (9 of 18). The frequencies of HLA A33, B44, B62, and B75 were lower in the lung cancer patients than in the control group. HLA-DR9 was higher in frequency in lung cancer patients than in the healthy controls (P<0.05), but HLA DR6 was lower in frequency in lung cancer patients than in controls (P<0.01). DRB1*0901 was significantly higher in frequency in lung cancer patients than in controls (P<0.05). On the other hand, DRB1*0802, DRB1*1302 and the DRB1*14 group (*1401, *1403, *1405, *1406, and *1407) were completely absent in lung cancer patients. The frequencies of HLA B35, B52, B62, DRB1*0404, and DRB1*0406 were higher in the ErbB-2-positive lung cancer patients than in the ErbB-2-negative lung cancer patients. However, these types of HLA were not included in significant frequencies in our group of lung cancers. Our results suggest that some HLA-antigens/alleles participate in the pathogenesis of lung cancer in Japanese patients. In addition, the relationship between HLA-associated genetic factors and ErbB-2 seems to be weak. These findings suggest that ErbB-2 is correlated with prognostic factors for lung cancer independently of HLA associated genetic factors. PMID- 10754211 TI - Preventive effect of epicatechin and ginsenoside Rb(2) on the inhibition of gap junctional intercellular communication by TPA and H(2)O(2). AB - The anticarcinogenic effects of epicatechin (EC) and ginsenoside Rb(2) (Rb(2)), which are major components of green tea and Korea ginseng, respectively, were investigated using a model system of gap junctional intercellular communication (GJIC) in WB-F344 rat liver epithelial cells. 12-O-tetradecanoylphorbol-13 acetate (TPA) and hydrogen peroxide, known as cancer promoters, inhibited GJIC in the epithelial cells as determined by the scrape loading/dye transfer assay, fluorescence redistribution assay after photobleaching, and immunofluorescent staining of connexin 43 using a laser confocal microscope. The inhibition of GJIC by TPA and H(2)O(2) was prevented with treatment of Rb(2) or EC. The effect of EC on GJIC was stronger in TPA-treated cells than in H(2)O(2)-treated cells, while the effect of Rb(2) was opposite to that of EC. EC, at the concentration of 27.8 microg/ml, prevented the TPA-induced GJIC inhibition by about 60%. Rb(2,) at the concentration of 277 microg/ml, recovered the H(2)O(2)-induced GJIC inhibition by about 60%. These results suggest that Rb(2) and EC may prevent human cancers by preventing the down-regulation of GJIC during the cancer promotion phase and that the anticancer effect of green tea and Korea ginseng may come from the major respective components, EC and Rb(2). PMID- 10754212 TI - Deoxycholic acid-induced apoptosis is switched to necrosis by bcl-2 and calphostin C. AB - We previously demonstrated that the cytotoxicity associated with exposure of HCT116 cells to deoxycholic acid was due to the induction of apoptosis. Here we show that this results in activation of caspase 3 and that over expression of bcl 2 can suppress this. Surprisingly, inhibition of apoptosis by over expression of bcl-2 or incubation with calphostin C, a PKC inhibitor, did not enhance cell survival, but instead caused a switchover to death by necrosis. Hence, DCA induced apoptosis requires caspase activity and both bcl-2 and PKC can determine the type of cell death induced by deoxycholic acid. PMID- 10754213 TI - Association of tenascin-R with murine brain myelin membranes: involvement of divalent cations. AB - In the central nervous system (CNS), tenascin-R (TN-R) is mainly expressed by oligodendrocytes and in white matter tracts. Here, we have examined the molecular association of TN-R with CNS myelin by incubation of myelin membranes (MM) purified from adult mouse brain under different ionic conditions. By Western blot analysis, the 160 kDa isoform was the main TN-R component detectable in MM as a dimer which became degraded to monomers of 160 kDa and major fragments of 125 and 80 kDa in the absence of protease inhibitors. In the presence of chelating agents, TN-R was completely extracted from MM. Calcium ions promoted the dissociation of TN-R while zinc or copper blocked it. TN-R release from MM was sensitive to heat suggesting the involvement of calcium-dependent myelin protease(s) in this process. In addition, 1,10-phenanthroline (a metalloprotease blocker) partially inhibited TN-R release in the presence of calcium ions. We conclude that divalent metal ions stabilize the association of TN-R with CNS myelin and upon damage, the protein can be released and degraded by endogenous proteases, suggesting the implication of myelin-derived TN-R in axon growth inhibition and demyelinating diseases. PMID- 10754214 TI - Effects on dystrophic (dy/dy) limb muscle of the K+ channel blocker tetraethylammonium. AB - K(+) channel blockers, such as tetraethylammonium (TEA) and 4-aminopyridine, increase force of normal skeletal muscle. To determine whether they also increase force of diseased muscle, effects of TEA were examined on limb muscles of dy/dy dystrophic mice in vitro. TEA significantly increased isometric twitch force of normal and dystrophic soleus muscle by 29.9+/-5.8 and 30.0+/-5.0%, respectively, and significantly increased force of normal and dystrophic extensor digitorum longus muscle by 18.3+/-1.9 and 34. 9+/-10.4%, respectively. Force increases could be attributed only partially to a prolongation of contraction time. These data indicate that TEA augments force of overtly diseased (dystrophic) limb muscle to an extent comparable with that of normal limb muscle. PMID- 10754215 TI - On the fractal properties of natural human standing. AB - We analyzed the temporal evolution of the displacement of the center of pressure (COP) during prolonged unconstrained standing (30 min) in non-impaired human subjects. The COP represents the collective outcome of the postural control system and the force of gravity and is the main parameter used in studies on postural control. Our analysis showed that the COP displacement during human standing displays fractal properties that were quantified by the Hurst exponent obtained from the classical rescaled adjusted range analysis. The average fractal or Hurst exponent (H) was 0.35+/-0.06. The presence of long-range correlations from a few seconds to several minutes due to the fractal characteristics of the postural control system has several important implications for the analysis of human balance. PMID- 10754216 TI - The human astrocytoma cell line U373MG produces monocyte chemotactic protein (MCP)-1 upon stimulation with beta-amyloid protein. AB - Astrocytes associated with beta-amyloid (Abeta) accumulate in senile plaques of Alzheimer's disease (AD). To investigate the biological effects of Abeta/astrocyte interaction, we examined chemokine production by the human astrocytoma cell line U373MG stimulated with Abeta peptides. Northern blot analysis and specific immunoassays demonstrate that Abeta [1-42] and Abeta [25 35] induce mRNA expression and release of monocyte chemotactic protein (MCP)-1 but not of gamma-interferon inducible protein (IP)-10 by U373MG cells. The observation that Abeta induces astrocyte production of the potent microglia chemoattractant MCP-1 contributes to understanding mechanism of damage exerted by Abeta in AD senile plaques. PMID- 10754217 TI - Distribution of heat shock protein 108 mRNA in the chicken central nervous system. AB - The constitutive expression of heat shock protein 108 (HSP108) mRNA is mapped in a normal chicken central nervous system using in situ hybridization technique. HSP108 mRNAs were found to be mainly localized in the small neuroglial cells of various regions of the brain, although some neuronal cells also showed positive signals. This tendency is observed to be more marked in the cerebellum; HSP108 signals were not found in the Purkinje cells, but in Bergmann glial cells and oligodendrocytes. Although neuronal cells in the deep cerebellar nuclei and the molecular layer showed occasional HSP108 signals, the expression pattern of HSP108 mRNA is different from homologous HSP90 that is mostly expressed in neurons, but rather similar to that of TfBP immunoreactivity, a new member of the HSP108 family. The constitutive neuroglial localization of HSP108 could suggest that HSP108 may play an important role in the normal metabolism of neuroglial cells in the chicken brain. PMID- 10754218 TI - Localisation of tachykinin NK1 and NK3 receptors in the human prefrontal and visual cortex. AB - The distribution of tachykinin NK(1) and NK(3) receptors in the prefrontal (Brodmann area 9) and visual cortex (Brodmann area 17) of formalin-fixed postmortem human brain tissue was studied by immunohistochemistry. NK(1)-like immunoreactivity (NK(1)-LI) was observed as a thin band at the cortical surface and dots of NK(1)-LI localised on small non-pyramidal cells and in the neuropil (layers I-III). NK(3)-LI was found in beaded fibres and cells with astrocyte-like morphology in the superficial cortical layers and white matter. Dots of NK(3)-LI were prominent in the neuropil and on pyramidal (layers III/V) and non-pyramidal (layers V/VI) cells. The NK(3)-LI was more abundant and widespread than the NK(1) LI. This is the first report of the distribution of the NK(1) receptor in the prefrontal and visual cortex of the human brain by immunohistochemistry. PMID- 10754219 TI - Differential activation of Fos-like immunoreactivity in the arcuate nucleus and amygdala after intracerebroventricular injection of sodium nitroprusside and N omega nitro-L-arginine in conscious and urethane-anesthetized lactating rats. AB - The effect of intracerebroventricular injection of a nitric oxide (NO) donor (sodium nitroprusside, SNP) or NO synthase inhibitor (N(omega) nitro-L-arginine, L-NA) on Fos-like immunoreactivity (FLI) in the arcuate nucleus (ARC) and the medial, cortical and central amygdaloid nuclei was examined in conscious and urethane-anesthetized lactating rats. In conscious dams, the number of FLI-cells in the medial and cortical amygdaloid nuclei were significantly larger after the injection of L-NA than after SNP injection. In anesthetized dams, the number of FLI-cells was decreased in the medial and cortical amygdaloid nuclei by L-NA injection, whereas the numbers were increased in the ARC and the medial amygdaloid nuclei after the injection of SNP. The nucleus-specific response to SNP or L-NA suggested possible involvement of NO in modulation of dynamics of neuronal activity within these nuclei. PMID- 10754220 TI - Extracellular toxicity of 6-hydroxydopamine on PC12 cells. AB - 6-hydroxydopamine (6-OHDA) is usually thought to cross cell membrane through dopamine uptake transporters, to inhibit mitochondrial respiration and to generate intracellular reactive oxygen species. In this study, we show that the anti-oxidants catalase, glutathione and N-acetyl-cysteine are able to reverse the toxic effects of 6-OHDA. These two latter compounds considerably slow down 6-OHDA oxidation in a cell free system suggesting a direct chemical interaction with the neurotoxin. Moreover, desipramine does not protect PC12 cells and 6-OHDA is also strongly toxic towards non-catecholaminergic C6 and NIH3T3 cells. These results thus suggest that 6-OHDA toxicity on PC12 cells mainly involves an extracellular process. PMID- 10754221 TI - Doxorubicin, an RNA synthesis inhibitor, prevents vasoconstriction and inhibits aberrant expression of endothelin-1 in the cerebral vasospasm model of the rat. AB - A vasoconstrictor peptide, endothelin-1 (ET-1), has been identified as one of the causative substances in cerebral vasospasm after subarachnoid hemorrhage. We investigated whether doxorubicin, an RNA synthesis inhibitor, effectively suppresses induction of ET-1 in the rat vasospasm model. Blood was injected around the right femoral artery and the left one was used as an internal control. Seven days later (day 7), diameters of the right femoral arteries narrowed to about 60% and this vasoconstriction was prevented by clinical dose (0.6 mg/kg) or one third of its dose of doxorubicin injected on day 1. Reverse transcriptase polymerase chain reaction analysis demonstrated that expression of ET-1 mRNA in the vasospastic artery was not detected in doxorubicin-treated rats. It is concluded that doxorubicin effectively inhibits aberrant expression of ET-1 in the vasospasm-destined artery in the rat. PMID- 10754222 TI - Individual differences in autonomic activity affects brainstem auditory frequency following response amplitude in humans. AB - Innervation of the cochlea by sympathetic fibers suggests that the autonomic nervous system (ANS) may influence auditory information processing. The brainstem frequency-following response (FFR) and spontaneous skin conductance activity (SCA) were measured while subjects discriminated between long (rare) and short (frequent) duration tones. When subjects were divided into three groups on the basis of SCA, those with low SCA variability had larger FFR amplitudes. These results agree with the only other study to report ANS effects on brainstem auditory evoked responses [28]. It is proposed that individual differences in autonomic response patterns may account for some of the amplitude variation reported in brainstem evoked potential studies. PMID- 10754223 TI - The difference in temporal distribution of c-Fos immunoreactive neurons between the medullary dorsal horn and the trigeminal subnucleus oralis in the rat following experimental tooth movement. AB - The difference in temporal distribution of c-Fos-immunoreactivity (Fos-IR) was assessed in the medullary dorsal horn (MDH) and in the dorsomedial part of the trigeminal subnucleus oralis (Vodm) following experimental tooth movement of the rat maxillary molars. The number of MDH c-Fos-immunoreactive neurons increased bilaterally at 2 h and decreased markedly by 12 h, and then increased again with a small peak at 48 h. In contrast, Vodm c-Fos expression was not up-regulated until 12 h, but increased in number after 24 h, which increase lasted until 72 h. These findings indicate that experimental tooth movement induced nociceptive c Fos response in a biphasic manner. Furthermore, the later response appeared after 24 h, and lasted for a few days, mainly manifested in the Vodm during experimental tooth movement. PMID- 10754224 TI - Binding and internalization of leptin by porcine choroid plexus cells in culture. AB - Leptin is an adipocyte derived hormone with profound behavioural and metabolic effects exerted by both central and peripheral sites of action. One of its targets in the central nervous system appears to be the epithelial cells of the choroid plexus where leptin receptor (OB-R) expression is particularly high. The most abundant receptor subtype at this site is OB-Ra which is truncated at its intracellular part and has been suggested to serve functions such as leptin transport or clearance. The choroid plexus may thus be a site where receptor mediated exchange of leptin between cerebrospinal fluid and blood takes place. The study here shows that porcine plexus epithelia preserve their ability of OB-R expression when grown in culture. In addition, our experiments suggest that leptin is rapidly internalized upon binding to these cells supporting the view of an OB-R mediated transport of leptin across the choroid plexus. PMID- 10754225 TI - Autoradiographic distribution of mu-, delta- and kappa 1-opioid stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate binding in human frontal cortex and cerebellum. AB - Opioid receptors are known to couple to G-proteins and to inhibit adenylyl cyclase. Receptor activation of G-proteins can be measured by agonist-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate (GTP gamma S-) binding in brain sections to localize neuroanatomically functional coupling of receptors to intracellular signal transduction mechanisms. In the present study the selective mu-, delta- and kappa 1-opioid agonists DAMGO ([D-Ala2,N-Me-Phe4, Gly-ol5] enkephalin), DPDPE ([D-Pen2,5]-enkephalin) and enadoline (CI-977) were used to stimulate [35S]GTP gamma S-binding in human brain sections of frontal cortex and cerebellum. In human frontal cortex mu- and delta- opioid stimulated [35S]GTP gamma S-binding was evenly distributed throughout the gray matter, while kappa(1) opioid stimulated [35S]GTP gamma S-binding was detected predominantly in lamina V and VI. In the cerebellar cortex stimulated [35S]GTP gamma S-binding revealed functional coupling of mu- and kappa 1-opioid receptors in the molecular layer. PMID- 10754226 TI - The pathogenic L392V mutation of presenilin 1 decreases the affinity to glycogen synthase kinase-3 beta. AB - Determination of the effects of presenilin 1 (PSEN1) mutations, involved in autosomal dominant early-onset Alzheimer's disease (ADEOAD), on the interaction between PSEN1 and binding proteins is essential to determine which interactions are involved in Alzheimer's disease (AD) pathogenesis. The PSEN1 binding protein glycogen synthase kinase-3 beta (GSK-3 beta) has been considered as a key protein in AD pathogenesis since GSK-3 beta phosphorylates tau and hyperphosphorylated tau is a main component of neurofibrillary tangles associated to AD. We show here, using surface plasmonic resonance, that the pathogenic L392V mutation, identified in a large French ADEOAD pedigree including 39 affected members, leads to a decreased affinity to GSK-3 beta. We conclude therefore that the increase of affinity of PSEN1 to GSK-3 beta reported in previous studies is not a common effect of pathogenic mutations associated to ADEOAD. PMID- 10754227 TI - Expression of prostaglandin E synthase mRNA is induced in beta-amyloid treated rat astrocytes. AB - Deposition of beta-amyloid (A beta) in the brain is considered to be one of the most critical events in the onset of Alzheimer's disease (AD). In order to identify factors involved in the exacerbation of AD, we investigated transcriptionally A beta-induced genes using a cDNA subtraction technique in rat astrocytes. One gene obtained was rat prostaglandin (PG) E synthase. In this report, we present the deduced sequence for rat PGE synthase for the first time and demonstrate the induction of PGE synthase mRNA by treatment of cells with A beta. Our results suggest a possibly significant role of this enzyme in the progression of AD. PMID- 10754228 TI - Enantio-selective occurrence of (S)-tetrahydropapaveroline in human brain. AB - Tetrahydropapaveroline is an endogenous complex alkaloid derived from dopamine through the oxidation by monoamine oxidase. This alkaloid is considered to be involved in the pathogenesis of alcoholism and to act as a false neurotransmitter. Recently the (S) enantiomer was proposed to be a precursor of morphine biosynthesis in the opium poppy. In this paper stereo-chemical characteristic of tetrahydropapaveroline in human brains was examined. In all four control human brains examined, only the (S)-tetrahydropapaveroline was detected. The concentrations were 0.12-0.22 pmol/g wet weight of brain tissue, and the presence of alcohol in blood did not affect the concentration. The results suggest that (S)-tetrahydropapaveroline may be enantio-selectively synthesized in human brain and it may be an intermediate of the de novo synthesis of morphine analogues. PMID- 10754229 TI - Decrease of adenosine A-1 receptor gene expression in cerebral cortex of aged rats. AB - Changes of adenosine A-1 receptor (A1-AR) gene expression in aging were investigated in cerebral cortex using the rat aged from 2 months (adult) to 24 months (aged). Quantification of A1-AR protein level by immunoblotting analysis showed an age-related decrease of A1-AR in cerebral cortex of Wistar rats. Compared to the preparations from 2-month-old animals, the levels of A1-AR in the 6-, 12-, and 24-month-old rats were reduced by 14.3+/-5.2, 32.5+/-4.5 and 28.2+/ 5.7%, respectively. Similar decrease of mRNA level in A1-AR was also obtained using Northern blotting analysis. Two representative spots of mRNA, a 3.4-kb transcript and a 5.6-kb transcript, were observed in X-ray film from cerebral cortex of rat hybridized with rat A1-AR cDNA probe. Compared to the 2 month-old rats, levels of the 5.6-kb transcript were decreased by 17.9+/-2.5, 27.4+/-3.2 and 23.1+/-2.1% in the 6-, 12- and 24-month-old rats, respectively. These results indicated an age-related decrease of A1-AR in cerebral cortex of the rat that seems responsible for the change of response to adenosine. PMID- 10754230 TI - Intracerebral injection of autologous whole blood in rats: time course of inflammation and cell death. AB - Intracerebral hemorrhage is associated with stroke and head trauma. The purpose of this study was to study brain inflammation and cell death in adult rats 1 h to 4 weeks after injection of blood into the striatum. Terminal dUTP nick-end labeling positive dying cells were evident 4 h to 4 weeks post-hemorrhage. Neutrophil infiltration was brief and peaked at 48 h. CD8a immunoreactive lymphocytes, possibly natural killer cells, became apparent at 48 h and persisted for 1 week. Microglial reaction was evident at 4 h and persisted for 4 weeks. We conclude that extravascular blood causes a mixed inflammatory cell reaction in brains that is maximal from 48-72 h following hemorrhage. This is associated with death of brain cells over a prolonged period of at least 4 weeks. PMID- 10754231 TI - Early kynurenergic impairment in Huntington's disease and in a transgenic animal model. AB - Several neuroactive metabolites of the kynurenine pathway of tryptophan degradation have been speculatively linked to the pathophysiology of Huntington's Disease (HD). Here we demonstrate that the levels of two of these metabolites, the free radical generator 3-hydroxykynurenine (3HK) and the neuroprotectant kynurenate (KYNA), are increased in the neostriatum of stage 1 HD patients and in the brain of mice transgenic for full-length mutant huntingtin. In both cases, the elevation in 3HK was far more pronounced, resulting in significant increases in the 3HK/KYNA ratios. These data suggest that abnormal kynurenine pathway metabolism may play a role during the early phases of the neurodegenerative process in HD. PMID- 10754232 TI - Invasion of a multitude of genetic niches by mobile endonuclease genes. AB - Persistence of a mobile DNA element in a population reflects a balance between the ability of the host to eliminate the element and the ability of the element to survive and to disseminate to other individuals. In each of the three biological kingdoms, several families of a mobile DNA element have been identified which encode a single protein that acts on nucleic acids. Collectively termed homing endonuclease genes (HEGs), these elements employ varied strategies to ensure their survival. Some members of the HEG families have a minimal impact on host fitness because they associate with genes having self-splicing introns or inteins that remove the HEGs at the RNA or protein level. The HEG and the intron/intein gene spread throughout the population by a gene conversion process initiated by the HEG-encoded endonuclease called 'homing' in which the HEG and intron/intein genes are copied to cognate alleles that lack them. The endonuclease activity also contributes to a high frequency of lateral transmission of HEGs between species as has been documented in plants and other systems. Other HEGs have positive selection value because the proteins have evolved activities that benefit their host organisms. The success of HEGs in colonizing diverse genetic niches results from the flexibility of the encoded endonucleases in adopting new specificities. PMID- 10754233 TI - Adhesion to cellulose by Ruminococcus albus: a combination of cellulosomes and Pil-proteins? AB - An obligatory step in cellulose degradation by anaerobic bacteria is the adhesion of the bacterium to the polysaccharide. In many anaerobic bacteria the adhesion protein, and the enzymes required for extensive polysaccharide hydrolysis, are organized into a complex and interesting structure called the cellulosome. The Gram-positive anaerobe Ruminococcus albus also produces a cellulosome-like complex, but the bacterium appears to possess other mechanism(s) for adhesion to plant surfaces and genes encoding functions relevant to growth on cellulose are conditionally expressed, as suggested by a combination of functional proteomics, differential display reverse-transcriptase PCR, and mutational analysis. A novel form of cellulose-binding protein has been identified and shown to belong to the Pil-protein family, being most similar to the type 4 fimbrial proteins of Gram negative, pathogenic bacteria. These studies have provided new insights into the adhesion of bacteria to plant surfaces, and call attention to the likely existence of genetically analogous adhesion determinants in both pathogenic and non-pathogenic bacteria. PMID- 10754234 TI - Homocysteine thiolactone is a positive effector of sigma(S) levels in Escherichia coli. AB - sigma(S) is a regulator of the stationary phase response in Escherichia coli. Multi-copy suppressors were sought in a strain with decreased levels of sigma(S) and one such suppressor was found to encode HsrA, a putative efflux pump. Multi copy expression of hsrA was shown to lead to accumulation of homocysteine, which is predicted to cause an increase in homocysteine thiolactone (HCTL) levels. A direct correlation between HCTL levels and sigma(S) accumulation was observed both in mutants and during normal cell growth, leading to the hypothesis that HCTL is a physiologically relevant positive effector of sigma(S) levels in vivo. PMID- 10754236 TI - Identification of Trichoderma strains from building materials by ITS1 ribotyping, UP-PCR fingerprinting and UP-PCR cross hybridization. AB - To study the role of Trichoderma in sick building syndrome, it is essential to be able to accurately identify species. Forty-four strains of Trichoderma spp. isolated from Danish buildings damaged by water leaks were identified using ITS1 ribotyping and universally primed PCR, UP-PCR. Ribotyping allowed the assignment of the strains into three distinct groups. High similarity of UP-PCR banding profiles of the strains allowed species designation for almost all strains (43 out of 44) when compared with the UP-PCR banding profiles obtained from reference strains of T. atroviride, T. citrinoviride, T. harzianum, T. longibrachiatum and T. viride. However, cross hybridization of UP-PCR products showed that the latter strain had high DNA homology to the ex-type strain of T. hamatum. The combined approach is a convenient way for reliable identification of Trichoderma strains. PMID- 10754235 TI - A new mutation in spo0A with intragenic suppressors in the effector domain. AB - Spo0A is a two domain response regulator, a key protein in the initiation of sporulation of Bacillus subtilis. This protein controls a number of changes in gene expression that occur during the transition from stationary phase to the onset of sporulation. The phosphorylated form of Spo0A influences the transcription of a specific set of genes. In addition to others, it represses abrB and activates spoIIA and spoIIE transcription. Although the N-terminal phosphoacceptor domain is well characterised, there is limited information on the C-terminal, DNA-binding domain. Comparisons of Spo0A homologues from a number of Bacillus and Clostridium species show that the C-terminal domain contains three highly conserved regions. In this study, we have investigated the influence of spo0A mutations mapping within the C-terminal domain on transcription from the abrB, spoIIA and spoIIE promoters using lacZ fusions. Our results indicate that described mutations can be part of signalling between N- and C-terminal domains of the protein. Also, the increased expression observed from the spoIIE promoter in some Spo0A mutants might result from a stabilising function of these mutations on the transcriptional apparatus utilising sigma(A). PMID- 10754237 TI - Restoration of effectiveness of beta-lactams on methicillin-resistant Staphylococcus aureus by tellimagrandin I from rose red. AB - We found that extract from petals of Rosa canina L. (rose red) strikingly reduced the minimum inhibitory concentration of beta-lactams in methicillin-resistant Staphylococcus aureus. We isolated two compounds that reduced the minimum inhibitory concentrations of beta-lactams from the extract, tellimagrandin I and rugosin B. Tellimagrandin I was very effective regarding the reduction of the minimum inhibitory concentration, and rugosin B showed some effect. Tellimagrandin I showed a weak bactericidal action when added together with oxacillin. Judging from the fractional inhibitory concentration index, the effect of tellimagrandin I and oxacillin was synergistic. Tellimagrandin I also significantly reduced the minimum inhibitory concentration of tetracycline in some strains of methicillin-resistant Staphylococcus aureus. PMID- 10754238 TI - Cloning and characterization of two hydrophobin genes differentially expressed during fruit body development in Lentinula edodes. AB - Hydrophobins play important roles in morphogenesis and pathogenesis in fungi and fruit development in mushrooms. Two genes encoding hydrophobins (Le.hyd1 and Le.hyd2) were isolated during sequencing of random clones from a primordial cDNA library of Lentinula edodes. The nucleotide sequences of these two genes were determined. These two genes are 760 and 738 bp in length and the deduced amino acid sequences are homologous to various fungal hydrophobins with characteristic cysteine spacing. These hydrophobin genes are Class I hydrophobins judging by their conserved domains and hydropathy patterns. The transcript level of Le.hyd1 is high in primordium and that of Le.hyd2 is high in dikaryotic mycelial tissues. Poor expression of these two genes in monokaryotic parents indicates that these two genes are under mating-type regulation. We thus suggest that differential expression of these two L. edodes hydrophobins during fruit development may contribute to their distinct roles in fruiting of this mushroom. PMID- 10754239 TI - Identification of a 49-kDa hydrophobic cell wall mannoprotein present in velum yeast which may be implicated in velum formation. AB - Analysis of velum-forming yeast cell wall components released by beta-1,3 glucanase treatment were compared with those of a non velum-forming yeast. SDS PAGE electrophoresis and Western blotting with ConA-peroxidase staining of mannoproteins allowed us to identify a 49-kDa mannoprotein present in the cell wall of the velum-forming yeast and hardly visible in the control. The cell wall nature of this protein was confirmed by labelling with the non-permeable sulfosuccinimydiyl-6-(biotinamido)hexanoate reagent. A partial purification of this mannoprotein by anion exchange HPLC followed by surface hydrophobicity determination revealed that the fraction containing the 49-kDa mannoprotein was the most hydrophobic. Since cell surface hydrophobicity plays an important role in aggregate formation, it is likely that this mannoprotein is involved in velum formation. PMID- 10754240 TI - Effect of Mg(2+) ion in protein secretion by magnesium-resistant strains of Pseudomonas aeruginosa and Vibrio parahaemolyticus isolated from the coastal water of Haldia port. AB - A rapidly growing industrial complex including oil refineries and chemical industries has developed around the coastal area of Haldia port in the district of Midnapore, West Bengal, India. The coastal water is highly polluted with industrial wastes along with petroleum hydrocarbons. The bacteria isolated from the different sites of the coastal waters were Escherichia coli, Alcaligenes, Acinetobacter, Klebsiella spp., Micrococcus spp., Vibrio spp., Pseudomonas aeruginosa and Vibrio parahaemolyticus. The salinity of the water during the time of collection of samples around the port area was 8. 2 ppt. Among the isolated organisms, only two isolates, P. aeruginosa and V. parahaemolyticus, showed growth at 300 mM Mg(2+) ion concentration. However, a 3 mM Mg(2+) concentration was detected in the coastal water whereas other metal ion concentrations were less than 3x10(-5) mM. Resistance to Mg(2+) (300 mM) was determined by a 5.5-kb plasmid. A large amount of a 40-kDa outer membrane protein, which was highly soluble in 1 M MgCl(2), was isolated from both V. parahaemolyticus and P. aeruginosa. The secretion of proteins in the culture supernatant of V. parahaemolyticus was highly increased when the cells were grown in the presence of 300 mM Mg(2+), whereas very low secretion was observed in the same concentration of Mg(2+) in the case of P. aeruginosa. Mg(2+) may act as a specific release factor in protein secretion by V. parahaemolyticus strains. PMID- 10754241 TI - Identification and nucleotide sequence of genes involved in the synthesis of lactocin 705, a two-peptide bacteriocin from Lactobacillus casei CRL 705. AB - The structural gene determinants of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705, have been amplified from a plasmid of approximately 35 kb and sequenced. Lactocin 705 is a class IIb bacteriocin, whose activity depends upon the complementation of two peptides (705alpha and 705beta) of 33 amino acid residues each. These peptides are synthesized as precursors with signal sequences of the double-glycine type, which exhibited high identities with the leader peptides of plantaricin S and J from Lactobacillus plantarum, brochocin C from Brochotrix campestris, sakacin P from Lactobacillus sake, and the competence stimulating peptides from Streptococcus gordonii and Streptococcus mitis. However, the two mature bacteriocins 705alpha and 705beta do not show significant similarity to other sequences in the databases. PMID- 10754242 TI - The polyester polyurethanase gene (pueA) from Pseudomonas chlororaphis encodes a lipase. AB - A gene (pueA, polyurethane esterase A) encoding an extracellular polyurethanase (PueA) was cloned from Pseudomonas chlororaphis into Escherichia coli. The enzyme secreted from E. coli showed esterase activity when assayed with p-nitrophenyl acetate. Subcloning of a 3. 2-kb SalI-EcoRI fragment into a T7 RNA polymerase expression vector (pT7-6) produced a (35)S-labeled protein of 65 kDa. Nucleotide sequencing of pueA showed an open reading frame encoding a 65-kDa protein of 617 amino acid residues, with the serine hydrolase consensus sequence GXSXG. PueA was over-expressed using the pT7-6 vector transformed into E. coli BL21(DE3) and was purified in one step using Sephadex G-75. PMID- 10754243 TI - Multilocus enzyme analysis in aerobic and anaerobic bacteria using gel electrophoresis-nitrocellulose blotting. AB - An optimized multilocus enzyme electrophoresis method, which involves polyacrylamide-agarose gel electrophoresis followed by electrophoretic transfers on nitrocellulose sheets, was developed for the analysis of enzyme polymorphism in several aerobic and anaerobic bacterial species including Staphylococcus aureus, Streptococcus pneumoniae, S. agalactiae, Klebsiella pneumoniae and K. oxytoca, Clostridium bifermentans and C. sordellii, and Prevotella bivia. Serial electrophoretic transfers (during 5-15 min each) from a single polyacrylamide gel could be achieved for most enzymes studied, and allowed an increased definition of enzyme bands on nitrocellulose as compared to migration gels. Four enzymes, which could not be blotted in such conditions, could still be stained in gels after blotting. Thus, the method allowed the combined analysis of several enzymes after a single gel electrophoresis separation. The analysis of enzyme polymorphism in the various species studied raised the interest of polymorphic loci such as esterase or glutamic-oxaloacetic transaminase for epidemiologic studies. The method characterized a genetic diversity of enzyme loci of S. pneumoniae higher than previously reported, and is thus convenient for the analysis of genetic relationships between related isolates. Since the present method reduces the tediousness of multilocus enzyme electrophoresis and requires experimental conditions that are not specific for the bacterial population studied, it may be proposed for rapid population genetics analysis of a wide variety of bacteria. PMID- 10754244 TI - Inactivation of the flagellin gene of Salmonella enterica serotype enteritidis strongly reduces invasion into differentiated Caco-2 cells. AB - A nonflagellated mutant of Salmonella enterica serotype Enteritidis was constructed by disrupting the flagellin gene (fliC). Northern blot analysis indicated that the mutation did not affect expression of the downstream fliU gene. Infection experiments with differentiated Caco-2 cells revealed that the mutant was about 50-fold less invasive than the wild-type strain, while bacterial adherence was unaffected. Complementation of the mutant with an intact fliC copy restored flagella formation and efficient bacterial invasion. Our data demonstrate that the fliC gene of S. enterica serotype Enteritidis is essential for the invasion of Caco-2 cells. PMID- 10754245 TI - Analysis of Leptospira isolates from mainland Portugal and the Azores islands. AB - From 228 recent Leptospira isolates from mainland Portugal and Azorean wild mammals, 149 were characterized at the serovar level by monoclonal antibodies (MAbs), a quick serological method in epidemiological studies. In order to compare this antigenic information with that from new genetic techniques, a sample of isolates was analyzed through pulsed-field agarose gel electrophoresis (PFGE) (n=71), mapped restriction site polymorphisms (MRSPs) in PCR-amplified rRNA genes (n=45, including 13 saprophytes) and arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting (n=32). MRSP and AP-PCR lead to species identification of the studied 32 pathogenic isolates: Leptospira interrogans (n=3), Leptospira kirschneri (n=8) and Leptospira borgpetersenii (n=21). MAbs and PFGE characterized pathogenic isolates at the serovar level and resulted mainly in agreement (64%) although many discrepancies (35%) were observed. PMID- 10754246 TI - The PCR amplification of non-tuberculous mycobacterial 16S rRNA sequences from soil. AB - Non-tuberculous mycobacteria are free living saprophytic organisms commonly found in soil and water. Some are major causes of opportunistic infection, particularly in immuno-compromised patients, and may influence the efficacy of bacille Calmette-Guerin vaccinations. Many of these organisms are not amenable to culture, so information about their distribution is limited. PCR primers designed to amplify part of the mycobacterial 16S rRNA gene were applied to DNA extracted from cultured organisms and soil. The PCR products from soil contained sequences with similarity to slow growing mycobacteria similar to Mycobacterium lentiflavum, and to fast growing mycobacteria such as the xenobiotic degraders PYR-I and RJGII. PMID- 10754249 TI - Characterization of a membrane-bound NADH-dependent Fe(3+) reductase from the dissimilatory Fe(3+)-reducing bacterium Geobacter sulfurreducens. AB - Geobacter sulfurreducens produces a single, membrane-associated Fe(3+) reductase activity when grown on fumarate or Fe(3+). The activity was initially isolated by solubilization of membranes with the non-ionic detergent dodecyl-beta-D maltoside, and partially purified by a combination of ion exchange chromatography and preparative non-denaturing gel electrophoresis. Molecular mass of the reductase, as determined by gel filtration chromatography, was approximately 300 kDa. Cofactor analysis of the purified reductase demonstrates that it contains a hemoprotein and flavin adenine dinucleotide. Kinetic and inhibitor studies show that the reductase is specific for NADH as electron donor, and confirm that the reductase enzymatically reduces Fe(3+). The cytochrome associated with the complex undergoes a reoxidation upon addition of Fe(3+) compounds, indicating an ability to pass reducing equivalents to Fe(3+). This is the first description of a purified NADH-dependent Fe(3+) reductase from a microorganism capable of coupling Fe(3+) reduction to growth. PMID- 10754248 TI - A Brevibacterium lactofermentum 16S rRNA gene used as target site for homologous recombination. AB - Genes for rRNA are highly conserved and present in multiple copies in most prokaryotic organisms increasing the number of theoretical sites for homologous recombination. They might be targets for integration events between unrelated microorganisms providing that an efficient genetic transfer is present. We have used a plasmid containing a portion of the 16S rRNA gene from the rrnD operon of Brevibacterium lactofermentum to transform the same strain resulting in non essential inactivation of various rrn operons. Integration of the transforming DNA occurs in all cases. The system may be used to test possible gene transfer at least among closely related strains and is of great interest for integration of foreign DNA and for mapping. PMID- 10754247 TI - Comparison of serum antibody titers to Helicobacter pylori lipopolysaccharides, CagA, VacA and partially purified cellular extracts in a Japanese population. AB - We examined the levels of antibody titers against Helicobacter pylori antigens, three types of lipopolysaccharides (LPSs), recombinant CagA antigen, recombinant VacA antigen and partially purified cellular antigens in the sera of Japanese volunteers. The three types of LPSs are LPS carrying the highly antigenic epitope, LPS carrying the weakly antigenic epitope and rough LPS, classified on the basis of antigenicity in humans. IgG titers against all H. pylori antigens tested were significantly different between gastroduodenal patients and healthy adults without H. pylori infection. IgG titers against LPS carrying the weakly antigenic epitope, rough LPS and VacA antigen, as well as IgA titers against the partially purified cellular extract were significantly higher in gastroduodenal patients than in H. pylori-positive healthy adults. However, IgG titers against LPS carrying the highly antigenic epitope, CagA antigen or the partially purified cellular extract showed no significant difference between patients and H. pylori positive healthy adults. The results indicated that increases in IgG titers against VacA antigen and the weakly antigenic and core epitopes of LPS, and in IgA titer against the partially purified cellular extract, were associated with disease state and may be useful in identifying active infection of H. pylori. PMID- 10754250 TI - Biochemical, electrophoretic and immunohistochemical aspects of malate dehydrogenase in truffles (Ascomycotina). AB - The malate dehydrogenase (MDH; EC 1.1.1.37; L-malate-NAD(+)-oxidoreductase) activities of truffles of the genus Tuber (Tuber melanosporum Vittad., Tuber brumale Vittad., Tuber aestivum Vittad., Tuber magnatum Pico, Tuber rufum Pico) have been characterized with regard to the K(m) and V(max) values in the direct and reverse reactions. The isoelectrofocusing has revealed bands showing pI values ranging from pH 5.85 to 7.8. The MDH of T. melanosporum has been partially purified by hydroxyapatite treatment, DEAE-cellulose and Sephadex G-75 columns. With the partially purified T. melanosporum MDH activity polyclonal anti-T. melanosporum MDH antibodies have been prepared and used to localize MDH in the mycorrhizae and ascocarps of T. melanosporum. These antibodies inhibit T. melanosporum MDH activity as well as that of T. magnatum but not that of rabbit liver; this supports the specificity of the MDH antibodies used to localize MDH in truffle tissues. PMID- 10754251 TI - The staphylokinase gene is located in the structural gene encoding N acetylmuramyl-L-alanine amidase in methicillin-resistant Staphylococcus aureus. AB - The nucleotide sequence of a 15600-bp DNA fragment containing the staphylokinase gene (sakNU3-1) of methicillin-resistant Staphylococcus aureus (MRSA) NU3-1 was determined. The sak gene was found within the ply gene encoding N-acetylmuramyl-L alanine amidase and thus the ply gene should be inactivated. In the flanking region of the sak gene, the tandem repeat sequences (GAAGTGTT and GAATGGTT) were present as possible junction points between the sak and ply genes. No sequences characteristic of the presence of an IS-like element were found. Upstream from the ply gene, the kdpA, kdpB and kdpC homologues were present. Downstream from the ply gene, the tagA, tagH and tagG homologues were present. The sak gene was inserted into the same position of ply in 5/6 of sak(+) MRSA isolates with different genotypes. In all of these sak(+) isolates, Sak was detected in the culture supernatant. PMID- 10754252 TI - A novel bacterium that utilizes monochlorobiphenyls and 4-chlorobenzoate as growth substrates. AB - A bacterial isolate, tentatively named SK-3, was isolated from tertiary lagoon sludge contaminated with polychlorinated biphenyls for over 25 years. SK-3 was able to grow on 2-chlorobiphenyl, 3-chlorobiphenyl, 4-chlorobiphenyl (4-CB), chloroacetate and 4-chlorobenzoic acid (4-CBA) as sole carbon and energy sources. During growth on 4-CB, a stoichiometric amount of chloride ion was produced and growth yields were comparable to those observed during growth on biphenyl. The production of 4-CBA as a metabolite was not observed. Protein yields during growth on 4-CB and biphenyl suggested that the presence of the chlorine did not impede use of carbons on both aromatic rings. Growth on 4-CBA also resulted in nearly stoichiometric production of chloride ion. Benzoate-grown SK-3 was also able to degrade several Aroclor 1242 congeners without the need for a primary substrate of previous growth on biphenyl. PMID- 10754253 TI - Characterization of the naphthalene-degrading bacterium, Rhodococcus opacus M213. AB - Bacterial strain M213 was isolated from a fuel oil-contaminated soil in Idaho, USA, by growth on naphthalene as a sole source of carbon, and was identified as Rhodococcus opacus M213 by 16S rDNA sequence analysis and growth on substrates characteristic of this species. M213 was screened for growth on a variety of aromatic hydrocarbons, and growth was observed only on simple 1 and 2 ring compounds. No growth or poor growth was observed with chlorinated aromatic compounds such as 2,4-dichlorophenol and chlorobenzoates. No growth was observed by M213 on salicylate, and M213 resting cells grown on naphthalene did not attack salicylate. In addition, no salicylate hydroxylase activity was detected in cell free lysates, suggesting a pathway for naphthalene catabolism that does not pass through salicylate. Enzyme assays indicated induction of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase on different substrates. Total DNA from M213 was screened for hybridization with a variety of genes encoding catechol dioxygenases, but hybridization was observed only with catA (encoding catechol 1,2-dioxygenase) from R. opacus 1CP and edoD (encoding catechol 2,3-dioxygenase) from Rhodococcus sp. I1. Plasmid analysis indicated the presence of two plasmids (pNUO1 and pNUO2). edoD hybridized to pNUO1, a very large (approximately 750 kb) linear plasmid. PMID- 10754254 TI - Escherichia coli O157:H7 induces attaching-effacing lesions in large intestinal mucosal explants from adult cattle. AB - Attaching-effacing (A/E) lesions following natural and experimental infection with Escherichia coli O157:H7 have been seen in neonatal and 3-4-month-old weanling but not older cattle. To test the hypothesis that the adult bovine large intestinal epithelium is resistant to the development of A/E lesions, colonic and rectal mucosal tissue explants from 18-month-old steers were inoculated with E. coli O157:H7 and examined. Epithelial cells of inoculated explants developed A/E lesions at the bacterial attachment sites, providing evidence that the large intestinal mucosal epithelium may be a site of infection that contributes to carriage of E. coli O157:H7 in adult cattle. PMID- 10754255 TI - Exopolysaccharide production by mucoid and non-mucoid strains of Burkholderia cepacia. AB - Thirteen strains of Burkholderia cepacia from various origins with mucoid and non mucoid phenotypes were assayed for exopolysaccharide (EPS) production. The EPS were characterized by glycosyl composition analysis and examination of the products resulting from lithium-ethylenediamine and Smith degradations. The results showed that all strains, including the non-mucoid strains, were able to produce EPS exhibiting the same structural features, i.e. presence of one rhamnosyl, three galactosyl, one mannosyl, one glucosyl and one glucuronosyl residues, suggesting that this EPS is representative of the B. cepacia species. PMID- 10754256 TI - Amino acid substitutions in the VanS sensor of the VanA-type vancomycin-resistant Enterococcus strains result in high-level vancomycin resistance and low-level teicoplanin resistance. AB - The vancomycin-resistant enterococci GV1, GV2 and GV3, which were isolated from droppings from broiler farms in Japan have been characterized as VanA-type VRE, which express high-level vancomycin resistance (256 or 512 microg ml(-1), MIC) and low-level teicoplanin resistance (1 or 2 microg ml(-1), MIC). The vancomycin resistances were encoded on plasmids. The vancomycin resistance conjugative plasmid pMG2 was isolated from the GV2 strain. The VanA determinant of pMG2 showed the same genetic organization as that of the VanA genes encoded on the representative transposon Tn1546, which comprises vanRSHAXYZ. The nucleotide sequences of all the genes, except the gene related to the vanS gene on Tn1546, were completely identical to the genes encoded on Tn1546. Three amino acid substitutions in the N-terminal region of the deduced VanS were detected in the nucleotide sequence of vanS encoded on pMG2. There were also three amino acid substitutions in the vanS gene of the GV1 and GV3 strains in the same positions as in the vanS gene of pMG2. Vancomycin induced the increased teicoplanin resistance in these strains. PMID- 10754257 TI - Interaction of the Neurospora crassa heat shock factor with the heat shock element during heat shock and different developmental stages. AB - The interaction of the heat shock factor (HSF) with the heat shock element (HSE) was determined by a non-radioactive electrophoretic mobility shift assay, in order to analyze HSF regulation in Neurospora crassa. HSF binds to HSE under normal, non-stress conditions and is thus constitutively trimerized. Upon heat shock, the HSF-HSE complex shows a retarded mobility. This was also observed in Saccharomyces cerevisiae, where this mobility shift was shown to be due to HSF phosphorylation [Sorger and Pelham (1988) Cell 54, 855-864]. In N. crassa, HSE dependent electrophoretic mobility shift is temperature- and time-dependent. Under normal growth conditions, the HSF is located in the cytoplasm as well as in the nucleus. In germinating conidia the HSF shows a retarded mobility typical for heat shock even at normal growth temperatures. No HSF-dependent mobility shift was detectable in aerial hyphae. PMID- 10754258 TI - Purification and partial characterization of Oenococcus oeni exoprotease. AB - The exoprotease from Oenococcus oeni produced in stress conditions was purified to homogeneity in two steps, a 14-fold increase of specific activity and a 44% recovery of proteinase activity. The molecular mass was estimated to be 33.1 kDa by gel filtration and 17 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These results suggest that the enzyme is a dimer consisting of two identical subunits. Optimal conditions for activity on grape juice were 25 degrees C and a pH of 4.5. Incubation at 70 degrees C, 15 min, destroyed proteolytic activity. The SDS-PAGE profile shows that the enzyme was able to degrade the grape juice proteins at a significantly high rate. The activity at low pH and pepstatin A inhibition indicate that this enzyme is an aspartic protease. The protease activity increases at acidic pH suggesting that it could be involved in the wine elaboration. PMID- 10754259 TI - Corrigendum to "I-ceuI fragment analysis of the shigella species: evidence for large-scale chromosome rearrangement in S. dysenteriae and S. flexneri". PMID- 10754260 TI - Oxidative stress during aging of stationary cultures of the yeast Saccharomyces cerevisiae. AB - Comparison of 5 d old stationary cultures of Saccharomyces cerevisiae and of cultures aged for 3 months revealed increased generation of reactive oxygen species assessed by 2', 7'-dichlorofluorescin oxidation, decreased activity of superoxide dismutase, decreased content of glutathione and increased protein carbonyl content during prolonged incubation of stationary yeast cultures. These results point to the occurrence of oxidative stress during aging of stationary cultures of the yeast. The magnitude of this stress was augmented in antioxidant deficient strains, devoid of superoxide dismutases and catalases, and of decreased glutathione content. PMID- 10754261 TI - Survival of hippocampal and cortical neurons in a mixture of MEM+ and B27 supplemented neurobasal medium. AB - Serum-free B-27 supplemented neurobasal (NB) and a 10% fetal bovine serum supplemented Eagle's minimum essential medium (MEM+) are used to culture rat embryonic hippocampal neurons for different purposes. Although NB medium leads to enhanced cell survival, it contains biological antioxidants and is not suitable for the study of free radical damage and oxidation in cultured neurons. MEM+ without additional antioxidants has been used widely in the study of free radical damage and oxidation, although it does not support optimum neuronal survival in culture. Serum in MEM+ leads to enhanced cell survival but also promotes glial cell proliferation. In this study, we used a new combination medium (NM-2) that consists of both NB and MEM+ for growing primary hippocampal and cortical neuronal cultures. NM-2 enhanced neuronal survival 78.9% for dissociated neurons at a density of 50 cells/mm(2) and 83.1% for 100 cells/mm(2), while decreasing glial cell proliferation to 2-3% and completely inhibiting oligodendrocytes. The NM-2 minimized the effectiveness of antioxidants in the medium to the neurotoxin 4-hydroxynonenal. It also decreased neuronal clumping and provided a more even distribution of neurons. Neurons survived for 4 weeks in NM-2 without changing the original medium. NM-2 provides a good environment for studies of free radical damage and oxidation of neurons. The combination incorporates the best of both NB and MEM+ that results in high neuron survival rate, low glial cell proliferation, reduced antioxidant level, and provides relatively pure cultures of hippocampal and cortical neurons. PMID- 10754262 TI - Pathways of phospholipid oxidation by HOCl in human LDL detected by LC-MS. AB - A wealth of evidence now indicates that low-density lipoprotein (LDL) must be modified to promote atherosclerosis, and that this may involve oxidants released by phagocytes. Many studies of oxidative damage in atherosclerosis previously have concentrated on damage by nonhalogenated oxidants, but HOCl is a highly toxic oxidant produced by myeloperoxidase in phagocytes, which is also likely to be important in the disease pathogenesis. Currently some controversy exists over the products resulting from reaction of HOCl with LDL lipids, in particular regarding whether predominantly chlorohydrins or lipid peroxides are formed. In this study LC-MS of phosphatidylcholines in human LDL treated either with HOCl or the myeloperoxidase system was used as a specific method to detect chlorohydrin and peroxide formation simultaneously, and with comparable sensitivity. Chlorohydrin products from lipids containing oleic, linoleic and arachidonic acids were detected, but no hydroperoxides of linoleoyl or arachidonoyl lipids could be observed. This study provides the first direct evidence that lipid chlorohydrins rather than peroxides are the major products of HOCl- or myeloperoxidase-treated LDL phospholipids. This in turn provides important information required for the study of oxidative damage in vivo which will allow the type and source of oxidants involved in the pathology of atherosclerosis to be investigated. PMID- 10754263 TI - The effect of age on cyclooxygenase-2 gene expression: NF-kappaB activation and IkappaBalpha degradation. AB - Increased oxidative stress resulting in the activation of NF-kappaB is thought to play a crucial role in the expression of the cyclooxygenase-2 (COX-2), which is the key enzyme in proinflammatory prostanoid synthesis. In the current study, we investigated whether the aging process affects the status of the redox-sensitive NF-kappaB in rat kidney, and how this age-related modulation is related to COX-2 gene expression and COX-derived reactive oxygen species (ROS). We found that the aging process strongly enhanced the activation of NF-kappaB and its DNA-binding activity with an increased ROS status. Accompanied with the change in the NF kappaB activity was a decreased IkappaBalpha as confirmed by the increased nuclear p65 protein. Thus, these data strongly indicated that the aging process increases NF-kappaB activity by downregulating IkappaBalpha. A closer examination further revealed that age-related oxidative status correlated with the increased COX-derived prostanoid biosynthetic process is mediated by the increased NF kappaB-regulated COX activity. This increase in NF-kappaB activity was accompanied by the increased COX-2 mRNA and protein levels. Based on these data, we concluded that the age-related increase in redox-sensitive NF-kappaB translocation and binding activities are associated with increased ROS, and further that this transactivation was modulated by the age-related decrease of IkappaBalpha. PMID- 10754264 TI - Protection by desferrioxamine and other hydroxamic acids against tetrachlorohydroquinone-induced cyto- and genotoxicity in human fibroblasts. AB - Tetrachlorohydroquinone (TCHQ) has been identified as a major toxic metabolite of the widely used wood preservative pentachlorophenol and has also been implicated in its genotoxicity. We have recently demonstrated that protection by the trihydroxamate iron chelator desferrioxamine (DFO) on TCHQ-induced single-strand breaks in isolated DNA was not the result of its chelation of iron but rather of its efficient scavenging of the reactive tetrachlorosemiquinone (TCSQ) radical. In this study, we extended our research from isolated DNA to human fibroblasts. We found that DFO provided marked protection against both the cyto- and genotoxicity induced by TCHQ in human fibroblasts when it was incubated simultaneously with TCHQ. Pretreatment of the cells with DFO followed by washing also provided marked protection, although less efficiently compared with the simultaneous treatment. Similar patterns of protection were also observed for three other hydroxamic acids (HAs): aceto-, benzo-, and salicylhydroxamic acid. Dimethyl sulfoxide, an efficient hydroxyl radical scavenger, provided only partial protection even at high concentrations. In vitro studies showed that the HAs tested effectively scavenged the reactive TCSQ radical and enhanced the formation of the less reactive and less toxic 2,5-dichloro-3, 6-dihydroxy-1,4 benzoquinone (chloranilic acid). The results of this study demonstrated that the protection provided by DFO and other HAs against TCHQ-induced cyto- and genotoxicity in human fibroblasts is mainly through scavenging of the observed reactive TCSQ radical and not through prevention of the Fenton reaction by the binding of iron in a redox-inactive form. PMID- 10754265 TI - Protein oxidation and degradation during proliferative senescence of human MRC-5 fibroblasts. AB - One of the highlights of age-related changes of cellular metabolism is the accumulation of oxidized proteins. The aging process on a cellular level can be treated either as the ongoing proliferation until a certain number of cell divisions is reached (the Hayflick limit) or as the aging of nondividing cells, that is, the age-related changes in cells without proliferation. The present investigation was undertaken to reveal the changes in protein turnover, proteasome activity, and protein oxidation status during proliferative senescence. We were able to demonstrate that the activity of the cytosolic proteasomal system declines dramatically during the proliferative senescence of human MRC-5 fibroblasts. Regardless of the loss in activity, it could be demonstrated that there are no changes in the transcription and translation of proteasomal subunits. This decline in proteasome activity was accompanied by an increased concentration of oxidized proteins. Cells at higher proliferation stages were no longer able to respond with increased degradation of endogenous [(35)S]-Met-radiolabeled proteins after hydrogen peroxide- or quinone-induced oxidative stress. It could be demonstrated that oxidized proteins in senescent human MRC-5 fibroblasts are not as quickly removed as they are in young cells. Therefore, our study demonstrates that the accumulation of oxidized proteins and decline in protein turnover and activity of the proteasomal system are not only a process of postmitotic aging but also occur during proliferative senescence and result in an increased half-life of oxidized proteins. PMID- 10754266 TI - A long-lived tyrosyl radical from the reaction between horse metmyoglobin and hydrogen peroxide. AB - The reaction between metmyoglobin (metMb) and hydrogen peroxide has been known since the 1950s to produce globin-centered free radicals. The direct electron spin resonance spectrum of a solution of horse metMb and hydrogen peroxide at room temperature consists of a multilined signal that decays in minutes at room temperature. Comparison of the direct ESR spectra obtained from the system under N(2)- and O(2)-saturated conditions demonstrates the presence of a peroxyl radical, identified by its g-value of 2.014. Computer simulations of the spectra recorded 3 s after the mixture of metMb and H(2)O(2) were calculated using hyperfine coupling constants of a(H2,6) = 1.3 G and a(H3,5) = 7.0 G for the ring and a(beta)(H1) = 16.7 G and a(beta)(H2) = 14.2 G for the methylene protons, and are consistent with a highly constrained, conformationally unstable tyrosyl radical. Spectra obtained at later time points contained a mixture of the 3 s signal and another signal that was insufficiently resolved for simulation. Efficient spin trapping with 3, 5-dibromo-4-nitrosobenzenesulfonic acid was observed only when the spin trap was present at the time of H(2)O(2) addition. Spin trapping experiments with either 5,5-dimethyl-1-pyrroline N-oxide (DMPO) or perdeuterated 2-methyl-2-nitrosopropane (MNP-d(9)), which have been shown to trap tyrosyl radicals, were nearly equally effective when the spin trap was added before or 10 min after the addition of H(2)O(2). The superhyperfine structure of the ESR spectra obtained from Pronase-treated MNP-d(9)/*metMb confirmed the assignment to a tyrosyl radical. Delayed spin trapping experiments with site directed mutant myoglobins in which either Tyr-103 or Tyr-146 was replaced by phenylalanine indicated that radical adduct formation with either DMPO or MNP d(9) requires the presence of Tyr-103 at all time points, implicating that residue as the radical site. PMID- 10754267 TI - Formation of isoprostane F(2)-like compounds (phytoprostanes F(1)) from alpha linolenic acid in plants. AB - Isoprostanes F(2) are biologically active prostaglandin F(2)-like compounds formed by free radical-catalyzed oxidation of arachidonic acid (C20:4). Here, we show that a series of dinor isoprostanes F(1), which we term phytoprostanes F(1) (PPF(1)s), are formed by nonenzymatic oxidation of linolenate (C18:3) in plants. Identification and quantification of PPF(1)s were achieved by a negative ion chemical ionization gas chromatography-mass spectrometry method using oxygen 18 labeled PPF(1)s as internal standards. PPF(1)s were found in leaves, flowers, and roots of taxonomically distinct plant species at concentrations ranging from 43 to 1380 ng/g of dry weight. In addition, esterified PPF(1)s were found at 10- to 150-fold higher concentrations. During the drying and storage of various plant organs, endogenous PPF(1) levels increased dramatically by 15- to 263-fold. Because the structurally related prostaglandin F(2alpha) and isoprostanes F(2) exert potent biological activities (i.e., broncho- and vasoconstriction) in the nanomolar range, PPF(1)s could potentially exert similar biological activities. Notably, fresh birch pollen, which can easily be inhaled, contains exceedingly high concentrations (32,440 ng/g) of free PPF(1)s. PMID- 10754268 TI - Oxidant-induced intestinal barrier disruption and its prevention by growth factors in a human colonic cell line: role of the microtubule cytoskeleton. AB - Reactive oxygen metabolites (ROM) are increased in the inflamed mucosa of inflammatory bowel disease (IBD) and may contribute to loss of intestinal barrier function in this disorder. Growth factors (GF) are protective. But the mechanisms of disruption and protection remain elusive. In the present investigation, we hypothesized that the microtubules (a critical cytoskeletal element) play a key role in the molecular mechanism of intestinal barrier dysfunction induced by ROM and in GF-mediated protection. Utilizing monolayers of a human colonic cell line (Caco-2), we evaluated the effects of ROM (H(2)O(2) or HOCl), in the presence or absence of GF (epidermal growth factor [EGF]; transforming growth factor-alpha [TGF-alpha]), on intestinal barrier function, tubulin (microtubule structural protein), and microtubule stability. Monolayers were also processed for two highly sensitive western immunoblots: fractionated polymerized tubulin (S2; an index of stability); monomeric tubulin (S1; an index of disruption) to detect the oxidation and disassembly/assembly of tubulin. ROM exposure led to a significant increase in the oxidation of tubulin, decrease in the stable S2 polymerized tubulin, and increase in the unstable S1 monomeric tubulin. In concert, each ROM in a dose dependent manner damaged the microtubule cytoskeleton and disrupted barrier function. GF pretreatment not only increased the S2 stable tubulin and decreased tubulin oxidation but also, concomitantly, prevented the disruption of microtubules and loss of barrier function in monolayers exposed to ROM. Antibody against the GF-receptor and inhibitors of GF-receptor tyrosine kinase abolished GF protection, indicating the involvement of epidermal growth factor receptor (EGFR) signaling pathway. As predicted, colchicine, an inhibitor of microtubule assembly, caused barrier dysfunction and prevented GF protection whereas taxol, a microtubule-stabilizing agent, mimicked the protective effects of GF. Thus, organization and stability of the microtubule cytoskeleton appears to be critical to both oxidant-induced mucosal barrier dysfunction and protection of intestinal barrier mediated by GF. Therefore, microtubules may be useful targets for development of drugs for the treatment of IBD. PMID- 10754269 TI - Electron-paramagnetic resonance spectroscopy using N-methyl-D-glucamine dithiocarbamate iron cannot discriminate between nitric oxide and nitroxyl: implications for the detection of reaction products for nitric oxide synthase. AB - Purified neuronal nitric oxide synthase (NOS) does not produce nitric oxide (NO) unless high concentrations of superoxide dismutase (SOD) are added, suggesting that nitroxyl (NO(-)) or a related molecule is the principal reaction product of NOS, which is SOD-dependently converted to NO. This hypothesis was questioned by experiments using electron paramagnetic resonance spectroscopy and iron N-methyl D-glucamine dithiocarbamate (Fe-MGD) as a trap for NO. Although NOS and the NO donor S-nitroso-N-acetyl-penicillamine produced an electron paramagnetic resonance signal, the NO(-) donor, Angeli's salt (AS) did not. AS is a labile compound that rapidly hydrolyzes to nitrite, and important positive control experiments showing that AS was intact were lacking. On reinvestigating this crucial experiment, we find identical MGD(2)-Fe-NO complexes both from S-nitroso N-acetyl-penicillamine and AS but not from nitrite. Moreover, the yield of MGD(2) Fe-NO complex from AS was stoichiometric even in the absence of SOD. Thus, MGD(2) Fe directly detects NO(-), and any conclusions drawn from MGD(2)-Fe-NO complexes with respect to the nature of the primary NOS product (NO, NO(-), or a related N oxide) are invalid. Thus, NOS may form NO(-) or related N-oxides instead of NO. PMID- 10754270 TI - Impairment with various antioxidants of the loss of mitochondrial transmembrane potential and of the cytosolic release of cytochrome c occuring during 7 ketocholesterol-induced apoptosis. AB - Previous investigations of our laboratory have shown that 7-ketocholesterol was a potent inducer of apoptosis involving a release of cytochrome c into the cytosol, and a lipid peroxidation process that could be the consequence of a production of radical oxygen species. According to these considerations, we asked whether some antioxidants were able to counteract 7-ketocholesterol-induced apoptosis, and whether prevention of cell death was associated with the impairment of mitochondrial events implied in the commitment to apoptosis, i.e., opening of the mitochondrial megachannels leading to the loss of the mitochondrial transmembrane potential (DeltaPsim), and release of cytochrome c from mitochondria into the cytosol. To this end, we studied the effects of glutathione (15 mM), N acetylcysteine (15 mM), vitamin E (100 microM), vitamin C (50 microM) and melatonin (1 mM) on U937 cells treated with 7-ketocholesterol (40 microg/ml). Only glutathione, N-acetylcysteine, and vitamin E prevented apoptosis measured by the occurrence of cells with condensed and/or fragmented nuclei, as well as the loss of DeltaPsim, and the release of cytochrome c. However, all the antioxidants used were potent inhibitors of the production of O(2)(*) occuring under treatment with 7-ketocholesterol. Collectively, our data demonstrate that impairment of apoptosis by glutathione, N-acetylcysteine, and vitamin E correlates with the prevention of mitochondrial dysfunctions, and they underline that the ability of antioxidants to counteract 7-ketocholesterol-induced apoptosis does not only depend on their capability to inhibit the production of O(2)(*). PMID- 10754272 TI - Evidence for a trypanothione-dependent peroxidase system in Trypanosoma cruzi. AB - Hydroperoxide metabolism in Crithidia fasciculata has recently been shown to be catalyzed by a cascade of three oxidoreductases comprising trypanothione reductase (TR), tryparedoxin (TXN1), and tryparedoxin peroxidase (TXNPx) (Nogoceke et al., Biol. Chem. 378, 827-836, 1997). The existence of this metabolic system in the human pathogen Trypanosoma cruzi is supported here by immunohistochemistry. Epimastigotes of T. cruzi display strong immunoreactivity with antibodies raised against TXN1 and TXNPx of C. fasciculata. In addition, a full-length open reading frame presumed to encode a peroxiredoxin-type protein in T. cruzi (Acc. Nr. AJ 012101) was heterologously expressed in Escherichia coli and shown to exhibit tryparedoxin peroxidase activity. With TXN, TXNPx, trypanothione and TR, T. cruzi possesses all components constituting the crithidial peroxidase system. It is concluded that the antioxidant defense of T. cruzi also depends on the NADPH-fuelled, trypanothione-mediated enzymatic hydroperoxide metabolism. PMID- 10754271 TI - Mitochondrial oxidative stress in mice lacking the glutathione peroxidase-1 gene. AB - Oxidative stress resulting from mitochondrially derived reactive oxygen species (ROS) has been hypothesized to damage mitochondrial oxidative phosphorylation (OXPHOS) and to be a factor in aging and degenerative disease. If this hypothesis is correct, then genetically inactivating potential mitochondrial antioxidant enzymes such as glutathione peroxidase-1 (Gpx1; EC 1.11.1.9) should increase mitochondrial ROS production and decrease OXPHOS function. To determine the expression pattern of Gpx1, isoform-specific antibodies were generated and mutant mice were prepared in which the Gpx1 protein was substituted for by beta galactosidase, driven by the Gpx1 promoter. These experiments revealed that Gpx1 is highly expressed in both the mitochondria and the cytosol of the liver and kidney, but poorly expressed in heart and muscle. To determine the physiological importance of Gpx1, mice lacking Gpx1 were generated by targeted mutagenesis in mouse ES cells. Homozygous mutant Gpx1(tm1Mgr) mice have 20% less body weight than normal animals and increased levels of lipid peroxides in the liver. Moreover, the liver mitochondria were found to release markedly increased hydrogen peroxide, a Gpx1 substrate, and have decreased mitochondrial respiratory control ratio and power output index. Hence, genetic inactivation of Gpx1 resulted in growth retardation, presumably due in part to reduced mitochondrial energy production as a product of increased oxidative stress. PMID- 10754273 TI - Hydrogen peroxide-induced gene expression in Arabidopsis thaliana. AB - Hydrogen peroxide (H(2)O(2)) is generated in plants after exposure to a variety of biotic and abiotic stresses, and has been shown to induce a number of cellular responses. Previously, we showed that H(2)O(2) generated during plant-elicitor interactions acts as a signaling molecule to induce the expression of defense genes and initiate programmed cell death in Arabidopsis thaliana suspension cultures. Here, we report for the first time the identification by RNA differential display of four genes whose expression is induced by H(2)O(2). These include genes that have sequence homology to previously identified Arabidopsis genes encoding a late embryogenesis-abundant protein, a DNA-damage repair protein, and a serine/threonine kinase. Their putative roles in H(2)O(2)-induced defense responses are discussed. PMID- 10754274 TI - DNA damage, repair, and antioxidant systems in brain regions: a correlative study. AB - 8-Hydroxy-2'-deoxyguanosine (oxo(8)dG) has been used as a marker of free radical damage to DNA and has been shown to accumulate during aging. Oxidative stress affects some brain regions more than others as demonstrated by regional differences in steady state oxo(8)dG levels in mouse brain. In our study, we have shown that regions such as the midbrain, caudate putamen, and hippocampus show high levels of oxo(8)dG in total DNA, although regions such as the cerebellum, cortex, and pons and medulla have lower levels. These regional differences in basal levels of DNA damage inversely correlate with the regional capacity to remove oxo(8)dG from DNA. Additionally, the activities of antioxidant enzymes (Cu/Zn superoxide dismutase, mitochondrial superoxide dismutase, and glutathione peroxidase) and the levels of the endogenous antioxidant glutathione are not predictors of the degree of free radical induced damage to DNA in different brain regions. Although each brain region has significant differences in antioxidant defenses, the capacity to excise the oxidized base from DNA seems to be the major determinant of the steady state levels of oxo(8)dG in each brain region. PMID- 10754275 TI - Modifications in heme iron of free and vesicle bound cytochrome c by tert-butyl hydroperoxide: a magnetic circular dichroism and electron paramagnetic resonance investigation. AB - To characterize changes to the heme and the influence of membrane lipids in the reaction of cytochrome c with peroxides, we studied the reaction of cytochrome c with tert-butyl hydroperoxide (tert-BuOOH) by magnetic circular dichroism (MCD) and direct electron paramagnetic resonance (EPR) in the presence and absence of different liposomes. Direct low-temperature (11 degrees K) EPR analysis of the cytochrome c heme iron on exposure to tert-BuOOH shows a gradual (180 s) conversion of the low-spin form to a high-spin Fe(III) species of rhombic symmetry (g = 4.3), with disappearance of a prior peroxyl radical signal (g(o) = 2.014). The conversion to high spin precedes Soret band bleaching, observable by UV/Vis spectroscopy and by magnetic circular dichroism (MCD) at room temperature, that indicates loss of iron coordination by the porphyrin ring. The presence of cardiolipin-containing liposomes delayed formation of the peroxyl radical and conversion to high-spin iron, while dicetylphosphate (DCP) liposomes accelerated these changes. Correspondingly, bleaching of cytochrome c by tert-BuOOH at room temperature was accelerated by several negatively charged liposome preparations, and inhibited by mitochondrial-mimetic phosphatidylcholinephosphatidylethanolaminecardiolipin (PCPECL) liposomes. Concomitant with bleaching, spin-trapping measurements with 5,5-dimethyl-1 pyroline-N-oxide showed that while the relative production of peroxyl, alkoxyl, and alkyl radicals was unaffected by DCP liposomes, PCPECL liposomes decreased the spin-trapped alkoxyl radical signal by 50%. The EPR results show that the primary initial change on exposure of cytochrome c to tert-BuOOH is a change to a high-spin Fe(III) species, and together with MCD measurements show that unsaturated cardiolipin-containing lipid membranes influence the interaction of tert-BuOOH with cytochrome c heme iron, to alter radical production and decrease damage to the cytochrome. PMID- 10754276 TI - Mechanism of oxidative DNA damage induced by carcinogenic allyl isothiocyanate. AB - Several isothiocyanates have been proposed as promising chemopreventive agents for human cancers. However, it has been reported that allyl isothiocyanate exhibit carcinogenic potential, and benzyl isothiocyanate and phenethyl isothiocyanate have tumor-promoting activities. We investigated whether these isothiocyanates could cause DNA damage, using (32)P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. Allyl isothiocyanate caused Cu(II)-mediated DNA damage and formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) more strongly than benzyl and phenethyl isothiocyanates. Catalase and bathocuproine, a Cu(I) specific chelator, inhibited Cu(II)-mediated DNA damage by these isothiocyanates, suggesting involvement of H(2)O(2) and Cu(I). Isothiocyanates induced DNA damage frequently at thymine and cytosine residues in the presence of Cu(II). A UV visible spectroscopic study revealed an association between the generation of superoxide and the yield of SH group from isothiocyanates. Furthermore, the yield of 8-oxodG formation was correlated with their superoxide-generating ability. Allyl isothiocyanate significantly induced 8-oxodG formation in HL-60 cells, but not in H(2)O(2)-resistant HP100 cells, suggesting the involvement of H(2)O(2) in cellular DNA damage. We conclude that oxidative DNA damage may play important roles in carcinogenic processes induced by allyl isothiocyanate. PMID- 10754277 TI - Evaluation of the postprandial effects of a fast-food meal on human plasma F(2) isoprostane levels. AB - Measurement of the F(2)-isoprostane, 8-epi-PGF(2alpha) is increasingly used as a sensitive and reliable marker of lipid peroxidation in vivo. Because the majority of 8-epi-PGF(2alpha) in plasma is associated with lipoproteins, it is possible that 8-epi-PGF(2alpha) derived from polyunsaturated fatty acid-rich food may become incorporated within these lipoproteins during synthesis and could contribute to the levels detected in plasma. In this study, we evaluated the postprandial effect of a single fast-food meal (McDonald's Big Mac meal, McDonald's Corp., London, England) on plasma total 8-epi-PGF(2alpha) in nine healthy subjects. Blood was collected before and 2 h postprandially. 8-Epi PGF(2alpha) was measured by immunoaffinity extraction and gas chromatography-mass spectrometry. Fasting plasma 8-epi-PGF(2alpha) (875 +/- 25 pM) increased postprandially (956 +/- 23 pM, p <.05), although no significant change was observed in the normalized concentrations (2. 78 +/- 0.1 vs. 2.95 +/- 0.3 nmol/mmol arachidonic acid). Plasma lipid hydroperoxides, fatty acids, vitamin E, total antioxidant status, cholesterol, and triglycerides were not altered. Plasma glucose increased postmeal (4.4 +/- 0.1 vs. 4.9 +/- 0.1 mM, p <.05). These results indicate that the overall contribution of this lipid-rich meal to plasma 8-epi-PGF(2alpha) and other lipid peroxidation markers was small. PMID- 10754278 TI - Study of vitamin E net mass transfer between alpha-tocopherol-enriched HDL and erythrocytes: application to asymptomatic hypercholesterolemic men. AB - We previously showed that hypercholesterolemic asymptomatic men had lower erythrocyte vitamin E content, despite normal plasma concentrations compared to normocholesterolemic men. We hypothesized that the reduced erythrocyte vitamin E concentration could be due to an impairment of transfer of vitamin E from plasma lipoproteins. We first developed a model for testing the ability of erythrocytes to accept vitamin E from high-density lipoproteins (HDL) pre-enriched in vitamin E, which allows to measure a net mass transfer of vitamin E from HDL to erythrocytes. Vitamin E-enriched HDL were obtained in controlled conditions of concentration and incubation time with a good reproducibility (CV 60% of the activity of these mutant Abs with low affinity for C1q. These data localize the C1q binding epicenter on human IgG1 and suggest that there are species-specific differences in the C1q binding site of Igs. PMID- 10754314 TI - Haemophilus influenzae stimulates ICAM-1 expression on respiratory epithelial cells. AB - Epithelial cells interact directly with bacteria in the environment and play a critical role in airway defense against microbial pathogens. In this study, we examined the response of respiratory epithelial cells to infection with nontypable Haemophilus influenzae. Using an in vitro cell culture model, we found that epithelial cell monolayers released significant quantities of IL-8 and expressed increased levels of ICAM-1 mRNA and surface protein in response to H. influenzae. In contrast, levels of IL-1beta, TNF-alpha, and MHC class I were not significantly affected, suggesting preferential activation of a specific subset of epithelial genes directed toward defense against bacteria. Induction of ICAM-1 required direct bacterial interaction with the epithelial cell surface and was not reproduced by purified H. influenzae lipooligosaccharide. Consistent with a functional role for this response, induction of ICAM-1 by H. influenzae mediated increased neutrophil adherence to the epithelial cell surface. Furthermore, in an in vivo murine model of airway infection with H. influenzae, increased epithelial cell ICAM-1 expression coincided with increased chemokine levels and neutrophil recruitment in the airway. These results indicate that ICAM-1 expression on human respiratory epithelial cells is induced by epithelial cell interaction with H. influenzae and suggest that an ICAM-1-dependent mechanism can mediate neutrophil adherence to these cells independent of inflammatory mediator release by other cell types. Direct induction of specific epithelial cell genes (such as ICAM-1 and IL-8) by bacterial infection may allow for rapid and efficient innate defense in the airway. PMID- 10754315 TI - IL-9 protects mice from Gram-negative bacterial shock: suppression of TNF-alpha, IL-12, and IFN-gamma, and induction of IL-10. AB - IL-9 is a T cell-derived cytokine that, similar to the Th2 cytokines IL-4 and IL 10, has been implicated in the response to parasitic infections, allergy, and inflammatory processes. Because both IL-4 and IL-10 can confer protection to mice from septic shock, we investigated whether IL-9 may also be capable of conferring resistance on recipients of an otherwise lethal challenge with Pseudomonas aeruginosa. Prophylactic injections of rIL-9 appeared to be most effective in preventing the onset of a lethal shock, according to a pattern that was both dose dependent and time dependent. The protective effect of IL-9 was correlated with marked decreases in the production of the inflammatory mediators TNF-alpha, IL 12, and IFN-gamma, as well as the induction of the anti-inflammatory cytokine IL 10. Sustained levels of IL-9-specific transcripts could be detected in the spleens of mice recovering from sublethal P. aeruginosa infection. Therefore, IL 9 may be protective in septic shock via a rather unique mechanism involving a complex modulation of inflammatory and anti-inflammatory mediators. PMID- 10754316 TI - Dendritic cells infected with a vaccinia vector carrying the human gp100 gene simultaneously present multiple specificities and elicit high-affinity T cells reactive to multiple epitopes and restricted by HLA-A2 and -A3. AB - To investigate the ability of human dendritic cells (DC) to process and present multiple epitopes from the gp100 melanoma tumor-associated Ags (TAA), DC from melanoma patients expressing HLA-A2 and HLA-A3 were pulsed with gp100-derived peptides G9154, G9209, or G9280 or were infected with a vaccinia vector (Vac Pmel/gp100) containing the gene for gp100 and used to elicit CTL from autologous PBL. CTL were also generated after stimulation of PBL with autologous tumor. CTL induced with autologous tumor stimulation demonstrated HLA-A2-restricted, gp100 specific lysis of autologous and allogeneic tumors and no lysis of HLA-A3 expressing, gp100+ target cells. CTL generated by G9154, G9209, or G9280 peptide pulsed, DC-lysed, HLA-A2-matched EBV transformed B cells pulsed with the corresponding peptide. CTL generated by Vac-Pmel/gp100-infected DC (DC/Pmel) lysed HLA-A2- or HLA-A3-matched B cell lines pulsed with the HLA-A2-restricted G9154, G9209, or G9280 or with the HLA-A3-restricted G917 peptide derived from gp100. Furthermore, these DC/Pmel-induced CTL demonstrated potent cytotoxicity against allogeneic HLA-A2- or HLA-A3-matched gp100+ melanoma cells and autologous tumor. We conclude that DC-expressing TAA present multiple gp100 epitopes in the context of multiple HLA class I-restricting alleles and elicit CTL that recognize multiple gp100-derived peptides in the context of multiple HLA class I alleles. The data suggest that for tumor immunotherapy, genetically modified DC that express an entire TAA may present the full array of possible CTL epitopes in the context of all possible HLA alleles and may be superior to DC pulsed with limited numbers of defined peptides. PMID- 10754317 TI - Suppression of endogenous IL-10 gene expression in dendritic cells enhances antigen presentation for specific Th1 induction: potential for cellular vaccine development. AB - A new paradigm for designing vaccines against certain microbial pathogens, including Chlamydia trachomatis, is based on the induction of local mucosal Th1 response. IL-10 is an anti-inflammatory cytokine that exerts negative immunoregulatory influence on Th1 response. This study investigated whether biochemical modulation of endogenous IL-10 expression at the level of APCs is a practical strategy for enhancing the specific Th1 response against pathogens controlled by Th1 immunity. The results revealed that the high resistance of genetically engineered IL-10-/- (IL-10KO) mice to genital chlamydial infection is a function of the predilection of their APCs to rapidly and preferentially activate a high Th1 response. Thus, in microbiological analysis, IL-10KO mice suffered a shorter duration of infection, less microbial burden, and limited ascending infection than immunocompetent wild-type mice. Also, IL-10KO were resistant to reinfection after 8 wk of the primary infection. Cellular and molecular immunologic evaluation indicated that IL-10KO mice induced greater frequency of chlamydial-specific Th1 response following C. trachomatis infection. Moreover, IL-10KO APCs or antisense IL-10 oligonucleotide-treated wild-type APCs were potent activators of Th1 response from naive or immune T cells. Furthermore, both Ag-pulsed dendritic cells from IL-10KO mice and IL-10 antisense-treated dendritic cells from wild-type mice were efficient cellular vaccines in adoptive immunotherapeutic vaccination against genital chlamydial infection. These findings may furnish a novel immunotherapeutic strategy for boosting the Th1 response against T cell-controlled pathogens and tumors, using IL-10-deficient APCs as vaccine delivery agents. PMID- 10754318 TI - Type I IFN modulates innate and specific antiviral immunity. AB - IFNs protect from virus infection by inducing an antiviral state and by modulating the immune response. Using mice deficient in multiple aspects of IFN signaling, we found that type I and type II IFN play distinct although complementing roles in the resolution of influenza viral disease. Both types of IFN influenced the profile of cytokines produced by T lymphocytes, with a significant bias toward Th2 differentiation occurring in the absence of responsiveness to either IFN. However, although a Th1 bias produced through inhibition of Th2 differentiation by IFN-gamma was not required to resolve infection, loss of type I IFN responsiveness led to exacerbated disease pathology characterized by granulocytic pulmonary inflammatory infiltrates. Responsiveness to type I IFN did not influence the generation of virus-specific cytotoxic lymphocytes or the rate of viral clearance, but induction of IL-10 and IL-15 in infected lungs through a type I IFN-dependent pathway correlated with a protective response to virus. Combined loss of both IFN pathways led to a severely polarized proinflammatory immune response and exacerbated disease. These results reveal an unexpected role for type I IFN in coordinating the host response to viral infection and controlling inflammation in the absence of a direct effect on virus replication. PMID- 10754319 TI - Nitric oxide mediates intestinal pathology but not immune expulsion during Trichinella spiralis infection in mice. AB - The relationship between intestinal pathology and immune expulsion of gastrointestinal (GI) nematodes remains controversial. Although immune expulsion of GI helminth parasites is usually associated with Th2 responses, the effector mechanisms directly responsible for parasite loss have not been identified. We have previously shown that while the intestinal pathology accompanying the expulsion of the GI parasite Trichinella spiralis may be dependent on IL-4 and mediated by TNF, parasite loss is independent of TNF. In contrast, intestinal pathology in other disease models has been attributed to Th1 cytokines, although it closely resembles that seen in helminth infections. Whereas production of inducible NO synthase (iNOS) in the gut is important for both homeostasis of the epithelial layer and in protection against pathogenic microorganisms, overproduction of NO has been implicated in the pathogenesis of a number of inflammatory conditions. We therefore investigated the role of NO in T. spiralis infection using iNOS-deficient mice. iNOS-/- and iNOS-/+ mice were infected with T. spiralis, and parasite expulsion and intestinal pathology were followed. Parasite expulsion proceeded similarly in both groups of animals, but significant intestinal pathology was only observed in the heterozygous mice. Thus it appears that, although the protective effects of Th2 responses in GI helminth infection do not require NO, this mediator contributes substantially to the associated enteropathy. NO may therefore be an important mediator of enteropathy in both Th1 and Th2-inducing conditions. PMID- 10754320 TI - Ongoing murine T1 or T2 immune responses to the hepatitis B surface antigen are excluded from the liver that expresses transgene-encoded hepatitis B surface antigen. AB - Different protein- or DNA-based vaccination techniques are available that prime potent humoral and cellular, T1 or T2 immune responses to the hepatitis B surface Ag (HBsAg) in mice. T1 and T2 are immune responses with isotype profile indicating Th1 and Th2 immunoregulation. We tested whether HBsAg-specific immune responses can be established in transgenic mice that express HBsAg in the liver (HBs-tg mice) using either these different vaccination techniques or an adoptive transfer system. HBsAg-specific responses could not be primed in HBs-tg mice with the established, potent vaccine delivery techniques. In contrast, adoptive transfers of T1- and T2-type HBsAg-immune spleen cells into congenic HBs-tg hosts (that were not conditioned by pretreatment) suppressed HBsAg antigenemia and gave rise to HBsAg-specific serum Ab titers. The establishment of continuously rising anti-HBsAg serum Ab levels with alternative isotype profiles (reflecting T1 or T2 polarization) in transplanted HBs-tg hosts required donor CD4+ T cell-dependent restimulation of adoptively transferred immune cells by transgene-derived HBsAg. Injections of HBsAg-specific Abs into HBs-tg mice did not establish stable humoral immunity. The expanding T1 or T2 immune responses to HBsAg in HBs-tg hosts did not suppress transgene-directed HBsAg expression in the liver and did not induce liver injury. In addition to priming functional antiviral effector cells, the conditioning of the liver microenvironment to enable delivery of antiviral effector functions to this organ are therefore critical for effective antiviral defense. A major challenge in the development of a therapeutic vaccine against chronic hepatitis B or C virus infection is thus the efficient targeting of specifically induced immune effector specificities to the liver. PMID- 10754321 TI - HLA-DQ tetramers identify epitope-specific T cells in peripheral blood of herpes simplex virus type 2-infected individuals: direct detection of immunodominant antigen-responsive cells. AB - Ag-specific CD4+ T cells are present in peripheral blood in low frequency, where they undergo recruitment and expansion during immune responses and in the pathogenesis of numerous autoimmune diseases. MHC tetramers, which constitute a labeled MHC-peptide ligand suitable for binding to the Ag-specific receptor on T cells, provide a novel approach for the detection and characterization of such rare cells. In this study, we utilized this technology to identify HLA DQ restricted Ag-specific T cells in the peripheral blood of human subjects and to identify immunodominant epitopes associated with viral infection. Peptides representing potential epitope regions of the VP16 protein from HSV-2 were loaded onto recombinant DQ0602 molecules to generate a panel of Ag-specific DQ0602 tetramers. VP16 Ag-specific DQ-restricted T cells were identified and expanded from the peripheral blood of HSV-2-infected individuals, representing two predominant epitope specificities. Although the VP16 369-380 peptide has a lower binding affinity for DQ0602 molecules than the VP16 33-52 peptide, T cells that recognized the VP16 369-380 peptide occurred at a much higher frequency than those that were specific for the VP16 33-52 peptide. PMID- 10754322 TI - IL-13-mediated worm expulsion is B7 independent and IFN-gamma sensitive. AB - B7 costimulation is a required component of many type 2 immune responses, including allergy and protective immunity to many nematode parasites. This response includes elevations in Th2 cytokines and associated effector functions including elevations in serum IgG1 and IgE and parasite expulsion. In studies of mice infected with Trichuris muris, blocking B7 ligand interactions inhibited protective immunity, suppressed IL-4 production, and enhanced IFN-gamma production, but unexpectedly did not inhibit production of the Th2 cytokine, IL 13. Blocking both IFN-gamma and B7 restored protective immunity, which was IL-13 dependent, but did not restore IL-4 or associated IgE responses. Although IL-13 was required for worm expulsion in mice in which both IFN-gamma and B7 were blocked, IL-4 could mediate expulsion in the absence of both IL-13 and IFN-gamma. These studies demonstrate that 1) B7 costimulation is required to induce IL-4, but not IL-13 responses; 2) IL-13 is elevated in association with the IFN-gamma response that occurs following inhibition of B7 interactions, but can only mediate IL-4-independent protection when IFN-gamma is also inhibited; and 3) increased IL-13 production, in the absence of increased IL-4 production, is not associated with an IgE response, even in the absence of IFN-gamma. PMID- 10754323 TI - Synthetic peptides of CD66a stimulate neutrophil adhesion to endothelial cells. AB - Four members of the carcinoembryonic Ag family, CD66a, CD66b, CD66c, and CD66d, are expressed on human neutrophils. CD66a, CD66b, CD66c, and CD66d Ab binding to the neutrophil surface triggers an activation signal that regulates the adhesive activity of CD11/CD18, resulting in an increase in neutrophil adhesion to HUVEC. To identify active sites on the CD66a Ag, molecular modeling was performed using IgG and CD4 as models, and 28 peptides of 14 aa in length were synthesized that were predicted to be present at loops and turns between beta-sheets. The peptides were tested for their ability to alter neutrophil adhesion to HUVEC. Three peptides, each from the N-terminal domain, increased neutrophil adhesion to HUVEC monolayers. This increase in neutrophil adhesion caused by CD66a peptides was associated with up-regulation of CD11/CD18 and down-regulation of CD62L on the neutrophil surface. Scrambled versions of these three peptides had no effect on neutrophil adhesion to the endothelial cells. The data suggest that peptide motifs from at least three regions of the N-terminal domain of CD66a are involved in the interaction of CD66a with other ligands and can initiate signal transduction in neutrophils. PMID- 10754324 TI - Cytokine-stimulated, but not HIV-infected, human monocyte-derived macrophages produce neurotoxic levels of l -cysteine. AB - Approximately one-quarter of individuals with AIDS develop neuropathological symptoms that are attributable to infection of the brain with HIV. The cognitive manifestations have been termed HIV-associated dementia. The mechanisms underlying HIV-associated neuronal injury are incompletely understood, but various studies have confirmed the release of neurotoxins by macrophages/microglia infected with HIV-1 or stimulated by viral proteins, including the envelope glycoprotein gp120. In the present study, we investigated the possibility that l -cysteine, a neurotoxin acting at the N-methyl-d aspartate subtype of glutamate receptor, could contribute to HIV-associated neuronal injury. Picomolar concentrations of gp120 were found to stimulate cysteine release from human monocyte-derived macrophages (hMDM) in amounts sufficient to injure cultured rat cerebrocortical neurons. TNF-alpha and IL 1beta, known to be increased in HIV-encephalitic brains, as well as a cellular product of cytokine stimulation, ceramide, were also shown to induce release of cysteine from hMDM in a dose-dependent manner. A TNF-alpha-neutralizing Ab and an IL-1betaR antagonist partially blocked gp120-induced cysteine release, suggesting that these cytokines may mediate the actions of gp120. Interestingly, hMDM infected with HIV-1 produced significantly less cysteine than uninfected cells following stimulation with TNF-alpha. Our findings imply that cysteine may play a role in the pathogenesis of neuronal injury in HIV-associated dementia due to its release from immune-activated macrophages but not virus-infected macrophages. Such uninfected cells comprise the vast majority of mononuclear phagocytes (macrophages and microglia) found in HIV-encephalitic brains. PMID- 10754325 TI - Stem cell factor plays a major role in the recruitment of eosinophils in allergic pleurisy in mice via the production of leukotriene B4. AB - The understanding of the mechanisms underlying eosinophil migration into tissue is an essential step in the development of novel therapies aimed at treating allergic diseases where eosinophil recruitment and activation are thought to play an essential role. In this study, we have examined the effects of the in vivo administration of stem cell factor (SCF) on eosinophil recruitment and tested whether endogenous SCF was involved in mediating eosinophil recruitment in response to Ag challenge in sensitized mice. The intrapleural injection of SCF induced a time- and concentration-dependent recruitment of eosinophils in mice. In allergic mice, SCF message was expressed early after Ag challenge and returned to baseline levels after 8 h. In agreement with the ability of SCF to induce eosinophil recruitment and its expression in the allergic reaction, an anti-SCF polyclonal Ab abrogated eosinophil recruitment when given before Ag challenge. SCF increased the levels of leukotriene B4 (LTB4) in the pleural cavity of mice and an LTB4 receptor antagonist, CP105,696, abrogated the effects of SCF on eosinophil recruitment. Similarly, recruitment of eosinophils in the allergic reaction was virtually abolished by CP105,696. Together, our data favor the hypothesis that the local release of SCF following Ag challenge may activate and/or prime mast cells for IgE-mediated release of inflammatory mediators, especially LTB4. The mediators released in turn drive the recruitment of eosinophils. Inhibition of the function of SCF in vivo may reduce the migration of eosinophils to sites of allergic inflammation and may, thus, be a relevant principle in the treatment of allergic diseases. PMID- 10754327 TI - Granulocyte-macrophage colony-stimulating factor delays neutrophil constitutive apoptosis through phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways. AB - Activated neutrophils play an important role in the pathogenesis of sepsis, glomerulonephritis, acute renal failure, and other inflammatory processes. The resolution of neutrophil-induced inflammation relies, in large part, on removal of apoptotic neutrophils. Neutrophils are constitutively committed to apoptosis, but inflammatory mediators, such as GM-CSF, slow neutrophil apoptosis by incompletely understood mechanisms. We addressed the hypothesis that GM-CSF delays neutrophil apoptosis by activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI 3-kinase) pathways. GM-CSF (20 ng/ml) significantly inhibited neutrophil apoptosis (GM-CSF, 32 vs 65% of cells p < 0. 0001). GM-CSF activated the PI 3-kinase/Akt pathway as determined by phosphorylation of Akt and BAD. GM-CSF-dependent Akt and BAD phosphorylation was blocked by the PI 3-kinase inhibitor LY294002. A role for the PI 3-kinase/Akt pathway in GM-CSF-stimulated delay of apoptosis was indicated by the ability of LY294002 to attenuate apoptosis delay. GM-CSF-dependent inhibition of apoptosis was significantly attenuated by PD98059, an ERK pathway inhibitor. LY294002 and PD98059 did not produce additive inhibition of apoptosis delay. To determine whether PI 3-kinase and ERK are used by other ligands that delay neutrophil apoptosis, we examined the role of these pathways in IL-8-induced apoptosis delay. LY294002 blocked IL-8-dependent Akt phosphorylation. PD98059 and LY294002 significantly attenuated IL-8 delay of apoptosis. These results indicate IL-8 and GM-CSF act, in part, to delay neutrophil apoptosis by stimulating PI 3-kinase and ERK-dependent pathways. PMID- 10754326 TI - TNF-alpha gene expression in macrophages: regulation by NF-kappa B is independent of c-Jun or C/EBP beta. AB - The interaction of transcription factors is critical in the regulation of gene expression. This study characterized the mechanism by which NF-kappa B family members interact to regulate the human TNF-alpha gene. A 120-bp TNF-alpha promoter-reporter, possessing binding sites for NF-kappa B (kappa B3), C/EBP beta (CCAAT/enhancer binding protein beta), and c-Jun, was activated by cotransfection of plasmids expressing the wild-type version of each of these transcription factors. Employing adenoviral vectors, dominant-negative versions of NF-kappa B p65, and c-Jun, but not C/EBP beta, suppressed (p < 0.05-0.001) LPS-induced TNF alpha secretion in primary human macrophages. Following LPS stimulation, NF-kappa B p50/p65 heterodimers bound to the kappa B3 site and c-Jun to the -103 AP-1 site of the TNF-alpha promoter. By transient transfection, NF-kappa B p65 and p50 synergistically activated the TNF-alpha promoter. In contrast, no synergy was observed between NF-kappa B p65, with or without NF-kappa B p50, and c-Jun or C/EBP beta, even in the presence of the coactivator p300. The contribution of the upstream kappa B binding sites was also examined. Following LPS stimulation, the kappa B1 site bound both NF-kappa B p50/p65 heterodimers and p50 homodimers. The binding by NF-kappa B p50 homodimers to the kappa B1, but not to the kappa 3, site contributed to the inability of macrophages to respond to a second LPS challenge. In summary, adjacent kappa B3 and AP-1 sites in the human TNF-alpha promoter contribute independently to LPS-induced activation. Although both the kappa B1 and kappa B3 sites bound transcriptionally active NF-kappa B p50/p65 heterodimers, only the kappa B1 site contributed to down-regulation by NF-kappa B p50 homodimers. PMID- 10754328 TI - Crucial role of the amino-terminal tyrosine residue 42 and the carboxyl-terminal PEST domain of I kappa B alpha in NF-kappa B activation by an oxidative stress. AB - Activation of transcription factor NF-kappa B involves the signal-dependent degradation of basally phosphorylated inhibitors such as I kappa B alpha. In response to proinflammatory cytokines or mitogens, the transduction machinery has recently been characterized, but the activation mechanism upon oxidative stress remains unknown. In the present work, we provide several lines of evidence that NF-kappa B activation in a T lymphocytic cell line (EL4) by hydrogen peroxide (H2O2) did not involve phosphorylation of the serine residues 32 and 36 in the amino-terminal part of I kappa B alpha. Indeed, mutation of Ser32 and Ser36 blocked IL-1 beta- or PMA-induced NF-kappa B activation, but had no effect on its activation by H2O2. Although I kappa B alpha was phosphorylated upon exposure to H2O2, tyrosine residue 42 and the C-terminal PEST (proline-glutamic acid-serine threonine) domain played an important role. Indeed, mutation of tyrosine 42 or serine/threonine residues of the PEST domain abolished NF-kappa B activation by H2O2, while it had no effect on activation by IL-1 beta or PMA-ionomycin. This H2O2-inducible phosphorylation was not dependent on I kappa B kinase activation, but could involve casein kinase II, because an inhibitor of this enzyme (5,6 dichloro-1-beta-D-ribofuranosyl-benzimidazole) blocks NF-kappa B activation. H2O2 induced I kappa B alpha phosphorylation was followed by its degradation by calpain proteases or through the proteasome. Taken together, our findings suggest that NF-kappa B activation by H2O2 involves a new mechanism that is totally distinct from those triggered by proinflammatory cytokines or mitogens. PMID- 10754329 TI - IL-1 receptor-associated kinase modulates host responsiveness to endotoxin. AB - Endotoxin triggers many of the inflammatory, hemodynamic, and hematological derangements of Gram-negative septic shock. Recent genetic studies in mice have identified the Toll-like receptor 4 as the transmembrane endotoxin signal transducer. The IL-1 intracellular signaling pathway has been implicated in Toll like receptor signal transduction. LPS-induced activation of the IL-1 receptor associated kinase (IRAK), and the influence of IRAK on intracellular signaling and cellular responses to endotoxin has not been explored in relevant innate immune cells. We demonstrate that LPS activates IRAK in murine macrophages. IRAK deficient macrophages, in contrast, are resistant to LPS. Deletion of IRAK disrupts several endotoxin-triggered signaling cascades. Furthermore, macrophages lacking IRAK exhibit impaired LPS-stimulated TNF-alpha production, and IRAK deficient mice withstand the lethal effects of LPS. These findings, coupled with the critical role for IRAK in IL-1 and IL-18 signal transduction, demonstrate the importance of this kinase and the IL-1/Toll signaling cassette in sensing and responding to Gram-negative infection. PMID- 10754330 TI - Calcitonin gene-related peptide induces IL-8 synthesis in human corneal epithelial cells. AB - Calcitonin gene-related peptide (CGRP), a neuropeptide with proinflammatory activities, is released from termini of corneal sensory neurons in response to pain stimuli. Because neutrophil infiltration of the clear corneal surface is a hallmark of corneal inflammation in the human eye, we determined whether CGRP can bind to human corneal epithelial cells (HCEC) and induce expression of the neutrophil chemotactic protein IL-8. It was found that HCEC specifically bound CGRP in a saturable manner with a Kd of 2.0 x 10-9 M. Exposure of HCEC to CGRP induced a significant increase in intracellular cAMP levels and enhanced IL-8 synthesis nearly 4-fold. The capacity of CGRP to stimulate cAMP and IL-8 synthesis was abrogated in the presence of the CGRP receptor antagonist CGRP8-37. CGRP stimulation had no effect on the half-life of IL-8 mRNA while increasing IL 8 pre-mRNA synthesis >2-fold. In contrast to IL-8, CGRP did not induce monocyte chemotactic protein-1 or RANTES synthesis, nor did the neuropeptide enhance detectable increases in steady state levels of mRNA specific for these two beta chemokines. The results suggest that HCEC possess CGRP receptors capable of initiating a signal transduction cascade that differentially activates expression of the IL-8 gene but not the genes for monocyte chemotactic protein-1 or RANTES. The capacity of CGRP to stimulate IL-8 synthesis in HCEC suggests that sensory neurons are involved in induction of acute inflammation at the eye surface. PMID- 10754331 TI - CX3C-chemokine, fractalkine-enhanced adhesion of THP-1 cells to endothelial cells through integrin-dependent and -independent mechanisms. AB - Leukocyte adhesion and trafficking at the endothelium requires both cellular adhesion molecules and chemotactic factors. A newly identified CX3C chemokine, fractalkine, expressed on activated endothelial cells, plays an important role in leukocyte adhesion and migration. We examined the functional effects of fractalkine on beta1 and beta2 integrin-mediated adhesion using a macrophage-like cell line, THP-1 cells. In this study, we report that THP-1 cells express mRNA encoding a receptor for fractalkine, CX3CR1, determined by Northern blotting. Scatchard analysis using fractalkine-SEAP (secreted form of placental alkaline phosphatase) chimeric proteins revealed that THP-1 cells express a single class of CX3CR1 with a dissociation constant of 30 pM and a mean expression of 440 sites per cell. THP-1 cells efficiently adhered, in a fractalkine-dependent manner, to full-length of fractalkine immobilized onto plastic and to the membrane-bound form of fractalkine expressed on ECV304 cells or TNF-alpha activated HUVECs. Moreover, soluble-fractalkine enhanced adhesion of THP-1 cells to fibronectin and ICAM-1 in a dose-dependent manner. Pertussis toxin, an inhibitor of Gi, inhibited the fractalkine-mediated enhancement of THP-1 cell adhesion to fibronectin and ICAM-1. Finally, we found that soluble-fractalkine also enhanced adhesion of freshly separated monocytes to fibronectin and ICAM-1. These results indicate that fractalkine may induce firm adhesion between monocytes and endothelial cells not only through an intrinsic adhesion function itself, but also through activation of integrin avidity for their ligands. PMID- 10754332 TI - Kinase pathways in chemoattractant-induced degranulation of neutrophils: the role of p38 mitogen-activated protein kinase activated by Src family kinases. AB - The aim of the present study was to investigate the role of tyrosine phosphorylation pathways in fMLP-induced exocytosis of the different secretory compartments (primary and secondary granules, as well as secretory vesicles) of neutrophils. Genistein, a broad specificity tyrosine kinase inhibitor, blocked the exocytosis of primary and secondary granules, but had only a marginal effect on the release of secretory vesicles. Genistein also inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen activated protein kinases (MAPK), raising the possibility that inhibition of ERK and/or p38 MAPK might be responsible for the effect of the drug on the degranulation response. Indeed, SB203580, an inhibitor of p38 MAPK, decreased the release of primary and secondary granules, but not that of secretory vesicles. However, blocking the ERK pathway with PD98059 had no effect on any of the exocytic responses tested. PP1, an inhibitor of Src family kinases, also attenuated the release of primary and secondary granules, and neutrophils from mice deficient in the Src family kinases Hck, Fgr, and Lyn were also defective in secondary granule release. Furthermore, activation of p38 MAPK was blocked by both PP1 and the hck-/-fgr-/-lyn-/- mutation. Taken together, our data indicate that fMLP-induced degranulation of primary and secondary granules of neutrophils is mediated by p38 MAPK activated via Src family tyrosine kinases. Although piceatannol, a reportedly selective inhibitor of Syk, also prevented degranulation and activation of p38 MAPK, no fMLP-induced phosphorylation of Syk could be observed, raising doubts about the specificity of the inhibitor. PMID- 10754333 TI - Expression of a functional high-affinity IgG receptor, Fc gamma RI, on human mast cells: Up-regulation by IFN-gamma. AB - Biologically relevant activation of human mast cells through Fc receptors is believed to occur primarily through the high-affinity IgE receptor Fc epsilon RI. However, the demonstration in animal models that allergic reactions do not necessarily require Ag-specific IgE, nor the presence of a functional IgE receptor, and the clinical occurrence of some allergic reactions in situations where Ag-specific IgE appears to be lacking, led us to examine the hypothesis that human mast cells might express the high-affinity IgG receptor Fc gamma RI and in turn be activated through aggregation of this receptor. We thus first determined by RT-PCR that resting human mast cells exhibit minimal message for Fc gamma RI. We next found that IFN-gamma up-regulated the expression of Fc gamma RI. This was confirmed by flow cytometry, where Fc gamma RI expression on human mast cells was increased from approximately 2 to 44% by IFN-gamma exposure. Fc epsilon RI, Fc gamma RII, and Fc gamma RIII expression was not affected. Scatchard plots were consisted with these data where the average binding sites for monomeric IgG1 (Ka = 4-5 x 108 M-1) increased from approximately 2,400 to 12,100-17,300 per cell. Aggregation of Fc gamma RI on human mast cells, and only after IFN-gamma exposure, led to significant degranulation as evidenced by histamine release (24.5 +/- 4.4%): and up-regulation of mRNA expression for specific cytokines including TNF-alpha, GM-CSF, IL-3 and IL-13. These findings thus suggest another mechanism by which human mast cells may be recruited into the inflammatory processes associated with some immunologic and infectious diseases. PMID- 10754334 TI - A role for complement in antibody-mediated inflammation: C5-deficient DBA/1 mice are resistant to collagen-induced arthritis. AB - Collagen-induced arthritis (CIA) represents an animal model of autoimmune polyarthritis with significant similarities to human rheumatoid arthritis that can be induced upon immunization with native type II collagen. As in rheumatoid arthritis, both cellular and humoral immune mechanisms contribute to disease pathogenesis. Genotypic studies have identified at least six genetic loci contributing to arthritis susceptibility, including the class II MHC. We have examined the mechanism of Ab-mediated inflammation in CIA joints, specifically the role of complement activation, by deriving a line of mice from the highly CIA susceptible DBA/1LacJ strain that are congenic for deficiency of the C5 complement component. We show that such C5-deficient DBA/1LacJ animals mount normal cellular and humoral immune responses to native type II collagen, with the activation of collagen-specific TNF-alpha-producing T cells in the periphery and substantial intra-articular deposition of complement-fixing IgG Abs. Nevertheless, these C5-deficient mice are highly resistant to the induction of CIA. These data provide evidence for an important role of complement in Ab triggered inflammation and in the pathogenesis of autoimmune arthritis. PMID- 10754335 TI - Neutrophil tethering on E-selectin activates beta 2 integrin binding to ICAM-1 through a mitogen-activated protein kinase signal transduction pathway. AB - On inflamed endothelium selectins support neutrophil capture and rolling that leads to firm adhesion through the activation and binding of beta 2 integrin. The primary mechanism of cell activation involves ligation of chemotactic agonists presented on the endothelium. We have pursued a second mechanism involving signal transduction through binding of selectins while neutrophils tether in shear flow. We assessed whether neutrophil rolling on E-selectin led to cell activation and arrest via beta 2integrins. Neutrophils were introduced into a parallel plate flow chamber having as a substrate an L cell monolayer coexpressing E-selectin and ICAM-1 (E/I). At shears >/=0.1 dyne/cm2, neutrophils rolled on the E/I. A step increase to 4.0 dynes/cm2 revealed that approximately 60% of the interacting cells remained firmly adherent, as compared with approximately 10% on L cells expressing E-selectin or ICAM-1 alone. Cell arrest was dependent on application of shear and activation of Mac-1 and LFA-1 to bind ICAM-1. Firm adhesion was inhibited by blocking E-selectin, L-selectin, or PSGL-1 with Abs and by inhibitors to the mitogen-activated protein kinases. A chimeric soluble E selectin-IgG molecule specifically bound sialylated ligands on neutrophils and activated adhesion that was also inhibited by blocking the mitogen-activated protein kinases. We conclude that neutrophils rolling on E-selectin undergo signal transduction leading to activation of cell arrest through beta 2 integrins binding to ICAM-1. PMID- 10754336 TI - Presentation of alpha B-crystallin to T cells in active multiple sclerosis lesions: an early event following inflammatory demyelination. AB - In the development of multiple sclerosis (MS), (re)activation of infiltrating T cells by myelin-derived Ags is considered to be a crucial step. Previously, alpha B-crystallin has been shown to be an important myelin Ag to human T cells. Since alpha B-crystallin is an intracellular heat shock protein, the question arises at what stage, if any, during lesional development in MS this Ag becomes available for CD4+ T cells. In 3 of 10 active MS lesions, alpha B-crystallin could be detected inside phagocytic vesicles of perivascular macrophages, colocalizing with myelin basic protein and myelin oligodendrocyte glycoprotein (MOG). Although the detectability of MOG in phagosomes is considered as a marker for very recent demyelination, MOG was detected in more macrophages and in more lesions than alpha B-crystallin. The disappearance of alpha B-crystallin from macrophages even before MOG was confirmed by in vitro studies; within 6 h after myelin-uptake alpha B-crystallin disappears from the phagosomes. Alpha B-crystallin-containing macrophages colocalized with infiltrating T cells and they were characterized by expression of MHC class II, CD40, and CD80. To examine functional presentation of myelin Ags to T cells, purified macrophages were pulsed in vitro with whole myelin membranes. These macrophages activated both myelin-primed and alpha B crystallin-primed T cells in terms of proliferation and IFN-gamma secretion. In addition, alpha B-crystallin-pulsed macrophages activated myelin-primed T cells to the same extent as myelin-pulsed macrophages, whereas myelin basic protein pulsed macrophages triggered no response at all. These data indicate that, in active MS lesions, alpha B-crystallin is available for functional presentation to T cells early during inflammatory demyelination. PMID- 10754337 TI - Mechanism for the isotype dependence of antibody-mediated toxicity in Cryptococcus neoformans-infected mice. AB - Ab-based therapies have undergone a renaissance in recent years, but infusion related reactions are a significant clinical problem. Administration of certain mAbs to Swiss Webster mice infected with Cryptococcus neoformans can result in acute lethal toxicity (ALT) characterized by cardiovascular collapse. The ability of a mAb to produce ALT is isotype dependent and occurs with IgG1 but not IgG3. To investigate this phenomenon, we measured spleen and liver cytokine responses and platelet-activating factor (PAF) content in mice given C. neoformans glucuronoxylomannan (GXM) followed by specific Ab of IgG1 or IgG3 isotype. We found no evidence to suggest that the differences in IgG1 and IgG3 toxicity were due to differences in chemokine or cytokine response. In contrast, liver and spleen tissue PAF content was significantly greater in mice IgG1. Furthermore, our results show differences in the response to IgG1- and IgG3-GXM complexes regarding: 1) macrophage-inflammatory protein-1alpha and monocyte chemoattractant protein-1 regulation, 2) splenic and hepatic PAF content, and 3) hepatic PAF content in infected mice. IgG1-associated ALT appears to be the result of greater production of PAF in response to IgG1-GXM complex formation. The results are consistent with the view that IgG1 and IgG3 interact with different Fc receptors. Our findings strongly suggest that the mechanism for Ab-mediated ALT is different from the cytokine release syndrome described after administration of other therapeutic mAbs. PMID- 10754338 TI - Differential expression of NK T cell V alpha 24J alpha Q invariant TCR chain in the lesions of multiple sclerosis and chronic inflammatory demyelinating polyneuropathy. AB - Human V alpha 24+ NK T cells are a unique subset of lymphocytes expressing the V alpha 24J alpha Q invariant TCR chain. Because they can rapidly produce large amounts of regulatory cytokines, a reduction of NK T cells may lead to the development of certain autoimmune diseases. Using a single-strand conformation polymorphism method, we demonstrate that a great reduction of V alpha 24J alpha Q NK T cells in the peripheral blood is an immunological hallmark of multiple sclerosis, whereas it is not appreciable in other autoimmune/inflammatory diseases such as chronic inflammatory demyelinating polyneuropathy. The chronic inflammatory demyelinating polyneuropathy lesions were often found to be infiltrated with V alpha 24J alpha Q NK T cells, but multiple sclerosis lesions only rarely expressed the V alpha 24J alpha Q TCR. It is therefore possible that the extent of NK T cell alteration may be a critical factor which would define the clinical and pathological features of autoimmune disease. Although the mechanism underlying the NK T cell deletion remains largely unclear, a remarkable contrast between the CNS and peripheral nervous system diseases allows us to speculate a role of tissue-specific elements such as the level of CD1d expression or differences in the CD1d-bound glycolipid. PMID- 10754339 TI - A tumor-infiltrating lymphocyte from a melanoma metastasis with decreased expression of melanoma differentiation antigens recognizes MAGE-12. AB - Twenty separate tumor infiltrating lymphocyte (TIL) bulk cultures and a tumor cell line were originated simultaneously from a fine needle aspiration biopsy of a metastasis in a patient with melanoma (F001) previously immunized with the HLA A*0201-associated gp100:209-217(210 M) peptide. None of the TIL recognized gp100. However, 12 recognized autologous (F001-MEL) and allogeneic melanoma cells expressing the HLA haplotype A*0201, B*0702, Cw*0702. Further characterization of F001-MEL demonstrated loss of gp100/PMel17, severely decreased expression of other melanoma differentiation Ags and retained expression of tumor-specific Ags. Transfection of HLA class I alleles into B*0702/Cw*0702-negative melanoma cell lines identified HLA-Cw*0702 as the restriction element for F001-TIL. A cDNA library from F001-MEL was used to transfect IFN-alpha-stimulated 293 human embryonal kidney (293-HEK) cells expressing HLA-Cw*0702. A 100-gene pool was identified that induced recognition of 293-HEK cells by F001-TIL. Subsequent cloning of the pool identified a cDNA sequence homologous, except for one amino acid (aa 187 D-->A), to MAGE-12. Among 25 peptide sequences from MAGE-12 with the HLA-Cw*0702 binding motif, MAGE-12:170-178 (VRIGHLYIL) induced IFN-gamma release by F001-TIL when pulsed on F001-EBV-B cells at concentrations as low as 10 pg/ml. Peptide sequences from MAGE-1, 2, 3, 4a, and 6 aligned to MAGE-12:170-178 were not recognized by F001-TIL. In summary a TIL recognizing a MAGE protein was developed from an HLA-A*0201 expressing tumor with strongly reduced expression of melanoma differentiation Ags. Persisting tumor-specific Ag expression maintained tumor immune competence suggesting that tumor-specific Ags/melanoma differentiation Ags may complement each other in the context of melanoma Ag specific vaccination. PMID- 10754340 TI - Characterization of T cell responses to Hev b 3, an allergen associated with latex allergy in spina bifida patients. AB - The prevalence of type I allergy to Hevea brasiliensis latex is particularly high among individuals with frequent exposure such as health care workers and patients with spina bifida (SB). Due to a birth defect of the spinal canal and the resulting neurological and orthopedic defects, these patients require multiple surgeries during childhood. SB patients display a unique pattern of sensitization: IgE-reactivity is preferentially directed against Hev b 3 and Hev b 1, two latex allergens with high sequence similarity. In this study, we analyzed the T cell response to Hev b 3 in latex-allergic SB patients using poly , oligo-, and monoclonal T lymphocyte cultures. All T cell clones (TCC) were CD3/CD4-positive and expressed the alphabeta TCR. According to their cytokine production pattern (IL-4 vs IFN-gamma), 12 of 21 TCC were classified as Th2-like, 2 of 21 were Th1-like, and 7 of 21 belonged to a Th0-like subset. Using 11 T cell lines and 21 TCC, nine T cell stimulating fragments were determined out of 52 overlapping 12-mer peptides representing the complete amino acid sequence of Hev b 3. Ag presentation of one dominant T cell epitope could be associated with a four-amino acid binding motif (YSTS, position 11-13) in the beta 1 chain of HLA DR molecules expressed by the respective patients. No reactivity was observed when Hev b 3-reactive T cell lines or TCC were incubated with peptides representing homologous parts of the Hev b 1 molecule, i.e., no cross-reactivity between Hev b 3 and Hev b 1 at the T cell level was evident. PMID- 10754341 TI - CD30+ T cells in rheumatoid synovitis: mechanisms of recruitment and functional role. AB - High serum levels of soluble CD30 (sCD30) have been reported to better predict the response to second line therapy in rheumatoid arthritis (RA). It is believed that sCD30 is released by CD30+ T cells present in the RA synovium. However, both the mechanism of recruitment to the joint and the functional role of this T cell subset in the pathogenesis of the disease remain unknown. This study confirmed higher levels of sCD30 in the serum and synovial fluid (SF) of RA patients compared with normal controls. However, analysis of mRNA and cell surface CD30 expression showed that CD30+ T cells are detectable in the SF, but not in the synovial membrane. In contrast, T cells expressing the CD30 transcript, but not the surface molecule, were found in the peripheral blood of both RA and normal controls. CD30 surface expression was up-regulated by adhesion and migration through endothelium in vitro and in a delayed-type hypersensitivity model in vivo. Although the great majority of fresh or cloned CD30+ T cells from SF produced both IFN-gamma and IL-4, CD30 expression strictly correlated with IL-4 synthesis in synovial T cell clones. In addition, CD30+ T cell clones also produced high amounts of the anti-inflammatory cytokine IL-10. On this basis, we would like to propose that synovial CD30+ cells may play a role in the control of the inflammatory response. Serum sCD30 may reflect such cell activity and, therefore, explain the previously demonstrated correlation between high sCD30 serum levels and positive response to therapy. PMID- 10754342 TI - Fine mapping of IGAD1 in IgA deficiency and common variable immunodeficiency: identification and characterization of haplotypes shared by affected members of 101 multiple-case families. AB - To limit the region containing a mutation predisposing to selective IgA deficiency (IgAD) and common variable immunodeficiency (CVID), 554 informative members of 101 multiple-case families were haplotyped at the IGAD1 candidate locus in the MHC. Microsatellite markers were placed onto the physical map of IGAD1 to establish their order and permit rapid haplotype analyses. Linkage analysis of this extended family set provided additional support for a strong susceptibility locus at IGAD1 with a maximum multipoint nonparametric linkage score in excess of 3. Although the transmission of maternal IGAD1 haplotypes from unaffected heterozygous parents to the affected offspring was in excess, this was not apparent in multiple-case families with a predominance of affected mothers, suggesting that this parental bias is influenced by the affection status of transmitting parents and supporting a maternal effect in disease susceptibility. Of 110 haplotypes shared by 258 affected family members, a single haplotype (H1) was found in 44 pairs of affected relatives, accounting for the majority of the IGAD1 contribution to the development of IgAD/CVID in our families. The H1 allelic variability was higher in the telomeric part of the class III region than in the distal part of the class II region in both single- and multiple-case families. Incomplete H1 haplotypes had most variant alleles in the telomeric part of the analyzed region in homozygous IgAD/CVID patients, whereas this was not observed in unaffected homozygotes. These data suggest that a telomeric part of the class II region or centromeric part of the class III region is the most likely location of IGAD1. PMID- 10754343 TI - A left atrial thrombus is not an absolute limitation to balloon mitral commissurotomy for patients with mitral stenosis. a serial transesophageal echocardiographic study. AB - Previous reports did not encourage balloon mitral commissurotomy (BMC) when left atrial (LA) thrombi were located beyond LA appendage. We hypothesize that LA thrombi may be resolved in some patients after anticoagulant therapy, and BMC can be performed subsequently. In the present study, we used transesophageal echocardiography (TEE) to evaluate the effects of anticoagulant on LA thrombi in patients with mitral stenosis, to compare the clinical differences between patients with (group A) and without (group B) resolution of LA thrombi, and to evaluate the safety of subsequent BMC in these patients. TEE was performed on 190 consecutive patients with mitral stenosis; 14 (7.4%) of them were found with LA thrombi. Serial TEE was performed and optimal anticoagulant therapy was achieved in all 14 patients. The thrombi were located within the LA appendage in 6 cases, attached to the LA posterior wall in 5, posterior wall and interatrial septum in 1, and LA appendage and posterior wall in 2. Based on the 6-month TEE follow-up, we found that the LA thrombi disappeared in 8 (57.2%) patients (group A) and persisted in 6 patients (group B). LA thrombi resolved within 3 months in 7 group A patients (87.5%). Furthermore, the resolution of LA thrombi was more frequently observed in patients either with a smaller LA dimension (51.9 +/- 3.4 vs. 57.8 +/ 4.8 mm, p = 0.02) or with their thrombi located inside their LA appendage (p = 0.03). No differences in other clinical and echocardiographic variables were noted between these two groups. Subsequently, group A patients underwent BMC without complications of systemic embolization. In conclusion, LA thrombi can be resolved after optimal anticoagulant therapy in a considerable proportion of patients with mitral stenosis. Serial TEE studies are helpful to observe LA thrombus resolution; they may also be useful in planning the treatment strategy. PMID- 10754345 TI - Pearls and pitfalls in the use and abuse of diuretics for chronic congestive heart failure. AB - The main purpose of using diuretics is usually lost sight of, i.e. it is for the relief of dyspnea by using the least amount of a diuretic. The production of a low output state and hypercoagulation in an attempt to achieve dry weight by lowering blood volume excessively are among the hazards of using more diuretic than is absolutely necessary to achieve the goal of relieving dyspnea. The use of jugular venous pressure measurement and the status of dyspnea should have precedence over body weight in determining diuretic dose adjustment. Often forgotten in using diuretics is that potassium without magnesium will not enter cells and that the almost universal preference for furosemide over thiazides threatens to increase the incidence of osteoporosis. Also, the tendency to ignore loss of the water-soluble vitamins thiamine and ascorbic acid may result in refractory edema and the inability to manage the stresses of congestive heart failure. PMID- 10754344 TI - Plasma apolipoprotein B levels predict platelet-dependent thrombosis in patients with coronary artery disease. AB - Elevated plasma apolipoprotein B is a known risk factor for atherosclerotic coronary artery disease (CAD), however its relationship to arterial thrombosis is unexplored. We prospectively assessed apolipoprotein B and platelet-dependent thrombosis (PDT) in 42 CAD patients (37 men, 5 women, mean age 68 +/- 9 years), by exposing porcine aortic media to their flowing unanticoagulated venous blood for 5 min using an ex vivo perfusion (Badimon) chamber. PDT was significantly correlated with apolipoprotein B (r = 0.41, p = 0.009), intracellular magnesium levels (r = -0.46, p = 0.003) fasting blood glucose (r = 0.47, p = 0.002), and total cholesterol (r = 0.43, p = 0.006). PDT did not correlate with serum total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, apolipoprotein A-I or fibrinogen levels. These findings suggest that the positive relationship of elevated apolipoprotein B to CAD may be, in part, related to its prothrombotic effects. PMID- 10754346 TI - Monitoring of extracellular aspartate aminotransferase and troponin T by microdialysis during and after cardioplegic heart arrest. AB - This study aims at developing per- and postopertive surveillance of the myocardium and focuses on ischemic damage following cardioplegic heart arrest. Levels of troponin T and total aspartate aminotransferase (ASAT) were analyzed in the myocardial interstitium of 10 patients with ischemic heart disease (IHD) who underwent coronary bypass surgery and in 12 patients with nonischemic heart disease (N-IHD) who underwent valvular surgery. Fluid from the myocardial interstitium of the anterior and the lateral wall of the heart was sampled by microdialysis probes that were implanted during surgery and extracted percutaneously 70-100 h later. There were no adverse reactions, and the equipment did not interfere with the surgical procedures. The peak in troponin T serum levels that occurred 4 h after cardiac arrest was preceded by a peak in troponin T levels in the microdialysates from the interstitium that occurred 1 h earlier. The concentration of troponin T in the microdialysate peak was 300 times higher than in the serum peak. The increase in serum ASAT levels during the first 7 h after cardiac arrest corresponded in time with a decrease in interstitial ASAT levels, which had already reached a maximum during cardiac arrest. The microdialysate/serum concentration ratio was considerably smaller for ASAT than for troponin T. Interstitial peak levels of troponin T correlated positively and significantly with peak levels of ASAT. Of the 22 patients, 15 had no postoperative events according to clinical outcome, ECG and serum tests. Fourteen of these had low to normal levels of interstitial ASAT and troponin T. Conversely, atrial fibrillation and/or premature atrial contractions were recorded in 8/22 patients, 7 of whom had elevated interstitial ASAT and/or troponin T concentrations in one or both of the sampled heart regions. The N-IHD patients had higher levels of troponin T in the interstitium 20-70 h following cardioplegia, while the peak levels did not differ between the groups. In conclusion, microdialysis sampling of troponin T and ASAT is safe and allows a highly sensitive analysis of the ischemic trauma exerted by the cardioplegic arrest. PMID- 10754347 TI - Histologic evidence of foreign body granulation tissue and de novo lesions in patients with coronary stent restenosis. AB - OBJECTIVES: We examined the relative contributions of foreign body granulation and de novo lesions to neointimal hyperplasia in atherectomized specimens of restenosis after coronary stenting. BACKGROUND: Clinicopathological studies have suggested that smooth muscle cell (SMC) hyperplasia is the most likely cause of restenosis after coronary stenting. It is not yet fully understood how SMC hyperplasia occurs or how SMCs stimulation can lead to intimal hyperplasia. Although inflammation has been postulated to be a major contributor to restenosis after coronary stenting, there is a paucity of data on the relationsip between inflammation and subsequent neointimal formation in humans. Only in a porcine experimental model of stent restenosis, foreign body granulation tissue as a cause of inflammation in stent restenosis was reported. METHODS: Tissue specimens were retrieved by directional atherectomy from 11 patients in whom stent restenosis developed after percutaneous revascularization of coronary artery disease. For specimens preserved in 10% buffered formalin, analysis of cellular composition was performed quantitatively after cell-specific immunostaining, i.e. CD68, UCHL-1, HLA-DR, smooth muscle actin, vimentin, desmin, PCNA and TGF-beta. RESULTS: Five of the 11 patients showed granulation tissues 3-6 months after stent implantation, of whom 3 patients revealed foreign body multinucleated giant cells around the stent struts where PCNA- and vimentin-positive SMCs were demonstrated. Calcification and de novo lesions in medial and adventitial tissues were observed in 3 other patients, and fresh and/or organized thrombi were documented in 3 of the 11 patients. CONCLUSIONS: These findings support the notion that stent restenosis results from SMC hyperplasia and suggest that the foreign body granulation tissue against metals of the stents and de novo lesions could play an important role in chronic inflammation leading to intimal hyperplasia and subsequently to stent restenosis in some patients. Clinicians should thus consider whether a patient may be allergic to stent components with unknown reaction, e.g. haptens. PMID- 10754348 TI - Cardiac involvement in Werdnig-Hoffmann's spinal muscular atrophy. AB - Despite the increasing interest in cardiac involvement (CI) of neuromuscular disorders, only few data are available on CI in spinal muscular atrophy (SMA). We tried to determine the cardiac manifestations of SMA, their incidence rates and the necessity of cardiac therapy in patients with SMA and CI. Eight patients with SMA, aged 10-79 years, underwent clinical cardiologic examination, ECG, echocardiography and ambulatory ECG. The most frequent findings were angina pectoris, palpitations, exertional dyspnea, ST abnormalities, couplets, thickened myocardium and diastolic dysfunction. In conclusion, cardiac manifestations of SMA mainly comprise ECG abnormalities and thickened myocardium. CI in SMA frequently necessitates cardiac therapy. PMID- 10754349 TI - Myocardial perfusion and angiographic findings in patients with paradoxical sinus deceleration during dobutamine technetium-99m sestamibi-gated SPECT imaging. AB - AIMS: Dobutamine-gated SPECT imaging allows simultaneous perfusion and function assessment and is an alternative stress method for evaluating coronary artery disease in patients unable to perform treadmill exercise. In some patients, a paradoxical decrease in heart rate has been observed during high dose dobutamine infusion. The purpose of this study was to describe the paradoxical decrease in heart rate observed during dobutamine perfusion scintigraphy and determine its relation to inducible ischemia and angiographic coronary artery disease. METHODS AND RESULTS: We studied 52 patients who underwent dobutamine-gated (99m)Tc sestamibi SPECT imaging and coronary angiography within 30 days. Paradoxical deceleration was defined as a decrease in heart rate >/=5 beats/min lasting at least 3 min during dobutamine infusion. Perfusion was graded on a five-point scale (0 = normal; 4 = absent uptake) and wall motion on a four-point scale (0 = akinesia/dyskinesia; 3 = normal) using the 20-segment model. Significant coronary artery disease was defined as >/=50% narrowing of lumen diameter of a major epicardial artery. Paradoxical deceleration occurred in 10 patients (19%, 95% CI 8-30%). A decrease in systolic blood pressure [8 patients (80%), 95% CI 56-92%] with angina [5 patients (50%), 95% CI 23-71%] often accompanied the decrease in heart rate. All 10 patients with sinus deceleration had an inferior wall perfusion defect and 8 of them had a corresponding wall motion abnormality on gated images. Significant coronary artery disease was present in all 10 patients with sinus deceleration, with an increased incidence of right coronary artery stenosis (p = 0.007). CONCLUSIONS: Paradoxical deceleration observed during dobutamine perfusion scintigraphy is associated with inducible ischemia and angiographic coronary artery disease and the underlying mechanism for this phenomenon may be the activation of the Bezold-Jarisch reflex. PMID- 10754350 TI - Detection of the earliest ventricular contraction site in patients with Wolff Parkinson-White syndrome using two-dimensional guided M-mode tissue Doppler echocardiography. AB - OBJECTIVE: The purpose of this study was to examine the feasibility of M-mode tissue Doppler imaging for localizing the accessory pathway in patients with Wolff-Parkinson-White (WPW) syndrome. METHODS: Two-dimensional guided tissue Doppler M-mode was recorded at the mitral and tricuspid annular levels in 13 WPW patients. Time intervals were measured from the onset of the delta wave or the R wave to the beginning of the ventricular systolic motion. The earliest contraction site was defined as the site demonstrating the shortest time interval, and compared with the earliest activated site determined by body surface mapping and the successful ablation site. RESULTS: In 6 patients with a left-sided pathway, tissue Doppler localization was identical to the ablation site. In 3 with a left-sided pathway and 3 with a right-sided pathway, localization was judged as an adjacent region of the ablation site. In 1 patient with a right lateral pathway, the pathway location was misdiagnosed. The tissue Doppler diagnosis for the left-sided pathways correlated well with the ablation site, in contrast to the right-sided pathways (p = 0.05). Prediction of the accessory pathway localization by tissue Doppler M-mode was equivalent to localization based on body surface mapping. CONCLUSIONS: In WPW syndrome, tissue Doppler M-mode can detect the earliest contraction sites and seems helpful in localizing the left-sided accessory pathways, but is of limited use for right sided pathways. PMID- 10754351 TI - On the interrelation of exercise blood pressure and heart rate. AB - Formulas are presented which establish a meaningful interrelationship between blood pressure and heart rate during exericse, describing both of them as functions of one variable and a set of common parameters, namely, a quantity interpreted as a no-load effort, and the rest values of heart rate and of systolic and diastolic blood pressure. The formulas can be used, in the place of graphic representations, to define the ranges of normal blood pressure and heart rate quite generally, or for specific populations. They can also be used to define the degree of hypertension of individuals or groups of individuals. The specific formulas and the ranges of normal values presented here, apply to men aged 20-50 years; they may have to be modified for other age groups. PMID- 10754352 TI - Attenuated responses of Doppler-derived hemodynamic parameters during supine bicycle exercise in heart transplant recipients. AB - The aim of this study was to characterize Doppler-derived hemodynamic parameters in heart transplant recipients at rest and during symptom-limited supine bicycle exercise. Eighteen sedentary patients aged 54.0 +/- 2 years, 1.6 +/- 1.0 years following cardiac transplantation, and 18 sedentary healthy volunteers aged 51.8 +/- 4 years were investigated. Basic hemodynamic parameters and Doppler-derived parameters were recorded at rest and at peak dynamic exercise. Resting heart rate, blood pressure and rate-pressure product were higher in the transplanted patients (p < 0.001). However, in comparison with the resting state, the increase in these parameters at exercise was lower in heart transplant recipients. In the healthy, dynamic exercise induced an increase in peak flow velocity, mean acceleration, flow velocity integral, stroke volume, cardiac output and cardiac index (p < 0.001 for all) while systemic vascular resistance, ejection time and acceleration time decreased (p < 0.001 for all). The following parameters increased in the transplanted patients at dynamic exercise: peak flow velocity, cardiac output and cardiac index (p < 0.001), mean acceleration (p < 0.01) and flow velocity integral (p < 0.05). Ejection time decreased (p < 0.05) and acceleration time and systemic vascular resistance remained unchanged. In conclusion, at rest peak flow velocity, mean acceleration, flow velocity integral and stroke volume are lower in the transplanted than in the healthy controls, while cardiac output, cardiac index and systemic vascular resistance are equal. Our study demonstrates attenuated responses of basic hemodynamic parameters and Doppler-derived cardiovascular indices at symptom-limited supine bicycle exercise in heart transplant recipients compared to healthy volunteers. PMID- 10754353 TI - Uni- and bipolar pacing threshold measurements with capturecontrol, a new automatic pacemaker function for capture verification. Comparison of the automatic and the manual pacing threshold measurement. AB - It is mandatory in pacemaker patients to determine the pacing threshold at each follow-up visit. To facilitate the pacing threshold measurements, complete automatic pacemaker tests are being developed. A new pacemaker algorithm for automatic capture verification (Capturecontrol) detects the presence of the evoked response signal 70-100 ms after the pacing pulse. The aim of this study was to determine the uni- and bipolar pacing thresholds using this automatic pacemaker function and compare them with the manually determined threshold. The study included 14 patients with the DDD pacemaker Logos (Biotronik) connected to the high-ohmic ventricular pacing lead Synox SX 60-BP (Biotronik). At predischarge and 8, 14, 20 and 26 weeks after implantation the uni- and bipolar pacing thresholds were assessed manually and with the automatic pacemaker function at 0.4 ms duration. Mean pacing thresholds determined with the automatic pacemaker function were not different from the manually measured values. This applied to uni- and bipolar pacing. Seventy percent of all unipolar and 67% of all bipolar measurements had no deviation. A deviation of 0.1 V between manual and automatic threshold measurement was observed in 25% (unipolar) and 28% (bipolar), respectively. A 0.2-volt difference occurred in 3% in the unipolar measurements. Deviations >/=0.3 V were found in 2% of all unipolar and in 5% of all bipolar measurements. In conclusion, automatic pacing threshold measurements using the Capturecontrol algorithm were similar compared to the manually determined thresholds. The excellent agreement between the two methods was observed for bipolar as well as unipolar pacing and on condition that all patients had a high-ohmic ventricular pacing lead. Therefore, fewer requirements are necessary for Capturecontrol than for presently available systems. Such pacemaker functions can help to speed up the measurements during follow-up visits. PMID- 10754354 TI - A case of constrictive pericarditis with local thickening of the pericardium without manifest ventricular interdependence. AB - This is the first case report of postsurgical constrictive pericarditis confined to the left ventricle in which the majority of diagnosis tests were not indicative of the disease. A 50-year-old woman with a past history of mitral valve replacement was admitted for right heart failure. Cardiac catheterization showed impaired diastolic filling but lacked the characteristic ventricular interdependence recently reported to be specific for the disease, without manifest radiological appearance of pericardial thickening. However, a new technique using magnetic resonance tagging cine revealed pericardial adhesion, limited to the left ventricle, which was confirmed during pericardiectomy. After the surgery, right heart failure and diastolic filling abnormality disappeared with restoration of normal heart pressures. PMID- 10754355 TI - Left ventricular pseudoaneurysm: diagnosis by cine magnetic resonance imaging. AB - We report a case that illustrates the difficulty in diagnosing left ventricular (LV) pseudoaneurysm and the potential value of cine magnetic resonance imaging (MRI). A 69-year-old man with a history of ischemic congestive heart failure had a mass found on routine chest X-ray, contiguous with the cardiac silhouette. Neither CT nor echocardiography accurately defined the mass. The diagnosis of LV pseudoaneurysm was definitely made by cine MRI. Although the accuracy of various imaging modalities for detecting LV pseudoaneurysm is not known, cine MRI was the most useful test in this case. Despite refusing surgery to correct the pseudoaneurysm, the patient survived for nearly 4 years, treated only with anticoagulation. PMID- 10754357 TI - Virtual colonoscopy: a gastroenterologist's perspective. AB - Virtual colonoscopy is an exciting new imaging technique with potential to alter current diagnostic approaches to colonic diseases, particularly colonic neoplasms. Although colonoscopy continues to offer the highest sensitivity and specificity for evaluation of the colon, virtual colonoscopy may offer greater safety, less discomfort, and consequently greater patient acceptance. In addition, virtual colonoscopy offers shorter procedure time, more accurate lesion location, and potentially lower cost as a screening test. Limited data are currently available to define virtual colonoscopy's full clinical role. This article describes the technical considerations, current clinical status, limitations, and potential indications of this new and exciting technology which gastroenterologists should not fear but embrace. PMID- 10754356 TI - Viagra, sexual intercourse and acute myocardial infarction. PMID- 10754358 TI - Evaluation and endoscopic palliation of cholangiocarcinoma. Management of cholangiocarcinoma. AB - Patients with a cholangiocarcinoma usually develop symptoms of biliary obstruction which dominate the course of their illness. At presentation, the diagnosis may be difficult to establish due to the desmoplastic nature of this tumor. Location and extent of the tumor, however, are more effectively evaluated by current radiologic techniques. Following these staging procedures, most patients are found to be unsuitable for curative resection. Therefore, establishing effective biliary drainage to relieve symptoms of obstruction becomes the most critical therapeutic goal in these patients. In the past, surgical biliary bypass procedures were advocated, but were associated with high perioperative morbidity and mortality rates. Endoscopic biliary stenting produces quick and effective relief of symptoms from biliary obstruction due to cholangiocarcinoma. Initially, successful biliary drainage is possible in most patients. However, subsequent stent occlusion or spread of tumor into nonstentable intrahepatic ducts leads to recurrent jaundice. Local therapies including (192)Ir brachytherapy and photodynamic therapy may prevent or postpone these problems. PMID- 10754359 TI - Inflammatory bowel disease and pregnancy: a review. AB - Inflammatory bowel disease often occurs during peak reproductive years. Rates of conception, pregnancy outcome and disease course during pregnancy should be discussed prior to attempted conception. The majority of patients whose disease is well controlled prior to pregnancy should expect a fertility rate comparable to the general population, and an uncomplicated pregnancy with a favorable outcome. The disease should continue to be pharmacologically or surgically controlled as necesssary during pregnancy; the majority of drug options available to pregnant patients being without detriment to the fetus. No predictable inheritance pattern has been established and, at this time, there is no ability to screen prenatally. PMID- 10754360 TI - Impact of inflammatory bowel disease on health-related quality of life. AB - Health-related quality of life (HRQOL) has a recognized importance to evaluate, manage and follow patients. Different types of instruments for measuring HRQOL have recently been introduced to evaluate HRQOL in patients with inflammatory bowel disease. Most questionnaires suggest that inflammatory bowel disease has a significant impact in HRQOL. It seems that ulcerative colitis has less profound effects on HRQOL than Crohn's disease and that some differences in HRQOL status are related to some factors such as the severity of the disease. When patients express their concerns, most important worries are related to surgery. Effective medical treatment appears to improve HRQOL. Effects of surgery on HRQOL can probably be different depending on the type of surgical technique and if it is curative or not. Recent studies suggest that inflammatory bowel disease not only impacts on patient's HRQOL but also on their caregivers. Features such as the impact on HRQOL of different disease variables, such as complications or patient personality, the role of medical or surgical treatments on different dimensions of HRQOL, the cost-utility evaluation of therapy are some of the fields that will probably focus the protagonism in the next years. PMID- 10754361 TI - Symptom priority ranking in the care of gastroesophageal reflux: a review of 1,850 cases. AB - BACKGROUND: Clinical history remains an important part of the medical evaluation of patients with gastroesophageal reflux disease (GERD). Heartburn, regurgitation, and dysphagia are considered typical symptoms of GERD. Priority rankings of these symptoms can be determined with a standardized questionnaire. OBJECTIVE: To determine whether symptom priority ranking and symptom severity grading can provide useful information in the evaluation of patients with GERD. METHODS: From 1,850 patients that were analyzed retrospectively, patients with dysphagia unrelated to GERD were excluded. A standardized questionnaire was applied before each patient underwent any esophageal diagnostic study. Priority of symptoms was determined to be primary, secondary, tertiary, or none based on the patient response to the questionnaire. Presence of a stricture was determined either by endoscopy, esophagraphy, or both studies. Stationary esophageal manometry and 24-hour pH monitoring were performed on all patients. Through bivariate and multivariate analysis, the relationships among typical GERD symptoms, esophageal reflux-related stenosis, lower esophageal sphincter pressure, and composite score were established. RESULTS: High priority ranking of the symptom dysphagia is predictive of the presence of an esophageal stricture, but has a negative association with abnormal manometric and pH studies. In contrast, high priority ranking of the symptom heartburn and regurgitation are positively associated with abnormal manometric and pH results. CONCLUSIONS: Priority ranking can be a valuable adjunct to objective testing in the evaluation of GERD. In certain clinical situations it can obviate the need for 24-hour pH monitoring. PMID- 10754362 TI - Frequency and clinicopathology associations of K-ras mutations in colorectal cancer in a northeast Mexican population. AB - Activation of the ras family gene has been implicated in colorectal tumorigenesis, K-ras being the most frequently altered gene. The frequency of K ras codon 12, 13 and 61 point mutations in patients with colorectal neoplasias was examined. We employed a polymerase chain reaction-restriction fragment length polymorphism assay and single-strand conformational polymorphism to detect mutations. We found that point mutations at codons 12 and 13 were present in 53% and 39% of the tumors, respectively, but none at codon 61. These results agree with previous reports. Point mutations were more frequent in adenomas than in carcinomas, with villous adenomas presenting a higher incidence of mutations than other adenomas. The association between clinical and histopathological parameters was investigated. Our study is the beginning of a new research line in molecular epidemiology of colorectal cancer and is the first to be carried out in one part of the Mexican population. PMID- 10754363 TI - A functional outcome swallowing scale for staging oropharyngeal dysphagia. AB - OBJECTIVE: To develop a simple and straightforward functional outcome swallowing scale (FOSS) for patients with oropharyngeal dysphagia to determine the severity of the disorder and the effectiveness of therapy or outcome. DESIGN: Five years ago, the author developed a dysphagia staging scale based on personal experience and a review of the English literature. This scale was shared with colleagues from the specialties of otolaryngology, speech pathology, neurology, and gastroenterology, both within and outside the author's institution. Minor modifications have been made. The scale has been used in clinical management and retrospective studies. SETTING: PATIENTS were seen in a multispecialty, tertiary care, academic center. PATIENTS: The patient population included the full spectrum of oropharyngeal dysphagia in adults, but was weighted heavily toward aging patients and patients with head and neck cancer, neurologic disorders, gastroesophageal conditions, and psychiatric problems. RESULTS: The stages are as follows: stage 0 = normal function and asymptomatic; stage I = normal function but with episodic or daily symptoms of dysphagia; stage II = compensated abnormal function manifested by significant dietary modifications or prolonged mealtime (without weight loss or aspiration); stage III = decompensated abnormal function with weight loss of 10% or less of body weight over 6 months due to dysphagia, or daily cough, gagging, or aspiration during meals; stage IV = severely decompensated abnormal function with weight loss of more than 10% of body weight over 6 months due to dysphagia, or severe aspiration with bronchopulmonary complications, nonoral feeding recommended for most of nutrition, and stage V = nonoral feeding for all nutrition. CONCLUSIONS: The FOSS was successful for staging various adult patients with dysphagia into clinically useful, overall performance categories. It has been applied successfully to retrospective outcome studies and to clinical management by clinicians from different specialties. PMID- 10754364 TI - Renal function in compensated hepatic cirrhosis: effects of an amino acid infusion and relationship with nitric acid. AB - AIMS: In order to assess the possible participation of nitric oxide (NO) in renal function during compensated hepatic cirrhosis, we studied renal function, the plasma and urinary levels of cGMP and the concentration of nitrates and nitrites, as markers of NO synthesis in blood and urine, in 10 patients with Child A hepatic cirrhosis as compared with 10 control subjects, both under basal conditions and during stimulation (amino acid-induced glomerular hyperfiltration). METHODS: To study renal function, the glomerular filtration rate (GFR), effective renal plasma flow (ERPF), renal functional reserve (RFR), renal venous resistance (RVR) and the filtration fraction (FF) were measured. Renin and aldosterone levels were determined to assess the possible involvement of these compounds in the renin-angiotensin-aldosterone axis. RESULTS: GFR and ERPF were significantly lower in the patients with cirrhosis than in the controls (mean GFR: 82+/-12.3 vs. 105+/-15 ml/min, p = 0. 01; ERPF 452+/-86 vs. 543+/-56 ml/min, p = 0.002). The RFR value was similar in both groups. In the basal situation cGMP levels were higher in plasma and urine in patients with cirrhosis than in the controls (plasma cGMP in cirrhosis 8.4+/-2.4 vs. 4.2+/-3.5 pmol/ml; urine cGMP in cirrhosis 1.2+/-2.1 vs. 0.68+/-0.1 pmol/ml). The NO levels were also higher in plasma and urine in patients with cirrhosis vs. controls (plasma NO in cirrhosis 45.5+/-9.2 vs. 30. 3+/-1.2 micromol/l; urinary NO in cirrhosis 6.2+/-1.3 vs. 3.1+/-2.3 micromol/ml). In both groups the amino acid perfusion increased GFR, ERPF, cGMP and NO levels in plasma and urine. In the patients with cirrhosis the RVR decreased significantly during perfusion and no noteworthy changes in FF were observed. The GFR values observed during amino acid perfusion were similar in patients with cirrhosis and portal hypertension to those observed in the controls (27.2+/-12 vs. 25.3+/-16%). However, the changes induced the ERPF were more marked in patients with cirrhosis (cirrhosis 35.3+/-15 vs. 22. 2+/-13%, p = 0.02). CONCLUSIONS: The present findings point to certain alterations in renal function in patients with hepatic cirrhosis and portal hypertension without ascitis, a clear difference being visible between the ERPF and GFR following amino acid-induced stimulation. The significant elevation in cGMP and NO levels in plasma and urine implies a maintained vasodilatory action that may at least partly compensate the vasoconstrictor effects of angiotensin II. PMID- 10754365 TI - Induced-hyperglycemia attenuates erythromycin-induced acceleration of hypertonic liquid-phase gastric emptying in type-I diabetic patients. AB - BACKGROUND: Erythromycin has been found to be a gastrointestinal prokinetic agent of hypertonic liquids, while acute hyperglycemia has been associated with delayed gastric emptying in diabetic patients. AIM: To investigate whether hyperglycemia, per se, reduces gastric motility during erythromycin-induced acceleration on gastric emptying of hypertonic liquids in diabetic patients. METHODS: In 12 type I diabetic patients following a hypertonic radiolabeled liquid meal, gastric emptying was measured scintigraphically during normoglycemia (5-8.9 mmol/l glucose) or hyperglycemia induced by intravenous (16-19 mmol/l) glucose infusion. The tests were performed on 4 separate days in random order after administering either placebo or 200 mg i.v. erythromycin. RESULTS: In the hyperglycemic state compared to normoglycemia, the gastric emptying of the hypertonic liquid was reduced after placebo or erythromycin administration. The lag-phase duration (17.8+/-5.5 and 7.8+/-4.5 vs. 10.8+/-3.4 and 3.7+/-2.5 min, respectively, p<0.001), the overall gastric emptying time of the half meal (52.8+/-13 and 24.9+/-5.5 vs. 42.5+/-10.5 min and 16.6+/-6 min, respectively, p<0.001) and the retained percentage of liquid meal in the stomach at 60 and 100 min postprandially (p<0.001) were significantly increased. CONCLUSIONS: The erythromycin-induced acceleration on gastric emptying of hypertonic liquids in diabetic patients is related to the plasma glucose level. The induced hyperglycemia reduces the erythromycin-induced acceleration of liquid-phase gastric emptying, decreasing the overall gastric emptying rate. In spite of the inhibitory effect of induced hyperglycemia on the gastric emptying of hypertonic liquids, erythromycin is still able to accelerate the emptying rate and could prove to be a useful prokinetic agent under hyperglycemic conditions. PMID- 10754366 TI - A large spontaneous splenorenal shunt in a patient with liver cirrhosis and uncomplicated portal hypertension. AB - The aim of this paper is to describe and discuss, on the basis of a thorough review of the literature, the case of a 70-year-old woman with probable cirrhosis secondary to chronic hepatitis B and C, uncomplicated portal hypertension (without ascites, encephalopathy or bleeding varices), splenomegaly and hypersplenism, and an unusual, spontaneous, large splenorenal shunt and recanalization of the umbilical vein. The tortuous and varicose splenorenal shunt was diagnosed by abdominal ultrasound and CT investigations. A duplex Doppler ultrasonography evaluation was performed to study shunt flow direction and velocity. No gastroesophageal varices were identified on endoscopic examination. The clinical relevance of spontaneous splenorenal shunt, often associated with fundic gastric varices, is discussed. PMID- 10754367 TI - Boerhaave's syndrome. PMID- 10754368 TI - Aged mice exhibit greater mortality concomitant to increased brain and plasma TNF alpha levels following intracerebroventricular injection of lipopolysaccharide. AB - BACKGROUND: Age-related defects in the development of peripheral inflammatory responses have been observed in rodents and humans. OBJECTIVE: We examined the effects of age on a centrally injected endotoxin-induced cytokine production and cellular activation in mice. METHODS: Male C57BL/6J (B6) mice, C3H/HeN mice, and C3H/HeJ mice received an intracerebroventricular injection of lipopolysaccharide (LPS) and were sacrificed at various times (2, 4, 8 h) thereafter. ELISA for IL 1beta, IL-6, IL-12, and TNF-alpha were conducted on forebrain tissue homogenates as well as plasma samples, and lectin staining to detect activated microglia was prepared for selected brain slices. RESULTS: Intracerebroventricular injection of LPS in B6 mice produced an age-associated increase in mortality which was paralleled with a significant increase in brain and plasma levels of TNF-alpha. AntiTNF-alpha- and IL-6-immunoreactive cells possessed macrophagelike morphologies and were observed along the LPS injection tract and scattered throughout the hilus of the dorsal hippocampus and cerebral cortices. This LPS mediated response was found to be specific in that the LPS-hyporesponsive mouse strain (C3H/HeJ) failed to demonstrate significant brain or plasma levels of TNF alpha after LPS administration compared to C3H/HeN mice. CONCLUSION: These results suggest that the age-related increases in TNF-alpha production and mortality following the intracerebroventricular administration of LPS may be due to an increased endotoxin hypersensitivity of brain microglia/macrophages within aged animals. PMID- 10754369 TI - Initial symptoms of Parkinson's disease with elderly onset. AB - BACKGROUND/OBJECTIVE: As the incidence of Parkinson's disease (PD) is related to aging, we consider it important to determine how the initial symptoms change with age in order to diagnose early elderly cases of PD accurately. METHODS: 84 patients (age at onset 70.7+/-9.0 years; mean +/-1 SD) were studied to see whether the initial symptoms change according to age. RESULTS: The prevalence of resting tremor was significantly lower in patients of advanced age (p = 0.041). In contrast, the incidence of postural and gait disorders increased significantly with aging (p = 0.032). The prevalences of rigidity and kinetic disorders, which are important clinical features of PD, were not influenced by aging. CONCLUSION: These findings suggest that the cause of PD is not related to the aging process itself, since the prevalences of all symptoms were not influenced by aging. Knowledge of the prevalence of the initial symptoms of PD may contribute to the accurate diagnosis in early and elderly cases. PMID- 10754370 TI - Use of in-patient hospital beds by people living in residential care. AB - BACKGROUND: There is concern that people living in residential care in Australia make significant and often inappropriate use of acute in-patient hospital services. To date, no factual information has been collected in Australia and its absence may allow myths and negative stereotypes to proliferate. OBJECTIVE: To determine how and why people living in residential care in Australia use in patient hospital beds. To determine the outcome of hospitalisation and functional status at 3 months following discharge. METHODS: Prospective study of 184 consecutive admissions to hospital following Emergency Department (ED) attendance involving people aged over 65 years and living in residential care in southern Adelaide, South Australia. Information was obtained from the facilities' transfer letters, and where these were inadequate or absent, telephone interviews were held with residential care staff. RESULTS: 153 people accounted for the 184 admissions. They had a mean age of 84 years and 69% were female. 61% came from hostels and 35% from nursing homes. They had a wide range of clinical problems and twice as many were admitted to medical than to surgical units. Their mean length of hospital stay was 7.9 days, 2.3 days higher than for non-same-day patients and was higher for hostel than for nursing home residents. All but two admissions were considered unavoidable though the provision of specialised care within residential care could have prevented a further 19 (10%) admissions. 96% of admissions resulted in survival to leave hospital and in 74%, people returned directly to their place of origin. At 3 months follow-up, a further 20% of the group had died while 5% were in hospital. In all, 14% of the original group were in a different long-term care facility while 56% were living at their former residence. CONCLUSIONS: People living in residential care are often hospitalised because of acute illness. In the vast majority of cases hospitalisation is both appropriate and unavoidable. Most did not require prolonged hospitalisation and were discharged alive, usually to their original residence. However, within 3 months many had died or had functionally declined. Strategies that prevent health breakdown in the residential care setting need to be developed and trialed. PMID- 10754371 TI - Nutritional status according to mini nutritional assessment in an institutionalized elderly population in Sweden. AB - BACKGROUND: In 1992, local municipalities in Sweden took over full responsibility for the long-term care of elderly. This has led to an increased care burden for the various assisted accommodation services run by the municipalities. OBJECTIVE: Since ageing and chronic diseases are risk factors for protein-energy malnutrition, we evaluated the nutritional status of all individuals in assisted accommodation, i.e., service flats (SF), old people's homes (OPH), group living for the demented (GLD), and nursing homes (NH), in three Swedish municipalities. METHODS: Of 994 eligible subjects, 872 were examined; the average age was 84.5+/ 8 years, and 69% were female. The Mini Nutritional Assessment (MNA) scale (0-30 points) was used, consisting of 18 point-weighted questions in four categories, i.e., anthropometry, global and dietary issues, and self-assessment. RESULTS: MNA <17, i.e., malnutrition, was noted in 36% of the study population. Divided according to accommodation type, the MNA scores were <17 in 21% of individuals in SF, 33% of those in OPH, 38% of those in GLD, and 71% of those in NH. The corresponding values for MNA scores 17-23.5 (risk for malnutrition) were 49, 51, 57, and 29%, respectively. Average body mass index (BMI) values were 24.2+/-5 (SF), 23.6+/-5 (OPH), 23.9+/-4 (GLD), and 22.3+/-4 (NH). BMI values < or =20 were found in 18% of those in SF, in 25% of those in OPH, in 19% of those in GLD, and in 33% of those in NH. Both MNA and BMI correlated with upper arm and calf circumference, with r values ranging from 0.4 to 0.7 (p<0.001). MNA and BMI correlated significantly (r = 0.52, p<0.001). Age correlated with MNA and BMI with r values of 0.1 (p<0.01) and 0.14 (p < 0.001), respectively. Subjects with significant help requirements during meals ate fewer whole meals per day than those who could feed themselves. CONCLUSIONS: Based on the MNA, one third of the study subjects living in assisted accommodation, and more than half of those living in NH, appeared to be malnourished. Further studies are necessary to assess to what extent these nutritional disturbances are reversible. PMID- 10754372 TI - Health and relicensing policies for older drivers in the European union. AB - BACKGROUND: The number of older drivers is increasing rapidly in the developed world. Many states of the European Union (EU) have initiated legislation to screen older drivers for age-related disease, despite evidence that screening may be harmful or have no positive effect. METHODS: We reviewed the current situation in the EU by sending a questionnaire to the Driver and Vehicle Licensing Agency in each member state. RESULTS: All fifteen countries replied. Regular medical screening occurs in ten. European Council Directive 91/439/EEC forms the basis for national guidelines on medical fitness to drive, but these are not specific for age-related illness, and interpretation of the directive varies between countries. Specific guidelines are not available in all countries for the age related conditions of dementia, stroke, and Parkinson's disease. In nine countries a medical colleague is available for consultation in difficult cases. Drivers are required to report illness to the Driver and Vehicle Licensing Agency only in the United Kingdom. In six countries, there is mandatory reporting by physicians of conditions which may pose a risk to driving. CONCLUSIONS: There is considerable heterogeneity within the EU on the processes of older driver screening. This diversity may facilitate research into the effect of different policies on safe mobility of older people. If changes in the screening process are considered, further knowledge is needed on issues such as older driver exposure and valid measures of safe mobility for the EU. More evidence-based research is required to help policy makers frame future guidelines and legislation, so as to promote safe mobility of older people. PMID- 10754373 TI - The adherent gastric antral and duodenal mucus gel layer thins with advancing age in subjects infected with Helicobacter pylori. AB - BACKGROUND: Peptic ulceration and Helicobacter pylori infection increase with advancing age. In the upper gastro-intestinal tract the first line of mucosal defence is the adherent mucus gel layer. OBJECTIVE: We have examined, using a novel histological fixation technique, the thickness of the adherent mucus gel layer (1) in the gastric antrum and (2) in the duodenum in relation to advancing age and H. pylori status. METHODS: The subjects had macroscopically normal stomach and duodenum at endoscopy. Measurement of the gastric antral mucus thickness was carried out on four antral biopsy specimens from within 2 cm of the pylorus (H. pylori positive n = 25, negative n = 21). The duodenal mucus thickness (D1) was measured from two biopsy specimens (H. pylori positive n = 7, negative n = 13). All specimens were snap frozen and cryostat sections stained using a modified PAS/AB stain. RESULTS: In all sections the mucus layer was continuous. In both duodenum and gastric antrum, the mucus thickness was not significantly different between H. pylori positive and H. pylori negative age matched samples. In duodenum and gastric antrum from H. pylori negative subjects, there was no correlation between mucus thickness and age. However, in H. pylori positive subjects, there was a significant thinning of the adherent gastric antral mucus gel layer (p = 0.005, r = -0.54) and the duodenal mucus thickness (p<0.001, r = -0.99) with advancing age. CONCLUSION: This study shows a significant thinning of the adherent mucus gel layer in H. pylori positive individuals, as stomach and duodenum age. In those without H. pylori, the mucus gel thickness is preserved in stomach and duodenum. PMID- 10754374 TI - Falls on integrated medical wards. AB - BACKGROUND: An effective approach to fall prevention should involve an assessment of environmental as well as patient-related characteristics. OBJECTIVE: To study the effect of age and ward design on fall characteristics among medical inpatients. METHODS: In a prospective open observational study over 1 year, we studied falls on three medical wards. Wards A and B are nuclear designed, and C is longitudinal. RESULTS: We recorded 199 falls involving 167 fallers. Fifty-four (27.1%) involved patients under 65 years. Most falls were intrinsic (60.8%) and involved elderly male patients (male/female ratio 97/48 vs. 24/30; p = 0.009). We identified no age differences in relation to location, activity, preceding fall, classification, time, consequences, and intervention required. On ward C, most falls occurred in the bed areas (bays and cubicles), but on wards A and B a higher proportion occurred in bathroom, corridor, and dayroom (C vs. A/B 87.9 vs. 73.7/62.0%; p = 0.04/p = 0.004). On ward C, activities of daily living around the bed significantly preceded falls (C vs. A/B 44.6 vs. 25.9/24.1%; p = 0.03/p = 0.01). Most falls were unwitnessed (C vs. A/B 10 vs. 21/20; p = 0.002/p = 0.0009). CONCLUSIONS: Intrinsic falls are the commonest; however, differences exist in fall demographics between wards, and this must be recognized to enhance the effectiveness of fall prevention programmes. PMID- 10754375 TI - Education, activity, health, blood pressure and apolipoprotein E as predictors of cognitive change in old age: a review. AB - BACKGROUND: Cross-sectional studies have identified a number of health and lifestyle factors that correlate with cognitive function in old age. It is important to know whether these factors also predict change in cognitive function over time. OBJECTIVE: To evaluate published findings on genetic, health and lifestyle predictors of cognitive change in late adulthood. METHODS: Studies reporting data on education, health, blood pressure, activity and apolipoprotein E (APOE) as predictors of cognitive change were reviewed. A total of 34 studies were identified that reported data on these predictors. The average follow-up period of studies reviewed was 7.68 years. RESULTS: Results showed that education, hypertension, objective indices of health and cardiovascular disease, and APOE were associated with cognitive change. Results regarding the effect of physical activity on cognitive change were inconclusive. CONCLUSION: Despite the lack of data on some predictors, the longitudinal trends are generally consistent with cross-sectional findings on predictors of cognitive performance in old age. More research is required to compare the influence of predictors on different types of cognitive abilities. PMID- 10754376 TI - Infusion phlebitis in post-operative patients: when and why. AB - BACKGROUND: The most common complication of intravenous therapy is infusion phlebitis. This study was done to prospectively assess its frequency in a series of consecutive patients who will undergo surgery, and to identify which variables may predict an increased risk for phlebitis. PATIENTS AND METHODS: 400 consecutive patients who will undergo surgery in a general surgery department were included. Only the first catheter, inserted the day before surgery, was taken into account. Eighteen variables (from the infusion, the catheter and from the patient) were prospectively evaluated for their contribution to the occurrence of phlebitis. RESULTS: 60/400 patients (15%) developed phlebitis, and most of them needed insertion of a further catheter. The univariate analysis showed that patients who developed phlebitis were older, and their pre-operative levels of both blood haemoglobin and neutrophil cound were significantly higher than those in patients who did not develop phlebitis. However, the multivariate analysis only confirmed the association with blood haemoglobin levels: the risk of phlebitis sharply increased in the patients with the highest haemoglobin levels. As to the influence of time on phlebitis development, there was a significant decrease in the day-specific risk, from the 5th day on. COMMENTS: In our series, blood haemoglobin levels were found to be the only variable associated to a higher risk of phlebitis. Besides, in contrast with the recommendations by the Centers for Disease Control, no significant increase in the day-specific risk of phlebitis was found. Thus, a guideline to select the type of catheter to be inserted in an individual patient is suggested. PMID- 10754377 TI - Effect of ticlopidine on platelet-derived microparticles in patients with connective tissue diseases. AB - We evaluated the plasma concentrations of platelet activation markers and platelet-derived microparticles (PMP) in patients with connective tissue diseases complaining of peripheral circulation disorders (n = 16) and studied the effect of ticlopidine hydrochloride (ticlopidine) on PMP generation. There were significant differences in the levels of PMP and a platelet activation marker between before and after treatment with ticlopidine (PMP: 695 +/- 393 vs. 354 +/- 206/10(4) platelets, p < 0. 01; platelet CD63: 9.13 +/- 5.64 vs. 5.22 +/- 2.74%, p < 0.05). On the other hand, markers of vascular endothelium, such as vascular endothelium-derived small vesicles and serum thrombomodulin levels, were not affected by the administration of ticlopidine. Levels of cytokines and soluble adhesion molecules remained unchanged by ticlopidine administration. These findings suggest that ticlopidine may be useful for the inhibition of PMP dependent vascular damage in patients with connective tissue diseases complaining of peripheral circulation disorders. PMID- 10754378 TI - Combined administration of dextran 70 and dalteparin does not increase perioperative blood loss compared to dextran 70 alone in major orthopedic surgery. AB - A prospective open-labeled clinical study was carried out to compare the safety of dextran 70 and low molecular weight heparin (dalteparin; DD group) versus dextran 70 alone (D group) in patients subjected to elective hip replacement surgery. Dalteparin, 5,000 IU/day and dextran 70, 500 ml during surgery and on the first postoperative day were administered to 214 patients. Dextran 70 alone was infused in 44 patients, 500 ml during surgery and on the 1st, 3rd and 5th postoperative day. Mean total blood loss during the operation and until the 2nd postoperative day was 1,708 ml in the DD group and 1,712 ml in the D group (p = 0.79). During the 1st postoperative week, no group differences were found in the relative number of patients that received packed red blood cells (p = 0.95), the amount of transfused packed red blood cells (p = 1.0) and changes in hemoglobin concentrations (p = 0.69). The present results suggest that dextran 70 and dalteparin can be combined in recommended doses without significantly increasing perioperative bleeding in patients undergoing hip replacement surgery. Bone traumatization and insufficient plugging of surgical traumatized bone surfaces with bone cement favor bleeding. Further well-designed studies are needed to evaluate the safety and efficacy of this regimen. PMID- 10754379 TI - Comparison of the effects of acetylsalicylic acid, ticlopidine and cilostazol on primary hemostasis using a quantitative bleeding time test apparatus. AB - We examined and compared the effects of aspirin (ASA), ticlopidine (TP) and cilostazol (CS) on bleeding time (BT) in 10 healthy adult male subjects using a newly developed quantitative bleeding time (QBT) test apparatus capable of simultaneously measuring total blood loss (Tv), maximum bleeding rate (Rmax), and bleeding pattern in addition to BT. All 3 drugs inhibited platelet aggregation response to ADP, collagen, epinephrine and arachidonic acid (p < 0.05), but not to ristocetin. Following oral administration of ASA (330 mg/day) or TP (300 mg/day) for 3 days, BT was significantly prolonged (mean BT increased from 359.3 to 646.0 s, p < 0.001, and from 323.3 to 528. 7 s, p < 0.01, respectively) and Tv was significantly increased (from 14.5 to 30.2 microl, p < 0.05, and from 12.5 to 19.2 microl, p < 0.01, respectively). Aspirin also increased Rmax (from 0.118 to 0. 159 microl/s, p < 0.05). The prolonged bleeding patterns after administration of ASA and TP were both type III, which has been reported to be less likely to lead to bleeding accidents. In contrast, none of these QBT parameters were altered by CS administration. PMID- 10754380 TI - Soluble P-selectin and proinflammatory cytokines in patients with polygenic type IIa hypercholesterolemia. AB - Plasma soluble P-selectin (sP-selectin), beta-thromboglobulin (beta-TG), von Willebrand Factor (vWF), prothrombin factor 1+2 (F1+2), IL-6 and IL-1beta levels were analyzed in 35 consecutive patients with polygenic type IIa hypercholesterolemia (HC) and 35 age- and sex-matched healthy subjects. sP selectin (p < 0.005), beta-TG (p < 0.05) and IL-1beta (p < 0.02) levels were higher in HC patients than healthy subjects whereas no significant difference was observed for vWF. sP-selectin directly correlated with beta-TG (p < 0.05) and IL 1beta levels (p < 0.005), but not with the other variables analyzed. A direct correlation was observed between F1+2 and IL-6 (p < 0.05), total cholesterol (p < 0.05) or LDL cholesterol (p < 0.05). We conclude that HC is associated with an increase of plasma sP-selectin levels, and that sP-selectin may be considered as a marker of in vivo platelet activation in type IIa polygenic HC. The correlations observed among the variables analyzed in the study suggest that proinflammatory cytokines might play a role in the prothrombotic state often associated with HC. PMID- 10754381 TI - Four cases of bleeding diathesis in children due to congenital plasminogen activator inhibitor-1 deficiency. AB - Congenital plasminogen activator inhibitor-1 (PAI-1) deficiency is an extremely rare disorder characterized by a bleeding diathesis that begins in childhood due to hyperfibrinolysis as a result of decreased PAI-1 activity. We now present 4 unrelated pediatric cases of congenital PAI-1 deficiency. All 4 patients had a history of recurrent episodes of subcutaneous bleeding beginning in early childhood. These episodes were characterized by abnormal prolonged bleeding after trauma, tooth extraction, and surgical procedures, as well as by rebleeding following initial hemostasis. The 2 female patients both had symptoms compatible with hypermenorrhea. The family history was positive in 2 of the 4 patients. Hemostatic screening studies in all 4 patients revealed no abnormalities. Testing for factor XIII antigen, von Willebrand factor antigen, ristocetin cofactor activity, alpha(2)-plasmin inhibitor (alpha2PI) activity, and plasminogen activity was normal. The euglobulin lysis times were shortened in all cases as compared with those in normal control subjects. None of the patients had elevated tissue plasminogen activator (tPA) antigen levels, but PAI activity was markedly decreased in all cases. Three of the patients also had reduced levels of PAI-1 antigen. There tended to be a reduction in tPA-PAI-1 complex in all cases. In addition, 2 patients had elevated PIC (plasmin-alpha2PI complex). Tourniquet tests were performed in 2 patients, with no appreciable rise in PAI-1 activity or PAI-1 antigen levels. The administration of tranexamic acid clearly improved hemorrhagic symptoms in these patients. We considered PAI-1 deficiency to be the likely etiology of the congenital bleeding diatheses in these 4 cases. PMID- 10754382 TI - The FVII activating protease cleaves single-chain plasminogen activators. AB - A serine protease isolated from plasma sharing structural characteristics with a hepatocyte growth factor activator-like protease has been demonstrated recently to activate FVII. Accordingly, it was named 'FVII activator'. Until now an impact of this protease on the fibrinolytic system has not been reported. We islolated the protease from cryo-poor plasma by subsequent ion exchange chromatography and adsorption to immobilized heparin and/or aprotinin. Incubation of single-chain plasminogen activators (sc-PAs) with the FVII activator revealed significant activation of urokinase sc-PA (scu-PA) and tissue sc-PA (sct-PA) in vitro. It was enhanced in the presence of calcium and heparin. Compared to kallikrein, a more efficient activation of scu-PA was observed, whereas sct-PA appeared to be a poorer substrate for the FVI activator. At low protease concentrations and in the presence of heparin the scu-PA activation was comparable to plasmin. Employing recalcified whole blood thrombelastography, the lysis of initially formed fibrin was observed after addition of a combination of scu-PA and the FVII activator, whereas the scu-PA alone had a negligible effect at the concentration used. The study results as presented demonstrate that the FVII activator is a potent activator of sc-PAs in vitro. Whether it plays a physiological role in fibrinolysis deserves further investigation. Its comparatively high affinity to heparin assumes a function in cell surface or matrix events. PMID- 10754383 TI - Genetic reassortment and patch repair by recombination in retroviruses. AB - Retroviral particles contain a diploid RNA genome which serves as template for the synthesis of double-stranded DNA in a complex process guided by virus-encoded reverse transcriptase. The dimeric nature of the genome allows the proceeding polymerase to switch templates during copying of the copackaged RNA molecules, leading to the generation of recombinant proviruses that harbor genetic information derived from both parental RNAs. Template switching abilities of reverse transcriptase facilitate the development of mosaic retroviruses with altered functional properties and thereby contribute to the restoration and evolution of retroviruses facing altering selective forces of their environment. This review focuses on the genetic patchwork of retroviruses and how mixing of sequence patches by recombination may lead to repair in terms of re-established replication and facilitate increased viral fitness, enhanced pathogenic potential, and altered virus tropisms. Endogenous retroelements represent an affluent source of functional viral sequences which may hitchhike with virions and serve as sequence donors in patch repair. We describe here the involvement of endogenous viruses in genetic reassortment and patch repair and review important examples derived from cell culture and animal studies. Moreover, we discuss how the patch repair phenomenon may challenge both safe usage of retrovirus-based gene vehicles in human gene therapy and the use of animal organs as xenografts in humans. Finally, the ongoing mixing of distinct human immunodeficiency virus strains and its implications for antiviral treatment is discussed. PMID- 10754384 TI - Role of dual pacemaker mechanisms in sinoatrial node discharge. AB - We investigated whether in the sinoatrial node (SAN) there are two different pacemaker mechanisms and whether either one can maintain spontaneous discharge. These questions were studied by means of an electrophysiological technique and of blockers of different diastolic currents in rabbit and guinea pig isolated SAN. In SAN subsidiary pacemakers of both species, Cs(+) (5-10 mM) or high [K(+)](o) (10-12 mM) decreased the maximum diastolic potential, abolished diastolic depolarization (DD) at polarized levels (subsidiary DD), unmasked a U-shaped dominant DD at depolarized levels, but did not stop the SAN. In rabbit SAN, E4031 (1 microM) and d-sotalol (100 microM) did not stop discharge, but did so after block of subsidiary DD by high [K(+)](o) or Cs(+). In guinea pig SAN, in Tyrode solution E4031, d-sotalol or indapamide (100 microM) did not stop SAN discharge. In the presence of Cs(+) or high [K(+)](o) indapamide (but not E4031 or d sotalol) stopped the SAN. Ba(2+) (1-5 mM) led to stoppage of discharge both in Tyrode solution and in high [K(+)](o) or Cs(+). Depolarization by blockers of DD unmasked sinusoidal fluctuations, which during recovery were responsible for resumption of discharge. We conclude that in rabbit and guinea pig SAN, two different pacemaker mechanisms (Cs(+)- and K(+)-sensitive subsidiary DD, and Cs(+)- and K(+)-insensitive dominant DD) can independently sustain discharge, but block of both mechanisms leads to quiescence. Abolition of dominant DD by blockers of I(K) is consistent with a decay of I(K) as the dominant pacemaking mechanism, I(Kr) being more important in rabbit and I(Ks) in guinea pig. Sinusoidal fluctuations appear to be an essential component of the pacemaking process. PMID- 10754385 TI - Investigations of the effects of ethanol on warfarin binding to human serum albumin. AB - Ethanol effects on warfarin binding to human serum albumin (HSA) have been studied by equilibrium dialysis and fluorescence methods at pH 7.4 in phosphate buffered saline at 37 degrees C. In the presence of various amounts of ethanol fluorescence intensity of bound warfarin decreased significantly but this intensity reduction was not solely from displacement of bound warfarin from HSA. By comparing fluorescence and equilibrium dialysis data we concluded that fluorescence intensity reduction of warfarin was mainly the result of changes in the surrounding environment of the warfarin binding site by ethanol interaction with HSA and that displacement of bound warfarin was not significant compared to the fluorescence intensity changes. The dissociation constant of warfarin binding to HSA decreased with an increasing amount of ethanol. From the changes in fluorescence intensity upon warfarin binding to HSA with the presence of ethanol ranging from 0 to 5.0% the following dissociation constants (Kd) were determined: 0% ethanol 5.39 +/- 0.2 microM, 0.1% ethanol 5.86 +/- 0.1 microM, 0.3% ethanol 5.83 +/- 0.2 microM, 0.5% ethanol 6.76 +/- 0.1 microM, 1% ethanol 7.01 +/- 0.1 microM, 3% ethanol 9.9 +/- 0.7 microM, 5% ethanol 13.01 +/- 0.1 microM. From the equilibrium dialysis with the same ranges of ethanol presence the following Kd values were obtained: 0% ethanol 6. 62 +/- 1.6 microM, 0.1% ethanol 6.81 +/- 1.1 microM, 0.3% ethanol 8. 26 +/- 2.5 microM, 0.5% ethanol 8.86 +/- 1.9 microM, 1% ethanol 11. 01 +/- 4.2 microM, 3% ethanol 20.75 +/- 2.4 microM, 5% ethanol 21.67 +/- 2.2 microM. The results suggest that warfarin bound to HSA was displaced by ethanol. These data indicate that ethanol influence on warfarin binding to HSA may alter the pharmacokinetics of warfarin. PMID- 10754386 TI - The role of endoplasmic reticular Ca(2+) stores in cell viability and tumor necrosis factor-alpha production of the murine macrophage RAW 264.7 cell line. AB - Thapsigargin (TG), an endoplasmic reticular (ER) Ca(2+)-ATPase inhibitor, can increase the intracellular calcium concentration and then deplete the TG sensitive intracellular Ca(2+) pool. In this study, we investigated the effects of TG on cell viability and tumor necrosis factor-alpha (TNF-alpha) production in the murine macrophage RAW 264.7 cell line. We found that treatment with TG (10 800 nM) induced apoptosis in RAW 264.7 cells in a dose-dependent manner (IC(50), 200 nM). Lipopolysaccharide (LPS, 1 microg/ml) markedly potentiated low concentrations of TG (10-75 nM) in inducing apoptosis (IC(50), 20 nM) as revealed by the DNA ladder. Polymycin B (an LPS receptor antagonist) inhibited the cytotoxic effect induced by LPS plus TG. Although TG, A23187 and ionomycin all definitely increased intracellular Ca(2+) concentrations, neither A23187 nor ionomycin mimicked TG in inducing apoptotic events in LPS-activated RAW 264.7 cells. Moreover, the production of TNF-alpha induced by LPS was profoundly potentiated by TG but not by A23187 or by ionomycin. We conclude from these combined results that TG-sensitive ER Ca(2+) stores play a pivotal role in modulating cell viability and TNF-alpha production. The mutual potentiation between the LPS receptor signaling pathway and the depletion of ER Ca(2+) stores implies the existence of cross-talk between these multiregulatory mechanisms in this murine macrophage RAW 264.7 cell line. PMID- 10754387 TI - Rapid full-length genomic sequencing of two cytopathically heterogeneous Australian primary HIV-1 isolates. AB - Two Australian HIV-1 isolates, derived from patient blood (HIV(MBC200)) and cerebrospinal fluid (HIV(MBC925)), were characterized after in vitro culture in peripheral blood mononuclear cells (PBMC). Although virus replication was similar, as measured by cell-free reverse transcriptase activity, only one of the two isolates (HIV-1(MCB200)) consistently induced cell syncytia and depleted the PBMC population of CD4+ cells by cell killing. A novel technique, devised for rapidly obtaining high-quality viral sequence data and the full-length genomic sequence of these two isolates, is presented. Analysis of the predicted sequence of the viral Env proteins provides correlates of the observed phenotypes. Phylogenetic analysis derived using near full-length sequence of these and other HIV-1 subtype B genomic sequences (including two other Australian isolates) shows a star-shaped phylogeny with each member having a similar genetic diversity. These data expand the database of genomic sequence available from well characterized primary clinical isolates of HIV-1 using a novel rapid technique. PMID- 10754388 TI - An Sp1 binding site involves the transcription of the Fas ligand gene induced by PMA and ionomycin in Jurkat cells. AB - The transcriptional regulation of the Fas ligand (FasL) gene in Jurkat cells was investigated. We demonstrated that an Sp1 binding site, located between -280 and 275 bp relative to the translational start site (+1) of the FasL gene, was important for the transcription of the FasL gene by deletion and mutation analysis in Jurkat cells after phorbol 12-myristate 13-acetate (PMA) and ionomycin treatment. Nuclear extract of Jurkat cells formed complexes with the oligonucleotides bearing the Sp1 site within -280 to -275 of the FasL promoter. Apart from the constitutive complexes, a new complex was observed after PMA and ionomycin stimulation. Plasmid containing the Sp1 site sequence with site directed mutation reduced the FasL promoter activity in driving the expression of reporter luciferase gene expression in transfected Jurkat cells after PMA and ionomycin stimulation. The binding of activated Jurkat cell nuclear extract to the mutated Sp1 binding site of the FasL promoter was ablated. In addition, the oligomer containing the Sp1 site of the FasL promoter could compete with oligomer with conserved Sp1 binding sequence in nuclear protein binding of activated Jurkat cells. The data presented in this study suggest that the transactivation of the FasL promoter via the Sp1 binding sequence (-280 to -275) involves the PMA and ionomycin-induced expression of the FasL gene. PMID- 10754389 TI - Sp1-like proteins function in the transcription of human ferredoxin genes. AB - We characterized a regulatory element located in the -76 to -62 region of the human ferredoxin gene. This region bound to Sp1-like proteins with low affinity, as shown using electrophoretic mobility shift, competition, antibody binding, and Southwestern experiments. The similarity of the regulatory element to Sp1 extends beyond its DNA-binding domain, as cloned Sp1 functioned equally well when fused to a peptide that bound to an irrelevant site. The function of these Sp1-binding sites is mediated through the cAMP-dependent protein kinase (PKA) signaling pathway, because reporter genes downstream of the Sp1-binding sites were not activated in a PKA-deficient cell line. Transfection of the catalytic subunit of PKA restored activated transcription. Similar Sp1-binding sites identified in the CYP11A1 and CYP21 genes also controlled cAMP-dependent transcription of the reporter gene. Our finding of the function of Sp1-like proteins in steroidogenic gene transcription adds one more role Sp1 plays in controlling physiological events. PMID- 10754390 TI - Identification of okadaic-acid-induced genes by mRNA differential display in glioma cells. AB - To identify novel genes associated with apoptosis in glioma cells, we treated T98G glioma cells with okadaic acid (OA). Differential display using 15 random primers was performed on RNA extracted from these cells. Upregulated bands were excised from polyacrylamide gels and cloned. Northern blots were used to confirm RNA expression in T98G cells. 18 RNA fragments corresponding to the untranslated region of genes were identified and sequenced. Three unknown gene fragments were used to screen a fetal brain cDNA library resulting in three complete cDNA sequences. The three sequences corresponded to a human gene homologous to the yeast translation initiation factor Sui-1, a cAMP-regulated phosphoprotein, ARPP 16/19, and a novel gene designated O48. Transcription of Sui-1 increased in response to all stress factors tested, whereas ARPP only responded to OA. 2-kb and 4-kb O48 RNA species were identified. OA and stress factors increased 2-kb expression while K252a (protein kinase inhibitor) increased 4-kb expression. Differential display is effective for identifying genes associated with apoptosis. Novel genes may be identified by further analysis of the gene fragments identified in this study. The function of O48 is unknown. PMID- 10754391 TI - Hepatitis B virus core protein interacts with the C-terminal region of actin binding protein. AB - Hepatitis B viral core protein is present in the nucleus and cytoplasm of infected hepatocytes. There is a strong correlation between the intrahepatic distribution of core protein and the viral replication state and disease activity in patients with chronic hepatitis. To understand the role of core protein in the pathogenesis of HBV, we used a yeast two-hybrid system to search for cellular proteins interacting with the carboxyl terminus of core protein, as this region is involved in a number of important functions in the viral replication cycle including RNA packaging and DNA synthesis. A cDNA encoding the extreme C-terminal region of human actin-binding protein, ABP-276/278, was identified. This interaction was further confirmed both in vitro and in vivo. In addition, the extreme C-terminal region of ABP-276/278 interacted with the nearly full-length HBV core protein. Since this region is present in both the core and the precore proteins, it is likely that both core and precore proteins of HBV can interact with the C-terminal region of ABP-276/278. The minimal region of ABP-276/278 which interacted with the HBV core protein was the C-terminal 199 amino acid residues which correspond to part of the 23rd repeat, the entire 24th repeat and the intervening hinge II region in ABPs. The potential functional outcome of ABP interaction in HBV replication and its contribution to the pathological changes seen in patients with chronic HBV infection are discussed. PMID- 10754392 TI - Spontaneous elastic tissue lesions in the rat abdominal aorta, a genetically determined phenotype. AB - In this study, phenotypic expression of spontaneous elastic laminae defects within the rat abdominal aorta was examined. Lesions in Brown Norway (BN) rats were compared with those of New Zealand genetically hypertensive (GH) rats. BN and GH rats were cross-bred to determine the phenotypic expression of these lesions in successive F(1) and F(2) generations. Lesions were assessed by distribution, number and a semiquantitative index of severity. All BN aortae contained numerous elastic tissue defects. In comparison, GH aortae contained only occasional elastic tissue lesions. F(1) aortae contained lesions in numbers similar to those of the parental BN strain; however, F(1) lesions were of significantly greater severity. Within the F(2) generation, a wide range in both lesion numbers and severity indices was observed, with approximately a quarter of animals having lesion numbers analogous to the GH parental strain. In conclusion, this study indicates that the spontaneous elastic tissue lesions observed within BN rats are consistent with an autosomal dominant, possibly single gene, effect. Moreover, epistatic effects, derived from the GH strain, may influence the severity of these lesions. The gene(s) responsible may be important in the development of conditions such as arteriosclerosis and aneurysms in humans. PMID- 10754393 TI - Hirulog-1 reduces expression of platelet-derived growth factor in neointima of rat carotid artery induced by balloon catheter injury. AB - Vascular restenosis is one of the major concerns for the treatment of atherosclerotic cardiovascular diseases using therapeutic vascular procedures. Hirulog-1, a synthetic thrombin inhibitor, effectively reduced ischemic events in coronary heart disease patients and caused less hemorrhagic complications compared to heparin. Thrombin stimulated the expression of platelet-derived growth factor (PDGF) in vascular cells. PDGF receptor blockers reduced angioplasty-induced restenosis in the swine model. The present study examined the effects of hirulog-1 on vascular stenosis, platelet deposition and the expression of PDGF in rat carotid arteries injured by balloon catheter. Multiple intravenous infusions of hirulog-1 (1 mg/kg/h for 4 h for 6 times), but not bolus injection or 1-2 times of infusion, reduced neointima/media ratio by 50% in balloon-injured carotid arteries compared to injured animals receiving saline alone. Activated partial thromboplastin time in hirulog-1-treated rats was significantly prolonged compared to saline controls but shorter than that in animals receiving heparin (50 U/kg/h). One of heparin-treated rat, but none of hirulog-1-treated, died from bleeding complication. Hirulog-1 injection transiently reduced platelet deposition on denuded intima visualized by scanning electron microscopy. Abundance of PDGF in neointima of injured carotid arteries detected by immunohistochemistry was significantly decreased following infusions of hirulog 1. The results suggest that balloon catheter injury induced neointima formation and the overexpression of PDGF in the neointima of rat carotid artery may be effectively suppressed by infusions with hirulog-1, a thrombin-specific inhibitor. PMID- 10754394 TI - Localisation of mRNA for JE/MCP-1 and its receptor CCR2 in atherosclerotic lesions of the ApoE knockout mouse. AB - MCP-1 has potent chemotactic activity for monocytes and is strongly implicated in the pathogenesis of atherosclerosis. In the present study, we have used in situ hybridisation to examine the gene expression of JE, the murine homologue of MCP 1, and its receptor, CCR2, during the development of atherosclerotic lesions in the ApoE knockout mouse. Interestingly, the earliest expression of JE detected during lesion development was found to be localised in mesenchymal cells in the adventitia and not in the intima. Macrophages were subsequently found to accumulate in these affected regions of the adventitia and these cells were found to express high levels of JE. At this stage, early macrophage-rich lesions with high expression of JE were also seen in the intima, but expression of mRNA for the receptor for JE (CCR2) was only found on adventitial macrophages and not in the intima. This sequence of events suggests that adventitial inflammation may be an important early event in lesion development and responsible for the subsequent accumulation of macrophages in the intima possibly by recruitment from the adventitia as well as via the vessel lumen. PMID- 10754395 TI - Static versus dynamic distensibility of the carotid artery in humans. AB - In clinical studies, the elastic behavior of central arteries is usually assessed by measuring dynamic distensibility. In this study, we aimed to investigate how dynamic and static distensibility of the common carotid artery (D(dyn) and D(stat), respectively) are related in 28 healthy volunteers of 20-71 years. The carotid diameter and its change with the pressure pulse were measured using an ultrasound echo-tracking device. Arterial blood pressure was measured by Finapres and carotid pressure was determined by applanation tonometry. D(dyn) was determined at rest using the pressure pulse, while D(stat) was determined during pressor responses induced by handgrip or cold pressor test. Data are given as mean +/- 1 SD. In younger subjects (<35 years), D(stat) did not differ from D(dyn) (7.0 +/- 3.4 vs. 6.5 +/- 2.1 x 10(-3) x mm Hg(-1), respectively), whereas in older subjects (>35 years), D(stat) was significantly higher than D(dyn) (3.8 +/- 1.4 vs. 2.1 +/- 0.9 x 10(-3) x mm Hg(-1), p < 0.001). For all subjects, D(stat) and D(dyn) decreased with increasing age and mean arterial pressure (MAP). Using stepwise multiple regression analysis, the strongest predictor of D(stat) proved to be MAP, while that of D(dyn) was age. D(stat) was found to be linearly related to the hysteresis loop area of the pressure-diameter relation (r = 0. 94), i.e. to vessel wall viscosity. It is concluded that, with increasing age, static distensibility overestimates the distension capacity of large arteries. PMID- 10754396 TI - Mean wall shear stress in the femoral arterial bifurcation is low and independent of age at rest. AB - In elastic arteries, mean wall shear stress appears to be close to 1. 5 Pa, the value predicted by the theory of minimal energy loss. This finding in elastic arteries does not necessarily represent the situation in muscular arteries. Elastic arteries have to store potential energy, while muscular arteries have mainly a conductive function. Therefore, we determined wall shear stress and its age dependency in the common and superficial femoral arteries, 2-3 cm from the flow divider in 54 presumed healthy volunteers between 21 and 74 years of age, using a non-invasive ultrasound system. Prior to the study, the reliability of this system was determined in terms of intrasubject variation. Mean wall shear stress was significantly lower in the common femoral artery (0.35 +/- 0.18 Pa) than in the superficial femoral artery (0.49 +/- 0.15 Pa). In all age categories, peak systolic wall shear stress and the maximal cyclic change in wall shear stress were not significantly different in the common and the superficial femoral arteries. Peak systolic wall shear stress in the common and the superficial femoral arteries was not significantly different from the value previously determined in the common carotid artery, but mean wall shear stress was lower in the common and superficial femoral arteries than in the common carotid artery by a factor of 2-4. In both the common and the superficial femoral arteries, mean, peak systolic and maximal cyclic change in wall shear stress did not change significantly with age, nor did diameter. We conclude that, as compared to elastic arteries, mean wall shear stress is low in the conductive arteries of a resting leg, due to backflow during the first part of the diastolic phase of the cardiac cycle and the absence of flow during the rest of the diastolic phase. Mean wall shear stress is lower in the common than in the superficial femoral artery due to additional reflections from the deep femoral artery. PMID- 10754397 TI - Cerebral hemodynamic response to CO(2) tests in patients with internal carotid artery occlusion: modeling study and in vivo validation. AB - The aim of this work was to analyze the cerebral hemodynamics in patients with internal carotid artery (ICA) occlusion during CO(2) dynamical tests. The study was performed using an original mathematical model of cerebral hemodynamics. The model includes the circle of Willis, the microvascular circulation in the territories of each cerebral artery, cortical anastomoses, intracranial pressure, the cerebrospinal fluid circulation, the brain venous circulation and two regulation mechanisms: autoregulation and CO(2) reactivity. Model predictions are compared with real data obtained on 20 healthy subjects (group A) and 14 patients with unilateral ICA stenosis (group B) during CO(2) tests. The percent changes in Doppler middle cerebral artery velocity per mm Hg of CO(2) pressure variations (reactivity index, RI) were separately computed during hypocapnia and hypercapnia. In group A, the reactivity index was practically the same in both sides. Patients in group B had a significantly lower CO(2) reactivity in the side ipsilateral to the occlusion (hypocapnia RI: 1.06 +/- 1.6 vs. 2.3 +/- 0.8%/mm Hg; hypercapnia: 0.9 +/- 0.8 vs. 2.4 +/- 1.0%/mm Hg). Our model can explain these results very well, assuming values for the diameter of the communicating arteries in the range reported in the clinical literature. Moreover, computer simulations suggest that patients with a small diameter of both communicating arteries (as low as 0.4-0.5 mm) exhibit a dramatic fall in CO(2) reactivity in the ipsilateral side compared with the contralateral one, with the appearance of paradoxical responses. A decrease in ipsilateral RI reactivity, however, may also depend on the presence of a significant stenosis of the contralateral ICA. PMID- 10754398 TI - Prevention of increases in blood pressure and left ventricular mass and remodeling of resistance arteries in young New Zealand genetically hypertensive rats: the effects of chronic treatment with valsartan, enalapril and felodipine. AB - The relative efficacy of three antihypertensive drugs in the prevention of further elevation of blood pressure (BP) and cardiovascular structural remodeling in 4-week-old genetically hypertensive (GH) rats was studied by means of two complementary methods, stereology and myography. Four to 10-week-old GH rats were treated with valsartan (10 mg/kg/day), enalapril (10 mg/kg/day) or felodipine (30 mg/kg/day). Untreated GH and normotensive control rats of Wistar origin served as controls. Tail-cuff systolic SBP was measured weekly and left ventricular (LV) mass determined at the end of the experiment. Mesenteric resistance arteries (MRA) were either fixed by perfusion, embedded in Technovit and sections stained for stereological analysis, or mounted on a wire myograph for structural and functional measurements. BP and LV mass were significantly reduced by all drugs; decreases in BP and LV mass were smaller after felodipine treatment. Valsartan and enalapril caused a decrease in BP to normotensive control values. Felodipine kept BP at the 4-week level and prevented further rise with age. Valsartan caused hypotrophic outward remodeling of MRA, enalapril eutrophic outward remodeling and felodipine hypotrophic remodeling. Myograph measurements showed remodeling of the same order. While all drugs lowered the media/lumen ratio in GH to normal, the outward remodeling after valsartan and enalapril indicates that valsartan and enalapril might be more effective in reversing the inward remodeling of resistance arteries found in essential hypertension. PMID- 10754399 TI - Local application of capsaicin into the draining lymph nodes attenuates expression of adjuvant-induced arthritis. AB - Adjuvant-induced experimental arthritis (AA) was examined in adult male Lewis rats after isolated capsaicin (CAPS)-induced loss of small, nonmyelinated, afferent fibers in lymph nodes draining the site of adjuvant challenge. AA was induced by intradermal injection of Freund's complete adjuvant (CFA) into the subplantar area of the right hind paw. Controls received similar injections of mineral oil, the vehicle for CFA. One day later, half of the CFA-treated rats and half of the mineral oil-treated rats received injections of CAPS bilaterally into the draining lymph nodes (DLN). The DLN of remaining rats were injected with 50:50 ethanol/sterile physiological saline, the vehicle for CAPS. This paradigm resulted in four groups designated: CFA/CAPS, CFA/vehicle, vehicle/CAPS and vehicle/vehicle. Since substance P (SP) is present in small, nonmyelinated, afferent fibers, the target of the neurotoxin, CAPS, a radioimmunoassay specific for SP was used to verify the loss of these nerve fibers. CAPS injections into the DLN resulted in a loss in SP concentration in the DLN, with no depletion of SP in the spleen or sciatic nerve. These findings support the destruction of SP containing nerves, which we interpret as verification of the selective loss of small, non-myelinated afferent nerves in the DLN with no significant spread of the neurotoxin to the nearby sciatic nerves which supply small, nonmyelinated, afferent fibers to the hind limb joints. Also, preservation of SP content in spleen indicates CAPS did not circulate via the lymphatic drainage. No chronic inflammation was observed in the fore or hind limbs from rats treated with the vehicle for CFA (vehicle/vehicle, vehicle/CAPS) at any time during the study. In CFA/vehicle-treated rats, bilateral, symmetrical inflammation of the hind limbs was apparent 14 days after challenge with CFA, and became progressively more inflamed through day 20. In contrast, hind limb inflammation in arthritic rats treated with CAPS was not symmetrical. On days 14 and 20 after challenge with CFA, the inflammatory response in the left hind limb, contralateral to the site of CFA injection, was significantly (p < 0.05) attenuated compared with the response seen on the right side of CFA/CAPS-treated rats, and with the response seen in left hind limb of CFA/vehicle-treated animals. In fact, the mean dorsoplantar width of contralateral hind limbs from CFA/CAPS-treated animals was not different from that measured in non-AA control groups. These findings support a role for small, nonmyelinated, sensory nerves that modulate immune responses in DLN in the development and progression of AA in Lewis rats. PMID- 10754400 TI - Characterization of the promoter-directing expression of growth hormone in a monocyte cell line. AB - Previous work from our laboratory has shown that cells of the immune system produce a growth hormone (GH) molecule similar to that produced by the pituitary. In the present study, using Southern analysis of RT-PCR products and sequencing of cloned cDNA molecules, we demonstrate that lymphoid cell lines utilize the same promoter and first exon as the pituitary somatotrope. To identify the cis elements involved in transcriptional regulation of immune cell-derived GH, we have coupled rat GH promoter fragments to a luciferase reporter gene and transfected a monocyte cell line (P-388) by electroporation. The results suggest the presence of both positive (-299/-193 bp) and negative (-193/-107 bp) regulatory elements. The same constructs transfected in the pituitary cell line, GH3, in contrast to the monocyte cell line, showed a gradual decrease in luciferase expression. The overexpression of GHF-1 or GHF-2 resulted in a modest but significant reduction in rat GH promoter activity in the P-388 cell line. Taken together, the data suggest that immune cells utilize the same first exon and promoter sequence for the expression of monocyte GH as that reported for the expression of pituitary GH. Further, it appears that sequences between -299 and 107 bp are important in the regulation of the promoter where different transcription factors may be recruited to promote GH expression in a monocyte cell line. PMID- 10754401 TI - Changes in the leukocyte distribution and surface expression of adhesion molecules accompanied with hypothalamically induced restlessness in the cat. AB - One type of emotional behavior called restlessness occurs when the anteromedial hypothalamus is stimulated in cats. We examined the changes in the distribution and surface expression of adhesion molecules in leukocytes accompanied with restlessness. Mature female cats were used for this study. The cats were stimulated with 60 Hz sine wave train pulses (20-90 microA, 10 s in duration, at 5-min intervals) for 60 min. Samples of blood were collected from 30 min before stimulation up to several hours after the final stimulation. The number of granulocytes increased just after stimulation, while at the same time the expression of L-selectin decreased. On the other hand, the number of CD4+ and CD8+ T lymphocytes decreased at 1-2 h after the end of the stimulation, while the expression of L-selectin increased. In addition, the expression of LFA-1 and VLA 4 did not change. These data suggest that hypothalamically elicited restlessness is thus accompanied by a leukocyte distribution change, which might be mediated by changes in the expression of L-selectin on leukocytes. Plasma cortisol increased during stimulation in restlessness. However, during in vitro culture experiments, cortisol did not alter the expression of leukocyte L-selectin which thus indicated that cortisol does not directly affect the surface expression of L selectin. These results thus suggest that hypothalamically induced restlessness is a useful stress model for psychoneuroimmunological studies. PMID- 10754402 TI - Human amylin stimulates inflammatory cytokine secretion from human glioma cells. AB - Chronic neurodegeneration in the brains of Alzheimer's disease (AD) patients may be mediated, at least in part, by the ability of amyloid beta (Abeta) to exacerbate inflammatory pathways in a conformation-dependent manner. In this regard, we previously reported that the Abeta-peptide-mediated potentiation of inflammatory cytokine secretion from interleukin-1beta (IL-1beta)-stimulated human astrocytoma cells was conformation dependent. Other amyloidogenic peptides, such as human amylin, which display similar conformation-dependent neurotoxic effects, may also elicit inflammatory cytokine secretion from glial cells. To test this hypothesis, we compared human and rat amylin for the effects on cytokine production in U-373 MG human astrocytoma cells. Human amylin alone stimulated U-373 MG cells to secrete IL-6 and IL-8 in a concentration-dependent manner with maximum effects seen at 10-25 microM peptide. In addition, human amylin markedly potentiated IL-1beta-stimulated cytokine production with a similar concentration dependence. In contrast, nonamyloidogenic rat amylin modestly stimulated cytokine secretion, either alone or combined with IL-1beta. Aging human amylin resulted in diminished cytokine secretion, probably due to the formation of large, less active aggregates. In agreement with our previous studies using Abeta, extracellular Ca(2+) was necessary for human amylin stimulation of cytokine secretion. Our data suggest that amyloidogenic peptides promote cytokine secretion through similar beta-sheeted secondary-structure- and extracellular-Ca(2+)-dependent mechanisms. PMID- 10754403 TI - Cytokine effects on glutamate uptake by human astrocytes. AB - Glutamate uptake by astrocytes has been postulated to play a neuroprotective role during brain inflammation. Using primary human fetal astrocyte cultures, we investigated the influence of selected cytokines on glutamate uptake activity. Interleukin (IL)-1beta and tumor necrosis factor-alpha dose-dependently inhibited astrocyte glutamate uptake, whereas interferon (IFN)-gamma alone stimulated this activity. The nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine, blocked IL-1beta-mediated inhibition of glutamate uptake, suggesting involvement of nitric oxide in the effect of IL-1beta. IL-1 receptor antagonist protein totally reversed the inhibitory effect of cytokines, suggesting a critical role of IL-1beta. The anti-inflammatory cytokine IFN-beta blocked cytokine (IL-1beta plus IFN-gamma)-induced inhibition of glutamate uptake with a corresponding reduction in nitric oxide generation. Taken together, these findings suggest that proinflammatory cytokines inhibit astrocyte glutamate uptake by a mechanism involving nitric oxide, and that IFN-beta may exert a therapeutically beneficial effect by blocking cytokine-induced nitric oxide production in inflammatory diseases of the brain. PMID- 10754404 TI - Natural killer cell activity and resistance to tumor metastasis in prepubescent rats: deficient baselines, but invulnerability to stress and beta-adrenergic stimulation. AB - Although young children and animals exhibit high rates of tumor development, little is known about natural killer (NK) cell activity in the very young. We recently provided direct evidence that reduced levels of NK activity in prepubescent rats underlie higher levels of susceptibility to metastasis. The aim of the current study was to further characterize NK activity and tumor resistance in prepubescent rats, specifically with respect to the effects of stress and sex, as these factors have been shown to modulate tumor development in adult populations. Two NK outcomes were assessed: levels of whole blood NK cytotoxicity and lung tumor retention of NK-sensitive MADB106 tumor cells which metastasize only to the lungs. The corticosterone (CS) response to surgery was also assessed. In the first set of experiments, prepubescent males and females (36 days of age) and mature males (98 days) were subjected to abdominal surgery and 5 h later were either tested for plasma CS levels or challenged with MADB106 tumor cells. The findings indicated that whereas surgery increased CS levels in the young rats to similar levels observed in mature animals, surgical stress did not increase lung tumor retention in the young animals, despite exerting marked and significant effects in the mature rats. These findings persisted when lower tumor loads were used in the young rats to compensate for the markedly reduced resistance to metastasis in this population. Because surgery involves activation of the sympathetic nervous system (SNS) which is known to regulate NK activity, we assessed the impact of the beta-adrenergic agonist, metaproterenol, on NK activity and on lung tumor retention. Metaproterenol (0.8 mg/kg, 1 h before testing) resulted in a large suppression of NK activity and resistance against MADB106 metastasis in mature males and females, but not in prepubescent animals. In mature, but not in young animals, males exhibited higher baseline levels of NK activity. Taken together, these findings indicate that NK cells of prepubescent rats are resistant to beta-adrenergic stimulation, and suggest that prepubescent rats are markedly less responsive to SNS-induced suppression of NK activity, which may underlie their invulnerability to the effects of surgery on MADB106 metastasis. PMID- 10754405 TI - Angiotensin-converting enzyme inhibition: facts and fiction. PMID- 10754406 TI - Importance of serotonergic mechanisms in the thrombotic complications in hemodialyzed patients treated with erythropoietin. AB - So far it is not clear how erythropoietin affects the anticoagulant properties of vascular endothelium in uremia. Since serotonin is also thought to play a role in the pathogenesis of thrombosis, the aim of the study was to evaluate major components of extrinsic coagulation pathway, markers of endothelial cell injury, lipoprotein (a) and peripheral serotonergic mechanisms during rHuEPO therapy in hemodialyzed patients. The study was performed on chronically hemodialyzed patients divided into two groups: with rHuEPO treatment and without rHuEPO therapy in relation to the control group. In uremic patients, thrombomodulin and von Willebrand factor, activity of factor VII, tissue factor pathway inhibitor (TFPI) activity, TFPI and tissue factor (TF) concentrations, lipoprotein (a) level were significantly higher when compared to healthy volunteers. Treatment with rHuEPO resulted in a further significant rise in markers of endothelial cell injury: thrombomodulin and von Willebrand factor and TFPI concentration. Extrinsic coagulation factors: activities of factor VII and X, TFPI activity and TF activity and concentration, lipoprotein (a) and vitronectin remained unchanged during rHuEPO therapy. Platelet serotonin content and whole blood serotonin were significantly lower in uremic patients relative to healthy volunteers and during rHuEPO treatment they increased significantly. Whole blood serotonin reached normal values. Plasma serotonin, significantly elevated in uremia, did not change during rHuEPO therapy. Serotonin uptake by uremic platelets was significantly impaired and remained unaltered during rHuEPO administration. Serotonin release by uremic platelets was also significantly depressed but a significant improvement was observed in rHuEPO-treated patients. Our data suggest that endothelial injury, TF pathway components and peripheral serotonergic system disturbances may predispose to thromboembolic complications and play a role in the pathogenesis of atherosclerosis in uremic patients, particularly treated with rHuEPO. Increase in TFPI may compensate the increase in TF in these patients. PMID- 10754407 TI - Cardiac autonomic neuropathy in patients with chronic renal failure on hemodialysis. AB - To characterize uremic cardiac autonomic neuropathy, we measured plasma catecholamines, analyzed the 24-hour heart rate variability (HRV), and acquired serial images with (123)I-metaiodobenzylguanidine (MIBG) in 44 patients with chronic renal failure on hemodialysis and in 14 controls. Time-domain measures were calculated using the Marquette HRV program. MIBG clearance rates from the heart and lung were evaluated on planar images, and the regional MIBG uptake in the left ventricular myocardium was evaluated with single-photon emission computed tomography. Compared with controls, plasma dopamine and norepinephrine levels were elevated (p < 0.001 and p = 0.03, respectively), and all the time domain measures of HRV were reduced in the patients (p < 0.001). The MIBG clearance rate from the heart was higher (p < 0.001), that from the lung was lower (p < 0.001), and the myocardial MIBG distribution was more heterogeneous in patients than in controls (total uptake score p /=93 cm) showed reduced HDL-C levels as compared with lean (waist girth <93 cm) patients (1.20 +/- 0.28 vs. 1.42 +/- 0.41 mmol/l, respectively, p < 0.01). Moreover, the HDL-C levels were markedly affected by the CETP TaqIB polymorphism in lean patients (+28 and +41% in B1B2 and B2B2 as compared with B1B1 patients, p < 0.05), but no significant difference was observed among obese patients. Significantly lower total cholesterol:HDL-C ratios were obtained in lean B2B2 homozygotes, suggesting that these patients could be less susceptible to atherosclerosis than lean B1B1 homozygotes. In addition, patients with the B1B1 genotype had more documented CHD as compared with patients carrying at least one B2 allele, supporting the protective effect of the B2 allele against CHD. In conclusion, considerable variation in HDL-C levels appears to be explained by the CETP TaqIB gene polymorphism in male renal transplant recipients, but this potential protective gene effect appears strongly reduced by concomitant abdominal obesity. PMID- 10754411 TI - Predictive value of repeat renal biopsies in children with nephrotic syndrome. AB - In children who exhibit a frequently relapsing course of minimal change disease (MCD), treatment is often difficult and frustrating to the physician and the family since the goal of a sustained remission remains elusive. The progression of the disease is often unpredictable from its clinical presentation since the lesion of MCD may evolve into a more severe form, such as mesangial IgM nephropathy or focal segmental glomerulosclerosis (FSGS), without alteration in signs and symptoms. Alkylating agents such as cyclophosphamide, or immunosuppressives such as cyclosporine can induce a more sustained remission, but are fraught with inherent toxicity, which makes difficult the decision to use these drugs in patients with MCD. Over a 10-year period we studied 49 patients who had more than one renal biopsy. Repeat biopsies were performed either to delineate the morphological lesion prior to change in therapy, or to confirm suspected drug toxicity, which would necessitate discontinuation of therapy. A total of 83 repeat biopsies were performed in these 49 patients. Of the 49 patients, 25 had MCD, and in 21 of these the lesion evolved into either IgM nephropathy (n = 7) or FSGS (n = 14). Of patients with IgM nephropathy (n = 12), 50% evolved into FSGS. The clinical diagnosis made prior to the repeat biopsy did not confirm with the histological diagnosis in 43% of cases, and a change in therapy or cessation of therapy was carried out in 43 of 83 repeat biopsy instances. Since the complications were mild and the ability of clinical findings to accurately predict the histological lesion limited, we conclude that repeat renal biopsies are a useful tool to fashion optimal therapy in children with frequently relapsing nephrotic syndrome. PMID- 10754412 TI - Therapeutic effects of ulinastatin on experimental crescentic glomerulonephritis in rats. AB - Ulinastatin is a potent protease inhibitor purified from the human urine that has been used clinically to treat acute pancreatitis and circulatory shock. In the current study, we evaluated the therapeutic effects of Ulinastatin in a rat model of crescentic glomerulonephritis (CrGN) and investigated its putative mechanisms. Wistar-Kyoto rats were injected with nephrotoxic serum and received daily intraperitoneal injection of Ulinastatin. Ulinastatin treatment significantly reduced proteinuria and glomerular crescentic formation. Moreover, glomerular infiltration of neutrophils and ED1+ cells (monocytes/macrophages) was significantly suppressed by Ulinastatin. In contrast, the glomerular deposition of heterologous (rabbit) and autologous (rat) antibodies was not changed. Neither serum complement activation nor the anti-rabbit immune response was affected by Ulinastatin administration. Our results suggest that Ulinastatin has preventive effects on rat experimental CrGN, mediated at least in part by inhibiting intraglomerular infiltration of inflammatory cells. PMID- 10754413 TI - Glomerular basement membrane anionic sites in adriamycin nephropathy: effect of saline loading and nitric oxide modulation. AB - BACKGROUND: In previous studies we found that experimental Adriamycin (ADR) nephropathy is associated with the loss of glomerular basement membrane (GBM) anionic sites provided by heparan sulfate proteoglycans. Chronic saline loading in normal rats resulted in a similar effect on the GBM anionic sites. The L arginine-nitric oxide synthase-nitric oxide system is involved in the pathogenesis of experimental chronic renal failure. The present study was performed to determine the combined effect of nitric oxide (NO) modulation and chronic saline loading in ADR nephropathy. The modulation of NO was done by chronic administration of L-arginine (NO donor) or N(w)-nitro-L-arginine, a known nitric oxide synthase inhibitor. METHODS: Systolic blood pressure was measured in awake rats by a tail-cuff method. Renal function was assessed by creatinine clearance, FeNa%, and daily protein excretion. The change of mean GBM widths and anionic sites distribution were assessed by electron microscopy. The localization of anionic sites was carried out by cationic colloidal gold. Plasma and urinary nitrates (NO(x)) were measured by nitrite (NO(2)) + nitrate (NO(3)), stable metabolites of NO. RESULTS: Two weeks after the ADR administration (3.5 mg/kg BW iv) the rats had severe renal failure (creatinine clearance 134 +/- 31 microl/min/100 g BW vs. initial values 670 +/- 29 microl/min/ 100 g BW, p < 0.001), high FeNa%, severe proteinuria, increased GBM width, significant reduction of GBM anionic sites and low urinary NO(x) excretion. The saline loading resulted in further reduction of GBM anionic sites count and blood pressure elevation. The inhibition of NO did not change the course of ADR nephropathy. The main finding of the present study is that chronic administration of L-arginine significantly alleviates the renal failure in the ADR (+/- saline loading) nephropathy. The L-arginine-treated rat had higher creatinine clearance, lower FeNa% and protein excretion and complete normalization of GBM anionic sites distribution. CONCLUSIONS: Sodium loading has a deleterious effect on GBM permselectivity. L-Arginine prevents the reduction of GBM anionic sites, decreases proteinuria and alleviates the renal insufficiency in ADR nephropathy. PMID- 10754414 TI - Protective effect of pyruvate upon cultured mesothelial cells exposed to 2 mM hydrogen peroxide. AB - Rat peritoneal mesothelial cells in culture have the capability of generating hydrogen peroxide. Exposure of these cells to glucose-enriched, lactated-buffered fluids for peritoneal dialysis significantly increases the production of H(2)O(2). Increased liberation of oxygen radicals also involves the risk of damaging the peritoneal membrane. Pyruvate being a natural oxidant scavenger abundantly present in mammalian cells, we hypothesized that its protective effects facing H(2)O(2) can eventually be of relevance for the mesothelial monolayer of patients on long-term peritoneal dialysis. So far, we designed an experimental study in which rat peritoneal mesothelial cells in culture were exposed to 2 mM H(2)O(2). Cell damage was estimated in terms of decreased capability of the mitochondrial dehydrogenases to reduce MTT. Addition of 2 mM sodium pyruvate to the medium prevented the negative effect of hydrogen peroxide. The MTT/protein values for the control group were 0.00357 +/- 0.00075. The ratio after exposure to 2 mM H(2)O(2) was 0. 00217 +/- 0.00028, whereas that detected in cells incubated in H(2)O(2) plus pyruvate was 0.00325 +/- 0.0082 (p < 0.05). These results indicate that pyruvate protected rat peritoneal mesothelial cells in culture against oxidant injury. These data are one more piece of evidence pointing at pyruvate as a potentially useful buffer for peritoneal dialysis solutions. PMID- 10754416 TI - Each of anthropometry, bioelectrical impedance analysis and dual-energy X-Ray absorptiometry methods can be used to assess lean body mass in hemodialysis patients. PMID- 10754415 TI - Cytomegalovirus infection in a seroendemic renal transplant population: a longitudinal study of virological markers. AB - BACKGROUND/AIMS: The detection of viremia by polymerase chain reaction (PCR) in cytomegalovirus (CMV) infection in renal allograft recipients has been shown to have a predictive value for disease. However, its diagnostic utility in a population with high background seropositivity has not been defined. This prospective study was undertaken to assess the relationship of CMV DNAemia, and/or IgM seropositivity to CMV disease in a seroendemic transplant population. METHODS: Consecutive patients undergoing renal transplantation between August 1997 and February 1998 were enrolled. Blood was sampled before transplantation from the donors and recipients for CMV serology and nested PCR for CMV DNA, and after transplantation from the recipients only at monthly intervals until 6 months. Patients were observed for the development of any CMV-like illness during follow-up. CMV DNA was quantitated using limiting dilution PCR on samples obtained from symptomatic patients at the time of illness and from asymptomatic patients at the end of their 6-month follow-up. RESULTS: A total of 57 recipient donor pairs were recruited. Immunosuppression was cyclosporine-based in 55 of 57 (95. 6%). The CMV serologic status was D+R+ in 55 of 57 and D+R- in 2 of 57 pairs. PCR positivity indicating viremia increased from 5% before transplantation to 95% at 6 months after transplantation. Similarly IgM positivity reached 80% at 3 months and thereafter; positivity for any marker was 100% by 6 months. Viremia was sustained in over half the patients. The incidence of CMV-attributable disease peaked at 3 months, and was predominantly mild and self-limiting. Tissue invasive disease appeared later in 4 patients (7%). Asymptomatic viremia was seen in 60-70% of patients at each sampling point. The positive predictive value (PPV) of PCR positivity for disease was 35-40%, and the negative predictive value (NPV), 90-100%. However, the high NPV was of use only in the early post transplant period, negativity for markers declining rapidly with time. Quantitative assay showed significantly higher levels of CMV DNA in symptomatic patients (p = 0.01). A cutoff of 0.001 microg had a specificity of 95% and a PPV of 92.3% for symptomatic CMV disease. CONCLUSION: Qualitative tests to detect CMV DNAemia and IgM, although useful markers of viremia and active infection, have limited utility for the diagnosis of disease in a seroendemic transplant population. Quantitation of CMV DNAemia may play an important role in diagnosis in such a setting. PMID- 10754417 TI - Maximal glomerular size as a predictor of recurrence in renal allograft recipients with focal segmental glomerulosclerosis. PMID- 10754418 TI - Hepatitis C virus genotypes in chronic hemodialysis patients. PMID- 10754419 TI - Vitamin D receptor gene polymorphisms and bone mineral density in patients on hemodialysis. PMID- 10754420 TI - Prolongation of survival after bilateral ureteral ligation in rats. PMID- 10754421 TI - Morbidity and mortality reduction associated with the use of erythropoietin. AB - AIM: The aim of this paper is to review the emerging evidence supporting erythropoietin therapy in improving the long-term prognosis of patients with renal failure, by correcting underlying anaemia. METHODS: This paper reviews and discusses data from several large retrospective databases containing the demographic and treatment details of many thousands of patients with renal insufficiency. Each investigation selected for review used appropriate statistical methodologies to determine the effects of erythropoietin on morbidity and mortality. RESULTS: There is a clear link between anaemia and the development of cardiovascular dysfunction. This is an important finding given that more than 90% of patients become anaemic as a consequence of renal failure. Database analysis has also shown that treatment of anaemic dialysis patients with erythropoietin significantly reduces mortality, often by more than 20%, primarily through effects on the cardiovascular system. In addition, improvements in the exercise capacity and quality of life of patients have been observed despite many patients entering dialysis with a considerable burden of ill health, particularly cardiovascular dysfunction. CONCLUSIONS: Retrospective database analyses have provided a clear understanding of the advantageous effects of erythropoietin treatment on morbidity and mortality in patients with end-stage renal disease. The implications of these findings for further improving treatment strategies in patients with renal insufficiency point to erythropoietin use before the onset of dialysis. PMID- 10754422 TI - Aspects of cardiovascular burden in pre-dialysis patients. AB - BACKGROUND: Cardiovascular disease (CVD) is a major cause of death in patients with chronic renal failure (CRF). It is well recognised that dialysis patients have a high burden of factors that predispose to CVD. What is less clear is the extent of this problem in the pre-dialysis patient. This is the subject of this review. METHODS: The role of potentially correctable cardiovascular risk factors in pre-dialysis patients has been examined using published data. RESULTS: Anaemia is a major cardiovascular risk factor in patients with CRF. Partial correction of renal anaemia with recombinant human erythropoietin leads to improvements in cardiac dysfunction in such patients, such as alleviation of left ventricular hypertrophy. Secondly, malnutrition and inflammation have also been recently identified as potentially correctable cardiovascular risk factors in these patients. CONCLUSIONS: Patients continue to enter dialysis with a considerable cardiovascular burden, many having already suffered a stroke or myocardial infarction. Many risk factors, including malnutrition and inflammation, account for the increased incidence of cardiovascular events. Anaemia is common in pre dialysis patients, and early corrective treatment may help to reduce the incidence of CVD and hence improve long-term outcome. PMID- 10754423 TI - How to improve survival in pre-dialysis patients. AB - AIMS: This survey was performed to determine how anaemia in pre-dialysis patients is currently managed. METHODS: A random sample of 200 nephrologists attending the 1999 ERA/EDTA Congress participated in an interactive survey session. Participants were questioned on their current prescribing attitudes and preferences, and asked to select a preferred answer from a number of alternatives by means of an electronic key-pad. RESULTS: Three quarters of the audience treated their pre-dialysis patients with erythropoietin. However, approximately 50% treated /=75%. In addition, more than half of the respondents said that very few of their patients currently self-administer erythropoietin. Increased healthcare expenditure was the main reason given for limiting treatment. CONCLUSIONS: The survey highlighted important gaps in long-term treatment strategies for pre-dialysis patients. It indicated that new approaches to the treatment of renal anaemia are needed to minimise disease progression and to prevent cardiac dysfunction and associated mortality. Erythropoietin has already been shown to significantly improve the survival of dialysis patients with renal anaemia, and new strategies might, therefore, include the prevention of anaemia in pre-dialysis patients by earlier initiation of erythropoietin therapy. PMID- 10754424 TI - Effect of posture and isoproterenol on beat-to-beat heart rate and QT variability. AB - RATIONALE: Recent literature emphasizes the utility of QT variability to study ventricular electrophysiologic function. In this investigation, we sought to test the hypothesis that beat-to-beat fluctuations in QT intervals are mediated by sympathetic activity in normal subjects using postural challenge and isoproterenol infusions. SUBJECTS AND METHODS: We obtained ECG in the supine and standing postures during spontaneous breathing, at 12, 15 and 20 per minute controlled breathing (n = 19), and before and after infusions of isoproterenol, a beta-adrenergic agent, in the supine posture during spontaneous breathing (n = 11) using lead II configuration in healthy human adult subjects. Heart rate (HR) and QT time series data were analyzed by spectral analysis of 256 s of real-time data. Beat-to-beat QT intervals were measured by automated analysis of ECG. A QT variability index (QT(vi)) was calculated for each subject as the logarithm of the ratio of normalized QT variance to normalized HR variance. We also calculated fractal dimensions of QT time series during spontaneous breathing. RESULTS: QT(vi) was significantly higher in the standing than in the supine posture (-1. 93 +/- 0.27 vs. -1.47 +/- 0.41; p = 0.0001), and also during isoproterenol infusions in the supine posture (-1.83 +/- 0.39 vs. -1. 27 +/- 0.43; p = 0.0001). Fractal dimensions of QT time series were also significantly higher during standing (p = 0.00001) and isoproterenol infusions (p = 0.0002). Respiratory rate or tidal volume did not account for the increased QT variability seen in the standing posture. CONCLUSIONS: A change from the supine to the standing posture as well as infusion of isoproterenol significantly increased the absolute as well as normalized QT variability, which appears to be due to an increase in sympathetic activity associated with these conditions. PMID- 10754425 TI - The human serotonin receptor gene (HTR2) MspI polymorphism in Japanese schizophrenic and alcoholic patients. AB - Epidemiological and genetic studies of alcoholism and schizophrenia have been performed, and in this study, the human serotonin receptor (HTR2) polymorphism was examined in 75 alcoholics and 31 schizophrenic patients. We found that there might not be a significant difference between these psychiatric disease patients and controls in the frequency of the C1/C2 HTR2 gene (MspI polymorphism). The results suggest that the human HTR2 MspI polymorphism might not be associated with a risk factor for developing alcohol dependence or susceptibility to schizophrenia. It is possible that there may be a racial difference in the frequency of the C1/C2 gene between Japanese and Caucasians. Further studies are required to determine whether or not the novel serotonin receptor polymorphism reflects the pathogenesis of alcoholism or schizophrenia. PMID- 10754426 TI - Clinical manifestations of psychiatric patients who are carriers of tay-sachs disease. Possible role of psychotropic drugs. AB - Patients with late-onset Tay-Sachs disease (TSD) may manifest with neuropsychiatric features. We hypothesized that the prevalence of TSD carriers in psychiatric patients is higher than in the general population and their clinical profile is different from that of their noncarrier counterparts. Among 500 Ashkenazi-Jewish psychiatric patients, 19 were found to be TSD carriers. Their prevalence in the study population is proportional to that in the general Ashkenazi population. However, abnormal neurological findings, especially cognitive impairment, were commoner among TSD carriers (47.4 vs. 26.2%, p = 0.04). It is possible that chronic use of some psychotropic drugs plays a role in this phenomenon. PMID- 10754427 TI - Season of birth variations in dimensions of functioning evaluated by the diagnostic interview for borderline patients. AB - In view of recent reports showing that cerebrospinal fluid (CSF) levels of monoamine metabolites exhibit season of birth variations, and that they are also associated with section II (impulse action patterns) of the diagnostic interview for borderline patients (DIB), we analyzed two samples of data to investigate the relationship between the season of birth and the DIB. The first sample comprised 202 patients participating in psychobiological research in Stockholm, and the second sample comprised 130 patients who had committed suicide in Vasterbotten in northern Sweden. Those with intermediate score for section II (impulse action patterns) were significantly more likely to have been born during the season October to January in the pooled data, and this tendency persisted in separate analyses for the two samples and for the two diagnostic groups mood disorders and schizophrenia, respectively. Those with high score for section IV (psychosis) were significantly more likely to have been born during February to April in the pooled sample and in the nonschizophrenic group. In the group with schizophrenia, those born during February to April had significantly high scores for section III (affects). These results throw further light on the role of season of birth in suicidology and in psychiatric morbidity. PMID- 10754428 TI - Placebo-controlled sleep laboratory studies on the acute effects of zolpidem on objective and subjective sleep and awakening quality in nonorganic insomnia related to neurotic and stress-related disorder. AB - Recent investigations in our sleep outpatient clinic demonstrated that 30% of patients exhibited organic and 70% nonorganic sleep disorders, with 41% showing as an additional diagnosis neurotic, stress-related, and somatoform disorders, 31% affective disorders and 15% mental and behavioral disorders due to psychoactive substance use. Thus, the aim of the study was to investigate the acute effects of the imidazopyridine zolpidem on objective and subjective sleep and awakening quality in the largest of the above-mentioned groups. In this single-blind, placebo-controlled cross-over study, 15 patients (9 females and 6 males aged 51.1 + 11. 3 years) diagnosed as having nonorganic insomnia (ICD-10: F 51.0) related to neurotic and stress-related disorders (F 1.1:12, F 41.2:2 and F 43.2:1) were included. Objective and subjective sleep and awakening quality measures were investigated in 3 subsequent nights in the sleep laboratory (adaptation, baseline/placebo and zolpidem 10 mg night), utilizing clinical, polysomnographic, psychometric and psychophysiological methods. The drug-free patients were matched according to age and sex with 15 normal healthy controls (age 51.2 + 11.8 years). Statistical analysis of polysomnographic variables demonstrated a significant lengthening of the total sleep period (TSP) and total sleep time (TST), an improvement in sleep efficiency and a shortening of sleep latencies after zolpidem as compared with placebo. These changes were opposite to the differences between patients and controls. Concerning sleep architecture, zolpidem increased the length of S4 and S3 + S4 as compared with placebo. Subjective sleep and awakening quality and the thymopsychic variables drive, mood, affectivity and wakefulness in the morning showed no significant changes, as a significant improvement had already occurred from the adaptation to the baseline/placebo night. Noopsychic variables (attention, concentration, attention variability, numerical memory, fine motor activity, reaction time measures) showed similar findings. Moreover, subjective sleep and awakening quality, thymopsychic and noopsychic measures during baseline/placebo recordings did not differ significantly from normative data (except for fine motor activity). Psychophysiological measures did not show any significant alterations either, except for a decrease in systolic blood pressure in the evening. CONCLUSION: As compared with placebo, zolpidem induced a significant improvement in objective sleep quality, mainly by increasing TSP, TST and sleep efficiency and shortening sleep latencies, thereby normalizing the disorder of initiating and maintaining sleep. Deep sleep stages S3 + S4 increased (although at baseline/placebo these stages did not differ from controls), while S1, S2 and SREM did not change significantly. Subjective sleep and awakening quality as well as thymopsychic and noopsychic performance in the morning mainly showed a placebo and 'first- night effect' phenomenon in these patients. Thus, the changes induced by zolpidem were somewhat different from those after classical benzodiazepines. PMID- 10754429 TI - Correlation dimension of the human electroencephalogram corresponds with cognitive load. AB - This study aimed at assessing the effects of cognitive activity and mental task load on the correlation dimension of the human electroencephalogram (EEG). Three experimental conditions were created: a baseline condition and two cognitive task conditions, a calculation task and a time estimation task. The calculation task was supposed to induce a higher mental load than the time estimation task, which is regarded as a less complex one. This was verified by a subjective rating scale. All conditions differed significantly in subjective estimated task load. The correlation dimension appeared to be higher in both task conditions compared to the baseline condition. A comparison of the two tasks indicated that the difference in correlation dimension between calculation and time estimation was also significant, with the highest value for calculation. It is concluded that cognitive and mental activity is associated with a higher correlation dimension in the EEG. This implies that the correlation dimension is a sensitive parameter in the analysis of electrical brain activity. PMID- 10754430 TI - The control of blepharospasm by essential fatty acids. AB - Dopamine depletion induced by administration of Ro4-1284 produces a condition of rapid and repeated eye blinking in rats. This condition mimics the human disorder, blepharospasm, which often accompanies parkinsonism and other dopamine deficiency disorders. When given a 3-week course of a compound (SR-3) developed from a specific ratio of two free polyunsaturated fatty acids - linoleic acid and alpha-linolenic acid - the eye blinking rate following administration of Ro4-1284 is reduced to saline and no drug control levels. These results suggest a favorable prospect for essential fatty acids in general, and SR-3 in particular, to provide an improved therapeutic option for the clinical management of benign essential blepharospasm. PMID- 10754431 TI - Policosanol, reaction time and event-related potentials. AB - The aim of the present study was to compare the results of a 1-week, double-blind placebo-controlled trial investigating the effects of isopolicosanol and octacosanol on reactivity and related brain activity. In particular, reaction time (RT) and event-related potentials such as contingent negative variations (CNV) and P300 (P3) have been studied. Thirty sedentary healthy students were tested before and after treatment (3.6 mg/die for 7 days) with orally administered tablets of placebo (group A), isopolicosanol (B) and octacosanol (C). RT were studied according to three procedures: simple RT (SRT), go/no-go RT (GRT) and choice RT (CRT). Results show that before treatment, there were no significant differences between groups A, B and C. After treatment, the RT of group A was unchanged, while the RT of groups B and C were reduced. In group B, in the SRT test, the reduction of RT was accompanied by electrical data exhibiting increased amplitudes of CNV and shorter latencies of P3. These results show that the main effect on reactivity and event-related potentials can be ascribed to policosanol and is mainly evident in the SRT test. PMID- 10754432 TI - Quantitative EEG in schizophrenic patients before and during pharmacotherapy. AB - The aim of the study was to determine the possible differences in quantitative EEG parameters of schizophrenic patients before and during therapy with neuroleptics. First EEG recordings were obtained from schizophrenic patients (n = 50) who had not been taking any medicaments during the preceding 2 months. Second EEG recordings were obtained during the administration of neuroleptic therapy. Amplitude values of particular spectral segment, i.e. delta, theta, alpha 1, alpha 2, beta 1 and beta 2 (after fast Fourier transformation) were analyzed. The F3, F4, C3, C4, T3, T4, P3, P4, O1 and O2 regions were observed. The effect of pharmacotherapy manifested as a decrease in delta and beta 2 activities. The alterations of the delta spectrum were recorded in each patient subgroup (regardless of the neuroleptic used). The changes in beta 2 activity were registered in patients on haloperidol and fluphenazine. PMID- 10754433 TI - Developmental expression of GABA(A) receptors in retinal transplants. AB - gamma-Aminobutyric acid (GABA) has transiently been found in certain retinal cells during development, and thus it has been suggested that besides its role as an inhibitory neurotransmitter, it also plays a role during the development of the retina. Further it has been suggested that this developmental role of GABA is mediated through GABA(A) receptors. Retinal cell transplants are being tried for the treatment of degenerative retinal disorders. Even though the donor tissue continues to proliferate, to develop and to differentiate after transplantation, its development is not entirely normal. Various neurotransmitters have been found in retinal transplants, but the receptors, which are needed for their action, have not been demonstrated. It was therefore of interest to see the expression of GABA(A) receptors during the development of the transplants. Embryonic day (E) 15 rabbit retinas were transplanted into the eyes of adult rabbits. Transplants were allowed to survive for various times so that the grafts attained the equivalent ages of (donor age + survival time) E 19, 21, 26, 29 and postnatal (PN) day 2, 5, 9, 19 and 95. On formaldehyde-fixed cryostat sections of the transplant tissue immunohistochemistry was performed. Antibodies against the alpha(1) and beta(2/3) subunits of the GABA(A) receptors were used to demonstrate these receptors. No immunoreactivity was detected in transplants of ages E 19 and 22. The GABA(A) receptor beta(2/3) subunit first appeared in E 29 transplants, whereas the GABA(A) receptor alpha(1) subunit was first detected at PN 2. At these ages faint immunoreactivity was detected in certain plexiform layers in the transplants. In older ages the immunoreactivity increased and also appeared in certain cells lying in between the rosettes along with the plexiform layers that are equivalent to the inner and the outer plexiform layers of the normal retina. The development of the GABA(A) receptor alpha(1) and beta(2/3) subunit immunoreactivity compared well with that during the development of the normal retina. The results suggest that GABA present in the retinal transplants can exert its action through its receptors. PMID- 10754434 TI - Subretinal injection of rod outer segments leads to an increase in the number of early-stage melanosomes in retinal pigment epithelial cells. AB - Our study was performed to test the hypothesis that subretinally injected protein can induce melanogenesis in the retinal pigment epithelium (RPE). Rod outer segments (ROS) were isolated from cattle eyes and injected into the subretinal space of Long Evans rats. Five days after surgery, the injected eyes were investigated by electron microscopy. The number of early-stage melanosomes and small melanin granules was compared in injected and noninjected eyes. It was found that the injected ROS were phagocytized by the RPE cells, and the number of early-stage melanosomes in the RPE was significantly increased in injected eyes compared to eyes without injection. The ROS-containing endosomes fused with melanolysosomes in which melanogenesis took place. The increased number of early stage melanosomes indicates new formation of melanin. PMID- 10754435 TI - Protein kinase C isoenzyme expression in retinal cells. AB - Protein kinase C (PKC) is involved in both the physiological and pathophysiological processes in the retina and plays an important role in signal transduction. The aim of this study was to determine the PKC isoenzyme profile in three retinal cell types in culture, namely RPE cells, pericytes and retinal microvascular endothelial cells. Confluent cultures were lysed and isoenzyme expression detected by Western blotting. PKC isoenzymes alpha, beta(2) and delta were observed for all three cell types while beta(1) was specific for RPE cells. This study has characterised the PKC isoenzyme profile in three retinal cell types and suggests that defining the cell-specific isoenzyme pattern is an important step in understanding their precise physiological role and regulation in the retina. PMID- 10754436 TI - Colocalization of aldehyde dehydrogenases and Fe/NADPH-induced lipid peroxidation in tissue sections of rat retina. AB - Epidemiological and experimental studies suggest the involvement of lipid peroxidation (LPO) in retinal diseases. Clinicians usually prescribe antioxidants to help in the treatment of proliferative diabetic vitreoretinopathy and age related macular degeneration. In spite of this, these processes inexorably induce visual impairment and may progress towards blindness. In addition to other pathogenic mechanisms not fully understood, it may be that peroxidic aldehydes from LPO occurring in the eyes, acting as cytotoxic chemicals, mediate in these chronic disorders. To test the mechanisms of removing peroxidic aldehydes from retinal cells and in an attempt to understand long-lasting changes induced by LPO, the distribution and activity of aldehyde dehydrogenases (ALDH) in the rat retina were studied and compared with the LPO sites induced by iron/nicotine adenine dinucleotide phosphate. Histochemical and immunocytochemical assays revealed the colocalization of LPO and ALDH, mainly in the photoreceptors and inner retinal layers. This suggests the involvement of ALDH in detoxifying peroxidic aldehydes from the retina. Any change in ALDH retinal expression and distribution might be of crucial importance in assessing the paths of LPO mediated vitreoretinopathies. Further research is needed to evaluate these findings and their application to new ophthalmic therapy. PMID- 10754437 TI - Blue-on-yellow perimetry to evaluate S cone sensitivity in diabetics. AB - Blue-on-yellow (B/Y) perimetry was performed on 31 patients with non-insulin dependent diabetes mellitus to study the loss of sensitivity to short wavelengths. Of these patients, 21 were without retinopathy (NDR) and 10 had early background retinopathy (SDR). Eleven normal subjects served as controls. The results were compared to white-on-white (W/W) perimetry. Foveal sensitivity determined by B/Y and W/W perimetry showed no significant difference between NDR, SDR and normals. However, the mean sensitivity in the central 30-degree area and that in the upper half of the central 20- to 30-degree concentric circular field were significantly decreased in B/Y perimetry in SDR patients (p < 0.05, p < 0.01, respectively). No significant sensitivity loss was detected in the W/W test. We conclude that there is a blue cone system sensitivity loss in the central 30-degree area, particularly in the upper half of the visual field and the paracentral area in diabetic patients with early background retinopathy. PMID- 10754438 TI - Nucleoside diphosphate kinase (nm23 protein) expression in retinoblastoma. AB - Forty-two enucleated eyes of 42 patients with unilateral retinoblastoma were studied histologically, including histochemically examination with anti-nm23 polyclonal antibody which does not recognise cDNA but its product. Primary tumours of >15 mm diameter with less evidence of apoptosis and with the most pleomorphic and anaplastic nuclei were associated with an increased risk of distant metastasis, but rosette formation did not discriminate. A high intensity of nm23 staining also indicated a tendency to metastasize, consistent with childhood neuroblastoma but in contrast to findings in carcinoma of the breast, colon and uterine cervix. PMID- 10754439 TI - Evaluation of the circulation in the retina, peripapillary choroid and optic disk in normal-tension glaucoma. AB - Computerized image analysis, including fluorescein angiography, was used to evaluate the retinal, choroidal and optic disk blood flow in 16 patients with normal-tension glaucoma (NTG) and to correlate this measurement with visual fields, retinal vessel width, optic disk pallor and blood pressure (BP). The angle of the ascending slope of the fluorescein dye curve was measured as an index of blood flow from the densitometric and time curves of the fluorescein angiograms in the optic disk, peripapillary choroid, retinal artery and vein for each quadrant. While the ascending slope as well as the retinal vessel width were most reduced in the inferior and nasal regions, the mean threshold was lowest in the superior and nasal quadrants. There were positive significant correlations between artery width and threshold value, between angles of slopes and pallor. In addition, systolic BP had a negative correlation with pallor, and diastolic BP had a positive one with slope in the choroid. These results indicated the probable association of a decrease in retinal, choroidal and optic disk blood flow in the inferior and nasal quadrants as well as vessel width in the inferior nasal quadrant with visual field loss in the superior quadrant, and also demonstrated an increasing blood flow for enlargement of pallor. A decrease in BP was found to be related to reduced blood flow in choroid and optic disk impairment. PMID- 10754440 TI - Linoleic acid cytotoxicity to bovine lens epithelial cells: influence of albumin on linoleic acid uptake and cytotoxicity. AB - The high cytotoxicity of linoleic acid (LA) to cultured bovine lens epithelial cells is correlated with high uptake rates for the fatty acid (FA). Both, LA uptake and LA cytotoxicity strongly increase with the increasing LA-to-albumin molar ratio in the culture medium. Cellular uptake and cytotoxicity of LA can be competitively inhibited with the noncytotoxic palmitic acid. The findings may be of interest in view of the low albumin concentration in aqueous humor, resulting in extremely low buffering capacities for free FAs including LA, oleic acid and other cytotoxic cis-unsaturated free FAs, which are strongly raised in pathological situations like diabetes mellitus. PMID- 10754441 TI - Effect of rabbit aqueous humor obtained after cataract surgery on collagen gel contraction induced by bovine lens epithelial cells. AB - We evaluated the effect of specimens of pre- and postoperative aqueous humor on the contraction of collagen gels, and the effect of transforming growth factor beta(2) (TGF-beta(2)) in postoperative aqueous humor. Rabbit aqueous humor was collected preoperatively and on postoperative day 7. Bovine lens epithelial cells (LECs) were cultured in collagen gel in F-12 nutrient mixture supplemented with 5% fetal bovine serum that contained 10% aqueous humor obtained under various conditions. Gel area was determined on day 4. Gels cultured with the medium that contained phosphate-buffered saline showed a statistically significant contraction after 4 days. Aqueous humor from aphakic or pseudophakic eyes significantly increased contraction, with both specimens having a similar effect. Approximately 60% of the contractile effect of the postoperative aqueous humor was neutralized by anti-TGF-beta(2) antibody. However, the promoting effect of the aqueous humor sampled postoperatively was less than that sampled preoperatively. Although the aqueous humor obtained postoperatively increased the contractility of the LECs, with the level of TGF-beta(2) apparently responsible for much of its effect, the effect was less than that observed in the aqueous humor obtained preoperatively. PMID- 10754442 TI - Plasma membrane calcium ATPase gene expression in bovine lens epithelium. AB - Plasma membrane calcium adenosine triphosphatase (Ca(2+)-ATPase) is an energy dependent protein responsible for transporting cytosolic calcium across the plasma membrane. Multiple plasma membrane Ca(2+)-ATPase isoforms are expressed from four genes (PMCA1-4) and alternative mRNA splicing. We have studied PMCA gene expression in bovine lens epithelium tissues by reverse transcription polymerase chain reaction, Southern blot, and Northern blot hybridization. All four PMCA genes are expressed in the lens epithelium, the PMCA3 transcript being the most abundant. The transcripts for PMCA1, PMCA2, and PMCA4 exist in decreasing order of abundance. There is no evidence for the expression of any novel PMCA genes in bovine lens epithelium. PMID- 10754443 TI - Quantification of nitric oxide and cytokines in rabbit aqueous humor after neodymium:YAG laser capsulotomy. AB - PURPOSE: The purpose of this study was to evaluate the presence of nitric oxide (NO) and cytokines such as interleukin 1 beta, interleukin 2 receptor (IL-2R), interleukin 6 and tumor necrosis factor alpha on aqueous humor after neodymium:YAG (Nd:YAG) laser posterior capsulotomy. METHODS: Fifteen rabbits (30 eyes) with dense posterior capsule opacities underwent Nd:YAG capsulotomy 4 months after phacoemulsification cataract surgery. After general and topical anesthesia, Nd:YAG capsulotomy was performed using an equal number of shots and power setting on all eyes. Aqueous humor specimens were collected from each eye preoperatively and at 12, 24 and 48 h after the surgery to determine NO and cytokine levels. RESULTS: The levels of NO and cytokines except IL-2R were significantly high as compared to preoperative levels in all eyes at 12 and 24 h postoperatively (p < 0.05). Although the levels of NO and cytokines were higher than preoperative levels at 48 h, the differences were not statistically significant (p > 0.05). CONCLUSION: These findings suggest that NO and cytokines appear to be potential inflammatory mediators in the occurrence of early inflammation following Nd:YAG posterior capsulotomy. PMID- 10754444 TI - Expanded polytetrafluoroethylene episcleral implants used as encircling scleral buckling. an experimental and histopathological study. AB - To investigate tissue changes induced by the implantation of expanded polytetrafluoroethylene (e-PTFE) used as encircling scleral buckling, an experimental and histopathological study was performed in rabbits. The eyes of 12 rabbits were implanted during 20-128 days with e-PTFE episcleral implants. Of these, 8 eyes were uncomplicated and enabled us to perform the histopathological study, 2 others were discarded for too short a time of implantation and, in 2 additional eyes, the episcleral implant was exposed with a wide conjunctival erosion. Surrounding the implant, a constant encapsulation was combined with numerous giant cells, forming a granuloma caused by irregularities of the implant outline. Its porous structure allowed a peripheral colonization by fibrovascular tissue. Scleral thinning and scleral invagination were the main scleral changes. No change of the conjunctiva made us suspect that a granuloma occurred before it was found histologically. The granulomatous changes did not seem to modify the apparently good experimental tolerance of the material after its implantation. PMID- 10754445 TI - Virucidal and chlamydicidal activities of eye drops with povidone-iodine liposome complex. AB - Povidone-iodine (PVP-I) is a broad-spectrum microbicide with in vitro activity against bacteria, viruses, fungi and protozoans. A 5% solution of PVP-I proved to be highly effective in ophthalmic surgery for the prophylaxis of endophthalmitis. For the antiseptic treatment of eye infections a novel application form of PVP-I has been developed by using a PVP-I liposome complex which demonstrated an excellent antimicrobial efficacy. In this study it could be shown that the novel liposomal formulations containing 2.5 or 5% PVP-I were as active as the aqueous solution against herpes simplex virus type 1, adenovirus type 8, coxsackievirus A9 and Chlamydia trachomatis in cell culture referring to equal PVP-I concentrations. Long-term cytotoxicity experiments demonstrated a moderate cytotoxicity for both formulations with a better tolerability of the liposomal PVP-I formulation compared with the aqueous solution. There is no evidence for a genotoxic activity of these agents. PMID- 10754446 TI - Intravitreal trovafloxacin against experimental Staphylococcus epidermidis endophthalmitis. AB - This study was designed to test the effects of intravitreal trovafloxacin on an experimental rabbit model of Staphylococcus epidermidis endophthalmitis. Out of 26 rabbits, 22 were given intravitreal S. epidermidis (100,000 CFU). At 24 h, group 1 (8 rabbits) and, at 48 h, group 2 (8 rabbits) received 100 microg intravitreal trovafloxacin. Group 3 (6 rabbits) was used as inoculated but untreated controls. Four rabbits (group 4) were used as uninfected controls. Clinical scores were calculated at 24, 48 and 72 h. Microbiological and histopathological scorings were made. Microbiological analysis showed that trovafloxacin administered at 24 or 48 h significantly reduced the number of bacteria compared to the untreated group. We conclude that trovafloxacin applied at 24 or 48 h is effective against S. epidermidis endophthalmitis in this experimental rabbit model. PMID- 10754447 TI - Kallikrein-kinin system in acute pancreatitis: potential of B(2)-bradykinin antagonists and kallikrein inhibitors. AB - The development of selective antagonists for bradykinin B(2) receptors has greatly advanced research on the role of the kallikrein-kinin system in acute pancreatitis. Kinins released during the course of the inflammatory injury are the major cause of the vascular symptoms, i.e. pancreatic oedema formation and its consequences, such as haemoconcentration, hypovolaemia and hypotension. Kinins are also involved in the accumulation of potentially cytotoxic factors in the pancreatic tissue. However, treatment with B(2) antagonists must begin prior to the formation of pancreatic oedema in order to inhibit or attenuate the vascular effects. Visceral pain as a possible target symptom for treatment with B(2) antagonists at later time points is suggested by the B(2) receptor-mediated activation of nociceptive afferents. PMID- 10754448 TI - Antinociceptive/anti-edema effects of liposomal morphine during acute inflammation of the rat paw. AB - We evaluated the anti-edema/antinociceptive effects of subcutaneous free and liposomal morphine in rats with carrageenan-induced inflammation of the paw. We assessed antinociception by the paw pressure test and edema by plethysmography. Unilamellar liposomes (150-200 nm) with 0.3% morphine hydrochloride were used; encapsulation significantly reduced the rate for release of morphine in vitro. During inflammation, the antinociceptive potency of free, but not liposomal morphine increased 2.5 times; moreover, duration of the effects was prolonged by encapsulation (p < 0.001). The anti-edema effects of liposomal morphine were more pronounced (p < 0. 001) and of longer duration (p < 0.05). All the effects were reversed by naloxone. The results show that morphine encapsulation enhances the anti-edema effects and prolongs antinociception. PMID- 10754449 TI - In vitro pharmacological profile of SK-896, a new human motilin analogue. AB - SK-896 ([Leu(13)]motilin-Hse) is a new human motilin analogue synthesized by Escherichia coli using a biotechnological method. We investigated the binding of SK-896 to motilin receptors and the contractile effect of SK-896 on smooth muscle preparations isolated from the gastrointestinal tract and various regional organs in order to clarify its in vitro pharmacological profile. SK-896 inhibited the binding of (125)I-human motilin to rabbit gastroduodenal motilin receptors with the same potency as unlabeled human motilin. The IC(50) values of SK-896 and human motilin were 3.5 +/- 1.5 and 3.1 +/- 1.8 nmol/l, respectively. The K(d) of human motilin was 3.0 +/- 1.5 nmol/l, and the Ki of SK-896 was 3.4 +/- 1.5 nmol/l. SK-896 induced contraction of smooth muscle preparations isolated from rabbit duodenum in a concentration-dependent manner. However, there was no effect of SK-896 on duodenal preparations isolated from the dog and the rat. SK-896 thus exhibited species specificity in its contractile effect. We next investigated the effect of SK-896 on various smooth muscle preparations isolated from rabbit gastrointestinal tract, trachea, bladder, gallbladder, uterus, vas deferens and artery. Results showed that SK-896 induced contraction of smooth muscle preparations isolated from gastrointestinal tract, with potencies in the order duodenum > gastric pylorus = jejunum = descending colon > ascending colon >/= ileum. However, there was no effect of SK-896 on smooth muscle preparations from gastric fundus and other regional organs. SK-896 thus exhibited regional specificity in its contractile effect. Moreover, the effects of SK-896 on smooth muscle preparations from rabbit duodenum were the same as those of human motilin, and were not inhibited by pretreatment with tetrodotoxin and atropine but were inhibited by verapamil. These findings indicate that SK-896 has the same pharmacological profile as human motilin. They suggest that SK-896 acts on gastrointestinal smooth muscle isolated from rabbit directly and specifically. PMID- 10754450 TI - Effect of nabumetone treatment on vascular responses of the thoracic aorta in rat experimental arthritis. AB - Nabumetone is a nonsteroidal anti-inflammatory (NSAI) drug which is known to cause less gastrointestinal damage than other NSAI drugs. This study was performed to evaluate whether nabumetone treatment might alter the vascular aberrations related to inflammation in a rat model of adjuvant-induced arthritis. Nabumetone treatment (120 or 240 mg x kg(-1) x day(-1), orally) was initiated on the 15th day of adjuvant inoculation and continued for 14 days. Arthritic lesions, vascular contractile and relaxant responses and gastroduodenal histopathological preparations were evaluated 29 days after adjuvant inoculation. The contractile responses of aortic rings to phenylephrine and KCl were increased in grade 2 arthritic rats. In grade 3 arthritis only the phenylephrine contractility was decreased. The relaxant responses to acetylcholine and sodium nitroprusside were decreased in grades 2 and 3. In healthy rats, nabumetone did not change the vascular responses. After treatment of arthritic rats with nabumetone, both the contractile and relaxant response of the aortic rings returned to normal, and arthritic score and paw swelling were reduced. Gastroduodenal histopathology did not show erosions or ulcers in any of the groups. In conclusion, nabumetone improved the systemic signs and vascular alterations in experimental arthritis without showing any gastrointestinal side effects. PMID- 10754452 TI - Influence of polyunsaturated fatty acids on vasoconstrictions induced by 8-iso PGF(2alpha) and 8-iso-PGE(2). AB - 8-iso-PGF(2alpha) and 8-iso-PGE(2), which are released in vivo by free radical catalyzed peroxidation of arachidonic acid, are equipotent vasoconstrictors in vivo and in vitro. It is assumed that they exert this effect via activation of the thromboxane A(2) (TP) receptor or a TP-receptor-like isoprostane receptor. Increased levels of 8-iso-PGF(2alpha) have been detected in human cardiovascular diseases. It has been found that polyunsaturated fatty acids (PUFAs) have many beneficial effects in cardiovascular diseases, including antivasoconstrictor actions. Therefore, we investigated the influence of perfusions with eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and dihomo-gamma linolenic acid (DGLA) at final concentrations of 3 and 30 micromol/l on vasoconstriction induced by 8-iso-PGF(2alpha), 8-iso-PGE(2) and the thromboxane A(2) mimetic U 46619 in the vasculature of the isolated perfused rabbit ear. Additionally, the effect of indomethacin (final concentration 3 micromol/l) on the effects of the PUFAs was investigated. Our results show that the PUFAs at a concentration of 30 micromol/l caused a significant inhibition of the vasoconstrictions induced by 8-iso-PGF(2alpha), 8-iso-PGE(2) and U 46619. Furthermore, it can be assumed that a part of the inhibitory effect of DGLA is due to the effect of a cyclooxygenase product, probably PGE(1), because indomethacin reduced the inhibitory effect of DGLA. PMID- 10754451 TI - Role of oxidative stress and glutathione in busulfan toxicity in cultured murine hepatocytes. AB - This study examines busulfan metabolism. Busulfan given in vivo or in vitro decreased hepatocyte glutathione (GSH) by 60 and 50%, respectively. In vitro, busulfan toxicity was prevented by glutathione S-transferase inhibitors or by antioxidants and led to increased production of oxidized GSH and thiobarbituric acid reactive substances. 'Rescue' from toxicity by GSH precursors was prevented by N,N-bis(2-chloroethyl)-N-nitrosourea (BCNU). Depletion of GSH exacerbated toxicity. In GSH-depleted hepatocytes, busulfan decreased GSH by 95% and BCNU did not prevent rescue by GSH precursors. CONCLUSIONS: (1) In hepatocytes with normal GSH: busulfan toxicity requires GSH conjugation, does not cause profound GSH depletion and is mediated by oxidative stress. We postulate that a GSH conjugate promotes oxidative stress. (2) In GSH-depleted hepatocytes: busulfan profoundly depletes GSH; toxicity is mediated by oxidative stress and is prevented by restoring GSH levels; cell death may be due to unopposed endogenous oxidative stress. PMID- 10754453 TI - Pharmacological properties of the gastric H(+)/K(+) ATPase inhibitor, AU-461. AB - AU-461 (1-(2-methyl-4-methoxyphenyl)-4-[(2-hydroxyethyl)amino]-6-beta,beta, beta trifluoroethoxy-2,3-dihydropyrrolo[3,2-c]quinoline) was tested for its ability to act as an anti-ulcer agent. AU-461 inhibited gastric H(+)/K(+) ATPase activities with IC(50) values of 12.15 and 4.20 micromol/l for rabbit and pig enzymes, respectively. The inhibition was reversible, and competitive with respect to the activating cation K(+). When AU-461 was examined for the in vivo antisecretory activity, we found that AU-461 reduced the histamine-stimulated acid secretion as well as the basal secretion in rat stomach. Duration of the antisecretory effect was about 6 h upon oral administration. AU-461 prevented dose-dependently the ulcer formation produced by either ethanol or NaOH. This protective effect was not altered by indomethacin pretreatment. In addition, the elevated plasma gastrin by the oral administration of AU-461 was returned to control by 12 h. Taken together, these results suggest that AU-461 could be developed as a new therapeutic agent for peptic ulcer disease. PMID- 10754454 TI - Inhibition of irritation and contact hypersensitivity by phenoxyacetic acid methyl ester in mice. AB - New anti-irritant treatments are required to prevent irritation and sensitization reactions to consumer medicines and dermatological drugs. We report here that phenoxyacetic acid methyl ester (PAME) is an effective agent to prevent and treat irritant and allergic contact dermatitis. Balb/c mice skin-treated with 1% PAME do not lose weight relative to vehicle-treated mice, nor is it irritating to mouse skin. Topical PAME prevents skin irritation to a wide variety of irritants including: arachidonic acid, capsaicin, sodium lauryl sulfate (SLS), disodium laureth sulfosuccinate and tetradecanoylphorbol-13-acetate. Histological studies showed that 1% PAME greatly diminished dermal neutrophilic infiltration and dermal capillary vessel dilation, and prevented epidermal hyperproliferation and hyperkeratosis that accompanies detergent (SLS)-induced skin irritation. Topical PAME inhibited ear swelling following ear challenge during the elicitation phase of contact hypersensitivity in mice sensitized with 1-chloro-2, 4 dinitrochlorobenzene (DNCB), oxazolone and the hair coloring dye rho phenylenediamine (PPD). Finally, topical administration of 1% PAME prior to PPD or DNCB sensitization prevented the induction phase of contact hypersensitivity. These results indicate that PAME represents a potential new category of potent topical anti-inflammatory agents. PMID- 10754455 TI - Pharmacological effects of a specific leukotriene B(4) receptor antagonist (VML 295) on blood leukocytes, cutaneous inflammation and epidermal proliferation. AB - VML 295 (LY 293111) is a potent and specific leukotriene(4) receptor antagonist. It has previously been shown in human volunteers that VML 295 at a dosage of 48 mg twice daily inhibits the ex vivo leukotriene B(4) (LTB(4))-induced upregulation of CD11b on peripheral blood neutrophils. A clear dose-response relatinship was shown. In addition, VML 295 inhibits various inflammatory aspects resulting from LTB(4) challenge of the skin, again showing a dose-response relationship. In view of the large variation in the elimination half-life of VML 295 (25-88.5 h) in individual human subjects, the present pharmacological study was designed to provide information on the pharmacodynamics of the drug by the assessment of VML 295 plasma concentrations, ex vivo LTB(4)-induced CD11b upregulation of neutrophils, neutrophil accumulation in the skin following epicutaneous application of LTB(4) and epidermal regeneration following standardized surface trauma. A group of 36 healthy volunteers were treated in a double-blind study with VML 295 at 200 mg twice daily, VML 295 at 200 mg once daily or placebo for 7 days. Before treatment, at the end of treatment and following discontinuation of treatment, VML 295 plasma concentrations and CD11b upregulation of blood neutrophils were assessed. In 18 subjects, the effects of the three treatments on LTB(4)-induced inflammatory were assessed before and at the end of treatment, and in the remaining 18 subjects the effects of these treatments on epidermal regeneration were assessed similarly. VML 295 at 200 mg either twice or once daily has a profound inhibitory effect on ex vivo LTB(4) induced CD11b upregulation of blood neutrophils, LTB(4)-induced neutrophil accumulation in the skin, trauma-induced hyperproliferation of the epidermis and regenerative keratinization. The twice daily dose schedule was significantly more effective than the once daily regimen in reducing ex vivo CD11b stimulation of neutrophils, in blood samples collected 24 h after discontinuation of VML 295 treatment. The twice daily schedule tended to be more efficient in skin biopsies, although this difference was not statistically significant in the number of subjects investigated. A plasma concentration of 100 ng/ml proved to be the threshold for these effects. The profound biological effects, both systemically and cutaneously, as well as the safety profile, make VML 295 a promising drug for skin disorders characterized by epidermal proliferation and neutrophil accumulation. PMID- 10754456 TI - Correlation between in vitro cyclic adenosine monophosphate phosphodiesterase inhibition and in vivo anti-inflammatory effect. AB - We tested the hypothesis that topical adenosine monophosphate phosphodiesterase (cAMP PDE) inhibitors are anti-inflammatory. These can be shown by a correlation between PDE inhibitory and anti-inflammatory function of a series of known PDE inhibitors. The effect of various cAMP PDE inhibitors on PDEs isolated from HaCaT cells was first investigated. These compounds were then tested as anti-irritants against topical 8% Balsam of Peru. A direct correlation was observed between the in vitro EC(50) values for PDE inhibition and the in vivo anti-inflammatory potential with a correlation coefficient of r = 0.79. These results stress the value of PDE inhibitors as anti-inflammatory agents in topical use, and also demonstrate that the in vitro PDE assay can be used to predict in vivo anti inflammatory potential. PMID- 10754457 TI - Cutaneous inflammation and proliferation in vitro: differential effects and mode of action of topical glucocorticoids. AB - The nonhalogenated double ester of prednisolone, prednicarbate (PC), is the first topical glucocorticoid with an improved benefit/risk ratio verified clinically and in vitro. To evaluate if this is due to unique characteristics of this steroid, a new compound created according to an identical concept, prednisolone 17-ethylcarbonate, 21-phenylacetate (PEP), and the new halogenated monoester desoximetasone 21-cinnamate (DCE) were tested and compared to PC, desoximetasone (DM) and betamethasone 17-valerate (BMV). Isolated foreskin keratinocytes served for in vitro investigations of anti-inflammatory processes in the epidermis, fibroblasts of the same origin were used to investigate the atrophogenic potential. Inflammation was induced by TNFalpha, resulting in an increased interleukin 1alpha (Il-1alpha) synthesis. As quantified by ELISA, all drugs significantly reduced Il-1alpha production. But PC and BMV appeared particularly potent, followed by DM and the two new congeners, which revealed minor anti inflammatory activity. Glucocorticoid esters including PEP are rapidly degraded in keratinocytes (85% within 12 h). Hence, a ribonuclease protection assay of Il 1alpha mRNA was performed allowing short incubation times and thus minimizing biodegradation. This assay confirmed the anti-inflammatory potency of native PC and BMV. In contrary DCE and PEP did not reduce Il-1alpha mRNA to a significant extent. Therefore PEP acts as a prodrug only. In fibroblasts, Il-1alpha and Il-6 syntheses indicate proliferation and inflammation, respectively. Whereas PC and PEP inhibited Il-1alpha and Il-6 production in fibroblasts only to a minor extent, cytokine synthesis was strongly affected by the conventional glucocorticoids BMV and DM, but also by DCE. The minor unwanted effect of PC and PEP on fibroblasts was also reflected by their low influence on cell proliferation as derived from (3)H-thymidine incorporation. Again, more pronounced antiproliferative features were seen with the halogenated glucocorticoids. In the following, the correlation between antiphlogistic effects in keratinocytes (suppression of Il-1alpha) and antiproliferative effects in fibroblasts (suppression of Il-1alpha and Il-6; (3)H-thymidine incorporation) was analyzed. Here, PC is revealed as the only glucocorticoid with an improved benefit/risk ratio. Native PEP is shown to be almost ineffective and DCE presents exactly the opposite features of PC. It is tempting to speculate if this is due to different glucocorticoid receptor subtypes or different signaling pathways in keratinocytes and fibroblasts. PMID- 10754458 TI - Diffusion of water across human skin in the presence of two barrier creams. AB - The in vitro permeability of tritiated water through fresh and frozen human skin was evaluated in the presence and absence of two different barrier creams. Treated (10 min) and untreated fresh and frozen human skin disks (4 mm in diameter) were mounted in flow cells of a continuous flow-through diffusion apparatus. Buffer/tritiated water was collected from the acceptor chambers at 2 hour intervals for a total of 20 h and counted in a liquid scintillation counter. The results indicated that both barrier creams lowered the average flux rates of tritiated water through fresh and frozen skin, but no significant differences could be detected between the two preparations. However, different results may be obtained when compounds with molecular weights much higher than water are used. PMID- 10754459 TI - Age and sex variation in lipid composition of human fingernail plates. AB - In contrast to epidermal lipid metabolism, the lipid biochemistry of nails has yet been poorly investigated. Our purpose was to define a reference population of healthy individuals as a base for the better understanding of nail diseases and age-induced changes. Therefore, we developed a method of processing and extracting the nail plates enabling us to assess the most relevant epidermal lipid classes by HPTLC. Our study revealed that nail plate lipid composition varies with age and sex: the lipid composition of the fertile years shows distinct profiles compared to that of childhood and old age, suggesting an influence of sex hormones on nail lipogenesis. Our results open the possibility in the future of an easier comparison between healthy and diseased nails and enable us to investigate factors influencing nail lipid composition such as drugs, metabolic diseases, toxic agents, cosmetics and nail therapeutics. PMID- 10754460 TI - Comparative study of the efficacy of various moisturizers on the skin of the ASR miniature swine. AB - The skin of the back of the ASR hairless miniature swine shows a rough and scaly fissured appearance. Clinically it resembles that of ichthyosis vulgaris or senile xerosis in humans. By using the skin of this animal, we developed an assay system to evaluate the efficacy of different moisturizers. The study was carried out in winter months from January to March. To establish this system, we studied various topical agents including such ordinary emollients as petrolatum and 10% salicylic acid in petrolatum as well as those available as pseudodrugs from cosmetic companies or prescription drugs from pharmaceutical companies such as heparinoid- containing creams (w/o and o/w) and 10% urea creams. Applying 0.01 g of each agent to 7 randomly selected skin areas (2x2 cm(2)) on the back of a 6 month-old swine once daily for various periods of time, we measured high frequency conductance, the parameter for the hydration state of the skin surface. As a result, we have established a simple assay system. It consists of applications of topical agents once daily for 5 days from Monday to Friday in the first week and of subsequent measurement of the skin surface hydration state once every week. Evaluation measurements were performed from day 14 to day 21 depending upon their moisturizing effects. We found that heparinoid-containing creams exerted highly effective moisturizing effects on the dry pig skin that lasted even over 3 weeks after discontinuation of the treatments that were carried out for the first 5 days. PMID- 10754461 TI - Dose-dependent inhibition of ribonuclease P activity by anthralin. AB - The effect of five different anthralin concentrations on tRNA biogenesis was investigated employing the ribonuclease P (RNase P) of the slime mold Dictyostelium discoideum as an in vitro cell-free experimental system. RNase P is an ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA precursors to produce the mature 5' end. Anthralin revealed a dose-dependent inhibition of RNase P activity indicating that this compound may have a direct effect on tRNA biogenesis. Taking into account that anthralin has no structural similarities to the substrate (pre-tRNA) of RNase P, it seems reasonable to suggest that this compound may bind to allosteric inhibition sites of the enzyme. PMID- 10754463 TI - Homozygous arginine at codon 72 of p53 has no prognostic significance in cervical cancer. AB - Mutation of p53, a tumour suppressor gene, is uncommon in cervical cancer but the detection of human papillomavirus (HPV) DNA in cervical cancer is common. The findings of increased susceptibility to degradation of p53 by E6 protein of HPV16/18 in cervical cancer with homozygous arginine at codon 72 (HA72) of p53 led to this study on whether cervical cancers with HA72 were more aggressive with the increase in the rate of loss of p53 function. In 102 cervical cancers, 76.5% were HPV16/18 positive and 30% had HA72. No survival difference was detected between HA72 and non-HA72 tumours irrespective of HPV16/18 status. Furthermore, the detection of HPV16/18 in cervical cancer was found not to be of prognostic significance in this study. PMID- 10754462 TI - Comparison of cytosolic p53 protein levels in the female genital tract and breast, and their tumors. AB - Under normal conditions, p53 protein is thought to maintain genomic stability. We measured this parameter in healthy tissues from female breast and genital tract using a quantitative, highly sensitive luminometric assay. An organ-specific pattern of p53 expression became evident: breast parenchyma (n = 40, median p53: 0.0346 ng/mg protein) and ovarian tissue (n = 12, 0.063 ng/mg) demonstrated markedly higher p53 levels than endometrium (n = 24, 0.0065 ng/mg), myometrium (n = 31, 0.005 ng/mg) or uterine cervix tissue (n = 25, 0. 002 ng/mg). Malignant tumors derived from these organs maintained the pattern of p53 expression with ovarian cancers (n = 14, median: 0.84 ng/mg) exceeding all other tissue types examined. Generally, p53 concentrations in malignant tumors, but also in uterine myomas were significantly higher than those in healthy controls. Breast cancer tissues, subgrouped according to prognostic parameters, demonstrated the highest p53 concentrations in samples with atypical histology, grading II-III, negative steroid receptors, and in cases of positive axillary lymph nodes. The frequency of elevated p53 concentrations in cancer cytosols, based on organ-specific normal concentrations, varied between 62% in breast cancers and 100% in cervical carcinomas. Uterine myomas showed 6% of elevated values. Grade II-III breast carcinomas overexpressed p53 more often than those with grading I (p < 0.05). In all carcinomas, the frequencies of overexpressed p53 protein markedly exceeded the frequencies of mutated p53 gene mutations reported in the literature. In conclusion, our data indicate that the extent of p53 expression and overexpression is organ dependent. When data of other studies on primary breast cancers are included, elevated levels of p53 protein in malignant tumors to some extent may indicate p53 gene mutations and worse prognosis if they exceed a higher threshold. PMID- 10754464 TI - Serum level of HER-2/neu in patients with gastric cancer: correlation with HER 2/neu overexpression in gastric carcinoma tissue. AB - Serum HER-2/neu (c-erbB-2) levels in patients with gastric cancer were evaluated by an enzyme-linked immunosorbent assay and tissue levels of HER-2/neu in the same cohort were determined by immunohistochemistry. Nine (16%) of 57 gastric carcinomas had an overexpression of HER-2/neu detected immunohistochemically. Of these 9 patients, 6 had elevated serum HER-2/neu levels, while 45 of 48 tissue samples with negative staining exhibited normal serum HER-2/neu levels. These results indicated that overall accuracy, positive predictive value, and negative predictive values of their serum measurements were 89, 67 and 94%, respectively. Serum levels of HER-2/neu were correlated with tissue overexpression of HER-2/neu in patients with gastric cancer. PMID- 10754465 TI - Passive immunotherapy of mice bearing Ehrlich ascites tumor expressing human, membrane-bound placental alkaline phosphatase. AB - The objective of our study was to test if a tumor expressing a transgene coding for a membrane-bound protein is amenable to immunotherapy by antibodies to the same protein. To this end, we have established an Ehrlich ascites tumor (EAT) cell line, EAT-DAP, stably expressing human, membrane-bound placental alkaline phosphatase (PLAP) by infecting EAT cells (EATC) with the retroviral vector DAP and selecting neomycin-resistant cells. EATC and EAT-DAP cells grew at similar rates in vitro, and produced ascites tumor in Swiss-Webster mice with similar efficiency. We have treated mice bearing EAT-DAP ascites tumor with a mouse monoclonal antibody to human PLAP or with a monoclonal antibody to human C proteins of the heterogenous ribonucleoprotein complex (hnRNP). The average survival of mice treated with anti-hnRNP was 16.4 +/- 1.1 days (n = 8). Treatment with anti-PLAP prolonged the survival of mice; in 4 mice average survival was 23.3 +/- 5.7 days. Four animals, however, survived for 60 days when they were killed and had no visible signs of tumor. These data support the notion that passive immunotherapy using antibodies against a membrane protein, expressed in tumor cells transduced by a viral vector coding for that protein, may be effective in controlling tumor growth. PMID- 10754467 TI - N-Glycans influence the in vitro adhesive and invasive behaviour of three metastatic cell lines. AB - Alterations in cellular glycosylation may play a key role in metastatic behaviour of tumour cells. We studied three metastatic cell lines, LOX (malignant melanoma), FEMX (malignant melanoma) and MA-11 (mammary carcinoma). These cell lines have a very different metastatic behaviour in vivo, and different glycans have been postulated to be partly responsible for these differences. To further investigate the functional role of carbohydrates, these three cell lines have been treated with tunicamycin, an inhibitor of the biosynthesis of N-glycans and benzyl- alpha-N-acetylgalactosamine (benzyl-alpha-GalNAc; BnGalNAc), an inhibitor of mature O-linked glycans. Various in vitro adhesion and invasion assays were undertaken for functional studies. Tunicamycin significantly inhibited adhesion to laminin, but only slightly affected cell adhesion to collagen IV. The same compound significantly decreased cellular invasiveness through a Matrigel invasion chamber. Moreover, tunicamycin reduced homotypic aggregation of cells. BnGalNAc had generally little effect on cell behaviour in in vitro assay. The effects of the inhibitors were, however, to some extent cell line-specific. We conclude that N-glycans, but probably not mature O-glycans have important in vitro functions in cell adhesion to laminin, cell invasion through Matrigel and cellular aggregation in the studied cell lines. These results support the view that carbohydrates are functionally involved in several steps of the metastatic process. PMID- 10754466 TI - Immunohistochemical analysis of sialic acid and fucose composition in human colorectal adenocarcinoma. AB - The expression of different sialoglycoconjugates and fucoglycoconjugates in normal mucosa and adenocarcinoma samples from 43 colorectal cancer patients was investigated by using specific lectins and applying a semiquantitative analysis. A pronounced decrease in the intracellular binding of the Maackia amurensis lectin, which recognizes alpha(2,3)-linked sialic acid residues, was found in the tumoral tissue. In contrast, a significant increase in the staining with the Sambucus nigra lectin (SNA I), which binds to alpha(2,6)-linked sialic acid residues, was detected in the epithelial cells as well as in the mucins from tumors. No significant differences in the reactivity with the Aleuria aurantia lectin, which recognizes the sequence Fuc(alpha1,6)GlcNAc, between normal and malignant colorectal tissues were detected. Furthermore, the correlation between lectin-binding profiles and the prognosis of colorectal cancer patients was examined. After an average postoperative follow-up period of 31 months, patients with tumors showing a strong SNA I staining presented a greater probability of disease recurrence. This result suggests that the intensity of staining with SNA I could be a valid parameter for predicting recurrence in colorectal cancer. PMID- 10754468 TI - Cell adhesion inhibition by glycoliposomes: effects of vesicle diameter and ligand density. AB - The Thomsen-Friedenreich antigen (TF) is a pancarcinoma marker which is involved in the development of liver metastasis by binding tumour cells to the asialoglycoprotein receptor on hepatocytes. Blocking of this receptor prevents metastasis under certain circumstances. We report on conditions for an effective inhibition of the adhesion of KG-1 leukaemia cells expressing TF by lactosylated liposomes. In order to reach strong inhibition, carbohydrate blocking probes must be multivalent. Glycoliposomes are able to carry a large number of glycolipids accommodated in the lipid bilayer. They should be able to adapt their glycolipid pattern in order to achieve multiple binding. We found that, in addition to the number of carbohydrates on the liposome surface, their size, and probably the arrangement of neutral glycolipids in clustered domains, determine the inhibitory properties of glycoliposomes. PMID- 10754469 TI - Defective nitric oxide synthesis: a link between metabolic insulin resistance, sympathetic overactivity and cardiovascular morbidity. AB - Epidemiological studies demonstrate an association between insulin resistance, hypertension and cardiovascular morbidity. In addition to its metabolic effects, insulin also has important cardiovascular actions. The sympathetic nervous system and the nitric oxide-l-arginine pathway have emerged as central players in the mediation of these actions. Over the past decade, the underlying mechanisms and the factors that may govern the interaction between insulin and these two major cardiovascular regulatory systems have been studied extensively in healthy people and insulin-resistant individuals. Here we summarize the current understanding and gaps in knowledge on these interactions. We propose that a genetic and/or acquired defect of nitric oxide synthesis could represent a central defect triggering many of the metabolic, vascular and sympathetic abnormalities characteristic of insulin-resistant states, all of which may predispose to cardiovascular disease. PMID- 10754470 TI - Treatment with retinoid X receptorgamma-selective ligand (bexarotene) may cause iatrogenic central hypothyroidism. PMID- 10754471 TI - Continuous medical education in endocrinology in Europe. A 1999 European Board of Endocrinology survey. PMID- 10754472 TI - Is inhibin a serum marker for ovarian cancer? PMID- 10754473 TI - Inhibin A, B and pro-alphaC in serum and peritoneal fluid in postmenopausal patients with ovarian tumors. AB - OBJECTIVE: To compare serum and peritoneal fluid concentrations of inhibin A, B, and pro-alphaC in women with ovarian tumors. METHODS: Serum and peritoneal fluid samples were taken from 41 postmenopausal women operated on for an ovarian tumor. Twenty-one patients with endometrial cancer formed a control group. Serum and peritoneal fluid inhibin A, B, and pro-alphaC concentrations, and serum FSH and tumor marker CA 125 (study group only) concentrations were analyzed. RESULTS: Inhibin A was found in low concentrations (median 4.1pg/ml, range <2-29pg/ml) in serum in most postmenopausal patients with epithelial ovarian carcinoma, whereas inhibin B was not measurable. Inhibin pro-alphaC circulated in high concentrations (median 125pg/ml, range 37->1000pg/ml). All inhibins were found in clearly greater concentrations in the peritoneal fluid than in serum. International Federation of Gynecology and Obstetrics (FIGO) stage III-IV and poor differentiation grade were associated with significantly lower concentrations of inhibin A and pro-alphaC in the peritoneal fluid compared with stages I-II or low grade. This correlation was not found in the serum concentrations of inhibin A or pro-alphaC. In the control group, no dimeric inhibins were found in serum, and pro-alphaC circulated in median concentrations of 47pg/ml (range 12-174pg/ml). CONCLUSIONS: Postmenopausal patients with epithelial ovarian tumors had low concentrations of inhibin A and relatively high concentrations of inhibin pro-alphaC in serum. The peritoneal fluid concentrations of all inhibins far exceeded those in the serum. Relatively low concentrations of inhibin A and pro-alphaC in the peritoneal fluid of patients with ovarian cancer seem to be associated with high stage and grade and, to a lesser degree, with positive peritoneal cytology. PMID- 10754474 TI - Semi-quantitative comparison of the differentiation markers and sodium iodide symporter messenger ribonucleic acids in papillary thyroid carcinomas using RT PCR. AB - OBJECTIVE: To investigate the levels of expression of the sodium iodide symporter (NIS) and three differentiation markers (thyroglobulin (Tg), thyroid peroxidase (TPO) and thyrotrophin receptor (TSH-R)) in 35 patients with primary (n=31) or recurrent (n=4) papillary thyroid carcinoma, and to compare the findings with clinical data. METHODS: We performed a multiplex semi-quantitative RT-PCR to analyse the relative levels of expression of Tg, TPO and TSH-R mRNAs, and a separate semi-quantitative RT-PCR for NIS mRNA. RESULTS: Tg, TPO and TSH-R mRNAs were expressed in all the patients, whereas NIS mRNA was expressed in all but eight. Analysis of the expression of the differentiation markers in all patients showed a significant correlation among Tg, TPO and NIS. With regard to the relationship between the expression of each gene and the MACIS score, there was significant correlation only for the Tg gene (P<0.05). CONCLUSIONS: The levels of expression of NIS mRNA correlated significantly with those of Tg and TPO mRNAs, but not with those of TSH-R mRNA. The relationship with clinical stage and prognostic score, however, varied among these differentiation markers. PMID- 10754475 TI - Comparisons among old and new provocative tests of GH secretion in 178 normal adults. AB - Classical provocative stimuli of GH secretion such as insulin-induced hypoglycaemia, arginine, clonidine, glucagon and levodopa have been widely used in clinical practice for approximately 30 years. On the other hand, in the last 10 years new potent stimuli of GH secretion have been proposed, but an extensive comparison with the classical ones has rarely been performed, at least in adults. In order to compare the GH-releasing activity of old and new provocative stimuli of GH secretion, and to define the normative values of the GH response, in 178 normal adults (95 males, 83 females; age range: 20-50 years, all within +/-15% of their ideal body weight), we studied the GH response to: insulin-induced hypoglycaemia (ITT, 0.1IU/kg i.v.), arginine (ARG, 0.5g/kg i.v.), clonidine (CLO, 300 microg/kg p.o.), glucagon (GLU, 1mg i.m.), pyridostigmine (PD, 120mg p.o.), galanin (GAL, 80pmol/kg per min), GH-releasing hormone (GHRH, 1 microg/kg i.v.), GHRH+ARG, GHRH+PD, hexarelin, a GH-releasing protein (HEX, 2 microg/kg i.v.) and GHRH+HEX (0.25 microg/kg i.v.). The mean (+/-s.e.m.) peak GH response to ITT (21.8+/-2.8, range: 3.0-84.0 microg/l) was similar to those to ARG (18.0+/-1.6, range: 2.9-39.5 microg/l) or GLU (20. 5+/-2.2, range: 10.6-36.9 microg/l) which, in turn, were higher (P<0. 001) than those to CLO (8.2+/-1.6, range: 0.3-21.5 microg/l), PD (9. 6+/-1.1, range: 2.2-33.0 microg/l) and GAL (9.3+/-1.1, range: 3.9-18. 3 microg/l). The GH response to GHRH (19.1+/-1.5, range: 2.7-55.0 microg/l) was similar to those after ITT, ARG or GLU but clearly lower than those after GHRH+ARG (65.9+/-5.5, range: 13.8-171.0 microg/l) and GHRH+PD (50.2+/-4.6, range: 17.7-134.5 microg/l) which, in turn, were similar. The GH response to HEX (55.3+/-5.5, range: 13.9-163.5 microg/l) was similar to those after GHRH+ARG and GHRH+PD but lower (P<0.001) than that after GHRH+HEX (86.0+/-4.3, range: 49. 0 125.0 microg/l) which was the most potent stimulus of GH secretion. In this adult population the third centile limits of peak GH response to various stimuli were the following: ITT: 5.3; ARG: 2.9; CLO: 1.5; GLU: 7.6; PD: 2.2; GAL: 4.0; GHRH: 5.0; GHRH+ARG: 17.8; GHRH+PD: 17.9; HEX: 21.6; GHRH+HEX: 57.1. These results confirm that, among classical provocative tests of GH secretion, ITT followed by ARG and GLU are the most potent ones and possess clear limits of normality. GHRH+ARG or PD and HEX are strong stimuli of GH secretion which, however, is maximally stimulated by a combination of GHRH and a low dose of HEX. It is recommended that each test is used with appropriate cut-off limits. PMID- 10754476 TI - Growth hormone isoforms in newborns and postpartum women. AB - The neonatal and postpartum periods are characterized by alterations in pituitary GH secretion. We have investigated the proportion of circulating non-22kDa GH isoforms in newborns, in women within the early postpartum phase (just after the disappearance of placental GH from the maternal circulation) and in women during late postpartum (during the somatotroph recovery phase). We studied 10 newborns (7 males; 3 females; median postnatal age, 45h), who had been admitted because of polycythaemia, 10 women in the early postpartum phase (median, 48h after delivery; range, 42-54h), 18 women in the late postpartum phase (median, 10 weeks after delivery; range, 3-25 weeks) and 9 healthy non-pregnant women. The proportion of non-22kDa GH isoforms was determined by the 22kDa GH exclusion assay, which is based on immunomagnetic extraction of 22kDa GH from serum, and quantitation of non-22kDa GH isoforms using a polyclonal GH assay. In newborns, non-22kDa GH isoforms were measured in two arterial blood samples obtained with a 5-6h interval. In the other groups, serum samples were obtained 40min after an i.v. bolus administration of the GH secretagogue, GH releasing peptide-1 (GHRP 1). In newborns, the median proportion of non-22kDa GH isoforms was 10% (range, 7. 2-19.4%) and the values were similar in samples collected at different times. In early postpartum women, total GH levels after GHRP-1 were lower and the proportion of non-22kDa GH isoforms was higher compared with the values in non pregnant and late-postpartum women. In late postpartum, there was a partial recovery of GH response to GHRP-1, as shown by an increment in total GH levels, which was associated with a decrease in the fraction of non-22kDa GH isoforms. In conclusion, we found that (i) the proportion of non-22kDa GH isoforms in the newborn is comparable to that in the adult (non-pregnant women), (ii) in early postpartum, the non-22kDa fraction is high within the small pool of readily releasable GH, (iii) in late postpartum, recovery of pituitary GH responsiveness is associated with a relative decrease in the release of non-22kDa GH isoforms. PMID- 10754477 TI - The GH response to low-dose bolus growth hormone-releasing hormone (GHRH(1 29)NH2) is attenuated in patients with longstanding post-irradiation GH insufficiency. AB - OBJECTIVE: Previous studies have suggested that post-irradiation GH insufficiency results from a loss of GHRH secretion, since many patients were able to release GH following exogenous GHRH stimulation. However, supramaximal doses of GHRH were used and the response may decline with time after radiotherapy. We re-evaluated the GHRH dose-response curve in patients post cranial irradiation and in controls. DESIGN: Randomized controlled study. METHODS: Five adult male long-term survivors of childhood brain tumours (median age 21.8 years (18.4-26.7); 13.7 years (11.4-15.7) post-radiotherapy, >30Gy) and five matched controls were studied. An intravenous bolus of GHRH(1-29)NH(2) was administered in doses at the lower (0.05 microg/kg) and upper (0.15 microg/kg) range of the dose-response curves for young males, as well as the standard supramaximal dose (1. 0 microg/kg). GH was measured before stimulation, every 2min for the first hour and every 5min for the second hour. All studies were conducted in a random fashion. RESULTS: Significantly lower peak and area under the curve (AUC) GH concentrations occurred in the irradiated group using 0.15 microg/kg (median peak Irradiated, 4. 5mU/l vs median Controls, 37.4mU/l; P<0.01) and 1.0 microg/kg (median peak Irradiated, 4.8mU/l vs median Controls, 15.2mU/l; P<0. 05) GHRH(1 29)NH(2). In irradiated subjects there was an incremental rise in GH output with increasing doses of GHRH(1-29)NH(2 )(median AUC: 122mU/l.min vs 179mU/l.min vs 268mU/l.min; P=0.007) reflecting altered pituitary sensitivity and reduced responsiveness. CONCLUSION: The GH response to bolus GHRH(1-29)NH(2) is attenuated in adult long-term survivors of childhood brain tumours. This may reflect direct pituitary damage and/or the loss of the tropic effects of chronic GHRH deficiency. PMID- 10754478 TI - Five cases with central diabetes insipidus and hypogonadism as first presentation of neurosarcoidosis. AB - OBJECTIVES: We retrospectively reviewed 5 patients with neurosarcoidosis, who all presented with central diabetes insipidus and hypogonadism. DESIGN: This was a single-centre, retrospective analysis of 5 cases with a minimum follow-up of 2 years. METHODS: Case analysis included clinical, biochemical, and endocrinological evaluation and frequent CT/MRI scans of involved organs as primary evaluation and in response to immunosuppressive therapy. RESULT: Neurosarcoidosis was diagnosed in all patients. Two patients had no proven extracerebral manifestation and had a stable disease over 3 and 5 years. One patient showed deterioration with corticosteroids alone but partial remission after additional cyclophosphamide. Pituitary dysfunction remained unchanged in all patients, despite total clinical and radiological remission in two patients. However, one of these patients died of acute granulomatous meningoencephalitis after two years of follow-up. CONCLUSION: Although the presenting symptoms of neurosarcoidosis may vary, the occurrence of central diabetes insipidus associated with typical radiological features is suggestive of neurosarcoidosis. However, there is an increasing number of case reports on lymphocytic hypophysitis. Without the bioptic diagnosis, the differentiation between potentially lethal isolated neurosarcoidosis and lymphocytic hypophysitis is difficult. These cases demonstrate the difficulties in diagnosing neurosarcoidosis and reflect experiences with follow-up parameters. PMID- 10754479 TI - Longitudinal changes of circadian leptin, insulin and cortisol plasma levels and their correlation during refeeding in patients with anorexia nervosa. AB - OBJECTIVE: To study the longitudinal changes in plasma levels of leptin, insulin and cortisol during the transition from the state of starvation to the state of refeeding focussing on diurnal secretion characteristics and their temporal relationships. DESIGN: Leptin, insulin and cortisol were measured every 2h for 24h during acute starvation (T1). Sampling was repeated after reaching half the target-body mass index (BMI) (T2) and again at target-BMI (17. 5kg/m(2); T3). The temporal relationships between the diurnal secretion patterns were assessed by cross-correlation analysis. RESULTS: Although BMIs at T1 were uniformly low, leptin levels varied widely within a range clearly below normal levels (0.03-1. 7microg/l). With increasing body fat during the course of refeeding, mean leptin levels increased from 0.64microg/l (range: 0.27-1. 73microg/l) (T1) to 1.61microg/l (range: 0.36-4.2microg/l) (T2) and to 3.67microg/l (range: 0.7 9.8microg/l) (T3). Circadian leptin secretion patterns showed maximal values uniformly around 0200h and minimal values around 0800h at all stages of the study. At all three weight levels, plasma leptin levels were highest between midnight and the early morning hours and lowest around the late morning hours. Refeeding neither profoundly changed secretion patterns of leptin nor did it change the positive, time-delayed relationship between leptin and insulin with increments in insulin secretion preceding those of leptin by 6h. A temporal relationship between leptin and cortisol could not be demonstrated in the state of semistarvation but emerged after a substantial weight gain; at that time, leptin increases preceded cortisol increases by 8h. CONCLUSIONS: Absolute leptin, insulin and cortisol levels are profoundly changed during starvation in anorectic patients, while refeeding, paralleled by a BMI gain, reverses these changes. During refeeding the relationship between leptin and cortisol changed profoundly, showing no significant correlation in the state of starvation, whereas at T3 after refeeding a strong inverse relationship could be observed. Leptin and insulin did not correlate significantly at any of the three stages studied. PMID- 10754480 TI - Genetic and clinical characterisation of maturity-onset diabetes of the young in Spanish families. AB - OBJECTIVE: To investigate the frequencies of the major maturity-onset diabetes of the young (MODY) subtypes in a panel of Spanish families and to assess phenotypic differences in patients with the different subtypes of MODY. METHODS: Forty-eight subjects from twenty families with clinical diagnosis of MODY were studied. They underwent a standardised clinical examination and a 75-g oral glucose tolerance test (OGTT) was performed. Estimations of insulin sensitivity (%S) and insulin secretion capacity (%B) were calculated by the computer-solved homeostasis model assessment (HOMA). Mutations in the coding regions of hepatocyte nuclear factor (HNF)-4alpha/MODY1, glucokinase (GCK/MODY2) and HNF-1alpha/MODY3 genes were investigated by single strand comformation polymorphism and sequencing analysis. RESULTS: Mutations in the GCK and HNF-1alpha genes were observed in 5 (25%) and 7 (35%) families respectively. Novel mutations included R385X, M238fsdelT, V226fsdelTinsAA and S418-7del11 in the GCK gene, and S121fsdelC, V133M, R159Q and V259D in the HNF-1alpha gene. No MODY1 families were found. Subjects which were neither MODY2 nor MODY3 (MODY-X) had a higher fasting glucose than subjects in the other groups. Insulin secretion capacity was similar in the three groups and the insulin sensitivity was decreased in MODY-X subjects. Glucose levels were significantly higher and insulin levels significantly lower, throughout the OGTT, in MODY3 compared with MODY2 subjects. CONCLUSIONS: Mutations in the GCK/MODY2 and HNF-1alpha/MODY3 genes account for the majority of cases in a panel of Spanish MODY families, with MODY3 being the most frequent subtype. The relative frequencies and the clinical characteristics of these MODY subtypes are in agreement with data previously reported in other European populations. MODY-X patients seem to present a heterogeneous clinical profile. PMID- 10754481 TI - Melatonin secretion from organ-cultured pineal glands of rats: modulation by gonadectomy and gonadotropin-releasing hormone agonist administration. AB - The objective of the study was to evaluate the effect of pretreatments such as gonadectomy in male and female rats, and gonadotropin-releasing hormone agonist (GnRHa) administration in female rats, on levels of secretion of melatonin, using an organ culture of pineal glands. Gonadectomy 2 weeks before the animal was killed increased the amount of melatonin secreted into the medium by the pineal glands of female rats but not of male rats. The increase in in vitro melatonin secretion after ovariectomy in female rats was prevented by estrogen replacement. Ovariectomy 3 and 4 weeks before death also significantly increased the amount of melatonin secretion. Administration of GnRHa 2 weeks before decapitation significantly decreased serum estradiol concentrations and significantly increased melatonin secretion by the pineal glands of female rat. GnRHa administration 3 or 4 weeks before decapitation also significantly decreased serum estradiol concentrations, but did not increase pineal secretion of melatonin. The results indicate that ovariectomy increases melatonin secretion from organ-cultured pineal glands and that this increase is suppressed by estrogen in adult female rats. In contrast, orchiectomy in male rats does not influence in vitro secretion of melatonin. These results suggest that the GnRH gonadotropin system may participate in the regulation of pineal melatonin secretion in adult female rats. PMID- 10754482 TI - Regulation of UT-OC-3 ovarian carcinoma cells by cytokines: inhibitory effects on cell proliferation and activation of transcription factors AP-1 and NF-kappaB. AB - The present study was designed to investigate the growth regulatory effects of cytokines in UT-OC-3 ovarian cystadenocarcinoma cells in vitro. The effects of interleukin-6 (IL-6), interferons alpha (IFN-alpha) and gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor alpha (TNF-alpha), and transforming growth factor beta1 (TGF-beta1) were investigated by (125)I-deoxyuridine ((125)IUdR) incorporation assay. In order to understand better the molecular mechanisms of the observed effects, the activation of DNA-binding proteins was studied by electrophoretic mobility shift assay. In addition, cellular DNA was tested by fragmentation analysis to determine if the most growth inhibitory cytokines are able to induce programmed cell death (apoptosis). After 48h in culture, TGF-beta1, TNF-alpha, IFN-alpha and IL-6 showed a clear inhibitory effect on (125)IUdR incorporation (P<0.005), and IFN-gamma and GM-CSF caused even more significant inhibition (P<0.001). IFN-alpha and IFN-gamma were both growth inhibitory after 72h in culture (P<0.005). Similarly, GM-CSF induced a slight inhibition (P<0.05), whereas TGF-beta1 and TNF alpha almost blocked DNA synthesis (P<0.001) after 72h. IL-6 had no statistically significant effect on cell proliferation after 72h. Transcription factors AP-1 and NF-kappaB were both constitutively expressed in UT-OC-3 cells. The binding activity of AP-1 was found to be stimulated by the growth inhibitory cytokines, TGF-beta1 and TNF-alpha, and the binding of NF-kappaB was stimulated by TNF alpha. Apoptosis does not seem to be induced by any of these cytokines in the UT OC-3 ovarian cancer cell model. PMID- 10754483 TI - Human chorionic gonadotropin with C-elongated alpha-subunit retains full receptor binding and partial agonist activity. AB - OBJECTIVE: To test whether extension of the C-terminus of human chorionic gonadotropin (hCG) alpha-subunit (halpha) alters the bioactivity of the recombined alphabeta heterodimer. DESIGN: The stop codon of halpha was mutated to produce a 24 amino acid extension. METHODS: The extended halpha (alpha(+24)) was co-expressed with hCGbeta in COS-7 cells and the receptor binding and in vivo bioactivity of the secreted hormone was compared with its wild-type counterpart. RESULTS: This extension did not impair the binding of hCG to rat LH/CG receptors and provoked a sixfold reduction in its stimulatory activity of testosterone secretion in rat Leydig cells. CONCLUSIONS: The extension of alpha by itself does not lead to inhibition of the alphabeta heterodimer to LH receptors but the structure of the extension appears to play an important role. It is thus possible that one-chain hCG chimeras with the beta N-terminus fused to the alpha C terminus might be active. PMID- 10754484 TI - In vitro pituitary and testicular effects of the leptin-related synthetic peptide leptin(116-130) amide involve actions both similar to and distinct from those of the native leptin molecule in the adult rat. AB - The obese gene (ob) product, leptin, has recently emerged as a key element in body weight homeostasis, neuroendocrine function and fertility. Identification of biologically active, readily synthesized fragments of the leptin molecule has drawn considerable attention, as they may provide a powerful tool for detailed characterization of the biological actions of leptin in different experimental settings. Recently, a fragment of mouse leptin protein comprising amino acids 116 130, termed leptin(116-130) amide, was shown to mimic the effects of the native molecule in terms of body weight gain and food intake, and to elicit LH and prolactin (PRL) secretion in vivo. As a continuation of our previous experimental work, the present study reports on the effects of leptin(116-130) amide on basal and stimulated testosterone secretion by adult rat testis in vitro. In addition, a comparison of the effects of human recombinant leptin and leptin(116-130) amide at the pituitary level on the patterns of LH, FSH, PRL and GH secretion is presented. As reported previously by our group, human recombinant leptin(10(-9) 10(-7)M) significantly inhibited both basal and human chorionic gonadotrophin (hCG)-stimulated testosterone secretion in vitro. Similarly, incubation of testicular tissue in the presence of increasing concentrations of leptin(116-130) amide (10(-9)-10(-5)M) resulted in a dose-dependent inhibition of basal and hCG stimulated testosterone secretion; a reduction that was significant from a dose of 10(-7)M upwards. In addition, leptin(116-130) amide, at all doses tested (10( 9)-10(-5)M), significantly decreased LH and FSH secretion by incubated hemi pituitaries from adult male rats. In contrast, in the same experimental protocol, recombinant leptin(10(-9)-10(-7)M) was ineffective in modulating LH and FSH release. Finally, neither recombinant leptin nor leptin(116-130) amide were able to change basal PRL and GH secretion in vitro. Our results confirm the ability of leptin, acting at the testicular level, to inhibit testosterone secretion, and map the effect to a domain of the leptin molecule that lies between amino acid residues 116 and 130. In addition, we provide evidence for a direct inhibitory action of leptin(116-130) amide on pituitary LH and FSH secretion, a phenomenon not observed for the native leptin molecule, in the adult male rat. PMID- 10754485 TI - Activities of 21-hydroxylase, 17alpha-hydroxylase and 17,20-lyase. PMID- 10754486 TI - DNA alterations in tumor scrapes vs. biopsies of squamous-cell carcinomas of the head and neck. AB - Genetic abnormalities in SCCHNs are frequent and may be useful for screening, follow-up and prognosis. A biopsy or resection generally is utilized to identify these alterations but analysis of scraped or exfoliated tumor cells has been proposed as simpler and more versatile. It is unknown how well genetic abnormalities in scrapes reflect those in the tumor. Therefore, we compared DNA alterations in tumor scrapes obtained prior to treatment with alterations in microdissected tumor biopsies. Eight primary squamous-cell carcinomas of the head and neck (SCCHNs) were examined at 14 loci to determine loss of heterozygosity (LOH) at sites on 3p, 9p, 11p, 11q and 17p and amplification of cyclin D1 (CCND1). All biopsies contained DNA alterations, but only 3/8 scrapes contained unequivocal abnormalities; 4/8 contained subtle alterations that could not have been definitively identified without comparison to the paired biopsies. Overall, 22 alterations were detected in the biopsies: 8/22 were found unequivocally in the scrapes; 7/22 were identifiable in scrapes only after the biopsy alterations were defined and 7/22 were absent from scrapes. One LOH in scrape, but not biopsy, DNA was found. Discrepancies between scrapes and tumors tended to increase if multiple tumor samples were examined. We conclude that DNA alterations can be detected in scrapes of SCCHNs but may inaccurately reflect the tumor's complex genetic abnormalities. This may be due to contamination of scrapes with normal cells or to genetic heterogeneity within the tumor not represented in the scrape. Although examining scrapes of SCCHNs is an attractive technique, its clinical utility may have limitations. PMID- 10754488 TI - Plasma levels of soluble CD105 correlate with metastasis in patients with breast cancer. AB - CD105 (endoglin), a receptor for transforming growth factor (TGF) beta1 and beta3 in vascular endothelial cells, is highly up-regulated in blood vessels of tissues where neovascularisation occurs. It modulates endothelial-mesenchymal signalling and is essential for angiogenesis. Indeed, CD105 knock-out mice die from malvascularisation by 11.5 day p.c. In the present study CD105, TGFbeta1 and CD105/TGFbeta1 complexes were quantified in plasma samples from 77 healthy individuals and 92 patients with early stage breast cancer prior to any treatment. When compared with normal controls, both CD105 and CD105/TGFbeta1 complex levels were significantly elevated in breast cancer patients, whereas TGFbeta1 levels were lower in cancer patients. The most important finding to emerge was that CD105 levels were significantly increased in patients who developed distant metastasis compared with disease-free patients. While there was no significant difference between CD105 levels in controls compared to disease free patients, it was significantly higher in patients with metastatic disease. Thus patients who had died following local relapse or distant metastases possessed the highest levels of CD105. Neither CD105/TGFbeta1 complex nor TGFbeta1 levels correlated with tumour progression. Our data indicate that CD105 might be a valuable novel angiogenic marker for identifying breast cancer patients who are at high risk of developing metastasis. PMID- 10754487 TI - Estrogen receptor (ER) and progesterone receptor (PgR), by ligand-binding assay compared with ER, PgR and pS2, by immuno-histochemistry in predicting response to tamoxifen in metastatic breast cancer: a Southwest Oncology Group Study. AB - Results of estrogen receptor (ER) and progesterone receptor (PgR) ligand-binding assays (LBAs) are strongly correlated with ER and PgR by immuno-histochemistry (IHC). To investigate whether ER and PgR by IHC are also strongly correlated with tamoxifen response, time to treatment failure (TTF) and overall survival (OS), the results of the 2 methods were directly compared in 205 patients with ER(+) metastatic breast cancer treated with daily tamoxifen (Southwest Oncology Group protocol 8228) with 9 years median follow-up. pS2, another estrogen-regulated molecule, was also analyzed. Tumors were scored for IHC from 0 to 5, according to the proportion of positively stained cells. These IHC scores for both ER and PgR were significantly associated with LBA levels (p < 0.001). There was a significant direct relationship between higher IHC ER, PgR and pS2 and increasing response to tamoxifen. TTF and OS were also significantly longer for patients with higher ER or PgR, but not pS2, IHC scores. Low, intermediate and high ER or PgR categories showed similar differences in response rates whether defined by LBA or IHC. In logistic regression models which included ER, PgR and pS2 by IHC; ER and PgR by LBA; and menopausal status, only ER (IHC) and pS2 (IHC) retained significance for predicting tamoxifen response (p = 0. 02 and p = 0.005, respectively), along with menopausal status (for PgR by IHC, p = 0.09). Increasing ER and PgR by IHC, as by LBA, are thus significantly associated with a progressively better response and longer survival in ER(+) metastatic breast cancer. pS2 is also predictive in this setting. PMID- 10754489 TI - Prognostic relevance of altered Fas (CD95)-system in human breast cancer. AB - The Fas ligand (FasL) and its receptor Fas (APO-1 or CD95) are members, respectively, of the tumor necrosis factor family that, upon interaction with each other, play a key role in the initiation of one apoptotic pathway. Faulty regulation of the Fas system has been described in a variety of human tumors with different histogenetic origin. Here, we describe the expression and distribution of Fas receptor and ligand pair antigens in surgical samples collected from a cohort of 186 patients bearing breast neoplasms (45 benign and 141 malignant lesions). Immunoperoxidase staining of formalin-fixed tissues showed that 91.1% of benign lesions expressed Fas, which was present in only 56.7% of malignant tumors. On the other hand, FasL was found positive in 22.2% of benign neoplasms and up-regulated in in situ as well as invasive carcinomas (53.9%). Moreover, in malignant tumors, the expression of receptor and ligand antigens appeared to be inversely related. When these findings were correlated with pathological parameters of prognostic relevance, a significant association was observed between FasL and the presence of metastatic lymph nodes and larger tumor size while Fas expression correlated to node-negative status and smaller tumor size. Patients with Fas positive tumors exhibited longer disease-free survival than those with Fas-negative carcinoma while FasL did not influence patient outcome. These relationships indicate that benign and malignant mammary lesions are characterized by differential cellular expression of Fas and FasL and suggest that a neoplastic Fas negative/FasL positive phenotype may be linked to breast cancer progression. PMID- 10754490 TI - Expression of p16(INK4A) and alterations of the 9p21-23 chromosome region in non small-cell lung carcinomas: relationship with tumor growth parameters and ploidy status. AB - The 9p21-23 chromosome region harbors a number of known and putative tumor suppressor genes (TSGs). The best characterized gene in this area is p16(INK4A) (CDKN2A). Alterations of its product have been observed in various malignancies, including non-small-cell lung carcinomas (NSCLCs). We earlier investigated the mechanisms underlying p16(INK4A) inactivation. In the present study, we examined, in a series of 87 NSCLCs, its relationship with the kinetic parameters [proliferation index (PI) and apoptotic index (Al)] and the ploidy status of the tumors. In addition, we extended our previous LOH analysis of the 9p21-23 region by examining flanking areas of p16(INK4A). Aberrant p16 expression was observed in 41.4% of the carcinomas. A significant association was found with increased PI (p = 0.037), but not with apoptosis. Aneuploid tumors were more frequently correlated with abnormal p16 staining (p = 0. 05). A high frequency of allelic imbalance (Alm) was noticed at the D9S161 (51.3%) and D9S157 (64.5%) loci, which lie approximately 4cM centromeric and 7cM telomeric, respectively, to CDKN2A. Abnormal p16(INK4A) expression was strongly correlated with Alm at D9S161 (p = 0.004). Allelic losses at D9S157 occurred more frequently in early stages (p = 0.018) and were significantly associated with deletions at D9S161 (p = 0.035). We conclude that, in a sub-set of NSCLCs, (i) abnormal p16 expression contributes to tumor growth mainly by increasing the proliferative activity in the initial stages of carcinogenesis; (ii) the association with aneuploidy merely reflects the impact of aberrant p16 on proliferative activity; and (iii) other putative TSGs possibly reside within the 9p21-23 region that possibly co-operate in certain cases with CDKN2A in the development of NSCLCs. PMID- 10754492 TI - cdc25a and the splicing variant cdc25b2, but not cdc25B1, -B3 or -C, are over expressed in aggressive human non-Hodgkin's lymphomas. AB - cdc25 is a family of phosphatases that activate the cyclin-dependent kinases at different points of the cell cycle. cdc25A and -B, but not -C, have been shown to have oncogenic potential. Three different splicing variants of the cdc25B gene, cdc25B1, -B2 and -B3, have also been identified. Experimental studies suggest that cdc25B2 may be more active in vivo than cdc25B3 and -B1, but the relative expression of these splicing variants in human tumors is not known. In this study, we have analyzed the expression of cdc25A, -B1, -B2, -B3 and -C mRNA in 9 non-neoplastic lymphoid samples, 89 non-Hodgki&ngrave;s lymphomas and 9 hematological cancer cell lines by semi-quantitative RT-PCR. cdc25A, -B and -C protein expression was examined by Western blot. Normal peripheral blood lymphocytes and reactive tissues expressed cdc25B1 and -B3 mRNA and very low or undetectable levels of cdc25A, -B2 and -C. High levels of cdc25A and cdc25B2 were found in 35% and 39% of the tumors, respectively, and they were more frequently observed in aggressive than in indolent lymphomas. cdc25B1 and -B3 splice variants were detected in virtually all tumors, and no significant differences were found between high- and low-grade lymphomas. cdc25A and -B protein expression was also higher in aggressive than in indolent lymphomas. cdc25C expression was relatively low in virtually all cases. In conclusion, these findings suggest that cdc25A and -B2, but not cdc25B1, -B3 and -C, are over expressed in a relatively large number of malignant lymphomas and may participate in the pathogenesis of aggressive variants. PMID- 10754491 TI - Bcl-2 with loss of apoptosis allows accumulation of genetic alterations: a pathway to metastatic progression in human breast cancer. AB - We have examined whether the extended life span of cells induced by Bcl-2 in T(1) ductal breast carcinomas might favor the acquisition and accumulation of genetic alterations that induce lymph node metastases. We analyzed the expression of c Myc, c-erbB-2 and epidermal growth factor receptor by immuno-histochemistry in a group of 142 T(1) (<2 cm) ductal breast carcinomas embedded in paraffin, previously studied for p53 mutation and Bcl-2 over-expression. We also measured the apoptotic status and estimated the excess risk (pOR) for lymph node metastasis according to the number of accumulated oncogene alterations and Bcl-2 and p53 expression. The linear relationship between number of oncogene alterations and presence of lymph node metastasis was statistically significant in Bcl-2-positive tumors (trend test, p = 0.03), p53-mutated tumors (trend test, p = 0.08) and tumors with loss of apoptosis (trend test, p = 0.08). Very large associations (pOR > 12) between the number of oncogene alterations and lymph node metastasis were observed among Bcl-2-positive tumors that showed increased loss of apoptosis (trend test, p = 0.03). Furthermore, in p53-negative tumors, a strong linear association was found between the number of oncogene alterations and risk of lymph node metastasis among Bcl-2-positive tumors (trend test, p = 0.03). In human T(1) ductal breast carcinoma, over-expression of Bcl-2 along with loss of apoptosis might render breast cancer cells susceptible to the acquisition of additional genetic lesions related to disease progression among p53-negative tumors. Thus, in breast cancer, there are at least 2 pathways to progression: Bcl 2- and p53-dependent mechanisms. PMID- 10754493 TI - Differential clinical significance of alpha(v)Beta(3) expression in primary lesions of acral lentiginous melanoma and of other melanoma histotypes. AB - Despite its potential clinical relevance, alpha(v)beta(3) expression has been analyzed only in a limited number of melanoma lesions, mostly nodular melanoma (NM) and superficial spreading melanoma (SSM). Therefore, in the present study, we have correlated alpha(v)beta(3) expression in 33 acral lentiginous melanomas (ALMs), 6 lentigo maligna melanomas, 7 mucosal melanomas, 12 NMs and 9 SSMs with their antigenic profile, with their histo-pathological characteristics and with the clinical course of the disease. Furthermore, we have compared alpha(v)beta(3) expression in ALM lesions with that in NM and SSM lesions since this information helps to clarify the relationship of the latter 2 histotypes with ALM. Such a relationship is uncertain since ALM has a clinical course similar to that of NM and SSM despite different antigenic profiles and biological characteristics. The level of alpha(v)beta(3) expression in primary lesions was not correlated with that of high-m. w. melanoma-associated antigen and intercellular adhesion molecule-1, with lesion thickness and with disease recurrence in ALM but was significantly correlated with these 4 parameters in the other melanoma histotypes analyzed. Therefore, alpha(v)beta(3) expression appears to have a differential clinical significance in ALM and in the other histotypes of melanoma we have analyzed since it appears to play a significant role in the progression of the disease only in non-ALM histotypes. PMID- 10754494 TI - DNA-adduct levels as a predictor of outcome for NSCLC patients receiving daily cisplatin and radiotherapy. AB - We aimed to investigate whether biological factors related to radiosensitivity and chemosensitivity have prognostic significance in non-small-cell-lung-cancer (NSCLC) patients treated with daily low doses of cisplatin and radiotherapy. We treated 27 NSCLC patients with concomitant daily low-dose cisplatin and radiotherapy between 1993 and 1995. Tumour specimens were analyzed for p53 and bcl-2 expression, and for cell proliferation using antibodies against ki-67. In addition, apoptosis was measured by an end-labeling technique (TUNEL). Finally, cisplatin-induced DNA modification in buccal cells was assessed immunocytochemically using a specific anti-serum. Univariate and multivariate analyses were performed to assess the association between the different variables and survival. The median follow-up was 41 months, and 21 patients (78%) have died. In a univariate analysis, age, tumour stage and cisplatin-DNA-adduct staining were the only factors significantly associated with survival (p < 0.05, log-rank test). p53, bcl-2, Ki-67 and apoptosis showed no relationship with outcome. Multivariate analysis revealed that cisplatin-DNA-adduct staining remained an independent prognostic factor (hazard ratio, 0.10, 95% CI, 0.02 0.49), with shorter survival times for patients with low adduct staining. PMID- 10754495 TI - VEGF overexpression in clinically localized prostate tumors and neuropilin-1 overexpression in metastatic forms. AB - Studies comparing tumor neovascularity with pathological findings suggest that angiogenesis contributes to the pathogenesis of prostate cancer. We have examined 42 primary sporadic prostate tumors at different clinical stages, together with 3 prostate cancer cell lines (DU145, PC3 and LNCaP), for expression of VEGF and the gene encoding the recently identified VEGF165 isoform-specific receptor neuropilin-1, by using a quantitative reverse transcription (RT)-PCR method. We also evaluated the VEGF transcription pattern. Upregulation of VEGF and neuropilin-1 was observed in 12 and 14 tumors, respectively. The VEGF165 isoform was slighly overrepresented in tumors that overexpressed VEGF. VEGF overexpression correlated with stage II disease (p < 0.05); neuropilin-1 overexpression correlated with advanced disease (p < 0. 01) and a high Gleason grade (p < 0.02). Our observations suggest that VEGF expression could be used as a prognostic marker in early-stage prostate tumors, whereas neuropilin-1 overexpression might be a marker of aggressiveness. PMID- 10754497 TI - Biological and clinical associations of c-jun activation in human breast cancer. AB - Sub-units and regulators of the activating protein-1(AP-1) complex have been implicated in breast-cancer biology, therapeutic response and prognosis. This study has immunocytochemically examined the impact of c-jun-protein activation on biological and clinical parameters in human primary breast cancers, employing an antibody specific for the serine 63-phosphorylated c-jun protein. Substantial nuclear immunostaining was commonly apparent, indicative of an activated c-jun pool, with associations with MAP-kinase-signalling elements, e.g., transforming growth factor-alpha (p = 0.04), epidermal growth factor receptor (p = 0.08), phosphorylated erk 1/2 MAP kinase (p = 0.001) and phosphorylated jun kinase (p = 0.05) Little association was noted with c-fos protein, perhaps indicating alternative AP-1 partners for c-jun with a diversity of cellular end-points. This may explain the lack of relationship with proliferation and grade, the imperfect association between increased c-jun activation and poorer survival (p = 0.061), and the apparent relationship with distant metastasis (p = 0.05). While increased c-jun activation related to poorer quality (p = 0.09) and shortened duration of endocrine response in oestrogen-receptor-positive patients (p = 0.018), no generalized effects on oestrogen-regulated gene products were noted, indicating that AP-1 influences on oestrogen-receptor/oestrogen-response element transactivation are unlikely to explain endocrine insensitivity. These data reinforce our belief that elevated AP-1 signalling influences aspects of the breast-cancer phenotype. PMID- 10754496 TI - htert expression correlates with MYC over-expression in human prostate cancer. AB - Expression of the telomerase catalytic sub-unit (htert) constitutes a key step in the development of human cancer. Although htert regulation is still unclear, several studies suggest that c-myc may activate its expression. Prostate cancer is one of the most common malignancies among men in Western countries. Since de regulated expression of myc as well as telomerase activation may contribute to the pathogenicity of this cancer, we investigated this pathway in prostate tumorigenesis. For this purpose, myc- and htert-mRNA expression was quantified in 33 sporadic prostate tumors using a real-time quantitative PCR assay based on TaqMan methodology. myc over-expression was observed in 19 (58%) of 33 tumors, whereas telomerase status evaluated by htert expression was observed in 22 (67%). There was no correlation between myc over-expression or htert expression level and tumor stage or Gleason grade. A significant association (p = 0.0024) was found between myc over-expression and elevated htert expression, indicating that the up-regulation of telomerase activity often observed in prostate tumors might be conferred through transactivation of htert by myc. It is likely that the ability of c-myc protein to stimulate expression of htert and thereby enhance telomerase activity represents an important step in prostate tumorigenesis. PMID- 10754498 TI - Clinical significance of p53 functional loss in squamous cell carcinoma of the oropharynx. AB - We examined the frequency of p53 mutations in 38 oropharyngeal squamous cell carcinomas (SCC), using both a yeast functional assay and a conventional immunohistochemical staining method (IHC) to detect p53 mutations. We also explored the clinical importance of p53 mutations in oropharyngeal SCC. An accumulation of p53 protein was detected in 17 of the 38 (45%) tumors by IHC, whereas the yeast-based assay detected 6 additional p53 mutations, for a total of 23 tumors (61%) with p53 mutations. The cDNA sequencing analysis revealed that the 6 mutations undetected by IHC consisted of 3 frameshift, 1 nonsense and 2 missense mutations. Thus, the yeast functional assay was more sensitive than conventional IHC for detecting p53 mutations. Subsequently, the relationship between p53 mutations and the clinico-pathological parameters in oropharyngeal SCC was evaluated using the results of the functional assay. Mutation of p53 was not associated with the patient age, sex, tumor stage or degree of tumor cell differentiation. Interestingly, heavy drinking had a significant positive correlation with the p53 mutation, but heavy smoking did not, suggesting that prolonged exposure to alcohol is more related to p53 mutation in oropharyngeal SCC than to tobacco consumption. Radiation sensitivity was examined by comparing tumor size on magnetic resonance images before and after completion of therapy with 45 Gy radiation, in the 18 cases of T2 oropharyngeal SCC that were initially treated by radiotherapy. The results showed that tumors with wild-type p53 decreased in size significantly compared to those with mutant p53. In 33 patients treated with curative intent, the overall survival after the completion of therapy was better in patients with a wild-type p53 tumor than in patients with a mutant p53 tumor. We conclude that p53 mutation is associated with radiation resistance and a decreased probability of survival in oropharyngeal SCC. PMID- 10754499 TI - Loss of heterozygosity analysis on chromosome 5p defines 5p13-12 as the critical region involved in tumor progression of bladder carcinomas. AB - We have previously observed loss of heterozygosity (LOH) at a single locus (del 27) on human chromosome 5p13-12 to correlate with bladder tumor progression. In this study, we examined 33 bladder tumors for their pattern of allelic loss on chromosome 5p using 7 microsatellite markers. In 14 of 15 bladder tumors with LOH at locus del-27, allelic loss was confined to chromosomal region 5p13-12. This region included the microsatellite marker D5S2025 that showed LOH in 5 of 11 (45%) informative cases with LOH at del-27. This suggests that D5S2025 and del-27 are located within a single critical region of LOH on 5p13-12 harboring a tumor suppressor gene involved in bladder tumor progression. Recurrent LOH at other loci was observed at microsatellite markers located at 5p15. However, these losses appeared to be independent of LOH at 5p13-12 and occurred predominantly in poorly differentiated (G3) and advanced (T3-T4) tumors. PMID- 10754500 TI - Comparison of the metastasis-inducing protein S100A4 (p9ka) with other prognostic markers in human breast cancer. AB - Our aim was to compare the occurrence and prognostic significance over 14-20 years of immunocytochemically detected S100A4 and other tumour variables in primary tumours from 349 patients with operable breast cancer. For a cut-off of 1% staining of the malignant cells, the antibody to S100A4 stains positively 56% of the carcinomas. There was a significant association of staining for S100A4 with tumours fixed to the chest wall, staining for c-erbB-2, c-erbB-3, pS2, cathepsin D and, inversely, at borderline levels with staining for estrogen receptor. Using Wilcoxon statistics in univariate analyses, staining for S100A4, nodal status, tumour class, histological grade and staining for c-erbB-2, p53 were associated negatively and staining for estrogen receptor, progesterone receptor were associated positively with patient survival times. The survival times of patients with S100A4-negative carcinomas with or without one of the other tumour variables showed no significant differences, whilst those of patients with S100A4-positive carcinomas showed significant differences in a negative or a positive way. Multivariate regression analysis for 137 patients showed that staining for S100A4 is most highly correlated with patient deaths, but involved lymph nodes, fixed tumours, high histological grade and staining for progesterone receptor were also significant independent prognostic variables. Our results suggest that in this set of patients, the tumour variable most tightly correlated with patient death is S100A4. PMID- 10754501 TI - A possible divergent role for the oestrogen receptor alpha and beta subtypes in clinical breast cancer. AB - We have examined the relative levels of oestrogen receptor beta (ERbeta) mRNA in 94 breast cancer specimens using a semi-quantitative RT-PCR procedure. We correlated its expression with ERalpha and various clinical, pathological and biochemical features of the disease. The level of ERbeta mRNA expression in these samples was found to be much lower than ERalpha. Although ERalpha mRNA species were found to be most frequently associated with histological grade I and II tumours, displaying tubular differentiation, low grades of nuclear pleomorphism and low mitotic activity, such features were not characteristic of ERbeta positive samples. Indeed, application of the Spearman rank correlation test revealed that there was an inverse association between ERbeta normalised levels and ERalpha protein HScore. Also ERbeta mRNA positive cancers were more frequently EGFR protein positive than their negative counterparts (p = 0.016), a feature normally associated with endocrine-insensitive disease. Our data suggest that although ERbeta levels are most likely lower than ERalpha, they may influence the biological behaviour of breast cancers containing low levels of ERalpha. PMID- 10754502 TI - Spatial convergence and divergence between cutaneous afferent axons and dorsal horn cells are not constant. AB - We have proposed a quantitative model of the development of dorsal horn cell receptive fields (RFs) and somatotopic organization (Brown et al. [1997] Somatosens. Motor Res. 14:93-106). One component of that model is a hypothesis that convergence and divergence of connections between low-threshold primary afferent mechanoreceptive axons and dorsal horn cells are invariant over skin location and dorsal horn location. The more limited, and more easily tested, hypothesis that spatial convergence and divergence between cutaneous mechanoreceptors and dorsal horn cell are constant was examined. Spatial divergence is the number of dorsal horn cells whose RFs overlap the RF center of a primary afferent, and spatial convergence is the number of afferent RF centers that lie within the RF of a dorsal horn cell. Innervation density was determined as a function of location on the hindlimb by using peripheral nerve recording and axon counting. A descriptive model of dorsal horn cell receptive fields (Brown et al. [1998] J. Neurophysiol. 31:833-848) was used to simulate RFs of the entire dorsal horn cell population in order to estimate RF area and map scale as a function of location on the hindlimb. Previously reported correlations among innervation density, map scale, and RF size were confirmed. However, these correlations were not linear. The hypothesis that spatial convergence and divergence are constant was rejected. The previously proposed model of development of dorsal horn cell somatotopy and RF geometries must be revised to take variable spatial convergence and divergence into account. PMID- 10754503 TI - Neurons immunoreactive for vasoactive intestinal polypeptide in the rat primary somatosensory cortex: morphology and spatial relationship to barrel-related columns. AB - Vasoactive intestinal polypeptide (VIP) in neocortex affects neuronal excitability as well as cortical blood flow and metabolism. Interneurons immunoreactive for VIP (VIP-IR neurons) are characterized by their predominantly bipolar appearance and the radial orientation of their main dendrites. In order to determine whether the morphology of VIP-IR neurons is related to the functional organization of the cortex into vertical columns, we combined both immunostaining of neurons containing VIP and cytochrome oxidase histochemistry for visualizing barrels, morphological layer IV correlates of functional columns, in the primary somatosensory (barrel) cortex of rats. VIP-IR neurons were localized in supragranular (48%), granular (16%), and infragranular layers (36%) as well as in the white matter. In the granular layer, a clear trend that more neurons were located in interbarrel septa rather than in barrels could be observed, resulting in a neuronal density which was about one-third higher in the septal area. VIP-IR neurons from the different cortical layers were three dimensionally reconstructed from serial sections by using a computer microscope system. The neurons were mostly bipolar. Striking morphological differences in both axonal and dendritic trees were found between neurons whose cell bodies were located in supragranular, granular, and the upper part of infragranular layers, and those whose cell bodies were located in the area below. The former had dendrites which often reached layer I, where they bifurcated several times, and axonal trees which were particularly oriented vertically, with a tangential extent smaller than the width of barrels. Therefore, these neurons were mostly confined to either a barrel- or septum-related column. By contrast, the dendrites of neurons of the latter group did not reach the granular layer. Furthermore, these neurons had axons with sometimes very long horizontal collaterals, which often spanned two, in one case three, barrel columns. It is proposed that the differential morphology of neurons with different locations as stated above parallels to some extent the divergence of input streaming into the corresponding layer-defined areas. As a possible consequence of this, VIP-IR neurons may be capable of adapting the excitability and metabolism of cortical compartments either in a spatially limited or more extensive way. PMID- 10754505 TI - Serotonergic nerve fibers in the primary olfactory pathway of the larval sea lamprey, Petromyzon marinus. AB - In this study, serotonin (5-hydroxytryptamine; 5HT)-immunoreactive (5HT-IR) neuronal fibers were identified in the primary olfactory pathway of the sea lamprey. These neurons are likely part of a nonolfactory neural system that innervates the olfactory sac. Cell bodies with 5HT immunoreactivity predominated in the lamina propria of the rostral portion of the nasal cavity and were less prevalent adjacent to the olfactory epithelium. The 5HT-IR fibers were parallel to axons of the olfactory receptor neurons in the lamina propria of the olfactory mucosa and in the olfactory nerve. Serotonergic fibers crossed from the olfactory nerve into the olfactory bulb or branched in the caudal portion of the olfactory nerve and terminated at the junction of the olfactory nerve with the olfactory bulb. In the dorsal olfactory bulb, 5HT-IR fibers coursed along the layer of olfactory fibers. Throughout the layer with glomeruli and mitral cells, 5HT-IR fibers were seen along the border of glomerular units. Experimental lesion of the olfactory nerve was used to determine the origin of 5HT-IR fibers rostral to the olfactory bulb. The loss of these fibers and their reappearance during outgrowth of olfactory receptor neurons inferred that they emanate from the cell bodies in the olfactory sac. The results from this study suggest that axons of olfactory receptor neurons in larval lampreys receive modulation by 5HT from these neuronal fibers. PMID- 10754506 TI - Up-regulation of pituitary adenylate cyclase-activating polypeptide in urinary bladder pathways after chronic cystitis. AB - These studies examined changes in the expression of pituitary adenylate cyclase activating polypeptide (PACAP) in micturition reflex pathways after chronic cystitis induced by cyclophosphamide (CYP). In control Wistar rats, PACAP immunoreactivity was expressed in fibers in the superficial dorsal horn at all segmental levels examined (L1, L2, and L4-S1). Bladder afferent cells (40-45%) in the dorsal root ganglia (DRG; L1, L2, L6, and S1) from control animals also exhibited PACAP immunoreactivity. After chronic, CYP-induced cystitis, PACAP immunoreactivity increased dramatically in spinal segments and DRG (L1, L2, L6, and S1) involved in micturition reflexes. The density of PACAP immunoreactivity was increased in the superficial laminae (I-II) of the L1, L2, L6, and S1 spinal segments. No changes in PACAP immunoreactivity were observed in the L4-L5 segments. Staining also increased dramatically in a fiber bundle extending ventrally from Lissauer's tract in lamina I along the lateral edge of the dorsal horn to the sacral parasympathetic nucleus in the L6-S1 spinal segments (lateral collateral pathway of Lissauer). After chronic cystitis, PACAP immunoreactivity in cells in the L1, L2, L6, and S1 DRG increased significantly (P 10(6) per cell). Dynamic flow cytometry allows the accurate determination of the k(on) and k(off) of antibody binding. The sensitivity of the method is two orders of magnitude better than with an optical biosensor. CONCLUSIONS: Although biosensors constitute a method of choice for measuring the interactions between soluble proteins, they are not well suited for measuring the interaction between soluble proteins and membrane-embedded proteins. On the contrary, flow cytometry is well suited for such an application, when it is used in a dynamic mode. PMID- 10754521 TI - Enzymatic amplification staining for flow cytometric analysis of cell surface molecules. AB - BACKGROUND: Flow cytometric analysis is a powerful technique for the single cell assessment of cell surface expression of selected molecules. The major deficiency of flow cytometry has been its relative insensitivity. Only molecules expressed in abundance have been readily observed. METHODS: We have developed an enzymatic amplification procedure for the analysis of cell surface molecules by flow cytometry. Transformed and nontransformed cells expressing MHC class I, CD5, CD3, CD4, CD6, CD7, CD34, CD45, MHC class II, Fas ligand, and phosphatidylserine were assessed. RESULTS: Our enzymatic amplification technology increased the fluorescence signal between 10 and 100-fold for all surface molecules tested. CONCLUSIONS: Enzymatic amplification staining produces a significant enhancement in the resolving power of flow cytometric analysis of cell surface molecules. Using this technique, we have been able to detect the presence of molecules that could not be observed by the standard procedure. PMID- 10754522 TI - Synthetic inhibitor of matrix metalloproteinases (batimastat) reduces prostate cancer growth in an orthotopic rat model. AB - BACKGROUND: Increased concentrations of metalloproteinases are associated with the invasive and metastatic behavior of several human malignant tumors. Normally, enzymatic activity is tightly regulated by nonspecific mechanisms and specific inhibitors. The aim of the study was to determine the potential of a synthetic metalloproteinase inhibitor, batimastat, to show its in vitro effect on MatLyLu cancer cells and its in vivo effect on tumor growth in orthotopic cancer (R3327 Dunning tumor) in rats. METHODS: In vitro, a dose response curve of batimastat was generated over 4 days using the MTT assay. Prostate cancer was injected in vivo in male Copenhagen rats by inoculating R3327 Dunning tumor cells (MatLyLu) into the ventral prostatic lobe of 30 rats. Each of 10 rats received batimastat (30 mg/kg body weight) or vehicle administered once a day by i.p. application beginning the day of cell inoculation. Ten rats remained untreated. The effect on local tumor growth was evaluated by measuring tumor weights 20 days after tumor cell inoculation. RESULTS: Significant inhibition of tumor cell proliferation in vitro occurred at 400 and 4,000 ng/ml batimastat. After orthotopic cell inoculation, tumors grew to mean weights of 18.9 g in the control group without treatment, to 22.3 g in the vehicle group, and to 11.1 g in the treated group. In comparison to the control group and to the vehicle group, tumor weights increased significantly less under treatment with batimastat. CONCLUSIONS: Batimastat is able to reduce tumor growth in the standard prostate cancer model. Using this model, activity against cancer progression of future inhibitory agents can be reliably assessed. PMID- 10754523 TI - Ornithine decarboxylase activity and its gene expression are increased in benign hyperplastic prostate. AB - BACKGROUND: Ornithine decarboxylase (ODC) is the first key enzyme in the polyamine biosynthesis pathway. Polyamine is believed to participate in cellular proliferation and differentiation. To study the relationship between ODC and the pathogenesis of benign prostatic hyperplasia (BPH), the polyamine levels, ODC activities, and expression of ODC mRNA in benign hyperplastic and normal human prostates were assayed. METHODS: Polyamine contents and ODC activities in tissue extracts were determined by reverse-phase high-performance liquid chromatography and spectrophotometric procedures, respectively. The ODC mRNA levels were assayed by Northern blot analysis. RESULTS: The contents of putrescine, spermidine, and spermine in BPH tissues were 2.2, 3.4, and 6.0 times higher than those in normal tissues, respectively; the ODC activity of BPH tissue was about 3.2 times higher than in normal tissue; the expression level of ODC mRNA in the BPH tissues was greater than that of normal tissues. CONCLUSIONS: The findings imply that 1) the increased ODC activity and polyamine content in prostatic tissue may correlate with the pathogenesis of BPH, and 2) the high level of ODC activity is induced by the overexpression of ODC mRNA. PMID- 10754524 TI - Generation of PSA-reactive effector cells after vaccination with a PSA-based vaccine in patients with prostate cancer. AB - BACKGROUND: JBT 1001 is a vaccine used for therapy of prostate cancer (CA), which consists of recombinant prostate-specific antigen (PSA) with lipid A formulated in liposomes. Patients with prostate CA were vaccinated with JBT 1001 emulsified in mineral oil (n = 5) or with the vaccine in combination with granulocyte macrophage colony-stimulating factor (GM-CSF) administered locally at the site of vaccination (n = 5). Frequency of PSA-reactive T cells was measured in peripheral blood mononuclear cells (PBMC) before and after immunization, using an interferon gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay with autologous dendritic cells (DC) as antigen-presenting cells. The hypothesis tested was that PSA-based vaccines induce T cell responses to human PSA. METHODS: In order to expand precursor cells, in vitro sensitization (IVS) was performed. Microcultures of peripheral blood lymphocytes (PBL) (1 x 10(5)/well) in medium supplemented with interleukin-2 (IL-2) (10 IU/ml) and interleukin-7 (IL-7) (10 ng/ml) were stimulated twice (day 0 and day 7) with monocyte-derived autologous DC, generated by culture with interleukin-4 (IL-4) and GM-CSF and pulsed with PSA (10 microg/ml) at an effector to stimulator ratio of 10:1. ELISPOT assays were performed on day 14 of culture. In addition, PBMC were separated on immunobeads into CD4(+) and CD8(+) subsets for ELISPOT assays performed without IVS. RESULTS: Two patients had PSA-reactive responses before vaccination (frequency range, 1/700-1/4,400). After vaccination, 8/10 patients had measurable PSA-reactive T cell frequencies, ranging from 1/200-1/1900, using IVS. In contrast, without IVS, but after immunoselection to enrich in CD8(+) and CD4(+) T cells, only 2/10 patients had detectable PSA-reactive T cells after vaccination, at a frequency ranging from 1/2,600-1/4,000. CONCLUSIONS: Vaccination with PSA formulated into liposomes induced T-cell responses in 8/10 patients with prostate carcinoma. The frequency of PSA-reactive precursor T cells was relatively low in the blood of these patients, and IVS, leading to amplification of the precursor cells prior to ELISPOT, was necessary for quantification of the PSA-responding T cells. Cellular responses to PSA were predominantly mediated by CD4(+) T lymphocytes. PMID- 10754525 TI - Fragile histidine triad gene expression in primary prostate cancer and in an in vitro model. AB - BACKGROUND: The fragile histidine triad (FHIT) is frequently deleted and altered in many human cancers. Replacement of the FHIT gene into cancer cell lines lacking FHIT expression results in loss of tumorigenicity and tumor growth. METHODS: We investigated the status and function of the FHIT gene in the etiology of prostate carcinoma, utilizing human prostate cancer tissues and cell lines and the multistep human prostate epithelial (HPE) cell tumor model. RESULTS: In primary cancers, either no FHIT protein expression or greatly reduced expression was observed in the tumor cells, while FHIT was expressed at high levels in the adjacent normal prostate epithelia. No aberrant FHIT transcripts were observed in normal HPE cells. Aberrant transcripts were observed in the immortalized and nontumorigenic HPV-18 C-1 cell line. A tumor cell line (129 Nu 5002-1) derived from chemical transformation of HPV-18 C-1 cells did not express the FHIT gene. Immunoblot analysis of FHIT protein levels confirmed the absence of FHIT expression in the 129 Nu 5002-1 tumor cell line. Among the metastatic prostate cancer cell lines, PC-3, DU-145, and S7 exhibited aberrant transcripts, but the LNCaP cell line (early passage) was normal. Upon cloning of the cDNA and determining the DNA sequence of the PCR fragments, we observed specific alterations such as deletions and insertions in the aberrant transcripts. A majority of prostate cancer cell lines expressed the normal-sized transcript in addition to the aberrant transcripts. CONCLUSIONS: Our results indicate that alterations in the FHIT gene represent an early event in prostate carcinogenesis. PMID- 10754526 TI - Efficient DNA-mediated gene transfer into prostate cancer cell line LNCaP. AB - BACKGROUND: Prostate cancer is the most commonly diagnosed cancer and the second leading cause of cancer death in men. In the beginning stages of the disease, prostate cancer is dependent on androgens for growth. The only common and readily available cell line to study this phase of prostate cancer in vitro is LNCaP, which was originally derived from a lymph node metastatis of a human prostatic adenocarcinoma. However, DNA-mediated gene transfer, a common and key procedure in cellular and molecular studies, is very inefficient for LNCaP cells, and this limits the utility of these cells in investigations of the molecular mechanisms of prostate carcinogenesis. METHODS: In search of a simple, reproducible, and cost-effective method for introducing DNA into LNCaP cells, we adopted and optimized two methods of transient transfection into LNCaP cells: a modified calcium phosphate (CaPO(4)) coprecipitation procedure and polyethylenimine (pEI) mediated transfection. RESULTS: When compared with the liposome-mediated transfection that was previously used for LNCaP cells, we find that the most efficient of these techniques is the modified CaPO(4) coprecipitation procedure. For experiments in which calcium exposure of the cells is not desirable, the pEI procedure provides a less efficient, but reproducible and cost-effective alternative. CONCLUSIONS: These two new DNA-mediated gene transfer methods should facilitate gene expression studies in LNCaP cells and thereby aid in the study of the androgen-dependent phase of prostate cancer. PMID- 10754527 TI - Absence of proximal duct apoptosis in the ventral prostate of transgenic mice carrying the C3(1)-TGF-beta type II dominant negative receptor. AB - BACKGROUND: Prostatic epithelial cells are sensitive to the inhibitory effects of TGF-beta. However, TGF-beta signaling in the prostate is dependent on androgenic status. Under the in vivo conditions, it is difficult to dissociate the effect of TGF-beta from that of androgen on the prostate. METHODS: The objective of the present study was to create and verify a transgenic mouse system in which epithelial cells of the ventral prostate are insensitive to the actions of TGF beta. By using a modified prostate-specific promoter, C3(1), the TGF-beta dominant negative receptor is only expressed in the epithelial cells of the ventral prostate, and these cells are resistant to TGF-beta. Morphology of transgenic animal prostates was compared to wild-type animal prostates by immunohistochemistry and microscopy. RESULTS: The prostate of transgenic mice exhibited an abnormal morphology with multiple layers of epithelial cells lining the proximal ducts, in contrast to the simple cuboidal monolayer of cells seen in the normal prostate. This observation was accompanied by a loss of apoptosis in this region, as seen by TUNEL assay. There was no significant difference in serum levels of testosterone between the wild-type and transgenic animals. CONCLUSIONS: These results demonstrated that a loss of sensitivity to TGF-beta results in the accumulation of multiple layers of epithelial cells in the proximal region of the ventral prostate. This abnormal growth illustrates that TGF-beta plays an important role in regulating prostate growth. The current transgenic system can be used as an experimental model to study the functional role of TGF-beta in prostatic growth and function. PMID- 10754528 TI - Metallothionein isoform 1 and 2 gene expression in the human prostate: downregulation of MT-1X in advanced prostate cancer. AB - BACKGROUND: Studies have shown an association of metallothionein (MT) overexpression with tumor type and grade. However, a family of genes underlies the expression of these proteins. The goals of this study were to define the expression of MT genes and protein in normal human prostate and to provide evidence that the expression of the MT isoforms is altered in prostate cancer. METHODS: Immunohistochemistry was used to localize MT protein, reverse transcription-polymerase chain reaction (RT-PCR) to determine the MT isoform specific mRNAs, and immunoblot analysis to determine MT protein levels. RESULTS: The localization of MT in the prostate was further defined using the E9 antibody. Using normal prostate tissue dissected from glands removed for prostate cancer, it was demonstrated that MT protein expression in the normal prostate is supported by mRNA from the MT-1A, MT-1E, MT-1X, and MT-2A genes. No expression of the MT-1X gene was demonstrated in cases of advanced prostate cancer. The expression of MT-1 and MT-2 isoform-specific mRNA varied among three commonly utilized prostate cancer cell lines. CONCLUSIONS: MT protein in the normal human prostate is supported by transcription of mRNA from the MT-1A, MT-1E, MT-1X, and MT-2A genes. Expression of MT-1X mRNA is downregulated in advanced prostate cancer. Variable expression of MT mRNA in prostate cell lines provides evidence that MT gene expression may be altered among individual prostate cancers. PMID- 10754529 TI - Body size, age at shaving initiation, and prostate cancer in a large, multiracial cohort. AB - BACKGROUND: The purpose of this study was to examine the potential relationship between body size, self-reported age at initiation of shaving, and subsequent risk of prostate cancer in a large, racially diverse cohort of men followed for up to 32 years. METHODS: The study population included 70,712 male subscribers to the Kaiser Permanente Medical Care Program who had received a multiphasic health checkup between 1964-1973. This general health checkup consisted of a number of laboratory tests and physical measurements, as well as a self-completed health questionnaire that included a request for men to record the age when they began shaving. Subjects were followed for the development of prostate cancer, using the local tumor registry. Cox regression was used to estimate relative risks (RR) and 95% confidence intervals (CI). RESULTS: Altogether, 2, 079 men in the study cohort were diagnosed with prostate cancer. There was a very strong positive association between prostate cancer risk and birth cohort. After adjusting for race, age, and birth year, there was no association between height, weight, body mass index, or several other anthropometric measures and prostate cancer risk in the full cohort. There was a suggestion of a very weak positive association between height and prostate cancer risk among white men. There also was no overall association between age at shaving initiation and prostate cancer risk, although nonwhite men who started shaving at a young age (20mm through stents. Is there a first choice strategy? AB - OBJECTIVE - This study compared the early and late results of the use of one single stent with those of the use of multiple stents in patients with lesions longer than 20mm. METHODS - Prospective assessment of patients electively treated with stents, with optimal stent deployment and followed-up for more than 3 months. From February '94 to January '98, 215 patients with lesions >20mm were treated. These patients were divided into 2 groups as follows: Group A - 105 patients (49%) with one stent implanted; Group B - 110 patients (51%) with multiple stents implanted. RESULTS - The mean length of the lesions was 26mm in group A (21-48mm) versus 29mm in group B (21-52mm) (p=0.01). Major complications occurred in one patient (0.9%) in group A (subacute thrombosis, myocardial infarctionand death) and in 2 patients (1.8%) in group B (one emergency surgery and one myocardial infarction) (p=NS). The results of the late follow-up period (>6 months) were similar for both groups (group A = 82% vs group B = 76%; p=NS), and we observed an event-free survical in 89% of the patients in group A and in 91% of the patients in group B (p=NS). Angina (group A = 11% vs group B = 7%) and lesion revascularization (group A = 5% vs group B = 6%; p=NS) also occurred in a similar percentage. No infarction or death was observed in the late follow-up period; restenosis was identified in 33% and 29% of the patients in groups A and B, respectively (p=NS). CONCLUSION - The results obtained using one stent and using multiple stents were similar; the greater cost-effectiveness of one stent implantation, however, seems to make this strategy the first choice. PMID- 10754589 TI - Exercise and heart failure. Relation of the severity of the disease to the anaerobic threshold and the respiratory compensation point. AB - OBJECTIVE - To identify, the anaerobic threshold and respiratory compensation point in patients with heart failure. METHODS - The study comprised 42 Men,divided according to the functional class (FC) as follows: group I (GI) - 15 patients in FC I; group II (GII) - 15 patients in FC II; and group III (GIII) - 12 patients in FC III. Patients underwent a treadmill cardiopulmonary exercise test, where the expired gases were analyzed. RESULTS - The values for the heart rate (in bpm) at the anaerobic threshold were the following: GI, 122+/-27; GII, 117+/-17; GIII, 114+/-22. At the respiratory compensation point, the heart rates (in bpm) were as follows: GI, 145+/-33; GII, 133+/-14; GIII 123+/-22. The values for the heart rates at the respiratory compensation point in GI and GIII showed statistical difference. The values of oxygen consumption (VO2) at the anaerobic threshold were the following (in ml/kg/min): GI, 13. 6+/-3.25; GII, 10.77+/-1.89; GIII, 8.7+/-1.44 and, at the respiratory compensation point, they were as follows: GI, 19.1+/-2. 2; GII, 14.22+/-2.63; GIII, 10.27+/-1.85. CONCLUSION - Patients with stable functional class I, II, and III heart failure reached the anaerobic threshold and the respiratory compensation point at different levels of oxygen consumption and heart rate. The role played by these thresholds in physical activity for this group of patients needs to be better clarified. PMID- 10754590 TI - Effects of lisinopril on experimental ischemia in rats. Influence of infarct size. AB - OBJECTIVE - Angiotensin-converting enzyme inhibitors (ACEIs) have gained importance in preventing or attenuating the process of ventricular remodeling after myocardial infarction. The significance of infarct size in regard to the response to ACEIs, however, is controversial. This study aimed to analyze the effects of lisinopril on mortality rate, cardiac function, degree of cardiac hypertrophy and fibrosis in rats with different infarct sizes. METHODS - Lisinopril (20 mg/kg/day) dissolved in drinking water was administered to rats immediately after coronary artery occlusion. After being sacrificed, the infarcted animals were divided into two groups: one group of animals with small infarcts (< 40% of the left ventricle) and another group of animals with large infarcts (> 40% of the left ventricle). RESULTS - The mortality rate was 31.7% in treated rats and 47% in the untreated rats. There was no statistical difference between the groups with small and large infarcts in regard to myocardial concentration of hydroxyproline. In small infarcts, the treatment attenuated the heart dysfunction characterized by lower levels of blood pressure and lower values of the first derivative of pressure and of the negative derivative of pressure. The degree of hypertrophy was also attenuated in small infarcts. In regard to large infarcts, no differences between the groups were observed. CONCLUSION - Treatment with the ACEIs had no effect on mortality rate and on the amount of fibrosis. The protective effect of lisinopril on heart function and on the degree of hypertrophy could only be detected in small infarcts PMID- 10754591 TI - Percutaneous mitral valvotomy in patients eighteen years old and younger. Immediate and late results. AB - OBJECTIVE - To analyze immediate and late results of percutaneous mitral valvotomy (PMV) in patients < or = 18 year. METHODS - Between August '87 and July '97, 48 procedures were performed on 40 patients. The mean age was 15.6 years; 68.7% were females four of whom were pregnant. RESULTS - Success was obtained in 91.7% of the procedures. Immediate complications were severe mitral regurgitation (6.3%) and cardiac tamponade (2.0%). Late follow-up was obtained in 88.8% of the patients (mean value=43.2+/-33.9 months). NYHA functional class (FC) I or II was observed in 96.2% of the patients and restenosis developed in five patients, at a mean follow-up of 29.7+/-11.9 months. Three patients presented with severe mitral insufficiency and underwent surgery. Two patients died. CONCLUSION - PMV represents a valid therapeutic option in young patients. In these patients, maybe because of subclinical rheumatic activity, restenosis may have a higher incidence and occur at an earlier stage than in others persons. PMID- 10754592 TI - Chylothorax after myocardial revascularization with the left internal thoracic artery. AB - A 38-year-old male underwent coronary artery bypass grafting (CABG). A saphenous vein graft was attached to the left marginal branch. The left internal thoracic artery was anastomosed to the left anterior descending artery (LAD). The early recovery was uneventful and the patient was discharged on the 5th postoperative day. After three months, he came back to the hospital complaining of weight loss, weakness, and dyspnea on mild exertion. Chest X-rays showed left pleural effusion. On physical examination, a decreased vesicular murmur was detected. After six days, the diagnosis of chylothorax was made after a milky fluid was detected in the plural cavity and total pulmonary expansion did not occur. On the next day, both anterior and posterior pleural drainage were performed by videothoracoscopy, and prolonged parenteral nutrition (PPN) was instituted for ten days. After seven days the patient was put on a low-fat diet for 8 days. The fluid accumulation ceased, the drains were removed and the patient was discharged with normal pulmonary expansion. PMID- 10754593 TI - Growth hormone for optimization of refractory heart failure treatment. AB - It has been reported that growth hormone may benefit selected patients with congestive heart failure. A 63-year-old man with refractory congestive heart failure waiting for heart transplantation, depending on intravenous drugs (dobutamine) and presenting with progressive worsening of the clinical status and cachexia, despite standard treatment, received growth hormone replacement (8 units per day) for optimization of congestive heart failure management. Increase in both serum growth hormone levels (from 0.3 to 0.8 microg/l) and serum IGF-1 levels (from 130 to 300ng/ml) was noted, in association with clinical status improvement, better optimization of heart failure treatment and discontinuation of dobutamine infusion. Left ventricular ejection fraction (by MUGA) increased from 13 % to 18 % and to 28 % later, in association with reduction of pulmonary pressures and increase in exercise capacity (rise in peak VO2 to 13.4 and to 16.2ml/kg/min later). The patient was "de-listed" for heart transplantation. Growth hormone may benefit selected patients with refractory heart failure. PMID- 10754594 TI - Syncope in a twenty-three-year-old male. PMID- 10754595 TI - Unique distribution of von Willebrand factor and thrombomodulin in endothelial cells of human pulmonary microvessels. PMID- 10754596 TI - [Primary immunodeficiency diseases]. PMID- 10754597 TI - Evaluation of glycosaminoglycans levels in normal joint fluid of the knee. AB - Glycosaminoglycans (GAGs) level in joint fluid have been investigated in various joint diseases as a joint maker. However, there are very few studies of normal joint fluid providing a baseline for a better understanding of altered GAGs level in pathological joint fluid. We investigated GAGs level in knee joint fluid for 25 healthy young volunteers with a mean age of 27.5 years (range: 18 to 36 years). Biochemical evaluations included the concentration of hyaluronic acid (HA), chondroitin 4-sulfate (C4S) and chondroitin 6-sulfate (C6S), and the C6S/C4S ratio. The unsaturated disaccharides derived from HA by Morgan-Elson methods, and those from CS were measured with high performance liquid chromatography. The mean HA concentration was 3.4+/-0.6 mg/ml. The mean concentration of C4S and C6S, and C6S/C4S ratio were 19.0+/-4.9 nmol/ml, 125.5+/ 44.4 nmol/ml and 6.5+/-1.1 respectively. PMID- 10754598 TI - A role of angiotensin-converting enzyme gene polymorphism in left ventricular remodeling after myocardial infarction. AB - BACKGROUND: Left ventricular remodeling (LVR) process is one of the important secondary sequele after acute myocardial infarction (AMI). However, little is known about the relationship between LVR and angiotensin converting enzyme (ACE) gene polymorphism as well as endothelial nitric oxide synthase (ecNOS) gene polymorphism. METHODS: Coronary angiography and left ventriculography were performed within 24 hours and 30+/-7 days after AMI onset. All consecutive 24 patients (57+/-6 years) had acute anterior MI with one vessel disease of left anterior descending artery and successful revascularization therapy during acute phase. Patients were divided into three groups according to the change of end diastolic volume index (EDVI)(Delta EDVI = EDVI 1 month-EDVI within 24 hrs); LVR(+) (Delta EDVI>7.0 ml/m(2), n = 5), LVR (-)(Delta EDVI<-7.0 ml/m(2), n = 13), and LVR (+/-)(-7.0 > > alpha2c mRNA in human spinal cord dorsal horn cell bodies. However, because 20% of dorsal horn alpha2ARs derive from cell bodies that reside in the associated dorsal root ganglion (DRG), it is important to evaluate alpha2AR expression in this tissue as well. Therefore, the authors evaluated the hypothesis that alpha2b mRNA, alpha2c mRNA, or both are present in human DRG. METHODS: Molecular approaches were used to determine alpha2AR expression in 28 human DRGs because of low overall receptor mRNA expression and small sample size. After creation of synthetic competitor cDNA and establishment of amplification conditions with parallel efficiencies, competitive reverse transcription polymerase chain reaction was performed using RNA isolated from human DRG. RESULTS: Overall expression of alpha2AR mRNA in DRG is low but reproducible at all spinal levels. alpha2b and alpha2cAR subtype mRNAs predominate (alpha2b approximately alpha2c), accounting for more than 95% of the total alpha2AR mRNA in DRG at all human spinal nerve root levels. CONCLUSIONS: Predominance of alpha2b and alpha2cAR mRNA in human DRG is distinct from alpha2AR mRNA expression in cell bodies originating in human spinal cord dorsal horn, where alpha2a and alpha2b predominate with little or absent alpha2c expression. These findings also highlight species heterogeneity in alpha2AR expression in DRG. If confirmed at a protein level, these findings provide an additional step in unraveling mechanisms involved in complex neural pathways such as those for pain. PMID- 10754616 TI - The incidence and mechanisms of pharyngeal and upper esophageal dysfunction in partially paralyzed humans: pharyngeal videoradiography and simultaneous manometry after atracurium. AB - BACKGROUND: Residual neuromuscular block caused by vecuronium alters pharyngeal function and impairs airway protection. The primary objectives of this investigation were to radiographically evaluate the swallowing act and to record the incidence of and the mechanism behind pharyngeal dysfunction during partial neuromuscular block. The secondary objective was to evaluate the effect of atracurium on pharyngeal function. METHODS: Twenty healthy volunteers were studied while awake during liquid-contrast bolus swallowing. The incidence of pharyngeal dysfunction was studied by fluoroscopy. The initiation of the swallowing process, the pharyngeal coordination, and the bolus transit time were evaluated. Simultaneous manometry was used to document pressure changes at the tongue base, the pharyngeal constrictor muscles, and the upper esophageal sphincter. After control recordings, an intravenous infusion of atracurium was administered to obtain train-of-four ratios (T4/T1) of 0.60, 0.70, and 0.80, followed by recovery to a train-of-four ratio of more than 0.90. RESULTS: The incidence of pharyngeal dysfunction was 6% during the control recordings and increased (P < 0.05) to 28%, 17%, and 20% at train-of-four ratios 0.60, 0.70, and 0.80, respectively. After recovery to a train-of-four ratio of more than 0.90, the incidence was 13%. Pharyngeal dysfunction occurred in 74 of 444 swallows, the majority (80%) resulting in laryngeal penetration. The initiation of the swallowing reflex was impaired during partial paralysis (P = 0.0081). The pharyngeal coordination was impaired at train-of-four ratios of 0.60 and 0.70 (P < 0.01). A marked reduction in the upper esophageal sphincter resting tone was found, as well as a reduced contraction force in the pharyngeal constrictor muscles. The bolus transit time did not change significantly. CONCLUSION: Partial neuromuscular paralysis caused by atracurium is associated with a four- to fivefold increase in the incidence of misdirected swallowing. The mechanism behind the pharyngeal dysfunction is a delayed initiation of the swallowing reflex, impaired pharyngeal muscle function, and impaired coordination. The majority of misdirected swallows resulted in penetration of bolus to the larynx. PMID- 10754617 TI - Reliability of pharmacodynamic analysis by logistic regression: a computer simulation study. AB - BACKGROUND: Many pharmacologic studies record data as binary yes-or-no variables, and analysis is performed using logistic regression. This study investigates the accuracy of estimation of the drug concentration associated with a 50% probability of drug effect (C50) and the term describing the steepness of the concentration-effect relation (gamma). METHODS: The authors developed a technique for simulating pharmacodynamic studies with binary yes-or-no responses. Simulations were conducted assuming either that each data point was derived from the same patient or that data were pooled from multiple patients in a population with log-normal distributions of C50 and gamma. Coefficients of variation were calculated. The authors also determined the percentage of simulations in which the 95% confidence intervals contained the true parameter value. RESULTS: The coefficient of variation of parameter estimates decreased with increasing n and gamma. The 95% confidence intervals for C50 estimation contained the true parameter value in more than 90% of the simulations. However, the 95% confidence intervals of gamma did not contain the true value in a substantial number of simulations of data from multiple patients. CONCLUSION: The coefficient of variation of parameter estimates may be as large as 40-50% for small studies (n < or = 20). The 95% confidence intervals of C50 almost always contain the true value, underscoring the need for always reporting confidence intervals. However, when data from multiple patients is naively pooled, the estimates of gamma may be biased, and the 95% confidence intervals may not contain the true value. PMID- 10754618 TI - Pharmacokinetics and arteriovenous differences in clevidipine concentration following a short- and a long-term intravenous infusion in healthy volunteers. AB - BACKGROUND: Clevidipine is an ultra-short-acting calcium antagonist developed for reduction and control of blood pressure during cardiac surgery. The objectives of the current study were to determine the pharmacokinetics of clevidipine after 20 min and 24-h intravenous infusions, and to determine the relation between the arterial and venous concentrations and the hemodynamic responses to clevidipine in healthy volunteers. METHODS: Four volunteers received clevidipine for 20 min, and eight subjects were administered clevidipine intravenously for 24 h at two different dose rates. Arterial and venous blood samples were drawn for pharmacokinetic evaluation, and blood pressure and heart rate were recorded. RESULTS: A triexponential disposition model described the pharmacokinetics of clevidipine. The mean arterial blood clearance of clevidipine was 0.069l/kg-1/min 1 and the mean volume of distribution at steady state was 0.19 l/kg. The duration of the infusion had negligible effect on the pharmacokinetic parameters, and the context-sensitive half-time for clevidipine, simulated from the mean pharmacokinetic parameters derived after 24 h infusion at the highest dose, was less than 1 min. The arterial blood levels reached steady state within 2 min of the start of infusion and were about twice as high as those in the venous blood at steady state. The peak response preceded the peak venous concentration and was slightly delayed from the peak arterial blood concentration. CONCLUSION: Clevidipine is a high clearance drug with a small volume of distribution, resulting in extremely short half-lives in healthy subjects. The initial rapid increase in the arterial blood concentrations and the short equilibrium time between the blood and the biophase suggest that clevidipine can be rapidly titrated to the desired effect. PMID- 10754619 TI - A dose-ranging study of rapacuronium in pediatric patients. AB - BACKGROUND: The aim of this study was to determine the dose or doses of the new rapid-onset, short-acting, neuromuscular blocking drug rapacuronium that would provide satisfactory conditions for tracheal intubation at 60 s in infants and children. METHODS: Sixty-five infants (< 1 yr), 51 younger children (1-6 yr), and 49 older children (7-12 yr) were studied. Anesthesia was induced with thiopental nitrous oxide-oxygen. Tracheal intubation was attempted 60 s after administration of one of five doses of rapacuronium (0.5, 1.0, 1.5, 2.0, or 2.5 mg/kg) and intubating conditions were assessed using a four-point scale. Following tracheal intubation, anesthesia was maintained with nitrous oxide-oxygen and alfentanil (12.5-50 microg/kg) as necessary. Neuromuscular transmission was monitored in an uncalibrated fashion using an acceleromyograph. RESULTS: Intubating conditions were good or excellent at 60 s in all infants after doses of 1.5 mg/kg or more and in all younger and older children after doses of 2.0 mg/kg or more. The duration of action of rapacuronium was dose- and age-dependent. Mean times to reappearance of the third twitch of the train-of-four (TOF; T3) were less than 10 min in infants at doses of 1.5 mg/kg or less and in younger and older children at doses of 2.0 mg/kg or less. Recovery of T3 after 1.0-2.0 mg/kg rapacuronium was significantly slower in infants compared with younger (P = 0.001) and older (P = 0.02) children. Five adverse experiences were related to rapacuronium administration: Bronchospasm (two instances), tachycardia (one instance), and increased salivation (two instances). None were serious. CONCLUSIONS: Doses of 1.5 and 2.0 mg/kg rapacuronium can produce satisfactory intubating conditions at 60 s in anesthetized infants and children, respectively, and are associated with a short duration of action. PMID- 10754620 TI - Maximum tolerated dose of nalmefene in patients receiving epidural fentanyl and dilute bupivacaine for postoperative analgesia. AB - BACKGROUND: This study investigated the ability of the modified continual reassessment method (MCRM) to determine the maximum tolerated dose of the opioid antagonist nalmefene, which does not reverse analgesia in an acceptable number of postoperative patients receiving epidural fentanyl in 0.075% bupivacaine. METHODS: In the postanesthetic care unit, patients received a single intravenous dose of 0.25, 0.50, 0.75, or 1.00 microg/kg nalmefene. Reversal of analgesia was defined as an increase in pain score of two or more integers above baseline on a visual analog scale from 0 through 10 after nalmefene administration. Patients were treated in cohorts of one, starting with the lowest dose. The maximum tolerated dose of nalmefene was defined as that dose, among the four studied, with a final mean probability of reversal of anesthesia (PROA) closest to 0.20 (ie., a 20% chance of causing reversal). The modified continual reassessment method is an iterative Bayesian statistical procedure that, in this study, selected the dose for each successive cohort as that having a mean PROA closest to the preselected target PROA of 0.20. RESULTS: The modified continual reassessment method repeatedly updated the PROA of each dose level as successive patients were observed for presence or absence of ROA. After 25 patients, the maximum tolerated dose of nalmefene was selected as 0.50 microg/kg (final mean PROA = 0.18). The 1.00-microg/kg dose was never tried because its projected PROA was far above 0.20. CONCLUSIONS: The modified continual reassessment method facilitated determination of the maximum tolerated dose ofnalmefene . Operating characteristics of the modified continual reassessment method suggest it may be an effective statistical tool for dose-finding in trials of selected analgesic or anesthetic agents. PMID- 10754621 TI - Investigation of effective anesthesia induction doses using a wide range of infusion rates with undiluted and diluted propofol. AB - BACKGROUND: The influence of infusion rate on the induction dose-response relation has not been investigated over a wide range of infusion rates. In this study, the authors defined the effect of different propofol infusion rates on the times and doses necessary to reach clinical induction of anesthesia. METHODS: The subjects of the study were 250 patients classified as American Society of Anesthesiologists physical status I or II aged 25-55 yr. For induction with undiluted propofol, 180 patients were allocated randomly to one of two groups of 90 patients each (A and B). Each group was further divided into nine subgroups (10 patients each) that were administered propofol infusion at rates of 10, 15, 20, 30, 40, 60, 100, 200, and 300 mg/kg-1/h-1. The remaining 70 patients (group C) were allocated randomly into seven subgroups (10 patients each), and these groups were induced with diluted propofol (0.5 mg/ ml) at the rates of 10, 15, 30, 60, 100, 200, and 300 mg/kg-1/ h-1. Group B was given crystalloid at the same infusion rates as group C via a catheter in the opposite arm. Induction time, induction dose, plasma arterial propofol concentration at loss of consciousness, and percentage decrease of systolic blood pressure were measured. A previously reported three-compartment model with an effect-site rate constant for propofol of 0.456/min was used to predict the induction time and dose at each infusion rate. RESULTS: The differences between predicted induction time and dose and the observed time and dose could be explained by factoring in the lag time from infusion site to central compartment (lag time circulation) and the amount of propofol in transit during this time (residual dose circulation). Residual dose circulation and lag time circulation correlated with infusion time from 20 to 60 s for undiluted and from 0 to 40 s for diluted propofol. At the infusion rates greater than 80 mg/kg-1/h-1, rapid circulation because of incomplete mixing in the central compartment decreased the excess induction time and dose. The use of diluted propofol significantly attenuated the decrease in systolic blood pressure provoked by the residual dose circulation. CONCLUSIONS: Induction dose and time are dependent on infusion rate in a complex manner, and residual dose circulation was a factor in overdose and hemodynamic depression. Hypotension during induction was attenuated by diluted propofol. PMID- 10754622 TI - Memory formation during general anesthesia for emergency cesarean sections. AB - BACKGROUND: Occurrence of explicit memory (i.e., conscious recall) has been reported especially after surgical procedures in which anesthesia is considered to be "light." In addition, previous research has shown that implicit memory (e.g., improved memory test performance in absence of conscious recall) decreases with increasing hypnotic state. The current study investigated explicit and implicit memory during emergency cesarean sections with consistently light levels of hypnotic state. METHOD: Words were presented via headphones, and the bispectral index was recorded throughout surgery. Memory for the presented words was tested after recovery with a word-stem completion test. Using both parts of the process dissociation procedure allowed separation of explicit and implicit memory. In the "inclusion" part of the process dissociation procedure, patients are asked to complete word stems, if possible, with the corresponding words recalled from the intraoperative presentation. In the "exclusion" part, patients are instructed to avoid the words presented intraoperatively and to use other words instead. In the absence of recall, patients are asked to use the first word that comes to mind. RESULTS: The mean bispectral index during word presentation was 76.3 (+/-3.0). On average, the 24 patients were able to make correct inclusion-exclusion decisions: In the inclusion part, hit rates (i.e., the probability of responding with a word presented during surgery) were higher than base rates (0.37 vs. 0.31), whereas in the exclusion part hit rates were lower (0.23 vs. 0.28). Importantly, the patients made these inclusion-exclusion decisions without being able to consciously recall the words presented during surgery. CONCLUSIONS: This study shows that if words are presented at relatively light levels of anesthesia, patients are able to control their inclusion exclusion decisions. This weak form of explicit memory can occur in the absence of conscious recall. PMID- 10754623 TI - Prophylactic ondansetron in prevention of postoperative nausea and vomiting following pediatric strabismus surgery: a dose-response study. AB - BACKGROUND: This study evaluated the antiemetic effectiveness, dose-response, and clinical usefulness of prophylactic ondansetron in the prevention of postoperative nausea and vomiting (PONV) in children undergoing strabismus repair. METHOD: The authors observed 180 children, American Society of Anesthesiologists physical status I or II, 2-12 yr of age, who were undergoing strabismus repair. After induction of anesthesia with halothane and nitrous oxide in oxygen or intravenous thiopental, children received either placebo (saline) or intravenous ondansetron in doses of 25, 50, 75, 100, and 150 /microg/kg (n = 30). The trachea was intubated and ventilation was controlled. Perioperative analgesic and fluid requirements were standardized. Episodes of nausea and vomiting were recorded for the first 24 h postoperatively. Data such as nonsurrogate (parental satisfaction scores and duration of postanesthesia care unit stay) and therapeutic (numbers needed to prevent and harm) outcome measures were collected. RESULTS: The incidences of PONV in the placebo and 25-, 50-, 75-, 100-, and 150 ,microg/kg ondansetron groups were 83, 77, 47, 30, 30, and 27%, respectively. The incidence was less in the 75(P = 0.002), 100- (P = 0.002), and 150-microg/kg (P < 0.001) ondansetron groups compared with placebo. Duration of stay in the postanesthesia care unit was shorter in the 75-, 100-, and 150-microg/kg ondansetron groups (P < 0.002) compared with the placebo group. Parental assessment scores for the child's perioperative experience and the positive number needed to prevent PONV were also better and favorable in the 75-, 100-, and 150-microg/kg ondansetron groups compared with the placebo group. The incidence (P > 0.99) and severity (P = 0.63) of PONV were similar in the 75- and 150-microg/kg ondansetron groups. Surrogate, nonsurrogate, and therapeutic outcome measures revealed that 75 microg/kg ondansetron provided the same benefits as did 100 and 150 microg/kg. CONCLUSION: The routine prophylactic use of ondansetron at a dose of 75 microg/kg is as effective as 150 microg/kg in preventing PONV and improving the "true" outcome measures after strabismus repair in children. PMID- 10754624 TI - Plasma concentration of fentanyl with xenon to block somatic and hemodynamic responses to surgical incision. AB - BACKGROUND: Although anesthesia with xenon has been supplemented with fentanyl, its requirement has not been established. This study was conducted to determine the plasma concentrations of fentanyl necessary to suppress somatic and hemodynamic responses to surgical incision in 50% patients in the presence of 0.7 minimum alveolar concentration (MAC) xenon. METHODS: Twenty-five patients were allocated randomly to predetermined fentanyl concentration between 0.5 and 4.0 ng/ml during 0.7 MAC xenon anesthesia. Fentanyl was administered using a pharmacokinetic model-driven computer-assisted continuous infusion device. At surgical incision each patient was monitored for somatic and hemodynamic responses. A somatic response was defined as any purposeful bodily movement. A positive hemodynamic response was defined as a more than 15% increase in heart rate or mean arterial pressure more than the preincision value. The concentrations of fentanyl to prevent somatic and hemodynamic responses in 50% of patients were calculated using logistic regression. RESULTS: The concentration of fentanyl to prevent a somatic response to skin incision in 50% of patients in the presence of 0.7 MAC xenon was 0.72 +/- 0.07 ng/ml and to prevent a hemodynamic response was 0.94 +/- 0.06 ng/ml. CONCLUSIONS: Comparing these results with previously published results in the presence of 70% nitrous oxide, the fentanyl requirement in xenon anesthesia is smaller than that in the equianesthetic nitrous oxide anesthesia. PMID- 10754625 TI - Both EMLA and placebo cream reduced pain during extracorporeal piezoelectric shock wave lithotripsy with the Piezolith 2300. AB - BACKGROUND: The objectives were to determine whether a eutectic mixture of local anesthetic (EMLA) or placebo cream reduces pain during extracorporeal piezoelectric shock wave lithotripsy (EPSWL), and to determine which of the components of the application (i.e., the occlusive dressing, the cream, or the local anesthetic) contributes to analgesia. METHODS: A randomized, double blind, crossover study (part 1) was performed in 12 patients who were scheduled for EPSWL procedures on an ambulatory basis who received the first treatment without any intervention and who had verbal pain scores of 70 or more (on a 0-to- 100 scale). For the next two treatments at 2-week intervals, patients were randomly assigned to receive either 10 g EMLA or 10 g placebo cream and then crossed over to receive the other. The cream and occlusive dressing were left in place and immersed in water throughout the procedure. Verbal numeric pain score was assessed at 5 min after receiving the maximal tolerable intensity of shock wave and at the end of the procedure. The study continued (part 2) in 202 ambulatory patients; 125 men and 77 women, American Society of Anesthesiologists physical status I and II, subjected to EPSWL were randomly allocated into five groups who received (1) nothing on the skin (control), (2) plastic occlusive dressing, (3) placebo cream and plastic occlusive dressing, (4) EMLA cream and plastic occlusive dressing, (5) EMLA cream and plastic occlusive dressing for 60 min to achieve cutaneous anesthesia, which was removed before EPSWL. Pain score was evaluated 10 min into the procedure and at the end of the procedure. RESULT: Both parts of the study showed that patients who received either EMLA or placebo cream with dressing throughout the procedure experienced less pain and tolerated higher energy levels compared with the control. Patients who received only pre-EPSWL cutaneous anesthesia of EMLA and who received only the occlusive dressing did not have a reduction in pain score. CONCLUSIONS: EMLA and placebo creams under occlusive dressing reduced pain during EPSWL. The presence of the cream itself as a coupling medium contributed to analgesia. This may be a useful, simple, safe, and economical adjuvant technique to reduce pain during immersion EPSWL. PMID- 10754626 TI - Contrasting synaptic actions of the inhalational general anesthetics isoflurane and xenon. AB - BACKGROUND: The mechanisms by which the inhalational general anesthetics isoflurane and xenon exert their effects are unknown. Moreover, there have been surprisingly few quantitative studies of the effects of these agents on central synapses, with virtually no information available regarding the actions of xenon. METHODS: The actions of isoflurane and xenon on gamma-aminobutyric acid-mediated (GABAergic) and glutamatergic synapses were investigated using voltage-clamp techniques on autaptic cultures of rat hippocampal neurons, a preparation that avoids the confounding effects of complex neuronal networks. RESULTS: Isoflurane exerts its greatest effects on GABAergic synapses, causing a marked increase in total charge transfer (by approximately 70% at minimum alveolar concentration) through the inhibitory postsynaptic current. This effect is entirely mediated by an increase in the slow component of the inhibitory postsynaptic current. At glutamatergic synapses, isoflurane has smaller effects, but it nonetheless significantly reduces the total charge transfer (by approximately 30% at minimum alveolar concentration) through the excitatory postsynaptic current, with the N methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor-mediated components being roughly equally sensitive. Xenon has no measurable effect on GABAergic inhibitory postsynaptic currents or on currents evoked by exogenous application of GABA, but it substantially inhibits total charge transfer (by approximately 60% at minimum alveolar concentration) through the excitatory postsynaptic current. Xenon selectively inhibits the NMDA receptor-mediated component of the current but has little effect on the AMPA/kainate receptor-mediated component. CONCLUSIONS: For both isoflurane and xenon, the most important targets appear to be postsynaptic. The authors' results show that isoflurane and xenon have very different effects on GABAergic and glutamatergic synaptic transmission, and this may account for their differing pharmacologic profiles. PMID- 10754627 TI - Effects of intravenous anesthetic agents on glutamate release: a role for GABAA receptor-mediated inhibition. AB - BACKGROUND: Many anesthetic agents are known to enhance the alpha1beta2gamma2S gamma-aminobutyric acid type A (GABAA) chloride current; however, they also depress excitatory neurotransmission. The authors evaluated two hypotheses: intravenous anesthetic agents inhibit glutamate release and any observed inhibition may be secondary to GABAA receptor activation. METHODS: Cerebrocortical slices were prepared from Wistar rats. After perfusion in oxygenated Krebs buffer for 60 min at 37 degrees C, samples for glutamate assay were obtained at 2-nmin intervals. After 6 min, a 2-min pulse of 46 mM K+ was applied to the slices (S1); this was repeated after 30 min (S2). Bicuculline (1 100 microM) was applied when the S1 response returned to basal level, and 10 min later, thiopental (1-300 micro/M), propofol (10 microM), or ketamine (30 microM) were also applied until the end of S2. Perfusate glutamate concentrations were measured fluorometrically, and the area under the glutamate release curves was expressed as a ratio (S2/S1). RESULTS: Potassium (46 mM) evoked a monophasic release of glutamate during S1 and S2, with a mean control S2/S1 ratio of 1.07 +/ 0.33 (mean +/- SD, n = 96). Ketamine and thiopental produced a concentration dependent inhibition of K+-evoked glutamate release with half-maximum inhibition of release values of 18.2 and 10.9 /microM, respectively. Release was also inhibited by propofol. Bicuculline produced a concentration dependent reversal of thiopental inhibition of glutamate release with a half-maximum reversal of the agonist effect of 10.3 microM. Bicuculline also reversed the effects of propofol but not those of ketamine. CONCLUSIONS: The authors' data indicate that thiopental, propofol, and ketamine inhibit K+-evoked glutamate release from rat cerebrocortical slices. The inhibition produced by thiopental and propofol is mediated by activation of GABAA receptors, revealing a subtle interplay between GABA-releasing (GABAergic) and glutamatergic transmission in anesthetic action. PMID- 10754628 TI - Mechanisms of nonimmunological histamine and tryptase release from human cutaneous mast cells. AB - BACKGROUND: If mast cells are stimulated they release multiple mediators that delineate markers for immunologic and nonimmunologic reactions; histamine and tryptase are the two best known. Although histamine can be assayed in plasma, it is a nonspecific marker with a very short half-life. Tryptase has a longer half life, but its release has not been proven to be specific for anaphylaxis. The authors investigated the mechanisms of nonimmunologic histamine release from human cutaneous mast cells to understand the mechanisms of mediator release and to determine whether tryptase was specific for allergic mediated activation. METHODS: Dispersed mast cell suspensions isolated from neonatal foreskins underwent challenge with vancomycin, calcium ionophore A23187, morphine, and atracurium, and histamine tryptase release was measured. The effects of calcium and magnesium, along with phospholipase C and phospholipase A2 inhibitors, also were investigated. RESULTS: Tryptase and histamine both were released by the known nonimmunologic stimuli (pharmacologic agents used in the current study; r2 = 0.6). Furthermore, vancomycin- and atracurium-induced histamine release was calcium dependent. Phospholipase C and phospholipase A2 inhibitors decreased vancomycin-induced histamine release, but not calcium ionophore A23187-induced release. CONCLUSIONS: Tryptase is not a specific marker of mast cell activation (ie., anaphylaxis), and signaling mechanisms for mast cell activation involve activation of phospholipase C and phospholipase A2 pathways that are also involved in other cellular activation mechanisms. PMID- 10754629 TI - Attenuation of ascending nociceptive signals to the rostroventromedial medulla induced by a novel alpha2-adrenoceptor agonist, MPV-2426, following intrathecal application in neuropathic rats. AB - BACKGROUND: In the current study, the potency and spread of the antinociception induced by MPV-2426, a novel alpha2-adrenoceptor agonist, was characterized in neuropathic and non-neuropathic animals. METHODS: Neuropathy was induced by unilateral ligation of two spinal nerves in the rat. After lumbar intrathecal or systemic administration of MPV-2426, thermally and mechanically evoked responses of nociceptive neurons of the rostroventromedial medulla were recorded during pentobarbitone anesthesia. To obtain a behavioral correlate of neurophysiologic findings, nocifensor reflex responses evoked by thermal and mechanical stimuli were assessed in unanesthetized neuropathic and control animals. RESULTS: After intrathecal administration, MPV-2426 and dexmedetomidine produced a dose-related antinociceptive effect, independent of the submodality of the noxious test stimulus or the pathophysiologic condition. This antinociceptive effect was spatially restricted to the inputs from the lower half of the body, and it was reversed by atipamezole, an alpha2-adrenoceptor antagonist. After systemic administration in non-neuropathic animals, MPV-2426 had no antinociceptive effect on responses to rostroventromedial medulla neurons, whereas systemically administered dexmedetomidine produced a dose-related suppression of nociceptive signals to the rostroventromedial medulla, independent of the site of test stimulation. In a behavioral study, intrathecal MPV-2426 produced a dose dependent suppression of nocifensor responses evoked by noxious mechanical or heat stimuli, whereas systemic administration of MPV-2426 had no effects. CONCLUSIONS: Intrathecal MPV-2426 has spatially limited antinociceptive properties in neuropathic and non-neuropathic conditions because of its action on spinal alpha2-adrenoceptors. These properties may be advantageous when designing therapy for spatially restricted pain problems. PMID- 10754630 TI - Volatile anesthetics differentially affect immunostimulated expression of inducible nitric oxide synthase: role of intracellular calcium. AB - BACKGROUND: Nitric oxide released by inducible nitric oxide synthase (iNOS) plays an important role in immune responses and systemic vasodilation in septic shock. Volatile anesthetics have been reported to interfere with signal transduction and gene expression. We studied the effect of volatile anesthetics on activity and expression of iNOS and potential mechanisms of action. METHODS: Nitrite release and iNOS expression were determined using the Griess reaction and Western and Northern blot techniques, respectively, in J774 murine macrophages stimulated with lipopolysaccharide and gamma-interferon in the absence and presence of various concentrations (0.25-2.0 minimum alveolar concentration [MAC]) of volatile anesthetics (i.e., halothane, enflurane, isoflurane, desflurane). Furthermore, potential interference of volatile anesthetics with specific signal transduction pathways was investigated. RESULTS: All volatile anesthetics, studied in a time- and dose-dependent manner, suppressed nitrite production and iNOS expression in J774 macrophages stimulated by lipopolysaccharide or gamma interferon at clinically relevant concentrations. The inhibition was completely antagonized by ionomycin but unaffected by diacylglycerol, phorbol myristate acetate, and C2-ceramide. In contrast, in cells costimulated by lipopolysaccharide plus gamma-interferon, volatile anesthetics significantly increased nitrite production and iNOS expression independent of ionomycin and other mediators studied. CONCLUSIONS: Volatile anesthetics strongly reduced the mRNA and protein levels of iNOS and NOS activity after a single stimulation with lipopolysaccharide or gamma-interferon, most likely by attenuating intracellular calcium increase. Costimnulation with lipopolysaccharide plus gamma-interferon, however, results in maximum iNOS expression and activity, which are no longer inhibited but are potentiated by volatile anesthetics by unidentified mechanisms. PMID- 10754631 TI - Direct coronary vasomotor effects of sevoflurane and desflurane in in situ canine hearts. AB - BACKGROUND: An extracorporeal system was used to investigate the direct coronary vasomotor effects of sevoflurane and desflurane in vivo. The role of the adenosine triphosphate-sensitive potassium channels (KATP channels) in these effects was evaluated. METHODS: Twenty-one open-chest, anesthetized (fentanyl midazolam) dogs were studied. The left anterior descending coronary artery was perfused at controlled pressure (80 mmHg) with normal arterial blood or arterial blood equilibrated with either sevoflurane or desflurane. Series 1 (n = 16) was divided into two groups of equal size on the basis of whether sevoflurane (1.2, 2.4, and 4.8%) or desflurane (3.6, 7.2, and 14.4%) was studied. The concentrations for the anesthetics corresponded to 0.5, 1.0, and 2.0 minimum alveolar concentration (MAC), respectively. Coronary blood flow (CBF) was measured with an ultrasonic, transit-time transducer. Local coronary venous samples were obtained and used to evaluate changes in myocardial oxygen extraction (EO2). In series 2 (n = 5), changes in CBF by 1 MAC sevoflurane and desflurane were assessed before and during intracoronary infusion of the KATP channel inhibitor glibenclamide (100 microg/min). RESULTS: Intracoronary sevoflurane and desflurane caused concentration-dependent increases in CBF (and decreases in EO2) that were comparable. Glibenclamide blunted significantly the anesthetic-induced increases in CBF. CONCLUSIONS: Sevoflurane and desflurane have comparable coronary vasodilative effects in in situ canine hearts. The KATP channels play a prominent role in these effects. When compared with data obtained previously in the same model, the coronary vasodilative effects of sevoflurane and desflurane are similar to those of enflurane and halothane but considerably smaller than that of isoflurane. PMID- 10754632 TI - Comparison of volatile anesthetic effects on actin-myosin cross-bridge cycling in neonatal versus adult cardiac muscle. AB - BACKGROUND: The neonatal myocardium is more sensitive to volatile anesthetics compared with adults. The greater myocardial sensitivity of neonates may be attributable to greater anesthetic effect on force regulation at the level of the cross-bridge. In the current study, the authors compared the effects of 1 and 2 minimum alveolar concentration (MAC) halothane and sevoflurane on cardiac muscle from 0- to 3-day-old (neonate) and 84-day-old (adult) rats. METHODS: Triton X-100 skinned muscle strips were maximally activated at pCa (negative logarithm of the Ca2+ concentration) of 4.0, and the following were measured in the presence or absence of anesthetic: Rate of force redevelopment after rapid shortening and restretching (ktr) and isometric stiffness at maximal activation and in rigor. The fraction of attached cross-bridges (alphafs) and apparent rate constants for cross-bridge attachment (fapp) and detachment (gapp) were calculated assuming a two-state model for cross-bridge cycling. Anesthetic-induced changes in the mean stiffness per cross-bridge were also estimated from values in rigor versus maximum activation in the presence or absence of anesthetic. RESULTS: Neonatal cardiac muscle displayed significantly smaller alphafs slower ktr and slower fapp compared with adult cardiac muscle; however, gapp was not significantly different. Halothane, and sevoflurane to a significantly lesser extent, decreased alphafs, fapp, and the mean force per cross-bridge and increased gapp to a greater extent in neonates. CONCLUSIONS: These data indicate that weaker force production in neonatal cardiac muscle involves, at least in part, less efficient cross-bridge cycling kinetics. The authors conclude that the greater myocardial sensitivity of neonates to volatile anesthetics reflects, at least in part, a direct inhibition of cross-bridge cycling, especially the rates of cross-bridge attachment and detachment. PMID- 10754633 TI - Antiallodynic effect of intrathecal gabapentin and its interaction with clonidine in a rat model of postoperative pain. AB - BACKGROUND: Systemic administration of gabapentin was shown previously to attenuate mechanical allodynia in a rat model of postoperative pain. Because intrathecal administration of gabapentin is effective in other hypersensitivity states, the authors tested its effect in the postoperative model, its interaction with another antiallodynic agent (clonidine), and a possible mechanism of gabapentin action (entry into sites of action via an L-amino acid transporter). METHODS: Male Sprague-Dawley rats were anesthetized with halothane, and an incision of the plantaris muscle of right hind paw induced punctate mechanical allodynia. Withdrawal threshold to von Frey filament application near the incision site was determined before and 2 h after surgery. Then, an intrathecal injection was performed and thresholds were determined every 30 min for 3 h thereafter. RESULTS: Paw incision induced a mechanical hypersensitivity (mechanical threshold > 25 g before incision and < 5 g after). Intrathecal gabapentin dose-dependently (10-100 microg) reduced mechanical allodynia. Intrathecal injection of an inhibitor of L-amino acid transporters or a competitor for this transporter, L-leucine, did not reverse the intrathecal effect of gabapentin. The ED50 of intrathecal gabapentin, clonidine, and their combination were 51, 31, and 9 microg, respectively, and isobolographic analysis showed synergy between gabapentin and clonidine. CONCLUSIONS: Intrathecal gabapentin is effective against tactile allodynia that occurs after paw incision, and interacts synergistically with clonidine. Unlike results in vitro, gabapentin does not obligatorily need to enter cells via the L-amino acid transporter mechanism to achieve its effects in vivo. PMID- 10754634 TI - Mechanisms of ventricular arrhythmias induced by myocardial contusion: a high resolution mapping study in left ventricular rabbit heart. AB - BACKGROUND: The aims of the Langendorff-perfused rabbit heart study were to evaluate the arrhythmogenic consequences of myocardial contusion and to determine the mechanism of arrhythmia. METHODS: Six hearts were in the control group, and 24 hearts (intact heart protocol) were submitted to one of four different contusion kinetic energies (75, 100, 150, or 200 millijoules [mJ]; n = 6). Occurrence of arrhythmia, of an electrically silent area (i.e., area with no electrical activity), and of line of fixed conduction block were reported before and for 1 h after contusion. In 16 hearts (frozen hearts) submitted to cryoprocedure and contusion impact of 100 or 200 mJ, ventricular conduction velocities, anisotropic ratio, wavelengths, ventricular effective refractory period, and its dispersion were measured before and for 1 h after contusion. Using high-resolution mapping, arrhythmias were recorded and analyzed. RESULTS: The intact heart study showed that the number and seriousness of contusion induced arrhythmias increased with increasing contusion kinetic energy, as did the number of electrically silent areas (five of six ventricular fibrillations and five of six electrically silent areas at 200 mJ). In the frozen heart study, immediately after contusion ventricular effective refractory periods were shortened and dispersed, and wavelengths were also shortened. The arrhythmia analysis showed that all ventricular tachycardias but one were based on reentry developed around an electrically silent area or a line of fixed conduction block. CONCLUSIONS: Myocardial contusion has direct arrhythmogenic effects, and the seriousness of arrhythmia increases with the level of contusion kinetic energy. The mechanism of arrhythmia was mainly based on reentrant circuit around a fixed obstacle. PMID- 10754635 TI - Subunit-dependent inhibition of human neuronal nicotinic acetylcholine receptors and other ligand-gated ion channels by dissociative anesthetics ketamine and dizocilpine. AB - Background The neuronal mechanisms responsible for dissociative anesthesia remain controversial. N-methyl-D-aspartate (NMDA) receptors are inhibited by ketamine and related drugs at concentrations lower than those required for anesthetic effects. Thus, the authors studied whether ligand-gated ion channels other than NMDA receptors might display a sensitivity to ketamine and dizocilpine that is consistent with concentrations required for anesthesia. METHODS: Heteromeric human neuronal nicotinic acetylcholine receptors (hnAChR channels alpha2beta2, alpha2beta4, alpha3beta2, alpha3beta4, alpha4beta2 and alpha4beta4), 5 hydroxytryptamine3 (5-HT3), alpha1beta2gamma2S gamma-aminobutyric acid type A (GABAA) and alpha1 glycine receptors were expressed in Xenopus oocytes, and effects of ketamine and dizocilpine were studied using the two-electrode voltage clamp technique. RESULTS: Both ketamine and dizocilpine inhibited hnAChRs in a noncompetitive and voltage-dependent manner. Receptors containing beta1 subunits were more sensitive to ketamine and dizocilpine than those containing beta2 subunits. The inhibitor concentration for half-maximal response (IC50) values for ketamine of hnAChRs composed of beta4 subunits were 9.5-29 microM, whereas those of beta2 subunits were 50-92 microM. Conversely, 5-HT3 receptors were inhibited only by concentrations of ketamine and dizocilpine higher than the anesthetic concentrations. This inhibition was mixed (competitive/noncompetitive). GABAA and glycine receptors were very resistant to dissociative anesthetics. CONCLUSIONS: Human nAChRs are inhibited by ketamine and dizocilpine at concentrations possibly achieved in vivo during anesthesia in a subunit-dependent manner, with beta subunits being more critical than alpha subunits. Conversely, 5-HT3, GABAA, and glycine receptors were relatively insensitive to dissociative anesthetics. PMID- 10754636 TI - Blockade of adenosine triphosphate-sensitive potassium channels by thiamylal in rat ventricular myocytes. AB - BACKGROUND: The adenosine triphosphate (ATP)-sensitive potassium (KATP) channels protect myocytes during ischemia and reperfusion. This study investigated the effects of thiamylal on the activities of KATP channels in isolated rat ventricular myocytes during simulated ischemia. METHODS: Male Wistar rats were anesthetized with ether. Single, quiescent ventricular myocytes were dispersed enzymatically. Membrane currents were recorded using patch-clamp techniques. In the cell-attached configuration, KATP channel currents were assessed before and during activation of these channels by 2,4-dinitrophenol and after administration of 25, 50, and 100 mg/l thiamylal. The open probability was determined from current-amplitude histograms. In the inside-out configuration, the current voltage relation was obtained before and after the application of thiamylal (50 mg/1). RESULTS: In the cell-attached configuration, 2,4-dinitrophenol caused frequent channel opening. 2,4-Dinitrophenol-induced channel activities were reduced significantly by glibenclamide, suggesting that the channels studied were KATP channels. Open probability of KATP channels was reduced by thiamylal in a concentration-dependent manner. KATP channels could be activated in the inside out configuration because of the absence of ATP. Thiamylal inhibited KATP channel activity without changing the single-channel conductance. CONCLUSIONS: The results obtained in this study indicate that thiamylal inhibits KATP channel activities in cell-attached and inside-out patches, suggesting a direct action of this drug on these channels. PMID- 10754637 TI - Modeling the uncertainty of surgical procedure times: comparison of log-normal and normal models. AB - BACKGROUND: Medical institutions are under increased economic pressure to schedule elective surgeries efficiently to contain the costs of surgical services. Surgical scheduling is complicated by variability inherent in the duration of surgical procedures. Modeling that variability, in turn, provides a mechanism to generate accurate time estimates. Accurate time estimates are important operationally to improve operating room utilization and strategically to identify surgeons, procedures, or patients whose duration of surgeries differ from what might be expected. METHODS: The authors retrospectively studied 40,076 surgical cases (1,580 Current Procedural Terminology-anesthesia combinations, each with a case frequency of five or more) from a large teaching hospital, and attempted to determine whether the distribution of surgical procedure times more closely fit a normal or a log-normal distribution. The authors tested goodness-of fit to these data for both models using the Shapiro-Wilk test. Reasons, in practice, the Shapiro-Wilk test may reject the fit of a log-normal model when in fact it should be retained were also evaluated. RESULTS: The Shapiro-Wilk test indicates that the log-normal model is superior to the normal model for a large and diverse set of surgeries. Goodness-of-fit tests may falsely reject the log normal model during certain conditions that include rounding errors in procedure times, large sample sizes, untrimmed outliers, and heterogeneous mixed populations of surgical procedure times. CONCLUSIONS: The authors recommend use of the log-normal model for predicting surgical procedure times for Current Procedural Terminology-anesthesia combinations. The results help to legitimize the use of log transforms to normalize surgical procedure times before hypothesis testing using linear statistical models or other parametric statistical tests to investigate factors affecting the duration of surgeries. PMID- 10754638 TI - Practice advisory for the prevention of perioperative peripheral neuropathies: a report by the American Society of Anesthesiologists Task Force on Prevention of Perioperative Peripheral Neuropathies. PMID- 10754639 TI - Real-time intraoperative monitoring of myocardial ischemia in noncardiac surgery. PMID- 10754640 TI - Bilateral lower extremity compartment syndromes following prolonged surgery in the low lithotomy position with serial compression stockings. PMID- 10754641 TI - Pregnant patient with primary pulmonary hypertension: inhaled pulmonary vasodilators and epidural anesthesia for cesarean delivery. PMID- 10754642 TI - Narcolepsy and anesthesia. PMID- 10754643 TI - Failure to detect CO2-absorbent exhaustion: seeing and believing. PMID- 10754644 TI - A Hairy Situation. PMID- 10754645 TI - Difficult Mask Ventilation. PMID- 10754647 TI - Aerosolization of Lidocaine. PMID- 10754646 TI - Aseptic meningitis after spinal anesthesia in an infant. PMID- 10754648 TI - Acetaminophen dosage in pediatric practice. PMID- 10754649 TI - What anesthesiologists should know about what neurologists should know about declaring brain death. PMID- 10754650 TI - Can brain death testing by perfect? PMID- 10754651 TI - Malignant hyperthermia as a cause for postoperative rhabdomyolysis. PMID- 10754652 TI - Analgesic effects of neostigmine in the periphery. PMID- 10754654 TI - An efficient technique for tracheal intubation using the StyletScope alone. PMID- 10754653 TI - Acute pulmonary edema caused by impaired switching from nasal to oral breathing in the emergence from anesthesia. PMID- 10754655 TI - Paper "preoperative computer". PMID- 10754656 TI - Solitary metastasis of renal cell carcinoma to the contralateral adrenal gland 22 years after nephrectomy. AB - A case of solitary metastasis to the contralateral adrenal 22 years after radical nephrectomy for renal cell carcinoma (RCC) is described. This case highlights the variable behavior of RCC, the tendency for adrenal metastasis, and the potential for prolonged survival after resection of late solitary metastases. PMID- 10754657 TI - Localized testicular infarction masquerading as a testicular neoplasm. AB - Localized testicular infarction appears to represent a relatively uncommon phenomenon. We describe a patient presenting with a testicular mass simulating a neoplasm, who proved to have a localized hemorrhagic infarction. PMID- 10754658 TI - Bilateral percutaneous nephrolithotomy for multiple cystine stones in an infant presenting with anuria. AB - We report the first case of simultaneous, bilateral percutaneous management of multiple urinary cystine stones in a 7.6-kg, 9-month-old infant who presented with anuria. A stone-free state was successfully achieved. PMID- 10754659 TI - Organ-sparing surgery for bilateral leydig cell tumor of the testis. AB - Leydig cell tumors of the testis are uncommon, and bilateral lesions are extremely rare. We report a case of bilateral Leydig cell tumor of the testis treated with radical orchiectomy and contralateral subtotal orchiectomy with the intent of preservation of hormonal function and fertility. PMID- 10754660 TI - Release of TNFalpha in response to SiC fibres: differential effects in rodent and human primary macrophages, and in macrophage-like cell lines. AB - Asbestos has been implicated in the pathogenesis of several lung diseases, but its mechanism of action is not fully understood. However, asbestos-induced oxidative stress and production of inflammatory cytokines may play a significant role. TNFalpha is an inflammatory cytokine which has a central role in inflammation and fibrosis due to its ability to stimulate fibroblasts and collagen deposition. In this study, a panel of fibres designated either pathogenic or non-pathogenic in recent animal studies, were utilized. The amount of TNFalpha released after a 16-hour exposure to the panel of fibres was compared in four different cell types; two primary macrophage cell types and two cell lines. TNFalpha release by cells exposed to the panel did not equate to pathogenicity, although the most pathogenic fibre caused three out of the four cell types tested, to produce the greatest amount of TNFalpha. Primary rat cells and primary human cells behaved in a similar manner as regards to TNFalpha production; the cell lines behaved quite differently to their primary counterparts with regards to TNFalpha production in this study. PMID- 10754661 TI - Frequent polymorphism of the human methylenetetrahydrofolate reductase. PMID- 10754662 TI - 48th Annual meeting of the Pacific Coast Reproductive Society. Rancho Mirago, California, USA. April 26-30, 2000. Abstracts. PMID- 10754663 TI - Concerned about control of coyotes and wolves. PMID- 10754665 TI - Veterinarians as bailees. PMID- 10754664 TI - What is your diagnosis? Kidney and ureteral injuries and subsequent adhesions and peritonitis in a dog hit by a car. PMID- 10754666 TI - Effects of insoluble and soluble dietary fiber on glycemic control in dogs with naturally occurring insulin-dependent diabetes mellitus. AB - OBJECTIVE: To evaluate the effects of diets differing in type and quantity of fiber on glycemic control in dogs with naturally occurring insulin-dependent diabetes mellitus. DESIGN: Prospective randomized crossover controlled trial. ANIMALS: 7 dogs with well-regulated naturally occurring insulin-dependent diabetes mellitus. PROCEDURE: Dogs were fed 1 of 3 diets for 1 month each in 1 of 6 randomized diet sequences. Diets included a low-fiber diet (LF) and 2 high fiber diets; 1 contained only insoluble fiber (HIF), and 1 contained soluble fiber in addition to insoluble fiber (HSF). Caloric intake was unchanged throughout the study. Glycemic control was assessed after each feeding trial by measuring serum fructosamine concentration and performing 5 serial measurements of blood glucose concentration every 2 hours after the morning feeding and insulin injection. RESULTS: Significant differences were not detected in body weight, required insulin dosage, or albumin concentration among dogs fed the HIF, HSF, and LF diets. Mean and maximum blood glucose concentrations and area under the blood glucose curve were significantly lower in dogs fed the HIF diet, compared with values in the same dogs fed the HSF or LF diet. Fructosamine concentration was significantly lower in dogs fed the HIF or HSF diet, compared with values in the same dogs fed the LF diet. CONCLUSIONS AND CLINICAL RELEVANCE: In dogs with naturally occurring insulin-dependent diabetes mellitus, a dry, high insoluble-fiber diet may aid in glycemic control. PMID- 10754667 TI - Effect of dietary insoluble fiber on control of glycemia in cats with naturally acquired diabetes mellitus. AB - OBJECTIVE: To evaluate effects of dietary insoluble fiber on control of glycemia in cats with naturally acquired diabetes mellitus. DESIGN: Randomized controlled crossover trial. ANIMALS: 16 cats with naturally acquired diabetes mellitus. PROCEDURE: Cats were fed a diet high in insoluble fiber (HF) containing 12% cellulose (dry-matter basis) or a diet low in insoluble fiber (LF) for 24 weeks; they were fed the other diet for the subsequent 24 weeks. Caloric intake and insulin treatment were adjusted to maintain stable body weight and control of glycemia, respectively. Cats were allowed an adaptation period of 6 weeks after initiation of a diet, after which control of glycemia was evaluated at 6-week intervals for 18 weeks. Variables assessed included serum glucose concentration measured during the preprandial state, blood glycated hemoglobin concentration, serum glucose concentration measured at 2-hour intervals for 12 hours beginning at the time of the morning insulin injection, 12-hour mean serum glucose concentration, and mean fluctuation in serum glucose concentration from the 12 hour mean serum glucose concentration. RESULTS: Mean daily caloric intake, body weight, or daily insulin dosage did not differ significantly between cats when fed HF and LF diets. Mean preprandial serum glucose concentration, most post prandial serum glucose concentrations, and the 12-hour mean serum glucose concentration were significantly lower when cats consumed the HF diet, compared with values when cats consumed the LF diet. CONCLUSIONS AND CLINICAL RELEVANCE: These results support feeding a commercially available diet containing approximately 12% insoluble fiber (dry-matter basis) to cats with naturally acquired diabetes mellitus. PMID- 10754668 TI - Effect of weight reduction on clinical signs of lameness in dogs with hip osteoarthritis. AB - OBJECTIVE: To determine the effect of weight reduction on clinical signs of lameness among overweight dogs with clinical and radiographic signs of hip osteoarthritis. DESIGN: Nonblinded prospective clinical trial. ANIMALS: 9 client owned dogs with radiographic signs of hip osteoarthritis that weighed 11 to 12% greater than their ideal body weight and were examined because of hind limb lameness. PROCEDURE: Dogs were weighed, and baseline body condition, hind limb lameness, and hip function scores were assigned. Severity of lameness was scored using a numerical rating scale and a visual analogue scale. Dogs were fed a restricted-calorie diet, with amount of diet fed calculated to provide 60% of the calories needed to maintain the dogs' current weights. Evaluations were repeated midway through and at the end of the weight-loss period. RESULTS: Dogs lost between 11 and 18% of initial body weight. Body weight, body condition score, and severity of hind limb lameness were all significantly decreased at the end of the weight-loss period. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that in overweight dogs with hind limb lameness secondary to hip osteoarthritis, weight reduction alone may result in a substantial improvement in clinical lameness. PMID- 10754669 TI - Modified perineal urethrostomy using preputial mucosa in cats. AB - A modified perineal urethrostomy was performed in 14 males cats with partial or complete urethral obstruction. Follow-up information was available for 2 to 18 months. By use of this method, the preputial tissues were preserved, and the penile urethra was anastomosed to the preputial mucosa to enlarge and lengthen the urethra. Urine flow was reestablished in all cats without evidence of urethral stenosis or other major complications. Appearance of the perineal region and prepuce was not substantially altered. PMID- 10754670 TI - Subcutaneous emphysema, pneumoperitoneum, and pneumoretroperitoneum after gastrostomy tube placement in a cat. AB - A 17-year-old spayed female domestic shorthair cat developed subcutaneous emphysema, pneumoperitoneum, and pneumoretroperitoneum during endoscopic placement of a gastrostomy feeding tube after gastric insufflation and cannula insertion. The cat underwent exploratory laparotomy to investigate the possibility of gastric rupture but only a 2- to 3-mm defect was found in the gastric fundus at the site of cannula insertion. Pasteurella multocida and Enterobacter spp were cultured aerobically from the peritoneal cavity. The cat recovered without complications. PMID- 10754671 TI - Laryngeal paralysis in cats: 16 cases (1990-1999). AB - OBJECTIVE: To determine clinical signs, physical examination findings, radiographic features, and concurrent diseases in cats with laryngeal paralysis, as well as evaluate the outcome of medical or surgical management. DESIGN: Retrospective study. ANIMALS: 16 cats. PROCEDURE: Medical records from January 1990 to April 1999 were examined for cats with laryngeal paralysis. Signalment, clinical signs, physical examination findings, cervical and thoracic radiographic findings, laryngeal examination results, and clinical outcome were reviewed. RESULTS: No breed or sex predilection was identified in 16 cats with laryngeal paralysis. The most common clinical signs included tachypnea or dyspnea, dysphagia, weight loss, change in vocalization, coughing, and lethargy. Clinical signs were evident for a median of 245 days. Airway obstruction was apparent on cervical and thoracic radiographic views in 9 cats. Examination of the larynx revealed bilateral laryngeal paralysis in 12 cats and unilateral laryngeal paralysis in 4 cats. The 4 cats with unilateral disease were managed with medical treatment, and 3 of these had acceptable long-term outcomes. Seven of 12 cats with bilateral paralysis underwent surgery; procedures performed included left arytenoid tie back, bilateral arytenoid tie back and ventriculo-cordectomy, and partial left arytenoidectomy. One cat was euthanatized as a result of complications from surgery. CONCLUSIONS AND CLINICAL RELEVANCE: Laryngeal paralysis is an uncommon cause of airway obstruction in cats. Cats with less severe clinical signs (often with unilateral paralysis) may be successfully managed with medical treatment, whereas cats with severe airway obstruction (often with bilateral paralysis) may benefit from surgical intervention. PMID- 10754672 TI - Risk factors affecting the outcome of surgery for atlantoaxial subluxation in dogs: 46 cases (1978-1998). AB - OBJECTIVE: To identify risk factors for successful surgical management of dogs with atlantoaxial subluxation (AAS). DESIGN: Retrospective study. ANIMALS: 46 dogs managed surgically for AAS. PROCEDURE: Age at onset of clinical abnormalities, duration of clinical abnormalities prior to surgery, radiographic appearance of the dens, type (dorsal or ventral procedure) and number (1 or 2) of surgeries performed, grade of postoperative atlantoaxial joint reduction, and neurologic status prior to surgery (preoperative), when dogs were discharged from the hospital (postoperative), and during a follow-up evaluation (final) were obtained from the dogs' medical records. Risk factors for surgical success and degree of neurologic improvement were identified and analyzed for predictive potential. RESULTS: Age at onset of clinical abnormalities < or = 24 months was significantly associated with greater odds of a successful first surgery and final outcome and a lower postoperative neurologic grade. Duration of clinical abnormalities < or = 10 months was significantly associated with greater odds of a successful final outcome and a lower final neurologic grade. A preoperative neurologic grade of 1 or 2 was significantly associated with a lower final neurologic grade. Potential risk factors that did not affect odds of a successful outcome included type of surgery performed, grade of atlantoaxial joint reduction, radiographic appearance of the dens, or need for a second surgery. CONCLUSIONS AND CLINICAL RELEVANCE: Age at onset of clinical abnormalities, duration of clinical abnormalities prior to surgery, and preoperative neurologic status are risk factors for success of surgical management of AAS in dogs. PMID- 10754673 TI - Clinical usefulness of cardiac event recording in dogs and cats examined because of syncope, episodic collapse, or intermittent weakness: 60 cases (1997-1999). AB - OBJECTIVE: To determine the clinical usefulness of cardiac event recording in evaluating dogs and cats with unexplained syncope, episodic collapse, or intermittent weakness. DESIGN: Retrospective study. ANIMALS: 58 dogs and 2 cats. PROCEDURE: Medical records and electrocardiographic rhythm strips obtained by cardiac event recordings were reviewed. Cardiac rhythm data from the event recordings were classified as diagnostic or nondiagnostic. Diagnostic yield was calculated by dividing the number of animals for which cardiac event recording was diagnostic by the total number of animals undergoing cardiac event recording. RESULTS: For 51 animals, cardiac event recording was classified as diagnostic; therefore, overall diagnostic yield was 85%. Diagnostic yield was lower for animals without underlying structural heart disease (75.5%) than for animals with structural heart disease (95.6%). A specific arrhythmia was identified as the cause of clinical signs in 18 of the 51 (35%) animals for which cardiac event recording was diagnostic. Cardiac arrhythmia was definitively excluded as the cause of clinical signs in the remaining 33 (65%) animals in which cardiac event recording was diagnostic. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that cardiac event recording had a high diagnostic yield in dogs and cats examined because of unexplained syncope, episodic collapse, or transient weakness and ataxia, regardless of whether animals did or did not have an underlying structural heart disease. Diagnostic yield of cardiac event recording was higher than that reported previously for Holter monitoring. PMID- 10754674 TI - Hypercalcemia and hypervitaminosis D in two lambs. AB - Twin 17-day-old crossbred male lambs were examined to determine the cause of weakness and failure to thrive. Hypercalcemia attributable to hypervitaminosis D was diagnosed. The milk replacer or an accidental overdose of an injectable vitamin D product was suspected to be the source, although a definite cause was not confirmed. Lambs responded favorably to palliative treatment (administration of saline [0.9% NaCl] solution to induce calcium diuresis) and changing the diet to another milk replacer. PMID- 10754675 TI - Oxidative reactivity of S-nitrosoglutathione with Hantzsch 1,4-dihydropyridine. AB - [formula: see text] S-Nitrosoglutathione oxidized 4-substituted Hantzsch 1,4 dihydropyridines in CH3CN/H2O or CH3CN/phosphate aqueous buffer solution to give aromatic products in various yields. PMID- 10754676 TI - (S)- or (R)-3-(E-enoyl)-4-phenyl-1,3-oxazolidin-2-ones: ideal Michael acceptors to afford a virtually complete control of simple and face diastereoselectivity in addition reactions with glycine derivatives. AB - [formula: see text] Enantiomerically pure (S)- or (R)-3-(E-enoyl)-4-phenyl-1,3 oxazolidin-2-ones were found to serve as ideal Michael acceptors in addition reactions with achiral Ni(II) complexes of glycine Schiff bases. Virtually complete control of simple and face diastereoselectivity, observed in these reactions, combined with quantitative chemical yields renders this methodology synthetically superior to the previous methods. PMID- 10754677 TI - Ready access to sialylated oligosaccharide donors. AB - [formula: see text] Numerous glycoconjugates contain the disaccharide Neu5Ac alpha (2-->3)DGalp. An efficient way to incorporate this disaccharide into synthetic glycoconjugates is to develop a disaccharide building block. This communication reports a chemoenzymatic route to such a building block which requires as few as four steps. Some examples using more chemical steps are also presented, which increase the flexibility. These disaccharide donors were used to prepare synthetic trisaccharides. PMID- 10754678 TI - Studies toward (-)-gymnodimine: concise routes to the spirocyclic and tetrahydrofuran moieties. AB - [formula: see text] (-)-Gymnodimine is a member of a unique class of potent marine toxins possessing imines within a spirocylic array. Herein we report the synthesis of the tetrahydrofuran fragment and a strategy toward the spirocyclic imine fragment of this family of toxins. Key reactions include an asymmetric anti aldol reaction to set the stereochemistry of the tetrahydrofuran and a formal, intermolecular Diels-Alder reaction involving an alpha-methylene-delta-lactam and a dienyne. PMID- 10754679 TI - Cryptocin, a potent tetramic acid antimycotic from the endophytic fungus Cryptosporiopsis cf. quercina. AB - [formula: see text] The endophytic fungus Cryptosporiopsis cf. quercina produces cryptocin in culture. Among other fungi, this unique tetramic acid displays antimycotic activity against Pyricularia oryzae, the causal agent of rice blast disease. Cryptocin also possesses activity against a wide variety of plant pathogenic but not human pathogenic fungi. The fine rhomboid-like crystals of cryptocin allowed structural elucidation by X-ray crystallography. The importance of cryptocin to the symbiotic relationship of C. quercina to its hosts is briefly discussed. PMID- 10754680 TI - Oligomeric building block approach to the synthesis of diastereomerically pure pentathymidine 3',5'-methanephosphonates. AB - [formula: see text] A method for a large-scale synthesis of stereodefined oligo(nucleoside 3',5'-methanephosphonates) has been developed, based on transient 3'-O protection, which allows for the conversion of the protecting chirally defined methanephosphonanilidate group, located at the 3' end of a stereoregular oligomer, into diastereomerically pure "oligomeric building blocks" for stereospecific coupling with the 5'-OH group of another oligonucleotide. PMID- 10754681 TI - Titanium enolates of thiazolidinethione chiral auxiliaries: versatile tools for asymmetric aldol additions. AB - [formula: see text] Asymmetric aldol additions using chlorotitanium enolates of thiazolidinethione propionates proceed with high diastereoselectivity for the "Evans" or "non-Evans" syn product depending on the nature and amount of the base used. With (-)-sparteine as the base, selectivities of 97:3 to > 99:1 were obtained for the Evans syn products with 2 equivalents of base and for the non Evans syn when 1 equiv of base was employed. The thiazolidinethione auxiliaries are easily removed, and the aldol adducts can be readily transformed to various functional groups. Even direct reduction to the aldehyde with diisobutylaluminum hydride is possible. PMID- 10754682 TI - A de novo synthesis of 2,6-dideoxy-C-aryl glycosides based on ring closing metathesis and diastereoselective epoxide cleavage/anomerization reactions. AB - [formula: see text] This paper describes a synthesis of enantiomerically pure 2,6 dideoxy-C-aryl glycosides, starting from non-carbohydrate precursors. The synthesis starts from homoallylic alcohols (obtained in enantiomerically pure form by enzymatic resolution), which are elaborated to dihydropyrans using ring closing metathesis as the key step. Epoxidation and epoxide cleavage complete the synthesis. The stereochemical outcome of the sequence depends on the conditions of the epoxide cleavage reaction. PMID- 10754683 TI - Understanding enediyne-protein interactions: diyl atom transfer results in generation of aminoacyl radicals. AB - [formula: see text] The origin of the protein modulating capacity of enediynes has been probed. A series of synthetic enediyne-derived diyls participated in atom transfer chemistry with a labeled amino acid. Subsequent experiments suggest that diyl radicals may modulate protein architecture via formation of captodatively stabilized radicals. PMID- 10754684 TI - Synthesis of 7-substituted tetracycline derivatives. AB - [formula: see text] The synthesis of 7-substituted tetracycline derivatives has been accomplished in high yield from 7-halotetracyclines by modified Suzuki and Stille coupling protocols. These novel derivatives may serve as a new class of tetracycline antibiotics effective against multi-antibiotic-resistant bacteria. PMID- 10754685 TI - Investigation of phenolic bioisosterism in opiates: 3-sulfonamido analogues of naltrexone and oxymorphone. AB - [formula: see text] The phenolic hydroxy group of opiate-derived ligands is of known importance for biological activity. On the basis of its putative role as a hydrogen-bonding donor in the interaction with opioid receptors, it was replaced with a sulfonamide group because of their similar pKa values. The first thebaine derived 3-amino (8a, 8b) and subsequent sulfonamide analogues (10a, 10b) were synthesized from naltrexone (1a) and oxymorphone (1b) in a linear nine-step synthesis. The sulfonamides were tested in vitro and found inactive. PMID- 10754686 TI - Stereoselectivity of methyl aryldiazoacetate cyclopropanations of 1,1 diarylethylene. Asymmetric synthesis of a cyclopropyl analogue of tamoxifen. AB - [formula: see text] Dirhodium tetrakis(S-(N-dodecylbenzenesulfonyl)prolinate) (Rh2(S-DOSP)4)-catalyzed decomposition of methyl phenyldiazoacetate in the presence of 1,1-diarylethylenes results in intermolecular cyclopropanation with high enantioselectivity (up to 99% ee) and moderate diastereoselectivity (up to 80% de). The reaction was applied to the asymmetric synthesis of a cyclopropyl analogue of tamoxifen. PMID- 10754687 TI - Addition of metallo enolates to chiral 1-acylpyridinium salts: total synthesis of (+)-cannabisativine. AB - [formula: see text] A novel route to the first asymmetric synthesis of (+) cannabisativine (1) is described. The total synthesis of 1 was accomplished with a high degree of regio- and stereoselectivity in 19 steps and 9% overall yield. PMID- 10754688 TI - Total synthesis of mycalamide A and 7-epi-mycalamide A. AB - [formula: see text] The final stages of a total synthesis of mycalamide A are described. A key step is the aldol reaction (mismatched) of imide 4 and aldehyde 5 which provided a ca. 5:4 mixture of aldols 10a and 10b, with incorrect C(7) stereochemistry. Elaboration of the 10a-10b mixture to mycalamide A required epimerization of C(7) at the stage of beta-keto imide 11. Alternatively, Swern oxidation of the 10a-10b mixture under conditions that minimize C(7) epimerization led to 7-epi-mycalamide A selectively. PMID- 10754689 TI - Sharpless asymmetric dihydroxylation of 5-aryl-2-vinylfurans: application to the synthesis of the spiroketal moiety of papulacandin D. AB - [formula: see text] Using the Sharpless catalytic asymmetric dihydroxylation reaction on 5-aryl-2-vinylfurans, diols are produced in high enantioexcess. The resulting diols can be efficiently transformed into the spiroketal ring precursor of the antifungal compound papulacandin D. Stereoselective reduction of this precursor followed by a diastereoselective dihydroxylation completes the synthesis of a mannopyranoside isomer of a papulacandin derivative. PMID- 10754690 TI - Enantioselective catalytic addition of HCN to ketoimines. Catalytic synthesis of quaternary amino acids. AB - [formula: see text] Highly enantioselective addition of HCN to ketoimines has been achieved for the first time using readily accessible and recyclable Schiff base catalysts. Essentially quantitative isolated yield and enantioselectivity of up to 95% ee was obtained. Furthermore, some of the Strecker adducts could be recrystallized in high recovery, yielding optically pure materials. Conversion of the alpha-aminonitrile adducts to the corresponding alpha-quaternary alpha-amino acids was effected in high yield by a formylation/hydrolysis sequence. PMID- 10754691 TI - Considerations for diffusive airflow integrity testing. PMID- 10754692 TI - Validation of a microwave sterilizer for injection ampules. AB - A program for validating the microwave sterilizer, which is a new type of sterilizer, was established and implemented. This program includes the following tests specific to microwave sterilizers: the internal pressure strength of ampules, the acceptable range of sterilizing temperature, performance and calibration of the infrared thermometer and the plane blackbody, maintaining of sterilizing temperature, the processing speed (sterilizing time), the unacceptable ampules selection mechanism, microwave leaks, and a microbiological challenge test of the ampule head space using a biological indicator. Injectable preparations of mecobalamin and diprophylline were used as models for validation of the microwave sterilizer. The bioburden approach was used for injectable mecobalamin preparation, and the over-kill approach was used for injectable diprophylline preparation. The basis for practical use of this microwave sterilizer has been confirmed by the establishment and implementation of this validation program. PMID- 10754693 TI - Evaluation of the ChemScan system for rapid microbiological analysis of pharmaceutical water. AB - The ChemScan system is a new method for the rapid detection and enumeration of viable microbial cells. It is based on the fluorescent labeling of viable microorganisms collected on a filter membrane and their subsequent automated detection and enumeration by a laser-scanning instrument. The new method was evaluated for the testing of pharmaceutical water by comparison with the standard plate count method. The ChemScan system appeared to be at least as sensitive as the standard method. In some cases the results were equivalent for both methods, but for most water samples the ChemScan results were higher than the standard plate count and sometimes exceeded the latter by an order of magnitude or more. PMID- 10754694 TI - Application of bacteriophages as surrogates for mammalian viruses: a case for use in filter validation based on precedents and current practices in medical and environmental virology. AB - Infectivity-based assays are the assays of choice for the detection of pathogenic mammalian viruses. While it is intuitively appropriate to conduct testing and validation studies with the known viral burden or a closely related mammalian species, logistic considerations often dictate otherwise. Consequently, bacteriophages have served as suitable surrogates for mammalian viruses in both medical and environmental virology applications. The wide range of bacteriophages available offers a powerful analytical tool amenable to several different applications: filter validation studies (where removal is based on size exclusion), investigations into virus contamination control issues, evaluation of barrier materials, etc. There is a considerable body of evidence to suggest and support the use of bacteriophages as surrogates for mammalian viruses. Use of appropriately sized bacteriophages provides an innocuous, efficacious and expeditious method for economical testing and validation of viral clearance capabilities of virus removal filters, thus facilitating performance of filter validation studies in biopharmaceuticals under product- and process-specific conditions in an overall effort towards ensuring the virological safety of biologicals. This paper discusses the limitations associated with mammalian virus assays and provides a rationale for the use of bacteriophages as surrogates for mammalian viruses. Data from published literature documenting applicability of bacteriophages in filter validation studies, especially when removal is based on size exclusion, is reviewed along with examples of studies from the fields of medical and environmental virology. PMID- 10754695 TI - The application of response surface methodology to sterilization process development and validation of a water cascade autoclave. AB - The application of Response Surface Methodology (RSM) to the performance validation of a water cascade sterilizer is described. The methodology was successfully used to 1) identify the cold and hot zones within maximum load configurations through three dimensional thermal mapping, 2) demonstrate the consistency of consecutive sterilization cycles in companion sterilizers, and 3) set target sterilizing values (F0) to achieve a high statistical assurance that any location will be above a minimum F0 (sterility assurance) and below a maximum F0 (for stability considerations). PMID- 10754696 TI - Training commentary. PMID- 10754697 TI - A globally harmonized glossary of terms for communicating computer validation key practices. AB - In the pharmaceutical, medical device, biotech, and blood bank industries, a computer validation vocabulary has emerged over time from many different sources. These sources include industry group publications, regulatory agency guidance documents, terminology from the computer industry, and articles written by individuals. Since these efforts were not harmonized, several independent glossaries exist today. Because these glossaries sometimes differ or contradict each other, terminology has proven to be a source of confusion. This article establishes a common global, harmonized glossary of terms required to understand and communicate computer validation key practices. PMID- 10754698 TI - The effect of validation on non-destructive particle inspection. PMID- 10754699 TI - Aseptic process monitoring--a better strategy. Validation Group, Inc. PMID- 10754701 TI - Ozone. PMID- 10754700 TI - Elements for a successful disinfection program in the pharmaceutical environment. PMID- 10754702 TI - Improved determination of organic volatile impurities in pharmaceutical materials by (USP-467) using automated static headspace GC/MS. PMID- 10754703 TI - Evaluation of intravenous flavopiridol formulations. AB - Flavopiridol is currently under consideration as a parenteral treatment of breast tumors. The solubility of this poorly water-soluble compound can be increased by a number of methods. However, because of its low water solubility, precipitation of the solubilized drug upon injection can be a problem. The potential of several flavopiridol formulations to precipitate is evaluated using a static serial dilution technique. It is shown that formulations containing up to 10 mg/mL of flavopiridol which produce negligible precipitation on dilution can be developed. PMID- 10754704 TI - PDA comments: PIC draft recommendations on the inspection of isolator technology. Parenteral Drug Association. PMID- 10754705 TI - The 1941 sulfathiazole disaster and the birth of good manufacturing practices. AB - The beginning of modern standards for good manufacturing practices can be traced to an incident that began in December 1940, when the Winthrop Chemical Company of New York put on the market sulfathiazole tablets contaminated with phenobarbital. Hundreds of deaths and injuries resulted. FDA's investigation into Winthrop's sulfathiazole production and the agency's efforts to retrieve the Winthrop drug remaining on the market revealed numerous control deficiencies in the plant and serious irregularities in the firm's attempt to recall the tainted tablets. The incident prompted FDA to require detailed controls in sulfathiazole production at Winthrop and throughout the industry, an approach that became the basis for production control standards for all pharmaceuticals. PMID- 10754706 TI - Who is the beneficiary? PMID- 10754708 TI - A comprehensive approach to compliance training in a pharmaceutical manufacturing facility. AB - A healthcare manufacturer seeking to ensure that dollars invested in training return value in performance improvements will want to consider each of these elements and develop a written training plan. Companies who have created a training plan to meet their business objectives are already reaping benefits of reduced turnover and increased productivity. PMID- 10754707 TI - Contributing factors to variability in biological indicator performance data. AB - In the past few years, there has been a change in emphasis by regulatory and compendial requirements to require that pharmaceutical manufacturers verify the accuracy of the thermal death time (D-value) and organism control counts for biological indicators used. Although the requirements were presented in draft form through compendial documents, only when the documents were officially issued, did many pharmaceutical manufacturers first initiate verification procedures. When implementing these procedures, numerous discrepancies were found between the labeled values and the verification values. This paper presents concepts on how these discrepancies may be resolved, as well as, how to prevent them in future verification studies. PMID- 10754709 TI - A novel self emulsifying parenteral drug delivery system. AB - The application of three polyhydroxy alcohols for improving parenteral emulsion formulations was investigated. A mixture of lecithin, as the primary emulsifier, and Span 20 as the secondary emulsifier, was used as the emulsifier system. The polyhydroxy alcohols selected were glycerol, propylene glycol and sorbitol. Soybean oil-in-water emulsions were prepared with the addition of increasing concentrations of each polyhydroxy alcohol. It was found that anhydrous mixtures of oil, surfactants and 30% or higher concentration of glycerol formed self emulsifying isotropic liquids, suitable for preparing Parenteral Self Emulsifying Drug Delivery Systems (PSEDDS). Spontaneous emulsification to submicron particle size of 0.4 micron occurred when these isotropic liquids were gently mixed with water. A PSEDDS formulation, containing 0.5% lidocaine, as the model drug showed similar spontaneous emulsification with particle size of 0.39 micron. Formulations containing propylene glycol, or sorbitol or lower concentrations of glycerol did not form self emulsifying mixtures. There were substantial differences in the particle size reduction pattern with each polyhydroxy alcohol. Glycerol was most effective, with minimum particle size obtained at 30% concentration. Addition of propylene glycol resulted in minimum particle size at 60% concentration. But there was increase in particle size at higher concentrations. Sorbitol was not very effective in reducing particle size. Alteration of the surfactant phase distribution at the interface was found to be the primary effect of polyhydroxy alcohols. PMID- 10754710 TI - Interaction of disinfectant residues on cleanroom substrates. AB - This study will determine the levels of disinfectant residues on stainless steel surfaces after simulated manual cleaning activities. Additionally, this study will determine if chemical interactions between different chemical agents, representative of commonly used cleanroom disinfectant technologies, subsequently applied to the same surfaces exist, and to what degree these interactions impact sporicidal performance of an oxidizing biocide against Bacillus subtilis. PMID- 10754711 TI - Determination of amylose/particulate relationship in hydroxyethylstarch. AB - Visual particulate was observed to form in parenteral hydroxyethylstarch over 24 months during stability studies. The particulate was identified as being amylose using FT-IR microscopy. Amylose values were measured in the bulk drug using the starch-iodine reaction. The amylose numbers measured in the bulk drug were found to correlate well with the measured particulate values in the parenteral formulation. PMID- 10754712 TI - Application of membrane filtration for removal of diminutive bioburden organisms in pharmaceutical products and processes. AB - In this report, we present results of a recent investigation in our laboratories demonstrated the effect of process conditions and/or drug product composition on the ability of 0.2 micron and 0.22 micron sterilizing grade filters to fully retain Ralstonia (formerly Burkholderia, formerly Pseudomonas) pickettii. R. pickettii is a opportunistic pathogen widely distributed in nature as well as clinical specimens and there have been several reports of nosocomial infections due to intrinsic manufacture-related R. pickettii contamination in filter sterilized parenteral fluids. This study documents the penetration of 0.2 micron nylon 66 and 0.22 micron modified PVDF sterilizing grade filters by R. pickettii (grown and challenged) in a drug solution under conditions that simulated a pharmaceutical filling operation. Penetration was not observed for every filter disc tested, and this may be explained, in part, by the stochastic nature (i.e., governed by the rules of probability) of the retention mechanisms involved. Scanning electron microscopy revealed significant changes in the microorganism's size and morphology as a result of exposure to the drug solution; these changes are consistent with those reported for bacteria subjected to nutrient deprivation. The SEM analyses of R. pickettii challenge suspensions in the drug solution showed that the average cell length decreased from 1.25 +/- 0.27 microns to 0.84 +/- 0.17 micron between zero and 24 hours. In addition, significant changes were observed in the size (length) distributions, with approximately 35% of the cells at 24 hours being smaller than any cell observed at the start of the challenge. These data suggest that the significant reduction in bioburden size and morphology that occurred as a result of exposure to the drug solution may play a role in the reduced ability of the 0.2 micron and 0.22 micron filters tested in this study to retain these organisms. Under the same test conditions where penetration of 0.2/0.22 micron filters was observed, 0.1 micron rated membrane filters qualified with both B. diminuta and Acholeplasma laidlawii mycoplasma consistently provided sterile effluent. Bacterial penetration of 0.2 (or 0.22) micron sterilizing grade filters was not observed under identical test conditions with either R. pickettii in a standardized solution (saline lactose broth) routinely used in challenge testing filters, or with the standard test organism, B. diminuta, in the drug solution. This study thus supports the renewed emphasis on both product- and process specific validation as well as routine bioburden monitoring expressed by regulatory agencies, and the use of enhanced bacterial removal efficiency 0.1 micron rated filters to provide enhanced sterility assurance in pharmaceutical processes. PMID- 10754713 TI - Evaluation of current compendial physiochemical test procedures for pharmaceutical elastomeric closures and development of an improved HPLC procedure. AB - The interaction between elastomeric container closures and the solutions they confine presents a potential hazard to the consumer due to extraction of closure ingredients into the dosage form. Each of the major Pharmacopeias, the United States, the European, and the Japanese, prescribe testing procedures for elastomeric closures. These consist of a series of non-specific wet chemical analyses performed on samples extracted into water or, in some cases, isopropanol (IPA) or the drug product vehicle. No consideration is given to the extracting potential of the drug product. Results from our testing on ten randomly selected closure samples indicated that these tests are not sensitive or specific enough to accurately measure the levels of extractables. Therefore, an HPLC gradient method was developed which had the required sensitivity and specificity. The prescribed compendial extractions, when performed on the various stopper types, proved inefficient and experiments were conducted in an attempt to improve them. These included increasing the time of the extractions, increasing the closure surface area, and increasing the strength of the extracting solvent (methylene chloride). The HPLC gradient method and the compendial wet chemical tests were then used to evaluate the stopper extractables. Results of the compendial analyses on the prescribed aqueous extractions were inconclusive as the number and relative amount of extractables in the closure could not be measured. The results of the compendial testing were only marginally improved using the stronger extraction conditions. Testing was dramatically improved, however, using the HPLC gradient method. As many as twenty extractables were detected in some of the samples and, unlike the compendial analysis, low level extractables were detected in the water samples. Identification of some of the extractables was accomplished via GC/MS. PMID- 10754714 TI - Pharmaceutical container/closure integrity. IV: Development of an indirect correlation between vacuum decay leak measurement and microbial ingress. AB - The rational development of a physical test method to evaluate the microbial barrier properties of sterile containers necessitates its correlation to microbiological exclusion. This can be accomplished by direct or indirect correlation. In the former, the proposed physical test is directly compared to microbial challenges using appropriate test units that stimulate container leaks at both high and low probabilities of microbial ingress. Previous work has demonstrated the development of a direct correlation using helium leak rate methods and microbial immersion challenges. An indirect correlation can be established by comparing the proposed physical method to well-defined leakage standards that represent various known levels of microbial ingress. Thus the quality assurance properties of a physical test method can be established by comparison to another physical test method that has been previously characterized. This approach has the distinct advantages of being faster, quantitatively rigorous, and less subject to the vicissitudes, of microbial testing. This approach was demonstrated by comparing the helium leak rate method to vacuum decay testing. Additionally it was demonstrated that vacuum decay testing was a fast and reproducible method for detecting leaks of about 1 to 2 mm. Leaks were simulated by affixing micropipettes into glass vials. PMID- 10754715 TI - PDA Technical Report No. 30: parametric release of pharmaceuticals terminally sterilized by moist heat. PDA Parametric Release Task Force. PMID- 10754716 TI - Baxa MicroMacrocompounder for parenteral nutrition solutions in a pediatric hospital. AB - The constant increase of parenteral nutrition (PN) manufacturing in our pediatric hospital led us to look for an automatic filling system. The aim of this study was to evaluate the MicroMacrocompounder (MM23) for compounding pediatric PN solutions. MM23 volumetric accuracy was tested with its different inlets (S, D, V) for volumes of sterile water for injection from 0.2 to 2900 mL. The influence of the solution viscosity and the source solution bottle replacements during the filling operation was also investigated. Manufacturing pediatric PN solutions was eventually assessed. Time to set up the system was 30 minutes. Maximum filling speeds with sterile water for injection were 860, 330, 154 mL/min for Vx, V and D inlets, respectively. Inlet S was not tested for MM23 filling speed. Minimal flush volume of 40 mL of sterile water for injection is necessary to clear the tube of residual ions. Average MM23 volumetric accuracy was < 5% for volumes > or = 0.5 mL for S and D inlets, and for volumes > or = 20 mL for V inlet. The volumetric accuracy was equal to 6.25% for 0.2 mL. In all experiments, volumetric accuracy was < 5%. The accuracy of electrolyte measurements performed on bag samples was less than 5% for 150 (74%) samples, between 5 and 10% for 27 (13%) samples and greater than 10% for 27 (13%) samples. Microbiological analysis showed no positive culture. The average manufacturing times were 56.8 +/- 4.5, 188.2 +/- 7.7 and 447.2 +/- 13.8 seconds for 130, 660 and 1800 mL bags, respectively. The MM23 compounder is suitable for compounding pediatric admixtures with source solutions volumes > or = 0.5 mL. This system has been used daily for five months in our department. PMID- 10754717 TI - Sterilization of API manufacturing equipment. PMID- 10754718 TI - Pharmaceutical container/closure integrity. V: An evaluation of the WILCO "LFC" method for leak testing pharmaceutical glass-stoppered vials. AB - The sensitivity, reliability, reproducibility and ease of use of the WILCO LFC package integrity test method was evaluated by preparing and testing a series of rubber-stoppered glass vials which were modified by affixing a glass micropipette through the vial side wall. The test units contained water, 50% aqueous ethanol, 20% lithium chloride or 20% aqueous glycerol. Leakage measurement obtained by LFC testing were compared to helium leak rate measurements. The LFC methods detected all leak > 0.0014 standard cubic centimeters per second (sccs), which represents a sensitivity about fourteen-fold greater than standard vacuum decay methods. The minimum detectable leak corresponded to a nominal micropipette internal diameter of between 1 and 2 microns. The effective detection range corresponded to a leak size associated with a 40 to 100% probability of microbial ingress based on a previously reported logistical regression model between helium leak rate and microbial immersion. The sensitivity did not vary with solvent or testing duration in range of 5 to 10 seconds. The coefficient of variation was about 3%. The LFC operation was rapid and without apparent mechanical or electronic problems over the two day testing period used in these studies. PMID- 10754719 TI - Competency-based training program design. PMID- 10754720 TI - The effect of glucose and copper on the stability of diatrizoic acid. AB - Hypaque Sodium Oral Solution is used as a X-ray contrast agent. The active ingredient is diatrizoate sodium. Several bottles of drug product were found to contain elevated levels of inorganic iodide, ranging from 0.001% (w/v) to 0.036% (w/v). The USP specification is no more than 0.02% w/v. The formulation contains sucrose as a sweetener and as a viscosity modifier. It was found that elevated levels of iodide were directly related to the amount of glucose found in the formulation. Glucose is a reducing sugar which can be generated by the decomposition of sucrose. Solution stress studies containing diatrizoate sodium and varying amounts of glucose and copper were conducted at three different pH values: 6, 7 and 8 in phosphate buffer at 60 degrees C. The decomposition of diatrizoate sodium was monitored by HPLC. The greatest amount of decomposition occurred in those solutions prepared in pH 8 phosphate buffer and containing both copper and glucose. It is suggested that glucose reacts with copper to form an activated species, which reacts to displace an iodide. PMID- 10754721 TI - Systematic screening of antioxidants for maximum protection against oxidation: an oxygen polarograph study. AB - The development of a simple method for rapid screening of antioxidants in the preformulation phase of drug development is reported. Using an easily oxidizable drug substance containing a tetrahydroisoquinoline nucleus, the relative antioxidant efficacies was determined by simultaneous measurement of dissolved oxygen depletion and drug disappearance rates in presence and absence of antioxidants by oxygen polarographic and high performance liquid chromatographic (HPLC) methods, respectively. Results showed an inverse correlation between oxygen depletion and drug disappearance rates (R2 > 0.85). In contrast, such a high correlation was not obtained when the standard redox potential of these antioxidants was used as a predictor of drug disappearance rates (R2 > 0.50). The rate at which sodium metabisulfite (BIS) and glutathione depleted dissolved oxygen was reduced in the presence of drug, indicating possible reaction between either of the two antioxidants and drug (OHM-11252). Hence, BIS and glutathione may not be suitable antioxidants for stabilizing OHM-11252 in solution. The oxygen depletion rate constants and percent of intact drug remaining were higher in presence of ascorbic acid (ASC) than BIS. Analysis of the reaction mixtures by HPLC diode array detection showed that the number and percent peak area of non drug peaks formed from ASC-stabilized drug were lower than for BIS. Hence, ASC provides the drug better protection from oxidative degradation. PMID- 10754722 TI - Resolving liposomal inhibition of quantitative LAL methods. AB - Bacterial endotoxin is composed of Lipid A as well as variable polysaccharide regions, and is capable of eliciting a pyrogenic response in vivo (1). NeXstar has developed a quantitative LAL assay at maximum sensitivity to provide assurance that its products are well below the endotoxin pyrogenic threshold. This test uses rigorous treatment of 1.0 mL liposome samples with sodium dodecyl sulfate, an anionic surfactant, or CHAPS, a zwitterionic surfactant to disrupt the lipids present in the liposome sample including endotoxin. This liposome disruption allows potentially undetectable endotoxin to become available to the LAL cascade for quantification and overcomes inhibition of the LAL test by liposomes. PMID- 10754723 TI - PDA comments on draft guidance for industry on changes to an approved NDA or ANDA. Parenteral Drug Association. PMID- 10754724 TI - PDA comments on proposed rule: supplements and other changes to an approved application. Parenteral Drug Applications. PMID- 10754725 TI - Network management in an FDA-regulated environment. PMID- 10754726 TI - Preparing workplace trainers to maximize ROI. PMID- 10754727 TI - The scientific basis for visible particle inspection. AB - The following paper was presented at the 1999 PDA International Conference in Tokyo, Japan February 24, 1999. The paper was written in response to the recent well-publicized incidents in Japan in which the presence of visible particulates in injectable products was observed. These incidents should be considered a wake up call to all concerned with injectable pharmaceutical products. The overview of visible particle inspection presented at the Tokyo PDA Meeting is reprinted here as a reminder that a global marketplace requires well-defined parameters and evaluation methods. In the absence of these basic requirements continuation of the present conflicting particle contamination evaluations will continue by default. At this time, visible particle inspection around the world lacks both direction and an accepted common language. The adoption of the probabalistic concepts introduced by Knapp and co-workers in 1980 can supply these basic requirements. The use of the probabalistic model and the statistically defined particle quality regions defined by Knapp and co-workers in 1980 was, at that time, a concept foreign to pharmaceutical quality. In the period since 1980, the methodology then introduced has been expanded and simplified. The analysis used by these workers was similar to that used in life insurance calculations and is just as reliable. The results obtained following the adoption of the framework and probabalistic concepts introduced by Pflug in sterile products since 1973 supports the use of tested and verified probabalistic concepts in the production of injectable products. Pflug's work has provided a basis for the secure communication of sterile production methods and results. This improvement in communication has potentiated a surge in sterile product improvement and a consequent reduction in product cost. The use of the probabalistic model and statistically defined particle contamination quality regions introduced by Knapp and co-workers in 1980 is believed to have a similar potential to improve product quality and reduce inspection costs in visible particle inspection procedures. Application of the probabalistic model results in a numerical evaluation of both the inspection security achieved in rejecting visible defects and the excess cost introduced by the false reject rate of good product. It has the ability to translate the U.S.P. language "essentially free" of particle contamination into objective, reproducible numerical results. Visible particle contamination data can be successfully analyzed only when its probabalistic nature is understood. The effect of disregarding the probabalistic nature of visible inspection data is well illustrated by the results of the landmark 1940 court case brought by Bristol Labs against the F.D.A. The F.D.A. contention that the containers were contaminated with visible particles was challenged by Bristol in a court case. When the experienced inspector, who reported the contamination, was asked to differentiate between good and contaminated ampules on the witness stand he failed to distinguish between them. Consequently, the case was dismissed. Any attempt to devise or analyze visible particle quality limits that is not based on the probabalistic nature of the accept/reject decision process can only result in the frustration and failure experienced by the F.D.A. expert in 1940. In the GMP philosophy, the introduction of any new device or system must be preceded by a validation demonstration. This is to ensure that the new device or system functions at least as well as the device or system to be replaced. The validation of semi- and fully automated systems for the detection of particle contamination rests upon the statistically replicable assessment of the security achieved with human inspection performance. Any attempt to validate an alternative inspection system without a knowledge of its probabalistic nature and the probabalistic nature of the human inspection benchmark performance PMID- 10754728 TI - Preformulation studies for the development of a parenteral liquid formulation of the immunomodulator, peldesine. AB - Peldesine (BCX-34) is a potent inhibitor of the enzyme purine nucleoside phosphorylase (PNP). The object of this study was to determine the preformulation parameters of BCX-34 and subsequently prepare a sterile liquid formulation for intravenous infusion. Ionization and solubility parameters were assessed prior to formulation development. Three pKa values of approximately 4.0, 5.2 and 10.3 were assigned for BCX-34. The pH-solubility profile showed an increase in solubility when the pH of the solution was less than the pKa1 value of 4.0 and when the pH was more than the pKa3 value of 10.3. BCX-34 has an octanol-water partition coefficient of approximately 7:1 (Log P = 0.8). The Van't Hoff temperature dependent solubility when measured over a range of 4-40 degrees C produced a heat of solution (delta Hc) of 7.68 kcal/mole. Based on these preformulation observations, two parenteral formulations containing 1 and 5 mg/mL of BCX-34 were prepared in an acidified saline solution and filled into 30 cc and 100 cc glass vials. Both formulation batches were sterile filtered, while only one 1 mg/mL batch was terminally autoclaved. The long-term stability of both solutions was performed and compared. Results of this study show that the drug product was stable. Following a screening study, Lactated Ringer's injection USP was selected as a suitable infusion medium for the dilution of the drug concentrate solutions. PMID- 10754729 TI - Effect of gamma-irradiation on peptide-containing hydrophilic poly (d,l-lactide co-glycolide) microspheres. AB - The effect of gamma-irradiation on the physicochemical properties of peptide containing hydrophilic poly (d,l-lactide-co-glycolide) (PLGA) microspheres was evaluated. PLGA (50/50, Mw: 8,600) with free carboxylic end groups was used to make drug-loaded and placebo microspheres by a solvent extraction evaporation method. Both formulated and non-formulated microspheres were gamma-irradiated at 0, 1, 1.5, and 2.5 Mrad doses. HPLC analysis based on extraction of peptide from the microspheres showed that peptide content of the microspheres was lowered upon irradiation and the reduction was more pronounced in formulated microspheres. The in-vitro release in 0.033M phosphate buffer, pH 7.0 at 37 degrees C (based on extraction of residual peptide) showed that the initial and subsequent release of peptide was higher in gamma-irradiated microspheres during the first 20 days. The difference became insignificant during the erosional controlled release of the peptide. There was no difference in release between the formulated and non formulated microspheres of the nonirradiated or irradiated forms. Molecular weights (Mw and Mn), determined by size exclusion chromatography, were reduced by gamma-irradiation for both formulated and non-formulated placebo microspheres. Differential scanning calorimetry showed a gradual reduction in Tg of placebo microspheres but no reduction in peptide-loaded microspheres. In-vivo evaluation of the nonirradiated and the 1.5 Mrad irradiated microspheres showed no marked differences through 28 days. Since irradiation caused a lowering of Mw and Mn with the appearance of a low amount of unidentified substances, seemingly catalyzed by the polymer and the formulation excipients, gamma-irradiation sterilization of these parenteral delivery systems requires careful investigation on an individual product basis. PMID- 10754730 TI - A biofunctional assay to study pRL-CMV plasmid DNA formulation stability. AB - The ability of a plasmid DNA formulation to code for a functional protein was assayed as a marker for plasmid DNA stability using a cotransfection method to measure transcription efficiency. This method shows increased sensitivity and reproducibility over single plasmid transfection methods. Method validation, by measuring DNA degradation rates, demonstrates that buffer choice may be of some importance in the pharmaceutical formulation of plasmid DNA. Degradation rates dependant on citrate buffer concentration were observed. This cotransfection method has proven superior to standard agarose gel electrophoresis in quantifying subtle pRL-CMV plasmid DNA damage and could be used to help predict stability of a final plasmid DNA dosage form. PMID- 10754732 TI - Parenteral formulations of small molecules therapeutics marketed in the United States (1999)--Part I. PMID- 10754731 TI - Effect of lyophilization on parenteral emulsions. AB - The feasibility of preparing lyophilized anhydrous products, for reconstitution in to emulsion dosage forms was investigated. Stable soybean o/w emulsions were prepared using a mixture of lecithin and Span 20 as the emulsifiers. Two series of emulsions were prepared for this study, each containing a polyhydroxy alcohol as a consurfactant for particle size reduction. Increasing concentrations of glycerol (10-30% w/w) were added to one group of emulsions and propylene glycol (20-60% w/w) was added to the second group of emulsions. All formulations were found to have good particle size stability. The emulsion formulation containing 30% glycerol could be successfully lyophilized into an anhydrous product. Reconstitution of this lyophilized product resulted in an emulsion essentially similar to the original emulsion prior to lyophilization. This is because the mixture of the oil phase and 30% w/w glycerol formed a self-emulsifying system. All other emulsion formulations were not suitable for lyophilization. These formulations cracked during lyophilization, separating into an upper oil layer and a lower layer of the continuous phase. The formation of an upper oil layer prevented complete drying of these emulsions. The particle size of these lyophilized emulsions, when reconstituted with the external phase was greater than the emulsion particle size prior to lyophilization. But the change in particle size was less with increasing concentrations of polyhydroxy alcohols. These results indicate that emulsions can be lyophilized to prepare a product suitable for reconstitution to a parenteral emulsion dosage form provided the formulation is designed to withstand temperature and phase changes during the lyophilization process. PMID- 10754733 TI - [Factors influencing transperitoneal transport of glucose in vitro]. AB - The effect of mixing fluid intensification, damage of mesothelial cells and gentamicin on the diffusive glucose transport across the peritoneal membrane were evaluated in the presented in vitro studies. A mathematical model of the mass transport was used to calculate the diffusive permeability, expressed as a diffusive permeability coefficient P [cm x s-1], for the investigated specimens. In the control conditions glucose transfer from the interstitial to the mesothelial side of membrane (I-->M) and in the opposite direction (M-->I) remained constant and P value at mean was 2.731 +/- 1.493 [cm x s-1 x 10(-4)]. The change of the stirring rate from 5.5 to 11 mL/min increased P values by about 74% for transport direction I-->M and 58% for M-->I, but change from 11 to 22 mL/min enhanced P at mean by about 42% for the both direction. The damage of the mesothelial layer, using of sodium deoxycholate (2.5 mmol/L; 103.6 mg%), increased the glucose transfer from the interstitial to the mesothelial side of the peritoneum by 41% and to the opposite direction by 57%. Furthermore, gentamicin did not change the I-->M transfer, but diminished M-->I transport by about 12%. IN CONCLUSION: 1. The reducing of unstirred fluid layer at the mesothelium and interstitium--fluid interface and the mesothelium damage increase the diffusive glucose transport in vitro; 2. The unstirred fluid layer restricts glucose transfer more than tissue barrier (mesothelium); 3. The peritoneal glucose transport, directed from the mesothelial to the interstitial side of the peritoneum (but not transfer to the opposite direction), decreases slightly after gentamicin introduction. PMID- 10754734 TI - [Echocardiographic assessment of pathologic geometry in the left left ventricle with aneurysm before and after endoventricular plasty]. AB - Large LV aneurysm (LVA) leads to progressive distortion of ventricular geometry. Endoventricular plasty (EVP) maintains LV original shape and size and may restrain this process. AIMS: Echocardiographic estimation of LVA size. Assessment of the results of EVP regarding operative technique. Assessment if the initial parameters of LV function and LVA size could predict the choice of surgical technique. MATERIAL AND METHOD: 33 patients with LVA underwent EVP: 18 with patch (group A), 15 without patch (group B). Echocardiography was performed before and after surgical procedure. LVA-area, LVA-volume, its proportion to LV area and volume (LVA-area/LV-EDA, LVA-volume/LV-EDV) were determined apart from routine parameters of LV function. RESULTS: Mean LVA-area was 15 cm2, LVA-volume was 50 mL. Mean LVA-areal LV-EDA was 0.38 LVA-volume/LV-EDV was 0.35. LVA exceeding 0.4 of LV area/volume was considered as a large. After surgery LV-EDV diminished +/- 40 mL, LVLd decreased +/- 1.5 cm. EF improved from 44% to 54%. Patients in group A had larger LVA-volume (60 vs 39 ml, p = 0.02) as well as LV-EDV (160 vs 120 ml, p = 0.03), and lower EF (41% vs 51% p = 0.002). Only 2 patients with LVA-volume greater than 56 ml were operated on without patch. After surgery echocardiographic parameters did not differ between the groups: in group A operation was much more extensive. SUMMARY: 1.) Echocardiographic parameters concerning size (LVA-area, LVA-area/LV-EDA) and volume (LVA-volume, LVA-volume/LV EDV) of LVA provides information about disturbances in LV geometry, are valuable in planning operative method. 2.) EVP with or without patch provides significant improvement of LV geometry and function in echocardiographic investigation. PMID- 10754735 TI - [The influence of thrombolytic therapy on selected parameters of left ventricular function in acute myocardial infarction]. AB - Our study aimed to evaluate the influence of thrombolytic therapy on some left ventricle (LV) function parameters in patients with acute myocardial infarction. The study was performed on 44 pts admitted to hospital due to acute myocardial infarction. The patients were divided into two groups: I group--30 pts (26 male, 4 female) at average age 57 +/- 10 who were treated with tissue plasminogen activator (t-PA) routinely and II group--14 pts (9 male, 5 female) at average age 62 +/- 10 in whom thrombolytic therapy was contraindicated for various reasons. Transthoracic echocardiography was performed just before treatment (0), 3.5 hours after the onset of drug administration (2 hours after the end of t-PA injection) (1) and on the 10th day of hospitalization (2). Control group consisted of 16 clinically healthy individuals (12 male, 4 female) at average age 54 +/- 9. The following parameters were evaluated: DT-E--wave of early diastolic transmitral flow deceleration time, IVRT--isovolumic relaxation time, E/A--early/atrial peak flow velocity ratio of transmitral flow, LATEF%--left atrial total emptying fraction, EF--left ventricle ejection fraction. In patients with acute myocardial infarction shortening of DT, prolongation of IVRT, lower E/A ratio and decrease of LATEF% compared to controls were observed. In group I EF was less than in clinically healthy individuals. E/A ratio was higher in pts from group I than from group II. In patients treated with t-PA 2 hours after treatment as well as on the 10th day significant prolongation of DT, shortening of IVRT and increase of LATEF% were observed. These changes were accompanied by the increase of EF. In patients with acute myocardial infarction not treated with t-PA significant increase in E/A ratio and EF on 10th day were observed. On the basis of the results were conclude: In patients with acute myocardial infarction LV diastolic function and with unproper relaxation as well as unproper compliance of LV myocardium is present. In patients with thrombolytic therapy LV filling pattern improves just two hours after t-PA administration (DT prolongation, IVRT shortening, LATEF% increase). Such tendency remains on the 10th day after treatment. In patients without thrombolytic therapy slight improvement occurs no sooner than on the 10th day of the MI. PMID- 10754736 TI - [Concentration of leptin in patients on hemodialysis and peritoneal dialysis]. AB - Leptin, the obese gene (ob) product, is a plasma protein synthesized exclusively in adipocytes and plays an important role in regulation of food intake and energy expenditure. The aim of the study was to compare serum leptin concentration in hemodialyzed (HD) patients and in patients undergoing continuous ambulatory peritoneal dialysis (CAPD), and to assess the relationship between leptinemia and parameters of nutritional state and inflammatory markers. We studied two group of patients with end-stage renal failure: 52 hemodialyzed individuals aged 24-74 years and 19 peritoneally dialyzed patients aged 20-70 years. Serum hemoglobin concentration, erythrocyte count, cholesterol, triglycerides, total protein, albumin, urea before and after HD, urea in CAPD patients, CRP, peritoneal protein loss were determined by standard laboratory methods. Serum leptin concentration before and after HD, in CAPD patients, leptin in ultrafiltrate and in peritoneal fluid was measured by commercially available radioimmunoassay. Plasma TNF-alpha and IL-1 concentration were measured by ELISA. We found that the serum leptin concentration was higher in hemodialyzed patients than in peritoneal dialyzed individuals (19.97 +/- 22.57 ng/mL vs 3.52 +/- 4.39 ng/mL, p < 0.01). There was a significant correlation between serum leptin levels and BMI in both studied groups (r = 0.81, p < 0.01 in HD group; r = 0.46, p < 0.05 in CAPD group). Concentrations of inflammatory markers (CRP, TNF-alpha, IL-1) were higher in hemodialyzed patients. Peritoneally dialyzed patients presented higher serum lipid concentrations (cholesterol and triglycerides) (p < 0.05). Neither inflammatory markers concentrations nor serum lipid concentrations correlate with the serum concentration of leptin. PMID- 10754737 TI - [Epidemiology of diabetes type 1 in the 0 to 29 year-old age group in Northeastern Poland, 1994-1998--prospective observations]. AB - It was recently suggested that there is a tendency of the rising incidence of diabetes type 1 in different countries in Europe. In our previous studies we observed the high frequency of ICA among first degree relatives of diabetic subjects, what could suggest a rapid increase in the incidence of diabetes type 1 also in the north-eastern region of Polan. The aim of the study was to estimate the annual incidence of diabetes type 1 in the north-eastern region of Poland and to evaluate the association of the incidence rates with the age, gender, urban rural differences and the season of the year of the disease onset. The register of new cases of IDDM was established in 1994 using 2 independent data sources. The degree of ascertainment was 98.9% for the combination of both data sources. We observed a rising trend in the incidence from 6.4 in 1994 to 9.9 cases per year per 100,000 in 1998 (p < 0.001). The highest incidence rate increase was recorded in boys aged 5 to 14 years old (16-17 cases per 100,000 in 1997-1998). The rising trend in this incidence was observed in the rural areas, while in urban populations the incidence was unchanged. Seasonal variation in the incidence was also recorded, with a peak in autumn and winter and nadir in summer. Our observations suggest the necessity of the continuation of the studies to identify the diabetogenic environmental factors and to try preventive procedures in the studied population. PMID- 10754738 TI - [Amyloid heart disease diagnosed with echocardiography]. AB - A sixty years old woman with amyloid heart disease has been presented. Postural hypotension was the main symptom in this case. Pneumonia and the developing congestive heart failure was the cause of death. Only the last echocardiographic examination: hypertrophied interventicular septum with a peculiar speckled appearance of myocardium, together with low voltage electrocardiography made possible to confirm this diagnosis. It was shown in this case, how difficult it is to diagnose amyloid heart disease. Low ejection fraction together with diastolic abnormalities is usually a forecast of bad prognosis. PMID- 10754739 TI - [A case of massive chylothorax during the course of malignant lymphoma]. AB - A 65 year old man with low grade malignant B cell lymphoma developed bilateral chylothorax, which reached the volume of 52.1. Chylothorax arised most probably as a result of pleural infiltration in the course of the disease. Chemotherapy and repeated drainage of chylus was successful. The resolution of massive bilateral chylothorax in our patient shows, that conservative measures (chemotherapy and repeated pleural drainage) should be tried even in very extensive chylothorax in the course of neoplastic disease. PMID- 10754740 TI - [The circulatory system and selected infectious diseases]. PMID- 10754741 TI - [Stroke and lipid disorders]. PMID- 10754742 TI - [Adverse effects of nonsteroidal anti-inflammatory drugs on the kidneys]. PMID- 10754743 TI - [Immunopathogenesis of glomerulonephritis--the importance of new concepts for clinical practice]. PMID- 10754744 TI - [Professor Ettore Bartoli awarded the Medal of the Polish Society of Internal Medicine]. PMID- 10754745 TI - Canadian Digestive Diseases Week. 39th Annual meeting of the Canadian Association of Gastroenterology in conjunction with the Canadian Association for the Study of the Liver. Quebec City, Quebec, Canada. March 5-12, 2000. Abstracts. PMID- 10754746 TI - [Diabetes. ALFEDIAM (French Language Association for the Study of Diabetes and Metabolic Diseases. Grenoble, France, 21-25 March 2000. Abstracts]. PMID- 10754747 TI - British Diabetic Association annual professional meeting. Brighton, United Kingdom, 15-17 March 2000. Abstracts. PMID- 10754748 TI - 14th European Histocompatibility Conference. Montpellier, France, April 4-7, 2000. Abstracts. PMID- 10754749 TI - The Physiological Society meetings in Pucon, Chile, 13-16 November 1999 and University of Birmingham, 20-22 December 1999. Abstracts. PMID- 10754751 TI - Oncology Nursing Society 25th annual Congress. San Antonio, Texas, USA. May 11 14, 2000. Abstracts. PMID- 10754750 TI - Advances in gene technology: signal transduction and therapeutic strategies. Proceedings of the 1999 Miami Bio/technology Winter Symposium. February 6-10, 1999. PMID- 10754752 TI - Oncology Nursing Society directories. PMID- 10754753 TI - Pediatric Academic Societies and the American Academy of Pediatrics joint meeting. Boston, Massachusetts, USA. May 12-16, 2000. Abstracts. PMID- 10754754 TI - 20th Annual Conference on Peritoneal Dialysis. San Francisco, California, USA. February 27-29, 2000. Abstracts. PMID- 10754755 TI - American Society of Regional Anesthesia annual meeting. Orlando, Florida, USA. March 30-April 2, 2000. Abstracts. PMID- 10754756 TI - Practitioners, scholars seek solutions in Harvard dialogue on medical error. PMID- 10754757 TI - ADE rate uncertain, reporting systems inadequate, GAO tells legislators. PMID- 10754758 TI - Alosetron approved for treatment of irritable bowel syndrome. PMID- 10754759 TI - NABP study for FDA shows most patients receive drug information but quality varies. PMID- 10754761 TI - Physician-industry interactions: greater awareness of downsides needed. PMID- 10754760 TI - Study weakens case for vitamin E supplementation. PMID- 10754762 TI - Huperzine A. PMID- 10754763 TI - Potentially remediable features of the medication-use environment in the United States. PMID- 10754764 TI - Considerations in designing an ideal medication-use system: lessons from caring for the elderly. PMID- 10754765 TI - Into the abyss: seven principles for identifying the causes of and preventing human error in complex systems. PMID- 10754766 TI - Information technology: the infrastructure for improvements to the medication-use process. PMID- 10754767 TI - A nurse's view of the medication-use system. PMID- 10754768 TI - Implementing yesterday's promises. PMID- 10754769 TI - Consensus-development exercise: recommended strategies for re-engineering the medication-use system. PMID- 10754770 TI - Observations on the conference: a pharmacist's perspective. PMID- 10754771 TI - Observations on the conference: a physician's perspective. PMID- 10754772 TI - JCPP plans for following up on the conference. Joint Commission of Pharmacy Practitioners. PMID- 10754773 TI - The influence of inert solids on ethanol production by Saccharomyces cerevisiae. AB - The catalytic role of various inert solid supports on acceleration of alcohol fermentation by Saccharomyces cerevisiae was investigated. The enhanced rate of alcohol production was dependent on the nature of the support as well as on the amount used. Among all the tested supports, chitosan flakes showed the maximum yield of alcohol (93% of theoretical yield). This higher rate of alcohol production was associated with the twofold increase in the activity of alcohol dehydrogenase over control. Our results suggest that the addition of a small fraction of solids in submerged fermentations to facilitate cell anchorage for enhanced metabolic activity is easier and more economical compared to cell immobilization processes. PMID- 10754774 TI - Maximizing management of patients with decompensated heart failure. AB - Patients with decompensated congestive heart failure can be categorized into those with either acute or chronic presentations. Patients with acute decompensated heart failure most often have an acute injury that affects either myocardial performance (i.e., myocardial infarction) or valvular/chamber integrity (mitral regurgitation, ventricular septal rupture), which leads to an acute rise in left ventricular (LV) filling pressures resulting in pulmonary edema and dyspnea. Therapy for these patients is aimed at treating the underlying cause of the myocardial injury as well as pharmacologic strategies to reduce LV filling pressures and to improve cardiac performance. In contrast, the therapy of patients presenting with decompensated heart failure in the setting of chronic LV systolic dysfunction, treated with angiotensin-converting enzyme inhibitors, digoxin, diuretics, and may be beta blockers, represent a poorly defined clinical entity that lacks clear guidelines for treatment. These patients can present with symptoms of volume overload and/or low cardiac output without evidence for a volume overloaded state. Potential diagnostic and therapeutic approaches include (1) a pulmonary artery catheter for invasive hemodynamic monitoring, (2) intravenous inotropic therapy, (3) LV mechanical assist device therapy, and (4) cardiac transplantation. This review presents some of the advantages and disadvantages of each of these interventions for patients with chronic systolic dysfunction who present with decompensated symptoms and require specialized management in the hospital setting. PMID- 10754775 TI - The economic burden of heart failure. AB - Heart failure, a major cause of morbidity and mortality among the elderly, is a serious public health problem. As the population ages and the prevalence of heart failure increases, expenditures related to the care of these patients will climb dramatically. As a result, the health care industry must develop strategies to contain this staggering economic burden. Strategies may include adopting approaches for preventing heart failure and implementing new treatment modalities with proven efficacy into large-scale clinical practice. Successful implementation of these strategies will require intensive physician and patient education and development of innovative approaches to fund support services. PMID- 10754776 TI - Inotropes in the beta-blocker era. AB - Beta-adrenergic blocking agents are now standard treatment for mild to moderate chronic heart failure (CHF). However, although many subjects improve on beta blockade, others do not, and some may even deteriorate. Even when subjects improve on beta blockade, they may subsequently decompensate and need acute treatment with a positive inotropic agent. In the presence of full beta blockade, a beta agonist such as dobutamine may have to be administered at very high (> 10 micrograms/kg/min) doses to increase cardiac output, and these doses may increase afterload. In contrast, phosphodiesterase inhibitors (PDEIs) such as milrinone or enoximone retain their full hemodynamic effects in the face of beta blockade. This is because the site of PDEI action is beyond the beta-adrenergic receptor, and because beta blockade reverses receptor pathway desensitization changes, which are detrimental to PDEI response. Moreover, when the combination of a PDEI and a beta-blocking agent is administered long term in CHF, their respective efficacies are additive and their adverse effects subtractive. The PDEI is administered first to increase the tolerability of beta-blocker initiation by counteracting the myocardial depressant effect of adrenergic withdrawal. With this combination, the signature effects of beta blockade (a substantial decrease in heart rate and an increase in left ventricular ejection fraction) are observed, the hemodynamic support conferred by the PDEI appears to be sustained, and clinical results are promising. However, large-scale placebo-controlled studies with PDEIs and beta blockers are needed to confirm these results. PMID- 10754777 TI - Human embryo development and pregnancies in an homologous granulosa cell coculture system. AB - PURPOSE: Our purpose was to determine the effects of the coculture of embryos on human granulosa cells (GCs) in patients in the first cycle of IVF-ET treatment and in patients with repeated implantation failures and to investigate the presence of specific proteins in a 48-hr GC conditioned medium and the GC ultrastructural characteristics. METHODS: Eighteen patients with tubal or idiopathic infertility were enrolled in this study: 7 patients (Trial 1) were in the first cycle of IVF-ET treatment and 11 patients (Trial 2) had repeated implantation failures (one to five). Embryos from each patient were cocultured randomly either on homologous granulosa cells or on a conventional culture medium. RESULTS: At the end of the coculture period (day 5 or 6), 50% of the embryos (Trial 1) reached the blastocyst stage, with respect to 35% in Trial 2. The pregnancy rate per retrieval was 14.2 and 9%, respectively, in Trial 1 and in Trial 2. Many conditioned media showed proteins of 24-29 kDa. and some of them showed additional proteins of 90 kDa. The ultrastructural analysis of GCs showed healthy, metabolically active, protein-synthesizing, and mostly steroidogenic cells. CONCLUSIONS: GC cultures improve embryo development but not pregnancy rates both in Trial 1 and in Trial 2. PMID- 10754778 TI - Ovarian reserve test with human menopausal gonadotropin as a predictor of in vitro fertilization outcome. AB - PURPOSE: Our purpose was to determine prospectively, using receiver-operating characteristic (ROC) analysis, whether the ovarian reserve test with hMG could improve the predictive value of a woman's age and basal levels of follicle stimulating hormone (FSH), E2, and inhibin or any combination of them regarding ovarian response and pregnancy rate in IVF treatment following pituitary desensitization. METHODS: The hMG test was performed within 3 months of IVF treatment in 80 women undergoing the first cycle of IVF and consisted of 2 ampoules of hMG daily for 5 days starting on cycle days 2 to 3. Hormone and ultrasound evaluation was performed on cycle days 2 to 3 and 7 to 8. RESULTS: The mean age and basal FSH levels were significantly higher in the canceled (n = 28) than in the control (n = 52) group, whereas the basal inhibin level was significantly higher in the latter. Regarding ovarian response, the combination FSH plus inhibin had the better diagnostic accuracy (predictive value of 70%) among basal variables. When post-hMG parameters (alone or in combination) were analyzed, E2 alone, with a 77% diagnostic accuracy, emerged as the best predictive variable of cancellation in IVF cycles. When ROC analysis was used, the area under the ROC curve for E2 post-hMG (diagnostic accuracy of 84.5%) was significantly higher than that for the estimates based on the combination of basal FSH and inhibin (diagnostic accuracy of 71.3%). However, woman's age was the only variable independently associated with pregnancy rate. CONCLUSIONS: The predictive power of the hMG test of ovarian reserve is better than that of age and basal hormone values (FSH and inhibin) and it is based mainly on the E2 response to hMG treatment. However, given that age is the only predictor of pregnancy and considering the cost and discomfort of the hMG test, the usefulness, if any, of the test in predicting IVF performance in the daily clinical setting remains to be established. PMID- 10754779 TI - Follicular development and hormonal levels following highly purified or recombinant follicle-stimulating hormone administration in ovulatory women undergoing ovarian stimulation after pituitary suppression for in vitro fertilization: implications for implantation potential. AB - PURPOSE: The main goal in the present study was to compare follicular development and estradiol levels after ovarian stimulation in pituitary suppressed normally ovulating women undergoing IVF, using highly purified urinary follicle stimulating hormone (FSH) (u-FSH-HP) and recombinant FSH (rec-FSH). A secondary variable in our study was embryo implantation potential, which is closely related to appropriate follicular development and oocyte competence. METHODS: For the main purpose of this study, 30 IVF patients (group 1) were treated during IVF consecutive cycles, using the same stimulation protocol, with u-FSH-HP in the first treatment study cycle and rec-FSH in the second one. As a control group (group 2) for implantation rates obtained in cycles treated with rec-FSH, 30 additional IVF patients were included who underwent a second IVF attempt again with u-FSH-HP. RESULTS: The total dose of FSH used and ovarian response obtained in terms of estradiol plasma levels and the total number of growing follicles on the day of human chronic gonadotropin (HCG) injection were similar in both treatment cycles in group 1 but better follicular dynamics and oocyte maturity were obtained with rec-FSH. The implantation rate was significantly higher in rec FSH treated cycles in patients in group 1 than in control women (group 2). CONCLUSIONS: rec-FSH is more efficacious than u-FSH-HP when used in the same patient in inducing multiple follicular development in down-regulated cycles as indicated by ovarian performance and oocyte maturity. In addition, rec-FSH yields significantly higher implantation rates than u-FSH-HP when used in patients undergoing their second IVF attempt. PMID- 10754780 TI - The effects of E2 supplementation from the early proliferative phase to the late secretory phase of the endometrium in hMG-stimulated IVF-ET. AB - PURPOSE: Our purpose was to determine if pregnancy rates (PRs) for hMG (human menopausal gonadotropin)-stimulated IVF-ET (in vitro fertilization--embryo transfer) can be increased by estradiol (E2) supplementation from the early proliferative phase to the late secretory phase of the endometrium. METHOD: Eighty-one infertile women with pure tubal factor were randomized into two groups. One group received no E2 supplementation (control group) and the other received oral E2 supplementation (2 mg two times daily) from the early proliferative phase starting on the third day of the menstrual cycle to the late secretory phase of the endometrium, with hMG stimulation for ovulation induction starting on the sixth day of the menstrual cycle. RESULTS: In 85 cycles, at least one embryo was transferred. Compared with the control group (n = 27 cycles), the E2 supplementation group (n = 58 cycles) had a significantly higher PR (control, 25.9%, versus E2 supplementation, 48.3%) and IR per ET (control, 10%, versus E2 supplementation, 26%), but FRs per retrieved oocytes were not statistically different between the two groups (control, 74%, versus E2 supplementation group, 73%). Four spontaneous abortions occurred in the E2 supplementation group, and one case in the control group. Ectopic pregnancy occurred in one case in the control group. CONCLUSIONS: Clinical PRs and IRs in the E2 supplementation group were significantly higher than in the control group, while FRs in the control group did not differ statistically from the E2 supplementation group. This suggests that E2 supplementation from the early proliferative phase to the late secretory phase of the endometrium in hMG-stimulated IVF-ET increases the receptivity of the endometrium for transferred embryos and clinical PRs. PMID- 10754781 TI - Is the timing of implantation affected by zona pellucida micromanipulation? AB - PURPOSE: Our purpose was to examine the timing of implantation and early embryo development following uterine transfer of oocytes/embryos previously subjected to zona pellucida micromanipulation. METHODS: A total of 68 singleton pregnancies resulting from IVF and embryo transfer with/without micromanipulation. Patients were divided into four groups according to the type of micromanipulation technique: assisted hatching, embryo biopsy, intracytoplasmic sperm injection, and no micromanipulation (control group). Serial serum beta-hCG levels were measured between 10 and 25 days after fertilization and log-transformed. Linear regression analyses were performed and extrapolated to hCG = 10 mIU/ml (hCG10) to estimate detectable implantation. The slopes of the regression lines were used to estimate the rising speed of hCG, an indirect sign of embryo development. RESULTS: There were no significant differences among groups with respect to hCG10, the slopes or intercepts of the regression lines. CONCLUSIONS: Various oocyte/embryo microsurgical procedures used in ART involving zona pellucida manipulation do not appear to affect the timing of implantation or early embryo development. PMID- 10754782 TI - In vitro maturation and fertilization of immature oocytes: a comparative study of fertilization techniques. AB - PURPOSE: Our purpose was to investigate the factors influencing maturation and fertilization of immature oocytes. METHODS: Immature oocytes were obtained from women undergoing cesarean section. They were cultured in the maturation medium either with or without cumulus cells. After maturation to metaphase II, they were randomly fertilized by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). RESULTS: After incubation for 48 hr, 441 oocytes (42.8%) reached metaphase II. Among them, 56.6% of cumulus-enclosed oocytes, but only 29.2% of denuded oocytes, reached metaphase II. Of the 289 cumulus-enclosed oocytes, the fertilization rates by IVF and ICSI were 56.3 and 84.1%, respectively (P < 0.01). Of the 152 denuded oocytes, the fertilization rates by IVF and ICSI were 39.5 and 84.5%, respectively (P < 0.01). The cleavage rates, however, were similar. CONCLUSIONS: Cumulus cells are beneficial in the maturation of human oocytes in vitro and that ICSI increases the fertilization rate for the in vitro matured oocytes. The developmental potential of the fertilized oocytes, however, is similar irrespective of the fertilization method or the presence or absence of cumulus cells. PMID- 10754783 TI - A quantitative and morphological analysis of oocytes collected during 438 IVF cycles. AB - PURPOSE: Our purpose was to determine if the number of retrieved oocytes, oocyte maturity, morphology, and other embryological parameters are related to the outcome of treatment. METHODS: This retrospective study on 438 IVF cycles analyzes the numbers of retrieved oocytes, fractured zona oocytes, germinal vesical-stage oocytes, normally and abnormally fertilized oocytes, pregnancy rate, age of female partner, ovarian stimulation protocol, day of hCG injection, and serum estradiol concentration. RESULTS: (1) Pregnancy rate increases with an increase in the number of retrieved oocytes, (2) a high incidence of fractured zona pellucida oocytes has a negative effect on fertilization rate but none on pregnancy rate, (3) a high incidence of immature oocytes is associated with improved fertilization and pregnancy rates, and (4) an inverse relationship between the presence of immature oocytes and oocytes with fractured zona pellucida is suggested. CONCLUSIONS: Precise oocyte assessment in IVF cycles provides informations useful for the analysis and improvement of ovarian stimulation protocols. PMID- 10754784 TI - The effects of age and abnormal sperm count on the nondisjunction of spermatozoa. AB - PURPOSE: The effect of paternal age on the nondisjunction of sex chromosomes is controversial. Also, the prevalence of chromosomal anomalies in infertile patients is controversial, it has been reported that the sex chromosomal aneuploidy rate following treatment with intracytoplasmic sperm injection (ICSI) is higher than in naturally conceived pregnancies. We investigated the influence of paternal age and oligozoospermia on the nondisjunction of spermatozoa. METHODS: We determined the rate of aneuploidy for gonosomes and autosomes, using two-color fluorescence in situ hybridization (FISH) of the X and Y chromosomes and chromosomes 12 and 18 in 10 donors under 25 years of age who had a normal sperm count (> or = 20 x 10(6)/ml), 10 donors over the age of 39 years with idiopathic infertility and normozoospermia (> or = 20 x 10(6)/ml), and 5 oligozoospermic donors (< 20 x 10(6)/ml). RESULTS: There was no obvious relationship between increasing age and autosomal disomy (disomy 12 and disomy 18). Neither autosomal disomy nor diploidy was increased in any group. The frequency of X-, Y-, XX-, and YY-bearing sperm did not differ significantly among groups, but the frequency of XY-bearing sperm was significantly higher in the older infertile group than in the control donors. CONCLUSIONS: The incidence of nondisjunction of paternal sex chromosome in meiosis I was higher in older men with idiopathic infertility. The present results suggest that the risk of producing XXY fetuses is higher among men > 39 years of age with idiopathic infertility. PMID- 10754785 TI - Chromomycin A3 staining as a useful tool for evaluation of male fertility. AB - PURPOSE: Our purpose was to investigate the association between percentage chromomycin A3 (CMA3) positivity of spermatozoa with some sperm parameters and in vitro fertilization rate. METHODS: Spermatozoa were collected from 139 men, washed in PBS, fixed in methanol/glacial acetic acid (3:1), and then spread on slides. CMA3 positivity is expressed as the percentage in 200 spermatozoa. RESULTS: Percentage of CMA3 positivity showed not only a negative correlation with fertilization rate but also a significant difference between fertilizing and nonfertilizing patients. Moreover, percentage of CMA3-positive spermatozoa showed a negative correlation with count and percentage motility and a positive correlation with percentage of abnormal morphology. Percentage of CMA3 positivity also had a positive correlation with some abnormalities of head such as amorphous and macrocephaly. Ultrastructural study showed chromatin unpackaging in high CMA3 positive semen samples in comparison with low CMA3-positive semen samples. CONCLUSION: There is a close relationship among fertilization rate, sperm parameters, and CMA3 positivity and CMA3 could be considered as a useful tool for evaluation of male fertility prior to infertility treatment. PMID- 10754786 TI - Study of the in vitro maturation of mouse oocytes induced by microinjection of maturation promoting factor (MPF). AB - PURPOSE: Maturation promoting factor (MPF) acts at the resumption of meiosis and nonspecifically throughout the animal species. There exists a considerable body of literature on MPF, but little work has been done to study the induction of maturation of mammalian oocytes by microinjection of extracted MPF. METHODS: Immature (GV-stage) mouse oocytes were microinjected MPF extracted from matured Xenopus eggs in the presence of dbcAMP. RESULTS: The rate of germinal vesicle, breakdown (GVBD) induced at 24 hr after MPF injection was significantly higher (90.5%) than that of the control (2.2%), which was injected with HTF medium containing dbcAMP (P < 0.0001). The rate of extrusion of the first polar body at 24 hr after MPF injection was significantly higher (84.1%) than that of the same control (1.1%) (P < 0.0001). CONCLUSIONS: From these results, it is concluded that the maturation of mammalian oocytes can be induced by the microinjection of MPF extracted from other species. PMID- 10754788 TI - Politics as usual. Health reform 2000. PMID- 10754787 TI - Shoulder pain in a child: a case presentation of ganglioneuroblastoma. AB - A four and one-half year-old child presented with a several month history of shoulder pain. Her workup revealed a large, homogeneous tumor in the apex of the chest. Surgical resection was performed demonstrating ganglioneuroblastoma. This case illustrates an unusual cause of joint discomfort in children. PMID- 10754789 TI - Carotenoid content in Lota lota (L.) individuals in various biological activity periods. AB - The authors investigated the carotenoid content in the particular parts of Lota lota in summer, autumn, and winter, i.e. when burbots exhibit the lowest and highest activity. By means of columnar and thin-layer chromatography, the following carotenoids were found to be present: alpha-carotene, beta-carotene, e carotene, beta-cryptoxanthin, neothxanthin, lutein, 3'-epilutein, zeaxanthin, tunaxanthin, antheraxanthin, lutein epoxide, echinenone, 3'-hydroxyechinenone, idoxanthin, canthaxanthin, alpha-doradexanthin, beta-doradexanthin, astaxanthin, diatoxanthin, parasiloxanthin, monadoxanthin, 7,8-dihydroparasiloxanthin, mutatoxanthin and rhodoxanthin. In the Lota lota individuals examined, the content of carotenoids was found to differ in winter and summer. The total carotenoid content ranged from 0.067 (gonads of males) of to 6.095 micrograms g-1 wet weight (fins of males from December). PMID- 10754790 TI - Direct effects of lithium chloride on the activities of delta 5-3 beta and 17 beta-hydroxysteroid dehydrogenase in the testis and Bidder's organ of the adult toad (Bufo melanostictus)--in vitro study. AB - Steroidogenic key enzymes, i.e. delta 5-3 beta and 17 beta-hydroxysteroid dehydrogenase (delta 5-3 beta and 17 beta-HSD) activities, in the testis and Bidder's organ of the toad were inhibited and ascorbic acid synthesis in these organs was decreased by a wide range of lithium concentration in in vitro study. A significant inhibition was noted at a concentration of 2.0 mM, which is easily achieved in the blood during the treatment of manic patients by lithium chloride. This experiment reflected that lithium exerts a direct inhibitory effect on hydroxysteroid dehydrogenase activities in the testis and Bidder's organ--a rudimentary ovary in Bufo. PMID- 10754791 TI - X-ray microanalysis of myotomal muscle in the sunbleak, Leucaspius delineatus (Heckel) and goldfish, Carassius auratus gibelio. AB - The functioning of a group of muscle fibres as a tissue that performs a well characterized type of contraction (slow or fast) depends on their biochemical and structural organization that is already well established. The biochemical and structural diversities between three types of fish muscle fibres found also a reflection in the content of light elements. The present work demonstrates significant differences in the content of diffusible elements (Cl, K, Na, and Mg) and bound elements (P and S) between the muscle fibres types. In general all muscle fibre types of goldfish (Carassius auratus gibelio) that belongs to stationary slow-swimming fish has lower K/Na ratios than those in all three fibre types of fast swimming sunbleak (Leucaspius delineatus). PMID- 10754792 TI - Preferential segregation of marker chromosomes 14 and 18 in mouse recombinant inbred strains derived from the KE and CBA/Kw strains. AB - The segregation pattern of chromosomes 14 and 18 were analyzed in recombinant inbred strains of mice developed from KE and CBA/Kw strains. The analysis was possible owing to the fact that the C-band on chromosomes 14 of the CBA/Kw strain and that of chromosome 18 of the KE strain show size polymorphism: while the CBA/Kw mice have a small sized C-band on chromosome 14, the KE mice show small C bands on chromosome 18. Chromosomes were identified by G-banding and FISH. The results show that the chromosomes with small centromeric chromatin segregate preferentially. PMID- 10754793 TI - Exoproteinases of the type A in pathogenesis of insect bacterial diseases. AB - Immune inhibitors produced in infected larvae of Galleria mellonella by such entomopathogens as Pseudomonas aeruginosa, Serratia marcescens and Heterorhabditis bacteriophora effectively blocked in vitro bactericidal activity of insect haemolymph against Escherichia coli D31, both in Galleria mellonella and Pieris brassicae pupae previously vaccinated with Enterobacter cloacae. Even at a trace concentration, the extracellular proteinases, by proteolytic degradation, totally destroyed the activity of cecropin peptides from Galleria and cecropin-like and attacin-family proteins from Pieris, but no ability to destroy antibacterial activity was shown by extracts obtained from Galleria larvae killed by massive doses of bacterial saprophytes. It is suggested that by blocking antibacterial immune response of the host, the proteinases help the bacteria to multiply in the haemolymph, thus they could be considered an important factor in the pathogenesis of bacterial diseases of insects. PMID- 10754794 TI - Biocidal activity of Bacillus species for Anopheles larvae. AB - It is generally accepted that Bacillus thuringiensis (B.t.) is specifically toxic to some insects but does not pose any threat to the environment, operators, or consumers. There are several other Bacillus species which can be used as effective bioinsecticides. In this study four different species of Bacillus, i.e., B. coagulans, B. megaterium, B. brevis, and B. sphaericus were isolated from soil samples collected from Kala Shah Kakoo and Kasur areas, in the suburbs of Lahore. Isolated Bacillus species were administered to mosquito larvae to evaluate their biocidal activity. B. coagulans I from Kala Shah Kakoo showed 93% mortality, while B. coagulans III from Kasur showed 70% mortality. Bacterial isolates most toxic to Anopheles larvae showed optimum growth at 37 degrees C and pH 7. These isolates have a great potency to controlling anopheline population. PMID- 10754795 TI - The policy context for community development practice in public health: a Canadian case study. AB - The Community Action in Public Health Study explored how public health managers, frontline staff, and community participants interpret, implement and receive policy guidelines urging collaboration with community groups in public health work. In-depth case studies of 6 public health units in Ontario, Canada focussed on 19 community development projects in 3 program areas (107 interviews). In the absence of formal policy guidelines on community development at provincial or local levels, informal policy predominated. Local senior management frequently set the tone, distinguishing health units in which community development was a basic philosophy underlying a broad spectrum of public health practice from those in which it was seen as only one among many possible strategies. Uncertainty and risk associated with informal policy lead many frontline staff to adopt strategies intended to preserve autonomy in community work, including "seeking forgiveness rather than permission" and maintaining relative "invisibility." PMID- 10754796 TI - Public health implications or urban agriculture. AB - The article presents the case for stronger public policies in support of urban gardening as a means to improve public health. It considers several beneficial aspects of gardening, such as food security, economic development, exercise, psychological and community well-being, and environmental stewardship. It also considers some of the public health problems associated with urban agriculture and suggests policies to ameliorate them. In the balance, urban gardening has potential as an important element of urban public health. PMID- 10754797 TI - The ethics of medical marijuana: government restrictions vs. medical necessity. AB - Marijuana is listed by the Drug Enforcement Agency (DEA) as an illegal Schedule I drug which has no currently accepted medical use. However, on March 17, 1999, 11 independent scientists appointed by the Institute of Medicine reported that medical marijuana was effective in controlling some forms of pain, alleviating nausea and vomiting due to chemotherapy, treating wasting due to AIDS, and combating muscle spasms associated with multiple sclerosis. There was also no evidence that using marijuana would increase illicit drug use or that it was a "gateway" drug. Despite this evidence the DEA refuses to reclassify marijuana as a Schedule II drug, which would allow physicians to prescribe unadulterated and standardized forms of marijuana. After reviewing the pertinent scientific data and applying the principle of double effect, there is a proportionate reason for allowing physicians to prescribe marijuana. Seriously ill patients have the right to effective therapies. To deny patients access to such a therapy is to deny them dignity and respect as persons. PMID- 10754798 TI - Developing a model law restricting the transporting of passengers in the cargo areas of pickup trucks. AB - Pickup trucks have become increasingly popular in the United States, accounting for about one in five vehicles involved in fatal motor-vehicle crashes. A critical factor in these deaths is the practice of carrying passengers in truck cargo areas, which are not designed for this purpose. Each year approximately 200 deaths occur to occupants riding in the back of pickup trucks. Over half the states have laws dealing with preventable injury problem, but these laws vary widely and most are too limited to be effective. We have reviewed existing laws, as well as crash injury data, and we have developed and recommend a model bill. The bill focuses on intended use, restricting passengers to only those portions of a vehicle designed for human transport. We have also conducted and report on a survey of legislative sponsors of pickup truck legislation. PMID- 10754800 TI - 2nd Croatian Congress on Atherosclerosis. Opatija, Croatia, May 21-24, 1999. Abstracts. PMID- 10754799 TI - Changes in the health care system of Vietnam in response to the emerging market economy. AB - This paper discusses the impact on the Vietnamese health care system of the change from a centralized socialist system to a market economy. It discusses recent policies based on expectations in relation to actual outcomes, and the impacts these changes have had on health care delivery and health infrastructure in Vietnam. It has become clear that the private medical sector is draining resources from the State rather than complementing the weakened national health system. Impacts on health education, pharmaceuticals, infrastructure support, geographic distribution of physicians, and equity are all discussed in terms of recent economic changes. It is suggested that adjustments must be made to ensure adequate health care for all Vietnamese including those in rural areas and the urban poor. The State must develop mechanisms to support the national health service before further deterioration occurs. PMID- 10754801 TI - Tobacco settlement dollars a "burning issue" for TMA. AB - More than 46 states and five U. S. territories have their collective hands outstretched as the tobacco industry digs into its deep pockets to make amends for the ill effects of its product. Even as Tennessee lawmakers debate the best use of an anticipated $215 million windfall, fears are growing that very few of the tobacco settlement dollars will go toward their intended purpose. PMID- 10754802 TI - Loss prevention case of the month. What will your colleagues say? PMID- 10754803 TI - Hamilton county physicians' experience with managed care. AB - BACKGROUND: Widespread physician participation in managed care over the last several years prompted this survey of the members of the Medical Society of Chattanooga and Hamilton County. METHODS: A 36-item questionnaire was mailed to all members of the Medical Society. RESULTS: Ninety-six responses from solo practitioners and groups representing 325 physicians were analyzed. More than 80% of respondents believed that managed care has affected the quality of patient care negatively and 71.8% believed managed care policies have compromised their patient care. More importantly, 90% said insurers have not secured their input in policy development. Referral processes were regarded as cumbersome or impossible by 78.2%. Regression analysis showed a significant relation between issues of too much managed care control, cumbersome referral processes, and physicians' opinion on impact of managed care on quality of patient care. CONCLUSIONS: Physician dissatisfaction with managed care in Hamilton County appears widespread and is mainly related to issues of too much managed care control over daily patient care, which physicians feel results in compromised quality of patient care. PMID- 10754804 TI - Natural history of isolated ventricular septal defect in the first five years of life. AB - BACKGROUND: Isolated ventricular septal defect (VSD) is the most common congenital heart defect. Incidence, prevalence, and clinical outcomes of VSD have been reported to vary significantly in different geographic areas. Spontaneous closure of VSD, in children, by various methods has been described. HYPOTHESIS: This prospective study was undertaken to evaluate natural history of patients with VSD in the first five years of life in the Northeast Tennessee and Southwest Virginia region. METHODS: Between December 1, 1998 and October 31, 1990, 124 infants were diagnosed clinically with isolated VSD. VSDs were classified as muscular, perimembranous, malalignment, or subpulmonic types by 2-dimensional echocardiogram with color flow mapping. Cardiac catheterization and angiocardiography were performed in 14 patients when clinically indicated. These patients were followed for at least five years. RESULTS: Overall spontaneous closure of VSD was 34% at one year and 67% at five years. Twenty-five percent of perimembranous and 4% of muscular VSDs required surgery by five years. The spontaneous closure rate of muscular VSD was twice that of the perimembranous type, though the relative distribution of both types was almost equal. Overall, 22% of children with VSD need follow-up after the fifth year of life. CONCLUSION: The overall clinical outcome of muscular VSD was consistently better than that of the perimembranous type, though 17% of muscular VSDs, irrespective of size, were open at 5 years of age and needed long-term follow-up. PMID- 10754806 TI - Department of Health report. Bioterrorism: a public health issue. PMID- 10754805 TI - Percutaneous transluminal angioplasty of an acute traumatic renal artery occlusion. PMID- 10754807 TI - Breast cancer--early detection key to survival. PMID- 10754808 TI - The Australian Midwifery Action Project. PMID- 10754809 TI - Research in midwifery--the relevance of a feminist theoretical framework. AB - As midwifery develops as a discipline and new models of care are introduced within maternity services, research activity and inquiry into practice and professional issues will be required. This paper suggests that an appropriate theoretical underpinning for research in midwifery is one that is based on feminist theory. It is argued that by adopting this approach, midwifery practice will focus on the needs of women and change in the provision of services for childbearing women will be in response to women's evaluation of existing services. By addressing the needs of women it is argued that the profession will also benefit, as the role of midwifery in the health care of childbearing women will be defined. PMID- 10754811 TI - The permanent location of the National Office of the Australian College of Midwives. National Executive Committee. PMID- 10754810 TI - The introduction of 'direct entry' midwifery courses in Australian universities: issues, myths and a need for collaboration. AB - This paper identifies some of the issues within the debate regarding the introduction of 'direct entry' midwifery education in Australia. It addresses questions that have been raised around terminology; the current midwifery education system; whether midwives also need to be nurses; how nurses who want to become midwives might enter the same programme with recognition of prior learning; and whether 'direct entry' midwifery education should become a mainstream option. A case is made for a collaborative initiative to consider all aspects of developing a national framework for Bachelor of Midwifery programmes. PMID- 10754812 TI - Midwives you've got m@il! PMID- 10754813 TI - The development of clinical indicators--the impact on midwifery practice in Queensland in the future. AB - Perineal trauma is the most frequently reported complication of labour and delivery in the Qld Perinatal Data Collection. It is documented that some individual accoucheurs seem to be particularly skillful in assisting at birth in a way that minimises perineal trauma. Recent trends within the Qld health industry have emphasised the importance of quality improvement activities focussing on health outcomes. Midwives accoucheur most of the "normal" deliveries in public hospitals, thus it is important that midwives play a leading part in the development and improvement of tools which identify quality outcomes and pinpoint areas for improvement. This study aims to benchmark current practices regarding perineal outcomes. Moreover, the authors aim to initiate discussion towards the development of the first clinical indicators appropriate to midwifery care. PMID- 10754814 TI - What do we read and why? PMID- 10754815 TI - Midwifery care for Australian Aboriginal women. AB - Australian Aboriginal women have twice the number of still born babies as Australian non Aboriginal women and have babies who are five times more likely to die within the neonatal period. The perinatal mortality rate is three times higher and the infant mortality is more than five times the overall rate for babies of Australian Aboriginal women compared to Australian non Aboriginal women. These are the stark statistics compiled by the Midwives' Notification System in Western Australia (1998). PMID- 10754817 TI - Midwives you've got m@il! PMID- 10754816 TI - ACMI competency standards for midwives--what they mean for your practice. AB - The introduction of the Australian College of Midwives Inc (ACMI) Competency Standards for Midwives in late 1998, has implications for every midwife in Australia. This paper will provide a brief history of the development of nursing and midwifery competencies in Australia, examine what the competencies mean for practice and suggest ways that the competencies can be used for assessment. It is well to remember that the competencies must be considered in context and midwifery legislation and education form a triad for the implementation and use of the competencies. PMID- 10754818 TI - Raspberry leaf and its effect on labour: safety and efficacy. AB - The purpose of this study was to examine the safety and efficacy of raspberry leaf products consumed by a group of mothers during their pregnancy, by comparison with a group of mothers who did not. A retrospective observational design was used. Subjects were women who birth their babies at Westmead Hospital between January 1998-July 1998. The sample consisted of 108 mothers; 57 (52.8%) consumed raspberry leaf products while 51 (47.2%) were in the control group. The findings suggest that the raspberry leaf herb can be consumed by women during their pregnancy for the purpose for which it is taken, that is, to shorten labour with no identified side effects for the women or their babies. The findings also suggest ingestion of the drug might decrease the likelihood of pre and post-term gestation. An unexpected finding in this study seems to indicate that women who ingest raspberry leaf might be less likely to receive an artificial rupture of their membranes, or require a caesarean section, forceps or vacuum birth than the women in the control group. PMID- 10754819 TI - A weight off my mind: the abandonment of routine antenatal weighing a change of practice research. AB - The 50 year old tradition of routinely weighing pregnant women, which has been identified as an obsolete practice, is still practiced by many. The antenatal clinic and community midwives, and medical staff from the Division of Obstetrics (Central Coast Area Health Service) as well as pregnant women attending the clinics have been surveyed to identify the impact on implementing evidence based practice (ceasing routine antenatal weighing). Using both quantitative and qualitative methods to provide a rich and detailed picture, the outcomes showed that the importance of weighing decreased for most of pregnant women. Midwives were surprised at the women's' acceptance to the change; and both health professional groups did not feel a loss of overall care. This research suggests that organised and planned change can achieve acceptance of evidence based practice. PMID- 10754820 TI - In search of the meaning of life? PMID- 10754821 TI - An introduction to catharsis and the healing crisis in reflexology. AB - This paper explores the concept of healing through catharsis in relation to the practice of reflexology. The literature will be reviewed to inform a critical analysis of the role of the reflexologist and specifically the therapeutic relationship. This area was chosen as I have had experiences of reflexology clients reporting feeling 'emotional', 'being tearful', and 'thinking more about their lives' past and present'. These observations have been illuminated by a recent study that suggests reflexology can be helpful for people with emotional needs (Trousdall 1997). Emotional and physical responses to reflexology are traditionally attributed to 'detoxification' and the signs of it occurring known as a 'healing crisis' (Sahai 1993, Griffiths 1995). Consideration will be given to the practitioner's skills and support, issues of boundaries to practice, and the effective management of patient's emotional and physical responses. The safety and potency of the therapeutic work will be key elements to the discussions. PMID- 10754822 TI - Lay perspectives of complementary medicine. AB - Whilst a tiny snapshot, it is clear that lay interpretations of complementary medicine vary considerably. You may wish to try this exercise to explore peoples' perceptions of this form of medicine and we would welcome responses to these questions. When practising CM it is easy to assume others share our health care perceptions and this may not always be the case. PMID- 10754823 TI - Challenging power, autonomy and politics in complementary therapies: a contentious view. PMID- 10754824 TI - Complementing your care: about the special interest groups. Part I--RCN complementary therapies forum. AB - Supports the integration of appropriate complementary therapies into healthcare. Encourages the use of evidence-based practice. Promotes the development of appropriate education for nurses wishing to use complementary therapies in their practice. Encourages its members to raise public awareness of the potential benefits and possible areas of concern relating to the use of complementary therapies. PMID- 10754825 TI - Complementing your care: about the special interest groups. Part 2--Complementary therapies in Maternity Care National Forum. PMID- 10754826 TI - Physiologic and psychodynamic responses to the administration of therapeutic touch in critical care. AB - Recent publications have questioned the efficacy of therapeutic touch (TT). The focus of attention has been on substantiating the existence of the recipient's energy field rather than on the physiologic and psychodynamic responses to TT. In this article the physiologic and psychodynamic responses during and following the administration of TT is described. The project involved the implementation of a time series design in which the physiologic and psychodynamic responses were measured. It is acknowledged that critical care environments are stressful for patients in terms of invasive medical and nursing procedures. Continuous bright lighting, and excessive noise prohibits the potential for relaxation and sleep. Within this context, the control of confounding variables was not possible, and therefore not an object of concern in the study. Rather the responses to TT in the natural setting were of importance to discern. Statistical repeated measures analysis of variance (one way) indicated there was no significant difference between pre-, during and post-physiologic variables in response to TT. However psychodynamic responses demonstrated significant correlation's in terms of relaxation and sleep. The non significance of physiologic change in variables pre , during and post-administration of TT indicates critically ill patients remained physiologically stable. Significant correlations of psychodynamic responses demonstrated it is possible for critically ill patients to experience periods of relaxation and sleep in an otherwise stressful environment. TT was found to be a useful therapy to enhance relaxation and sleep in critically ill patients. PMID- 10754827 TI - Healing, energy and the complementary therapies. PMID- 10754828 TI - Trends in nursing and midwifery research and the need for change in complementary therapy research. AB - In recent years there has been a change in nursing and midwifery research. Whilst many of the subjects being studied remain the same, nurses and midwives have started to employ a range of data collection methods that are relatively new to the profession. Predominantly quantitative research, which concentrates on reduction, objectivity, manipulation, categorization, passivity, control, prediction, causality and generalizability (Munhall & Oiler 1986), is starting to be replaced by other approaches perhaps more congruent with nursing, midwifery and caring. As Moody (1990) stated, 'the 1980s ushered in an array of diverse, sophisticated research methods...' with other authors adding that 'nursing is just beginning to authenticate new territory that incorporates a plurality of methods' (Nagle & Mitchell 1991). The following is an exploration of the recent apparent shift away from a focus on quantitative research in nursing and midwifery towards the use of qualitative methods which emphasize a greater degree of individuality, humanism, participation and interaction. It is suggested that the traditional quantitative research paradigm still exists in the field of complementary therapy research and that the shift that has taken place in nursing and midwifery research needs to be considered more seriously in the field of research in complementary therapies. PMID- 10754829 TI - The integration of complementary therapies in North and South Thames Regional Health Authorities' critical care units. AB - Subsequent to the rising interest in complementary therapies, a survey was conducted to determine the extent of integration of complementary therapies in critical care units in the North and South Thames Regional Health Authorities in the Greater London area. In total, 45 critical care units were surveyed at random. The results of the survey indicated Neonatal Intensive Care Units showed the greatest interest and provision (75%) of complementary therapies. This was primarily in the application of baby massage. In contrast, only 10% of Coronary Care Units surveyed provided complementary therapies. Results further indicated that of 51.1% of critical care units which claimed to provide complementary therapies, only 7% provided interventions on a routine, systematic basis. PMID- 10754830 TI - Research issues in complementary therapies. AB - Complementary medicine views health as a balance of forces to achieve optimum wellbeing of body, mind and spirit, whilst conventional healthcare focuses on the prevention, diagnosis and treatment of disease. The World Health Organisation estimates that globally 80% of primary consultations occur within holistic therapies (Lewith 1995). Numerous reasons explain this, e.g. dissatisfaction with technological medicine, increasing individual responsibility for health, and more client involvement in treatment. The increased use of complementary therapies in the UK has stimulated debate about health, illness and care which poses issues for the overall delivery of contemporary healthcare. Rising standards of accountability create expectations that research is available which informs safe and effective practice. This article reflects on the particular issue of research methodology into the effectiveness of healthcare. PMID- 10754831 TI - Complementary medicine is holism in practice: the view of the ICM. PMID- 10754832 TI - The Healing Touch programme: passion into action. PMID- 10754833 TI - From medical herbalism to phytomedicine: back to the future? PMID- 10754834 TI - A holistic framework for maternity care. AB - This paper explores the concept of holism and its implications for maternity care. The integration of all aspects which affect the care of an individual mother and her baby is considered a holistic framework for maternity care is proposed. PMID- 10754835 TI - Polarity and unity in caring: the healing power of symptoms. AB - This article explores the idea that suffering is a key aspect of the healing process. Suffering often manifests itself as 'disease' within the body in the form of physical symptoms. It is posited here that this is the psyche's unconscious way of drawing attention to the inner tension of the conflict of opposites. Whilst this tension is often the seat of discomfort, it also holds the key to the individual's own creative potential, including that of healing. Drawing upon the work of contemporary nursing theorists Margaret Newman, Rosemary Parse, depth psychologists Thomas Moore and James Hillman this article argues that caring and suffering are unnecessarily polarized in nursing. A method of working with symptoms is discussed that honours both the poetics of the suffering and the individual's inclination towards health. PMID- 10754836 TI - Music as therapy: a brief history. PMID- 10754837 TI - What are the reasons for nurses using complementary therapy in practice? AB - The increasing popularity of complementary therapies has generated many surveys to establish how many nurses are involved in its use. This paper explores reasons why nurses are using complementary therapy in orthodox nursing practice to determine the catalysts influencing its use. The following areas are addressed: definitions, terminology and historical aspects of complementary medicine relationship with orthodox medicine, examining dominance, conflict, partnerships and socialization rise in popularity nurse involvement and implications to the profession methodology and findings conclusion. Information about nurses practising complementary therapies was obtained from the King's Fund London and from hospitals within the researcher's local area. Questionnaires for the pilot study were self administered locally, while those for the main study were sent through the postal system. PMID- 10754838 TI - Is it time for a new injury score? PMID- 10754839 TI - Research, publication & funding sources--Part I. PMID- 10754840 TI - Fat embolism syndrome. PMID- 10754842 TI - President's message. The sounds of nursing leadership. PMID- 10754841 TI - Trauma registrar training: integrating registry functions into the trauma program -Part 2. AB - Effective health-information management is heavily reliant upon the data collection process of the health care program. This is also true in trauma-care systems, where healthcare providers have come to rely on timely access to complete and accurate information to support many critical functions of a trauma program. The process by which data are collected and analyzed deserves constant attention and reassessment. Data obtained in reports and analyses are only as accurate as the data abstraction and validation processes allow. The University of Pennsylvania Health System Trauma Network recognizes this, and as a result, developed this training and orientation guideline. This guideline supports the importance of the registry role in the overall trauma program, but most importantly, it fosters and supports a team approach to data collection, which in our experience has allowed us to develop and maintain trauma registry databases which provides valid, useful and current information for all. PMID- 10754843 TI - Lateness is not greatness. PMID- 10754844 TI - The development of objective techniques to measure nursing knowledge, 1941-1952. AB - An indirect by-product of A Curriculum Guide for Schools of Nursing, released by the National League of Nursing Education (NLNE) in 1937, was the attention it focused on evaluating the nursing student objectively (1). The first such guide had been issued 20 years earlier and revised in 1927, with only a slight modification. During the NLNE annual convention one year after publication of the final revision, a Joint Committee on Nursing Test was appointed to consider the possibility of developing objective techniques to measure nursing on a cooperative basis. Representatives from the League, the Association of Collegiate Schools of Nursing, and the Department of Nursing Education, Teachers College, Columbia University, were charged with taking steps to initiate such a project and seeking funds to support it (1). At the time, Isabel M. Stewart, who initiated the effort, was the director of the Teachers College Department of Nursing Education as well as an active participant in several educational NLNE endeavors. PMID- 10754845 TI - Thinking in nursing education. Part I. A student's experience learning to think. AB - Learning to think critically is a central commitment of nursing education. There is a substantial body of literature describing nursing educators' attempts to define critical thinking (1-3) to differentiate critical thinking from other kinds of thinking (1,4), and to measure students' ability (and changes in ability) to think critically (2,5-7). These efforts were facilitated when the National League for Nursing Accrediting Commission (NLNAC) identified critical thinking as an outcome criterion for the accrediation of undergraduate and graduate nursing programs. This change in accreditation led to the proliferation of framework (8,9) and strategies (10,11) for nursing educators to use in demonstrating compliance with this criterion. Describing strategies and frameworks for teaching critical thinking is helpful. However, explicating how teachers teach and students learn critical thinking in actual clinical situations illuminates the contextual aspects of practice that influence learning to think (12). Conventional strategies teachers use to assist students to learn critical thinking include individual and group activities, discussions and interactions between students and teachers, clinical simulations, and problem-solving encounters. Although such strategies are commonly thought to be effective in teaching critical thinking in classroom or laboratory situations, little research has been conducted to evaluate the relationship between specific teaching strategies and students' ability to think critically in specific situations (1). A further limitation of laboratory and classroom strategies is that they need to be supplemented with contextual experiences. Providing students with opportunities to practice critical thinking in actual clinical situations is difficult because the context of care is rapidly changing and schools of nursing continue to allocate limited resources to practice education. This two-year study, which was undertaken to reveal common contemporary approaches to teaching and learning critical thinking in clinical courses, analyzes the lived experiences of 45 students and teachers. Part I describes a typical student's experiences of learning "nurse thinking" in the context of clinical practice. Part II describes a typical teacher's experiences creating opportunities for students to learn and practice critical thinking in a community clinical course. PMID- 10754846 TI - Thinking in nursing education. Part II. A teacher's experience. AB - Across academia, educators are investigating teaching strategies that facilitate students' abilities to think critically. Because may these strategies require low teacher-student ratios or sustained involvement over time, efforts to implement them are often constrained by diminishing resources for education, faculty reductions, and increasing number of part-time teachers and students. In nursing, the challenges of teaching and learning critical thinking are compounded by the demands of providing care to patients with increasingly acute and complex problems in a wide variety of settings. To meet these challenges, nurse teachers have commonly used a variety of strategies to teach critical thinking (1). For instance, they often provide students with case studies or simulated clinical situations in classroom and laboratory settings (2). At other times, students are taught a process of critical thinking and given structured clinical assignments, such as care plans or care maps, where they apply this process in anticipating the care a particular patient will require. Accompanying students onto clinical units, teachers typically evaluate critical thinking ability by reviewing a student's preparation prior to the experience and discussing it with the student during the course of the experience. The rationales students provide for particular nursing interventions are taken as evidence of their critical thinking ability. While this approach is commonly thought to be effective, the evolving health care system has placed increased emphasis on community nursing (3,4), where it is often difficult to prespecify learning experiences or to anticipate patient care needs. In addition, teachers are often not able to accompany each student to the clinical site. Thus, the traditional strategies for teaching and learning critical thinking common to hospital-based clinical courses are being challenged, transformed, and extended (5). Part II of this article describes findings that suggest how many teachers and students are challenging the conventional approaches to schooling and creating pedagogies that are more responsive to the contemporary context of health care. PMID- 10754847 TI - Critical thinking skills of baccalaureate nursing students at program entry and exit. AB - Most nurse educators would agree that critical thinking is an essential competency for the professional nurse in today's ever-changing health care environment. In fact, critical thinking has been identified as an integral component of professional nursing practice and has been incorporated in accreditation guidelines (1-3). As a result of this imperative, professional programs of nursing must explicate a clear definition of critical thinking, identify specific learning outcomes reflective of critical thinking abilities, and select appropriate ways to measure the achievement of these outcomes in graduating students. Although much has been written about the need for critical thinking skills, the concept and measurement of critical thinking within the context of nursing education has not yet been clearly defined (4-6). As a result, nursing programs are developing their own conceptual definitions of critical thinking and using a variety of methods to measure outcomes (5). The authors caution that to ensure validity of findings, the instruments used must reflect the individual program's definition of the concept. In most cases, outcomes have been assessed using a cross-sectional design that compares students at different levels or types of programs or are measured as an end-of-program outcome. While such designs can provide educators with knowledge of students' critical thinking abilities at a particular point in the educational process, longitudinal data are needed to assess the effects of the educational program. PMID- 10754848 TI - Use of the Watson-Glaser Critical Thinking Appraisal with BSN students. AB - The complexity and changing nature of nursing today requires proficiency in thinking skills to ensure knowledgeable, confident, creative, and sensitive decisions regarding client care. Nurse educators are faced with the task of promoting educational strategies to develop the abilities of nursing students to think critically in all health care settings (1). However, a lack of consensus on what characterizes critical thinking leads to difficulty in the development of instruments for adequate measurement. It is important to decide on a definition of critical thinking that faculty support and are willing to use in practice. The term is diversely defined in the literature. For example, Alfaro-LeFevre states that critical thinking in nursing "entails purposeful, goal directed thinking; aims to make judgments based on evidence (fact) rather than conjecture (guesswork); is based on principles of science and scientific method; requires strategies that maximize human potential and compensate for problems caused by human nature" (2). The authors of this study use a definition by Paul: "thinking about your thinking while you are thinking in order to make your thinking better, more clear, more accurate, more defensible" (3). The authors believe that the development and/or enhancement of critical thinking ability must be the result of conscious, deliberate activity throughout the nursing program. As the student matures, the ability to think critically will be manifested in decision making that reflects accurate assessment and resolution of problems. The nursing faculty selected an instrument to evaluate the critical thinking abilities of baccalaureate nursing students that had strong reliability and validity documented in the nursing literature: the total and subtest scores of the Watson Glaser Critical Thinking Appraisal (WGCTA), Form A (4). The instrument was deemed to be congruent with the definition of critical thinking supported by the faculty. PMID- 10754849 TI - Focus groups and vulnerable populations. Insight into client strengths and needs in complex community health care environments. AB - Focus groups are a useful qualitative research technique to assist in interpreting quantitative community assessment data. Data obtained from focus groups can provide sociological and psychological insights into the perceptions of population subgroups and suggest answers to the "why" questions raised by descriptive data about such issues as teen pregnancy, poverty, immunization levels, or lifestyle-related morbidity and mortality. Application of these insights can lead to the better use of community strengths and the creation of community-specific responses to barriers to health care. Focus groups work well for involving hard-to-reach members of a community in program development, planning, and evaluation. They may be more effective than face-to-face interviews and questionnaires because people often have not thought about how they feel and tend not to form opinions in isolation (1). The information sought through the use of focus groups is not randomly distributed in the population. Thus, groups are not randomly selected, and data are not gathered with the intent to generalize to all populations. PMID- 10754851 TI - A site visitor's first year. AB - CHAP--The Community Health Accreditation Program--is a wholly owned subsidiary of NLN. In this regular column, employees and associates of CHAP, and representatives of CHAP-accredited agencies will introduce readers to various aspects of accreditation of home and community health care organizations. Here, Jim Kahler, who is involved in many facets of community health care in Hawaii talks about his impressions after a year as a part-time CHAP site visitor. PMID- 10754850 TI - Nursing leadership for the new millennium. Essential knowledge & skills. AB - The major plenary sessions at the NLN 24th Biennial Convention in Miami Beach, Florida, addressed the subject of leadership as an important component of the convention's theme--The Nursing Renaissance: New Ways of Being, Learning, and Leading. Dr. Maryann F. Fralic spoke to the essential knowledge and skills of nursing leaders. PMID- 10754852 TI - How does NLNAC support the concept of differentiated practice? PMID- 10754853 TI - The National Agenda for Nursing Education Research. PMID- 10754854 TI - President's message. Coming full circle with the Nightingale legacy. PMID- 10754855 TI - The power of positive thinking & other important new age messages from the past. PMID- 10754856 TI - One hundred years ago. Nursing education at the dawn of the 20th century. AB - The late 19th and early 20th centuries ushered in a number of significant events that helped advance the nursing profession in its early development. Nursing leaders began to show interest in the reform movements under way, such as woman's suffrage, settlement houses, and labor activities. They knew that nurses were not alone in their struggle to improve the health of the public. Philanthropic groups abounded, working for social betterment as well as for changes needed in overcrowded tenements, food inspection, and information to the nation about healthy living (1). In addition, serious concerns surfaced in the profession relating to the proliferation of training schools and reforms required in the educational system. PMID- 10754857 TI - Challenging traditional assumptions about minority students in nursing education. Outcomes from Project IMPART. AB - In Becoming Gentlemen, Lani Guinier describes the experience of women who sit on the margin of the male dominant culture of law school. "It is time," she contends, "to use the information from the margin to rethink the whole" (I, p. 17). Application of Guinier's premise to nursing suggests that minority students exist on the margins of nursing and that understanding their experiences may yield valuable insights into nursing education and nursing practice. One possible implication for nurse educators is the importance of ensuring that nurses--the largest group of health care Providers--reflect America's rapidly changing demographics and have the experience, research expertise, and professional socialization to address the critical health care issues facing minority persons. PMID- 10754858 TI - A model to restructure nursing education. Vision on 22nd street. AB - Since 1993, the Cleveland State University Department of Nursing and the Visiting Nurse Association of Cleveland have been involved in an innovative partnership known as Vision on 22nd Street. This partnership has produced a model to restructure the nursing curriculum and the practice setting in such a way that a seamless process, fully integrated within university and agency operations, prepares undergraduate nursing students for community-based practice. The groundwork supporting the model, the development of the curriculum, the preparation of participants, and the implementation of the model for program evaluation are the subject of this article. PMID- 10754860 TI - A student's experiences in El Salvador. PMID- 10754859 TI - Public health education for Liberian refugees. PMID- 10754861 TI - Nursing leadership for the new millennium. Claiming the wisdom & worth of clinical practice. PMID- 10754862 TI - OASIS: a welcoming patch of green? PMID- 10754863 TI - How does emphasis on core criteria make clear that different levels of nursing education lead to differences in practice, as opposed to homogenizing evaluation of programs? PMID- 10754864 TI - Crossroad considerations. PMID- 10754865 TI - Choosing nutrition support: how and when to initiate. AB - Surveys have shown that malnutrition is present in approximately 50% of all hospitalized patients. With the continued reduction in length of stay in acute care facilities, it is critical that ALL patients receive a nutrition screen and, when appropriate, a comprehensive nutritional assessment. Once the nutrition evaluation has been completed, the next step is to determine the most appropriate route of nutrition intervention. This article provides some guidelines that can be used to determine the type of nutritional support to be implemented. It also contains information on access routes, benefits, and complications with interventions for both enteral tube feeding and parenteral nutrition. PMID- 10754866 TI - Breaking the quality barrier: critical thinking and conflict resolution. AB - Breaking the quality barrier requires case manages to possess the skills of critical thinking and conflict resolution. After a brief focus on habits that create barriers to critical thinking, a critical thinking assessment is offered. Strategies for critical thinking and how to create a culture of openness and flexibility are discussed. Critical thinkers bring to conflict situations skills that foster win-win conflict resolution. An example is used to discuss the skills of assertiveness and negotiation. An example is given that demonstrates how to deal with defensiveness by first stating agreement and then disagreement. PMID- 10754867 TI - An acute-care model in the management of end-of-life issues. AB - Healthcare professionals who provide direct patient care have the knowledge and skill to discuss the biophysiologic aspects of a patient's condition, but the discussion of the management of end-of-life and bioethical issues is often problematic. In this article, we discuss practices and procedures that have proved helpful in encouraging open discussion of end-of-life issues, as well as appropriate management. PMID- 10754868 TI - Hospital-based nursing case management: role clarification. AB - Hospital-based nursing case management as a model of healthcare delivery has substantially grown over the last 10 years. Nursing case management as a viable professional role has developed along with this trend. In an era of decreasing reimbursements and increasing accreditation requirements, hospital administrators view nurse case managers as one answer to balancing cost and quality. However, the actual role and practice of nurse case managers within the hospital setting is inconsistent and often depends on the needs and expectations of the organization, as well as the level of experience and educational preparation of the nurse case manager. PMID- 10754869 TI - UKCC report on the future of nurse education, Fitness for Practice. PMID- 10754870 TI - Management opportunities. PMID- 10754871 TI - Papers for health. PMID- 10754872 TI - Prison nursing. PMID- 10754873 TI - Education for healthcare. PMID- 10754874 TI - Caring for African clients with HIV. PMID- 10754875 TI - Midwives' choice. Interview by Tom Keighley. PMID- 10754876 TI - Discipline at work. Part Two, The formal process. PMID- 10754877 TI - A century of achievement. PMID- 10754878 TI - A vision for nursing. PMID- 10754879 TI - The ICN story, 1899-1999. PMID- 10754880 TI - Family health. PMID- 10754881 TI - Good news for nurses and midwives. PMID- 10754882 TI - [Patient admission: hospitality in the hospital--concept and evaluation]. AB - Welcoming is an everyday act, performed all day long, in different places. But, when it comes to welcoming a patient, it is a matter of health care, for which we are the only responsible. Since this person arrives in a place where his (her) body and mind are going to be ill-treated (s)he will be all the more receptive to his (her) new environment. Faced with feelings of fear, anguish, stress, the patient and ourselves try to establish a relationship made of confidence and a feeling of safety, which are necessary to his (her) integration. This will be achieved by means of verbal and non-verbal communication, by reducing or even getting round the obstacles. By studying this moment, this meeting, with different evaluation tools, I have been able to sort out the strong and weak points of my Institution, in order to propose adequate means of improvements, (creation of a team dynamics on this subject, working out of a definition, evaluation grids and application). The staff will thus be able to readjust his care as well as possible, at the level of the information to be given, and above all the attitude to adopt, in order to improve the quality of welcoming, as, eventually, it is what the patient will keep in mind and what he will use for his evaluation. PMID- 10754883 TI - [Use of research in clinical practice: support program for cardiac patients]. AB - The research aims at the acquisition of knowledge and its application in the practice. The knowledge which most interests the nurses is the one pertaining to the improvement of nursing practice. Before initiating a change in the clinical practice on the basis of the research results, it is important to consider some rigorous criteria which will provide guidance to an enlightened decision as to whether such results should be adopted or not. With the help of an example of nursing care concerning a support programme for the benefit of heart patients, this article presents the application of a model of use of research results. The model consists of six phases in support of a critical judgement on the value of a scientific work: preparation, validation, comparative evaluation, decision making, adoption/application and evaluation. Each of these phases states the application of the model criteria, not only to the results, but to the whole research processes followed by the author. This model of research use can provide the nurse with means of proposing changes in the practice, based on the knowledge issuing from research. PMID- 10754884 TI - [Alternating training approach: comparative study of faculty education at the faculty training college and nurses' education]. AB - The hypothesis of this essay asserts that sandwich courses which are the object of this study in the framework of vocational training in the I.S.F.I. (nurses' school) and I.U.F.M (teacher training), will pertain to an educational logic if it facilitates the construction of the link between theory and practice, the emergence of practice knowledge, the construction of professional skills and if it enables the student to have access to a form of professional socialization. Today, the device lacks coherence and efficiency, there is not much time devoted to analysis and concentration. The presence of more performing trainers, involved in a real partnership would be necessary as well as the reorientation of these training on the professional skills. However, the sandwich courses help the students to distance themselves from practice and enable them to live through their own experiences. The study of the characteristics of these two training enables to make proposals aiming at improving the existing situation: they concern the development of new pedagogical methods, the creation of adapted tools, the improvement of the existing partnership and the training of the trainers. PMID- 10754885 TI - [Measure of parental behavior during children's ambulatory surgery]. AB - A new instrument, l'Inventaire des conduites parentales, was validated by the main author during her doctoral studies. The purpose of her research was to measure the effect of an educational intervention on parent's behaviors. This instrument, based on an educational theoretical framework, was composed of 16 behaviors divided into 3 categories: verbal information, cognitive strategies and attitudes. The reliability of the initial version of the instrument was measured with a sample of 142 parents. The interrater agreement was of 94.3%. The factor analysis showed that 14 of the 16 items were closely related to 4 clusters of variables. Following the factor analysis results, and the utilization of this instrument by research assistants, it was revised and is now described in this article. PMID- 10754886 TI - [How to do a bibliographic search on the Internet?]. PMID- 10754887 TI - [The concept of relationships in social psychology]. PMID- 10754888 TI - [The nursing diagnosis of "spiritual distress", a necessary re-evaluation]. AB - The subject of the study consisted in identifying the spiritual needs of the terminally ill patients, so as to investigate and specify the implementation field of the diagnosis of "spiritual distress" by the French nurses relative to the values and beliefs. The key moments, when the spiritual needs can express themselves also had to be spotted, in order to investigate the possible role of the nurse faced with the spiritual needs of the patients. The study was carried out on a population of 27 AIDS and cancer patients, hospitalized in two units of palliative cares of the Paris region and on a population of 20 nurses of these same units. It was made during the year 1996 by means of interviews with patients, comprising 51 open or half-open questions and questionnaires for nurses, comprising 20 questions. These tools have been structured on the basis of 4 main lines namely spirituality, religion, the ill being called "spiritual distress", nursing diagnosis (for the nurses). The results from the patients show that nearly all of them have had a feeling of ill being which would be of spiritual nature, where existential questioning prevails concerning the meaning of life, of death, of pain, of illness whereas only slightly more than a third of the nurses think that it happens frequently to the patients. The spiritual distress can be described as the failure of giving a meaning to one's life. Religion is mainly evoked in terms of rites with "mosaic" beliefs specific to each individual. The big majority of the nurses concerned by this study have an intuitive knowledge of the main features of the nursing diagnosis of "spiritual distress". This knowledge does not seem to be linked really to the training they had, but rather to their professional experiences and maybe to the personal life story. This nursing diagnosis does not seem to be adapted to our western culture. Its lack of discriminatory power and of flexibility does not make it very useful for the caretaker who needs to clarify beforehand the concepts of spiritual needs and of religious needs. As for the actions, the appeal to the religion representatives or to the psychologist is not always what the patients want even though the ill being appears mainly when the patients feel lonely ... we therefore resort to actions of relation of help with an active listening and help to the rereading of life for which the nurse is in a rather good position, according to the patients. PMID- 10754889 TI - [Concepts and methods in ethnology]. PMID- 10754890 TI - [Caring, it's the experience of understanding yourself through the detour of another]. PMID- 10754891 TI - [Analysis of the bulletins of the "Society of Nurses of Public Health Hospitals", 1903-1913]. PMID- 10754892 TI - Where is rehabilitation nursing documentation going? PMID- 10754893 TI - Bridging the gap. PMID- 10754894 TI - Job characteristics related to job satisfaction in rehabilitation nursing. AB - While much research is available on the job satisfaction of nurses in general, little is known about the job satisfaction of rehabilitation nurses. The purpose of this quantitative descriptive study was to identify job characteristics that are related to job satisfaction in rehabilitation nursing. The target population was rehabilitation nurses throughout the United States. The McCloskey-Muller Satisfaction Scale was used to measure three dimensions of job satisfaction: safety rewards (potential against dangerous threats), social rewards (the need to belong), and psychological rewards (autonomy, responsibility, recognition, and appreciation). Respondents rated their satisfaction with aspects of their current job by selecting a response from a 5-point Likert scale for 31 items in these three dimensions. The results suggest that rehabilitation nurses are satisfied with scheduling, extrinsic rewards, coworkers, social interactions, professional opportunities, delivery of care, praise, recognition, and responsibility. They are, however, less satisfied with child care facilities, balance between family and work, opportunities to participate in organizational decision making, control in the work setting, and career advancement. Opportunities to participate in nursing research or to write and publish neither satisfied nor dissatisfied the majority of respondents. Nursing administrators need to examine the factors that provide job satisfaction to rehabilitation nurses so as to retain capable nurses in the practice environment. PMID- 10754895 TI - A collaborative assessment of workload and patient care needs in four rehabilitation facilities. AB - This article describes the process used by nurse executives at four freestanding rehabilitation facilities to implement and validate an interactive patient classification system. The research process involved defining critical indicators, measuring workloads by level of staff, and validating the number of care hours for the levels of patient classification. The database enabled the four consortium members to share their knowledge, resources, and costs of implementing a patient classification system, and it provides a benchmark of rehabilitation services. The study data are being used in making staffing decisions, preparing and defending budgets, and identifying the cost of care by disability classification. PMID- 10754896 TI - Using the Functional Independence Measure instrument to predict stroke rehabilitation outcomes. AB - The purpose of the study described in this article was to identify the factors that have an impact on stroke patients' discharge destination. Two hundred thirty four stroke patients admitted to a rehabilitation facility over a 2-year period were examined. Functional Independence Measure (FIM) data were used to examine functional status, demographic characteristics, and the discharge destination of patients admitted to the facility's program. The relationship between patients' FIM scores at discharge and their discharge locations was analyzed using the chi square statistic. The results showed that a discharge FIM score of 80 or above had a high specificity and sensitivity with patients' discharge to their homes. In addition, outliers were analyzed, and the results showed that family members of only 20% of the patients who were discharged to their homes were working, in contrast to 65% of the family members of patients who were discharged to a skilled nursing facility. The availability of a nonworking family member and the ability of a family to provide supervision and physical assistance were more likely to be factors related to discharge of patients to their homes. Ninety percent of the families of patients discharged to their homes were able to provide supervision and to provide physical assistance. Thus, both functional status and social factors, such as family availability and support, are critical elements in predicting the discharge destination of this patient population. PMID- 10754897 TI - Research supporting the congruence between rehabilitation principles and home health nursing practice. AB - A grounded-theory study of 30 home health nurses conducted in the Washington, DC, metropolitan area used unstructured audiotaped interviews to elicit data as to how home health nurses define their practice. The purpose of the study was to develop a beginning substantive research-based theory of home health nursing practice. The model that emerged consists of three stages by which nurses attain autonomy in their practice. Adaptation was found to be the core category, in that nurses cannot function effectively or successfully in the home health arena unless they are or learn to be adaptable. Data also revealed that home health nurses either knowingly or unknowingly use rehabilitation nursing principles in their practice, thereby lending credence to the supposition that home health nursing practice is congruent with rehabilitation nursing principles. PMID- 10754898 TI - Mechanical restraints, rehabilitation therapies, and staffing adequacy as risk factors for falls in an elderly hospitalized population. AB - This case-control study of fall-related risk factors for elderly hospitalized patients hypothesized that the use of mechanical restraints, participation in a rehabilitation program, and staffing inadequacy increase the risk of falls. The study included 252 patients, also known as "cases," aged 60 to 85 years, who fell during the period between March 1 and December 31, 1993, in a large metropolitan hospital; and 250 "controls," randomly selected patients aged 60 to 85 years, who were matched with the cases in terms of length of stay to the day of the fall. Analysis using conditional logistic regression revealed that individuals who had been placed in a mechanical restraint at any point during their hospital stay prior to the fall (for the cases) or the selected day (for the controls) had approximately twice the risk of falling as did patients who had not been placed in restraints. Cases and controls showed no significant differences in terms of their participation in occupational therapy, physical therapy, or cardiac rehabilitation, and staffing adequacy was similar for both groups. While the finding of 2 to 4.7 times the risk for falls for those placed in a mechanical restraint only approached statistical significance, the results indicate that the risk of falling is highest soon after a patient has had to be placed in a mechanical restraint. PMID- 10754899 TI - Latex allergy: another real Y2K issue. PMID- 10754900 TI - Meeting the emotional needs of a patient. PMID- 10754901 TI - Home cardiac rehabilitation for congestive heart failure: a nursing case management approach. AB - As the only major cardiovascular disease increasing in incidence and prevalence, congestive heart failure (CHF) is a major health threat. Progression of the disease often leads to severe disability and requires intensive medical and psychological management. Cardiac rehabilitation for CHF can improve a patient's functional ability, alleviate activity-related symptoms, improve quality of life, and restore and maintain physiological, psychological, and social status. The expansion of home care services and advances in technology allow cardiac rehabilitation to take place in the patient's home. Because of their training in health promotion and prevention, assessment, and coordination of services, nurses are the ideal providers of comprehensive home cardiac rehabilitation. Financially, physically, and psychologically beneficial for CHF patients and their families, home cardiac rehabilitation is also cost-effective for society. This article substantiates the benefits of home cardiac rehabilitation for patients with CHF and explains why nurses are the ideal case managers for such programs. PMID- 10754902 TI - Identifying and overcoming barriers to providing sexuality information in the clinical setting. AB - While the scope of the clinician-patient relationship in rehabilitation includes the opportunity to provide information on sexuality, all too often this does not occur. This article identifies barriers to providing information on sexuality and makes suggestions for overcoming them. At a preconference workshop, "Sexuality After Spinal Cord Injury: Understanding the Effects of Knowledge and Attitude on Dissemination Practices," held during the Association of Rehabilitation Nurses 22nd Annual Educational Conference, participants explored barriers to providing sexuality information to their patients and ways for nurse managers and staff members to ameliorate their own discomfort and that of their patients. The barriers identified included lack of time, lack of knowledge, personal attitudes about sexuality, and patient lack of readiness. Approaches that can improve the comfort levels of nurse managers and staff include using educational films and written materials, conducting role playing to explore typical patient questions, and initiating conversation about sexuality with patients. Participants' suggestions for increasing patient comfort level included adopting an open and nonjudgmental listening mode, distinguishing between the physical and emotional aspects of sexual issues, and promoting peer counseling. PMID- 10754903 TI - An exploratory study of nurses' perceptions of their role in neurological rehabilitation. AB - The role of the nursing profession in the multidisciplinary specialty of neurological rehabilitation has not been clearly defined in the United Kingdom. A qualitative exploratory study was performed in a highly specialized neurological rehabilitation unit. Nine registered nurses having at least 1 year of experience in this setting were interviewed in a semistructured manner. Transcripts of the interviews were subjected to a theme analysis. Three main themes emerged in relation to the role of the nurse. The first theme referred to actions directly related to patient care. This theme included four concepts: the promotion of patient independence, routine tasks, the management of patient care, and counseling. The second theme involved the organizational issues that influence practice. Nurse opinions and views of their roles formed the third theme. The subjects' views identified skills specific to neurological rehabilitation nursing and areas for further exploration. PMID- 10754904 TI - Personal perceptions and women's participation in cardiac rehabilitation. AB - The purpose of this study was to determine whether there were differences in personal perceptions between women who participated in cardiac rehabilitation (CR) and women who did not. This cross-sectional study used a convenience sample of 370 female patients who had been treated and discharged between April 1, 1995, and September 30, 1995. Three standardized scales, a self-report instrument, and medical records reviews were used to collect data at five hospitals and four CR centers in three midsized northeastern Ohio cities. Differences in personal perception and demographic variables between participants and nonparticipants were examined. Education, history of coronary heart disease (CHD), and specific cardiac diagnoses showed statistically significant differences between the groups. Logistic regression tested the model for participation. No variable or group of variables was found to predict CR participation in this sample of women. All cardiac patients need information about CR. Patients with less education, a history of CHD, and a diagnosis of stable angina need special instructions and closer follow-up. PMID- 10754905 TI - Conducting biological research to advance rehabilitation nursing practice. AB - Rehabilitation nursing practice is concerned with many clinical manifestations that have an underlying biological impairment. Advances in managing these manifestations will depend in part on research that incorporates the biological dimension. The purpose of this article is to encourage more rehabilitation nurses to engage in biological research. To achieve this aim, several different categories of biological nursing research are described using rehabilitation nursing examples, biological measures and approaches are discussed, and possible general clinical outcomes, with examples from previously published biological nursing research, are described. Biological nursing research may enhance professional competence, improve patient care, and improve patient safety. PMID- 10754906 TI - How can we make nursing more visible in the media? PMID- 10754907 TI - Outcomes: what's all the fuss about? AB - Rehabilitation programs in a wide variety of settings must be able to use information to respond to internal and external demands for positive outcomes. Providers need information to plan and manage outcomes, oversee resource utilization, monitor efficiency and effectiveness, and communicate to stakeholders. Outcomes are useful for consumers (the person served, the community), stakeholders (those who have an interest in the rehabilitation program or services), referrers, and payers. Data must be gathered and analyzed in order to measure and manage outcomes. The information gathered should reflect the values and mission of the organization and meet the needs of all stakeholders. Analyzed information should be used to improve performance in identified areas. Information that accurately reflects the performance of the organization and its rehabilitation programs is also essential. Information about rehabilitation program outcomes data should be at a level that the target audience understands. These concepts are essential if rehabilitation programs are to ensure a successful, viable future. The rehabilitation nurse has the knowledge and expertise not only to assist with data collection, but also to participate in the analysis and evaluation of the outcomes data. Implementing practice changes to promote quality care and positive outcomes for our rehabilitation patients is an essential element of the role of the rehabilitation nurse. By understanding the importance of monitoring outcomes, the rehabilitation nurse acts as an invaluable team member who is instrumental in facilitating change to promote the best possible outcome for the patient. PMID- 10754908 TI - Colors, symbols, and other communication ideas. PMID- 10754909 TI - Quality of life for family caregivers of people with chronic health problems. AB - The trend toward community-based care, along with advances in medical technology, has resulted in increased numbers of individuals with complex healthcare needs being cared for at home by their families. This shift from hospital to community care places increased demands on family caregivers. Families are now providing long-term care for chronically ill people with a variety of conditions. Caregiver research has, for the most part, explored burden, stress, and depression as outcomes of caregiving. There is little research assessing the quality of life (QOL) of long-term caregivers. In the research on quality of life, the patient is most frequently the focus. With the increased demand on families as caregivers, quality of life needs to be included as a variable in studying family caregivers for chronically ill individuals. The purpose of this paper is to discuss issues in studying quality of life in family caregivers of persons with chronic conditions. Conceptualization of quality of life is explored, measurement of quality of life examined and the research on quality of life reviewed, with a focus on the quality of life of the family caregiver. PMID- 10754910 TI - A nursing practice model for chronic illness. AB - As the 21st century approaches, one very important issue for nursing is the increased prevalence of chronic conditions such as arthritis. Self-management is one way that individuals can cope with the uncertainty and many changes chronic illness brings. The purpose of this article is to describe a nursing model that addresses the need for enhanced self-management skills and the rehabilitation nurse's role in this process. The model is based on the concept of intentional action and uses Orem's (1995) self-care deficit nursing theory as its conceptual structure. The expected outcomes of care for clients are effective self management skills and subsequently improved health status and quality of life. The model will be useful in the design of rehabilitation nursing research as well as in the planning of care for individuals with chronic illness. PMID- 10754912 TI - A multimodal approach to treatment of aggression in a severely brain-injured adolescent. AB - This case study presents a multimodal treatment approach to the rehabilitation of a severely brain-injured adolescent exhibiting significant aggressive and noncompliant behaviors. All treatments were mass-rehearsed in an overly concrete manner, given her cognitive deficits. Specific interventions included contingency management, stimulus control, problem solving, social skills training, relaxation training, anger management, and parent training. Results indicated a significant reduction in delinquent and aggressive behaviors and in social and attention problems. This success was maintained at an 8-month follow-up. These findings may assist nurses, rehabilitation specialists, and families to better manage aggressive and noncompliant behaviors that frequently follow a traumatic brain injury. PMID- 10754911 TI - 'Getting up from here': frail older women's experiences after falling. AB - Few researchers have explored older persons' experiences of falling. As part of a descriptive phenomenological study of older widows' experience of home care, 25 interviews were conducted with nine frail women who had fallen at least once at home. Some women were able to get up on their own or tried to do so. For them, the phenomenon of "getting up from here" and its component phenomena, such as "finding something solid [on which to pull up]," were primarily focused on maximizing the opportunities afforded by the home's environmental features. When the women needed help to get up, the phenomenon of "getting up from here" was understood as an exemplar of the home care experience; the women's intentions were focused on contacting or interacting with people who were already assisting them as they lived alone at home. Understanding the variability in the experience of falling is an essential basis both for sensitive interaction with elders who have fallen and for appropriate assessment of frail elders who are at risk of falling in their homes. PMID- 10754913 TI - Using focus groups in rehabilitation nursing. AB - The purpose of this paper is to describe the research method of focus groups and give an example of how it could be used to advance rehabilitation nursing in the area of poststroke adaptation or recovery. Stroke is a leading cause of death and disability in elderly Americans. Although much is known about the physical effects and treatments of stroke, little has been published on the adaptive behaviors or recovery process of stroke survivors. The focus-group technique can provide an "insider's" view of the adaptive behaviors of stroke survivors and is a cost-effective way to generate rich qualitative data to guide nursing practice. PMID- 10754914 TI - Management of antibiotic-resistant organisms in the rehabilitation setting. AB - Preventing the spread of infection is a team effort. Development and use of rehabilitation-based infection control practices for control of ARO nosocomial infections must be a priority for rehabilitation research. Ongoing infection control surveillance of ARO presence, along with monitoring of resistance patterns, equips infection control practitioners with scientific data to identify appropriate barriers for use in the rehabilitation setting. Modification of antimicrobial usage may offer hope for reversing some of the damage done. With the assistance of physicians, infection control practitioners, laboratory personnel and others, we can prevent the spread of these dangerous organisms. PMID- 10754915 TI - Using contingency management to reduce the incidence of pressure ulcers in a patient with a history of related surgeries. PMID- 10754916 TI - Cardiac rehabilitation: participating in an exercise program in a quest to survive. AB - Cardiovascular diseases are the major cause of death in the United States. The American Heart Association reports that approximately 500,000 persons die each year from heart attacks. Cardiac rehabilitation programs are based on objectives that would, when adhered to, extend and improve an individual's quality of life after experiencing a cardiac event. The purpose of this qualitative study was to explore and describe strategies that individuals who experienced a cardiac event used to overcome barriers associated with an exercise program. The sample consisted of six subjects (four females and two males) who were enrolled in a cardiac exercise program for 1 year or longer, and who continued to actively participate in at least two cardiac exercise classes per year. This study revealed that individuals who consistently participated in a cardiac exercise program did not use strategies to overcome barriers; rather, in their quest to survive, they used strategies to ensure their participation in the program. PMID- 10754917 TI - Continuity and discontinuity: the quality of life following stroke. AB - The purpose of this existential-phenomenological study was to investigate the experience of life after stroke rehabilitation, to provide a holistic view for nurses and others in providing care. Analysis involved a part-to-whole dialectic; selected transcripts, including the bracketing interview, were analyzed in an interpretive research group. There were 14 participants, with a 2-year median length of time since stroke; participants exhibited various disabilities. The world of the stroke survivor is grounded in a life of loss and effort from which emerged interrelated themes: independence/dependence, in control/out of control, and connection/disconnection with others. A fundamental aspect of these themes is a sense of continuity that coexists with discontinuity in the experience of self. The findings have implications for how nurses interact with stroke survivors, for goal-setting, and for specific strategies that focus on meanings for clients. PMID- 10754918 TI - Outcomes of nurse caring as perceived by individuals with spinal cord injury during rehabilitation. AB - A study was undertaken to describe the meaning, process, and consequences of nurse caring from the perspective of persons with spinal cord injuries (SCI) during rehabilitation. The constant comparative method, or grounded theory, was used in this prospective, exploratory project. In-depth tape-recorded interviews were conducted with 22 participants throughout their initial rehabilitation stay. A process of knowledge acquisition and decision making, facilitated by nurses and therapists in special caring relationships with the persons with SCI, resulted in outcomes of well-being, self-care, autonomy in decision making, independence, and hope, all of which contributed to the persons with SCI's reintegration of self before their discharge from inpatient rehabilitation. PMID- 10754919 TI - Social problem-solving partnerships with family caregivers. AB - The goal of this pilot study was to compare the effectiveness of home and telephone social problem-solving partnerships on primary family caregiver outcomes and to determine whether certain caregiver and stroke survivor characteristics influenced these outcomes. Thirty primary family caregivers were assigned to either a home visit, telephone contact, or control group. A registered nurse trained caregivers in the intervention groups in a series of seven telephone calls or home visits during a 12-week period to use social problem-solving skills in managing caregiving problems. Primary family caregiver outcomes were compared before the intervention, during the intervention (at 2 and 5 weeks after discharge), and after the intervention (at 13 weeks after discharge). Compared to the home and control groups, the telephone group had a significant reduction in depression, more positive problem-solving skills, and greater caregiver preparedness during the intervention, and improved, but nonsignificant depression, problem-solving, and caregiver preparedness scores postintervention. Race, age, and education were significant for selected outcomes. PMID- 10754920 TI - A dream awakened. PMID- 10754921 TI - Perceived accessibility versus actual physical accessibility of healthcare facilities. AB - This study addressed how healthcare clinics perceive themselves in regard to accessibility for persons with spinal cord injuries (SCI). All 40 of the clinics surveyed reported that they were wheelchair accessible; however, there was significant variability in the number of sites that actually met the guidelines of the Americans with Disability Act. In general, a person using a wheelchair could enter the building, the examination room, and the bathroom. The majority of sites did not have an examination table that could be lowered to wheelchair level. Most reported limited experience in working with persons with (SCI), yet they claimed to be able to assist with difficult transfers. Only one site knew about autonomic dysreflexia. Problems of accessibility appeared to be seriously compounded by the clinics' perception of how they met physical accessibility guidelines without consideration of the actual needs of persons with SCI. This study addressed the perception of accessibility as reported by clinic managers versus actual accessibility in healthcare clinics in a Midwestern metropolitan area for persons using wheelchairs. PMID- 10754922 TI - The roles of the pastor in the interdisciplinary rehabilitation team. AB - People with chronic illnesses and functional limitations may face a lifetime of changes and adjustments. Often, the onset of a long-term illness or disease requires a person to rethink values and develop new coping strategies in order to adapt to a life-changing event. At such times, people may draw on sources of spiritual support, finding comfort from a pastor or other clergy. This article describes key roles taken by the clergy who provide these services. Patients discharged from inpatient rehabilitation units have reported using faith and prayer as effective coping strategies. Religious faith can have a positive influence on emotions and may be directly related to improved functional ability. Disciplines of faith, such as solitude, silence, and meditation, may promote mental health. Although this article presents information from a Christian church perspective, readers should note that services should be considered from a wide range of spiritual representatives. PMID- 10754923 TI - The biopsychosocial adjustment of disabled elderly: a 1-year follow-up. AB - The objectives of this study were to identify and compare changes in the physical, social, and structural functions and in the family relationships of elderly patients upon hospitalization in a geriatric rehabilitation unit, and 1 year after discharge to the community (in the Negev region of Israel). The study was conducted on 88 elderly people with a mean age of 74 years. The Assessment of Dependency for Long-Term Care Benefits test was used twice as the study instrument for measuring the given objectives, once during the first week following admission to the unit and once 1 year later. At the end of the year there was improvement in most activities of daily living (ADL), a decrease in social roles, an increase in the number of illnesses, greater restrictions in diet, and an increase in use of social services. Changes were observed in the structure of the family social networks, the source of the primary caregiver, and living arrangements. Changes were not observed in the perceived family relationships. In both time periods, married elderly people evaluated family relations as better than did unmarried people. However, unmarried subjects demonstrated greater improvement in physical and social functions. Also, time had a significant effect on social function. Elderly people who were ill for less than 2 months prior to admission demonstrated greater social improvement than those whose illness lasted longer. PMID- 10754924 TI - Nurses' perspectives of encouraging clients' care-of-self in a short-term rehabilitation unit within a long-term care facility. AB - This article presents a study conducted on a short-term rehabilitation unit in a long-term care facility. The purpose of the study was to explore, through qualitative methods, nurses' perspectives of encouraging clients to care for themselves. Although the literature suggested that encouraging self-care does not occur, the findings of this study showed that nurses have a broader view than what is currently known in nursing as "self-care." Potential impediments to nursing practice were overcome by the development of nurse-client relationships through which the nurses encouraged clients in what is described throughout this study as "care-of-self." Rehabilitation nurses play an important role in helping clients to integrate and reclaim the care of their emerging new selves. Care-of self may be critical to clients not only in reaching their outcome goals for discharge but also in maintaining outcomes beyond discharge. PMID- 10754925 TI - Autonomic hyperreflexia. 1977. PMID- 10754926 TI - Designing a laparoscopic operating room. AB - This article focuses on 4 major aspects in the design of a laparoscopic suite. These areas include: Preliminary overview of the project: Planning, budgeting for CON (certificate of need), and justifying the project. Preconstruction phase and design of the suite: Interpreting blueprints, essential equipment, addressing standards and codes. Construction phase: Developing a project team, life safety, OSHA, JCAH issues, coordination of equipment purchases, infection control. Postconstruction of the project: Certification of gases, inservicing of personnel, addressing policy revisions. PMID- 10754927 TI - Renovation of a twelve-room OR suite. AB - Managing the renovation of an operating room is both challenging and rewarding. Good organizational and negotiating skills are needed for dealing with all of the various individuals and groups you will encounter as the project unfolds. Developing an operational plan will assist you to ensure your department continues to function in an organized and efficient manner throughout the project. PMID- 10754928 TI - Reading and understanding blueprints. AB - There is a common body of knowledge that must be familiar to all when interpreting architectural plans. Although health care practitioners may be experts in their own field, they may be unfamiliar with facility planning and design and therefore lack understanding of how to communicate effectively with the architectural team. The information provided is intended to familiarize nurses and other health care professionals with basic terminology and to provide an understanding of how to identify areas, structures, and dimensions on architectural plans. PMID- 10754929 TI - Planning capital equipment purchases for renovation: a practical guide. AB - The article stresses the importance of planning equipment acquisitions with the architect as well as with the nursing and medical staff. Relationships between the medical facility, the architect, the construction contractors, equipment vendors, and the purchasing department are explored. Pitfalls in planning, funding, writing specifications, and bid response evaluations are discussed and remedied. PMID- 10754930 TI - Safety considerations during construction and renovation. AB - As renovations occur in hospitals, it is important to implement proper controls to reduce hazards such as fire, air contaminants, unauthorized access, and potential infections. It is difficult to control all hazards in facilities with 500 beds or more. Planning, incorporating safety actions, educating, and invoking staff commitment make it possible to incorporate the controls necessary to maintain a safe environment for patients, staff, and visitors during all construction. PMID- 10754931 TI - Infection control during construction and renovation in the operating room. AB - This article describes a case study of reconstruction of an operating room suite. Suggestions for completing the renovations while controlling microorganisms are supplied. A comprehensive Infection Control Program is discussed, which includes essential environment controls in the operating room during any construction and renovation. A policy designed by the authors to minimize surgical infections during reconstruction is shared with the readers. PMID- 10754932 TI - New construction and renovation: lessons learned. AB - Construction and renovation of an operating room suite is an expensive and challenging project. Perioperative managers must use their knowledge and expertise to ensure that the vision of the facility is realized. Managers may be unfamiliar with facility planning and design and lack understanding of how to communicate effectively with the architectural team. Important items to consider and how to avoid costly errors are discussed. PMID- 10754933 TI - Funding for renovation: a rural hospital's experience. AB - A small rural hospital identifies the need for renovation of the Obstetric Department. A Women's Health group is formed and, together with hospital staff and community service groups, raises not only the necessary funds but also awareness of the need for the community hospital. PMID- 10754934 TI - HIV prevention and homosexual men: should we be optimistic about the new millennium? PMID- 10754935 TI - Are STIs underreported in rural Australia? PMID- 10754937 TI - Avoiding HIV transmission: women need more options. PMID- 10754936 TI - Why is Trichomonas vaginalis ignored? PMID- 10754938 TI - Sexual dysfunction associated with treatment of cervical cancer. PMID- 10754940 TI - Impact of HIV on adult (15-54) mortality in London: 1979-96. AB - OBJECTIVE: To describe the impact of HIV on mortality in men and women aged 15-54 in London. DESIGN: Combination of routine mortality statistics with reports of AIDS deaths adjusted for underreporting and change in address from time of report to time of death. Calculation of standardised mortality ratios (SMRs) for males including and excluding HIV comparing inner London and outer London with the rest of England and Wales. METHODS: Comparison of trends in all cause mortality and SMRs in males over time. Comparison of trends in HIV related deaths with other main causes of deaths in males and females in London. RESULTS: Age standardised rates for the rest of England and Wales showed a continual decline from 1979 to 1996 but rates in inner London males (ages 15-54) stopped declining around 1984-5 leading to a considerable increase in the SMR for inner London from 127 for 1985 7 to 171 for 1994-6. SMRs excluding HIV related deaths for inner London, however, showed no significant change over this time. There was a fall in HIV related mortality in 1996, though HIV was still the leading cause of death in males and second leading cause of death in females in inner London, and the fourth commonest cause of death in males in outer London. CONCLUSION: These data are the first to indicate the impact of HIV on mortality within a significant population in England and Wales. They show that public health priorities in London are different from the rest of the country. Analyses of trends of all cause mortality in people under 65 may mislead unless they take account of HIV. PMID- 10754941 TI - Factors associated with the time elapsed between the initial detection of HIV-1 antibodies and a diagnosis of AIDS among patients followed in Lyons University Hospitals. CISIH Collaborators. AB - OBJECTIVE: To identify the factors associated with a short period between the initial detection of HIV-1 antibodies and AIDS diagnosis among patients from Lyons, France. DESIGN AND METHOD: Prospective hospital based cohort study of patients diagnosed with AIDS in Lyons University Hospitals from 1994 to 1997. Cox regression was used to identify the variables independently associated with a short period between the first positive HIV-1 detection test and AIDS. RESULTS: 466 patients were studied, the mean period between the detection of HIV-1 antibodies and AIDS was 48 months and did not change across calendar years. Age < 46 years (hazard ratio (HR) 0.77, 95% confidence interval (CI) 0.58-1.00), HIV-1 transmission by heterosexual contact (HR 1.93, 95% CI 1.49-2.51), Pneumocystis carinii pneumonia (HR 1.67, 95% CI 1.28-2.17), or Kaposi's sarcoma (HR 1.42, 95% CI 1.06-1.90) as the first AIDS defining event, and CD4+ count < 100 x 10(3)/ml (HR 1.25, 95% CI 1.02-1.55) were associated with a short time interval between detection of HIV-1 antibodies and AIDS. CONCLUSION: Educational interventions focused on heterosexuals and those aged over 45 are needed to promote the early detection of HIV infection, in the hope of reducing transmission and improving individual prognosis. PMID- 10754939 TI - Increasing prevalence of genital herpes in developing countries: implications for heterosexual HIV transmission and STI control programmes. AB - BACKGROUND: The recognition that sexually transmitted infections (STI) facilitate HIV transmission among heterosexuals has led to a rejuvenated focus on improving STI control as a component of HIV prevention programmes in developing countries. While efforts so far have focused mainly on all STI, there is increasing evidence that genital ulcers facilitate a considerable proportion of HIV transmission among heterosexuals and that this effect has been underestimated. This paper focuses on the epidemiology of genital herpes in developing countries past and present. OBJECTIVES: To review the scientific literature about the epidemiology of genital herpes in developing countries and discuss the implications of the findings for STI control and HIV prevention programmes. SEARCH METHODOLOGY: A Medline search for June 1966 to August 1999 using the keywords, genital herpes, STD and developing countries, and genital ulcers in MeSH and free text. Abstract books from recent international AIDS conferences and other international STD conferences were reviewed. The annual reports of the medical officers of heath for Harare 1982-1998 and Durban 1989-1997 were reviewed to detect trends in genital herpes diagnoses. FINDINGS: Genital herpes, formerly regarded as a minor STI in most developing countries, has now emerged as a leading cause of genital ulceration in many countries where syphilis and chancroid were more prevalent previously. This increased recognition of genital herpes reflects both a change in the pattern of STI epidemiology through a decline in syphilis and chancroid as a response to HIV control programmes and improved techniques for diagnosing herpetic infection. Countries with significant heterosexual HIV epidemics also appear to have rapidly increasing numbers of genital herpes cases. CONCLUSIONS: The emergence of this herpes epidemic must be addressed through innovative strategies that will be viable, sustainable, acceptable, and effective in developing countries. In countries where genital herpes is a significant problem, local adaptation of WHO treatment algorithms should be made. STI service providers should be trained about issues around the transmission of herpes and how best to advise clients about dealing with, and recognising, recurrences. The effectiveness of antiviral treatment for genital herpes should be investigated in core groups at high risk of HIV. PMID- 10754942 TI - Viral subtype and heterosexual acquisition of HIV infections diagnosed in Scotland. AB - OBJECTIVE: As at December 1998, 87% of the estimated 33 million people living with HIV throughout the world resided in Africa and South East Asia. In Scotland (and the United Kingdom), a major public health concern has been that non-B subtypes of HIV which predominate in the regions above might enter the country and spread heterosexually among the indigenous population. The authors conducted an investigation to determine if, and to what extent, such transmission had occurred. METHODS: Stored blood samples from people who were diagnosed as HIV positive in central Scotland during 1995-7 and who were reported to have acquired their infection heterosexually, were identified. Sequence data were sought from each sample and, where obtained, viral subtype was assigned. For each case, viral subtype was linked to corresponding epidemiological details on heterosexual risk. RESULTS: Viral sequence was obtained from specimens for 53 of 59 cases. For 43 of the 53 cases, information on region of sexual contact was known. All 19 cases who had a sexual risk in Africa or Asia had a non-B subtype (A, C, or E) while 20 of 24 cases who did not report sexual contact in these regions had a B subtype (p < 0.0001). Of the remaining 10 cases, nine had a subtype B and one a subtype C virus. CONCLUSION: There is no evidence that non-B viral strains from developing countries have yet disseminated appreciably among indigenous heterosexual men and women within Scotland. Continuing to collect both demographic and molecular data from indigenous heterosexuals who are newly diagnosed with HIV would improve the chances of detecting rapidly any appreciable dissemination of non-B subtypes among this population if it were to occur. Such information would be helpful in informing HIV prevention strategies. PMID- 10754943 TI - Rapid detection of glycoprotein G gene for the diagnosis and typing of herpes simplex virus infection in genital herpes. AB - OBJECTIVE: To develop a new, rapid, and convenient technique for the diagnosis and typing of herpes simplex virus (HSV) in genital herpes (GH). METHODS: Using samples from skin vesicle fluid and urogenital mucosal swabs of subjects with GH, conventional polymerase chain reaction (PCR) (directed to polymerase gene: PCRpG) were compared with a newly developed PCR (directed to HSV glycoprotein gene: PCRgG). Both PCR methods were compared with virus isolation culture (VI) with indirect immunofluorescent staining (IIF). RESULTS: 80 samples from 40 GH patients (25 males) were tested. Positive results were seen in 52.5% (42/80) using PCRgG compared with 40% (32/80) by VI. Most of PCRgG positive samples (95.1%) were caused by HSV-2 infection. In samples from healing lesions, HSV was detected more often by PCRgG, than by VI. The results of typing by PCRgG and IIF were highly consistent. CONCLUSION: PCRgG is more sensitive than VI and PCRgG in detecting HSV in urogenital samples from subjects with GH. PCRgG is a convenient technique for the rapid detection and typing of GH. PMID- 10754944 TI - Impact of suppressive antiviral therapy on the health related quality of life of patients with recurrent genital herpes infection. AB - OBJECTIVE: To investigate whether suppressive antiviral therapy improves health related quality of life in patients with recurrent genital herpes. METHODS: Health related quality of life was measured using the disease specific recurrent genital herpes quality of life questionnaire (RGHQoL) as part of a randomized, double blind, 52 week, placebo controlled, dose ranging study of once and twice daily valaciclovir or aciclovir for the suppression of recurrent genital herpes in patients with six or more recurrences per year. RESULTS: Of 1479 participants, 1349 patients completed the baseline questionnaire. There were no significant baseline differences in RGHQoL score between any of the treatment groups. After 3 months there were significantly greater improvements in mean RGHQoL scores for all active treatment groups compared with placebo (p < 0.05). Mean RGHQoL score improvements from baseline remained significantly higher in the active treatment groups than in the placebo group after 6 and 12 months, indicating that the improved health related quality of life in patients receiving suppressive antiviral therapy was sustained over a prolonged period of time. CONCLUSION: Suppressive antiviral therapy is an effective strategy for improving the quality of life of patients with recurrent genital herpes. These improvements in quality of life are sustained over time, thus enhancing the clinical benefit in the long term management of this condition. PMID- 10754945 TI - British Co-operative Clinical Group national survey on diagnostic issues surrounding genital herpes. MSSVD Special Interest Group on Genital Herpes and the British Co-operative Clinical Group. AB - OBJECTIVES: To investigate the current use of diagnostic methods for genital herpes simplex virus (HSV) infection, to determine how information from these tests influences clinical practice, and to identify areas for future guideline development within genitourinary medicine (GUM) clinics in the United Kingdom. METHODS: National survey of 173 consultants in UK GUM clinics. RESULTS: Completed questionnaires were returned by 146 (84%) consultants. Cell culture was the first line diagnostic method for 133 (91%) respondents, the remaining 13 (9%) used antigen detection tests. Typing of isolates (HSV-1 or HSV-2) was available in their local laboratory to 109 (75%) clinicians; however, less than two thirds routinely pass this information on to their patients. Although 74 (51%) respondents had access to serological diagnosis, the majority of methods described were non-specific; only three (2%) had access to type specific tests. Only 81 (56%) respondents frequently (> 90% of the time) recommend notification of recent sexual partners of genital herpes patients. CONCLUSIONS: While access to culture based diagnosis is widespread, type specific serology has limited availability. Information on typing of isolates as HSV-1 or 2, although available in three quarters of centres, is underutilized in counselling patients. As HSV type influences both clinical and subclinical reactivation rates and may also affect probability of transmission, this is an important omission. Future guidelines need to address the optimal use of viral typing and new diagnostic tests to optimise health gain; there is also a need for evidence based recommendations about partner notification in genital herpes. PMID- 10754946 TI - Association of oncogenic human papillomavirus DNA with high grade cervical intraepithelial neoplasia: the role of cigarette smoking. PMID- 10754947 TI - Knowledge of Chlamydia trachomatis genital infection and its consequences in people attending a genitourinary medicine clinic. AB - OBJECTIVES: To assess knowledge of Chlamydia trachomatis infections, with a comparison of knowledge of Neisseria gonorrhoeae infections. METHODS: A cross sectional survey, by self completed questionnaire, of 200 subjects attending a genitourinary outpatient clinic. RESULTS: The response rate was 82% (90 male and 73 female). 51% of men (60% of females) had heard of chlamydia. 65 (82%) were unaware of the potential consequences of infection. 66% were unaware that the disease could be asymptomatic. Significantly more men (77%) than women (60%) had heard of gonorrhoea. Most participants (68%-82%) knew little of the possible consequences of this infection, and only 26% were aware that it could be asymptomatic. Knowledge was higher regarding fertility topics. There was no correlation between knowledge and either age or socioeconomic status. However, greater knowledge was displayed by those who read health information leaflets always or often. For both men and women, the preferred source of health information was the doctor. Other popular sources were health information leaflets, women's magazines, and television. CONCLUSIONS: Barely half the participants had heard of chlamydia and gonorrhoea. Further knowledge of either infection was very poor. There are serious implications for public health. The reasons for this are unclear and require exploration before targeted health promotion. Doctors and the popular media are acceptable, and potentially effective, sources of information. Acquisition of knowledge is important, both to reduce sexual risk taking behaviour and its consequences, and to allow for informed consent for chlamydia screening programmes. PMID- 10754948 TI - High prevalence of genital Chlamydia trachomatis infection in women presenting in different clinical settings in Jamaica: implications for control strategies. AB - OBJECTIVE: To determine the prevalence of genital Chlamydia trachomatis infection and risk factors in women attending family planning, gynaecology, and sexually transmitted disease (STD) clinics in Jamaica. METHODS: Endocervical specimens from 645 women including 238 family planning, 170 gynaecology, and 237 STD clinic attendees were examined for C trachomatis using a direct fluorescence assay (DFA) and culture. Investigations were carried out for the presence of other STD pathogens and demographic, behavioural, historical, and clinical data recorded for each participant. RESULTS: The prevalence of C trachomatis infection was 35%, 47%, and 55% in family planning, gynaecology, and STD clinic clients, respectively. The performance of the DFA was comparable to that of culture in screening for C trachomatis. Logistic regression analysis revealed that the independent risk factors for C trachomatis infection were non-barrier contraceptive methods in family planning clients (OR = 2.1; 95% confidence interval (CI) = 1.2-3.9; p = 0.0110), cervical ectopy in gynaecology clients (OR = 3.9; 95% CI = 1.4-10.6; p = 0.0076) and concomitant Trichomonas vaginalis infection in STD clients (OR = 3.5; 95% CI = 1.8-6.8; p = 0.003). Age, number of sex partners, and reason for visit were not identified as risk factors for C trachomatis infection. CONCLUSIONS: Consistently high prevalence of C trachomatis infection occurs in Jamaican women. Universal screening or presumptive treatment should be evaluated as prevention and control measures for C trachomatis infection in this population where all women appear to be at risk. PMID- 10754949 TI - Microscopic features of vaginal candidiasis and their relation to symptomatology. AB - OBJECTIVES: To document the microscopic features of vaginal candidiasis and to examine the relation between yeast morphology and patient symptomatology. METHOD: The study population comprised women undergoing screening for genital infection at a department of genitourinary medicine. RESULTS/CONCLUSION: Data were collected on 267 women of whom 234 were found to have vaginal candidiasis by vaginal culture. The remaining 33 patients had microscopic features of candidiasis (spores and/or hyphae) but were culture negative. Of the culture positive women, microscopy was positive in 182 (78%). "Spores only" were identified in 65 (28%), "hyphae only" in 16 (7%), and both "spores and hyphae" in 101 (43%). 68% of culture positive women were symptomatic, the commonest symptoms being irritation alone (27%) or irritation plus vaginal discharge (25%). No association was found between yeast morphology (spores, budding/non-budding; hyphae, branching/non-branching) as identified on microscopy of vaginal secretions and symptomatology. PMID- 10754950 TI - Safety and acceptability of vaginal disinfection with benzalkonium chloride in HIV infected pregnant women in west Africa: ANRS 049b phase II randomized, double blinded placebo controlled trial. DITRAME Study Group. AB - OBJECTIVES: To study the tolerance and acceptability in Africa of a perinatal intervention to prevent vertical HIV transmission using benzalkonium chloride disinfection. DESIGN: A randomized, double blinded phase II trial. SETTING: Prenatal care units in Abidjan (Cote d'Ivoire) and Bobo-Dioulasso (Burkina Faso). PATIENTS: Women accepting testing and counselling who were seropositive for HIV-1 and under 37 weeks of pregnancy were eligible. A total of 108 women (54 in each group) enrolled from November 1996 to April 1997, with their informed consent. INTERVENTION: Women self administered daily a vaginal suppository of 1% benzalkonium chloride or matched placebo from 36 weeks of pregnancy, and a single intrapartum dose. The neonate was bathed with 1% benzalkonium chloride solution or placebo within 30 minutes after birth. MAIN OUTCOME MEASURES: Adverse events were recorded weekly, with a questionnaire and speculum examination in women through delivery, and examination of the neonate through day 30. The incidence of genital signs and symptoms in the women and cutaneous or ophthalmological events in newborns were compared between groups on an intent to treat basis. RESULTS: The median duration of prepartum treatment was 21 days (range 0-87 days). Compliance was 87% for prepartum and 69% for intrapartum treatment, and 88% for the neonatal bath, without differences between the two groups. In women, the most frequent event was leucorrhoea; the incidence of adverse events did not differ between treatment groups. In children, the incidence of dermatitis and conjunctivitis did not differ between the benzalkonium chloride and placebo groups (p = 0.16 and p = 0.29, respectively). CONCLUSION: Vaginal disinfection with benzalkonium chloride is a feasible and well tolerated intervention in west Africa. Its efficacy in preventing vertical HIV transmission remains to be demonstrated. PMID- 10754952 TI - Estimating the prevalence of Trichomonas vaginalis, Chlamydia trachomatis, Neisseria gonorrhoeae, and human papillomavirus infection in indigenous women in northern Australia. AB - OBJECTIVE: To estimate more accurately the age specific prevalence of Trichomonas vaginalis, Chlamydia trachomatis, Neisseria gonorrhoeae, and human papillomavirus infection (HPV) in indigenous women living in urban, rural, and remote areas of the "Top End" of the Northern Territory (NT). DESIGN: Analysis of data obtained from two community based studies using self administered tampon specimens tested by polymerase chain reaction for sexually transmitted disease (STD). Data pertaining to the notifiable STDs (N gonorrhoeae and C trachomatis) were obtained from the NT health department. PATIENTS: 1090 indigenous women (age range 12-73 years) were enrolled when they attended local community health centres, family planning clinics, and STD clinics. The majority attended clinics in their home community in the course of "well women's checks" which encourage women to undergo screening for a variety of general medical conditions. RESULTS: The overall prevalence of T vaginalis, C trachomatis, N gonorrhoeae, and HPV was 0.25 (95% CI: 0.22-0.28), 0.11 (0.09-0.13), 0.17 (0.15-0.19), and 0.42 (0.37-0.48) respectively. Of the women found to be infected (excluding HPV), 25.5% had two or more of the above organisms detected. There was a statistically significant increase in the age specific prevalence of T vaginalis but a significant decrease with age for C trachomatis and HPV infection. There was no statistically significant change for N gonorrhoeae with age. CONCLUSIONS: STDs are hyperendemic in this population of indigenous women and the notification data significantly underestimate their prevalence. Distinct patterns of age specific prevalence were demonstrated, highlighting the need to tailor control strategies to specific epidemiological features. PMID- 10754951 TI - Syphilis serology and HIV infection in Harare, Zimbabwe. AB - OBJECTIVE: To determine the reliability of serological tests in detecting syphilis in a factory worker cohort and examine the impact of concurrent HIV infection on serological tests for syphilis. METHOD: Reactions to non-treponemal and treponemal antigens were tested using sera from a cohort of 3401 factory workers in Harare, Zimbabwe. The participants consented to regular testing for syphilis, by VDRL, and HIV using two ELISAs. All sera from men who were VDRL positive, and a random sample of VDRL negative sera, were tested by RPR, TPHA, and where appropriate FTA-Abs. From the results, men were defined as having no syphilis, active syphilis, incident syphilis, historic syphilis, or giving biological false positive reactions. RESULTS: 709 sera were examined from 580 men. There were 78 cases of active syphilis in the cohort, giving a prevalence of 2.3%, and the seroincidence was 0.25 per 100 person years of follow up. The prevalence of HIV in the cohort was 19.8%. There was a strong association between syphilis, whether active, incident or historic, and HIV seropositivity. With both HIV positive and negative sera the negative predictive values of VDRL and RPR were > 99.9% while the positive predictive value for VDRL (30%) was lower than for RPR (39%). Biological false positive reactions were detected in 0.5% of the cohort, with in most cases a transient rise in VDRL titres up to < 1/16. Higher false positive titres occurred in five men, each of whom was HIV positive. CONCLUSIONS: The VDRL is reliable in detecting possible cases of syphilis even in a community with a high prevalence of heterosexually transmitted HIV. There is need, however, for confirmatory tests. The prevalence of syphilis in this cohort is very low in comparison with other countries in southern Africa, but is consistent with recent data from Harare. Despite a strong association between syphilis and HIV it was clear that syphilis could not be counted as a major factor fueling the HIV epidemic in Zimbabwe. PMID- 10754953 TI - Penile intraepithelial neoplasia--a veiled lesion in genitourinary medicine. AB - Penile intraepithelial neoplasia (PIN) is a clinically well known condition. However, its diagnosis is often difficult. We present four cases of PIN, seen in our department. Various histological patterns ranging from PIN I to PIN III were noted in these cases. PMID- 10754954 TI - Sexually transmitted infections in a basic healthcare clinic in Dhaka, Bangladesh: syndromic management for cervicitis is not justified. PMID- 10754955 TI - Cinderella and the glass slipper: the growth and modernization of a specialty. PMID- 10754956 TI - Dry sex practices and HIV infection in the Dominican Republic and Haiti. PMID- 10754957 TI - Late presentation of HIV infected patients at Amsterdam outpatient HIV clinics. PMID- 10754958 TI - Biliary atresia in an infant born to an HIV positive mother. PMID- 10754959 TI - Trends in the incidence of syphilis and gonorrhoea in Novosibirsk region, western Siberia. PMID- 10754960 TI - Sexually transmitted infections in elderly people. PMID- 10754961 TI - Treatment of non-specific urethritis should be two weeks, not 1. PMID- 10754962 TI - Management of difficult anogenital warts. PMID- 10754963 TI - The global burden of injuries. AB - The traditional view of injuries as "accidents", or random events, has resulted in the historical neglect of this area of public health. However, the most recent estimates show that injuries are among the leading causes of death and disability in the world. They affect all populations, regardless of age, sex, income, or geographic region. In 1998, about 5.8 million people (97.9 per 100,000 population) died of injuries worldwide, and injuries caused 16% of the global burden of disease. Road traffic injuries are the 10th leading cause of death and the 9th leading cause of the burden of disease; self-inflicted injuries, falls, and interpersonal violence follow closely. Injuries affect mostly young people, often causing long-term disability. Decreasing the burden of injuries is among the main challenges for public health in the next century--injuries are preventable, and many effective strategies are available. Public health officials must gain a better understanding of the magnitude and characteristics of the problem, contribute to the development and evaluation of injury prevention programs, and develop the best possible prehospital and hospital care and rehabilitation for injured persons. PMID- 10754964 TI - Suffocated prone: the iatrogenic tragedy of SIDS. AB - Epidemiologic research has shown that prone sleeping is a major risk factor for sudden infant death syndrome (SIDS). In a public health review from Sweden, we explored the historical background of the SIDS epidemic, starting with the view of the Catholic Church that sudden infant deaths were infanticides and ending with the slowly disseminated recommendation of a prone sleeping position during the 1960s, 1970s, and 1980s. The story of the SIDS epidemic illustrates a pitfall of preventive medicine--the translation of health care routines for patients to general health advice that targets the whole population. False advice, as well as correct advice, may have a profound effect on public health because of the many individuals concerned. Preventive measures must be based on scientific evidence, and systematic supervision and evaluations are necessary to identify the benefits or the harm of the measures. The discovery of the link between prone sleeping and SIDS has been called a success story for epidemiology, but the slow acceptance of the causal relationship between prone sleeping and SIDS illustrates the weak position of epidemiology and public health within the health care system. PMID- 10754965 TI - Homicide prosecutions and progress toward workplace safety. PMID- 10754966 TI - When does a worker's death become murder? AB - During the past 2 decades, a growing number of manslaughter and even murder charges have been brought against employers in cases involving the death of workers on the job. In this commentary, the author reviews some of these recent cases and looks at other periods in American history when workers' deaths were considered a form of homicide. He examines the social forces that shape how we define a worker's death: as an accidental, chance occurrence for which no individual is responsible, or as a predictable result of gross indifference to human life for which management bears criminal responsibility. He asks whether there is a parallel between the conditions of 19th-century laissez-faire capitalism that led to popular movements promoting workplace safety and the move in recent decades toward deregulation and fewer restraints on industry that has led state and local prosecutors to criminalize some workplace accidents. Despite an increased federal presence, the activities of state and local district attorneys perhaps signal a redefinition of the popular understanding of employers' responsibility in maintaining a safe workplace. PMID- 10754967 TI - Work-related death: a continuing epidemic. AB - Worldwide, work-related illnesses and injuries kill approximately 1.1 million people per year. In 1992, an estimated 65,000 people in the United States died of occupational injuries or illness. Most estimates indicate that occupational diseases account for far more fatalities than occupational injuries. However, an accurate enumeration of occupational disease fatalities is hampered by a paucity of data, owing to underdiagnosis of occupational diseases and inadequacy of current surveillance systems. In this commentary, the authors review the epidemiology of death due to occupational disease and injury in the United States and discuss vulnerable populations, emerging trends, and prevention strategies for this ongoing public health problem. PMID- 10754968 TI - The effect of economic sanctions on the mortality of Iraqi children prior to the 1991 Persian Gulf War. AB - OBJECTIVES: This study examined the effect of sanctions on mortality among Iraqi children. METHODS: The effects of economic sanctions on health are not well known. Past studies on the effect of economic sanctions on mortality have suffered from unreliable data sources and the collinearity of sanctions with other negative economic events. We overcame these weaknesses by using individual child records from a retrospective survey of mothers conducted after the 1991 Persian Gulf War to examine the effect of sanctions on mortality among Iraqi children. Multivariate proportional hazards analysis was used to assess the effect of economic sanctions prior to war (from August through December 1990). RESULTS: We found that after controlling for child and maternal characteristics, when economic sanctions were entered into the proportional hazards equation, the risk of dying increased dramatically. This increase was highly significant statistically. CONCLUSIONS: Innovative application of robust epidemiologic research tools can contribute to assessments of health and well-being even under the methodological and practical constraints of comprehensive economic sanctions, but more research is needed. PMID- 10754969 TI - Frequency and correlates of intimate partner violence by type: physical, sexual, and psychological battering. AB - OBJECTIVES: This study estimated the frequency and correlates of intimate partner violence by type (physical, sexual, battering, or emotional abuse) among women seeking primary health care. METHODS: Women aged 18 to 65 years who attended family practice clinics in 1997 and 1998 took part. Participation included a brief in-clinic survey assessing intimate partner violence. Multiple polytomous logistic regression was used to assess correlates of partner violence by type. RESULTS: Of 1401 eligible women surveyed, 772 (55.1%) had experienced some type of intimate partner violence in a current, most recent, or past intimate relationship with a male partner; 20.2% were currently experiencing intimate partner violence. Among those who had experienced partner violence in any relationship, 77.3% experienced physical or sexual violence, and 22.7% experienced nonphysical abuse. Alcohol and/or drug abuse by the male partner was the strongest correlate of violence. CONCLUSIONS: Partner substance abuse and intimate partner violence in the woman's family of origin were strong risk factors for experiencing violence. Efforts to universally screen for partner violence and to effectively intervene to reduce the impact of such violence on women's lives must be a public health priority. PMID- 10754970 TI - Domestic violence and childhood sexual abuse in HIV-infected women and women at risk for HIV. AB - OBJECTIVES: The purpose of this study was to determine the prevalence and effect of domestic violence and childhood sexual abuse in women with HIV or at risk for HIV infection. METHODS: Participants with HIV or at risk for HIV infection enrolled in the Women's Interagency HIV Study. Childhood sexual abuse; all physical, sexual, and coercive violence by a partner; HIV serostatus; demographic data; and substance use and sexual habits were assessed. RESULTS: The lifetime prevalence of domestic violence was 66% and 67%, respectively, in 1288 women with HIV and 357 uninfected women. One quarter of the women reported recent abuse, and 31% of the HIV-seropositive women and 27% of the HIV-seronegative women reported childhood sexual abuse. Childhood sexual abuse was strongly associated with a lifetime history of domestic violence and high-risk behaviors, including using drugs, having more than 10 male sexual partners and having male partners at risk for HIV infection, and exchanging sex for drugs, money, or shelter. CONCLUSIONS: Our data support the hypothesis of a continuum of risk, with early childhood abuse leading to later domestic violence, which may increase the risk of behaviors leading to HIV infection. PMID- 10754971 TI - Early predictors of adolescent violence. AB - OBJECTIVES: This study sought to identify early predictors of adolescent violence and to assess whether they vary by sex and across different types and levels of violence. METHODS: Data from a 5-year longitudinal self-report survey of more than 4300 high school seniors and dropouts from California and Oregon were used to regress measures of relational, predatory, and overall violence on predictors measured 5 years earlier. RESULTS: Deviant behavior in grade 7, poor grades, and weak bonds with middle school predicted violent behavior 5 years later. Attending a middle school with comparatively high levels of cigarette and marijuana use was also linked with subsequent violence. Early drug use and peer drug use predicted increased levels of predatory violence but not its simple occurrence. Girls with low self-esteem during early adolescence were more likely to hit others later on; boys who attended multiple elementary schools were also more likely to engage in relational violence. CONCLUSIONS: Violence prevention programs for younger adolescents should include efforts to prevent or reduce troublesome behavior in school and poor academic performance. Adolescent girls may also profit from efforts to raise self-esteem; adolescent boys may need extra training in resisting influences that encourage deviant behavior. Programs aimed at preventing drug use may yield an added violence-reduction bonus. PMID- 10754972 TI - Lifetime prevalence of suicide symptoms and affective disorders among men reporting same-sex sexual partners: results from NHANES III. AB - OBJECTIVES: This study examined lifetime prevalence of suicide symptoms and affective disorders among men reporting a history of same-sex sexual partners. METHODS: In the third National Health and Nutrition Examination Survey, men aged 17 to 39 years were assessed for lifetime history of affective disorders and sexual behavior patterns. The study classified this subset of men into 3 groups: those reporting same-sex sexual partners, those reporting only female sexual partners, and those reporting no sexual partners. Groups were compared for histories of suicide symptoms and affective disorders. RESULTS: A total of 2.2% (95% confidence interval [CI] = 1.3%, 3.1%) of men reported same-sex sexual partners. These men evidenced greater lifetime prevalence rates of suicide symptoms than men reporting only female partners. However, homosexually/bisexually experienced men were no more likely than exclusively heterosexual men to meet criteria for lifetime diagnosis of other affective disorders. CONCLUSIONS: These data provide further evidence of an increased risk for suicide symptoms among homosexually experienced men. Results also hint at a small, increased risk of recurrent depression among gay men, with symptom onset occurring, on average, during early adolescence. PMID- 10754973 TI - Social context and geographic patterns of homicide among US black and white males. AB - OBJECTIVES: The recently published Atlas of United States Mortality depicted striking regional differences in homicide rates for Black and White males in the United States. This study examined these rates to gain an understanding of the contribution of social context to geographic variability in homicide. METHODS: Homicide rates were calculated by health service area for the years 1988 to 1992. The contributions of age, geographic location, urbanization, and sociostructural characteristics were evaluated by means of a weighted linear mixed effects model. RESULTS: Regional differences in urbanization explained much of the geographic variation in homicide rates, but sociostructural factors also had a significant impact. The results suggest that these effects operate similarly for White and Black males, although differences were found in the magnitudes of the effects for the 2 groups. CONCLUSIONS: Results point to a strong association between homicide and urbanization and socioeconomic conditions in all regions of the country for both Black and White males. These findings shed light on the potential correlates of high homicide rates in the United States in the near future. PMID- 10754974 TI - Firearm storage patterns in US homes with children. AB - OBJECTIVES: This study determined the prevalence and storage patterns of firearms in US homes with children. METHODS: We analyzed data from the 1994 National Health Interview Survey and Year 2000 objectives supplement. A multistage sample design was used to represent the civilian noninstitutionalized US population. RESULTS: Respondents from 35% of the homes with children younger than 18 years (representing more than 22 million children in more than 11 million homes) reported having at least 1 firearm. Among homes with children and firearms, 43% had at least 1 unlocked firearm (i.e., not in a locked place and not locked with a trigger lock or other locking mechanism). Overall, 9% kept firearms unlocked and loaded, and 4% kept them unlocked, unloaded, and stored with ammunition; thus, a total of 13% of the homes with children and firearms--1.4 million homes with 2.6 million children--stored firearms in a manner most accessible to children. In contrast, 39% of these families kept firearms locked, unloaded, and separate from ammunition. CONCLUSIONS: Many children live in homes with firearms that are stored in an accessible manner. Efforts to prevent children's access to firearms are needed. PMID- 10754975 TI - Effects of pool-fencing ordinances and other factors on childhood drowning in Los Angeles County, 1990-1995. AB - OBJECTIVES: This study estimated the effects of local pool-fencing ordinances and other factors on the rate of childhood drowning in Los Angeles County, California. METHODS: Stage 1 was a retrospective dynamic cohort study of all drownings among children younger than 10 years that occurred in residential swimming pools in Los Angeles County between 1990 and 1995. Stage 2 was a matched case-control study that compared pools in which childhood drownings occurred (cases) with randomly selected pools in which drownings did not occur (controls). RESULTS: The drowning rate was relatively high among toddlers (aged 1-4 years), boys, and African Americans and in areas with a high density of residential swimming pools. Pool-fencing ordinances were not associated with a reduced overall rate of childhood drowning. CONCLUSIONS: Local ordinances enacted in Los Angeles County before 1996 do not appear to have been effective in reducing the rate of childhood drowning in residential pools. Possible reasons for this ineffectiveness are insufficient building codes for isolating pools from homes, inadequate enforcement of the ordinances, and inadequate operation or maintenance of fencing equipment by pool owners. PMID- 10754976 TI - Psychiatric morbidity, service use, and need for care in the general population: results of The Netherlands Mental Health Survey and Incidence Study. AB - OBJECTIVES: This study examined the use of primary health care, mental health care, and informal care services, as well as unmet care needs, by individuals with different psychiatric diagnoses. METHODS: Data were derived from the Netherlands Mental Health Survey and Incidence Study and were based on a representative sample (n = 7147) of the general population (aged 18-64 years). RESULTS: In a 12-month period, 33.9% of those with a psychiatric disorder used some form of care; 27.2% used primary care, and 15.3% used mental health care. Patients with mood disorders were the most likely to enlist professional care; those with alcohol- and drug-related disorders were the least likely to do so. Higher educated persons who live alone, single parents, unemployed persons, and disabled persons were more likely to use mental health care. Unmet need for professional help was reported by 16.8% (men 9.9%, women 23.9%) of those with a disorder. CONCLUSIONS: Care use varies widely by diagnostic category. The role of general medical practitioners in treating persons with psychiatric disorders is more limited than was anticipated. Patients in categories associated with extensive use of professional care are more likely to have unmet care needs. PMID- 10754977 TI - Lifetime prevalence of and risk factors for psychiatric disorders among Mexican migrant farmworkers in California. AB - OBJECTIVES: In this study, the prevalence of and risk factors for 12 psychiatric disorders were examined by sex and ethnicity (Indian vs non-Indian) among Mexican migrant farm-workers working in Fresno County, California. METHODS: Subjects aged 18 through 59 years were selected under a cluster sampling design (n = 1001). A modified version of the Composite International Diagnostic Interview was used for case ascertainment. The effects of sociodemographic and acculturation factors on lifetime psychiatric disorders were tested. RESULTS: Lifetime rates of any psychiatric disorder were as follows: men, 26.7% (SE = 1.9); women, 16.8% (SE = 1.7); Indians, 26.0% (SE = 4.5); non-Indians, 20.1% (SE = 1.3). Total lifetime rates were as follows: affective disorders, 5.7%; anxiety disorders, 12.5%; any substance abuse or dependence, 8.7%; antisocial personality, 0.2%. Lifetime prevalence of any psychiatric disorder was lower for migrants than for Mexican Americans and for the US population as a whole. High acculturation and primary US residence increased the likelihood of lifetime psychiatric disorders. CONCLUSIONS: The results underscore the risk posed by cultural adjustment problems, the potential for progressive deterioration of this population's mental health, and the need for culturally appropriate mental health services. PMID- 10754978 TI - The relation of residential segregation to all-cause mortality: a study in black and white. AB - OBJECTIVES: This study investigated the influence of an aggregate measure of the social environment on racial differences in all-cause mortality. METHODS: Data from the National Longitudinal Mortality Study were analyzed. RESULTS: After adjustment for family income, age-adjusted mortality risk increased with increasing minority residential segregation among Blacks aged 25 to 44 years and non-Blacks aged 45 to 64 years. In most age/race/gender groups, the highest and lowest mortality risks occurred in the highest and lowest categories of residential segregation, respectively. CONCLUSIONS: These results suggest that minority residential segregation may influence mortality risk and underscore the traditional emphasis on the social underpinnings of disease and death. PMID- 10754979 TI - Local labor unions' positions on worksite tobacco control. AB - OBJECTIVES: This report describes local unions' positions on tobacco control initiatives and factors related to these positions. METHODS: A national random sample of local union leaders was surveyed by telephone. RESULTS: Forty-eight percent of local unions supported worksite smoking bans or restrictions, and only 8% opposed both a ban and a restriction. CONCLUSIONS: Support for tobacco control initiatives among local unions was higher than might be expected on the basis of previous evidence. Engaging unions in smoking policy formation is likely to contribute to the larger public health goal of reducing smoking and exposure to second-hand smoke among workers. PMID- 10754980 TI - Revisiting the effect of the Pap test on cervical cancer. AB - OBJECTIVES: This report documents the effect of not having had a Papanicolaou (Pap) test on survival with uterine cervical squamous carcinoma. METHODS: Data were derived from Charity Hospital of Louisiana at New Orleans Tumor Registry reports for 1984-1987 and 1996. RESULTS: During the 5 study years, 101 of 213 women (47%) with invasive carcinoma had not undergone a previous Pap test. From 1984 to 1987, the observed 5-year survival rate for 171 patients with invasive carcinoma was 43%. The observed 5-year rate for 107 patients with carcinoma in situ from 1984 to 1986 was 99%. CONCLUSIONS: The goal of a yearly Pap test for all women can be approached by a number of different routes, with the use of all health facilities augmented with collection of specimens by trained nonphysician personnel. PMID- 10754981 TI - Reproductive health services in rural Washington State: scope of practice and provision of medical abortions, 1996-1997. AB - OBJECTIVES: This study explored reproductive health care in rural Washington State, reasons given by providers for not offering abortions, and providers' willingness to use medical abortifacients. METHODS: Physicians, midwives, nurse practitioners, and physician assistants in rural Washington completed an inventory of reproductive health services that they provide, whether and why they do not perform abortions, and whether they would use medical abortifacients. RESULTS: Of the respondents, 89.2% reported providing reproductive health care. Only 1.2% reported performing surgical abortions, and 26.1% indicated that they would probably prescribe medical abortifacients. CONCLUSIONS: Few providers offer surgical abortions in rural Washington. Greater numbers report a willingness to prescribe medical abortifacients. PMID- 10754982 TI - Relative effect of genetic and environmental factors on body height: differences across birth cohorts among Finnish men and women. AB - OBJECTIVES: This study examined the change in heritability of adult body height across birth cohorts in Finland. METHODS: In 1981, cross-sectional questionnaires were completed by 10,968 twin pairs born before 1958. The effect of genetic factors was estimated via genetic modeling. RESULTS: Heritability increased from the cohort born before 1929 (0.76, 95% confidence interval [CI] = 0.65, 0.88 in men; 0.66, 95% CI = 0.55, 0.77 in women) to that born in 1947 through 1957 (0.81, 95% CI = 0.73, 0.87 in men; 0.82, 95% CI = 0.75, 0.89 in women). CONCLUSIONS: Heritability of height increased across Finnish birth cohorts born in the first half of this century and leveled off after World War II. Environmental factors, compared with genetic factors, appear to be more important among women than men. PMID- 10754983 TI - Community education efforts enhance postabortion care program in Ghana. PMID- 10754984 TI - OSHA matters: workers and managers say it backs them up. PMID- 10754985 TI - Ethics and schools of public health. PMID- 10754986 TI - Prognostic role of K-ras in patients with progressive colon cancer who received treatment with Marimastat (BB2516). AB - We determined the prognostic role of K-ras mutation in tumor tissue of patients with refractory colon cancer who received Marimastat (BB2516). DNA was extracted from paraffin-stored tumor tissue of 27 patients who previously failed 5 fluorouracil and were treated with BB2516. The presence of K-ras mutation was characterized by Polymerase Chain Reaction using ras- and p53-specific primers. ras and p53 oncoprotein expression was analyzed by an automated biotin-avidin immunoproxidase technique. Seventeen patients had a normal K-ras sequence and 10 patients had a K-ras mutation. Median survival of patients with a normal ras sequence was 330 days from the time of BB2516 treatment compared with 160 days for patients with a K-ras mutation (p = 0.0442, Wilcoxon; 0.0130 Log-Rank). No differences in age, sex, cancer stage, surgical treatment, or chemotherapy treatment were observed. Abnormalities involving ras expression did not affect survival. By comparison, median survival for patients with p53 mutation or p53 overexpression was both 158 days after BB2516 treatment. Patients having both K ras and p53 mutations had the poorest median survival of 113 days (p = 0.035). There is a suggestion by univariate analysis that the presence of a K-ras mutation may predict survival in patients with progressive colon cancer. Further assessment with larger patient numbers and multivariate analysis is indicated. PMID- 10754988 TI - Hepatic enzyme induction with phenobarbital and doxorubicin metabolism and myelotoxicity in the rabbit. AB - Doxorubicin (DOX) undergoes extensive liver metabolism. This study was designed to compare the pharmacokinetic and myelotoxicity profiles of DOX and metabolites with and without phenobarbital-associated hepatic enzyme induction. DOX was administered i.v. to eight rabbits with and without 7 prior days of oral phenobarbital, with venous blood samples collected between 0 and 72 hr for determination of plasma DOX and metabolite concentrations by high-performance liquid chromatography and complete blood counts obtained on days 1, 5, 7, 8, and 9. DOX AUC infinity, t1/2 beta and CLT values were significantly reduced by phenobarbital induction (PBI), while only the formation clearance of DOX metabolites was significantly changed. PBI had no effect on nadir neutrophil counts but was associated with significantly accelerated neutrophil recovery. Hepatic enzyme induction with phenobarbital significantly reduces plasma DOX exposure while increasing the rate of metabolite formation. These effects result in significant acceleration of neutrophil recovery. PMID- 10754987 TI - Reversion of primary hyperfibrinogenolysis in patients with hormone-refractory prostate cancer using docetaxel. AB - Tumor-associated proteases play a major role in determining the biologic behavior and aggressiveness of prostate cancer. Several authors have described the association between the increased levels of urokinase plasminogen activator in the plasma and in the malignant prostatic tissue with the metastatic potential of prostate cancer. However, the direct effect of this activity in producing fibronogenolysis in patients with prostate cancer has not been addressed. To evaluate the role of chemotherapy in reversing fibrinogenolysis in patients with prostate cancer, eight patients with hormone-refractory prostate cancer, bleeding, and laboratory evidence of primary hyperfibrinogenolysis were treated with docetaxel. The drug was given 48 hr after initiation of all supportive measures. Laboratory data, including plasminogen, alpha 2-antiplasmin, and fibrinogen, were recorded before and after treatment. Prostate-specific antigen (PSA) was measured at the time of referral and before subsequent cycles (3 weeks). Five patients had resolution of the fibrinolytic process after one cycle of treatment with docetaxel. This was demonstrated by improvement in both the laboratory parameters and the bleeding episodes. Further follow-up showed stabilization of the hematologic parameters and reduction in PSA values in these patients. Two patients died from uncontrolled bleeding despite all supportive measures. One patient did not demonstrate response to this treatment in terms of normalization of the fibrinolytic indicators or reduction in PSA. Primary fibrinogenolysis associated with metastatic prostate cancer is a serious complication. Docetaxel appears to be effective in reversing this process in some hormone-refractory patients. Although this response appears to be due to antitumor activity, a direct effect on the fibrinolytic pathway induced by the tumor cannot be excluded. Further work in this area is warranted. PMID- 10754990 TI - Experimental basis for cancer prevention by vitamin E. AB - Recent clinical trials have demonstrated the significant cancer preventive potential of vitamin E in many different cancer sites, ranging from oral and pharyngeal cancer to prostate cancer. There is an extensive experimental basis for this clinical cancer inhibition. The experimental background includes animal studies (experimental pathology, immunology and molecular biology, synergism, selectivity and safety), in vitro biochemical studies, and human studies (epidemiology and biomarkers, prevention of many pathologic entities other than cancer). PMID- 10754989 TI - Antitumor effects on mouse melanoma elicited by local secretion of interleukin-12 and their enhancement by treatment with interleukin-18. AB - To investigate the mechanism of the antitumor effect of locally secreted interleukin-12 (IL-12), we introduced the IL-12 p35 and p40 cDNAs into mouse B16 melanoma cells. IL-12 gene-transfected B16 melanoma (B16/IL12) showed marked retardation of tumor growth when implanted subcutaneously into syngeneic mice. In these mice, depletion of not only Natural Killer (NK) cells but also CD8+ T cells diminished the antitumor effect of locally secreted IL-12. Immunohistochemical analysis showed that NK cells and macrophages accumulated more densely at the center and periphery of B16/IL12 tumors than that of parental B16 tumors, whereas CD4+ T cells and CD8+ T cells accumulated sparsely only at the periphery of both transfected and untransfected tumors. Systemic treatment with interleukin-18 (IL 18) markedly inhibited the growth of B16/IL12 but did not influence the tumor growth of parental B16 cells in vivo. These results suggest that local IL-12 secretion can retard the growth of B16 melanoma mediated primarily by NK cells and indirectly by CD8+ T cells and that its antitumor effect is augmented by systemic treatment with the novel cytokine IL-18. PMID- 10754991 TI - Progress in the diagnosis and treatment of rhabdomyosarcoma and related soft tissue sarcomas. AB - Advances in the diagnosis and treatment of rhabdomyosarcoma and related soft tissue sarcomas continue in the Intergroup Rhabdomyosarcoma Study Group (IRSG) and European cooperative groups. The use of molecular biology techniques in soft tissue sarcomas are redefining the classic pathology of these small blue cell tumors. Improvements in imaging, radiotherapy, and surgery, in part, deserve credit for the better survival seen in all cooperative trials. These advances confound the interpretation of consecutively run chemotherapy trials using historical comparisons. The IRSG has reported improvement in the prognosis of both nonmetastatic and metastatic embryonal rhabdomyosarcoma as attributable to three, three-drug regimens that use cyclophosphamide at 2.2 g/m2 in either maintenance or induction and maintenance therapy. Patients of any age with metastatic, nonembryonal, and those over 10 years of age with metastatic embryonal rhabdomyosarcoma continue to have a poor prognosis, which even megatherapy has failed to change. The doublet of ifosfamide and etoposide in combination with vincristine, actinomycin D, and cyclophosphamide at 2.2 g/m2 achieved a remarkable 3-year survival of 58% in patients with metastatic rhabdomyosarcoma and undifferentiated soft tissue sarcoma. The topoisomerase I inhibitor, topotecan, has recently been found by the IRSG to have a 57% overall response rate in patients with metastatic alveolar rhabdomyosarcoma. Topotecan has completed testing with cyclophosphamide in a phase II window study in newly diagnosed patients with metastatic disease and has been incorporated into a randomized trial in intermediate risk patients in IRSG-V. Molecular studies in IRSG-V will be applied in the detection of occult bone marrow metastases and the evaluation of resection margins at initial and second-look surgery. Long-term follow-up will be required in patients with gross residual sarcoma randomized to conventional and hyperfractionated radiotherapy in IRSG-IV to assess late effects. Although older patients with unfavorable histology and metastatic disease continue to have a poor prognosis, the overall 5-year survival of children and adolescents with nonmetastatic and metastatic rhabdomyosarcoma is approaching 80%. As molecular discoveries advance the diagnosis and detection of rhabdomyosarcoma, it is hoped that the futuristic molecular based treatment strategies in development and early testing will further improve survival in high risk patients with metastatic soft tissue sarcoma. PMID- 10754992 TI - Taxol in the management of cancers of the breast and the ovary. PMID- 10754993 TI - Comparing cost-effectiveness analyses for the clinical oncology setting: the example of the Gynecologic Oncology Group 111 trial. AB - For the practicing oncologist, balancing quality of care with cost containment has become an unavoidable challenge. The development of new technologies, increased patient awareness, growth of managed care, and aging of our population represent conflicting interests in this endeavor. Medical literature has recently been inundated with economic analyses in an effort to approach some of these difficult questions, but often times it is difficult to see how this research applies to any particular oncologist's practice. This article identifies many of the key issues raised in the critical evaluation of cost-effectiveness analyses as they relate to the practicing oncologist. We offer suggestions on the interpretation of these studies to the clinical setting, using the recently published Journal of Clinical Oncology articles on cost-effectiveness analyses of paclitaxel-cisplatin as first-line therapy for ovarian cancer as examples. PMID- 10754994 TI - Telomerase: therapeutic potential in cancer. PMID- 10754995 TI - On the safety of silicone gel breast implants. PMID- 10754996 TI - Are silicone gel breast implants safe? PMID- 10754997 TI - On the safety of silicone gel breast implants. PMID- 10754998 TI - Are silicone gel breast implants safe? PMID- 10754999 TI - Molecular markers for colon cancer: where do we go from here? PMID- 10755000 TI - Radioimmunoscintigraphy in the management of colorectal and ovarian cancer: does identification improve outcome? PMID- 10755002 TI - [Imaging in the diagnosis of neonatal necrotizing enterocolitis and its complications]. PMID- 10755001 TI - Drugs for asthma. PMID- 10755003 TI - [Computed tomography in dental implantation. Technical and methodological aspects]. PMID- 10755004 TI - [Synovial healing in reconstructed cruciate ligaments. Our personal experience compared in single interventions and combined reconstructions]. AB - INTRODUCTION: The healing process of tendon grafts used in cruciate ligament reconstruction is called ligamentization. The tendon structural architecture changes progressively into the histological appearance of normal ligament. Amiel and Clancy have demonstrated that this process is time-dependent in anterior cruciate ligament (ACL) reconstruction, the tendon graft taking one year to become similar to a normal cruciate ligament. Three different maturation phases of anterior cruciate graft ligamentization can be seen at MRI: periligamentous proliferation, intraligamentous proliferation and definitive healing. We report our MR findings in the reconstruction of single ACL, posterior cruciate ligament (PCL), and of both ACL and PCL. MATERIAL AND METHODS: January 1995 to January 1999 we selected 60 patients submitted to arthroscopic cruciate ligament reconstruction. The ACL was reconstructed with the patellar tendon in 22 cases and the PCL in 23; fifteen patients underwent double reconstruction of ACL, with hamstring tendons, and PCL, with patellar tendons. Fifty-five patients were followed-up with MRI: 45 with a dedicated permanent magnet (Artroscan, Esaote Biomedica, Genoa, Italy) and 10 with a permanent low-field unit (AIRIS, Hitachi, Japan); the same technical parameters were used in all cases. Forty of 55 patients were examined at 1, 3, 6, 9 and 12 postoperative months, while 15 were followed-up longer (12 to 36 months postoperatively). At the first follow-up MR examination, 5 patients (2 ACL and 3 PCL) were excluded for incorrect tunnel position (1 case), hypertrophic scarring (2 cases) and new trauma (2 cases). MR findings were compared with clinical data of joint stability. RESULTS: MRI showed the different stages of the healing process in 20 ACL patients: proliferating soft tissue around the graft with the low signal intensity typical of tendons (stage I, 1-3 months postoperatively), the graft becomes progressively hyperintense (stage II, 3-9 months postoperatively), and finally the low signal intensity indicating completed ligamentization (stage III, 12 months postoperatively). As for PCL reconstructions, MR findings were similar but the process took longer, even 24 months. Finally, in the 15 cases of double reconstruction, both grafts were difficult to distinguish on T1-weighted images for a very long time (24-36 months postoperatively). DISCUSSION AND CONCLUSIONS: Relative to the ACL, ligamentization takes longer for PCL and combined ACL and PCL reconstructions, probably because the morphological changes in PCL and double grafts may be impaired by many factors, such as gravity, long bone tunnels, hemarthrosis, hyperplastic synovial reaction in the intercondylar notch; also, rehabilitation protocols differ in ACL from PCL patients. No signs of instability were found at physical or arthrometric examinations. MRI demonstrates the different stages of ligamentization and thus proves a useful tool for postoperative follow-up in cruciate ligament reconstructions. Contrast-enhanced (Gd) studies are reserved to the cases of graft impingement or if other abnormalities are suspected. PMID- 10755005 TI - [The role of magnetic resonance in the diagnosis of the os trigonum syndrome]. AB - PURPOSE: We investigated the yield of Magnetic Resonance Imaging (MRI) in hindfoot conditions, particularly the os trigonum syndrome, which are very difficult to diagnose clinically. MATERIAL AND METHODS: We examined 7 consecutive patients complaining of hindfoot pain for more than 4 months (male:female = 1:6; age range 16-22 years, average 18.6). Three patients practiced competitive sports and 4 ballet. We performed conventional radiography (orthogonal projections) and then MRI with a 0.5 T superconductive unit with surface coils; MR images were acquired with T1-weighted spin-echo (SE), T2-weighted gradient-echo (GRE), and fast inversion recovery (FIR) fat-suppressed sequences with 4 mm thickness and 0 mm gap. RESULTS: In 2 cases the os trigonum had irregular margins with subchondral sclerosis and widened synchondrosis. In 3 patients we found flexor hallucis longus tenosynovitis, likely caused by tendon compression and displacement within its sheath; there were neither os trigonum marrow edema nor synchondrosis widening. One patient had os trigonum hypertrophy, mild synchondrosis widening and marrow edema, in the os trigonum and the posterior aspect of talus. One patient had the os trigonum, but no signs referable to the os trigonum syndrome. CONCLUSIONS: In the posterior impingement syndrome, our objective is to show inflammatory changes in the posterior capsule of the ankle joint, adjacent ligaments, tendons and chondrosynovial surface. In these cases, the yield of conventional radiography and CT is rather poor, while MRI provides important information on soft tissues involvement, synovial reaction, chondral and subchondral bone injuries and the association of flexor hallucis longus synovitis, if present. MRI also yields detailed information for correct therapeutic approach. In conclusion, for the (differential) diagnosis of hindfoot pain in clinically suspected os trigonum syndrome, MRI appears to be the technique of choice, after conventional radiography, thanks to its noninvasiveness, multiplanarity, and high spatial and contrast resolution. PMID- 10755006 TI - [The topicality and use of the radiological exam in gunshot wounds of the limbs. An assessment of 132 cases]. AB - INTRODUCTION: Gunshot wounds of limbs are frequent injuries especially in Western countries. They can be single or associated with other penetrating gunshot wounds, for instance to the chest, abdomen and skull. PURPOSE: We investigated the current role and usefulness of conventional radiography in the assessment of gunshot injuries to limbs because, despite major advances in diagnostic imaging, this method remains the examination of choice in this condition. We stress the valuable contribution of conventional radiography to detection of bone blow-out fractures, multifocal traumatic bone changes, bone and joint injuries, bullet retention, and finally subcutaneous emphysema. MATERIAL AND METHODS: We retrospectively reviewed 132 cases of firearm injuries of limbs submitted to radiography March 1996 to July 1999. All the patients were men ranging in age 17 66 years (mean: 35). Radiography followed a preliminary physical examination, and follow-ups were carried out in the following days after orthopedic reduction of bone fractures or surgery with metal osteosynthesis. Emergency CT was performed first when chest, abdomen, or skull were involved. RESULTS: The lower limbs were involved three times as much as the upper ones; the leg was most frequently involved (61%), followed by thigh (61%), forearm and hands (24%), and feet (15%). The right lower limb was wounded in 65% of cases, especially tibia (55 cases) and femur (46 cases). Spiral injuries to bone diaphysis were the most frequent ones, followed by mixed fractures caused by cortical bone sinking from bullet impact. Bullets were retained in 60% of cases; subcutaneous emphysema was found in 78% of cases and vascular injuries in 25 cases in relation to disarranged fractures. We observed 4 arteriovenous fistulas during 3 years' follow-up. DISCUSSION AND CONCLUSIONS: Gunshot wounds to the limbs need a different clinical, diagnostic and therapeutic approach than thoracoabdominal and skull injuries, which require immediate and quick diagnosis and emergency treatment. The cases with injury to a primary artery from open and splintered fractures require emergency surgical reconstruction with vascular anastomosis and reduction of compound fractures, to prevent necrosis and amputation. Conventional radiography does depict the bullet and its site, subcutaneous emphysema, blow-out fractures, and the location of bone splinters. This permits adequate emergency surgery and an efficacious orthopedic approach, as well as selection of the cases to be submitted to clinical monitoring. PMID- 10755007 TI - [Changes in the blood-labyrinth barrier. Assessments by magnetic resonance]. AB - PURPOSE: Recent progress in magnetic resonance imaging (MRI), with contrast enhanced and steady-state sequences, allows fine depiction of labyrinth abnormalities related to neoplastic, inflammatory, ischemic, degenerative or traumatic disorders. We examined 488 patients with sensorineural hearing loss, vertigo or dizziness, but normal CT findings, to evaluate MR capabilities in showing labyrinth conditions. MATERIAL AND METHODS: January 1994 to May 1998, four hundred and eighty-eight patients with labyrinthine symptoms were submitted to CT. Sixty-eight of them, with normal CT findings, were also examined with MRI, which was performed using quadrature head or surface coils and a single dose (0.1 mmol/kg) Gd-DTPA administration. Conventional T1 and T2 high resolution SE images were acquired. The labyrinth was studied of 52 patients with normal CT findings and no abnormalities in the cerebello-pontine angle or internal auditory canal. RESULTS: Fourteen of 52 patients (27%) exhibited labyrinth enhancement from tumor (5%), hemorrhage (3%), infection (15%), surgical (2%) or radiosurgical (2%) procedures. GRASS sequences allowed differentiation of mass lesions (e.g., tumors, clots) from other conditions. CONCLUSION: Generally the labyrinth exhibits no contrast enhancement even after a triple Gd-DTPA dose. In inflammatory conditions, enhancement is not always diffuse, as expected, but may be focal. Spontaneous hemorrhages can account for labyrinth enhancement. In neoplastic conditions, enhancement may persist for as many as 6 months, and a mass effect against labyrinthine fluids may appear on GRASS images. Although there are no reports on labyrinthine degeneration after radiation therapy, one of our patients submitted to irradiation 7 years previously, had focal bilateral cochlear enhancement, which suggested a correlation with previous treatment. PMID- 10755008 TI - [The angiogram sign in pulmonary atelectases studied by spiral computed tomography. Its incidence and semeiologic value]. AB - PURPOSE: We investigated the yield of Helical CT in the study of lung vessels, the incidence of the angiogram sign and its actual value. MATERIAL AND METHODS: July 1997 to December 1998, we studied 30 cases of pulmonary collapse of different origin. We found 15 cases of passive collapse (10 from pleural effusion, 2 from diaphragmatic compression, 2 from traumatic pneumothorax, and 1 from isthmic aortic aneurysm), 12 cases of obstructive collapse (9 from bronchogenic carcinoma, 1 from mucoid obstruction, 1 from hilar lymphadenopathy, and 1 from mediastinal cancer), 2 cases of adhesive collapse and 1 case of round atelectasis. All the examinations were performed with the Helical technique during nonionic iodinated contrast agent injection (bolus, 300-350 mg/mL); we used an automatic injector set at 2-3 mL/s. We studied the images for the angiogram sign, that is hyperdense bands, usually longer than 2 cm, through the collapsed lung, which correspond to normal pulmonary vascular branches. RESULTS: The angiogram sign was found in 95% of passive (14/15) and in 80% of obstructive (10/12) collapses. However in 2 of those we also found some hyperdense spots referable to vessels seen on transverse slices. The angiogram sign was missing in 1 of 2 adhesive collapses because of vascular distortion from irradiation-related fibrosis. The "comet-tail" vascular pattern was seen in round atelectasis. We had no cases of cicatricial collapse. CONCLUSIONS: Thanks to its fast acquisition capabilities, Helical CT well depicts peak vascular enhancement, which permitted identification of the angiogram sign in several pulmonary collapse cases. Thus, this sign becomes even less specific, and just one of the signs of pulmonary consolidation. PMID- 10755009 TI - [Cine magnetic resonance with dobutamine following a myocardial infarct]. AB - PURPOSE: Dobutamine cine MRI is a new diagnostic imaging technique in the pretreatment (revascularization) assessment of myocardial infarction patients. We report the results of a comparative study of the diagnostic yield of dobutamine cine MRI with that of stress echocardiography in the assessment of viable myocardium. We also propose a new method for analysis of cine MR images, employing digital subtraction, aimed at decreasing subjectivity in the quantitative assessment of myocardial wall thickening. MATERIAL AND METHODS: Twenty-six patients (21 men and 5 women) with a history of myocardial infarction who were scheduled for revascularization were submitted to stress echocardiography and dobutamine cine MRI to evaluate contractile recovery of the segments considered akinetic or hypokinetic at baseline echocardiography. Dobutamine was administered in growing doses (5, 10, 15 gamma/kg/min). We considered 16 segments of the left ventricle in each patient. We performed a quantitative analysis of systolic wall thickening on individual cine MR frames both by manual measurements and by digital subtraction. RESULTS: In the 416 segments studied, we found 307 normokinetic, 64 scarred and 45 viable segments with stress echocardiography, versus 302 normokinetic, 83 scarred and 31 viable segments with dobutamine MRI. Wall thickening analysis on Cine MR images showed 268 normal, 68 scarred and 80 viable segments, versus 274 normal, 58 scarred and 84 viable segments with digital subtraction. Three months after revascularization 15 patients were examined to check contractile recovery of the segments considered as viable. Echocardiography had 79% sensitivity and 97% specificity, while cine MRI had 96% and 86%, respectively. Quantitative assessment of systolic wall thickening by cine MRI and digital subtraction had 96% sensitivity and 91% specificity, with no statistically significant differences between the two techniques. In patients with anteroseptal wall myocardial infarction stress echocardiography had 75% sensitivity and 97% specificity. In the subgroup of 13 patients with diaphragmatic or inferior wall infarction echocardiography sensitivity dropped to 68%, versus 96% of cine MRI, but its specificity was higher, namely 97 versus 86%. CONCLUSIONS: In anteroseptal infarction, echocardiography permits to distinguish viable myocardium and scarred myocardial tissue with good sensitivity and specificity, but cine MRI performs better. In inferolateral or diaphragmatic infarction, cine MRI has much higher sensitivity than stress echocardiography and thus makes the technique of choice to evaluate viable myocardium in these sites. The digital subtraction technique is as accurate as manual measurements, but reduces the error rate and permits quicker evaluation, particularly in subendocardial thickening. PMID- 10755010 TI - [An assessment of abdominal fatty tissue distribution in obese children. A comparison between echography and computed tomography]. AB - PURPOSE: Computed tomography (CT) and, more recently, ultrasound (US), have proved excellent tools for quantifying adipose tissue distribution. Body fat distribution is an important factor in the treatment of obesity and its complications. We investigated the correlation between CT and US measurements in pediatric obesity. MATERIAL AND METHODS: Forty obese children and adolescents aged 4.1-14.8 years were submitted to CT and US. Intra-abdominal, subcutaneous and total body fat were calculated (in cm2), with the CT image analysis software. The rectus muscle-spine and rectus muscle-aorta distances, as indicative of visceral fat thickness, were measured on US images with(out) compression. The distance between skin-fat and fat-rectus muscle interfaces was measured as subcutaneous fat thickness. We also compared US-CT findings with other morphometric variables--i.e., patient's (ideal) body weight and skin fold measures. RESULTS: At US, the rectus muscle-aorta and rectus muscle-spine distances ranged 2.4-7.5 cm (mean: 4.47 cm) and 3.6-8.9 cm (mean: 5.79 cm), respectively. The skin-rectus muscle distance ranged 1.2-7.5 cm (mean: 3.14 cm). A statistically significant correlation was found between the CT measurement of visceral fat and the aorta-rectus muscle and rectus muscle-spine distances (r = 0.80 and 0.74, respectively). The US measurements of subcutaneous fat were correlated with CT subcutaneous fat area (r = 0.82). No correlation was found between overweight, as calculated by body mass index, and CT or US fat. CONCLUSION: Our findings indicate that US is as useful as CT in evaluating body fat distribution in pediatric obesity. PMID- 10755011 TI - [The role of radiology in assessing patients with pathological obesity submitted to modified Mason's vertical gastroplasty. A study of 322 cases]. AB - PURPOSE: Mason's vertical banded gastroplasty (VBG) is one the most popular surgical techniques for the treatment of morbid obesity because it is effective and easy to perform and presents a low incidence of long-term complications. We report our personal results in 322 Mason's VBG patients who were followed-up radiologically. MATERIAL AND METHODS: 322 patients submitted to modified Mason's VBG underwent radiological follow-up, preferably with single contrast, to assess the shape and volume of the pouch and the presence of postoperative complications. The patients were 272 women and 50 men whose average weight was 124 kg (range: 78 to 218 kg). The women's average age was 37 years (range: 17 to 69) and the men's 36 (range: 19 to 64). We performed a double contrast examination, adapting the dosage of effervescent powders to the gastric pouch capacity, only in particular cases where a more detailed study of pouch surface was required. The examinations were performed at 1 month and 12 months postoperatively to evaluate the pouch shape and volume, in order to correlate surgical results with weight loss. Premature or unscheduled examinations were necessary only in some cases due to suspected complications or unsatisfactory weight loss. First we perform right anterior oblique projections with the patient standing, with a direct preliminary study of the epigastric region to locate the two metal clips. The patient is given barium in small swallows because large amounts might obscure the pouch by filling the fundus of the excluded stomach. Then we take left anterior oblique views to depict the neopylorus and the staple lines, and left posterior oblique projections in lateral and supine recumbency. RESULTS: We observed early postoperative complications such as pseudopylorus edema (8 patients) and intragastric hemorrhage (1 patient), and late complications such as pseudopylorus adherence to the gallbladder bed (1 patient), pseudodiverticular extroversion (1 patient), and axial enlargement (4 patients) of the pouch. The only two cases of staple-line dehiscence were both identified radiographically. In 305 patients with satisfactory weight loss no complications were observed with a pouch size of 45-70 mL (at 1 month) and 58-70 mL (at 12 months). DISCUSSION AND CONCLUSIONS: Upper gastrointestinal radiological studies permit to detect both early and late postoperative complications in Mason's VBG patients. They also provide data on the neostomach and permit to correlate the pouch volume with weight loss. PMID- 10755012 TI - [Multiphasic spiral computed tomography in hepatocarcinoma. An evaluation following different percutaneous ablative procedures]. AB - PURPOSE: To report our personal experience with helical CT evaluation of hepatocellular carcinoma treated with various percutaneous interventional procedures. We assessed both nodular response and the spectrum of changes within normal parenchyma. MATERIAL AND METHODS: December 1996 to September 1998 we examined with helical CT 41 patients (73 nodules in all) with hepatocellular carcinoma treated with percutaneous ablation therapies: conventional ethanol injection in 18 subjects (31 nodules), one-shot ethanol injection in 3 (8 nodules), radiofrequency thermal ablation in 16 (25 nodules), and combined chemoembolization and ethanol injection in 4 (9 nodules). CT performed was 4-27 days after the last session, acquiring biphasic volumetric images in 14 patients and triphasic volumetric images in 27. A second treatment with subsequent CT study was performed for 28 lesions; 15 underwent 3 serial studies and 6 underwent 4 studies. RESULTS: Compared with pretreatment findings, the diameter was unchanged in 62% of the nodules and increased in 38%. Morphology was unchanged in 63% of the lesions while in 37% a mild deformation toward the needle path or a more regular and round shape was evident. Borders were unchanged in 37% of the cases and modified in 63%, appearing well-defined in 73% and ill-defined in 27%. The necrotic portion had a low attenuation with a nodule-to-parenchyma gradient more evident on delayed than on venous and finally arterial acquisitions; 8% of the lesions were not recognizable on unenhanced scans. Residual viable tissue was identified in 44% of the nodules and quantified as 100% in 1% of all lesions, > 75% in 3%, > 50% in 4%, > 25% in 12%, < 25% in 23%. It was located centrally in 6% of the cases, peripherally in 12%, and eccentrically in 81%, and the shape was crescent in 66% of the cases, oval in 19%, and different in 16%. During the arterial phase the residual tumor appeared hyperdense in 97% of the nodules and isodense in 3%, while during the portal phase it was hyperdense in 22%, isodense in 28% and hypodense in 50%, and during the delayed phase hypodense in 100%. For what concerns the uninvolved parenchyma, lobar atrophy was present in 4% of the nodules, segmental atrophy in 8%, and subsegmental in 16%; caval thrombosis was found in 1 patient, portal thrombosis in 4. Arterioportal fistulas were identified in 5% of the nodules and areas of transient inhomogeneous attenuation in several cases. Subsegmental biliary dilation was detected in 4% of the lesions. CONCLUSION: Multiple-phase helical CT allows optimal depiction of primitive liver nodules treated with percutaneous interventional procedures and has a central role in the assessment of tumor response. Accurate is also the evaluation of changes or complications involving the surrounding parenchyma. Regarding the ablation effect, two typical features can be identified: after total necrosis nodular volume is unchanged or increased, borders are well defined, and density is low and homogeneous (especially in delayed phases), while after partial necrosis the diameter is not modified, margins are ill-defined, attenuation is less decreased, and peripheral tumor maintains high density in arterial phase and low density in delayed phases. As for the treatment procedure, we found that thermal ablation with radiofrequency and single-session ethanol injection cause more evident alterations within both nodule and hepatic parenchyma, while after multi-session ethanol injection changes are less dramatic and consequently more difficult to assess. PMID- 10755013 TI - [An orange a day keeps the scurvy away]. PMID- 10755014 TI - [From a lesion of the internal meniscus to the diagnosis of systemic mastocytosis. The magnetic resonance aspects in a case]. PMID- 10755015 TI - [Eosinophilic granuloma of the cranial vault: a color and power Doppler echographic study in a case]. PMID- 10755016 TI - [Lipoma of the psoas muscle. A rare case]. PMID- 10755017 TI - [Superficial siderosis of the central nervous system associated with Escherichia coli meningoencephalitis. A case report]. PMID- 10755018 TI - [Neurinosarcoma of the neck. A study of a rare case using color Doppler echography]. PMID- 10755019 TI - Rapidly growing bronchogenic adenocarcinoma in native lung following unilateral lung transplantation. CT findings in a case. PMID- 10755021 TI - [Cavernous angioma of the kidney. The color Doppler echographic, computed tomographic and magnetic resonance aspects in a case]. PMID- 10755020 TI - [Subdiaphragmatic extralobar pulmonary sequestration. A report of 3 neonatal cases]. PMID- 10755022 TI - [An endodermal sinus tumor in an adolescent female. A case report]. PMID- 10755023 TI - [Sialolithiasis with an unusual dilatation of the submandibular duct. A color Doppler echographic study of a case]. PMID- 10755024 TI - [It was not a breast cancer!]. PMID- 10755025 TI - [The rareness of modesty ... of imaging]. PMID- 10755026 TI - Experience in treating persons with HIV/AIDS and the legalization of assisted suicide: the views of Canadian physicians. AB - The objective of this study is to determine the opinions of a random sample of Canadian family physicians and a population of non-specialist physicians known to provide care to persons with HIV/AIDS about the legalization of physician assisted suicide for persons with HIV disease. In addition, we have attempted to ascertain the physician characteristics that may be associated with a favourable or negative opinion. Self-administered, anonymous questionnaires were mailed to 2,890 family physicians across Canada. Logistic regression analysis was used to determine whether physician characteristics were predictive of agreement with the legalization of physician assisted suicide. Of the respondents who had an opinion, 60% agreed with the legalization of physician assisted suicide. Multivariate analyses indicated that physicians who were living in the provinces of British Columbia (BC), Ontario, or Quebec (OR = 1.63, 95% CI: 1.10, 2.43) and who provided routine follow-up care (OR = 1.85, 95% CI: 1.30, 2.63) or palliative care (OR 1.66, 95% CI: 1.13, 2.44) to those with HIV disease were more likely to agree with legalization of physician assisted suicide. This analysis demonstrates a strong support for the legalization of physician assisted suicide for persons with HIV disease among physicians experienced in providing care to those affected. PMID- 10755028 TI - Symptom interpretation: implications for delay in HIV testing and care among HIV infected late middle-aged and older adults. AB - Physical symptoms and the attributions assigned to them are fundamental aspects of individuals' illness representations and influence health behaviours. The effects of the presence or absence of symptoms and the interpretation of these symptoms on the initiation of HIV testing and medical care are explored using data from a psychosocial study of HIV illness in late middle-aged and older men and women. The absence of symptoms negatively influenced willingness both to seek testing and to seek medical care. While the presence of symptoms would be expected to lead to testing and the initiation of medical care, the effect of symptoms was dependent on causal interpretations of the symptoms. Symptoms attributed (or misattributed) to other illnesses or to normal aging did not lead to initiation of testing or care. These results appear to be due to people's lay belief that illness must include symptoms and due to a resistance against accepting an illness identity. This research suggests that HIV education and counselling should emphasize the need for individuals at risk for HIV to seek testing and medical care even if symptoms of the disease are absent. PMID- 10755027 TI - Factors influencing marital stability among HIV discordant couples in northern Thailand. AB - The burden of HIV in stable relationships places emotional, economic and physical stresses on families. We compared the influence of HIV notification on marital partnerships in northern Thailand among a cohort of HIV discordant couples, and identified factors associated with marital disruption. Data were collected using in-depth interviews with both members of six separated or divorced couples and 13 couples whose relationship remained intact. Five factors influenced marital stability following HIV notification: longer duration of relationship; economic constraints, extended family members' opinions, especially parents; the existence of children from the marriage; and fear of stigmatization by community members. Social influences, both overt and perceived, are important in shaping marital behaviour and decision-making in HIV epidemic areas. HIV counselling needs to be extended beyond the individual seeking testing to include stable partners (and perhaps further, to include the extended family), although it is recognized that this is not the norm for most HIV testing centres. PMID- 10755029 TI - Is care and support associated with preventive behaviour among people with HIV? AB - Care and support should play a critical role in assisting people who are HIV positive to understand the need for prevention and to enable them to protect others. Differences in sexual risk reduction among 154 newly diagnosed HIV positive individuals from semi-urban Tanzania were examined using a randomized control design, which assigned a control group to regular health services and an experimental group to enhanced care and support. Data were collected at baseline, three months and six months on self-reported sexual risk behaviours, disclosure of serostatus, reproductive health and psychosocial support. Over the six-month period, significant risk reduction occurred among both groups, with most of the behaviour change occurring during the first three months, e.g. 86 respondents (56%) reported condom use at last intercourse at 3 months compared with 24(16%) at baseline (p = 0.05). Extra care and support did not lead to increased risk reduction among the experimental group. The study population as a whole significantly changed their behaviour, suggesting that in the short term, learning one is HIV-positive has an impact on risk reduction. PMID- 10755030 TI - Knowledge of prophylaxis treatment therapy among HIV-positive prisoners. AB - The present study examined the level of knowledge and understanding of Pneumocystis Carinii Pneumonia (PCP) treatment therapy among heterosexual and non heterosexual inmates (prisoners) with HIV-infection. This study seeks to determine if any differences exist between HIV-positive inmates based on sexual orientation, with reference to PCP treatment therapy. It is our contention that this effort will provide health professionals with valuable insight regarding delivering expanded care for HIV-infected individuals in incarcerated settings. The participants for this study were 99 HIV-positive inmates recruited between May and June 1995. These individuals were drawn from a list of all HIV/AIDS prisoners at the facility. More than 56% of the sample admitted their preferred sexual orientation as heterosexual compared to 43.4% non-heterosexual. More non heterosexual seropositive inmates were likely to report PCP was preventable (OR = 1.17, 95% CI = 0.56, 2.42), as well as noting its contagious attributes (OR = 1.41, 95% CI = 0.90, 2.21). Furthermore, non-heterosexual inmates were more likely to report they were taking prescribed medications for their infections (OR = 1.34, 95% CI = 0.76, 2.36) and that they knew the names of the prescriptions they were taking (OR = 1.13, 95% CI = 0.59, 2.14). Regardless of sexual orientation, it is consistent with the research that HIV-positive individuals may engage in risky sexual behaviour that may place non-infected individuals at risk of contracting the virus. Prison provides an opportunity to provide education to multiple at-risk populations. PMID- 10755031 TI - A night with Venus: partner assessments and high-risk sexual encounters. AB - This study examined partner assessments and high-risk sexual encounters among STD clinic patients. Participants were 42 English-speaking attendees of a public STD clinic in Southern California with ages ranging between 18 and 60. Twelve focus groups were conducted. Qualitative data from the focus groups were analyzed using grounded theory. The analysis resulted in delineating five temporally-ordered stages of causal sexual encounters. Participants reported that they did not use condoms when they perceived new sexual partners as being safe. Instead of directly addressing their partner's sexual history, they relied heavily on both visual and verbal cues to judge if their partner was clean, or disease-free. This assessment process reflects an error in judgement, particularly because most of the participants had already contracted a STD. Suggestions are provided for incorporating the information about the partner assessment process into AIDS educational programmes. PMID- 10755032 TI - Perception of vulnerability to HIV infection in a cohort of homosexual/bisexual men in Rio de Janeiro, Brazil. Oswaldo Cruz Foundation STD/HIV Prevention Group. AB - The present study addresses sociodemographic characteristics, knowledge and attitudes regarding HIV/AIDS, as well as risk behaviour and perception of vulnerability to HIV infection, in the first 295 homosexual and bisexual volunteers (excluding male commercial sex workers and transvestites) taking part in a cohort study in Rio de Janeiro. The sample has a higher socio-economic level than the remainder of the cohort, and than comparable strata of the general population, and a sound knowledge about how to protect themselves against HIV infection. In addition, an association between perception of vulnerability to infection and the practice of unprotected anal sex was observed. An association was also observed between higher levels of formal education and perception of vulnerability; however no association was found with markers for past and recent exposure to sexually transmitted infections (hepatitis B, HbsAG and syphilis). The results of the present study suggest that knowledge and awareness of risk do not easily translate into behaviour change, since significant proportions of the cohort continue to practise unprotected sex with both principal and casual partners, despite their awareness of HIV infection risks as well as preventive measures. PMID- 10755033 TI - Changing sexual attitudes and behaviour in China: implications for the spread of HIV and other sexually transmitted diseases. AB - In Imperial China sexual behaviour was regarded as an indispensable activity to reach harmony with the universe, through the unity of the interaction of two opposing forces: yin and yang. Sexual intercourse was accepted when linked to procreation within a family context, while an individual's sexuality was not considered important. Homosexuality was tolerated although not advocated, while masturbation was denounced. Since the One Child Family and Open Door policies in the 1970s and the economic reforms of the 1980s, attitudes towards sexuality in China have changed. Premarital sex has become widely accepted among young people and people in China are now more tolerant toward extramarital sex. Nowadays young people consider that love should dominate marriage and the quality of an individual's sex life is currently more valued than it used to be. Attitudes towards masturbation have become more tolerant and though homosexuality has been hidden by society for a long time, in recent years it has begun to be considered as a legitimate lifestyle choice. Attitudes towards sex and sexual behaviour have become recognized as an individuals' responsibility as long as no offence occurs against society or the interests of other individuals, resulting in the recognition of diversity of sexual behaviour. As part of the changing attitudes to sex and sexual behaviour, heterosexual transmission is becoming the most important route of HIV transmission in China. This is complicated by the internal migration of an estimated 120 million labourers moving from the countryside to the cities as the result of economic reforms, most of whom are sexually active young men. Unless addressed directly, these factors may add to the estimated 300,000 HIV-infected Chinese, further fuelling an already rapidly spreading epidemic. The ramifications of the Chinese HIV epidemic will not only be felt within China, but also within the surrounding Asian countries thereby fuelling the HIV pandemic. PMID- 10755034 TI - Implementation of a comprehensive AIDS education programme for schools in Masaka District, Uganda. AB - As part of a large IEC (Information, Education and Communication)/STD intervention trial, a 19-lesson, comprehensive school-based AIDS education programme was implemented and evaluated in 50 primary and 16 secondary schools in 12 parishes of Masaka District, Uganda. A series of three teacher-training and evaluation workshops spread over a year was held in each parish, between which teachers implemented the programme in the classroom. One hundred and forty-eight teachers were trained and about 3,500 students were subsequently exposed to the programme. Both teachers and students responded positively, which suggests that this type of programme has much to offer young people who attend school. However, some problems were encountered: language, programme content, community resistance to teaching about condoms, and several practical issues. Proposed solutions include flexibility with the English language policy, alternative approaches to role play activities, targeting influential individuals with information about the need for young people to learn about safer sex, and a parallel community based IEC programme to facilitate community acceptance of the need for the programme. In addition, implementation may be incomplete unless comprehensive AIDS education is fully incorporated into the curriculum, and properly examined. These findings are placed in the context of other life skills/AIDS education programmes being introduced both in Uganda and elsewhere in Africa. PMID- 10755035 TI - Current concepts of the mechanisms of menstruation. AB - Menstruation is the process whereby the superficial or functionalis layer of the endometrium lining the uterine cavity disintegrates and is removed from the uterine lumen towards the end of the luteal phase of a non-pregnant cycle. Within a period of 5-6 days the old lining is removed and a new lining regenerated without scarring occurring, a remarkable example of controlled tissue remodelling unparalleled in other organs. This chapter describes the phenomenon of menstruation and tissue repair and reviews the vasoconstrictor and inflammatory hypotheses for the initiation of menstruation. Particular emphasis is placed on the roles of steroids, prostaglandins, endothelin, lysosomal enzymes and matrix metalloproteinases, and the involvement of the fibrinolytic system and apoptosis. Repair of the endometrium is compared to the processes and mechanisms of wound healing. PMID- 10755036 TI - Health care implications of dysfunctional uterine bleeding. AB - Objective menorrhagia (measured menstrual blood loss of > 80 ml) affects approximately 10% of the population, while the subjective complaint of menorrhagia is experienced by almost a third of the female population of childbearing age. Consequently, large numbers of women consult their general practitioners annually and many are subsequently referred on for gynaecological opinion. Ineffective utilization of medical therapies may result in inappropriate referral and subsequently may result in operative intervention. The introduction of many minimally invasive techniques to manage menorrhagia has not brought about the expected reduction in surgical intervention rates but has merely increased the overall operative burden on the National Health Service (NHS). Recent advances in medical therapy may, for the first time, offer a rational and effective alternative to surgical therapy but long-term clinical and cost effectiveness studies are required. At present menorrhagia continues to be a major drain on health care resources. PMID- 10755037 TI - Medical management of dysfunctional uterine bleeding. AB - Complaints of excessive menstrual bleeding (menorrhagia) have a substantial impact on gynaecological services and in most cases no organic pathology is identified. Up to 50% of women who present with menorrhagia have blood losses within the normal range. Medical therapy is indicated for patients who do not wish surgery, or for whom surgery is unsuitable. Nonsteroidal anti-inflammatory drugs and tranexamic acid offer a simple therapy to be taken during menses, with reductions in menstrual blood loss (MBL) of 25-35% and 50% respectively. Danazol and the gonadatrophin-releasing hormone analogues are highly effective, but their side-effects make them suitable only for short-term use. The combined oral contraceptive pill and the levonorgestrel intrauterine system give reductions in MBL of 50% and 80%, with additional contraceptive cover. Cyclical progestogens are the most commonly prescribed therapy in the United Kingdom but they are ineffective for the management of ovulatory menorrhagia unless taken at high doses (10-15 mg daily) for 3 weeks out of 4. PMID- 10755038 TI - Bleeding arising from the use of exogenous steroids. AB - A range of hormonal contraceptives and hormone replacement therapies are widely and increasingly used in most societies. These preparations result in a wide variety of scheduled and unscheduled effects on uterine bleeding patterns, some of which may be quite disconcerting for the women who use them. There is now an extensive literature on the analysis of these bleeding patterns, the way in which different hormonal methods affect these patterns and the influence which they may have on women's choice to continue or discontinue use of a method. This review has concentrated particularly on the issues related to bleeding patterns with hormonal contraceptives, but many of the issues are also relevant to menopausal hormone replacement therapy. This is particularly the case with research into the complex vascular and molecular mechanisms underlying disturbances of bleeding, where there may well be substantial overlap. Perception and tolerance are important in determining a woman's approach to a change in her menstrual pattern, and her possible acceptance of counselling and treatment approaches which may improve her symptoms. PMID- 10755039 TI - Fibroids: pathophysiology and current medical treatment. AB - Uterine leiomyomas, or fibroids, are a major cause of abnormal uterine bleeding in women. These benign tumours develop during the reproductive years and their growth has been shown to be dependent on the ovarian steroid hormones oestradiol and progesterone. The growth promoting effects of these steroid hormones appear to be mediated through the local production of specific growth factors. Traditional treatment for leiomyomas has been surgical removal through either hysterectomy or myomectomy. Newer surgical techniques, such as hysteroscopic removal of leiomyomas, endometrial ablation, or uterine arterial embolization, are now being tested as effective but less invasive methods of treatment. Non surgical treatment of leiomyomas has been primarily through the use of gonadotrophin-releasing hormone agonists which suppress circulating oestradiol and progesterone levels by shutting down the pituitary-ovarian axis. The suppression in steroid hormone levels results in significant fibroid shrinkage, but long-term use of these compounds is not recommended because patients suffer significant bone loss. New antisteroidal compounds, such as the antiprogestin RU 486 and the selective oestrogen-receptor modulator raloxifene, are now being tested as possible therapeutic agents for fibroids. PMID- 10755040 TI - Dysfunctional uterine bleeding in adolescence. AB - Dysfunctional uterine bleeding in adolescence is usually not a pathological process but a physiological adjustment in the achievement of normal reproductive status. The source of the problem in the vast majority of patients lies in the development of an intact hypothalamo-pituitary ovarian axis which exhibits normal pulsatile gonadotrophin release. The achievement of this in point usually resolves the dysfunctional bleeding problem. Management of these problems is dependent on the clinical impact of blood loss and only requires medication when menstrual loss needs to be controlled. Special circumstances, such as mentally retarded children, require special attention in order to fulfil a number of clinical difficulties and almost always require medical intervention. PMID- 10755041 TI - Hysterectomy as treatment for dysfunctional uterine bleeding. AB - The surgical treatment of patients with dysfunctional uterine bleeding (DUB) is discussed in this chapter, including indications, techniques and complications. Hysterectomy is the definitive treatment for DUB; in most studies it has a higher rate of patient satisfaction than does hysteroscopic endometrial ablation. The vaginal or laparoscopic approach should be selected in this group of patients with small uteri, while laparotomy is seldom indicated. In general, the indications for vaginal hysterectomy and endometrial ablation differ from those of laparoscopic hysterectomy. Ablation and vaginal hysterectomy are done for hypermenorrhoea, while laparoscopic hysterectomy is best when pathology is present, usually adhesions, endometriosis or fibroids. PMID- 10755043 TI - Bioethics and genetic research in psychiatry. PMID- 10755042 TI - The alternatives to hysterectomy. AB - Hysterectomy is one of the commonest major operations, with 72,362 procedures performed in England in 1993. However, for women with dysfunctional uterine bleeding other less invasive surgical options have been developed. In particular, hysteroscopic endometrial ablation has been shown to be an effective therapy. Prospective randomized trials have shown that hysteroscopic surgery is associated with a shorter operating time, fewer complications, less analgesic requirement, a faster resumption of normal activities and work and savings in treatment costs. Psychological and social functioning also improves with no significant differences compared to hysterectomy. There is, however, a tendency for women randomized to hysterectomy to report higher rates of satisfaction. Several non hysteroscopic ablative techniques are currently being developed and tested. While attractive, as they do not require significant hysteroscopic skills and will probably prove to have fewer operative risks than hysteroscopic procedures, the results of these procedures are still in the evaluation phase. PMID- 10755044 TI - Patients with schizophrenia who smoke. Private disaster, public resource. PMID- 10755045 TI - Doctors on tribunals. A confusion of roles. AB - BACKGROUND: Mental health review tribunals are required to apply legal criteria within a clinical context. This can create tensions within both law and psychiatry. AIMS: To examine the role of the medical member of the tribunal as a possible mediator between the two disciplines. METHOD: Observation of tribunal hearings and panel deliberations and interviews with tribunal members were used to describe the role of the medical member. RESULTS: The dual roles imposed on the medical member as witness and decision-maker and as doctor and legal actor create formal demands and ethical conflicts that are hard, in practice, either to meet or to resolve. CONCLUSIONS: The structure for providing tribunals with access to expert psychiatric input and advice requires reconsideration. PMID- 10755046 TI - Role of risk assessment in reducing homicides by people with mental illness. AB - BACKGROUND: Improved risk assessment has been stressed as the way to reduce homicides by people with mental illness. The feasibility of predicting rare events needs examining. AIMS: To examine the findings of public inquiries into homicides by people with mental illness to see if they support the claim that better risk assessment would have averted the tragedy. METHOD: Analysis was made of the findings of the public inquiries between 1988 and 1997 in relation to the predictability and preventability of the homicides. RESULTS: Of the homicides considered by the inquiry panels, 27.5% were judged to have been predictable, 65% preventable and 60% of the patients had a long-term history containing violence or substantial risk factors for violence. CONCLUSIONS: Improved risk assessment has only a limited role in reducing homicides. More deaths could be prevented by improved mental health care irrespective of the risk of violence. If services become biased towards those assessed as high risk, then ethical concerns arise about the care of both violent and non-violent patients. PMID- 10755047 TI - Adolescent psychiatric in-patients. A high-risk group for premature death. AB - BACKGROUND: Research has demonstrated increased mortality rates in adolescent psychiatric in-patients. AIMS: To investigate this excess mortality by calculating standardised mortality ratios (SMRs) relative to cause of death, diagnosis, cohort and age. METHOD: A nationwide Norwegian sample of 1095 former adolescent psychiatric in-patients were followed up 15-33 years after first hospitalisation by record linkage to the National Death Cause Registry. RESULTS: The SMR was significantly increased for almost all causes of death investigated. In males, all psychiatric diagnoses had significantly increased SMRs, whereas in females, organic mental disorder, anxiety disorder and affective disorder had non significantly increased SMRs. The SMR was significantly elevated for all age spans and cohorts investigated. CONCLUSIONS: A broad prevention strategy is needed to combat the increased mortality rates found in adolescent psychiatric in patients. PMID- 10755048 TI - Assessing young people who deliberately harm themselves. AB - BACKGROUND: Many young people who harm themselves have chronic mental health or social problems or are at risk of future self-harm or even suicide. The accident and emergency (A&E) clinic is an important gateway to treatment. AIMS: To describe the psychosocial assessment of 12- to 24-year-old patients attending A&E clinics following deliberate self-harm (DSH) and to identify features of service management and provision which maximise specialist assessment. METHOD: A postal questionnaire was sent to a sample of one in three A&E departments in England. In a representative sample of 18 of these hospitals, staff were interviewed and 50 case notes per hospital were examined. RESULTS: Psychosocial assessment by non specialist doctors in A&E departments tended to be of variable quality, focused on short-term risk. Around 43% of patients aged 12-24 were assessed by a specialist; specialist assessment was associated with high admission rates and the presence of on-site psychiatric departments and DSH teams. CONCLUSIONS: Young DSH patients at risk often go unidentified; as a result their psychological problems may not be treated. Hospitals are frequently unaware of the proportion of patients discharged without adequate assessment. PMID- 10755049 TI - Parental high concern and adolescent-onset anorexia nervosa. A case-control study to investigate direction of causality. AB - BACKGROUND: Robust evidence that anorexia nervosa is preceded rather than accompanied by high-concern (overprotective) parenting is limited. AIMS: To look for evidence of parental high concern occurring before any onset of disorder. METHOD: Forty consecutive referrals of adolescent girls with DSM-III-R anorexia nervosa were compared with matched controls using obstetric records and maternal interviews. RESULTS: Index mothers reported higher rates of: near-exclusive child care (P = 0.02), infant sleep difficulties (P = 0.018), severe distress at first regular separation (P = 0.048), high maternal trait anxiety levels (P = 0.008) and later age for first sleeping away from home (P = 0.009). More index families had experienced a severe obstetric loss prior to their daughter's birth (P = 0.066). CONCLUSIONS: This study lends evidence to the clinical contention that high-concern parenting in infancy is associated with the later development of anorexia nervosa. This may derive, in part, from aspects of unresolved grief. PMID- 10755050 TI - Impact of hospitalisation on the outcome of adolescent anorexia nervosa. AB - BACKGROUND: Owing to the lack of controlled trials of treatment setting in adolescent anorexia nervosa, the benefits and costs of in-patient treatment are not established. AIMS: To clarify the relationship between a range of presenting features, treatment received and medium- to long-term outcome in adolescent anorexia nervosa. METHOD: A range of presenting variables were rated for 75 cases of DSM-III-R anorexia nervosa at presentation to an adolescent service, including the Morgan-Russell Global Assessment Score. Cases were followed up at 2-7 years and outcome rated according to reliable methods. Setting of treatment received was also recorded. RESULTS: Two out of 75 cases had died by the time of follow up. Adequate data for 72 enabled an outcome category to be assigned. The 21 who had received inpatient treatment had a significantly worse outcome than the 51 never admitted to hospital. Multivariate analysis suggests admission to be the major predictor of poor outcome. CONCLUSIONS: The benefits and costs of admission to hospital require further investigation, ideally in a randomised-controlled trial. The negative consequences of in-patient treatment are neglected in research. PMID- 10755051 TI - First-episode major depression in adolescents. Affective, cognitive and endocrine characteristics of risk status and predictors of onset. AB - BACKGROUND: There is little information on whether patterns of steroids precede and are associated with depressive onset. AIMS: To establish whether there is an association between salivary cortisol and/or dihydroepiandrosterone (DHEA) levels and depression independent of psychosocial risk. METHOD: Two subgroups of adolescents in the community at high (n = 181) and low (n = 65) risk for psychopathology were interviewed for recent psychiatric disorder at entry and again at 12 months. Salivary samples (08.00 and 20.00 h) for hormone estimations and self-reports on current mood and cognitive style were obtained at both assessments. RESULTS: Neither hormone was associated with risk status, current mood or cognitive style at entry. Of 31 onsets of major depression that occurred over the next 12 months, 30 came from the high-risk group but were not associated with any particular pattern of risk. Increased negative mood and feelings and DHEA (08.00 h) hypersecretion at entry were associated with subsequent major depression. CONCLUSIONS: Both negative mood and feelings and alterations in adrenal steroid function precede the onset of first-episode major depression in adolescents. Variation in levels of hormones may arise from more distal origins than recent life events and current ongoing difficulties. PMID- 10755052 TI - Cardiff depression study. A sib-pair study of life events and familiality in major depression. AB - BACKGROUND: An excess of both depression and undesirable life events in first degree relatives of probands with depression as compared with controls has been reported. This association may have reflected a familial factor in common. AIMS: To examine the familiality of life events and depression and whether there may be a common familial factor influencing vulnerability to depression and the experiencing of life events. METHOD: In a sib-pair design, 108 probands with depression and their siblings were compared with 105 healthy controls and their siblings for psychopathology and life events. RESULTS: The lifetime relative risk of depressive disorder in the siblings of depressed subjects as compared with siblings of controls was 9.74, although these groups did not differ in the life events measures. Several categories of events showed significant sibling correlations, but this was due to the same event affecting both members of the pair. CONCLUSIONS: Although depressive disorder was strongly familial, the familial effects on life events were largely explained by shared experiences. There was no evidence for a common factor influencing both depression and life events. PMID- 10755053 TI - Familial influence on variation in age of onset and behavioural phenotype in Alzheimer's disease. AB - BACKGROUND: Alzheimer's disease manifests considerable heterogeneity, the cause of which is unknown. AIMS: To determine the familial (genotypic) influence on phenomenology (phenotype) in Alzheimer's disease. METHOD: Affected sibling pairs with Alzheimer's disease were assessed for a range of cognitive and non-cognitive symptoms. Resemblance for phenotypic characteristics was estimated using intraclass correlations for continuous traits and by pairwise concordance for dichotomous traits. The relationship between age of onset and APOE genotype was examined using linear regression analysis. RESULTS: Significant familial effects on age of onset (intraclass correlation 0.41) and mood state (intraclass correlation 0.26), and a relatively high pairwise concordance for agitation (excess concordance 0.1) were found. The APOE locus was found to account for 4% of the variance in age of onset. CONCLUSIONS: Substantial familial influence on age of onset, depression and agitation suggests that genotype does influence phenotype in Alzheimer's disease. Establishing the molecular basis for this phenotypic variation may prove relevant to other neuropsychiatric disorders. PMID- 10755054 TI - Prospective controlled study of psychiatric out-patient non-attendance. Characteristics and outcome. AB - BACKGROUND: Psychiatric clinics have high non-attendance rates and failure to attend may be a sign of deteriorating mental health. AIMS: To investigate why psychiatric out-patients fail to attend, and the outcome of attenders and non attenders. METHOD: Prospective cohort study of randomly selected attenders and non-attenders at general adult psychiatric out-patient clinics. Subjects were interviewed at recruitment and severity of mental disorder and degree of social adjustment were measured. Six and 12 months later their engagement with the clinic and any psychiatric admissions were ascertained. RESULTS: Of the 365 patients included in the study, 30 were untraceable and 224 consented to participate. Follow-up patients were more psychiatrically unwell than new patients. For follow-up patients, non-attenders had lower social functioning and more severe mental disorder than those who attended. At 12-month follow-up patients who missed their appointment were more likely to have been admitted than those who attended. CONCLUSIONS: Those who miss psychiatric follow-up out-patient appointments are more unwell and more poorly socially functioning than those who attend. They have a greater chance of drop-out from clinic contact and subsequent admission. PMID- 10755055 TI - Extent and contributing factors of drug expenditure of injectors in Glasgow. Multi-site city-wide cross-sectional study. AB - BACKGROUND: Recent concern about drug use has focused attention on the illegal income generated by users. AIMS: To investigate factors associated with drugs expenditure and to estimate the cost of illegal acquisitions used to pay for drugs. METHOD: We collected self-report data from 954 current injectors, interviewed at multiple street, needle/syringe exchange and drug treatment sites throughout Glasgow. RESULTS: Injectors' mean weekly drug spending was 324 Pounds. The mean annual illegal drugs spend was estimated to be 11,000 Pounds per injector. We provide a central estimate--194 million Pounds per annum--of the retail value of goods acquired illegally by injectors in Glasgow in order to pay for drugs. Higher drug spends were associated with having been imprisoned more often and with those reporting acquisitive crime, drug dealing and prostitution. Treatment with methadone, among individuals who injected in the previous two months, was associated with a 20% reduction in a typical spend on drugs. CONCLUSIONS: Treatment effectiveness needs to be measured both in terms of health benefit and in terms of reduction in drugs expenditure and recidivism. PMID- 10755056 TI - Determinants of quality of life at first presentation with schizophrenia. AB - BACKGROUND: Quality of life (QOL) has gained importance as a global measure of social and clinical outcome in schizophrenia. AIMS: To identify the clinical correlates of QOL at the time of first presentation with schizophrenia. METHOD: Over two years, consecutive first-episode psychosis patients presenting to a catchment area psychiatric service underwent validated clinical assessments of premorbid adjustment, illness duration, symptoms and QOL. RESULTS: At presentation, subjects already had a diminished QOL. Although independent of gender and age at onset of psychosis, QOL was influenced by premorbid adjustment, duration of untreated psychosis and symptoms. CONCLUSIONS: Reducing the duration of untreated psychosis may have a beneficial effect on the subsequent QOL of patients presenting with schizophrenia. First-episode patients with a protracted duration of untreated psychosis or impaired premorbid adjustment may warrant specific treatment interventions to prevent the development of secondary handicaps. PMID- 10755057 TI - Phobic nature of social difficulty in facially disfigured people. AB - BACKGROUND: Over 390,000 people in the UK are disfigured. Facial disfigurement distresses sufferers markedly but has been studied little. AIMS: To compare fearful avoidance of people with a facial disfigurement with that of a group of patients with phobia. METHOD: Comparison of Fear Questionnaire agoraphobia, social phobia and anxiety depression sub-scale scores of 112 facially disfigured people (who scored high on Fear Questionnaire problem severity in three survey studies) with those of 66 out-patients with agoraphobia and 68 out-patients with social phobia. RESULTS: Facially disfigured people and patients with social phobia had similar Fear Questionnaire scores. In contrast, facially disfigured people scored lower on the agoraphobia sub-score but higher on the social phobia sub-score than did patients with agoraphobia. CONCLUSIONS: Facially disfigured people with psychological difficulties resembled people with social phobia on Fear Questionnaire social phobia, agoraphobia and anxiety/depression sub-scores but were less agoraphobic and more socially phobic than were people with agoraphobia. Facially disfigured people thus appeared to be socially phobic and to deserve the cognitive--behavioural therapy that is effective for such phobias. PMID- 10755058 TI - Effect of acute tryptophan depletion on CO2-induced anxiety in patients with panic disorder and normal volunteers. AB - BACKGROUND: Uncertainties remain about the role of serotonin in the aetiology and treatment of panic disorder. AIMS: To investigate the effect of reducing brain serotonin function on anxiety at rest, and following 5% CO2 provocation in normal controls and patients with panic disorder. METHOD: Twenty drug-free patients with DSM-III-R panic disorder and 19 controls received a tryptophan-free amino acid drink on one occasion and a control drink on the other in a double-blind, balanced protocol. 5% CO2 was given as a panic challenge after 270 minutes. RESULTS: Plasma tryptophan fell by more than 80% both patients and controls after the tryptophan-free drink. Tryptophan depletion did not alter resting anxiety. In patients alone, tryptophan depletion caused a greater anxiogenic response and an increased rate of panic attacks (9 v. 2, P < 0.05) after 5% CO2 challenge. No normal volunteers panicked. CONCLUSIONS: Serotonin may directly modulate panic anxiety in patients with panic disorder. This may underlie the efficacy of serotonergic antidepressants in treating panic disorder. PMID- 10755059 TI - Rapid eye movement sleep behaviour disorder, depression and cognitive impairment. Case study. AB - BACKGROUND: Rapid eye movement (REM) sleep behaviour disorder is a relatively new diagnostic category. It has never before been associated with a treatable depressive condition. AIMS: To report on a 74-year-old man with a history of depression and REM sleep behaviour disorder, associated with mild cognitive impairment. METHOD: Assessment using brain CT, MRI, PET, electroencephalography, neuropsychological testing and nocturnal polysomnography. RESULTS: Depression was treated with sertraline. Sleep laboratory studies supported a diagnosis of REM sleep behaviour disorder, which was treated with clonazepam. Sleep apnoea, revealed later, was treated with nasal continuous positive airways pressure. Brain MRI showed mild atrophy, but neuropsychological testing indicated no progressive cognitive deterioration. CONCLUSIONS: This case draws attention to REM sleep behaviour disorder and its potential interaction with depression and cognitive impairment, producing symptoms which can be mistaken for early dementia. The diagnosis of REM sleep behaviour disorder is easily missed, and it requires careful history-taking and sleep investigation in all suspected sufferers. Associated neurological, sleep and psychiatric conditions (including depression and cognitive impairment) may confound the diagnosis. PMID- 10755060 TI - Rabbit syndrome treated with olanzapine. PMID- 10755061 TI - Serotonin transporters in ecstasy users. PMID- 10755063 TI - One hundred years ago. Glasgow District Asylum, Gartloch (report for the year ending May 5th 1899) PMID- 10755062 TI - Substance misuse in first-episode psychosis. PMID- 10755064 TI - Magnetic resonance spectroscopy. Bridging the neurochemistry and neuroanatomy of schizophrenia. PMID- 10755065 TI - Stalking--a contemporary challenge for forensic and clinical psychiatry. AB - BACKGROUND: Stalking is causing pervasive and intense personal suffering and is an area of psychiatry that is currently overlooked. AIMS: To review demographic and clinical characteristics of stalkers as well as the psychological consequences for victims of stalking. METHOD: A Medline and PsycLit search was conducted on stalking, forensic psychiatry, personality disorders, de Clerambault syndrome and erotomania, with respect to the relevance of the articles selected for stalking. RESULTS: Stalkers are best thought of as a heterogeneous group whose behaviour can be motivated by different forms of psychopathology, including psychosis and severe personality disorders. CONCLUSIONS: There is a clear need to arrive at a consensus on a typology of stalkers and associated diagnostic criteria. The effectiveness of psychological and pharmacological treatments have not yet been investigated. Treatment may need to be supplemented with external incentives provided by the legal system. PMID- 10755066 TI - Three-year outcome of first-episode psychoses in an established community psychiatric service. AB - BACKGROUND: Changes in service provision, secular trends in substance misuse and changing social structures might affect outcome in psychosis. AIMS: To assess the three-year outcome of an inception cohort of first-episode psychoses treated in a modern, community-oriented service; to compare outcomes with an earlier cohort treated in hospital-based care; and to examine the predictive validity of ICD-10 diagnostic criteria. METHOD: Three-year follow-up (1995-1997) of an inception cohort of first-episode psychoses and comparison with two-year follow-up (1980 1982) of the Determinants of Outcome of Severe Mental Disorders (DOSMED) Nottingham cohort. RESULTS: On most outcome measures, non-affective psychoses had a worse outcome than affective psychoses. Affective psychoses had better outcome than previously reported. Substance-related psychoses had very poor occupational outcome. Similar proportions of the current and DOSMED cohort were in remission but the former were rated as having greater disability. CONCLUSIONS: In a modern community service, 30-60% of patients with first-episode psychoses experience a good three-year outcome. The ICD-10 criteria have good predictive validity. PMID- 10755067 TI - Long-stay patients discharged from psychiatric hospitals. Social and clinical outcomes after five years in the community. The TAPS Project 46. AB - BACKGROUND: There have been no large-scale prospective studies evaluating the transfer of care from psychiatric hospitals to district-based services. AIMS: We aimed to compare the quality of life of patients in two north London hospitals scheduled for closure with that in the community homes to which they were discharged. METHOD: The total long-stay population of Friern Hospital and several hundred long-stay patients in Claybury Hospital were assessed with a batch of eight schedules while in hospital. They were followed up after one year in the community and then at five years. RESULTS: Of the 670 discharged patients, 126 died before the five-year follow-up. Data were obtained on 523 (97%) of the survivors. There was no change in the patients' clinical state or in their problems of social behaviour. However, they gained domestic and community living skills. They also acquired friends and confidants. They were living in much freer conditions and the great majority wanted to remain in their current homes. CONCLUSIONS: Community care has enhanced the quality of life of this group of patients, involved in a well-planned and adequately resourced reprovision programme. PMID- 10755068 TI - Outcome of hospital-treated depression at 4.5 years. An elderly and a younger adult cohort compared. AB - BACKGROUND: Direct comparisons of the prognosis for treated depression in adult and elderly cohorts are few, but suggest higher morbidity and mortality in the elderly. AIMS: To examine outcome in two such groups after 4.5 years and compare results with those reported elsewhere. METHOD: Fifty-six adults (aged under 65) and 54 elderly people (over 65) with primary depression were assessed 4.5 years after receiving hospital treatment, and factors influencing the outcome were explored. RESULTS: Recovery rates were higher in the adults than in the elderly (42.8% v. 24%), largely due to higher rates of death (33%) and dementia (14.8%) in the latter group, who also suffered more serious health problems (62.9% v. 28.5%). Survival analysis showed no difference in the recovery time between cohorts, with over 90% recovered after 25 weeks. After deducting the natural deaths, melancholic illness proved a poor outcome predictor in the adults. CONCLUSIONS: The outlook for elderly depressed patients is poorer than for younger patients because of concurrent physical disease, a higher death rate and the development of dementia. PMID- 10755069 TI - Lifetime panic-depression comorbidity in the National Comorbidity Survey. Association with symptoms, impairment, course and help-seeking. AB - BACKGROUND: Most prior studies of panic-depression comorbidity have been limited methodologically by use of small clinical samples and incomplete analyses. AIMS: General population data were used to study the association of lifetime and recent (12 months) panic-depression comorbidity with symptom severity, impairment, course and help-seeking in the National Comorbidity Survey (NCS). METHOD: The NCS is a nationally representative survey of the prevalences and correlates of major DSM-III-R disorders in the US household population. RESULTS: Strong lifetime and current comorbidity were found between panic and depression. Comorbidity was associated with greater symptom severity, persistence, role impairment, suicidality and help-seeking, with many findings persisting after controlling for additional comorbid diagnoses. Findings did not differ according to which disorder was chronologically primary. CONCLUSIONS: Both lifetime and current panic-depression comorbidity are markers for more severe, persistent and disabling illness. Neither additional comorbid diagnoses nor the primary secondary distinction were important moderators of these associations. PMID- 10755070 TI - Synaptophysin gene expression in schizophrenia. Investigation of synaptic pathology in the cerebral cortex. AB - BACKGROUND: Decreased expression of proteins such as synaptophysin in the hippocampus and prefrontal cortex in schizophrenia is suggestive of synaptic pathology. However, the overall profile of changes is unclear. AIMS: To investigate synaptophysin gene expression in the cerebral cortex in schizophrenia. METHOD: The dorsolateral prefrontal (Brodmann area [BA] 9/46), anterior cingulate (BA 24), superior temporal (BA 22) and occipital (BA 17) cortex were studied in two series of brains, totalling 19 cases and 19 controls. Synaptophysin was measured by immunoautoradiography and immunoblotting. Synaptophysin messenger RNA (mRNA) was measured using in situ hybridisation. RESULTS: Synaptophysin was unchanged in schizophrenia, except for a reduction in BA 17 of one brain series. Synaptophysin mRNA was decreased in BA 17, and in BA 22 in the women with schizophrenia. No alterations were seen in BA 9/46. CONCLUSIONS: Synaptophysin expression is decreased in some cortical areas in schizophrenia. The alterations affect the mRNA more than the protein, and have an unexpected regional distribution. The characteristics of the implied synaptic pathology remain to be determined. PMID- 10755071 TI - Neighbourhood variation in incidence of schizophrenia. Evidence for person environment interaction. AB - BACKGROUND: Neighbourhood characteristics may influence the risk of psychosis, independently of their individual-level equivalents. AIMS: To examine these issues in a multi-level model of schizophrenia incidence. METHOD: Cases of schizophrenia, incident between 1986 and 1997, were identified from the Maastricht Mental Health Case Register. A multi-level analysis was conducted to examine the independent effects of individual-level and neighbourhood-level variables in 35 neighbourhoods. RESULTS: Independent of individual-level single and divorced marital status, an effect of the proportion of single persons and proportion of divorced persons in a neighbourhood was apparent (per 1% increase respectively: RR = 1.02; 95% CI 1.00-1.03; and RR = 1.12, 95% CI 1.04-1.21). Single marital status interacted with the neighbourhood proportion of single persons, the effect being stronger in neighbourhoods with fewer single-person households. CONCLUSIONS: The neighbourhood environment modifies the individual risk for schizophrenia. Premorbid vulnerability resulting in single marital status may be more likely to progress to overt disease in an environment with a higher perceived level of social isolation. PMID- 10755072 TI - Unpublished rating scales: a major source of bias in randomised controlled trials of treatments for schizophrenia. AB - BACKGROUND: A recent review suggested an association between using unpublished scales in clinical trials and finding significant results. AIMS: To determine whether such an association existed in schizophrenia trials. METHOD: Three hundred trials were randomly selected from the Cochrane Schizophrenia Group's Register. All comparisons between treatment groups and control groups using rating scales were identified. The publication status of each scale was determined and claims of a significant treatment effect were recorded. RESULTS: Trials were more likely to report that a treatment was superior to control when an unpublished scale was used to make the comparison (relative risk 1.37 (95% CI 1.12-1.68)). This effect increased when a 'gold-standard' definition of treatment superiority was applied (RR 1.94 (95% CI 1.35-2.79)). In non-pharmacological trials, one-third of 'gold-standard' claims of treatment superiority would not have been made if published scales had been used. CONCLUSIONS: Unpublished scales are a source of bias in schizophrenia trials. PMID- 10755073 TI - Trends in special (high-security) hospitals. 1: Referrals and admissions. AB - BACKGROUND: Special hospitals in England provide psychiatric care and treatment in high security. Their future is often questioned. AIMS: To test for variation in demand for high-security psychiatric services over one 10-year period. METHOD: This study was from the special hospitals' case registers and hospital records. The main measures were numbers and annual rates for referrals and beds offered; the Mental Health Act 1983 (MHA) classification of mental disorder; adjusted population rates by health region; admission episodes; legal category of detention; admission source and type of offence. RESULTS: Referrals to special hospitals showed no decrease during the 10 years; an apparent increase may reflect underrecording before 1992. Admissions fell by about 16% over the 10 years, but with regional variation. Women, civil cases, admissions under the MHA classifications of psychopathic disorder or mental impairment and directly from a court on a hospital order were most affected. There was an increase in admissions of pre-trial and sentenced male prisoners, and of transferred hospital order patients from other hospitals. CONCLUSIONS: There is continuing demand from all parts of the country for high-security hospital beds. The smaller numbers admitted appear to include more demanding cases. PMID- 10755074 TI - Trends in special (high-security) hospitals. 2: Residency and discharge episodes, 1986-1995. AB - BACKGROUND: It has been argued that many patients in special hospital beds do not need to be there. In the 1990s there were initiatives to discharge women and people with learning difficulties. AIMS: To test for trends in special hospital discharges and to examine annual resident cohorts. METHOD: This study was from case registers and hospital records. The main measures were numbers and annual rates for referrals and beds offered; the Mental Health Act 1983 (MHA) classification of mental disorder; adjusted population rates by region; admission episodes; legal category of detention; admission source and type of offence. RESULTS: The median annual number of residents was 1859 (range 1697-1910), with an 8% fall for the period. This particularly affected people in mental impairment categories. Numbers were sustained in the male mental illness groups. Discharges, mainly to other institutions, increased. There was no overall change over the 10 years in length of stay for treatment, but successive admission cohorts from 1986 did show some reduction, even with solely remand order cases excluded. CONCLUSIONS: Service planners need a longitudinal perspective on service use. Trends over 10 years to both fewer admissions and more discharges have reduced the special hospital population, but despite new treatments for schizophrenia, men under mental illness classification, as well as transfer from other secure settings, have gone against this trend. PMID- 10755075 TI - Suicide in psychiatric hospital in-patients. Risk factors and their predictive power. AB - BACKGROUND: Psychiatric hospital in-patients are known to be at high risk of suicide, yet there is little reliable knowledge of risk factors or their predictive power. AIMS: To identify risk factors for suicide in psychiatric hospital in-patients and to evaluate their predictive power in detecting people at risk of suicide. METHOD: Using a case-control design, 112 people who committed suicide while in-patients in psychiatric hospitals were compared with 112 randomly selected controls. Univariate analysis and multivariate analyses were used to estimate odds ratios and adjusted likelihood ratios. RESULTS: The rate of suicide in psychiatric in-patients was 13.7 (95% CI 11.7-16.1) per 10,000 admissions. There were five predictive factors with likelihood ratios > 2, following adjustment: planned suicide attempt, 4.1; actual suicide attempt, 4.9; recent bereavement, 4.0; presence of delusions, 2.3; chronic mental illness, 2.2; and family history of suicide, 4.6. On this basis, only two of the patients who committed suicide had a predicted risk of suicide above 5%. CONCLUSIONS: Although several factors were identified that were strongly associated with suicide, their clinical utility is limited by low sensitivity and specificity, combined with the rarity of suicide, even in this high-risk group. PMID- 10755076 TI - Childhood predictors of adult medically unexplained hospitalisations. Results from a national birth cohort study. AB - BACKGROUND: It has been suggested that adults with medically unexplained physical symptoms experienced greater ill-health then others (either in themselves or their families) during childhood. AIMS: To test these hypotheses. METHOD: We used data from the Medical Research Council (MRC) National Survey of Health and Development, a population-based cohort study established in 1946 (n = 5362). Subjects were followed from birth in 1946 until 1989 (age 43 years). As outcome, we used operationally defined medically unexplained hospital admissions at age 15 43 years. Exposure variables included childhood illness, and illness in parents during the childhood of the subjects. RESULTS: The risk set (n = 4603) comprised individuals still in the Survey at age 15. Ninety-five unexplained hospital admissions were identified. Subjects whose mothers reported below-average health in the father were at increased risk of subsequent unexplained admissions. Below average reported health in the mother was not associated with this increased risk. Defined physical diseases in childhood were not associated, but persistent abdominal pain at age 7-15 years was. CONCLUSIONS: Unexplained hospital admissions are associated with certain childhood experiences of illness, but defined physical illness in childhood is not a risk factor. PMID- 10755077 TI - Obsessive-compulsive disorder and delusions revisited. AB - BACKGROUND: The concept of fixed, unshakeable (delusional) beliefs within the context of obsessive-compulsive disorder (OCD) is one that has received varying amounts of attention in the literature, and has not yet received universal acknowledgement. There are good grounds for including these cases within the diagnostic concepts of OCD, with significant implications for clinical management. AIMS: To present cases with unusual OCD, in order to re-evaluate the issue of delusions and OCD. METHOD: The cases of five subjects with delusions in the course of obsessive-compulsive disorder are presented to illustrate 'delusional' OCD. The management and outcome of these cases are discussed. RESULTS: Fixity and bizarreness of beliefs in OCD occur on a continuum from 'none' to 'delusional intensity' and may fluctuate within subjects. CONCLUSIONS: The idea that these cases may represent a form of OCD has implications for management, as, if this is correct, they should be able to respond to appropriate behavioural and/or pharmacological strategies used in OCD. PMID- 10755078 TI - Psychiatric symptoms among clients seeking treatment for drug dependence. Intake data from the National Treatment Outcome Research Study. AB - BACKGROUND: Many people with substance use disorders are vulnerable to other psychiatric disorders and present to addiction treatment services with comorbid psychiatric symptoms. AIMS: To describe the prevalence of recent psychiatric treatment and current psychiatric symptoms and explore links between substance misuse, personal/social functioning and symptom severity. METHOD: Subjects were 1075 adults recruited to the National Treatment Outcome Research Study (NTORS), of whom 90% were opiate-dependent. Psychiatric symptoms at intake were recorded using sub-scales from the Brief Symptom Inventory. RESULTS: Recent psychiatric treatment was reported by one in five subjects. Psychiatric symptom levels were high and females had elevated scores on all scales. Symptoms were elevated among opiate users who were also frequent users of benzodiazepines, alcohol and, in particular, stimulants. Gender, physical health, drug dependence and personal relationship problems were more powerful predictors of psychiatric symptoms than substance use. CONCLUSIONS: Addictions service providers should be vigilant to psychiatric problems among their clients at intake to treatment. Psychiatric symptoms are more closely linked to polydrug use than to opiate use in this population. PMID- 10755079 TI - Smoking habits and plasma lipid peroxide and vitamin E levels in never-treated first-episode patients with schizophrenia. AB - BACKGROUND: Lipid peroxidation may be increased in schizophrenia, due to the illness, lifestyle or medication. AIMS: To determine plasma lipid peroxide levels and serum vitamin E and A levels in first-episode never-treated people with schizophrenia and in controls. METHOD: Thirty in-patients with a first episode of schizophrenia or schizophreniform psychosis were recruited, as were controls matched for gender, age, smoking and dietary status. Blood samples were taken, smoking status was recorded and body mass index measured. RESULTS: There were no significant differences between patients and controls in plasma peroxide levels. Seventy-three per cent of the patients smoked. Patients who smoked had a higher mean lipid peroxide level than non-smokers. Seventy-seven per cent of patients and 70% of controls had a ratio of vitamin E to cholesterol of less than 5. Body mass index was lower in patients than in controls. CONCLUSIONS: As a result of the high prevalence of smoking this group shows increased lipid peroxidation. Low serum ratios of vitamin E to cholesterol in both patients and controls suggest an unsatisfactory diet. PMID- 10755080 TI - Authorship of clinical trial reports. PMID- 10755081 TI - Psychological model of post-stroke major depression. PMID- 10755082 TI - Psychiatry and civil unrest in Northern Ireland. PMID- 10755083 TI - Studying grief in adults with learning disabilities. PMID- 10755084 TI - Maternal eating disorder and mother-child conflict. PMID- 10755085 TI - Compulsory treatment in anorexia nervosa. PMID- 10755086 TI - Resource allocation for mental health care. PMID- 10755087 TI - Fluoxetine-induced anaesthesia of vagina and nipples. PMID- 10755088 TI - Spontaneous orgasms--an epileptic case without structural correlate. PMID- 10755089 TI - One hundred years ago. 1900. PMID- 10755090 TI - The Heart Outcomes Protection Evaluation (HOPE) study: relevance to ophthalmological practice? PMID- 10755091 TI - Severe sclerokeratitis due to Pseudomonas aeruginosa in noncontact-lens wearers. AB - PURPOSE: To review the clinical presentation, treatment and outcome in four cases of severe anterior segment infection by Pseudomonas aeruginosa unrelated to contact lens wear. METHODS: Four cases presenting over an 18 month period were reviewed. RESULTS: The cases had variable presenting features and outcomes. Complications such as persistent infection, corneal thinning and phthisis bulbi were noted. Possible factors influencing adherence and tissue disruption are discussed. CONCLUSIONS: Suspicion of infection by P. aeruginosa and prompt isolation of the organism is needed early in the course of disease. Intensive and prolonged treatment with parenteral and topical antibiotics combined with judicious use of topical steroid gives the best chance of a favourable outcome. PMID- 10755092 TI - Decreased visual acuity in patients with cytomegalovirus retinitis and AIDS. AB - PURPOSE: To investigate the causes of decreased visual acuity in patients with cytomegalovirus (CMV) retinitis in the acquired immunodeficiency syndrome (AIDS). METHODS: All human immunodeficiency virus (HIV)-positive patients seen in two ophthalmology units over a 15 month period from September 1996 were included in this retrospective study. A detailed ophthalmic examination was performed on all patients and in addition those with CMV retinitis underwent serial fundus photography. Decreased visual acuity was defined as a best corrected visual acuity < or = 6/12. CMV and retroviral treatment, CD4+ count and HIV viral load were also documented for each patient. RESULTS: Of 110 patients seen over the 15 month period, 26 (41 eyes) had a diagnosis of CMV retinitis. Twelve patients (16 eyes) with CMV retinitis had decreased visual acuity. The decreased visual acuity in 7 eyes was initially due to the CMV retinitis involving the macula and the optic nerve. Retinal detachment was responsible in 2 eyes and optic nerve atrophy in 1 eye. In 6 eyes (4 patients) the decreased visual acuity was due to a maculopathy--cystoid macular oedema and/or an epiretinal membrane in the presence of an inactive zone 2 or 3 CMV retinitis--with all these patients exhibiting a vitritis of varying grade. The decreased visual acuity in the maculopathy subgroup was irreversible in all except 1 eye, and 2 eyes in this category later developed a cataract. CONCLUSION: In this series, CMV-retinitis-'related' maculopathy was a major (38%) cause of decreased visual acuity, occurring in the absence of zone 1 retinitis and despite inactive peripheral CMV retinitis. A varying degree of vitritis was an associated feature in all these patients. This study therefore highlights maculopathy as an important and previously unrecognised significant cause of visual morbidity in CMV retinitis. PMID- 10755093 TI - Familial thrombophilia and retinal vein occlusion. AB - PURPOSE: To perform a pilot study on the prevalence of familial thrombophilia in all cases of retinal vein occlusion with no known risk factors. METHODS: Over the 1 year study period 71 patients presented with a new diagnosis of retinal vein occlusion (age 28-90 years). Fifty-five (77%) were excluded because of local predisposing factors. The remaining 16 (23%) had a full risk factor history taken and blood investigations of rheological factors and thrombophilia including tests for the factor V Leiden mutation, prothrombin G20210A allele and hyper homocystinemia. RESULTS: Of those with no local predisposing factors, 3 patients had antiphospholipid antibodies, 3 had raised fibrinogen levels, 4 had hyper homocystinemia and 1 was heterozygous for the Leiden mutation. Other lifestyle risk factors such as obesity, smoking and a positive family history of venous thrombosis were not uncommon. No patient had the prothrombin G20210A variant. CONCLUSIONS: It seems likely that several risk factors, both genetic and acquired, need to be present for thrombosis to occur. In investigating a new patient with a retinal vein occlusion one should test for hypertension, glaucoma and diabetes mellitus. Estimation of plasma viscosity and a full blood count are cheap investigations which may reveal neoplasia or vasculitis, and lipid levels should be estimated. In a young patient or one with an unexpected vein occlusion and a personal or family history of thrombosis, a hypercoagulable state may rarely be identified. This additional testing should include testing for antiphospholipid antibodies and a full thrombophilia screen including the factor V Leiden mutation, homocysteine and the prothrombin variant as part of a clinical trial. Until the role of these markers in thrombosis is better defined in relation to causation of retinal vein occlusion and treatment has been shown to improve outcome, we can not recommend them for routine testing. If a hereditary defect is found, referral should be made to a hematologist and consideration given to anticoagulation and screening of family members to prevent further thrombotic episodes. Retinal vein occlusions are multifactorial in origin except in rare cases. PMID- 10755095 TI - The influence of retinopathy of prematurity on ocular growth. AB - PURPOSE: Retinopathy of prematurity (ROP) stage 3 eyes that require treatment have a greater tendency to myopia compared with eyes with mild ROP. As the mechanisms controlling this myopia are as yet ill understood, we undertook this study to investigate what effect the initial stage of ROP and modality of treatment had on ocular growth. METHODS: Eighty-five children were assessed. All children were refracted and underwent 'through-the-lid' biometry using the Zeiss Humphrey biometer 820. The printout obtained was then recorded on video so that the scan could be captured on computer for formal calibration and measurement by a masked observer. Differences in the distribution of variables between the stages of ROP were analysed by one-way analysis of variance, non-parametric Kruskal-Wallis one-way analysis of variance or Mann-Whitney U-test as appropriate. RESULTS: A difference between the stages of ROP was apparent only for posterior segment length (PSL) (R: p = 0.03; L: p = 0.05) and a borderline difference for anterior chamber depth (ACD) (R: p = 0.06; L: p = 0.06). However, if stage 3 was divided into categories of treated and untreated, axial length (AL) achieved borderline significance (R: p = 0.07; L: p = 0.05) but with no difference between laser-treated and the other stages for AL. Lens thickness (LT) also appears to be influenced by type of treatment (R: p = 0.06; L: p = 0.13). Myopia was associated with stage 3 (R + L: p = 0.0001) and if stage 3 was subdivided the significance was maintained only for the laser- and cryotherapy treated eyes. CONCLUSIONS: Laser-treated eyes were less myopic than those treated with cryotherapy. AL does not explain all the myopia found in stage 3 treated eyes. The study confirms the tendency towards anterior segment arrest in stage 3 ROP. PMID- 10755094 TI - Congenital hypertrophy of the retinal pigment epithelium and APC mutations in two Chinese families with familial adenomatous polyposis. AB - PURPOSE: Congenital hypertrophy of the retinal pigment epithelium (CHRPE) exists almost exclusively among familial adenomatous polyposis (FAP) patients with adenomatous polyposis coli (APC) mutations between codon 413 in exon 9 and codon 1387 in exon 15. We investigated the locality of APC mutations in relationship to the occurrence of CHRPE in two Chinese families with FAP. METHODS: Genomic DNA of available members of two unrelated Chinese FAP families was investigated for sequence alteration in the APC gene by polymerase chain reaction and direct sequencing. All subjects were examined by binocular indirect ophthalmoscopy (BIO) for CHRPE. RESULTS: A mutation in exon 6, Arg216Stop, was identified in one patient with FAP and CHRPE. An Arg283Stop mutation in exon 8 was found in 5 members in another family; 4 of them had FAP and all had small hypopigmented white lesions, probably a new type of CHRPE. CONCLUSIONS: We found two mutations, Arg216Stop and Arg283Stop, upstream of codon 413 in FAP patients presenting with CHRPE. Arg283Stop has not previously been reported in such patients. A large scale study on CHRPE and APC mutations in Chinese FAP patients is required to affirm their inter-relationships and the significance of the hypopigmented white lesions. PMID- 10755096 TI - Should all patients with candidaemia have an ophthalmic examination to rule out ocular candidiasis? AB - PURPOSE: To determine the incidence of ocular candidiasis and length of ophthalmic follow-up required to rule out ocular candidiasis in candidemia patients. METHODS: We prospectively studied patients with candidemia at our institution. Eligibility criteria included a dilated ophthalmological examination within 72 h of fungemia. Patients without ocular candidiasis on initial examination had follow-up dilated ophthalmoscopy performed at 1, 2, 4, 12 and 24 weeks. RESULTS: Between May 1996 and March 1997 a total of 50 patients with fungemia were identified of whom 31 were included in the study; 15 excluded patients died before an initial examination was performed. The overall incidence of ocular candidiasis was 26% (8/31 patients), all manifested as chorioretinitis. Five patients (16%) had ocular candidiasis on their initial examination. One of 21 patients (5%) without ocular candidiasis on initial examination developed ocular candidiasis within 1 week. Two of 16 patients (13%) without ocular candidiasis on initial examination or at 1 week follow-up developed ocular candidiasis within 2 weeks. No evidence of ocular candidiasis occurred in the 12 patients with follow-up at 4 weeks, the 8 patients with follow-up at 12 weeks and the 4 patients with follow-up at 24 weeks. CONCLUSION: The incidence of ocular candidiasis among hospitalized patients is clinically significant. We recommend ophthalmological follow-up for development of ocular candidiasis for at least 2 weeks after an initial negative eye examination. PMID- 10755097 TI - Passing the DVLA field regulations following bilateral macular photocoagulation in diabetics. AB - PURPOSE: At present the Driver and Vehicle and Licensing Agency (DVLA) of the UK requires all diabetic patients with laser treatment in both eyes to undergo a binocular driving field assessment. Increasingly we find that many such patients have undergone only macular laser treatment without panretinal photocoagulation. We aimed to ascertain the frequency and causes of failure of the binocular driving field test after bilateral macular laser treatment performed according to a set protocol. METHODS: A retrospective case notes study was combined with a prospective recall of patients for visual field assessment. All diabetic patients under the care of one consultant who had had bilateral macular laser treatment (without panretinal photocoagulation) over a 4 year period were identified and the case notes studied. Patients who could not achieve a corrected binocular acuity of 6/9 were excluded, as were patients with glaucoma. Patients who had not had the DVLA test were recalled to perform the test, whether or not they had an intention to drive. Failure was defined utilising strict criteria on the Esterman Programme of the Humphrey Analyser. RESULTS: A total of 138 patients were identified. Fifty-four were not eligible (21 because of poor acuity, 29 had died or were too ill to participate and 4 for other reasons). Of the 84 who were eligible, 64 had visual fields performed and 20 declined to attend. Sixty-three of 64 tested passed the Esterman test, the one failure having a previously unknown and relatively mild homonymous field defect. CONCLUSION: Patients undergoing bilateral macular laser can be counselled that, provided their acuity remains adequate, they have a very low chance of failing the DVLA field regulations. PMID- 10755098 TI - A comparison between laser interferometric measurement of fundus pulsation and pneumotonometric measurement of pulsatile ocular blood flow. 1. Baseline considerations. AB - PURPOSE: Several methods have been proposed for the investigation of the human choroidal circulation. The aim of the present study was to compare laser interferometric measurements of cardiac synchronous fundus pulsations with pneumotonometric measurements of intraocular pressure pulse and pulsatile ocular blood flow in humans. METHODS: The association between fundus pulsation amplitude as assessed with laser interferometry and pulse amplitude (PA) and pulsatile ocular blood flow (POBF) as assessed with pneumotonometry was investigated in 28 healthy subjects. Additionally, we investigated the distribution of fundus pulsation amplitude (FPA) in a region of -15 degrees to +15 degrees around the macula (n = 18) and the influence of accommodation paralysis with cyclopentolate on FPA (n = 10). RESULTS: There was a high association between FPA and PA (r = 0.86, p < 0.001) and FPA and POBF (r = 0.70, p < 0.001). Fundus pulsations in the macula were significantly smaller than in the optic disc, but significantly larger than those in peripheral regions of the retina. Administration of cyclopentolate did not influence FPA. CONCLUSIONS: On the basis of the strong correlation between laser interferometric measurements of FPA and pneumotonometric measurements of PA and POBF, we conclude that the FPA is a valid index of pulsatile choroidal perfusion in humans. PMID- 10755099 TI - A comparison between laser interferometric measurement of fundus pulsation and pneumotonometric measurement of pulsatile ocular blood flow. 2. Effects of changes in pCO2 and pO2 and of isoproterenol. AB - PURPOSE: We have shown in the companion paper that, under baseline conditions, there is a high degree of association between laser interferometrically measured fundus pulsation amplitude (FPA) and pneumotonometrically measured pulse amplitude (PA) and pulsatile ocular blood flow (POBF). The present study investigated the effect of high pCO2, of high pO2 and of isoproterenol on POBF as assessed with laser interferometry and pneumotonometry. METHODS: Pneumotonometry and laser interferometry were performed in young healthy subjects during breathing of 100% O2 (n = 10; hyperoxia) and of 5% CO2 + 95% air (n = 8; hypercapnia). In addition these parameters were studied during stepwise increasing doses of isoproterenol, a beta-receptor agonist (n = 8). RESULTS: Inhalation of 5% CO2 + 95% air increased FPA (24 +/- 12%, p < 0.001), PA (26 +/- 13%, p < 0.001) and POBF (15 +/- 8%, p = 0.002). Inhalation of 100% O2 decreased FPA (-5 +/- 7%, p = 0.027), but did not change PA or POBF. The effect of 100% O2 inhalation on FPA in the optic disc was more pronounced (-11% to -20%) than in the macula. Isoproterenol caused a dose-dependent increase in FPA, PA and POBF (p < 0.001). The association between the induced changes in FPA and PA or POBF was highly significant. CONCLUSIONS: The present study shows that FPA can be taken as a valid relative measure of pulsatile choroidal blood flow. Our results in the optic disc indicate that FPA at the neuroretinal rim and at the cup is influenced by retinal and choroidal circulation. PMID- 10755101 TI - Angular change in backscattering of light from the human lens with nuclear cataract. AB - PURPOSE: To determine how many meridians are needed to optimise detection of lens nuclear opacities in Scheimpflug photography utilising the Nidek EAS-1000 system. METHODS: Ten eyes with nuclear cataract from 10 patients (age 53-83 years) were selected in this study. For each eye, the lens was imaged with the Nidek EAS-1000 system at the 0 degree, 30 degrees, 60 degrees, 90 degrees, 120 degrees and 150 degrees meridians, respectively. The intensity of the backscattering of light within a common lens nuclear area on the lens image was measured. In addition an aqueous suspension of polystyrene spheres was employed as a standard to evaluate the possible angular change caused by the camera system. Analysis of variance and the polynomial regression was used for data analysis. RESULTS: The intensity of the backscattered light differs significantly among meridians. The highest intensity is found at the 90 degrees meridian. The intensity decreases towards the nasal and temporal side. There is also an angular variation caused by the camera system. CONCLUSIONS: There is a regular change in the backscattered light documented by the Nidek EAS-1000 system. It is sufficient to take one meridian to estimate the lens nuclear cataract. The variation in the backscattered light at different angles is caused partly by the variation of the camera. PMID- 10755100 TI - Phacoemulsification cataract surgery: is routine review necessary on the first post-operative day? AB - PURPOSE: To determine the value of routine review on the first post-operative day following phacoemulsification cataract surgery. METHODS: A prospective study was performed of 238 consecutive patients who underwent phacoemulsification cataract surgery. Local anaesthesia was used for 97% of patients and surgery was performed as a day-case procedure for 93% of patients. The findings at the first day post operative review were analysed separately for patients who had undergone uncomplicated surgery and patients who had suffered an intraoperative complication. Four patients were excluded because of incomplete data collection. RESULTS: A total of 227 patients underwent uncomplicated phacoemulsification cataract surgery. Thirteen (5.7%, 95% confidence interval (CI) 3.1-9.6%) of these were found to have post-operative complications at their first day review which comprised corneal oedema (4.4%, 95% CI 2.1-8.0%), raised intraocular pressure > or = 30 mmHg (1.3%, 95% CI 0.3-3.8%), hyphaema (0.9%, 95% CI 0.1-3.1%), corneal abrasion (0.4%, 95% CI 0.0-2.4%) and anterior uveitis (0.4%, 95% CI 0.0-2.4%). These findings led to the standard post-operative management being altered for 5 (2.2%) patients. Intraoperative complications occurred in 7 (2.9%) patients during phacoemulsification cataract surgery. Five (71%) of these patients had post-operative complications at their first day review. CONCLUSIONS: Routine review on the first post-operative day following uncomplicated phacoemulsification cataract surgery could safely be withdrawn. A single post operative review at 1-2 weeks after surgery would then be required, supplemented by patient-initiated post-operative review in the interim. PMID- 10755102 TI - Long-term disorders of the blood-aqueous barrier after small-incision cataract surgery. AB - PURPOSE: To determine the long-term function of the blood-aqueous barrier after small-incision cataract surgery with implantation of a foldable intraocular lens. METHODS: The blood-aqueous barrier function in 74 eyes of 62 patients who underwent cataract surgery was examined using a laser flare-cell meter. The measurements were performed pre-operatively and post-operatively between 12 and 35 months after surgery. For statistical analysis a linear regression was used. The study was designed as a single cohort study, with comparison of pre- and post operative values. RESULTS: Highly statistically significant differences (p < 0.0001) were found between pre-operative flare values and those measured at the final visit. The linear regression model showed significantly higher flare values post-operatively compared with those measured pre-operatively. Other variables such as incision technique, sex, operation time, phaco time and systemic disease had no influence on this outcome. CONCLUSION: The results suggest that there is persistent blood-aqueous barrier dysregulation even several years after cataract surgery. PMID- 10755103 TI - Ocular sequelae of pneumatic drills. AB - PURPOSE: To determine the ocular sequelae of rock-drilling. METHODS: Nineteen rock-drillers and 20 age-, sex- and race-matched control subjects underwent ocular examination in a prospective manner. RESULTS: Subepithelial corneal opacities were found in 12 drillers (63%) and none of the controls; pigmented trabecular meshwork was noted in 13 drillers (68%) and 1 control (5%); low tension glaucoma was diagnosed in 1 driller (5%); vitreous liquefaction was present in 17 of 17 drillers (100%) examined by a retina specialist and 9 of 20 controls (45%). CONCLUSIONS: The sequelae of drilling include corneal scars from projectile pieces of drilled stone as well as vibration-induced pigment deposition in the trabecular meshwork and vitreous liquefaction. Wearing of safety glasses is recommended. PMID- 10755104 TI - The acute effects of cigarette smoking on human optic nerve head and posterior fundus circulation in light smokers. AB - PURPOSE: To study the acute effects of cigarette smoking on tissue circulation in the optic nerve head (ONH) and posterior fundus in smokers with a short smoking history. METHODS: Ten healthy smokers whose length of smoking history was within 2 years (age 25 +/- 1 years; smoking index (number of cigarettes smoked per day x length of smoking history in years) 16 +/- 4, mean +/- SE) were included in the study. Using the laser speckle method, normalised blur (NB) value, a quantitative index of tissue blood velocity, was measured every 0.125 s and averaged over three pulses across an area located in the temporal site of the ONH free of visible surface vessels (NBONH) and across an area located halfway between the macula and the ONH with no discrete vessels visible (NBch-ret). NBONH, NBch-ret and intraocular pressure (IOP) in one randomly chosen eye, and blood pressure (BP) and pulse rate (PR) were measured before, and 1, 5, 10, 15, 20, 25, 30, 45, 60 and 90 min after sham smoking. One week later, NBONH, NBch-ret and IOP in the same eye, and BP and PR were measured after cigarette smoking according to the same time schedule as in the control experiment. RESULTS: After sham smoking, no parameter showed a significant change during the experiment. Differences in NBONH from the baseline were not significant between the smoking experiment and sham smoking experiment, while NBch-ret showed a significant difference at 30 min. Inter-group difference in the time course of the difference from baseline was significant (ANOVA, p = 0.0246, 0.0021). BP and PR were significantly increased between 1 and 20 min after smoking while IOP showed no significant change at any time of measurement. CONCLUSIONS: In light smokers, cigarette smoking showed little effect on tissue blood velocity in the ONH and slightly decreased that in the posterior fundus, suggesting a significant increase in vascular resistance in these tissues. PMID- 10755105 TI - Twenty-four hour control of intraocular pressure with dorzolamide and timolol maleate in exfoliation and primary open-angle glaucoma. AB - PURPOSE: To determine the efficacy and safety of adding dorzolamide 2% twice daily to timolol maleate solution 0.5% twice daily when treating exfoliation glaucoma or primary open-angle glaucoma. METHODS: This was a single-centre, crossover intra-individually controlled comparison. Sixty-two consecutive patients (31 with exfoliation glaucoma and 31 with primary open angle glaucoma) chronically treated with timolol maleate twice daily were included in this trial. Patients then had added dorzolamide 2% twice daily (08:00 and 20:00), instilled approximately 10 min after timolol maleate. Patients underwent diurnal intraocular pressure (IOP) testing (six measurements over 24 h), first on timolol maleate monotherapy and 2 months later following the addition of dorzolamide 2% as adjunctive therapy. RESULTS: On timolol monotherapy patients with exfoliation glaucoma had a higher mean IOP at 02:00, 06:00, 10:00, 14:00 and 22:00 hour time points as well as a higher maximum, minimum and range of IOP throughout the day compared with the primary open-angle glaucoma group (p < 0.05). Following the addition of dorzolamide as adjunctive therapy to timolol maleate there was a significant reduction in IOP (p < 0.05) at all time points in both glaucomas, but mean IOP at 10:00, 14:00, 18:00 and 22:00 hour time points, as well as the peak and range of IOP, remained higher in the exfoliation glaucoma group. No serious adverse events were noted with dorzolamide. Bitter taste, the most common symptom, was noted in 30% of patients. CONCLUSIONS: These data suggest that dorzolamide 2% used adjunctively with timolol maleate 0.5% solution is effective in reducing diurnal IOP in patients with primary open-angle and exfoliation glaucoma but does not alter the characteristics of higher IOP levels in the latter disease. PMID- 10755106 TI - Ocular abnormalities associated with cerebral palsy after preterm birth. AB - PURPOSE: To document the ocular abnormalities in children with cerebral palsy (CP) after premature birth. METHODS: All the children born before 32 weeks gestation between 1 January 1990 and 31 December 1991 in a geographically defined population of approximately 3 million, were examined by an ophthalmologist and a paediatrician at 2 years old. RESULTS: Five hundred and fifty-eight children (98.8% of the study group) were examined, of whom 41 had disabling CP and 13 had non-disabling CP. Children with CP had a higher incidence of abnormalities compared with children without CP: cicatricial retinopathy of prematurity occurred in 8 children with CP (14.8%) compared with 8 without CP (1.6%, p < 0.0001), cortical visual impairment occurred in 6 with CP (11.1%) compared with 1 child without CP (0.2%, p < 0.0001) and concomitant strabismus in 28 with CP (51.9%) compared with 42 without CP (8.4%, p < 0.0001). The prevalence of refractive error without other ocular abnormalities was similar for children with CP (4/54, 7.4%) and those without CP (54/504, 10.7%, p = 0.90). Significant ocular abnormalities had been previously unrecognised in 8 children with CP (14.8%). CONCLUSION: There are some differences between these results and previous series. These differences probably reflect the fact that previous work has studied severe CP of more diverse aetiology. The high frequency of abnormalities highlights the importance of ocular assessment of these children. PMID- 10755107 TI - Ophthalmological and intracranial anomalies in patients with clinical anophthalmos. AB - PURPOSE: To better describe the clinical and neuroimaging spectrum of abnormalities in clinical anophthalmos. METHODS: We performed a retrospective review of all 17 patients admitted to the King Khaled Eye Specialist Hospital with clinical anophthalmos over a 15 year period who had a complete ophthalmological examination documented and received computed tomographic (CT) imaging of the orbits and brain. RESULTS: Patients with clinical anophthalmos had a high incidence of developmental abnormalities involving both eyes (15/17 patients, 88%), the brain (12/17 patients, 71%) and the body (7/12, 58%). The incidence of central nervous system anomalies reached 100% in patients with bilateral small optic nerves on CT scan. CONCLUSIONS: Patients with clinical anophthalmos share a similar constellation of neurological, somatic and neuroradiological abnormalities as patients with microphthalmos, septo-optic dysplasia and clinical optic nerve hypoplasia. This fact may provide insight into developmental abnormalities of the afferent visual system and brain. PMID- 10755108 TI - Protection of corneal endothelium from irrigation damage: a comparison of sodium hyaluronate and hydroxypropylmethylcellulose. AB - PURPOSE: To compare the endothelial protection of sodium hyaluronate and hydroxypropylmethylcellulose against endothelial damage induced by irrigation. METHODS: An in vitro assay with freshly excised porcine eyes was developed using the Janus green photometry technique. Irrigation and aspiration technique was standardised. Forty pairs of porcine eyes were used. One randomly chosen eye was filled with sodium hyaluronate (SH) and the other with hydroxypropylmethylcellulose (HPMC). Irrigation and aspiration was carried out with balanced salt solution for 5 min. Twenty additional pairs of porcine eyes served as controls. Student's t-test was used for statistical analysis. RESULTS: Both viscoelastic agents protected the endothelium as compared with controls. The endothelial protection, determined with the Janus green photometric technique, was significantly greater with HPMC than with SH. CONCLUSIONS: Viscoelastic agents are effective in protecting the endothelium from irrigation damage in porcine eyes in vitro. HPMC provided greater protection than SH in this particular model. PMID- 10755109 TI - Retinal maturation is delayed by repeated, but not single, maternal injections of betamethasone in sheep. AB - PURPOSE: The safety and efficacy of prescribing a single maternal course of corticosteroid during pregnancy has been documented in human trials. However, the current trend is to prescribe repeated courses of corticosteroid. We investigated an aspect of the safety of this practice in an animal model. METHODS: Date-mated ewes received saline, single or four corticosteroid injections between days 104 and 124 of gestation (term = 150). Lambs were delivered on day 125 or 145 by caesarian section after spinal anaesthesia. Eye diameters were measured and semi thin toluidine-blue-stained transverse sections of retinae were analysed using an Optimus Image Analysis program. RESULTS: At 125 days, retinal measures in the ventral periphery and area centralis were significantly thinner than control (p = 0.0001). At 145 days, total eye size was significantly reduced compared with control (p = 0.03), and retinal measures in the ventral periphery (p = 0.0001), but not the area centralis (p = 0.19), remained significantly different from control. CONCLUSION: Repeated maternal administration of corticosteroid may affect retinal maturation in the fetus. PMID- 10755110 TI - Corneal autografts: are the theoretical advantages achieved in practice? PMID- 10755111 TI - Scleral dellen complicating primary pterygium excision. PMID- 10755112 TI - An effective therapeutic modality for the management of hyperaemic conjunctival margins following the Hughes' procedure. PMID- 10755113 TI - Conjunctivitis--sometimes more than meets the eye! PMID- 10755114 TI - Keratoconjunctivitis sicca associated with lichen sclerosus at atrophicus. PMID- 10755115 TI - A transsexual patient with bilateral central retinal vein occlusions. PMID- 10755117 TI - Accelerated radiation retinopathy in diabetes and pregnancy. PMID- 10755116 TI - Vasoproliferative tumour of the ocular fundus associated with Waardenburg's syndrome. PMID- 10755118 TI - Single-site Langerhans cell histiocytosis (eosinophilic granuloma) of the orbit. PMID- 10755119 TI - Ganciclovir implant: clinicopathological study. PMID- 10755120 TI - Broken suture--predisposing factor for Nocardia keratitis. PMID- 10755121 TI - Isolated post-operative Aspergillus niger endophthalmitis. PMID- 10755122 TI - Incomplete bitemporal hemianopia without headache: an unusual case of pituitary apoplexy. PMID- 10755123 TI - Role of aspirin in reducing the frequency of second eye involvement in patients with non-arteritic anterior ischaemic optic neuropathy. PMID- 10755124 TI - Biometry of the silicone oil-filled eye. PMID- 10755125 TI - Domiciliary post-operative assessment following cataract surgery carried out by specialist nurses. PMID- 10755126 TI - Management of monocular congenital cataracts. PMID- 10755128 TI - Polymer additive migration to foods--a direct comparison of experimental data and values calculated from migration models for high density polyethylene (HDPE). AB - To reduce the amount of compliance testing for food contact polymers the use of migration modelling has been proposed. This study was conducted to provide valid data for the independent evaluation of two such diffusion-based models using a range of different high density polyethylene (HDPE) polymers and plastics additives. Seventy-two experimental migration data have been obtained in triplicate and used to evaluate two Fickian-based migration models in the prediction of specific migration of four HDPE additives into olive oil. All tests were conducted using olive oil, representing the most severe case for fatty foods with test conditions of 2 h at 70 degrees C, 6 h at 70 degrees C, 10 days at 40 degrees C representing short term exposures at high temperatures and room temperature storage. Predicted migration values were calculated by inserting the measured initial concentration of additive in the polymers (Cp,0) into the equations together with known variables such as additive molecular weight, temperature and exposure time. The results indicate that both models predict migration values into olive oil close to, or in excess of, the experimental results. The Piringer migration model, using the 'exact' calculations of the Migratest Lite program, gave an overestimation for 83% of the migration values generated in this study. The highest overestimation was 3.7 times the measured value. For all measurements, the predicted migration from the Migratest Lite program was greater than 50% of the observed value. The FDA model was found more accurately to predict migration in most situations but underestimated migration more frequently. Differences in the polymer specification had little effect on specific migration of the additives investigated. PMID- 10755127 TI - The effects of cereal substrate and temperature on production of beauvericin, moniliformin and fusaproliferin by Fusarium subglutinans ITEM-1434. AB - One strain of Fusarium subglutinans (ITEM-1434) isolated from maize ear rot in Poland was tested for the ability to synthesize moniliformin (MON), beauvericin (BEA) and fusaproliferin (FP) on six cereal substrates (wheat, rye, barley, oat, maize and rice kernels) for 3 weeks at 25 degrees C and on rice at three different temperatures (20, 25 and 30 degrees C). Most MON (497 micrograms/g) was produced on rice; most BEA (704 micrograms/g) on wheat or rice, and most FP (422 micrograms/g) on rye. When cultured on rice, F. subglutinans produced the highest levels of BEA and FP at 20-25 degrees C, while MON production was best at 30 degrees C. PMID- 10755129 TI - Determination of benzidine in the food colours tartrazine and sunset yellow FCF, by reduction and derivatization followed by high-performance liquid chromatography. AB - Free and bound benzidine, a non-sulphonated aromatic amine (NSAA), were determined in the food colours tartrazine and sunset yellow FCF. Bound amines were released by reducing with sodium dithionite, then total NSAAs were extracted into chloroform, transferred to aqueous acid solution and diazotized with sodium nitrite before coupling with 2-naphthol-3,6-disulphonic acid, disodium salt (R salt). Coloured benzidine and aniline derivatives (BZDRS and ANRS) were analysed using reversed-phase ion pair high-performance liquid chromatography (HPLC) and an absorbance detector set at 548 nm. Levels of total benzidine were similar to those reported in studies conducted in the USA, and ranged from < 5 to 270 ng/g. Total aniline was also determined (0.2-188 micrograms/g). Recoveries of benzidine with this method were found to be lower than expected (average ca 46%), but were reproducible. Detection limits were 15-20 ng BZDRS/g (3-4 ng benzidine/g). Mass spectrometry (LC-MS) was evaluated for identifying and determining purity of the standards, but difficulties were encountered when the methodology was applied to commercial samples. PMID- 10755131 TI - Tylosin depletion in edible tissues of turkeys. AB - The depletion of tylosin residues in edible turkey tissues was followed after 3 days of administration of tylosin tartrate at 500 mg l-1 in drinking water, to 30 turkeys. Immediately after the end of the treatment (day 0) and at day 1, 3, 5 and 10 of withdrawal, six turkeys (three males and three females) per time were sacrificed and samples of edible tissues were collected. Tissue homogenates were extracted, purified and analysed by HPLC according to a method previously published for the analysis of tylosin residues in pig tissues. In all tissues, tylosin residues were already below the detection limits of 50 micrograms kg-1 at time zero. However, in several samples of tissues (skin + fat, liver, kidney, muscle), from the six turkeys sacrificed at that time, one peak corresponding to an unknown tylosin equivalent was detected at measurable concentrations. The identification of this unknown compound was performed by LC-MS/MS analysis of the extracts from incurred samples. The mass fragmentation of the compound was consistent with the structure of tylosin D (the alcoholic derivative of tylosin A), the major metabolite of tylosin previously recovered and identified in tissues and/or excreta from treated chickens, cattle and pigs. PMID- 10755130 TI - Dietary exposure estimates of 30 elements from the UK Total Diet Study. AB - Dietary exposures of consumers to 30 elements (aluminium, antimony, arsenic, barium, bismuth, boron, cadmium, calcium, chromium, cobalt, copper, germanium, gold, iridium, iron, lead, lithium, manganese, mercury, molybdenum, nickel, palladium, platinum, rhodium, ruthenium, selenium, strontium, thallium, tin and zinc) estimated from the UK 1994 Total Diet Study are reported, and compared with those from previous UK Total Diet Studies and those from other countries. Dietary exposure estimates were generally low and, where comparisons are possible, similar to those from other countries and below the relevant Provisional Tolerable Weekly Intakes and Provisional Maximum Tolerable Daily Intakes. For most of those elements included in previous UK Total Diet Studies, dietary exposures have declined. PMID- 10755132 TI - DDT and HCH residues in dairy milk samples collected from different geographical regions of India: a multicentre study. AB - Under a multicentre study conducted by the Indian Council of Medical Research, 2205 samples of dairy milk were collected from rural and urban areas of 12 states representing different geographical regions of India. These samples were analysed for residues of DDT and different isomers of HCH by gas-liquid chromatography. Analytical quality assurance between various participating laboratories was ensured through analysis of check samples. The residues of DDT and HCH were detected in more than 80% of samples analysed. Concentrations of DDT residues, alpha-HCH, beta-HCH, gamma-HCH and delta-HCH exceeded their maximum residue limits prescribed by the Ministry of Health and Family Welfare of the Indian Government in 37, 21, 42, 28 and 4% of the samples, respectively. Median values of DDT and HCH found in dairy milk in India were more than the corresponding values reported from most other countries. The results showed significant variations in the incidence as well as level of these contaminants in dairy milk from different regions of the country. PMID- 10755133 TI - A study on the equivalence of olive oil and the EU official substitute test media for migration testing at high temperatures. AB - As a consequence of an increasing awareness and findings about the technical and analytical difficulties related to the employment of triglycerides as fatty food simulants in migration testing, EU Directive 82/711/EEC on the basic rules of migration has been amended for the second time and adapted with the recent Commission Directive 97/48/EC. The major merit of this important amendment is to authorize alternative tests such as extraction tests and to allow substitute tests for the fat test with concrete indication of corresponding test conditions to be applied. As a novelty in food law compliance testing it authorizes a modified polyphenylene oxide (MPPO), well-known as Tenax, a thermostable and highly adsorptive porous polymer, as a 'solid' matrix to substitute fat simulant D at temperatures equal to or higher than 100 degrees C. In the study presented here, comparative migration testing--overall and specific--was carried out between fat simulants olive oil and 14C-labelled HB 307 and the substitute test media iso-octane, 95% ethanol and MPPO using polypropylene materials as examples at test temperatures of 100 degrees C to 120 degrees C and prescribed corresponding substitute test conditions. As a consequence, a number of conclusions were drawn which are relevant for overall and/or specific migration testing under the investigated test conditions. One of the major findings was that MPPO was generally more equivalent to the fat test than the other two substitute test media. The presence of volatile hydrocarbons in the test samples was found to play a crucial role with respect to the equivalence of the iso octane-based substitute test. At high volatile concentrations in the test sample the iso-octane test underestimated the olive oil benchmark whereas low volatile concentrations led to satisfying equivalence. It is therefore recommended that the procedure is modified and GC-FID screening of the isooctane extract is carried out. As a logical consequence, the possibility of quantitative determination of the overall extract by GC-FID is recommendable and should be investigated in more depth. Concerning the overall migration test procedure in olive oil, the vacuum pre-conditioning at 60 degrees C should be given re consideration to avoid the possibility of loss of volatile migrants whose determination should be within the scope of the overall migration test method itself. Finally, the specific migration related equivalence between olive oil, MPPO and iso-octane needs further investigations to clarify the net effect of strongly increased diffusion rates at the regular high temperature versus the substitute test solvent accelerated extraction effect at 60 degrees C. Stability aspects of migrants at the different test temperatures deserve further consideration. PMID- 10755134 TI - Effects of food preservatives on Alternaria alternata growth and tenuazonic acid production. AB - The effects of different organic acids on Alternaria alternata growth and tenuazonic acid production (TeA) were evaluated. Both TeA pure toxin solution and TeA production in solid medium were considered. Sodium benzoate, potassium sorbate and sodium propionate, all preservatives commonly used by food industry in Argentina, were tested. TeA was stable as pure toxin solution when was treated with the salts of organic acids used. A differential effect was observed when the preservatives were evaluated in relation to A. alternata growth and TeA production in solid medium. Levels above 10 mg/kg of sodium benzoate and potassium sorbate produced a total inhibition of fungal development and toxin biosynthesis. Sodium propionate produced a 59% decrease in A. alternata growth and total inhibition of TeA production only at the highest concentration of preservatives used. PMID- 10755135 TI - Agaritine content in processed foods containing the cultivated mushroom (Agaricus bisporus) on the Nordic and the Czech market. AB - The level of agaritine was measured in fresh and canned cultivated mushroom (Agaricus bisporus) as well as in other food products containing A. bisporus, by reversed phase high performance liquid chromatography. The two fresh samples were purchased on the open market and contained 212 and 229 mg/kg, respectively. Of the 35 different trademarks of canned mushroom products studied, 25 were based on cut mushrooms and 10 on whole mushrooms. On average, whole mushrooms contained 14.9 +/- 6.7 mg agaritine per kg product whereas cut mushrooms contained 18.1 +/- 7.8 mg/kg. There was no statistically significant difference between these two values. Agaritine levels in brine were generally slightly lower than the levels detected in canned mushrooms. Thus, the level of agaritine in A. bisporus is reduced more than 10 times during the wet canning process, resulting in low levels in canned products. On a portion basis, somewhat higher amounts of agaritine may be found in some other food products (mushroom soup and pasta sauce) containing A. bisporus. PMID- 10755136 TI - Analysis of ochratoxin A in pig kidney and rye flour using liquid chromatography tandem mass spectrometry (LC/MS/MS). AB - A liquid chromatography electrospray tandem mass spectrometric (LC/MS/MS) method is described for analysis and confirmation of ochratoxin A in pig kidney and rye flour using derivatization of ochratoxin A to the methyl ester. Ochratoxin A methyl(d3)ester is used as internal standard. The method works well, the detection limit is 0.02 microgram/kg and the repeatability (coefficient of variation) is between 6% and 16% in the contamination range 0.5 to 8 micrograms/kg. PMID- 10755137 TI - Estimation of intake of bisphenol-A-diglycidyl-ether (BADGE) from canned fish consumption in Europe and migration survey. AB - The exposure to bisphenol-A-diglycidyl-ether (BADGE) from canned fish in oil was assessed from consumption data collected for each Member State of the European Union and Switzerland, and migration data from a European survey on 382 samples. Trade figures were used when no consumption data were available. The average consumption of canned fish in Europe was 2.3 kg per person per year, with values ranging from 0.2 kg per person per year in the United Kingdom to 5.1 kg per person per year in Denmark. The exposure to BADGE was calculated as microgram per person per day. The data indicated that exposure to BADGE was in the range below 4 mg per person per year, i.e. 9 micrograms per person per day, hence a fairly low exposure in part due to the fact that canned fish is a relatively minor dietary item. An approximation assuming the general figure of a 60 kg adult, would thus be 0.15 microgram/kg body weight per day. This is a fairly limited exposure considering the provisional limit in food had been set a 1 mg/kg and assumed 1 kg of food ingested. In countries for which increased exposure was found, the reason was mainly caused by one individual sample exhibiting a high concentration rather than a larger number of samples with mildly elevated concentrations. PMID- 10755138 TI - Dietary arsenic intakes in the United States: FDA Total Diet Study, September 1991-December 1996. AB - The FDA has conducted the Total Dietary Study (TDS), a yearly market basket programme, since 1961. It is designed to monitor the levels of toxic chemical contaminants (pesticide residues, industrial and elemental contaminants) and essential nutrients in the US food supply. It also provides information on trends in dietary concentrations and exposures for the general population. Foods are collected from retail stores once a year from each of four geographic areas of the US and are analysed either after preparation/cooking or as ready-to-eat. The latest TDS (1991-1997) data show that arsenic (inorganic and organic, > or = 0.03 ppm) was found in 63 (24%) of the 261-264 foods/mixed dishes analysed. The highest concentration was found in seafood, followed by rice/rice cereal, mushrooms, and poultry. Based on the United States Department of Agriculture's 1987-1988 Nationwide Food Consumption Survey, the estimated daily total arsenic average intakes, in microgram/day, are: 2 for infants, 23 for toddlers, 20 for 6 year-old children, 13 for 10-year-old children, 15 for 14-16-year-old boys, 21 for 14-16-year-old girls, 57 for 25-30-year-old men, 28 for 25-30-year-old women, 47 for 40-45-year-old men, 37 for 40-45-year-old women, 92 for 60-65-year-old men, 72 for 60-65-year-old women, 69 for 70-year-old men, and 42 for 70-year-old women. Of the estimated total arsenic intakes for infants, 42% arise from seafood and 31% from rice/rice cereals. Of the estimated total arsenic intakes, seafood contributes 76-90% for children (2-10-year olds), 79-85% for 14-16-year olds, and 89-96% for adults (> or = 25-30-year olds); rice/rice cereals contributes 4-8% for children, 8% for 14-16-year olds, and 1-4% for adults (> or = 25-30-year olds). PMID- 10755139 TI - Dietary intake of pesticides: State of Kuwait total diet study. AB - The State of Kuwait conducted a total diet study (TDS) to determine the dietary intakes of selected pesticides. This paper reports the results of this study. A national food consumption survey was performed and core food lists for different population groups were established representing the total diet of the Kuwaiti population. Food items (table-ready) were purchased/prepared and analysed for their contents of organophosphate/organochlorine carbamate, benzimidazole and phenylurea pesticides according to the US FDA Pesticide Analytical Manual (PAM I). Dietary intakes of these pesticides are presented for 19 population groups, which range from infants to elderly adults. The intakes of selected population groups are compared with representative findings from the US FDA/TDS and data published from other countries. In general, the average daily intakes were well below acceptable limits, but higher than those reported from developed countries. PMID- 10755140 TI - Comparison of two methods to assess the intake of flavouring substances. AB - It is important to assess the intake of flavouring substances in order to be confident that exposure to the substance from its intended use presents no significant risk. A number of methods exist to estimate intake of food ingredients. Two such methods, one using a detailed dietary analysis based on food consumption and composition and one using 10 times the annual volume of use on a per capita basis (per capita x 10), were compared for their precision and practicality in assessing the intake of 10 flavouring substances. The detailed dietary analysis method of determining exposure resulted in good estimates of the distribution of intakes across the population, as well as patterns of intake of individuals. This method is both expensive and labour intensive. The per capita x 10 method yields results that, compared with those obtained by detailed dietary analysis, tend consistently to overstate exposure. Thus, this method is a conservative and practical approach to assessing exposure to flavouring substances and other food ingredients. PMID- 10755141 TI - Seeds of creation and disaster. PMID- 10755142 TI - The roles of vision and eye movements in the control of activities of daily living. AB - The aim of this study was to determine the pattern of fixations during the performance of a well-learned task in a natural setting (making tea), and to classify the types of monitoring action that the eyes perform. We used a head mounted eye-movement video camera, which provided a continuous view of the scene ahead, with a dot indicating foveal direction with an accuracy of about 1 deg. A second video camera recorded the subject's activities from across the room. The videos were linked and analysed frame by frame. Foveal direction was always close to the object being manipulated, and very few fixations were irrelevant to the task. The first object-related fixation typically led the first indication of manipulation by 0.56 s, and vision moved to the next object about 0.61 s before manipulation of the previous object was complete. Each object-related act that did not involve a waiting period lasted an average of 3.3 s and involved about 7 fixations. Roughly a third of all fixations on objects could be definitely identified with one of four monitoring functions: locating objects used later in the process, directing the hand or object in the hand to a new location, guiding the approach of one object to another (e.g. kettle and lid), and checking the state of some variable (e.g. water level). We conclude that although the actions of tea-making are 'automated' and proceed with little conscious involvement, the eyes closely monitor every step of the process. This type of unconscious attention must be a common phenomenon in everyday life. PMID- 10755143 TI - Revisiting the global-dominance phenomenon outside the focus of attention. AB - Previous research has demonstrated global dominance for attended and unattended stimuli. In this paper, this phenomenon is shown to be restricted to small compound stimuli. As a first step, local dominance was obtained with large (8 deg in height) attended stimuli when a single stimulus was displayed. Next, dominance in attended and unattended stimuli was investigated by displaying two large compound stimuli, one surrounded by a square (attended compound stimulus), the other one enclosed in a circle (unattended compound stimulus). The way attention was directed to the attended stimulus was varied. No dominance was observed when subjects were instructed to process the stimulus appearing in the square (experiment 2). However, when a rapid-onset cue pre-directed attention to the attended stimulus, local dominance emerged for attended, but not for unattended stimuli (experiment 3). This latter result was obtained whether or not subjects were more experienced at local than global processing (experiment 4). The implications of the results for the locus of processing dominance are discussed. PMID- 10755144 TI - Surface formation and depth in monocular scene perception. AB - The visual perception of monocular stimuli perceived as 3-D objects has received considerable attention from researchers in human and machine vision. However, most previous research has focused on how individual 3-D objects are perceived. Here this is extended to a study of how the structure of 3-D scenes containing multiple, possibly disconnected objects and features is perceived. Da Vinci stereopsis, stereo capture, and other surface formation and interpolation phenomena in stereopsis and structure-from-motion suggest that small features having ambiguous depth may be assigned depth by interpolation with features having unambiguous depth. I investigated whether vision may use similar mechanisms to assign relative depth to multiple objects and features in sparse monocular images, such as line drawings, especially when other depth cues are absent. I propose that vision tends to organize disconnected objects and features into common surfaces to construct 3-D-scene interpretations. Interpolations that are too weak to generate a visible surface percept may still be strong enough to assign relative depth to objects within a scene. When there exists more than one possible surface interpolation in a scene, the visual system's preference for one interpolation over another seems to be influenced by a number of factors, including: (i) proximity, (ii) smoothness, (iii) a preference for roughly frontoparallel surfaces and 'ground' surfaces, (iv) attention and fixation, and (v) higher-level factors. I present a variety of demonstrations and an experiment to support this surface-formation hypothesis. PMID- 10755145 TI - The framing effect with rectangular and trapezoidal surfaces: actual and pictorial surface slant, frame orientation, and viewing condition. AB - The perceived slant of a surface relative to the frontal plane can be reduced when the surface is viewed through a frame between the observer and the surface. Aspects of this framing effect were investigated in three experiments in which observers judged the orientations-in-depth of rectangular and trapezoidal surfaces which were matched for pictorial depth. In experiments 1 and 2, viewing was stationary-monocular. In experiment 1, a frontal rectangular frame was present or absent during viewing. The perceived slants of the surfaces were reduced in the presence of the frame; the reduction for the trapezoidal surface was greater, suggesting that conflict in stimulus information contributes to the phenomenon. In experiment 2, the rectangular frame was either frontal or slanted; in a third condition, a frame was trapezoidal and frontal. The conditions all elicited similar results, suggesting that the framing effect is not explained by pictorial perception of the display, or by assimilation of the surface orientation to the frame orientation. In experiment 3, viewing was moving monocular to introduce motion parallax; the framing effect was reduced, being appreciable only for a trapezoidal surface. The results are related to other phenomena in which depth perception of points in space tends towards a frontal plane; this frontal-plane tendency is attributed to heavy experimental demands, mainly concerning impoverished, conflicting, and distracting information. PMID- 10755146 TI - Glass patterns: grouping by contrast similarity. AB - Recent work has shown that certain contrast phenomena associated with Glass patterns can be accounted for by filtering mechanisms applied within the luminance or energy domain. Hitherto, these phenomena were regarded as problematic for energy-processing models, and were taken as evidence in support of symbolic-processing accounts. An additional, and controversial, contrast effect is investigated. It is shown that in a Glass pattern consisting of simultaneous vertical and horizontal translations of different contrast strength, the most salient structure is determined by grouping (pairing) of the two low contrast elements of the pattern. The finding that grouping in such patterns is by contrast similarity, as opposed to energy, is consistent with the symbolic processing approach, but has yet to be accounted for by filtering mechanisms. PMID- 10755147 TI - Color filling-in under steady fixation: behavioral demonstration in monkeys and humans. AB - Color filling-in is a phenomenon in which the color of an object appears to be filled-in by the color of the surrounding field. We have studied the question of whether monkeys perceive color filling-in with near-foveal stimuli under steady fixation. Two monkeys were trained to fixate steadily and to attend to a disk, surrounded by an annulus of the complementary color, in parafoveal vision. Using displays in which the color of the disk was gradually changed to that of the annulus, we trained the animals to signal when they perceived a uniform color field. During the experiment, we introduced a small percentage of trials in which the disk color remained constant, and looked for 'filling-in' responses in these trials. Three human subjects were also tested for comparison. All subjects produced 'filling-in' responses with frequencies that were significantly higher for static disks with blurred borders than for moving disks or disks with sharp borders. This indicates that the monkeys' responses reflected perceptual filling in, rather than random behavior. The time course of filling-in was similar in monkeys and humans. For the blurred static disks, responses occurred first after 3-4 s of fixation, reaching a probability of 0.2-0.8 by the end of 6 s, depending on the subject. PMID- 10755148 TI - Motion aftereffect of combined first-order and second-order motion. AB - When, after prolonged viewing of a moving stimulus, a stationary (test) pattern is presented to an observer, this results in an illusory movement in the direction opposite to the adapting motion. Typically, this motion aftereffect (MAE) does not occur after adaptation to a second-order motion stimulus (i.e. an equiluminous stimulus where the movement is defined by a contrast or texture border, not by a luminance border). However, a MAE of second-order motion is perceived when, instead of a static test pattern, a dynamic test pattern is used. Here, we investigate whether a second-order motion stimulus does affect the MAE on a static test pattern (sMAE), when second-order motion is presented in combination with first-order motion during adaptation. The results show that this is indeed the case. Although the second-order motion stimulus is too weak to produce a convincing sMAE on its own, its influence on the sMAE is of equal strength to that of the first-order motion component, when they are adapted to simultaneously. The results suggest that the perceptual appearance of the sMAE originates from the site where first-order and second-order motion are integrated. PMID- 10755149 TI - The illusory-letters phenomenon: an illustration of graphemic restoration in visual word recognition. AB - We present a demonstration of word perception in which stimuli containing very few letters (just 50% of their original number) are presented for unlimited durations and yet are seen unequivocally as complete words. The phenomenon suggests that recognition of words can be achieved even when perception of their component letters is prevented. PMID- 10755150 TI - Spatial and contextual factors in human performance on the travelling salesperson problem. AB - The travelling salesperson problem (TSP) provides a realistic and practical example of a visuo-spatial problem-solving task. In previous research, we have found that the quality of solutions produced by human participants for small TSPs compares well with solutions from a range of computer algorithms. We have proposed that the ability of participants to find solutions reflects the natural properties of human perception, solutions being found through global perceptual processing of the problem array to extract a best figure from the TSP points. In this paper, we extend the study of human performance on the task in order to understand further how human abilities are utilised in solving real-world TSPs. The results of experiment 1 show that high levels of solution quality are maintained in solving larger TSPs than had been investigated previously with human participants, and that the presence of an implied real-world context in the problems has no effect upon performance. Experiment 2 demonstrated that the presence of regularity in the point layout of a TSP can facilitate performance. This was confirmed in experiment 3, where effects of the internality of point clusters were also found. All three experiments were consistent with a global, perceptually based approach to the problem by participants. We suggest that the role of perceptual processing in spatial problem-solving is an important area for further research in both theoretical and applied domains. PMID- 10755151 TI - Foveal and peripheral thresholds for detection and resolution of vanishing optotype tumbling E's. AB - We measured detection and resolution acuity for vanishing optotype tumbling E stimuli in both the fovea and at 30 degrees in the periphery to determine if peripheral resolution is sampling limited for this stimulus. In the fovea, where acuity is optically limited, detection and resolution were the same. At 30 degrees, however, detection was markedly better than resolution indicating that peripheral resolution is sampling limited for this stimulus. Detection acuity was higher when contrast was 90% rather than 40%, but resolution did not change with contrast. The vanishing optotype is a legitimate perimetric stimulus to measure retinal ganglion cell density provided the task is resolution and not detection. PMID- 10755152 TI - Ethambutol affects the spectral and polarisation sensitivity of on-responses in the optic nerve of rainbow trout. AB - Juvenile rainbow trout (Oncorhynchus mykiss) were given ethambutol (900 mg kg-1 body mass per day) for 4 weeks to examine its effects on vision. Using multi-unit recording from the optic nerve, spectral sensitivity of the on-responses were significantly affected in two regions, 340-440 nm and 600-660 nm. Off-responses were statistically unaffected. Changes in sensitivity to polarised light were also observed with on-responses to vertically-polarized light decreasing relative to horizontally-polarised light. In contrast, off-responses were less affected. The treatment effects were attributed to changes in the relative contribution of the photoreceptor channels as recorded at the level of the optic nerve. PMID- 10755153 TI - Factors limiting contrast sensitivity in experimentally amblyopic macaque monkeys. AB - Contrast detection is impaired in amblyopes. To understand the contrast processing deficit in amblyopia, we studied the effects of masking noise on contrast threshold in amblyopic macaque monkeys. Amblyopia developed as a result of either experimentally induced strabismus or anisometropia. We used random spatiotemporal broadband noise of varying contrast power to mask the detection of sinusoidal grating patches. We compared masking in the amblyopic and non amblyopic eyes. From the masking functions, we calculated equivalent noise contrast (the noise power at which detection threshold was elevated by square root of 2) and signal-to-noise ratio (the ratio of threshold contrast to noise contrast at high noise power). The relation between contrast threshold and masking noise level was similar for amblyopic and non-amblyopic eyes. Although in most cases there was some elevation in equivalent noise for amblyopic compared to fellow eyes, signal-to-noise ratio showed greater variation with the extent of amblyopia. These results support the idea that the contrast detection deficit in amblyopia is a cortical deficit. PMID- 10755154 TI - Detecting collinear dots in noise. AB - We estimated the sensitivity for detecting a row of collinear target elements (usually dots) by measuring the maximum density of randomly positioned noise elements that allowed 75% correct detection of the orientation of alignment (binary choice: horizontal versus vertical) of the target elements. We varied the number of target elements, their mode of generation, and their accuracy of positioning. As reported previously (Moulden (1994) Higher-order processing in the visual system. Ciba Foundation Symposium 184. Chichester: Wiley), target detection improved rapidly until the number of target elements reached about seven, and then improved more slowly beyond this point. However, this break was reduced (and often removed entirely) when the target array was formed by repositioning pre-existing noise elements lying close to the target location, rather than by superimposition of additional target elements onto the noise array. This almost linear slope of improvement, coupled with the observation that target detection was disrupted more by random jitter of target elements at right angles to their axis of alignment than by jittering along this axis, argues against a two-stage process of perceptual grouping (Moulden, 1994) and supports instead an explanation based on the operation of a single mechanism. This single mechanism explanation is further supported by the observation that intrinsic positional uncertainty (estimated from the results of jitter experiments) was independent of target element number. Additional experiments showed that target detection is facilitated by aperiodic noise dots that fall close to the target axis. The results are discussed in relation to alternative explanations of perceptual grouping. PMID- 10755155 TI - Position-based motion perception for color and texture stimuli: effects of contrast and speed. AB - Motion can be perceived either through low-level, motion-energy detection or through tracking the change in position of features. Previously we have shown that, while luminance-based motion likely is detected with velocity-sensitive motion-energy units, patterns defined by texture or binocular disparity ('second order' stimuli) were tracked by a position-sensitive mechanism (Seiffert & Cavanagh (1998) Vision Research, 38, 3569-3582). Here, we use the same technique, measuring motion amplitude thresholds of oscillating gratings over a range of temporal frequencies and find that the motion of low-contrast equiluminant red/green gratings is also detected with position tracking. In addition, we find that as contrast or speed increases these results change: high-contrast or high speed equiluminant color or texture-based motion is detected by velocity sensitive mechanisms. These results help resolve the dispute over the processes which detect the motion of non-luminance based stimuli. Both systems are available, but their relative efficiency changes as a function of contrast and speed. A position-tracking process is more sensitive at low contrasts and low speeds whereas a motion-energy system is more sensitive at high contrasts and high speeds. PMID- 10755156 TI - Detection of flickering edges: absence of a red-green edge detector. AB - Kelly ((1975) Science, 188, 371-372) showed that a centrally-fixated, contrast reversing edge has a very different effect on the detection of luminance and red green flicker. Red-green flicker sensitivity was approximately 3-fold greater for a uniform field than for a 'split' field with the two sides flickering out-of phase. Just the opposite effects were observed for luminance flicker--the split field yielded a 7-fold advantage over the uniform field at 2 or 4 Hz and a 3-fold advantage at 12 Hz. Contrary to Kelly, we find that the split field offers only a very small advantage of 40% for luminance flicker at 2 Hz and virtually no advantage at 4 Hz and above. Kelly's chromatic results are surprising since one might expect that the larger color difference (or step) across the central edge would aid chromatic discrimination rather than strongly suppressing sensitivity. We show that the central chromatic edge only weakly impairs detection. Further results show that the two sides of the chromatic split field are detected essentially independently by red or green 'blob' detectors, which do not take advantage of the color difference across the edge. This has a remarkable implication: when wavelength discrimination is measured with a bipartite field whose two side are slowly modulated in opposite directions, then one side may be deleted with little adverse effect. PMID- 10755157 TI - Adaptation of torsional eye alignment in relation to head roll. AB - The coordination of head tilt, ocular counter-roll and vertical vergence is maintained by adaptive mechanisms; the desired outcome being clear single vision. A disruption or imbalance in otolith-ocular pathways may result in diplopia which stimulates these adaptive processes. In the present experiment, dove prisms were used to create cyclodisparities that varied with head tilt about a naso-occipital axis (roll). A stimulus for incyclovergence was presented with the head rolled 45 degrees to one side and a stimulus for an excyclovergence was presented with the head rolled 45 degrees to the other side. At the end of 1 h of training, all subjects demonstrated a change in open-loop cyclovergence that would help to correct for the cyclodisparities experienced during the closed-loop training period. The change appeared to be a simple gain change in the ocular counter-roll of one or both eyes. PMID- 10755158 TI - Express averaging saccades in monkeys. AB - When monkeys are presented simultaneously with multiple stimuli, they can make one of two types of response. Either they make averaging saccades, that land at intermediate locations between the targets, or target-directed saccades, that land close to one of the targets. The two types of saccades occur at different latencies and are thought to reflect different processes; fast reflexive averaging and slower target selection. We investigated the latency of averaging saccades in five monkeys, with particular emphasis on 'express' latency saccades, which are thought to be inhibited by target selection. Express averaging saccades were made prolifically by the two monkeys that made both express and regular latency saccades, but only when no specific instruction was given regarding the saccade target. When these monkeys had to choose one of the targets, on the basis of its color, they still made averaging saccades. However, the endpoints formed two distributions close to the targets as opposed to one single distribution centered between the targets, as was the case when targets were identical; also, express saccades were almost entirely absent. We conclude that express averaging saccades are a form of spatial and temporal optimization of gaze shifting. PMID- 10755159 TI - Contrast facilitation in motion detection: evidence for a Reichardt detector in human vision. AB - We measured contrast thresholds for detecting the direction of movement of a grating in a two-frame sequence with a quadrature shift between frames. The threshold contrast for the first frame was determined for a range of contrasts of the second frame. As the contrast of the second frame was increased, the threshold contrast of the first frame initially fell, and then remained constant at a value that was approximately 0.3 log units below the threshold contrast when the two frames had the same contrast (the yoked threshold). We show that this motion facilitation effect is a specific prediction of a Reichardt detector, with additive noise before the multiplication site. When the order of the two frames was reversed, the motion facilitation effect was found for contrast of the second frame, but at sufficiently high contrasts of the first frame, masking was found. The temporal order asymmetry is not found at near yoked-threshold contrasts of the first frame. The Motion Facilitation Effect was smaller than the contrast facilitation in a classical contrast discrimination experiment. PMID- 10755160 TI - Effects of biased feedback on learning and deciding in a vernier discrimination task. AB - We investigate the influence of biased feedback on decision and learning processes in a vernier discrimination task. Subjects adjust their decision criteria and hence their responses according to biased external feedback. However, they do not use learning processes to encode incorrectly classified stimuli. As soon as correct feedback is restored observers regain their original performance indicating an involvement of internal criteria. If the external feedback is switched off instead of being corrected, the rebound is less vigorous. The findings contradict predictions of supervised neural network models. PMID- 10755161 TI - The role of convexity in perceptual completion: beyond good continuation. AB - Since the seminal work of the Gestalt psychologists, there has been great interest in understanding what factors determine the perceptual organization of images. While the Gestaltists demonstrated the significance of grouping cues such as similarity, proximity and good continuation, it has not been well understood whether their catalog of grouping cues is complete--in part due to the paucity of effective methodologies for examining the significance of various grouping cues. We describe a novel, objective method to study perceptual grouping of planar regions separated by an occluder. We demonstrate that the stronger the grouping between two such regions, the harder it will be to resolve their relative stereoscopic depth. We use this new method to call into question many existing theories of perceptual completion (Ullman, S. (1976). Biological Cybernetics, 25, 1-6; Shashua, A., & Ullman, S. (1988). 2nd International Conference on Computer Vision (pp. 321-327); Parent, P., & Zucker, S. (1989). IEEE Transactions on Pattern Analysis and Machine Intelligence, 11, 823-839; Kellman, P. J., & Shipley, T. F. (1991). Cognitive psychology, Liveright, New York; Heitger, R., & von der Heydt, R. (1993). A computational model of neural contour processing, figure-ground segregation and illusory contours. In Internal Conference Computer Vision (pp. 32-40); Mumford, D. (1994). Algebraic geometry and its applications, Springer, New York; Williams, L. R., & Jacobs, D. W. (1997). Neural Computation, 9, 837-858) that are based on Gestalt grouping cues by demonstrating that convexity plays a strong role in perceptual completion. In some cases convexity dominates the effects of the well known Gestalt cue of good continuation. While convexity has been known to play a role in figure/ground segmentation (Rubin, 1927; Kanizsa & Gerbino, 1976), this is the first demonstration of its importance in perceptual completion. PMID- 10755162 TI - Testing for glaucoma with the spatial frequency doubling illusion. AB - We examined the performance of tests for glaucoma based on the spatial frequency doubling (FD) illusion. Contrast thresholds for seeing the FD illusion in four large visual field regions were measured from 340 subjects who were tested up to seven times over 2 years. Median sensitivities of 91% at specificities of 95% were obtained. Test-retest variability for the worst hemifield thresholds averaged 2.22 db +/- 0.09 S.E. for all tested groups, and significant progression was observed for glaucoma suspects over the seven visits, indicating that tests based on the FD illusion can detect diffuse early glaucomatous loss. PMID- 10755163 TI - The effects of simulated cataract on reading with normal vision and simulated central scotoma. AB - Reading rates are slower for persons with low vision than for normally-sighted persons. This study investigated the change in reading performance and reading eye movements when we simulated the two most common causes of low vision--central field loss and cataract--and their combination (scotoma + cataract). Three subjects read sentences with each of these simulated impairments at five different letter sizes. They required larger letters to read with the cataract or scotoma than they did with normal vision, and larger still to read with scotoma + cataract; the change in eye movements relative to normal vision was similar across conditions. When reading large letters (1.61 degrees), the cataract had almost no effect, while the scotoma and scotoma + cataract reduced reading rate for two of the subjects. The cataract had a greater impact on performance relative to normal vision for these same two subjects, while for the third subject the cataract had a greater impact with the scotoma in place. Cataract extraction tends to be postponed in patients with central field loss because it is not perceived to be beneficial. The findings from this study, as well as others, suggest that patients with central field loss would benefit from cataract extraction. PMID- 10755164 TI - Nystagmus induced by pharmacological inactivation of the brainstem ocular motor integrator in monkey. AB - A common cause of pathological nystagmus is malfunction of the mechanism by which the brain integrates eye velocity signals to produce eye position commands. For horizontal gaze, neurons in the nucleus prepositus hypoglossi-medial vestibular nucleus region (NPH-MVN) play a vital role in this neural integrator function. We studied the effects on gaze stability of pharmacological intervention in the NPH MVN of monkeys by microinjections of eight drugs. Agents with agonist or antagonist actions at gamma-aminobutyric acid (GABA), glutamate, and kainate receptors all caused gaze-evoked nystagmus with centripetal eye drifts; glycine and strychnine had no effect. When the GABAA-agonist muscimol was injected near the center of MVN, the eyes drifted away from the central position with increasing-velocity waveforms, implying an unstable neural integrator. The observed effects of these drugs on gaze stability may be related to inactivation either of neurons within NPH-MVN or the cerebellar projections to them that control the fidelity of neural integration. Drugs that influence GABA or glutamine transmission may have a role in the treatment of nystagmus due to an abnormal neural integrator. PMID- 10755165 TI - Doppler echocardiographic examinations in the assessment of the athletic heart. AB - Doppler echocardiography is a method with the help of which flow velocity and the duration of different intervals can be estimated. The ratio between early and late peak velocities (E/A) is linearly proportional to diastolic function, i.e. to ventricular compliance. Data of 179 athletes and of 42 nontrained young healthy men indicated that the E/A quotient was higher in athletes than in the sedentary controls (2.086 +/- 0.505 vs. 1.905 +/- 0.384) in young adult age, but of the different athletes it was the only group of endurance athletes that showed a significant increase. Regular physical training seems to protect against an age dependent impairment of left ventricular compliance, as an increased E/A ratio can be observed at the age of 31-45 years (1.77 +/- 0.46 vs. 1.43 +/- 0.276) as well as in men above 45 years of age (1.61 +/- 0.36 vs. 1.24 +/- 0.36). Bradycardia of the athletic heart resulted in a significantly longer duration of the cardiac cycle in athletes than in non-athletes. Different phases of the cardiac cycle, however, were not equally modified. There were periods the absolute duration of which were slightly decreased, unchanged or slightly increased, but the relative ones are strongly decreased: such as isovolumetric contraction time (ICT), acceleration of the transaortic flow (AOAT), deceleration of the transaortic flow (AODT), acceleration period of the early diastolic filling (EACC), and deceleration period of the early diastolic filling (EDT). There were periods the absolute duration of which increased proportionally to the increase of the whole cardiac cycle, while relative duration was not changed: isovolumetric contraction time (IVRT) and the atrial systole (A). There was one period that showed the greatest variability in the different subjects and both its absolute and relative duration was definitely increased in the athletes: this was the EA period, i.e. the period from the end of early filling to the beginning of the atrial systole. PMID- 10755166 TI - Effect of digoxin imprinting in adolescence on the sexual behavior of adult rats. AB - Four-time 3 micrograms digoxin treatment of male rats at puberty (in six weeks old rats) significantly increased the libido of rats (number of intromissions) and reduced the number of ejaculations, two months after the treatments (in three and a half months old rats). In female rats the Meyerson index and lordosis quotient were not significantly decreased. The experiment calls attention to the wide-ranging imprinting effect of digoxin which was also demonstrated earlier after prenatal (maternal) treatment. The experiment also supports the male sexual potency influencing effect of digoxin treatment, previously supposed in men. PMID- 10755167 TI - Direct and transgenerational effect of benzpyrene treatment at adolescent age on the uterine estrogen receptor and thymic glucocorticoid receptor of the adult rat. AB - Hormonal imprinting develops perinatally at the first encounter between the maturing receptor and the target hormone, helping the normal accomplishment of receptor maturation. In the presence of hormone excess or foreign molecules able to bind to the maturing receptor, faulty imprinting takes place, which disturbs the normal receptor function for life. Earlier experiments demonstrated that the effect of faulty perinatal benzpyrene imprinting of the steroid hormone receptors is transmitted to the progeny generations. In certain organs which are maturing later (such as the uterus) imprinting can be executed at adolescence. In the present experiments pubertal benzpyrene imprinting caused a durable decrease in female's estrogen receptor density. The transgenerational effect of this type of imprinting was also studied. The pubertal imprinting of the parents was transgenerationally transmitted to the offspring generation in which--without further treatment--the density (Bmax) of the uterine estrogen receptors was significantly higher than that in the controls. There were measurable effects neither in the affinity (Kd) of uterine estrogen receptors nor in the Kd and Bmax of the male thymus glucocorticoid receptors. The experiments call attention to the profound and comprehensive imprinting effect of the environmental pollutant benzpyrene. PMID- 10755168 TI - Effects of cue information on response production and inhibition measured by event-related potentials. AB - The aim of this study was to investigate how information carried by a cue stimulus modulate event-related potentials (ERPs) to a subsequent target stimulus which either calls for an overt response (Go stimulus) or no response (Nogo stimulus). One of the cues predicted the likely appearance of the Go stimulus (Go cue) whereas the other cue predicted the likely appearance of the Nogo stimulus (Nogo cue). Our results showed that unpredicted Nogo stimuli elicited enlarged N200 component. This finding supports the notion that Nogo N200 reflects response inhibition processes, i.e., the amplitude of the N200 is a function of the difficulty of response inhibition. In other words, increased N200 to Nogo stimuli following Go cues might be related to increased efforts in activating the response inhibition system thereby interrupting preparations to respond. PMID- 10755169 TI - Visual event-related potentials evoked by using a virtual reality display. AB - This study aimed at investigating whether a virtual reality display (VRD) is an appropriate tool for evoking visual event-related potentials (VEPs). VEPs evoked by VRD stimuli were highly similar in form to VEPs evoked by using a computer monitor, both having two dominant peaks, labeled P100 and N200. Monitor and VRD N200 latencies and amplitudes were highly correlated. However, peak latencies were longer and the peaks were broader when stimuli were presented on the VRD. Besides, VRD P100 amplitude was smaller, and an N75 peak could be seen usually only on monitor VEPs. PMID- 10755170 TI - Experimental hypertriglyceridaemia and hypercholesterolaemia in rats. AB - Lipid disorders and cardiovascular diseases have been related in many studies. We here studied the influence of acute ingestion of a long chain triglyceride (LCT) emulsion (rich in triglycerides) on plasma triglyceride (TG) and total cholesterol (TC) levels in laboratory animals (Wistar rats), comparing it with the induction of hypertriglyceridaemia and hypercholesterolaemia by Triton WR 1339 injection. The results show that Triton would be suitable for inducing hypercholesterolaemia but not hypertriglyceridaemias similar to those in humans. The LCT emulsion intake, however, provoked transitory hyperlipaemia with values similar to those often found in hyperlipaemic subjects, and would thus be suitable for testing possible antilipaemic treatments. Our study also presents a model of hypertriglyceridaemia, hypercholesterolaemia and obesity in experimental animals, provoked by a chronic intake of an LCT emulsion. This model may be useful in investigating the mechanisms of the pathogenesis of atherosclerosis and the pharmacological treatment of obesity and dyslipaemias. PMID- 10755171 TI - Experimental data proving the presence of inhibitors of amylase activity in biliary and pancreatic juice. AB - Two parallel studies on albino male rats are performed. In the first study, there is a group which underwent resection of the proximal third of the small intestine. While the other group despite resection of the same segment also has a ligated common biliary and pancreatic duct. In the second study, one group of the experimental animals is only with ligated pancreatic duct and in the other group the same duct is implanted in the initial part of the ileum. On the 15th day after the surgical interventions the amylase activity and the absorption of glucose in the small intestine are studied by the method of turned sacs "in vitro". It is established that the glucose transport does not change after the four surgical interventions. However, the amylase activity increases about twice times after resection of the upper third of the small intestine and more than 4 times after resection of the same segment with simultaneous ligature of the common biliary and pancreas duct. Only at ligating the duct, the amylase activity is decreased in the jejunum and is significantly increased in the ileum, while its implantation in the initial part of the ileum does not change its activity in both studied segments of the small intestine. It is concluded that there are unknown inhibitors for the amylase activity in the biliary and pancreatic juice. The discussed issue is why they inhibit only the enzymatic compensatory processes without influencing the transport systems of the small intestine. PMID- 10755172 TI - Neuropsychological function in mild hyperphenylalaninemia. AB - Whether specific cognitive deficits related to frontal-lobe dysfunction that have been reported in individuals with phenylketonuria (PKU) are also characteristic of mild hyperphenylalaninemia (MHP) was investigated. Tests of executive function and control tasks not assessing executive function were administered to a group of individuals with MHP and a group without MHP, similar in age, gender, and IQ. Tests of academic skills and behavior-rating questionnaires were also administered to the group with MHP. No group differences were found for any measure, suggesting that the mild elevations of phenylalanine in individuals with MHP are not sufficient to produce behavioral and cognitive impairments characteristic of PKU. PMID- 10755173 TI - Quality and costs of community-based residential supports, village communities, and residential campuses in the United Kingdom. AB - The costs, nature, and benefits of residential supports were examined for 86 adults with mental retardation living in village communities, 133 adults living in newly built residential campuses, and 281 adults living in dispersed housing schemes (small community-based group homes and supported living). Results indicated that (a) the adjusted comprehensive costs of provision in dispersed housing schemes were 15% higher than in residential campuses and 20% higher than in village communities; (b) dispersed housing schemes and village communities offered a significantly greater quality of care than did residential campuses; and (c) there appeared to be distinct patterns of quality of life benefits associated with dispersed housing schemes and village communities, with both approaches offering a greater quality of life than did residential campuses. PMID- 10755174 TI - Long-term effects of naltrexone on self-injurious behavior. AB - A subgroup of self-injuring patients responds positively to the opiate-blocking agent naltrexone in acute, double-blind studies. In this study we examined the effects of naltrexone after acute treatment and the long-term effects of naltrexone on SIB. Rates of SIB were collected from pretreatment baseline; a second baseline a year after the acute trial; and a subsequent 12-month double blind, placebo-controlled treatment. A subgroup of patients decreased SIB for a year without treatment after acute exposure to naltrexone. Five participants who decreased SIB by 70% after acute treatment increased SIB to the long-term treatment with naltrexone. In contrast, those for whom SIB increased over the one year treatment hiatus decreased their SIB after the first long-term treatment. Discussion of these complex effects considered the role of background opioid levels, dosing, and treatment regimen of naltrexone and other factors limiting receptor adaptation among patients who exhibit SIB. PMID- 10755175 TI - Auditory integration training for children with autism: no behavioral benefits detected. AB - Auditory integration training and a control treatment were provided for 16 children with autism in a crossover experimental design. Measures, blind to treatment order, included parent and teacher ratings of behavior, direct observational recordings, IQ, language, and social/adaptive tests. Significant differences tended to show that the control condition was superior on parent rated measures of hyperactivity and on direct observational measures of ear occlusion. No differences were detected on teacher-rated measures. Children's IQs and language comprehension did not increase, but adaptive/social behavior scores and expressive language quotients decreased. The majority of parents (56%) were unable to report in retrospect when their child had received auditory integration training. No individual child was identified as benefiting clinically or educationally from the treatment. PMID- 10755176 TI - Some relationships between informant assessment and functional analysis of problem behavior. AB - The strengths and limitations of informant assessment of problem behavior was investigated through examination of such behavior across seven situations and 3 participants. The results of informant assessment were compared to those obtained from functional analysis to determine some of the parameters under which informant assessment might provide a practical alternative to functional analysis. The results showed that informant hypotheses about the function of problem behavior were validated by subsequent functional analyses only when informants identified their hypotheses as involving situations likely to evoke problem behavior. Hypotheses involving situations that informants rated as less likely to evoke problem behavior were not validated by subsequent functional analyses. The implications of these findings for improving the validity of informant assessment are discussed. PMID- 10755177 TI - Efferent connections of the ectostriatal core. An anterograde tracer study. AB - In the present study the efferent connections of the ectostriatal core were investigated with biotinylated dextran-amine anterograde tracer in the chicken using light microscopy. The efferents of the ectostriatal core were labelled anterogradely, but retrograde labelling was also observed, which displayed the afferents of the same region. The ectostriatal belt received a few thin varicose fibres; retrogradely labelled cells also appeared. The most extended projection ended in the surrounding neostriatum, which turned out to be reciprocally connected to the ectostriatal core. On the basis of these connections, the neostriatum is said to be an important visual associative center. Efferent fibres reached the motor areas as well. A significant projection entered the paleostriatum augmentatum, especially the ventral part. The paleostriatum primitivum also received a few fibres. The other motor center, the medial part of the anterior archistriatum, was proved to be directly connected to the ectostriatal core as well. Considering that the archistriatum is also connected indirectly to the Wulst, the movements are able to be guided by well processed visual information. PMID- 10755178 TI - The combined retrograde transport and unbiased stereological study of the claustrocortical connections in the rabbit. AB - The quantitative analysis of the claustrocortical connections in the rabbit, labeled with the fluorescent retrograde tracer Fluoro-Gold (FG), was conducted by means of unbiased stereology. The FG was injected into selected regions of the motor, somatosensory, auditory and visual cortices and then a comparison of the various claustrocortical projections was carried out. This was achieved by comparing (1) the numerical densities of projecting neurones for each claustral projection zone and (2) the distribution of the labeled neurones throughout the rostro-caudal extent of the claustrum. No significant differences between the numerical densities of labeled neurones in the various projection zones are reported. The motor and primary somatosensory projections dominated in the anterior and central parts of the claustrum, whereas the secondary somatosensory, auditory and visual projections--in the posterior part. The difference in the distributions was significant (p < 0.001). Summarizing, the cortical projections in the claustrum, although varying topographically, do not reveal a quantitative differentiation. This may speak in favour of the integrative and modulating function of this structure in relationship to the neocortex. PMID- 10755179 TI - Acellular nerve graft re-seeded by Schwann cells migrating from the nerve stump can stimulate spinal motoneurons for functional reinnervation of the rat muscle. AB - The acellular nerve graft was utilised to restore a functional reinnervation of the biceps brachii muscle from the motoneuron pool of the cervical spinal cord. The musculocutaneous nerve stump was sutured to an acellular nerve graft, the opposite end of which was inserted into the cervical spinal cord cranial to the avulsed C5 ventral root. The acellular nerve graft was repopulated by Schwann cells heavily immunostained for NGFr within 90 days. The Schwann cells migrating from the nerve stump reached the spinal cord grey matter, where they stimulated the motoneurons to send axonal sprouts. The functional reinnervation of the biceps brachii muscle was assessed by means of the behavioural (grooming) test and EMG, the presence of myelinated and unmyelinated axons was demonstrated by light and electron microscopy. The axonal reconnection of the musculocutaneous nerve stump was verified by horseradish peroxidase retrograde labelling of the spinal motoneurons. Moreover, the motoneurons on the operated side of the C5 spinal segment displayed increased immunostaining for GAP-43 in comparison to the contralateral side, whereas the pattern of AChE histochemical reaction was similar on both the operated and contralateral side, of the C5 segment 150 days after acellular graft implantation. The regenerated axons bridged a 4-cm long originally acellular nerve graft to reach and reinnervate the biceps brachii muscle. The reinnervation of the neuromuscular junctions was morphologically determined by immunofluorescence for neurofilaments. The number of myelinated axons in the acellular nerve graft was significantly higher than those growing over the cellular graft, but their diameter was smaller. The results of experiments presented here demonstrate functional recovery of the biceps muscle reinnervation through the acellular nerve graft repopulated by migrating Schwann cells. The process of reinnervation by acellular nerve graft is however delayed and worse in comparison with the cellular graft. PMID- 10755180 TI - Automatic image analysis of the postnatal growth of axons and myelin sheaths in the tibial and peroneal nerves of the rabbit. AB - The numerous morphometric studies on the myelinated fibers in the peripheral nerves have presented varying results. Only a few studies deal with the peripheral nerves from rabbits. In this work, a morphometric study was carried out on the tibial and peroneal nerves of new-born, 10-, 15-, 20-, 30-, 60-, 90- and 240-day-old rabbits. The bilateral proximal segments of both nerves were investigated. Negatives of semi-thin cross sections were used for myelinated fiber morphometric analysis, carried out by an OLYMPUS Video image analyser. Two morphometric parameters, the average axon diameter (AD) (the average length of Feret's diameters) and the specific width of the myelin sheaths (SWMS) (specifying the total width of the myelin sheath), were evaluated for every age group. In the tibial and peroneal nerves a bimodal distribution of the average AD appeared on the 20th day, and of the SWMS on the 10th day postnatum. A tight correlation was obtained when comparing mean AD (mAD) and mean SWMS (mSWMS) in new-born and 240-day-old rabbits. From birth to adulthood the mAD increased in both nerves by about 270% and the mSWMS by about 280%. The mAD/mSWMS ratio in both new-born and 8-month-old rabbits was found to be 4 in the tibial and 5 in the peroneal nerves. According to the available data, an approach to the measurement of AD as an average length of Feret's diameters and the measurement of the myelin thickness as a SWMS has not yet been employed. An extension of this methodological approach could help to understand the growth and myelinization of peripheral nerve fibers. PMID- 10755182 TI - Lectin histochemistry of the canine anal glands. AB - The distribution and selectivity of complex carbohydrates in the canine anal glands were studied by means of lectin histochemistry, using PO-labeled lectins. The secretory epithelium of the anal glands and the excretory duct system exhibited large amounts of mainly neutral glycoproteins with various terminal sugars (alpha-D-mannose, beta-N-acetyl-D-glucosamine, alpha-N-acetyl-D galactosamine, alpha-D-galactose, alpha-L-fucose, N-acetyl-neuraminic acid). Distinctly prominent in the secretion were alpha-L-fucose residues. This relatively hydrophobic sugar may in particular modify or control the viscoelastic properties of the anal gland mucus, so that a stable mucous coat of the rather dry faeces can be formed. In addition, it was obvious that the major part of the excretory duct system is also involved in secretion production, and that the essential function of the saccular dilatations of the excretory ducts is to ensure secretion maturation. PMID- 10755181 TI - The antiovulatory potential of progesterone antagonists correlates with a down regulation of progesterone receptors in the hypothalamus, pituitary and ovaries. AB - These studies analyze the regulation of progesterone receptors (PRs) in central and peripheral tissues with the aim of further understanding mechanistically the inhibition of ovulation by progesterone antagonists (PA). Therefore, it was of interest to investigate the influence of the progesterone receptor antagonist, Onapristone (ON), on PRs in the ovary, pituitary (PT), and hypothalamus (HYP), since ON effectively inhibits ovulation in rats. For this study PMSG/hCG-primed immature and adult female rats were treated with ON. Immunohistochemistry was used for the detection of PRs. Progesterone (P4) and estradiol (E2) levels were determined by RIA. PR expression in the ovaries of immature rats was not detectable until after hCG administration. In these animals, ON caused a reduction in the staining intensity of PR in the tertiary follicles at the time when the preovulatory P4-surge was inhibited (6 h post hCG). Adult rats treated for 15 days with ON showed a decreased PR expression in PT and HYP. At this time (proestrus, 7 p.m.) the P4 and E2 levels are significantly lowered. These results suggest that after treatment with ON the expression of PR is reduced in the ovary, PT and HYP. The regulation of PR in the ovary seems to be less dependent on estrogens than on LH. Thus, it is conceivable that the reduced PR expression after ON treatment may be a result of decreased LH sensitivity in the ovary. In the pituitary and hypothalamus, PR expression is stimulated by estrogens and progesterone, and therefore the fall in the P4 and E2 levels in ON-treated animals may be responsible for the reduced PR expression in PT and HYP, and may contribute to the antiovulatory effect of ON. We therefore conclude that the mechanism of the antiovulatory potency of progesterone antagonists is based on a reduced preovulatory P4-production and PR expression in the ovary and also on the down-regulation of PR in the anterior pituitary and hypothalamus. PMID- 10755183 TI - Morphology of the copulatory apparatus of the spotted tinamou Nothura maculosa (Aves: Tinamiformes). AB - The components of the copulatory apparatus of Nothura maculosa were identified and studied by means of dissections, parenteral latex injections and standard histological and histochemical techniques. N. maculosa possesses an intromittent phallus with a tubular cavity, within which a fixed base and a tubular portion can be distinguished. An ejaculatory groove, which is supported by a fibrous body, runs dorsally at the base of the phallus. The tubular portion of the phallus inserts into the fibrous body, the former possessing two continuous but morphologically distinct parts, one of which is eversible and is extruded during erection while the other is a fixed non-eversible glandular portion. The walls of both parts are formed by the mucosa, an intermediate layer of connective tissue containing a wide lymphatic space which surrounds the whole perimeter of the tube, and an external layer of dense connective tissue. The mucosa of the eversible portion, which lodges the phallic groove, is lined by a non-keratinized stratified squamous epithelium. In the fixed tubular portion, the tubular lumen is reduced in size, the phallic groove disappears, the mucosa becomes folded and there occurs an abrupt change to a pseudostratified columnar secretory epithelium. The copulatory apparatus of N. maculosa includes the vascular bodies of the phallus, which show morphofunctional continuity with the phallic structures, thus forming a single system involved in erection. On erection, the eversible portion of the phallus evaginates and emerges from the cloacal opening as a spiral shaped structure directed towards the left. PMID- 10755184 TI - Ovariuterus of Pandinus imperator, Koch (Scorpiones; Scorpionidae): comparison of virgin female with mother. AB - The ovariuterus of the female Pandinus imperator Koch (Scorpiones; Scorpionidae), was compared in a virgin female and a female that had previously given birth at least twice (in the laboratory). The virgin female did not have any embryonic diverticulae (Ed) nor did it have any degenerated, post-partum diverticulae (Dd), whereas in the mother scorpion several Dd were clearly seen on the ovariuterus. This latter female lacked any embryonic diverticulae (Ed). The number of the Dd corresponds well with the number of juveniles in the last brood born to that female during the previous year. Based on the total number of diverticula observed, and the average known litter size, it is suggested that these long lived scorpions are potentially able to breed at least six times during their lifetime. Since they apparently do not breed in consecutive years (as is evident from the lack of Ed in the female that had bred a year before) and perhaps only every alternate year, they are capable of breeding for 12 years. If a litter amounts to about 25 young, a female is capable of producing 150 young. As it takes about three years to mature from nymph to adult, the life expectancy in this species is therefore about 15 years, by a conservative estimate. PMID- 10755185 TI - Ultrastructural study of the root dentine surface resuming resorption on human deciduous teeth. AB - Resorption of deciduous teeth is not continuous, but alternates with periods of repair or rest. Dentine surfaces in periods of rest or repair resume resorption by odontoclasts during physiological root resorption of the deciduous teeth. However, no observations of such dentine surfaces have been shown. The characteristic feature of the dentine surfaces resuming resorption remains unknown. Tartrate-resistant acid phosphatase activity (TRAP) was detected on human deciduous teeth. The root resorbing surfaces on these teeth were photographed with a whole-mount light microscope, and the photographed areas were serially sectioned into 0.5 micron semithin sections. Preodontoclasts and odontoclasts were three-dimensionally reconstructed. On root resorbing surfaces, areas with small scattered TRAP-positive cells were observed among areas with many TRAP-positive resorbing odontoclasts and TRAP-negative areas. The sections showed that areas with small scattered TRAP-positive cells have features similar to those of TRAP-negative areas, but there were three kinds of characteristic TRAP-positive cells: preodontoclasts, odontoclasts forming small lacunae, and preodontoclasts, and odontoclasts with cytoplasmic processes extending to the dentine surface, which is covered with cells. These results suggest that the areas with small scattered TRAP-positive cells could be at the stage of resuming resorption, and show that the presence of preodontoclasts and odontoclasts with cytoplasmic processes extending to the covered dentine surface is a characteristic feature of the dentine surface at this stage. PMID- 10755186 TI - The structure of the cemento-dentinal junction in rat molars. AB - The cemento-dentinal junction was observed in the acellular and cellular cementum of rat molars by light and scanning electron microscopy. Scanning electron microscopy, combined with NaOH maceration, was used to observe the fibrous architecture directly in this region. Light microscopy revealed that the cemento dentinal junction contains fewer collagen fibrils and more proteoglycans than the cementum and dentin. Scanning electron microscopy also showed that fibril intermingling is found only in some regions of the fibril-poor junction in macerated specimens. Prolonged maceration breaks down the cemento-dentinal junction in spite of the fibril intermingling. Only macerated specimens showed detachment here. It was established that NaOH maceration removes interfibrillar substances effectively, and does not damage the fibril structure or architecture. This suggests that the adhesion of proteoglycans is more important than fibril intermingling for preserving the cemento-dentinal attachment in the rat molar. PMID- 10755187 TI - A case of a persistent left vena cava superior with atresia of the right atrial ostium of the coronary sinus. AB - A persistent left vena cava superior with an atretic ostium of the coronary sinus was found during the routine dissecting course in the embalmed cadaver of an 83 year-old woman who had died from cardiac infarction. The left vena cava superior was very narrow in diameter (4 mm), originated at the lateral part of the left vena brachiocephalica and ran down between the venae pulmonales sinistrae and the auricula sinistra. The vena cava opened into the sinus coronarius of the heart, which terminated as a blind sac due to an atretic ostium. The vena coronaria sinistra as well as the vena interventricularis posterior drained into the sinus coronarius. Congenital atresia of the coronary opening is a rare malformation and is usually associated with other anomalies. The congenital ostial atresia could be the cause of a persistent left vena cava superior, which then takes over the drainage of the cardiac veins. PMID- 10755188 TI - The caliber of the human long saphenous vein and its congenital variations. AB - The purpose of the present study is to evaluate the caliber of the normal human long saphenous vein (LSV) in order to verify the occurrence of congenital narrowings. The LSV morphology was evaluated by the dissection of 32 cadaveric limbs, and by ultrasonography of 102 healthy living subjects. The LSV caliber was constant in most of the limbs, showing only a mild and progressive increase from the ankle to the groin. Furthermore, great individual variation in LSV caliber was found. A segmental narrowing of the LSV was present in 39.8% of limbs. The narrow segment was visible with the naked eye during dissection or by ultrasonography in 22.4% of cases (LSV hypoplasia). In the remaining 17.4% the caliber was so reduced that it could only be detected microscopically (LSV aplasia). In relation to the narrow segments, the main ascending flow was shunted in a collateral vein running within the superficial hypodermis. The narrow segments of the LSV had a weaker and less muscular wall than did those of normal caliber. Hypoplasia and aplasia of the LSV are probably due to segmental failure in the development of the vessel, and represent a risk factor for varicosis. In fact, the ascending flow is shunted from the LSV in a collateral vein that runs in the yielding superficial fatty layer of the hypodermis. Furthermore, the high incidence of LSV segmental hypoplasia and aplasia has also to be considered whenever this vein is used as an arterial graft, because of the marked anatomical remodelling. PMID- 10755189 TI - CFTR gene mutations and male infertility. AB - Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are a relatively frequent cause of male infertility. Depending on their molecular consequences, CFTR mutations may either result in typical cystic fibrosis (CF), one of the most common autosomal recessive disorders, which is characterized by chronic lung disease, pancreatic exocrine insufficiency, an increase in the concentration of sweat electrolytes and male infertility, due to obstructive azoospermia, or in atypical (often monosymptomatic) forms of CF such as congenital absence of the vas deferens (bi- or unilateral), bilateral ejaculatory duct obstruction or bilateral obstructions within the epididymides. All males with idiopathic obstructive azoospermia bear an increased risk for CF offspring. Couples requesting microsurgical epididymal sperm aspiration and in vitro fertilization, e.g. intracytoplasmic sperm injection, should be offered genetic counselling and molecular genetic analysis of the CFTR gene, if male infertility due to obstructive azoospermia is the underlying cause. PMID- 10755190 TI - Effect of hypothyroidism on the testes in mature rats and treatment with levothyroxine and zinc. AB - This study was performed to investigate the effect of propylthiouracil (PTU) on rat testis, and to compare the results of the different treatment regimens of levothyroxine and zinc. Twenty sexually mature Wistar albino rats were subjected to PTU for 14 days intraperitoneally to make them hypothyroidic. The effect of PTU on testicular function was assessed histopathologically after unilateral orchiectomy on day 15, and treatment was evaluated by measuring serum thyroid stimulating hormone (TSH), T3, T4, and zinc levels on days 0, 7, and 15. The rats were then divided into five groups which were given levothyroxine and/or zinc treatment for 15 days. Orchiectomies were repeated on day 30, and specimens were evaluated histopathologically. Although serum T3, T4 and zinc levels decreased, serum TSH levels increased in PTU-treated rats, and the difference to the control group was statistically significant (P < 0.001). Maturation arrest of spermatogenesis, a reduced number of Sertoli and Leydig cells, a decreased tubular diameter, interstitial oedema, and thickening of basal membrane were observed in hypothyroidic testicles. After treatment, testicular histology and spermatogenesis gradually recovered in all groups with hypothyroidism, but maximum improvement was achieved in the levothyroxine + zinc sulphate replacement group, indicating a possible role of zinc in testicular function. PMID- 10755191 TI - Infertility treatment in autosomal dominant polycystic kidney disease (ADPKD)--a case report. AB - Autosomal dominant polycystic kidney disease (ADPKD) is a frequently occurring inherited condition with cysts in many organs including the kidneys. However, a combination of seminal vesicle cysts, cystic obstruction of ejaculatory duct and ADPKD is rarely encountered. The following case report presents an infertile ADPKD patient who had seminal vesicle cysts and ejaculatory duct cyst, and describes the treatment by transurethral resection of the ejaculatory duct. PMID- 10755192 TI - Inhibition of motility of hamster spermatozoa by protein tyrosine kinase inhibitors. AB - Genistein, tyrphostin and piceatannol, which are specific inhibitors of protein tyrosine kinase, were screened for their effects on the motility of intact and demembranated hamster spermatozoa. Of the three inhibitors only piceatannol inhibited the motility of intact spermatozoa. None of the inhibitors had any inhibitory effect on the reactivation of motility of demembranated hamster spermatozoa. Taken together these results indicated that a protein tyrosine kinase associated with the membrane of hamster spermatozoa was probably involved in sustenance of hamster sperm motility. Therefore in the present study a membrane-associated protein tyrosine kinase was purified from a detergent-soluble extract of plasma membranes of mature hamster spermatozoa. The purification involved cation exchange chromatography on fast protein liquid chromatography (FPLC) followed by affinity chromatography either on an antiphosphotyrosine antibody agarose or poly glu-tyr agarose column. The pure protein tyrosine kinase had an apparent molecular mass of 45 kDa. The enzyme was not inhibited by genistein or herbimycin but was inhibited by piceatannol. This is the first report on the purification of a sperm plasma membrane-associated protein tyrosine kinase, an enzyme which has also been implicated in hamster sperm motility. PMID- 10755193 TI - The effect of melatonin implants on blood testosterone and acrosin activity in spermatozoa of the ram. AB - In a series of consecutive blood sampling in 15 days intervals over 15 weeks after implantation of melatonin in rams an increased mean value, basal level and number of peaks of testosterone was observed in samples of the third fortnight (45th day). This increase was greater in the autumn (breeding season) than in spring (non-breeding season). Total acrosin activity in spermatozoa was increased between days 35-56 (autumn) and days 49-70 (spring) after implantation and the relative increase was higher in autumn than in spring. The increase of acrosin activity was independent of the changes of testosterone. An increase of acrosin activity by melatonin, in cases of low activity, might improve fertilization rates in sheep not only during the breeding season, but also during the non breeding season (after oestrus induction). PMID- 10755194 TI - Purification of proteins from rat sperm membranes that interact with ligands other than phosphomannosyl residues. AB - In this study proteins were purified from rat sperm membranes which might be the high affinity sites for ligands of epididymal fluid other than the mannose-6 phosphate receptors. The sperm membrane proteins were solubilized and passed over an affinity column containing epididymal fluid proteins coupled to a matrix. Two bands in the range of 45-55 kDa were eluted from the column with fructose-6 phosphate but not with mannose-6-phosphate. Although the molecular weight of these proteins are similar to those of the cation-dependent phosphomannosyl receptors they are not related. These two proteins may correspond either to two different receptors or to forms of the same receptor that recognize ligands from rat epididymal fluid. Sequencing and identification of these proteins will be the aim of future studies. PMID- 10755195 TI - Role of K+ATP channels in ischemic preconditioning and cardioprotection. AB - Since the phenomenon of ischemic preconditioning was first described some 15 years ago, interest in strategies aimed at reducing infarct size has increased. During the past 10 years, investigations into the mechanism of ischemic preconditioning have clearly demonstrated the cardioprotective effect of K+ATP channel opening. Thus, K+ATP channel activation has been shown to be involved in cardioprotection by a variety of stimuli, including a brief period of complete ischemia (classical ischemic preconditioning) or a partial coronary artery occlusion. In addition, ischemia in remote organs and nonischemic stimuli in the heart such as ventricular pacing, stretch, and heat stress also confer protection via K+ATP channel activation. Pharmacological agents that open K+ATP channels reduce infarct size, but K+ATP channel opening must occur prior to or early during the sustained infarct-producing coronary artery occlusion, while the degree and memory of cardioprotection are less than those produced by classical ischemic preconditioning. Although the exact mechanism by which K+ATP channel activation protects is still incompletely understood, recent studies indicate a role for the mitochondrial K+ATP channels. Before K+ATP channel opening can be employed in patients at increased risk of developing myocardial infarction (e.g., unstable angina), it is mandatory to determine whether tolerance (tachyphylaxia) occurs with repeated administration of K+ATP channel openers in a fashion similar to what occurs with ischemic preconditioning. PMID- 10755196 TI - KATP channel openers, myocardial ischemia, and arrhythmias--should the electrophysiologist worry? PMID- 10755197 TI - Vascular ATP-dependent potassium channels, nitric oxide, and human forearm reactive hyperemia. AB - Vascular ATP-dependent potassium (K+ATP) channels open and contribute to reactive hyperemia (RH) in animals. The contribution of K+ATP channels to ischemic vasolidation during RH and interactions with endothelium-derived nitric oxide have not been well characterized in human subjects. RH blood flow responses (mL/dL) following 5 minutes of cuff occlusion were measured using strain-gauge plethysmography in 22 normal human subjects age 42 +/- 2 years. Measurements were obtained at baseline and following intra-arterial administration of the K+ATP channel closer glibenclamide, the nitric oxide synthase inhibitor L-N-monomethyl arginine (L-NMMA), or both drugs simultaneously. Glibenclamide (100 micrograms/min) did not change basal flow (2.7 +/- 0.3 to 2.7 +/- 0.3 mL/min/dL), but L-NMMA (8 mumol/min) and combined glibenclamide and L-NMMA significantly (p < 0.05) decreased basal flow (3.0 +/- 0.5 to 2.0 +/- 0.2 and 3.3 +/- 0.5 to 2.5 +/- 0.3, respectively). Glibenclamide significantly (p < 0.01) decreased RH flow (18.2 +/- 1.3 to 14.8 +/- 1.3) and excess flow (5.3 +/- 1.2 to 1.3 +/- 1.3). L NMMA significantly (p < 0.05) decreased RH flow (21.2 +/- 1.8 to 18.9 +/- 1.9) and tended to decrease excess flow (6.1 +/- 2.2 to 3.9 +/- 2.5). Combined drug infusion significantly (p < 0.1) decreased RH flow (21.6 +/- 2.2 to 18.0 +/- 2.4) and excess flow (6.3 +/- 1.6 to 1.6 +/- 1.6), with reductions in RH and excess flow similar to those following glibenclamide infusion alone. We conclude that forearm vascular K+ATP channels are closed at baseline. They open and contribute to RH vasodilation. The addition of nitric oxide inhibition to K+ATP channel blockade does not result in additive or synergistic inhibition of RH. PMID- 10755200 TI - Benefit of antihypertensive treatment in the diabetic patients enrolled in the Systolic Hypertension in Europe (Syst-Eur) trial. AB - In this review we attempt to determine the role of calcium channel blockers in preventing cardiovascular sequelae in patients with both hypertension and diabetes mellitus. The data have been collected from three sources: post-hoc analyses of subgroups of diabetic patients in placebo-controlled hypertension trials (SHEP, Syst-Eur, Syst-China); stepped-care blood pressure-oriented trials (HOT); and comparative trials focusing primarily on metabolic aspects and intermediate endpoints (ABCD, FACET). On balance, the data seem to indicate that long-acting calcium channel blockers score remarkably well in preventing cardiovascular complications in diabetic hypertensive patients. PMID- 10755198 TI - Coordinate regulation of metabolic enzyme encoding genes during cardiac development and following carvedilol therapy in spontaneously hypertensive rats. AB - Fuel substrate utilization is highly regulated during cardiac development and with the onset of cardiac hypertrophy. Glucose and lactate are the predominant fuel substrates utilized during cardiac development. Postnatally, a switch occurs so that fatty acids become the chief energy substrate in the nonfed adult mammalian heart. A reversion back towards fetal energy metabolism occurs with the development of cardiac hypertrophy. To evaluate the role of this substrate preference switch in the development of cardiac hypertrophy, the molecular regulation directing these switches is being explored. Thus, we have begun by defining the temporal expression patterns of genes encoding key rate-controlling enzymes directing major fuel substrate metabolism during cardiac development, with pressure-overload-induced cardiac hypertrophy, and following antihypertensive therapy in spontaneously hypertensive rats. The genes encoding the fatty acid and adult enriched rate-controlling glycolytic enzymes are expressed at low levels in the fetal and neonatal rat heart. The genes encoding these enzymes are significantly and coordinately upregulated (> or = 70%) in adult rat hearts compared to the fetal expression patterns. A reciprocal and coordinate downregulation (> or = 40% reduction) of the fatty acid and adult enriched glycolytic enzyme encoding genes are observed with the induction of pressure-overload-induced hypertrophy in spontaneously hypertensive rats compared to Wistar-Furth normotensive control rats. Antihypertensive therapy with carvedilol, a vasodilating alpha- and beta-adrenoreceptor antagonist, attenuates this reversion of the metabolic gene expression pattern towards fetal levels compared to placebo-treated littermate controls. This coordinate developmental and hypertrophy-induced regulation of genes that encode enzymes controlling both fatty acid and glycolytic catabolic pathways in the heart implicates potential mutual/overlapping regulatory signaling proteins within their gene regulatory programs. These gene regulatory pathways need to be identified and modulated in order to characterize the functional role of fuel substrate metabolism in cardiac development and with the induction of cardiac hypertrophy. PMID- 10755199 TI - Mesenteric cyclooxygenase products after combined antihypertensive treatment in uninephrectomized SHRs. AB - Unilateral nephrectomy in the spontaneously hypertensive rat (SHR) does not produce any change in blood pressure but does induce humoral alterations that might influence the antihypertensive action of some drugs. In this study, the antihypertensive effect of treatment (5 weeks) with placebo (control), verapamil, trandolapril, or their combination (verapamil plus trandolapril) was investigated in SHRs with half renal mass ablation, regarding the structure and function of small mesenteric arteries. Arterial pressure was followed during the period of treatment. Trandolapril and veratran returned pressure to normal, while verapamil was ineffective. Statistically significant differences in the parameters of vessel structure were not observed among groups; thus, the alterations in functionality cannot be attributed to morphologic changes. The noradrenaline induced contraction was reduced similarly by the three treatments as compared to controls. This difference involved a higher participation of nitric oxide in the trandolapril group, while in the verapamil group the origin of the difference might be due to the abolishment of a cyclooxygenase product. Veratran retained both effects. Acetylcholine-evoked relaxation of vessels precontracted with noradrenaline was improved with treatments versus controls. The abolishment of a contracting prostanoid or an NO scavenger from the cyclooxygenase pathway, due to the treatments implemented, is probably the cause of this. PMID- 10755201 TI - Angiotensin II formation in human vasculature after chronic ACE inhibition: a prospective, randomized, placebo-controlled study. QUO VADIS Investigators. AB - The QUO VADIS (the effects of QUinapril On Vascular Ace and Determinants of ISchemia) study was a randomized, double-blind, placebo-controlled trial designed to evaluate the effects of long-term angiotensin-converting enzyme (ACE) inhibition on angiotensin II formation in human vasculature. Patients (n = 187) scheduled for coronary artery bypass surgery used study medication 27 +/- 1 days before surgery. Segments of internal mammary arteries were exposed to increasing doses (0.1 nM-1 microM) of angiotensin I and II in organ baths. The rate of local angiotensin II formation is a function of the reciprocal of the difference between the pEC50's of the dose response curves to angiotensin I and II ( log/mol) and of the area between the curves (units). Quinapril (40 mg) and captopril (3 x 50 mg) similarly and significantly reduced mean blood pressure compared with placebo (p = 0.04). Difference between pEC50's was 0.90 +/- 0.08 in quinapril patients compared with 0.60 +/- 0.08 for placebo (p = 0.01); the area between curves was 91 +/- 8 for quinapril patients compared with 67 +/- 8 for placebo (p = 0.03). Angiotensin II formation was decreased to a lesser extent with captopril and was not statistically different from placebo (p = 0.3); the difference between pEC50's was 0.83 +/- 0.15; the area between curves was 84 +/- 12. This is the first randomized study to demonstrate that long-term oral treatment with an ACE inhibitor reduces vascular angiotensin II formation in humans. PMID- 10755202 TI - Guanabenz combination therapy inhibits sympathetic nerve activity and regresses left ventricular hypertrophy. AB - The cardiovascular and sympatholytic effects of combination therapy with guanabenz were examined in 26 patients (48 +/- 13 years old [mean +/- SD]) with stage 2 and 3 hypertension. Included in the study were patients under treatment with conventional antihypertensive drugs whose systolic and diastolic blood pressure was above 140 and 90 mmHg, respectively. Blood pressure, heart rate, and sympathetic parameters such as plasma concentration of norepinephrine and muscle sympathetic nerve activity at rest as well as during ambulatory conditions, 24 hour urinary excretion of norepinephrine, and low frequency (LF: 0.04-0.15 Hz)/high frequency (HF: 0.15-0.4 Hz) power ratio as a marker of cardiac sympathetic activity during 24 hours were examined before and after guanabenz (4 8 mg/d) combination therapy with first-line antihypertensive drugs such as diuretics. Left ventricular mass index (LVMI) was also calculated by conventional echocardiography. After 32 weeks of guanabenz combination therapy, systolic and diastolic blood pressure, heart rate, plasma and urinary excretion of norepinephrine, muscle sympathetic nerve activity, and LF/HF power ratio were significantly decreased, while neither LF nor HF power was changed. LVMI was also significantly decreased (270 +/- 81 vs. 236 +/- 83 g/m2, p < 0.005). These results indicate that guanabenz combination therapy inhibits sympathetic nerve activity under resting conditions as well as during ambulatory conditions and may accelerate regression of left ventricular hypertrophy in patients with moderate to severe hypertension. PMID- 10755203 TI - Long-term celiprolol therapy lowers fasting plasma leptin levels. Celiprolol Multicenter Study Group. AB - The effects of celiprolol on fasting plasma leptin levels, glucose tolerance, and insulin sensitivity were studied in a randomized, investigator-masked, and parallel clinical trial. Modified oral glucose tolerance tests (OGTT) were performed during the previous antihypertensive monotherapy (beta- or Ca-blocker, or ACE inhibitor), and 6 and 12 months after randomization to celiprolol (200-400 mg daily) or to control group, where the therapy was kept unchanged. One hundred sixty-nine dyslipidemic and hypertensive nondiabetics with an age range of 42-65 years and an average body mass index of 28.4 kg/m2 completed the study according to the protocol. The mean circulating leptin level decreased from 7.5 to 6.6 ng/mL in men (p < 0.05) and from 23.0 to 19.7 ng/mL in women during the 12-month celiprolol treatment. The incremental glucose area under the curve (AUC) in the 2 hour OGTT decreased from 3.8 to 3.0 h* mmol/L (p < 0.01), and insulin AUC decreased from 134 to 99 h* mU/L (p < 0.01). The insulin sensitivity index increased by 22% (p < 0.01) and the serum triglyceride level decreased by 15% in the celiprolol group. Changes in serum cholesterol were clinically insignificant. In the control group, no significant change was seen in any measured variable. A decrease in leptin levels in the celiprolol group was associated with improved insulin sensitivity, while the weight of the moderately obese patients did not change. The clinical significance of a 14% decrease in fasting plasma leptin level remains to be elucidated. The results suggest amelioration of leptin resistance during long-term celiprolol therapy. PMID- 10755204 TI - Hemodynamic effects of a novel sodium channel activator in dogs with chronic heart failure. AB - The use of positive inotropic agents, such as sympathomimetics and phosphodiesterase inhibitors, in heart failure (HF) is limited by proarrhythmic and positive chronotropic effects. In the present study, we compared the hemodynamic effects of intravenous LY366634 (LY), a Na+ channel enhancer, with dobutamine (DOB), in eight dogs with HF produced by intracoronary microembolizations. We also determined whether intravenous LY has synergistic effects when combined with digoxin. After baseline measurements, infusion of DOB was initiated at a dose of 2 micrograms/kg/min and increased until an increase of heart rate (HR) 30% of baseline or ventricular arrhythmias developed. Once hemodynamics returned to baseline, LY was infused at a dose of 2 micrograms/kg/min and increased until the LV fractional area of shortening (FAS), determined echocardiographically, reached a similar level as with DOB. Both drugs increased FAS equivalently compared to baseline (DOB, 24 +/- 3 to 47 +/- 2; LY, 27 +/- 2 to 46 +/- 2%). DOB increased HR from 78 +/- 4 min-1 at baseline to 107 +/- 7 min-1 at maximal dose (p < 0.05) and provoked serious arrhythmias in one dog. In contrast, LY infusion did not increase HR (82 +/- 7 vs. 80 +/- 8 min-1) or elicit arrhythmias. After 1 week of oral digoxin, dogs were infused again with LY. A lower dose of LY was needed to achieve the same increase in FAS compared to LY alone, but this was not statistically significant. The combination of LY with digoxin did not increase HR or evoke arrhythmias. We conclude that in dogs with HF, intravenous LY improves LV function to the same extent as DOB without increasing HR or evoking ventricular arrhythmias. The combination of LY with digoxin elicits a safe positive inotropic response. PMID- 10755205 TI - Efficacy of rescue thrombolysis in patients with acute myocardial infarction: preliminary findings. AB - Thrombolysis reduces mortality in patients with acute myocardial infarction (AMI) who are hospitalized within 6 hours from the onset of symptoms. AMIs involving a small area of myocardium show a lower mortality in comparison with AMI involving a large area. The present study was aimed at evaluating the safety and efficacy of rescue thrombolysis in patients with large AMI who had failed thrombolysis. Ninety patients (69 Males and 21 Females), mean age 56.7 +/- 9 years, hospitalized for suspected AMI within 4 hours from the onset of symptoms, suitable for thrombolysis (First episode), and showing pain and persistent ST segment elevation 120 minutes after starting thrombolysis, were randomized (double-blind) into two groups. Group A (45 patients: 10 females and 35 males) received an additional thrombolytic treatment (rTPA 50 mg), 10 mg as bolus plus 40 mg in 60 minutes. Group B (45 patients: 11 females and 34 males) received placebo. Positive noninvasive markers were defined as follows: (1) resolution of chest pain, (2) > or = 50% reduction in ST segment elevation, (3) double marker of creatine kinase (CK) and CK-MB activity 2 hours after the start of thrombolysis, and (4) occurrence of reperfusion arrhythmias within the first 120 minutes of thrombolytic therapy. Blood pressure, heart rate, and ECG were continuously monitored. An echocardiogram was carried out at entry, and before discharge, to control ejection fraction and segmentary kinetics. Adverse events such as death, re-AMI, recurrent angina, incidence of major and minor bleeding, and emergency CABG/PTCA were checked. The groups were similar in terms of age, sex, diabetes, smoking habits, hypertension, and adjuvant therapy (beta blockers). No significant difference was observed between the two groups regarding the time elapsed from the onset of symptoms to thrombolysis and AMI localization. Thirty-five patients (77.7%) showed reperfusion (10-50 minutes) after commencement of additional rTPA. Of the patients receiving placebo, 12 (26.6%) showed reperfusion within 35-85 minutes. Group A showed an earlier and lower CK and CK-MB peak than the control group, (respectively, p = 0.0001-0.009 and 0.002). Mortality (17.7%, 16 patients) was higher in group B than in the additional rTPA group, i.e., 6.6% (3 patients) in group A versus 28.8% (13 patients) in Group B (p = 0.041). Seven patients from group A showed nonfatal re AMI. Angina was observed in 18 patients (40%) from group A and 3 (6.6%) from group B (p = 0.006). Ten of these patients underwent urgent PTCA (9 from group A and 1 from group B), and 3 from group A underwent urgent CABG. Minor bleeding was higher in group A than in group B (44.4% versus 15.5%, p = 0.047). Major bleeding was observed in group A (nonfatal stroke). At predischarge, the echocardiogram ejection fraction was higher in group A than in group B (46 +/- 8% versus 38 +/- 7%, p = 0.0001). Our data suggest that an additional dose of thrombolytic drug in patients with unsuccessful thrombolysis is feasible and also that the bleeding increase is an acceptable risk in comparison with the advantages obtained in reducing AMI extension. Rescue thrombolysis can allow a gain in time to perform mechanical revascularization in patients admitted to hospital without an interventionist cardiology laboratory or in those who have to be referred to another hospital for urgent CABG. PMID- 10755206 TI - Calcium channel antagonist controversy: symposium at the Congress of the International Society of Cardiovascular Pharmacotherapy on March 30, 1999, in Amsterdam. PMID- 10755207 TI - Low-dose glucose-insulin-potassium is ineffective in acute myocardial infarction: a negative study with a positive view. PMID- 10755208 TI - Effects of the Ayurvedic drug Cholesteronil on cardiovascular risk factors. PMID- 10755209 TI - Verapamil in acute myocardial infarction. The rationales of the VAMI and DAVIT III trials. AB - Verapamil is well tolerated in patients with stable and unstable angina pectoris, as well as in patients with threatened infarction. No data exist documenting verapamil to be inferior to beta-blockers in these stages of coronary heart diseases. In the pre-thrombolytic era i.v. intervention with verapamil did not add any benefit in the early phase of AMI. However, a retrospective analysis suggests the hypothesis that i.v. verapamil given in combination with thrombolysis may improve the prognosis, and an ongoing study (VAMI trial) examines this hypothesis. When given in the late in-hospital phase of AMI to patients without heart failure verapamil significantly reduces mortality and morbidity. When given in the late in-hospital phase to patients with heart failure verapamil did not cause the course or prognosis to deteriorate and might even improve it in patients with residual ischaemia, especially when given in combination with an ACE-inhibitors. A planned study (DAVIT III study) has to confirm these preliminary data. PMID- 10755210 TI - The use and development of retroviral vectors to deliver cytokine genes for cancer therapy. AB - In this review, we describe technical advancements of retroviral vectors to address issues of safety, titer, and clinical scale manufacturing to produce high quality retroviral vector preparations that have made direct intratumoral administration of cytokine encoding recombinant vectors a feasible cancer therapy in the clinic. We also review possible further advances in retroviral vector design, which may prove important in expanding these clinical applications. PMID- 10755211 TI - Fast-dissolve drug delivery systems. AB - Fast-dissolve drug delivery is a rapidly growing area in the pharmaceutical industry. This paper defines the technology, discusses its benefits, and reviews and compares various fast-dissolve technologies currently available on the market. PMID- 10755212 TI - Karyotypic characterization and constitutive heterochromatin in the grasshopper Stiphra robusta (Orthoptera proscopiidae). AB - Conventional analysis, C-banding, silver nitrate and base specific fluorochrome staining with chromomycin A3 (CMA3) were used to analyse the meiotic chromosomes of the grasshopper Stiphra robusta. Diploid numbers of 2n = 19 in the males and 2n = 20 in the females were observed. The chromosome complement comprised a graded series of uniarmed chromosomes, and the X chromosome was medium sized. The nucleolar organizer regions, restricted to the bivalent chromosomes 6, 7 and 8, were CMA3 positive. PMID- 10755213 TI - The prevalence of type II diabetes mellitus is haptoglobin phenotype-independent. AB - Haptoglobin (Hp) phenotype distribution and the association between Hp polymorphism and type II diabetes mellitus was investigated in a Jordanian sample population consisting of 618 nondiabetics and 265 diabetics. In nondiabetics, Hp 2-2 was the most predominant type occurring at a frequency of 0.529 followed by Hp 2-1 occurring at a frequency of 0.387. In diabetics, the Hp 2-2 frequency was 0.540 while that of Hp 2-1 was 0.381. No statistically significant variation was detected in Hp type distribution between the two groups. The Hp2 allele occurred at a frequency of 0.722 in nondiabetics and 0.730 in diabetics. In both groups, the Hp type distribution was in agreement with the Hardy-Weinberg equilibrium calculations. These results suggest that type II diabetes mellitus is Hp phenotype-independent. PMID- 10755214 TI - Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and soluble interleukin-2 receptor (sIL-2R) concentrations in peripheral blood as markers of pituitary tumours. AB - Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and soluble interleukin-2 receptor (sIL-2R) are important cytokines. They are secreted by normal pituitary glands and those with all types of adenomas and may be involved in pituitary tissue growth. The peripheral blood concentrations of VEGF, bFGF and sIL-2R in nineteen patients (17-70 years) with pituitary tumours and ten healthy subjects (23-34 years) were studied. Hypersecretion of prolactin (five cases), human growth hormone (four cases), and thyroid stimulating hormone (one case) was recorded in some patients, and the remaining subjects were diagnosed as having nonfunctional pituitary tumours. Increased peripheral blood plasma levels of VEGF (310.82 +/- 59.17 pg/ml) compared with controls (40.32 +/- 11.80 pg/ml; p < 0.01), as well as bFGF (87.27 +/- 7.58 pg/ml) versus controls (11.14 +/- 2.43 pg/ml; p < 0.001) were recorded. The levels of sIL-2R did not differ between the pituitary tumour patients (4,490.58 +/- 581.50 pg/ml) and control subjects (3,617.01 +/- 1,397.18 pg/ml; p > 0.05). The concentrations of VEGF and bFGF in the peripheral blood are useful additional markers of the presence of tumours. PMID- 10755215 TI - Characterization of constitutive heterochromatin of Callithrix geoffroyi (Callitrichidae, Primates) by restriction enzymes and fluorochrome bands. AB - The neotropical primate genus Callithrix comprises two groups of species, jacchus and argentata, which inhabit distinct geographical regions and manifest different fur coloration and constitutive heterochromatin (CH) markers in their karyotypes. In this investigation the CH of a representative of the jacchus group, Callithrix geoffroyi, was analysed using fluorochromes and restriction enzymes in situ. To clarify the source of the constitutive heterochromatin of both groups, the data obtained in the jacchus group were compared with those published in the argentata group obtained by the same techniques. The C-bands of C. geoffroyi (four specimens, 2n = 46) were centromeric in all chromosomes, and distally located in pairs 6 and 22. The Alu I, Hae III, Hin fI, Rsa I, Dde I, Mbo I, and Msp I restriction endonucleases and CMA3 and DAPI fluorochromes produced different bands, which allowed the characterization of four distinct types of constitutive heterochromatin in the C. geoffroyi genome. Several of these types of heterochromatin were present in the ancestor of the two groups of species, jacchus and argentata, while others originated after their cladogenesis. PMID- 10755216 TI - 'Sloughing-off' of heterochromatin in Werner's syndrome cells during high temperature phosphate incubation. AB - Previous investigations of cells undergoing rapid division revealed the presence of heterochromatic 'dots' in chromosomes as well as numerous chromocentres in interphase nuclei. Such structures were seen in human embryonic cells, as well as cells from organisms capable of regeneration, and cells from various malignancies. Cells with a reduced capacity for reproduction were found to be virtually devoid of nuclear chromocentres and chromosome dots after incubation in phosphate buffer at high temperature. The lack of heterochromatin in such cells (Werner's syndrome) thereby explained their reduced capacity for cell division and the resultant rapid rate of aging in individuals afflicted. Re-examination of such slides containing these cells revealed that chromocentres and chromosome dots were present initially, but the incubation process resulted in a 'sloughing off' of such structures. The incubation process left these heterochromatic structures intact in malignant and control cells, inferring a link between cell proliferation and stable intact heterochromatin. These findings implicate heterochromatin as the object of the purported chromosomal instability factor characteristic of Werner's syndrome. The loss of heterochromatin did not result in chromosome breakage, suggesting that heterochromatin may not be an integral part of chromosome structure, but rather a surface feature or covering. PMID- 10755217 TI - Impact of diazepam on pineal-adrenal axis in an avian model. AB - Diazepam, a benzodiazepine derivative, better known as a melatonin blocker in mammals, was injected into pigeons at a dose of 3 mg/kg body weight/day for 1 h, 1 day, 7 days and 15 days. This was done to investigate whether diazepam-induced changes in the pineal gland were reflected in the functioning of the adrenal gland. The results indicated that diazepam caused inhibition of pineal function and the degree of inhibition was very much time dependent. In addition, the pineal gland was unable to modulate the adrenomedullary hormonal titre yet it considerably influenced the physiology of the adrenal cortex. PMID- 10755218 TI - Effects of aging on the useful field of view. AB - Previous research has shown that the useful field of view (UFOV) is a useful tool in predicting driving ability, and the UFOV also seems to decline with age. The goals of the current study were first, to examine UFOV changes systematically as a function of age (15-84), and, second, to determine the effect of dividing attention on the UFOV. Our results show that the deterioration in the UFOV begins early in life (by 20 years, or younger). This deterioration is best conceptualized as a decrease in the efficiency with which observers can extract information from a cluttered scene, rather than by shrinking of the field of view per se. The diminished efficiency among elderly observers is exacerbated when conditions require the division of attention between central and peripheral tasks. PMID- 10755219 TI - Memorability functions in verbal memory: a longitudinal approach. AB - Middle-aged (N = 252, M = 39.91), young-old (N = 486, M = 60.77), and old-old (N = 137, M = 74.42) participants in the Seattle Longitudinal Study were tested on two occasions on their recall of a 20-item word list. Proportions of participants in each group correctly recalling each word-unit served as the dependent measure. Word-unit scores obtained in 1991 were regressed on those from 1984, yielding linear functions that varied by age group. Each set of word-unit scores (three groups on two occasions) was then regressed on word familiarity, imageability, primacy, and recency. The relative influence of each of these variables on memorability was then compared, both between and within cohorts. Primacy and familiarity were consistently strong predictors. Imageability and recency were predictive of memorability in the middle-aged group, but less so in the young old, and not at all in the old-old group. Results and possible implications are discussed. PMID- 10755220 TI - Estimation of short temporal intervals in Alzheimer's disease. AB - This study investigated the estimation of short temporal intervals in Alzheimer's disease (AD). Eight patients with dementia of the Alzheimer type, and eight age matched controls were evaluated in a time-estimation task. The task consisted in the production of three short empty intervals (5, 10, and 25 s). Results indicated that AD patients show deficits both in the accuracy and precision of time judgments: in the three intervals evaluated, the magnitude of absolute error and the variability in time judgments were significantly greater in AD patients than healthy respondents (p < .01). These findings are discussed taking into account the contribution of attentional processes during the performance of temporal tasks. It is concluded that the estimation of short temporal intervals could be useful as an objective indicator of cognitive decline in AD. PMID- 10755221 TI - Gains in fluid ability performance and their relationship to cortisol. AB - The present analyses examined the relationship between cortisol and performance gains in crystallized (Gc) and fluid (Gf) ability, as well as the relationship to measures of everyday cognitive functioning in a sample of 73 older adults. The analyses were part of a larger study examining the impact of anxiety-reduction training on everyday tasks. Cortisol was assessed via urine sampling. Results indicated that there was a significant negative correlation between cortisol and performance gains for Gf, replicating earlier findings reported by our laboratory. These findings also extend the relationship to cortisol measured in urine; previous analyses were conducted using serum-based cortisol. Cortisol was also significantly related to performance gains in some measures of everyday cognitive functioning. PMID- 10755222 TI - Stride parameters in healthy young and old women--measurement variability on a simple walkway. AB - Gait analysis in disabled and old probands may be considerably altered by applied devices and the artificial surrounding of a gait laboratory. To circumvent distractions from such factors, we evaluated the gait pattern of healthy women on a simple walkway and supplemented the analysis by reliability measurements. In order to consider age-related changes of gait parameters in health, healthy young (YHW) and old women (OHW) were investigated. Five gait parameters were assessed during 10 independent runs of each individual on the walkway. Multiple-regression analysis revealed a significant relationship between age and gait velocity, stride length, and the coefficient of variation (CV) of step width. Standard errors of mean were uniformly low across all parameters and groups, except for some increase in step width (OHW) and cadence (YHW). Across groups, CV was small for stride length, larger for foot angle, and largest for step width. An acceptably low within-session variability of the stride parameters was found. PMID- 10755223 TI - Age differences in the expression of manual asymmetry. AB - Manual dexterity declines in older adults; however, the degree to which the decline is symmetrical in the two hands is less clear. In this investigation the differences between young (aged 18 to 24) and old (aged 62 to 72) men and women in the expression of manual asymmetry across several motor tasks were examined. Five motor tasks provided a broad measurement of several motor characteristics necessary for instrumental activities of daily living. The main effect of sex was not significant; therefore data from men and women were pooled. Little support was found for generalized asymmetrical performance in old adults compared to young adults across motor tasks. An age-by-hand group analysis indicated that age was related to preferred hand performance on two tasks, but when a within-subject analysis of hand differences was performed, and the speed accuracy trade-off considered, differential asymmetry was seen only in the most complex, speeded task. PMID- 10755224 TI - New 1,4-anthracene-9,10-dione derivatives as potential anticancer agents. AB - The amino-substituted anthracene-9,10-dione (9,10-anthraquinone) derivatives represent one of the most important classes of potential anticancer agents. To better understand the basic rules governing DNA sequence specificity, we have recently synthesized a new class of D- and L-aminoacyl-anthraquinone derivatives. We have tested these new compounds as cytotoxic agents, and we have correlated their activity with the configuration of the chiral aminoacyl moiety. Molecular modeling studies have been performed to compare the test drugs in terms of steric overlapping. PMID- 10755225 TI - Synthesis and pharmacological properties of 1-(4-substituted)butyl derivatives of amides of 7-methyl-3-phenyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimi dine 5 -carboxylic acid. AB - The synthesis of 1-(4-substituted)butyl derivatives of amides of 7-methyl-3 phenyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimid ine-5- carboxylic acid and the results of the preliminary pharmacological screening are described in this paper. Some of them showed a weak analgesic action and caused suppression of the spontaneous locomotor activity of mice. PMID- 10755226 TI - Electrochemical study of cefetamet-Na and its polarographic determination. AB - Polarographic behavior of cephalosporin cefetamet-Na (Cef-Na) in aqueous solutions of pH ranging from 1.7 to 12.5 was investigated by applying direct current (dc) polarography, differential pulse polarography (dpp), alternating current (ac) polarography, cyclic voltammetry and electrolysis at constant potential. The characteristics of the corresponding electrode reaction are presented and discussed. The electrode reaction was found to be affected by strong adsorption, strongly and slightly pronounced in acidic and alkaline media, respectively. The methoxyimino group electroreduction was carried out and the mechanistic scheme was suggested. In addition, a sensitive dpp method was proposed for analytical determination of a very low concentrations of Cef-Na. PMID- 10755227 TI - Activity of some 3-formylchromone derivatives on the induction of chloroplast free mutants in Euglena gracilis. AB - The hereditary bleaching test on Euglena gracilis was used for detecting extranuclear mutations. The highest bleaching activity (induction of the chloroplast-free mutants) was shown by the 6-R-3-formylchromones. On the other hand, bleaching-inactive 6-R-3-formylchromone acylhydrazones (derived from gallic and salicylic acids), added at sufficient concentrations in the case of chloroplast mutagenesis in E. gracilis, act as a potent antimutagen. This effect appeared to be a unique feature of chromone derivatives, but was dependent on the type of mutagen. These substances were very effective against the bleaching activity of acridine orange, and were less effective against N-methyl-N'-nitro-N nitrosoguanidine. The genotoxic effects of these mutagens was reduced, especially during the first stages of induction of this specific cytoplasmic mutation. The experimental study of mutagenicity and antimutagenicty of 3-formylchromone hydrazones was reinforced by data obtained by the semi-empirical AM1 method and lipophilicity values. PMID- 10755228 TI - Synthesis and biological assays of new H3-antagonists with imidazole and imidazoline polar groups. AB - New histamine H3-receptor antagonists were synthesised and tested on rat brain membranes and on electrically stimulated guinea-pig ileum. The new compounds have a central polar group represented by a 2-alkylimidazole or a 2-thioimidazoline nucleus. The effect of the polar group basicity on the optimal length of the alkyl chain, connecting this group to a 4(5)-imidazolyl ring, was investigated. The best affinity values, obtained by displacement of [3H]-RAMHA from rat brain, were obtained for the 2-alkylimidazole derivatives (2a-f) with tetramethylene chain (pKi 8.03-8.97), having an intermediate basicity between that of the previously reported 2-thioimidazoles (1a-i) and that of 2-alkylthioimidazolines (3a-h). In contrast, a general lowering of affinity (pKi 5.90-7.63) was observed for compounds of the last series (3a-h), with a complex dependence on the terminal lipophilic group and chain length. PMID- 10755229 TI - Synthesis and antiviral assays of some 2-substituted benzimidazole-N-carbamates. AB - Some 2-substituted benzimidazole-N-carbamates were synthesized and tested in vitro for antiviral activity. Two derivatives were active at noncytotoxic concentrations. The results confirmed the importance of the substituents at the 2 position of benzimidazole; an isopropylcarboxamide group led to the best activity. PMID- 10755230 TI - Synthesis of (Z)-2-(2-formamido-4-thiazolyl)-2-(substituted alkoxyimino) acetic acids. AB - (Z)-2-(2-formamido-4-thiazolyl)-2-(substituted alkoxyimino) acetic acids were synthesized by a new method based on the following sequence of reactions: treatment of the tert-butyl acetoacetate with sodium nitrite, alkylation of the oxime formed with an appropriate alkyl halide, halogenation of methyl alpha-keto group and simultaneous cleavage of tert-butyl ester with sulfuryl chloride, protection of the obtained acid function with diphenyldiazomethane, formation of the 2-aminothiazole ring by the Hantzsch method with thiourea, formylation of the amino group and selective final cleavage of the diphenylmethyl ester by treatment with trifluoroacetic acid and anisol. The developed procedure allows the synthesis of (Z)-2-(2-formamido-4-thiazolyl)-2-(substituted alkoxyimino) acetic acids, with an ester function in the alkoxyimino group employing a simple method and obtaining higher yields in comparison with the habitually used classic method. PMID- 10755231 TI - Some new 1,2,3,4-tetrahydroquinoline derivatives. AB - Two 1,2,3,4-tetrahydoquinoline-based compounds were synthesized and evaluated for antinociceptive properties. Both compounds displayed no significant analgesic activity and at the higher dose showed no characterized CNS depressant activity. PMID- 10755232 TI - Mixed oxo-hydroxy bile acids as actual or potential impurities in ursodeoxycholic acid preparation: a 1H and 13C NMR study. AB - Some distinctive unprecedented 1H NMR signals and the complete 13C NMR resonances are assigned for the entire set of mixed oxo-hydroxy bile acid isomers, obtained by selective oxidation of the hydroxy groups at positions (3,7), (3,12) and (3,7,12) of chenodesoxycholic acid, desoxycholic acid and cholic acid, respectively. Partially or totally oxidized products are the major actual or potential impurities formed during the preparation of the pharmaceutically active ursodeoxycholic and chenodeoxycholic acids. PMID- 10755233 TI - Synthesis, structure and antiaggregatory effects of some N-(4,5-dihydro-1H imidazol-2-yl)indoles. AB - A series of 1-(4,5-dihydro-1H-imidazol-2-yl)indole derivatives was prepared in order to evaluate their antiaggregatory activity in human platelets. The compounds 4a-m were prepared by reacting N-aryl-N-(4,5-dihydro-1H-imidazol-2 yl)hydroxylamines (2a-d) with monosubstituted acetylene derivatives 3a-b. Imidazoline derivatives 4 were further acetylated or sulfonylated to give amides 5a-c and sulfonamides 6a-c and 7a-c, respectively. Eight compounds were taken as representative aryliminoimidazoline analogs. Among them only one, 4m, showed a good concentration-dependent action against the primary or alpha 2-adrenoreceptor mediated phase of noradrenaline-induced aggregation in platelets. PMID- 10755234 TI - Transformation of the sydnone ring into oxadiazolinones. A convenient one-pot synthesis of 3-aryl-5-methyl-1,3,4-oxadiazolin-2-ones from 3-arylsydnones and their antimicrobial activity. AB - 3-Arylsydnones (Ia-u) have been converted into the corresponding 3-aryl-5-methyl 1,3,4-oxadiazolin-2-ones (IIIa-u) by a single-step reaction with bromine in acetic anhydride. In the preliminary screening of all these compounds, the halogen-substituted derivatives have shown antimicrobial activities equal to those of the standard drugs used. PMID- 10755235 TI - Receptor chemistry towards the third millennium. PMID- 10755236 TI - New pharmacodynamic parameters for antimicrobial agents. AB - The application of pharmacodynamic theories to antimicrobial chemotherapy has greatly improved the prediction of the time course of activity expressed by antibiotics. Being a major component of the antibiotic-bacterium interaction system, pharmacodynamics, when properly integrated with the pharmacokinetics established for the antibiotic, allow better evaluation of the dosage regimen in conjunction with its clinical response. Before this approach becomes effective, detailed background information on the complex antibiotic-bacterium interactions have to be secured. To achieve this, proper characterization of a time-kill curve is a prerequisite. The use of susceptibility endpoints such as the MIC with respect to the antibiotic concentrations achievable in vivo represent the conventional approach to clinical dosing of antimicrobial agents, i.e. by maintaining concentrations above the MIC. Recently, a number of surrogate markers have been proposed by combining suitable pharmacokinetic parameters and susceptibility data, e.g. peak/MIC ratio, AUC>MIC, time above MIC, AUIC etc. to enhance the prediction of clinical outcomes. Attempts have been made to apply these pharmacokinetic/pharmacodynamic markers to antibiotics of the same class as well as to antibiotics from different classes. This review aims to discuss the various microbial dynamic responses in relation to antibiotic exposure and the development of different pharmacokinetic/pharmacodynamic markers for use in current antimicrobial chemotherapy. PMID- 10755237 TI - Helicobacter pylori: cultivability and antibiotic susceptibility of coccoid forms. AB - Helicobacter pylori is an actively dividing helical bacterium that changes to coccoid morphology as the culture ages. It has been suggested that the coccoid forms may be involved in transmission of infection and in relapses following antimicrobial therapy. The aim of this investigation was to determine the survival and susceptibility of the coccoid forms to amoxycillin, erythromycin, gentamicin and metronidazole. Colony counts and microscopic examination were performed after 1-4 weeks of culture. At 2 and 4 weeks, identical cultures were treated with the antibiotics for 24 h. Our results showed that 4-week cultures of coccoid forms were cultivable after antibiotic treatment. PMID- 10755238 TI - A comparison of the activities of three amphotericin B lipid formulations against experimental visceral and cutaneous leishmaniasis. AB - The polyene antibiotic, amphotericin B, the gold standard for systemic fungal infections is also a recommended second line treatment for visceral, cutaneous and mucocutaneous leishmaniasis. Acute toxicity has limited the use of amphotericin B but less toxic lipid formulations, AmBisome, Amphocil and Abelcet, have shown potential for the treatment of clinical visceral and mucocutaneous leishmaniasis. This study compares the in vitro and in vivo anti-leishmanial activity of Fungizone and the three lipid formulations. AmBisome and Amphocil were more active (ED50 values 0.3 and 0.7 mg/kg, respectively) than Abelcet (ED50 2.7 mg/kg) against L. donovani in a mouse model. Against L. major in vivo, AmBisome at a dose of 25 mg/kg was the most successful at reducing lesion size, with Amphocil also showing activity while Abelcet was inactive. In the L. donovani--peritoneal macrophage (PEM) model Fungizone and Amphocil were significantly more active (ED50 values 0.013 and 0.02 microg/ml, respectively) than AmBisome and Abelcet (ED50 values 1.5 and 2.6 microg/ml). This trend was similar in the L. major--PEM model (Fungizone > Amphocil > AmBisome > Abelcet). THP-1 macrophages infected with L. donovani amastigotes showed a different profile with Amphocil = Abelcet > AmBisome > Fungizone. Differences could be due to the interaction of the formulations with the biological milieu and uptake into different cell types. PMID- 10755239 TI - Randomized controlled trial of doxycycline prophylaxis against leptospirosis in an endemic area. AB - Leptospirosis occurs as seasonal outbreaks, lasting for about 3 weeks during October-November in North Andaman. A randomized controlled trial was undertaken to assess the efficacy of doxycycline prophylaxis in the prevention of infection and clinical disease due to leptospires during the outbreak period. A sample population of 782 persons, randomized into two groups was given doxycycline 200 mg/week and a placebo. The microscopic agglutination test was done on blood samples collected on day zero, after 6 weeks and after 12 weeks. Infection rates and attack rates of clinical illness were calculated in the two groups based on the serological results. Statistically there was no difference in the infection rates among the two groups. However, a statistically significant difference was observed in the clinical disease attack rates (3.11 vs. 6.82%) between study group and control group. The results of the study indicate that doxycycline prophylaxis does not prevent leptospiral infection in an endemic area, but has a significant protective effect in reducing the morbidity and mortality during outbreaks. PMID- 10755240 TI - Trends in antimicrobial susceptibility of bacterial pathogens isolated from patients with bloodstream infections in the USA, Canada and Latin America. SENTRY Participants Group. AB - From January through June of 1998, 4579 bloodstream infections (BSI) due to bacterial pathogens were reported from SENTRY hospitals in Canada, the USA and Latin America. Staphylococcus aureus, Escherichia coli, and coagulase-negative staphylococcus (CoNS) were the most common pathogens, together accounting for 55.2% of all BSI during this time period. Compared with the 5794 BSI reported from SENTRY from January through June of 1997, no major change was seen in the frequencies of occurrence of the most common bacterial causes of BSI. Between 1997 and 1998, the major change in antimicrobial resistance was an increase in oxacillin-resistance in both S. aureus and CoNS in all regions. These data demonstrate widespread antimicrobial resistance in Canada, Latin America and the USA, with a notable increase in oxacillin-resistance among staphylococci. Ongoing surveillance remains essential, and will enhance efforts to limit the scope of this worldwide problem. PMID- 10755242 TI - The effects of alternative treatments for HIV disease on recommended pharmacological regimens. AB - The use of alternative treatments for HIV disease was assessed before and after the introduction of highly active antiretroviral therapy (HAART) by the use of a standardised questionnaire. These data were related to epidemiological, clinical and laboratory parameters and compliance levels to recommended antiretroviral and anti-Pneumocystis carinii regimens. Compared with the 476 evaluable patients interviewed during the first 9 months of 1996, the 549 evaluable subjects screened in January-September 1998 showed less frequent recourse to alternative treatments (22.8 vs. 35.7% of patients; P < 0.001). A significant correlation between use of alternatives, poor compliance to antiretroviral drugs and anti-P. carinii chemoprophylaxis and clinical and immunological progression of HIV disease was shown in 1996, but was not maintained in 1998. No relevant differences were found in the selection of most non-conventional treatments and in the number of strategies followed and their duration of use. Unorthodox treatments were used by most patients concurrently rather than instead of official therapeutic regimens. No correlation was found between the use of alternative treatment and the patients' age, gender, type of risk for HIV disease and duration of HIV seropositivity. The correlations between alternative and official treatments for HIV disease before and during the HAART era shows that a considerable percentage of patients still resort to alternatives in 1998 compared with 1996 but that this does not interfere with compliance with recommended pharmacological regimens or the progression of the disease. PMID- 10755241 TI - Detection of genes encoding aminoglycoside-modifying enzymes in staphylococci by polymerase chain reaction and dot blot hybridization. AB - Dot blot hybridization and the polymerase chain reaction (PCR) were used to study aminoglycoside-modifying enzymes in aminoglycoside-resistant staphylococci isolated in hospitals in Kuwait. DNA encoding the acetyltransferase (AAC) (6') phosphotransferase (APH) (2"), nucleotidyltransferase (ANT) (4') and APH (3') enzymes were detected in Staphylococcus aureus and coagulase negative staphylococci. ANT (4') was the most common enzyme detected. The majority of isolates contained genes for all three modifying enzymes, AAC (6')-APH (2"), ANT (4') and APH (3'); only few isolates carried genes for a single modifying enzyme. Genes encoding the AAC (6')-APH (2") were detected in all except two gentamicin resistant isolates. In these isolates the genes for the AAC (6')-APH (2") enzyme could not be detected by PCR and dot blot hybridization. Whereas antibiotic resistance testing could be used to predict the presence of the AAC (6')-APH (2") enzyme it was not useful in predicting the presence of the ANT (4') or APH (3') enzymes in gentamicin-resistant isolates. Results obtained with dot blot hybridization were comparable to those obtained with PCR. However, PCR was fast and results were obtained within the same day. Therefore PCR would be preferred for the detection and confirmation of the presence of aminoglycoside-modifying enzymes in clinical microbiology laboratories. PMID- 10755243 TI - Proton translocating ATPase mediated fungicidal activity of a novel complex carbohydrate: CAN-296. AB - CAN-296 is a complex carbohydrate (approximately 4300 Da) isolated from the cell wall of Mucor rouxii. It exhibits excellent in vitro fungicidal activity against a wide spectrum of pathogenic yeasts, including isolates resistant to azoles and polyenes. The rapid irreversible action of CAN-296 on intact fungal cells and protoplasts suggested a membrane-located target for its action. The proton translocating ATPase (H+-ATPase) of fungi is an essential enzyme required for the regulation of intracellular pH and nutrient transport. Inhibition of H+-ATPase leads to intracellular acidification and cell death. We therefore investigated the effect of CAN-296 on H+-ATPase-mediated proton pumping by intact cells of Candida and Saccharomyces species by measuring the glucose-induced acidification of external medium. CAN-296 inhibited proton pumping of Candida albicans, Candida glabrata, Candida krusei, Candida guilliermondii and Saccharomyces cerevisiae at low concentrations (0.078-1.25 mg/l). Other commonly used antifungal agents such as amphotericin B, itraconazole and fluconazole had no effect on H+-ATPase mediated proton pumping. A clinical isolate of C. glabrata with reduced in vitro susceptibility (MIC = 10 mg/l) to CAN-296 also showed resistance to CAN-296 inhibition of proton pumping. Purified membrane fractions rich in H+-ATPase activity were not inhibited by CAN-296 suggesting that the effect on the H+ ATPase-mediated proton pumping in intact yeast cells is an indirect effect, perhaps mediated by local or global disruption of the plasma membrane. These results suggest that the inhibition of fungal H+-ATPase is at least partly responsible for the antifungal activity of CAN-296. PMID- 10755244 TI - Susceptibility of Polish clinical strains of Yersinia enterocolitica serotype O3 to antibiotics. AB - A total of 199 clinical strains of Yersinia enterocolitica serotype O3, biotype 4 were tested for their susceptibility to antibiotics (158 strains carried the virulence plasmid pYV and 41 strains did not). A total of 114 isolates were tested by a standard disk diffusion method for 21 antibiotics. Almost all strains tested were resistant to ampicillin and cefazolin and susceptible to amoxycillin/clavulanate, cefaclor, cefamandole, cefuroxime, cefotaxime, ceftriaxone, aztreonam, imipenem, gentamicin, amikacin, netilmicin, tetracycline, doxycycline, chloramphenicol, ciprofloxacin, sulphamethoxazole, trimethoprim, co trimoxazole and furazolidone. In addition, minimal inhibitory concentrations of 15 antibiotics were determined by the agar dilution method for all 199 strains (158 carrying plasmid pYV and 41 strains that did not). Third-generation cephalosporins such as cefotaxime and ceftriaxone and a fluoroquinolone (ciprofloxacin) were the most active antimicrobial agents tested followed by aztreonam, imipenem, trimethoprim, tetracycline, gentamicin, chloramphenicol, amoxycillin/clavulanate, cefaclor, cefuroxime, amikacin, furazolidone and sulphamethoxazole. The present study demonstrated a high susceptibility of clinical strains of Y. enterocolitica to most of the tested antibiotics. In general there was no significant difference between susceptibility to antibacterial agents of strains with or without plasmid pYV. PMID- 10755245 TI - Serum and lung levels of thiamphenicol after administration of its glycinate N acetylcysteinate ester in experimentally infected guinea pigs. AB - Thiamphenicol is an analogue of chloramphenicol and is characterised by a broad spectrum of action. In this study, serum and lung levels of thiamphenicol (TAP) were studied in infected guinea pigs after the administration of thiamphenicol glycinate N-acetylcysteinate (TGA). Animals received a single dose of TGA (15 mg/kg, subcutaneously) immediately after intra-tracheal infection with Haemophilus influenzae (about 10(7) CFU/animal). Serum and lung concentrations of TAP were determined at 0, 1, 3, 6, 12 and 24 h after drug administration by means of HPLC. TAP serum levels were elevated at 1 h and remained detectable for 24 h after drug administration. Tissue lung levels were comparable to peak serum concentrations but remained higher and decreased more slowly than serum concentrations. PMID- 10755246 TI - Ofloxacin for the treatment of urinary tract infections and biofilms in spinal cord injury. AB - Forty two paraplegic and quadriplegic hospitalized spinal cord injured patients with urinary tract infections (UTI) were included in a double blind, randomized treatment study comparing 7 days ofloxacin (300 mg bd) with trimethoprim sulphamethoxazole (TMPSMX; 160-800 mg bd) or an alternative, chosen because of resistance to TMPSMX. The 4-day clinical cure rate, defined as an asymptomatic patient with sterile urine, was 90% (19/21) with ofloxacin, significantly greater than 48% (10/21) for the comparison group (P=0.003) and the rate at end of therapy was 90% (19/21) with ofloxacin, against 57% (12/21) (P=0.015). Bacterial biofilms were detected on bladder epithelial cells in 39/41 (95%) patients. The biofilm score fell significantly following ofloxacin therapy (P < 0.001) or alternative therapy (P < 0.001). Ofloxacin treatment led to significantly more biofilm eradication than the other antibiotic group on day 4 (62 vs. 24%) (P=0.005) and day 7 (67 vs. 35%) (P=0.014). The study showed that ofloxacin was better than TMPSMX and alternatives at relieving clinical infection and eradicating bladder cell biofilms. PMID- 10755247 TI - Gallstones in the elderly. PMID- 10755248 TI - Diagnosing collagenous colitis: does it make a difference? AB - Collagenous colitis is part of the spectrum of disorders known as microscopic colitis, which also include lymphocytic colitis and a mixed disorder with both a collagen band present and also intra-epithelial lymphocytes. Collagenous colitis appears to be caused by mucosal injury from toxins in the faecal stream. The collagen band is a result of the underlying inflammatory process and not the cause of symptoms. The diagnosis is based on colonic mucosal pathology and a compatible clinical presentation. Treatment is empiric and ranges from no therapy, to anti-inflammatory or anti-secretory agents, to diverting ileostomy. PMID- 10755249 TI - HTLV-I infection and the low prevalence of Helicobacter pylori infection in Japan. AB - HTLV-I retrovirus infection and Helicobacter pylori infection are common in Japan; however a report in this issue shows a significantly low prevalence of H. pylori in patients infected with HTLV-I. The reasons for this may be due to differences in genetic susceptibility of the infections, the gastric milieu in HTLV-I being unable to support H. pylori, or because antimicrobial therapy in HTLV-I infection has eradicated H. pylori in this susceptible population. Similarities between HIV-1, another retroviral infection, and the prevalence of H. pylori are noted. PMID- 10755250 TI - Gallstone disease in an elderly population: the Silea study. AB - BACKGROUND: Little is known on gallbladder emptying and gallstone composition in the elderly. AIMS AND SUBJECTS: Cross-sectional survey on the prevalence of gallstone disease and associated factors, gallstone characteristics and gallbladder emptying in a population aged > or = 60 years. METHODS: Gallstone number and size as well as gallbladder motor function were assessed by ultrasound. Gallstone composition and pattern were evaluated by conventional radiology and computed tomography (CT) based on Hounsfield units (HU). RESULTS: Gallstones were found in 148/1,065 subjects (13.9%), while 136 subjects (12.8%) were cholecystectomized with an overall prevalence of gallstone disease of 26.7% (sex ratio: F > M). Multiple gallstones (62.7%) and small gallstones (52%, diameter < or = 15 mm) were seen; silent gallstones accounted for 93.9% of the total. Only diabetes mellitus in women was significantly associated with cholelithiasis. Gallbladder fasting volumes were larger in gallstone carriers than in controls (P < 0.01); residual and ejection volumes were also significantly greater in gallstone carriers, whereas ejection fractions were similar in the two groups (50.3% +/- 2.4 versus 54.9% +/- 3.0; not significant). Gallstone calcifications were detected in 29/91 gallstone carriers by X-ray and in another 20 by CT (HU > 90). Moreover, 35 gallstone carriers had a score < or = 50 HU and six had attenuation values between 50 and 90 HU. Six gallstone patterns were identified: hypo-isodense, homogeneously dense, rimmed, laminated, core hyperdense, gas-containing. CONCLUSIONS: In the elderly, the prevalence of gallstone disease is very high, especially in women, but gallstone size, number and pattern and gallbladder emptying do not differ from those reported in the middle-aged gallstone population. Advanced age is associated with a high rate of calcified, probably pigment stones. PMID- 10755251 TI - Collagenous colitis: a long-term follow-up study. AB - OBJECTIVE: Collagenous colitis was first described in 1976. It is a rare disorder and the long-term course and prognosis of the disease are unknown. The aim of this study was to evaluate the course of the disease more than 5 years after the diagnosis. DESIGN: A retrospective follow-up of patients with collagenous colitis diagnosed during the period 1979-1990. METHODS: All examinations performed at the time of diagnosis were reviewed and the clinical courses of the patients were determined by evaluation of the medical records. At a follow-up visit in 1996 complete histories were obtained and conventional laboratory tests were performed. RESULTS: Two men and 22 women aged 20-82 years with collagenous colitis were identified. At the time of diagnosis, extensive investigation did not reveal other gastrointestinal diseases. At the time of follow-up, six patients had died from diseases unrelated to collagenous colitis, 10 patients suffered from chronic or intermittent diarrhoea, and four patients had been without gastrointestinal symptoms for the last 2-10 years. None of the patients developed colorectal cancer or chronic inflammatory bowel disease. Four patients were lost to follow-up. CONCLUSION: Collagenous colitis is a benign disease, most often with a chronic course. No association with other gastrointestinal diseases was found. PMID- 10755252 TI - Low prevalence of Helicobacter pylori in individuals with HTLV-I infection. AB - BACKGROUND: The prevalence of Helicobacter pylori in HIV-positive individuals is significantly lower than in HIV-negative controls. However, its prevalence in individuals infected with human T-cell leukaemia virus type I (HTLV-I), another important member of the human retrovirus family, has not been previously investigated. OBJECTIVE: To establish the prevalence of H. pylori in HTLV-I positive individuals in the Nagasaki Prefecture, which is an area endemic for HTLV-I. METHODS: We examined sera from 146 HTLV-I-positive individuals with a mean age of 56.7 years, consisting of 45 adult T-cell leukaemia (ATL) patients, 13 HTLV-I-associated myelopathy (HAM) patients and 88 healthy carriers. Serum samples of 292 age- and sex-matched HTLV-I-negative controls were also examined. Serum anti-H. pylori immunoglobulin (Ig) G antibody was examined using an enzyme linked immunosorbent assay kit. Twenty-eight HTLV-I-positive patients were examined endoscopically, assessed for H. pylori by culture, histology and CLO test using gastric biopsy specimens, and gastritis in these patients was also graded histologically. RESULTS: The seroprevalence of H. pylori was 48% in HTLV-I positive individuals versus 64% in HTLV-I-negative controls (P < 0.01). In the three HTLV-I-positive groups, ATL patients and carriers had significantly lower seroprevalence of H. pylori than the HTLV-I-negative controls (P < 0.05). Assessment of H. pylori using gastric biopsy specimens also showed a significantly lower prevalence of H. pylori infection in HTLV-I-positive patients than controls (46% versus 70%, P < 0.05). Histological examination showed a significantly higher degree of activity, inflammation and glandular atrophy in the antrum and corpus in H. pylori-positive patients compared to H. pylori negative patients. H. pylori-positive patients with HTLV-I infection had a more severe degree of glandular atrophy in the corpus than H. pylori-positive controls without HTLV-I infection. CONCLUSION: We have found a reduced prevalence of H. pylori in HTLV-I-positive individuals. Whatever the explanation, infection with HTLV-I does not predispose to the risk of H. pylori infection. PMID- 10755253 TI - Helicobacter pylori-associated antibodies in patients with duodenal ulcer, gastric and oesophageal adenocarcinoma. AB - OBJECTIVE: To associate Helicobacter pylori-associated antibodies with clinical disease in groups of patients with duodenal ulcer, gastric adenocarcinoma, oesophageal adenocarcinoma and normal mucosa. DESIGN: Prospective observational sero-epidemiology study. Identification of consecutive in-patients with duodenal ulcer, gastric adenocarcinoma, oesophageal adenocarcinoma and normal mucosa. Analyses of sera for antibodies to whole H. pylori, Cag A and Vac A antigens using ELISA and Western blot. Statistical analyses. SETTING: Walsgrave Hospital, Coventry, a district general hospital that serves a population of 350,000. PARTICIPANTS: Consecutive in-patients with an endoscopic diagnosis of duodenal ulcer (n = 31), gastric adenocarcinoma (n = 31), oesophageal adenocarcinoma (n = 40) and normal mucosa (n = 46). MAIN OUTCOME MEASURES: A profile of antibodies was constructed for each patient group and between-group comparisons were made. A logistic regression model determined the H. pylori-associated antibody that could best predict a patient's diagnosis. A discriminatory power for each antibody was calculated. RESULTS: Whole H. pylori, Cag A and Vac A antibodies are found more commonly in duodenal ulcer patients when compared to oesophageal adenocarcinoma (P < 0.003) and normal mucosa patients (P < 0.015). Similarly, gastric adenocarcinoma patients have antibodies to whole H. pylori, Cag A and Vac A more frequently than oesophageal adenocarcinoma (P< 0.002) and normal mucosa patients (P < 0.006). Vac A antibodies discriminate between duodenal ulcer/gastric adenocarcinoma and oesophageal adenocarcinoma/normal mucosa patients (odds ratio 5.56, log likelihood -90.06, P < 0.001) more effectively than Cag A antibodies (odds ratio 4.17, log likelihood -91.88, P < 0.001). CONCLUSIONS: Similar profiles of H. pylori-associated antibodies are seen in patients with duodenal ulcer and gastric adenocarcinoma, confirming that virulent H. pylori are involved in the pathogenesis of both diseases. Antibodies to Vac A could be used to identify patients at increased risk of developing H. pylori-associated disease. PMID- 10755254 TI - Proximal gastric tone in gastro-oesophageal reflux disease. AB - BACKGROUND/OBJECTIVE: Abnormal gastric function may be involved in the pathogenesis of several gastrointestinal functional disorders. This study evaluated gastric tone in gastro-oesophageal reflux disease (GORD). METHODS: Proximal gastric tone was measured with an electronic barostat in fasting conditions and after oral ingestion of a 200 ml/200 kcal liquid meal in 10 patients with GORD, with control groups consisting of 10 patients with dysmotility-like dyspepsia and 16 healthy subjects. RESULTS: Minimal distending pressure was increased in GORD patients compared to dyspeptic patients (P < 0.04) and controls (P< 0.001). Maximal postprandial gastric relaxation was significantly increased in GORD patients (430 +/- 95 ml) compared to dyspeptic patients (200 +/- 152 ml, P < 0.0001) and controls (342 +/- 88 ml, P= 0.05). Endoscopy-negative and mild oesophagitis patients had more profound maximal relaxation than patients with moderate or severe oesophagitis, whereas those with dyspepsia had significantly reduced gastric relaxation compared to GORD patients and controls (P < 0.002). CONCLUSIONS: In GORD, the postprandial gastric relaxation is more pronounced than in normal and dyspeptic patients. The pathophysiological relevance of this abnormal motility pattern remains to be determined. PMID- 10755255 TI - Irritable bowel syndrome in patients with dyspepsia: a community-based study in southern Europe. AB - OBJECTIVES: Dyspepsia and irritable bowel syndrome (IBS) share aetiopathogenic factors, and may therefore be part of a single disorder. This study was intended to determine their prevalence in the general population, and the degree of overlap between these two digestive disorders. DESIGN: Descriptive study. METHODS: A sample of 264 subjects chosen randomly from the population census of a city in Spain, and considered representative of the general population in this city, was surveyed by questionnaire. RESULTS: The prevalence of dyspepsia was 23.9%, and that of IBS was 13.6%. Of the subjects with dyspepsia, 31.6% had IBS, and of the subjects with IBS, 55.6% reported symptoms of dyspepsia. The prevalence of IBS was higher among subjects with dyspepsia (31.7%) than among those who reported no symptoms of dyspepsia (7.9%; P < 0.05). Moreover, the prevalence of IBS was similar in three subgroups identified according to the type of dyspepsia described (ulcer-like, reflux-like or dysmotility-like). When we compared subjects with both dyspepsia and IBS and those with dyspepsia alone, we found no significant differences in clinical characteristics except for abdominal pain and fear of cancer, which were more frequent in the former. Of the entire sample, 27.7% of the subjects sought medical attention for IBS and 17% missed work because of IBS. CONCLUSION: Our findings suggest that functional dyspepsia and IBS are two manifestations of a single, more extensive digestive system disorder. PMID- 10755256 TI - Clinical relevance of antroduodenal manometry. AB - OBJECTIVE: To evaluate the outcome of antroduodenal manometry studies and their effect on the clinical treatment of patients. DESIGN: A retrospective review of clinical antroduodenal manometric studies performed between September 1990 and March 1997 (n = 109). SETTING: Tertiary referral centre. MAIN OUTCOME MEASURES: The predominant symptom, the indication for the study, the outcome and the clinical impact were scored. A positive impact was defined as an outcome that resulted in an alteration of the management of the patient (medication, surgery, feeding), established a new diagnosis, or resulted in new investigations or in referral to another specialist. RESULTS: Full records were obtained from 91 studies in 85 patients (mean age 43 years). Nausea and vomiting were the most predominant symptoms (37.4%). In 49.5% of the cases, the test was performed due to suspicion of a generalized motor disorder. A normal outcome was found in 37 studies. Non-specific motor abnormalities were reported in 72% of the studies with an abnormal outcome. Pseudo-obstruction was diagnosed in 20%. The manometric studies resulted in a new therapy in 12.6%, a new diagnosis in 14.9%, and referral to another specialist in 8%. A positive clinical impact was found in 28.7% of the patients. CONCLUSION: Antroduodenal manometry can be a helpful diagnostic technique in a specialized centre. More research is needed to gain insight into the significance of the large number of non-specific abnormalities that are often found. PMID- 10755257 TI - Gluten-induced nitric oxide and pro-inflammatory cytokine release by cultured coeliac small intestinal biopsies. AB - OBJECTIVES: To determine whether there was increased nitric oxide (NO) production from coeliac small intestinal biopsies cultured in vitro with gluten and whether the inhibition of NO production could prevent gluten-induced enterotoxicity. The relationship between NO production with the pro-inflammatory cytokines interferon gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) was evaluated. DESIGN: Small intestinal biopsies from ten patients with treated coeliac disease and six controls were studied. METHODS: Small intestinal biopsies were taken from each patient and set up in culture with Frazer's fraction III (FFIII), a peptic/tryptic digest of gluten, FFIII plus L NMMA and L-NMMA alone, culture medium alone and ovalbumin which served as a control protein. The biopsies were cultured for 20 h at 37 degrees C. The supernatants were then collected and analysed for nitrite using the Greiss reaction; cytokine levels were determined using ELISA kits. Enterocyte height was determined by microscopy using a calibrated eyepiece graticule and cryostat sections of the cultured biopsies. RESULTS: Coeliac biopsies cultured with FFIII produced significantly greater nitrite compared to culture medium alone (P< 0.05) and this could be blocked with L-NMMA (P< 0.01). A reduction in enterocyte height was seen in coeliac biopsies cultured with FFIII compared to culture medium alone (P < 0.01) and this was ameliorated but not completely blocked when FFIII was cultured with L-NMMA. These changes were not seen in the controls. There was a significant reduction in IL-1beta levels in the supernatant of coeliac biopsies cultured with FFIII compared to culture medium alone (P< 0.05), but when cultured with FFIII and L-NMMA there was a significant increase in IL-1beta levels (P< 0.05). An increase in IFN-gamma levels was also seen when coeliac biopsies were cultured with FFIII and L-NMMA (P< 0.05). This pattern was not seen with TNF alpha. CONCLUSIONS: Increased levels of NO can be found when coeliac biopsies are cultured with gluten in an in vitro small intestinal culture system, and NO may play a role in the observed enterotoxicity as the inhibition of NO production ameliorates the enterocyte damage. The results suggest that NO is involved in the regulation of pro-inflammatory cytokines, particularly IL-1beta. This is likely to be one of many pathways leading to the observed mucosal pathology in coeliac disease and demonstrates the close interactions between them. PMID- 10755258 TI - Bone mineral density in patients with inflammatory bowel disease. AB - BACKGROUND: Studies have shown an association between inflammatory bowel disease (IBD) and low bone density. Previous publications, however, measured only a single parameter, either T or Z score, making comparison of data difficult. OBJECTIVE: To assess the effect of disease factors on both T and Z scores in a population of patients with IBD. METHODS: Risk factors for development of low bone density were recorded in IBD patients with confirmed diagnosis and disease extent. Bone density was then measured at the spine and neck of femur using dual energy X-ray absorptiometry. RESULTS: Ninety-one patients (49 male, 42 female) with a mean age of 46.6 years (range 22-84) were studied. Forty-eight patients had ulcerative colitis and 43 had Crohn's disease. Mean Z scores were -0.60 at the hip and -0.61 at the spine, whilst mean T scores were - 1.61 at the hip and 1.15 at the spine. Univariate analysis of Z scores identified Crohn's disease, high steroid use and low BMI as significantly associated with low bone density. An identical analysis using T scores failed to show any significant relationships. On multivariate analysis of Z scores, only disease type and BMI remained significant. CONCLUSIONS: Low bone density is associated with IBD particularly in patients with Crohn's disease and low BMI. This large UK study is the first to report both T and Z scores in patients with IBD and shows that Z scores are the most reliable guide to the effect of IBD on bone density. PMID- 10755259 TI - Asian ethnic origin and the risk of inflammatory bowel disease. AB - OBJECTIVE: To assess whether inflammatory bowel disease (IBD) is more prevalent in young Asians than Europeans living in Great Britain. DESIGN: Longitudinal birth cohort study of all those born 5-11 April 1970 in Great Britain--the 1970 British Cohort Study (BCS70). METHODS: The relationship of a diagnosis of ulcerative colitis or Crohn's disease by age 26 years with ethnic origin was investigated among 8,432 cohort members with complete data using multiple logistic regression. We adjusted for potential confounding factors, household crowding and sex, as well as for a family history of IBD. RESULTS: Young Asians born in Britain were significantly more likely than indigenous Europeans to have a diagnosis of IBD by age 26 years, with relative odds of 6.10 (95% CI 2.14 17.33). This group of cohort members had ethnic origins in India, Pakistan or Bangladesh (although none of those from Bangladesh had IBD). This relationship remained statistically significant after adjustment for the potential confounding factors and family history of IBD. CONCLUSION: Young Asians who were born in Britain are at a significantly higher risk of developing IBD than the indigenous European population. This may reflect a greater genetic predisposition to IBD that is uncovered by exposure to environmental factors. PMID- 10755260 TI - p53 protein accumulation and colonic adenoma recurrence. AB - OBJECTIVES: The prognostic value of p53 protein accumulation in colonic adenomas is still controversial. The aim of the present study was to determine whether the evaluation of p53 protein accumulation in newly diagnosed colonic adenomas could predict the development of metachronous adenomas. DESIGN/METHODS: Fifty-five patients who underwent prior endoscopic polypectomy for colonic adenomas were colonoscopically re-evaluated at 24-38 months after index colonoscopy. In cases with more than one adenoma, the one with the greatest diameter and the most serious histology was taken into account. p53 protein expression was immunohistochemically examined using specific monoclonal antibody. RESULTS: p53 protein was detected in 41.8% of the 55 index adenomas. Recurrent adenomas were present in 21 patients (38.2%). Metachronous adenomas were present in 56.5% of patients with p53-positive index adenomas and in 25% of those with p53-negative index adenomas (odds ratio 3.90, P = 0.018). Among patients with 1 or 2 index adenomas, metachronous adenomas were found in 50% of those with p53-positive index adenomas and in 22.6% of those with p53-negative index adenomas (odds ratio 3.43, P= 0.042). Multivariate stepwise logistic regression analysis revealed that number of index adenomas per patient (1 or 2 versus > 2) and p53 expression (positive versus negative) in index adenomas contain independent prognostic information for adenoma recurrence (chi2 = 8.2, P= 0.004 and chi2 = 4.08, P = 0.04 respectively). Patients aged < 60 years developed recurrent adenomas relatively more frequently if they had a p53-positive index adenoma (P= 0.068). In the subgroup of patients aged < 60 years with 1 or 2 index adenomas, the recurrence of adenomas was more frequent in those with a p53-positive index adenoma but the difference did not reach statistical significance (P= 0.13). CONCLUSIONS: Our data suggest that p53 expression in index adenomas is associated with recurrent colonic adenomas. PMID- 10755261 TI - Chronic liver disease related to hepatitis C virus: age of patients seems to be a determinant of severity independently of viral genotype. AB - BACKGROUND: Hepatitis C virus infection accounts for varying severity of chronic liver disease. Clinical manifestations of infection have been related to different virus genotypes, with conflicting results. DESIGN: We performed a cross sectional study on a Northern-Italian group of patients with chronic hepatitis, cirrhosis and hepatocellular carcinoma related to hepatitis C virus infection in order to verify the association of different viral strains and the outcomes of viral disease. METHODS: Two hundred and seventy-one patients referred to our unit for liver disease were studied and clinical, biochemical, histological, and functional parameters were investigated. RESULTS: Different viral genotypes were not associated with peculiar findings in any of the degrees of liver disease. However, a progressive age increase was associated with disease severity, although clinical and functional staging of cirrhotic patients with hepatocellular carcinoma was better compared to tumour-free cirrhotic patients. There was an increased prevalence of genotype 1b related to the age of the patients. In multivariate regression analysis the patients' age and apparent duration of infection were independently associated with the presence of cirrhosis and only the age of patients was associated to hepatocellular carcinoma. CONCLUSIONS: In the population we studied age of the patients seemed to be a determinant conditioning disease severity, likely reflecting older infections and long-standing liver disease. The prevalence of certain genotypes in varying degrees of liver disease could be an epiphenomenon which might also be explained by the changing prevalence of infecting strains over the past decades. PMID- 10755262 TI - Can the MEGX test and serum bile acids improve the prognostic ability of Child Pugh's score in liver cirrhosis? AB - BACKGROUND: Liver transplantation is nowadays the therapeutic option for end stage liver disease. Correct disease staging is the main step towards improving the timing of listing for liver transplantation so as to avoid premature or late entry. The need for correct prognostic evaluation is due to the limited number of donors and to the increasing number of patients awaiting transplantation. Our aim was to verify whether Child-Pugh's score might be improved by adding the monoethylglycinexylidide (MEGX) formation test and/or serum bile acid determination. METHODS: We evaluated 182 cirrhotic patients (44 Child-Pugh class A, 97 class B, and 41 class C) of mixed aetiology referring to a tertiary care centre for functional staging of liver disease. These patients were prospectively followed-up for 12-72 months. During this period, 45 patients died, 46 received a transplant, and 91 survived without transplantation. The end-point of analysis was either survival or liver disease-related death at the 6th, 12th, 18th and 24th months of follow-up. The 46 transplanted patients were excluded from the study upon transplantation. RESULTS: In our study, a cut-off for Child-Pugh's score < 8 confirmed its usefulness, especially in short-term prognostic prediction, while mid- and long-term prediction improved by almost 10% by using the combination of a Child- Pugh's score > 8 and an MEGX value < 15 mg/l. Cox's multi-variate regression analysis indicated that MEGX values either with Child Pugh's score or with prothrombin activity and ascites were independent prognostic variables. CONCLUSIONS: Besides confirming that Child-Pugh's score as the basis of prognostic evaluation of cirrhotic patients, these results suggest that the MEGX test might be a complement to the original score when a patient is being evaluated for a liver transplantation programme. PMID- 10755263 TI - Protein-losing enteropathy with an endoscopic feature of 'the watermelon colon'. AB - A watermelon lesion involving the gastrointestinal tract other than the stomach has not been reported previously except for two cases with rectal involvement and another case with caecal involvement. This report describes a case of protein losing enteropathy with endoscopic features of 'the watermelon lesion' involving nearly the entire colon. A 57-year-old man presented with a six-month history of generalized oedema and diarrhoea. Laboratory findings included elevated erythrocyte sedimentation rate and decreased serum total protein and albumin. Serum fluorescent anti-nuclear antibody, anti-dsDNA antibody and anti-La antibody were positive. Upper gastrointestinal endoscopy was unremarkable. Colonoscopy showed seven or eight longitudinal reddish stripes along the entire length of the colon and rectum sparing the caecum. Biopsies from the duodenum and colon revealed mildly ectatic lymphatics in the submucosa. Colonic biopsy also exhibited some haemosiderin-laden macrophages in the muscularis mucosae and submucosa. 99mTc-human serum albumin scintigraphy demonstrated a leak into the intestinal lumen. Oral prednisolone therapy was started and his symptoms and signs gradually improved over months. Steroid could be withdrawn after 7 months of therapy, and the patient has remained free of symptoms for 1 year of follow-up after withdrawal of the steroid. PMID- 10755264 TI - Autoimmune liver disease in patients with neoplastic diseases. AB - BACKGROUND: Development of de novo autoimmune liver disease has not been well documented in patients with malignant diseases. METHODS/RESULTS: In this paper we report on a series of six patients with neoplastic disorders who acquired liver disease with autoimmune features. Five patients had suffered from haematological neoplasms and one from colonic cancer. In two patients, liver disease was detected at the time of presentation with malignancy. In the remaining four, all of whom were successfully treated for malignancies, features of liver disease presented at intervals 24-72 months after the cancer diagnosis. Twelve liver specimens (11 biopsies and one hepatectomy specimen) were obtained at time intervals of 1-76 months after initial presentation of neoplastic disease. Biopsies from three patients showed features of hepatitis (one acute, one sub acute, one chronic). Two patients had histological features suggestive of an overlap syndrome (one autoimmune hepatitis/primary biliary cirrhosis, one autoimmune hepatitis/primary sclerosing cholangitis). The sixth patient had features of autoimmune cholangiopathy. All but one responded well to steroid therapy with complete clinical and biochemical remission obtained 4 weeks to 8 months after steroid introduction. We discuss briefly possible aetiologies of autoimmune liver disease in these patients. CONCLUSIONS: Autoimmune liver disease may be precipitated by therapy for neoplastic disease or malignant disease itself. The unusually heterogeneous clinicopathological findings in this group as well as the response to treatment support the concept of a wide spectrum of manifestations of autoimmune liver disease. The results may also suggest that autoimmune liver disease may be possibly added to the list of paraneoplastic syndromes. Further prospective studies are required to confirm a causal association and to determine whether the mechanisms involved are disease- or treatment-related. PMID- 10755265 TI - The clinical spectrum of posthypoxic myoclonus. PMID- 10755266 TI - Physiology of human posthypoxic myoclonus. AB - There are two types of posthypoxic myoclonus, acute and chronic. The acute type has not been intensively studied but is likely to be brain stem in origin. Chronic posthypoxic myoclonus is most commonly cortical reflex myoclonus, but reticular reflex myoclonus and exaggerated startle may also occur. These three conditions have characteristic physiological features. In assessing the relevance of an animal model, it would be appropriate to identify the myoclonus with one of these three patterns. PMID- 10755267 TI - Early electrophysiological and histologic changes after global cerebral ischemia in rats. AB - INTRODUCTION: Cerebral anoxia is fundamental to morbidity and mortality after resuscitation from cardiac arrest. With no proven effective primary therapy for post-anoxic brain injury, the goal of neurologic care are supportive, to provide prognosis and prevention of further complications. With the multifaceted approach using electroencephalography (EEG), somatosensory evoked potentials (SEP), multiunit recordings, behavioral and histologic assessment, we investigated the hyperacute recovery period after resuscitation from cardiac arrest in a rat model to define the value of EEG and SEP in assessing neurologic injury. METHODS: Two cohorts of rats were subjected to sham and graded asphyxic-cardiac arrest. EEG was collected during baseline, at injury, and 90 minutes into recovery in the first rat cohort. EEG bursting during the first 90 minutes of recovery was visually analyzed and correlated with the neurologic recovery at 24 hours after injury. The neurologic recovery was assessed using a neurodeficit score (NDS) with 80 as normal and 0 as brain dead. The next rat cohort subjected to asphyxic cardiac arrest was studied using SEP and multiunit recording in the VPL; brain histologic studies were performed at 4 hours after the asphyxia. RESULTS: The first rat cohort subjected to graded asphyxic-cardiac arrest emerged from EEG isoelectricity by burst-suppression pattern during the first 90 minutes after asphyxia. Six rats in the good outcome group (NDS >60) showed increased frequency of bursting, leading to return of EEG background activity. Six rats with a bad outcome (NDS <60) had low-intensity and persistent bursting without return of EEG background activity within 90 minutes of observation. Visual assessment showed increased EEG peak burst counts during the first 90 minutes of recovery for the rats with a good outcome compared with the rats with a bad outcome. In the second cohort, the rats were subjected to 3 minutes, 5 minutes, and 7 minutes of asphyxia. The N20 recovered to 60% of baseline in all three cases. The recovery profile of VPL is similar to that of cortical N2O for the animal with 3 minutes of asphyxia. However, VPL response is suppressed after 7 minutes of asphyxia leading to a divergence in the rate of recovery of the cortical N20 and VPL response. In both the animals (with mild and intermediate injury) in which the early response in VPL recovered to more than 50% of baseline, the recovery profile was similar to the N20 in cortical evoked potential (EP). The rats were killed 4 hours after asphyxia and the hematoxylin and eosin stain performed on the brains showed evidence of neuronal injury in the thalamic reticular nucleus (TRN) which seemed to correlate with the duration of asphyxia. CONCLUSION: We present a multimodality assessment of early neurologic recovery following resuscitation from cardiac arrest. The recovery of bursting and high-frequency oscillations may be regulated by interneurons in the TRN. The early selective vulnerability of these interneurons in the TRN may be crucial to the early neurologic recovery as assessed by EP, multiunit recording, EEG, and neurologic behavioral recovery. PMID- 10755268 TI - Animal models of myoclonus: an overview. AB - Attempts to characterize the mechanism(s) associated with myoclonus have led to the development of several naturally occurring and pharmacologically based animal models of myoclonus. Congenital disorders in animals that result in myoclonic seizures have been found in subpopulations of baboons that exhibit photoresponsive myoclonus and in herds of Hereford cattle that possess a fatal, autosomal-inherited imbalance in spinal glycine neurotransmission. Pharmacologically based models of myoclonus use a variety of approaches to product myoclonic seizures in test animals. PMID- 10755269 TI - Animal models of posthypoxic myoclonus: I. Development and validation. PMID- 10755270 TI - Animal model of posthypoxic myoclonus: II. Neurochemical, pathologic, and pharmacologic characterization. AB - The sudden, brief, shock-like, involuntary movements caused by active muscular contractions or inhibitions characterize myoclonus. It is manifested in a wide variety of pathologic conditions affecting the brain, spinal cord, or peripheral nerves, and is thought to be related to neuronal hyperexcitability. The pathology, physiology, and pharmacology of myoclonus are not well understood as a result of the rarity of the disorder in people and the lack of a suitable animal model. Posthypoxic myoclonus is a major myoclonus syndrome which occurs as a result of severe cerebral ischemia/hypoxia. There has been tremendous interest in the development of a suitable animal model that reflects the etiology and clinical pathology of posthypoxic myoclonus. Recently, we have developed a new animal model of posthypoxic myoclonus in which rats were subjected to a mechanically induced cardiac arrest procedure. Herein, we describe the neurochemical, pharmacologic, and pathologic characteristics of this animal model of posthypoxic myoclonus. PMID- 10755271 TI - Posthypoxic myoclonus in the rat: natural history, stability, and serotonergic influences. PMID- 10755272 TI - Involvement of GABA(A) receptors in myoclonus. AB - Alterations in multiple neurochemical systems have been reported in animal and human studies of posthypoxic myoclonus. It is impossible, however, to establish causative relationships between the observed changes and the myoclonic movements from these studies. Therefore, to establish causative links between neurochemical changes and myoclonus, ligands that target neurotransmitter systems that are altered in posthypoxic myoclonus were microinjected into the lateral ventricles of normal rats to identify the changes that can produce myoclonus. Of the ligands that were tested, only the GABA(A) antagonists produced myoclonus after intracerebroventricular administration, suggesting the importance of disinhibition of GABAergic systems in myoclonus. To further examine the role of GABA in myoclonus, GABAergic antagonists were microinjected into the nucleus reticularis of the thalamus (NRT), an area of the brain in which extensive pathologic changes are seen in posthypoxic animals. GABA(A), but not GABA(B), antagonists produced myoclonus after microinjection into the NRT. Earlier investigators have further reported the ability of GABA(A) antagonists to produce myoclonus after microinjection into the caudate. The data therefore suggest that disruption of activity at GABA(A) receptors at any one of a number of levels in the neural axis can produce myoclonus. PMID- 10755273 TI - Investigation of the role of the cerebellum in the myoclonic-like movement disorder exhibited by tottering mice. AB - Recently it has been discovered that defects in neuronal ion channels can result in seizure disorders. The tottering mouse is a genetic animal model carrying a mutation in the alpha1A calcium channel subunit that causes these mice to exhibit generalized petit mal-like epilepsy, cerebellar ataxia, and an intermittent movement disorder that has some characteristics similar to myoclonus or myoclonic epilepsy. We postulate that abnormal cerebellar Purkinje cell output to the deep cerebellar nuclei results in the intermittent movement disorder observed in these mice. The frequency and duration of seizure activity were measured in tottering mice before and 2 weeks after surgical or chemical lesioning of the cerebellum. Surgical lesions in the anterior cerebellar vermis of tottering mice produced significant reductions in seizure duration and frequency. Surgical lesioning of the posterior cerebellar vermis had no significant effect. Chemical lesions of the same cerebellar regions, using a locally applied neurotoxin, NMD-L-A, appear to produce effects similar to the surgical lesions. These data indicate that anterior vermal cerebellar output is important for production of the seizures associated with the intermittent movement disorder observed in tottering mice. PMID- 10755274 TI - Basic science of knee joint injury mechanisms. PMID- 10755275 TI - Antagonist muscle coactivation during isokinetic knee extension. AB - The aim of the present study was to quantify the amount of antagonist coactivation and the resultant moment of force generated by the hamstring muscles during maximal quadriceps contraction in slow isokinetic knee extension. The net joint moment at the knee joint and electromyographic (EMG) signals of the vastus medialis, vastus lateralis, rectus femoris muscles (quadriceps) and the biceps femoris caput longum and semitendinosus muscles (hamstrings) were obtained in 16 male subjects during maximal isokinetic knee joint extension (KinCom, ROM 90-10 degrees, 30 degrees x s(-1)). Two types of extension were performed: [1] maximal concentric quadriceps contractions and [2] maximal eccentric hamstring contractions Hamstring antagonist EMG in [1] were converted into antagonist moment based on the EMG-moment relationships determined in [2] and vice versa. Since antagonist muscle coactivation was present in both [1] and [2] a set of related equations was constructed to yield the moment/EMG relationships for the hamstring and quadriceps muscles, respectively. The equations were solved separately for every 0.05 degrees knee joint angle in the 90-10 degrees range of excursion (0 degrees = full extension) ensuring that the specificity of muscle length and internal muscle lever arms were incorporated into the moment/EMG relationships established. Substantial hamstring coactivation was observed during quadriceps agonist contraction. This resulted in a constant level of antagonist hamstring moment of about 30 Nm throughout the range of motion. In the range of 30-10 degrees from full knee extension this antagonist hamstring moment corresponded to 30-75% of the measured knee extensor moment. The level of antagonist coactivation was 3-fold higher for the lateral (Bfcl) compared to medial (ST) hamstring muscles The amount of EMG crosstalk between agonist antagonist muscle pairs was negligible (Rxy2<0.02-0.06). The present data show that substantial antagonist coactivation of the hamstring muscles may be present during slow isokinetic knee extension. In consequence substantial antagonist flexor moments are generated. The antagonist hamstring moments potentially counteract the anterior tibial shear and excessive internal tibial rotation induced by the contractile forces of the quadriceps near full knee extension. In doing so the hamstring coactivation is suggested to assist the mechanical and neurosensory functions of the anterior cruciate ligament (ACL). PMID- 10755276 TI - Motor pattern of the knee joint muscles during side-step cutting in European team handball. Influence on muscular co-ordination after an intervention study. AB - The incidence of anterior cruciate ligament (ACL) injuries is reported to be high in many popular team sports The most risky movement in European team handball is reported to be the side-step cutting manoeuvre. Well-rehabilitated ACL-deficient athletes have been shown to have increased co-contraction about the knee joint with this manoeuvre. The aim of this study was to examine the effect of a prophylactic training programme on the co-contraction of the knee joints of non injured European team handball players The subjects performed 'match-like' cutting manoeuvres on a force platform. EMG-signals from the involved knee joint muscles and force platform signals were simultaneously stored on a personal computer. The data collected before and after a 12-week prophylactic training program were compared. The results demonstrated that rehabilitation exercises cannot increase co-contraction about the knee joint in healthy athletes during side-step cutting. This is contradictory to the reported effect on ACL-deficient athletes. PMID- 10755277 TI - Can the hamstring muscles protect the anterior cruciate ligament during a side cutting maneuver? AB - Because anterior cruciate ligament (ACL) injuries are common in European handball the present study assessed knee joint shear forces to estimate ACL loading in six elite female handball players during a side-cutting maneuver. A pilot investigation in three dimensions showed that peak moments occurred in the sagittal plane at a high velocity. Therefore, analysis of the movement was performed in two dimensions using high-speed cinematography, ground reaction forces, and electromyography (EMG). Film and force plate data allowed for calculation of net joint moments (inverse dynamics), estimates of instantaneous muscle-tendon lengths, contraction velocities, and peak loading of the ACL. During the breaking phase of the maneuver the peak knee joint moment was 239 Nm (99-309), which yielded an ACL-load of 520 N (215-673). The corresponding peak EMG amplitudes for the hamstring muscles were 34-39% of maximum EMG. During the breaking phase the quadriceps muscle contracted eccentrically with a velocity of 216-253% fiber length/s. In contrast, the hamstring muscles contracted concentrically with a velocity of 222-427% fiber length/s. These results suggest that a side-cutting maneuver produces loads that are insufficient to rupture the ACL. Furthermore, the rapid concentric hamstring contraction suggests that even during maximal activation, the ability of the hamstrings to reduce the ACL load is marginal. PMID- 10755278 TI - Proprioceptive sensitivity and performance in anterior cruciate ligament deficient knee joints. AB - We studied the performance and proprioception of the knee joint in a group of non reconstructed anterior cruciate ligament (ACL)-deficient (n=20) patients and compared them with a group of ACL-reconstructed patients (n=18) and a group of healthy controls (n=20). Each patient was scored according to Lysholm and Tegner and was then asked to subjectively evaluate the performance of the injured knee and the degree of retropatellar discomfort. The knee joint laxity was measured. The performance was assessed based on the performance in a triple jump test and a one-leg one-step leap test. The proprioception in the knee was measured as the threshold when passive movement was detected and as the ability to reproduce a flexion angle from a start position of 60 degrees of flexion or from full extension of the knee. All tests were performed on both legs. The scoring systems and the subjective evaluation showed significant differences between the reconstructed and the non-reconstructed patients. No significant difference in knee joint laxity was found between the two groups. In the triple jump test and the one-step leap test, both groups performed significantly worse on the leg with the injured knee joint than on the non-injured leg. The proprioceptive tests showed decreased ability to recognize and reproduce a prior angle from a start position of 60 degrees. The threshold to detection of passive movement with the injured knee was significantly increased in both groups of patients. No difference was found between the dominant and non-dominant knee in the control group. When reproduction of the same angles started from full extension, the groups did not differ. These data show that decreased performance and changes in the proprioception of the knee joint accompany ACL rupture. PMID- 10755279 TI - A 7-year study on risks and costs of knee injuries in male and female youth participants in 12 sports. AB - Knee injuries are common and account in various sports for 15-50% of all sports injuries. The cost of knee injuries is therefore a large part of the cost for medical care of sports injuries. Furthermore, the risk of acquiring a knee injury during sports is considered higher for females than for males. The nationwide organization "Youth and Sports" represents the major source of organized sports and recreation for Swiss youth and engages annually around 370000 participants in the age group of 14 to 20 years. The purpose of this study was to combine data on knee injuries from two sources, the first being data on the exposure to risk found in the activity registration in "Youth and Sports" and the second injuries with their associated costs resulting from the activities and filed at the Swiss Military Insurance. This allowed calculation of knee injury incidences, to compare risks between males and females and to estimate the costs of medical treatment. The study comprises 3864 knee injuries from 12 sports during 7 years. Females were significantly more at risk in six sports: alpinism, downhill skiing, gymnastics, volleyball, basketball and team handball. The incidences of knee injuries and of cruciate ligament injuries in particular, together with the costs per hour of participation, all displayed the same sports as the top five for both females and males: ice hockey, team handball, soccer, downhill skiing and basketball. Female alpinism and gymnastics had also high rankings. Knee injuries comprised 10% of all injuries in males and 13% in females, but their proportional contribution to the costs per hour of participation was 27% and 33%, respectively. From this study it can be concluded that females were significantly more at risk for knee injuries than males in six sports and that knee injuries accounted for a high proportion of the costs of medical treatment. PMID- 10755281 TI - Effects of walking on bone quality as determined by ultrasound in the elderly. AB - In the present study, measurements of broadband ultrasound attenuation (BUA) were used as indications of bone quality in elderly residents of an old people's home. To investigate the possibility of using number of steps walked as an indicator of the role played by exercise on bone quality, we studied the relationship between BUA and the number of steps, taken as a measure of the number of impacts against the ground placing a direct mechanical stress on the bones. The subjects were 59 healthy elderly women with a mean age of 78+/-8 years who had no impairments in walking or daily life. A statistical analysis of the relationship between age, number of steps, BUA, stride length, and walking speed revealed that number of steps and BUA declined with age, and that BUA increased with a greater number of steps. Among other factors, age was found to be negatively correlated with weight (r= -0.343, P<0.01), BUA (r=-0.542, P<0.001), total number of steps (r= -0.524, P<0.001) and past exercise habits (r=-0.425, P<0.001). There were positive correlations between BUA and total number of steps (r=0.606, P<0.001), walking speed (r=0.460, P<0.001), stride length (r=0.373, P<0.01) and past exercise habits (r=0.429, P<0.001). These results show that the benefit to bone quality increases the more a person walks in daily life. The above demonstrates that degree of walking activity is positively correlated to bone quality among the elderly. These results also suggest that, when investigating the relationship between exercise and bone quality, one should look not only at distance and walking speed, but also number of steps as the number of impacts against the ground that place a direct mechanical stress on the bones. This may prove to be a useful indicator of bone quality in future use. PMID- 10755280 TI - Changes in hemoglobin values in elite cross-country skiers from 1987-1999. AB - Hemoglobin data have been available from ski teams beginning from 1987, and from 1989 to 1999 we have followed hemoglobin values in elite cross-country skiers in international competitions. The mean values at the 1989 World Nordic Ski Championships were lower than population reference values, as would be expected from plasma volume expansion associated with endurance training. However, an increase, particularly in the maximal values, became obvious in 1994 and rose further in 1996. These extreme values provide both a health risk to the individual athlete and unfair competition. After a rule limiting hemoglobin values was introduced, the drop of the highest values was remarkable: among men 15 g/l (0.23 mmol/l) and among women 42 g/l (0.65 mmol/l). It would appear that the rule had achieved its goal of limiting extreme hemoglobin values. Yet the mean hemoglobin concentrations in men and women have continued to rise, suggesting the continued use of artificial methods to increase total hemoglobin mass. PMID- 10755282 TI - Motor performance status in 10 to 17-year-old Estonian girls. AB - The improvement of motor abilities is associated with the periodical acceleration of changes in adolescents of both sexes. The present cross-sectional study is aimed at establishing smooth curves of motor performance status in 10 to 17-year old girls. Motor performance was tested in 902 girls with the aid of 30 m dash, standing long jump, vertical jump, pushing a stuffed ball (2 kg), standing quintuplet jump, isometric strength of back extensor muscles, trunk forward flexion and 1-min ergocycling at the highest possible rate. Statistically significant differences of all studied motor abilities between the age groups of 10-12 were indicated. In height and body mass the most pronounced differences (on average 6.5 cm and 7.7 kg, respectively) appeared between the age groups of 12 and 13. At the age of 13 the group results were statistically higher than those at 12 in pushing a stuffed ball, vertical jump, quintuplet jump, strength of back extensors muscle, 30 m dash and ergocycling test, but not in standing long jump and trunk forward flexion. At the age of 14 the performance was not higher than at 13, except in the vertical jump and quintuplet jump. From 14 to 16 years of age differences reappeared in the results of vertical jump, quintuplet jump, pushing a stuffed ball, 1-min cycling and trunk forward flexion but not in the 30 m dash and standing long jump. The lack of significant differences between the age groups of 16 and 17 indicated the final stabilization of tested motor abilities. The obtained results suggest the existence of several periods in motor performance status in 10 to 17-year-old Estonian girls: 1) The biggest differences in the mean results of the tests on motor abilities occurred between ages 10-11, 11-12 and 12-13, which coincide with the biggest differences in height and weight at the same age. 2) The differences in the mean results of most tests on motor abilities stabilized between the age groups of 13 and 14. The mean results of 14-year-old girls were lower in some tests compared to the results of 13-year-olds. 3) The positive differences in the mean results remained between the age groups of 14-15 and 15-16 (excluding the sprint velocity and standing long jump). 4) The final stabilization of motor abilities occurred at the age of 16 to 17. PMID- 10755283 TI - Anterior cruciate ligament injury in indoor ball games. AB - Three videorecorded incidents of knee injuries inflicted during indoor ball games are reported. Injuries and especially anterior cruciate ligament ruptures seemed to be triggered in varus loaded knees by femural external rotation, or in valgus loaded knees by femural internal rotation with the pivot shifted to the lateral femurotibial compartment. The observations suggest that it may be to the players' advantage to be trained in not letting their knees sag medially or laterally during side-stepping or sudden changes in speed. PMID- 10755284 TI - Item analyses of age relations on reasoning tests. AB - Analyses at the level of individual items were conducted on 11 data sets representing various combinations of participant samples and tests of reasoning. The magnitude of the relations between age and solution accuracy did not vary systematically across a wide range of item difficulty, although there was some evidence for independent age-related influences on the more difficult items. The results were tentatively interpreted as reflecting the operation of at least 2 types of age-related effects on tests of reasoning, 1 common to all items and 1 sensitive to the greater processing demands associated with more difficult items. PMID- 10755285 TI - Further evidence that negative priming in the Stroop color-word task is equivalent in older and younger adults. AB - In 2 experiments, possible adult age differences in negative priming were explored using several variants of the Stroop color-word task. Negative priming was at least as high in the older adults as in the younger adults in every variant. Negative priming varied as a function of condition, but the age equivalence was unaffected. This result was true even when the possibility of general slowing was taken into account. Across conditions, interference and negative priming were positively correlated. The results do not permit a clear choice between the 2 major theoretical explanations of negative priming, inhibition and memory retrieval; they do show that negative priming can be systematically manipulated within an experimental paradigm. PMID- 10755287 TI - Complex semantic processing in old age: does it stay or does it go? AB - Old adults' (N = 24) and young adults' (N = 24) speed of producing exemplars of semantic categories (i.e., semantic fluency) varying in difficulty was assessed both in a standard condition and in a "set-switching" condition where exemplars had to be produced from 2 categories in an alternating manner. "Retrieval position function" parameters were used to assess speed of semantic access independent of nonsemantic factors. Results suggested age effects in nonsemantic components but not in semantic retrieval per se. Also, age deficits in set switching were relatively subtle. Findings are discussed with relation to issues of domain specificity of age effects as well as of the role of executive control during semantic retrieval and age differences therein. PMID- 10755286 TI - Person-specific paths of cognitive decline in Alzheimer's disease and their relation to age. AB - Change in global and specific measures of cognitive function was studied in a cohort of 410 persons with Alzheimer's disease. Persons completed up to 5 annual evaluations; follow-up participation among survivors exceeded 90%. Average annual decline was 0.57 standard score units (95% confidence interval [CI]: -0.51 to 0.62) on a composite measure based on 17 individual tests and 3.26 points (95% CI: -3.06 to 3.46) on the Mini-Mental State Examination, but substantial heterogeneity was apparent. On both global and specific measures, rate of cognitive decline was reduced in older persons compared with younger persons. A similar effect was observed for estimated age of disease onset. The effect of age was approximately linear and was not attributable to education, sex, race, other conditions that impair cognition, or mortality. The results indicate that person specific paths of cognitive decline in Alzheimer's disease vary substantially and suggest that in clinical settings some of this variability is related to age. PMID- 10755288 TI - Structural constraints on process explanations in cognitive aging. AB - Much of the current research in the area of cognitive aging has been focused on investigating specific processes presumed to be responsible for the age differences observed in particular cognitive tasks. A central thesis of this article is that age-related effects on cognitive variables seldom occur in isolation, and hence, they are best interpreted in the context of the structural interrelations that exist among variables and the relations of age on that organizational structure. Results from analyses of 2 separate data sets suggest that large proportions of the age-related effects across a wide range of cognitive variables are shared and that independent, or unique, age-related effects often contribute relatively little to the age differences observed in many cognitive variables. These findings imply that it is important to consider the structure within which a variable occurs when attempting to investigate the processes responsible for age-related differences on that variable. PMID- 10755289 TI - Contribution of age, genes, and environment to the relationship between perceptual speed and cognitive ability. AB - The aim of this study was to examine genetic influences on cognitive ability in adulthood in the context of the relationship between perceptual speed and cognitive aging. Quantitative genetic analysis of data from the Swedish Adoption/Twin Study of Aging allowed for estimation of the contribution of age, genetic, and environmental effects to the variance in a latent cognitive factor and to the covariance between the cognitive factor and perceptual speed. The sample included 292 pairs of monozygotic and dizygotic twins. both reared together and reared apart, ranging in age from 40 to 84 years. Analysis of components of total variance in the cognitive factor indicated that 90% of the age-related variance in the cognitive factor was shared with perceptual speed and 70% of the genetic variance in the cognitive factor was shared with perceptual speed. The correlation between the speed and cognitive factors was primarily genetically mediated. PMID- 10755290 TI - Effect of off-target verbosity on communication efficiency in a referential communication task. AB - The referential communication task was used to see if high off-target verbosity (OTV), defined as excessive speech that is lacking in focus, negatively affects communication of nonautobiographical information. The task required 1 individual (the director) to communicate descriptions of abstract figures to another (the matcher). Out of 455 adults aged 63 to 93 who were screened for OTV, 27 directors were drawn from each of the top and bottom 15% of the range of OTV scores and 26 directors and all 80 matchers from the middle 50%. High OTV directors were less efficient communicators about the figures and showed poorer inhibitory control but did not intrude personal information in their speech. The implications of the findings for the inhibitory deficit and pragmatic change explanations of OTV are discussed. PMID- 10755291 TI - Age-related changes in the control of attention in depth. AB - In 2 experiments, the authors examined the control of spatial attention in depth in advanced age. Observers viewed 2 sets of lines that overlapped in two dimensional (2-D) space but that were presented at different depth locations. An exogenous cue indicated the depth at which a colored target line would appear. On some trials, a distracting colored element was also presented. The luminance of this distracter varied from high to low. For both older and younger adults, distractors slowed reaction time less if they were at a different depth from the target. This effect was more robust for older adults with low-luminance distractors, indicating an important role for target features as well as attentional control. Adding another feature (i.e., color) that distinguished between target and distracter reduced the effects of slowing shown by the older observers and eliminated the cost of low-luminance distractors for all observers. The results suggest that attentional control in depth is maintained in aging. PMID- 10755292 TI - The effects of talker familiarity on spoken word identification in younger and older listeners. AB - The present study was designed to examine age differences in the ability to use voice information acquired intentionally (Experiment 1) or incidentally (Experiment 2) as an aid to spoken word identification. Following both implicit and explicit voice learning, participants were asked to identify novel words spoken either by familiar talkers (ones they had been exposed to in the training phase) or by 4 unfamiliar voices. In both experiments, explicit memory for talkers' voices was significantly lower in older than in young listeners. Despite this age-related decline in voice recognition, however, older adults exhibited equivalent, and in some cases greater, benefit than young listeners from having words spoken by familiar talkers. Implications of the findings for age-related changes in explicit versus implicit memory systems are discussed. PMID- 10755293 TI - Longitudinal relationships between depressive symptoms and health in normal older and middle-aged adults. AB - Comorbidity between health and depression is salient in late life, when risk for physical illness rises. Other community studies have not distinguished between the effects of brief and long-standing depressive symptoms on excess morbidity and mortality. S. Cohen and M. S. Rodriguez's (1995) differential hypothesis of pathways between depression and health was used to examine the relationships between health and depression in a prospective probability sample of 1,479 community-resident middle-aged and older adults. Findings suggest that different durations of depressive symptoms have different relationships to health. Health had an impact on short-term increases in depressive symptoms but depressive symptoms had a weaker impact on health. The reciprocal impact was indistinguishable from the health influence on depression. In contrast, longer term depressive symptoms had a clear impact on health. The results imply that physical illness can affect depressive states; depressive traits but not states can affect illness. PMID- 10755294 TI - Listening to discourse in distracting settings: the effects of aging. AB - Younger and older adults listened to discourse in quiet and in conversational noise, before answering questions concerning the material. Some questions required listeners to recall specific details; others were of a more integrative nature. When the listening situation was adjusted for individual differences in hearing, younger and older adults were equally adept at remembering the gist of the passages in both quiet and in two levels of noise. The two age groups also did not differ with respect to memory for specific details when listening in quiet or in a moderate level of noise, even when required to perform a concurrent task. Only at the loudest noise level did younger adults tend to recall more detail than older adults. However, when no adjustments were made to compensate for the poorer hearing of older adults (all participants tested under identical listening conditions), older adults could not recall as much detail as younger adults, either in quiet or in noise. The results indicate that the speech comprehension difficulties of older adults primarily reflect declines in hearing rather than in cognitive ability. PMID- 10755295 TI - Adult age differences in task switching. AB - Age differences in 2 components of task-set switching speed were investigated in 118 adults aged 20 to 80 years using task-set homogeneous (e.g., AAAA ...) and task-set heterogeneous (e.g., AABBAABB ... ) blocks. General switch costs were defined as latency differences between heterogeneous and homogeneous blocks. whereas specific switch costs were defined as differences between switch and nonswitch trials within heterogeneous blocks. Both types of costs generalized over verbal, figural, and numeric stimulus materials; were more highly correlated to fluid than to crystallized abilities; and were not eliminated after 6 sessions of practice, indicating that they reflect basic and domain-general aspects of cognitive control. Most important, age-associated increments in costs were significantly greater for general than for specific switch costs, suggesting that the ability to efficiently maintain and coordinate 2 alternating task sets in working memory instead of 1 is more negatively affected by advancing age than the ability to execute the task switch itself. PMID- 10755296 TI - Centrality of women's multiple roles: beneficial and detrimental consequences for psychological well-being. AB - Theorists have proposed that greater centrality (personal importance) of a social role is associated with better psychological well-being but that role centrality exacerbates the negative effects of stress in that same social role on well being. The present study found evidence to support both hypotheses in a sample of 296 women who simultaneously occupied the roles of parent care provider, mother, wife, and employee. Greater centrality of all four roles was related to better psychological well-being. As predicted, wife centrality exacerbated the effects of wife stress on life satisfaction, and employee centrality exacerbated the effects of employee stress on depressive symptoms. Contrary to prediction, centrality of the mother role buffered women from the negative effects of mother stress on depressive symptoms. These findings point to an aspect of role identity that can benefit well-being but that has complex effects in the context of role stress. PMID- 10755297 TI - Converging evidence that visuospatial cognition is more age-sensitive than verbal cognition. AB - In 3 separate experiments, the same samples of young and older adults were tested on verbal and visuospatial processing speed tasks, verbal and visuospatial working memory tasks, and verbal and visuospatial paired-associates learning tasks. In Experiment 1, older adults were generally slower than young adults on all speeded tasks, but age-related slowing was much more pronounced on visuospatial tasks than on verbal tasks. In Experiment 2, older adults showed smaller memory spans than young adults in general, but memory for locations showed a greater age difference than memory for letters. In Experiment 3, older adults had greater difficulty learning novel information than young adults overall, but older adults showed greater deficits learning visuospatial than verbal information. Taken together, the differential deficits observed on both speeded and unspeeded tasks strongly suggest that visuospatial cognition is generally more affected by aging than verbal cognition. PMID- 10755298 TI - Cloning and disruption of pgx4 encoding an in planta expressed exopolygalacturonase from Fusarium oxysporum. AB - Fusarium oxysporum f. sp. lycopersici, the causal agent of tomato vascular wilt, produces an array of pectinolytic enzymes, including at least two exo-alpha1,4 polygalacturonases (exoPGs). A gene encoding an exoPG, pgx4, was isolated with degenerate polymerase chain reaction primers derived from amino acid sequences conserved in two fungal exoPGs. pgx4 encodes a 454 amino acid polypeptide with nine potential N-glycosylation sites and a putative 21 amino acid N-terminal signal peptide. The deduced mature protein has a calculated molecular mass of 47.9 kDa, a pI of 8.0, and 51 and 49% identity with the exoPGs of Cochliobolus carbonum and Aspergillus tubingensis, respectively. The gene is present in a single copy in different formae speciales of F. oxysporum. Expression of pgx4 was detected during in vitro growth on pectin, polygalacturonic acid, and tomato vascular tissue and in roots and stems of tomato plants infected by F. oxysporum f. sp. lycopersici. Two mutants of F. oxysporum f. sp. lycopersici with a copy of pgx4 inactivated by gene replacement were as virulent on tomato plants as the wild-type strain. PMID- 10755299 TI - Transgenic resistance to PVY(O) associated with post-transcriptional silencing of P1 transgene is overcome by PVY(N) strains that carry highly homologous P1 sequences and recover transgene expression at infection. AB - Resistance to Potato virus Y (PVY) has been obtained in our previous studies through expression of the PVY P1 gene in sense or antisense orientation in potato cv. Pito. In the present study, the mechanism and strain specificity of the resistance were analyzed. Several features including low steady-state P1 mRNA expression in the resistant P1 plants indicated that resistance was based on post transcriptional gene silencing (PTGS). Resistance was specific to PVY(O) isolates, the PVY strain group from which the P1 transgene was derived. However, according to group analyses, there was no distinguishing characteristic between the PVY(O) and PVY(N) strains P1 gene sequences. Therefore, the ability of the PVY(N) strains to overcome resistance could not be explained solely based on their P1 gene sequences. Infection with PVY(N) of the PVY(O)-resistant transgenic lines led to a recovery of expression of the P1 transgene. These data suggested that factors other than sequence homology are required in determination of the resistance specificity. PMID- 10755300 TI - The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis. AB - The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization. PMID- 10755301 TI - Quorum sensing in the plant pathogen Erwinia carotovora subsp. carotovora: the role of expR(Ecc). AB - The production of the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, the extracellular cell wall-degrading enzymes, is partly controlled by the diffusible signal molecule N-(3-oxohexanoyl)-L homoserine lactone (OHHL). OHHL is synthesized by the product of the expI/carI gene. Linked to expI we found a gene encoding a putative transcriptional regulator of the LuxR-family. This gene, expR(Ecc), is transcribed convergently to the expI gene and the two open reading frames are partially overlapping. The ExpR(Ecc) protein showed extensive amino acid sequence similarity to the repressor EsaR from Pantoea stewartii subsp. stewartii (formerly Erwinia stewartii subsp. stewartii) and to the ExpR(Ech) protein of Erwinia chrysanthemi. Inactivation of the E. carotovora subsp. carotovora expR(Ecc) gene caused no decrease in virulence or production of virulence determinants in vitro. In contrast, there was a slight increase in the maceration capacity of the mutant strain. The effects of ExpR(Ecc) were probably mediated by changes in OHHL levels. Inactivation of expR(Ecc) resulted in increased OHHL levels during early logarithmic growth. In addition, overexpression of expR(Ecc) caused a clear decrease in the production of virulence determinants and part of this effect was likely to be caused by OHHL binding to ExpR(Ecc). ExpR(Ecc) did not appear to exhibit transcriptional regulation of expI, but the effect on OHHL was apparently due to other mechanisms. PMID- 10755302 TI - Mutants of Xanthomonas oryzae pv. oryzae deficient in general secretory pathway are virulence deficient and unable to secrete xylanase. AB - Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight, a serious disease of rice. A virulence- and xylanase-deficient mutant of Xoo was isolated following ethyl methane sulfonate (EMS) mutagenesis. A cosmid clone that restored virulence and xylanase secretion was obtained from a genomic library by functional complementation. Transposon mutagenesis and marker exchange studies revealed genes on the cloned DNA that were required for xylanase production and virulence. Sequence analysis with transposon-specific primers revealed that these genes were homologues of xps F and xps D, which encode components of a protein secretion system in Xanthomonas campestris pv. campestris. Enzyme assays showed xylanase accumulation in the periplasmic space and cytoplasm of the xps F mutant and the complementing clone restored transport to the extracellular space. PMID- 10755303 TI - Recessive and dominant genes interfere with the vascular transport of Potato virus A in diploid potatoes. AB - Resistance to Potato virus A (PVA) was examined in a diploid cross involving Solanum tuberosum subsp. andigena as a resistance source. Hypersensitive resistance (HR) to PVA cosegregated with extreme resistance (ER) to Potato virus Y conferred by the dominant gene Ry(adg) on chromosome XI. Hence, HR to PVA was controlled by a novel, dominant resistance gene closely linked to Ry(adg), or Ry(adg) recognized both viruses but conferred a different type of resistance to each virus. The HR prevented systemic infection with PVA following mechanical inoculation but not following graft inoculation. Another, recessive gene, ra, that may be linked or even allelic with Ry(adg) fully blocked vascular transport of PVA in graft-inoculated plants. Hence, a possibility exists that the genes for the three types of resistance to potyviruses may reside at the same, resistance gene-rich chromosome region syntenic in solanaceous species and might be related. The gene ra acted against all of the three PVA strains tested and, therefore, the avirulence determinants could not be mapped. However, also, PVA strain-specific resistance was found in the progeny. It was overcome by mutations introduced into the viral genome-linked protein and the helper component proteinase and/or the coat protein. PMID- 10755304 TI - Involvement of diamine oxidase and peroxidase in insolubilization of the extracellular matrix: implications for pea nodule initiation by Rhizobium leguminosarum. AB - Rhizobium leguminosarum colonizes host cells and tissues through infection threads, which are tubular in-growths of the plant cell wall. Monoclonal antibody MAC265 recognizes a plant matrix glycoprotein (MGP) associated with the lumen of these infection threads. This glycoprotein is also released in soluble form from the root tips of pea seedlings. In the presence of hydrogen peroxide, release of glycoprotein from root tips was not observed. Extractability from root tips was therefore used as the basis for investigating the peroxide-driven insolubilization of MGP and the possible involvement of two extracellular enzymes, peroxidase (POD) and diamine oxidase (DAO), was investigated. Release of MGP from root tips was enhanced by application of POD and DAO inhibitors (salicylhydroxamic acid and o-phenanthroline, respectively). Furthermore, release of MGP was inhibited by pretreatment of roots with putrescine (the substrate of DAO) and also by application of a partially purified extract of DAO from pea shoots. Following inoculation of pea roots with R. leguminosarum, elevated levels of DAO transcript were observed by reverse transcriptase-polymerase chain reaction (RT-PCR), but these then dropped to a low level from 4 to 10 days post inoculation, rising again in more mature nodules. In situ hybridization studies indicated that the bulk of the transcription was associated with the infected tissue in the center of the nodule. On the basis of these observations, we postulate that DAO may be involved in the peroxide-driven hardening of MGP in the lumen of infection threads and in the intercellular matrix. PMID- 10755305 TI - Evidence against a direct antimicrobial role of H2O2 in the infection of plants by Erwinia chrysanthemi. AB - We have investigated the role of bacterial resistance to oxidative stress in pathogenesis. The oxyR gene from the pathogenic bacterium Erwinia chrysanthemi has been characterized. It is closely related to that found in Escherichia coli (88% overall amino acid identity). An E. chrysanthemi oxyR mutant strain was constructed by marker exchange. After induction with a sublethal dose of H2O2, this mutant was more sensitive to H2O2 and showed reduced levels of catalase and glutathione reductase activities, compared with the wild type. The oxyR mutant was unable to form individual colonies on agar plates unless catalase was added exogenously. However, it retained full virulence in potato tubers and tobacco leaves. These results suggest that the host-produced H2O2 has no direct antimicrobial effect on the interaction of E. chrysanthemi with the two plant species. PMID- 10755306 TI - Interacting signal pathways control defense gene expression in Arabidopsis in response to cell wall-degrading enzymes from Erwinia carotovora. AB - We have characterized the role of salicylic acid (SA)-independent defense signaling in Arabidopsis thaliana in response to the plant pathogen Erwinia carotovora subsp. carotovora. Use of pathway-specific target genes as well as signal mutants allowed us to elucidate the role and interactions of ethylene, jasmonic acid (JA), and SA signal pathways in this response. Gene expression studies suggest a central role for both ethylene and JA pathways in the regulation of defense gene expression triggered by the pathogen or by plant cell wall-degrading enzymes (CF) secreted by the pathogen. Our results suggest that ethylene and JA act in concert in this regulation. In addition, CF triggers another, strictly JA-mediated response inhibited by ethylene and SA. SA does not appear to have a major role in activating defense gene expression in response to CF. However, SA may have a dual role in controlling CF-induced gene expression, by enhancing the expression of genes synergistically induced by ethylene and JA and repressing genes induced by JA alone. PMID- 10755307 TI - Agroinfiltration is a versatile tool that facilitates comparative analyses of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis. AB - The avirulence genes Avr9 and Avr4 from the fungal tomato pathogen Cladosporium fulvum encode extracellular proteins that elicit a hypersensitive response when injected into leaves of tomato plants carrying the matching resistance genes, Cf 9 and Cf-4, respectively. We successfully expressed both Avr9 and Avr4 genes in tobacco with the Agrobacterium tumefaciens transient transformation assay (agroinfiltration). In addition, we expressed the matching resistance genes, Cf-9 and Cf-4, through agroinfiltration. By combining transient Cf gene expression with either transgenic plants expressing one of the gene partners, Potato virus X (PVX)-mediated Avr gene expression, or elicitor injections, we demonstrated that agroinfiltration is a reliable and versatile tool to study Avr/Cf-mediated recognition. Significantly, agroinfiltration can be used to quantify and compare Avr/Cf-induced responses. Comparison of different Avr/Cf-interactions within one tobacco leaf showed that Avr9/Cf-9-induced necrosis developed slower than necrosis induced by Avr4/Cf-4. Quantitative analysis demonstrated that this temporal difference was due to a difference in Avr gene activities. Transient expression of matching Avr/Cf gene pairs in a number of plant families indicated that the signal transduction pathway required for Avr/Cf-induced responses is conserved within solanaceous species. Most non-solanaceous species did not develop specific Avr/Cf-induced responses. However, co-expression of the Avr4/Cf 4 gene pair in lettuce resulted in necrosis, providing the first proof that a resistance (R) gene can function in a different plant family. PMID- 10755308 TI - A two-component regulatory system, pehR-pehS, controls endopolygalacturonase production and virulence in the plant pathogen Erwinia carotovora subsp. carotovora. AB - Genes coding for the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, the plant cell wall-degrading enzymes, are under the coordinate control of global regulator systems including both positive and negative factors. In addition to this global control, some virulence determinants are subject to specific regulation. We have previously shown that mutations in the pehR locus result in reduced virulence and impaired production of one of these enzymes, an endopolygalacturonase (PehA). In contrast, these pehR strains produce essentially wild-type levels of other extracellular enzymes including pectate lyases and cellulases. In this work, we characterized the pehR locus and showed that the DNA sequence is composed of two genes, designated pehR and pehS, present in an operon. Mutations in either pehR or pehS caused a Peh-negative phenotype and resulted in reduced virulence on tobacco seedlings. Complementation experiments indicated that both genes are required for transcriptional activation of the endopolygalacturonase gene, pehA, as well as restoration of virulence. Structural characterization of the pehR-pehS operon demonstrated that the corresponding polypeptides are highly similar to the two-component transcriptional regulators PhoP-PhoQ of both Escherichia coli and Salmonella typhimurium. Functional similarity of PehR-PehS with PhoP-PhoQ of E. coli and S. typhimurium was demonstrated by genetic complementation. PMID- 10755309 TI - Heterologous expression of functional Ptr ToxA. AB - Ptr ToxA, a proteinaceous host-selective toxin (HST) produced by the fungus Pyrenophora tritici-repentis, was expressed in Escherichia coli and purified as a polyhistidine-tagged, fusion protein (NC-FP). NC-FP, consisting of both the N and C domains of the ToxA open reading frame (ORF), is produced as an insoluble protein in E. coli at approximately 10 to 16 mg per liter of culture. Following in vitro refolding, NC-FP elicits cultivar-specific necrosis in wheat, with a specific activity similar to that of native Ptr ToxA. A fusion protein consisting of only the C domain has approximately 10 to 20% of the activity of native Ptr ToxA. These data suggest that (i) the N domain is important for maximal activity of Ptr ToxA, (ii) the N domain does not function to eliminate activity of the protoxin, and (iii) post-translational modifications of Ptr ToxA are not essential for activity. A C domain construct with a cysteine residue mutated to glycine is inactive. This, plus the observation that toxin activity is sensitive to reducing agents, provides evidence that the two cysteine residues in Ptr ToxA are involved in a disulfide bond that is essential for activity. The heterologous expression of Ptr ToxA provides a valuable tool for addressing a number of issues such as receptor binding studies, structure/function studies, and screening wheat cultivars for disease resistance. PMID- 10755310 TI - Comparison of the hypersensitive response induced by the tomato Cf-4 and Cf-9 genes in Nicotiana spp. AB - We have previously shown that tomato Cf-9 induces an Avr9-dependent hypersensitive response (HR) in Nicotiana tabacum and potato. We show here that Cf-4 also induces an Avr4-dependent HR in two tobacco species (N. tabacum and N. benthamiana). The HR induced by Cf-4 and Cf-9 was compared in stable tobacco transgenics by a seedling lethal assay and resistance to recombinant Potato virus X expressing Avr4 or Avr9. We also compared HR induction with Agrobacterium mediated transient expression. The Cf-4/Avr4 combination induced a more rapid HR than Cf-9/Avr9. Sensitive assays for Cf-9 and Cf-4 function should prove useful for structure/function analyses of these resistance proteins in tobacco. PMID- 10755311 TI - Identification of rice blast fungal elicitor-responsive genes by differential display analysis. AB - In order to study molecular interactions that occur between rice and rice blast fungus upon infection, we isolated fungal elicitor-responsive genes from rice (Oryza sativa cv. Milyang 117) suspension-cultured cells treated with fungal elicitor prepared from the rice blast fungus (Magnaporthe grisea) employing a method that combined mRNA differential display and cDNA library screening. Data base searches with the isolated cDNA clones revealed that the OsERG1 and OsERG2 cDNAs share significant similarities with the mammalian Ca2+-dependent lipid binding (C2) domains. The OsCPX1 cDNA is highly homologous to peroxidases. The OsHin1 cDNA exhibits homology to the tobacco hin1 gene, whose expression is induced by avirulent pathogens. The OsLPL1 and OsMEK1 cDNAs share homologies with lysophospholipases and serine/threonine mitogen-activated protein (MAP) kinase kinases, respectively. The OsWRKY1 and OsEREBP1 cDNAs are homologous to transcription factors, such as the WRKY protein family and the AP2/EREBP family, respectively. Transcripts of the OsERG1, OsHin1, and OsMEK1 genes were specifically elevated only in response to the avirulent race KJ301 of the rice blast fungus. Our study yielded a number of elicitor-responsive genes that will not only provide molecular probes, but also contribute to our understanding of host defense mechanisms against the rice blast fungus. PMID- 10755312 TI - A Lotus japonicus nodulation system based on heterologous expression of the fucosyl transferase NodZ and the acetyl transferase NoIL in Rhizobium leguminosarum. AB - Heterologous expression of NodZ and NolL proteins in Rhizobium leguminosarum bv. viciae led to the production of acetyl fucosylated lipo-chitin oligosaccharides (LCOs), indicating that the NolL protein obtained from Mesorhizobium loti functions as an acetyl transferase. We show that the NolL-dependent acetylation is specific for the fucosyl penta-N-acetylglucosamine species. In addition, the NolL protein caused elevated production of LCOs. Efficient nodulation of Lotus japonicus by the NodZ/NolL-producing strain was demonstrated. Nodulation efficiency was further improved by the addition of the ethylene inhibitor L-alpha (2-aminoethoxyvinyl) glycine (AVG). PMID- 10755313 TI - A pharmacodynamic analysis method to determine the relative importance of drug concentration and treatment time on effect. AB - PURPOSES: The pharmacodynamics of most drugs follow the empirical relationship, C(n) x T = h, where C is drug concentration, T is exposure time and h is drug exposure constant. The value of n indicates the relative importance of C and T in determining the effect. An n value greater than 1.0 indicates that for two infusions that produce the same C x T, a short infusion that delivers high concentrations over a short duration will produce a greater C(n) x T and therefore a greater effect, compared to a long infusion that delivers lower concentrations. The reverse is true for an n value less than 1.0 and would support the use of a slow infusion. Hence, it is important to determine the n values and whether the n value significantly differs from 1.0. This report describes a three-step method for this purpose. METHODS: First, we obtained experimental data on the relationship between drug concentration, treatment time and effect, and analyzed the data with a three-dimensional surface response method to obtain the pharmacodynamic model parameters and the magnitude of data variability. The experiments used mitomycin C and two human cancer cell lines, i.e. bladder RT4 and pharynx FaDu cells. The n values obtained from four experiments ranged from 1.04 to 1.16 for FaDu cells and from 1.14 to 1.46 for RT4 cells. The variability in the effect data decreased from 11.9% at 0% effect to 6.14% at 100% effect. Second, these results were used with Monte Carlo simulations to generate 100 concentration-time-effect data sets, which contained randomly and normally distributed data variability comparable to the experimentally observed variability, for each experimentally determined n value. This is analogous to performing 100 experiments under the same experimental conditions. Third, we analyzed the simulated data sets to obtain 100 estimated n values. The frequency with which these estimated n values fell above or below 1.0 indicated the probability that the experimentally determined n value used in the Monte Carlo simulations was truly different from 1.0. We defined this frequency for individual experiments as F(one), and calculated the overall probability for multiple experiments (F(multiple)). A probability of greater than 97.5% (i.e. P < 0.05 for a two-tailed test) was considered statistically significant. RESULTS: Analysis of the mitomycin C pharmacodynamic data yielded F(one) and F(multiple) of 99% to 100% for FaDu and RT4 cells, indicating that the n values for these cells were significantly higher than 1.0. A comparison of the statistical significance of the n value analyzed by the three-step pharmacodynamic analysis method, a conventional statistical method such as the Student's t-test and nonlinear regression analysis, indicated two advantages for the pharmacodynamic method: fewer experiments were required (theoretically only one experiment with three replicates would be sufficient) and a higher statistical significance of the n value was obtained. CONCLUSIONS: In summary, the three-step pharmacodynamic study design and analysis method can be used to define the relative importance of drug concentration and treatment time on drug effect. PMID- 10755314 TI - Human intestinal es nucleoside transporter: molecular characterization and nucleoside inhibitory profiles. AB - PURPOSE: To clone and sequence the equilibrative nitrobenzylthioinosine (NBMPR) sensitive nucleoside transporter (es) from the human small intestine and to examine the capacities of nucleosides and nucleoside analogs to inhibit the uptake of uridine by this transporter. METHODS: Using PCR, es was cloned from a cDNA library of the human small intestine. The uptake of 3H-uridine (10 microM) by the recombinant es, expressed in Xenopus oocytes, was measured in the presence (2 mM) and absence of nucleosides and nucleoside analogs. RESULTS: The amino acid sequence of this es transporter was identical to that of the human placental es transporter. Uptake of 3H-uridine by this es transporter was inhibitable by 1 microM NBMPR. Removal of the oxygen from the 3' position or from both the 2' and 3' positions, but not from 2' or 5' position, resulted in a partial or total loss of the capacity of the nucleosides to inhibit 3H-uridine uptake. No modifications of the adenosine base or of the uridine base (except for 3 and 6 positions on uracil) affected nucleoside inhibitory capacity. CONCLUSION: The es transporters of the human intestine and placenta are identical in their amino acid sequences. Moreover, the inhibitory profiles of various nucleoside analogs in inhibiting the uptake of uridine by the intestinal es transporter are similar to those obtained with the as-yet-uncloned human erythrocyte es transporter. Collectively, these findings suggest that the es transporter does not appear to be functionally variant in the human placenta, small intestine or erythrocytes. PMID- 10755315 TI - Phase I/II study of cisplatin, ifosfamide and irinotecan with rhG-CSF support in patients with stage IIIB and IV non-small-cell lung cancer. AB - PURPOSE: We conducted a phase I/II study in previously untreated patients with stage IIIB or IV non-small-cell lung cancer (NSCLC) to: (1) determine the maximum tolerated dose (MTD) of cisplatin combined with a fixed schedule of ifosfamide and irinotecan with rhG-CSF support; and (2) to determine the overall response rate and median survival of patients entered on this study. METHODS: Ifosfamide (1.5 g/m2) and irinotecan (60 mg/m2) were administered at fixed doses on days 1-4 and on days 1, 8 and 15, respectively. Cisplatin was given on day 1 at 60 mg/m2 and was increased in 10-mg/m2 increments. This regimen was repeated every 4 weeks. rhG-CSF (nartograstim) was administered subcutaneously at a dose of 1 microg/kg on days 5-18 except on the day of irinotecan treatment. RESULTS: Between June 1995 and April 1998, 46 patients were registered onto this phase I/II study. The MTD of cisplatin was defined according to toxicity and the dose during three courses was increased. Since at the 80 mg/m2 dose level more than one-third of the patients were treated with dose modification, the dose of 70 mg/m2 was recommended for phase II study. The dose-limiting toxicity was leukopenia. The overall response rate was 62.2% (95% CI 48.0-76.4%, the median response duration was 144 days, and the median survival time was 393 days. CONCLUSION: For phase II study, we recommend doses of cisplatin 70 mg/m2 on day 1 combined with ifosfamide and irinotecan with rhG-CSF support. Both the response rate and preliminary survival data in this study suggest a high degree of activity of this combination in previously untreated NSCLC. PMID- 10755316 TI - Pharmacokinetics of carboplatin administered in combination with the bradykinin agonist Cereport (RMP-7) for the treatment of brain tumours. AB - INTRODUCTION: Cereport (RMP-7) is a novel bradykinin agonist which is being developed as a modulator of the blood-brain barrier (BBB). In order to investigate the pharmacokinetics of carboplatin in combination with Cereport, we performed pharmacological studies in conjunction with early clinical trials. METHODS: Pharmacokinetic samples were collected from eight patients in a phase I study (Cereport 100-300 ng/ kg) and ten patients in a phase II study (Cereport 300 ng/kg). Pharmacokinetic parameters for carboplatin were compared with respect to the dose of Cereport and with historical controls. RESULTS: Cereport combined with carboplatin was well-tolerated, with mild haematological toxicities consistent with the target area under the concentration time curve (AUC) of 7 mg/ml x min. Although the clearance of carboplatin was within the range reported for this drug alone, the addition of Cereport resulted in a higher than expected carboplatin AUC. This effect was related to the dose of Cereport in the phase I study (AUC values 104-133% of target, Spearman rank correlation coefficient = 0.71, P < 0.001). The higher than expected AUC value was confirmed in the phase II study (AUC values 106-189% of target). CONCLUSIONS: Co-administration of Cereport with carboplatin may result in a greater than predicted AUC. The mechanism of this possible interaction remains to be determined, although this did not result in any increased toxicity. Thus, the clinical potential of this combination in the treatment of brain tumours warrants further investigation. PMID- 10755317 TI - Preferential activation of capecitabine in tumor following oral administration to colorectal cancer patients. AB - PURPOSE: [corrected] Capecitabine (Xeloda) is a novel fluoropyrimidine carbamate rationally designed to generate 5-fluorouracil (5-FU) preferentially in tumors. The purpose of this study was to demonstrate the preferential activation of capecitabine, after oral administration, in tumor in colorectal cancer patients, by the comparison of 5-FU concentrations in tumor tissues, healthy tissues and plasma. METHODS: Nineteen patients requiring surgical resection of primary tumor and/or liver metastases received 1,255 mg/m2 of capecitabine twice daily p.o. for 5-7 days prior to surgery. On the day of surgery, samples of tumor tissue, adjacent healthy tissue and blood samples were collected simultaneously from each patient, 2 to 12 h after the last dose of capecitabine had been administered. Concentrations of 5-FU in various tissues and plasma were determined by HPLC. The activities of the enzymes (CD, TP and DPD) involved in the formation and catabolism of 5-FU were measured in tissue homogenates, by catabolic assays. RESULTS: The ratio of 5-FU concentrations in tumor to adjacent healthy tissue (T/H) was used as the primary marker for the preferential activation of capecitabine in tumor. In primary colorectal tumors, the concentration of 5-FU was on average 3.2 times higher than in adjacent healthy tissue (P = 0.002). The mean liver metastasis/healthy tissue 5-FU concentration ratio was 1.4 (P = 0.49, not statistically different). The mean tissue/plasma 5-FU concentration ratios exceeded 20 for colorectal tumor and ranged from 8 to 10 for other tissues. CONCLUSIONS: The results demonstrated the preferential activation of capecitabine to 5-FU in colorectal tumor, after oral administration to patients. This is explained to a great extent by the activity of TP in colorectal tumor tissue, (the enzyme responsible for the conversion of 5'-DFUR to 5-FU), which is approximately four times that in adjacent healthy tissue. In the liver, TP activity is approximately equal in metastatic and healthy tissue, which explains the lack of preferential activation of capecitabine in these tissues. PMID- 10755318 TI - N,N-diethyl-2-[4-(phenylmethyl)phenoxy] ethanamine (DPPE) a chemopotentiating and cytoprotective agent in clinical trials: interaction with histamine at cytochrome P450 3A4 and other isozymes that metabolize antineoplastic drugs. AB - PURPOSE: N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl (DPPE), an intracellular histamine (HA) antagonist with chemopotentiating and cytoprotective properties, is currently in phase 2 and 3 clinical trials in breast and prostate cancer. DPPE modulates growth at in vitro concentrations that antagonize HA binding to cytochromes P450 in rat liver microsomes. HA inhibits P450 metabolism of some drugs. Recent in vitro studies in human colon cancer cells have linked DPPE enhancement of paclitaxel, doxorubicin and vinblastine cytotoxicity to inhibition of the P-glycoprotein (P-gp) pump. Many substrates of P-gp are also substrates of CYP3A4, a P450 isozyme that metabolizes a variety of antineoplastic agents and is highly expressed in some malignant tissues. Therefore, we assessed whether (a) DPPE and HA interact at CYP3A4 and other P450 human isozymes, and (b) DPPE inhibits the catalytic activity of CYP3A4. METHODS: Using spectral analysis, we measured DPPE and HA binding to insect microsomes that express human P450 isozymes 1A1, 2B6, 2D6 or 3A4. Employing thin-layer chromatography, we assessed the metabolism of DPPE by each isozyme and DPPE inhibition of testosterone metabolism by CYP3A4 and by rat liver microsomes. RESULTS: (1) DPPE evoked "type I" (substrate site binding) absorbance-difference spectra with CYP2D6 (K(S) = 4.1 +/- 0.4 microM), CYP3A4 (K(S) = 31 +/- 15 microM) and CYP1A1 (K(S) = 40 +/- 9 microM), but not with CYP2B6. (2) In correspondence with the binding studies, DPPE was metabolized by CYP2D6, CYP3A4 and CYP1A1; no metabolism occurred with CYP2B6. (3) HA evoked "type II" (heme iron binding) absorbance-difference spectra with all four isozymes, with K(S) values in the range 80-600 microM. DPPE inhibited HA (600 microM) binding to CYP2D6 (IC50 = 4 microM, 95% CI= 1.8-8.9 microM) and CYP1A1 (IC50 = 135 microM: 95% CI = 100-177 microM), but stimulated HA (500 and 1000 microM) binding to CYP3A4 (EC50 = 155 microM, 95% CI = 104-231 microM). DPPE did not affect HA binding to CYP2B6. (4) DPPE inhibited the metabolism of testosterone by CYP3A4. The concentration/effect curve was biphasic: DPPE inhibited metabolism by 30% at the first site (IC50 = 3 microM, 95% CI = 0.5-25.5 microM), and an additional 70% inhibition occurred at the second site (IC50 = 350 microM, 95% CI = 215-570 microM). A similar result was observed with rat liver microsomes. CONCLUSION: DPPE is a substrate for CYP3A4, CYP2D6 and CYP1A1, but not CYP2B6. DPPE inhibits testosterone metabolism by interacting at two sites on CYP3A4, the first correlating with its K(S) value to bind the substrate site and the second, with its EC50 value to enhance HA binding to the heme iron. We postulate that (1) the inhibitory effect of DPPE on CYP3A4 activity is mediated directly at the substrate site and indirectly by its enhancement of the binding of HA to the heme moiety; (2) in tumor cells that express high constitutive levels of CYP3A4, potentiation of chemotherapy cytotoxicity by DPPE results, in part, from inhibition of CYP3A4-mediated metabolism and P-gp-mediated efflux of antineoplastic drugs; (3) in normal cells that express low constitutive levels of the isozyme, cytoprotection by DPPE results, in part, from induction of CYP3A4 and P-gp, resulting in an increase both in metabolism and efflux of antineoplastic drugs. PMID- 10755319 TI - Effect of high-dose cyclosporine on etoposide pharmacodynamics in a trial to reverse P-glycoprotein (MDR1 gene) mediated drug resistance. AB - PURPOSE: The consequences of using cyclosporine (CsA) therapy to modulate P glycoprotein-mediated multidrug resistance include increased myelosuppression, hyperbilirubinemia, and altered disposition of the cytotoxin. The purpose of this study was to analyze further the relationship between the degree of leukopenia, and etoposide pharmacokinetic factors. METHODS: Each patient initially received intravenously-administered etoposide alone (150-200 mg/m2/d x 3). Later it was given in combination with CsA administered at escalating loading doses (range 2-7 mg/kg) as a 2 hour intravenous (IV) infusion followed by a 3 day continuous infusion, at doses ranging from 5 to 21 mg/ kg/day. Serial plasma etoposide concentration-time samples were assayed by high-performance liquid chromatography (HPLC). The area under the curve (AUC) of unbound etoposide was calculated from the total plasma etoposide AUC using a previous published equation [22] where % unbound etoposide = (1.4 x total bilirubin) - (6.8 x serum albumin) + 34.4. The percent decrease in white blood cell (WBC) count and the total or unbound etoposide AUC relationship was fitted to a sigmoid Emax model adapted for paired observations, where: % Decrease in WBC count =E(max) x PDRV(H+Z x delta)/(PDRV50 + Z x beta) + PDRVH + Z x delta In this equation, Z was the variable describing the two treatment groups (0 = no CsA and 1 = CsA). The fitted parameters were PDRV50, the pharmacodynamic response variable (PDRV) producing 50% of the maximal response; parameter beta, which describes the effect of the treatment group on the PDRV50; parameter H (Hill constant), which defines the slope of the response curve and parameter delta, which describes the effect of the treatment group on parameter H. RESULTS: CsA at a median concentration of 1,938 microg/ml resulted in a median increase in the total plasma etoposide AUC by 103% and the calculated unbound plasma etoposide AUC by 104%. This paralleled a 12% greater median percent decrease in WBC count during etoposide + CsA treatment (72% vs. 84%, P = 0.03). The percent decrease in WBC count and total or unbound etoposide AUC relationship was fitted to the sigmoid Emax model. The model using the unbound etoposide AUC described the data adequately (r = 0.790) and was precise, with a mean absolute error of 6.4% (95% confidence interval: -4.9, 7.8). The fitted parameter-estimates suggested that at equivalent unbound etoposide AUC values above 10 microg x h/ml, the sigmoid Emax model predicted a 5% greater WBC count suppression when CsA was added to the treatment regimen. CONCLUSION: These findings suggest that a small degree of the enhanced myelosuppression observed with CsA combined with etoposide might be attributable to inhibition of P glycoprotein in bone marrow precursor cells. However, the majority of the effect observed appears to be due to pharmacokinetic interactions, which result in increases in unbound etoposide. PMID- 10755320 TI - Ara-C affects formation of cancer cell DNA synthesome replication intermediates. AB - PURPOSE: An intact and fully functional multiprotein DNA replication complex (DNA synthesome) from human as well as from murine mammary carcinoma cells was first isolated and characterized in our laboratory. The human cell synthesome supports the in vitro origin-specific simian virus 40 (SV40) DNA replication reaction in the presence of the viral large T-antigen using a semiconservative mechanism and has been shown to contain all the proteins and enzymes required to support DNA synthesis. We are currently using the DNA synthesome as a unique model for analyzing the mechanism of action of anticancer drugs affecting DNA replication. The purpose of this study was to further investigate the mechanism of action of ara-C using the DNA synthesome isolated from the human breast cancer cell line MDA MB-468. METHODS: Synthesome-mediated SV40 DNA replication was performed in the presence of various concentrations of ara-CTP (the active metabolite of ara C) and the types of daughter DNA molecules produced were analyzed lusing neutral and alkaline gel electrophoresis. We also examined the effect of ara-C on intact MDA MB-468 cell DNA synthesis and on cell proliferation. In addition, we studied the effect of ara-CTP on the activity of some of the synthesome target proteins (the DNA polymerases alpha and delta). RESULTS: Full-length daughter DNA molecules were obtained in the presence of low concentrations of ara-CTP while at higher concentrations, there was an inhibition of full-length daughter DNA synthesis. The findings suggest that specifically the initiation phase of DNA synthesis was inhibited by ara-CTP since the production of the short Okazaki fragments was suppressed at all concentrations of the drug above 10 microM. In addition, it was found that the IC50 of ara-CTP for inhibition of synthesome mediated in vitro DNA replication was comparable to that required to inhibit intact cell DNA synthesis. Further experimentation has shown that ara-CTP preferentially inhibits the activity of the synthesome-associated DNA polymerase alpha enzyme while the DNA polymerase delta seems to be resistant to the inhibitory effect of that drug. CONCLUSIONS: Our results indicate that ara-C's action on DNA replication is mediated primarily through DNA polymerase alpha and suggest that this enzyme plays a key role in DNA synthetic initiation events. The results also provide definitive support for the use of the DNA synthesome as a unique and powerful model for analyzing the mechanism of action of anticancer drugs which directly affect DNA replication. PMID- 10755321 TI - Effects of gemcitabine and araC on in vitro DNA synthesis mediated by the human breast cell DNA synthesome. AB - PURPOSE: Gemcitabine (dFdC) and cytarabine (araC) are both analogs of deoxycytidine. Gemcitabine is a relatively new drug that has been shown in both clinical trials and in vitro systems to have more potent antitumor activity than araC. We have previously isolated a fully functional multiprotein DNA replication complex from human cells and termed it the DNA synthesome. Using the DNA synthesome, we have successfully examined the mechanism of action of several anticancer drugs that directly affect DNA synthesis. In this study, we compared the effects of dFdC and araC on in vitro DNA synthesis mediated by the DNA synthesome with the effects of these drugs on intact MCF7 cell DNA synthesis. METHODS: We examined the effects of dFdC and araC on intact MCF7 cell DNA synthesis and clonogenicity. We also performed in vitro SV40 replication assays mediated by the MCF7 cell-derived DNA synthesome in presence of dFdCTP and araCTP. The types of daughter molecules produced in the assay were analyzed by neutral and alkaline agarose gel electrophoresis. Finally, we examined the effects ofdFdCTP and araCTP on the synthesome-associated DNA polymerase alpha and delta activities. RESULTS: Our results showed that dFdC was more potent than araC at inhibiting intact MCF7 cell DNA synthesis and clonogenicity. [3H]Thymidine incorporation was inhibited by 50% at a dFdC concentration of 10 microM, which was about tenfold lower than the concentration of araC required to inhibit intact cell DNA synthesis by the same amount. As examined by clonogenicity assay, dFdC was also significantly more cytotoxic than araC after a 24-h incubation. In vitro SV40 replication assays using the DNA synthesome derived from MCF7 cells demonstrated that the formation of full-length DNA along with replication intermediates were inhibited by dFdCTP in a concentration-dependent manner. Full length DNA was produced in the in vitro DNA replication assay even when the dFdCTP was incubated in the assay at concentrations of up to 1 mM. We observed that in the presence of 10 microM dCTP, 3 microM dFdCTP and 60 microM araCTP were required to inhibit in vitro SV40 DNA synthesis by 50%. Although dFdCTP is more potent than araCTP at inhibiting in vitro SV40 DNA synthesis, there was no significant difference between the inhibitory effect of these two drugs on the activity of the MCF7 synthesome-associated DNA polymerases alpha and delta. It was found that the drug concentrations required to inhibit 50% of the synthesome associated DNA polymerase delta activity were much higher than those required to inhibit 50% of DNA polymerase alpha activity for both dFdCTP and araCTP. CONCLUSION: Taken together, our results demonstrated that: (1) dFdC is a more potent inhibitor of intact cell DNA synthesis and in vitro SV40 DNA replication than araC; (2) the decrease in the synthetic activity of synthesome-mediated in vitro SV40 origin-dependent DNA synthesis by dFdCTP and araCTP correlates with the inhibition of DNA polymerase alpha activity; and (3) the MCF7 cell DNA synthesome can serve as a unique and relevant model to study the mechanism of action of anticancer drugs that directly affect DNA synthesis. PMID- 10755322 TI - Comparison of the protective effects of amifostine and dexrazoxane against the toxicity of doxorubicin in spontaneously hypertensive rats. AB - PURPOSE: To compare the protective effects of amifostine and dexrazoxane against the chronic toxicity induced by doxorubicin in spontaneously hypertensive rats (SHR). METHODS: The animals were pretreated with amifostine (200 mg/kg. i.p.), dexrazoxane (25 mg/kg, i.p.) or saline 30 min before the administration of doxorubicin (1 mg/kg, i.v.), once-weekly for 12 weeks. Control animals received similar amounts of amifostine or saline. The SHR underwent necropsy examination 1 week after the last dosing, and cardiac, renal, and gastrointestinal lesions were graded semiquantitatively. RESULTS: Amifostine and dexrazoxane provided equal degrees of protection against the renal toxicity of doxorubicin. However, dexrazoxane was more cardioprotective than amifostine, and prevented the mortality induced by doxorubicin. This mortality was not decreased by pretreatment with amifostine. The loss of body weight caused by doxorubicin was actually worsened by coadministration of amifostine. CONCLUSIONS: Compared to dexrazoxane, amifostine provided a comparable degree of protection against the nephrotoxicity of doxorubicin, but was less cardioprotective and did not prevent the mortality and loss of body weight produced by doxorubicin. These differences may be related to the fact that amifostine may act as a scavenger of reactive oxygen species, whereas dexrazoxane may prevent their formation. PMID- 10755323 TI - Evidence for a role of chloroethylaziridine in the cytotoxicity of cyclophosphamide. AB - A number of investigators have observed that the use of 4 hydroperoxycyclophosphamide (4-HC) in multiwell plate cytotoxicity assays can be associated with toxicity to cells in wells that contain no drug. Previous reports have implicated diffusion of 4-HC decomposition products, and acrolein in particular, as the active species. PURPOSE: The purpose of this study was to elucidate the species responsible for the airborne cytotoxicity of 4-HC, and to devise ways to minimize such effects in chemosensitivity assays. METHODS: To this end, analogues of 4-HC were synthesized to identify the contributions of individual cyclophosphamide metabolites to cytotoxicity. The analogues were then tested for activity against three human breast tumor cell lines (including a line resistant to 4-HC), and one non-small-cell lung carcinoma line. Cytotoxicity was evaluated by assays that quantitate cellular metabolism and nucleic acid content. RESULTS: Didechloro-4-hydroperoxycyclophosphamide, a compound that generates acrolein and a nontoxic analogue of phosphoramide mustard, gave no cross-well toxicity. In contrast, a significant neighboring well effect was observed with phenylketophosphamide, a compound that generates phosphoramide mustard but not acrolein. Addition of authentic chloroethylaziridine reproduced the airborne toxicity patterns generated by 4-HC and phenylketophosphamide. Increasing the buffering capacity of the growth medium and sealing the microtiter plates prevented airborne cytotoxicity. CONCLUSION: Since it is unlikely that phosphoramide mustard is volatile, these findings implicate chloroethylaziridine rather than acrolein as the volatile metabolite of 4-HC that is responsible for airborne cytotoxicity. The fact that chloroethylaziridine is generated in amounts sufficient to volatilize, diffuse across wells and cause cytotoxicity indicates that it is an important component in the overall cytotoxicity of 4-HC in vitro. Furthermore, these findings suggest that chloroethylaziridine may also contribute to the toxicity of cyclophosphamide in vivo. PMID- 10755325 TI - Photodynamic therapy in gastrointestinal cancer: a realistic option? AB - Photodynamic therapy is now a useful and practical option of the local treatment of gastrointestinal cancers. There is increasing screening and surveillance of patients at risk of oesophageal and gastric cancers. The early detection of disease is often unhelpful if an elderly or frail patient needs to be subjected to radical resectional surgery. Photodynamic therapy can eradicate and cure early mucosal disease following a single endoscopic treatment. If the disease is more advanced good local control and palliation is often possible. Overall, palliation can often be achieved using simpler methods which are highly effective and not associated with the problems of prolonged photosensitisation. It is rapidly becoming clear that the ideal indication is for the treatment of dysplastic lesions in the oesophagus associated with columnar-lined oesophagus (Barrett's oesophagus). In these circumstances a heterogeneous field change often with multifocal dysplasia or cancer can be widely eradicated. Similar areas of squamous dysplasia in the upper oesophagus can be treated. At present a major complication of stricture formation is associated with the use of some photosensitisers. The treatment of cancer at the ampulla of Vater and choriocarcinoma is also proving very effective. The treatment can be performed at endoscopy and is well tolerated and safe. PMID- 10755324 TI - Schedule-dependent activity of irinotecan plus BCNU against malignant glioma xenografts. AB - PURPOSE: To further evaluate the activity of irinotecan (CPT-11) plus 1,3-bis (chloroethyl)-1-nitrosourea (BCNU) in the treatment of central nervous system tumor-derived xenografts in athymic nude mice. METHODS: We report studies evaluating the schedule-dependence of this regimen in the treatment of the malignant glioma xenograft D-54 MG. RESULTS: The combination of BCNU and CPT-11 showed the highest enhancement index (2.0-3.3) when BCNU was given on day 1 and CPT-11 was given on days 1-5 and 8-12. Delay of CPT-11 administration to day 3 or day 5 substantially decreased activity with enhancement indices of 1.6-1.8 and 0.6-1.0, respectively. Delay of BCNU administration to day 8 also reduced the CPT 11 activity with enhancement indices of 1.2-1.4. CONCLUSIONS: These results suggest that the presence of a BCNU-induced adduct or possibly crosslink prior to administration of CPT-11 is critical for enhanced activity. Although the mechanism of this enhancement is not currently known, a phase I trial of CPT-11 plus BCNU for adults with recurrent malignant glioma based on these results is in progress. PMID- 10755326 TI - Optimising management of patients with advanced heart failure: the importance of preventing progression. AB - Heart failure is a highly complex, progressive and deadly disease. When incorrectly treated, it results in irreversible structural damage to the myocardium and resists any conventional treatment. This stage has been arbitrarily termed refractory heart failure. However, with timely and sufficiently applied neurohumoral antagonists, progression can be prevented, or at least delayed. In contrast, as soon as heart failure has become moderate or severe due to advanced left ventricular dysfunction, polypharmacy is the rule. Physicians should make every effort to maintain or reconsider optimal neurohumoral antagonist therapy in such patients, even if symptomatic improvement from these agents may be slow. Proper use of diuretics is essential not only for symptom relief but also to achieve full benefit from angiotensin converting enzyme inhibitors and beta-blockers. Digitalis may be particularly indicated in severe heart failure, irrespective of rhythm. Adjunctive regimens can be helpful in specific patients, but evidence of their salutary effects to prolong life is lacking. In the decompensated state, tailoring intravenous therapy to haemodynamic goals followed by (re-)institution of optimal oral therapy is an option. Only if these strategies fail is heart transplantation justified. While waiting for a donor, patients have been bridged with various intravenous agents, most often inotropes, but symptom relief is associated with risk of increased mortality due to these drugs. New hope emerges from drugs interfering with endothelin and the cytokines, and from research into increasing contractility with calcium sensitising agents. Even though these developments follow established routes, they may enable a more effective approach to prevent worsening heart failure in every single patient. PMID- 10755327 TI - Recognition and treatment of dysthymia in elderly patients. AB - This review focuses on recent literature concerning dysthymia in the elderly population. Epidemiological data and clinical picture, diagnostic and therapeutic issues are evaluated and discussed. Although depressive syndromes are common in older patients, prevalence rates of dysthymia in the elderly are lower than in younger adults. This finding may be the consequence of the diagnostic criteria provided by the Diagnostic and Statistical Manual of Mental Disorders (DSM) which are not specific for older adults. Other factors that complicate making diagnoses of dysthymia in older individuals are comorbid general conditions, cognitive deterioration and disorders, and frequent adverse life events (e.g. bereavement). The effects of these factors should be better defined to clarify whether elderly dysthymia is underestimated and if modified diagnostic criteria should be provided. A few researchers have identified a series of clinical features that are clearly different in the elderly and in young adult patients with dysthymia. These features are particularly related to the late onset and to the peculiar comorbidity of this disorder and suggest that dysthymia is a different disorder in the elderly. Drug treatment of depressive conditions in the elderly is currently based on new antidepressants [selective serotonin (5-hydroxytryptamine; 5-HT) reuptake inhibitors, norepinephrine (noradrenaline) reuptake inhibitors, benzamides]. These agents have an improved adverse effect profile compared with some of the older agents. Moreover, very few systematic studies have been performed using these drugs in samples of older patients with dysthymia and available data do not allow conclusions on drug choice and dosage. Besides, no specific data are available concerning the psychotherapy of dysthymia in this age group. All these topics need to be further investigated in studies comparing the elderly with control groups of younger patients with dysthymia. PMID- 10755330 TI - Extended-release oxybutynin. AB - Extended-release oxybutynin (Ditropan XL) uses an osmotic system (OROS) to deliver a controlled amount of oxybutynin chloride into the gastrointestinal tract over a 24-hour period when taken once daily. Oxybutynin binds to M3 muscarinic receptors on the detrusor muscle of the bladder, preventing acetylcholinergic activation and relaxing the muscle. Mean peak plasma concentrations are lower with extended-release oxybutynin 15mg once daily than with conventional immediate-release oxybutynin 5mg taken 3 times daily. Relative bioavailabilities of parent drug and metabolite N-desethoxybutynin are 153 and 69%, respectively, for extended-release oxybutynin when compared with immediate release oxybutynin. In short (< or =6 weeks) randomised, double-blind clinical trials of patients with detrusor instability, extended-release oxybutynin 5 to 30mg once daily significantly reduced the mean weekly number of urge incontinence episodes by 84 to 90%. Extended-release oxybutynin had similar efficacy to immediate-release oxybutynin. Adverse events reported by patients taking extended release oxybutynin were dose-related anticholinergic effects, most frequently dry mouth, somnolence, constipation, blurred vision and dizziness. A large noncomparative study demonstrated that approximately two thirds of the patients prescribed extended-release oxybutynin for detrusor instability were still taking the medication 6 months later. PMID- 10755329 TI - Verteporfin. AB - Verteporfin, a benzoporphyrin derivative monoacid ring A, is a photosensitising drug for photodynamic therapy (PDT) activated by low-intensity, nonheat generating light of 689nm wavelength. Activation generates cytotoxic oxygen free radicals. The specificity and uptake of verteporfin for target cells with a high expression of low density lipoprotein (LDL) receptors, such as tumour and neovascular endothelial cells, is enhanced by the use of a liposomal formulation and its rapid uptake by plasma LDL. Verteporfin therapy (at light doses < 150 J/cm) selectively damages neovascular endothelial cells leading to thrombus formation and specific occlusion of choroidal neovascular vessels in subfoveal lesions in patients with age-related macular degeneration (AMD). Repeated applications of verteporfin therapy 6 mg/m2 improved or maintained visual acuity in the majority of patients with some classic subfoveal choroidal neovascularisation (CNV) secondary to AMD at 1 year's follow-up in 2 large multicentre, placebo-controlled, double-blind trials. Furthermore. in a subgroup of these patients with predominantly classic CNV secondary to AMD, there was a significantly more marked visual acuity (VA) benefit with 67.3% of verteporfin treated eyes experiencing less than a 15-letter loss of VA versus 39.3% with placebo treatment. Multiple applications of verteporfin therapy were well tolerated in patients with subfoveal CNV secondary to AMD. The most common adverse events were visual disturbances, injection site reactions, photosensitivity reactions and infusion-related back pain. PMID- 10755328 TI - Cholinesterase inhibitors for the treatment of Alzheimer's disease in the elderly. AB - The treatment of Alzheimer's disease is of increasing importance as the population ages and the number of people with the disease increases. The aetiology of Alzheimer's disease is complex and therefore treatment strategies rely on generalised pathological findings. Cholinesterase inhibitors enhance a generalised deficit of central nervous system acetylcholine and are the first class of agents specifically approved for the treatment of Alzheimer's disease. The clinical efficacy of the different cholinesterase inhibitors is similar; however, differences in pharmacodynamic and pharmacokinetic parameters can influence tolerability and safety in the elderly population. Concomitant disease states, significant drug interactions and the altered kinetics and dynamics seen in elderly patients can also affect treatment outcome. Although cholinesterase inhibitors are not 'curative' for Alzheimer's disease, clinical evidence indicates that these drugs can significantly delay the progress of cognitive impairment. Consequently, they represent a useful treatment for the symptoms of Alzheimer's disease in the elderly. PMID- 10755331 TI - Management of vascular injuries using endovascular techniques. AB - Vascular surgery is in a transitional phase. Standard open repair is being replaced by endovascular and minimally invasive techniques. The techniques developed for elective cases are also applicable for injured patients. The introduction of endovascular treatment for emergencies has consequences for both institutional and regional organisation. Success can be achieved only by the prepared team trained with wide elective experience. Endovascular repair of vascular injuries has many advantages, such as remote approach and limited exposure with less surgical stress to the patient. Because the long term effects are only partially known a word of caution is justified. Endovascular repair of vascular trauma is still a developing form of treatment. PMID- 10755332 TI - Clinical recurrence of papillary thyroid cancer in the remnant lobe. AB - OBJECTIVE: To find out which factors predict recurrence of cancer in the remnant after unilateral thyroid lobectomy for patients with papillary cancer. DESIGN: Retrospective study. SETTING: Teaching hospital, Japan. SUBJECTS: One hundred thirty-three patients with a clinically solitary papillary thyroid cancer who had unilateral lobectomy for the primary disease between 1966 and 1990 and were followed up for more than 60 months. Twelve patients had developed recurrences in the remnant gland by the time of the second operation and 121 patients had not as judged by a second operation, an ultrasound examination, or by palpation. RESULT: The primary tumour size in those who developed recurrences was significantly larger than in those who did not (p < 0.0001), and clinical signs of regional lymph node involvement or distant metastases were also significantly more common (p = 0.006). No patient died of their cancer. CONCLUSION: Size of the primary tumour is an important prognostic factor for recurrence of solitary papillary thyroid cancer in the remnant after unilateral lobectomy. Such recurrences are unlikely to be lethal. PMID- 10755333 TI - Relation between functional dysphagia and vocal cord palsy after transhiatal oesophagectomy. AB - OBJECTIVE: To assess the incidence, natural course, and possible pathogenesis of dysphagia that is not caused by anastomotic stricture, after transhiatal oesophagectomy and gastric tube reconstruction. DESIGN: Prospective study. SETTING: District teaching hospital, The Netherlands. SUBJECTS: 22 patients who had transhiatal oesophagectomy and gastric tube reconstruction for cancer. MAIN OUTCOME MEASURES: Incidence of dysphagia that is not caused by anastomotic stricture one week after operation, and presence of this functional dysphagia and correlation with vocal cord palsy at 4, 8, 12, and 16 weeks postoperatively. RESULTS: The incidence of functional dysphagia was 7 out of 22 (32%); it was self limiting in 5 out of 7 (71%) of the cases and associated with the incidence of vocal cord palsy (p = 0.0006). CONCLUSION: Functional dysphagia after transhiatal oesophagectomy occurs frequently, but is self-limiting in most patients. Injury to branches of the recurrent laryngeal nerve is a likely cause. PMID- 10755334 TI - Laparoscopic hernia repair in patients with bilateral groin hernias. AB - OBJECTIVE: To compare outcome of unilateral and bilateral laparoscopic hernia repair. DESIGN: Prospective consecutive trial. SETTING: University hospital, Sweden. SUBJECTS: 380 patients who had unilateral hernias repaired laparoscopically and 64 patients who had bilateral hernias repaired. The median (range) age in the two groups was 56 (21-86) and 61 (30-85) years, respectively and the median (range) follow-up was 42 (24-58) months. MAIN OUTCOME MEASURES: Operating time, hospital stay, complications, and time to recovery. RESULTS: The median (range) operating time was 70 (25-240) minutes in the unilateral and in the bilateral group 113 (55-330) minutes. The complication rate, recurrence rate, and time to full recovery did not differ between the groups. CONCLUSION: The laparoscopic approach seems to be a good option for patients with bilateral inguinal hernias. PMID- 10755335 TI - Comparison of laparoscopically assisted and conventional ileocolic resection for Crohn's disease. AB - OBJECTIVE: To compare safety, outcome, and feasibility of laparoscopic assisted and conventional laparotomy for ileocolic resection in Crohn's disease. DESIGN: Retrospective study. SETTING: Private clinic, USA. SUBJECTS: 74 patients who had ileocolic resection and anastomosis for Crohn's disease between August 1991 and July 1996, 48 through conventional laparotomy and 26 in whom it was laparoscopically assisted. MAIN OUTCOME MEASURES: Age, operating time, duration of hospital stay, early and late morbidity, and patients' subjective assessment. RESULTS: The mean age was 42 (+/- 17) in the conventional group and 40 (+/- 15) in the laparoscopically assisted group. The mean operating time was significantly shorter in the conventional group, 90.5 +/- 3.7 minutes, compared with 150 +/- 1.2 minutes in the laparoscopic-assisted group (p < 0.0001), but they stayed in hospital significantly longer, 9.6 +/- 0.6 days in the conventional group, compared with 7 +/- 0.8 days in the laparoscopic-assisted group (p < 0.0001). There were no differences between the groups in the incidence of early complications or the cost of admission, but at a mean follow up of 30 months (range 2-59) significantly more patients in the conventional group had developed symptomatic bowel obstruction (15/48 compared with 2/26, p = 0.02). 31 patients in the conventional group (65%) and 16 in the laparoscopically assisted group (62%) returned their subjective assessments. There were no differences between the groups in the number with changed bowel habits, use of drugs for bowel movement, or restricted diet, but patients in the laparoscopically assisted group returned to work more quickly (3.7 +/- 1.2 weeks) compared with 8.2 +/- 1.1 weeks in the conventional group, had better cosmetic results (14/16 compared with 13/31, p = 0.004), and were more likely to have improved social and sexual lives (8/16 compared with 5/31, p = 0.02). CONCLUSION: Laparoscopically assisted ileocolic resection for Crohn' s disease is safe and has less morbidity than conventional laparotomy. PMID- 10755336 TI - Gracilis transposition in complicated perianal fistula and unhealed perineal wounds in Crohn's disease. AB - OBJECTIVE: To assess the efficacy of transposition of gracilis muscle in the treatment of chronic recurrent fistulas and unhealed perineal wounds after proctectomy in patients with Crohn's disease. DESIGN: Retrospective study. SETTING: Academic clinic, United States. SUBJECTS: 7 patients with Crohn's disease: 3 had unhealed perineal wounds and persistent sinuses; 2 had had several attempts to repair rectovaginal fistulas; 1 had a rectourethral fistula; and 1 a pouch vaginal fistula. INTERVENTION: Transposition of the gracilis muscle. MAIN OUTCOME MEASURE: Healing. RESULTS: Mean follow up was 18 months (range 3-30). All patients operated on for unhealed perineal wounds had healed completely within 3 6 months. The patients with a rectovaginal fistula and a rectourethral fistula had both healed by 1 month postoperatively. Two fistulas recurred, and the small pouch-vaginal fistula remained but was asymptomatic. CONCLUSIONS: Transposition of the gracilis is a viable option for the treatment of persistent sinus and unhealed perineal wound after proctectomy for Crohn's disease. It could also be an option before proctectomy for patients with other types of Crohn's-related or complicated fistulas for whom other treatments have failed. A larger series will be required before a definite conclusion can be drawn. PMID- 10755337 TI - Lateral internal sphincterotomy together with haemorrhoidectomy for treatment of haemorrhoids: a randomised prospective study. AB - OBJECTIVE: To investigate anorectal manometric findings in patients with haemorrhoids and to evaluate the clinical effects and physiological consequences of adding a lateral internal sphincterotomy (LIS) to haemorrhoidectomy. DESIGN: Randomised prospective study. SETTING: Teaching hospital, Naples. PATIENTS: 48 consecutive patients with prolapsed piles who had anorectal manometry; 10 healthy volunteers served as controls. INTERVENTIONS: Resting and squeeze pressures, sphincter length and rectoanal inhibitory reflex were recorded. 6 patients were excluded because anal pressures were not raised, so 42 patients were randomised. 22 patients had haemorrhoidectomy plus LIS; and 20 had haemorrhoidectomy alone. MAIN OUTCOME MEASURES: Morbidity, continence, and anorectal manometry. RESULTS: Sphincter anomalies were found in 87.5% (n = 42) of patients. Haemorrhoidectomy alone did not affect anal pressures, which returned to the normal ranges after sphincterotomy. Those who had LIS did better postoperatively than those who had did not. 4 patients who did not have a sphincterotomy developed anal strictures. No patient who had LIS developed incontinence of faeces. CONCLUSIONS: High anal pressures are common in patients with haemorrhoids suggesting that they may have a pathogenetic role; anorectal manometry is useful in the investigation of anal pressure patterns; and when indicated, lateral sphincterotomy avoids pain, urinary retention, and stenosis, and is safe. PMID- 10755338 TI - Effect of hydrocolloid dressings on healing by second intention after excision of pilonidal sinus. AB - OBJECTIVE: To assess the efficacy of hydrocolloid dressings in wound management after excision of pilonidal sinus. DESIGN: Prospective randomised trial. SETTING: District hospital, Spain. PATIENTS: 38 patients with chronic pilonidal sinus. INTERVENTIONS: Open excision with healing by second intention. Divided into three groups: conventional gauze dressing (control, n = 15), Comfeel (n = 12) and Varihesive (n = 11). MAIN OUTCOME MEASURES: Median healing time, infection rate, intolerance, pain, comfort, ease of management, leakage, and recurrence. RESULTS: Median healing time was 68 days (range 33-168) in the control group, compared with 65 days (range 40-137) in the two hydrocolloid groups combined. There were no differences between the hydrocolloid groups. There were no recurrences during the 74 months of follow-up. A third of the postoperative cultures in the control group grew pathogens compared with 1/23 of the patients treated with hydrocolloid dressings (p = 0.03). This was of no clinical relevance. 14/23 in the hydrocolloid group developed leaks. Pain was significantly less in the first four postoperative weeks among the patients in the hydrocolloid group than in the control group (p < 0.05). CONCLUSIONS: Hydrocolloid dressings lessen pain and increase comfort for patients after excision of pilonidal sinus, though time to healing is no shorten than when a conventional gauze dressing is used. PMID- 10755339 TI - Prognostic variables for patients with stage III malignant melanoma. AB - OBJECTIVE: To evaluate the prognostic factors for patients with stage III malignant melanoma and to identify patients at high risk of developing recurrent disease who may benefit from adjuvant therapy. DESIGN: Retrospective study. SETTING: Specialist hospital, USA. SUBJECTS: 130 patients with stage III malignant melanoma (according to the TNM classification), treated at the Roswell Park Cancer Institute between 1970 and 1992. MAIN OUTCOME MEASURES: Survival and prognostic factors on multivariate analysis. RESULTS: Four factors were independent prognostic indicators for patients with stage III malignant melanoma: age >51 years (p = 0.008), >3 involved lymph nodes, (p = 0.03), the site of the primary tumour on head or trunk, (p = 0.007), and the presence of palpable lymph nodes (p = 0.004). CONCLUSION: These prognostic factors help us to stratify patients into low and high-risk groups. High-risk patients may benefit from more aggressive adjuvant therapy in future trials of treatment of melanoma. PMID- 10755340 TI - The long gamma nail in the treatment of 329 subtrochanteric fractures with major extension into the femoral shaft. AB - OBJECTIVE: To study the outcome of treating extensive proximal femoral fractures with the long Gamma nail. DESIGN: Retrospective study, with a minimum follow-up of a year. SETTING: 24 hospitals throughout The Netherlands. SUBJECTS: 329 patients with primary proximal femoral fractures. MAIN OUTCOME MEASURES: Functional results and complications. RESULTS: 19 patients died in hospital and 6 were lost to follow-up. Of the remaining 304 who were fully documented, 241 (80%) became fully mobile, 59 (19%) moderately mobile, and 4 remained bedridden. 264 patients had no pain, 38 had moderate pain and used oral painkillers, and 2 had severe pain that was difficult to treat. Major technical complications occurred in 17 patients: alignment and locking gave rise to most problems, 4 patients developed deep infections, and 7 patients needed additional measures before the fracture was solid. CONCLUSIONS: The long Gamma nail in the treatment of extensive proximal femoral fractures gave good functional results with acceptable rates of complications and union problems. PMID- 10755341 TI - Treatment of impending and actual pathological femoral fractures with the long Gamma nail in The Netherlands. AB - OBJECTIVE: To compare the results of stabilising impending and actual pathological femoral fractures using the long Gamma nail with the published results of other methods. DESIGN: Retrospective study. SETTING: 20 hospitals throughout The Netherlands. PATIENTS: 101 patients with metastases in 110 femurs, which were stabilised with a long Gamma nail. MAIN OUTCOME MEASURES: Functional results and complications. RESULTS: Minimum follow-up was 1.5 years or until death, Mean survival was 12 months (range 0-82). 14 patients died in hospital; 5 had technical complications and fat embolism was suspected in 3 patients. 92% of the patients became mobile and pain was absent or acceptable in 93%. CONCLUSION: The use of the long Gamma nail produces better functional results, fewer technical complications, and an incidence of general complications no different from those reported for other methods. PMID- 10755342 TI - Continuous or intermittent vascular clamping during hemihepatectomy in pigs: hyaluronic acid kinetics in the assessment of early microvascular liver damage. AB - OBJECTIVE: To assess the uptake of hyaluronic acid (HA) as a marker of microvascular damage in a model of hemihepatectomy in pigs having continuous or intermittent vascular inflow occlusion. DESIGN: Prospective, animal study. SETTING: Laboratory for experimental surgery, University hospital, The Netherlands. INTERVENTIONS: Total liver ischaemia was achieved during 90 minutes by continuous (n = 5) or intermittent (n = 5) occlusion of the portal vein and hepatic artery followed by 120 minutes of reperfusion. In a second series of pigs (n = 8) a left hemihepatectomy was added to the protocol. MAIN OUTCOME MEASURES: Uptake of exogenous HA was assessed before ischaemia and after 120 minutes of reperfusion, together with the galactose elimination capacity. Plasma activities of aspartate aminotransferase (AST), alanine amino transferase, and lactate dehydrogenase were measured and specimens of liver were obtained for histopathological examination. RESULTS: HA uptake was slightly reduced after reperfusion in unresected livers compared with uptake before ischaemia. After hemihepatectomy HA uptake after reperfusion was significantly reduced after both continuous and intermittent occlusion, but more HA was taken up after continuous occlusion (p = 0.02). Release of AST after reperfusion was increased only after hemihepatectomy. CONCLUSIONS: Microvascular damage, as assessed by HA uptake capacity, significantly contributed to normothermic ischaemia and reperfusion injury in porcine liver. Vascular inflow occlusion during 90 minutes in combination with hemihepatectomy resulted in less liver damage when vascular occlusion was continuous rather than intermittent. PMID- 10755343 TI - Rupture of mesenteric cyst after blunt abdominal trauma. PMID- 10755344 TI - Abdominal wall ruptured by blunt trauma in a child. PMID- 10755345 TI - Tuberculosis in an intramammary lymph node. PMID- 10755346 TI - Role of simple V-Y advancement flap in the treatment of complicated pilonidal sinus. PMID- 10755347 TI - Urologic injuries in the Gulf War. AB - This study aims to compare the urologic injuries treated at Kuwait during the Gulf War with those recently reported by the United States urologic surgeons. Gunshot caused the majority of the injuries (58%) on our side of the battle compared with fragmentation injuries (83%) on the other side. We had statistically significant higher renal and ureteric injuries (14/35 compared with 5/30; p<0.04, Chi-square). This difference can be explained by the use of the flak jackets on the other side or the difference of the type of weapons used. Our management had a similar preservative approach. Only 4/12 of renal injuries (33%) had nephrectomy. Two ureteric injuries were missed by general surgeons which were successfully managed by the urologists. This study supports the presence of urologists within military surgical teams. PMID- 10755348 TI - Relationship between clear cell/compact cell ratio and computed tomographic attenuation number in adrenocortical adenoma. AB - Clear cell/compact cell ratio in histologic sections from 21 resected adrenocortical adenomas was assessed. Seven patients had Cushing's syndrome another 7 had primary aldosteronism caused by adrenal adenomas and 7 had non hyperfunctioning adenomas. The results were correlated with the corresponding unenhanced CT attenuation numbers. There was a negative correlation between clear cell/compact cell ratios and unenhanced CT attenuation numbers. However, CT attenuation numbers did not show significant differences between cortisol producing, aldosterone-producing and nonfunctioning adenomas. These facts suggest that clear cell/compact cell ratio in adrenocortical adenomas accounts for their attenuation number on unenhanced CT scans. However, it is difficult with the use of unenhanced CT attenuation numbers to differentiate cortisol-producing adenomas from aldosterone-producing and nonhyperfunctioning adenomas. PMID- 10755349 TI - Renal stone formation and development. AB - A concise account of formation mechanisms of attached (papillary) and unattached renal stones is presented. Urinary conditions prevailing at least during the stone forming period are indicated. Ten main categories of renal stones, covering over 95% of all conceivable calculi, are distinguished based on their composition and structure. Aetiologic factors leading to stone formation of every category are specified and general outlines of recommended treatment procedures indicated. PMID- 10755350 TI - Collecting (Bellini) duct carcinoma of the kidney--clinical, radiologic and immunohistochemical findings. AB - We present clinical and radiological findings in a case of collecting (Bellini) duct carcinoma (CDC). This is a rare and aggressive kidney cancer originating from the distal renal tubule. The patient underwent radical nephrectomy and the pathological report showed trabeculopapillary, partially solid adenocarcinoma infiltrating the renal capsule and sinus. Immunohistochemical, as well as mucinocarminic and PAS staining studies are necessary for diagnosis of CDC. Surprisingly, one year follow-up studies were negative for recurrent disease. Chromosomal findings are usually different from most often diagnosed clear cell renal cell cancers. Findings from the literature are briefly discussed. PMID- 10755351 TI - Alteration of epidermal growth factor receptor expression following ischaemia of renal tissue. AB - This study was aimed to investigate Epidermal Growth Factor Receptor (EGF-R) expression after ischaemic injury in renal tissue and the effects of calcium channel blockers in the prevention of damage due to ischaemic insult. Simple nephrectomy was performed in a group of Sprague-Dawley rats, and kidneys were grouped according to cold ischaemia time (1, 6, 12, 24 and 48 hours, respectively) and to the type of calcium channel blockers (diltiazem and verapamil) used. EGF-R expression status was investigated in each group by immunohistochemistry on paraffin sections. Overall expression of EGF-receptor was detected in 8 (22.8%) kidneys. In terms of localization of EGF-receptor expression cortical tubular staining was detected in 8 (100%) kidneys, medullar tubular staining in (62.5%) kidneys and glomerular mesangial staining in 5 (62.5%) kidneys. There was no difference between various ischaemia times and different calcium channel blockers used. It has been concluded that hypoxia and cold ischaemia causes widespread down-regulation of EGF-receptor expression in renal tissue regardless of treatment with calcium channel blockers. PMID- 10755352 TI - Report on 59 patients with renal amyloidosis. AB - We studied a group of 59 patients with renal amyloidosis. Mean age (45 male, 14 female) was 33.05+/-13.04 years. All of the cases had secondary amyloidosis. The causes of secondary amyloidosis were as follows: familial Mediterranean fever (FMF) 18 (30.5%), pulmonary tuberculosis 12 (20.33%), chronic oseomyelitis 8 (13.55%), bronchiectasia 9 (15.25%), rheumatic diseases 4 (6.4%), Castleman's disease 1 (1.6%), unknown aetiology 7 (11.86%). Hypertension was detected in 15.3% of the cases. In patients with less than 20 ml/min creatinine clearance (Ccr) hypertension was found in 20%. Hypotension was detected in 6 patients and all of these cases had severe hypoalbuminaemia (<2.1 g/dl). Nephrotic range proteinuria (>3.5 g/day) was found in 75% of cases. Daily proteinuria was correlated with serum levels of albumin, total lipid and cholesterol, haematocrit and duration of disease. The mean Ccr was 51.03+/-40.60 ml/min. Twenty-nine per cent of patients had Ccr less than 20 ml/min. Renal, subcutaneous fat and rectal biopsies demonstrated amyloid in 100%, 20% and 57.6%, respectively, of patients tested. Patients with secondary amyloidosis were treated with colchicine in addition to the therapy of primary disease (in 6 patients). Nine patients died, and end-stage renal disease developed in 12 patients during four years of follow up. Proteinuria disappeared or decreased in patients with secondary amyloidosis except secondary to collagen tissue disease, without advanced renal failure. Colchicine did not affect amyloid deposition in 2 patients with normal renal function and negative proteinuria, who were rebiopsied. It can be questioned that "Colchicine may have effect(s) for decrement on proteinuria". At least colchicine can be of use in secondary amyloidosis. PMID- 10755353 TI - Primary obstructive megaureter in adults: need for an aggressive management strategy. AB - PURPOSE: Primary obstructive megaureter (POM) is an uncommon disease in adults. We reviewed our experience with this disease to determine the clinical profile, management and prognosis of this disease in adults. METHODS: We studied 37 adults with POM who presented from January 1989 to December 1998. Their clinical presentation, renal function, radiologic data, complications, treatment as well as the results and follow-up were studied. RESULTS: The patients' age ranged from 13 to 52 years. Male:female ratio was 27:10. Seven patients had bilateral disease. All patients were symptomatic excepting 2. Complications at presentation were loin pain (26 cases), urinary infection (15 cases), calculus disease (17 cases), azotaemia (5 cases), and obstructive jaundice (1 case). Associated congenital anomalies included contralateral renal agenesis (2 cases), posterior urethral valve (1 case) and exstrophy of bladder (1 case). Thirty-four patients required surgical intervention. Of these, 26 patients underwent ureteroneocystostomy (UNC) with ureteral tailoring in 18 patients; 4 patients were treated endoscopically by ureteric meatotomy and stenting, 2 patients with nonfunctioning kidney by nephroureterectomy, 2 patients in advanced renal failure by percutaneous nephrostomy alone. In 4 out of 5 patients uraemia did not improve despite adequate drainage. CONCLUSION: The majority of adults with POM are symptomatic, have complications and require surgical correction. Complications of stone formation (46%) and renal failure (13.5%) are unusually common in adults. Once renal failure is advanced, intervention appears futile, therefore, it is imperative to treat these patients as soon as possible. Surgical correction by ureteric re-implantation is effective and has low morbidity. PMID- 10755354 TI - Vesicostomy in adult meningomyelocele patients. Reappraisal of an old technique. AB - Intermittent catheterization is the standard treatment of neurogenic bladder dysfunction. In adult meningomyelocele patients, self-catheterization is not always possible or is not accepted. In these patients, urinary diversion is frequently hampered by anatomical abnormalities, preventing the use of bowel. Vesicostomy as a safe procedure in selected adult patients was employed at our department. PMID- 10755355 TI - Our changing strategies on bladder neck suspension operations. AB - With currently changing strategies, we retrospectively reviewed our operative results in female stress incontinence to find a suitable solution to correct both urethral hypermobility and intrinsic sphincter dysfunction with reasonable success rates. No Incision Bladder Neck Suspension (NIBNS), Modified Four Corner Bladder Neck Suspension (MFCBNS) and in situ vaginal wall sling operation with bone fixation were performed on 24, 26, 25 patients from 1992 to 1994, 1994 to 1996, 1996 to 1998, respectively, with pre- and postoperative evaluations and success rate determinations. Cure rates for NIBNS operations were 90.47, 72.2 and 50 per cent after 6 months, 2 years and 5 years, respectively. For MFCBNS operations cure rates were 96 and 75 per cent after 6 months and 2 years; for in situ vaginal wall sling by bone fixation the rates were 100 per cent in primary cases and 80 per cent in secondary cases after one year of follow-up. After two years the cure rate was 88.8 per cent in primary cases. By fixation and elevation of the urethrovesical junction and by external compression from under the urethra, in situ vaginal wall sling by bone fixation offers a better solution to urethral hypermobility and intrinsic sphincter dysfunction for urinary stress incontinence. PMID- 10755356 TI - Transitional cell carcinoma of the bladder in patients under 40 years of age. AB - A review of our records between 1993 and 1998 identified 25 patients with transitional cell carcinoma of the bladder who were less than 40 years old, 22 males and 3 females. The youngest patient was 19 years old. At the time of diagnosis 16 patients had superficial (Ta/T1) and 9 had invasive disease. Twenty four patients were followed up for a period of 3-71 months (mean: 19.2 months). The recurrence rate for patients with superficial disease was 12.5%, and the progression rate for patients with invasive disease was 77.7%. We concluded that the patients under 30 years of age presented with lower grade and lower stage disease than those over 30. Transitional cell carcinoma of the bladder in young adults has a natural history similar to that seen in older patients. Accordingly, all patients, regardless of age, should be treated as aggressively as necessary on the basis of the stage and the grade of the tumour. PMID- 10755357 TI - Investigations of urinary lead concentration in patients with urinary bladder carcinoma. AB - Authors investigated the lead content of urine in 24 patients with urinary bladder carcinoma using atomic spectrometry. It was found that in 40% of these patients it was increased. These results might indicate an involvement of lead in carcinogenesis. PMID- 10755358 TI - Metabolic changes and urodynamic findings after continent urinary diversion. AB - Methods of creating continent urinary diversions were developed in the mid-1980s (neobladder, pouch) providing patients with continence and anatomically appropriate voluntary urine discharge. In a follow-up investigation on 18 patients, the question to be clarified was whether continent urinary diversion meets the demands of an ideal bladder substitute. Follow-up examination showed normal clinical test values almost without exception. Only five out of 18 patients had discrete acidosis. Neither malabsorption syndrome nor any disorder of vitamin D3 metabolism was found. Ultrasonography and X-ray diagnostics showed normal conditions, without stones and reflux. Urodynamic investigations revealed that bladder emptying was almost free of residual urine, and continence was largely undisturbed. In accordance with these data, there is almost perfect patient acceptance. At present, there are still no sufficient data on the risk of tumour induction, since the latency period required (about 20 years) in most cases has not yet been reached. Since there are currently numerous publications on "urinary diversion carcinoma", consistent follow-up is necessary comprising not only the metabolic, but also the oncological risks of urinary diversion via intestinal segments. PMID- 10755359 TI - The clinical value of the ratio of free prostate specific antigen to total prostate specific antigen. AB - OBJECTIVE: To examine the usefulness of the ratio of free prostate specific antigen (FPSA) to total prostate specific antigen (TPSA) in men with serum TPSA concentration of 4 to 10 ng/mL by using the cut off value of 0.15 for avoiding unnecessary biopsies. PATIENTS AND METHODS: Two hundred thirty-six men aged between 52 and 91 with symptoms of prostatism were evaluated with digital rectal examination (DRE), FPSA and TPSA measurements. Patients with TPSA values under 4 ng/mL were biopsied if they had positive DRE and/or a FPSA/TPSA ratio lower than 0.15. All patients with TPSA values higher than 4 ng/mL were also biopsied. The predictive value and sensitivity of FPSA/TPSA ratio and TPSA alone were calculated. RESULTS: Eleven patients out of 170 with a TPSA value lower than 4 ng/mL were biopsied. Fifty-five patients had a value between 4.1 and 10 ng/mL. We performed transrectal ultrasound (TRUS) and prostate biopsy in these men except one patient. Biopsy proven prostate cancer was detected only in 12 patients. In this group of patients the predictive value of TPSA was 21%, but the predictive value of FPSA/TPSA ratio of 0.15 was 78% maintaining at least 90% sensitivity. Eleven of the patients had a prostate specific antigen (PSA) value higher than 10 ng/mL. In 6 of these patients the biopsy result was prostate cancer and 10 of these patients had a FPSA/TPSA ratio lower than 0.15. CONCLUSION: In patients with TPSA values between 4-10 ng/mL the cut off value of FPSA/TPSA ratio of 0.15 can be used to eliminate unnecessary biopsies with minimal loss of cancer patients. PMID- 10755360 TI - Multiple transrectal ultrasound guided prostatic biopsies: morbidity and tolerance. AB - OBJECTIVE: Early detection of prostate cancer has become a matter of vital importance in modern societies. Ultrasound guided transrectal biopsy is the current standard urological procedure to detect prostate cancer. In this study our intention was to confirm the high tolerance and low complication rate of this procedure, facts already established in urologists' minds. MATERIAL AND METHODS: In order to evaluate the morbidity as well as the acceptance of the procedure, we investigated 120 patients who underwent ultrasound guided transrectal biopsies of the prostate, in our department from September 1995 to January 1996. All patients at each biopsy underwent 6 needle passes and took periprocedural antibiotic therapy. Alongside with recording the periprocedural side effects of this method patients answered a questionnaire in order to evaluate the pain they experienced by this procedure. RESULTS: Twenty patients were found to have prostate cancer at various stages. All patients tolerated well the whole procedure. Pain was the most common complaint among patients. Several complications were recorded; the most common of all was haematuria. Only two patients required admission to hospital because they developed fever after the procedure. CONCLUSION: Ultrasound guided transrectal biopsy of the prostate is a well-tolerated and effective method for obtaining multiple biopsy specimens from the prostate with low incidence of serious complications. Its is also accurate enough, allowing its use in everyday urology, as a diagnostic procedure. PMID- 10755361 TI - Prostatic intraepithelial neoplasia in prostate specimens: frequency, significance and relationship to the sampling of the specimen (a retrospective study of 121 cases). AB - OBJECTIVES: To determine the frequency of PIN (prostatic intraepithelial neoplasia) in prostate specimens and the relationship of PIN with PCA (prostatic carcinoma) and amount of sampling of the specimen. MATERIALS AND METHODS: All the haematoxylin-eosin stained slides of 121 cases diagnosed between 1990 and 1995 were re-examined retrospectively. The amount of sampling of prostate specimens was also re-examined. RESULTS: PIN was observed in 47.9% of all prostate specimens. The frequency of incidental PIN was 71.4% in cystoprostatectomy specimens. PIN was present in 58.3% of the cases with PCA. We observed foci of high-grade PIN adjacent to sites of invasive carcinoma in 100.0% of prostatectomy specimens with PCA. PIN was high-grade in 100.0% of the carcinomatous prostates with PIN. It was multifocal in 53.4% of 58 cases with PIN. Incidental PCA was identified in 14.3% of cytoprostatectomies for bladder cancer. The average number of paraffin blocks of prostatic tissue was 4.1 (+/-2.6) in cases with PIN and 3.2 (+/-1.4) in cases without PIN. CONCLUSION: In prostate specimens, the determination of PIN is very important since it is the most likely precursor of PCA. The probability of detecting PIN and PCA in a prostate specimen is directly related to the amount of sampling. PMID- 10755362 TI - Penile urethral fistula caused by prolonged urethral catheterization in a paraplegic child. PMID- 10755363 TI - Core through internal urethrotomy in the management of post-traumatic isolated bladder neck and prostatic urethral strictures in adults. A report of 4 cases. PMID- 10755364 TI - Bilateral cryptorchidism associated with 47,XYY karyotype. AB - We describe an 11-month-old boy with karyotype of 47,XYY who presented with bilateral cryptorchidism, and discuss the hormonal condition of the patient. PMID- 10755365 TI - Effect of prolonged restriction of motor activity on primates hydration homeostasis. AB - It has been assumed that restriction of motor activity (hypokinesia) induces significant changes in body hydration homeostasis. Thus, the objective of this study was to measure body hydration level during prolonged hypokinesia (HK). The studies were done on 12 male Macaca Mulatta (rhesus monkeys) aged three to five years (4.75 to 6.96 kg) during a 15-day period of pre HK and a 90-day period of HK. All primates were divided equally into two groups: monkeys placed under vivarium conditions served as vivarium control primates (VCP) and monkeys subjected to HK served as hypokinetic primates (HKP). For simulation of the HK effect, the HKM group was kept for 90 days in small individual cages that restricted their movements in all directions without hindering food and water intakes. During the pre HK period of 15 days and the HK period of 90 days the following parameters were measured: total body water (TBW), extracellular fluid volume (EFV), intracellular fluid volume (IFV), circulating plasma volume (CPV) and interstitial fluid volume (IsFV), urinary and plasma sodium and potassium, fluid consumption and elimination in urine, and body weight. Significant (p < or = 0.01) decrease in the TBW, IFV, and CPV was observed in the HKP group when compared with the VCP group. In the HKP group EFV and IsFV decreased significantly (p < or = 0.01) when compared with the VCP group only in the initial seven days of the HK period, while after the 7th day progressive increase could be observed. Fluid loss, urinary electrolyte excretion and plasma electrolyte concentration increased significantly (p < or = 0.01), while fluid intakes decreased significantly (p < or = 0.01) in the HKP group when compared with the VCP group. In the HKP group body weight decreased significantly (p < or = 0.01) when compared with the VCP group. In the VCP group the measured parameters did not change significantly when compared with the baseline control values. It was concluded that prolonged exposure to HK induces significant changes in body hydration homeostasis while body dehydration in monkeys caused primarily due to decreased CPV. PMID- 10755366 TI - Enzymatic markers of cyclosporine nephrotoxicity in patients after renal transplantation. AB - Toxicity of cyclosporine (CsA), an immunosuppressive drug widely used in transplantation, to the transplanted kidney creates a serious side effect. Therefore, searching for sensitive indicators of nephrotoxic action is well worth the effort. In this work we describe the results of estimation of urine concentration of lysosomal enzymes widely present in the kidney: N-acetyl-beta-D glucosaminidase (NAG), its isoenzyme NAG-B and beta-glucuronidase (beta-Gr). The studies were conducted in various periods after transplantation of kidneys, on patients under various treatments and receiving different doses of CsA. The results indicate a substantial dependence of the activity of NAG and NAG-B on CsA doses and the period after transplantation. The enzyme proved to be also a sensitive indicator of graft rejection. No such dependence was observed in the case of beta-Gr. PMID- 10755367 TI - Rapid, simple and sensitive high-performance liquid chromatographic method for detection and determination of acyclovir in human plasma and its use in bioavailability studies. AB - A rapid, simple and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the measurement of acyclovir concentrations in human plasma and its use in bioavailability studies is evaluated. Unchanged acyclovir has been quantified without the introduction of an internal standard using the present method. Human plasma proteins were selectively precipitated by the addition of 7% perchloric acid to spiked plasma samples or to the plasma samples obtained after acyclovir administration to human volunteers and the mixture was spun at 1000 g for 10 min. The supernatant was directly injected into a Novaflex C18 column and detected at 254 nm. The mobile phase consisted of octane sulfonic acid buffer (pH 2.5) and methanol (92:08). The limit of quantitation for acyclovir in plasma was 20 ng/ml, which enabled the determination of the area under the curve (AUC) more precisely, that is, it is much closer to its extrapolated value. The present method has been successfully applied to samples from bioavailability studies. PMID- 10755368 TI - Quantitative high-performance liquid chromatographic determination of acrolein in plasma after derivatization with Luminarin 3. AB - A rapid, sensitive and specific high-performance liquid chromatographic method for the quantification of acrolein (1), one of the toxic metabolites of oxazaphosphorine alkylating agents (cyclophosphamide and ifosfamide) was developed. Condensation of acrolein with Luminarin 3 afforded a fluorescent derivative that could be specifically detected and quantified. Chromatographic conditions involved a C18 RP column Uptisphere and a gradient elution system to optimize resolution and time analysis. The method showed high sensitivity with a limit of detection of 100 pmol/ml and a limit of quantification of 300 pmol/ml. This technique is particularly suitable for pharmacokinetic studies on plasma of oxazaphosphorine-receiving patients. PMID- 10755369 TI - Neutral amino acids monitoring in phenylketonuric plasma microdialysates using micellar electrokinetic chromatography and laser-induced fluorescence detection. AB - Neutral and non-polar amino acids such as phenylalanine (Phe), valine (Val), tyrosine (Tyr), threonine (Thre) and GABA are hard to resolve by capillary zone electrophoresis (CZE). Their separation is possible by adding a surfactant to the mobile phase. This method is called micellar electrokinetic chromatography (MEKC). We used MEKC with laser-induced fluorescence detection (LIFD) to separate and quantitate these amino acids in plasma microdialysates of patients with phenylketonuria (PKU). This disease is an inborn enzymatic defect with decreased conversion of Phe to Tyr that causes severe neurological damage and mental deterioration, which is diagnosed by measuring plasma Phe and Phe/Tyr ratio. The amino acids tested had linear concentration-signal relation. PKU patients had significantly higher Phe, lower Tyr, 21 times higher Phe/Tyr ratio and decreased values of Val and Thre than controls. These results show that microdialysis of biological fluids coupled with MEKC-LIFD is a convenient technique to measure neutral amino acids in clinical disorders such as PKU. PMID- 10755370 TI - Liquid chromatographic-electrospray tandem mass spectrometric method for the simultaneous quantitation of the prodrug fosinopril and the active drug fosinoprilat in human serum. AB - A sensitive, specific, accurate and reproducible LC-MS-MS method was developed and validated for the simultaneous quantitation of the prodrug fosinopril and its active drug fosinoprilat in human serum. The method employed acidification of the serum samples to minimize the hydrolysis of fosinopril to fosinoprilat prior to purification by solid-phase extraction to isolate the two analytes and the two internal standards from human serum. The extracted samples were analyzed by turbo ionspray LC-MS-MS in the positive ion mode. Chromatography was performed on a polymer-based C18 column (Asahipak ODP PVA-C18, 2x50 mm) using gradient elution with methanol and 10 mM ammonium acetate, pH 5.5. The calibration curve, 1.17 to 300 ng/ml, was fitted to a weighted (1/x) linear regression model. Serum quality control (QC) samples used to gauge the accuracy and precision of the method were prepared at concentrations of 5.00, 100, 250 and 500 ng/ml of each analyte. The inter-assay accuracies were within 6% (DEV) for both analytes. The intra- and inter-assay precisions were within 7% and 11% (RSD), respectively, for both analytes. The hydrolysis of fosinopril to fosinoprilat during sample processing was < or = 6%. This degree of conversion would cause little error in the analysis of post-dose serum samples since such samples are known to contain low levels of the prodrug compared to the drug. PMID- 10755371 TI - Bisphenol derivative of allysine for high-performance liquid chromatographic analysis of allysine residue of proteins. AB - Allysine is the most important precursor of physiologically essential cross-links formation in collagen and elastin and is formed by enzymatic oxidative deamination of lysine residues. Because it is a highly reactive aldehyde, many cross-linking amino acid residues may arise from its reaction with other allysine residues or lysine or even histidine residues. We purified and isolated an allysine bisphenol derivative, 1-amino-1-carboxy-5,5-bis-p-hydroxyphenylpentane (ACPP), from the reaction products of phenol and allysine residue of bovine ligamentum nuchae by acid hydrolysis in 6 M HCl. The structure of ACPP was verified by UV, fast atom bombardment-MS, 1H- and 13C-nuclear magnetic resonance spectroscopies. The optimal reaction condition for ACPP synthesis accompanied by hydrolysis of such proteins was investigated and an ion-paired high-performance liquid chromatographic method for determination of allysine as ACPP was also developed. PMID- 10755372 TI - Simultaneous determination of the peptide-mitomycin KW-2149 and its metabolites in plasma by high-performance liquid chromatography. AB - A gradient high-performance liquid chromatographic (HPLC) method is described for the quantification of KW-2149 and its two major metabolites in plasma. The method involves a sample clean-up by solid-phase extraction on C18 columns, separation of the respective compounds by HPLC on a YMC ODS-AQ column (5-microm particle size, 150x6 mm I.D.), using a methanol-water gradient system as an eluent, and measurement by UV absorbance detection at 375 nm. The limits of quantitation were 10 ng/ml for KW-2149 and M-16, and 15 ng/ml for M-18. Recoveries from plasma were higher than 92% on C18 extraction columns. Intra-day precision, expressed as %C.V., was between 1.4 and 6.5%. Intra-day accuracy ranged from 94 to 107%. Precision and accuracy of variability of inter-assays increased somewhat; however, were still within acceptable ranges. The ability of the method to quantify KW-2149 and two major metabolites simultaneously, with precision, accuracy and sensitivity, make it useful in monitoring the fate of this new mitomycin in cancer patients. PMID- 10755373 TI - Determination of lycopene in tissues and plasma of rats by normal-phase high performance liquid chromatography with photometric detection. AB - An analytical method for the determination of lycopene in tissues and plasma of rats is described. The method was validated for the determination of lycopene in liver and plasma with respect to selectivity, linearity, accuracy, recovery and precision. Following precipitation of proteins with water-ethanol plasma was extracted with hexane; tissues were extracted with acetone followed by precipitation of proteins with water-ethanol and extraction of lycopene with hexane. Separation and quantification of geometrical isomers of lycopene was achieved by normal-phase HPLC with UV/VIS detection at 471 nm. The method proved to be selective and specific for lycopene in plasma and liver. Detector response was linear in the range from 2 ng/g to 10 microg/g liver and 0.5 ng/ml to 2 microg/ml plasma, respectively. Average recoveries ranged from 96 to 101% in spiked liver samples and from 91 to 94% in spiked plasma samples. Intra-day variability (C.V.) was < or = 6% and < or = 5% in liver and plasma, respectively. Inter-day precision was < or = 9% for liver samples and < or = 6% for plasma samples. The procedures were successfully applied to the sample analysis of pharmacokinetic and metabolism studies. PMID- 10755374 TI - Validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue by gas chromatography-mass spectrometry. AB - A selective and extremely sensitive procedure has been developed and optimized, using high-performance liquid chromatography (HPLC), specific derivatization and gas chromatography-mass spectrometry (GC-MS), to simultaneously quantify very small amounts of different neurosteroids from rat brain. Unconjugated and sulfated steroids in brain extracts were separated by solid-phase extraction. The unconjugated fraction was further purified by HPLC, the steroids being collected in a single fraction, and the sulfated fraction was solvolyzed. All steroids were derivatized with heptafluorobutyric acid anhydride and analyzed by GC-MS (electron impact ionization) using selected-ion monitoring. High sensitivity and accuracy were obtained for all steroids. The detection limits were 1 pg for pregnenolone (PREG), dehydroepiandrosterone (DHEA) and their sulfate esters PREG S and DHEA-S, 2 pg for progesterone (PROG) and 5 pg for 3alpha,5alpha tetrahydroprogesterone (3alpha,5alpha-THP). In a pilot study on a rat brain, the concentrations of PREG-S and DHEA-S were 8.26+/-0.80 and 2.47+/-0.27 ng/g, respectively. Those of PREG, DHEA and PROG were 4.17+/-0.22, 0.45+/-0.02 and 1.95+/-0.10 ng/g, respectively. Good linearity and accuracy were observed for each steroid. The methodology validated here, allows femtomoles of neurosteroids, including the sulfates, found in small brain samples (at least equal to 10 mg) to be quantified simultaneously. PMID- 10755375 TI - Enantioselective analysis of ketone bodies in patients with beta-ketothiolase deficiency, medium-chain acyl coenzyme A dehydrogenase deficiency and ketonemic vomiting. AB - Enantioselective multidimensional gas chromatography-mass spectrometry (enantio MDGC-MS) is a valuable tool for the differentiation of enantiomers from complex matrices when present in trace amounts. The separation of chiral compounds provides further information on the diagnosis of diseases, and on normal and abnormal biochemical pathways. The formation of the normal urinary metabolite 3 hydroxy-2-methylbutanoic acid (HMBA), excreted in abnormally high amounts in beta ketothiolase deficiency, is not absolutely clarified. Metabolic pathways involving this metabolite are isoleucine catabolism, as well as presumably beta oxidation of fatty acids and ketogenesis. The latter two pathways are distinguishable in their enantioselectivity. Enantioselective analysis gives further information on interfering metabolic pathways and the selectivity of the enzyme(s) forming HMBA. Different ratios of the stereoisomers of HMBA in control urine samples and patients with beta-ketothiolase deficiency were detected. Analogous to HMBA urinary 3-hydroxybutanoic acid (HBA) was investigated in several diseases. The formation of HBA and HMBA is expected to result from the same or similar metabolic pathways. Differences in the enantiomeric ratio of HMBA may originate from the enantioselectivity of different enzyme systems. PMID- 10755376 TI - Gas chromatographic-mass spectrometric procedures for determination of the catechol-O-methyltransferase (COMT) activity and for detection of unstable catecholic metabolites in human and rat liver preparations after COMT catalyzed in statu nascendi derivatization using S-adenosylmethionine. AB - A procedure is presented for determination of the catechol-O-methyltransferase (COMT) activity in liver cytosolic preparations using 3,4-dihydroxyphenethylamine as substrate and by quantifying the product 3-methoxy-4-hydroxyphenethylamine (3 MHP). For quantification of 3-MHP in liver cytosolic preparations a gas chromatographic-mass spectrometric procedure after liquid-liquid extraction and acetylation was established and validated. The intra- and inter-day accuracy and precision were better than 15% and 20%, respectively. Extraction efficiency and selectivity were also sufficient. For in statu nascendi derivatization of unstable catecholic metabolites in liver microsome preparations, cytosolic preparations with COMT activities of at least 1 nmol product/min/mg protein were used after addition of S-adenosylmethionine. Such catecholic metabolites, which are claimed to be responsible for toxic effects in vivo, e.g., neurotoxicity or carcinogenesis, must not be overlooked in in vitro metabolism studies. Using this trick, gas chromatography-mass spectrometry (GC-MS) was suitable for the determination of catecholic metabolites in human and rat liver preparations after the same sample preparation as for 3-MHP quantification. The applicability was exemplified for the antidepressant paroxetine. PMID- 10755377 TI - Analysis of N(alpha)-methylhistamine by gas chromatography-mass spectrometry. AB - A gas chromatography-electron capture mass spectrometry assay has been developed for the histamine H3 receptor agonist, N(alpha)-methylhistamine (N(alpha)-MH). The assay is linear from 50 pg-10 ng, with a limit of detection of 50 pg/ml for gastric juice and plasma, and 50 pg/sample for bacteria (10(7)-10(8) CFU) and gastric tissue (5-10 mg wet weight). The limits of quantification are 100 pg/ml for gastric juice (%RSD=1.4) and plasma (%RSD=9.4), and 100 pg/sample for bacteria (%RSD=3.9) and tissue (%RSD=5.8). N(alpha)-MH was not present in human plasma, but low levels (1.4 ng/ml and 0.4 ng/ml) were detected in two samples of human gastric juice obtained from patients infected with Helicobacter pylori. PMID- 10755378 TI - Allelic discrimination by denaturing high-performance liquid chromatography. AB - Ion-pair reversed-phase high-performance liquid chromatography on alkylated non porous poly(styrene-divinylbenzene) particles allows the resolution of single stranded DNA molecules of identical size (<100 nucleotides) that differ in a single base. Allelic discrimination is obtained by injecting short DNA amplicons containing the genetic variants of interest into an adequately preheated mobile phase that results in the instantaneous complete denaturation of the PCR products. All possible transitions and transversions other than C-->G can be typed accurately. The method complements the discovery of single-nucleotide polymorphisms by means of HPLC based heteroduplex detection under partially denaturing conditions and allows their rapid genotyping without the need of adding a reference chromosome. PMID- 10755379 TI - Measurement of both protein-bound and total S-2-(3-aminopropylamino)ethanethiol (WR-1065) in blood by high-performance liquid chromatography. AB - A high-performance liquid chromatographic method for automated analysis of both protein-bound and total S-2-(3-aminopropylamino)ethanethiol (WR-1065) in blood has been developed in our laboratory. WR-1065 is the active thiol metabolite of the radio- and chemo-protector drug amifostine (WR-2721). Using WR-1065 quality control levels over the experimental range: 7.0, 45.0 and 85.0 micromol/l spiked into plasma, method validation for total WR-1065 included between-run assessment of imprecision (SD/C.V.%: 1.11/16.7%, 6.58/15.5% and 9.24/11.3%, respectively) and % accuracy (94.7, 106.0 and 97.2%). PMID- 10755380 TI - Determination of alpha- and beta-trenbolone in bovine muscle and liver by liquid chromatography with fluorescence detection. AB - A sensitive and rapid high-performance liquid chromatographic screening method is described for the determination of anabolic steroid trenbolone in bovine muscle and liver. Trenbolone was analyzed as alpha- and beta-trenbolone. Samples were extracted with ethyl acetate and cleaned up on a silica solid-phase extraction (SPE) cartridge. Liver samples were cleaned up on a multifunctional SPE cartridge before using a silica SPE cartridge. Analysis of alpha- and beta-trenbolone was performed by reversed-phase high-performance liquid chromatography (HPLC) with a fluorescence detector. The detection limits for this method were estimated to be 0.2 and 1.0 ng/g in bovine muscle and liver, respectively. The mean recoveries spiked in muscle at 2 ng/g and in liver at 10 ng/g were over 80%. PMID- 10755381 TI - Anticlastogenic potential of 1alpha,25-dihydroxyvitamin D3 in murine lymphoma. AB - Vitamin D3, having gained scientific interest for so long because of its role in mineral homeostasis, has now received great importance as a possible antitumor agent. This study was undertaken in an attempt to visualize the possible anticlastogenic potential of the vitamin in an ascitic mouse lymphoma model namely, Dalton's lymphoma. Frequencies of structural type chromosomal aberrations, sister chromatid exchanges and micronucleus assays have been chosen as the genotoxic endpoints in the proposed investigation. All these cytogenetic markers have been found to be markedly elevated during the progression of lymphoma in bone marrow cells. Vitamin D3 effectively suppressed the frequencies of chromosomal aberrations and sister chromatid exchanges in the lymphoma-bearing mice during the entire phase of tumor growth that significantly coupled with almost two-fold increase in survival time (37 +/- 2 and 68 +/- 2 days in lymphoma controls and vitamin D3-treated lymphoma-bearing mice, respectively), thus substantiating the antineoplastic efficacy of this secosteroid. The outcome of this study also is clearly reflected in the depletion of circulating (serum) vitamin D3 levels in the lymphoma control mice compared with normal (vehicle) controls while a still higher level was maintained in the VD3-treated lymphoma mice. This anticlastogenic property of the vitamin has so far been neglected and this is the first attempt to unravel the vitamin D3's effect in combating tumor development in vivo by limiting the frequencies of chromosomal aberrations, sister chromatid exchanges and micronuclei at least in transplantable murine model studied herein. PMID- 10755382 TI - Activation of matrix metalloproteinase-2 in head and neck squamous cell carcinoma: studies of clinical samples and in vitro cell lines co-cultured with fibroblasts. AB - We undertook this present study to investigate the activation of matrix metalloproteinase-2 (MMP-2) in human head and neck squamous cell carcinomas (HNSCC) tissues and cell lines. Gelatinolytic activities of active MMP-2 were significantly higher in carcinoma samples than in normal portions. Furthermore, the activation ratio of proMMP-2 significantly correlated with cervical lymph node metastasis. In vitro studies revealed an HNSCC cell line, HEp-2, to produce neither the pro form nor the active form of MMP-2, but human fibroblasts were found to produce proMMP-2. However, coculture of HEp-2 cells with fibroblasts resulted in the production of not only proMMP-2 but also activeMMP-2 in the culture medium. Northern blot analysis revealed a stronger expression of membrane type 1 matrix metalloproteinase (MT1-MMP),which is a specific activator of MMP-2, mRNA in HEp-2 cells than in fibroblasts. These results suggest the activation of proMMP-2 as an important event in the process of HNSCC metastasis. They also suggest MMP-2 is secreted in its pro form by stromal fibroblasts surrounding the cancer cells and activated by MT1-MMP localized on the cancer cells. PMID- 10755383 TI - Progression of human breast cancers to the metastatic state is linked to genotypes of catechol-O-methyltransferase. AB - There is increasing evidence that catecholestrogens may contribute to the development of breast cancer. Specifically, inactivation of catecholestrogens may prevent the genesis and arrest the progression of the disease. Catechol-O methyltransferase (COMT), Glutathione S-transferase (GST) M1 and GSTP1 are responsible for the detoxification of catecholestrogens, and are polymorphic in the human population. In this study, a PCR-based restriction fragment length polymorphism analysis was performed to determine genotypes of the COMT, GSTM1 and GSTP1 genes. We investigated the relationship between the germline polymorphism of these genes and clinico-pathological characteristics in 140 patients with breast cancer. Among 73 patients with the low activity COMT allele, 49 (67%) had regional lymph node metastasis. On the other hand, only 27 (40%) of 67 patients without the low activity allele had lymph node metastasis. The COMT genotype was significantly associated with clinical stage and the extent of regional lymph node metastasis of breast cancer (P<0.05). However, polymorphisms of the GSTM1 and GSTP1 gene were not associated with clinico-pathological factors. Our findings suggest that the allele encoding for low activity COMT may contribute to the progression of breast cancer. PMID- 10755384 TI - Lobe specific effects of testosterone and estrogen on 3,2'-dimethyl-4 aminobiphenyl-induced rat prostate carcinogenesis. AB - We have previously shown that chronic administration of a pharmacological dose of testosterone propionate (TP) after treatment with the carcinogen, 3,2'-dimethyl-4 aminobiphenyl (DMAB), results in development of invasive and metastatic adenocarcinomas arising from the dorso-lateral and anterior prostate, as well as the seminal vesicles. Co-administration of ethinyl estradiol (EE) with TP increased the yield of carcinomas in the lateral and anterior lobes. In the present experiment, male F344 rats were treated with DMAB for 20 weeks and then co-administered a pharmacological dose of TP together with various doses of EE for 40 weeks. Without hormone(s) administration, carcinomas were confined to the ventral prostate and all were of intra-acinar type. TP administration suppressed development of the ventral prostate carcinomas but caused invasive carcinomas of the lateral and anterior lobes and of seminal vesicles and intra-acinar carcinomas in the dorsal prostate. The appearance of carcinomas in the lateral and anterior prostate was increased by co-administration of EE in a dose-related fashion but carcinomas of the seminal vesicles were inversely reduced. The suppressive influence of TP on ventral carcinoma development was overcome by only the highest dose of EE. It is concluded that estrogen can modify the enhancing effects of TP on induction of rat prostate and seminal vesicle carcinomas in a dose-related fashion with lobe specificity. PMID- 10755386 TI - Effects of phenethyl isothiocyanate and benzyl isothiocyanate, individually and in combination, on lung tumorigenesis induced in A/J mice by benzo[a]pyrene and 4 (methylnitrosamino)-1-(3-pyridyl)-1-butanone. AB - Phenethyl isothiocyanate (PEITC) is an effective inhibitor of lung tumorigenesis induced in rats and mice by the tobacco-specific carcinogen 4-(methylnitrosamino) 1-(3-pyridyl)-1-butanone (NNK) while benzyl isothiocyanate (BITC) inhibits lung tumorigenesis induced in mice by another tobacco smoke carcinogen, benzo[a]pyrene (BaP). However, little is known about the inhibitory effects of PEITC and BITC in combination, or about the effects of PEITC or BITC on tumorigenesis by a mixture of NNK and BaP. In this study, we carried out a series of experiments pertinent to these questions. In Experiment 1, treatment of A/J mice with PEITC (6 micromol), BITC (6 micromol), or a combination of the two (6 micromol each) by gavage, 2 h prior to each of eight weekly gavage treatments with a mixture of BaP and NNK (3 micromol of each), had no effect on lung tumor multiplicity. In Experiment 2, we evaluated the inhibitory potential of four different mixtures of PEITC and BITC, administered by gavage 2 h prior to each of eight weekly doses of BaP and NNK, as given in Experiment 1. Mixtures of PEITC and BITC (12 micromol of each, or 12 micromol PEITC and 9 micromol BITC) significantly reduced lung tumorigenesis induced by a mixture of BaP and NNK. In Experiment 3, we investigated the effects of dietary PEITC (3 micromol/g diet), BITC (1 micromol/g diet), or a mixture of PEITC (3 micromol/g diet) and BITC (1 micromol/g diet). These compounds were started 1 week before, and continued through to 1 week after the eight weekly treatments with BaP and NNK. PEITC, and PEITC plus BITC, both significantly inhibited lung tumor multiplicity; inhibition was due mainly to PEITC. In Experiment 4, we tested dietary PEITC (3, 1, or 0.3 micromol/g diet) as an inhibitor of lung tumorigenesis induced by BaP, NNK, or BaP plus NNK using a protocol identical to that in Experiment 3. PEITC was an effective inhibitor of lung tumor multiplicity induced by NNK and a mixture of BaP plus NNK, but not by BaP. Dietary PEITC, or PEITC plus BITC, was more effective in these experiments than the compounds given by gavage. The results of this study demonstrate that proper doses of dietary PEITC and dietary as well as gavaged PEITC plus BITC are effective inhibitors of lung tumorigenesis induced in A/J mice by a mixture of BaP and NNK. PMID- 10755385 TI - Antioxidant and anti-tumor promoting activities of the methanol extract of heat processed ginseng. AB - Heat treatment of Panax ginseng C.A. Meyer at a temperature higher than that applied to the conventional preparation of red ginseng yielded a mixture of saponins with potent antioxidative properties. Thus, the methanol extract of heat processed neoginseng (designated as 'NGMe') attenuated lipid peroxidation in rat brain homogenates induced by ferric ion or ferric ion plus ascorbic acid. Furthermore, the extract protected against strand scission in phiX174 supercoiled DNA induced by UV photolysis of H2O2, and was also capable of scavenging superoxide generated by xanthine-xanthine oxidase or by 12-O-tetradecanoylphorbol 13-acetate (TPA) in differentiated human promyelocytic leukemia (HL-60) cells. Topical application of NGMe onto shaven backs of female ICR mice 10 min prior to TPA, significantly ameliorated skin papillomagenesis initiated by 7,12 dimethylbenz[a]anthracene. Moreover, TPA-induced enhancement of epidermal ornithine decarboxylase (ODC) activity and ODC mRNA expression was abolished by a topical dose (0.68 mg) of NGMe. Likewise, TPA-induced production of tumor necrosis factor- in mouse skin was inhibited by NGMe pretreatment. PMID- 10755387 TI - Methylation of exon 2 of p16 is associated with late stage oesophageal cancer. AB - Methylation of the p16 gene was studied in 16 oesophageal tumours. Five (31%) of the tumours were found to be methylated in exon 1 and eight (50%) were methylated in exon 2. The loss of p16 protein correlated with methylation of exon 1 (P = 0.005). However, methylation of exon 2, but not exon 1, was found to be associated with late stage tumours (P = 0.01). We conclude that the methylation of exon 2 of p16 may have effects on the progression of oesophageal tumours that are independent of the expression of the p16 protein. PMID- 10755388 TI - Delay of DNA-adduct repair and severe toxicity in xeroderma pigmentosum group A gene (XPA) deficient mice treated with 2-amino-1-methyl-6-phenyl-imidazo [4,5-b] pyridine (PhIP). AB - Group-A xeroderma pigmentosum (XPA) gene-deficient mice are defective in nucleotide-excision repair and highly susceptible to ultraviolet-B-, and 9,10 dimethyl-1,2-benz[a]anthracene (DMBA)-induced skin carcinogenesis. In this study, changes of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP)-DNA adduct formations in the liver, colon and lung, as assessed by the 32P-postlabeling method and immunohistochemical analysis, and carcinogenic and/or toxic susceptibility of both sexes of XPA-deficient mice (XPA-/-) to PhIP, which is a carcinogenic heterocyclic amine, was examined. Levels of PhIP-DNA adduct formations in the liver, colon and lung, were almost twice as high in XPA-/- as in wild type mice (XPA+/+) mice, 7 days after a single i.g. administration of PhIP, and their delay in recovery was observed in XPA-/- mice. For the long-term experiment, XPA-/- and XPA+/+ type mice were treated with 80 ppm PhIP in the diet for the first 4 weeks followed by 40 ppm after a 2-week recovery period (long term experiment I), or 40 ppm PhIP throughout the experiment (long-term experiment II). Severe toxicity, as evidenced by body weight retardation and poor survival, was observed in the PhIP treated XPA-/- mice of both sexes, but not in the XPA+/+. At week 40 the experiments were terminated and histopathological examinations were performed after complete autopsy. Only lymphomas/leukemias were observed as neoplastic lesions, but no significant differences were observed between the groups. As non-neoplastic lesions, degenerating changes, for example in the pancreatic acinar cells, were observed with XPA-/- mice tending to be more sensitive than XPA+/+ mice. The present study demonstrated that PhIP-DNA adduct formations in the liver, colon and lung of XPA-/- mice were demonstrated and their recovery rate was more delayed than XPA+/+ mice, and furthermore, more severe toxicity to PhIP in XPA-deficient mice was observed, but they were not susceptible to PhIP carcinogenicity under the conditions of the experiment. PMID- 10755389 TI - Inhibitory effect of luteinising hormone-releasing hormone analogues on human endometrial cancer in vitro. AB - We studied the effects of luteinising hormone-releasing hormone (LHRH) agonist leuproreline (1 microM for 96 h) and LHRH antagonist cetrorelix on the cell growth of primary cultures from nine human endometrial cancers using the sulphorhodamine colorimetric test. Histological examinations and reverse transcription and polymerase chain reaction amplification (RT-PCR) for LHRH receptors were also performed. The endometrial cancers examined had a medium to high degree of proliferative activity and a low degree of apoptotic power; furthermore, they expressed the LHRH receptor RNA variably, detectable in 71% of cases. The addition of leuproreline or cetrorelix to cell cultures inhibited growth in a statistically significant way compared to untreated control cells; nevertheless, the percentage of cell growth inhibition obtained was very variable. These data suggest that LHRH analogues can exert differential inhibitory effects on the growth of endometrial cancer, which seems to be independent of the expression of specific LHRH receptors. PMID- 10755391 TI - The antimitotic effects of Ukrain, a Chelidonium majus alkaloid derivative, are reversible in vitro. AB - Ukrain is alleged to be an effective chemotherapeutic drug which causes minimal side-effects as a result of selective toxicity towards malignant cells only. We previously failed to confirm this claim and found Ukrain to be equally toxic to normal, transformed and malignant cell lines by causing a metaphase arrest. In this study we have found the antimitotic actions of Ukrain to be reversible in low doses in vitro, as shown by flow cytometry and concurrent haematoxylin and eosin stains. We hypothesize that the lack of side-effects found in vivo may be due to the lack of therapeutically effective dosages being administered, therefore enabling cells to overcome the metaphase arrest and survive. PMID- 10755390 TI - Mammaglobin B gene as a novel marker for lymph node micrometastasis in patients with abdominal cancers. AB - Mammaglobin B is a recently-isolated gene speculated to belong to the uteroglobin gene family and is overexpressed in primary breast cancers. We investigated mammaglobin B mRNA expression in various cancers of the digestive system. Given the absence of mammaglobin B expression in normal lymph nodes, we also assessed the usefulness of mammaglobin B as a marker for lymph node micrometastases in cancer patients. Mammaglobin B gene transcripts were frequently detected by reverse transcriptase-polymerase chain reaction (RT-PCR) assay in primary tumors of the esophagus (2/3), stomach (7/7), colon (15/15), pancreas (4/6), common bile duct (6/6), cholangioma (2/2) and gall bladder (1/1). Mammaglobin B overexpression was observed in three of 15 cases (20%) of colon cancer, suggesting its possible contribution to colon carcinogenesis. Down-regulated mammaglobin B expression was observed in hepatoma cells in comparison with corresponding non-cancerous livers (3/3). RT-PCR assay of mammaglobin B detected 14 of 15 histologically positive lymph nodes from patients with gastric cancer, colon cancer and cholangioma. Seven of 32 (22%), three of nine (33%), and three of seven (43%) histologically negative nodes from patients with gastric, colon and cholangiocellular carcinoma, respectively, were found to express mammaglobin B mRNA. Our results showed that expression of mammaglobin B was frequently detected in cancers originating in digestive organs, especially adenocarcinomas, and that mammaglobin B gene detected by RT-PCR may be a potentially useful molecular marker for lymph node micrometastases of various digestive organ cancers. PMID- 10755392 TI - Reduction of murine mammary tumor metastasis by conjugated linoleic acid. AB - Recent studies have shown that conjugated linoleic acid (CLA) can inhibit the initiation and thus, incidence of mammary tumors in rodents. The concentration of CLA required for these effects was as low as 0.1% of the diet, with no increased effects above 1%. To date, there is little evidence that CLA has any effect on growth or metastasis of mammary tumors. In this report, we demonstrate that CLA, at the concentrations used in previous studies, had a significant effect on the latency, metastasis, and pulmonary tumor burden of transplantable murine mammary tumors grown in mice fed 20% fat diets. The latency of tumors from mice fed CLA was significantly increased when compared with the 0% CLA control diet. The volume of pulmonary tumor burden, as a result of spontaneous metastasis, decreased proportionately with increasing concentrations of dietary CLA. With 0.5 and 1% CLA, pulmonary tumor burden was significantly decreased compared to mice treated with the eicosanoid inhibitor, indomethacin and fed diets containing no CLA. Tumors of mice fed as little as 0.1% CLA and as much as 1% had significantly decreased numbers of pulmonary nodules when compared with diets containing no CLA. The decrease in the number of pulmonary nodules by CLA was nearly as effective as indomethacin, a known suppressor of tumor growth and metastasis in this malignant model. These data suggest that effects of CLA on mammary tumorigenesis may go beyond the reported alterations in tumor incidence and effect later stages, especially metastasis. PMID- 10755393 TI - Modulation of murine mammary tumor vasculature by dietary n-3 fatty acids in fish oil. AB - We have previously shown that mice fed a high (n-3) fatty acid-containing diet with 20% (w/w) total fat had significantly slower mammary tumor growth, decreased numbers of metastatic pulmonary nodules, and decreased total metastatic load. In this study we sought to determine whether tumor vascularization was altered in mice fed diets varying in concentrations of (n-3) and (n-6) fatty acids. Several direct or indirect parameters of vascularization were tested. With 20% dietary fat, fish oil (FO) or a mixture of FO and safflower oil (FS) significantly reduced blood vascular area, mast cell number and macrophage infiltration in solid mammary tumors compared to tumors grown in mice fed safflower oil (SO). A decreasing trend was seen in the percent area of vessels positive for CD31 and vascular endothelial growth factor (VEGF) in the 20% FO and 20% FS compared to the 20% SO dietary groups. VEGF concentrations were twice as high in smaller tumors (100 mm3) from all dietary groups as compared to larger tumors (500 mm3). A two-fold increase in VEGF levels was found in the 20% SO dietary group compared to the 20% FO group in 100-mm3 but not larger tumors. We conclude that at 20% total fat, the n-3 fatty acids found in fish oil may inhibit primary mammary tumor growth through modulation of select determinants of vascularization. PMID- 10755394 TI - Photodynamic therapy efficacy and tissue distribution of hypericin in a mouse P388 lymphoma tumor model. AB - The phototherapeutic properties and tissue distribution of hypericin were investigated in DBA/2 mice bearing subcutaneously transplanted P388 lymphoma cells. The efficacy of the photodynamic therapy (PDT) 2 h after administration of hypericin (2, 5, or 20 mg/kg, i.p., 120 J/cm2, 595 nm) was substantially greater than the efficacy after a 24 h interval. PDT with Photofrin (5 mg/kg, i.p., 24-h interval, 120 J/cm2, 630 nm) showed no significant antitumoral effect. The hypericin uptake in some tissues was measured after administration of hypericin (5 or 20 mg/kg, i.p.) up to 168 h. A comparison of the distribution data and the PDT efficacy at various intervals suggests that the plasma concentration of hypericin, and to a lesser extent the tumor uptake, determines the tumor response to PDT with hypericin. PMID- 10755395 TI - A polymorphic tetranucleotide repeat in the CYP19 gene and male breast cancer. AB - The CYP19 gene codes for the aromatase enzyme that is involved in the synthesis of oestrogens. This case-control study examines the relationship between a tetranucleotide repeat sequence in the CYP19 gene and the development of male breast cancer. No significant differences were found between male breast cancer cases and controls. PMID- 10755396 TI - Attitude towards prophylactic surgery and effects of genetic counselling in families with BRCA mutations. Austrian Hereditary Breast and Ovarian Cancer Group. AB - The intent of this study was to evaluate the effect that an awareness of being a BRCA1 or BRCA2 mutation carrier has on the attitude towards prophylactic surgery and on developing depression symptoms. Thirty-five families were selected on the basis of previously detected BRCA1 or 2 mutations and 90 family members were given the appropriate questionnaires. Prophylactic mastectomy (PM) was considered by 21% of the Austrian mutation carriers (29% affected and 8% non-affected carriers). The majority of affected and non-affected carriers expected PM to impair the quality of their life. Fifty per cent would undergo prophylactic oophorectomy (53% affected and 46% non-affected carriers). The self-rating depression scale indicated that following mutation result disclosure the depression scores of carriers decreased (40 baseline vs 38 after result disclosure, P = 0.3), whereas, for non-carriers, scores increased (36 baseline vs 40 after result disclosure, P = 0.05). We conclude that information about carrier status is not associated with increased depression symptoms in mutation carriers. In non-carriers, depression scores increased slightly, probably reflecting survivor guilt. The option of having PM was associated with a negative impact on the quality of life and was declined by the majority of Austrian mutation carriers. PMID- 10755397 TI - Long-term survival of stage I multiple myeloma given chemotherapy just after diagnosis or at progression of the disease: a multicentre randomized study. Cooperative Group of Study and Treatment of Multiple Myeloma. AB - We conducted a randomized trial to evaluate whether melphalan-prednisone (MPH-P) treatment administered just after diagnosis improves survival of stage I multiple myeloma (MM). Between January 1987 and March 1993, 145 consecutive previously untreated patients with stage I MM were randomized between treatment with MPH-P (administered for 4 days every 6 weeks) just after diagnosis and treatment only at disease progression. Survival was not influenced by MPH-P treatment either administered just after diagnosis or at disease progression (64 vs 71 months respectively). Comparing the first with the second group the odds ratio of death is 1.17 (95% confidence interval 0.57-2.42; P = 0.64). Disease progression occurred within a year in about 50% of patients who were initially untreated. Response rate was similar in both groups, but duration of response was shorter in patients who were treated at disease progression (48 vs 79 months, P = 0.044). Patients actually treated at disease progression (34/70) survived shorter than those who had neither disease progression nor treatment (56 vs > 92 months; P = 0.005). Starting MPH-P just after diagnosis does not improve survival and response rate in stage I MM, with respect to deferring therapy until disease progression. However, patients with stage I MM randomized to have treatment delayed and who actually progressed and were treated had shorter survival than those with stable disease and no treatment. Biologic or other disease features could identify these subgroups of patients. PMID- 10755398 TI - Immunogenicity of vaccination against influenza, Streptococcus pneumoniae and Haemophilus influenzae type B in patients with multiple myeloma. AB - Vaccination against influenza and Streptococcus pneumoniae is recommended for elderly and immunocompromised individuals. However, there is little information concerning the efficacy of vaccination in specific groups of patients. In this study, 52 patients underwent vaccination against influenza, S. pneumoniae and Haemophilus influenzae type b (Hib) as they attended hospital outpatient clinics. Serum was analysed prior to vaccination and 4-6 weeks afterwards. Antibody titres against S. pneumoniae and Hib were compared with reference values corresponding to the geometric mean titres of a healthy UK population. For influenza vaccination, haemagglutination inhibition (HI) titres were measured against three inactivated strains; a titre of > or = 1/40 was considered protective. No patient had protective titres to all three antigens prior to vaccination and 41 patients (85%) had titres < 1/40 to all 3 strains. Post vaccination only 9/48 patients (19%) achieved protective antibody titres. Resistance to S. pneumoniae and response to Pneumovax II was also poor: prevaccination, 45 patients (93%) had suboptimal antibody titres and in 26/43 patients (61%) titres remained low post vaccination. Resistance to Hib and response to vaccination was comparable with the healthy adult UK population. These results question the practice of routine influenza and pneumococcal vaccination in myeloma patients. PMID- 10755399 TI - Molecular analysis of the BRCA1 and BRCA2 genes in 32 breast and/or ovarian cancer Spanish families. AB - It is estimated that about 5-10% of breast cancer cases may be due to inherited predisposition. Until now, two main susceptibility genes have been identified: BRCA1 and BRCA2. The first linkage and mutational studies suggested that mutations in these two genes would account for the majority of high-risk breast cancer families, but recent studies show how the proportion of families due to BRCA1 or BRCA2 mutations strongly depends on the population and the types of family analyzed. It is now clear that, in the context of families with a modest cancer profile, which are the most commonly found in the clinical practice, the percentage of mutations found is much lower than that suggested by the first studies. In the present study, we analyze a group of 32 Spanish families, which contained at least three cases of female breast cancer (at least one of them diagnosed before the age of 50 years), for the presence of mutations in the BRCA genes. The total proportion of mutations was low (25%), although the percentage of mutations in the BRCA1 and BRCA2 genes was higher, considering the breast and ovarian cancer families and the male breast cancer families respectively. Our results are in agreement with the idea that a great proportion of moderate-risk cancer families could be due to low penetrance susceptibility genes distinct from BRCA1 or BRCA2. PMID- 10755400 TI - The expression of MDM2/CDK4 gene product in the differential diagnosis of well differentiated liposarcoma and large deep-seated lipoma. AB - Ordinary lipomas are cytogenetically characterized by a variety of balanced rearrangements involving chromosome segment 12q13-15, whereas well differentiated liposarcomas (WDL) show supernumerary ring and giant marker chromosomes, known to contain amplified 12q sequences. The tight correlation between the presence of ring chromosomes and both amplification and overexpression of MDM2 and CDK4 genes suggests the exploration of the possibility that immunocytochemistry (ICC) might assist in the differential diagnosis of lipoma-like well differentiated liposarcomas (LL-WDL) and large deep-seated lipomas (LDSL). For this purpose, 21 cases of the former and 19 cases of the latter tumours were analysed by ICC and, according to the availability of material, by molecular and cytogenetic approaches. All lipomas displayed a null MDM2/CDK4 phenotype, whereas all LL-WDL showed MDM2/CDK4 or CDK4 phenotypes. Southern blot analysis performed on 16 suitable cases, complemented by fluorescence in situ hybridization and classical cytogenetic analysis in 11 cases, was consistent with, and further supported the immunophenotyping data. In conclusion, MDM2/CDK4 product-based immunophenotyping appears to represent a valuable method for the categorization of arguable LDSL. PMID- 10755401 TI - Genetic analysis of multiple synchronous lesions of the colon adenoma-carcinoma sequence. AB - The colorectal adenoma-carcinoma sequence represents a well-known paradigm for the sequential development of cancer driven by the accumulation of genomic defects. Although the colorectal adenoma-carcinoma sequence is well investigated, studies about tumours of different dignity co-existent in the same patient are seldom. In order to address the distribution of genetic alterations in different lesions of the same patient, we coincidently investigated carcinomas, adenomas and aberrant crypt foci in patients with sporadic colon cancer. By utilizing polymerase chain reaction, single-strand conformation polymorphism, heteroduplex analysis, restriction fragment length polymorphism, protein truncation test and sequencing techniques we looked for mutations and microsatellite instability of APC, H-ras, K-ras, p53, DCC and the DNA repair genes hMLH1/hMSH2. In accordance with the suggested adenoma-carcinoma sequence of the colon, four patients reflected the progressive accumulation of genetic defects in synchronously appearing tumours during carcinogenesis. However, two patients with non hereditary malignomas presented different genetic instabilities in different but synchronously appearing tumours suggesting non-clonal growth under almost identical conditions of the environment. Thus, sporadically manifesting multiple lesions of the colon were not necessarily driven by similar genetic mechanisms. Premalignant lesions may transform into malignant tumours starting from different types of genetic instability, which indicates independent and simultaneous tumorigenesis within the same organ. PMID- 10755402 TI - Limitations of CD44v6 amplification for the detection of tumour cells in the blood of colorectal cancer patients. AB - Based on the important role of CD44 splice variants in colorectal cancer progression and metastasis, we evaluated the use of CD44v6 expression to detect and assess the metastatic potential of colorectal tumour cells circulating in peripheral blood. A nested amplification was designed that allowed to detect 10 100 colon cancer cells. This assay was applied to blood samples from healthy donors. Strong signals were detected in all cases, indicating that it cannot be used to detect colorectal carcinoma cells in whole blood. We then included an enrichment step based on the use of an anti-epithelial cells monoclonal antibody (BerEP4) coupled to magnetic beads. The CD44v6 reverse transcription polymerase chain reaction (RT PCR) assay was performed on cDNA synthesized from blood samples treated with these beads. We analysed 18 samples from 12 patients with a gastrointestinal disease, and 36 samples from ten patients with a colorectal cancer. None of the patients used as negative controls were found to contain epithelial cells in their blood as determined by cytokeratin 19 RT-PCR. By contrast, CD44 transcripts containing exon v6 were detected in nine out of the 18 samples tested (50%). For the colorectal cancer patients, six out of the seven samples (85.7%) that were cytokeratin 19-positive were CD44v6-negative, whereas ten samples out of the 29 not containing epithelial cells were CD44v6-positive (34.5%). This is probably due to the persistence of CD8+ leucocytes in the enriched preparations, as determined by PCR analysis of the CD8 alpha-chain. We conclude that detection of CD44v6 transcripts using a sensitive nested RT-PCR assay has no potential value to detect and characterize colorectal cancer micrometastases from blood, even following an initial enrichment step. PMID- 10755403 TI - Selective interactions between epithelial tumour cells and bone marrow mesenchymal stem cells. AB - This work is a comparative study on the features displayed by an epithelial metastatic breast cancer cell line (MCF-7) when set in co-culture with human bone marrow mesenchymal stem cells (MSC) or a feeder layer of 3T3 fibroblasts. MSC, a subset of non-haematopoietic cells in the marrow stroma, display a potential for self-renewal, proliferation and differentiation into precursors for bone, cartilage, connective and muscular tissue. Adhesion of MCF-7 cells to monolayers of MSC or 3T3 was high (95 and 85% respectively). Once attached, MCF-7 grow well on both monolayers. Morphology of MCF-7 cells, as analysed by light and epifluorescence microscopy, revealed that MCF-7 cells grow in clusters on 3T3, but disperse on MSC. Concomitant with the lost of their aggregation status, MCF-7 on MSC express low levels of the intercellular adhesion molecules, E-cadherin and epithelial-specific antigen (ESA). These results suggest that MSC represent an appropriate cell target to investigate the cellular and molecular events occurring at the interface of epithelial-marrow stromal interactions. Together, the model here described should permit to further evaluate the significance and prognostic impact of the shift of micrometastatic cells from a cluster-aggregated into a single-cell status. PMID- 10755404 TI - Treatment of canine oral squamous cell carcinomas with photodynamic therapy. AB - Eleven dogs with naturally occurring oral squamous cell carcinomas were treated with photodynamic therapy (PDT) using Photochlor (HPPH) as the photosensitizer. The largest length of the tumours measured in a two-dimensional plane ranged from 0.9 to 6.8 cm. Seven of the tumours invaded underlying bone as determined by radiograph appearance. Photochlor was injected intravenously at a dose of 0.3 mg kg(-1). Forty-eight hours later the tumours were treated. Tumours with a surface to base depth of greater than 1 cm were surgically reduced to less than 1 cm. Irradiation with 665 nm light with an energy density of 100 J cm(-2) was administered. Eight dogs were considered cured with no tumour recurrence for at least 17 months after treatment. Local treatment of oral squamous cell carcinomas with PDT appears to give results similar to those obtained with surgical removal of large portions of the mandible or maxilla. The cosmetic results with PDT are superior to those of radical surgical removal. The new sensitizer, Photochlor, appears effective for oral squamous carcinomas with results similar to those reported for other sensitizers. PMID- 10755405 TI - Mitoxantrone and ametantrone induce interstrand cross-links in DNA of tumour cells. AB - We show here that mitoxantrone and ametantrone induce interstrand DNA cross-links in HeLa S3 cells. These cross-links were observed only in cellular system suggesting that metabolism of the drugs is a necessary step leading to DNA cross linking. Biologically inactive analogue of mitoxantrone, compound NSC 321458, did not induce cross-links in DNA of tumour cells which suggests that DNA cross linking is associated with the cytotoxic and anti-tumour activity of these compounds. PMID- 10755406 TI - A unique cytosolic activity related but distinct from NQO1 catalyses metabolic activation of mitomycin C. AB - Mitomycin C (MMC) is a prototype bioreductive drug employed to treat a variety of cancers including head and neck cancer. Among the various enzymes, dicoumarol inhibitable cytosolic NAD(P)H:quinone oxidoreductase1 (NQO1) was shown to catalyse bioreductive activation of MMC leading to cross-linking of the DNA and cytotoxicity. However, the role of NQO1 in metabolic activation of MMC has been disputed. In this report, we present cellular and animal models to demonstrate that NQO1 may play only a minor role in metabolic activation of MMC. We further demonstrate that bioreductive activation of MMC is catalysed by a unique cytosolic activity which is related but distinct from NQO1. Chinese hamster ovary (CHO) cells were developed that permanently express higher levels of cDNA-derived NQO1. These cells showed significantly increased protection against menadione toxicity. However, they failed to demonstrate higher cytotoxicity due to exposure to MMC under oxygen (normal air) or hypoxia, as compared to the wild-type control CHO cells. Disruption of the NQO1 gene by homologous recombination generated NQO1 /- mice that do not express the NQO1 gene resulting in the loss of NQO1 protein and activity. The cytosolic fractions from liver and colon tissues of NQO1-/- mice showed similar amounts of DNA cross-linking upon exposure to MMC, as observed in NQO1+/+ mice. The unique cytosolic activity that activated MMC in cytosolic fractions of liver and colon tissues of NQO1-/- mice was designated as cytosolic MMC reductase. This activity, like NQO1, was inhibited by dicoumarol and immunologically related to NQO1. PMID- 10755407 TI - Direct activation of oestrogen receptor-alpha by interleukin-6 in primary cultures of breast cancer epithelial cells. AB - Interleukin 6 (IL-6) is secreted by breast tumours and shows synergistic activity with 17beta-oestradiol (E2), leading to increases in reductive 17beta hydroxysteroid dehydrogenase activity in breast cancer epithelial cells. However, the mechanisms involved are poorly understood. Using short-term epithelial cultures established from primary breast tumours, we have examined whether IL-6 could directly affect transcriptional activity of oestrogen reception alpha (ERalpha). Tumour epithelial cultures were established from 15 breast tumours, grown to 70% confluence and transiently transfected with a plasmid reporter containing the vitellogenin oestrogen response element and the luciferase coding sequence (ERE-TK-LUC). Following transfection, cells were incubated with E2, IL 6, the pure anti-oestrogen ZM 182780 or combinations of these substances for 48 h. Luciferase activity was then measured in cell lysates. E2 caused a dose dependent increase in luciferase expression, causing a maximum threefold stimulation at 100 pM. In the presence of IL-6, transcriptional activity was increased by up to 2.5-fold in ERalpha+ cultures (11/15). In combination with E2, synergistic effects were observed with increases in luciferase activity of up to sixfold over controls. This effect could be blocked by treatment with ZM 182780. Pre-incubation of cells with an antibody directed against the signalling component of IL-6, gp130, was ineffective in blocking the E2 response. This antibody reduced, but did not completely block the effect of IL-6 either alone or in combination with E2, suggesting cross-talk between the two signalling pathways. In conclusion, these results provide evidence for direct transcriptional activation of ERalpha by IL-6. PMID- 10755408 TI - Alterations in cathepsin H activity and protein patterns in human colorectal carcinomas. AB - Our analyses of cathepsin H activity levels and protein forms in human colorectal cancers compared to matched control mucosa support the concept that altered proteinase expression patterns may reflect both cancer stage and site. Cathepsin H-specific activity was significantly increased in colorectal cancers compared to control mucosa (P = 0.003; n = 77). Highest specific activities and cancer/normal ratios (C/N) for activity were measured in Dukes' B and C stage carcinomas, cancers involved in local spread and invasion to lymph nodes. In contrast, cathepsin B and L activities analysed in the same paired extracts had been shown to be most frequently elevated in earlier stage carcinomas (Dukes' A and B), confirming that cathepsin H demonstrates a distinct pattern of expression during colorectal cancer progression. Although cathepsin H activities were most commonly elevated in Dukes' C cancers at all colon sites, both specific activity and C/N ratios were significantly higher for cancers of the left colon compared to other colon locations. A subset of 43 paired extracts analysed on Western blots also revealed consistent changes in cathepsin H protein forms in cancers. Normal mucosa typically showed a strong protein doublet at 31 and 29 kDa while cancers demonstrated decreased expression or total loss of the 31 kDa protein (90% of cases), equal or increased expression of the 29-kDa protein (67% of cases) and the new appearance or up-regulation of a cathepsin H band at 22 kDa (78% of cases). C/N ratios for cathepsin H enzyme activity correlated significantly with C/N ratios for the 29 kDa mature single-chain protein form (P < 0.001), with increased activity most commonly associated with elevated expression of 29-kDa cathepsin H but also with up-regulation of the 22-kDa band, suggesting a shift to more fully processed, mature active cathepsin H protein forms in cancers. Changes in cathepsin H expression were also detected by immunohistochemistry as elevated cathepsin H staining in tumour epithelial cells. PMID- 10755409 TI - Osteoblastic differentiation and P-glycoprotein multidrug resistance in a murine osteosarcoma model. AB - A recent study of multidrug resistance (MDR) 1 gene transfected osteosarcoma cells found a cause-effect relationship between increased expression of P glycoprotein (P-gp) and a low aggressive phenotype. However, several experimental and clinical studies have observed contradictory findings in that P-gp expression has been associated with tumour progression. In the present study, we characterized P-gp-positive and P-gp-negative single-cell clones of a murine osteosarcoma, to further investigate the relationship between P-gp expression and changes in cell phenotype. Although these clones were all selected by doxorubicin (DOX) exposure, they were heterogeneous with respect to MDR1 gene expression. The P-gp-positive clones revealed MDR phenotype, whereas the P-gp-negative clones showed no resistance to drugs. Morphological and functional analysis showed that both the P-gp-positive and P-gp-negative clones were more differentiated than the parent cells in terms of enhanced activity of cellular alkaline phosphatase, an increase in well-organized actin stress fibres and enhanced osteogenic activity. Moreover, these subclones all displayed a decrease in malignant potential such as oncogenic activity, tumour growth rate and metastatic ability, regardless of their P-gp status. These results indicate that the observed osteoblastic differentiation and less aggressive phenotype in DOX-selected osteosarcoma cells may not only be explained by the direct effect of P-gp, and accordingly, consideration of the effect of DOX, as well as P-gp, appears to be important. PMID- 10755411 TI - Ethnic group and survival from childhood cancer: report from the UK Children's Cancer Study Group. AB - Survival following cancer was analysed in relation to ethnic group among children diagnosed in Britain during 1981-1996 and treated at paediatric oncology centres by members of the UK Children's Cancer Study Group. Survival was analysed for 11 diagnostic groups: acute lymphoblastic leukaemia (ALL), acute non-lymphocytic leukaemia, Hodgkin's disease, non-Hodgkin's lymphoma, astrocytoma, primitive neuroectodermal tumour, neuroblastoma, Wilms' tumour, osteosarcoma, Ewing's sarcoma and rhabdomyosarcoma. There were no significant differences in survival between White and non-White children over the study period as a whole. Among children with ALL, however, the relative risk of death allowing for period of diagnosis, age and white blood count was 1.25 for those of South Asian ethnic origin compared with Whites (P = 0.057). PMID- 10755410 TI - Smoking, diet, pregnancy and oral contraceptive use as risk factors for cervical intra-epithelial neoplasia in relation to human papillomavirus infection. AB - Smoking, nutrition, parity and oral contraceptive use have been reported as major environmental risk factors for cervical cancer. After the discovery of the very strong link between human papillomavirus (HPV) infection and cervical cancer, it is unclear whether the association of these environmental factors with cervical cancer reflect secondary associations attributable to confounding by HPV, if they are independent risk factors or whether they may act as cofactors to HPV infection in cervical carcinogenesis. To investigate this issue, we performed a population-based case-control study in the Vasterbotten county of Northern Sweden of 137 women with high-grade cervical intra-epithelial neoplasia (CIN 2-3) and 253 healthy age-matched women. The women answered a 94-item questionnaire on diet, smoking, oral contraceptive use and sexual history and donated specimens for diagnosis of present HPV infection (nested polymerase chain reaction on cervical brush samples) and for past or present HPV infections (HPV seropositivity). The previously described protective effects of dietary micronutrients were not detected. Pregnancy appeared to be a risk factor in the multivariate analysis (P < 0.0001). Prolonged oral contraceptive use and sexual history were associated with CIN 2-3 in univariate analysis, but these associations lost significance after taking HPV into account. Smoking was associated with CIN 2-3 (odds ratio (OR) 2.6, 95% confidence interval (CI) 1.7 4.0), the effect was dose-dependent (P = 0.002) and the smoking-associated risk was not affected by adjusting for HPV, neither when adjusting for HPV DNA (OR 2.5, CI 1.3-4.9) nor when adjusting for HPV seropositivity (OR 3.0, CI 1.9-4.7). In conclusion, after taking HPV into account, smoking appeared to be the most significant environmental risk factor for cervical neoplasia. PMID- 10755412 TI - Second primary neoplasms following non-Hodgkin's lymphoma in New South Wales, Australia. AB - The incidence of non-Hodgkin's lymphoma (NHL) has been increasing rapidly over the last three decades. The reasons for this trend are not known although increasing exposure to sunlight has been postulated. We used data from the New South Wales Central Cancer Registry to analyse second primary neoplasms following NHL diagnosed between 1972 and 1995, to identify possible common causal agents. A total of 12,452 patients contributed 54,308 person-years of follow-up during which time there were 705 second primary neoplasms compared to 592.99 expected (standardized incidence ratio (SIR = 1.19, 95% confidence interval (CI) 1.10 1.28). There were excesses of melanomas of skin (SIR = 2.38, 95% CI 1.92-2.91), lip cancer (SIR = 2.74, 95% CI 1.59-4.38), tongue cancer (SIR = 2.53, 95% CI 1.09 4.99) and bladder cancer (SIR = 1.64, 95% CI 1.19-2.21). There was also over a threefold excess in soft tissue sarcomas (SIR = 3.61, 95% CI 1.80-6.45) and in thyroid cancer (SIR = 3.42, 95% CI 1.56-6.49). The SIR for myeloid leukaemia was 0.78 (95% CI 0.28-1.69). The increases in melanoma of the skin and cancer of the lip and tongue among patients with NHL strongly suggest sunlight exposure as a shared causal agent. The increase in soft tissue sarcomas might be due to shared effects of exposure to chemicals such as phenoxy acid herbicides. The increases in bladder and thyroid cancers are likely to be explained by effects of treatment for NHL. We did not find a chemotherapy related increased risk of myeloid leukaemia among NHL patients. PMID- 10755413 TI - Association between high-risk HPV types, HLA DRB1* and DQB1* alleles and cervical cancer in British women. AB - Cervical scrapes from 116 British women referred with cervical cancer were tested for the presence of high oncogenic risk human papillomavirus (HPV) genotypes (HPV(hr)). Ninety-four per cent of the scrapes had one or more of these virus types and 66% were HPV16-positive. HPV18 was more frequent in adenocarcinoma. No evidence was found for an increased cancer risk associated with the HPV16 E6 350G variant. The HLA DRB1* and DQB1* alleles in these women and in 155 women with normal cytology and negative for HPV(hr) DNA were compared. DQB1*0301 alone (2P = 0.02) and in combination with DRB1*0401 (2P = 0.02) was found to be associated with cervical cancer. This was more marked in cancers positive for HPV types other than HPV16. In contrast, DRB1*1501 alone and in combination with DQB1*0602 was not significantly elevated in cancers overall, but did show some excess in HPV16-positive cancers (2P = 0.05), associated with HPV16-positive cervical cancers. Taking all cancers together, a marginally significant protective effect was found for DQB1*0501 (2P = 0.03) but no protective effect could be seen for DRB1*1301. PMID- 10755414 TI - Cancer among children of parents with autoimmune diseases. AB - Many different aetiologies for childhood cancer have been suggested, but few are well established. One is that parental autoimmune disease is linked with susceptibility for haematopoietic malignancies in their offspring during childhood. The present study is the first to investigate this hypothesis using a follow-up design. A cohort of 53,811 children of more than 36,000 patients diagnosed with a systemic, organ-specific or suspected autoimmune disease were followed up for cancer incidence in the Danish Cancer Registry during 1968-1993. The parents were identified through the National Registry of Patients, while their children were traced in the Central Population Register. Cancer incidence among the offspring was compared with that in the corresponding childhood population of Denmark. In total, 115 cancers were observed among children aged 0 19 years, yielding a non-significant standardized incidence ratio of 1.07. Lymphomas contributed 21 cases to the overall number of tumours, 60% more than expected (95% confidence interval (CI) 1.0-2.4); leukaemia contributed 37 cases representing an excess of 30% (95% CI 0.9-1.8). Our results give some support to the hypothesis that parental autoimmune disease is associated with childhood lymphoma and leukaemia. PMID- 10755415 TI - Socio-economic and lifestyle factors associated with the risk of prostate cancer. AB - International and interethnic differences in prostate cancer incidence suggest an environmental aetiology, and lifestyle and socio-economic factors have been studied, but with divergent results. Information on a cohort of 22,895 Norwegian men aged 40 years and more was obtained from a health examination and two self administered questionnaires. Information on incident cases of prostate cancer was made available from the Cancer Registry. We used the Cox proportional hazards model to calculate incidence rate ratios as estimates of the relative risk (RR) with 95% confidence interval (CI). Reported P-values are two-sided. During a mean follow-up of 9.3 years, 644 cases were diagnosed. Risk was elevated among men in occupations of high compared to low socio-economic status (RR = 1.30; 95% CI 1.05 1.61), and among men with high education compared to the least educated (RR = 1.56; 95% CI 1.11-2.19). A RR of 1.56 (95% CI 0.97-2.44) suggests a higher risk among divorced or separated men, compared with married men. We also found indications of a weak negative association with leisure-time physical activity (RR = 0.80; 95% CI 0.62-1.03 for high vs low activity), a weak positive association with increasing number of cigarettes (P = 0.046), while alcohol consumption was not related to the risk of prostate cancer. These results show that high socio-economic status is associated with increased risk of prostate cancer, and that divorced or separated men might be at higher risk than married men. Data from this study also indicate that high levels of physical activity may reduce prostate cancer risk. PMID- 10755416 TI - Non-steroidal anti-inflammatory drugs and bladder cancer prevention. AB - Inclusion of phenacetin among 'proven' human carcinogens by the IARC in 1987, raised concerns about the carcinogenic potential of acetaminophen, its major metabolite. Acetaminophen has been implicated as a possible causal agent in the development of cancer of the renal pelvis. The bladder and renal pelvis, which derive from the same embryological structure, share the same transitional type of epithelium. Past studies have been inconclusive on the possible relationship among these analgesics and bladder cancer but no large, highly detailed study of this association has been conducted. A population-based case-control study conducted in Los Angeles, California, involved 1514 incident bladder cancer cases and an equal number of controls who were matched to the index cases by sex, date of birth (within 5 years) and race. Detailed information on medication use and prior medical conditions was collected through in-person interviews. Regular use of analgesics was not associated with an increased risk of bladder cancer in either men or women. In fact, compared with non- or irregular users, regular analgesic users were at a decreased risk of bladder cancer overall (odds ratio (OR) = 0.81, 95% confidence interval (CI) = 0.68-0.96). However, there were clear differences in both the direction and strength of the associations between the different formulation classes of analgesics and bladder cancer risk. Intake of phenacetin was positively related to bladder cancer risk in a dose-dependent manner while intake of its major metabolite in humans, acetaminophen, was unrelated to risk. Intake of all classes of NSAIDs, except pyrazolon derivatives, were negatively associated with bladder cancer risk, with suggestive evidence that the protective effect varies in strength by subcategories of formulation. Acetic acids seemed to exhibit the strongest protective effect, whereas aspirin/other salicylic acids and oxicam showed the weakest protection. PMID- 10755417 TI - The knee after meniscectomy. PMID- 10755418 TI - At the crossroads--neonatal detection of developmental dysplasia of the hip. PMID- 10755419 TI - Articular cartilage--to repair or not to repair. PMID- 10755421 TI - Arrest of the growth plate after arterial cannulation in infancy. AB - Seven children who had partial arrest of the growth plate after neonatal arterial cannulation, developed obvious skeletal changes in adolescence. Cannulation of the femoral artery produced ischaemia which led to four cases of ipsilateral shortening of the lower limb and one of partial arrest of the proximal femoral physis with subsequent coxa valga. The two arrests in the upper limb affected the humerus, ulna and radius, and the radius alone, after cannulation of the brachial and radial arteries, respectively. These late effects of cannulation are not widely appreciated, and may occur as a result of thrombosis rather than extravasation. PMID- 10755422 TI - Cemented total hip arthroplasty with acetabular bone graft for developmental dysplasia. Long-term results and survivorship analysis. AB - Although the technique of autogenous acetabular bone grafting has been widely used to augment containment of the acetabulum in total hip arthroplasty (THA) for developmental dysplasia, the role of this technique in improving long-term results remains controversial. We present the long-term results of cemented THA with acetabular bone grafting in 112 patients (133 hips) in order to clarify the factors which affect the outcome. The mean follow-up was for 12.3 years (8 to 24). Kaplan-Meier survivorship analysis predicted a rate of survival of the acetabular component at 15 years of 96% (95% confidence interval (CI) 92 to 99) with revision for aseptic loosening as the endpoint, and of 75% (95% CI 65 to 85) when radiological loosening was used. Parametric survivorship analysis using the Cox proportional-hazards model indicated that trochanteric nonunion, lateral placement of the socket, and delayed trabecular reorientation of the bone graft were risk factors for loosening of the acetabular component. Our findings have shown that autologous acetabular bone grafting is of value for long-term success provided that the risk factors are reduced. PMID- 10755420 TI - Deprivation, urbanisation and Perthes' disease in Northern Ireland. AB - It has been suggested that Perthes' disease is more prevalent in urban areas, and that the risk increases with deprivation. We present the findings of a preliminary analysis of Perthes' disease in Northern Ireland, which is shown to have one of the highest national annual rates of incidence in the world (11.6 per 100000). Of the 313 children diagnosed over a seven-year period, 311 were allocated to the enumeration districts of the 1991 census, thus allowing the incidence to be calculated using both spatial and non-spatial aggregation. The cases were grouped according to the size of the settlement from highly urbanised to open countryside and by level of area deprivation. While the incidence of Perthes' disease was found to be associated with indicators of the level of deprivation for areas, there was no evidence to suggest that there was an increased risk in urban areas; the highest rate was found in the most deprived rural category. PMID- 10755423 TI - Poor results of the Freeman uncemented metal-backed acetabular component. Five-to nine-year results. AB - We reviewed 120 consecutive primary total hip replacements in 109 patients in whom a Freeman uncemented metal-backed component had been used. Acetabular components were used with a Freeman neck-retaining stem in one of three configurations: cemented smooth stem, uncemented smooth stem or uncemented ridged stem. After a mean follow up of 72 months (62 to 113) there were nine cases of aseptic loosening; a survivorship at eight years of 83%. In addition, more than one-third of the remaining surviving cups showed loosening radiologically. The Freeman acetabular component demonstrated a characteristic pattern of loosening on radiographs. The high incidence of aseptic loosening can be explained by poor design and material failure. PMID- 10755424 TI - The medium-term results of a cemented Freeman femoral neck-retaining prosthesis. AB - We have reviewed 202 consecutive primary hip replacements using a Freeman cemented femoral component after a mean period of follow-up of 64 months (23 to 113). There was only one revision for aseptic loosening. Retention of the femoral neck may act to reduce the torsional and shear forces at the implant-cement interface and may provide a seal against the migration of polyethylene-laden joint fluid in the potential joint space. The cemented Freeman femoral component with retention of the femoral neck was successful for up to nine years. PMID- 10755425 TI - Ionogran in revision arthroplasty. AB - Glass ionomer cement (Ionocem) was developed for use in bone surgery and is reported to be notably biocompatible. Between 1991 and 1994 we performed revision operations for aseptic loosening of arthroplasties of the hip on 45 patients using this material in its granulate form (Ionogran) mixed with homologous bone as a bone substitute. Of these 45 patients, 42 were followed up for a mean of 42 months. Early reloosening of the acetabular component has occurred in ten after a mean of 30 months. Histological examination showed large deposits of aluminium in the adjacent connective tissue and bone. Osteoblastic function and bone mineralisation were clearly inhibited. The serum levels of aluminium were also increased. The toxic damage at the bone interface caused by high local levels of aluminium must be seen as an important factor in the high rate of early reloosening. Our findings cast doubt on the biocompatibility of this material and we do not recommend continuation of its further use in orthopaedic surgery. PMID- 10755426 TI - Acute compartment syndrome. Who is at risk? AB - We have analysed associated factors in 164 patients with acute compartment syndrome whom we treated over an eight-year period. In 69% there was an associated fracture, about half of which were of the tibial shaft. Most patients were men, usually under 35 years of age. Acute compartment syndrome of the forearm, with associated fracture of the distal end of the radius, was again seen most commonly in young men. Injury to soft tissues, without fracture, was the second most common cause of the syndrome and one-tenth of the patients had a bleeding disorder or were taking anticoagulant drugs. We found that young patients, especially men, were at risk of acute compartment syndrome after injury. When treating such injured patients, the diagnosis should be made early, utilising measurements of tissue pressure. PMID- 10755427 TI - Displaced supracondylar fractures of the humerus in children. Audit changes practice. AB - We performed an audit of 71 children with consecutive displaced, extension-type supracondylar fractures of the humerus over a period of 30 months. The fractures were classified according to the Wilkins modification of the Gartland system. There were 29 type IIA, 22 type IIB and 20 type III. We assessed the effectiveness of guidelines proposed after a previous four-year review of 83 supracondylar fractures. These recommended that: 1) an experienced surgeon should be responsible for the initial management; 2) closed or open reduction of type IIB and type-III fractures must be supplemented by stabilisation with Kirschner (K-) wires; and 3) K-wires of adequate thickness (1.6 mm) must be used in a crossed configuration. The guidelines were followed in 52 of the 71 cases. When they were observed there were no reoperations and no malunion. In 19 children in whom they had not been observed more than one-third required further operation and six had a varus deformity. Failure to institute treatment according to the guidelines led to an unsatisfactory result in 11 patients. When they were followed the result of treatment was much better. We have devised a protocol for the management of these difficult injuries. PMID- 10755428 TI - Calcaneal fractures in children. Long-term results of treatment. AB - The late results of treatment of calcaneal fractures in 17 children (19 fractures) were reviewed at a mean of 16.8 years after injury. With the exception of one patient, all fractures had been treated conservatively. At follow-up there were few complaints. All but two patients had full or slightly reduced mobility of the subtalar joint and unrestricted foot function, including the ability to walk comfortably on uneven surfaces. Minor radiological abnormalities of the hindfoot were common; there were two cases of post-traumatic osteoarthritis. Clinical scoring of the ankle and hindfoot using the American Orthopaedic Foot and Ankle Society rating system averaged 96.2 points. The results suggest that up to 16.8 years after injury almost all children achieve excellent long-term functional results with conservative treatment of fractures of the os calcis. Open management may only be appropriate for adolescents with severe displacements. PMID- 10755429 TI - Total meniscectomy in adolescence. A thirty-year follow-up. AB - We have carried out a prospective, longitudinal 30-year review of 95 adolescents who underwent total meniscectomy in one knee, and have compared the results with those observed 13 years earlier. All the medical records were scrutinised. Of the 63 patients reviewed clinically, 47 reported decreased sporting activity, although subjective satisfaction rose by 3% to 71%. The scores on the WOMAC osteoarthritis index differed significantly between patients grouped by subjective global assessment. Satisfactory function scores increased from 48% to 60%. In the 53 patients consenting to bilateral radiography of the knee, the incidence of narrowing of the articular cartilage in the operated knee increased significantly between the reviews (19% to 36%). Progression of degenerative change paralleled reduction in activity. Outcome measures were best after medial, intermediate after lateral and worst after double meniscectomy. PMID- 10755430 TI - Estimation of the migration of tibial components in total knee arthroplasty. A roentgen stereophotogrammetric analysis. AB - Accurate quantitative measurements of micromovement immediately after operation would be a reliable indicator of the stability of an individual component. We have therefore developed a system for measuring micromovement of the tibial component using three non-contact displacement transducers attached to the tibial cortex during total knee arthroplasty (TKA). Using this system we measured the initial stability in 31 uncemented TKAs. All the tibial components were fixed by a stem and four screws. The initial stability was defined as the amount of displacement when a load of 20 kg was applied. The mean subsidence was 60.7 microm and the mean lift-off was 103.3 microm. We also studied the migration of the tibial component using roentgen stereophotogrammetric analysis (RSA) for up to two years after operation. Most migration occurred during the first six months, after which all prostheses remained stable. We defined migration as the maximum total point motion (MTPM) at two years after operation. The mean migration was 1.29 mm at two years. Our results show that there was a significant correlation between the initial stability and migration (p < 0.05) and emphasise the importance of the initial stability of the tibial component. PMID- 10755431 TI - The effect of the patellar tendon-bearing cast on loading. AB - We assessed the unloading effect of the patellar tendon-bearing (PTB) cast in five healthy volunteers using a new system for analysis of dynamic plantar pressure. We devised a method to improve the unloading effect of the PTB cast, and tested this using the same system. Our findings showed that the conventional PTB cast only achieved unloading of 30% of the body-weight and that the part of the cast on the leg had a more important role in the unloading than that which was in contact with the patellar tendon. When the depth of the free space under the foot inside the PTB cast was 1, 2 and 3 cm, the unloading effect was 60%, 80% and 98%, respectively. The unloading effect of the conventional PTB cast was disappointing at only 30% of body-weight. It was improved by producing a space between the sole of the foot and the cast, and was adjustable by altering the depth of this space. PMID- 10755432 TI - Treatment of the mobile, painful arthritic elbow by distraction interposition arthroplasty. AB - Between 1986 and 1994, 13 patients with mobile painful arthritic elbows were treated by distraction interposition arthroplasty using fascia lata. The mean period of follow-up was 63 months. An elbow distractor/fixator was applied for three to four weeks to separate the articular surfaces and to protect the fascial graft. Nine of the 13 patients (69%) had satisfactory relief from pain; eight (62%) had an excellent or good result by the objective criteria of the Mayo Elbow Performance score. Four have required revision to total elbow arthroplasty at a mean of 30 months with good results to date. Instability of the elbow, both before and after surgery, was found to be associated with unsatisfactory results. The rate of success when the procedure was performed for inflammatory arthritis was similar to that for post-traumatic arthritis, about 67%. Eight complications occurred in six patients, all in the group with post-traumatic arthritis. Two of these required further surgical procedures such as transposition of the ulnar nerve or repair of hernia of the fascia lata. Although less reliable than prosthetic replacement, distraction interposition arthroplasty is a useful option in the treatment of young, high-demand patients with arthritis of the elbow. It is rarely indicated in the presence of generalised inflammatory arthritis, but may be of value in those patients in whom the disease is limited primarily to the elbow. PMID- 10755433 TI - Lengthening of congenital below-elbow amputation stumps by the Ilizarov technique. AB - Patients with short congenital amputations below the elbow often function as if they have had a disarticulation of the elbow. We have reviewed the results in six patients who had lengthening of such stumps by the Ilizarov technique to improve the fitting of prostheses. The mean lengthening was 5.6 cm (3.4 to 8.4), and in two patients flexion contractures of the elbows were corrected simultaneously. Additional lateral distraction was used in one patient to provide a better surface on the stump. There were no major complications. All six patients were able to use their prosthesis at the latest follow-up after 39 to 78 months. PMID- 10755434 TI - Kinematics of the wrist. Evidence for two types of movement. AB - We enrolled 34 normal volunteers to test the hypothesis that there were two types of movement of the wrist. On lateral radiographs two distinct patterns of movement emerged. Some volunteers showed extensive rotation of the lunate with a mean range of dorsiflexion of 65 degrees, while others had a mean range of 50 degrees. The extensive rotators were associated with a greater excursion of the centre of articulation of the wrist. It is suggested that dynamic external fixation of a fracture of the distal radius carries with it the risk of stretching the ligaments or causing volar displacement at the site of the fracture. PMID- 10755435 TI - Functional outcome of surgery for fractures of the ankle. A prospective, randomised comparison of management in a cast or a functional brace. AB - We randomised prospectively 60 consecutive patients who were undergoing internal fixation of similar fractures of the ankle into two groups, one of which was treated by immobilisation in a below-knee cast and the other by a functional brace with early movement. All were instructed to avoid weight-bearing on the affected side. They were seen at 6, 12, 26 and 52 weeks. The functional rating scale of Mazur et al was used to evaluate the patients at each follow-up and we recorded the time of return to work. After one year the patients completed the SF 36 questionnaire. By then 55 patients remained in the study, 28 (mean age 45.5 years) in group 1 and 27 (mean age 39.5 years) in group 2. Those in group 2 had higher functional scores at each follow-up but only at six weeks was this difference significant (p = 0.02). They also had higher mean SF-36 scores, but this difference was significant only for two of the eight aspects investigated. For patients gainfully employed, not on workers' compensation, the mean time from surgery to return to work was 53.3 days for group 2 and 106.5 days for group 1; this difference was significant (p = 0.01). No patient developed a problem with the wound or had loss of fixation. Our findings support the use of a functional brace and early movement after surgery for fractures of the ankle. PMID- 10755436 TI - Function after correction of a clawed great toe by a modified Robert Jones transfer. AB - We carried out a cross-sectional study in 51 patients (81 feet) with a clawed hallux in association with a cavus foot after a modified Robert Jones tendon transfer. The mean follow-up was 42 months (9 to 88). In all feet, concomitant procedures had been undertaken, such as extension osteotomy of the first metatarsal and transfer of the tendon of the peroneus longus to peroneus brevis, to correct the underlying foot deformity. All patients were evaluated clinically and radiologically. The overall rate of patient satisfaction was 86%. The deformity of the hallux was corrected in 80 feet. Catching of the big toe when walking barefoot, transfer lesions and metatarsalgia, hallux flexus, hallux limitus and asymptomatic nonunion of the interphalangeal joint were the most frequent complications. Hallux limitus was more likely when elevation of the first ray occurred (p = 0.012). Additional transfer of the tendon of peroneus longus to peroneus brevis was a significant risk factor for elevation of the first metatarsal (p < 0.0001). The deforming force of extensor hallucis longus is effectively eliminated by the Jones transfer, but the mechanics of the first metatarsophalangeal joint are altered. The muscle balance and stability of the entire first ray should be taken into consideration in the management of clawed hallux. PMID- 10755437 TI - Extendible replacements of the proximal tibia for bone tumours. AB - Limb salvage is now customary in the treatment of primary bone tumours. The proximal tibia is a frequent site for these neoplasms but reconstruction, especially in children, is a formidable challenge. We reviewed 20 children with extendible replacements of the proximal tibia, all with a minimum follow-up of five years. Five died from their disease and, of the remaining 15, four had above knee amputations for complications. Infection occurred in seven patients; in five it was related to the lengthening procedure. Aseptic loosening is inevitable in the younger children and only two have avoided a revision, amputation or other major complication; both were aged 12 years at the time of the initial surgery. Despite this, 11 children are alive with a functioning leg and a mean Musculoskeletal Tumour Society functional score of 83%. The lengthening mechanisms used in our series required extensive open operations. We are now using a simpler, minimally invasive, technique which we hope will decrease the incidence of complications. At present, the use of extendible prostheses of the proximal tibia remains an experimental procedure. PMID- 10755439 TI - A foreign body in the spinal canal. A case report. AB - An 18-year-old man who presented with weakness in his lower limbs, had an upper motor neurone lesion at the D12-L1 level. At laminectomy two stone-like objects were found which proved to be bundles of tiny pieces of wood. They are thought to have entered the cord through an abdominal penetrating injury sustained six years previously. PMID- 10755438 TI - Chondrosarcoma in a family with multiple hereditary exostoses. AB - Multiple hereditary exostoses is an autosomal dominant skeletal disorder in which there are numerous cartilage-capped excrescences in areas of actively growing bone. The condition is genetically heterogeneous, and at least three genes, ext1, ext2 and ext3 are involved. The reported risk for malignant transformation to chondrosarcoma has been from 0.6% to 2.8%. We have reviewed six generations of a family with 114 living adult members, 46 of them with multiple exostoses. Four have had operations for chondrosarcoma, giving the risk for malignant transformation as 8.3% in this family. Clinical and radiological examination revealed two additional patients with a suspicion of malignancy, but in whom the histological findings were benign. Reported elsewhere in detail, genetic linkage analysis mapped the causative gene to chromosome 11 and molecular studies revealed a guanine-to-thymine transversion in the ext2 gene. Patients with multiple hereditary exostoses carry a relatively high risk of malignant transformation. They should be informed of this possibility and regularly reviewed. PMID- 10755440 TI - Intraoperative electroneurography in the assessment of the level of operation for cervical spondylotic myelopathy in the elderly. AB - We treated 31 patients aged 65 years or more with cervical spondylotic myelopathy by microsurgical decompression and fusion at a single most appropriate level, in spite of MRI evidence of compression at several levels. Spinal cord potentials evoked at operation localised the level responsible for the principal lesion at C3-4 in 18 patients, C4-5 in 11 and at C5-6 in two. Despite the frequent coexistence of other age-related conditions, impairing ability to walk, the average Nurick grade improved from 3.5 before operation to 2.2 at a mean follow up of 48 months. There was also good recovery of finger dexterity and sensitivity. Operation at a single optimal level, as opposed to several, has the advantage of minimising complications, of particular importance in this age group. PMID- 10755441 TI - Influence of extracorporeal irradiation on the reintegration of autologous grafts of bone and joint. Study in a canine model. AB - We studied the effects of irradiation on the reintegration of autologous osteoarticular grafts over a period of 24 weeks in a canine model. In 16 foxhounds the medial femoral condyle was resected, irradiated and immediately replanted. In the control group resection and replantation were performed without irradiation. Reintegration was assessed by macroscopic analysis, histology, radiography and gait analysis. Reintegration was equal at 12 weeks, but significantly inferior in the irradiated group after 24 weeks with delayed bone remodelling. The articular cartilage showed modest degeneration. Conventional radiography and histology showed corresponding changes. Limb function was adequate but the gait was inferior in the treated group. PMID- 10755442 TI - Human osteoblast-like cells phagocytose metal particles and express the macrophage marker CD68 in vitro. AB - Periprosthetic osteolysis is a major cause of aseptic loosening in artificial joint replacement. It is assumed to occur in conjunction with the activation of macrophages. We have shown in vitro that human osteoblast-like cells, isolated from bone specimens obtained from patients undergoing hip replacement, phagocytose fine particles of titanium alloy (TiAlV). The human osteoblast-like cells were identified immunocytochemically by the presence of bone-specific alkaline phosphatase (BAP). With increasing duration of culture, a variable number of the osteoblastic cells became positive for the macrophage marker CD68, independent of the phagocytosis of particles, with a fine granular cytoplasmic staining which was coexpressed with BAP as revealed by immunodoublestaining. The metal particles were not toxic to the osteoblastic cells since even in culture for up to four weeks massively laden cells were vital and had a characteristic morphology. Cells of the human osteosarcoma cell line (HOS 58) were also able to phagocytose metal particles but had only a low expression of the CD68 antigen. Fluorescence-activated cell scanning confirmed our immunocytochemical results. Additionally, the cells were found to be negative for the major histocompatibility complex-II (MHC-II) which is a marker for macrophages and other antigen-presenting cells. Negative results of histochemical tests for tartrate-resistant acid phosphatase excluded the contamination by osteoclasts or macrophages in culture. Our observations suggest that the osteoblast can either change to a phagocytosing cell or that the phagocytosis is an underestimated property of the osteoblast. The detection of the CD68 antigen is insufficient to prove the monocytic lineage. In order to discriminate between macrophages and osteoblasts additional markers should be used. To our knowledge, this is the first demonstration of cells of an osteoblastic origin which have acquired a mixed phenotype of both osteoblasts and macrophages. PMID- 10755443 TI - Osseointegration of Ti6Al4V alloy implants coated with titanium nitride by a new method. AB - Coating titanium alloy implants with titanium nitride (TiN) by the method of Powder Immersion Reaction Assisted Coating (PIRAC) produces a stable layer on their surface. We have examined the ability of the new TiN coating to undergo osseointegration. We implanted TiN-coated and uncoated Ti6Al4V alloy pins into the femora of six-month-old female Wistar rats. SEM after two months showed a bone collar around both TiN-coated and uncoated implants. Morphometrical analysis revealed no significant differences between the percentage of the implant-bone contact and the area and volume of the bone around TiN-coated compared with uncoated implants. Electron-probe microanalysis indicated the presence of calcium and phosphorus at the implant-bone interface. Mineralisation around the implants was also confirmed by labelling with oxytetracycline. Strong activity of alkaline phosphatase and weak activity of tartrate-resistant acid phosphatase were shown histochemically. Very few macrophages were detected by the non-specific esterase reaction at the site of implantation. Our findings indicate good biocompatibility and bone-bonding properties of the new PIRAC TiN coatings which are comparable to those of uncoated Ti6Al4V alloy implants. PMID- 10755444 TI - The fixation of the cemented femoral component. Effects of stem stiffness, cement thickness and roughness of the cement-bone surface. AB - After cemented total hip arthroplasty (THA) there may be failure at either the cement-stem or the cement-bone interface. This results from the occurrence of abnormally high shear and compressive stresses within the cement and excessive relative micromovement. We therefore evaluated micromovement and stress at the cement-bone and cement-stem interfaces for a titanium and a chromium-cobalt stem. The behaviour of both implants was similar and no substantial differences were found in the size and distribution of micromovement on either interface with respect to the stiffness of the stem. Micromovement was minimal with a cement mantle 3 to 4 mm thick but then increased with greater thickness of the cement. Abnormally high micromovement occurred when the cement was thinner than 2 mm and the stem was made of titanium. The relative decrease in surface roughness augmented slipping but decreased debonding at the cement-bone interface. Shear stress at this site did not vary significantly for the different coefficients of cement-bone friction while compressive and hoop stresses within the cement increased slightly. PMID- 10755445 TI - Prevention of deep-vein thrombosis after total knee replacement. PMID- 10755446 TI - Polyethylene wear, osteolysis and acetabular loosening with an HA-coated hip prosthesis. PMID- 10755447 TI - Is diagnostic arthroscopy of the hip worthwhile? PMID- 10755448 TI - Osteoid osteoma. PMID- 10755449 TI - Infections associated with dental procedures in total hip arthroplasty. PMID- 10755450 TI - Degenerative spondylolisthesis: developmental or acquired. PMID- 10755451 TI - Idiopathic scoliosis in twins studied by DNA fingerprinting. PMID- 10755452 TI - Radiological factors influencing femoral and acetabular failure in cemented Charnley total hip arthroplasties. PMID- 10755453 TI - Functional outcome at 5 years in children with obstetrical brachial plexus palsy with and without microsurgical reconstruction. AB - Between 1987 and 1998, 470 patients visited the national clinic for obstetrical brachial plexus palsies at the Karolinska Hospital, Stockholm, Sweden. This study reports on 247 children examined at 5 years of age using a special protocol for testing the sensory and motor function of the hand. The children were distributed in various groups depending on the number of injured nerves, whether they had some muscle activity in their biceps or deltoid muscles at 3 months of age, and whether any operations with nerve reconstruction had been performed. The shoulder range of movement in C5-6 palsies was significantly better in the group which had been operated on (operated group), but otherwise there were no differences between children who had been operated on (non-operated group) and those who had not. A decrease in grip strength and bimanual function in level C5-6 palsies was found, though these roots should not innervate the distal hand. Outcome was not influenced by the number of avulsions in upper-plexus palsies or whether the operation took place before or after the age of 6 months. The group with extensive lesions (C5-Th1) had the most root avulsions, showing a correlation between increased avulsions and decreased hand function. This study does not support operating on children with no activity of the biceps and deltoid muscles at 3 months of age, as other authors have concluded. Rather, it favours waiting for a late recovery. PMID- 10755454 TI - The findings in paediatric obstetric brachial palsy differ from those in older patients: a suggested explanation. AB - An EMG and nerve-conduction-study protocol has been developed and used in all patients with brachial plexus lesions since 1985. The findings of EMG and nerve conduction studies in obstetric brachial palsy (OBP) mostly suggest a falsely optimistic prognosis. To analyse this, all subjects were selected with complete avulsion of both roots C5 and C6 and/or complete rupture of the upper trunk verified during operation from a group of 162 infants with OBP (aged 4 to 14 months) and a group of 184 child and adult patients (aged 6 to 74 years) with a traumatic brachial plexus lesion. Fourteen infants, approximately 4 months old, with OBP, and 19 adults (aged 16 to 30 years) met the selection criteria. The infants from the group with OBP had at 4 months a nearly normal recruitment pattern of motor units in the biceps brachii and deltoid muscles, with little or no denervation. However, in the older group with traumatic brachial palsy, the same lesion caused complete denervation of both muscles. From the group with OBP, a third group (N=3) with the same lesion plus an additional lesion of C7 or a rupture of the middle trunk was selected. This additional lesion resulted in nearly complete denervation of both muscles. This means that C7 largely contributes to the innervation of both muscles at the time of birth, but this innervation is lost during later life in normally developing individuals (apoptosis). A central mechanism might be responsible for the clinical palsy and later spontaneous improvement which is always found in the infants with OBP. PMID- 10755455 TI - Identification of malnutrition in children with cerebral palsy: poor performance of weight-for-height centiles. AB - This study aims to evaluate the use of the United States National Center for Health Statistics (NCHS) weight-for-height centiles (WHC) in screening children with cerebral palsy (CP) for depleted body fat and to identify an alternate screening method. Growth data from 276 children aged from 3 to 12 years with CP were analyzed retrospectively. Height or a proxy for height, mid-upper arm circumference, weight, and skinfold thicknesses were recorded. Mid-upper arm fat area was calculated for each participant. The sensitivities and specificities of WHC and a number of alternative anthropometric screening methods for identifying participants with severely depleted fat stores were determined. WHC <10th centile failed to identify 45% of children with severely depleted fat stores. Triceps skinfold thickness <10th centile identified 96% of malnourished children. WHC standards lack adequate sensitivity for identification of severely depleted fat stores in children with CP. Use of triceps skinfold thickness, using cut-off value of <10th centile for age and sex, is recommended to screen for suboptimal fat stores in children with CP. PMID- 10755456 TI - Respiratory sinus arrhythmia during feeding: a measure of vagal regulation of metabolism, ingestion, and digestion in preterm infants. AB - Respiratory sinus arrhythmia (RSA), a non-invasive indicator of vagal regulation of the heart, and heart period (HP) were monitored before, during, and after oral or gastric-tube bolus feedings in 32 preterm infants. Group 1 infants (n=15) were < or =30 weeks gestational age (GA) at birth (mean 28.3 weeks) and group 2 infants (n=17) were > or =31 weeks GA at birth (mean=33.2 weeks). Mean postmenstrual ages at the time of study were 33.5 +/- 2.3 (SD) weeks in group 1 and 33.9 +/- 1.6 (SD) weeks in group 2. RSA and HP decreased in both groups during feeding. However, postfeeding RSA and HP increased toward prefeed levels only for group 2 infants. In addition, RSA and HP changes during feeding were correlated only for group 2 infants. The results suggest that the preterm infant may experience a maturational lag in vagal function and in the influence of vagal activity on metabolic mechanisms (i.e. heart rate) related to ingestive needs. This maturational lag may contribute to continued feeding difficulties and may be a measurable marker of subtle neurodevelopmental problems. PMID- 10755457 TI - A randomized controlled trial of early dietary supply of long-chain polyunsaturated fatty acids and mental development in term infants. AB - The effects of dietary docosahexaenoic acid (DHA) supply during infancy on later cognitive development of healthy term infants were evaluated in a randomized clinical trial of infant formula milk supplemented with 0.35% DHA or with 0.36% DHA and 0.72% arachidonic acid (AA), or control formula which provided no DHA or AA. Fifty-six 18-month-old children (26 male, 30 female) who were enrolled in the trial within the first 5 days of life and fed the assigned diet to 17 weeks of age were tested using the Bayley Scales of Infant Development, 2nd edition (BSID II) (Bayley 1993) at the Retina Foundation of the Southwest, Dallas, TX. These children had also been assessed at 4 months and 12 months of age for blood fatty acid composition, sweep visual evoked potential (VEP) acuity, and forced-choice preferential looking (FPL) acuity (Birch et al. 1998). Supplementation of infant formula with DHA+AA was associated with a mean increase of 7 points on the Mental Development Index (MDI) of the BSID-II. Both the cognitive and motor subscales of the MDI showed a significant developmental age advantage for DHA- and DHA+AA supplemented groups over the control group. While a similar trend was found for the language subscale, it did not reach statistical significance. Neither the Psychomotor Development Index nor the Behavior Rating Scale of the BSID-II showed significant differences among diet groups, consistent with a specific advantage of DHA supplementation on mental development. Significant correlations between plasma and RBC-DHA at 4 months of age but not at 12 months of age and MDI at 18 months of age suggest that early dietary supply of DHA was a major dietary determinant of improved performance on the MDI. PMID- 10755458 TI - Sensitivity of the pendulum test for assessing spasticity in persons with cerebral palsy. AB - The sensitivity of the pendulum test to variation in spasticity in persons with spastic cerebral palsy (CP) was tested in 30 participants with CP and 10 participants without CP (controls) (mean age 13.8 years). The participants with CP were classified into three groups, with normal (mean age 15.9 years), mild/moderate (13.0 years), or severe (23.0 years) muscle tone, as assessed clinically using a modified Ashworth scale. Joint motion during the pendulum test was measured with an electrogoniometer. Muscle relaxation was confirmed using surface EMG. Outcome measures from the pendulum test were (1) number of oscillations, (2) duration of oscillations, (3) excursion of the first backward swing, and (4) relaxation index (first swing excursion/difference between the starting and resting angles). Data were assessed using one-way analysis of variance. Outcome measures 1 to 3 differed significantly between control participants and participants with CP (p<0.05). The first swing excursion was the best predictor of the degree of spasticity in persons with CP, being significantly different between all groups (p<0.05). The number of oscillations and their duration differentiated between control participants and all participants with CP (p<0.05) but not between participants with CP who had mild/moderate versus severe spasticity (p>0.05). The relaxation index was not a sensitive measure (p>0.05 between most study groups). We conclude that the pendulum test is a valid tool for assessing spasticity in persons with CP and that the first swing excursion is the most sensitive outcome measure. PMID- 10755459 TI - The feasibility of universal screening for primary speech and language delay: findings from a systematic review of the literature. AB - This paper reports on a systematic review of the literature commissioned to examine the feasibility of universal screening for speech and language delay. The results, based on an examination of productivity figures, including positive predictive ability and likelihood ratio, indicate that a number of screening tests are adequate. Sensitivity was generally lower than specificity, and study quality was inversely related to both sensitivity and likelihood ratio, suggesting that it is easier to identify accurately children who do not have language and speech problems than those who do. The review concluded that there is insufficient evidence to warrant the introduction of universal screening at this stage. This paper discusses the type of data that would be needed to address this issue further and recommendations are made for alternative approaches to early identification. PMID- 10755461 TI - Nutritional supplementation in Down syndrome: theoretical considerations and current status. PMID- 10755460 TI - Neurodevelopmental profile of a new dysmorphic syndrome associated with submicroscopic partial deletion of 1p36.3. AB - We describe four children with dysmorphic syndrome with severe learning disability (SLD). Their chromosomes had been normal on conventional cytogenetic examination. However, screening using a multiprobe fluorescence in situ hybridisation (FISH) technique for subtelomeric abnormalities revealed a deletion of the p arm of chromosome 1. The physical features include body asymmetry, microcephaly, distinctive facies with deep-set eyes, sharply defined eye sockets, and mid-face hypoplasia; the neurodevelopmental profile was characterised by SLD, motor delay with hypotonia, markedly delayed visual maturation, and postural asymmetry together with epilepsy. This phenotype is consistent with that described for partial monosomy for 1p36.3. PMID- 10755462 TI - 'Developmental dysmusia (developmental musical dyslexia)'. PMID- 10755463 TI - Effects of phytoestrogens on aromatase, 3beta and 17beta-hydroxysteroid dehydrogenase activities and human breast cancer cells. AB - Isoflavones and others phytoestrogens have been suggested to be anticarcinogenic. Anti-aromatase, antiestrogenic or antiproliferative actions of these compounds have been postulated and related to the observation that there is a reduced incidence of breast cancer associated with diet. In this study, we explored some mechanisms by which they can exert cancer-preventive effects. Phytoestrogens were tested for estimating anti-aromatase, anti-3beta-hydroxysteroid dehydrogenase delta5/delta4 isomerase (3beta-HSD) and anti-17beta-hydroxysteroid dehydrogenase (17beta-HSD) activities in human placental microsomes. We found that isoflavonoids and compounds which presented the phenolic B ring in the 3 position on the pyran ring preferentially inhibited 3beta-HSD and/or 17beta-HSD activities than aromatase activity. We also evaluated their interactions with the estrogen receptor using a stably transfected human breast cancer cell line (MVLN). On the other hand phytoestrogens were evaluated for their effects on the proliferation in estrogen-dependent (MCF-7) and independent (MDA-MB231) human breast cancer cells. We established a relationship structure-activity and determined regions or/and substituents essential for these different activities. However, at high concentrations it seems that some phytoestrogens exert their protection against breast cancer through other estrogen-independent mechanisms. PMID- 10755464 TI - Effect of buprenorphine on CYP3A activity in rat and human liver microsomes. AB - Buprenorphine is a partial opioid agonist available in France as an alternative to methadone in the treatment of opiate-dependent individuals. Twenty deaths have been reported in patients who have ingested buprenorphine in combination with benzodiazepines. Since buprenorphine and many benzodiazepines are CYP3A substrates, the effect of buprenorphine on CYP3A activity was examined in order to assess the likelihood of a pharmacokinetic interaction. The formation of 6beta hydroxytestosterone was measured in dexamethasone-induced rat liver microsomes and in human liver microsomes under control conditions and in the presence of buprenorphine. Buprenorphine was found to be a weak inhibitor of CYP3A with a 50% decrease in enzyme activity occurring at a concentration of 118 microM (IC50) in human liver microsomes. IC50 was 0.3 microM for ketoconazole in the same system. Since the IC50 for buprenorphine is roughly 2000 times higher than typical plasma concentrations, this drug is unlikely to cause clinically significant inhibition of CYP3A in patients. Excessive CNS depression due to the combination of buprenorphine and benzodiazepines is most likely due to additive or synergistic pharmacologic effect unrelated to a pharmacokinetic interaction between the drugs. PMID- 10755465 TI - Exposure to a hypogeomagnetic field or to oscillating magnetic fields similarly reduce stress-induced analgesia in C57 male mice. AB - Previous studies have shown that exposure to altered magnetic fields alters analgesic responses in a variety of species, including humans. Here we examined whether deprivation of the normally occurring geomagnetic field also affects stress-induced analgesia, by measuring the nociceptive responses of C57 male mice that were restraint-stressed in a hypogeomagnetic environment (inside a mu-metal box). Stress-induced analgesia was significantly suppressed in a manner comparable to that observed in mice that were either exposed to altered oscillating magnetic fields or treated with the prototypic opiate antagonist naloxone. These results represent the first piece of evidence that a period in a hypogeomagnetic environment inhibits stress-induced analgesia. PMID- 10755467 TI - Testicular hormonal regulation of the renin-angiotensin system in the rat epididymis. AB - Evidence for the existence of an intrinsic angiotensin system based on locally formed angiotensinogen as a precursor for angiotensin production has been demonstrated in the rat epididymis. The data strongly support the presence of an epididymal renin-angiotensin system (RAS) which may be important for epididymal and sperm functions. In the present study, the effects of castration and testicular hormonal replacement on the expression of RAS components from the rat epididymis are investigated at the gene and protein levels. Results from northern blot and western blot analyses consistently showed that the expression of angiotensinogen mRNA and protein was apparently abolished by castration whereas their expression was completely restored to control levels when the castrated rats were hormonally replaced with either testosterone alone or with combined testosterone and estradiol. Northern blot did not detect any signal for angiotensinogen mRNA while western blot could detect a weak signal for angiotensinogen protein when the castrated rats were replaced with estradiol alone. Renin could be detected neither in control, castrated nor hormonally replaced rats. Moreover, the expression of angiotensin II receptor, type I (AT1) was almost abolished by castration as demonstrated by northern blot and reverse transcription-polymerase chain reaction. These data indicate that the expression of RAS by the rat epididymis at the levels of its precursor angiotensinogen and its receptor AT1, is subject to the regulation of testicular hormones and its expression appears to be predominantly testosterone-dependent. PMID- 10755466 TI - A secretin releasing peptide exists in dog pancreatic juice. AB - Canine pancreatic juice has been shown to stimulate exocrine pancreatic secretion in the dog. In the present study we investigated whether there is a secretin releasing peptide in canine pancreatic juice. Pancreatic juice was collected from the dogs with Thomas gastric and duodenal cannulas while pancreatic secretion was stimulated by intravenous administration of secretin at 0.5 microg/kg/h and CCK-8 at 0.2 microg/kg/h, respectively. The pancreatic juice was separated into three different molecular weight (MW) fractions (Fr) by ultrafiltration (Fr 1; MW > 10,000, Fr 2; MW=10,000-4,000 and Fr 3; MW < 4,000), respectively. All the fractions were bioassayed in anesthetized rats. Fraction 3 dose-dependently and significantly stimulated pancreatic juice flow volume from 78.0% to 99.4% (p<0.05) and bicarbonate output from 128.9% to 202.1% (p<0.01), respectively. Plasma secretin concentration also increased from 1.2 +/- 0.5 pM to 5.0 +/- 0.8 pM and 6.0 +/- 1.0 pM (p<0.05). None of these fractions increased pancreatic protein secretion or plasma CCK level. The stimulatory effect of Fraction 3 on pancreatic secretion and the release of secretin was completely abolished by treatment with trypsin (1 mg/ml for 60 min at 37 degrees C) but not by heating (100 degrees C, 10 min). Intravenous injection of a rabbit anti-secretin serum, which rendered plasma secretin almost undetectable in rat plasma, also abolished Fr 3-stimulated pancreatic secretion of fluid and bicarbonate secretion. These observations suggest that a secretin-releasing peptide exists in the canine pancreatic juice. It is trypsin-sensitive and heat-resistant. This peptide may play a significant physiological role on the release of secretin and regulation of exocrine pancreatic secretion. PMID- 10755469 TI - Acute ethanol administration and transient ischemia: a behavioral and neuropathological study. AB - A pressing clinical question is how acute ethanol exposure might alter the outcome of a simultaneous transient ischemic attack (TIA), since ethanol is known to dysregulate key intermediary metabolites post-ischemia. Mongolian gerbils were administered ethanol (1 or 4 g/kg, s.c.) 1 hour before induction of transient ischemia, via bilateral carotid occlusions of 5 minutes duration. A control group was administered isotonic saline and rendered ischemic. All animals were maintained normothermic during the ischemic procedure. Subjects underwent behavioral assay of acquisition to the water maze 7 days after recovery from the surgery, and neuropathological examination 1-month after the ischemic brain insult. There were no behavioral or neuropathological between-group differences suggesting that mechanisms other than adverse ethanol-induced perturbations of ischemic processes predominate in mediating epidemiological findings of elevated stroke morbidity with high ethanol consumption. PMID- 10755468 TI - Tumor necrosis factor alpha enhances the cytotoxicity induced by nitric oxide in cultured cerebral endothelial cells. AB - It has been shown that independent sources of nitric oxide (NO) and the inflammatory cytokine tumor necrosis factor alpha (TNFalpha) contribute to the breakdown of the blood-brain barrier (BBB) in the pathogenesis of a number of brain disorders. However, the interaction of NO and TNFalpha has not been elucidated. The present study was designed to determine whether the toxicity induced by NO is altered by TNFalpha in brain capillary endothelial cells (BCECs), and if so, whether it is related to the generation of superoxide. TNFalpha (50-400 U/ml) did not produce toxicity until at a concentration of 800 U/ml. This toxic effect was completely blocked by copper-zinc superoxide dismutase (SOD)/catalase or N(omega)-nitro-L-arginine methyl ester (L-NAME) or oxyhemoglobin (HbO2). Sodium nitroprusside (SNP) reduced with 0.4 mM ascorbate (SNP/Vc) significantly increased Lactate dehydrogenase (LDH) efflux in a concentration-dependent manner. This cytotoxicity of SNP/Vc was also completely inhibited by SOD/catalase or HbO2. When SNP/Vc used in combination with 400 U/ml TNFalpha, a more remarkable LDH efflux was induced than SNP/Vc alone, even as little as 0.01 mM SNP/Vc was toxic, although a dose of 400 U/ml TNFalpha alone had no effect on LDH efflux. In addition, either 0.4 mM SNP/Vc and 800 U/ml TNFalpha alone or 0.4 mM SNP/Vc and 400 U/ml TNFalpha in combination significantly increased malondialdehyde (MDA) content, but nitric oxide synthase (NOS) activity was inhibited only by SNP/Vc and TNF in combination. These results suggest that TNFalpha enhances the toxicity of NO in BCECs and that at least part of this enhancement involves the generation of superoxide. PMID- 10755470 TI - Mitochondrial ultrastructure and density in a primate model of persistent tardive dyskinesia. AB - The use of neuroleptic drugs to treat schizophrenia is almost invariably associated with extrapyramidal movement disorders. One of these disorders, tardive dyskinesia (TD), can persist long after neuroleptic withdrawal suggesting that permanent neurological damage is produced. However, there appears to be no convincing pathology of TD and its pathogenesis remains unknown. Findings that neuroleptics interfere with normal mitochondrial function and produce mitochondrial ultrastructural changes in the basal ganglia of patients and animals suggest that mitochondrial dysfunction plays a role in TD. We have established a model for persistent TD in baboons that appears to involve compromised mitochondrial function. In this study, we evaluated two animals treated for 41 weeks with a derivative of haloperidol and two treated with vehicle only. Treatment was then withdrawn and the animals observed for a further 17-18 weeks. Treated animals developed abnormal orofacial signs that were consistent with TD. These symptoms persisted during the drug-free period. The animals were euthanased, the brains perfused-fixed then post-fixed in 4% paraformaldehyde and the caudate and putamen prepared for electron microscopy. Regardless of whether mitochondria were located in neural soma, excitatory terminals, glia or in non-somal neuropil there was no consistent difference either in size or number between treated and control animals. Thus, even if mitochondria in striatal neurons undergo ultrastructural alterations during neuroleptic therapy, these changes do not persist after drug withdrawal. PMID- 10755472 TI - Mycelial protoplast isolation and regeneration of Lentinus lepideus. AB - Generation of fungal protoplast is essential for fusion and transformation systems. Protoplast fusion offers great potential for the improvement of industrially important microorganisms. To establish conditions for the protoplast isolation and regeneration of the mycelia of Lentinus lepideus, various enzymes and osmotic stabilizers were examined. To investigate suitable medium for the culture of L. lepideus, the mycelia were grown in ten different media at 28 degrees C for 10 days. Among them potato dextrose agar (PDA) medium was found to be the best for colony growth. When Novozym 234, cellulase and beta-glucuronidase were added to the mycelia in combination or alone, Novozym 234 alone at the concentration of 10 mg/ml was the most effective for the protoplast yield. Purified spherical protoplasts of the mycelia were osmotically hypersensitive and further incubation of the mycelia with the lytic enzyme resulted in the older parts of the hyphae swollen. When we applied various osmotic stabilizers at the fixed concentration of 0.6 M on the protoplasts, the yields of protoplasts were increased until 4-hr incubation. However application of sucrose or MgSO4 led to further protection of protoplasts after that time and reached a plateau on 5- and 7-hr incubations, respectively. The suitable incubation time and optimal pH with the lytic enzyme for the maximum release of protoplasts were 6 hrs of incubation and pH 5, respectively. When we examined various osmotic stabilizers for the regeneration of the protoplast, the complete medium containing 0.6 M sucrose induced highest hyphal growth with regeneration frequency of 3.28%. PMID- 10755471 TI - Increase in receptor binding affinity for nimodipine in the rat brain with permanent occlusion of bilateral carotid arteries. AB - The permanent occlusion of bilateral common carotid arteries (2VO) in rats has been shown to cause progressive and long-lasting cognitive deficits which may be due to impairment of memory retention and/or memory recall process. To clarify the function of voltage dependent calcium channels and the receptor binding of nimodipine by chronic cerebral ischemia, we examined specific (+)-[3H]PN 200-110 binding and the effect of oral administration of nimodipine in brain regions and hearts of rats, at 2 weeks to 4 months after permanent 2VO. There was no significant difference in either dissociation constant (Kd) or maximal number of binding sites (Bmax) for (+)-[3H]PN 200-110 in the cerebral cortex, hippocampus, corpus striatum and thalamus between 2VO and sham rats. In addition, in vitro inhibitory effect of nimodipine on cerebral cortical (+)-[3H]PN 200-110 binding in 2VO rats was similar to that in sham rats. Compared to control rats, oral administration of nimodipine to both 2VO and sham rats at 2 months after permanent 2VO brought about a significant increase in Kd values of specific (+) [3H]PN 200-110 binding in the cerebral cortex, hippocampus, thalamus and myocardium, and the increase in Kd values was much larger in brain regions of 2VO rats than sham rats. However, the increase in Kd values in the myocardium did not differ between 2VO and sham rats. This observation suggests an increased in vivo binding affinity for nimodipine in chronic ischemic brain. In conclusion, the present study has shown that oral administration of nimodipine may cause a greater occupation in vivo of 1,4-dihydropyridine (DHP) calcium channel antagonist receptors in brains of permanent 2VO rats than in sham rats. Thus, nimodipine may be pharmacologically effective in preventing brain dysfunction due to cerebral ischemia in vivo. PMID- 10755473 TI - Protein constituent contributes to the hypotensive and vasorelaxant activities of Cordyceps sinensis. AB - Cordyceps sinensis is a herb medicine in China for the treatment of general debility after sickness and for persons of advanced age. In the present study, cordyceps sinensis was extract by phosphate buffer saline (PBS) and dialyzed overnight against PBS using a membrane cut off at 3,500 dalton molecular weight. The resulting macromolecule fraction (defined as CS) was assayed in anesthetized rats for hypotensive effects and in isolated aorta for vasorelaxant effects. Intravenous injection of CS (8,16, 24 and 32 mg/kg, respectively) suppressed significantly the mean arterial pressure (MAP) in a dose-dependent manner. 32 mg/kg of CS induces the maximal hypotensive response with a 58 +/- 4 mm Hg (from 107 +/- 6 to 49 +/- 3 mm Hg) change in MAP and a over 45 min action duration. In aortic rings precontracted with phenylephrine treatment with CS between 0.5 and 500 microg/ml induced dose dependent relaxation. Maximal vasorelaxant response evoked by 150 microg/ml CS was 68.9 +/- 7.3%. Furthermore, CS-induced vasorelaxation is mediated by the endothelium possibly by stimulating the release of the nitric oxide and endothelium-derived hyperpolarizing factor. In conclusion, the present study revealed that presence of a constituent in CS which reduces MAP by relaxing the vascular beds directly. However, the effect may be caused by a single active ingredient or by the combined action of many active agents found in the extract. PMID- 10755474 TI - Genetic variations of S-mephenytoin 4'-hydroxylase (CYP2C19) in the Chinese population. AB - Most of phenotyping studies have shown that Chinese populations have a higher incidence of poor metabolizers (PMs) of S-mephenytoin 4'-hydroxylation compared with populations of African and European descent. The present study was aimed at defining an exact population frequency of the genetic defect of S-mephenytoin 4' hydroxylase (CYP2C19) in native and overseas Chinese healthy populations. All the related data were systematically summarized and re-analyzed using meta-analysis method, and consistency between phenotypic and genotypic frequencies of the PM was tested. A statistically significant homogeneity was across all 11 phenotyping studies (chi2 = 15.17, d.f. = 10; P > 0.05) and also across the remaining 4 genotyping studies (chi2 = 2.61, d.f. = 3; P > 0.05) except for a non-randomly selected population analysis. An approximate estimate of the PM phenotypic and genotypic frequencies was 13.6% (212 of 1555; 95% CI: 11.9%-15.3%) and 13.8% (79 of 573; 95%CI: 11.0%-16.6%), respectively. There was a good consistency between phenotyped and genotyped PM frequencies. The half of all genotyped EMs (50.3%, 276 of 549) were heterozygotes. The data estimate that 14% of Chinese would be homozygotes of CYP2C19 defective alleles, and that 176 million Chinese would be slow metabolizers of CYP2C19 substrates. PMID- 10755475 TI - On the activity of trifluoperazine and palmitoylcarnitine in mice: delayed hypersensitivity models. AB - The effect of pre- and post-challenge treatments with trifluoperazine and palmitoylcarnitine, two protein kinase C (PKC) inhibitors characterised by their interaction with the phospholipid enzyme cofactor, on the inflammation caused by delayed hypersensitivity (DTH) to dinitrofluorobenzene (DNFB) and sheep red blood cells (SRBC) in mice is reported. The activity of dexamethasone and two immunosuppressors, azathioprine and methotrexate, is also evaluated. The effectiveness of pre-treatment with each of the test drugs diminished when the DNFB challenge dose increased, whereas trifluoperazine and azathioprine were more active when administered after the challenge at the high DNFB dose. Trifluoperazine, which is also a calmodulin-antagonist, was the more effective of the PKC inhibitors tested on DNFB-DTH (39% and 59% inhibition swelling 24 and 96 h after challenge, respectively). SRBC-DTH was sensitive only to the action of the drugs given after challenge. In this test, PKC inhibitors showed a moderate effect, in the same range as methotrexate, whereas dexamethasone suppressed the reaction. The ability of trifluoperazine in inhibiting cutaneous DTH reaction, depending on the treatment schedule and the hapten challenge dose, has been determined. PMID- 10755476 TI - Suppression of paraquat-induced wet dog shakes by nitric oxide synthase inhibitors in rats. AB - We have found that paraquat (PQ), a widely used herbicide, causes wet dog shakes (WDS), which involve the central opioid system, in rats. A non-selective nitric oxide (NO) synthase (NOS) inhibitor, N(omega)-nitro-L-arginine (L-NA), but not its less active enantiomer, N(omega)-nitro-D-arginine, decreased the PQ-induced WDS in a dose-related manner. A selective neuronal NOS inhibitor in vivo, 7 nitroindazole, also decreased the PQ-induced WDS. Although an opioid receptor antagonist, naloxone, reversed the suppressive effect of these NOS inhibitors on the PQ-induced WDS, L-arginine, an NO precursor, had no effect on it. These findings suggest that the suppression of the PQ-induced WDS by NOS inhibition is associated with the central opioid system and is insusceptible to exogenous L arginine. PMID- 10755477 TI - A piece of my mind. Faith and doubt. PMID- 10755478 TI - National Library of Medicine to help consumers use online health data. PMID- 10755479 TI - Research on children's anxiety needed. PMID- 10755481 TI - From the Food and Drug Administration. PMID- 10755480 TI - Ensuring ethical internet information. PMID- 10755482 TI - Medicine in uniform PMID- 10755483 TI - From the Centers for Disease Control and Prevention. Update: influenza activity- United States, 1999-2000 season. PMID- 10755484 TI - From the Centers for Disease Control and Prevention. Developing and expanding contributions of the global laboratory network for poliomyelitis eradication, 1997-1999. PMID- 10755485 TI - Reasons physicians do not follow clinical practice guidelines. PMID- 10755487 TI - Reasons physicians do not follow clinical practice guidelines. PMID- 10755486 TI - Reasons physicians do not follow clinical practice guidelines. PMID- 10755488 TI - Tourism and smoke-free restaurant ordinances. PMID- 10755489 TI - Clinical breast examination for detecting breast cancer. PMID- 10755490 TI - Clinical breast examination for detecting breast cancer. PMID- 10755491 TI - Clinical breast examination for detecting breast cancer. PMID- 10755492 TI - School-based scoliosis screening. PMID- 10755493 TI - Early-onset familial Alzheimer disease with coexisting beta-amyloid and prion pathology. PMID- 10755494 TI - Does cranberry juice have antibacterial activity? PMID- 10755495 TI - Effect of metformin and rosiglitazone combination therapy in patients with type 2 diabetes mellitus: a randomized controlled trial. AB - CONTEXT: Most antidiabetic agents target only 1 of several underlying causes of diabetes. The complementary actions of the antidiabetic agents metformin hydrochloride and rosiglitazone maleate may maintain optimal glycemic control in patients with type 2 diabetes; therefore, their combined use may be indicated for patients whose diabetes is poorly controlled by metformin alone. OBJECTIVE: To evaluate the efficacy of metformin-rosiglitazone therapy in patients whose type 2 diabetes is inadequately controlled with metformin alone. DESIGN: Randomized, double-blind, placebo-controlled trial from April 1997 and March 1998. SETTING: Thirty-six outpatient centers in the United States. PATIENTS: Three hundred forty eight patients aged 40 to 80 years with a mean fasting plasma glucose level of 12.0 mmol/L (216 mg/dL), a mean glycosylated hemoglobin level of 8.8%, and a mean body mass index of 30.1 kg/m2 were randomized. INTERVENTIONS: Patients were assigned to receive 2.5 g/d of metformin plus placebo (n = 116); 2.5 g/d of metformin plus 4 mg/d of rosiglitazone (n = 119); or 2.5 g/d of metformin and 8 mg/d of rosiglitazone (n = 113) for 26 weeks. MAIN OUTCOME MEASURES: Glycosylated hemoglobin levels, fasting plasma glucose levels, insulin sensitivity, and beta cell function, compared between baseline and week 26, by treatment group. RESULTS: Glycosylated hemoglobin levels, fasting plasma glucose levels, insulin sensitivity, and beta-cell function improved significantly with metformin rosiglitazone therapy in a dose-dependent manner. The mean levels of glycosylated hemoglobin decreased by 1.0% in the 4 mg/d metformin-rosiglitazone group and by 1.2% in the 8 mg/d metformin-rosiglitazone group and fasting plasma glucose levels by 2.2 mmol/L (39.8 mg/dL) and 2.9 mmol/L (52.9 mg/dL) compared with the metformin-placebo group (P<.001 for all). Of patients receiving 8 mg/d of metformin-rosiglitazone, 28.1% achieved a glycosylated hemoglobin level of 7% or less [corrected]. Dose-dependent increases in body weight and total and low density lipoprotein cholesterol levels were observed (P<.001 for both rosiglitazone groups vs placebo). The proportion of patients reporting adverse experiences was comparable across all groups. CONCLUSIONS: Our data suggest that combination treatment with once-daily metformin-rosiglitazone improves glycemic control, insulin sensitivity, and beta-cell function more effectively than treatment with metformin alone. PMID- 10755496 TI - Valvular abnormalities and cardiovascular status following exposure to dexfenfluramine or phentermine/fenfluramine. AB - CONTEXT: Fenfluramine and dexfenfluramine were voluntarily withdrawn from the market in September 1997 because of reports of an association with heart valve abnormalities. Studies have been limited by lack of comparison with untreated controls. OBJECTIVE: To evaluate cardiovascular status and the prevalence of valvular abnormalities, as assessed by clinical cardiovascular parameters and echocardiography, in patients treated for obesity with dexfenfluramine or phentermine/fenfluramine. DESIGN: Reader-blinded controlled study completed in February 1998. SETTING AND PARTICIPANTS: Twenty-five clinical centers in the United States. Of 1640 enrolled subjects, 1473 were eligible (479 and 455 had taken dexfenfluramine and phentermine/fenfluramine, respectively, continuously for 30 days or more in the previous 14 months, and 539 were untreated matched controls) and provided clinical and echocardiographic data. Mean (SD) age was 47.4 (11.4) years, mean body mass index was 35.0 (7.4) kg/m2, and 74% were women. Mean (SD) duration of therapy was 6.0 (3.3) months (range, 1-18.4 months) in the dexfenfluramine group, and 11.9 (10.4) months (range, 1.4-63 months) in the phentermine/fenfluramine group, while the untreated group had no anorexigen use during the previous 5 years. MAIN OUTCOME MEASURES: Cardiovascular signs and symptoms; echocardiographic evidence of aortic (AR) or mitral (MR) regurgitation according to US Food and Drug Administration (FDA) criteria (AR > or = mild or MR > or = moderate) and by grade; tricuspid and pulmonic valve regurgitation; and aortic, mitral, and tricuspid valve leaflet mobility and thickness, for treated vs untreated subjects. RESULTS: Cardiovascular signs and symptoms were similar among anorexigen-treated and untreated subjects. Prevalence rates and relative risk (RR) of AR were significantly increased in anorexigen-treated patients and were 8.9% in the dexfenfluramine group (RR, 2.18; 95% confidence interval [CI], 1.32-3.59), 13.7% in the phentermine/fenfluramine group (RR, 3.34; 95% CI, 2.09 5.35), and 4.1% in the untreated group (P<.001). No statistically significant differences in prevalence were observed for MR, thickening or decreased mobility of any valve leaflet, calculated pulmonary artery systolic pressure, or left ventricular ejection fraction. Serious cardiac events (including myocardial infarction, congestive heart failure, or ventricular arrhythmia) occurring at any time were not statistically different in treated and untreated subjects (dexfenfluramine, 9.0%; phentermine/fenfluramine, 4.0%; and untreated, 8.4%); and following anorexigen treatment were uncommon (dexfenfluramine, 2.3%; phentermine/fenfluramine, 2.4%, and untreated, 3.3%, when adjusted for the median start date of anorexigen use). CONCLUSIONS: Our data indicate that use of dexfenfluramine and phentermine/fenfluramine is associated with an increase in the prevalence of AR using FDA echocardiographic criteria, but was not associated with an increase in the prevalence of MR using FDA criteria or with serious cardiac events. PMID- 10755497 TI - Trends in medical use and abuse of opioid analgesics. AB - CONTEXT: Pain often is inadequately treated due in part to reluctance about using opioid analgesics and fear that they will be abused. Although international and national expert groups have determined that opioid analgesics are essential for the relief of pain, little information has been available about the health consequences of the abuse of these drugs. OBJECTIVE: To evaluate the proportion of drug abuse related to opioid analgesics and the trends in medical use and abuse of 5 opioid analgesics used to treat severe pain: fentanyl, hydromorphone, meperidine, morphine, and oxycodone. DESIGN AND SETTING: Retrospective survey of medical records from 1990 to 1996 stored in the databases of the Drug Abuse Warning Network (source of abuse data) and the Automation of Reports and Consolidated Orders System (source of medical use data). PATIENTS: Nationally representative sample of hospital emergency department admissions resulting from drug abuse. MAIN OUTCOME MEASURES: Medical use in grams and grams per 100,000 population and mentions of drug abuse by number and percentage of the population. RESULTS: From 1990 to 1996, there were increases in medical use of morphine (59%; 2.2 to 3.5 million g), fentanyl (1168%; 3263 to 41,371 g), oxycodone (23%; 1.6 to 2.0 million g), and hydromorphone (19%; 118,455 to 141,325 g), and a decrease in the medical use of meperidine (35%; 5.2 to 3.4 million g). During the same period, the total number of drug abuse mentions per year due to opioid analgesics increased from 32,430 to 34,563 (6.6%), although the proportion of mentions for opioid abuse relative to total drug abuse mentions decreased from 5.1% to 3.8%. Reports of abuse decreased for meperidine (39%; 1335 to 806), oxycodone (29%; 4526 to 3190), fentanyl (59%; 59 to 24), and hydromorphone (15%; 718 to 609), and increased for morphine (3%; 838 to 865). CONCLUSIONS: The trend of increasing medical use of opioid analgesics to treat pain does not appear to contribute to increases in the health consequences of opioid analgesic abuse. PMID- 10755498 TI - Comparison of vignettes, standardized patients, and chart abstraction: a prospective validation study of 3 methods for measuring quality. AB - CONTEXT: Better health care quality is a universal goal, yet measuring quality has proven to be difficult and problematic. A central problem has been isolating physician practices from other effects of the health care system. OBJECTIVE: To validate clinical vignettes as a method for measuring the competence of physicians and the quality of their actual practice. DESIGN: Prospective trial conducted in 1997 comparing 3 methods for measuring the quality of care for 4 common outpatient conditions: (1) structured reports by standardized patients (SPs), trained actors who presented unannounced to physicians' clinics (the gold standard); (2) abstraction of medical records for those same visits; and (3) physicians' responses to clinical vignettes that exactly corresponded to the SPs' presentations. Setting Outpatient primary care clinics at 2 Veterans Affairs medical centers. PARTICIPANTS: Ninety-eight (97%) of 101 general internal medicine staff physicians, faculty, and second- and third-year residents consented to be randomized for the study. From this group, 10 physicians at each site were randomly selected for inclusion. MAIN OUTCOME MEASURES: A total of 160 quality scores (8 cases x 20 physicians) were generated for each method using identical explicit criteria based on national guidelines and local expert panels. Scores were defined as the percentage of process criteria correctly met and were compared among the 3 methods. RESULTS: The quality of care, as measured by all 3 methods, ranged from 76.2% (SPs) to 71.0% (vignettes) to 65.6% (chart abstraction). Measuring quality using vignettes consistently produced scores closer to the gold standard of SP scores than using chart abstraction. This pattern was robust when the scores were disaggregated by the 4 conditions (P<.001 to <.05), by case complexity (P<.001), by site (P<.001), and by level of physician training (P values from <.001 to <.05). The pattern persisted, although less dominantly, when we assessed the component domains of the clinical encounter -history, physical examination, diagnosis, and treatment. Vignettes were responsive to expected directions of variation in quality between sites and levels of training. The vignette responses did not appear to be sensitive to physicians' having seen an SP presenting with the same case. CONCLUSIONS: Our data indicate that quality of health care can be measured in an outpatient setting by using clinical vignettes. Vignettes appear to be a valid and comprehensive method that directly focuses on the process of care provided in actual clinical practice. Vignettes show promise as an inexpensive case-mix adjusted method for measuring the quality of care provided by a group of physicians. PMID- 10755500 TI - American medical malpractice litigation in historical perspective. AB - Medical malpractice and the problems associated with it remain an important issue in the US medical community. Yet relatively little information regarding the long term history of malpractice litigation can be found in the literature. This article addresses 2 questions: (1) when and why did medical malpractice litigation originate in the United States and (2) what historical factors best explain its subsequent perpetuation and growth? Medical malpractice litigation appeared in the United States around 1840 for reasons specific to that period. Those reasons are discussed in the context of marketplace professionalism, an environment that provided few quality controls over medical practitioners. Medical malpractice litigation has since been sustained for a century and a half by an interacting combination of 6 principal factors. Three of these factors are medical: the innovative pressures on American medicine, the spread of uniform standards, and the advent of medical malpractice liability insurance. Three are legal factors: contingent fees, citizen juries, and the nature of tort pleading in the United States. Knowledge of these historical factors may prove useful to those seeking to reform the current medical malpractice litigation system. PMID- 10755499 TI - E5 murine monoclonal antiendotoxin antibody in gram-negative sepsis: a randomized controlled trial. E5 Study Investigators. AB - CONTEXT: Knowledge and understanding of gram-negative sepsis have grown over the past 20 years, but the ability to treat severe sepsis successfully has not. OBJECTIVE: To assess the efficacy and safety of E5 in the treatment of patients with severe gram-negative sepsis. DESIGN: A multicenter, double-blind, randomized, placebo-controlled trial conducted at 136 US medical centers from April 1993 to April 1997, designed with 90% power to detect a 25% relative risk reduction, incorporating 2 planned interim analyses. SETTING: Intensive care units at university medical centers, Veterans Affairs medical centers, and community hospitals. PATIENTS: Adults aged 18 years or older, with signs and symptoms consistent with severe sepsis and documented or probable gram-negative infection. INTERVENTION: Patients were assigned to receive 2 doses of either E5, a murine monoclonal antibody directed against endotoxin (n = 550; 2 mg/kg per day by intravenous infusion 24 hours apart) or placebo (n = 552). MAIN OUTCOME MEASURES: The primary end point was mortality at day 14; secondary end points were mortality at day 28, adverse event rates, and 14-day and 28-day mortality in the subgroup without shock at presentation. RESULTS: The trial was stopped after the second interim analysis. A total of 1090 patients received study medication and 915 had gram-negative infection confirmed by culture. There were no statistically significant differences in mortality between the E5 and placebo groups at either day 14 (29.7% vs 31.1%; P = .67) or day 28 (38.5% vs 40.3%; P = .56). Patients presenting without shock had a slightly lower mortality when treated with E5 but the difference was not significant (28.9% vs 33.0% for the E5 and placebo groups, respectively, at day 28; P = .32). There was a similar profile of adverse event rates between E5 and placebo. CONCLUSIONS: Despite adequate sample size and high enrollment of patients with confirmed gram-negative sepsis, E5 did not improve short-term survival. Current study rationale and designs should be carefully reviewed before further large-scale studies of patients with sepsis are conducted. PMID- 10755501 TI - Heart valve disorders and appetite-suppressant drugs. PMID- 10755502 TI - The quest to quantify quality. PMID- 10755503 TI - A public health approach to reducing error: medical malpractice as a barrier. PMID- 10755505 TI - National interest waiver eligibility for international medical graduates. PMID- 10755504 TI - Continuing controversy over the international medical graduate. PMID- 10755506 TI - High-stakes medical performance testing: the Clinical Skills Assessment program. PMID- 10755507 TI - The posttraining plans of international medical graduates and US medical graduates in New York State. PMID- 10755508 TI - Overcoming cultural barriers: international medical graduates in the United States. PMID- 10755509 TI - JAMA Patient Page: pain management. PMID- 10755510 TI - N-acetylcysteine: not simply a glutathione precursor. PMID- 10755512 TI - Split liver transplantation. PMID- 10755511 TI - Some problems with posttransplant lymphoproliferative disease. PMID- 10755513 TI - Towards protection of the islands in the (blood)stream. PMID- 10755514 TI - A potential revolution in pretransplant histocompatibility testing: selective omission of the donor crossmatch. PMID- 10755516 TI - Selective omission of the donor cross-match before renal transplantation: efficacy, safety and effects on cold storage time. AB - BACKGROUND: A donor lymphocyte cross-match (XM) test performed before renal transplantation is considered mandatory but may delay the transplant and increase the cold storage time. With careful documentation of sensitizing events and with knowledge of previous antibody screening results, it is often possible to predict the XM result for a given donor HLA mismatch. In this study, a policy was adopted of omitting the pretransplant XM in patients in whom a negative result was predicted with absolute confidence. METHODS: Recipients were selected for cadaveric donor kidney transplantation using a computer algorithm based on HLA match, sensitization status, time on the waiting list and donor and recipient age. The immediate pretransplant cross-match test was omitted in non-sensitized recipients and in sensitized recipients where antibody specificities were precisely defined and not against donor HLA. RESULTS: From October 1997 to May 1999, 53 of 96 (55%) consecutive cadaveric kidney donor transplants were performed without a pretransplant XM. In all cases, a negative donor HLA-specific antibody XM was confirmed after transplantation. Omission of the pre-transplant XM was associated with a significant reduction in cold ischemic time (15.0 hr vs. 18.2 hr, P=0.01) and a reduced incidence of delayed graft function (13% vs. 33%, P=0.03). However, there was no difference in transplant outcome at 1 year. CONCLUSION: Rigorous attention to priming events together with careful antibody screening allows the pre-transplant XM test to be safely omitted in approximately half the patients awaiting renal transplantation. This policy allows a modest reduction in cold ischemia time, but it remains to be seen whether this is of clinical benefit. PMID- 10755515 TI - Damage to porcine islets of Langerhans after exposure to human blood in vitro, or after intraportal transplantation to cynomologus monkeys: protective effects of sCR1 and heparin. AB - BACKGROUND: Porcine islets offer an attractive alternative to human islets in clinical islet transplantation. The preferred method of islet transplantation is intra-portal injection into the liver. We have recently shown, both in vitro with human islets and in vivo with porcine islets, that islets exposed to allogeneic blood trigger an injurious inflammatory reaction characterized by activation of both coagulation and the complement systems. We have now tested whether a similar reaction is triggered when xenogeneic porcine islets are exposed to human blood in vitro and after intraportal transplantation into primates. Furthermore, we investigated the effect of inhibiting the complement and coagulation systems. METHOD: Islets isolated from adult and fetal porcine pancreas were perfused with fresh human blood in surface heparinized PVC tubings for 5-60 min. Blood cell counts and parameters related to coagulation and the complement system were analyzed, and islets were retrieved after the perifusion was examined by immunohistochemical method. Heparin and soluble complement receptor 1 (sCR1; TP10, 100 microg/ml) were added to the system in some experiments. Furthermore, adult porcine islets were transplanted intraportally into untreated and sCR1- (40 mg/kg BW i.v.) treated cynomolgus monkeys, and plasma insulin concentration was monitored during 60 min after transplantation. RESULTS: Porcine islets perifused with human blood triggered an immediate inflammatory reaction, characterized by a rapid consumption and activation of platelets, consumption of neutrophils and monocytes, activation of the coagulation and complement systems, and release of large amounts of insulin. Islet morphologic analysis revealed damaged islets embedded in clots and infiltrated with CD11+ leukocytes. C3a and C5b-9 was deposited on the islet surface, but human immunoglobulin was not. Complement inhibition with sCR1 reduced insulin release significantly. Intraportal islet transplantation into untreated cynomolgus monkeys resulted in a marked and rapid increase in plasma insulin concentration indicative of islet damage. Pretreatment of the monkeys with sCR1 resulted in significantly less insulin release than in untreated control monkeys. CONCLUSION: Exposure of isolated xenogeneic islets of Langerhans to blood, both in vitro and in vivo, resulted in acute islet damage. Complement and platelets seem to have a central role in the reactions described. Strategies to efficiently inhibit these reactions will be crucial for clinical intraportal islet xenotransplantation to be successful. PMID- 10755517 TI - Additive efficacy of CTLA4Ig and OX40Ig secreted by genetically modified grafts. AB - BACKGROUND: The use of systemic immunosuppressive drugs have been paramount in the success in transplantation, but there are serious deleterious effects. Genetic modification of grafts to secrete immunomodulators locally may be a way to reduce the need for systemic immunosuppression. METHODS AND RESULTS: An insulinoma cell line, NIT, having the nonobese diabetic (NOD) genotype but also expressing the SV40 large T Ag, was transfected with CTLA4Ig or OX40Ig in an attempt to block signals in the costimulatory/adhesion pathways. The extracellular domains of these molecules have been fused to the Fc of IgG2c derived from the NOD mouse strain. This resulted in secreted and dimerized proteins. SV40 T Ag is potent at inducing graft rejection. Test and control transfectants were transplanted subcutaneously into young NOD mice to determine whether secretion of CTLA4Ig and OX40Ig would promote survival of the insulinoma graft. In immunodeficient mice, cell growth was similar for all transfectants. However, in immunocompetent NOD mice, the survival/growth of test grafts was significantly better than that of controls. By combining test transfectants, we found that graft survival was enhanced in an additive and significant fashion. In vitro, there was a significant reduction in immune responses-compared with control-when purified fusion proteins were added to mixed leukocyte reaction cultures. CONCLUSIONS: We conclude that blockade of individual costimulatory/adhesion signals by graft manipulation can contribute to transplantation success and that blockade of combinations of signals in these pathways enhances this success. Successful immunomodulation by the graft itself can be achieved. PMID- 10755518 TI - Establishment of fully xenogeneic (mouse-->rat) bone marrow chimeras: evidence for normal development and clonal deletion of mouse T cells. AB - BACKGROUND: Xenotransplantation is a potential solution to the critical shortage of transplantable organs. However, conventional immunosuppressive agents do not control the vigorous cellular and humoral rejection across species disparities. The induction of donor specific tolerance via bone marrow chimerism may be a method to avoid xenograft rejection. In xenogeneic chimeras, T cell repertoire selection plays an important role in the induction of tolerance. Until now a model of mouse-->rat multilineage chimerism has not been reported. This study reports the establishment of fully xenogeneic mouse-->rat multilineage chimeras and evaluates the role of T cell development and repertoire selection in tolerance induction in a xenogeneic environment. METHODS: Recipient rats were irradiated at a dose of total body irradiation ranging between 800-1100 cGy and injected with 120-300x10(6) donor mouse bone marrow cells. Chimeras were typed for engraftment at 4 weeks and then monthly thereafter. T cell repertoire was evaluated in chimeras using two-color flow cytometry and monoclonal antibodies directed against the variable portion of the beta chain of the T cell receptor. RESULTS: Fully xenogeneic multilineage bone marrow chimerism was produced in a mouse-->rat model by using ablative radiation and a high dose of donor cells. Mouse T cells develop in a phenotypically normal fashion in chimeric rats and the host rat is capable of deleting T cells that are reactive to the donor mouse strain. CONCLUSION: Long-term multilineage bone marrow chimerism can be produced in a mouse-->rat bone marrow transplant model. Mouse T cells develop in a phenotypically normal fashion and negative selection of specific T cell receptor Vbeta occurs in a xenogeneic environment in a predictable fashion paralleling that for syngeneic or allogeneic transplantation. PMID- 10755519 TI - Oral efficacy of the macrolide immunosuppressant SDZ RAD and of cyclosporine microemulsion in cynomolgus monkey kidney allotransplantation. AB - BACKGROUND: 40-O-(2-Hydroxyethyl)-rapamycin (SDZ RAD) is a novel, potent, macrolide immunosuppressant. Its efficacy in rodent transplantation models provided the rationale for us to evaluate the compound in a more relevant, large animal transplantation model. METHODS: Life-supporting kidney allotransplantation was performed in cynomolgus monkeys: rejection was inferred from a rise in serum creatinine or urea and was subsequently confirmed by histopathology. This model was validated with the microemulsion formulation of cyclosporine (i.e., Neoral). Two studies with a microemulsion formulation of SDZ RAD were performed. First, in a dose-finding study, the SDZ RAD dose was reduced in a stepwise fashion until rejection occurred, either with SDZ RAD as monotherapy, or in combination with a fixed, suboptimal dose of cyclosporine. Second, an efficacy study was performed in which two fixed SDZ RAD doses (0.75 and 1.50 mg/kg/ day) were evaluated in monotherapy and compared with the same doses of rapamycin (sirolimus). All immunosuppressants were administered once daily by gastric gavage. RESULTS: Untreated control animals rejected their grafts between 4 and 8 days after transplantation. Cyclosporine (initially at 150 mg/kg/day, reduced to 100 mg/kg/day 2 weeks after transplantation) yielded long-term (>100 days) rejection free allograft survival in four of five animals. A 10 mg/kg/day dose of cyclosporine led to rejection between 10 and 27 days after transplantation and was considered suboptimal. In the dose-finding study with SDZ RAD monotherapy, rejection occurred in most of the cases (four of six animals) when a dose level of 0.63 mg/kg/day had been reached. Combined with suboptimal cyclosporine, this threshold SDZ RAD dose was about 2-fold lower. In the efficacy study, median graft survival with histologically proven rejection was 32 days (range 8-91 days, n=6) for 0.75 mg(kg/day SDZ RAD and 59 days (range 28-85 days, n=6) for 1.50 mg/kg/day SDZ RAD. For sirolimus, median graft survival was 43 days (range 5-103 days, n=7) for the 0.75 mg/kg/day dose and 56 days (range 8-103 days, n=8) for the 1.50 mg/kg/day dose. There was no statistically significant difference in efficacy between SDZ RAD and sirolimus. CONCLUSION: SDZ RAD, in the absence of any other immunosuppressant and at doses that do not show any overt toxicity, considerably prolongs rejection-free survival of cynomolgus monkeys after life supporting kidney allotransplantation. PMID- 10755521 TI - A surface-bound form of human C1 esterase inhibitor improves xenograft rejection. AB - BACKGROUND: The purpose of the present study was to investigate the effect of the C1 esterase inhibitor (C1-INH) molecule against human complement attack on a swine endothelial cell (SEC) membrane. Human C1-INH functions as an inhibitor for complement reaction in the first step of the classical pathway in the fluid phase. METHODS: A surface-bound form of human C1-INH (C1-INH-PI) consisting of a full-length coding sequence of C1-INH and a glycosylphosphatidylinositol (GPI) anchor of the decay-accelerating factor (CD55) was constructed, and stable Chinese hamster ovarian tumor (CHO) cell lines and SEC lines expressing C1-INH-PI were then prepared by transfection of the constructed cDNA. The basic function of the transfected molecules on the xenosurface was investigated using CHO transfectants for the sake of convenience. The efficacy of C1-INH-mediated protection of SEC from human complement was then assessed as an in vitro hyperacute rejection model of a swine-to-human discordant xenograft. RESULTS: Flowcytometric profiles of the stable CHO and SEC transfectants with C1-INH-PI showed a medium level of expression of these molecules. The C1-INH levels were significantly reduced as a result of phosphatidylinositol-specific phospholipase C (PI-PLC) treatment, suggesting that the molecules were present as the PI-anchor form. Approximately 51.3 x 10(4) and 13.3 x 10(4) molecules of C1-INH-PI blocked human complement-mediated cell lysis by approximately 75% on the CHO cell and by 60-65% on the SEC cell, respectively. In addition, the complement-inhibiting activity of human C1-INH molecules is not homologously restricted. CONCLUSIONS: The results suggest that the surface-bound form of C1-INH represents a good candidate as a safeguard against hyperacute rejection of xenografts. PMID- 10755520 TI - Blocking the CD28-B7 T-cell costimulatory pathway abrogates the development of obliterative bronchiolitis in a murine heterotopic airway model. AB - BACKGROUND: CTLA4IgG that binds to B7 effectively inhibits the signaling of CD28/B7 pathway and induces antigen-specific T-cell unresponsiveness in vitro and in vivo. We examined whether the development of obliterative bronchiolitis in a murine heterotopic airway transplantation model is T cell dependent and whether CTLA4IgG abrogates the development of obliterative bronchiolitis. METHODS: Tracheae with main bronchi from C3H/He (H2k), BALB/C (H2d), or C57BL/6 (H2b) mice were transplanted heterotopically into subcutaneous pockets on the backs of BALB/C or BALB/C nu/nu mice on day 0. Recipient mice were untreated or intraperitoneally treated with either CTLA4IgG or human IgG with different time and dose schedules. RESULTS: The development of obliterative bronchiolitis, which leads to luminal obliteration by fibrous tissue in a murine heterotopic airway transplantation model, was T cell dependent and the development of obliterative bronchiolitis was significantly abrogated by the CTLA4IgG treatment. However, the normal ciliated columnar respiratory epithelial cells in allografts were lost and replaced by flattened attenuated epithelial cells even after the CTLA4IgG treatment. We further demonstrated that CTLA4IgG treatment did not result in the induction of donor-specific unresponsiveness. CONCLUSIONS: We demonstrated that the development of obliterative bronchiolitis in a murine heterotopic airway model involves both CD28/B7-dependent and -independent processes. The luminal obliteration by fibrous tissue is clearly CD28/B7 dependent and can be inhibited by CTLA4IgG. The luminal obliteration of allografted trachea by fibrous tissues and the loss of ciliated columnar respiratory epithelial cells represent distinct disease processes. PMID- 10755522 TI - Exocrine contamination of isolated islets of Langerhans deteriorates the process of revascularization after free transplantation. AB - BACKGROUND: We investigated the influence of exocrine tissue contamination on the process of vascularization of isolated pancreatic islets of Langerhans after free transplantation into Syrian golden hamsters. METHODS: After isolation by a modified collagenase digestion technique, a total of 45 individual islets contaminated with exocrine tissue were transplanted syngeneically into striated muscle of dorsal skinfold chambers of nine animals. Sixty-six purified islet grafts transplanted into nine additional animals served as controls. Islet engraftment as well as the process of angiogenesis and revascularization were analyzed at days 3, 5, 9, and 13 after transplantation using intravital fluorescent microscopic techniques. RESULTS: Islet grafts contaminated with exocrine tissue showed a significantly (P<0.05) lower take rate initially after transplantation when compared with purified islet grafts. In addition, functional capillary density, which served as an indicator of graft vascularization, was found significantly (P<0.05) decreased in contaminated compared with purified islets. Exocrine tissue contamination was further associated with marked leukocyte-endothelial cell interaction within the grafts' postcapillary venules, which finally resulted in six of nine animals in an overwhelming unspecific inflammation of the transplantation site with successive loss of the islet transplants. CONCLUSIONS: These findings support the view that exocrine tissue contamination is detrimental for the process of angiogenesis and vascularization of freely transplanted pancreatic islets when grafted to a confined site such as striated muscle tissue. PMID- 10755523 TI - Chronically decreased oxygen tension in rat pancreatic islets transplanted under the kidney capsule. AB - BACKGROUND: A factor of potential importance in the failure of islet grafts is poor or inadequate engraftment of the islets in the implantation organ. This study measured the oxygen tension and blood perfusion in 1-, 2-, and 9-month-old islet grafts. METHODS: The partial pressure of oxygen was measured in pancreatic islets transplanted beneath the renal capsule of diabetic and nondiabetic recipient rats with a modified Clark electrode (outer tip diameter 2-6 microm). The size of the graft (250 islets) was by purpose not large enough to cure the diabetic recipients. The oxygen tension in islets within the pancreas was also recorded. Blood perfusion was measured with the laser-Doppler technique. RESULTS: Within native pancreatic islets, the partial pressure of oxygen was approximately 40 mm Hg (n=8). In islets transplanted to nondiabetic animals, the oxygen tension was approximately 6-7 mm Hg 1, 2, and 9 months posttransplantation. No differences could be seen between the different time points after transplantation. In the diabetic recipients, an even more pronounced decrease in graft tissue oxygen tension was recorded. The mean oxygen tension in the superficial renal cortex surrounding the implanted islets was similar in all groups (approximately 15 mm Hg). Intravenous administration of glucose (0.1 gxkg( 1)x min(-1)) did not affect the oxygen tension in any of the investigated tissues. The islet graft blood flow was similar in all groups, measuring approximately 50% of the blood flow in the kidney cortex. CONCLUSION: The oxygen tension in islets implanted beneath the kidney capsule is markedly lower than in native islets up to 9 months after transplantation. Moreover, persistent hyperglycemia in the recipient causes an even further decrease in graft oxygen tension, despite similar blood perfusion. To what extent this may contribute to islet graft failure remains to be determined. PMID- 10755524 TI - Modification of oxidative stress in response to intestinal preconditioning. AB - Previous studies have demonstrated that intestinal preconditioning protects the organ from ischemia reperfusion damage. Xanthine oxidase mediating free radical generation contributes to the development of injury associated to ischemia reperfusion. Thus, any process able to modulate the oxygen free radical generation system could attenuate the injury. Also, it is known that nitric oxide is implicated in the preconditioning response. The aim of this work is to determine: (1) the effect of intestinal preconditioning on the xanthine oxidase system, (2) the relevance of this system in the development of injury, and (3) its relationship with nitric oxide. For this purpose, we have determined the activity of the xanthine dehydrogenase/xanthine oxidase system, the levels of its substrate (xanthine), and end-product (uric acid) and oxidant stress status in rat small intestine subjected to ischemic pre-conditioning. The effects of nitric oxide inhibition have also been evaluated. Results show that the percentage of xanthine dehydrogenase to xanthine oxidase conversion, xanthine, uric acid concentration, lipoperoxides, and reduced glutathione were significantly reduced in preconditioned rats irrespectively of nitric oxide inhibition. In summary, this work shows that oxidative stress in intestinal preconditioning is reduced as consequence of the diminished conversion of xanthine dehydrogenase to xanthine oxidase, and also as a consequence of the reduced availability of xanthine. PMID- 10755525 TI - Inhibition of chronic rejection of aortic allografts by dietary glycine. AB - BACKGROUND: Chronic rejection is influenced by a variety of risk factors, including histoincompatibility and ischemia. Glycine, a cytoprotective agent, has been shown to protect against ischemia-reperfusion injury in the liver, inactivate hepatic resident macrophages, minimize cyclosporin A-induced nephrotoxicity, and exhibit immunosuppressive properties in vitro. The aim of this study was to investigate whether dietary glycine could reduce development of chronic rejection. METHODS: Lewis recipients of Fisher-344 abdominal aortic allografts received diets that contained either 5% glycine plus 15% casein or 20% casein as control for 10 weeks. Vascular lesions of aortic isografts and allografts were evaluated quantitatively with image analysis and cell counting. RESULTS: No significant vascular changes were observed in isografts (mean medial areas of 3.3 +/- 0.3x0(5) microm2). However, dramatic intimal thickening (neointimal area 2.1+/-0.3) and medial thinning (1.5+/-0.3) were observed in allografts from rats fed control diet. In contrast, glycine significantly reduced the neointima by 45% (1.2+/-0.3) and protected the media (3.5+/-0.2). This led to intima to media area ratios almost twice as large in the control group as in glycine-fed rats (2.2+/-0.4 vs. 1.1+/-0.3, P<0.05). Moreover, infiltrating leukocytes, especially macrophages, were reduced significantly in the adventitia by glycine. In addition, glycine inhibited proliferation and migration of rat aortic smooth muscle cells in culture by 45 and 60%, respectively. CONCLUSION: These results indicate that dietary glycine minimizes histopathological changes of chronic rejection by reducing the immune response and, in part, by minimizing proliferation and migration of smooth muscle cells. PMID- 10755527 TI - Renal transplantation in older adults: is graft survival affected by age? A case control study. AB - BACKGROUND: The demand for kidney allografts in older patients is growing continually. Previously published data indicate that the higher rate of graft losses resulting from the age-related increased mortality in older transplant recipients is balanced by a significantly lower number of graft losses from immunological problems (acute and chronic rejection) in old patients. This single center study was performed to scrutinize these results with the methods of a case control analysis. METHODS: Ninety-one patients, 65 years and older (mean age 67), were included in the case group. Their data were compared with those obtained from two control groups, 40-55 and 18-35 years old, respectively (mean ages 48 and 29, respectively). Apart from age, the groups were matched with regard to HLA mismatches and date of transplantation. RESULTS: The number of initially non functioning grafts and donor age did not differ significantly between the case and the control groups. During the follow-up of 5 years, acute rejections were significantly more frequent in the older control group. In contrast to previous studies, however, graft losses caused by rejections were not significantly more frequent in younger patients than in transplant recipients over age 65 years. Thus, as a consequence of increased patient mortality, the total graft survival in the case group was significantly worse than in the control groups. CONCLUSIONS: In the presence of organ shortage, an indication for kidney transplantation in patients over 65 years has to be considered carefully because age did not prove to have a beneficial effect on graft survival. Nevertheless, patients of this age group should not be excluded from renal transplantation, because not only medical, but also ethical, issues are involved. PMID- 10755526 TI - Long-term medical complications in patients surviving > or = 5 years after liver transplant. AB - BACKGROUND: Short-term outcomes of liver transplantation are well reported. Little is known, however, about long-term results in liver recipients surviving > or =5 years. We sought to analyze long-term complications in liver recipients surviving > or =5 years after transplant, to assess their medical condition and to compare findings to the general population. METHODS: We analyzed the chart and database records of all patients (n=139) who underwent liver transplantation at a major transplant center before January 1, 1991. Outcome measures included the presence of diabetes, hypertension, heart, renal or neurological disease, osteoporosis, incidence of de novo malignancy or fracture, or other pathology, body mass index, serum cholesterol and glucose, liver function, blood pressure, frequency of laboratory and clinic follow-up, current pharmacological regimen, and late rejection episodes. RESULTS: Ninety-six patients (70%) survived > or =5 years. Compared to numbers expected based on U.S. population rates, transplant recipients had significantly higher overall prevalences of hypertension (standardized prevalence ratio [SPR]=3.07, 95% confidence interval [CI], 2.35 3.93) and diabetes (SPR=5.99, 95% CI, 4.15-8.38), and higher incidences of de novo malignancy (standardized incidence ratio [SIR]=3.94, 95% CI, 2.09-6.73), non Hodgkin's lymphoma (SIR=28.56, 95% CI, 7.68-73.11), non-melanoma skin cancer (estimated SIR> or =3.16) and fractures in women (SIR=2.05, 95% CI, 1.12-3.43). Forty-one of 87 (47.1%) patients were obese, and 23 patients (27.4%) had elevated serum cholesterol levels (> or =240 mg/dl, 6.22 mmol/L), compared to 33% and 19.5% of U.S. adults, respectively. Prevalences of heart or peptic ulcer disease were not significantly higher. CONCLUSIONS: Liver transplantation is being performed with excellent 5-year survival. Significant comorbidities exist, however, which appear to be related to long-term immunosuppression. PMID- 10755528 TI - A comparison of the effects of dialysis and renal transplantation on the survival of older uremic patients. AB - BACKGROUND: Patients over age 60 constitute half of all new patients accepted into the renal replacement therapy programs in Australia. However, the optimal treatment of their end-stage renal disease remains controversial. The aim of the present study was to compare survival for dialysis and renal transplantation in older patients who were rigorously screened and considered eligible for transplantation. METHODS: The study cohort consisted of 174 consecutive patients over 60 who were accepted on to the Queensland cadaveric renal transplant waiting list between January 1, 1993 and December 31, 1997. Follow-up was terminated on October 1, 1998. Data were analyzed on an intention-to-transplant basis using a Cox regression model with time-varying explanatory variables. An alternative survival analysis was also performed, in which patients no longer considered suitable for transplantation were censored at the time of their removal from the waiting list. RESULTS: There were 67 patients receiving a renal transplant, whereas the other 107 continued to undergo dialysis. These two groups were well matched at baseline with respect to age, gender, body mass index, renal disease etiology, comorbid illnesses, and dialysis duration and modality. The overall mortality rate was 0.096 per patient-year (0.131 for dialysis and 0.029 for transplant, P<0.001). Respective 1-, 3- and 5-year survivals were 92%, 62%, and 27% for the dialysis group and 98%, 95%, and 90% (P<0.01) for the transplant group. Patients in the transplant group had an adjusted hazard ratio 0.16 times that of the dialysis group (95% confidence interval 0.06-0.42). If patients were censored at the time of their withdrawal from the transplant waiting list, the adjusted hazard ratio was 0.24 (95% confidence interval 0.09-0.69). CONCLUSIONS: Renal transplantation seems to confer a substantial survival advantage over dialysis in patients with end-stage renal failure who are rigorously screened and considered suitable for renal transplantation. PMID- 10755529 TI - Low dose antithymocyte globulins in renal transplantation: daily versus intermittent administration based on T-cell monitoring. AB - BACKGROUND: Despite the long history of use of antithymocyte globulins (ATG) in renal transplantation, ideal doses and duration of ATG administration based on the monitoring of T lymphocytes have yet to be defined. METHODS: Two immunosuppressive regimens based on low-dose rabbit ATG (Thymoglobuline; Imtix Sang-stat, Lyon, France) were assessed during the first year after transplantation: daily ATG (DATG; n=23) where 50 mg of ATG was given every day and intermittent ATG (IATG; n=16) where similar doses of ATG were given for the first 3 days and then intermittently only if CD3+ T lymphocytes (measured by flow cytometry) were > 10/mm3. Both groups received steroids, azathioprine, and cyclosporine. RESULTS: ATG-induced depletion was similar for peripheral blood lymphocytes and T cells in both groups: it began at day 1 after transplantation, was submaximal at day 3, and reached maximum intensity between days 6 and 8, from which time cell counts progressively increased. However, T-cell depletion was still present at day 20. The total ATG dose per patient (381.5+/-121 vs. 564+/ 135 mg/patient) and the mean cumulative daily dose of ATG (0.60+/-0.17 vs. 0.80+/ 0.14 mg/kg/day) were significantly lower in the IATG group (P=0.0001 and 0.0006, respectively). The overlap of ATG and cyclosporine treatment was 6.7+/-3 vs. 7.4+/-4.3 days (P=NS), and the mean duration of ATG therapy was 11.3+/-3.2 vs. 11.6+/-2.7 days in the IATG and DATG groups, respectively (P=NS). ATG was given in an average of one dose every 1.6 days in the IATG group compared with one dose daily in the DATG group (P=7 x 10(-7)). There was no significant difference in renal graft function, the number of acute graft rejections, or ATG-related side effects and complications. Despite the daily immunological follow-up, there was a net saving of $760/patient in the cost of treatment in the IATG group. CONCLUSION: IATG had the advantage of a reduction in the dose of ATG and in the cost of treatment, while offering similar T-cell depletion and effective immunosuppression. This approach could be proposed as an induction protocol, particularly for patients with poor graft function in whom cyclosporine introduction has to be delayed or those with increased risk of cytomegalovirus infections or secondary malignancies. PMID- 10755530 TI - Increased levels of GALbeta1-4GLCNACalpha2-6 sialyltransferase pretransplant predict delayed graft function in kidney transplant recipients. AB - BACKGROUND: Galbeta1-4GlcNAcalpha2-6 sialyltransferase (ST6GalI) is an acute phase reactant whose release from cells can be induced by proinflammatory cytokines. Because patients with chronic renal failure have high circulating levels of proinflammatory cytokines, we hypothesized that patients on the renal transplant waiting list would have high circulating levels of ST6GalI, which might adversely affect post-transplant events. METHODS: Levels of ST6GalI were measured in the serum of 70 patients immediately before renal transplant; these were correlated with posttransplant events, such as delayed graft function and rejection. RESULTS: The mean serum level of ST6GalI was significantly higher in the patients (3162+/-97 U) than in 19 controls (2569 +/- 125 U; P<0.003). Patients who required dialysis posttransplant for treatment of delayed graft function (n=20) had significantly higher levels of ST6GalI pretransplant (3735+/ 228 U) than patients (n=50) who did not require dialysis (2933+/-83 U; P<0.0001). In a multivariate analysis the ST6GalI level and cold ischemic time were found to be independent risk factors for the development of delayed graft function. CONCLUSIONS: ST6GalI levels are high in renal failure patients awaiting a renal transplant and may be a risk factor for the development of delayed graft function. The assessment and perhaps modulation of a potential transplant recipient's ST6GalI systemic level may be beneficial. PMID- 10755531 TI - Postransplant lymphoproliferative disorder localized near the allograft in renal transplantation. AB - BACKGROUND: Posttransplant lymphoproliferative disorder (PTLD), a complication of immunosuppression, develops in approximately 1% of renal allograft recipients. Typically, PTLD is a proliferation of B-cells associated with Epstein-Barr virus (EBV) infection; it is said to be most often a systemic disease. Involvement occasionally is localized near the allograft. METHODS: This is a retrospective analysis of all cases of PTLD in recipients of 1474 renal transplants performed at University of Alabama at Birmingham between 1993 and 1997. RESULTS: Of 14 patients developing PTLD, 10 had disease localized near the allograft. The mean interval from transplantation to diagnosis was 221 +/- 70 days. All patients presented with renal dysfunction; an ultrasound examination revealed a hilar mass, with hydronephrosis in five and stenosis of renal vessels in eight. No patient had lymphadenopathy, according to computerized tomographic or magnetic resonance imaging findings. After reduction of immunosuppressive therapy, seven required a nephrectomy because of rejection, progressive dysfunction, or mass enlargement. Tissue recovered in four patients was consistent with PTLD; the tumors in the remaining three patients were unresectable and regressed. One patient died 1 month after a nephrectomy, and another died 4 years after surgery; neither had evidence of PTLD when they died. Three patients retain functional grafts without clinical or radiographical evidence of progression. All patients with disseminated disease died. CONCLUSIONS: In a large cohort of renal allograft recipients, PTLD affected 1%. Disease localized near the allograft was the most common variant. For most patients with localized disease, the outcome was graft loss, and the mortality was low. Localized PTLD should be considered in the differential diagnosis of allograft dysfunction in the 1st posttransplant year. PMID- 10755532 TI - Detection of anti-HLA antibody by flow cytometry in patients with a left ventricular assist device is associated with early rejection following heart transplantation. AB - BACKGROUND: Patients with a left ventricular assist device (LVAD) as a bridge to heart transplantation (HT) often have elevated levels of panel reactive antibodies (PRA). The clinical significance of anti-human histocompatibility leukocyte antigen (HLA) antibodies detected by flow cytometry in PRA negative patients remains unclear. METHODS: Eighteen patients who underwent LVAD placement as a successful bridge to HT had standard anti-human globulin complement dependent cytotoxicity and retrospective flow cytometry assays performed to detect class I anti-HLA antibodies. A positive flow result was defined as a fluorescent ratio of 23:1 versus a negative control. RESULTS: Six patients had anti-HLA antibodies detected by flow cytometry. Univariate analysis demonstrated more moderate-severe rejection episodes (ISHLT > or = IIIA) at 2 months (0.83+/ 0.75 vs. 0; P=0.04) and a trend toward decreased time to first rejection (61+/-17 vs. 225+/-62 days; P=0.06) in these patients. No differences were observed in donor-recipient HLA mismatch or 1 year Kaplan-Meier survival between patients with or without anti-HLA antibodies. CONCLUSION: Despite a negative PRA, LVAD patients with class I anti-HLA antibodies detected by flow cytometry have a greater incidence of moderate-severe rejection in the first 2 months after HT. Flow cytometry may be a useful clinical tool in screening PRA negative LVAD patients before transplantation. Patients with positive anti-HLA antibody screening by flow cytometry may require more intensive immunosuppression in the early post-HT period. PMID- 10755533 TI - Critical threshold of azathioprine dosage for maintenance immunosuppression in kidney graft recipients. Collaborative Transplant Study. AB - BACKGROUND: There are conflicting reports in the literature concerning the efficacy of maintenance immunosuppression in renal transplantation with a regimen of azathioprine and steroids. METHODS: The daily dosage (mg/kg) of azathioprine administered 1 year after transplantation was analyzed in relation to subsequent long-term graft outcome. Transplants performed from 1985 to 1996 and reported to the Collaborative Transplant Study were analyzed. RESULTS: In patients on maintenance immunosuppression without cyclosporine, the daily dosage of azathioprine had a highly significant influence on long-term graft outcome. Patients who received > 1.5 mg/kg/ day of azathioprine had a 69% graft survival rate at 7 years, compared with a 55% rate in patients receiving 1.01-1.5 mg/kg/day (P<0.0001) and a 45% rate in patients receiving < or = 1.00 mg/kg/day (P<0.0001). This observation was valid for patients who were taken off cyclosporine during the first year as well as for patients who were treated with azathioprine and steroids (without cyclosporine) from the beginning. CONCLUSION: Maintenance immunosuppression with azathioprine and steroids results in good long term kidney graft survival, provided azathioprine is administered at a daily dose of >1.5 mg/kg. PMID- 10755534 TI - Subjective xerostomia in long-term surviving children and adolescents after pediatric bone marrow transplantation. AB - BACKGROUND: The aim of the present investigation was to evaluate whether the subjective symptoms of dry mouth in long-term-surviving pediatric bone marrow transplant (BMT) patients are associated with low unstimulated salivary secretion rates (USSR) and with stimulated whole salivary secretion rates (SSSR). METHODS: Fifty-three patients surviving > or =2 years after pediatric allogeneic BMT were included. USSR, SSSR, and the change in salivary secretion rates since the previous year were estimated. A questionnaire regarding subjective symptoms of xerostomia was answered. RESULTS: The mean USSR and SSSR were 0.24+/-0.17 and 0.90 +/- 0.58 ml/min, respectively. Salivary gland dysfunction, defined as USSR < or =0.1 ml/min or SSSR < or =0.5 ml/min, was present in 35% of the patients. Seventy-nine percent of the patients expressed one or more symptom of dry mouth, and 49% gave at least two answers indicating dry mouth. The number of complaints increased with age at examination (P<0.05). Both USSR (P<0.01) and SSSR (P<0.01) were inversely correlated to the total number of complaints of xerostomia. A reduction in SSSR compared with the year before was correlated to two or more complaints of xerostomia (P<0.01). The presence of dry mouth at night or on awakening was indicative of both low USSR (P<0.01) and SSSR (P<0.001). Patients reporting dryness during the day had significantly lower SSSR (P<0.05). CONCLUSION: The expression of subjective complaints of xerostomia among long-term surviving pediatric BMT patients is correlated to salivary gland dysfunction and age. It is very important to identify these patients with salivary gland dysfunction to relieve their symptoms and prevent secondary complications. PMID- 10755535 TI - Epstein-Barr virus-negative lymphoproliferate disorders in long-term survivors after heart, kidney, and liver transplant. AB - BACKGROUND: Solid organ transplant patients undergoing long-term immunosuppression have high risk of developing lymphomas. The pathogenesis of the late-occurring posttransplantation lymphoproliferative disorders (PTLD) have not yet been extensively investigated. METHODS: We studied 15 patients who developed PTLD after a median of 79 months (range 22-156 months) after organ transplant. Clonality, presence of Epstein-Barr virus (EBV) genome, and genetic lesions were evaluated by Southern blot analysis or polymerase chain reaction. RESULTS: All monomorphic PTLD and two of three polymorphic PTLD showed a monoclonal pattern. Overall, 44% of samples demonstrated the presence of the EBV genome. Within monomorphic PTLD, the EBV-positive lymphomas were even lower (31%). A c-myc gene rearrangement was found in two cases (13%), whereas none of the 15 samples so far investigated showed bcl-1, bcl-2, or bcl-6 rearrangement. The modulation of immunosuppression was ineffective in all patients with monomorphic PTLD independent of the presence of the EBV genome. The clinical outcome after chemotherapy was poor because of infectious complications and resistant disease. With a median follow-up of 4 months, the median survival time of these patients was 7 months. CONCLUSIONS: Late occurring lymphomas could be considered an entity distinct from PTLD, occurring within 1 year of transplant, because they show a histological and clinical presentation similar to lymphomas of immunocompetent subjects, are frequently negative for the EBV genome, are invariably clonal, and may rearrange the c-myc oncogene. New therapeutic strategies are required to reduce the mortality rate, and new modalities of long-lasting immunosuppression are called for. PMID- 10755536 TI - Randomized trial of tacrolimus (Prograf) in combination with azathioprine or mycophenolate mofetil versus cyclosporine (Neoral) with mycophenolate mofetil after cadaveric kidney transplantation. AB - BACKGROUND: Our clinical trial was designed to investigate the optimal combination of immunosuppressants for renal transplantation. METHODS: A randomized three-arm, parallel group, open label, prospective study was performed at 15 North American centers to compare three immunosuppressive regimens: tacrolimus + azathioprine (AZA) versus cyclosporine (Neoral) + mycophenolate mofetil (MMF) versus tacrolimus + MMF. All patients were first cadaveric kidney transplants receiving the same maintenance corticosteroid regimen. Only patients with delayed graft function (32%) received antilymphocyte induction. A total of 223 patients were randomized, transplanted, and followed for 1 year. RESULTS: There were no significant differences in baseline demography between the three treatment groups. At 1 year the results are as follows: acute rejection 17% (95% confidence interval 9%, 26%) in tacrolimus + AZA; 20% (confidence interval 11%, 29%) in cyclosporine + MMF; and 15% (confidence interval 7%, 24%) in tacrolimus + MMF. The incidence of steroid resistant rejection requiring antilymphocyte therapy was 12% in the tacrolimus + AZA group, 11% in the cyclosporine + MMF group, and 4% in the tacrolimus + MMF group. There were no significant differences in overall patient or graft survival. Tacrolimus-treated patients had a lower incidence of hyperlipidemia through 6 months posttransplant. The incidence of posttransplant diabetes mellitus requiring insulin was 14% in the tacrolimus + AZA group, 7% in the cyclosporine + MMF and 7% in the tacrolimus + MMF groups. CONCLUSIONS: All regimens yielded similar acute rejection rates and graft survival, but the tacrolimus + MMF regimen was associated with the lowest rate of steroid resistant rejection requiring antilymphocyte therapy. PMID- 10755537 TI - The trouble with kidneys derived from the non heart-beating donor: a single center 10-year experience. AB - BACKGROUND: The demand for renal transplantation has increasingly outstripped the supply of donor organs especially over the past 10 years. Although related and unrelated live donation is being promoted as one option for increasing the donor pool, it is unlikely that this will in itself be able to bridge the gap. Non heart beating donors (NHBD) can provide an alternative supply of organs, which should substantially increase the donor pool. METHODS: In Newcastle, NHBD kidneys have been used for transplantation for a period of 10 years. In the early period (1988-1993) excellent results were obtained (90.5% success); however, these donors were controlled NHBD, Maastricht category III. In the second phase (1994 1998) increasing numbers of donors were obtained from the Accident and Emergency Department unit. These were failed resuscitation for cardiac arrest (category II). The rates of success in this period were poor (45.5% success) and the program was halted. The third phase of the program used machine perfusion of the kidneys and glutathione S transferase enzyme analysis to assess viability. RESULTS: Using such approaches renal transplants from largely category II donors produced a success rate of 92.3% which was significantly better than the phase II period of the program (P=0.023, Fisher two-tail test). CONCLUSION: Machine perfusion and viability assessment of NHB kidneys in phase III of the program has increased our donor pool as well as improved the graft survival. This is particularly relevant for the use of the category II NHB donor where the incidence of primary nonfunction was high, illustrated by phase II where machine perfusion/viability assessment was not used. PMID- 10755538 TI - Incidence, progression and functional significance of cardiac allograft vasculopathy after heart transplantation. AB - BACKGROUND: Cardiac allograft vasculopathy after heart transplantation leads to an accelerated form of atherosclerosis with marked and often diffuse vessel wall changes that limit long-term survival. Previous studies showed contradictory results relating vessel wall changes to endothelial vasodilator response. METHODS: A total of 30 cardiac transplant recipients were studied 3, 12, and 24 months after heart transplantation. Coronary angiography was performed at rest, during supine bicycle ergometry, and after 1.6 mg sublingual nitroglycerin. Coronary cross-sectional area (biplane coronary angiography) and coronary artery wall changes (intravascular ultrasound) were assessed and extent of intimal changes correlated to vasodilator responses to nitroglycerine and bicycle ergometry. RESULTS: Intravascular ultrasound showed significant intimal thickening in 43, 64, and 58% of patients at 3, 12, and 24 months. Intimal thickening 3 months after transplantation was related to donor age (r=0.70, P<0.01) but did not predict progression of disease that manifested itself angiographically as a decrease in coronary cross-sectional area at 12 and 24 months (P<0.005) and significant coronary stenosis in 12% of patients after 24 months. Endothelium-independent vasodilatation after nitroglycerin (33+/-15, 44+/ 20, and 43+/-24%) was normal. Endothelium-dependent, flow-induced vasodilatation during exercise was decreased (14+/-11, 18+/-14, and 16+/-17%) but did not correlate to intimal changes assessed by ultrasound. CONCLUSIONS: The study confirms the high incidence of intimal thickening after heart transplantation as assessed by intravascular ultrasound. Impaired exercise-induced vasodilatation suggests diminished bioavailability of endothelium-derived nitric oxide to physiological stimulation but the lack of relationship between coronary wall changes and this functional impairment suggests intermittent and presumably reversible endothelial injury in graft atherosclerosis. PMID- 10755539 TI - N-acetyl-L-cysteine for preventing lung reperfusion injury after liver ischemia reperfusion: a possible dual protective mechanism in a dose-response study. AB - BACKGROUND: Acute lung reperfusion injury (ALI) frequently follows an ischemic event in another organ, such as organ transplantation. We recently demonstrated that lung priming with N-acetyl-L-cysteine (NAC) prevented liver ischemia reperfusion (IR)-induced ALI pending on reduced glutathione (GSH) amount of replenishment. We now assessed the therapeutic effect of NAC-in preventing ALI caused by liver IR-if administered to the lung during liver reperfusion. PROCEDURES: Rat isolated livers were stabilized (30 min) and then perfused with modified Krebs-Henseleit solution (control, n=20) or made globally ischemic (IR, n=20) for 2 hr. Rat lungs were isolated separately, ventilated, and stabilized (30 min) with Krebs plus 5% bovine albumin. Pairs of liver and lung were then reperfused together for 15 min, followed by only lung recirculation with the liver effluent for another 45 min. Three more controls (n=20 each) and three ischemic groups (n=20 each) included lungs which were treated with 100, 150 or 225 mg x kg(-1) NAC (0.5, 0.74, or 1.1 mmol, respectively) during the 15-min liver and lung reperfusion period. RESULTS: Pulmonary artery and ventilatory pressures and vascular resistance increased by 60-80% of baseline, compliance decreased, and bronchoalveolar lavage volume and content were abnormally high in the IR-nontreated and the IR-100 lungs. Most parameters in IR-150 and IR-225 lungs remained almost similar to controls. Postinsult GSH content in IR-100, 150, and -225 lungs was at 20%, 110%, and 90% above the IR-nontreated lungs, respectively. CONCLUSIONS: Lung treatment with NAC during its reperfusion with IR liver effluent prevented ALI. Lung GSH replenishment accounted for lung protection, but its content did not correlate directly with grade of protection; NAC itself seemingly afforded lung protection as well. PMID- 10755540 TI - National sharing of cadaveric isolated intestinal allografts for human transplantation: a feasibility study. AB - Most isolated intestinal graft losses are immunological. We conducted a pilot study to evaluate the feasibility of national sharing of HLA no-mismatch allografts for cadaveric isolated intestinal transplantation. METHODS: UNOS data were analyzed in a theoretical model. Part I: All solid organ donors between 1/95 8/97 who would have met criteria for bowel donation were considered potential donors for all recipients who actually received isolated intestinal transplants during this period. We then determined how many donor intestines could have been directed to no-mismatch candidates had national sharing been in place. Donor exclusion criteria were CMV+ donors to CMV- recipients, hemodynamic instability, age >50, size mismatch (donor weight greater than recipient), and obesity. Mean and median waits for transplants, as well as theoretical mean and median waits for transplants that would have occurred given national sharing, were calculated. Part II: We estimated, based on registry graft survival data, the number of intestinal transplants necessary to demonstrate a no-mismatch graft survival advantage at 2 years. RESULTS: Part I: Although no actual cadaveric no-mismatch transplant was performed, 12-17% of patients could have received no-mismatch allografts had sharing been in place, using various donor acceptance criteria. The impact on waiting time was variable. Part II: Accepting a 15% rate of no mismatch cases and a survival advantage of 10% at 2 years, 793 transplants would be required to prove an advantage to HLA matching at P<0.05. If the graft survival advantage were 20% at 2 years, the time to show significance would be approximately 5 years. Using early acute rejection as an endpoint could require fewer transplants (93), and only a few years to complete the study. CONCLUSIONS: National sharing of cadaveric isolated intestinal allografts is feasible. Median waits would not be significantly increased. The time necessary to prove graft survival advantage would be considerable, but a difference in the rate of acute rejection could be seen within 2 years. Additionally, a national sharing arrangement might improve the overall outcome of isolated intestinal transplantation. PMID- 10755541 TI - Renal cortical neoplasms in long term survivors of solid organ transplantation. AB - BACKGROUND: Renal cortical neoplasms have been reported after organ transplantation, but the level of risk as well as the histological features are poorly defined. METHODS: A retrospective autopsy-based study was performed to evaluate renal neoplasms occurring in patients who underwent solid organ transplantation, died, and received an autopsy from 1981 to 1997 (383 liver, 125 heart, 52 lung, 39 heart/lung, 98 kidney, 4 bowel). Patients were divided into those with short (less than 101 days), medium (101 days to 5 years), and long term survival (more than 5 years). The control group consisted of hospital autopsies on nontransplanted patients from the odd-numbered years, 1983 through 1997. RESULTS: Renal cortical neoplasms were identified in 32/1325 of nontransplanted patients and 15/701 transplanted patients. In transplanted patients, neoplasms were identified in 14 native and 1 allograft kidney: 2/391 in short-term survivors, 3/234 in medium, and 10/76 in long term survivors. While transplant patients with short and medium length survival had no increased risk for neoplasms, patients with long-term survival showed a 9-fold increase in cortical neoplasms. Transplant patients with neoplasms averaged 47 years of age at death, significantly younger than the average age of 70 for nontransplanted control patients with renal neoplasms. The neoplasms in transplanted patients were all tubulopapillary, except for one clear cell neoplasm and ranged in size from 0.1 to 2 cm. CONCLUSIONS: Long-term survivors of solid organ transplants have an 9-fold increased risk of developing tubulopapillary renal cortical neoplasms. PMID- 10755543 TI - Safety and efficacy of tacrolimus in combination with mycophenolate mofetil (MMF) in cadaveric renal transplant recipients. FK506/MMF Dose-Ranging Kidney Transplant Study Group. AB - BACKGROUND: Tacrolimus (FK506) is a safe and effective treatment for the prevention of rejection of renal allografts. Mycophenolate mofetil (MMF) has been used as adjunct immunosuppressive therapy with cyclosporine and corticosteroids for the same purpose. The objective of this study was to investigate the safety and efficacy of FK506 and MMF in renal transplant recipients. METHODS: After cadaveric renal transplant, patients were randomized to receive tacrolimus in combination with either azathioprine (AZA, n=59), MMF 1 g/day (n=59), or MMF 2 g/day group (n=58). Patients were followed for 1 yr posttransplant for the incidence of biopsy-confirmed acute rejection, patient and graft survival, and adverse events. RESULTS: Tacrolimus doses and trough concentrations were similar between treatment groups at all time points; 80% of patients were maintained within a range of 5.0-13.9 ng/ml at 12 months posttransplant. The mean dose of MMF decreased in the 2 g/day group to 1.5 g/day by 6 months posttransplant, primarily due to gastrointestinal GI-related disorders. The incidence of biopsy confirmed acute rejection at 1 year was 32.2%, 32.2%, and 8.6% in the AZA, MMF 1 g/day, and MMF 2 g/day groups, respectively (P<0.01). The use of antilymphocyte antibodies for the treatment of rejection was comparable across treatment groups. The incidence of most adverse events was similar across treatment groups and comparable with previous reports. The overall incidence of posttransplant diabetes mellitus was 11.9%, with the lowest rate observed in the MMF 2 g/day group (4.7%), and was reversible in 40% of patients. The incidence of malignancies and opportunistic infections was low and not different across treatment groups. CONCLUSION: Tacrolimus in combination with an initial dose of MMF 2 g/day is a very effective and safe regimen in cadaveric kidney transplant recipients. PMID- 10755542 TI - Pretransplant systemic inflammation and acute rejection after renal transplantation. AB - BACKGROUND: There are presently no established pre-transplant tests that consistently identify patients who may be at increased risk for acute rejection episodes after renal transplantation. We studied whether pretransplant serum levels of C-reactive protein (CRP), a marker for the presence of systemic inflammation, would predict the occurrence of acute rejection episodes after renal transplantation. METHODS: Pretransplant serum was tested for CRP level in 97 consecutive renal transplant recipients. Time to acute rejection after transplantation was stratified by CRP level and compared using the Kaplan-Meier method. In addition, Cox regression multivariate analysis was performed to assess whether any pretransplant covariates could independently predict the subsequent occurrence of acute rejection episodes. RESULTS: Pretransplant mean CRP levels were higher in patients who subsequently had a rejection episode versus those who had no rejection (22.2+/-2.9 vs. 11.7+/-1.8 microg/ml, respectively, P=0.003). Patients less than the median CRP value had a significantly longer time to rejection compared to those with higher CRP levels (P=0.002). Similarly, patients within the lowest CRP quartile had longer times to rejection when compared with the highest quartile (P=0.006). Cox proportional hazards regression multivariate analysis identified CRP level as the only independent pretransplant risk factor for rejection identified (P=0.044). CONCLUSIONS: Pretransplant systemic inflammation as manifested by elevated serum CRP level independently predicts the risk of acute rejection after renal transplantation and may be useful in stratifying patients at the time of transplantation according to immunological risk. Thus, assessment of pretransplant systemic inflammatory status may be helpful in prospective individualization of immunosuppression therapy after renal transplantation. PMID- 10755544 TI - Tumor necrosis factor genetic polymorphisms correlate with infections after renal transplantation. AB - BACKGROUND: Nonimmunosuppressed individuals possessing a NcoI restriction enzyme site in the tumor necrosis factor (TNF) gene locus produce less TNF-alpha in vitro and in vivo than do individuals lacking this site. We have previously shown that this NcoI+/low TNF-alpha genotype is independently associated with increased rates of infection for liver transplant recipients. METHODS: In this study, we performed polymerase chain reaction amplification and restriction fragment length polymorphism analysis of the TNF locus from 45 renal transplant recipients to determine whether the presence of the NcoI site is associated with the frequency of rejection, infection, time to rejection or infection, and patient or graft survival. RESULTS: Twenty-six recipients were typed with the NcoI+/low TNF-alpha genotype, whereas 19 recipients had the NcoI-/high TNF-alpha genotype. Age, sex, donor type, secondary immunosuppression, use of anti-lymphocyte preparations, graft ischemia time, and year of transplant were evenly distributed in the two groups. There was no difference between the genotype groups in the rate of, or time to, rejection. In contrast, significantly more patients with the NcoI+/low TNF-alpha site developed infections (46% vs. 10% P=0.01). In bivari able models, each controlling for donor type, ischemia time, recipient age, use of antilymphocyte agents, and secondary immunosuppression, the NcoI+/low TNF-alpha genotype was still independently associated with increased numbers of infections (relative risk, 5.38; confidence interval, 1.20-23.8). Conclusion. We conclude that in individuals genetically predetermined to be low TNF-alpha producers, the additional inhibition of TNF-alpha production by routine immunosuppression may be excessive, rendering these individuals less able to respond to infectious stimuli. These patients may benefit from lower doses or withdrawal of corticosteroids, which are known inhibitors of TNF-alpha transcription. PMID- 10755545 TI - Increased immunosuppressive vulnerability in elderly renal transplant recipients. PMID- 10755546 TI - Mycophenolic acid-induced GTP depletion also affects ATP and pyrimidine synthesis in mitogen-stimulated primary human T-lymphocytes. AB - BACKGROUND: Mycophenolate mofetil (MMF) is an effective immunosuppressant developed for use in organ transplantation. It specifically targets lymphocyte purine biosynthesis. However, side effects do occur. Understanding how the active metabolite of MMF, mycophenolic acid (MPA) affects the normally integrated interaction between intracellular purine and pyrimidine pathways might aid the development of improved therapeutic regimes. METHODS: We used a primary human T lymphocyte model to study how preincubation with MPA (0.1-50 microM) affected normal ribonucleotide pool responses to phytohemagglutinin using radiolabeled precursors. RESULTS: MPA not only restricted the mitogen-induced expansion of GTP pools, but actually induced a severe drop in both GTP (10% of unstimulated cells) and GDP-sugar pools, with a concomitant fall in ATP (up to 50%). These effects were concentration dependent. By contrast, uridine pools expanded whereas CTP pools remained at resting levels. These changes were confirmed by the altered incorporation of [14C]-bicarbonate and [14C]-glycine into nucleotides. Restriction of [14C]-hypoxanthine incorporation and reduction of [14C]-uridine uptake comparable to that of unstimulated cells indicated that MPA also inhibited both salvage routes of nucleotide synthesis. CONCLUSION: MPA affects pyrimidine as well as purine responses to mitogens in T-lymphocytes, but not in an integrated way. The molecular mechanisms underlying these disproportionate changes can best be explained by MPA-related inhibition of amidophosphoribosyltransferase, catalysing the first step in purine biosynthesis. This would increase phosphoribosylpyrophosphate availability, thereby stimulating UTP biosynthesis. Such imbalances, coupled with ATP-depletion, could underlie reported side effects and might be overcome by appropriate combination therapies. PMID- 10755547 TI - Risk of lymphoid neoplasia after cardiothoracic transplantation. a cohort study of the relation to Epstein-Barr virus. AB - BACKGROUND: Organ transplantation is associated with a greatly increased risk of Epstein-Barr virus (EBV)-associated lymphoproliferative disease (LPD), which is often fatal. There has been little epidemiological analysis, however, of the risk factors for LPD in transplant patients and none on whether the risks of non-EBV associated lymphoid neoplasms are also increased. METHODS: The risk of lymphoid neoplasia was assessed in a cohort of 1563 patients who underwent cardiothoracic transplantation at Harefield Hospital, UK from 1980 to 1994 and were followed until December 1995. EBV antibody was assessed in the patients before transplantation, and lymphoid neoplasms were assessed for EBV RNA and latent EBV gene expression. RESULTS: Thirty cases of LPD occurred during follow-up. One lymphoma of unknown EBV status occurred. There were also six cases of EBV negative non-Hodgkin's lymphoma (EBV-negative NHL), a highly significant excess over expectations from the general population rates of NHL (standardized incidence ratio 10.2 [95% confidence interval, 4.6-22.8]). The risk of LPD was significantly 10-fold raised in individuals who were EBV seronegative before transplantation; independently of this, it decreased steeply with age at transplantation and was greatest in the first year after transplantation. The risk was significantly raised in young seronegative recipients if the donor was older than the recipient. EBV-negative NHL occurred entirely in men 45 years old and older who were EBV seropositive before transplantation, and risk was not related to duration since transplantation. CONCLUSIONS: The risk factors found for LPD accord with EBV etiology and with greater hazard from primary infection than from reactivation. A second non-Hodgkin's lymphoid neoplasm, not related to EBV, seems also to be a consequence of transplantation and immunosuppression but is unlikely to be due to first infection by a ubiquitous agent. Its etiology and prevention need investigation separately from LPD. PMID- 10755549 TI - In vivo T-cell depletion enhances production of anti-GALalpha1,3GAL natural antibodies in alpha1,3-galactosyltransferase-deficient mice. AB - BACKGROUND: It has been reported that T-cell depletion by in vivo treatment with monoclonal antibodies results in polyclonal B-cell activation. However, its effects on B cells responding to Galalpha1,3Gal (Gal) epitopes remain unknown. METHODS: alpha1,3-Galactosyltransferase-deficient (GalT-/-) mice were treated with depleting anti-CD4 and CD8 monoclonal antibodies. The kinetics of anti-Gal natural antibodies (NAb) and total immunoglobulin levels in their sera were evaluated. The frequencies of anti-Gal NAb-producing cells were determined in the various tissues of GalT-/- mice by enzyme-linked immunospot assay. RESULTS: In vivo T-cell depletion led to significant increases in both anti-Gal IgM and total IgM levels in sera of GalT-/- mice, but did not influence either anti-Gal IgG or total IgG levels. An increased frequency of anti-Gal and total IgM-producing cells was observed in the spleens and bone marrow of T-cell-depleted GalT-/-mice but not in peritoneal cavity cells. CONCLUSION: In vivo T-cell depletion facilitates anti-Gal IgM production, suggesting that T cells deliver inhibitory signals to B cells responding to Gal. PMID- 10755548 TI - Absence of host B7 expression is sufficient for long-term murine vascularized heart allograft survival. AB - CD28 antagonists have been shown to promote long-term graft survival and induce donor-specific tolerance. In this study, the role of CD28/B7 costimulation and the relative importance of host versus donor B7 expression in allograft rejection was assessed in a murine abdominal vascularized heterotopic heart transplant model. Wild-type, CD28-deficient, or B7-1/B7-2-deficient C57BL/6 (B6) mice were grafted with allogeneic wild type or B7-1/B7-2-deficient hearts. The results demonstrate allogeneic heart grafts survive long-term in mCTLA4Ig-treated B6 and untreated B7-1/B7-2-deficient B6 recipients but not CD28KO B6 mice. B7-1/B7-2KO B6 recipients treated with anti-CD28 (PV-1) or recombinant human IL-2 rejected the heart transplants indicating that these mice are immunologically competent to reject grafts if costimulatory signals are supplied or bypassed. Finally, there was no difference in rejection between normal animals transplanted with wild-type versus B7-1/B7-2-deficient hearts. These results support a critical role for B7 expressing host antigen presenting cells in the rejection of heart allografts in mice and differences among B7KO and CD28KO animals. PMID- 10755550 TI - Anti-Galalpha1-3Gal antibody levels in organ transplant recipients receiving immunosuppressive therapy. AB - The effect of long-term pharmacologic immunosuppression (PI) on anti-Galalpha1 3Gal (alphaGal) antibody (Ab) levels has not been determined previously in humans. In this study, we measured alpha Gal Ab levels by ELISA in 14 healthy volunteers (controls) and in 70 patients with grafts (kidney, heart, liver) who had received different combinations of PI (including cyclosporine, tacrolimus, azathioprine, mycophenolate mofetil, and steroids) for >3 months. There was great variation in Gal IgM (<80-fold) and IgG (<160-fold). There was no difference in Gal IgM or Gal IgG between any one group and any other. In kidney patients with either high (mean 68%) or low (mean 6%) panel-reactive alloantibodies, there was no difference in alpha Gal Ab level or serum cytotoxicity to pig cells. In vitro immunoadsorption of alphaGal Ab from the serum did not change panel-reactive alloantibody positivity. Therapy with OKT3, a mouse product that might stimulate alphaGal Ab production, led to no significant change in patient Ab levels. We conclude that long-term (>3 months) PI does not reduce Gal Ab levels sufficiently to be of clinical value in xenotransplantation. PMID- 10755551 TI - Detection of HLA class I and II antibodies by ELISA and complement-dependent cytotoxicity before and after transplantation. AB - BACKGROUND: Anti-class I IgG can be detected by complement-dependent cytotoxicity (CDC) and by ELISA. We compared ELISA and CDC for both class I and class II antibodies on method agreement and relation to rejection-free and graft survival. METHODS: Peak, current, and posttransplant sera (n=429) of 143 renal allograft patients were tested by National Institutes of Health technique (NIHT), two-color fluorescence (TCF), and ELISA. Method agreement was assessed by intraclass correlation coefficient (ICC). Rejection and graft survival were analyzed by uni- and multivariate techniques. The screening results for each serum were compared, as was the change in result of current to posttransplant serum. RESULTS: The ICC of ELISA and NIHT was insufficient; it was lower for TCF than NIHT. Graft survival was not related to the result of any assay. Rejection-free survival was related to ELISA and NIHT in current and posttransplant serum. With the NIHT, the change in percent panel-reactive antibody (%PRA) correlated better with rejection than it did with ELISA. The combined antibody status of current and posttransplant serum was a risk factor for rejection in all assays, and for TCF also in multivariate analysis. The rejection rate was higher if the posttransplant serum was ELISA-negative/CDC-positive, rather than ELISA positive/CDC-negative. For ELISA, class I specificities (and not %PRA) in peak and current sera were related to rejection, even if the antibodies were not donor directed. In the case of the National Institutes of Health technique (NIHT), %PRA and not specificity was related to rejection. Class II antibodies were never related to rejection. CONCLUSIONS: ELISA and NEIT are complementary screening techniques in this patient population. They are of equal predictive value for rejection. The optimal strategy in combining these techniques must be determined. PMID- 10755552 TI - Conditions that enable human hematopoietic stem cell engraftment in all NOD-SCID mice. AB - BACKGROUND: Transplantation of human hematopoietic stem cells is the only true test of their long-term repopulation potential. Models are readily available to evaluate murine hematopoietic stem cells, but few exist that allow reliable quantification of human stem cells. The non-obese diabetic-severe combined immunodeficient (NOD-SCID) mouse model enables quantification of human hematopoietic stem cells, but the conditions that permit human engraftment in all animals have yet to be defined. The aims of the project were, therefore, to describe the variables that allow human engraftment in the NOD-SCID mouse model and the techniques that accurately quantify this engraftment. METHODS: NOD-SCID mice that had or had not received 250, 325, or 400 cGy irradiation received cord blood (CB) mononuclear or CD34+ cells i.v. or i.p. Mice were killed 6 weeks after transplantation, and the bone marrow, spleen, and thymus were harvested. Four color flow cytometric analysis, semi-quantitative PCR, myeloid and erythroid progenitor, and stem cell assays were used to monitor human engraftment. RESULTS: A 250 or 325 cGy and i.v. injection of CB mononuclear or CD34+ cells is required to detect multilineage human engraftment in the bone marrow, spleen, or thymus of NOD-SCID mice. Four-color flow cytometric analysis and semi-quantitative PCR enable accurate detection of 0.1% human cells. Progenitor and stem cell assays provide functional information about the engrafted cells. CONCLUSIONS: Successful development of the NOD-SCID mouse model and techniques to assess human engraftment now allow it to be used reliably to analyze the effects of short-term cytokine exposure on the long-term repopulating capacity of CB stem cells. PMID- 10755553 TI - High sequence homology between human and pig CD40 with conserved binding to human CD154. AB - BACKGROUND: Understanding the molecular interactions between pig tissues and human immune cells is fundamental to achieving long-term pig to human xenograft survival. CD40 has been shown to be central in the interaction of T cells with many antigen-presenting cells including B cells, and dendritic cells. It has been clearly shown in vitro that human T cells can effectively recognize pig major histocompatibility complex proteins, and that various accessory molecule interactions are compatible between these species, including human CD28 with pig B7 family members (CD80/CD86). The importance of CD40 in transplantation has been established using blocking antibodies to its ligand, CD154, which prolong allograft survival in mouse and primate models. METHODS: Pig CD40 was cloned from a porcine spleen cDNA library and subsequently sequenced. Expression of pig CD40 was detected by flow cytometry using soluble human CD154 (hCD154-Ig). Results. Comparison of the derived amino acid sequence of pig with human shows 74% identity. Significantly, there is conservation between pig and human at 5 residues shown by mutagenesis studies to be essential for binding of human CD40 to CD154. hCD154Ckappa was shown to bind pig B cell lines and a proportion of human and pig lymphocytes and further confirmed by staining of COS cells transfected with pig CD40. Conclusions. Recipient human cells bearing CD154 will, therefore, be able to bind donor pig CD40, and these interactions might modulate effector functions and hence influence xenograft survival. Further investigation is necessary to ascertain the exact nature of these interactions and their implications for xenograft survival. PMID- 10755554 TI - The induction of major histocompatibility complex class II expression is sufficient for the direct activation of human CD4+ T cells by porcine vascular endothelial cells. AB - BACKGROUND: The role played by major histocompatibility complex (MHC) class II positive vascular endothelial cells in organ graft rejection is unknown but potentially very important. Methods. The MHC class II-negative porcine vascular endothelial cell line PIEC was stably transfected with the human class II transactivator CIITA, in order to induce MHC class II expression without the coinduction of T-cell costimulatory ligands. These PIEC cells were compared with interferon gamma-treated PIEC cells for their capacity to stimulate the proliferation of pure human CD4+ T cells. Results. The CIITA-transfected PIECs were as effective as interferon y-treated PIECs for stimulating unprimed human CD4+ T cells, the peak response with the CIITA-transfected cells in fact occurring earlier (day 3 instead of day 5). Monoclonal antibodies to SLA-DR substantially inhibited the CD4+ T-cell responses in both cases. However, whereas the response to interferon gamma-treated PIEC was partially inhibited by CTLA4 Ig, that to CIITA-transfected PIEC was not. Conclusions. The strong stimulation of CD4+ T cells by the specific induction of MHC class II antigens demonstrates that PIEC cells constitutively express functionally effective levels of costimulatory ligands. This finding strengthens the case that vascular endothelial cells are professional antigen-presenting cells and that MHC class II positive vascular endothelial cells might play a role in the rejection of organ allografts. PMID- 10755555 TI - Association of glucocorticoids and cyclosporin A or rapamycin prevents E-selectin and IL-8 expression during LPS- and TNFalpha-mediated endothelial cell activation. AB - BACKGROUND: Endothelial cell (EC) activation plays an important role in inflammation, hemostasis, and organ rejection of allogeneic and xenogeneic transplantation. These processes leads to rapid and transient up-regulation of proinflammatory molecules, such as the adhesion molecule E-selectin and the chemotactic cytokine IL-8. The purpose of this study was to investigate the specific effects of several major and potentially synergistic immunosuppressive drugs-cyclosporin A (CsA), rapamycin (Rap), and glucocorticoids (GC)-on lipopolysaccharide (LPS)- or tumor necrosis factor (TNF)alpha-induced EC activation METHODS: The ability of immunosuppressive drugs, used alone or in combination, to prevent in vitro TNFalpha- and LPS-induced expression of E selectin and interleukin 8 on porcine ECs, as well as their effect on leukocyte EC interaction, were investigated. In addition, we studied the in vivo effect of these drugs after i.v. administration of recombinant TNFalpha to rats. RESULTS: At high concentrations, which correspond to the acceptable experimental levels in primate xenograft recipients, CsA, Rap, and GC individually inhibited E-selectin protein induction in a dose-dependent manner in cultured porcine ECs treated with LPS with an additive effect when the drugs were associated. The pattern of drug mediated inhibition was related to the stimulus used to activate ECs (i.e., LPS vs. TNFalpha). Reduced expression of E-selectin on ECs activated in the presence of the tested immunosuppressive drugs correlated with a weaker adhesion of human U937 cells to ECs. Messenger RNA analysis demonstrated that the presence of CsA, Rap, and GC during EC activation inhibited E-selectin and interleukin 8 at the gene expression level. LPS-mediated induction of IbetaBalpha expression was not observed in ECs treated with CsA, whereas GC reduced its transcripts by approximately 50%. It is interesting that in vivo studies confirmed that CsA and GC inhibited EC activation at therapeutic doses (1 mg/kg and 10 mg/kg for GC and CsA, respectively) and showed that the combination of CsA and GC efficiently prevents TNFalpha-mediated induction of E-selectin on cardiac ECs. CONCLUSION: Our data show that, besides their specific immunosuppressive effects on T cells, CsA, Rap, and GC can efficiently contribute to the attenuation of EC activation in vivo and the resulting inhibition is enhanced by the association of CsA with GC. PMID- 10755556 TI - Quantitation of Epstein-Barr virus DNA in the blood of adult liver transplant recipients. AB - BACKGROUND: Posttransplant lymphoproliferation is most often observed in pediatric transplant recipients who experience primary Epstein-Barr virus (EBV) infection at the time of or after transplantation. Lymphoproliferation is believed to be caused by impaired control of EBV-infected cells, which may be of recipient or donor origin. Most studies of EBV infection and lymphoproliferation have focused on the pediatric age group. METHODS: We have undertaken a prospective study of EBV infection in adult liver transplant recipients. Sequentially collected peripheral blood lymphocytes were examined with a recently developed quantitative polymerase chain reaction assay. The assay quantitates EBV DNA genomic titre over a 5 log10 range. RESULTS: Compared with healthy EBV seropositive people not undergoing immunosuppressive therapy, blood EBV DNA titre is elevated in patients with liver disease before transplantation. Overall, highest titres were observed during the first posttransplant month, and in the context of antilymphocyte therapy. In one patient, lymphoproliferation was associated with high titres which fell during reduction of immunosuppressive therapy. In another patient with lymphoproliferation of donor lymphocyte origin, blood EBV DNA titre was not as high. CONCLUSIONS: EBV proliferation is seen in the context of advanced liver disease and after liver transplantation. EBV DNA quantitation is a useful tool to examine the effects of immunosuppression on EBV associated lymphoproliferation, and may be an essential technique for programs exploring the merits of EBV adoptive immunotherapy. PMID- 10755557 TI - Prominent and sustained up-regulation of gp130-signaling cytokines and the chemokine MIP-2 in murine renal ischemia-reperfusion injury. AB - Ischemia-reperfusion injury (IRI) is a major cause of renal dysfunction in both native kidneys and renal allografts. To broaden our understanding of the inflammatory mediators involved in IRI, we used multi-probe RNase protection assays to examine the expression of 26 different cytokine genes in a murine model of renal IRI. We observed that, in addition to up-regulation of IL-1beta and to a lesser extent TNF-alpha, IRI was associated with an intense and sustained up regulation of three gp130-signaling cytokines, IL-6, IL-11, and leukemia inhibitory factor (LIF), as well as with up-regulation of the neutrophil chemotactic and activating mediator macrophage inflammatory protein (MIP)-2. Macrophage colony-stimulating factor (M-CSF) and monocyte chemoattractant protein (MCP)-1 were also moderately up-regulated after IRI, whereas mRNA levels of several other inflammatory mediators including IL-1alpha, IL-2, IL-4, interferon (IFN)-gamma, GM-CSF, and RANTES were minimally increased or remained undetectable. These findings identify MIP-2 as an attractive target for inhibition of leukocyte recruitment in renal IRI and also suggest a potentially novel role for gp130-mediated signals in IRI. PMID- 10755558 TI - Area under the serum creatinine time-curve is a strong predictor of chronic renal allograft rejection. AB - BACKGROUND: A number of risk factors for chronic renal allograft rejection have been identified; in particular the number and severity of acute rejections, hypertension, hyperlipidemia, and insufficient immunosuppression. METHODS: In a retrospective case control study, all histologically confirmed cases of chronic rejection (n=45) that occurred between 1985 and 1993 among patients transplanted at Huddinge Hospital were compared with twice as many controls. Determinants such as donor age and sex, HLA-mismatch, cold ischemia time, recipient age and sex, body mass index, cause of renal disease, time undergoing dialysis, condition of blood vessels at surgery, time of onset, number of acute rejection episodes during the first 3 months, area under the serum creatinine versus time curve (AUC(Creatinine)), blood pressure, blood lipids, and cyclosporine concentrations at various times after the transplantation were also compared. Additional data were obtained from a questionnaire, concerning 79% of the cases and controls. RESULTS: Cases and controls were similar with regard to most determinants, that is, blood pressure, blood lipids, and average cyclosporine concentrations. The main outstanding risk factor for chronic rejection was the time-averaged creatinine (AUC(Creatinine)) value between day 22 and 3 months after transplantation. The adjusted odds ratio for chronic rejection increased stepwise from 1.1 to 9.2, when AUC(Creatinine) increased from < 150 to >300 micromol/l. The number of acute rejection episodes and number of HLA-mismatches also had a significant effect on the risk of chronic rejection. CONCLUSIONS: To reduce the risk of developing chronic rejection after renal transplantation acute rejection episodes during the first 3 months should be avoided as much as possible. PMID- 10755559 TI - Early increased chemokine expression and production in murine allogeneic skin grafts is mediated by natural killer cells. AB - BACKGROUND: Increased expression of chemokine mRNA is observed in allogeneic but not syngeneic skin grafts 3-4 days after transplantation. The recipient cells mediating this early inflammatory response in allografts remain unidentified. METHODS: Isogeneic and allogeneic skin grafts were transplanted to euthymic and athymic nude mice. mRNA expression and protein production of macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and the murine homolog of Gro(alpha), i.e. KC, from graft homogenates retrieved 3-4 days posttransplantation was tested by Northern blot hybridization and ELISA. To deplete NK cells, recipients were treated with antiasialo GM1 (ASGM1) antisera or with anti-NK1.1 mAb before transplantation. RESULTS: Expression of KC, MIP 1alpha, and MIP-1beta mRNA was equivalent in C57BL/6 allogeneic skin grafts and BALB/c isografts at day 2 posttransplant. At day 3 posttransplant, chemokine mRNA levels decreased in isografts but were maintained at high levels in the allografts. Increased early chemokine mRNA was also observed in C57BL/6, but not BALB/c++ grafts on BALB/c athymi(nu/nu) recipients. Treatment of allograft recipients with ASGM1 or with anti-NK1.1 antibody eliminated NK cells from the spleen and allograft infiltrating cell populations and decreased early chemokine mRNA levels in allografts 60-70%. Analyses of allograft homogenates indicated increased levels of KC, MIP-1alpha, and MIP-1beta protein at day 4 posttransplant that were decreased in recipients depleted of NK cells. Early chemokine mRNA levels were equivalent in isogeneic and semiallogeneic F1 grafts. CONCLUSIONS: Early chemokine mRNA expression and protein production in allogeneic skin grafts is amplified by recipient natural killer (NK) cells. These results indicate a novel function for infiltrating NK cells in mediating early increased intra allograft chemokine production and inflammation during the initiation of acute rejection. PMID- 10755560 TI - Postprandial hepatic glycogen synthesis in liver transplant recipients. AB - BACKGROUND: The liver plays a central role in glucose homeostasis by releasing glucose in the fasting state and by taking up and converting into glycogen part of the glucose absorbed from the gastrointestinal tract after meal ingestion. METHODS: To determine whether the hepatic denervation that accompanies liver transplantation interferes with these functions, we assessed glucose tolerance to an oral glucose load in seven patients at 2-6 weeks after orthotopic liver transplantation, in six patients after kidney transplantation, and in six healthy controls. Hepatic glycogen synthesis was non-invasively assessed over the 4 hours after ingestion of a glucose load by monitoring hepatic uridine diphosphoglucose turnover with 13C galactose and acetaminophen. RESULTS: Liver and kidney transplant recipients had increased postprandial glucose concentrations but normal hepatic uridine diphosphoglucose turnover, indicating an unaltered hepatic glycogen synthesis. CONCLUSIONS: These results indicate that denervated liver transplants have an adequate glucoregulatory function. Postprandial hyperglycemia in liver transplant recipients is therefore not due to alterations of liver glucose metabolism. PMID- 10755561 TI - Indications and results of chemotherapy in children with posttransplant lymphoproliferative disease after liver transplantation. AB - Among 39 posttransplant lymphoproliferative diseases (PTLD) in a cohort of 450 pediatric liver transplant recipients, 3 had a malignant lymphoma, unresponsive to arrest of immunosuppression and to gancyclovir, interferon, and anti interleukin 6 antibodies. Lymphoma appeared 20, 46, and 96 months posttransplantation and 16, 43, and 90 months after primary Epstein-Barr virus infection. In one case, the patient had histological progression from plasmacytic hyperplasia PTLD, concomitant with symptomatic primary infection, to Burkitt-like lymphoma 43 months later. These three patients received five courses of chemotherapy, after a cyclophosphamide, doxorubicin, vincristine, and prednisone regimen for Burkitt-like or LH 89 scheme for Hodgkin-like PTLDs. Chemotherapy was well tolerated, and all three were free of disease and without immunosuppression 19, 14, and 4 months after chemotherapy. In Burkitt-like or Hodgkin-like PTLDs, immunomodulatory or antiviral drugs were inefficient. Chemotherapy is indicated and can be safely and successfully used. Long-term arrest of immunosuppression seems feasible without graft rejection. PMID- 10755562 TI - Prediction and diagnosis of cytomegalovirus disease in renal transplant recipients using qualitative and quantitative polymerase chain reaction. AB - BACKGROUND: Preemptive antiviral therapy against cytomegalovirus (CMV) disease after transplantation requires information from suitable laboratory markers. We examined the use of qualitative and quantitative polymerase chain reaction (PCR) to monitor renal transplant recipients. METHODS: A cohort of 77 renal transplant recipients was monitored using an in-house and a commercial (Amplicor; Roche Diagnostic, Basel, Switzerland) PCR on leukocytes and plasma. Quantitative plasma viral load was determined using a commercial PCR kit (CMV Monitor; Roche Diagnostic). Patients were analyzed according to their pretransplantation CMV serological status (R- or R+). RESULTS: Seventeen patients developed CMV disease after transplantation. Qualitative leukocyte PCRs had the best overall sensitivity (54-69%) and specificity (75-87%) in identifying R- recipients with CMV disease before onset. The specificities of qualitative PCRs for R+ recipients were poor and, if used, could result in unnecessary preemptive treatment in up to 50% of patients. Symptomatic and asymptomatic R+, but not R-, recipients could be distinguished using a plasma viral load of 25,000 copies/ml. An increase in viral load of >0.7 log (fivefold) per week also distinguished between symptomatic and asymptomatic R+ recipients with high sensitivity (100%) and specificity (95%). CONCLUSIONS: Qualitative leukocyte PCRs were the best assays to predict CMV disease for R- recipients who received R+ kidneys. None of the qualitative assays could be used to guide preemptive therapy of R+ recipients, but plasma viral load and its incremental rate could be used as diagnostic tools in R+ recipients. PMID- 10755563 TI - Changes in hepatitis C virus RNA viremia concentrations in long-term renal transplant patients after introduction of mycophenolate mofetil. AB - BACKGROUND: Mycophenolate mofetil (MMF) is a potent immunosuppressive agent and might inhibit chronic rejection, at least in primates. The prevalence of chronic hepatitis C virus (HCV) infection is high in renal transplant (RT) patients. To date, it has not been demonstrated whether MMF has any effect upon HCV viremia. METHODS: Fourteen long-term HCV(+) RT patients with chronic allograft dysfunction whose maintenance immunosuppression was based on cyclosporine, were given MMF therapy either in place of azathioprine (n=11) or in addition to baseline therapy (n=3). HCV viremia levels were measured by the Amplicor HCV-Monitor RT-PCR assay (Roche Diagnostic Systems) on two separate occasions before the introduction of MMF, and 1 year after changing to MMF or at the last follow-up visit. RESULTS: MMF therapy was associated with a significant rise in HCV viremia, i.e., 5.8+/ 0.5 vs. 5.2+/-0.7 log copies/ml (P=0.01), although there were no significant changes in liver enzymes. The increase in HCV viremia was not related to HCV genotypes either. At the patient level, HCV RNA concentrations changed in only seven patients (group B), i.e. >1 log copies/ml, whereas it remained stable in the others (group A). Before conversion, the only significant difference between group A and B was the level of HCV RNA, i.e., 5.5+/-0.4 log copies/ml in group A and 4.9+/-0.7 log copies/ml in group B (P=0.05). CONCLUSION: Our study suggests that MMF should be used with caution in stable HCV RT patients whose maintenance immunosuppressive therapy is based on cyclosporine, at least in the case of patients with a low HCV RNA titer. PMID- 10755564 TI - p53 codon 72 polymorphism and susceptibility to skin cancer after renal transplantation. AB - BACKGROUND: Infection with human papillomavirus (HPV) is an important risk factor for the development of skin cancer after renal transplantation. It has recently been suggested that degradation of the tumor suppressor gene p53 is an important mechanism for human papillomavirus-induced carcinogenesis. A common genomic polymorphism occurs at codon 72 of the p53 gene, and in vitro the codon 72Arg variant appears to be particularly susceptible to degradation. METHODS: To test the hypothesis that this polymorphism predisposes to the development of human papillomavirus-associated tumors, we studied p53 codon 72 genotype in 222 long term survivors of renal transplantation, of whom 55 had developed at least one skin tumor. RESULTS: No differences in allele or genotype frequency were detected between individuals who had or had not developed skin tumors after transplantation, or any subgroup thereof. CONCLUSIONS: The p53 codon 72Arginine allele does not confer susceptibility to the development of skin tumors after renal transplantation. PMID- 10755565 TI - Cellular allograft rejection affecting the donor aorta after combined heart-lung transplantation. AB - BACKGROUND: Infectious pseudoaneurysms of the ascending aorta are a recognized major complication after heart-lung transplantation. METHOD: This report describes an unusual and previously unreported complication, that of cellular allograft rejection, which caused a pseudoaneurysm of the donor's ascending aorta in a patient who underwent combined heart-lung transplantation. Repair was performed by primary suture after mobilization of the aortic segments. RESULT: On histological examination the resected aneurysm showed evidence of proliferative vasculitis with perivascular infiltration of the vasa vasorum by mononuclear cells. The mononuclear cells were identified as CD4+ and CD8+ by immunohistological staining. CONCLUSIONS: This report shows that cellular allograft rejection may affect the donor aorta after heart-lung transplantation and may result in pseudoaneurysm formation, even under triple-drug immunosuppression after ABO-compatible allograft transplantation. PMID- 10755566 TI - In situ prosthetic graft repair of a mycotic aneurysm of the aorta after orthotopic liver transplantation. AB - BACKGROUND: Vascular complications after liver transplant are associated with a high incidence of graft failure and mortality. Mycotic aneurysms, although uncommon, carry the additional risk of infection and rupture. METHODS: We report a case of a 51-year-old woman who developed a mycotic aneurysm of the aorta secondary to construction of an infrarenal donor iliac artery graft during a retransplant. We evaluated risk factors for the aneurysm, appropriate diagnosis, and potential treatments. RESULTS: The aneurysm was repaired with an in situ prosthetic graft. The patient is alive with good liver function 31 months posttreatment. CONCLUSIONS: The use of in situ prosthetic grafts for repair of mycotic aneurysms is appropriate in certain situations and may be life-saving. PMID- 10755567 TI - TGF-beta expression in renal transplant biopsies: a comparative study between cyclosporin-A and tacrolimus. AB - BACKGROUND: Chronic rejection is a major cause of graft dysfunction after kidney transplantation. This fibroproliferative disease may be promoted by overproduction of transforming growth factor beta (TGF-beta). Previous studies have suggested that CsA might increase production of this growth factor. The current study was designed to measure the expression of TGF-beta(b) in renal transplant biopsy specimens from patients undergoing immunosuppressive therapy with either CsA or tacrolimus (FK506). METHOD: Paraffin-embedded renal biopsy specimens were sectioned, dewaxed, and incubated with primary antibody against TGF-beta(b)1 latency-associated protein and active TGF-beta(b1). After washing, the sections were treated with secondary antibody conjugated with FITC. In each case, the sections were assessed by semi-quantitative scanning laser confocal microscopic method. RESULTS: There was no significant difference in latent TGF beta(b) expression between biopsy specimens from patients receiving CsA and patients receiving FK506. However, biopsy specimens from patients receiving CsA expressed significantly more active TGF-beta(b1) than biopsy specimens from patients receiving FK506 (P<0.0001, Mann-Whitney test). DISCUSSION: The increased level of active TGF-beta1 expression in renal biopsy specimens of patients receiving CsA may indicate a mechanism of chronic rejection. However, these biopsies were performed to assess deranged renal function; therefore, the specimens may reflect events rather than differences in medication. PMID- 10755568 TI - In situ split liver transplantation for two adult recipients. AB - BACKGROUND: Modifications of the in situ split liver technique are needed for safe transplantation in two adult recipients with a single donor. METHODS: The graft from a brain-dead donor, 187 cm tall and weighing 89 kg, was split in situ with a transection performed along the main portal fissure retaining the middle hepatic vein with the left graft. The right and left grafts, which weighed 985 and 760 g, respectively, were transplanted in two adult recipients weighing 70 and 56 kg, respectively. RESULTS: Both recipients had minor intraoperative blood loss and were discharged from intensive care on day 3. Both grafts were rapidly functional, and the two patients were in excellent condition with normal liver function tests 9 months after surgery. CONCLUSION: In situ split liver transplantation can be performed with the middle hepatic vein retained in the left graft to obtain a sufficient volume of the two grafts suitable for two adult recipients. This modification of the technique could expand the donor pool for adult recipients. PMID- 10755569 TI - ICAM-1 deficiency suppresses host allosensitization and rejection of MHC disparate corneal transplants. AB - BACKGROUND: We used a murine model of orthotopic corneal transplantation to determine whether host deficiency in ICAM-1 promotes survival of corneal grafts with different degrees of allodisparity. METHODS: ICAM-1-/- and wild-type C57BL/6 (ICAM-1+/+) received corneal grafts from the following strains of mice: BALB/c (fully mismatched), BALB.b (mismatched at multiple minor H only), or B10.D2 [including major histocompatibility complex (MHC) mismatch]. Graft rejection, induction of allospecific delayed-type hypersensitivity (DTH) responses, and leukocytic infiltration of grafts were measured. RESULTS: There were no differences in long-term survival of allografts that were either fully mismatched or had only minor H disparity in ICAM-1+/+ vs. ICAM-1-/-hosts. However, whereas B10.D2 grafts were accepted in only 58% of the ICAM-1+/+ hosts, graft survival in ICAM-1-/- recipients was 100% (P=0.006). Moreover, none of the ICAM-1-/- mice receiving B10.D2 grafts developed allospecific DTH. CONCLUSIONS: Prolonged survival seen in MHC-mismatched grafts in ICAM-1-/- mice, along with a suppressed DTH response to donor alloantigens after transplantation, suggest that ICAM-1 is associated with recipient sensitization to MHC alloantigens. PMID- 10755570 TI - Telomerase activity in rat liver allografts. AB - BACKGROUND: Telomerase activity in grafts may be involved in the alteration of cellular senescence after transplantation or its relevant immunological events. METHODS: At the age of 20 weeks, donor livers harvested from DA (RT1a) were orthotopically transplanted into PVG (RT1c) or LEW (RT1(1)) rats. Rats having undergone orthotopic liver transplantation (OLT; DA-PVG) naturally overcome rejection, whereas all OLT (DA-LEW) rats die from acute rejection within 14 days. Telomerase activity in liver allografts was measured at various intervals post OLT. RESULTS: At day 7 when the most severe rejection episode was observed in OLT (DA-LEW) and OLT (DA-PVG), the telomerase activity was significantly higher than in syngeneic OLT (DA-DA) rats, in which no rejection occurred. Telomerase activity in tolerogenic OLT (DA-PVG) livers remained elevated for at least 2 months. CONCLUSION: These results suggest that telomerase activity in allogeneic OLT livers may reflect regenerating hepatocytes or activation of lymphocytes and/or hematopoietic stem cells associated with rejection or tolerance. PMID- 10755571 TI - CD4+ T cells responsive through the indirect pathway can mediate skin graft rejection in the absence of interferon-gamma. AB - BACKGROUND: T cells responding through the indirect pathway can induce allograft rejection, but mechanisms of rejection are not known. Interferon-y (IFN-gamma) may be an important mediator of rejection under these circumstances. METHODS: We transferred CD4+ T cells from IFN-gamma-deficient (IFN-gamma-/-) mice into SCID recipients of MHC II-deficient (MHC II-/-) skin grafts. Under these conditions, rejection can only occur via the indirect pathway and cannot be mediated by T cell production of IFN-gamma. RESULTS: IFN-gamma-/- CD4+ T cells rejected MHC II /- skin grafts. Flow cytometry revealed only CD4+ T cells in the recipients. Cytokine enzyme-linked immunosorbent spot assays confirmed only indirect recognition with an associated expansion of an alloreactive population of IL-2-, IL-4-, and IL-5-secreting T cells. CONCLUSION: CD4+ T cells recognizing alloantigens via the indirect pathway can mediate skin graft rejection in the absence of IFN-gamma. PMID- 10755572 TI - Enhanced allograft survival induced by posttransplant donor spleen cell infusion occurs via a mechanism that is distinct from the mechanism of enhancement by donor bone marrow. AB - BACKGROUND: Our previous studies have shown that the ability of donor bone marrow to augment skin graft survival in antithymocyte serum (ATS)-treated recipients is dependent on the presence of functional CD95-ligand (Fas-ligand) molecules on donor cells. Because donor spleen cells can augment graft survival to a similar degree in the same model, we investigated whether the donor spleen cell effect was also dependent on the presence of CD95-ligand on donor cells and CD95 on recipient cells. METHODS: Mutant mice bearing defects in the expression of CD95 (lpr mutation) and CD95-ligand (gld mutation) were used as recipients and cell donors, respectively. Recipients were injected with rabbit ATS on days -1 and +2, and then were injected with 5x10(7) spleen cells on day +7. Skin graft survival was compared and correlated with the use of mutant mice as recipients and cell donors. RESULTS: The combination of ATS and infusions of wild-type [median survival (MST)=44 days, P=0.0004] and gld (mutant CD95-ligand, MST=37 days, P=0.02) donor spleen cells enhanced C3H graft survival, compared with (C57BL/6 x A)F1 recipients treated with ATS alone (MST=27 days). Furthermore, C57BL/6 lpr (CD95-deficient) strain recipients treated with ATS and donor spleen cells demonstrated enhanced B10.D2(R107) strain skin graft survival (MST=44 days, P=0.003), compared with C57BL/6 lpr recipients treated with ATS alone (MST=31 days). Wild-type C57BL/6 recipients treated in the same manner also exhibited an extension of graft survival (MST=64 days) versus controls treated with ATS alone (MST=31 days). CONCLUSION: The data demonstrate that the ability of donor spleen cells to augment allograft survival is not dependent on the CD95/CD95-ligand pathway; therefore the deletion of allospecific cells by donor spleen cells may be induced via a pathway other than deletion by donor bone marrow cells. PMID- 10755573 TI - Genetic susceptibility to renal ischemia reperfusion injury revealed in a murine model. AB - BACKGROUND: The development of genetically engineered mice has led to increased use of mouse models to study renal ischemic reperfusion injury (IRI). We hypothesized that susceptibility to IRI could result from strain differences due to genetic factors. METHODS: Our study compared recovery subsequent to renal IRI in NIH Swiss, C57BL/6, and BALB/c mice. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were evaluated postischemia. We also conducted reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of renal cytokines and adhesion molecules postischemia. RESULTS: At 48 hr postischemia, renal dysfunction in NIH Swiss mice was significantly reduced, compared with other groups (P<0.01). BUN measurements confirmed renal protection at 48 hr in the NIH Swiss group. RT-PCR analysis of mRNA postischemia demonstrated that, between strains, there was little difference in mRNA expression for renal cytokines and adhesion molecules. CONCLUSIONS: NIH Swiss mice appear to be resistant in susceptibility to renal IRI. Early expression of pro-inflammatory genes was not associated with resistance to IRI, thus genetic factors could be important in outcome after renal IRI. PMID- 10755574 TI - Use of grafts procured from transplant recipients. PMID- 10755575 TI - Cytomegaloviral infections in renal transplant recipients treated with daclizumab. PMID- 10755576 TI - Pilot study of growth hormone administration during the refeeding of malnourished anorexia nervosa patients. AB - OBJECTIVE: In anorexia nervosa (AN), medical stabilization and nutritional repletion are pivotal steps toward physical and psychological recovery. Nutritional stabilization is often difficult in this patient group. Recombinant human growth hormone (rhGH) has been safely used as adjuvant therapy in other groups of malnourished patients. We hypothesize that rhGH treatment will hasten medical stabilization in AN patients. STUDY DESIGN: Fifteen patients admitted for inpatient treatment for AN, ages 12-18 years, were enrolled in a 28-day randomized, double-blind, placebo-controlled study. Patients received rhGH (0.05 mg/kg subcutaneously) or an equivalent volume of placebo daily. Outcome measures included time to reach medical/cardiovascular stability, rate of weight gain, and duration of hospitalization. All patients received a standard refeeding protocol. RESULTS: Mean admission body mass index was 14.5 kg/m2. The rhGH and placebo groups did not differ significantly in admission weight, BMI or daily caloric intake. Patients treated with rhGH reached medical/cardiovascular stability more rapidly than those treated with placebo (median 17 vs. 37 days, p = 0.02). Numerical but not statistically significant improvements were seen in weight gain and length of hospitalization in the rhGH group. CONCLUSION: Patients treated with rhGH achieved medical/cardiovascular stability more rapidly than those treated with placebo, and this, in turn, decreased the length of stay. PMID- 10755577 TI - Therapeutic effects and tolerability of fluvoxamine treatment in adolescents with dysthymia. AB - OBJECTIVE: The aim of the study was to evaluate therapeutic effects and tolerability of fluvoxamine (150-200 mg daily) in 21 dysthymic adolescents. SUBJECTS: Twenty-one adolescents, attending psychiatric clinics, who met DSM-III R criteria for dysthymia, without significant co-morbidity were the subjects. METHODS: Axis I and II diagnoses were made by using SCID-P and SCID II. Score A of >13 on HAMD-S at baseline was mandatory. The HAMD-S was completed after 4, 8, and 26 weeks. Adverse effects were recorded after 1, 2, 4, and 8, weeks. Tolerability was assessed by using CGI-T after 1, 2, and 4 weeks. Adverse effects caused three subjects to withdraw from the study. RESULTS: Good clinical response (decrease of HAMD-S score >50%) was observed after 4 weeks in 48% of patients; after 8 weeks in 56% of patients, and after 26 weeks in 44% patients. Relapse occurred in 34% of subjects. Fluvoxamine was well tolerated in 76.2% of the adolescents; poor toleration resulted in its discontinuation in 14.2% adolescents. PMID- 10755578 TI - Open-label treatment with risperidone of 26 psychiatrically-hospitalized children and adolescents with mixed diagnoses and aggressive behavior. AB - Open-label risperidone was administered to 26 subjects (24 boys: 19 with borderline IQ and 5 with mild mental retardation), 10-18 years old, who were hospitalized for treatment of psychiatric disorders associated with aggressive behavior. Risperidone was given in daily doses ranging from 0.5 to 4 mg for periods of 2-12 months. Treatment response was monitored by means of the improvement scale of the CGI and the modified OAS. Extrapyramidal side effects were measured on the ESRS. Fourteen (54%) of 26 subjects had a marked reduction in aggression; 10 subjects had a moderate reduction; two subjects had mild changes; and none worsened. Two subjects had a marked weight gain in the first 8 weeks of treatment. In seven of the 22 children who continued taking risperidone after week 8, tiredness and sedation that necessitated dose reduction emerged between weeks 8 and 16. These results suggest that risperidone may be useful when treating severe aggressive behavior in children and adolescents. Weight gain and sedation can be troublesome side effects. PMID- 10755579 TI - Fluoxetine-related death in a child with cytochrome P-450 2D6 genetic deficiency. AB - The clinical course of a 9-year-old diagnosed with attention-deficit hyperactivity disorder, obsessive-compulsive disorder, and Tourette's disorder and treated with a combination of methylphenidate, clonidine, and fluoxetine is described. The patient experienced over a 10-month period, signs and symptoms suggestive of metabolic toxicity marked by bouts of gastrointestinal distress, low-grade fever, incoordination, and disorientation. Generalized seizures were observed, and the patient lapsed into status epilepticus followed by cardiac arrest and subsequently expired. At autopsy, blood, brain, and other tissue concentrations of fluoxetine and norfluoxetine were several-fold higher than expected based on literature reports for overdose situations. The medical examiner's report indicated death caused by fluoxetine toxicity. As the child's adoptive parents controlled medication access, they were investigated by social welfare agencies. Further genetic testing of autopsy tissue revealed the presence of a gene defect at the cytochrome P450 CYP2D locus, which results in poor metabolism of fluoxetine. As a result of this and other evidence, the investigation of the adoptive parents was terminated. This is the first report of a fluoxetine-related death in a child with a confirmed genetic polymorphism of the CYP2D6 gene that results in impaired drug metabolism. Issues relevant to child and adolescent psychopharmacology arising from this case are discussed. PMID- 10755580 TI - Bleeding and selective serotonin reuptake inhibitors in childhood and adolescence. AB - The selective serotonin reuptake inhibitors (SSRIs) are becoming widely used in children and adolescents, with possible unexpected side effects being observed over time. SSRIs have been associated with bleeding in adults who have unremarkable routine hematologic laboratory results except abnormal bleeding time or platelet counts in few cases. Given the increase of pediatric SSRI prescriptions, in this article we describe five children, ages 8 through 15, who developed bruising or epistaxis 1 week to 3 months after starting SSRI treatment. It is possible that the effects SSRI on platelet functioning are causing the bleeding observed in some patients and/or that a separate coagulopathy is present and contributing to bleeding. The subject matter deserves future investigation. PMID- 10755581 TI - Adverse response to methylphenidate in combination with valproic acid. AB - The use of methylphenidate (MPH) in combination with antiepileptic drugs is gaining acceptance for children with epilepsy who have the symptoms of attention deficit hyperactivity disorder (ADHD). We report two cases of an adverse effect of dyskinesia and bruxism when MPH was given to children maintained on valproic acid. These adverse effects were rapid and severe. Because of the potential for serious and persistent side effects from this combination of medications, caution is warranted in the treatment of ADHD with MPH for children taking valproic acid. Further prospective studies of the use of valproic acid and MPH in combination are required. PMID- 10755583 TI - Science, politics and public opinion: tackling 'the modern dilemma'. PMID- 10755582 TI - Mood stabilizer augmentation with olanzapine in acutely manic children. AB - We report on three cases of acutely manic prepubertal children diagnosed with bipolar disorder who were treated with olanzapine in addition to their existing mood stabilizer regimens. All three had marked improvement of their manic symptoms within 3-5 days of beginning olanzapine therapy as measured by clinician rated instruments. Adverse effects included sedation and weight gain. These results suggest that olanzapine may have an antimanic or mood stabilizing effect in acutely manic children with bipolar disorder. PMID- 10755584 TI - Tough times, tough decisions. PMID- 10755585 TI - Education and progression--is there really a crisis? PMID- 10755586 TI - Antigenic and genetic characterisation of Newcastle disease viruses isolated from outbreaks in domestic fowl and turkeys in Great Britain during 1997. AB - Antigenic and genetic analyses of viruses from the 11 outbreaks of Newcastle disease in Great Britain, 12 of the outbreaks in Northern Ireland and the single outbreak in the Republic of Ireland which occurred in 1997, indicated that they were all essentially similar. In addition, the viruses from the British Isles were very similar to viruses isolated from three outbreaks in pheasants in Denmark between August and November 1996, from a goosander in Finland in September 1996, from an outbreak in chickens in Norway in February 1997, and from an outbreak in chickens in Sweden in November 1997. Viruses from outbreaks in other countries during 1995 to 1997 could be distinguished antigenically and/or genetically from the 1996 to 1997 Scandinavian/British Isles isolates, as could viruses responsible for two separate outbreaks in caged birds in quarantine premises in Great Britain in March 1997. Minor nucleotide differences in the 413 base region of the fusion gene and the 187-base region of the haemagglutinin neuraminidase gene sequenced in this study allowed the 1996 to 1997 Scandinavian/British Isles isolates to be divided into groups. These groups broadly corresponded to the clusters of disease outbreaks, but suggested that the discrete outbreak in Scotland was probably the result of virus spread from Northern Ireland. Overall, the antigenic and genetic analyses of these viruses were consistent with the theory that the virus was introduced into the British Isles by migratory birds moving from north-east Europe. However, it was not possible to rule out other sources, such as the movement of pheasants from Denmark. PMID- 10755587 TI - Behavioural and physiological responses of pigs to being transported for up to 24 hours followed by six hours recovery in lairage. AB - Ninety-six pure-bred Large White pigs weighing 80 to 100 kg were either not transported or transported for eight, 16 or 24 hours, and then either slaughtered immediately on arrival at the abattoir or kept in lairage for six hours before being slaughtered. The pigs travelling for eight, 16 or 24 hours lost 2.2, 2.0 and 4.3 per cent of liveweight, respectively, and the pigs travelling for 24 hours lost 2.6 per cent of hot carcase weight Both liveweight and hot carcase weight recovered during the lairage period although there was a net liveweight loss in comparison with the control animals. Only transport for 24 hours led to losses in carcase weight The concentration of non-esterified fatty acids increased with the time spent travelling. The concentrations of cortisol, creatine phosphokinase and lactate were all low in comparison with the levels found in commercially slaughtered animals. Plasma albumin and protein concentrations indicated that the animals were becoming dehydrated during the longer journeys. The animals lay down for most of the journey and appeared to be asleep. During the period in lairage, the animals transported for eight hours had two distinct periods of feeding and drinking but spent most of the time lying down, but those transported for 16 and 24 hours showed far more eating and drinking activity. All the groups appeared tired, but the urge to eat of the groups transported for 16 hours, and especially 24 hours appeared to be more important. There were behavioural and the physiological differences between the transported groups and the controls. Six hours in lairage with access to food and water allowed most of the physiological parameters to return to pretransport levels. PMID- 10755589 TI - Heart rate and behavioural responses of crib-biting horses to two acute stressors. AB - The heart rate and behaviour of 14 adult saddle horses, eight crib-biters and six normal controls, were investigated. Initially, the relationship between crib biting and heart rate was investigated while the horses were undisturbed. The horses were tested when restrained with a lip twitch, and assessed when they were exposed suddenly to the rapid inflation of a balloon. The heart rate of the crib biters during crib-biting was lower than during other behaviours. The crib-biters had a higher overall mean heart rate (P<0.05) suggesting that they may have had a higher basal sympathetic activity. After the application of the twitch, all the horses had a transient increase in heart rate which returned to basal values more rapidly in the crib-biters. The crib-biters were less reactive to the lip twitch, five of the six investigated remaining calm, and after the release of the twitch, they spent more time nibbling (P<0.05) than the control horses. The crib-biters reacted more strongly to the inflation of the balloon (three of the six reacted), and after it had been inflated they spent more time walking in the box. PMID- 10755588 TI - Identification of seven serotypes of bluetongue virus from the People's Republic of China. AB - Seven serotypes (1, 2, 3, 4, 12, 15 and 16) of bluetongue virus were isolated from the blood of sheep and cattle in the People's Republic of China between 1986 and 1996. Six of these viruses were isolated in Yunnan province. The sheep from which serotypes 1 and 16 were isolated showed obvious signs of bluetongue disease, whereas the cattle from which serotypes 2, 3, 4, 12 and 15 were isolated were clinically normal. Phylogenetic analyses of these viruses indicate that they are more closely related to one another, and to an Australian strain of serotype 1, than they are to prototype strains of bluetongue virus serotypes 2, 10, 11, 13 and 17 from the USA. PMID- 10755590 TI - Isolation of Mycoplasma ovine/caprine serogroup 11 from infertile sheep in Britain. PMID- 10755591 TI - Pathogenicity of Streptococcus suis serotype 4 in piglets. PMID- 10755592 TI - Susceptibility of yak (Bos grunniens) to copper deficiency. PMID- 10755593 TI - In-feed copper for cattle. PMID- 10755594 TI - Use of Fevaxyn Pentofel. PMID- 10755595 TI - Arthritis in pigs. PMID- 10755596 TI - Neurological and urological condition in young boxer dogs. PMID- 10755597 TI - Pathological assessment of sentinel lymph-node biopsies in patients with breast cancer. PMID- 10755598 TI - Prognostic significance of apoptosis and associated factors in oral squamous cell carcinoma. AB - Tumour progression is characterised by an imbalance between cell proliferation and apoptosis. The aim of our study was to estimate the importance of proliferation and apoptosis associated parameters in primary squamous cell carcinomas (SCCs) of the oral cavity and oropharynx. For determination of apoptosis, the enzymatic labelling of DNA fragmentation with a terminal transferase reaction was used in 156 tissue samples of 107 patients, including corresponding lymph-node metastases in nine cases. P53, bcl-2, and Ki-67 were determined immunohistologically. P53 was detectable in 50.5% of the cases. Positive staining was associated significantly with decreased apoptosis (P<0.003). Bcl-2 was upregulated in 31.8% of the cases depending on the tumour grading (P<0.001) and correlated negatively with apoptosis (P<0.001). Proliferation (P<0.006) and apoptosis (P<0.03) were enhanced in larger tumours, though a direct correlation between these two parameters was not proven. Nevertheless, in contrast to the conventional tumour staging and grading, neither the expression of p53 or bcl-2 nor the apoptosis or Ki-67 measurements were able to predict survival or recurrence-free survival of the patients suffering from a SCC in the oral cavity or oropharynx. Our observations suggest that the function of wild-type p53 to induce apoptosis is lost in at least half of the SCCs under study and that the physiological function of bcl-2 as potent inhibitor of apoptosis is widely preserved in oral SCC. PMID- 10755599 TI - Increased in situ expression of nitric oxide synthase in human colorectal cancer. AB - There is growing evidence that nitric oxide (NO) has an important role in tumor growth. However, information on the expression of NO synthase (NOS) in colorectal cancers is scanty. We therefore investigated the distribution and expression of NOS in human colorectal cancers. The expression of three types of NOS, inducible (iNOS), endothelial (eNOS) and neuronal (nNOS), was examined by immunohistochemistry in 25 cases of colorectal cancer. The expression of iNOS was also investigated at the mRNA level using the reverse transcriptase polymerase chain reaction (RT-PCR) in 6 cases. Correlations were made between iNOS expression and the histopathological findings. Immunoreactive iNOS was detected in the tumor cells in 22 cases (88%) with diffuse cytoplasmic reactions. Expression of iNOS-mRNA detected by RT-PCR in three tumor tissues was over five fold that in normal mucosa. Intensified immunoreactivity of iNOS was associated with vascular invasion. iNOS expression did not correlate with pathological staging, tumor size, lymph node metastasis, p53 expression or tumor vessel density. Immunoreactive eNOS stained more strongly in the endothelial cells of microvessels within and around the tumor than in the areas remote from the tumor. There is enhanced expression of iNOS and eNOS in human colorectal cancers, which may correlate with tumor growth and vascular invasion. PMID- 10755600 TI - Mucosal humoral immune response to CagA shows a high prevalence in patients with gastric MALT-type lymphoma. AB - In the pathogenesis of gastric mucosa-associated lymphoid tissue (MALT)-type lymphoma, CagA-positive Helicobacter pylori strains have been suspected of making a significant contribution. To investigate this hypothesis in more detail, the mucosal humoral immune response of 15 patients with gastric MALT-type lymphoma was examined in the tumor and in the tumor-free gastritis of the same patient. Mononuclear cells from different sites (antrum, corpus, lymphoma) were cultured. Culture supernatant and serum of the same patient were used for immunodetection of CagA. All patients displayed an immune response to CagA in the tissue-culture supernatants. Although the humoral immune response in the tumor was restricted to a very few H. pylori antigens, antibodies directed against CagA protein were found in most patients. The immune response to CagA in nearly all lymphoma patients--not only in the serum, but also in the mucosa, including the tumor site -support the hypothesis that CagA is involved in the pathogenesis of gastric MALT type lymphoma. PMID- 10755601 TI - Expression of MUC1, Thomsen-Friedenreich-related antigens, and cytokeratin 19 in human renal cell carcinomas and tubular clear cell lesions. AB - The expression of MUC1, MUC2, mucin-associated Thomsen-Friedenreich-related antigens (TF, sialosyl-TF, Tn, and sialosyl-Tn), and cytokeratin 19 (CK19) was systematically investigated in situ in 58 resected human kidney tumours, surrounding tissue of normal appearance, and two normal kidneys obtained at autopsy, using monoclonal antibodies. In kidney tissues of normal appearance, TF, s-TF, MUC1 and CK19 were positive in distal tubules and collecting ducts but negative in proximal tubules. In contrast, MUC2, Tn, and s-Tn were negative throughout the normal renal tubular system. Almost all renal cell carcinomas (RCCs) showed strong immunoreactivity for MUC1, but all were negative for MUC2. Some RCCs expressed TF, Tn, s-Tn, and CK19. In addition, the immunomorphological characteristics of the majority of clear-cell RCCs and clear/granular RCCs with anti-MUC1 and anti-CK 19 closely resembled those of the collecting duct and the distal tubule rather than the proximal tubule. In the renal tissue of otherwise normal appearance adjacent to clear-cell RCCs and clear/granular RCCs, clear cells with excessive storage of glycogen were often found in the collecting duct system, but only rarely in the proximal tubules. These results suggest that the majority of clear-cell RCCs and clear/granular RCCs may originate from the collecting duct system. PMID- 10755602 TI - The prevalence of human thymic lymphoid follicles is lower in suicides. AB - In an autopsy study we determined the prevalence of thymic lymphoid follicles in 311 accident victims in whom the time interval between accident and death was known. We found that the prevalence decreased abruptly in those surviving 48 h or more (P=0.000008). We then compared the prevalence in 271 accident and 168 suicide victims, all of whom had died less than 48 h after the incident and found that the prevalence was significantly lower in the suicide group (P=0.03). We conclude that this difference may be related to the effect on the thymus of high levels of psychological stress likely to have been experienced by the suicides in the days prior to the act. The use of the term hyperplasia to indicate the presence of lymphoid follicles in the thymus and the methodology appropriate for determining the prevalence of thymic lymphoid follicles are discussed. PMID- 10755603 TI - Expression of beta2 integrins and macrophage-associated antigens in meningeal tumours. AB - This study assessed the expression of leukocyte integrins and macrophage associated antigens in meningiomas. Fourteen benign meningiomas, ten atypical/anaplastic meningiomas, two hemangiopericytomas and one solitary fibrous tumour (SFT) were included. Frozen sections were immunostained using antibodies directed against leukocyte integrins, CD68, CD14, CD2, CD1a, DRC1 and CD34. Their expression was evaluated semi-quantitatively. Ki67 positive cells were counted. Arachnoid membranes served as controls. Arachnoid cells expressed the beta2 integrin subunit and KP1. Beta2 was detected in the tumour cells of 14 meningiomas. In nine cases, this was associated with an alpha-integrin subunit. There was no statistical difference in the expression of beta2 between benign and atypical/anaplastic meningiomas. KP1 was constantly expressed by the tumour cells of meningiomas. It was not expressed by other meningeal tumours. CD34 was detected in the fibrous meningiomas, hemangiopericytomas and the SFT. In each tumour, macrophages were more numerous than T lymphocytes. There was no statistical difference in the density of macrophages and T lymphocytes between the benign and atypical/anaplastic meningiomas. There was no correlation between the Ki67 proliferation index and macrophage infiltration. Meningiomas, through the expression of leukocyte antigens, have a very particular phenotype. The expression of beta2 integrins could play a role in the attraction of immunocompetent cells in the stroma of meningiomas. PMID- 10755605 TI - A method for culturing and transplanting biliary epithelial cell from syrian golden hamster. AB - The present paper describes the establishment of a method for simultaneous culturing of biliary epithelial cells (BECs) from the gall bladder (GB), extrahepatic bile duct (EBD) and intrahepatic bile duct (IBD) of the hamster. GB, EBD and IBD were cut from the biliary tree after collagenase perfusion of the liver. These biliary segments were minced into fragments. The fragments were embedded in collagen gel and cultured in Dulbecco's modified Eagle medium/HamF12 medium containing 10% fetal bovine serum. The various cells subsequently spread from the fragments and formed cellular sheets. After the fragments and flattened cells were removed with the aid of a Pasteur pipette under phase-contrast microscopy, the sheets remaining were found to be composed of cuboidal cells. These cuboidal cells were shown to express gamma glutamyl transpeptidase and cytokeratin 7, which are known to be specific markers of BECs. Ultrastructurally, a large number of microvilli were observed on the luminal surface and junctional complex and interdigitation was identifiable on the lateral surfaces. BEC cultures were subcultured by digestion with collagenase and dispase and then dissociated by subsequent digestion in trypsin and ethylenediaminetetraacetic acid and then maintained on collagen gel for up to 8 weeks. After several passages, the BECs in culture eventually increased in size and showed vacuoles in the cytoplasm. They demonstrated irreversible growth arrest at 9 weeks. The BECs tended to form cystic structures when the BECs with collagen gel were transplanted into the interscapular fat pads of syngeneic hamsters. We established a method for culturing and transplanting biliary cells from syrian golden hamsters. This method may help to clarify the mechanism of hepatobiliary diseases. PMID- 10755604 TI - Splenic smooth-muscle tumors in children with acquired immunodeficiency syndrome: report of two cases of this unusual location with evidence of an association with Epstein-Barr virus. AB - Smooth-muscle neoplasms are rarely located in the spleen. They have been previously reported in five cases of children with human immunodeficiency virus (HIV) infection/acquired immunodeficiency syndrome (AIDS). Two cases of children with HIV infection/AIDS with autopsy and surgical pathology evidence of multiple smooth-muscle neoplasms with splenic involvement are presented. DNA was extracted from histology slides in both cases for analysis for Epstein Barr (EB) virus. In both cases, the presence of EB virus was confirmed. This paper documents two additional cases of the unusual phenomenon of splenic involvement by smooth muscle neoplasms in the setting of AIDS in childhood and further supports the role of EB virus in the development of these neoplasms. PMID- 10755606 TI - The T-type calcium channel blocker mibefradil reduced interstitial and perivascular fibrosis and improved hemodynamic parameters in myocardial infarction-induced cardiac failure in rats. AB - Fibrillar collagen accumulates within the interstitium and around coronary arteries following cardiac failure and is responsible for abnormal myocardial stiffness and reduced coronary performance associated with impaired cardiac function. The aim of the study was to determine the effects of long-term treatment with the T-type calcium channel antagonist mibefradil on myocardial remodeling and cardiac function after chronic myocardial infarction (MI). MI was induced by permanent ligation of the left coronary artery in male Wistar rats. Animals were assigned to sham-operated, placebo-treated or mibefradil-treated (10 mg/kg per day p.o.) MI groups. Treatment with mibefradil was started either 7 days before, 24 h after, or 7 days after ligation and continued for 6 weeks after MI. At this time point, mean arterial blood pressure (MAP), heart rate (HR), left ventricular end-diastolic pressure (LVEDP) and cardiac contractility (dP/dt(max)) were measured in conscious rats. Morphometric parameters were determined in picrosirius red-stained hearts: total heart weight (THW), interstitial and perivascular collagen volume fraction (ICVF, PCVF), myocardial infarct size (IS), vascular perimeter (VP), inner vascular diameter (IVD) and media thickness (MT). Six weeks after MI, MAP and dP/dt(max) were decreased, and LVEDP was increased in placebo-treated animals. In mibefradil-treated animals whose treatment started 7 days before or 24 h after MI, MAP and dP/dt(max) were higher, and LVEDP was lower than in placebo-treated controls. THW, ICVF, PCVF and MT were higher in placebo treated animals. Mibefradil treatment resulted in higher ICVF and IS, higher VP and IVD (when started 7 days before MI) and lower PCVF and MT (when started 7 days before or 24 h after MI) than were observed in placebo-treated controls. Chronic treatment with mibefradil reduced interstitial and perivascular fibrosis and improved cardiac function in MI-induced heart failure in rats. Cardiac remodeling was best prevented when treatment was begun before the ischemic event. PMID- 10755607 TI - Malignant adenomyoepithelioma of the breast with metastasis in the thyroid gland 12 years after excision of the primary tumor. Case report and review of the literature. AB - We describe a patient who was admitted to our hospital with an enlarged left lobe of the thyroid gland. Since fine-needle aspiration showed atypical follicular cells, a surgical exploration followed. Owing to extensive tumor infiltration into the surrounding tissues curative surgery was not possible, and only an incisional biopsy was taken. Histological examination of this biopsy revealed a mixed tumor composed of epithelial and myoepithelial cells. A primary thyroid tumor, metastasis of a salivary gland, and a skin appendage tumor could be excluded based on clinical examination, conventional histology, and immunohistochemistry. A tumor of the left breast treated 12 years earlier had originally been classified as an intraductal/intracystic carcinoma with focal invasion, but was re-examined. Using immunohistochemistry, the breast tumor was reclassified as a malignant adenomyoepithelioma. The current tumor was apparently a metastasis from this primary breast tumor. An updated review of the literature is given, including current knowledge on histological and immunohistochemical features of adenomyoepithelioma of the breast, with special attention to the reported pathological characteristics of recurrent and malignant tumors. Based on the reported pathological characteristics of recurrent and metastatic tumors we offer a diagnostic tool for identifying potentially malignant and recurrent tumors. PMID- 10755608 TI - Chorangioma with trophoblastic proliferation. AB - Only two examples of the entity of chorangiocarcinoma, in which there is a proliferation of both the vascular and epithelial components of the placental villi, have been reported in the literature. To test the hypothesis that chorangiocarcinomas are actually more common than implied by the literature, histological sections of chorangiomas were reviewed. Syncytiotrophoblast and cytotrophoblast proliferation, with nuclear atypia, similar to that found in trophoblastic neoplasia, were seen in 15 of 23 cases. Thus, 65% of chorangiomas fulfilled the diagnostic criteria to warrant re-assignment as "chorangiocarcinoma". The proliferation index of the cytotrophoblast in the tumor as measured by MIB-1 (Ki-67) immunostaining was significantly higher in "chorangiocarcinoma" than chorangioma (35.4% vs 15.7%, P<0.02). The following factors had no relationship to the presence or absence of trophoblastic proliferation: vascularity, cellularity, infarction, size or location of the chorangioma, or age of the patient. Five of the 15 chorangiomas with trophoblastic proliferation were of the chorangiomatosis variety. No formal follow-up was performed, as this was a retrospective study, but there is no recorded case of persistent gestational trophoblastic disease in this cohort, although one woman with "chorangiocarcinoma" had a history of previous hydatidiform molar pregnancies. An apparently benign clinical course is seen. These lesions, best described as chorangiomas with trophoblastic proliferation, are more common than suggested by the rarity of reported cases. PMID- 10755609 TI - Simultaneous appearance of an adenomyoma and pancreatic heterotopia of the stomach. AB - Adenomyomas of the stomach are rare tumours characterised by duct/gland-like structures embedded within a smooth muscle stroma. Although the histogenesis of adenomyomas remains unclear, the histological appearance has justified the assumption that these are abortive forms of pancreatic heterotopia. We report an unusual case with simultaneous and independent appearance of both adenomyoma and pancreatic heterotopia of the stomach including immunohistochemical characterisation, supporting the concept of a common histiogenetic origin of both lesions. PMID- 10755610 TI - Proteolysis in MHC class II antigen presentation: who's in charge? AB - Antigen-presenting cells (APC) degrade proteins intracellularly to generate peptides, which are then bound by products of the major histocompatibility complex (MHC) and exposed on the surface of the APC for recognition by T cells. The supply of antigenic peptides and their association with MHC molecules requires the concerted action of a cohort of accessory molecules that includes chaperones, transporters of peptides, and the proteases that degrade the antigens. PMID- 10755611 TI - The relationship of MHC-peptide binding and T cell activation probed using chemically defined MHC class II oligomers. AB - A series of novel chemically defined soluble oligomers of the human MHC class II protein HLA-DR1 was constructed to probe the molecular requirements for initiation of T cell activation. MHC dimers, trimers, and tetramers stimulated T cells, as measured by upregulation of the activation markers CD69 and CD25, and by internalization of activated T cell receptor subunits. Monomeric MHC-peptide complexes engaged T cell receptors but did not induce activation. For a given amount of receptor engagement, the extent of activation was equivalent for each of the oligomers and correlated with the number of T cell receptor cross-links induced. These results suggest that formation or rearrangement of a T cell receptor dimer is necessary and sufficient for initiation of T cell signaling. PMID- 10755612 TI - A functional hot spot for antigen recognition in a superagonist TCR/MHC complex. AB - A longstanding question in T cell receptor signaling is how structurally similar ligands, with similar affinities, can have substantially different biological activity. The crystal structure of the 2C TCR complex of H-2Kb with superagonist peptide SIYR at 2.8 A elucidates a structural basis for TCR discrimination of altered peptide ligands. The difference in antigen potency is modulated by two cavities in the TCR combining site, formed mainly by CDRs 3alpha, 3beta, and 1beta, that complement centrally located peptide residues. This "functional hot spot" allows the TCR to finely discriminate amongst energetically similar interactions within different ligands for those in which the peptide appropriately stabilizes the TCR/pMHC complex and provides a new structural perspective for understanding differential signaling resulting from T cell cross reactivity. PMID- 10755613 TI - A proposed mechanism for the induction of cytotoxic T lymphocyte production by heat shock fusion proteins. AB - A 65 kDa mycobacterial heat shock protein (hsp65), fused to a polypeptide that contains an octapeptide (SIYRYYGL) agonist for a particular T cell receptor (2C TCR), stimulated C57BL/6 mice as well as CD4-deficient mice to produce CD8+ cytolytic T lymphocytes (CTL) to the fusion partner's octapeptide. This and other hsp65 fusion proteins but not native hsp65 itself stimulated dendritic cells in vitro and in vivo to upregulate the levels of MHC (class I and II) and costimulatory (B7.2) molecules. The results suggest a mechanism for the general finding that hsp fusion proteins, having fusion partners of widely differing lengths and sequences, elicit CD8 CTL to peptides from the fusion partners without requiring exogenous adjuvants or the participation of CD4+ T cells. PMID- 10755614 TI - The ST3Gal-I sialyltransferase controls CD8+ T lymphocyte homeostasis by modulating O-glycan biosynthesis. AB - T lymphocyte activation evokes distinct changes in cell surface O-glycans. CD8+ T cells undergo an elimination of sialic acid on core 1 O-glycans and an induction of core 2 O-glycans until either apoptotic death or differentiation into memory cells. We find that the ST3Gal-I sialyltransferase is required for core 1 O glycan sialylation and its deficiency induces core 2 O-glycan biosynthesis. Apoptosis ensues with the loss of peripheral CD8+ T cells in the absence of immune stimulation. Cell surface ligation of the ST3Gal-I substrate CD43 recapitulates this phenotype by a caspase 3-independent mechanism. Control of core 1 O-glycan sialylation in T lymphocytes by ST3Gal-I comprises a homeostatic mechanism that eliminates CD8+ T cells by apoptosis while facilitating the production of viable CD8+ memory T cells. PMID- 10755615 TI - Amphiphysin IIm, a novel amphiphysin II isoform, is required for macrophage phagocytosis. AB - Phagocytosis of pathogens by macrophages initiates the innate immune response, which in turn orchestrates the adaptive immune response. Amphiphysin II participates in receptor-mediated endocytosis, in part, by recruiting the GTPase dynamin to the nascent endosome. We demonstrate here that a novel isoform of amphiphysin II associates with early phagosomes in macrophages. We have ablated the dynamin-binding site of this protein and shown that this mutant form of amphiphysin II inhibits phagocytosis at the stage of membrane extension around the bound particles. We define a signaling cascade in which PI3K is required to recruit amphiphysin II to the phagosome, and amphiphysin II in turn recruits dynamin. Thus, amphiphysin II facilitates a critical initial step in host response to infection. PMID- 10755616 TI - Sequential involvement of NFAT and Egr transcription factors in FasL regulation. AB - The critical function of NFAT proteins in maintaining lymphoid homeostasis was revealed in mice lacking both NFATp and NFAT4 (DKO). DKO mice exhibit increased lymphoproliferation, decreased activation-induced cell death, and impaired induction of FasL. The transcription factors Egr2 and Egr3 are potent activators of FasL expression. Here we find that Egr2 and Egr3 are NFAT target genes. Activation of FasL occurs via the NFAT-dependent induction of Egr3, as demonstrated by the ability of exogenously provided NFATp to restore Egr dependent FasL promoter activity in DKO lymph node cells. Further, Egr3 expression is enriched in Th1 cells, suggesting a molecular basis for the known preferential expression of FasL in the Th1 versus Th2 subset. PMID- 10755617 TI - Recruitment of the IKK signalosome to the p55 TNF receptor: RIP and A20 bind to NEMO (IKKgamma) upon receptor stimulation. AB - The adapter protein RIP plays a crucial role in NF-kappaB activation by TNF. Here we show that triggering of the p55 TNF receptor induces binding of RIP to NEMO (IKKgamma), a component of the I-kappa-B-kinase (IKK) "signalosome" complex, as well as recruitment of RIP to the receptor together with the three major signalosome components, NEMO, IKK1 and IKK2, and some kind of covalent modification of the recruited RIP molecules. It also induces binding of NEMO to the signaling inhibitor A20, and recruitment of A20 to the receptor. Enforced expression of NEMO in cells revealed that NEMO can both promote and block NF kappaB activation and dramatically augments the phosphorylation of c-Jun. The findings suggest that the signaling activities of the IKK signalosome are regulated through binding of NEMO to RIP and A20 within the p55 TNF receptor complex. PMID- 10755618 TI - Lck activity controls CD4/CD8 T cell lineage commitment. AB - Thymocytes carrying MHC class I-restricted TCRs differentiate into CD8 T cells, while those recognizing MHC class II become CD4 T cells. The mechanisms underlying how MHC class recognition, coreceptor expression, and effector function are coordinated are not well understood. Since the tyrosine kinase Lck binds with more affinity to CD4 than CD8, it has been proposed as a candidate to mediate this process. By using transgenic mice with altered Lck activity, we show that thymocytes carrying a class II-restricted TCR develop into functional CD8 T cells when Lck activity is reduced. Conversely, thymocytes carrying a class I restricted TCR develop into functional CD4 T cells when Lck activity is increased. These results directly show that quantitative differences in the Lck signal control the CD4/CD8 lineage decision. PMID- 10755619 TI - ROG, repressor of GATA, regulates the expression of cytokine genes. AB - GATA-3 is a T cell-specific transcription factor and is essential for the development of the T cell lineage. Recently, it was shown that the expression of GATA-3 is further induced in CD4+ helper T cells upon differentiation into type 2 but not type 1 effector cells. Here, we report the molecular cloning of a GATA-3 interacting protein, repressor of GATA (ROG). ROG is a lymphoid-specific gene and is rapidly induced in Th cells upon stimulation with anti-CD3. In in vitro assays, ROG represses the GATA-3-induced transactivation. Furthermore, overexpression of ROG in Th clones inhibits the production of Th cytokines. Taken together, our results suggest that ROG might play a critical role in regulating the differentiation and activation of Th cells. PMID- 10755622 TI - Leprosy in the new millennium. PMID- 10755621 TI - The RasGAP-binding protein p62dok is a mediator of inhibitory FcgammaRIIB signals in B cells. AB - The low affinity receptor for IgG, FcgammaRIIB, functions to dampen the antibody response and reduce the risk of autoimmunity. This function is reportedly mediated in part by inhibition of B cell antigen receptor (BCR)-mediated p21ras activation, though the basis of this inhibition is unknown. We show here that FcgammaRIIB-BCR coaggregation leads to increased tyrosine phosphorylation of the RasGAP-binding protein p62dok, with a concomitant increase in its binding to RasGAP. These effects require the recruitment and tyrosine phosphorylation of the phosphatidylinositol 5-phosphatase SHIP, which further recruits p62dok via the latter's phosphotyrosine-binding domain. Using chimeric FcgammaRIIB containing the RasGAP-binding domain of p62dok, we demonstrate that p62dok contains all structural information required to mediate the inhibitory effect of FcgammaRIIB on Erk activation. PMID- 10755620 TI - Functional assessment of precursors from murine bone marrow suggests a sequence of early B lineage differentiation events. AB - Most lineage marker-negative (Lin-)TdT+ cells from murine marrow lack CD34 but display c-kit at low density as well as IL-7Ralpha and Flk-2/Flt-3 receptors. Single cells with these characteristics generated CD45RA+CD19- as well as CD19+ lymphocytes in culture. CD45RA+CD19- marrow cells were resolved into three nonoverlapping subsets. One subset, lacking DX5 and Ly-6C antigens, yielded CD19+ cells in culture. Further analysis demonstrated CD24 on most Lin-TdT+ cells and all CD45R+CD19-DX5-Ly-6C- cells. Mac-1/CD11b was absent from these two subsets of B lineage precursors, while IL-7Ralpha was retained during subsequent differentiation to a CD19+ and stromal cell-independent stage. These findings contrast with previous descriptions of B lymphocyte precursors and suggest a sequence of early differentiation events. PMID- 10755623 TI - HLA molecules, bacteria and autoimmunity. AB - It has been well established that many diseases are linked to HLA antigens. Two of the most interesting HLA associations may provide some insight into the pathogenesis of rheumatic inflammatory conditions. In ankylosing spondylitis (AS), 96% of patients possess HLA-B27, whilst the frequency of this marker in the general population is c. 8%. In rheumatoid arthritis (RA), >90% of patients possess either HLA-DR1 or some subtypes of HLA-DR4, whilst the frequency of this marker in the general population is c. 35%. The association between HLA-B27 and reactive arthritis (ReA) has also been well established. Furthermore, it has been shown that ReA is triggered by infection via the gastrointestinal tract due to Yersinia, Salmonella or Campylobacter spp. and in the genitourinary tract due to chlamydia. In a similar way, microbiological and immunological studies have revealed an association between Klebsiella pneumoniae in AS and Proteus mirabilis in RA. This article reviews the possible pathological implications of the associations between HLA-B27, K. pneumoniae and AS, as well as HLA-DR1/DR4, P. mirabilis and RA. PMID- 10755624 TI - Weak bases affect late stages of Mayaro virus replication cycle in vertebrate cells. AB - This paper describes the effect of two weak bases (ammonium chloride and chloroquine) on the morphogenesis of Mayaro virus. When Mayaro virus-infected TC7 (monkey kidney) cells were treated with these agents it was observed that weak bases caused a significant reduction in virus yield. Also, cellular protein synthesis, which is inhibited by Mayaro virus infection, recovered to nearly normal levels. However, the synthesis of Mayaro virus proteins was affected. These phenomena were dose-dependent. The process of Mayaro virus infection in vertebrate cells is very rapid. Virus precursors are not observed in cell cytoplasm and budding through the plasma membrane seems to be the only way of virus release. Electron microscopy of cells infected with Mayaro virus and treated with weak bases revealed an accumulation of virus structures in cell cytoplasm. The study also noted an inhibition of budding through the plasma membrane and the appearance of virus particles inside intracytoplasmic vacuoles. These observations indicate an impairment at the final stages of the virus replication cycle. PMID- 10755625 TI - Enhancement of hepatocyte growth factor (scatter factor) production by human gingival fibroblasts in culture stimulated with Porphyromonas gingivalis fimbriae. AB - Hepatocyte growth factor (HGF), also known as scatter factor (SF), is a motogen, mitogen and morphogen produced by mesenchymal cells that mainly acts on epithelial cells and is involved in osteoclast stimulation. This study examined the possible enhanced production of HGF/SF by human gingival fibroblasts upon stimulation with killed cells of Porphyromonas gingivalis strain 381 and its representative bioactive cellular components, fimbriae and lipopolysaccharide (LPS). P. gingivalis whole cells enhanced the production of HGF/SF detected by ELISA in culture supernates of the fibroblasts. Fimbriae prepared from P. gingivalis exhibited powerful HGF/SF-inducing activity in a concentration dependent manner, with peak activity observed at 100-200 microg/ml. The fimbriae induced HGF/SF mRNA expression by the cells was also detected by reverse transcription-PCR. P. gingivalis LPS exhibited weak HGF/SF-inducing activity. The study also examined the HGF/SF-inducing activity of seven synthetic peptides corresponding to the segments of P. gingivalis fimbrial subunit protein. The peptides of residues 282-301 and 302-321, which exhibited antagonistic effects against P. gingivalis fimbriae-binding to human gingival fibroblasts in a previous study, showed weak activity, whereas other non-antagonistic peptides showed no significant activity. These findings indicated that P. gingivalis fimbriae enhanced production of HGF/SF by human gingival fibroblasts, whereas synthetic peptide segments of fimbrial subunit protein were not sufficient to exert the activity. PMID- 10755626 TI - The role of fimbriae and flagella in the adherence of avian strains of Escherichia coli O78:K80 to tissue culture cells and tracheal and gut explants. AB - To investigate the role of fimbriae and flagella in the pathogenesis of avian colibacillosis, isogenic insertionally inactivated mutant strains of Escherichia coli O78:K80 strain EC34195 defective in the elaboration of type-1 and curli fimbriae and flagella were constructed by allelic exchange. Single and multiple non-fimbriate and non-flagellate mutant strains were compared to the wild-type in vitro in adherence assays with a HEp-2 cell line, a mucus-secreting cell line HT2916E, a non-mucus-secreting cell line HT2919A, tracheal explant and proximal gut explant. Mutant strains defective in the elaboration of type-1 fimbriae were significantly less adherent--in the order of 90% reduction--than the wild-type strain in all assays. Mutant strains defective in the elaboration of flagella were generally as adherent as the wild-type strain except when assayed with the mucus-secreting cell line HT2916E, for which a significant reduction of adherence -of the order of 90%--compared with the wild-type strain was observed. Mutant strains defective for the elaboration of curli fimbriae adhered as well as the wild-type strain in all assays, except when assayed in tests with gut explant tissue for which a significant reduction of adherence--of the order of 80%- compared with the wild-type strain was observed. Adherence to explants was to epithelial, not serous, surfaces and was 10-fold greater to tracheal than to gut explants. Together, these data support the hypothesis that type-1 fimbriae are significant factors in adherence, aided by flagella for penetration of mucus and curli fimbriae for adherence to the gut. PMID- 10755627 TI - Regulation by protein kinase of phagocytosis of Mycobacterium leprae by macrophages. AB - Mycobacterium leprae multiplies within host macrophages. The mechanism of internalisation of the bacteria by the phagocytic cells is unknown. In this study, M. leprae was purified from the foot pads of experimentally infected nu/nu mice. Peritoneal macrophages were harvested from BALB/c mice or C57 beige (bg/bg) mice. The effect of protein kinase inhibitors (erbstatin, genistein or staurosporine for BALB/c and bg/bg mice, plus herbimycin for bg/bg mice) on phagocytosis of the mycobacteria by the macrophage monolayers was tested. The untreated (control) macrophages phagocytosed M. leprae. Phagocytosis by BALB/c macrophages was inhibited by erbstatin and staurosporine but not by genistein; all the protein kinase inhibitors prevented uptake of M. leprae by bg/bg cells. The results demonstrate that protein kinase regulates phagocytosis of M. leprae by macrophages. The mechanism might prove to be a rational drug target for mycobacteria that multiply intracellularly. PMID- 10755628 TI - Quantitative culture of Helicobacter pylori from gastric juice: the potential for transmission. AB - The transmission of Helicobacter pylori may occur by spread of organisms from gastric juice which has been introduced into the mouth by gastro-oesophageal reflux. The aim of this study was to quantify the load of H. pylori present in gastric juice available for transmission. Gastric antral biopsy and gastric juice samples were collected from 108 adult dyspeptic patients undergoing routine upper gastroscopy and the presence of H. pylori was determined. In all, 54 (50%) of 108 patients gave positive results in the gastric antral biopsy rapid urease test and for H. pylori histology. The gastric juice of 40 (37%) of patients gave positive results for the urease A gene by PCR assay; 34 (31%) of patients were positive by these three tests and H. pylori was cultured from the gastric juice of 13 (38%) of these patients. The median count of H. pylori in gastric juice was 1.75 x 10(1) cfu/ml. Viable organisms in gastric juice may lead to transmission of H. pylori when refluxed or vomited into the mouth. PMID- 10755629 TI - Characteristic distribution pattern of Helicobacter pylori in dental plaque and saliva detected with nested PCR. AB - The precise mode of transmission and the natural reservoir for Helicobacter pylori are unknown. PCR assays have proved to be highly sensitive and specific and are regarded as the method of choice for detecting H. pylori DNA in the oral cavity. The aim of this study was to investigate the prevalence and distribution of H. pylori in the oral cavity. Forty-two patients undergoing gastroscopy were investigated for the presence of H. pylori in dental plaque and saliva by nested PCR, and in the stomach by the 13C-urea breath test. Samples tested comprised dental plaque from molars, premolars and incisors and saliva. Two sets of primers homologous to the 860-bp fragment of H. pylori DNA, which have been shown previously to be highly sensitive and specific, were used for nested PCR. Eleven patients (26.2%) were infected with H. pylori in the stomach. H. pylori DNA was identified in dental plaque samples from 41 patients (97%) and in 23 saliva samples (55%). The prevalence in dental plaque from molars, premolars and incisors was 82%, 64% and 59%, with an odds ratio of 3.18, 1.24 and 1 (reference), respectively. In conclusion, H. pylori was present in the oral cavity of 97% of tested patients, with a characteristic distribution that was independent of the infection status of the stomach. Thus H. pylori may belong to the normal oral microflora. PMID- 10755630 TI - Molecular analysis of Shigella sonnei isolated from three well-documented outbreaks in school children. AB - Fifty-eight isolates of Shigella sonnei from three outbreaks in school children and eight control isolates from epidemiologically unrelated sporadic clinical infections in Taiwan were compared by antibiotic susceptibility testing and molecular typing. Antibiotic susceptibility testing showed that all strains except one sporadic isolate were multi-resistant. Ribotyping after restriction endonuclease digestion with SalI, PvuII and HindII generated the same ribosomal pattern in 65 of the 66 isolates. Plasmid profile analysis and pulsed-field gel electrophoresis (PFGE) produced eight and nine distinct patterns, respectively, and were in agreement with the epidemiological relationship of the outbreak strains. Nevertheless, some of the sporadic isolates could be discriminated only by a combination of these two methods. This study showed that plasmid profiling in combination with PFGE may be superior to ribotyping in molecular epidemiological investigations of S. sonnei. PMID- 10755631 TI - Clinical and microbiological epidemiology of Streptococcus pneumoniae bacteraemia. AB - A survey of Streptococcus pneumoniae bacteraemia in the Grampian region of Scotland was carried out over a 2-year period. One hundred and four bacteraemic episodes were identified in 103 patients, an incidence of 9.8/100,000 population/year, and the mortality was 24%. Clinical information was abstracted from 92 sets of patient notes and 98 isolates of S. pneumoniae were available for further study. The incidence of S. pneumoniae bacteraemia was highest at the extremes of age and peaked at 78 cases/100,000 population/year in those over 80 years old. Many patients had predisposing conditions, of which chronic lung disease (23%), chronic alcohol abuse (10%) and malignant disease (10%) were the commonest. Age was the highest risk factor for mortality, with 20 of the 22 deaths in those over 65 years old. The commonest serotype of S. pneumoniae isolated was serotype 14 (23.5%). Only one isolate (serotype 6A) showed intermediate resistance to penicillin, but 12 isolates (12.2%) were resistant to erythromycin. Nine of these 12 isolates were of serotype 14 and had MICs clustered in the range 12-24 mg/L. Examination of all serotype 14 isolates by pulsed-field gel electrophoresis (PFGE) showed the presence of two distinct genetic clusters, with all the erythromycin-resistant isolates in the same cluster. These isolates had similar PFGE profiles to erythromycin-resistant serotype 14 strains isolated elsewhere in the UK and they were positive for the mefE gene by PCR, confirming that resistance was of the M phenotype. The recent increase in erythromycin resistance in S. pneumoniae may be due, at least in part, to the spread of a serotype 14 clone of the M phenotype which appears to be an important cause of invasive disease. PMID- 10755632 TI - Origins of Staphylococcus epidermidis and Streptococcus oralis causing bacteraemia in a bone marrow transplant patient. AB - Coagulase-negative staphylococcal bacteraemia in immunocompromised patients is often associated with the use of central venous catheters, while the proposed origin of viridans streptococci causing bacteraemia in this patient group is the oral cavity. This report describes an episode of polymicrobial bacteraemia caused by Staphylococcus epidermidis and Streptococcus oralis followed by several further episodes of S. epidermidis bacteraemia in a 15-year-old boy after bone marrow transplantation. Pulsed-field gel electrophoresis (PFGE) of SmaI chromosomal DNA digests was used to compare blood culture and oral isolates of S. epidermidis and Str. oralis. The results indicated that the mouth was the source of both S. epidermidis and Str. oralis causing the first episode of bacteraemia. PFGE further demonstrated that the central venous catheter was the origin of a second strain of S. epidermidis responsible for subsequent episodes of staphylococcal bacteraemia. Both the oral mucosa and central venous lines should be considered as potential sources of organisms, including coagulase-negative staphylococci, associated with bacteraemia in immunocompromised patients. PMID- 10755633 TI - An outbreak of skin sepsis in abattoir workers caused by an 'unusual' strain of Streptococcus pyogenes. AB - An outbreak of indolent skin infections due to an 'unusual' serological type of Streptococcus pyogenes that lasted for 3 months and affected eight workers in an abattoir is described. The group A streptococcal (GAS) isolates were serotyped as M-type 59; however, they possessed a T-protein pattern (T5/27/44) that is not commonly associated with M-type 59. Further genotypic characterisation studies revealed that all eight isolates were indistinguishable by pulsed-field gel electrophoresis (PFGE) and possessed the emm gene encoding for the M-type 59. Once identified, and after a combination of penicillin treatment, exclusion of workers with lesions and reinforcement of standard hygiene precautions, no further cases developed. Although common in the 1970s and 1980s, streptococcal infections in this situation are now reported infrequently. This report serves to highlight the issues surrounding working practices in abattoirs. PMID- 10755634 TI - Multilocus enzyme electrophoresis analysis of Aspergillus fumigatus strains isolated from the first clinical sample from patients with invasive aspergillosis. EBGA Network. European Research Group on Biotype and Genotype of Aspergillus. AB - The genotypes of 50 isolates of Aspergillus fumigatus from 11 patients with invasive aspergillosis, obtained from three hospitals in different geographical areas, were determined by multilocus enzyme electrophoresis (MLEE). The study analysed the genetic polymorphism of multiple isolates from the first sample. Seven of the 14 enzymic loci studied were polymorphic, giving rise to eight different electrophoretic types. For nine of 11 patients studied, no polymorphism was observed in isolates within the first clinical sample. Analysis of genetic distance between electrophoretic types demonstrated a genetic heterogeneity within each geographical site. Moreover, some genotypes were preferentially found in a given area and this revealed a population structure within these geographical sites. Therefore, the epidemiology of A. fumigatus should be considered separately for each of these areas. The multiple discriminatory markers of MLEE seem to provide a powerful tool for increasing the understanding of the biology of this fungus. PMID- 10755635 TI - Identification and characterisation of Escherichia coli strains of O157 and non O157 serogroups containing three distinct Shiga toxin genes. AB - Three Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from patients with diarrhoea were identified, each of which contained three distinct stx genes (stx1, stx2 and stx2c). The strains belonged to the serotypes O52:H19, O75:H- and O157:H- and harboured eae and EHEC-hly sequences. Colony-blot immunoassay was used to demonstrate that both major types of Stx were expressed. The association of stx genes with either phage or phage DNA was demonstrated in all three strains. Isolated phage DNA from all strains contained stx1 sequences, but stx2 sequences were found only in phage DNA of two of these strains. The presence of three distinct stx genes may enhance the virulence of STEC strains and should be monitored. The observations demonstrate not only the potential of stx genes to spread within different serotypes, but also their capacity to accumulate within a single strain. PMID- 10755636 TI - A method of decontaminating Strongyloides venezuelensis larvae for the study of strongyloidiasis in germ-free and conventional mice. AB - To study the possible influence of intestinal micro-organisms on the course of strongyloidiasis in mice, a method was developed to obtain axenic infective larvae of Strongyloides venezuelensis. Cultured larvae from conventional mice were treated with sodium hypochlorite 0.25% for 10 min, washed in distilled water and then exposed to various combinations of antibiotics for 30 or 60 min. Success was achieved with a combination of penicillin 180 mg/L and ceftazidime 1 mg/ml. Decontamination of the larvae was determined by aerobic and anaerobic culture and by inoculation into gnotobiotic mice. Viability was established by subcutaneous inoculation of larvae into germ-free and conventional mice. Preliminary results showed that gnotobiotic mice were more susceptible than conventional mice to infection with axenic S. venezuelensis larvae as judged by faecal egg excretion, recovery of worms in the small intestine and histopathological examination of the duodenal mucosa. These results suggest that the normal intestinal flora protects the host against experimental infection with S. venezuelensis. PMID- 10755637 TI - Chlamydia pneumoniae infection during an influenza virus A epidemic: preliminary report. PMID- 10755638 TI - Isolation of plasmids from Mycobacterium avium-intracellulare complex (MAC) strains from India. PMID- 10755639 TI - Markedly increased plasma (1-->3)-beta-D-glucan is a diagnostic and therapeutic indicator of Pneumocystis carinii pneumonia in a non-AIDS patient. PMID- 10755640 TI - 18F-labeled FECNT: a selective radioligand for PET imaging of brain dopamine transporters. AB - Fluorine-18 labeled 2beta-carbomethoxy-3beta-(4-chlorophenyl)-8-(2 fluoroethyl)nort ropane (FECNT) was synthesized in the development of a dopamine transporter (DAT) imaging ligand for positron emission tomography (PET). The methods of radiolabeling and ligand synthesis of FECNT, and the results of the in vitro characterization and in vivo tissue distribution in rats and in vivo PET imaging in rhesus monkeys of [18F]FECNT are described. Fluorine-18 was introduced into 2beta-carbomethoxy-3beta-(4-chlorophenyl)-8-(2-fluoroethyl)nort ropane (4) by preparation of 1-[18F]fluoro-2-tosyloxyethane (2) followed by alkylation of 2beta-carbomethoxy-3beta-(4-chlorophenyl)nortropane (3) in 21% radiochemical yield (decay corrected to end of bombardment [EOB]). Competition binding in cells stably expressing the transfected human DAT serotonin transporter (SERT) and norepinephrine transporter (NET) labeled by [3H]WIN 35428, [3H]citalopram, and [3H]nisoxetine, respectively, indicated the following order of DAT affinity: GBR 12909 > CIT >> 2beta-carbomethoxy-3beta-(4-chlorophenyl)-8-(3-fluoropropyl) nortropane (FPCT) > FECNT. The affinity of FECNT for SERT and NET was 25- and 156 fold lower, respectively, than for DAT. Blocking studies were performed in rats with a series of transporter-specific agents and demonstrated that the brain uptake of [18F]FECNT was selective and specific for DAT-rich regions. PET brain imaging studies in monkeys demonstrated high [18F]FECNT uptake in the caudate and putamen that resulted in caudate-to-cerebellum and putamen-to-cerebellum ratios of 10.5 at 60 min. [18F]FECNT uptake in the caudate/putamen peaked in less than 75 min and exhibited higher caudate- and putamen-to-cerebellum ratios at transient equilibrium than reported for 11C-WIN 35,428, [11C]CIT/RTI-55, or [18F]beta-CIT-FP. Analysis of monkey arterial plasma samples using high performance liquid chromatography determined that there was no detectable formation of lipophilic radiolabeled metabolites capable of entering the brain. In equilibrium displacement experiments with CIT in rhesus monkeys, radioactivity in the putamen was displaced with an average half-time of 10.2 min. These results indicate that [18F]FECNT is a radioligand that is superior to 11C-WIN 35,428, [11C]CIT/RTI-55, [18F]beta-CIT-FP, and [18F]FPCT for mapping brain DAT in humans using PET. PMID- 10755642 TI - In vivo [125I]-iodobenzovesamicol binding reflects cortical cholinergic deficiency induced by specific immunolesion of rat basal forebrain cholinergic system. AB - In this study, radiolabeled iodobenzovesamicol (IBVM), which is known to bind with high affinity to the vesicular acetylcholine transporter, was tested for its usefulness in imaging cortical cholinergic deficits in vivo. To induce reductions in cortical cholinergic input, the cholinergic immunotoxin 192IgG-saporin was employed. This has been shown to selectively and efficiently destroy basal forebrain cholinergic neurons in rats. The efficiency of the immunolesion was verified by histochemical acetylcholinesterase staining. [125I]-IBVM binding before and after lesioning was measured using autoradiography. Basal forebrain cholinergic cell loss resulted in a considerable reduction in [125I]-IBVM binding in the cholinoceptive target regions, but not in the striatum and cerebellum, brain regions that do not receive a cholinergic input by the basal forebrain cholinergic nuclei, suggesting that [123I]-IBVM has potential in imaging cortical cholinergic deficits in vivo, at least in animals. PMID- 10755643 TI - Demonstration of [11C] 5-hydroxy-L-tryptophan uptake and decarboxylation in carcinoid tumors by specific positioning labeling in positron emission tomography. AB - In three patients with carcinoid liver and/or lymph node metastases, we studied the process of tumor tracer uptake and decarboxylation by means of positron emission tomography (PET) using 5-hydroxy-L-tryptophan (5-HTP) 11C-labeled in the beta-position (HTP) and later the same day with 5-HTP 11C-labeled in the carboxyl group (HTC). With HTP, in which the 11C-label follows the molecule through decarboxylation to form 11C-serotonin, a high tumor accumulation of the tracer was found. With HTC, in which the label is rapidly eliminated from the tissues as 11CO2 if decarboxylation takes place, there was virtually no uptake by the tumors. By utilizing data from PET scanning with both tracers, we could quantify the decarboxylation rate and tissue accumulation of [11C]-serotonin and hence the enzymatic action of aromatic amino acid decarboxylase. PMID- 10755641 TI - [123I]IPCIT and [123I]beta-CIT as SPECT tracers for the dopamine transporter: a comparative analysis in nonhuman primates. AB - [123I]2beta-carbomethoxy-3beta-(4-iodophenyl)tropane ([123I]-CIT) and its isopropylester analog [123I]PCIT, both of which are phenyltropane derivatives of cocaine with high affinity for the dopamine (DA) transporter, were compared using single photon emission computed tomography in nonhuman primates. Although IPCIT is significantly more selective for the DA transporter than beta-CIT, striatal distribution volumes of specifically bound tracer were similar for both tracers. Compartmental modeling results were compared with a simple peak equilibrium method used previously by this group. The peak equilibrium method is shown to overestimate striatal distribution volumes, primarily due to a difference in the calculated time of peak specific uptake. PMID- 10755644 TI - Reproducibility of repeated measures of deuterium substituted [11C]L-deprenyl ([11C]L-deprenyl-D2) binding in the human brain. AB - The purpose of this study was to assess the reproducibility of repeated positron emission tomography (PET) measures of brain monoamine oxidase B (MAO B) using deuterium-substituted [11C]L-deprenyl ([11C]L-deprenyl-D2) in normal subjects and to validate the method used for estimating the kinetic constants from the irreversible 3-compartment model applied to the tracer binding. Five normal healthy subjects (age range 23-73 years) each received two PET scans with [11C]L deprenyl-D2. The time interval between scans was 7-27 days. Time-activity data from eight regions of interest and an arterial plasma input function was used to calculate lambda k3, a model term proportional to MAO B, and K1, the plasma to brain transfer constant that is related to blood flow. Linear (LIN) and nonlinear least-squares (NLLSQ) estimation methods were used to calculate the optimum model constants. A comparison of time-activity curves for scan 1 and scan 2 showed that the percent of change for peak uptake varied from -18.5 to 15.0% and that increases and decreases in uptake on scan 2 were associated with increases and decreases in the value of the arterial input of the tracer. Calculation of lambda k3 showed a difference between scan 1 and scan 2 in the global value ranging between -6.97 and 4.5% (average -2.1 +/- 4.7%). The average percent change for eight brain regions for the five subjects was -2.84 +/- 7.07%. Values of lambda k3 for scan 1 and scan 2 were highly correlated (r2 = 0.98; p < 0.0001; slope 0.955). Similarly, values of K1 showed a significant correlation between scan 1 and scan 2 (r2 = 0.61; p < 0.0001; slope 0.638) though the values for scan 2 were generally lower than those of scan 1. There was essentially no difference between the values of model constants calculated using the NLLSQ or LIN methods. Regional brain uptake of [11C]L-deprenyl-D2 varied between scan 1 and scan 2, driven by the differences in arterial tracer input. Application of a 3-compartment model to regional time-activity data and arterial input function yielded lambda k3 values for scan 1 and scan 2 with an average difference of -2.84 +/- 7.07%. Linear regression applied to values of lambda k3 from the LIN and NLLSQ methods validated the use of the linear method for calculating lambda k3. PMID- 10755645 TI - 6-[18F]Fluoro-A-85380: an in vivo tracer for the nicotinic acetylcholine receptor. AB - 6-[18F]Fluoro-3-(2(S)-azetidinylmethoxy)pyridine (6-[18F]fluoro-A-85380 or 6 [18F]FA), a new tracer for positron emission tomography, was synthesized by no carrier-added [18F] fluorination of 6-iodo-3-((1-tert-butoxycarbonyl-2(S) azetidinyl)methoxy)pyridine followed by acidic deprotection. 6-[18F]FA followed the regional densities of brain nicotinic acetylcholine receptors (nAChRs) reported in the literature. Evidence of binding to nAChRs and high specificity of the binding in vivo was demonstrated by inhibition with nAChR selective ligands as well as with unlabeled 6-FA. A preliminary toxicology study of the 6-FA showed a relatively low biological effect. PMID- 10755646 TI - Effect of chronic (-)-nicotine treatment on rat cerebral benzodiazepine receptors. AB - The purpose of this study was to clarify the effect of (-)-nicotine on cerebral benzodiazepine receptors (BzR) with radiotracer methods. The effect of (-) nicotine on BzR was examined in in vitro studies using chronic (-)-nicotine treated rats using 3H-diazepam. The in vitro radioreceptor assay showed a 14% increase in the maximum number of binding sites of BzR in chronic (-)-nicotine treated rats in comparison with the control rats. Moreover, a convenient in vivo uptake index of 125I-iomazenil was calculated and a higher uptake of the radioactivity was observed in the chronic (-)-nicotine-treated group than in the control group. Although further studies of the mechanism of (-)-nicotine on such BzR changes are required, an increase in the amount of BzR in the cerebral cortex was found in rats that underwent chronic (-)-nicotine treatment, and this result contributed to the understanding of the effects of (-)-nicotine and smoking on neural functions. PMID- 10755647 TI - Synthesis, radiolabeling, and biodistribution of putative metabolites of iodoazomycin arabinoside. AB - Scintigraphic evaluation of patients with advanced oncological disease showed uptake of radioactivity in the brain following administration of the hypoxic imaging agent 123I-iodoazomycin arabinoside (123I-IAZA). Three proposed metabolites of IAZA--methyl 5-deoxy-5-iodo-D-arabinofuranoside, methyl 2,3-di-O acetyl-5-deoxy-5-iodo-alpha-D-arabinofuranoside, and 1-(5-deoxy-5-iodo-alpha-D arabinofuranosyl)-2-aminoimidazole (IAIA)--were synthesized, radiolabeled with 125I, and investigated in normal and tumor-bearing murine models for their contribution to this unusual phenomenon. The three compounds were readily radiolabeled by melt or solvent exchange procedures. Biodistribution data indicated rapid blood clearance, rapid excretion, and little tissue accumulation in the brain. IAIA showed significant tumor to blood ratios at 4 h (4.3:1) and liver to blood ratios at 24 h (30:1). PMID- 10755648 TI - The preclinical pharmacologic study of dopamine transporter imaging agent [99mTc]TRODAT-1. AB - The purpose of this study was to investigate the pharmacologic characteristics of TRODAT-1 (2beta-((N,N'-bis(2-mercaptoethyl)ethylene diamino)methyl), 3beta-(4 chlorophenyl)tropane) labeled with [99mTc] as an imaging agent for dopamine transporter (DAT). Radiochemical purity of [99mTc]TRODAT-1 was over 90%. The partition coefficients in octanol and buffer were 2.12 and 2.19 at pH 7.0 and 7.4, respectively. Animal studies have been performed in rats, rabbits, and normal and hemi-Parkinsonian model monkeys. Biodistribution displayed moderate uptake in rat brain (0.28 %ID/organ at 2 min) and the striatal uptake was 0.193, 0.142, and 0.136 %ID/g at 2, 60, and 120 min, respectively. The ratios of striatal/cerebellar (ST/CB) uptake were 2.4, 4.45, and 2.45 %ID/g at 60, 120, and 240 min, respectively. The major radioactivity was excreted by the hepatobiliary system. Blood clearance kinetics was performed in rabbits, and the initial half life of 1.18 min and late half-life of 367.8 min were obtained. Brain single photon emission computed tomography imaging studies in normal monkeys showed the ratios of ST/CB uptake were 1.56-2.0 %ID/g and indicated that both uptake and retention in the striatal area were associated with the DAT. The imaging of hemi Parkinsonian model monkeys also displayed the expected selectivity, the highest uptake being observed in the basal ganglia area of the normal side. Thereby, it is suggested that [99mTc]TRODAT-1 is a safe and useful imaging agent for localization of the presynaptic DAT in the brain. PMID- 10755649 TI - 55Co-EDTA for renal imaging using positron emission tomography (PET): a feasibility study. AB - The feasibility of imaging renal function with 55Co-ethylene diamine tetraacetic acid (EDTA) and dynamic positron emission tomography was investigated. A group of normal Wistar rats was injected intravenously with 55Co-EDTA and underwent dynamic positron emission tomography (PET) imaging in order to study the biodistribution. The time-activity curves of the heart (blood pool), both kidneys, liver, and bladder were observed. In two animals, blood and urinary clearances of 55Co-EDTA were compared with those for 51Cr-EDTA. In one animal, unilateral reduction in kidney function was induced and the right/left ratio for the kidneys was determined. The time-activity curves showed that 55Co-EDTA cleared rapidly from the blood pool (heart), whereas prompt and high target-to background ratios for both kidneys were obtained. The entire tracer was cleared from the renal parenchyma by urinary excretion and collection of the activity in the bladder. No specific activity uptake was noticed in any other organ or tissue. The clearances of 55Co-EDTA and 51Cr-EDTA in blood were not significantly different, showing that the nature of the M++ has no influence on the in vivo behavior of EDTA. 55Co can be produced easily by cyclotron irradiation and 55Co EDTA is a promising physiological tracer for nephrological research using PET. PMID- 10755650 TI - A comprehensive study on the blockage of thyroid and gastric uptakes of 188Re perrhenate in endovascular irradiation using liquid-filled balloon to prevent restenosis. AB - 188Re-perrhenate has been reported effective in preventing restenosis after percutaneous transluminal coronary angioplasty. However, if the balloon ruptures, 188Re-perrhenate is released into the circulation, causing high radiation dosing to the thyroid and stomach. In this study, we evaluated the effects of perchlorate or iodide given at different times and in different ways for blocking the uptake of 188Re-perrhenate in the thyroid glands and the stomach to find the best method to apply clinically to reduce the radiation dose in case of balloon rupture. Sodium perchlorate, sodium iodide, or potassium iodide was given orally or intravenously to rats before, during, and after the injection of 188Re perrhenate. The rats were sacrificed and we calculated the concentration of 188Re perrhenate in various organs to evaluate the preblocking, mixed formula, and postblocking effects of perchlorate or iodide. Our data showed that the preblocking method effectively reduced the uptake of 188Re-perrhenate in both the thyroid and the stomach. The mixed formula method also demonstrated good blocking effect. The postblocking method showed obvious depression of thyroid uptake of perrhenate but its blocking effect on the stomach was not satisfactory. PMID- 10755651 TI - Synthesis and characterization of nido-carborane-cobalamin conjugates. AB - Three vitamin B12 (cyanocobalamin) conjugates bearing one nido-carborane molecule or two nido-carborane molecules linked to the propionamide side chains via a four carbon linker have been synthesized. Reaction of o-carboranoylchloride with 1,4 diaminobutane in pyridine produced nido-carboranoyl(4-amidobutyl)amine, which was linked to the b- and d-monocarboxylic acids and the b,d-dicarboxylic acid of cyanocobalamin. Mass spectrometry analysis as well as 11B nuclear magnetic resonance demonstrated that during the reaction of o-carboranonylchloride with diaminobutane one of the boron atoms was eliminated. In vitro biological activity of the cyanocobalamin-nido-carborane conjugates was assessed by the unsaturated vitamin B12 binding capacity assay. When compared with 57Co cyanocobalamin, the biological activity of cyanocobalamin-b-nido-carborane, cyanocobalamin-d-nido carborane, and cyanocobalamin-b-d-bis-nido-carborane conjugates were 92.93%, 35.75%, and 37.02%, respectively. These findings suggest that the 10B cobalamin conjugates might be useful agents in treating malignant tumors via neutron capture therapy. PMID- 10755652 TI - Synthesis, characterization, and evaluation of a novel bifunctional chelating agent for the lead isotopes 203Pb and 212Pb. AB - Radioisotopes of Pb(II) have been of some interest in radioimmunotherapy and radioimmunoimaging (RII). However, the absence of a kinetically stable bifunctional chelating agent for Pb(II) has hampered its use for these applications. 203Pb (T(1/2) = 52.02 h) has application potential in RII, with a gamma-emission that is ideal for single photon emission computerized tomography, whereas 212Pb (T(1/2) = 10 h) is a source of highly cytotoxic alpha-particles via its decay to its 212Bi (T(1/2) = 60 min) daughter. The synthesis of the novel bifunctional chelating agent 2-(4-isothiocyanotobenzyl)-1,4,7,10-tetraaza 1,4,7,10-tetra- (2-carbamoyl methyl)-cyclododecane (4-NCS-Bz-TCMC) is reported herein. The Pb[TCMC]2+ complex was less labile to metal ion release than Pb[DOTA]2- at pH 3.5 and below in isotopic exchange experiments. In addition to increased stability to Pb2+ ion release at low pH, the bifunctional TCMC ligand was found to have many other advantages over the bifunctional 1,4,7,10 tetraazacyclodocane-1,4,7,10-tetraacetic acid (DOTA) ligand. These include a shorter and more straightforward synthetic route, a more efficient conjugation reaction to a monoclonal antibody (mAb), with a higher chelate to protein ratio, a higher percent immuroreactivity, and a more efficient radiolabeling reaction of the mAb-ligand conjugate with 203Pb. PMID- 10755653 TI - Preclinical evaluation of radiopharmaceutical toxicology prerequisites. PMID- 10755654 TI - Biomechanical and metabolic effects of varying backpack loading on simulated marching. AB - Twelve healthy, male Army recruits performed three, 40-min treadmill marches at 6 km/h, under three load carriage conditions: 0%-body weight (BW) backpack load, 15%-BW load and 30%-BW load. Kinematic and kinetic data were obtained, immediately before and after each treadmill march, for computing ankle, knee and hip joint rotations and moments. Metabolic data (oxygen uptake (VO2), expired ventilation (VE), respiratory exchange ratio (RER)), heart rate (HR) and ratings of perceived exertion (RPE) were collected continuously during marching. Significant differences (p < or = 0.05) were observed between each load for VO2, HR and VE throughout the marches. At 40 min, relative energy costs for 0%-BW, 15% BW and 30%-BW loads were 30, 36 and 41% VO2max, respectively. RPE responses during marching significantly differed for only the 30%-BW load and were greater than responses at 0%-BW and 15%-BW loads. During load carriage trials prior to treadmill marches (pre-march), peaks in internal, hip extension, knee extension and ankle plantar flexion moments increased with increasing backpack load. Relative to 0%-BW load, percentage increases in knee moments, due to 15%-BW and 30%-BW loads, pre-march, were substantially larger than the percentage increases for hip extension and plantar flexion moments, pre-march. Pre-march and post march peaks in hip extension and ankle plantar flexion moments were similar with all loads, while notable pre-march to post-march declines were observed for knee extension moment peaks, at 15%-BW and 30%-BW load. Pre-march joint loading data suggests that the knee may be effecting substantial compensations during backpack loaded marching, perhaps to attenuate shock or reduce load elsewhere. Post-march kinetic data (particularly at 15%-BW and 30%-BW load), however, indicates that such knee mechanics were not sustained and suggests that excessive knee extensor fatigue may occur prior to march end, even though overall metabolic responses, at 15%-BW and 30%-BW load, remained within generally recommended limits to prevent fatigue during prolonged work. PMID- 10755655 TI - Validity of self-reporting and video-recording for measuring knee-straining work postures. AB - The aim of this study was to compare the validity of self-reporting and video recording as methods of measuring the duration of knee-straining work postures, and to evaluate the reproducibility of timings of the video-recordings. Thirty nine carpenters and 33 floorlayers were video-filmed while working, and were asked immediately afterwards to quantify the amount of time spent in knee straining positions. The investigators recorded the periods of knee-straining work with a stopwatch during playback of the video-film, and the agreement between the two investigators' measurements was studied in 13 pairs of measurements. The video-observation method was very effective for timing knee strain work. Furthermore, Spearman's correlation coefficient (0.88) indicated a good association between observed and self-reported knee-straining work. Estimation of self-reported knee-straining work collected by interview showed good correlation with measurements of video-recordings. When judging the repeatability of timings of knee-straining work from the video-recordings, a high level of agreement was seen between the two observers in the 13 dual measurements of time spent in knee-straining work positions. PMID- 10755656 TI - Contrast thresholds and fixation disparity during 5-Hz sinusoidal single- and dual-axis (vertical and lateral) whole-body vibration. AB - The present study assumed that whole-body vibration, transmitted through the seat, impairs spatial retinal resolution and oculomotor alignment parallel to the vibration axis. More specifically, that the decrement increases gradually from single-axis lateral via single-axis vertical and dual-axis linear to dual-axis circular motion. Twenty participants (19-26 years of age) with good vision volunteered for the experiment where in three sessions one of the following three conditions, contrast threshold, nonius bias or fixation disparity, for vertically and horizontally oriented test patterns was determined during five experimental conditions. The latter comprised a control (a(z) = a(y) = 0) and four conditions where 5-Hz sinusoidal motion of 1.2 ms(-2) rms were applied separately, either in the vertical or in the lateral direction, or simultaneously in both directions, once without and once with a phase shift of 90 degrees, thus causing dual-axis linear or circular motion. Contrast thresholds for horizontal gratings and the variability of vertical fixation disparity increased significantly whenever the participants were exposed to vertical motion (alone or combined with lateral motion). These effects may result in an increased difficulty in properly recognizing characters and graphic patterns containing horizontal lines and in the development of asthenopic complaints. PMID- 10755657 TI - Trunk muscle activation and low back loading in lifting in the absence of load knowledge. AB - People who know the actual mass of an object to be lifted normally prepare themselves before attempting a lift to control the movement and to minimize low back loading. In this study, the trunk muscular reactions and low back torque were investigated in the situation in which the individual did not know the actual mass but only had some idea of the range within which the mass lay. Nine males lifted boxes weighing 6.5 or 16.5 kg under the condition in which they knew the actual mass before attempting a lift (the 'known' condition) and the condition in which they only had the information that the mass would be within the range of 6.5-16.5 kg (the 'unknown' condition). The ground reaction forces and body movements were measured in the trials and, from these, the L5/S1 torques were calculated. The activation of back and abdominal muscles was also measured. For the 6.5 kg weight, a higher (16%) back muscle activation in grasping the box and a higher (10%) peak L5/S1 torque in actual lifting were observed in the 'unknown' compared with the 'known' weight condition. For the 16.5 kg weight, the back muscle activation was lower (10%) during grasping, and higher (10%) during lifting in the 'unknown' compared with the 'known' weight condition. Knowledge of the load had no effect on the activation of the abdominal muscles. It was concluded that in the so-called 'unknown' conditions, the risks of low back injury were increased in comparison with the conditions where the actual weight was known in advance. PMID- 10755658 TI - Posture, discomfort and performance in a VDT task. AB - Improvements in workplace, working posture, and discomfort need to be justified in terms of improvements in performance. Previously, a visual inspection task has been investigated. The objective of the current study was to demonstrate the interactions between workplace, work duration, discomfort, working posture, as well as performance in a 2-h typing task. Three levels of keyboard heights were used to change working posture (e.g. joint angles and postural shifts), and thus presumably discomfort (e.g. rating of perceived discomfort and body part discomfort), and performance (e.g. typing speed, error rate and error correction rate). The results indicated that the hypothesized posture-comfort-performance interrelationships were partially supported. Keyboard height had effects on working posture adopted. As in previous studies, the rate of postural shift was a good indication of discomfort in a VDT task. Discomfort and postural shift rate had adverse effects on performance (e.g. error rate). However, these effects on error rate may not be strong. PMID- 10755659 TI - Effects of a target movement direction cue on head-tracking performance. AB - A review of the literature has shown that most investigations of head-tracking performance have used symmetrically shaped targets. This paper identifies a problem in using circular targets to represent the movement of complex targets (i.e. targets giving directional cues). Two experiments investigated the effects of a target direction cue on head-tracking performance. In the first experiment, practice did not improve performance when tracking either with or without a 'look ahead trace' showing all target positions 160 ms into the future. A second experiment utilized a 'look-ahead trace' showing target positions with eight different lead-times (0-560 ms). With lead-times of 160 ms or more, significant improvements in tracking performance and subjective difficulty ratings were obtained. Tracking responses were also significantly affected. The results suggest caution when performance data obtained with a symmetrical target are generalized to predict tracking performance with a real target giving cues to the direction of movement. The look-ahead trace offers a systematic means of bridging the gap between a symmetrical target and a real target having direction of movement cues. PMID- 10755660 TI - Force direction and physical load in dynamic pushing and pulling. AB - In pushing and pulling wheeled carts, the direction of force exertion may, beside the force magnitude, considerably affect musculoskeletal loading. This paper describes how force direction changes as handle height and force level change, and the effects this has on the loads on the shoulder and low back. Eight subjects pushed against or pulled on a stationary bar or movable cart at various handle heights and horizontal force levels while walking on a treadmill. The forces at the hands in the vertical and horizontal direction were measured by a force-transducer. The forces, body movements and anthropometric data were used to calculate the net joint torques in the sagittal plane in the shoulder and the lumbosacral joint. The magnitudes and directions of forces did not differ between the cart and the bar pushing and pulling. Force direction was affected by the horizontal force level and handle height. As handle height and horizontal force level increased, the pushing force direction changed from 45 degrees (SD 3.3 degrees) downward to near horizontal, while the pulling force direction changed from pulling upward by 14 degrees (SD 15.3 degrees) to near horizontal. As a result, it was found that across conditions the changes in force exertion were frequently reflected in changes in shoulder torque and low back torque although of a much smaller magnitude. Therefore, an accurate evaluation of musculoskeletal loads in pushing and pulling requires, besides a knowledge of the force magnitude, knowledge of the direction of force exertion with respect to the body. PMID- 10755661 TI - Ordinal depth information from accommodation? AB - The ability to judge egocentric distance was assessed in two groups of six observers using a manual pointing task. The purpose of the study was to determine the extent to which blur-driven accommodation can provide information on target distance in the absence of any retinal cues to distance. Observers were extremely accurate when carrying out the pointing task in a 'full-cue' condition. In contrast, observers were extremely poor at carrying out the task when accommodation was the only distance cue available. Responses on individual trials bore little relationship to the actual target distance in any of the observers. On the other hand, accommodation weakly biased the mean responses in some observers. This bias appears to be due to the observers' effective use of accommodation to determine whether the target presented in one trial was nearer or further away than the target presented in the previous trial. Accommodation therefore appears to provide ordinal information, although the distance signal may actually arise from accommodation-driven vergence. The poverty of accommodation as a source of metric information was highlighted in a second group of observers who all demonstrated a strong bias when perceiving distance in the presence of an initially ambiguous retinal cue. It is concluded that accommodation can act as a source of ordinal distance information in the absence of other cues to distance but the contribution of accommodation to normal distance perception in full-cue conditions is questioned. PMID- 10755662 TI - Endurance time, pain and resumption in passive loading of the elbow joint. AB - This study investigated reactions in passively loaded, fully extended elbow joints in 13 men. Pain reactions during and after loading were studied, as were endurance time, T(end), and resumption time, Tr. The loading moment on the elbow joint, Mn, varied between 7 and 100% of maximum elbow moment. Discomfort/ pain was estimated with Borg's CR-10 scale. T(end) decreased with increasing load level. The opposite was found for the resumption time: the higher the load, held until T(end), the shorter the Tr. The pain limiting the working capability originated mainly from muscle tissue and not from the joint itself. It is concluded that the relation between load and endurance time for passively loaded, fully extended elbow joints resembles that for muscular loading in more 'normal' postures. Equations for estimation of endurance and resumption times (in minutes) for fully extended joints are proposed: T(end) = 20.6e(-6.04Mn) and Tr = 0.0167e(8.84/(1.46+0.346Mn)), respectively. PMID- 10755663 TI - Dynamic leg volume changes when sitting in a locked and free floating tilt office chair. AB - It is well established that prolonged sitting may lead to swelling of the lower extremities. However, activation of the vein pump system by repeated walking breaks or dynamic tiltable foot-rests have been shown to reduce foot and leg oedema. Some office chairs incorporate tilt mechanisms facilitating movements of the body from the feet up. The present study was undertaken to establish whether a beneficial effect on the transcapillary fluid balance of the legs by enabling such mechanisms could be documented. An office chair where the tilt mechanism could be locked or open was used for the study. The seat position and seat activity level was recorded by a transducer system developed for the study. Calf volume and calf muscle pump activity was detected by mercury strain gauge plethysmography. In the locked position there was a steady increase in volume of mean = 1.2% (range = 0.8-1.8%) for all participants in the 30-min study period. On the other hand, for all participants there was a decrease in calf volume (mean = 0.7%, range = 0.1-1.2%, p = 0.008) when the tilt mechanism was open (30-min period), irrespective of what study period came first. The study showed that upward seat deflection was not associated with concomitant venous obstruction, since such obstruction was detected in less than 2% of the time period with more than 50% of maximal upward deflection. A locked seat mechanism does not prevent activation of the vein pump mechanism, but the study indicates that office chairs that permit variation in seat angle per se stimulate movements of the leg. This, in turn, activates vein pumps and counteracts local oedema formation in seated working postures. PMID- 10755664 TI - Ku autoantigen: a multifunctional DNA-binding protein. AB - Ku is a heterodimeric protein composed of approximately 70- and approximately 80 kDa subunits (Ku70 and Ku80) originally identified as an autoantigen recognized by the sera of patients with autoimmune diseases. Ku has high binding affinity for DNA ends and that is why originally it was known as a DNA end binding protein, but now it is known to also bind the DNA structure at nicks, gaps, hairpins, as well as the ends of telomeres. It has been reported also to bind with sequence specificity to DNA and with weak affinity to RNA. Ku is an abundant nuclear protein and is present in vertebrates, insects, yeast, and worms. Ku contains ssDNA-dependent ATPase and ATP-dependent DNA helicase activities. It is the regulatory subunit of the DNA-dependent protein kinase that phosphorylates many proteins, including SV-40 large T antigen, p53, RNA-polymerase II, RP-A, topoisomerases, hsp90, and many transcription factors such as c-Jun, c-Fos, oct 1, sp-1, c-Myc, TFIID, and many more. It seems to be a multifunctional protein that has been implicated to be involved directly or indirectly in many important cellular metabolic processes such as DNA double-strand break repair, V(D)J recombination of immunoglobulins and T-cell receptor genes, immunoglobulin isotype switching, DNA replication, transcription regulation, regulation of heat shock-induced responses, regulation of the precise structure of telomeric termini, and it also plays a novel role in G2 and M phases of the cell cycle. The mechanism underlying the regulation of all the diverse functions of Ku is still obscure. PMID- 10755665 TI - Induction, regulation, degradation, and biological significance of mammalian metallothioneins. AB - MTs are small cysteine-rich metal-binding proteins found in many species and, although there are differences between them, it is of note that they have a great deal of sequence and structural homology. Mammalian MTs are 61 or 62 amino acid polypeptides containing 20 conserved cysteine residues that underpin the binding of metals. The existence of MT across species is indicative of its biological demand, while the conservation of cysteines indicates that these are undoubtedly central to the function of this protein. Four MT isoforms have been found so far, MT-1, MT-2, MT-3, and MT-4, but these also have subtypes with 17 MT genes identified in man, of which 10 are known to be functional. Different cells express different MT isoforms with varying levels of expression perhaps as a result of the different function of each isoform. Even different metals induce and bind to MTs to different extents. Over 40 years of research into MT have yielded much information on this protein, but have failed to assign to it a definitive biological role. The fact that multiple MT isoforms exist, and the great variety of substances and agents that act as inducers, further complicates the search for the biological role of MTs. This article reviews the current knowledge on the biochemistry, induction, regulation, and degradation of this protein in mammals, with a particular emphasis on human MTs. It also considers the possible biological roles of this protein, which include participation in cell proliferation and apoptosis, homeostasis of essential metals, cellular free radical scavenging, and metal detoxification. PMID- 10755666 TI - Evidence-based psychosocial treatment for schizophrenia. AB - Current recommendations for evidence-based schizophrenia treatment support a comprehensive, individualized approach that integrates advances in psychopharmacology with psychosocial strategies for disease management. In this issue of the Schizophrenia Bulletin, we invited clinician investigators to summarize new empirical data concerning the efficacy of psychosocial interventions that target common and particularly problematic aspects of schizophrenia. A rich formulary of psychosocial interventions with demonstrated efficacy is now available. With new neuroleptic medications, these interventions should define the current standard of care for schizophrenia. PMID- 10755667 TI - Update on family psychoeducation for schizophrenia. AB - The Schizophrenia Patient Outcomes Research Team and others have previously included family psychoeducation and family support in best practices guidelines and treatment recommendations for persons with schizophrenia. In this article we review in detail 15 new studies on family interventions to consider issues around the implementation of family interventions in current practice. The data supporting the efficacy of family psychoeducation remain compelling. Such programs should remain as part of best practices guidelines and treatment recommendations. However, assessment of the appropriateness of family psychoeducation for a particular patient and family should consider (1) the interest of the family and patient; (2) the extent and quality of family and patient involvement; (3) the presence of patient outcomes that clinicians, family members, and patients can identify as goals; and (4) whether the patient and family would choose family psychoeducation instead of alternatives available in the agency to achieve outcomes identified. PMID- 10755668 TI - Psychosocial skills training for schizophrenia: lessons from the laboratory. AB - A large body of research supports the efficacy of psychosocial treatments for schizophrenia, particularly learning-based therapies. The Schizophrenia Patient Outcomes Research Team recommended that cognitive-behavioral therapies be used in schizophrenia, and skills training was included in the practice guideline for treating patients with schizophrenia published by the American Psychiatric Association. This article provides an updated review of empirical studies of psychosocial skills training, showing its value in treating patients with schizophrenia as well as its broader clinical effectiveness. Data supporting the efficacy of psychosocial skills training continue to accumulate. Such programs should continue to be included in best practices guidelines and treatment recommendations for schizophrenia. Future clinical service research could be directed toward integration of skills training with other psychosocial treatment methods. PMID- 10755669 TI - Evolving perspectives on individual psychotherapy for schizophrenia. AB - Some form of individual psychotherapy, in combination with the prescription of antipsychotic medications, is likely the most common treatment for patients with schizophrenia. In the absence of empirical data supporting the efficacy of a particular approach, psychotherapy has often been guided by ideology and deference to authority. In recent years, a reformulation of schizophrenia as a disorder requiring individualized, comprehensive treatment has allowed the development and empirical testing of new targeted and illness-phase-specific individual psychotherapies. This report reviews randomized clinical trials that have evaluated individual psychotherapy of schizophrenia in the context of changing contemporaneous beliefs about the disorder's etiology and treatment. A general approach to individual treatment, termed "flexible psychotherapy," derived from historical approaches but consistent with available clinical and research perspectives, is outlined. PMID- 10755670 TI - Cognitive-behavioral therapy for medication-resistant symptoms. AB - Cognitive-behavioral therapy for psychosis is described. It draws on the cognitive models and therapy approach of Beck and colleagues, combined with an application of stress-vulnerability models of schizophrenia and cognitive models of psychotic symptoms. There is encouraging evidence for the efficacy of this approach. Four controlled trials have found that cognitive-behavioral therapy reduces symptoms of psychosis, and there is some evidence that it may contribute to relapse reduction. Studies that have examined factors that predict treatment response are reviewed. There is preliminary evidence that a good outcome is partially predicted by a measure of cognitive flexibility or a "chink of insight." People who present with only negative symptoms may show poorer outcome. However, there is no evidence that intelligence or symptom severity is associated with outcome. Implications for selecting patients and for optimal duration of treatment are discussed. Finally, the importance of taking account of the heterogeneity of people with psychosis, so that individual treatment goals are identified, is discussed. PMID- 10755671 TI - Vocational rehabilitation for persons with schizophrenia: recent research and implications for practice. AB - This article presents research-based principles of vocational rehabilitation that have emerged from the study of diagnostically heterogeneous populations of persons with severe mental illness. Employment and vocational functioning outcomes of people with schizophrenia from recently published followup studies are described. In addition, we present research conducted over the past decade concerning differential outcomes of vocational rehabilitation services for people with schizophrenia versus other psychotic and nonpsychotic disorders. We then explore studies of people with schizophrenia that may illuminate the links between specific features of this disorder--including symptomatology, social skills, and neuropsychological impairments--and poorer vocational outcome. We conclude with a set of recommendations for clinical practice that draw upon the most recent discoveries and insights in this field. PMID- 10755672 TI - Psychosocial approaches to dual diagnosis. AB - Recent research elucidates many aspects of the problem of co-occurring substance use disorder (SUD) in patients with severe mental illness, which is often termed dual diagnosis. This paper provides a brief overview of current research on the epidemiology, adverse consequences, and phenomenology of dual diagnosis, followed by a more extensive review of current approaches to services, assessment, and treatment. Accumulating evidence shows that comorbid SUD is quite common among individuals with severe mental illness and that these individuals suffer serious adverse consequences of SUD. The research further suggests that traditional, separate services for individuals with dual disorders are ineffective, and that integrated treatment programs, which combine mental health and substance abuse interventions, offer more promise. In addition to a comprehensive integration of services, successful programs include assessment, assertive case management, motivational interventions for patients who do not recognize the need for substance abuse treatment, behavioral interventions for those who are trying to attain or maintain abstinence, family interventions, housing, rehabilitation, and psychopharmacology. Further research is needed on the organization and financing of dual-diagnosis services and on specific components of the integrated treatment model, such as group treatments, family interventions, and housing approaches. PMID- 10755674 TI - Commentary: what happens to psychosocial treatments on the way to the clinic? PMID- 10755673 TI - Neurocognitive deficits and functional outcome in schizophrenia: are we measuring the "right stuff"? AB - There has been a surge of interest in the functional consequences of neurocognitive deficits in schizophrenia. The published literature in this area has doubled in the last few years. In this paper, we will attempt to confirm the conclusions from a previous review that certain neurocognitive domains (secondary verbal memory, immediate memory, executive functioning as measured by card sorting, and vigilance) are associated with functional outcome. In addition to surveying the number of replicated findings and tallying box scores of results, we will approach the review of the studies in a more thorough and empirical manner by applying a meta-analysis. Lastly, we will discuss what we see as a key limitation of this literature, specifically, the relatively narrow selection of predictor measures. This limitation has constrained identification of mediating variables that may explain the mechanisms for these relationships. PMID- 10755675 TI - Commentary: can intensive psychosocial treatments make a difference in a time of atypical antipsychotics and managed care? PMID- 10755676 TI - Commentary: psychological and social treatments for schizophrenia: not just old remedies in new bottles. PMID- 10755677 TI - Editor's concluding remarks: psychosocial treatment: more than compassion and common sense? PMID- 10755678 TI - At issue: a default analysis for schizophrenia research. AB - The concept that there are at least three clinical syndromes within schizophrenia has been influential, and the significance of these syndromes is supported by a growing number of studies. However, the meaning of these syndromes is not clear. The yield of research studies could be increased by the application of a "default" data analysis, in which the differential association--if any--of the three syndromes with the dependent variable(s) of a study is determined. PMID- 10755679 TI - Psychopathological syndromes of schizophrenia: evaluation of the dimensional structure of the positive and negative syndrome scale. AB - The Positive and Negative Syndrome Scale (PANSS) was originally designed as a rating system that provides balanced representation of positive and negative symptom features. Evidence from recent factor-analytic studies suggests that a five-dimensional solution appears to best fit the psychopathological data as assessed with the PANSS. To investigate the dimensional structure, we administered the PANSS to 253 inpatients with schizophrenia. In accordance with former studies, principal components analyses yielded five orthogonal dimensions: hostile excitement; negative, cognitive, and positive syndrome; and depression. When compared with questionnaires measuring subjective nonpsychotic experiences of schizophrenia, paranoid mood, and depression, the correlation pattern verifies the PANSS components. In addition, we investigated a subsample of 70 male patients with a Continuous Performance Test (CPT), a Span of Apprehension Task, and a Modality Shift Effect (MSE) paradigm; the CPT was significantly associated with the cognitive syndrome, and the MSE correlated with the negative syndrome. PMID- 10755680 TI - Substance use disorder in hospitalized severely mentally ill psychiatric patients: prevalence, correlates, and subgroups. AB - The prevalence and demographic and clinical correlates of lifetime substance use disorders were examined in a cohort of 325 recently hospitalized psychiatric patients (53% schizophrenia or schizoaffective disorder). Alcohol use was the most common type of substance use disorder, followed by cannabis and cocaine use. Univariate analyses indicated that gender (male), age (younger), education (less), history of time in jail, conduct disorder symptoms, and antisocial personality disorder symptoms were predictive of substance use disorders. Lifetime cannabis use disorder was uniquely predicted by marital status (never married) and fewer psychiatric hospitalizations during the previous 6 months. Optimal classification tree analysis, an exploratory, nonlinear method of identifying patient subgroups, was successful in predicting 74 percent to 86 percent of the alcohol, cannabis, and cocaine use disorders. The implications of this method for identifying specific patient subgroups and service needs are discussed. PMID- 10755681 TI - Insight, symptoms, and neurocognition in schizophrenia and schizoaffective disorder. AB - There are conflicting reports in the literature regarding the relationships among impaired insight, symptoms, and neurocognition in schizophrenia. The inconsistent findings likely reflect the multidimensionality of insight in this population, along with variations in study design. We examined 46 individuals with chronic schizophrenia or schizoaffective disorder who were recently discharged from an inpatient unit. Insight was operationalized as awareness of having a mental disorder and awareness and attribution of both current and past symptoms. Positive, negative, disorganized, and depression symptoms were rated, and a neurocognition battery, including measures of visual processing, memory, visuo spatial ability, and executive functions, was administered. Poor awareness of symptoms was moderately associated with core schizophrenia symptoms, and higher levels of depression were strongly associated with good awareness. Symptom misattribution, more so than symptom unawareness, was associated with deficits in frontal lobe functioning. Finally, different patterns of associations between symptoms, neurocognition, and insight were noted for current symptoms versus past symptoms. The data suggest that insight deficits in schizophrenia are multidimensional, and that investigators should pay careful attention to the choice of measures as well as to phase of illness characteristics in future studies. PMID- 10755682 TI - Fine volumetric analysis of the cerebral ventricular system in schizophrenia: further evidence for multifocal mild to moderate enlargement. AB - We used traditional volumetric regional analysis and a finer anterior-posterior (AP) profile volumetric analysis to examine the cerebral ventricular system in an all-male, demographically matched sample of schizophrenia patients (n = 73) and normal controls (n = 29) using 2.8-mm-thin coronal T1-weighted magnetic resonance images from a 1.5 tesla scanner. Traditional regional analysis was performed on various regions using absolute volumes after adjusting for intracranial volume (ICV) and age. The fine AP profile analysis was done by intrasubject "stacking" of contiguous coronal cross-sectional volumes (adjusted for ICV and age) across the AP plane, intersubject AP alignment of all slices relative to the mammillary bodies, and plotting of slice volumes along the AP plane with 95 percent t-test based confidence intervals. Schizophrenia subjects had mild to moderate multifocal ventricular enlargement (overall effect size d = 0.48), which was especially prominent in the right posterior temporal horn and, more generally, in the central to posterior portions of the lateral and third ventricles. Schizophrenia subjects also had milder enlargement in the left frontal horn, but no significant differences were found in the anterior temporal horns and the right frontal horn. Post hoc analyses of demographic, clinical, and neuropsychological variables did not account for much variance in the ventriculomegaly observed in the schizophrenia group. The lack of a single locus in the observed ventricular enlargement, the nonsignificant results from schizophrenia subtypes based on regional distributions, and the strong positive correlations among the ventricular regions for the schizophrenia group suggest that the ventriculomegaly seen in this chronic population reflects a single brainwide disease process leading to a multifocal or patchy loss of integrity in brain structure. PMID- 10755683 TI - The initial prodrome in schizophrenia: searching for naturalistic core dimensions of experience and behavior. AB - The scientific exploration of prepsychotic detection and intervention in psychosis has just commenced. To identify developing psychosis at prodromal stages, it is important to learn how patients and families perceive initial prodromes naturalistically. We must understand better what we are going to detect, because the essential components of this phase, particularly the subjective experiences, remain unsettled. In a series of 19 first episode DSM-IV schizophrenia patients, we explore prodromal phenomena in depth and identify potential core dimensions. On the basis of our findings, we describe experiences and behaviors that appear to be essential components of initial prodromes. The subjects reported serious difficulties interpreting and talking about prodromal experiences at the time these occurred, causing delayed identification. We report detailed reasons for this, pointing out vulnerable aspects of at-risk assessments. From eight proposed groups of experiences, two are highlighted as tentative core dimensions: "disturbance of perception of self" and "extreme preoccupation by and withdrawal to overvalued ideas." Four potential dimensions of prodromal behavior are also identified: (1) quit school, university, or job, or major school truancy, (2) marked and lasting observable shift of interests, (3) marked and lasting social passivity, withdrawal, or isolation, and (4) marked and lasting change in global appearance or behavior. We argue that the findings, the phenomena, and their significance in prodromes are valid because they are logical and coherent in light of clinical experience as well as the empirical literature of a full century. PMID- 10755684 TI - Risk factors for the deficit syndrome of schizophrenia. AB - Previous studies have found two risk factors associated with the deficit syndrome of schizophrenia: an increase in summer births, compared to others with schizophrenia; and a higher risk of schizophrenia in relatives. In data from the Camberwell Register Psychosis Series, a population-based sample that approximated a treated-incidence sample, the deficit/nondeficit categorization was made using a previously validated proxy method. Associations were found between the deficit syndrome and both summer birth and a family history of schizophrenia. In contrast, nondeficit schizophrenia was associated with a family history of psychiatric problems other than schizophrenia. The deficit group also had poorer insight. An early age of onset was associated with disorganization, but not with the deficit or nondeficit group. The deficit/nondeficit differences could not be attributed to confounding by demographic features or the severity of hallucinations, delusions, or formal thought disorder. PMID- 10755685 TI - First person account: understanding health as a continuum. PMID- 10755686 TI - Regulation of cholecystokinin secretion in humans. PMID- 10755687 TI - Effect of germinated barley foodstuff administration on mineral utilization in rodents. AB - Germinated barley foodstuff (GBF), which is derived from the aleurone and scutellum fractions of germinated barley, is rich in water-insoluble dietary fiber, which reportedly modulates mineral absorption in vivo. This study was conducted to examine whether GBF affects mineral absorption. We performed three experiments. In experiment 1, female ICR mice (4 weeks old) were fed a diet supplemented with 10% GBF or cellulose (CE; as a control) for 24 weeks. Body weight and food intake were recorded twice weekly. On the last experimental day, serum biochemical parameters, including electrolyte parameters, were determined, and major digestive organs were examined histopathologically. In experiment 2, male Sprague-Dawley rats (5 weeks old) were fed the CE and GBF diets, with the mineral content of the CE diet adjusted to the same level as that in the GBF diet, for 14 days, and the intake and fecal excretion of minerals (calcium, Ca; magnesium, Mg: Iron, Fe; Phosphate, P) were determined for the last 3 days. In experiment 3, the binding of Ca and Mg to GBF or cellulose and the release of Ca and Mg from GBF or cellulose was measured in phosphate buffer (pH 6.8). Long-term administration of GBF did not affect the growth rate of mice. GBF did not inhibit the absorption of Ca and Mg, and rather promoted the absorption of Ca and Mg by the gastrointestinal tract. The absorption of Fe and P was not attenuated by GBF administration, and GBF contained only a low level of phytic acid. GBF did not inhibit mineral absorption, differing from the effect of other dietary fibers, and is expected to be useful as a dietary source of Ca and Mg. PMID- 10755688 TI - Predictive value of histology at the invasive margin in the prognosis of early invasive colorectal carcinoma. AB - To accurately select patients with malignant colorectal polyps who are at high risk of adverse outcome, we examined the predictive value of clinicopathological factors, with special attention paid to the histology at the invasive margin. We examined 75 submucosal carcinomas from 75 patients, initially resected by polypectomy, including endoscopic, trans-anal, trans-sacral, and trans sphincteric local excision. The associations between clinicopathological features such as sex and age; tumor size, location, shape, depth of submucosal invasion, vascular invasion, histology at the central part, and histology at the invasive margin; and the presence or absence of a residual adenomatous component and adverse outcome were examined by univariate and multivariate logistic regression analyses. Lymph node metastases were found in 2 patients, local recurrence in 4, and distant metastases in 2. Univariate logistic regression analysis showed that unfavorable histology at the invasive margin was significantly associated with lymph node metastasis or local recurrence (P = 0.0373), whereas the association of lymphatic invasion and vascular (lymphatic or venous) invasion with lymph node metastasis or local recurrence had marginal significance (P = 0.0785; P = 0.0990). Multivariate logistic regression analysis, with unfavorable histology at the invasive margin and lymphatic invasion as independent variables, showed that unfavorable histology alone had significance (P = 0.0373) in predicting adverse outcome. Widely accepted criteria such as massive submucosal invasion, positive vascular invasion, and poorly differentiated histology, were less useful in predicting adverse outcome. These results suggest that unfavorable histology at the invasive margin is a useful risk factor for predicting lymph node metastasis or local recurrence in patients with malignant colorectal polyps. PMID- 10755689 TI - Spontaneous loss of hepatitis B surface antigen in chronic carriers, based on a long-term follow-up study in Goto Islands, Japan. AB - Annual mass examination was performed between 1972 and 1997 in Tomie-town, Goto Islands, Japan, where hepatitis B virus (HBV) infection is very prevalent. In the present study, the incidence of spontaneous loss of hepatitis B surface antigen (HBsAg) in HBsAg carriers was determined in this area. Three thousand and nineteen inhabitants were tested for HBsAg two or more times in our annual surveys. Among them, 131 (4.3%) were defined as chronic HBsAg carriers based on the persistence of HBsAg for 1 or more years. These 131 subjects were followed for 12.2 +/- 7.6 years. During the follow-up period, spontaneous loss of HBsAg occurred in 38 (29%) of the 131 carriers, with a yearly incidence of 2.5%. This loss was seen more frequently in carriers aged 40 years or more on enrollment than in those aged less than 40 years during the same observation periods (P = 0.0141), irrespective of sex or the results of liver function tests. The values for liver function test results were similar before and after loss of HBsAg in these carriers. Stored serum samples were available for later analysis of HBV-DNA by polymerase chain reaction in 32 carriers with loss of HBsAg. The HBV-DNA sequence was detected in 26 (81%) and 2 of the 32 carriers (6%) before and after loss of HBsAg, respectively. These results indicate that spontaneous loss of HBsAg, largely attributable to clearance of viremia, occurs age-dependently in chronic carriers. PMID- 10755690 TI - Viral serostatus and coexisting inflammatory activity affect metachronous carcinogenesis after hepatectomy for hepatocellular carcinoma. A further report. AB - Little data are available regarding the effects of hepatitis virus serostatus and the severity of coexisting chronic inflammation on intrahepatic recurrence after hepatectomy for hepatocellular carcinoma (HCC). We investigated the extent to which these factors modified the prognosis of hepatectomized patients. A total of 274 patients treated in the period January 1981 to December 1996 were divided into three groups: antihepatitis C-positive (HCV; n = 144), hepatitis B surface antigen-positive and HCV antibody (Ab)-negative (HBsAg; n = 106), and HBsAg negative and HCV Ab-negative (NBNC; n = 20). Positivity for HBV-related antibody in the HCV group was 76%. Histologic grading of inflammatory activity from coexisting hepatitis was determined according to Knodel's histological activity index (HAI) scoring system. Post-hepatectomy crude survival rates and disease free survival (DFS) rates were compared, according to tumor characteristics, between the three groups. In the patients overall and also in the patients with a single nodular HCC, the HCV group had significantly higher HAI scores and preoperative serum aspartate aminotransaminase (AST) levels than the other two groups. When the patients were limited to those with a single nodular HCC, the crude survival was similar in the three groups with comparable tumor characteristics; however, the DFS was different (NBNC > HBsAg > HCV). When the patients were further limited to those with a single nodular HCC without microscopic extracapsular spread, in whom removal of the tumor was expected to be microscopically complete, the difference in the DFS became more marked. Irrespective of the viral serostatus, better crude and disease-free survivals were observed in the patients with lower AST levels (< or =50 IU/ 1) than in those with higher AST levels (>50IU/l). In contrast, there were no differences in survivals and HAI scores according to the presence or absence of HBV-related antibody in the HCV group. From our univariate analysis, we can conclude that the severity of virally induced inflammation, which was well correlated with viral serostatus, may be a factor that affects intrahepatic recurrence, which is more likely to originate from metachronous carcinogenesis. Prior co-infection of HBV in HCV patients may not be an adverse risk factor for intrahepatic recurrence. PMID- 10755691 TI - Induction of hepatic stellate cell proliferation by LPS-stimulated peripheral blood mononuclear cells from patients with liver cirrhosis. AB - We studied hepatic stellate cell proliferation in vitro. Peripheral blood mononuclear cells (PBMC) from patients with chronic active hepatitis C (CAH) and liver cirrhosis (LC) were cultured for 24h in the presence or absence of Escherichia coli lipopolysaccharides (LPS). Hepatic stellate cell proliferation induced by the culture supernatants was measured, and interleukin-1 (IL-1) and IL 6 levels in the culture supernatants were quantified. Culture supernatants of LPS stimulated PBMC from LC patients induced rat hepatic stellate cell proliferation by almost 2.8-fold (stimulation index, 2.83 +/- 1.41) compared with when the cells were cultured without addition of PBMC culture supernatants. Production of IL-1beta was significantly higher in the culture supernatants of both CAH and LC patients than in those of ten healthy controls (P < 0.01 and P < 0.05, respectively). But there was no significant correlation between IL-1 production and the induction of hepatic stellate cell proliferation by the culture supernatants. Although there were no significant differences in IL-6 production by LPS-stimulated PBMC among healthy controls and CAH and LC patients, we observed a significant correlation between IL-6 production and the induction of hepatic stellate cell proliferation in the culture supernatants of LC patients. Rat hepatic stellate cells themselves produced IL-6, and treatment with IL-6 antisense oligodeoxynucleotides suppressed the cell proliferation, suggesting that IL-6 is an autocrine growth factor for hepatic stellate cells. The addition of human recombinant IL-6 (hrIL-6) augmented rat hepatic stellate cell proliferation, indicating that excessive IL-6 may further facilitate cell proliferation. These findings suggest that a cytokine cascade including IL-6 may participate in hepatic stellate cell proliferation in LC patients when they are exposed to endotoxin. PMID- 10755692 TI - Fluctuation patterns of HCV-RNA serum level in patients with chronic hepatitis C. AB - The serum level of hepatitis C virus (HCV)-RNA is clinically important as a predictor of the response to interferon (IFN) therapy in patients with chronic hepatitis C. If serum HCV-RNA levels fluctuate during follow-up, and IFN therapy is begun at the time of a low HCV-RNA level, the IFN therapy may be more effective. We evaluated the fluctuation of HCV-RNA serum levels for 2 years in 212 patients with chronic hepatitis C, untreated with IFN who had HCV genotype 1b and an HCV-RNA level of 10 Meq/ml or more at first consultation. The HCV-RNA level was measured monthly for 2 years with an HCV branched DNA probe assay (b DNA probe assay). We classified HCV-RNA patterns into three types by the ratio of maximum HCV-RNA level (a) to minimum HCV-RNA level (b). In pattern 1 (constant type, 151 patients; 71.2%) the a/ b ratio was 1-5. In pattern 2 (slight fluctuation type, 46 patients; 21.7%) the a/b ratio was 5-10. In pattern 3 (severe fluctuation type, 15 patients; 7.1%), the a/b ratio was 10 or more. Next, we evaluated the factors associated with the three patterns. Acute exacerbation of chronic hepatitis was regarded as an increase in serum alanine aminotransferase (ALT) level to more than 250 IU/l. The incidence of acute exacerbation for a 2-year follow-up was 13.9% (21/151) in pattern 1, 19.6% (9/46) in pattern 2, and 53.3% (8/15) in pattern 3. Multivariate analysis showed that acute exacerbation was the most important factor in the manifestation pattern 3. In conclusion, we found that: (1) about 70% of patients had a constant HCV-RNA levels for 2 years. (2) A few patients had severe fluctuation of serum HCV-RNA level after acute exacerbation of chronic hepatitis. PMID- 10755694 TI - Idiopathic portal hypertension associated with systemic lupus erythematosus. AB - A case of idiopathic portal hypertension (IPH) associated with systemic lupus erythematosus (SLE) is reported in a 38-year-old man who had been diagnosed with SLE and treated for 18 years. Esophageal varices. found in 1994 on endoscopic examination, had been followed up for 2 years. On July 16, 1996, he was admitted to Nagoya University Hospital because there was a high risk of bleeding from the esophageal varices due to severe thrombocytopenia. As partial splenic embolization had temporarily controlled the thrombocytopenia, splenectomy and devascularization of the stomach vessels were performed after endoscopic ligation of the esophageal varices. Histological specimens of wedge biopsied liver showed chronic inactive hepatitis without cirrhosis. The presence of anticardiolipin antibody, indicated by positivity for lupus anticoagulant, was suggestive of the presence of a common immunological mechanism in the etiology of SLE and IPH. PMID- 10755693 TI - Autoimmune reactivity of sera to hepatocyte plasma membrane in type 1 autoimmune hepatitis. AB - Type 1 autoimmune hepatitis (AIH-1) is an organ-specific autoimmune liver disease for which no tissue-specific autoantigen has yet been identified. We examined the reactivity by sensitive immunoblotting with enhanced chemiluminescence (IB-ECL) of 43 sera from patients with AIH-1 and 182 sera from patients with other diseases on hepatocyte plasma membrane derived from rat or human liver (RHPM, HHPM) and separated by aqueous two-phase partition. The sera studied were from patients with AIH-1, primary biliary cirrhosis, chronic viral hepatitis, and systemic lupus erythematosus (SLE); and from normal subjects. Specificity of reactivity by IB-ECL was sought: (i) by testing sera on human or rat liver membrane; (ii) by testing sera on liver or kidney membrane; (iii) by serial titration of reactive sera; and (iv) by testing reactive sera from AIH-1 before and after successful treatment with prednisolone. The results were that in AIH-1 there were multiple reactive components which were not species-specific, since they were detected with both RHPM and HHPM, but were mostly tissue-specific for liver. There was no significant correlation between antinuclear antibodies (ANA) titer and the frequencies of sera reactivities against RHPM. Most of these reactive components were demonstrable at a lesser frequency in other liver diseases and in SLE. There was a striking decrease in reactivity by IB-ECL of AIH 1 sera with liver membrane after clinical remission, further suggesting that differences between AIH-1 and other inflammatory liver diseases and SLE are predominantly quantitative rather than qualitative. However, our study did point to candidate liver membrane antigens with molecular sizes of 136, 116, 81, and 49 kDa, additional to components previously described by others. The molecular identification of these prominent reactants with AIH-1 sera could prove informative for ascertaining pathogenesis. PMID- 10755695 TI - Hepatocellular carcinoma complicated by hemothorax. AB - Although hemoperitoneum is a well-known cause of death induced by rupture of hepatocellular carcinoma (HCC). hemothorax caused by rupture of HCC has not been adequately described. We report here a 64-year-old woman who died of bleeding from pleural wall metastasis of HCC. She presented with liver failure and sudden onset hypotension, and on the same day, she died of hypovolemic shock associated with liver failure. Autopsy revealed HCC metastatic to the lungs and right pleura and 21 of bloody pleural effusion in the right side. The cut surface of the metastatic nodule in the right pleura was filled with coagulated blood and necrotic tissue. We concluded that the pleural metastasis had ruptured and caused the right hemothorax. We discuss reported cases of HCC complicated by hemothorax. PMID- 10755696 TI - A patient with primary biliary cirrhosis associated with autoimmune hemolytic anemia. AB - Primary biliary cirrhosis is often associated with autoimmune conditions, such as thyroid disease, sicca complex, and rheumatoid arthritis. However, an association with autoimmune hemolytic anemia has rarely been reported. We present a case of primary biliary cirrhosis associated with warm type autoimmune hemolytic anemia, and we review prior reports. PMID- 10755697 TI - Does IL-6 regulate liver fibrosis/cirrhosis directly and indirectly? PMID- 10755698 TI - Autoantibodies to hepatocyte plasma membrane antigens in autoimmune hepatitis: old and new subjects; what do we need? PMID- 10755699 TI - Childhood duodenal ulcer: is there any pathogenetic association with Helicobacter pylori urease? PMID- 10755700 TI - Detection of beryllium sensitivity using a flow cytometric lymphocyte proliferation test: the Immuno-Be-LPT. AB - Measurement of lymphocyte proliferation to detect hypersensitivity to beryllium (Be-LPT) in vitro is done presently using a method based on tritiated thymidine incorporation. Although this method is sensitive it gives no information on cell viability or responding lymphocyte subsets. We have developed reliable and simple flow cytometric assays for lymphocyte proliferation testing (Immuno-Be-LPT) by combining immunophenotyping with bromodeoxyuridine (BrdU) incorporation or DNA content using propidium iodide (PI) or 4'6'-diimidazolin-2-phenylindole (DAPI). Evaluation of beryllium-induced lymphocyte proliferation in blood cells from seven patients with chronic beryllium disease (CBD) and 120 beryllium workers by both the Bc-LPT and the Immuno-Be-LPT showed agreement between the tests. The Immuno-Bc-LPT provided additional information about the specific type of lymphocytes responding. CD4+ lymphocytes proliferated in response to beryllium in blood samples from all seven CBD individuals and CD8+ lymphocytes proliferated in six of the seven. Four beryllium workers without CBD had positive responses to beryllium primarily in the CD8+ cells. The use of the individual's own plasma supported a greater beryllium or tetanus-induced proliferation of CD4+ lymphocytes when compared to commercial human serum. The response of CD4+ lymphocytes measured in the Immuno-Be-LPT may provide a new marker for the diagnosis of CBD. PMID- 10755701 TI - Allergen-triggered airway hyperresponsiveness and lung pathology in mice sensitized with the biopesticide Metarhizium anisopliae. AB - Metarhizium anisopliae is an entomopathogenic fungus recently licensed for indoor control of cockroaches, a major source of allergens. While M. anisopliae has been shown to be non-infectious and non-toxic to mammals there has been only limited research on potential allergenicity. Using a mouse model, we previously demonstrated allergic immune and inflammatory responses to this agent. The present study was designed to determine whether these responses were associated with changes in pulmonary responses, lung pathology, and the cytokine profile in bronchoalveolar lavage fluid (BALF). Soluble factors from fungal components were combined in equal protein amounts to form M. anisopliae crude antigen (MACA). BALB/C mice were intratracheally (i.t.) challenged with 10 microg MACA 14 days post intraperitoneal sensitization with 25 microg fungal antigen in aluminum hydroxide adjuvant. Physiological and cellular changes were examined. The mice were tested for airway hyperresponsiveness before (No Chal) and after (1, 3, and 8 days post challenge (DPIT)) MACA IT challenge. Subsequently, serum, BALF and the lungs were harvested. All treatment groups concurrently demonstrated significant non-specific pulmonary inflammation (neutrophil influx) and increased pulmonary sensitivity to methacholine (Mch) at 1 DPIT MACA challenge. Where as both adjuvant treated and naive mice airway responses had returned to near normal levels by 3 DPIT, mice which were previously sensitized with MACA were still hyperresponsive to Mch challenge at 3 and 8 DPIT. This hyperresponsiveness correlates with eosinophil and lymphocyte influx, which is maximal at 3 DPIT and still elevated at 8 DPIT. Interleukin (IL) 5 was elevated for all treatment groups at 1 DPIT but only the MACA sensitized mice maintained elevated levels for both 3 and 8 DPIT. Furthermore, MACA sensitized mice had a more extensive inflammatory histopathology at all examined time points with peribronchial and perivascular infiltrates, like those associated with allergic responsiveness, peaking at 3 DPIT. These pulmonary pathologic changes appeared to be consistent with elevated levels of serum and BALF total IgE, BALF IL-4, eosinophils, and lymphocytes following MACA IT challenge in MACA sensitized mice. There were no significant differences among the three treatment groups with regard to BALF interferon (IFN) gamma. The cytokines profiled indicate a Th2-type response, which is reflected in the cellular influx and total IgE induction. These data further indicate that immune inflammatory responses, observed in mice following MACA sensitization and challenge, are associated with physiologic changes and histopathology characteristic of allergic disease. PMID- 10755702 TI - Dose-dependent effects on tissue distribution and metabolism of dimethylarsinic acid in the mouse after intravenous administration. AB - Most mammals methylate inorganic arsenic to dimethylarsinic acid (DMA). This organic arsenical causes organ-specific toxicity and is a multi-organ tumor promoter. The objective of this study was to examine whether dose could affect the distribution and metabolism of DMA. Female B6C3F1 mice (3-4/time point) were administered 1.11 or 111 mg/kg of DMA (1 microCi of [14C] or unlabeled) intravenously and killed serially (5-480 min). Blood was separated into plasma and red blood cell fractions and liver, kidney and lung were removed, weighed and homogenized. Tissue samples were oxidized and analyzed for DMA-derived radioactivity. Blood and several organs of the non-radioactive DMA-treated animals were digested in acid and analyzed by hydride generation atomic absorption spectrophotometry for DMA and metabolites. Concentration-time profiles showed a biexponential decrease of DMA-derived radioactivity in all tissues examined. Kidney had the highest concentration (1-20% dose/gm) of radioactivity of all tissues up to 60 min post-administration. Concentration of radioactivity was greater in plasma than red blood cells at 5 and 15 min and then was similar for the remaining time points. A dose-dependent effect on the concentration of radioactivity was observed in the lung. The retention of radioactivity in the lung was altered compared with liver and kidney, with a much longer t1/2beta and a disproportionate increase in area under the curve with increased dose. No methylated or demethylated products of DMA were detected in blood or any organ up to 8 h post-exposure. The dose-dependent distribution of DMA in the lung may have a role in the toxic effects DMA elicits in this organ. PMID- 10755703 TI - Stimulated pulmonary cell hyperplasia underlies resistance to alpha naphthylthiourea. AB - The rodenticide alpha-naphthylthiourea (ANTU) causes pulmonary edema and pleural effusion that leads to death via pulmonary insufficiency. Rats become resistant to the lethal effect of ANTU if they are first exposed to a small, nonlethal dose of ANTU. Young rats are also resistant to ANTU. The mechanism by which rats develop resistance by a prior, small dose exposure has yet to be determined. Growth factor induced-pulmonary hyperplasia has been demonstrated to attenuate ANTU-induced lung leak. We hypothesized that a small dose of ANTU protects against a large dose through pulmonary cell hyperplasia induced by the protective dose. Furthermore, we hypothesized that this hyperplasia is associated with altered transcription of growth factors. Male Sprague-Dawley rats (175-225 g) were treated with a low dose of ANTU (5 mg ANTU/kg; ANTU(L)) 24 h before challenge with a 100% lethal dose of ANTU (70 mg ANTU/kg; ANTU(H)) resulting in 100% protection against the lethal effect of ANTU(H). ANTU(L) protection against ANTU(H) lasted for 5 days, slowly phased out, all being lost by day 20. Injury was assessed by estimating pulmonary vascular permeability and through histopathological examination. ANTU(H) alone resulted in an increase in pulmonary edema leading to animal death. However, injury was prevented if the rats were first treated with ANTU(L). There was a stimulation of pulmonary cell hyperplasia in the lungs of ANTU(L) treated rats as measured by [3H]-thymidine and bromodeoxyuridine incorporation. Treatment with the antimitotic agent colchicine abolished ANTU(L)-induced resistance to ANTU(H). ANTU resistant rats were also resistant to the lethal effect of paraquat. Paraquat is not taken up by pneumocytes if they are undergoing hyperplasia. ANTU(L) administration resulted in an up regulation of gene transcription for keratinocyte growth factor, transforming growth factor-beta, keratinocyte growth factor receptor and epidermal growth factor receptor as determined through reverse transcription polymerase chain reaction. A significant increase in transforming growth factor alpha was not observed. These findings collectively suggest that ANTU(L)-induced pulmonary cell hyperplasia underlies resistance to ANTU(H). Furthermore, the stimulation of hyperplasia may be due to altered growth factor and growth factor receptor expressions. PMID- 10755706 TI - Relationship between blood/air partition coefficients of lipophilic organic solvents and blood triglyceride levels. AB - The blood/air partition coefficient is one of the important parameters for understanding the pharmacokinetics of organic solvents. In conventional pharmacokinetic models, the partition coefficient has been used as a constant value for each solvent. However, blood triglyceride content varies according to a diet, and the variation may affect the partition coefficient of lipophilic organic solvents. In this study, the relationship between the blood/air partition coefficient of lipophilic organic solvents and plasma triglyceride concentrations was clarified. Corn oil (4.0 ml/kg) was administered orally to male Wistar rats, and blood samples were collected 3 h after the administration, when the plasma triglyceride level reached a peak. Control rats received the same amount of distilled water via the same route. The blood/air partition coefficients of five types of lipophilic solvents (m-xylene, trichloroethylene, 1,1,1-trichloroethane, chloroform and carbon tetrachloride) were determined in these two groups of blood samples. The hematological and biochemical parameters were also examined in the same blood samples. Although the mean plasma triglyceride level in the oil group increased about 3.3 times compared with that in the control group, the increase in the blood/air partition coefficients of the five lipophilic solvents was relatively low (approximately 1.2-1.5 times). We concluded that the diet has no significant effect on the blood/air partition coefficients of organic solvents, and the value can be used as a constant parameter for each solvent in a physiological simulation model. PMID- 10755704 TI - Tolerance to fumonisin toxicity in a mouse strain lacking the P75 tumor necrosis factor receptor. AB - Fumonisin B1 (FB1), a potent mycotoxin prevalent in corn and cereals, causes a variety of toxic effects in different mammalian species. The biochemical responses of FB1 involve inhibition of ceramide synthase leading to accumulation of free sphingoid bases and a possible involvement of tumor necrosis factor alpha (TNFalpha). To further characterize the role of TNFalpha, toxic response to FB1 was investigated in male C57BL/6J mice (WT) and a corresponding TNFalpha receptor knockout (TRK) strain, genetically modified to lack the TNFalpha1b receptor. The hepatotoxic effects of 5 daily injections of 2.25 mg/kg per day of FB1 were observed in WT but were reduced in TRK, evidenced by circulating alanine aminotransferase and aspartate aminotransferase levels and histopathological evaluation of the tissue. FB1 induced TNFalpha expression in the livers of both WT and TRK mice to a similar extent (3-4 fold over control); however, a corresponding increase of cellular NFkappaB, expected after the downstream cellular signaling of TNFalpha, was noted only in the WT. Accumulation of liver sphingosine after FB1 treatment was similar in both WT and TRK, but the FB1 induced increases in liver sphinganine and kidney sphingosine and sphinganine were lower in TRK than in WT. Results emphasized the role of TNFalpha in FB1 induced hepatotoxicity in mice and the possible relationship of sphingoid base accumulation and TNFalpha induction. Moreover, the presence of TNFalpha receptor 1b appears to be important in mediating the hepatotoxic responses of TNFalpha and FB1 in mice. PMID- 10755705 TI - Inhibition of gap-junctional-intercellular communication in thyroid-follicular cells by propylthiouracil and low iodine diet. AB - Propylthiouracil (PTU) or low-iodine diet (LID) treatment increases thyroid follicular-cell proliferation, possibly by disrupting the movement of small molecules (< 1.2 kD) through membrane channels called gap junctions. Numerous tumor promoters and proliferative disease states exhibit inhibited gap-junctional intercellular communication (GJIC) prior to the induction of cell proliferation, yet the association between GJIC and apoptosis is unclear. In the present study, we used an ex vivo method to examine whether GJIC is inhibited in the thyroid of PTU- or LID-treated rats. In addition, the effect of these models of hypothyroidism on thyroid-follicular-cell proliferation and apoptosis was examined to determine the association between GJIC and cell homeostasis. After 14 days of treatment of either PTU or LID (plus 1% KClO4 in the drinking water), serum tri-iodothyronine (T3) and thyroxine, (T4) was decreased to nearly undetectable levels and serum TSH was increased in PTU- and LID-treated rats. At the same time point, GJIC was decreased 30-35% in PTU- and LID-treated rats while thyroid-follicular-cell proliferation increased nearly threefold in both treatment groups. Interestingly, apoptosis increased twofold in both hypothyroid treatment groups. These data suggest that PTU or LID treatment inhibit thyroid GJIC during a state of increased thyroid-follicular-cell proliferation and apoptosis. While the increase in proliferation was anticipated, the paradoxical increase in apoptosis during decreased GJIC in thyroid-follicular cells warrants further examination. PMID- 10755707 TI - Overcoming the pitfalls of Web search engines--Toxicology abstracts. PMID- 10755708 TI - The influence of thymoquinone on doxorubicin-induced hyperlipidemic nephropathy in rats. AB - The effect of thymoquinone (TQ), the main constituent of the volatile oil of Nigella sativa seeds, on the nephropathy and oxidative stress induced by doxorubicin (DOX) in rats was investigated. A single intravenous injection of DOX (6 mg/kg) induced a severe nephrotic syndrome (after 5 weeks) associated with hypoalbuminemia, hypoproteinemia, elevated serum urea, hyperlipidemia, and a high urinary excretion of protein, albumin and N-acetyl-beta-D-glucosaminidase (NAG). In the kidney, DOX induced a significant increase in total triglycerides (TG), total cholesterol (TC), and lipid peroxides and a significant decrease in non protein sulfhydryl (NPSH) content and catalase (CAT) activity. Treatment of rats with TQ (10 mg/kg per day) supplemented with the drinking water for 5 days before DOX, and daily thereafter, significantly lowered serum urea, TG, and TC. Similarly, TG, TC and lipid peroxides in the kidneys of TQ-treated rats were decreased significantly compared with DOX alone. Moreover, NPSH content and CAT activity in the kidneys of TQ-treated DOX group were significantly elevated compared with DOX alone. Treatment with TQ significantly suppressed DOX-induced proteinuria, albuminuria, and urinary excretion of NAG. The results confirm the involvement of free radicals in the pathogenesis of nephropathy induced by DOX. Likewise, the study demonstrates the high antioxidant potential of TQ and its marked effect on the suppression of DOX-induced nephropathy. The data suggest that TQ might be applicable as a protective agent for proteinuria and hyperlipidemia associated with nephrotic syndrome. PMID- 10755709 TI - Differential calcium transport disturbances in renal membrane vesicles after cadmium-metallothionein injection in rats. AB - Cadmium-metallothionein (CdMT) induced calciuria may result from disturbed calcium (Ca) transport through the renal tubular epithelium. The present study aimed at defining time of onset and the degree of disturbed calcium transport. Kidneys were obtained from rats at 4, 12 and 24 h after a single injection of CdMT (dose 0.4 mg Cd/kg b.w.), and compared to saline injected controls. Rapid filtration 45Ca-assays were performed on basolateral and luminal membrane vesicles, isolated from kidney cortex using a sequential ultracentrifugation procedure. Luminal 45Ca uptake was increased at 4 h and then declined to about 80% of controls, suggesting an early phase perturbation of Ca absorption. Basolateral 45Ca uptake was reduced to less than 50% of controls, starting already at 4 h while 45Ca binding was reduced at 8 h. This may reflect an inhibited basolateral Ca pump mechanism after the binding step. Since the Ca pump normally expels Ca from the cell, an accumulation of intracellular calcium was indicated. Metal analysis verified a four-fold increase of Ca in kidney cortex at 24 h. This suggests that Cd impact on tubular cells involves disturbances on cellular absorption as well as expulsion of Ca. PMID- 10755710 TI - Beryllium-stimulated production of tumor necrosis factor-alpha by a mouse hybrid macrophage cell line. AB - Chronic beryllium disease (CBD) results from exposure to the light-weight metal beryllium (Be). In vitro stimulation of bronchoalveolar lavage cells from CBD subjects causes the production of high levels of TNF-alpha, IFN-gamma and IL-6. We tested the hypothesis that Be-stimulation might induce the production of TNF alpha by macrophage cell lines. We observed that H36.12j cells (12j), a mouse hybrid macrophage cell line, but not other mouse and human macrophage cell lines, produced TNF-alpha upon Be-stimulation. The response was maximal at 100 microM BeSO4 and did not occur when 12j cells were stimulated with either aluminum sulfate or cobalt sulfate. Beryllium-stimulated the production of 725+/-25 pg/ml (mean +/- SEM) TNF-alpha protein by 12j cells as measured by ELISA of culture supernatants after 24 h. As measured by RT-PCR, Be-stimulated 12j cell TNF-alpha protein production was accompanied by an increased intracellular TNF-alpha mRNA at 3 and 24 h. The addition of 10U or 100U of rMu-IFN-gamma to Be-stimulated 12j cells further increased TNF-alpha production 1.5-4 fold (1.6+/-0.1 ng/ml) respectively. Bacterial lipopolysaccharide (LPS, 1 microg/ml) stimulated production of TNF-alpha in 12j culture supernatants after 6 h (515+/-151 pg/ml). This early versus late TNF-alpha production suggests that LPS and Be both stimulate 12j cell TNF-alpha synthesis, but through different pathways. We report for the first time, the direct effects of Be stimulation on the ability of 12j cells to produce TNF-alpha. The 12j cell line, contrasted with other macrophage hybrids that do not respond to Be-stimulation, may provide a useful tool to evaluate the mechanisms by which Be stimulates macrophage cytokine production, and by which T cell derived IFN-gamma amplifies TNF-alpha production in granulomatous diseases. PMID- 10755711 TI - Beryllium-stimulation does not activate transcription factors in a mouse hybrid macrophage cell line. AB - We tested the hypothesis that beryllium (Be) could stimulate H36.12j cell (12j) TNF-alpha production by transcription factor-mediated pathways similar to those induced by either LPS- or IFN-gamma stimulation. Unstimulated 12j cells produce constitutive levels of TNF-alpha (175+/-18 pg/ml, mean +/- SEM) detected by ELISA of culture supernatants after 24 h. Beryllium-stimulated (100 microM BeSO4) 12j cell TNF-alpha (724+/-47 pg/ml) was observed after 24 h while LPS-stimulated (1 microg/ml) TNF-alpha (515+/-151 pg/ml) after 6 h. Recombinant-Mu-IFN-gamma (10 U) stimulated 12j cell TNF-alpha at lower levels (284+/-31 pg/ml) while rMu-IFN gamma + Be-stimulated 12j cells produced 1195+/-225 pg/ml TNF-alpha. Constitutive levels of transcription factors were observed in unstimulated 12j cell nuclei. In LPS-stimulated 12j cells IkappaBalpha was degraded in the cytoplasm and increased levels of NF-kappaB were found in nuclei after 30 min. After 3 h there were increased levels of AP-1 and CREB, with increased amounts of Fos family, Jun B and Jun D transcription factors. In contrast, Be-stimulation failed to increase the levels of any transcription factor tested, NF-kappaB, AP-1, AP-2, CREB, C/EBP, Sp-1, Egr-1, Ets, NF-Y or Oct-1, in 12j cells. A pattern of increased transcription factors, similar to that observed for LPS-stimulation, was found in 12j cell nuclei after stimulation with rMu-IFN-gamma. However, NF-kappaB was increased at 3 h while AP-1 (Jun B and Jun D) and CREB were increased at 15 h. Co stimulation of 12j cells with rMu-IFN-gamma + Be increased the levels of NF-KB in 12j cell nuclei at 3 h, and the levels of AP-1 and CREB at 15 h, however, only Jun B was increased. Our data show 12j cell TNF-alpha production was associated with increased levels of transcription factors present in nuclei with disparate kinetics and patterns of expression depending on the trigger. We reject our initial hypothesis and conclude that Be-stimulation signals 12j cell TNF-alpha synthesis via a transcription factor-independent pathway. Beryllium may induce novel pathways of macrophage cytokine gene regulation. PMID- 10755712 TI - A comparison of hepatocyte cytotoxic mechanisms for Cu2+ and Cd2+. AB - The molecular cytotoxic mechanisms of hepatocyte cell death induced by CuCl2, an essential redox transition metal has been compared with CdCl2, an environmental toxin. The ED50 concentrations found for Cu2+ and Cd2+ (i.e. 50% membrane lysis in 2 h) were 50 and 20 microM respectively. However reactive oxygen species ('ROS') formation, GSH oxidation and lipid peroxidation were induced by Cu2+ at these concentrations much more rapidly than by Cd2+. The decline of mitochondrial membrane potential though occurred at the same time and to the same extent for both metals. Furthermore the cytotoxicity and decline of mitochondrial membrane potential induced by these metals was prevented by the 'ROS' scavengers dimethyl sulfoxide, mannitol, catalase or SOD, as well as by desferoxamine, N,N diphenylphenylenediamine or alpha-tocopherol succinate. Hepatocyte GSH was protective as GSH depleted hepatocytes were much more susceptible to Cu2+ and Cd2+ than normal hepatocytes. It is concluded that Cu2+-induced cytotoxicity occurs as a result of a mitochondrial 'ROS' formation independently of cytosolic 'ROS' formation due to redox cycling. PMID- 10755713 TI - Arachidonic acid release and prostaglandin E2 synthesis as irritant index of surfactants in 3T6 fibroblast cultures. AB - Surface-active agents are components of many drugs and cosmetics. [3H]arachidonic acid ([3H]AA) is rapidly incorporated into cell membrane phospholipids. As a result of membrane disintegration or enzymatic catalysis, it is released to the medium and [3H]AA metabolites can be measured by the scintillation technique. In order to validate the use of this test to predict the irritant and toxic effects of surfactants in vitro, various surfactants were examined using this methodology and the neutral red test. Benzalkonium chloride and sodium dodecylsulfate were more irritant and cytotoxic than amphoteric and non-ionic surfactants such as cocoamidopropylbetaine and Tween 80, respectively. The rank order of the toxicity/irritancy of these agents was similar to that given by other tests in vivo and in vitro. Moreover, our results confirm that the measurement of [3H]AA release in 3T6 fibroblast cultures is a sensitive assay of membrane damage. PMID- 10755714 TI - Effects of vitamin E on dolichol content of rats acutely treated with 1,2 dichloroethane. AB - Previous investigations have demonstrated that 1,2-dichloroethane (DCE) poisoning affects dolichol (Dol) concentration in rat liver. Dol, a long-chain polyprenol, is considered an important membrane component: as dolichyl phosphate, it is rate limiting for the synthesis of glycoprotein; as free or fatty acid, it is highly concentrated in the Golgi apparatus (GA) where it can increase membrane fluidity and permeability, required glycoprotein maturation and secretion. DCE biotransformation may stimulate pro-oxidant events through hepatocellular glutathione depletion. Since the molecules of Dol are susceptible to oxidative degradation, the aim of this investigation is to verify whether vitamin E (vit. E) supplementation in rats is able to prevent Dol breakdown during acute DCE treatment. Before acute DCE administration (628 mg/kg body weight), a group of male Wistar rats were pretreated with vit. E (33 mg/kg body weight) for 3 days. High-performance liquid chromatography analysis has shown that within 5-60 min after DCE administration, the Dol concentration decreased in liver homogenate, cytosol, microsomes and GA. Particularly, 60 min after the treatment, Dol levels in the trans Golgi fraction were 71% lower than in controls. Rat pre-treatment with vit. E prevented the DCE-induced decrease in Dol concentrations of all liver fractions considered, in particular the reduction of total-Dol observed in the trans Golgi fraction 60 min after treatment was only 40%. These data suggest that hepatic metabolism of DCE is able to promote peroxidative attacks which lead to the degradation of Dol molecules. The pre-treatment of rats with vit. E results in a good, although not complete, prevention of total-Dol depletion after DCE poisoning. PMID- 10755715 TI - Receptor radiopharmaceuticals: biomolecular nuclear medicine. PMID- 10755716 TI - Dose escalation study with rhenium-188 hydroxyethylidene diphosphonate in prostate cancer patients with osseous metastases. AB - The aim of this study was to determine the maximum tolerated dose of rhenium-188 hydroxyethylidene diphosphonate (HEDP) in prostate cancer patients with osseous metastases who are suffering from bone pain. Twenty-two patients received a single injection of escalating doses of carrier-added 188Re-HEDP [1.3 GBq (35 mCi), 2.6 GBq (70 mCi), 3.3 GBq (90 mCi) and 4.4 GBq (120 mCi)]. Blood counts and biochemical parameters were measured weekly over a period of 8 weeks. Haematological toxicity (WHO grading) of grade 3 or 4 was considered unacceptable. Clinical follow-up studies including methods of pain documentation (medication, pain diary) were performed for 6 months after treatment. In the 1.3 GBq group, no haematological toxicity was observed. First haematotoxic results were noted in those patients with a dose of 2.6 GBq 188Re-HEDP. In the 3.3-GBq group, one patient showed a reversible thrombopenia of grade 1, one a reversible thrombopenia of grade 2 and three a reversible leukopenia of grade 1. In the 4.4 GBq group, thrombopenia of grades 3 and 4 was observed in one and two patients (baseline thrombocyte count <200x10(9)/l), respectively, and leukopenia of grade 3 was documented in one patient. The overall nadir of thrombopenia was at week 4. The individual, maximum percentage decrease in thrombocytes in the 1.3-, 2.6-, 3.3- and 4.4-GBq groups was 17%, 40%, 60% and 86%, respectively. In two patients, a transient increase in serum creatinine was observed (max. 1.6 mg/dl). Pain palliation was reported by 64% of patients, with a mean duration of 7.5 weeks. The response rate seemed to increase with higher doses, reaching 75% in the 4.4 GBq group. It is concluded that in prostate cancer patients, the maximum tolerated dose of 188Re-HEDP is 3.3 GBq if the baseline thrombocyte count is below 200x10(9)/l. In patients with thrombocyte counts significantly above 200x10(9)/l, a dose of 4.4 GBq might be tolerable. Thrombo- and leukopenia are the most important side-effects. Pain palliation can be achieved in 60%-75% of patients receiving a dose of 2.6 GBq or more of 188Re-HEDP. Studies in a larger patient population are warranted to evaluate further the palliative effect of 188Re-HEDP. PMID- 10755717 TI - A novel phantom design for emission tomography enabling scatter- and attenuation "free" single-photon emission tomography imaging. AB - A newly designed technique for experimental single-photon emission tomography (SPET) and positron emission tomography (PET) data acquisition with minor disturbing effects from scatter and attenuation has been developed. In principle, the method is based on discrete sampling of the radioactivity distribution in 3D objects by means of equidistant 2D planes. The starting point is a set of digitised 2D sections representing the radioactivity distribution of the 3D object. Having a radioactivity-related grey scale, the 2D images are printed on paper sheets using radioactive ink. The radioactive sheets can be shaped to the outline of the object and stacked into a 3D structure with air or some arbitrary dense material in between. For this work, equidistantly spaced transverse images of a uniform cylindrical phantom and of the digitised Hoffman rCBF phantom were selected and printed out on paper sheets. The uniform radioactivity sheets were imaged on the surface of a low-energy ultra-high-resolution collimator (4 mm full width at half-maximum) of a three-headed SPET camera. The reproducibility was 0.7% and the uniformity was 1.2%. Each rCBF sheet, containing between 8.3 and 80 MBq of 99mTcO4- depending on size, was first imaged on the collimator and then stacked into a 3D structure with constant 12 mm air spacing between the slices. SPET was performed with the sheets perpendicular to the central axis of the camera. The total weight of the stacked rCBF phantom in air was 63 g, giving a scatter contribution comparable to that of a point source in air. The overall attenuation losses were <20%. A second SPET study was performed with 12-mm polystyrene plates in between the radioactive sheets. With polystyrene plates, the total phantom weight was 2300 g, giving a scatter and attenuation magnitude similar to that of a patient study. With the proposed technique, it is possible to obtain "ideal" experimental images (essentially built up by primary photons) for comparison with "real" images degraded by photon scattering and attenuation losses. The method can serve as a tool for experimental validation and intercomparison of attenuation and scatter correction methods. Moreover, the large flexibility of this phantom design will allow investigations of arbitrary activity distributions and autoradiography or other imaging techniques such as PET, x-ray computed tomography or magnetic resonance imaging. PMID- 10755718 TI - Interest of the ordered subsets expectation maximization (OS-EM) algorithm in pinhole single-photon emission tomography reconstruction: a phantom study. AB - Pinhole single-photon emission tomography (SPET) has been proposed to improve the trade-off between sensitivity and resolution for small organs located in close proximity to the pinhole aperture. This technique is hampered by artefacts in the non-central slices. These artefacts are caused by truncation and by the fact that the pinhole SPET data collected in a circular orbit do not contain sufficient information for exact reconstruction. The ordered subsets expectation maximization (OS-EM) algorithm is a potential solution to these problems. In this study a three-dimensional OS-EM algorithm was implemented for data acquired on a single-head gamma camera equipped with a pinhole collimator (PH OS-EM). The aim of this study was to compare the PH OS-EM algorithm with the filtered back projection algorithm of Feldkamp, Davis and Kress (FDK) and with the conventional parallel-hole geometry as a whole, using a line source phantom, Picker's thyroid phantom and a phantom mimicking the human cervical column. Correction for the angular dependency of the sensitivity in the pinhole geometry was based on a uniform flood acquisition. The projection data were shifted according to the measured centre of rotation. No correction was made for attenuation, scatter or distance-dependent camera resolution. The resolution measured with the line source phantom showed a significant improvement with PH OS-EM as compared with FDK, especially in the axial direction. Using Picker's thyroid phantom, one iteration with eight subsets was sufficient to obtain images with similar noise levels in uniform regions of interest to those obtained with the FDK algorithm. With these parameters the reconstruction time was 2.5 times longer than for the FDK method. Furthermore, there was a reduction in the artefacts caused by the circular orbit SPET acquisition. The images obtained from the phantom mimicking the human cervical column indicated that the improvement in image quality with PH OS-EM is relevant for future clinical use and that the improvements obtained using the OS-EM algorithm are more significant for the pinhole geometry than for the conventional parallel-hole geometry. We conclude that PH OS-EM is a practical and promising alternative for pinhole SPET reconstruction. PMID- 10755719 TI - Validation of fully automatic brain SPET to MR co-registration. AB - Fully automatic co-registration of functional to anatomical brain images using information intrinsic to the scans has been validated in a clinical setting for positron emission tomography (PET), but not for single-photon emission tomography (SPET). In this paper we evaluate technetium-99m hexamethylpropylene amine oxime to magnetic resonance (MR) co-registration for five fully automatic methods. We attached six small fiducial markers, visible in both SPET and MR, to the skin of 13 subjects. No increase in the radius of SPET acquisition was necessary. Distortion of the fiducial marker distribution observed in the SPET and MR studies was characterised by a measure independent of registration and three subjects were excluded on the basis of excessive distortion. The location of each fiducial marker was determined in each modality to sub-pixel precision and the inter-modality distance was averaged over all markers to give a fiducial registration error (FRE). The component of FRE excluding the variability inherent in the validation method was estimated by computing the error transformation between the arrays of MR marker locations and registered SPET marker locations. When applied to the fiducial marker locations this yielded the surface registration error (SRE), and when applied to a representative set of locations within the brain it yielded the intrinsic registration error (IRE). For the best method, mean IRE was 1.2 mm, SRE 1.5 mm and FRE 2.4 mm (with corresponding maxima of 3.3, 4.3 and 5.0 mm). All methods yielded a mean IRE <3 mm. The accuracy of the most accurate fully automatic SPET to MR co-registration was comparable with that published for PET to MR. With high standards of calibration and instrumentation, intra-subject cerebral SPET to MR registration accuracy of <2 mm is attainable. PMID- 10755720 TI - Towards an optimal reference region in single-photon emission tomography difference images in epilepsy. AB - There is marked variability in the cerebral blood flow (CBF) between the ictal and interictal state in epilepsy, and it would therefore be desirable to increase the reliability of ictal/interictal single-photon emission tomography (SPET) difference images. We aimed to improve the step of quantitative normalization of images by finding the best possible reference region. In 16 patients (11 with lateralization of the epileptogenic focus, five with bilateral foci) both ictal and inter-ictal SPET scans were performed after injection of technetium-99m labelled tracer. Then, each region among a selected set (brain+cerebellum, brain, cerebellum, hemispheres, and for patients with an expected lateralization, cortical lobe containing the focus and symmetrical contralateral lobe) was investigated by comparison of the regional ictal/inter-ictal variance in counts. Among patients with a suspected lateralized focus, the distribution of CBF in the contralateral cortical lobe appeared to vary less between ictal and inter-ictal states than in other investigated areas. As a consequence, this latter region constitutes the best choice as a reference region. For patients with bilateral foci, the cerebellum appears to be a good compromise even though it presents with significant CBF changes. PMID- 10755721 TI - Recovery correction for quantitation in emission tomography: a feasibility study. AB - In emission tomography, the spread of regional tracer uptake to surrounding areas caused by limited spatial resolution of the tomograph must be taken into account when quantitating activity concentrations in vivo. Assuming linearity and stationarity, the relationship between imaged activity concentration and true activity concentration is only dependent on the geometric relationship between the limited spatial resolution of the tomograph in all three dimensions and the three-dimensional size and shape of the object. In particular it is independent of the type of object studied. This concept is characterized by the term "recovery coefficient". Recovery effects can be corrected for by recovery coefficients determined in a calibration measurement for lesions of simple geometrical shape. This method works on anatomical structures that can be approximated to simple geometrical objects. The aim of this study was to investigate whether recovery correction of appropriate structures is feasible in a clinical setting. Measurements were done on a positron emission tomography (PET) scanner in the 2D and 3D acquisition mode and on an analogue and digital single-photon emission tomography (SPET) system using commercially available software for image reconstruction and correction of absorption and scatter effects. The results of hot spot and cold spot phantom measurements were compared to validate the assumed conditions of linearity and stationarity. It can be concluded that a recovery correction is feasible for PET scanners down to lesions measuring about 1.5xFWHM in size, whereas with simple correction schemes, which are widely available, an object-independent recovery correction for SPET cannot be performed. This result can be attributed to imperfections in the commercially available methods for attenuation and scatter correction in SPET, which are only approximate. PMID- 10755722 TI - Hepatobiliary scintigraphy in the assessment of biliary obstruction after hepatic resection with biliary-enteric anastomosis. AB - We investigated the usefulness of hepatobiliary scintigraphy (HBS) for diagnosing biliary obstruction after curative hepatic resection with biliary-enteric anastomosis. The study population consisted of 54 patients who underwent surgery for benign (n=18) or malignant (n=36) biliary disease. We analysed 68 technetium 99m DISIDA scintigrams which were performed at least 1 month after the surgery (median: 9 months). Final diagnosis was made by operative exploration, other invasive radiological studies or clinical and radiological follow-up for at least 6 months after the surgery. Diagnostic accuracy was analysed according to the pretest likelihood of biliary obstruction. There were two total and 15 segmental biliary obstructions. In patients with symptoms of biliary obstruction and abnormal liver function, HBS always allowed correct diagnosis (two instances of total obstruction, seven of segmental obstruction and seven of non-obstruction). Among the patients with nonspecific symptoms or isolated elevation of serum alkaline phosphatase, HBS diagnosed segmental biliary obstruction in seven of the eight instances, and non-obstruction in 22 of 23 instances. There were no cases of biliary obstruction and no false-positive results of HBS in 21 instances with no clinical signs or symptoms of biliary obstruction. The diagnostic sensitivity and specificity of HBS for biliary obstruction were 94% (16/17) and 97% (50/51), respectively. In conclusion, HBS is a highly accurate modality for the diagnosis of segmental biliary obstruction during long-term follow-up after hepatic resection with biliary-enteric anastomosis. PMID- 10755723 TI - Clinical relevance of gallium-67 scintigraphy in lymphoma before and after therapy. AB - The clinical impact of gallium-67 scintigraphy before and after therapy for lymphoma remains controversial. The aims of this study were: (1) to compare the staging of lymphoma by 67Ga scintigraphy only with staging by clinical examination and conventional imaging (CI), and (2) to analyse the clinical relevance of both 67Ga imaging and CI after treatment. From March 1995 to November 1998, 86 67Ga scintigraphy studies were performed in 62 patients with Hodgkin's disease (n=52) or non-Hodgkin's lymphoma (n=10). 67Ga scintigraphy was performed at diagnosis (n=44) or after therapy (n=42) using 185-220 MBq 67Ga citrate and planar and single-photon emission tomography (SPET) studies. Treatment comprised radiotherapy, chemotherapy or combined modalities. CI included plain chest radiography, computed tomography (CT) of the chest and abdomen/pelvis, ultrasound of the abdomen, lymphography, bone marrow biopsy and, when necessary, magnetic resonance imaging (MRI) and bone scintigraphy. For individual suspected sites of disease before treatment, complete agreement between clinical examination and CI on the one hand and 67Ga scintigraphy on the other hand was observed in 25/44 patients (57%; 95% confidence interval 41%-72%). Clinical examination and CI showed more sites than did 67Ga scintigraphy in 12/44 patients (27%) and 67Ga imaging demonstrated more sites than CI in 6/44 patients (11%). The clinical stage of the disease as assessed using 67Ga scintigraphy only was in agreement with that using all diagnostic procedures in 34/44 patients (77%; 95% confidence interval 62%-89%). Compared with CI staging, 67Ga scintigraphy downstaged seven patients (16%) and upstaged three (7%). 67Ga scintigraphy downstaged mainly because of the limited value of the technique below the diaphragm and upstaged owing to the good sensitivity in the lung. After therapy, both CI and 67Ga scintigraphy were normal in 11 patients. All but one of these patients were in complete remission after a median follow-up of 31 months. In contrast, radiological residual mass was observed in 31/42 patients. 67Ga imaging was normal in 22/31 (71%); 17 of these 22 patients, including nine with a large residual mass (> or =2 cm), were in complete remission after a median follow-up of 32 months, while four suffered relapses 8-45 months later. The cause of death remained unknown in one patient. 67Ga scintigraphy showed abnormal uptake in 9 of the 31 patients with a large residual mass. Active disease was demonstrated in eight patients and one patient was in complete remission 30 months thereafter. Our data show that 67Ga imaging cannot replace CI in initial staging but can demonstrate additional individual sites of disease in more than 10% of patients and can lead to clinical upstaging with potential prognostic and therapeutic consequences. After therapy, 67Ga scintigraphy has a clinical impact when radiological abnormalities persist because it can either avoid unnecessary complementary treatment or confirm the need to change treatment modalities. PMID- 10755724 TI - Limited performance of quantitative assessment of myocardial function by thallium 201 gated myocardial single-photon emission tomography. AB - We investigated the reproducibility between thallium-201 and technetium-99m methoxyisobutylisonitrile (MIBI) gated single-photon emission tomography (SPET) for the assessment of indices of myocardial function such as end-diastolic and end-systolic volume (EDV, ESV), ejection fraction (EF) and wall motion. Rest 201Tl (111 MBq) gated SPET was sequentially performed twice in 20 patients. Rest 201Tl gated SPET and rest 99mTc-MIBI (370 MBq) gated SPET were performed 24 h apart in 40 patients. Wall motion was graded using the surface display of the Cedars quantitative gated SPET (QGS) software. EDV, ESV and EF were also measured using the QGS software. The reproducibility of functional assessment on rest 201Tl gated SPET was compared with that on 99mTc-MIBI gated SPET, and also with that between 201Tl gated SPET and 99mTc-MIBI gated SPET performed on the next day. The two standard deviation (2 SD) values for EDV, ESV and EF on the Bland Altman plot were 29 ml, 19 ml and 12%, respectively, on repeated 201Tl gated SPET, compared with 14 ml, 11 ml and 5.3% on repeated 99mTc-MIBI gated SPET. The correlations were good (r=0.96, 0.97 and 0.87) between the two measurements of EDV, ESV and EF on repeated rest studies with 201Tl and 99mTc-MIBI gated SPET. However, Bland-Altman analysis revealed that the 2 SD values between the two measurements were 31 ml, 23 ml and 12%. We were able to score the wall motion in all cases using the 3D surface display of the QGS on 201Tl gated SPET. The kappa value of the wall motion grade on the repeated 201Tl study was 0.35, while that of the wall motion grade on the repeated 99mTc-MIBI study was 0.76. The kappa value was 0.49 for grading of wall motion on repeated rest studies with 201Tl and 99mTc-MIBI. In conclusion, QGS helped determine EDV, ESV, EF and wall motion on 201Tl gated SPET. Because the EDV, ESV and EF were less reproducible on repeated 201Tl gated SPET or on 201Tl gated SPET and 99mTc-MIBI gated SPET on the next day than on repeated 99mTc-MIBI gated SPET, functional measurement on 201Tl gated SPET did not seem to be interchangeable with that on 99mTc-MIBI gated SPET. PMID- 10755725 TI - Quantitative assessment of regional myocardial blood flow using oxygen-15 labelled water and positron emission tomography: a multicentre evaluation in Japan. AB - Recently, a method has been proposed for the quantitative measurement of regional myocardial blood flow (MBF) using oxygen-15-labelled water and positron emission tomography (PET). A multicentre project was organized with the intention of evaluating the accuracy of this method, particularly as a multicentre clinical investigative tool. Each of seven institutions performed PET studies on more than five normal volunteers following a specified protocol. The PET study included a transmission scan, a 15O-carbon monoxide static scan and a 15O-water dynamic scan, thereby yielding MBF values which should have been independent of the spatial resolution of the PET scanner employed. Fifty-three subjects (aged 20-63 years, mean+/-SD 36+/-12 years) were studied at rest, and 31 of these subjects were also studied after dipyridamole in five institutions. Inter-institution consistency and intra-subject variation in MBF values were then evaluated. MBF averaged for all subjects was 0.93+/-0.34 ml min(-1) g(-1) at rest and 3.40+/ 1.73 ml min(-1) g(-1) after the administration of dipyridamole, and the flow reserve (defined as the ratio of the two MBF values) was 3.82+/-2.12; these values are consistent with previous reports. Resting MBF values were significantly correlated with the heart rate-blood pressure product (RPP) (y=0.31+6.56E-5x, P<0.010), and RPP was in resting MBF observed in all institutions was well explained by the age-dependent RPP. No significant difference was observed in resting MBF among the institutions. Except in one institution, no significant difference was seen in dipyridamole MBF or myocardial flow reserve. No significant difference was found among the myocardial segments. Regional variation was reasonably small in five institutions, but was not acceptable in two institutions, which was attributed to the scanner performance. These observations suggest that the 15O-water PET technique is useful for a multicentre clinical study if the PET scanner can provide time-activity data with good count statistics. PMID- 10755726 TI - Transmission imaging for registration of ictal and interictal single-photon emission tomography, magnetic resonance imaging and electroencephalography. AB - A method developed for registration of ictal and interictal single-photon emission tomography (SPET), magnetic resonance imaging (MRI) and electroencephalography (EEG) is described. For SPET studies, technetium-99m ethyl cysteinate dimer (ECD) was injected intravenously while the patient was monitored on video-EEG to document the ictal or interictal state. Imaging was performed using a triple-head gamma camera equipped with a transmission imaging device using a gadolinium-153 source. The images (128x128 pixels, voxel size 3.7x3.7x3.6 mm3) were reconstructed using an iterative algorithm and postfiltered with a Wiener filter. The gold-plated silver electrodes on the patient's scalp were utilized as markers for registration of the ictal and interictal SPET images, as these metallic markers were clearly seen on the transmission images. Fitting of the marker sets was based on a non-iterative least squares method. The interictal SPET image was subtracted from the ictal image after scaling. The T1-weighted MPRAGE MR images with voxel size of 1.0x1.0x1.0 mm3 were obtained with a 1.5-T scanner. For registration of MR and subtraction SPET images, the external marker set of the ictal SPET study was fitted to the surface of the head segmented from MR images. The SPET registration was tested with a phantom experiment. Registration of ictal and interictal SPET in five patient studies resulted in a 2 mm RMS residual of the marker sets. The estimated RMS error of registration in the final result combining locations of the electrodes, subtraction SPET and MR images was 3-5 mm. In conclusion, transmission imaging can be utilized for an accurate and easily implemented registration procedure for ictal and interictal SPET, MRI and EEG. PMID- 10755728 TI - Interferon-alpha induced changes in CEA expression in patients with CEA-producing tumours. AB - Enhancement of antigen expression could result in improved tumour targeting using antibodies directed to the antigen. In this study we performed radioimmunoscintigraphy using 99mTc-CEA-Scan to analyse the effect of interferon alpha (IFN-alpha) in enhancing the expression of carcinoembryonic antigen (CEA) in ten patients with CEA-producing tumours. Furthermore, we investigated the feasibility of a future therapeutic study with this antibody fragment labelled with rhenium-186. Although IFN-alpha gave rise to a significant increase in antibody uptake by the tumour, the absolute antibody uptake in the tumour appeared to be poor, with a mean of 0.475% of injected dose (ID) in the tumour before IFN-alpha, rising to 0.562% ID in the tumour after IFN-alpha. Pharmacokinetic analysis demonstrated no significant alterations after IFN-alpha. In conclusion, the administration of IFN-alpha is an attractive way to achieve enhanced tumour targeting, although the increase was of little clinical significance in this patient population and using this antibody fragment. PMID- 10755727 TI - Metabolism of [123I]epidepride may affect brain dopamine D2 receptor imaging with single-photon emission tomography. AB - Iodine-123 labelled epidepride is a novel radiopharmaceutical for the study of cerebral dopamine D2 receptors using single-photon emission tomography (SPET). A lipophilic labelled metabolite of [123I]epidepride which may enter the brain and hamper the quantitation of receptors has been observed in human plasma. In the present study, gradient high-performance liquid chromatography (HPLC) was used to investigate the plasma concentration of the lipophilic labelled metabolite and its correlation to SPET imaging of striatal dopamine D2 receptors. A linear regression fit showed a negative correlation between the amount of the lipophilic labelled metabolite and the striatum to cerebellum ratio (n=16, R=-0.58, P<0.02), suggesting that plasma metabolite analysis is essential when imaging dopamine D2 receptors with SPET using [123I]epidepride. PMID- 10755729 TI - What is the message of a meeting? Analysis and comparison of the 2458 abstracts of the EANM/WCNMB congress in Berlin and the SNM meeting in Toronto in 1998. European Association of Nuclear Medicine/World Federation of Nuclear Medicine and Biology, and the Society of Nuclear Medicine. AB - The abstracts of the joint congress of EANM/WCNMB in Berlin 1998 and of the 45th Annual Meeting of the Society of Nuclear Medicine in Toronto 1998 have been analysed and compared in terms of comprehensibility, composition, questions at issue, methods, patient/subject number, type of conclusion and duplication of information between the meetings. All 1362 and 1096 abstracts, respectively, were analysed from the abstract books with regard to ten "hard" and four "soft" variables. The dominant topics were new radiopharmaceuticals, methods of synthesis, examination methods, evaluation of examinations, investigation algorithms, technical devices and novel use of radiopharmaceuticals. In addition to these topics, there were numerous reports about established radiopharmaceuticals and techniques, often without a specific merit mentioned. There were also many abstracts with questions outside nuclear medicine, but using such techniques. Few papers reported negative findings or dealt with quality assurance, dosimetry, and cost-effectiveness. Many of the conclusions contained hyperbole. Some abstracts were very extensive and detailed. Sixty-seven contributions conveyed identical information at both meetings. Structured and/or paragraphed abstracts promote clarity and reduce the number of lines that need to be read in order to comprehend the background and aim of the abstract. Such contributions were more frequent at the EANM/WCNMB congress while the SNM meeting covered a wider field with a greater representation of radiophysics, instrumentation, and computer evaluations. PMID- 10755730 TI - Endovascular brachytherapy for the prevention of restenosis after angioplasty. AB - Restenosis is an unsolved clinical and financial limitation of angioplasty. Local irradiation is a new approach for the reduction of restenosis. Several animal studies have demonstrated the effective inhibition of arterial neointimal proliferation by percutaneous or endovascular irradiation. High-dose-rate irradiation from gamma and beta sources can be applied from radioactive wires or seeds and from liquid beta-emitter-filled balloon catheters. Dosimetric calculations have been performed for all relevant radionuclides. An effective dose can be applied within 10 min to the treated arteries. Beta-emitters are characterized by a low tissue penetration, which simplifies radiation protection but complicates the achievement of a homogeneous dose distribution without centering of the irradiation source. Gamma-emitters are characterized by deep tissue penetration and delivery of almost the same dose to all vessel layers; however, considerable care with regard to radiation protection of the environment is required if gamma-emitters are used. The liquid-filled balloon ensures a homogeneous dose delivery due to the self-centring irradiation source but entails the possibility of radioactivity incorporation in the event of balloon rupture. The most attractive radionuclide for this purpose is rhenium-188, which is available from the 188W/188Re generator system. Radiation exposure after accidental incorporation can be limited by chelation with mercaptoacetyltriglycine or by subsequent oral administration of perchlorate. Initial clinical trials have demonstrated the feasibility of the various irradiation techniques and yielded encouraging results. The use of unsealed radioactivity in a balloon catheter involves the nuclear medicine physician in this new field of therapy. This review discusses the concepts, the radiotracers and the results of animal experiments and early clinical trials in the field of endovascular irradiation employed as a possible means to prevent restenosis after angioplasty. PMID- 10755731 TI - Reduction of contamination risks during clinical studies with Technegas. PMID- 10755732 TI - The parieto-occipital region--confusions at the boundary? PMID- 10755733 TI - Blood transfusions and blood sampling during red blood cell mean life span determinations. PMID- 10755734 TI - Repeated ECS and fluoxetine administration have equivalent effects on hippocampal synaptic plasticity. AB - RATIONALE: Recent studies have implicated intracellular transduction pathways and neurotrophic factors in the action of antidepressants. Adaptation in these pathways may ultimately affect electrophysiological and morphological properties of neurones. We have previously shown that repeated electroconvulsive stimulation, a safe and effective antidepressant treatment, has profound effects on hippocampal synaptic connectivity and plasticity in the rat. Here, we investigated whether these electrophysiological properties were shared by the chemical antidepressant, fluoxetine. OBJECTIVES: To compare the electrophysiological and cognitive effects of two very different antidepressant treatments: repeated electroconvulsive stimulation (rECS); and chronic administration of the serotonin specific re-uptake inhibitor (SSRI), fluoxetine. METHODS: Rats were exposed to either rECS or daily fluoxetine administration for 15 days. The animals were then anaesthetised and dentate field excitatory post synaptic potential (fEPSP) characteristics were measured before and after the induction of long-term potentiation (LTP) by high frequency perforant path stimulation. In a separate experiment, the effects of rECS and chronic fluoxetine administration on acquisition and retention of a spatial learning task in the Morris watermaze were determined. RESULTS: Chronic fluoxetine administration and rECS produced equivalent increases in dentate fEPSP compared to respective control groups. LTP induction was attenuated in both groups. Spatial learning was, in contrast, unaffected by fluoxetine treatment but significantly impaired following rECS. CONCLUSIONS: Given that fluoxetine and rECS share antidepressant properties, but differ in their effects on learning and memory, we propose that the common effects on dentate connectivity and synaptic plasticity described here are more likely to relate to affective rather than cognitive function. This result is consistent with other experiments showing that a reduction in dentate connectivity correlates with stress susceptibility in animals. PMID- 10755735 TI - The discriminative stimulus properties of the atypical antipsychotic olanzapine in rats. AB - RATIONALE: Analysis of the preclinical behavioral effects of atypical antipsychotic agents will provide a better understanding of how they differ from typical antipsychotics and aid in the development of future atypical antipsychotic drugs. OBJECTIVES: The present study was designed to provide information about the discriminative stimulus properties of the atypical antipsychotic olanzapine. METHODS: Rats were trained to discriminate the atypical antipsychotic olanzapine (either 0.5 mg/kg OLZ or 0.25 mg/kg OLZ, i.p.) from vehicle in a twolever drug discrimination procedure. The atypical antipsychotic clozapine fully substituted for olanzapine in both the 0.5-mg/kg OLZ group (99.3% drug lever responding [DLR]) and the 0.25-mg/kg OLZ group (99.9% DLR). The typical antipsychotic chlorpromazine also substituted for olanzapine in both the 0.5-mg/kg OLZ group (87.5% DLR) and in the 0.25-mg/kg OLZ group (98.9% DLR); whereas, haloperidol displayed partial substitution for olanzapine in the 0.5 mg/kg OLZ group (56.1% DLR) and in the 0.25-mg/kg OLZ group (76.4% DLR). The 5.0 mg/kg dose of thioridazine produced olanzapine-appropriate responding in the 0.5 mg/kg OLZ group (99.6% DLR), but only partial substitution was seen with the 0.25 mg/kg OLZ training dose (64.0% DLR). The atypical antipsychotics raclopride (53.9% DLR) and risperidone (60.1% DLR) displayed only partial substitution in the 0.5-mg/kg OLZ group. Both the muscarinic cholinergic antagonist scopolamine (90.0% DLR) and the 5-HT2A/2C serotonergic antagonist ritanserin (86.0% DLR) fully substituted for olanzapine in the 0.5-mg/kg OLZ group. CONCLUSIONS: In contrast to previous discrimination studies with clozapine-trained rats, the typical antipsychotic agents chlorpromazine and thioridazine and the serotonin antagonist ritanserin substituted for olanzapine. These results demonstrate that there are differences in the mechanisms underlying the discriminative stimulus properties of clozapine and olanzapine. Specifically, olanzapine's discriminative stimulus properties appear to be meditated in part by both cholinergic and serotonergic mechanisms. PMID- 10755736 TI - Effects of the competitive nicotinic antagonist erysodine on behavior occasioned or maintained by nicotine: comparison with mecamylamine. AB - RATIONALE: The cellular effects of nicotine underlying its addictive liability are thought to be mediated by neuronal nicotinic receptors (nACHRs) in the central nervous system. It is believed that densely expressed beta32-containing nACHRs in the central nervous system are responsible for these actions, but few data are available that can directly assess subtype mediation of nicotine's acute subjective and reinforcing effects. OBJECTIVE: The present study compared the effects of the competitive nACHR antagonist erysodine and the noncompetitive antagonist mecamylamine in rats trained to discriminate or self-administer nicotine. METHODS: Adult male rats were trained to discriminate 0.4-mg/kg injections of nicotine from vehicle in a two-lever procedure of food-maintained behavior, or to self-administer 0.03-mg/kg injections of nicotine under fixed ratio 5 or progressive-ratio schedules of reinforcement. Additional rats were trained under a food-maintained procedure of lever pressing. RESULTS: Erysodine (0.3-10 mg/kg) and mecamylamine (0.1-1.0 mg/kg) blocked nicotine discrimination, although only erysodine produced the rightward shift that would be predicted of a competitive antagonist. Erysodine (0.32-32 mg/kg) and mecamylamine (0.32-3.2 mg/kg) also selectively reduced nicotine self-administration on a fixed-ratio schedule and lowered break points on a progressive-ratio schedule. CONCLUSIONS: Based on the known affinity of erysodine for alpha4beta2 nACHRs and its selectivity relative to alpha7 and alpha1beta1gammadelta receptors, the present data support a critical role of beta2-containing nACHR constructs in the discriminative and reinforcing actions of nicotine. PMID- 10755737 TI - The role of nicotinic and muscarinic acetylcholine receptors in attention. AB - RATIONALE: This study tried to determine the relative roles of muscarinic and nicotinic cholinergic receptors in attentional processing. METHODS: The effects of cholinoceptor agonists and antagonists, and of an anticholinesterase, were studied on performance of rats in a five-choice serial reaction time task. RESULTS: Scopolamine (0.1 mg/kg) and mecamylamine (5.0 mg/kg) produced deficits in accuracy and reaction time, respectively. This may suggest a differential role for the two types of cholinoceptors in information processing. Combinations of sub-threshold doses of scopolamine (0.01-0.03 mg/kg) and mecamylamine (0.5-1.6 mg/kg), which alone did not affect accuracy or reaction time, did not produce significant deficits in attention. However, the pattern of effects after combined treatment suggested that the differential deficits seen with these drugs alone remained. The anticholinesterase physostigmine (0.1 mg/kg) and the non-selective muscarinic agonist oxotremorine (0.03 mg/kg) induced severe behavioural disruption at doses that appeared to be relatively well tolerated in previous studies; this precluded the derivation of accuracy and response time data at these doses. At lower doses, neither physostigmine (0.05 mg/kg) nor oxotremorine (0.003 mg/kg) significantly affected any performance measure; this may reflect the ability of both drugs to indirectly or directly activate presynaptic muscarinic receptors that inhibit acetylcholine release, respectively. CONCLUSIONS: Both muscarinic and nicotinic cholinoceptors may be important in attention but they may serve different roles in information processing; this hypothesis could be tested using tasks that place different emphasis on different stages of information processing. PMID- 10755738 TI - Evaluation of anti-cocaine antibodies and a cocaine vaccine in a rat self administration model. AB - RATIONALE: Previous pre-clinical studies with an anti-cocaine monoclonal antibody left open several issues critical to assessing the effectiveness of a vaccine for altering cocaine self-administration behavior. OBJECTIVES: The objectives of this study were to determine, first, whether changes in self-administration behavior would be systematically related to antibody level and, second, how the antibody affected the self-administration of different doses of cocaine. METHODS: Two experiments were conducted using a second-order schedule of drug delivery in rats. The first was a passive-administration study using the anti-cocaine monoclonal antibody MO240 to examine the relationship between antibody level and cocaine self-administration behavior, and the second was an active-immunization study to examine the efficacy of the cocaine vaccine IPC-1010 for blocking various doses of self-administered cocaine. RESULTS: The passive-administration experiment with control and 4-mg or 12-mg MO240 treatments showed that antagonism of the 1 mg/kg cocaine training dose was dependent on antibody level. In animals whose serum antibody levels were sustained above 0.05 mg/ml, there was a sufficient amount of antibody to reduce drug-seeking behavior and drug intake. In the active-immunization experiment, the cocaine vaccine IPC-1010 induced average serum antibody levels of 0.08 mg/ml and reduced the reacquisition of behavior by 1 mg/kg cocaine. Antagonism of cocaine self-administration after immunization was evident across a range of doses of cocaine and was only apparent in animals whose serum antibody levels exceeded 0.05 mg/ml. Furthermore, there was no evidence that the antagonism was surmountable within the dose range examined (up to 5.6 mg/kg). CONCLUSIONS: Antagonism of cocaine self-administration across a range of doses is feasible after immunization with a cocaine vaccine as long as antibody levels are of a sufficient concentration. PMID- 10755739 TI - Acute opioid but not benzodiazepine dependence in rats responding for intracranial self-stimulation. AB - RATIONALE: Four-hour pretreatment with a single dose of morphine or related opioids sensitizes rats responding for intracranial self-stimulation (ICSS) to the rate-decreasing effect of naltrexone, indicative of antagonist-precipitated withdrawal from acute opioid dependence. OBJECTIVES: To determine whether sensitization to naltrexone could be observed in morphine-pretreated rats responding under a progressive ratio (PR) schedule of ICSS and to determine whether acute pretreatment with benzodiazepines produces similar sensitization to flumazenil. METHODS: Rats with an electrode in the medial forebrain bundle were trained to respond under an ICSS PR schedule, in which the number of responses required for a 250-ms stimulus started at one, then increased gradually. If no responding occurred for 30 s, the response requirement reverted to a single response and the break point was operationally defined. RESULTS: Pretreatment (4 h) with 3.0 mg/kg or 5.6 mg/kg morphine reduced the ED25 values of naltrexone for decreasing response rate from 18+/-6.7 mg/kg to 0.021+/-0.006 mg/kg and 0.006+/ 0.001 mg/kg, respectively. Changes in break point usually paralleled changes in response rate. In contrast, 4- to 24-h pretreatment with the benzodiazepines chlordiazepoxide (30 mg/kg and 100 mg/kg) or diazepam (3.0 mg/kg and 10 mg/kg), behaviorally-active doses, did not significantly alter sensitivity to the effects of flumazenil (1.0-30 mg/kg). CONCLUSIONS: These results show that PR ICSS provides a stable behavioral baseline for testing drugs in rats and extend to this procedure the generality of the phenomenon of acute opioid dependence. There was no comparable evidence of acute benzodiazepine dependence, suggesting that there are differences in the ways that opioid and benzodiazepine agonists initiate the adaptive changes that underlie the state of physical dependence. PMID- 10755740 TI - Reinforcing and subjective effects of morphine in human opioid abusers: effect of dose and alternative reinforcer. AB - RATIONALE: Although most opioid self-administration research has been conducted with laboratory animals, such research with humans is necessary to answer questions unique to human drug-taking behavior. OBJECTIVE: We investigated the influence of morphine dose and an alternative non-drug reinforcer on choice between morphine versus money and examined the relationship between drug reinforced behavior and subjective euphoria. METHODS: Five male opioid users participated in the 7-week study. During the first 5 weeks, a single dose of morphine (0, 4, 8, 16, or 32 mg/70 kg) was available each week. On Monday, subjects received an IM injection of the dose tested that week. On Tuesday, Thursday, and Friday, subjects could work for morphine or money under a second order, progressive ratio schedule. For each primary ratio completed on the drug lever, subjects earned one-ninth of the available drug dose, and for each ratio completed on the money lever, subjects earned $1. Total amount of drug earned was administered in a single IM injection at the end of the session; money earned was credited to the subject's account. RESULTS: As morphine dose increased, responding for drug increased in an orderly manner and responding for money decreased. During the final phase of the study, the lowest and highest doses that maintained drug responding for each subject were repeated, and the value of the alternative reinforcer was increased to $2 per ratio. This manipulation was associated with decreased drug-maintained responding at the lowest, but not the highest, reinforcing dose of morphine. CONCLUSION: The progressive ratio, concurrent access procedure may be useful in predicting the outcome of drug abuse treatment interventions that use alternate reinforcement strategies. PMID- 10755741 TI - Phencyclidine-induced abnormal behaviors in rats as measured by the hole board apparatus. AB - RATIONALE: Phencyclidine (PCP) and methamphetamine (MAP) are known as psychotomimetic agents. Both agents produce behavioral alterations in animals. OBJECTIVE: The present study investigated the difference in behavioral alterations in rats induced by these two psychotomimetic agents using the hole board apparatus (HBA). In addition, mechanisms underlying PCP-induced behavioral changes were also investigated. METHODS: After the administration of PCP (1-4 mg/kg SC) or MAP (1-4 mg/kg SC), locomotor activity and dipping behavior were assessed using HBA. Effect of selective NMDA antagonists, (+)MK801 and 3-((+/-)-2 carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), on rat behaviors were also assessed. The effects of D-alanine (D-Ala), a coagonist of NMDA receptors, or neuroleptics, haloperidol, clozapine and risperidone, on PCP-induced behavioral changes were investigated. RESULTS: PCP increased locomotor activity and decreased exploratory behaviors of rats in HBA. On the other hand, MAP increased locomotor activity but did not decrease exploratory behaviors. (+)MK-801 produced hyperactivity as well as decreased exploratory behaviors, eliciting behavioral changes very similar to those of PCP. CPP decreased the exploratory behavior but failed to produce hyperactivity. D-Ala attenuated both behavioral changes induced by PCP. Three neuroleptics tested here inhibited hyperactivity but did not attenuate decreases in exploratory behavior. CONCLUSION: These results suggest that PCP-induced decrease in exploratory behavior are attributable to antagonism of NMDA receptors and may not involve dopaminergic transmission via D2 receptors. PMID- 10755742 TI - Cocaine self-administration "binges": transition from behavioral and autonomic regulation toward homeostatic dysregulation in rats. AB - BACKGROUND: An essential feature of cocaine addiction is the breakdown to control or regulate drug intake. OBJECTIVE: The present studies aimed to examine the transition from regulated intravenous cocaine reinforcement to a more unpredictable, chaotic pattern of cocaine self-administration in rats that were given continuous access to the drug. Autonomic activity was continuously monitored via biotelemetry senders for heart-rate and core temperature before, during and after the cocaine "binges", in an attempt to characterize the breakdown of homeostatic regulation. METHODS: After Long-Evans rats were fitted with intravenous catheters and intraperitoneal telemetry senders, they acquired cocaine self-administration with each fifth lever press being reinforced by a 0.25 mg cocaine infusion. Rats self-administered 15 cocaine infusions daily at stable rates for ca. 2-3 weeks, when continuous access periods ("binges") of 26 and 72 h were scheduled, with a 3-week cocaine-free period between and following the two "binges". RESULTS: A distinctive pattern of cocaine self-administration emerged during the "binges" that consisted of (1) an initial loading phase, (2) stable, predictable inter-infusion intervals for up to 8-10 h, termed "regulatory phase", (3) increased variability in inter-fusion intervals, mostly beginning at 22-24 h of continuous access. During the first half of the 72-h "binge", the autonomic activities remained elevated, showed a greatly constrained variability, and the characteristic circadian rhythmicity was substantially decreased. The average cocaine intake (6.8+/-0.5 mg/kg per hour) during the "regulatory" phase did not change during the subsequent phases. Following the 72-h "binge", the amplitude of autonomic circadian rhythms remained attenuated for more than 2 weeks. In a separate set of animals, the dose effect of inter-infusion intervals following the self-administered infusion was similar during a variable dose protocol, scheduled in an early and a late phase of a 30-h long "binge". CONCLUSIONS: The homeostatic dysregulation during the "binge" as evidenced by the diminished capacity of autonomic functions to vary is accompanied by emerging irregularities in the pattern of cocaine self-administration. PMID- 10755743 TI - Cocaine-induced changes in extracellular dopamine determined by microdialysis in awake squirrel monkeys. AB - RATIONALE: The behavioral effects of cocaine have been linked to brain dopamine systems. Extending the findings to neurochemical studies in the squirrel monkey would enhance our understanding of the behavioral pharmacology of cocaine in nonhuman primates. OBJECTIVES: The present studies characterized the effects of cocaine and the selective dopamine uptake inhibitor GBR 12909 on extracellular dopamine in the caudate nucleus of awake squirrel monkeys through microdialysis experiments. METHODS: Guide cannulae were implanted in the caudate nucleus of four monkeys using a stereotaxic apparatus and coordinates obtained from a standard squirrel monkey brain atlas. Accurate probe placement was confirmed in all subjects with magnetic resonance imaging. RESULTS: Collectively, the results support the feasibility of a repeated-measures design. Stability of tissue integrity after repeated probe insertion was supported by measurement of consistent basal levels of dopamine and its metabolites across several experiments, observation of potassium-induced dopamine release and absence of significant glial proliferation as assessed by GFAP (glial fibrillary acidic protein) immunochemistry. Moreover, peak drug effects and time-course of action were similar when multiple probes were positioned in the same anatomical site over several experiments. Cocaine (1.0 mg/kg i.m.) and GBR 12909 (3.0 mg/kg i.m.) elevated extracellular dopamine to approximately 300% of basal levels, but GBR 12909 produced a slower, more sustained elevation than cocaine. CONCLUSIONS: The results validate the use of microdialysis in awake primates using repeated sampling of the same anatomical site and demonstrate orderly changes in extracellular dopamine following administration of dopamine uptake inhibitors. PMID- 10755744 TI - Amphetamine-induced activation of forebrain EEG is prevented by noradrenergic beta-receptor blockade in the halothane-anesthetized rat. AB - RATIONALE: Amphetamine (AMPH)-like stimulants represent an intensively studied class of psychoactive drugs. Despite the well-known and potent arousal-enhancing effects of these drugs, the neurobiological substrates of AMPH-induced arousal have rarely been examined explicitly. Activity of the locus coeruleus noradrenergic system is causally and positively related to behavioral and electroencephalographic (EEG) indices of arousal. For example, activation of locus coeruleus neurons or stimulation of medial basal forebrain noradrenergic beta-receptors elicits activation of forebrain EEG in the anesthetized rat. Further, stimulation of noradrenergic beta-receptors within the medial basal forebrain elicits a substantial increase in alert, active waking. These and other observations suggest that at least some of the arousal-enhancing actions of AMPH like stimulants derive from AMPH-induced increases in noradrenergic neurotransmission at beta-receptors. The current study examines the extent to which AMPH-induced activation of cortical EEG is dependent on actions of central beta-receptors. METHODS: The effects of intracerebroventricular (ICV; 2 microl) pretreatment with either vehicle (artificial extracellular fluid) or the beta antagonist, timolol (25, 50 or 100 microg), on the cortical EEG activating effects of intravenous AMPH (0.15 mg/kg) were examined in the halothane anesthetized rat. EEG was recorded on polygraph and video recording tape and later analyzed using power spectral analyses (PSA). AMPH-induced alteration in cortical EEG activity was measured using PSA in vehicle- and timolol-pretreated animals. RESULTS: Neither vehicle nor timolol ICV infusions altered cortical EEG activity patterns. In vehicle-pretreated animals, AMPH elicited a robust activation of cortical EEG, characterized by the substantial decrease in large amplitude, slow-wave activity. Timolol pretreatment dose-dependently prevented AMPH-induced cortical EEG activation. This effect of timolol was statistically significant at the 50 microg and 100 microg dose. CONCLUSIONS: These observations indicate that, under these experimental conditions, AMPH-induced activation of the forebrain is dependent on actions of noradrenergic beta-receptors. Combined with previous observations, these observations support the hypothesis that AMPH induced increases in arousal involve noradrenergic neurotransmission at beta receptors. PMID- 10755745 TI - Effect of baclofen on cocaine self-administration in rats reinforced under fixed ratio 1 and progressive-ratio schedules. AB - RATIONALE: Recent reports have indicated that the gamma-aminobutyric acid (GABA)B agonist baclofen attenuates the reinforcing effects of cocaine. OBJECTIVES: To further evaluate the effect of baclofen on cocaine self-administration under a fixed ratio (FR) and progressive ratio (PR) schedule of reinforcement. METHODS: In the first series of experiments, three dose-response curves were generated that examined the effect of three doses of baclofen (1.8, 3.2, or 5.6 mg/kg, i.p.) against four unit-injection doses of cocaine (0.19, 0.38, 0.75, and 1.5 mg/kg per injection) reinforced under a FRI schedule. For comparison, an additional group of rats was pretreated with haloperidol (32, 56, or 100 microg/kg, i.p.). A separate experiment examined the effect of baclofen (1.8, 3.2, or 5.6 mg/kg, i.p.) on responding for concurrently available cocaine or food reinforcement. RESULTS: Under the FR1 schedule, baclofen suppressed intake of low but not high unit injection doses of cocaine. In contrast to haloperidol, baclofen had no effect on the distribution of inter-injection intervals and, instead, produced long pauses in cocaine self-administration. Baclofen dose dependently reduced cocaine-reinforced responding on a PR schedule; concurrent access to a food-reinforced lever demonstrated that the animals retained the capacity to respond at high rates. CONCLUSION: The effect of baclofen pretreatment on cocaine self-administration is dependent on the unit injection dose of cocaine and on the response requirements of the schedule. PMID- 10755746 TI - Memory disturbances in "Ecstasy" users are correlated with an altered brain serotonin neurotransmission. AB - RATIONALE: Methylenedioxymethamphetamine (MDMA) is known to damage brain pre synaptic serotonin (5-HT) neurons. Since loss of 5-HT neurons has been implicated in memory loss, it is important to establish whether MDMA use may produce changes in postsynaptic 5-HT receptors and memory function in humans. OBJECTIVES: To investigate whether MDMA use leads to compensative alterations in post-synaptic 5 HT2A receptors and whether there is a relation with memory disturbances. METHODS: Brain cortical 5-HT2A receptor densities were studied with [123I]-5-I-R91150 SPECT in five abstinent MDMA users and nine healthy controls. Memory performance was assessed using RAVLT. RESULTS: [123I]-5-I-R91150 binding ratios were significantly higher in the occipital cortex of MDMA users than in controls, indicating up-regulation. Mean cortical 5-HT2A receptor binding correlated positively with RAVLT-recall in MDMA users. CONCLUSION: Our preliminary results may indicate altered 5-HT neuronal function with correlated memory impairment in abstinent MDMA users. PMID- 10755747 TI - Does mirtazapine enhance serotonergic neurotransmission in depressed patients? PMID- 10755748 TI - Imaging findings of hemorrhagic cystitis in pediatric oncology patients. AB - In pediatric oncology patients, hemorrhagic cystitis may be a life-threatening complication of bone-marrow transplantation, chemotherapy, and/or radiation therapy. The inciting agent in urine can affect the entire urothelium from the renal collecting system to the bladder, and the severity of disease can vary. The radiologist often plays a key role in the diagnosis, follow-up, and occasionally the treatment of hemorrhagic cystitis and its complications. This review discusses the imaging findings in the kidneys and bladder in patients with hemorrhagic cystitis both before and after treatment for this disease. Findings on two-dimensional sonography, color Doppler and power Doppler sonography, computed tomography, magnetic resonance imaging, antegrade pyleography, and cystography are presented. PMID- 10755749 TI - Single-system ureteroceles in infants and children: imaging features. AB - PURPOSE: The purpose of this manuscript is to describe the clinical and imaging findings in children who have single-system ureteroceles. MATERIALS AND METHODS: We reviewed the urology records and imaging studies in 32 consecutive infants and children who were diagnosed in our department with single-system ureteroceles. RESULTS: There were 35 ureteroceles in the 32 patients-- 29 were unilateral (14 right-sided, 15 left-sided) and 3 were bilateral. Twenty-five patients were boys (78 %) and 7 girls. Mean age at presentation was 0.7 years (0-9.2 years). Prenatally detected hydronephrosis or cystic renal dysplasia was the most common presentation (24 patients). Four presented with urinary infection, 2 with abdominal mass, 1 had myelomeningocele, and 1 had hypospadias. Three patients also had multiple non-urologic, congenital anomalies. Thirty-three ureteroceles were intravesical, and 2 were ectopic to the bladder neck. Twenty-four ureteroceles were associated with ipsilateral hydroureteronephrosis and 10 with ipsilateral multicystic dysplastic kidney. One patient had a normal ipsilateral kidney and a contralateral multicystic dysplastic kidney. The ureterocele was identified on at least one imaging study in each patient. Sixteen ureteroceles (47%) everted at VCUG, mimicking paraureteral diverticula. Other variations included ureterocele prolapse and inadvertent ureterocele catheterization (1 each). CONCLUSIONS: Single-system ureterocele is an important, although uncommon cause of hydronephrosis and renal dysplasia in infants and children. Single system ureterocele is distinguished clinically from the more common duplex-system ureterocele by its frequent occurrence in boys and its association with multicystic dysplastic kidney. Because these ureteroceles are frequently small and have a propensity to evert at VCUG, they can be mistaken for paraureteral diverticula. PMID- 10755750 TI - Mesoblastic nephroma: prenatal ultrasonographic and MRI features. AB - Antenatal detection of mesoblastic nephroma by US is possible. Reviewing the literature, we found 19 previously reported cases, only 1 of which underwent prenatal MRI. We present a further case diagnosed by US and confirmed with MRI. The imaging findings and differential diagnoses are discussed. Early and correct detection of this rare entity is of great interest, as it may facilitate prevention and management of severe obstetric and neonatal complications such as polyhydramnios and prematurity. MRI can help to evaluate the origin and the morphological features of a fetal abdominal mass. PMID- 10755751 TI - Ultrasonographic late results after surgically treated cryptorchidism. AB - BACKGROUND: Because uncorrected cryptorchidism is accompanied by the high risk of later disturbed testicular function and cancer, early surgery in the second year of life is recommended. OBJECTIVE: To evaluate testicular morphology, the sonomorphologic testicular long-term outcome and additional complications, as well as possible differences depending on varying ages at surgery. MATERIALS AND METHODS: Seventy-five previously maldescended testes in 68 boys were studied with US, 2-11 years after intrascrotal orchidopexy. Nineteen had been operated on in the first or second year of life, while the other 49 boys underwent surgery at a later age (up to 7 years). Each examination utilised conventional B-mode and colour Doppler (7-MHz linear ART probe, Acuson XP 128) to examine the scrotal and inguinal regions on both sides; testicular volume and perfusion were assessed. Ultrasound changes in testicular volume, architecture and Doppler flow rates were regarded as the most valid indirect indices of testicular damage. Histopathological correlation was not obtained, for ethical reasons, in any of the probands. RESULTS: Thirty-five of the surgically fixed testes were normal with regard to position, volume, structure and perfusion. The other 40 (53%) showed abnormalities of one or more of these parameters without any correlation with the patient's age at surgery or the time interval between surgery and US. Additional relevant findings, which were also found on the non-operated side, were: microlithiasis (n = 6), inguinally retained testis (n = 6), hydrocoele (n = 5), hydatid (n = 5) and varicocoele (n = 1). CONCLUSIONS: Ultrasound, including colour Doppler, enables an exact morphological analysis of the late results after surgically corrected cryptorchidism. The spectrum of findings does not show any correlation with the time of surgery. Thus, the value of even early surgery has to be questioned. Pre-existent primary damage (dysplasia) seems more important for long-term outcome of the testis. Additionally, US was of high value in demonstrating additional unexpected anomalies, the majority of which needed sonographic follow-up or even surgery. PMID- 10755752 TI - Appearance of autosomal recessive polycystic kidney disease in magnetic resonance imaging and RARE-MR-urography. AB - PURPOSE: To describe the appearance of autosomal recessive polycystic kidney disease (ARPKD) on MRI and RARE-MR urography. MATERIALS AND METHODS: Seven boys and one girl (aged 3 months to 14 years, median 2.5 years) were evaluated. Images were obtained with 0.23-T and 1.5-T MR systems using T1-weighted (T1-W) spin echo, T2-weighted (T2-W) turbo-spin-echo and RARE-MR-urography sequences. Signal intensities, morphological appearance of the affected kidneys and, specifically, the picture of the urinary tract on RARE-MR-urography were evaluated. RESULTS: All children showed kidney enlargement, reniform but humpy kidney shape, homogeneously grainy renal parenchyma, normal renal pelvis and normal calyces. Signal intensity was hyperintense in T2-W images in all cases. In six cases (n = 7), T1-W images were hypointense. On RARE-MR urography a hyperintense, linear radial pattern was seen in the cortex and medulla which represents the characteristic microcystic dilatation of collecting ducts in ARPKD. Three boys and the girl presented with a few circumscribed small subcapsular cysts. CONCLUSIONS: In order to confirm the diagnosis of ARPKD, RARE-MR urography seems to be a non-invasive imaging tool that shows directly the microcystic dilated water-filled collecting ducts. PMID- 10755753 TI - Regression of multiple hepatic masses in a young patient with thalassaemia. AB - BACKGROUND: We report a young girl with thalassaemia who showed development and regression of multiple hepatic masses. RESULTS: The tumours, detected 3 years after allogeneic bone-marrow transplantation, showed progressive reduction in size and number following a phlebotomy program to treat iron overload. CONCLUSION: The detailed CT, MRI and histological findings are described. PMID- 10755754 TI - Small intestinal obstruction by remnants of the omphalomesenteric duct: findings on contrast enema. AB - OBJECTIVE: We reviewed the contrast enema examinations and medical records of six patients with small intestinal obstruction due to omphalomesenteric duct remnant to evaluate for characteristic imaging findings. RESULTS: In five out of the six patients the point of obstruction was demonstrated on the enema; in three patients, the characteristic "beak" of a volvulus was seen, either in the terminal ileum or cecum. In three patients, there was medial deviation of the cecum. CONCLUSION: The characteristic radiographic features of volvulus at the cecum or terminal ileum and medial deviation of the cecum should suggest persistence of an omphalomesenteric duct remnant as the etiology of obstruction in a child less than 2 years of age. The appearance of omphalomesenteric duct remnant obstruction on enema examination, though not specific, is characteristic and should be familiar to pediatric radiologists. PMID- 10755755 TI - Acute hemoperitoneum in children: prevalence of low-attenuation fluid. AB - BACKGROUND: Recent evidence indicates that acute hemoperitoneum may have lower than expected attenuation values at CT. OBJECTIVE: To characterize the attenuation of acute hemoperitoneum at CT in children following blunt abdominal trauma and to assess the prevalence of low-attenuation fluid. MATERIALS AND METHODS: The CT scans of 19 consecutive children with isolated hepatic or splenic injury and associated peritoneal fluid were retrospectively analyzed. The attenuation value of peritoneal fluid was assessed in all peritoneal spaces. RESULTS: Fluid was noted in 53 peritoneal spaces (27 abdominal, 26 pelvic). Fluid attenuation ranged from 20 to 64 HU. The mean fluid attenuation in pelvic spaces (37.5 +/- 9.4 HU) was significantly lower than in abdominal spaces (444.9 +/- 10.2 HU) (P = 0.008). Fluid in 8/26 (31 %) pelvic spaces and 2/27 (7 %) abdominal spaces had attenuation values < or = 30 HU. Fluid surrounding the site of injury (perihepatic or perisplenic space) was significantly higher in attenuation than fluid at other sites (P < 0.001). There was no correlation between the mean attenuation value of peritoneal fluid in each patient and the admission hematocrit (r = -0.14, P = 0.55). CONCLUSIONS: There is great variability in the attenuation of acute hemoperitoneum. Blood in pelvic spaces has significantly lower attenuation than blood in abdominal spaces. Hemoperitoneum in the pelvis has values of < or = 30 HU in approximately one-third of spaces. The attenuation of acute hemoperitoneum does not correlate with hematocrit. PMID- 10755756 TI - Proton MR spectroscopy in connatal Pelizaeus-Merzbacher disease. AB - BACKGROUND: Pelizaeus-Merzbacher disease (PMD) is a rare dysmyelinating disorder characterised by early pendular nystagmus, often rotatory and muscular hypotonia with subsequent ataxia, spasticity and mental retardation. Various point mutations or duplications in the PLP gene on the X chromosome are responsible for PMD in the majority of patients. Autosomal recessive inheritance, particularly in the connatal form, cannot be excluded. Three different forms of the disease have been identified based on their onset, progression and severity of myelin pathology indicated by MRI features. Objective. To determine if MR spectroscopy is useful in the diagnosis of the connatal form of PMD. MATERIALS AND METHODS: Proton MR spectroscopy was performed on two children with connatal PMD. RESULTS: Our patients showed a markedly decreased peak of Cho. This alteration is well represented by quantitative analysis of the NAA-to-Cho ratio, which is the most important ratio affected. A significant decrease of the Cho-to-Cr ratio is also present. In the connatal form of PMD, global lack of myelination may be relevant, as demonstrated by a significant Cho peak reduction. CONCLUSIONS: Proton MR spectroscopy may be of diagnostic value in metabolic and destructive disorders of the brain. A greater number of patients with connatal PMD is needed in order to elucidate the significance of reduction of the Cho peak. PMID- 10755757 TI - Malignant peripheral nerve-sheath tumor arising in a previously irradiated neuroblastoma: report of 2 cases and a review of the literature. AB - BACKGROUND: Only ten cases of the rare occurrence of a malignant peripheral nerve sheath tumor (MPNST) arising in a ganglioneuroma either de novo or at a site of previous irradiation have been reported. PATIENTS AND METHODS: We present two children who at the age of 19 months and 6 months were diagnosed with a cervicothoracic ganglioneuroblastoma and a retroperitoneal neuroblastoma, respectively. They both received radiation therapy as part of the treatment of their disease. RESULTS: Following a 12-year interval, MPNST arose inside a benign ganglioneuroma in both patients. We illustrate the imaging findings in these two cases and review the cases reported in the literature to increase awareness of this association among radiologists. PMID- 10755759 TI - A variant of Reinhardt-Pfeiffer mesomelic skeletal dysplasia. AB - Mesomelic bone dysplasias are characterised by disproportionate shortness of the middle segment of the extremities and short stature. Various patterns of this dysplasia have been described in the literature. The type involving mainly the ulna and fibula was described by Reinhardt and Pfeiffer as the ulno-fibular mesomelic dysplasia affecting the distal ulna and proximal fibula. Involvement of the distal fibula in mesomelic dysplasia has not been described to our knowledge. We report here a case of mesomelic dysplasia affecting mainly the distal fibula and distal ulna, with severe ankle deformity requiring surgical correction. PMID- 10755758 TI - Synovial osteochondromatosis of the sternoclavicular joint. AB - A 9-year-old boy with a rare localisation of synovial osteochondromatosis at the left sternoclavicular joint is presented. Synovial osteochondromatosis at this site and at this age has not been previously reported. Joint loose bodies were removed surgically and partial synovectomy was performed. Follow-up showed no evidence of recurrence over a period of 8 years. PMID- 10755760 TI - Is the open-mouth odontoid view necessary in children under 5 years? AB - OBJECTIVES: Questionnaires (984) were submitted to pediatric radiologists to determine how often odontoid fractures were missed on lateral views and detected on open-mouth odontoid views in children under 5 years. Other questions relating to imaging protocols of the odontoid also were included, and there were 432 respondents. RESULTS: Of these, 161 respondents indicated that an open-mouth odontoid view was not routinely included in their imaging protocol. CONCLUSIONS: There was considerable variability on how the problem of imaging of the odontoid was addressed, but overall there were enough data to begin to consider the concept that the open-mouth odontoid might not be needed in patients under 5 years of age if a lateral view is normal. PMID- 10755761 TI - Reference values for radiological evaluation of cervical vertebral body shape and spinal canal. AB - BACKGROUND: Defining normal values is essential for reliable evaluation of growth disturbances. Previous studies of the cervical spine have mainly focused on the sagittal canal diameter and interpedicular distances. Values for vertebral body height and depth have been published only in adult men and cadavers. OBJECTIVES: To define normal values for vertebral body height (H)/vertebral body depth (D) ratio (H/D ratio) and sagittal canal diameter (S)/ vertebral body depth ratio (S/D ratio) in C2-7. MATERIALS AND METHODS: Lateral cervical spine radiographs were available from 441 children and 192 adults. Subjects' ages varied from newborn to 39 years. Vertebral body height and depth and sagittal canal diameter were measured and ratios were calculated. This was a cross-sectional and retrospective study. RESULTS: Vertebral bodies grow relatively more in height than in depth, most actively at puberty. At all levels, the H/D ratio remains below 1, indicating that vertebral body depth is greater than height. The SD ratio is quite stable until 7-8 years of age and then it starts to decline slowly. CONCLUSIONS: When estimating platyspondyly, the age of the patient must be taken into consideration because vertebral body height is lower in children. Growth of the spinal canal declines after 7-8 years of age. PMID- 10755763 TI - Tracheal agenesis: evaluation by helical computed tomography. AB - We present a newborn infant with type II tracheal agenesis. Helical CT was performed and confirmed the diagnosis. Retrospective coronal and sagittal multiplanar reconstructions clearly demonstrated the complex anatomy of the malformation. The absence of a significant portion of healthy trachea did not present any possibility for surgical correction. PMID- 10755762 TI - Unusual findings in two cases of Langerhans' cell histiocytosis. AB - We report unusual findings in two patients with Langerhans' cell histiocytosis (LCH), namely a cervical mass lesion with extensive destruction of the posterior elements of a cervical vertebra and gastrointestinal lesions as part of multisystem involvement. The aim of our report is threefold. Firstly, LCH can be responsible for osteolysis of the vertebral posterior arches, with or without involvement of the vertebral body, and should be included in the differential diagnoses of cervical mass lesions. Secondly, in a patient with confirmed LCH and additional protein-losing enteropathy, gastrointestinal involvement should be considered as a possibility since it is an important factor for establishing prognosis. Thirdly, LCH lesions can be very extensive and yet have a good response to therapy, whereas less spectacular lesions may not respond or respond only partially to therapy. Thus, an important factor in establishing prognosis is the presence of multisystem involvement at diagnosis, regardless of the extent of a lesion at a particular site. PMID- 10755764 TI - Treatment of pentobarbitol sodium (Nembutal) hyperactivity: a new approach. PMID- 10755765 TI - Asphyxia, cerebral palsy and litigation. PMID- 10755766 TI - Maternal work and pregnancy. PMID- 10755767 TI - Maternal height as a risk factor for Caesarean section due to failure to progress in labour. AB - We examined for a regional sample of the New Zealand population, the relationship between maternal height and an increased risk of emergency Caesarean section due to arrested labour, to identify a height below which the risk of Caesarean section increases markedly and to quantify the risk of a Caesarean section for a range of maternal heights. The data of nulliparous singleton pregnancies over the period 1994-1998 was sorted into 2 study groups, one resulting in emergency Caesarean section for arrested labour and the other a group of women who had normal vaginal delivery requiring no intervention. The means and standard deviations of these 2 groups were found and 99% confidence intervals calculated. They were analysed for statistical difference and then a logistical regression calculation tried to identify a height at which the risk of a Caesarean section increased suddenly. There were 81 women in the Caesarean section group and 997 in the normal vaginal delivery group. Mean heights and confidence intervals were 161.0 cm (158.9-163.1) and 164.6 cm (164.0-165.2) respectively. There was a statistically significant difference between these means (p<0.001) but logistic regression analysis showed that risk of Caesarean section increased gradually with decreasing height, and even then did not reach more than 30% risk until a height of less than 140 cm. Low maternal height was associated with increased risk of Caesarean section due to labour arrest. Because the likelihood of having a normal vaginal delivery was still very good (>80 %) at modest degrees of short stature, this risk factor alone is unlikely to affect management. However the combination of other risk factors with maternal height may be of clinical use. PMID- 10755768 TI - Emergency cervical cerclage. Is there a role for amnioreduction? PMID- 10755769 TI - Measurement of free thyroxine (T4) levels in pregnancy. AB - Free T4 and TSH were measured in 119 pregnant women. Twenty of these women had the free T4 measured a second time during the pregnancy; 35 of the 119 had the free T4 measured by 2 different methods. The results showed that free T4 fell as pregnancy progressed but even at the early stages of pregnancy many measured free T4 concentrations were below the lower end of the nonpregnant normal range. This remained the case when the measurement was made by a dialysis kit method. These results challenge the current widely-accepted statement that free T4 concentrations remain within the normal nonpregnant range during pregnancy. This study highlights the need for each laboratory to develop its own normal range for free T4 levels in pregnancy. PMID- 10755770 TI - A trial of simple versus intensified dietary modification for prevention of progression to diabetes mellitus in women with impaired glucose tolerance. AB - Women with impaired glucose tolerance are at high risk of developing noninsulin dependent diabetes mellitus (NIDDM). The Mercy Hospital for Women has a long-term follow-up programme for women with gestational diabetes, which identifies many women with impaired glucose tolerance. Two hundred of these women were entered into a randomized controlled trial of intensive versus routine dietary advice. Seven women were lost to follow-up. The annual incidence rates of diabetes mellitus for the 2 groups were 6.1% (intervention) and 7.3% (control), an incident rate ratio of 0.83, 95% confidence interval 0.47-1.48, p = 0.50. Overall, there was a return to normal glucose tolerance in 44% of patients. Multivariate analysis showed that body mass index, fasting and 2-hour plasma glucose levels at trial entry were significantly associated with an increased risk of diabetes mellitus. Impaired glucose tolerance is an important condition that should be treated with advice about lifestyle modification (diet and/or exercise). We consider that future trials in the management of women with previous gestational diabetes who have impaired glucose tolerance should investigate the effect of pharmacological intervention in addition to diet and/or exercise, the latter providing a therapy that it would be unethical to exclude on the evidence presently available. PMID- 10755771 TI - Eating disorders and reproduction. AB - Eating disorders are common and characteristically affect young women at what would otherwise be their peak of reproductive functioning. Anorexia nervosa and bulimia nervosa impinge on reproduction both behaviourally and physiologically, with effects on menstruation, ovarian function, fertility, sexuality and pregnancy. This review presents a summary of current knowledge and makes suggestions for future research, along with some clinical recommendations for the management of eating disorders in pregnancy. PMID- 10755772 TI - Adnexal torsion. AB - We performed a retrospective audit of 24 cases of adnexal torsion managed at National Women's Hospital from 1996 to 1997 inclusive. There have been several reports in the recent literature of ovarian conservation in cases of ovarian infarction secondary to torsion in women desiring further fertility (1-3). In our review 50% (12 of 24) of patients were treated by oophorectomy or salpingo oophorectomy and most of these women desired further fertility. In spite of torsion being suspected in 73% (17 of 24), the time interval between admission and operation varied from 0.5 to 52 hours with a mean of 8 hours; 46% (11 of 24) were treated laparoscopically without recourse to laparotomy. As expertise increases, we consider that adnexal torsion will be increasingly managed laparoscopically. These results suggest that more urgent surgery should be scheduled if ovarian torsion is suspected, and in view of recent reports in the literature greater consideration should be given to conservation of infarcted ovaries if further fertility is required. PMID- 10755773 TI - Incidence and management of gynaecological-related ureteric injuries. AB - We report a 5-year review of all ureteric injuries at a major Sydney teaching hospital as a result of gynecological procedures. A retrospective analysis was made of all hospital medical records and consultant follow-up notes from January,1990 to May, 1995. Injuries were recorded in 22 patients with a mean age 52 years (range 31-88). Of these, 17 occurred at our institution, while 5 were referred from peripheral hospitals. Two injuries were bilateral, and 6 were discovered intraoperatively. Patients were managed acutely where possible. Treatment options included cystoscopy and retrograde stenting, deligation, neoureterocystostomy, transureteroureterostomy or nephrectomy. Follow-up imaging was available in 16 patients, of those, 15 were normal, and 1 showed bilateral obstruction secondary to radiotherapy. The overall risk of ureteric injury for all methods of hysterectomy was 0.44% (0.24% for total abdominal hysterectomy). PMID- 10755774 TI - Laparoscopically-assisted vaginal hysterectomy for fibroid uteri weighing at least 500 grammes. AB - We retrospectively reviewed the medical records of 13 women who underwent laparoscopically-assisted vaginal hysterectomy (LAVH) where the uterus weighed 500 g or more. LAVH was successfully performed in 10 of these 13 women for whom the mean uterine weight was 619 g, mean operating time 99 minutes, and mean postoperative hospital stay 3.7 days. One of the 3 women who underwent abdominal hysterectomy required blood transfusion for intraoperative bleeding. There was no febrile or other operative morbidity associated with any patient. As up to 75% of hysterectomies are performed abdominally, LAVH may replace many abdominal hysterectomies for large fibroid uteri when vaginal hysterectomy is not feasible. PMID- 10755775 TI - Tubal ectopic pregnancy: an evaluation of laparoscopic surgery versus laparotomy in 614 patients. AB - We performed a prospective nonrandomized multicentre study to compare laparoscopic surgery and laparotomy in the immediate surgical outcome of tubal ectopic pregnancy (TEP), at 9 teaching hospitals in Hong Kong with a laparoscopic surgical service, on all patients with the operative diagnosis of tubal ectopic pregnancy between July 1, 1996 and June 30, 1997. In the period studied, 630 patients were recruited of which 614 were suitable for analysis. In them, 382 (62.2%) had laparoscopic surgery while the rest had laparotomy with or without diagnostic laparoscopy. Significantly more cases of shock ended in laparotomy (86.1% versus 13.9%). After exclusion of patients with shock, laparoscopic surgery offered a significantly shorter postoperative hospital stay (mean 2.7 days versus 5.3 days), a slightly lower perioperative complication rate (8.1% versus 13.9%) and more conservative surgery (90.1% of all salpingotomies) than laparotomy. A longer operating time was needed for laparoscopic surgery (1.2 hours versus 1.01 hours). PMID- 10755776 TI - A case report and review of pregnancies in rudimentary noncommunicating uterine horns. AB - A liveborn and subsequently thriving child was delivered abdominally from a pregnancy in a rudimentary noncommunicating uterine horn. This is the 13th English language report of neonatal survival from such an unusual and life threatening situation. The case presents some 'clues' in the history and a review of the literature suggests that the previously reported bleak outlook for these pregnancies can now be tempered with cautious optimism. Ultrasound diagnosis of Mullerian abnormalities during pregnancy remains difficult but has been reported in several other cases in the literature and may become more common in the future. PMID- 10755777 TI - Catheterization prior to evacuation of uterus and diagnostic curettage. AB - Dilatation and curettage and evacuation of the uterus are the 2 most common minor gynaecological procedures that trainees perform. Some trainees are taught to catheterize the patient before these procedures. This study was carried out with 2 aims, firstly to establish the prevalence of significant residual urine volume prior to these procedures and secondly to gain information on the practices among members and fellows of the RNZCOG residing in New Zealand with regard to catheterization prior to these operations. The measured residual urine volume was greater than 50 mL in 37.5% of patients and greater than 100 mL in 12.1%. About 92% of those surveyed did not catheterize prior to either procedure. The risk of infection does not appear to be a valid argument for not catheterizing. Routine urinary catheterization before these procedures is unnecessary. PMID- 10755778 TI - Treatment of acute myocardial infarction in pregnancy with coronary artery balloon angioplasty and stenting. AB - Acute myocardial infarction in pregnancy is a rare condition with substantial risk of maternal and fetal mortality. We present a case of myocardial infarction during pregnancy which was treated by percutaneous coronary artery balloon angioplasty and stenting with excellent pregnancy outcome. PMID- 10755779 TI - Influence of seasonal variation on pregnancy-induced hypertension and/or preeclampsia. AB - Seasonal variation of temperature and humidity are said to influence the incidence of hypertensive disorders of pregnancy. The aim of this study was to determine if temperature and humidity exert any influence on the incidence of pregnancy-induced hypertension and preeclampsia in Kuwait. This is a retrospective study performed in the Maternity Hospital, Kuwait (MHK) in the years 1992-1994. Monthly distribution of all deliveries and those in which the patients had pregnancy-induced hypertension and preeclampsia were recorded. There are some studies including ours which do not show any significant correlation between meteorological factors and hypertensive disorders of pregnancy. We found that the incidence of pregnancy-induced hypertension per 1,000 deliveries was high in June when the temperature was very high and the humidity at its lowest. The reverse was true for the incidence of preeclampsia per 1,000 deliveries, which was high in November when the temperature was low and the humidity high. PMID- 10755780 TI - 'White coat hypertension' in pregnancy. PMID- 10755781 TI - Phaeochromocytoma in pregnancy. AB - The diagnosis of phaeochromocytoma during pregnancy requires a high index of suspicion and is essential for the favourable outcome of mother and offspring. Delayed diagnosis is associated with considerable morbidity and mortality. The cases of 4 patients presenting with phaeochromocytoma during pregnancy over an 11 year period are presented. PMID- 10755782 TI - Patient preference the leading indication for elective Caesarean section in public patients--results of a 2-year prospective audit in a teaching hospital. AB - In response to a Western Australian Ministerial enquiry into the levels of intervention in childbirth, a 2-year prospective audit was undertaken of the indication and timing of all public Caesarean section deliveries at King Edward Memorial hospital. During the study period, a total of 9,138 deliveries were performed at the hospital, of which 1,624 were by Caesarean section, an overall rate of 17.8%. Of these, 633 (39%) were elective and 911 (61%) were nonelective. The most common primary indication for elective Caesarean section was maternal choice, largely due to a refusal of the patient to consent to a trial of scar following a single previous Caesarean section or a refusal to attempt a trial of vaginal breech delivery. The most common indications for nonelective Caesarean section birth were an intrapartum diagnosis of suspected fetal distress or failure to progress in labour. Strategies to reduce the incidence of Caesarean section birth need to focus on the pathways involved with maternal decision making in the birth process, and on improving the diagnosis of intrapartum fetal compromise. PMID- 10755783 TI - Registrar experience in vaginal breech delivery. How much is occurring? AB - The debate surrounding the safety of vaginal breech delivery compared to Caesarean delivery may be close to resolution, with a large randomized trial underway. However, trends in the management of vaginal breech delivery over recent years have led to suggestions that training in the technique of such deliveries may be inadequate. We have attempted to quantify the level of training in vaginal breech delivery available to registrars. This was accomplished by surveying obstetric training schemes in comparable regions of England and South Australia. Our findings suggest that registrar exposure to this important obstetric skill may be insufficient to guarantee expertise. PMID- 10755784 TI - Problems in the detection of intrapartum fetal asphyxia with intermittent auscultation. AB - We present 4 cases of severe intrapartum fetal asphyxia occurring during spontaneous unaugmented labours at term in low-risk women. In each case the baseline heart rate was completely normal, and the only indication of asphyxia was markedly decreased variability detected with electronic fetal heart rate monitoring. Correct action was taken in 3 cases that probably prevented fetal death or reduced neonatal morbidity. In no case would intermittent auscultation have been able to identify the compromised fetus. PMID- 10755785 TI - Vulval perineal haematomas in the immediate postpartum period and their management. PMID- 10755786 TI - A new method for rapid measurement of lactate in fetal and neonatal blood. AB - A prospective trial to determine the accuracy and precision of the Boehringer Mannheim Accusport handheld lactate meter in measuring plasma lactate levels in umbilical cord blood and neonatal blood microsamples was performed in the labour ward and the neonatal intensive care unit of the NepeanHospital. Specimens were collected from the umbilical artery of 160 consecutive deliveries covering gestations from 26 to 42 weeks, and from 110 umbilical artery catheters covering a range of gestations from 26 to 41 weeks. Serum was also obtained from an exchange transfusion for coefficient of variation analysis. Blood was simultaneously tested for lactic acid concentration on the Boehringer Mannheim (BM) Accusport held lactate meter and the Radiometer ABL 625 blood-gas machine. Clinical data from the mother and baby were recorded together with the full blood gas analysis for comparison with the lactate measures. Coefficients of variation for the BM Accusport lactate meter were established by a further 120 samples of plasma lactate at 6 concentrations from 1 to 20 mmol/L. The stability of measurements with the BM lactate meter over a wide range of temperatures was ascertained by repeated sampling of known concentrations of plasma lactate from 0.5 degrees C to 37 degrees C. The BM Accusport lactate meter was found to be accurate from 1 mmol/L to 20 mmol/L with a Passing Bablok regression line y = 0.004 + 0.915 x (95% CI of slope of 0.889 to 0.946 and intercept -0.138 to 0.094) for whole blood, and y = 0.200 + 1.000 x (95% CI of slope 0.989 to 1.018 and intercept 0.080 to 0.222) for plasma. Between run coefficient of variation (CV) was calculated to be 1.23% to 5.53% over the clinically significant range (2.2 19.3 mmol/L). The BM lactate meter was accurate from 5 to 37 degrees C. At 0.5 degrees C the BM lactate meter significantly underestimated the plasma lactate concentration. There was no significant effect of haematocrit (41.5 to 62%), gestation, or operator on the accuracy of the BM lactate meter. The Accusport handheld lactate meter is an accurate, commercially available, method of measuring plasma lactate levels in only 60 seconds at the point-of-care. It requires only 15 microL of blood and is significantly cheaper than other assay methods. The BM lactate meter is well suited to assess lactic acidaemia of fetal scalp and neonatal blood samples to help quantify hypoxic stress in the perinatal period. PMID- 10755787 TI - Home within 24 hours of laparoscopic hysterectomy. AB - We assessed the feasibility of safe discharge home within 24 hours following laparoscopic hysterectomy in 30 patients who met the inclusion criteria and consented to be enrolled in the study group. Patients were admitted on the day of their surgery with the expectation of discharge within 24 hours. Appropriate home nursing follow-up and phone contact by the surgical team were organized preoperatively. Inclusion criteria were: age 30-65 years, absence of any major medical history that would require prolonged hospitalization, availability of home support for the first 48 hours after discharge and presence of a working telephone line and an address within the area of the Community Home Nursing service. All 30 operative procedures were completed without incident. Six patients underwent total laparoscopic hysterectomy (TLH) (all the procedures of hysterectomy being performed laparoscopically including the suturing of uterine arteries, colpotomy and closure of the vaginal vault. The uterus was removed vaginally) and 24 patients underwent laparoscopic hysterectomy (LH) (this techniques differs from TLH in that the colpotomy was performed laparoscopically but the uterosacral ligaments were divided vaginally and the vault also was closed vaginally after the uterus was removed vaginally). The average operating time was 115 minutes (range 85-150 minutes) and the average blood loss was 97 mL (20-250 mL). There were no intraoperative complications, no requirement for transfusion and no readmission to hospital for any of the patients in the study. Postoperative complications were minor (umbilical cellulitis (1), intestinal colic (1)) and both were treated with resolution of the symptoms. Ninety per cent of patients in the study were discharged within 24 hours of their surgery, the average duration of stay being 22.9 hours (20-24 hours). Three patients were not fit for discharge at 24 hours postoperatively due to general lethargy, migraine and nausea; their average discharge time was 53.5 hours. The study showed that laparoscopic hysterectomy can be associated with a reduction in length of in patient stay compared to traditional laparotomy. Furthermore this reduction could be safely reduced to 24 hours following laparoscopic hysterectomy. There was also an associated cost saving in terms of inpatient bed days. Patient satisfaction with this protocol was high in this selected and motivated group. PMID- 10755788 TI - The effects of abdominal hysterectomy on bladder neck and urinary incontinence. AB - We assessed the bladder neck (BN) by perineal ultrasonography of 39 patients before and 1 year after hysterectomy, and we compared them with 30 control cases in terms of stress urinary incontinence. We evaluated the BN position and mobility in the downwards and backwards directions. The bladder neck was found to be significantly lower at rest, in the downwards direction, in the postoperative period. The stress position of the BN was not significantly different. Its downwards mobility decreased, but not significantly. There was no significant difference in the location of the BN with respect to the pubis, at rest and during stress, in the backwards direction, between the preoperative and postoperative periods. Backwards mobility of the BN decreased significantly following hysterectomy. Stress incontinence was not significantly different between the study group and the control group after one year. We concluded that hysterectomy did not weaken urethral support and did not increase the rate of stress incontinence. PMID- 10755789 TI - Fertility and ovarian function after conservative surgery for germ cell tumours of the ovary. AB - Malignant ovarian germ cell tumours (MOGCT) principally occur in girls and young women and are generally unilateral. Effective combination chemotherapy with conservative surgery has seen a dramatic improvement in survival rates. This increase has shifted the focus to long-term fertility and reproductive outcome. The present study describes 45 patients with MOGCT treated with conservative surgery to preserve fertility, with or without the addition of chemotherapy. The age range was 10 to 32 years with a mean of 20 years. The majority of the subjects had Stage 1 tumours; 44 underwent unilateral salpingo-oophorectomy and 1 patient ovarian cystectomy. Adjuvant chemotherapy was administered in 29 patients. Overall mean follow-up was 58.7 months. There were 4 recurrences and 2 deaths. Survival of those with Stage 1 disease was 97% and for advanced stages 87%. During chemotherapy 50% became amenorrhoeic but 96% resumed normal menstrual function on completion. Seven healthy babies were recorded in the chemotherapy group and no documented birth defects occurred in any of these. There was no case of persistent infertility; 3 patients experienced temporary problems. It is concluded that conservative fertility-sparing surgery is the treatment of choice in these young women and advanced disease is not necessarily a contraindication. The majority can anticipate normal menstrual function and fertility. PMID- 10755790 TI - Uterine leiomyosarcoma--a Singapore experience. AB - Uterine leiomyosarcoma is an aggressive tumour. In our retrospective series of 27 patients, there were 25 with nonmyxoid high-grade leiomyosarcoma of the uterus. The stage distribution was Stage 1, 16; Stage 3, 5 and Stage 4, 4. In the patients with Stage 1 disease, 3 of the 8 patients who received adjuvant chemotherapy subsequently developed recurrent disease. In contrast, 6 of the 8 patients who did not receive adjuvant chemotherapy subsequently developed recurrent disease; 2 of the patients in the latter group also received adjuvant radiotherapy. Six of the 9 women with recurrences were distant 'failures' alone, 2 were both distant and pelvic 'failures' and 1 was pelvic 'failure' alone. All the patients with advanced-stage disease eventually succumbed to the disease despite the therapies given. This study is small and retrospective but it suggests that there might be a role for adjuvant chemotherapy in the management of the early stage of this disease. PMID- 10755791 TI - Low maternal serum oestriol at mid-trimester may indicate a fetal disorder of cholesterol biosynthesis. PMID- 10755792 TI - Splenic infarction in pregnancy. AB - We present a case of splenic infarction in pregnancy, secondary to acute bacterial endocarditis. Left upper quadrant pain in pregnancy can be due to a variety of causes and in the septic or unwell patient, splenic infarct should be considered in the differential diagnosis. The diagnosis of splenic infarct should be considered especially in those at increased risk of bacterial endocarditis. Acute bacterial endocarditis can occur even in patients without any risk factors. Bacterial endocarditis is rare in pregnancy and splenic infarction is even rarer. However when it occurs, rapid diagnosis and management are necessary to minimize embolic phenomena. With the increasing use of intravenous drugs and with increasing numbers of Pacific Islanders in our pregnant population, it is important to be alert to the risk of bacterial endocarditis and to avoid serious sequelae. Patient education to the importance of medical follow-up in order to prevent such a life-threatening condition, and to avoid more complicated acute treatment, is imperative. PMID- 10755793 TI - Small bowel volvulus complicating pregnancy. PMID- 10755794 TI - A case of maternal bradycardia at therapeutic doses of magnesium sulphate in preeclampsia. PMID- 10755795 TI - Streptococcus viridans intra-amniotic infection associated with antecedent cunnilingus. AB - Introduction of bacteria into the amniotic cavity has been associated with oral sex through an ascending infectious route. Previous reports have implicated both Capnocytophaga and Fusobacterium nucleatum via this process. We report a case of Streptococcus viridans intra-amniotic infection occurring at 25 weeks' gestation. Patient questioning revealed a close temporal relationship between the onset of symptoms and previous episodes of cunnilingus. The diagnosis of subclinical bacterial colonization of the amniotic fluid should be considered in patients presenting with preterm labour and no apparent aetiology. A history of recent cunnilingus may be associated with the presence of Streptococcus viridans in the amniotic fluid. PMID- 10755796 TI - A case of delayed diagnosis of the cauda equina syndrome in pregnancy. PMID- 10755797 TI - Anterior sacral meningocele as a pelvic complication of Marfan syndrome. AB - Anterior sacral meningocele (ASM) is well recognized in Marfan syndrome as a consequence of dural ectasia. Two cases presenting as nongynaecological pelvic masses are described highlighting the clinical difficulty in diagnosis and the classical radiological findings. The classification of ASM and associated anomalies of the sacrum are reviewed. PMID- 10755798 TI - The therapeutic dilemma of an ectopic pregnancy in the setting of the severe ovarian hyperstimulation syndrome. AB - Severe ovarian hyperstimulation syndrome as a result of assisted reproductive therapy occurs rarely. However, this iatrogenic condition can result in a life threatening illness with difficult management dilemmas for the attending physicians. A patient with severe adult respiratory distress syndrome and septicaemia after in vitro fertilization required prolonged intensive care treatment and subsequently had a probable ectopic pregnancy treated with systemic methotrexate as an alternative to surgical management. A satisfactory outcome was obtained, followed by a spontaneous successful pregnancy some months after these events. PMID- 10755799 TI - Extremely elevated serum CA125 due to endometriosis. AB - Endometriosis can mimic cancer in a pre-menopausal woman (1). In this case an ultrasound showed a complex ovarian cyst with a low resistance blood flow and the serum CA 125 level was 6,114 iu/mL (normal <35), more than twice the highest level previously reported due to benign disease. PMID- 10755800 TI - An unusual presentation of gestational trophoblastic disease. AB - We report a case of gestational trophoblastic disease leading to an acute abdomen due to a haemoperitoneum in a young multipara. PMID- 10755802 TI - The misplace of litigation in medical practice. PMID- 10755801 TI - A case of massive fetomaternal haemorrhage at term associated with choriocarcinoma. PMID- 10755803 TI - Managing insomnia in the primary care setting: raising the issues. AB - The optimal management of insomnia in the primary care setting should be viewed as a public health problem that will require specific attention. Important recent strides in the understanding of insomnia, its consequences, and its treatment do not always provide a basis for management strategies in a setting with distinct practical limitations. A somewhat different research focus will be needed if the scientific advances are to be translated into practical improvements in therapy. In primary care today, multiple agendas compete for the physician's time. Therefore, it is necessary to view diagnosis and management in terms of both what is efficient and what is optimally effective. Much can be learned from experience with medical risk factors of broad prevalence, such as hypercholesterolemia and hypertension. Large outcome trials demonstrating the benefits of drug therapy were required before pharmacologic management became standard care in the primary care setting. For insomnia, specific issues that must be addressed include the components of diagnosis that will guide therapy and affect prognosis. How can the 10% of adults with insomnia in the primary care practice be subdivided to identify those most in need of therapy? Stated another way, what are the features of insomnia that predict risk? Is duration important? Severity? Frequency? Which treatments are most effective? Which are most efficient in terms of the time required of patient and practitioner? Do treatments for insomnia produce patient satisfaction? Do they prevent adverse outcomes, such as depression and automobile accidents? Studies are now addressing many of these questions. In selecting research priorities, however, the practical application of this information in the clinical setting is important if the ultimate goal is to reduce the number of patients suffering from insomnia and its consequences. PMID- 10755804 TI - The need for flexibility in dosing of hypnotic agents. AB - Although a variety of medical and psychiatric disorders are known to cause insomnia, there are many patients for which a cause cannot be determined. When the etiology is unknown, treatment of insomnia must be symptomatic. Epidemiologic studies suggest that insomnia does not occur on a regular basis in most people. In addition, the presentation of insomnia relative to the time of night is often variable, with waking in the middle of the night and initiating sleep upon going to bed being the most common. The intermittent occurrence of most insomnia suggests that treatment is best accomplished by using hypnotics on an "as needed" basis when difficulties with sleep occur. When pharmacological treatment of insomnia is warranted, benzodiazepine receptor agonists (BzRAs) are often the preferred class of agents. Agents with a shorter duration of action and rapid onset of action are preferred for flexible administration, providing an option for middle of the night dosing if this is when insomnia occurs. Of the available hypnotic agents in the BzRA class, triazolam, zolpidem, and zaleplon have rapid onsets of action and short half-lives. However, with a half-life of 1 hour, only zaleplon appears to be suited for middle of the night administration. Other important factors that affect selection of an agent for the treatment of intermittent insomnia include psychomotor or cognitive impairment and rebound insomnia after discontinuation of therapy. In one placebo-controlled trial, residual sedation was seen after flurazepam, but not with zaleplon, following middle-of-the-night administration. In addition, rebound insomnia was not apparent in a 4-week, placebo-controlled trial of zaleplon. In this same study, transient rebound insomnia was apparent with zolpidem compared to placebo. More data are needed on long-term therapy with hypnotic agents given intermittently on nights during which insomnia occurs. PMID- 10755805 TI - Insomnia in the elderly: a review for the primary care practitioner. AB - The elderly are known to have a high prevalence of insomnia. Causes of insomnia include: medical, psychiatric, and drug issues; circadian rhythm changes; sleep disorders; and psychosocial factors. The elderly frequently use sleeping aids. Risks associated with elderly patients' use of hypnotic drugs are attributable to concomitant comorbid conditions, use of multiple medications, altered pharmacokinetics, and increased central nervous system sensitivity to these drugs. Treatment options for insomnia include behavior modification and pharmacotherapy. The choice of hypnotics is based on matching the nature of the insomnia to the hypnotic agent. Benzodiazepine receptor agonists are common hypnotics prescribed for insomnia in the elderly. The ideal agent has rapid onset, duration of action that lasts through the night but no residual daytime effects, and no adverse effects. The longer-acting agents have been shown to result in a higher risk of falls and hip fractures in the elderly. This relationship is not apparent with short-acting agents. Zaleplon, the newest benzodiazepine receptor agonist, has the shortest half-life of available agents. Studies have demonstrated that zaleplon is effective in improving sleep latency, duration, and sleep quality in the elderly. Zaleplon does not appear to cause rebound insomnia, residual sedation, or adversely affect psychomotor function. The key for the healthcare professional is finding the appropriate treatment or treatment combination, including behavioral modification and pharmacotherapy. When hypnotics are indicated, the most appropriate short-acting agent should be considered. PMID- 10755806 TI - Perspectives on the management of insomnia in patients with chronic respiratory disorders. AB - Complaints of poor sleep are very common in people with chronic respiratory disorders. In patients with chronic obstructive pulmonary disease (COPD), poor sleep may be due to many causes, including cough, excess mucous production, and frequent arousals from sleep caused by hypercapnia, as well as secondary to medications used to manage the lung disease. Patients with obstructive sleep apnea (OSA) also complain of excessive daytime sleepiness and fatigue due to poor quality sleep, although the mechanism of sleep disruption is somewhat different from that in patients with COPD. Although benzodiazepines are often the drugs of choice for the management of insomnia, caution is suggested with the use of these agents in patients with chronic obstructive respiratory disease due to the reduction in upper airway muscle tone and blunting of the arousal response to hypercapnia. However, controlled trials with short-acting benzodiazepine receptor antagonists, including triazolam, zolpidem, and zaleplon, suggest that these agents may be safely used in selected patients who have mild to moderate COPD without daytime hypercapnia. Less data are available on the use of these agents for patients with OSA, but a preliminary trial using zaleplon suggests that respiratory function is not adversely affected in patients with mild to moderate OSA. Studies are needed to further define the benefit-risk ratio of the use of benzodiazepine receptor agonists for the management of insomnia in patients with chronic obstructive lung disease. PMID- 10755807 TI - Nonselective and selective benzodiazepine receptor agonists--where are we today? AB - Insomnia is problematic for many individuals, causing them to seek treatment. There is a long history of therapies aimed at restoring normal sleep patterns, each having its advantages and disadvantages. This review traces the history of insomnia drug therapies from chloral hydrate and the barbiturates through the benzodiazepines and explores the newest selective benzodiazepine receptor agonists, including zolpidem and zaleplon. The mechanisms of action of the benzodiazepine receptor agonists are compared and contrasted. A pharmacokinetic comparison is presented showing the importance that parameters such as dose, onset of action, lipophilicity, metabolites, half-life, and receptor-binding affinity have on clinical effects. The possible adverse effects of sleep aids are discussed, including residual sedation and psychomotor impairment, daytime anxiety, anterograde amnesia and cognitive impairment, rebound insomnia, and drug tolerance and dependence. Effects on sleep efficiency and staging are also discussed. Recommendations for the primary care physician on the selection of hypnotics are also provided. Benzodiazepine receptor agonists are often appropriate agents in the treatment of insomnia; however, individual drug and patient considerations are important in matching the most appropriate agent to the individual patient. Zolpidem and zaleplon, newer selective benzodiazepine receptor agonists, offer additional treatment options. PMID- 10755808 TI - Similarities between severe tinnitus and chronic pain. AB - The symptoms and signs of severe tinnitus and chronic pain have many similarities and similar hypotheses have been presented regarding how the symptoms are generated. Pain and tinnitus have many different forms. The severity of the symptoms of both varies within wide limits, and it is not likely that all forms have the same pathology. Some individuals with severe tinnitus perceive sounds to be unpleasant or painful. This may be similar to what is known as allodynia, which is a painful sensation of normally innocuous stimulation of the skin. Many individuals with chronic pain experience a worsening of their pain from repeated stimulation (the "wind-up" phenomenon). This is similar to the increasingly unpleasant feeling from sounds that are repeated that many individuals with severe tinnitus experience. There are also similarities in the hypotheses about the generation of pain and tinnitus. Although less severe tinnitus may be generated in the ear, it is believed that severe tinnitus in many cases is caused by changes in the nervous system that occur as a result of neural plasticity. Acute pain caused by tissue injury is generated at the site of injury but chronic pain is often generated in the central nervous system, yet another similarity between chronic pain and severe tinnitus. The changes in the nervous system consist of altered synaptic efficacy including opening of dormant synapses. For pain, this is believed to occur in the wide dynamic range neurons of the spinal cord and brain stem. Less is known about the anatomic location of the changes that cause severe tinnitus but there are indications that it may be the inferior colliculus. It is also possible that other auditory systems than the classical ascending pathways may be involved in severe tinnitus. PMID- 10755809 TI - Neurophysiologic mechanisms of tinnitus. AB - Research over the past decade has provided new insights into the neural mechanisms likely to produce the false percepts of sound associated with tinnitus. These insights have emerged mainly as a result of electrophysiologic studies, examining changes in brain activity, and behavioral studies, examining changes in perception, in animals that have been treated with well-known tinnitus inducers such as salicylates, quinine, and intense sound. The available evidence, based on electrophysiologic studies, suggests that tinnitus is associated with disturbances in spontaneous neural activity in the auditory system. These abnormalities include increases in spontaneous activity (hyperactivity), changes in the timing of neural discharges (i.e., the temporal firing properties of neurons), and an increase in bursting activity of neurons. Parallel studies using behavioral testing methods have demonstrated that agents, which produce these neural changes, also cause tinnitus in animals. This article reviews the literature concerned with both behavioral evidence for tinnitus in animal models and the associated changes that occur at peripheral and central levels of the auditory system. PMID- 10755810 TI - Psychoacoustic measures of tinnitus. AB - This report reviews research from the 1930s to the present that has extended our understanding by investigating the characteristics of tinnitus that can be studied using psychoacoustic techniques. Studies of tinnitus masking and residual inhibition began in the 1970s, leading to the therapeutic use of tinnitus masking and a consequent increase in research devoted to tinnitus measurement. In 1981, the CIBA Foundation symposium on tinnitus advocated general adoption of four tinnitus measures: (1) pitch, (2) loudness, (3) maskability, and (4) residual inhibition. Since then, psychoacoustic research into all four topics has proliferated, yielding many valuable insights and controversies concerning the details of measurement techniques. A consensus has emerged that neither the loudness nor other psychoacoustic measures of tinnitus bear a consistent relation to the severity or perceived loudness of tinnitus. Nevertheless, quantification is needed in clinical trials of proposed treatments and in a variety of other types of tinnitus research. Standardization of techniques for specifying the acoustic parameters of tinnitus thus continues to be an important research goal. PMID- 10755811 TI - A re-evaluation of tinnitus reliability testing. AB - The purposes of these investigations were to (1) evaluate tinnitus loudness measures of unskilled normal listeners asked to imagine that they are experiencing a constant monaural tonal tinnitus and (2) compare the performance of these listeners to that of a sample of patients with tinnitus. Subgroups of 24 patients participated in two investigations. Results suggest that (1) normal subjects asked to imagine a high-pitched, tonal tinnitus show significantly greater tinnitus loudness matching levels (a) when they do not have an external reference, (b) after a 1-week interval, and (c) for low-frequency matching tones; (2) patients with high-pitched tinnitus did not demonstrate statistically significant differences in tinnitus loudness judgments within or between sessions or frequencies; (3) tinnitus patients do not differ significantly from normals feigning tinnitus in the variability of tinnitus loudness matching levels within a single session (two measures) or after a 1-week interval (one measure); and (4) normals feigning tinnitus tended to choose significantly greater loudness matching levels than did tinnitus subjects. PMID- 10755812 TI - Tinnitus Retraining Therapy (TRT) as a method for treatment of tinnitus and hyperacusis patients. AB - The aim of this paper is to provide information about the neurophysiologic model of tinnitus and Tinnitus Retraining Therapy (TRT). With this overview of the model and therapy, professionals may discern with this basic foundation of knowledge whether they wish to pursue learning and subsequently implement TRT in their practice. This paper provides an overview only and is insufficient for the implementation of TRT. PMID- 10755813 TI - Functional analysis of genes involved in skeletal development. PMID- 10755815 TI - Electronic media takes on paper publishing. PMID- 10755814 TI - Drs. Haackert Prize 1999 awarded to Prof. Juri Wladimiroff, The Netherlands. PMID- 10755816 TI - Avoiding the sharks. PMID- 10755817 TI - 4th European Peritoneal Dialysis Meeting. Madrid, Spain, April 15-18, 2000. Abstracts. PMID- 10755818 TI - Kozak sequence polymorphism in the platelet GPIbalpha gene is not associated with risk of myocardial infarction. PMID- 10755819 TI - The V410I (G1228A) variant of the caspase-10 gene is a common polymorphism of the Danish population. PMID- 10755820 TI - HIV-associated dysfunction of in vitro IL-12 production depends on the nature of the stimulus and on the CD4 T-cell count of the patient. PMID- 10755821 TI - Autoantibodies neutralizing thrombopoietin in a patient with amegakaryocytic thrombocytopenic purpura. PMID- 10755822 TI - The preferential expression of CD7 and CD34 in myeloid blast crisis in chronic myeloid leukemia. PMID- 10755823 TI - Effects of GP IIb/IIIa receptor antagonists on the activating clotting time of heparinized blood. PMID- 10755824 TI - Skeletal muscle phospholipid metabolism in streptozotocin-diabetic rats. PMID- 10755825 TI - Antioxidant status in vegetarians versus omnivores: a mechanism for longer life? PMID- 10755827 TI - TOUCHINGbase. Honey, I shrunk the chips! PMID- 10755828 TI - MacDibbs Mouth Assessment. A new tool to evaluate mucositis in the radiation therapy patient. AB - PURPOSE: The purposes of this descriptive, longitudinal study were to evaluate the MacDibbs Mouth Assessment instrument for the assessment of mucositis in the radiation therapy patient being treated for head and neck cancer and to describe the course of radiation-induced mucositis in these patients. DESCRIPTION OF STUDY: This pilot study used self-report and provider assessment, as well as medical record review, to obtain data about ambulatory radiation therapy outpatients (n = 10). The participants were primarily male, white, middle-aged, married or partnered, unemployed, edentulous, currently smoking, and using alcohol. RESULTS: One or more mucositis ulcers were observed in all patients and occurred at an average of 2858.2 cGy. The corresponding Mouth Symptom Score was 5.9 (instrument range 0-21). Interrater reliability for 13 of the 14 items was 100%. The one difficulty encountered with the MacDibbs was in the measurement of ulcers longer than the periodontal probe used to measure them. CLINICAL IMPLICATIONS: The MacDibbs should prove useful for clinicians and researchers because it is efficient, easy to use, emphasizes accurate diagnosis of oral changes, has an easily discernible endpoint, and assesses signs and symptoms. PMID- 10755826 TI - Regulation of extracellular matrix proteins by transforming growth factor beta1 in cultured pulmonary endothelial cells. AB - Transforming growth factor beta-1 (TGF-beta1), which is present in lung tissue, has been suggested to play a role in modulating vascular cell function in vivo. The action of TGF-beta1 in vivo, especially at the local site of application to connective tissue, is anabolic and leads to pulmonary fibrosis and angiogenesis, strongly indicating that TGF-beta may have practical applications in repair of tissue injury caused by burns, trauma, or surgery. In the present study, we have used cultured bovine pulmonary artery endothelial (BPAE) cells as a model system. Expression of various proteins, including SPARC (secreted protein acidic and rich in cysteines), type IV procollagen and fibronectin (FN) was examined by radiolabeling the cells with [3H]proline, immunoprecipitation with specific antibodies, and Northern blot analyses by using specific cDNA probes. Cultured cells were labeled with [3H]proline for 24 h in either the absence or in the presence of TGF-beta1 (0-20 ng/ml). Incorporation of radioactivity was observed in a concentration-dependent manner, maximal at 5 ng/ml. Northern blot hybridization demonstrated that TGF-beta1 (5 ng/ml) treatment of BPAE cells caused an increase in steady-state levels PMID- 10755829 TI - On odds ratio and relative risk. PMID- 10755830 TI - [Vladimir Grigor'evich Eliseev (on the centenary of his birth)]. PMID- 10755831 TI - Lack of subsensitivity to albuterol after treatment with salmeterol in patients with asthma. PMID- 10755832 TI - New and old ways of understanding microbial pathogenesis. PMID- 10755833 TI - Pseudomonas: global bacteria. PMID- 10755834 TI - Association of Upper Gastrointestinal Surgeons of Great Britain and Ireland. 23 24 September 1999. Abstracts. PMID- 10755835 TI - The missing mammographic abnormality. PMID- 10755836 TI - Laparoscopy extends the indications for liver resesction in patients with cirrhosis. PMID- 10755837 TI - [Public health management as a science, specialty and training discipline]. PMID- 10755838 TI - 12th National Meeting of the Italian Society for Pediatric Endocrinology and Diabetology (SIEDP). Taormina, 13-16 October 1999. Abstracts. PMID- 10755839 TI - [IX Congress of the Catalan Society of Digestology. Tortosa, Tarrangona, Spain. 27-29 January 2000. Abstracts]. PMID- 10755840 TI - Dentistry: a multifaceted profession. PMID- 10755841 TI - Licensure and mobility. PMID- 10755842 TI - Protocol for the dental office. PMID- 10755843 TI - Use of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside for the isolation of beta-galactosidase-positive bacteria from municipal water supplies. AB - A new medium, mX-Gal, has been developed for the membrane filter enumeration of beta-galactosidase-positive bacteria in municipal water supplies. mX-Gal medium contains the chromogenic beta-galactosidase substrate 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal). All Aeromonas, Citrobacter, and Enterobacter strains isolated from raw water on mX-Gal medium were beta-galactosidase positive. In contrast, only 10 to 20% of these strains produced a red colony with a metallic sheen on m-Endo agar LES medium. Of 674 chlorinated water samples analyzed for total coliforms on m-Endo agar LES medium and for beta-galactosidase positive bacteria on mX-Gal medium, 18 that were negative for coliforms on m-Endo agar LES showed beta-galactosidase-positive bacteria on mX-Gal. Of a total of 50 beta-galactosidase-positive bacteria isolated from these samples, 76% were identified as Aeromonas hydrophila. PMID- 10755844 TI - Web Alert. Apoptosis. PMID- 10755846 TI - Proceedings of the 2nd International Conference on Hemorheology and Tissue Oxygenation in Hypertension and Vascular Diseases. Bari, Italy, 29-31 October 1998. PMID- 10755845 TI - The relationship between gut microflora and intestinal inflammation. PMID- 10755847 TI - Cholinesterase inhibitors and Gingko extracts--are they comparable in the treatment of dementia? Comparison of published placebo-controlled efficacy studies of at least six months' duration. AB - The efficacy of four cholinesterase inhibitors (tacrine, donepezil, rivastigmine, metrifonate) and Ginkgo special extract EGb 761 in Alzheimer's disease were compared. The differences in the effects of the active substance and placebo on cognition were measured on the ADAS-Cog scale, taking into account the different degrees of dementia in the various studies and the dropout rate due to adverse drug reactions. Efficacy, expressed as the delay in symptom progression or the difference in response rate between active substance and placebo, showed no major differences between the four cholinesterase inhibitors and the Ginkgo special extract. Only tacrine exhibited a high dropout rate due to adverse drug reactions. In view of this, the subject of new prescriptions should be critically reviewed. Second-generation cholinesterase inhibitors (donepezil, rivastigmine, metrifonate) and Ginkgo special extract EGb 761 should be considered equally effective in the treatment of mild to moderate Alzheimer's dementia. PMID- 10755849 TI - Current literature in diabetes. PMID- 10755848 TI - Findings emerging from complete microbial genome sequences. AB - Sixteen microorganisms, including one eukaryote, four archaeons, and 11 eubacteria, have been completely sequenced and published. More than 50 genomes are scheduled to be completed by the year 2000. This explosive growth of information is forcing change in many scientific disciplines (e.g. bioinformatics and molecular genetics), spawning new fields, and even changing the way scientific information is used and shared. Novel, global genome sequence comparisons seem slow to appear but the infrastructure for these projects is being built, and we expect exciting developments in the near future. PMID- 10755850 TI - 26th Meeting of the Scandinavian Neuropediatric Society. Bornholm, Denmark, 13-16 May 1998. Abstracts. PMID- 10755851 TI - Proceedings of the 2nd European Stavudine Symposium. Cannes, France, 25 April 1998. PMID- 10755852 TI - Research Institute for Genetic and Human Therapy (RIGHT) 1998 annual meeting. Pavia, Italy, 19-20 April 1998. Abstracts. PMID- 10755853 TI - [Essential hyperhidrosis]. PMID- 10755854 TI - [Non-invasive pressure ventilation (NPPV) in chronic respiratory insufficiency]. PMID- 10755855 TI - Bilateral central pain with unilateral brain lesion. PMID- 10755857 TI - Annual Conference of the International Embryo Transfer Society. Boston, Massachusetts, USA. January 18-20, 1998. Proceedings and abstracts. PMID- 10755856 TI - Papilledema and proteinuria in cerebral venous thrombosis. PMID- 10755858 TI - Annual Conference of the International Embryo Transfer Society. Quebec City, Quebec, Canada. January 10-12, 1999. Proceedings and abstracts. PMID- 10755859 TI - [Extensive duodenal stenosis related to parietal cystic dystrophy in heterotopic pancreas: efficacy of octreotide treatment]. PMID- 10755860 TI - [Non-hemorrhagic and erythematous colitis associated with Klebsiella oxytoca after treatment with erythromycin]. PMID- 10755861 TI - [Metastatic goiter from colonic cancer]. PMID- 10755862 TI - [Chemoembolization followed by systemic chemotherapy with dacarbazine for a metastatic glucagonoma]. PMID- 10755863 TI - [Cyclo 3 Fort-induced lymphocytic colitis associated with ileal villous atrophy]. PMID- 10755864 TI - [Lack of association between hepatitis C virus infection and diabetes mellitus in Gabon]. PMID- 10755865 TI - [Eradication of hepatitis C virus after 3 months of interferon treatment in a patient with chronic hepatitis C]. PMID- 10755866 TI - [CECED 2000. XVIII Meeting of the Study Group of Digestive Epithelial Cells. Paris, France, 13-14 January 2000. Abstracts]. PMID- 10755867 TI - Image of the month. PMID- 10755868 TI - Colon polyps in Beckwith-Wiedmann syndrome: role of imprinted genes. PMID- 10755869 TI - Aortic thrombosis with splenic and renal embolization. PMID- 10755870 TI - Refecoxib versus placebo ulcer rates. PMID- 10755871 TI - The clinical role of COX-2 inhibitors. PMID- 10755872 TI - COX-2 selectivity and lack of gastrointestinal toxicity: true, true and unrelated. PMID- 10755874 TI - Quantitative genetics. A special issue in honour of Professor Douglas Falconer. PMID- 10755873 TI - Development of a tensostat for gastric perception studies. PMID- 10755875 TI - Regarding Shikama et al. IJROBP 1999;44;991-6. PMID- 10755876 TI - Regarding Robinson et al. IJROPB 1999;44(3):497-506. PMID- 10755877 TI - Is prolonged survival possible for patients with supraclavicular node metastases in NSCLC treated with chemoradiotherapy? IJROBP 1999;44(4): 847-853. PMID- 10755878 TI - Stereotactic radiosurgery plus whole brain radiotherapy versus radiotherapy alone for patients with multiple brain metastases: regarding Kondziolka et al. IJROBP 1999;45:427-434. PMID- 10755879 TI - Toxicity with accelerated hyperfractionated pelvic radiotherapy. PMID- 10755880 TI - Regarding Chen et al. IJROBP 1999;44(5):1057-1063. PMID- 10755881 TI - Welfare of equids ever-evolving. PMID- 10755882 TI - Final rule extends E coli testing in slaughter operations. PMID- 10755883 TI - Very low frequence of Pneumocystis carinii DNA detection by PCR in specimens from patients with lung damage. PMID- 10755884 TI - Pneumolysin detection identifies atypical isolates of Streptococcus pneumoniae. PMID- 10755886 TI - Modeling the effects of continuous risk factors. PMID- 10755887 TI - [Diagnosis and therapy of malignant lymphoma]. PMID- 10755885 TI - Identical T cell clones are located within the mouse gut epithelium and lamina propia and circulate in the thoracic duct lymph. AB - Murine gut intraepithelial (IEL) T cell receptor (TCR)-alpha/beta lymphocytes bearing CD8alpha/13 or CD8alpha/alpha coreceptors have been shown previously to express different oligoclonal TCR beta chain repertoires in the same mouse, in agreement with other evidence indicating that these two populations belong to different ontogenic lineages, with only CD8alpha/beta+ IELs being fully thymus dependent. CD8alpha/beta+, but not CD8alpha/alpha+, T lymphocytes are also present in the lamina propria. Here, we show that CD8alpha/beta+ lymphocytes from the lamina propria and the epithelium are both oligoclonal, and that they share the same TCR-beta clonotypes in the same mouse, as is also the case for CD4alpha T cells. Furthermore, identical T cell clones were detected among CD8alpha/beta IELs and CD8alpha/beta+ blasts circulating into the thoracic duct (TD) lymph of the same mouse, whereas TD small lymphocytes are polyclonal. These findings must be considered in light of previous observations showing that T blasts, but not small T lymphocytes, circulating in the TD lymph have the capacity of homing into the gut epithelium and lamina propria. These combined observations have interesting implications for our understanding of the recirculation of gut thymus dependent lymphocytes and their precursors, and of the events leading up to the selection of their restricted TCR repertoire. PMID- 10755889 TI - Acute Leukemia Forum '99: Advances and Controversies in the Therapy of Acute Myeloid Leukemia. San Francisco, California, USA. 16 April 1999. Proceedings. PMID- 10755890 TI - HTLV Molecular Biology and Pathogenesis. Warrenton, Virginia, USA. March 17-19, 2000. Abstracts. PMID- 10755888 TI - Toward a more accurate quantitation of the activity of recombinant retroviruses: alternatives to titer and multiplicity of infection. AB - In this paper, we present a mathematical model with experimental support of how several key parameters govern the adsorption of active retrovirus particles onto the surface of adherent cells. These parameters, including time of adsorption, volume of virus, and the number, size, and type of target cells, as well as the intrinsic properties of the virus, diffusion coefficient, and half-life (t1/2), have been incorporated into a mathematical expression that describes the rate at which active virus particles adsorb to the cell surface. From this expression, we have obtained estimates of Cvo, the starting concentration of active retrovirus particles. In contrast to titer, Cvo is independent of the specific conditions of the assay. The relatively slow diffusion (D = 2 x 10(-8) cm2/s) and rapid decay (t1/2 = 6 to 7 h) of retrovirus particles explain why Cvo values are significantly higher than titer values. Values of Cvo also indicate that the number of defective particles in a retrovirus stock is much lower than previously thought, which has implications especially for the use of retroviruses for in vivo gene therapy. With this expression, we have also computed AVC (active viruses/cell), the number of active retrovirus particles that would adsorb per cell during a given adsorption time. In contrast to multiplicity of infection, which is based on titer and is subject to the same inaccuracies, AVC is based on the physicochemical parameters of the transduction assay and so is a more reliable alternative. PMID- 10755891 TI - [The orthodontist in Italy or, "The wandering shepherd in Asia"]. PMID- 10755892 TI - Caring for the dying--congressional mischief. PMID- 10755893 TI - Caring for the dying--congressional mischief. PMID- 10755894 TI - Recurrent tuberculosis due to exogenous reinfection. PMID- 10755895 TI - Recurrent tuberculosis due to exogenous reinfection. PMID- 10755896 TI - Beta-blockade for patients undergoing vascular surgery. PMID- 10755897 TI - Beta-blockade for patients undergoing vascular surgery. PMID- 10755898 TI - Ownership of dialysis facilities and patients' survival. PMID- 10755899 TI - Ownership of dialysis facilities and patients' survival. PMID- 10755900 TI - Ownership of dialysis facilities and patients' survival. PMID- 10755901 TI - Ownership of dialysis facilities and patients' survival. PMID- 10755902 TI - Ownership of dialysis facilities and patients' survival. PMID- 10755903 TI - Beauty is ova-rated. PMID- 10755904 TI - Australian jailed for removal of fossilized footprints. PMID- 10755905 TI - US energy agency pulls plug on role in genome project. PMID- 10755906 TI - Biology back issues free as publishers walk highwire. PMID- 10755907 TI - Entomology. Love is not puffed up. PMID- 10755908 TI - Impossibility of deleting an unknown quantum state. AB - A photon in an arbitrary polarization state cannot be cloned perfectly. But suppose that at our disposal we have several copies of a photon in an unknown state. Is it possible to delete the information content of one or more of these photons by a physical process? Specifically, if two photons are in the same initial polarization state, is there a mechanism that produces one photon in the same initial state and the other in some standard polarization state? If this could be done, then one would create a standard blank state onto which one could copy an unknown state approximately, by deterministic cloning or exactly, by probabilistic cloning. This could in principle be useful in quantum computation, where one could store new information in an already computed state by deleting the old information. Here we show, however, that the linearity of quantum theory does not allow us to delete a copy of an arbitrary quantum state perfectly. Though in a classical computer information can be deleted (reversibly) against a copy, the analogous task cannot be accomplished, even irreversibly, with quantum information. PMID- 10755910 TI - [AOAR-SIGerO IVth National Congress: Tumors in the Aged. Rome, Italy, 13-14 December 1999. Proceedings]. PMID- 10755909 TI - [CBO guidelines for diagnosis and treatment of acute ankle injury]. PMID- 10755911 TI - Papers from the Malariology Centenary Conference. Rome, Italy, 16-19 November 1998. PMID- 10755912 TI - Discolouration of permanent teeth and enamel hypoplasia due to tetracycline. PMID- 10755913 TI - New weakness in a critically ill patient. PMID- 10755914 TI - [Updates on the role of photochromogenic mycobacteria in human pathology (review of literature)]. PMID- 10755915 TI - CT demonstration of the 1996 AJCC-UICC regional lymph node classification for lung cancer staging. AB - The new international lymph node classification adopted by the American Joint Committee on Cancer (AJCC) and the Union Internationale Contre le Cancer (UICC) is described and illustrated with computed tomography (CT). Anatomic landmarks for 14 hilar, intrapulmonary, and mediastinal lymph node stations are designated. Main differences between the new international classification and the American Thoracic Society (ATS) one are emphasized. In particular, mediastinal pleural reflection is now used to differentiate N2 from N1 nodes. The ATS 10L (left peribronchial nodes) and 10R (right tracheobronchial nodes) stations are now replaced by the AJCC-UICC station 10 (hilar nodes) and the AJCC-UICC station 4 (lower paratracheal, including azygos, nodes), respectively. This very important difference from the ATS classification helps classify the 4 lower paratracheal nodes as N2 nodes, even though the pleural reflection is not seen with CT. The 5 AJCC-UICC nodes are renamed subaortic nodes instead of aortopulmonary ATS nodes. Paraortic nodes, which previously were classified as 5 ATS nodes, are now included with the 6 AJCC-UICC nodes (now renamed paraaortic nodes instead of anterior mediastinal ATS nodes). This change helps accurate labeling because the border between 5 and 6 ATS nodes was not always clear on CT scans. Radiologists should be familiar with this new classification to be able to more accurately compare the lung cancer staging done in different institutions around the world. PMID- 10755916 TI - Computer simulations of the mechanical properties of metals. AB - Atomic-scale computer simulations can be used to gain a better understanding of the mechanical properties of materials. In this paper we demonstrate how this can be done in the case of nanocrystalline copper, and give a brief overview of how simulations may be extended to larger length scales. Nanocrystline metals are metals with grain sizes in the nanometre range, they have a number of technologically interesting properties such as much increased hardness and yield strength. Our simulations show that the deformation mechanisms are different in these materials than in coarse-grained materials. The main deformation is occurring in the grain boundaries, and only little dislocation activity is seen inside the grains. This leads to a hardening of the material as the grain size is increased, and the volume fraction of grain boundaries is decreased. PMID- 10755917 TI - Asymmetric catalysis of 1,3-dipolar cycloaddition reactions--the concepts of activation and induction of asymmetry. PMID- 10755918 TI - Genome sequencing. Talks of public-private deal end in acrimony. PMID- 10755919 TI - Planetary science. Buried channels may have fed Mars ocean. PMID- 10755920 TI - Space station biology. Panel says NASA must show results--fast. PMID- 10755921 TI - Geomicrobiology. Hardy microbe thrives at pH 0. PMID- 10755922 TI - Environmental disasters. Wildlife deaths are a grim wake-up call in Eastern Europe. PMID- 10755923 TI - Obesity research. Tracing leptin's partners in regulating body weight. PMID- 10755924 TI - Gene therapist, heal thyself. PMID- 10755925 TI - Salmonella enteritidis epidemic. PMID- 10755926 TI - Salmonella enteritidis epidemic. PMID- 10755927 TI - Ecology. Can protected areas be expanded in Africa? PMID- 10755928 TI - Cell cycle. Piecing together the p53 puzzle. PMID- 10755930 TI - Genomics: journey to the center of biology. PMID- 10755929 TI - Microbiology. A furtive pathogen revealed. PMID- 10755931 TI - Kentucky 31, far from home. PMID- 10755932 TI - Human genome research. SNP mappers confront reality and find it daunting. PMID- 10755933 TI - Scientific community: Oxford wins a crown synchrotron jewel. PMID- 10755934 TI - Scientific misconduct. Cancer researcher sacked for alleged fraud. PMID- 10755935 TI - Genome sequencing. Clinton and Blair back rapid release of data. PMID- 10755936 TI - Biomedicine. Congress investigates fetal tissue sales. PMID- 10755937 TI - On the hunt for a wolf in sheep's clothing. PMID- 10755938 TI - Too much of a good thing? PMID- 10755939 TI - Transgenic crops: a cautionary tale. PMID- 10755940 TI - Transgenic crops: a cautionary tale. PMID- 10755941 TI - Clinical research. PMID- 10755942 TI - Science under the Nazis. PMID- 10755943 TI - Policy forum: intellectual property rights. Reforming the patent system. PMID- 10755944 TI - Perspectives: signal transduction. Inositol phosphates in the nucleus. PMID- 10755945 TI - Perspectives: evolutionary biology. Limbless tetrapods and snakes with legs. PMID- 10755946 TI - Perspectives: neuroscience. Strengthening visual connections. PMID- 10755947 TI - Techview: drug discovery. Integrating combinatorial synthesis and bioassays. PMID- 10755948 TI - When Pharma merges, R&D is the dowry. PMID- 10755949 TI - Structural genomics offers high-speed look at proteins. PMID- 10755950 TI - Malaria researchers wait for industry to join fight. PMID- 10755951 TI - U.S., Europe, Japan look to speed up drug reviews. PMID- 10755952 TI - Genomic medicine and the future of health care. AB - Genomic technologies and computational advances are leading to an information revolution in biology and medicine. Simulations of molecular processes in cells and predictions of drug effects in humans will advance pharmaceutical research and speed up clinical trials. Computational prognostics and diagnostics that combine with genotyping and molecular profiling may soon cause fundamental changes in the practice of health care. PMID- 10755953 TI - The global drug gap. AB - Global inequities in access to pharmaceutical products exist between rich and poor countries because of market and government failures as well as huge income differences. Multiple policies are required to address this global drug gap for three categories of pharmaceutical products: essential drugs, new drugs, and yet to-be-developed drugs. Policies should combine "push" approaches of subsidies to support targeted drug development, "pull" approaches of financial incentives such as market guarantees, and "process" approaches aimed at improved institutional capacity. Constructive solutions are needed that can both protect the incentives for research and development and reduce the inequities of access. PMID- 10755954 TI - Current awareness on yeast. PMID- 10755955 TI - Frequency doubling technology perimetry for the detection of glaucomatous visual field loss. PMID- 10755956 TI - Corneal endothelial deposits in children positive for human immunodeficiency virus receiving rifabutin prophylaxis for Mycobacterium avium complex bacteremia. PMID- 10755957 TI - Photic maculopathy after pterygium excision. PMID- 10755958 TI - Granular corneal dystrophy with homozygous mutations in the kerato-epithelin gene. PMID- 10755959 TI - Laryngoscopy grades and percentage glottic opening. PMID- 10755960 TI - Mechanical failure of the McCoy laryngoscope during difficult intubation. PMID- 10755961 TI - The effect of steam sterilisation on fibrelight Macintosh laryngoscopes. PMID- 10755962 TI - Reinforced laryngeal mask severed by biting. PMID- 10755963 TI - Aerosol administration into tracheal tubes. PMID- 10755964 TI - Guidance on the use of breathing system filters. PMID- 10755965 TI - Severe bronchospasm relieved by papaverine? PMID- 10755966 TI - Connector design for oxygen therapy tubing. PMID- 10755967 TI - A method for monitoring end-tidal carbon dioxide levels for patients breathing spontaneously. PMID- 10755968 TI - Comparison of sevoflurane and propofol. PMID- 10755969 TI - Postoperative nausea and vomiting following 8% sevoflurane anaesthesia. PMID- 10755970 TI - Maternal mortality in Africa. PMID- 10755971 TI - Benzodiazepines at Caesarean section. PMID- 10755972 TI - Management of disconnection between epidural filters and catheters: a dilemma for the anaesthetist. PMID- 10755973 TI - An unusual epidural-related pressure sore. PMID- 10755974 TI - An awake patient may not detect spinal cord puncture. PMID- 10755975 TI - Decubitus ulcers after instituting epidural analgesia for pain relief in labour. PMID- 10755976 TI - Epidural anaesthesia and foot-drop. PMID- 10755977 TI - VEID. Vein Entry Indicator Device. PMID- 10755978 TI - Ear pain and central venous catheters. PMID- 10755979 TI - Ear pain and central venous catheters. PMID- 10755980 TI - An unusual case of hypertension. PMID- 10755981 TI - Postoperative crying in infants. PMID- 10755982 TI - The patient-at-risk team. PMID- 10755983 TI - Burns following day-case surgery. PMID- 10755984 TI - An old injury with a modern twist. PMID- 10755986 TI - A cautionary tale. PMID- 10755985 TI - Unusual complication of tracheostomy. Mallinckrodt Medical. PMID- 10755987 TI - 'Sulpha' hypersensitivity. PMID- 10755988 TI - Caution with ropivacaine ampoules. PMID- 10755989 TI - Syringe drug labels. PMID- 10755990 TI - An unusual cause of airway compromise during percutaneous dilatational tracheostomy. PMID- 10755991 TI - [Minimizing anaerobic infections]. PMID- 10755992 TI - [Treatment of pain in a young child]. PMID- 10755993 TI - [How far should we go when informing patients?]. PMID- 10755994 TI - A point mutation associated with bacterial macrolide resistance is present in both 23S rRNA genes of an erythromycin-resistant Treponema pallidum clinical isolate. PMID- 10755995 TI - Urinary glycosaminoglycan excretion in urolithiasis. PMID- 10755997 TI - Cysteine proteinases and the pathogenesis of amebiasis. AB - Amebiasis is a major cause of morbidity and mortality throughout the tropical world. Entamoeba histolytica is now recognized as a separate species from the morphologically identical E. dispar, which cannot invade. Cysteine proteinases are a key virulence factor of E. histolytica and play a role in intestinal invasion by degrading the extracellular matrix and circumventing the host immune response through cleavage of secretory immunoglobulin A (sIgA), IgG, and activation of complement. Cysteine proteinases are encoded by at least seven genes, several of which are found in E. histolytica but not E. dispar. A number of new animal models, including the formation of liver abscesses in SCID mice and intestinal infection in human intestinal xenografts, have proven useful to confirm the critical role of cysteine proteinases in invasion. Detailed structural analysis of cysteine proteinases should provide further insights into their biochemical function and may facilitate the design of specific inhibitors which could be used as potential chemotherapeutic agents in the future. PMID- 10755996 TI - Applications of flow cytometry to clinical microbiology. AB - Classical microbiology techniques are relatively slow in comparison to other analytical techniques, in many cases due to the need to culture the microorganisms. Furthermore, classical approaches are difficult with unculturable microorganisms. More recently, the emergence of molecular biology techniques, particularly those on antibodies and nucleic acid probes combined with amplification techniques, has provided speediness and specificity to microbiological diagnosis. Flow cytometry (FCM) allows single- or multiple microbe detection in clinical samples in an easy, reliable, and fast way. Microbes can be identified on the basis of their peculiar cytometric parameters or by means of certain fluorochromes that can be used either independently or bound to specific antibodies or oligonucleotides. FCM has permitted the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity in a rapid, accurate, and highly reproducible way. Furthermore, this technique allows the monitoring of in vitro antimicrobial activity and of antimicrobial treatments ex vivo. The most outstanding contribution of FCM is the possibility of detecting the presence of heterogeneous populations with different responses to antimicrobial treatments. Despite these advantages, the application of FCM in clinical microbiology is not yet widespread, probably due to the lack of access to flow cytometers or the lack of knowledge about the potential of this technique. One of the goals of this review is to attempt to mitigate this latter circumstance. We are convinced that in the near future, the availability of commercial kits should increase the use of this technique in the clinical microbiology laboratory. PMID- 10755998 TI - Drug resistance in human helminths: current situation and lessons from livestock. AB - In this review the available reports on drug resistance in human helminths, particularly hookworms and schistosomes, are critically analyzed. The experiences with helminths of livestock are then reviewed, in particular the factors contributing to the development of anthelmintic resistance, the mechanisms and genetics of resistance to various anthelmintic classes, and the methods available for detection. These experiences appear to be worryingly similar and relevant to the potential development of drug resistance in human helminths. Recommendations to reduce its risks are suggested. PMID- 10755999 TI - Clinical significance of hepatitis C virus genotypes. AB - On the basis of phylogenetic analysis of nucleotide sequences, multiple genotypes and subtypes of hepatitis C virus (HCV) have been identified. Characterization of these genetic groups is likely to facilitate and contribute to the development of an effective vaccine against infection with HCV. Differences among HCV genotypes in geographic distributions have provided investigators with an epidemiologic marker that can be used to trace the source of HCV infection in a given population. HCV genotype 1 may represent a more aggressive strain and one that is less likely to respond to interferon treatment than HCV genotype 2 or 3. However, these observations require confirmation before HCV genotyping can be used in clinical settings. PMID- 10756001 TI - Worldwide Haemophilus influenzae type b disease at the beginning of the 21st century: global analysis of the disease burden 25 years after the use of the polysaccharide vaccine and a decade after the advent of conjugates. AB - Vaccination against Haemophilus influenzae type b (Hib) diseases began a quarter of a century ago with a polysaccharide vaccine; this vaccine was followed by four different conjugates 10 years later. In this review, the burden of global Hib disease is quantified following this 25-year period of vaccine availability to determine the potential impact of conjugate vaccines. This task was accomplished by analysis of data available in 10 languages in 75 geographical regions of over 50 countries. All severe Hib diseases, not only meningitis, were characterized, and special attention was paid to the most vulnerable age group, i.e., children aged 0 to 4 years. Prior to vaccination, the weighted worldwide incidence of meningitis in patients younger than 5 years was 57/100,000, and for all Hib diseases except nonbacteremic pneumonia, it was 71/100,000, indicating 357,000 and 445,000 cases per year, respectively. At least 108,500 of these children died. For all age groups combined, there were 486,000 cases of Hib disease, excluding pneumonia, with 114,200 deaths and probably an equal number of sequelae per annum. If the figures for nonbacteremic pneumonia are included, a conservative estimate is that over 2.2 million cases of infection and 520,000 deaths from Hib disease occurred worldwide, but the true numbers might have been greater. Despite these large numbers and availability of safe and efficacious vaccines, only 38,000 cases annually are prevented-a meager 8% or less than a 2% reduction in cases, depending on whether nonbacteremic pneumonia is included in the calculations. Although vaccination has had great success in some affluent countries, the current level of activity has had a very small impact globally. The use of conjugates, preferably with a reduced number of doses and in combination with other vaccines or perhaps in fractional doses, should be extended to less privileged countries, where most Hib disease occurs. PMID- 10756002 TI - Pathogenesis of intestinal amebiasis: from molecules to disease. AB - In spite of a wealth of knowledge on the biochemistry and cellular and molecular biology of Entamoeba histolytica, little has been done to apply these advances to our understanding of the lesions observed in patients with intestinal amebiasis. In this review, the pathological and histological findings in acute amebic colitis are related to the molecular mechanisms of E. histolytica pathogenicity described to date. Infection of the human colon by E. histolytica produces focal ulceration of the intestinal mucosa, resulting in dysentery (diarrhea with blood and mucus). Although a complete picture has not yet been achieved, the basic mechanisms involved in the production of focal lytic lesions include complex multifactorial processes in which lectins facilitate adhesion, proteases degrade extracellular matrix components, porins help nourish the parasite and may also kill incoming polymorphonuclear leukocytes and macrophages, and motility is used by the parasite to invade deeper layers of the colon. In addition, E. histolytica has developed mechanisms to modulate the immune response during acute infection. Nevertheless, much still needs to be unraveled to understand how this microscopic parasite has earned its well-deserved histolytic name. PMID- 10756004 TI - Chimeric matrix proteins encoded by defective proviruses with large internal deletions in human T-cell leukemia virus type 1-infected humans. AB - Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T cell leukemia/lymphoma (ATLL), HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and other diseases. The mechanisms of virus pathogenesis are still obscure. The occurrence of defective proviruses in HTLV-1-infected cell lines and the peripheral blood mononuclear cells (PBMC) of infected individuals is a frequent feature of virus infection. We detected defective proviruses with large internal deletions in PBMC from ATLL and HAM/TSP patients and in asymptomatic HTLV-1 carriers. Seventeen PCR-amplified defective proviruses were sequenced, and three types of deletions were found. Besides truncated MA and the 5' end of the genome, truncated CA, truncated SU, and more frequently truncated TM linked to the pX region were detected. Reverse transcription-PCR analysis of PBMC from ATLL patients and asymptomatic carriers also revealed RNA transcripts with large internal deletions. Analysis of two RT-PCR cDNA clones confirmed a Gag TM-pX structure of the transcripts. Most defective proviruses contained numerous internal stop codons, but some were capable of coding for the truncated MA linked to a variable out-of-frame peptide. Cloned defective proviruses with long open reading frames were subjected to in vitro transcription-translation followed by radioimmunoprecipitation, which showed expression of chimeric proteins between 8 and 12 kDa. Possible roles of defective proviruses and chimeric proteins are discussed, although there is no firm association with pathogenesis. PMID- 10756005 TI - Adenovirus vector-induced expression of the C-X-C chemokine IP-10 is mediated through capsid-dependent activation of NF-kappaB. AB - The use of adenovirus vectors for gene therapy has been limited by well-defined cellular and humoral immune responses. We have previously shown that adenovirus vectors rapidly induce the expression of the C-X-C chemokine, interferon inducible protein 10 (IP-10), in vivo. Various first-generation, type 5 adenovirus vectors, including adCMVbetagal and UV-psoralen-inactivated adenovirus, equally induced the expression of IP-10 mRNA as early as 3 h following infection in mouse renal epithelial cells (REC). Luciferase reporter experiments using deletional mutants of the murine IP-10 5'-flanking region revealed that transcriptional activation of the IP-10 promoter by adCMVbetagal was dependent on the -161- to -96-bp region upstream of the transcription start site. In electrophoretic mobility shift assays, adCMVbetagal, adCMV-GFP, FG140, and transcription-defective adenovirus induced protein binding to oligonucleotides containing a consensus sequence for NF-kappaB at position -113 of the IP-10 promoter. Supershift assays confirmed an increase in binding activity of NF-kappaB p65 but not p50 or cRel in REC cells infected with various replication-deficient adenoviruses. Coinfection of REC cells with adCMVbetagal and an adenoviral vector expressing IkappaBalpha resulted in suppression of adCMVbetagal-induced expression of IP-10 at 6 and 16 h, further strengthening the conclusion that adenovirus-induced activation of IP-10 is dependent on NF-kappaB. The induction of IP-10 appeared to be direct because infection with adenovirus vectors failed to induce the expression of the potent IP-10 stimulators, interferon gamma and tumor necrosis factor alpha. Together, these findings demonstrate that adenovirus vectors directly induce the expression of IP-10 through capsid dependent activation of NF-kappaB. PMID- 10756006 TI - Generation of cytomegalovirus-specific human T-lymphocyte clones by using autologous B-lymphoblastoid cells with stable expression of pp65 or IE1 proteins: a tool to study the fine specificity of the antiviral response. AB - Cytotoxic T lymphocytes (CTLs) play a central role in the control of persistent human cytomegalovirus (HCMV) infection in healthy virus carriers. Previous analyses of the specificity of HCMV-reactive CD8(+) CTLs drawn from in vitro models in which antigen-presenting cells were autologous fibroblasts infected with laboratory HCMV strains have shown focusing of CTL responses against the major tegument protein, pp65. By contrast, the 72-kDa major immediate-early protein (IE1) was identified as a minor target for this response. Here we have studied the fine specificity and T-cell-receptor features of T-cell clones generated against autologous B lymphoblastoid cell lines stably transfected with HCMV cDNA coding for either pp65 or a natural variant of IE1. This strategy allowed efficient generation of T-cell clones against IE1 and pp65 and led to the identification of several new IE1 and pp65 epitopes, including some located in polymorphic regions of IE1. Such an approach may provide relevant information about the characteristics of the CTL response to IE1 and the effect of viral polymorphism on the immune response against HCMV. PMID- 10756007 TI - Roles of adeno-associated virus Rep protein and human chromosome 19 in site specific recombination. AB - Adeno-associated virus type 2 (AAV) is the only known eucaryotic virus capable of targeted integration in human cells. AAV integrates preferentially into human chromosome (ch) 19q13.3qter. The nonstructural proteins of AAV-2, Rep78 and Rep68, are essential for targeted integration. Rep78 and Rep68 are multifunctional proteins with diverse biochemical activities, including site specific binding to AAV and ch-19 target sequences, helicase activity, and strand specific, site-specific endonuclease activities. Both a Rep DNA binding element (RBE) and a nicking site essential for AAV replication present within the viral terminal repeats are also located on ch-19. Recently, identical RBE sequences have been identified at other locations in the human genome. This fact raises numerous questions concerning AAV targeted integration; specifically, how many RBE sequences are in the human genome? How does Rep discriminate between these and the ch-19 RBE sequence? Does Rep interact with all sites and, if so, how is targeted integration within a fixed time frame facilitated? To better characterize the role of Rep in targeted integration, we established a Rep dependent filter DNA binding assay using a highly purified Rep-68 fusion protein. Electron microscopy (EM) analysis was also performed to determine the characteristics of the Rep-RBE interaction. Our results determined that the Rep affinity for ch-19 is not distinct compared to other RBEs in the human genome when utilizing naked DNA. In fact, a minimum-binding site (GAGYGAGC) efficiently associated with Rep, suggesting that as many as 2 x 10(5) sites may exist. In addition, such sites also exist frequently in nonprimate mammalian genomes, although AAV integrates site specifically into primate genomes. EM analysis demonstrated that only one Rep-DNA complex was formed on ch-19 target DNA. Surprisingly, identically sized complexes were observed on all substrates containing a RBE sequence, but never on DNA lacking an RBE. Rep-DNA complexes involved a multimeric protein structure that spanned ca. 60 bp. Immunoprecipitation of AAV latently infected cells determined that 1,000 to 4,000 copies of Rep78 and Rep68 protein are expressed per cell. Comparison of the Rep association constant with those of established DNA binding proteins indicates that sufficient molecules of Rep are present to interact with all potential RBE sites. Moreover, Rep expression in the absence of AAV cis-acting substrate resulted in Rep-dependent amplification and rearrangement of the target sequence in ch-19. This result suggests that this locus is a hot spot for Rep-dependent recombination. Finally, we engineered mice to carry a single 2.7-kb human ch-19 insertion containing the AAV ch-19 target locus. Using cells derived from these mice, we demonstrated that this sequence was sufficient for site-specific recombination after infection with transducing vectors expressing Rep. This result indicates that any host factors required for targeting are conserved between human and mouse. Furthermore, the human ch-19 cis sequences and chromatin structure required for site-specific recombination are contained within this fragment. Overall, these results indicate that the specificity of targeted recombination to human ch-19 is not dictated by differential Rep affinities for RBE sites. Instead, specificity is likely dictated by human ch-19 sequences that serve as a Rep protein-mediated origin of replication, thus facilitating viral targeting through Rep-Rep interactions and host enzymes, resulting in site specific recombination. Control of specificity is clearly dictated by the ch-19 sequences, since transfer of these sequences into the mouse genome are sufficient to achieve Rep-dependent site-specific integration. PMID- 10756000 TI - Zygomycetes in human disease. AB - The Zygomycetes represent relatively uncommon isolates in the clinical laboratory, reflecting either environmental contaminants or, less commonly, a clinical disease called zygomycosis. There are two orders of Zygomycetes containing organisms that cause human disease, the Mucorales and the Entomophthorales. The majority of human illness is caused by the Mucorales. While disease is most commonly linked to Rhizopus spp., other organisms are also associated with human infection, including Mucor, Rhizomucor, Absidia, Apophysomyces, Saksenaea, Cunninghamella, Cokeromyces, and Syncephalastrum spp. Although Mortierella spp. do cause disease in animals, there is no longer sufficient evidence to suggest that they are true human pathogens. The spores from these molds are transmitted by inhalation, via a variety of percutaneous routes, or by ingestion of spores. Human zygomycosis caused by the Mucorales generally occurs in immunocompromised hosts as opportunistic infections. Host risk factors include diabetes mellitus, neutropenia, sustained immunosuppressive therapy, chronic prednisone use, iron chelation therapy, broad-spectrum antibiotic use, severe malnutrition, and primary breakdown in the integrity of the cutaneous barrier such as trauma, surgical wounds, needle sticks, or burns. Zygomycosis occurs only rarely in immunocompetent hosts. The disease manifestations reflect the mode of transmission, with rhinocerebral and pulmonary diseases being the most common manifestations. Cutaneous, gastrointestinal, and allergic diseases are also seen. The Mucorales are associated with angioinvasive disease, often leading to thrombosis, infarction of involved tissues, and tissue destruction mediated by a number of fungal proteases, lipases, and mycotoxins. If the diagnosis is not made early, dissemination often occurs. Therapy, if it is to be effective, must be started early and requires combinations of antifungal drugs, surgical intervention, and reversal of the underlying risk factors. The Entomophthorales are closely related to the Mucorales on the basis of sexual growth by production of zygospores and by the production of coenocytic hyphae. Despite these similarities, the Entomophthorales and Mucorales have dramatically different gross morphologies, asexual reproductive characteristics, and disease manifestations. In comparison to the floccose aerial mycelium of the Mucorales, the Entomophthorales produce a compact, glabrous mycelium. The asexually produced spores of the Entomophthorales may be passively released or actively expelled into the environment. Human disease with these organisms occurs predominantly in tropical regions, with transmission occurring by implantation of spores via minor trauma such as insect bites or by inhalation of spores into the sinuses. Conidiobolus typically infects mucocutaneous sites to produce sinusitis disease, while Basidiobolus infections occur as subcutaneous mycosis of the trunk and extremities. The Entomophthorales are true pathogens, infecting primarily immunocompetent hosts. They generally do not invade blood vessels and rarely disseminate. Occasional cases of disseminated and angioinvasive disease have recently been described, primarily in immunocompromised patients, suggesting a possible emerging role for this organism as an opportunist. PMID- 10756008 TI - Clinical isolates of measles virus use CD46 as a cellular receptor. AB - Laboratory strains of measles viruses (MV), such as Edmonston and Halle, use the complement regulatory protein CD46 as a cell surface receptor. The receptor usage of clinical isolates of MV, however, remains unclear. Receptor usage by primary patient isolates of MV was compared to isolates that had been passaged on a variety of tissue culture cell lines. All of the isolates could infect cells in a CD46-dependent manner, but their tropism was restricted according to cell type (e.g., lymphocytes versus fibroblasts). The results indicate that patient isolates that have not been adapted to tissue culture cell lines use CD46 as a receptor. In addition, passaging primary MV patient isolates in B95-8 cells selected variants that had alternate receptor usage compared to the original isolate. Thus, changes in receptor usage by MV are dependent upon the cell type used for isolation. Furthermore, our results confirm the relevance of the CD46 receptor to natural measles infection. PMID- 10756009 TI - The viral nucleocapsid protein of transmissible gastroenteritis coronavirus (TGEV) is cleaved by caspase-6 and -7 during TGEV-induced apoptosis. AB - The transmissible gastroenteritis coronavirus (TGEV), like many other viruses, exerts much of its cytopathic effect through the induction of apoptosis of its host cell. Apoptosis is coordinated by a family of cysteine proteases, called caspases, that are activated during apoptosis and participate in dismantling the cell by cleaving key structural and regulatory proteins. We have explored the caspase activation events that are initiated upon infection of the human rectal tumor cell line HRT18 with TGEV. We show that TGEV infection results in the activation of caspase-3, -6, -7, -8, and -9 and cleavage of the caspase substrates eIF4GI, gelsolin, and alpha-fodrin. Surprisingly, the TGEV nucleoprotein (N) underwent proteolysis in parallel with the activation of caspases within the host cell. Cleavage of the N protein was inhibited by cell permeative caspase inhibitors, suggesting that this viral structural protein is a target for host cell caspases. We show that the TGEV nucleoprotein is a substrate for both caspase-6 and -7, and using site-directed mutagenesis, we have mapped the cleavage site to VVPD(359) downward arrow. These data demonstrate that viral proteins can be targeted for destruction by the host cell death machinery. PMID- 10756010 TI - Cospeciation and horizontal transmission of avian sarcoma and leukosis virus gag genes in galliform birds. AB - In a study of the evolution and distribution of avian retroviruses, we found avian sarcoma and leukosis virus (ASLV) gag genes in 26 species of galliform birds from North America, Central America, eastern Europe, Asia, and Africa. Nineteen of the 26 host species from whom ASLVs were sequenced were not previously known to contain ASLVs. We assessed congruence between ASLV phylogenies based on a total of 110 gag gene sequences and ASLV-host phylogenies based on mitochondrial 12S ribosomal DNA and ND2 sequences to infer coevolutionary history for ASLVs and their hosts. Widespread distribution of ASLVs among diverse, endemic galliform host species suggests an ancient association. Congruent ASLV and host phylogenies for two species of Perdix, two species of Gallus, and Lagopus lagopus and L. mutus also indicate an old association with vertical transmission and cospeciation for these ASLVs and hosts. An inference of horizontal transmission of ASLVs among some members of the Tetraoninae subfamily (grouse and ptarmigan) is supported by ASLV monophyletic groups reflecting geographic distribution and proximity of hosts rather than host species phylogeny. We provide a preliminary phylogenetic taxonomy for the new ASLVs, in which named taxa denote monophyletic groups. PMID- 10756011 TI - The role of alpha/beta and gamma interferons in development of immunity to influenza A virus in mice. AB - During influenza virus infection innate and adaptive immune defenses are activated to eliminate the virus and thereby bring about recovery from illness. Both arms of the adaptive immune system, antibody neutralization of free virus and termination of intracellular virus replication by antiviral cytotoxic T cells (CTLs), play pivotal roles in virus elimination and protection from disease. Innate cytokine responses, such as alpha/beta interferon (IFN-alpha/beta) or IFN gamma, can have roles in determining the rate of virus replication in the initial stages of infection and in shaping the initial inflammatory and downstream adaptive immune responses. The effect of these cytokines on the replication of pneumotropic influenza A virus in the respiratory tract and in the regulation of adaptive antiviral immunity was examined after intranasal infection of mice with null mutations in receptors for IFN-alpha/beta, IFN-gamma, and both IFNs. Virus titers in the lungs of mice unable to respond to IFNs were not significantly different from congenic controls for both primary and secondary infection. Likewise the mice were comparably susceptible to X31 (H3N2) influenza virus infection. No significant disruption to the development of normal antiviral CTL or antibody responses was observed. In contrast, mice bearing the disrupted IFN alpha/beta receptor exhibited accelerated kinetics and significantly higher levels of neutralizing antibody activity during primary or secondary heterosubtypic influenza virus infection. Thus, these observations reveal no significant contribution for IFN-controlled pathways in shaping acute or memory T cell responses to pneumotropic influenza virus infection but do indicate some role for IFN-alpha/beta in the regulation of antibody responses. Recognizing the pivotal role of CTLs and antibody in virus clearance, it is reasonable to assume a redundancy in IFN-mediated antiviral effects in pulmonary influenza. However, IFN-alpha/beta seems to be a valid factor in determining tissue tropism and replicative rates of highly virulent influenza virus strains as reported previously by others, and this aspect is discussed here. PMID- 10756003 TI - The ins and outs of DNA fingerprinting the infectious fungi. AB - DNA fingerprinting methods have evolved as major tools in fungal epidemiology. However, no single method has emerged as the method of choice, and some methods perform better than others at different levels of resolution. In this review, requirements for an effective DNA fingerprinting method are proposed and procedures are described for testing the efficacy of a method. In light of the proposed requirements, the most common methods now being used to DNA fingerprint the infectious fungi are described and assessed. These methods include restriction fragment length polymorphisms (RFLP), RFLP with hybridization probes, randomly amplified polymorphic DNA and other PCR-based methods, electrophoretic karyotyping, and sequencing-based methods. Procedures for computing similarity coefficients, generating phylogenetic trees, and testing the stability of clusters are then described. To facilitate the analysis of DNA fingerprinting data, computer-assisted methods are described. Finally, the problems inherent in the collection of test and control isolates are considered, and DNA fingerprinting studies of strain maintenance during persistent or recurrent infections, microevolution in infecting strains, and the origin of nosocomial infections are assessed in light of the preceding discussion of the ins and outs of DNA fingerprinting. The intent of this review is to generate an awareness of the need to verify the efficacy of each DNA fingerprinting method for the level of genetic relatedness necessary to answer the epidemiological question posed, to use quantitative methods to analyze DNA fingerprint data, to use computer assisted DNA fingerprint analysis systems to analyze data, and to file data in a form that can be used in the future for retrospective and comparative studies. PMID- 10756012 TI - Role of the cytoplasmic tail of pseudorabies virus glycoprotein E in virion formation. AB - Glycoproteins M (gM), E (gE), and I (gI) of pseudorabies virus (PrV) are required for efficient formation of mature virions. The simultaneous absence of gM and the gE/gI complex results in severe deficiencies in virion morphogenesis and cell-to cell spread, leading to drastically decreased virus titers and a small-plaque phenotype (A. Brack, J. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). Serial passaging in noncomplementing cells of a virus mutant unable to express gM, gE, and gI resulted in a reversion of the small-plaque phenotype and restoration of infectious virus formation to the level of a gM(-) mutant. Genetic analyses showed that reversion of the phenotype was accompanied by a genomic rearrangement which led to the fusion of a portion of the gE gene encoding the cytoplasmic domain to the 3' end of the glycoprotein D gene, resulting in expression of a chimeric gD-gE protein. Since this indicated that the intracytoplasmic domain of gE was responsible for the observed phenotypic alterations, the UL10 (gM) gene was deleted in a PrV mutant, PrV-107, which specifically lacked the cytoplasmic tail of gE. Regarding one-step growth, plaque size, and virion formation as observed under the electron microscope, the mutant lacking gM and the gE cytoplasmic tail proved to be very similar to the gE/I/M triple mutant. Thus, our data indicate that it is the cytoplasmic tail of gE which is responsible for the observed phenotypic effects in conjunction with deletion of gM. We hypothesize that the cytoplasmic domain of gE specifically interacts with components of the capsid and/or tegument, leading to efficient secondary envelopment of intracytoplasmic capsids. PMID- 10756013 TI - Evidence for viral virulence as a predominant factor limiting human immunodeficiency virus vaccine efficacy. AB - Current strategies in human immunodeficiency virus type 1 (HIV-1) vaccine development are often based on the production of different vaccine antigens according to particular genetic clades of HIV-1 variants. To determine if virus virulence or genetic distance had a greater impact on HIV-1 vaccine efficacy, we designed a series of heterologous chimeric simian/human immunodeficiency virus (SHIV) challenge experiments in HIV-1 subunit-vaccinated rhesus macaques. Of a total of 22 animals, 10 nonimmunized animals served as controls; the remainder were vaccinated with the CCR5 binding envelope of HIV-1(W6.1D). In the first study, heterologous challenge included two nonpathogenic SHIV chimeras encoding the envelopes of the divergent clade B HIV-1(han2) and HIV-1(sf13) strains. In the second study, all immunized animals were rechallenged with SHIV(89. 6p), a virus closely related to the vaccine strain but highly virulent. Protection from either of the divergent SHIV(sf13) or SHIV(han2) challenges was demonstrated in the majority of the vaccinated animals. In contrast, upon challenge with the more related but virulent SHIV(89.6p), protection was achieved in only one of the previously protected vaccinees. A secondary but beneficial effect of immunization on virus load and CD4(+) T-cell counts was observed despite failure to protect from infection. In addition to revealing different levels of protective immunity, these results suggest the importance of developing vaccine strategies capable of protecting from particularly virulent variants of HIV-1. PMID- 10756014 TI - Establishment and characterization of molecular clones of porcine endogenous retroviruses replicating on human cells. AB - The use of pig xenografts is being considered to alleviate the shortage of allogeneic organs for transplantation. In addition to the problems overcoming immunological and physiological barriers, the existence of numerous porcine microorganisms poses the risk of initiating a xenozoonosis. Recently, different classes of type C porcine endogenous retoviruses (PERV) which are infectious for human cells in vitro have been partially described. We therefore examined whether completely intact proviruses exist that produce infectious and replication competent virions. Several proviral PERV sequences were cloned and characterized. One molecular PERV class B clone, PERV-B(43), generated infectious particles after transfection into human 293 cells. A second clone, PERV-B(33), which was highly homologous to PERV-B(43), showed a G-to-A mutation in the first start codon (Met to Ile) of the env gene, preventing this provirus from replicating. However, a genetic recombinant, PERV-B(33)/ATG, carrying a restored env start codon, became infectious and could be serially passaged on 293 cells similar to virus clone PERV-B(43). PERV protein expression was detected 24 to 48 h posttransfection (p. t.) using cross-reacting antiserum, and reverse transcriptase activity was found at 12 to 14 days p.t. The transcriptional start and stop sites as well as the splice donor and splice acceptor sites of PERV mRNA were mapped, yielding a subgenomic env transcript of 3. 1 kb. PERV-B(33) and PERV B(43) differ in the number of copies of a 39-bp segment in the U3 region of the long terminal repeat. Strategies to identify and to specifically suppress or eliminate those proviruses from the pig genome might help in the production of PERV-free animals. PMID- 10756015 TI - Subgenomic negative-strand RNA function during mouse hepatitis virus infection. AB - Mouse hepatitis virus (MHV)-infected cells contain full-length and subgenomic length positive- and negative-strand RNAs. The origin and function of the subgenomic negative-strand RNAs is controversial. In this report we demonstrate that the synthesis and molar ratios of subgenomic negative strands are similar in alternative host cells, suggesting that these RNAs function as important mediators of positive-strand synthesis. Using kinetic labeling experiments, we show that the full-length and subgenomic-length replicative form RNAs rapidly accumulate and then saturate with label, suggesting that the subgenomic-length negative strands are the principal mediators of positive-strand synthesis. Using cycloheximide, which preferentially inhibits negative-strand and to a lesser extent positive-strand synthesis, we demonstrate that cycloheximide treatment equally inhibits full-length and subgenomic-length negative-strand synthesis. Importantly, following treatment, previously transcribed negative strands remain in transcriptionally active complexes even in the absence of new negative-strand synthesis. These findings indicate that the subgenomic-length negative strands are the principal templates of positive-strand synthesis during MHV infection. PMID- 10756016 TI - A group B coxsackievirus/poliovirus 5' nontranslated region chimera can act as an attenuated vaccine strain in mice. AB - The linear, single-stranded enterovirus RNA genome is flanked at either end with a nontranslated region (NTR). By replacing the entire 5' NTR of coxsackievirus B3 (CVB3) with that from type 1 poliovirus, a progeny virus was obtained following transfection of HeLa cells. The chimeric virus, CPV/49, replicates like the parental CVB3 strain in HeLa cells but is attenuated for replication and yield in primary human coronary artery endothelial cell cultures, in a human pancreas tumor cell line, and in primary murine heart fibroblast cultures. Western blotting analyses of CPV/49 replication in murine heart fibroblast cultures demonstrate that synthesis of CPV/49 proteins is significantly slower than that of the parental CVB3 strain. CPV/49 replicates in murine hearts and pancreata, causing no disease in hearts and a minor pancreatic inflammation in some mice that resolves by 28 days postinoculation. A single inoculation with CPV/49 induces protective anti-CVB3 neutralizing antibody titers that completely protect mice from both heart and pancreatic disease when mice are challenged 28 days p.i. with genetically diverse virulent strains of CVB3. That a chimeric CVB3 strain, created from sequences of two virulent viruses, is sufficiently attenuated to act as an avirulent, protective vaccine strain in mice suggests that chimeric genome technology merits further evaluation for the development of new nonpoliovirus enteroviral vectors. PMID- 10756017 TI - The interaction of bacteriophage P2 B protein with Escherichia coli DnaB helicase. AB - Bacteriophage P2 requires several host proteins for lytic replication, including helicase DnaB but not the helicase loader, DnaC. Some genetic studies have suggested that the loading is done by a phage-encoded protein, P2 B. However, a P2 minichromosome containing only the P2 initiator gene A and a marker gene can be established as a plasmid without requiring the P2 B gene. Here we demonstrate that P2 B associates with DnaB. This was done by using the yeast two-hybrid system in vivo and was confirmed in vitro, where (35)S-labeled P2 B bound specifically to DnaB adsorbed to Q Sepharose beads and monoclonal antibodies directed against the His-tagged P2 B protein were shown to coprecipitate the DnaB protein. Finally, P2 B was shown to stabilize the opening of a reporter origin, a reaction that is facilitated by the inactivation of DnaB. In this respect, P2 B was comparable to lambda P protein, which is known to be capable of binding and inactivating the helicase while acting as a helicase loader. Even though P2 B has little similarity to other known or predicted helicase loaders, we suggest that P2 B is required for efficient loading of DnaB and that this role, although dispensable for P2 plasmid replication, becomes essential for P2 lytic replication. PMID- 10756018 TI - Isolation and characterization of an equine foamy virus. AB - Foamy viruses (FVs) are complex retroviruses which have been isolated from different animal species including nonhuman primates, cattle, and cats. Here, we report the isolation and characterization of a new FV isolated from blood samples of horses. Similar to other FVs, the equine foamy virus (EFV) exhibits a highly characteristic ultrastructure and induces syncytium formation and subsequent cell lysis on a large number of cell lines. Molecular cloning of EFV reveals that the general organization is that of other known FVs, whereas sequence similarity with its bovine FV counterpart is only 40%. Interestingly, EFV buds exclusively from the plasma membrane and not from the endoplasmic reticulum (ER), as previously shown for other FVs. The absence of the ER retrieval dilysine motif in EFV Env is likely responsible for this unexpected sorting pathway. PMID- 10756019 TI - Expression of functional influenza virus RNA polymerase in the methylotrophic yeast Pichia pastoris. AB - Influenza virus RNA polymerase with the subunit composition PB1-PB2-PA is a multifunctional enzyme with the activities of both synthesis and cleavage of RNA and is involved in both transcription and replication of the viral genome. In order to produce large amounts of the functional viral RNA polymerase sufficient for analysis of its structure-function relationships, the cDNAs for RNA segments 1, 2, and 3 of influenza virus A/PR/8, each under independent control of the alcohol oxidase gene promoter, were integrated into the chromosome of the methylotrophic yeast Pichia pastoris. Simultaneous expression of all three P proteins in the yeast P. pastoris was achieved by the addition of methanol. To purify the P protein complexes, a sequence coding for a histidine tag was added to the PB2 protein gene at its N terminus. Starting from the induced P. pastoris cell lysate, we partially purified a 3P complex by Ni(2+)-agarose affinity column chromatography. The 3P complex showed influenza virus model RNA-directed and ApG primed RNA synthesis in vitro but was virtually inactive without addition of template or primer. The kinetic properties of model template-directed RNA synthesis and the requirements for template sequence were analyzed using the 3P complex. Furthermore, the 3P complex showed capped RNA-primed RNA synthesis. Thus, we conclude that functional influenza virus RNA polymerase with the catalytic properties of a transcriptase is formed in the methylotrophic yeast P. pastoris. PMID- 10756020 TI - The vaccinia virus A14.5L gene encodes a hydrophobic 53-amino-acid virion membrane protein that enhances virulence in mice and is conserved among vertebrate poxviruses. AB - A short sequence, located between the A14L and A15L open reading frames (ORFs) of vaccinia virus, was predicted to encode a hydrophobic protein of 53 amino acids that is conserved in orthopoxviruses, leporipoxviruses, yatapoxiruses, and molluscipoxviruses. We constructed a recombinant vaccinia virus with a 10-codon epitope tag appended to the C terminus of the A14.5L ORF. Synthesis of the tagged protein occurred at late times and was blocked by an inhibitor of DNA replication, consistent with regulation by a predicted late promoter just upstream of the A14.5L ORF. Hydrophobicity of the protein was demonstrated by extraction into the detergent phase of Triton X-114. The protein was associated with purified vaccinia virus particles and with membranes of immature and mature virions that were visualized by electron microscopy of infected cells. Efficient release of the protein from purified virions occurred after treatment with a nonionic detergent and reducing agent. A mutant virus, in which the A14.5L ORF was largely deleted, produced normal-size plaques in several cell lines, and the yields of infectious intra- and extracellular viruses were similar to those of the parent. In contrast, with a mouse model, mutant viruses with the A14.5L ORF largely deleted were attenuated relative to that of the parental virus or a mutant virus with a restored A14.5L gene. PMID- 10756021 TI - Polyomavirus-infected dendritic cells induce antiviral CD8(+) T lymphocytes. AB - CD8(+) T cells are critical for the clearance of acute polyomavirus infection and the prevention of polyomavirus-induced tumors, but the antigen-presenting cell(s) involved in generating polyomavirus-specific CD8(+) T cells have not been defined. We investigated whether dendritic cells and macrophages are permissive for polyomavirus infection and examined their potential for inducing antiviral CD8(+) T cells. Although dendritic cells and macrophages both supported productive polyomavirus infection, dendritic cells were markedly more efficient at presenting the immunodominant viral epitope to CD8(+) T cells. Additionally, infected dendritic cells, but not infected macrophages, primed anti-polyomavirus CD8(+) T cells in vivo. Treatment with Flt3 ligand, a hematopoietic growth factor that dramatically expands the number of dendritic cells, markedly enhanced the magnitude of virus-specific CD8(+) T-cell responses during acute infection and the pool of memory anti-polyomavirus CD8(+) T cells. These findings suggest that virus-infected dendritic cells induce polyomavirus-specific CD8(+) T cells in vivo and raise the potential for their use as cellular adjuvants to promote CD8(+) T cell surveillance against polyomavirus-induced tumors. PMID- 10756022 TI - Protection of the villus epithelial cells of the small intestine from rotavirus infection does not require immunoglobulin A. AB - Immunoglobulin A (IgA) is the primary immune response induced in the intestine by rotavirus infection, but vaccination with virus-like particles induces predominantly IgG, not IgA. To definitively assess the role of IgA in protection from rotavirus infection, IgA knockout mice, which are devoid of serum and secretory IgA, were infected and then rechallenged with murine rotavirus at either 6 weeks or 10 months. Following primary rotavirus infection, IgA knockout mice cleared virus as effectively as IgA normal control mice. Rotavirus-infected IgA knockout mice produced no serum or fecal IgA but did have high levels of antirotavirus serum IgG and IgM and fecal IgG, whereas IgA normal control mice made both serum IgA and IgG and fecal IgA. Both IgA normal and IgA knockout mice were totally protected from rotavirus challenge at 42 days. Ten months following a primary infection, both IgA normal and knockout mice still had high levels of serum and fecal antirotavirus antibody and were totally protected from rotavirus challenge. To determine if compensatory mechanisms other than IgG were responsible for protection from rotavirus infection in IgA knockout mice, mice were depleted of CD4(+) T cells or CD8(+) T cells. No changes in the level of protection were seen in depleted mice. These data show that fecal or systemic IgA is not essential for protection from rotavirus infection and suggest that in the absence of IgA, IgG may play a significant role in protection from mucosal pathogens. PMID- 10756023 TI - Induction of indolamine 2,3-dioxygenase in primary human macrophages by human immunodeficiency virus type 1 is strain dependent. AB - Increased kynurenine pathway metabolism has been implicated in the etiology of AIDS dementia complex (ADC). The rate-limiting enzyme for this pathway is indolamine 2,3-dioxygenase (IDO). We tested the efficacy of different strains of human immunodeficiency virus type 1 (HIV1-BaL, HIV1-JRFL, and HIV1-631) to induce IDO in cultured human monocyte-derived macrophages (MDM). A significant increase in both IDO protein and kynurenine synthesis was observed after 48 h in MDM infected with the brain-derived HIV-1 isolates, laboratory-adapted (LA) HIV1 JRFL, and primary isolate HIV1-631. In contrast, almost no kynurenine production or IDO protein was evident in MDM infected with the highly replicating macrophage tropic LA strain HIV1-BaL. The induction of IDO and kynurenine synthesis by HIV1 JRFL and HIV1-631 declined to baseline levels by day 8 postinfection. Abundant HIV-1 replication did not reduce the ability of exogenous gamma interferon (IFN gamma) to induce IDO and kynurenine synthesis in HIV-infected MDM. The addition of anti-IFN-gamma antibody to MDM infected with HIV1-JRFL resulted in an absence of detectable IDO protein after 48 h and a decrease of 64% +/- 1% in supernatant kynurenine concentration. Together, these results indicate that only selected strains of HIV-1 are capable of inducing IDO synthesis and subsequent kynurenine metabolism in MDM. The induction of IDO, while apparently independent of replication capacity, appears to be mediated by a transient production of IFN gamma in MDM responding to the initial infection with selected strains of HIV-1. PMID- 10756024 TI - Establishment of monoclonal anti-retroviral gp70 autoantibodies from MRL/lpr lupus mice and induction of glomerular gp70 deposition and pathology by transfer into non-autoimmune mice. AB - Several strains of mice, including MRL/MpJ mice homozygous for the Fas mutant lpr gene (MRL/lpr mice), F(1) hybrids of New Zealand Black and New Zealand White mice, and BXSB/MpJ mice carrying a Y-linked autoimmune acceleration gene, spontaneously develop immune complex-mediated glomerulonephritis. The involvement of the envelope glycoprotein gp70 of an endogenous xenotropic virus in the formation of circulating immune complexes and their deposition in the glomerular lesions have been demonstrated, as has the pathogenicity of various antinuclear, antiphospholipid, and rheumatoid factor autoantibodies. In recent genetic linkage studies as well as in a study of cytokine-induced protection against nephritis development, the strongest association of serum levels of gp70-anti-gp70 immune complexes, rather than the levels of antinuclear autoantibodies, with the development and severity of glomerulonephritis has been demonstrated, suggesting a major pathogenic role of anti-gp70 autoantibodies in the lupus-prone mice. However, the pathogenicity of anti-gp70 autoantibodies has not yet been directly tested. To examine if anti-gp70 autoantibodies induce glomerular pathology, we established from unmanipulated MRL/lpr mice hybridoma clones that secrete monoclonal antibodies reactive with endogenous xenotropic viral env gene products. Upon transplantation, a high proportion of these anti-gp70 antibody producing hybridoma clones induced in syngeneic non-autoimmune and severe combined immunodeficiency mice proliferative or wire loop-like glomerular lesions. Furthermore, deposition of gp70 in glomeruli and pathological changes were observed after intravenous injection of representative clones of purified anti-gp70 immunoglobulin G, demonstrating pathogenicity of at least some anti gp70 autoantibodies. PMID- 10756026 TI - Virus entry is a major determinant of cell tropism of Edmonston and wild-type strains of measles virus as revealed by vesicular stomatitis virus pseudotypes bearing their envelope proteins. AB - The Edmonston strain of measles virus (MV) that utilizes the human CD46 as the cellular receptor produced cytopathic effects (CPE) in all of the primate cell lines examined. In contrast, the wild-type MV strains isolated in a marmoset B cell line B95a (the KA and Ichinose strains) replicated and produced CPE in some but not all of the primate lymphoid cell lines. To determine the mechanism underlying this difference in cell tropism, we used a recently developed recombinant vesicular stomatitis virus (VSV) containing as a reporter the green fluorescent protein gene in lieu of the VSV G protein gene (VSVDeltaG*). MV glycoproteins were efficiently incorporated into VSVDeltaG*, producing the VSV pseudotypes. VSVDeltaG* complemented with VSV G protein efficiently infected all of the cell lines tested. The VSV pseudotype bearing the Edmonston hemagglutinin (H) and fusion (F) protein (VSVDeltaG*-EdHF) infected all cell lines in which the Edmonston strain caused CPE, including the rodent cell lines to which the human CD46 gene was stably transfected. The pseudotype bearing the wild-type KA H protein and Edmonston F protein (VSVDeltaG*-KAHF) infected all lymphoid cell lines in which the wild-type MV strains caused CPE as efficiently as VSVDeltaG* EdHF, but it did not infect any of the cell lines resistant to infection with the KA strain. The results indicate that the difference in cell tropism between these MV strains was largely determined by virus entry, in which the H proteins of respective MV strains play a decisive role. PMID- 10756025 TI - Anti-human immunodeficiency virus type 1 (HIV-1) CD8(+) T-lymphocyte reactivity during combination antiretroviral therapy in HIV-1-infected patients with advanced immunodeficiency. AB - The long-term efficacy of combination antiretroviral therapy may relate to augmentation of anti-human immunodeficiency virus type 1 (HIV-1) CD8(+) T-cell responses. We found that prolonged treatment of late-stage HIV-1-infected patients with a protease inhibitor and two nucleoside reverse transcriptase inhibitors failed to restore sustained, high levels of HIV-1-specific, HLA class I-restricted, cytotoxic-T-lymphocyte precursors and gamma interferon (IFN-gamma) production by CD8(+) T cells. In some patients, particularly those initiating three-drug combination therapy simultaneously rather than sequentially, there were early, transient increases in the frequency of anti-HIV-1 CD8(+) T cells that correlated with decreases in HIV-1 RNA and increases in T-cell counts. In the other patients, HIV-1-specific T-cell functions either failed to increase or declined from baseline during triple-drug therapy, even though some of these patients showed suppression of plasma HIV-1 RNA. These effects of combination therapy were not unique to HIV-1 specific T-cell responses, since similar effects were noted for CD8(+) T cells specific for the cytomegalovirus pp65 matrix protein. The level and breadth of CD8(+) cell reactivity to HLA A*02 HIV-1 epitopes, as determined by IFN-gamma production and HLA tetramer staining after combination therapy, were related to the corresponding responses prior to treatment. There was, however, a stable, residual population of potentially immunocompetent HIV-1-specific T cells remaining after therapy, as shown by tetramer staining of CD8(+) CD45RO(+) cells. These results indicate that new strategies will be needed to target residual, immunocompetent HIV-1-specific CD8(+) T cells to enhance the effectiveness of antiretroviral therapy in patients with advanced immunodeficiency. PMID- 10756027 TI - Mutations in the 2C region of poliovirus responsible for altered sensitivity to benzimidazole derivatives. AB - MRL-1237, [1-(4-fluorophenyl)-2-(4-imino-1,4-dihydropyridin-1-yl) methylbenzimidazole hydrochloride], is a potent and selective inhibitor of the replication of enteroviruses. To reveal the target molecule of MRL-1237 in viral replication, we selected spontaneous MRL-1237-resistant poliovirus mutants. Of 15 MRL-1237-resistant mutants obtained, 14 were cross-resistant to guanidine hydrochloride (mrgr), while 1 was susceptible (mrgs). Sequence analysis of the 2C region revealed that the 14 mrgr mutants contained a single nucleotide substitution that altered an amino acid residue from Phe-164 to Tyr. The mrgs mutant, on the other hand, contained a substitution of Ile-120 to Val. Through the construction of a cDNA-derived mutant, we confirmed that the single mutation at Phe-164 was really responsible for the reduced susceptibility to MRL-1237. MRL 1237 inhibited poliovirus-specific RNA synthesis in HeLa cells infected with a wild strain but not with an F164Y mutant. We furthermore examined the effect of mutations of the 2C region on the drug sensitivity of cDNA-derived guanidine resistant and -dependent mutants. Two guanidine-resistant mutants were cross resistant to MRL-1237 but remained susceptible to another benzimidazole, enviroxime. Either MRL-1237 or guanidine stimulated the viral replication of two guanidine-dependent mutants, but enviroxime did not. These results indicate that MRL-1237, like guanidine, targets the 2C protein of poliovirus for its antiviral effect. PMID- 10756028 TI - Simian immunodeficiency virus containing mutations in N-terminal tyrosine residues and in the PxxP motif in Nef replicates efficiently in rhesus macaques. AB - SIVmac Nef contains two N-terminal tyrosines that were proposed to be part of an SH2-ligand domain and/or a tyrosine-based endocytosis signal and a putative SH3 ligand domain (P(104)xxP(107)). In the present study, we investigated the effects of combined mutations in these tyrosine and proline residues on simian immunodeficiency virus (SIV) Nef interactions with the cellular signal transduction and endocytic machinery. We found that mutation of Y(28)F, Y(39)F, P(104)A, and P(107)A (FFAA-Nef) had little effect on Nef functions such as the association with the cellular tyrosine kinase Src, downregulation of cell surface expression of CD4 and class I major histocompatibility complex, and enhancement of virion infectivity. However, mutations in the PxxP sequence reduced the ability of Nef to stimulate viral replication in primary lymphocytes. Three macaques infected with the SIVmac239 FFAA-Nef variant showed high viral loads during the acute phase of infection. Reversions in the mutated prolines were observed between 12 and 20 weeks postinfection. Importantly, reversion of A(107)- >P, which restored the ability of Nef to coprecipitate a 62-kDa phosphoprotein in in vitro kinase assays, did not precede the development of a high viral load. The Y(28)/Y(39)-->F(28)/F(39) substitutions did not revert. In conclusion, mutations in both the tyrosine residues and the putative SH3 ligand domain apparently do not disrupt major aspects of SIV Nef function in vivo. PMID- 10756029 TI - Intrahepatic induction of alpha/beta interferon eliminates viral RNA-containing capsids in hepatitis B virus transgenic mice. AB - We have previously shown that hepatitis B virus (HBV) replication is abolished in the liver of HBV transgenic mice by stimuli that induce alpha/beta interferon (IFN-alpha/beta) in the liver. The present study was done to identify the step(s) in HBV replication that is affected by this cytokine in transgenic mice treated with the IFN-alpha/beta inducer polyinosinic-polycytidylic acid [poly(I-C)]. Here we show that the pool of cytoplasmic HBV pregenomic RNA (pgRNA)-containing capsids is reduced 10-fold within 9 h after poly(I-C) administration, while there is no change in the abundance of HBV mRNA or in the translational status of cytoplasmic HBV transcripts. In addition, we show that the pool of HBV DNA containing capsids is not reduced to the same degree until at least 15 h posttreatment, and we show that virus export is not accelerated and the half-life of virions in the serum is unchanged. These results indicate that IFN-alpha/beta triggers intracellular events that either inhibit the assembly of pgRNA containing capsids or accelerate their degradation, and that maturation and secretion of virus is responsible for clearance of HBV capsids and their cargo of replicative intermediates from the cytoplasm of the hepatocyte. PMID- 10756030 TI - Microarray analysis identifies interferon-inducible genes and Stat-1 as major transcriptional targets of human papillomavirus type 31. AB - Human papillomaviruses (HPVs) infect keratinocytes and induce proliferative lesions. In infected cells, viral gene products alter the activities of cellular proteins, such as Rb and p53, resulting in altered cell cycle response. It is likely that HPV gene products also alter expression of cellular genes. In this study we used microarray analysis to examine the global changes in gene expression induced by high-risk HPV type 31 (HPV31). Among 7,075 known genes and ESTs (expressed sequence tags) tested, we found that 178 were upregulated and 150 were downregulated twofold or more in HPV31 cells compared to normal human keratinocytes. While no specific pattern could be deduced from the list of genes that were upregulated, downregulated genes could be classified to three groups: genes that are involved in the regulation of cell growth, genes that are specifically expressed in keratinocytes, and genes whose expression is increased in response to interferon stimulation. The basal level of expression of several interferon-responsive genes was found to be downregulated in HPV31 cells by both microarray analysis and Northern blot analysis in different HPV31 cell lines. When cells were treated with alpha or gamma interferon, expression of interferon inducible genes was impaired. At high doses of interferon, the effects were less pronounced. Among the genes repressed by HPV31 was the signal transducer and activator of transcription (Stat-1), which plays a major role in mediating the interferon response. Suppression of Stat-1 expression may contribute to a suppressed response to interferon as well as immune evasion. PMID- 10756031 TI - A global neutralization resistance phenotype of human immunodeficiency virus type 1 is determined by distinct mechanisms mediating enhanced infectivity and conformational change of the envelope complex. AB - We have described previously genetic characterization of neutralization resistant, high-infectivity, and neutralization-sensitive, low-infectivity mutants of human immunodeficiency virus type 1 (HIV-1) MN envelope. The distinct phenotypes of these clones are attributable to six mutations affecting functional interactions between the gp120 C4-V5 regions and the gp41 leucine zipper. In the present study we examined mechanisms responsible for the phenotypic differences between these envelopes using neutralization and immunofluorescence assays (IFA). Most monoclonal antibodies (MAbs) tested against gp120 epitopes (V3, CD4 binding site, and CD4-induced) were 20 to 100 times more efficient at neutralizing pseudovirus expressing sensitive rather than resistant envelope. By IFA cells expressing neutralization sensitive envelope bound MAbs to gp120 epitopes more, but gp41 epitopes less, than neutralization-resistant envelope. This binding difference appeared to reflect conformational change, since it did not correlate with the level of protein expression or gp120-gp41 dissociation. This conformational change was mostly attributable to one mutation, L544P, which contributes to neutralization resistance but not to infectivity enhancement. The V420I mutation, which contributes a major effect to both high infectivity and neutralization resistance, had no apparent effect on conformation. Notably, a conformation-dependent V3 neutralization epitope remained sensitive to neutralization and accessible to binding by MAbs on neutralization-resistant HIV 1 envelope. Sensitivity to sCD4 did not distinguish the clones, suggesting that the phenotypes may be related to post-CD4-binding effects. The results demonstrate that neutralization resistance can be determined by distinguishable effects of mutations, which cause changes in envelope conformation and/or function(s) related to infectivity. A conformation-dependent V3 epitope may be an important target for neutralization of resistant strains of HIV-1. PMID- 10756032 TI - Dysregulation of cyclin E gene expression in human cytomegalovirus-infected cells requires viral early gene expression and is associated with changes in the Rb related protein p130. AB - We have previously shown that many cell cycle regulatory gene products are markedly affected by infection of primary fibroblasts with human cytomegalovirus (HCMV) (F. M. Jault, J. M. Jault, F. Ruchti, E. A. Fortunato, C. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697-6704, 1995). One of these proteins, cyclin E, is a key determinant of cell cycle progression during G(1), and its mRNA levels are significantly increased in HCMV-infected fibroblasts (B. S. Salvant, E. A. Fortunato, and D. H. Spector, J. Virol. 72:3729 3741, 1998). To determine the molecular basis of this effect, we have examined the events that occur at the endogenous cyclin E promoter during the course of infection. In vivo dimethyl sulfate footprinting of the cyclin E promoter revealed several regions of protection and hypersensitivity that were unique to infected cells. In accord with this observation, we find that the virus-induced cyclin E transcripts initiate downstream of the start site identified in mock infected cells, in regions where these newly appearing protected and hypersensitive sites occur. Viral gene expression is required for this induction. However, the viral immediate-early proteins IE1-72 and IE2-86, either alone or in combination, cannot induce expression of the endogenous cyclin E. The virus must progress past the immediate-early phase and express an early gene product(s) for activation of cyclin E expression. Moreover, IE1-72 does not appear to be required, as infection of cells with an HCMV mutant containing a deletion in the IE1-72 gene leads to full upregulation of cyclin E expression. Using electrophoretic mobility shift assays with infected cell extracts and a region of the cyclin E promoter that includes two previously defined E2F sites as the probe, we detected the appearance of an infection-specific banding pattern. One of the infection-specific bands contained the proteins E2F-4, DP-1, and p130, which were maintained in the infected cells as uniquely phosphorylated species. These results suggest that an altered E2F-4-DP-1-p130 complex along with viral early gene expression may play a role in the transcriptional regulation of cyclin E mRNA during HCMV infection. PMID- 10756033 TI - A single amino acid change in nsP1 attenuates neurovirulence of the Sindbis-group alphavirus S.A.AR86. AB - S.A.AR86, a member of the Sindbis group of alphaviruses, is neurovirulent in adult mice and has a unique threonine at position 538 of nsP1; nonneurovirulent members of this group of alphaviruses encode isoleucine. Isoleucine was introduced at position 538 in the wild-type S.A.AR86 infectious clone, ps55, and virus derived from this mutant clone, ps51, was significantly attenuated for neurovirulence compared to that derived from ps55. Intracranial (i.c. ) s55 infection resulted in severe disease, including hind limb paresis, conjunctivitis, weight loss, and death in 89% of animals. In contrast, s51 caused fewer clinical signs and no mortality. Nevertheless, comparison of the virus derived from the mutant (ps51) and wild-type (ps55) S.A.AR86 molecular clones demonstrated that s51 grew as well as or better than the wild-type s55 virus in tissue culture and that viral titers in the brain following i.c. infection with s51 were equivalent to those of wild-type s55 virus. Analysis of viral replication within the brain by in situ hybridization revealed that both viruses established infection in similar regions of the brain at early times postinfection (12 to 72 h). However, at late times postinfection, the wild-type s55 virus had spread throughout large areas of the brain, while the s51 mutant exhibited a restricted pattern of replication. This suggests that s51 is either defective in spreading throughout the brain at late times postinfection or is cleared more rapidly than s55. Further evidence for the contribution of nsP1 Thr 538 to S.A.AR86 neurovirulence was provided by experiments in which a threonine residue was introduced at nsP1 position 538 of Sindbis virus strain TR339, which is nonneurovirulent in weanling mice. The resulting virus, 39ns1, demonstrated significantly increased neurovirulence and morbidity, including weight loss and hind limb paresis. These results demonstrate a role for alphavirus nonstructural protein genes in adult mouse neurovirulence. PMID- 10756034 TI - Frequent homologous recombination events between molecules of one RNA component in a multipartite RNA virus. AB - Brome mosaic bromovirus (BMV), a tripartite plus-sense RNA virus, has been used as a model system to study homologous RNA recombination among molecules of the same RNA component. Pairs of BMV RNA3 variants carrying marker mutations at different locations were coinoculated on a local lesion host, and the progeny RNA3 in a large number of lesions was analyzed. The majority of doubly infected lesions accumulated the RNA3 recombinants. The distribution of the recombinant types was relatively even, indicating that both RNA3 counterparts could serve as donor or as acceptor molecules. The frequency of crossovers between one pair of RNA3 variants, which possessed closely located markers, was similar to that of another pair of RNA3 variants with more distant markers, suggesting the existence of an internal recombination hot spot. The majority of crossovers were precise, but some recombinants had minor sequence modifications, possibly marking the sites of imprecise homologous crossovers. Our results suggest discontinuous RNA replication, with the replicase changing among the homologous RNA templates and generating RNA diversity. This approach can be easily extended to other RNA viruses for identification of homologous recombination hot spots. PMID- 10756035 TI - A single deletion in the membrane-proximal region of the Sindbis virus glycoprotein E2 endodomain blocks virus assembly. AB - The envelopment of the Sindbis virus nucleocapsid in the modified cell plasma membrane involves a highly specific interaction between the capsid (C) protein and the endodomain of the E2 glycoprotein. We have previously identified a domain of the Sindbis virus C protein involved in binding to the E2 endodomain (H. Lee and D. T. Brown, Virology 202:390-400, 1994). The C-E2 binding domain resides in a hydrophobic cleft with C Y180 and W247 on opposing sides of the cleft. Structural modeling studies indicate that the E2 domain, which is proposed to bind the C protein (E2 398T, 399P, and 400Y), is located at a sufficient distance from the membrane to occupy the C protein binding cleft (S. Lee, K. E. Owen, H. K. Choi, H. Lee, G. Lu, G. Wengler, D. T. Brown, M. G. Rossmann, and R. J. Kuhn, Structure 4:531-541, 1996). To measure the critical spanning length of the E2 endodomain which positions the TPY domain into the putative C binding cleft, we have constructed a deletion mutant, DeltaK391, in which a nonconserved lysine (E2 K391) at the membrane-cytoplasm junction of the E2 tail has been deleted. This mutant was found to produce very low levels of virus from BHK-21 cells due to a defect in an unidentified step in nucleocapsid binding to the E2 endodomain. In contrast, DeltaK391 produced wild-type levels of virus from tissue-cultured mosquito cells. We propose that the phenotypic differences displayed by this mutant in the two diverse host cells arise from fundamental differences in the lipid composition of the insect cell membranes which affect the physical and structural properties of membranes and thereby virus assembly. The data suggest that these viruses have evolved properties adapted specifically for assembly in the diverse hosts in which they grow. PMID- 10756036 TI - The phage infection process: a functional role for the distal linker region of bacteriophage protein 3. AB - The filamentous bacteriophage infects Escherichia coli by interaction with the F pilus and the TolQRA complex. The virus-encoded protein initiating this process is the gene 3 protein (g3p). The g3p molecule can be divided into three different domains separated by two glycine-rich linker regions. Though there has been extensive evaluation of the importance of the diverse domains of g3p, no proper function has so far been assigned to these linker regions. Through the design of mutated variants of g3p that were displayed on the surface of bacteriophage, we were able to elucidate a possible role for the distal glycine-rich linker region. A phage that displayed a g3p comprised of only the N1 domain, the first linker region, and the C-terminal domain was able to infect cells at almost the same frequency as the wild-type phage. This infection was proven to be dependent on the motif between amino acid residues 68 and 86 (i.e., the first glycine-rich linker region of g3p) and on F-pilus expression. PMID- 10756037 TI - Protective immunity in macaques vaccinated with a modified vaccinia virus Ankara based measles virus vaccine in the presence of passively acquired antibodies. AB - Recombinant modified vaccinia virus Ankara (MVA), encoding the measles virus (MV) fusion (F) and hemagglutinin (H) (MVA-FH) glycoproteins, was evaluated in an MV vaccination-challenge model with macaques. Animals were vaccinated twice in the absence or presence of passively transferred MV-neutralizing macaque antibodies and challenged 1 year later intratracheally with wild-type MV. After the second vaccination with MVA-FH, all the animals developed MV-neutralizing antibodies and MV-specific T-cell responses. Although MVA-FH was slightly less effective in inducing MV-neutralizing antibodies in the absence of passively transferred antibodies than the currently used live attenuated vaccine, it proved to be more effective in the presence of such antibodies. All vaccinated animals were effectively protected from the challenge infection. These data suggest that MVA FH should be further tested as an alternative to the current vaccine for infants with maternally acquired MV-neutralizing antibodies and for adults with waning vaccine-induced immunity. PMID- 10756039 TI - Detection of hepatitis B virus infection in wild-born chimpanzees (Pan troglodytes verus): phylogenetic relationships with human and other primate genotypes. AB - Infection with hepatitis B virus (HBV) was detected by serological testing for HBV surface antigen and by PCR assay for HBV DNA in serum samples from two common chimpanzees (Pan troglodytes subsp. verus) born in West Africa. The complete genome sequences obtained by nucleotide sequencing of overlapping DNA fragments amplified by PCR were compared with HBV variants recovered from other primates and with human genotypes A to F. Both chimpanzee sequences were 3, 182 nucleotides in length, and the surface gene sequence predicted the existence of a, d, and w serological determinants. Neither sequence contained stop codons in the precore region. On phylogenetic analysis, the HBV variants infecting the chimpanzees clustered together with a third chimpanzee HBV isolate independently obtained from an infected captive animal (A. J. Zuckerman, A. Thornton, C. R. Howard, K. N. Tsiquaye, D. M. Jones, and M. R. Brambell, Lancet ii:652-654, 1978), with an overall sequence similarity of >94%. This provides strong evidence for a chimpanzee-specific genotype of HBV which circulates in nature. These findings add to the recent evidence for infection in the wild of other Old and New World primates (gibbon, orangutan, and woolly monkey) with species-specific variants of HBV. There is no evidence for close phylogenetic clustering of variants found so far in primates with any of the established HBV genotypes from humans. With the new evidence for the widespread distribution of HBV in primates, hypotheses for the origins of human infection are reviewed. PMID- 10756038 TI - A single intramuscular injection of recombinant plasmid DNA induces protective immunity and prevents Japanese encephalitis in mice. AB - Plasmid vectors containing Japanese encephalitis virus (JEV) premembrane (prM) and envelope (E) genes were constructed that expressed prM and E proteins under the control of a cytomegalovirus immediate-early gene promoter. COS-1 cells transformed with this plasmid vector (JE-4B clone) secreted JEV-specific extracellular particles (EPs) into the culture media. Groups of outbred ICR mice were given one or two doses of recombinant plasmid DNA or two doses of the commercial vaccine JEVAX. All mice that received one or two doses of DNA vaccine maintained JEV-specific antibodies 18 months after initial immunization. JEVAX induced 100% seroconversion in 3-week-old mice; however, none of the 3-day-old mice had enzyme-linked immunosorbent assay titers higher than 1:400. Female mice immunized with this DNA vaccine developed plaque reduction neutralization antibody titers of between 1:20 and 1:160 and provided 45 to 100% passive protection to their progeny following intraperitoneal challenge with 5,000 PFU of virulent JEV strain SA14. Seven-week-old adult mice that had received a single dose of JEV DNA vaccine when 3 days of age were completely protected from a 50, 000-PFU JEV intraperitoneal challenge. These results demonstrate that a recombinant plasmid DNA which produced JEV EPs in vitro is an effective vaccine. PMID- 10756040 TI - The use of chimeric Venezuelan equine encephalitis viruses as an approach for the molecular identification of natural virulence determinants. AB - Venezuelan equine encephalitis (VEE) virus antigenic subtypes and varieties are considered either epidemic/epizootic or enzootic. In addition to epidemiological differences between the epidemic and enzootic viruses, several in vitro and in vivo laboratory markers distinguishing the viruses have been identified, including differential plaque size, sensitivity to interferon (IFN), and virulence for guinea pigs. These observations have been shown to be useful predictors of natural, equine virulence and epizootic potential. Chimeric viruses containing variety IAB (epizootic) nonstructural genes with variety IE (enzootic) structural genes (VE/IAB-IE) or IE nonstructural genes and IAB structural genes (IE/IAB) were constructed to systematically analyze and map viral phenotype and virulence determinants. Plaque size analysis showed that both chimeric viruses produced a mean plaque diameter that was intermediate between those of the parental strains. Additionally, both chimeric viruses showed intermediate levels of virus replication and virulence for guinea pigs compared to the parental strains. However, IE/IAB produced a slightly higher viremia and an average survival time 2 days shorter than the VE/IAB-IE virus. Finally, IFN sensitivity assays revealed that only one chimera, VE/IAB-IE, was intermediate between the two parental types. The second chimera, containing the IE nonstructural genes, was at least five times more sensitive to IFN than the IE parental virus and greater than 50 times more sensitive than the IAB parent. These results implicate viral components in both the structural and nonstructural portions of the genome in contributing to the epizootic phenotype and indicate the potential for epidemic emergence from the IE enzootic VEE viruses. PMID- 10756041 TI - The nucleotide sequence of koala (Phascolarctos cinereus) retrovirus: a novel type C endogenous virus related to Gibbon ape leukemia virus. AB - A novel retrovirus, morphologically consistent with mammalian C-type retroviruses, was detected by electron microscopy in mitogen-stimulated peripheral blood mononuclear cell cultures from 163 koalas and in lymphoma tissue from 3 koalas. PCR amplified provirus from the blood and tissues of 17 wild and captive koalas, and reverse transcriptase-PCR demonstrated viral mRNA, viral genomic RNA, and reverse transcriptase activity in koala serum and cell culture supernatants. Comparison of viral sequences derived from genomic DNA and mRNA showed identity indicative of a single retroviral species-here designated koala retrovirus (KoRV). Southern blot analysis of koala tissue genomic DNA using labelled KoRV probes demonstrated banding consistent with an endogenous retrovirus. Complete and apparently truncated proviruses were detected in DNA of both clinically normal koalas and those with hematopoietic disease. KoRV-related viruses were not detected in other marsupials, and phylogenetic analysis showed that KoRV paradoxically clusters with gibbon ape leukemia virus (GALV). The strong similarity between GALV and KoRV suggests that these viruses are closely related and that recent cross-host transmission has occurred. The complete proviral DNA sequence of KoRV is reported. PMID- 10756042 TI - Rescue of multiple viral functions by a second-site suppressor of a human immunodeficiency virus type 1 nucleocapsid mutation. AB - Human immunodeficiency type 1 (HIV-1) bearing the nucleocapsid (NC) mutation R10A/K11A is replication defective. After serial passage of the mutant virus in tissue culture, we isolated a revertant that retained the original mutation. It had acquired, in addition, a new mutation (E21K) that was formally demonstrated to be sufficient for restoration of viral replication. Detailed analysis of the replication defect of R10A/K11A revealed a threefold reduction in virion yield and a fivefold reduction in packaging of viral genomic RNA. Real-time PCR was then used to quantitate viral DNA synthesis following infection of Jurkat T cells. After adjustment for the assembly and packaging defects, a minor (twofold) reduction in synthesis of either strong-stop, full-length linear DNA or 2-LTR circles was observed with R10A/K11A virions, indicating that reverse transcription and nuclear transport of the viral genome were largely intact. However, after adjustment for the amounts of full-length or 2-LTR circles produced, R10A/K11A virions were at least 10-fold less infectious than wild type, indicating that viral DNA produced by the R10A/K11A mutant failed to integrate. Each of the above-mentioned defects was corrected by introduction of the second site compensatory mutation E21K. These results demonstrate that the replication defect of mutant R10A/K11A can be explained by impairment at multiple steps in the viral life cycle, most important among them being integration and RNA packaging. The E21K mutation is predicted to restore positive charge to the face of the R10A/K11A mutant NC protein that interacts with the HIV-1 SL3 RNA stem loop, emphasizing the importance of NC basic residues for HIV-1 replication. PMID- 10756043 TI - Apoptosis in coxsackievirus B3-caused diseases: interaction between the capsid protein VP2 and the proapoptotic protein siva. AB - Coxsackievirus B3 (CVB3) is a common factor in human myocarditis. Apoptotic events are present in CVB3-induced disease, but it is unclear how CVB3 is involved in apoptosis and which viral proteins may induce the apoptotic pathway. In this report we demonstrate that the human and murine proapoptotic protein Siva specifically interact with the CVB3 capsid protein VP2. Furthermore, the transcription of Siva is strongly induced in tissue of CVB3-infected mice and is present in the same area which is positively stained for apoptosis, CD27, and CD70. It has been proposed that Siva is involved in the CD27/CD70-transduced apoptosis. Therefore, we suggest a molecular mechanism through which apoptotic events contributes to CVB3-caused pathogenesis. PMID- 10756045 TI - Nucleic acid-dependent cross-linking of the nucleocapsid protein of Sindbis virus. AB - The assembly of the alphavirus nucleocapsid core is a multistep event requiring the association of the nucleocapsid protein with nucleic acid and the subsequent oligomerization of capsid proteins into an assembled core particle. Although the mechanism of assembly has been investigated extensively both in vivo and in vitro, no intermediates in the core assembly pathway have been identified. Through the use of both truncated and mutant Sindbis virus nucleocapsid proteins and a variety of cross-linking reagents, a possible nucleic acid-protein assembly intermediate has been detected. The cross-linked species, a covalent dimer, has been detected only in the presence of nucleic acid and with capsid proteins capable of binding nucleic acid. Optimum nucleic acid-dependent cross-linking was seen at a protein-to-nucleic-acid ratio identical to that required for maximum binding of the capsid protein to nucleic acid. Identical results were observed when cross-linking in vitro assembled core particles of both Sindbis and Ross River viruses. Purified cross-linked dimers of truncated proteins and of mutant proteins that failed to assemble were found to incorporate into assembled core particles when present as minor components in assembly reactions, suggesting that the cross-linking traps an authentic intermediate in nucleocapsid core assembly. Endoproteinase Lys-C mapping of the position of the cross-link indicated that lysine 250 of one capsid protein was cross-linked to lysine 250 of an adjacent capsid protein. Examination of the position of the cross-link in relation to the existing model of the nucleocapsid core suggests that the cross-linked species is a cross-capsomere contact between a pentamer and hexamer at the quasi-threefold axis or is a cross-capsomere contact between hexamers at the threefold axis of the icosahedral core particle and suggests several possible assembly models involving a nucleic acid-bound dimer of capsid protein as an early step in the assembly pathway. PMID- 10756044 TI - Virus-specific cofactor requirement and chimeric hepatitis C virus/GB virus B nonstructural protein 3. AB - GB virus B (GBV-B) is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species), making it an attractive surrogate virus for in vivo testing of anti-HCV inhibitors in a small monkey model. It has been reported that the nonstructural protein 3 (NS3) serine protease of GBV-B shares similar substrate specificity with its counterpart in HCV. Authentic proteolytic processing of the HCV polyprotein junctions (NS4A/4B, NS4B/5A, and NS5A/5B) can be accomplished by the GBV-B NS3 protease in an HCV NS4A cofactor-independent fashion. We further characterized the protease activity of a full-length GBV-B NS3 protein and its cofactor requirement using in vitro-translated GBV-B substrates. Cleavages at the NS4A/4B and NS5A/5B junctions were readily detectable only in the presence of a cofactor peptide derived from the central region of GBV-B NS4A. Interestingly, the GBV-B substrates could also be cleaved by the HCV NS3 protease in an HCV NS4A cofactor-dependent manner, supporting the notion that HCV and GBV-B share similar NS3 protease specificity while retaining a virus-specific cofactor requirement. This finding of a strict virus-specific cofactor requirement is consistent with the lack of sequence homology in the NS4A cofactor regions of HCV and GBV-B. The minimum cofactor region that supported GBV B protease activity was mapped to a central region of GBV-B NS4A (between amino acids Phe22 and Val36) which overlapped with the cofactor region of HCV. Alanine substitution analysis demonstrated that two amino acids, Val27 and Trp31, were essential for the cofactor activity, a finding reminiscent of the two critical residues in the HCV NS4A cofactor, Ile25 and Ile29. A model for the GBV-B NS3 protease domain and NS4A cofactor complex revealed that GBV-B might have developed a similar structural strategy in the activation and regulation of its NS3 protease activity. Finally, a chimeric HCV/GBV-B bifunctional NS3, consisting of an N-terminal HCV protease domain and a C-terminal GBV-B RNA helicase domain, was engineered. Both enzymatic activities were retained by the chimeric protein, which could lead to the development of a chimeric GBV-B virus that depends on HCV protease function. PMID- 10756046 TI - Brome mosaic virus polymerase-like protein 2a is directed to the endoplasmic reticulum by helicase-like viral protein 1a. AB - Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes RNA replication proteins 1a and 2a. 1a contains a C-terminal helicase-like domain and an N-terminal domain implicated in viral RNA capping, and 2a contains a central polymerase-like domain. 1a and 2a colocalize in an endoplasmic reticulum (ER)-associated replication complex that is the site of BMV specific RNA-dependent RNA synthesis in plant and yeast cells. 1a also localizes to the ER in the absence of 2a or viral RNA replication templates. To investigate the determinants of 2a localization, we fused 2a to the green fluorescent protein (GFP), creating a functional GFP-2a fusion that supported BMV RNA replication and subgenomic mRNA transcription. In the absence of 1a, the GFP-2a fusion was found to be diffused throughout the cytoplasm and in punctate spots not associated with any cytoplasmic organelle so far tested. Formation of these spots was dependent on the C-terminal half of 2a and may represent aggregation of a fraction of 2a. When coexpressed with 1a, GFP-2a colocalized with 1a and ER-resident protein Kar2p in a partial or complete ring around the nucleus. Consistent with these results, cell fractionation showed that both the GFP-2a fusion and wild-type (wt) 2a remained soluble when expressed alone, while in cells coexpressing 1a, most of the GFP-2a fusion or wt 2a cofractionated with 1a in the rapidly sedimenting membrane fraction. Deletion analysis showed that the N-terminal 120-amino-acid segment of 2a, containing one of two 2a regions previously shown to interact with 1a, was necessary and sufficient for 1a-directed localization of GFP-2a derivatives to the ER. These results suggest that 1a, which also interacts independently with the ER and viral RNA, is a key organizer of RNA replication complex assembly. PMID- 10756047 TI - Infectious bronchitis virus E protein is targeted to the Golgi complex and directs release of virus-like particles. AB - The coronavirus E protein is a poorly characterized small envelope protein present in low levels in virions. We are interested in the role of E in the intracellular targeting of infectious bronchitis virus (IBV) membrane proteins. We generated a cDNA clone of IBV E and antibodies to the E protein to study its cell biological properties in the absence of virus infection. We show that IBV E is an integral membrane protein when expressed in cells from cDNA. Epitope specific antibodies revealed that the C terminus of IBV E is cytoplasmic and the N terminus is translocated. The short luminal N terminus of IBV E contains a consensus site for N-linked glycosylation, but the site is not used. When expressed using recombinant vaccinia virus, the IBV E protein is released from cells at low levels in sedimentable particles that have a density similar to that of coronavirus virions. The IBV M protein is incorporated into these particles when present. Indirect immunofluorescence microscopy showed that E is localized to the Golgi complex in cells transiently expressing IBV E. When coexpressed with IBV M, both from cDNA and in IBV infection, the two proteins are colocalized in Golgi membranes, near the coronavirus budding site. Thus, even though IBV E is present at low levels in virions, it is apparently expressed at high levels in infected cells near the site of virus assembly. PMID- 10756048 TI - Dominant role of host selective pressure in driving hepatitis C virus evolution in perinatal infection. AB - The dynamics of the genetic diversification of hepatitis C virus (HCV) populations was addressed in perinatal infection. Clonal sequences of hypervariable region 1 of the putative E2 envelope protein of HCV were obtained from four HCV-infected newborns (sequential samples spanning a period of 6 to 13 months after birth) and from their mothers (all samples collected at delivery). The data show that the variants detected between birth and the third month of life in samples from the four newborns were present in the HCV populations of their mothers at delivery. In the newborns, a unique viral variant (or a small group of closely related variants) remained stable for weeks despite active viral replication. Diversification of the intrahost HCV population was observed 6 to 13 months after birth and was substantially higher in two of the four subjects, as documented by the intersample genetic distance (GD) (P = 0.007). Importantly, a significant correlation between increasing GD and high values for the intersample K(a)/K(s) ratio (the ratio between anoffymous and synonymous substitutions; an index of the action of selective forces) was observed, as documented by the increase of both parameters over time (P = 0.01). These data argue for a dominant role of positive selection for amino acid changes in driving the pattern of genetic diversification of HCV populations, indicate that the intrahost evolution of HCV populations is compatible with a Darwinian model system, and may have implications in the designing of future antiviral strategies. PMID- 10756049 TI - Reevaluation of amino acid variability of the human immunodeficiency virus type 1 gp120 envelope glycoprotein and prediction of new discontinuous epitopes. AB - To elucidate the evolutionary mechanisms of the human immunodeficiency virus type 1 gp120 envelope glycoprotein at the single-site level, the degree of amino acid variation and the numbers of synonymous and nonsynonymous substitutions were examined in 186 nucleotide sequences for gp120 (subtype B). Analyses of amino acid variabilities showed that the level of variability was very different from site to site in both conserved (C1 to C5) and variable (V1 to V5) regions previously assigned. To examine the relative importance of positive and negative selection for each amino acid position, the numbers of synonymous and nonsynonymous substitutions that occurred at each codon position were estimated by taking phylogenetic relationships into account. Among the 414 codon positions examined, we identified 33 positions where nonsynonymous substitutions were significantly predominant. These positions where positive selection may be operating, which we call putative positive selection (PS) sites, were found not only in the variable loops but also in the conserved regions (C1 to C4). In particular, we found seven PS sites at the surface positions of the alpha-helix (positions 335 to 347 in the C3 region) in the opposite face for CD4 binding. Furthermore, two PS sites in the C2 region and four PS sites in the C4 region were detected in the same face of the protein. The PS sites found in the C2, C3, and C4 regions were separated in the amino acid sequence but close together in the three-dimensional structure. This observation suggests the existence of discontinuous epitopes in the protein's surface including this alpha-helix, although the antigenicity of this area has not been reported yet. PMID- 10756050 TI - Dominant-negative inhibition of prion formation diminished by deletion mutagenesis of the prion protein. AB - Polymorphic basic residues near the C terminus of the prion protein (PrP) in humans and sheep appear to protect against prion disease. In heterozygotes, inhibition of prion formation appears to be dominant negative and has been simulated in cultured cells persistently infected with scrapie prions. The results of nuclear magnetic resonance and mutagenesis studies indicate that specific substitutions at the C-terminal residues 167, 171, 214, and 218 of PrP(C) act as dominant-negative, inhibitors of PrP(Sc) formation (K. Kaneko et al., Proc. Natl. Acad. Sci. USA 94:10069-10074, 1997). Trafficking of substituted PrP(C) to caveaola-like domains or rafts by the glycolipid anchor was required for the dominant-negative phenotype; interestingly, amino acid replacements at multiple sites were less effective than single-residue substitutions. To elucidate which domains of PrP(C) are responsible for dominant-negative inhibition of PrP(Sc) formation, we analyzed whether N-terminally truncated PrP(Q218K) molecules exhibited dominant-negative effects in the conversion of full-length PrP(C) to PrP(Sc). We found that the C-terminal domain of PrP is not sufficient to impede the conversion of the full-length PrP(C) molecule and that N terminally truncated molecules (with residues 23 to 88 and 23 to 120 deleted) have reduced dominant-negative activity. Whether the N-terminal region of PrP acts by stabilizing the C-terminal domain of the molecule or by modulating the binding of PrP(C) to an auxiliary molecule that participates in PrP(Sc) formation remains to be established. PMID- 10756052 TI - Cultured cell sublines highly susceptible to prion infection. AB - Cultured cell lines infected with prions produce an abnormal isoform of the prion protein (PrP(Sc)). In order to derive cell lines producing sufficient quantities of PrP(Sc) for most studies, it has been necessary to subclone infected cultures and select the subclones producing the largest amounts of PrP(Sc). Since postinfection cloning can introduce differences between infected and uninfected cell lines, we sought an approach to generate prion-infected cell lines that would avoid clonal artifacts. Using an improved cell blot technique, which permits sensitive and rapid comparison of PrP(Sc) levels in multiple independent cell cultures, we discovered marked heterogeneity with regard to prion susceptibility in tumor cell sublines. We exploited this heterogeneity to derive sublines which are highly susceptible to prion infection and used these cells to generate prion-infected lines without further subcloning. These infected sublines can be compared to the cognate uninfected cultures without interference from cloning artifacts. We also used susceptible cell lines and our modified cell blot procedure to develop a sensitive and reproducible quantitative cell culture bioassay for prions. We found that the sublines were at least 100-fold more susceptible to strain RML prions than to strain ME7 prions. Comparisons between scrapie-susceptible and -resistant cell lines may reveal factors that modulate prion propagation. PMID- 10756051 TI - Unusual polymorphisms in human immunodeficiency virus type 1 associated with nonprogressive infection. AB - Factors accounting for long-term nonprogression may include infection with an attenuated strain of human immunodeficiency virus type 1 (HIV-1), genetic polymorphisms in the host, and virus-specific immune responses. In this study, we examined eight individuals with nonprogressing or slowly progressing HIV-1 infection, none of whom were homozygous for host-specific polymorphisms (CCR5 Delta32, CCR2-64I, and SDF-1-3'A) which have been associated with slower disease progression. HIV-1 was recovered from seven of the eight, and recovered virus was used for sequencing the full-length HIV-1 genome; full-length HIV-1 genome sequences from the eighth were determined following amplification of viral sequences directly from peripheral blood mononuclear cells (PBMC). Longitudinal studies of one individual with HIV-1 that consistently exhibited a slow/low growth phenotype revealed a single amino acid deletion in a conserved region of the gp41 transmembrane protein that was not seen in any of 131 envelope sequences in the Los Alamos HIV-1 sequence database. Genetic analysis also revealed that five of the eight individuals harbored HIV-1 with unusual 1- or 2-amino-acid deletions in the Gag sequence compared to subgroup B Gag consensus sequences. These deletions in Gag have either never been observed previously or are extremely rare in the database. Three individuals had deletions in Nef, and one had a 4-amino-acid insertion in Vpu. The unusual polymorphisms in Gag, Env, and Nef described here were also found in stored PBMC samples taken 3 to 11 years prior to, or in one case 4 years subsequent to, the time of sampling for the original sequencing. In all, seven of the eight individuals exhibited one or more unusual polymorphisms; a total of 13 unusual polymorphisms were documented in these seven individuals. These polymorphisms may have been present from the time of initial infection or may have appeared in response to immune surveillance or other selective pressures. Our results indicate that unusual, difficult-to-revert polymorphisms in HIV-1 can be found associated with slow progression or nonprogression in a majority of such cases. PMID- 10756053 TI - Consequences of Fas-mediated human dendritic cell apoptosis induced by measles virus. AB - Mortality from measles virus (MV) infection is caused mostly by secondary infections associated with a pronounced immunosuppression. Dendritic cells (DCs) represent a major target of MV and could be involved in immunosuppression. In this study, human monocyte-derived DCs were used to demonstrate that DC apoptosis in MV-infected DC-T-cell cocultures is Fas mediated, whereas apoptotic T cells could not be rescued by blocking the Fas pathway. Two novel consequences of DC apoptosis after MV infection were demonstrated. (i) Fas-mediated apoptosis of DCs facilitates MV release, while CD40 activation enhances MV replication in DCs. Indeed, detailed studies of infectious MV release and intracellular MV nucleoprotein (NP) showed that inhibition of CD40-CD40L ligand interaction blocks NP synthesis. We conclude that the CD40 ligand expressed by activated T cells first enhances MV replication in DCs, and then Fas ligand produced by activated T cells induces Fas-mediated apoptosis of DCs, thus facilitating MV release. (ii) Not only MV-infected DCs but also bystander uninfected DCs undergo a maturation process confirmed by CD1a, CD40, CD80, CD86, CD83, and major histocompatibility complex type II labeling. The bystander maturation effect results from contact and/or engulfment of MV-induced apoptotic DCs by uninfected DCs. A model is proposed to explain how both a specific immune response and immunosuppression can simultaneously occur after MV infection through Fas-mediated apoptosis and CD40 activation of DCs. PMID- 10756054 TI - Stable high-level expression of heterologous genes in vitro and in vivo by noncytopathic DNA-based Kunjin virus replicon vectors. AB - Primary features of the flavivirus Kunjin (KUN) subgenomic replicons include continuous noncytopathic replication in host cell cytoplasm and the ability to be encapsidated into secreted virus-like particles (VLPs). Previously we reported preparation of RNA-based KUN replicon vectors and expression of heterologous genes (HG) in cell culture after RNA transfection or after infection with recombinant KUN VLPs (A. N. Varnavski and A. A. Khromykh, Virology 255:366-375, 1999). In this study we describe the development of the next generation of KUN replicon vectors, which allow synthesis of replicon RNA in vivo from corresponding plasmid DNAs. These DNA-based vectors were able to direct stable expression of beta-galactosidase (beta-Gal) in several mammalian cell lines, and expression remained high ( approximately 150 pg per cell) throughout cell passaging. The applicability of these vectors in vivo was demonstrated by beta Gal expression in the mouse lung epithelium for at least 8 weeks after intranasal inoculation and induction of anti-beta-Gal antibody response after intramuscular inoculation of the beta-Gal-encoding KUN replicon DNA. The noncytopathic nature of DNA-based KUN replicon vectors combined with high-level and stability of HG expression in a broad range of host cells should prove them to be useful in a variety of applications in vitro and in vivo. PMID- 10756056 TI - A mutation in human immunodeficiency virus type 1 protease, N88S, that causes in vitro hypersensitivity to amprenavir. AB - Amprenavir (Agenerase, 141-W94, VX-478) is a human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PRI) recently approved for the treatment of HIV-1 infection in the United States. A major cause of treatment failure is the development of resistance to PRIs. One potential use for amprenavir is as salvage therapy for patients for whom treatment that includes one (or more) of the other four currently approved PRIs-saquinavir, indinavir, ritonavir, and nelfinavir-has failed. We evaluated the cross-resistance to amprenavir of viruses that evolved during treatment with the two most commonly prescribed PRIs, nelfinavir and indinavir. Unexpectedly, a dramatic increase in susceptibility (2.5- to 12. 5 fold) was observed with 20 of 312 (6.4%) patient viruses analyzed. The most pronounced increases in susceptibility were strongly associated with an N88S mutation in protease. All viruses that carried the N88S mutation were hypersensitive to amprenavir. Site-directed mutagenesis studies confirmed the causal role of N88S in determining amprenavir hypersensitivity. The presence of the N88S mutation and associated amprenavir hypersensitivity may be useful in predicting an improved clinical response to amprenavir salvage therapy. PMID- 10756055 TI - N-linked glycosylation of CXCR4 masks coreceptor function for CCR5-dependent human immunodeficiency virus type 1 isolates. AB - The chemokine receptors CXCR4 and CCR5 are the principal coreceptors for infection of X4 and R5 human immunodeficiency virus type 1 (HIV-1) isolates, respectively. Here we report on the unexpected observation that the removal of the N-linked glycosylation sites in CXCR4 potentially allows the protein to serve as a universal coreceptor for both X4 and R5 laboratory-adapted and primary HIV-1 strains. We hypothesize that this alteration unmasks existing common extracellular structures reflecting a conserved three-dimensional similarity of important elements of CXCR4 and CCR5 that are involved in HIV envelope glycoprotein (Env) interaction. These results may have far-reaching implications for the differential recognition of cell type-dependent glycosylated CXCR4 by HIV 1 isolates and their evolution in vivo. They also suggest a possible explanation for the various observations of restricted virus entry in some cell types and further our understanding of the framework of elements that represent the Env coreceptor contact sites. PMID- 10756057 TI - Retroviral vector targeting to human immunodeficiency virus type 1-infected cells by receptor pseudotyping. AB - We report the generation of retroviral vectors based on Moloney murine leukemia virus that specifically transduce cells infected with T-cell-tropic human immunodeficiency virus type 1 (HIV-1). This vector was pseudotyped with T-cell tropic HIV-1 receptors CD4 and CXCR4. We demonstrate that transduction is contingent upon HIV-1 gp120 and gp41 expression. PMID- 10756058 TI - Expression and characterization of the Borna disease virus polymerase. AB - Borna disease virus is the prototype of a new family, Bornaviridae, within the order Mononegavirales, that is characterized by nuclear transcription, splicing, low level replication, and neurotropism. The products of five open reading frames predicted from the genomic sequence have been confirmed; however, expression of the sixth, corresponding to the putative viral polymerase (L), has not been demonstrated. Here, we describe expression and characterization of a 190-kDa protein proposed to represent L. Expression of this protein from the third transcription unit of the viral genome is dependent on a splicing event that fuses a small upstream open reading frame in frame with the larger downstream continuous open reading frame. The protein is detected by serum antibodies from infected rats and is present in the nucleus, where it colocalizes with the phosphoprotein. L is also shown to be phosphorylated by cellular kinases and to interact with the viral phosphoprotein in coimmunoprecipitation studies. These findings are consistent with the identity of the 190-kDa protein as the viral polymerase and provide insights and describe reagents that will be useful for Bornavirus molecular biology and pathobiology. PMID- 10756059 TI - High frequency of virus-specific interleukin-2-producing CD4(+) T cells and Th1 dominance during lymphocytic choriomeningitis virus infection. AB - Analysis of C57BL/6 mice acutely infected with lymphocytic choriomeningitis virus (LCMV) by using intracellular cytokine staining revealed a high frequency (2 to 10%) of CD4(+) T cells secreting the Th1-associated cytokines interleukin-2 (IL 2), gamma interferon (IFN-gamma), and tumor necrosis factor alpha, with no concomitant increase in the frequency of CD4(+) T cells secreting the Th2 associated cytokines IL-4, IL-5, and IL-10 following stimulation with viral peptides. In LCMV-infected C57BL/6 CD8(-/-) mice, more than 20% of the CD4(+) T cells secreted IFN-gamma after viral peptide stimulation, whereas less than 1% of the CD4(+) T cells secreted IL-4 under these same conditions. Mice persistently infected with a high dose of LCMV clone 13 also generated a virtually exclusive Th1 response. Thus, LCMV induces a much more profound virus-specific CD4(+) T cell response than previously recognized, and it is dramatically skewed to a Th1 phenotype. PMID- 10756060 TI - Envelope glycoprotein determinants of increased fusogenicity in a pathogenic simian-human immunodeficiency virus (SHIV-KB9) passaged in vivo. AB - Changes in the envelope glycoprotein ectodomains of a nonpathogenic simian-human immunodeficiency virus (SHIV-89.6) that was serially passaged in vivo have been shown to be responsible for the increased pathogenicity of the resulting virus, SHIV-KB9 (G. B. Karlsson, et al., J. Exp. Med. 188:1159-1171, 1998). The 12 amino acid changes in the envelope glycoprotein ectodomains resulted in increased chemokine receptor-binding and syncytium-forming abilities. Here we identify the envelope glycoprotein determinants of these properties. A single amino acid change in the gp120 third variable (V3) loop was both necessary and sufficient for the observed increase in the binding of the SHIV-KB9 gp120 glycoprotein to the CCR5 chemokine receptor. The increased syncytium-forming ability of SHIV-KB9 involved, in addition to the V3 loop change, changes in the second conserved (C2) region of gp120 (residue 225) and in the gp41 ectodomain (residues 564 and 567). The C2 and gp41 ectodomain changes influenced syncytium formation in a cooperative manner. Changes in the V1/V2 gp120 variable loops exerted a negative effect on syncytium formation and chemokine receptor binding, supporting a previously described role of these changes in immune evasion. The definition of the passage-associated changes that determine the efficiency of chemokine receptor binding and membrane fusogenicity will allow evaluation of the contribution of these properties to in vivo CD4-positive lymphocyte depletion. PMID- 10756061 TI - High levels of TIMP-2 correlate with adverse prognosis in breast cancer. AB - TIMP-2 is an endogenous inhibitor of MMPs. Most data from model systems suggest that high levels of this inhibitor prevent metastasis. In human breast cancers, however, we show that high levels of TIMP-2 correlate with both shortened disease free interval and overall survival. In primary breast cancers, TIMP-2 levels showed no significant relationship with either tumor size or axillary node status but correlated inversely with estrogen receptor levels. TIMP-2 levels also correlated significantly with those for TIMP-1. We conclude that high levels of endogenous TIMP-2, like other protease inhibitors such as PAI-1 and TIMP-1, correlate with progression of human breast cancer. PMID- 10756062 TI - Stage-specific effects of bone morphogenetic proteins on the oligodendrocyte lineage. AB - Oligodendrocyte maturation is regulated by multiple secreted factors present in the brain during critical stages of development. Whereas most of these factors promote oligodendrocyte proliferation and survival, members of the bone morphogenetic protein family (BMPs) recently have been shown to inhibit oligodendrocyte differentiation in vitro. Oligodendrocyte precursors treated with BMPs differentiate to the astrocyte lineage. Given that cells at various stages of the oligodendrocyte lineage have distinct responses to growth factors, we hypothesized that the response to BMP would be stage-specific. Using highly purified, stage-specific cultures, we found that BMP has distinct effects on cultured oligodendrocyte preprogenitors, precursors, and mature oligodendrocytes. Oligodendrocyte preprogenitors (PSA-NCAM+, A2B5-) treated with BMP2 or BMP4 developed a novel astrocyte phenotype characterized by a morphological change and expression of glial fibrillary acidic protein (GFAP) but little glutamine synthetase expression and no labeling with A2B5 antibody. In contrast, treating oligodendrocyte precursors with BMPs resulted in the accumulation of cells with the traditional type 2 astrocyte phenotype (GFAP+, A2B5+). However, many of the cells with an astrocytic morphology did not express GFAP or glutamine synthetase unless thyroid hormone was present in the medium. The addition of fibroblast growth factor along with BMP to either oligodendrocyte preprogenitor or the oligodendrocyte precursor cells inhibited the switch to the astrocyte lineage, whereas platelet-derived growth factor addition had no effect. Treatment of mature oligodendrocytes with BMP elicited no change in morphology or expression of GFAP. These data suggest that as cells progress through the oligodendrocyte lineage, they show developmentally restricted responses to the BMPs. PMID- 10756063 TI - Spatial and temporal changes in natural and target deprivation-induced cell death in the mouse inferior olive. AB - The survival of inferior olive neurons is dependent on contact with cerebellar Purkinje cells. There is evidence that this dependence changes with time. Because inferior olivary axons, called climbing fibers, already show significant topographical ordering in cerebellar target zones during late embryogenesis in mice, the question arises as to whether olive neurons are dependent on target Purkinje cells for their survival at this early age. To better characterize this issue, inferior olive development was studied in two transgenic mouse mutants, wnt-1 and L7ADT, with embryonic and early postnatal loss of cerebellar target cells, respectively, and compared to that in the well-studied mutant, Lurcher. Morphological criteria as well as quantitative measures of apoptosis were considered in this developmental analysis. Survival of inferior olive neurons is observed to be independent of Purkinje cells throughout embryogenesis, but dependence begins immediately at birth in both wild types and mutants. Thereafter, wild types and mutants show a rapid increase in olive cell apoptosis, with a peak at postnatal day 4, followed by a period of low-level, but significant, apoptosis that continues to at least postnatal day 11; the main difference is that apoptosis is quantitatively enhanced in the mutants compared to wild types. The multiphasic course of these effects roughly parallels the known phases of climbing fiber synaptogenesis. In addition, despite significant temporal differences among the mutants with respect to absolute numbers of dying cells, there are common spatial features suggestive of distinct intrinsic programs linking different olivary subnuclei to their targets. PMID- 10756064 TI - Progenitor cells with the capacity to differentiate into sympathetic-like neurons are transiently detected in mammalian embryonic dorsal root ganglia. AB - We have identified a population of progenitor cells in embryonic dorsal root ganglia (DRG) of the mouse. These cells can be induced into proliferating and differentiating into neurons that display multiple characteristics of the sympathetic lineage by a brief exposure to a defined medium. The sympathetic neuronal characteristics include immunoreactivity for the transcription factors MASH-1 and Phox2a and for the neurotransmitter markers tyrosine hydroxylase, choline acetyltransferase, and NPY. The progenitor cells are detected in DRG from embryonic day 11.5 (E11.5) to E15. An identical time course is observed in primary cultures of neural crest, suggesting that the competency of these cells is intrinsically regulated. We also present evidence that the environment of the sensory ganglion, unlike that of a sympathetic ganglion, prevents these cells from differentiating into neurons. PMID- 10756065 TI - Cutaneous overexpression of neurotrophin-3 (NT3) selectively restores sensory innervation in NT3 gene knockout mice. AB - Neurotrophin-3 (NT3) is essential for development of sensory innervation to the skin. NT3 supports the postnatal survival of primary sensory neurons that mediate mechanoreception and their Merkel cell containing touch dome end organs (Airaksinen et al., 1996). In this study we determined whether NT3 overexpressed in the skin could restore innervation lost when endogenous NT3 levels were reduced. Hybrid mice that overexpress NT3 in basal keratinocytes but lack one endogenous NT3 allele (K14-NT3/NT3(+/-)) were compared to NT3 overexpresser (K14 NT3) mice, heterozygous knockout (NT3(+/-)) mice, and littermate control mice. In line with previous analyses, NT3(+/-) mice lost 63% of the Merkel cells associated with touch domes, 67% of touch dome units and the associated SAI innervation. All of these parameters were restored to overexpresser levels in K14 NT3/NT3(+/-) mice. Knockout NT3(+/-) mice also had a 31% reduction of L4/L5 dorsal root ganglion cells and a 24% reduction of myelinated axons in the saphenous cutaneous nerve. These losses were also restored in hybrid K14 NT3/NT3(+/-) mice, though only to control mouse values. These results indicate that overexpression of NT3 in skin of NT3(+/-) knockout mice rescued most cutaneous neurons lost in NT3(+/-) mice, but was unable to rescue NT3-dependent neurons that project to noncutaneous sensory targets. PMID- 10756066 TI - Cell turnover in the vomeronasal epithelium: evidence for differential migration and maturation of subclasses of vomeronasal neurons in the adult opossum. AB - Previous investigations of cell turnover in the mammalian vomeronasal sensory epithelium (VN-SE) raised two issues. First, if, in addition to the already demonstrated vertical migration, horizontal migration from the edges of the VN-SE participates in neuronal replacement. Second, whether or not migration and maturation is differential in upper and lower populations of vomeronasal neurons, since these two cell populations are chemically, physiologically, functionally, and perhaps evolutionarily different. By injecting bromodeoxyuridine (BrdU) into adult opossum (Monodelphis domestica) and permitting different survival times, the pattern of distribution of BrdU-labeled cells was analyzed. No evidence of horizontal migration in neuronal replacement was found. To investigate vertical migration and maturation of subclasses of vomeronasal neurons, double immunohistochemistry of BrdU and markers of the lower (G(oalpha) protein) and upper [G(i2alpha) protein and olfactory marker protein (OMP)] cell populations were performed. Three days after administration of BrdU, some mature neurons were observed in both lower and upper layers of the VN-SE, as demonstrated by coexpression of BrdU with G(oalpha) protein and OMP, respectively. The data on vertical distribution, however, indicate that most of the daughter cells enter the G(oalpha)-protein-expressing zone of the VN-SE by day 5, whereas most daughter cells do not reach the G(i2alpha)-protein-expressing zone until day 7, suggesting that these two populations mature at slightly different rates. These results are the first evidence of differential neurogenesis of subclasses of vomeronasal neurons. PMID- 10756067 TI - Estrogen-regulated developmental neuronal apoptosis is determined by estrogen receptor subtype and the Fas/Fas ligand system. AB - Adult sexual dimorphism in neuronal cell number is controlled by estrogen exposure during a tightly defined period of rat brain development. The mechanisms of estrogen's effect are unknown; one possibility is regulation of programmed cell death (apoptosis). In this study we have shown that estradiol can function as a neuroprotective agent or an inducer of apoptosis, depending on the estrogen receptor-subtype present in the cell. Thus, ERalpha has a neuroprotective effect, while ERbeta mediates the induction of apoptosis in neuronal cells. Moreover, we show that estrogen-induced apoptosis through ER-beta requires the expression of Fas- and Fas ligand (FasL) proteins, since the absence of FasL in neurons prevents this effect. Furthermore, we demonstrate that microglia-secreted products induce the expression of FasL necessary to mediate estradiol-ERbeta apoptotic effect. These findings may explain the dichotomous effect of fetal estradiol on the adult neuronal number. PMID- 10756068 TI - The relationship between rates of HVc neuron addition and vocal plasticity in adult songbirds. AB - In adulthood, songbird species vary considerably in the extent to which they rely on auditory feedback to maintain a stable song structure. The continued recruitment of new neurons into vocal motor circuitry may contribute to this lack of resiliency in song behavior insofar as new neurons that are not privy to auditory instruction could eventually corrupt established neural function. In a first step to explore this possibility, we used a comparative approach to determine if species differences in the rate of vocal change after deafening in adulthood correlate positively with the extent of HVc neuron addition. We confirmed previous reports that deafening in adulthood changes syllable phonology much more rapidly in bengalese finches than in zebra finches. Using [(3)H]thymidine autoradiography to identify neurons generated in adulthood, we found that the proportion of new neurons in the HVc one month after labeling was nearly twice as great in bengalese than in zebra finches. Moreover, among the subset of HVc vocal motor neurons that project to the robust nucleus of the archistriatum, the incidence of [(3)H]thymidine-labeled neurons was nearly three times as great in bengalese than in zebra finches. This correlation between the proportion of newly added neurons and the rate of song deterioration supports the hypothesis that HVc neuron addition may disrupt stable adult song production if new neurons cannot be "trained" via auditory feedback. PMID- 10756069 TI - Synaptic ultrastructure in nerve terminals of Drosophila larvae overexpressing the learning gene dunce. AB - We investigated synaptic ultrastructure of individual nerve ending varicosities at the Drosophila larval neuromuscular junction in transgenic larvae overexpressing the learning gene dunce (dnc) in the nervous system. It was previously shown that cAMP is reduced to one-third normal in these larvae and that they have fewer nerve terminal varicosities and smaller junction potentials, although transmitter release from individual nerve ending varicosities is not significantly altered. We tested the hypothesis that synaptic ultrastructure is modified to compensate for possible reduced efficacy of synaptic transmission resulting from lower than normal cAMP. Synaptic size and number of presynaptic dense bodies (active zone structures) per synapse are modestly enhanced in transgenic larvae overexpressing the dnc gene product and in rutabaga (rut(1)) mutant larvae, which have reduced adenylyl cyclase activity and reduced neural cAMP. The incidence of complex synapses (possessing 2 or more presynaptic dense bodies) was not consistently different in experimental larvae compared to controls. The observations suggest that chronic reduction of cAMP levels in the nervous system of Drosophila larvae, although leading to a modest compensatory change in synaptic structure, does not markedly alter several synaptic ultrastructural parameters which are thought to influence the strength of transmitter release; thus, homeostatic mechanisms do not act to maintain normal sized junction potentials by altering synaptic structure. PMID- 10756070 TI - Neuron/target matching between chorda tympani neurons and taste buds during postnatal rat development. AB - During postnatal development, a relationship is established between the size of individual taste buds and number of innervating neurons. To determine whether rearrangement of neurons that innervate taste buds establishes this relationship, we labeled single taste buds at postnatal day 10 (P10) and again at either P15, P20, or P40 with retrograde fluorescent neuronal tracers. The number of single- and double-labeled geniculate ganglion cells was counted, and the respective taste bud volumes were measured for the three groups of rats. The current study replicates findings from an earlier report demonstrating that the larger the taste bud, the more geniculate ganglion cells that innervate it. This relationship between taste bud size and number of innervating neurons is not apparent until P40, when taste bud size reaches maturity. These findings are extended here by demonstrating that the number of neurons that innervate taste buds at P10, when taste bud size is small and relatively homogeneous, predicts the size that the respective taste bud will become at maturity. Moreover, while there is some neural rearrangement of taste bud innervation from P10 to P40, rearrangement does not impact the relationship between taste bud size and innervating neurons. That is, the neurons that maintain contact with taste buds from P10 through P40 accurately predict the mature taste bud size. Therefore, the size of the mature taste bud is determined by P10 and relates to the number of sensory neurons that innervate it at that age and the number of neurons that maintain contact with it throughout the first 40 days of postnatal development. PMID- 10756072 TI - Preferential expression of myelencephalon-specific protease by oligodendrocytes of the adult rat spinal cord white matter. AB - Myelencephalon-specific protease (MSP) is a novel serine protease that is expressed predominantly in the nervous system. In the adult rat spinal cord, MSP mRNA expression was dramatically upregulated, in both the white and gray matter, after systemic exposure to the glutamate receptor agonist, kainic acid (KA) (Scarisbrick et al. J Neurosci 17: 8156-8168, 1997b). To determine the cell specific expression patterns of MSP, we generated MSP-specific monoclonal antibodies. These have been used in immunohistochemical and in situ hybridization colocalization studies, to demonstrate that MSP mRNA and protein are produced predominantly by CNP-immunoreactive oligodendroglia, but not by GFAP immunoreactive astrocytes, in the white matter of the normal adult cord. In vitro, the soma of oligodendrocytes were also densely MSP immunoreactive, as were their growth tips, while astrocytes were associated with lower levels. These findings suggest that the enzymatic activity of MSP is likely to be important in the biology of oligodendrocytes and/or in the maintenance of the nerve fiber tracts of the adult spinal cord. PMID- 10756071 TI - Transplantation of human olfactory ensheathing cells elicits remyelination of demyelinated rat spinal cord. AB - Human olfactory ensheathing cells (OECs) were prepared from adult human olfactory nerves, which were removed during surgery for frontal base tumors, and were transplanted into the demyelinated spinal cord of immunosuppressed adult rats. Extensive remyelination was observed in the lesion site: In situ hybridization using a human DNA probe (COT-1) indicated a similar number of COT-1-positive cells and OEC nuclei within the repaired lesion. The myelination was of a peripheral type with large nuclei and cytoplasmic regions surrounding the axons, characteristic of Schwann cell and OEC remyelination. These results provide evidence that adult human OECs are able to produce Schwann cell-like myelin sheaths around demyelinated axons in the adult mammalian CNS in vivo. PMID- 10756073 TI - Fibroblast growth factors-5 and -9 distinctly regulate expression and function of the gap junction protein connexin43 in cultured astroglial cells from different brain regions. AB - Astroglial cells contribute to neuronal maintenance and function in the normal and diseased brain. Gap junctions formed predominantly by connexin43 (cx43) provide important pathways to coordinate astroglial responses. We have previously shown that fibroblast growth factor (FGF)-2, which occurs ubiquitously in the CNS, downregulates gap junction communication in cortical and striatal, but not in mesencephalic astroglial cells in vitro (Reuss et al. Glia 22:19-30, 1998). Other members of the FGF family expressed in the CNS include FGF-5 and FGF-9. We show that both FGF-5 and FGF-9, like FGF-2, downregulate astroglial gap junctions and functional coupling. However, their effects are strikingly different from different brain regions, with regard to astroglial cells. FGF-5 specifically affects mesencephalic astroglial cells without changing coupling of cortical and striatal astroglia, while FGF-9 reduces gap junctional coupling in astroglia from all three brain regions. Both cx43 mRNA and protein levels as well as functional coupling assessed by dye spreading are affected. To clarify whether brain region specific effects of FGFs on astroglial coupling are due to differential expression of FGF receptors (FGFR), we monitored expression of the four known FGFR mRNAs in astroglial cultures by RT-PCR. Irrespective of their regional origin, astroglial cells express mRNAs for FGFR-2 and FGFR-3. In summary, our results provide evidence for an important role of FGF-2, -5, and -9 in a distinct, CNS region-specific regulation mechanism of astroglial gap junction communication. The molecular basis underlying the regionally distinct responsiveness of astrocytes to different FGFs may be sought beyond distinct FGFR expression. PMID- 10756074 TI - Evidence for cell specific regulation by PACAP38 of the proenkephalin gene expression in neocortical cells. AB - During the first postnatal week, glial cell production for the neocortex continues in the neocortical subventricular zone. During this time, the proenkephalin gene (PEnk) is expressed in numerous cells of the subventricular zone and of the adjacent neocortex. When neocortical astroglial cells are brought into dissociation culture, they also produce PEnk mRNA. We have investigated the effect of pituitary adenylate cyclase activating peptide-38 (PACAP38) on PEnk gene expression in dissociation cultures as well as in slice cultures, which contained the subventricular zone and the adjacent neocortex. PACAP38 enhanced the levels of PEnk mRNA in both culture systems. In dissociated astroglial cells, inhibition of protein kinase A, of p44,42 mitogen-activated protein kinase as well as of the EGF-receptor tyrosine kinase by H89, PD98059 and AG1478, respectively, reduced the PACAP38-induced expression in a synergistic manner. In the neocortical part of the slice cultures, the effect of PACAP38 on PEnk gene expression was inhibited only by H89 and PD98059. Here, protein kinase A and p44,42 MAP kinases shared a mechanism which increased the gene expression. Surprisingly, the expression of the PEnk gene in the glial progenitors of the subventricular zone as induced by PACAP38 was not affected by any of the three protein kinase inhibitors, but was blocked by the unspecific kinase inhibitor H7. It is concluded that PACAP38 induced the PEnk gene expression in both culture systems in a cell-type specific manner. PMID- 10756075 TI - Activation of the JAK/STAT pathway following transient focal cerebral ischemia: signaling through Jak1 and Stat3 in astrocytes. AB - JAK/STAT is one of the pathways bearing signals from the cell membrane to the nucleus in response to extracellular growth factors and cytokines. In the present study, we examined the cellular distribution of Jak1 and Stat3, and activation of the JAK/STAT pathway following transient focal cerebral ischemia in the rat. Jak1 was mainly seen in white matter astrocytes and in certain neurons. Notably, large pyramidal neurons of cortical layer V showed the highest neuronal Jak1 expression within cerebral cortex and, in addition, expressed Stat3 indicating that the JAK/STAT pathway is involved in signaling in the corticofugal projection system. Shortly following ischemia, Jak1 immunoreactive astrocytes located in the ipsilateral neighbouring white matter and ischemic cortex and striatum showed nuclear translocation of Stat3. These features were maintained in large reactive astrocytes that surrounded the infarct from 3 to 7 days. At these later times, the abundant reactive microglia/macrophages were strongly immunoreactive to Stat3 and, to a lesser extent, Jak1. Two main protein complexes showing DNA binding activity at the sis-inducible element site were found under basal conditions, followed by changes in this pattern following ischemia concomitant with neuronal cell loss and activation of glia. This study showed basal cerebral activity of JAK/STAT signaling pathway, involving Jak1 and Stat3 proteins, and selective activation following ischemia. It is suggested that the kinase activity of Jak1 mediates nuclear translocation of Stat3 in astrocytes, and that this signaling pathway is involved in the astroglial response to focal cerebral ischemia. PMID- 10756076 TI - Neurons and astrocytes express EPO mRNA: oxygen-sensing mechanisms that involve the redox-state of the brain. AB - Erythropoietin (Epo), the major hormone controlling the hypoxia-induced increase in the number of erythrocytes, has also a functional role in the brain. However, few data exist as to the cellular source of brain-derived Epo as well as to the molecular mechanisms that control Epo expression in the central nervous system. Using patch-clamp and RT-PCR methods, we provide direct evidence that, besides astrocytes, neurons are a source of Epo in the brain. Both the astrocytic and neuronal expression of Epo mRNA are induced not only by hypoxia, but also by desferrioxamine (DFX) and cobalt chloride (CoCl(2)), two agents known to mimic the hypoxic induction of Epo in hepatoma cells. This induction is blocked by cycloheximide suggesting that de novo protein synthesis is required. Furthermore, the addition of H(2)O(2) decreases the hypoxia-induced Epo mRNA levels. These data indicate that, following hypoxia, a common oxygen sensing and signaling pathway leads to increased Epo gene expression in both nervous and hepatoma cells; this pathway would be dependent on the redox-state of the brain. Furthermore, we show that the in vivo administration of CoCl(2) and DFX to mice induces an increased Epo mRNA level in the neocortex. As Epo protects the brain against ischemia, our in vivo experiments suggest that the use of molecules such as CoCl(2) or DFX, that provoke an increased Epo gene expression in the brain, could be useful in the development of potential therapeutic strategies for the treatment of hypoxic or ischemic brain injury. PMID- 10756077 TI - Interleukin-1beta-induced expression of monocyte chemotactic protein-1 in the rabbit retina: an in situ and immunohistochemical study. AB - In this study, the temporal and spatial expression of the chemotactic factor monocyte chemotactic protein-1 (MCP-1) was examined in the rabbit retina after challenge with the proinflammatory cytokine interleukin-1beta (IL-1). In these tissues, IL-1 induces an acute inflammatory response of the epiretinal vessels that peaks approximately 24 h postintraocular injection (pi) with the cytokine. At 2 h after challenge with IL-1, MCP-1 mRNA was expressed by perivascular microglial cells and astrocytes that form the glial limitans. Protein analysis at 3 h pi with IL-1 confirmed these sites of MCP-1 expression. The intensity of the mRNA and protein signals increased at 6 h and at 24 h. At these time points, MCP 1 message and protein also were detected in infiltrating macrophages and, at the latest time point, in endothelial cells as well. These data support the conclusion that IL-1 provides a strong stimulus for the rapid expression of MCP-1 mRNA and protein in retinal tissues, and they further support the role of endogenous glial cells as important sources of mediators involved in the regulation of inflammation occurring within the nervous system. PMID- 10756078 TI - Platelet-derived growth factor-BB supports the survival of cultured rat Schwann cell precursors in synergy with neurotrophin-3. AB - To understand the intimate axon-Schwann cell relationship required for the accurate development and regeneration of the peripheral nervous system (PNS), it is important to elucidate the repertoire of growth factors involved in this tightly regulated bi-directional dialogue. We focused on the identification and functional characterization of receptor tyrosine kinases (RTKs) in Schwann cells to gain insights into the corresponding growth factor ligands, which may be regulating the highly controlled differentiation of the Schwann cell lineage. Using an RT-PCR based differential display approach, we have identified 17 tyrosine kinases in embryonic rat sciatic nerves during the crucial transition from Schwann cell precursors to early Schwann cells. In this study, we have examined the expression and function of TrkC and the platelet-derived growth factor (PDGF) receptors alpha and beta on Schwann cell precursor cells. These receptors are expressed on freshly isolated Schwann cell precursors, and we show that PDGF-BB is able to rescue a subpopulation of these cells from apoptotic cell death in vitro. Furthermore, the TrkC-ligand neurotrophin-3 (NT-3) can act synergistically to potentiate this effect. However, PDGF-BB and NT-3 do not induce Schwann cell precursor proliferation or differentiation. Our data are consistent with a model suggesting that a combination of growth factors that include PDGF-BB and NT-3 are acting in concert and in synergy to regulate early Schwann cell development. PMID- 10756079 TI - Formation of myelin after transplantation of neural precursor cells into the retina of young postnatal mice. AB - We have isolated neural precursors from the striata of embryonic wild-type and transgenic mice ubiquitously expressing enhanced green fluorescent protein. Cells were expanded in vitro in the presence of epidermal growth factor and transplanted into the retina of young postnatal mice. One month after transplantation, cells showed widespread integration into the host tissue and differentiated into a variety of morphologically distinct cell types. A fraction of cells was identified as oligodendrocytes exclusively located in the immediate vicinity to the nerve fiber layer. Similar results were obtained with neural precursors isolated from embryonic spinal cord. Differentiated oligodendrocytes and myelin were still detectable in the host tissue 4 months after transplantation, the latest time point investigated. Remarkably, prolonged survival periods of experimental animals resulted in a significant increase in the number of donor-derived oligodendrocytes and the area of the nerve fiber layer being myelinated. The presence of high numbers of oligodendrocytes and their location close to the retinal nerve fiber layer suggest that the differentiation of transplanted neural precursors into distinct neural cell types is influenced by host-derived environmental cues. PMID- 10756081 TI - B cell apoptosis in the central nervous system in experimental autoimmune encephalomyelitis: roles of B cell CD95, CD95L and Bcl-2 expression. AB - The role and fate of B cells in the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE) are unknown. Using enzyme-linked immunospot assays we now show that B cells reactive to myelin basic protein (MBP) accumulate in the CNS of Lewis rats with acute EAE induced by immunization with MBP and adjuvants. We also report that B cells are eliminated from the CNS by apoptosis during spontaneous recovery from this disease. Apoptotic B cells were identified by flow cytometry of inflammatory cells extracted from the spinal cord and by histological sections of the spinal cord using light and electron microscopic immunocytochemistry. B cell apoptosis occurred preferentially in the CNS rather than in the peripheral lymphoid organs and was maximal just prior to the onset of spontaneous clinical recovery. Three colour flow cytometry indicated that B cells expressing CD95 (Fas) or CD95 ligand (CD95L) were highly vulnerable to apoptosis, whereas B cells expressing Bcl-2 were relatively protected from apoptosis. We propose that B cells are eliminated from the CNS by the interaction of CD95L and CD95 on the same B cell and that this contributes to the spontaneous resolution of CNS inflammation and clinical recovery in acute EAE. PMID- 10756080 TI - Upregulation of phospholipase D in astrocytes in response to transient forebrain ischemia. AB - Previous in vitro studies using cell cultures or brain slices have demonstrated that phospholipase D (PLD) in the nervous system is involved in the signaling mechanism in response to a variety of agonists. However, little is known about the pathophysiological role of PLD-mediated signaling in the adult brain. We examined the changes in the expression of a PLD isozyme, PLD1, in the adult rat hippocampus, using immunological approaches and an assay for PLD activity after transient forebrain ischemia (four-vessel occlusion model) that results in the selective delayed death of CA1 pyramidal cells and induces reactive astrocytes in the CA1 subfield. In the control hippocampus, PLD1 the level of immunoreactivity was very low. After ischemia, in parallel with the results of Western blot analysis and the PLD activity assay, immunohistochemical analysis of PLD1 demonstrated that the immunoreactive proteins peaked at 7-14 days and were most prominent in the CA1 and the dentate hilar region. The temporal and spatial patterns of immunoreactivity of both PLD1 and glial fibrillary acidic protein (GFAP) were very similar, indicating that reactive astrocytes express PLD1, confirmed by double staining for PLD1 and GFAP. These results demonstrate that reactive astrocytes upregulate PLD in vivo after injury in the adult rat hippocampus. PMID- 10756082 TI - Treatment of established relapsing experimental autoimmune encephalomyelitis with the proteasome inhibitor PS-519. AB - PLP139-151-induced relapsing experimental autoimmune encephalomyelitis (R-EAE) in SJL mice is a Th1-mediated autoimmune demyelinating disease model for multiple sclerosis (MS) in which the primary disease relapse is mediated by T cells specific for the endogenous PLP178-191 epitope. This complex inflammatory process requires the co-ordinated expression of a wide variety of immune-related genes active at a variety of stages of the autoimmune process which are regulated, in part, by the transcription factor nuclear factor (NF)-kappaB which is activated via the ubiquitin-proteasome pathway. We asked if in vivo administration of a selective inhibitor of the ubiquitin-proteasome pathway, PS-519, which downregulates activation of NF-kappaB, could downregulate ongoing R-EAE. Administration of PS-519 during the remission phase, following acute clinical disease was effective in significantly reducing the incidence of clinical relapses, CNS histopathology, and T cell responses to both the initiating and relapse-associated PLP epitopes. The inhibition of clinical disease was dependent upon continuous administration of PS-519 in that recovery of T cell function and onset of disease relapses developed within 10-14 days of drug withdrawal. The data suggest that targeting the ubiquitin proteasome pathway, in particular NF kappaB, may offer a novel and efficacious approach for the treatment of progressive autoimmune diseases, including MS. PMID- 10756083 TI - TGF-beta1 inhibits protracted-relapsing experimental autoimmune encephalomyelitis by activating dendritic cells. AB - Protracted-relapsing experimental autoimmune encephalomyelitis (PR-EAE) in DA rats is an animal model closely related to multiple sclerosis (MS). Previous studies showed that nasal administration of TGF-beta1 suppressed the development and relapse of PR-EAE clinically and pathologically. Here we demonstrate that this suppressive effect was associated with activation of dendritic cells (DC), showing elevated proliferative response and IFN-gamma and nitric oxide (NO) production by DC. DC derived from TGF-beta1-treated rats with PR-EAE also showed high expression of nitric oxide synthase 2 (NOS2) at both mRNA and protein levels. Apoptotic cells were increased in spleen sections of TGF-beta1-treated rats compared to control rats. In studying mechanisms of apoptosis in TGF-beta1 treated rats, in vitro experiments demonstrated that TGF-beta1-treated DC induced apoptosis of CD4(+)T cells by a NO pathway after co-culture with T cells. These results support the hypothesis that TGF-beta1-induced suppression of PR-EAE is associated with apoptosis of CD4(+)T cells induced by DC-derived NO. PMID- 10756084 TI - Phosphatidylserine expression on cell surfaces promotes antibody-dependent aggregation and thrombosis in beta2-glycoprotein I-immune mice. AB - Beta-2-glycoprotein I (beta2GP1) has been implicated as the primary antigenic target in antiphospholipid syndrome. To study the role beta2GP1 antibodies play in thrombosis associated with this syndrome, the clearance and binding of phosphatidylserine (PS)-containing target membranes were monitored in beta2GP1 immune mice. Clearance in immune mice (T(1/2)4.8 min) was faster than in normal mice (T(1/2)11.0 min). Analysis of PS vesicles recovered from immune mice by sequencing and Western blotting showed the presence of bound beta2GP1 and autologous antibody, respectively. Bleeding times in immune mice were approximately 30% shorter than in control mice. In vitro clotting times, however, were the same in both populations. To determine if the in vivo results could be attributed to the interaction of autoantibodies with the vascular endothelium, the binding of PS-containing target membranes to normal and apoptotic endothelial cells was studied. While endothelial cells bound PS vesicles, beta2GP1 reduced uptake by approximately 50% in both normal and apoptotic endothelium. In the presence of beta2GP1 antibodies, however, uptake in apoptotic cells, but not normal cells, increased by more than two-fold. These results suggest that thrombosis in antiphospholipid syndrome could, in part, be due to antibody dependent cross-linking of beta2GP1 bound to PS-expressing cells and the vascular endothelium. PMID- 10756085 TI - Dual overlapping peptides recognized by insulin peptide B:9-23 T cell receptor AV13S3 T cell clones of the NOD mouse. AB - T cells isolated from islets of non-obese diabetic (NOD) mice are enriched for insulin-reactive cells. The great majority of these T cells recognize insulin B chain peptide (B:9-23). B:9-23 reactive T cell clones are diabetogenic and show a dramatic TCR alpha -chain restriction (predominant AV13S3). We have studied the reactivity of five different B:9-23 reactive T cell clones to truncated peptides and alanine substituted analogues of B:9-23. Amongst these AV13S3 T cell clones, one reacted with peptide B:9-16 and four with B:13-23. The two peptides have in common only four amino acids (B:13-16; EALY). Having defined minimal peptide epitopes, we evaluated a mutant insulin sequence (B:13 glutamine) which retains metabolic activity. As predicted, this single amino acid change abrogated T cell reactivity. In addition, we have created a modified I-A(g7)gene with the B:9-23 peptide covalently linked to I-A(g7). Antigen presenting cells transfected with this construct were excellent presenting cells for all clones studied. The definition of dual peptide motifs and creation of bioactive covalent I-A(g7)-B:9 23 should facilitate studies of the pathogenic significance and antigen recognition by B:9-23 reactive diabetogenic T cells. PMID- 10756086 TI - Immune-mediated cholangiohepatitis in neonatally thymectomized mice: the role of T cells and analysis of T-cell receptor Vbeta usage. AB - We have previously reported the induction of immune-mediated cholangiohepatitis following injection of a hybrid recombinant proteins containing the E2 of the pyruvate dehydrogenase (PDC-E2) and the branched-chain keto-acid dehydrogenase (BCOADC-E2) to neonatally thymectomized (Tx) A/J mice. Further, we demonstrated that intrahepatic infiltrating mononuclear cells could transfer pathology to other Tx mice. To further our observations, we examined intrahepatic infiltrating mononuclear cells by flow cytometry and used cell transfer experiments to identify the phenotype involved. Interestingly, following immunization of neonatally Tx A/J mice and immunization with the bihybrid molecule, the number of CD3+infiltrating mononuclear cells were significantly higher (77.8%) compared with the control group. There was a small although not significant increase among intrahepatic infiltrating mononuclear cells and splenic cells of Vbeta 5.1,5.2+, Vbeta7+and Vbeta17+. In addition, Vbeta14+cells accounted for 20.4% of the infiltrating T-cells (P<0.01 vs. the control group). In further experiments, CD3+, CD4+or CD8+cells were isolated and removed from intrahepatic infiltrating mononuclear cells and subpopulations of mononuclear cells transferred to Tx mice. Both CD3+CD4+cells and CD3+CD8+cells are required for development of the lesion, and the damage is mediated by CD3+Vbeta14+cells. PMID- 10756087 TI - Nucleotide variations amongst V(H)Genes of AMA-producing B cell clones in primary biliary cirrhosis. AB - Primary biliary cirrhosis, a chronic liver disease characterized by progressive inflammatory destruction of intrahepatic bile ducts, is also characterized by the presence of antimitochondrial antibodies (AMA). The predominant autoantibody is directed at the E2 component of pyruvate dehydrogenase (PDC-E2). Recent studies of this autoantibody response have analysed immunoglobulin-variable regions of human monoclonal antibodies and provided evidence for antigen-driven clonal selection. However, the number of clones analysed has been very limited and the presence of somatic mutations not formally proven. In this study, we took advantage of three stable B cell lines producing human IgG anti-PDC-E2 mAbs from a patient with PBC. We analysed the V(H)and V(L)gene structure of these reagents and, in addition, analysed 10 V(H)-D and D-J(H)sequences over a period of nearly 3 years. The expressed Ig V regions of the heavy chain (V(H)) and the light chain (V(L)) genes of mAb18, mAb37, and mAb82 utilized the V(H)III-VlambdaI, V(H)IV VlambdaIII, and V(H)IV-V(k)IV gene families, respectively. The utilized gene elements were Ig gene elements that were found frequently in other antibodies with different specificity and affinity. Presence of somatic point-mutations was confirmed in mAb82 by comparison of the expressed V(H)gene sequence with that of corresponding germline V(H)gene obtained from the granulocyte genomic DNA of the same patient. Interestingly, clonally related B cells were consistently found throughout the observation period and nucleotide variations among the V(H)genes were very few, ranging from 0.19 to 0.72% per base. These findings suggest that long-lived B cell clones can exist and may contribute, at least in part, to maintenance of autoantibodies in humans. PMID- 10756088 TI - Prevalence and clinical significance of anti-phospholipid antibodies in multiple sclerosis: a study of 89 patients. AB - The prevalence of serum anti-phospholipid antibodies (aPL) was evaluated in multiple sclerosis (MS) patients to search for a possible association with a distinct form of the disease. Anti-cardiolipin antibodies (Ab) (aCL) and anti beta 2 glycoprotein I Ab (abeta2GPI) were measured together with antinuclear Ab (ANA), anti-double-stranded DNA Ab (anti-ds DNA) and anti-myelin Ab in 89 patients. Twenty-nine (32.6%) patients had serum aPL, 19xaCL (15 of the IgG and four of the IgM isotype), 14 abeta2GPI (two IgG and 12 IgM) (four of these patients had both Ab). Prevalence of aCL correlated with that of ANA, which were positive in 52 patients (P=0. 005) and with anti-myelin Ab detected in two patients (P=0.046) but not with that of anti-ds DNA (mostly of the IgM class) detected in 28% of case by ELISA. No correlation could be found between aPL and age, sex, duration of the disease from diagnosis, category of MS, clinical course, clinical symptoms, serum IgM levels nor atypical lesions by magnetic resonance imaging. Hence, aCL and abeta2GPI are neither rare in MS nor associated with a specific clinical form of the disease and they cannot be a diagnosis exclusion criteria. PMID- 10756089 TI - Kinetics of anti-fibrillin-1 autoantibodies in MCTD and CREST syndrome. AB - Using a highly sensitive Radioimmunoassay (RIA), the kinetics of synthesis of anti-fibrillin (Fbn-1) autoantibodies were studied in 17 patients with mixed connective tissue disease (MCTD) and two with CREST syndrome calcinosis, Raynaud's oesophageal dismotility, sclerodectyly and teleangiectasis who were found to be positive for this autoimmune response. IgG autoantibodies specific for recombinant Fbn-1 (rFbn-1) (aa 369-425) were found in all patients excepting one with MCTD, multiple sclerosis, and dermatomyositis. IgM were found in fewer cases. Several kinetics patterns of anti-Fbn-1 autoantibodies were observed: a) long lasting persistence of IgG and IgM autoantibodies up to 14 years; b) fluctuation of antibodies during various periods up to 16 years; c) disappearance of antibody response after several years, and d) patients producing IgG but not IgM autoantibodies. No differences in the synthesis of autoantibodies were observed between MCTD patients with a stable disease, and those developing during the course features of systemic sclerosis (SSc), Sjogren's syndrome, or rheumatoid-like arthritis. In one patient displaying a lupus-like syndrome for 3 years, the appearance of anti-Fbn-1 autoantibodies coincided with the occurrence of MCTD and scleroderma. While the detection of anti-Fbn-1 autoantibodies may be clinically useful in differential diagnosis or eventual prognosis of patients with connective tissue diseases, their role in the pathogenesis of scleroderma syndromes requires further investigation. PMID- 10756090 TI - Characterization of a highly complex region in Xq13 and mapping of three isodicentric breakpoints associated with preleukemia. AB - The chromosomal abnormality represented by an isodicentric X chromosome [idic(X)(q13)] is associated with a subset of acute myeloid leukemia (AML) and preleukemia observed in elderly females. A previous study localized the breakpoints of two acquired isodicentric X chromosomes associated with myelodysplasia to a 450-kb region proximal to the XIST gene. Here we report the construction and extensive characterization of a reliable 1-Mb P1 artificial chromosome and bacterial artificial chromosome contig covering a highly problematic region in Xq13 that includes the previously described isodicentric breakpoint region. In addition to mapping of the brain-specific gene (NAP1L2) and the phosphoglyceryl kinase alpha subunit 1 gene (PHKA1) and generation and mapping of a large number of STSs throughout the contig, we have mapped a putative transcriptional regulatory protein (HDACL1), and 35 ESTs. Sequencing data, Southern blot analysis, and fiber-FISH analysis have permitted characterization of extensive region-specific duplications and triplications in addition to an unusually high concentration of long interspersed repeat elements, both of which could be implicated in isodicentric chromosome formation and other Xq13 chromosome aberrations. FISH analysis of metaphase chromosomes from two previously unpublished AML patients and one preleukemic patient using cosmid clones and selected subclones allowed mapping of the idic(X)(q13) breakpoints to a 100-kb interval, consistent with the involvement of an X-linked gene in the genesis of this form of preleukemia, disruption of which may represent a preliminary step in progression to AML. Assembly and physical mapping of this complex 1-Mb contig establish a foundation for ongoing sequencing and gene identification projects in the region. PMID- 10756091 TI - Cloning and characterization of the murine toll-like receptor 5 (Tlr5) gene: sequence and mRNA expression studies in Salmonella-susceptible MOLF/Ei mice. AB - Toll-like receptors (TLRs) are a group of evolutionarily conserved pattern recognition receptors involved in the activation of the immune system in response to various pathogens. In this paper, we describe the cloning and characterization of the mouse homologue of human TLR5. Mouse Tlr5 encodes a 859-amino-acid protein that contains an N-terminal signal sequence, a leucine-rich repeat extracellular domain, a short transmembrane domain typical of type I transmembrane proteins, and a Toll/interleukin-1R signaling domain characteristic of all TLR proteins. The mouse Tlr5 protein shows 81% homology to human TLR5 and approximately 40% similarity to other TLR family members. Northern blot analysis reveals that Tlr5 is expressed predominantly in liver and lung with low-level expression in most other tissues examined. We have mapped Tlr5 to distal chromosome 1 using the (C57BL/6J x Mus spretus) x C57BL/6J Jackson BSB panel as well as a (C57BL/6J x MOLF/Ei)F(2) panel with the following position: D1Mit112-8.0 cM-Tlr5-9.6 cM D1Mit17. The presence of a quantitative trait locus for susceptibility to Salmonella typhimurium on distal chromosome 1 prompted the examination of Tlr5 in susceptible MOLF/Ei mice. Polymorphic sequence variants in Tlr5 allowed us to identify a unique 4-allele haplotype in MOLF/Ei. Furthermore, using both Northern blot analysis and reverse transcription-polymerase chain reaction, we have shown a reduced expression of Tlr5 during infection of MOLF/Ei mice with Salmonella. The assignment of Tlr5 to a chromosomal region known to harbor a Salmonella susceptibility locus together with decreased expression of Tlr5 mRNA in liver of susceptible MOLF/Ei mice suggests the possibility that, as with other members of this family, Tlr5 may play a role in host response to bacterial gram-negative infections. PMID- 10756092 TI - Identification and characterization of a novel polycystin family member, polycystin-L2, in mouse and human: sequence, expression, alternative splicing, and chromosomal localization. AB - Polycystins-1, -2, -L, and -REJ are the four known members of the polycystin family of proteins. In this study, we describe a fifth member of the family, polycystin-L2, encoded by PKD2L2 in human and Pkd2l2 in mouse. Full-length cDNA sequences for both mouse and human polycystin-L2 were obtained from testis cDNA. Sequence analysis predicts that the mouse and human polycystin-L2 proteins consist of 621 and 624 amino acid residues, respectively. Polycystin-L2 has significant homology with polycystins-L and -2, with similarities of 58 and 59%, respectively. Both human and murine polycystin-L2 proteins are predicted to have seven putative transmembrane (TM) domains, and, by comparison with transient receptor potential channels, the six carboxyl-terminal TM domains are likely to constitute an ion channel subunit. Northern blot analysis indicated that mouse Pkd2l2 has an abundant approximately 2.5-kb transcript in testis and an approximately 2.2-kb transcript in heart. RT-PCR analysis showed that the full length transcript is expressed in human brain, kidney, testis, and HepG2 cells, and there are three alternatively spliced variants that were differentially expressed. PKD2L2 consists of 17 exons spanning approximately 50 kb of genomic DNA. PKD2L2 was mapped to human chromosome 5q31 and Pkd2l2 to mouse chromosome 18 in band C. PMID- 10756094 TI - An integrated map of human 6q22.3-q24 including a 3-Mb high-resolution BAC/PAC contig encompassing a QTL for fetal hemoglobin. AB - Genetic studies have previously assigned a quantitative trait locus (QTL) for hemoglobin F and F cells to a region of approximately 4 Mb between the markers D6S408 and D6S292 on chromosome 6q23. An initial yeast artificial chromosome contig of 13 clones spanning this region was generated. Further linkage analysis of an extended kindred refined the candidate interval to 1-2 cM, and key recombination events now place the QTL within a region of <800 kb. We describe a high-resolution bacterial clone contig spanning 3 Mb covering this critical region. The map consists of 223 bacterial artificial chromosome (BAC) and 100 P1 artificial chromosome (PAC) clones ordered by sequence-tagged site (STS) content and restriction fragment fingerprinting with a minimum tiling path of 22 BACs and 1 PAC. A total of 194 STSs map to this interval of 3 Mb, giving an average marker resolution of approximately one per 15 kb. About half of the markers were novel and were isolated in the present study, including three CA repeats and 13 single nucleotide polymorphisms. Altogether 24 expressed sequence tags, 6 of which are unique genes, have been mapped to the contig. PMID- 10756093 TI - A new gene family including DSCR1 (Down Syndrome Candidate Region 1) and ZAKI-4: characterization from yeast to human and identification of DSCR1-like 2, a novel human member (DSCR1L2). AB - A new gene family has been identified on the basis of in-depth bioinformatics analysis of the Down syndrome candidate region 1 (DSCR1) gene, located on 21q22.1. We have determined the complete coding sequences of similar genes in Saccharomyces cerevisiae and Caenorhabditis elegans, as well as that of a novel human gene, named DSCR1L2 (DSCR1-like 2). Peripheral blood leukocyte cDNA sequencing predicts as its product a 241-amino-acid protein highly similar to products of the human genes DSCR1 and ZAKI-4 (HGMW-approved symbol DSCR1L1). The highest level of expression of DSCR1L2 mRNA was found by Northern blot analysis in heart and skeletal muscles, liver, kidney, and peripheral blood leukocytes (three transcripts of 3.2, 5. 2, and 7.5 kb). The gene consists of four exons and spans about 22 kb on chromosome 1 (1p33-p35.3) (Human Chromosome 1, Sanger Centre). Exon/intron organization is highly conserved between DSCR1 and DSCR1L2. Two alternative DSCR1L2 mRNA splicing forms have been recognized, with one lacking 10 amino acids in the middle of the protein. Analysis of expressed sequence tags (ESTs) shows DSCR1L2 expression in fetal tissues (heart, liver, and spleen) and in adenocarcinomas. ESTs related to the murine DSCR1L2 orthologue are found in the 2-cell stage mouse embryo, in developing brain stem and spinal cord, and in thymus and T cells. The most prominent feature identified in the protein family is a central short, unique serine-proline motif (including an ISPPXSPP box), which is strongly conserved from yeast to human but is absent in bacteria. Moreover, homology with the RNA-binding domain was weakly but consistently detected in a stretch of 80 amino acids at the amino-terminus by fine sequence analysis based on tools utilizing both hidden Markov models and BLAST. The identification of this new gene family should allow a better understanding of the functions of the genes belonging to it. PMID- 10756095 TI - The human homolog of insect-derived growth factor, CECR1, is a candidate gene for features of cat eye syndrome. AB - Cat eye syndrome (CES) is a developmental disorder with multiple organ involvement, associated with the duplication of a 2-Mb region of 22q11.2. Using exon trapping and genomic sequence analysis, we have isolated and characterized a gene, CECR1, that maps to this critical region. The protein encoded by CECR1 is similar to previously identified novel growth factors: IDGF from Sarcophaga peregrina (flesh fly) and MDGF from Aplysia californica (sea hare). The CECR1 gene is alternatively spliced and expressed in numerous tissues, with most abundant expression in human adult heart, lung, lymphoblasts, and placenta as well as fetal lung, liver, and kidney. In situ hybridization of a human embryo shows specific expression in the outflow tract and atrium of the developing heart, the VII/VIII cranial nerve ganglion, and the notochord. The location of this gene in the CES critical region and its embryonic expression suggest that the overexpression of CECR1 may be responsible for at least some features of CES, particularly the heart defects. PMID- 10756096 TI - Statistical analysis of the 5' untranslated region of human mRNA using "Oligo Capped" cDNA libraries. AB - We constructed 34 types of human "full-length enriched" and "5'-end enriched" cDNA libraries based on the "Oligo-Capping" method. We randomly picked and sequenced 10,000 clones from these libraries. BLAST analysis showed that about 50% of the cDNAs were identical to known genes. Among them, we selected 954 species of cDNA that should represent the entire sequence from the mRNA start sites. Compared with previously reported sequences, they were on average 45 bp longer in the 5'-end. Using these cDNA data, we statistically analyzed the sequence features of the 5'UTR. The average length of the 5'UTR was 125 bp, and there was little correlation with the corresponding mRNA length (correlation coefficient = 0.26). Of the 954 species of 5'UTR, 459 contained no in-frame terminator codon, which is against the common belief. Two hundred seventy-eight species contained at least one ATG codon upstream of the initiator ATG codon. We identified 569 upstream ATGs, in total, 63% of which adequately satisfied Kozak's criteria. These findings are contrary to the typical translation initiation model, which states that translation is initiated from the "first" ATG codon. PMID- 10756097 TI - The structure and expression of the murine wildtype p53-induced phosphatase 1 (Wip1) gene. AB - The human wildtype p53-induced phosphatase 1 (Wip1; GenBank symbol Ppm1d) gene encodes a type 2C protein phosphatase (PP2C) that is induced by ionizing radiation in a p53-dependent manner. We have cloned and sequenced the mouse Wip1 gene and its encoded mRNA. The mouse Wip1 gene is composed of six exons and spans over 36 kb of DNA. The mouse cDNA sequence predicts a 598-amino-acid protein with a molecular mass of roughly 66 kDa. Comparison of human and mouse Wip1 sequences revealed 83% overall identity at the amino acid level. The 5'-flanking region of exon 1 had promoter elements characteristic of a housekeeping gene. The Wip1 coding sequences share conserved functional regions with other PP2Cs from a diverse array of species. Expression of Wip1 mRNA was detected ubiquitously in adult and embryonic tissues, though expression in the testis was much higher than in other tissues. Wip1 has been mapped near the p53 gene on mouse chromosome 11. PMID- 10756098 TI - Lhx9 and Lhx9alpha LIM-homeodomain factors: genomic structure, expression patterns, chromosomal localization, and phylogenetic analysis. AB - Lhx9 is a LIM-homeodomain (LIM-hd) transcription factor expressed in the embryonic mouse brain. We report the isolation of Lhx9alpha, a cDNA encoding a truncated isoform of Lhx9 that lacks the recognition helix of the homeodomain and differs from Lhx9 cDNA in its 3'-coding and 3'-UTR sequences. Isolation of the Lhx9 gene showed that Lhx9 and Lhx9alpha are coded by six exons spanning 10 kb of genomic sequence and that Lhx9alpha is an isoform generated by alternative splicing of the fifth exon. Lhx9 was mapped to the subtelomeric region of chromosome 1. Further molecular analysis showed that Lhx9 is a new candidate gene for the unidentified dreher (dr) mutation in mouse. The comparison of genomic structure and molecular phylogenetic analysis led to the identification of six groups of LIM-hd proteins, a basis for further classification and knowledge of their evolutionary relationships. To investigate a possible role for Lhx9alpha, the expression patterns of Lhx9 and Lhx9alpha were compared during embryogenesis. Lhx9alpha was expressed at lower levels than Lhx9, with a similar but distinct pattern in the brain, especially in the neocortex. We suggest that Lhx9alpha could function as an endogenous dominant-negative form of Lhx9 during development, both to regulate in space and time the transcriptional effects of Lhx9 and to add a degree of refinement to the LIM-hd code. PMID- 10756099 TI - Peptide YY-2 (PYY2) and pancreatic polypeptide-2 (PPY2): species-specific evolution of novel members of the neuropeptide Y gene family. AB - Several gene duplication events have led to the creation of at least five distinct members of the neuropeptide Y gene family. We now reveal that the most recent of these events, involving the PYY-PPY gene cluster on chromosome 17q21.1, has led to the creation of novel PYY- and PP-like genes on chromosome 17q11 in the human genome. Sequence analysis of the novel human PYY2 and PPY2 genes shows an extensive homology to the peptide YY-pancreatic polypeptide genes, at the level of gene structure, nucleotide sequence, and primary amino acid sequence. The extremely high degree of homology between the PYY-PPY and the PYY2-PPY2 gene clusters, in both coding regions and especially noncoding regions, suggests that the PYY2 and PPY2 genes have arisen by a very recent gene duplication. Similar gene duplication events of the PYY-PPY gene cluster have also occurred in other species, including cow and baboon, but have not been confirmed in the rat and mouse genomes. Interestingly, despite the greater than 92% nucleotide sequence identity between these new genes, a few specific mutations have resulted in significantly altered peptide sequences. These altered sequences are accompanied by acquisition of new functions apparently unrelated to the neurotransmitter/endocrine role of PYY and PPY, as demonstrated by the major involvement of bovine PYY2, also known as seminal plasmin, in the fertilization process. PMID- 10756101 TI - Hierarchical unfolding of the alpha-lactalbumin molten globule: presence of a compact intermediate without a unique tertiary fold. AB - The difference between the framework model and the hydrophobic collapse model of protein folding largely rests on whether a secondary-structure framework can exist independently of native tertiary interactions. Here, we used circular dichroism and disulfide exchange experiments to examine the unfolding mechanism of alpha-LA(alpha), a two- disulfide variant of human alpha-lactalbumin (alpha LA) that adopts a molten globule conformation under near physiological conditions. Our results show that as the concentration of denaturant increases, the alpha-LA molten globule first loses its ability to form a specific, native like tertiary fold. Subsequently, at a higher denaturant concentration, the protein loses its secondary structure and adopts an extended conformation. A compact, non-native disulfide bond isomer, which does not form significantly under both native and strongly denaturing conditions, was found to be moderately populated in approximately 2 M guanidine hydrochloride (GuHCl). Qualitatively the same result was also obtained in urea. These results suggest that formation of secondary structure is a necessary, but not sufficient condition for formation of the native-like tertiary fold and support a hierarchical model of protein folding. PMID- 10756100 TI - cDNA cloning and mapping of a novel islet-brain/JNK-interacting protein. AB - IB1/JIP-1 is a scaffold protein that regulates the c-Jun NH(2)-terminal kinase (JNK) signaling pathway, which is activated by environmental stresses and/or by treatment with proinflammatory cytokines including IL-1beta and TNF-alpha. The JNKs play an essential role in many biological processes, including the maturation and differentiation of immune cells and the apoptosis of cell targets of the immune system. IB1 is expressed predominantly in brain and pancreatic beta cells where it protects cells from proapoptotic programs. Recently, a mutation in the amino-terminus of IB1 was associated with diabetes. A novel isoform, IB2, was cloned and characterized. Overall, both IB1 and IB2 proteins share a very similar organization, with a JNK-binding domain, a Src homology 3 domain, a phosphotyrosine-interacting domain, and polyacidic and polyproline stretches located at similar positions. The IB2 gene (HGMW-approved symbol MAPK8IP2) maps to human chromosome 22q13 and contains 10 coding exons. Northern and RT-PCR analyses indicate that IB2 is expressed in brain and in pancreatic cells, including insulin-secreting cells. IB2 interacts with both JNK and the JNK-kinase MKK7. In addition, ectopic expression of the JNK-binding domain of IB2 decreases IL-1beta-induced pancreatic beta-cell death. These data establish IB2 as a novel scaffold protein that regulates the JNK signaling pathway in brain and pancreatic beta-cells and indicate that IB2 represents a novel candidate gene for diabetes. PMID- 10756102 TI - The Bacillus subtilis AddAB helicase/nuclease is regulated by its cognate Chi sequence in vitro. AB - The AddAB enzyme is important to homologous DNA recombination in Bacillus subtilis, where it is thought to be the functional counterpart of the RecBCD enzyme of Escherichia coli. In vivo, AddAB responds to a specific five-nucleotide sequence (5'-AGCGG-3' or its complement) in a manner analogous to the response of the RecBCD enzyme to interaction with chi sequences. Here, we show that purified AddAB enzyme is able to load at a double-stranded DNA end and is both a DNA helicase and nuclease, whose combined action results in the degradation of both strands of the DNA duplex. During translocation, recognition of the properly oriented sequence 5'-AGCGG-3' causes attenuation of the AddAB enzyme nuclease activity that is responsible for degradation of the strand 3'-terminal at the entry site. Therefore, we conclude that 5'-AGCGG-3' is the B. subtilis Chi site and it is hereafter referred to as chi(Bs). After encountering chi(Bs), both the degradation of the 5'-terminal strand and the helicase activity persist. Thus, processing of a double-stranded DNA end by the AddAB enzyme produces a duplex DNA molecule with a protruding 3'-terminated single-stranded tail, a universal intermediate of the recombination process. PMID- 10756103 TI - Structure of a molluscan hemocyanin didecamer (HtH1 from Haliotis tuberculata) at 12 A resolution by cryoelectron microscopy. AB - A 12 A resolution three-dimensional density map of the Haliotis tuberculata hemocyanin type 1 (HtH1) didecamer has been obtained by cryoelectron microscopy of unstained molecules and angular reconstitution. The dyad symmetry of the 8 MDa D5 HtH1 didecamer, formed by the pairing of two asymmetric 4 MDa ring-like C5 decamers, is emphasised. The major and minor surface helical grooves of the didecamer are well defined, in agreement with earlier data on molluscan hemocyanins. The location of the obliquely orientated repeating unit, a subunit dimer, within the decamer has been defined. Following interactive extraction of this dimer, several new structural features of the dimer and of the subunit have now emerged with improved detail. The subunit dimer possesses pseudo 2-fold symmetry, resulting from the steric arrangement of the wall domains/functional units (FUs-abcdef) of the two subunits. The arc and collar FUs (g and h) depart from this inherent 2-fold symmetry and are thereby responsible for the asymmetry of the C5 decamer, with the internalised collar/arc complex at one edge of the decamer. The FU heterodimers forming the wall morphological units have a hollow centre, and thus create a series of repeating channels that extend within the wall through all three tiers of the decamer. The connections between the wall and the arc are defined with improved clarity, and evidence is provided to indicate that the arc and collar FU pairs have a homodimeric composition (gg and hh, respectively). Two possibilities for the subunit path within the subunit dimer are presented, which correlate with the available structural, immunolabelling and protease cleavage data from HtH1 and other molluscan hemocyanins. PMID- 10756105 TI - Structural basis for the network of functional cooperativities in cytochrome c(3) from Desulfovibrio gigas: solution structures of the oxidised and reduced states. AB - Cytochrome c(3) is a 14 kDa tetrahaem protein that plays a central role in the bioenergetic metabolism of Desulfovibrio spp. This involves an energy transduction mechanism made possible by a complex network of functional cooperativities between redox and redox/protolytic centres (the redox-Bohr effect), which enables cytochrome c(3) to work as a proton activator. The three dimensional structures of the oxidised and reduced Desulfovibrio gigas cytochrome c(3) in solution were solved using 2D (1)H-NMR data. The reduced protein structures were calculated using INDYANA, an extended version of DYANA that allows automatic calibration of NOE data. The oxidised protein structure, which includes four paramagnetic centres, was solved using the program PARADYANA, which also includes the structural paramagnetic parameters. In this case, initial structures were used to correct the upper and lower volume restraints for paramagnetic leakage, and angle restraints derived from (13)C Fermi contact shifts of haem moiety substituents were used for the axial histidine ligands. Despite the reduction of the NOE intensities by paramagnetic relaxation, the final family of structures is of similar precision and accuracy to that obtained for the reduced form. Comparison of the two structures shows that, although the global folds of the two families of structures are similar, significant localised differences occur upon change of redox state, some of which could not be detected by comparison with the X-ray structure of the oxidised state: (1) there is a redox-linked concerted rearrangement of Lys80 and Lys90 that results in the stabilisation of haem moieties II and III when both molecules are oxidised or both are reduced, in agreement with the previously measured positive redox cooperativity between these two haem moieties. This cooperativity regulates electron transfer, enabling a two-electron step adapted to the function of cytochromes c(3) as the coupling partner of hydrogenase; and (2) the movement of haem I propionate 13 towards the interior of the protein upon reduction explains the positive redox-Bohr effect, establishing the structural basis for the redox linked proton activation mechanism necessary for energy conservation, driving ATP synthesis. PMID- 10756104 TI - The 3D arrangement of the 23 S and 5 S rRNA in the Escherichia coli 50 S ribosomal subunit based on a cryo-electron microscopic reconstruction at 7.5 A resolution. AB - The Escherichia coli 23 S and 5 S rRNA molecules have been fitted helix by helix to a cryo-electron microscopic (EM) reconstruction of the 50 S ribosomal subunit, using an unfiltered version of the recently published 50 S reconstruction at 7.5 A resolution. At this resolution, the EM density shows a well-defined network of fine structural elements, in which the major and minor grooves of the rRNA helices can be discerned at many locations. The 3D folding of the rRNA molecules within this EM density is constrained by their well-established secondary structures, and further constraints are provided by intra and inter-rRNA crosslinking data, as well as by tertiary interactions and pseudoknots. RNA protein cross-link and foot-print sites on the 23 S and 5 S rRNA were used to position the rRNA elements concerned in relation to the known arrangement of the ribosomal proteins as determined by immuno-electron microscopy. The published X ray or NMR structures of seven 50 S ribosomal proteins or RNA-protein complexes were incorporated into the EM density. The 3D locations of cross-link and foot print sites to the 23 S rRNA from tRNA bound to the ribosomal A, P or E sites were correlated with the positions of the tRNA molecules directly observed in earlier reconstructions of the 70 S ribosome at 13 A or 20 A. Similarly, the positions of cross-link sites within the peptidyl transferase ring of the 23 S rRNA from the aminoacyl residue of tRNA were correlated with the locations of the CCA ends of the A and P site tRNA. Sites on the 23 S rRNA that are cross-linked to the N termini of peptides of different lengths were all found to lie within or close to the internal tunnel connecting the peptidyl transferase region with the presumed peptide exit site on the solvent side of the 50 S subunit. The post transcriptionally modified bases in the 23 S rRNA form a cluster close to the peptidyl transferase area. The minimum conserved core elements of the secondary structure of the 23 S rRNA form a compact block within the 3D structure and, conversely, the points corresponding to the locations of expansion segments in 28 S rRNA all lie on the outside of the structure. PMID- 10756106 TI - The solution structure of Rhodobacter sphaeroides LH1beta reveals two helical domains separated by a more flexible region: structural consequences for the LH1 complex. AB - Here, the solution structure of the Rhodobacter sphaeroides core light-harvesting complex beta polypeptide solubilised in chloroform:methanol is presented. The structure, determined by homonuclear NMR spectroscopy and distance geometry, comprises two alpha helical regions (residue -34 to -15 and -11 to +6, using the numbering system in which the conserved histidine residue is numbered zero) joined by a more flexible four amino acid residue linker. The C-terminal helix forms the membrane spanning region in the intact LH1 complex, whilst the N terminal helix must lie in the lipid head groups or in the cytoplasm, and form the basis of interaction with the alpha polypeptide. The structure of a mutant beta polypeptide W(+9)F was also determined. This mutant, which is deficient in a hydrogen bond donor to the bacteriochlorophyll, showed an identical structure to the wild-type, implying that observed differences in interaction with other LH1 polypeptides must arise from cofactor binding. Using these structures we propose a modification to existing models of the intact LH1 complex by replacing the continuous helix of the beta polypeptide with two helices, one of which lies at an acute angle to the membrane plane. We suggest that a key difference between LH1 and LH2 is that the beta subunit is more bent in LH1. This modification puts the N terminus of LH1beta close to the reaction centre H subunit, and provides a rationale for the different ring sizes of LH1 and LH2 complexes. PMID- 10756107 TI - Aminoglycoside binding in the major groove of duplex RNA: the thermodynamic and electrostatic forces that govern recognition. AB - We use a combination of spectroscopic, calorimetric, viscometric and computer modeling techniques to characterize the binding of the aminoglycoside antibiotic, tobramycin, to the polymeric RNA duplex, poly(rI).poly(rC), which exhibits the characteristic A-type conformation that is conserved among natural and synthetic double-helical RNA sequences. Our results reveal the following significant features: (i) CD-detected binding of tobramycin to poly(rI).poly(rC) reveals an apparent site size of four base-pairs per bound drug molecule; (ii) tobramycin binding enhances the thermal stability of the host poly(rI).poly(rC) duplex, the extent of which decreases upon increasing in Na(+) concentration and/or pH conditions; (iii) the enthalpy of tobramycin- poly(rI).poly(rC) complexation increases with increasing pH conditions, an observation consistent with binding induced protonation of one or more drug amino groups; (iv) the affinity of tobramycin for poly(rI).poly(rC) is sensitive to both pH and Na(+) concentration, with increases in pH and/or Na(+) concentration resulting in a concomitant reduction in binding affinity. The salt dependence of the tobramycin binding affinity reveals that the drug binds to the host RNA duplex as trication. (v) The thermodynamic driving force for tobramycin- poly(rI).poly(rC) complexation depends on pH conditions. Specifically, at pH< or =6.0, tobramycin binding is entropy driven, but is enthalpy driven at pH > 6.0. (vi) Viscometric data reveal non-intercalative binding properties when tobramycin complexes with poly(rI).poly(rC), consistent with a major groove-directed mode of binding. These data also are consistent with a binding-induced reduction in the apparent molecular length of the host RNA duplex. (vii) Computer modeling studies reveal a tobramycin-poly(rI). poly(rC) complex in which the drug fits snugly at the base of the RNA major groove and is stabilized, at least in part, by an array of hydrogen bonding interactions with both base and backbone atoms of the host RNA. These studies also demonstrate an inability of tobramycin to form a stable low energy complex with the minor groove of the poly(rI).poly(rC) duplex. In the aggregate, our results suggest that tobramycin-RNA recognition is dictated and controlled by a broad range of factors that include electrostatic interactions, hydrogen bonding interactions, drug protonation reactions, and binding-induced alterations in the structure of the host RNA. These modulatory effects on tobramycin-RNA complexation are discussed in terms of their potential importance for the selective recognition of specific RNA structural motifs, such as asymmetric internal loops or hairpin loop-stem junctions, by aminoglycoside antibiotics and their derivatives. PMID- 10756108 TI - High-resolution crystal structure of S. cerevisiae Ypt51(DeltaC15)-GppNHp, a small GTP-binding protein involved in regulation of endocytosis. AB - Ypt/Rab proteins are membrane-associated small GTP-binding proteins which play a central role in the coordination, activation and regulation of vesicle-mediated transport in eukaryotic cells. We present the 1.5 A high-resolution crystal structure of Ypt51 in its active, GppNHp-bound conformation. Ypt51 is an important regulator involved in the endocytic membrane traffic of Saccharomyces cerevisiae. The structure reveals small but significant structural differences compared with H-Ras p21. The effector loop and the catalytic loop are well defined and stabilized by extensive hydrophobic interactions. The switch I and switch II regions form a well-defined epitope for hypothetical effector protein binding. Sequence comparisons between the different isoforms Ypt51, Ypt52 and Ypt53 provide the first insights into determinants for specific effector binding and for fine-tuning of the intrinsic GTP-hydrolysis rate. PMID- 10756109 TI - The three-dimensional structure of a Plasmodium falciparum cyclophilin in complex with the potent anti-malarial cyclosporin A. AB - Cyclosporin A (CsA) is a potent anti-malarial compound in vitro and in vivo in mice though better known for its immunosuppressive properties in humans. Crystal structures of wild-type and a double mutant Plasmodium falciparum cyclophilin (PfCyP19 and mPfCyP19) complexed with CsA have been determined using diffraction terms to a resolution of 2.1 A (1 A=0.1 nm). The wild-type has a single PfCyP19/CsA complex per asymmetric unit in space group P1 and refined to an R work of 0.15 and R-free of 0.19. An altered cyclophilin, with two accidental mutations, Phe120 to Leu in the CsA binding pocket and Leu171 to Trp at the C terminus, presents two complexes per asymmetric unit in the orthorhombic space group P2(1)2(1)2. This refined to an R-work of 0.18 and R-free 0.21. The mutations were identified from the crystallographic analysis and the C-terminal alteration helps to explain the different crystal forms obtained. PfCyP19 shares approximately 61 % sequence identity with human cyclophilin A (hCyPA) and the structures are similar, consisting of an eight-stranded antiparallel beta-barrel core capped by two alpha-helices. The fold creates a hydrophobic active-site, the floor of which is formed by side-chains of residues from four antiparallel beta strands and the walls from loops and turns. We identified C-H.O hydrogen bonds between the drug and protein that may be an important feature of cyclophilins and suggest a general mode of interaction between hydrophobic molecules. Comparisons with cyclophilin-dipeptide complexes suggests that a specific C-H.O hydrogen bonding interaction may contribute to ligand binding. Residues Ser106, His99 and Asp130, located close to the active site and conserved in most cyclophilins, are arranged in a manner reminiscent of a serine protease catalytic triad. A Ser106Ala mutant was engineered to test the hypothesis that this triad contributes to CyP function. Mutant and wild-type enzymes were found to have similar catalytic properties. PMID- 10756110 TI - Metal-dependent conformational changes in a recombinant vWF-A domain from human factor B: a solution study by circular dichroism, fourier transform infrared and (1)H NMR spectroscopy. AB - Factor B is a key component of the alternative pathway of complement and is cleaved by factor D into the Ba and Bb fragments when complexed with the activated form of C3, namely C3b. The Bb fragment contains a von Willebrand factor type A (vWF-A) domain, which is composed of an open twisted almost parallel beta-sheet flanked on both sides by seven alpha-helices A1 to A7, with a metal coordination site at its active-site cleft. Homology modelling of this vWF A domain shows that the metal-binding site was present. Two recombinant vWF-A domains (Gly229-Ile444 and Gly229-Gln448) were examined by circular dichroism and Fourier transform infrared spectroscopy and indicated a significant conformational transition in the presence and absence of Mg(2+). Two upfield shifted signals in the (1)H NMR spectrum were used as sensitive probes of the vWF A protein structure, one of which was assigned to a methyl group and demonstrated metal- and pH-dependent properties between two distinct conformations. Temperature denaturation studies followed by spectroscopy showed that metal binding caused the vWF-A structure to become significantly more stable. Ring current calculations based on a homology model for the vWF-A structure correlated one upfield-shifted signal with a methyl group on the alpha-helices in the vWF-A structure and the other one with individual single protons. An allosteric property of the vWF-A domain has thus been identified, and its implications for factor B activation were examined. Since the vWF-A domain after alpha-helix A7 is connected by a short link to the catalytic serine protease domain in the Bb fragment, the identification of a metal-free and a more stable metal-bound conformation for the vWF-A domain implies that the vWF-A interaction with C3b may alter its Mg(2+)-bound coordination in such a way as to induce conformational changes that may regulate the proteolytic activity of factor B. PMID- 10756111 TI - Mechanisms for auto-inhibition and forced product release in glycine N methyltransferase: crystal structures of wild-type, mutant R175K and S adenosylhomocysteine-bound R175K enzymes. AB - Glycine N-methyltransferase (S-adenosyl-l-methionine: glycine methyltransferase, EC 2.1.1.20; GNMT) catalyzes the AdoMet-dependent methylation of glycine to form sarcosine (N-methylglycine). Unlike most methyltransferases, GNMT is a tetrameric protein showing a positive cooperativity in AdoMet binding and weak inhibition by S-adenosylhomocysteine (AdoHcy). The first crystal structure of GNMT complexed with AdoMet showed a unique "closed" molecular basket structure, in which the N terminal section penetrates and corks the entrance of the adjacent subunit. Thus, the apparent entrance or exit of the active site is not recognizable in the subunit structure, suggesting that the enzyme must possess a second, enzymatically active, "open" structural conformation. A new crystalline form of the R175K enzyme has been grown in the presence of an excess of AdoHcy, and its crystal structure has been determined at 3.0 A resolution. In this structure, the N-terminal domain (40 amino acid residues) of each subunit has moved out of the active site of the adjacent subunit, and the entrances of the active sites are now opened widely. An AdoHcy molecule has entered the site occupied in the "closed" structure by Glu15 and Gly16 of the N-terminal domain of the adjacent subunit. An AdoHcy binds to the consensus AdoMet binding site observed in the other methyltransferase. This AdoHcy binding site supports the glycine binding site (Arg175) deduced from a chemical modification study and site-directed mutagenesis (R175K). The crystal structures of WT and R175K enzymes were also determined at 2.5 A resolution. These enzyme structures have a closed molecular basket structure and are isomorphous to the previously determined AdoMet-GNMT structure. By comparing the open structure to the closed structure, mechanisms for auto-inhibition and for the forced release of the product AdoHcy have been revealed in the GNMT structure. The N-terminal section of the adjacent subunit occupies the AdoMet binding site and thus inhibits the methyltransfer reaction, whereas the same N-terminal section forces the departure of the potentially potent inhibitor AdoHcy from the active site and thus facilitates the methyltransfer reaction. Consequently GNMT is less active at a low level of AdoMet concentration, and is only weakly inhibited by AdoHcy. These properties of GNMT are particularly suited for regulation of the cellular AdoMet/AdoHcy ratio. PMID- 10756112 TI - Dynamics of early contact formation in cultured adult rat cardiomyocytes studied by N-cadherin fused to green fluorescent protein. AB - We investigated dynamic events during the formation of intercalated disc-like structures of adult rat cardiomyocytes (ARC) in long-term culture. Given the complexity of ARC cytoIarchitecture after de- and re-differentiation, and the non uniform morphological development of individual cells, green fluorescent protein (GFP) technology was used to track N-cadherin in living cells. Sorting and functionality of the GFP fusion protein was tested in ARC. Isolated ARC were micro-injected with the expression construct at the onset of spreading in culture, and the fluorescence signals were tracked during contact formation and in fully redifferentiated living cells. The first contact sites were found to be established by cellular protrusions, which were marked by an ultrastructure similar to microspikes and probably have a role as exploratory units in the spreading phase. Subsequently, initial contact sites served as anchorage for the most prominent stress fibre-like structures. The fusion protein appeared before connexin-43 at newly established cell-cell contacts. Membrane invaginations at the sarcolemma facing the substratum of cultured ARC may be responsible for the appearance of a striped pattern of N-cadherin and other adherens junction proteins away from intercalated disc-like structures. The stripes were immobile in redifferentiated cells, while the distinct small fluorescent particles in the cell body were found to move directionally at speeds around 10 micro m/min. These results contribute to the understanding of the mechanisms of cell-cell contact formation of adult cardiomyocytes, which is a prerequisite for any future implantation technology. PMID- 10756113 TI - Study of V(1)-vascular vasopressin receptor gene microsatellite polymorphisms in human essential hypertension. AB - Vasopressin (AVP) actions on vascular tone and blood pressure are mainly mediated by the V(1)-vascular receptor (V(1)R). We recently reported the structure and functional expression of the human V(1)R cDNA and described the genomic characteristics, tissue expression, chromosomal localization, and regional mapping of the human V(1)R gene, AVPR1A. To test whether the V(1)R is a marker for human essential hypertension, we sequenced the human AVPR1A gene and its 5; upstream region and found several DNA microsatellite motifs. One (GT)(14) (GA)(13)-(A)(8)microsatellite is located 2983 bp downstream of the transcription start site, within a 2.2 kbp intron interrupting the coding sequence of the receptor. Three other microsatellites are present in the 5; flanking DNA of the AVPR1A gene: a (GT)(25)dinucleotide repeat, a complex (CT)(4)-TT-(CT)(8) (GT)(24)motif and a (GATA)(14)tetranucleotide repeat located respectively 3956 bp, 3625 bp and 553 bp upstream of the transcription start site. Analysis of these polymorphisms in 79 hypertensive and 86 normotensive subjects for the (GT)(14)-(GA)(13)-(A)(8)and the (GT)(25)motifs revealed a high percentage of heterozygosity but no difference in alleles frequencies between the two groups. A linkage study using the affected sib pair method and the (GT)(25)repeat in 446 hypertensive sib pairs from 282 French Caucasian pedigrees showed no excess of alleles sharing at the AVPR1A locus. No linkage was found in the subgroups of patients with early onset hypertension (diagnosis before age 40) or severe hypertension (diastolic blood pressure >/=100 mmHg or requirement for >/=two medications). These findings suggest that molecular variants of the V(1)R gene are not involved in unselected forms of essential hypertension. PMID- 10756114 TI - Endothelin-1 stimulates cardiac fibroblast proliferation through activation of protein kinase C. AB - After myocardial ischemia, circulating levels of the mitogen endothelin-1 (ET-1) increase. The effects of ET-1 on cardiac fibroblasts are poorly characterized. Therefore we examined the influence of ET-1 on cardiac fibroblast proliferation with a view to elucidating the signal transduction mechanisms underlying this effect. ET-1 (10 n m) stimulated [(3)H]thymidine incorporation and cell proliferation in cultured neonatal rat cardiac fibroblasts, consistent with its activity as a mitogen. We examined the role of protein kinase C (PKC) on this function. Inhibition of PKC activation with either chelerythrine (1 microm) or staurosporine (1 n m) attenuated ET-1-induced increases in DNA synthesis and cell number. Downregulation of PKC by chronic pretreatment with 10 n m phorbol 12 myristate 13-acetate (PMA) also prevented ET-1-induced mitogenesis. In contrast to previous reports that cardiac fibroblast proliferation stimulated by angiotensin II acts independently of PKC, the ET-1 mediated mitogenic effect requires activation of PKC in these cells. Findings in adult rat cardiac fibroblasts were identical. In addition, we noted that concurrent treatment with the pro-inflammatory cytokine interleukin 1 beta which, like ET-1, is released after myocardial ischemia, attenuated the ET-1-induced increases in DNA synthesis and cell number. This effect was not mediated through a nitric oxide synthase pathway. PMID- 10756115 TI - Cardiac unloading alters contractility and calcium homeostasis in ventricular myocytes. AB - Altered cardiac workload has an important effect on myocyte structure and function. Cardiac hypertrophy resulting from an increase in load has been studied extensively in the past. However, the effects of unloading and atrophy have recently become of more interest since devices for mechanical left ventricular unloading have been introduced into clinical practice for the treatment of patients with terminal heart failure, and a resulting improved cardiac and myocyte contractility have been reported. We used the heterotopic abdominal mouse heart transplant model in order to study the effects of 5 days of unloading on cell size (confocal microscopy), contractility (fractional shortening: video motion), calcium homeostasis ([Ca(2+)](i)transients, SR Ca(2+)content); and L type Ca(2+)and sodium/calcium exchanger currents (whole cell patch clamp technique). We found unloading caused decreased cell volume consistent with atrophy. An increased fractional shortening and [Ca(2+)](i)transient were observed in myocytes from unloaded hearts as compared with controls. Transsarcolemmal I(Ca,L)and I(Na/Ca)densities, and SR Ca(2+)content were unaltered, as was membrane capacitance. A reduction in cell volume with mainteinance of internal and surface membrane areas, and/or a decrease in concentration of cellular protein Ca(2+)buffers, may contribute to the increase in the [Ca(2+)](i)transient in this model. PMID- 10756116 TI - Distant upstream regulatory domains direct high levels of beta -myosin heavy chain gene expression in differentiated embryonic stem cells. AB - Eukaryotic gene transcription takes place in the context of chromatin. In order to study the expression of the beta -myosin heavy chain (MyHC) gene in its appropriate cardiac environment in vitro, embryonic stem cell lines were generated and induced to differentiate into the cardiac lineage. We show that the upstream region of the beta -MyHC gene (-5518 to -2490 relative to the transcriptional start site) directed high levels of transcriptional activity only when stably integrated, but not when expressed extrachromosomally in transient assays. These results are consistent with earlier findings using an in vivo transgenic approach. The expression of beta -MyHC reporter gene constructs was strictly correlated to differentiation status and coincided with the expression of endogenous cardiac marker genes and with morphological differentiation of embryoid bodies in vitro. Using populations of stably transfected cell clones, two domains important for high level expression were identified. The analysis of individual cell clones suggested that the positive regulatory domains act according to the graded model of enhancement. These results show that chromosomal integration is necessary for the appropriate function of the beta -MyHC gene's upstream regulatory region. PMID- 10756117 TI - Epidermal growth factor induces hypertrophic responses and Stat5 activation in rat ventricular cardiomyocytes. AB - Epidermal growth factor (EGF) was tested for its ability to promote hypertrophic responses in neonatal rat ventricular cardiomyocytes. Exposure of these cells to 100 n m EGF for 2-18 h resulted in a time-dependent increase in protein synthesis reaching 174+/-18% of control values at 18 h. After 30 min stimulation, the mRNA levels of c-jun and c-fos were also increased 20- and 36-fold, respectively. We also investigated EGF-induced activation of Stat (signal transducers and activators of transcription) proteins as well as the possible interactions of this signaling pathway with the p38 and p42/44 MAP kinases cascades. EGF did not activate Stat1 and Stat3, but did induce a rapid and transient activation of Stat5, which corresponded mainly to Stat5b DNA-binding. The EGF-promoted Stat5 DNA-binding was decreased in a concentration-dependent manner by the p38 MAPK inhibitor SB 203580 (IC(50)=1.2 microm), whereas it was tripled by 50 micro m PD 98059, an inhibitor of the p42/44 MAPK cascade. This is the first demonstration that EGF increases protein synthesis and early response gene expression in cardiomyocytes, responses considered as markers of hypertrophy in these cells. The results further show that EGF activates Stat5, that this response requires p38 MAPK stimulation, and it is negatively modulated by p42/44 MAPK. PMID- 10756118 TI - Inhomogeneous transmural conduction during early ischaemia in patients with coronary artery disease. AB - Electrical inhomogeneity and conduction slowing are critical factors in the initiation and maintenance of ventricular arrhythmias during early ischaemia. Studies in animal models have shown delay in epicardial activation compared to endocardial activation. Epicardial activation delay has been attributed to either enhanced sensitivity of epicardium to ischaemia or to mid-myocardial conduction delay. No information is available in humans and in particular in patients with chronic ischaemia due to coronary artery disease who may have altered electrophysiological properties. Twenty-three patients undergoing routine coronary surgery were studied. All had severe two or three vessel coronary artery disease and a documented history of angina for a mean of 2.4 years. On cardiopulmonary bypass a 3 min period of ischaemia was created by cross clamping the aorta between the input from the pump oxygenator and the coronary arteries. During atrial pacing (normal endocardial to epicardial activation) intramyocardial activation time within the left ventricular free wall between subendocardial and subepicardial plunge electrode terminals, increased from 12.7+/-1.5 ms (control) to 28.2+/-3.2 ms after 3 min ischaemia at the base. At the apex, the activation time increase (over the same distance) was less (19.5+/ 2 ms at 3 min ischaemia). This difference in increase in activation time at the base and apex was significant (P<0.05). At the apex the ischaemia induced activation delay occurred primarily over the endocardial half of the wall, whereas the opposite was observed at the base of the heart. Using an epicardial electrode array stimulation along the long axis of the epicardial fibres showed minimal conduction delay during ischaemia whereas stimulation transverse to the epicardial fibres resulted in substantial conduction time prolongation, as was the case with intramural conduction. Intramural conduction during ischaemia was similar in non-infarcted regions of infarcted hearts compared to hearts with no previous MI. To conclude, in patients with coronary artery disease epicardial activation delay early during ischaemia is caused primarily by intramural delay and not by delay along the epicardium. Moreover, the ischaemia-induced transmural activation delay is inhomogeneous. PMID- 10756119 TI - Role of MIP-2 in coxsackievirus B3 myocarditis. AB - Interleukin-8 (IL-8) is a chemotactic cytokine for neutrophils and lymphocytes. Macrophage inflammatory protein-2 (MIP-2) is a murine counterpart of IL-8. The present study was performed to determine the role of MIP-2 in murine myocarditis. We examined (1) the MIP-2 producing activity of Coxsackievirus B3 (CB3)-infected cultured macrophages, (2) serial plasma MIP-2 levels in CB3-induced mice by enzyme-linked immunosorbent assay (ELISA), and (3) the effects of anti-mouse MIP 2 monoclonal antibody (mAb) in vivo upon myocarditis. The production of MIP-2 increased in an infection dose- and time-dependent manner in virus-infected RAW 264.7 macrophages. Three-week-old C(3)H/He mice were inoculated with CB3. Plasma MIP-2 levels were significantly elevated in mice on days 7, 10 and 14 post infection. Mice were injected subcutaneously with anti-MIP-2 mAb at 10 microg/day (Group 2) or 100 microg/day (Group 3) on days 0-7, and were observed until day 14. Uninfected control mice (Group 1) were injected with saline. Survival rate was higher in the anti-MIP-2-treated group (Group 3), but not in Group 2, than in the control group. Histopathological analysis revealed that cellular infiltration and myocardial necrosis with macrophage and T cell accumulation were less prominent in the anti-MIP-2 mAb-treated groups as compared to the controls. MIP-2 is an important naturally occurring inflammatory cytokine in CB3 myocarditis, and anti-MIP-2 mAb treatment may prevent the inflammatory response. PMID- 10756120 TI - Increased sodium-calcium exchange current in right ventricular cell hypertrophy induced by simulated high altitude in adult rats. AB - Ventricular hypertrophy is associated with an increase in action potential (AP) duration which is potentially arrhythmogenic. The implication of the Na-Ca exchange current (I(Na-Ca)) in the lengthening of the AP is controversial. The role of this current in the increased duration of the low plateau of the AP in hypertrophied adult rat ventricular myocytes by simulated chronic high-altitude exposure ( approximately 4500 m) was evaluated. Electrophysiological experiments were carried out on isolated right ventricular myocytes from exposed and control rats with the perforated patch or the conventional whole-cell technique in current or in voltage clamp condition. With the two techniques, a significant increase of the low plateau duration was observed in hypertrophied myocytes as compared to controls. The low plateau in hypertrophied myocytes was depressed when Na was replaced by Li and was no longer recorded when intracellular Ca was buffered with EGTA. Inward tail currents, evoked either on repolarization to -80 mV following a depolarizing pulse to +10 mV or by interrupted AP technique, were greater in hypertrophied than in control myocytes and were abolished when Na was replaced by Li or when intracellular Ca was buffered with EGTA, indicating an increased Na-Ca exchange activity. The Li-sensitive current-voltage curves, obtained by a voltage clamp ramp protocol with an intracellular calcium buffered solution, were not significantly different in both hypertrophied and control myocytes, suggesting no modification in the density of the Na-Ca exchange protein. This was corroborated by the lack of difference in NCX1 mRNA levels between right ventricles from control and exposed rats. We conclude that increased duration of the low plateau of rat ventricular AP in altitude cardiac hypertrophy may be attributed to an increase of the inward I(Na-Ca). This augmented I(Na-Ca)may result from a modification in the intracellular Ca homeostasis. PMID- 10756121 TI - Diminished expression of sarcoplasmic reticulum Ca(2+)-ATPase and ryanodine sensitive Ca(2+)Channel mRNA in streptozotocin-induced diabetic rat heart. AB - The diabetic heart has an abnormal intracellular calcium ([Ca(2+)]i) metabolism. However, the responsible molecular mechanisms are unclear. The present study aimed to investigate mRNAs expressed in the proteins which regulate heart [Ca(2+)]i metabolism in streptozotocin (STZ)-induced diabetic rats. Expression of sarcoplasmic reticulum Ca(2+)-adenosine triphosphatase (SR Ca(2+)-ATPase) mRNA was significantly less in the heart 3 weeks after STZ injection than that in the age-matched controls. Together with the down-regulation of SR Ca(2+)-ATPase, expression of ryanodine sensitive Ca(2+)channel (RYR) mRNA was also decreased 12 weeks after STZ injection. Insulin supplementation fully restored the decreased mRNAs expression of SR Ca(2+)-ATPase and RYR. The diminished expression and restoration with insulin supplementation of SR Ca(2+)-ATPase was further confirmed at the protein level. In contrast, expression of mRNAs coding the L type Ca(2+)channel, Na(+)-Ca(2+)exchanger, or phospholamban were not affected 3 or 12 weeks after STZ injection. These results can be taken to indicate that the down-regulation of SR Ca(2+)-ATPase and RYR mRNAs is a possible underlying cause of cardiac dysfunction in STZ-induced diabetic rats. PMID- 10756122 TI - Altered cardiac endothelin receptors and protein kinase C in deoxycorticosterone salt hypertensive rats. AB - The aim of the present study was to assess the status of ET-1 receptor subtypes (ET(A)and ET(B)) in ventricular myocytes and fibroblasts and to determine the role of PKC-dependent pathways in ET-1-stimulated cardiac cells in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Systolic blood pressure and relative heart to body weight were significantly increased in DOCA salt rats. In unilaterally nephrectomized (Uni-Nx) control rats, more than 90% of cardiomyocyte ET receptors were of the ET(A)subtype, whereas in fibroblasts ET(A)and ET(B)receptors were present in a 1:3 ratio. In DOCA-salt rats, the density of the ET(A)receptor subtype was reduced by 31% in cardiomyocytes and in cardiac fibroblasts only ET(B)receptor density was decreased by 29%. Affinity was unchanged. The relative expression of immunoreactive PKC alpha, gamma and epsilon was significantly increased, whereas PKC delta was not altered in cardiac extracts of DOCA-salt rats. In cardiac fibroblasts from DOCA-salt rats PKC delta was significantly increased and PKC epsilon was not translocated after ET-1 stimulation. The hearts of DOCA-salt hypertensive rats are thus characterized by: (1) decreased density of cardiomyocyte ET(A)receptors and fibroblast ET(B)receptors; (2) cell-specific enhanced expression of some PKC isoenzymes (alpha, gamma, delta and epsilon); and (3) unresponsiveness of PKC epsilon to translocate in the presence of ET-1. Together with alterations of ET-1-induced Ca(2+)handling in cardiac myocytes and fibroblasts, which we previously reported, results from the present study indicate a marked modification of the cardiac ET-1 system of DOCA-salt hypertensive rats. PMID- 10756123 TI - ATP-Sensitive potassium channels: a review of their cardioprotective pharmacology. AB - ATP-sensitive potassium channels (K(ATP)) have been thought to be a mediator of cardioprotection for the last ten years. Significant progress has been made in learning the pharmacology of this channel as well as its molecular regulation with regard to cardioprotection. K(ATP)openers as a class protect ischemic/reperfused myocardium and appear to do so by conservation of energy. The reduced rate of ATP hydrolysis during ischemia exerted by these openers is not due to a cardioplegic effect and is independent of action potential shortening. Compounds have been synthesized which retain the cardioprotective effects of first generation K(ATP)openers, but are devoid of vasodilator and cardiac sarcolemmal potassium outward currents. These results suggest receptor or channel subtypes. Recent pharmacologic and molecular biology studies suggest the activation of mitochondrial K(ATP)as the relevant cardioprotective site. Implications of these results for future drug discovery and preconditioning are discussed. PMID- 10756124 TI - Reversal of cardiac hypertrophy in transgenic disease models by calcineurin inhibition. AB - Heart disease remains one of the leading causes of morbidity and mortality in the industrialized nations of the world. Intense investigation has centered around identifying and manipulating intracellular signaling pathways that direct hypertrophic and myopathic responses in an attempt to intervene in the progression or reverse certain forms of heart disease. We show here that cyclosporin A-mediated inhibition of the calcium-regulated phosphatase, calcineurin (PP2B), reverses cardiac hypertrophy and myopathic dilation in two transgenic mouse models of cardiomyopathy. Reversal was demonstrated by gravimetric analysis, echocardiography, histological analysis, and molecular analysis of hypertrophy-associated gene expression. In contrast, a third mouse model of hypertrophic cardiomyopathy due to activated NFAT3 cardiac-specific expression was not affected by cyclosporin A. These results suggest that calcineurin may function in the long-term maintenance of cardiac hypertrophy or myopathic disease states. PMID- 10756125 TI - Quantitative analysis of acid-base balance in show jumpers before and after exercise. AB - The acid-base status of venous blood was studied in 17 show jumpers before and after exercise using both a traditional and a quantitative approach. Partial pressure of carbon dioxide (PCO(2)), pH, haemoglobin, and plasma concentrations of sodium (Na(+)), chloride (Cl(-)), potasium (K(+)), ionized calcium (Ca(2+)), total proteins, albumin, lactate and phosphorus were measured in jugular venous blood samples obtained before and immediately after finishing a show jumping competition. Bicarbonate, anion gap and globulin concentration were calculated from the measured parameters. 'Quantitative analysis' of acid-base balance was performed utilising values for three independent variables: PCO(2), strong ion difference [SID = (Na(+)+ K(+)+ Ca(2+)) - (Cl(-)+ Lact)] and total concentration of weak acids [A(T)= Alb (1 paragraph sign23 pH - 6 paragraph sign31) + Pi (0 paragraph sign309 pH - 0 paragraph sign469) 10/30 paragraph sign97]; plasma concentrations of hydrogen ion ([H(+)]) were also calculated from these variables using Stewart's equation. No significant changes in blood pH were detected after the show jumping competition. Exercise resulted in a significant increase in lactate, Na(+), K(+), haemoglobin, total proteins, albumin, globulin and anion gap, and a decrease in bicarbonate, Cl(-)and Ca(2+). PCO(2)decreased after exercise while SID and A(T)increased. A significant correlation between measured and calculated [H(+)] was found both before and after exercise. However, individual [H(+)] values were not accurately predicted from Stewart's equation. In conclusion, even though pH did not change, significant modifications in the acid-base balance of horses have been found after a show jumping competition. In addition, quantitative analysis has been shown to provide an adequate interpretation of acid-base status in show jumpers before and after exercise. PMID- 10756126 TI - Canine leptin: cDNA cloning, expression and activity of recombinant protein. AB - Leptin, the product of the ob gene, is one of the key molecules for the regulation of appetite and whole-body energy balance, and thereby for the pathogenesis of obesity. In an attempt to clarify the roles of leptin in obesity and/or related diseases in companion animals, canine leptin c DNA was cloned by amplifying reverse-transcriptase products of RNA extracted from the adipose tissue of the beagle. A c DNA clone of about 3 kbp contained a 501 bp open reading frame coding a 167-amino acid protein with a 21-amino acid signal peptide. The sequence of a 146-amino acid mature leptin was more than 79 per cent identical to those of other mammals. Northern blot analysis revealed abundant expression of leptin m RNA in adipose tissue, but not in other tissues, in adult beagles. When Chinese hamster ovary cells expressing the rat leptin receptor were stimulated with recombinant canine leptin produced by E. coli, some intracellular signal transduction proteins were phosphorylated, indicating that the recombinant leptin was biologically active. The data reported herein will be helpful for further studies of leptin of the dog in health and disease. PMID- 10756127 TI - Bovine FcgammaRIII with a single extracellular domain. AB - The full length of gene encoding Fc receptor types III for bovine immunoglobulin G (boFcgammaRIII, CD16) was obtained with PCR from the c DNA library of bovine alveolar macrophages. Through molecular cloning and sequencing it is shown that the coding gene has 507 bp and codes for 168 amino acids. There are signal peptide, transmembrane spanning domain and three potential N-linked glycosylation sites in the sequence. Compared with the boFcgammaRIII cDNA sequence cloned by Collins et al, it deletes 82 amino acids and has only one extracellular domain. It is suggested that this cDNA fragment is a special form of the gene structure of bovine FcgammaRIIIA (CD16-II). PMID- 10756128 TI - Type II and type III monodeiodinase activities in the skin of untreated and propylthiouracil-treated cashmere goats. AB - The presence or absence of types I, II and III iodothyronine monodeiodinase enzymes (MDI, MDII and MDIII) and their levels of activity in the skin of goats, which were orally dosed for 60 days with 0, 1.1, 2.2, 4.4, 8.8, 17.5, or 35 mg( 1)kg liveweight day(-1)of the anti-thyroid, enzyme-inhibiting drug, propylthiouracil (PTU), were determined. Contrary to our earlier report that PTU did not influence skin MDII activity, the currect more thorough investigation (in terms of numbers of observations and the efficiency of the enzyme extraction procedure) indicated that doses of 1.1.to 17.5 mg kg(-1)liveweight induced a 2 to 3 fold increase (P = 0.01) in MDII activity. However, in three of the four goats treated with 35 mg kg(-1)group, activity was similar to that of control animals. There were no significant differences between treatments in MDIII activity but there was a trend towards lower levels of activity in the goats dosed with 17.5 and 35 mg kg(-1). It is concluded that there is significant MDII and MDIII activity in the skin of goats and that although there is none of the PTU sensitive MDI enzyme, synthesis of T3 within the skin could nevertheless be modified through increases in MDII activity induced by lower T4 concentrations in the circulation caused by PTU. Changes in pattern of fibre moult induced by treatment with low doses of MD-inhibiting drugs may therefore be achieved through this effect. Since MDII and MDIII enzyme activity may be reduced by high doses of PTU, prolonged treatment with high doses of PTU may have adverse effects on skin tissue. PMID- 10756129 TI - Effects of food withdrawal and transport on 5- to 10-day-old calves. AB - The effects of food withdrawal for 30 hours and transport for up to 12 hours on 5 to 10-day-old calves were determined by monitoring plasma concentrations of glucose, beta-hydroxybutyrate, urea, lactate and creatine phosphokinase. In addition gamma-glutamyl transferase activity, packed cell volume, total plasma protein concentration, bodyweight and rectal temperature were recorded. Food withdrawal for 30 hours caused hypoglycemia but calves maintained normothermia and although their beta-hydroxybutyrate level increased indicating lipid mobilisation, the changes in blood urea level were minimal indicating low amino acid catabolism. When calves were transported for 12 hours in addition to having food withdrawn for 30 hours, they remained normoglycemic for six hours longer than non-transported calves, probably due to an effect of muscular activity during bracing against truck movements. Stocking calves at a lower density during transport apparently had a beneficial effect, as calves were able to lie down throughout the journey and therefore produced a similar metabolic profile to resting, non-transported animals. Transport and food withdrawal had no obvious effects on calf hydration. The results of this study suggest that food withdrawal for up to 30 hours and transport for up to 12 hours had no detrimental effects on the metabolism of healthy and clinically normal calves. With correct feeding regimes and transport protocols, welfare compromise in young, healthy calves being transported for up to 12 hours can be minimised when they are slaughtered within 30 hours of the start of transport. PMID- 10756130 TI - Active spermiophagy in the initial part of the proximal efferent duct of the epididymis of normal domestic fowl (Gallus domesticus). AB - In a study of the absorptive activities of the excurrent ducts of the testis of birds, six adult cocks of the Ovambo breed of domestic fowl were deeply anaesthetised and intravascularly perfused with buffered 3 per cent glutaraldehyde. Epididymal tissue was prepared conventionally for electron microscopy. In favourable sections in three of these birds avid ingestion of spermatozoa was revealed in the non-ciliated (Type I) cells of the epithelial lining of the initial part of the proximal efferent duct, at and a little beyond its junction with the rete testis. No obvious defects were detected in luminal spermatozoa. The testicular excurrent ducts were neither distended nor obstructed, and mononuclear cell mobilisation was absent in both the ductal and periductal tissue. The significance of this observation with regard to the specific segment of the efferent duct system involved and how certain spermatozoa are identified for elimination from the duct lumen, must await precise studies. PMID- 10756131 TI - Effect of single bouts of moderate and high intensity exercise and training on equine peripheral blood neutrophil function. AB - The effects of single bouts of moderate (30 to 40 per cent VO(2)max) and high (115 per cent VO(2)max) intensity exercise on equine peripheral blood leucocyte function were evaluated by determining neutrophil phagocytosis and oxidative burst activity before and after treadmill exercise and training. Prior to all exercise tests, the possible effect of diurnal variation was evaluated in samples obtained from four resting horses. Subsequently eight horses underwent moderate and high intensity exercise protocols and then commenced a 17-week training period. High intensity exercise tests were repeated in week 10, after 7 weeks of endurance training, and in week 17, after a further 6 weeks of high intensity training. Time of sampling had a significant effect on neutrophil function for resting, untrained horses. Prior to training, moderate intensity exercise was associated with improved neutrophil phagocytosis and oxidative burst activity. High intensity exercise was associated with transient impairment of these responses. A similar reduction was not demonstrable following high intensity exercise in weeks 10 or 17 of training. Neutrophil function in week 17 was suppressed at all sampling times relative to results obtained in week 10, suggesting that high intensity training may have been associated with a general reduction in neutrophil function. PMID- 10756132 TI - Development and use of monoclonal antibodies to chicken fibronectin to show that the chicken hepatocellular carcinoma cell line, LMH, constitutively expresses fibronectin. AB - Fibronectin (Fn) is a high molecular weight glycoprotein and acute phase reactant that contributes to a variety of cellular activities including proliferation and wound healing. Production of Fn is influenced by cytokines such as IL-1alpha, IL 6 and TNF -alpha, and in serum Fn levels can function as an indicator of sepsis and reticulo-endothelial function. Here we describe the production of a panel of mAb to chicken Fn and give evidence that a chicken hepatocellular carcinoma cell line, LMH, constitutively expresses Fn. A capture ELISA to measure chicken Fn was developed using an IgG1 mAb (AV62) as the capture Ab, and biotinylated AV63 (IgG2b) as the detecting Ab. This study identified a single commercially available mAb directed against human Fn that also recognised chicken Fn. By contrast, the anti-chicken Fn mAbs did not cross-react with either human or bovine Fn. PMID- 10756133 TI - Comparison of the performance of linear resistance and ultrasonic pneumotachometers at rest and during lobeline-induced hyperpnoea. AB - The performance of a Fleisch No. 5 pneumotachometer (F), and two commercial ultrasonic pneumotachometers, the BRDL (B) and the Spiroson (S) systems were compared in respect to their use for determination of ventilatory parameters at rest and during lobeline-induced hyperpnoea. Five clinically healthy Thoroughbred horses were tested with the three pneumotachometers in random order. Respiratory airflow, respired gas concentrations, oesophageal pressures, pressures within the mask systems and arterial blood gases were determined before and during lobeline induced hyperpnoea. Because measured peak expiratory airflow rates exceeded the stated linear range of the Fleisch pneumotachometer ( approximately +/- 25 l s( 1)) differential pressure-flow curves were determined in vitro over the range of flows recorded in vivo. Expired flows greater than the linear range were corrected according to the derived regression equation. No differences in any of the measured variables among the three systems were present at rest. At peak ventilation of lobeline-induced hyperpnoea mask pressures [Delta P(mask)(mean (SEM)): F: 9.6 (2.8) cm H(2)O, B: 0.8 (0.4) cm H(2)O, S: 1.4 (0.8) cm H(2)O] and end tidal carbon dioxide [ ET CO(2)(mean (SEM)): F: 2. 6 (0.1)%, B: 2.1 (0.2)%, S: 2.1 (0.1)%] were significantly higher in system F. Despite a tendency for respiratory frequency and peak inspired and expired flows, to be lower with system F, no significant differences in the measurements of ventilatory mechanics were detected. In conclusion, the ultrasonic flowmeters pose significantly lower resistive loads onto the respiratory system during ventilation above resting levels than Fleisch No 5 pneumotachometers. However, at the flowrates achieved during lobeline-induced hyperpnoea an in vitro calibration of the differential pressure-flow relationship allows correction for expiratory alinearity in system F. In addition, the performance of the Spiroson flowmeter is accurate in determining ventilatory mechanics at rest and during lobeline-induced hyperpnoea. PMID- 10756134 TI - Equine osteoclast-like cells generated in vitro demonstrate similar characteristics to directly isolated mature osteoclasts. AB - We report on novel methods to isolate osteoclasts (OC s) and generate osteoclast like cells (OCL s) from the bone and bone marrow of the equine femur. OC s were successfully isolated from bone scrapings taken from the endosteal surface of the femurs of three horses. OCL s were generated from bone marrow cells taken from the same animals. The validity of using the formation of OCL s as a method for studying OC differentiation and activity was confirmed by the similar characteristics of these two cells. In particular, they both were multinuclear, expressed the enzyme tartrate resistant acid phosphatase and the vitronectin receptor. Most importantly, both were able to resorb bone as demonstrated by the formation of extensive resorption pits when cultured on dentine slices. The generation of OCL s from bone marrow obtained from the equine femur can therefore be used to study equine OC differentiation and for studies requiring the generation of large numbers of these cells. OC s isolated directly from the same bones may be used to examine the effect of a variety of factors on bone resorption in vitro and to continually reaffirm the validity of using OCL s for large-scale studies on OC biology. Such research is essential for improved understanding of bone turnover and endochondral ossification in the horse. PMID- 10756135 TI - Fat infiltration in liver and activity of lecithin:cholesterol acyltransferase in serum of dry and lactating dairy cows. AB - To study the relationship between fat infiltration in the liver and the decrease in the activity of serum lecithin:cholesterol acyltransferase (LCAT), blood and liver samples were taken from 14 multiparous (MP) and 16 primiparous (PP) dairy cows four weeks and one week before, and one, four and eight weeks after calving. During the dry period, half the animals received a protein supplement. LCAT activity decreased significantly in all groups before parturition, but returned to the initial level within 1-4 weeks after parturition. There were no differences between the feeding groups, but after calving, the LCAT activity in the MP cows was significantly lower than in the PP cows. Fat infiltration was not detected in the PP cows, and in the MP cows fat infiltration was mild and fell within the physiological range. There was no correlation between liver fat content and the activity of LCAT in serum. These results suggest that shortly before and after parturition, the activity of LCAT in serum is limited by post transcriptional regulation, possibly by the availability of amino acid for export protein synthesis in the liver. PMID- 10756136 TI - Actions and interactions of ADP, 5- HT, histamine and PAF on equine platelets. AB - Platelets are thought to play a role in equine diseases such as acute laminitis and verminous arteritis and may be involved in allergic disease. Mediators implicated in the pathophysiology of these conditions activate platelets and responses may be enhanced by interactions between mediators. The present study compared platelet aggregation, thromboxane production and release of radiolabelled [(3)H]5- HT in response to 5- HT, histamine, ADP and PAF alone and in combination in vitro.PAF caused concentration-related aggregation, [(3)H]5- HT release and thromboxane production. In contrast, ADP caused aggregation and 5- HT induced the release of [(3)H]5- HT with little effect on other platelet functions. Histamine had little or no effect on equine platelets. Addition of 5- HT (10 microM) prior to ADP significantly displaced the aggregation response curve to the left. The profile of responses to PAF, ADP and 5- HT suggests differential activation of intracellular signalling pathways regulating these events. The enhanced response to ADP in the presence of 5- HT may have implications in thromboembolic disease in the horse. PMID- 10756137 TI - Immunohistochemical localisation of rabbit haemorrhagic disease virus VP-60 antigen in early infection of young and adult rabbits. AB - This study evaluated the time course distribution of rabbit haemorrhagic disease virus (RHDV) structural protein VP60 in tissues from experimentally infected rabbits from three different age groups. Viral VP60 antigen could not be detected in tissue samples from animals under four weeks, and only a few hepatocytes (0.01 to 0.2 per cent) were stained in the 6-week-old animals. A 6-week-old rabbit euthanised at 72 hpi showed VP60-labelling in hepatocytes and macrophages close to areas of inflammation. Viral VP60 antigen was detected as early as 12 hpi in a few hepatocytes (0.03 per cent) from adult animals. Within this age group, the extent of hepatocyte labelling considerably increased at 18 (3.0 per cent), 24 (25.5 per cent), 36 (50 per cent) and 48 (60 per cent) hpi. Extrahepatic viral VP60 antigen was also detected at 36 and 48 hpi in spleen macrophages and lymphocytes from adult rabbits. These findings support the hypothesis that the hepatocyte is the only cell type in the liver able to support RHDV replication almost immediately after viral infection. PMID- 10756138 TI - Cell proliferation and apoptosis in the pathogenesis of oesophagogastric lesions in pigs. AB - Recent studies have stressed the importance of epithelial hyperproliferation in the pathogenesis of early lesions (parakeratotic hyperkeratosis) of the porcine gastric pars oesophagea (PO). In this study, immunohistochemical staining with Ki67 (clone MIB1) and AgNOR proteins silver staining were used to evaluate, by means of image analysis, cell proliferation in normal and parakeratotic (parakeratotic hyperkeratosis) epithelia of the PO. Apoptotic activity was also assessed with the TUNEL assay and compared with cell proliferative parameters. Early lesions of the PO were characterised by a significant increase in epithelial proliferative activity while there was no difference in the apoptotic activity between normal and parakeratotic epithelia. Our data confirm the hyperproliferative nature of epithelial changes preceding degeneration and erosion/ulcer of the PO and suggest that an underlying feature of gastric ulcers in pigs is an imbalance between cell proliferation and programmed cell death. PMID- 10756139 TI - Effect of parainfluenza-3 virus challenge on cell-mediated immune function in parainfluenza-3 vaccinated and non-vaccinated calves. AB - A group of four conventional, colostrum-fed calves was vaccinated with live parainfluenza type 3 (PI-3) virus vaccine at 1 and 5 weeks of age. A group of four control calves was treated with cell culture medium at the same time. Two weeks after the second vaccination, both groups of calves were challenged with PI 3 virus by a combined respiratory route. Blood and nasal mucus samples were collected at intervals, and alveolar macrophages were recovered before and after challenge by bronchoalveolar lavage. The results demonstrated that clearance of virus, as indicated by presence of virus antigen was more rapid in previously vaccinated calves. Several alveolar macrophage functions were markedly reduced in all calves 5 to 7 days following virus challenge, although microbicidal activity was unaffected, compared to the controls. The production of neutrophil chemotactic factors by alveolar macrophages occurred more rapidly after virus challenge in the previously vaccinated calves and this correlated with a more rapid neutrophil influx into the lungs in these animals. PMID- 10756141 TI - The mutation rate in the human mtDNA control region. AB - The mutation rate of the mitochondrial control region has been widely used to calibrate human population history. However, estimates of the mutation rate in this region have spanned two orders of magnitude. To readdress this rate, we sequenced the mtDNA control region in 272 individuals, who were related by a total of 705 mtDNA transmission events, from 26 large Icelandic pedigrees. Three base substitutions were observed, and the mutation rate across the two hypervariable regions was estimated to be 3/705 =.0043 per generation (95% confidence interval [CI].00088-.013), or.32/site/1 million years (95% CI.065 .97). This study is substantially larger than others published, which have directly assessed mtDNA mutation rates on the basis of pedigrees, and the estimated mutation rate is intermediate among those derived from pedigree-based studies. Our estimated rate remains higher than those based on phylogenetic comparisons. We discuss possible reasons for-and consequences of-this discrepancy. The present study also provides information on rates of insertion/deletion mutations, rates of heteroplasmy, and the reliability of maternal links in the Icelandic genealogy database. PMID- 10756142 TI - Rumors of disease in the global village: outbreak verification. AB - Emerging infectious diseases and the growth of information technology have produced new demands and possibilities for disease surveillance and response. Increasing numbers of outbreak reports must be assessed rapidly so that control efforts can be initiated and unsubstantiated reports can be identified to protect countries from unnecessary economic damage. The World Health Organization has set up a process for timely outbreak verification to convert large amounts of data into accurate information for suitable action. We describe the context and processes of outbreak verification and information dissemination. PMID- 10756143 TI - Malaria on the move: human population movement and malaria transmission. AB - Reports of malaria are increasing in many countries and in areas thought free of the disease. One of the factors contributing to the reemergence of malaria is human migration. People move for a number of reasons, including environmental deterioration, economic necessity, conflicts, and natural disasters. These factors are most likely to affect the poor, many of whom live in or near malarious areas. Identifying and understanding the influence of these population movements can improve prevention measures and malaria control programs. PMID- 10756145 TI - Vaccines for mucosal immunity to combat emerging infectious diseases. AB - The mucosal immune system consists of molecules, cells, and organized lymphoid structures intended to provide immunity to pathogens that impinge upon mucosal surfaces. Mucosal infection by intracellular pathogens results in the induction of cell- mediated immunity, as manifested by CD4-positive (CD4 + ) T helper-type 1 cells, as well as CD8 + cytotoxic T-lymphocytes. These responses are normally accompanied by the synthesis of secretory immunoglobulin A (S-IgA) antibodies, which provide an important first line of defense against invasion of deeper tissues by these pathogens. New-generation live, attenuated viral vaccines, such as the cold-adapted, recombinant nasal influenza and oral rotavirus vaccines, optimize this form of mucosal immune protection. Despite these advances, new and reemerging infectious diseases are tipping the balance in favor of the parasite; continued mucosal vaccine development will be needed to effectively combat these new threats. PMID- 10756144 TI - The bdr gene families of the Lyme disease and relapsing fever spirochetes: potential influence on biology, pathogenesis, and evolution. AB - Species of the genus Borrelia cause human and animal infections, including Lyme disease, relapsing fever, and epizootic bovine abortion. The borrelial genome is unique among bacterial genomes in that it is composed of a linear chromosome and a series of linear and circular plasmids. The plasmids exhibit significant genetic redundancy and carry 175 paralogous gene families, most of unknown function. Homologous alleles on different plasmids could influence the organization and evolution of the Borrelia genome by serving as foci for interplasmid homologous recombination. The plasmid-carried Borrelia direct repeat (bdr) gene family encodes polymorphic, acidic proteins with putative phosphorylation sites and transmembrane domains. These proteins may play regulatory roles in Borrelia. We describe recent progress in the characterization of the Borrelia bdr genes and discuss the possible influence of this gene family on the biology, pathogenesis, and evolution of the Borrelia genome. PMID- 10756147 TI - Vibrio cholerae O139 in Calcutta, 1992-1998: incidence, antibiograms, and genotypes. AB - We report results of surveillance for cholera caused by Vibrio cholerae O139 from September 1992, when it was first identified, to December 1998. V. cholerae O139 dominated as the causative agent of cholera in Calcutta during 1992-93 and 1996- 97, while the O1 strains dominated during the rest of the period. Dramatic shifts in patterns of resistance to cotrimoxazole, neomycin, and streptomycin were observed. Molecular epidemiologic studies showed clonal diversity among the O139 strains and continuous emergence of new epidemic clones, reflected by changes in the structure, organization, and location of the CTX prophages in the V. cholerae O139 PMID- 10756146 TI - Competence of American robins as reservoir hosts for Lyme disease spirochetes. AB - To explore the competence of American robins as a reservoir for Lyme disease spirochetes, we determined the susceptibility of these birds to tickborne spirochetes and their subsequent infectivity for larval vector ticks. Robins acquired infection and became infectious to almost all xenodiagnostic ticks soon after exposure to infected nymphal ticks. Although infectivity waned after 2 months, the robins remained susceptible to reinfection, became infectious again, and permitted repeated feeding by vector ticks. In addition, spirochetes passaged through birds retained infectivity for mammalian hosts. American robins become as infectious for vector ticks as do reservoir mice, but infectivity in robins wanes more rapidly. PMID- 10756148 TI - Multivariate Markovian modeling of tuberculosis: forecast for the United States. AB - We have developed a computer-implemented, multivariate Markov chain model to project tuberculosis (TB) incidence in the United States from 1980 to 2010 in disaggregated demographic groups. Uncertainty in model parameters and in the projections is represented by fuzzy numbers. Projections are made under the assumption that current TB control measures will remain unchanged for the projection period. The projections of the model demonstrate an intermediate increase in national TB incidence (similar to that which actually occurred) followed by continuing decline. The rate of decline depends strongly on geographic, racial, and ethnic characteristics. The model predicts that the rate of decline in the number of cases among Hispanics will be slower than among white non-Hispanics and black non-Hispanics a prediction supported by the most recent data. PMID- 10756149 TI - Serologic response to culture filtrate antigens of Mycobacterium ulcerans during Buruli ulcer disease. AB - Buruli ulcer (BU) is an emerging necrotic skin disease caused by Mycobacterium ulcerans. To assess the potential for a serodiagnostic test, we measured the humoral immune response of BU patients to M. ulcerans antigens and compared this response with delayed-type hypersensitivity responses to both Burulin and PPD. The delayed-type hypersensitivity response generally supported the diagnosis of BU, with overall reactivity to Burulin in 28 (71.8%) of 39 patients tested, compared with 3 (14%) of 21 healthy controls. However, this positive skin test response was observed primarily in patients with healed or active disease, and rarely in patients with early disease (p=0.009). When tested for a serologic response to M. ulcerans culture filtrate, 43 (70.5%) of 61 BU patients had antibodies to these antigens, compared with 10 (37.0%) of 27 controls and 4 (30. 8%) of 13 tuberculosis patients. There was no correlation between disease stage and the onset of this serum antibody response. Our findings suggest that serologic testing may be useful in the diagnosis and surveillance of BU. PMID- 10756150 TI - Haemophilus influenzae type b and Streptococcus pneumoniae as causes of pneumonia among children in Beijing, China. AB - To determine if Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae could be identified more often from the nasopharynx of patients with pneumonia than from control patients, we obtained nasopharyngeal swab specimens from 96 patients with chest x-ray-confirmed pneumonia and 214 age-matched control patients with diarrhea or dermatitis from the outpatient department at Beijing Children's Hospital. Pneumonia patients were more likely to be colonized with Hib and S. pneumoniae than control patients, even after the data were adjusted for possible confounding factors such as day-care attendance, the presence of other children in the household, and recent antibiotic use. In China, where blood cultures from pneumonia patients are rarely positive, the results of these nasopharyngeal cultures provide supporting evidence for the role of Hib and S. pneumoniae as causes of childhood pneumonia. PMID- 10756151 TI - Bacteroides fragilis enterotoxin gene sequences in patients with inflammatory bowel disease. AB - We identified enterotoxigenic Bacteroides fragilis in stool specimens of patients with inflammatory bowel disease and other gastrointestinal disorders. The organism was detected in 11 (13.2%) of 83 patients with inflammatory bowel disease. Of 57 patients with active disease, 19.3% were toxin positive; none of those with inactive disease had specimens positive for enterotoxigenic Bacteroides fragilis gene sequences. PMID- 10756152 TI - Outbreak among drug users caused by a clonal strain of group A streptococcus. AB - We describe an outbreak among drug users of severe soft-tissue infections caused by a clonal strain of group A streptococcus of M-type 25. Cases (n = 19) in drug users were defined as infections (mainly needle abscesses) due to the outbreak strain. Comparison with controls showed that infected drug users bought drugs more often at a specific place. Drug purchase and use habits may have contributed to this outbreak. PMID- 10756153 TI - Erythromycin resistance in Streptococcus pyogenes in Italy. AB - In a prospective study of acute pharyngitis in Italian children, 69 (38.3%) of 180 isolates of Streptococcus pyogenes were resistant to macrolides. S. pyogenes was eradicated in 12 (63.1%) of 19 patients with erythromycin-resistant S. pyogenes treated with clarithromycin and in 22 (88%) of 25 patients with erythromycin-susceptible strains. The constitutive-resistant phenotype was correlated with failure of macrolide treatment. PMID- 10756154 TI - Sin nombre virus (SNV) Ig isotype antibody response during acute and convalescent phases of hantavirus pulmonary syndrome. AB - Serum samples from 22 hantavirus pulmonary syndrome (HPS) patients were tested for Sin Nombre virus (SNV)-reactive antibodies. In the acute phase of HPS, 100% and 67% of the samples tested positive for SNV-specific immunoglobulin (Ig) M and IgA, respectively. Among the virus-specific IgG antibodies, the most prevalent were IgG3 (in 97% of samples), followed by IgG1 (70%), IgG2 (30%), and IgG4 (3%). PMID- 10756155 TI - Bovine tuberculosis and the endangered Iberian lynx. AB - We report the first case of bovine tuberculosis in a free-living Iberian lynx (Lynx pardina), an extremely endangered feline, from Donana National Park in Spain. The isolate (Mycobacterium bovis) correlates by molecular characterization with other isolates from wild ungulates in the park, strongly suggesting an epidemiologic link. Mycobacterium bovis infects many animal species, with wild and free-ranging domestic ungulates being the main reservoirs in nature (1). PMID- 10756156 TI - Haff disease: from the Baltic Sea to the U.S. shore. AB - Haff disease, identified in Europe in 1924, is unexplained rhabdomyolysis in a person who ate fish in the 24 hours before onset of illness. We describe a series of six U.S. patients from 1997 and report new epidemiologic and etiologic aspects. Although Haff disease is traditionally an epidemic foodborne illness, these six cases occurred in two clusters and as one sporadic case. PMID- 10756157 TI - Chlamydia pneumoniae infection in a breeding colony of African clawed frogs (Xenopus tropicalis). AB - More than 90% of a breeding colony of clawed frogs (Xenopus tropicalis) imported to the United States from western Africa died in an epizootic of chlamydiosis. Chlamydial inclusions were observed by light and electron microscopy in the liver of an infected frog. Chlamydia pneumoniae was isolated in cell cultures from four frogs. A cutaneous infection by a chytridiomycete fungus observed in two frogs could have been a cofactor in the die-off.ous Diseases PMID- 10756158 TI - The impact of health communication and enhanced laboratory-based surveillance on detection of cyclosporiasis outbreaks in California. AB - We investigated the timing of diagnosis, influence of media information on testing for Cyclospora, and the method used to identify cases during eight cyclosporiasis outbreaks in California in spring of 1997. We found that Internet information, media reports, and enhanced laboratory surveillance improved detection of these outbreaks. PMID- 10756159 TI - Norwalk-like viral gastroenteritis outbreak in U.S. Army trainees. AB - An outbreak of acute gastroenteritis hospitalized 99 (12%) of 835 U. S. Army trainees at Fort Bliss, El Paso, Texas, from August 27 to September 1, 1998. Reverse transcriptase polymerase chain reaction tests for Norwalk-like virus were positive for genogroup 2. Gastroenteritis was associated with one post dining facility and with soft drinks. PMID- 10756160 TI - Preventing zoonotic diseases in immunocompromised persons: the role of physicians and veterinarians. PMID- 10756162 TI - Acute endotoxemia protracts leukocyte / endothelium interaction after local ischemia and reperfusion in striated muscle. AB - Postischemic reperfusion injury and endotoxemia have been shown to promote multiple organ failure in polytraumatized patients. Both pathomechanisms comprise endothelial injury, leukocyte activation, and enhanced leukocyte/endothelium interaction in the microcirculation. Using a dorsal skinfold chamber model for intravital microscopy in striated muscle of awake hamsters, we investigated whether acute endotoxemia enhances postischemic leukocyte/endothelium interaction. - In control animals (n = 8), reperfusion after 2 hours of pressure induced ischemia elicited the rolling and adhesion of fluorescently stained leukocytes to the endothelium of postcapillary venules with a maximum at 0.5 and 2 hours after reperfusion and a decline towards preischemic values after 24 hours. Postischemic leukocyte adhesion was enhanced and protracted in animals where acute endotoxemia was induced through intravenous injection of endotoxin (Salmonella abortus equi, 0.1 microg?kg superset-1) either 10 minutes prior to ischemia (n = 8) or to reperfusion (n = 8). - These results suggest that acute endotoxemia has the potential to aggravate the leukocyte-triggered reperfusion damage to striated muscle and presumably to vital organs and thus favours the development of multiple organ failure after primarily successful reperfusion. PMID- 10756163 TI - Immunostimulatory activity of the bacterial extract OM-8. AB - The bacterial extract OM-89 used for the prevention and treatment of recurrent urinary tract infections constitutes an effective immunostimulant in vitro and in vivo. Here we demonstrate that OM-89 shows mitogenic properties towards murine spleen cell cultures from LPS responder and non-responder mice. In macrophages the extract induces the translocation of NF-kappaB into the cell nucleus and RNI (radical nitrogen intermediates) release, which could be attributed to single fractions of the extract. Our findings on the in vitro immunostimulatory effect of OM-89, as well as its immunogenic and adjuvant properties, are of importance for understanding its therapeutic efficacy as demonstrated in clinical studies. PMID- 10756164 TI - Biliary atresia: which factors predict the success of a Kasai operation? An analysis of 36 patients. AB - Biliary atresia (BA) is one of the most frequent causes of neonatal cholestasis. Portoenterostomy is one therapeutic option in these patients with a success rate of 30-40%. To answer the question of therapy liver transplantation or Kasai operation - we analyzed 36 consecutive patients being followed in our center during the past 7 years. Two groups were formed: group I : patients developing cirrhosis within the first 2 years of life with the need for liver transplantation (n = 21). Group II: patients without need for transplantation within the first 2 years of life (n = 15). The two groups were compared regarding birth weight, age at diagnosis, age at Kasai-procedure, liver histology. The following biochemical parameters were analyzed at the time of diagnosis, 1 week and 5 weeks after Kasai: AST, ALT, gammaGT, and bilirubin. - RESULTS: Clinical characteristics were similar in both groups. However BA was diagnosed in group I 8.2 weeks after birth compared to 5.6 wk in group II. gammaGT, ALT, AST, and bilirubin were similar in both groups at the time of diagnosis and 1 wk after Kasai. However 5 wk after Kasai gammaGT was 276 U/l in group I compared to 72 U/l in group II (p <0.001), bilirubin was 6.3mg/dl in group I compared to 2. 3mg/dl in group II (p <0.001). - CONCLUSION: Kasai operation before the 7th wk of life increases the success rate of this technique significantly. Children with cirrhosis at the time of diagnosis should be evaluated for primary liver transplantation. gammaGT and bilirubin 5 weeks after Kasai operation may be useful markers for the success of this procedure. Patients with a gammaGT > 100 U/l and a bilirubin level >5mg/dl should be followed closely and should be evaluated for liver transplantation early. PMID- 10756165 TI - A hepatitis C virus (HCV) core protein derived peptide inhibits HCV specific lymphocyte proliferation. AB - T helper lymphocytes are important regulatory cells for the immune response in chronic hepatitis C. They recognize peptides, which are generated from the viral proteins by antigen processing and are bound to MHC (major histocompatibility complex) class II molecules. However, antigen processing might also result in non immunogenic peptide fragments that can modify T cell activation. - To identify such peptide fragments in hepatitis C, we studied binding of 15 synthetic HCV core derived peptides to MHC class II molecules of 9 human homozygous typing B cell lines (HT-BCLs) as well as T cell proliferation in 41 HLA-typed patients with chronic hepatitis C. - We identified a peptide (HCV core aa 59-83) which bound to 7 HT-BCLs, whereas PBMC of only 2 out of 36 patients with the corresponding HLA-DR alleles proliferated in response to this peptide. Competition experiments indicated that small amounts of peptide aa 59-83 specifically inhibited the proliferative response to the recombinant core protein but not to core derived immunogenic peptides. Our data show that a peptide fragment from the HCV core region aa 59-83 can interfere in vitro with immune recognition of the HCV core protein. PMID- 10756166 TI - Pet-fish food lung- a new form of extrinsic allergic alveolitis. AB - We report a female patient, who developed extrinsic allergic alveolitis due to pet-fish food inhalation. Some ingredients of pet-fish food are known triggers of type I allergy. To our knowledge, this is the first case in which pet-fish food is reported as causative agent inducing type III allergy. PMID- 10756167 TI - Bird Keeper's lung without bird keepinge AB - The identification of disease inducing allergens in hypersensitivity pneumonitis can be very problematic, and only by a thorough analysis of anamnestic data the source of allergen can be identified. We report a case of a 32-year-old female diagnosed with hypersensitivity pneumonitis caused by the inhalation of budgerigar antigen in her home. She had been living there for two years and had never been a bird keeper at all. The former proprietor of the house was a budgerigar keeper for years. When we detected precipitating antibodies against different antigens including pigeon and budgerigar antigens as well as hay and Aureobasidium pullulans, the source of antigen exposition was not definitely clear. In the serum of our patient we found precipitating antibodies against protein structures extracted from dust samples from the patient's home, which were not detected in the serum of her husband. Using Western blots of budgerigar serum and of the dust sample from the patient's home we could demonstrate an IgG reactive banding pattern in our patient's serum. The banding pattern against budgerigar serum correlated very closely to that of a control patient, who was a budgerigar keeper with hypersensitivity pneumonitis. The patient's husband reacted neither against budgerigar serum nor against the dust sample, while he and his wife showed double banding at about 9 kDA, when their serum was exposed to dust from a home free of bird keeping. These results point to the fact, that the house dust sample of our patient contained budgerigar antigen, leading to an indirect antigen expositon causing hypersensitivity alveolitis. However, the positive reaction of the patient serum against the protein extract from the dust sample of her home needs further confirmation by inhibition experiments using budgerigar antigen. - Our patient received a prolonged treatment with corticosteroids, and after about one year the vital capacity of the lungs, which was reduced by 50% at the beginning of the treatment, returned to normal. The patient is still living in her home. Although she has been off medication for one year, lung function has not deteriorated. This fact points to a reduction of the amount of antigen in the patient's home. PMID- 10756168 TI - Extrinsic allergic alveolitis in domestic environments (Domestic allergic alveolitis) caused by mouldy tapestry AB - Extrinsic allergic alveolitis often occures as bird fancier's lung or is caused by occupational treatment with antigenic materials. In housing environments antigens of allergic alveolitis are also found, especially mould fungi. lf a source of antigens is absent in the anamnesis and the clinical picture as well as the clinical findings are ambiguous, the diagnosis of extrinsic allergic alveolits is delayed or unobtained. The following example shows that in spite of a detailed allergic anamnesis the source of antigens may remain occult and only an inspection of the dwelling rooms leads to an elucidation. - A sixty-one year old non-smoking women was twice admitted to hospital with a temperature of more than 39 degrees C, intense dyspnea on exertion and a strong dry cough under the persumed diagnosis pneumonia. The laboratory values showed nonspecific signs of inflammation, the blood gas analysis was changed to a heavy hypoxemia (pO subset2 49.2 mmHg) and in the chest x-ray there were seen miliary and partly reticular alterations. The chest computed tomography showed extensive densities in both upper and lower parts of the lungs and the pulmonary function test corresponded with a low-grade to middle-grade restriction (VC subsetin = 67%, TLC = 69%). A high dose of corticosteroids produced an improvement of the radiological findings and of the pulmonary function. Also the specialized diagnostic in a hospital for pulmonary diseases yielded no new knowledge, and an interstitial pneumonia was diagnosed. Only the new formation of an acute pneumonic clinical picture on the day of returning to the patients own habitation suggested a noxious substance in the domestic environment. The inspection of the rooms finally showed the source of antigen to be from a condensation water soaked, moulded tapestry on the embrasure of a small unopenable window in the bedroom just on the head of the bed. The nutritive mediums left in the rooms furnished evidence of the mould species Penicillium, Cladosporium and Botrytis, in the bedroom however mostly Penicillium. The RAST of specific IgG proved a positive result of all three mould species (Penicillium sp. 1 : 100, Cladosporium sp. 1 : 200 and Botrytis sp. 1 : 200). The diagnosis of an extrinsic allergic alveolitis caused by mould was confirmed by a controlled re-exposure test, which promptly gave rise to a relapse and forced the patient to change habitation with the result of no further ailments. - Characteristic for the extrinsic allergic alveolitis in the described case is the inefficiency of antibiotic therapy, the immediate recovery by corticosteroids and furthermore the outbreak of relapses caused by re-exposure. If the source of antigens is unclear, the inspection of the habitation by an experienced allergologist may lead to success. PMID- 10756169 TI - Workplace-related complaints due to exposure to contaminated humidifier water and the VDI guideline 6022 AB - We describe a case report of one patient with hypersensitivity pneumonitis (humidifier lung) due to exposure to contaminated humidifier water and another four patients with related complaints. The setting was a building with photographic laboratories and corresponding office rooms that were supported with air by a humidification, ventilation, and air conditioning (HVAC) system. In the humidifier water, we found different fungi, particularly species of the genus Verticillium, as well as gramnegative bacteria, but no Legionella species. Colony counts were done according to the German Drinking Water Regulations and the VDI Guideline 6022 at 20 degrees C and 36 degrees C; the results exceeded the recommended level of 103 cfu/ml. The hygienic role of the isolated microorganisms and their products (e.g., antigens, endotoxins) is discussed. The case report emphasizes the importance of careful maintenance and control of HVAC systems. The new VDI guideline 6022 (issue July 1998) gives detailed information on this subject and pays special attention to qualifications and training of the HVAC operations staff. PMID- 10756170 TI - Hypersensitivity alveolitis induced by endogenous candida species AB - We report the case of a 64-year old woman with severe attacks of allergic alveolitis occurring frequently independent of a concrete place. These attacks were regularely preceded by diarrhoe. Laboratory data showed marked leukopenia and circulating Candida albicans (C.a.)-antigen. Bronchoalveolar lavage revealed an increase in neutrophils (13%). Transbronchial biopsies showed focal alveolitis and focal septal fibrosis with bronchiolitis obliterans and an epitheloid cell granuloma. Immunohistochemical examination revealed C.a.-antigen in alveolar macrophages. In the faeces a high amount of C.a. and C. glabrata was detected. In serum IgG and IgA were positive against C.a., IgE against C.a. was negative. Lymphocyte proliferation assay with C.a.-antigen was positive. Intradermal skin test with C.a. showed positive immediate and late phase reaction. Furthermore inhalation challenge with C.a.-antigen was positive. A febrile reaction with chills, dyspnea and hypoxemia and leukocytosis in peripheral blood occured after 6 hours. Lung function showed a predominantly obstructive impairment of ventilation. An extensive search for other IgG- or IgE-mediated allergies (other fungi, environmental or food allergens) was completely negative. Investigation of the gastrointestinal tract did not show any abnormality exept the detection of lactose intolerance. There was no fungal growth in the patients flat. After initiation of antimycotic treatment the symptoms resolved completely. - We conclude that the disease was induced by C.a.-antigen reaching the lungs from the intestinal tract via the bloodstream. It is still a matter of debate whether an additional factor is necessary for the antigen to penetrate the intestinal mucosa. PMID- 10756171 TI - Histologically proven extrinsic allergic alveolitis with severe obstructive pulmonary emphysema AB - Anamnesis: 61-year old man with progressive shortness of breath on exercise. Cough and expectoration during the last 6 years. - Exposure: Driver of cereals, massive exposure to mouldy and pest contaminated grains. Gave up his profession in 1979 due to dyspnea with short (2-3 h) latency after exposure. Since 1980 intermittent exposure during occasional jobs; renewed symptomatology. Aspergillus fumigatus detected on agar plates inoculated with material from wet areas in bathroom and kitchen. - Clinical symptoms: Barely audible vesicular breathing, barrel-shaped thorax, inspiratory-intercostal retraction. - Bodyplethysmography: Obstructive pulmonary emphysema with FEV1 0.8 l, TLC 7.8 l, RV/TLC relation 67%. Precipitin-detection: Significantly increased IgG against Fusarium. Other moulds including Aspergillus: negative; thermophilic actinomycetes: negative; pigeon and chicken: negative; Ouchterlony with native material from patients flat: negative. - CT including HR-CT: Bilateral-substantial emphysema, no bullae, no ground glass opacity, no signs for interstitial lung diseases, no mediastinal enlargement of lymph nodes. - Alpha-1-Antitrypsin: 1.67 to 2.3 g/l (normal range), phenotype M1. - Histology: In resected material from right-side lung-volume-resection detection of pulmonary emphysema as well as lymphocyte infiltration and numerous epitheloid cell granulomas with Langhans'giant cells without caseation assessed as residues of an exogenous allergic alveolitis. - Conclusion: In a patient with lung volume reduction surgery due to severe emphysema histologically a persistent exogenous allergic alveolitis was detected, which might have caused the emphysema. PMID- 10756172 TI - IgG subclass responses to pigeon intestinal mucin in pigeon Fanciers' lung AB - Pigeon fanciers' lung (PFL) is a form of extrinsic allergic alveolitis caused by exposure to avian antigens. Although the mechanisms involved in the pathogenesis of PFL are unclear it has been suggested that immune complexes formed on the alveolar epithelial surface of the lung initiate the symptoms of the acute stages of discase. Pigeon intestinal mucin has been implicated as an important antigen in the pathogenesis of PFL, and affected fanciers have very high antibody titres against this antigen. In this study we investigated the IgG and IgG subclass responses to both intact and papain digested mucin in 250 pigeon fanciers classified according to the presence of absence of precipitating antibodies and clinical status. Sera were screened by quantitative ELISA for IgG and IgG subclass activity to these antigens. There was no significant difference in the median IgG titre to pigeon mucin in individuals with PFL and asymptomatic antibody positive individuals. IgG1 and IgG2 were the major antibody subclasses against pigeon intestinal mucin with lower titres of IgG3 and negligible IgG4. Patients with PFL had significantly higher titres of anti-mucin IgG1 than asymptomatic antibody positive individuals (p = 0.0019) whilst there was no significant differences in IgG2, IgG3 and IgG4 titres between these two groups. Papain digestion of mucin resulted in a 600 fold decrease in IgG3 titres whilst IgG1 and IgG2 titres were essentially unaffected as compared to undigested mucin. This suggests that both IgG1 and IgG2 are directed against the O-linked oligosaccharides in the papain resistant regions of mucin whilst the majority of IgG3 is directed against papain sensitive protein epitopes (or possibly N-Iinked sugars in the sparsely glycosylated regions). - The difference between symptomatic and asymptomatic sera in the IgG subclass profiles to pigeon intestinal mucin together with the very high titres of anti-mucin antibody in all individuals with PFL confirm pigeon mucin as a major antigen in disease. Differences in antibody subclass profiles are likely to affect the composition and properties of the immune complexes, with consequences for the developmcnt of disease. PMID- 10756173 TI - Child neglect: guidance for pediatricians. PMID- 10756174 TI - Neonatal seizures. PMID- 10756175 TI - Diabetes insipidus. PMID- 10756176 TI - Addressing common pediatric concerns through children's books. PMID- 10756177 TI - Index of suspicion. Case 1. Neisseria gonorrhoeae. PMID- 10756178 TI - Increased expression of osteonectin/SPARC mRNA and protein in age-related human cataracts and spatial expression in the normal human lens. AB - PURPOSE: We have previously reported increased levels of Osteonectin/SPARC transcript in age-related cataractous compared to normal human lenses. The purpose of the present study was to evaluate the corresponding levels of osteonectin/SPARC protein in age-related cataractous relative to normal lenses and to evaluate the levels of osteonectin/SPARC transcript in specific types of age-related human cataracts. The spatial expression of osteonectin/SPARC was also evaluated in normal human lenses. METHODS: Specific types of age-related cataracts were collected and graded. Normal human lenses were microdissected into epithelia and fibers. Osteonectin/SPARC protein levels were monitored by Western immunoblotting, and transcript levels were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). Osteonectin/SPARC expression patterns were examined by RT-PCR and by immunostaining. RESULTS: Higher levels of osteonectin/SPARC protein were detected in age-related cataractous relative to normal human lenses. Increased levels of osteonectin/SPARC transcript were also detected in posterior-subcapsular and nuclear cataractous lenses relative to normal lenses. Osteonectin/SPARC transcripts were detected in the lens epithelium but not fibers. Osteonectin/SPARC protein levels were highest in the peripheral lens epithelium. CONCLUSIONS: Consistent with our previous studies on osteonectin/SPARC mRNA levels, osteonectin/SPARC protein levels were also elevated in cataractous compared to normal human lenses. Increased levels of osteonectin/SPARC mRNA were also found in nuclear and posterior-subcapsular cataracts relative to normal lenses. Osteonectin/SPARC expression is confined to the lens epithelium, and osteonectin/SPARC levels are highest in the peripheral lens epithelium. PMID- 10756179 TI - Prediction of structural and functional relationships of Repeat 1 of human interphotoreceptor retinoid-binding protein (IRBP) with other proteins. AB - PURPOSE: We compared the structure and function of interphotoreceptor retinoid binding protein (IRBP) related proteins and predicted domain and secondary structure within each repeat of IRBP and its relatives. We tested whether tail specific protease (Tsp), which bears sequence similarity to IRBP Domain B, binds fatty acids or retinoids, and whether IRBP possessed protease activity resembling Tsp's catalytic function. These tests helped us to learn whether the primary sequence similarities of family members extended to higher order structural and functional levels. METHODS: Predictions derived from multiple sequence alignments among IRBP and Tsp family members and secondary structure computer programs were carried out. The first repeat of human IRBP (EcR1) and Tsp were expressed, purified, and tested for binding properties. Tsp was examined for fluorescence enhancement of retinol or 16-anthroyloxy-palmitic acid (16-AP) to test for ligand binding. IRBP was tested for protease activity. RESULTS: Tsp did not exhibit fluorescence enhancement with retinol or 16-AP. IRBP did not exhibit protease activity. The positions of critical residues needed for the ligand binding properties of retinol were predicted. Primary sequence and three-dimensional similarity was found between Domain A of IRBP Repeat 3 and eglin c. CONCLUSIONS: The sequence similarity of Tsp and IRBP raised the possibility that each might share the function of the other protein: IRBP might possess protease activity or Tsp might possess retinoid or fatty acid binding activity. Our studies do not support such a shared function hypothesis, and suggest that the sequence similarity is the result of maintenance of structure. The finding of similarity to eglin c in Domain A suggests the possibility of a tight interaction between Domain A and Domain B, possibly implying the need for Domain A in retinoid binding, and suggesting that both Domains should be present in testing mutations. The positions of predicted critical amino acids suggest models in which a large binding pocket holds the retinoid or fatty acid ligand. These predictions are tested in a companion paper. PMID- 10756180 TI - Effects of dispersed point substitutions in Repeat 1 of human interphotoreceptor retinoid binding protein (IRBP). AB - PURPOSE: The purpose of this study was to measure the effects of mutations on the retinol binding capability of human Repeat 1 of interphotoreceptor retinoid binding protein (IRBP). First, we predicted important functional amino acids by several computer programs. We also noted the lack of shared functions between Tail-specific protease (Tsp) and IRBP, which bear sequence similarity, and this aided in predicting functional residues. We analyzed the effects of point substitutions on the retinol and fatty acid binding properties of Repeat 1 of human IRBP at 25 and 50 degrees C. METHODS: To find residues critical to retinol binding that might affect function, a series of thirteen mutations were created by site-specific mutagenesis between positions 140 and 280 in Repeat 1 of human IRBP. These mutants were expressed, purified, and tested for binding properties. The conformations of the proteins were examined by circular dichroism (CD) scans. RESULTS: Seven of the mutations exhibited reduced binding capacity, and five were not expressed at high enough levels to assess binding activity. Four of the mutants were purified, and their CD scans were very similar to those of Repeat 1. Only one of the mutations did not affect binding, folding, or expression when compare to wild type Repeat 1. CONCLUSIONS: Several IRBP mutants containing point mutations retained native structure but lost retinol binding function. The data suggest that retinol binding is affected by many different amino acid substitutions in or near a binding pocket. That even a single point substitution can profoundly affect binding without affecting overall conformation suggests that much of Domain B (from amino acid positions 80 to 300) is involved with ligand binding. This excludes three previously proposed IRBP-retinol binding mechanisms: (1) retinol binds to a small portion of the protein repeat, (2) retinol can bind to any hydrophobic patch in the protein, and (3) native conformation is not required for retinol binding to the repeat. PMID- 10756181 TI - G239T mutation in Repeat 1 of human IRBP: possible implications for more than one binding site in a single repeat. AB - PURPOSE: Interphotoreceptor retinoid-binding protein(IRBP) is a four-repeat protein found in the interphotoreceptor space. Each repeat can bind retinoids and fatty acids. The purpose of this study was to examine the effects of the single amino acid substitution, G239T, versus the wild type sequence of human IRBP Repeat 1, on ligand binding at equilibrium, ligand off rates, and protection of retinol from degradation. METHODS: G239T was created by site-specific mutagenesis, expressed in E. coli, and purified. E. coli expressed wild type Repeat 1 (EcR1) and G239T were subjected to thermal denaturation and analyzed by circular dichroism spectroscopy. We compared the ligand binding properties by fluorescence enhancement of retinol and 16-anthroyloxy-palmitate, tryptophan quenching of the proteins by different ligands, binding competition assays, protection of retinol from degradation, and stopped-flow kinetics to measure transfer of ligands to and from model membranes. RESULTS: Circular dichroism, fluorescence, and absorbance spectroscopy of G239T and EcR1 showed similar wavelength scans. G239T exhibited about three-fold less fluorescence of bound all trans-retinol or 13-cis-retinol versus EcR1. Retinol quenching of intrinsic protein fluorescence was reduced by 37% in G239T versus EcR1. Other retinoids used as quenchers produced no difference between intrinsic protein fluorescence of either G239T or EcR1; all exhibited saturable high affinity binding to each protein. Docosahexaenoic acid (DHA) served as a competitive inhibitor of retinol fluorescence enhancement with EcR1. However, DHA did not alter retinol fluorescence with G239T. 16-anthroyloxy-palmitate (16-AP) exhibited about 30% higher levels of fluorescence enhancement when bound to G239T versus EcR1. EcR1 prevented oxidative damage of all-trans-retinol, whereas G239T provided much less protection. Each protein could accept 9-cis-retinal from small unilamellar vesicles (SUVs) as measured by stopped flow kinetics. Off rates were the same in comparing G239T and EcR1 as acceptors. CONCLUSIONS: Despite the general similarity in shape between G239T and EcR1 and the nearly identical binding behavior with some ligands, distinct differences exist in the ligand binding properties of G239T and EcR1. Fluorescence enhancement/quenching and retinol protection experiments suggest that retinol binding is reduced by about 50% in G239T versus EcR1. The data suggest that either: (1) EcR1 contains two binding sites for retinol and G239T has lost one site or (2) EcR1 has a single binding site that is altered in G239T to reduce retinol binding. Results of all the experiments were consistent with the first model while some of the data were not consistent with the second model. Thus, it is possible that position 239, found in Domain B2 of IRBP Repeat 1, is located in or near one of two retinol binding sites. PMID- 10756182 TI - RNA-protein crosslinking to AMP residues at internal positions in RNA with a new photocrosslinking ATP analog. AB - A new photocrosslinking purine analog was synthesized and evaluated as a transcription substrate for Escherichia coli RNA polymerase. This analog, 8-[(4 azidophenacyl)thio]adenosine 5'-triphosphate (8-APAS-ATP) contains an aryl azide photocrosslinking group that is attached to the ATP base via a sulfur-linked arm on the 8 position of the purine ring. This position is not involved in the normal Watson-Crick base pairing needed for specific hybridization. Although 8-APAS-ATP could not replace ATP as a substrate for transcription initiation, once stable elongation complexes were formed, 8-APAS-AMP could be site-specifically incorporated into the RNA, and this transcript could be further elongated, placing the photoreactive analog at internal positions in the RNA. Irradiation of transcription elongation complexes in which the RNA contained the analog exclusively at the 3' end of an RNA 22mer, or a 23mer with the analog 1 nt from the 3' end, produced RNA crosslinks to the RNA polymerase subunits that form the RNA 3' end binding site (beta, beta'). Both 8-APAS-AMP and the related 8-azido AMP were subjected to conformational modeling as nucleoside monophosphates and in DNA-RNA hybrids. Surprisingly, the lowest energy conformation for 8-APAS-AMP was found to be syn, while that of 8-azido-AMP was anti, suggesting that the conformational properties and transcription substrate properties of 8-azido-ATP should be re-evaluated. Although the azide and linker together are larger in 8 APAS-ATP than in 8-N(3)-ATP, the flexibility of the linker itself allows this analog to adopt several different energetically favorable conformations, making it a good substrate for the RNA polymerase. PMID- 10756183 TI - Flexible non-nucleotide linkers as loop replacements in short double helical RNAs. AB - Ethylene glycol oligomers have been studied systematically as non-nucleotide loop replacements in short hairpin oligoribonucleotides. Structural optimization concerns the length of the linkers and is based on the thermodynamic stabilities of the corresponding duplexes. The optimum linker is derived from heptakis (ethylene glycol) provided that the duplex end to be bridged comprises solely the terminal base pair; the optimum linker is derived from hexakis(ethylene glycol) if a dangling unpaired nucleotide is incorporated into the loop. Moreover, these linkers have been compared to other commonly used linker types which consist of repeating units of tris- or tetrakis(ethylene glycol) phosphate, or of 3 hydroxypropane-1-phosphate. In all cases, the correlation between linker length and duplex stability is independent of the kind of counter ions used (Na(+), Na(+)/Mg(2+), K(+)or Li(+)). Furthermore, all duplexes with non-nucleotide loop replacements are less stable than those with the corresponding standard nucleotide loop. The results corroborate that the linkers are solvent-exposed and do not specifically interfere with the terminal nucleotides at the bridged duplex end. PMID- 10756184 TI - DNA sequence elements located immediately upstream of the -10 hexamer in Escherichia coli promoters: a systematic study. AB - We have made a systematic study of how the activity of an Escherichia coli promoter is affected by the base sequence immediately upstream of the -10 hexamer. Starting with an activator-independent promoter, with a 17 bp spacing between the -10 and -35 hexamer elements, we constructed derivatives with all possible combinations of bases at positions -15 and -14. Promoter activity is greatest when the 'non-template' strand carries T and G at positions -15 and -14, respectively. Promoter activity can be further enhanced by a second T and G at positions -17 and -16, respectively, immediately upstream of the first 'TG motif'. Our results show that the base sequence of the DNA segment upstream of the -10 hexamer can make a significant contribution to promoter strength. Using published collections of characterised E.coli promoters, we have studied the frequency of occurrence of 'TG motifs' upstream of the promoters' -10 elements. We conclude that correctly placed 'TG motifs' are found at over 20% of E.coli promoters. PMID- 10756185 TI - Functional analysis of the homeodomain protein SIX5. AB - SIX5 (previously known as myotonic dystrophy associated homeodomain protein - DMAHP ) is a member of the SIX [ sine oculis homeobox (Drosophila ) homologue ] gene family which encodes proteins containing a SIX domain adjacent to a homeo domain. To investigate the DNA binding specificities of these two domains in SIX5, they were expressed as GST fusion proteins, both separately and together. Affinity purified recombinant proteins and cell lysates from bacteria expressing the recombinant proteins were used in gel retardation assays with double stranded oligonucleotides representing putative DNA binding sites. The putative sites included two in the promoter region of DMPK (dystrophia myotonica protein kinase ) and the previously characterised murine Six4 DNA binding site in the Na(+)/K(+) ATPase alpha 1 subunit gene ( ATP1A1 ) regulatory element (ARE). None of the recombinant proteins showed any affinity for the two putative sites in DMPK. However, the two recombinant proteins containing the homeodomain both formed at least one specific complex with the ARE. The recombinant protein containing both domains formed a second specific complex with the ARE, assumed to be a dimer complex. Finally, a whole genome PCR-based screen was used to identify genomic DNA sequences to which SIX5 binds, as an initial stage in the identification of genes regulated by SIX5. PMID- 10756186 TI - Whole genome sequence-enabled prediction of sequences performed for random PCR products of Escherichia coli. AB - The sequence of an unknown PCR product generated by random (and conventional) PCR could be determined without sequencing when it is provided with the template DNA sequence. Theoretically, this was based on formerly established ideas which assert that the amount of random PCR product mainly depends on the stability of the primer-binding structures and that the dynamic solution structure of DNA is essentially governed by the Watson-Crick base pairing. However, it has not been clear whether this holds true for larger genomes of mega- to gigabase size, beside the lambda phage genome (of 50 kb) used previously, nor has it been ascertained to uniquely specify the sequence of a random PCR product. Here, we jointly use two computer programs together with experimental data from Genome Profiling (i.e. TGGE analysis of random PCR products). The first procedure carried out by a newly remodeled computer program (PCRAna-A1) was shown to be competent to calculate a set of random PCR products from Escherichia coli genome DNA (4.7 Mb). The other procedure performed with another program (Poland-H) played a critical role in determining the final candidate sequence by theoretically offering the initial melting temperature and the melting pattern of unspecified candidate sequences. The success attained here not only proved our method to be useful for sequence prediction but also confirmed the above mentioned ideas as rational. We believe that this is the first case to computer utilize a genome sequence as a whole. PMID- 10756187 TI - Characterization of Candida albicans RNA triphosphatase and mutational analysis of its active site. AB - The RNA triphosphatase component (CaCet1p) of the mRNA capping apparatus of the pathogenic fungus Candida albicans differs mechanistically and structurally from the RNA triphosphatase of mammals. Hence, CaCet1p is an attractive antifungal target. Here we identify a C-terminal catalytic domain of CaCet1p from residue 257 to 520 and characterize a manganese-dependent and cobalt-dependent NTPase activity intrinsic to CaCet1p. The NTPase can be exploited to screen in vitro for inhibitors. The amino acids that comprise the active site of CaCet1p were identified by alanine-scanning mutagenesis, which was guided by the crystal structure of the homologous RNA triphosphatase from Saccharomyces cerevisiae (Cet1p). Thirteen residues required for the phosphohydrolase activity of CaCet1p (Glu287, Glu289, Asp363, Arg379, Lys396, Glu420, Arg441, Lys443, Arg445, Asp458, Glu472, Glu474 and Glu476) are located within the hydrophilic interior of an eight-strand beta barrel of Cet1p. Each of the eight strands contributes at least one essential amino acid. The essential CaCet1p residues include all of the side chains that coordinate manganese and sulfate (i.e., gamma phosphate) in the Cet1p product complex. These results suggest that the active site structure and catalytic mechanism are conserved among fungal RNA triphosphatases. PMID- 10756188 TI - DNA strand transfer catalyzed by vaccinia topoisomerase: ligation of DNAs containing a 3' mononucleotide overhang. AB - The specificity of vaccinia topoisomerase for transesterification to DNA at the sequence 5'-CCCTT and its versatility in strand transfer have illuminated the recombinogenic properties of type IB topoisomerases and spawned topoisomerase based strategies for DNA cloning. Here we characterize a pathway of topoisomerase mediated DNA ligation in which enzyme bound covalently to a CCCTT end with an unpaired +1T nucleotide rapidly and efficiently joins the CCCTT strand to a duplex DNA containing a 3' A overhang. The joining reaction occurs with high efficiency, albeit slowly, to duplex DNAs containing 3' G, T or C overhangs. Strand transfer can be restricted to the correctly paired 3' A overhang by including 0.5 M NaCl in the ligation reaction mixture. The effects of base mismatches and increased ionic strength on the rates of 3' overhang ligation provide a quantitative picture of the relative contributions of +1 T:A base pairing and electrostatic interactions downstream of the scissile phosphate to the productive binding of an unlinked acceptor DNA to the active site. The results clarify the biochemistry underlying topoisomerase-cloning of PCR products with non-templated 3' overhangs. PMID- 10756189 TI - Site-specific modification and RNA crosslinking of the RNA-binding domain of PKR. AB - RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates and inhibits the function of the translation initiation factor eIF-2. PKR is activated in vitro by binding RNA molecules with extensive duplex structure. To further define the nature of the RNA regulation of PKR, we have prepared and characterized site-specifically modified proteins consisting of the PKR 20 kDa RNA-binding domain (RBD). Here we show that the two cysteines found naturally in this domain can be altered by site-directed mutagenesis without loss of RNA binding affinity or the RNA-regulated kinase activity. Introduction of cysteine residues at other sites in the PKR RBD allows for site specific modification with thiol-selective reagents. PKR RBD mutants reacted selectively with a maleimide to introduce a photoactivatable cross-linking aryl azide at three different positions in the protein. RNA crosslinking efficiency was found to be dependent on the amino acid modified, suggesting differences in access to the RNA from these positions in the protein. One of the amino acid modifications that led to crosslinking of the RNA is located at a residue known to be an autophosphorylation site, suggesting that autophosphorylation at this site could influence the RNA binding properties of PKR. The PKR RBD conjugates described here and other similar reagents prepared via these methods are applicable to future studies of PKR-RNA complexes using techniques such as photocrosslinking, fluorescence resonance energy transfer and affinity cleaving. PMID- 10756190 TI - Structural basis for uracil DNA glycosylase interaction with uracil: NMR study. AB - Two dimensional (2D) NMR and molecular dynamics simulations have been used to determine the three dimensional (3D) structure of a hairpin DNA, d-CTA-GAGGATCC TUTT-GGATCCT (22mer; abbreviated as U2-hairpin), which has uracil at the second position from the 5' end of the tetraloop. The(1)H resonances of this hairpin have been assigned almost completely. NMR restrained molecular dynamics and energy minimization procedures have been used to describe the 3D structure of U2 hairpin. This study establishes that the stem of the hairpin adopts a right handed B-DNA conformation, while the T(12)and T(15)nucleotides stack upon 3' and 5' ends of the stem, respectively. Further, T(14)stacks upon both T(12)and T(15). Though U(13)partially stacks upon T(14), no stacking interaction is observed between U(13)and T(12). All the individual nucleotide bases belonging to the stem and T(12)and T(15)of the loop adopt ' anti ' conformation with respect to their sugar moiety, while the U(13)and T(14)of the loop are in ' syn ' conformation. The turning phosphate in the loop is located between T(13)and T(14). This study and a concurrent NMR structural study on yet another hairpin DNA d-CTAGAGGAATAA TTTU-GGATCCT (22mer; abbreviated as U4-hairpin), with uracil at the fourth position from the 5' end of the tetraloop throw light upon various interactions which have been reported between Escherichia coli uracil DNA glycosylase (UDG) and uracil containing DNA. The epsilon of T(12)and alpha, beta, gamma, epsilon and zeta of U(13)and gamma of T(14), which partially influence the local conformation of U(13)in U2-hairpin are all locked in ' trans ' conformation. Such stretched out backbone conformation in the vicinity of U(13)could be the reason as to why the U2-hairpin is found to be the poor substrate for its interaction with UDG compared to the other substrates in which the uracil is at first, third and fourth positions of the tetraloop from its 5' end, as reported earlier by Vinay and Varshney. This study shows that UDG actively promotes the flipping of uracil from a stacked conformation and rules out the possibility of UDG recognizing the flipped out uracil bases. PMID- 10756191 TI - Intramolecular interaction of yeast TFIIB in transcription control. AB - The general transcription factor TFIIB is a key component in the eukaryotic RNA polymerase II (RNAPII) transcriptional machinery. We have previously shown that a yeast TFIIB mutant (called YR1m4) with four amino acid residues in a species specific region changed to corresponding human residues affects the expression of genes activated by different activators in vivo. We report here that YR1m4 can interact with several affected activators in vitro. In addition, YR1m4 and other mutants with amino acid alterations within the same region can interact with TATA binding protein (TBP) and RNAPII normally. However, YR1m4 is defective in supporting activator-independent transcription in assays con-ducted both in vitro and in vivo. We further demonstrate that the interaction between the C-terminal core domain and the N-terminal region is weakened in YR1m4 and other related TFIIB mutants. These results suggest that the intramolecular interaction property of yeast TFIIB plays an important role in transcription regulation in cells. PMID- 10756192 TI - MeCP2 driven transcriptional repression in vitro: selectivity for methylated DNA, action at a distance and contacts with the basal transcription machinery. AB - The pathways for selective transcriptional repression of methylated DNA templates by the methyl-CpG-binding protein MeCP2 have been investigated using a purified in vitro transcription system that does not assemble chromatin. MeCP2 selectively inhibits transcription complex assembly on methylated DNA but does not destabilize a pre-assembled transcription complex. MeCP2 functions to repress transcription at a distance of >500 bp from the transcription start site. The transcription repression domain (TRD) of MeCP2 will repress transcription in vitro when fused to a heterologous Gal4 DNA-binding domain. The TRD associates with TFIIB. Exogenous TFIIB does not relieve transcriptional repression established by either intact MeCP2 or a Gal4-TRD fusion protein under these in vitro conditions, nor does the addition of histone deacetylase inhibitors. We find that the transcriptional repression established by both MeCP2 and the Gal4 TRD fusion protein in vitro also correlates with selective assembly of large nucleoprotein complexes. The formation of such complexes reflects a local concentration of DNA-bound transcriptional repressor that may stabilize a state of repression even in the presence of exogenous transcriptional machinery. PMID- 10756193 TI - Thermodynamic parameters for DNA sequences with dangling ends. AB - The thermodynamic contributions to duplex formation of all 32 possible single nucleotide dangling ends on a Watson-Crick pair are reported. In most instances, dangling ends are stabilizing with free energy contributions ranging from +0.48 (GT(A)) to-0.96 kcal/mol (). In comparison, Watson-Crick nearest-neighbor increments range from -0. 58 (TA/AT) to -2.24 (GC/CG) kcal/mol. Hence, in some cases, a dangling end contributes as much to duplex stability as a Watson-Crick A T base pair. The implications of these results for DNA probe design are discussed. Analysis of the sequence dependence of dangling-end stabilities show that the nature of the closing base pair largely determines the stabilization. For a given closing base pair, however, adenine dangling ends are always more or equally as stable as the other dangling nucleotides. Moreover, 5' dangling ends are more or equally as stabilizing as their 3' counterparts. Comparison of DNA with RNA dangling-end motifs shows that DNA motifs with 5' dangling ends contribute to stability equally or more than their RNA counterparts. Conversely, RNA 3' dangling ends contribute to stability equally or more than their DNA counterparts. This data set has been incorporated into a DNA secondary structure prediction algorithm (DNA MFOLD) (http://mfold2.wustl.edu/mfold/dna/for m1.cgi) as well as a DNA hybridization prediction algorithm (HYTHERtrade mark) (http://jsl1.chem.wayne.edu/Hyther/hythermenu .html). PMID- 10756194 TI - Surface plasmon resonance kinetic studies of the HIV TAR RNA kissing hairpin complex and its stabilization by 2-thiouridine modification. AB - Surface plasmon resonance (BIACORE) was used to determine the kinetic values for formation of the HIV TAR-TAR* ('kissing hairpin') RNA complex. The TAR component was also synthesized with the modified nucleoside 2-thiouridine at position 7 in the loop and the kinetics and equilibrium dissociation constants compared with the unmodified TAR hairpin. The BIACORE data show an equilibrium dissociation constant of 1.58 nM for the complex containing the s(2)U modified TAR hairpin, which is 8-fold lower than for the parent hairpin (12.5 nM). This is a result of a 2-fold faster k(a) (4.14x10(5) M(-1) s(-1) versus 2.1x10(5) M(-1) s(-1)) and a 4-fold slower k(d) (6.55x10(-4) s(-1) versus 2.63x10(-3) s(-1)). (1)H NMR imino spectra show that the secondary structure interactions involved in complex formation are retained in the s(2)U-modified complex. Magnesium has been reported to significantly stabilize the TAR-TAR* complex and we found that Mn(2+) and Ca(2+) are also strongly stabilizing, while Mg(2+) exhibited the greatest effect on the complex kinetics. The stabilizing effects of 2-thiouridine indicate that this base modification may be generally useful as an antisense RNA modification for oligonucleotide therapeutics which target RNA loops. PMID- 10756195 TI - Identification of the Saccharomyces cerevisiae RNA:pseudouridine synthase responsible for formation of psi(2819) in 21S mitochondrial ribosomal RNA. AB - So far, four RNA:pseudouridine (Psi)-synthases have been identified in yeast Saccharomyces cerevisiae. Together, they act on cytoplasmic and mitochondrial tRNAs, U2 snRNA and rRNAs from cytoplasmic ribosomes. However, RNA:Psi-synthases responsible for several U-->Psi conversions in tRNAs and UsnRNAs remained to be identified. Based on conserved amino-acid motifs in already characterised RNA:Psi synthases, four additional open reading frames (ORFs) encoding putative RNA:Psi synthases were identified in S.cerevisiae. Upon disruption of one of them, the YLR165c ORF, we found that the unique Psi residue normally present in the fully matured mitochondrial rRNAs (Psi(2819)in 21S rRNA) was missing, while Psi residues at all the tested pseudo-uridylation sites in cytoplasmic and mitochondrial tRNAs and in nuclear UsnRNAs were retained. The selective U-->Psi conversion at position 2819 in mitochondrial 21S rRNA was restored when the deleted yeast strain was transformed by a plasmid expressing the wild-type YLR165c ORF. Complementation was lost after point mutation (D71-->A) in the postulated active site of the YLR165c-encoded protein, indicating the direct role of the YLR165c protein in Psi(2819)synthesis in mitochondrial 21S rRNA. Hence, for nomenclature homogeneity the YLR165c ORF was renamed PUS5 and the corresponding RNA:Psi-synthase Pus5p. As already noticed for other mitochondrial RNA modification enzymes, no canonical mitochondrial targeting signal was identified in Pus5p. Our results also show that Psi(2819)in mitochondrial 21S rRNA is not essential for cell viability. PMID- 10756197 TI - Human Kruppel-like factor 8: a CACCC-box binding protein that associates with CtBP and represses transcription. AB - CACCC-boxes are recognised by transcription factors of the Sp/Kruppel-like Factor (Sp1/KLF) family. Here we describe one member of this family, KLF8/ZNF741/BKLF3 (KLF8). KLF8 contains a characteristic C-terminal DNA-binding domain comprised of three Kruppel-like zinc fingers, but also has limited homology to another family member, KLF3/Basic Kruppel-like Factor (KLF3/BKLF), in its N-terminus. Most significantly, it shares with KLF3/BKLF a Pro-Val-Asp-Leu-Ser/Thr motif. In KLF3/BKLF this motif mediates contact with the co-repressor protein C-terminal Binding Protein (CtBP). We demonstrate that the KLF8 Pro-Val-Asp-Leu-Ser motif also contacts CtBP. We show that the N-terminus of KLF8 functions as a repression domain and that its activity relies on the integrity of the CtBP recognition motif. We demonstrate that the zinc fingers of KLF8 recognize CACCC elements in DNA and that full-length KLF8 can repress a CACCC-dependent promoter. Finally we determine that KLF8 is broadly expressed in human tissues. These results establish KLF8 as a CACCC-box binding protein that associates with CtBP and represses transcription. PMID- 10756196 TI - Sensitivity of human type II topoisomerases to DNA damage: stimulation of enzyme mediated DNA cleavage by abasic, oxidized and alkylated lesions. AB - Type II topoisomerases are essential enzymes that are also the primary cellular targets for a number of important anticancer drugs. These drugs act by increasing levels of topoisomerase II-mediated DNA cleavage. Recent studies indicate that endogenous forms of DNA damage, such as abasic sites and base mismatches, also stimulate the DNA scission activity of the enzyme. To extend our understanding of how type II topoisomerases react to DNA damage, the effects of abasic sites, and oxidized and alkylated bases on DNA cleavage mediated by human topo-isomerase IIalpha and beta were determined. Based on experiments that incorporated random abasic sites into plasmid DNA, human type II enzymes can locate lesions even within a background of several thousand undamaged base pairs. As determined by experiments that utilized site-specific forms of DNA lesions, oxidized or monoalkylated purines that allow base pairing and induce little distortion in the double helix have modest effects on topoisomerase II-mediated DNA cleavage. In contrast, 1,N(6)-ethenoadenine, a bulky lesion that disrupts base pairing, enhanced DNA cleavage approximately 10-fold. 1,N(6)-Ethenoadenine is the first lesion found to rival the stimulatory effects of apurinic sites on the DNA scission activity of eukaryotic type II topoisomerases. PMID- 10756198 TI - Interaction of three-way DNA junctions with steroids. AB - DNA aptamers that bind to cholic acid were previously isolated by an in vitro selection method. Secondary structural prediction and deletion-mutant experiments suggested that the cholic-acid binding regions of 19 sequenced clones could form three-way-junction structures. In this article, the secondary structures of the sequenced clones and the structural requirements for binding to cholic acid were evaluated. A course of mutational-analysis and chemical-modification experiments provided strong support for the predicted secondary structure and also indicated that the binding site is located at the branching point of the three-way junction. Sequence analysis revealed that the sequences of the three base pairs flanking the junction of the three stems are highly conserved among selected clones. The evaluation of the relative binding of several bile acids and structurally related steroids with the aptamer was also carried out. The results revealed a broad range of selectivity and preference for hydrophobic steroids rather than for cholic acid upon binding, indicating that the binding is driven by a hydrophobic interaction. The experimental results reported here allowed us to propose a structural model of a binding site formed by three Watson-Crick base pairs. PMID- 10756199 TI - Structures of the potassium-saturated, 2:1, and intermediate, 1:1, forms of a quadruplex DNA. AB - Potassium can stabilize the formation of chair- or edge-type quadruplex DNA structures and appears to be the only naturally occurring cation that can do so. As quadruplex DNAs may be important in the structure of telomere, centromere, triplet repeat and other DNAs, information about the details of the potassium quadruplex DNA interactions are of interest. The structures of the 1:1 and the fully saturated, 2:1, potassium-DNA complexes of d(GGTTGGTGTGGTTGG) have been determined using the combination of experimental NMR results and restrained molecular dynamics simulations. The refined structures have been used to model the interactions at the potassium binding sites. Comparison of the 1:1 and 2:1 potassium:DNA structures indicates how potassium binding can determine the folding pattern of the DNA. In each binding site potassium interacts with the carbonyl oxygens of both the loop thymine residues and the guanine residues of the adjacent quartet. PMID- 10756200 TI - Accurate and rapid modeling of iron-bleomycin-induced DNA damage using tethered duplex oligonucleotides and electrospray ionization ion trap mass spectrometric analysis. AB - Bleomycin B(2)(BLM) in the presence of iron [Fe(II)] and O(2)catalyzes single stranded (ss) and double-stranded (ds) cleavage of DNA. Electrospray ionization ion trap mass spectrometry was used to monitor these cleavage processes. Two duplex oligonucleotides containing an ethylene oxide tether between both strands were used in this investigation, allowing facile monitoring of all ss and ds cleavage events. A sequence for site-specific binding and cleavage by Fe-BLM was incorporated into each analyte. One of these core sequences, GTAC, is a known hot spot for ds cleavage, while the other sequence, GGCC, is a hot-spot for ss cleavage. Incubation of each oligo-nucleotide under anaerobic conditions with Fe(II)-BLM allowed detection of the non-covalent ternary Fe-BLM/oligonucleotide complex in the gas phase. Cleavage studies were then performed utilizing O(2) activated Fe(II)-BLM. No work-up or separation steps were required and direct MS and MS/MS analyses of the crude reaction mixtures confirmed sequence-specific Fe BLM-induced cleavage. Comparison of the cleavage patterns for both oligonucleotides revealed sequence-dependent preferences for ss and ds cleavages in accordance with previously established gel electrophoresis analysis of hairpin oligonucleotides. This novel methodology allowed direct, rapid and accurate determination of cleavage profiles of model duplex oligonucleotides after exposure to activated Fe-BLM. PMID- 10756201 TI - Identification of a transcription factor IIIA-interacting protein. AB - Transcription factor IIIA (TFIIIA) activates 5S ribosomal RNA gene transcription in eukaryotes. The protein from vertebrates has nine contiguous Cys(2)His(2)zinc fingers which function in nucleic acid binding, and a C-terminal region involved in transcription activation. In order to identify protein partners for TFIIIA, yeast two-hybrid screens were performed using the C-terminal region of Xenopus TFIIIA as an attractor and a rat cDNA library as a source of potential partners. A cDNA clone was identified which produced a protein in yeast that interacted with Xenopus TFIIIA but not with yeast TFIIIA. This rat clone was sequenced and the primary structure of the human homolog (termed TFIIIA-intP for TFIIIA interacting protein) was determined from expressed sequence tags. In vitro interaction of recombinant human TFIIIA-intP with recombinant Xenopus TFIIIA was demonstrated by immuno-precipitation of the complex using anti-TFIIIA-intP antibody. Interaction of rat TFIIIA with rat TFIIIA-intP was indicated by co chromatography of the two proteins on DEAE-5PW following fractionation of a rat liver extract on cation, anion and gel filtration resins. In a HeLa cell nuclear extract, recombinant TFIIIA-intP was able to stimulate TFIIIA-dependent transcription of the Xenopus 5S ribosomal RNA gene but not TFIIIA-independent transcription of the human adenovirus VA RNA gene. PMID- 10756202 TI - The human LEF-1 gene contains a promoter preferentially active in lymphocytes and encodes multiple isoforms derived from alternative splicing. AB - Lymphoid Enhancer Factor-1 (LEF-1) is a member of a family of transcription factors that function as downstream mediators of the Wnt signal transduction pathway. In the absence of Wnt signals, specific LEF/TCF isoforms repress rather than activate gene targets through recruitment of the co-repressor CtBP. Characterization of the full-length human LEF-1 gene locus and its complete set of mRNA products shows that this family member exists as a unique set of alternatively spliced isoforms; none are homologous to TCF-1E/TCF-4E. Therefore LEF-1 is distinct from its TCF family members in that it cannot engage in activities specific to this isoform such as recruitment of the co-repressor CtBP. Expression of alternatively spliced LEF-1 isoforms are driven by a promoter that is highly active in lymphocyte cell lines. Transcription initiates within a TATA less core promoter region that contains consensus binding sites for Sp1, an E box, an Initiator element and a LEF/TCF binding site, all juxtaposed to the start sites of transcription. The promoter is most active in a B lymphocyte cell line (Raji) in which the endogenous LEF-1 gene is silent, suggesting that the promoter region is actively repressed by a silencing mechanism. PMID- 10756203 TI - Fission yeast hrp1, a chromodomain ATPase, is required for proper chromosome segregation and its overexpression interferes with chromatin condensation. AB - Hrp1 of Schizosaccharomyces pombe is a member of the CHD protein family, characterized by a chromodomain, a Myb-like telobox-related DNA-binding domain and a SNF2-related helicase/ATPase domain. CHD proteins are thought to be required for modification of the chromatin structure in transcription, but the exact roles of CHD proteins are not known. Here we examine the sub-cellular localization and biochemical activity of Hrp1 and the phenotypes of hrp1 Delta and Hrp1-overexpressing strains. Fluorescence microscopy revealed that Hrp1 protein is targeted to the nucleus. We found that Hrp1 exhibited DNA-dependent ATPase activity, stimulated by both single- and double-stranded DNA. Overexpression of Hrp1 caused slow cell growth accompanied by defective chromosome condensation in anaphase resulting in a 'cut' (celluntimelytorn) phenotype and chromosome loss. The hrp1 Delta mutation also caused abnormal anaphase and mini-chromosome loss phenotypes. Electron micrographs demonstrated that aberrantly shaped nucleoli appeared in Hrp1-overexpressing cells. Therefore, these results suggest that Hrp1 may play a role in mitotic chromosome segregation and maintenance of chromatin structure by utilizing the energy from ATP hydrolysis. PMID- 10756204 TI - Functional characterization of Caenorhabditis elegans DNA topoisomerase IIIalpha. AB - To investigate the function of a DNA topoisomerase III enzyme in Caenorhabditis elegans, the full-length cDNA of C.elegans DNA topoisomerase IIIalpha was cloned. The deduced amino acid sequence exhibited identities of 48 and 39% with those of human DNA topoisomerase IIIalpha and Saccharomyces cerevisiae DNA topoisomerase III, respectively. The overexpressed polypeptide showed an optimal activity for removing negative DNA supercoils at a relatively high temperature of 52-57 degrees C, which is similar to the optimum temperatures of other eukaryotic DNA topoisomerase III enzymes. When topoisomerase IIIalpha expression was interfered with by a cognate double-stranded RNA injection, pleiotropic phenotypes with abnormalities in germ cell proliferation, oogenesis and embryo-genesis appeared. These phenotypes were well correlated with mRNA expression localized in the meiotic cells of gonad and early embryonic cells. PMID- 10756205 TI - Detection and mapping of mismatched base pairs in DNA molecules by atomic force microscopy. AB - Attempts were made to apply atomic force microscopy (AFM) imaging to the detection and mapping of the sites of base substitutions in DNA molecules. In essence, DNA fragments to be examined for possible base substitutions were mixed with an equal amount of a corresponding DNA standard and subjected to heat denaturation and subsequent annealing. The reassociated DNA was incubated with MutS protein, a protein that recognizes and binds to mismatched base pairs in duplex DNA. Bound MutS protein molecules were then detected by AFM and their positions along the DNA molecules were determined by calculating the distance from one of the DNA termini, which had been tagged with a biotin-avidin complex. Base substitutions present in DNA molecules >1 kb were effectively detected by this procedure, and the positions determined were in good agreement with the actual mutation sites. This method is quite simple, has virtually no limitations on the size of DNA fragments to be examined and requires only a very small amount of DNA sample. PMID- 10756206 TI - AFM characterization of single strand-specific endonuclease activity on linear DNA. AB - The specificity of nucleases for nicked and un-nicked double-stranded DNA has been characterized using atomic force microscopy (AFM). We have found that AFM has advantages over the usual macroscopic analyses, such as sucrose gradient centrifugation or electrophoresis, in characterizing nuclease digestion. In particular, short DNA fragments resulting from non-specific digestion were detected and, thus, the true length distribution of digested DNA was revealed. A simple numerical method is proposed to estimate the number of nicked sites per DNA molecule based on AFM images. PMID- 10756207 TI - Highly efficient solid phase synthesis of oligonucleotide analogs containing phosphorodithioate linkages. AB - A triester method for the synthesis of deoxynucleoside phosphorodithioate dimers is described. The phosphorodithioate linkage is introduced using a new dithiophosphorylating reagent DPSE-SP(S)Cl(2)where DPSE = 2 diphenylmethylsilylethyl. This group is removed quickly using tetra-butylammonium fluoride leading to the quantitative formation of phosphorodithioate diesters uncontaminated with the corresponding phosphorothioates. The utility of this group is demonstrated by the synthesis of a penta-decathymidylic acid, [T(PS(2))T(PO(2))](7)T, which contains alternating phosphorodithioate/phosphate diester internucleotide linkages. PMID- 10756208 TI - Retro-recombination screening of a mouse embryonic stem cell genomic library. AB - Targeted gene disruption is an important tool in molecular medicine, allowing for the generation of animal models of human disease. Conventional methods of targeting vector (TV) construction are difficult and represent a rate limiting step in any targeting experiment. We previously demonstrated that bacteriophage are capable of acting as TVs directly, obviating the requirement for 'rolling out' plasmids from primary phage clones and thus eliminating an additional, time consuming step. We have also developed methods which facilitate the construction of TVs using recombination. In this approach, modification cassettes and point mutations are shuttled to specific sites in phage TVs using phage-plasmid recombination. Here, we report a further improvement in TV generation using a recombination screening-based approach deemed 'retro-recombination screening' (RRS). We demonstrate that phage vectors containing specific genomic clones can be genetically isolated from a lambdaTK embryonic stem cell genomic library using a cycle of integrative recombination and condensation. By introducing the gam gene of bacteriophage lambda into the probe plasmid it is possible to select for positive clones which have excised the plasmid, thus returning to their native conformation following purification from the library. Rapid clone isolation using the RRS protocol provides another method by which the time required for TV construction may be further reduced. PMID- 10756209 TI - Branch migration inhibition in PCR-amplified DNA: homogeneous mutation detection. AB - A novel method for detection of any mutation located within a PCR-amplified DNA sequence was demonstrated. The method is based on the inhibition of spontaneous DNA branch migration. Partial duplexes produced by PCR amplification of a test and a reference genomic DNA sample anneal to form four-stranded cruciform structures. Spontaneous DNA branch migration results in dissociation of these structures when the test and reference sequences are identical. Any base substitution, deletion or insertion inhibits branch migration and produces stable cruciform structures. When suitable ligands are attached to the PCR primers, the cruciform structures can be detected by standard immunochemical methods. This approach was tested using several commonly occurring mutations within the human cystic fibrosis gene. New methods for increasing the specificity of PCR amplifications are described that were used for successful mutation analysis. PMID- 10756210 TI - Microchip electrophoresis: a method for high-speed SNP detection. AB - As a trial practical application, we have applied optimized microfabricated electrophoresis devices, combined with enzymatic mutation detection methods, to the determination of single nucleotide polymorphism (SNP) sites in the p53 suppressor gene. Using clinical samples, we have achieved robust assays with quality factors as good as conventional electrophoresis in approximately 100 s. This is 10 and 50 times faster than capillary and slab gel electro-phoresis, respectively. The method was highly accurate with an average error of mutation site measurement of only +/-5 bp. No clean-up of the digestion mixtures was needed prior to injection. This greatly simplifies sample handling relative to capillary instruments, which is important for high-throughput screening applications. Following identification, absolute mutation determination of the screened samples was achieved in a second microdevice optimized for four-color DNA sequencing. Total run time was 25 min in this second device and sequencing data were in full agreement with ABI Prism 377 sequencing runs which required 3.5 h. The tandem application of microdevices for location then full characterization of SNPs appears to confirm many of the improvements claimed for future application of microdevices in practical scaled screening for mutational analysis. PMID- 10756211 TI - A sensitive scanning technology for low frequency nuclear point mutations in human genomic DNA. AB - Knowledge of the kinds and numbers of nuclear point mutations in human tissues is essential to the understanding of the mutation mechanisms underlying genetic diseases. However, nuclear point mutant fractions in normal humans are so low that few methods exist to measure them. We have now developed a means to scan for point mutations in 100 bp nuclear single copy sequences at mutant fractions as low as 10(-6). Beginning with about 10(8) human cells we first enrich for the desired nuclear sequence 10,000-fold from the genomic DNA by sequence-specific hybridization coupled with a biotin-streptavidin capture system. We next enrich for rare mutant sequences 100-fold against the wild-type sequence by wide bore constant denaturant capillary electrophoresis (CDCE). The mutant-enriched sample is subsequently amplified by high fidelity PCR using fluorescein-labeled primers. Amplified mutant sequences are further enriched via two rounds of CDCE coupled with high fidelity PCR. Individual mutants, seen as distinct peaks on CDCE, are then isolated and sequenced. We have tested this approach by measuring N-methyl N'-nitro-N-nitrosoguanidine (MNNG)-induced point mutations in a 121 bp sequence of the adenomatous polyposis coli gene (APC) in human lymphoblastoid MT1 cells. Twelve different MNNG-induced GC-->AT transitions were reproducibly observed in MNNG-treated cells at mutant fractions between 2 x 10(-6) and 9 x 10(-6). The sensitivity of this approach was limited by the fidelity of Pfu DNA polymerase, which created 14 different GC-->TA transversions at a mutant fraction equivalent to approximately 10(-6) in the original samples. The approach described herein should be general for all DNA sequences suitable for CDCE analysis. Its sensitivity and capacity would permit detection of stem cell mutations in tissue sectors consisting of approximately 10(8) cells. PMID- 10756213 TI - Mechanisms of eosinophil-associated inflammation. AB - Increasing evidence supports a critical role for the eosinophil in disease. Here, the mechanisms underlying eosinophil-associated inflammation are reviewed including eosinophil constituents, eosinophil maturation and release from the bone marrow, and eosinophil tissue recruitment and activation. Eosinophil effector functions in bronchial asthma are summarized with particular attention to pulmonary M2 muscarinic receptors and bronchial hyperreactivity. Recent findings supporting roles for IL-5, the eosinophil, and its ribonucleases in viral immunity are presented. Overall, this information supports an expanded view of eosinophil participation in health and disease. PMID- 10756214 TI - Chronic urticaria. AB - Chronic urticaria remains a major problem in terms of etiology, investigation, and management. It is important to identify patients in whom physical urticaria is the principal cause of disability. Once confirmed by appropriate challenge testing, no further investigation is required. Urticarial vasculitis (UV) is a major differential diagnosis of "idiopathic" urticaria (CIU). I perform biopsy of most patients in this category because UV cannot be considered confirmed in the absence of histologic evidence. Patients with confirmed UV need to be thoroughly investigated for paraproteins, lupus erythematosus hepatitis B and C, and inflammatory bowel disease. Of patients with CIU, a few (<5%) prove to have food additive reactivity confirmed by placebo-controlled challenge testing. There is no convincing evidence of the involvement of Helicobacter pylori or parasite infestation as a cause of chronic urticaria, although H pylori could have an indirect role. Recently it has become clear that 27% to 50% of patients with CIU have functional autoantibodies directed against the alpha-chain of the high affinity IgE receptor or less commonly against IgG. These antibodies, whose involvement has now been independently confirmed in several centers, are identified by autologous serum skin testing and confirmed by histamine release studies or immunoblotting. Their removal (by intravenous Ig or plasmapheresis) or treatment by cyclosporine has proved highly beneficial in severely affected patients. However, the routine treatment of all CIU patients, irrespective of etiology, remains the judicious use of H(1) antihistamines. PMID- 10756215 TI - Immune mechanisms of smooth muscle hyperreactivity in asthma. AB - Asthma is characterized by bronchial hyperresponsiveness to a variety of bronchospasmogenic stimuli. To study the pathophysiologic mechanisms underlying the increased sensitivity and degree of maximal airway narrowing, various in vivo and in vitro models have been developed with methods of active and passive sensitization. These studies indicated a major role for alterations in the smooth muscle itself rather than neural dysfunction or airway inflammation as the underlying cause for the development of bronchial hyperresponsiveness. During the last years smooth muscle cells were found to exhibit not only the "classical" contractile phenotype but also a proliferative-synthetic phenotype, which is capable of producing proinflammatory cytokines, chemotaxins, and growth factors. Allergic sensitization can alter both contractile and secretory functions, thereby indicating that the smooth muscle cell could contribute directly to the persistence of airway inflammation in asthma. A better understanding of the changes within the smooth muscle cells and of the mechanisms that lead to their induction could contribute to the development of novel therapeutic approaches for the treatment of asthma. PMID- 10756216 TI - Expanding habitat of the imported fire ant (Solenopsis invicta): a public health concern. AB - Residents in the southeastern United States would hardly describe life with the aggressive imported fire ant as peaceful coexistence. The continued spread of these insects has produced agricultural problems, changes in the ecosystem, and increasing numbers of subjects with sting sequelae, including hypersensitivity reactions, secondary infections, and rare neurologic sequelae. Evolutionary changes have facilitated their expansion northward into Virginia and westward into California, and increasing urbanization will likely permit further expansion. Recent reports of building invasion with sting attacks inside occupied dwellings, including health care facilities, heighten public health concerns. This article reviews the medically important entomology, clinical aspects of stings, and the current approaches to chemical control of fire ants. We also propose directions for future research and treatment. PMID- 10756217 TI - Rhinovirus-induced PBMC responses and outcome of experimental infection in allergic subjects. AB - BACKGROUND: The immune response to rhinovirus (RV) infections is considered to contribute to upper respiratory symptoms and may also be an important contributor to lower airway dysfunction in patients with asthma. OBJECTIVE: This study was conducted to determine the relationship of RV-specific responses in PBMCs to the outcome of experimentally induced infection with RV16. METHODS: Twenty-two subjects with either allergic rhinitis or asthma were inoculated with RV16: virus induced proliferation and cytokine production were determined on PBMCs obtained before and then again 7 and 28 days after inoculation. RESULTS: Several subjects had proliferative responses to RV16 before inoculation, and precold RV-specific proliferative responses were inversely correlated (r(s) = -0.62, P <. 005) with RV shedding after inoculation. In addition, there was a negative correlation (r(s) = -0.58, P = 0.01) between precold RV-induced IFN-gamma secretion ex vivo and peak RV shedding during the cold. CONCLUSIONS: Certain RV-specific lymphocyte responses before the cold (vigorous proliferation or IFN-gamma secretion) were associated with reduced viral shedding after inoculation. These findings suggest that variations in mononuclear cell responses to RV could contribute to the individual variability in viral shedding during experimentally induced, and perhaps naturally acquired, RV infections in subjects with respiratory allergy or asthma. PMID- 10756218 TI - Inhaled budesonide for the treatment of acute wheezing and dyspnea in children up to 24 months old receiving intravenous hydrocortisone. AB - BACKGROUND: Inhaled corticosteroids are highly effective in the treatment of asthma at all ages, and their use in younger children is increasing. There are no data currently available on the treatment of infants with acute wheeze and dyspnea with nebulized budesonide. OBJECTIVE: Our purpose was to assess the clinical effect of nebulized budesonide in infants with acute wheeze and dyspnea. METHODS: A prospective study was performed comparing the addition of nebulized budesonide 0.25 mg every 6 hours (group A, n = 32) and nebulized ipratropium bromide 0.1 mg every 6 hours (group B, n = 39) with the normal treatment regimen with intravenous fluid, hydrocortisone, and nebulized fenoterol. A clinical score was made at admission and every 12 hours. The score included wheezing and costal retraction (0-6) and respiratory rate (counts per minute). RESULTS: Seventy-one infants aged 3 to 24 months were studied (42 boys). A statistically significant reduction was seen in clinical score and respiratory rate in both groups 12 hours after admission. The children who received budesonide improved significantly faster than the children who received ipratropium bromide, and the hospitalization period was significantly lower in the budesonide group (66.4 hours) compared with the ipratropium bromide group (93 hours) (P <.01). Three patients from the budesonide group and 2 from the ipratropium bromide group were readmitted within the first 4 weeks. CONCLUSION: Treatment of infants with acute wheeze with nebulized budesonide is associated with faster clinical improvement and reduction in hospital stay period. PMID- 10756219 TI - A randomized, double-blind, placebo-controlled trial of the effect of zafirlukast on upper and lower respiratory responses to cat challenge. AB - BACKGROUND: Zafirlukast, a leukotriene antagonist, has been shown to have protective effects against a variety of asthma triggers. OBJECTIVE: Our purpose was to evaluate zafirlukast's effects on upper and lower airway responses to cat allergen exposure with use of a well-characterized cat exposure model. METHODS: In a double-blind, placebo-controlled, cross-over trial 18 subjects with cat induced asthma were randomly assigned to receive 1 week each of zafirlukast or placebo followed by a 1-hour cat challenge. Upper and lower respiratory symptoms were rated and spirometry and acoustic rhinometry were performed. Challenges were stopped early if the subject was too uncomfortable or had a >50% decrease in FEV(1). RESULTS: Overall changes in FEV(1) were significantly different with zafirlukast treatment (P = .02). Significant differences in FEV(1) change were detected at 15 and 30 minutes (P = .027 and .05, respectively) but not at 45 and 60 minutes. Changes in acoustic rhinometry were also significantly different at 15 and 30 minutes (P =.05 and .0005, respectively) but not at 45 and 60 minutes. Challenge length was significantly longer with zafirlukast versus placebo after adjustment for differences in allergen exposure (P = .022). Respiratory symptom scores were significantly different (lower respiratory, P < .001; upper respiratory, P = .038) through the first 30 minutes of the challenge after adjustment for allergen exposure. CONCLUSIONS: Zafirlukast was significantly more effective than placebo in preserving pulmonary function and nasal anatomy and extending challenge length when cat-sensitive asthmatic subjects were exposed to high levels of cat allergen. PMID- 10756220 TI - 15-Lipoxygenase expression and 15(S)-hydroxyeicoisatetraenoic acid release and reincorporation in induced sputum of asthmatic subjects. AB - BACKGROUND: Recent evidence shows that 15(S)-hydroxy-eicoisatetraenoic acid (15[S]-HETE) can be released and rapidly reincorporated into cellular lipids. These mechanisms exert several immunoregulatory functions that may be relevant in airway inflammation. OBJECTIVE: Our purpose was to evaluate the levels of both soluble and cell-associated 15(S)-HETE and to examine 15-lipoxygenase (15-LO) messenger RNA (mRNA) expression in sputum samples obtained from 10 control and 18 asthmatic subjects. METHODS: Levels of 15(S)-HETE were measured by reverse-phase HPLC separation followed by RIA in supernatants and in cell membrane-extracted phospholipids after acid hydrolysis. 15-LO mRNA was evaluated by primed in situ hybridization (PRINS). Combined immunocytochemistry and PRINS was used to identify the phenotype of cells bearing 15-LO transcripts. RESULTS: Levels of both soluble and cell-associated 15(S)-HETE were higher in asthmatic than in control subjects (P <.0001). The percentage of cells expressing 15-LO mRNA was higher in asthmatic than in control subjects (P <.01). On double staining for specific cell-type markers and 15-LO mRNA, macrophages were the major source for 15-LO. CONCLUSION: This study shows that the induced sputum technique allows the evaluation of 15-LO activity and that soluble, cell-associated 15(S)-HETE and 15 LO levels are higher in asthmatic than in control subjects. In addition, this study indicates that, in induced sputum, airway macrophages are the major source of 15(S)-HETE in asthma. PMID- 10756221 TI - Neurocognitive functioning in children with mild and moderate asthma in the childhood asthma management program. The Childhood Asthma Management Program (CAMP) Research Group. AB - BACKGROUND: The Childhood Asthma Management Program (CAMP) is a multicenter double-blind, randomized, placebo-controlled, clinical trial of two anti inflammatory agents and placebo in children with mild and moderate asthma. OBJECTIVE: The interrelationship between asthma severity and neurocognitive functioning among 1041 children (age range, 5-12 years) enrolled in the CAMP trial was examined. METHODS: Asthma severity was established at baseline with a clinical history of asthma symptomatology and measures of lung function (spirometry and methacholine challenge). Diary cards were used in a screening to record nighttime awakenings and doctor contacts caused by asthma symptoms, symptom severity, and number of puffs from a rescue inhaler. All children received a comprehensive neurocognitive assessment at the end of the 28-day screening period (before randomization), including measures of intelligence, attention, memory, and academic achievement. RESULTS: Significant differences were found between children with mild and moderate asthma on lung function and symptom outcome variables (log(e)FEV(1)PC(20), DeltaFEV(1) percent predicted, change in peak flow percent predicted, nighttime awakenings caused by asthma, average symptom severity score, and average daily number of puffs from a rescue inhaler) but not on neurocognitive variables. Multiple regression analyses revealed that asthma outcomes could not be predicted by neurocognitive variables despite controlling for socioeconomic status. The prevalence of neurocognitive dysfunction, as indicated by the use of psychostimulant medication, was found to be consistent with that found in the existing literature. CONCLUSION: Mild and moderate asthma symptoms are not related to neurocognitive functioning in the children enrolled in CAMP. Mean performance on neurocognitive variables was found to be similar to that of national normative data. PMID- 10756222 TI - A new method for simultaneous immunodetection and morphologic identification of individual sources of pollen allergens. AB - BACKGROUND: Exposure to outdoor allergens has commonly been estimated by collecting airborne particles with a Hirst-type spore trap and then using morphologic criteria to identify the intact pollen grains and fungal spores that are recognized as allergen sources. Several antibody-based blotting or fixation methods have also been developed that enable the counting of amorphous airborne particles carrying allergen, but none of these methods allow the ready association of the released allergen with the morphologically identifiable particle of origin. A method has been developed that uses pressure-sensitive adhesive tape to sample the airborne particles and then allows the immunoidentification of the specific particles that are the allergen sources. OBJECTIVE: Our purpose was to visualize and immunostain the particles carrying pollen allergen that are collected with a volumetric spore trap. METHODS: A Burkard sampler was used to collect airborne particles onto pressure-sensitive adhesive tapes. The particles were permanently fixed between the tape and a protein-binding membrane when the tape was laminated with the membrane. Allergens that elute from the particles onto the membrane were detected with a range of antibodies. Both the particle and associated immunostained allergen were viewed through the transparent tape for final microscopic identification. RESULTS: Polyclonal and monoclonal antibodies and IgE from allergic patients stained allergens in the periphery of particles collected on the tapes. Individual pollen grains and paucimicronic particles were seen with halos of immunostained allergen surrounding them. When IgE was used, the density of immunostaining in the halo surrounding Lolium perenne pollen grains was found to be proportional to the level of Lolium-specific IgE. The method is highly sensitive and can be used to detect different airborne particles that carry allergen. Both the particle and the immunostaining can be subjected to a range of simple measurement techniques. CONCLUSION: Individual particles carrying allergens and antigens were visualized. These particles included intact pollen grains, paucimicronic particles, and fungal spores. PMID- 10756223 TI - As-needed use of fluticasone propionate nasal spray reduces symptoms of seasonal allergic rhinitis. AB - BACKGROUND: The daily use of intranasal corticosteroids is approved for the treatment of seasonal allergic rhinitis. OBJECTIVE: Our objective was to test the effectiveness of as-needed use of intranasal corticosteroids in the treatment of seasonal allergic rhinitis. METHODS: A double-blind, placebo-controlled, randomized, parallel-group study of the as-needed usage of fluticasone propionate nasal spray in the management of seasonal allergic rhinitis was performed. Outcome measures were symptom score, Rhinitis Quality of Life Questionnaire (RQLQ), and the number of eosinophils and the level of eosinophilic cationic protein (ECP) in nasal lavage. RESULTS: Twenty-six subjects in each group completed the 4-week study. The median symptom score over the duration of the study in the placebo group was 8.5 versus 4.5 in the active group. The active group had significant improvement on the interim visit in the sleep, non nose/eye, activities, nasal, practical, and overall domains (P <.05) of the RQLQ and on the final visit in the nasal symptom domain. The number of eosinophils was significantly lower in the active than in the placebo group at the final visit. Changes in ECP were not significant. CONCLUSION: As-needed fluticasone propionate nasal spray is efficacious in the treatment of seasonal allergic rhinitis. PMID- 10756224 TI - Suplatast tosilate inhibits goblet-cell metaplasia of airway epithelium in sensitized mice. AB - BACKGROUND: IL-4 and IL-13 play a putative role in mucus hypersecretion in asthma. Suplatast tosilate prevents the synthesis of T(H2) cytokines. OBJECTIVE: Because suplatast tosilate inhibits T(H2) cytokines but does not inhibits IFN gamma production, we examined the effect of suplatast on IL-4- or IL-13- and ovalbumin (OVA)-induced mucin synthesis in NCI-H292 cells in vitro and in bronchi of pathogen-free BALB/c mice in vivo. METHODS: In vitro, NCI-H292 cells were preincubated with suplatast tosilate (0.1-100 microgram/mL) 1 hour before adding human recombinant IL-4 (10 ng/mL). In vivo, mouse recombinant IL-4 or IL-13 (250 ng per/mouse) was instilled intranasally in mice pretreated with suplatast tosilate (50 mg.kg(-1).d(-1)). Mucous glycoconjugates were stained with Alcian blue (AB)/periodic acid-Schiff (PAS) stain. To evaluate effects of suplatast tosilate on goblet-cell metaplasia in OVA-sensitized mice, animals were pretreated with suplatast tosilate (1-50 mg.kg(-1).d(-1)) intragastrically. IL-4 and IL-13 were measured, and allergic inflammatory cells were analyzed in bronchoalveolar lavage fluid of OVA-sensitized mice. RESULTS: Pretreatment with suplastast did not prevent IL-4- or IL-13-induced increase in mucous glycoconjugate production in NCI-H292 cells or in mice. OVA sensitization increased AB/PAS-stained area of the epithelium (48.1% +/- 2.4%, P <.01 compared with control mice). Suplatast tosilate inhibited OVA-induced goblet-cell metaplasia in airway epithelium in a dose-dependent fashion; 50 mg.kg(-1).d(-1) decreased the AB/PAS area to 22.7% +/- 2.7% (P <.05 compared with OVA sensitization alone). Pretreatment with suplatast tosilate also prevented OVA induced increase in IL-4 and IL-13 levels and decreased the number of lymphocytes and eosinophils in bronchoalveolar lavage fluid (P <.05 compared with values of mice given OVA alone). CONCLUSION: These results indicate that suplatast tosilate prevents allergen-induced goblet-cell metaplasia and the recruitment of eosinophils and lymphocytes into the airways. These results suggest that this effect is due to the prevention of the production of T(H2) cytokines in airways. PMID- 10756225 TI - Prognosis and prediction of response to surgery in allergic patients with chronic sinusitis. AB - BACKGROUND: Chronic rhinosinusitis (CRS) occurs frequently in patients with atopy, but little is known of the prognosis after surgery and of factors that may predict a poor outcome. OBJECTIVE: Our purpose was to assess the long-term prognosis in atopic patients with CRS who undergo surgery and whether certain immune markers could predict a worse prognosis in this setting. METHODS: Fifteen patients with diffuse involvement of the sinuses on computed tomographic (CT) scan but without nasal polyposis underwent ethmoidectomy with middle meatotomy for CRS when it was clinically indicated. All patients had a biopsy of the inferior turbinate and of the most inflamed areas of the maxillary and ethmoid sinuses at the time of surgery. Follow-up was performed by video endoscopy and by assessment of 2 chronic sinusitis questionnaires at 0, 6, and 24 months postoperatively. The number of lymphocyte subsets (CD3, CD4, CD8), mast cells and eosinophils, and cells expressing IL-4 and IL-5 messenger RNA (mRNA) in all 3 biopsy sites at the time of surgery were compared with the clinical response after surgery. RESULTS: Seven patients had persistent improvement after surgery, with a decrease in pain, rhinorrhea, or nasal obstruction and a decrease in the need for medication. Eight patients were unchanged or worsened after surgery with disabling rhinorrhea and repeated sinusitis. We found no difference in the number of inflammatory cells, lymphocyte subsets, or IL-4 mRNA-positive cells in the sinus mucosa between responders and nonresponders. However, an increased number of cells expressing IL-5 mRNA was found in the ethmoid sinus at the time of surgery in patients who did not respond to the surgical intervention (P =.007). CONCLUSION: More than 50% of patients with perennial rhinitis and CRS do not improve after surgery, a response that may be predicted by more cells expressing IL-5 mRNA in the ethmoid sinuses. The increased number of cells expressing IL-5 mRNA may have the potential to be used as a marker for prediction of the response to surgery. The worsening of symptoms in some patients with CRS after sinus surgery could be a result of the disturbance of the anatomy of the sinuses and exposure to the environmental allergens. PMID- 10756226 TI - Airway mast cells and eosinophils correlate with clinical severity and airway hyperresponsiveness in corticosteroid-treated asthma. AB - BACKGROUND: The relationship between airway inflammation and asthma severity in corticosteroid-treated asthma is unclear. OBJECTIVES: Our purpose was to characterize the inflammatory cell profile of the airway lumen and epithelium in corticosteroid-treated asthma and to relate these findings to clinical and physiologic markers of asthma severity. METHODS: Adults (n = 20) with asthma received standardized high-dose inhaled corticosteroid therapy with beclomethasone 2000 microgram per day for 8 weeks. Airway responsiveness to methacholine and hypertonic (4.5%) saline solution was then assessed, followed by sputum induction and, 1 week later, bronchoscopy with bronchoalveolar lavage and bronchial brush biopsy to assess inflammatory cells. RESULTS: Clinical asthma severity was associated with airway hyperresponsiveness. Metachromatic cells were the main granulocyte present in bronchial brush biopsy specimens and correlated with airway responsiveness to saline solution (r = -0.75), methacholine (r = 0.74), peak flow variability (r = 0.59), and clinical asthma severity (r = 0.57). Eosinophils were the main granulocyte present in sputum and correlated with airway responsiveness to saline solution (r = -0.63) but not with other clinical markers of asthma severity. Bronchoalveolar lavage cell counts were not related to clinical asthma severity. CONCLUSIONS: In asthmatic patients treated with cortico-steroids, the dominant inflammatory effector cell in the epithelium is the metachromatic cell, and in sputum it is the eosinophil. These cells correlate with the degree of airway hyperresponsiveness. Clinical asthma severity correlates with airway responsiveness and epithelial metachromatic cells. Induced sputum eosinophils and airway responsiveness to hypertonic saline solution may be useful markers of airway inflammation for clinical practice. PMID- 10756227 TI - Identification of cytokine-regulated genes in human leukocytes in vivo. AB - BACKGROUND: Human polymorphic nuclear granulocytes (PMNs) such as neutrophils and eosinophils play a critical role in mediating inflammatory responses to microbial and parasitic infections. Exposure of these leukocytes to cytokines leads to an amplification of granulocyte effector functions by a mechanism termed "priming." Although many studies have investigated the effects of granulocyte priming, little is known concerning the molecular mechanisms that lead to this phenomenon. OBJECTIVE: The purpose of this study was to identify potential markers for granulocyte priming and thus also to gain further insight into the pathogenesis of inflammatory responses. METHODS: We used a modified differential display technique, random arbitrary primed-PCR to identify genes regulated during the priming of human polymorphic nuclear granulocytes by GM-CSF in vitro. Genes identified were validated by Northern blot analysis of in vitro and in vivo primed leukocytes. RESULTS: Several genes were identified and their expression characterized in vitro. One of these genes, 5-lipoxygenase-activating protein, was also found to be up-regulated in leukocytes isolated after allergen challenge of allergic asthmatic patients. CONCLUSION: The use of differential display technology is a rapid and effective means of identifying genes whose expression is regulated by priming in vitro and in vivo. Further analysis will lead to a better understanding of the priming phenotype and may provide further insight into the pathologic mechanisms of inflammatory processes. PMID- 10756228 TI - Inhibition of adhesion of human neutrophils and eosinophils to P-selectin by the sialyl Lewis antagonist TBC1269: preferential activity against neutrophil adhesion in vitro. AB - BACKGROUND: Leukocyte rolling on vascular endothelium is mediated by selectins and their carbohydrate-containing counterligands. The tetrasaccharide sialyl Lewis(x) (sLe(x)) binds to all 3 selectins, so compounds that mimic sLe(x) are potential antagonists. OBJECTIVE: Our purpose was to examine the ability of the sLe(x) mimetic TBC1269 to inhibit binding of human neutrophils and eosinophils to P-selectin. METHODS: Expression of the primary P-selectin ligand, P-selectin glycoprotein ligand-1 (PSGL-1), was examined on neutrophils and eosinophils, and their adhesion to immobilized P-selectin was examined under both static and dynamic conditions in the presence and absence of TBC1269. RESULTS: Neutrophils and eosinophils expressed PSGL-1, with eosinophils expressing about twice as much as neutrophils. In the absence of TBC1269, both cell types adhered avidly to P selectin under static and dynamic conditions. For neutrophils, preincubation of P selectin-coated plates with TBC1269 (1 to 1000 microgram/mL) resulted in concentration-dependent decreases in neutrophil adhesion, with significant inhibition seen at concentrations >/=100 microgram/mL. Eosinophil adhesion to P selectin was more refractory to inhibition by TBC1269 and was only partially inhibited at the highest concentration tested (1000 microgram/mL). Two structurally related control compounds, TBC1900 and TBC746, had no effect when tested at similar concentrations. CONCLUSION: These data indicate that an sLe(x) mimetic can exhibit cell type-specific differences in potencies with respect to antagonism of P-selectin adhesion. Although this may in part be the result of differences in PSGL-1 expression, the discrepancy in potencies may also be due to other differences, including carbohydrate composition and binding affinity of PSGL-1. PMID- 10756229 TI - Sequential patterns of inflammatory events during developing and expressed skin late-phase reactions. AB - BACKGROUND: Although there has been much study of the histologic features of the late-phase reactions (LPR) seen 6 to 24 hours after intradermal injection of allergens, much less is known about the events occurring during development of such LPR. OBJECTIVE: Our purpose was to compare sequential gross and histologic inflammatory responses during developing skin LPR within 6 hours after challenge. METHODS: Gross reactions were measured and biopsy specimens obtained at 20 minutes and 1, 2, and 6 hours after intradermal allergen (Ag) and buffer diluent control (B) injections in 7 atopic subjects with known immediate and LPR. Inflammatory cell responses were compared, as detected by immunohistochemistry in Ag and B sites. These findings were then compared with those at 24 hours. RESULTS: Gross LPR evolved without a hiatus from the immediate wheal responses over the next 6 hours (P =.04 vs that in B sites) and then decreased by 24 hours. Prominent PMN accumulation started by 20 minutes, peaking at 1 hour (P <.01). Eosinophil accumulation was significant, starting at 1 hour (P <.001) and peaking at 6 hours (P < .001). Many eosinophils were activated (EG(2)(+)). T-cell accumulation started at 2 hours (P =.01) and was most prominent at 24 hours. The frequency of vessels expressing E-selectin increased at 1 hour (P <.005), correlating with the degree of local PMN accumulation. The frequency of vessels expressing vascular cell adhesion molecules started increasing at 6 hours (P = .02), well after eosinophil accumulation was prominent. CONCLUSIONS: Skin LPR is characterized by evolution of a gross indurated reaction from the immediate whealing response over the first 6 hours after intradermal Ag challenge, with an early accumulation of PMN and eosinophils, not directly attributable to lymphocyte entry or vascular cell adhesion molecule expression. Likely, multiple other factors may also play roles in the complex pathogenesis of LPR. PMID- 10756230 TI - Induction of corticosteroid insensitivity in human PBMCs by microbial superantigens. AB - BACKGROUND: Microbial superantigens have been described to contribute to the pathogenesis of chronic inflammatory diseases often complicated by insensitivity to glucocorticoid therapy. In bronchial asthma glucocorticoid insensitivity has been associated with increased expression of glucocorticoid receptor beta, an endogenous inhibitor of the classic glucocorticoid receptor alpha. OBJECTIVE: To study a potential mechanism by which superantigens could contribute to poor disease control, we examined their capacity to alter steroid sensitivity and expression of glucocorticoid receptor beta. METHODS: The capacity of dexamethasone to inhibit stimulation of PBMCs from 7 healthy subjects with the prototypic superantigens, staphylococcal enterotoxin (SE) B, toxic shock syndrome toxin (TSST)-1 and SEE, versus PHA, was tested. The expression of glucocorticoid receptor beta in normal PBMCs after stimulation with SEB, versus PHA, was assessed by immunocytochemistry. RESULTS: Dexamethasone 10(-6) mol/L caused a 99% inhibition of PHA-induced PBMC proliferation but only a 19% inhibition of the SEB induced, 26% inhibition of the TSST-1, and 29% inhibition of the SEE-induced PBMC proliferation (P <.01 for all superantigens versus PHA) demonstrating that superantigens can induce steroid insensitivity. Stimulation of normal PBMCs with SEB induced a significant increase of glucocorticoid receptor beta compared with PHA and unstimulated cells (P <.01). CONCLUSION: We have demonstrated the capacity of microbial superantigens to induce glucocorticoid insensitivity, which should be considered in the diagnosis and treatment of patients with superantigen triggered diseases. These data suggest that superantigens may contribute to glucocorticoid insensitivity through induction of glucocorticoid receptor beta. PMID- 10756231 TI - Serum IgE response to orally ingested antigen: a novel IgE response model with allergen-specific T-cell receptor transgenic mice. AB - BACKGROUND: The mechanism by which orally ingested allergens elicit an IgE response remains unclear because there are few animal models available for investigation of this response. OBJECTIVE: We tried to develop a murine model suitable for investigation of the IgE response to orally ingested allergens, which would allow us to identify T cells that could promote IgE production. METHODS: Ovalbumin (OVA)-specific T-cell receptor transgenic mice were fed a diet containing OVA, and both the serum antibody response and cytokine production by splenocytes were examined. RESULTS: Oral administration of OVA to transgenic mice led to an increase in the levels of both antigen-specific IgE and total IgE in the sera. Subsequent intravenous challenge of OVA-fed transgenic mice with OVA resulted in anaphylactic shock. Analysis of cytokine production by splenocytes revealed that high IL-4-producing T cells appeared in the spleen 1 week after the start of feeding the OVA diet. T cells from these mice were found to promote IgE secretion by BALB/c B cells in vitro. This helper activity and the levels of IL-4 secretion were diminished after long-term feeding. These findings suggest the possibility that the orally ingested antigen elicited a response by a subpopulation of T cells that produce high levels of T(H2)-type cytokines and that promote IgE secretion, and these same T cells were tolerized by the orally ingested antigen. CONCLUSION: This experimental model with transgenic mice may be a useful tool for further studies of the cellular and molecular mechanisms of the T-cell and IgE responses to orally ingested antigens. PMID- 10756232 TI - The environmental pollutant pyrene induces the production of IL-4. AB - BACKGROUND: Epidemiologic studies and experiments with mouse models suggest that polyaromatic hydrocarbons contained in, among others, diesel exhaust particles can promote the development of allergy. OBJECTIVE: Because IL-4 organizes allergic responses in vivo, we have investigated whether pyrene, a major compound of diesel exhaust particles, can affect the production of IL-4. METHODS: IL-4 production by primary human T cells was assessed by ELISA and messenger RNA transcription was detected by Northern blotting. Activation of the IL-4 promoter was tested in reporter gene assays with transiently transfected cell lines. RESULTS: Pyrene induced transcription of IL-4 messenger RNA and expression of IL 4 protein in primary human T cells. Pyrene, but not related polyaromatic hydrocarbons, enhanced basal transcription of the human and mouse IL-4 promoter. CONCLUSION: Our results suggest that pyrene may promote allergic diseases by inducing the production of IL-4. PMID- 10756233 TI - Dissociation of allergen-specific IgE and IgA responses in sera and tears of pollen-allergic patients: a study performed with purified recombinant pollen allergens. AB - BACKGROUND: Trees and grass pollen allergens represent potent elicitors of allergic rhinoconjunctivitis and asthma. Little is known regarding the presence of allergen-specific IgA antibodies in sera and tears and their association with IgE responses in patients with allergic conjunctivitis. OBJECTIVE: The purpose of this study was to compare the specificities of IgE and IgA antibodies in sera and tears of pollen-allergic patients with conjunctivitis by using purified recombinant pollen allergens. METHODS: Sera and tears collected from 23 pollen allergic and from 23 nonatopic individuals were analyzed for IgE and IgA reactivity to nitrocellulose-blotted birch and timothy grass pollen extracts. In addition, we determined the specificities of IgE, IgG(1-4), and IgA antibodies with use of a panel of purified recombinant pollen allergens (timothy grass: rPhl p 1, rPhl p 2, rPhl p 5; birch: rBet v 1, rBet v 2) in serum and tear samples by immunoblotting and ELISA. Statistical analyses of data were performed by t test and Mann Whitney U test. RESULTS: Serum and tears of many of the pollen-allergic individuals with conjunctivitis exhibited specificity for the very same pollen allergens. No allergen-specific IgE antibodies were detected in tears of nonatopic individuals. IgA antibodies in sera and tears of patients with allergic conjunctivitis were mainly directed against nonallergenic moieties and showed specificities that were significantly different from those of IgE antibodies. CONCLUSION: The dissociation of IgE and IgA responses and the lack of allergen specific IgA antibodies in mucosal secretions (eg, tears) may contribute to allergic manifestations in target organs of atopy. Induction of allergen-specific IgA antibodies may hence be considered as a promising strategy for the treatment of mucosal forms of atopy. PMID- 10756234 TI - Evidence for a disease-promoting effect of Staphylococcus aureus-derived exotoxins in atopic dermatitis. AB - BACKGROUND: The skin of patients with atopic dermatitis (AD) exhibits a striking susceptibility to colonization with Staphylococcus aureus. Some strains of S aureus secrete exotoxins with T-cell superantigen activity (toxigenic strains), and abnormal T-cell functions are known to play a critical role in AD. OBJECTIVE: Our purpose was to examine the impact of superantigen production by skin colonizing S aureus on disease severity. METHODS: In a cross-sectional study of 74 children with AD, the presence and density of toxigenic and nontoxigenic strains of S aureus was correlated with disease severity. In a subgroup of patients the T-cell receptor Vbeta repertoire of peripheral blood and lesional T cells was investigated and correlated with individual superantigen activity of skin-colonizing S aureus. RESULTS: Fifty-three percent of children with AD were colonized with toxigenic strains of S aureus producing staphylococcal enterotoxin C, staphylococcal enterotoxin A, toxic shock syndrome toxin-1, staphylococcal enterotoxin B, and staphylococcal enterotoxin D in decreasing frequency. Children colonized with toxigenic S aureus strains had higher disease severity compared with the nontoxigenic and S aureus-negative groups. Patients colonized with toxigenic S aureus exhibited shifts in the intradermal T-cell receptor Vbeta repertoire that correspond to the respective superantigen-responsive T-cell subsets. CONCLUSION: The data demonstrate that S aureus-released exotoxins can modulate disease severity and dermal T-cell infiltration. PMID- 10756235 TI - Application of Staphylococcal enterotoxin B on normal and atopic skin induces up regulation of T cells by a superantigen-mediated mechanism. AB - BACKGROUND: The skin of patients with inflammatory skin diseases such as atopic dermatitis is frequently colonized with Staphylococcus aureus. Colonization with S aureus has been reported to exacerbate atopic dermatitis. Recent studies have demonstrated that S aureus isolated from the skin of patients with atopic dermatitis releases bacterial toxins that act as superantigens. We have previously applied the staphylococcal superantigen staphylococcal enterotoxin B (SEB) on intact human skin and found that the application led to induction of dermatitis. OBJECTIVE: The purpose of the study was to determine whether superantigen-induced dermatitis is primarily due to a T cell-superantigen mediated reaction or represents nonspecific cytokine-driven inflammation. METHODS: We applied SEB, vehicle, and sodium lauryl sulfate on normal skin in healthy (n = 6) and atopic subjects (n = 6) and biopsy specimens were taken from all treated areas. The biopsy specimens from all subjects and peripheral blood from the atopic subjects were analyzed for the T-cell receptor (TCR) Vbeta repertoire with mAbs against TCR Vbeta 2, 3, 8.1, 12, 14, and 17. RESULTS: From all subjects, both healthy and patients with atopic dermatitis, skin biopsy specimens from SEB-treated areas demonstrated selective accumulation of T cells expressing SEB-reactive TCR Vbeta 12 and 17 (P <.05). This selective up regulation was not found in the sodium lauryl sulfate-treated areas. CONCLUSION: Our data strongly support that superantigen-induced T-cell activation is involved in the dermatitis seen after experimental application of SEB on intact skin. PMID- 10756236 TI - Complementary DNA cloning and immunologic characterization of a new Penicillium citrinum allergen (Pen c 3). AB - BACKGROUND: Penicillium citrinum has been identified as the most prevalent airborne Penicillium species in the Taipei area. It is important to understand the allergenic composition of this ubiquitous fungal species. OBJECTIVE: The complementary DNA (cDNA) clone of an allergen from P citrinum was isolated and expressed in Escherichia coli as a fusion protein. mAbs were prepared with the recombinant protein as antigen. The corresponding natural allergen in the fungal extracts was identified with the mAbs. METHODS: A Uni-Zap XR P citrinum cDNA library was screened with sera from asthmatic patients. An IgE-binding cDNA clone was isolated and expressed as a glutathione-S-transferase fusion protein. The frequency of IgE binding to the expressed protein was analyzed by immunoblotting. Spleen cells from BALB/c mice immunized with the recombinant protein were fused with NS-1 cells for mAb generation. RESULTS: A P citrinum cDNA library was screened with a mixture of serum samples from 4 asthmatic patients. An IgE binding cDNA clone was obtained and designated as PCE2. PCE2 has a 694-bp insert that contains a 167 amino acids open reading frame. The deduced amino acid sequence of the encoded protein has 82.6% (138 amino acids) identity with an Aspergillus fumigatus peroxisomal membrane protein allergen (Asp f 3). PCE2 was expressed in E coli as a fusion protein and designated as Pen c 3. Sera from 13 (46%) of the 28 Penicillium-sensitized asthmatic patients demonstrated IgE binding to Pen c 3. In addition, 11 of the 13 Pen c 3-positive serum samples have IgE immunoblot reactivity to recombinant Asp f 3. The presence of IgE cross reactivity between Pen c 3 and Asp f 3 was also detected by immunoblot inhibition. Four of the 6 mAbs generated against Pen c 3 cross-react with Asp f 3. The presence of the corresponding 18-k natural allergens in the crude extracts of P citrinum and A fumigatus were detected by immunoblot with use of the mAbs and sera from asthmatic patients. CONCLUSION: Results obtained suggest that the peroxisomal membrane protein (Pen c 3) is an important allergen of P citrinum. PCE2 is a full-length cDNA clone encoding this allergen. In addition, the mAbs generated may be useful in standardizing the diagnostic allergenic extracts. PMID- 10756237 TI - Allergy to eggs from duck and goose without sensitization to hen egg proteins. AB - BACKGROUND: Eggs are among the foods most frequently causing allergy. Hen eggs are the most important. Those of other birds are of lesser significance. OBJECTIVE: We report an unusual case of food allergy after consumption of eggs from duck and goose in an adult patient without hen egg allergy. METHODS: Skin prick tests were performed with fresh white and yolk from eggs of duck and goose and egg white, egg yolk, ovalbumin, and ovomucoid from hen egg. Specific serum IgE was measured to hen egg proteins. SDS-PAGE and IgE immunoblotting were carried out with egg white extracts from hen, duck, and goose. RESULTS: Skin tests were positive to egg whites from duck and goose. The skin tests and specific serum IgE were negative to hen egg proteins. Immunoblotting demonstrated the presence of specific IgE to a proteic band of molecular weight around 45 kd. CONCLUSIONS: We report a patient with an IgE-mediated allergy to egg white from duck and goose without hen egg allergy. Ovalbumin seems to be the responsible protein. The antigenic determinant of this protein seems to be specific of order Anseriforme and it is not present in the ovalbumin of order Galliforme. PMID- 10756238 TI - The accuracy of references of three allergy journals. PMID- 10756239 TI - Impact of personal avoidance practices on health care workers sensitized to natural rubber latex. PMID- 10756242 TI - Main coronary artery and coronary ostial stenosis in children: detection by transthoracic color flow and pulsed Doppler echocardiography. AB - Coronary artery stenosis (CAS) and coronary ostial stenosis (COS) are potentially life-threatening conditions. The echocardiographic diagnosis of CAS and COS in children has not been described. We report on the transthoracic echocardiography (TTE) findings of CAS and COS in children. Six patients, aged 1 week to 12 years, with clinically confirmed COS (n = 5) and CAS (n = l) were diagnosed by TTE. Their echocardiographic findings were compared with 26 healthy control subjects of a similar age range. Left COS was associated with an aberrant left coronary artery (CA) from the contralateral aortic sinus (n = 2), an intramural left CA with d-transposition of the great vessels (n = l), and supravalvular aortic stenosis (n = l). Right COS was present in a patient with aortic valvular stenosis. Acquired left main CAS was diagnosed in the sixth patient 3 years after orthotopic heart transplantation. Coronary ostial stenosis was recognized when a color flow acceleration signal was present proximal to and extending into the coronary ostium (CO). Coronary artery stenosis was detected when a coarctated color flow stream was present within the stenosed CA segment with turbulent distal flow. These findings were not detected in the control cohort who demonstrated laminar CA and CO flow signals. All patients had increased spectral velocity in the CA distal to the stenosed segment (patients = 50 +/- 5 cm/s, controls = 24 +/- 6 cm/sec; P <.01). Delayed peak diastolic velocity seemed to indicate severe stenosis. We conclude that (1) CO acceleration signals and turbulent coarctated CA flow signals are abnormal findings in TTE coronary Doppler assessment. They indicate COS and CAS, respectively. (2) Knowledge of the normal TTE CA flow velocity patterns is essential so that abnormal velocity signals such as seen with CAS and COS can be recognized and a timely diagnosis made. PMID- 10756241 TI - Dramatic augmentation of a food allergy by acetylsalicylic acid. PMID- 10756243 TI - Assessment of coronary flow reserve with transthoracic Doppler echocardiography: comparison among adenosine, standard-dose dipyridamole, and high-dose dipyridamole. AB - Coronary flow reserve (CFR), defined as a ratio of hyperemic-to-basal coronary flow velocity, provides important information about the functional aspect of coronary circulation. However, it usually is determined by invasive methods during catheterization. Recent studies have shown that transthoracic Doppler echocardiography (TTDE) may be useful in the measurement of coronary flow velocity in the distal portion of the left anterior descending coronary artery (LAD). The vasodilators used for hyperemia are adenosine and dipyridamole. However, the coronary vasodilative response and systemic hemodynamic effects of the two agents have not been directly compared with TTDE. We assessed blood flow velocity and vascular resistance in the distal LAD by TTDE during an intravenous 2-minute adenosine infusion (140 microg/kg/min) and low- (0.56 mg/kg) and high dose dipyridamole (0. 84 mg/kg) infusion in 25 patients with patent LAD. Coronary flow velocity was successfully recorded in 20 patients (80%) during baseline and the consecutive vasodilator-infusion period. Compared with low-dose dipyridamole, adenosine infusion induced a higher CFR (3.7 +/- 0.87 vs 2.73 +/- 0.65; P <.05) and a lower coronary resistance index (0.31 +/- 0.04 vs 0.35 +/- 0.08; P <.05). But by increasing the dipyridamole dose to 0.84 mg/kg, the values of the CFR and coronary resistance index became comparable to those of adenosine infusion (2.85 +/- 0.78 vs 3.03 +/- 0.7, P = not significant [NS]; 0.33 +/- 0.04 vs 0.32 +/- 0.09, P = NS; respectively). We conclude that adenosine seems to be a favorable vasodilator for the measurement of CFR with TTDE. PMID- 10756244 TI - Transesophageal echocardiography assessment of left atrial appendage function in untreated systemic hypertensive patients in sinus rhythm. AB - To assess left atrial appendage (LAA) function in hypertensive patients without treatment, transesophageal echocardiography (TEE) was performed in 46 hypertensive patients in sinus rhythm, aged 40 to 55 years, and in 16 control subjects (group I) without cardiovascular disease, aged 41 to 54 years. The hypertensive patients were divided into 2 groups according to left ventricular (LV) systolic function: group II, the group with normal LV systolic function (ejection fraction 0.63 +/- 0.08), and group III, the group with LV systolic dysfunction (ejection fraction 0.39 +/- 0.05). The LAA late emptying velocities (EVs) were significantly reduced in the hypertensive subgroups compared with the control group (P <.001), but no significant difference in the LAAEV was found between groups II and III. The LAAEV in the hypertensive patients had a significant negative correlation with diastolic blood pressure, systolic blood pressure, and left atrial (LA) diameter. The maximal LAA areas were significantly larger in the hypertensive subgroups than in the control group (P <.05). No significant difference in LAA maximal area existed between groups II and III. The maximal LAA area in the hypertensive patients had a significant positive correlation with diastolic blood pressure, systolic blood pressure, and LA diameter, but a significant negative correlation with LV ejection fraction. With TEE, LA spontaneous echocardiographic contrast (SEC) was present in 6 (43%) of 14 patients in group III (P <.01) and in 7 (22%) of 32 patients in group II (P <.05). No significant difference in the occurrence of LASEC was found between groups II and III. Left atrial appendage thrombi by TEE were observed in 4 (29%) of 14 patients in group III (P <.05) and in 4 (13%) of 32 patients in group II (P = not significant). No significant difference in the occurrence of LAA thrombus existed between groups II and III. In conclusion, in patients with untreated hypertension, marked elevation of afterload imposed on the left atrium may involve both the left atrium and the LAA, resulting in impairment of LAA function. This condition may worsen with subsequent occurrence of SEC and later, thrombus formation. Therefore assessment of LAA function may be important even in the hypertensive patient in sinus rhythm. PMID- 10756245 TI - Analysis of shape and motion of the mitral annulus in subjects with and without cardiomyopathy by echocardiographic 3-dimensional reconstruction. AB - The shape and dynamics of the mitral annulus of 10 patients without heart disease (controls), 3 patients with dilated cardiomyopathy, and 5 patients with hypertrophic obstructive cardiomyopathy and normal systolic function were analyzed by transesophageal echocardiography and 3-dimensional reconstruction. Mitral annular orifice area, apico-basal motion of the annulus, and nonplanarity were calculated over time. Annular area was largest in end diastole and smallest in end systole. Mean areas were 11.8 +/- 2.5 cm(2) (controls), 15.2 +/- 4.2 cm(2) (dilated cardiomyopathy), and 10.2 +/- 2.4 cm(2) (hypertrophic cardiomyopathy) (P = not significant). After correction for body surface, annuli from patients with normal left ventricular function were smaller than annuli from patients with dilated cardiomyopathy (5.9 +/- 1.2 cm(2)/m(2) vs 7.7 +/- 1.0 cm(2)/m(2); P <.02). The change in area during the cardiac cycle showed significant differences: 23.8% +/- 5.1% (controls), 13.2% +/- 2.3% (dilated cardiomyopathy), and 32.4% +/- 7.6% (hypertrophic cardiomyopathy) (P <.001). Apico-basal motion was highest in controls, followed by those with hypertrophic obstructive and dilated cardiomyopathy (1.0 +/- 0.3 cm, 0.8 +/- 0.2 cm, 0.3 +/- 0.2 cm, respectively; P <.01). Visual inspection and Fourier analysis showed a consistent pattern of anteroseptal and posterolateral elevations of the annulus toward the left atrium. In conclusion, although area changes and apico-basal motion of the mitral annulus strongly depend on left ventricular systolic function, nonplanarity is a structural feature preserved throughout the cardiac cycle in all three groups. PMID- 10756246 TI - Premature ventricular contractions during triggered imaging with ultrasound contrast. AB - BACKGROUND: Premature ventricular contractions (PVCs) were observed during triggered second harmonic imaging of a contrast agent for myocardial perfusion assessment, with continuous infusion of the contrast agent. Further investigation into the relation of this phenomenon to both ultrasound energy and the contrast agent was carried out during a subsequent bolus-versus-infusion study. METHODS AND RESULTS: Two open-label studies in healthy male volunteers were performed. The initial study was a dose-response study in 10 subjects, which compared 3 infusion rates. Each volunteer received 3 continuous infusions with different infusion rates of the contrast agent for either 10 (n = 6) or 20 (n = 4) minutes. End-systolic triggered imaging with a mechanical index (MI) of 1.5 was used throughout this part of the study. The second study compared bolus injection with a continuous infusion in 9 volunteers, with a single-dose level but different imaging modalities: end-systolic and end-diastolic triggered imaging at MIs of both 1.1 and 1.5. Spontaneous baseline PVCs were uncommon: 10 in 344 minutes (0.03 PVC/min, maximal 1 PVC/min) of baseline imaging. During end-diastolic triggering, no increase in PVCs was seen, irrespective of MI. A significant increase to 1.06 PVC/min (P <.001) was seen during end-systolic imaging with an MI of 1.5, but not with an MI of 1.1. The increase in PVC rate was dose-dependent in the initial study. CONCLUSION: Imaging of contrast agents with high acoustic pressures can cause PVCs if end-systolic triggering is used. This effect is related to both the dose of contrast agent and acoustic pressure. It does not occur during end-diastolic triggered imaging. Precautionary measures would include using lower MIs or end-diastolic triggering. PMID- 10756247 TI - Significant beat-to-beat hemodynamic changes in fetal circulation: a consequence of abrupt intrathoracic pressure variation induced by hiccup. AB - During routine fetal echocardiographic studies, we incidentally observed abrupt beat-to-beat changes in blood flow velocity and direction during bouts of hiccups in fetuses with a normal heart and regular and synchronized atrioventricular cardiac rhythm. The effect of hiccups on blood flow velocity and direction varied depending on the time of occurrence of hiccups during the cardiac cycle. In systole, a significant transient reduction of peak flow velocity occurred at the aortic and pulmonic valves, and brief tricuspid regurgitation appeared synchronously with each hiccup. In diastole, a transient reversal of flow direction was recorded simultaneously with the hiccup at the aorta and ductus arteriosus, and acceleration of peak flow velocity was observed across the tricuspid and mitral valves. Throughout the entire cardiac cycle, marked blood flow acceleration was observed in the superior vena cava, inferior vena cava, and ostium secundum simultaneously with the hiccup. A direct transmission of briefly augmented, negative intrathoracic pressure to a compliant aorta and systemic veins appears to be a reasonable explanation for most of our observations. PMID- 10756248 TI - Validity and reproducibility of echocardiographic measurement of left ventricular ejection fraction by acoustic quantification with tissue harmonic imaging technique. AB - The tissue harmonic imaging technique can enhance detection of the cardiac endocardial border. When combined with an acoustic quantification (AQ) method, an improvement of accuracy and reproducibility of real-time measurement of left ventricular (LV) function might be expected. However, few data exist regarding the measurement of LV function by AQ with the harmonic imaging technique. Therefore, we evaluated the validity and reproducibility of AQ measurement of LV ejection fraction with or without harmonic imaging technique. A total of 50 patients (mean age 58 +/- 10 years) who underwent left ventriculography were included in our study. The LV end-diastolic and end-systolic volumes by ventriculography were 131 +/- 52 mL and 72 +/- 43 mL, respectively, and were underestimated by both conventional (70 +/- 32 mL and 36 +/- 25 mL) and harmonic (67 +/- 30 mL and 34 +/- 22 mL) AQ obtained in the apical 4-chamber view. The calculated ejection fraction by ventriculography was 0.49 +/- 0. 11 and correlated with that by conventional AQ (0.51 +/- 0.11; y = 0. 72x + 0.152; r = 0.73). This was a marked improvement when compared with the ejection fraction by harmonic AQ (0.50 +/- 0.11; y = 0.89x + 0.065; r = 0.91). Interestingly, interobserver and intraobserver variabilities of conventional AQ, which were 15.6% and 8.6%, respectively, were much improved by harmonic AQ (8.9% and 4.5%, respectively). These results indicate the feasibility of real-time measurement of LV ejection fraction by harmonic imaging, although absolute LV volume can be underestimated even by this technique. PMID- 10756249 TI - Are changes in myocardial integrated backscatter restricted to the ischemic zone in acute induced ischemia? An in vivo animal study. AB - Integrated backscatter (IB) from a myocardial region, calculated from radiofrequency echocardiographic data, has been proposed as a useful parameter for investigating changes in myocardial tissue induced by ischemia. In 10 closed chest dogs, 5 minutes of myocardial ischemia was induced by either a proximal occlusion of the circumflex coronary artery (CX) (5 dogs), resulting in extensive ischemia in the posterior wall, or by occluding the distal CX vessel (5 dogs) to produce a small localized ischemic zone in the posterior wall. High-resolution digital radiofrequency data from the whole left ventricular myocardium, in the imaging plane during one complete heart cycle, were acquired with a whole-image real-time acquisition approach. Regions in the septum and posterior wall (both ischemic tissue and, in the case of distal occlusions, tissue surrounding the ischemic zone) were chosen for analysis, and IB and cyclic variation (CV) of IB were calculated. Post occlusion, an increase in mean IB values was found in the ischemic segment. However, an increase in CV was also observed in the peri ischemic zone for the distal CX occlusion and in the septum after proximal CX occlusion. These findings show that changes in CV are not restricted to the ischemic zone but may also occur in distal myocardium. This may be explained by changes in the regional contractile state and loading conditions of the "normal" myocardium, which are altered in response to the distal ischemia. PMID- 10756250 TI - Unusual eustachian valve function. AB - The eustachian valve directs oxygen-rich blood from the inferior vena cava toward the foramen ovale and away from the tricuspid valve during fetal development. Ordinarily, it does not prevent reflux of right atrial blood back into the inferior vena cava because it does not function as a true valve. Here we describe an unusual adult patient with severe tricuspid valve regurgitation in whom the eustachian valve did function as a true, albeit regurgitant, valve. PMID- 10756251 TI - Transformation of mitral valve prolapse to dynamic left ventricular outflow tract obstruction and back again in a patient with acute transient myocardial depression. AB - We describe an unusual case of transient resolution of preexisting mitral valve (MV) prolapse during acute cardiac dysfunction and the development of dynamic left ventricular (LV) outflow tract obstruction. The patient presented with lightheadedness, chest pain, and compromised hemodynamic status. Echocardiography revealed akinesis and deformation of the LV anterior wall and apex, hyperdynamic activity in the bases, anterior MV leaflet systolic anterior motion without prolapse, and a dynamic outflow tract gradient. Myocardial function fully recovered over 1 month. Repeat ultrasonography showed posterior MV leaflet prolapse and no anterior MV leaflet systolic anterior motion. Elongated MV leaflets may have contributed to dynamic outflow tract obstruction and life threatening hemodynamic compromise during LV conformational change. PMID- 10756252 TI - The vegetation that was not a vegetation: excusable misinterpretation of transesophageal echocardiography findings in late infective endocarditis of a prosthetic mitral valve. AB - The echocardiographic detection of a valvar mobile mass with independent mobility is one of the major Duke criteria for diagnosing infective endocarditis.(1) We report a case in which a mobile mass seen by transesophageal echocardiography on a prosthetic mitral valve with proven endocarditis was not a vegetation but pannus. This is an important clinical message for the interpretation of mobile masses on artificial valves. PMID- 10756253 TI - Echocardiographic features of genetic diseases: part 4. Connective tissue. PMID- 10756254 TI - Contrast echocardiography: current and future applications. AB - Recent updates in the field of echocardiography have resulted in improvements in image quality, especially in those patients whose ultrasonographic (ultrasound) evaluation was previously suboptimal. Intravenous contrast agents are now available in the United States and Europe for the indication of left ventricular opacification and enhanced endocardial border delineation. The use of contrast enables acquisition of ultrasound images of improved quality. The technique is especially useful in obese patients and those with lung disease. Patients in these categories comprise approximately 10% to 20% of routine echocardiographic examinations. Stress echocardiography examinations can be even more challenging, as the image acquisition time factor is critically important for accurate detection of coronary disease. Improvements in image quality with intravenous contrast agents can facilitate image acquisition and enhance delineation of regional wall motion abnormalities at the peak level of exercise. Recent phase III clinical trial data on the use of Optison and several other agents (currently under evaluation) have revealed that for approximately half of patients, image quality substantively improves, which enables the examination to be salvaged and/or increases diagnostic accuracy. For the "difficult-to-image" patient, this added information results in (1) enhanced laboratory efficiency, (2) a reduction in downstream testing, and (3) possible improvements in patient outcome. In addition, substantial research efforts are underway to use ultrasound contrast agents for assessment of myocardial perfusion. The detection of myocardial perfusion during echocardiographic examinations will permit the simultaneous assessment of global and regional myocardial structure, function, and perfusion all of the indicators necessary to enable the optimal noninvasive assessment of coronary artery disease. Despite the added benefit in improved efficacy of testing, few data exist regarding the long-term effectiveness of these agents. Currently under evaluation are the clinical and economic outcome implications of intravenous contrast agent use for daily clinical decision making in a variety of patient subsets. Until these data are known, this document offers a preliminary synthesis of available evidence on the value of intravenous contrast agents for use in rest and stress echocardiography. At present, it is the position of this guideline committee that intravenous contrast agents demonstrate substantial value in the difficult-to-image patient with comorbid conditions limiting an ultrasound evaluation of the heart. For such patients, the use of intravenous contrast agents should be encouraged as a means to provide added diagnostic information and to streamline early detection and treatment of underlying cardiac pathophysiology. As with all new technology, this document will require updates and revisions as additional data become available. PMID- 10756255 TI - It's all in the details. PMID- 10756257 TI - Make no apologies. PMID- 10756256 TI - Revisiting root resorption. PMID- 10756259 TI - In response: PMID- 10756258 TI - Another Ricketts contribution. PMID- 10756260 TI - Muscle response to the Twin-block appliance. PMID- 10756261 TI - Follow-up on distraction osteogenesis in the mandible. PMID- 10756262 TI - Clinical applications of composite intramembranous bone grafts. AB - The aim of this article is to introduce the composite intramembranous bone graft mixed with demineralized bone matrix to the clinician and to demonstrate its various clinical applications in the field of clinical orthodontics in the form of case reports. Understanding the mechanism of healing of this composite bone graft provides sound experimental precedent that allows this graft material to become a predictable part of our future orthodontic management. The cases highlighted here took advantage of the several properties of the composite intramembranous demineralized bone matrix graft. An accidental loss of the buccal plate of bone occurred during extraction of a buccally placed premolar for orthodontic purposes. The defect was repaired using chin bone mixed with demineralized bone matrix, and the teeth were successfully moved into the grafted area. A 5-year follow-up showed a stable gingival condition at the grafted area. In this report, ridge augmentation with intramembranous demineralized bone matrix as an adjunct to orthodontic treatment allowed successful placement of endosseous implants. In conclusion, the same graft material was successfully used in the repair of a massive alveolar cleft. Long-term follow-up of these cases showed that this graft is a promising graft material that could be integrated into our orthodontic practice. PMID- 10756263 TI - Muscle activity with the mandibular lip bumper. AB - This prospective study was designed to measure muscle activity with lip bumper treatment. Surface electromyography was used to measure upper and lower lip activity with and without the appliance in place. Measurements were taken on a sample of 25 adolescent patients (age range, 10 to 17 years) before and after 12 months of lip bumper treatment. Recordings included a closed lip rest position, swallowing of water, and speech of the words "church," "phone," and "pop." Insertion of the lip bumper at the pretreatment recording resulted in significantly increased activity of the upper and lower lips at rest (P <.001) and while swallowing (P <.05), whereas the response for speech was variable. Pretreatment and posttreatment muscle activity measurements of the lower lip were compared to determine whether 12 months of treatment with the appliance resulted in muscle adaptation. The initial increase of muscle activity with insertion of the appliance for the closed lip rest position and for swallowing was not followed by a decrease during the treatment period. There were significant differences in muscle activity when comparing the "new" equilibrium introduced by the lip bumper to the "old" pretreatment equilibrium without the appliance. Although it was shown that the insertion of the appliance had a significant influence on muscle activity of the lips, the results of this study did not indicate adaptation of the lower lip to the appliance over the treatment period. PMID- 10756264 TI - Rapid orthodontic tooth movement into newly distracted bone after mandibular distraction osteogenesis in a canine model. AB - Orthodontic tooth movement through recently distracted fibrous bone tissue has not been investigated previously. We hypothesized that a tooth can be moved into the fibrous new bone created by the distraction process at a rapid rate. Four mature beagle dogs were used in this study. An edentulous space was created in 2 weeks by using a bone-borne intraoral distraction device on each side of the mandibular body between the third and fourth premolars. Calibrated elastic threads with 50 g of orthodontic force were applied to move the fourth premolar into the edentulous space for 5 weeks. On one side, the tooth was moved simultaneously with distraction; and on the opposite side, it was initiated immediately after the cessation of distraction. The fourth premolars were moved 1.2 mm per week. The results indicated that the best time to initiate tooth movement was immediately after the end of distraction. With this approach, most of the periodontal support was preserved after orthodontic tooth movement. In contrast, moderate to severe alveolar bone loss was noted in the fourth premolars moved simultaneously with distraction. This is one of the first experimental studies to demonstrate successful rapid orthodontic tooth movement into an edentulous space newly created by distraction osteogenesis. Clinical implications of these results may be applied to relieve severe dental crowding and to correct sagittal or transverse dental arch discrepancies. PMID- 10756265 TI - Alveolar bone resorption and the center of resistance modification (3-D analysis by means of the finite element method). AB - The main goal of this research was to study the behavior of initial tooth displacements associated with alveolar bone loss situations when loaded by a force of 1 N. The analysis of displacements was carried out by the finite element method. Six 3-dimensional models of an upper central incisor (designated Geramy 391 to 396) with 1 to 8 mm of alveolar bone loss were formulated and used by the author. Center of rotation and center of resistance were located for the various stages of alveolar bone loss. The results revealed that the moment/force ratio (at the bracket level) required to produce bodily movement increases in association with alveolar bone loss. Bone loss causes center of resistance movement toward the apex, but its relative distance to the alveolar crest decreases at the same time. Greater amounts of displacements of incisal edge and apex were observed with increased alveolar bone loss for a constant applied force. Center of rotation of the tipping movement also shifted toward the cervical line. Among the many differences between orthodontic treatment of an adolescent and an adult patient is the presence of alveolar bone loss in the adult cases. Alveolar bone loss causes center of resistance changes as a result of the alterations in bone support. This necessitates modifications in the applied force system to produce the same movement as in a tooth with a healthy supporting structure. PMID- 10756266 TI - Relationship of natural head position to craniofacial morphology. AB - This investigation aimed at discussing the utility of natural head position-based cephalometric variables and to evaluate the relationship between natural head position and craniofacial morphology. Lateral facial photographs and cephalograms of 284 young adult males taken in a natural head position were analyzed. The average inclination of the intracranial reference planes, Frankfurt horizontal, and palatal plane, in relation to the true horizontal were nearly similar and smaller than 1 degrees. Variables based on the true vertical to describe mandibular sagittal position like B-N(vert) and Pg-N(vert) had very high variances. To study the topographic error, flexors and extenders were identified on basis of four positional variables: NSL/VER; FH/VER; PP/VER; and PMvert/VER. Only 15 measurements were different, according to at least 2 of the 4 positional variables. The 3 that were different in all categories were: facial axis (NBa/PmGn), lower face height (ANS-Me), and the facial ratio (N-ANS/ANS-Me). The "extenders" had higher values for the facial axis and lower face height, and smaller for the face height ratio. Besides these 3 measurements, there was a tendency for the extenders to have increased anterior vertical height, distal sagittal relations, and smaller and retrognathic mandibles. Correlation coefficients between postural and morphologic variables tended to confirm these observations. PMID- 10756267 TI - For four sixes. AB - This article reviews the literature on orthodontic treatment involving extraction of first molars and highlights many of the clinical considerations when treating such cases. Case reports illustrate the potential problems and indicate some solutions. PMID- 10756268 TI - Useful data from application of the HLD (CalMod) Index. PMID- 10756269 TI - Shear bond strengths of orthodontic plastic brackets. AB - The purpose of the present study was to evaluate the shear bond strengths of plastic brackets and the influences of the bracket filler contents on the bonding. The shear bond strengths of 4 plastic brackets (Spirit; Spirit MB; Clear Bracket; Aesthetic-Line) bonded to enamel with 4 orthodontic adhesives (Orthomite Superbond; System 1+; Transbond XT; and Kurasper-F) were compared with the strength of a conventional metal bracket. The findings of this study indicated the following: (1) shear bond strength of the 4 plastic brackets was significantly lower than that of the conventional metal brackets (P <.05), with most of the values ranging from 3 MPa to 6 MPa; (2) when comparing the bond strengths of plastic brackets, Aesthetic-Line had the largest value followed by Spirit MB, Spirit, and Clear Bracket, and when the plastic brackets were bonded with Orthomite Superbond, they showed relatively stronger bond strengths than when bonded with the other adhesives. Clear Bracket showed relatively lower values especially when bonded with System 1+; (3) the application of primer did not increase the durability of the bond strengths when bonding Spirit and Clear brackets; and (4) fillers contained in each plastic bracket ranging from 9.18% to 19. 52% were fairly well distributed and showed the same morphology of a fiber type 10 microm in diameter with different lengths. The filler concentration tended to correlate with the bond strength. The exposed fillers on the bracket base surface may play a more important role in plastic bracket adhesion than the macro-morphology of the base surface. PMID- 10756270 TI - Angle, the innovator, mechanical genius, and clinician. PMID- 10756271 TI - Treatment of a patient with a Class I malocclusion with bialveolar protrusion, mild upper and lower crowding, and mild mandibular prognathism. PMID- 10756272 TI - Treatment of a Class II, Division 1, malocclusion with the extraction of maxillary canines and mandibular first premolars. AB - This is the case report of a patient whose healthy but malformed maxillary canines and normal mandibular first premolars were extracted in order to correct a Class II, Division 1, malocclusion. Maxillary premolars were substituted for the extracted canines. PMID- 10756273 TI - A histologic and histomorphometric evaluation of pulpal reactions following rapid palatal expansion. AB - The aim of this study was to investigate the effects of the heavy forces exerted by the rapid palatal expansion (RPE) appliance on the pulpal tissue of anchor premolar teeth and to evaluate these effects by histologic and histomorphometric methods. Materials consisted of 34 sound upper premolars, extractions of which were required as part of orthodontic treatment. Twenty-three teeth were extracted after RPE; the remaining 11 teeth, which had not undergone any orthodontic force, were taken as controls. After extraction, the teeth were prepared for histologic examination under light microscopy. Histomorphometric measurements were performed by using image analysis. The data gained were statistically evaluated by nonparametric tests. The null hypothesis was that there was no difference between pulpal tissues of control and test teeth with respect to different durations of RPE. Our results reveal that the null hypothesis was rejected. Of the parameters evaluated, vessel area and minimum and maximum vessel diameters showed significant differences among the groups. Especially the difference between the control and 3-month groups had marked statistical significance. In conclusion, forces applied by RPE appliances caused an adaptive vascular tissue response, as well as fibrotic changes, in the "affected" upper premolars. PMID- 10756274 TI - Basal nitric oxide production is enhanced by hydraulic pressure in cultured human periodontal ligament fibroblasts. AB - To understand orthodontic tooth movement and determine optimal orthodontic force from a biological viewpoint, nitric oxide production in cultured human periodontal ligament fibroblasts was measured at varying levels of hydraulic pressure. The fibroblasts in a culture flask were exposed to the controlled change in hydraulic pressure, and intracellular nitric oxide levels were measured in real time by a nitric oxide-binding fluorescent dye, diaminofluorescein-2. The fibroblasts produced a significantly larger amount of nitric oxide at the pressure of 75 and 100 mmHg, compared with the pressure of 0, 25, and 50 mmHg (P <.0001, one-way ANOVA, and P <.05, Tukey-Kramer test). Immunohistochemically, the cultured fibroblasts expressed brain nitric oxide synthase. The pressure level to enhance nitric oxide production was comparable to the magnitude of clinically used orthodontic force (80 g/cm(2)). Nitric oxide might be a key regulator in orthodontic tooth movement. PMID- 10756275 TI - Cephalometric comparisons between Chinese and Caucasian patients with obstructive sleep apnea. AB - The purpose of this study was to compare two groups of adult men from different ethnic backgrounds and with obstructive sleep apnea; they were selected by matching age, gender, skeletal pattern, body mass index, and respiratory disturbance index. Pretreatment cephalometric radiographs and overnight polysomnograms of 30 Chinese and 43 Caucasian patients with Class II, Division 1 malocclusions were analyzed to investigate if there were craniofacial and upper airway structural differences between the two ethnic groups. The Chinese group, when compared with the group of Caucasian patients, revealed more severe underlying craniofacial skeletal discrepancies with significantly smaller maxilla and mandibles, more severe mandibular retrognathism, proclined lower incisors, increased total and upper facial heights, and steeper and shorter anterior cranial bases. However, no significant differences were found between the two groups in posterior facial height, ratio of upper to lower anterior facial height, and the position of hyoid bone, maxilla, and upper incisors. With regard to soft tissue and upper airway measurements, there were no significant ethnic differences in tongue and soft palate size, vertical length of oropharynx, and anteroposterior dimensions of the upper airway at most of the levels except for a larger super-posterior airway space, a larger nasopharynx and oropharynx cross sectional area, and a smaller tongue height in the Chinese group. We conclude that there are a number of craniofacial and upper airway structures that differ between the two ethnic groups that may be relevant to the treatment of obstructive sleep apnea in various ethnic groups. PMID- 10756277 TI - Lester levern merrifield, feb 1, 1921 to jan 15, 2000 PMID- 10756276 TI - Vertical components of overbite change: a mathematical model. AB - The purpose of this study was to better understand the multidimensional nature of overbite changes that occur during adolescence. The study used longitudinal cephalograms of 181 untreated children (102 males, 79 females) taken at ages 10 and 15. Four major components that directly affect overbite were measured: (1) maxillary vertical displacement, (2) mandibular vertical displacement, (3) upper incisor vertical change within the bone, (4) lower incisor vertical change within the bone. Cranial base, maxillary, and mandibular superimpositions were performed for each subject to assess the vertical changes that occurred in these 4 components and to assess overbite. A multiple regression analysis was used to develop a mathematical model describing the relationships of these components to changes in overbite. The model was validated with an independent subsample and a comparison of subjects whose overbites decreased and those whose overbites increased. The results showed that overbite changed minimally (0.2 mm) over the 5 year period; variation ranged from a 2.4 mm decrease to a 5.6 mm increase. The regression model indicated that the mandibular skeletal changes were twice as important as the mandibular dental changes and about 2.5 times as important as the maxillary changes in effecting overbite change. Within the mandibular skeletal component, vertical growth was more important than mandibular rotation in determining overbite change. The model demonstrated that a multivariate approach is necessary to understand overbite changes. More effective orthodontic treatment might be achieved by focusing on the primary components effecting overbite change, especially those with the greatest potential for therapeutic modification. PMID- 10756278 TI - Maurice samuel berman, 1914-1999 PMID- 10756279 TI - Stanley jacobs, 1939-2000 PMID- 10756280 TI - Bernhard Wolf Weinberger (1885-1960). PMID- 10756281 TI - Computer voice recognition. PMID- 10756283 TI - Canadian academy of restorative dentistry and prosthodontics-founded in 1993 PMID- 10756282 TI - Litigation, legislation, and ethics. Self-incrimination in the civil arena. PMID- 10756284 TI - Carl O. Boucher prosthodontic conference-founded in 1966 PMID- 10756285 TI - Northeastern gnathological society-founded in 1967 PMID- 10756286 TI - How to evaluate a diagnostic test. AB - Tests are used in dentistry to establish, confirm, or reject the clinical impression of a diagnosis. Not all tests are equal in their ability to establish a diagnosis, with some tests demonstrating positive results when no disease is present (false positive) or negative results when disease is present (false negative). Using simple mathematical computations, it is possible to determine the extent to which a test can reliably establish the presence or absence of disease. This article describes the concepts of sensitivity, specificity, positive predictive value, negative predictive value, and likelihood ratios through the evaluation of a clinically relevant paper on vital staining for oral carcinoma. This article also describes methods for literature evaluation to determine whether a test conveys meaningful diagnostic information. PMID- 10756287 TI - Removable occlusal overlay splint in the management of tooth wear: a clinical report. PMID- 10756288 TI - Clinical assessment of high-strength all-ceramic crowns. AB - STATEMENT OF PROBLEM: All-ceramic crowns are being used extensively. Little data are available on their clinical performance. PURPOSE: This study evaluated the clinical performance of In-Ceram (Vita Zahnfabrik) crowns. MATERIAL AND METHODS: Forty-one patients (16 men, 25 women; mean age 47.3 years, range 18 to 77 years) were examined with a total of 80 In-Ceram all-ceramic crowns fabricated at the University of Iowa College of Dentistry from 1994 to 1997. The percentage distribution for crowns included: 67% anterior single crowns, 26% posterior single crowns, 6% anterior implant crowns, and 1% posterior implant crowns. This study documented the integrity of the junction between crown and tooth, color match to adjacent teeth, secondary dental caries, wear of crown and opposing dentition, and visible cracks in the crown. Alpha, Bravo, and Charlie ratings were assigned with a modified USPHS criteria. The patients were also surveyed with respect to oral hygiene and satisfaction of treatment. Estimations of 4-year success rates and corresponding confidence intervals were calculated by fitting a constant hazard function model with the SAS procedure GENMOD. RESULTS: Marginal integrity for 88% of artificial crowns was rated Alpha or Bravo. Shade match for 99% was Bravo or better. Only 1% of the crowns were carious, and 1 crown exhibited occlusal wear. One premolar crown had a small fracture of veneering porcelain. One molar crown was remade after core fracture. All patients (100%) expressed satisfaction with their crowns. The estimated 4-year success rates (Alpha or Bravo), with 95% confidence intervals in parentheses were calculated as: 83.5% (65.7%-94.6%) for marginal integrity, 95.8% (82.9%-99.8%) for shade match, and 95.5% (81.6%-99.7%) for secondary caries, 100% (88%-100%) for wear, and 100% (88%-100%) for cracks. PMID- 10756289 TI - Soft tissue correction of a single-tooth pontic space: a comparative quantitative volume assessment. AB - STATEMENT OF PROBLEM: Various augmentation procedures are recommended for the correction of localized alveolar ridge defects. However, no study has quantitatively evaluated the results of these procedures to date. PURPOSE: This study compared 2 soft tissue augmentation surgeries commonly used to alter contours of single-tooth pontic space by quantifying 3-dimensional volume changes with the optical projection Moire method at 1 and 3.5 months after surgery. MATERIAL AND METHODS: Twenty-four patients required surgery. Each patient had a localized alveolar ridge defect, corresponding to a mesial-distal width of 1 single tooth. The defect of 12 patients was corrected with a subepithelial connective tissue graft; the remaining 12 patients were treated by receiving a free full-thickness gingival graft, which included epithelium and connective tissue with fatty tissue. Six unoperated defects of 6 patients formed the control group. For each defect, an impression was made before treatment, at 1 and 3.5 months after surgery to measure the volume changes on the dental casts with a validated projection Moire system. Volume change was assessed relative to the preoperative dimension at the buccal and crestal aspects of the single-tooth pontic space. RESULTS: At 3.5 months postsurgery, mean volumetric gain for the connective tissue group with 159 mm(3) (SD +/- 80) was significantly greater (P =.027) than for free full-thickness gingival graft group with 104 mm(3) (SD +/- 31). CONCLUSION: The applied projection Moire method proved its applicability in assessing 3-dimensional volume changes of pontic spaces with a single-tooth width. Volumetric assessment after 1 and 3.5 months revealed significantly greater volume gain with the subepithelial connective tissue graft in comparison to the free full-thickness gingival graft. PMID- 10756290 TI - Influence of ceramic and cement thickness on the masking of various types of opaque posts. AB - STATEMENT OF PROBLEM: Dark-colored posts may negatively affect the esthetics of all-ceramic single unit crowns as the thin layers of luting cement may not be sufficiently opaque. PURPOSE: This in vitro study evaluated the influence of the color of 2 commercially available nonmetallic opaque posts (carbon fiber and zirconia) and an experimental esthetic post, and the shade and thickness of luting cements on the esthetics of all-ceramic restorations. MATERIAL AND METHODS: Sample disks at several thickness values were made in glass-ceramic (IPS Empress), an experimental ceramic, a zirconia, a carbon fiber post material, a resin composite material (Z100) as reference, and a luting cement (Variolink II). A laboratory procedure, with 3 possible combinations of stapling the disks, was used. This was performed for 4 substrates, 3 cement colors at 2 thickness values, and 3 heights of ceramic disks. For each combination, the shift in color was measured with a spectrophotometer. Readings were performed for 3 conditions: (1) ability of ceramic to mask the aspect of the abutment in relation to its thickness (1.0, 1.5, or 2.0 mm); (2) effect of a change in cement color (W, Y, or B) on the final color of the ceramic; and (3) influence of cement film thickness (0.1 or 0.2 mm) on the final color of the ceramic. RESULTS: When ceramic thickness was 1 mm, all other variables were visually appreciable. For ceramic thickness of 1.5 mm, color differences decreased and most differences were appreciable only with laboratory instruments. For ceramic thickness of 2.0 mm, there were no detectable, clinically relevant differences. CONCLUSION: The final esthetic result of the all-ceramic IPS-Empress glass-ceramic restoration was not affected by the presence of different substrates with different colors when the thickness was more than 2.0 mm. When ceramic thickness decreases to 1.5 mm, it is advised to take the substrate aspects into consideration. If the ceramic thickness is less than 1.0 mm, the use of a full ceramic crown is contraindicated because color matching of the abutment is required to ensure an acceptable esthetic result. Differences in cement thickness (0.1 or 0.2 mm) may slightly affect the final result. As this parameter can be controlled by the operator only to a certain extent, it cannot be considered as a procedure to correct color. Availability of different cement shades allows only minor esthetic corrections, which might be instrumentally detectable but are clinically not relevant. PMID- 10756291 TI - Color and translucency of in vivo natural central incisors. AB - STATEMENT OF PROBLEM: The range of shades in shade guides is not consistent with natural teeth, and there is no information on the color and translucency of the natural tooth for all age groups. Therefore, it is important to obtain a database of the characteristics of the natural tooth from a wide age group. PURPOSE: This study obtained information on color and translucency of natural teeth for all age groups, and attempted to clarify the difference of the color and translucency between natural teeth and VITA Lumin Vacuum shade guide. MATERIAL AND METHODS: Natural central incisors of 87 subjects (42 men and 45 women; age range from 13 to 84 years) and 16 shades of VITA Lumin Vacuum shade guide were evaluated. Color and translucency of 5 sites, each 1.0 mm in diameter, on the surfaces of individual teeth were measured for L*, a*, and b*, using a color computer according to CIELAB color spaces. RESULTS: At the center site, negative correlation was found between age and L*, with positive correlation between age and b*. Both a* and b* of the natural tooth increased when moving in the direction of the cervical site, but translucency decreased in the direction of the root. The a* value for the natural tooth was significantly higher than those for VITA Lumin Vacuum shade guide. CONCLUSION: This study found that the older the subject, the darker and more yellow the color at the center site of the natural tooth. Both reddish and yellowish colors of natural teeth tend to increase from the incisal to cervical, whereas translucency decreases. Red-green chromaticity of VITA Lumin Vacuum shade guide was not distributed to cover the natural tooth. PMID- 10756292 TI - Treatment outcomes of fixed or removable implant-supported prostheses in the edentulous maxilla. Part I: patients' assessments. AB - STATEMENT OF PROBLEM: Distinct clinical parameters determine whether fixed or removable implant-supported prostheses are indicated to restore the edentulous maxilla. However, there is a strong belief that fixed implant prostheses meet with greater patient acceptance and satisfaction, but this may differ from the patients' perceptions, their psychological responses to treatment, and their assessments of the treatment outcome. PURPOSE: This prospective clinical study compared the treatment outcomes of fixed and removable implant-supported restorations in the edentulous maxilla with the main emphasis on the patient's point of view. MATERIAL AND METHODS: Twenty patients who requested an implant supported superstructure to restore the edentulous maxilla were asked to complete a questionnaire measuring their satisfaction with the present situation and the psychologic impact of their oral health status with their responses marked on a Visual Analog Scale (VAS). Ten patients were treated with a fixed, screw-retained implant prosthesis (group 1), and 10 were treated with a removable, implant supported and bar-retained overdenture (group 2). Six months after prosthetic rehabilitation, patients were again given the questionnaire to assess their psychologic well-being and satisfaction with the implant-supported restoration. RESULTS: Both prosthesis designs were associated with significant improvements in comfort and retention, function, esthetics and appearance, taste, speech, and self-esteem. No difference was found between the 2 groups with respect to how the patients assessed the implant therapy. However, the results indicated that patients in group 2 experienced greater differences between pretreatment and posttreatment scores for the parameters esthetics, taste, and speech. Treatment costs per unit were significantly higher in group 1 than in group 2. CONCLUSION: Patients in groups 1 and 2 were similarly satisfied with their implant-supported prostheses in the edentulous maxilla with regard to their well-being and the cost utility, irrespective of whether the restoration was fixed or removable. PMID- 10756293 TI - Treatment outcomes of fixed or removable implant-supported prostheses in the edentulous maxilla. Part II: clinical findings. AB - STATEMENT OF PROBLEM: There is a widespread belief that maxillary overdenture prostheses are associated with a higher frequency of complications and require more maintenance than fixed implant prostheses. PURPOSE: This prospective clinical study compared the treatment outcomes of fixed and removable implant supported restorations in the edentulous maxilla with the main emphasis on the clinician's point of view. MATERIAL AND METHODS: Ten patients were treated with fixed screw-retained implant prostheses (group 1), and 10 patients were treated with removable implant-supported overdentures (group 2) in the edentulous maxilla. Recall was scheduled at 6-month intervals to investigate the prosthodontic treatment outcomes, including implant survival, prosthesis time until retreatment, and maintenance issues. Clinical parameters gingival index (GI), plaque index (PI), the clinical attachment level, and radiographic marginal bone levels measured, along with any biologic and mechanical complications were recorded. RESULTS: Patients were followed over a mean period of 39 months (SD=7; group 1) and 27 months (SD=10; group 2) after implant placement. Cumulative implant survival was 97.6% for group 1 and 94.4% for group 2 after an 18-month observation period. The mean time until retreatment after prostheses insertion was 23.4 months for group 1 and 19.8 months for group 2 (n.s.). In both groups, the increase over time in the radiographically investigated bone level was found to be significant. The indices given for the mucosal health and oral hygiene status (GI and PI) were highly correlated in both groups at each recall appointment, but no significant differences were found between groups 1 and 2. CONCLUSION: In groups 1 and 2, comparable prosthodontic treatment outcomes were achieved. The majority of mechanical complications could be managed chairside during recall visits and did not require additional appointments, so that the time and costs involved in providing maintenance were kept down. PMID- 10756294 TI - Altered sensation associated with implants in the anterior mandible: a prospective study. AB - STATEMENT OF PROBLEM: Reported incidences of altered sensation after placement of mandibular implants range from a low of 0% to almost 44%, making it difficult to advise subjects regarding risk. PURPOSE: This study involved presurgical and postsurgical assessment of sensation in the lips and chins of 75 subjects, all of whom objectively demonstrated normal sensation before surgery. MATERIAL AND METHODS: Objective and subjective tests were administered before, and at planned intervals for 12 months after, the placement of 2 implants in the anterior mandible. RESULTS: Objective changes were observed in only 1 subject and then only at the 2-week postsurgery interval. There were significant differences for all subjects in subjective sensation changes from baseline to 2 weeks after surgery (P =.007) and from 2 weeks to 6 months (P =.003) or 12 months (P =. 003). There was a higher percentage of subjects with normal sensation before surgery who reported sensory changes 2 weeks after implant placement (34% vs 24% for all subjects), but only 1% of subjects still reported altered sensation 1 year later. Although there were no significant differences between men and women in reports of altered sensation, more women than men noted sensation changes 2 weeks after implants were placed (25% vs 13%). There appeared to be no relationships among gender, the degree of ridge resorption, and reports of altered sensation. CONCLUSION: Thus, although approximately 24% of subjects may report altered sensation in the short-term after implant surgery in the anterior mandible, the problem appears to be a transient one, with only about 1% experiencing sensation changes 1 year after implant surgery. PMID- 10756295 TI - Effect of an antirotation resistance form on screw loosening for single implant supported crowns. AB - STATEMENT OF PROBLEM: The gold screw of the single tooth implant-supported restoration has frequently been reported to exhibit the problem of screw loosening. PURPOSE: This in vitro study considered an antirotation resistance form with an increased moment arm length as a strategy to increase the net effect of the retaining screw preload. MATERIAL AND METHODS: Three groups examined included (a) standard abutments (3 mm), (b) modified standard abutments (3 mm) with 4 milled (1 x 1 mm) notches placed equidistant around the periphery, and (c) Estheticone abutments (1 mm). Crown castings of 2% gold-palladium-based alloy were made using plastic and resin patterns molded to fit inside a quarter-inch socket wrench. For testing, each specimen consisted of a 15-mm endosseous threaded implant analog, its abutment (20 N force) and crown casting (10 N force) screwed together and securely clamped in a vise. A clockwise shear (rotational) force was applied using a manual torque driver and the torque recorded to failure of the gold screw or loosening of the abutment screw. RESULTS: Analysis revealed that the dislodging force for groups (b) and (c) was similar (69.6 and 71.0 N.cm, respectively), whereas group (a) (standard implant external hex) demonstrated that the smallest moment arm required a significantly smaller dislodging force (21.3 N.cm). CONCLUSION: These findings suggest that the addition of an antirotation resistance form increased the length of the moment arm, thereby increasing the effect of preload and reducing the problem of screw loosening. PMID- 10756296 TI - Prosthetic alternative treatment for choanal atresia: a clinical report. PMID- 10756297 TI - Effect of dentin desensitizers and cementing agents on retention of full crowns using standardized crown preparations. AB - STATEMENT OF PROBLEM: Past research has not controlled preparation surface area when examining the influence of dentin desensitizers on the retentive strength of cemented cast crowns, leading to inconsistent results. PURPOSE: This research controlled crown preparation surface area and evaluated the effect of various dentin desensitizers and conventional cementing agents on the in vitro retentive strength of cast crowns. METHODS AND MATERIAL: Freshly extracted human molars were prepared for a standardized crown preparation (26 degrees total convergence, 4 mm axial height) with a custom-made pantograph. Dentin desensitizers included none (control), a polymerizable material (All-Bond 2), and a nonpolymerizable desensitizer (Gluma Desensitizer). Cementing agents included zinc phosphate (Fleck's), glass ionomer (Ketac-Cem), resin-modified glass ionomer (Fuji II), and resin cement (Panavia 21). Twelve teeth were prepared for each test condition (144 teeth total). Individual castings were made from a base metal alloy (Rexillium III). Crowns were removed after storage at 26 degrees C for 48 hours at 100% relative humidity using a universal testing machine at a crosshead speed of 1.27 mm/min. The proportion of cement retained on the tooth and casting after debonding was quantified according to treatment. Statistical treatment included 1 and 2-way ANOVAs, followed by the Tukey-Kramer post hoc test at a preset alpha of 0.05.Results. Resin cement exhibited the highest retentive strength and all dentin treatments resulted in significantly different retentive values (All-Bond 2 (5.68 +/- 0.70 MPa) > control (4.67 +/- 0.48 MPa) > Gluma (4.12 +/- 0.37 MPa)). Retention of resin-modified glass ionomer was between the resin cement and glass ionomer groups: All-Bond 2 (3.46 +/- 0.26 MPa) > Gluma (2.81 +/- 0.15 MPa) = control (2.96 +/- 0.18 MPa). Conventional glass ionomer values were between those of Fuji Plus and zinc phosphate groups: All Bond 2 (2.23 +/- 0. 20 MPa) = control (2.36 +/- 0.20 MPa) > Gluma (1.98 +/- 0.23 MPa). Zinc phosphate had the lowest retention values: control (1.68 +/- 0. 08 MPa) > Gluma (0.81 +/- 0.11 MPa) > All Bond 2 (0.67 +/- 0.14 MPa). The majority of cement was retained on the debonded tooth surface versus the casting, with the exception of zinc phosphate when used with dentin pretreatments. CONCLUSION: Controlled crown surface areas reduced the variation in strength values permitting high discrimination among retention values of desensitizer/cement combinations. In all but 1 combination, Gluma desensitizer significantly decreased crown retention. With resin cement and resin modified glass ionomer, use of All-Bond 2 desensitizer significantly increased crown retention values. PMID- 10756299 TI - Use of orthodontic expansion screw in fabricating section custom trays. PMID- 10756298 TI - Study of the physical properties of type IV gypsum, resin-containing, and epoxy die materials. AB - STATEMENT OF PROBLEM: Little published information is available comparing the properties of recently developed resin-modified gypsum and epoxy resin die materials, which are claimed to be superior to conventional type IV gypsum die materials. PURPOSE: This study compared the properties of 3 new die materials and 2 conventional type IV gypsum products. METHODS AND MATERIAL: The linear dimensional change, detail reproduction, surface hardness, abrasion resistance, and transverse strength of 2 recently introduced, resin-modified gypsum die materials (Resinrock and Milestone), a new epoxy resin die material (Epoxy-Die), and 2 conventional type IV gypsum die materials (Silky-Rock and Die-Stone) were studied. RESULTS: All gypsum products expanded, whereas the epoxy resin material contracted during setting. The epoxy resin exhibited much better detail reproduction, abrasion resistance, and transverse strength than the gypsum materials, which were similar in these properties. A conventional type IV gypsum exhibited the highest surface hardness, whereas the epoxy resin had the lowest value. CONCLUSION: The resin-modified gypsum products were not significantly superior to the conventional type IV gypsum die materials. In general, the epoxy resin exhibited the best properties of the materials studied; however, its setting shrinkage may necessitate alterations in technique to achieve well adapted castings. PMID- 10756300 TI - Fabrication and use of a metal reinforcing frame in a fracture-prone mandibular complete denture. AB - A procedure to reinforce mandibular complete dentures with a rigid internal horseshoe-shaped frame is described. Sequential clinical and laboratory procedures to incorporate a metal frame at a predetermined, controlled position within the prosthesis are presented. This procedure provides not only strength but ensures adequate space for a resilient liner if required. PMID- 10756301 TI - Radiographic stent for a quick and precise bone height analysis. PMID- 10756302 TI - Locking acrylic resin dental stent for image-guided surgery. AB - This article presents a procedure for fabricating a locking acrylic resin dental stent for use in image-guided base-of-skull surgery and neurosurgery. The stent offers advantages over conventional bone screw-anchored systems to surgeons and patients. In view of the increasing use of image guidance in base-of-skull surgery and neurosurgery, prosthodontists will meet a growing demand for this type of device in the future. PMID- 10756303 TI - Easy way to prepare plastic sprues for dowel fabrication. PMID- 10756304 TI - Patterned poly(chlorotrifluoroethylene) guides primary nerve cell adhesion and neurite outgrowth. AB - Central nervous system (CNS) neurons, unlike those of the peripheral nervous system, do not spontaneously regenerate following injury. Recently it has been shown that in the developing CNS, a combination of cell-adhesive and cell repulsive cues guide growing axons to their targets. We hypothesized that by mimicking these guidance signals, we could guide nerve cell adhesion and neurite outgrowth in vitro. Our objective was to direct primary nerve cell adhesion and neurite outgrowth on poly(chlorotrifluoroethylene) (PCTFE) surfaces by incorporating alternating patterns of cell-adhesive (peptide) and nonadhesive (polyethylene glycol; PEG) regions. PCTFE was surface-modified with lithium PEG alkoxide, demonstrating the first report of metal-halogen exchange with an alkoxide and PCTFE. Titanium and then gold were sputtered onto PEG-modified films, using a shadow-masking technique that creates alternating patterns on the micrometer scale. PCTFE-Au regions then were modified with one of two cysteine terminated laminin-derived peptides, C-GYIGSR or C-SIKVAV. Hippocampal neuron cell-surface interactions on homogeneously modified surfaces showed that neuron adhesion was decreased significantly on PEG-modified surfaces and was increased significantly on peptide-modified surfaces. Cell adhesion was greatest on CGYIGSR surfaces while neurite length was greatest on CSIKVAV surfaces and PLL/laminin positive controls, indicating the promise of peptides for enhanced cellular interactions. On patterned surfaces, hippocampal neurons adhered and extended neurites preferentially on peptide regions. By incorporating PEG and peptide molecules on the surface, we were able to simultaneously mimic cell-repulsive and cell-adhesive cues, respectively, and maintain the biopatterning of primary CNS neurons for over 1 week in culture. PMID- 10756305 TI - Effect of porosity and surface hydrophilicity on migration of epithelial tissue over synthetic polymer. AB - The relative effects of porosity and surface chemistry on the migration of epithelial tissue over the surface of a polymer were determined in vitro. These studies compared nonporous polymers with those having 0.1-microm diameter track etched pores and were conducted on polycarbonate and polyester. Epithelial tissue migration over the polymer surface was stimulated by the presence of these pores. The surface chemistries of the polymers were modified by deposition of various polymer films using radio frequency gas deposition, giving a range of surfaces that varied in air:water sessile contact angle (SCA) of between 26 and 100 degrees. Tissue migration on the nonporous surfaces was affected by the surface chemistry, being generally linear as a function of the SCA and higher on hydrophilic than on hydrophobic surfaces but reduced if the hydrophilic surface had a mobile chemistry. The effects of the 0.1-microm diameter pores and the surface hydrophilicity were additive with the maximal level of epithelial tissue migration occurring on a porous, hydrophilic polymer surface. PMID- 10756306 TI - Cytokine expression in vitro by cultured human endothelial cells in contact with polyethylene terephthalate coated with pyrolytic carbon and collagen. AB - In order to evaluate whether or not polyethylene terephthalate coated with pyrolytic carbon and collagen (PET+PC) favors inflammatory or hyperplastic reactions, the expression of mRNAs specific for interleukin-6 (IL-6), platelet derived growth factor-A (PDGF-A), PDGF-B, transforming growth factor-beta1 (TGF beta1), and TGF-beta2 were tested in vitro by cultured human umbilical vein endothelial cells (HUVEC). The cultures were put in contact with PET+PC for 1, 24, 48, and 72 h. The same cells cultured on polystyrene without biomaterials were tested as negative controls; cultures incubated with LPS were the positive control. The expression of mRNAs was evaluated by RT-PCR with specific primers. PET+PC did not determine any differences in the expression of IL-6-specific mRNA at any of the incubation times compared to the negative control while LPS (the positive control) induced expression after 24, 48, and 72 h. PET+PC induced a more precocious expression of mRNA specific for PDGF-A than did the negative control; however, the expression no longer was present after 48 h while in the negative control the expression stopped after 72 h. PET+PC induced a less frequent expression of PDGF-B-specific mRNA than did the negative control and LPS, especially after 24 h. PET+PC induced a later expression of TGF-beta2 specific mRNA than did the negative control and a less frequent expression of mRNA specific for TGF-beta1 after 24 and 72 h. PMID- 10756307 TI - Human monocyte adhesion and activation on crystalline polymers with different morphology and wettability in vitro. AB - This study evaluated the effects of crystalline polyamide (Nylon-66), poly(ethylene-co-vinyl alcohol) (PEVA), and poly(vinylidene fluoride) (PVDF) polymers with nonporous and porous morphologies on the ability of monocytes to adhere and subsequently activate to produce IL-1beta, IL-6, and tumor necrosis factor alpha. The results indicated monocyte adhesion and activation on a material might differ to a great extent, depending on the surface morphology and wettability. As the polymer wettability increases, the ability of monocytes to adhere increases but the ability to produce cytokines decreases. Similarly, these polymers, when prepared with porous surfaces, enhance monocyte adhesion but suppress monocyte release of cytokines. Therefore, the hydrophobic PVDF with a nonporous surface stimulates the most activity in adherent monocytes but shows the greatest inhibition of monocyte adhesion when compared with all of the other membranes. In contrast, the hydrophilic Nylon-66, which has a porous surface, is a relatively better substrate for this work. Therefore, monocyte behavior on a biomaterial may be influenced by a specific surface property. Based on this result, we propose that monocyte adhesion is regulated by a different mechanism than monocyte activation. Consequently, the generation of cytokines by monocytes is not proportional to the number of cells adherent to the surface. PMID- 10756308 TI - A comparison of the surface characteristics and ion release of Ti6Al4V and heat treated Ti6Al4V. AB - This work seeks to investigate the nanosurface characteristics and ion release for a Ti6Al4V alloy prepared by various methods (as received and heat treated at 1300 degrees C for 2 h) with three different passivation treatments (34% nitric acid passivation, 400 degrees C heating in air, and aging in 100 degrees C deionized water). The surface and nanosurface composition are not related to the surface passivation treatments and experimental materials as evaluated by energy dispersive spectroscopy and X-ray photoelectron spectroscopy (XPS) analyses. After passivation and autoclaving treatments, the specimens were immersed in 8.0 mM ethylenediaminetetraacetic acid (EDTA) in Hank's solution and maintained at 37 degrees C for periods of time up to 16 days. The 400 degrees C treated specimens exhibit a substantial reduction in constituent release, which may be attributed to the thicker thickness and rutile structure of the surface oxides. After soaking in Hank's-EDTA solution, a significant time-related decrease in constituent release rate is observed for all kinds of specimens throughout the 0 16 day experimental period. The thicker oxides may be a factor in the improved dissolution resistance. Upon immersion, nonelemental Ca and P are both detected on the surfaces of all kinds of specimens by XPS analysis, and this could be explained by the existence of two types of hydroxyl groups (acidic and basic OH groups) on the oxide surface of the specimens. PMID- 10756309 TI - Surface modification of poly(ethylene-co-vinyl alcohol) (EVA). Part I. Introduction of carboxyl groups and immobilization of collagen. AB - To enhance the surface biocompatibility of poly(ethylene-co-vinyl alcohol) (EVA) and high-density polyethylene (HDPE), carboxyl groups were introduced by ozone exposure. Type I collagen was immobilized onto the surface through polyion complexing. The carboxyl groups on the EVA were characterized by electron spectroscopy for chemical analysis and neutralization. The amounts of the carboxylic group and collagen increased with increases in time and temperature of exposure. Water-soluble fragments were produced by ozone exposure to EVA, and they acted as collagen crosslinkers. The differences in charge distribution of carboxyl groups affected the amount of collagen immobilization. Graft polymerization of acrylic acid was also carried out onto EVA and HDPE surfaces. The amount of collagen immobilized by graft polymerization was much higher than that by ozone exposure despite the introduction of almost the same amounts of carboxylic groups. It was suggested that the negative charge distribution influences the amount of collagen immobilized onto films. PMID- 10756310 TI - Formation of calcium phosphate/collagen composites through mineralization of collagen matrix. AB - Several types of calcium phosphate/collagen composites, including noncrystalline calcium phosphate/collagen, poorly crystalline carbonate-apatite (PCCA)/collagen, and PCCA + tetracalcium phosphate/collagen composites, were prepared through the mineralization of collagen matrix. The type I collagen was presoaked with a PO(3 )(4) containing solution and then immersed in a Ca(2+) containing solution to allow mineral deposition. The solution of 0.56 M sodium dibasic phosphate (Na(2)HPO(4)) with a pH of nearly 14 was metastable and its crystallization produced Na(2)HPO(4) and sodium tripolyphosphate hexahydrate (Na(5)P(3)O(10)). 6H(2)O), leading to a controlled release of orthophosphate ions during the subsequent mineral precipitation. The development of the composites was investigated in detail. The mineral contributed up to 60-70% of the weight of the final composites. The strength and Young's modulus of the composites in tensile tests overlapped the lower range of values reported for bone. When implanted in muscle tissue, the composite showed biodegradability that was partly through a multinucleated giant cell mediated process. In a bone explant culture model it was observed that bone-derived cells deposited mineralizing collagenous matrix on the composite. PMID- 10756311 TI - Regulation of biodegradability and drug release behavior of aliphatic polyesters by blending. AB - Polyester blending of poly(epsilon-caprolactone) (PCL) with poly(D, L-lactide) (PLA) and their random copolymers (R(CL/LA)) was found to be a convenient approach to regulate the degradation and drug release behaviors of the polyesters. The blend composition and compatibility both affected its degradation and drug release behavior. A DSC study showed that PCL was compatible with 50:50 poly(CL-CO-D,L-LA) (R(50/50)) but incompatible with 25:75 poly(CL-CO-LA) (R(25/75)) and PLA homopolymer. The hydrolysis experiments indicated that with the same CL/LA segment proportion, compatible blends (PCL/R(50/50)) had higher water content and faster weight loss than incompatible blends (PCL/PLA, PCL/R(25/75)). In the compatible blends the PCL degradation rate was increased while that of R(50/50) was decreased. The controlled release kinetics, diffusion constants, and permeation coefficients of the polymer blends were measured by using northindrone (NTD) as a model. The NTD release rates from the polyester blends increased as the CL unit fraction increased but decreased with increasing the LA unit fraction in the blends. With the same CL/LA unit ratios, the NTD release rate from the compatible blend was slower than that from the incompatible blend. The NTD release from the polyester blend was controlled by the diffusion process in the early stage, but the degradation-caused NTD release was later involved. By tailoring the blend composition to such an extent that the degradation-caused release compensated the decline of the diffusion-caused release, a zero-order NTD release was achieved. PMID- 10756312 TI - Physical, chemical, and biological characterization of pulsed laser deposited and plasma sputtered hydroxyapatite thin films on titanium alloy. AB - The physical, chemical, and biological properties of pulsed laser deposited (PLD) and plasma sputtered (PS) hydroxyapatite (HA) coatings were compared. Human osteoblast-like cell responses to these coatings in vitro were assayed for proliferation and phenotypic expression. PS coatings formed smooth and continuous thin films that followed the contours of the substrate surface. PLD coatings consisted of numerous spheroidal micro- and macroparticles. The crystallinity of all coatings was quantified by comparison with the HA target used for both the PS and PLD processes. The XRD and FTIR results indicated that unannealed PLD coatings deposited at room temperature had X-ray spectra consistent with an amorphous structure and were found to dissolve after only a few hours in saline solution. Annealing at 400 degrees C increased the crystallinity (87-98%), which resulted in improved stability and cell activity. The PS coatings showed greater chemical stability than the unannealed PLD coatings and contained an approximate 15% crystalline phase, increasing to 65% postannealing. Cell proliferation and alkaline phosphatase production were significantly higher on unannealed PS specimens than the other coating treatments. There may be benefits in engineering the presence of a minor percentage of a microcrystalline phase in an amorphous or nanometer scale polycrystalline HA structure. PMID- 10756313 TI - Subcutaneous abscess formation around catheters induced by viable and nonviable Staphylococcus epidermidis as well as by small amounts of bacterial cell wall components. AB - The use of catheters is often complicated by infection, mainly due to Staphylococcus epidermidis. Recently, a novel poly(vinylpyrrolidone)-grafted silicone elastomer catheter (SEpvp) was introduced. Less bacteria adhered to SEpvp than to conventional SE catheters in vitro. The frequency of S. epidermidis infection associated with SEpvp and SE was assessed in a rabbit model. Unexpectedly, abscesses were induced by the injection of low numbers of S. epidermidis along subcutaneously inserted SEpvp. No abscesses were seen around SE, even when very high numbers of S. epidermidis were injected. This bioincompatibility reaction observed around the SEpvp was independent of the host, bacterial strain, and method of inoculation. Abscesses were also induced by nonviable S. epidermidis and by bacterial cell wall components. Because these incompatibility reactions were not observed in the absence of bacteria, biocompatibility testing should include experiments in which the inflammatory effects of the combination of catheter and (non)viable bacteria are tested. PMID- 10756314 TI - Surface modification of poly(lactic acid) nanospheres using hydrophobically modified dextrans as stabilizers in an o/w emulsion/evaporation technique. AB - Sterically stabilized biocompatible poly(lactic acid) (PLA) nanospheres were prepared by an o/w emulsion/evaporation technique, using hydrophobically modified dextrans (DexP) as the emulsion stabilizer. Photon correlation spectroscopy, zetametry, and differential scanning calorimetry studies corroborated that interfacial adhesion between immiscible dextran and PLA chains was achieved by compatibilization of polymer segments via hydrophobic groups grafted onto dextran and thus leading to the formation of entanglements between the hydrophobic dextran parts and the PLA matrix. The presence of dextran exposed at the particle surface was confirmed by X-ray photoelectron spectroscopy and by the fact that the suspensions showed an increased stability in concentrated NaCl solutions and a reduction of bovine serum albumin adsorption compared to uncoated PLA nanoparticles. A comparison of the characteristics of PLA nanospheres DexP-coated via the emulsion procedure (NS(em)) with those of PLA particles coated by DexP adsorption (NS(ad)) suggests that the conformation of the polymer in the superficial layers may be different. However, both DexP layers behave similarly in terms of stability and protein adsorption. PMID- 10756315 TI - A viscous bioerodible poly(ortho ester) as a new biomaterial for intraocular application. AB - The biocompatibility of a viscous, hydrophobic, bioerodible poly(ortho ester) (POE) intended for intraocular application was investigated. POE was evaluated as a blank carrier and as containing modulators of degradation. Each formulation was injected intracamerally and intravitreally in rabbit eyes, and clinical and histological examinations were performed postoperatively for 2 weeks. In the case of intracameral injections, polymer biocompatibility appeared to depend on the amount injected in the anterior chamber. When 50 microL was administered, the polymer degraded within 2 weeks, and clinical observations showed good biocompatibility of POE with no toxicity to the ocular tissues or increase in intraocular pressure. The injection of a larger volume, 100 microL, of POE, appeared inappropriate because of direct contact of polymeric material with the corneal endothelium, and triggered reversible edema and inflammation in the anterior chamber of the eye that regressed after a few days. After intravitreal administration, POE was well tolerated and no inflammatory reaction developed during the observation period. The polymer degraded slowly, appearing as a round whitish bubble in the vitreous cavity. The presence of modulators of degradation both improved POE biocompatibility and prolonged polymer lifetime in the eye. POE appears to be a promising biomaterial for clinical intraocular application. PMID- 10756316 TI - Synthesis and characterization of polypyrrole-hyaluronic acid composite biomaterials for tissue engineering applications. AB - New tissue engineering technologies will rely on biomaterials that physically support tissue growth and stimulate specific cell functions. The goal of this study was to create a biomaterial that combines inherent biological properties which can specifically trigger desired cellular responses (e.g., angiogenesis) with electrical properties which have been shown to improve the regeneration of several tissues including bone and nerve. To this end, composites of the biologically active polysaccharide hyaluronic acid (HA) and the electrically conducting polymer polypyrrole (PP) were synthesized and characterized. Electrical conductivity of the composite biomaterial (PP/HA) was measured by a four-point probe technique, scanning electron microscopy was used to characterize surface topography, X-ray photoelectron spectroscopy and reflectance infrared spectroscopy were used to evaluate surface and bulk chemistry, and an assay with biotinylated hyaluronic acid binding protein was used to determine surface HA content. PP/HA materials were also evaluated for in vitro cell compatibility and tissue response in rats. Smooth, conductive, HA-containing PP films were produced; these films retained HA on their surfaces for several days in vitro and promoted vascularization in vivo. PP/HA composite biomaterials are promising candidates for tissue engineering and wound-healing applications that may benefit from both electrical stimulation and enhanced vascularization. PMID- 10756317 TI - Two different types of nonthrombogenic surfaces: PEG suppresses platelet adhesion ATP-independently but HEMA-St block copolymer requires ATP consumption of platelets to prevent adhesion. AB - Poly(ethylene glycol) (PEG) and a hydrophobic-hydrophilic microdomain structured block copolymer comprising poly(2-hydroxyethyl methacrylate) and polystyrene (HEMA-St) have been reported to show good blood compatibility owing to inhibition of platelet activation. By using a computer-assisted novel technique to analyze platelet behavior on the surfaces, we found two different mechanisms to prevent platelet adhesion. Platelets were prevented from adhesion and spreading on the microdomain surface and retained cell movement for a long time. The platelet movement velocity was not significantly different between PEG-grafted surfaces and HEMA-St block copolymer-cast surfaces. However, platelet motion was qualitatively different. Platelets on HEMA-St block copolymer-cast surfaces moved with rolling, spinning, and vibrating, whereas platelet movement was limited to oscillatory vibration on PEG-grafted surfaces. When platelets were treated with NaN(3), an adenosine triphosphate (ATP) synthesis inhibitor, before contacting the surfaces, platelets movement velocity was decreased only on HEMA-St block copolymer-cast surfaces. Such an inhibitory effect was hardly observed with platelets on PEG-grafted surfaces. We propose two different mechanisms to prevent platelet adhesion onto surfaces. One is ATP-independent as observed with PEG, and the other is ATP-dependent for HEMA-St block copolymer, where platelets consume ATP to prevent adhesion. PMID- 10756318 TI - Cytoplasmic delivery of a macromolecular fluorescent probe by poly(d, l-lactic-co glycolic acid) microspheres. AB - A macromolecular fluorescent probe encapsulated in poly(d, l-lactic-co-glycolic acid) (PLGA) microspheres was used as a model for studying cytoplasmic delivery of antigens. We hypothesized that Texas red dextran loaded in PLGA microspheres would be delivered to the cytoplasm and that cytoplasmic delivery would be affected by polymer molecular weight. Cellular localization of the Texas red dextran was investigated at two different molecular weights of PLGA: 6000 and 60,000 g/mol. Intracellular degradation and processing of Texas red dextran loaded PLGA microspheres by mouse peritoneal macrophages was monitored both in vitro and in vivo for a 7-day period using confocal laser scanning microscopy (CLSM). The results revealed cytoplasmic delivery of the fluorescent probe at both molecular weights of PLGA. Furthermore, the CLSM images showed that both in vitro and in vivo, the kinetics of microsphere degradation and cytoplasmic delivery were more rapid for the 6000 g/mol PLGA microspheres than the 60,000 g/mol PLGA microspheres. Hence, this study provides physical evidence that PLGA microspheres are capable of cytoplasmic delivery and that delivery to the cytosol can be controlled by modifying formulation parameters such as polymer molecular weight. PMID- 10756320 TI - Initial stability of a new hybrid fixation hip stem: experimental measurement of implant-bone micromotion under torsional load in comparison with cemented and cementless stems. AB - A new hybrid fixation stem, named cemented-locked uncemented (CLU), for total hip arthroplasty was developed to achieve good initial stability. Primary stability is guaranteed by the cement which is injected into two pockets in the lateral area. This leaves a large surface available for long-term biologic fixation (direct bone attachment on implant). This study evaluates in vitro the initial stability of the CLU prototype under torsional load, in comparison with cemented and cementless stems. The results show that the CLU stem is very stable in simulated stair climbing. Its micromotions are comparable to those of a cemented prosthesis, and significantly less (80-90% lower) than those for a cementless stem. These findings confirm the optimal initial stability expected from the CLU prototype. This new design, which employs hybrid fixation, should improve bone formation on the implant and reduce the risk of stem loosening. PMID- 10756319 TI - An investigation of fibroblast mitochondria enzyme activity and respiration in response to metallic ions released from dental alloys. AB - Most cellular functions evaluated for biocompatibility are high-energy processes such as proliferation and therefore are not usually affected before a decrease in energy production is observed. Several studies have shown that metabolic functions are altered at much lower concentrations than several normally used biocompatibility tests such as viability. Therefore, the purpose of this study was to provide an in-depth evaluation of metallic ion effects on mitochondria function and thereby biocompatibility. These studies evaluated the mitochondrial function of human gingival fibroblasts exposed to the salt solutions of ions released from nickel-based dental alloys, particularly beryllium (Be(2+)), chromium (Cr(6+) and Cr(3+)), nickel (Ni(2+)), and molybdenum (Mo(6+)). Mitochondrial function was examined by NADH:CoQ reductase activity, succinate dehydrogenase activity, and oxygen consumption. PMID- 10756321 TI - Examination of the physical state of chlorhexidine within viscoelastic, bioadhesive semisolids using raman spectroscopy. AB - This study examined the effects of polymeric components on the physical state of chlorhexidine within bioadhesive, semisolid formulations using Raman spectroscopy. Semisolid formulations were prepared in which chlorhexidine base (CHX, 5%w/w, particle size <63 microm) was dispersed in aqueous (phosphate buffered saline, pH 6.8) polymer matrices consisting of one or more polymeric components, namely HEC (3%w/w), PVP (3%), and PC (PC, 3%). Raman spectra were recorded using 785-nm excitation and were typically accumulated for 360 s. The Raman spectra were dominated by the presence of CHX. The spectra of CHX in HEC and in HEC/PVP gels were indistinguishable from that for solid CHX as a result of the insolubility of CHX in these formulations. However, in systems containing PC and CHX, there was a shift in the strongest band from 1564 cm(-1) to 1608 cm(-1), which may be accredited to protonation of the basic CHX by the numerous carboxylic acidic groups on PC. Identical shifts in the band positions were observed when this protonation was modeled using ethanoic acid, supporting the view that there was a simple acid base reaction between PC and CHX. However, there were notable differences in the relative intensities of the peaks from these samples, with the spectrum of CHX in the PC matrix displaying properties intermediate between those of CHX dissolved in ethanoic acid and solid CHX diacetate. This may be accredited to the limited solubility of the CHX-PC ion pair. In matrices containing HEC and PC, no peak was observed at 1564 cm(-1), whereas the intensity of the peak at 1608 cm(-1) was increased. Therefore, in these formulations CHX was completely converted to the di-cation as a result of the synergistic effects of PC (which protonated CHX) and HEC (which solubilized the di-cation). In the absence of either HEC or PC, complete protonation was not achieved. It is suggested that this enhancement of solubility of H(2)CHX(2+) may be due to hydrogen bonding, given the hydroxylated nature of HEC. In conclusion, this study has shown the applicability of Raman spectroscopy for both the analysis of opaque, semisolid formulations and, additionally, for the examination of the state of therapeutic agents within such matrices. In particular, using Raman spectroscopy, it was uniquely possible to identify the roles of various polymeric components on both the ionization and solubilization of CHX within aqueous semisolid systems. PMID- 10756322 TI - Vitreal elimination kinetics of large molecular weight FITC-labeled dextrans in albino rabbits using a novel microsampling technique. AB - A novel sampling technique that allowed for continuous vitreal sampling of high molecular weight compounds was developed. This technique generated consistent and reproducible results. Fluorescein isothiocyanate-linked dextrans (FITC-dextrans) with average molecular weights of 4.4, 9.3, and 38.9 kD were selected for the study. A 100 microgram dose was administered into the vitreous by a short-term infusion (100 microL) over a period of 45 s, and sampling was carried out for 10 h. The vitreal elimination of these dextrans was found to follow apparent first order elimination kinetics, having half-lives of 246 min, 275 min, and 484 min, respectively. Aqueous levels were also determined at the end of 10 h and were correlated with vitreal dextran concentrations. The FITC-dextrans displayed an initial equilibration phase of about 200 min followed by linear first-order elimination. Apparent diffusion coefficients in the vitreous have been calculated to be 7.56 x 10(-6) and 6.18 x 10(-6) cm(2)/s for 4.4 and 9.3 kD dextrans, respectively. Furthermore, it became evident that with progressively higher molecular weight FITC-dextrans the vitreal elimination rate constant gradually decreased. The elimination rate constant was found to be inversely related to the logarithm of molecular weight with a correlation coefficient of 0.983. Results obtained suggest an elimination mechanism primarily involving the transretinal route possibly with some involvement of the aqueous pathway. PMID- 10756323 TI - Determination of the dosage of recombinant hirudin to inhibit arterial thrombosis in baboons. AB - Recombinant hirudin, a potent and direct inhibitor of thrombin, effectively inhibits platelet-dependent thrombosis. Our aim was to establish the plasma concentration at which r-hirudin expresses its optimal antithrombotic effect. We measured the extent of inhibition of (111)In-labeled platelet deposition onto 0.6 cm(2) segments of Dacron vascular grafts. These grafts were incorporated as extension segments into exteriorized permanent femoral arteriovenous shunts in baboons. In six control studies a mean of 1.99 +/- 0.26 x 10(9) platelets were deposited at the end of 120 min. In the treatment studies, a thrombus was allowed to form for 10 min in six animals. Treatment for 30 min with r-hirudin at dosages of 140, 70, and 35 microgram/kg/min, but not 14 microgram/kg/min, dose dependently interrupted platelet deposition. The relationship between the percent inhibition of platelet deposition caused by r-hirudin and the plasma concentration of hirudin was exponential (i.e., % Inhibition = 95(1-e(0.23 x [r hirudin])) (R(2) = 0.76). From this, we estimated that 50% inhibition of platelet deposition will occur at a plasma concentration of approximately 3.3 microgram r hirudin/mL and 80% at 8.1 microgram/mL. The relationship between the inhibition of platelet deposition and the plasma concentration of hirudin makes it possible to estimate the dose of hirudin that will result in a given level of inhibition of platelet deposition. PMID- 10756324 TI - Neonatal hepatic propranolol elimination: studies in the isolated perfused neonatal sheep liver. AB - Using the isolated perfused neonatal sheep liver model, we examined the disposition of propranolol (n = 8, age 0.25-10 days) and compared our findings with our previous study from the perfused near-term fetal sheep liver (Ring JA, et al. 1995. Drug Metab Dispos 23:190-196). Within 45 min of dosage, perfusate propranolol levels had fallen by three orders of magnitude to be less than the limit of detection. Perfusate disappearance curves were monoexponential in six experiments and biexponential in two experiments. The mean shunt-corrected hepatic extraction ratio was 0.92 +/- 0.09, much greater than that seen in the fetal sheep liver (0.26 +/- 0.13, P < 0.0001) but still less than values in the adult sheep (0.97). At the conclusion of the perfusion, 4-hydroxypropranolol was the major metabolite present and 5-hydroxypropranolol and N desisopropylpropranolol were minor metabolites. We conclude that the isolated perfused neonatal sheep liver is a useful model with which to study the maturation of neonatal hepatic drug oxidation. Our study shows that propranolol is rapidly eliminated by the neonatal liver to form several metabolites at rates far greater than in the fetal liver, but rates of elimination have not yet reached that reported in the adult sheep liver. PMID- 10756325 TI - Synthesis and in vitro/in vivo evaluation of 5-aminosalicyl-glycine as a colon specific prodrug of 5-aminosalicylic acid. AB - A simple synthetic route for the preparation of amino acid conjugate of 5 aminosalicylic acid (5-ASA) was exploited and prepared 5-aminosalicyl-glycine (5 ASA-Gly) in good yield. In vitro and in vivo properties of 5-ASA-Gly as a colon specific prodrug of 5-ASA were investigated using rats as the test animal. Incubation of 5-ASA-Gly with cecal or colonic contents at 37 degrees C released 5 ASA in 65 or 27% of the dose in 8 h, respectively. No 5-ASA was detected from the incubation of 5-ASA-Gly with the homogenates of stomach or small intestine. Plasma concentration of 5-ASA-Gly decreased rapidly after intravenous administration of 5-ASA-Gly, and no 5-ASA was detected in the blood, which indicated 5-ASA-Gly was not degraded in the plasma. After oral administration of 5-ASA-Gly, about 50% of the administered dose was recovered as 5-ASA and N-acetyl ASA and 3% as 5-ASA-Gly from feces and 14% as 5-ASA-Gly and 28% as 5-ASA and N acetyl-ASA from urine in 24 h. These results suggested that a large fraction of 5 ASA-Gly was delivered to the large intestine and activated to liberate 5-ASA. For comparison, total recovery of 5-ASA and N-acetyl-5-ASA from feces after oral administration of 5-ASA-Gly was greater than that from sulfasalazine, which is one of the most commonly prescribed prodrugs of 5-ASA. PMID- 10756326 TI - Use of nonlinear mixed effect modeling for the meta-analysis of preclinical pharmacokinetic data: application to S 20342 in the rat. AB - The standard two-stage analysis of separate preclinical pharmacokinetic (PK) and toxicokinetic (TK) studies may lead to good information on the bioavailability in the rat at a low (pharmacologic) dose but only an idea on the dose/exposure relationship, on gender, and on time effect. In view of these drawbacks, we decided therefore to explore the usefulness of the implementation of a meta analysis in preclinical studies in a given species (the rat in this case) taking as an example S 20342, an investigational new drug with potential antipsychotic properties. A nonlinear mixed-effect PK model was built from all intravenous (IV) and oral (PO) data collected until the completion of the 4-week toxicity study. The database included data from 201 Wistar rats (161 males and 40 females). Forty animals received the drug IV and 161 PO. The treatment duration ranged from 1 day to 4 weeks. IV doses were 3, 5, and 20 mg/kg, and 11 different oral doses were tested in the range of 5 to 200 mg/kg. Three different salts were administered PO: hydrochloride, sulfate, and mesylate. The modeling was performed with NONMEM IV. The best pharmacokinetic model was a two-compartment model with simultaneous first-order and zero-order absorption. The combination of these two input functions allowed the model to fit the peak plasma concentrations observed in the first hour (first order), especially after oral administration of low doses, and to take into account the prolonged absorption phase when the dose increased (zero order). A significant gender effect was found on CL. In addition, significant positive correlations were found between weight and CL, weight and Vc, and dose and the dose fraction after a zero-order absorption. No covariate significantly influenced the other parameters. In conclusion, the meta-analysis of preclinical data allowed for an objective assessment of statistically significant effects throughout the model-building process, leading to a better knowledge (and thus a better understanding) of preclinical PK in the rat. Moreover, the model obtained could be used to interpret further preclinical specific studies involving a sparse sampling design (e.g., further TK studies and PK/PD studies). Although this meta-analysis is more complicated than the noncompartmental approach and requires a case-by-case effort, it could be very useful to integrate this approach in the preclinical development process. PMID- 10756327 TI - Poly(D,L-lactide) nanocapsules prepared by a solvent displacement process: influence of the composition on physicochemical and structural properties. AB - Nanocapsules (NC) were prepared by interfacial deposition of preformed biodegradable polymer (PLA(50)) after a solvent displacement process. The influence of the composition used for the preparation of NC was evaluated in terms of particle size, polydispersity, zeta potential, homogeneity, and structural characteristics of the systems. The nature of the oil phase, polymer molecular weight, type and concentration of different surfactants were investigated to optimize the formulation to obtain NC suitable for intravenous administration. The influence of the physicochemical properties of the different oils used in NC preparation on the NC size was evaluated. The interfacial tension between the oil and water phases seems to have a greater effect on NC size than the oil viscosity. Miglyol 810 and ethyl oleate lead to the formation of smaller NC, probably because of the reduced interfacial tension. The polymer molecular weight plays only a small role in NC surface charge in the presence of lecithin, whereas NC surface charge, size, polydispersity, and short-term stability were highly influenced by lecithin purity. It appears that the absence of poloxamer 188 leads to smaller polydispersity, less contamination with nanospheres, and reduced formation of structures other than NC. Furthermore, electron microscopy and density gradient density techniques were used to examine the structure of the particles formed and their homogeneity. NC formation was evidenced by the bands with intermediate density between nanoemulsion and nanospheres; however, other bands of low intensity were observed. The presence of liposomes and multilayers in NC preparation was confirmed by electron microscopy. The percentage of carboxyfluorescein entrapped in different NC formulations allowed us to estimate the contamination by liposomes. It has been show that, under our experimental conditions, an excess of lecithin is an essential prerequisite for a stable preparation of PLA NC. PMID- 10756328 TI - Cationic phosphonolipids as nonviral vectors: in vitro and in vivo applications. AB - Since the development of the concept of gene therapy using cationic lipids as nonviral vectors by Felgner's group in 1987, numerous molecules have been synthesized. Such vectors were first proposed to avoid viral vector-induced drawbacks. But, it quickly became clear that a thorough knowledge of their physical and chemical characteristics was fundamental to use them under optima conditions. Over the last years our laboratory has developed a family of cationic lipids called phosphonolipids whose structure is based on that of natural phosphonolipids; compared with other vectors, these compounds had to be well tolerated by biologic membranes. Some of our synthesized molecules exhibited an interesting potential for gene transfer, both in vitro and in vivo. Structural changes in the different parts (hydrophobic, hydrophilic, and intermediary domains) of these vectors were evaluated in vitro on different cell-lines; these studies led us to select some of these molecules to carry out in vivo tests. So, the plasmid/phosphonolipid complexes were first administered to mice by intratracheal and aerosol routes with a beta-galactosidase plasmid as reporter gene. In a second set of experiments, we explored the possibilities offered by intravenous injection; in these studies, we used a luciferase plasmid as reporter gene because of its high sensibility. These experiments revealed a transgene expression essentially localized in the lungs. In a further study, we compared systemic administration with local ones; we, then, observed that the optimum formulation of a given molecule depended on its route of administration. PMID- 10756329 TI - Phosphonocationic lipids in protein delivery to mice lungs. AB - Cationic liposomes constitute one of the main approaches currently investigated to introduce a gene with therapeutic properties into a cell. Another alternative consists in directly introducing the normal protein of concern to, for example, restore the deleted function. We report here on in vitro and in vivo results obtained with GLB73, one of the phosphonolipids investigated as gene transfer agents. In previous studies this cationic lipid had shown its DNA-transfer efficacy in vitro and in vivo. We also confirmed the feasibility of protein/cationic lipid delivery in epithelial cells of mice lungs after intratracheal administration by use of a reporter gene (beta-galactosidase). Two quantitative tests (i.e., a chemiluminescent assay and a flow cytometry assay) were used to determine the amount of beta-galactosidase found in the lungs and the percentage of transfected cells. They showed that 50% of the cells of mice lungs were still positive at day 4 after protein/GLB73 delivery. Immunohistochemistry and electron microscopy studies allowed us to determine the spatial distribution and visualize the penetration of our complex into the lungs. PMID- 10756330 TI - Protection of bovine serum albumin from aggregation by Tween 80. AB - In an attempt to explain the mechanism of protein stabilization conferred by detergents, we investigated the effect of Tween 80 on aggregation of bovine serum albumin (BSA) using circular dichroism (CD) and native gel electrophoresis. CD thermal scans showed that BSA denatures at about 54 degrees C in 20 mM Tris, pH 7.2, forming soluble aggregates. Because of this aggregation, thermal unfolding of BSA under these conditions was only partially reversible, as indicated by reduced signal changes in the second scan. On the basis of this observation, BSA was thermally stressed by incubating at 50, 60, or 70 degrees C for 15 min and then analyzed by native gel electrophoresis. There was no change at 50 degrees C before and after heating, whereas at 60 degrees C the intensity of the original monomer and dimer bands decreased and that of aggregate bands increased, with much greater changes at 70 degrees C. Addition of Tween 80 before heating reduced aggregation and increased the monomer content. These effects of Tween 80 were greater as its concentration was increased from 0.001 to 1%. There was no correlation between the protective effects and the critical micelle concentration (CMC) of Tween 80. Addition of Tween 80 after 15 min incubation at 70 degrees C, or after 70 degrees C heating followed by cooling to room temperature, had no effect, demonstrating that Tween 80 must be present during the 70 degrees C heating step to be protective. Native gel electrophoresis run at 60 degrees C showed multiple aggregate bands and new bands migrating around the dimer and monomer positions, which may correspond to precursors of aggregates. Tween 80 reduced formation of these new bands and aggregates, further demonstrating that it must be present during heating. Finally, CD thermal scans showed that 0.1% Tween 80 only slightly increased the apparent melting temperature. The observed stabilization of BSA against heat treatment is, therefore, due to Tween 80 altering aggregation behavior rather than inducing significant stabilization of the native state. PMID- 10756331 TI - Use of poly(ethylene glycol)-lipid conjugates to regulate the surface attributes and transfection activity of lipid-DNA particles. AB - We evaluated the use of poly(ethylene glycol) (PEG)-modified lipids to control the surface properties of a lipid-based gene transfer system. The lipid-DNA particles (LDPs) used form spontaneously when plasmid DNA is added to mixed detergent lipid micelles consisting of the non-ionic detergent n-octyl-D glucopyranoside, the cationic lipid dioleyldimethylammonium chloride (DODAC), the zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and selected PEG-modified phosphatidylethanolamines. The inclusion of DODAC is required to form the hydrophobic lipid-DNA complex. DOPE is included to facilitate dissociation of DNA from the cationic lipid and the PEG-modified lipids are added in an effort to stabilize the surface attributes of the resulting lipid-DNA particles. We used PEG-lipids that varied in acyl chain composition because of recent results demonstrating acyl chain dependent transfer of PEG-lipids from lipid vesicles, providing the potential to allow a transformation of the surface properties due to loss of surface grafted PEG. The addition of PEG-modified lipids does not interfere in LDP formation and its presence favors formation of smaller particles (75 nm in contrast to 130 nm in the absence of the PEG-modified lipid). PEG-lipid incorporation causes a concentration dependent reduction in LDP-mediated transfection of B16/BL6 melanoma cells, a result that can be partially attributed to a reduction in particle binding to cells. However, significant LDP binding to B16/BL6 cells was still observed under conditions where LDP transfection activity was reduced by more than 85%. The potential for PEG to interfere with LDP processing following cell binding is discussed. PMID- 10756332 TI - A novel heparin/protamine-based pro-drug type delivery system for protease drugs. AB - Previously we proposed a heparin/protamine-based system for delivery of protease drugs such as tissue-specific plasminogen activator (tPA). To demonstrate the feasibility of this approach as well as its pro-drug and triggered release features, positively charged peptides [(Arg)(7)Cys] were successfully linked to tissue-specific plasminogen activator (tPA) using the crosslinking agent N succinimidyl-3-(2-pyridyldithio)- propionate. This cation-modified tPA showed much stronger heparin affinity than the parent tPA. The complex formed by mtPA and heparin was stable in human plasma, and the activity of mtPA in such a complex was inhibited by the appended heparin. Similarly, the activity of mtPA could also be inhibited by a heparin-antifibrin IgG conjugate in which heparin was linked, via endpoint attachment, to the sugar moieties in the F(c) region of anti-fibrin IgG. Aside from this pro-drug feature exhibited by the binding of the macromolecule heparin to mtPA, results from chromogenic and in vitro clot lysis assay demonstrated that the heparin-induced inhibition of the mtPA activity could be easily reversed by the addition of an adequate amount of protamine. These findings suggest the applicability of the heparin/protamine delivery system to abort the potential bleeding risks associated with clinical use of tPA. In addition to the chemical conjugation method, modified tPA could also be produced by the recombinant DNA method. The expressed modified tPA (EmtPA) thus prepared retained the full catalytic activity of the parent tPA, and this activity could also be inhibited by heparin, and the heparin-induced inhibition could be reversed following the addition of protamine. PMID- 10756333 TI - Targeted transfer of polyethylenimine-avidin-DNA bioconjugates to hematopoietic cells using biotinylated monoclonal antibodies. AB - Here we examine whether attachment of biotinylated antibodies to proteins on the cell surface increases the transfection efficiency of polyethylenimine-avidin-DNA bioconjugate gene transfer. Preliminary experiments were performed to compare avidin endocytosis into cells incubated with biotinylated antibodies. Antibody biotinylation resulted in the endocytosis of avidin-FITC into nearly 100% of cells compared with no detectable binding or entry into unbiotinylated cells. Gene transfer was accomplished with avidin conjugated to polyethylenimine (PEI) at a molar ratio of 4:1 (PA4). Plasmid DNA encoding the green fluorescent protein (GFP) gene was condensed on the PA4, and transfection efficiencies were measured by flow cytometry as the percentage of cells that fluoresced at levels greater than two standard deviations above the negative control. Gene transfer efficiencies were compared among K562, HEL, and Jurkat leukemia cell lines. Control transfections with DNA alone or untargeted PEI-DNA resulted in qter) with no visible translocation. By using microsatellite markers, the deletion breakpoint was mapped to a 350-kb region between D13S274 and D13S1311 and was paternal in origin. An analysis of 13q deletions with NTDs, including the present case, suggests that a deletion in 13q33-34 is sufficient to cause an NTD. The deletions associated with NTDs are distal to and nonoverlapping with the previously defined critical region in 13q32 for the major malformation syndrome [Brown et al., 1999: Am J Hum Genet 57: 859 866]. Our analysis also suggests that one or more genes in 13q33-34 produces NTDs by haploinsufficiency. PMID- 10756349 TI - Sibs with anencephaly, anophthalmia, clefts, omphalocele, and polydactyly: hydrolethalus or acrocallosal syndrome? AB - Major characteristics of the acrocallosal syndrome include severe mental retardation, agenesis or hypoplasia of the corpus callosum, and polydactyly of fingers and toes. In the past few years, anencephaly has also been noted, together with other midline defects. We report on a nonconsanguineous, Norwegian couple with a history of two pregnancies with a male and a female fetus, respectively, with anencephaly, median cleft lip and palate, omphalocele, and preaxial polydactyly, suggesting the diagnosis of the acrocallosal syndrome. Both fetuses also lacked eyes and nose, a finding not previously reported in the acrocallosal syndrome. Microphthalmia has been reported in the hydrolethalus syndrome, which may be caused by mutations in the same gene as the acrocallosal syndrome. The present report adds support to the hypothesis that the acrocallosal and hydrolethalus syndromes may be allelic conditions. The family history is consistent with autosomal recessive inheritance. PMID- 10756350 TI - Agnathia-holoprosencephaly-situs inversus. PMID- 10756351 TI - Silver-Russell syndrome and cystic fibrosis associated with maternal uniparental disomy 7. PMID- 10756352 TI - Complex chromosomal translocation in a patient with Kallmann syndrome. PMID- 10756353 TI - Human homologue of the murine bare patches/striated gene is not mutated in incontinentia pigmenti type 2. PMID- 10756354 TI - Thrombotic thrombocytopenic purpura: now is the time for clinical trials. PMID- 10756355 TI - HFE, iron homeostasis and genetic hemochromatosis. PMID- 10756356 TI - Cell surface expression of HFE protein in epithelial cells, macrophages, and monocytes. AB - BACKGROUND AND OBJECTIVE: Most patients with hereditary hemochromatosis are homozygous for a Cys282AETyr mutation in the HFE gene. This mutation has been shown to impair the association of the HFE gene product with b(2)-microglobulin and to prevent its cell surface presentation in transfected COS-7 and 293 cells. This study was performed to examine the expression of HFE protein in epithelial cells, macrophages, and circulating leukocytes obtained from normal subjects and patients with hereditary hemochromatosis. DESIGN AND METHODS: Antisera against two different peptides of the HFE protein were used to immunostain tissue sections and isolate granulocytes, lymphocytes and monocytes. RESULTS: Immunocytochemical staining showed that the HFE protein is expressed in gastric epithelial cells, tissue macrophages, and circulating monocytes and granulocytes. The cell surface associated signal, which was seen in normal gastric epithelial cells, monocytes and macrophages, was also present in C282Y mutant cells from patients with hereditary hemochromatosis, although at apparently reduced amounts in these cells. INTERPRETATION AND CONCLUSIONS: From these studies, it is clear that the C282Y mutation reduces but does not completely prevent presentation of the HFE protein on the cell surface of human monocytes, tissue macrophages, and gastric epithelial cells. PMID- 10756357 TI - Immunohistochemistry of HFE in the duodenum of C282Y homozygotes with antisera for recombinant HFE protein. AB - BACKGROUND AND OBJECTIVE: HFE is a class-I MHC related protein which carries the C282Y mutation in most patients with hereditary hemochromatosis, an iron overload disease. HFE protein is expected to have a relevant role in the regulation of duodenal iron absorption, and HFE protein was immunohistochemically identified in the crypt cells. The aim of the work was to analyze whether the C282Y mutation affects HFE accumulation in the duodenum. DESIGN AND METHODS: We developed antisera for the extracellular portion of recombinant human HFE protein expressed in E. coli. The antisera were specific for HFE protein and the C282Y mutant in immunoblotting, immunoprecipitation and immunocytochemistry experiments of transfected cells, and they did not cross react with HLA antigens in various analyses. The antisera gave positive results in the staining of paraffin-fixed sections of duodenal slices of subjects with hemochromatosis. RESULTS: The antisera stained evident supranuclear granules in all enterocytes of 7 C282Y homozygous subjects, and a dark area in the same region in 3 other C282Y homozygotes. Granular bodies were absent from the duodenal sections of 8 C282Y negative subjects, from 2 C282Y heterozygotes and 3 C282Y homozygotes, with or without hemochromatosis. INTERPRETATION AND CONCLUSIONS: The detection of HFE protein in granular bodies in the enterocytes of the large majority (77%) of C282Y homozygotes and not in other subjects suggests that the mutation facilitates protein accumulation in the duodenum. PMID- 10756358 TI - Functional differences between dendritic cells derived from CD34+ bone marrow and peripheral blood stem cells. AB - BACKGROUND AND OBJECTIVE: It has been previously demonstrated that dendritic cells (DCs) are characterized by an immature stage with high antigen internalization capacity, followed by a mature stage with predominantly immunostimulatory ability. The shift from the immature to the mature state can be induced in vitro by the addition of tumor necrosis factor-a (TNFa). The aim of our study was to investigate the maturation steps of DCs obtained from CD34(+) cells from peripheral blood stem cells (PBSC) and bone marrow (BM). DESIGN AND METHODS: DCs were generated in vitro from PBSC and BM CD34(+) selected cells. The endocytic activity of the cells was measured by means of dextran-FITC uptake and alloreactivity evaluated with mixed leukocyte reactions. Immunophenotypic analysis was performed by flow cytometry. RESULTS: We observed that DCs from PBSC, in contrast to the BM derived DCs, were never able to take up soluble antigens. Mixed leukocyte reactions (MLR) performed both on PBSC and BM CD34(+) derived DCs showed an allo-stimulatory activity comparable to normal controls at day 10, but significantly higher at day 14 after the addition of TNFa. Immunophenotypic analysis showed typical dendritic markers in all the samples and, after treatment with TNFa, enhanced expression of co-stimulatory molecules. INTERPRETATION AND CONCLUSIONS: Our data seem to indicate that, in our culture conditions, BM-derived DCs could be efficiently used for pulsing with specific peptides, while PBSC-derived DCs, being functionally mature, should be more suitable for gene therapy. PMID- 10756359 TI - Peripheral blood neutrophils from hepatitis C virus-infected patients are replication sites of the virus. AB - BACKGROUND AND OBJECTIVE: Hepatitis C virus (HCV) is able to cause not only acute and chronic liver disease, but also immunologic and hematologic disorders. In order to clarify the extra-hepatic tropism of HCV, and to understand the pathogenetic mechanisms of HCV infection, we evaluated viral replication in peripheral blood mononuclear cells. DESIGN AND METHODS: The presence of genomic and antigenomic (replicative) forms of HCV in B- and T-lymphocytes, monocytes, and polymorphonuclear leukocytes (PML) was determined by reverse transcriptase polymerase chain reaction in 54 HCV-RNA positive patients and, as control groups, in 10 patients who had recovered from HCV infection without evidence of serum HCV RNA, and in 10 HCV-negative subjects. RESULTS: In HCV-RNA positive patients, the genomic RNA was found in 94% of B-cells, in 14% of T-cells, in 40% of monocytes and in 77% of PML, while only 1 of the HCV-RNA negative subjects showed positivity in B-cells. The anti-genomic form of HCV-RNA was found in 52% of B cells, in 3% of monocytes, and in 31% of PML. By contrast, it was never detected in T-cells and in HCV-RNA negative subjects. Neither genomic nor anti-genomic forms were found in HCV-negative cases. INTERPRETATION AND CONCLUSIONS: These data suggest that PML are replication sites of HCV. Whether the infection occurs at the level of the stem cells or subsequently during myeloid cell differentiation is, as yet, unknown. The absence of correlation between the presence of replicative forms and any clinical and/or laboratory data opens the question of the role of HCV replication in extra-hepatic sites. PMID- 10756360 TI - AML1 gene amplification: a novel finding in childhood acute lymphoblastic leukemia. AB - BACKGROUND AND OBJECTIVE: We previously found a high-level amplification in chromosomal region 21q22 in two children with acute lymphoblastic leukemia (ALL) using comparative genomic hybridization. The same region harbors the AML1 gene. The aim of the present study was to investigate whether AML1 is a target gene in these amplifications. DESIGN AND METHODS: Bone marrow samples were obtained from 112 childhood ALL patients. The copy number of AML1 was studied using fluorescent in situ hybridization with a dual color DNA probe specific for the AML1 and TEL genes. RESULTS: Three of the patients had 3-to-8 fold amplification of AML1 and showed a high-level amplification of 21q22 by comparative genomic hybridization. In two of them the extra copies were shown to be located tandemly in a derivative of chromosome 21. Thirty-seven of the patients (33%) had 1-to-2 extra copies of AML1, most probably reflecting the incidence of trisomy 21 and tetrasomy 21. The TEL-AML1 fusion was less frequent in the patients with extra copies of AML1 (7/40; 18%) than in the patients with no extra copy (24/72; 33%). None of the three patients with 3-to-8 fold amplification of AML1 showed the fusion or loss of TEL. INTERPRETATION AND CONCLUSIONS: Our findings suggest that the AML1 gene is a target gene in the 21q22 amplicon in childhood ALL. To understand the role, if any, of the AML1 amplification in leukemogenesis, further studies are needed. PMID- 10756361 TI - Amifostine in the treatment of low-risk myelodysplastic syndromes. AB - BACKGROUND AND OBJECTIVE: The phosphorylated aminothiol agent amifostine (Ethyol) protects bone marrow and other tissues from toxicity due to ionizing radiation and antineoplastic drugs, and stimulates progenitors from normal and myelodysplastic bone marrow. Contrasting results have been published so far on the effectiveness of amifostine in correcting cytopenia in patients with myelodysplastic syndromes (MDS). DESIGN AND METHODS: In a pilot phase II study we treated 26 patients with low risk MDS (13 RA, 2 RARS, 2 CMML, 9 RAEB with blasts < 10%) with amifostine (200 mg/m(2 )x 3/week for 4 weeks). RESULTS: Hemoglobin concentration, reticulocyte, neutrophil and platelet counts increased respectively in 6 (23%), 11 (42%), 13 (50%) and 9 (34%) of patients. Red cell transfusions were reduced (> 50%) in 4/26 patients and abolished in 1/26. Unexpectedly a significant decrease in soluble transferrin receptor level at week 4 of therapy, compared to the basal level (p<0.04), was observed in the whole population of patients. INTERPRETATION AND CONCLUSIONS: Amifostine can ameliorate cytopenia in some patients with MDS, with few and mild side effects. Neutropenia is more likely to be corrected than anemia or thrombocytopenia. Mechanisms underlying this biological effect remain to be clarified. PMID- 10756362 TI - The role of surgery in the treatment of gastrointestinal lymphomas other than low grade MALT lymphomas. AB - BACKGROUND AND OBJECTIVE: A bias in clinical investigations on gastrointestinal lymphomas is the lack of testing the intention to treat as to resection, emergency conditions at presentation and selection brought about by the evaluation of feasibility of surgery. DESIGN AND METHODS: A prospective study involved 154 patients with gastrointestinal nodular or high-grade MALT lymphomas, 111 with a gastric and 43 with an intestinal presentation. The decision to resect or treat conservatively was left to clinicians, on condition that it was previously defined for each patient. RESULTS: Failure-free survival was significantly higher in the 106 resected patients than in the 48 unresected ones but did not differ according to either primary intention to treat or emergency surgery/elective treatment. Survival was similar in patients operated on by choice and in those because of an emergency. Intentionally unresected patients had a significantly better survival than those not undergoing surgery despite the initial intention, for a number of clinical reasons. Patients with gastric lymphoma survived longer than those with intestinal disease and prognostic factors were analyzed separately in the two groups. The best predictors of prognosis were performance status and serum lactic dehydrogenase level in gastric lymphomas, resection alone in intestinal ones. INTERPRETATION AND CONCLUSIONS: The prognosis of gastric lymphomas depends on lymphoma-related factors and not on surgical treatment. The prognosis of intestinal ones is exclusively related to surgery. These data support the appropriateness of different clinical approaches to gastric and intestinal lymphomas. PMID- 10756363 TI - Platelet function during cardiopulmonary bypass not changed by two different doses of aprotinin. AB - BACKGROUND AND OBJECTIVE: Bleeding is one of the major complications of cardiopulmonary bypass (CBP) during cardiac surgery. A platelet function defect seems to be the main cause of the hemostatic problems associated with CBP. Controversial results have been reported concerning the possible protective mechanism of action of aprotinin on platelets. DESIGN AND METHODS: In this study we investigated the effect of two different dosages of aprotinin (high and pump prime-only dose) on platelet reactivity in vitro and adhesion, activation and aggregation receptors on the platelet surface. RESULTS: The results obtained from 53 patients undergoing CBP showed a significantly deficient platelet aggregation in response to agonist in all groups without differences between aprotinin treated or not treated patients. No changes in platelet surface expression of glycoprotein (GP) IIb-IIIa, GPIb, GPIV and P-selectin, were observed during CBP between patients treated with aprotinin or not. INTERPRETATION AND CONCLUSIONS: These data suggest that inadequate platelet function induced by CBP is not a defect intrinsic to the platelet. We conclude that the hemostatic effect of aprotinin, regardless of the dose employed, is not mediated by protection of platelet function. PMID- 10756364 TI - Factor V leiden increases plasma F1+2 levels both in normal and deep venous thrombosis subjects. AB - BACKGROUND AND OBJECTIVE: A simple approach to understanding molecular mechanisms leading to thrombosis is the definition of how genetic factors influence biochemical parameters of coagulation. Conflicting data have been reported regarding the role that the genotype of factor V plays in the control of plasma F1+2 levels. The aim of this study was to test whether the factor V Leiden mutation affects F1+2 levels. DESIGN AND METHODS: We studied the effect of factor V Leiden mutation (detected by the polymerase chain reaction technique) on plasma F1+2 levels in 418 normal subjects and 39 subjects affected by deep venous thrombosis. RESULTS: In both normal subjects and those with venous thrombosis, heterozygotes for the Leiden mutation showed significantly higher plasma levels of F1+2 (p<0.0001 and p<0.005, respectively). Subjects with venous thrombosis had a higher allelic frequency of the Leiden mutation than normal subjects (11.5% and 3.1%, respectively). INTERPRETATION AND CONCLUSIONS: The results indicate that the genotype of factor V is a determinant of plasma F1+2 concentration. The allelic frequency of Leiden mutation in our normal subjects is higher than that found in other Italian populations but similar to that reported for populations of north- and middle-Europe. This finding is consistent with the peculiar ancestry and history of Friuli (the area in which subjects for this study were recruited), with respect to other Italian regions. PMID- 10756365 TI - Quality analysis of blood components obtained by automated buffy-coat layer removal with a top & bottom system (Optipress (R)II). AB - BACKGROUND AND OBJECTIVE: There are Council of Europe recommendations for the quality of blood components. We analyzed the quality of blood components processed by a top & bottom system (Optipress((R)) II), the routine method used in our blood bank, to test whether the components reached the recommended quality. DESIGN AND METHODS: Blood was collected in triple CPD-SAGM bags (Optipac((R)) Baxter). Whole blood (WB) was centrifuged at 4,158 g for 14 min before separation by an automated top & bottom system (Optipress((R) )II). Platelet concentrate (PC) was prepared by pooling four isogroup buffy-coat (BC) units before low-speed centrifugation, and transferring the supernatant (4 BC-PC) to a 5-day storage bag (PL732, Baxter). An alternative approach involved PC preparation from a single BC unit by adding approximately 70 mL of plasma before centrifugation, followed by transfer of the platelet concentrate (1BC-PC) to a 300 mL Teruflex((R)) transfer bag. Both 4 BC-PC and 1 BC-PC were stored in a flat agitator at 22 degrees C for up to 5 days after collection. Cell counts were determined, along with hemoglobin and hematocrit in a Sysmex K-800 cell counter. The pH was determined on day 5 at 22 degrees C. Weights were measured and volumes were calculated based on specific gravity. Statistical analyses were carried out using the Kolmogorov-Smirnov test as a normality distribution test, the t-test for parametric values and Wilcoxon's test as a non-parametric test. Statistical significance between samples was considered to have been reached when p<0.05. RESULTS: The best parameters for configuring the system were: strength 25; BC volume 33-55; level of BC 5.5. Red blood cell (n = 1,434) volume was 279+/-20 mL, with 54.92+/-7.16 g of hemoglobin. More than 96% of units had fewer than 1.2x10(9) white blood cells. Fresh plasma volume (n = 803) averaged 279+/-19 mL, with a white blood cell contamination of fewer than 0.1x10(9)/L in all samples examined (n = 23). Platelet recovery in BC was 92+/-9% of platelets present in WB; the percentage of removed leukocytes was 74+/-10%, and between 13 and 15% of RBCs were lost in the BC (95% confidence interval). The BC volume (n = 1,037) fitted the target volume of 60 mL, except for some devices, when Optipress II((R)) lost the configuration for this parameter. Of 4 BC-PCs 80.3% yielded more than 0.6x10(11) platelets per unit, whereas this criterion was only met by 59.7% of 1 BC-PCs, and a greater proportion of 1 BC-PCs (58.8%) showed pH values within the range of 6.5-7.4 after 5 days of storage in comparison with 4 BC-PCs (44.25%). INTERPRETATION AND CONCLUSIONS: Optipress II((R)) provides standardized, leukocyte-poor blood components. Council of Europe requirements were met in a large percentage of red-cell concentrates, with less than 92 and 74% of the original platelets and leukocytes, respectively, and a small hemoglobin loss per unit. The system gave an optimal yield in terms of plasma volume. The top & bottom technique allowed us to reduce the number of blood units per platelet concentrate from 6 to 4 units, with similar platelet yields compared with traditional procedures. Nevertheless, the storage conditions must be improved to satisfy all Council of Europe requirements for platelet concentrates. PMID- 10756366 TI - Relationships between total CD34+ cells reinfused, CD34+ subsets and engraftment kinetics in breast cancer patients. AB - BACKGROUND AND OBJECTIVE: The aim of the present study was to evaluate the correlation between the number of CD34+ cells transfused and the duration of hypoplasia, and the relationship between various CD34+ subsets (CD34+/33-; CD34+/38-; CD34+/ HLA-DR-; CD34+/Thy-1+) and engraftment kinetics in a series of patients with breast cancer treated with high doses of thiotepa and melphalan. DESIGN AND METHODS: We treated 42 consecutive patients: 19 in an adjuvant context (>= 4 positive axillary nodes) and 23 for metastatic disease. A combination of thiotepa 600 mg/m(2) and melphalan 140-160 mg/m(2) was administered as the conditioning regimen. All patients received peripheral blood progenitor cells (PBPC) and growth factors for hematopoietic rescue. RESULTS: In univariate analysis, we found a significant relationship between the number of CD34+ cells reinfused and the time to hematologic recovery and the duration of hospital stay. We observed an inverse correlation between the number of CD34+ cells reinfused and the units of platelets transfused. Cox multivariate analysis confirmed that the number of CD34+ cells reinfused is the most effective predictor of time to hematologic recovery. CFU-GM resulted to be a better predictor of the duration of hospitalization. INTERPRETATION AND CONCLUSIONS: We found a significant relationship between the number of PBPC reinfused and the time to hematologic recovery after high doses of thiotepa and melphalan. In our experience, the numbers of subsets of CD34+ cells infused did not give compared additional information to that provided by the total number of CD34+ cells infused. PMID- 10756367 TI - Incidence and characterization of secondary myelodysplastic syndromes following autologous transplantation. AB - BACKGROUND AND OBJECTIVE: Secondary myelodysplastic syndromes (sMDS) and secondary acute myeloid leukemias (sAML) have been observed after conventional chemo/radiotherapy and autologous hematopoietic stem cell transplantation. The aim of the present study was to analyze Spanish experience regarding the incidence and characteristics of sMDS and sAML following autologous transplantation. DESIGN AND METHODS: We obtained information from 7 institutions which perform autologous transplantation in Spain. Data from 1,081 and 1,411 patients who had received allogeneic and autologous transplantation, respectively, were available. RESULTS: None of the allografted patients had developed a sMDS/sAML so far. Thirteen cases of sMDS/sAML following autologous transplantation were reported. The mean age of these 13 patients at the time of transplantation was 40 years (range 16-58). Five had non-Hodgkin's lymphoma, 6 had Hodgkin's disease, 1 had acute myeloblastic leukemia and 1 had multiple myeloma. The crude overall incidence of sMDS/sAML was 0.9%. The incidence did not differ according to the source of progenitor cells (1% and 0.8% for bone marrow and peripheral blood, respectively). Cytogenetic analysis showed clonal abnormalities in 11 of the 13 cases. Patients with sMDS/sAML had received more doses of alkylating agents than non-sMDS patients (p = 0.0015). The median time between transplantation and diagnosis of sMDS/sAML was 28 months (range 1.5-63). This time was significantly longer for patients who received bone marrow than for those who received peripheral blood (45 versus 18 months, p = 0.01). Median overall survival after diagnosis of sMDS/sAML was 13 months. INTERPRETATION AND CONCLUSIONS: The crude incidence of sMDS/sAML in our series was similar to other published incidences. We did not find any difference in incidence between patients who had received bone marrow or peripheral blood; however, the medi an time elapsed between transplantation and sMDS diagnosis was shorter when peripheral blood was infused. Higher doses of alkylating agents were associated with the appearance of sMDS/AML. PMID- 10756368 TI - Thrombotic thrombocytopenic purpura treatment in year 2000. AB - BACKGROUND AND OBJECTIVE: For several decades clinicians worldwide considered TTP a severe and frustrating therapeutic problem. Fortunately, however, the prognosis of TTP patients has greatly benefited from the use of plasma manipulation techniques, particularly plasma-exchange (PE), so that the overall rate of complete responses currently ranges between 70-85% and may even exceed these figures. Despite this dramatic improvement, a number of questions remain concerning the best treatment for TTP patients. Analyzing acquired data and discussing future perspectives, this review will address the following key issues: is PE really the treatment of choice for TTP and what is the role of PE with cryosupernatant? what is the role of all the drugs which are commonly combined with PE, antiplatelet drugs and steroids in particular? what, if any, is the role of cytotoxic agents, especially vincristine? is there a treatment for PE resistant patients? does secondary TTP need different treatments? DESIGN AND METHODS: The authors have been involved in the study and treatment of TTP for years; furthermore, they extensively searched the PubMed database of the National Library of Congress through the Internet. INTERPRETATION AND CONCLUSIONS: PE remains the treatment of choice for TTP. A large randomized trial now in progress will assess whether exchange with cryosupernatant plasma can improve treatment efficacy. The administration of antiplatelet drugs in combination with PE was fiercely debated over the past years but seems indicated both in acute TTP and as a prophylactic treatment to prevent relapses. It appears that steroids cannot be avoided, especially in light of the latest findings on TTP pathogenesis, but only specific trials will assess the optimal cortisone type and dose. Presently, different treatments can be suggested only to patients failing to respond to PE, while no specific therapy can be indicated for secondary TTP, which usually has a very poor prognosis. Finally, we would like to stress that only international co operative (multicenter) trials on large series of patients will be able to shed light on a still obscure, if fascinating, disease. Our hope and wish is that the new century will see TTP among the diseases defeated by man's clever mind and heart. PMID- 10756369 TI - Acute childhood idiopathic thrombocytopenic purpura: AIEOP consensus guidelines for diagnosis and treatment. Associazione Italiana di Ematologia e Oncologia Pediatrica. AB - BACKGROUND AND OBJECTIVE: A recent evaluation carried out by the Associazione Italiana di Ematologia e Oncologia Pediatrica (AIEOP) about practice management of acute childhood idiopathic thrombocytopenic purpura (ITP) revealed a remarkable difference of behaviors among the different AIEOP centers. A need for common practice guidelines for this frequent illness arose from this observation. Our aim was to make the diagnosis and treatment of childhood ITP uniform. In the future we will evaluate the influence of these guidelines on practice behaviors. DATA SOURCES AND METHODS: Our main reference was the 1996 document produced by the American Society of Hematology (ASH). Their recommendations were updated with information from literature searched for in the MEDLINE database (June 1996 October 1998); search terms included: thrombocytopenia, ITP, diagnosis, therapy, children. The computerized search retrieved 83 articles. DATA EXTRACTION: the scientific validity of the literature was evaluated by a panel of members using published guidelines. The strength of the evidence was assessed using level of evidence criteria. Only data from level I and level II studies were taken in account. Only one study out of the 83 retrieved articles met these selection criteria and it was considered in addition to the 11 out of 581 articles selected in the ASH ITP guidelines. This preliminary work pointed out each issue about ITP not addressed by clinical studies and all participants in a Consensus Conference expressed their opinion about these issues. RESULTS: Diagnosis is essentially based on history, physical examination, a complete blood count and an examination of the peripheral blood smear. Treatment is recommended taking into account the clinical picture and number of platelets. The main difference between these guidelines and those from ASH are: AIEOP guidelines rely on the opinion of the members of the consensus conference, ASH ones on a panel of experts; therapeutic options include only products available in Italy; the indications to treatment rely more on clinical picture than on platelet number. INTERPRETATION AND CONCLUSIONS: These are explicitly developed, evidence-based practice guidelines to assist Italian pediatricians in making decisions about diagnosis and appropriate health care for patients with acute childhood ITP. PMID- 10756370 TI - Intracardiac thrombosis in a case of Behcet's disease associated with the prothrombin 20210G-A mutation. AB - Thrombosis occurs in 20 to 30% of patients with Behcet's disease (BD), but the precise pathogenic mechanism underlying the thrombotic tendency in these patients is not well known. Venous thromboses are commonly located in the lower extremities, but right intracardiac thrombi are extremely rare. We report for the first time on a young patient with BD associated the 20210G-A prothrombin gene mutation and right intracardiac thrombosis. We suggest that the association of BD with this newly recognized prothrombotic genetic mutation may have contributed to the development of the thrombotic event in this patient. PMID- 10756371 TI - Budd-Chiari syndrome in chronic myeloid leukemia. PMID- 10756372 TI - Human-platelet-antigen and neutrophil-antigen gene frequency in the Italian population determined by polymerase chain reaction with sequence specific primers. PMID- 10756373 TI - Cell cycle analysis in the diagnosis of Fanconi's anemia. PMID- 10756374 TI - Near-tetraploid acute myeloid leukemia after allogeneic bone marrow transplantation. PMID- 10756375 TI - A case of atypical myelodysplastic syndrome with a novel reciprocal translocation t(1;12)(q21;p13) PMID- 10756376 TI - Cytogenetic and molecular studies of variant Ph' translocations. PMID- 10756377 TI - Complete molecular remission induced by rituximab in a patient with diffuse large cell lymphoma. PMID- 10756378 TI - A brief intensive chemotherapy in T-prolymphocytic leukemia. PMID- 10756379 TI - Fulminant hemophagocytic syndrome as presenting feature of T-cell lymphoma and Epstein-Barr virus infection. PMID- 10756380 TI - Splenectomy in patients with refractory or relapsing thrombotic thrombocytopenic purpura. PMID- 10756381 TI - b-thalassemia intermedia resulting from compound heterozigosity for an IVSI-1 (G A) and a silent 5' UTR +33 (C-G) mutations. PMID- 10756382 TI - Peri-operative use of recombinant human erythropoietin in Jehovah's Witnesses. PMID- 10756383 TI - Methylenetetrahydrofolate reductase gene polymorphism and coronary artery disease in Taiwan Chinese. PMID- 10756384 TI - Cyclic oscillations of neutrophils, monocytes, and CD8-positive lymphocytes in a healthy subject. PMID- 10756385 TI - Just the Facts, Ma'am. PMID- 10756386 TI - Chemotherapy of Testis Cancer. AB - BACKGROUND: Prior to the use of cisplatin, durable complete remissions of metastatic testis cancer were rare. In 1977, a treatment program including a chemotherapy program of cisplatin, vinblastine, and bleomycin (PVB) led to high response rates and acceptable toxicities in patients with disseminated testis cancer. Since then, the BEP (bleomycin, etoposide, and cisplatin) regimen has been established as standard therapy for good- and poor-risk disease and ifosfamide-based regimens or high-dose chemotherapy with stem cell rescue as salvage therapy. METHODS: The results of those prospective, randomized clinical trials that have markedly improved the outlook of patients with this type of cancer have been reviewed. RESULTS: Categories of risk have been defined. Standard therapy for both good-risk and poor-risk disease remains BEP therapy. High-dose chemotherapy with autologous bone marrow or peripheral stem cell rescue transplantation is being investigated to overcome chemotherapy resistance. CONCLUSIONS: While the present state of the art for treating metastatic testicular cancer is promising, approximately one third of patients with "poor risk" disease will not achieve a remission. Trials of new agents and approaches are needed to increase patient survival. PMID- 10756387 TI - Current Status of Partial Nephrectomy in the Management of Kidney Cancer. AB - BACKGROUND: The technique of partial nephrectomy for managing renal cancers is well recognized, but guidelines regarding indications for its use are not generally accepted. METHODS: The authors review the indications for partial nephrectomy in various clinical situations, and they include their own experience to clarify the utility of the technique. RESULTS: Intraoperative renal ultrasound and helical computed tomography can assist the surgeon in technical decisions. Partial nephrectomy is considered when nephrectomy would render the patient anephric and dependent on dialysis. CONCLUSIONS: The technical and operative advances in partial nephrectomy make the approach increasingly attractive for patients with kidney cancer in a variety of clinical circumstances. PMID- 10756388 TI - The Mainz Classification of Renal Cell Tumors. AB - BACKGROUND: Tumors arising from the renal tubular epithelium have variable characteristics and have been subject to a variety of histologic classifications. METHODS: The authors describe the distinct clinical, pathologic, phenotypic, and genotypic features of different types of renal tumors. RESULTS: The Mainz classification is now widely accepted because characteristic genetic alterations have been demonstrated in each tumor type. CONCLUSIONS: The increasing emphasis on utilizing genetic characteristics of specific tumors is reflected by the more widespread use of the Mainz classification for renal cell tumors. PMID- 10756389 TI - Nutrients in the Chemoprevention of Prostate Cancer: Current and Future Prospects. AB - BACKGROUND: External factors such as diet and lifestyle may be important in the etiology of invasive prostate cancer. Specific features of prostate cancer, including high prevalence, long latency, and significant mortality and morbidity, provide the opportunities for chemoprevention. METHODS: The authors examine the experimental and epidemiological data demonstrating the chemopreventive activity, safety, and toxicity of chronic administration of these specific nutrients as chemopreventive agents in prostate cancer. RESULTS: Several nutrients have been identified as agents that inhibit mutagenesis and hyperproliferation or induce apoptosis or differentiation, which are critical characteristics for chemoprevention. Successful chemopreventive strategies require well-characterized agents, suitable cohorts, and reliable intermediate biomarkers of cancer for evaluating efficacy. Phytoestrogens/isoflavones, vitamins D and E, selenium, and lycopene have been identified as promising nutrients in the role of chemoprevention of prostate cancer. CONCLUSIONS: Clinical studies to evaluate the safety and effectiveness of these agents as future prospects in cancer chemoprevention, both individually and in combination, are warranted.